Pestaloficiols Q–S from the plant endophytic fungus Pestalotiopsis fici

Pestaloficiols Q–S from the plant endophytic fungus Pestalotiopsis fici

Fitoterapia 85 (2013) 114–118 Contents lists available at SciVerse ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote Pesta...

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Fitoterapia 85 (2013) 114–118

Contents lists available at SciVerse ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Pestaloficiols Q–S from the plant endophytic fungus Pestalotiopsis fici Shuchun Liu a, Liangdong Guo a, Yongsheng Che a, b,⁎, Ling Liu a,⁎ a b

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, PR China Beijing Institute of Pharmacology & Toxicology, Beijing 100850, PR China

a r t i c l e

i n f o

Article history: Received 24 October 2012 Accepted in revised form 5 January 2013 Available online 23 January 2013 Keywords: Pestaloficiol Cytotoxic activity Endophytic fungus Structure elucidation

a b s t r a c t Two new isoprenylated chromone derivatives, pestaloficiols Q (1) and R (2), and one new benzofuran derivative, pestaloficiol S (3), along with three known metabolites, anofinic acid (4), siccayne (5), and pyrenophorol (6) were isolated from solid cultures of the plant endophytic fungus Pestalotiopsis fici. Their structures were elucidated primarily by NMR spectroscopy, and the absolute of the C-6 secondary alcohol in 1 was deduced on the basis of circular dichroism (CD) data. Compound 5 showed cytotoxic activity against the human cancer cell lines, HeLa and HT29, with IC50 values of 48.2 and 33.9 μM, respectively. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Many Pestalotiopsis spp. are either endophytic or pathogenic on living plant leaves and twigs [1]. Chemical studies of the fungal genus Pestalotiopsis have afforded a variety of bioactive natural products [2,3]. During an ongoing search for new bioactive natural products from this fungal genus, a subculture of Pestalotiopsis fici (AS 3.9138 = W106-1), isolated from the branches of the tea plant Camellia sinensis (Theaceae) in the suburb of Hangzhou, People's Republic of China, was grown in different solid-substrate fermentation cultures. Chemical studies of the resulting crude extracts had afforded structurally unique natural products showing inhibitory effects on replication of HIV-1 virus in C8166 cells including chloropupukeananin [4], the first chlorinated pupukeanane analogue with a highly functionalized tricyclo-[4.3.1.0 3,7]decane skeleton, and pestaloficiols A–E, new anti-HIV-1 cyclopropane derivatives isolated under different fermentation conditions [5]. Subsequent chemical investigations of the extract from a larger scale fermention of this fungus led to the isolation of other biologically active metabolites with interesting ⁎ Corresponding authors at: No. 8, Beiertiao, Zhongguancun, Haidian District, Beijing 100190, PR China. Tel.: +86 10 82618783; fax: +86 10 82618785. E-mail addresses: [email protected] (Y. Che), [email protected] (L. Liu). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.01.010

structural features [6–12]. In addition, two new isoprenylated chromone derivatives, and one new benzofuran derivative, which we named pestaloficiols Q–S (1–3), along with three known metabolites, anofinic acid (4) [13], siccayne (5) [14], and pyrenophorol (6) [15] were isolated from the crude extract. Details of the isolation, structure elucidation, and biological acitivities of these compounds are reported herein.

2. Experimental 2.1. General Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on Shimadzu Biospec1601 spectrometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. 1H and 13C NMR data were acquired with Varian Mercury-400 and 500 spectrometers using solvent signals (acetone-d6: δH 2.05/δC 29.8, 206.1) as references. The HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. ESIMS data and HRESIMS data were obtained using an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped with an electrospray ionization (ESI) source. The fragmentor and capillary voltages were kept

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at 125 and 3500 V, respectively. Nitrogen was supplied as the nebulizing and drying gas. The temperature of the drying gas was set at 300 °C. The flow rate of the drying gas and the pressure of the nebulizer were 10 L/min and 10 psi, respectively. All MS experiments were performed in positive ion mode. Full-scan spectra were acquired over a scan range of m/z 100–1000 at 1.03 spectra/s. 2.2. Fungal material The culture of P. fici was isolated from the branches of C. sinensis (Theaceae) in a suburb of Hangzhou, People's Republic of China, in April, 2005. The isolate was identified as P. fici by one of the authors (L.G.) based on sequence (GenBank accession number DQ812914) analysis of the ITS region of the ribosomal DNA and assigned the accession number AS 3.9138 (= W106-1) in the China General Microbial Culture Collection (CGMCC) at the Institute of Microbiology, Chinese Academy of Sciences, Beijing. The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 °C for 10 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm 3) under aseptic conditions and 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract); the final pH of the media was adjusted to 6.5 and sterilized by autoclave. Three flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 170 rpm for five days to prepare the seed culture. Spore inoculum was prepared by suspension in sterile, distilled H2O to give a final spore/cell suspension of 1 × 10 6/mL. Fermentation was carried out in 12 Fernbach flasks (500 mL), each containing 80 g of rice. Distilled H2O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 40 days.

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25 min), and pyrenophorol (6; 11.0 mg, tR 11.7 min; 30–70% MeOH in H2O for 25 min). Pestaloficiol Q (1): pale yellow oil; [α] 25D −189 (c 0.1, MeOH); UV (MeOH) λmax (logε) 302 (4.25) nm; CD (c 1.5× 10−4 M, MeOH) λmax (Δε) 298 (−0.39), 224 (+1.75) nm; IR (neat) νmax 3453 (br), 2977, 2928, 1765, 1641, 1582, 1370, 1154 cm−1; 1H, 13C NMR, and HMBC data see Table 1; HRESIMS m/z 343.1518 [M+ Na]+ (calcd for C18H24O5Na, 343.1516). Pestaloficiol R (2): pale yellow oil; UV (MeOH) λmax (logε) 217 (3.65) nm; IR (Neat) νmax 3339 (br), 2978, 1713, 1687, 1472, 1332, 1254, 1167 cm −1; 1H, 13C NMR, and HMBC data see Table 1; HRESIMS m/z 251.0918 [M + H] + (calcd for C13H15O5, 251.0914). Pestaloficiol S (3): pale yellow oil; UV (MeOH) λmax (logε) 217 (3.62), 260 (3.31) nm; IR (Neat) νmax 3388 (br), 2974, 2201, 1723, 1610, 1505, 1447, 1307, 1140 cm −1; 1H, and 13C NMR, and HMBC data see Table 1; HRESIMS m/z 229.1243 [M + H] + (calcd for C15H17O2, 229.1223). 2.4. MTT assay [9] The assay was run in triplicate. In 96-well plates, each well was plated with 10 4 cells. After cell attachment overnight, the medium was removed, and each well was treated with 50 μL of medium containing 0.2% DMSO, or appropriate concentrations of the test compounds (10 mg/mL as a stock solution of a compound in DMSO and serial dilutions). Cells were treated at 37 °C for 4 h in a humidified incubator at 5% CO2 first, and were allowed to grow for another 48 h after the medium was changed to fresh Dulbecco's Modified Eagle Medium (DMEM). MTT (Sigma) was dissolved in serum-free medium or PBS at 0.5 mg/mL and sonicated briefly. In the dark, 50 μL of MTT/ medium was added into each well after the medium was removed from wells, and incubated at 37 °C for 3 h. Upon removal of MTT/medium, 100 μL of DMSO was added to each well, and agitated at 60 rpm for 5 min to dissolve the precipitate. The assay plate was read at 540 nm using a microplate reader.

2.3. Extraction and isolation 3. Results and discussion The fermented material was extracted with EtOAc (4×1.0 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (10.0 g), which was fractionated by silica gel vacuum liquid chromatography (VLC) using petroleum ether/EtOAc gradient elution. The fraction (200 mg) eluted with 8% EtOAc was separated by Sephadex LH-20 column chromatography (CC) using 1:1 CH2Cl2–MeOH as eluents. The resulting subfractions were combined and further purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4× 250 mm; 40–80% MeOH in H2O for 35 min; 2 mL/min) to afford pestaloficiol S (3; 2.0 mg, tR 34.8 min) and anofinic acid (4; 2.5 mg, tR 29.5 min). The fraction eluted with 11% EtOAc (90 mg) was purified by HPLC (70–89% MeOH in H2O for 20 min; 2 mL/min) to afford pestaloficiol Q (1; 2.5 mg; tR 12.7 min). The fractions eluted with 20% (69 mg), 28% (300 mg), and 30% (160 mg) EtOAc were combined and fractionated again by Sephadex LH-20 CC eluting with 1:1 CH2Cl2–MeOH. Purification of these subfractions with different gradients afforded pestaloficiol R (2; 3.0 mg, tR 20.6 min; 20–80% MeOH in H2O for 25 min), siccayne (5; 1.2 mg, tR 18.5 min; 50–80% MeOH in H2O for

The molecular formula of pestaloficiol Q (1) was established as C18H24O5 (seven degrees of unsaturation) by its HRESIMS (m/z 343.1518 [M+ Na] +; Δ −0.2 mmu) and NMR data (Table 1). The 1H and 13C NMR spectra of 1 showed the signals for one exchangeable proton (δH 4.14), five methyl groups, three methylene units, one oxymethine, one oxygenated sp 3 quaternary carbon, three olefins, one carboxyl carbon (δC 168.7), and one α,β-unsaturated ketone carbon (δC 198.7). Interpretation of the 1H–1H COSY NMR data led to the identification of two isolated proton spin-systems, which were C-5–C-6 (including OH-6) and C-9–C-10. HMBC correlations from H2-5 to C-4a, C-6, C-7, and C-8a, from H-6 to C-5 and C-7, from OH-6 to C-5 and C-7, and from H2-9 to C-7, C-8, and C-8a completed an α,β-conjugated cyclohexenone ring with the isoprenyl group attached to C-8. Those from H2-5 to C-4, from H2-3 to C-4 and C-4a, and from H3-14 and H3-15 to C-2 and C-3 established the C-2–C-4a substructure, with C-14 and C-15 directly attached to C-2. Considering the 13C NMR chemical shifts of C-2 (δC 78.5) and C-8a (δC 161.6), as well as the unsaturation requirement for 1, these two oxygenated

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Table 1 NMR data for 1–3 in Acetone-d6. Pos

Pestaloficiol Q (1) a δH

2 3a 3b 4 4a 5a 5b 6 7 8 8a 9a 9b 10 11 12 13 14 15 16 17 6-OH 8-OH a b

Pestaloficiol R (2) b

δC , mult.

(J in Hz)

2.11, dddd (14, 12, 3.5, 2.5) 3.13, dd (14, 6.5) 3.95, dd (12, 6.5)

2.95, dd (14, 7.0) 3.03, dd (14, 7.0) 5.05, t (7.0) 1.70, 1.61, 1.36, 1.47,

HMBC

78.5, qC 39.0, CH2

2.49, d (17, 2.5) 2.74, d (17, 3.5)

2, 4, 4a, 14, 15 2, 4, 4a, 14, 15

148.2, qC 114.5, qC 29.6, CH2

4, 4a, 6, 7, 8a 4, 4a, 6, 7, 8a 5, 7

70.7, CH 198.7, qC 117.3, qC 161.6, qC 22.8, CH2

7, 8, 8a, 10, 11 7, 8, 8a, 10, 11 9, 12, 13

2.25, s 4.14, br s

Pestaloficiol S (3)

b

δH (J in Hz)

δC , mult.

2.68, s

79.8, qC 49.2, CH2

7.13, d (2.5)

192.4, qC 121.3, qC 109.6, CH

7.05, d (2.5)

123.0, CH 131.5, qC 17.9, CH3 25.8, CH3 26.0, CH3 28.4, CH3 168.7, qC 20.6, CH3

s s s s

a

3.58, s

151.3, qC 126.4, CH 124.9, qC 152.5, qC 35.4, CH2

HMBC 2, 4, 4a, 11, 12

δHa (J in Hz)

δCb, mult.

HMBC

3.05, s

87.8, qC 43.9, CH2

2, 4, 5, 9, 15, 16

131.7, qC 4, 6, 7, 8a

111.9, qC 6.75, d (8.0) 6.66, d (8.0)

5, 6, 8a, 9

124.8, CH 116.6, CH 143.4, qC

7, 8, 8a, 10

147.0, qC

172.1, qC

10, 11, 13 10, 11, 12 2, 3, 15 2, 3, 14

1.39, s 1.39, s

16 5, 7

8.10, s

26.5, CH3 26.5, CH3

4, 8, 10 5, 8, 9

5.26, 1.93, 1.45, 1.45,

2, 3, 12 2, 3, 11

88.0, qC 92.2, qC 128.2, qC 121.4, CH2 23.9, CH3 28.5, CH3 28.5, CH3

s s s s

8.18, s

11, 12, 14 11, 12, 13 2, 3, 16 2, 3, 15

7, 8, 9

Recorded at 500 MHz. Recorded at 125 MHz.

The absolute configuration of the C-6 in 1 was assigned using Snatzke's sector rule for the nonplanar enones (Fig. 2), which was established by the Cotton effect in the 320–350 nm (R band) region of the CD spectrum [16]. The negative Cotton effect observed in the R band region of the CD spectrum of 1 (Fig. 3) suggested C-6 has S configuration.

carbons were connected to the same oxygen to form a reduced dihydropyranone moiety. In addition, NMR resonances for an acetyl group (δH 2.25; δC 20.6 and 168.7) were observed in the spectra of 1, indicating that the C-4 (δC 148.2) oxygen was acylated. Collectively, these data permitted assignment of structure 1 to pestaloficiol Q (Fig. 1).

12

13 11

9

14

O 15

7

2

16

14 12

HOOC

O

4 17

13 10

5

10

O

15

OH

O

4

6

2

OH

16

O

9

8

O

O 1

OH 2

3

OH

9

O

O O

COOH 4

11

OH

O

O

OH 5

13

12

O

OH

OH O

6

Fig. 1. Structure for compounds 1–7.

7

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Fig. 2. Application of Snatzke's enone sector rule for prediction of the sign of the n→π* transition in CD spectra (320–350 nm) to 1.

The elemental composition of pestaloficiol R (2) was established as C13H14O5 (seven degrees of unsaturation) by its HRESIMS (m/z 251.0918 [M+ H]+; Δ −0.4 mmu) and NMR data (Table 1). Its 1H and 13C NMR spectra showed resonances for one exchangeable proton (δH = 8.10), two methyl groups, two methylene units, one oxygenated sp3 quaternary carbon, six aromatic carbons (two of which are protonated), one carboxylic carbon (δC = 172.1), and one α,β-unsaturated ketone carbon (δC = 192.4). These data, together with one unobserved exchangeable proton, accounted for all the 1H and 13 C NMR resonances for 2. The 1H and 13C NMR data of 2 closely resembled those of pestaloficiol J (7) which was isolated from the same fungus [7], except that the resonances for the double bond C-10/C-11 (δH = 5.26; δC = 123.0, 133.1) and two methyl groups Me-12/13 (δH =1.71; δC = 17.9/25.9) were replaced by the resonance for carboxylic carbon C-10 (δC = 172.1). Such observations were confirmed by HMBC correlations from H2-9 to C-7, C-8, C-8a, and C-10. Considering the chemical shift of C-10 (δC = 172.1), the remaining hydroxyl group should be attached to C-10 to form carboxylic acid unit. Therefore, the structure of 2 was determined as shown. Pestaloficiol S (3) gave a pseudomolecular ion (m/z 229.1243 [M + H] +; Δ − 2.0 mmu) by HRESIMS, consistent with a molecular formula of C15H16O2 (eight degrees of unsaturation). Analysis of its 1H and 13C NMR data (Table 1) revealed one exchangeable proton (δH = 8.18), three methyl groups, one methylene, one oxygenated sp 3 quaternary carbon, eight aromatic/olefinic carbons (three of which are protonated), and two sp quaternary carbons (δC = 88.0 and

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92.2) possibly due to the presence of an ethynyl unit [17]. These data accounted for all the resonances observed in the NMR spectra of 3. HMBC correlations from H2-13 to C-11, C-12, and C-14, and from H3-14 to C-11, C-12, and C-13 established the C-10–C-14 substructure. HMBC cross-peaks from H-6 to C-4, C-8, and C-10, from H-7 to C-5, C-8, and C-9, and from 8-OH to C-7, C-8, and C-9, together with those from H2-3 to C-4, C-5, and C-9, established a tetrasubstituted aryl ring with C-3, C-10, and a hydroxyl group directly attached to C-4, C-5, and C-8, respectively. The correlations from H2-3 to C-2, and C-15/C-16, and from H3-15/H3-16 to C-2, and C-3 indicated that C-3, C-15, and C-16 were directly attached to C-2. Considering the chemical shifts of C-2 (δC = 87.8) and C-9 (δC = 147.0) and the unsaturation requirement for 3, the two carbons should be attached to the remaining oxygen atom to form a reduced dihydrofurane moiety. On the basis of these data, the gross structure of 3 was established as shown. The known metabolites, anofinic acid (4), siccayne (5), and pyrenophorol (6) were readily identified by comparison of the NMR and MS data with those reported in the literature [13–15]. Compounds 1–6 were evaluated for cytotoxicity against the human cancer cell lines, HeLa and HT29. Compound 5 showed cytotoxic activity against the two cell lines, with IC50 values of 48.2 and 33.9 μM, respectively (5-fluorouracil as the positive control with IC50 values of 8.0 and 12.0 μM, respectively). Pestaloficiols Q (1) and R (2), two new isoprenylated chromone derivatives, are structurally related to pestaloficiol J (7) [7], but differs in having different substituents. Pestaloficiol S (3) is analogous of the known compound cycloarthropsone produced by Arthropsis truncata [18], but differs in having different substituents at C-2, C-3, and C-5. Biogenetically, these compounds could be derived from two units of prenoids and a polyketide. The discovery of these new secondary metabolites further expanded the structural diversity of the bioactive products produced by the plant endophytic fungus P. fici. Acknowledgments We gratefully acknowledge financial support from the Key Program of National Hi-Tech Research and Development (2012AA021703), the National Natural Science Foundation of China (21002120), the Chinese Academy of Sciences (KSCX2-EW-G-6), the Ministry of Science and Technology of China (2010ZX09401-403, 2012ZX09301-002-003), and the

2.5 2

CD [Mdeg]

1.5 1 0.5 0 200

250

300

-0.5 -1

Wavelength [nm] Fig. 3. Experimental CD spectrum of 1 in MeOH.

350

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