Identification of Oxaphenalenone Ketals from the Ascomycete Fungus

Article pubs.acs.org/jnp

Identification of Oxaphenalenone Ketals from the Ascomycete Fungus Neonectria sp. Jinwei Ren,† Shubing Niu,‡ Li Li,§ Zhufeng Geng,⊥ Xingzhong Liu,† and Yongsheng Che*,‡ †

State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ‡ State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, AMMS, Beijing 100850, People’s Republic of China § Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ⊥ Analytical and Testing Center, Beijing Normal University, Beijing 100875, People’s Republic of China S Supporting Information *

ABSTRACT: Neonectrolides B−E (4−7), four new oxaphenalenone ketals incorporating the new furo[2,3-b]isochromeno[3,4,5-def ]chromen11(6aH)-one skeleton, were isolated from the fermentation extract of the ascomycete fungus Neonectria sp. in an in-depth investigation guided by HPLC fingerprint and a cytotoxicity assay. The previously identified oxaphenalenone spiroketal neonectrolide A (1) and its putative biosynthetic precursors (2 and 3) were also reisolated in the current work. The structures of 4−7 were primarily elucidated by interpretation of NMR spectroscopic data, and the absolute configurations were deduced by electronic circular dichroism calculations. Compound 6 showed cytotoxic effects against four of the six human tumor cell lines tested. Biosynthetically, compounds 4−7 could be derived via the Diels−Alder reaction cascades starting from derivatives of the co-isolated metabolites 2 and 3.

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precursors, corymbiferan lactone E (2) and 3-dehydroxy-4-Oacetylcephalosporolide C (3).8 In cytotoxicity evaluations against six human tumor cell lines, HeLa (cervical epithelium), A549 (lung carcinoma epithelial), MCF-7 (breast cancer), HCT116 (colon carcinoma), SW480 (colon carcinoma), and T24 (bladder carcinoma), the crude extract showed inhibitory effects on the proliferation of A549 and T24 cells. However, the previously isolated compounds 1 and 3 were cytotoxic only to T24 cells,8 suggesting the presence of an active unidentified component active against the A549 cells. In addition, the HPLC chromatogram of the crude extract revealed several peaks with similar UV characteristics to neonectrolide A (1),8 but the corresponding metabolites could not be isolated in sufficient amounts for structure elucidation and cytotoxic evaluation. Therefore, the strain was refermented in a larger scale on rice (3 kg) using conditions in which compounds 1−3 were initially isolated, and the EtOAc extract was prepared for further fractionation and purification. In the current work, four additional new oxaphenalenone ketals, named neonectrolides B−E (4−7), were isolated from the crude extract as guided by HPLC fingerprint and cytotoxicity activity, together with neonectrolide A (1) and its putative

ungi from special habitats have a proven track record in the production of structurally unique secondary metabolites with diverse biological effects, presumably due to their highly adapted metabolic systems that evolved during the natural selection process.1−4 The Qinghai-Tibetan plateau, with an average elevation of over 4000 m,5 is such an environment that harbors many microorganisms, including fungi.6 Recently, we have initiated a project targeting the chemistry of those fungal species isolated from the soil samples collected in alpine regions of the Qinghai-Tibetan plateau and discovered a variety of secondary metabolites with cytotoxic effects against human tumor cell lines.7 Neonectria is a fungal genus in the Nectriaceae family, and the species of this genus have been frequently found in the soil samples on the Qinghai-Tibetan plateau. However, the chemistry of Neonectria spp. remained underexplored, with only a few documented examples.8−10 In an ongoing search for new cytotoxic secondary metabolites from fungi inhabiting this unique environment, a strain of Neonectria sp. isolated from a soil sample that was collected on the Qinghai-Tibetan plateau, Chayu, People’s Republic of China, was chemically investigated, leading to the isolation of neonectrolide A (1), a unique oxaphenalenone spiroketal possessing the new 4,5-dihydro3H,3′H-spiro[furan-2,2′-isochromeno[3,4,5-def ]chromen]5′(3a′H)-one skeleton, together with its putative biosynthetic © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 15, 2015

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DOI: 10.1021/acs.jnatprod.5b00159 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

The 1H and 13C NMR spectra for 4 showed resonances for one exchangeable proton (δH 11.45), three methyl groups including one O-methyl, four methylene units, four methines with three oxygenated, one doubly oxygenated sp3 quaternary carbon (δC 109.4), 10 olefinic/aromatic carbons (two of which were protonated), and two carboxylic carbons (δC 169.8 and 176.6, respectively). Analysis of its NMR data (Table 1) indicated that the skeleton of 4 incorporates the same moiety (rings A−D) derived from the isochromeno[3,4,5-def ]chromen-5(2H)-one skeleton as found in neonectrolide A (1).8 The presence of such a tetracyclic partial structure was further confirmed by relevant HMBC correlations. Interpretation of the 1H−1H COSY NMR data established the second isolated proton spinsystem of C-2′−C-5′ (Figure 1). In the HMBC spectrum of 4, correlations from H-4′ and H2-5′ to C-6′ were observed, indicating that C-5′ is attached to the tetracycle at C-6′. HMBC cross-peaks from H-2′ and H-3′ to the carboxylic carbon C-1′ (δC 176.6), plus a key correlation from H-4′ to C-1′, established the γ-lactone moiety in 4 (ring F). Considering the doubly oxygenated nature of C-6′ (δC 109.4) and the chemical shift value for C-9′ (δC 75.6), the only remaining oxygen atom was connected to both C-6′ and C-9′ by default, forming a THF moiety (ring E) fused to the tetracycle at C6′/ C7′ to satisfy the unsaturation requirement. However, no

biosynthetic precursors, corymbiferan lactone E (2) and 3dehydroxy-4-O-acetylcephalosporolide C (3).8 Details of the isolation, structure elucidation, cytotoxicity, and putative biogenesis of 4−7 are reported herein.

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RESULTS AND DISCUSSION Neonectriolide B (4) was isolated as pale yellow, amorphous powder. Its molecular formula was determined to be C24H24O8 (13 degrees of unsaturation) on the basis of HRESIMS (m/z 441.1542 [M + H]+; Δ −0.7 mmu) and NMR data (Table 1). Table 1. NMR Data for 4−7 4 pos.

δC,a mult.

1 3

169.8, qC 73.0, CH

4 5 6 7 7a 8 9 10 11 11a 12 13 1′ 2′ 3′

96.8, 151.6, 95.4, 160.3, 113.6, 147.9, 118.4, 163.2, 97.6, 132.5, 25.4, 55.6, 176.6, 28.8, 29.3,

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

4′ 5′

76.5, CH 41.9, CH2

6′ 7′ 8′

109.4, qC 46.0, CH 32.9, CH2

9′ 10′ 10OH a

75.6, CH2 21.6, CH3

δHb (J in Hz) 6.01, d (5.0)

5 HMBCb 1, 4, 5, 11a, 6′, 7′, 8′

δC,a mult. 170.0, qC 72.7, CH

6.35, s

3, 4, 5, 7, 7a

6.76, s

1, 7a, 10, 11, 12

2.80, s 3.91, s

7a, 8, 9 7

2.56, overlap 2.52, overlap 2.08, m 4.87, m 2.40, dd (14.5, 5.0) 2.30, dd (14.5, 5.0)

1′, 3′, 4′ 1′, 2′, 4′, 5′

96.0, qC 151.0, qC 95.2, CH 160.4, qC 113.5, qC 147.8, qC 118.3, CH 163.3, qC 97.8, qC 132.6, qC 25.3, CH3 55.5, CH3 176.0 qC 28.6, CH2 29.1, CH2

1′, 6′ 3′, 4′, 6′, 7′

76.6, CH 42.7, CH2

3.28, m 2.27, overlap 1.91, m 4.32, m 1.28, d (6.0) 11.45, s

3, 4, 6′, 8′, 9′ 6′, 7′, 9′, 10′ 7′ 8′, 9′ 9, 10, 11

110.0, qC 43.3, CH 31.8, CH2 75.8, CH2 21.8, CH3

6 δHb (J in Hz)

5.91, d (5.5)

6.28, s

6.75, s

2.80, s 3.90, s 2.56, overlap 2.52, overlap 1.99, m 4.83, m 2.35, dd (15.0, 4.0) 2.27, dd overlap

3.40, m 2.28, overlap 1.93, m 4.47, m 1.33, d (6.0) 11.20, s

δC,a mult. 169.9, qC 73.1, CH 96.7, 150.2, 95.7, 160.4, 113.7, 148.0, 118.4, 163.3, 96.5, 132.5, 25.4, 55.5, 176.5, 28.8, 29.4,


76.5, CH 45.0, CH2

109.0, qC 47.3, CH 34.0, CH2 76.4, CH2 22.1, CH3

7 δHb (J in Hz)

6.00, d (6.0)

6.34, s

6.79, s

2.82, s 3.92, s 2.56, overlap 2.51, overlap 2.06, m 4.86, m 2.37, dd (14.5, 5.5) 2.24, dd (14.5, 6.5) 3.21, m 2.42, m 1.70, m 4.36, m 1.29, d (6.0) 11.54, s

δC,a mult. 170.2, qC 72.7, CH 95.9, 150.0, 95.3, 160.5, 113.6, 148.0, 118.3, 163.4, 97.9, 132.6, 25.4, 55.5, 176.5, 28.7, 29.8,


77.3, CH 43.0, CH2

109.7, qC 45.1, CH 33.2, CH2 76.7, CH2 22.6, CH3

δHb (J in Hz) 5.91, d (6.0)

6.29, s

6.78, s

2.81, s 3.91, s 2.56, overlap 2.51, overlap 1.97, m 4.85, m 2.36, dd (15.0, 4.0) 2.25, dd (15.0, 7.5) 3.39, m 2.44, m 1.74, m 4.50, m 1.37, d (6.5) 11.54, s

Recorded at 125 MHz in CDCl3. bRecorded at 500 MHz in CDCl3. B



Article

Figure 1. 1H−1H COSY and key HMBC correlations for compound 4.

evidence for this connectivity was provided by the HMBC data. Therefore, the planar structure for 4 was established. The relative configuration of 4 was proposed by selected NOE experiments. In the NOE difference (NOED) spectrum of 4, correlations (Figure 2) of H-3 with H-5′a and of H-7′ with H-5′a, H-5′b, and H3-10′ placed these protons on the same face of the ring system. NOED correlations from H-3′a to H-5′b and H-10′ and from H-4′ to H-6 revealed their proximity in space. Therefore, the relative configuration of 4 was proposed as shown (Figure 2). The absolute configuration of 4 was deduced by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by time-dependent density functional theory (TDDFT).12 In view of the established relative configuration, one of the two enantiomers, (3R,4′R,6′R,7′S,9′R)-4 (4a) or (3S,4′S,6′S,7′R,9′S)-4 (4b), should represent the absolute configuration of 4. Considering the facts that the C-5′/C-6′ bond is flexible and the rigid skeleton of 4 (rings A−E) is the major contributor to its CD, a simplified structure 9 was used for CD calculation. A systematic conformational analysis was performed for (3R,6′R,7′S,9′R)-9 (9a) and (3S,6′S,7′R,9′R)-9 (9b) by the Molecular Operating Environment (MOE) software package using the MMFF94 molecular mechanics force field calculation. The MMFF94 conformational search followed by reoptimization using TDDFT at the B3LYP/6-31G(d) basis set level afforded two lowest-energy conformers for 9a and 9b (Figure S16). The overall calculated ECD spectra of 9a and 9b were then generated by Boltzmann-weighting of the two conformers with 99.86% and 99.99% populations, respectively, by their relative free energies (Figure 3). The absolute configuration of 4 was then extrapolated by comparison of the experimental and calculated ECD spectra of 9a and 9b (Figure 3). The experimental CD spectrum of 4 was nearly identical to the calculated ECD spectrum of (3R,6′R,7′S,9′R)-9 (9a) (Figure 3), leading to the deduction of the 3R, 6′R, 7′S, 9′R absolute configuration for 9. Therefore, neonectriolide B (4) was deduced to have the 3R, 4′R, 6′R, 7′S, 9′R absolute configuration. Neonectriolides C−E (5−7) were all determined to have the same molecular formula C24H24O8 as 4 by HRESIMS data, and their 1H and 13C NMR spectra resembled those for 4, implying their isomeric relationships. Analysis of their 1H and 13C NMR data (Table 1) suggested that the structural variations were likely caused by different configurations for the stereogenic centers on rings D−E. Interpretation of the 2D NMR data for 5−7 confirmed the above postulation and allowed determination of their gross strcutures as shown.

Figure 2. Key NOESY correlations for compounds 4−6 in 3D models.

The relative configuration of the partial structure for 5 (rings D and E) was determined to be the same as that of 4 on the basis of NOED data (Figure 2). A key NOED correlation between H-4′ and H3-10′ indicated that they adopt the same orientation in space, establising the relative configuration of 5 (Figure 2). Since the CD spectrum of 5 (Figure 3) was nearly identical to that of 4, both showing positive Cotton effects (CEs) in the regions of 200−220 and 240−260 nm and negative CEs at 220−240 and 260−400 nm (Figure 3), indicating that the absolute configurations for C-3, C-6′, C-7′, and C-9′ in 5 are the same as those in 4. Considering the relative configuration proposed for C-4′, the absolute configuration of neonectriolide C (5) was deduced to be 3R, 4′S, 6′R, 7′S, 9′R. The relative configuration of 6 was also proposed on the basis of NOED data (Figure 2). NOED correlations of H-3 with H-5′b and of H-7′ with H-5′b and H-9 suggested that these protons adopted the same orientation, whereas those of H-5′b with H-3′a and H-3′b, of H-7′ with H-4′ and H-9′, and C



Article

did not show detectable cytotoxicity against any of the six cell lines at 50 μM. Compounds 4−7 are new additions to the class of oxaphenalenone-derived natural products,12−17 although they are related to the previously identified neonectriolide A (1)8 and share the same putative biosynthetic precursors 2 and 3 as illustrated in a proposed biosynthetic pathway in Scheme 1. The hypothetical intermediate 10 was likely derived from precursor 2 via oxidation and dehydration reactions, and intermediates 13 and 15 were presumably generated from precursor 3 via two different reaction cascades. Diels−Alder addition of 10 with 13 and 15 led to the formation of compounds 1 and 4−7, respectively. Compounds 4−7 incorporate the new frame of furo[2,3-b]isochromeno[3,4,5def ]chromen-11(6aH)-one, with a C-6′-attached γ-lactone derivative, which is different from the core structure of 3H,5′H-spiro[furan-2,2′-isochromeno[3,4,5-def ]chromen]-5′one found in 1.

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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 Unico UV-2802H 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 a Bruker Avance-500 spectrometer using solvent signals (CDCl3: δH 7.26/δC 77.36) 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 1200 instrument (Agilent, USA) equipped with a variable-wavelength UV detector. Fungal Material. The culture of Neonectria sp. (Nectriaceae) was isolated from a soil sample collected from Qinghai-Tibetan plateau (N: 28°27′, E: 97°02′), Chayu, Tibet, People’s Republic of China, in May 2004. The isolate was identified by one of the authors (X.L.) based on morphology and sequence (GenBank Accession No. JX566703) analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar at 15 °C for 10 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm3) under aseptic conditions, and 15 of these pieces were used to inoculate five 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). Five flasks of the inoculated media were incubated at 15 °C on a rotary shaker at 170 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 36 Fernbach flasks (500 mL) each containing 80 g of

Figure 3. Experimental CD spectra of 4−7 in MeOH and the calculated ECD spectra of (3R,6′R,7′S,9′R)-9 (9a) and (3S,6′S,7′R,9′R)-9 (9b).

of H3-10′ with H-6 revealed their proximity in space, thereby completing the relative configuration of 6. Since the CD spectrum of 6 (Figure 3) showed negative Cotton effects in the regions of 200−220 and 240−260 nm and positive CEs at 220−240 and 260−400 nm (Figure 3). This was nearly identical to that of 9b, indicating that C-3, C-6′, C-7′, and C-9′ in 6 possess the same absolute configuration as in 9b. Therefore, the absolute configuration of neonectriolide D (6) was deduced to be 3S, 4′S, 6′S, 7′R, 9′R. For neonectriolide E (7), NOE correlations from H-3 to H5′a and H-5′b and from H-7′ to H-5′b and H-9′ placed these protons on the same face of the ring system, while no crosspeak was available to propose the relative configuration for C4′. Considering the CEs of 7 were nearly identical to those of 6, with the same relative configuration for C-3, C-6′, C-7′, and C9′, this suggests that 7 is a C-4′ epimer of 6. Therefore, neonectriolide E (7) was deduced to have the 3S, 4′R, 6′S, 7′R, 9′R absolute configuration. Compounds 4−7 were tested for cytotoxicity against a panel of six human tumor cell lines, HeLa, A549, MCF7, HCT116, SW480, and T24 (Table 2). In addition to its cytotoxicity against the T24 cells, compound 6 was found to be cytotoxic to the A549 cells, showing an IC50 value of 10.7 μM (the positive control cisplatin showed an IC50 value 4.29 μM). Compound 4 Table 2. Cytotoxicity of Compounds 5−7a

IC50 (μM)

a

compound

HeLa

A549

MCF7

5 6 7 cisplatin

>50 >50 32.1 ± 0.94 10.7 ± 0.29

>50 10.7 ± 1.7 39.8 ± 3.2 4.29 ± 0.17

>50 >50 36.7 ± 0.71 11.2 ± 0.78

HCT116 48.4 17.8 32.8 14.1

± ± ± ±

0.64 0.80 1.3 0.85

SW480

T24

± ± ± ±

>50 10.9 ± 0.73 45.3 ± 1.8 2.17 ± 0.22

48.4 18.1 31.6 11.0

1.1 0.82 1.6 1.25

4 was inactive at 50 μM. D



Article

Scheme 1. Hypothetical Biosynthetic Pathways for 4−7

3 ↔ H-5′b; H-7′ ↔ H-5′b, H-4′, H-9′; H-9′ ↔ H-4′; H-6 ↔ H3-10; H-5′a ↔ H-3′b; HRESIMS m/z 441.1543 (calcd for C24H25O8, 441.1549). Neonectrolide E (7): pale yellow powder; mp 148−150 °C; [α]25.0 D +46 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (2.48), 230 (sh, 2.39), 257 (sh, 1.47), 277 (1.74), 342 (0.68) nm; CD (c 2.27 × 10−4 M, MeOH) λmax (Δε) 209 (−4.83), 230 (+4.44), 255 (−1.75), 277 (+1.60), 345 (+2.89) nm; IR (neat) νmax 2929, 2360, 2341, 1775, 1662, 1457, 1379, 1286, 1263, 1172, 1063, 1029, 1001 cm−1; 1H and 13 C NMR, HMBC data see Table 1; NOESY correlations (CDCl3, 500 MHz) H-3 ↔ H-4′, H-5′a; H-7′ ↔ H-5′a, H-5′b, H-9′; HRESIMS m/ z 441.1546 (calcd for C24H25O8, 441.1549). Computational Details. Systematic conformational analyses for 6 were performed via the 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 TDDFT at the B3LYP/631G(d) 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 30 lowest electronic transitions were calculated, 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 correlation. All quantum computations were performed using the Gaussian 03 package,18 on an IBM cluster machine located at the High Performance Computing Center of Peking Union Medical College. MTS Assay.19 The assay was run in triplicate. 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 an appropriate concentrations of the test compounds and the positive control cisplatin. The plate was incubated for 48 h at 37 °C in a humidified, 5% CO2 atmosphere. Proliferation was assessed by adding 20 μL of MTS (Promega) to each well in the dark, incubating for 90 min at 37 °C, and measuring the OD at 490 nm using a microplate reader.

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 15 °C for 40 days. Extraction and Isolation. The fermented material was extracted repeatedly with EtOAc (4 × 2.4 L), and the organic solvent was evaporated to dryness under vacuum to afford the crude extract (47.0 g), which was fractionated by silica gel vacuum liquid chromatography using a petroleum ether−acetone−MeOH gradient elution. The fraction (563 mg) that eluted with 5:1 petroleum ether−acetone was further separated by Sephadex LH-20 column chromatography eluting with 1:1 CH2Cl2−MeOH, and the resulting subfractions were purified by semipreparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 56% MeCN in H2O for 50 min; 2 mL/min) to afford a mixture of 4 and 5 (38.0 mg, tR 28.03 min) and a mixture of 6 and 7 (10.5 mg, tR 32.63 min). The above mixtures were again separated by semipreparative HPLC using a Cellucota-RP column (9.4 × 250 mm, 59% MeCN in H2O for 2 min, followed by 59−64% over 45 min; 1 mL/min) to afford 4 (13.7 mg, tR 13.87 min), 5 (11.2 mg, tR 15.19 min), 6 (3.7 mg, tR 14.77 min), and 7 (2.5 mg, tR 16.04 min). Neonectrolide B (4): pale yellow powder; mp 145−147 °C; [α]25.0 D −104.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (2.43), 230 (sh, 2.20), 257 (sh, 1.45), 277 (1.71), 342 (0.66) nm; CD (c 2.27 × 10−4 M, MeOH) λmax (Δε) 210 (5.64), 230 (−3.84), 257 (3.18), 279 (−1.77), 341 (−0.94) nm; IR (neat) νmax 2933, 1776, 1656, 1619, 1592, 1458, 1446, 1379, 1210, 1116, 1060, 1026, 1001 cm−1; 1H NMR, 13 C NMR, and HMBC data see Table 1; NOESY correlations (CDCl3, 500 MHz) H-3 ↔ H-4′, H-5′a; H-7′ ↔ H-5′a, H-5′b, H-10′; H-4′ ↔ H-6; H-3′a ↔ H-5′b; HRESIMS m/z 441.1542 (calcd for C24H25O8, 441.1549). Neonectrolide C (5): pale yellow powder; mp 142−144 °C; [α]25.0 D −108.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (3.85), 230 (sh, 3.56), 257 (sh, 1.97), 277 (2.41), 342 (0.92) nm; CD (c 2.27 × 10−4 M, MeOH) λmax (Δε) 208 (+8.05), 228 (−7.09), 256 (5.84), 278 (−3.09), 341 (−1.97) nm; IR (neat) νmax 2971, 2158, 1975, 1777, 1664, 1457, 1381, 1208, 1167, 1062, 1001 cm−1; 1H NMR, 13C NMR, and HMBC data see Table 1; NOESY correlations (CDCl3, 500 MHz) H-3 ↔ H-5′a, H-5′b; H-7′ ↔ H-5′b, H-10′; H-4′ ↔ H-10′; H-5′a ↔ H-3′b; HRESIMS m/z 441.1555 (calcd for C24H25O8, 441.1549). Neonectrolide D (6): pale yellow powder; mp 145−147 °C; [α]25.0 D +38.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 220 (1.22), 230 (sh, 1.17), 257 (sh, 0.73), 277 (0.86), 342 (0.33) nm; CD (c 2.27 × 10−4 M, MeOH) λmax (Δε) 207 (−8.26), 228 (+5.36), 253 (−6.71), 280 (+2.24), 337 (+2.09) nm; IR (neat) νmax 2928, 1776, 1768, 1654, 1614, 1457, 1380, 1209, 1171, 1027 cm−1; 1H NMR, 13C NMR, and HMBC data see Table 1; NOESY correlations (CDCl3, 500 MHz) HE



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Article

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ASSOCIATED CONTENT

S Supporting Information *

1

H, APT, and NOED NMR spectra of 4−7, HPLC chromatograms of 4−7, and CD calculations for 4. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00159.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +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 (81001380) and the National Program of Drug Research and Development (2012ZX09301-003).

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