Chlorotheolides A and B, Spiroketals Generated ... - ACS Publications

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Chlorotheolides A and B, Spiroketals Generated via Diels−Alder Reactions in the Endophytic Fungus Pestalotiopsis theae Ling Liu,† Yu Han,‡,§ Junhai Xiao,‡ Li Li,⊥ Liangdong Guo,† Xuejun Jiang,† Lingyi Kong,*,§ and Yongsheng Che*,‡ †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China ‡ State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, People’s Republic of China § State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, People’s Republic of China ⊥ Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Chlorotheolides A (1) and B (2), two new spiroketals possessing the unique [4,7]methanochromene and dispirotrione skeletons, respectively, and their putative biosynthetic precursors, 1-undecen-2,3-dicarboxylic acid (3) and maldoxin (4), were isolated from the solid substrate fermentation of the plant endophytic fungus Pestalotiopsis theae (N635). Their structures were elucidated based on NMR spectroscopic data and electronic circular dichroism calculations. Biogenetically, compounds 1 and 2 could be generated from the co-isolated 3 and 4 via Diels−Alder reactions. Chlorotheolide (2) showed an antiproliferative effect against the human tumor cell line HeLa and induced an autophagic process in the cells.

revealed that the major adducts were those generated from the Diels−Alder reactions occurring at the carboxylate face of maldoxin (4).6 However, efforts to isolate 4 from the fermented rice culture of P. f ici were unsuccessful, possibly due to its high reactivity, which remained an unsolved puzzle. In a continuing search for new bioactive metabolites from this genus, a strain of Pestalotiopsis theae (N635), also endophytic to Camellia sinensis, was first cultured on rice. Fractionation of the EtOAc extract afforded chlorotheolides A (1) and B (2), two new spiroketals with unique skeletons, and 1-undecene-2,3-dicarboxylic acid (3), a new methylenesuccinic acid derivative as one of the putative biosynthetic precursors. The fungus was then cultured on PDA medium for comparison. Surprisingly, compounds 1−3 were not detected in the EtOAc extract, but the long missing precursor, maldoxin (4), was found and subsequently isolated. Herein, we report the structure elucidation, bioactivity, and plausible biogenesis for compounds 1 and 2.

Pestalotiopsis spp. are a class of the most widely distributed plant endophytic fungi and have attracted much attention recently due to their ability to produce bioactive secondary metabolites with diverse structures and bioactivities.1 Our previous chemical investigations on Pestalotiopsis f ici (W106-1), a strain endophytic to Camellia sinensis, led to the discovery of over 70 new bioactive natural products with unique structural features,2−6 demonstrating its huge potential for the production of bioactive secondary metabolites. Representatives isolated from the fungus include chloropupukeananin and the chloropupukeanolides2−4 and the chloropestolides,5,6 featuring new skeletons derived from the chlorinated tricyclo[4.3.1.03,7]decane (pupukeanane) and bicyclo[2.2.2]octenone moieties, respectively. Biogenetically, these compounds were presumably generated via Diels−Alder reactions from the co-isolated precursors, pestheic acid (PA) and iso-A82775C.6 Their unique structures and bioactivities have attracted the attention of synthetic chemists,7 and their endeavors enabled clarification of the proposed biogenesis for these compounds, in which PA was first oxidized to maldoxin (4),7,8 with a masked benzoquinone as the reactive diene,9 and then reacted with iso-A82775C via the reverse electron demanding Diels−Alder (REDDA) routes to form the core skeletons of these metabolites.6 Our study has © 2016 American Chemical Society and American Society of Pharmacognosy

Received: June 15, 2016 Published: October 12, 2016 2616

DOI: 10.1021/acs.jnatprod.6b00550 J. Nat. Prod. 2016, 79, 2616−2623

Journal of Natural Products

Article

methyl groups with three oxygenated, seven methylene units, 10 olefinic/aromatic carbons including four protonated, two quaternary carbons, three heteroatom-bonded tertiary carbons, and three carboxylic carbons (δC 170.0, 169.6, and 163.5, respectively). These data accounted for all the NMR resonances of 1 except for one chlorine atom. Analysis of the 1 H−1H COSY NMR data for 1 (Figure 1) defined the isolated

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RESULTS AND DISCUSSION Chlorotheolide A (1) was assigned the molecular formula C30H35ClO11 (13 degrees of unsaturation) by HRESIMS. Its NMR data (Table 1) revealed the presence of two exchangeable protons at 3.49 and 9.86 ppm, respectively, five

Figure 1. Key 1H−1H COSY and HMBC correlations for 1 and 2.

spin system of C-20−C-26. HMBC cross-peaks from H3-27 to C-14, C-15, and C-16, OH-13 to C-12, C-13, and C-14, H-14 to C-11 and C-12, and H-16 to C-11, C-12, and C-17 (Figure

Table 1. NMR Data for 1 and 2 in CDCl3 1

a

position

δC , mult.

1 2a 2b 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OH-13 OH-6

49.2, qC 39.1, CH2

a

52.5, 114.5, 136.0, 98.4, 71.0, 151.3, 93.6, 106.4, 163.5, 96.9, 160.8, 111.3, 150.3, 107.2, 156.8, 169.6, 170.0, 29.9, 28.0, 29.6, 29.1, 31.8, 22.5, 14.0, 22.7, 56.6, 53.2, 52.7,

qC qC CH qC qC qC CH qC qC qC qC CH qC CH qC qC qC CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH3 CH3 CH3 CH3

δH (J in Hz) b

2.64, d (14.0) 2.68, d (14.0)

2 δC , mult. c

HMBC 1, 3, 4, 7, 9, 10, 18, 19 1, 3, 4, 7, 9, 10, 18, 19

6.12, t (1.2)

3, 4, 6, 20

5.49, s

1, 2, 3, 6, 7, 8, 10, 18

6.42, s

11, 12, 13, 16, 27

6.33, s

11, 12, 14, 17, 27

2.02, m; 2.20, m 1.52, m 1.31−1.34, m 1.31−1.34, m 1.31−1.34, m 1.31−1.34, m 0.99, t (7.0) 2.28, s 3.72, s 3.80, s 3.74, s 9.86, s 3.49, s

3, 4, 5, 21, 22 20

24, 25 14, 15, 16 8 18 19 12, 13, 14 6, 10

51.6, qC 34.5, CH2 55.5, 122.9, 138.8, 187.6, 74.3, 148.0, 97.0, 97.1, 162.1, 96.1, 160.9, 112.3, 150.7, 107.7, 154.8, 167.8, 176.6, 23.7, 26.1, 29.1, 29.0, 31.7, 22.6, 14.1, 22.6, 58.6, 53.4,

qC qC CH qC qC qC CH qC qC qC qC CH qC CH qC qC qC CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH3 CH3 CH3

δH (J in Hz) d

HMBC

2.34, d (14.0) 2.99, d (14.0)

1, 3, 4, 6, 7, 9, 18, 19 1, 3, 4, 7, 9, 18, 19

6.65, t (2.1)

3, 4, 7, 19, 20

5.75, s

1, 2, 3, 6, 7, 8, 10, 18

6.53, s

12, 13, 16, 27

6.31, s

12, 14, 17, 27

1.69, m; 2.30, m 1.54, m; 1.42, m 1.34−1.36, m 1.34−1.36, m 1.34−1.36, m 1.34−1.36, m 0.94, t (7.0) 2.33, s 3.88, s 3.84, s

3, 4, 5, 21 4, 20, 22

24, 25 14, 15, 16 8 18

9.75, s

12, 13, 14

Recorded at 150 MHz. bRecorded at 600 MHz. cRecorded at 100 MHz. dRecorded at 400 MHz. 2617



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(1R,3S,6S,7S,10S)-, (1R,3S,6S,7S,10R)-, and (1S,3R,6R,7R,10S)-1′ (1′a−1′d). An MMFF94 conformational search and DFT reoptimization at the B3LYP/6-31+G(d,p) level yielded eight and seven lowest energy conformers for 1′a and 1′c, respectively (Figure S20). The overall calculated ECD spectra of 1′a−1′d were then generated by Boltzmann weighting of the conformers (Figure 3). The experimental ECD curve of 1 was nearly identical to the calculated ECD spectrum of 1′a, suggesting the 1S,3R,6R,7R,10R absolute configuration for 1. Chlorotheolide B (2) was assigned a molecular formula of C29H31ClO10 (14 degrees of unsaturation) by HRESIMS. Analysis of its NMR data (Table 1) revealed the same aliphatic chain and bicyclo[2.2.2]oct-2-ene moiety spirally joined to the HMBD unit at C-10 as appears in 1. However, the C-6 ketal carbon at 98.4 ppm in the spectrum of 1 was replaced by a ketone carbon at 187.6 ppm in 2, similar to that found in the chloropestolides (e.g., 6).5,6 This observation was supported by the weak but distinctive four-bond HMBC correlations from H2a and H-9 to C-6 (Figure 1). A key HMBC cross-peak from H-5 to C-19 and the downfield chemical shift values for H-5/C5 (δH/C 6.65/138.8 in 2 vs 6.12/136.0 in 1) and C-19 (δC 176.6 in 2 vs 170.0 in 1) suggested that the C-19 carboxylic carbon acylated the C-5 oxygen to form a furan-2(3H)-one moiety spirally joined to the bicyclo[2.2.2]oct-2-en-5-one unit at C-3. On the basis of these data, the gross structure of 2 was established. The relative configuration of 2 was also assigned based on NOESY data (Figure 2) and by analogy to chloropestolides A− D.5,6 NOESY correlations of H-2a with H-9 and H-20a established the relative configurations for C-1, C-3, and C-7, which are the same as their counterparts in chloropestolides A− D.5,6 The absolute configuration of 2 was deduced by comparison of the experimental ECD spectrum with the simulated ECD spectra generated for a simplified structure 2′ ( F i g u r e 4 ). F o ur enantiome rs, ( 1 S ,3 S, 7R ,1 0R )- , (1R,3R,7S,10S)-, (1R,3R,7S,10R)-, and (1S,3S,7R,10S)-2′ (2′a−2′d; Figure 4), were calculated, followed by MMFF94 conformational search and DFT reoptimization at the B3LYP/ 6-31+G(d,p) level, affording the three lowest energy conformers for 2′a and 2′c, respectively (Figure S21). The experimental ECD spectrum of 2 matched the calculated ECD curve of 2′a, suggesting the 1S,3S,7R,10R absolute configuration for 2. The molecular formula of 3 was determined as C13H22O4 (three degrees of unsaturation) by HRESIMS. Literature search on this formula readily identified 1-undecene-2,3-dicarboxylic acid,11 a synthetic intermediate of maleic anhydrides, possessing the same planar structure, which was confirmed by interpretation of the 2D NMR data (Table S1). The absolute configuration of 3 was also deduced by comparison of its experimental ECD spectrum with the calculated ECD spectra generated for the enantiomers (3R)-3′ (3′a) and (3S)-3′ (3′b) (Figure 5). The MMFF94 conformational search followed by DFT reoptimization at the B3LYP/6-31+G(d,p) basis set level afforded the 12 lowest energy conformers for 3′a (Figure S22). The experimental ECD spectrum of 3 resembled the calculated ECD curve of 3′a, suggesting the 3R absolute configuration. The known compound maldoxin (4) was characterized by comparison of its MS and NMR data (Table S1) with those reported8 and was assigned the 2R absolute configuration by comparing its experimental ECD spectrum with the simulated

1) revealed the presence of a dihydroxymethylbenzoic acid moiety, as found in the chloropupukeanolides (e.g., 5).3,4 Further correlations from H-5 to C-3, C-4, and C-6 and from H2-20 to C-3, C-4, and C-5 connected both C-3 and C-20 to the C-4/C-5 olefin at C-4, while those from H2-2 and H-9 to C1, the C-10 ketal carbon (δC 106.4), and the C-18 carboxylic carbon linked C-1 to C-2, C-9, C-10, and C-18. In turn, HMBC correlations from H2-2 to C-3, C-4, C-7 (δC 71.0), and the C19 carboxylic carbon and from H-9 to C-7 and C-8 indicated that C-7 is allylic to the C-8/C-9 olefin, and C-2, C-7, and C-19 are all attached to C-3. The cross-peaks from H3-28, H3-29, and H3-30 to C-8, C-18, and C-19, respectively, established the locations of these methoxy groups. Additional correlations from H-5 to C-6 and from OH-6 to C-6 and C-10, plus the chemical shifts for C-5 (δC 136.0) and C-6 (δC 98.4), suggested that C-5 and C-6 were both connected to the same oxygen atom to form a dihydro-2H-pyran ring with the hydroxy group bearing C-6 attached to C-10, establishing a partial structure with the [4,7]methanochromene unit. The shift values for C-11 (δC 163.5) and C-17 (δC 156.8) suggested that each carbon was connected to one of the two C-10 linked oxygen atoms via an ester and an ether linkage, respectively, completing the 5hydroxy-7-methyl-4H-benzo[d][1,3]dioxin-4-one (HMBD) unit as found in chloropupukeanolides C and D,4 which formed a C-10 spiral junction with the [4,7]methanochromene moiety. In addition, the chemical shift for C-7 (δC 71.0) and the pentacyclic nature of 1 required that C-6 and the remaining chlorine atom be directly attached to C-7 to form the chlorinated spiro[benzo[d][1,3]dioxine-2,8′-[4,7]methanochromen]-4-one skeleton. Collectively, the planar structure of 1 was proposed. The relative configuration of 1 was assigned by analysis of NOESY data (Figure 2) and by analogy to chloropupukeano-

Figure 2. Key NOESY correlations for 1 and 2.

lides C and D.4 The C-4/C-5 and C-8/C-9 olefins were assigned the Z- and E-geometry, respectively, based on NOESY correlations of H-5 with H-20b and of H-9 with H3-28. Rigidity for the core [4,7]methanochromene skeleton predetermined the relative configurations for C-1, C-3, C-6, and C-7, which are the same as their counterparts in the chloropupukeanolides.4 This was further confirmed by a NOESY correlation observed between H-2a and H-9. However, the relative configuration of C-10 could not be assigned due to the lack of relevant NOESY correlations. The absolute configuration of 1 was deduced by comparison of the experimental electronic circular dichroism (ECD) spectrum with the simulated ECD spectra (Figure 3) generated by the time-dependent density functional theory (TDDFT).10 Due to insignificant contribution to the ECD property of 1 by the aliphatic chain, a simplified structure 1′ (Figure 3) was used to calculate the four enantiomers (1S,3R,6R,7R,10R)-, 2618



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Figure 3. Experimental ECD spectrum of 1 in MeOH and the calculated ECD spectra of 1′a−1′d.

Figure 4. Experimental ECD spectrum of 2 in MeOH and the calculated ECD spectra of 2′a−2′d.

for such an effect in HeLa cells. Compound 2 was found to inhibit cell viability in time- and dose-dependent manners by the MTS assay (Figure 7A). Under electron microscopy, accumulation of membrane vacuoles was observed at the 2 h time point in the treated cells, indicating that 2 enhances the autophagic process in HeLa cells (Figure 7B). In the immunofluorescence assay, an increase in punctuate LC3 and further accumulation in the presence of chloroquine (CQ; a compound that blocks the fusion of autophagosomes and lyosomes and is often used in the detection of autophagic flux12) also suggested that 2 promotes autophagy (Figure 7C). Utilizing immunoblotting, the ratios of LC3-II to actin were

ECD spectra of (2S)-4 (4a) and (2R)-4 (4b) (Figures 6 and S23). Compounds 1−4 were tested for inhibitory effects on the proliferation of two human tumor cell lines, HeLa (cervical carcinoma) and MCF-7 (breast adenocarcinoma). Compounds 1 and 2 displayed weak inhibitory effects, with IC50 values of 13.3−73.2 μM, compared to the positive control cisplatin (IC50 values of 4.7 and 4.9 μM, respectively). Compounds 3 and 4 did not show detectable activity against the tested cell lines at 20 μg/mL. Since natural products with autophagy induction may correlate to its cytotoxic activity, compound 2 was evaluated 2619



Article

Diels−Alder reactions form carbon−carbon bonds in both organic synthesis and natural product biosynthesis.13 However, only a few enzymes have been found to catalyze such reactions in nature.14 Three types of Diels−Alderases have been found in the biosyntheses of spinosyn A and pyrroindomycins.15,16 From a biogenetic perspective, compounds 1 and 2 could be generated from the co-isolated 3 and 4 via similar REDDA routes to those occurring in P. f ici.2−6 Specifically, addition from the carboxylate face of 4 with 3 first afforded the hypothetical intermediates a and b, and then 1 and 2 could be generated from a and b, respectively (Scheme 1). Failure to isolate adducts c and d in our study, which would be formed by addition from the ether face of 4, suggested the stereopreference for the involved Diels−Alder reactions. In addition, a ratio of 1:4 for the generated 1 and 2 indicated that the endo adduct is predominant, implying that the reactions could be catalyzed by a Diels−Alderase. In summary, chlorotheolides A (1) and B (2) represent the first examples of spiroketals incorporating unusual [4,7]methanochromene and dispiro-trione skeletons, respectively. Isolation of the long missing maldoxin (4) from P. theae provided evidence for its involvement in the Diels−Alder reactions as a reactive diene, which filled the gap in the previously proposed biogenesis.

Figure 5. Experimental ECD spectrum of 3 in MeOH and the calculated ECD spectra for enantiomers 3′a and 3′b.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Research Analytical automatic polarimeter, and UV data were obtained on a Shimadzu Biospec-1601 spectrophotometer. 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, -500, and -600 spectrometers using solvent signals (CDCl3: δH 7.28/δC 77.0; 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 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 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. HPLC separations were performed on an Agilent 1260 instrument equipped with a variable-wavelength UV detector. Column chromatography (CC) was carried out on ODS (50 μm; YMC). Chloroquine diphosphate salt (C6628) and polyclonal antibodies against LC3 (L7543) were purchased from Sigma-Aldrich. P62 (sc-28359) was acquired from Santa Cruz Biotechnology. Antibody against actin (TA-09) was obtained from ZhongShanJinQiaoBiocompany. MTS reagent powder (G1111) was acquired from Promega Corporation. Fungal Material. The culture of P. theae (N635) was isolated from Camellia sinensis in Hangzhou, People’s Republic of China. The isolate was identified by one of the authors (L.G.) based on sequence (GenBank Accession No. KF641183) analysis of the ITS region of the rDNA. The 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 cm3) 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 5 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 × 106/

Figure 6. Experimental ECD spectrum of 4 in MeOH and the calculated ECD spectra for enantiomers 4a and 4b.

increased upon treatment with 2, and more LC3-II was accumulated in the presence of CQ. In addition, the level of SQSTM1/p62, a selective substrate and marker for autophagy, 12 was decreased by 2 (Figure 7D). Collectively, chlorotheolide B (2) was demonstrated to activate the autophagic process in HeLa cells at a concentration of 10 μM. Chlorotheolides A (1) and B (2) are new spiroketals with skeletons differing significantly from those previously found in P. f ici.2−6 They incorporate the typical 4H-spiro[benzo[d][1,3]dioxine-2,7′-bicyclo[2.2.2]octane]-4,8′-dione unit, which further fused with the 3,4-dihydro-2H-pyran-2-ol at C-3, C-6, and C-7 to complete the pentacyclic core of 1 with a C-10 spiro-junction between the [4,7]methanochromene and HMBD moieties, while a C-3 spiro-junction between the furan-2(3H)-one and 4H-spiro[benzo[d][1,3]dioxine-2,7′bicyclo[2.2.2]octane]-4,8′-dione completed the skeleton of 2 featuring the unique dispiro-trione moiety. This is the first time compound 3 was demonstrated to be a natural product, and the absolute configuration for 3 and 4 was deduced by ECD calculations. 2620



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Figure 7. Chlorotheolide B (2) induces autophagy in HeLa cells. (A) Cell viability analyzed by MTS after treatment with 10 μM 2 for up to 24 h (single asterisk denotes the group statistically different from the control; p < 0.05). (B) Electron microscopy for cells treated for 2 h. (C) Punctuates for LC3 counted after 2 h treatment in the presence/absence of 15 μM chloroquine (CQ). (D) Immunoblotting of cells with the indicated antibodies after 2 h treatment and lyses. Ratios of LC3-II and p62 to actin (A) were calculated and are presented below the blots.

Scheme 1. Hypothetical Biosynthetic Pathways for 1 and 2

mL. Fermentation was carried out in 24 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. In addition, the strain was cultured on 50 plates of PDA at 25 °C for 7 days. Extraction and Isolation. The fermented rice material was extracted repeatedly with EtOAc (4 × 4.0 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (15 g), which was fractionated by silica gel vacuum liquid chromatography (VLC) using petroleum ether−EtOAc gradient elution. The fractions (1.1 g) eluted with 20−30% EtOAc were combined and separated by ODS CC using MeOH−H2O gradient elution. A 70 mg subfraction eluted with 80% MeOH was purified by

RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 90− 100% MeOH in H2O for 30 min; 2.0 mL/min) to afford 1 (1.0 mg, tR 10.05 min) and 2 (2.7 mg, tR 13.03 min). The fractions (3.2 g) eluted with 35−55% EtOAc were combined and separated by ODS CC using MeOH−H2O gradient elution. A 240 mg subfraction eluted with 65% MeOH was separated by Sephadex LH-20 CC eluting with MeOH, and the resulting subfractions were purified by RP HPLC to afford 3 (8.4 mg, tR 27.25 min; 70−76% MeOH in H2O for 30 min; 2.0 mL/ min). The fermented PDA material was also extracted with EtOAc (3 × 1.0 L) to afford the crude extract of 0.99 g, which was separated by ODS CC using MeOH−H2O gradient elution. A 78 mg fraction eluted with 60% MeOH was purified by RP HPLC to afford 4 (4.1 mg, tR 23.65 min; 60−70% MeOH in H2O for 30 min; 2.0 mL/min). 2621



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Chlorotheolide A (1): pale yellow oil; [α]25D +4.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (3.51), 266 (2.88); 323 (2.46) nm; CD (c 1.7 × 10−3 M, MeOH) λmax (Δε) 219 (+17.5), 274 (−3.95) nm; IR (neat) νmax 3280 (br), 2955, 2854, 1737,1705, 1644, 1464, 1362, 1147, 1037 cm−1; 1H and 13C NMR and HMBC data see Table 1; NOESY correlations (CDCl3, 600 MHz) H-2a ↔ H-9; H-5 ↔ H-20b; H-9 ↔ H-2a, H3-28; HRESIMS m/z 607.1945 (calcd for C30H36ClO11, 607.1941). Chlorotheolide B (2): pale yellow oil; [α]25D −106 (c 0.27, MeOH); UV (MeOH) λmax (log ε) 220 (4.22), 267 (3.88), 323 (3.83) nm; CD (c 6.3 × 10−4 M, MeOH) λmax (Δε) 221 (+23.4), 276 (−25.4), 321 (+7.2), 360 (−10.4) nm; IR (neat) νmax 3271 (br), 2928, 2856, 1799, 1761, 1743, 1710, 1641, 1582, 1465, 1357, 1202, 1098 cm−1; 1H and 13 C NMR and HMBC data see Table 1; NOESY correlations (CDCl3, 400 MHz) H-2a ↔ H-9; H-5 ↔ H-20a; H-9 ↔ H-2a; H3-28; HRESIMS m/z 575.1695 (calcd for C29H32ClO10, 575.1679). 1-Undecene-2,3-dicarboxylic acid (3): yellow oil; [α]25D −22.1 (c 0.44, MeOH); UV (MeOH) λmax (log ε) 210 (3.40) nm; CD (c 2.1 × 10−3 M, MeOH) λmax (Δε) 203 (−0.57), 223 (+0.55) nm; IR (neat) νmax 2955, 2912, 1690, 1626, 1420, 1286, 958 cm−1; 1H and 13C NMR and HMBC data see Table S1; HRESIMS m/z 243.1598 (calcd for C13H22O4, 243.1591). Maldoxin (4): [α]25D −31.7 (c 0.12, MeOH); CD (c 3.4 × 10−4 M, MeOH) λmax (Δε) 217 (+13.9), 270 (−3.2), 341 (−1.6), 429 (−0.70) nm; NMR (Table S1) and MS data were consistent with literature values.8 Computational Details. Systematic conformational analyses for 1′a, 1′c, 2′a, 2′c, 3′a, and 4a were performed via the Molecular Operating Environment (MOE) ver. 2009.10. (Chemical Computing Group, Canada) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational analyses were further optimized using DFT at the B3LYP/631+G(d,p) basis set level. The stationary points have been checked as the true minima of the potential energy surface by verifying they do not exhibit vibrational imaginary frequencies. The 80 lowest electronic transitions were calculated at the Cam-B3LYP/6-31+G(d,p) level, and the rotational strengths of each electronic excitation were given using both dipole length and dipole velocity representations. ECD spectra were stimulated using a Gaussian function with a half-bandwidth of 0.3 eV. Equilibrium populations of conformers at 298.15 K were calculated from their relative free energies (ΔG) using Boltzmann statistics. The overall ECD spectra were then generated according to Boltzmann weighting of each conformer. The systematic errors in the prediction of the wavelength and excited-state energies are compensated for by employing UV correction. MTS Assay.17 In a 96-well plate, each well was plated with (2−5) × 103 cells (depending on the cell multiplication rate). After cell attachment overnight, the medium was removed, and each well was treated with 100 μL of medium containing 0.1% DMSO or appropriate concentrations of the test compounds and the positive control cisplatin (100 mM as stock solution of a compound in DMSO and serial dilutions; the test compounds showed good solubility in DMSO and did not precipitate when added to the cells). The plate was incubated at 37 °C for 48 h in a humidified, 5% CO2 atmosphere. Proliferation was assessed by adding 20 μL of MTS (Promega) to each well in the dark, followed by incubation at 37 °C for 90 min. The assay plate was read at 490 nm using a microplate reader. The assay was run in triplicate. Cell Culture and Immunoblotting Analysis. HeLa cells were grown in DMEM medium containing 10% fetal bovine serum (GIBCO) with the antibiotics. Cells were grown to 70% confluence before adding the test compounds. Whole cell lysates were prepared with lysis using Triton X-100/glycerol buffer, containing 50 mM TrisHCl, pH 7.4, 4 mM EDTA, 2 mM EGTA, and 1 mM dithiothreitol, supplemented with 1% Triton X-100, 1% SDS, and protease inhibitors, separated on a SDS-PAGE gel, and then transferred to a PVDF membrane. Immunoblotting was performed using appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies, followed by detection with enhanced chemiluminescence (Pierce

Chemical). Ratios of LC3-II and p62 to actin (A) were calculated and presented below the blots. Immunofluorescence. HeLa cells were plated on glass coverslips and then underwent the indicated treatment. Cells were washed with Ca2+- and Mg2+-free PBS (CMF-PBS), fixed with freshly prepared 4% paraformaldehyde, and permeabilized by incubation with CMF-PBS containing 0.1% TritonX-100 and 0.5% bovine serum albumin (BSA). Then cells were washed three times with CMF-PBS and incubated with the indicated antibodies in the presence of 0.1% TritonX-100 and 0.5% BSA. After washing three times, cells were incubated with the secondary antibodies (Alexa Fluor 594 goat anti-rabbit and Alexa Fluor 488 goat anti-mouse) diluting in CMF-PBS containing 0.5% BSA and then immersed in VECTASHIELD with 4′,6-diamidino-2-phenylindole to visualize the nuclei after washing three times. Images were acquired via fluorescence microscopy. Punctates for LC3 were counted after treatment with 10 μM 2 for 2 h in the presence/absence of 15 μM CQ. Electron Microscopy. Electron microscopy was performed as previously described.17 Briefly, samples (10 μM) were washed three times with PBS, trypsinized, and collected by centrifuging. Cell pellets were fixed with 4% paraformaldehyde at 4 °C overnight, postfixed with 1% OsO4 incacodylate buffer at room temperature (RT) for 1 h, and dehydrated stepwise with ethanol. The dehydrated pellets were rinsed with propylene oxide at RT for 30 min and embedded in Spurr resin for sectioning. Images of thin sections were observed under a transmission electron microscope (JEM1230, Japan). Statistical Analysis. Several X-ray films were analyzed to verify the linear range for the chemiluminescence signals, and quantifications were carried out using densitometry. Normally distributed data are shown as mean ± SD and were analyzed using one-way analysis of variance and the Student−Newman−Keuls post hoc test. Data are shown as mean ± SD in graphs.

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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.6b00550. NMR spectra of 1−4, NMR data of 3 and 4, and ECD calculations for 1−4 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail (L. Kong): [email protected]. *Tel/Fax (Y. Che): +86 10 66932679. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (81273395 and 21372004), the National Program of Drug Research and Development (2012ZX09301-003), and the Program of the Excellent Young Scientists of Chinese Academy of Sciences.

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REFERENCES

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Chlorotheolides A and B, Spiroketals Generated ... - ACS Publications

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