Meroterpenoid and Diphenyl Ether Derivatives from Penicillium sp. MA

Article pubs.acs.org/jnp

Meroterpenoid and Diphenyl Ether Derivatives from Penicillium sp. MA-37, a Fungus Isolated from Marine Mangrove Rhizospheric Soil Yi Zhang,†,‡ Xiao-Ming Li,† Zhuo Shang,† Chun-Shun Li,† Nai-Yun Ji,*,§ 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 ‡ Graduate University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, People’s Republic of China § Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Chunhui Road 17, Yantai 264003, People’s Republic of China S Supporting Information *

ABSTRACT: Penicillium sp. MA-37, which was obtained from the rhizospheric soil of the mangrove plant Bruguiera gymnorrhiza, exhibited different chemical profiles in static and shaken fermentation modes. Three new meroterpenoid derivatives, 4,25-dehydrominiolutelide B (1), 4,25-dehydro-22deoxyminiolutelide B (2), and isominiolutelide A (3), together with three known ones were characterized from its static fermentation, while three new diphenyl ether derivatives, namely, Δ1′,3′-1′-dehydroxypenicillide (4), 7-O-acetylsecopenicillide C (5), and hydroxytenellic acid B (6), along with five related metabolites were isolated from the shaken culture. The structures of these compounds were elucidated on the basis of spectroscopic analysis, and the structure of compound 2 was confirmed by X-ray crystallographic analysis. The absolute configurations of 1−3 and 6 were determined by ECD and modified Mosher’s method, respectively. All isolated compounds were evaluated for brine shrimp lethality and antibacterial activity.

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3-methylbutyl)benzoic acid (7) (CAS registry number 1174387-50-8), penicillide (8),10 secopenicillide C (9),11 dehydroisopenicillide (10), 12 and 3′-O-methyldehydroisopenicillide (11).12 It should be noted that although compound 7 is commercially available, there is no published NMR data for this compound. The fully assigned 1H and 13C NMR data were reported for the first time in the present report. This paper describes the isolation, structure elucidation, and biological activity of the isolated compounds.

arine microorganisms, especially marine fungi, are known to be a promising reservoir of biologically active natural products for drug discovery.1 There has been an increasing number of reports of bioactive metabolites from mangrove-derived endophytic fungi.2−4 Rhizospheres of mangrove plants, another unique habitat with a diversity of associated microorganisms, also showed potential in finding structurally interesting and biologically active secondary metabolites.5−7 During our ongoing search for bioactive fungal metabolites from marine-derived fungi,2−4 a fungal strain, Penicillium sp. MA-37, which was isolated from the rhizospheric soil samples of the mangrove plant Bruguiera gymnorrhiza, attracted our attention. Primary screening of optimized cultivation conditions for this strain indicated that the alteration of fermentation modes greatly affected its chemical profiles (see Scheme S1 in the Supporting Information). From its static culture, six meroterpenoid derivatives including three new compounds, 4,25-dehydrominiolutelide B (1), 4,25-dehydro22-deoxyminiolutelide B (2), and isominiolutelide A (3), and three known congeners, berkeleyacetal A,8 berkeleyacetal B,8 and 22-epoxyberkeleydione,9 were identified. In contrast, chemical investigation of the shaken culture led to the isolation

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RESULTS AND DISCUSSION The EtOAc extract of a static culture of Penicillium sp. MA-37 was fractionated and purified by a combination of column chromatography involving normal- and reversed-phase silica gel, Sephadex LH-20, and semipreparative reversed-phase HPLC, to yield six meroterpenoid derivatives, while a shaken culture, subjected to similar extraction and separation procedures, gave rise to eight diphenyl ether derivatives. Compound 1 was isolated as a colorless crystalline solid. HRESIMS established its molecular formula as C26H30O10 with 12 degrees of unsaturation. The 1H NMR spectrum (Table 1) along with HSQC experiment displayed resonances characteristic for five methyl singlets (H-17, H-18, H-19, H-24, and H-

of three new diphenyl derivatives, Δ1′,3′-1′-dehydroxypenicillide (4), 7-O-acetylsecopenicillide C (5), and hydroxytenellic acid B (6), along with five related derivatives, namely, 6-[2-hydroxy-6(hydroxymethyl)-4-methylphenoxy]-2-methoxy-3-(1-methoxy© XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 4, 2012

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dx.doi.org/10.1021/np300377b | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. 1H and 13C NMR Data for Compounds 1−3 1a no.

162.3, C 118.8, CH

3 4 5 6

155.4, 144.6, 43.5, 36.6,

C C CH CH2

7 8 9 10 10-OH 11 12 13 14 15 16 17 18 19 20 21 22 22-OH 23 24 25

50.5, 179.4, 76.8, 102.8,

C C CH C

65.0, 46.4, 79.7, 128.1, 133.0, 82.4, 26.7, 25.8, 16.0, 174.8, 13.3, 78.9,

C C CH CH C C CH3 CH3 CH3 C CH3 C

26 a

δC

1 2

2a δH (J in Hz)

5.96, br s

2.00, m β 2.20, t (13.3) α 2.00, m

3.90, q (6.4)

δC 162.3, C 118.7, CH 155.4, 145.1, 45.6, 32.6,

C C CH CH2

45.3, 180.1, 76.3, 102.2,

C C CH C

60.7, 46.0, 80.4, 127.5, 133.0, 82.3, 26.7, 25.7, 16.3, 174.3, 13.4, 43.2,

C C CH CH C C CH3 CH3 CH3 C CH3 CH

8.03, s

107.5, CH 19.5, CH3 118.6, CH2 51.8, CH3

4.66, d (1.9) 6.04, br s

1.42, s 1.57, s 0.75, s 1.29, d (6.4) 6.19, s 5.40, s 1.31, s a 5.43, s b 5.08, s 3.74, s

3b δH (J in Hz)

δC 167.8, C 34.4, CH2

5.98, br s

1.97, m β 1.97, m α 1.58, m

3.67, q (6.4)

127.6, 136.1, 46.0, 28.9,

C C CH CH2

48.5, 173.9, 74.6, 207.1,

C C CH C

49.4, 55.7, 74.9, 125.3, 146.9, 81.5, 28.1, 25.8, 11.5, 155.2, 16.1, 78.2,

CH C CH CH C C CH3 CH3 CH3 C CH3 C

3a δH (J in Hz)

δC 168.8, C 33.7, CH2

3.30, s

2.26, d (14.2) β 1.79, dd (14.2, 3.0) α 1.29, m

3.93, q (7.0)

125.6, 136.8, 45.6, 28.6,

C C CH CH2

48.6, 175.4, 73.9, 208.6,

C C CH C

49.2, 55.6, 75.1, 126.4, 144.5, 81.9, 27.9, 26.5, 11.0, 154.4, 15.9, 77.4,

CH C CH CH C C CH3 CH3 CH3 C CH3 C

δH (J in Hz) β 3.63, d (20.8) α 3.11, d (20.8)

2.03, dd (12.3, 3.9) 1.74, m

4.22, q (7.0)

6.55, s

98.6, CH 25.9, CH3 118.7, CH2 52.4, CH3

4.67, d (2.4) 6.04, br s

1.43, s 1.58, s 0.75, s 1.26, d (6.4) 3.30, d (7.5) 5.70, d (7.5) 1.46, s a 5.46, s b 5.10, s 3.75, s

3.06, s 5.68, d (6.8) 6.19, d (6.8)

1.04, s 1.07, s 1.33, s 1.29, d (7.0)

2.88, s 5.12, d (6.9) 6.55, d (6.9)

1.29, s 1.45, s 1.13, s 1.30, d (7.0)

101.3, CH 19.4, CH3 14.5, CH3

4.89, s 0.66, s 1.32, s

100.2, CH 20.2, CH3 14.8, CH3

5.96, s 1.21, s 1.74, s

54.0, CH3

3.17, s

54.4, CH3

3.59, s

Measured at DMSO-d6. bMeasured at C6D6.

the NMR data revealed that 1 belonged to the meroterpenoid class and possessed a carbon skeleton similar to miniolutelide B (12), a recently described polyketide-terpenoid hybrid meroterpenoid from the sea-mud-derived fungus Penicillum minioluteum 03HE3-1.9 The main differences in their NMR

26) and one methyl doublet (H-21), three oxymethine protons (H-9, H-13, and H-23), and four olefinic protons (H-2, H-14, and H2-25). The 13C and DEPT NMR spectra demonstrated the presence of six methyls, two methylenes, six methines, and 12 quaternary carbons (Table 1). Comprehensive analysis of B



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data were the presence of signals in the 13C NMR spectrum of 1 for the exocyclic double bond at δC 144.6 for the quaternary carbon C-4 and at δC 118.6 for the methylene C-25 (Table 1), which replaced the signals at δC 42.9 for the methine C-4 and at δC 18.6 for the methyl C-25, respectively, in that of 12.9 Accordingly, the corresponding signals for the methine (H-4, at δH 2.22) and methyl (H3-25, at δH 0.61) groups in the 1H NMR spectrum of 12 disapperared in that of 1. Instead, terminal olefinic proton signals (H2-25, at δH 5.43 and 5.08) were observed. The above evidence suggested that 1 was the 4,25-dehydro derivative of 12. This change was supported by the observed HMBC correlations from H2-25 to C-3 and C-5 (Figure 1). Analysis of other key NMR data as well as the observed key HMBC and 1H−1H COSY correlations resulted in the elucidation for the planar structure of 1.

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

The relative configuration of 1 was determined by a NOESY experiment. The observed NOE correlations from H-23 to H-9, OH-10, and OH-22, from H3-19 to OH-10 and H3-26, and from H3-26 to H3-24 indicated their cofacial orientation. Meanwhile, the correlations from H-13 to H-5 and H3-21 placed these groups on the opposite face (Figure 2). The structure of compound 1 was determined, and the trivial name 4,25-dehydrominiolutelide B was assigned. It is noteworthy that the cross-conjugated triene moiety (C-2/C-3, C-4/C-25, and C-14/C-15) in 1 is new to the family of meroterpenoids. To determine the absolute configuration of 1, the electronic circular dichroism (ECD) spectrum was experimentally recorded, and it showed a negative Cotton effect at 283 nm (Figure 3). The theoretical ECD was then calculated,13 and the calculated curve was in good agreement with that of the experimental one (Figure 3), which indicated that the absolute configuration of 1 was 5S, 7R, 9S, 10S, 11R, 12S, 13R, 22S, and 23R. Compound 2, named 4,25-dehydro-22-deoxyminiolutelide B, was isolated as colorless crystals. It was assigned the molecular formula C26H30O9 (12 unsaturations) by HRESIMS, with one less oxygen atom than 1. Detailed comparison of the NMR data of 2 with those of 1 (Table 1) indicated that the O-bearing quaternary carbon at δC 78.9 (C-22) in 1 was replaced by a nonoxygenated methine unit at δC 43.2 (C-22) and δH 3.30 (H22), suggesting that the OH substitution at C-22 in 1 was missing in 2. This deduction was supported by the observed COSY correlation from H-22 to H-23 and HMBC correlations from H-22 to C-6, C-8, C-10, C-12, C-20, and C-24 (Figure 1). The relative configuration of 2 was determined from the NOESY spectrum. The key NOE correlations from H-13 to H5 and H3-21 indicated the cis relationship of these protons. On the other face of the molecule, NOE correlations from OH-10 to H-9, H3-19, and H3-26, from H3-24 to H-22 and H3-26, and from H-9 to H-23 were observed (Figure 2). An X-ray crystallographic experiment confirmed the structure and the relative configuration of 2 (Figure 4). The above data indicated

Figure 2. Key NOESY correlations of compounds 1−3.

that 2 possessed the same relative configuration as that of 1. However, compound 2 showed a positive Cotton effect at 264 nm, which was different from that of 1, thus implying that 2 might possess a different absolute configuration compared to that of 1. Compound 2 was then submitted for theoretical ECD calculation,13 and the calculated curve displayed a positive Cotton effect and matched well with that of the experimental one, although a slight peak shift was observed (Figure 3). The absolute configuration of 2 was thus assigned as 5R, 7S, 9R, 10R, 11R, 12R, 13S, 22S, and 23S, which was different from that of 1 and therefore suggested that compounds 1 and 2 might be derived from different biosynthetic pathways, or the key step for the biosynthetic pathway of these two compounds might be controlled by different enzyme. The HRESIMS of compound 3 gave it a molecular formula of C26H32O10, with 11 degrees of unsaturation. Detailed comparison of the 1H and 13C NMR spectral data of 3 C



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(Table 1) with those of miniolutelide A (13)9 revealed that the structures of these two compounds were very similar, with the double bond at C-2/C-3 in 13 moved to C-3/C-4 in 3. This was supported by the fact that the olefinic methine C-2 at δC 116.7 and the saturated methine C-4 at δC 37.4 of 13 were replaced by a methylene carbon at δC 34.4 (C-2) and an olefinic quaternary carbon at δC 136.1 (C-4), respectively, in the 13C NMR spectrum of 3. The downfield chemical shift of the lactone carbonyl carbon C-1 (from δC 162.8 in 13 to δC 167.8 in 3) and the upfield chemical shift of the olefinic quaternary carbon C-3 (from δC 154.4 in 13 to δC 127.6 in 3) also supported this deduction. The observed HMBC correlations from H2-2 (δH 3.30) to C-4 and C-15 and from the singlet H325 (δH 1.32) to C-3 and C-5 (Figure 1) confirmed this. The relative configuration of all chiral centers of 3, except for C-22 due to the undetected OH proton signal, was established by a NOESY experiment. The observed NOE correlations from H-5 to H-11 and H3-21 and from H-13 to H-11 and H3-19 implied their cofacial character. Mutual correlations of H-23 and H-9 and of H-23 and H3-24 suggested their cis relationships (Figure 2). The relative configuration at C-11 and C-12 was substantiated by the significant differences in the 13C NMR spectra (δC 58.2 for C-11 and δC 43.0 for C-12 in 13 versus δC 49.4 for C-11 and δC 55.7 for C-12 in 3). The configuration at C-22 was assigned the α-orientation by comparasion of the 13C NMR shifts (δC 83.0 in 13 versus δC 78.4 in 3) and from

Figure 3. Experimental and calculated ECD spectra of 1 and 2 (in MeOH).

Figure 4. X-ray crystallographic structure and the asymmetric unit of compound 2. D



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Table 2. 1H and 13C NMR Data for Compounds 4−6 (DMSO-d6) 4 no.

δC

1 2 3 4 4-OMe 4a 5 7 7a 8 9 9-Me 10 11 11-OH 11a 12a 1′ 2′

118.2, 130.3, 128.4, 154.3, 62.2, 120.4, 166.7, 68.6, 127.0, 119.9, 134.0, 20.3, 118.3, 148.4,

CH CH C C CH3 C C CH2 C CH C CH3 CH C

141.5, 151.3, 121.1, 133.5,

C C CH CH

3′ 4′

141.6, C 118.7, CH2

5′ 1″ 2″

18.1, CH3

5 δH (J in Hz) 6.96, d (8.6) 7.88, d (8.6)

δC 106.6, 129.8, 122.3, 158.6,

CH CH C C

112.0, 171.7, 61.1, 129.1, 118.9, 134.4, 20.6, 117.7, 150.8,

C C CH2 C CH C CH3 CH C

141.5, 155.5, 27.5, 122.9,

C C CH2 CH

6 δH (J in Hz)

δC

6.08, d (8.3) 6.86, d (8.3)

3.83, s

5.12, s 6.44, d (1.5) 2.16, s 6.78, d (1.5)

5.07, s 6.61, d (1.5) 2.21, s 6.58,, d (1.5)

112.7, 124.5, 134.2, 152.4, 61.1, 127.8, 169.4, 58.4, 135.4, 117.4, 133.6, 20.8, 117.0, 150.6,

CH CH C C CH3 C C CH2 C CH C CH3 CH C

140.8, 153.3, 64.3, 48.3,

C C CH CH2

δH (J in Hz) 6.44, d (8.5) 7.09, d (8.5)

3.80, s

4.56, s 6.64, br s 2.18, s 6.39, br s

9.68, s

6.70, d (16.3) 7.03, d (16.3)

a 5.22, br s b 5.16, br s 1.94, s

3.13, d (7.3) 5.23, t (7.3)

130.9, C 17.5, CH3 25.4, CH3 170.1, C 20.6, CH3

1.64, s

24.4, CH 23.5, CH3

4.83, dd (9.0, 3.9) a 1.44, ddd (14.0, 9.0, 4.8) b 1.27, m 1.72, m 0.87, d (6.6)

1.65, s

21.9, CH3

0.91, d (6.6)

2.05, s

Figure 5. Key HMBC (arrows) and 1H−1H COSY (bold lines) correlations of compounds 4−6.

methylbuta-1,3-dienyl group and locating it at C-3 (Figure 5). The large coupling constant (J = 16.3 Hz) for H-1′ and H-2′ indicated the E-geometry for the double bond at C-1′/C-2′.

biogenetic considerations. The absolute configuration of 3 was determined by an ECD experiment.9 The torsion angle of the cis-homodiene moiety in ring B was estimated to be 335° (absolute value, see S12 in the Supporting Information) on the basis of its relative configuration and an MM2 calculation.14 The ECD spectrum exhibited a positive Cotton effect (Δε = +6.1) at 268 nm. The above evidence enabled the assignment of the absolute configuration of 3 as 5S, 7R, 9S, 11R, 12R, 13R, 22R, and 23R. The trivial name isominiolutelide A was assigned to 3. Compound 4 was isolated as a colorless, amorphous solid. HRESIMS established its molecular formula as C21H20O5 (12 degrees of unsaturation). Detailed analysis of its NMR spectroscopic data (Table 2) indicated that 4 was an analogue of penicillide (8).10 The main difference between them was ascribed to the C5 unit side chain. The observed COSY correlations from the olefinic H-1′ (δH 6.70) to the olefinic H2′ (δH 7.03) and HMBC correlations from H-1′ to C-2 (δC 130.3), C-4 (δC 154.3), and the olefinic quaternary C-3′ (δC 141.6), from H-2′ to C-3 (δC 128.4), C-4′ (δC 118.7), and C-5′ (δC 18.1), from the terminal olefinic H2-4′ (δH 5.22 and 5.16) to C-2′ (δC 133.5) and C-5′, and from the methyl H3-5′ (δH 1.94) to C-2′ and C-4′ allowed for the construction of a 3-

The trivial name Δ1′,3′-1′-dehydroxypenicillide (4) was proposed for compound 4. Compound 5 was assigned the molecular formula C22H24O7 (11 degrees of unsaturation) on the basis of HRESIMS. The 1H and 13C NMR data of 5 (Table 2) were very similar to those of secopenicillide C (9),11 except for the presence of signals corresponding to an acetyl group (δC 170.1 for C-1″, δH 2.05 for H-2″ and δC 20.6 for C-2″) in 5. The observed HMBC correlation from H2-7 (δH 5.07) to the carbonyl carbon C-1″, to the aromatic methine carbon C-8 (δC 118.9), and to the aromatic quaternary carbon C-11a (δC 141.5) indicated the acylation of the OH at C-7 (Figure 5). Compound 5 was named 7-O-acetylsecopenicillide C. The molecular formula of 6 was determined to be C21H26O7 (9 unsaturations) on the basis of HRESIMS. Detailed analysis of the 1H and 13C NMR data of 6 (Table 2) revealed that its chemical structure was very similar to tenellic acid B (14), a diphenyl ether isolated from the aquatic fungus Dendrospora tenella.15 The only difference was the replacement of the aldehyde carbon signal at δC 189.9 (C-7) of 14 with an E



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oxygenated methylene carbon signal at δC 58.4 (C-7) in the 13C NMR spectrum of 6. This deduction was strongly supported by the observation of the methylene proton signal at δH 4.56 (H27) as well as its HMBC correlations to C-8 and C-11a (Figure 5). The absolute configuration of 6 was determined by the modified Mosher’s method.16 Treatment of 6 with (R)-(−)and (S)-(+)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) gave the (S)- and (R)-MTPA esters 6s and 6r, respectively. The 1H NMR signals of the two MTPA esters were assigned, and the ΔδH(S−R) values were then calculated (Figure 6). The results indicated that the absolute configuration of C-1′ was S. The trivial name hydroxytenellic acid B was assigned to compound 6.

commercial silica gel (200−300 mesh, Qingdao Haiyang Chemical Co.), 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 Co.). Fungal Material. The fungus Penicillium sp. MA-37 was isolated from the rhizospheric soil of Bruguiera gymnorrhiza collected from Hainan Island, P. R. China, in July 2010. The fungus was identified by sequence (GenBank accession number JQ693497) analysis of the ITS region of its rDNA as described previously.17 The strain is preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences. Fermentation, Extraction, and Isolation of Secondary Metabolites in Static Cultivation. For static cultivation, the fungus was cultured in 30 × 1 L Erlenmeyer flasks, each containing 300 mL of liquid medium (2% mannitol, 2% maltose, 1% glucose, 1% monosodium glutamate, 0.3% yeast extract, 0.1% corn steep liquor, 0.05% KH2PO4, 0.03% MgSO4·7H2O, seawater, pH 6.5−7.0). After incubation for 30 days at 28 °C, the whole fermented culture was filtered to separate the broth from the mycelia. The broth was extracted three times with EtOAc, while the mycelia were extracted three times with a mixture of 80% acetone in H2O. The acetone extract was then evaporated under reduced pressure to yield an aqueous solution, which was then extracted three times with EtOAc. Since the chemical profiles of the two EtOAc extracts were almost identical, they were combined and concentrated to afford the crude extract (16.0 g) for further separation. The crude extract was subjected to silica gel vacuum liquid chromatography (VLC) eluting with mixed solvents of increasing polarity [petroleum ether−EtOAc (from 100:1 to 1:1) and CHCl3− MeOH (from 20:1 to 0:1)] to yield 12 fractions (Fr.1 to Fr.12). Fr.7 (2.2 g) was further fractionated by column chromatography (CC) on silica gel eluting with a CHCl3−MeOH gradient to afford five subfractions (Fr.7-1 to Fr.7-5). Fr.7-3 (750.0 mg) was purified by CC on Lobar LiChroprep C18 eluting with a H2O−MeOH gradient (from 1:1 to 0:1) and then by semipreparative HPLC (Elite ODS-BP column, 10 μm; 300 × 10.0 mm; 45% MeCN−H2O, 3.0 mL/min, UV detection at 215 nm) to yield berkeleyacetal A (4.3 mg, tR 23.3 min), berkeleyacetal B (3.8 mg, tR 19.0 min), and 22-epoxyberkeleydione (5.0 mg, tR 17.3 min). Fr.7-4 (650.0 mg) was purified by CC on Lobar LiChroprep C18 eluting with a H2O−MeOH gradient (from 1:1 to 0:1) and on silica gel eluting with a CHCl3−MeOH gradient (from 100:1 to 20:1) to yield compounds 1 (45.5 mg), 2 (34.1 mg), and 3 (6.2 mg). 4,25-Dehydrominiolutelide B (1): colorless crystalline solid; mp 217−219 °C; [α]25D −106.9 (c 0.80, MeOH); UV (MeOH) λmax (log ε) 200 (3.49), 269 (3.32) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 525.1733 [M + Na]+ (calcd for C26H30O10Na, 525.1737). 4,25-Dehydro-22-deoxyminiolutelide B (2): colorless crystal; mp 203−205 °C; [α]25D −36.9 (c 0.33, MeOH); UV (MeOH) λmax (log ε) 201 (3.80), 274 (3.68) nm; 1H and 13C NMR data, see Table 1; HRESIMS m/z 509.1782 [M + Na]+ (calcd for C26H30O9Na, 509.1787). Isominiolutelide A (3): colorless crystalline solid; mp 218−220 °C; [α]25D −44.4 (c 0.36, MeOH); UV (MeOH) λmax (log ε) 201 (4.07), 257 (3.53) nm; CD (c 0.42, MeOH) λmax (Δε) 209 (−27.0), 268 (+6.1), 304 (−6.2) nm; 1H and 13C NMR data, measured in C6D6 and DMSO-d6, see Table 1; HRESIMS m/z 527.1896 [M + Na]+ (calcd for C26H32O10Na, 527.1893). Fermentation, Extraction, and Isolation of Secondary Metabolites in Shaking Cultivation. The shaken fermentation growth of the fungus was accomplished on a rotary shaker at 120 rpm at 28 °C for 7 days in 120 × 500 mL Erlenmeyer flasks containing the same liquid medium as that for static fermentation (200 mL/flask). The fermented cultures were exhaustively extracted with EtOAc by ultrasonic processor (three times). The EtOAc extract was filtered and concentrated to give the crude extract (8.0 g). The extract was fractionated by VLC on silica gel to give eight fractions (Fr.A to Fr.H). Fr.C (2.7 g) was further separated by CC on

Figure 6. Values of ΔδH(S−R) (measured in DMSO-d6) of the MTPA esters of 6.

The molecular formula of 7 was determined to be C22H28O7 by HRESIMS, having one more CH2 unit than that of 6. The presence of the singlet resonance at δH 3.16 corresponding to the methoxyl group at C-1′ and its HMBC correlation to C-1′ suggested that the OH group at C-1′ in 6 was replaced by a methoxyl group in 7. The structure of 7 was thus assigned as 6[2-hydroxy-6-(hydroxymethyl)-4-methylphenoxy]-2-methoxy3-(1-methoxy-3-methylbutyl)benzoic acid. This compound is listed in the CAS registry file (registry number 1174387-50-8), and its NMR data are reported for the first time (Experimental Section). All isolated compounds were evaluated in the brine shrimp lethality assay and for activity against Micrococcus luteus and Escherichia coli. In the brine shrimp lethality assay, 3′-Omethyldehydroisopenicillide (11) was found to be active, with an LD50 of 72.6 μM, comparable to the positive control, colchicine (LD50 71.1 μM). Δ1′,3′-1′-Dehydroxypenicillide (4), penicillide (8), and berkeleyacetal B were also active, with LD50 values of 135.9, 158.5, and 160.0 μM, respectively. In the broth microdilution test, compound 5 was active against bacteria M. luteus and E. coli with MICs of 64 and 16 μg/mL, respectively, while compound 7 showed MICs of 256 and 32 μg/mL, respectively. The positive control chloromycetin showed an MIC value of 4 μg/mL to both bacteria.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined with an SGW X-4 micro-melting-point apparatus. Optical rotations were measured on an Optical Activity AA-55 polarimeter. UV spectra were recorded on a Gold Spectrumlab 54 UV−vis spectrophotometer. ECD spectra were acquired on a Chirascan spectropolarimeter. NMR spectra were recorded on a Bruker Avance 500 MHz spectrometer. Mass spectra were determined on a Q-Tof Ultima Global GAA076-LC mass spectrometer. Semipreparative HPLC was performed using a Dionex HPLC system equipped with a P680 pump, an ASI-100 automated sample injector, and a UVD340U multiple wavelength detector controlled with Chromeleon software, version 6.80. Column chromatography was performed with F



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by direct methods with the SHELXTL software package.20 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.21 Crystal data for compound 2: C53H64O19, fw = 1005.04, two molecules containing a solvent molecule (MeOH) in the unit, monoclinic space group P2(1), unit cell dimensions a = 12.06(1) Å, b = 11.48(1) Å, c = 18.64(2) Å, V = 2570.3(5) Å3, α = γ = 90°, β = 95.5010(1)°, Z = 2, dcalcd = 1.229 mg/m3, crystal dimensions 0.46 × 0.43 × 0.40 mm, μ = 0.099 mm−1, F(000) = 1068. The 12 938 measurements yielded 4771 independent reflections after equivalent data were averaged, and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0414 and wR2 = 0.1023[I > 2σ(I)]. Computational Details. The energy-minimized conformers of 1 and 2 were generated via the Dreiding force field in MarvinSketch regardless of rotations of methyl and hydroxyl groups,22 and the geometries were further optimized at the B3LYP/6-31G(d) level in methanol with the integral equation formalism variant polarizable continuum model (IEF-PCM) without vibrational imaginary frequencies. The predominant conformers (Figure 7) were subjected to the

Lobar LiChroprep C18 eluting with a H2O−MeOH gradient (from 2:1 to 0:1) to afford five subfractions (Fr.C-1 to Fr.C-5). Fr.C-2 (600.0 mg) was subjected to CC on Sephadex LH-20 (MeOH) and then preparative TLC with CHCl3−MeOH eluant to yield 6 [7.3 mg, CHCl3−MeOH (5:1, 1.0% HAc), Rf = 0.6] and secopenicillide C (9) [20.3 mg, CHCl3−MeOH (20:1, 1.0% HAc), Rf = 0.4]. Fr.C-3 (98.0 mg) was purified by CC on silica gel eluting with CHCl3−MeOH (30:1, 1.0% HAc) to yield 5 (3.7 mg) and 7 (8.8 mg). Fr.C-4 (480.0 mg) was subjected to CC on silica gel eluting with a CHCl3−MeOH gradient (from 50:1 to 2:1, 1.0% HAc) and on Sephadex LH-20 (MeOH) to yield penicillide (8) (59.0 mg) and dehydroisopenicillide (10) (18.7 mg). Preparative TLC with CHCl3−MeOH eluant was employed for purification to Fr.C-5 (210.0 mg), yielding 4 [5.3 mg, CHCl3−MeOH (100:1), Rf = 0.6] and 3′-O-methyl dehydroisopenicillide (11) [7.5 mg, CHCl3−MeOH (20:1), Rf = 0.7]. Δ1′,3′-1′-Dehydroxypenicillide (4): colorless solid; UV (MeOH) λmax (log ε) 202 (4.53), 280 (3.98) nm; 1H and 13C NMR data, see Table 2; HRESIMS m/z 375.1203 [M + Na]+ (calcd for C21H20O5Na, 375.1208). 7-O-Acetylsecopenicillide C (5): colorless solid; UV (MeOH) λmax (log ε) 203 (4.92), 302 (3.71) nm; 1H and 13C NMR data, see Table 2; HRESIMS m/z 399.1443 [M − H]− (calcd for C22H23O7, 399.1444). Hydroxytenellic acid B (6): colorless solid; [α]25D +5.0 (c 0.53, MeOH); UV (MeOH) λmax (log ε) 204 (4.56), 280 (3.23) nm; 1H and 13C NMR data in DMSO-d6, see Table 2; 1H NMR data (in CD3OD) δH (at 500 MHz) 6.29 (d, J = 8.6 Hz, H-1), 7.15 (d, J = 8.6 Hz, H-2), 3.95 (s, 4-OMe), 4.52 (s, H2-7), 6.79 (d, J = 1.6 Hz, H-8), 2.29 (s, 9-Me), 6.66 (d, J = 1.6 Hz, H-10), 5.03 (dd, J = 8.6, 4.9 Hz, H1′), 1.60 (ddd, J = 14.0, 8.6, 5.6 Hz, H-2′a), 1.45 (ddd, J = 14.0, 8.2, 4.9, H-2′b), 1.73 (m, H-3′), 0.93 (d, J = 6.6 Hz, H3-4′), 0.94 (d, J = 6.6 Hz, H3-5′); 13C NMR data (in CD3OD at 125 MHz) δC 110.6 (C-1, CH), 126.7 (C-2, CH), 133.6 (C-3, C), 154.9 (C-4, C), 62.3 (4-OMe, CH3), 126.2 (C-4a, C), 167.4 (C-5, C), 60.2 (C-7, CH2), 136.3 (C-7a, C), 120.7 (C-8, CH), 136.9 (C-9, C), 21.2 (9-Me, CH3), 118.2 (C-10, CH), 150.9 (C-11, C), 139.2 (C-11a, C), 154.9 (C-12a, C), 66.9 (C1′, CH), 48.9 (C-2′, CH2), 26.0 (C-3′, CH), 23.8 (C-4′, CH3), 22.5 (C-5′, CH3); HRESIMS m/z 389.1605 [M − H]− (calcd for C21H25O7, 389.1600). Compound 7: colorless solid; [α]25D +3.1 (c 0.64, MeOH); UV (MeOH) λmax (log ε) 204 (4.68), 280 (3.33) nm; 1H NMR data (in CD3OD at 500 MHz) δH 6.31 (d, J = 8.6 Hz, H-1), 7.05 (d, J = 8.6 Hz, H-2), 3.95 (s, 4-OMe), 4.52 (s, H2-7), 6.80 (d, J = 1.6 Hz, H-8), 2.29 (s, 9-Me), 6.67 (d, J = 1.6 Hz, H-10), 4.61 (dd, J = 8.8, 4.5 Hz, H-1′), 3.16 (s, 1′-OMe), 1.62 (ddd, J = 14.0, 8.8, 5.5 Hz, H-2′a), 1.36 (ddd, J = 14.0, 8.2, 4.5 Hz, H-2′b), 1.74 (m, H-3′), 0.92 (d, J = 6.5 Hz, H3-4′), 0.94 (d, J = 6.5 Hz, H3-5′); 13C NMR data (in CD3OD at 125 MHz) δC 110.8 (C-1, CH), 126.8 (C-2, CH), 130.4 (C-3, C), 156.0 (C-4, C), 62.2 (4-OMe, CH3), 126.2 (C-4a, C), 165.9 (C-5, C), 60.2 (C-7, CH2), 136.3 (C-7a, C), 120.8 (C-8, CH), 137.0 (C-9, C), 21.2 (9-Me, CH3), 118.2 (C-10, CH), 150.9 (C-11, C), 139.1 (C-11a, C), 155.2 (C-12a, C), 77.1 (C-1′, CH), 56.8 (1′-OMe, CH3), 48.1 (C-2′, CH2), 26.1 (C-3′, CH), 23.7 (C-4′, CH3), 22.5 (C-5′, CH3); HRESIMS m/z 403.1759 [M − H]− (calcd for C22H27O7, 403.1757). Preparation of the (R)- and (S)-MTPA Ester Derivatives of 6 (ref 16). To a stirred solution of 6 (1.5 mg) in pyridine (400 μL) was added 4-(dimethylamino)pyridine (2 mg) and (S)-(+)-α-methoxy-α(trifluoromethyl)phenylacetyl chloride (MTPA-Cl, 10 μL). The mixture was stirred at room temperature for 10 h, and the reaction stopped by adding 0.2 mL of H2O. The reaction mixture was then subjected to preparative TLC with PE−EtOAc (5:1) eluant to give the respective (R)-Mosher ester 6r (1.3 mg). Treatment of 6 (1.7 mg) with (R)-MTPA-Cl (10 μL) with the same procedure yielded the corresponding (S)-Mosher ester 6s (1.4 mg). X-ray Crystallographic Analysis of 2 (ref 18). All crystallographic data were collected on a Bruker Smart-1000 CCD diffractometer equipped with graphite-monochromatic Mo Kα radiation (λ = 0.71073 Å) at 298(2) K. The data were corrected for absorption by using the program SADABS.19 The structure was solved

Figure 7. Energy-minimized conformers of 1 and 2 (in MeOH).

theoretical calculations of ECD spectra (Figure 3) at the B3LYP/631G(d) level using the time-dependent density functional theory (TDDFT) method, which were drawn via SpecDic software with sigma = 0.25 and UV shift = −15 nm (magnified by 0.3 times for 2).23 Brine Shrimp Lethality Assay. The assay was performed by a procedure reported in the literature.24 DMSO and colchicine were used as negative and positive controls, respectively. Antibacterial Assays. The experiments were performed using the disk diffusion method25 and broth microdilution method.26 Chloromycetin was used as positive control.

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ASSOCIATED CONTENT

S Supporting Information *

HPLC profiles of crude extracts from different cultivation, Xray crystallographic file of compound 2 (in CIF format), MM2 calculation of compound 3, and selected 1D and 2D NMR spectra for compounds 1−7. 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] (N.Y.J.); [email protected] (B.-G.W.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Natural Science Foundation of China (30910103914 and 30970293) and from the Ministry of Science and Technology of China (2010CB833800) is gratefully acknowledged. G



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REFERENCES

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Meroterpenoid and Diphenyl Ether Derivatives from Penicillium sp. MA

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