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Cytotoxic cassaine diterpenoid–diterpenoid amide dimers and diterpenoid amides from the leaves of Erythrophleum fordii
Phytochemistry 71 (2010) 1749–1755
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Cytotoxic cassaine diterpenoid–diterpenoid amide dimers and diterpenoid amides from the leaves of Erythrophleum fordii Dan Du, Jing Qu, Jia-Ming Wang, Shi-Shan Yu *, Xiao-Guang Chen, Song Xu, Shuang-Gang Ma, Yong Li, Guang-Zhi Ding, Lei Fang Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education and Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 2 November 2009 Received in revised form 22 April 2010 Available online 2 August 2010 Keywords: Erythrophleum fordii Leguminosae Cassaine diterpenoid–diterpenoid amide dimers Cassaine diterpenoid amides Cytotoxicity
a b s t r a c t Detailed phytochemical investigation from the leaves of Erythrophleum fordii resulted in the isolation of 13 compounds, including three cassaine diterpenoid–diterpenoid amide dimers (1, 3 and 5), and seven cassaine diterpenoid amides (6 and 8–13), together with three previously reported ones, erythrophlesins D (2), C (4) and 3b-hydroxynorerythrosuamide (7). Compounds 1, 3 and 5 are further additions to the small group of cassaine diterpenoid dimers represented by erythrophlesins A–D. Their structures were determined by analysis of extensive one- and two-dimensional NMR experiments and ESIMS methods. Cytotoxic activities of the isolated compounds were tested against HCT-8, Bel-7402, BGC-823, A549 and A2780 human cancer cell lines in the MTT test. Results showed that compounds 1 and 3–5 exhibited significantly selective cytotoxic activities (IC50 < 10 lM) against these cells, respectively. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Erythrophleum (Leguminosae) is a genus composed of about 15 species of large trees found in the tropical parts of Africa, tropical and subtropical parts of Eastern Asia and North Australia, but only one species, Erythrophleum fordii, is native to China (Chen et al., 1985). E. fordii is a species with medicinal and poisonous properties (Chen and Zhen, 1987). It is known by the name ‘‘Gemu’’ and used by native Chinese as invigoration and promoting blood circulation agents (Cui and Ran, 1993). Previously, various components including alkaloids (cassaine diterpenoid amines and amides) (Culvenor et al., 1971; Cronlund, 1973; Qu et al., 2006b) and terpenoids (Li et al., 2004; Yu et al., 2005; Tsao et al., 2008) were reported from the genus Erythrophleum. The alkaloids showed a digitalis-like action on the heart (Cronlund and Sandberg, 1976; Verotta et al., 1995) and cytotoxic activity against some tumor cell lines (Loder et al., 1974; Loder and Nearn, 1975; Qu et al., 2006a). Four new cassaine diterpenoid dimers, erythrophlesins A–D, have recently been reported from Erythrophleum succirubrum, possessing an unsymmetrical dimeric structure through an ester bond between two diterpenoid units not reported in the earlier studies (Miyagawa et al., 2009). In continuation of our interest in the chemical investigation of Chinese medicinal and toxic plants (Fu et al., 2006; Su et al., * Corresponding author. Tel.: +86 10 63165324/60212125; fax: +86 10 63017757. E-mail address: [email protected] (S.-S. Yu). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.07.004
2008; Liu et al., 2009), we have identified E. fordii as one of the major plants for chemical and biological investigations. Our research on its bark has been reported (Qu et al., 2006a). In the present paper, we describe the isolation, structure elucidation and biotesting of three novel cassaine diterpenoid–diterpenoid amide dimers (1, 3 and 5) and seven new cassaine diterpenoid amides (6 and 8–13) from the leaves of E. fordii that also afforded the known compounds 2, 4 and 7.
2. Results and discussion Leaves of E. fordii were collected in Guangxi Province, China. The air-dried powdered leaves material were extracted with EtOH/ water (95:5, v/v). The crude extract was submitted to a diatomite column, eluting successively with petroleum, EtOAc and MeOH. The EtOAc extract after evaporation of the solvent was repeatedly subjected to polyamide, ODS, Sephadex LH-20 and preparative reversed-phase HPLC, which permitted the isolation of 1–13. Compound 1 was isolated as a white powder. A quasi-molecular ion at m/z 798.4429 [M+H]+ in the positive HR-ESIMS indicated a molecular formula, C44H63NO12. Its IR spectrum showed bands at 3383 and 1721 cm1 indicative of the presence of hydroxyl and carbonyl group in the molecule. The 13C NMR spectrum displayed resonances for six carbonyls (dC 166.0, 168.0, 172.7, 175.4, 208.1 and 208.5), four olefinic carbons (dC 113.1, 115.6, 160.7 and 166.4) and three oxymethines (dC 75.6, 75.8 and 78.2). The
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D. Du et al. / Phytochemistry 71 (2010) 1749–1755
sessed the same skeleton. The only difference with erythrophlesin D (2) was the absence of a methyl at the nitrogen atom. This was supported by C-13 (d4 +3.6), C-16 (d4 1.9) and C-21 (d4 8.8) in 1 relative to 2. Correlations from the N–H (dH 5.85) to C-16 (dC 168.0) and C-21 (dC 42.3) in the HMBC spectrum together with cross-peaks from H2-21 (dH 3.45) to H2-22 (dH 3.74) and N–H (dH 5.85) in the COSY spectrum further confirmed the N–H linkage
resonances for six methyls (dH 0.89, 0.98, 1.20, 1.21, 1.21 and 1.21), two methoxyls (dH 3.69 and 3.76), two exocyclic olefinic protons (dH 5.64 and 5.66), three oxymethines (dH 3.89, 3.94 and 4.66) and a secondary amide N–H (dH 5.85) were observed in the 1H NMR spectrum. All the signals in the above spectra indicated a striking resemblance to those of co-occurring erythrophlesin D (2) (Miyagawa et al., 2009), and the two compounds clearly pos-
O 12 1
2
O 12’
11’ 20’ 1’ 9’
2’
10’ 4’ 5’
3’
H
O
8’
6’
16’
3
R1
15’ 14’
7’
O
H
H
8
6
18
R2
19
24
19
24
OH
15 13 14
7
21
17
R3
17’
OH
O
19’ 18’
10 4 5
H
16
22
13’
H
H
O
11 20 9
R4 N
1 R1 = CO2CH3; R2 = O; R3 = (β) OH, H; R4 = H
21’
23
2 R1 = CO2CH3; R2 = O; R3 = (β) OH, H; R4 = CH3 3 R1 = H; R2 = O; R3 = (β) OH, H; R4 = H 23
4 R1 = H; R2 = O; R3 = (β) OH, H; R4 = CH3 19
24
5 R1 = CO2CH3; R2 = (α) OH, H; R3 = O; R4 = H
R N
O
16
12 1
2
HO 3 O
2011 9
10 4 5
H
19 18
O
H 6
H 8
21
O
OH
17
O
OH
O
H
O 1’ 2’
H
O O
23
7 R = CH3 R N
O
OH O 8 R=H
23
9 R = CH3
O
OH
H 5’ 4’
3’
2’
1’
O
H
O O
H N
H
H
O
OH
H
6 R=H
24
R N
15 13 14
7
22
OH O
H
R O
H O
O OH
10 R = H
12 R = OH
11 R = CH3
13 R = H
23
Fig. 1. Chemical structures of compounds 1–13.
OH
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D. Du et al. / Phytochemistry 71 (2010) 1749–1755
groups, two methoxyl groups, two exocyclic olefinic protons, three oxymethines and six carbonyls (dC 166.1, 167.9, 174.1, 175.4, 208.6 and 209.5), which suggested that, 5 is also a cassaine diterpenoid dimer. Compared with 1, the H-5 proton signal in 5 was shifted upfield to dH 1.48 (1H, d, J = 12.5 Hz) and coupled with a proton H-6 at dH 4.38 (1H, d, J = 12.5 Hz). In addition, the H-8 proton signal was shifted downfield to dH 2.38 (1H, dd, J = 12.5, 3.0 Hz). Thus, the hydroxyl group is located at C-6 and the ketone at C-7. The relative configuration of compound 5 was deduced from a NOESY experiment. The methyl protons Me-20 correlated with the methine proton H-8 and the oxymethine proton H-6, and the methine proton H-80 correlated with the methyl protons Me-200 and the methine proton H-140 , indicating a b-orientation for H-6, H-8 and H-80 . In addition, the a-configurations of H-3, H-5, H-9, H3-18, H-50 , H-70 , H-90 and H3-180 were deduced from NOESY correlations of H-3 to H-5 and H3-18, and H-50 to H-70 , H-90 and H3-180 . The olefinic protons dH 5.63 (H-15) and dH 5.74 (H-150 ) showed NOESY cross-peaks to the methines resonating at dH 2.94 (H-14) and dH 2.82 (H-140 ), respectively, indicating the E geometry of the two double bonds. Thus erythrophlesin G has structure 5. Compound 6 was isolated as a white powder. Its molecular formula was established as C23H35NO7 by the positive-ion mode
instead of the N–CH3 linkage. Thus, structure 1 (Fig. 1) was assigned to this compound which was given the name erythrophlesin E. NOESY correlations confirmed that it has the same relative stereochemistry as erythrophlesin D (2). Compound 3 was isolated as a white powder. The HR-ESIMS exhibited a quasi-molecular ion peak at m/z 740.4401 [M+H]+, supporting the molecular formula of C42H61NO10, indicating 14 mass units less than the co-occurring erythrophlesin C (4) (Miyagawa et al., 2009). Inspection of its 1D (1H and 13C) and 2D (HSQC, COSY and HMBC) NMR data (Table 1) established the presence of nearly identical structural features to those found in 4, except that resonances for the methyl group at nitrogen atom were absent. This was confirmed by HMBC correlations from the N–H (dH 5.84) to C-16 (dC 168.1) and C-21 (dC 42.3). Thus, structure 3 was deduced for this compound which was called erythrophlesin F. Again NOESY data confirmed that the relative stereochemistry was the same as in 1 and 2. Compound 5, named erythrophlesin G, was isolated as a white powder. Its molecular formula was deduced as C44H63NO12 due to the appearance of an [M+H]+ ion at m/z 798.4405 in the positive HR-ESIMS. The 1H and 13C NMR spectroscopic data (Table 1) showed the presence of characteristic signals for six methyl
Table 1 NMR spectroscopic data of compounds 1–5 in CDCl3 (500 MHz for 1H NMR and 125 MHz for Position
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 N–H
1
2
13
C NMR, d in ppm, J in Hz).
3
4
5
dH (J)
dC
dH (J)
dC
dH (J)
dC
dH (J)
dC
dH (J)
dC
1.89 m/1.42 m 2.26 m/1.76 m 4.66 dd (12.0, 4.0)
36.5 23.0 78.2 45.7 64.7 208.1 75.8 51.3 46.4 43.0 26.2 23.2 160.7 40.3 115.6 168.0 13.7 25.8 172.7 14.4 42.3 62.6
1.85 m/1.42 m 2.26 m/1.78 m 4.66 dd (12.0, 4.0)
36.5 23.8 78.2 45.7 64.6 208.1 75.8 51.2 46.4 43.0 26.2 24.3 157.1 39.8 115.0 169.9 13.8 25.8 172.6 14.4 51.1 61.7 37.3 51.8 38.6 18.9 38.3 41.9 64.5 208.5 75.6 50.2 46.3 42.9 26.2 23.7 166.4 40.4 113.1 166.0 13.6 28.6 175.4 15.1 51.6
1.84 1.92 4.46 2.14 2.13
1.83 1.89 4.46 2.14 2.13
36.5 26.4 76.6 31.0 60.6 209.6 75.6 52.3 43.7 43.0 26.4 24.5 157.4 39.9 114.9 170.0 13.7 16.1
1.90 m/1.32 m 2.24 m/1.79 m 4.61 dd (12.0, 4.0)
1.21 d (7.0) 0.88 d (7.0)
36.5 26.4 76.6 30.9 60.6 209.6 75.5 52.4 43.7 43.0 26.4 23.4 160.9 40.3 115.5 168.1 13.5 16.1
0.68 s 3.45 m 3.74 t (5.0)
13.6 42.3 62.7
37.1 23.9 78.1 49.4 57.9 75.1 209.5 51.7 47.0 37.4 27.2 23.3 159.9 39.4 115.5 167.9 14.9 26.4 174.1 13.6 42.3 62.7
2.44 s 3.94 d (10.5) 1.86 m 1.70 m 1.90 m/1.17 m 3.87 m/1.93 m 2.73 m 5.64 br s 1.20 d (6.0) 1.21 s 0.98 s 3.45 m 3.74 t (5.0) 3.76 1.76 1.72 2.03
s m/1.18 m m/1.52 m m/1.15 m
2.24 s 3.89 d (11.0) 1.86 m 1.72 m 1.90 m/1.15 m 3.73 m/2.01 m 2.81 m 5.66 br s 1.21 d (5.0) 1.21 s 0.89 s 3.69 s 5.85 br s
51.8 38.6 18.9 38.3 41.9 64.5 208.5 75.6 50.2 46.3 42.9 26.2 23.7 166.4 40.4 113.1 166.0 13.6 28.6 175.4 15.1 51.6
2.44 s 3.94 d (10.5) 1.86 m 1.75 m 1.88 m/1.18 m 3.15 m/2.09 m 2.83 m 5.93 br s 1.21 d (6.0) 1.21 s 0.98 3.57 3.80 3.06 3.76 1.78 1.72 2.03
s m t (5.0) s s m/1.19 m m/1.52 m m/1.15 m
2.24 s 3.89 d (10.5) 1.87 m 1.71 m 1.93 m/1.18 m 3.73 m/2.00 m 2.81m 5.66 br s 1.20 d (6.0) 1.21 s 0.89 s 3.69 s
m/1.41 m m/1.47 m m m m
4.00 d (8.0) 1.75 m 1.73 m 1.92 m/1.10 m 3.86 m/2.01 m 2.77 m 5.64 br s
1.80 m/1.19 m 1.74 m/1.52 m 2.03 m/1.15 m 2.24 s 3.90 d (11.0) 1.89 m 1.73 m 1.94 m/1.18 m 3.79 m/2.06 m 2.83 m 5.73 br s 1.21 d (7.0) 1.22 s 0.90 s 3.70 s 5.84 br s
38.6 18.9 38.3 41.9 64.5 208.6 75.6 50.3 46.4 42.8 26.2 23.7 165.8 40.3 113.4 166.6 13.7 28.6 175.4 15.1 51.6
m/1.39 m m/1.44 m m m m
4.01 d (8.0) 1.77 m 1.70 m 1.89 m/1.10 m 3.15 m/2.05 m 2.85 m 5.91 br s 1.22 d (7.0) 0.88 d (7.0) 0.68 3.58 3.79 3.07
s m t (5.0) s
1.80 m/1.20 m 1.75 m/1.50 m 2.05 m/1.14 m 2.24 s 3.90 d (10.5) 1.89 m 1.72 m 1.95 m/1.18 m 3.76 m/2.03 m 2.82 m 5.73 br s 1.21 d (7.0) 1.22 s 0.90 s 3.69 s
13.6 51.2 61.8 37.3 38.6 19.0 38.3 42.0 64.5 208.6 75.6 50.3 46.4 42.8 26.2 23.7 165.8 40.4 113.4 166.6 13.7 28.6 175.4 15.1 51.6
1.48 d (12.5) 4.38 d (12.5) 2.38 dd (12.5, 3.0) 1.65 m 1.92 m/1.18 m 3.86 m/1.98 m 2.94 m 5.63 br s 1.11 d (6.0) 1.50 s 1.07 s 3.45 m 3.73 t (5.0) 3.75 1.75 1.73 2.06
s m/1.18 m m/1.53 m m/1.15 m
2.25 s 3.90 d (10.5) 1.87 m 1.70 m 1.96 m/1.21 m 3.79 m/2.03 m 2.82 m 5.74 br s 1.20 d (7.5) 1.22 s 0.90 s 3.70 s 5.88 br s
51.7 38.6 19.0 38.3 42.0 64.5 208.6 75.6 50.3 46.4 43.0 26.2 23.7 166.4 40.3 113.5 166.1 13.6 28.6 175.4 15.1 51.6
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HR-ESIMS showing a pseudo-molecular ion peak at m/z 438.2489 [M+H]+. IR absorptions at 3353 and 1721 cm1 showed the presence of hydroxyl and carbonyl groups, respectively. Based on the 13 C NMR and DEPT spectra, compound 6 included three carbonyl (dC 168.1, 174.1 and 208.0), and two olefinic carbons (dC 115.5 and 160.9). Analysis of its 1H NMR spectrum indicated the presence of three methyl groups at dH 0.90, 1.19 and 1.35, a methoxyl group at dH 3.75, an exocyclic vinyl proton at dH 5.64, and a secondary amide proton at dH 5.89. These signals were similar to those of 3b-hydroxynorerythrosuamide (7) (Verotta et al., 1995), except for the lack of an N-methyl group, suggesting that 6 was a demethyl derivative of 7. This conclusion was confirmed by the COSY correlations of the NH, 2H-22 and 2H-23 and the corresponding carbon shifts. Thus, compound 6 is 3b-hydroxydinorerythrosuamide. Analysis of the HMBC spectrum permitted the assignment of all the proton and carbon resonances (see tables) and the relative stereochemistry was confirmed from the NOESY spectrum. Compound 8, was purified as a white powder. The HR-ESIMS gave a molecular ion peak at m/z 480.2622 [M+H]+ giving the molecular formula C25H37NO8. The NMR spectroscopic data of 8 was virtually identical with that of 6, apart from the presence of an acetate group which was readily assigned to the b position at C-3 (H-3, dd, 4.65, J = 12.0, 4.0 Hz) on the basis of an HMBC correlation from H-3 to the acetate carbonyl group. Thus, compound 8 is 3bacetoxydinorerythrosuamide. Compound 9 was purified as a white powder whose HR-ESIMS showed a peak at m/z 494.2754 [M+H]+, indicating the molecular formula of C26H39NO8. Interpretation of the 1H and 13C NMR spectra of 8 and 9 indicated that 9 was a methylation product of 8, and this was supported by the presence of 1H–13C NMR long-range correlations of the methyl protons dH 3.06 (H3-23) with the amide carbonyl dC 169.9 (C-16) and methylene dC 51.1 (C-21). Thus, compound 9 is 3b-acetoxynorerythrosuamide. Compound 10 was isolated as a white powder. The molecular formula was deduced as C28H41NO8 from the ion peak at m/z 520.2927 [M+H]+ obtained by positive HR-ESIMS. The 1H and 13C NMR spectroscopic data (Tables 2 and 3) of 10 closely resembled
Table 3 13 C NMR spectroscopic data (125 MHz, CDCl3) for compounds 6–13 (d in ppm). Position
6
7
8
9
10
11
12
13
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 10 20 30 40 50
37.1 27.6 78.1 47.7 64.4 208.0 75.8 50.9 46.1 43.2 26.2 23.2 160.9 40.3 115.5 168.1 13.7 25.6 174.1 14.4 42.4 62.8
37.1 27.5 78.0 47.7 64.3 208.0 75.8 50.8 46.1 43.2 26.2 24.4 157.3 39.8 114.9 169.9 13.8 25.6 174.0 14.4 51.1 61.7 37.3 51.8
36.5 23.7 78.7 45.7 64.6 208.1 75.8 51.3 46.4 43.0 26.2 23.2 160.6 40.2 115.6 168.0 13.7 25.7 172.6 14.4 42.3 62.6
36.5 23.7 78.8 45.7 64.6 208.1 75.8 51.2 46.5 43.0 26.2 24.3 157.1 39.8 115.0 169.9 13.8 25.7 172.6 14.4 51.1 61.7 37.3 51.8 170.4 21.0
36.5 23.7 78.7 45.8 64.7 208.1 75.8 51.3 46.5 43.0 26.3 23.2 160.7 40.3 115.6 168.0 13.7 25.8 172.7 14.4 42.3 62.7
36.5 23.7 78.7 45.8 64.6 208.2 75.8 51.2 46.5 43.0 26.2 24.3 157.1 39.9 115.0 169.9 13.9 25.8 172.7 14.4 51.1 61.7 37.3 51.7 167.1 128.3 137.9 14.4 11.8
37.9 27.8 78.0 50.3 58.1 75.4 210.1 51.1 46.3 37.6 27.3 23.3 160.0 39.4 115.4 167.8 14.9 25.3 178.0 13.5 42.3 62.6
39.8 19.2 39.6 45.3 58.5 75.8 210.4 51.5 47.1 37.8 27.3 23.5 160.4 39.5 115.2 168.0 14.9 31.5 177.4 13.9 42.3 62.7
51.7
51.6
51.8
51.8 170.4 21.0
51.7 167.1 128.3 137.9 14.4 11.8
those of 6, with the only difference being the presence of an additional fragment of C5H7O. The signals of dC 167.1, dC 128.3, dC 137.9, dC 14.4, dC 11.8, dH 1.77 (3H, d, J = 5.5) and dH 1.79 (3H, s) were diagnostic of a tigloyl moiety (Yang et al., 1999). An HMBC correlation of H-3 to the tigloyl carbonyl group confirmed its attachment to C-3. Thus, compound 10 is 3b-tigloyloxydinorerythrosuamide. Compound 11 was purified as a white powder. The HR-ESIMS indicated an ion peak at m/z 534.3082 [M+H]+, which suggested a molecular formula C29H43NO8. The 1H and 13C NMR spectroscopic
Table 2 1 H NMR spectroscopic data (500 MHz, CDCl3) for compounds 6–13 (d in ppm, J in Hz). Position
6
7
8
1 2 3
1.87 m/1.35 m 2.14 m/1.75 m 3.35 m
1.87 m/1.32 m 2.14 m/1.75 m 3.35 m
5 6 7 8
2.33 s
2.33 s
1.87 2.22 4.65 4.0) 2.42
3.92 d (10.5) 1.81 m
3.92 d (10.5) 1.84 m
3.93 d (10.5) 1.83 m
1.67 4.0) 1.97 3.87 2.75 5.64 1.19 1.35 0.90 3.47 3.73
1.66 3.0) 2.01 3.15 2.82 5.91 1.20 1.35 0.91 3.57 3.78 3.07 3.74
1.68 3.5) 1.86 3.86 2.73 5.64 1.18 1.19 0.96 3.44 3.72
9 11 12 14 15 17 18 20 21 22 23 24 20 30 40 50 N–H
dt (12.0, m/1.22 m m/2.00 m m br s d (7.0) s s m m
3.75 s
5.89 br s
dt (12.0, m/1.18 m m/2.06 m m br s d (7.5) s s m m s s
9
10
11
m/1.40 m m/1.70 m dd (11.5,
1.86 m/1.43 m 2.21 m/1.70 m 4.68 dd (12, 4.5)
s
2.44 s
1.85 2.22 4.68 4.0) 2.44
3.94 d (10.0) 1.86 m
3.94 d (10.5) 1.83 m
3.94 d (10.0) 1.87 m
dt (12.0,
1.66 m
m/1.16 m m/2.00 m m br s d (6.0) s s m t (4.5)
1.85 3.14 2.82 5.90 1.21 1.19 0.97 3.57 3.78 3.06 3.76 2.02
1.70 3.5) 1.88 3.87 2.74 5.64 1.19 1.20 0.99 3.44 3.73
1.70 3.5) 1.89 3.15 2.83 5.91 1.21 1.20 1.00 3.57 3.79 3.07 3.76
m/1.40 m m/1.70 m dd (12.0, s
3.75 s 2.02 s
6.05 br s
1.84 2.22 4.64 3.5) 2.42
m/1.14 m m/2.02 m m br s d (7.5) s s m t (5.0) s s s
dt (12.0, m/1.21 m m/2.01 m m br s d (6.0) s s m t (4.5)
3.76 s 6.77 1.77 1.79 5.95
d (5.5) d (5.5) s br s
12 m/1.42 m m/1.70 m dd (11.5, s
dt (12.0, m/1.16 m m/2.05 m m br s d (6.0) s s m t (4.5) s s
13
1.87 1.91 3.10 4.0) 1.36 4.58
m/1.23 m m/1.25 m dt (11.5,
1.85 m/1.07 m 1.67 m/1.53 m 2.26 m/1.09 m
d (12.0) d (12.0)
1.37 d (12.5) 4.76 d (12.5)
2.38 3.0) 1.64 3.0) 1.91 3.84 2.90 5.62 1.12 1.70 0.88 3.45 3.74
dd (13.0, dt (13.5,
2.39 dd (11.5, 3.5) 1.68 m
m/1.28 m m/2.03 m m br s d (7.0) s s m t (4.5)
2.00 3.84 2.91 5.62 1.12 1.45 0.87 3.44 3.73
m/1.26 m m/2.03 m m br s d (7.0) s s m t (5.0)
3.75 s
3.71 s
5.91 br s
5.97 br s
6.77 d (7.0) 1.77 d (7.0) 1.79 s
D. Du et al. / Phytochemistry 71 (2010) 1749–1755
data were almost identical with those of 10, apart from the presence of a N-methyl group. This was confirmed by correlations from the methyl protons H3-23 (dH 3.07, s) to the carbonyl C-16 (dC 169.9) and the methylene C-21 (dC 51.1) in the HMBC spectrum. Thus, compound 11 is 3b-tigloyloxynorerythrosuamide. Compound 12 was purified as a white powder. The ion peak in HR-ESIMS at m/z 438.2506 [M+H]+ indicated the same molecular formula as compound 6, C23H35NO7. A comparison of the 1H and 13 C NMR spectroscopic data of 12 with those of 6 suggested they shared the same skeleton, except for the 6a-hydroxy-7-keto group in 12 instead of the 6-keto-7b-hydroxy group. The notable upfield shift of H-5 and the downfield shift of H-8 (vide infra) supported this conclusion. The presence of a NOE interaction between H-6 and Me-20 confirmed the a configuration of the C-6 hydroxyl group. Thus, compound 12 is 6a-hydroxydinorerythrophlamide. Compound 13 was purified as a white powder. The HR-ESIMS indicated a molecular formula C23H35NO6 deduced from the ion peak at m/z 422.2573 [M+H]+. The NMR features of 13 were analogous to those of 12, apart from the absence of an oxygen substituent at C-3, confirmed by HSQC, COSY and HMBC data (see tables). Hence, compound 13 is 6a-hydroxydinorcassamide. Three known alkaloids, erythrophlesin D (2) (Miyagawa et al., 2009), erythrophlesin C (4) (Miyagawa et al., 2009) and 3bhydroxynorerythrosuamide (7) (Verotta et al., 1995), were identified by the comparison of their NMR spectroscopic data with those reported in the literature, respectively. The isolated alkaloids 1–13 were screened in an in vitro cytotoxicity assay. Camptothecin was used as a positive control, and the data are shown in Table 4. The cassaine diterpenoid–diterpenoid amide dimers (1 and 3–5) indicated significantly selective cytotoxicity against HCT-8, Bel-7402, BGC-823, A549 and A2780 cell lines. Compound 1 exhibited cytotoxicity against BGC-823 and A2780 cell lines with IC50 values of 3.47 and 0.91 lM, respectively. Compounds 3 and 5 demonstrated strongly selective cytotoxic activity against A2780 with IC50 values of 4.54 and 7.40 lM, respectively. Compound 4 displayed inhibitory effects on Bel-7402, BGC-823, A549 and A2780. However, no significant activity was observed among the monomers 6–11 and 13, and only compound 12, 6ahydroxydinorerythrophlamide, showed activity against all the cell lines tested. Compound 12 was the only cassaine diterpenoid amide tested in this study that possessed a hydroxy at C-3 and a 6a-hydroxy-7-keto group, suggesting that these residues may be involved in the cytotoxic activity. Structure–activity studies of cassaine diterpenoid amides in our earlier research have demonstrated that substituents at C-3 might influence cytotoxic activity and compounds with 6-keto-7-hydroxy groups showed no activity (Qu et al., 2006a). In combination with the data obtained in the present study, we speculate that the C-3 hydroxyl group and the C-7 ketone in cassaine diterpenoid amides might be necessary for cytotoxic activity. Moreover, the analysis of the IC50 values indiTable 4 Cytotoxicity of compounds 1, 3–5 and 12 against five human cancer cell lines. Compounda
1 3 4 5 12 Camptothecinb
IC50 (lM) HCT-8
Bel-7402
BGC-823
A549
A2780
>10 >10 >10 >10 2.77 3.20
>10 >10 2.77 >10 0.52 12.51
3.47 >10 0.67 >10 1.25 9.72
>10 >10 3.82 >10 8.67 3.11
0.91 4.54 0.49 7.40 1.35 0.29
HCT-8 = human colon cancer cell line; Bel-7402 = human hepatoma cell line; BGC823 = human gastric carcinoma cell line; A549 = human lung epithelial cell line; A2780 = human epithelial carcinoma cell line. ‘‘>10’’ = inactive. a Compounds 2, 6–11 and 13 were inactive (IC50 > 10 lM) for all cells. b Positive control.
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cated that dimers showed better selectivities than monomers. These findings encourage further studies on the exact mechanism by which compounds 1, 3–5 and 12 exert their cytotoxic activity. 3. Conclusions Phytochemical investigation of the toxic plant, E. fordii, led to the isolation of thirteen compounds (1–13), including three new cassaine diterpenoid–diterpenoid amide dimers, erythrophlesins E–G, seven new cassaine diterpenoid amides and three known related compounds. All of the compounds were tested for cytotoxicity against HCT-8, Bel-7402, BGC-823, A549 and A2780 cell lines. Compounds 1, 3–5 and 12 had significant cytotoxicity properties with IC50 values ranging from 0.49 to 8.67 lM. 4. Experimental 4.1. General experimental procedures The NMR spectra were recorded with an Inova 500 FT-NMR spectrometer. ESIMS and HR-ESIMS were run on an Agilent 1100 Series LC/MSD Trap mass spectrometer. Analytical HPLC was performed on an Agilent 1100 Series instrument with a DAD detector, using a YMC-Pack ODS (100 4.6 mm, 5 lm). Preparative HPLC was performed on a Shimadazu instrument equipped with a LC6AD pump, a SPD-10A detector, using a YMC-Pack ODS-A column (250 20 mm, 5 lm). IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer by a microscope transmission method. The UV spectra were obtained on a V650 spectrometer. Melting points were conducted on a XT-5B micromelting point apparatus and are uncorrected. Optical rotations were measured with a P2000 polarimeter. Polyamide (30–60 mesh, Jiangsu Linjiang Chemical Reagents Factory, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Sweden) and ODS (50 lm, Merck) were used for column chromatography (CC). Silica gel GF-254 (Qingdao Marine Chemical Factory) was used for TLC. Solvents (CHCl3, MeOH and EtOH) were of analytical grade and purchased from Beijing Chemical Company, Beijing, China. 4.2. Plant materia Leaves of E. fordii Oliver (Leguminosae) were harvested in August 2007 in Guangxi Province, China, and identified by Prof. Songji Wei (Guangxi College of Chinese Traditional Medicine). A voucher specimen (No. 07089) has been deposited in the Herbarium of the Department of Medicinal plants, Institute of Materia Medica, Chinese Academy of Medical Sciences. 4.3. Extraction and isolation Powdered air-dried leaves of E. fordii (5.2 kg) were extracted with EtOH/water (95:5, v/v) under conditions of reflux (3 20 L), this being maintained for 3h. After filtration and removal of the solvent under reduced pressure, the residue (806 g) was diluted in MeOH (1.5 L), filtered and adsorbed on a diatomite column. The latter was eluted successively with petroleum (60–90 °C) (10 L), EtOAc (10 L), and MeOH (20 L), to afford petroleum ether (90 g), EtOAc (45 g) and MeOH extracts (532 g), respectively. The EtOAc extract was subjected to a polyamide column, eluting with H2O, EtOH–H2O (3:7), EtOH–H2O (6:4) and EtOH–H2O (9:1) to yield Fr.1 (20.0 g), Fr.2 (7.6 g), Fr.3 (5.1 g) and Fr.4 (1.6 g). Fr.1 and Fr.2 were then combined and submitted to repeated Sephadex LH-20 (MeOH as eluent) and ODS (MeOH–H2O as eluent) to give a total alkaloid portion (17.8 g), which was subjected to an ODS (50 lm, 300 g) CC using a gradient of MeOH–H2O (5:95–90:10)
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as eluents. According to the TLC profiles, 12 major fractions (1–12) were obtained. Fraction 3 (1.93 g) was submitted to ODS (MeOH– H2O, from 20:80 to 60:40), followed by a preparative C-18 column using a mobile phase consisting of MeCN–H2O 15:85, leading to the isolation of 6 (35 mg; w/w 0.0043%) and 7 (20 mg; w/w 0.0025%). Fraction 4 (1.09 g) was purified using the same column (MeCN–H2O 17:83) to give 12 (14 mg; w/w 0.0017%). Fraction 6 (1.44 g) was separated by a preparative C-18 column (MeCN–H2O 24:76) to yield 8 (43 mg; w/w 0.0053%) and 9 (48 mg; w/w 0.0060%). Fraction 8 (1.66 g) was purified by an ODS column (50 lm, 100 g) and preparative HPLC using MeCN–H2O 30:70 to provide 10 (30 mg; w/w 0.0037%), 11 (27 mg; w/w 0.0033%) and 13 (28 mg; w/w 0.0035%). Fraction 10 (0.25 g) using MeCN–H2O 48:52 on the preparative C-18 column, afforded 1 (27 mg; w/w 0.0033%), 2 (27 mg; w/w 0.0033%), 3 (18 mg; w/w 0.0022%), 4 (8 mg; w/w 0.0010%) and 5 (7 mg; w/w 0.00086%). 4.3.1. Erythrophlesin E (1) White powder; mp 194–196 °C; ½a20 D 65.2 (c 0.08, EtOH); UV (EtOH) kmax nm: 222; IR mmax cm1: 3383, 2937, 1721, 1693, 1660, 1537, 1466, 1393, 1374, 1275, 1257, 1165, 1042, 1026, 969, 868; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 798.4429 [M+H]+ (calcd. for C44H64NO12, 798.4428); ESIMS (positive-ion mode) m/z: 798 [M+H]+, 820 [M+Na]+, 836 [M+K]+; ESIMS (negative-ion mode) m/z: 796 [MH], 832 [M+Cl]. 4.3.2. Erythrophlesin D (2) White powder; mp 174–176 °C; ½a20 D 81.1 (c 0.08, EtOH); UV (EtOH) kmax nm: 222; IR mmax cm1: 3436, 2942, 1717, 1645, 1608, 1458, 1394, 1374, 1256, 1163, 1042, 969, 868; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 812.4597 [M+H]+ (calcd. for C45H66NO12, 812.4585); ESIMS (positive-ion mode) m/z: 812 [M+H]+, 834 [M+Na]+; ESIMS (negative-ion mode) m/z: 810 [MH], 846 [M+Cl]. 4.3.3. Erythrophlesin F (3) White powder; mp 178–180 °C; ½a20 D 58.4 (c 0.08, EtOH); UV (EtOH) kmax nm: 223; IR mmax cm1: 3378, 2941, 2875, 1714, 1642, 1532, 1460, 1391, 1321, 1258, 1197, 1157, 1042, 1026, 967, 868; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 740.4401 [M+H]+ (calcd. for C42H62NO10, 740.4373); ESIMS (positive-ion mode) m/z: 740 [M+H]+, 762 [M+Na]+; ESIMS (negative-ion mode) m/z: 738 [MH], 774 [M+Cl]. 4.3.4. Erythrophlesin C (4) White powder; mp 164–166 °C; ½a20 D 63.4 (c 0.08, EtOH); UV (EtOH) kmax nm: 221; IR mmax cm1: 3419, 2941, 2876, 1714, 1646, 1607, 1460, 1393, 1257, 1196, 1044, 1025, 968, 867; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 754.4564 [M+H]+ (calcd. for C43H64NO10, 754.4530); ESIMS (positive-ion mode) m/z: 754 [M+H]+, 776 [M+Na]+; ESIMS (negative-ion mode) m/z: 752 [MH], 788 [M+Cl]. 4.3.5. Erythrophlesin G (5) White powder; mp 162–164 °C; ½a20 D 57.1 (c 0.06, EtOH); UV (EtOH) kmax nm: 222; IR mmax cm1: 3381, 2942, 2880, 1714, 1641, 1533, 1457, 1393, 1321, 1257, 1227, 1165, 1041, 1013, 870, 720; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 798.4405 [M+H]+ (calcd. for C44H64NO12, 798.4428); ESIMS (positive-ion mode) m/z: 798 [M+H]+, 820 [M+Na]+; ESIMS (negative-ion mode) m/z: 796 [MH], 832 [M+Cl].
4.3.6. 3b-Hydroxydinorerythrosuamide (6) White powder; mp 128–130 °C; ½a20 D 92.8 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3353, 2942, 2879, 1721, 1656, 1629, 1537, 1452, 1394, 1372, 1322, 1251, 1208, 1165, 1118, 1062, 1037, 967, 867; for 1H NMR (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 438.2489 [M+H]+ (calcd. for C23H36NO7, 438.2486); ESIMS (positive-ion mode) m/z: 438 [M+H]+, 460 [M+Na]+. 4.3.7. 3b-Hydroxynorerythrosuamide (7) White powder; mp 112–114 °C; ½a20 D 48.3 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3384, 2941, 2881, 1722, 1649, 1601, 1456, 1401, 1372, 1252, 1195, 1163, 1119, 1065, 1039, 968, 938, 864, 729; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; for 1H (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) spectroscopic data see supplementary data; ESIMS (positive-ion mode) m/z: 452 [M+H]+, 474 [M+Na]+. 4.3.8. 3b-Acetoxydinorerythrosuamide (8) White powder; mp 127–129 °C; ½a20 D 76.4 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3365, 2943, 2879, 1701, 1660, 1631, 1457, 1394, 1267, 1157, 1119, 1056, 981, 865; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 480.2622 [M+H]+ (calcd. for C25H38NO8, 480.2591); ESIMS (positive-ion mode) m/z: 480 [M+H]+, 502 [M+Na]+, 514 [M+K]+; ESIMS (negative-ion mode) m/z: 478 [MH], 514 [M+Cl]. 4.3.9. 3b-Acetoxynorerythrosuamide (9) White powder; mp 106–108 °C; ½a20 D 58.6 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3391, 2943, 2880, 1730, 1650, 1605, 1458, 1401, 1369, 1239, 1168, 1121, 1079, 1034, 969, 867, 733; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 494.2754 [M+H]+ (calcd. for C26H40NO8, 494.2748); ESIMS (positive-ion mode) m/z: 494 [M+H]+, 516 [M+Na]+, 528 [M+K]+; ESIMS (negative-ion mode) m/z: 492 [MH], 528 [M+Cl]. 4.3.10. 3b-Tigloyloxydinorerythrosuamide (10) White powder; mp 126–128 °C; ½a20 D 47.6 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3340, 2944, 2879, 1712, 1651, 1536, 1441, 1392, 1346, 1268, 1253, 1209, 1164, 1120, 1075, 1041, 968, 871, 733; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 520.2927 [M+H]+ (calcd. for C28H42NO8, 520.2904); ESIMS (positive-ion mode) m/z: 520 [M+H]+, 542 [M+Na]+; ESIMS (negative-ion mode) m/z: 554 [M+Cl]. 4.3.11. 3b-Tigloyloxynorerythrosuamide (11) White powder; mp 108–110 °C; ½a20 D 46.5 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3413, 2943, 2877, 1709, 1649, 1605, 1454, 1438, 1398, 1346, 1265, 1251, 1197, 1163, 1120, 1074, 1045, 1027, 968, 733; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESMS m/z: 534.3082 [M+H]+ (calcd. for C29H44NO8, 534.3061); ESIMS (positive-ion mode) m/z: 534 [M+H]+, 556 [M+Na]+, 572 [M+K]+; ESIMS (negative-ion mode) m/z: 532 [MH], 568 [M+Cl]. 4.3.12. 6a-Hydroxydinorerythrophlamide (12) White powder; mp 113–115 °C; ½a20 D 42.7 (c 0.09, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3375, 2944, 2879, 1701, 1660, 1631, 1537, 1457, 1394, 1334, 1267, 1249, 1218, 1157, 1119, 1097, 1056, 1021, 981, 865, 716; for 1H NMR (500 MHz,
D. Du et al. / Phytochemistry 71 (2010) 1749–1755
CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 438.2506 [M+H]+ (calcd. for C22H36NO7, 438.2486); ESIMS (positive-ion mode) m/z: 438 [M+H]+, 460 [M+Na]+, 476 [M+K]+. 4.3.13. 6a-Hydroxydinorcassamide (13) White powder; mp 96–98 °C; ½a20 D 34.2 (c 0.09, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3380, 2941, 2876, 1716, 1659, 1632, 1536, 1459, 1393, 1329, 1265, 1222, 1155, 1097, 1070, 1021, 978, 869; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 422.2573 [M+H]+ (calcd. for C23H36NO6, 422.2537); ESIMS (positive-ion mode) m/z: 422 [M+H]+, 444 [M+Na]+, 456 [M+K]+; ESIMS (negative-ion mode) m/z: 420 [MH], 437 [M+Cl]. 4.4. Cytotoxicity assay The cytotoxicity assay against HCT-8 (human colon cancer), Bel7402 (human hepatoma cancer), BGC-823 (human gastric cancer), A549 (human lung epithelial) and A2780 (human ovarian cancer) cells (IC50) was assessed using the MTT method as described in the literature (Alley et al., 1988). Camptothecin was used as a control compound. Acknowledgments The research presented in this paper was supported by the National Found for Distinguished Young Scholars (No. 30625040), National Natural Science Foundation of China (No. 90713039) and National Science and Technology Project of China (No. 2009ZX09311-004). We are very grateful to Professor Songji Wei in Guangxi College of Chinese Traditional Medicine for his assistance in the plant collection and identification, the Department of Medicinal Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College for their assistance in measurements of IR, NMR, ESIMS and HR-ESIMS spectra. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2010.07.004.
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Cytotoxic cassaine diterpenoid–diterpenoid amide dimers and diterpenoid amides from the leaves of Erythrophleum fordii Dan Du, Jing Qu, Jia-Ming Wang, Shi-Shan Yu *, Xiao-Guang Chen, Song Xu, Shuang-Gang Ma, Yong Li, Guang-Zhi Ding, Lei Fang Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education and Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 2 November 2009 Received in revised form 22 April 2010 Available online 2 August 2010 Keywords: Erythrophleum fordii Leguminosae Cassaine diterpenoid–diterpenoid amide dimers Cassaine diterpenoid amides Cytotoxicity
a b s t r a c t Detailed phytochemical investigation from the leaves of Erythrophleum fordii resulted in the isolation of 13 compounds, including three cassaine diterpenoid–diterpenoid amide dimers (1, 3 and 5), and seven cassaine diterpenoid amides (6 and 8–13), together with three previously reported ones, erythrophlesins D (2), C (4) and 3b-hydroxynorerythrosuamide (7). Compounds 1, 3 and 5 are further additions to the small group of cassaine diterpenoid dimers represented by erythrophlesins A–D. Their structures were determined by analysis of extensive one- and two-dimensional NMR experiments and ESIMS methods. Cytotoxic activities of the isolated compounds were tested against HCT-8, Bel-7402, BGC-823, A549 and A2780 human cancer cell lines in the MTT test. Results showed that compounds 1 and 3–5 exhibited significantly selective cytotoxic activities (IC50 < 10 lM) against these cells, respectively. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Erythrophleum (Leguminosae) is a genus composed of about 15 species of large trees found in the tropical parts of Africa, tropical and subtropical parts of Eastern Asia and North Australia, but only one species, Erythrophleum fordii, is native to China (Chen et al., 1985). E. fordii is a species with medicinal and poisonous properties (Chen and Zhen, 1987). It is known by the name ‘‘Gemu’’ and used by native Chinese as invigoration and promoting blood circulation agents (Cui and Ran, 1993). Previously, various components including alkaloids (cassaine diterpenoid amines and amides) (Culvenor et al., 1971; Cronlund, 1973; Qu et al., 2006b) and terpenoids (Li et al., 2004; Yu et al., 2005; Tsao et al., 2008) were reported from the genus Erythrophleum. The alkaloids showed a digitalis-like action on the heart (Cronlund and Sandberg, 1976; Verotta et al., 1995) and cytotoxic activity against some tumor cell lines (Loder et al., 1974; Loder and Nearn, 1975; Qu et al., 2006a). Four new cassaine diterpenoid dimers, erythrophlesins A–D, have recently been reported from Erythrophleum succirubrum, possessing an unsymmetrical dimeric structure through an ester bond between two diterpenoid units not reported in the earlier studies (Miyagawa et al., 2009). In continuation of our interest in the chemical investigation of Chinese medicinal and toxic plants (Fu et al., 2006; Su et al., * Corresponding author. Tel.: +86 10 63165324/60212125; fax: +86 10 63017757. E-mail address: [email protected] (S.-S. Yu). 0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.07.004
2008; Liu et al., 2009), we have identified E. fordii as one of the major plants for chemical and biological investigations. Our research on its bark has been reported (Qu et al., 2006a). In the present paper, we describe the isolation, structure elucidation and biotesting of three novel cassaine diterpenoid–diterpenoid amide dimers (1, 3 and 5) and seven new cassaine diterpenoid amides (6 and 8–13) from the leaves of E. fordii that also afforded the known compounds 2, 4 and 7.
2. Results and discussion Leaves of E. fordii were collected in Guangxi Province, China. The air-dried powdered leaves material were extracted with EtOH/ water (95:5, v/v). The crude extract was submitted to a diatomite column, eluting successively with petroleum, EtOAc and MeOH. The EtOAc extract after evaporation of the solvent was repeatedly subjected to polyamide, ODS, Sephadex LH-20 and preparative reversed-phase HPLC, which permitted the isolation of 1–13. Compound 1 was isolated as a white powder. A quasi-molecular ion at m/z 798.4429 [M+H]+ in the positive HR-ESIMS indicated a molecular formula, C44H63NO12. Its IR spectrum showed bands at 3383 and 1721 cm1 indicative of the presence of hydroxyl and carbonyl group in the molecule. The 13C NMR spectrum displayed resonances for six carbonyls (dC 166.0, 168.0, 172.7, 175.4, 208.1 and 208.5), four olefinic carbons (dC 113.1, 115.6, 160.7 and 166.4) and three oxymethines (dC 75.6, 75.8 and 78.2). The
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D. Du et al. / Phytochemistry 71 (2010) 1749–1755
sessed the same skeleton. The only difference with erythrophlesin D (2) was the absence of a methyl at the nitrogen atom. This was supported by C-13 (d4 +3.6), C-16 (d4 1.9) and C-21 (d4 8.8) in 1 relative to 2. Correlations from the N–H (dH 5.85) to C-16 (dC 168.0) and C-21 (dC 42.3) in the HMBC spectrum together with cross-peaks from H2-21 (dH 3.45) to H2-22 (dH 3.74) and N–H (dH 5.85) in the COSY spectrum further confirmed the N–H linkage
resonances for six methyls (dH 0.89, 0.98, 1.20, 1.21, 1.21 and 1.21), two methoxyls (dH 3.69 and 3.76), two exocyclic olefinic protons (dH 5.64 and 5.66), three oxymethines (dH 3.89, 3.94 and 4.66) and a secondary amide N–H (dH 5.85) were observed in the 1H NMR spectrum. All the signals in the above spectra indicated a striking resemblance to those of co-occurring erythrophlesin D (2) (Miyagawa et al., 2009), and the two compounds clearly pos-
O 12 1
2
O 12’
11’ 20’ 1’ 9’
2’
10’ 4’ 5’
3’
H
O
8’
6’
16’
3
R1
15’ 14’
7’
O
H
H
8
6
18
R2
19
24
19
24
OH
15 13 14
7
21
17
R3
17’
OH
O
19’ 18’
10 4 5
H
16
22
13’
H
H
O
11 20 9
R4 N
1 R1 = CO2CH3; R2 = O; R3 = (β) OH, H; R4 = H
21’
23
2 R1 = CO2CH3; R2 = O; R3 = (β) OH, H; R4 = CH3 3 R1 = H; R2 = O; R3 = (β) OH, H; R4 = H 23
4 R1 = H; R2 = O; R3 = (β) OH, H; R4 = CH3 19
24
5 R1 = CO2CH3; R2 = (α) OH, H; R3 = O; R4 = H
R N
O
16
12 1
2
HO 3 O
2011 9
10 4 5
H
19 18
O
H 6
H 8
21
O
OH
17
O
OH
O
H
O 1’ 2’
H
O O
23
7 R = CH3 R N
O
OH O 8 R=H
23
9 R = CH3
O
OH
H 5’ 4’
3’
2’
1’
O
H
O O
H N
H
H
O
OH
H
6 R=H
24
R N
15 13 14
7
22
OH O
H
R O
H O
O OH
10 R = H
12 R = OH
11 R = CH3
13 R = H
23
Fig. 1. Chemical structures of compounds 1–13.
OH
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D. Du et al. / Phytochemistry 71 (2010) 1749–1755
groups, two methoxyl groups, two exocyclic olefinic protons, three oxymethines and six carbonyls (dC 166.1, 167.9, 174.1, 175.4, 208.6 and 209.5), which suggested that, 5 is also a cassaine diterpenoid dimer. Compared with 1, the H-5 proton signal in 5 was shifted upfield to dH 1.48 (1H, d, J = 12.5 Hz) and coupled with a proton H-6 at dH 4.38 (1H, d, J = 12.5 Hz). In addition, the H-8 proton signal was shifted downfield to dH 2.38 (1H, dd, J = 12.5, 3.0 Hz). Thus, the hydroxyl group is located at C-6 and the ketone at C-7. The relative configuration of compound 5 was deduced from a NOESY experiment. The methyl protons Me-20 correlated with the methine proton H-8 and the oxymethine proton H-6, and the methine proton H-80 correlated with the methyl protons Me-200 and the methine proton H-140 , indicating a b-orientation for H-6, H-8 and H-80 . In addition, the a-configurations of H-3, H-5, H-9, H3-18, H-50 , H-70 , H-90 and H3-180 were deduced from NOESY correlations of H-3 to H-5 and H3-18, and H-50 to H-70 , H-90 and H3-180 . The olefinic protons dH 5.63 (H-15) and dH 5.74 (H-150 ) showed NOESY cross-peaks to the methines resonating at dH 2.94 (H-14) and dH 2.82 (H-140 ), respectively, indicating the E geometry of the two double bonds. Thus erythrophlesin G has structure 5. Compound 6 was isolated as a white powder. Its molecular formula was established as C23H35NO7 by the positive-ion mode
instead of the N–CH3 linkage. Thus, structure 1 (Fig. 1) was assigned to this compound which was given the name erythrophlesin E. NOESY correlations confirmed that it has the same relative stereochemistry as erythrophlesin D (2). Compound 3 was isolated as a white powder. The HR-ESIMS exhibited a quasi-molecular ion peak at m/z 740.4401 [M+H]+, supporting the molecular formula of C42H61NO10, indicating 14 mass units less than the co-occurring erythrophlesin C (4) (Miyagawa et al., 2009). Inspection of its 1D (1H and 13C) and 2D (HSQC, COSY and HMBC) NMR data (Table 1) established the presence of nearly identical structural features to those found in 4, except that resonances for the methyl group at nitrogen atom were absent. This was confirmed by HMBC correlations from the N–H (dH 5.84) to C-16 (dC 168.1) and C-21 (dC 42.3). Thus, structure 3 was deduced for this compound which was called erythrophlesin F. Again NOESY data confirmed that the relative stereochemistry was the same as in 1 and 2. Compound 5, named erythrophlesin G, was isolated as a white powder. Its molecular formula was deduced as C44H63NO12 due to the appearance of an [M+H]+ ion at m/z 798.4405 in the positive HR-ESIMS. The 1H and 13C NMR spectroscopic data (Table 1) showed the presence of characteristic signals for six methyl
Table 1 NMR spectroscopic data of compounds 1–5 in CDCl3 (500 MHz for 1H NMR and 125 MHz for Position
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 N–H
1
2
13
C NMR, d in ppm, J in Hz).
3
4
5
dH (J)
dC
dH (J)
dC
dH (J)
dC
dH (J)
dC
dH (J)
dC
1.89 m/1.42 m 2.26 m/1.76 m 4.66 dd (12.0, 4.0)
36.5 23.0 78.2 45.7 64.7 208.1 75.8 51.3 46.4 43.0 26.2 23.2 160.7 40.3 115.6 168.0 13.7 25.8 172.7 14.4 42.3 62.6
1.85 m/1.42 m 2.26 m/1.78 m 4.66 dd (12.0, 4.0)
36.5 23.8 78.2 45.7 64.6 208.1 75.8 51.2 46.4 43.0 26.2 24.3 157.1 39.8 115.0 169.9 13.8 25.8 172.6 14.4 51.1 61.7 37.3 51.8 38.6 18.9 38.3 41.9 64.5 208.5 75.6 50.2 46.3 42.9 26.2 23.7 166.4 40.4 113.1 166.0 13.6 28.6 175.4 15.1 51.6
1.84 1.92 4.46 2.14 2.13
1.83 1.89 4.46 2.14 2.13
36.5 26.4 76.6 31.0 60.6 209.6 75.6 52.3 43.7 43.0 26.4 24.5 157.4 39.9 114.9 170.0 13.7 16.1
1.90 m/1.32 m 2.24 m/1.79 m 4.61 dd (12.0, 4.0)
1.21 d (7.0) 0.88 d (7.0)
36.5 26.4 76.6 30.9 60.6 209.6 75.5 52.4 43.7 43.0 26.4 23.4 160.9 40.3 115.5 168.1 13.5 16.1
0.68 s 3.45 m 3.74 t (5.0)
13.6 42.3 62.7
37.1 23.9 78.1 49.4 57.9 75.1 209.5 51.7 47.0 37.4 27.2 23.3 159.9 39.4 115.5 167.9 14.9 26.4 174.1 13.6 42.3 62.7
2.44 s 3.94 d (10.5) 1.86 m 1.70 m 1.90 m/1.17 m 3.87 m/1.93 m 2.73 m 5.64 br s 1.20 d (6.0) 1.21 s 0.98 s 3.45 m 3.74 t (5.0) 3.76 1.76 1.72 2.03
s m/1.18 m m/1.52 m m/1.15 m
2.24 s 3.89 d (11.0) 1.86 m 1.72 m 1.90 m/1.15 m 3.73 m/2.01 m 2.81 m 5.66 br s 1.21 d (5.0) 1.21 s 0.89 s 3.69 s 5.85 br s
51.8 38.6 18.9 38.3 41.9 64.5 208.5 75.6 50.2 46.3 42.9 26.2 23.7 166.4 40.4 113.1 166.0 13.6 28.6 175.4 15.1 51.6
2.44 s 3.94 d (10.5) 1.86 m 1.75 m 1.88 m/1.18 m 3.15 m/2.09 m 2.83 m 5.93 br s 1.21 d (6.0) 1.21 s 0.98 3.57 3.80 3.06 3.76 1.78 1.72 2.03
s m t (5.0) s s m/1.19 m m/1.52 m m/1.15 m
2.24 s 3.89 d (10.5) 1.87 m 1.71 m 1.93 m/1.18 m 3.73 m/2.00 m 2.81m 5.66 br s 1.20 d (6.0) 1.21 s 0.89 s 3.69 s
m/1.41 m m/1.47 m m m m
4.00 d (8.0) 1.75 m 1.73 m 1.92 m/1.10 m 3.86 m/2.01 m 2.77 m 5.64 br s
1.80 m/1.19 m 1.74 m/1.52 m 2.03 m/1.15 m 2.24 s 3.90 d (11.0) 1.89 m 1.73 m 1.94 m/1.18 m 3.79 m/2.06 m 2.83 m 5.73 br s 1.21 d (7.0) 1.22 s 0.90 s 3.70 s 5.84 br s
38.6 18.9 38.3 41.9 64.5 208.6 75.6 50.3 46.4 42.8 26.2 23.7 165.8 40.3 113.4 166.6 13.7 28.6 175.4 15.1 51.6
m/1.39 m m/1.44 m m m m
4.01 d (8.0) 1.77 m 1.70 m 1.89 m/1.10 m 3.15 m/2.05 m 2.85 m 5.91 br s 1.22 d (7.0) 0.88 d (7.0) 0.68 3.58 3.79 3.07
s m t (5.0) s
1.80 m/1.20 m 1.75 m/1.50 m 2.05 m/1.14 m 2.24 s 3.90 d (10.5) 1.89 m 1.72 m 1.95 m/1.18 m 3.76 m/2.03 m 2.82 m 5.73 br s 1.21 d (7.0) 1.22 s 0.90 s 3.69 s
13.6 51.2 61.8 37.3 38.6 19.0 38.3 42.0 64.5 208.6 75.6 50.3 46.4 42.8 26.2 23.7 165.8 40.4 113.4 166.6 13.7 28.6 175.4 15.1 51.6
1.48 d (12.5) 4.38 d (12.5) 2.38 dd (12.5, 3.0) 1.65 m 1.92 m/1.18 m 3.86 m/1.98 m 2.94 m 5.63 br s 1.11 d (6.0) 1.50 s 1.07 s 3.45 m 3.73 t (5.0) 3.75 1.75 1.73 2.06
s m/1.18 m m/1.53 m m/1.15 m
2.25 s 3.90 d (10.5) 1.87 m 1.70 m 1.96 m/1.21 m 3.79 m/2.03 m 2.82 m 5.74 br s 1.20 d (7.5) 1.22 s 0.90 s 3.70 s 5.88 br s
51.7 38.6 19.0 38.3 42.0 64.5 208.6 75.6 50.3 46.4 43.0 26.2 23.7 166.4 40.3 113.5 166.1 13.6 28.6 175.4 15.1 51.6
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HR-ESIMS showing a pseudo-molecular ion peak at m/z 438.2489 [M+H]+. IR absorptions at 3353 and 1721 cm1 showed the presence of hydroxyl and carbonyl groups, respectively. Based on the 13 C NMR and DEPT spectra, compound 6 included three carbonyl (dC 168.1, 174.1 and 208.0), and two olefinic carbons (dC 115.5 and 160.9). Analysis of its 1H NMR spectrum indicated the presence of three methyl groups at dH 0.90, 1.19 and 1.35, a methoxyl group at dH 3.75, an exocyclic vinyl proton at dH 5.64, and a secondary amide proton at dH 5.89. These signals were similar to those of 3b-hydroxynorerythrosuamide (7) (Verotta et al., 1995), except for the lack of an N-methyl group, suggesting that 6 was a demethyl derivative of 7. This conclusion was confirmed by the COSY correlations of the NH, 2H-22 and 2H-23 and the corresponding carbon shifts. Thus, compound 6 is 3b-hydroxydinorerythrosuamide. Analysis of the HMBC spectrum permitted the assignment of all the proton and carbon resonances (see tables) and the relative stereochemistry was confirmed from the NOESY spectrum. Compound 8, was purified as a white powder. The HR-ESIMS gave a molecular ion peak at m/z 480.2622 [M+H]+ giving the molecular formula C25H37NO8. The NMR spectroscopic data of 8 was virtually identical with that of 6, apart from the presence of an acetate group which was readily assigned to the b position at C-3 (H-3, dd, 4.65, J = 12.0, 4.0 Hz) on the basis of an HMBC correlation from H-3 to the acetate carbonyl group. Thus, compound 8 is 3bacetoxydinorerythrosuamide. Compound 9 was purified as a white powder whose HR-ESIMS showed a peak at m/z 494.2754 [M+H]+, indicating the molecular formula of C26H39NO8. Interpretation of the 1H and 13C NMR spectra of 8 and 9 indicated that 9 was a methylation product of 8, and this was supported by the presence of 1H–13C NMR long-range correlations of the methyl protons dH 3.06 (H3-23) with the amide carbonyl dC 169.9 (C-16) and methylene dC 51.1 (C-21). Thus, compound 9 is 3b-acetoxynorerythrosuamide. Compound 10 was isolated as a white powder. The molecular formula was deduced as C28H41NO8 from the ion peak at m/z 520.2927 [M+H]+ obtained by positive HR-ESIMS. The 1H and 13C NMR spectroscopic data (Tables 2 and 3) of 10 closely resembled
Table 3 13 C NMR spectroscopic data (125 MHz, CDCl3) for compounds 6–13 (d in ppm). Position
6
7
8
9
10
11
12
13
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 10 20 30 40 50
37.1 27.6 78.1 47.7 64.4 208.0 75.8 50.9 46.1 43.2 26.2 23.2 160.9 40.3 115.5 168.1 13.7 25.6 174.1 14.4 42.4 62.8
37.1 27.5 78.0 47.7 64.3 208.0 75.8 50.8 46.1 43.2 26.2 24.4 157.3 39.8 114.9 169.9 13.8 25.6 174.0 14.4 51.1 61.7 37.3 51.8
36.5 23.7 78.7 45.7 64.6 208.1 75.8 51.3 46.4 43.0 26.2 23.2 160.6 40.2 115.6 168.0 13.7 25.7 172.6 14.4 42.3 62.6
36.5 23.7 78.8 45.7 64.6 208.1 75.8 51.2 46.5 43.0 26.2 24.3 157.1 39.8 115.0 169.9 13.8 25.7 172.6 14.4 51.1 61.7 37.3 51.8 170.4 21.0
36.5 23.7 78.7 45.8 64.7 208.1 75.8 51.3 46.5 43.0 26.3 23.2 160.7 40.3 115.6 168.0 13.7 25.8 172.7 14.4 42.3 62.7
36.5 23.7 78.7 45.8 64.6 208.2 75.8 51.2 46.5 43.0 26.2 24.3 157.1 39.9 115.0 169.9 13.9 25.8 172.7 14.4 51.1 61.7 37.3 51.7 167.1 128.3 137.9 14.4 11.8
37.9 27.8 78.0 50.3 58.1 75.4 210.1 51.1 46.3 37.6 27.3 23.3 160.0 39.4 115.4 167.8 14.9 25.3 178.0 13.5 42.3 62.6
39.8 19.2 39.6 45.3 58.5 75.8 210.4 51.5 47.1 37.8 27.3 23.5 160.4 39.5 115.2 168.0 14.9 31.5 177.4 13.9 42.3 62.7
51.7
51.6
51.8
51.8 170.4 21.0
51.7 167.1 128.3 137.9 14.4 11.8
those of 6, with the only difference being the presence of an additional fragment of C5H7O. The signals of dC 167.1, dC 128.3, dC 137.9, dC 14.4, dC 11.8, dH 1.77 (3H, d, J = 5.5) and dH 1.79 (3H, s) were diagnostic of a tigloyl moiety (Yang et al., 1999). An HMBC correlation of H-3 to the tigloyl carbonyl group confirmed its attachment to C-3. Thus, compound 10 is 3b-tigloyloxydinorerythrosuamide. Compound 11 was purified as a white powder. The HR-ESIMS indicated an ion peak at m/z 534.3082 [M+H]+, which suggested a molecular formula C29H43NO8. The 1H and 13C NMR spectroscopic
Table 2 1 H NMR spectroscopic data (500 MHz, CDCl3) for compounds 6–13 (d in ppm, J in Hz). Position
6
7
8
1 2 3
1.87 m/1.35 m 2.14 m/1.75 m 3.35 m
1.87 m/1.32 m 2.14 m/1.75 m 3.35 m
5 6 7 8
2.33 s
2.33 s
1.87 2.22 4.65 4.0) 2.42
3.92 d (10.5) 1.81 m
3.92 d (10.5) 1.84 m
3.93 d (10.5) 1.83 m
1.67 4.0) 1.97 3.87 2.75 5.64 1.19 1.35 0.90 3.47 3.73
1.66 3.0) 2.01 3.15 2.82 5.91 1.20 1.35 0.91 3.57 3.78 3.07 3.74
1.68 3.5) 1.86 3.86 2.73 5.64 1.18 1.19 0.96 3.44 3.72
9 11 12 14 15 17 18 20 21 22 23 24 20 30 40 50 N–H
dt (12.0, m/1.22 m m/2.00 m m br s d (7.0) s s m m
3.75 s
5.89 br s
dt (12.0, m/1.18 m m/2.06 m m br s d (7.5) s s m m s s
9
10
11
m/1.40 m m/1.70 m dd (11.5,
1.86 m/1.43 m 2.21 m/1.70 m 4.68 dd (12, 4.5)
s
2.44 s
1.85 2.22 4.68 4.0) 2.44
3.94 d (10.0) 1.86 m
3.94 d (10.5) 1.83 m
3.94 d (10.0) 1.87 m
dt (12.0,
1.66 m
m/1.16 m m/2.00 m m br s d (6.0) s s m t (4.5)
1.85 3.14 2.82 5.90 1.21 1.19 0.97 3.57 3.78 3.06 3.76 2.02
1.70 3.5) 1.88 3.87 2.74 5.64 1.19 1.20 0.99 3.44 3.73
1.70 3.5) 1.89 3.15 2.83 5.91 1.21 1.20 1.00 3.57 3.79 3.07 3.76
m/1.40 m m/1.70 m dd (12.0, s
3.75 s 2.02 s
6.05 br s
1.84 2.22 4.64 3.5) 2.42
m/1.14 m m/2.02 m m br s d (7.5) s s m t (5.0) s s s
dt (12.0, m/1.21 m m/2.01 m m br s d (6.0) s s m t (4.5)
3.76 s 6.77 1.77 1.79 5.95
d (5.5) d (5.5) s br s
12 m/1.42 m m/1.70 m dd (11.5, s
dt (12.0, m/1.16 m m/2.05 m m br s d (6.0) s s m t (4.5) s s
13
1.87 1.91 3.10 4.0) 1.36 4.58
m/1.23 m m/1.25 m dt (11.5,
1.85 m/1.07 m 1.67 m/1.53 m 2.26 m/1.09 m
d (12.0) d (12.0)
1.37 d (12.5) 4.76 d (12.5)
2.38 3.0) 1.64 3.0) 1.91 3.84 2.90 5.62 1.12 1.70 0.88 3.45 3.74
dd (13.0, dt (13.5,
2.39 dd (11.5, 3.5) 1.68 m
m/1.28 m m/2.03 m m br s d (7.0) s s m t (4.5)
2.00 3.84 2.91 5.62 1.12 1.45 0.87 3.44 3.73
m/1.26 m m/2.03 m m br s d (7.0) s s m t (5.0)
3.75 s
3.71 s
5.91 br s
5.97 br s
6.77 d (7.0) 1.77 d (7.0) 1.79 s
D. Du et al. / Phytochemistry 71 (2010) 1749–1755
data were almost identical with those of 10, apart from the presence of a N-methyl group. This was confirmed by correlations from the methyl protons H3-23 (dH 3.07, s) to the carbonyl C-16 (dC 169.9) and the methylene C-21 (dC 51.1) in the HMBC spectrum. Thus, compound 11 is 3b-tigloyloxynorerythrosuamide. Compound 12 was purified as a white powder. The ion peak in HR-ESIMS at m/z 438.2506 [M+H]+ indicated the same molecular formula as compound 6, C23H35NO7. A comparison of the 1H and 13 C NMR spectroscopic data of 12 with those of 6 suggested they shared the same skeleton, except for the 6a-hydroxy-7-keto group in 12 instead of the 6-keto-7b-hydroxy group. The notable upfield shift of H-5 and the downfield shift of H-8 (vide infra) supported this conclusion. The presence of a NOE interaction between H-6 and Me-20 confirmed the a configuration of the C-6 hydroxyl group. Thus, compound 12 is 6a-hydroxydinorerythrophlamide. Compound 13 was purified as a white powder. The HR-ESIMS indicated a molecular formula C23H35NO6 deduced from the ion peak at m/z 422.2573 [M+H]+. The NMR features of 13 were analogous to those of 12, apart from the absence of an oxygen substituent at C-3, confirmed by HSQC, COSY and HMBC data (see tables). Hence, compound 13 is 6a-hydroxydinorcassamide. Three known alkaloids, erythrophlesin D (2) (Miyagawa et al., 2009), erythrophlesin C (4) (Miyagawa et al., 2009) and 3bhydroxynorerythrosuamide (7) (Verotta et al., 1995), were identified by the comparison of their NMR spectroscopic data with those reported in the literature, respectively. The isolated alkaloids 1–13 were screened in an in vitro cytotoxicity assay. Camptothecin was used as a positive control, and the data are shown in Table 4. The cassaine diterpenoid–diterpenoid amide dimers (1 and 3–5) indicated significantly selective cytotoxicity against HCT-8, Bel-7402, BGC-823, A549 and A2780 cell lines. Compound 1 exhibited cytotoxicity against BGC-823 and A2780 cell lines with IC50 values of 3.47 and 0.91 lM, respectively. Compounds 3 and 5 demonstrated strongly selective cytotoxic activity against A2780 with IC50 values of 4.54 and 7.40 lM, respectively. Compound 4 displayed inhibitory effects on Bel-7402, BGC-823, A549 and A2780. However, no significant activity was observed among the monomers 6–11 and 13, and only compound 12, 6ahydroxydinorerythrophlamide, showed activity against all the cell lines tested. Compound 12 was the only cassaine diterpenoid amide tested in this study that possessed a hydroxy at C-3 and a 6a-hydroxy-7-keto group, suggesting that these residues may be involved in the cytotoxic activity. Structure–activity studies of cassaine diterpenoid amides in our earlier research have demonstrated that substituents at C-3 might influence cytotoxic activity and compounds with 6-keto-7-hydroxy groups showed no activity (Qu et al., 2006a). In combination with the data obtained in the present study, we speculate that the C-3 hydroxyl group and the C-7 ketone in cassaine diterpenoid amides might be necessary for cytotoxic activity. Moreover, the analysis of the IC50 values indiTable 4 Cytotoxicity of compounds 1, 3–5 and 12 against five human cancer cell lines. Compounda
1 3 4 5 12 Camptothecinb
IC50 (lM) HCT-8
Bel-7402
BGC-823
A549
A2780
>10 >10 >10 >10 2.77 3.20
>10 >10 2.77 >10 0.52 12.51
3.47 >10 0.67 >10 1.25 9.72
>10 >10 3.82 >10 8.67 3.11
0.91 4.54 0.49 7.40 1.35 0.29
HCT-8 = human colon cancer cell line; Bel-7402 = human hepatoma cell line; BGC823 = human gastric carcinoma cell line; A549 = human lung epithelial cell line; A2780 = human epithelial carcinoma cell line. ‘‘>10’’ = inactive. a Compounds 2, 6–11 and 13 were inactive (IC50 > 10 lM) for all cells. b Positive control.
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cated that dimers showed better selectivities than monomers. These findings encourage further studies on the exact mechanism by which compounds 1, 3–5 and 12 exert their cytotoxic activity. 3. Conclusions Phytochemical investigation of the toxic plant, E. fordii, led to the isolation of thirteen compounds (1–13), including three new cassaine diterpenoid–diterpenoid amide dimers, erythrophlesins E–G, seven new cassaine diterpenoid amides and three known related compounds. All of the compounds were tested for cytotoxicity against HCT-8, Bel-7402, BGC-823, A549 and A2780 cell lines. Compounds 1, 3–5 and 12 had significant cytotoxicity properties with IC50 values ranging from 0.49 to 8.67 lM. 4. Experimental 4.1. General experimental procedures The NMR spectra were recorded with an Inova 500 FT-NMR spectrometer. ESIMS and HR-ESIMS were run on an Agilent 1100 Series LC/MSD Trap mass spectrometer. Analytical HPLC was performed on an Agilent 1100 Series instrument with a DAD detector, using a YMC-Pack ODS (100 4.6 mm, 5 lm). Preparative HPLC was performed on a Shimadazu instrument equipped with a LC6AD pump, a SPD-10A detector, using a YMC-Pack ODS-A column (250 20 mm, 5 lm). IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer by a microscope transmission method. The UV spectra were obtained on a V650 spectrometer. Melting points were conducted on a XT-5B micromelting point apparatus and are uncorrected. Optical rotations were measured with a P2000 polarimeter. Polyamide (30–60 mesh, Jiangsu Linjiang Chemical Reagents Factory, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Sweden) and ODS (50 lm, Merck) were used for column chromatography (CC). Silica gel GF-254 (Qingdao Marine Chemical Factory) was used for TLC. Solvents (CHCl3, MeOH and EtOH) were of analytical grade and purchased from Beijing Chemical Company, Beijing, China. 4.2. Plant materia Leaves of E. fordii Oliver (Leguminosae) were harvested in August 2007 in Guangxi Province, China, and identified by Prof. Songji Wei (Guangxi College of Chinese Traditional Medicine). A voucher specimen (No. 07089) has been deposited in the Herbarium of the Department of Medicinal plants, Institute of Materia Medica, Chinese Academy of Medical Sciences. 4.3. Extraction and isolation Powdered air-dried leaves of E. fordii (5.2 kg) were extracted with EtOH/water (95:5, v/v) under conditions of reflux (3 20 L), this being maintained for 3h. After filtration and removal of the solvent under reduced pressure, the residue (806 g) was diluted in MeOH (1.5 L), filtered and adsorbed on a diatomite column. The latter was eluted successively with petroleum (60–90 °C) (10 L), EtOAc (10 L), and MeOH (20 L), to afford petroleum ether (90 g), EtOAc (45 g) and MeOH extracts (532 g), respectively. The EtOAc extract was subjected to a polyamide column, eluting with H2O, EtOH–H2O (3:7), EtOH–H2O (6:4) and EtOH–H2O (9:1) to yield Fr.1 (20.0 g), Fr.2 (7.6 g), Fr.3 (5.1 g) and Fr.4 (1.6 g). Fr.1 and Fr.2 were then combined and submitted to repeated Sephadex LH-20 (MeOH as eluent) and ODS (MeOH–H2O as eluent) to give a total alkaloid portion (17.8 g), which was subjected to an ODS (50 lm, 300 g) CC using a gradient of MeOH–H2O (5:95–90:10)
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as eluents. According to the TLC profiles, 12 major fractions (1–12) were obtained. Fraction 3 (1.93 g) was submitted to ODS (MeOH– H2O, from 20:80 to 60:40), followed by a preparative C-18 column using a mobile phase consisting of MeCN–H2O 15:85, leading to the isolation of 6 (35 mg; w/w 0.0043%) and 7 (20 mg; w/w 0.0025%). Fraction 4 (1.09 g) was purified using the same column (MeCN–H2O 17:83) to give 12 (14 mg; w/w 0.0017%). Fraction 6 (1.44 g) was separated by a preparative C-18 column (MeCN–H2O 24:76) to yield 8 (43 mg; w/w 0.0053%) and 9 (48 mg; w/w 0.0060%). Fraction 8 (1.66 g) was purified by an ODS column (50 lm, 100 g) and preparative HPLC using MeCN–H2O 30:70 to provide 10 (30 mg; w/w 0.0037%), 11 (27 mg; w/w 0.0033%) and 13 (28 mg; w/w 0.0035%). Fraction 10 (0.25 g) using MeCN–H2O 48:52 on the preparative C-18 column, afforded 1 (27 mg; w/w 0.0033%), 2 (27 mg; w/w 0.0033%), 3 (18 mg; w/w 0.0022%), 4 (8 mg; w/w 0.0010%) and 5 (7 mg; w/w 0.00086%). 4.3.1. Erythrophlesin E (1) White powder; mp 194–196 °C; ½a20 D 65.2 (c 0.08, EtOH); UV (EtOH) kmax nm: 222; IR mmax cm1: 3383, 2937, 1721, 1693, 1660, 1537, 1466, 1393, 1374, 1275, 1257, 1165, 1042, 1026, 969, 868; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 798.4429 [M+H]+ (calcd. for C44H64NO12, 798.4428); ESIMS (positive-ion mode) m/z: 798 [M+H]+, 820 [M+Na]+, 836 [M+K]+; ESIMS (negative-ion mode) m/z: 796 [MH], 832 [M+Cl]. 4.3.2. Erythrophlesin D (2) White powder; mp 174–176 °C; ½a20 D 81.1 (c 0.08, EtOH); UV (EtOH) kmax nm: 222; IR mmax cm1: 3436, 2942, 1717, 1645, 1608, 1458, 1394, 1374, 1256, 1163, 1042, 969, 868; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 812.4597 [M+H]+ (calcd. for C45H66NO12, 812.4585); ESIMS (positive-ion mode) m/z: 812 [M+H]+, 834 [M+Na]+; ESIMS (negative-ion mode) m/z: 810 [MH], 846 [M+Cl]. 4.3.3. Erythrophlesin F (3) White powder; mp 178–180 °C; ½a20 D 58.4 (c 0.08, EtOH); UV (EtOH) kmax nm: 223; IR mmax cm1: 3378, 2941, 2875, 1714, 1642, 1532, 1460, 1391, 1321, 1258, 1197, 1157, 1042, 1026, 967, 868; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 740.4401 [M+H]+ (calcd. for C42H62NO10, 740.4373); ESIMS (positive-ion mode) m/z: 740 [M+H]+, 762 [M+Na]+; ESIMS (negative-ion mode) m/z: 738 [MH], 774 [M+Cl]. 4.3.4. Erythrophlesin C (4) White powder; mp 164–166 °C; ½a20 D 63.4 (c 0.08, EtOH); UV (EtOH) kmax nm: 221; IR mmax cm1: 3419, 2941, 2876, 1714, 1646, 1607, 1460, 1393, 1257, 1196, 1044, 1025, 968, 867; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 754.4564 [M+H]+ (calcd. for C43H64NO10, 754.4530); ESIMS (positive-ion mode) m/z: 754 [M+H]+, 776 [M+Na]+; ESIMS (negative-ion mode) m/z: 752 [MH], 788 [M+Cl]. 4.3.5. Erythrophlesin G (5) White powder; mp 162–164 °C; ½a20 D 57.1 (c 0.06, EtOH); UV (EtOH) kmax nm: 222; IR mmax cm1: 3381, 2942, 2880, 1714, 1641, 1533, 1457, 1393, 1321, 1257, 1227, 1165, 1041, 1013, 870, 720; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Table 1; HR-ESIMS m/z: 798.4405 [M+H]+ (calcd. for C44H64NO12, 798.4428); ESIMS (positive-ion mode) m/z: 798 [M+H]+, 820 [M+Na]+; ESIMS (negative-ion mode) m/z: 796 [MH], 832 [M+Cl].
4.3.6. 3b-Hydroxydinorerythrosuamide (6) White powder; mp 128–130 °C; ½a20 D 92.8 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3353, 2942, 2879, 1721, 1656, 1629, 1537, 1452, 1394, 1372, 1322, 1251, 1208, 1165, 1118, 1062, 1037, 967, 867; for 1H NMR (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 438.2489 [M+H]+ (calcd. for C23H36NO7, 438.2486); ESIMS (positive-ion mode) m/z: 438 [M+H]+, 460 [M+Na]+. 4.3.7. 3b-Hydroxynorerythrosuamide (7) White powder; mp 112–114 °C; ½a20 D 48.3 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3384, 2941, 2881, 1722, 1649, 1601, 1456, 1401, 1372, 1252, 1195, 1163, 1119, 1065, 1039, 968, 938, 864, 729; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; for 1H (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) spectroscopic data see supplementary data; ESIMS (positive-ion mode) m/z: 452 [M+H]+, 474 [M+Na]+. 4.3.8. 3b-Acetoxydinorerythrosuamide (8) White powder; mp 127–129 °C; ½a20 D 76.4 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3365, 2943, 2879, 1701, 1660, 1631, 1457, 1394, 1267, 1157, 1119, 1056, 981, 865; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 480.2622 [M+H]+ (calcd. for C25H38NO8, 480.2591); ESIMS (positive-ion mode) m/z: 480 [M+H]+, 502 [M+Na]+, 514 [M+K]+; ESIMS (negative-ion mode) m/z: 478 [MH], 514 [M+Cl]. 4.3.9. 3b-Acetoxynorerythrosuamide (9) White powder; mp 106–108 °C; ½a20 D 58.6 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3391, 2943, 2880, 1730, 1650, 1605, 1458, 1401, 1369, 1239, 1168, 1121, 1079, 1034, 969, 867, 733; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 494.2754 [M+H]+ (calcd. for C26H40NO8, 494.2748); ESIMS (positive-ion mode) m/z: 494 [M+H]+, 516 [M+Na]+, 528 [M+K]+; ESIMS (negative-ion mode) m/z: 492 [MH], 528 [M+Cl]. 4.3.10. 3b-Tigloyloxydinorerythrosuamide (10) White powder; mp 126–128 °C; ½a20 D 47.6 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3340, 2944, 2879, 1712, 1651, 1536, 1441, 1392, 1346, 1268, 1253, 1209, 1164, 1120, 1075, 1041, 968, 871, 733; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 520.2927 [M+H]+ (calcd. for C28H42NO8, 520.2904); ESIMS (positive-ion mode) m/z: 520 [M+H]+, 542 [M+Na]+; ESIMS (negative-ion mode) m/z: 554 [M+Cl]. 4.3.11. 3b-Tigloyloxynorerythrosuamide (11) White powder; mp 108–110 °C; ½a20 D 46.5 (c 0.08, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3413, 2943, 2877, 1709, 1649, 1605, 1454, 1438, 1398, 1346, 1265, 1251, 1197, 1163, 1120, 1074, 1045, 1027, 968, 733; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESMS m/z: 534.3082 [M+H]+ (calcd. for C29H44NO8, 534.3061); ESIMS (positive-ion mode) m/z: 534 [M+H]+, 556 [M+Na]+, 572 [M+K]+; ESIMS (negative-ion mode) m/z: 532 [MH], 568 [M+Cl]. 4.3.12. 6a-Hydroxydinorerythrophlamide (12) White powder; mp 113–115 °C; ½a20 D 42.7 (c 0.09, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3375, 2944, 2879, 1701, 1660, 1631, 1537, 1457, 1394, 1334, 1267, 1249, 1218, 1157, 1119, 1097, 1056, 1021, 981, 865, 716; for 1H NMR (500 MHz,
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CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 438.2506 [M+H]+ (calcd. for C22H36NO7, 438.2486); ESIMS (positive-ion mode) m/z: 438 [M+H]+, 460 [M+Na]+, 476 [M+K]+. 4.3.13. 6a-Hydroxydinorcassamide (13) White powder; mp 96–98 °C; ½a20 D 34.2 (c 0.09, EtOH); UV (EtOH) kmax nm: 243; IR mmax cm1: 3380, 2941, 2876, 1716, 1659, 1632, 1536, 1459, 1393, 1329, 1265, 1222, 1155, 1097, 1070, 1021, 978, 869; for 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectroscopic data, see Tables 2 and 3; HR-ESIMS m/z: 422.2573 [M+H]+ (calcd. for C23H36NO6, 422.2537); ESIMS (positive-ion mode) m/z: 422 [M+H]+, 444 [M+Na]+, 456 [M+K]+; ESIMS (negative-ion mode) m/z: 420 [MH], 437 [M+Cl]. 4.4. Cytotoxicity assay The cytotoxicity assay against HCT-8 (human colon cancer), Bel7402 (human hepatoma cancer), BGC-823 (human gastric cancer), A549 (human lung epithelial) and A2780 (human ovarian cancer) cells (IC50) was assessed using the MTT method as described in the literature (Alley et al., 1988). Camptothecin was used as a control compound. Acknowledgments The research presented in this paper was supported by the National Found for Distinguished Young Scholars (No. 30625040), National Natural Science Foundation of China (No. 90713039) and National Science and Technology Project of China (No. 2009ZX09311-004). We are very grateful to Professor Songji Wei in Guangxi College of Chinese Traditional Medicine for his assistance in the plant collection and identification, the Department of Medicinal Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College for their assistance in measurements of IR, NMR, ESIMS and HR-ESIMS spectra. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2010.07.004.
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