4-Phenyl-3,4-dihydroquinolone Derivatives from Aspergillus

Measured in DMSO-d6 at 500 and 125 MHz for 1H and 13C, respectively. b ...... Pockrandt , D.; Ludwig , L.; Fan , A.; Konig , G. M.; Li , S. M. ChemBio...
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4‑Phenyl-3,4-dihydroquinolone Derivatives from Aspergillus nidulans MA-143, an Endophytic Fungus Isolated from the Mangrove Plant Rhizophora stylosa Chun-Yan An,†,‡ Xiao-Ming Li,† Han Luo,†,‡ Chun-Shun Li,† Ming-Hui Wang,†,‡ Gang-Ming Xu,† and Bin-Gui Wang*,† †

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, People’s Republic of China ‡ University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Six new 4-phenyl-3,4-dihydroquinolone derivatives (1−6) along with the related aflaquinolone A (7) were isolated and identified from the cultures of Aspergillus nidulans MA-143, an endophytic fungus obtained from the fresh leaves of the marine mangrove plant Rhizophora stylosa. Their structures including absolute configurations were determined by spectroscopic analysis and electronic circular dichroism experiments, and the structure of compound 1 was confirmed by single-crystal X-ray crystallographic analysis. In bioscreening experiments, none of the isolated compounds showed potent antibacterial or cytotoxic activity. However, compounds 2, 3, and 7 exhibited lethality against brine shrimp (Artemia salina), with LD50 values of 7.1, 4.5, and 5.5 μM, respectively.

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ndophytic fungi derived from mangrove plants have been attracting considerable attention owing to their ability to produce bioactive secondary metabolites.1−3 As part of our ongoing research to discover structurally interesting and biologically active secondary metabolites from mangrovederived fungi,4−6 we recently focused on the endophytic fungal strain Aspergillus nidulans MA-143 that was isolated from the fresh leaves of the mangrove plant Rhizophora stylosa. As a model fungal strain, the secondary metabolism of A. nidulans has been explored mainly by genetic modulation. 7−12 Preliminary screening of the culture extract of this fungal strain indicated brine shrimp (Artemia salina) lethality and antibacterial activity. An organic extract of the fermentation medium of the fungus led to the isolation of six new 4-phenyl3,4-dihydroquinolone derivatives, namely, aniduquinolones A− C (1−3), 6-deoxyaflaquinolone E (4), isoaflaquinolone E (5), and 14-hydroxyaflaquinolone F (6), as well as one related known compound, aflaquinolone A (7). These compounds incorporate a 4-phenyl-3,4-dihydroquinolin-2-one moiety and are new members of the fungal quinoline alkaloids, which include the aflaquinolones, aspoquinolones, and penigequinolones.13−16 Quinoline alkaloids have been widely isolated from plants, but few examples of such compounds have been described from filamentous fungi.15 The isolation, structure elucidation, and bioactivity evaluation of the isolated compounds are described in this report. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The culture broth and mycelia of A. nidulans MA-143 were separated by filtration and then exhaustively extracted with EtOAc and MeOH, respectively. The combined extracts were further purified by a combination of column chromatography (CC) including Si gel, reversed-phase Si gel C18, Sephadex LHReceived: June 11, 2013

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fungicolous isolates of Aspergillus spp.13 In the HMBC spectrum, observed correlations from H-3 to C-2, C-4, and C-5, from the exchangeable amide NH proton H-1 to C-3, C-5, and C-9, from the methoxy protons H3-27 to C-3, and from the aromatic protons H-12/H-16 to C-4 (Figure 1) established the quinolone portion of compound 1 was the same as that of aflaquinolone A (7).13

20, and semipreparative HPLC, to yield seven dihydroquinolones (1−7). Compound 1, initially obtained as an amorphous solid, showed a pseudomolecular ion peak in the low-resolution negative ESIMS spectrum at m/z 434 [M − H]−. Its molecular formula was determined as C26H29NO5 based on HRESIMS data, requiring 13 degrees of unsaturation. The 1H NMR spectrum (Table 1) showed well-dispersed signals over a wide Table 1. NMR Spectroscopic Data for Compounds 1 and 2a 1 position 1-NH 2 3 4 5 6 7 8 9 10 11 12/16 13/15 14 17 18 19 20 21 22 23 24 25 26 27 4-OH a

δC

2 δH (J in Hz)

δC

10.29, br s 166.1, 84.2, 78.6, 111.1, 155.0, 119.5, 126.7, 106.6, 136.0, 140.0, 126.1, 128.5, 128.5, 120.3, 134.0, 82.9, 37.2,

C CH C C C C CH CH C C CH CH CH CH CH C CH2

30.3, CH2 81.1, 146.2, 17.9, 109.6,

CH C CH3 CH2

27.3, CH3 58.3, CH3

3.60, s

7.35, m 6.44, d (8.3)

7.22, 7.35, 7.35, 6.67, 6.24,

d (7.6) m m d (16.2) d (16.2)

α 1.92, m β 1.76, m α 1.72, m β 2.01, m 4.33, t (6.7) 1.67, 4.75, 4.96, 1.34, 3.45,

s s s s s

δH (J in Hz) 10.24, br s

166.1, 84.2, 78.6, 111.0, 155.3, 119.5, 126.6, 106.4, 135.9, 140.1, 126.1, 128.4, 128.4, 120.2, 133.9, 82.6, 37.5,

C CH C C Cb C CH CH C C CH CH CH CH CH C CH2

25.9, CH2 85.2, 70.1, 24.9, 26.7,

CH C CH3 CH3

27.1, CH3 58.3, CH3

3.60, s

7.33, m 6.40, d (8.3)

7.21, 7.33, 7.33, 6.64, 6.20,

d (6.2) m m d (16.2) d (16.2)

1.66, 1.87, 1.76, 1.82, 3.70,

m m m m t (6.7)

Figure 1. Key HMBC (arrows) and COSY (bold lines) correlations from compounds 1−3.

1.05, s 1.06, s

However, the terpenoid side chain of compound 1 was significantly different from that of aflaquinolone A (7). Specifically, the cyclohexane moiety present in 7 was absent in 1. Instead, signals for a substituted tetrahydrofuran unit and a terminal double bond were observed (Table 1). The COSY correlations from H-21 to H-20 and H-22 as well as HMBC correlations from the terminal olefinic protons H2-25 to C-22, C-23, and C-24, from H3-24 to C-22, and from H3-26 to C-20 and C-18 supported the above deduction (Figure 1). The relative configuration of 1 was determined by analysis of J-values and NOESY data (Figure 2). The observed key NOE correlations from H-3 to H-12/H-16 indicated the cofacial orientation of H-3 and the phenyl ring (Figure 2), while the 3OMe and 4-OH groups were placed on the opposite side. This assignment was consistent with the corresponding configuration of aflaquinolone A (7).13 As for the terpenoid portion of the molecule, the typical large coupling constant for H-17 and H-18 (J = 16.2 Hz) indicated an E-geometry for the double bond at C-17/C-18, while the NOESY correlations from H-22 to H-17 and H-18 and from H3-26 to one of the two protons in the terminal double bond H-25 (at δH 4.96) indicated the same orientation of the 19-Me and 22-allyl moieties. On the basis of the above evidence, the relative configurations for the dihydroquinoline-2-one and terpenoid units were determined, respectively. However, the stereochemical relationship between these two units could not be correlated based on the NOESY

1.30, s 3.44, s 4.06, s

Measured in DMSO-d6 at 500 and 125 MHz for 1H and respectively. bA weak signal, but was confirmed by HMBC.

13

C,

field range, which disclosed the presence of an amide proton (H-1), a monosubstituted phenyl group (H-12−H-16), a 1,2,3,4-tetrasubstituted aromatic unit (H-8 and H-9), and trans (H-17 and H-18) and terminal (H2-25) double bonds at lower field. In addition, two oxymethine protons (H-3 and H22), a methoxy group (H3-27), and signals indicative of two methylenes (H2-20 and H2-21) and two methyls (H3-24 and H3-26) were present at higher field. These findings were in agreement with the 13C NMR and DEPT data (Table 1), which exhibited 26 carbon signals as three methyls (with one oxygenated), three methylenes (with one terminal), two oxygenated sp3 and nine sp2 methines, and nine quaternary carbons (with two oxygenated sp3 and six sp2 as well as one amide/ester carbon). Detailed analysis of the above NMR data as well as 2D NMR correlations indicated that the structure of compound 1 was similar to that of aflaquinolone A (7), a 3,4-dihydroquinolone derivative that was very recently obtained from marine and B

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reported that the ECD spectra of aflaquinolones and related analogues are affected largely by the absolute configuration of the dihydroquinolin-2-one unit, regardless of the configuration for the terpenoid side chain.13 The ECD spectrum of 1 (Experimental Section and S39 in the Supporting Information) showed positive Cotton effects (CEs) at 221, 280, 286, and 322 nm and a strong negative CE at 251 nm, which were nearly identical to those observed for aflaquinolone A (7).13 Thus, the 3S, 4S-absolute configuration was assigned to compound 1 and, subsequently, the 19R, 22R-configuration was deduced based on the relative configuration as revealed by X-ray crystallographic analysis. Because the terpenoid portion of compound 1 was significantly different from that of aflaquinolone A (7), the trivial name aniduquinolone A was assigned to 1. Aniduquinolone B (2) was obtained as a colorless solid. The structure elucidation of 2 was relatively straightforward due to the close relationship with compound 1. On the basis of negative HRESIMS data, compound 2 was assigned the molecular formula C26H31NO6 (12 unsaturations), having one H2O unit more than that of 1. Its structure was assigned by independent and detailed analysis of the 1D and 2D NMR data as well as by comparison with the data for 1. The same 3methoxy-4-phenyl-4,6-dihydroxy-3,4-dihydroquinoline-2-one moiety as in 1 was readily identified for 2 (Tables 1 and 2). However, the side chain slightly differed from that of 1, with the signals for the terminal double bond at C-23(25) missing in the NMR spectra of 2. Instead, signals for an oxygenated quaternary carbon at δC 70.1 (C-23) and for an additional methyl group at δH 1.06/δC 26.7 (CH3-25) as well as associated changes in chemical shifts of the nearby proton and carbon signals were observed in the NMR spectra of 2 (Table 1). The above observations suggested that the terminal double bond at C-23(25) in 1 was hydrated in 2. This deduction was further confirmed by the HMBC correlations from H3-24/H3-25 to C22 and C-23 (Figure 1). The relative configuration of 2 was determined as shown in Figure 2 based on NOE correlations from H-3 to the aromatic protons H-12/H-16 as well as from H3-26 to H3-25 and from H-22 to H-17 and H-18. The absolute configuration of the dihydroquinolone portion in 2 was also assigned as 3S, 4S by the ECD spectrum, which was almost identical to that of 1. However, the absolute configuration for the terpenoid unit remains unassigned. Aniduquinolone C (3) was shown to have the molecular formula C21H23NO4 as determined by HRESIMS. Analysis of the 1H and 13C NMR spectra of 3 (Table 2) indicated the presence of a 3-methoxy-4-phenyl-4,6-dihydroxy-3,4-dihydrodihydroquinolone unit in the molecule, the same as that of 1 and 2. In addition, signals for two methyls (δH 1.65/δC 17.5 and δH 1.68/δC 25.4, CH3-21 and CH3-22), one methylene (δH 3.19 and 3.09/δC 27.4, CH2-17), and two olefinic resonances including one methine (δH 5.24/δC 122.8, CH-18) and one quaternary carbon (δC 131.1, C-19) were also observed (Table 2). These signals implied the presence of a 3-methyl-2-butenyl group in 3, and this group was verified by the COSY correlations between H2-17 and H-18, as well as by the HMBC correlations from H2-17 to C-19 and from H3-21 to C18 and C-19 (Figure 1). The attachment of the 3-methyl-2butenyl group at the aromatic carbon C-7 was established by the HMBC cross-peak from H2-17 to C-7 (Figure 1). The NOE correlation from H-3 to the phenyl protons H-12/H-16 (Figure S2 in the Supporting Information) enabled the assignment of the relative configuration at C-3 and C-4 to be the same as that of compounds 1 and 2. The ECD spectrum of

Figure 2. Key NOESY correlations of compounds 1 and 2.

experiment, as no diagnostic NOE cross-peak could be detected between these two units. To unambiguously assign the structure and relative configuration, a single X-ray diffraction study was performed. However, compound 1 was initially isolated as an amorphous solid and it was difficult to get a suitable crystal for X-ray analysis, but after many attempts, X-ray quality crystals were obtained by slow evaporation of a solution of 1 in MeOH. The results from the X-ray diffraction crystallographic analysis confirmed the structure and established the relative configuration for 1 as shown (Figure 3). The absolute configuration of compound 1 was determined by analysis and comparison of the electronic circular dichroism (ECD) data with those of aflaquinolone derivatives.13 It was

Figure 3. X-ray crystallographic structure of compound 1, with one molecule containing a CH3OH solvent molecule in the unit cell. (Note: a different numbering system is used for the structure in the text.) C

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Table 2. NMR Spectroscopic Data for Compounds 3−6a 3 position 1-NH 2 3 4 5 6 7 8 9 10 11 12/16 13/15 14 17 18 19 20 21 3-OMe 3-OH 4-OH 6-OH 7-OH 14-OH a

δC

4 δH (J in Hz)

δC

10.16, br s 165.9, 84.3, 78.6, 110.8, 154.8, 122.8, 129.1, 106.2, 134.9, 140.0, 126.1, 128.4, 128.4, 27.4,

C CH C C C C CH CH C C CH CH CH CH2

122.8, 131.1, 17.5, 25.4, 58.2,

CH C CH3 CH3 CH3

3.58, d (1.1)

6.96, d (8.1) 6.38, d (8.1)

7.19, 7.33, 7.33, 3.19, 3.09, 5.24,

d (8.0) m m dd (15.3, 7.2) dd (15.3, 7.2) t (7.2)

1.65, s 1.68, s 3.43, s

5 δH (J in Hz)

δC

10.22, br s 168.1, 83.8, 76.6, 129.1, 122.0, 127.2, 127.3, 115.0, 136.7, 142.2, 126.6, 127.7, 128.5,

C CH C C CH CH CH CH C C CH CH CH

59.0, CH3

d (6.9) t (6.9) t (6.9) d (6.9)

7.34, m 7.34, m 7.28, m

3.32, s

169.0, 85.4, 78.1, 131.8, 115.8, 154.0, 116.5, 117.5, 130.1, 143.8, 128.2, 129.2, 128.6,

C CH C C CH C CH CH C C CH CH CH

60.5, CH3

5.81, br s

δC

δH (J in Hz)

9.94, br s

4.19, s

6.89, 6.89, 7.22, 6.89,

6 δH (J in Hz)

4.06, s

6.37, d (2.5) 6.61, dd (8.5, 2.5) 6.72, d (8.5)

7.32, m 7.32, m 7.28, m

10.21, br s 170.3, 74.4, 76.4, 129.6, 128.5, 115.1, 127.2, 121.9, 136.9, 132.5, 128.0, 114.4, 156.2,

C CH C C CH CH CH CH C C CH CH C

4.41, d (5.2)

6.80, 6.89, 7.20, 6.89,

d (7.6) d (7.6) t (7.6) d (7.6)

7.13, d (8.5) 6.71, d (8.5)

3.31, s 5.71, br s

5.15, d (5.2) 5.40, br s

9.59, br s 9.05, br s 9.32, br s

Measured in DMSO-d6 at 500 and 125 MHz for 1H and 13C, respectively.

and 2.5 Hz), and H-9 (d, J = 8.5 Hz) in the 1H NMR spectrum (Table 2) implied the presence of a 1,2,4-trisubstituted aromatic moiety in the molecule. The above data suggested that the 6-OH in aflaquinolone E was a 7-OH in 5. The COSY correlation from H-8 to H-9 and the HMBC correlation from H-9 to C-7 (Figure S1 in the Supporting Information) confirmed the presence of the OH group at C-7. The structure for compound 5 was thus determined, and it was named isoaflaquinolone E. Compound 6 was shown to have the molecular formula C15H13NO4 by means of negative HRESIMS. Comparing the NMR data (Table 2) with that of aflaquinolone F13 suggested the similarity of the structures for the two compounds. However, the phenyl group in aflaquinolone F was replaced by a p-hydroxyphenyl group in 6, as evidenced by the NMR spectra of 6 (Table 2). The COSY and HMBC correlations (Figure S1 in the Supporting Information) agreed with this deduction. The structure for compound 6 was thus determined, and it was named 14-hydroxyaflaquinolone F. Besides the six new compounds 1−6, aflaquinolone A (7)13 was also isolated from the culture extracts of A. nidulans MA143. The structure of 7 was elucidated by comparing its NMR data and specific rotation with those reported in the literature.13 Compounds 1−6 were examined for antibacterial activity against Escherichia coli and Staphyloccocus aureus, cytotoxicity against four tumor cell lines (BEL-7402, MDA-MB-231, HL-60, and K562), and brine shrimp lethality against Artemia salina. None of them showed potent antibacterial or cytoxic activities. However, compounds 2, 3, and 7 exhibited modest brine shrimp lethality, with LD50 values of 7.1, 4.5, and 5.5 μM, respectively.

compound 3 was very similar to that of 1 and 2 and thus allowed the assignment of the 3S, 4S-absolute configuration for 3. Different from that of aniduquinolones A−C (1−3), compounds 4−6 lacked the terpenoid side chain and only had the 4-phenyl-3,4-dihyroquinolone unit in their molecules, with various substitutions at C-3, C-7, and/or C-14. On the basis of the NOESY and the CD measurements, the relative and absolute configurations at C-3 and C-4 of compounds 4−6 were assigned to be same as that of aniduquinolones A−C (1− 3). In the CD spectra (Experimental Section) of 4−6, a slight shift of the wavelengths was observed, which might be caused by the absence of the side chain olefin in these molecules.13 Compound 4 was obtained as a pale yellow solid. Its molecular formula was established as C16H15NO3 by negative HRESIMS. Comprehensive analysis of the 1H and 13C NMR data (Table 2) suggested that 4 also possessed a similar 3methoxy-4-phenyl-4-hydroxy-3,4-dihydroquinolin-2-one unit to that in compounds 1−3 and aflaquinolone E,13 but it had no substitutions at C-6 and C-7. The 6-OH group in aflaquinolone E was not observed in the structure of 4, and the COSY and HMBC correlations (Figure S1 in the Supporting Information) verified the above deduction. These data along with the difference in the molecular formula suggested that compound 4 is the 6-deoxy derivative of aflaquinolone E. The structure for 3 was thus assigned, and it was named 6-deoxyaflaquinolone E. Compound 5, a pale yellow solid, had a molecular formula of C16H15NO4 based on positive HRESIMS. Detailed analysis of the NMR data revealed that compound 5 has the same skeleton as that of 4 as well as aflaquinolone E.13 The splitting pattern of the aromatic protons for H-6 (d, J = 2.5 Hz), H-8 (dd, J = 8.5 D

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MeOH (from 20:1 to 0:1)] to yield seven fractions (Fr.1−Fr.7). Fr.2 (11.0 g), eluted by petroleum ether−EtOAc, 1:1, was further fractionated by CC on silica gel eluting with a CHCl3−MeOH gradient to afford 10 subfractions (Fr.2.1−Fr.2.10). Fr.2.1 (7.0 g) was subjected to CC on Lobar LiChroprep C18 eluting with a MeOH− H2O gradient (from 1:9 to 1:0) and then further purified by semipreparative HPLC eluting with 85% aqueous MeOH to afford compounds 1 (45.7 mg, tR 10.5 min) and 3 (4.4 mg, tR 11.8 min). Fr.2.2 (3.0 g) was purified by CC on silica gel eluting with CHCl3− MeOH (from 50:1 to 5:1) and Sephadex LH-20 (petroleum ether− CHCl3−MeOH, 5:5:1) and then subjected to semipreparative HPLC eluting with 75% aqueous MeOH to yield compounds 2 (2.6 mg, tR 32.0 min), 4 (3.0 mg, tR 28.0 min), and 5 (3.0 mg, 23.7 min). Fr.3 (1.2 g) was purified by CC on Lobar LiChroprep C18 eluting with a MeOH−H2O gradient (from 2:8 to 1:0) and then further purified by semipreparative HPLC eluting with 55% MeOH to obtain compounds 6 (3.0 mg, tR 25.8 min) and 7 (50.0 mg, tR 9.4 min). Aniduquinolone A (1): colorless crystals; mp 125−127 °C; [α]20 D +50 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 200 (4.80), 228 (4.57), 275 (4.47), 318 (4.46) nm; CD (0.70 mM, MeOH) λmax (Δε) 221 (+18.45), 251 (−19.21), 280 (+7.57), 286 (+7.02), 322 (+5.04) nm; 1H and 13C NMR data, see Table 1; ESIMS m/z 434 [M − H]−; HRESIMS m/z 434.1980 (calcd for C26H28NO5, 434.1973). Aniduquinolone B (2): colorless solid; [α]20 D +31 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 200 (4.81), 202 (4.78), 229 (4.60), 273 (4.43), 316 (4.42) nm; CD (0.79 mM, MeOH) λmax (Δε) 221 (+20.81), 251 (−23.46), 280 (+7.28), 286 (+6.83), 320 (+4.83) nm; 1 H and 13C NMR data, see Table 1; ESIMS m/z 452 [M − H]−; HRESIMS m/z 452.2088 (calcd for C26H30NO6, 452.2079). Aniduquinolone C (3): pale yellow solid; [α]20 D +11 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 200 (4.33), 244 (3.77), 301 (3.77) nm; CD (0.59 mM, MeOH) λmax (Δε) 221 (+18.51), 246 (−16.76), 272 (+5.97), 284 (+2.50), 300 (+4.27) nm; 1H and 13C NMR data, see Table 2; ESIMS m/z 352 [M − H]−; HRESIMS m/z 352.1493 (calcd for C21H22NO4, 352.1554). 6-Deoxyaflaquinolone E (4): pale yellow solid; [α]20 D −80 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 201 (5.00), 247 (4.28), 272 (3.97) nm; CD (0.59 mM, MeOH) λmax (Δε) 222 (+12.56), 251 (−8.10), 270 (+5.14), 290 (−1.16) nm; 1H and 13C NMR data, see Table 2; ESIMS m/z 268 [M − H]−; HRESIMS m/z 268.0991 (calcd for C16H14NO3, 268.0979). Isoaflaquinolone E (5): pale yellow solid; [α]20 D −26 (c 0.35, MeOH); UV (MeOH) λmax (log ε) 202 (4.18), 234 (3.46), 265 (3.71), 285 (3.42), 301 (3.33) nm; CD (1.47 mM, MeOH) λmax (Δε) 201 (+0.44), 208 (−3.07), 221 (+26.92), 248 (−11.06), 271 (+6.68), 310 (−1.0) nm; 1H and 13C NMR data, see Table 2; ESIMS m/z 308 [M + Na]+; HRESIMS m/z 308.0886 (calcd for C16H15NO4Na, 308.0893). 14-Hydroxyaflaquinolone F (6): yellowish solid; [α]20 D −33 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 200 (5.07), 243 (4.14), 271 (3.86) nm; CD (1.11 mM, MeOH) λmax (Δε) 205 (−4.26), 238 (+1.63), 258 (−2.49), 274 (+1.87), 292 (−0.60), 300 (−0.41) nm; 1H and 13C NMR data, see Table 2; ESIMS m/z 270 [M − H]−; HRESIMS m/z 270.0798 (calcd for C15H12NO4, 270.0772). Aflaquinolone A (7): colorless crystals; mp 234−236 °C; [α]20 D +20 (c 0.5, MeOH) (literature value: +14 (c 0.19, MeOH)).13 X-ray Crystallographic Analysis of Compound 1 (ref 19). All crystallographic data were collected on a Bruker Smart-1000 CCD diffractometer equipped with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) at 298(2) K. The data were corrected for absorption by using the program SADABS.20 The structure was solved by direct methods with the SHELXTL software package.21 All nonhydrogen atoms were refined anisotropically. The H atoms were located by geometrical calculations, and their positions and thermal parameters were fixed during the structure refinement. The structure was refined by full-matrix least-squares techniques.22 Crystal data for compound 1: C26H29NO5·CH3OH, fw = 467.54, one molecule containing a CH3OH solvent molecule in the unit cell, monoclinic space group P2(1), unit cell dimensions a = 8.3638(8) Å, b = 13.8405(12) Å, c = 11.1316(11) Å, V = 1284.1(2) Å3, α = γ = 90°, β

Bioactivity examinations for quinolones showed these compounds have selective activities owing to different moieties in the molecules. Neff and co-workers reported that aflaquinolones lacked inhibitory activity against five tumor cell lines (K562, B16F10, HL-60, MDA-MB-231, and Hep3B),13 while Scherlach and Hertweck indicated that aspoquinolones A and B exhibited cytotoxicity against L-929 mouse fibroblast and K-562 human leukemia cell lines.15 Structurally, the aspoquinolones differ from the aflaquinolones as well as compounds 1 and 2 by the presence of a 14-OMe and a cyclopropane in the side chain, which might affect the cytotoxic activity of this class of compounds. Peniprequinolone and the penigequinolones, which also have a 14-OMe and with various terpenoid side chains, showed nematicidal activity toward Pratylenchus penetrans.17 On the basis of the reported data available, however, none of these activities are potent. Broadened screening in various models is thus necessary, and this might be helpful to find potent biological activity for the quinolone derivatives as well as for understanding structure− activity relationships.

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting point was determined with an SGW X-4 micro-melting-point apparatus. Optical rotations were measured on an Optical Activity A-55 polarimeter. UV data were obtained on a Lengguang Gold S54 spectrophotometer. ECD spectra were acquired on a Chirascan spectropolarimeter. The 1 H, 13C, and 2D NMR spectroscopic data were recorded on a Bruker Avance 500 spectrometer. Mass spectra were measured on a VG Autospec 3000 mass spectrometer. HPLC analysis was performed on a Dionex HPLC system equipped with a P680 pump, an ASI-100 automated sample injector, a TCC-100 column oven, a UV-DAD 340U detector, and a Dionex Acclaim ODS column (4.6 × 250 mm, 5 μm). Semipreparative HPLC was accomplished on a Dionex UltiMate U3000 system using an Agilent RP-18 column (21.2 × 250 mm, 10 μm), eluting with aqueous MeOH at a flow rate 16 mL/min. Column chromatography was performed with silica gel (200−300 mesh, Qingdao Haiyang Chemical Factory), Lobar LiChroprep RP-18 (40− 63 μm; Merck), and Sephadex LH-20 (18−110 μm, Merck). TLC analysis and preparative TLC were conducted on precoated silica gel plates (GF-254, Qingdao Haiyang Chemical Factory). Fungal Material. The fungus Aspergillus nidulans MA-143 was isolated from fresh leaves of the mangrove plant Rhizophora stylosa by using the procedures provided in an earlier report.18 The fungus grew slowly on potato dextrose agar plates and turned from white to green mycelia within five days. Fungal identification was performed using a molecular biological protocol by DNA amplification and sequencing of the ITS region as described previously.18 The resulting sequence data obtained from the fungal strain has been deposited at GenBank (with accession no. JQ839285). The result from the BLAST search indicated that the sequence was the same (100%) as that for the sequence of Aspergillus nidulans (compared to AY 452983). The strain is preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS). Fermentation, Extraction, and Isolation. The fermentation was statically carried out in liquid potato-dextrose broth medium (1000 mL naturally sourced and filtered seawater from the Huiquan gulf of the Yellow Sea near the campus of IOCAS, 20 g glucose, 5 g peptone, 3 g yeast extract, pH 6.5−7.0) in 1 L Erlenmeyer flasks (300 mL/flask) for 30 days at room temperature. The fermented whole broth (100 flasks) was filtered through cheesecloth to separate the culture broth and mycelia, which were extracted exhaustively with EtOAc and MeOH, respectively. As the chemical profiles of the two extracts were almost identical, they were combined and concentrated to obtain 31.0 g of an organic extract. The extract was subjected to silica gel vacuum liquid chromatography eluting with mixed solvents of increasing polarity [petroleum ether−EtOAc (from 5:1 to 1:1) and CHCl3− E

dx.doi.org/10.1021/np4004646 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

= 94.7550(10)°, Z = 2, dcalcd = 1.209 mg/m3, crystal dimensions 0.40 × 0.35 × 0.11 mm, μ = 0.085 mm−1, F(000) = 500. The 6507 measurements yielded 4305 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0496 and wR2 = 0.0992 [I > 2σ(I)]. Antibacterial Assays. Antibacterial assays against E. coli and S. aureus were carried out using the well diffusion method.23 Chloromycetin was used as a positive control. Cytotoxicity Assays. The cytotoxic activities against BEL-7402 (human hepatocellular carcinoma), MDA-MB-231(human breast carcinoma), HL-60 (human promyelocytic leukemia cells), and K562 (human acute myelocytic leukemia cell line) cell lines were determined according to previously reported methods.24 Brine Shrimp Assays. Evaluation of the extract and pure compounds for brine shrimp (A. salina) lethality was determined as described previously.25 Colchicine was used as a positive control.

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(15) Scherlach, K.; Hertweck, C. Org. Biomol. Chem. 2006, 4, 3517− 3520. (16) Kimura, Y.; Kusano, M.; Koshino, H.; Uzawa, J.; Fujioka, S.; Tani, K. Tetrahedron Lett. 1996, 37, 4961−4964. (17) Kusano, M.; Koshino, H.; Uzawa, J.; Fujioka, S.; Kawano, T.; Kimura, Y. Biosci. Biotechnol. Biochem. 2000, 64, 2559−2568. (18) Wang, S.; Li, X. M.; Teuscher, F.; Li, D. L.; Diesel, A.; Ebel, R.; Proksch, P.; Wang, B. G. J. Nat. Prod. 2006, 69, 1622−1625. (19) Crystallographic data of compound 1 have been deposited in the Cambridge Crystallographic Data Centre as CCDC 916585. The data can be obtained free of charge via http://www.ccdc.cam.ac.uk/ data_request/cif (or from the CCDC, 12 Union Road, Cambridge CB21EZ, U.K.; fax: +44-1223-336-033; e-mail: [email protected]. uk). (20) Sheldrick, G. M. SADABS, Software for Empirical Absorption Correction; University of Göttingen: Germany, 1996. (21) Sheldrick, G. M. SHELXTL, Structure Determination Software Programs; Bruker Analytical X-ray System Inc.: Madison, WI, 1997. (22) Sheldrick, G. M. SHELXL-97 and SHELXS-97, Program for X-ray Crystal Structure Solution and Refinement; University of Göttingen: Germany, 1997. (23) Al-Burtamani, S. K. S.; Fatope, M. O.; Marwah, R. G.; Onifade, A. K.; Al-Saidi, S. H. J. Ethnopharmacol. 2005, 96, 107−112. (24) Bergeron, R. J.; Cavanaugh, P. F., Jr.; Kline, S. J.; Hughes, R. G., Jr.; Elliott, G. T.; Porter, C. W. Biochem. Biophys. Res. Commun. 1984, 121, 848−854. (25) Meyer, B. N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.; McLaughlin, J. L. Planta Med. 1982, 45, 31−34.

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel and Fax: ++86-532-82898553. E-mail: [email protected]. ac.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Ministry of Science and Technology of China (2013AA092901 and 2010CB833802) and from the Natural Science Foundation of China (31270403 and 30910103914) is gratefully acknowledged.

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REFERENCES

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