Triterpenes from the Aerial Parts of Cimicifuga yunnanensis and Their

Apr 26, 2013 - Di-Fan Zhu , Guo-Lei Zhu , Ling-Mei Kong , Ni-Man Bao , Lin Zhou , Yin Nian , Ming-Hua Qiu. Natural Products and Bioprospecting 2015 5,...
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Triterpenes from the Aerial Parts of Cimicif uga yunnanensis and Their Antiproliferative Effects on p53N236S Mouse Embryonic Fibroblasts Yin Nian,† Hui Zhu,‡ Wen-Ru Tang,‡ Yin Luo,‡ Jiang, Du,§ and Ming-Hua Qiu*,† †

State Key Laboratory of Phytochemistry, Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ Laboratory of Molecular Genetics of Aging & Tumor, Faculty of Medicine, Kunming University of Science & Technology, Kunming, 650500, People’s Republic of China § Research Institute of Yunnan Biovalley, Dengzhanhua Pharmaceutical Co. Ltd, Kunming, 650224, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new triterpene derivatives, yunnanterpenes A−F (1−6), 15,16-seco-cimiterpenes A and B (7, 8), and cimilactone C (9), and 15 known analogues (10−24) were isolated from the aerial parts of Cimicif uga yunnanensis. The new structures were established using a combination of MS, NMR, and single-crystal X-ray diffraction techniques. WT MEFs (wildtype mouse embryonic fibroblasts) and tumorigenic cell lines p53−/−+H-RasV12 and p53−/−+p53N236S+H-RasV12 were used for evaluating active structures, targeting p53N236S (corresponding to p53N239S in humans) mutation. Compound 5 showed nonselective activities against these cell lines, with IC50 values of 5.8, 8.6, and 6.0 μM, respectively. Compound 4 exhibited greater selectivity against the p53−/−+p53N236S+H-RasV12 cells (IC50 5.5 μM) than against the WT MEFs cells (IC50 14.3 μM).

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pathway.9 However, there have been no reports on the chemical constituents of the aerial parts of C. yunnanensis. Thus, we initiated a study on the aerial parts of C. yunnanensis from Daocheng County. As a result, nine new triterpenes, yunnanterpenes A−F (1−6), 15,16-seco-cimiterpenes A and B (7, 8), and cimilactone C (9), and 15 known compounds (10− 24) were isolated and identified (Supporting Information, Figure 1S). All of these compounds were evaluated for their antiproliferative effects on the WT MEFs, p53−/−+H-RasV12, and p53−/−+p53N236S+H-RasV12 cell lines by the MTT method. Described herein are the isolation, structure elucidation, and biological activities of these compounds.

current strategy in cancer treatment is to apply personalized treatment aimed at different p53 mutants.1 The IARC TP53 database (version R14, November 2009) shows that human p53N239S (corresponding to p53N236S in the mouse) has been reported as a somatic mutation in 32 tumor cases and tumor origin tissues including breast, colon, stomach, hematopoietic and reticuloendothelial systems, liver and intrahepatic bile ducts, bronchus and lung, and brain.2 The wide tumor spectrum harboring p53N239S suggested its importance in tumorigenesis. Previous investigations on the gained functions of the mutation indicated that p53N236S per se was not oncogenic enough to form tumors. However, p53N236S can interact with H-RasV12 and reduce the cellular stress response to oncogenic signals to facilitate cell growth and tumorigenesis.1 Thus, using nontumorigenic and p53 knockout mouse embryonic fibroblasts (cell line p53−/− MEFs) as background, H-RasV12 was introduced into p53−/− MEFs with or without p53N236S, and two tumorigenic cell lines, p53−/−+HRasV12 and p53−/−+p53N236S+H-RasV12, were established for evaluation of active structures, targeting p53N236S mutation. As a part of our program to explore potential antitumor constituents from traditional Chinese medicine, a series of cytotoxic 9,19-cycloartane-type triterpenes were isolated from Cimicif uga spp. (Ranunculaceae), such as C. foetida, C. yunnanensis, C. dahurica, and C. heracleifolia.3−8 Three active compounds isolated from the roots of C. yunnanensis induced apoptosis of MCF-7 cells via the p53-dependent mitochondrial © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was obtained as colorless crystals. The HREIMS of 1 gave an [M]+ ion peak at m/z 502.3287 (calcd 502.3294), consistent with the molecular formula C30H46O6, requiring eight rings or sites of unsaturation. The IR spectrum showed absorptions for OH (3383 cm−1) and carbonyl (1706 cm−1) groups. The 1H NMR spectrum (Table 1) showed characteristic cyclopropane methylene signals at δH 0.47 and 0.72 (each 1H, d, J = 4.1 Hz), a secondary methyl signal at δH 1.46 (d, J = 6.5 Hz), and five tertiary methyl groups at δH 0.82−1.76. The Received: January 16, 2013

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CH3-27 (δH 1.76) and H-26α (δH 4.10). However, it was not possible to identify the stereochemistry of C-23 by ROESY experiment. To confirm the structure and determine the absolute configuration of C-23, 1 was crystallized from MeOH to afford a crystal of the orthorhombic space group P212121, which was analyzed by X-ray crystallography. On the basis of six oxygen atoms in the molecule, the final refinement of the Cu Kα data resulted in a Flack parameter of 0.06(11) and allowed unambiguous assignment of the absolute configuration (Figure 2). According to the projection, the absolute configuration at C-23 was S. Therefore, the structure of 1, named yunnanterpene A, was determined to be as shown. Yunnanterpene B (2) and yunnanterpene C (3) were determined to have the molecular formulas C30H48O6 and C32H50O7 by HREIMS (m/z 504.3444 [M]+ and 546.3557 [M]+, respectively). The spectroscopic data of 2 (Tables 1 and 2) were similar to those of 1 except that the carbonyl group was replaced by a hydroxy group at C-3 in 2. The deduction was confirmed by HMBC correlations of H-1 (δH 1.55 and 1.24) and H-2 (δH 1.95 and 1.83) with C-3 (δC 77.52), as well as a COSY correlation of H-2/H-3. The diagnostic ROESY correlation of H-3/H-5 suggested the β-orientation of the OH group at C-3. In addition, the same relative configurations at C-16, C-17, C-20, C-24, and C-25, as well as the conformations of rings E and F in 2 as in 1, were deduced from the similar NMR chemical shifts, proton coupling constants, and ROESY correlations (H-16/CH3-28, H-17/ CH3-28 H-22β, H-26β/H-24, and H-26α/CH3-27) found in 2. Thus, the structure of 2 was established as shown, and it was named yunnanterpene B. When the spectroscopic data of 3 (Tables 1 and 2) were compared with those of 2, the resonance of a hydroxy group was absent in 3, showing instead an Oacetyl group functionality at C-12. The deduction was confirmed by the correlation of H-12 (δH 5.11) with the acetyl carbonyl carbon at δC 170.57, as well as correlations of H-11 (δH 2.75 and 1.15) with H-12 in the H1−H1 COSY spectrum. Significant ROESY correlations of H-12 with H-17 and CH3-28 indicated β-orientation of the substituent at C-12. Accordingly, the structure of 3 was determined as shown. Yunnanterpene D (4) was assigned the molecular formula C30H48O6, as determined by HREIMS (m/z 504.3441 [M]+). The NMR data of compounds 4 and 2 were very similar, with the main differences being that the signals ascribed to the characteristic cyclopropane methylene were absent. However, an additional methyl group was observed. On the basis of the evidence, we deduced that the 9,19-cyclopropane ring of 4 was open and the methyl group was attached at C-9 or C-10. In the 13 C NMR spectrum (Table 2), two quaternary olefinic carbons at δC 134.31 and 137.56 were observed. HMBC correlations of H-7 (δH 1.88 and 1.09) and CH3-28 (δH 0.85) with the unsaturated carbon signal at δC 134.31 (C-8) and of H-11 (δH 2.64 and 2.07) with the unsaturated carbon signal at δC 137.56 (C-9) located the double bond at C-8 and C-9. Thus, the methyl group (CH3-19) was attached at C-10, which was further supported by the HMBC correlation of the methyl signal at δH 0.92 with C-1 at δC 36.59. The orientation of CH319 was assigned as β by the ROESY correlation of CH3-19 (δH 0.92) and CH3-18 (δH 1.05). Thus, the structure of 4 was determined as shown. Yunnanterpene E (5) was determined to have the molecular formula C30H46O5 by HREIMS (m/z 486.3344 [M]+). The UV and IR spectra of 5 showed absorption bands for hydroxy groups (νmax 3425 cm−1) and a unit of conjugated double

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C NMR and DEPT (Table 2) spectra displayed signals for one carbonyl group, seven quaternary carbons (including two oxygenated ones), seven methines (including three oxygenated ones), nine methylenes (including an oxygenated one), and six methyls. The data suggested that 1 was a highly oxygenated 9,19-cycloartane-type triterpene, and a seven-ring structure was required to fulfill the unsaturation requirement. The 1H−1H COSY (Figure 1) and HMQC spectra disclosed that 1 had partial structures −CH2−CH2− (for C-1 to C-2), −CHCH2CH2CH− (for C-5 to C-8), −CH2CH(OH)− (for C-11 to C-12), and −CH2CHCHCH(CH3)CH2− (for C-15 to C-17, C-20 to C-22) and a pair of geminal signals for CH2-26 at δH 4.10 and 4.32 (each 1H, d, J = 8.6 Hz) that were typical of rings A, B, C, D, and part of E and F of a 9,19-cycloartane-type triterpene skeleton.22 HMBC correlations observed from H-26 at δH 4.10 and 4.32 to the two quaternary carbons at δC 110.87 (C-23) and 79.78 (C-25), from the methyl resonance at δH 1.76 (Me-27) to the quaternary carbon resonance at δC 79.78 (C25), the methylene carbon resonance at δC 77.19 (C-26), and the methine resonances at δC 83.67 (C-24), and from H-16 at δH 4.71 (dd, J = 14.7, 7.8 Hz) to the quaternary carbon at δC 110.87 (C-23) were similar to those of 23-epi-26-deoxyactein22 and indicated that rings E and F of 1 each contained one oxygen unit. Since compound 1 had a carbonyl group (δC 214.97), no more rings were required. Thus, we deduced that OH groups were attached to C-24 and C-25 in 1. This conclusion was supported by the molecular weight, as well as by the signals of C-24 at δC 83.67 and 79.78 in the 13C NMR spectrum of 1. In the HMBC spectrum, the correlations of H-2 (δH 1.43 and 1.71) and CH3-30 (δH 1.12) with the carbonyl carbon at δC 214.97 placed the carbonyl group at C-3. An OH was located at C-12 by the 1H−1H COSY correlations of H-12 at δH 4.06 and H-11 at δH 2.57 and 1.48. The relative configuration of the core structure of 1 was established by the ROESY experiment (Figure 1). The crosspeaks of H-12, H-16, and H-17 with CH3-28 in the ROESY spectrum assigned their orientations as α. In addition, H-22β (δH 2.85) and H-26β (δH 4.32) showed correlations with H-24, indicating an α-orientation of the OH group at C-24. The orientation of CH3-27 was assigned as α by the correlation of B

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Table 1. 1H NMR Data of Compounds 1−9 (δ in ppm, J in Hz) 1b

position

2b

3b

4b

5b

1

1.71 m

1.55 m

1.47 m

1.55 m

2.01 m

2

1.43a 2.62 m 2.28 m

1.24a 1.95 m 1.83 dd (12.3, 3.4) 3.52 m

1.04a 1.91 m 1.80 m

1.09 m 1.72 m 1.12a

1.48 m 1.96 m 1.22a

3.47 m

3.32 m

3.48 m

1.28 m

1.21a

1.55 m 0.83 m

1.51 m 0.77 dd (24.6, 12.3) 1.26a 0.95 dd (24.7, 11.9) 1.55 m

1.02 brd (12.4) 1.59 m 1.40 m 1.88 m 1.09a

3 4 5 6

1.56 dd (12.1, 4.1) 1.37 m 0.84a

7

1.30 m 0.97a

8

1.65a

1.33 m 1.02 brd (14.6) 1.66 m

2.57 dd (15.0, 8.4) 1.48a

2.60 dd (15.4, 8.6) 1.50a

2.75 m

4.06 m

4.08 m

5.11 m

9 10 11

12 13 14 15

16 17 18 19 20 21 22

23 24 25 26

27 28 29 30 3-sugar 1′ 2′ 3′

1.15 brd (16.0)

7b

3.24 dd (11.6, 3.7)

1.24 dd (11.9, 5.0) 2.11 m 1.22a

1.55a

1.49 m

1.51a

0.96a

2.04 m 1.33a

1.29 m 0.85 m

1.38 m 0.91 m

5.54 brd (5.0)

3.84 m

1.36 m 1.16 m

1.38 m 1.17 m

1.27 m 0.51 dd (25.3, 12.8) 0.98a 0.70 brd (12.7)

2.18 brd (7.7)

2.43 m

2.41 m

1.36 dd (12.1, 5.4)

1.61

2.51 dd (16.0, 9.0) 0.92 dd (16.1, 3.5) 4.83a

2.64 dd (18.0, 7.9) 2.07 m

5.39a

2.37 m

1.42 m (2H)

1.47 m (2H)

4.24 brd (12.4)

2.21 d (17.6)

1.70 dd (15.4, 6.0) 4.10 t (7.5)

1.55a

1.63a

1.38 m

1.40 m

9.95 s

9.93 s

1.78 m

2.76 d (5.7)

2.75 d (5.2)

1.60 dd (13.6, 5.4) 4.57 dd (13.5, 8.0 1.92a

1.52 s 0.60 d (4.3) −0.03 d (4.5) 2.06 m

1.02 0.36 0.00 1.78

0.99 d (6.0) 2.04 m

0.74 d (6.4) 2.23 dd (14.7, 3.3) 2.03 t (14.1)

1.84 m

2.04 m

1.68 m

1.63 m

1.72 m

4.73 dd (14.8, 7.5) 1.93 m

4.65 q (7.5)

4.66 dd (14.5, 7.4) 1.80 m

1.86 dd (12.2, 6.7) 4.77 dd (14.9, 7.6) 1.62 m

1.39 0.66 0.31 2.12

1.29 0.60 0.24 1.97

2.62dd (13.4, 8.2) 2.35 m 4.84 dd (14.4, 7.7) 1.99 t (8.5)

1.46 d (6.5) 2.85 dd (13.8, 2.7) 1.70 m

1.48 d (6.5) 2.85 dd (13.8, 2.3) 1.70 m

1.02 d (6.4) 2.77 m 1.61 m

1.44 d (6.4) 2.75 dd (13.8, 2.5) 1.59 m

4.60 d (6.1)

4.59 brs

4.58 d (5.5)

4.52 brd (4.0)

4.62 s

4.63 s

1.51 s 0.60 d (4.2) −0.04 d (4.2) 2.04 dd (12.3, 5.9) 0.98 d (6.3) 2.55 dd (14.3, 5.2) 1.79 m 5.01 d (11.1) 3.70 s

4.32 d (8.6)

4.33d (8.6)

4.29 d (8.6)

4.34 d (8.6)

4.34 brd (8.4)

1.58 s

1.55 s

4.10 1.76 0.82 0.98 1.12

4.11 1.76 0.88 1.05 1.22

4.07 1.75 0.86 1.04 1.20

4.24 brd (12.4) 4.00 d (8.6) 1.66 s 0.85 s 0.94 s 1.12 s

4.09 1.77 0.93 1.12 1.21

4.10 1.78 1.08 1.00 1.33

1.63 1.69 0.93 1.21

1.59 1.60 0.97 1.23

d (8.6) s s s s

d (8.6) s s s s

s d (3.0) d (3.6) m

1.32 m 2.32 brd (9.6) 1.89 t (13.8) 3.49 dd (11.6, 4.1)

1.88 m

s d (3.7) d (3.9) m

1.29 m 2.34 brd (9.9) 1.91 m

1.21 dd (13.2, 4.0) 0.80a 1.67 m 1.56 m

3.49 dd (11.6, 3.8)

1.91 m

1.80 m

9b

a

1.58

1.92 m 1.88 dd (12.7, 8.1) 1.64 dd (11.2, 5.4) 4.71 dd (14.7, 7.8) 1.90 dd (15.2, 7.0) 1.39 s 0.72 d (4.1) 0.47 d (4.1) 2.13 m

8b

a

1.57 dd (14.2, 3.5) 1.33a 2.35 m 1.90 qd (12.9, 4.0) 3.49 dd (11.7, 4.2)

d (8.6) s s s s

1.05 s 0.92 s

0.79 s

2.00 m

1.97 m

1.44 0.91 0.20 2.18

0.98 d (6.5) 2.78 dd (13.8, 2.2) 1.58 m

1.54 d (6.4) 2.88 dd (13.8, 2.7) 1.73 brd (13.3)

d (8.6) s s s s

s d (4.1) d (4.4) brd (7.7)

d (8.4) s s s s

4.80 d (7.2) 4.48 t (7.5) 4.19 dd (9.0, 3.2) 4.35 brs 4.32 brd (11.2, 2.4) 3.83 brd (10.9)

4′ 5′

AcO-12

6c

2.09 s

s s s s

4.83 d (7.2) 4.49 t (8.0) 4.19 dd (7.9, 2.9) 4.35 brs 4.37 brd (12.9) 3.87 d (11.6)

1.87 m 5.04 d (11.5) 3.73 s

s s s s

s d (3.9) d (4.2) m

0.61 s 0.80 s 0.96 s

4.90 d (7.6) 4.06 t (8.1) 4.20 t (8.8) 4.26 m 4.43 dd (11.2, 5.2) 3.83 t (10.7) 1.90 s

C

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Table 1. continued a

Signals overlapped. bRecorded at 500 MHz in pyridine-d5. cRecorded at 600 MHz in pyridine-d5.

Table 2. 13C NMR Data of Compounds 1−9 (δ in ppm) position

1a

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 3-sugar 1′ 2′ 3′ 4′ 5″ AcO-12

33.30 37.40 214.97 50.07 48.04 21.00 25.64 45.64 21.71 26.34 40.58 72.14 50.46 47.59 44.22 72.84 57.01 12.97 29.07 26.37 21.75 37.47 110.87 83.67 79.78 77.19 20.83 19.58 20.78 22.58

a

2a CH2 CH2 C C CH CH2 CH2 CH C C CH2 CH C C CH2 CH CH CH3 CH2 CH CH3 CH2 C CH C CH2 CH3 CH3 CH3 CH3

31.98 30.82 77.52 40.71 46.69 20.54 25.57 45.38 20.51 26.61 40.25 72.08 50.15 47.51 43.96 72.61 56.73 12.61 29.34 26.09 21.52 37.09 110.62 83.32 79.54 76.97 20.46 19.37 14.48 25.93

3a CH2 CH2 CH C CH CH2 CH2 CH C C CH2 CH C C CH2 CH CH CH3 CH2 CH CH3 CH2 C CH C CH2 CH3 CH3 CH3 CH3

32.24 31.11 77.73 41.01 46.98 20.81 25.97 46.01 20.82 27.06 36.98 77.30 47.85 48.84 43.80 72.53 56.45 13.61 30.04 26.13 21.15 37.33 110.58 83.85 79.68 77.13 20.88 19.71 14.81 26.19

4b CH2 CH2 CH C CH CH2 CH2 CH C C CH2 CH C C CH2 CH CH CH3 CH2 CH CH3 CH2 C CH C CH2 CH3 CH3 CH3 CH3

36.59 29.11 78.42 39.96 51.53 19.10 27.07 134.31 137.56 37.98 35.21 71.38 49.67 50.55 41.04 73.68 55.58 12.10 19.84 27.07 22.73 37.68 111.29 84.38 80.22 77.84 21.40 25.44 16.86 29.11

5a CH2 CH2 CH C CH CH2 CH2 C C C CH2 CH C C CH2 CH CH CH3 CH3 CH CH3 CH2 C CH C CH2 CH3 CH3 CH3 CH3

36.39 28.62 78.14 39.36 49.85 23.47 121.49 146.67 142.07 37.94 116.44 38.11 43.44 48.26 40.63 72.68 55.25 18.68 23.08 26.41 20.83 37.04 110.97 83.92 79.70 77.03 20.87 25.83 16.69 28.89

170.57 C 21.66 CH3

6b CH2 CH2 CH C CH CH2 CH C C C CH CH2 C C CH2 CH2 CH CH3 CH3 CH CH3 CH2 C CH C CH2 CH3 CH3 CH3 CH3

7a

8a

9a

31.72 30.14 88.51 41.35 45.97 31.78 69.86 53.02 22.03 27.24 40.63 72.26 51.09 48.57 45.95 73.71 56.96 12.78 27.42 26.78 22.54 38.02 111.31 84.33 80.22 77.49 21.38 19.81 15.58 26.24

CH2 CH2 CH C CH CH2 CH CH C C CH2 CH C C CH2 CH CH CH3 CH2 CH CH3 CH2 C CH C CH2 CH3 CH3 CH3 CH3

30.49 29.44 87.93 41.31 43.93 18.39 21.44 38.52 20.81 25.54 26.96 32.63 47.32 55.34 207.56 175.02 55.19 18.03 21.83 28.49 24.82 36.74 78.57 79.88 72.45 26.00 28.49 14.69 14.46 25.35

CH2 CH2 CH C CH CH2 CH2 CH C C CH2 CH2 C C C C CH CH3 CH2 CH CH3 CH2 CH CH C CH3 CH3 CH3 CH3 CH3

30.42 CH2 29.49 CH2 88.00 CH 41.30 C 44.15 CH 18.55 CH2 21.68 CH2 38.70 CH 20.79 C 25.71 C 26.95 CH2 32.64 CH2 47.20 C 55.30 C 207.38 C 174.86 C 55.49 CH 18.21 CH3 22.07 CH2 28.48 CH 24.80 CH3 36.71 CH2 78.57 CH 79.86CH 72.43 C 26.02 CH3 28.98 CH3 14.73 CH3 14.38 CH3 25.35 CH3

108.06 73.43 75.17 70.15 67.46

CH CH CH CH CH2

107.52 72.99 74.68 69.66 67.15

CH CH CH CH CH2

107.48 75.60 78.61 71.31 67.14

31.06 30.48 77.75 41.00 46.93 20.70 25.80 46.16 20.16 27.24 36.61 76.79 48.35 48.69 43.88 80.46 53.77 13.29 29.86 26.90 21.99 38.63 173.60

CH2 CH2 CH C CH CH2 CH2 CH C C CH2 CH C C CH2 CH CH CH3 CH2 CH CH3 CH2 C

19.63 CH3 14.72 CH3 26.15 CH3

CH CH CH CH CH2 170.65 C 21.42 CH3

Recorded at 125 MHz in pyridine-d5. bRecorded at 150 MHz in pyridine-d5.

respectively. These observations could be explained by the conjugated double bonds located at C-7/C-8 and C-9/C-11 and absence of an OH group at C-12. In the 1H−1H COSY spectrum, the correlations of the proton signals at δH 1.51 and 0.77 (H-6) with the olefinic signal at δH 5.54 (H-7) and of the proton signals at δH 2.12 and 1.92 (H-12) with the olefinic signal at δH 5.54 (H-11) further supported this deduction. Accordingly, the structure of 5 was determined as shown. Yunnanterpene F (6) gave the molecular formula C35H56O11 from its HREIMS data at m/z 652.3833 [M]+. The 1H NMR spectrum of compound 6 showed signals for a cyclopropane methylene at δH 0.20 (1H, d, J = 4.1 Hz) and 0.91 (1H, d, J = 4.4 Hz), six methyl groups at δH 1.00−1.78, and an anomeric proton resonance at δH 4.80 (d, J = 7.2 Hz), suggesting that 6 was a 9,19-cycloartane-type triterpene glycoside. An HMBC correlation between the anomeric proton at δH 4.80 (d, J = 7.2

Figure 1. Major HMBC (→), 1H−1H COSY (−), and ROESY (↔) correlations of compound 1.

bonds (λmax 242 nm; νmax 1631 cm−1). The NMR spectroscopic data of 5 closely resembled those of 4, except for the presence of four olefinic carbon signals at δC 116.44, 121.49, 142.07, and 146.67 and the absence of resonances of two methylenes and the presence of a hydroxymethine due to C-7, C-11, and C-12, D

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Figure 2. X-ray crystal structures of 1 and 7.

Hz) and the methine signal at δC 88.51 (C-3) located the sugar moiety at C-3. The sugar obtained after acid hydrolysis was identified as L-arabinose by comparing its TLC and specific rotation with a standard. The NMR data (Tables 1 and 2) of the aglycone part of 6 resembled those of 2 with the exception of an additional OH group, which was assigned to C-7 on the basis of the correlations of H-6 (δH 2.04 and 1.33) and H-8 (δH 2.18) with the hydroxymethine proton (δH 3.84) in the 1H−1H COSY spectrum. In the ROESY spectrum, correlations of H-3/ H-5 and CH3-28/H-7 suggested the β-orientation of the substituents at C-3 and C-7. Therefore, the structure of 6 was determined as shown. Compounds 7 and 8 (15,16-seco-cimiterpenes A and B) both had the molecular formula C35H56O10, as determined by HREIMS. The NMR data of 7 were similar to the resonances of 15,16-seco-14-formyl-16-oxo-7-en-hydroshengmanol-3-O-β-Dxylopyranoside,23 with the major differences at ring B and the sugar unit. In the 13C spectrum of 1, a pair of double-bond signals due to C-7 at δC 122.2 (d) and C-8 at δC 140.4 (s) were absent, showing instead a methylene and a methine at δC 21.44 and 38.52, respectively. The sugar obtained after acid hydrolysis was identified as L-arabinose by comparing its TLC and specific rotation with a standard. In the ROESY spectrum, CH3-18 revealed a correlation with H-8, while CH3-28 showed a correlation with H-7α, suggesting that CH3-18 was β-orientated and that CH3-28 was α-orientated. However, it was difficult to identify the stereochemistry of C-17, C-20, C-23, and C-24 by ROESY experiments, due to the cleavage between C-15 and C16. To confirm the structure and determine the absolute configuration of the chiral centers, 7 was crystallized from MeOH to afford a crystal of the orthorhombic space group P212121, which was analyzed by X-ray crystallography. On the basis of 10 oxygen atoms in the molecule, the final refinement of the Cu Kα data resulted in a Flack parameter of 0.06(10), which allowed unambiguous assignment of the absolute configuration (Figure 2). According to the projection, the absolute configurations of C-17, C-20, C-23, and C-24 were assigned as R, R, R, and S. Accordingly, the structure of 7 was determined as shown. The NMR data of 8 were quite similar to those of 7 except for the sugar moiety at δC 107.48 (d), 75.60 (d), 78.61 (d), 71.31 (d), and 67.14 (t). The sugar obtained after acid hydrolysis was identified as D-xylose by comparing its TLC and specific rotation with a standard. On the basis of the ROESY correlations of H-17 and Me-21/H-22α, H-23/H-22β, and H-23/H-24, identical NMR data, and the coupling constants of H-24, the same configurations of C-17 (R), C-20 (R), C-23 (R), and C-24 (S) of 7 and 8 can be concluded. Thus, the structure of 8 was determined as shown.

Cimilactone C (9) was determined to have the molecular formula C28H42O5 by HREIMS (m/z 458.3027, [M]+). The IR spectrum of 9 showed absorptions for OH (3485 cm−1) and carbonyl (1729 and 1705 cm−1) groups. 1H NMR signals for a cyclopropane methylene at δH 0.36 (1H, d, J = 3.9 Hz) and 0.00 (1H, d, J = 4.2 Hz), an acetyl methyl group at δH 1.90, four singlet methyl groups at δH 0.61, 0.80, 0.96, and 1.02, and a secmethyl group at δH 0.74 (d, J = 6.4 Hz) were observed. Analysis of the data obtained led to the conclusion that 9 was a tetranortriterpenoid with an acetoxy group. 13C NMR and DEPT spectroscopic data of 9 were identical to the aglycone resonances of cimilactone A24 except for an upfield shift of the C-3 signal by 9.95 ppm, which was consistent with the absence of a sugar unit at C-3. Significant ROESY correlations of H-3/ H-5, H-12/CH3-28, H-16/CH3-28, and H-17/CH3-28 suggested the β-orientation of the substituents at C-3, C-12, C-16, and C-17. Therefore, the structure of 9 was established as shown. The known compounds 12β-hydrocimilactone (10),10 cimigenol (11),11 12β-O-acetylcimigenol (12),12 12β-hydrocimigenol (13),13 12β-hydrocimigenol-7(8)-en (14),7 12βhydrocimigenol-7(8)-3-one (15),14 24-epi-7(8)-en-cimigenol3-O-α-L-arabinopyranoside (16),15 24-epi-7(8)-en-cimigenol-3O-β-D-xylosepyranoside (17),15 7(8)-en-cimigenol-3-O-α-L-arabinopyranoside (18),16 25-anhydrocimigenol (19),17 25-anhydrocimigenol-3-O-α- L -arabinopyranoside (20), 6 acerinol (21),18 acteol (22),19 12β-O-acetylcimiracemonol (23),20 and dahurinol (24)21 were identified (see Supporting Information for structures of 10−24) by comparing their physical and spectroscopic data with reported data. All isolated compounds (1−24) were evaluated for their inhibitory activities against the WT MEF, p53−/−+H-RasV12, and p53−/−+p53N236S+H-RasV12 cells, a featured approach for screening active structures targeting p53N236S mutation. Compounds 4 and 5 showed the most potent activities against the p53−/−+p53N236S+H-RasV12 cells, having IC50 values of 5.5 and 8.6 μM, respectively (Supporting Information, Table 1S). However, 5 indicated nonselective cytotoxicity against the WT MEFs, with an IC50 value of 6.0 μM. Compound 4 exhibited approximately 3-fold higher selectivity against the WT MEFs cells (IC50 14.5 μM) than 5. Thus, the mechanism of action of compound 4 is worth studying in a more advanced way. Compounds 9, 14, 19, 21, and 22 showed greater selective activities against the p53−/−+H-RasV12 cells (IC50 7.5, 9.8, 6.2, 10.2, and 7.4 μM) than against the WT MEFs and p53−/−+p53N236S+H-RasV12 cell line. The phenomenon may be due to the following: (1) p53N236S can interact with HRasV12 in the p53−/−+p53N236S+H-RasV12 cells and facilitate cell proliferation; (2) the normal function of P53 in the WT E

dx.doi.org/10.1021/np4000262 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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Article

(3β,12β,23S,24R,25S)-16,23:23,26-Diepoxy-3,12,24,25-tetrahydroxy-9,19-cycloartane (2): white powder; [α]27 D −89.3 (c 0.08, MeOH); IR (KBr) νmax 3422, 2953, 2871, 1447, 1379, 1165, 1089, 1020, 957 cm−1; 1H (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectra see Tables 1 and 2; ESIMS m/z 527 [M + Na]+; HREIMS m/z 504.3444 (calcd for C30H48O6, 504.3451). (3β,12β,23S,24R,25S)-16,23:23,26-Diepoxy-12-acetoxy-3,24,25trihydroxy-9,19-cycloartane (3): white powder; [α]27 D −112.1 (c 0.09, MeOH); IR (KBr) νmax 3448, 2935, 2873, 1715, 1455, 1382, 1248, 1095, 983 cm−1; 1H (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectra see Tables 1 and 2; ESIMS m/z 569 [M + Na]+; HREIMS m/z 546.3557 (calcd for C32H50O7, 546.3557). (3β,12β,23S,24R,25S)-16,23:23,26-Diepoxy-3,12,24,25-tetrahydroxylanosta-8(9)-ene (4): white powder; [α]27 D −71.1 (c 0.05, MeOH); IR (KBr) νmax 3452, 2961, 2874, 1630, 1453, 1377, 1168, 1034, 961 cm−1; 1H (C5D5N, 500 MHz) and 13C NMR (C5D5N, 150 MHz) spectra see Tables 1 and 2; ESIMS m/z 527 [M + Na]+; HREIMS m/z 504.3454 (calcd for C30H48O6, 504.3451). (3β,23S,24R,25S)-16,23:23,26-Diepoxy-3,24,25-trihydroxylanosta-7(8),9(11)-diene (5): white powder; [α]27 D −136.8 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 242 (0.51), 196 (0.19). IR (KBr) νmax 3425, 2958, 2874, 1631, 1452, 1374, 1156, 1033, 948 cm−1; 1H (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectra see Tables 1 and 2; ESIMS m/z 509 [M + Na]+; HREIMS m/z 486.3344 (calcd for C30H46O5, 486.3345). (3β,6β,12β,23S,24R,25S)-16,23:23,26-Diepoxy-6,12,24,25-tetrahydroxy-9,19-cycloart-3-O-β-D-xylopyranoside (6): white powder; [α]27 D −21.6 (c 0.10, MeOH); IR (KBr) νmax 3441, 2948, 2874, 1711, 1456, −1 1 13 1383, 1142, 1085, 1010 cm ; H (C5D5N, 600 MHz) and C NMR (C5D5N, 150 MHz) spectra see Tables 1 and 2; ESIMS m/z 675 [M + Na]+; HREIMS m/z 652.3833 (calcd for C35H56O11, 652.3823). (3β,17R,23R,24S)-15,16-seco-14-Formyl-16-oxo-16,23-epoxy9,19-cycloart-3-O-α-L-arabinopyranoside (7): colorless crystals; mp 213-216 °C; [α]27 D −65.4 (c 0.05, MeOH); IR (KBr) νmax 3457, 2939, 2884, 1711, 1640, 1458, 1384, 1257, 1142, 1069, 995 cm−1; 1H (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectra see Tables 1 and 2; ESIMS m/z 659 [M + Na]+; HREIMS m/z 636.3871 (calcd for C35H56O10, 636.3873). (3β,17R,23R,24S)-15,16-seco-14-Formyl-16-oxo-16,23-epoxy9,19-cycloart-3-O-β-D-xylosepyranoside (8): white powder; [α]D27 −66.0 (c 0.06, MeOH); IR (KBr) νmax 3424, 2962, 2870, 1711, 1640, 1460, 1378, 1239, 1070, 9789 cm−1; 1H (C5D5N, 500 MHz) and 13 C NMR (C5D5N, 125 MHz) spectra see Tables 1 and 2; ESIMS m/z 659 [M + Na]+; HREIMS m/z 636.3876 (calcd for C35H56O10, 636.3873). (3β,12β,16β)-3-Hydroxy-12-acetoxy-24,25,26,27-tetranor-9,19cycloart-23,16-olide (9): white powder; [α]D27 −111.5 (c 0.12, MeOH); IR (KBr) νmax 3485, 2924, 2860, 1729, 1705, 1439, 1378, 1262, 1126, 1031, 987 cm−1; 1H (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz) spectra see Tables 1 and 2; ESIMS m/z 481 [M + Na]+; HREIMS m/z 458.3027 (calcd for C28H42O5, 458.3032). X-ray Crystal Structure Analysis. Colorless crystals of 1 and 7 were obtained from CH3OH. Intensity data were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97.25 Refinements were performed with SHELXL-97, using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The H atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystallographic data (excluding structure factor tables) for the structures reported have been deposited with the Cambridge Crystallographic Data Center as supplementary publications no. CCDC 918339 for 1 and CCDC 918340 for 7. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44(0) (1223) 336 033; e-mail: [email protected]]. Yunnanterpene A (1): C30H46O6, M = 502.67, orthorhombic, a = 10.1345(2) Å, b = 15.4301(3) Å, c = 16.8872(3) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 2640.76(9) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.689 mm−1, 21 112 reflections measured,

MEFs can regulate the cellular stress response to noxious signals and promote cell growth.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in MeOH with a Horiba SEAP-300 polarimeter. 1H and 13 C NMR spectra were recorded in pyridine-d5 on Bruker DRX-500 and Avance III-600 MHz spectrometers (Bruker, Zű rich, Switzerland). Unless otherwise specified, chemical shifts (δ) were expressed in ppm with respect to the solvent signals. Mass spectra were performed on a VG Autospec-3000 spectrometer. Infrared spectra were recorded on a Shimadzu IR-450 instrument with KBr pellets. Thin-layer chromatography was performed on precoated TLC plates (200−250 μm thickness, silica gel 60 F254, Qingdao Marine Chemical, Inc.), and spots were visualized by heating after spraying with 10% aqueous H2SO4. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a YMC-Pack Pro C18 RS 10 mm × 250 mm column. Silica gel (200−300 mesh, Qingdao Marine Chemical, Inc.), Lichroprep RP-18 (40−63 μm, Merck), and Sephadex LH-20 (20−150 μm, Pharmacia) were used for column chromatography (CC). Plant Material. The aerial parts of Cimicif uga yunnanensis (1.5 kg) were collected from Daocheng County, Sichuan Province, China, in September 2008 and were identified by Prof. Shengji Pei, Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (KUN No. 200809007) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, China. Extraction and Isolation. The dried and milled aerial parts of C. yunnanensis (1.5 kg) were extracted with MeOH (3 × 5 L; 24 h each) at room temperature to give a residue (137.8 g) after evaporating under vacuum at 50 °C. The extract was subjected to silica gel CC (3 kg, 10 × 150 cm) and eluted with CHCl3−MeOH [100:0 (3 L), 50:1 (6 L), 20:1 (8 L), 10:1 (5 L), 0:100 (4 L)] to afford fractions A (16.7 g), B (22.7 g), C (18.5 g), D (19.3 g), and E (20.7). Fraction B was divided into five subfractions (B.1−B.5) after performing RP-18 CC (500 g, 6 × 50 cm), eluting with MeOH−H2O (gradient from 50:50 to 100:0). Fraction B.3 (4.6 g) was subjected to repeated silica gel CC (60 g, 5 × 40 cm), eluted with CHCl3−Me2CO (gradient from 20:1 to 10:1), and then repeated semipreparative HPLC (eluted with CH3CN−H2O, gradient from 60:40 to 85:15) to yield 1 (6.0 mg), 2 (7.2 mg), 3 (6.9 mg), 12 (4.8 mg), 13 (3.2 mg), 15 (4.3 mg), and 19 (4.7 mg). Compounds 11 (4.8 mg), 14 (3.5 mg), 21 (4.3 mg), 22 (5.7 mg), 23 (3.5 mg), and 24 (3.6 mg) were isolated from fraction B.4 (3.8 g) by conducting silica gel CC (40 g, 5 × 40 cm), eluting with CHCl3−Me2CO (20:1, 7 L), followed by repeated semipreparative HPLC (eluted with CH3CN−H2O, gradient from 60:40 to 90:10). Fraction B.5 (3.9 g) was applied to a silica gel column (30 g, 4 × 40 cm), eluted with CHCl3−Me2CO (20:1, 8 L), then subjected to semipreparative HPLC (eluted with CH3CN−H2O, gradient from 65:35 to 90:10) to afford 4 (3.7 mg), 5 (3.2 mg), 9 (5.9 mg), and 10 (6.6 mg). Fraction C (18.5 g) was separated into four subfractions (C.1−C.4) by performing RP-18 CC (300 g, 5 × 40 cm), eluting with MeOH−H2O (gradient from 50:40 to 90:10). Fraction C.3 (2.3 g) was subjected to silica gel CC (50 g, 4 × 40 cm) eluted with CHCl3− Me2CO (gradient from 10:1 to 5:1), then repeated semipreparative HPLC (eluted with CH3CN−H2O, gradient from 60:40 to 75:25) to obtain 6 (4.5 mg), 16 (3.8 mg), 20 (3.3 mg), and 18 (2.8 mg). Fraction C.2 (4.8 g) was subjected to silica gel CC (50 g, 4 × 40 cm), eluting with CHCl3−Me2CO (10:1, 13 L), to yield 7 (7.2 mg), 8 (5.0 mg), and 18 (4.2 mg). (12β,23S,24R,25S)-16,23:23,26-Diepoxy-12,24,25-trihydroxy9,19-cycloart-3-one (1): colorless crystals; mp 226−228 °C; [α]27 D −71.7 (c 0.09, MeOH); IR (KBr) νmax 3439, 2965, 2857, 1706, 1455, 1416, 1343, 1172, 1020, 961 cm−1; 1H (C5D5N, 500 MHz) and 13C NMR (C5D5N, 150 MHz) spectra see Tables 1 and 2; ESIMS m/z 525 [M + Na]+; HREIMS m/z 502.3287 (calcd for C30H46O6, 502.3294). F

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4789 independent reflections (Rint = 0.0274). The final R1 values were 0.0282 (I > 2σ(I)). The final wR(F2) values were 0.0726 (I > 2σ(I)). The final R1 values were 0.0283 (all data). The final wR(F2) values were 0.0726 (all data). The goodness of fit on F2 was 1.046. Flack parameter = 0.06(11). The Hooft parameter is 0.02(3) for 2029 Bijvoet pairs. 15,16-seco-Cimiterpene A (7): C35H56O10, M = 636.80, orthorhombic, a = 8.3726(2) Å, b = 10.4589(2) Å, c = 36.9890(8) Å, α = 90.00°, β = 90.00°, γ = 90.00°, V = 3239.06(12) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.769 mm−1, 15 921 reflections measured, 5572 independent reflections (Rint = 0.0287). The final R1 values were 0.0294 (I > 2σ(I)). The final wR(F2) values were 0.0765 (I > 2σ(I)). The final R1 values were 0.0295 (all data). The final wR(F2) values were 0.0766 (all data). The goodness of fit on F2 was 1.063. Flack parameter = 0.06(10). The Hooft parameter is 0.07(3) for 2177 Bijvoet pairs. Hydrolysis and Identification of the Sugar Moieties in Compounds 6−8. Compounds 6, 7, and 8 (4 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 monosaccharide from compounds 6 and 7 had an Rf (EtOAc− CHCl3−MeOH−H2O, 3:2:2:1) and specific rotation ([α]20 D +82.8 (c 0.09, MeOH)) corresponding to those of L-arabinose (Sigma-Aldrich), while the monosaccharide from compound 8 had an Rf (EtOAc− CHCl3−MeOH−H2O, 3:2:2:1) and specific rotation ([α]20 D +30.2 (c 0.13, H2O)) corresponding to those of D-xylose (Sigma-Aldrich). Cytotoxicity Bioassay. WT MEF, p53−/−+H-RasV12 MEF, and p53−/−+p53N236S + H-RasV12 MEF cell lines were used in the cytotoxic assay. Cells were cultured in DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA), in 5% CO2 at 37 °C. The assay was performed according to the MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method in 96-well microplates.26,27 Briefly, 100 μL of cells was seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before addition of 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 concentrations of 0.064, 0.32, 1.6, 8, and 40 μM in triplicates for 48 h, with cisplatin (Sigma, USA) as a positive control. After compound treatment, cell viability was detected and a cell growth curve was graphed. IC50 values were calculated by Reed and Muench’s method.28

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ASSOCIATED CONTENT

S Supporting Information *

This material (1H, 13C, HSQC, HMBC, COSY, ROESY, ESIMS, and HREIMS spectra of compounds 1−9, X-ray data of compounds 1 and 7, structures of compounds 10−24, and bioassay results of compounds 1−24) is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (86) 871-65223257. Fax: (86) 871-65223255. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. U1132604), the Knowledge Innovation Program of the CAS (Grant Nos. KZCX2-XB2-1503, KSCX2-EW-R-15), the Top Talents Program of Yunnan Province (2009C1120), and the Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2010-ZZ14). G

dx.doi.org/10.1021/np4000262 | J. Nat. Prod. XXXX, XXX, XXX−XXX