Bioactive Spirobisnaphthalenes from the Endophytic Fungus

Sep 19, 2014 - Palmarumycins B1−B4 (1−4) are uncommon 2,3,4-trihy- droxy-substituted spirobisnaphthalenes, among which palmar- umycins B1 (1), B3 ...
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Bioactive Spirobisnaphthalenes from the Endophytic Fungus Berkleasmium sp. Tijiang Shan,†,‡ Jin Tian,†,‡ Xiaohan Wang,† Yan Mou,† Ziling Mao,† Daowan Lai,† Jungui Dai,§ Youliang Peng,† Ligang Zhou,*,† and Mingan Wang*,⊥ †

MOA Key Laboratory of Plant Pathology, Department of Plant Pathology, College of Agronomy and Biotechnology, and Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, People’s Republic of China § State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, China ⊥

S Supporting Information *

ABSTRACT: Nine new spirobisnaphthalenes, palmarumycins B1−B9 (1−9), along with 13 known compounds (10−22), were isolated from cultures of the fungus Berkleasmium sp., an endophyte isolated from the medicinal plant Dioscorea zingiberensis C. H. Wright. The structures of the new compounds were elucidated by analysis of the 1D and 2D NMR and HRESIMS spectra and by comparison with known compounds. Compounds 7−9 contain an uncommon 2,3-dihydro-1H-inden-1-one unit. All isolated compounds were evaluated for their antibacterial activities against Bacillus subtilis, Staphylococcus hemolyticus, Agrobacterium tumefaciens, Pseudomonas lachrymans, Ralstonia solanacearum, and Xanthomonas vesicatoria and for their antifungal effects against the spore germination of Magnaporthe oryzae. Palmarumycin C8 (22) exhibited the best antibacterial and antifungal effects. In addition, diepoxin δ (11) and palmarumycin C8 (22) showed pronounced cytotoxic activities against five human cancer cell lines (HCT-8, Bel-7402, BGC-823, A 549, A 2780) with IC50 values of 1.28−5.83 μM. spirobisnaphthalenes, palmarumycins B1−B9 (1−9), and 13 known congeners (10−22) (Figure S1; see the Supporting Information). To the best of our knowledge, phytochemical investigations on Berkleasmium species had been so far reported only by our group11 and the Isaka group.16 Isaka et al. had found several cytotoxic and antimalarial eremophilane sesquiterpenoids from a saprobic fungus, Berkleasmium nigroapicale BCC 8220, isolated from dead pseudostems of Amomum siamense (Zingiberaceae).16 Herein, we describe the isolation, structure elucidation, and biological activity of the isolated compounds.

E

ndophytic fungi reside in the healthy tissue of their host harmoniously without causing any obviously negative effect.1 They have been reported to be promising sources of bioactive natural products with unique structures and exhibiting diverse biological activities.1−3 In recent years, growing numbers of fungal endophytes have been isolated from plants, and bioprospecting for bioactive compounds from the fungal endophytes has attracted considerable interest.1−3 Spirobisnaphthalenes are fungal secondary metabolites that consist of one 1,8-dioxynaphthalene unit and one decalin unit linked through a spiro-ketal bridge.4,5 Compounds of this family were reported to possess antibacterial,6−9 antifungal,6,7,10,11 algicidal,7,9 antiplasmodial,12 antileishmanial,13 cytotoxic,12 and anti-invasive14 activities. These intriguing fungal metabolites have been the target for total synthesis.4,15 In our search for new bioactive substances from endophytic fungi, a fungal endophyte that showed good antimicrobial activity was isolated from the medicinal plant Dioscorea zingiberensis C. H. Wright (Dioscoreaceae). Our previous investigation on this fungal extract had led to the isolation of several known spirobisnaphthalenes.11 In continuation of our interest in searching for minor spirobisnaphthalenes from this fungus, the endophyte was fermented in a large scale in potato dextrose broth (PDB) medium. Subsequent fractionation of the obtained EtOAc extract has afforded the isolation of nine new © XXXX American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION An EtOAc extract of the Berkleasmium sp. was subjected to column chromatography over silica gel and Sephadex LH-20, followed by further purification by semipreparative HPLC to yield nine new compounds (1−9), as well as 13 known palmarumycin derivatives (10−22). The structures of the new compounds are similar to those of palmarumycins first discovered in 1994 by Krohn et al.,6,7 and these new derivatives are named palmarumycins B1−B9 (1−9). Palmarumycins B1− Received: November 28, 2013

A

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Chart 1

shown in Figure 1. The requirement of one more degree of unsaturation and only one oxygen atom remaining to be assigned as indicated by the molecular formula has permitted the assignment of an epoxy linkage between C-4a and C-8a, and the planar structure of 1 was established. The relative configuration was determined by analysis of the NOESY spectrum and of the coupling constants (Figure 2). The large 3JH‑2, H‑3 (10.0 Hz), and 3JH‑3, H‑4 (7.8 Hz) indicated that H-2, H-3, and H-4 all adopted axial orientations with H-2 and H-4 oriented to the same face (tentatively assigned as the α-face). The NOESY correlations of H-2/H-4 and OH-5/H-4 suggested OH-5 was situated on the α-face, while the correlation between OH-4 and H-5 indicated that H-5 was βoriented. It is interesting that compound 1 is structurally related to diepoxin ζ (13). These compounds differ in C-2 and C-3, in which an epoxide is present in the latter. The structural relationship has led to an assumption that compound 1 is possibly an artifact generated by opening the epoxide (C-2/C3) of the parent compound (i.e., diepoxin ζ (13)) during the isolation and/or purification procedure. However, 1 was not detectable by HPLC after incubation of diepoxin ζ (13) with silica gel in air at RT for 2 weeks, suggesting that compound 1 is a natural product. Moreover, diepoxin ζ (13) did not change when incubated with PDB medium (without inoculation of the fungus) after 2 weeks (Figure S3). Further, this compound was incubated only with PDB medium with the pH adjusted to 5.7, which was the lowest value observed during the fermentation process in a 2-week period (Figure S2). Diepoxin ζ (13) was stable, and there was no production of 1 after 7 days (Figure S4). Since the absolute configuration of diepoxin ζ was established by using the exciton coupling CD method,17 the absolute configuration of 1 was deduced as depicted from a biogenetic consideration. The molecular formula of palmarumycin B2 (2) was determined as C20H18O8 by HRESIMS, which contained two more hydrogen atoms than that of 1. A comparison of the 1H and 13C NMR data (Table 1) indicated that compound 2 was closely related to 1. Both compounds showed resonances for one 1,8-dioxynaphthalene ring, four oxygenated methines, one keto group, and three oxygenated quaternary carbons; the only differences were attributed to the presence of two methylene groups (δC 26.5, 34.3) in 2 instead of two sp2 methine groups

B9 possess a 1,8-dioxynaphthalene ring in the lower part, as inferred from their 1H and 13C NMR spectra (Tables 1 and 2), and only differ in the upper ring. Therefore, the focus of the structure elucidation is to establish the structure of the upper moiety. Palmarumycin B1 (1) was isolated as a colorless solid. Its molecular formula was determined as C20H16O8, as a prominent pseudomolecular ion peak was observed at m/z 385.09121 [M + H]+ in the HRESIMS spectrum. The IR spectrum indicated the presence of hydroxy (3378 cm−1) and keto (1692 cm−1) functional groups. The 1H and 13C NMR spectra of 1 (Table 1) suggested it to be a palmarumycin derivative.7 Except for the signals attributed to the 1,8-dioxynaphthalene ring, the resonances for one keto group (δC 188.2), two sp2 carbons (δC 145.5, 125.1), and six oxygenated sp3 carbons including four methines (δC 77.7, 69.4, 68.6, 60.3) and three quaternary carbons (δC 99.1, 70.4, 61.6) were also clearly seen in the 13C NMR spectrum. Similarly, the 1H NMR spectrum showed resonances for six aromatic protons owing to the 1,8dioxynaphthalene ring, and those for two olefinic protons (δH 6.76, dd; 5.84, d), and four oxygenated methine protons (δH 3.52, dd; 3.46, m; 4.22, dd; 4.71, dd). In addition, four H2Oexchangeable protons (δH 5.03, 5.14, 5.59, 6.06, each d) were observed in the 1H NMR spectrum, which each correlated to one of the four aforementioned oxygenated methine protons in the COSY spectrum, thus suggesting the presence of four hydroxy-bearing methine groups in the molecule. The above units were linked together by analysis of the COSY, HSQC, and HMBC spectra. Analysis of the COSY spectrum suggested the presence of two spin systems, which included CH(2)− CH(3)−CH(4) and CH(5)−CH(6)−CH(7) (Figure 1). The HMBC correlations seen from H-2 (δH 3.52, dd) to C-1 (δC 99.1), especially from OH-2 (δH 5.14, d) to C-1 and C-3 (δC 69.4), suggested the spiro ketal carbon was positioned at C-1 (Figure 1). The key HMBC correlations between OH-4 (δH 5.59, d) and OH-5 (δH 6.06, d) and the quaternary carbon resonating at δC 70.4 (C-4a) established the linkage of C-4 and C-5 via C-4a. The correlations from H-6 (δH 6.76, dd) and H-7 (δH 5.84, d) to the keto group (δC 188.2, C-8) in the HMBC spectrum allowed the establishment of an α,β-unsaturated keto group. Further HMBC correlations from H-2, H-4 (δH 4.22, dd), H-5 (δH 4.71, dd), and H-7 to C-8a (δC 61.6) were used to establish a bicyclic carbon skeleton in the upper unit of 1, as B

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a

125.1, CH

188.2, 61.6, 148.2, 106.5, 127.3, 118.8, 133.3, 119.2, 127.1, 106.5, 147.0, 111.4,

1 2 3 4 4a 5 6

7

8 8a 1′ 2′ 3′ 4′ 4a′ 5′ 6′ 7′ 8′ 8a′ OH-2 OH-3 OH-4 OH-5 OH-6 OH-8 OH-8a

C

5.14, 5.03, 5.59, 6.06,

d d d d

(6.0) (5.0) (6.3) (8.3)

7.36−7.46a 7.36−7.46a 6.78, br d (7.8)

6.83, dd (6.6, 1.4) 7.36−7.46a 7.36−7.46a

5.84, d (10.5)

4.71, dd (7.8, 4.9) 6.76, dd (10.5, 4.8)

3.52, dd (10.0, 5.9) 3.46, m 4.22, dd (7.8, 6.3)

δH (J in Hz)

δC, type

200.3, qC 64.9, qC 149.2, qC 108.44, CH 128.3, CH 120.8, CH 135.3, qC 120.5, CH 128.0, CH 108.41, CH 148.2, qC 113.4, qC

34.3, CH2

100.5, qC 78.6, CH 72.3, CH 70.58, CHb 70.55, qCb 63.2, CH 26.5, CH2 t (3.1) m m ddd (16.3, 12.2, 6.8) ddd (16.3, 5.3, 2.8)

7.33−7.44a 7.33−7.44a 6.79, d (7.3)

6.88, br d (6.5) 7.33−7.44a 7.33−7.44a

4.75, 1.94, 1.88, 2.55, 2.21,

3.63a 3.65, m 4.28, d (7.1)

δH (J in Hz)

2 (CD3OD)

62.9, 65.9, 148.8, 106.3, 127.1, 118.8, 133.1, 118.2, 127.0, 106.1, 148.1, 112.0,

CH qC qC CH CH CH qC CH CH CH qC qC

129.6, CH

qC CH CH CH qC CH CH

δC, type 99.7, 77.1, 69.1, 69.3, 66.8, 60.8, 125.2,

4.73, d (8.3)

4.92, d (4.8) 4.88a 5.22, d (6.3) 5.10, d (8.0)

7.34−7.39a 7.34−7.39a 6.82, ddc

6.73, ddc 7.34−7.39a 7.34−7.39a

4.89, m

5.38, dd (10.4, 2.2)

4.40, dd (7.9, 5.2) 5.59, ddd (10.3, 5.2, 2.2)

3.44, dd (10.2, 4.8) 3.48, m 4.08, dd (7.5, 6.3)

δH (J in Hz)

3 (DMSO-d6) qC CH CH CH qC CH CH2

62.1, 66.5, 148.3, 107.3, 127.3, 118.7, 133.3, 119.3, 127.1, 107.1, 147.6, 112.3,

CH qC qC CH CH CH qC CH CH CH qC qC

25.2, CH2

100.8, 76.0, 71.3, 69.4, 67.7, 62.4, 24.8,

δC, type

m m m m m m

br s br s s br s 4.17, br s

4.86, 4.91, 5.13, 4.95,

7.43, d (8.0) 7.38−7.42a 6.86, d (7.0)

6.80, ddc 7.38−7.42a 7.38, da

4.19, 1.75, 1.23, 1.67, 1.20, 4.36,

3.36, d (8.4) 3.48, t (8.4) 4.01, d (8.2)

δH (J in Hz)

4 (DMSO-d6)

Overlapped signals. bAssignments within the same row may be interchanged. cNot a first-order split; the coupling constants were not given.

qC qC qC CH CH CH qC CH CH CH qC qC

qC CH CH CH qC CH CH

δC, type

99.1, 77.7, 69.4, 68.6, 70.4, 60.3, 145.5,

position

1 (DMSO-d6)

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data for 1−5

202.9, 82.5, 149.3, 106.5, 127.2, 118.5, 133.0, 118.4, 127.0, 106.2, 149.2, 112.3,

qC qC qC CH CH CH qC CH CH CH qC qC

44.0, CH2

qC CH CH CH qC CH CH

δC, type 102.2, 76.3, 69.5, 127.8, 136.0, 68.1, 70.1,

6.33, s

4.98, d (6.5) 4.47, d (2.2)

5.32, d (5.5) 5.06, d (5.7)

7.32−7.37a 7.32−7.37a 6.74, ddc

6.73, ddc 7.32−7.37a 7.32−7.37a

3.38, dd (12.8, 3.8) 2.09, dd (12.8, 2.6)

4.77, m 4.10a

4.09, dd (7.9, 5.4) 4.26, m 5.74, br s

δH (J in Hz)

5 (DMSO-d6)

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Table 2. 1H (400 MHz) and 13C (100 MHz) NMR Data for 6−9 6 (CDCl3) position 1 2 3 4 4a 5 6 7 8 8a 1′ 2′ 3′ 4′ 4a′ 5′ 6′ 7′ 8′ 8a′ −OCH3 −COCH3 OH-2 OH-5 OH-8 a

δC, type 98.2, qC 29.5, CH2 34.3, CH2 203.4, 116.3, 158.1, 117.2, 137.1, 139.6, 124.2, 147.3, 109.6, 127.7, 121.2, 134.4, 121.2, 127.7, 109.6, 147.3, 113.4,

qC qC qC CH CH qC qC qC CH CH CH qC CH CH CH qC qC

δH (J in Hz) 2.49, t (6.5) 2.88, t (6.5)

7.42, d (8.3) 7.70, d (8.3)

6.97, d (7.5) 7.46, dd (7.5, 8.3) 7.54, d (8.3) 7.54, d (8.3) 7.46, dd (7.5, 8.3) 6.97, d (7.5)

7 (DMSO-d6) δC, type

8 (DMSO-d6)

δH (J in Hz)

δC, type

δH (J in Hz)

9 (DMSO-d6) δC, type

102.2, qC 85.7, qC 167.9, qC

102.8, qC 85.8, qC 167.7, qC

103.1, qC 82.0, qC 49.4, CH2

195.2, 122.0, 148.6, 120.3, 126.3, 147.8, 130.6, 149.0, 107.7, 127.5, 119.5, 133.6, 120.3, 127.1, 108.1, 147.2, 112.2, 52.2,

198.0, 132.1, 156.4, 113.3, 133.8, 123.9, 136.9, 148.7, 107.8, 127.5, 119.7, 133.6, 120.4, 127.1, 108.2, 147.1, 112.1, 52.3,

199.5, 121.7, 148.1, 119.8, 125.5, 147.6, 130.0, 149.2, 108.0, 127.7, 119.3, 133.7, 120.1, 127.3, 107.5, 148.0, 112.1,

qC qC qC CH CH qC qC qC CH CH CH qC CH CH CH qC qC CH3

7.01, d (8.8) 7.19, d (8.8)

6.88, d (7.3) 7.45, dd (7.3, 8.8) 7.51, d (8.8) 7.54, d (8.4) 7.43, dd (7.5, 8.4) 6.82, d (7.5)

3.49, s

qC qC qC CH CH CH qC qC CH CH CH qC CH CH CH qC qC CH3

7.33, d (7.8) 7.63, t (7.8) 7.35, d (7.8)

6.91, d (7.4) 7.46, dd (7.9, 7.4) 7.54, d (7.9) 7.56, d (7.8) 7.44, dd (7.8, 7.4) 6.85, d (7.4)

qC qC qCa CH CH qC qC qC CH CH CH qC CH CH CH qCa qC

6.66, s 10.12, br s 9.57, s

2.77, d (16.4), 2.50, d (16.4)

6.95, d (8.7) 7.11, d (8.7)

6.85, d (7.3) 7.45, dd (7.3, 8.4) 7.51, d (8.4) 7.57, d (8.3) 7.49, dd (7.4, 8.3) 6.91, d (7.4)

3.48, s 204.6, qC; 30.1, CH3

13.01, s

δH (J in Hz)

6.86, s 10.64, br s

1.95, s 6.13, s 9.72, br s 9.39, s

Assignments within the same row may be interchanged.

in 1. The methylene groups were assigned to C-6 (δC 26.5) and C-7 (δC 34.3), respectively, as the keto group (δC 200.3) was significantly shifted downfield compared to that of 1. This was corroborated by the COSY correlations seen from H-5 (δH 4.75, t) to H2-6 (δH 1.94, 1.88, m) and from H2-6 to H2-7 (δH 2.55, and 2.21, each ddd) and by the HMBC correlations of H26/C-4a (δC 70.55), H2-6/C-8 (δC 200.3), H2-7/C-8, and H2-7/ C-8a (64.9). Thus, compound 2 was determined as the 6,7dihydro derivative of 1. The relative configuration of the upperright ring (C-1−C-4) in 2 was determined to be the same as

Figure 1. Selected COSY and HMBC correlations of 1, 5, and 7.

Figure 2. Key NOESY correlations of 1 and 5. D

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by utilizing the observed COSY correlations of H-5 (δH 4.77, m)/H-6 (δH 4.10, m) and H-6/H2-7 (δH 3.38, 2.09, each dd) (Figure 1). The above moieties were connected by analysis of the HMBC spectrum. The correlations from OH-2 (δH 5.32, d) to C-2 (δC 76.3), C-3 (δC 69.5), and the spiro carbon (δC 102.2, C-1) ascertained that the naphthalene ring was connected to C-1. The correlations from the olefinic proton (H-4, δH 5.74) to C-4a (δC 136.0) and C-5 (δC 68.1) were used to connect the C-1−C-4 and C-5−7 moieties via C-4a. The 1H chemical shifts of the methylene protons (H2-7) (δH 3.38, 2.09, each dd) indicated that the carbonyl group was located at the neighboring position (C-8) of this methylene group. The HMBC correlations from H2-7 to this carbonyl group (δC 202.9, C-8) and the oxygenated quaternary carbon (δC 82.5, C8a) and from OH-8a (δH 6.33, s) to C-8a, C-8, C-1, and C-4a not only confirmed the position of C-8 but also established the linkage between C-1, C-4a, C-8, and C-8a; thus the planar structure of 5 was determined as shown in Figure 1. The relative configuration was established by analysis of the J values and the NOESY spectrum. The large vicinal coupling constant between H-2 and H-3 (7.9 Hz) indicated the pseudoaxial orientations for both protons (Figure 2), while the small coupling constants between H-6 and H2-7 (J = 2.6, 3.8 Hz) suggested that H-6 must adopt a pseudoequatorial orientation. In the NOESY spectrum, the correlations observed from H-5 to H-6, H-7a (δH 3.38), and OH-8a and from H-7a to OH-8a indicated that these protons were on the same face (β). The pseudoequatorial orientation of OH-5 was corroborated by the NOE interaction between H-4 and OH-5. No NOESY correlation was found between OH-8a and H-2, such that H-2 has to be α-oriented. Thus, the relative configuration of 5 was determined as shown in Figure 2. A new metabolite palmarumycin B6 (6) was likewise isolated as a colorless solid. The negative ESIMS spectrum of 6 exhibited a pair of isotopic peaks at m/z 351.1 and 353.0 with a ratio of ca. 3:1, indicating the presence of one chlorine atom in the molecule. The high-resolution ESIMS established the molecular formula C20H13O4Cl, as a prominent ion peak was observed at m/z 351.04092 [M − H]−. The NMR data of 6 closely resembled those of palmarumycin CP17, a metabolite isolated from the Panamanian endophytic fungus Edenia sp.,13 except that the signal for OH-8 was missing in 6, and H-6 (δH 7.42, d) and H-7 (δH 7.70, d) were significantly shifted downfield compared to those of palmarumycin CP17. This could only be explained by the substitution of 8-Cl for the 8OH in 6, and palmarumycin B6 (6) was determined as the 8chloro derivative of palmarumycin CP17. Palmarumycin B7 (7) was isolated as a light yellow solid. Its molecular formula was determined to be C21H14O8 by HRESIMS, indicating 14 degrees of unsaturation. The IR spectrum displayed absorptions for ester (1756 cm−1) and keto (1707 cm−1) functionalities. The 13C NMR spectrum exhibited a total of 21 carbon signals, except the 10 signals assigned to a 1,8-dioxynaphthalene ring, the remaining 11 being incorporated into one tetrasubstituted benzene ring (δC 122.0, qC; 148.6, qC; 120.3, CH; 126.3, CH; 147.8, qC; 130.6, qC), one keto group (δC 195.2), one ester carbonyl (δC 167.9), two oxygenated quaternary carbons (δC 102.2, 85.7), and one methoxyl group (δC 52.2, q). In the 1H NMR spectrum, the presence of a pair of doublets at δH 7.01 (d, J = 8.8 Hz) and 7.19 (d, J = 8.8 Hz) that were assigned to two vicinal aromatic protons and of the downfield resonances at δH 9.57 (s) and 10.12 (br s) attributed to two phenolic groups was reminiscent

that of 1, as inferred by their similar coupling constants and NOEs, whereas the configuration for C-5 was opposite in 2, as suggested by the NOE correlation observed between H-4 (δH 4.28, d) and H-5 (δH 4.75, t). Palmarumycin B3 (3) was obtained as a colorless solid. The NMR and HRESIMS data suggested its molecular formula as C20H18O8, containing two more hydrogen atoms than that of 1. The NMR data of 3 were quite similar to those of 1 (Table 1), except that the signals for a hydroxy-bearing methine (δH 4.89, m; δC 62.9, CH) in 3 replaced those of a keto group in 1, suggesting that 3 was a 8-hydrogenated derivative of 1. This was confirmed by the COSY correlation seen between the olefinic proton (H-7: δH 5.38, dd) and H-8 (δH 4.89, m) and by the HMBC correlations observed from H-8 to C-8a (δC 65.9), C-1 (δC 99.7), C-4a (δC 66.8), C-6 (δC 125.2), and C-7 (δC 129.6). The relative configuration for all the chiral centers, except C-8, was deduced to be the same as that of 1, on the basis of their similar coupling constants and NOEs. The weak NOE correlation between H-5 and H-8 suggested the cisconfiguration of OH-5 and OH-8. Palmarumycin B4 (4) was isolated as a congener of palmarumycin B3 (3). The molecular formula of compound 4 was established as C20H20O8, containing two more hydrogen atoms than that of 3. A comparison of the NMR data suggested that the double bond (C-6/7) in 3 was replaced by two methylene groups (δC 24.8, 25.2) in 4, which can explain the difference in their molecular mass. This is confirmed by the COSY spectrum, in which H-5 (δH 4.19, m) showed a correlation to H2-6 (δH 1.75, 1.23, m), which in turn correlated with H2-7 (δH 1.67, 1.20 m), and this methylene group further correlated with H-8 (δH 4.36, m) to establish a CH(5)− CH2(6)−CH2(7)−CH(8) spin system. The relative configuration of 4 in all chiral centers was determined to be the same as that of 3 by analysis of the NOESY spectrum and 1H−1H coupling constants. Palmarumycin B5 (5) was isolated as a colorless, amorphous solid. It showed pseudomolecular ion peaks at m/z 409.08861 [M + Na]+ and 425.06238 [M + K]+ in the HRESIMS spectrum, consistent with the molecular formula C20H18O8, indicating 12 degrees of unsaturation. In the 13C NMR spectrum, 20 carbon signals were observed, which were assignable to one carbonyl group (δC 202.9), one trisubstituted double bond (δC 127.8, CH; 136.0, qC), two oxygenated quaternary carbons (δC 102.2, 82.5), four oxygenated methines (δC 76.3, 70.1, 69.5, 68.1), one methylene group (δC 44.0), and 10 carbons attributable to a 1,8-dioxynaphthalene ring. Similarly, the 1H NMR spectrum (DMSO-d6) exhibited signals for one olefinic proton (δH 5.74, br s), four oxygenated methine protons (δH 4.09, dd; 4.10, m; 4.26, m; 4.77, m), and two methylene protons (δH 3.38 dd, 2.09 dd), as well as protons belonging to five hydroxys (δH 5.32, d; 5.06, d; 4.98, d; 4.47, d; 6.33, s). The above functionalities account for 9 degrees of unsaturation. Apart from one ring that connected the spiro ketal carbon and the naphthalene ring, there must be two more rings present in the upper unit to fulfill the remaining degrees of unsaturation required by the molecular formula. The bicyclic upper unit was constructed by analysis of the 2D NMR spectra (COSY, HSQC, HMBC). In the COSY spectrum, the olefinic proton (δH 5.74, H-4) showed a correlation to one oxygenated methine proton resonating at δH 4.26 (H-3), which in turn correlated with the second methine proton (δH 4.09, H-2), thus furnishing the C2−C-4 moiety. The C-5−C-7 moiety was likewise established E

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Scheme 1. Proposed Biosynthetic Pathway for Compounds 7−9

pseudomolecular ion peak at m/z 415.07795 [M + Na]+ in the HRESIMS spectrum. The NMR data of 9 were similar to those of 7 (Table 2), indicating both compounds share a parahydroquinone-fused cyclopentone moiety in the upper units. The differences were ascribed to the side chain, in which a 2oxo-propyl group in 9 replaced the methoxycarbonyl group in 8, as evidenced by the HMBC correlations observed from OH2 (δH 6.13, s) to the methylene group (C-3: δC 49.4; δH 2.77, 2.50, each d), C-1 (δC 103.1), C-2 (δC 82.0), and C-4 (δC 199.5) and from the terminal methyl group (δH 1.95, s; δC 30.1) to C-3 and the keto group (δC 204.6) located at the side chain. Compounds 7 and 8 contain only one chiral center (C-2), and their absolute configurations were thus determined by comparison of the specific optical rotatory values with those reported for similar structures in the literature. The (S)-methyl 2-hydroxy-1-oxo-2,3-dihydro-1H-indene-2-carboxylate displayed positive optical rotatory power ([α]25D = +52.1 (c 1.0, CHCl3)),18 while its enantiomer showed negative value ([α]20D = −45.2 (c 1.0, CHCl3)).19 Assuming that the substitution of an additional naphthalene ring to C-3 of methyl 2-hydroxy-1-oxo2,3-dihydro-1H-indene-2-carboxylate does not change the sign of the optical rotation, the absolute configuration of palmarumycins B7 (7) and B8 (8) was then deduced as shown [7: [α]20D = +19.8 (c 0.1, MeOH); 8: [α]20D = +18.8 (c 0.1, MeOH)]. From biogenetic considerations, the absolute configuration for 9 was assumed to be the same as those of 7 and 8. Palmarumycins B1−B4 (1−4) are uncommon 2,3,4-trihydroxy-substituted spirobisnaphthalenes, among which palmarumycins B1 (1), B3 (3), and B4 (4) are structurally related to the co-occurring diepoxin ζ (13),17 palmarumycin C15 (14),7 and palmarumycin C16 (15),7 respectively. Although these new palmarumycins (i.e., B1, B3, and B4) and the latter diepoxins only differed in C-2 and C-3, the possibility that the former compounds were artifacts derived from the latter by opening the C-2/3 epoxide during the isolation and purification

of a 1,2,3,4-tetrasubstituted benzene ring with two hydroxys substituted at the para position if the 13C chemical shifts were taken into consideration. The observation of a singlet (δH 6.66, OH-2) for the OH group in the 1H NMR spectrum (DMSOd6) proved to be crucial for the structure elucidation, as this proton (OH-2) showed key HMBC correlations to the spiro carbon (C-1, δC 102.2), C-2 (δC 85.7, qC), the keto carbon (C4, δC 195.2), and the ester carbonyl (C-3, δC 167.9) (Figure 1). The HMBC correlation observed between the methoxyl group (δH 3.49, s) and the ester carbonyl allowed the establishment of a methyl ester. Then, the tetrasubstituted benzene ring has to fuse the above moiety via C-4a and C-8a to complete the molecule. This was confirmed by the observation of long-range 1 H−13C correlations from H-6 (δH 7.01, d) to C-4 and from H7 (δH 7.19, d) to C-1. Palmarumycin B8 (8) was isolated as an analogue of palmarumycin B7 (7), as indicated by their similar UV, IR, and NMR spectra. The molecular formula of 8 was determined to be C21H14O7, bearing one oxygen atom less than 7. The 1H NMR spectrum of 8 showed resonances for one phenolic OH (δH 10.64, br s), instead of the two found in 7, and for a set of signals including one triplet (δH 7.63) and two doublets (δH 7.35, 7.33) that comprised an ABC spin system, suggesting that one of the phenolic OH groups in 7 was substituted by a hydrogen atom in 8. These changes were also reflected in the 13 C NMR spectra, in which both compounds showed almost superimposed resonances except for the aromatic ring and the adjacent keto group in the upper units (Table 2). An HMBC experiment was then used to determine the substitution pattern of the aromatic ring. In the HMBC spectrum, key correlations observed from H-8 (δH 7.35, d) to C-1 (δC 102.8) and longrange correlations seen from H-6 (δH 7.33, d) to the keto group (C-4, 198.0) suggested the OH is attached to C-5. Therefore, palmarumycin B8 (8) was elucidated as the 8-deoxy derivative of palmarumycin B7 (7). The molecular formula of palmarumycin B9 (9) was determined to be C22H16O7, as this compound exhibited a F

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Table 3. Antibacterial Activity (MIC, μg/mL)a

a

compound

B. subtilis

A. tumefaciens

S. hemolyticus

R. solanacearum

P. lachrymans

X. vesicatoria

1 11 12 13 16 17 18 21 22 streptomycin sulfateb

200 75 37.5 50 25 100 37.5 50 50 5

200 75 50 50 37.5 250 37.5 37.5 25 5

200 75 50 50 25 250 50 37.5 25 5

200 75 37.5 50 37.5 250 37.5 37.5 25 5

150 75 37.5 50 37.5 200 37.5 25 25 7.5

150 75 37.5 50 37.5 200 50 37.5 25 10

The other isolated compounds were inactive with MICs > 250 μg/mL. bPositive control.

procedure could be ruled out, as incubation of diepoxin ζ (13), palmarumycin C15 (14), and palmarumycin C16 (15) with silica gel did not lead to the formation of 1, 3, and 4, respectively. The fact that compounds 13−15 were stable when incubated with PDB medium or PDB medium with pH adjusted to 5.7 (Figures S3−S6) suggested that 1, 3, and 4 were not the degraded products of 13, 14, and 15, respectively, and should be natural products. Palmarumycins B7−B9 (7−9) are examples of spirobisnaphthalenes that contain a rare bicyclic (6/5) unit (i.e., 2,3dihydro-1H-inden-1-one) bridged to a 1,8-dioxynaphthalene ring through a spiroketal linkage. To the best of our knowledge, only one analogue of this class, palmarumycin C6, had been reported previously.7 A plausible biosynthetic pathway was proposed for compounds 7−9 (Scheme 1). The basic skeleton of spirobisnaphthalenes was proposed to be biosynthesized from suitable phenolic precursors followed by oxidative coupling.7 The phenolic precursors could be derived from a polyketide pathway involving one acetyl-CoA and four (or five) malonyl-CoA units, which are then oxidized, followed by radical−radical coupling to yield the spirobisnaphthalene skeleton. The oxidative coupling between radicals S1a and S2b would afford the 3-acetylpalmarumycin CP1, although this metabolite has not been reported so far. This intermediate could be oxidized at C-2 to give a 2-hydoxylated derivative. The electron transfer accompanied by hydrogen migration in this derivative would result in the production of the ring-contracted derivative (compound 9). Similarly, the coupling of S1a and S1b could produce palmarumycin CP1,7 which could be oxidized at C-2 and C-3 to give a 3,4-diketo derivative. The oxidation cleavage of the C-3/4 bond within this diketone, accompanied by the formation of a new σ bond between C-2 and C-4, would yield the carboxylic precursors, which could be further converted to the methyl esters (compounds 7 and 8) by Omethylation with S-adenosylmethionine (SAM). The known compounds (10−22) were identified by comparison of the spectroscopic data with the literature, which included diepoxins γ (10),17 δ (11),17 κ (12),17 and ζ (13),17 palmarumycins C15 (14),7 C16 (15),7 C11 (16),7 and C12 (17),7 cladospirone B (18),9 palmarumycin CP17 (19),13 palmarumycin C6 (20),7 1,4,7β-trihydroxy-8-(spirodioxy-1′,8′naphthyl)-7,8-dihydronaphthalene (21),20 and palmarumycin C8 (22).7 It is worth mentioning that compound 21, previously obtained as a synthetic product by treatment of cladospirone bisepoxide (diepoxin ζ) with lithium iodide in the course of exploring the reactivity of the epoxy and enone functional groups in this bisepoxide,20 was isolated as a natural product for the first time in the present study.

All isolated compounds were evaluated for their antibacterial activities against Bacillus subtilis (ATCC 11562), Staphylococcus hemolyticus (ATCC 29970), Agrobacterium tumefaciens No.8 (ATCC 11158), Pseudomonas lachrymans (ATCC 11921), Ralstonia solanacearum (ATCC 11696), and Xanthomonas vesicatoria (ATCC 11633). Among them, palmarumycin B1 (1), diepoxins δ (11), κ (12), and ζ (13), palmarumycins C11 (16) and C12 (17), cladospirone B (18), 1,4,7β-trihydroxy8(spirodioxy-1′,8′-naphthyl)-7,8-dihydronaphthalene (21), and palmarumycin C8 (22) showed antibacterial activities with MIC values of 25−250 μg/mL; in particular, palmarumycin C8 (22) showed the most promising antibacterial effect against the six bacteria tested (Table 3). The isolated compounds were also evaluated for their antifungal activities by using the spore germination inhibition assay. Among the test compounds, compounds 13, 16−18, and 20−22 inhibited spore germination of Magnaporthe oryzae with IC50 (μg/mL) values in the range 9.1−124.5 (Table 4). The Table 4. Inhibitory Activities against the Spore Germination of M. oryzaea compound 13 16 17 18 20 21 22 carbendazimb

IC50 (μg/mL) 105.0 32.3 76.7 64.8 124.5 35.9 9.1 6.3

± ± ± ± ± ± ± ±

1.2 2.8 3.3 2.3 1.3 2.6 0.4 0.2

The other compounds were inactive (IC50 >200 μg/mL). bPositive control.

a

other compounds were not active against the spore germination (IC50> 200 μg/mL). Palmarumycin C8 (22) showed the best inhibitory activity (IC50 9.1 μg/mL) among the compounds tested, although not as active as the positive control carbendazim (IC50 6.3 μg/mL). The cytotoxicities of all the isolated compounds were evaluated against five human cancer cell lines (HCT-8, Bel7402, BGC-823, A 549, A 2780) using the MTT assay (Table 5). Diepoxin δ (11) and palmarumycin C8 (22) showed pronounced activities against all the tested cell lines, with IC50 values of 1.28−5.83 μM, while diepoxins κ (12), and ζ (13) selectively inhibited the growth of Bel-7402 and A 549 with moderate to weak activities. The other compounds were inactive against the tested cells (IC50 >10 μM). Diepoxin ζ was G

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Table 5. Cytotoxic Activities (IC50, μM)a compound

HCT-8

Bel-7402

BGC-823

A 549

A 2780

11 12 13 22 camptothecinb

1.7 >10 >10 4.2 3.6

3.3 6.4 5.1 2.5 6.3

3.3 >10 >10 2.6 0.04

3.2 8.7 8.8 1.6 1.0 × 10−3

5.8 >10 >10 1.3 0.9

The other compounds were inactive (IC50> 10 μM). control.

a

b

ether−acetone (100:0−0:100) to afford three subfractions, B1−B12. Palmarumycin CP17 (19) (12.1 mg) was crystallized from subfraction B2 and further purified by recrystallization. Subfraction B3 was chromatographed over Sephadex LH-20 (eluted with CHCl3−MeOH, 1:1) to afford palmarumycin C6 (20) (18.5 mg). Subfraction B4 was subjected to column chromatography over Sephadex LH-20 (CHCl3− MeOH, 1:1) and further purified by preparative HPLC (MeOH−H2O, 65:35) to yield a light yellow powder (52.2 mg, palmarumycin C8 (22)). Similarly, subfractions B5 and B6 were processed in the same manner as that of B4 to afford cladospirone B (18) (13.1 mg) and palmarumycin C11 (16) (18.4 mg), respectively. A mixture of 7 (14.5 mg) and 9 (9.1 mg) was crystallized from subfraction B7, which was further purified by preparative HPLC eluting with MeOH−H2O (35:65). Subfraction B8 was fractionated by column chromatography over Sephadex LH-20 to give two further subfractions (B8a and B8b), which were purified by preparative HPLC to afford 1,4,7β-trihydroxy8-(spirodioxy-1′,8′-naphthyl)-7,8-dihydronaphthalene (21) (13.8 mg, from B8a) and palmarumycin C12 (17) (29.2 mg, from B8b), respectively. 8 (8.8 mg) was likewise obtained from subfraction B10 by chromatography over Sephadex LH-20 (CHCl3−MeOH, 1:1) and a preparative HPLC column. Fraction C was similarly fractionated by column chromatography over Sephadex LH-20 (eluted with CHCl3−MeOH, 1:1) and preparative HPLC to yield diepoxin γ (10) (189.6 mg), diepoxin ζ (13) (576.0 mg), and diepoxin δ (11) (257.0 mg). Fraction D was subjected to column chromatography over silica gel (200−300 mesh) eluting with a gradient of CHCl3−MeOH (0:100− 100:0) to obtain 10 subfractions (D1−D10). Subfraction D3 was subjected to gel filtration over Sephadex LH-20 (CHCl3−MeOH, 1:1) to yield diepoxin κ (12) (17.0 mg). Subfraction D5 was purified by preparative HPLC to give palmarumycin C15 (14) (25.6 mg) and palmarumycin C16 (15) (20.8 mg). Fraction E was also subjected to column chromatography over silica gel (200−300 mesh) eluting with a gradient of CHCl3−MeOH (0:100−100:0, v/v) to obtain 13 subfractions. Subfraction E7 was subjected to gel permeation chromatography over Sephadex LH-20 (eluting with CHCl3−MeOH, 1:1), followed by preparative HPLC purification to give 1 (24.5 mg). Subfraction E8 was processed in a similar way to obtain 2 (24.0 mg). Subfraction E9 was subjected to column chromatography over Sephadex LH-20 using CHCl3−MeOH (1:1) as eluent to yield two subsubfractions (E9a and E9b). Subsubfraction E9a was further purified by preparative HPLC to afford 4 (10.4 mg). Similarly, 3 (11.6 mg) and 5 (11.9 mg) were obtained from subsubfraction E9b by HPLC fractionation. Palmarumycin B1 (1): colorless solid (MeOH); mp 140−142 °C; [α]20D +20.1 (c 0.1, MeOH); UV (MeOH) λmax 202.8, 220.8 (sh), 286.4 nm; CD (MeOH) 212 (Δε −9.60), 230 (Δε +10.27), 285 (Δε −0.31), 324 (Δε +2.22) nm; IR νmax 3378, 3063, 2974, 2923, 1692, 1640, 1608, 1590, 1418, 1382, 1281, 1206, 1143, 1067, 1045, 816, 757 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6, 100 MHz) data, see Table 1; HRESIMS m/z 385.09121 [M + H]+ (calcd for C20H17O8, 385.09179). Palmarumycin B2 (2): colorless solid (MeOH); mp 132−134 °C; [α]20D +18.9 (c 0.1, MeOH); UV (MeOH) λmax 203.8, 223.6 (sh), 283.4 nm; CD (MeOH) 208 (Δε +5.69), 224 (Δε +14.26), 252 (Δε −0.61), 291 (Δε +3.49), 320 (Δε −0.74), 364 (Δε +0.11) nm; IR νmax 3383, 3062, 2939, 1724, 1640, 1608, 1590, 1418, 1381, 1279, 1204, 1142, 1053, 824, 758 cm−1; 1H NMR (CD3OD, 400 MHz), 13C NMR (CD3OD, 100 MHz) data, see Table 1; HRESIMS m/z 387.10690 [M + H]+ (calcd for C20H19O8, 387.10744), 409.08882 [M + Na]+ (calcd for C20H18O8Na, 409.08939). Palmarumycin B3 (3): colorless, amorphous solid; [α]20D +18.4 (c 0.1, MeOH); UV (MeOH) λmax 208.8 (sh), 224.2, 298.6 nm; CD (MeOH) 213 (Δε +1.41), 225 (Δε +4.96) nm; IR νmax 3358, 2926, 1640, 1606, 1419, 1382, 1283, 1195, 1138, 1025, 818, 762 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6, 100 MHz) data, see Table 1; HRESIMS m/z 404.13336 [M + NH4]+ (calcd for C20H22NO8, 404.13399). Palmarumycin B4 (4): colorless, amorphous solid; [α]20D +17.7 (c 0.1, MeOH); UV (MeOH) λmax 224.8, 298.6 nm; CD (MeOH) 213

Positive

reported to inhibit the growth of HT 1080 human fibrosarcoma cells in the antitumor invasion chamber assay with an IC50 of 0.37 μM.14

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points (uncorrected) were measured on an XT4-100B microscopic melting-point apparatus (Beijing Ke Yi Instrument Factory). The optical rotations were measured on a JASCO P-2000 polarimeter. The UV spectra were scanned by a JASCO V650 spectrophotometer. CD spectra were recorded on a JASCO J-815 CD spectrometer. IR spectra were recorded on a Nicolet 5700 spectrometer by an FT-IR microscope transmission method. HRESIMS spectra were measured on a Bruker Apex IV FTMS. Standard 1D and 2D NMR spectra were recorded on a Bruker Avance DRX-400 NMR spectrometer (1H at 400 MHz and 13 C at 100 MHz). The chemical shifts were referred to the solvent peaks (δH 2.50, δC 39.5 for DMSO-d6; δH 3.31, δC 49.0 for CD3OD; and δH 7.26, δC 77.2 for CDCl3). Silica gel (100−200 and 200−300 mesh, Qingdao Marine Chemical Inc.), Sephadex LH-20 (Pharmacia), and C18 reversed-phase silica gel (YMC) were used for column chromatography (CC). Semipreparative HPLC separation was carried out on a Lumtech instrument equipped with an HPLC K-501 pump and a K-2501 UV detector, using an XB-C18 column (250 mm × 21.2 mm, 5 μm, Welch Materials Inc.). Precoated silica gel GF-254 plates (Qingdao Marine Chemical Inc.) were used for analytical TLC. Spots were visualized under UV light (254 or 356 nm) or by spraying with 10% H2SO4 in 95% EtOH followed by heating. Fungal Source. The endophytic fungus Dzf12 was isolated from the healthy rhizomes of D. zingiberensis collected in Hubei Province in June 2011. The isolate was identified as a species of Berkleasmium sp. (DQ280463) by analysis of its morphological characteristics and the rRNA gene internal transcribed spacer (ITS) sequence (GenBank accession no. EU543255). The living culture (voucher no. CGMCC 2476) has been deposited in the China General Microbiological Culture Collection Center (CGMCC). The fungus is also stored on potato dextrose agar (PDA) slants at 4 °C and in 40% glycerol at −70 °C in the Herbarium of the College of Agronomy and Biotechnology, China Agricultural University. Fermentation, Extraction, and Isolation. The endophytic fungus was cultured on PDA (potato 200 g/L, dextrose 20 g/L, and agar 20 g/L) medium in Petri dishes at 25 °C for 10 days. Then, three agar plugs (0.5 × 0.5 cm) were inoculated in a 1000 mL Erlenmeyer flask containing 300 mL of potato dextrose broth (potato 200 g/L and dextrose 20 g/L) medium and incubated on a rotary shaker at 150 rpm and 25 °C for 7 days. After 7 days, the fungal cultures were harvested. The fermentation was performed in several batches to afford a total of 150 L of broth. The mycelia and broth were separated, and each was extracted with ethyl acetate (EtOAc). Then, the EtOAc extracts were combined and concentrated under vacuum at 40 °C on a rotary evaporator to obtain a brownish residue (153.8 g). The EtOAc extract was subjected to column chromatography over silica gel (200−300 mesh) eluted with CHCl3−MeOH (20:1, v/v) to obtain five fractions (Frs. A−E). After solvent evaporation, a light yellow crystal was crystallized from fraction A, which was further purified by preparative HPLC to give a light yellow solid (12.0 mg, 6). Fraction B was subjected to medium-pressure liquid chromatography on silica gel (200−300 mesh) eluting with a gradient of petroleum H

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(Δε −3.63), 246 (Δε +0.43) nm; IR νmax 3357, 2936, 1638, 1608, 1589, 1416, 1381, 1278, 1026, 825, 761 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6, 100 MHz) data, see Table 1; HRESIMS m/z 411.10512 [M + Na]+ (calcd for C20H20O8Na, 411.10504), 427.07883 [M + K]+ (calcd for C20H20O8K, 427.07898). Palmarumycin B5 (5): colorless, amorphous solid; [α]20D +20.6 (c 0.1, MeOH); UV (MeOH) λmax 209.4, 221.8, 299.6 nm; CD (MeOH) 211 (Δε −12.52), 230 (Δε +12.06), 262 (Δε −0.29), 308 (Δε +1.26) nm; IR νmax 3347, 2907, 1733, 1639, 1606, 1419, 1382, 1284, 1063, 1046, 1026, 1000, 819, 762 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13 C NMR (DMSO-d6, 100 MHz) data, see Table 1; HRESIMS m/z 409.08861 [M + Na]+ (calcd for C20H18O8Na, 409.08939), 425.06238 [M + K]+ (calcd for C20H18O8K, 425.06333). Palmarumycin B6 (6): colorless solid (CHCl3); mp 191−192 °C; UV (MeOH) λmax 203.8, 239.6, 286.4 nm; IR νmax 3084, 3059, 3006, 2961, 2910, 1640, 1608, 1584, 1433, 1410, 1375, 1337, 1288, 1268, 1177, 1147, 1109, 1042, 926, 900, 820, 760 cm−1; 1H NMR (CDC13, 400 MHz), 13C NMR (CDC13, 100 MHz) data, see Table 2; HRESIMS m/z 351.04092 [M − H]− (calcd for C20H12O435Cl, 351.04296). Palmarumycin B7 (7): light yellow solid (MeOH); mp 220−222 °C; [α]20D +19.8 (c 0.1, MeOH); UV (MeOH) λmax 203.6, 223.8 (sh), 285.8 nm; CD (MeOH) 211 (Δε +2.93), 224 (Δε +10.51), 257 (Δε −0.19), 306 (Δε +0.41) nm; IR νmax 3563, 3363, 3069, 2955, 2840, 1756, 1707, 1611, 1587, 1511, 1466, 1413, 1378, 1307, 1261, 1169, 1130, 1086, 1044, 871, 842, 816, 755, 710 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6; 100 MHz) data, see Table 2; HRESIMS m/z 417.05739 [M + Na]+ (calcd for C21H14O8Na, 417.05809). Palmarumycin B8 (8): colorless solid (MeOH); mp 230−232 °C; [α]20D +18.8 (c 0.1, MeOH); UV (MeOH) λmax 203.2, 221.6, 286.8 nm; CD (MeOH) 210 (Δε −3.41), 220 (Δε −1.01), 232 (Δε −2.10) nm; IR νmax 3409, 3058, 2954, 1760, 1707, 1607, 1586, 1472, 1415, 1380, 1311, 1296, 1264, 1188, 1102, 1048, 996, 816, 750 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6, 100 MHz) data, see Table 2; HRESIMS m/z 379.08183 [M + H]+ (calcd for C21H15O7, 379.08123), 401.06387 [M + Na]+ (calcd for C21H14O7Na, 401.06317). Palmarumycin B9 (9): light yellow solid (MeOH); mp 217−219 °C; [α]20D +18.0 (c 0.1, MeOH); UV (MeOH) λmax 208.8, 224.4, 301.0 nm; CD (MeOH) 231 (Δε +0.94), 242 (Δε +1.79) nm; IR νmax 3383, 3067, 3001, 2962, 1714, 1607, 1587, 1506, 1480, 1414, 1377, 1304, 1270, 1063, 1032, 882, 838, 815, 759, 753 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6, 100 MHz) data, see Table 2; HRESIMS m/z 415.07795 [M + Na]+ (calcd for C22H16O7Na, 415.07882). Antibacterial Assay. The antibacterial activities were tested against Gram-positive bacteria (B. subtilis ATCC 11562 and S. hemolyticus ATCC 29970) and Gram-negative bacteria (A. tumefaciens No.8 ATCC 11158, P. lachrymans ATCC 11921, R. solanacearum ATCC 11696, and X. vesicatoria ATCC 11633). Streptomycin sulfate was used as the positive control. The minimum inhibitory concentrations (MIC) of the compounds and positive control were determined in sterile 96-well plates by the modified broth dilution test.21 Antifungal Assay. The antifungal activity of the isolated compounds was tested against M. oryzae using a spore germination assay as described previously.11 The spores were prepared from 7-dayold cultures of M. oryzae. The test compound dissolved in 10% aqueous ethanol (25 μL) was mixed with an equal volume of fungal spore suspension (containing 2 × 106 spores per mL) on a concave glass slide. Slides containing the spores were incubated in a dark moist chamber at 25 °C for 8 h. Each slide was then observed under a microscope for spore germination status. Carbendazim was used as the positive control, while 10% ethanol was used as the negative control. The percentage (%) of spore germination inhibition was determined as [(Gc − Gt)/Gc] × 100, where Gc is the average of the germinated spore numbers in the negative control (n = 3), and Gt is the average of the germinated spore numbers in the treated sets (n = 3). Three replicates were used for each treatment. The half-inhibition

concentration (IC50) of each sample was determined by linear regression. Cytotoxic Assay. Cytotoxicity was tested against five human carcinoma cell lines (HCT-8, Bel-7402, BGC-823, A549, and A2780) using the microculture tetrazolium (MTT) assay as described previously.22 Camptothecin was used as the positive control.

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ASSOCIATED CONTENT

S Supporting Information *

Copies of IR, 1D (1H, 13C) and 2D NMR, MS, and CD spectra for compounds 1−9 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(L. Zhou) Tel: +86 10 62731199. Fax: +86 10 6273 1062. Email: [email protected]. *(M. Wang) Tel: +86 10 62734093. E-mail: [email protected]. cn. Author Contributions ‡

T. Shan and J. Tian contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank the Hi-Tech R&D Program of China (2011AA10A202), the National Basic Research Program of China (2010CB126105), the National Natural Science Foundation of China (31071710), the program for Changjiang Scholars and Innovative Research Team in University of China (IRT1042), and the Chinese Universities Scientific Fund (2014BH026) for their financial support in this research.

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REFERENCES

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