Sordarin Diterpene Glycosides with an Unusual 1,3-Dioxolan

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Sordarin Diterpene Glycosides with an Unusual 1,3-Dioxolan-4-one Ring from the Zoanthid-Derived Fungus Curvularia hawaiiensis TA26-15 Meng-Qi Zhang,†,‡ Kai-Xia Xu,†,‡ Ying Xue,†,‡ Fei Cao,†,‡,§ Lu-Jia Yang,†,‡ Xue-Mei Hou,†,‡ Chang-Yun Wang,*,†,‡,⊥ and Chang-Lun Shao*,†,‡

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†

Key Laboratory of Marine Drugs, The Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, People’s Republic of China § Chinese Center for Chirality, Key Laboratory of Medicinal Chemistry and Molecular Diagnostics of the Ministry of Education, College of Pharmacy, Hebei University, Baoding 071002, Hebei, People’s Republic of China ⊥ Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, People’s Republic of China S Supporting Information *

ABSTRACT: Six new sordarin tetracyclic diterpene glycosides, moriniafungins B−G (1−6), and a new sordaricin tetracyclic diterpene, sordaricin B (8), together with two known analogues, moriniafungin (7) and sordaricin (9), were isolated from the zoanthid-derived fungus Curvularia hawaiiensis TA26-15. The structures of the new compounds were elucidated by comprehensive analyses of spectroscopic data, including 1D and 2D NMR and MS data. Compounds 1−6 represent the first case of sordarins from marine-derived fungi possessing a sordarose with a spiro 1,3-dioxolan-4-one ring, which is rare in the nature. Compound 4 showed antifungal activity against Candida albicans ATCC10231 with an MIC value of 2.9 μM.

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study, chemical investigation of the EtOAc extract of C. hawaiiensis TA26-15 resulted in the isolation of seven sordarin glycosides, moriniafungins B−G (1−6)19 and the known moriniafungin (7),7 and a new sordaricin, sordaricin B (8),19 in addition to sordaricin (9).6 Herein, we reported the isolation, structure determination, and antifungal activity evaluation of these compounds.

arine-derived fungi are increasingly recognized as abundant sources of new bioactive and unique structural compounds.1−3 Terpenoids from marine-derived fungi reveal structural diversity and potential biological activities.4 Sordarin, a tetracyclic diterpene glycoside, was first isolated in 1969 from fermentations of the fungus Sordaria araneosa.5 Sordarin and its derivatives, diterpenes with a unique 5/6/5/5 fused tetracyclic ring system, have mainly been obtained from fungal cultures6−9 and by chemical synthesis.10,11 Sordarins have proven to be potent inhibitors of EF2 function in fungal protein synthesis with an extremely high level of selectivity for fungi while being ineffective against higher eukaryotes.12,13 A literature survey revealed that sordarins have mainly been obtained from terrestrial fungi,8−15 with only four sordarins from marine-derived fungi.14−16 During our ongoing search for new bioactive secondary metabolites from marine-derived fungi, a series of bioactive natural products with antifungal, antibacterial, and antiviral activities have been discovered.17 The zoanthid-derived fungus Curvularia hawaiiensis TA26-15 (Cochliobolus hawaiiensis)18 attracted our attention because the EtOAc extract of this fungus exhibited potent antifungal activities. In the present © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The marine-derived fungus C. hawaiiensis TA26-15, isolated from the inner tissue of the zoanthid Palythoa haddoni, was cultivated in rice medium supplemented with sea salt. The EtOAc extract (28.5 g) was separated by repeated column chromatography and purified by semipreparative HPLC to give compounds 1−9. The structures of known compounds moriniafungin (7) and sordaricin (9) were identified by comparison of their spectroscopic data (1H and 13C NMR, MS, [α]D, and IR) with those reported in the literature.6,7 Received: February 19, 2019

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DOI: 10.1021/acs.jnatprod.9b00164 J. Nat. Prod. XXXX, XXX, XXX−XXX

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molecular formula and NMR spectroscopic data, indicating that a 2-hydroxyoctanedioic acid side chain in 1 replaced the 2hydroxysebacic acid side chain in 7 (Tables 1 and 2). Additionally, in the negative ion ESIMS spectrum of 1, an ion at m/z 331.2 [M − C15H23O8]− revealed the sordaricin aglycone residue. The ion at m/z 489.2 [M − H − C8H12O4]− supported the sordarin residue (Figure S12). As was observed for moriniafungin,7 the direct evidence, a 1 H−13C correlation of H-2″ to C-3′, to connect the modified sordarose residue and the aliphatic acid side chain was lacking. However, the unusual carbonyl stretch at 1802 cm−1 in the IR spectrum revealed the presence of a carbonyl in a small ring, consistent with the connection of the sordarose residue and the aliphatic acid side via a 1,3-dioxolan-4-one ring at C-3′. Thus, the planar structure of 1 was determined. The general relative configuration of 1 was determined to be the same as that of 7 by 1D and 2D NOE spectroscopic data and coupling constants (Figure 1, Table 1). The 1,3-dioxolan4-one ring formed a spiro ring at C-3′ containing two stereogenic centers, resulting in four diastereomeric possibilities for this ring. The observation of an NOE correlation between H3-7′ (C-4′ O-methyl protons) and H-2″ in both 2D NOESY and 1D NOE difference experiments (Figure 2) suggested that only two possible isomers with C3′(R)/C-2″(S) or C3′(S)/C-2″(R) configurations may be present in 1. With consideration of the biogenetic origin, the absolute config-

Moriniafungin B (1) was obtained as a colorless solid with a molecular formula of C35H50O12 established from the (−)-HRESIMS spectrum, indicating 11 degrees of unsaturation. The signal at 3498 cm−1 in the IR spectrum suggested the presence of hydroxy groups, and the signals at 1802 and 1715 cm−1 indicated the appearance of carbonyl groups in 1. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) revealed that compound 1 was a sordarin glycoside with a modified sordarose residue (Figure 1) and that it closely resembled the known compound moriniafungin (7), originally from the plant endophytic fungus Morinia pestalozzioides,7 except for the aliphatic acid side chain. The aliphatic acid side chain in 1 was two methylenes shorter than that in 7, according to the

Table 1. 1H NMR Data for 1−6 and 8 (500 MHz, δ in ppm, J in Hz) no. 3 4 5 6 7 8 10 11 14 15 16 17 18 19 20 1′ 2′ 4′ 5′ 6′ 7′ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ COOCH3

δH (1)a

δH (2)a

δH (3)b

δH (4)c

2.01, m 1.83, m; 1.20, m 2.05, m; 1.28, m 2.10, m 1.79, m 1.90, m; 1.85, m 2.77, t (3.0) 6.11, d (3.0) 1.94, m; 1.22, m 9.75, s 0.79, d (7.0) 3.96, d (9.5); 3.79, d (9.5) 2.32, m 1.02, d (6.5) 0.97, d (6.5) 4.56, brs 3.74, s 3.35, d (10.0) 3.53, qd (6.0, 10.0) 1.28, d (6.0) 3.46, s 4.60, dd (7.0, 4.5) 1.86, m; 1.63, m 1.42, m 1.27, m 1.63, m 2.28, t (7.5)

2.02, m 1.89, m; 1.17, m 2.08, m; 1.29, m 2.10, m 1.80, m 1.96, m; 1.87, m 2.77, t (3.0) 6.11, d (3.0) 1.98, m; 1.31, m 9.75, s 0.79, d (7.0) 3.97, d (9.5); 3.80, d (9.5) 2.32, m 1.02, d (6.5) 0.97, d (6.5) 4.58, brs 3.74, s 3.35, d (10.0) 3.53, qd (6.0, 10.0) 1.28, d (6.0) 3.46, s 4.63, dd (7.0, 4.5) 1.87, m; 1.68, m 1.68, m 2.39, t (7.0)

2.06, m 1.87, m; 1.15, m 2.15, m; 1.32, m 2.13, m 1.82, m 1.90, m; 1.88, m 2.77, t (3.0) 6.09, d (3.0) 2.01, m; 1.27, m 9.74, s 0.82, d (6.0) 3.94, d (9.5); 3.77, d (9.5) 2.30, m 1.04, d (6.5) 0.98, d (6.5) 4.49, brs 3.74, s 3.35, d (10.0) 3.52, qd (6.0, 10.0) 1.33, d (6.0) 3.48, s 4.64, dd (7.0, 4.5) 1.88, m; 1.69, m 1.68, m 2.42, t (7.0)

1.96, m 1.88, m; 0.99, m 2.06, m; 1.20, m 2.08, m 1.78, m 1.98, m; 1.81, m 2.70, t (3.0) 6.07, d (3.0) 1.95, m; 1.30, m 9.72, s 0.78, d (7.0) 4.00, d (9.5); 3.73, d (9.5) 2.32, m 1.03, d (6.5) 0.97, d (6.5) 4.54, brs 3.71, s 3.36, d (10.0) 3.54, qd (6.0, 10.0) 1.33, d (6.0) 3.48, s 4.60, dd (6.5, 4.5) 1.82, m; 1.63, m 1.46, m 1.23, m 1.29, m 1.28, m 1.62, m 2.29, t (7.5) 3.66, s

3.67, s

δH (5)a 2.03, 1.85, 2.12, 2.09, 1.73, 1.98, 2.74, 6.14, 1.96,

m m; 1.13, m; 1.21, m m m; 1.84, t (3.0) d (3.0) m; 1.22,

δH (6)a m m

m

m

0.78, d (7.0) 4.00, d (9.5); 3.79, d (9.5) 2.42, m 1.03, d (6.5) 1.02, d (6.5) 4.55, brs 3.72, s 3.35, d (10.0) 3.52, qd (6.0, 10.0) 1.28, d (6.0) 3.46, s 4.59, dd (7.0, 4.5) 1.83, m; 1.67, m 1.48, m 1.36, m 1.36, m 1.36, m 1.61, m 2.29, t (7.5)

2.01, 1.90, 2.05, 2.09, 1.80, 2.03, 2.71, 6.11, 1.99,

m m; 1.11, m; 1.20, m m m; 1.86, t (3.0) d (3.0) m; 1.30,

δH (8)b m m

m

m

0.78, d (6.5) 4.00, d (9.5); 3.78, d (9.5) 2.43, m 1.03, d (6.5) 1.01, d (6.5) 4.55, brs 3.72, s 3.35, d (10.0) 3.51, qd (6.0, 10.0) 1.28, d (6.0) 3.46, s 4.59, dd (7.0, 4.5) 1.84, m; 1.67, m 1.48, m 1.42, m 1.35, m 1.35, m 1.62, m 2.29, t (7.5) 3.60, s

2.06, 1.86, 2.14, 2.17, 1.82, 1.95, 2.53, 6.09, 1.98,

m m; 1.12, m; 1.28, m m m; 1.82, t (3.0) d (3.0) m; 1.25,

m m

m

m

0.81, d (7.0) 3.79, d (11.0); 3.64, d (11.0) 2.45, m 1.04, d (6.5) 1.02, d (6.5)

a

Measured in acetone-d6, bMeasured in CD3OD, cMeasured in CDCl3 B

DOI: 10.1021/acs.jnatprod.9b00164 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C NMR Data for 1−6 and 8 (125 MHz, δ in ppm)

no.

δC (1)a

δC (2)a

δC (3)b

δC (4)c

δC (5)a

δC (6)a

δC (8)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 10″ COOCH3

72.9, C 59.2, C 42.2, CH 26.9, CH2 32.8, CH2 32.0, CH 42.2, CH 29.1, CH2 66.5, C 46.9, CH 131.4, CH 149.2, C 173.8, C 29.7, CH2 204.0, CH 17.8, CH3 75.3, CH2 28.4, CH 21.5, CH3 23.0, CH3 99.6, CH 74.8, CH 109.3, C 83.9, CH 70.2, CH 17.9, CH3 61.9, CH3 173.4, C 76.4, CH 32.8, CH2 25.4, CH2 29.0, CH2 25.2, CH2 34.0, CH2 174.5, C

72.9, C 59.2, C 42.2, CH 26.9, CH2 32.4, CH2 31.9, CH 42.1, CH 29.9, CH2 66.5, C 46.9, CH 131.4, CH 149.1, C 173.9, C 29.4, CH2 204.0, CH 17.8, CH3 75.3, CH2 28.4, CH 21.5, CH3 23.0, CH3 99.6, CH 74.8, CH 109.4, C 83.8, CH 70.2, CH 17.9, CH3 61.9, CH3 173.2, C 76.3, CH 32.8, CH2 21.2, CH2 33.6, CH2 174.3, C

73.8, C 59.7, C 42.7, CH 27.2, CH2 32.6, CH2 32.4, CH 42.6, CH 29.7, CH2 66.9, C 47.4, CH 131.7, CH 149.9, C 174.0, C 30.4, CH2 206.3, CH 17.8, CH3 76.3, CH2 28.9, CH 21.5, CH3 23.1, CH3 100.3, CH 75.0, CH 110.0, C 84.2, CH 71.0, CH 17.9, CH3 62.2, CH3 174.0, C 76.3, CH 33.1, CH2 21.5, CH2 34.1, CH2 175.2, C

72.4, C 59.0, C 41.8, CH 26.4, CH2 32.1, CH2 31.1, CH 41.4, CH 29.3, CH2 65.7, C 46.3, CH 130.8, CH 148.4, C 172.7, C 29.4, CH2 204.8, CH 17.5, CH3 74.5, CH2 27.8, CH 21.3, CH3 22.7, CH3 98.1, CH 74.0, CH 108.0, C 82.8, CH 69.8, CH 17.6, CH3 62.1, CH3 172.7, C 76.0, CH 32.4, CH2 25.0, CH2 29.1, CH2 29.1, CH2 29.1, CH2 24.7, CH2 34.1, CH2 174.5, C 51.7, CH3

72.1, C 56.0, C 44.8, CH 27.7, CH2 33.0, CH2 32.6, CH 41.9, CH 29.6, CH2 67.2, C 45.7, CH 132.5, CH 149.3, C 173.4, C 32.2, CH2 173.4, C 17.8, CH3 76.0, CH2 28.7, CH 22.0, CH3 23.1, CH3 99.7, CH 74.8, CH 109.2, C 83.9, CH 70.2, CH 17.9, CH3 61.9, CH3 173.4, C 76.4, CH 33.6, CH2 25.6, CH2 30.2, CH2 30.1, CH2 29.9, CH2 25.5, CH2 34.2, CH2 174.7, C

72.1, C 56.4, C 44.8, CH 27.7, CH2 33.0, CH2 32.7, CH 41.9, CH 29.6, CH2 67.2, C 45.7, CH 132.3, CH 149.5, C 173.4, C 32.3, CH2 173.4, C 17.9, CH3 76.2, CH2 28.7, CH 22.1, CH3 23.2, CH3 99.7, CH 74.9, CH 109.2, C 83.9, CH 70.2, CH 17.9, CH3 61.9, CH3 173.4, C 76.4, CH 33.6, CH2 25.6, CH2 30.2, CH2 30.1, CH2 29.9, CH2 25.4, CH2 34.3, CH2 174.1, C 51.5, CH3

74.9, C 59.1, C 45.3, CH 28.1, CH2 34.0, CH2 33.4, CH 42.4, CH 29.0, CH2 68.2, C 46.7, CH 132.5, CH 149.8, C 174.4, C 32.6, CH2 176.9, C 17.9, CH3 68.1, CH2 29.0, CH 22.1, CH3 23.2, CH3

52.1, CH3

a

Measured in acetone-d6, bMeasured in CD3OD, cMeasured in CDCl3

Figure 1. Key 2D NMR correlations for 1 and 8.

by cocrystallization with EF2.7 The very similar chemical shifts near the dioxolanone rings of 1 and 7 (Table S1) support a shared configuration. Correspondingly, the absolute configuration of the modified sordarose residue could be assigned. To determine the absolute configuration of the aglycone, 1 together with 7 were hydrolyzed to obtain the sordaricin units. The similarity of the specific rotations of the sordaricin units (1: [α]20D −56.2 (c 0.200, MeOH); 7: [α]20D −59.8 (c 0.200, MeOH)) to that of co-isolated sordaricin (9) ([α]20D −53.6 (c 0.221, MeOH); lit. [α]20D −62.0 (c 0.330, MeOH)6)

Figure 2. Two possible configurations for the dioxolanone ring in 1.

uration of the 1,3-dioxolan-4-one ring in 1 was deduced to be C3′(R)/C-2″(S), the same as that in the co-isolated known compound 7, of which the absolute configuration was assigned C

DOI: 10.1021/acs.jnatprod.9b00164 J. Nat. Prod. XXXX, XXX, XXX−XXX

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(Table 2). A carboxyl (δC 176.9) was assigned at C-2 in 8, replacing the aldehyde group in 9. The relative configuration of 8 was determined to be the same as that of 9 by a NOESY spectrum (Figure 1). Compound 8 displayed a similar specific rotation to that of 9. By biogenetic consideration, the absolute configuration of 8 could be proposed as 1R,2S,3R,6R,7R,9S,10R, the same as in 9. From the marine-derived fungus C. hawaiiensis TA26-15, nine tetracyclic diterpenes have been obtained. Among them, moriniafungins B−G (1−6) were identified as sordarin derivatives containing a modified sordarose. Their structures varied in the aldehyde group linked to C-2, the chain length of the aliphatic acid side chain, and a free acid or methyl ester at the terminus of the aliphatic acid side chain. This is the second report of sordarins containing a sordarose with a spiro 1,3dioxolan-4-one ring, which is rare in nature, and this is the first discovery of sordarins from marine-derived fungi possessing a sordarose with this unprecedented ring. Interestingly, the first sordarin with a sordarose incorporating a spiro 1,3-dioxolan-4one ring, moriniafungin (7), was obtained from a terrestrial Morinia fungus7 belonging to the Sordariomycetes class, while moriniafungins B−G (1−6) were discovered from a marinederived Curvularia fungus belonging to another class, Dothidiomycetes. All of the isolated compounds were tested for their antifungal, antibacterial, and antiviral activities. Compound 4 displayed strong antifungal activity against C. albicans ATCC10231 with an MIC value of 2.9 μM (Table 3).

confirmed the same absolute configuration of the sordaricin units in 1 and 7 to that of 9, the absolute configuration of which was determined by chemical syntheses.20 Therefore, the absolute configuration of 1, named moriniafungin B, was proposed as 1R,2S,3R,6R,7R,9S,10R,1′R,2′R,3′R,4′R,5′R,2″S. Moriniafungin C (2) was assigned the molecular formula C33H46O12 by HRESIMS. The spectroscopic features suggested that 2 also is a sordarin derivative (Tables 1 and 2). The aliphatic acid side chain in 2 was four methylenes shorter than that in 7,7 according to the molecular formula and NMR spectroscopic data, indicating that a 2-hydroxyhexanedioic acid side chain in 2 took the place of the 2-hydroxysebacic acid side chain in 7. The molecular formula of moriniafungin D (3) was established as C34H48O12 from HRESIMS data. The 1D NMR data of 3 were very close to those of 2 (Tables 1 and 2). The major difference between 3 and 2 was the appearance of a signal for an additional methoxy in 3 (δH 3.67, δC 52.1) (Tables 1 and 2). The HMBC correlations from H-4″ to C-6′′ and the new OCH3 to C-6″ indicated that a methyl ester appeared at C-6′′ of 3 (Figure S1). Moriniafungin E (4) possessed the molecular formula C38H56O12 based on the HRESIMS spectrum. The spectroscopic features (Tables 1 and 2) suggested that its structure was very similar to that of 7.7 One more methoxy (δH 2.29, δC 51.7) appeared in 4, according to the NMR spectroscopic data. The HMBC correlations of H-8″ to C-10′′ and OCH3 to C10″ indicated that the methyl ester appeared at C-10′′ in 4 (Figure S1). Moriniafungin F (5) was assigned the molecular formula C37H54O13 with a molecular mass of 706, 16 units higher than that of 7. Detailed analysis of the 1H and 13C NMR spectroscopic data (Tables 1 and 2) revealed that the structure of 5 was similar to that of 7,7 except for the absence of the aldehyde group (δH 9.68, δC 205.1). In the 13C NMR spectrum, the shielded chemical shift of C-2 (δC 56.0) and the deshielded C-3 (δC 44.8) and C-14 (δC 32.2) in 5 compared with those in 7 in the same solvent revealed that a carboxyl at C-2 in 5 replaced the aldehyde group in 7. The molecular formula of moriniafungin G (6) was determined to be C38H56O13 by HRESIMS data. The 1D NMR data of 6 were very close to those of 5 (Tables 1 and 2). The major difference between these two compounds was the appearance of a second methoxy signal in 6 (δH 3.60, δC 51.5). The HMBC correlations from H-8″ to C-10′′ and from OCH3 to C-10″ indicated a methyl ester at C-10′′ in 6 (Figure S1). The relative configurations of 2−6 were determined to be the same as that of 1 based on the 1D and 2D NOE spectroscopic data and coupling constants (Table 1, Figure S1). Compounds 2−6 had the same skeleton and similar specific rotations to that of 1. By biogenetic consideration, the absolute configurations of 2−6 could be proposed as 1R,2S,3R,6R,7R,9S,10R,1′R,2′R,3′R,4′R,5′R,2″S, the same as in 1 and 7. Sordaricin B (8) possessed the molecular formula C20H28O5 with a molecular mass of 348, 16 units higher than that of 9. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) revealed that the structure of 8 was similar to that of the known compound sordaricin (9), the aglycone of sordarin previously reported from Sordaria araneosa,6 except for the absence of the aldehyde group. In the 13C NMR spectrum, the chemical shifts of C-3 (δC 45.3) and C-14 (δC 32.6) were deshielded compared with those in 9 in the same solvent

Table 3. Antifungal Activity of 1−9 against C. albicans ATCC10231 compd

MIC (μM)

compd

MIC (μM)

1 2 3 4 5

8.7 12 9.8 2.9 15

6 7 8 9 carbendazim

13 3.7 24 18 0.16

Compounds 1−4 and 7 exhibited stronger activities than 9, and compounds 5 and 6 had stronger activities than 8, indicating that the glycosyl moiety may be the essential group for the antifungal activity. These results were consistent with the findings reported in the literature.9,21 The antifungal activity order of the compounds, 7 > 1 > 2 and 4 > 3, indicated that the length of the aliphatic acid side chain may contribute to the effect on activity. The antifungal activities of 4 and 7 were stronger than those of 6 and 5, respectively, indicating that the C-2 carboxylic acid had a detrimental effect on activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Nicolet-Nexus-470 spectrometer using KBr pellets (Thermo Electron). NMR spectra were acquired using a JEOL JEMECP NMR spectrometer at 500 MHz for 1H and 125 MHz for 13 C, using tetramethylsilane as an internal standard. ESIMS and HRESIMS spectra were measured on a Micromass Q-TOF spectrometer (Waters Corp.). HPLC separation was performed using a Hitachi L-2000 HPLC system (Hitachi High Technologies) coupled with a Hitachi L-2455 photodiode array detector. A Kromasil C18 semipreparative HPLC column (250 × 10 mm, 5 μm) (Eka Nobel) was used. Silica gel (200−300 mesh; Qingdao Marine Chemical Group Co.) and Sephadex LH-20 (Amersham Biosciences D

DOI: 10.1021/acs.jnatprod.9b00164 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Sordaricin B (8): colorless solid; [α]20D −43.2 (c 0.454, MeOH); IR (KBr) νmax 2955, 1700, 1399 cm−1; 1H and 13C NMR see Tables 1 and 2; (−)-HRESIMS m/z 347.1858 [M − H]− (calcd for C20H27O5, 347.1853). Moriniafungin (7): colorless solid; [α]20D −47.6 (c 0.333, MeOH) (lit. [α]25D −39.9 (c 0.539, MeOH)).7 Sordaricin (9): colorless solid; [α]20D −53.6 (c 0.221, MeOH) (lit. [α]20D −62.0 (c 0.330, MeOH)).6 Bioassays. Antifungal Bioassays. The antifungal bioassays were conducted following the National Committee for Clinical Laboratory Standards (NCCLS) recommendations.22 Nine pathogenic fungal strains, Candida albicans ATCC10231, Aspergillus niger van Tiegh ATCC16888, Thielaviopsis paradoxa ACCC36964, Pestalotiopsis theae Steyaert ACCC38043, Gloeosporium musarum ATCC16011, Alternaria oleracea ACCC39304, Bipolaris oryzae ACCC36967, Exserohilum turcicum ACCC37867, Pestalotia calabae, were grown on potato dextrose agar. Targeted microbes (three to four colonies) were prepared from broth culture (28 °C for 48 h), and the final spore suspensions of fungi were 104 mycelial fragments/mL. The test compounds (100 μM as stock solution in DMSO and serial dilutions) were transferred to a 96-well clear plate in triplicate, and the fungal spore suspensions were added to each well, achieving a final volume of 200 μL. Carbendazim was used as a positive control. After incubation, the absorbances at 492 nm of the tested solutions were measured with a microplate reader. The minimum inhibitory concentration (MIC) values were measured and calculated. Antibacterial Assays. The antibacterial activity was evaluated by the conventional broth dilution assay.23 Nine pathogenic bacterial strains, Bacillus cereus ATCC11077, B. subtilis ATCC6633, Staphylococcus epidermidis ATCC12228, S. aureus ATCC27154, Tetragenococcus halophilus ATCC13623, Pseudomonas putida ATCC17485, Vibrio parahemolyticus ATCC17802, Kocuria rhizophila ATCC9341, and Nocardia brasiliensis ATCC19019, were used, and ciprofloxacin was used as a positive control. Antiviral Assays. The antiviral activities against respiratory syncytial virus (RSV), coxsackievirus B3 (CoxB3), influenza virus H1N1, herpes simplex virus (HSV), and enterovirus 71 (EV71) were determined by the cytopathic effects assays, according to established procedures.24 Ribavirin was used as a positive control.

Inc.) were used for column chromatography. Precoated silica gel GF254 plates (Yantai Zifu Chemical Group Co.) were used for analytical TLC. Fungal Material. The fungal strain Curvularia hawaiiensis TA2615 was isolated from a piece of fresh tissue from the inner part of the zoanthid Palythoa haddoni, collected from the Weizhou coral reefs (109°10′ E, 20°54′ N) in the South China Sea in April 2010. The fungus was identified as C. hawaiiensis according to its morphological traits and a molecular protocol by amplification and sequencing of the DNA sequences of the ITS region of the rRNA gene. The 520 base pair ITS sequence had 100% sequence identity to that of C. hawaiiensis (MN173132). The strain (GenBank accession JF819139) was deposited in the Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, P.R. China. Extraction and Isolation. The fermentation was carried out in 80 Erlenmeyer flasks (500 mL), each containing 45 g of rice. Distilled H2O (75 mL) and natural sea salt (2.25 g; Yangkou saltern, China) were added into each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 45 days. After 45 days, the fermentated rice sample was harvested and extracted repeatedly using EtOAc (2 × 8 L) followed by CH2Cl2/MeOH (1:1) (2 × 8 L). The solvents in the extracts were evaporated in a vacuum, respectively. Then the extracts were combined and further extracted by EtOAc/ H2O (1:1) to provide an EtOAc extract (28.5 g). The EtOAc extract was subjected to a silica gel column chromatography (CC) eluting with petroleum ether/EtOAc in a gradient from 100:0 to 0:100, followed by CH2Cl2/MeOH (from 100:0 to 0:100), to obtain eight fractions (Fr.1−Fr.8). Fr.5 was separated on a Sephadex LH-20 column with a mixture of CH2Cl2/ MeOH (v/v, 1:1) to afford three subfractions (Fr.5.1−Fr.5.3). Fr.5.1 was purified on an ODS column eluting with 50% MeOH/H2O and further separated by semipreparative HPLC eluting with 50% MeOH/ (H2O + 0.1% trifluoroacetic acid (TFA)) to give 5 (8.9 mg), 8 (3.8 mg), and 9 (18.3 mg). Fr.5.2 was subjected to an ODS column eluting with 50% MeOH/H2O and finally purified by semipreparative HPLC eluting with 42% CH3CN/(H2O + 0.1% TFA) to yield 2 (15.6 mg), 3 (3.2 mg), and 1 (8.0 mg). Fr.5.3 was chromatographed on an ODS column eluting with 50% MeOH/H2O and then purified by semipreparative HPLC eluting with 60% CH3CN/(H2O + 0.1% TFA) to obtain 4 (19.8 mg), 6 (6.5 mg), and 7 (32.0 mg). Moriniafungin B (1): colorless solid; [α]20D −64.4 (c 0.475, MeOH); IR (KBr) νmax 3498, 2952, 2868, 1802, 1715, 1459, 1200,1139 cm−1; 1H and 13C NMR see Tables 1 and 2; (−)-ESIMS m/z 489.2 [M − H − C8H12O4]−, m/z 331.2 [M − C15H23O8]−; (−)-HRESIMS m/z 661.3224 [M − H]− (calcd for C35H49O12, 661.3219). Moriniafungin C (2): colorless solid; [α]20D −40.4 (c 0.428, MeOH); IR (KBr) νmax 3600, 3128, 2958, 2869, 1400, 1199, 1905 cm−1; 1H and 13C NMR see Tables 1 and 2; (−)-HRESIMS m/z 633.2912 [M − H]− (calcd for C33H45O12, 633.2906). Moriniafungin D (3): colorless solid; [α]20D −35.1 (c 0.435, MeOH); IR (KBr) νmax 3600, 2958, 1798, 1683, 1399, 1207, 1096 cm−1; 1H and 13C NMR see Tables 1 and 2; (−)-HRESIMS m/z 647.3063 [M − H]− (calcd for C34H47O12, 647.3062). Moriniafungin E (4): colorless solid; [α]20D −45.1 (c 0.367, MeOH); IR (KBr) νmax 2937, 2868, 1803, 1717, 1201, 1096 cm−1; 1H and 13C NMR see Tables 1 and 2; (+)-HRESIMS m/z 705.3846 [M + H]+ (calcd for C38H57O12, 705.3845). Moriniafungin F (5): colorless solid; [α]20D −50.1 (c 0.375, MeOH); IR (KBr) νmax 3674, 2935, 1799, 1701 cm−1; 1H and 13C NMR see Tables 1 and 2; (−)-HRESIMS m/z 705.3489 [M − H]− (calcd for C37H53O13, 705.3481). Moriniafungin G (6): colorless solid; [α]20D −50.5 (c 0.364, MeOH); IR (KBr) νmax 3675, 3651, 2951, 1799, 1704, 1201 cm−1; 1H and 13C NMR see Tables 1 and 2; (−)-HRESIMS m/z 719.3646 [M − H]− (calcd for C38H55O13, 719.3637).

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00164. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (C.-Y.W.): [email protected]. Tel: +86-5328203-1536. Fax: +86-532-8203-1536. *E-mail (C.-L.S.): [email protected]. Tel: +86-5328203-1381. ORCID

Fei Cao: 0000-0002-5676-3176 Chang-Yun Wang: 0000-0002-0236-1606 Chang-Lun Shao: 0000-0001-7230-188X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 41830535; U1606403), the National Key Research and Development Program of China (No. 2018YFC0310900), the Scientific and Technological E

DOI: 10.1021/acs.jnatprod.9b00164 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology (No. 2016ASKJ08), the Fundamental Research Funds for the Central Universities of China (No. 201762017), and the Taishan Scholars Program, China.

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REFERENCES

(1) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311−335. (2) Zhu, A.; Zhang, X. W.; Zhang, M.; Li, W.; Ma, Z. Y.; Zhu, H. J.; Cao, F. Mar. Drugs 2018, 16, 312. (3) Blunt, J. W.; Carroll, A. R.; Copp, B. R.; Davis, R. A.; Keyzers, R. A.; Prinsep, M. R. Nat. Prod. Rep. 2018, 35, 8−53. (4) Elissawy, A. M.; El-Shazly, M.; Ebada, S. S.; Singab, A. N. B.; Proksch, P. Mar. Drugs 2015, 13, 1966−1992. (5) Sigg, H. P.; Binningen, S. C. U.S. 3,432,598,196 Antibiotic SL 1969, 2266. (6) Hauser, D.; Sigg, H. P. Helv. Chim. Acta 1971, 54, 1178−1190. (7) Basilio, A.; Justice, M.; Harris, G.; Bills, G.; Collado, J.; Cruz, M. de la; Diez, M. T.; Hernandez, P.; Liberator, P.; Nielsen kahn, J.; Pelaez, F.; Platas, G.; Schmatz, D.; Shastry, M.; Tormo, J. R.; Andersen, G. R.; Vicente, F. Bioorg. Med. Chem. 2006, 14, 560−566. (8) Pongcharoen, W.; Rukachaisirikul, V.; Phongpaichit, S.; Kuhn, T.; Pelzing, M.; Sakayaroj, J.; Taylor, W. C. Phytochemistry 2008, 69, 1900−1902. (9) Chang, Y. C.; Lu, C. K.; Chiang, Y. R.; Wang, G. J.; Ju, Y. M.; Kuo, Y. H.; Lee, T. H. J. Nat. Prod. 2014, 77, 751−757. (10) Kato, N.; Kusakabe, S.; Wu, X.; Kamitamari, M.; Takeshita, H. J. Chem. Soc., Chem. Commun. 1993, 12, 1002−1004. (11) Schulé, A.; Liang, H.; Vors, J. P.; Ciufolini, M. A. J. Org. Chem. 2009, 74, 1587−1597. (12) Justice, M. C.; Ku, T. M.; Hsu, J.; Carniol, K.; Schmatz, D.; Nielsen, J. J. Biol. Chem. 1999, 274, 4869−4875. (13) Chakraborty, B.; Mukherjee, R.; Sengupta, J. J. Comput.-Aided Mol. Des. 2013, 27, 173−184. (14) Daferner, M.; Mensch, S.; Anke, T.; Sterner, O. Z. Naturforsch., C: J. Biosci. 1999, 54, 474−480. (15) Tanaka, M.; Moriguchi, T.; Kizuka, M.; Ono, Y.; Miyakoshi, S. I.; Ogita, T. J. Antibiot. 2002, 55, 437−441. (16) Zhou, Q. H.; Xu, D.; Fan, C. Q.; Yang, Y. P.; Li, H. R.; Chen, B. Chin. J. Mar. Drugs 2013, 32, 26−30. (17) (a) Shao, C. L.; Wu, H. X.; Wang, C. Y.; Liu, Q. A.; Xu, Y.; Wei, M. Y.; Qian, P. Y.; Gu, Y. C.; Zheng, C. J.; She, Z. G.; Lin, Y. C. J. Nat. Prod. 2011, 74, 629−633. (b) Liu, Q. A.; Shao, C. L.; Gu, Y. C.; Blum, M.; Gan, L. S.; Wang, K. L.; Chen, M.; Wang, C. Y. J. Agric. Food Chem. 2014, 62, 3183−3191. (c) Chen, M.; Shao, C. L.; Meng, H.; She, Z. G.; Wang, C. Y. J. Nat. Prod. 2014, 77, 2726−2730. (d) Jia, Y. L.; Wei, M. Y.; Chen, H. Y.; Guan, F. F.; Wang, C. Y.; Shao, C. L. Org. Lett. 2015, 17, 4216−4219. (e) Shi, T.; Qi, J.; Shao, C. L.; Zhao, D. L.; Hou, X. M.; Wang, C. Y. Mar. Drugs 2017, 15, 146. (18) Qin, X. Y.; Yang, K. L.; Li, J.; Wang, C. Y.; Shao, C. L. Mar. Biotechnol. 2015, 17, 99−109. (19) The original moriniafungin and sordaricin, which were not formally named with an A designation as the first representative in the structure class, are considered to be moriniafungin A and sordaricin A. (20) Mander, L. N.; Thomson, R. J. Org. Lett. 2003, 5, 1321−1324. (21) Liang, H. Beilstein J. Org. Chem. 2008, 4, 31. (22) National Committee on Clinical Laboratory Standards (NCCLS). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidial Forming Filamentous Fungi, NCCLS document M38-A; NCCLS: Wayne, PA, USA, 2002. (23) Appendino, G.; Gibbons, S.; Giana, A.; Pagani, A.; Grassi, G.; Stavri, M.; Smith, E.; Rahman, M. M. J. Nat. Prod. 2008, 71, 1427− 1430. (24) Grassauer, A.; Weinmuellner, R.; Meier, C.; Pretsch, A.; Priesc hl-Grassauer, E.; Unger, H. Virol. J. 2008, 5, 107.

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DOI: 10.1021/acs.jnatprod.9b00164 J. Nat. Prod. XXXX, XXX, XXX−XXX