Minor cytotoxic cardenolide glycosides from the root of Streptocaulon juventas

Steroids 93 (2015) 39–46

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Minor cytotoxic cardenolide glycosides from the root of Streptocaulon juventas Chun Ye a, Hua Wang a, Rui Xue a, Na Han a, Lihui Wang b, Jingyu Yang b, Yu Wang c, Jun Yin a,⇑ a Development and Utilization Key Laboratory of Northeast Plant Materials, Key Laboratory of Northeast Authentic Materials Research and Development in Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China b Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China c The People’s Liberation Army 463 Hospital, Shenyang 110042, China

a r t i c l e

i n f o

Article history: Received 24 April 2014 Received in revised form 16 October 2014 Accepted 29 October 2014 Available online 18 November 2014 Keywords: Asclepiadaceae Streptocaulon juventas Cardenolide glycoside Antitumor Structure–activity relationship

a b s t r a c t In order to determine new minor natural cardenolide glycosides as cytotoxic candidates, we isolated six new cardenolide glycosides together with four known ones, which had never previously been reported in the genus, by bioassay-guided separation from the 75% ethanol extract of Streptocaulon juventas (Asclepiadaceae). Their structures were elucidated on the basis of spectroscopic analysis, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC). The cytotoxic activities of these compounds were evaluated against A549 and NCI-H460 cell lines by MTT assay and compound 7 exhibited inhibitory activity against the two cell lines, while other compounds displayed a range of inhibitory activity against NCI-H460 and A549 cells. Their structure–activity relationships were also discussed. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Streptocaulon is a small genus of the Asclepiadaceae family which contains only five species. It has not been studied in detail until recently when it attracted interest because it exhibited strong anti-proliferative activity [1]. A survey of the literature showed that cardenolide glycosides were the biologically active compounds in this genus along with the discovery of a class of cardiac glycosides with preventive and therapeutic effects on proliferative diseases such as cancer [2–5]. Moreover, these cardenolide glycosides were associated with typical genins, such as digitoxigenin, acovenosigenin A, periplogenin, 17b-H-periplogenin, oleandrigenin, 16-O-acetylacovenosigenin, 16-O-acetylperiplogenin, 3b,5b,14b-trihydroxyl-card-16,20(22)-dienolide, 1b,3b,14b-trihydroxy-5b-card-16,20(22)-dienolide, 1a,14b-dihydroxy-5b-card20(22)-enolide and evonogenin, and their sugar moieties included b-D-cymarose, b-D-digitoxose, b-D-digitalose and b-D-glucose [3–5]. Among these compounds, most of the novel cardenolide glycosides contained the skeleton of acovenosigenin A. Despite this, statistical data from SciFinder showed that only ten cardenolides with the acovenosigenin A moiety have been identified in the natural world, ⇑ Corresponding author at: School of Traditional Chinese Materia Medica 48#, Shenyang Pharmaceutical University, Wenhua Road 103, Shenhe District, Shenyang 110016, China. Tel./fax: +86 24 23986491. E-mail address: [email protected] (J. Yin). http://dx.doi.org/10.1016/j.steroids.2014.10.005 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

in the Streptocaulon, Saussutea, Euonymus and Acokanthera genus [5–8]. Streptocaulon juventas, has a root which has been used as a folk medicine for the treatment of rheumatism, neurasthenia and dyspepsia patients since ancient times in the Guangxi and Yunnan provinces of China [9]. In 2002, Ueda et al. reported that the MeOH extract of the roots of S. juventas possessed potent antiproliferative activity against the human HT-1080 fibrosarcoma cell line in a study involving 231 extracts from 77 Vietnamese medicinal plants [3]. We have previously reported that the most active fraction, the 75% ethanol extract, obtained from the roots of S. juventas strongly inhibited the A549 cell line in vitro (IC50 value, 0.89 lg/mL) and also tumor growth in A549 tumor-bearing mice (inhibition rate: 58%) [2], 39 cardenolide glycosides have already been isolated from this plant [4,5]. It appears to be a good raw medicinal material due to the fact that it has a total product yield of cardenolides up to 2.35 mg/g (analyzed by the content of digitoxigenin) [10]. As part of our continuing search to discover novel cardenolides, a further phytochemical analysis has been carried out on the minor bioactive compounds with interesting structures obtained from the active fraction of the plant, so that 1-hydroxylated cardenolides present in trace amounts (< 0.01%) were isolated and identified, illustrating the structural diversity of cardiac glycosides [11]. In this paper, TLC, MPLC and HPLC techniques were mainly used and these allowed us to discover six new cardenolides (1–6),

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C. Ye et al. / Steroids 93 (2015) 39–46

together with four known ones (7–10) which had never been found in the genus before (Fig. 1). Using bioactivity-guided isolation, we obtained four new compounds possessing the aglycone of acovenosigenin A. The structures of the new cardenolides were elucidated by analysis of 1 and 2D NMR and MS data. Compounds 1–3, 5–10 exhibited potent anti-proliferative activity in the NCI-H460 and A549 cell lines.

2.2. Plant material The roots of S. juventas were collected in Yunnan Province, China, in 2009. The plant material was identified by Prof. Jun Yin (Division of Pharmacognosy, Shenyang Pharmaceutical University), and a voucher sample (SPU 2162–2165) was deposited at the herbarium of Shenyang Pharmaceutical University, Shenyang, China. 2.3. Extraction and isolation

2. Experimental 2.1. General Optical rotations were determined on a Perkin–Elmer 241MC polarimeter using a 0.1 dm cell and using MeOH as a blank. NMR spectra were obtained on Bruker ARX-300 and ARX-600 spectrometers, using TMS as an internal standard. HR-TOFMS was obtained on a Bruker microTOF mass spectrometer, equipped with an electrospray ionization (ESI) ion source. Ultraviolet–visible spectra was recorded on UV-2201 Shimadzu UV–vis scanning spectrophotometer using MeOH as a blank. Column chromatography was peformed with macroporous resin HPD100 (Tianjin Resin Co., Ltd.), silica gel (100–200 mesh and 200–300 mesh; Qingdao Marine Chemical Co., Ltd.), ODS-A (50 lM; YMC), and Sephadex LH-20 (GE Healthcare). Preparative HPLC was conducted on a YMC ODS-A column (250  30 mm I.D., 5 lM) equipped with a pump and a single-wavelength UV detector. Analytical HPLC was carried out on a Shimadzu LC10-A HPLC system with an LC-10ATVP pump, a Shimadzu LC-10AVP UV–vis detector (Shimadzu Co., Ltd.), and an N-2000 chromatographic work station (Intelligent Information Engineering Co., Ltd.) using a C18 column (250  4.6 mm). Cisplatin was purchased from Sigma Co., Ltd. with a purity of 99%. All chemical reagents used in the studies were produced by Yuwang Chemicals Industries, Ltd. The human lung A549 adenocarcinoma cell line and large cell carcinoma NCI-H460 cell line were purchased from American Type Culture Collection (ACTT, Manassas, U.S.A.).

R119

11

9H

1 2

R2O

1 2 3 4 5 6 7 8 9 10

R1 H H OH OH OH OH H H H H

3

10

H

R3

6

5 4

12

8

13 14

OH

O

23

O 21 18

20

22

17 16

R4

15

7

R2 (2-O-Ac-Dtl)4-Glc6-Glc Dig4-Glc4-Glc Cym4-Glc6-Glc (2-O-Ac-Dtl)4-Glc6-Glc (2-O-Ac-Dtl)4-Glc6-Glc Cym4-Dtl4 -Glc6-Glc Dtl4-Glc6-Glc Glc4-Glc (3-O-Ac-Dig)4-Glc H

R3 OH OH H H H H H H H OH

R4 H H H H OAc H H H H OH

Cym:β-D-cymarose Dig: β-D-digitoxose Dtl: β-D-digitalose Glc: β-D-glucose Fig. 1. Chemical structures of compounds 1–10.

The milled roots of S. juventas (30 kg, air-dried) were extracted with 75% EtOH (300 L, reflux, 1.5 h  3). The residue was concentrated under reduced pressure, then separated on a HPD100 macroporous resin column and eluted with a gradient mixture of H2O/EtOH (100:0, 70:30, 40:60, 10:90 v/v) to give four fractions (H2O, 30%, 60%, 90% EtOH). Fr. 60% EtOH (315 g) which showed significant activity in the human lung A549 adenocarcinoma cell line with an IC50 value of 1.44 lg/mL [10], was subjected to open-column chromatography on a silica gel column (200–300 mesh, 8.5  20 cm, 650 g) with stepwise gradient elution with a mixture of petroleum ether and EtOAc (5:1, 2:1, 1:1) followed by CH2Cl2/ MeOH (30:1, 20:1, 10:1, 5: 1, 3:1, 1:1, each 5 L) to give five fractions (Fr. 1-Fr. 5). Fr. 5 (95.5 g) was further separated into three fractions (Fr. 5-1- Fr. 5–3) by MPLC on a silica gel column (200– 300 mesh, 6.5  32 cm, 530 g), eluting with CH2Cl2/MeOH/H2O (5:1:1, 3:1:1, 1:1:1; 3 L each). A portion of Fr. 5–1 (8.0 g) was further chromatographed on an ODS column (120 g) using a MeOH/ H2O gradient as the mobile phase to give fraction A [MeOH/H2O (3:7)] and fraction B [MeOH/H2O (7:13)] by MPLC. The fractions collected were monitored and recombined based on their TLC profiles. Fr. A (634 mg) was purified using a Sephadex LH-20 column and elution with MeOH followed by preparative HPLC (MeOH/ H2O 50:50, 9.0 mL/min) to yield 5 (10 mg, tR 27.0 min). Fr. B (3.1 g) was loaded onto a Sephadex LH-20 column and eluted with MeOH to obtain six fractions (Fr. B1–B6). Fr. B1 (102 mg) was purified by preparative HPLC (27% Acetonitrile–H2O, 9.0 mL/min) to yield 1 (48 mg, tR 100.0 min). Fr. B2 (148 mg) was purified by preparative HPLC (23% Acetonitrile–H2O, 9.0 mL/min) and silica gel column chromatography (EtOAc/MeOH/H2O 5:1:0.9) to yield 2 (6 mg). Fr. B3 (112 mg) was purified by preparative HPLC (23% Acetonitrile–H2O, 9.0 mL/min) to yield 3 (32 mg, tR 73.0 min). Fr. B4 (38 mg) was purified by silica gel column chromatography (EtOAc/MeOH/H2O 5:1:0.9) to yield 4 (26 mg). Fr. B5 (108 mg) was purified by preparative HPLC (23% Acetonitrile–H2O, 9.0 mL/ min) to yield 6 (10 mg, tR 224 min) and 7 (14 mg, tR 94 min). Fr. B6 (111 mg) was purified by preparative HPLC (25% Acetonitrile– H2O, 9.0 mL/min) followed by silica gel column chromatography (EtOAc/MeOH/H2O 5:1:0.9) to yield 8 (11 mg). A portion of Fr. 5–2 (7.0 g) was subjected to ODS column chromatography and gradient elution with MeOH/H2O to give fraction C [MeOH/H2O 60:40], and Fr. C (480 mg) was purified by preparative HPLC (50% MeOH–H2O, 9.0 mL/min) and silica gel column chromatography (EtOAc/Acetone/H2O 1:1:0.1) to yield 9 (4.2 mg) and 10 (1 mg). 2.4. Periplogenin 3-O-[O-b-D-glucopyranosyl-(1?6)-O-b-Dglucopyranosyl-(1?4)-2-O-acetyl-b-D-digitalopyranoside] (1) White powder (MeOH); ½a23 D 5.6 (c 0.49, MeOH); UV (MeOH) kmax 218; HR–ESI-MS m/z 939.4183 [M+Na]+ (calcd for C44H68O20Na, 939.4196); 1H NMR and 13C NMR, see Table 1. 2.5. Periplogenin 3-O-[O-b-D-glucopyranosyl-(1?4)-O-bDglucopyranosyl-(1?4)-b-D-digitoxopyranoside] (2) White powder (MeOH); ½a23 D +17.2 (c 0.29, MeOH); UV (MeOH) kmax 213; HR–ESI–MS m/z 889.4030 [M+HCOO] (calcd for C42H65O20, 889.4075); 1H NMR and 13C NMR, see Table 1.

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C. Ye et al. / Steroids 93 (2015) 39–46 Table 1 H and 13C NMR data of compounds 1, 2 and 3 (CD3OD).

1

Position

1a dH (J in Hz)

dC

dH (J in Hz)

dC

dH (J in Hz)

dC

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

1.35 m, 1.70 m 1.64 m, 1.77 m 4.13 br s 1.51 m, 2.19 m

26.5 26.9 78.6 36.1 75.0 35.6 24.8 41.6 40.1 41.8 22.6 40.9 50.9 86.3 33.3 28.0 51.9 16.3 17.2 178.3 75.3

1.39 m, 1.72 m 1.66 m, 1.78 m 4.16 br s 1.58 m, 2.18 m

26.6 26.8 77.3 35.9 75.7 35.5 24.8 41.7 40.2 41.8 22.7 40.9 50.9 86.3 33.3 28.0 51.9 16.3 17.2 178.4 75.3

3.70 br s 1.90 m, 2.09 m 4.17 br s 1.51 m, 1.97 m 2.10 m 1.35 m, 1.85 m 1.29 m, 1.34 m 1.68 m 1.59 m

74.0 32.1 76.6 31.2 32.1 27.3 22.3 42.7 38.4 41.2 22.1 40.8 50.9 86.2 33.3 27.3 52.0 16.4 19.2 178.3 75.3

22 23 10 20 30 40 50 60 3-O-Me 2-O-Ac

2b

1.31 m, 1.71 m 1.19 m, 1.92 m 1.61 m 1.61 m 1.31 m, 1.45 m 1.48 m, 1.51 m

1.71 m, 2.13 m 1.87 m, 2.16 m 2.83 m 0.88 s 0.92 s 4.91 dd (1.5, 18.3) 5.00 dd (1.3, 18.6) 5.90 s

117.8 177.2 102.5 72.7 83.4 75.4 71.8 17.4 58.7 172.2 21.1 104.6 75.8 77.8 71.8 77.4 70.4

4.53 d (8.0) 5.09 dd (8.0, 10.1) 3.47 dd (2.9, 10.1) 4.25 d (2.4) 3.69 m 1.31 d (6.4) 3.44 s

10 0 20 0 30 0 40 0 50 0 60 0

2.09 s 4.54 d (7.5) 3.26 m 3.37 m 3.29 m 3.46 m 3.76 dd (6.6, 11.8) 4.15 m

10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0

4.40 d (7.7) 3.19 m 3.33 m 3.27 m 3.24 m 3.66 m, 3.87 m

105.1 75.2 78.1 71.7 78.1 62.9

a 1

13

b 1

13

H NMR data were measured at 300 MHz, and H NMR data were measured at 600 MHz, and

3a

1.34 m, 1.71 m 1.21 m, 1.95 m 1.64 d (3.1) 1.62 m 1.32 m, 1.47 m 1.52 m

1.71 m, 2.15 m 1.87 m, 2.16 m 2.84 m 0.88 s 0.93 s 4.91 dd (1.7, 18.5) 5.03 dd (1.5, 18.5) 5.90 s 4.94 dd (1.8, 9.7) 1.71 m, 1.97 m 4.29 dd (2.9, 6.5) 3.27 m 3.88 dt (2.1, 11.8) 1.29 d (6.2)

4.41 3.28 3.50 3.60 3.41 3.65 3.87

117.8 177.2 98.0 38.9 68.4 84.2 69.7 18.5

1.81 m 1.29 m, 1.49 m

1.73 m, 2.17 m 1.35 m, 1.85 m 2.81 m 0.88 s 1.07 s 4.91 dd (1.5, 17.1) 5.03 dd (1.5, 18.5) 5.90 s 4.82 (overlap) 1.65 m, 2.08 m 3.98 d (2.8) 3.36 m 3.88 m 1.31 d (6.2) 3.46 s

117.8 177.2 97.8 36.9 78.6 83.8 70.4 18.6 58.7

d (7.8) dd (2.4, 9.6) t (9.0) t (9.2) dt (9.6, 3) dd (5.6, 11.9) dt (2.1, 11.8)

105.6 74.8 76.3 80.0 76.2 62.4

4.35 d (7.7) 3.24 m 3.34 m 3.25 m 3.49 m 3.71 m, 4.17 m

106.2 75.3 77.9 71.9 76.9 70.6

4.41 d (7.8) 3.22 m 3.36 m 3.30 m 3.33 dd (2.2, 5.7) 3.82 dd (2.3, 12.0) 3.90 m

104.6 74.9 77.8 71.4 78.1 61.4

4.35 d (7.7) 3.19 m 3.31 m 3.32 m 3.25 m 3.65 m, 3.87 m

105.1 75.2 78.1 71.7 78.1 62.9

C NMR data were measured at 75 MHz. C NMR data were measured at 150 MHz.

2.6. Acovenosigenin A 3-O-[O-b-D-glucopyranosyl-(1?6)-O-b-Dglucopyranosyl-(1?4)-b-D-cymaropyranoside] (3)

2.8. 16-O-acetyl-hydroxyacovenosigenin 3-O-[O-b-D-glucopyranosyl(1?6)-O-b-D-glucopyranosyl-(1?4)-2-O-acetyl-b-Ddigitalopyranoside] (5)

White powder (MeOH); ½a23 15.6 (c 0.18, MeOH); UV D (MeOH) kmax 216; HR–ESI–MS m/z 881.4141 [M+Na]+ (calcd for C42H66O18Na, 881.4141); 1H NMR and 13C NMR, see Table 1.

White powder (MeOH); ½a23 D 37.6 (c 0.19, MeOH); UV (MeOH) kmax 212; HR–ESI–MS m/z 997.4228 [M+Na]+ (calcd for C46H70O22Na, 997.4251); 1H NMR and 13C NMR, see Table 2.

2.7. Acovenosigenin A 3-O-[O-b-D-glucopyranosyl-(1?6)-O-b-Dglucopyranosyl-(1?4)-2-O-acetyl-b-D-digitalopyranoside] (4)

2.9. Acovenosigenin A 3-O-[O-b-D-glucopyranosyl-(1?6)-O-b- Dglucopyranosyl-(1?4)-O-b-D-digitalopyranosyl-(1?4)-b-Dcymaropyranoside] (6)

White powder (MeOH); ½a23 D 33.1 (c 0.16, MeOH); UV (MeOH) kmax 218; HR–ESI–MS m/z 939.4188 [M+Na]+ (calcd for C44H68O20Na, 939.4196); 1H NMR and 13C NMR, see Table 2.

White powder (MeOH); ½a23 D 16.2 (c 0.21, MeOH); UV (MeOH) kmax 216; HR–ESI–MS m/z 1041.4900 [M+Na]+ (calcd for C49H78O22Na, 1041.4877); 1H NMR and 13C NMR, see Table 2.

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C. Ye et al. / Steroids 93 (2015) 39–46

2.10. Odoroside G (7) 24 White power (MeOH); ½a20 D 11.0 (c 0.27, MeOH) [[12] ½aD 17.0 ± 2 (c 1.060, MeOH)].

2.11. Digitoxigenin 3-O-b-D-cellobioside (8) 1 White power (MeOH); ½a20 D +8.1 (c 0.12, MeOH); H NMR (CD3OD, 300 MHz): d 5.89 (1H, s, H-22), 5.00 (1H, d, J = 18.6 Hz, H-21), 4.91 (1H, dd, J = 18.3, 1.5 Hz, H-21), 4.41 (1H, d, J = 7.8 Hz, H-10 ), 4.35(1H, d, J = 7.7 Hz, H-10 0 ); 4.06 (1H, br s, H-3), 2.83 (1H, m, H17), 0.96 (3H, s, H-19), 0.88 (3H, s, H-18), 13C NMR (CD3OD, 75 MHz): d 178.5 (C-20), 177.3 (C-23), 117.8 (C-22), 104.6 (C-10 ), 102.6 (C-100 ), 86.5 (C-14), 80.9 (C-40 ), 78.1 (C-300 ), 77.9 (C-50 0 ), 76.6 (C-30 ), 76.4 (C-50 ), 75.8 (C-200 ), 75.4 (C-21), 75.0 (C-20 ), 74.9 (C-3), 71.4 (C-400 ), 62.5 (C-600 ), 62.0 (C-60 ), 52.2 (C-17), 51.1 (C13), 42.7 (C-8), 41.0 (C-12), 37.5 (C-5), 36.9 (C-9), 36.3 (C-10), 33.4 (C-15), 31.3 (C-1), 30.9 (C-4), 28.1 (C-16), 27.8 (C-2), 27.5 (C-6), 24.1 (C-19), 22.6 (C-11), 22.4 (C-7), 16.4 (C-18).

2.12. Digitoxigenin-3-O-b-D-glucosyl-(1?4)-3-O-acetyl-b-Ddigitoxoside (9) 25 White power (MeOH); ½a20 D +20.0 (c 0.17, MeOH) [(13) ½aD 1 +29.1 (c 0.75, MeOH)]; H NMR (CD3OD, 300 MHz): d 5.89 (1H, s, H-22), 5.49 (1H, m, H-10 ), 5.00 (1H, d, J = 18.3 Hz, H-21), 4.93 (1H,d, J = 1.5 Hz, H-21), 4.36 (1H, d, J = 7.7 Hz, H-100 ); 4.01 (1H, br s, H-3), 2.83 (1H, m, H-17), 2.08 (3H, s, 3-O-Me), 1.32 (3H, d, J = 6.2 Hz, H-10 ), 0.94 (3H, s, H-19), 0.88 (3H, s, H-18), 13C NMR (CD3OD, 150 MHz): d 178.5 (C-20), 177.3 (C-23), 172.8 (C-2-OAc), 117.8 (C-22), 105.8 (C-100 ), 97.6 (C-10 ), 86.5 (C-14), 81.6 (C40 ), 78.0 (C-300 ), 78.0 (C-500 ), 75.4 (C-200 ), 75.3 (C-21), 75.1 (C-3), 72.0 (C-30 ), 71.5 (C-400 ), 70.6 (C-50 ), 63.0 (C-600 ), 52.2 (C-17), 51.1 (C-13), 42.7 (C-12), 41.0 (C-8), 38.1 (C-9), 37.5 (C-5), 36.9 (C-10), 36.4 (C-20 ), 33.4 (C-15), 31.4 (C-1), 31.4 (C-4), 28.1 (C-16), 27.9 (C-6), 27.6 (C-2), 24.3 (C-19), 22.6 (C-7), 22.4 (C-11), 21.3 (C-3-OMe), 18.6 (C-60 ), 16.4 (C-18).

2.13. 5b-Hydroxygitoxigenin (10) White power (MeOH); ½a20 +13.7 (c 0.26, MeOH) [[14] D ½a20 D +48.6 (c 0.62, MeOH)]. 2.14. Identification of the monosaccharide of compounds 1–6 A solution containing compound 1 (1 mg) in 1, 4-dioxane (0.5 mL) and 1 N HCl (4.5 mL) was heated at 90 °C for 6 h. After cooling, the mixture was extracted with EtOAc (3  5 mL), followed by neutralizing the aqueous layer with 1 N KOH. Compounds 2–6 were also treated in the same way. The monosaccharides were purified by preparative TLC, then identified using n-BuOH-Me2CO-H2O (8:5:1) and the Rf values were 0.45, 0.60, 0.80 and 0.85 for glucose, digitalose, digitoxose and cymarose, respectively. The absolute configurations were determined by measurement of the optical rotations as D-glucose [½a23 D +51.3 (c 0.1, H2O)], D-digitalose [½a23 D +105.0 (c 0.1, H2O)], D-digitoxose 23 [½a23 D +42.1 (c 0.03, H2O)] and D-cymarose [½aD + 55.9 (c 0.07, H2O)]. We figured out the position of the sugar attachments by the H–C long range correlations in the HMBC spectrum and 1H and 13C NMR chemical shifts, and mentioned it in detail in the structure analysis of results and discussion part. 2.15. In vitro anti-proliferation bioassay A549 and NCI-H460 cells were cultured in RPMI-1640 medium (GIBCO, NY, U.S.A.) and maintained at 37 °C in 5% CO2. After

incubation for 24 h at 37 °C at the appropriate density of 8  104/ mL in 96-well tissue culture plates (100 lL per well), compounds 1–3, 5–10 were added after being dissolved in dimethyl sulfoxide (DMSO) and diluted at a concentration of 100 lmol/L (the final concentration of DMSO was 0.1%), then the cells continued to be incubated with the nine compounds for 48 h. Cell growth was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay incubated for 3–4 h at 37 °C. Then, the medium was removed, and the cells were dissolved in 100 lL dimethyl sulfoxide and the absorbance at 492 nm was measured. Cisplatin (100 lmol/L) was used as a positive control. The percentage of cell growth inhibition was calculated as follows: Cell growth inhibition ð%Þ ¼ ½A549 ðcontrolÞ  A549 ðcompoundÞ=A549 ðcontrolÞ  100

The IC50 values of compounds that inhibited cell viability by over 50% at a concentration of 100 lM were calculated. All cytotoxic activity data were analyzed by SPSS (16.0) and expressed as mean ± S.E. 3. Results and discussion Compound 1, a white amorphous powder, with the formula C44H68O20 having an HR–ESI–MS at m/z 939.4183 [M+Na]+ ion (calcd for 939.4196). The 1H NMR and 13C NMR spectra displayed characteristic signals of a cardenolide [dH 5.90 (1H, s, H-22), 5.00 (1H, dd, J = 18.6, 1.3 Hz, H-21), 4.91 (1H, dd, J = 18.3, 1.5 Hz, H-21), dC 178.3 (C20), 75.3 (C-21), 117.8 (C-22), 177.2 (C-23)] representative of an a,b-unsaturated c-lactone, and two methyl groups [dH 0.88 (s, 18-Me), 0.92 (s, 19-Me), dC 16.3 (C-18), 17.2 (C-19)]. In addition, one oxymethyl [dH 3.44 (s), dC 58.7] and one acetyl [dH 2.09 (s), dC 172.2, 21.1] group were observed. The aglycone part of 1 was identified as periplogenin by the above analysis and comparison with the1H and 13C NMR resonances in the Ref. [15]. The presence of three sugar units in 1 was indicated by three anomeric proton signals at dH 4.53 (1H, d, J = 8.0 Hz, H-10 ), dH 4.40 (1H, d, J = 7.7 Hz, H-1000 ) and dH 4.54 (1H, d, J = 7.5 Hz, H-10 0 ). It was also evident from the 1H and 13C NMR data that one of the sugar units was 6-deoxysugar due to the proton signal displayed at 1.31 (d, J = 6.4 Hz, H-60 ) [16]. The HMBC correlation between the methoxy protons (dH 3.44) and C0 -3 (dC 83.4), showed that the methoxy group was located at C0 -3. The methyl protons (dH 2.10, dC 21.1) and the carbonyl carbon (dC 172.2) indicated the presence of an acetyl group, which was located at C-2’ of digitalose by the connectivity from dH 5.09 (H-20 ) to dC 172.2 in HMBC (Fig. 2). Complete assignment of the sugar protons and carbons by 1H–1H COSY and HSQC experiments showed characteristic signals of one 2-O-acetyl b-digitalopyranosyl unit (dC 102.5, 72.7, 83.4, 75.4, 71.8, 17.4, 58.7, 172.2, 21.1) and two b-glucopyranosyl (dC 104.6, 75.8, 77.8, 71.8, 77.4, 70.4; 105.1, 75.2, 78.1, 71.7, 78.1, 62.9) units (Table 1). An unambiguous determination of the sugar sequencing and linkage sites was made from the HMBC spectrum. The HMBC correlations from H-1000 (dH 4.40) to C-600 (dC 70.4), H-100 (dH 4.54) to C-40 and H-10 (dH 4.53) to C-3 (dC 78.6) of the aglycone moiety, suggested the connections of the sugar units, and the connection between the glycosides and aglycone. On the basis of this evidence, the structure of compound 1 was established as periplogenin 3-O[O-b-D-glucopyranosyl-(1?6)-O-b-D-glucopyranosyl-(1?4)-2-Oacetyl-b-D-digitalopyranoside]. Compound 2, obtained as a white amorphous powder, had a molecular formula of C41H64O18 from the HR–ESI–MS at m/z 889.4030 [M+HCOO] ion in negative mode (calcd for 889.4075). Compared with the 1D and 2D NMR spectroscopic data of compound 1, the aglycone of 2 was identical to that of 1. The rest of the

43

C. Ye et al. / Steroids 93 (2015) 39–46 Table 2 H and 13C NMR data of compounds 4, 5 and 6 (CD3OD).

1

Position

4a

1 2

3.66 m 1.97 m

73.7 32.4

3.65 m 1.98 m

73.7 32.4

3 4

4.22 br s 1.55 m, 1.94 m

75.8 29.9

4.23 br s 1.55 m, 1.94 m

75.8 29.9

5 6 7 8 9 10 11 12 13 14 15

1.76 m 1.35 m, 1.85 m 1.33 m, 1.81 m 1.68 m 1.59 m

32.1 27.3 22.3 42.7 38.4 41.3 22.1 40.7 50.9 86.1 33.3

1.75 m 1.36 m, 1.85 m 1.25 m, 1.84 m 1.67 td (3.5, 11.9) 1.56 m

32.1 27.2 21.8 42.7 38.2 41.3 21.9 39.8 51.2 84.8 41.2

16 17 18 19 20 21

1.87 m, 2.17 m 2.82 m 0.88 s 1.06 s

dH (J in Hz)

22 23 16-OAcc 10 20 30 40 50 60 3-O-Me 2-O-Acc 10 0 20 0 30 0 40 0 50 0 60 0 3-O-Me 10 0 0 20 0 0 30 0 0 40 0 0 50 0 0 60 0 0

1.29 m, 1.84 m 1.45 m, 1.47 m

1.73 m, 2.17 m

4.91 dd (1.8, 16.5) 5.03 dd (1.5, 18.0) 5.90 br s

4.49 d (8.1) 5.10 m 3.50 dd (2.9, 10.2) 4.27 d (2.5) 3.69 m 1.32 d (6.3) 3.45 s 2.06 s 4.54 d (7.5) 3.25 m 3.36 m 3.30 m 3.47 m 3.76 dd (6.6, 11.7) 4.16 dd (1.5, 11.7) 4.40 d (7.8) 3.20 m 3.30 m 3.30 m 3.26 m 3.66 m 3.87 d (12.3)

5b dC

28.1 52.0 16.4 19.3 178.3 75.3 117.8 177.2

99.6 72.9 83.1 75.2 71.8 17.5 58.8 172.1 21.1 104.6 75.7 77.8 71.9 77.4 70.5

105.1 75.2 78.1 71.7 78.0 62.8

6b

dH (J in Hz)

dC

1.22 m, 1.35 m 1.41 m, 1.53 m

1.79 m 2.76 dd (9.8, 15.5) 5.45 dt (2.3, 9.0) 3.25 d (9.0) 0.94 s 1.05 s

75.9 57.3 16.4 19.3 171.5 77.5

4.95 dd (1.5, 18.5) 5.01 dd (1.5, 18.5) 5.98 br s

1.94 s 4.49 d (8.4) 5.09 t (9.3) 3.49 d (3.0) 4.26 d (3.0) 3.69 m 1.32 d (6.4) 3.45 s 2.07 s 4.54 d (7.8) 3.25 m 3.25 m 3.29 m 3.46 d (1.8) 3.75 dd (6.8, 12.0) 4.15 dd (1.8, 12.0) 3.51 s 4.39 d (7.8) 3.19 m 3.32 m 3.27 m 3.25 m 3.65 m 3.86 dd (1.9, 12.0)

121.8 176.7 172.1 20.9 99.6 72.9 83.1 75.2 71.8 17.3 58.7 172.1 21.1 104.6 75.7 78.0 71.9 77.4 70.5 58.9 105.2 75.2 77.8 71.7 78.0 62.8

10 0 0 0 20 0 0 0 30 0 0 0 40 0 0 0 50 0 0 0 60 0 0 0

dH (J in Hz) 3.70 br s 1.89 m 2.09 dd (2.4, 15.0) 4.17 br s 1.50 m 1.95 dd (2.4, 13.8) 2.08 m 1.35 m, 1.84 m 1.34 m, 1.81 m 1.68 d (3.0) 1.59 d (3.6) 1.29 m 1.29 m, 1.47 m

1.73 m, 2.17 m 1.87 m, 2.17 m 2.82 m 0.88 s 1.07 s 4.91 dd (1.2, 18.6) 5.03 dd (1.2, 18.6) 5.89 s

4.83 dd (1.8, 9.6) 1.55 m, 2.13 m 3.83 q (3.0) 3.30 m 3.89 m 1.31 d (6.0)

dC 74.0 32.1 76.6 31.2 32.1 27.3 22.3 42.7 38.4 41.2 22.1 40.8 50.9 86.2 33.3 28.0 52.1 16.4 19.2 178.3 75.3 117.8 177.2

97.7 36.5 78.4 83.8 70.4 18.7

4.30 d (7.8) 3.64 m 3.23 m 4.15 m 3.63 m 1.29 d (6.6)

106.5 71.7 85.5 76.1 71.4 17.7

3.51 s 4.55 d (7.8) 3.22 m 3.35 m 3.28 m 3.45 m 3.77 dd (6.5, 11.8) 4.13 m 4.39 d (7.8) 3.19 m 3.35 m 3.28 m 3.22 m 3.65 m 3.86 dd (2.0, 11.9)

58.9 104.8 75.9 78.0 71.8 77.4 70.3 105.1 75.2 78.1 71.8 78.0 62.8

a 1

H NMR data were measured at 300 MHz, and 13C NMR data were measured at 75 MHz. H NMR data were measured at 600 MHz, and 13C NMR data were measured at 150 MHz. Assignments may be interchanged in each column.

b 1 c

1

H and 13C NMR data of the sugar moieties were found to be similar by comparison with previously published data [17], except for the linkage between the two glucose units. The HMBC correlation from dH 4.41 (H-1000 ) of the outer unit to dC 80.0 (C-400 ) of the inner glucose unit, indicated that the two sugar moieties were (1?4) linked. Thus, the structure of compound 2 was determined to be

periplogenin 3-O-[O-b-D-glucopyranosyl-(1?4)-O-b-D-glucopyranosyl-(1?4) -b-D-digitoxopyranoside]. Compound 3, obtained as a white amorphous powder, had a molecular formula of C42H66O18 as determined by the HR–ESI– MS at m/z 881.4141 [M+Na]+ ion in positive mode (calcd for 881.4141).

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C. Ye et al. / Steroids 93 (2015) 39–46

The 1H NMR spectrum of compound 3 showed two sets of three proton singlet signals at 0.88 (3H, s, 18-Me), and 1.07 (3H, s, 19Me)], one olefinic proton signal at dH 5.90 (1H, s, H-22), and a pair of double doublet signals at 5.03 (dd, J = 18.5, 1.5 Hz, H-21) and 4.91 (dd, J = 17.1, 1.5 Hz, H-21), which were characteristic of a cardenolide skeleton. Two oxymethine signals at dH 3.70 (1H, br s), and 4.17 (1H, br. s) indicated that the aglycone had two O-substituted protons. The aglycone of 3 was determined to be acovenosigenin A by comparison with the data in the Ref. [5]. Also, the three resonances for the anomeric protons at dH 4.82 (overlap), dH 4.35 (d, J = 7.7 Hz) and dH 4.35 (d, J = 7.7 Hz) suggested the presence of three sugar units, and the set of signals at dC 97.8, 36.9, 78.6, 83.8, 70.4, 18.6, and 58.7 suggested the presence of one b-cymaropyranose and the rest of values matched the chemical shifts of two b-glucopyranose units [18]. The HMBC correlations between H-1000 (dH 4.35) and C-600 (dC 70.6), H-100 (dH 4.35) and C-4 0 (dC 83.8), and H-10 (dH 4.82) and C-3 (dC 76.6) confirmed the connectivity of the sugar units. Thus, 3 was concluded to be acovenosigenin A 3-O[O-b-D-glucopyranosyl-(1?6)-O-b-D-glucopyranosyl-(1?4)-O-b-Dcymaropyranoside]. Compound 4, obtained as a white amorphous powder, exhibited an [M+Na]+ ion at m/z 939.4188 in positive mode of the HR–ESI– MS, establishing that its molecular formula was C44H68O20 (calcd for 939.4196). Comparison of the 13C NMR data of compound 4 with that of 3 [C-1 (dC 73.7), C-5 (dC 32.1) C-19 (dC 19.3)], it was concluded that the aglycone of 4 was acovenosigenin A. However, analyses of the 13C NMR and 1H NMR signals of the sugar moiety of 4, showed that it consisted of one 2-O-acetyl b-digitalopyranosyl unit and two b-glucopyranosyl units, similar to the sugar moiety of 1. The (1?6) linkage between the two glucoses, and the (1?4) linkage between the inner glucose and digitalose was established by the correlation between H-1000 (dH 4.40) and C-600 (dC 70.5), and the correlation between H-100 (dH 4.54) and C-40 (dC 75.2) in the HMBC spectrum. A correlation between H-10 (dH 4.49) of the digitalose unit and C3 (dC 75.8) of cardenolide aglycone was also identified in the HMBC spectrum. Thus, 4 was determined to be acovenosigenin A 3-O-[Ob-D-glucopyranosyl-(1?6)-O-b-D-glucopyranosyl-(1?4)-2-Oacetyl-b-D-digitalopyranoside]. Compound 5, obtained as a white amorphous powder, with an [M+Na]+ ion at m/z 997.4228 in positive HR–ESI–MS, was assigned the molecular formula of C46H70O22 (calcd for 997.4251). By comparing the 1D and 2D NMR spectroscopic data, the aglycone of 5 was found to be similar to that of 4 except for one additional acetyl group (Table 2). Considering the low field shifting of 1 H and 13C of C-16 [(dC 75.9, dH 5.45 dt (2.3, 9.0 Hz)], the acetyl group was deduced to connected to C-16. The same coupling constants of H-16 and H-17 (9.0 Hz) indicated that the acetyl group connected at C-16 had a b-axial orientation, so the aglycone of 5 was deduced to be 16-O-acetyl-hydroxyacovenosigenin, which was also confirmed by comparation with reference data [5]. Furthermore, the 1H and 13C NMR data of the sugar portions were in agreement with those of compounds 1 and 4. Thus, 5 was concluded to be 16-O-acetyl-hydroxyacovenosigenin 3-O-[O-b-D-glucopyranosyl-(1?6)-O-b-D-glucopyranosyl-(1?4)-2-O-acetyl-b-Ddigitalopyranoside]. Compound 6, a white amorphous powder, showed an [M+Na]+ ion at m/z 1041.4900 by HR–ESI–MS in positive mode, corresponding to a molecular formula of C49H78O22 (calcd for 1041.4877). Comparing the 1H and 13C NMR data of 6 with those of 3, the aglycone was confirmed as Acovenosigenin A. Then, four anomeric proton signals [dH 4.83 (dd, J = 9.6, 1.8 Hz), dH 4.30 (d, J = 7.8 Hz), dH 4.55 (d, J = 7.8 Hz), and dH 4.39 (d, J = 7.8 Hz)] confirmed that 6 was acovenosigenin A tetrasaccharide. By analysis of the HSQC and HMBC spectra and comparison with the reference data [3], the sugar moiety was deduced to consist of one b-cymaropyranose,

one b-digitalopyranose and two b-glucopyranose units. The HMBC correlation from dH 4.39 (H-1000 0 ) to dC 70.3(C-6000 ), dH 4.55 (H-1000 ) to dC 76.1 (C-400 ) and dH 4.30 (H-100 ) to dC 83.8 (C-40 ), suggested a linkage of the four sugars from C-100 to C-40 , C-1000 to C-400 , and C-10000 to C-6000 . The location of the sugar moiety was determined to be C-3 from an HMBC correlation between dH 4.83 (H-10 ) of the cymarose unit and dC 76.6 (C-3) of the aglycone moiety. Thus, the structure of compound 6 was determined to be acovenosigenin A 3-O-[O-b-Dglucopyranosyl-(1?6)-O-b- D-glucopyranosyl-(1?4)-O-b-D-digitalopyranosyl-(1?4)-b-D-cymaropyranoside]. The monosaccharides of compounds 1–6 obtained by acid hydrolysis of each compound were identified as glucose, cymarose, digitalose and digitoxose by TLC comparison with authentic samples. Also, the absolute configurations of D-glucose, D-cymarose, D-digitalose and D-digitoxose were determined by measurement of their optical rotations after separation by preparative TLC. The relatively large 3JH-1,H-2 values of glucose, digitalose (7.8 Hz), digitoxose and cymarose (9.6 and 1.2 Hz) in their pyranose form indicated their b anomeric orientation [3,19,20]. The known compounds 8 and 9 were identified as digitoxigenin 3-O-b-D-cellobioside (8) [21] and digitoxigenin-3-O-b-D-glucosyl(1?4)-3-O-acetyl-b-D-digitoxoside (9) [13] by analyses of their 1 H and 13C NMR spectra, while the 1H and 13C NMR spectroscopic data of these two compounds were reported for the first time. The other two known compounds were identified as odoroside G (7) [22] and 5b-hydroxygitoxigenin (10) [23] by comparing the 13C NMR data with reference data. The anti-proliferative activities of all the isolated compounds, except for compound 4 (it was not present in a high enough amount for activity investigation), were evaluated in vitro against human lung A549 adenocarcinoma and large cell carcinoma NCIH460 cell lines by MTT-assay at a concentration of 100 lM, while cisplatin was used as a positive control (Fig. 3). The compounds with the inhibition rate over 50% towards either of the cell lines were measured and the IC50 values as shown in Table 3. Except the compounds 1, 7, 9 and the positive control cisplatin, most compounds displayed much stronger anti-proliferative activities against A549 cell line than those against the NCI-H460 cell line. Only compound 7 inhibited both of cancer cell lines, displaying wide cytotoxicity and more prospective potential. We previously reported the cytotoxicities of cardenolide glycosides from S. juventas against the two above NSCLC cell lines and have discussed their structure–activity relationships [24]. Comparing with the previous report, some new structure–activity relationships against A549 cells are summarized as far as the new compounds were found and investigated in this manuscript. We found that for 3b, 14b-dihydroxyl cardiac glycosides, the hydroxyl group at C-1, C-5 or C-16 all decreased the inhibiting activities, the acetyl group at C-16 could increase (4 < 1 < 5 < 17⁄) or decrease (3 < 14⁄ < 40⁄ < 27⁄, 38⁄ < 15⁄ < 13⁄ < 21⁄) the inhibition depending on whether there were of other substituents on the aglycone. The decrease of cytotoxicity induced by the acetyl group at C-30

O

O

OH O HO HO

O H

OH

O

HO HO OH H3CO

O

H O

O

OH

OH

OCOCH3 Fig. 2. Selected COSY (1H–1H— and HMBC (1H–13Cy) correlations of compound 1.

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C. Ye et al. / Steroids 93 (2015) 39–46

Cell growth inhibition(%)

100

NCI-H460 A549

80 60 40 20

10

9

8

7

6

5

3

2

1

Ci sp la t in

Bl an k

0

Fig. 3. The cell growth inhibition (%) of A549 and NCI-H460 cells after treatment with 9 compounds (100 lmol/L), as measured by MTT-assay (mean ± S.E.). Cisplatin (100 lmol/L) was used as a positive control.

Acknowledgements

Table 3 Antitumor activities of compounds 1–3, 5–10. Compounds

1 2 3 5 6 7 8 9 10 Cisplatin

IC50 (lM) NCI-H460

A549

38.308 – – – – 0.873 – 8.206 – 2.564

– 0.016 0.38 21.497 3.159 9.048 6.876 – 1.845 35.635

‘‘–’’: Not tested, IC50 values of compounds were calculated if they inhibited a tumour cell proliferation of over 50% at a concentration of 100 lM. Using Cisplatin as a positive control.

This work was financially supported by grants from the National Natural Science and Technology Major Project ‘‘Key New Drug Creation and Manufacturing Program’’ (No. 2010ZX09401304) and the Natural Science Foundation of China (No. 30973958).

Appendix A. Supplementary data 1

H-NMR, 13C-NMR, COSY, HMBC, HR-ESI-MS and UV spectra for compounds 1–6, as well as the 13C NMR data of compound 7 and 10 are available as Supporting Information. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.steroids.2014. 10.005. References

reported by Xue would be weakened accompanied with the extension of carbohydrate chain (9 < 28⁄ < 29⁄ < 21⁄). The acetylation at the C-20 of sugar moiety would enhance the inhibiting activity for triglycosylation (7 < 17⁄). The connection way of saccharides also affect the activity, the order of activity is 1?4 < 1?2 < 1?6 connection (8 < 25⁄ < 23⁄) (⁄ means this compounds from Ref. [24]). Forty years ago, when cardiac glycosides were first reported to exhibit antitumor activity, their toxicity limited their development [25,26]. Our results show that these compounds might be modestly selective for different cancer cells. The possibility of using cardiac glycosides to treat lung cancer depends on the identification of a leading compound effective against a range of different types of lung cancers, which would provide us with a new strategy to develop in future studies. 4. Conclusions In summary, the chemical constituents of the active antitumor fraction were investigated and 10 cardenolide glycosides (1–10) were obtained, including six new ones (1–6) and four known ones, isolated from the Streptocaulon genus for the first time. Moreover, all the 13C and part of the 1H NMR spectroscopic data of compounds 8 and 9 were reported for the first time. Most new compounds possessed the skeleton of acovenosigenin A, which increased our understanding of this kind of cardenolide glycoside. In addition, almost all these compounds exhibited different effects against NCI-H460 and A549 cells as shown by MTT assay, while compound 7 potently inhibited the two cell lines.

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