Bioactive Phenanthrene and Bibenzyl Derivatives from the Stems of

Article pubs.acs.org/jnp

Bioactive Phenanthrene and Bibenzyl Derivatives from the Stems of Dendrobium nobile Xue-Ming Zhou,†,# Cai-Juan Zheng,†,# Li-She Gan,‡ Guang-Ying Chen,*,† Xiao-Peng Zhang,† Xiao-Ping Song,*,† Gao-Nan Li,† and Chong-Ge Sun§ †

Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, People’s Republic of China ‡ College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058, People’s Republic of China § Hainan Boying Orchid Industrial Development Co., LTD, Haikou 570105, People’s Republic of China S Supporting Information *

ABSTRACT: A new enantiomeric pair of spirodiketones, (+)- and (−)-denobilone A (1 and 2), three new phenanthrene derivatives (3−5), and three new biphenanthrenes (22−24), along with 11 known phenanthrene derivatives (6−16), five known bibenzyl derivatives (17−21), and four known biphenanthrenes (25−28), were isolated from Dendrobium nobile. The structures of 1−5 and 22−24 were elucidated using comprehensive spectroscopic methods. (+)-Denobilone and (−)-denobilone A (1 and 2) were isolated as a pair of enantiomers by chiral HPLC. The absolute configurations of (+)- and (−)-denobilone A (1 and 2) were determined by comparing their experimental and calculated electronic circular dichroism spectra. The absolute configuration of denobilone B (3) was determined by X-ray crystallographic analysis. The inhibitory activities of all compounds against nine phytopathogenic fungi and three cancer cell lines were evaluated.

Dendrobium nobile (Orchidaceae), widely distributed in China, is not only an ornamental but also a medicinal plant. The stems of several Dendrobium species are used as both traditional medicine and folk remedies for the treatment of various conditions, such as diabetes, chronic atrophic gastritis, and cardiovascular and skin aging diseases.1−6 D. nobile has been reported to contain phenanthrenes, bibenzyls, alkaloids, fluorenones, sesquiterpenoids, and caffeoylglucose.7−15 Especially, phenanthrene derivatives exhibit a wide range of biological activities such as anti-inflammatory, antiallergic, antimicrobial, cytotoxic, antiplatelet aggregation, phytotoxic, antifungal, spasmolytic, antifibrotic, and inhibitory activities on NO production.13,16−22 However, many farmers grow D. nobile as an ornamental plant in China. After its use as an ornamental plant becomes redundant, the stems and flowers of D. nobile are discarded as rubbish. To add value for D. nobile, an investigation of active constituents from these stems was explored. The investigation of antifungal and cytotoxic compounds from D. nobile led to the isolation of a new enantiomeric pair of spirodiketones, (−)- and (+)-denobilone A (1 and 2), three new phenanthrene derivatives (3−5), three new biphenan© XXXX American Chemical Society and American Society of Pharmacognosy

threnes (22−24), 11 known phenanthrene derivatives (6−16), five known bibenzyl derivatives (17−21), and four known biphenanthrenes (25−28). These compounds were tested for their antifungal activity against nine phytopathogenic fungi and cytotoxic activity on three cancer cell lines.

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RESULTS AND DISCUSSION Eight new and 20 known compounds were isolated from the 70% EtOH extract of the stems of D. nobile by repeated octadecylsilyl silica gel (ODS) column chromatography (CC), silica gel CC, Sephadex LH-20 CC, and semipreparative HPLC. These were identified as (+)- and (−)- denobilone A (1 and 2), denobilones B and C (3 and 4), 7-hydroxy-9,10-dihydro-1,4phenanthrenedione (5), hircinol (6),21 ephemeranthol-A (7),23 erianthridin (8),24 4,5-dihydroxy-2-methoxy-9,10-dihydrophenanthrene (9),25 flavanthridin (10),26 lusianthridin (11),27 6,7dihydroxy-2-methoxy-1,4-phenanthrenedione (12),28 moscatin (13),29 confusarin (14),30 nudol (15),31 lusianthrin (16),27 Received: March 20, 2016

A

DOI: 10.1021/acs.jnatprod.6b00252 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Structure of compounds 1−28.

Figure 2. Key partial structures of compounds 1−5 from HMBC and 1H−1H COSY data.

3′,4-dihydroxy-3,5′-dimethoxybibenzyl (17),32 3-hydroxy-5-methoxybibenzyl (18),33 batatasin III (19),34 tristin (20),35 3,3′,5trihydroxybibenzyl (21),36 denthyrsinol A (22), denthyrsinol B (23), denthyrsinol C (24), denthyrsinol (25),37 phochinenin G (26),38 phochinenin D (27),39 and 4,4′,7,7′-tetrahydroxy-2,2′dimethoxy-9,9′,10,10′-tetrahydro-1,1′-phenanthrene (28).40 Denobilone A (1/2) possesses a molecular formula of C15H14O4 (nine indices of hydrogen deficiency), as deduced from HRESIMS (m/z 281.0798 for 1 and 281.0795 for 2 [M + Na]+; calcd 281.0790) and 13C NMR data. The 1H and 13C (1D) NMR and 2D HSQC data revealed the presence of two carbonyls (δC 195.2 and 192.8), eight olefinic carbons (δC 162.1, 153.3, 145.9, 129.5, 129.3, 115.8, 113.2, and 112.2), three methylenes (δC 46.6, 38.1, and 30.5), one methoxy (δC 55.7),

and one sp3 quaternary carbon (δC 59.0) (Figures S3−S5, Supporting Information). The COSY correlations of H-6 to H7 and H-7 to H-8, combined with the HMBC correlations from H-6 to C-5a/C-5 and H-8 to C-5a/C-8a, indicated the presence of a benzene moiety in 1 (Figure 2). The COSY correlation of H-3 to H-4a combined with the HMBC correlations from H-3 to C-1/C-2/C-4/C-4a and H-4a to C-1/C-3/C-4/C-10a indicated the presence of the C-1/C-2/C-3/C-4/C-4a/10a unit in 1 (Figure 2). The HMBC correlations from H-9 to C5a/C-8/C-8a and H-10 to C-1/C-4a/C-5a/C-10a indicated that the two units were linked by C-9/C-10, and C-10a was directly linked with C-5a (Figure 2). The location of the methoxy group at C-2 was confirmed by the HMBC cross-peak of 2-OCH3 with C-2. The 2D structure of denobilone A was B



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Figure 3. Key partial structures of compounds 22−24 from HMBC and 1H−1H COSY data.

thus constructed, the first example of a phenanthrene with a unique benzo[b]-5a(4a→10a)-abeo-spirodiketone 6/5/6 ring system. The specific rotation and electronic circular dichroism (ECD) spectra of denobilone A showed [α]24D and ECD Δε values near zero, reminiscent of a racemic mixture. Chiral HPLC analysis of denobilone A indicated a ratio of approximately 1:1 (Figure S1, Supporting Information). Chiral HPLC separation afforded compounds 1 and 2, and their specific rotations (1, +159; 2, −162) and ECD curves reflected their enantiomeric relationship (Figure 4).

from HRESIMS (m/z 283.0947 [M + Na]+; calcd 283.0946) and 13C NMR data. The 1D NMR and 2D HSQC data revealed the presence of one carbonyl, eight olefinic carbons, three methylenes, an sp3 methine, a methoxy, and an oxygenated tertiary carbon (Figures S13−S15, Supporting Information). The COSY correlations of H-1 to H-10a, H-10a to H-10, and H-10 to H-9, combined with the HMBC correlations from H-3 to C-1/C-2/C-4a/C-4 and H-1 to C-2/C-3, indicated the presence of the C-4a/C-4/C-3/C-2/C-1/C-10a/C-10/C-9 fragment in 3 (Figure 2). The COSY correlations of H-6 to H-7 and H-7 to H-8, combined with the HMBC correlations from H-6 to C-5/C-7/C-8/C-5a and H-8 to C-7/C-5a/C-8a, indicated the presence of the C-5a/C-5/C-6/C-7/C-8/8a fragment in 3 (Figure 2). The HMBC correlations from H-9 to C-5a/C-8/C-8a indicated that C-9 was linked to C-8a. The HMBC correlations from H-10a to C-4/C-4a/C-5a indicated that C-4, C-5a, and C-10a were linked to C-4a (Figure 2). The HMBC correlation from 4-OMe to C-4 revealed that the methoxy group was linked to C-4 (Figure 2). The 2D structure of 3 was thus constructed as shown in Figure 1. The relative configuration of 3 could, however, not be confirmed unambiguously. Crystallization of 3 from MeOH/HOAc (20:1) afforded colorless crystals, which gave an X-ray crystal structure with a Flack parameter of 0.07(19). Thus, the absolute configuration of 3 was defined as (4aR, 10aR) (Figure 5). Thus, compound 3 (denobilone B) was elucidated as (4aR,10aR)-4a,5-dihydroxy-4-methoxy-1,9,10,10a-tetahydro-2(4aH)-phenanthreneone.

Figure 4. Experimental ECD spectra of 1 (red solid line) and 2 (black solid line) and calculated ECD spectra for 1 (red dashed line) and 2 (black dashed line).

The experimental ECD spectrum (Figure 4) of (−)-denobilone A (2) exhibited negative (237 nm), positive (276 nm), and negative (349 nm) Cotton effects. The calculated ECD spectrum for the R enantiomer showed a similar ECD curve. Both the calculated and experimental ECD spectra for (S)-1 exhibited sequential positive, negative, and positive Cotton effects. The above information permitted the determination of the S and R absolute configurations of 1 and 2, respectively. To confirm the above conclusion, the specific rotation of (S)-1 was calculated (Supporting Information S57)41−43 as +210. Compared to the experimental specific rotation of 1 (+159) and 2 (−162), the S and R configurations, respectively, of (+)and (−)-denobilone A (1 and 2) were confirmed. Thus, compounds 1 and 2 were assigned as (10aS)-5-hydroxy-2methoxy-5a(4a→10a)-abeo-4a,9,10,10a-tetahydro-1,4-phenanthrenedione and (10aR)-5-hydroxy-2-methoxy-5a(4a→10a)abeo-4a,9,10,10a-tetahydro-1,4-phenanthrenedione, respectively. Denobilone B (3) possesses a molecular formula of C15H16O4 (eight indices of hydrogen deficiency), as deduced

Figure 5. ORTEP drawing of 3.

Denobilone C (4) possesses a molecular formula of C15H14O5 (nine indices of hydrogen deficiency), as deduced from HRESIMS (m/z 297.2578 [M + Na]+; calcd 297.2584) and 13C NMR data. The 1D NMR data of 4 were very similar to those of denobilone B (3), except for the presence of a C-9 carbonyl signal (δC 197.4) in 4 and the absence of a C-9 methylene signal (δC 29.9) in 3. This was corroborated by the C



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and a lusianthridin (11)27 subunit. The moscatin and lusianthridin subunits were connected by a C-1−C-3′ linkage, which was confirmed by the deshielded resonances of C-1/3′ (δC 107.5 to 116.5, 113.5 to 120.2) and the absence of H-1 and H-3′ in the moscatin and lusianthridin subunits, respectively. This assignment was corroborated by the HMBC correlations from H-4′ to C-1 (Figure 3). Thus, denthyrsinol B (23) was identified as a new C-1−C-3′ linked phenanthrene dimer with a C-1−C-3′ linkage. Denthyrsinol B (24) possesses a molecular formula of C31H26O7 as deduced from HRESIMS (m/z 531.1423 [M + Na]+; calcd 531.1420) and 13C NMR data. The 1D NMR data indicated that the structure of 24 comprised a 9,10dihydrophenanthrene and a moscatin (13)29 subunit. The two subunits were connected by a C-1−C-1′ linkage, as confirmed by the deshielded resonance of C-1′ (δC 107.5 to 117.3), the absence of H-1 and H-1′ in the moscatin, and the HMBC correlations from H-2′ to C-1 and from H-3/10 to C-1 (Figure 3). Thus, denthyrsinol C (24) was identified as a new C-1−C-1′ linked phenanthrene dimer. The [α]24D and ECD Δε values of compounds 22−26 were near zero, reminiscent of racemic mixtures. However, compounds 22−26 could not be resolved by chiral HPLC (MeOH/H2O v/v 5:95, 10:90, 20:80, and 30:70; CH3CN/H2O v/v 5:95, 10:90, 20:80, and 30:7; temperature: 25 °C; chiral column: ULTRON ES-OVM column), a problem that may be caused by an insufficient barrier to free rotation of the biphenyl axes.45 Cytotoxic Activities. All compounds were tested for cytotoxic activities against HeLa, MCF-7, and A549 cells. However, only compounds 1 and 2 showed moderate inhibitory effects on HeLa, MCF-7, and A549 cells, with IC50 values of 9.8, 9.4, and 9.9 μM, respectively. The IC50 values of other compounds higher than 10 μM were regarded as inactive. Antifungal Activities. All compounds were evaluated against the phytopathogenic fungi Alternaria brassicicola, Phytophthora parasitica var. nicotianae, Colletotrichum capsici, Bipolaris oryzae, Diaporthe medusaea nitschke, Ceratocystis paradoxa moreau, Exserohilum turcicum, Pestallozzia theae, and Alternaria citri, and the results are shown in Table 5. Compounds 17−21 exhibited broad-spectrum antifungal activitiy against these phytopathogenic fungi (Table 5). Compounds 18−21 exhibited especially antifungal activity against Bipolaris oryzae, with MIC values ranging from 96.2 to 109.6 μM, compared to 133.3 μM for prochloraz. Compound 17 exhibited antifungal activity against Diaporthe medusaea nitschke, with an MIC value of 91.2 μM, compared to 133.3 μM for prochloraz.

HMBC correlations from H-8 and H-10 to C-9 (Figure 2). The relative configuration of 4 could not be confirmed via the NMR data. Owing to a putative shared biosynthesis pathway with 3, the absolute configuration of 4 was tentatively assigned as (4aR, 10aS). The ECD spectra of 3 and 4 showed similar features in the 200−500 nm range (Figure 6). Thus, the absolute

Figure 6. ECD spectra of 3 and 4.

configuration of denobilone C (4) was defined as (4aR, 10aS). Thus, compound 4 (denobilone C) was elucidated as (4aR,10aS)-4a,5-dihydroxy-4-methoxy-10,10a-dihydro-2,9(1H,4aH)-phenanthrenedione. Compound 5 possesses a molecular formula of C14H10O3 (10 indices of hydrogen deficiency), as deduced from HRESIMS (m/z 249.0533 [M + Na]+; calcd 249.0528) and 13C NMR data. Its 1D NMR spectra closely resembled those of 7methoxy-9,10-dihydro-1,4-phenanthrenequinone,44 except for the absence of the methoxy signal at δH 3.85. This was corroborated by the HMBC correlations from H-6 and H-8 to C-7 (Figure 2). Thus, the structure of 5 was defined as 7hydroxy-9,10-dihydro-1,4-phenanthrenedione. Denthyrsinol A (22) possesses a molecular formula C31H26O7 as deduced from HRESIMS (m/z 533.1578 [M + Na]+; calcd 533.1576) and 13C NMR data. The 1D NMR data indicated that the structure of 22 comprised a 9,10dihydrophenanthrene and a phenanthrene subunit. The 1D NMR spectroscopic data of the 9,10-dihydrophenanthrene subunit closely resembled those of lusianthridin (11),27 except for the deshielded resonances of C-3′ (δC 113.5 to 120.6) and the absence of an aromatic H-3′ signal in the lusianthridin subunit. These data indicated that H-3′ was replaced by a phenanthrene subunit. In addition, the HMBC spectrum showed that H-4′ correlated to C-1, C-2′, and C-3′, and H-3 and H-10 correlated to C-1 (Figure 3). These data indicated that the two monomeric moieties were linked via a C-1−C-3′ bond. The location of the methoxy groups at C-2 and C-4 and hydroxyl groups at C-5 and C-10, respectively, in the phenanthrene subunit was confirmed by the HMBC correlations from 2-OCH3 to C-2, 4-OCH3 to C-4, H-6/7 to C-5, and H-8/10 to C-9 (Figure 3). Thus, denthyrsinol A (22) was identified as a new C-1−C-3′ linked phenanthrene dimer. Denthyrsinol B (23) possesses a molecular formula of C30H24O6 as deduced from HRESIMS (m/z 503.1466 [M + Na]+; calcd 503.1471) and 13C NMR data. The 1D NMR data indicated that the structure of 23 comprised a moscatin (13)29

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EXPERIMENTAL SECTION

General Experimental Procedures. Semipreparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDB-C18 column (9.4 × 250 mm, 5 μm) and an ULTRON ES-OVM column (150 × 10 mm, 5 μm). The other experimental procedures were performed as reported previously.46 Plant Material. The stems of D. nobile were provided by the Hainan Boying Orchid Industrial Development Co., LTD, in June 2014. A voucher specimen (No. GFM20140612) has been deposited at the Key Laboratory of Tropical Chemistry of Medicinal Plant of the Ministry of Education, Hainan Normal University (Hainan, China). Extraction and Isolation. The air-dried and powdered stems (5 kg) of D. nobile were extracted with 70% EtOH (3 × 20 L, 5 days each) at room temperature. After concentration under reduced pressure, the water-soluble residue was partitioned successively with D



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Table 1. 1H NMR Data (δ) for 1−5 (400 MHz) (δ in ppm, J in Hz) 1/2a

3b

4b

3.16, dd (17.2, 5.2) 2.23, dd (17.2, 2.0)

3.13, dd (17.6, 5.2) 2.63, dd (17.6, 2.8)

6.08, d (0.8)

5.31, s

5.48, s

6.61, d (8.0)

7.11, dd (8.0, 1.2) 7.35, dd (8.0, 8.0) 7.56, dd (8.0, 1.2)

9

6.66, dd (8.0, 0.8) 7.07, dd (8.0, 7.6) 6.74, dd (7.6, 0.8) 2.99, m

10

2.13, m

position 1

2 3 5 6 7 8

7.06, dd (8.0, 7.6) 6.61, d (7.6) 2.89, m 2.78, m 1.85, m

position 3 6 7 8 9 10 2-OCH3 4-OCH3 1′ 2′ 3′ 4′ 6′ 7′ 8′ 9′ 10′ 7′-OCH3

6.79, d (7.6) 6.80, d (7.6) 7.93, d (9.2) 6.78, dd (9.2, 2.8)

6.77, d (2.8)

2.77, dd (17.6, 4.8) 2.63, dd (17.6, 12.8)

2.61, m

3.52, d (16.4) 2.68, dd (16.4, 1.2)

5a 8a 10a 2-OCH3 4-OCH3 a

5a

2.74, dd (15.6, 7.2)

2.34, m 4a

Table 3. 1H NMR Data (δ) for 22−24 in Acetone-d6 (400 MHz) (δ in ppm, J in Hz) 23

s dd (7.6, 2.4) dd (8.0, 7.6) dd (8.0, 2.4)

6.91, 3.76, 4.14, 6.94,

s s s s

7.12, 7.11, 7.44, 7.40, 7.55, 7.39,

24

s dd (7.6, 1.6) dd (7.6, 7.6) dd (7.6, 1.6) d (9.2) d (9.2)

4.19, s 6.92, s

7.04, 7.18, 7.46, 7.81,

s dd (7.6, 1.2) dd (8.0, 7.6) dd (8.0, 1.2)

6.38, s 3.48, s 4.23, s 7.44, d (8.0) 7.27, d (8.0)

8.30, s 6.40, d (2.8) 6.41, 2.79, 2.79, 3.74,

8.27, s 6.39, d (2.4) 6.42, d (2.4)

d (2.8) m m s

7.04, d (2.8) 7.04, d (2.8) 7.37, d (8.8) 7.22, d (8.8)

2.80, m 2.80, m 3.74

Table 4. 13C NMR Data (δ) for 22−24 in Acetone-d6 (100 MHz) (δ in ppm) 2.53, m

3.01, m

3.83, s 3.67, s

3.71, s

Acetone-d6. bMethanol-d4.

Table 2. 13C NMR Data (δ) for 1−5 (100 MHz) (δ in ppm)

a

22 6.97, 7.14, 7.44, 7.83,

position

1/2a

3b

4b

5a

1 2 3 4 5 6 7 8 9 10 4a 5a 8a 10a 2-OCH3 4-OCH3

192.8 162.1 112.2 195.2 153.3 113.2 129.3 115.8 30.5 38.1 46.6 129.5 145.9 59.0 55.7

40.3 201.0 100.7 179.7 159.3 115.0 130.1 121.3 29.9 27.3 76.6 123.1 139.4 43.1

39.1 199.7 102.3 176.9 159.1 123.6 130.9 119.7 197.4 41.7 75.4 129.0 133.9 42.6

187.1 137.8 136.6 188.1 132.2 114.4 160.0 115.8 20.9 28.0 137.0 121.9 142.1 139.0

57.0

57.3

b

Acetone-d6. Methanol-d4.

petroleum ether and EtOAc. The EtOAc extract (95 g) was separated using silica gel CC (petroleum ether, EtOAc, MeOH v/v, gradient) to generate seven fractions (Frs. 1−7). Fr. 3 (12 g) was separated by silica gel CC and eluted with petroleum ether/EtOAc (from 9:1 to 2:1) to afford three subfractions (3a−3c). Subfractions 3a−3c were further separated by semipreparative HPLC (MeOH/H2O, 55:45 for subfraction 3a, 49:51 for subfraction 3b, and 49:51 for subfraction 3c, v/v) to obtain 5 (4 mg) and 20 (11 mg) from subfraction 3a; 18 (8 mg), 19 (12 mg), and 21 (15 mg) from subfraction 3b; and 12 (10 mg) and 17 (13 mg) from subfraction 3c. Fr. 4 (18 g) was separated by silica gel CC and eluted with petroleum ether/EtOAc (from 6:1 to 1:1) to afford four subfractions (4a−4d). Subfraction 4a was isolated by CC on silica gel eluting with CHCl3/acetone (50:1, v/v) and then

position

22

23

24

1 2 3 4 5 6 7 8 9 10 4a 5a 8a 10a 2-OCH3 4-OCH3 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 4a′ 5a′ 8a′ 10a′ 7′-OCH3

115.6 154.6 100.3 155.5 155.2 117.5 127.0 114.2 154.6 101.5 110.4 121.3 128.8 136.7 55.5 58.3 116.2 154.6 120.6 133.4 156.0 101.5 159.4 106.1 31.5 30.6 126.3 115.7 141.4 139.8 55.3

116.5 155.1 102.4 156.0 155.3 116.9 127.4 120.8 129.1 125.4 100.9 114.3 134.7 136.0

117.3 154.9 99.8 155.5 155.3 117.8 127.1 114.3 155.2 101.2 110.3 121.2 128.7 136.9 55.4 58.4 120.7 131.5 117.2 155.7 156.5 102.3 157.5 107.5 127.2 127.3 126.0 114.1 137.1 134.3 58.4

58.5 116.0 154.6 120.2 133.4 155.6 101.6 159.5 106.1 31.5 30.6 126.5 115.6 141.5 139.9 55.3

separated by Sephadex LH-20 CC eluting with CHCl3/MeOH (1:1, v/ v) and further purified by using ODS eluting with MeOH/H2O (40:60, v/v) to obtain 6 (18.5 mg), 8 (12.5 mg), 13 (23.5 mg), and 14 (6.3 mg). Subfractions 4b−4d were further separated by semipreparative HPLC (MeOH/H2O, 45:55 for subfraction 4b, 40:60 for E



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Table 5. Antifungal Activity of Isolated Compoundsa MIC (μM)

a

compound

A. brassicicola

P. parasitica var. nicotianae

C. capsici

B. oryzae

D.medusaea Nitschke

C. paradoxa Moreau

E. turcicum

P. theae

A. citri

17 18 19 20 21 prochlorazb

182.4 >400 102.5 192.3 >400 33.3

365.0 219.3 204.9 384.6 217.7 133.3

91.2 109.6 102.5 96.2 108.7 33.3

182.4 109.6 102.5 96.2 108.7 133.3

91.2 219.3 204.9 192.3 217.4 133.3

91.2 109.6 102.5 96.2 108.7 66.7

182.4 109.6 102.5 384.6 108.7 33.3

91.2 219.3 204.9 96.2 217.4 66.7

91.2 109.6 102.5 96.2 108.7 66.7

Compounds 1−16 and 22−28 were inactive for all phytopathogenic fungals used (MIC > 400 μM). bProchloraz was used as a positive control.

subfraction 4c, and 40:60 for subfraction 4c, v/v) to obtain 7 (9 mg), 9 (7 mg), and 11 (14 mg) from subfraction 4b; 10 (9 mg), 15 (21 mg), and 16 (25 mg) from subfraction 4c; and 1/2 (6 mg), 3 (6 mg), and 4 (3 mg) from subfraction 4d. Fr. 5 (5 g) was separated by silica gel CC eluted with petroleum ether/EtOAc (from 5:1 to 1:3) to afford two subfractions (5a, 5b). Subfractions 5a and 5b were further isolated by semipreparative HPLC (CH3CN/H2O, 30:70 for subfraction 5a and 25:75 for subfraction 5b v/v) to obtain 26 (15 mg) and 28 (12 mg) from subfraction 5a and 22 (16 mg), 23 (11 mg), 24 (14 mg), 25 (9 mg), and 27 (13 mg) from subfraction 5b. (+)-Denobilone A (1): colorless oil; [α]24D +159 (c 0.2, MeOH); ECD (c 2.5 × 10−4 mol/L, MeOH) λmax (Δε) 221 (−2.98), 278 (3.42), 351 (0.81); UV (MeOH) λmax (log ε) 271 nm; IR (KBr) νmax 3428, 3011, 2985, 2935, 2838, 1716, 1658, 1607, 1460 cm; 1D NMR data, see Tables 1 and 2; HRESIMS m/z 281.0798 [M + Na]+ (calcd for C15H14O4Na, 281.0790). (−)-Denobilone A (2): colorless oil; [α]24D −162 (c 0.2, MeOH); ECD (c 2.5 × 10−4 mol/L, MeOH) λmax (Δε) 220 (2.49), 278 (−3.02), 352 (0.75); UV (MeOH) λmax (log ε) 271 nm; IR (KBr) νmax 3428, 3011, 2985, 2935, 2838, 1716, 1658, 1607, 1460 cm; 1D NMR data: see Tables 1 and 2; HRESIMS m/z 281.0795 [M + Na]+ (calcd for C15H14O4Na, 281.0790). Denobilone B (3): colorless oil; [α]24D +48 (c 0.25, MeOH); mp 176.1−178.4 °C (crystal from MeOH/HOAc, 20:1, v/v); ECD (c 1 × 10−4 mol/L, MeOH) λmax (Δε) 249 (7.62), 288 (0.91), 325 (−0.43); UV (MeOH) λmax (log ε) 256 nm; IR (KBr) νmax 3439, 2968, 1715, 1643, and 1458 cm; 1D NMR data, see Tables 1 and 2; HRESIMS m/ z 283.0947 [M + Na]+ (calcd for C15H16O4Na, 283.0946). Denobilone C (4): colorless oil; [α]24D +210 (c 0.35, MeOH); ECD (c 5 × 10−5 mol/L, MeOH) λmax (Δε) 253 (18.35), 306 (1.24), 339 (−1.20); UV (MeOH) λmax (log ε) 248 and 312 nm; IR (KBr) νmax 3431, 2983, 2928, 1718, 1637, and 1451 cm−1; 1D NMR data, see Tables 1 and 2; HRESIMS m/z 297.2578 [M + Na]+ (calcd for C15H14O5Na, 297.2584). 7-Hydroxy-9,10-dihydro-1,4-phenanthrenedione (5): red, amorphous powder; UV (MeOH) λmax (log ε) 213, 254, and 455 nm; IR (KBr) νmax 3421, 1645, and 1608 cm−1; 1D NMR, see Tables 1 and 2; HRESIMS m/z 249.0533 [M + Na]+ (calcd for C14H10O3Na, 249.0528). Denthyrsinol A (22): white, amorphous powder; [α]24D 0 (c 0.3, MeOH); λmax (log ε) 262 and 283 nm; IR (KBr) νmax 3373, 2940, 2836, 1614, 1466, and 1357 cm−1; 1D NMR data, see Tables 3 and 4; HRESIMS m/z 533.1578 [M + Na]+ (calcd for C31H26O7Na, 533.1576). Denthyrsinol B (23): white, amorphous powder; [α]24D 0 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 259 and 282 nm; IR (KBr) νmax 3301, 2928, 2831, 1610, 1456, and 1353 cm−1; 1D NMR, see Tables 1 and 2; HRESIMS m/z 503.1466 [M + H]+ (calcd for C30H24O6Na, 503.1471). Denthyrsinol C (24): white, amorphous powder [α]24D 0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 260, 287, and 325 nm; IR (KBr) νmax 3172, 2838, 1605, 1463, and 1349 cm−1; 1D NMR data, see Tables 3 and 4; HRESIMS m/z 531.1423 [M + Na]+ (calcd for C31H24O7Na, 531.1420). X-ray Crystal Structure of Compound 3. Compound 3 was crystallized form MeOH/HOAc (v/v = 20:1) at room temperature.

Diffraction intensity data collection, structure solution, hydrogen atom treatment, and refinement for 3 were performed as reported previously.46 The crystallographic data have been deposited with the Cambridge Crystallographic Data Centre: CCDC reference number 1422109. Crystal Data for Denobilone B (3). 2(C15H16O4), 3(H2O) M = 574.60, space group C2221, a = 11.0841(2) Å, b = 20.4600(4) Å, c = 12.6447(2) Å, α = β = γ = 90°, V = 2867.57(9) Å3, Z = 4, T = 293(2) K, Dc = 1.331 g/cm3, μ(Cu Kα) = 0.846 mm−1, F(000) = 1224, 7171, independent reflections 2473 (Rint = 0.0217). The final R1 = 0.0336 [I > 2σ(I)]. The final wR2 (F2) = 0.0918 [I > 2σ(I)]. The final R1 = 0.0345 (all data). The final wR2 (F2) = 0.0926 (all data). Flack parameter = 0.07(19). Cells. HeLa, MCF-7, and A549 were provided by College of Pharmacy, Hebei University, and maintained in DMEM medium (Gibco) containing 5% fetal bovine serum (Gibco) at 37 °C in air with 5% CO2. Fungal Strains. Alternaria brassicicola, Phytophthora parasitica var. nicotianae, Colletotrichum capsici, Bipolaris oryzae, Diaporthe medusaea nitschke, Ceratocystis paradoxa moreau, Exserohilum turcicum, Pestallozzia theae, and Alternaria citri were grown on PDA (potato dextrose agar). Biological Assays. Cytotoxic activities of all compounds against HeLa, MCF-7, and A549 cell lines were evaluated by the MTT method.47 Epirubicin was used as a positive control. The antifungal activities of all compounds against Alternaria brassicicola, Phytophthora parasitica var. nicotianae, Colletotrichum capsici, Bipolaris oryzae, Diaporthe medusaea nitschke, Ceratocystis paradoxa moreau, Exserohilum turcicum, Pestallozzia theae, and Alternaria citri were evaluated by the National Center for Clinical Laboratory Standards (NCCLS) recommendations (Supporting Information S58). Prochloraz was used as a positive control.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00252. 1D and 2D NMR and HRESIMS spectra of the new compounds (1−5, 22−24); calculation procedure (ECD) for 1 and 2; antifungal bioassay (PDF) X-ray crystallographic data for 3 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for G.-Y. Chen: [email protected]. Tel: 86-898 65730237. Fax: 86-898 65889422. *E-mail for X.-P. Song: [email protected]. Author Contributions #

X. M. Zhou and C. J. Zheng contributed equally to this study.

Notes

The authors declare no competing financial interest. F



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Article

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ACKNOWLEDGMENTS This work was supported by The Industry-University-Research Cooperation Integration Program of Hainan Province (SQ20150CXY0064), 973 Program, Ministry of Science and Technology of China (Grant No. 2014CB560714), and the Hainan Province Natural Science Foundation of Innovative Research Team Project (2016CXTD007).

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