Griseofulvin Derivative and Indole Alkaloids from Penicillium

Jan 24, 2017 - A new griseofulvin derivative, 4′-demethoxy-4′-N-isopentylisogriseofulvin (1), three new indole alkaloids, 2-demethylcyclopiamide E...
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Griseofulvin Derivative and Indole Alkaloids from Penicillium griseof ulvum CPCC 400528 Dewu Zhang,† Lili Zhao,† Lining Wang,†,‡ Xiaomei Fang,† Jianyuan Zhao,† Xinwei Wang,† Li Li,§ Hongyu Liu,† Yuzhen Wei,† Xuefu You,† Shan Cen,† and Liyan Yu*,† †

Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Tian Tan Xi Li, Beijing 100050, People’s Republic of China ‡ College of Herbal Medicine, Tianjin University of Traditional Chinese Medicines, 88 Yuquan Road, Tianjin 300193, People’s Republic of China § Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: A new griseofulvin derivative, 4′-demethoxy-4′N-isopentylisogriseofulvin (1), three new indole alkaloids, 2demethylcyclopiamide E (2), 2-demethylsperadine F (3), and clopiamine C (4), and five known metabolites (5−9) were isolated from Penicillium griseof ulvum CPCC 400528. Compound 1 is the first reported griseofulvin analogue with an Nisopentane group and the first example of a naturally occurring N-containing griseofulvin analogue. Their structures and absolute configurations were elucidated through extensive spectroscopic analyses, calculated ECD, and single-crystal Xray diffraction (Cu Kα). The possible biogenetic pathway of 1− 3 was proposed. Compounds 1, 2, and 5 exhibited anti-HIV activities with IC50 values of 33.2, 20.5, and 12.6 μM, respectively.

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RESULTS AND DISCUSSION 4′-Demethoxy-4′-N-isopentylisogriseofulvin (1) was isolated as a pale yellow powder, and HRESIMS provided a molecular formula of C21H26ClNO5 with nine degrees of unsaturation. The IR spectrum indicated the presence of NH (3268 cm−1), carbonyl (1700 cm−1), and aromatic (1613, 1582 cm−1) groups. The 1H NMR data displayed the presence of one NH signal [δH 7.57 (1H, t, J = 6.0 Hz, NH)], two olefinic protons [δH 6.43 (1H, s, H-5) and 4.90 (1H, s, H-3′)], two methines [δH 2.65 (1H, m, H-6′) and 1.65 (1H, m, H-3″)], three methylenes [δH 3.05 (2H, dd, J = 14.4, 6.0 Hz, H-1″); δH 2.92 (1H, dd, J = 16.2, 12.0 Hz, Ha-5′), 2.42 (1H, dd, J = 16.2, 4.8 Hz, Hb-5′); and δH 1.44 (2H, dd, J = 14.4, 7.2 Hz, H-2″)], and five methyls [δH 4.02 (3H, s, H-9), 3.90 (3H, s, H-8), 0.91 (3H, d, J = 7.2 Hz, H-4″), 0.90 (3H, d, J = 7.2 Hz, H-5″), and 0.83 (3H, d, J = 6.6 Hz, H-7′)]. The 13C NMR and DEPT data (Table 1) exhibited 21 carbon resonances, including nine nonprotonated carbons (δC 193.0, 184.6, 169.6, 164.8, 164.2, 157.6, 105.4, 95.5, and 95.0), four methine carbons (δC 92.2, 91.0, 35.3, and 25.8), three methylene carbons (δC 41.1, 37.0, and 32.2), and five methyl carbons (δC 57.9, 56.8, 22.8, 22.8, and 15.0). Its 1H

ungi inhabiting special and competitive environments have been proven to be a rich source for the discovery of bioactive natural products with diverse structural features.1−5 Fungi produce a wide range of nitrogen-containing metabolites,6−8 and these display a variety of biological properties such as cytotoxic,9−11 antibacterial,12,13 and antidepressant14 activities. A number of bioactive N-containing natural products that showed impressive biological activities have been isolated from the genus Penicillium.15−21 As part of our ongoing search for structurally novel metabolites with interesting biological activities from fungi, bioassay-guided fractionation of the extract of the fungus Penicillium griseof ulvum CPCC 400528 led to the isolation of one new griseofulvin derivative, 4′demethoxy-4′-N-isopentylisogriseofulvin (1), with an N-isopentane unit, three indole alkaloids, 2-demethylcyclopiamide E (2), 2-demethylsperadine F (3), and clopiamine C (4), and five known metabolites, cyclopiamide (5),22 cyclopiamide E (6),23 griseofulvin (7),24 dechlorogriseofluvin (8),25 and 1,6-dihydroxy-3-methoxy-8-methylxanthone (9).26 Compound 1 is the first griseofulvin analogue bearing an N-isopentane moiety and the first example of a naturally occurring N-containing griseofulvin derivative. Herein, we report the isolation, structural elucidation, plausible biogenetic pathway, and biological activities of these metabolites. © 2017 American Chemical Society and American Society of Pharmacognosy

Received: September 13, 2016 Published: January 24, 2017 371

DOI: 10.1021/acs.jnatprod.6b00829 J. Nat. Prod. 2017, 80, 371−376

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Chart 1

The critical HMBC correlations from the proton at δH 7.57 (NH) to C-3′, C-5′, and C-1″ and from H-1″ to C-4′ suggested that the isopentane moiety was attached to C-4′ by an NH moiety. Furthermore, the HMBC correlations (Figure 1) of H3′/C-2, C-2′, and C-5′, H-5′/C-2, C-3′, C-4′, C-6′, and C-7′, and H-6′/C-2, C-3, C-5′, and C-7′, along with the 1H−1H COSY correlations (Figure 1) of H-5′/H-6′/H-7′, demonstrated the presence of a cyclohexenone ring. Accordingly, the planar structure of 1 was determined to be a new griseofulvin derivative bearing an N-isopentane group. Electronic circular dichroism (ECD) calculations were used to determine the absolute configuration of 1 by the timedependent density functional theory (TDDFT) method at the B3LYP/6-31G(d) level in methanol (see S4).27 The conformationally flexible side chain has no effect on the CD spectrum of 1, and a simplified structure of 1A (Figure S1), in which a methyl moiety replaced the isopentane unit in 1, was used for the ECD calculations. Four stereoisomers, (2S,6′R)-1Aa, (2R,6′S)-1Ab, (2S,6′S)-1Ac, and (2R,6′R)-1Ad, existed on the basis of the relative configuration (Figure S1). Comparison of the theoretically calculated and experimental ECD curves (Figure 2) showed that the calculated curves of 1Aa and 1Ac were both similar to the experimental data, indicating the absolute configuration of 1 as 2S. Thus, more evidence was necessary to determine the absolute configuration of C-6′. The coupling constant values of 3JH‑5′,H‑6′ (12.0 Hz, 4.8 Hz) were similar to reported coupling constant values of 3JH‑5′,H‑6′ of griseofulvin derivatives with 6′R configuration [such as griseofulvin (7)24 (13.2, 4.8 Hz) and (2S,6′R)-(7-chloro-4,6dimethoxybenzofuran-3-one)-2-spiro-1′-(3′-benzyl-6′-methylcyclohex-2′,4′-dione)28 (11.5, 5.2 Hz)] and different from those of the griseofulvin derivative with 6′S configuration [such as epidechlorogriseofulvin29 (8.3, 8.3 Hz)], suggesting the R configuration of C-6′. Therefore, the structure of 1 was determined to be (2S,6′R)-4′-demethoxy-4′-N-isopentylisogriseofulvin. 2-Demethylcyclopiamide E (2) was obtained as a yellow powder; its molecular formula of C19H15N3O2 was deduced by HRESIMS. The 1H and 13C NMR data (Table 2) of 2 were similar to those of cyclopiamide E (6).23 A proton at δH 10.93 (NH) was present for 2 rather than a methyl moiety (δH 3.49, δC 26.6) for 6. The location of the NH moiety was determined by the HMBC correlations (Figure 3) for H-2/C-1, C-3, C-8, and C-19. On the basis of the above data and extensive 1D and

Table 1. NMR Spectroscopic Data (600 MHz, DMSO-d6) of 1 position 2 3 3a 4 5 6 7 7a 8 9 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ NH

δC, type 95.0, 193.0, 105.4, 157.6, 91.0, 164.2, 95.5, 169.6, 56.8, 57.9, 184.6, 92.2, 164.8, 32.2,

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

35.3, 15.0, 41.1, 37.0, 25.8, 22.8, 22.8,

CH CH3 CH2 CH2 CH CH3 CH3

δH (J in Hz)

6.43, s

3.90, s 4.02, s 4.90, s 2.92, 2.42, 2.65, 0.83, 3.05, 1.44, 1.65, 0.91, 0.90, 7.57,

dd (16.2, dd (16.2, m d (6.6) dd (14.4, dd (14.4, m d (7.2) d (7.2) t (6.0)

12.0) 4.8)

6.0) 7.2)

and 13C NMR spectroscopic data were similar to those of griseofulvin (7), except for the absence of the methyl moiety (δH 3.62, δC 56.6) and the presence of an isopentane group (δH 3.05, δC 41.1; δH 1.44, δC 37.0; δH 1.65, δC 25.8; δH 0.91, δC 22.8; δH 0.90, δC 22.8), which was determined by the HMBC (Figure 1) cross-peaks for H-1″/C-2″ and C-3″ and H-3″/C1″, C-2″, C-4″, and C-5″, together with the spin system from H-1″ to H-5″ on the basis of the 1H−1H COSY correlations.

Figure 1. COSY (bold) and key HMBC (→) correlations of 1. 372

DOI: 10.1021/acs.jnatprod.6b00829 J. Nat. Prod. 2017, 80, 371−376

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Figure 2. Calculated ECD spectra of 1Aa−d and the experimental CD spectrum of 1.

Table 2. NMR Spectroscopic Data (600 MHz, DMSO-d6) for 2−4 2-demethylcyclopiamide E (2) position

δC, type

1 2 3 4 5 6 7 8 9

165.7, CH

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 14-OH 15-OH 16-OH

151.1, C 68.3, C

δH (J in Hz)

2-demethylsperadine F (3) δC, type

10.93, s 139.2, 106.0, 131.1. 119.3, 131.0, 126.8, 124.0,

C CH CH CH C C CH

160.4, C 112.0, CH 164.3, C 155.2, 127.2, 123.4, 24.2, 24.2, 23.4,

C C C CH3 CH3 CH3

δH (J in Hz)

179.9, C

6.97, d (6.6) 7.58, dd (8.4, 6.6) 7.62, d (8.4)

8.50, s

10.98, s 140.9, 108.3, 131.6, 121.0, 138.4, 122.9, 26.8,

C CH CH CH C C CH2

6.73, d (7.8) 7.25, dd (7.8, 7.8) 6.89, d (7.8)

2.61, dd (13.2, 4.8) 2.39, dd (13.2, 12.6) 2.32, m

54.3, CH 68.4, C

6.24, s

1.92, s 1.92, s 2.32, s

169.8, 87.8, 106.9, 102.4, 53.7, 83.0, 21.2, 30.0, 208.9, 26.9,

C C C C CH C CH3 CH3 C CH3

3.20, d (7.8) 1.62, s 1.43, s 2.21, s

clopiamine C (4) δC, type

δH (J in Hz)

60.2, C 180.2, C 56.0, 52.4, 189.4, 106.7, 158.4, 103.7,

C CH2 C C C CH

132.0, 118.0, 148.0, 48.8, 44.0, 26.8, 61.2, 30.9, 20.7, 52.9,

CH C C C CH CH2 CH CH2 CH2 CH2

64.0, 94.6, 41.3, 25.6, 23.2, 55.8, 22.8, 22.8,

CH2 C CH2 CH3 CH3 CH3 CH3 CH3

2.87, d (15.0); 2.42, d (15.0)

6.64, d (8.4) 7.56, d (8.4)

3.54, 1.85, 1.92, 1.90, 1.63, 2.87,

d (9.0) 1.71, m m 1.21, m m 1.97, m

3.64, d (12.6); 2.70, d (12.6) 2.68, 1.57, 1.26, 3.79, 0.97, 0.87,

d (15.6); 2.61, d (15.6) s s s s s

6.48, s 6.18, s 5.31, s

2-Demethylsperadine F (3) was isolated as a pale yellow powder. Its molecular formula was established as C20H20N2O7 by HRESIMS, requiring 12 degrees of unsaturation. The 1H and 13C NMR data (Table 2) of 3 were similar to those of speradine F30 except for the absence of a methyl (δH 3.17, δC 26.6) group and the presence of a proton (NH, δH 10.98). The HMBC correlations (Figure 4) from the proton at δH 10.98 (2NH) to C-1, C-2, C-8, and C-18 assigned the location of 2-NH. The NOESY correlations of H-17/H-10, H-20, 15-OH, and 16OH indicated that these protons were at the same side. Furthermore, the almost identical carbon and proton chemical shifts of 3 compared with those of speradine F30 in the same

Figure 3. Key HMBC (→) correlations of 2.

2D NMR experiments (1H NMR, 13C NMR, DEPT, HSQC, HMBC), the structure of 2 was established and named 2demethylcyclopiamide E. 373

DOI: 10.1021/acs.jnatprod.6b00829 J. Nat. Prod. 2017, 80, 371−376

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Figure 4. COSY (bold), key HMBC (→), and NOESY (↔) correlations of 3.

Figure 6. Single-crystal X-ray diffraction of 4.

solvent (Table S1) suggested that they shared the same relative configurations of chiral centers. The CD spectrum (Figure S36) and optical rotation of 3 were similar to those of speradine F,23,30 suggesting the same absolute configuration of these two compounds. Thus, the structure of 3 was elucidated as (10R,14S,15R,16S,17R,18R)-2-demethylsperadine F. Clopiamine C (4) was obtained as colorless bulk crystals (MeOH−H 2 O), and it gave an HRESIMS ion peak corresponding to a molecular formula of C26H33N3O5 with 12 degrees of unsaturation. The 13C NMR and DEPT data (Table 2) showed 26 carbon resonances, which consisted of 10 nonprotonated carbons (including two carbonyl, four olefinic), four methines (including two olefinic), seven methylenes, and five methyls. The HMBC correlations (Figure 5) for H-9/C-7,

To the best of our knowledge, 4′-demethoxy-4′-Nisopentylisogriseofulvin (1) represents the first example of griseofulvin derivatives with an N-isopentane group, which could plausibly be derived from griseofulvin.32 As shown in Scheme 1, griseofulvin could be transformed through doubleScheme 1. Proposed Biogenetic Pathway of 1

Figure 5. COSY (bold) and key HMBC (→) correlations of 4.

bond migration and demethylation to afford griseofulvic acid,28 which undergoes nucleophile substitution reaction with NH3 to produce N-substituted intermediate a. The subsequent prenylation and reduction lead to the generation of 1. The possible biogenetic pathway of 2 and 3 was also proposed as shown in Scheme S1. Intriguingly, the proposed biosynthetic pathway showed that 2-demethylcyclopiamide E (2), with a highly reduced pentacyclic ring system, shared the same precursor as 2-demethylsperadine F (3), featuring a highly oxygenated hexacyclic ring system. Compounds 1−9 were evaluated for anti-HIV and antibacterial activities. Compounds 1, 2, and 5 displayed moderate anti-HIV activities, as shown in Table 3. None of them showed antimicrobial activity against Staphylococcus aureus and Escherichia coli, with MIC values of >64 μg/mL.

C-8, and C-11; H-10/C-8 and C-12; H-5/C-4, C-6, C-7, C-24, and C-25; H-24/C-4, C-5, and C-25, and H-26/C-8 indicated the presence of a 2,2-dimethyl-2,3-dihydroquinolin-4(1H)-one group, which features a 1,2,3,4-tetrasubstituted benzene moiety. The HMBC cross-peaks of H-14/C-1, C-13, C-15, C-16, C-21, C-22, C-27, and C-28; H-19/C-16; H-21/C-14, C-16, C-22, and C-23; H-23/C-1, C-13, C-21, and C-22; and H-27/C-1, C13, C-14, and C-28, together with the spin system from H-14 to H-19 on the basis of the 1H−1H COSY correlations and the chemical shifts of C-16 (δC 61.2), C-19 (δC 52.9), and C-21 (δC 64.0), established the presence of an 8,8-dimethyldecahydro1H-cyclopenta[f ]indolizine unit. Furthermore, the critical HMBC correlations from H-10 to C-1 and from H-23 to C11 suggested a direct connection between C-1 and C-11, and the HMBC cross-peak from H-23 to C-2 and the chemical shifts of C-2 (δC 180.2), C-4 (δC 56.0), and C-12 (δC 148.0) demonstrated the presence of a five-membered lactam ring, which was composed by C-1, C-2, C-11, C-12, and N-3. There remained a NO2 fragment to be assigned. The NO2 moiety was attached to C-22, a tertiary carbon at δC 94.6. Thus, the planar structure of 4 was established and similar to known compounds, cyclopiamines A and B,31 indicating that these compounds were stereoisomers. The relative and absolute configurations were determined by single-crystal X-ray diffraction using the anomalous scattering of Cu Kα radiation with a Flack parameter of −0.02(19) (Figure 6). Therefore, its absolute configuration was unambiguously assigned as 1S,14R,16S,22R.

Table 3. Anti-HIV Activities of 1−9 and Efavirenz

374

compound

IC50 (μM)

1 2 3 4 5 6 7 8 9 efavirenz

33.2 20.5 70.1 57.7 12.6 77.9 87.1 89.7 79.4 1.4 × 10−3 DOI: 10.1021/acs.jnatprod.6b00829 J. Nat. Prod. 2017, 80, 371−376

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4′-Demethoxy-4′-N-isopentylisogriseofulvin (1): pale yellow powder; [α]25D +163.1 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 210 (0.47), 293 (0.73) nm; IR (νmax) 3268, 2957, 1700, 1613, 1582, 1468, 1351, 1220, 1001, and 824 cm−1; CD (MeOH) Δε (nm) −4.08 (212.5), +10.99 (233.5), −16.63 (290), +20.90 (321); ESIMS m/z 408.2 [M + H]+; HRESIMS m/z 408.1570 [M + H]+ (calcd for C21H27ClNO5, 408.1572); 1H and 13C NMR data, see Table 1. 2-Demethylcyclopiamide E (2): yellow powder; UV (MeOH) λmax (log ε) 203 (0.53), 222 (0.48), 275 (0.29) nm; IR (νmax) 3543, 3366, 1708, 1647, 1578, 1401, 1363, 1169, 923, 845, and 773 cm−1; ESIMS m/z 318.2 [M + H]+; HRESIMS m/z 318.1239 [M + H]+ (calcd for C19H16N3O2, 318.1237); 1H and 13C NMR data, see Table 2. 2-Demethylsperadine F (3): pale yellow powder; [α]25D −19.7 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 217 (0.37) nm; IR (νmax) 3258, 2917, 1702, 1624, 1232, 1065, 902, and 767 cm−1; CD (MeOH) Δε (nm) +24.41 (225.5), −2.53 (269.5), −2.96 (304); ESIMS m/z 401.1 [M + H]+; HRESIMS m/z 401.1342 [M + H]+ (calcd for C20H21N2O7, 401.1343); 1H and 13C NMR data, see Table 2. Clopiamine C (4): colorless bulk crystals (CH2Cl2−CH2OH); [α]25D +117.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 206 (0.82), 232 (0.37), 262 (0.23), 349 (0.12) nm; IR (νmax) 2973, 2943, 1711, 1687, 1611, 1490, 1367, 1246, and 818 cm−1; CD (MeOH) Δε (nm) +8.53 (219), −10.70 (243), +5.20 (268), −2.31 (355.5); ESIMS m/z 468.3 [M + H]+; HRESIMS m/z 468.2505 [M + H]+ (calcd for C26H34N3O5, 468.2493); 1H and 13C NMR data, see Table 2. X-ray Crystallographic Analyses of Clopiamine C (4). Colorless bulk crystals of 4 were obtained in CH2Cl2−CH2OH (1:2). Crystal data were collected on a Rigaku MicroMax 002+ diffractometer with Cu Kα radiation using the ω and κ scan technique to a maximum 2θ value of 144.36°. The crystal structures were solved by direct methods with SHELXS-97, and all non-hydrogen atoms were refined anisotropically using the least-squares method. All hydrogen atoms were positioned by geometric calculations and difference Fourier overlapping calculations. The absolute configuration was determined based on the Flack parameter, −0.02(19). Crystallographic data for the structure of 4 have been submitted to the Cambridge Crystallographic Data Centre as supplementary publication CCDC 1501806. Copies of these data can be obtained free of charge via www. ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44-(0)1223-336033 or e-mail: [email protected]). Crystallographic data for clopiamine C (4): C26H33N3O5, M = 467.57, orthorhombic system, space group P212121, crystal dimensions 0.31 × 0.52 × 0.56 mm, Cu Kα radiation, a = 10.831(2) Å, b = 14.3307(19) Å, c = 16.615(3) Å, V = 2579.0(7) Å3, Z = 4, Dcalcd = 1.287 g·cm−3. The total number of independent reflections measured was 4845, of which 4513 were observed (|F|2 ≥ 2σ|F|2). The final indices were R1 = 0.0411, wR2 = 0.1079 (w = 1/σ|F|2), S = 1.033. HIV-Inhibitory Bioassay.33 293T cells (2 × 105/mL) were cotransfected with 0.6 μg of pNL-Luc-E and 0.4 μg of pHIT/G. After 48 h, the VSV-G pseudotyped viral supernatant (HIV-1) was harvested by filtration through a 0.45 μm filter, and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux). SupT1 cells were exposed to VSV-G pseudotyped HIV-1 (MOI = 1) at 37 °C for 48 h in the absence or presence of test compounds (Efavirenz was used as positive control). The inhibition rate was determined by using a firefly Luciferase Assay System (Promega). Antibacterial Assays.34 The minimal inhibitory concentrations (MICs) of the isolated compounds were determined by the broth microdilution method in 96-well plates according to Clinical and Laboratory Standards Institute. All the test strains used in this study were standard strains obtained from American Type Culture Collection (ATCC), and levofloxacin was used as positive control. The final concentrations of compounds ranged from 0.125 to 256 μg/ mL. Culture plates were incubated at 37 °C for 18 h. The MICs were defined as the lowest concentration that prevented visible growth of the bacteria.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on an Autopol IV automatic polarimeter. The CD spectra were measured on a JASCO J-815 spectropolarimeter. UV spectra were obtained on an Agilent 8453 UV−vis spectrometer. IR spectra were acquired on a Nicolet 5700 FT-IR microscope spectrometer (FTIR Microscope Transmission). 1D and 2D NMR spectra were performed at 600 MHz for 1H NMR and 150 MHz for 13C NMR on a Bruker ARX-600 spectrometer. Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in hertz (Hz). ESIMS and HRESIMS data were measured using a Thermo LTQ Orbitrap XL mass spectrometer. Column chromatography (CC) was carried out with silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, China). Semipreparative HPLC was performed on a Shimadzu HPLC instrument equipped with a Shodex RI102 detector and a Grace Adsorbosphere C18 column (250 mm × 10 mm, i.d., 5 μm) by eluting with CH3OH−H2O. Analytical TLC was carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Industry, Qingdao, China), and spots were visualized under UV light or by spraying with 10% H2SO4 in 90% EtOH followed by heating at 120 °C. Fungal Material. The fungal strain CPCC 400528 was isolated from the soil, collected in Kanas Lake, Xinjiang Uygur Autonomous Region, China, in July 2009. The sequence data of this strain were deposited in GenBank with accession number KY419198. A BLAST search showed that the sequence was the same (100%) as the sequence of Penicillium griseof ulvum NRRL 3523 (accession no. DQ339549). The strain was deposited at the China Pharmaceutical Culture Collection (No. CPCC 400528), Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College. Fermentation, Extraction, and Isolation. The fungal strain was spread onto slants of PDA medium (potato extract 0.4%, dextrose 2.0%, agar 1.5%; autoclaved at 121 °C for 15 min) and incubated at 28 °C for 8−10 days. A loop of spores of the strain was inoculated into 500 mL Erlenmeyer flasks containing 100 mL of sterile fermentation medium (glucose 2.0%, malt extract 2.0%, yeast 0.5%, peptone 1.0%, corn steep liquor 1.0%, KH2PO4 0.05%, MgSO4 0.03%, vitamin B 0.5 × 10−5%, pH 6.0) at 28 °C on a rotary shaker (200 rpm) for 48 h to prepare the seed culture. Then 100 mL of seed culture was transferred into 5000 mL flasks containing 1000 mL of the same medium at 28 °C on a rotary shaker (200 rpm) for 4 days. The cultures (3 L) were filtered under reduced pressure to afford the filtrate and mycelia. The filtrate was extracted with EtOAc three times. The EtOAc extract was evaporated under reduced pressure to yield 5.6 g of residue, which was subjected to silica gel CC eluting with a petroleum ether−acetone gradient (100:0−0:100) to produce 10 fractions on the basis of TLC analysis. Fraction Fr.2 (205 mg) was isolated by reversed-phase semipreparative HPLC eluting with CH3CN−H2O (50:50) at 4 mL/min to give 9 (2.4 mg, tR = 16.2 min). Fr.4 (300 mg) was applied to Sephadex LH-20 CC eluting with CHCl3−CH3OH (50:50) to afford four fractions (Fr.4.1−Fr.4.4). Fr.4.2 (28 mg) was separated by reversed-phase semipreparative HPLC eluting with CH3OH−H2O (60:40) at 4 mL/min to give 6 (1.0 mg, tR = 15.1 min). Fr.4.3 (173 mg) was purified by reversed-phase semipreparative HPLC eluting with CH3OH−H2O (60:40) at 4 mL/ min to yield 7 (15.0 mg, tR = 10.9 min) and 8 (2.6 mg, tR = 7.0 min). Fr.4.4 (62 mg) was isolated by reversed-phase semipreparative HPLC eluting with CH3OH−H2O (65:35) at 4 mL/min to yield 3 (1.5 mg, tR = 6.7 min). Fr.5 (478 mg) was initially subjected to Sephadex LH-20 CC to give five fractions (Fr.5.1−Fr.5.5). Fr.5.2 (55 mg) was further separated via reversed-phase semipreparative HPLC eluting with CH3OH−H2O (65:35) at 4 mL/min to obtain 4 (18.0 mg, tR = 31.6 min). Fr.5.3 (44 mg) was purified by reversed-phase semipreparative HPLC eluting with CH3OH−H2O (65:35) at 4 mL/min to yield 1 (0.8 mg, tR = 11.3 min) and 5 (5.8 mg, tR = 16.7 min). Fr.5.4 (121 mg) was isolated by reversed-phase semipreparative HPLC eluting with CH3OH−H2O (65:35) at 4 mL/min to yield 2 (1.3 mg, tR = 11.1 min). 375

DOI: 10.1021/acs.jnatprod.6b00829 J. Nat. Prod. 2017, 80, 371−376

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00829. HRESIMS, IR, UV, 1D and 2D NMR spectra for 1−4, CD spectra of 1, 3, and 4, ECD calculations of 1, proposed biosynthetic pathway of 2 and 3 (PDF) X-ray crystallographic data for 4 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (L. Yu): +86-10-63187118. Fax: +86-10-63187118. Email: [email protected]. ORCID

Dewu Zhang: 0000-0001-6289-0617 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Infrastructure of Microbial Resources (No. NIMR-2016-3), the National Natural Science Foundation of China (No. 81402835), 973 Program (No. 2012CB911102), and Beijing Key Laboratory of Anti-infective Agents. L.Y. is supported by Xiehe Scholar.

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DOI: 10.1021/acs.jnatprod.6b00829 J. Nat. Prod. 2017, 80, 371−376