Accepted Manuscript Topoisomerase II inhibitors from the roots of Stellera ch amaejasme L Li-Ping Liu, Kun Han, Wei Chen, Ying-Ying Zhang, Lin-Jiang Tong, Ting Peng, Hua Xie, Jian Ding, Hong-Bing Wang PII: DOI: Reference:

S0968-0896(14)00400-3 http://dx.doi.org/10.1016/j.bmc.2014.05.042 BMC 11603

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

24 March 2014 19 May 2014 20 May 2014

Please cite this article as: Liu, L-P., Han, K., Chen, W., Zhang, Y-Y., Tong, L-J., Peng, T., Xie, H., Ding, J., Wang, H-B., Topoisomerase II inhibitors from the roots of Stellera ch amaejasme L, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.05.042

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Topoisomerase II inhibitors from the roots of Stellera chamaejasme L. Li-Ping Liu a†, Kun Hanb†, Wei Chena, Ying-Ying Zhanga, Lin-Jiang Tongb, Ting Pengb, Hua Xieb*, Jian Dingb*, Hong-Bing Wanga*

a

School of Life Science & Technology, Tongji University, Shanghai, 200092, P. R.

China b

Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China

*Corresponding Author. Tel.: +86 21 65983693; Fax: +86 21 65983693. E-mail address: [email protected] (H.-B. Wang); [email protected] (H. Xie); [email protected] (J. Ding) † These authors contributed equally to this work.

1

Abstract Three new compounds, including one daphnane diterpene (1), one sesquiterpene (6), and one lignan (7) have been isolated from the Stellera chamaejasme L., together with five other known compounds, including four daphnane diterpenenoids (2–5) and one lignan (8). The structures of the new compounds were elucidated by spectroscopic analysis. The cytotoxicities of compounds 1–8 towards human lung adenocarcinoma cells (A549 cells) were evaluated using a sulforhodamine B assay. All of the compounds displayed significant cytotoxicity, with IC50 values in the ranging of 0.2 nM to 2.0 µM. Mechanistic studies revealed that the antitumor activities of compounds 1–3 and 7 were derived from their inhibition of topoisomerase II (Topo II). Furthermore, as a Topo II inhibitor, compound 1 was found to effectively induced G2-M phase cell cycle arrest and apoptosis in cancer cells.

Keywords: Stellera chamaejasme L.; cytotoxic activity; topoisomerase II; apoptosis

2

1. Introduction Stellera chamaejasme L. is a toxic perennial herb belonging to the Thymelaeaceae family of flowering plants, and its root is used in traditional Chinese medicine, where it is known as “langdu”. The root of S. chamaejasme L. possesses several interesting biological effects, and has been reported as a therapeutic agent for the treatment of leucocythemia and stomach cancer.1Extracts of S. chamaejasme L. have also shown insecticidal activity towards some pests.2 Gnidimacrin, which is a daphnane-type diterpene, was isolated from the root of S. chamaejasme L. in 1995,3 and has been reported to exhibit significant levels of anticancer and anti-HIV-1 activity.3,4 Several studies have been conducted towards determining the chemical constituents in the root of S. chamaejasme L., which have led to the identification of several diterpenoids,5,6 biflavonoids,7-11 and lignans,12and these compounds have been shown to exhibit antitumor, antimalarial, and antibacterial activities. As part of our ongoing research towards the identification compounds with anticancer activity from Chinese medicinal plants, we became interested in investigating the chemical constituents present in the dried roots of S. chamaejasme L. Following the extraction of the plant roots with ethanol, we identified three new compounds, including one daphnane diterpene (1), one sesquiterpene (6), and one lignan (7), as well as five known compounds, including four daphnane diterpene (2–5) and one lignan (8) (Fig.1). The known compounds were identified as wikstroelide J (2),13huratoxin(3),1412β-acetoxy-huratoxin(4),15simplexin(5)16, and (–)-haplomyrfolin (8)17 through a comparison of their 1H and 13C NMR and mass spectra data with those 3

described in the literature. Herein, we describe our work towards the isolation and structural elucidation of the three unknown compounds isolated from the root of S. chamaejasme L, as well as an evaluation of their in vitro antitumor activities and their mechanism of action. 2. Results and discussion 2.1. Structure elucidation of new compounds Compound 1 was isolated as colorless oil, and its molecular formula was determined to be C34H50O9 by high-resolution electrospray ionization mass spectrometry (HRESIMS), which gave an m/z of 625.3338 for [M+Na]+ (calcd. for C34H50O9Na, 625.3353). The 1H and 13C NMR data of 1 (Table 1) were very similar to those of a recently reported daphnane-type diterpene that had been isolated from S. chamaejasme L.,6 which suggested that compound 1 contained a daphnane-type diterpene moiety and an unsaturated fatty acid with a diene system. Compound 1 was 14 mass units (i.e., a methylene unit) smaller than the previously reported daphnane-type diterpene compound, which suggested that the fatty acid unit in 1 was tetradecadienoic acid. Analysis of compound 1 by heteronuclear multiple-bond correlation spectroscopy (HMBC) suggested that its ester moiety was linked to the 13-hydroxyl group, which was confirmed through a comparison of the 13C NMR shift of C-13 with data from the literature (Fig. 2). The stereochemistry of the diene system was determined to be 2'E, 4'E, based on the large coupling constants (15Hz) between H-2' and H-3', and H-4' and H-5'. On the basis of these observations, as well as analysis of the 1H-1H COSY, heteronuclear single quantum correlation (HMQC), and 4

HMBC spectra and a comparison with known compounds from the same plant, the structure of 1 was determined to be that shown in Fig. 1. Compound 6 was isolated as a colorless oil, and its molecular formula was determined to be C15H22O3 by HRESIMS, which gave m/z peak of 250.1562 for [M]+ (calcd. for C15H22O3, 250.1570). The 1H and

13

C NMR spectroscopic data of 6 (Table

1) indicated the presence of three methyl groups, as well as four methylene (one oxygenated), three methane (one olefinic), and five quaternary (one olefinic and one carbonyl) carbons, which suggested that 6 was a guaiane-type sesquiterpene.18,19 A review of the literature revealed that the spectroscopic data for compound 6 were very similar to those of chamaejasmone A, which is guaiane-type sesquiterpene isolated from the same plant,20 except for the absence of carbonyl moiety at C-3. The degree of unsaturation of compound 6 was calculated and confirmed the absence of one of carbonyl group. The planar structure of 6 was confirmed by analysis of its 1H-1H COSY, HMBC (Fig. 2), and HMQC spectra. The NOESY spectrum of compound 6 revealed a correlation between H-5 and H-4, but no correlation between H-5 and the methyl group at C-4. These NOSEY data indicated that H-5 and the C-4 methyl were trans-orientated. Correlations were also observed in the ROESY spectrum between the methyl groups at C-10 and C-11 and the methylene moiety at C-11, which suggested that the methyl and methylene at C-11 were syn-oriented (Fig. 3). Based on these data, as well as the HMBC, HMQC and 1H-1H COSY spectra, the structure of compound 6 was determined to as shown in Fig. 1, and the material was named chamaejasmone D. 5

Compound 7 was isolated as a colorless oil, and its molecular formula was determined to be C20H22O5 by HRESIMS, which gave an m/z value of 365.1359 for [M+Na]+ (calcd. for C20H22O5Na, 365.1366). The 1H and 13C NMR data (Table 2) for compound 7 indicated that this material was a lignan. Analysis of the 1H-1H COSY, HMBC, and HMQC spectra of 7 allowed for all of the signals from the 1H and

13

C

NMR spectra to be fully assigned, and revealed that the structure of 7 was very similar to lariciresinol, which is a lignan that was isolated from Wikstroemia elliptica.21 In contrast to lariciresinol, the

13

C NMR spectrum of 7 contained a

methylene signal at δ 37.0 for C-9' as opposed to oxygenated methylene at a much lower field. Furthermore, the degree of unsaturation of compound 7 suggested that this material contained an extra degree of unsaturation compared with lariciresinol. The HMBC spectrum (Fig. 2) of compound 7 revealed that there were correlations between H-9' (δ 2.59, 2.72) and C-5 (δ 113.6) and C-1 (δ 132.4), which suggested that C-9' was a methylene unit located in a ring system and not a methylene unit bearing a free hydroxy group, as in lariciresinol. The NOESY spectrum of compound 7 showed a correlation between H-8 and H-8', but no correlation between H-7' and H-8/H-8', which indicated that H-8 and H-8' were in the α-orientation, whereas H-7' was β-oriented (Fig. 3). Consideration of the 1H-1 H COSY, HMBC, and HMQC spectra allowed for all of the signals in the 1H and

13

C NMR spectra of compound 7 to be

fully assigned. Taken together, these data indicated that the structure of 7 was as shown in Fig. 1, and the material was subsequently named as stelleralignan. 2.2. Inhibition of cell proliferation in vitro 6

The cytotoxicities of compounds 1–8 towards A549 human lung adenocarcinoma epithelial cells were evaluated using a sulforhodamine B (SRB) assay, which showed that all of the compounds displayed high levels of cytotoxicity with IC50 values in the range of 0.2 nM to 2.0 µM (Table 3). Notably, compounds 3–5 showed the most potent inhibitory activities, with IC50 values of less than 1.0 nM (Table 3). These results demonstrated that the compounds isolated from S. chamaejasme possessed potent anti-tumor activity. 2.3. Inhibitory activities towards topoisomerase I and topoisomerase II The inhibitory activities of compounds 1–7 towards Topo II were evaluated in an in vitro biochemical assay to develop a deeper understanding of the mechanism of action of their antitumor activities. The results of this assay showed that compounds1–3 and 7 exhibited significant inhibitory activity towards Topo II. As shown in Figure 4A, compared with the relaxation of a negatively supercoiled plasmid caused by the Topo II enzyme (i.e., the lane including no drug), treatment with compounds 1–3 and 7 resulted in the pBR322 plasmid existing predominantly in a supercoiled state. Furthermore, the effects of compounds 1–3 and 7 were much stronger than that of the positive control compound VP-16 at the same concentration. To further corroborate the effects of compounds 1–3 and 7 towards Topo II activity, we evaluated the activity of these compounds in another specific Topo II-mediated kDNA decatenation assay. As shown in Figure 4B, Topo II catalyzed the decatenation of kDNA to minicircles in the presence of ATP (lane 2 vs. lane 1). Treatment with compounds 1–3 and 7 resulted in all of the minicircles being pushed back into the 7

gelaperture at a concentration of 100 µM, provided further evidence that compounds 1–3 and 7 possessed Topo II inhibitory activity (Fig. 4B). To exclude the possibility that Topo I inhibition was also involved in the antitumor activity of these compounds, we also performed a Topo I-mediated supercoiled PBR322 relaxation assay both in the presence and in the absence of compounds 1–3 and 7. Camptothecin (CPT), which is a well known Topo I inhibitor, and H-1, which is a novel Topo I inhibitor identified in our lab (not published), were used as positive controls in this assay. The results revealed that all eight of the compounds showed no obvious effect on Topo I activity at concentrations up to 100 µM (Fig. 4C), which indicated that compounds 1–3 and 7 exhibited their antitumor activities through the inhibition of Topo II, and not Topo I. The mechanism of action for the antitumor activity of other compounds isolated in this work, including compounds 4, 5, 6 and 8, remains unclear and further studies will therefore be required to develop a deeper understanding of the way in which these compounds exert their antitumor activity. 2.4. Compounds 1 induced G2-M arrest and apoptosis in A549 cells Given that the occurrence of Topo II inhibition could result in cell cycle arrest and apoptosis, the decision was taken to further investigate the ability of these compounds to induce cell cycle arrest and apoptosis in A549 cancer cells, using flow cytometry and an Annexin-V-propidium iodide (PI) staining assay, respectively. As compound 1 was identified as one of the novel Topo II inhibitors possessing potent anti-tumor activity, it was selected for these experiments. The flow cytometry results showed that the treatment of A549 cells with compound 1 for 24 h lead to an increase in the 8

number of A549 cells occupying the G2-M phase (Fig. 5A). For example, treatment with 300 nM of compound 1 caused more than 50% of the cells to go into the G2 phase, compared with 18% in the untreated control group. Furthermore, the results of the Annexin-V and PI staining assay22 showed that the exposure of A549 cells to compound 1 for 48 h acted as a trigger for the cells to enter into an early apoptotic process (Fig. 5B). For example, 19.9% of the A549 cells were observed with well-characterized apoptosis following their treatment with 30 nM of compound 1, compared with 5.75% in the untreated control cells. These results therefore demonstrated that compound 1 exhibited its antitumor activity through the induction of apoptosis and G2-M cell cycle arrest. 3. Experimental 3.1. General experimental procedures NMR spectra were recorded on a Bruker AM-400 spectrometer using TMS as an internal reference standard. Low resolution electrospray ionization mass spectroscopy (LRESIMS) was conducted on a Finnian LCQ-DECA instrument, and HRESIMS data were obtained on Micromass LCT and Mariner

spectrometers. Purification by

column chromatography was performed on silica gel H-60 (Qingdao Haiyang Chemical Group Corporation, Qingdao, China), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), and LiChroprep RP-18 (40-63 µm, Merck). HSGF254 silica gel TLC plates (Yantai Chemical Industrial Institute, Yantai, China) were used for analytical TLC. 3.2. Plant material 9

The dried plant material was collected in the February of 2010 from the Qinhai Province, People’s Republic of China. This material was identified by Dr. Zhi-Bing Guan from the Yunnan Branch Institute of Medicinal Plants, Chinese Academy of Medical Sciences. Voucher specimens (No. 10-02-09) were collected and stored in the “Herbarium” of the Institute. 3.3. Extraction and isolation A 95% EtOH extract of the dried plant roots (5.0 kg) was sequentially extracted with petroleum ether, chloroform, ethyl acetate, and n-butanol. The CHCl3 extract (150 g) was applied to a silica gel column, which was subsequently eluted with CHCl3 containing increasing amounts of MeOH to give six fractions (fractions 1-6). Fraction 2 was applied to a silica gel column and eluted with a solvent mixture of CHCl3 and MeOH of increasing polarity (i.e., 50:1–1:1, v/v) to afford eight subfractions (fractions 2.1–2.8). Fraction 2.3 was purified over RP-18 eluting with MeOH containing increasing amounts of H2O (3:1–1:2, v/v), followed by purification over Sephadex LH-20 eluting with MeOH to give compounds 1 (8 mg) and 2 (10 mg). Fraction 2.4 was subjected to repeated column chromatography over silica gel, followed by sequential purification over Sephadex LH-20 eluting with MeOH and RP-18 eluting with a mixture of MeOH and H2O (5:1–1:1, v/v) to afford compounds 3 (12 mg), 4 (10 mg) and 5 (14 mg). Compounds 6 (5 mg), 7 (7 mg), and 8 (10 mg) were obtained from fraction 2.7 by repeated chromatographic purification over silica gel eluting with a mixture of CHCl3 and MeOH (20:1-1:1,v/v) followed by purification over Sephadex LH-20 eluting with MeOH. 10

3.4. Cell lines and culture Human lung adenocarcinoma cells (A549 cells) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). This cell line was maintained in F12 medium (GIBCO, Grand Island, NE, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO) at pH 7.4 in a humidified atmosphere of 95% air plus 5% CO2 at 37°C. 3.5. Cell proliferation assay Cell proliferation was evaluated using an SRB assay.23 Cells were seeded into 96-well plates and cultured over night at 37°C in a 5% CO2 incubator. The cells were then treated with compounds 1–8 for 72 h. The medium was removed immediately after the drug treatment (the suspended cells were span at 3,000 ×g for 10 min to allow for the medium to be carefully extracted), and replaced with 10% precooled trichloroacetic acid (TCA) (100 µL in each well). The cells were fixed for 1 h at 4 °C, and the plate was then washed five times with distilled water and dried. One-hundred microliters of a 4 mg/mL solution of SRB (Sigma, St. Louis, MO, USA) in 1% acetic acid were added to each well and the plate was incubated for 15 min. The plate was then washed five times with 1% acetic acid solution and dried. The SRB in the cells was subsequently dissolved in 150 µL of 10 mM Tris-HCl and measured at 560 nm using a multiwell VersaMax spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The rate of inhibition towards cell proliferation was calculated as [1 – (A560treated/A560control)] ×100%. The IC50 values were obtained using the Logit method and were determined based on the average results of at least three independent tests. 11

3.6. Topo I orTopo II-mediated supercoiled PBR322 relaxation The DNA relaxation assays used in the current study were conducted in accordance with a previously reported procedure.24,25 The reaction buffer contained 10 mM Tris-HCl (pH 7.9), 50 mM KCl, 50 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 15 mg/mL of bovine serum albumin (BSA), 1 mM ATP (ATP was omitted in the Topo I-mediated DNA relaxation assay), 12.5 µg/mL supercoiled pBR322 for the Topo I assay (6.67 µg/mL for the Topo II assay) and 0.01 unit/µL of Topo I (0.0467 units/µL Topo IIα) (TopoGEN, Columbus, OH, USA). Relaxation was employed at 37 °C for 15 min and was stopped by the addition of 2 µL of 10% SDS. Electrophoresis was carried out in a 1% agarose gel with 1×TAE (40 mM Tris base, 40 mM acetate acid and 1 mM EDTA) at 4 V/cm for 1 hr. The DNA bands were stained with 0.5 mg/mL of ethidium bromide solution and photographed using a Chemi Genius 2 Gel Documentation System (Syngene, Cambridge, UK). 3.7. Topo II–mediated kDNA decatenation assay Topo II activity was measured by the ATP-dependent decatenation of kDNA.26,27 The standard reaction mixture consisted of 50 mM Tris-HCl (pH 7.7), 50 mM KCl, 5 mM MgCl2, 1 mM ATP, 0.5 mM dithiothreitol, 0.5 mM EDTA, 50 mg/mL of BSA, 20 µg/mL of kDNA and 1 unit of Topo IIα in a total volume of 15 µL. After incubation at 37 °C for 15 min, the reaction was terminated by the addition of 1 µL of 10% SDS. The DNA samples were electrophoresed in 1% agarose gel in 1×TAE buffer for 50 min at 120 V. The gel was stained with ethidium bromide at room temperature and photographed on a Gel Doc XR+ System (Bio-Rad, Hercules, CA USA). 12

3.8. PI staining for flow cytometry A549 cells (3×105 each well) were seeded into 6-well plates and treated with the compounds for 6 h. The cells were then harvested and washed once with cold phosphate buffer saline (PBS) before being fixed in 70% ethanol on ice for 15 min. The staining process was conducted in PBS containing 40 µg/mL RNase and 10 µg/mL PI at room temperature in the absence of light for 30 min. The cells were then analyzed using an FACSC alibur cytometer (Becton Dickinson, San Jose, CA, USA). The cells undergoing apoptosis were obtained from the distinct subdiploidregion of the DNA distribution histograms. At least 10,000 events were counted for each sample.28 3.9. Annexin-V-PI staining assay Quantification of early apoptosis by measuring the level of phosphatidylserine (PS) exposure was carried out by Annexin-V (Sigma) staining. Briefly, after being treated with the agents for 48 h, the cells were resuspended with in cold binding buffer and treated with 5 µL of Annexin-VFITC and 5 µL of PI. The cells were then incubated for 10 min in absence of light at room temperature before being analyzed by flowcytometry using a

FACS-Calibur

cytometer

(Becton Dickinson).

Annexin-V+/PI– cellswere defined as early apoptotic cells.29

13

The

Acknowledgements Financial support from the National Natural Science Foundation (Grant nos. 81001369, 31170327, 81173080 and 81321092) and the Fundamental Research Funds for the Central Universities of the People’s Republic of China is gratefully acknowledged.

14

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Bates, R.-B.; Klenck, R.-E.; Tempesta, M.-S. J. Nat. Prod.1983, 46, 675. 15. Shiryo, Y.; Kazuhiko, K.; Hiroya, H. Phytochemistry, 1993, 32, 141. 16. Powell, R.-G.; Weisleder, D.; Smith, C.-R. J. Nat. Prod.1985, 48, 102. 17. Gozler, B.; Rentsch, D.; Gozler, T.; Unver, N.; Hesse, M.Phytochemistry, 1996, 42, 695. 18. Sinstad, E.-E.; Cataln, C.-A.-N.; Gutirrez, A.-B.; Diaz, J.-G.; Goedken,V.-L.; Herz, W. Phytochemistry 1991, 30, 1933. 19. Bohlmann, F.; Zdero, G. Phytochemistry 1982, 21, 2543. 20. Qiao, L.-R.; Zou, J.-H.; Li, L.; Sun, H.; Si, Y.-K.; Zhang, D.; Chen, X.-G.; Dai, J.-G. Planta Med. 2012, 78, 711. 21. Duh, C.-Y.; Jr, C.-H.-P.; Pezzuto, J.-M.; Kinghorn, A.-D.; Farnsworth, N.-R. J. Nat. Prod. 1986, 49, 706. 22. Van, E.-M.; Nieland, L.-J.; Ramaekers, F.-C.; Schutte, B.; Reutelingsperger, C.-P. Cytometry1998, 31,1. 23. Yang, F.; Chen, Y.; Duan, W.; Zhang, C.; Zhu, H.; Ding, J. Int. J. Cancer2006, 119, 1184. 24. Zhu, H.;Huang, M.;Yang, F.; Chen, Y.;Miao, Z.-H.; Qian, X.-H.;Xu, Y.-F.;Qin, Y.-X.; Luo, H.-B.;Shen, X.;Geng, M.-Y.;Cai, Y.-J.; Ding, J. Mol.Cancer Ther. 2007, 6, 484. 25. Shi, Z.-Y.; Li, Y.-Q.; Kang, Y.-H.; Hu, G.-Q.; Huang-fu, C.-S.; Deng, J.-B.; Liu, B. Acta Pharmacol. Sin. 2012, 33, 271. 26. Tanabe, K.; Ikegami, Y.; Ishida, R.; Andoh, T. CancerRes. 1991, 51, 4903. 27. Wang, X.; Chen, Z.; Tong, L.-J.; Tan, S.-Y.; Zhou, W.; Peng, T.; Han, K.; Ding, J.; 16

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17

Table1 1

H (400MHz,

position

, J/Hz) and 13C NMR (100 MHz) data of 1 and 6 1 δH 7.65 (br s)

1 2 3 4 5 6

δC 162.7 134.4 209.5 72.4 70.9 62.0

δC 150.4 119.5 41.9 34.7 43.9 33.8

7 8 9

63.6 39.2 76.8

3.18 (s) 3.64 (br s)

10 11 12 13 14 15 16 17 18 19 20

49.9 37.5 35.1 84.3 78.3 142.4 117.2 19.6 18.0 9.9 64.9

3.93 (br s) 2.15 (m) 2.49, 2.01 (m)

1' 2' 3'

165.5 119.2 145.9

4.25 (s)

83.9 221.3 48.5

5.65 (br s)

42.7 50.4 12.8 65.6 16.9 17.8

6 δH 5.29 (d, 2.4) 1.95, 2.62 (m) 2.44 (m) 2.70 (m) 1.94 (m), 1.45 (dd, 3.9, 8.0)

1.98 (d, 18.7), 2.63 (d, 18.7)

0.78 (s) 3.64 (d, 1.1) 1.27 (s) 0.92 (d, 7.2)

5.19, 5.28 (br s) 1.81 (d, 1.2) 1.01 (d, 5.2) 1.77 (d, 0.8) 3.69 (d, 12.0), 3.86 (d, 12.0)

5.76 (d, 15.0) 7.20 (dd, 11.0, 15.0) 4' 128.1 6.16 (dd, 11.0, 15.0) 5' 145.7 6.14 (m) 6' 34.0 1.26 (s) 7' 33.0 1.26 (s) 8'-12' 29.9-30.6 1.26 (s) 13' 23.7 1.27 (overlap) 14' 14.2 0.88 (t, 7.0) Values in parentheses are J values in Hz. 1 recorded in CDCl3, 6 recorded in CD3OD.

18

Table 2 1

H (400MHz, CDCl3,

Position 1 2 3 4 5 6 7 8 9 1' 2' 3' 4' 5' 6' 7' 8' 9' 3-OMe 3'-OMe

δC 132.4 114.4 146.6 144.1 113.6 125.7 38.1 38.4 71.3 133.3 111.7 146.5 143.7 111.7 121.8 79.8 48.8 37.0 56.1 56.2

, J/Hz) and 13C NMR(100 MHz,CDCl3) data of 7 7 δH 6.84 (br s)

6.64 (br s) 2.78 (m) 2.44 (m) 3.68, 4.05 (m) 6.71 (br s)

6.61 (br s) 6.70 (m) 4.45 (br s) 2.31 ( m) 2.59, 2.72(m) 3.87 (s) 3.84 (s)

19

R3 R1 18 11

OH

1 10 19

6

HO 14

R2

20

R2 OH R

O H

13 10 1

O

4

OH

R3 H OAc

O R=

13

O

O HO HO 1 2

O R2

8

4

R1 R OH

R1

15

OHO OH R1 R R (CH2)8CH3

3 4 5

(CH2)8CH3

O CH2OH

12

O 8

11

OH

H

6

6

15

R2 H OAc H

O H3CO

1

8 4

HO

H

7

6

7

9'

H3CO

9

O

8' 7' O

H

1'

6'

HO O 2'

8 OCH3

O

4'

O

OH

Figure 1.Structures of compounds 1-8

H3CO O HO OCH3

7

OH

Figure2.Key HMBC correlations of 1, 6, and 7

20

Figure 3. Key NOESY correlations of 6 and 7

21

Table 3. Cytotoxicities of compounds 1–8 towards A549 cells Compound.

Cytotoxicity (IC50, µM) X±SD

1

0.0027±0.0015

2

0.024±0.008

3

0.0007±0.0005

4

0.0004±0.0003

5

0.0003±0.0002

6

1.951±0.061

7

1.531±0.495

8

0.273±0.076

22

Figure 4. Effects of different compounds on activities of topoisomerase II (A and B) and topoisomerase I activity(C) at a concentration of 100µM. (A) Supercoiled PBR322 relaxation assay for topoisomerase II. (B) kDNA decatenation assay for topoisomerase II. (C) Supercoiled PBR322 relaxation assay for Topoisomerase I. RLX: relaxed form of pBR322 DNA, SC: supercoiled form of pBR322DNA.

23

A

Percentage (%)

100% 80% 60%

G2-M S G1

40% 20% 0%

NC

10

30

100

1 (nM)

B

300

300 VP16 (nM)

1 (10 nM)

Con

17.57 %

5.75 %

1 (30 nM)

19.94 %

Figure 5. Effect of compound 1 on inducing G2-M phase cell cycle arrest and apoptosis in A549 cells. (A) A549 cells were treated with compound 1 for 24h at the indicated concentrations, and were subsequently evaluated by flow cytometry. (B) Cells were treated with compound 1 for 48 h at the indicated concentrations and then collected. Apoptosis was detected by Annexin-V and PI staining. The cells in the upperright section (Annexin-V+/PI–) were considered to be early apoptotic cells.

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Graphical abstract

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