Cycloartane Triterpenoids and Their Glycosides from the Rhizomes of

Article pubs.acs.org/jnp

Cycloartane Triterpenoids and Their Glycosides from the Rhizomes of Cimicif uga fetida Ji-Yong Chen,†,§,∇ Ping-Lin Li,†,∇ Xu-Li Tang,*,‡ Shu-Jiang Wang,§ Yong-Tao Jiang,⊥ Li Shen,⊥ Ben-Ming Xu,⊥ Yong-Liang Shao,∥ and Guo-Qiang Li*,† †

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100, People’s Republic of China § Shandong Luye Pharmaceutical Co., Ltd., Yantai 264003, People’s Republic of China ⊥ School of Pharmacy, Yantai University, Yantai 264005, People’s Republic of China ∥ State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, People’s Republic of China S Supporting Information *

ABSTRACT: A phytochemical study on the rhizomes of Cimicif uga fetida resulted in the isolation of two new cycloartane triterpenoids (1 and 2), eight new cycloartane glycosides (3−10), and six known cycloartane glycoside analogues (11−16). The structures of 1−10 were determined by application of spectroscopic methods, with the absolute configuration of 1 determined by X-ray crystallography. Compounds 1−6, as three pairs of epimers at C-10 and C-24, belong to a seven-membered-ring variant of 9,10-seco-9,19-cycloartane triterpenoids, and glycosides 3−10 were found to be 3-O-β-D-xylopyranosides. The cytotoxicity of the isolates was evaluated against five selected human tumor cell lines, and the known compounds 15 and 16 showed cytotoxicity against the hepatocellular carcinoma SMMC-7721 cell line with IC50 values of 5.5 and 6.3 μM, respectively. Cimicif uga species (known as “Sheng-ma”) (Ranunculaceae) have been used in traditional medicine in mainland China since the first medicinal description in an ancient Chinese medical book “Shennong Bencao Jing”.1 Currently, three Cimicif uga species, namely, C. heracleifolia, C. dahurica, and C. fetida, are listed officially in the Chinese Pharmacopoeia 2010 and available clinically for the treatment of headache, sore throat, toothache, and uterine prolapse.2 During the past two decades, many phytochemical and biological studies have been performed on medicinally used Cimicif uga species,3−7 showing that cyclolanostane triterpenoids and their glycosides are the main bioactive constituents. These compounds are used also as markers to standardize medicinal Cimicif uga species extracts.1,8 However, previous studies on Chinese Cimicif uga species have focused mainly on the samples growing in southwestern and northeastern areas of the country, having humid subtropical and cold-temperate monsoon climates, respectively. In the present study, the rhizomes of C. fetida in another chinese region, Gansu Province, in the northwest, having a characteristic continental plateau climate, were investigated. Two new cycloartane triterpenoids, cimifetidanols A and B (1 and 2), and eight new glycosides, cimifetidanosides A−H © XXXX American Chemical Society and American Society of Pharmacognosy

(3−10), together with six known glycosides (11−16), were isolated from an EtOH extract of the rhizomes of C. fetida. The structures of the new compounds were elucidated by a combination of NMR spectroscopy, MS, single-crystal X-ray diffraction analysis, experimental and calculated ECD spectroscopy, and induced CD (ICD) spectroscopy. Structurally, all the new compounds reported herein were found to possess a cycloartane skeleton and the isolated glycosides to contain a 3O-β-D-xylopyranoside moiety. Compounds 1−6 were obtained as three pairs of epimers featuring a 9,10-seco-seven-membered B-ring, with compounds 3−6 isomerized at the C-10 position. Compound 7 is a dehydrated product of cimicifugoside H-2 that was obtained from different Cimicif uga species.9,10 Compounds 8 and 9 possess different oxygenated patterns in the side chain relative to the analogues of asiaticoside A from Actaea asiatica.11 In turn, compound 10 is the first cimiracemoside C-23/C-24 isomer from C. racemosa.12 The cytotoxic activities were evaluated for all compounds isolated against five selected human tumor cell lines. Received: March 18, 2014

A

dx.doi.org/10.1021/np500249v | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. HMBC correlations from H3-29/30 to C-3, C-4, and C-5 and from H3-26/27 to C-24 and C-25 confirmed the

Figure 1. Key COSY (−) and HMBC (→) correlations of 1, 7, and partial structures of 8−10.

locations of the hydroxy groups at C-3, C-24, and C-25, respectively. The positions of three double bonds (C-1−C-10, C-7−C-8, C-9−C-11) were supported by COSY correlations of H-1/H2-2/H-3, H2-6/H-7, and H-11/H2-12 and HMBC correlations from H-1 to C-2, C-3, and C-5, from H-7 to C5, C-6, C-9, and C-14, from H-11 to C-8 and C-13, and from H2-19 to C-1, C-8, C-9, C-10, and C-11. The two carbonyl groups at C-16 and C-23 were determined by HMBC correlations from H2-15 to C-16 and from H2-22/H-24 to C23, respectively. The relative configuration of 1 was determined on the basis of proton coupling constants and NOESY data. The hydroxy group at C-3 could be assigned as β-oriented according to the large coupling constants of H-3 (δH 3.78, dd, J = 9.5, 6.6 Hz).13 The NOESY correlations of H-3/H3-29 and H-3/H-5 supported the assignments of H3-29 and H-5α. The absolute configuration of 1 was determined by single-crystal Xray diffraction analysis using Cu Kα radiation (Figure 2) and

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RESULTS AND DISCUSSION The molecular formula of compound 1 was determined as C30H44O5 by HRESIMS, indicating nine degrees of unsaturation. IR absorption bands at 3443, 1731, and 1713 cm−1 indicated the presence of hydroxy and carbonyl groups. In the 13C NMR (DEPT) spectrum, 30 carbon signals could be resolved as seven methyls, six methylenes, eight methines, and nine quaternary carbons. The typical signals for six tertiary methyl groups in the 1H NMR spectrum at δH 0.75, 1.67, 1.55, 1.03, 1.30, and 0.95 and a secondary methyl at δH 1.11 (d, J = 6.7 Hz) suggested compound 1 to be a 9,19-cyclolanostane triterpenoid.5 Additionally, the 1H NMR spectrum showed three olefinic methine resonances at δH 5.38 (1H, d, J = 5.3 Hz, H-11), 5.46 (1H, t, J = 6.5 Hz, H-7), and 5.54 (1H, s, H-1), in accordance with the occurrence of three pairs of double bonds at δC 120.7 (C-1) and 139.0 (C-10), 125.6 (C-7) and 142.5 (C8), and 121.1 (C-11) and 137.9 (C-9) in the 13C NMR spectrum of 1. Also observed in this spectrum were two carbonyl signals at δC 218.2 (C-16) and 213.7 (C-23), as well as three oxygen-bearing carbon signals at δC 73.6 (C-3), 84.0 (C24), and 72.5 (C-25). Such evidence suggested that compound 1 is a highly oxygenated four-ring-containing 9,10-seco-9,19cyclolanostane. This was supported by the downfield-shifted H2-19 at δH 3.22 and 3.13 (each 1H, d, J = 13.5 Hz) and the absence of saturated quaternary carbons (C-9 and C-10).13 Analysis of key COSY and HMBC correlations was used to establish the planar structure of compound 1, as shown in

Figure 2. ORTEP diagram of 1.

was assigned as 3S, 5R, 13R, 14R, 17R, 20R, 24R based on a Flack absolute structure parameter of 0.0(2). Thus, compound 1 was finally established as (3S,5R,13R,14R,17R,20R,24R)3,24,25-trihydroxy-9,10-seco-9,19-cyclolanost-1(10),7,9(11)-triene-16,23-dione and named cimifetidanol A. Cimifetidanol B (2) gave the same molecular formula as compound 1 by HRESIMS. The IR and 1D NMR spectra of compound 2 showed close resemblances to those of 1 except for slight differences of the 1H and 13C NMR signals around C24 (Tables 1 and 2), which supported the assignment of 2. On HRESIMS, compound 3 gave the molecular formula C35H54O10, and in the 13C NMR (DEPT) spectrum 35 signals B



Article

Table 1. 1H NMR Data of Compounds 1−10 (Recorded at 400 MHz in Pyridine-d5, δ in ppm, J in Hz) position 1

2 3 5 6 7

8 11

1 5.54 br s

2 5.55 br s

2.52 m 2.27 m 3.78 dd (6.6, 9.5) 2.36 m

2.53 m 2.26 m 3.78 dd (6.2,10.1) 2.36 m

2.64 m 2.38 m 5.46 t (6.5)

2.64 m 2.38 m 5.46 t (6.4)

5.38 br d (5.3)

5.38 br d (5.2)

3

4

5

6

1.93 br d

2.26 m

1.93 br d

(13.9)

1.75 m

(13.9)

1.69 dt (3.5, 13.9) 2.48 m 2.30 m 3.53 dd (3.5, 11.7) 1.42 d (12.0) 2.79 m 2.25 m 5.46 br d (7.5)

5.36 br s

2.56 m 2.08 m 4.03a 1.96 d (8.6) 2.42 m 5.55 t (6.2)

5.40 d (5.2)

1.71 dt (3.5, 13.9) 2.52 m 2.31 m 3.55 dd (3.3, 11.7) 1.42 d (12.0) 2.82 m 2.25 m 5.48 br d (7.6)

5.38 br s

7

8 1.55 m

1.50 m

1.54 m

1.70 m

1.14 m

1.13 m

1.10 m

2.58 m 2.08 m 4.05a

2.42 m 2.08 m 3.58 dd (4.0, 11.6) 1.33 m

2.29 m 1.88 m 3.47 dd (3.9, 11.5) 1.28 m

2.28 m 1.86 m 3.46 dd (4.0, 11.6) 1.25 m

2.29 m 1.87 m 3.47 dd (11.5, 4.0) 1.24 m

1.95 m 1.71 m 5.13 br d (7.9)

1.51 m 0.78 m 1.30a

1.47 m 0.74 m 1.25 m

1.44 m 0.68 m 0.93 m

0.97 m 1.63 m 2.74 dd (8.6, 15.9) 1.18 dd (3.2, 16.2) 5.11 dd (2.9, 8.4)

0.94 m 1.65 m 2.65 m 1.16 m

1.25 m 1.58 m 2.75 dd (16.0, 9.1) 1.16 m

5.30 dd (3.7, 9.0)

5.18 dd (3.2, 8.8)

1.95 dd (7.7, 14.0) 1.85 m

1.92 m

2.29 d (18)

1.99 dd (8.1, 12.8) 1.77 m

4.2 m 2.09 m 1.32 s 0.59 d (4.1)

4.99 ddd (8.1, 8.1, 8.1) 1.85 m 1.38 s 0.22 d (4.0)

1.97 d (8.0) 2.43 m 5.57 t (6.7)

5.43 d (5.4)

4.54 dd (3.0, 9.2)

2.31 br d

2.31 br d

2.30 m

2.30 m

2.31 m

2.35 m

2.81 m

(17.4) 2.16 dd (17.4, 6.0) 2.41 d (17.8) 2.10 d (17.8)

2.14 m

2.13 m

2.15 m

2.15 m

2.19 m

15

(17.4) 2.16 dd (6.0, 17.4) 2.41 d (17.8) 2.10 d (17.8)

2.55 d (17.9) 2.06 d (17.9)

2.38 d (9.1)

2.59 d (17.8) 2.11 d (17.8)

2.41 d (18.3) 2.15 d (18.0)

2.48 d (18)

16 17 18 19

20 21 22

2.39 d (8.4) 0.75 s 3.22 d (13.5) 3.13 d (13.5) 2.72 m 1.11 d (6.7) 3.81a

2.39 d (9.0) 0.76 s 3.22 d (13.5) 3.13 d (13.5) 2.73 m 1.10 d (6.6) 4.09 br d

3.42 dd (8.9, 18.5)

(17.1) 3.18

a

a

3.42 dd (9.0, 18.6) 4.50 s

2.35 d (9.2) 0.77 s 2.97 d (14.1) 2.48 d (14.7) 2.73 m 1.08 d (6.5) 3.88 d (18.0) 3.41 dd

2.34 d (8.9) 0.81 s 2.54 d (14.2) 2.37 d (14.2) 2.60 m 0.99 d (6.1) 3.67 d (15.2) 2.63 dd (8.4, 16.9)

2.32a 0.77 s 2.99 d (14.6), 2.50 d (14.6) 2.60 m 1.00 d (6.0) 3.68a

2.42 m 1.21 s 1.95 d (3.5) 0.95 d (3.3)

0.23 d (4.0)

0.19 d (4.1)

0.54 d (4.0)

2.66 m 1.04 d (6.6) 3.68 m

1.78 m 1.02 d (6.0) 2.78 d (13.3)

2.45 m 1.12 d (7.2) 5.19 d (3.1)

1.88 m 1.35 d (5.8) 3.83 d (9.6)

2.60 m

2.92 dd (8.8, 17.9)

1.63 m

3.77 s

3.78 s

5.04 s

4.32 d (4.6)

4.30 s

1.65 s

1.66 s

1.60 s 0.87 s 1.32 s 1.02 s 2.12 s 4.85 d (7.5) 4.04 m 4.16 t (7.7) 4.23 m 4.36 dd (4.8, 11.0) 3.74 t (10.5)

1.53 s 0.86 s 1.32 s 1.00 s 2.09 s 4.86 d (7.4) 4.04 t (8.0) 4.17 t (8.6) 4.23 m 4.37 dd (5.0, 11.2) 3.74 t (10.5)

(9.0,18.5)

4.50 s 1.67 s

1.62 s

1.66 s

1.68 s

1.36 s

1.37 s

27 28 29 30 AcO-12 1′ 2′ 3′ 4′ 5′

1.55 1.03 1.30 0.95

1.58 1.03 1.30 0.95

1.55 1.05 1.47 1.38

1.56 0.99 1.52 1.29

1.35 1.09 1.49 1.40

1.36 1.05 1.52 1.30

s s s s

s s s s

4.90 d (7.5) 4.04 t (8.1) 4.18 t (8.6) 4.23 m 4.39 dd (5.1, 11.3) 3.77 t (11.0)

1.78 m

4.80 ddd (7.3, 7.3, 7.3) 1.86 m 1.32 s 0.59 d (3.6)

24 25 26

s s s s

4.41 s

2.37 d (8.4) 0.81 s 2.53 d (14.0) 2.36 d (14.0) 2.71 m 1.09 d (6.6) 3.87 dd (2.4, 18.6)

10

2.78 m

12

2.10 m

9

2.26 dd (6.5) 1.76 dd (5.2)

4.52 s

s s s s

4.83 d (7.4) 4.06 t (7.8) 4.16 t (8.7) 4.24 m 4.34 dd (5.0, 11.0) 3.69 t (10.5)

s s s s

4.92 d (7.5) 4.06 t (8.1) 4.19 t (8.6) 4.25 m 4.40 dd (5.1, 11.3) 3.80 t (11.0)

s s s s

4.84 d (7.4) 4.06 t (8.0) 4.16 t (8.7) 4.25 m 4.34 dd (5.1, 11.1) 3.70 t (10.8)

5.47 5.15 1.85 1.19 1.41 1.14

3.76 m 1.31 d (6.4)

s s s s s s

4.89 d (7.5) 4.04 t (7.8) 4.16 t (8.6) 4.21 m 4.34 dd (5.0, 11.2) 3.74 t (10.4)

1.28 d (6.4) 0.86 s 1.33 s 1.04 s 2.11 s 4.86 d (7.5) 4.04 t (8.0) 4.16 t (8.6) 4.22 m 4.36 dd (4.9, 11.2) 3.74 m

Signals overlapped. C



Article

Table 2. 13C NMR Data of Compounds 1−10 (Recorded at 100 MHz in Pyridine-d5, δ in ppm) position

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 AcO-12

120.7 33.2 73.6 39.0 51.1 25.3 125.6 142.5 137.9 139.0 121.1 36.6 43.4 45.8 47.9 218.2 60.0 16.7 43.8 27.5 20.6 47.6 213.7 84.0 72.5 28.1 25.7 25.1 25.1 14.6

120.7 33.2 73.6 39.0 51.1 25.3 125.6 142.5 137.9 139.0 121.1 36.6 43.4 45.8 47.9 218.3 60.0 16.6 43.8 27.4 20.4 47.4 214.3 84.4 72.4 27.5 26.3 25.1 25.1 14.6

41.0 26.8 88.5 40.5 53.1 24.5 123.9 143.3 134.5 72.0 126.9 38.2 44.3 46.0 47.4 218.5 59.8 17.5 54.9 27.5 20.6 47.4 213.7 84.0 72.5 28.0 25.7 22.8 27.3 15.8

32.7 25.6 84.2 38.9 57.4 27.4 128.9 144.0 135.1 74.6 124.8 36.8 43.2 45.8 47.6 218.4 60.0 16.9 51.7 27.6 20.5 47.7 213.8 84.1 72.5 28.1 25.7 25.8 30.0 23.0

41.0 26.8 88.5 40.5 53.1 24.6 124.1 143.2 134.6 72.0 126.8 38.3 44.2 46.0 47.5 218.6 59.5 17.3 54.9 27.6 20.5 47.2 205.6 65.7 60.7 18.3 24.5 22.8 27.3 15.8

32.8 25.6 84.2 39.0 57.4 27.4 129.1 143.9 135.2 74.7 124.7 36.9 43.1 45.8 47.7 218.5 59.9 16.9 51.7 27.7 20.5 47.5 205.6 65.8 60.7 18.3 24.5 25.8 30.0 23.0

27.4 29.8 88.3 40.7 43.8 22.0 115.3 147.2 27.5 29.3 62.9 47.2 44.4 46.1 49.7 218.4 61.2 20.1 18.5 27.7 20.2 43.7 210.6 82.5 144.4 114.8 17.9 27.8 25.9 14.5

107.6 75.5 78.6 71.2 67.1

107.0 75.7 78.7 71.2 67.1

107.7 75.5 78.6 71.2 67.2

107.0 75.7 78.7 71.2 67.1

107.5 75.5 78.6 71.2 67.1

31.9 29.9 88.1 41.2 47.1 20.5 25.7 45.8 20.1 26.7 36.6 77.0 48.8 48.0 43.9 72.2 59.4 13.5 29.6 25.9 21.1 39.1 99.4 210.5 33.7 20.4 20.2 19.5 25.7 15.3 170.5 21.6 107.5 75.6 78.6 71.2 67.1

31.9 29.8 88.0 41.2 47.0 20.3 25.7 46.5 20.5 27.1 36.5 76.9 48.9 47.8 45.7 74.6 52.3 13.0 30.1 24.6 25.6 105.8 153.6 79.4 72.9 27.4 25.9 20.8 25.6 15.3 170.6 21.1 107.5 75.5 78.6 71.2 67.1

32.0 29.9 88.0 41.2 47.0 20.4 25.8 45.6 19.9 26.7 36.8 77.0 49.4 47.9 42.8 71.9 51.4 13.8 29.8 35.9 17.3 84.9 103.0 81.8 79.9 21.6 28.9 19.8 25.6 15.3 170.5 21.6 107.5 75.6 78.6 71.2 67.1

1′ 2′ 3′ 4′ 5′

(δH 2.53, d, J = 14.0 Hz) as β-oriented. A molecular modeling analysis of the comparative C-10 epimers (10S* and 10R*) of 3 further indicated that the NOESY correlations mentioned above could only be accommodated by the 10S* relative configuration. The result was supported by comparison of the NMR data of 3 with those of the known compound podocarpaside D.13 Thus, compound 3 was established as (10β,24R)-10,24,25-trihydroxy-9,10-seco-9,19-cyclolanost7,9(11)-diene-16,23-dione-3-O-β-D-xylopyranoside and has been named cimifetidanoside A. Cimifetidanoside B (4) gave the same molecular formula as 3 by HRESIMS. 2D NMR experiments on 4 were used to establish the same planar structure. A 1D NMR data comparison (Tables 1 and 2) between 3 and 4 showed their close resemblance except for some differences in rings A−C. Compounds 3 and 4 were similar to the cycloartane triterpenoid alkaloids kleinhospitines A−D from Kleinhovia hospita with isomerized C-9 and C-10 functionalities,14 indicating the possible configurational changes at C-3 and/or C-10 in 4. As the chemical shift of H-3 overlapped with H-2′ in the 1H NMR spectrum of 4 (Table 1 and Figures S32 and S33, Supporting Information), the aglycone (4a) obtained on the

were resolved as seven methyls, eight methylenes, 11 methines, and nine quaternary carbons (Table 1). A comparative 1H NMR study with 1 showed their general structural resemblance except for the occurrence of a xylose moiety in 3. The monosaccharide obtained after acid hydrolysis of 3 was identified as D-xylose by comparing its Rf value and optical rotation with an authentic sample. The coupling constant of the anomeric proton at δH 4.90 (H-1′, d, J = 7.5 Hz) in the 1H NMR spectrum of 3 indicated the D-xylose unit to be in the βconfiguration. The xylose unit was shown to be attached at C-3 by observation of HMBC correlations from H-1′ to C-3 and from H-3 to C-1′. From the HMQC spectrum, 1H NMR signals for a methylene group at δH 1.69 (dt, J = 13.9, 3.5 Hz, H-1) and 1.93 (br d, J = 13.9 Hz, H-1) were observed, instead of the olefinic methine at δH 5.54 (br s, H-1) for 1. This corresponded to the presence of a hydroxylated carbon at C-10 (δC 72.0), which was confirmed by HMBC correlations from H2-19 to C-1, C-5, C-7, C-8, C-9, and C-10. The NOESY correlations (Figure 3) of H3-29/H-3, H-1′/H-3, H-3/H-5, Hb19 (δH 2.36, d, J = 14.0 Hz)/H-5, H2-19/H2-1, H3-18/H-20, and H-17/H3-21 indicated that H-3, H-5, Hb-19, H3-29, and H3-21 could be assigned as α-oriented and H3-30 and Ha-19 D



Article

Figure 4. Experimental CD spectra of compounds 3, 4a, 5, and 6.

Figure 3. Key NOESY correlations of cmpounds 3, 4a, and 10.

enzymatic hydrolysis of 4 showed clearly the proton signal of H-3 at δH 4.16 (t, J = 5.4 Hz) (Figures S41 and S42, Supporting Information), which suggested a possible β-orientation of H-3. However, the NOE correlations of H3-29/H-3, H-5 (δH 2.00, d, J = 9.6 Hz)/H-3, and H3-29/H-5 in the NOESY spectrum of 4a indicated H-3 to be α-oriented (Figures 3 and S44, Supporting Information). A molecular modeling analysis followed by geometry optimization at the B3LYP/DGDZVP level for all four possible candidate stereoisomers 3S/10S, 3S/10R, 3R/ 10R, and 3R/10S marked as 4a-1−4a-4 (Figures SC1−SC4, Supporting Information)15,16 indicated that the conformational change of ring A caused by the change of C-10 configuration could result in the small coupling constant of H-3 with an αorientation and that only in a 3S configuration could the spatial distances between H-3 and H-5 be close enough to observe an NOE correlation. Furthermore, an ECD method was used to determine the configuration of C-10 in 3 and 4. The experimental ECD spectra of compounds 3 and 4a showed a notable difference (Figure 4). ECD curves for all four possible candidate stereoisomers 4a-1−4a-4 were calculated with the TD-DFT theory method at the B3LYP/DGDZVP level, as reported previously (Figures SC1−SC4, Supporting Information).15,16 Visual inspection suggested that the 10S isomer (4a1 and 4a-4) curves displayed a pronounced positive Cotton effect (CE) at 240 nm, consistent with the experimental curve of 3, in contrast to that of its mirror image. In turn, the 10R isomers (4a-2 and 4a-3) showed a weak positive CE at 242 nm similar to the experimental curve of 4a (Figure 5). Accordingly, the configurations at C-3 and C-10 of compounds 3 and 4 were established finally as 3S/10S and 3S/10R, respectively. An HRESIMS experiment gave for compound 5 a molecular formula of C35H52O9, 18 atomic units (H2O) less than that of 3.

Figure 5. Experimental CD spectra of 3 and 4a overlaid with calculated spectra for the candidate stereostructures.

The 1D NMR data (Tables 1 and 2) of 5 showed a close similarity to 3 except for an upfield shift of the protons and carbons around C-24, indicating the presence of a 24,25epoxide ring in 5. This was supported by HMBC correlations from H3-26 and H3-27 to C-24 and C-25 and from H2-22 and H-24 to C-23. The chiral centers of the nucleus in 5 were suggested to have the same relative configurations as in 3 on the basis of the comparison of the coupling constants, NOESY, ECD, and other NMR data for these two compounds (Figure 4 and Supporting Information). The 24R configuration could be determined by the characteristic chemical shift for C-24 at δC 65.7,17 which was further supported by comparing the 1H and 13 C NMR chemical shifts for the side chain of compound 5 with that of the analogue cimicifugoside H-1.9 Thus, compound 5 (cimifetidanoside C) was established as (3S,5S,10S,13R,14R,17R,20R,24R)-10-hydroxy-24,25-expoxy9,10-seco-9,19-cyclolanost-7,9(11)-diene-16,23-dione-3-O-β-Dxylopyranoside. Cimifetidanoside D (6) exhibited the same molecular formula as 5 by HRESIMS. Similar to the NMR relationships between 3 and 4, compound 6 also showed changes in rings A−C when comparing its 1D NMR data with those of 5 (Tables 1 and 2). The 1D NMR data of the nucleus and side chain in 6 showed close similarities in the corresponding data for 4 and 5. The similar rotation values between the two pairs of comparable compounds 3/5 and 4/6 indicated their stereochemical consistency. The evidence available suggested that compound 6 is the 10R isomer of 5. E



Article

from H-16 to C-23, together with the spin system of H2-15/H16/H-17/H-20(H3-21)/H-22, as deduced by COSY correlations, revealed the presence of a 16,23-epoxy-24,25-dihydroxy22-ene moiety in the side chain of 9. The relative configuration of the tetracyclic nucleus in 8 was determined to be the same as that of 9 on the basis of the coupling constants and NOESY experiments. Thus, compound 9 was proposed as (3β,12β,16β,24ξ)-12-acetoxy-16:23-expoxy-24,25-dihydroxy9,19-cyclolanost-22-ene-3-O-β-D-xylopyranoside and named cimifetidanoside G. Compound 10 was isolated as a white powder, and the HRESIMS gave a molecular formula of C37H58O11. The 1H NMR spectrum displayed resonances for cyclopropanemethylene protons (δH 0.22, 0.54, J = 4.0 Hz), an acetyl group (δH 2.09, s), and six tertiary (δH 1.66, 1.53, 1.38, 1.32, 1.00, and 0.86) and a secondary methyl (δH 1.35, d, J = 5.8 Hz) group, as well as an anomeric proton signal at δH 4.86 (d, J = 7.4 Hz), which were characteristic of a 9,19-cyclolanostane-type triterpene monoglycoside. On the basis of 1H−1H COSY, HSQC, and HMBC spectroscopic data (Figures 1 and S98− S100, Supporting Information), the planar structure of 10 was determined to be the same as that of cimiaceroside H.12 The 1 H and 13C NMR data of 10 were also very similar to those of cimiracemoside H with slight differences at C-22−C-27. The chemical shifts for these positions were at δC 84.9 (C-22), 103.0 (C-23), 81.8 (C-24), 79.9 (C-25), 21.6 (C-26), and 28.9 (C27), instead of δC 86.9 (C-22), 105.8 (C-23), 83.6 (C-24), 83.8 (C-25), 28.1 (C-26), and 25.2 (C-27) in cimiaceroside H. The key NOESY correlations between H-3/H3-29, H3-18/12β-OAc, H3-28/H-12, H3-28/H-16, H3-18/H-20, H-22/H3-21, H-22/ H3-26, and H-24/H3-27 indicated that compound 10 should have the same configurations as cimiracemoside H at the C-3, C-12, C-16, and C-22 positions. However, the cross-peak between H-24 and H3-27 implied that the OH-24 is α-oriented. The absolute configuration of the 23,24-diol of compound 10 was determined through the induced CD spectrum of a Mo2(OAc)4 complex with aglycone 10a produced.19 The metal complex of 10a in DMSO gave an ICD (Figure 6), in which the

Compound 7 gave a molecular formula of C35H52O9 by HRESIMS, and the 1H NMR spectrum displayed characteristic cycloartane proton signals, including a cyclopropane methylene of CH2-19 at δH 0.95 (1H, d, J = 3.3 Hz) and 1.95 (1H, d, J = 3.5 Hz), and six methyl proton signals at δH 1.14, 1.19, 1.21, 1.41, 1.85 (each 3H, s, 5 × CH3) and 1.04 (3H, d, J = 6.6 Hz). A β-D-xylopyranoside unit was observed by comparison of the 1D NMR data with those of compounds 3−6 (Tables 1 and 2). Additionally, the 1H and 13C NMR data of compound 7 showed a close resemblance to those of cimicifugoside H-2,9 except for the presence of a terminal double bond at C-25 and C-26 instead of an oxygenated quaternary carbon (C-25) and methyl group (C-26). Thus, it was reasonable to infer that 7 is a 25-dehydrated derivative of cimicifugoside H-2, which was confirmed by combined analysis of its COSY, HMQC, and HMBC spectra (Figures S72−S74, Supporting Information). Key HMBC correlations from H3-27 (δH 1.85) to C-24, C-25, and C-26, from H2-26 to C-24 and C-25, from H-19 to C-1, C5, C-7, C-8, C-9, C-10, and C-11, and from H3-29 and H3-30 to C-3, C-4, and C-5 were observed (Figure 1). In turn, H-3 and H-11 were both determined as being α-oriented from their coupling constants. Also, the hydroxy group at C-24 could be proposed as α-oriented on the basis of biogenic considerations and comparison of its optical rotation value and other cimicifugoside analogues.9 On the basis of the above analysis, the structure of compound 7 (cimifetidanoside E) was determined as 25-dehydrocimicifugoside H-2. HRESIMS of compound 8 gave a molecular formula of C37H58O10. Apart from the signals for an acetyl group and xylose unit, resonances for a cyclopropane methylene (H2-19) at δH 0.23 and 0.59 (each 1H, d, J = 4.1 Hz), four tert-methyl singlet signals at δH 0.86, 1.04, 1.32, and 1.33, and three methyl groups at δH 1.02 (3H, d, J = 6.0 Hz), 1.28 (3H, d, J = 6.4 Hz), and 1.31 (3H, d, J = 6.4 Hz) for a cycloartane skeleton were observed in the 1H NMR spectrum (Table 1). The 13C NMR (DEPT) spectrum (Table 2) showed four additional oxygenated carbons at δC 88.1 (CH), 77.0 (CH), 72.2 (C), and 99.4 (C) as well as signals for the xylose moiety. Detailed comparison of the 1H and 13C NMR data of 8 with those of its analogue asiaticoside A11 indicated these compounds to have identical partial structures in rings A−D, but differing in ring E. HMBC correlations from H-22 to C-17, C-20, C-21, C-23, and C-24, from H3-21 to C-17, C-20, and C-22, and from H-16 to C-23, together with the spin system of H2-15/H-16/H-17/H20(H3-21)/H2-22 deduced from a COSY experiment (Figure 1), suggested the presence of a 23-hydroxy-16:23-epoxy moiety in 8. H-3, H-12, and H-16 were all determined as being αoriented from their chemical shifts and coupling patterns compared with the same protons of asiaticoside A and actein.11,18 The NOESY correlations of H-3 with H3-29, of H-12, and H-16 with H3-28, and of H-16 with H3-21 further supported the assignments. Additionally, NOESY correlations of Hα-15 (δH 1.99, dd, J = 8.1, 12.8 Hz) with H3-28 and of Hβ15 (δH 1.77, m) with H3-26/H3-27 indicated that the hydroxy group at C-23 is α-oriented. Compound 8 (cimifetidanoside F) was therefore established as (3β,12β,16β,23α)-12-acetoxy16:23-expoxy-23-hydroxy-9,19-cyclolanost-24-one-3-O-β-D-xylopyranoside. Compound 9 gave a molecular formula of C37H58O10 by HRESIMS, and detailed comparison of the 1H and 13C NMR data of 9 and 8 showed these molecules to differ structurally in ring E (Tables 1 and 2). HMBC correlations from H3-26 and H3-27 to C-24 and C-25, from H-24 to C-22 and C-23, and

Figure 6. Mo2(OAc)4 complexes-induced CD spectrum (ICD) of 10a.

positive Cotton effect permitted the assignment of a 23S,24Sconfiguration for 10a, according to the Snatzke rule.19 Therefore, compound 10 (cimifetidanoside H) was proposed as (22R,23S,24S)-12β-acetoxy-16β:23;22:25-diepoxy-3β,23,24trihydroxy-9,19-cycloanostane-3-O-β-D-xylopyranoside. The occurrence of the new 9,10-seco-9,19-cyclolanostane triterpenoids (1−6) may be able to be used as marker compounds of C. fetida when obtained from northwestern mainland China. Additionally, six known 9,19-cyclolanostanetype triterpene monoglycosides were also isolated from C. fetida. Comparison of their spectroscopic data with those in the F



Article

Fraction D1 (3.2 g) was purified by passage over an ODS silica gel column with MeOH−H2O (60:40 to 85:15) and recrystallized from MeOH to yield 8 (55 mg). Repeated silica gel column chromatography of fraction D2 (6.3 g) using CHCl3−MeOH (80:1 to 10:1) and recrystallization yielded 11 (165 mg) and 12 (108 mg). Repeated silica gel column chromatography of fraction D4 (3.3 g) with CHCl3− MeOH (50:1 to 10:1) and recrystallization afforded 13 (45 mg) and 14 (38 mg). Fraction D6 (2.8 g) was purified using an ODS silica gel column with MeOH−H2O (40:60 to 80:20), followed by purification using preparative HPLC eluted with CH3CN−H2O (40:60), furnished 3 (30 mg), 4 (12 mg), 5 (22 mg), 6 (8 mg), and 9 (20 mg). Fraction D8 (1.5 g) was purified with an ODS silica gel column with CH3CN− H2O (25:75 to 40:60), followed by purification using preparative HPLC with CH3CN−H2O (35:65) and then separation over a column containing Sephadex LH-20 (MeOH), to yield 7 (15 mg) and 10 (18 mg). Cimifetidanol A (1): white crystals from MeOH; [α]20D −48.1 (c 0.02, MeOH); IR (KBr) νmax 3443, 2973, 1731, 1713, 1466, 1385, 1038 cm−1; 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 507.3086 [M + Na]+ (calcd for C30H44O5Na, 507.3081). Cimifetidanol B (2): white crystals from MeOH; [α]20D −20.8 (c 0.02, MeOH); IR (KBr) νmax 3441, 2970, 1731, 1696, 1469, 1384, 1037 cm−1; 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 507.3079 [M + Na]+ (calcd for C30H44O5Na, 507.3081). Cimifetidanoside A (3): white, amorphous powder; [α]20D +73.6 (c 0.02, MeOH); CD (c 0.0004 M, MeOH) λmax (mdeg) 238.0 (56.7), 298.5 (−12.0) nm; IR (KBr) νmax 3435, 2970, 1729, 1647, 1366, 1044 cm−1; 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 657.3613 [M + Na]+ (calcd for C35H54O10Na, 657.3609). Cimifetidanoside B (4): white, amorphous powder; [α]20D −29.7 (c 0.04, MeOH); IR (KBr) νmax 3423, 2928, 1732, 1635, 1385, 1042 cm−1; 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 657.3612 [M + Na]+ (calcd for C35H54O10Na, 657.3609). Cimifetidanoside C (5): white, amorphous powder; [α]20D +95.2 (c 0.02, MeOH); CD (c 0.0005 M, MeOH) λmax (mdeg) 239.0 (57.0), 301.5 (−12.1) nm; IR (KBr) νmax 3434, 2966, 1726, 1636, 1384, 1046 cm−1; 1HNMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 639.3513 [M + Na]+ (calcd for C35H52O9Na, 639.3504). Cimifetidanoside D (6): white, amorphous powder; [α]20D −24.1 (c 0.08, MeOH); CD (c 0.001 M, MeOH) λmax (mdeg) 241.5 (3.1), 301.5 (−15.2) nm; IR (KBr) νmax 3423, 2930, 1732, 1654, 1381, 1045 cm−1; 1HNMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 639.3511 [M + Na]+ (calcd for C35H52O9Na, 639.3504). Cimifetidanoside E (7): white, amorphous powder; [α]20D −51.4 (c 0.04, MeOH); IR (KBr) νmax 3434, 2933, 2873, 1729, 1635, 1381, 1043 cm−1; 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 617.3684 [M + H]+ (calcd for C35H53O9, 617.3684). Cimifetidanoside F (8): white, amorphous powder; [α]20D −44.0 (c 0.05, MeOH); IR (KBr) νmax 3446, 2966, 2874, 1720, 1459, 1381, 1243, 1043 cm−1; 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 685.3932 [M + Na]+ (calcd for C37H58O10Na, 685.3922). Cimifetidanoside G (9): white, amorphous powder; [α]20D −60.2 (c 0.03, MeOH); IR (KBr) νmax 3432, 2951, 1733, 1383, 1245, 1045 cm−1; 1HNMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 685.3828 [M + Na]+ (calcd for C37H58O10Na, 685.3922). Cimifetidanoside H (10): white, amorphous powder; [α]20D −27.0 (c 0.02, MeOH); IR (KBr) νmax 3423, 2971, 2881, 1735, 1708, 1640, 1461, 1380, 1045 cm−1; 1H NMR (pyridine-d5, 400 MHz) and 13C NMR (pyridine-d5, 100 MHz), see Tables 1 and 2; HRESIMS m/z 701.3874 [M + Na]+(calcd for C37H58O11Na, 701.3871).

literature (Supporting Information) led to these compounds being identified as actein (11),18 23-epi-26-deoxyactein (12),20 12β-acetylcimigenol-3-O-β-D-xylopyranoside (13),21 12β-hydroxycimigenol-3-O-β-D-xylopyranoside (14),22 25,3′-O-diacetylcimigenol-3-β-D-xylopyranoside (15),5 and 25,4′-O-diacetylcimigenol-3β-D-xylopyranoside (16).5,23 The compounds isolated in the present study were evaluated for their cytotoxicity against five human cancer cell lines using the MTT method, with cisplatin as the positive control.24 All compounds were inactive (IC50 > 10 μM) for all cell lines, except for compounds 15 and 16, which showed cytotoxicity against the SMMC-7721 cell line, with IC50 values of 5.5 and 6.3 μM, respectively. This is the first report of activity 15 and 16 against the SMMC-7721 cell line. In previous studies, compounds 15 and 16 also showed cytotoxicity against the HepG2 cell line, with respective IC50 values of 0.71 and 2.8 μM.5 These data showed that compounds having an acetyl group attached to both the cimigenol skeleton and a sugar unit are more potent than those without such substituents.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol IV polarimeter with a 1 dm cell. UV spectra were obtained on a Shimadzu UV-2401PC spectrophotometer. The CD spectrum was obtained on a JASCO 815 spectrometer. IR spectra were recorded on a PerkinElmer Spectrum One FT-IR spectrometer with KBr pellets. 1D and 2D NMR experiments were performed on a Bruker AV 400 MHz instrument in pyridine-d5, with TMS as an internal standard. HRESIMS was performed on a Micromass Q-TOF Ultima Global GAA076 LC-MS spectrometer and Thermo Scientific LTQ Orbitrap Discovery. A Varian Prostar preparative HPLC was used for isolation and purification with a Phenomenex Luna C18 (2) preparative HPLC column (250 × 21.2 mm, 5 μm). An Agilent 1100 HPLC (DAD and Alltech ELSD 2000ES) was used to detect the purities of all compounds with a Supelco Discovery C18 column (250 × 4.6 mm, 5 μm). Silica gel (200−300 mesh, Qingdao Ocean Chemical Company), RP-18 silica gel (50 μm, YMC), and Sephadex LH-20 (20−150 μm, Pharmacia) were used for column chromatography (CC). Fractions were monitored by TLC (HSG, Yantai, People’s Republic of China), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. A plastic ball-and-stick model combined with a computer-based model on Spartan 10 software was used to create the molecular models. Plant Material. Rhizomes of C. fetida, about six years old, were collected at Longnan, Gansu Province, People’s Republic of China, in May 2008, and identified by one of the authors (B.-M.X.). A voucher specimen (No. 2008018) was deposited in the Specimen Laboratory of the School of Pharmacy, Yantai University. Extraction and Isolation. Air-dried rhizomes of C. fetida (6 kg) were pulverized and extracted three times with EtOH under reflux conditions. The EtOH extract was concentrated under reduced pressure, yielding a viscous residue (450 g), which was suspended in H2O and extracted successively with petroleum ether (60−90 °C), EtOAc, and n-BuOH. The EtOAc fraction (220 g) was subjected to silica gel CC using a stepwise gradient elution of CHCl3−MeOH (100:1, 80:1, 50:1, 30:1, 10:1, 5:1, and 2:1) to afford five subfractions (A−E). Fraction B (60 g) was chromatographed on a silica gel column using n-hexane−acetone as eluent (20:1 to 1:1, v/v) to give eight subfractions (B1−B8). Fraction B4 (2.2 g) was further separated on an ODS silica gel column by eluting with CH3CN−H2O (4:6 to 7:3, v/v), to afford subfractions B4-1−B4-4. Subfraction B4-3 was purified using preparative HPLC with CH3CN−H2O (60:40) to yield 1 (78 mg) and 2 (15 mg). Repeated silica gel column chromatography of fraction B2 (2.5 g) using CHCl3−MeOH (100:1 to 50:1) yielded 15 (50 mg) and 16 (48 mg). Fraction D (45 g) was separated on silica gel eluted with n-hexane−acetone (20:1 to 1:3) to give nine subfractions (D1−D9). G



Article

Single-Crystal X-ray Diffraction Analysis and Crystallographic Data of Compound 1. X-ray diffraction data were collected at 292.8(3) K on a Bruker SMART APEX II diffractometer with Cu Kα radiation (λ = 1.541 84 Å). The structure was solved by direct methods and refined by full-matrix least-squares techniques (SHELXL97). All non-hydrogen atoms were refined with anisotropic thermal parameters. Hydrogen atoms were located by geometrical calculations and from positions in the electron density maps. The view of compound 1 was generated with ORTEP-1 (Figure 2). Crystallographic data (excluding structure factors) for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1013638. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 12 23336033 or e-mail: [email protected]). Main parameters: C30H44O5·CH3OH, Mr = 516.69, monoclinic, space group P21, a = 10.413(9) Å, b = 7.530(6) Å, c = 18.670(18) Å, β = 99.68(8)°, V = 1443.0(2) Å3, Z = 2, F(000) = 564, Dcalc = 1.189 g/ cm3, and μ(Cu Kα) = 0.643 mm−1. Altogether, 3029 reflections were measured (4.30° ≤ θ ≤ 70.40°), of which 4538 were unique (Rint = 0.0313), and, of these, 3997 had I > 2σ(I). The final refinement gave R1 = 0.0545, wR2 = 0.1392. Hydrolysis and Identification of the Sugar Moieties in Compounds 3−10. Compounds 3−10 (5 mg of each) were dissolved in MeOH (5 mL) and refluxed with 0.5 N HCl (3 mL) for 4 h. Each reaction mixture was diluted with H2O and extracted with CHCl3 (3 × 10 mL). Each aqueous layer was then neutralized by Ag2CO3, and the precipitate filtered to give a monosaccharide. The monosaccharides from compounds 3−10 had the same Rf (EtOAc− CHCl3−MeOH−H2O, 3:2:2:1) and specific rotation value of [α]20D +24.8 (c 0.11, H2O), corresponding to that of D-xylose (SigmaAldrich). Hydrolysis of 4 and 10 with Cellulase. Compound 4 (6 mg) and compound 10 (10 mg) were each dissolved in MeOH (1 and 2 mL), and a 0.03% AcOH solution (50 and 100 mL) was added with stirring, followed by addition of cellulose (from Trichoderma viride, 100 and 200 mg) with stirring. The reaction was continued for 5 days at 45 °C. The reaction solution was then shaken with EtOAc (50 mL × 3 and 100 mL × 3), and the EtOAc solvent was evaporated in vacuo. The residue of 4 was purified using preparative HPLC with CH3CN− H2O (40:60) to yield 4a (3.5 mg), while that of 10 was purified using preparative HPLC with CH3CN−H2O (35:65) to yield 10a (6.0 mg). Cytotoxicity Assays. Five human cancer cell lines, HL-60 human myeloid leukemia, SMMC-7721 hepatocellular carcinoma, H460 lung cancer, MCF-7 breast cancer, and HeLa cervical cancer, were used in the cytotoxicity assay. Cells were cultured in DMEM medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Invitrogen), in 5% CO2 at 37 °C. The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) method in 96-well microplates. Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before addition of the test compounds, while suspended cells were seeded just before drug addition with an initial density of 1 × 105 cells/mL. Each tumor cell line was exposed to the test compound at different concentrations in triplicate for 48 h, with cisplatin (Sigma, St Louis, MO, USA) used as a positive control giving IC50 values of 3.7, 10.1, 6.7, 5.6, and 2.4 μM for the HL-60, SMMC-7721, H460, MCF-7, and HeLa cell lines, respectively. After compound treatment, the absorbance of each group was measured by using a microplate reader at a wavelength of 570 nm, using 630 nm as the reference wavelength. IC50 values were calculated by Reed and Muench’s method.25

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4a, are available free of charge via the Internet at http://pubs. acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(X.-L. Tang) Tel: +86-532-82032323. Fax: +86-53282033054. E-mail: [email protected]. *(G.-Q. Li) E-mail: [email protected]. Author Contributions ∇

J.-Y Chen and P.-L Li contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Hi-tech Research and Develoment Program of China (No. 2013AA093002, No. 2013AA092902). Special thanks are given to Prof. F. Zhao and her research group (School of Pharmacy, Yantai University, Yantai, People’s Republic of China) for the cytotoxicity data.

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REFERENCES

(1) Li, J.-X.; Yu, Z.-Y. Curr. Med. Chem. 2006, 13, 2927−2951. (2) Pharmacopoeia of the People’s Republic of China Vol.1; Medicine Science and Technology Press of China: Beijing, 2010; p 68. (3) Liu, B. B.; Chen, S. H.; Chen, S. B. Central South Pharm. 2012, 10, 53−58. (4) Sun, L.-R.; Qing, C.; Zhang, Y.-L.; Jia, S.-Y.; Li, Z.-R.; Pei, S.-J.; Qiu, M.-H.; Cross, M. L.; Qiu, S. X. Beilstein J. Org. Chem. 2007, 3, 1− 7. (5) Nian, Y.; Zhang, Y.-L.; Chen, J.-C.; Lu, L.; Qiu, M.-H.; Qing, C. J. Nat. Prod. 2010, 73, 93−98. (6) Lu, L.; Chen, J.-C.; Song, H.-J.; Li, Y.; Nian, Y.; Qiu, M.-H. Chem. Pharm. Bull. 2010, 58, 729−733. (7) Nian, Y.; Zhang, X.-M.; Li, Y.; Wang, Y.-Y.; Chen, J.-C.; Lu, L.; Zhou, L.; Qiu, M.-H. Phytochemistry 2011, 72, 1473−1481. (8) Zhou, L.; Wu, Q.-L.; Simon, J. E.; Liu, C.-Y.; Yang, J.-S.; Liao, Y.H. ACS Symp. Ser. 2008, 987, 59−99. (9) Koeda, M.; Aoki, Y.; Sakurai, N.; Nagai, M. Chem. Pharm. Bull. 1995, 43, 771−776. (10) Kadota, S.; Li, J. X.; Tanaka, K.; Namba, T. Tetrahedron 1995, 51, 1143−1166. (11) Gao, J. C.; Huang, F.; Zhang, J. C.; Zhu, G. Y.; Yang, M. S.; Xiao, P. G. J. Nat. Prod. 2006, 69, 1500−1502. (12) Shao, Y.; Harris, A.; Wang, M.; Zhang, H.; Cordell, G. A.; Bowman, M.; Lemmo, E. J. Nat. Prod. 2000, 63, 905−910. (13) Ali, Z.; Khan, S. I.; Fronczek, F. R.; Khan, I. A. Phytochemistry 2007, 68, 373−382. (14) Zhou, C.-X.; Zou, L.; Gan, L.-S.; Cao, Y.-L. Org. Lett. 2013, 15, 2734−2737. (15) Zhang, J.; Tang, X.; Li, J.; Li, P.; de Voogd, N. J.; Ni, X.; Jin, X.; Yao, X.; Li, P.; Li, G. J. Nat. Prod. 2013, 76, 600−606. (16) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.;

ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra and HRESIMS of compounds 1−10 and spectroscopic data for aglycons (4a and 10a) and the known compounds 11−16, as well as computational details for H



Article

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