Caesalpins A–H, Bioactive Cassane-Type Diterpenes from the Seeds

Jun 12, 2013 - Guoxu Ma†, Jingquan Yuan‡, Haifeng Wu†, Li Cao†, Xiaopo Zhang†, ... Zheng†, Liyong Li†, Lijing Zhang†, Junshan Yang†,...
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Caesalpins A−H, Bioactive Cassane-Type Diterpenes from the Seeds of Caesalpinia minax Guoxu Ma,† Jingquan Yuan,‡ Haifeng Wu,† Li Cao,† Xiaopo Zhang,† Lijia Xu,† Hua Wei,§ Lizhen Wu,† Qingxia Zheng,† Liyong Li,† Lijing Zhang,† Junshan Yang,† and Xudong Xu*,† †

Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, People’s Republic of China ‡ National Engineering Laboratory of Southwest Endangered Medicinal Resource Development, National Development and Reform Commission, Guangxi Botanical Garden of Medicinal Plants, Nanning 530023, People’s Republic of China § Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, People’s Republic of China S Supporting Information *

ABSTRACT: Eight new cassane-type diterpenes, caesalpins A−H (1−8), were isolated from the ethyl acetate extract of Caesalpinia minax. Compound 1 displayed significant antiproliferative activity against HepG-2 (IC50 4.7 μM) and MCF-7 (IC50 2.1 μM) cells, and compounds 2 and 4 exhibited selective cytotoxic activities against MCF-7 (IC50 7.9 μM) and AGS (IC50 6.5 μM) cells.

T

he genus Caesalpinia (Fabaceae), which is widely distributed in the tropical and subtropical regions of Southeast Asia, is a rich source of cassane-type diterpenes. The structures of these diterpenes comprise tetracyclic frameworks with a fused furan ring or butenolide moiety.1−3 Some cassanetype diterpenes display significant bioactivity, including antiproliferative,4 antimalarial,5 antibacterial,6 antihelmintic, and antineoplastic effects.7,8 In particular, escobarine A exhibits significant antiproliferative activity against the K-562 cancer cell line, with an IC50 value of 2.52 μM.9 The seeds of Caesalpinia minax Hance, which is known as Kushilian, are widely used in the Chinese traditional system of medicine for the treatment of anemopyretic colds, dysentery, and skin itching and sores.10 To identify potential bioactive cassane-type diterpenes, an EtOAc extract of Kushilian afforded eight new compounds, designated caesalpins A−H (1−8), together with four known cassane-type diterpenes, neocaesalpin W,11 neocaesalpin C,12 neocaesalpin O,13 and neocaesalpin D (Figure 1).12 The antiproliferative effects of the diterpenoids on the AGS, A549, HepG-2, and MCF-7 human cancer cell lines were determined.

Compound 1 was obtained as an amorphous, white powder, and its molecular formula, C25H34O8, was deduced by HRESIMS (m/z 485.2133 [M + Na]+). Its IR spectrum contained absorption bands for hydroxy (3549 cm−1) and α,βunsaturated carbonyl (1,733 cm−1) groups. The 1H NMR spectrum (Table 1) displayed four methyl signals at δH 1.15 (H3-18), 1.16 (H3-20), 1.22 (H3-19), and 2.03 (H3-17). A methoxy signal at δH 3.66 and two acetoxy methyl signals at δH 2.09 and 2.19 indicated the presence of a carboxymethyl and two acetoxy substituents. An olefinic proton signal evident at δH 5.83 (d, J = 2.4) revealed the presence of a double bond. The 13 C APT (Table 1) data for compound 1 indicated the presence of 25 carbons, including seven methyl, four methylene, four methine (one olefinic carbon at δC 127.0), and 10 quaternary carbons (three olefinic carbons at δC 130.4, 136.4, and 149.9; three ester carbonyls at δC 169.2, 170.6, and 171.4; and one ketocarbonyl at δC 197.2). The NMR data indicated that compound 1 was a tricyclic cassane diterpene with a conjugated unsaturated carbonyl moiety.2,8,13 Analysis of the 1D and 2D NMR (HSQC, 1H−1H COSY, HMBC) data allowed the structure of compound 1 to be determined. The proton signals were assigned to the corresponding carbons through direct 1H and 13C correlations in the HSQC spectrum. From the 1H−1H



RESULTS AND DISCUSSION The air-dried and powdered seeds of C. minax were extracted three times with MeOH. The resulting extract was separated via column chromatography (CC) and semipreparative HPLC purification, affording 12 cassane-type diterpenes (1−12). © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 31, 2012

A

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Figure 1. Structures of compounds 1−8.

Table 1. NMR Data (600 MHz, in CDCl3) for Compounds 1−3 1

2

3

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

1 2

75.0, CH 22.6, CH2

75.0, CH 22.3, CH2

32.2, CH2

4.81, t (2.4) 1.72−1.75, m 1.86−1.88, m 1.05−1.07, m 1.70−1.73, m

75.1, CH 22.4, CH2

3

4.85, t (1.8) 1.78−1.83, m 1.87−1.90, m 1.11−1.14, m 1.84−1.86, m

4.82, t (2.4) 1.74−1.77, m 1.88−1.92, m 1.02−1.05, m 1.71−1.73, m

4 5 6 7 8 9 10 11 12 13 14 15

38.5, C 76.2, C 72.3, CH 127.0, CH 136.4, C 36.9, CH 44.9, C 35.5, CH 197.2, C 130.4, C 149.9, C 31.8, CH2

16 17 18 19 20 1-OCOCH3 1-OCOCH3 6-OCOCH3 6-OCOCH3 7-OCOCH3 7-OCOCH3 16-OCH3

171.4, C 16.5, CH3 30.2, CH3 26.0, CH3 18.0, CH3 169.2, C 21.5, CH3 170.6, C 21.9, CH3

position

5-OH

52.2, CH3

5.82, d (2.4) 5.83, d (2.4) 3.46, m 2.28, m

3.36, d (16.8) 3.62, d (16.8) 2.03, 1.15, 1.22, 1.16,

s s s s

2.09, s 2.19, s

3.66, s

32.5, CH2 38.6, C 79.2, C 75.6, CH 75.3, CH 43.4, CH 37.9, CH 44.2, C 37.6, CH 196.3, C 130.7, C 158.6, C 31.3, CH2 171.6, C 18.6, CH3 29.9, CH3 24.6, CH3 17.5, CH3 169.0, C 21.9, CH3 170.6, C 21.4, CH3 170.9, C 21.7, CH3 52.2, CH3

5.54, 5.37, 2.89, 2.87,

d (9.0) t (9.0) d (3.6) s

2.11−2.20, m

3.28, d (15.6) 3.52, d (15.6) 1.89, 1.11, 1.12, 12.6,

s s s s

2.07, s 2.02, s 2.08, s 3.65, s

32.6, CH2 38.7, C 79.2, C 75.5, CH 75.6, CH 43.6, CH 38.1, CH 44.1, C 38.1, CH 197.4, C 132.3, C 158.0, C 30.4, CH2 104.7, CH 18.4, CH3 30.6, CH3 24.6, CH3 17.6, CH3 169.0, C 21.9, CH3 170.6, C 21.3, CH3 170.9, C 21.8, CH3 54.5, CH3 53.9, CH3

5.54, 5.35, 2.88, 2.81,

d (8.4) t (8.4) s m

2.12−2.16, m

2.67, d (4.8) 4.32, 1.98, 1.11, 1.12, 1.25,

t (4.8) s s s s

2.06, s 2.02, s 2.08, s 3.29, s 3.32, s

2.86, br s

B

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basis of the correlation of H2-15 (δH 3.36, d, J = 16.8; 3.62, d, J = 16.8) with C-16 (δC 171.4, −COOCH3). The relative configuration of compound 1 was established through analysis of its NOESY spectrum. The β-orientations of H-1 and H-6 were evident from the NOE correlation of H-1 and H-6 with H3-20. The interaction of H3-18 with H-9 suggested that they were α-oriented. The ECD spectrum (Supporting Information, Figure S49) of compound 1 exhibited a positive Cotton effect at 329 (Δε 3.9) nm and a negative effect at 284 (Δε −18.3) nm due to the conjugated dienone chromophore. However, the absence of proper model compounds to use as references made the assignment of the absolute configuration at C-7 unreliable. Therefore, an ECD analysis was conducted for the Rh-complex of 1 (Supporting Information, Figure S50), which displayed a negative Cotton effect at 350 (Δε −0.5) nm, suggesting an R absolute configuration for C-5.14,15 Thus, the structure of 1 was assigned as 1α,6α-diacetoxy-5α-hydroxy-15-carboxymethyl-12oxocassa-7(8),13(14)-diene, and this compound was designated the trivial name caesalpin A. This is the first time that this type of unsaturated carbonyl moiety has been found among cassane diterpenes obtained from the genus Caesalpinia. Compound 2, which was also obtained as an amorphous, white powder, was assigned as C27H38O10 on the basis of its

COSY analysis, three substructures (drawn with bold bonds in Figure 2) were established. In the HMBC spectrum (Figure 2),

Figure 2. Key 1H−1H COSY (bonds) and HMBC (arrows) correlations for compound 1.

the correlations of H-7 (δH 5.83, d, J = 2.4) with C-8 (δC 136.4) and C-14 (δC 149.9); H2-15 (3.36, d, J = 16.8; 3.62, d, J = 16.8) with C-12 (δC 197.2), C-13 (δC 130.4), and C-14 (δC 149.9); and H3-17 (δH 2.03, s) with C-8 (δC 136.4), C-12 (δC 197.2), C-13 (δC 130.4), and C-14 (δC 149.9) implied an α,β,γ,δunsaturated carbonyl moiety. Furthermore, the HMBC correlations of H3-OAc (δH 2.09) with C-1 (δC 75.0) and δC 169.2 and of H3-OAc (δH 2.19) with C-6 (δC 72.3) and δC 170.6 indicated that the acetoxy groups were attached to C-1 and C-6. The carboxymethyl group was located at C-15 on the Table 2. NMR Data (600 MHz) for Compounds 4−6 4a

6a

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

1 2

75.8, CH 23.0, CH2

77.4, CH 22.7, CH2

5.21, d (1.8) 5.27, m

33.9, CH2

5.66, t (1.8) 1.87−1.90, m 2.13−2.16, m 1.16−1.20, m 1.92−1.94, m

74.5, CH 67.3, CH

3

5.77, t (3.0) 1.99−2.04, m 2.28−2.31, m 1.51−1.53, m 1.79−1.84, m

36.0, CH2

1.28−1.32, m 1.97−1.99, m

position

a

5b

4 5 6

36.6, C 167.3, C 128.2, CH

7

188.0, C

8 9 10 11

124.7, 148.7, 46.3, 104.9,

C C C CH

12 13 14 15 16 17 18 19 20 1-OCOCH3 1-OCOCH3 2-OCOCH3 2-OCOCH3 6-OCOCH3 6-OCOCH3 12-OCH3 5-OH

156.4, 127.9, 135.5, 106.1, 145.8, 19.6, 32.4, 29.9, 33.0, 170.4, 21.0,

C C C CH CH CH3 CH3 CH3 CH3 C CH3

6.53, s

7.41, s

6.90, 7.61, 2.93, 1.34, 1.39, 1.58,

d (1.8) d (1.8) s s s s

1.67, s

33.8, CH2 39.7, C 81.0, C 81.3, CH

5.87, d (6.0)

40.4, C 76.7, C 25.6, CH2

74.7, CH

5.03, d (6.0)

23.5, CH2

7.00, s

39.7, 32.3, 45.1, 37.5,

CH CH C CH2

107.9, 171.2, 36.0, 115.9, 170.5, 11.9, 28.4, 25.8, 17.0, 169.1, 21.1, 170.1, 20.9,

C C CH CH C CH CH3 CH3 CH3 C CH3 C CH3

128.3, 140.9, 50.5, 104.9,

C C C CH

155.9, 131.6, 132.6, 106.3, 146.5, 17.1, 31.0, 25.3, 29.8, 171.4, 21.2,

C C C CH CH CH3 CH3 CH3 CH3 C CH3

173.1, C 22.3, CH3

6.86, 7.66, 2.57, 1.24, 1.24, 1.62,

d (2.4) d (2.4) s s s s

1.86, s

δH (J in Hz)

1.56−1.61, m 1.65−1.68, m 1.25−1.28, m 1.94−1.96, m 1.41, dd (13.2, 4.2) 2.60, td (12.0, 2.4) 1.21−21.25, m 2.08−2.12, m

2.94, q (7.2) 5.80, s 1.17, 1.08, 1.09, 1.14,

d (7.2) s s s

2.16, s 1.97, s

2.21, s 51.0, CH3

3.12, s 3.05, br s

Spectra were recorded in CDCl3. bSpectra were recorded in methanol-d4. C

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positive HRESIMS results (m/z 545.2390 [M + Na]+). The NMR data for this compound (Table 1) were similar to those of 1, except for the absence of one double bond and the presence of an additional acetoxy group. In the HSQC spectrum of 2, H-7 (δH 5.37, t, J = 9.0) and H-8 (δH 2.89, d, J = 3.6) displayed direct correlations with C-7 (δC 75.3) and C8 (δC 43.4), respectively. Taken together with the correlation of the extra acetoxy methyl signal at δH 2.08 with δC 75.3 in the HMBC spectrum, these data fully confirmed the above assumption. On the basis of the ECD rules for α,β-unsaturated carbonyl derivatives, the Cotton effects observed at 325 (Δε −0.6) and 250 nm (Δε 1.7) (Supporting Information, Figure S51) suggested an R absolute configuration for C-8.16,17 Therefore, the structure of 2 was established as 1α,6α,7βtriacetoxy-5α-hydroxy-15-carboxymethyl-12-oxocassa-13(14)diene, and the compound was named caesalpin B. Compound 3, an amorphous, white powder, exhibited the molecular formula C28H42O10 according to HRESIMS (m/z 561.2642 [M + Na]+). A comparison of the NMR data (Table 1) of 3 and 2 indicated that the carboxymethyl group in 2 was replaced by a dimethylacetal moiety in 3. This difference was confirmed by the HMBC correlations of H2-15 (δH 2.67, d, J = 4.8), 16-OCH3 (δH 3.29, s), and 16-OCH3 (δH 3.32, s) with C16 (δC 104.7) and the 1H−1H COSY correlation of H2-15 (δH 2.67, d, J = 4.8) with H-16 (δH 4.32, t, J = 4.8). The similar NOE and ECD spectra (Supporting Information, Figures S12; S18; S51; S52) of 3 and 2 suggested that their absolute configurations were identical. Accordingly, the structure of 3 was identified as 1α,6α,7β-triacetoxy-5α-hydroxy-16,16-dimethoxy-12-oxocassa-13(14)-diene and named caesalpin C. Compound 4, an amorphous, white powder, had a molecular formula of C22H24O4 on the basis of HRESIMS analysis (m/z [M + Na]+ 375.1588). The IR absorptions revealed the presence of hydroxy (3434 nm), unsaturated ketocarbonyl (1733 nm), and aromatic (3010 nm, 1610 nm) functionalities. The 1H NMR spectrum (Table 2) showed four methyl signals at δH 1.34, 1.39, 1.58, and 2.93. Two mutually coupled olefinic protons at δH 6.90 (H-15, d, J = 1.8) and 7.61 (H-16, d, J = 1.8) suggested the presence of a fused furan ring.2 The 13C APT data (Table 2) showed 22 carbon signals, including 10 olefinic and aromatic carbons (δC 104.9, 106.1, 124.7, 127.9, 128.2, 135.5, 145.8, 148.7, 156.4, and 167.3) and one carbonyl carbon (δC 188.0). These data suggested that 4 was a dehydrogenated tetracyclic cassane diterpene with a fused furan ring. The conjugation of the benzene ring with the fused furan ring was confirmed by the HMBC correlations (Figure 3) of H3-11 (δH

acetoxy group was located at C-1 on the basis of the correlations of −OCOCH3 (δH 1.67, s) with C-1 (δC 75.8) and −OCOCH3 (δC 170.4). The NOE enhancements from H1 (δH 5.77, t, J = 3.0) to H3-19 (δH 1.39, s) and H3-20 (δH 1.58, s) indicated that the acetoxy group at C-1 was α-oriented. The ECD spectrum of 4 (Supporting Information, Figure S53) displayed negative Cotton effects at 286 (Δε −1.7) and 325 nm (Δε −0.8) and a positive Cotton effect at 242 (Δε 7.4) nm, corresponding to the n → π* and π → π* transitions of the cross-conjugated dienone. On the basis of the ECD exciton chirality method, C-10 possessed the S absolute configuration.18 Therefore, compound 4 was defined as 1α-acetoxy-7oxo-14-methylvoucapane-5(6),8(14),9(11)-diene and named caesalpin D. Compound 5 exhibited a molecular formula of C24H30O7, as determined by HRESIMS (m/z 453.1907 [M + Na]+). The NMR data (Table 2) revealed that 5 possessed the same partial structure as 4, except that the α,β-unsaturated carbonyl moiety in 4 was absent in 5. In the 1H−1H COSY spectrum, the correlation of H-6 (δH 5.87, d, J = 6.0) with H-7 (δH 5.03, d, J = 6.0) indicated that both C-6 (δC 81.3) and C-7 (δC 74.7) were oxygenated. The correlations of H-6 (δH 5.87) with C-5 (δC 81.0) and C-7 (δC 74.7) and of −OCOCH3 (δH 2.21, s) with C-6 (δC 81.3) in the HMBC spectrum, together with the above molecular formula, suggested that an acetoxy group was present at C-6, whereas hydroxy groups were positioned at C-5 and C7. A (7R, 8S) absolute configuration for this compound was supported by the Cotton effects observed in the ECD spectrum (Supporting Information, Figure S54).18 Thus, compound 5 was identified as 1α,6α-diacetoxy-5α,7β-dihydroxy-14-methylvoucapane-8(14),9(11)-diene and named caesalpin E. Compound 6 was obtained as an amorphous, white powder. HRESIMS analysis showed a quasi-molecular ion peak at m/z 487.2325 [M + Na]+ (calcd 487.2308) in the positive-ion mode. In conjunction with the 1H NMR and 13C APT (Table 2) data, the molecular formula was deduced as C25H36O8. The IR spectrum indicated a hydroxy absorption at 3593 cm−1 and a carbonyl absorption at 1757 cm−1 (lactone). The UV absorption maximum at 212 nm along with the IR absorption band at 1757 cm−1 suggested the presence of an α,βunsaturated butenolide moiety.19 The olefinic proton signal at δH 5.80 (H-15, s) and downfield carbon signals at δC 171.2 (C13), 115.9 (C-15), and 170.5 (C-16) also confirmed the presence of such a moiety, and combined with the four methyl signals at δH 1.08 (H3-18, s), 1.09 (H3-19, s), 1.14 (H3-20, s), and 1.17 (H3-20, d, J = 7.2), which are typical signals for cassane-type diterpenes, indicated that 6 was a tetracyclic cassane diterpene possessing a fused butenolide unit. The carbon-bonded protons of 6 were assigned from the HSQC spectrum. The HMBC correlations (Figure 3) of H-1 (δH 5.21, d, J = 1.8) with −OCOCH3 (δC 169.1), H-2 (δH 5.27, m) with −OCOCH3 (δC 170.1), and 12-OCH3 (δH 3.12, s) with C-12 (δC 107.9) indicated that the acetoxy groups were attached to C-1 and C-2 and that a methoxy group was attached to C-12. The relative configuration of compound 6 was determined from the NOESY spectrum. The NOEs from H3-20 to H-1 (δH 5.21, d, J = 1.8), H-2 (δH 5.27, m), H-8, H-11ax (δH 2.08−2.12, m), and H3-19 (δH 1.09, s); from H3-18 to 5-OH (δH 3.05, br s) and H-9 (δH 2.60, td, J = 12.0, 2.4); and from H-9 to 12-OCH3 (δH 3.12 s) and 14-CH3 (δH 1.17, d, J = 7.2) indicated that rings A and B are in chair conformations with a trans-fused ring junction, thus confirming the relative configurations at C-1, C2, C4, C-5, C10, C-12, and C-14. The high-amplitude ECD

Figure 3. Key HMBC (arrows) correlations for compounds 4 and 6.

7.41, s) with C-12 (δC 156.4) and C-13 (δC 127.9) and of H317 (δH 2.93, s) with C-8 (δC 124.7), C-13 (δC 127.9), C-14 (δC 135.5), and C-15 (δC 106.1). In addition, the existence of an α,β-unsaturated carbonyl moiety conjugated with a benzene ring was supported by the HMBC correlations of H-6 (δH 6.53, s) with C-5 (δC 167.3), C-7 (188.0), and C-8 (δC 124.7) and of H3-17 (δH 2.93, s) with C-7 (δC 188.0). Furthermore, one D

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Table 3. NMR Data (600 MHz in CDCl3) for Compounds 7 and 8 7 position

δC, type

1 2

74.9, CH 22.9, CH2

3

30.1, CH2

4 5 6

38.5, C 78.4, C 35.8, CH2

7 8 9 10 11

68.3, 48.7, 37.4, 43.4, 35.9,

CH CH CH C CH2

12 13 14 15 16 17

107.5, 166.8, 141.1, 115.8, 169.3, 114.9,

C C C CH C CH2

18 19 20 1-OCOCH3

28.2, 25.0, 17.3, 169.9, 21.3,

CH3 CH3 CH3 C CH3

8 δH (J in Hz) 4.94, t (2.4) 1.71−1.73, m 1.93−1.97, m 1.14−1.19, m 1.74−1.78, m

50.2, CH3

4.89, s 1.71−1.76, 1.90−1.96, 1.20−1.22, 1.83−1.87,

38.8, C 79.6, C 77.2, CH

1.42−1.46, m 2.06−2.08, m

5.93, s d (1.8) d (1.8) s s s

2.12, s

6-OCOCH3 12-OCH3 5-OH

δH (J in Hz)

75.2, CH 22.0, CH2 32.5, CH2

1.60−1.63, m 2.16−2.18, m 4.39, m 2.03−2.05, m 2.66, td (12.0, 1.8)

5.38, 5.62, 1.08, 1.09, 1.10,

δC, type

3.11, s 2.91, br s

72.4, 48.4, 36.9, 44.2, 35.7,

CH CH CH C CH2

107.0, 166.3, 139.9, 115.6, 169.7, 116.2,

C C C CH C CH2

30.8, 24.6, 16.9, 169.1, 21.2, 172.4, 22.0, 50.2,

CH3 CH3 CH3 C CH3 C CH3 CH3

m m m m

5.34, d (8.4) 4.28, d (8.4) 2.15−2.19, m 2.70, t (12.0) 1.41−1.48, m 2.08−2.10, m

6.00, s 5.60, 5.40, 1.16, 1.11, 1.19,

d (1.8) d (1.8) s s s

2.11, s 2.21, s 3.11, s 2.98, br s

Table 4. In Vitro Antiproliferative Activity of Compounds 1−12 IC50 (μM) compound 1 2 3 4 5 6 7 8 9 10 11 12 doxorubicinb a

AGS >50 48.3 45.8 6.5 >50 >50 >50 >50 29.1 >50 >50 >50 1.2

± 1.2 ± 0.8 ± 0.5

± 2.2

± 0.02

A549

HepG-2

MCF-7

14.6 ± 2.8a 25.2 ± 0.6 31.8 ± 1.4 24.8 ± 1.3 >50 >50 >50 >50 >50 31.9 ± 3.0 >50 >50 0.74 ± 0.01

4.7 ± 0.7 18.5 ± 0.3 10.6 ± 2.2 >50 >50 >50 34.7 ± 2.6 38.9 ± 1.7 47.2 ± 3.0 >50 >50 >50 0.59 ± 0.13

2.1 ± 0.4 7.9 ± 1.4 15.6 ± 2.3 >50 >50 46.1 ± 2.6 >50 >50 >50 36.1 ± 3.2 41.2 ± 1.0 >50 0.94 ± 0.08

The values presented are the means ± SD of triplicate experiments. bPositive control substance.

Cotton effects (Supporting Information, Figure S55) for the γlactone chromophore at 230 nm (Δ ε −13.2) indicated that C12 possessed the R absolute configuration.20 Therefore, the structure of compound 6 was established as 1α,2α-diacetoxy5α-hydroxy-12α-methoxy-14α-methylcassa-13(15)-en-16,12olide, and the compound was named caesalpin F. Compound 7, an amorphous, white powder, exhibited the molecular formula C23H32O7 through HRESIMS analysis (m/z

[M + Na]+ 443.2030). The 1H NMR and 13C APT spectra (Table 3) were similar to those of 6, except for the absence of one acetoxy and one methyl group and the presence of a second double bond (δC 141.1, 114.9) in 7. In the HSQC and HMBC spectra, the proton signals at δH 5.38 (d, J = 1.8) and 5.62 (d, J = 1.8) were directly linked to the carbon signal at δC 114.9 and showed long-distance correlations with C-14 (δC 141.1) and C-13 (δC 166.8), suggesting that the double bond E

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employed were analytical grade (Beijing Chemical Plant, People’s Republic of China). Plant Material. C. minax seeds were collected in Nanning, Guangxi Province, China, in November 2009 and identified by one of the authors (J.-Q.Y.). A voucher specimen (No. 21650) was deposited at the Guangxi Botanical Garden of Medicinal Plants. Extraction and Isolation. The air-dried and powdered seeds of C. minax (1.0 kg) were extracted with MeOH (3 × 8 L). Removal of the MeOH under reduced pressure yielded the extract (21.3 g). The residue was subjected to CC over silica gel (100−200 mesh) eluting with n-hexane, CHCl3, EtOAc (3 × 500 mL each, 3 × 60 cm), and MeOH (1000 mL). The ether−EtOAc fraction (5.4 g), which showed the most significant activity (IC50 = 21.6 μg/mL), was subjected to CC over silica gel (100−200 mesh, 2 × 60 cm) eluting with CHCl3− MeOH (from 1:0 to 0:1, each 400 mL), to provide three fractions (Fr. A−C). Fr. A (0.8 g) was subjected to CC over silica gel (300−400 mesh, 1 × 60 cm) eluting with petroleum ether−CHCl3 (1:1, 1:1.5, 1:2, 1:2.5, 1;3, 1:4, 1:5, 0:1) followed by CHCl3−MeOH (100:1, 80:1, 40:1), yielding compounds 4 (5.7 mg, 0.000 57%) and 5 (4.2 mg, 0.000 42%). Fr. B (2.6 g) was purified using a Sephadex LH-20 column (2 × 135 cm) eluting with CHCl3−MeOH (40:60) and then subjected to chromatography using ODS MPLC elution with MeOH−H2O (60:40; 80:20; 100:0), to yield three fractions (Fr. B1−B3). Fr. B1 (0.6 g) was subjected to CC on silica gel (300−400 mesh) eluting with petroleum CHCl3−MeOH (100:1; 80:1; 60:1; 50:1; 40:1; 30:1; 20:1; 0:1), to afford compounds 1 (5.3 mg, 0.000 53%) and 6 (3.4 mg, 0.000 34%). Fr. B2 (1.1 g) was separated via RP flash chromatography over C18 silica gel eluting with MeOH−H2O (50:50; 80:20) to give two fractions (Fr. B21−2). Fr. B21−2 were purified through semipreparative HPLC elution using a MeOH−H2O gradient solvent system (from 40% to 100% MeOH) and a Kromasil RP-18 column. Finally, compounds 2 (1.5 mg, 0.000 15%) and 3 (1.2 mg, 0.000 12%) were obtained from Fr. B21 at tR 22.4 and 28.6 min, respectively, using a MeOH−H2O (60:40) solvent system. For Fr. B22, the application of a MeOH−H2O (58:42) solvent system yielded compound 12 (4.2 mg, 0.000 42%) at tR 32.2 min. Fr. B3 (0.5 g) was subjected to CC over silica gel (300−400 mesh, 1 × 60 cm) eluting with CHCl3−MeOH (100:0, 100:1, 80:1, 60:1, 50:1, 40:1, 20:1) to afford compounds 9 (8.2 mg, 0.000 82%) and 10 (7.6 mg, 0.000 76%). Fr. C (1.2 g) was separated through Sephadex LH-20 elution with CHCl3−MeOH (40:60), and then subjected to CC over silica gel (100−200 mesh) eluting with CHCl3−MeOH (40:1; 10:1; 0:100), to give three fractions (Fr. C1−C3). Fr. C1 and C2 were separated via semipreparative HPLC with a MeOH−H2O gradient solvent system (from 40% to 100% MeOH) using a Kromasil RP-18 column. Finally, compounds 7 (6.5 mg, 0.000 65%) and 8 (1.8 mg, 0.000 18%) were obtained at tR 27.5 and 30.4 min, respectively, using a MeOH−H2O (62:38) system, and compound 11 was obtained at tR 33.5 min using a MeOH−H2O (56:44) solvent system from Fr. C3. Caesalpin A (1): amorphous, white powder; [α]20D +27.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 285 (3.34), 203 (2.14) nm; ECD (CDCl3) 329 (Δε +3.9), 284 (Δε −18.3) nm; IR (film) νmax 3549 (OH), 1733 (CO) cm−1; 1H NMR and 13C APT see Table 1; HRESIMS m/z 485.2133 (calcd for C25H34O8 Na [M + Na]+, 485.2151). Caesalpin B (2): amorphous, white powder; [α]20D +26.2 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 239 (3.01), 207 (2.53) nm; ECD (MeOH) 325 (Δε −0.6), 250 (Δε +1.7) nm; IR (film) νmax 3549 (OH), 1740 (CO) cm−1; 1H NMR and 13C APT see Table 1; HRESIMS m/z 545.2390 (calcd for C27H38O10 Na [M + Na]+, 545.2363). Caesalpin C (3): amorphous, white powder; [α]20D +12.5 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 244 (3.26), 204 (2.37) nm; ECD (MeOH) 327 (Δε −0.7), 250 (Δε +1.9) nm; IR (film) νmax 3545 (OH), 1744 (CO) cm−1; 1H NMR and 13C APT see Table 1; HRESIMS m/z 561.2642 (calcd for C28H42O10 Na [M + Na]+, 561.2676). Caesalpin D (4): amorphous, white powder; [α]20D +74.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 285 (3.38), 243 (4.72), 208 (4.16) nm; ECD (MeOH) 325 (Δε −0.8), 286 (Δε −1.7), 242 (Δε +7.5)

was between C-14 and C-17. The typical upfield-shifted C-2 (δC 22.9) and downfield-shifted C-7 (δC 68.3), together with the absence of an HMBC correlation between H-7 (δH 4.39, m) and an ester carbonyl, indicated that C-7 was hydroxylated. The ECD spectrum of 7 (Supporting Information, Figure S56) displayed a positive Cotton effect at 260 nm (Δε 10.2) and a negative effect at 219 nm (Δε −5.8), confirming the (8R, 12R) absolute configuration for 7 based on the exciton chirality method and the δ-lactone rules.16,21,22 Accordingly, the structure of 7 was established as 1α-acetoxy-5α,7β-dihydroxy12α-methoxycassa-14(17),13(15)-dien-16,12-olide and named caesalpin G. Compound 8 exhibited an [M + Na]+ ion peak at m/z 501.2125 using HRESIMS, corresponding to the molecular formula C25H34O9. The obtained 1H NMR and 13C APT signals (Table 3) were closely related to those of 7, with the exception of an extra acetoxy group. The downfield-shifted C-6 (δC 77.2) of 8, in contrast to the C-6 methylene (δC 35.8) of 7, as well as the HMBC correlation of H-6 (δH 5.34, d, J = 8.4) with −OCOCH3 (δC 172.4), suggested that the additional acetoxy group was at C-6. The similar NOE and ECD spectra (Supporting Information, Figures S42; S48; S56; S57) of 8 and 7 suggested that their absolute configurations were identical. Therefore, the structure of 8 was identified as 1α,6α-diacetoxy5α,7β-dihydroxy-12α-methoxycassa-14(17),13(15)-dien-16,12olide, and the compound was named caesalpin H. Compounds 1−12 were tested for their cytotoxicity against four human cancer cell lines (AGS, A549, HepG-2, and MCF7) using the MTT method. The obtained IC50 values were in the range 2.1−48.3 μM, and doxorubicin was used as a positive control (Table 4). Compound 1 showed significant activity against HepG-2 (IC50 4.7 μM) and MCF-7 (IC50 2.1 μM) cells, whereas compounds 2 and 4 exhibited selective cytotoxic activities against MCF-7 (IC50 7.9 μM) and AGS (IC50 6.5 μM) cells. Compound 3 displayed moderate activity against HepG-2 (IC50 10.6 μM) and MCF-7 (IC50 15.6 μM), whereas the other compounds showed only low activity or were inactive against all of the tested cell lines (IC50 > 20 μM). On the basis of these biological results, we hypothesize that the presence of an α,βunsaturated carbonyl moiety in the tricyclic cassane diterpenes and the tetracyclic framework with a fused furan ring in the cassane diterpenes may enhance the cytotoxic activities of the compounds.



EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation data were obtained using a Perkin-Elmer 341 digital polarimeter. UV data were recorded with a Shimadzu UV2550 spectrometer. ECD spectra were obtained using a JASCO J-815 spectropolarimeter. IR data were recorded using an FTIR-8400S spectrometer. NMR spectra were obtained using a Bruker AV III 600 NMR spectrometer (the chemical shift values are presented as the δ values with TMS as an internal standard). HRESIMS was performed using an LTQ-Obitrap XL spectrometer. HPLC separation was conducted using a Lumiere K1001 pump, a Lumiere K-2501 single λ absorbance detector, and a Kromasil (250 × 10 mm) semipreparative column packed with C18 (5 μm, AkzoNobel, Amsterdam, Holland). Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel (CHP 20P, 75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), C-18 reversed-phase silica gel (40−63 μm, Merck, Darmstadt, Germany), and silica gel (100−200 and 300−400 mesh, Qingdao Marine Chemical Plant, Qingdao, People’s Republic of China) were used for CC, and precoated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, People’s Republic of China) were used for TLC. All solvents F

dx.doi.org/10.1021/np300918q | J. Nat. Prod. XXXX, XXX, XXX−XXX

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nm; IR (film) νmax 3434 (OH), 1733 (CO), 3010 (CH), 1610 (CC) cm−1; 1H NMR and 13C APT see Table 2; HRESIMS m/z 375.1588 (calcd for C22H24O4 Na [M + Na]+, 375.1572). Caesalpin E (5): amorphous, white powder; [α]20D −2.1 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 290 (2.05), 279 (2.86), 251 (3.57), 216 (4.94) nm; ECD (DMSO) 326 (Δε −1.7), 277 (Δε +2.3) nm; IR (film) νmax 3430 (OH), 1738 (CO), 3100 (CH), 1608 (CC) cm−1; 1H NMR and 13C APT see Table 2; HRESIMS m/z 453.1907 (calcd for C24H30O7 Na [M + Na]+, 453.1889). Caesalpin F (6): amorphous, white powder; [α]20D −41.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (4.75) nm; ECD (MeOH) 230 (Δε −13.2) nm; IR (film) νmax 3593 (OH), 1757 (CO) cm−1; 1 H NMR and 13C APT see Table 2; HRESIMS m/z 487.2325 (calcd for C25H36O8 Na [M + Na]+, 487.2308). Caesalpin G (7): amorphous, white powder; [α]20D +27.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (4.37) nm; ECD (MeOH) 260 (Δε +10.2), 219 (Δε −5.8) nm; IR (film) νmax 3508 (OH), 1761 (CO) cm−1; 1H NMR and 13C APT see Table 3; HRESIMS m/z 443.2030 (calcd for C23H32O7 Na [M + Na]+, 443.2046). Caesalpin H (8): amorphous, white powder; [α]20D +39.1 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 218 (4.98) nm; ECD (MeOH) 260 (Δε +10.3), 216 (Δε −6.0) nm; IR (film) νmax 3510 (OH), 1740 (CO) cm−1; 1H NMR and 13C APT see Table 3; HRESIMS m/z 501.2125 (calcd for C25H34O9 Na [M + Na]+, 501.2101). Rh-Complex of 1. Compound 1 (0.5 mg) was dissolved in a dry solution of [Rh2(OCOCF3)4] (1.0 mg) in CDCl3 (300 μL). The first ECD spectrum was recorded immediately after mixing, and the time was monitored until stabilization of the spectrum. The inherent ECD was then subtracted, and the observed sign of the E band at 350 nm in the induced ECD spectrum was correlated with the absolute configuration of the C-5 tertiary alcohol moiety.14,15 Cytotoxicity Assay. The cytotoxicity of compounds 1−12 was assessed via the MTT method using the AGS, A549, HepG-2, and MCF-7 cancer cell lines. The cells were grown in DMEM supplemented with 10% fetal bovine serum and cultured at a density of 6 × 104 cells/mL in a 96-well microtiter plate. Five different concentrations of each compound in DMSO were subsequently added to the wells. Each concentration was tested in triplicate. After incubation under 5% CO2 at 37 °C for 48 h, 10 μL of MTT (4 mg/ mL) was added to each well, and the cells were incubated for another 4 h. Then, the liquid in each well was removed, and DMSO (200 μL) was added. The absorbance was recorded using a microplate reader at a wavelength of 570 nm.



ments Transformation Project (No. 1298009-22), and Nanning Science Research and Technology Development Program (No. 201102088C).



ASSOCIATED CONTENT

* Supporting Information S

1D and 2D NMR, and CD spectra of compounds 1−8 (Figures S1−S57) are available free of charge via the Internet at http:// pubs.acs.org.



REFERENCES

(1) He, S. Z. Chin. Tradit. Herb. Drugs 2000, 31, 225−226. (2) Pranithanchai, W.; Karalai, C.; Ponglimanont, C.; Subhadhirasakul, S.; Chantrapromma, K. Phytochemistry 2009, 70, 300−304. (3) Huang, M. J.; Chen, Y. D.; Wei, D. Z. Mod. Chin. Med. 2010, 12, 11−14. (4) Hou, Y.; Cao, S.; Brodie, P.; Miller, J. S.; Birkinshaw, C.; Ratovoson, F.; Rakotondrajaona, R.; Andriantsiferana, R.; Rasamison, V. E.; Kingston, D. G. I. J. Nat. Prod. 2008, 71, 150−152. (5) Kalauni, S. K.; Awale, S.; Tezuka, Y.; Banskota, A. H.; Linn, T. Z.; Asih, P. B. S.; Syafruddin, D.; Kadota, S. Biol. Pharm. Bull. 2006, 29, 1050−1052. (6) Dickson, R. A.; Houghton, P. J.; Hylands, P. J. Phytochemistry 2007, 68, 1436−1441. (7) Jabbar, A.; Zaman, M. A.; Iqbal, Z.; Yaseen, M.; Shamim, A. J. Ethnopharmacol. 2007, 114, 86−91. (8) Yadav, P. P.; Maurya, R.; Sarkar, J.; Arora, A.; Kanojiya, S.; Sinha, S.; Srivastava, M. N.; Raghubir, R. Phytochemistry 2009, 70, 256−261. (9) Dimayuga, R. E.; Espinoza, J. A.; Garcia, A.; Delgado, G.; Salinas, G. M. M.; Fernandez, S. S. Planta Med. 2006, 72, 757−761. (10) Wu, Z. H.; Wang, L. B.; Gao, H. Y.; Sun, B. H.; Huang, J.; Wu, L. J. China J. Chin. Mat. Med. 2008, 10, 1145−1147. (11) Wu, Z. H.; Wang, Y. Y.; Huang, J.; Sun, B. H.; Wu, L. J. Asian J. Tradit. Med. 2007, 2, 135−139. (12) Kinoshita, T. Chem. Pharm. Bull. 2000, 48, 1375−1377. (13) Yin, Y. H.; Ma, L.; Hu, L. H. Helv. Chim. Acta 2008, 91, 972− 977. (14) Frelek, J.; Szczepek, W. J. Tetrahedron: Asymmetry 1999, 10, 1507−1520. (15) Gerards, M.; Snatzke, G. Tetrahedron Asymmetry 1990, 1, 221− 236. (16) Ye, X. L. Stereochemistry; Beijing University Express: Beijing, 1999; Vol. 2, Chapter 4, pp 257−265. (17) Lin, L. C.; Shen, C. C.; Wu, Y. C.; Tsai, T.-H. J. Nat. Prod. 2006, 69, 842−844. (18) Lin, S.; Zhang, Y. L.; Liu, M. T.; Yang, S.; Gan, M. L.; Zi, J. C.; Song, W. X.; Fan, X. N.; Wang, S. J.; Liu, Y.; Yang, Y. C.; Chen, X. G.; Guo, Y.; Wang, W. J.; Shi, J. G. J. Nat. Prod. 2010, 73, 1914−1921. (19) Kinoshita, T.; Haga, Y.; Narimatsu, S.; Shimada, M.; Goda, Y. Chem. Pharm. Bull. 2005, 53, 717−720. (20) Matsuno, Y.; Deguchi, J.; Hirasawa, Y.; Ohyama, K.; Toyoda, H.; Hirobe, C.; Ekasari, W.; Widyawaruyanti, A.; Zaini, N. C.; Morita, H. Bioorg. Med. Chem. Lett. 2008, 18, 3774−3777. (21) Wolf, H. Tetrahedron Lett. 1966, 7, 5151−5156. (22) Beecham, A. F. Tetrahedron Lett. 1968, 9, 2355−2360.

AUTHOR INFORMATION

Corresponding Author

*Tel: +86-010-57833296. Fax: +86-010-57833296. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This study was financially supported by the Technological Large Platform for Comprehensive Research and Development of New Drugs in the Eleventh Five-Year ‘‘Significant New Drugs Created’’ Science and Technology Major Projects (No. 2009ZX09301-003), National Natural Science Foundation of China (No. 30973626), Innovation Capacity-Building in Guangxi Science and Technology Agency (No. 10100027-3), National Science and Technology Support Program (No. 2012BA127B06), Guangxi Science and Technology AchieveG

dx.doi.org/10.1021/np300918q | J. Nat. Prod. XXXX, XXX, XXX−XXX