Cassane Diterpenoids from the Pericarps of Caesalpinia bonduc

Jan 12, 2016 - School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, People's Republic of China. ⊥Scho...
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Cassane Diterpenoids from the Pericarps of Caesalpinia bonduc Panpan Zhang,†,‡ Chunping Tang,†,§ Sheng Yao,†,§ Changqiang Ke,†,§ Ge Lin,§,∥ Hui-Ming Hua,*,‡ and Yang Ye*,†,§,⊥ †

State Key Laboratory of Drug Research and Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China ‡ Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, Liaoning, People’s Republic of China § Joint Research Laboratory for Promoting Globalization of Traditional Chinese Medicines between Shanghai Institute of Materia Medica, Chinese Academy of Sciences and The Chinese University of Hong Kong, Hong Kong, People’s Republic of China ∥ School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, People’s Republic of China ⊥ School of Life Science and Technology, ShanghaiTech University, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: Ten new cassane-type diterpenoids, caesalbonducins D−F (1−3), 6-deacetoxybonducellpin B (4), 3-acetoxyα-caesalpin (5), 2(3)-en-α-caesalpin (6), 1α-hydroxycaesalpinin J (7), 1α-hydroxy-6-decaetoxysalpinin J (8), 6α-hydroxycaesall M (9), and 6α-hydroxy-14(17)-dehydrocaesalpin F (10), along with eight known compounds (11−18), were isolated from the pericarps of Caesalpinia bonduc. Compounds 1−3 and 11 are methyl-migrated cassane-type diterpenoids with a 19(4→3)-cassane skeleton. The structures of 1−10 were elucidated on the basis of 1D and 2D NMR methods and other spectroscopic analysis. The neuroprotective effects of the isolated compounds were evaluated.

C

assane furanoditerpenes, mainly distributed in various genera of the family Fabaceae (especially the genus Caesalpinia), are a group of structurally diverse natural products characterized by a tetracyclic framework with a fused furan ring or butenolide moiety.1−3 Some of these diterpenoids have shown significant antiviral,4 antiproliferative,2 antioxidant,5 antimalarial,6 antibacterial,5 antihelmintic,7 and antineoplastic8 activities. Caesalpinia bonduc (L.) Roxb. is a stout climber distributed throughout the tropical and subtropical regions of Southeast Asia. The seeds of this plant have long been used as a traditional Chinese medicine to treat common colds, fever, and dysentery.9 Previous investigations on the chemical constituents of the seeds have resulted in the isolation of an array of cassane-type diterpenes.10−16 In 1997, a rearranged 19(4→3)abeo-cassane furanoditerpene, caesalpinin, was reported.11 In a continuing effort to search for bioactive constituents from natural sources, a systematic investigation of the pericarps of C. bonduc was carried out, leading to the isolation and structure elucidation of 10 new (1−10) and eight known cassane diterpenoids (11−18). Like compound 11, compounds 1−3 were found to possess a rare methyl-migrated 19(4→3)-cassane skeleton. Herein are described the isolation and structure elucidation of compounds 1−10, as well as the in vitro neuroprotective effects of these compounds. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 10, 2015

A

DOI: 10.1021/acs.jnatprod.5b00520 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. 1H NMR Spectroscopic Data (400 MHz, in CDCl3) for Compounds 1−5 (δ in ppm, J in Hz) position 1 2

1

2

3

4.91, t (2.4) 1.89, m 2.03, m

5.07, d (3.6) 2.12, m 2.50, m

4.70, 1.61, 1.78, 1.99,

t (3.2) m m m

2.66, 5.47, 5.61, 2.30, 2.75, 2.39, 2.48,

q (7.6) br s br s m dd (11.2, 6.4) dd (16.0, 6.4) dd (16.0, 11.2)

5.42, 5.67, 2.24, 2.57, 2.43, 2.46,

br d (8.8) dd (10.0, 8.8) dd (11.2, 10.0) dd (11.2, 5.6) m m

5.55, 5.53, 2.11, 2.45, 1.32, 2.13,

br s dd (13.2, 10.0) m td (12.4, 2.8) m m

6.38, 7.24, 1.49, 1.40, 4.67, 5.05, 1.07, 2.08,

d (2.0) d (2.0) s d (7.6) s s s s

6.37, 7.23, 1.53, 1.77, 1.63,

d (1.6) d (1.6) s s s

6.09, s

3 4 6 7 8 9 11 14 15 16 17 18 19 20 OCOCH3-1 OCOCH3-3 OCOCH3-6 OCOCH3-7 OCH3-17 OCH2CH3-12

■

2.08, s 2.01, s

1.55, s 1.45, s 1.14, d (7.2)

1.12, s 2.04, s

1.15, s 2.08, s

2.08, s 1.96, s

2.00, s 2.04, s

4

5

2.50, 2.52, 1.65, 2.00,

m m m m

2.79, m 2.85, m 5.00, dd (4.8, 4.8)

3.84, 3.60, 2.26, 2.63, 2.43, 3.45, 3.50, 6.14, 7.22,

br d (9.2) dd (10.4, 9.2) br d (10.4) dd, (11.6, 4.8) m dd, (16.0, 4.8) d (9.2) d (1.6) d (1.6)

5.60, 5.60, 2.13, 2.82, 2.45, 3.14,

br s br s m br d (4.8) dd (16.0, 11.6) dd (16.0, 4.8)

1.40, s 1.27, s

6.34, 7.22, 1.49, 1.18, 1.28,

d (2.0) d (2.0) s s s

1.39, s

1.54, s 2.10, s 1.99, s 2.06, s

3.74, s 3.17, q (7.2) 3.53, q (7.2) 1.16, t (7.2)

OCH2CH3-12

RESULTS AND DISCUSSION A 95% EtOH extract of the pericarps of C. bonduc was concentrated and then suspended in water. The solution was partitioned with petroleum ether and EtOAc, successively. The EtOAc fraction was separated by repeated column chromatography and preparative HPLC to afford 10 new diterpenoids (1−10) and eight known compounds (11−18). The known compounds were identified as caesalpinin MN (11),17 bonducellpin B (12),10 caesalpinin J (13),6 bonducellpin E (14),14 norcaesalpinin F (15),6 1-deacetoxy-1-oxocaesalmin C (16),17 caesalpin G (17),18 and 14(17)-dehydrocaesalpin F (18),19 by measuring their 1H and 13C NMR spectroscopic data and by comparison with literature values. Compound 1 was isolated as a white, amorphous powder, and its molecular formula was deduced to be C26H34O9 from the HRESIMS ion at m/z 513.2095 [M + Na]+. The IR spectrum showed the presence of hydroxy (3478 cm−1) and carbonyl (1746 cm−1) groups. The 1H NMR (Table 1) spectrum displayed signals attributable to three methyls (δH 1.07, 1.40, 1.49), three acetoxy methyls (δH 2.01, 2.08 × 2), three oxygenated methines (δH 4.91, 5.47, 5.61), an exomethylene (δH 4.67, 5.05), and two protons (δH 6.38, 7.24) of a 1,2-disubstituted furan ring. The 13C NMR data (Table 2) revealed the occurrence of 26 carbon resonances, consisting of six methyls, three methylenes (one olefinic at δC 113.3), eight methines (two olefinic at δC 107.4, 142.0 and three oxygenated at δC 74.1, 74.9, 75.1), and nine quaternary carbons (three ester carbonyls at δC 170.2, 170.8, 170.9, three olefinic at δC 125.2, 147.6, 148.5, and two oxygenated at δC 72.7, 79.4). These data indicated that 1 is a tetracyclic cassane diterpene with three acetoxy substituents. Analysis of the 1D and 2D NMR (HSQC,

HMBC, ROESY) 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. The HMBC correlations of H-1/C-2, C-10, C-20, H-6/C-7, and H-7/C-6 indicated that the three acetoxy groups are attached to C-1, C-6, and C-7, respectively. The significantly downfield shift of C-5 (δC 79.4) and the HMBC correlations from OH-5 to C-5, C-6, and C-10 implied that one hydroxy group is located at C-5. Another hydroxy group substituent was determined to be at C-14 based on the HMBC correlations between H3-17 and C-8, C-13, and C-14 (an oxygen-substituted quaternary carbon at δC 72.7). The HMBC correlations of H-19/C-3, C-4, C-5, C-18, H-4/C-2, C3, C-5, and H3-18/C-3, C-4 suggested that an exocylic double bond occurred between C-3 and C-19. All of the evidence obtained suggested that 1 is a rearranged cassane-type diterpene having a methyl group that has migrated to C-3 from C-4. The relative configuration of 1 was inferred from a ROESY experiment. The correlations of H-1/H3-20, H3-20/H8, H-8/H-6, and H-6/H3-20 indicated the β-orientation of H-1, H-6, H-8, and CH3-20, while the cross-peaks of H3-18/OH-5, OH-5/H-7, H-7/H-9, and H-9/H3-17 suggested that these protons and substituent groups are α-oriented. Therefore, the structure of 1 was determined as shown, and the compound has been named caesalbonducin D. Compound 2, also obtained as a white, amorphous powder, was designated with a molecular formula of C26H34O9 on the basis of its HRESIMS. The NMR data (Tables 1 and 2) of 2 were similar to those of 1, except for the absence of an exomethylene and the presence of an additional methyl group and an olefinic double bond. The double bond between C-3 B

DOI: 10.1021/acs.jnatprod.5b00520 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Spectroscopic Data (125 MHz, in CDCl3) for Compounds 1−10 (δ in ppm) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OCOCH3-1 OCOCH3-1 OCOCH3-2 OCOCH3-2 OCOCH3-3 OCOCH3-3 OCOCH3-6 OCOCH3-6 OCOCH3-7 OCOCH3-7 OCH3-17 OCH2CH3-12 OCH2CH3-12

1 75.1, 32.5, 148.5, 38.2, 79.4, 74.9, 74.1, 47.2, 33.4, 44.7, 23.2, 147.6, 125.2, 72.7, 107.4, 142.0, 25.2, 23.2, 113.3, 16.7, 170.2, 21.4,

170.9, 21.7, 170.8, 21.7,

2 d t s d s d d d d s t s s s d d q q t q s q

s q s q

73.1, 34.9, 126.6, 128.5, 75.6, 75.2, 73.4, 48.4, 33.8, 42.9, 23.5, 147.9, 128.5, 72.7, 107.5, 142.0, 24.8, 15.0, 20.3, 16.7, 169.5, 21.5,

171.1, 21.7, 170.8, 21.5,

3 d t s s s d d d d s t s s s d d q q q q s q

s q s q

73.8, 27.5, 32.1, 65.8, 68.0, 69.7, 71.7, 51.4, 33.9, 41.0, 37.3, 106.1, 172.0, 74.8, 117.0, 168.7, 20.5, 20.9, 17.2, 18.9, 170.7, 21.4,

170.2, 21.3, 169.7, 21.3,

4 d d d s s d d d d s t s s s d s q q q q s q

213.3, 35.4, 37.3, 38.7, 81.1, 75.0, 78.0, 40.7, 38.1, 55.2, 24.3, 151.7, 112.0, 46.1, 108.5, 141.3, 176.6, 27.7, 28.7, 14.9,

5 s t t s s d d d d s t s s d d d s q q q

s q s q

6

7

207.3, 41.1, 75.9, 43.7, 81.4, 75.0, 73.2, 48.4, 36.9, 55.6, 25.4, 149.2, 124.5, 73.0, 107.2, 141.8, 24.6, 25.7, 20.5, 16.7,

s t d s s d d d d s t s s s d d q q q q

201.9, 122.9, 152.5, 41.8, 80.4, 75.5, 73.1, 48.5, 36.4, 53.1, 26.0, 149.3, 124.1, 73.1, 107.3, 141.7, 24.7, 27.3, 24.4, 17.9,

s d d s s d d d d s t s s s d d q q q q

169.8, 21.2, 170.9, 21.6, 169.9, 21.6,

s q s q s q

169.8, 21.6, 170.9, 21.7,

s q s q

52.6, q

72.4, 25.8, 32.1, 38.9, 81.0, 74.6, 77.4, 38.3, 36.1, 45.1, 21.6, 150.5, 113.1, 45.9, 108.4, 141.7, 174.4, 30.4, 24.5, 17.3,

8 d t t s s d d d d s t s s d d d s q q q

72.7, 25.9, 32.1, 38.9, 80.8, 76.7, 77.5, 41.5, 35.9, 45.2, 21.8, 150.6, 113.5, 46.4, 108.7, 141.6, 175.8, 30.9, 24.6, 17.3,

9 d t t s s d d d d s t s s d d d s q q q

74.7, 67.2, 37.3, 41.0, 79.2, 74.7, 79.2, 38.1, 36.4, 46.2, 21.7, 149.7, 113.3, 46.0, 108.4, 141.9, 174.3, 31.3, 25.7, 16.9, 169.2, 21.3, 170.6, 21.1,

10 d d t s s d d d d s t s s d d d s q q q s q s q

73.5, 65.8, 77.1, 43.0, 77.1, 25.7, 29.5, 72.2, 42.3, 47.0, 19.1, 151.5, 118.2, 142.1, 106.9, 142.2, 104.5, 23.6, 24.7, 17.9, 169.5, 21.0, 169.9, 20.7, 168.4, 21.1,

d d d s s t t s d s t s s s d d t q q q s q s q s q

171.3, s 21.9, q

52.3, q

52.4, q

172.1, s 21.2, q 52.3, q

59.1, t 14.9, q

C-4 and C-5. The locations of three acetoxy groups at C-1, C-6, and C-7 were inferred from the long-range correlations of H-1 (δH 4.70), H-6 (δH 5.55), and H-7 (δH 5.53) to the respective carbonyl carbon of the acetoxy groups. The ethoxy group was attached to C-12, as inferred from the long-range correlations between the methylene protons (δH 3.53, 3.17) and C-12 (δC 106.1). The correlations from H3-17 (δH 1.55, s) to C-8 (δC 51.4), C-13 (δC 172.0), and C-14 (δC 74.8) implied that a hydroxy group is located at C-14. The ROESY correlations of H-1/H3-20, H3-20/H-6, H-6/H3-18, H3-18/H3-19, and H-8/ H3-20 indicated that H-1, H-6, H-8, CH3-18, and CH3-19 are in the β-orientation, while the ROESY cross-peaks of H-7/H-9, H-9/H3-17, and H3-17/OCH2CH3-12 showed that H-7, H-9, CH3-17, and OCH2CH3-12 are α-oriented. Thus, the structure of 3 (caesalbonducin F) was proposed as shown. The molecular formula of compound 4, a white, amorphous powder, was established as C21H28O7 according to the quasimolecular ion at m/z 415.1727 [M + Na]+ in the HRESIMS. The IR spectrum indicated the presence of hydroxy (3536 cm−1) and carbonyl (1740, 1704 cm−1) groups. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) were similar to those of bonducellpin B (12), except for the absence of an acetyl group in 4. The three hydroxy groups were located at C5, C-6, and C-7 by the analysis of its HSQC and HMBC data. The relative configuration of 4 was determined to be the same as 12 from the coupling constants and the ROESY correlations.

and C-4 was inferred from the HMBC correlations between H22 (δH 2.12, 2.50) and C-1 (δC 73.1), C-3 (δC 126.6), and C-4 (δC 128.5), H3-19 (δH 1.63, s) and C-2 (δC 34.9), C-3, and C-4, and H3-18 (δH 1.77) and C-3, C-4, and C-5 (δC 75.6). The ROESY spectrum showed that the relative configuration of 2 is identical to that of 1. Accordingly, the structure of 2 (caesalbonducin E) was constructed as shown. Compound 3, a white, amorphous powder, gave a molecular formula of C28H38O11 according to its HRESIMS. The IR spectrum showed absorption bands at 3454 and 1750 cm−1, indicative of hydroxy and carbonyl groups, respectively. The 1H NMR spectrum (Table 1) displayed signals for three tertiary methyls, a secondary methyl, three acetoxy methyls, an ethoxy group, and an olefinic proton. The olefinic proton signal at δH 6.09 (H-15) and downfield carbon signals at δC 117.0 (C-15), 168.7 (C-16), and 172.0 (C-13) confirmed the presence of an α,β-unsaturated butenolide moiety. Altogether, 28 carbon resonances were observed in the 13C NMR and DEPT spectra (Table 2). The carbon-bonded protons of 3 were assigned from the HSQC spectrum. In the HMBC spectrum, the correlations from H3-19 (δH 1.14, d, J = 7.2 Hz) to C-4 (δC 65.8) and from H3-18 (δH 1.45, s) to C-3 (δC 32.1), C-4 (δC 65.8), and C-5 (δC 68.0) suggested that both C-4 and C-5 are oxygenated. The chemical shift of C-5 (δC 68.0), appearing at a higher field than those for the hydroxylated C-5 (δC 75.6−82.8) in normal cassane-type diterpenes,18,20 indicated an epoxide ring between C

DOI: 10.1021/acs.jnatprod.5b00520 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 3. 1H NMR Spectroscopic Data (400 MHz, in CDCl3) for Compounds 6−10 (δ in ppm, J in Hz) position

6

7

8

5.80, d (10.4)

3

6.12, d (10.4)

6

5.51, br s

3.67, 1.69, 2.02, 1.06, 2.01, 5.50,

7

5.65, br s

5.36, br s

3.82, dd (10.8, 9.2)

5.27, m

8 9 11

2.17, 2.81, 2.49, 3.32,

2.61, 2.95, 2.48, 2.87, 3.42, 6.10, 7.23,

2.40, 2.87, 2.48, 2.80, 3.49, 6.17, 7.24,

2.54, 2.58, 2.38, 2.46, 3.40, 6.09, 7.22,

m m dd (15.6, 4.8) m br d (7.6) d (2.0) d (2.0)

1.33, 1.33, 1.33, 2.15, 1.98,

overlap overlap overlap s s

14 15 16 17 18 19 20 OCOCH3-1 OCOCH3-2 OCOCH3-3 OCOCH3-6 OCOCH3-7 OCH3-17

m td (11.6, 4.8) d (16.0, 11.6) d (16.0, 4.8)

6.35, d (2.0) 7.22, d (2.0) 1.47, s

m m m m m br d (9.6)

3.70, 1.68, 2.03, 1.05, 2.00, 5.34,

dd (11.2, 10.4) dd (12.0, 5.6) m dd (15.6, 5.6) d (8.8) d (1.6) d (1.6)

m m m m m d (9.2)

9

1 2

m dd (12.0, 6.0) m dd (16.0, 6.0) br d (8.0) d (2.0) d (2.0)

1.29, s 1.28, s 1.44, s

1.02, s 1.13, s 1.19, s

1.09, s 1.13, s 1.17, s

2.11, s 2.01, s

2.03, s 1.94, s 3.69, s

2.21, s 3.73, s

10

5.24, br d (2.0) 5.30, m

5.34, d (4.0) 5.50, dd (4.0, 4.0)

1.37, m 1.93, m 3.90, t (9.2)

5.11, d (4.0) 1.61, 1.71, 2.31, 2.34,

m m br d (4.4) dd (12.8, 4.4)

3.24, br d (8.0) 2.49, dd (18.4, 4.8) 2.93, dd (18.4, 8.0) 6.44, 7.27, 5.17, 5.24, 1.12, 1.11, 0.95, 2.13, 1.95, 2.15,

d (2.0) d (2.0) br s br s s s s s s s

2.05, s 3.71, s

Compound 7 was obtained as an amorphous powder. The quasimolecular ion at m/z 501.2095 [M + Na]+ in the HRESIMS and the 13C NMR data suggested a molecular formula of C25H34O9. The 1D NMR data (Tables 2 and 3) of 7 showed close similarities to those of caesalpinin J (13). Compared with 13, a hydroxy group rather than a carbonyl group was located at C-1 for 7, which was deduced from the HMBC correlations from H-1 (δH 3.67) to C-3 (δC 32.1) and C-5 (δC 81.0) and from H2-2 (δH 2.02, 1.69) and H3-20 (δH 1.19) to C-1 (δC 72.4). The relative configuration of 7 was confirmed by the ROESY experiment. The structure of 7 (1αhydroxycaesalpinin J) was established as shown. Compound 8 showed an [M + Na]+ ion peak at m/z 459.1989 in the HRESIMS, corresponding to a molecular formula of C23H32O8. The 1H and 13C NMR signals observed (Tables 2 and 3) were closely related to those of 7, except that a hydroxy group rather than an acetoxy group was observed at C-7 in 8. The ROESY experiment indicated that 8 has the same relative configuration as 7. Therefore, the structure of 8 (1αhydroxy-6-decaetoxysalpinin J) was established as shown. Compound 9, a white, amorphous powder, gave the molecular formula C27H36O11, from the [M]+ peak at m/z 536.2261 in the HREIMS. The 1H and 13C NMR signals of 9 obtained (Tables 2 and 3) were closely related to those of the known caesall M,20 except for the signals of an additional hydroxy group. The location of the hydroxy group was determined to be at C-6 based on the downfield shift of H-6 (δH 3.90) and the HMBC correlation between H-6 and C-7. The relative configuration of 9 was confirmed to be the same as that of caesall M by a ROESY experiment, with the hydroxy group at C-6 being β-oriented. Thus, the structure of 9 (6αhydroxycaesall M) was determined as shown.

The structure of 4 (6-deacetoxybonducellpin B) was established as shown. Compound 5, a white, amorphous powder, exhibited a molecular formula of C26H34O10 through HREIMS analysis (m/ z [M]+ 506.2168). The IR absorptions showed the presence of hydroxy (3569 cm−1) and carbonyl (1750 cm−1) groups. The 1 H and 13C NMR signals obtained (Tables 1 and 2) were closely related to those of the known compound α-caesalpin,18 with the exception of an additional acetoxy group in 5. The downfield-shifted C-3 (δC 75.9) of 5, in contrast to CH2-3 (δC 39.4) of α-caesalpin, suggested that the additional acetoxy group is at C-3. Such an elucidation was supported by the HMBC correlations of H2-2/C-1 (δC 207.3), C-3 (δC 75.9), H3/C-18 (δC 25.7), C-19 (δC 20.5), OCOCH3-3 (δC 169.8), and H3-18(19)/C-3, C-4 (δC 43.7), C-5 (δC 81.4). The relative configuration of 5 was identical to that of α-caesalpin with an αoriented H-3 inferred from the ROESY correlation of H-3/H318. Compound 5 (3-acetoxy-α-caesalpin) was established as shown. Compound 6 was isolated as a white, amorphous powder and gave the molecular formula C24H30O8, as established by HREIMS analysis (m/z [M]+ 446.1934). The NMR data (Tables 2 and 3) also were similar to those of α-caesalpin,18 except for the presence of one double bond in 6. The double bond was determined to be between C-2 and C-3 by the HMBC correlations from H-2 (δH 5.80) to C-1 (δC 201.9) and C-3 (δC 152.5) and from H-3 (δH 6.12) to C-1, C-2 (δC 122.9), and C-4 (δC 41.8). The relative configuration of 6 was assigned by the ROESY spectrum and the NMR data when compared with those of α-caesalpin. The structure of 6, 2(3)-en-αcaesalpin, was established as shown. D

DOI: 10.1021/acs.jnatprod.5b00520 J. Nat. Prod. XXXX, XXX, XXX−XXX

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petroleum ether−acetone mixtures (8:1, 6:1, 4:1, 2:1, 1:1) to give seven fractions, 2A−2G. Fraction 2A was subjected to chromatography on a Sephadex LH-20 column (eluted with MeOH) to give subfractions 2A1−2A4. Subfraction 2A3 was finally separated by preparative HPLC (MeCN−H2O, 40−60%, 0−100 min) to afford 7 (19 mg) and 13 (155 mg). Subfraction 2A3a was separated on a silica gel column (eluted with CH2Cl2−MeOH, 100:1, 80:1, 60:1) to yield 9 (8.4 mg), 12 (18 mg), and 14 (52 mg). Fraction 2B was applied to a Sephadex LH-20 column (eluted with MeOH) to give fractions 2B1− 2B4. Fraction 2B3 was purified by semipreparative HPLC (MeCN− H2O, 35−75%, 0−90 min) to yield 10 (2 mg), 15 (3 mg), and 16 (19 mg). Fraction 2C was separated on a Sephadex LH-20 column (eluted with MeOH) and then purified by preparative HPLC (MeCN−H2O, 40−60%, 0−100 min) to give 1 (16 mg), 11 (515 mg), and 18 (49 mg). Fraction 2D was applied to a Sephadex LH-20 column (eluted with MeOH) to give fractions 2D1−2D3. Fraction 2D1 was subjected to chromatography on a silica gel column (eluted with CH2Cl2− MeOH, 100:1, 80:1, 60:1) to afford subfractions 2D1a−2D1c. Subfraction 2D1c was further purified by preparative HPLC (MeCN−H2O, 35−55%, 0−100 min) to yield 2 (109 mg). Fraction 2E was chromatographed on a Sephadex LH-20 column (eluted with MeOH), giving fractions 2E1−2E3. Fraction 2E1 was separated by preparative HPLC (MeCN−H2O, 35−70%, 0−100 min) to afford 4 (6 mg), 5 (2 mg), and 6 (9 mg). Fractions 2E1a and 2E1b were subjected to silica gel column chromatography (eluted with CH2Cl2− MeOH, 100:1, 80:1, 60:1, 50:1) to yield 8 (55 mg) and 17 (9 mg), respectively. Fraction 2F was applied to a Sephadex LH-20 column (eluted with MeOH) to give fractions 2F1−2F4. Fraction 2F1 was separated by silica gel column chromatography (eluted with CH2Cl2− MeOH, 80:1, 60:1, 50:1, 30:1) to afford subfractions 2F1a−2F1c. Subfraction 2F1a was purified by preparative HPLC (MeCN−H2O, 35−65%, 0−100 min) to yield 3 (4 mg). Caesalbonducin D (1): white, amorphous powder; [α]20D +26.5 (c 0.12, CHCl3); IR (KBr) νmax 3478, 2962, 1746, 1258 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; HRESIMS m/z 513.2095 [M + Na]+ (calcd for C26H34O9Na, 513.2101). Caesalbonducin E (2): white, amorphous powder; [α]20D +25.0 (c 0.23, CHCl3); IR (KBr) νmax 3467, 2987, 1742, 1233, 1041 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 513.2095 [M + Na]+ (calcd for C26H34O9Na, 513.2101). Caesalbonducin F (3): white amorphous powder; [α]20D −56.7 (c 0.10, CHCl3); UV (MeOH) λmax (log ε) 220 (3.92) nm; IR (KBr) νmax 3454, 2963, 1750, 1261 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 573.2306 [M + Na]+ (calcd for C28H38O11Na, 573.2312). 6-Deacetoxybonducellpin B (4): white, amorphous powder; [α]20D +14.5 (c 0.10, CHCl3); UV (MeOH) λmax (log ε) 205 (3.94) nm; IR (KBr) νmax 3536, 3420, 1740, 1704 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 415.1727 [M + Na]+ (calcd for C21H28O7Na, 415.1733). 3-Acetoxy-α-caesalpin (5): white, amorphous powder; [α]20D +18.6 (c 0.28, CHCl3); IR (KBr) νmax 3569, 1750, 1253 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HREIMS m/z 506.2168 [M]+ (calcd for C26H34O10, 506.2152). 2(3)-En-α-caesalpin (6): white, amorphous powder; [α]20D +45.4 (c 0.15, CHCl3); UV (MeOH) λmax (log ε) 225 (4.0) nm; IR (KBr) νmax 3453, 1749, 1260 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HREIMS m/z 446.1934 [M]+ (calcd for C24H30O8, 446.1941). 1α-Hydroxycaesalpinin J (7): white, amorphous powder; [α]20D −20.9 (c 0.27, CHCl3); IR (KBr) νmax 3440, 2950, 1757, 1245 cm−1; 1 H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 501.2095 [M + Na]+ (calcd for C25H34O9Na, 501.2101). 1α-Hydroxy-6-decaetoxysalpinin J (8): white, amorphous powder; [α]20D +3.6 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 218 (3.94), 278 (3.06) nm; IR (KBr) νmax 3442, 1746 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 459.1989 [M + Na]+ (calcd for C23H32O8Na, 459.1995). 6α-Hydroxycaesall M (9): white, amorphous powder; [α]20D −28.3 (c 0.12, CHCl3); IR (KBr) νmax 2963, 2924, 1741, 1261 cm−1; 1H and

Compound 10 was obtained as a white, amorphous powder, and its molecular formula was assigned as C23H32O8 on the basis of its HREIMS (m/z 490.2199 [M]+). A comparison of the 1H and 13C NMR data (Tables 2 and 3) of 10 and 14(17)dehydrocaesalpin F (18) indicated their structures to be closely related with 10, having an additional hydroxy group located at C-8. The structure proposed was confirmed by the HMBC correlations from H2-7, H-9, and H2-14 to C-8 (δC 72.2). The ROESY correlations of OH-8/H-9 and H-9/OH-5 suggested further an α-orientation for OH-8. Thus, the structure of 10 (6α-hydroxy-14(17)-dehydrocaesalpin F) was established as shown. Some of the isolated compounds were evaluated for their neuroprotective effects against hydrogen peroxide (H2O2)-, amyloid-β25−35 (Aβ25−35)-, and oxygen-glucose deprivation (OGD)-induced neurotoxicity in SH-SY5Y cells by the MTT method. The results showed that all compounds were inactive against all of the tested cells at concentrations of 1 and 10 μM (for details see Tables S1−S3 in the Supporting Information). N-Acetylcysteine (NAC) and epigallocatechin gallate (EGCG) were used as positive controls.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a Rudolph Autopol VI automatic polarimeter. UV spectra were measured with a Varian Cary 50 spectrophotometer. IR spectra were obtained on a Nicolet Magna FTIR-750 spectrometer using KBr disks. 1H, 13C, and 2D NMR spectra were recorded on Bruker AM-400 and INVOR-600 NMR spectrometers. Chemical shifts are reported in ppm (δ) with TMS as internal standard, and coupling constants (J) in hertz. EIMS and HREIMS were measured on a Finnigan MAT-95 mass spectrometer, while ESIMS and HRESIMS were obtained on a Micromass Q-TOF Global mass spectrometer. All HPLC analyses were carried out on a Waters Sunfire RP C18, 3.5 μm, 4.6 mm × 100 mm column, eluted with a gradient of CH3CN−H2O (5 to 95%) with 0.1% HOAc. Preparative HPLC was performed on a Varian PrepStar system with an Alltech 3300 ELSD. Chromatographic separations were performed on a Waters Sunfire RP C18, 5 μm, 30 mm × 150 mm column and a Waters Sunfire RP C18, 5 μm, 19 mm × 150 mm column, using a gradient solvent system composed of H2O and CH3CN, with a flow rate of 25.0 and 10.0 mL/min, respectively. Column chromatographic separations were carried out using silica gel (200−300 mesh, Qingdao Marine Chemical Industrials, Qingdao, People’s Republic of China), MCI gel CHP20P (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and Sephadex LH20 (Pharmacia Biotech AB, Uppsala, Sweden). TLC was carried out on precoated silica gel GF254 plates (Yantai Chemical Industrials, Yantai, People’s Republic of China), and the TLC spots were viewed at 254 nm and visualized with 5% sulfuric acid in alcohol containing 10 mg/ mL vanillin. Plant Material. The pericarps of C. bonduc were collected in Zhanjiang City, Guangdong Province, People’s Republic of China, in April 2014 and identified by Prof. Jin-Gui Shen from the Shanghai Institute of Materia Medica. A voucher specimen (no. 20140416) has been deposited at the Herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. The air-dried, powdered pericarps of C. bonduc (9.8 kg) were extracted with 95% EtOH (3 × 25 L) at room temperature (72 h each). Concentration of the combined percolates under reduced pressure yielded a dark brown crude extract (360 g). The extract was suspended in H2O and partitioned with petroleum ether and EtOAc, successively, to yield a petroleum ether (115 g) and a EtOAc (40 g) extract, respectively. The EtOAc fraction (40 g) was applied to an MCI gel column (eluted with EtOH in H2O, 50%, 80%, and 95% in a step gradient manner) to afford three fractions, 1−3. Fraction 2 (17.3 g) was chromatographed on a silica gel column (200−300 mesh) eluted with E

DOI: 10.1021/acs.jnatprod.5b00520 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C NMR data, see Tables 2 and 3; HREIMS m/z 536.2261 [M]+ (calcd for C23H32O8, 536.2258). 6α-Hydroxy-14(17)-dehydrocaesalpin F (10): white, amorphous powder; [α]20D −40.7 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 203 (3.78), 216 (3.64) nm; IR (KBr) νmax 3584, 2943, 1752 cm−1; 1H and 13 C NMR data, see Tables 2 and 3; HREIMS m/z 490.2199 [M]+ (calcd for C23H32O8, 490.2203). Neuroprotective Assay. SH-SY5Y cells were used as high passages from the American Type Culture Collection and maintained at 37 °C in a humidified atmosphere containing 5% CO2. For H2O2 exposure, the cells were pretreated with each test compound (1 or 10 μM) or N-acetylcysteine (300 μM) for 2 h and then suffered cell injury by 100 μM H2O2 for another 24 h. For Aβ exposure, the cells were pretreated with compounds (1 or 10 μM) or epigallocatechin gallate (10 μM) for 2 h, followed by exposure to 10 μM Aβ25−35 in the presence of compounds for another 24 h. For OGD-induced injury, SH-SY5Y cells, pretreated with each test compound for 2 h, were exposed to 1 mg/mL OGD for 1 h and cultured for another 24 h under normal conditions, and SH-SY5Y cells cultured with glucose under normal condition served as controls. Cell viability was evaluated by incubating with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) at a final concentration of 0.5 mg/mL for 4 h at 37 °C. The medium was replaced with 100 μL of DMSO; then absorbance was read at 490 nm. Data were analyzed by one-way analysis of variance (ANOVA) and expressed as means ± SD with p < 0.05 as the level of significance.

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(6) Awale, S.; Linn, T. Z.; Tezuka, Y.; Kalauni, S. K.; Banskota, A. H.; Attamimi, F.; Ueda, J.; Kadota, S. Chem. Pharm. Bull. 2006, 54, 213− 218. (7) Jabbar, A.; Zaman, M. A.; Iqbal, Z.; Yaseen, M.; Shamim, A. J. 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) Jiangsu New Medical College. Dictionary of Chinese Traditional Medicine; Shanghai Science and Technology Publishing House: Shanghai, 1986; pp 1289−1290. (10) Peter, R. S.; Tinto, W. F.; Mclean, S.; Reynolds, W. F.; Yu, M. J. Nat. Prod. 1997, 60, 1219−1221. (11) Peter, R. S.; Tinto, W. F.; McLean, S.; Reynolds, W. F.; Tay, L. L. Tetrahedron Lett. 1997, 38, 5767−5770. (12) Lyder, D. L.; Peter, R. S.; Tinto, W. F.; Bissada, S. M.; McLean, S.; Reynolds, W. F. J. Nat. Prod. 1998, 61, 1462−1465. (13) Kinoshita, T. Chem. Pharm. Bull. 2000, 48, 1375−1377. (14) Pudhom, K.; Sommit, D.; Suwankitti, N.; Petsom, A. J. Nat. Prod. 2007, 70, 1542−1544. (15) Wu, Z. H.; Wang, Y. Y.; Huang, J.; Sun, B. H.; Wu, L. J. Asian J. Tradit. Med. 2007, 2, 135−139. (16) Wu, L.; Luo, J.; Zhang, Y. M.; Wang, X. B.; Yang, L.; Kong, L. Y. Fitoterapia 2014, 93, 201−208. (17) Kalauni, S. K.; Awale, S.; Tezuka, Y.; Banskota, A. H.; Linn, T. Z.; Kadota, S. Chem. Pharm. Bull. 2005, 53, 1300−1304. (18) Peter, R. S.; Tinto, W. F.; McLean, S.; Reynolds, W. F.; Tay, L. L.; Yu, M.; Chan, W. R. Magn. Reson. Chem. 1998, 36, 124−127. (19) Pascoe, K. O.; Burke, B. A.; Chan, W. R. J. Nat. Prod. 1986, 49, 913−915. (20) Wu, L.; Wang, X. B.; Shan, S. M.; Luo, J.; Kong, L. Y. Chem. Pharm. Bull. 2014, 62, 729−733.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00520. 1D and 2D NMR data of compounds 1−10 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: 86-24-23986465. E-mail: [email protected] (H.-M. Hua). *Tel: 86-21-50806726. Fax: 86-50806726. E-mail: yye@mail. shcnc.ac.cn (Y. Ye). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Science and Technology Major Project “Key New Drug Creation and Manufacturing Program” (Nos. 2012ZX09301001-001, 2015ZX09103002), the National Natural Science Funds of China (Nos. 81302657, 81573305, 81473112), the Ministry of Science and Technology (2010DFA30980), the Chinese Academy of Sciences (KSZD-EW-Z-004-01), and the Shanghai Commission of Science and Technology (11DZ1970700, 12JC1410300) is gratefully acknowledged.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00520 J. Nat. Prod. XXXX, XXX, XXX−XXX