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7 oxygen heterocycle from Stachybotrys chartarum
Phytochemistry Letters 35 (2020) 73–77
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Bistachybotrysins W-Y, three new phenylspirodrimane dimers with a 6/7 oxygen heterocycle from Stachybotrys chartarum
T
Jimei Liua,b,c,1, Jiamin Fenga,b,c,1, Xiaona Jiaa,b,c, Jinlian Zhaoa,b,c, Ridao Chena,b,c, Kebo Xiea,b,c, Dawei Chena,b,c, Yan Lia,b,c, Jungui Daia,b,c,* a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China b CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China c NHC Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China
ARTICLE INFO
ABSTRACT
Keywords: Bistachybotrysin Phenylspirodrimane dimer Stachybotrys chartarum Cytotoxicity
Bistachybotrysins W-Y (1–3), three new phenylspirodrimane dimers with a 6/7 oxygen heterocycle as the linkage, were isolated from Stachybotrys chartarum CGMCC 3.5365. The structures of 1–3 were elucidated through extensive spectroscopic data analysis, including 1D/2D NMR, HR-ESI-MS, and ECD spectra. Compounds 1 and 3 displayed moderate cytotoxic activity against human tumor cell lines HCT116, Daoy, and HepG2 with IC50 values ranging from 5.9 to 9.8 μM.
1. Introduction Stachybotrys are known for the production of a variety of secondary metabolites including trichothecene mycotoxins, diterpenes, phenylspirodrimanes, and the prenylated phenol derivatives (Wang et al., 2015). Among of them, phenylspirodrimanes were discovered as the major class of secondary metabolites produced by this fungus, exhibiting a wide range of bioactivities including anticomplement and antiviral effects, endothelin receptor antagonism, and antibacterial, anti-inflammatory, and cytotoxic activities (Ma et al., 2013; Wang et al., 2015; Chunyu et al., 2016; Kim et al., 2016; Zhang et al., 2019a). Our previous chemical investigation for the S. chartarum CGMCC 3.5365 (China General Microbiological Culture Collection Center, CGMCC) have led to identification of several new monomeric phenylspirodrimanes (Zhao et al., 2017a) and rare dimeric phenylspirodrimanes (Zhao et al., 2018; Zhang et al., 2019b; Feng et al., 2019). Further biological studies revealed most of phenylspirodrimane dimers exhibited significant cytotoxicity against the human tumor cells (Zhao et al., 2018; Zhang et al., 2019b; Feng et al., 2019). As part of our ongoing search for structurally novel phenylspirodrimane dimers with potent biological activities, a recent chemical investigation on this fungal strain has contributed to the isolation of bistachybotrysins W-Y
(1-3) (Fig. 1), three new members for phenylspirodrimane dimer family. Herein, we report the isolation, structural elucidation, and biological activities of the three phenylspirodrimane dimers. 2. Results and discussion The fermentation, extraction, and isolation of broth and mycelia of the fungal strain S. chartarum were performed as described previously (Zhao et al., 2017a, 2017b). The EtOAc extract (137.0 g) was subjected to silica gel column chromatography (CC), Sephadex LH-20 CC, and semipreparative HPLC to afford 1 (1.1 mg), 2 (1.1 mg), and 3 (21.4 mg), respectively. Bistachybotrysin W (1) was obtained as a white amorphous powder. It displayed an HR-ESI-MS ion peak at m/z 833.4464 [M+H]+ (calcd for C48H65O12, 833.4471), consistent with a molecular formula of C48H64O12 with 17 degrees of unsaturation. The IR spectrum showed characteristic absorption for hydroxyl (3396 cm–1) and carbonyl (1714 cm–1), and the UV spectrum displayed maximum absorptions at 213 and 287 nm. In the 1H NMR spectrum (Table 1), the clearly recognized signals were assigned for two sets of typical phenylspirodrimane, including four pairs of methyl resonances (H3-12/12′, H3-13/13′, H3-14/ 14′, and H3-15/15′), one pair of aromatic singlet protons (H-18/18′),
Corresponding author at: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China. E-mail address: [email protected] (J. Dai). 1 These authors made equal contributions to this work. ⁎
https://doi.org/10.1016/j.phytol.2019.11.011 Received 2 September 2019; Received in revised form 10 November 2019; Accepted 13 November 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of compounds 1–3.
one pair of methylene protons (H2-11/11′), and two pairs of oxygenated methine protons (H-2/H-2′ and H-3/3′). From 13C NMR and DEPT spectroscopic data (Table 1), a total of 48 carbon resonances were assigned, most of which appeared in pairs except signals at δC 93.8 (C-23), δC 77.6 (C-23′), δC 71.1 (C-22), δC 69.7 (C-22′), δC 171.0 (C-1′′), and 21.0 (C-2′′). Considering the previously reported phenylspirodrimane dimers from this fungal strain, the above spectroscopic data indicated that 1 was a dimeric structure with two similar phenylspirodrimanes (unit A and unit B). Further analysis of the 1D and 2D NMR spectra demonstrated that 1 shared the same backbone with reported bistachybotrysins A–C (Zhao et al., 2018). And the general spectroscopic features (Table 1) were similar to those of bistachybotrysin C except for the presence of an additional acetyl group (δH-2′′ 2.10/δC-2′′ 21.0, δC-1′′ 171.0), the HMBC correlation from H-3 to C-1′′ (Fig. 2) as well as the downfield shift of H-3 (δH 3.32→δH 4.83) unambiguously established the attachment of acetyl group at C-3. Therefore, 1 was identified as an acetyl derivative of bistachybotrysin C. The relative configuration of 1 was deduced by 1D-NOE correlations (Figs. 3 and S7). The NOE correlations of H-2/H-3, H3-15, and H3-14; H3-14/H3-15, H-2, and H-3; H3-13/H-5; H3-15/H-8 and H-11 in unit A, as well as H-2′/H-3′, H3-15′, and H3-14′; H3-15′/H3-14′, H-8′, and H-11′; H-5′/H3-13′ in unit B, together with the small coupling constant of JH2,H-3 and JH-2′,H-3′, indicating that both units A and B of 1 possessed same relative configuration as those of bistachybotrysins A–J (Zhao et al., 2018; Zhang et al., 2019b; Feng et al., 2019). In addition, signal enhancement of H-22 and H-23 were observed after irradiation of H23′, suggesting H-22, H-23, and H-23′ in unit C had the same orientation. The small coupling constant of JH-22/H-23′ (3.8 Hz) further confirmed the cis-orientation of H-22 and H-23′ (Ramana et al., 2008), which was in great agreement with the orientation of that in bistachybotrysins A–C (Zhao et al., 2018). From the biosynthetic standpoint, the bistachybotrysin C was synthesized through the intact incorporation of two monomers (mer-NF5003B and K-76) and maintained their natural absolute configuration (Zhao et al., 2018). Thus, the unit A and unit B of 1 might have the same absolute configuration at C-2, C-3, C-5, C-8, C-9, and C-10 as mer-NF5003B and K-76. Furthermore, comparison of the ECD spectrum revealed that 1 has similar Cotton effects [202.5 (–21.72), 221 (31.85), and 284.5 (3.36) nm, Fig. S11] to those of bistachybotrysins A–C, thus the absolute configuration of 1 was determined as 2R, 3S, 5S, 8R, 9R, 10S, 2′R, 3′S, 5′S, 8′R, 9′R, 10′S, 22S, 23S, and 23′S. Compounds 2 and 3 were isolated as white amorphous powders. Their molecular formulas were deduced as C48H64O12 and C50H66O13 from the HR-ESI-MS ion peaks at m/z 833.4446 [M+H]+ (calcd for C48H65O12, 833.4471) and 875.4551 [M+H]+ (calcd for C50H67O13, 875.4576), respectively. Their IR, UV, 1H, and 13C NMR spectroscopic data (Table 1) showed close similarity to those of 1. In compound 2, the general features of its 1H and 13C NMR spectroscopic data closely resembled those of 1 except the different chemical shifts for H-3, C-3, and H-3′. Analysis of DEPT, 1H−1H COSY, and HMBC correlations suggested that the acetyl group was attached at C-3′ instead of C-3 in 2, which was further supported by the downfield shift of H-3′ (δH 3.40→
δH 4.94). For 3, the only difference from 1 was the observation of an additional acetyl group (δC-3′′ 171.9, δC-4′′ 21.5/δH-2′′ 2.31), and the downfield shift of H-3′ (δH 3.39→δH 4.93) as well as the HMBC correlation of H-3′/C-3′′ (Fig. 2), indicating the another acetyl group attaching at C-3′. Moreover, 2 and 3 were all assigned to have the same 2R, 3S, 5S, 8R, 9R, 10S, 22S, 23S, 23′S, 2′R, 3′S, 5′S, 8′R, 9′R, and 10′S configurations as 1, due to their consistent NOEs (Figs. 3, S18, and S29) and similar ECD Cotton effects (Figs. S17 and S25). To the best of our knowledge, there are only three members in phenylspirodrimane dimer family with 6/7 oxygen heterocycle linkage reported so far, including bistachybotrysins A–C (Zhao et al., 2018). From a biosynthetic standpoint, compounds 1-3 were proposed to have the same biosynthetic pathway as we have reported before (Zhao et al., 2018), and the downstream regio-selective acetylation greatly enriches the diversity of structures. Compounds 1–3 were evaluated for in vitro cytotoxicity (paclitaxel as the positive control) against five human tumor cell lines (colorectal carcinoma HCT116, lung carcinoma NCI-H460, gastric carcinoma BGC823, medulloblastoma Daoy, and liver carcinoma HepG2). Compounds 1 and 3 displayed selective cytotoxicity against the HCT116, Daoy, and HepG2 cell lines with IC50 values ranging from 5.9 to 9.8 μM (Table 2). 3. Experimental section 3.1. General experimental procedures Optical rotations were measured on a Perkin-Elmer Model-343 digital polarimeter (PerkinElmer Inc., Waltham, Massachusetts, USA). The ECD and UV absorption spectra were recorded on a Jasco J-815 spectropolarimeter (Jasco Corporation, Tokyo, Japan). IR spectra were acquired on a Nicolet 5700 FT-IR microscope spectrometer (FTIR Microscope Transmission, Thermo Electron Scientific Instrument Crop., Madison, Wisconsin, USA). 1D/2D NMR spectra were obtained at 600 MHz for 1H NMR and 150 MHz for 13C NMR on VNOVA SYSTEM-600 spectrometer (Varian Inc., Palo Alto, California, USA). Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in Hertz (Hz). HR-ESI-MS data were measured using an LTQ-FT Ultra ESI-FTICR-MS spectrometer (ThermoFisher Scientific, CA, USA). Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, PR China) and Sephadex LH-20 gel (Amersham Biosciences, Sweden) were used for column chromatography (CC). Semi-preparative HPLC was performed on a Shimadzu HPLC instrument equipped with a Shimadzu RID-10A detector (Shimadzu Corporation, Tokyo, Japan) and a Shiseido Capcell Pak C18 MG II S5 column (250 mm × 10 mm, i.d., 5 μm, Shiseido Co., Tokyo, Japan) or a grace Allsphere silica gel column (250 mm × 10 mm, i.d., 5 μm, W.R. Grace Co., Columbia, Maryland, USA). Analytical TLC was carried out on pre-coated silica gel GF254 plates (Qingdao Marine Chemical Industry, Qingdao, China), and spots were visualized under UV light or by spraying with 10 % H2SO4 in 90 % EtOH followed by heating at 120 °C. 74
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Table 1 1 H (600 MHz) and No.
1
13
Table 1 (continued)
C (150 MHz) NMR data of 1–3 in acetone-d6.
1
2
No.
3
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
34.9, CH2
1.37, dd (12.1, 4.3) 1.83c 4.04, m 4.83, d (2.2)
34.0, CH2
1.31c 1.77c
34.9, CH2
3.96, m 3.33, brs
65.3, CH 80.6, CH
1.40, dd (12.0, 4.2) 1.85c 4.04, m 4.87, d (2.3)
2 3
65.3, CH 80.7, CH
4 5 6
38.9, C 41.2, CH 21.3, CH2
7
66.4, CH 79.2, CH
2.12c 1.50c; 1.56c
38.9, C 39.9, CH 21.5, CH2
31.9, CH2
1.52c; 1.61c
31.9, CH2
8 9 10 11
37.4, 97.7, 44.3, 32.4,
1.84c
37.5, 97.7, 44.1, 32.4,
12
15.9, CH3
13 14 15 16 17 18 19 20 21 22 23 1′
28.5, CH3 22.0, CH3 17.4, CH3 111.4, C 154.2, C 106.1, CH 139.3, C 109.5, C 158.3, C 71.1, CH 93.8, CH 34.3, CH2
2′ 3′
66.3, CH 80.2, CH
4′ 5′
38.8, C 39.8, CH
6′
CH C C CH2
3.22, d (16.5) 2.84c 0.75, d (6.5) 0.90, s 0.99, s 1.10, s 6.61, s
4.62, m 6.01, s 1.31c 1.68, dd (12.2, 12.2) 3.94, m 3.39, brs
CH C C CH2
16.1, CH3 29.2, CH3 22.5, CH3 17.4, CH3 112.8, C 154.3, C 107.6, CH 138.7, C 106.7, C 159.4, C 70.0, CH 93.5, CH 35.3, CH2
65.2, CH 80.3, CH 38.9, C 42.0, CH
21.6, CH2
2.32, dd (12.3, 2.1) 1.48c; 1.55c
21.5, CH2
7′
32.2, CH2
1.52c; 1.61c
31.7, CH2
8′ 9′ 10′ 11′
36.6, 97.3, 44.5, 32.0,
1.87c
37.9, 97.6, 44.1, 32.8,
12′
16.0, CH3
13′ 14′ 15′ 16′ 17′ 18′ 19′ 20′ 21′ 22′
29.3, CH3 22.4, CH3 16.9, CH3 111.6, C 152.4, C 105.8, CH 141.0, C 117.0, C 159.9, C 69.7, CH2
23′
77.6, CH
1′′ 2′′
171.0,C 21.0, CH3
CH C C CH2
3.19, d (16.4) 2.87, d (16.4) 0.81, d (6.5) 1.09, s 0.92, s 1.07, s 6.08, s
4.93, d (14.2) 4.47, d (14.2) 5.21, d (3.8) 2.10, s
CH C C CH2
16.0, CH3 28.3, CH3 22.4, CH3 17.5, CH3 111.2, C 152.6, C 106.3, CH 141.9, C 115.2, C 160.3, C 69.3, CH2
77.5, CH
2.16,m 1.50c; 1.57c 1.51c; 1.59c 1.83c 3.22, d (16.4) 2.79c 0.74, d (6.5) 1.04, s 0.89, s 1.06, s 6.50, s
4.53, m 6.01, s 1.47c 1.79c 4.08, m 4.94, d (2.8)
31.8, CH2
1.52c; 1.61c
37.7, 97.8, 44.2, 32.6,
1.87c
CH C C CH2
16.1, CH3 28.4, CH3 22.1, CH3 17.3, CH3 112.5, C 154.4, C 106.7, CH 138.5, C 107.6, C 159.1, C 70.8, CH 93.7, CH 35.3, CH2
21.3, CH2
1.49c; 1.55c
31.8, CH2
1.52c; 1.61c
37.8, 97.8, 43.4, 32.7,
CH C C CH2
1.86c
d
15.9, CH3
s s s
28.3, CH3 22.4, CH3 17.5, CH3 111.4, C 152.6, C 106.4, CH 141.5, C 115.7, C 160.2, C 69.3, CH2
0.75, (6.4) 0.94, 1.01, 1.12,
6.08, s
4.63, d (15.2) 4.56, d (15.2) 5.37, d (2.5)
77.2, CH 171.2, C 21.0,CH3
δH (J in Hz)
δC, type 172.0, C 21.6, CH3
3 δH (J in Hz) 2.34, s
δC, type
δH (J in Hz)
171.9, C 21.5, CH3
2.31, s
Overlapped signals.
3.2. Fungal material, fermentation, extraction and isolation The fungal strain S. chartarum CGMCC 3.5365 was purchased from CGMCC (Beijing, PR China). The fermentation, extraction, and isolation of broth and mycelia were performed as described previously (Zhao et al., 2017a, 2017b). The EtOAc extract (137.0 g) was initially separated on silica gel CC eluting with a petroleum ether–acetone gradient (100:0, 5:1, 3:1, 1:1, 1:3, 1:5, and 0:100) as well as 100 % methanol to give nine fractions (Fr1-Fr9) based on TLC analysis. Then, each fraction was analysed by the means of HPLC-MS and the results showed that fraction 6 contained phenylspirodrimane dimers. Fraction 6 (9.7 g) was firstly subjected to silica gel CC eluting with a petroleum ether–acetone gradient (10:1, 6:1, 5:1, 4:1, 3:1, 2:1, 10:1, 1:1, 1:2 and 0:100) to obtain ten fractions (Fr6.1–Fr6.10). Fr6.8 (2.0 g) was then fractionated on Sephadex LH-20 CC with methanol to generate nine fractions (Fr6.8.1–Fr6.8.9). Fr6.8.6 (1.26 g) was further purified on gel CC to generate six fractions (Fr6.8.6.1–Fr6.8.6.6). Fr6.8.6.4 (400 mg) and Fr6.8.6.5 (430 mg) were separated through normal phase semi-preparative HPLC eluting with n-hexane–isopropanol (6:1, v/v) at 4 mL/min to yield fractions (Fr6.8.6.4.1–Fr6.8.6.4.6) and (Fr6.8.6.5.1–Fr6.8.6.5.6), respectively. Fr6.8.6.4.3 (58 mg) and Fr6.8.6.5.3 (80 mg) were together submitted to reverse phase semi-preparative HPLC eluting with CH3CN–H2O (72:28, v/v) at 4 mL/min to yield 1 (1.1 mg, tR =18.3 min), 2 (1.1 mg, tR =10.4 min), and 3 (21.4 mg, tR =35.4 min).
4.62, m 6.08, s 1.49c 1.79, dd (12.3, 12.3)
2.03c
3.26, d (16.4) 2.82c
c
6.55, s
38.8, C 42.1, CH
2
3′′ 4′′
3.22, d (16.3) 2.86c 0.73, d (6.4) 0.91, s 0.99, s 1.10, s
4.08, m 4.93,d (2.6)
1.48c; 1.55c 1.51c; 1.61c 1.85c
δC, type
2.10c 1.50c; 1.58c
65.1, CH 80.3, CH
2.06c
0.72, (6.5) 0.93, 1.01, 1.12,
39.0, C 41.4, CH 21.5, CH2
1
3.2.1. Bistachybotrysin W (1) White amorphous powder; [α]25 D +36.4, (c 0.22, MeOH); UV (MeOH) λmax (log ε): 213 (4.22) and 287 (3.65) nm; ECD (MeOH) λmax (Δε): 202.5 (–21.72), 221 (31.85), and 284.5 (3.36) nm; IR (vmax): 3396, 2938, 1714, 1623, 1455, 1260, and 1044 cm–1; 1H and 13C NMR spectroscopic data, see Table 6; HR-ESI-MS: m/z 833.4464 [M+H]+ (calcd for C48H65O12, 833.4471). 3.2.2. Bistachybotrysin X (2) White amorphous powder; [α]25 D +80, (c 0.22, MeOH); UV (MeOH) λmax (log ε): 213 (4.86) and 287 (3.84) nm; ECD (MeOH) λmax (Δε): 202.5 (–26.36), 222.5 (47.82), and 284.5 (8.48) nm; IR (vmax): 3368, 2936, 1714, 1623, 1457, 1259, and 1045 cm–1; 1H and 13C NMR spectroscopic data, see Table 6; HR-ESI-MS: m/z 833.4446 [M+H]+ (calcd for C48H65O12, 833.4471).
3.28, d (16.2) 2.83c d s s s
3.2.3. Bistachybotrysin Y (3) White amorphous powder; [α]25 +120, (c 0.48, MeOH); UV D (MeOH) λmax (log ε): 213 (4.78) and 287 (3.76) nm; ECD (MeOH) λmax (Δε): 205 (–30.8), 223 (53.7), and 284 (8.37) nm; IR (vmax): 3389, 2935, 1739, 1714, 1626, 1456, 1258, and 1047 cm–1; 1H and 13C NMR spectroscopic data, see Table 6; HR-ESI-MS: m/z 875.4551 [M+H]+ (calcd for C50H67O13, 875.4576).
6.11, s
4.72, d (14.9) 4.49, d (14.9) 5.31, d (3.1)
3.3. Cytotoxicity bioassay Cytotoxicity of the compounds against five human tumor cell lines (colorectal carcinoma HCT116, lung carcinoma NCI-H460, gastric carcinoma BGC823, medulloblastoma Daoy, and liver carcinoma HepG2)
2.12, s
75
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Fig. 2. The 1H―1H COSY ( of compounds 1−3.
) and HMBC (
) correlations
Fig. 3. The NOESY correlations of compounds 1−3.
Major Project (No. 2018ZX09711001-001-001).
Table 2 Cytotoxicity of compounds 1―3. Compounds
1 2 3 paclitaxel
Appendix A. Supplementary data
Cytotoxicity IC50 (μM) HCT116
NCI-H460
BGC823
Daoy
HepG2
12.1 22.6 5.9 3.8 nM
11.5 10.8 13.0 0.4 nM
13.2 15.1 14.1 2.0 nM
8.8 21.5 6.4 0.2 nM
7.0 11.5 9.8 10.2 nM
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.11.011. References Carmichael, J., Degraff, W.G., Gazdar, A.F., Minna, J.D., Mitchell, J.B., 1987. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 47, 936–943. Chunyu, W.X., Ding, Z.G., Li, M.G., Zhao, J.Y., Gu, S.J., Gao, Y., Wang, F., Ding, J.H., Wen, M.L., 2016. Stachartins A–E, phenylspirodrimanes from the tin mine tailings–associated fungus Stachybotrys chartarum. Helv. Chim. Acta 99, 583–587. https://doi.org/10.1002/hlca.201600020. Feng, J., Zhang, M., Jia, X., Zhao, J., Chen, R., Xie, K., Chen, D., Li, Y., Liu, J., Dai, J., 2019. Bistachybotrysins F−J, five new phenylspirodrimane dimers with a central cyclopentanone linkage from Stachybotrys chartarum. Fitoterapia 136, 104158. https://doi.org/10.1016/j.fitote.2019.04.013. Kim, J.W., Ko, S.K., Kim, H.M., Kim, J.G.H., Son, S., Kim, G.S., Hwang, G.J., Jeon, E.S., Shin, K.S., Ryoo, I.J., Hong, Y.S., Oh, H., Lee, K.H., Soung, N.K., Hashizume, D., Nogawa, T., Takahashi, S., Kim, B.Y., Osada, H., Jang, J.H., Ahn, J.S., 2016. Stachybotrysin, an osteoclast differentiation inhibitor from the marine-derived fungus Stachybotrys sp. KCB13F013. J. Nat. Prod. 79, 2703–2708. https://doi.org/10. 1021/acs.jnatprod.6b00641. Ma, X., Li, L., Zhu, T., Ba, M., Li, G., Gu, Q., Guo, Y., Li, D., 2013. Phenylspirodrimanes with anti-HIV activity from the sponge-derived fungus Stachybotrys chartarum MXHX73. J. Nat. Prod. 76, 2298–2306. https://doi.org/10.1021/np400683h. Ramana, C.V., Reddy, C.N., Gonnade, R.G., 2008. An expeditious one-step entry to the tetracyclic core of integrastatins. Chem. Commum. 27, 3151–3153. https://doi.org/ 10.1039/B801755G. Wang, A., Xu, Y., Gao, Y., Huang, Q., Luo, X., An, H., Dong, J., 2015. Chemical and bioactive diversities of the genera Stachybotrys and Memnoniella secondary metabolites. Phytochem. Rev. 45, 623–655. https://doi.org/10.1007/s11101-014-9365-1. Zhao, J., Feng, J., Tan, Z., Liu, J., Zhao, J., Chen, R., Xie, K., Zhang, D., Li, Y., Yu, L., Chen, X., Dai, J., 2017a. Stachybotrysins A–G, phenylspirodrimane derivatives from the fungus Stachybotrys chartarum. J. Nat. Prod. 80, 1819–1826. https://doi.org/10. 1021/acs.jnatprod.7b00014. Zhao, J., Liu, J., Shen, Y., Tan, Z., Zhang, M., Chen, R., Zhao, J., Zhang, D., Yu, L., Dai, J., 2017b. Stachybotrysams A–E, prenylated isoindolinone derivatives with anti-HIV activity from the fungus Stachybotrys chartarum. Phytochem. Lett. 20, 289–294. https://doi.org/10.1016/j.phytol.2017.04.031.
was measured using the MTT assay (Carmichael et al., 1987). The cells were maintained in a RRMI S7 1640 medium supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, and 100 g/mL streptomycin. Cultures were incubated at 37 °C in a humidified atmosphere of 5 % CO2. Tumor cells were seeded in 96-well microliter plates at 1200 cells per well. After 24 h, compounds were added to the wells. After incubation for 96 h, cell viability was determined by measuring the metabolic conversion of MTT into purple formazan crystals by viable cells. The MTT assay results were read using an MK3 Wellscan (Labsystem Dragon, Helsinki, Finland) plate reader at 570 nm. All compounds were tested at five concentrations (10―4, 10―5, 10―6, 10―7, and 10―8 M) in 100 % DMSO with a final concentration of DMSO of 0.1 % (v/v) in each well. Paclitaxel was used as a positive control. IC50 values were calculated using Microsoft Excel software. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements This work was financially supported by National Natural Science Foundation of China (No. 81803403), CAMS Innovation Fund for Medical Sciences (No. CIFMS-2018-I2M-3-005), the Drug Innovation
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J. Liu, et al. Zhao, J., Feng, J., Tan, Z., Liu, J., Zhang, M., Chen, R., Xie, K., Chen, D., Li, Y., Chen, X., Dai, J., 2018. Bistachybotrysins A–C, three phenylspirodrimane dimers with cytotoxicity from Stachybotrys chartarum. Bioorg. Med. Chem. Lett. 28, 355–359. https:// doi.org/10.1016/j.bmcl.2017.12.039. Zhang, H., Yang, M.H., Zhuo, F.F., Gao, N., Cheng, X.B., Wang, X.B., Pei, Y.H., Kong, L.Y., 2019a. Seven new cytotoxic phenylspirodrimane derivatives from the endophytic
fungus Stachybotrys chartarum. RSC Adv. 9, 3520–3531. https://doi.org/10.1039/ c8ra10195g. Zhang, M., Feng, J., Jia, X., Zhao, J., Liu, J., Chen, R., Xie, K., Chen, D., Li, Y., Zhang, D., Dai, J., 2019b. Bistachybotrysins D and E, one stereoisomeric pair of cytotoxic phenylspirodrimane dimers from Stachybotrys chartarum. Chin. Chem. Lett. 30, 435–438. https://doi.org/10.1016/j.cclet.2018.04.031.
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Bistachybotrysins W-Y, three new phenylspirodrimane dimers with a 6/7 oxygen heterocycle from Stachybotrys chartarum
T
Jimei Liua,b,c,1, Jiamin Fenga,b,c,1, Xiaona Jiaa,b,c, Jinlian Zhaoa,b,c, Ridao Chena,b,c, Kebo Xiea,b,c, Dawei Chena,b,c, Yan Lia,b,c, Jungui Daia,b,c,* a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China b CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China c NHC Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China
ARTICLE INFO
ABSTRACT
Keywords: Bistachybotrysin Phenylspirodrimane dimer Stachybotrys chartarum Cytotoxicity
Bistachybotrysins W-Y (1–3), three new phenylspirodrimane dimers with a 6/7 oxygen heterocycle as the linkage, were isolated from Stachybotrys chartarum CGMCC 3.5365. The structures of 1–3 were elucidated through extensive spectroscopic data analysis, including 1D/2D NMR, HR-ESI-MS, and ECD spectra. Compounds 1 and 3 displayed moderate cytotoxic activity against human tumor cell lines HCT116, Daoy, and HepG2 with IC50 values ranging from 5.9 to 9.8 μM.
1. Introduction Stachybotrys are known for the production of a variety of secondary metabolites including trichothecene mycotoxins, diterpenes, phenylspirodrimanes, and the prenylated phenol derivatives (Wang et al., 2015). Among of them, phenylspirodrimanes were discovered as the major class of secondary metabolites produced by this fungus, exhibiting a wide range of bioactivities including anticomplement and antiviral effects, endothelin receptor antagonism, and antibacterial, anti-inflammatory, and cytotoxic activities (Ma et al., 2013; Wang et al., 2015; Chunyu et al., 2016; Kim et al., 2016; Zhang et al., 2019a). Our previous chemical investigation for the S. chartarum CGMCC 3.5365 (China General Microbiological Culture Collection Center, CGMCC) have led to identification of several new monomeric phenylspirodrimanes (Zhao et al., 2017a) and rare dimeric phenylspirodrimanes (Zhao et al., 2018; Zhang et al., 2019b; Feng et al., 2019). Further biological studies revealed most of phenylspirodrimane dimers exhibited significant cytotoxicity against the human tumor cells (Zhao et al., 2018; Zhang et al., 2019b; Feng et al., 2019). As part of our ongoing search for structurally novel phenylspirodrimane dimers with potent biological activities, a recent chemical investigation on this fungal strain has contributed to the isolation of bistachybotrysins W-Y
(1-3) (Fig. 1), three new members for phenylspirodrimane dimer family. Herein, we report the isolation, structural elucidation, and biological activities of the three phenylspirodrimane dimers. 2. Results and discussion The fermentation, extraction, and isolation of broth and mycelia of the fungal strain S. chartarum were performed as described previously (Zhao et al., 2017a, 2017b). The EtOAc extract (137.0 g) was subjected to silica gel column chromatography (CC), Sephadex LH-20 CC, and semipreparative HPLC to afford 1 (1.1 mg), 2 (1.1 mg), and 3 (21.4 mg), respectively. Bistachybotrysin W (1) was obtained as a white amorphous powder. It displayed an HR-ESI-MS ion peak at m/z 833.4464 [M+H]+ (calcd for C48H65O12, 833.4471), consistent with a molecular formula of C48H64O12 with 17 degrees of unsaturation. The IR spectrum showed characteristic absorption for hydroxyl (3396 cm–1) and carbonyl (1714 cm–1), and the UV spectrum displayed maximum absorptions at 213 and 287 nm. In the 1H NMR spectrum (Table 1), the clearly recognized signals were assigned for two sets of typical phenylspirodrimane, including four pairs of methyl resonances (H3-12/12′, H3-13/13′, H3-14/ 14′, and H3-15/15′), one pair of aromatic singlet protons (H-18/18′),
Corresponding author at: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China. E-mail address: [email protected] (J. Dai). 1 These authors made equal contributions to this work. ⁎
https://doi.org/10.1016/j.phytol.2019.11.011 Received 2 September 2019; Received in revised form 10 November 2019; Accepted 13 November 2019 1874-3900/ © 2019 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
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Fig. 1. Structures of compounds 1–3.
one pair of methylene protons (H2-11/11′), and two pairs of oxygenated methine protons (H-2/H-2′ and H-3/3′). From 13C NMR and DEPT spectroscopic data (Table 1), a total of 48 carbon resonances were assigned, most of which appeared in pairs except signals at δC 93.8 (C-23), δC 77.6 (C-23′), δC 71.1 (C-22), δC 69.7 (C-22′), δC 171.0 (C-1′′), and 21.0 (C-2′′). Considering the previously reported phenylspirodrimane dimers from this fungal strain, the above spectroscopic data indicated that 1 was a dimeric structure with two similar phenylspirodrimanes (unit A and unit B). Further analysis of the 1D and 2D NMR spectra demonstrated that 1 shared the same backbone with reported bistachybotrysins A–C (Zhao et al., 2018). And the general spectroscopic features (Table 1) were similar to those of bistachybotrysin C except for the presence of an additional acetyl group (δH-2′′ 2.10/δC-2′′ 21.0, δC-1′′ 171.0), the HMBC correlation from H-3 to C-1′′ (Fig. 2) as well as the downfield shift of H-3 (δH 3.32→δH 4.83) unambiguously established the attachment of acetyl group at C-3. Therefore, 1 was identified as an acetyl derivative of bistachybotrysin C. The relative configuration of 1 was deduced by 1D-NOE correlations (Figs. 3 and S7). The NOE correlations of H-2/H-3, H3-15, and H3-14; H3-14/H3-15, H-2, and H-3; H3-13/H-5; H3-15/H-8 and H-11 in unit A, as well as H-2′/H-3′, H3-15′, and H3-14′; H3-15′/H3-14′, H-8′, and H-11′; H-5′/H3-13′ in unit B, together with the small coupling constant of JH2,H-3 and JH-2′,H-3′, indicating that both units A and B of 1 possessed same relative configuration as those of bistachybotrysins A–J (Zhao et al., 2018; Zhang et al., 2019b; Feng et al., 2019). In addition, signal enhancement of H-22 and H-23 were observed after irradiation of H23′, suggesting H-22, H-23, and H-23′ in unit C had the same orientation. The small coupling constant of JH-22/H-23′ (3.8 Hz) further confirmed the cis-orientation of H-22 and H-23′ (Ramana et al., 2008), which was in great agreement with the orientation of that in bistachybotrysins A–C (Zhao et al., 2018). From the biosynthetic standpoint, the bistachybotrysin C was synthesized through the intact incorporation of two monomers (mer-NF5003B and K-76) and maintained their natural absolute configuration (Zhao et al., 2018). Thus, the unit A and unit B of 1 might have the same absolute configuration at C-2, C-3, C-5, C-8, C-9, and C-10 as mer-NF5003B and K-76. Furthermore, comparison of the ECD spectrum revealed that 1 has similar Cotton effects [202.5 (–21.72), 221 (31.85), and 284.5 (3.36) nm, Fig. S11] to those of bistachybotrysins A–C, thus the absolute configuration of 1 was determined as 2R, 3S, 5S, 8R, 9R, 10S, 2′R, 3′S, 5′S, 8′R, 9′R, 10′S, 22S, 23S, and 23′S. Compounds 2 and 3 were isolated as white amorphous powders. Their molecular formulas were deduced as C48H64O12 and C50H66O13 from the HR-ESI-MS ion peaks at m/z 833.4446 [M+H]+ (calcd for C48H65O12, 833.4471) and 875.4551 [M+H]+ (calcd for C50H67O13, 875.4576), respectively. Their IR, UV, 1H, and 13C NMR spectroscopic data (Table 1) showed close similarity to those of 1. In compound 2, the general features of its 1H and 13C NMR spectroscopic data closely resembled those of 1 except the different chemical shifts for H-3, C-3, and H-3′. Analysis of DEPT, 1H−1H COSY, and HMBC correlations suggested that the acetyl group was attached at C-3′ instead of C-3 in 2, which was further supported by the downfield shift of H-3′ (δH 3.40→
δH 4.94). For 3, the only difference from 1 was the observation of an additional acetyl group (δC-3′′ 171.9, δC-4′′ 21.5/δH-2′′ 2.31), and the downfield shift of H-3′ (δH 3.39→δH 4.93) as well as the HMBC correlation of H-3′/C-3′′ (Fig. 2), indicating the another acetyl group attaching at C-3′. Moreover, 2 and 3 were all assigned to have the same 2R, 3S, 5S, 8R, 9R, 10S, 22S, 23S, 23′S, 2′R, 3′S, 5′S, 8′R, 9′R, and 10′S configurations as 1, due to their consistent NOEs (Figs. 3, S18, and S29) and similar ECD Cotton effects (Figs. S17 and S25). To the best of our knowledge, there are only three members in phenylspirodrimane dimer family with 6/7 oxygen heterocycle linkage reported so far, including bistachybotrysins A–C (Zhao et al., 2018). From a biosynthetic standpoint, compounds 1-3 were proposed to have the same biosynthetic pathway as we have reported before (Zhao et al., 2018), and the downstream regio-selective acetylation greatly enriches the diversity of structures. Compounds 1–3 were evaluated for in vitro cytotoxicity (paclitaxel as the positive control) against five human tumor cell lines (colorectal carcinoma HCT116, lung carcinoma NCI-H460, gastric carcinoma BGC823, medulloblastoma Daoy, and liver carcinoma HepG2). Compounds 1 and 3 displayed selective cytotoxicity against the HCT116, Daoy, and HepG2 cell lines with IC50 values ranging from 5.9 to 9.8 μM (Table 2). 3. Experimental section 3.1. General experimental procedures Optical rotations were measured on a Perkin-Elmer Model-343 digital polarimeter (PerkinElmer Inc., Waltham, Massachusetts, USA). The ECD and UV absorption spectra were recorded on a Jasco J-815 spectropolarimeter (Jasco Corporation, Tokyo, Japan). IR spectra were acquired on a Nicolet 5700 FT-IR microscope spectrometer (FTIR Microscope Transmission, Thermo Electron Scientific Instrument Crop., Madison, Wisconsin, USA). 1D/2D NMR spectra were obtained at 600 MHz for 1H NMR and 150 MHz for 13C NMR on VNOVA SYSTEM-600 spectrometer (Varian Inc., Palo Alto, California, USA). Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in Hertz (Hz). HR-ESI-MS data were measured using an LTQ-FT Ultra ESI-FTICR-MS spectrometer (ThermoFisher Scientific, CA, USA). Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, PR China) and Sephadex LH-20 gel (Amersham Biosciences, Sweden) were used for column chromatography (CC). Semi-preparative HPLC was performed on a Shimadzu HPLC instrument equipped with a Shimadzu RID-10A detector (Shimadzu Corporation, Tokyo, Japan) and a Shiseido Capcell Pak C18 MG II S5 column (250 mm × 10 mm, i.d., 5 μm, Shiseido Co., Tokyo, Japan) or a grace Allsphere silica gel column (250 mm × 10 mm, i.d., 5 μm, W.R. Grace Co., Columbia, Maryland, USA). Analytical TLC was carried out on pre-coated silica gel GF254 plates (Qingdao Marine Chemical Industry, Qingdao, China), and spots were visualized under UV light or by spraying with 10 % H2SO4 in 90 % EtOH followed by heating at 120 °C. 74
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Table 1 1 H (600 MHz) and No.
1
13
Table 1 (continued)
C (150 MHz) NMR data of 1–3 in acetone-d6.
1
2
No.
3
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
34.9, CH2
1.37, dd (12.1, 4.3) 1.83c 4.04, m 4.83, d (2.2)
34.0, CH2
1.31c 1.77c
34.9, CH2
3.96, m 3.33, brs
65.3, CH 80.6, CH
1.40, dd (12.0, 4.2) 1.85c 4.04, m 4.87, d (2.3)
2 3
65.3, CH 80.7, CH
4 5 6
38.9, C 41.2, CH 21.3, CH2
7
66.4, CH 79.2, CH
2.12c 1.50c; 1.56c
38.9, C 39.9, CH 21.5, CH2
31.9, CH2
1.52c; 1.61c
31.9, CH2
8 9 10 11
37.4, 97.7, 44.3, 32.4,
1.84c
37.5, 97.7, 44.1, 32.4,
12
15.9, CH3
13 14 15 16 17 18 19 20 21 22 23 1′
28.5, CH3 22.0, CH3 17.4, CH3 111.4, C 154.2, C 106.1, CH 139.3, C 109.5, C 158.3, C 71.1, CH 93.8, CH 34.3, CH2
2′ 3′
66.3, CH 80.2, CH
4′ 5′
38.8, C 39.8, CH
6′
CH C C CH2
3.22, d (16.5) 2.84c 0.75, d (6.5) 0.90, s 0.99, s 1.10, s 6.61, s
4.62, m 6.01, s 1.31c 1.68, dd (12.2, 12.2) 3.94, m 3.39, brs
CH C C CH2
16.1, CH3 29.2, CH3 22.5, CH3 17.4, CH3 112.8, C 154.3, C 107.6, CH 138.7, C 106.7, C 159.4, C 70.0, CH 93.5, CH 35.3, CH2
65.2, CH 80.3, CH 38.9, C 42.0, CH
21.6, CH2
2.32, dd (12.3, 2.1) 1.48c; 1.55c
21.5, CH2
7′
32.2, CH2
1.52c; 1.61c
31.7, CH2
8′ 9′ 10′ 11′
36.6, 97.3, 44.5, 32.0,
1.87c
37.9, 97.6, 44.1, 32.8,
12′
16.0, CH3
13′ 14′ 15′ 16′ 17′ 18′ 19′ 20′ 21′ 22′
29.3, CH3 22.4, CH3 16.9, CH3 111.6, C 152.4, C 105.8, CH 141.0, C 117.0, C 159.9, C 69.7, CH2
23′
77.6, CH
1′′ 2′′
171.0,C 21.0, CH3
CH C C CH2
3.19, d (16.4) 2.87, d (16.4) 0.81, d (6.5) 1.09, s 0.92, s 1.07, s 6.08, s
4.93, d (14.2) 4.47, d (14.2) 5.21, d (3.8) 2.10, s
CH C C CH2
16.0, CH3 28.3, CH3 22.4, CH3 17.5, CH3 111.2, C 152.6, C 106.3, CH 141.9, C 115.2, C 160.3, C 69.3, CH2
77.5, CH
2.16,m 1.50c; 1.57c 1.51c; 1.59c 1.83c 3.22, d (16.4) 2.79c 0.74, d (6.5) 1.04, s 0.89, s 1.06, s 6.50, s
4.53, m 6.01, s 1.47c 1.79c 4.08, m 4.94, d (2.8)
31.8, CH2
1.52c; 1.61c
37.7, 97.8, 44.2, 32.6,
1.87c
CH C C CH2
16.1, CH3 28.4, CH3 22.1, CH3 17.3, CH3 112.5, C 154.4, C 106.7, CH 138.5, C 107.6, C 159.1, C 70.8, CH 93.7, CH 35.3, CH2
21.3, CH2
1.49c; 1.55c
31.8, CH2
1.52c; 1.61c
37.8, 97.8, 43.4, 32.7,
CH C C CH2
1.86c
d
15.9, CH3
s s s
28.3, CH3 22.4, CH3 17.5, CH3 111.4, C 152.6, C 106.4, CH 141.5, C 115.7, C 160.2, C 69.3, CH2
0.75, (6.4) 0.94, 1.01, 1.12,
6.08, s
4.63, d (15.2) 4.56, d (15.2) 5.37, d (2.5)
77.2, CH 171.2, C 21.0,CH3
δH (J in Hz)
δC, type 172.0, C 21.6, CH3
3 δH (J in Hz) 2.34, s
δC, type
δH (J in Hz)
171.9, C 21.5, CH3
2.31, s
Overlapped signals.
3.2. Fungal material, fermentation, extraction and isolation The fungal strain S. chartarum CGMCC 3.5365 was purchased from CGMCC (Beijing, PR China). The fermentation, extraction, and isolation of broth and mycelia were performed as described previously (Zhao et al., 2017a, 2017b). The EtOAc extract (137.0 g) was initially separated on silica gel CC eluting with a petroleum ether–acetone gradient (100:0, 5:1, 3:1, 1:1, 1:3, 1:5, and 0:100) as well as 100 % methanol to give nine fractions (Fr1-Fr9) based on TLC analysis. Then, each fraction was analysed by the means of HPLC-MS and the results showed that fraction 6 contained phenylspirodrimane dimers. Fraction 6 (9.7 g) was firstly subjected to silica gel CC eluting with a petroleum ether–acetone gradient (10:1, 6:1, 5:1, 4:1, 3:1, 2:1, 10:1, 1:1, 1:2 and 0:100) to obtain ten fractions (Fr6.1–Fr6.10). Fr6.8 (2.0 g) was then fractionated on Sephadex LH-20 CC with methanol to generate nine fractions (Fr6.8.1–Fr6.8.9). Fr6.8.6 (1.26 g) was further purified on gel CC to generate six fractions (Fr6.8.6.1–Fr6.8.6.6). Fr6.8.6.4 (400 mg) and Fr6.8.6.5 (430 mg) were separated through normal phase semi-preparative HPLC eluting with n-hexane–isopropanol (6:1, v/v) at 4 mL/min to yield fractions (Fr6.8.6.4.1–Fr6.8.6.4.6) and (Fr6.8.6.5.1–Fr6.8.6.5.6), respectively. Fr6.8.6.4.3 (58 mg) and Fr6.8.6.5.3 (80 mg) were together submitted to reverse phase semi-preparative HPLC eluting with CH3CN–H2O (72:28, v/v) at 4 mL/min to yield 1 (1.1 mg, tR =18.3 min), 2 (1.1 mg, tR =10.4 min), and 3 (21.4 mg, tR =35.4 min).
4.62, m 6.08, s 1.49c 1.79, dd (12.3, 12.3)
2.03c
3.26, d (16.4) 2.82c
c
6.55, s
38.8, C 42.1, CH
2
3′′ 4′′
3.22, d (16.3) 2.86c 0.73, d (6.4) 0.91, s 0.99, s 1.10, s
4.08, m 4.93,d (2.6)
1.48c; 1.55c 1.51c; 1.61c 1.85c
δC, type
2.10c 1.50c; 1.58c
65.1, CH 80.3, CH
2.06c
0.72, (6.5) 0.93, 1.01, 1.12,
39.0, C 41.4, CH 21.5, CH2
1
3.2.1. Bistachybotrysin W (1) White amorphous powder; [α]25 D +36.4, (c 0.22, MeOH); UV (MeOH) λmax (log ε): 213 (4.22) and 287 (3.65) nm; ECD (MeOH) λmax (Δε): 202.5 (–21.72), 221 (31.85), and 284.5 (3.36) nm; IR (vmax): 3396, 2938, 1714, 1623, 1455, 1260, and 1044 cm–1; 1H and 13C NMR spectroscopic data, see Table 6; HR-ESI-MS: m/z 833.4464 [M+H]+ (calcd for C48H65O12, 833.4471). 3.2.2. Bistachybotrysin X (2) White amorphous powder; [α]25 D +80, (c 0.22, MeOH); UV (MeOH) λmax (log ε): 213 (4.86) and 287 (3.84) nm; ECD (MeOH) λmax (Δε): 202.5 (–26.36), 222.5 (47.82), and 284.5 (8.48) nm; IR (vmax): 3368, 2936, 1714, 1623, 1457, 1259, and 1045 cm–1; 1H and 13C NMR spectroscopic data, see Table 6; HR-ESI-MS: m/z 833.4446 [M+H]+ (calcd for C48H65O12, 833.4471).
3.28, d (16.2) 2.83c d s s s
3.2.3. Bistachybotrysin Y (3) White amorphous powder; [α]25 +120, (c 0.48, MeOH); UV D (MeOH) λmax (log ε): 213 (4.78) and 287 (3.76) nm; ECD (MeOH) λmax (Δε): 205 (–30.8), 223 (53.7), and 284 (8.37) nm; IR (vmax): 3389, 2935, 1739, 1714, 1626, 1456, 1258, and 1047 cm–1; 1H and 13C NMR spectroscopic data, see Table 6; HR-ESI-MS: m/z 875.4551 [M+H]+ (calcd for C50H67O13, 875.4576).
6.11, s
4.72, d (14.9) 4.49, d (14.9) 5.31, d (3.1)
3.3. Cytotoxicity bioassay Cytotoxicity of the compounds against five human tumor cell lines (colorectal carcinoma HCT116, lung carcinoma NCI-H460, gastric carcinoma BGC823, medulloblastoma Daoy, and liver carcinoma HepG2)
2.12, s
75
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Fig. 2. The 1H―1H COSY ( of compounds 1−3.
) and HMBC (
) correlations
Fig. 3. The NOESY correlations of compounds 1−3.
Major Project (No. 2018ZX09711001-001-001).
Table 2 Cytotoxicity of compounds 1―3. Compounds
1 2 3 paclitaxel
Appendix A. Supplementary data
Cytotoxicity IC50 (μM) HCT116
NCI-H460
BGC823
Daoy
HepG2
12.1 22.6 5.9 3.8 nM
11.5 10.8 13.0 0.4 nM
13.2 15.1 14.1 2.0 nM
8.8 21.5 6.4 0.2 nM
7.0 11.5 9.8 10.2 nM
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was measured using the MTT assay (Carmichael et al., 1987). The cells were maintained in a RRMI S7 1640 medium supplemented with 10 % (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, and 100 g/mL streptomycin. Cultures were incubated at 37 °C in a humidified atmosphere of 5 % CO2. Tumor cells were seeded in 96-well microliter plates at 1200 cells per well. After 24 h, compounds were added to the wells. After incubation for 96 h, cell viability was determined by measuring the metabolic conversion of MTT into purple formazan crystals by viable cells. The MTT assay results were read using an MK3 Wellscan (Labsystem Dragon, Helsinki, Finland) plate reader at 570 nm. All compounds were tested at five concentrations (10―4, 10―5, 10―6, 10―7, and 10―8 M) in 100 % DMSO with a final concentration of DMSO of 0.1 % (v/v) in each well. Paclitaxel was used as a positive control. IC50 values were calculated using Microsoft Excel software. Declaration of Competing Interest The authors declare no competing financial interest. Acknowledgements This work was financially supported by National Natural Science Foundation of China (No. 81803403), CAMS Innovation Fund for Medical Sciences (No. CIFMS-2018-I2M-3-005), the Drug Innovation
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