Caesalminaxins A–L, Cassane Diterpenoids from the Seeds of

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Caesalminaxins A−L, Cassane Diterpenoids from the Seeds of Caesalpinia minax Yong Zheng, Shu-Wei Zhang, Hai-Jian Cong, Yu-Jie Huang, and Li-Jiang Xuan* State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Fourteen new cassane diterpenoids, caesalminaxins A−L (1−14), and three known compounds were isolated from the seeds of Caesalpinia minax. Among the new diterpenoids, compounds 3 and 4 possess a rare spiro C/D ring system. The C-16 epimeric mixture 1/2 has an unprecedented carbon skeleton, presumably derived from 3 by cleavage of the C-13−C-14 bond. Compound 5 is the first example of a cassane diterpenoid with a spiro A/B ring system. The structures of the compounds were elucidated on the basis of 1D and 2D NMR analysis, and the absolute configurations of 3, 4, 9, and 11 were determined by singlecrystal X-ray crystallography. Biosynthesis pathways for 1/2, 3, and 5 are postulated. Compounds 4, 8, and the known bonducellpin D exhibited moderate activity against four tested human cancer cell lines, HepG-2, K562, HeLa, and Du145.

C

bonducellpin D exhibited moderate activity against four tested human cancer cell lines, HepG-2, K562, HeLa, and Du145.

assane diterpenoids, which are characterized by a molecular skeleton constructed of three fused cyclohexane rings and a furan ring or an α,β-butenolide moiety, are mainly distributed in Caesalpinia plants, such as Caesalpinia sappan Linn.,1 C. mimosoides Lamk.,2 C. pulcherrima,3−7 C. crista,8−11 and C. bonduc.12,13 Some of these diterpenoids have been found to exhibit antiviral,3,16 anti-inflammatory,6 antimalarial,11 and antiproliferative activities.5 C. minax Hance (Fabaceae) is a shrub widely growing in the tropical and subtropical regions of Southeast Asia. The seeds of this plant, which are called “Kushilian” in Chinese, have been used in Chinese folk medicine for the treatment of common cold, fever, and dysentery.14 Previous phytochemical studies afforded a series of cassane diterpenoids, caesalmins A−G,15,16 spirocaesalmin,17 macrocaesalmin,18 neocaesalpins J−N,19 neocaesalpins S−U,20 neocaesalpins AA−AE,21 and sucutiniranes G−I.22 In the course of our ongoing search for bioactive diterpenoids with unique structures, we investigated the chemical constituents of the seeds of C. minax. This investigation led to the isolation of 14 new cassane diterpenoids, caesalminaxins A−L (1−14), and three known compounds. Among the new diterpenoids, compounds 3 and 4 possess a rare spiro C/D ring system. The C-16 epimeric mixture 1/2 has an unprecedented carbon skeleton, presumably derived from 3 by cleavage of the C-13−C-14 bond. Compound 5 is the first example of a cassane diterpenoid with a spiro A/B ring system. The structures and absolute configurations of 3, 4, 9, and 11 were elucidated by singlecrystal X-ray crystallography. Biosynthesis pathways for 1/2, 3, and 5 are postulated. The new compounds are an important addition to the structurally diverse and complex class of cassane diterpenoids. Compounds 4, 8, and the known compound © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compounds 1/2 were obtained as a 1:1 mixture of C-16 epimers. The molecular formula of 1/2 was determined to be C26H36O11 by the HRESIMS ion at m/z 547.2159 [M + Na]+, indicating nine indices of hydrogen deficiency. The IR spectrum showed the presence of hydroxy (3470 cm−1) and carbonyl (1754, 1729 cm−1) groups. The 1H NMR spectrum (Table 1) exhibited signals of an olefinic proton at δH 7.25/7.32 (s, H-15); four oxymethines at δH 5.16 (br s, H-1), 5.82/5.83 (d, J = 8.7 Hz, H-6), 6.16/6.24 (dd, J = 11.5, 8.7 Hz, H-7), and Received: July 10, 2013 Published: December 4, 2013 2210

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Table 1. 1H NMR (500 MHz) Data for Compounds 1−7 (δH in ppm, J in Hz) position 1 2 3 6 7 8 9 11 14 15 16 17 18 19 20 1-OAc 6-OAc 7-OAc 5-OH

1/2a

3b

4b

5.16, br s 1.78, m 1.91, m 1.02, br d (14.2) 2.11, m 5.82/5.83, d (8.7) 6.16/6.24, dd (11.5, 8.7) 3.21, dd (11.5, 11.5) 3.76/3.77, m 2.32, m 2.55/2.56, m

4.73, br s 1.61, m 1.98, m 1.11, m 1.82, td (14.5, 3.9) 5.51, br s n. d.d 2.04, m 3.32, m 1.37, t (11.8) 1.67, dd (11.8, 5.6)

4.77, br s 1.61, m 2.00, m 1.11, m 1.85, td (14.5, 3.9) 5.42, br s n. d.d 1.90, m 3.43, td (13.2, 4.6) 1.35, t (12.5) 1.58, m

7.25/7.32, 6.40/6.48, 2.30/2.41, 1.33, s 1.15, s 1.15, s 2.02, s 1.99, s 2.21, s 4.70/4.80,

3.51, 5.58, 1.44, 1.07, 1.12, 1.18, 2.12, 2.01, 2.07, 3.06, 3.73,

3.41, 5.53, 1.49, 1.09, 1.10, 1.15, 2.12, 1.96, 2.06, 3.21,

s br s s

s

d (2.2) d (2.2) s s s s s s s s s (14-OH)

1′ 2′ a

d (2.3) d (2.3) s s s s s s s s

5b 10.03, s 5.79, d (6.3) 1.73, d (13.1) 2.57, dd (13.1, 6.3) 5.30, d (9.6) 4.59, dd (11.4, 9.6) 2.14, m 2.62, m 2.30, m 2.47, m 3.26 d (13.1) 6.56, d (1.9) 7.28, d (1.9) 1.11, s 1.20, s 1.26, s 2.16, s

6/7c 4.71, br s 1.66, m 1.94, m 1.04, m 1.87, m 5.46/5.48, d (9.1) 5.72/5.76, dd (9.1, 9.1) 2.93/3.00, dd (12.2, 10.3) 2.47, dt (13.7, 3.7) 2.11, m 2.66/2.72, t (13.7) 3.80, d (4.2)/3.88, s 5.38, d (4.2)/5.19, s 1.24/1.27, s 1.12/1.13, s 1.12/1.14, s 1.26, s 2.07, s 2.04, s 1.98/2.01, s

3.16, s (2-OH) 2.96, m 3.28, m 1.00, t (6.9)

Recorded in pyridine-d5. bRecorded in CDCl3. cRecorded in methanol-d4. dNot detected.

6.40/6.48 (br s, H-16); two methines at δH 3.21 (dd, J = 11.5, 11.5 Hz, H-8) and 3.76/3.77 (m, H-9); one acetyl methyl at δH 2.30/2.41 (s, H-17); three acetoxy methyls; and three methyl singlets at δH 1.33 (H-18) and 1.15 (H-19 and H-20). The 13C NMR spectrum (Table 2) revealed 26 carbon signals, consisting of seven methyls, three methylenes, seven methines (one olefinic and four oxygenated), and nine quaternary carbons (one ketocarbonyl, four ester carbonyls, one olefinic, and one oxygenated). These data indicated that 1 was a tricyclic diterpenoid with three acetoxy substituents. The COSY correlations of H-1/H-2/H-3 and H-6/H-7/H-8/H-9/H-11, together with the HMBC correlations of H-1/C-3, C-5; Me-18, Me-19/C-4, C-5; Me-20/C-1, C-9, C-10; H-6/C-5; H-7/C-14; H-8/C-6, C-10, C-14; and Me-17/C-14 (Figure 1) enabled the construction of rings A and B. Ring C was deduced to be a γhydroxy-α,β-unsaturated γ-lactone moiety by the HMBC correlations of H-15/C-12, C-16 and the COSY cross-peaks of H-15/H-16. The COSY correlations of H-9/H-11, combined with the HMBC correlations of H-8/C-11 and H-11/C-12, C13, C-15 showed that ring B was connected to ring C through C-11. Thus, the gross structures of 1/2 were determined. In the ROESY spectrum, the correlations of H-1/Me-20, Me20/H-8, H-8/H-6, and H-6/Me-19 indicated the β-orientations of H-1, H-6, H-8, H-19, and H-20, while the cross-peaks of Me18/5-OH, 5-OH/H-9, and H-9/H-7 revealed the α-orientations of these protons. Therefore, the structures of 1/2 were determined and named caesalminaxins A1/A2. Compound 3 was obtained as colorless needles, and its molecular formula was determined to be C26H36O11 by the HRESIMS ion at m/z 547.2156 [M + Na]+. Only 35 proton and 25 carbon signals were observed in the 1H and 13C NMR spectra (Tables 1 and 2), respectively, including seven methyls

(three acetoxy methyls), three methylenes, six methines (four oxygenated), nine quaternary carbons (four ester carbonyls and two oxygenated), and two hydroxy groups at δH 3.06 (s, 5-OH) and 3.73 (s, 14-OH). One proton (H-7) and one carbon (C-7) signal were not detected in several different deuterated solvents. The 1D NMR data of 3 showed similarities to those of spirocaesalmin.17 Compared with spirocaesalmin, the C-17 methoxycarbonyl group was replaced by a methyl group [δH 1.44 (s); δC 26.0] in 3, as deduced from the HMBC correlations of Me-17/C-8, C-13, and C-14. The downfield shift of C-14 (δC 80.7) and the HMBC correlations of 14-OH/ C-8, C-13, and C-14 implied that a hydroxy group was located at C-14. Furthermore, an acetoxy group was positioned at C-6 based on the downfield shift of H-6 [δH 5.51 (br s)]. The X-ray diffraction data of 3 not only confirmed the gross structure but also defined the absolute configuration (Figure 2). Therefore, 3 was named caesalminaxin B. The rare skeleton of compound 3 is proposed to be rearranged from a cassane diterpenoid through oxidation of the double bond in the furan ring and migration of the C-11−C-12 bond to C-13. In addition, compounds 1/2 represent an unprecedented cassane diterpene skeleton, presumaly generated from 3 through cleavage of the C-13−C-14 bond. The proposed biosynthesis pathways of 1/2 and 3 are shown in Scheme 1. Compound 4 had the molecular formula C28H40O11 by HRESIMS. The 1D NMR data (Tables 1 and 2) of 4 were similar to those of 3, except for the presence of an ethoxy rather than a hydroxy group at C-14, which was confirmed by the HMBC correlation of H-1′ [δH 2.96 (m) and 3.28 (m)]/C-14. The absolute configuration of 4 was determined by X-ray crystallographic analysis (Figure 2), and the compound was named caesalminaxin C. 2211

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Table 2. 13C NMR (125 MHz) Data for Compounds 1−12 (δC in ppm) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1-OAc 6-OAc 7-OAc 1′ 2′ 12-OMe 14-OMe 16-OMe a

1/2a

3b

4b

5b

6/7c

8b

9b

10b

11b

12b

75.1 22.3/22.6 33.0 39.4 80.3 74.9/75.0 74.5/74.8 57.5/57.8 36.4/36.9 46.8/46.9 25.5/25.7 172.2/172.3 135.7/135.8 209.8 147.3/147.8 98.3/98.5 31.3/31.6 31.2 24.8 17.4/17.6 170.7 21.8 170.7 21.0 170.4/170.5 21.9

75.5 23.0 32.6 38.7 80.4 75.3 n. d.d 51.1 39.3 43.1 32.5 177.1 58.4 80.7 56.7 76.7 26.0 30.3 24.2 16.9 169.7 21.3 170.7 21.7 170.4 21.2

75.5 23.1 32.7 38.7 80.3 75.8 n. d.d 53.4 38.9 43.0 34.5 174.5 58.0 85.3 57.4 76.8 23.6 30.5 24.2 16.3 169.3 21.0 171.0 21.8 169.3 21.0 59.3 15.0

201.1 98.6 51.0 45.6 95.6 74.2 82.2 44.5 32.7 56.8 21.7 151.5 113.3 41.6 107.7 142.2 172.7 29.8 24.9 9.8

76.3 23.3 33.2 39.4 79.9 77.0/76.2 74.4/74.2 45.4/43.6 36.5/35.8 45.9/46.0 41.5 210.5 88.3/87.7 91.2/93.4 79.0/88.6 99.2/106.3 14.9 30.9 24.9 17.2 171.6 21.0 172.1 21.8 172.6/173.0 21.7

75.5 21.8 32.4 38.6 79.3 75.1 73.1 50.9 34.6 44.5 39.1 111.6 150.9 74.8 122.6 107.5 20.7 30.6 24.6 16.8 169.0 21.1 170.5 21.8 170.8 21.6

75.5 22.5 32.4 38.6 79.2 75.2 72.1 45.0 34.1 44.6 38.5 111.8 146.4 79.9 123.2 107.7 19.0 30.6 24.5 16.8 169.0 21.2 170.5 21.8 170.2 21.2

75.6 21.8 32.4 38.6 79.3 75.2 73.4 50.5 34.4 44.5 39.0 109.1 150.5 74.6 121.6 105.6 21.3 30.6 24.6 16.8 169.0 21.2 170.5 21.6 170.8 21.6

75.5 22.5 32.4 38.9 79.1 75.2 72.0 47.9 33.0 44.5 38.6 109.5 147.2 79.8 121.7 105.5 17.5 30.6 24.5 16.7 169.3 21.2 170.4 21.7 170.4 21.3

75.3 22.5 32.4 38.6 79.5 77.2 72.2 48.2 37.5 44.7 37.6 109.9 146.0 141.0 121.7 106.3 112.5 29.8 24.6 16.8 169.3 21.2 172.2 22.1

49.1

49.0 51.5 55.2

50.7

50.5 51.3 56.5

49.9

169.0 21.8

55.5

56.6

56.7

Recorded in pyridine-d5. bRecorded in CDCl3. cRecorded in methanol-d4. dNot detected.

hemiacetal methine (δC 98.6) in 5. This deduction was supported by the COSY correlations of H-2/H-3, along with the HMBC correlations of H-2/C-4; H-1/C-10; and Me-20/C1 (Figure 3). The HMBC correlations of H-2/C-4, C-5 and H3/C-5 indicated that C-2 was linked to C-5 through an oxygen atom. Therefore, the gross structure of 5 was established. The ROESY correlations of Me-20/H-6, H-6/H-8, and H-8/Me-20 suggested the β-orientations of these protons, while the crosspeaks of H-7/H-9, H-9/H-1, and H-7/H-14 suggested the αorientations of H-7, H-9, H-14, and H-1. The relative configuration of C-2 was deduced from the correlations of 2OH/H-1 and H-2/H-7 shown in Figure 3. It is interesting that compound 5, named caesalminaxin D, was obtained as a single product, while 1/2 were isolated as a mixture, suggesting that the α-orientation of 2-OH in 5 was stabilized by hydrogen bond formation with the formyl group. Compound 5 is most likely derived from caesalpinin D via cleavage of the C-1−C-2 bond, followed by formation of the spiro A/B ring system. The proposed biosynthesis pathway for 5 is shown in Scheme 2. Compounds 6/7 were obtained as a mixture of C-16 epimers, and the molecular formula was determined to be C26H38O12 by HRESIMS. The IR spectrum indicated the presence of hydroxy (3370 cm−1) and carbonyl groups (1745, 1733, 1702 cm−1). The 1D NMR data of 6/7 (Tables 1 and 2) were similar to those of magnicaesalpin,23 except for the signals due to the lactone carbonyl at C-16 and methylene at C-15 being replaced by signals of two oxymethines [δH 5.38 (d, J =

Figure 1. Selected COSY () and HMBC (H→C) correlations of 1/ 2.

The molecular formula of compound 5 was determined to be C22H26O8 from HRESIMS. The 1H NMR spectrum (Table 1) indicated the presence of a formyl group at δH 10.03 (s, H-1); two protons of a 1,2-disubstituted furan ring at δH 6.56 (d, J = 1.9 Hz, H-15) and 7.28 (d, J = 1.9 Hz, H-16); three oxymethines at δH 4.59 (dd, J = 11.4, 9.6 Hz, H-7), 5.30 (d, J = 9.6 Hz, H-6), and 5.79 (d, J = 6.3 Hz, H-2); three methines at δH 2.14 (m, H-8), 2.62 (m, H-9), and 3.26 (d, J = 13.1 Hz, H14); an acetoxy methyl; three methyl singlets at δH 1.11 (H18), 1.20 (H-19), and 1.26 (H-20); and a hydroxy group at δH 3.16 (s, 2-OH). The 13C NMR spectrum (Table 2) displayed 22 carbon signals including four methyls, two methylenes, nine methines (one formyl, two olefinic, and three oxygenated), and seven quaternary carbons (two ester carbonyls, two olefinic, and one oxygenated). The 1D NMR data of 5 were similar to those of caesalpinin D.10 Compared with caesalpinin D, the signals of the C-1 oxymethine and C-2 methylene were replaced by a C-1 formyl group (δC 201.1) and a C-2 2212

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Figure 3. Selected COSY (), HMBC (H→C), and ROESY (H↔H) correlations of 5 (R = OAc).

configuration of C-13 and C-15. Therefore, X-ray diffraction was performed on a single crystal of 7 obtained from MeOH. X-ray crystallographic analysis (Figure 4) determined the relative configuration of 7, and the 6/7 mixture was named caesalminaxins E1/E2. Compound 8 was obtained as colorless crystals, with a molecular formula of C28H42O11 by HRESIMS. The 1H NMR spectrum (Table 3) showed the presence of an olefinic proton at δH 5.82 (s, H-15); four oxymethines at δH 5.72 (s, H-16), 5.61 (dd, J = 8.6, 8.6 Hz, H-7), 5.44 (d, J = 8.6 Hz, H-6), and 4.84 (br s, H-1); two methoxy groups at δH 3.06 (s, 12-OMe) and 3.42 (s, 16-OMe); three acetoxy methyls; four methyl singlets at δH 1.11 (H-18 and H-19), 1.13 (H-20), and 1.47 (H20); and a hydroxy group at δH 2.92 (d, J = 1.3 Hz, 5-OH). The 13 C NMR spectrum (Table 2) displayed 28 carbon signals, due to nine methyls, three methylenes, seven methines (one olefinic and four oxygenated), and nine quaternary carbons (three ester carbonyls, one olefinic, and three oxygenated). The 1D NMR data of 8 closely resembled those of neocaesalpin L.6 The major difference was that C-16 of 8 was acetalic (δC 107.5) rather than the lactone carbonyl in neocaesalpin L, which was supported by the COSY correlations of H-15/H-16, along with the HMBC correlations of H-16/C-13; H-15/C-12, C-16; and 16-OMe/C-16 (Figure 5). Furthermore, two additional methoxy groups appeared at C-12 and C-16, judging from the HMBC correlations of 12-OMe/C-12 and 16-OMe/C-16, respectively. The ROESY correlations of H-1/Me-20, H-20/H6, and Me-20/H-8 indicated that H-1, H-20, H-6, and H-8 were in β-orientations, while the ROESY cross-peaks of 5-OH/H-9,

Figure 2. X-ray crystallographic structures of 3 and 4 (Cu Kα).

4.2 Hz)/5.19 (s); δC 99.2/106.3 and δH 3.80 (d, J = 4.2 Hz)/ 3.88 (s); δC 79.0/88.6]. The HMBC correlations of H-15, H16/C-14 confirmed the location of the oxygenated methines at C-15 and C-16, respectively. The coupling constant of H-15 and H-16 appeared as two doublets (J = 4.2 Hz) in 6 but as singlets in 7, suggesting that 6/7 were C-16 epimers. It was not possible to use ROESY data to determine the relative Scheme 1. Proposed Biosynthesis Pathways for 1/2 and 3

2213

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Scheme 2. Proposed Biosynthesis Pathway for 5

Figure 5. Selected COSY (), HMBC (H→C), and ROESY (H↔H) correlations of 8 (R = OAc). Figure 4. X-ray crystallographic structure of 7 (Mo Kα).

3) of 9 were similar to those of 8, except for the presence of an O-methyl group at C-14, which was supported by the correlations of 14-OMe/C-14. The ROESY spectrum showed that the relative configuration of 9 was identical to that of 8. The absolute configuration of 9, named caesalminaxin G, was determined by X-ray crystallographic analysis (Figure 6).

H-9/Me-17, Me-17/H-7, Me-17/12-OMe, and 12-OMe/H-16 showed that these protons were α-oriented. Accordingly, 8 was named caesalminaxin F. The molecular formula of compound 9 was determined to be C29H44O11 from HRESIMS. The 1D NMR data (Tables 2 and

Table 3. 1H NMR (500 MHz) Data for Compounds 8−12 in CDCl3 (δH in ppm, J in Hz) position 1 2 3 6 7 8 9 11 15 16 17 18 19 20 1-OAc 6-OAc 7-OAc 12-OMe 14-OMe 16-OMe 5-OH

8

9

10

11

4.84, 1.69, 1.85, 1.07, 1.71, 5.44, 5.61, 1.89, 2.63, 1.35, 1.85, 5.82, 5.72, 1.47,

br s m m m m d (8.6) dd (8.6, 8.6) m td (12.6, 2.3) t (12.6) m s s s

4.85, 1.71, 1.90, 1.06, 1.76, 5.40, 5.63, 2.12, 2.68, 1.31, 1.80, 5.70, 5.74, 1.48,

br s m m m m d (9.0) dd (9.0, 9.0) m td (12.5, 2.1) t (12.5) dd (12.5, 2.1) s s s

4.85, 1.73, 1.90, 1.07, 1.75, 5.45, 5.61, 1.81, 2.62, 1.16, 1.89, 5.83, 5.33, 1.51,

br s m m m m d (8.5) dd (8.5, 8.5) m td (12.4, 2.3) t (12.4) m d (1.1) d (1.1) s

4.83, 1.76, 1.90, 1.09, 1.73, 5.39, 5.62, 1.92, 2.66, 1.18, 1.89, 5.73, 5.32, 1.53,

br s m m m m d (8.8) dd (8.8, 8.8) m td (12.4, 2.1) m m d (1.2) d (1.2) s

1.11, 1.11, 1.13, 2.10, 2.07, 1.97, 3.06,

s s s s s s s

1.10, 1.11, 1,14, 2.09, 2.03, 1.97, 3.05, 3.28, 3.41, 2.95,

s s s s s s s s s d (1.0)

1.11, 1.11, 1.12, 2.11, 2.06, 1.98, 3.13,

s s s s s s s

1.10, 1.10, 1.11, 2.11, 2.04, 1.97, 3.13, 3.28, 3.48, 2.94,

s s s s s s s s s s

3.42, s 2.92, d (1.3)

3.48, s 2.95, s 2214

12 4.84, 1.71, 1.86, 0.92, 1.77, 5.32, 4.21, 2.04, 2.60, 1.22, 1.92, 5.73, 5.37, 5.26, 5.32, 1.13, 1.08, 1.11, 2.08, 2.17,

br s m m m m d (9.4) dd (9.4, 9.4) m td (12.1, 2.3) m m d (1.4) d (1.4) s s s s s s s

3.11, s 3.50, s 3.03, s

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the acetoxy group at C-7 in 9 was replaced by a hydroxy group in 12. The relative configuration of 12, named caesalminaxin J, was assigned by the ROESY spectrum, along with 1D NMR data compared with those of 9. Compound 13 was determined to have the molecular formula C26H36O10 by HRESIMS. The 13C NMR spectrum (Table 4) showed the presence of seven methyls (three acetoxy Table 4. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data for Compounds 13 and 14 in Methanol-d4 13 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Figure 6. X-ray crystallographic structures of 9 and 11 (Cu Kα).

Compound 10 was found to possess the same molecular formula as 8 by HRESIMS. Comparison of the 1D NMR data of 10 and 8 (Tables 2 and 3) revealed that they had similar structures, which was further verified by the COSY and HMBC correlations. The major differences between 10 and 8 were that signals of H-16 and C-16 in 10 shifted upfield by ΔδH 0.39 and ΔδC 1.9 compared with those of 8. These differences indicated that 10 and 8 were C-16 epimers, suggesting a β-orientation of H-16 in 10. Furthermore, the absence of the ROESY correlations of H-16/12-OMe also supported a β-orientation of H-16. Compound 10 was named caesalminaxin H. Compound 11 had the molecular formula C29H44O11 by HRESIMS. Comparison of the 1D NMR data of 11 (Tables 2 and 3) with those of 10 showed that they had similar structures, except for the presence of an O-methyl group at C-14 in 11. This deduction was further confirmed by the HMBC correlations of 14-OMe/C-14. The absolute configuration of 11, named caesalminaxin I, was determined by X-ray crystallographic analysis (Figure 6). Compound 12 was assigned the molecular formula C26H38O9 from HRESIMS. The 1D NMR data of 12 (Tables 2 and 3) were similar to those of 9. The major difference was that the C14 methoxy and C-17 methyl groups in 9 were replaced by an exocylic double bond [δC 141.0 (C-14) and 112.5 (C-17)] in 12, as deduced from the HMBC correlations of H-17 [δH 5.26 (s) and 5.32 (s)]/C-8, C-13. Furthermore, the upfield shift of H-7 of 12 from δH 5.61 to 4.21 compared with 9 suggested that

4.80, 1.68, 1.96, 1.06, 1.88,

5.60, 5.38, 3.02, 2.98,

br s m m m m

3.34, d (17.2) 3.43, d (17.2)

6-OAc

2.04, s

7-OAc

2.00, s

2′ 3′ 4′

33.4

2.12, dd (15.8, 13.7) 2.30, dd (15.8, 2.6)

1.92, 1.11, 1.11, 1.28, 2.05,

s s s s s

14 δC 77.6 23.2

d (9.1) dd (9.1, 9.1) dd (11.5, 11.5) td (11.5, 2.6)

16 17 18 19 20 1-OAc

1′

a

δH (J in Hz)

39.4 79.8 76.6 75.6 45.3 39.2 44.6 38.6 199.4 131.9 161.0 32.0 176.0a 18.6 31.0 24.7 17.8 171.5 21.7 172.3 21.0 172.3 21.8

δH (J in Hz) 4.86, 1.72, 1.96, 1.10, 1.95,

5.50, 5.80, 2.45, 2.78,

br s m m m m

d (8.8) dd (8.8) m td (11.7, 5.2)

2.24, dd (15.9, 5.2) 2.51, m

6.23, d (1.8) 7.30, 1.50, 1.18, 1.18, 1.34, 2.10,

d (1.8) s s s s s

2.06, s 2.00, s 2.96, 3.42, 1.44, 1.34, 0.90,

dd (15.6, 7.8) m m (2H) m (2H) t (7.3)

δC 77.6 23.2 33.2 39.4 79.9 77.3 74.7 41.2 37.4 45.9 23.7 151.6 123.4 79.2 108.8 143.0 25.8 31.1 25.3 17.9 171.7 21.1 172.2 21.4 171.9 21.7 63.1 33.8 20.5 14.5

The data were obtained from the HMBC spectrum.

methyls), four methylenes, five methines (three oxygenated), and seven quaternary carbons (one conjugated ketocarbonyl, one hydroxycarbonyl, three ester carbonyls, two olefinic, and one oxygenated). The NMR data indicated that 13 was a tricyclic cassane diterpenoid with a cleaved furan ring. The position of the conjugated carbonyl group was determined to be at C-12 (δC 199.4) based on the HMBC correlations of H11, H-15/C-12. The tetrasubstituted double bond was located between C-13 (δC 131.9) and C-14 (δC 161.0), which was confirmed by the HMBC correlations of H-15/C-13, C-14 and Me-17, H-7/C-14. The hydroxycarbonyl group was positioned at C-16 (δC 176.9) by the HMBC correlation of H-15/C-16. The ROESY correlations of H-1/Me-20, H-6/Me-20, and H-8/ 2215

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Table 5. Cytotoxic Activities of the Isolated Compounds against Four Cancer Linesa,b

a

compound

HepG-2

K562

HeLa

DU145

4 8 bonducellpin D camptothecinc

>50 11.5 + 2.9 8.0 + 1.3 0.6 + 0.1

9.9 + 1.7 9.2 + 0.9 >50 0.4 + 0.1

12.1 + 0.8 >50 >50 0.5 + 0.1

17.1 + 1.4 >50 12.0 + 0.8 0.8 + 0.1

Results are expressed as IC50 values in μM. bResults are represented as mean ± SD based on three independent experiments. cPositive control.

Me-20 showed that H-1, H-6, H-8, and Me-20 were in βorientations. The large coupling constant between H-6 and H-7 (J = 9.1 Hz) revealed an antiplanar relationship between H-6 and H-7, suggesting that H-7 was α-oriented. Likewise, H-9 was deduced to be α-oriented based on the large coupling constant between H-8 and H-9 (J = 11.5 Hz). Compound 13 was named caesalminaxin K. Compound 14 was obtained as a white powder, and its molecular formula was determined to be C30H44O9 by HRESIMS. The 1D NMR data (Table 4) closely resembled those of caesalmin E,16 except for the presence of an n-butoxy group at C-14 in 14. This deduction was confirmed by the key HMBC correlations of H-1′ [δH 2.96 (dd, J = 15.6, 7.8 Hz) and 3.42 (m)]/C-14. The ROESY spectrum revealed that the relative configuration of 14, named caesalminaxin L, was identical to that of caesalmin E. Compounds 8−12, bearing O-methyls at C-12 and C-16, may be artifacts formed from use of methanol during the extraction and purification process. LC-TOFMS analysis showed that compounds 8−12 were absent in the acetone crude extract. Compounds 4 and 14 are genuine natural products, which were confirmed to be present in the acetone crude extract by TLC and LC-TOFMS analyses. The known compounds bonducellpin D,24 caesalmin D,16 and neocaesalpin L19 were identified by comparison of their spectroscopic data with reported data. The isolated compounds were tested for cytotoxicity against the human cancer cell lines HepG-2, K562, HeLa, and DU145 using the MTT method, and the results are shown in Table 5. Compounds 4, 8, and the known bonducellpin D exhibited moderate activity against K562 (IC50 9.9 μM), K562 (IC50 9.2 μM), and HepG-2 (IC50 8.0 μM) cells, respectively.

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Extraction and Isolation. The air-dried and powdered seeds of C. minax (20 kg) were extracted with 80% aqueous acetone three times at room temperature and concentrated under vacuum to yield a residue, which was partitioned between H2O and CHCl3. The CHCl3 fraction was subjected to silica gel CC and eluted with petroleum ether− acetone (20:1 to 0:1) to yield fractions 1−9. Fraction 4 was fractionated on an MCI column eluted with MeOH−H2O (4:6 to 10:0) to yield six fractions, 4A−4F. Fraction 4C was chromatographed on a silica gel CC eluted with CHCl3−MeOH (200:1 to 0:1) to give fractions 4C1−4C5. Fraction 4C3 was subjected to silica gel CC eluted CHCl3−MeOH (200:1 to 160:1) to give fractions 4C31−4C35. Fraction 4C35 was purified by HPLC (MeOH−H2O, 53:47) to yield 1/2 (15 mg). Fraction 4C5 was chromatographed on a silica gel CC eluted with petroleum ether−acetone (2.5:1 to 0:1) to give fractions 4C51−4C53. Fraction 4C52 was purified by HPLC (CH3CN−H2O, 30:70) to yield 6/7 (28 mg). Fraction 4E was subjected to silica gel CC eluted with petroleum ether−EtOAc (4:1 to 1:3) to give fractions 4E1−4E13. Fraction 4E2 was purified by HPLC (MeOH−H2O, 75:25) to yield 14 (70 mg). Fraction 4E4 was separated by silica gel CC eluted with petroleum ether−EtOAc (3.5:1) to afford fractions 4E41−4E46. Fraction 4E43 was repeatedly subjected to Sephadex LH20 CC eluted with CHCl3−MeOH (1:1) to give fractions 4E431− 4E434. Fraction 4E431 was purified by HPLC (CH3CN−H2O, 55:45) to yield 9 (20 mg) and 11 (12 mg). Fraction 4E44 was purified by HPLC (CH3CN−H2O, 40:60) to yield 12 (13 mg). Fraction 4E8 was applied to Sephadex LH-20 CC (MeOH) to obtain 5 (9 mg). Fraction 4D was chromatographed on a silica gel CC eluted with CHCl3− MeOH (200:1 to 9:1) to afford fractions 4D1−4D7. Fraction 4D3 was separated by Sephadex LH-20 CC (MeOH) to give fractions 4D311− 4D314. Fraction 4D311 was purified by HPLC (MeOH−H2O, 55:45) to yield 8 (30 mg) and 10 (27 mg). Fraction 4D7 was subjected to silica gel CC eluted with CHCl3−MeOH (50:1) to give 13 (23 mg). Compound 3 (18 mg) was obtained through recrystallization in MeOH from fraction 4D4. Compound 4 (16 mg) was recrystallized in CHCl3−MeOH from fraction 4D3. Caesalminaxins A1(1)/A2 (2): colorless crystals (MeOH); [α]23 D +45 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 208 (3.02) nm; IR (KBr) νmax 3470, 1754, 1729, 1204 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 547.2159 [M + Na]+ (calcd for C26H36O11Na, 547.215). Caesalminaxin B (3): colorless needles (MeOH−CHCl3); [α]23 D +16 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 207 (2.98) nm; IR −1 1 13 (KBr) νmax 3461, 1767, 1729, 1279 cm ; H and C NMR data, see Tables 1 and 2; HRESIMS m/z 547.2156 [M + Na]+ (calcd for C26H36O11Na, 547.215). Caesalminaxin C (4): colorless needles (MeOH−CHCl3); [α]23 D +68 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 208 (2.94) nm; IR (KBr) νmax 3424, 1765, 1720, 1246 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 575.2468 [M + Na]+ (calcd for C28H40O11Na, 575.2463). Caesalminaxin D (5): colorless crystals (MeOH); [α]23 D −3 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 217 (3.60) nm; IR (KBr) νmax 3440, 2939, 1762, 1732, 1714, 1236 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 441.1523 [M + Na]+ (calcd for C22H26O8Na, 441.152). Caesalminaxins E1 (6)/E2 (7): colorless crystals (MeOH); [α]23 D +54 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 205 (3.03) nm; IR (KBr) νmax 3370, 1745, 1733, 1702, 1238, 1224 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 565.2249 [M + Na]+ (calcd for C26H38O12Na, 565.2255).

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 341 polarimeter. UV and IR spectra were recorded on a Shimadzu UV-2450 and a Perkin-Elmer 577 spectrophotometer, respectively. NMR spectra were recorded on a Varian Mercury NMR spectrometer operating at 500 MHz for 1H and 125 MHz for 13C. HRESIMS was measured on an Agilent G6224A TOF spectrometer. TLC was performed on precoated silica gel GF254 plates (Qingdao Marine Chemical Factory). Column chromatography (CC) was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Factory), Sephadex LH-20 (20−80 μm, Amersham Pharmacia Biotech AB), and MCI gel CHP-20P (70−150 μm, Mitsubishi Chemical Industries Co. Ltd.). Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph (YMC-Pack ODS-C18, 5 μm, 250 × 10 mm, detection: UV 210 nm). All solvents were spectroscopic grade (Adamas-beta Reagent Co. Ltd.) or distilled prior to use. Plant Material. The seeds of C. minax were collected in Guangxi Province, People’s Republic of China, in December 2011, and were authenticated by Prof. He-Ming Yang. A voucher specimen (SIMMXLJ-145) has been deposited at the Shanghai Institute of Materia Medica. 2216

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Caesalminaxin F (8): colorless crystals (MeOH); [α]23 D −47 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 206 (2.89) nm; IR (KBr) νmax 3570, 1739, 1230, 1033 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 577.2620 [M + Na]+ (cald for C28H42O11Na, 577.2619). Caesalminaxin G (9): colorless crystals (MeOH); [α]23 D +2 (c 0. 5, CHCl3); UV (MeOH) λmax (log ε) 207 (2.57) nm; IR (KBr) νmax 3544, 1736, 1232, 1097 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 591.2778 [M + Na]+ (calcd for C29H44O11Na, 591.2776). Caesalminaxin H (10): colorless crystals (MeOH); [α]23 D −48 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 207 (3.07) nm; IR (KBr) νmax 3580, 1734, 1247, 1103 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 577.2625 [M + Na]+ (calcd for C28H42O11Na, 577.2619). Caesalminaxin I (11): colorless crystals (MeOH); [α]23 D −9 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 207 (2.88) nm; IR (KBr) νmax 3523, 1735, 1212, 1057 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 591.2770 [M + Na]+ (calcd for C29H44O11Na, 591.2776). Caesalminaxin J (12): colorless crystals (MeOH); [α]23 D +101 (c 0.4, CHCl3); UV (MeOH) λmax (log ε) 216 (4.97) nm; IR (KBr) νmax 3370, 1739, 913 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 517.2407 [M + Na]+ (calcd for C26H38O9Na, 517.2408). Caesalminaxin K (13): colorless crystals (MeOH); [α]23 D +10 (c 0.3, CHCl3); UV (MeOH) λmax (log ε) 205 (2.79) nm; IR (KBr) νmax 3587, 1734, 1203 cm−1; 1H and 13C NMR data, see Table 4; HRESIMS m/z 531.2189 [M + Na]+ (calcd for C26H36O10Na, 531.2201). Caesalminaxin L (14): white powder; [α]23 D +11 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 217 (4.10) nm; IR (KBr) νmax 3562, 1727, 1279, 1232 cm−1; 1H and 13C NMR data, see Table 4; HRESIMS m/z 571.2880 [M + Na]+ (calcd for C30H44O9Na, 571.2878). Crystal data of 3: C26H36O11, M = 524.56, colorless needle, size 0.2 mm × 0.16 mm × 0.05 mm, orthohombic, space group P2(1)2(1)2(1), a = 11.2384(2) Å, b = 12.0287(3) Å, c = 19.6185(4) Å, α = 90°, β = 90°, γ = 90°, V = 2652.09(10) Å3, T = 140(2) K, Z = 4, d = 1.359 g/cm3, λ(Cu Kα) = 1.541 78 Å, F(000) = 1160, reflections collected/ unique 16 999/4750 [R(int) = 0.0327], h (−13/12), k (−13/14), l (−23/23), theta range 4.31−69.7°, completeness 99.0%, data/ restraints/parameters 4750/0/352, final R indices R1 = 0.0346 and wR2 = 0.1110 (I > 2σ(I)), R1 = 0.0352 and wR2 = 0.1118 (all data), GOF = 0.994, largest diff peak and hole 0.330 and −0.193 e Å−3, absolute structure parameter 0.12(12). Crystal data of 4: C28H40O11, M = 552. 6, colorless needle, size 0.25 mm × 0.16 mm × 0.08 mm, orthohombic, space group P2(1)2(1)2(1), a = 11.6379(2) Å, b = 11.7388(2) Å, c = 19.9846(3) Å, α = 90°, β = 90°, γ = 90°, V = 2730.62(8) Å, T = 140(2) K, Z = 4, d = 1.344 g/cm3, λ(Cu Kα) = 1.541 78 Å, F(000) = 1184, reflections collected/unique 14 553/4900 [R(int) = 0.0249], h (−13/14), k (−14/13), l (−24/24), theta range 4.37−69.69°, completeness 98.2%, data/restraints/parameters 4900/0/361, final R indices R1 = 0.0339 and wR2 = 0.0912 (I > 2σ(I)), R1 = 0.0345 and wR2 = 0.0920 (all data), GOF = 1.033, largest diff peak and hole, 0.229 and −0.238 e Å−3, absolute structure parameter 0.00(12). Crystal data of 7: C26H38O12, M = 542.56, colorless crystal, size 0.180 mm × 0.190 mm × 0.200 mm, orthohombic, space group P2(1)2(1)2(1), a = 11.3438(8) Å, b = 14.0817(9) Å, c = 16.2703(10) Å, α = 90°, β = 90°, γ = 90°, V = 2599.0(3) Å, T = 296(2) K, Z = 4, d = 1.387 g/cm3, λ(Mo Kα) = 0.71073 Å, F(000) = 1160, 11 277 reflections in h (−6/12), k (−16/16), l (−19/18), range 1.91−25.00°, completeness 99.0%, 4427 independent reflections, R(int) = 0.0327, data/restraints/parameters 4427/7/363, final R indices, R1 = 0.0369, wR2 = 0.0874 (I > 2σ(I)), R1 = 0.0421, wR2 = 0.0968 (all data), GOF = 1.076, largest diff peak and hole, 0.544 and −0.203 e Å−3. Crystal data of 9: C29H44O11, M = 568.64, colorless crystal, size 0.20 mm × 0.10 mm × 0.04 mm, orthohombic, space group P2(1)2(1)2(1), a = 11.35390(10) Å, b = 11.88610(3) Å, c = 21.7292(2) Å, α = 90°, β = 90°, γ = 90°, V = 2932.43(4) Å3, T =

140(2) K, Z = 4, d = 1.288 g/cm3, λ(Cu Kα) = 1.54178 Å, F(000) = 1224, reflections collected/unique 5413/5413 [R(int) = 0.0000], h (−13/13), k (0/15), l (0/28), theta range 4.07−69.54°, completeness 99.2%, data/restraints/parameters 5413/0/372, final R indices R1 = 0.0318 and wR2 = 0.0862 (I > 2σ(I)), R1 = 0.0322 and wR2 = 0.0865 (all data), GOF = 1.022, largest diff peak and hole, 0.265 and −0.205 e Å−3, absolute structure parameter 0.03(11). Crystal data of 11: C29H44O11, M = 568. 64, colorless crystal, size 0.25 mm × 0.10 mm × 0.02 mm, monoclinic, space group C2, a = 20.3979(7) Å, b = 9.6961(3) Å, c = 16.0481(6) Å, α = 90°, β = 90°, γ = 90°, V = 3131.11(19) Å3, T = 140(2) K, Z = 4, d = 1.206 g/cm3, λ(Cu Kα) = 1.54178 Å, F(000) = 1224, reflections collected/unique 10 723/4638 [R(int) = 0.0350], h (−24/24), k (−11/10), l (−18/18), theta range 4.81−69.46°, completeness 97.7%, data/restraints/ parameters 4638/1/372, final R indices R1 = 0.0660 and wR2 = 0.1793 (I > 2σ(I)), R1 = 0.0674 and wR2 = 0.1810 (all data), GOF = 1.052, largest diff peak and hole, 0.507 and −0.338 e Å−3, absolute structure parameter 0.0(2). Crystallographic data for 3 (deposition no. CCDC 945518), 4 (deposition no. CCDC 945519), 7 (deposition no. CCDC 930053), 9 (deposition no. CCDC 945520), and 11 (deposition no. CCDC 945521) have been deposited at the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44 (0) 1223−336033 or e-mail: [email protected]. uk]. Biological Assays. The cytotoxic activities against the cancer cell lines HepG-2, K562, HeLa, and DU145 were determined by the MTT assay. Briefly, 100 μL of each cell line (5 × 104 cells mL−1) was seeded in 96-well microplates and incubated at 37 °C for 24 h. Then, 100 μL of various concentrations of the test chemical in DMSO was added. Camptothecin was used as a positive control. After 24, 48, and 72 h, the cells were stained with MTT. The optical density of each well was measured at 490 nm compared to the negative control. The data obtained were presented graphically by plotting the test chemical concentrations versus the percent cell viability, where the concentration causing cell death by 50% was determined as the half-maximal inhibitory concentration (IC50).

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ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra of compounds 1−14 and X-ray crystallographic data (CIF files) of 3, 4, 7, 9, and 11. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/fax: +86 21 20231968. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported financially by the National Science & Technology major project “Key New Drug Creation and Manufacturing Program”, China (No. 2009ZX09301-001), and the National Natural Sciences Foundation of China (No. 30901851).

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REFERENCES

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