Cytotoxic Isoprenylated Epoxycyclohexanediols from the Plant Endophyte Pestalotiopsis fici

Cytotoxic Isoprenylated Epoxycyclohexanediols from the Plant Endophyte Pestalotiopsis fici

Chinese Journal of Natural Medicines 2011, 9(5): 0374−0379 Chinese Journal of Natural Medicines doi: 10.3724/SP.J.1009.2011.00374 Cytotoxic Isopren...

390KB Sizes 0 Downloads 96 Views

Chinese Journal of Natural Medicines 2011, 9(5): 0374−0379

Chinese Journal of Natural Medicines

doi: 10.3724/SP.J.1009.2011.00374

Cytotoxic Isoprenylated Epoxycyclohexanediols from the Plant Endophyte Pestalotiopsis fici LIU Shu-Chun1, YE Xin2, GUO Liang-Dong1, LIU Ling1* 1

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, China; Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, China 2

Available online 20 Sep. 2011

[ABSTRACT] AIM: To search for new cytotoxic secondary metabolites from solid cultures of the plant endophytic fungus Pestalotiopsis fici. METHOD: Pestalofones F–H (1–3) and pestalodiols A–D (4–7) were isolated from the crude extract using silica gel, Sephadex LH-20 column chromatography, and reversed-phase HPLC. Their structures were elucidated by NMR spectroscopy. The new metabolites were tested for cytotoxicity against the HeLa and MCF-7 cells using the MTT method. RESULTS: Compounds 1–3 and 6 showed cytotoxicity against HeLa and MCF-7 cells. CONCLUSION: Seven new cytotoxic isoprenylated epoxycyclohexanediol derivatives were identified from the plant endophytic fungus Pestalotiopsis fici. [KEY WORDS] Pestalotiopsis fici; Endophytic fungus; Cytotoxic; Secondary metabolites; Epoxycyclohexanediol

[CLC Number] R93, R965.1 [Document code] A [Article ID] 1672-3651(2011)05-0374-06

1

Introduction

As one of the most widely distributed endophytic fungal genera, Pestalotiopsis (Amphisphaeriaceae) has attracted much attention in recent years due to its ability to produce a variety of bioactive secondary metabolites including anticancer agent taxol [1-12]. Our previous work on the fungus P. fici (AS 3.9138 = W106-1) grown in different solid-substrate fermentation cultures afforded unique metabolites showing significant anti-HIV-1 effects [13-14]. Subsequent chemical investigations of the extract from a larger scale fermentation of this fungus led to the isolation of other biologically active metabolites with interesting structural features [15-19]. In addition, seven new isoprenylated epoxycyclohexanediol derivatives including three heterodimers (1–3) named pestalofones F–H (1–3), and pestalodiols A–D (4–7), were isolated from the crude extract. Some of these metabolites showed modest cytotoxicity against human tumor cell lines, HeLa and [Received on] 29-Jun.-2011 [Research funding] This project was supported by the National Natural Science Foundation of China (No. 21002120) and the Ministry of Science and Technology of China (No. 2010ZX09401- 403). [*Corresponding author] LIU Ling: Prof., Tel: 86-10-82618783; E-mail: [email protected] These authors have no any conflict of interest to declare. Copyright © 2011, China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

MCF-7. Details of the isolation, structural elucidation, and cytotoxicity of these compounds are reported herein.

2 2.1

Materials and Methods

General Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. 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. ESI-MS data were recorded on a Bruker Esquire 3000plus spectrometer, and HRESI-MS data were obtained using Bruker APEX III 7.0 T and APEX II FT-ICR spectrometers, respectively. 2.2 Fungal material The culture of P. fici was isolated from the branches of Camellia sinensis (Theaceae) in suburb of Hangzhou, China, in April, 2005. The strain was identified by one of the authors (L.G.) based on sequence (GenBank accession No. DQ812914) analysis of the ITS region of the ribosomal DNA and assigned the accession number AS 3.9138 in the China General Microbial Culture Collection (CGMCC) at the Institute of Microbiology, Chinese Academy of Sciences, Beijing. The fungus was cultured on slants of potato dextrose agar at 25 °C for 10 days. Agar plugs were cut into small pieces (about 0.5 cm × 0.5 cm × 0.5 cm) under aseptic conditions.

LIU Shuchun, et al. /Chinese Journal of Natural Medicines 2011, 9(5): 374−379

Fifteen pieces were used to inoculate in three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract), and the final pH of the media was adjusted to 6.5. After sterilization, three flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 170 r·min−1 for five days to prepare the seed culture. Spore inoculum was prepared by suspending the seed culture in sterile, distilled H2O to give a final spore/cell suspension of 1 × 106/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. 2.3 Extraction and isolation 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 (108 mg) eluted with 35% 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 mm × 250 mm; 40% MeOH in H2O for 2 min, followed by 40%–50% MeOH in H2O over 30 min; 2 mL·min−1) to afford a mixture of 2 and 3 (5.0 mg, tR 28.12 min). The fraction eluted with 42% (80 mg) EtOAc was purified by HPLC (65% MeOH in H2O for 2 min, followed by 65%–80% MeOH in H2O over 30 min; 2 mL·min−1) to afford 1 (2.0 mg, tR 18.25 min). The fractions eluted with 12% (102 mg), 22% (98 mg), 40% (75 mg) and 42% (60 mg) EtOAc were fractionated again by Sephadex LH-20 CC eluting with CH2Cl2–MeOH (1 : 1). Purification of the resulting subfractions with different gradients afforded pestalodiols A (4; 2.2 mg, tR 23.66 min; 40% MeOH in H2O for 2 min, followed by 40%–60% MeOH in H2O over 30 min), B (5; 3.0 mg, tR 19.16 min; 35% MeOH in H2O for 2 min, and followed by 35%–50% MeOH in H2O over 25 min), C (6; 2.5 mg, tR 20.84 min; 65% MeOH in H2O for 2 min, followed by 65%–78% MeOH in H2O over 20 min), and D (7; 2.3 mg, tR 14.38 min; 35% MeOH in H2O for 2 min, followed by 35%–60% MeOH in H2O over 30 min). Pestalofone F (1): colorless oil; [α]D25 +20.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 233 (4.01), 273 (3.98) nm; IR (neat) νmax 3 498 (br), 2 924, 2 852, 1 722, 1 608, 1 574, 1 426, 1 383, 1 292, 1 146, 1 033 cm–1; 1H, 13C NMR, HMBC, and NOESY data see Table 1; HRESI-MS m/z 647.210 1 (calcd. for C33H36O12Na, 647.209 9). Pestalofones G (2) and H (3): pale yellow oil; 1H and 13C NMR data see Table 2; HMBC correlations (acetone-d6, 400 MHz) H3-1 → C-2, 3, 4; H3-2 → C-1, 3, 4; H-4 → C-1, 2, 5, 6; H2-5 → C-3, 4, 6, 7, 16; H-7 → C-5, 6, 8, 9; H-8 → C-7, 9;

Table 1 NMR data for 1 (400 MHz, acetone-d6) Position 1 2 3 4 5a 5b 6 7 8 9 10 11 12a 12b 13 14 15 16 1′ 2′ 3′ 4′ 4′a 5′ 6′ 7′ 8′ 8′a 9′ 9′a 10′a 11′ 12′ 13′ 14′ OH-8 OH-10 OH-16 OH-1′

δC, mult. 18.0, q 25.8, q 135.5, s 118.7, d 32.6, t

δH (J in Hz) 1.59, s 1.64, s

HMBCa 2, 3, 4 1, 3, 4

NOESY

5.18, t (6.5) 2.27, dd (15, 6.5) 2.80, dd (15, 6.5)

1, 2 3, 4, 6, 7, 16 3, 4, 6, 7, 16

7,16 7,16 7, 16

5, 6, 8, 9, 16

4, 5

7, 8, 10, 11, 16

12,16

3′, 4′, 4′a, 10, 11 3′, 4′, 4′a, 10, 11

9, 11′

1′, 2′, 3′, 13 13, 14 5, 9, 10

15, 11′ 14 4, 5, 9

65.9, s 62.3, d 3.34, d (3.5) 64.9, d 4.25, ddd (6.0, 5.5, 3.5) 30.3, t 1.99, d (5.5) 81.8, s 211.4, s 36.3, t 4.38, d (19) 4.45, d (19) 205.6, s 40.9, t 3.92, s 29.2, q 2.19, s 70.5, d 3.96, d (10) 158.5, s 117.4, s 148.6, s 113.4, s 153.2, s 102.4, d 7.27, d (2.0) 166.1, s 113.4, d 6.96, d (2.0) 136.1, s 111.3, s 180.9, s 106.7, s 159.0, s 17.2, q 2.19, s 169.1, s 53.0, q 3.91, s 57.0, q 4.00, s 4.32, d (6.0) 4.42, s 4.05, d (10) 12.82, s

6′, 7′, 8′a, 10′a 5′, 6′, 8′a, 12′

2′, 3′, 4′

12, 14

12′ 6′

a

HMBC correlations, optimized for 8 Hz, are from proton(s) stated to the indicated carbon.

H2-9 → C-7, 8, 10, 11, 16; H2-12 → C-10, 11, 13, 14, 15; H2-14 → C-4′, 5′, 6′, 12, 13, 15; H3-15 → C-12, 13, 14; H-16 →C-5, 9, 10; H-3′ → C-1′, 2′, 5′, 8′; H3-8′ → C-3′, 4′, 5′; H-4″→ C-2″, 3″, 5″, 6″; H-6″→ C-7′, 2″, 4″, 5″, 7″; H3-8″ → C-7″; H3-9″ → C-5″; OH-2′ → C-1′, 2′, 3′; NOESY correlations (acetone-d6, 500 MHz ) 2: H-4 ↔ H-7, H-16; H2-5 ↔ H-7, H-16; H-7 ↔ H-4, H2-5; H-9a ↔ H3-15; H-12a ↔ H3-15, H-16; H-12b ↔ H3-15; H-14 ↔ H3-15; H3-15 ↔ H-9a, H2-12, H-14; H-16 ↔ H-4, H2-5, H-12a; 3: H-4 ↔ H-7, H-16; H2-5 ↔ H-7, H-16; H-7 ↔ H-4, H2-5; H-12a ↔ H3-15; H-12b ↔ H3-15, H-16; H-14 ↔ H3-15; H3-15 ↔ H2-12, H-14; H-16 ↔ H-4, H2-5, H-12b; HRESI-MS m/z 649.225 7 (calcd. for C33H38O12Na, 649.225 5).

LIU Shuchun, et al. /Chinese Journal of Natural Medicines 2011, 9(5): 374−379 Table 2 NMR data for 2 and 3 (400 MHz, acetone-d6) Position 1 2 3 4 5a 5b 6 7 8 9a 9b 10 11 12a 12b 13 14a 14b 15 16 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ OH-8 OH-10 OH-16 OH-2′ OH-3″

δC, mult. 18.0, q 25.9, q 135.4, s 118.8, d 34.4, t

2 δH (J in Hz) 1.60, s 1.69, s 5.14, t (7.5) 2.24, dd (15, 7.5) 2.59, dd (15, 7.5)

64.7, s 63.7, d 3.27, d (1.5) 65.5, d 4.18, ddd (11, 5.5, 1.5) 30.1, t 2.09, dd (15, 5.5) 2.27, dd (15, 11) 80.4, s 209.3, s 47.2, t 2,64, d (19) 3.11, d (19) 89.7, s 39.5, t 2.74, d (16) 2.87, d (16) 26.4, q 1.20, s 71.9, d 3.61, br s 107.6, s 162.8, s 109.3, d 6.20, s 143.7, s 118.0, s 160.0, s 199.3, s 19.6, q 2.11, s 130.9, s 125.6, s 156.1, s 106.4, d 6.72, d (2.0) 161.5, s 106.7, d 7.05, d (2.0) 166.7, s 52.4, q 3.69, s 56.0, q 3.85, s 4.00, br s 4.39, br s 3.65, br s 12.4, s 8.60, br s

δC, mult. 18.0, q 25.9, q 135.4, s 118.8, d 34.4, t

3 δH (J in Hz) 1.64, s 1.71, s 5.13, t (7.5) 2.21, dd (15, 7.5) 2.56, dd (15, 7.5)

64.7, s 63.7, d 3.27, d (1.5) 65.5, d 4.18, ddd (11, 5.5, 1.5) 30.1, t 1.66, dd (15, 5.5) 1.81, dd (15, 11) 80.4, s 209.3, s 47.3, t 2,79, d (19) 2.97, d (19) 89.7, s 39.3, t 2.73, d (16) 2.96, d (16) 26.3, q 1.16, s 72.0, d 3.63, br s 107.6, s 163.1, s 109.4, d 6.20, s 143.7, s 118.0, s 160.0, s 199.3, s 19.6, q 2.12, s 131.9, s 125.6, s 156.1, s 106.4, d 6.71, d (2.0) 161.5, s 106.7, d 7.04, d (2.0) 166.7, s 52.4, q 3.69, s 56.0, q 3.84, s 4.00, br s 4.39, br s 3.65, br s 12.4, s 8.60, br s

a

HMBC correlations, optimized for 8 Hz, are from proton(s) stated to the indicated carbon.

Pestalodiol A (4): pale yellow oil; [α]D25 +173.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 205 (3.94) nm; IR (neat) νmax 3 356 (br), 2 973, 2 926, 1 971, 1 648, 1 439, 1 033 cm–1; 1 H NMR (acetone-d6, 400 MHz) δ: 5.31 (1H, d, J = 2.5 Hz, H-12), 5.15 (1H, br d, J = 5.0 Hz, OH-8), 5.13 (1H, t, J = 8.0 Hz, H-4), 4.97 (1H, br d, J = 7.5 Hz, OH-16), 4.65 (1H, br s, OH-13), 4.21 (1H, d, J = 7.5 Hz, H-16), 4.06 (1H, ddd, J = 11, 5.5, 5.0 Hz, H-8), 3.21 (1H, d, J = 1.5 Hz, H-7), 2.75 (1H, dd, J = 15, 8.0 Hz, H-5a), 2.28 (1H, ddd, J = 16, 11, 2.5 Hz, H-9a), 2.09 (1H, dd, J = 16, 5.5 Hz, H-9b), 2.03 (1H, dd, J =

15, 8.0 Hz, H-5b), 1.69 (3H, s, H3-2), 1.63 (3H, s, H3-1), 1.24 (6H, s, H3-14/H3-15); 13C NMR (acetone-d6, 100 MHz) δ: 200. 0 (C, C-11), 135.4 (C, C-3), 119.3 (CH, C-4), 103.6 (C, C-10), 102.3 (CH, C-12), 69.3 (C, C-13), 68.7 (CH, C-16), 68.5 (CH, C-8), 65.8 (C, C-6), 63.3 (CH, C-7), 33.5 (CH2, C-5), 33.5 (CH2, C-9), 30.3 (CH3, C-15), 30.1(CH3, C-14), 25.9 (CH3, C-2), 18.0 (CH3, C-1); HRESI-MS m/z 303.156 5 (calcd. for C16H24O4Na, 303.156 7). Pestalodiol B (5): yellow oil; [α] D25 +209.0 (c 0.1, MeOH); UV (MeOH) λmax 208 (ε 9 600) nm; IR (neat) νmax 3 360 (br), 2 974, 2 927, 1 972, 1 596, 1 436, 1 034 cm–1; 1H NMR (acetone-d6, 400 MHz) δ: 5.30 (1H, d, J = 1.5 Hz, H-12), 5.14 (1H, t, J = 7.5 Hz, H-4), 4.25 (1H, d, J = 8.5 Hz, H-16), 3.99 (1H, ddd, J = 9.0, 5.5 Hz, H-8), 3.97 (1H, br s, OH-13), 3.81 (1H, br d, J = 8.5 Hz, OH-16), 3.70 (2H, br s, OH-8/OH-14), 3.40 (1H, d, J = 10 Hz, H-14a), 3.34 (1H, d, J = 10 Hz, H-14b), 3.21 (1H, s, H-7), 2.80 (1H, dd, J = 15, 7.5 Hz, H-5a), 2.24 (1H, ddd, J = 16, 9.0, 1.5 Hz, H-9a), 2.13 (1H, dd, J = 16, 5.5 Hz, H-9b), 2.10 (1H, dd, J = 15, 7.5 Hz, H-5b), 1.69 (3H, s, H3-2), 1.63 (3H, s, H3-1), 1.18 (3H, s, H3-15); 13C NMR (acetone-d6, 100 MHz) δ: 200. 8 (C, C-11), 135.4 (C, C-3), 119.2 (CH, C-4), 103.3 (C, C-10), 99.3 (CH, C-12), 72.5 (C, C-13), 70.8 (CH2, C-14), 68.8 (CH, C-16), 68.2 (CH, C-8), 65.8 (C, C-6), 63.1 (CH, C-7), 33.2 (CH2, C-5), 31.6 (CH2, C-9), 25.9 (CH3, C-2), 24.7 (CH3, C-15), 18.0 (CH3, C-1); HMBC coreelations (acetone-d6, 400 MHz) H3-1 → C-2, 3, 4; H3-2 → C-1, 3, 4; H-4 → C-1, 2; H2-5 → C-3, 4, 6, 16; H-7 → C-5, 6, 8, 9; H2-9 → C-7, 8, 11; H-12 → C-10; H2-14 → C-12, 13, 15; H3-15 → C-12, 13, 14; H-16 → C-9, 10, 11; HRESI-MS m/z 319.151 9 (calcd. for C16H24O5Na, 319.151 6). Pestalodiol C (6): yellow oil; [α]D25 +46.0 (c 0.1, MeOH); UV (MeOH) λmax 218 (ε 8 600) nm; IR (neat) νmax 3 409 (br), 2 974, 2 926, 1 956, 1 739, 1 629, 1 433, 1 371, 1 228, 1 037 cm–1; 1H NMR (acetone-d6, 400 MHz) δ: 5.99 (1H, s, H-12), 4.12 (1H, br s, OH-8), 5.63 (1H, s, H-16), 5.11 (1H, t, J = 8.0 Hz, H-4), 4.94 (1H, s, H-14a), 4.85 (1H, s, H-14b), 4.06 (1H, dd, J = 9.0, 5.5 Hz, H-8), 3.25 (1H, s, H-7), 2.63 (1H, dd, J = 15, 8.0 Hz, H-5a), 2.28 (1H, dd, J = 16, 9.0 Hz, H-9a), 2.24 (1H, dd, J = 16, 5.5 Hz, H-9b), 2.05 (1H, dd, J = 15, 8.0 Hz, H-5b), 2.02 (3H, s, H3-18), 1.68 (6H, s, H3-2/H3-15), 1.60 (3H, s, H3-1); 13C NMR (acetone-d6, 100 MHz) δ: 205. 1 (C, C-11), 170.4 (C, C-17), 139.6 (C, C-13), 136.0 (C, C-3), 118.7 (CH, C-4), 114.9 (CH2, C-14), 100.5 (C, C-10), 99.3 (CH, C-12), 70.2 (CH, C-16), 67.3 (CH, C-8), 63.6 (C, C-6), 62.0 (CH, C-7), 33.4 (CH2, C-5), 32.5 (CH2, C-9), 25.9 (CH3, C-2), 21.8 (CH3, C-18), 19.6 (CH3, C-15), 18.0 (CH3, C-1); HRESI-MS m/z 327.157 1 (calcd. for C18H24O4Na, 327.156 7). Pestalodiol D (7): yellow oil; [α]D25 +8.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 240 (4.18) nm; IR (neat) νmax 3 406 (br), 2 917, 1 679, 1 616, 1 422, 1 047 cm–1; 1H NMR (acetone-d6, 400 MHz) δ: 6.18 (1H, s, H-12), 3.64 (1H, br d, J = 6.0 Hz, OH-8), 5.13 (1H, t, J = 7.0 Hz, H-4), 3.73 (1H, br d, J = 8.0 Hz, OH-16), 4.06 (1H, dd, J = 8.0, 7.5 Hz, H-16), 3.98

LIU Shuchun, et al. /Chinese Journal of Natural Medicines 2011, 9(5): 374−379

(1H, dddd, J = 6.0, 5.5, 4.0, 3.5 Hz, H-8), 3.17 (1H, d, J = 4.0 Hz, H-7), 2.85 (H, ddd, J = 11, 7.5, 3.0 Hz, H-10), 2.78 (1H, dd, J = 15, 7.0 Hz, H-5a), 1.98 (1H, ddd, J = 16, 3.5, 3.0 Hz, H-9a), 1.52 (1H, ddd, J = 16, 11, 5.5 Hz, H-9b), 2.15 (1H, dd, J = 15, 7.0 Hz, H-5b), 1.70 (3H, s, H3-2), 1.63 (3H, s, H3-1), 2.08 (3H, s, H3-15), 1.88 (3H, s, H3-14); 13C NMR (acetone-d6, 100 MHz) δ: 202.3 (C, C-11), 155.9 (C, C-13), 135.0 (C, C-3), 124.6 (CH, C-12), 119.3 (CH, C-4), 69.8 (CH, C-16), 65.4 (CH, C-8), 64.0 (C, C-6), 61.9 (CH, C-7), 50.6 (CH, C-10), 32.9 (CH2, C-5), 31.8 (CH2, C-9), 27.5 (CH3, C-14), 26.0 (CH3, C-2), 20.6 (CH3, C-15), 18.0 (CH3, C-1); HMBC coreelations (acetone-d6, 400 MHz) H3-1 → C-2, 3, 4; H3-2 → C-1, 3, 4; H-4 → C-1, 2; H2-5 → C-3, 4, 6, 7; H-8 → C-7, 9; H2-9 → C-7, 8, 10, 11; H-12 → C-11, 14, 15; H3-14 → C-12, 13, 15; H3-15 → C-12, 13, 14; NOESY correlations (acetone-d6, 500 MHz ) H-4 ↔ H-7, H-16; H2-5 ↔ H-7, H-16; H-7 ↔ H-4, H2-5; H-12 ↔ H-16; H-16 ↔ H-4, H2-5, H-12; HRESI-MS m/z 303.157 1 (calcd. for C16H24O4Na, 303.156 7). 2.4 MTT assay [18] The assay was run in triplicate. In 96-well plates, each well was plated with 104 cells. After cell attachment overnight, the Dulbecco’s Modified Eagle Medium (DMEM) 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−1 as a stock solution of a compound in DMSO and serial dilutions). Cells were treated at 37 ºC 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−1 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 ºC

for 3 h. Upon removal of MTT/medium, 100 μL of DMSO was added to each well, and agitated at 60 r·min−1 for 5 min to dissolve the precipitate. The assay plate was read at 540 nm using a microplate reader.

3

Results and Discussion

Pestalofone F (1) was assigned the molecular formula C33H36O12 (16 degrees of unsaturation) by HRESI-MS (m/z 647.210 1 [M + Na]+; Δ –0.2 mmu). Analysis of its NMR spectroscopic data (Table 1) revealed four exchangeable protons, six methyl groups including two O-methyls, four methylenes, three oxymethines, two oxygenated sp3 quaternary carbons, 14 olefinic/aromatic carbons (three of which are protonated), one carboxylic carbon (δC 169.1), and three ketone carbons (δC 180.9, 205.6, and 211.4). Interpretation of its 1H–1H COSY and HMBC data established a heterodimeric gross structure identical to that of peatalofone E (8)[15], indicating that 1 is a stereoisomer of 8. The relative configuration of 1 was assigned by analysis of its 1H–1H coupling constants and NOESY data (Figure 1), and by comparison to 8. The isoprenylated epoxycyclohexanediol moiety possesses the same configuration as 8 except for C-10. The small coupling constants of 3.5 Hz between H-7 and H-8, and 5.5 Hz between H-8 and H 2 -9 indicated that H-7 and H-8 are pseudo-equatorially orientated. NOESY correlations of H2-9 with H2-12 revealed their proximity in space, thereby establishing the relative configuration of C-10 as shown. The absolute configuration of 1 was presumed to be analogous to that of 8. Compounds 2 and 3 were obtained as an inseparable mixture of two isomers in a 6 : 5 ratio, as determined by the integration of some well-resolved 1H NMR resonances for each compound. Structural elucidation of 2 and 3 was performed on this mixture due to unsuccessful efforts to separate

LIU Shuchun, et al. /Chinese Journal of Natural Medicines 2011, 9(5): 374−379

Fig. 1 Key NOESY correlations for 1

Fig. 2 Key NOESY correlations for 2 and 3

the two metabolites. Compounds 2 and 3 were each assigned the molecular formula C33H38O12 (15 degrees of unsaturation) by HRESI-MS (m/z 649.225 7 [M + Na]+; Δ –0.2 mmu). The 1 H and 13C NMR spectra of both compounds displayed resonances for five exchangeable protons, six methyl groups (two O-methyls), four methylenes, three oxymethines, three oxygenated sp3 quaternary carbons, 14 olefinic/aromatic carbons (four of which are protonated), one carboxylic carbon (δC 166.7), and two ketone carbons (δC 199.3 and 209.3, respectively). Analysis of the 2D NMR data of 2 and 3 (Table 2) established the same isoprenylated epoxycyclohexanediol moiety as found in 1. Further interpretation of the 2D NMR data indicated that the bottom half of 2 and 3 was identical to that of pestalofone D (9) [15], except that the resonances for the allenyl unit in 9 were replaced by those for a ketone-containing C10–C12 unit. This structural variation was confirmed by HMBC correlations from H2-9 to the C-11 (δC 209.3) and from H2-12 to C-10 (δC 80.4) and C-11, thereby completing the gross structures of 2 and 3 as shown. The relative configurations of 2 and 3 were assigned on the basis of the 1H–1H coupling constants and NOESY data, and by analogy to the known compounds 8 and 9. The isoprenylated epoxycyclohexanediol moiety in 2, 3, and 8 possessed the same relative configuration, suggesting that 2 differs from 3 by having different configuration at the C-13 stereogenic center. The relative configuration of C-13 in 2 was determined based on the NOESY correlations of H-9a with H3-15 in the major isomer 2, whereas that in 3 was deduced to be opposite to 2 on the basis of biosynthetic considerations, which was partially supported by the absence of a NOESY correlation of H-9a with H3-15. The absolute con-

figurations of 2 and 3 was also deduced as shown by analogy to 9. Pestalodiol A (4) was assigned a molecular formula of C16H24O4 (five degrees of unsaturation) on the basis of HRESI-MS data (m/z 303.156 5 [M + Na]+; Δ +0.2 mmu), which is 18 mass units more than the known compound 10 [13] . The NMR data of 4 closely resembled those of 10, except that the resonances for the terminal olefin (δH/δC 4.84; 4.93/114.2; 139.4) were replaced by those for a methyl group (δH/δC 1.24/30.1), one oxygenated sp3 quaternary carbon (δC 69.3), and one exchangeable proton (δH 4.65) Therefore, the gross structure of pestalodiol A was established as 4, with its absolute configuration deduced by analogy to 10. The molecular formula of 5 was assigned as C16H24O5 (five degrees of unsaturation) by HRESI-MS (m/z 319.151 9 [M + Na]+; Δ –0.3 mmu), 16 mass units more than that of 4. Analysis of its NMR data revealed structural features similar to those of 4, except that one methyl group (δH/δC 1.24/30.1) was replaced by an oxymethylene (δH/δC 3.34; 3.40/70.8) and one exchangeable proton (δH 3.70) in 5, which was consistent with the HRESI-MS data of 5. The above-mentioned structural variation was confirmed by corresponding HMBC data. Therefore, 5 was established as the oxidative product of 4, with its absolute configuration deduced by analogy to 4. The elemental composition of 6 was determined to be C18H24O4 (seven degrees of unsaturation) by HRESI-MS (m/z 327.157 1 [M + Na]+; Δ –0.4 mmu). The extra 42 mass units compared to 10 suggested the presence of an acetyl group. Analysis of its NMR spectroscopic data revealed nearly identical structural features to 10, except that the oxymethine proton (H-16) was significantly downfield to δH 5.63 in 6 compared to δH 4.26 in 10. In addition, NMR resonances characteristic of an acetyl group (δH 2.02; δC 21.8 and 170.4) were observed, indicating that the C-16 oxygen of 6 was acylated. The absolute configuration of 6 was also deduced by analogy to 10. Pestalodiol D (7) gave a pseudomolecular ion [M + Na]+ peak at m/z 303.157 1 (Δ –0.4 mmu) by HRESI-MS, consistent with the molecular formula C16H24O4 (five degrees of unsaturation), which is 18 mass units more than that of 10. Interpretation of its 1D and 2D NMR data established an isoprenylated 2, 3-epoxycyclohexan-1, 4-diol moiety identical to that presented in 10. HMBC correlations from H2-9 to C-10, and the C-11 (δC 202.3) ketone carbon, H-12 to C-11, C-14, and C-15, and from H3-14/H3-15 to C-12 and C-13 completed the C-11–C-15 substructure of 7 with C-11 directly attached to C-10. On the basis of these data, the gross structure of 7 was established. The relative configuration of 7 was also deduced by analogy to 10, except that for C-10, which was assigned by a NOESY correlation of H-12 with H-16. The absolute configuration of 7 was also presumed to be analogous to that of 10. Compounds 1–7 were tested for cytotoxicity against two human tumor cell lines, HeLa (cervical epithelium) and MCF-7 (breast adenocarcinoma) (Table 3). Compound 1 displayed cytotoxicity against the HeLa and MCF-7 cells,

LIU Shuchun, et al. /Chinese Journal of Natural Medicines 2011, 9(5): 374−379

with IC50 values of 14.4 and 11.9 µmol·L−1, respectively; while 6 also showed cytotoxic effect against the two cell lines, with IC50 values of 16.7 and 57.5 µmol·L−1, respectively (the positive control 5-fluorouracil showed IC50 values of 10.0 and 15.0 µmol·L−1, respectively).

[6]

Table 3 Cytotoxicity of compounds 1–7 Compound 1 2 and 3 4 5 6 7 5-fluorouracil

IC50/µmol·L HeLa 14.4 36.4 > 142.8 > 135.1 16.7 107.1 10.0

[5]

−1

MCF-7 11.9 33.6 > 142.8 > 135.1 57.5 107.1 15.0

Compounds 1–7 are new isoprenylated epoxycyclohexanediol derivatives. Structurally, 1–3 could be the heterodimeric metabolites derived from the known precursors iso-A82775C (10) [13] and isosulochrin (11) [15] via a series of reactions. Compound 1 is a new C-10 stereoisomer of pestalofone E (8) [15], which was presumed to be derived from 2 and 3 through further reactions. While 2 and 3 could be the oxidative products of pestalofone D (9) [15]. Pestalodiols A–D (4–7) could be derived from iso-A82775C (10) via the reactions including oxidation, reduction, acetylation. Biogenetically, 4–7 could be derived from two units of prenoids and a polyketide. The discovery of these new bioactive metabolites imdicates that further efforts should be made to fully explore the metabolic potential of this fungus.

References

[7]

[8] [9] [10] [11] [12] [13] [14]

[15] [16]

[1] Strobel GA, Yang X, Sears J, et al. Taxol from Pestalotiopsis microspora, an endophytic fungus of Taxus wallochiana [J]. Microbiology, 1996, 142(2): 435-440. [2] Strobel GA, Hess WM, Li JY, et al. Pestalotiopsis guepinii, a taxol-producing endophyte of the wollemi pine, Wollemia nobilis [J]. Aust J Bot, 1997, 45(6): 1073-1082. [3] Harper JK, Arif AM, Ford EJ, et al. Pestacin: a 1, 3-dihydro isobenzofuran from Pestalotiopsis microspora possessing antioxidant and antimycotic activities [J]. Tetrahedron, 2003, 59(14): 2471-2476. [4] Strobel G, Ford E, Worapong J, et al. Isopestacin, an isobenzo-

[17] [18]

[19]

furanone from Pestalotiopsis microspora, possessing antifungal and antioxidant activities[J]. Phytochemistry, 2002, 60(2): 179-183. Xu J, Kjer J, Sendker J, et al. Chromones from the endophytic fungus Pestalotiopsis sp. isolated from the Chinese mangrove plant Rhizophora mucronata [J]. J Nat Prod, 2009, 72(4): 662-665. Pulici M, Sugawara F, Koshino H, et al. Pestalotiopsins A and B: New saryophyllenes from an endophytic fungus of Taxus brevifolia [J]. J Org Chem, 1996, 61(6): 2122-2124. Lee JC, Strobel GA, Lobkovsky E, et al. Torreyanic acid: a selectively cytotoxic quinine dimmer from the endophytic fungus Pestalotiopsis microspora [J]. J Org Chem, 1996, 61 (10): 3232-3233. Pulici M, Sugawara F, Koshino H, et al. A new isodrimeninol from Pestalotiopsis sp. [J]. J Nat Prod, 1996, 59(1): 47-48. Li JY, Strobel GA. Jesterone and hydroxy-jesterone antioomycete cyclohexenone epoxides from the endophytic fungus Pestalotiopsis jesteri [J]. Phytochemistry, 2001, 57(2): 261-265. Li E, Jiang L, Guo L, et al. Pestalachlorides A–C, antifungal metabolites from the plant endophytic fungus Pestalotiopsis adusta [J]. Bioorg Med Chem, 2008, 16(17): 7894-7899. Liu L, Gao H, Chen X, et al. Brasilamides A–D, sesquiterpenoids from the plant endophytic fungus Paraconiothyrium brasiliense [J]. Eur J Org Chem, 2010, 2010 (17): 3302-3306. Ding G, Jiang L, Guo L, et al. Pestalazines and pestalamides, bioactive metabolites from the plant pathogenic fungus Pestalotiopsis theae [J]. J Nat Prod, 2008, 71(11): 1861-1865. Liu L, Liu S, Jiang L, et al. Chloropupukeananin, the first chlorinated pupukeanane derivative, and its precursors from Pestalotiopsis fici [J]. Org Lett, 2008, 10(7): 1397-1400. Liu L, Tian R, Liu S, et al. Pestaloficiols A–E, bioactive cyclopropane derivatives from the plant endophytic fungus Pestalotiopsis fici [J]. Bioorg Med Chem, 2008, 16(11): 60216026. Liu L, Liu S, Chen X, et al. Pestalofones A–E, bioactive cyclohexanone derivatives from the plant endophytic fungus Pestalotiopsis fici[J]. Bioorg Med Chem, 2009, 17(2): 606-613. Liu L, Li Y, Liu S, et al. Chloropestolide A, an antitumor metabolite with an unprecedented spiroketal skeleton from Pestalotiopsis fici [J]. Org Lett, 2009, 11(13): 2836-2839. Liu L, Liu S, Niu S, et al. Isoprenylated chromone cerivatives from the plant endophytic fungus Pestalotiopsis fici [J]. J Nat Prod, 2009, 72(8): 1482-1486. Liu L, Niu S, Lu X, et al. Unique metabolites of Pestalotiopsis fici suggest a biosynthetic hypothesis involving a Diels-Alder reaction and then mechanistic diversification [J]. Chem Commun, 2010, 46(3): 460-462. Liu L, Bruhn T, Guo L, et al. Chloropupukeanolides C–E, cytotoxic pupukeanane chlorides with a spiroketal skeleton from Pestalotiopsis fici [J]. Chem Eur J, 2011, 17(9): 2604-2613.

源于植物内生无花果拟盘多毛孢真菌的新环氧环己二醇衍生物 刘述春 1, 叶

昕 2, 郭良栋 1, 刘

玲 1*

1

中国科学院微生物研究所真菌学国家重点实验室(筹), 北京 100190;

2

中国科学院微生物研究所中国科学院病原微生物与免疫学重点实验室, 北京, 1001900

【摘 要】从一株植物内生无花果拟盘多毛孢真菌的放大发酵产物中获得了 7 个新结构的异戊二烯取代环氧环己二醇衍 生物,分别命名为 pestalofones F–H (1–3) 和 pestalodiols A–D (4–7), 并应用 MS、NMR 等光谱技术鉴定了其结构。化合物 1–3, 6 和 7 对 HeLa 和 MCF-7 肿瘤细胞株具有中等程度的细胞毒活性。 【关键词】 Pestalotiopsis fici; 内生真菌; 细胞毒; 次生代谢产物; 环氧环已二醇 【基金项目】国家自然科学基金项目(No. 21002120), 科技部项目(No. 2010ZX09401-403)