Cytotoxic and Antibacterial Marfuraquinocins from the Deep South

Nov 19, 2013 - ABSTRACT: Four new sesquiterpenoid naphthoquinones, marfuraquinocins A−D (1−4), and two new geranylated phenazines, phenaziterpenes...
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Cytotoxic and Antibacterial Marfuraquinocins from the Deep South China Sea-Derived Streptomyces niveus SCSIO 3406 Yongxiang Song,† Hongbo Huang,† Yuchan Chen,‡ Jie Ding,† Yun Zhang,† Aijun Sun,† Weimin Zhang,‡ and Jianhua Ju*,† †

CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China ‡ Guangdong Institute of Microbiology, 100 Central Xianlie Road, Guangzhou 510070, China S Supporting Information *

ABSTRACT: Four new sesquiterpenoid naphthoquinones, marfuraquinocins A−D (1−4), and two new geranylated phenazines, phenaziterpenes A (5) and B (6), were isolated from the fermentation broth of Streptomyces niveus SCSIO 3406, which originated from a South China Sea sediment sample obtained from a depth of 3536 m. The structures of 1−6 were elucidated on the basis of extensive MS and one-dimensional and two-dimensional NMR spectroscopic analyses. In a panel of cytotoxicity and antibacterial assays, 1 and 3 were found to inhibit a NCI-H460 cancer cell line with IC50 values of 3.7 and 4.4 μM, respectively. Compounds 1, 3, and 4 exhibited antibacterial activities against Staphylococcus aureus ATCC 29213 with equivalent MIC values of 8.0 μg/mL; compounds 3 and 4 each showed antibacterial activity against methicillin-resistant Staphylococcus epidermidis (MRSE) shhs-E1 with MIC values of 8.0 μg/mL.

A

naphthoquinones, named marfuraquinocins A−D (1−4), and two geranylated phenazines, named phenaziterpenes A (5) and B (6). In this paper, we report the fermentation, isolation, structure elucidation, as well as cytotoxic and antibacterial activities of these compounds.

ctinomycetes play an important role in supplying bioactive natural products with clinical or pharmaceutical applications.1 In the last several decades, a greater focus has been placed on studying actinomycetes from marine environments,2 and they have proven to be a productive new source for novel anticancer and antibacterial agents.3−6 In our efforts to explore anti-infective and cytotoxic metabolites from marine-derived actinomycetes originating from the South China Sea, we have reported antimalarial alkaloids and a cytotoxic cyclopeptide from Marinactinospora thermotolerans SCSIO 00652,7,8 cytotoxic angucyclines from Streptomyces lusitanus SCSIO LR32,9 and an unusual pregnene-based steroid from Streptomyces sp. SCSIO 03219.10 Recently, we found that the fermentation extract of another deep South China Sea sediment-derived actinomycete, identified as Streptomyces niveus SCSIO 3406, showed antibacterial activity against methicillin-resistant Staphylococcus epidermidis (MRSE) shhs-E1 and cytotoxic activity against a panel of human tumor cell lines. Subsequent chemical investigation of the fermentation broth of this strain led to the isolation of six new compounds, including four sesquiterpenoid © 2013 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Strain S. niveus SCSIO 3406 was fermented on a 24 L scale in individual 1 L Erlenmeyer flasks. The fermentation extract was subjected to silica gel column chromatography and semipreparative HPLC, affording compounds 1−6. Marfuraquinocin A (1) was isolated as a yellow solid. Its molecular formula (C26H32O5) was determined upon analysis of the HRESIMS peak at m/z 425.2313 [M + H]+; this molecular formula afforded 11 degrees of unsaturation. The UV spectrum of 1 showed absorption peaks at 415, 301, 264 and 221 nm, indicating the presence of a highly conjugated system. Its IR Received: July 25, 2013 Published: November 19, 2013 2263

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C13 methyl groups in 1. The NOESY correlation between H-12 and H-14b also supported the cis configuration (Figure 2). We were unable to determine the configuration at C-16 after a series of unsuccessful NOESY experiments and crystallization attempts. Marfuraquinocin B (2) was obtained as a yellow powder. Its molecular formula C26H32O5 was established from the HRESIMS peak at m/z 425.2318 [M + H]+, which is the same as that of 1. The 1H and 13C NMR spectroscopic data of 2 closely resembled those of 1 (Table 1), indicating that the structural features were almost identical to those found in 1. Inspection of HSQC and HMBC NMR data revealed that 2 possesses the same planar structure as 1 (Figure 1). Detailed comparisons of the NMR data of 2 and 1 revealed that the chemical shifts of C-11 (δH 1.51, δC 13.8), C-12 (δH 4.55, δC 91.9), and C-25 (δH 1.44, δC 22.6) in 2 differed from those found in 1. The chemical shifts of the methyl protons H3-11 and H3-25 in 2 closely resembled those of the trans-oriented methyl groups in (−)-furaquinocin C (δH 1.51 and 1.47).12 The NOESY correlations between H-12 and H3-25 and between H3-11 and H-14b also supported the trans configuration of C12and C13- methyl groups (Figure 2). The structure of 2 was determined to be the C-13 epimer of 1. Marfuraquinocin C (3) was isolated as a yellow powder. The molecular formula C26H32O6 was determined upon analysis of the HRESIMS peak at m/z 441.2270 [M + H]+, which indicated the presence of one additional oxygen atom in 3 relative to 1. The 1H and 13C NMR spectroscopic data exhibited similarities to those of 1 (Table 1), although signals for the C12 methyl group (noted in 1) were missing, whereas additional signals for an oxygen-bearing methylene group were observed (δH 4.00, 1H, dd, J = 12.5, 8.0 Hz, H-11a; δH 3.89, 1H, dd, J = 12.5, 2.0 Hz, H-11b, δC 62.2, C-11) in 3. Further analyses of COSY, HMQC, and HMBC data of 3 confirmed the presence of an OH group with connectivity to C-11 (Figure 1). Consequently, the structure of 3 is a C-11 hydroxylated congener of 1. Marfuraquinocin D (4) had a molecular formula of C26H32O6, as determined by HRESIMS. The 1H and 13C NMR spectroscopic data were similar to those obtained for compound 2 (Table 1), although signals representative of the C12 methyl group (as noted in 2) were missing, whereas additional signals for an oxygen-bearing methylene (δH 3.99, 1H, dd, J = 12.5, 9.5 Hz and 3.73, 1H, dd, J = 12.5, 1.5 Hz, δC 60.5, C-11) were apparent in compound 4. HMBC correlations from H-11 to C-12 supported the initial notions that the C12 methyl group in 2 was replaced by a hydroxymethylene group (Figure 1). Interestingly, the relationship of compound 2 to 4 was found to be the same as the relationship of compound 1 to 3. Similar to compounds 1 and 2, the relative configurations of the CH2OH group attached to the C12 and C13 methyl group were elucidated to be cis in 3 and trans in 4, respectively, according to the 1H NMR chemical shifts of the C13 methyl group (δH 1.35 in 3 and δH 1.46 in 4), which correspond to δH 1.30 in the synthetic product (+)-3-epi-furaquinocin C and δH 1.47 in (−)-furaquinocin C, respectively.12 The NOESY correlation observed between H-11b and H3-25 in 3 confirmed the cis configuration; the NOESY correlations between H-11a/ b and H-14b/a, and between H-12 and the C13 methyl group in 4 confirmed the trans configuration (Figure 2). The configuration at C-16 in 3 and 4 remains unassigned due to reasons previously described.

spectrum exhibited absorption bands at 3271 cm−1 (OH), 1633 and 1620 cm−1 (CO), and 1573 cm−1 (CC). Analysis of the 1H and 13C NMR spectroscopic data for 1 (Table 1) revealed four C-methyl groups, one methoxy group, five aliphatic and one olefinic methylene groups, four methine groups, including two aromatic methines, and eleven quaternary carbons, including two carbonyls. Two carbonyl signals at δC 180.2 (C-2) and 183.6 (C-9), six quarternary carbon signals between δC 161.1 and 109.4 (C-1, 3, 5−8), and two aromatic methines (δH 7.20, δC 109.5, C-4; δH 6.03, δC 111.3, C-10) indicated the presence of a naphthoquinone moiety in the structure.11 In the HMBC spectrum, the correlations of H-4/C-2, C-6, and C-8 and H-10/C-1, C-2, C-8, and C-9 confirmed the presence of the naphthoquinone moiety. The HMBC correlation from the methoxy protons (δH 3.84, 3H, s, δC 56.2) to C-1 confirmed the position of OMe-26 (Figure 1). The 1H NMR spectrum of 1 also showed a pair of terminal double bond proton signals at δH 4.73 (1H, s, H-24a) and 4.55 (1H, d, J = 2.5 Hz, H-24b); the 13C NMR spectrum confirmed the presence of the double bond [δC 149.2 (C-17) and 109.3 (C-24)]. COSY correlations of H-14a/H2-15/H-16, H2-18/H2-19/H220, and H3-11/H-12 established the three major aliphatic spin systems, as shown in Figure 1. The HMBC correlations from H3-11 to C-12, C-13, from H3-25 to C-6, C-12, C-13, and from H-12 to C-6, C-7, C-13 suggested that a 1,2-dimethylfuran moiety shared connectivity to the naphthoquinone moiety at C6 and C-7. Further HMBC correlations of H-12/C-14 and of H3-25/C-14 indicated linkage of the two-carbon aliphatic chain to the quaternary C-13 of the furan ring. HMBC correlations of H3-22/C-23 and H3-23/C-22 suggested that a gem-dimethyl moiety was present. HMBC correlations from these methyl protons to C-16, C-20, and C-21, as well as HMBC correlations from the terminal olefinic protons H-24a and H-24b to C-16 and C-18, established a 1,1-dimethyl-3-methylenecyclohexane. Finally, the 13C NMR chemical shift of C-5 (δC 156.9) in addition to the remaining atoms in the molecular formula (OH) indicated the presence of a C-5 OH group. Hence, the planar structure of 1 was established. The chemical shifts of the methyl protons H3-11 (δH 1.45) and H3-25 (δH 1.26) in 1 were nearly identical to those of the cis-oriented methyl groups in (+)-3-epifuraquinocin C (δH 1.47 and 1.30)12 and neomarinone (δH 1.47 and 1.27)13 rather than the trans oriented methyl groups in (−)-furaquinocin C (δH 1.51 and 1.47),12 supporting the cis configuration of the C12 and 2264

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Table 1. 1H (500 MHz) and 13C NMR (125 HMz) Spectroscopic Data of Marfuraquinocins A−D (1−4) Marfuraquinocin A (1)a pos. 1 2 3 4 5 6 7 8 9 10 11

δC, type 159.3, C 180.2, C 132.9, C 109.5, CH 156.9, C 129.0, C 161.1, C 109.4, C 183.6, C 111.3, CH 15.6, CH3

12

88.4, CH

13 14a

46.9, C 36.9, CH2

14b 15a

21.4, CH2

16

54.8, CH

17 18

149.2, C 32.2, CH2 23.6, CH2 36.0, CH2

20a 20b 21 22 23 24a 24b 25 26 a

7.20, s

6.03, s 1.45, d (6.5)

4.85, q (6.5)

1.94, td (13.5, 4.5) 1.38, md 1.51, md

δC, type 159.3, C 180.2, C 132.8, C 109.6, CH 157.3, C 129.3, C 161.5, C 109.4, C 183.5, C 111.4, CH 13.8, CH3

91.9, CH 46.8, C 34.0, CH2 21.4, CH2

1.21, md

15b

19

δH, mult. (J in Hz)

Marfuraquinocin B (2)a

1.65, dd (11.0, 3.0) 1.99, m 1.50, m 1.36, m

54.8, CH 149.1, C 32.4, CH2 23.6, CH2 36.2, CH2

1.16, m 35.0, C 28.3, CH3 26.6, CH3 109.3, CH2 19.7, CH3 56.2, CH3

0.88, s 0.77, s 4.73, s 4.55, d (2.5) 1.26, s 3.84, s

δH, mult. (J in Hz)

7.23, s

6.03, s 1.51, d (6.5)

4.55, q (6.5)

1.65, td (13.0, 4.5) 1.38, md 1.46, md 1.22, td (13.0, 4.5) 1.58, dd (11.0, 3.0) 1.97, m 1.48, m 1.35, m

Marfuraquinocin C (3)a δC, type 159.5, C 180.1, C 132.7, C 110.3, CH 157.9, C 128.8, C 160.7, C 108.7, C 184.0, C 111.2, CH 62.2, CH2

92.7, CH 46.3, C 37.7, CH2 21.2, CH2

54.6, CH 149.1, C 32.2, CH2 23.6, CH2 36.0, CH2

1.15, m 34.9, C 28.3, CH3 26.3, CH3 109.1, CH2 22.6, CH3 56.2, CH3

0.83, s 0.71, s 4.71, d (2.5) 4.47, d (2.5) 1.44, s 3.84, s

δH mult. (J in Hz)

7.24, s

5.99, s a: 4.00, dd (12.5, 8.0) b: 3.89, dd (12.5, 2.0) 4.77, dd (8.0, 2.0)

1.93, td (12.5, 3.5) 1.47, md 1.54, dd (13.0, 3.5) 1.21, dd (13.0, 3.5) 1.65, dd (11.5, 3.0) 2.02, m 1.46, m 1.38, m

Marfuraquinocin D (4)b δC, type 159.5, C 180.4, C 132.4, C 109.9, CH 159.0, C 128.4, C 160.9, C 108.0, C 184.1, C 111.1, CH 60.5, CH2

97.2, CH 46.3, C 33.8, CH2

18.9, CH3 56.3, CH3

0.87, s 0.76, s 4.71, s 4.52, d (2.0) 1.35, s 3.83, s

7.04, s

5.97, s a: 3.99, dd (12.5, 9.5) b: 3.73, dd (12.5, 1.5) 4.44, dd (9.5, 1.5) 1.53, md

21.6, CH2

54.6, CH 148.8, C 32.2, CH2 23.5, CH2 36.1, CH2

1.15, m 35.0, C 28.3, CH3 26.6, CH3 109.3, CH2

δH, mult. (J in Hz)

1.22, md 1.37, dt (12.5, 3.0) 1.17, dd (12.5, 3.0) 1.48, md

1.90, m 1.44, m 1.31, m

Marfuraquinocin D (4)c δC, type 161.4, C 182.3, C 134.3, C 111.2, CH 160.6, C 129.1, C 162.1, C 109.2, C 185.7 C 111.8, CH 61.4, CH2

97.5, CH 47.6, C 35.0, CH2 23.2, CH2

22.7, CH3 56.2, CH3

0.78, s 0.66, s 4.66, s 4.41, d (2.0) 1.46, s 3.81, s

7.16, s

6.10, s a: 4.05, dd (12.5, 8.5) b: 3.87, dd (12.5, 3.0) 4.47, dd (8.5, 3.0) 1.72, td (13.0, 4.5) 1.34, md 1.43, md 1.18, md

56.0, CH 150.4, C 33.3, CH2 24.7, CH2 37.2, CH2

1.12, m 34.8, C 28.1, CH3 26.1, CH3 109.0, CH2

δH mult. (J in Hz)

1.63, dd (11.0, 3.0) 1.99, m 1.49, m 1.37, m 1.15, m

35.7, C 28.8, CH3 26.8, CH3 109.8, CH2 24.0, CH3 56.9, CH3

0.87, s 0.74, s 4.75, s 4.51, d (2.0) 1.55, s 3.88, s

Recorded in CDCl3. bRecorded in CDCl3/CD3OD (4:1). cRecorded in CD3OD. dOverlapped.

which can be grouped into two ABX spin systems with the aid of the COSY correlations of H-2/H-3/H-4 and H-7/H-8/H-9. This indicated that 5 has a 1,6- or 1,9-disubstituted phenazine moiety.14 In the HMBC spectrum, the correlations of H-2 to C1, C-10a, C-4, H-3 to C-1, C-4a, H-7 to C-6, C-5a, C-9 and H-8 to C-6 and C-9a confirmed the presence of the phenazine moiety. The COSY spectrum revealed two additional spin systems of H2-11/H-12 and H2-14/H2-15/H-16. Moreover, the HMBC correlations of H-12 to C-13, C-14 and C-18, the methyl protons of H3-18 to C-12, C-13, and C-14; H-16 to C14, C-15, C-19, and C-20; and the methyl protons of H3-19 and H3-20 to C-17 indicated the connectivity of the two isoprene units. An HMBC correlation of H2-11 (δH 4.96, 2H, d, J = 6.0

Phenaziterpene A (5) was isolated as a yellow solid. The molecular formula C22H24N2O2 was established upon analysis of the HRESIMS peak at m/z 349.1918 [M + H]+; this molecular formula afforded 12 degrees of unsaturation. The UV absorptions at 233, 274, 371, and 433 nm implied the presence of an extended conjugated system. The 1H and 13C NMR spectroscopic data suggested the presence of three methyl groups, three methylene groups, eight methine, and eight quaternary carbons. The 1H NMR spectroscopic data of 5 (Table 2) revealed six aromatic protons at δH 7.05 (1H, d, J = 7.5 Hz, H-2), 7.69 (1H, dd, J = 8.0, 8.0 Hz, H-3), 7.76 (1H, d, J = 8.5 Hz, H-4), and at δH 7.23 (1H, d, J = 7.5 Hz, H-7), 7.73 (1H, dd, J = 8.0, 8.0 Hz, H-8), 7.92 (1H, d, J = 8.5 Hz, H-9), 2265

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Hz, δC 66.6) to C-1 (δC 154.4) indicated placement of the terpene unit at C-1. The NOESY correlations of H2-11 to H-9, H-2, and H3-18 confirmed that 5 is a 1,6-disubstituted phenazine and that the configuration of the double bond (C12/C-13) is E. The hydroxy group was placed at C-6 (δC 151.4) to meet the requirement of the molecular formula. This new compound was named phenaziterpene A (Figure 3).

Figure 1. COSY and HMBC key correlations of compounds 1−4.

Figure 3. COSY, HMBC, and NOESY correlations of compound 5.

Phenaziterpene B (6) was obtained as a yellow solid. Its molecular formula, C23H26N2O2, was determined by HRESIMS; the molecular formula afforded 12 degrees of unsaturation. The 1H and 13C NMR data (Table 2) obtained for 6 were similar to those obtained for 5. However, signals for one additional methoxy group (δH 4.19, 3H, s, δC 56.5) were present in the NMR data for compound 6. The HMBC correlation from the methoxy protons to C-6 indicated the placement of this methoxy group at C-6. Consequently, the structure of 6 was established, and its name assigned as phenaziterpene B. Thus far, furaquinocin analogues have been reported from soil-derived microorganisms, such as furaquinocins A−H from Streptomyces sp. KO-3988,11,15,16 furaquinocins I−J from Streptomyces reveromyceticus SN-593,17 and furanonaphthoquinone from Streptomyces cinnamonensis ATCC 15413,18 as well as neomarinone from the marine-derived actinomycete CNH099.19,13 Due to their cytotoxicity against human cancer cells,11,16,19 total chemical syntheses of furaquinocins A− E20−23 and neomarinone24 have been completed. Natural phenazines have been primarily isolated from the Pseudomonas and Streptomyces species and were found to possess antibiotic, antitumor, antimalarial, and antiparasitic activities; consequently, their derivatization and total syntheses have been explored.25 Compounds 5 and 6 are structurally related to geranylphenazinediol that was isolated recently from the Streptomyces sp. strain LB173, exhibiting inhibitory activity toward human acetylcholinesterase. Geranylphenazinediol bears C-geranylation, while compounds 5 and 6 both bear Ogeranylation.26 In this study, compounds 1−6 were evaluated for potential cytotoxic activities against the human glioblastoma cell line SF268, the human breast adenocarcinoma cell line MCF-7, the human lung cancer cell line NCI-H460, and the human hepatocarcinoma cancer cell line HepG2, using the SRB method and with cis-dichlorodiamineplatinum (DDP) as the positive control (Table 3).27 Compounds 1 and 3 showed cytotoxic activity against the NCI-H460 cancer cell line with IC50 values of 3.7 and 4.4 μM, respectively. Compounds 1−6 were evaluated for their antibacterial activities using a

Figure 2. Key NOESY correlations of compounds 1−4.

Table 2. 1H (500 MHz) and 13C NMR (125 HMz) Spectroscopic Data of Phenaziterpenes A (5) and B (6) in CDCl3 phenaziterpene A (5) pos.

δC, type

1 2 3 4 4a 5a 6 7 8 9 9a 10a 11 12 13 14 15 16 17 18 19 20 6-OMe

154.4, C 108.3, CH 130.6, CH 120.6, CH 141.9,C 134.5,C 151.4,C 109.3, CH 131.1, CH 120.7, CH 142.6,C 137.9,C 66.6, CH2 119.4, CH 140.8,C 26.2, CH2 39.5, CH2 123.8, CH 131.8, C 16.9, CH3 25.7, CH3 17.7, CH3

δH, mult. (J in Hz) 7.05, d (7.5) 7.69, dd (8.0, 8.0) 7.76, d (8.5)

7.23, d (7.5) 7.73, dd (8.0, 8.0) 7.92, d (8.5)

4.96, d (6.0) 5.66, t (6.0) 2.17, m 2.13, m 5.09, t (6.0) 1.80, s 1.64, s 1.58, s

phenaziterpene B (6) δC, type 155.0, C 106.8, CH 129.8, CH 121.9, CH 143.1,C 136.9,C 154.2,C 108.5, CH 130.1, CH 122.3, CH 143.2,C 137.4,C 66.7, CH2 119.7, CH 140.7,C 26.3, CH2 39.6, CH2 123.9, CH 131.8, C 16.9, CH3 25.7, CH3 17.7, CH3 56.5, CH3

δH, mult. (J in Hz) 7.09, d (7.0) 7.73, dd (7.5, 7.5) 8.00, d (8.0)

7.08, d (7.0) 7.74, dd (7.5, 7.5) 8.00, d (8.5)

4.98, d (6.5) 5.68, t (6.5) 2.16, m 2.13, m 5.11, t (6.0) 1.81, 1.66, 1.60, 4.19,

s s s s

2266

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inoculations by transfer into each of the 250 mL Erlenmeyer flasks (×100) containing 50 mL of modified-AM2ab medium (0.5% soybean flour, 0.5% soluble starch, 0.2% yeast extract, 0.2% peptone, 2% glucose, 3% sea salt, 0.05% K2HPO4, 0.05% MgSO4·7H2O, 0.4% NaCl, 0.2% CaCO3, pH 7.2) and were incubated at 28 °C on rotary shakers (200 rpm) for 36 h. Each of the seed cultures was aseptically transferred to 1 L Erlenmeyer flasks containing 200 mL of modifiedAM2ab medium, respectively. These flasks were then incubated at 28 °C on rotary shakers (200 rpm) for 9 days. After fermentation, the culture (24 L) was centrifuged (3600 rpm, 10 min) to yield the supernatant and a mycelia cake. The supernatant was extracted with equal volumes of butanone three times; the organic phase was concentrated under reduced pressure to give a residue. The mycelia cake was extracted with 3 L of acetone, evaporated to dryness, and the resulting residues were combined (25.3 g). Isolation of Secondary Metabolites. The combined extract was subjected to silica gel CC using gradient elution with a CHCl3/MeOH mixture (100/0, 98/2, 95/5, 92/8, 90/10, 80/20, 50/50 v/v) to give seven fractions (A1−A7). Fraction A1 was subjected to silica gel CC using a mixture of petroleum ether/EtOAc with a 4% step gradient increasing in EtOAc concentration to afford B3−B5 (92/8−84/16) and B6−B8 (80/20−72/28). Fractions B3−B5 and B6−B8 were further purified by repetitive semipreparative RP-C18 HPLC eluting with CH3CN/H2O (9:1) at a flow rate of 2.5 mL/min to afford compounds 5 (tR = 23.7 min, 20.5 mg) and 6 (tR = 22.8 min, 12.4 mg), respectively. Fractions A2−A6 were subjected to silica gel CC with petroleum ether/EtOAc to give fractions C9−C10 (68/32−64/36) and C15−C16 (44/56−40/60). The C9−C10 fractions were repeatedly applied to semipreparative RP-C18 HPLC and eluted with a linear gradient from 80% to 89% aqueous CH3CN over the course of 21 min and gave compounds 1 (tR 18.8 min, 5.4 mg) and 2 (tR 18.2 min, 6.8 mg). Similarly, compounds 3 (7.0 mg) and 4 (5.6 mg) were obtained from fractions C15−C16 by semipreparative RP-C18 HPLC using a linear gradient elution of CH3CN/H2O from 63% to 83% aqueous CH3CN over the course of 21 min at tR = 19.6 and tR = 19.9 min, respectively. Marfuraquinocin A (1): yellow powder; [α]25 D + 72 (c 0.40, MeOH); UV (MeOH) λmax (log ε) 221 (4.40), 264 (4.17), 301 (3.93), 415 (3.54) nm; IR (ATR) νmax 3271, 2928, 1633, 1620, 1573, 1296, 1242 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; (−)-ESIMS m/z 423.4 [M − H]−, (+)-ESIMS m/z 425.2 [M + H]+; (+)-HRESIMS m/z 425.2313 [M + H]+ (calcd for C26H33O5, 425.2323). Marfuraquinocin B (2): yellow powder; [α]25 D −12 (c 0.42, MeOH); UV (MeOH) λmax (log ε) 221 (4.31), 264 (4.09), 301 (3.84), 407 (3.42) nm; IR (ATR) νmax 3354, 2927, 1634, 1618, 1574, 1294, 1242 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; (−)-ESIMS m/z 423.4 [M − H] −, (+)-ESIMS m/z 425.2 [M + H]+; (+)-HRESIMS m/z 425.2318 [M + H]+ (calcd for C26H33O5, 425.2323). Marfuraquinocin C (3): yellow powder; [α]25 D +101 (c 0.40, MeOH); UV (MeOH) λmax (log ε) 221 (4.42), 264 (4.19), 302 (3.95), 414 (3.58) nm; IR (ATR) νmax 3437, 2934, 1634, 1618, 1574, 1296, 1242 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; (−)-ESIMS m/z 439.5 [M − H]−, (+)-ESIMS m/z 441.2 [M + H]+;

Table 3. IC50 Values (μM) of Compounds 1−6 against Human Tumor Cell Linesa SF-268 1 2 3 4 5 6 DDP a

26.7 27.9 17.9 18.3 10.2 30.4 4.8

± ± ± ± ± ± ±

0.2 0.4 0.3 0.8 0.7 1.6 0.3

MCF-7 12.9 18.7 18.9 14.8 16.8 21.9 4.1

± ± ± ± ± ± ±

NCI-H460

0.7 0.4 1.0 0.3 1.7 0.7 0.2

3.7 18.8 4.4 8.8 68.9 >100 2.9

± ± ± ± ±

0.1 0.4 0.7 0.3 2.2

± 0.1

HepG-2 17.5 18.5 11.9 15.5 22.3 52.7 2.5

± ± ± ± ± ± ±

0.7 0.2 0.3 1.2 1.2 1.8 0.1

Compounds are considered cytotoxic when the IC50 value is ≤10 μM.

previously described method;8,28 ampicillin and kanamycin were used as antibacterial controls. Staphyloccocus aureus ATCC 29213, Escherichia coli ATCC 25922, Acinetobacter baumannii ATCC 19606, Aeromonas hydrophila ATCC 7966, Micrococcus luteus,8 methicillin-resistant S. aureus (MRSA) shhs-A1, and methicillin-resistant S. epidermidis (MRSE) shhs-E1 were used as test microorganisms (Table 4). Compounds 1, 3, and 4 all showed inhibitory activities against S. aureus ATCC 29213 with MIC values of 8.0 μg/mL. Compounds 3 and 4 both exhibited inhibitory activities against MRSE with MICs of 8.0 μg/mL.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained with an MCP-500 polarimeter (Anton Paar). UV spectra were measured with a UV-2600 spectrometer (Shimadzu). NMR spectra were acquired using an Avance 500 spectrometer (Bruker) at 500 MHz for 1H and 125 MHz for 13C. Low-resolution and highresolution mass spectrometric data were determined using an amaZon SL ion trap mass spectrometer and a MaXis quadrupole-time-of-flight mass spectrometer (Bruker), respectively. Column chromatography (CC) was carried out on silica gel (100−200 mesh, Yantai Jiangyou Silica Gel Development Company, Ltd.). Semipreparative reversed phase-high-performance liquid chromatography (RP-HPLC) was performed using a ProStar 210 solvent delivery system equipped with a ProStar 335 PDA detector (Varian) and using a YMC-Pack ODS-A column (250 × 10 mm, 5 μm). Bacterial Strain. Strain SCSIO 3406 was isolated from a sediment sample collected in the South China Sea at E 120°0.250′ and N 20°22.971′ at a depth of 3536 m. It was identified as S. niveus SCSIO 3406 on the basis of morphological characteristics29 and 16S RNA sequence analysis, compared with other sequences in the GenBank database. The DNA sequence has been deposited in GenBank with accession no. KF613004. The strain has been preserved at the RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences and at the China General Microbiological Culture Collection Center (CGMCC) with the no. CGMCC 7863. Fermentation and Extraction. The strain was grown on modified ISP-4 agar plates and incubated at 28 °C. The mycelium was used for

Table 4. Antibacterial Activities (MIC, μg/mL) of Compounds 1−6

1 2 3 4 5 6 Ampicillin Kanamycin

E. coli ATCC 25922

S. aureus ATCC 29213

A. baumannii ATCC 19606

A. hydrophila ATCC 7966

M. luteus

MRSA (clinical isolate shhs-A1)

MRSE (clinical isolate shhs-E1)

128.0 128.0 128.0 128.0 128.0 128.0 2.0 0.5

8.0 64.0 8.0 8.0 128.0 128.0 0.5 0.5

>128.0 >128.0 >128.0 >128.0 >128.0 >128.0 ≥128.0 4.0

128.0 128.0 128.0 128.0 64.0 128.0 128.0 0.5

>128.0 >128.0 >128.0 >128.0 >128.0 >128.0 2.0 0.5

>128.0 >128.0 >128.0 >128.0 >128.0 >128.0 >128.0 0.5

64.0 32.0 8.0 8.0 128.0 128.0 2.0 128.0

2267

dx.doi.org/10.1021/np4006025 | J. Nat. Prod. 2013, 76, 2263−2268

Journal of Natural Products

Article

(+)-HRESIMS m/z 441.2270 [M + H]+ (calcd for C26H33O6, 441.2272). Marfuraquinocin D (4): yellow powder; [α]25 D −39 (c 0.40, MeOH); UV (MeOH) λmax (log ε) 221 (4.40), 265 (4.19), 302 (3.95), 408 (3.56) nm; IR (ATR) νmax 3420, 2926, 1634, 1618, 1576, 1294, 1242 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; (+)-HRESIMS m/z 441.2269 [M + H]+ (calcd for C26H33O6, 441.2272). Phenaziterpene A (5): yellow powder; UV (CHCl3) λmax (log ε) 233 (4.06), 274 (4.76), 371 (3.43), 433 (3.23) nm; IR (ATR) νmax 3566, 2965, 2926, 2864, 1533, 1485, 802, 737 cm−1; 1H and 13C NMR spectroscopic data, see Table 2; (+)-HRESIMS m/z 349.1918 [M + H]+ (calcd for C22H25N2O2, 349.1911). Phenaziterpene B (6): yellow powder; UV (CHCl3) λmax (log ε) 233 (4.11), 274 (4.74), 367 (3.53), 426 (3.43) nm; IR (ATR) νmax 2955, 2922, 2851, 1487, 1150, 804, 739 cm−1; 1H and 13C NMR spectroscopic data, see Table 2; (+)-HRESIMS m/z 363.2074 [M + H]+ (calcd for C23H27N2O2, 363.2067). Cytotoxicity Assay. Compounds 1−6 were evaluated for their cytotoxic activities against four human cancer cell lines, SF-268, MCF7, NCI-H460, and HepG-2, using a previously reported SRB method.27 Antibacterial Assay. Compounds 1−6 were tested for their antibacterial activities against S. aureus ATCC 29213, E. coli ATCC 25922, A. baumannii ATCC 19606, A. hydrophila ATCC 7966, M. luteus,8 methicillin-resistant S. aureus (clinical isolate shhs-A1 from Shanghai Huashan Hospital), and methicillin-resistant S. epidermidis (clinical isolate shhs-E1 from Shanghai Huashan Hospital) using a broth dilution method.28 Each of the reported MIC values in Table 4 is the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in microdilution wells as detected by the unaided eye.28

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(7) Huang, H.; Yao, Y.; He, Z.; Yang, T.; Ma, J.; Tian, X.; Li, Y.; Huang, C.; Chen, X.; Li, W.; Zhang, S.; Zhang, C.; Ju, J. J. Nat. Prod. 2011, 74, 2122−2127. (8) Zhou, X.; Huang, H.; Chen, Y.; Tan, J.; Song, Y.; Zou, J.; Tian, X.; Hua, Y.; Ju, J. J. Nat. Prod. 2012, 75, 2251−2255. (9) Huang, H.; Yang, T.; Ren, X.; Liu, J.; Song, Y.; Sun, A.; Ma, J.; Wang, B.; Zhang, Y.; Huang, C.; Zhang, C.; Ju, J. J. Nat. Prod. 2012, 75, 202−208. (10) Zhang, Y.; Zhou, X.; Huang, H.; Tian, X.; Song, Y.; Zhang, S.; Ju, J. J. Antibiot. 2013, 66, 327−331. (11) Funayama, S.; Ishibashi, M.; Anraku, Y.; Komiyama, K.; O̅ mura, S. Tetrahedron Lett. 1989, 30, 7427−7430. (12) Smith, A. B.; Sestelo, J. P.; Dormer, P. G. J. Am. Chem. Soc. 1995, 117, 10755−10756. (13) Kalaitzis, J. A.; Hamano, Y.; Nilsen, G.; Moore, B. S. Org. Lett. 2003, 5, 4449−4452. (14) Yun, B. S.; Ryoo, I. J.; Kim, W. G.; Kim, J. P.; Koshino, H.; Seto, H.; Yoo, I. D. Tetrahedron Lett. 1996, 37, 8529−8530. (15) Komiyama, K.; Funayama, S.; Anraku, Y.; Ishibashi, M.; Takahashi, Y.; O̅ mura, S. J. Antibiot. 1990, 43, 247−252. (16) Ishibashi, M.; Funayama, S.; Anraku, Y.; Komiyama, K.; O̅ mura, S. J. Antibiot. 1991, 44, 390−395. (17) Panthee, S.; Takahashi, S.; Takagi, H.; Nogawa, T.; Oowada, E.; Uramoto, M.; Osada, H. J. Antibiot. 2011, 64, 509−513. (18) Sedmera, P.; Pospíšil, S.; Novák, J. J. Nat. Prod. 1991, 54, 870− 872. (19) Hardt, I. H.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 2000, 41, 2073−2076. (20) Saito, T.; Morimoto, M.; Akiyama, C.; Matsumoto, T.; Suzuki, K. J. Am. Chem. Soc. 1995, 117, 10757−10758. (21) Saito, T.; Suzuki, T.; Morimoto, M.; Akiyama, C.; Ochiai, T.; Takeuchi, K.; Matsumoto, T.; Suzuki, K. J. Am. Chem. Soc. 1998, 120, 11633−11644. (22) Trost, B. M.; Thiel, O. R.; Tsui, H. C. J. Am. Chem. Soc. 2002, 124, 11616−11617. (23) Trost, B. M.; Thiel, O. R.; Tsui, H. C. J. Am. Chem. Soc. 2003, 125, 13155−13164. (24) Peña-López M, M.; Martínez, M. M.; Sarandeses, L. A.; Sestelo, J. P. Chem.Eur. J. 2009, 15, 910−916. (25) Laursen, J. B.; Nielsen, J. Chem. Rev. 2004, 104, 1663−1686. (26) Ohlendorf, B.; Schulz, D.; Erhard, A.; Nagel, K.; Imhoff, J. F. J. Nat. Prod. 2012, 75, 1400−1404. (27) Chen, Z.; Song, Y.; Chen, Y.; Huang, H.; Zhang, W.; Ju, J. J. Nat. Prod. 2012, 75, 1215−1219. (28) CLSI; Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, Approved Standard, 8th ed.; Clinical and Laboratory Standards Institute: Villanova, PA, 2009; pp M07−A8. (29) Kämpfer, P. In Order XIV. Streptomycetales ord. nov. Bergey’s Manual of Systematic Bacteriology; Goodfellow, M. et al.Eds.; Springer: New York, 2012, 1446−1804.

ASSOCIATED CONTENT

* Supporting Information S

Spectra of one-dimensional and two-dimensional NMR for compounds 1−6. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +86-20-89023028. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China (Grants 41206135 and 41106138), National High Technology Research and Development Program of China (Grant 2012AA092104), and the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant KSCX2-EW-G-12). We thank Prof. M. Wang, Institute of Antibiotics, Huashan Hospital of Fudan University, for providing us MRSA isolate shhs-A1 and MRSE isolate shhsE1. We additionally thank the analytical facility center (Ms. Xiao and Mr. Li) of the South China Sea Institute of Oceanology for acquisition of NMR data.

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REFERENCES

(1) Berdy, J. J. Antibiot. 2005, 58, 1−26. (2) Goodfellow, M.; Fiedler, H. P. Antonie Van Leeuwenhoek 2010, 98, 119−142. (3) Olano, C.; Méndez, C.; Salas, J. A. Mar. Drugs 2009, 7, 210−248. (4) Bull, A. T.; Stach, J. E. M. Trends Microbiol. 2007, 15, 491−499. (5) Williams, P. G. Trends Biotechnol. 2009, 27, 45−52. (6) Gerwick, W. H.; Moore, B. S. Chem. Biol. 2012, 19, 85−98. 2268

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