Cytotoxic and Antibacterial Eremophilane Sesquiterpenes from the

(1,2) As part of an ongoing search for new and bioactive natural products from ... (3) The strain was then fermented on a large scale (50 L), and its ...
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Article Cite This: J. Nat. Prod. 2018, 81, 1405−1410

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Cytotoxic and Antibacterial Eremophilane Sesquiterpenes from the Marine-Derived Fungus Cochliobolus lunatus SCSIO41401 Wei Fang,†,‡ Jianjiao Wang,‡ Junfeng Wang,‡ Liqiao Shi,† Kunlong Li,‡ Xiuping Lin,‡ Yong Min,† Bin Yang,‡ Lan Tang,§ Yonghong Liu,‡ and Xuefeng Zhou*,‡ †

Hubei Biopesticide Engineering Research Center, Hubei Academy of Agricultural Science, Wuhan 430064, China CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China § Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China

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‡

S Supporting Information *

ABSTRACT: Three new eremophilane sesquiterpenes, dendryphiellins H−J (1−3), and three new phthalide natural products (4−6) were isolated from the marine-derived fungus Cochliobolus lunatus SCSIO41401. Their structures including absolute configurations were determined by spectroscopic and calculated ECD analyses. Dendryphiellin I (2) showed cytotoxic and antibacterial activities against five cancer cell lines (IC50 1.4 to 4.3 μM) and three bacterial species (MIC 1.5 to 13 μg/mL), respectively. Dendryphiellin J (3), a rare naturally occurring aldoxime analogue, displayed cytotoxicities against ACHN and HepG-2 cells with IC50 values of 3.1 and 5.9 μM, respectively. Further studies indicated that 3 induced apoptosis in ACHN cells in a dose- and time-dependent manner. and had growth inhibition of 62% at a concentration of 50 μg/ mL (p < 0.05 compared with control).3 The strain was then fermented on a large scale (50 L), and its EtOAc extract was fractionated and purified by chromatographic methods to yield nine pure compounds.

M

arine-derived microorganisms have shown promising potential to produce diverse bioactive metabolites with new chemical structures, and endophytic fungi from various marine algal hosts are increasingly being considered as sources of pharmaceutical compounds.1,2 As part of an ongoing search for new and bioactive natural products from marine microorganisms, an organic extract prepared from a culture of marine-derived strain SCSIO41401 was found to be active against human HepG2 cells. The strain was assigned as Cochliobolus lunatus SCSIO41401, according to the ITS sequence. In this study, three eremophilane sesquiterpenes (1−3) and three phthalides derivatives (4−6) were isolated from the culture extract of this fungus, together with purpureone (7), emodin (8), and 2,5-dimethyl-7-hydroxychromone. Compounds 1−3, 5, and 6 were identified as new compounds, while 4 was obtained as a new natural product. Those compounds were tested for their cytotoxicities against five cancer cell lines and for antibacterial activities against four bacterial species. Described herein are the isolation, structure determination, and biological evaluation of these compounds.

Compound 1 was isolated as a colorless oil. Its molecular formula was established by HRESIMS to be C15H20O3. Comparison of the 1H and 13C NMR data for 1 (Table 1) with those of reported dendryphiellin G4 or dihydrobipolaroxin 5 suggested that they had the same eremophilane sesquiterpene skeleton. The major difference between them was that the ketone carbonyl at the 3-position in dendryphiellin

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RESULTS AND DISCUSSION The EtOAc extracts of the small-scale liquid fermentation (1 L) of several marine microorganism strains were prepared and screened for their bioactivities. The extract of strain SCSIO41401 showed obvious activities against HepG2 cells © 2018 American Chemical Society and American Society of Pharmacognosy

Received: January 3, 2018 Published: May 22, 2018 1405

DOI: 10.1021/acs.jnatprod.8b00015 J. Nat. Prod. 2018, 81, 1405−1410

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Table 1. NMR Spectroscopic Data (1H 700 MHz, 13C 175 MHz, CD3OD) for 1−3 1 δC, type 1 2 3

128.9, CH 140.7, CH 33.4, CH2

4 5 6

40.0, CH 37.7, C 47.2, CH2

7 8 9 10 11 12

77.8, C 199.8, C 122.8, CH 166.7, C 154.2, C 111.7, CH2 63.1, CH2

13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ OCH3

19.0, CH3 14.7, CH3

2 δH (J in Hz) 6.24, d (9.8) 6.32, m a: 2.25, m b: 2.14, m 1.72, m a: 2.03, d (14.6) b: 2.08, d (14.6)

5.76, s

a: 5.40, brs b: 5.34, brs a: 4.05, d (14.2) b: 3.98, d (14.2) 1.26, s 0.98, d (6.9)

3

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

132.2, CH 134.0, CH 70.6, CH

6.51, d (9.8) 6.28, dd (9.8, 5.0) 5.46, dd (5.0, 5.0)

132.4, CH 133.6, CH 70.6, CH

6.49, d (9.8) 6.27, dd (9.8, 5.0) 5.46, dd (5.0, 5.0)

42.8, CH 37.5, C 46.4, CH2

1.99, dq (5.0, 7.1)

43.0, CH 37.5 46.5, CH2

1.98, dq (5.0, 7.1)

75.9, C 198.0, C 125.0, CH 163.5, C 156.4, C 136.9, CH2 194.9, CH 23.0, CH3 9.2, CH3 168.6, C 116.0, CH 151.9, CH 133.1, C 150.2, CH 36.2, CH 31.0, CH2 12.3, CH3 20.5, CH3 12.5, CH3

a: 2.01, d (14.3) b: 2.09, d (14.3)

5.92, s

a: 6.83, brs b: 6.41, brs 9.51, s 1.56, s 1.02, d (7.1) 5.82, d (15.6) 7.32, d (15.6) 5.71, d (9.9) 2.50, m a: 1.44, m b: 1.32, m 0.87, t (7.4) 1.01, d (6.6) 1.82, s

76.7, C 198.8, C 125.6, CH 162.6, C 148.3, C 123.1, CH2 149.4, CH 23.2, CH3 10.6, CH3 168.7, C 116.0, CH 151.9, CH 133.1, C 150.2, CH 36.2, CH 31.0, CH2 12.3, 20.5, 12.5, 62.2,

CH3 CH3 CH3 CH3

a: 2.03, d (14.3) b: 2.45, d (14.3)

5.92, s

5.99, brs 5.60, brs 7.67, s 1.55, s 1.04, d (7.1) 5.82, d (15.6) 7.32, d (15.6) 5.71, d (9.8) 2.50, m a: 1.44, m b: 1.32, m 0.87, t (7.4) 1.01, d (6.7) 1.82, s 3.70, s

Figure 1. Key HMBC, COSY, and NOESY correlations of 1−6.

G4 was replaced by a methylene in 1. This was further confirmed by observed HMBC correlations from H2-3 (δH 2.25, 2.14) to C-1 (δC 128.9), C-2 (δC 140.7), C-4 (δC 40.0), C-5 (δC 37.7), and C-15 (δC 14.7) and COSY correlations of H-1 (δH 6.24, d, J = 9.8 Hz)/H-2 (δH 6.32, m)/H2-3/H-4 (δH 1.72, m)/H3-15 (δH 0.98, d, J = 6.9 Hz) (Figure 1). Thus, the planar structure of 1 was determined.

The NOESY correlations (Figure 1) of H3-15/H3-14 (δH 1.26, s)/H-6b (δH 2.08, d, J = 14.6 Hz) and H-4 (δH 1.72, m, H-4)/H-6a (δH 2.03, d, J = 14.6 Hz)/H2-13 (δH 4.05, d, J = 14.2 Hz; δH 3.98, d, J = 14.2 Hz) indicate that Me-15, Me-14, and OH-7 have a cis configuration. So, the relative configuration of 1 was established as (4S*,5R*,7R*). In order to discriminate between (4S,5R,7R)-1 and (4R,5S,7S)-1, the electronic circular dichroism (ECD) spectrum of 1 was compared with those 1406

DOI: 10.1021/acs.jnatprod.8b00015 J. Nat. Prod. 2018, 81, 1405−1410

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of 2 were nearly identical to those of reported chaetopenoid B,6 except the COOH-13 (δC 170.5) in chaetopenoid B was replaced by CHO-13 (δH 9.51, s; δC 194.9) in 2. The planar structure of 2 was confirmed by HMBC and COSY correlations (Figure 1). The 1H−1H coupling constant between H-3 and H4 (J = 5.0 Hz) and the NOESY correlations of H-3/H-4 suggested that they were on the same side of the ring system.5,7 Other NOESY correlations (Figure 1) of H3-15/H3-14/H-6b and H-4/H-6a/H-12a indicated that the sesquiterpene moiety of 2 has the same relative configuration as 1. The large coupling constant between H-2′ and H-3′ (J = 15.6 Hz) in 2 and the NOESY correlations of H-5′/H-3′ and H3-10′/H-2′ indicated that both olefins on the side-chain were in a trans configuration. The absolute configuration of C-6′ on the side-chain was proposed to be S by the specific rotation of the hydrolyzed product 4,6-dimethyl-(2E,4E)-octadienoic acid ([α]20 D +40) of 2, compared to that of the reported hydrolyzed product 6methyl-(2E,4E,6S)-octadienoic acid ([α]20 D +45.4) of chaetopenoid A.7,8 The ECD spectra of 2 showed positive CEs at about 232 and 297 nm and negative CEs at about 213 and 251 nm (Figure S2), which were identical to that of reported KM-01.8 Therefore, the absolute configuration of 2 was proposed as 3S,4R,5R,7R,6′S and given the trivial name dendryphiellin I. Compound 3 had the molecular formula C26H35NO5, as established by HRESIMS. Analysis of the 1H and 13C NMR data (Table 1) suggested 3 is also an eremophilane sesquiterpene with a fatty acid ester side-chain. The NMR data of 3 were similar to those of 2, except the signals of CHO13 (δH 9.51, s; δC 194.9) in 2 were replaced with δH 7.67 (s) and δC 149.4 in 3, as well as the presence of an additional Omethyl in 3. The O-methyl (δH 3.70, s, 3H; δC 62.2) was located on 7-OH, which was confirmed by HMBC correlation (Figure 1). The group on position 13 was suggested to be a CH2NO moiety by the molecular formula. The HSQC and HMBC correlations from H-13 to C-7 (δC 76.7), C-11 (δC 148.3), and C-12 (δC 123.1) confirmed it to be an aldoxime moiety (―CHNOH), rather than an amide moiety (―CONH2). The E configuration of the aldoxime was confirmed by NMR shifts (δC 149.4, δH 7.67, CH-13), compared to those of the reported E- or Z-configured aldoxime moiety.9 Compound 3 had the same side-chain moiety as 2, because the NMR data of the side-chain moiety of 3 were identical to those of 2 (Table 1). Compound 3, named dendryphiellin J, shared the same relative and absolute configuration of sesquiterpene and side-chain moieties as those of 2, determined by the NOESY correlations (Figure 1) and the ECD spectrum (Figure S2). Although naturally

calculated for each enantiomer using the time-dependent density functional theory (TDDFT) method, as ECD calculations have been reported for eremophilane sesquiterpenes.5,6 Briefly, conformational analyses of (4S,5R,7R)-1 and (4R,5S,7S)-1 were carried out in the SYBYL-x 2.0 program using the MMFF94 force field, within a 6 kcal/mol energy window (Figure S1, Table S1). All conformers obtained for each enantiomer were optimized using DFT calculations at the B3LYP/6-31G(d) level in MeOH. The theoretical ECD spectrum of each enantiomer was then calculated using TDDFT at the same level (Table S2). The calculated ECD spectrum for enantiomer 4S,5R,7R showed a good fit with the experimental curve of 1, which displayed one strong negative Cotton effect (CE) at 229 nm and two weak positive CEs at 282 and 370 nm (Figure 2). On the other hand, the calculated

Figure 2. Comparison between calculated and experimental ECD spectra of 1.

ECD spectrum of (4R,5S,7S)-1 displayed one strong positive CE at 231 nm and two weak negative CEs at 310 and 368 nm. Hence, the 4S,5R,7R configuration of 1 was confirmed, which is in agreement with the reported configurations of dendryphiellin G4 and dihydrobipolaroxin.5 Taken together, the complete structure of 1 was resolved and given the trivial name dendryphiellin H. Compound 2 had the molecular formula of C25H32O5, as established by HRESIMS. Comprehensive analysis of the 1H and 13C NMR data (Table 1) suggested 2 is an eremophilane sesquiterpene with a fatty acid ester side chain. The NMR data

Table 2. NMR Spectroscopic Data (1H 700 MHz, 13C 175 MHz, CD3OD) for 4−6 4 δC, type 1 3 3a 4 5 6 7 7a 8 9

173.1, C 84.1, CH 142.5, C 124.2, CH 123.9, CH 160.0, C 110.8, CH 128.4, C 28.7, CH2 8.9, CH3

5 δH (J in Hz) 5.46, dd (6.9, 4.2) 7.39, d (8.2) 7.18, dd (8.2, 2.2) 7.15, d (2.2) 2.10, m; 1.77, m 0.94, t (7.4)

δC, type

6 δH (J in Hz)

172.3, C 83.4, CH 142.6, C 113.4, CH 123.2, CH 146.7, C 145.8, C 113.4, C 28.9, CH2 8.8, CH3 1407

5.38, dd (6.8, 4.3) 6.79, d (7.8) 7.12, d (7.8)

2.06, m; 1.75, m 0.94, t (7.4)

δC, type 173.7, C 83.4, CH 145.3, C 108.5, CH 154.1, C 148.2, C 110.5, CH 117.8, C 28.6, CH2 8.8, CH3

δH (J in Hz) 5.35, dd (6.8, 4.2) 6.9, s

7.1, s 2.05, m; 1.72, m 0.94, t (7.4) DOI: 10.1021/acs.jnatprod.8b00015 J. Nat. Prod. 2018, 81, 1405−1410

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Table 3. Cytotoxic and Antibacterial Activities of Compounds 1−8a 1 Cytotoxic (IC50, μM) ACHN >50 786-O >50 OS-RC-2 >50 HepG-2 >50 SGC7901 >50 Antibacterial (MIC, μg/mL) E. coli >100 S. aureus >100 E. rhusiopathiae >100 P. multocida >100

control

2

3

4

5

6

7

8

4.3 2.5 3.7 1.4 1.4

3.1 27 >50 5.9 17

>50 >50 >50 >50 >50

>50 >50 >50 >50 >50

34 28 >50 >50 >50

>50 46 >50 >50 >50

/b / / 1.7 5.6

3.5c 5.3c 15c 12d 12d

>100 1.5 13 13

>100 >100 >100 >100

>100 >100 >100 >100

>100 >100 >100 >100

>100 >100 >100 >100

>100 50 25 13

>100 100 >100 >100

25e 13f 13f 13f

a f

2,5-Dimethyl-7-hydroxychromone was also tested, but was found to be inactive. b/ = not detected. cSorafenib. d5-Fluorouracil; eStreptomycin. Penicillin.

Figure 3. Cell apoptosis of ACHN cells induced by 3.

occurring aldoxime derivatives are rare,10 the aldoxime moiety in 3 is well understood to be biosynthesized from the formyl group in 2. Compound 4 was isolated as a colorless oil. Its molecular formula was established by HRESIMS to be C10H10O3. Comprehensive analysis of the 1H and 13C NMR data (Table 2) suggested 4 is a phthalide derivate,10 similar to isoochracein.12 The 1H NMR spectrum of 4 showed the presence of three aromatic protons at δH 7.39 (1H, d, J = 8.2 Hz, H-4), 7.18 (1H, dd, J = 8.2, 2.2 Hz, H-5), and 7.15 (1H, d, J = 2.2 Hz, H-6), indicating a typical 1,2,4-trisubstituted benzene moiety with a hydroxy substituted on C-6, which was confirmed by HMBC correlations (Figure 1). The absolute configuration at C-3 in 4 was proposed to be S by specific rotations (4: [α]20 D −97), compared to that reported for (S)-3-ethylphthalide 11 Thus, compound 4 was identified as (S)-3([α]23 D −73.5). ethyl-6-hydroxyphthalide. This compound was obtained in this study as a new natural product. Compound 5 had the molecular formula C10H10O4, as established by HRESIMS. The NMR data of 5 (Table 2) were similar to those of 4, except for the presence of an additional

hydroxy substituted on C-7 of the benzene moiety, which was confirmed by HMBC correlations (Figure 1). The absolute configuration at C-3 in 5 was also determined to be S by the specific rotation (5: [α]20 D −75). Thus, the structure of 5 was identified as (S)-3-ethyl-6,7-dihydroxyphthalide. Compound 6 also had the molecular formula C10H10O4. The NMR data of 6 (Table 2) suggested 6 is also a phthalide derivative, with two hydroxy groups substituted on C-5 and C-6 of the benzene moiety. The planar structure was confirmed by HMBC correlations, and the absolute configuration was determined to be S by the specific rotation (6: [α]20 D −71). The structure of 6 was identified as (S)-3-ethyl-5,6-dihydroxyphthalide. The structures of the other three known compounds were characterized by comparison of their NMR and MS data with literature data (Supporting Information) and determined to be purpureone (7),13 emodin (8),14 and 2,5-dimethyl-7-hydroxychromone.14 The cytotoxicities of all compounds were evaluated against three renal cancer cell lines (ACHN, 786-O, and OS-RC-2), a human liver cancer cell line (HepG-2), and a human gastric 1408

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broth from 200 flasks (about 50 L total) was harvested and extracted with EtOAc, to yield an organic extract. Isolation and Purification. The organic extract (55 g) was subjected to silica gel column chromatography (CC) and was separated by a linear gradient of petroleum ether/EtOAc (50:0, 50:1, 20:1, 5:1, 2:1, 1:1, and 0:1) to yield seven fractions (fr.a−fr.g). Fraction fr.b was chromatographed over Sephadex LH-20 (CHCl3/ MeOH, 1:1), followed by repeated silica gel CC (CHCl3/MeOH, gradient elution 100:1−10:1) purification, to afford compounds 2 (12.5 mg) and 3 (9.6 mg). Compound 1 (18.8 mg) was purified and obtained by repeated silica gel CC (CHCl3/MeOH, 50:1) of fr.c. Fr.d was dissolved in MeOH and separated by semipreparative HPLC (2.5 mL/min) with a gradient solvent system from 20% to 60% CH3CN/ H2O to yield compounds 4 (11 mg), 5 (7.2 mg), and 6 (9.2 mg). Fractions fr.e and fr.f were divided into six subfractions (fr.e1−e6) and five subfractions (fr.f1−f5), respectively, by Sephadex LH-20 (CHCl3/ MeOH, 1:3) CC. Compounds 7 (17.8 mg), 8 (15.1 mg), and 2,5dimethyl-7-hydroxychromone (6.8 mg) were purified by repeated semipreparative HPLC (25% to 40% MeCN/H2O, 2 mL/min) of fr.f2, fr.f4, and fr.e5, respectively. Dendryphiellin H (1): colorless oil; [α]20 D +130 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 282 (2.22) nm; IR (KBr) νmax 3364, 2926, 1651, 1616, 1456, 1373, 1236, 1015, 893 cm−1; ECD (c 0.02, MeOH) λmax (Δε) 223 (−4.24), 275 (+ 0.94), 302 (− 0.84), 360 (+ 2.91); 1H and 13 C NMR data, Table 1; HRESIMS m/z 271.1307 [M + Na]+ (calcd for C15H20O3Na, 271.1305). Dendryphiellin I (2): colorless oil; [α]20 D +180 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 273 (2.46) nm; IR (KBr) νmax 3364, 2963, 1690, 1616, 1456, 1293, 1240, 1161, 1026, 989 cm−1; ECD (c 0.02, MeOH) λmax (Δε) 213 (−13.02), 232 (+4.98), 251 (−10.83), 297 (+20.69), 354 (+5.24); 1H and 13C NMR data, Table 1; HRESIMS m/z 435.2145 [M + Na]+ (calcd for C25H32O5Na, 435.2142). Dendryphiellin J (3): colorless oil; [α]20 D +280 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 273 (2.82) nm; IR (KBr) νmax 3364, 2963, 2926, 1714, 1668, 1628 1456, 1292, 1240, 1161, 1109, 1059, 991 cm−1; ECD (c 0.02, MeOH) λmax (Δε) 214 (−30.21), 234 (+21.45), 252 (−3.28), 296 (+47.08), 357 (+11.89); 1H and 13C NMR data, Table 1; HRESIMS m/z 464.2413 [M + Na]+ (calcd for C26H35NO5Na, 435.2407). (S)-3-Ethyl-6-hydroxyphthalide (4): colorless oil; [α]20 D −97 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.32), 235 (3.66), 323 (3.55) nm; IR (KBr) νmax 3292, 1732, 1499, 1464, 1306, 1065, 961 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 201.0524 [M + Na]+ (calcd for C10H10O3Na, 201.0522). (S)-3-Ethyl-6,7-dihydroxyphthalide (5): colorless oil; [α]20 D −75 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.12), 238 (3.67), 323 (3.56) nm; IR (KBr) νmax 3364, 1732, 1518, 1281, 968 cm−1; 1H and 13 C NMR data, Table 2; HRESIMS m/z 193.0509 [M − H]− (calcd for C10H9O4, 193.0506). (S)-3-Ethyl-5,6-dihydroxyphthalide (6): colorless oil; [α]20 D −71 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.23), 242 (3.65), 323 (3.54) nm; IR (KBr) νmax 3150, 1715, 1605, 1476, 1337, 1304, 1057, 959 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 217.0473 [M + Na]+ (calcd for C10H10O4Na, 217.0471). Hydrolysis of Compound 2. Compound 2 (1.4 mg) was dissolved in MeOH (1 mL) and 0.1 N KOH (1 mL) and then stirred for 1.5 h at room temperature (15 °C). The reaction products were extracted with EtOAc (2 mL) three times. Then, 0.1 N HCl was added and theh pH of the solution was adjusted to 7. The solution was extracted with diethyl ether (2 mL) three times. The organic layers were collected and vacuum evaporated, and 4,6-dimethyl-(2E,4E,6S)octadienoic acid (0.15 mg) was obtained after preparative HPLC purification. 4,6-Dimethyl-(2E,4E,6S)-octadienoic acid: [α]D20 +40 (c 0.1, MeOH); 1H NMR (175 MHz, CDCl3) δH 0.86 (3H, t, J = 7.4 Hz), 1.00 (3H, d, J = 6.6 Hz), 1.31 (1H, m), 1.43 (1H, m), 1.81 (3H, s), 2.48 (1H, m), 5.70 (1H, d, J = 9.9 Hz), 5.80 (1H, d, J = 15.6 Hz), 7.29 (1H, d, J = 15.6 Hz); ESIMS m/z 191.1 [M + Na]+. Theory and Calculation Details. The calculations were performed by using the TDDFT method as carried out in Gaussian

cancer cell line (SGC7901) (Table 3). Dendryphiellin I (2) showed significant cytotoxicities to all the tested cancer cell lines, with IC50 values of 1.4 to 4.3 μM. Dendryphiellin J (3) showed cytotoxicities only to ACHN and HepG-2 cells, with IC50 values of 3.1 and 5.9 μM, respectively. It is suggested that the α,β-unsaturated aldehyde moiety in the eremophilane sesquiterpene contributed broad-spectrum cytotoxic activities, while the α,β-unsaturated oxime moiety might contribute selective activity. Because of its unusual oxime moiety and its cytotoxicity to ACHN cells, the abilities of dendryphiellin J (3) to arrest cell cycle and induce cell apoptosis were further investigated. The results showed that 3 could not arrest the cell cycle of ACHN cells (Figure S3). The apoptotic cells induced by 3 were quantified by flow cytometry using annexin V (AV)-FITC/ propidium iodide (PI) double staining. ACHN cells were treated with 3 (1, 2, and 4 μM) for 24, 48, and 72 h and then analyzed by flow cytometry. The results showed that 3 induced apoptosis in ACHN cells (93.3% for early and late apoptotic cells, 72 h) significantly, at a concentration of 4 μM. The other data in Figure 3 suggest that 3 induced apoptosis in ACHN cells in a dose- and time-dependent manner. Moreover, the antibacterial activities of all the obtained compounds against four bacterial species (Escherichia coli, Staphylococcus aureus subsp. aureus Rosenbach, Erysipelothrix rhusiopathiae, Pasteurella multocida subsp. Multocida) were tested. Dendryphiellin I (2) showed activity against S. aureus, with an MIC value of 1.5 μg/mL. Compounds 2 and 7 also showed antibacterial activities against two pathogenic bacteria of swine disease, E. rhusiopathiae and P. multocida, with MIC values of 13 to 25 μg/mL (Table 3).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter. UV and ECD spectra were obtained with a Chirascan circular dichroism spectrophotometer (Applied Photophysics). NMR spectra were recorded on a Bruker AC 700 MHz spectrometer using tetramethylsilane as standard. HRESIMS spectra were recorded with a Bruker maXis Q-TOF in positive/ negative ion mode. Column chromatography was performed on silica gel (Cosmosil) and Sephadex LH-20 (Amersham Biosciences). Semipreparative HPLC (Agilent, 1260 infinity) was performed using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm). The artificial sea salt was purchased from Guangzhou Haili Aquarium Technology Company. Fungal Material. The fungal strain SCSIO41401 was isolated from a marine alga Coelarthrum sp. collected in Yongxing Island, South China Sea. The strain was identified according to the ITS region sequence. The resulting sequence data (Supporting Information), which were most similar (99.999%) to the sequence of Cochliobolus lunatus (accession no. DQ337381.1), have been deposited in GenBank (accession no. MG706143). This fungus, identified as Cochliobolus lunatus SCSIO41401, was stored on Medium B (bacto agar 15 g, malt extract 15 g, artificial sea salt 24.4 g, distilled H2O 1000 mL, pH 7.4− 7.8) agar slants at 4 °C and deposited in the Center for Marine Microbiology, South China Sea Institute of Oceanology, CAS, China. Fermentation and Extraction. The seed medium (consisting of 6.25 g maltose, 6.25 g malt extract, 1 g yeast extract, 6.25 g peptone, 1.25 g potassium dihydrogen phosphate, and 1000 mL distilled H2O, pH 7.0) in 500 mL Erlenmeyer flasks (200 mL/flask) was inoculated with strain SCSIO41401 and incubated at 25 °C for 3 days on a rotating shaker (180 rpm). Production medium (the same as the seed medium) in 500 mL flasks was inoculated with 10% seed solution. The flasks were incubated at 28 °C statically for 7 days. In the stage of bioactive screening of the strains, four flasks (about 250 mL broth/ flask) were used and incubated. For isolation of the active substances, 1409

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Journal of Natural Products

Article

03.15,16 Conformational analysis of 1 was initially performed in the SYBYL-x 2.0 program using the MMFF94 method (Table S1). The obtained conformers (within a 6 kcal/mol energy window) based on Boltzmann distribution were optimized at the B3LYP/6-31G(d) level in MeOH (Table S2, Figure S1), using the SCRF/PCM method.17 TDDFT18 at the B3LYP/6-31G(d) level in MeOH was employed to calculate the electronic excitation energies and rotational strengths. The ECD spectra of different conformers were generated by SpecDis and weighted by Boltzmann distribution after UV correction. Cytotoxicities and Apoptosis Assay. The human cancer cell lines, ACHN, OS-RC-2, 786-O, HepG-2, and SGC7901, were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. The inhibitory activities of the extracts against the HepG2 cells were assessed by the MTT method as reported in our previous study.3 The CCK-8 assay was used to evaluate the cytotoxicities of those obtained compounds against three renal cancer cell lines (ACHN, 786-O, and OS-RC-2),19 with sorafenib, a drug approved for the treatment of advanced renal cell carcinoma, as the positive control. The activities of the compounds against HepG-2 and SGC7901 were determined by the MTT method,20 with 5-fluorouracil as the positive control. Cell cycle arrest and cell apoptosis by 3 in ACHN cells were analyzed using flow cytometry, as reported in our previous study.19 Antibacterial Assay. The antibacterial activities against four bacterial strains, Escherichia coli (ATCC25922), Staphylococcus aureus subsp. aureus Rosenbach (ATCC25923), Erysipelothrix rhusiopathiae (ATCC 19414), and Pasteurella multocida subsp. Multocida (ATCC 43137), were determined by a serial dilution technique using 96-well microtiter plates.21 Tested compounds and positive controls (streptomycin or penicillin) were dissolved in DMSO to give a stock solution. Bacterial species were cultured overnight at 37 °C in Luria−Bertani broth (S. aureus and E. coli) and trypticase soy broth (P. multocida and E.rhusiopathiae, add 10% calf serum) and diluted to 106 cfu/mL when used. The plates were incubated at 37 °C for 24 h. The results were observed with a Multiskan Mk3 (Thermo) at 630 nm.

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Open Project of Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation.

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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.8b00015. Conformational analysis of 1; ITS region sequence of the strain; experimental ECD spectra of 2 and 3; cell cycle arrest data of 3; and HRESIMS, IR, 1H, 13C and 2D NMR spectra of 1−6 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-20-89023174. E-mail: [email protected]. ORCID

Lan Tang: 0000-0002-2345-0886 Yonghong Liu: 0000-0001-8327-3108 Xuefeng Zhou: 0000-0001-9601-4869 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (81741154, 31601681, 41376162, 41476135), National Major Scientific and Technological Special Project for “Significant New Drugs Development” (2018ZX09735001), Pearl River S&T Nova Program of Guangzhou Scientific Research Project (201610010017, 201710010136), Science and Technology Project of Guangdong Province (2016A020222009, 2016A020222010), and the 1410

DOI: 10.1021/acs.jnatprod.8b00015 J. Nat. Prod. 2018, 81, 1405−1410