Hypercohones D-G, New Polycyclic Polyprenylated Acylphloroglucinol Type Natural Products from Hypericum cohaerens

Abstract

Four new polycyclic polyprenylated acylphloroglucinol type metabolites, hypercohones D-G (1-4), along with four known analogues (5-8), were isolated from the aerial parts of Hypericum cohaerens. The structures of these isolates were elucidated by extensive spectroscopic methods. The inhibitory activities of these isolates against five human cancer cell lines in vitro were also tested.

[Figure not available: see fulltext.]

Nat. Prod. Bioprospect. (2014) 4:7379 DOI 10.1007/s13659-014-0007-5

ORIGINAL ARTICLE

Hypercohones DG, New Polycyclic Polyprenylated Acylphloroglucinol Type Natural Products from Hypericum cohaerens

Jing-Jing Zhang Xing-Wei Yang Jun-Zeng Ma

Xia Liu Li-Xin Yang Sheng-Chao Yang

Gang Xu

Received: 13 January 2014 / Accepted: 24 February 2014 / Published online: 18 March 2014 The Author(s) 2014. This article is published with open access at Springerlink.com

Abstract Four new polycyclic polyprenylated acylphloroglucinol type metabolites, hypercohones DG (14), along with four known analogues (58), were isolated from the aerial parts of Hypericum cohaerens. The structures of these isolates were elucidated by extensive spectroscopic methods. The inhibitory activities of these isolates against ve human cancer cell lines in vitro were also tested.

Keywords Guttiferae Hypericum cohaerens Acylphloroglucinol Hypercohones DG

1 Introduction

Polycyclic polyprenylated acylphloroglucinols (PPAPs), with a highly oxygenated and densely substituted bicyclo[3.3.1]nonane-2,4,9-trione or other related core structures decorated with prenyl or geranyl side chains, are a kind of natural products from Hypericum, Clusia, and Garcinia plants in family Guttiferae [13]. This kind of

Electronic supplementary material The online version of this

article (doi:http://dx.doi.org/10.1007/s13659-014-0007-5

Web End =10.1007/s13659-014-0007-5 ) contains supplementary

material, which is available to authorized users.

J.-J. Zhang X.-W. Yang J.-Z. Ma X. Liu L.-X. Yang

G. Xu (&)

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Peoples Republic of China e-mail: [email protected]

J.-J. Zhang X.-W. Yang

University of Chinese Academy of Sciences, Beijing 100049, Peoples Republic of China

S.-C. Yang (&)

Institute of Chinese Medicinal Materials, Yunnan Agricultural University, Kunming 650201, Peoples Republic of China e-mail: [email protected]

metabolites showed a wide variety of biological activities such as antitumor, antimicrobial, anti-HIV, antioxidant, and especially in the central nervous system as modulators of neurotransmitters associated with neuronal damage and depression [49]. Hyperforin was the most famous one in acylphloroglucinol family isolated from H. perforatum (St. Johns Wort) [11], and was reported to possess signicant antidepressant, anticancer, and antibiotic activities [10].

The plants of genus Hypericum, occurring widely in temperate regions, have been used as traditional medicines in many countries all over the world [12]. H. cohaerens N. Robson was an endemic plant distributing in Guizhou and Yunnan provinces, P. R. China [13]. A polyprenylated acylphloroglucinol derivative with a novel bicyclo[5.3.1]-hendecane core (hypercohin A) and twelve other PPAP type natural products have been reported from this plant previously [1416]. In our systematic search for new and bioactive PPAPs, a phytochemical investigation on the aerial parts of H. cohaerens was carried out and four new PPAP type metabolites (hypercohones DG, 14) were isolated along with four known metabolites 58. It was notable that 4 was a spiro-cyclic type PPAP, and also was the fth natural product with such a complex ring system. Reported herein, was the isolation, structural elucidation, and the cytotoxic bioassay of these PPAP type metabolites.

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37

36

38

14

26

13

35

34

13

24

25

34

31

33

15

16

33

11

O

O

38

37

11

12

32

29 HO

29

27

12

27

1

22

23

35

28

O

HO

20

8

O

30

31

6

7

12 8 9

10

10

36

2

33 3 4 5

17

18

O

7

28

O

O

19

1

O

2

3 4 5

6

9

32

O

21

20

18

14

H

22

19

17

O

O

15 16

13

O

O

1 2

O

O

O

23

26

H

21

OH

24

25

11

3

4

OH

O O HO

OH

O

O

O

HO

O

O

O

O

O

O

H

O

O

O

O

H

OH

5

HO

6

7

8

2 Results and Discussion

The MeOH extract of the air-dried and powdered aerial parts of H. cohaerens (10.0 kg) was subjected to a silica gel column to afford ve fractions AE. Fraction B was subjected to a series of chromatographic methods, and led to the isolation of four new acylphloroglucinol derivatives, hypercohones DG (14), together with four known ones, uralodin A (5), [17] uralodin B (6), [18] oxepahyperforin (7) [19], and tomoeone E (8) [20].

Compound 1 was obtained as a light yellow gum. Its molecular formula C35H50O4 was deduced on the basis of its positive HRESIMS peak at m/z 557.3606 [M?Na]?

(calcd for 557.3606), indicating 11 indices of hydrogen deciency. The IR spectrum showed 1731 and 1665 cm-1

due to carbonyl functionalities. The 13C NMR and DEPT

spectra of 1 exhibited signals for 35 carbons, including twelve quaternary carbons (including three carbonyl carbons, ve olenic ones, and one oxygenated sp2 carbon), seven methines (including four unsaturated ones and an oxygenated one), ve methylenes, and eleven methyls. These data indicated the characteristic signals for an acylphloroglucinol core with two nonconjugated carbonyl groups (dC 208.3 C-9; dC 210.7, C-10), an enolized 1,3-diketo group (dC 170.4, C-2; dC 133.9, C-3; dC 196.8, C-4), and two quaternary carbons at dC 73.9 (C-1), and dC 66.9 (C-5) [1719] The 1H NMR spectrum of 1 showed the presence of four isoprenyl groups [dH 5.08 (1H, m, H-18), dH 5.06 (1H, m, H-23), dH 5.05 (1H, m, H-28), dH 5.18 (1H, d, J = 8.3 Hz, H-35)] and an isopropyl group [dH2.38 (1H, m, H-11), dH 0.97 (3H, d, J = 6.6 Hz, H-12), dH

1.03 (3H, d, J = 6.6 Hz, H-13)] (Table 2). Based on these data, 1 was considered as an acylphloroglucinol derivative having four isoprenyl groups and an isopropyl group. The HMBC correlations from H-17 at dH 3.15 (1H, dd,

J = 13.8, 8.4 Hz, H-17a), dH 3.08 (1H, dd, J = 13.8,6.6 Hz, H-17b) to C-2 (dC 170.4), C-3 (dC 133.9), and C-4 (dC 196.8), from H-22 (dH 2.41, 2H, m) to C-4 (dC 196.8),

C-5 (dC 66.9), C-6 (dC 41.71), and C-9 (dC 208.3), and the spinspin system of H2-6/H-7/H-27/H-28 obtained from the 1H1H COSY spectrum suggested the three isoprenyls were located at C-3, C-5, and C-7, respectively. Then, the remained isobutyryl was deduced to locate at C-1.

Further analysis of the NMR data of 1 with those of hyperforin revealed that they were structurally similar to each other except that the signals for the methylene at C-34 in hyperforin was replaced by an oxygenated methine (dC80.9) in 1, [10, 11] as evidenced by the H2-33/H-34/H-35 unit observed in the 1H1H COSY spectrum (Fig. 1). The existence of the epoxy group between C-34 and C-2 (dC 170.4) can be fully conrmed by the 11 indices of hydrogen deciency. In addition, the HMBC correlations from Me-32 (dH 1.08, 3H, s) to C-1 (dC 73.9), C-7 (dC39.5), C-8 (dC 49.0), and C-33 (dC 37.3) conrmed the structures furthermore.

The relative conguration of compound 1 was determined on the basis of a ROESY experiment (Fig. 1). The ROESY correlations of Me-32/H-27, Me-32/H-33b, H-34/

H-33a, H-34/H-7, and Me-12/H-35, indicated that H-7 and H-34 were both a-oriented and Me-32 was b-oriented. Furthermore, the relative congurations of C-1 and C-5 in 1 were elucidated to be the same with those of hyperforin,

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New Polycyclic Polyprenylated Acylphloroglucinol Type Natural Products 75

Table 1 13C NMR data for compounds 13 in CD3OD (d in ppm)

Position 1a 2a 3b

1 73.9, C 77.8, C 73.3, C2 170.4, C 167.1, C 169.0, C3 133.9, C 134.4, C 128.5, C4 196.8, C 199.0, C 196.0, C5 66.9, C 66.8, C 65.9, C6 41.71, CH2 43.2, CH2 41.6, CH2 7 39.5, CH 38.0, CH 38.2, CH8 49.0, C 46.7, C 47.6, C9 208.3, C 209.6, C 207.9, C10 210.7, C 210.5, C 197.5, C11 41.66, CH 42.3, CH 138.6, C12 21.3, CH3 21.9, CH3 129.8, CH 13 20.7, CH3 21.6, CH3 129.8, CH 14 129.0, CH 15 133.7, CH 16 129.0, CH 17 23.4, CH2 23.8, CH2 23.3, CH2 18 122.0, CH 121.7, CH 121.8, CH 19 131.5, C 134.5, C 133.5, C20 26.0, CH3 25.9, CH3 25.97, CH3 21 18.0, CH3 18.3, CH3 18.2, CH3 22 30.2, CH2 30.6, CH2 30.3, CH2 23 121.1, CH 120.8, CH 121.0, CH 24 134.9, C 135.0, C 135.5, C25 26.2, CH3 26.2, CH3 26.2, CH3 26 18.2, CH3 18.2, CH3 18.3, CH3 27 28.9, CH2 27.7, CH2 27.9, CH2 28 123.4, CH 123.3, CH 123.5, CH 29 134.5, C 134.6, C 134.4, C30 26.1, CH3 26.1, CH3 26.00, CH3 31 17.9, CH3 18.0, CH3 18.0, CH3 32 15.3, CH3 15.8, CH3 17.7, CH3 33 37.3, CH2 32.3, CH2 34.9, CH2 34 80.9, CH 22.8, CH2 24.6, CH2 35 125.2, CH 88.3, CH 88.2, CH 36 138.7, C 73.4, C 72.3, C37 25.8, CH3 23.7, CH3 27.5, CH3 38 18.6, CH3 28.2, CH3 22.7, CH3

a Recorded at 150 MHz

b Recorded at 100 MHz

O

O

O

O

Fig. 1 Key HMBC ( ), 1H-1H COSY ( ), and ROESY ( ) correlations of 1. (Color gure online)

as evidenced by the ROESY correlations of H-6b/H-23, H-6b/H-22, and H-6a/H-7. Thus, the structure of 1 was elucidated and named as hypercohone D.

Compound 2 was isolated as colorless oil. Based on the positive HRESIMS (m/z 553.3888 [M?H]?, calcd for 553.3893), the molecular formula was dened as C35H52O5.

The IR spectrum showed absorption bands at 3441

(hydroxyl) and 1724, 1663 cm-1 (carbonyl groups). Extensive analysis of the 1D NMR spectroscopic data (Tables 1 and 2) of 2 exhibited a close resemblance with oxepahyperforin (7) [19]. The differences in the 1D spectral data of 2 compared to 7 were that the chemical shifts of C-32 (dC 15.8),

C-33 (dC 32.3), C-34 (dC 22.8), C-35 (dC 88.3), and C-38 (dC28.2) were all little deviated from those of 7, which indicated that the conguration of C-35 might be differently. Carefully analysis of the ROESY spectrum revealed that H-35 was a-oriented, as determined by the correlations of Me-32/H-27, H-7/H-34a, and H-35/H-34a. Therefore, 2 was elucidated to be as the 35-epimer of 7 and named hypercohone E (Fig. 2).

Compound 3 possessed a molecular formula of C38H50O5,

inferred by HRESIMS at m/z 587.3720 [M?H]? (calcd for 587.3736). Its UV absorption exhibited maximum at 278, 250, and 205 nm. The IR spectrum displayed peaks at 3440 cm-1 (hydroxyl), 1723 cm-1 (conjugated ketone) and 1653 cm-1 (double-bond). Comparison of the 1D and 2D NMR data indicated that the structures of 3 and 7 were similar (Tables 1 and 2) [19]. However, the signals for the isopropyl group in 7 were replaced by signals for a phenyl group in 3, which was conrmed by HMBC correlations from both H-12 (dH 7.59, d, J = 8.2 Hz) and H-13 (dH 7.59, d,

J = 8.2 Hz) to C-10 (dC 197.5) and the proton spin system of H-13/H-14/H-15/H-16/H-12 observed from the 1H1H COSY spectrum. The ROESY correlations of Me-32/H-27, Me-32/H-33b, H-35/H-33b, and H-12 or H-13/H-35 deduced that 3 had the same relative congurations as 7 at C-7, C-8 and C-35 and all assigned as b-orientation. Ultimately, the structure of compound 3 was deduced and named as hyper-cohone F.

Hypercohone G (4) was isolated as a light yellow gum. The molecular formula, C33H40O5, was determined by

HREIMS (m/z 516.2871 [M]?, calcd for 516.2876). The IR spectrum revealed characteristic bands corresponding to the hydroxyl (3451 cm-1), conjugated carbonyl (1715 and 1679 cm-1), and double-bond (1630 cm-1) groups. Detailed comparison of the 1D-NMR data of 4 with those of Sampsonol C disclosed that they were closely related and shared a same spiro-cyclic skeleton, except for the

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76 J.-J. Zhang et al.

Table 2 1H NMR data for compounds 13 in CD3OD (600 MHz, d

in ppm, J in Hz)

No. 1 2 3

6 Ha 1.81, dd (5.4,

13.8)

Ha 1.88, m Ha 1.82, dd (4.9,

13.5)

Hb 1.51, dd (12.6,

13.8)

HO

O

O

Hb 1.41, t (13.2) Hb 1.69, m

7 2.18, m 1.91, m 2.10, m11 2.38, m 2.33, m12 0.97, d (6.6) 1.08, d (6.6) 7.59, d (8.2)13 1.03, d (6.6) 1.10, m 7.59, d (8.2)14 7.31, dd (7.5, 8.2) 15 7.50, t (7.5)16 7.31, dd (7.5, 8.2) 17 3.15, dd (8.4, 13.8) 3.18, dd (7.2,14.4)

3.16, dd (7.2, 13.9)

O

O

Fig. 2 Key HMBC ( ), 1H-1H COSY ( ), and ROESY ( ) correlations of 2. (Color gure online)

O

O

2.99, dd (6.8, 13.9)

18 5.08, m 5.09, t (7.2) 4.90, t (6.8) 20 1.68, m 1.67, s 1.632, s21 1.68, s 1.68, s 1.627, s22 2.41, m 2.44, dd (7.2,13.8)

2.47, m

3.08, dd (6.6, 13.8) 3.12, dd (6.6,14.4)

O

O

OH

Fig. 3 Key HMBC ( ), 1H-1H COSY ( ), and ROESY ( ) correlations of 4. (Color gure online)

2.37, dd (7.2,13.8)

23 5.06, m 4.89, t (7.8) 5.12, t (7.1) 25 1.64, s 1.59, s 1.67, s26 1.69, s 1.66, s 1.67, s27 Ha 2.05, m Ha 1.94, m Ha 2.16, m

Hb 1.63, m Hb 1.62, m Hb 1.73, m 28 5.05, m 4.99, t (7.8) 5.02, t (7.2) 30 1.68, s 1.68, s 1.68, s31 1.58, s 1.57, s 1.59, s32 1.08, s 1.03, s 1.25, s33 Ha 1.70, m Ha 1.65, m Ha 2.06, m

Hb 2.21, m Hb 2.63, t (14.4) Hb 2.33, m 34 4.97, m Hb 2.04, m 2.02, m

Ha 1.77, m 1.45, m35 5.18, d (8.3) 4.11, d (8.4) 3.73, d (9.0) 37 1.76, s 1.11, s 0.72, s38 1.69, s 1.32, s 1.00, s

signal of Me-17 in sampsonol C was replaced by an isoprenyl group in 4 [21]. This can be revealed by the presence of ve carbon signals ascribable for a prenyl at dC41.4 (t, C-17), 119.8 (d, C-18), 137.0 (s, C-19), 26.3 (q, C-20), and 17.8 (q, C-21) (Table 3). The mentioned isoprenyl group was located at C-6 by the HMBC correlations of H-17 to C-1 (dC 199.3), C-5 (dC 209.8), and C-6 (dC65.4) coupled with the correlation of H-17/H-18 observed in 1H-1H COSY spectrum. The ROESY interactions between Me-15/H-12, H-12/H-11a, H-11b/H-8, and H-8/

Me-16 suggested that 4 had the same relative congurations as Sampsonol C at C-8, C-9, C-12, and C-13 (Fig. 3). Therefore, the structure of 4 was established as illustrated and named hypercohone G.

Compounds 18 were tested for in vitro inhibitory activities against HL-60, SMMC-7721, A549, MCF-7 and SW480 human tumor cell lines using the MTT method described previously [22]. The results indicated that all the compounds were inactive with IC50 [ 30 lM.

3 Experiment Section

3.1 General Experimental Procedures

Optical rotations were obtained with a Jasco P-1020 polarimeter. UV spectra were measured on Shimadzu UV-2401A spectraphotometer. IR spectra were detected on a Bruker Tensor-27 infrared spectrophotometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker AV-400, and Avance III-600 MHz spectrometers with TMS as the internal standard. Chemical shifts (d) were expressed in ppm with reference to the solvent signals. HRESIMS analysis and HREIMS were determined on API QSTAR time-of-ight spectrometer and on Waters Auto spec Premier P776 mass spectrometer. Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatography with a Zorbax SB-C18 (9.4 mm 9 25 cm)

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New Polycyclic Polyprenylated Acylphloroglucinol Type Natural Products 77

Table 3 1H and 13C NMR data for compound 4 in CD3OD

Position dCa dH (J in Hz)b Position dCa dHb

1 199.3, C 17 41.4, CH2 2.72, dd (9.0, 13.2)

2 114.7, C 2.34, dd (7.2, 13.2)

3 179.7, C 18 119.8, CH 5.03, m

4 60.1, C 19 137.0, C

5 209.8, C 20 26.3, CH3 1.70, s

6 65.4, C 21 17.8, CH3 1.52, s

7 34.1, CH2 2.08, m 22 36.6, CH2 2.63, d (7.5)1.61, m

8 50.8, CH 1.16, m 23 121.2, CH9 77.3, C 24 136.7, C 5.04, m 10 40.6, CH2 1.71, m 25 26.4, CH3 1.72, s 11 23.4, CH2 Ha 1.63, m 26 18.1, CH3 1.58, s

Hb 0.99, m12 48.6, CH 2.04, dt (6.4, 12.0) 27 194.1, C13 93.9, C 28 139.1, C14 45.0, CH2 2.12, m 29 (33) 130.2, CH 7.81, d (8.3)

1.87, dd (1.9, 11.7)15 21.9, CH3 1.38, s 30 (32) 129.7, CH 7.49, t (7.5, 8.3)

16 26.6, CH3 1.29, s 31 134.8, CH 7.63, t (7.5)

a Recorded at 150 MHz

b Recorded at 600 MHz

column. Column chromatography was performed on Sephadex LH-20 (GE Healthcare), Silica gel (100200 and 200300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China), and Amphichroic RP-18 gel (4063 lm,

Merck, Darmstadt, Germany) and MCI gel (75150 lm, Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10 % H2SO4 in

EtOH.

3.2 Plant Material

The aerial parts of H. cohaerens N. Robson were collected in Daguan prefecture, Yunnan Province, China, in October 2009. The plant was identied by Dr. En-De Liu, Kunming Institute of Botany, Kunming, China. A voucher specimen was deposited with Kunming Institute of Botany with identication number 200910H01.

3.3 Extraction and Isolation

The aerial parts of the air-dried H. cohaerens (10.0 kg) were powdered and percolated with MeOH at room temperature and ltered. The ltrate was evaporated in vacuo to be concentrated. The crude extract (1.5 kg) was subjected to

silica gel column chromatography eluted with a petroleum ether-acetone gradient (1:0, 8:1, 4:1, 2:1, and 0:1) to produce ve fractions, AE. Fraction B (86.4 g) was separated over a MCI-gel column (MeOH-H2O from 8:2 to 10:0) to obtain ve fractions (Fr. B1B5). Fr. B2 (22.0 g) was isolated over an MCI gel column (MeOH-H2O from 85:15 to 100:0) to obtain four fractions (Fr. B2a-B2d). Fr. B2a(5.0 g) was separated on a silica gel column, eluted with petroleum ether-acetone (from 50:1 to 8:2), to yield six fractions (B2a1-B2a6). Fr. B2a2 was puried by repeated silica gel columns and semipreparative HPLC (RP-18, 93 % CH3CN-H2O) to afford 1 (25 mg), 2 (5 mg), 3 (12 mg), oxepahyperforin (7, 14 mg). Fr. B3 (13 g) was separated over a MCI-gel column (MeOH-H2O from 85:15 to 100:0)

to obtain ve fractions (Fr. B3aB3e). Fr. B3b was then chromatographed on a silica gel column, eluted with petroleum ether-acetone (from 9:1 to 7:3), to yield seven fractions (Fr. B3b1B3b7). Subfraction B3b3 (200 mg) was chromatographed by semipreparative HPLC (90 % MeOHH2O) to afford three fractions (Fr. B3b3aFr. B3b3c). Fr.

B3b3a was separated by a silica gel column, using ether-ethyl acetate (9:1) as solvent system to obtain uralodin A (5, 16 mg) and uralodin B (6, 21 mg). Fr. B3b3b and Fr. B3b3c were puried by semipreparative TLC to yield 4 (5 mg) and tomoeone F (8, 13 mg), respectively.

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78 J.-J. Zhang et al.

3.4 Hypercohone D (1)

Light yellow gum; [a]D24-97.0 (c 0.07, MeOH); UV (MeOH) kmax (log e) 270.0 (3.69) nm; IR (KBr) mmax 2963, 2927, 2856, 1731, 1665, 1634, 1450, 1380, 1287, 1262, 1121, 1100, 1077, 803 cm-1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 557 [M?Na]?; positive HRESIMS m/z 557.3606 (calcd for C35H50O4Na [M?Na]?, 557.3606).

3.5 Hypercohone E (2)

Colorless oil; [a]D21-42.8 (c 0.12, MeOH); UV (MeOH) kmax (log e) 260.0 (3.82) nm; IR (KBr) mmax 3441, 2969, 2927, 2857, 1724, 1663, 1628, 1449, 1382, 1121, 1094 cm-1; 1H

and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 575 [M?Na]?; positive HRESIMS m/z 553.3888 (calcd for C35H53O5 [M?H]?, 553.3893).

3.6 Hypercohone F (3)

Colorless oil; [a]D23-240.8 (c 0.17, MeOH); UV (MeOH) kmax (log e) 278.2 (4.00), 250.0 (4.13) nm; IR (KBr) mmax 3440, 2967, 2927, 1723, 1693, 1653, 1600, 1448, 1383, 1252, 1217, 1113, 1087 cm-1; 1H and 13C NMR data see Tables 1 and 2; positive ESIMS m/z 609 [M?Na]?; positive HRESIMS m/z 587.3720 (calcd for C38H51O5

[M?H]?, 587.3736).

3.7 Hypercohone G (4)

Light yellow gum; [a]D18-56.1 (c 0.16, MeOH); UV (MeOH) kmax (log e) 253.0 (4.32) nm; IR (KBr) mmax 3451, 2967, 2931, 2873, 1715, 1679, 1630, 1449, 1382, 1353, 1315, 1267, 1241, 1183, 1173, 1126, 1042, 764 cm-1; 1H

and 13C NMR data see Table 3; positive ESIMS m/z 539 [M?Na]?; positive HREIMS m/z 516.2871 (calcd for C33H40O5 [M]?, 516.2876).

3.8 Cytotoxicity Assays

The following human tumor cell lines were used: HL-60, SMMC-7721, A-549, MCF-7, and SW-480, which were obtained from ATCC (Manassas, VA, USA). All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10 % fetal bovine serum (FBS, Hyclone) at 37 C in a humidied atmosphere with 5 % CO2.

Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA). Briey, 100 lL of adherent cells was seeded into each well of a 96-well cell culture plate and

allowed to adhere for 12 h before test compound addition, while suspended cells were seeded just before test compound addition, both with an initial density of 1 9 105 cells/mL in 100 lL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cis-platin and paclitaxel (Sigma) as positive control. After the incubation, MTT (100 lg) was added to each well, and the incubation continued for 4 h at 37 C. The cells were lysed with 100 lL of 20 % SDS-50 % DMF after removal of 100 lL of medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muenchs method [22].

Acknowledgments The work was nancially supported by the foundations from NSFC (20972167), the Young Academic Leader Raising Foundation of Yunnan Province (No. 2009CI073), and the foundation from CAS to Dr G. Xu.

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

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