Antiproliferative Cardiac Glycosides from the Latex ... - ACS Publications

Sep 13, 2013 - The effects of the cardiac glycosides on apoptosis and the expression of orphan nuclear receptor Nur77 were examined in human NIH-H460 ...
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Antiproliferative Cardiac Glycosides from the Latex of Antiaris toxicaria Qian Liu,†,∥ Jin-Shan Tang,†,∥ Meng-Jie Hu,‡ Jie Liu,‡ Hai-Feng Chen,‡ Hao Gao,† Guang-Hui Wang,‡ Shun-Lin Li,§ Xiao-Jiang Hao,§ Xiao-Kun Zhang,*,‡,⊥ and Xin-Sheng Yao*,† †

Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China ‡ School of Pharmaceutical Sciences, Xiamen University, Xiamen 361005, People’s Republic of China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China ⊥ Sanford-Burnham Medical Research Institute, Cancer Center, 10901 N. Torrey Pines Road, La Jolla, California 92037, United States S Supporting Information *

ABSTRACT: Phytochemical investigation of the latex of Antiaris toxicaria resulted in the isolation of 15 new [antiarosides J−X (1−15)] and 17 known cardiac glycosides. The effects of the cardiac glycosides on apoptosis and the expression of orphan nuclear receptor Nur77 were examined in human NIH-H460 lung cancer cells. Several of the cardiac glycosides induced apoptosis in lung cancer cells, which was accompanied by induction of Nur77 protein expression. Treatment of cancer cells with the cardiac glycosides resulted in translocation of the Nur77 protein from the nucleus to the cytoplasm and subsequent targeting to mitochondria. The results show that the cardiac glycosides exert their apoptotic effect through the Nur77-dependent apoptotic pathway.

protein subsequently translocates from the nucleus to the cytoplasm, where it targets mitochondria through interaction with Bcl-2, leading to cytochrome c release and apoptosis. Thus, subcellular localization of Nur77 plays a critical role in the survival and death of cancer cells.6,7 In our previous search for anticancer agents from plants, 11 cardiac glycosides were isolated from the stem of A. toxicaria by using bioassay-guided fractionation.8 The compounds were observed to inhibit the growth of various cancer cell lines at nanomolar concentrations. Also, inhibition of cancer cell growth by the cardiac glycosides was accompanied by induction of Nur77 expression, suggesting that Nur77 might mediate the anticancer effects.6,8 In the study described below, we evaluated the effects of 15 new and 17 known cardiac glycosides, isolated from the latex of A. toxicaria, on the induction of apoptosis and Nur77 protein expression in human NIH-H460 lung cancer cells. In addition, the cardiac glycosides found to induce Nur77 expression were also examined for their modulation of the subcellular localization of Nur77 protein. The results of this

Antiaris toxicaria (Pers.) Lesch. (Moraceae), known as the “upas tree”, is widespread over the tropical rain forests of Southeast Asia. It has been the main source of the principle component of dart and arrow poison because it contains a complex mixture of cardiac glycosides.1−4 Cardiac glycosides are clinically used in the treatment of congestive heart failure and as antiarrhythmic agents due to their strong inhibitory activity toward the ubiquitous cell surface enzyme Na+/K+ATPase. Recently, analysis of epidemiological data along with results arising from in vitro and in vivo studies demonstrate that cardiac glycosides exhibit potent antiproliferative and apoptotic effects on cancer cells through complex signal transduction mechanisms, and the first cardiac glycoside-based substances are now undergoing clinical trials for cancer treatment.5 The exact mechanisms underlying these effects of cardiac glycosides have not been fully elucidated. Nur77, also known as TR3 or NGFI-B, is an immediate-early response gene and an orphan member of the nuclear receptor superfamily. In addition to its regulation of transcription in the nucleus, Nur77 has extranuclear effects in response to a variety of apoptotic agents. Upon stimulation by apoptosis-inducing agents, Nur77 is usually induced and the induced Nur77 © 2013 American Chemical Society and American Society of Pharmacognosy

Received: June 26, 2013 Published: September 13, 2013 1771

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antiarigenin 3-O-β-4,6-dideoxy-D-allopyranoside, and it was named antiaroside J. Cardiac glycoside 2, obtained as a colorless, amorphous powder, displayed a HRESIMS that contained a quasimolecular ion at m/z 567.2805 [M + H]+, suggesting that it had a molecular formula of C29H42O11, which was the same as that of β-antiarin (16).9 The 1H and 13C NMR signals were similar to those of 16 except for obvious differences of chemical shifts of C-1, C-2, and C-5, indicating that 2 was a stereoisomer of 16. The downfield shifts of C-1 and C-2 resonances from δ 19.3 and 25.8 to δ 22.9 and 28.0 and upfield shift of C-5 from δ 74.1 to 71.7 indicated that the orientation of the 3-OH group in 2 was different from that of 16. The α-orientation of the C-3 substituent was demonstrated by using the NOE correlation between H-3 and 5-OH. Key HMBC correlations between H-1′ and C-3 indicated that the rhamnose unit present in 2 was linked to C-3. The L-configuration of rhamnose was defined via acid hydrolysis and appropriate derivatization of the resulting sugar.10 Thus, 2 was identified as antiarigenin 3α-O-α-Lrhamnopyranoside, and it was named antiaroside K. In the same way, the new cardiac glycosides 3 and 4 were identified as C-3 epimers of antialloside (17)11 and α-antiarin (18),4 respectively, and were named antiaroside L (3) and antiaroside M (4). Cardiac glycoside 5 was isolated as a colorless syrup. The HRESIMS showed a quasimolecular ion at m/z 567.2811 [M + H]+, suggesting a molecular formula of C29H42O11. The UV spectrum indicated the presence of an α,β-unsaturated carbonyl group (λmax = 217 nm). 1H−1H COSY, HSQC, and HMBC spectra showed that 5 had the same planar structure as that of β-antiarin (16) (Figure S2, Supporting Information). A comparison of the 13C NMR data of 5 with that of 16 showed differences of the chemical shifts of C-18 and C-9. The downfield shifts of C-18 from δ 10.4 (16) to 17.8 (5) and upfield shifts of C-9 from δ 37.1 (16) to 33.9 (5) were ascribed to the γ-gauche effect, suggesting that the C-12 substituent was α-orientated in 5. ROESY correlation between H-12 and CH318 in 5 supported this conclusion, and 5 was named antiaroside N. Compound 6, obtained as a colorless powder, had the molecular formula C29H42O10, as determined on the basis of the HRESIMS. Analyses of 13C NMR with DEPT 135 signals revealed that 6, having one oxygen atom less than 5, possessed five quaternary carbons (one less than compounds 1−5). The absence of a quaternary carbon signal at δ 74.4 (C-5 in 5) and the presence of a carbon signal at δ 30.3 (C-5 in 6) suggested that 6 was a 5-deoxy derivative of 5, a conclusion supported by upfield shifts observed for C-4 and C-6 from δ 37.2 and 35.6 (5) to δ 30.7 and 22.6 (6). In the ROESY spectrum, a correlation between H-5 (δ 2.58) and 19-CHO (δ 9.55) suggested that H-5 was β-orientated. The carbon signals at δ 101.6 (CH, C-1′), 69.6 (CH, C-2′), 74.4 (CH, C-3′), 74.0 (CH, C-4′), 70.0 (CH, C-5′), and 17.4 (CH3, C-6′) indicated the presence of a 6-deoxy-β-D-gulose unit in 6. HMBC correlation between H-1′ and C-3 placed the 6-deoxy-β-Dgulose unit at C-3. Therefore, 6 was named antiaroside O. Comparisons of the 1H and 13C NMR signals for 6 with those of 7 and 8 revealed that the latter substances contained the same aglycones, but differed in the nature of the attached sugar units. The sugar units of 7 and 8 were identified as C-3-linked 6-deoxy-β-D-allose and α-L-rhamnose, respectively, through analysis of their 1H and 13C NMR signals and HMBC correlations (Figure S2, Supporting Information). Thus,

effort demonstrate that translocation of Nur77 from the nucleus to the cytoplasm and its mitochondrial targeting play a critical role in mediating the apoptotic effect of cardiac glycosides in cancer cells.

■

RESULTS AND DISCUSSION The procedure for isolation of the cardiac glycosides began with suspension of the latex of A. toxicaria in H2O, followed by successive partitioning between CHCl3 and n-BuOH. The extracts containing cardiac glycosides were subjected to a series of column chromatographic separations including silica gel, Sephadex LH-20, ODS, and (semi)preparative RP HPLC. This process led to the isolation of 15 new and 17 known cardiac glycosides. The Keddle reagent and vanillin/H2SO4 solution were used to monitor cardiac glycosides on TLC, the former showing red spots and the latter green spots.

Cardiac glycoside 1 was obtained as colorless crystals (MeOH). The molecular formula was established as C29H42O10 by using HRESIMS. The IR spectrum of 1 contained prominent absorption maxima at 3420, 1715, and 1739 cm−1, indicating the presence of hydroxy and carbonyl functionalities. The 1H and 13C NMR signals for 1 were assigned using 1D and 2D NMR experiments (Tables 1 and 2). The 1H NMR spectrum showed resonances characteristic of butenolide ring protons at δ 6.25 (1H, s, H-22), 5.23 (1H, dd, J = 18.0, 1.2 Hz, H-21a), and 5.11 (1H, dd, J = 18.0, 1.2 Hz, H21b). The presence of signals at δ 10.41 (1H, s, H-19) and 1.23 (3H, s, H-18) suggested that 1 had a cardenolide skeleton carrying 19-CHO and 18-CH3 groups. The cardenolide steroidal tetracyclic ring system was confirmed by using the key HMBC correlations between 19-CHO (δ 10.41, s, H-19) and both C-1 and C10 and between CH3-18 (δ 1.23, s, H-18) and C-12, C-13, C-14, and C-17. 1H−1H COSY, HSQC, and HMBC experiments permitted full assignment of the resonances in the aglycone as those corresponding to antiarigenin.3 The anomeric proton resonance at δ 5.30 (1H, d, J = 8.0 Hz, H-1′) and carbon resonance at δ 99.5 (CH, C-1′) suggested the presence of a sugar unit in the structure of 1. Analyses of 1 H−1H COSY, HSQC, and HMBC data revealed that the sugar moiety was 4,6-dideoxyallose and that, based on the large coupling constant of the anomeric proton (J = 8.0 Hz), it was a β-glycoside. HMBC correlation between H-1′ and C-3 was used to establish the position of the sugar unit. The X-ray diffraction analysis (Figure 1) revealed the D-configuration of the 4,6-dideoxyallose. The combined results showed that 1 was 1772

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Table 1. 13C NMR Data (δ in ppm) of Antiarosides J−X (1−15) in Pyridine-d5 position

1a

2a

3a

4a

5a

6c

7b

8c

9a

10a

11a

12b

13a

14a

15a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 1′ 2′ 3′ 4′ 5′ 6′

19.3 26.5 73.2 38.2 74.1 35.2 25.5 42.0 37.1 55.6 32.0 74.7 56.9 85.4 32.9 28.2 46.7 10.5 209.2 176.8 74.4 117.9 175.1 99.5 72.7 69.1 41.1 67.2 21.7

22.9 28.0 72.8 43.1 71.7 35.8 26.1 43.0 39.4 56.3 31.4 74.5 56.9 85.4 33.4 31.1 46.8 10.4 208.7 176.8 74.3 117.9 175.1 100.2 73.1 73.2 74.5 70.2 18.9

22.9 28.0 72.8 43.3 71.8 35.9 26.1 42.9 39.4 56.4 31.3 74.5 56.9 85.3 33.4 31.3 46.8 10.4 208.6 176.8 74.3 117.8 175.1 100.1 73.2 74.0 74.7 70.5 19.0

23.0 28.0 73.7 43.4 71.8 35.9 26.2 43.0 39.4 56.4 31.4 74.5 56.9 85.4 33.4 31.4 46.8 10.4 208.7 176.8 74.3 117.8 175.1 100.6 69.6 73.9 74.2 71.0 19.1

19.1 25.9 74.1 37.2 74.4 35.6 25.1 42.8 33.9 55.3 30.1 75.3 54 85 34.7 31.7 46.7 17.8 209.3 177.6 74.4 118 175 100.8 72.8 73.1 73.2 71.1 18.9

28.1 25.8 73.7 30.7 30.3 22.6 22.6 42.2 32.6 51.5 30.0 74.7 57.2 85.4 33.1 28.9 46.6 10.5 207.0 177.0 74.4 117.8 175.1 101.6 69.6 74.4 74.0 70.0 17.4

28.2 25.9 73.7 30.1 30.4 22.6 22.6 42.3 32.7 51.9 30.8 74.8 57.3 85.5 33.2 29.0 46.7 10.5 206.9 177.0 74.4 117.8 175.1 101.0 72.9 73.5 74.9 70.7 19.1

28.1 25.6 71.4 29.5 30.5 22.8 22.6 42.3 32.5 51.4 30.7 74.7 57.2 85.4 33.2 28.9 46.6 10.5 206.7 177.0 74.4 117.8 175.1 100.3 73.1 73.2 74.3 70.6 18.9

24.6 27.9 67.2 37.5 75.1 40.0 24.4 41.1 40.1 56.5 21.3 40.5 51.6 85.7 32.9 28.9 50.7 16.6 174.0 176.4 74.1 118.1 174.9 96.4 74.8 79.0 71.6 79.9 62.6

22.2 27.3 73.9 36.2 73.9 37.9 25.1 40.8 36.7 55.2 33.3 75 57.2 85.7 33.5 28.4 46.8 10.8 178.0 177.1 74.4 117.8 175.1 99.6 73.1 73.0 74.7 71.0 19.1

19.2 24.7 73.4 37.3 74.4 35.7 24.4 41.4 36.8 55.2 30.9 69.4 56.9 85.7 32.7 25.7 48.5 13.0 208.8 176.3 75.0 114.9 175.2 101.0 72.8 73.2 74.1 71.1 18.9

19.2 24.9 73.6 38.0 74.0 35.2 24.4 41.3 36.8 55.4 30.9 69.4 56.9 85.8 32.7 26.1 48.5 13.0 209.2 176.3 75.0 114.9 175.2 99.5 72.8 73.2 74.6 70.9 19.0

19.2 24.9 73.3 38.1 74.1 35.2 24.4 41.4 36.8 55.5 30.9 69.4 57.0 85.7 32.7 26.2 48.5 14.0 209.3 176.4 75.0 114.8 175.3 99.9 69.8 74.1 74.2 70.3 18.9

26.1 31.4 73.4 35.9 71.6 43.4 22.7 43.2 42.2 56.6 22.3 39.8 50.2 84.7 33.1 27.4 51.6 16.3 208.8 176.2 74.1 118.5 174.9 100.6 69.6 73.9 74.2 69.8 17.4

28.3 25.4 75.3 31.0 31.5 22.5 27.5 42.1 33.3 40.1 31.6 75.5 57.3 86.0 33.8 27.4 47.1 10.7 66.4 177.1 74.5 117.8 175.2 102.1 69.8 74.5 74.2 70.2 17.5

a

100 MHz. b125 MHz. c75 MHz.

Table 2. 1H NMR Data (δ in ppm, J in Hz) of Antiarosides J−N (1−5) in Pyridine-d5 position

1a

2a

3a

4a

5a

1a/b 2a/b 3 4a/b 6a/b 7a/b 8 9 11a/b 12 15a/b 16a/b 17 18 19 21a/b

2.65, td (16.0, 4.0)/1.90, m 2.04, m/1.76, m 4.45, br s 2.36, m/1.81, m 2.13, m/1.87, m 2.45, m/1.44, qd (13.6, 4.0) 2.40, m 1.90, m 2.12, m/1.79, m 3.68, m 1.93, m/1.91, m 2.12, m/2.10, m 3.73, t (7.6) 1.23, s 10.41, s 5.23, dd (18.0, 1.2) 5.11, dd (18.0, 1.2) 6.25, s 5.30, d (8.0) 3.79, dd (8.0, 2.8) 4.45, m 2.01, m/1.61, td (13.6, 2.0) 4.38, m 1.24, d (6.4)

2.32, m/2.18, m 2.05, m 4.66, m 2.38, m/1.67, m 2.42, m/1.82, m 2.37, m/2.07, m 1.95, m 2.39, m 2.24, m/1.82, m 3.66, m 2.03, m/1.95, m 2.23, m/1.53, m 3.71, t (8.0) 1.20, s 10.17, s 5.23, dd (18.0, 1.2) 5.08, dd (18.0, 1.2) 6.22, s 5.38, br s 4.45, m 4.45, m 4.24, m 4.25, m 1.59, d (5.6)

2.31, m/2.16, m 2.05, m 4.80, m 2.40, m/1.76, m 2.36, m/1.75, m 2.31, m/2.04, m 1.93, m 2.37, m 2.28, m/1.80, m 3.63, m 2.01, m/1.93, m 2.22, m/1.60, m 3.70, m 1.18, s 10.11, s 5.22, dd (18.0, 1.2) 5.07, dd (18.0, 1.2) 6.22, s 5.27, d (8.0) 3.88, dd (7.8, 3.0) 4.63, t (3.0) 3.64, m 4.13, m 1.52, d (6.0)

2.31, m/2.16, m 2.04, m 4.85, m 2.42, m/1.74, m 2.38, m/1.75, m 2.34, m/2.08, m 1.93, m 2.38, m 2.35, m/1.80, m 3.64, m 2.00, m/1.93, m 2.24, m/1.62, m 3.71, t (7.6) 1.19, s 10.13, s 5.23, dd (18.0, 1.2) 5.08, dd (18.0, 1.2) 6.23, s 5.31, d (8.0) 4.42, dd (8.5, 3.5) 4.74, t (3.2) 4.10, d (2.8) 4.40, q (6.5) 1.48, d (6.4)

2.44, m/1.91, m 1.92, m/1.79, m 4.28, br s 2.34, m/1.80, m 2.22, m/1.72, m 2.50, m/1.57, m 2.38, m 2.41, m 2.32, m/2.08, m 3.93, m 2.66, m/1.97, m 1.94, m/1.82, m 3.78, m 1.08, s 10.43, s 5.28, dd (16.4, 1.2) 5.09, dd (16.4, 1.2) 6.14, s 5.47, br s 4.49, m 4.42, dd (9.2, 3.2) 4.29, m 4.19, m 1.64, d (6.0)

22 1′ 2′ 3′ 4′ 5′ 6′ a

400 MHz.

1773

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quite similar to those of antialloside (17),11 except for the absence of aldehyde signals at δ 10.38 (1H, s, H-19) and 208.8 (C-19) in 17 and the presence of a carbonyl carbon signal at δ 178.0 (C-19). The 1H and 13C NMR signals for the sugar residue in 10 indicated that it was 6-deoxy-β-D-allose. In view of the molecular formula, it appeared that in 10 the aldehyde group of 17 was replaced by a carboxylic acid group. Thus, 10 was named antiaroside S. Cardiac glycoside 11 had the molecular formula C29H42O11 by analysis of the HRESIMS. The 1H, 13C, 1H−1H COSY, HSQC, and HMBC spectra revealed that 11 had the same planar structure as β-antiarin (16). Comparison of the 13C NMR data of 16 and 11 showed that signals for C-12, C-16, C17, C-18, and C-22 were shifted from δ 74.6, 28.1, 46.7, 10.4, and 117.9 in 16 to δ 69.4, 25.7, 48.5, 13.0, and 114.9 in 11. The observations indicated that the C-17 substituent in 11 was αorientated.12 The L-configuration of the rhamnose moiety in 11 was determined using acid hydrolysis and appropriate derivatization of the resulting sugar.10 Thus, 11 was named antiaroside T. Comparisons of the 1H and 13C NMR signals of 12 and 13 with those of 11 suggested that the latter substances contained the same aglycones but differed in the nature of their sugar units. The sugar units of 12 and 13 were identified to be 6deoxy-β-D-allose and 6-deoxy-β-D-gulose, based on characteristic 1H and 13C NMR data, and linked to C-3 of the aglycones based on HMBC correlations. Thus, compounds 12 and 13 were named antiaroside U and antiaroside V, respectively. Cardiac glycoside 14 had the molecular formula C29H42O10. Analyses of 1H and 13C NMR signals indicated that 14 was a stereoisomer of desglucocheirotoxin (22). The 1H−1H COSY, HSQC, HMBC, and ROESY spectra suggested that the C-3

Figure 1. Plot of the X-ray crystallographic data of 1.

compounds 7 and 8 were named antiaroside P and antiaroside Q, respectively. Cardiac glycoside 9 was obtained as a white, amorphous powder, and the HRESIMS indicated that it had the molecular formula C29H42O12. Assignment of the NMR signals using 1D and 2 D NMR experiments showed that a carbonyl carbon signal at δ 174.0 existed in 9, and it was assigned to C-19. In the HMBC spectrum of 9, correlation between H-1′ and C-19 illustrated that the sugar unit was linked to C-19 instead of C-3 as observed for related substances. 1H−1H COSY and HSQC spectra indicated that the sugar unit was glucose with a βorientation, assigned based on the large J value of H-1′ (J = 8.0 Hz). The D-configuration of the glucose unit was defined via acid hydrolysis and appropriate derivatization of the resulting sugar.10 Thus, compound 9 was named antiaroside R. Compound 10 had the molecular formula C29H42O12 based on the HRESIMS. The 1H and 13C NMR signals of 10 were

Table 3. 1H NMR Data (δ in ppm, J in Hz) of Antiarosides O−S (6−10) in Pyridine-d5 6c

position 1a/b 2a/b 3 4a/b 5 6a/b 7a/b 8 9 11a/b 12 15a/b 16a/b 17 18 19 21a/b 22 1′ 2′ 3′ 4′ 5′ 6′ a

2.18, 2.13, 4.35, 2.30, 2.58, 1.67, 2.10, 2.25, 1.99, 1.83, 3.71, 2.07, 1.50, 3.78, 1.34, 9.55, 5.27, 5.12, 6.25, 5.35, 4.51, 4.79, 4.15, 4.60, 1.56,

m/2.14, m m/1.62, m br s m/2.02, m m m/1.29, m m m m m m m/1.92, m m/1.23, m t (7.8) s s dd (16.8, 1.2) dd (16.8, 1.2) s d (8.1) d (6.9) t (3.3) br s qd (6.3, 0.9) d (6.6)

7b 2.15, 2.09, 4.32, 1.81, 2.60, 1.58, 2.10, 2.22, 1.97, 2.31, 3.70, 1.97, 1.50, 3.78, 1.35, 9.57, 5.26, 5.12, 6.25, 5.32, 3.99, 4.68, 3.71, 4.32, 1.61,

m/2.11, m m/1.63, m br s m/1.27, m m m/1.30, m m m m m/2.02, m m m/1.89, m m/1.25, m t (7.5) s s dd (18.0, 1.2) dd (18.0, 1.2) s d (7.5) d (7.5, 2.7) t (2.7) m m d (6.0)

8c 2.33, 1.83, 4.16, 2.01, 2.29, 1.58, 2.14, 2.23, 1.96, 2.17, 3.72, 2.02, 1.77, 3.78, 1.34, 9.57, 5.26, 5.12, 6.25, 5.40, 4.53, 4.48, 4.28, 4.22, 1.64,

m/2.11, m m/1.54, m br s m/1.50, m m m/1.30, m m/1.81, m m m m/2.05, m m m/1.22, m m/1.26, m t (7.5) s s dd (18.0, 1.2) dd (18.0, 1.2) s br s m m t (9.3) m d (6.0)

9a

10a

2.44, 2.13, 4.41, 3.10,

d (3.2)/1.44, m m/2.00, m br s m/1.67, m

3.21, 2.12, 4.49, 2.20,

m/2.25, m m/1.90, m br s m/2.02, m

1.84, 1.82, 2.80, 2.28, 3.16, 1.44, 2.13, 1.94, 2.78, 1.13,

m m m m m/2.28, m m m/1.91, m m/1.75, m m s

3.10, 2.47, 3.04, 2.00, 2.36, 3.78, 2.14, 2.16, 3.77, 1.28,

m/1.70, m/1.47, m m m/2.01, m m/2.00, m t (7.8) s

5.30, 5.05, 6.09, 6.44, 4.13, 4.22, 4.29, 3.99, 4.37,

dd (18.0, 1.2) dd (18.0, 1.2) s d (8.0) t (8.4) t (8.4) m m m/4.32, m

5.30, 5.14, 6.26, 5.43, 3.96, 4.67, 3.70, 4.37, 1.62,

dd (18.0, 1.2) dd (18.0, 1.2) s d (7.8) dd (7.8, 2.7) t (2.7) dd (9.6, 2.7) m d (6.0)

m m

m m

400 MHz. b500 MHz. c300 MHz. 1774

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Table 4. 1H NMR Data (δ in ppm, J in Hz) of Antiarosides T−X (11−15) in Pyridine-d5 11a

position 1a/b 2a/b 3 4a/b 5 6a/b 7a/b 8 9 11a/b 12 15a/b 16a/b 17 18 19

2.44, 2.48, 4.32, 2.35,

2.21, m/1.79, m 2.15, m 2.42, m 1.87, m 2.11, m/1.92, m 3.76, m 2.06, m/1.76, m 1.99, m 3.52, t (8.8) 1.45, s 10.39, s

2.16, m/1.89, 2.11, m/2.01, 2.40, m 1.87, m 1.99, m/1.85, 3.73, m 2.03, m/1.71, 2.03, m/1.73, 3.51, t (8.4) 1.44, s 10.45, s

21a/b

5.47, 5.03, 6.12, 5.47, 4.49, 4.40, 4.26, 4.18, 1.63,

5.46, 5.03, 6.10, 5.36, 3.91, 4.64, 3.68, 4.31, 1.58,

22 1′ 2′ 3′ 4′ 5′ 6′ a

m/1.92, m m/2.14, m br s m

12b

dd (16.4, 1.2) dd (16.4, 1.2) br s br s m dd (9.2, 3.2) t (9.2) m d (6.0)

2.68, 2.45, 4.47, 2.38,

13a

m/1.93, m m/1.42, m br s m/1.81, m m m

m m m

dd (16.4, 1.2) dd (16.4, 1.2) br s d (8.0) dd (8.0, 2.8) t (2.8) m m d (6.4)

2.69, 2.47, 4.54, 2.39,

m/1.95, m m/1.43, m br s m/1.85, m

2.20, m/1.94, 2.13, m/2.01, 2.42, m 1.87, m 1.90, m 3.75, m 2.07, m/1.75, 2.13, m/1.81, 3.52, t (8.8) 1.45, s 10.46, s 5.48, 5.03, 6.13, 5.44, 4.50, 4.77, 4.15, 4.62, 1.56,

14a 2.39, 2.34, 4.88, 2.42,

m m

m/2.06, m m/1.65, m m m/1.86, m

2.25, m/1.71, 2.31, m/1.76, 2.20, m 2.41, m 1.80, m/1.27, 1.36, m 1.89, m/1.22, 2.11, m/1.99, 2.73, m 0.94, s 10.09, s

m m

dd (16.8, 1.2) dd (16.8, 1.2) s d (8.0) dd (8.4, 3.2) t (3.2) d (3.2) q (6.8) d (6.8)

5.29, 5.00, 6.11, 5.33, 4.45, 4.77, 4.12, 4.41, 1.49,

15a

m m

m m m

dd (18.0, 1.2) dd (18.0, 1.2) br s d (8.0) dd (8.0, 3.2) t (2.8) br d (3.2) m d (6.4)

2.16, 2.56, 4.41, 2.78, 2.01, 2.21, 2.14, 2.16, 2.05, 1.92, 3.74, 1.99, 1.80, 3.77, 1.28, 4.10, 3.89, 5.28, 5.13, 6.26, 5.39, 4.51, 4.78, 4.15, 4.63, 1.57,

m/2.08, m m/1.77, m br s m/2.12, m m m/1.48, m m/1.97, m m m m/1.83, m dd (11.0, 4.5) m/1.95, m m/1.24, m t (7.5) s d (11.0) d (11.0) dd (18.0, 1.2) dd (18.0, 1.2) s d (8.0) dd (8.0, 3.0) t (3.0) br d (3.0) qd (6.0, 0.5) d (6.5)

400 MHz. b500 MHz.

substituent was α-orientated, which was supported by observation of a coupling constant for the H-3 resonance (1H, m).13 A doublet for the anomeric proton at δ 5.33 (J = 8.0 Hz, H-1′), four oxymethine protons between δ 4.12 and 4.77, and a methyl proton signal at δ 1.49 (3H, d, J = 6.4 Hz) all indicated the presence of a β-orientated deoxyhexose unit in 14, which was identified as β-6-deoxygulose based on analysis of 13 C NMR data. The sugar unit was located at C-3 of the aglycone as determined by HMBC correlation between H-1′ of the β-6-deoxy-gulose unit and C-3 of the aglycone moiety. Therefore, 14 was named antiaroside W. Compound 15 had the molecular formula C29H44O10. Comparison of the 1H and 13C NMR signals of 15 with those of 6 showed that the former lacked an aldehyde group but possessed a hydroxymethyl group (δ 66.4). The 1H and 13C NMR data indicated the presence of a β-6-deoxygulose unit connected to C-3 of the aglycone based on an HMBC correlation between the anomeric proton (H-1′) and C-3. ROESY correlations between H-19a and H-8; H-8 and CH3-18; H-19b and H-5; and H-9 and H-12 provided further evidence for the relative orientation of the aglycone, which indicated that the A/B and C/D ring junctions were cis, with H-5 and the CH2OH-10, OH-14, and CH3-18 groups all having β orientations. Thus, compound 15 was named antiaroside X. The 17 known cardiac glycosides, isolated in this effort, were β-antiarin (16),9 antialloside (17),11 α-antiarin (18),4 toxicarioside B (19),4 convallatoxin (20),14 strophalloside (21),14 desglucocheirotoxin (22),15 antiaroside C (23),13 19-(glucosyloxy)-3β,5,14-trihydroxy-5β-card-20(22)-enolide (24),16 convallatoxol (25),17 deglucocheirotoxol (26)18 cannogenol-3-Oα-L-rhamnoside (27),19 al-dihydro-α-antiarin (28),20 al-dihydro-β-antiarin (29),21 antioside (30),22 α-antioside (31),2 and

antiogoside (32) (Figure S1, Supporting Information).11 The identities of these substances were established by comparison of their physical and spectroscopic data with those reported previously. The cytotoxic activities of cardiac glycosides 1−32 toward human NIH-H460 lung cancer cells were evaluated using MTT assays (Table 5). Compounds 1, 5−8, 15−17, 19−22, and 25− Table 5. Inhibitory Rates of 1−32a compound

inhibitory rate (%)

compound

inhibitory rate (%)

1 2 3 4 5 6 7 8 9 10 11

81.6 6.3 6.9 3.2 82.8 82.3 78.9 81.9 11.1 7.8 6.2

13 14 15 16 17 18 19 20 21 22 23

9.3 6.8 63.8 81.4 70.7 45.0 71.4 82.1 65.0 76.9 18.9

compound

inhibitory rate (%)

24 25 26 27 28 29 30 31 32

29.4 80.7 67.9 82.4 64.2 60.2 75.7 33.9 34.3

a

NIH-H460 cells were incubated for 48 h with compounds 1−32 (50 nM), and cell viability was then evaluated by the MTT assay.

30 significantly inhibited the viability of NIH-H460 cells at concentrations of 50 nM. Analysis of the structure−activity relationship suggested that the orientation of C-3 and C-17 substituents in these substances was a crucial factor in determining their cytotoxic activities. This conclusion was based on the observation that 2−4, and 11−13, which 1775

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Figure 2. Induction of Nur77 expression by 1−32. NIH-H460 cells were incubated for 3 h with compounds 1−32 (20 nM), and Nur77 expression was determined by Western blotting using anti-Nur77 antibody.

Figure 3. Dose-dependent and time-dependent induction of Nur77. NIH-H460 cells were treated with vehicle or with 7, 9, or 10 at 2.5, 5, 10, 20, or 40 nM for 3 h or at 20 nM for the indicated period of time. Nur77 expression was determined by using Western blotting with anti-Nur77 antibody.

contained α-oriented C-3 and C-17 substituents, displayed weak inhibitory activities. Consistent with a previous report,23 substances possessing an α-L-rhamnose moiety at C-3 showed the strongest cytotoxicity. The position at which the sugar unit was linked also had an effect on cytotoxicity. Substances in which the sugar was attached to C-19 (9 and 24) exhibited lower cytotoxicities than those in which this moiety was linked to C-3. The results of our previous study revealed that inhibition of cancer cell proliferation by cardiac glycosides was accompanied by induction of the expression of the orphan nuclear receptor Nur77. This observation suggested that induction of Nur77 expression could contribute to the apoptotic activities of these substances.8 To explore the structure−activity relationships of 1−32 toward Nur77 induction, protein level induction of Nur77 was determined by using Western blotting (Figure 2). Cells treated with most of the cardiac glycosides at concentrations of 20 nM exhibited strong induction of Nur77 expression in 3 h. Among the substances explored, strophalloside (21) was the strongest inducer of Nur77 expression,

consistent with our previous observations.8 Structure−activity relationship analyses indicated that the configuration at C-3 and C-17 also influenced the induction of Nur77 expression because only the substances having β-orientation but not those with α-orientation of substituents at these positions displayed significant activity. Moreover, substances with 5β-H substitution had stronger Nur77 induction activity than those containing a 5β-OH substituent (16, 17, 18/6, 7, 8, and 25/ 27). Finally, it appeared that 6-deoxy-β-D-allose substitution at C-3 exerted a stronger effect on Nur77 induction activity for compounds bearing the same aglycones. Interestingly, although Nur77 expression was strongly induced by substances in the series that had sugar units attached to C-19 (e.g., 9 and 24), these compounds failed to show cytotoxicity toward NIH-H460 cells (Table 5). Because the subcellular localization of Nur77 is important for its apoptotic effect, studies were carried out with 9 and 10 to determine their ability to promote translocation of the induced Nur77 protein from the nucleus to the cytoplasm. For comparison purposes, 7, a substance that caused both 1776

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Figure 4. Effect of 7, 9, and 10 on subcellular localization of Nur77. NIH-H460 cells were treated with 20 nM 7, 9, or 10 for 3 h and then stained with 25 nM MitoTraker Red (Invitrogen) for 15 min. Endogenous Nur77 expression was immunostained with anti-Nur77 antibody. Cells were costained with DAPI to visualize the nuclei.

Figure 5. Apoptotic effects of 7, 9, and 10. NIH-H460 cells were treated with 7, 9, or 10 for 12, 24, and 36 h and examined for apoptotic cell formation by using DAPI staining. Apoptotic cells were compared following different treatments.

cytotoxicity and Nur77 expression, was also studied. The results showed that 7 had a potent inhibitory effect on the viability of NIH-H460 cells with an IC50 value of 95 nM, while 9 and 10 had no effect (IC50 >1000 nM) on the viability of this cell line (Figure S3, Supporting Information). Despite their different cytotoxic behaviors, 7, 9, and 10 all rapidly induced potent Nur77 expression at low nM concentrations (2−5 nM for 1−2 h) (Figure 3). However, a striking difference was observed when their effect on subcellular localization of Nur77 was examined by using immunostaining. As shown in Figure 4, these substances enhanced Nur77 immunostaining in cells, suggesting that they induced Nur77 protein expression. However, cells treated with 7 displayed a diffused distribution of Nur77 in both the nucleus and cytoplasm and most of the cytoplasmic Nur77 targeted to mitochondria, as revealed by the results of costaining with MitoTracker Red (a red-fluorescent dye that stains mitochondria in live cells). This finding

demonstrated that 7 induced nuclear export and mitochondrial targeting of Nur77. In contrast, Nur77 protein induced by 9 and 10 occurred exclusively in the nucleus of the treated cells, indicating that these substances could not induce Nur77 nuclear export. Pro-apoptotic effects were also evaluated by using DAPI staining of NIH-H460 cells treated with 20 nM 7, 9, and 10 for 36 h (Figure 5). A significant number of the cells (ca. 22.3%) underwent apoptosis when treated with 7, while only 6.8% and 3.4% apoptotic cells were produced upon treatment with 9 and 10, respectively. The apoptotic effect of 7 was also examined by determining its induction of PARP cleavage, a sensitive apoptotic marker occurring early in the apoptotic response.24 Induction of PARP cleavage by 7 was visible at 24 h posttreatment (Figure S4A, Supporting Information). The ability of 7 to induce apoptosis was further demonstrated by its ability to activate caspase 3, a major executioner caspase known to cleave 1777

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PARP.24 The results of immunostaining with an antibody recognizing cleaved caspase-3 showed that treatment of NIHH460 cells with 7 resulted in activation of caspase 3, while treatment with 9 and 10 did not (Figure S4B, Supporting Information). Together, the observations made in this effort demonstrated that both induction of Nur77 expression and its subsequent translocation from the nucleus to the cytoplasm are critical events in apoptosis induction by cardiac glycosides in cancer cells.

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to obtain compounds 2 (34.6 mg) and 3 (13.4 mg). B4 was applied to semipreparative RP-HPLC to afford compounds 4 (22.9 mg), 11 (18.0 mg), and 12 (2.2 mg). B4 was chromatographed over ODS (⦶ 3.5 × 12 cm) MPLC using a MeOH/H2O gradient to afford four subfractions (B4.1−B4.4). B4.1 was subjected to semipreparative RP-HPLC to afford compounds 5 (4.7 mg) and 10 (4.1 mg). B4-3 was applied to semipreparative RP-HPLC to yield compounds 13 (9.0 mg) and 29 (10.0 mg). B6 was chromatographed on an ODS column (⦶ 3.2 × 60 cm) using MeOH/H2O (25:75) to obtain five subfractions (B6.1−B6.5). B6.2 was subjected to semipreparative RP-HPLC, which yielded compounds 7 (3.5 mg) and 8 (23.5 mg). B6.3 was applied to semipreparative RP-HPLC eluted by MeOH/H2O (30:70) to obtain 28 (20.0 mg) and 32 (15.0 mg). B7 was chromatographed on ODS MPLC (⦶ 3.5 × 13 cm) and eluted with MeOH/H2O (28:72) to afford compounds 6 (12.0 mg), 9 (25.0 mg), 15 (2.0 mg), and 24 (10.0 mg). B8 was applied to semipreparative RP-HPLC to yield compounds 14 (1.1 mg), 23 (1.2 mg), and 25 (10.8 mg). Antiaroside J (1): colorless crystals (MeOH); mp 309−315 °C; [α]28 D −7.6 (c 0.87, MeOH); IR (KBr) νmax 3420, 2937, 1739, 1715, 1034 cm−1; 1H NMR (pyridine-d5, 400 MHz), see Table 2; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 573 [M + Na]+, 549 [M − H]−; HRESIMS m/z 551.2864 [M + H]+ (calcd for C29H43O10, 551.2856). Antiaroside K (2): colorless powder, [α]27 D −18.3 (c 0.70, MeOH); IR (KBr) νmax 3471, 2955, 1750, 1710, 1059 cm−1; 1H NMR (pyridine-d5, 400 MHz), see Table 2; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 567 [M + H]+, 1167 [2 M + Cl]−; HRESIMS m/z 567.2805 [M + H]+ (calcd for C29H43O11, 567.2805). Antiaroside L (3): colorless syrup; [α]27 D −19.7 (c 0.11, MeOH); IR (KBr) νmax 3471, 2938, 1725, 1626, 1170, 1079, 1029 cm−1; 1H NMR (pyridine-d5, 400 MHz) see Table 2; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 589 [M + Na]+, 1167 [2 M + Cl]−; HRESIMS m/z 567.2807 [M + H]+ (calcd for C29H43O11, 567.2805). Antiaroside M (4): colorless powder, [α]28 D −25.6 (c 0.80, MeOH); 1 H NMR (pyridine-d5, 400 MHz), see Table 2; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 567 [M + H]+, 1167 [2 M + Cl]−; HRESIMS m/z 567.2807 [M + H]+ (calcd for C29H43O11, 567.2805). Antiaroside N (5): colorless syrup; [α]28 D +4.4 (c 0.70, MeOH); UV (MeOH) λmax (log ε) 217 (4.18) nm; 1H NMR (pyridine-d5, 400 MHz), see Table 2; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 589 [M + Na]+, 1167 [2 M + Cl]−; HRESIMS m/z 567.2811 [M + H]+ (calcd for C29H43O11, 567.2805). Antiaroside O (6): colorless powder; [α]28 D −40.0 (c 1.00, MeOH); 1 H NMR (pyridine-d5, 300 MHz), see Table 3; 13C NMR (pyridine-d5, 75 MHz), see Table 1; ESIMS m/z 551 [M + H]+, 549 [M − H]−; HRESIMS m/z 551.2841, [M + H]+ (calcd for C29H43O10, 551.2856). 1 Antiaroside P (7): colorless syrup; [α]28 D −16.7 (c 1.05, MeOH); H NMR (pyridine-d5, 500 MHz), see Table 3; 13C NMR (pyridine-d5, 125 MHz), see Table 1; ESIMS m/z 1123 [2 M + Na]+, 1135 [2 M + Cl]−; HRESIMS m/z 551.2856, [M + H]+ (calcd for C29H43O10, 551.2856). Antiarodside Q (8): colorless syrup; [α]28 D −25.3 (c 0.98, MeOH); 1 H NMR (pyridine-d5, 300 MHz), see Table 3; 13C NMR (pyridine-d5, 75 MHz), see Table 1; ESIMS m/z 1123 [2 M + Na]+, 1135 [2 M + Cl]−; HRESIMS m/z 551.2841, [M + H]+ (calcd for C29H43O10, 551.2856). Antiaroside R (9): colorless powder, [α]28 D +33.2 (c 1.06, MeOH); IR (KBr) νmax 3408, 2936, 1729, 1630, 1071 cm−1; 1H NMR (pyridine-d5, 400 MHz), see Table 3; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 1187 [2 M + Na]+, 1199 [2 M + Cl]−; HRESIMS m/z 605.2576 [M + Na]+ (calcd for C29H42O12Na, 605.2574). Antiaroside S (10): colorless powder; [α]28 D +44.8 (c 0.60, MeOH); 1 H NMR (pyridine-d5, 400 MHz), see Table 3; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 605 [M + Na]+, 581 [M − H]−; HRESIMS m/z 583.2755 [M + H]+ (calcd for C29H43O12, 583.2755). Antiaroside T (11): colorless powder; [α]28 D +5.1 (c 0.90, MeOH); 1 H NMR (pyridine-d5, 400 MHz), see Table 4; 13C NMR (pyridine-d5,

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-5 micromelting point apparatus (Tech, Beijing, P. R. China). Optical rotations were obtained on a P-1020 digital polarimeter (Jasco Corporation). UV spectra were measured on a Jasco V-550 UV/vis spectrophotometer. IR spectra were recorded on a Jasco FTIR-480 Plus spectrometer. NMR spectra were measured on Bruker AV 300, AV 400, and 500 instruments. Chemical shifts were given in ppm (δ) relative to chemical shifts of solvent resonances (pyridine-d5: 7.58 and 135.9 ppm). HRESIMS spectra were obtained on a Micromass Q-TOF mass spectrometer. HPLC was performed on a Cosmosil C18 column (4.6 × 250 mm, 5 μm) and a HPLC system equipped with a Dionex Ultimate 3000 pump, a Dionex Ultimate 3000 diode array detector, a Dionex Ultimate 3000 column compartment, and a Dionex Ultimate 3000 autosampler (Dionex, USA). Semipreparative HPLC was performed on a Shimadzu LC-6AD liquid chromatograph with SPD-20A detector, using an ODS column [YMCPack ODS-A (10.0 × 250 mm, 5 μm, 220 and 254 nm)]. Open column chromatography (CC) was performed using silica gel (200− 300 mesh, Qingdao Haiyang Chemical Group Corp, Qingdao), ODS (50 μm, YMC), and Sephadex LH-20 (Pharmacia). TLC analysis was performed on precoated silica gel GF254 plates (Qingdao Haiyang Chemical Group Corp, Qingdao). Plant Material. Latex of Antiaris toxicaria was collected from the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Yunnan Province, P. R. China, in March 2011. The plant was authenticated by Professor Yu Chen of the Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (ANTO201103) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University. Extraction and Isolation. The latex of A. toxicaria (300 mL, 84.0 g) was suspended in H2O and then partitioned between CHCl3/H2O and n-BuOH/H2O successively to yield a CHCl3-soluble fraction (3.6 g) and a n-BuOH-soluble fraction (11.0 g). The CHCl3 fraction was subjected to open silica gel CC (⦶ 3.3 × 25 cm) using a CHCl3/ MeOH gradient to give 10 subfractions (C1−C10). C8 (79.0 mg) was applied to preparative RP-HPLC and eluted with MeOH/H2O (40:60) to obtain compound 1 (30.4 mg). C9 (0.53 g) was chromatographed over ODS (⦶ 3.5 × 12 cm) medium-pressure liquid chromatography (MPLC) using MeOH/H2O (40:60) to afford five subfractions (C9-1−C9-5). C9-1 was subjected to preparative RPHPLC and eluted with MeOH/H2O (48:52) to afford compounds 20 (77.6 mg) and 22 (100.0 mg). C9-2 was applied to semipreparative RP-HPLC to afford compounds 19 (1.0 mg) and 21 (28.6 mg). C10 (0.93 g) was chromatographed on ODS (⦶ 3.5 × 12 cm) MPLC and eluted with MeOH/H2O (45:55) to give four subfractions (C10.1− C10.4). C10.1 was separated by RP-HPLC on a YMC-Pack ODS-A column (10.0 × 250 mm, 5 μm) with MeOH/H2O (30:70) as the eluent to yield compounds 16 (100.0 mg) and 30 (5.0 mg). C10.2 was applied to semipreparative RP-HPLC eluted by MeOH/H2O (30:70) to obtain compounds 17 (85.0 mg) and 18 (80.5 mg). C10.3 was subjected to RP-HPLC eluted with MeOH/H2O (40:60) to yield compounds 26 (18.4 mg) and 31 (8.9 mg). C10.4 was applied to preparative RP-HPLC and eluted with MeOH/H2O (45:55) to obtain compound 27 (2.3 mg). The n-BuOH fraction was subjected to ODS (⦶ 3.3 × 25 cm) MPLC eluting with a MeOH/H2O gradient to give 10 fractions (B1− B10). B3 was chromatographed on an ODS column (⦶ 3.2 × 60 cm) 1778

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(MTT) at a final concentration of 0.5 mg/mL in RPMI 1640 medium containing 10% FBS for 2 h in the dark at 37 °C. Then 200 μL of DMSO was added to the wells. Cultures were incubated at room temperature (RT) for 15 min and read at 570 nm. Western Blotting. Equal amounts of the lysates were electrophoresed on an 8% SDS-PAGE gel and transferred onto polyvinylidene difluoride membranes, which were then blocked with 5% nonfat milk in TBST [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/ L NaCl, and 0.1% Tween 20] for 1 h, incubated with various primary antibodies overnight at 4 °C, and incubated with secondary antibodies for 1 h. Immunoreactive products were detected by using chemiluminescence with an enhanced chemiluminescence system (ECL, Amersham Biosciences). The dilutions of the primary antibodies were anti-Nur77 (Cell Signal, 3960) in 1:1000 and antiPARP (BD Biosciences, 556494) in 1:1000. The blots were reprobed with anti-β-actin antibody for loading control. Immunofluorescence Microscopy. Cells mounted on glass slides were treated with different compounds, permeabilized with PBS containing 0.01% digitonin for 30 min, and blocked with 1% bovine serum albumin in PBS for 30 min at room temperature, followed by incubation with Nur77 antibodies (1:200) or cleaved caspase-3 (1:300) for 3 h at RT and detection by FITC/Cy3-labeled anti-rabbit IgG (1:200) at RT for 1 h. Cells were costained with 4′6′diamidino-2-phenylindole (DAPI) to visualize nuclei. For mitochondria staining, after treatment of the compounds, medium was removed, prewarmed medium containing MitoTracker Red probe (25 nM) was added, and the mixtures were incubated for 15 min. The images were recorded using a LSM-510 confocal laser scanning microscope system. Apoptosis Assays. For DAPI staining, cells were incubated with vehicle or with 20 nM 7, 9, and 10 in serum-free medium for 12, 24, and 36 h. Detached and attached cells were collected and incubated in PBS containing 50 μg/mL DAPI and 100 μg/mL DNase-free RNase A at 37 °C for 20 min. Apoptotic cells were identified with typical morphology of shrinkage of the cytoplasm, membrane blebbing, and nuclear condensation and/or fragmentation. At least 300 cells from more than five random microscopic fields were counted by two independent investigators.

100 MHz), see Table 1; ESIMS m/z 589 [M + Na]+, 1167 [2 M + Cl]−; HRESIMS m/z 567.2804 [M + H]+ (calcd for C29H43O11, 567.2805). Antiaroside U (12): colorless powder; [α]28 D +30.7 (c 0.30, MeOH); UV (MeOH) λmax (log ε) 215 (4.28) nm; IR (KBr) νmax 3430, 2955, 1720, 1635, 1086, 1033 cm−1; 1H NMR (pyridine-d5, 500 MHz), see Table 4; 13C NMR (pyridine-d5, 125 MHz), see Table 1; ESIMS m/z 589 [M + Na]+, 565 [M − H]−; HRESIMS m/z 567.2809 [M + H]+ (calcd for C29H43O11, 567.2805). Antiaroside V (13): colorless syrup; [α]28 D +1.9 (c 0.26, MeOH); IR (KBr) νmax 3436, 2937, 1724, 1626, 1068, 1033 cm−1; 1H NMR (pyridine-d5, 400 MHz), see Table 4; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 1155 [2 M + Na]+, 1167 [2 M + Cl]−; HRESIMS m/z 567.2800 [M + H]+ (calcd for C29H43O11, 567.2805). Antiaroside W (14): colorless powder; [α]28 D −33.2 (c 0.50, MeOH); IR (KBr) νmax 3439, 1716, 1630, 1128, 1064 cm−1; 1H NMR (pyridine-d5, 400 MHz), see Table 4; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 1123 [2 M + Na]+, 1135 [2 M + Cl]−; HRESIMS m/z 551.2856 [M + H]+ (calcd for C29H43O10, 551.2856). Antiaroside X (15): colorless, amorphous powder; [α]28 D −66.8 (c 0.25, MeOH); 1H NMR (pyridine-d5, 400 MHz), see Table 4; 13C NMR (pyridine-d5, 100 MHz), see Table 1; ESIMS m/z 1127 [2 M + Na]+, 587 [M + Cl]−; HRESIMS m/z 553.3015 [M + H]+ (calcd for C29H45O10, 553.3013). X-ray Crystallographic Analysis of 1. Colorless blocks, C29H42O10 2(H2O), M = 586.66, orthorhombic, space group P212121, a = 8.99910(10) Å, b = 10.3062(2) Å, c = 30.5548(5) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 2833.85(8) Å3, Z = 4, dx = 1.375 Mg/m3, F(000) = 1264, μ(Cu Kα) = 0.887 mm−1. Data collection was performed on a Sapphire CCD using graphite-monochromated Cu Kα radiation (λ = 1.54184 Å) at 173.0 K; 7292 reflections were collected to θmax = 62.78°; 4070 independent reflections were obtained, with 3908 reflections above the designated intensity threshold for observation [F2 > 4σ(F2)]. The structures were solved by direct methods (SHELXS 97) and refined by full-matrix least-squares on F2. In the structure refinements, non-hydrogen atoms were placed on the geometrically ideal positions by the “ride on” method. Hydrogen atoms bonded to oxygen were located by the structure factors with isotropic temperature factors. The final R = 0.0369, Rw = 0.1044, and S = 1.103, and Flack = 0.10 (18). Crystallographic data for compound 1 reported in this paper have been deposited with the Cambridge Crystallographic Data Centre (CCDC 924893). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44-(0)1223-336033 or e-mail: [email protected]]. Acid Hydrolysis Reactions of 2, 5, 8, 9, and 11. The cardiac glycosides (1.0 mg each) were hydrolyzed using 2 mL of 2 N HCl for 1 h at 80−90 °C. The resulting mixtures were extracted with EtOAc (2 × 2 mL). The aqueous layers were concentrated and heated with Lcycteine methyl ester in pyridine at 60 °C for 1 h. Sugar (D/L) standards were also derivatized using L-cycteine methyl ester in the same manner. Then arylisothiocyanates were added to the reaction mixtures, which were then stirred at 60 °C for 1 h. The reaction mixtures were analyzed using C18 HPLC with a UV detector (at 250 nm).10 The retention times (min) of the derivatized standards were as follows: D-glucose (21.04), L-glucose (19.12), L-rhamnose (28.23). By comparing the retention times with those of the standards, the sugar in compounds 2, 5, 8, and 11 was determined to be L-rhamnose, and the sugar in 9 was identified as D-glucose. Cell Culture. The NIH-H460 cell line was purchased from the American Type Culture Collection and maintained in RPMI 1640 medium containing 10% FBS and 1% L-glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin. MTT Assay. Cells were counted using a hemocytometer, equally distributed in 96-well plates (1 × 104 cells per well) and treated with 1−32 (50 nM) for 48 h, and then cell proliferation was evaluated with an MTT assay procedure as previously described.25,26 To determine cell viability, the medium was removed and cells were incubated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide

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ASSOCIATED CONTENT

S Supporting Information *

Structures of 16−32 (Figure S1). Key HMBC and ROESY correlations of 5 and 8 (Figure S2). IC50 values of 7, 9, and 10 (Figure S3). Apoptotic effects of 7, 9, and 10 (Figure S4). NMR spectra of 1−15 (Figures S5−S34). X-ray data of 1. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +86 20 85225849. E-mail: [email protected]. *Tel: +86 59 22181851. Fax: +86 59 22181879. E-mail: [email protected]. Author Contributions ∥

Q. Liu and J.-S. Tang contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Mr. T. Shi, Mr. J. Geng, and Dr. Y. Yu for the HRESIMS and NMR measurements. This work was supported by grants from National Natural Science Foundation of China (81001373 and 91129302) and the Fund of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2010-KF04). 1779

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