Chemical Constituents Isolated from the Root Bark of Cudrania

Article pubs.acs.org/jnp

Chemical Constituents Isolated from the Root Bark of Cudrania tricuspidata and Their Potential Neuroprotective Effects Jaeyoung Kwon,†,‡ Nguyen Tuan Hiep,† Dong-Woo Kim,§ Sungeun Hong,§ Yuanqiang Guo,⊥ Bang Yeon Hwang,∥ Hak Ju Lee,# Woongchon Mar,*,§ and Dongho Lee*,†,‡ †

Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, and ‡Institute of Life Science and Natural Resources, Korea University, Seoul 02841, Republic of Korea § Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea ⊥ State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People’s Republic of China ∥ College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea # Korea Forest Research Institute, Seoul 02455, Republic of Korea S Supporting Information *

ABSTRACT: Seventy-five compounds, including 21 new compounds (1−21), were isolated from the root bark of Cudrania tricuspidata. The structures of the isolated compounds were elucidated by interpretation of their spectroscopic data. All isolated compounds were evaluated for their neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced cell death, and nine compounds had activities with EC50 values of 1.9−30.2 μM. The 75 isolated compounds along with 34 previously reported xanthones were tested also for neuroprotective effects against the 1-methyl-4-phenylpyridinium ion (MPP+) and oxygen glucose deprivation (OGD)-induced cell death. Three compounds were active against MPP+-induced cell death with EC50 values of 0.2− 10.3 μM, and 23 compounds were active in the OGD model with EC50 values of 2.9−35.5 μM.

I

Cudrania tricuspidata (Carr.) Bureau, belonging to the family Moraceae, is a small deciduous tree distributed in Eastern Asia, and it has previously been reported that several extracts and identified constituents from this plant have been shown to have neuroprotective,5,6 anti-inflammatory,7,8 and antioxidant properties.9 Recently, we reported that extracts and various constituents such as xanthones and isoflavones from the root bark and fruits of C. tricuspidata have neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced cell death in SH-SY5Y cells.10,11 In this ongoing program, 21 new compounds along with 54 known compounds have been further isolated from the root bark of C. tricuspidata, and nine of these compounds were found to have neuroprotective effects in a 6-OHDA model with EC50 values of 1.9−30.2 μM. This investigation led to an evaluation of various neuroprotective

mprovements in the quality of life and public health systems have led to an increased life expectancy in the past century.1 Three-quarters of the world’s population older than 60 years of age are expected to live in developing countries by 2025 according to the World Health Organization.1 Concurrently, the incidence of neurodegenerative diseases such as ischemia and Parkinson’s disease (PD) has been steadily increasing with advancing age, and the financial and societal costs associated with these diseases are gradually increasing.2 Neurodegenerative diseases are characterized by a progressive degeneration of the structure and function of neurons and glial cells in the central nervous system.2 Although the exact pathogenesis of neurodegenerative diseases has not been fully elucidated, it is characterized by the promotion of apoptosis, or the deliberate suicide of the cell to protect nearby neurons. Several therapeutic methods are expected to slow the progression of these disorders, but a radical cure is yet to be found.3,4 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: March 7, 2016

A

DOI: 10.1021/acs.jnatprod.6b00204 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

cudraxanthone R, 12 except that the 2-(1-hydroxy-1methylethyl)dihydrofuran group is fused at C-7 and C-8, as indicated by the HMBC cross-peaks of H-16/C-7 (δC 146.2), and the hydroxy group at C-5 was transposed to C-7, as evidenced by the signal at δH 6.78 (1H, s, H-5) and δC 146.2 (C-7). Further analysis of the 2D NMR spectroscopic data (Figure 1) permitted the assignment of the structure of compound 1, which was termed cudratrixanthone P. The absolute configuration of compound 1 was established by comparing its experimental ECD spectrum with that calculated using the time-dependent density functional theory (TDDFT) method. After conformational searches using the MMFF force field in Spartan’14 software,13 selected conformers were optimized at the B3LYP/6-31+G(d,p) level in Gaussian 09 software.14 After optimization, the theoretical ECD spectrum was calculated at the CAM-B3LYP/TZVP level with a CPCM solvent model in MeCN. The calculated ECD spectrum showed a good fit with the experimental result (Figure 2), confirming the absolute configuration of 1 as 17R. Results from HRESIMS analysis suggested that compound 2 has the same molecular formula of C23H24O7 as that of 1. In addition, the 1H and 13C NMR data (Table 1) indicated a structural similarity to 1, which has a 1,3,6,7-tetraoxygenated xanthone moiety with a 1,1-dimethylallyl group and a 2-(1hydroxy-1-methylethyl)dihydrofuran group. The differences between the two compounds were evident in the substituent patterns on the aromatic rings. The HMBC cross-peaks of Me12 and Me-13/C-2 (δC 115.0) and H-17/C-5 (δC 115.8) suggested that the 1,1-dimethylallyl group is attached at C-2 and the 2-(1-hydroxy-1-methylethyl)dihydrofuran group is fused at C-5 and C-6, respectively. Further analysis of the 2D NMR spectroscopic data helped assign the structure of compound 2 as shown, which was named cudratrixanthone Q. Compound 3 was formulated as C23H24O8 from HRESIMS analysis. The 1D NMR data (Table 1) indicated its structural

effects of these 75 isolated compounds as well as 34 previously reported xanthones using two other neurodegenerative disease models. The results indicated three compounds to have neuroprotective effects against 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell death with EC50 values of 0.2−10.3 μM, while 23 compounds showed activities on oxygen glucose deprivation (OGD)-induced neurotoxicity in the EC50 range of 2.9−35.5 μM. Described herein are the isolation, structure determination, and biological evaluation of these compounds.

■

RESULTS AND DISCUSSION Compound 1 was isolated as a yellow, amorphous solid, and its molecular formula of C23H24O7 was determined by HRESIMS analysis, corresponding to 12 degrees of unsaturation. The IR spectrum revealed the presence of hydroxy groups (3244 cm−1) and a carbonyl group (1626 cm−1). The 1H NMR data (Table 1) exhibited a sharp hydroxy group signal at δH 13.41 (1H, s, OH-1) and two aromatic methine signals at δH 6.78 (1H, s, H5) and 6.25 (1H, s, H-2), which were characteristic of a 1,3,6,7tetraoxygenated xanthone skeleton. Moreover, a 2-(1-hydroxy1-methylethyl)dihydrofuran moiety at δH 4.79 (1H, t, J = 9.5 Hz, H-17) and 3.72 (2H, d, J = 9.5 Hz, H-16) and a 1,1dimethylallyl group at δH 6.36 (1H, dd, J = 17.5, 10.5 Hz, H14), 5.02 (1H, d, J = 17.5 Hz, H-15a), 4.91 (1H, d, J = 10.5 Hz, H-15b), and 1.67 (6H, s, Me-12 and Me-13) were observed. The 13C NMR data (Table 1) showed 23 carbon signals, which were assigned to four methyl carbons at δC 29.7 (C-12 and C13), 25.9 (C-20), and 25.4 (C-19), two methylene carbons at δC 108.0 (C-15) and 33.3 (C-16), four methine carbons at δC 151.6 (C-14), 102.9 (C-5), 99.4 (C-2), and 92.0 (C-17), one carbonyl carbon at δC 182.1 (C-9), six oxygenated tertiary carbons at δC 163.9 (C-3), 162.0 (C-1), 156.9 (C-4a), 153.1 (C-6), 148.7 (C-4b), and 146.2 (C-7), and four quaternary carbons at δC 126.9 (C-8), 111.7 (C-4), 110.8 (C-8a), and 104.2 (C-9a). These 1D NMR data were similar to those of B


C

108.0

33.3 92.0

71.5 25.4 25.9

16 17

18 19 20 OH OMe

162.0 99.4 163.9 111.7 156.9 148.7 102.9 153.1 146.2 126.9 110.8 182.1 104.2 41.8 29.7 29.7 151.6

C CH3 CH3

CH2 CH

CH2

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

δC, type

15

1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 11 12 13 14

position

1

1.29, s 1.26, s 13.41, s

1.67, s 1.67, s 6.36, dd (17.5, 10.5) 5.02, d (17.5) 4.91, d (10.5) 3.72, d (9.5) 4.79, t (9.5)

6.78, s

6.25, s

δH, mult (J in Hz)

71.6 25.6 25.9

28.5 93.2

108.6

164.4 115.0 163.8 95.1 156.4 148.1 115.8 156.3 140.2 110.7 115.6 180.9 103.2 41.7 29.3 29.3 150.9

C CH3 CH3

CH2 CH

CH2

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

δC, type

2

1.34, s 1.28, s 14.33, s

1.63, s 1.63, s 6.38, dd (17.5, 10.5) 4.97, d (17.5) 4.86, d (10.5) 3.44, m 4.95, dd (10.0, 2.5)

7.46, s

6.41, s

δH, mult (J in Hz)

71.1, 25.9, 25.5,

71.6, 100.8,

108.6,

164.3, 115.2, 163.4, 95.3, 156.8, 148.8, 118.3, 156.4, 140.5, 111.8, 115.5, 180.7, 103.3, 41.7, 29.3, 29.3, 150.9,

C CH3 CH3

CH CH

CH2


δC, type

3

1.33, s 1.30, s 14.29, s

1.63, s 1.63, s 6.37, dd (17.5, 10.5) 4.95, d (17.5) 4.85, d (10.5) 5.80, d (3.5) 4.55, d (3.5)

7.55, s

6.45, s

δH, mult (J in Hz)

Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1−6 in Acetone-d6

C CH3 CH3 CH3

57.1,

CH CH

CH3


71.0, 26.5, 25.5,

80.7, 98.2,

14.6,

159.1, 117.5, 166.5, 90.0, 158.4, 149.0, 116.1, 157.5, 140.8, 112.1, 115.2, 180.8, 104.2, 44.1, 25.0, 20.9, 91.7,

δC, type

4

1.36, s 1.24, s 13.50, s 3.58, s

5.47, d (3.0) 4.62, d (3.0)

1.39, d (6.5)

1.49, s 1.25, s 4.54, q (6.5)

7.59, s

6.37, s

δH, mult (J in Hz)

71.1, 25.8, 25.6,

71.5, 100.7,

14.6,

159.0, 117.3, 166.3, 90.0, 158.5, 149.0, 118.4, 156.8, 140.7, 111.4, 115.0, 180.8, 104.1, 44.0, 25.4, 20.9, 91.6,

C CH3 CH3

CH CH

CH3


δC, type

5

1.34, s 1.31, s 13.57, s

5.83, d (3.5) 4.56, d (3.5)

1.39, d (6.5)

1.49, s 1.24, s 4.53, q (6.5)

7.54, s

6.35, s

δH, mult (J in Hz)

56.2

69.3 30.1 30.1

103.4 168.9

107.3

162.7 96.2 165.8 113.6 155.8 149.8 99.0 156.2 141.1 128.1 106.7 181.9 104.4 41.9 29.2 29.2 151.9

CH3

C CH3 CH3

CH C

CH2


δC, type

6

1.67, s 1.67, s 13.68, s 3.92, s

1.66, s 1.66, s 6.34, dd (17.5, 10.5) 4.96, d (17.5) 4.87, d (10.5) 7.52, s

6.86, s

6.45, s

δH, mult (J in Hz)

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Figure 1. HMBC and COSY correlations of compounds 1, 13, and 16−19.

at C-5 and C-6, respectively. The relative configuration at C-16 and C-17 was established to be in the trans form, given the coupling constant (J = 3.0 Hz) between H-16 and H-17, as further supported by the NOESY cross-peaks of H-16/Me-20 and H-17/Me-19. Therefore, compound 4 (16-methoxycudratrixanthone M) was assigned as shown. The molecular formula of compound 5 was determined to be C23H24O8 on the basis of HRESIMS analysis. The 1D NMR data of 5 (Table 1) were closely similar to those of 4, except for the lack of a methoxy group signal at C-16. The gross structure of compound 5 (16-hydroxycudratrixanthone M) was deduced from additional 2D NMR studies including the COSY, HSQC, and HMBC spectra. The relative configuration at C-16 and C17 was in the trans form, as observed in 3 and 4. The molecular formula of compound 6 was determined as C24H24O7 by HRESIMS analysis. The 1H and 13C NMR spectroscopic data (Table 1) were similar to those of cudratrixanthone O,10 with a 1,3,6,7-tetraoxygenated xanthone skeleton with two C5 groups. Differences between the two compounds included the replacement of a 2-(1-methylethenyl)furan group by a 2-(1-hydroxy-1-methylethyl)furan-2-yl group [δH 7.52 (1H, s, H-16) and 1.67 (6H, s, Me-19 and Me-20)] as well as the presence of a methoxy signal at δH 3.92 (3H, s, OMe-3). The HMBC cross-peaks of Me-12 and Me-13/C-4 (δC 113.6), H-16/C-7 (δC 141.1), and OMe-3/C-3 (δC 165.8) suggested that the 1,1-dimethylallyl group is at C-4, the 2-(1hydroxy-1-methylethyl)furan-2-yl group is at C-7 and C-8, and a methoxy group is at C-3, respectively. Consequently, compound 6 (cudratrixanthone R) was assigned as shown. The molecular formula of compound 7 was determined to be C23H22O7, using HRESIMS analysis. Detailed analysis of its 1H and 13C NMR spectroscopic data (Table 2) showed that 7 possesses the same 1,3,6,7-tetraoxygenated skeleton as 6, except that a methoxy signal at C-3 was absent and the substituent patterns of the aromatic rings were changed. A 1,1-dimethylallyl and a 2-(1-hydroxy-1-methylethyl)furan-2-yl group were located at C-2, and C-5 and C-6, respectively, as indicated by the HMBC cross-peaks of Me-12 and Me-13/C-2 (δC 116.2) and H-16/C-6 (δC 149.1). Accordingly, the new compound 7 (cudratrixanthone S) was determined as shown.

Figure 2. Calculated and experimental ECD spectra of compound 1.

similarity with 2, except for the presence of a hydroxy group at C-16, as evidenced by the chemical shift at δH 4.55 (1H, d, J = 3.5 Hz, H-16). The relative configuration of the two oxygenated methine protons on the dihydrofuran ring was determined to be in the trans form based on a comparison of the coupling constant between H-16 and H-17 (J = 3.5 Hz), as further supported by the NOESY cross-peaks of H-16/Me-19 and H-17/Me-20.8 Accordingly, compound 3 (16-hydroxycudratrixanthone Q) was assigned as shown. Compound 4 gave the molecular formula C24H26O8, as deduced by HRESIMS analysis. The 1H and 13C NMR spectroscopic data (Table 1) were similar to those of cudratrixanthone M,10 having a 1,3,6,7-tetraoxygenated xanthone framework with two C5 groups, but there was found to be a methoxy group present at C-16, which was confirmed by the HMBC cross-peak of OMe-16/C-16 (δC 80.7). The HMBC cross-peaks of Me-12, Me-13, and OH-1/C-2 (δC 117.5) and of H-16/C-5 (δC 116.1) and C-6 (δC 157.4) indicated that a 2,3,3trimethyldihydrofuran group is fused at C-2 and C-3 and that a 2-(1-hydroxy-1-methylethyl)-3-methoxydihydrofuran is located D


E

29.3,

20 21

CH3

C CH3

14.24, s

1.68, s

1.68, s 18.1

145.7 123.6

198.5

37.3

CH3

C CH2

C

CH2

CH2

13.47, s

6.22, s 5.85, s 1.90, s

4.84, s

4.90, d (10.5)

1.67, s 1.67, s 6.36, dd (17.5, 10.5) 5.01, d (17.5)

6.91, s

6.21, s

18.2

148.0 110.6

76.4

30.6

108.7

163.5 115.4 164.3 95.0 156.5 150.4 114.4 152.5 144.0 107.4 113.7 181.2 103.2 41.7 29.2 29.2 150.9

CH3

C CH2

CH

CH2

CH2


δC, type

14.27, s

4.87, s 4.73, s 1.88, s

3.27, dd (14.0, 5.0) 3.18, dd (14.0, 7.5) 4.55, dd (7.5, 4.5)

4.86, d (10.5)

1.64, s 1.64, s 6.39, dd (17.5, 10.5) 4.98, d (17.5)

7.48, s

6.43, s

δH, mult (J in Hz)

18.3,

148.0, 110.6,

76.2,

30.6,

14.6,

158.6, 117.2, 166.3, 89.7, 158.9, 150.7, 114.4, 152.5, 144.1, 107.1, 113.7, 181.2, 104.0, 44.0, 25.5, 20.9, 91.6,

CH3

C CH2

CH

CH2

CH3


δC, type

13.56, s

4.85, s 4.71, s 1.89, s

3.29, dd (14.0, 4.5) 3.19, dd (14.0, 8.0) 4.55, dd (8.5, 5.0)

1.39, d (6.5)

1.49, s 1.24, s 4.52, q (6.5)

7.47, s

6.35, s

δH, mult (J in Hz)

29.0, 150.8,

41.9, 29.0,

130.2,

115.7,

156.3,

160.3, 111.9, 160.8, 110.0, 153.3, 152.7, 100.9, 154.2, 141.8, 128.9, 111.6, 183.8, 104.5, 27.7, 132.5, 130.2, 115.7,

11

CH2 CH C CH3 CH3

CH2

CH3 CH

C CH3

C

C

C

C C C C C C CH C C C C C C CH2 C CH CH

δC, type

26.4, 124.4, 131.4, 18.3, 26.1,

69.2, 29.3,

18 19

C

7.05, s

108.0


δH, mult (J in Hz)

10

23 24 25 26 27 OH

167.2,

17

CH

4.86, d (10.5)

1.65, s 1.65, s 6.40, dd (17.5, 10.5) 4.98, d (17.5)

7.51, s

6.55, s

162.0 99.6 162.3 112.1 156.2 152.7 101.6 153.2 142.4 123.0 112.2 183.2 104.3 41.7 29.5 29.5 151.6

δC, type

9

112.0,

99.0,

16

CH2


δH, mult (J in Hz)

8

22

108.7,

163.7, 116.2, 164.7, 95.2, 156.5, 145.1, 120.2, 149.1, 141.2, 104.4, 116.6, 181.6, 103.7, 41.8, 29.2, 29.2, 150.9,

δC, type

15

1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 11 12 13 14

position

7

Table 2. 1H and 13C NMR Spectroscopic Data for Compounds 7−12 in Acetone-d6

1.83, s 1.64, s 14.33, s

1.69, s 6.47, dd (18.0, 10.5) 5.35, d (17.5) 5.24, d (11.0) 4.18, d (6.5) 5.32, t (6.5)

1.69, s

7.11, d (8.5)

6.68, d (8.5)

7.11, d (8.5) 6.68, d (8.5)

3.93, s

6.89, s

δH, mult (J in Hz)

23.2, 122.7, 132.8, 18.1, 25.7,

129.9,

156.4, 115.9,

129.9, 115.9,

132.2,

28.2,

113.1,

161.8, 114.2, 161.4, 107.6, 154.4, 151.9, 116.7, 150.7, 143.1, 106.5, 116.0, 181.6, 103.4, 42.1, 28.1, 28.1, 150.6,

CH2 CH C CH3 CH3

CH

C CH

CH CH

C

CH2

CH2

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

δC, type

12

1.73, s 1.58, s 14.49, s

3.60, d (6.5) 5.21, t (6.5)

7.06, d (8.5)

6.69, d (8.5)

7.06, d (8.5) 6.69, d (8.5)

1.65, s 1.65, s 6.50, dd (17.5, 10.5) 5.42, dd (17.5) 5.31, dd (10.5) 4.11, s

7.50, s

δH, mult (J in Hz)

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Table 3. 1H and 13C NMR Spectroscopic Data for Compounds 13−16 in Acetone-d6 13 position

δC, type

2

75.1,

CH

3

42.7,

CH2

4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′

197.8, 162.8, 106.8, 166.6, 96.4, 162.9, 103.0, 118.8, 155.2, 104.2, 156.1, 114.8,

C C C C CH C C C C CH C C

6′ 1″ 2″ 3″ 4″

126.0, 122.6, 128.7, 77.1, 28.3,

CH CH CH C CH3

5″ 1‴

28.3, 29.4,

CH3 CH2

2‴

76.6,

CH

3‴ 4‴

148.3, 110.4,

C CH2

5‴ OH

18.4,

CH3

14 δH, mult (J in Hz)

5.68, dd (13.5, 3.0) 3.16, dd (17.0, 13.5) 2.75, dd (17.0, 3.0)

6.00, s

6.37, s

7.19, s 6.37, d (9.5) 5.58, d (9.5) 1.40, s 1.40, s 2.97, dd (14.5, 3.5) 2.83, dd (14.5, 8.0) 4.38, dd (8.0, 3.0) 4.93, s 4.77, s 1.82, s 12.69, s

δC, type 75.4,

CH

42.6,

CH2

197.8, 159.5, 106.8, 169.8, 90.7, 165.1, 103.7, 118.7, 159.2, 104.2, 155.2, 114.8,


126.0, 122.6, 128.7, 77.1, 28.3,

CH CH CH C CH3

28.3, 26.7,


5.68, dd (13.5, 3.0) 3.15, dd (17.0, 13.5) 2.73, dd (17.0, 3.0)

δC, type 75.4,

CH

42.6,

CH2

197.8, 159.5, 106.8, 169.7, 90.7, 165.1, 103.6, 118.6, 159.2, 104.2, 155.2, 114.8,


1.40, s

126.0, 122.6, 128.7, 77.1, 28.4,

CH CH CH C CH3

CH3 CH2

1.40, s 3.05, m

28.4, 26.7,

92.8,

CH

4.77, dd (9.5, 2.5)

71.5, 25.9,

C CH3

25.4,

CH3

5.93, s

6.38, s

7.19, s 6.35, d (10.0) 5.58, d (10.0)

1.26, s 1.23, s 12.36, s

Compound 8 showed the molecular formula C23H22O7 as determined by HRESIMS analysis. The 1H and 13C NMR data (Table 2) exhibited the characteristic signals of a 1,3,6,7tetraoxygenated xanthone framework with two C5 groups similar to those of cudratrixanthone C,10 except for the lack of a methoxy signal at C-7. Further analysis of the COSY, HSQC, and HMBC spectra suggested the structure of compound 8 as 7-O-demethylcudratrixanthone C. The molecular formula of compound 9 was determined to be C23H24O7 by HRESIMS analysis. The 1D NMR data (Table 2) were comparable to those of cudratrixanthone G,10 with the exception of the substituent patterns of the aromatic rings. In contrast to cudratrixanthone G, the HMBC cross-peaks of Me12 and Me-13/C-2 (δC 115.4) and of H-16a/C-4b (δC 150.4) and C-6 (δC 152.5) indicated that a 1,1-dimethylallyl and a 2hydroxy-3-methylbut-3-enyl group are attached at C-2 and C-5, respectively. Therefore, compound 9 (cudratrixanthone T) was assigned as shown. The 1H and 13C NMR analysis of compound 10 (Table 2) suggested a similar structure to that of 9, except that a 1,1dimethylallyl group was replaced by a 2,3,3-trimethyldihydrofuran group. The HMBC cross-peaks of Me-12 and Me-13/C-2


5.68, dd (13.5, 3.0) 3.15, dd (17.0, 13.5) 2.73, dd (17.0, 3.0)

δC, type

δH, mult (J in Hz)

163.6,

C

117.6,

C

182.6, 156.8, 105.7, 160.2, 95.5, 158.4, 105.3, 112.1, 157.4, 103.9, 161.9, 108.2,

C C C C CH C C C C CH C CH

1.40, s

132.1, 35.3, 198.2, 145.0, 125.0,

CH CH2 C C CH2

CH3 CH2

1.40, s 3.06, m

17.8, 115.9,

CH3 CH

6.07, 5.80, 1.81, 6.67,

92.8,

CH

4.77, dd (9.5, 3.0)

129.3,

CH

5.75, d (10.0)

71.4, 25.9,

C CH3

78.7, 28.3,

C CH3

1.46, s

25.4,

CH3

28.3,

CH3

5.92, s

6.38, s

7.19, s 6.37, d (10.0) 5.58, d (10.0)

1.27, s 1.22, s 12.35, s

6.32, s

6.55, d (2.0) 6.47, dd (8.5, 2.5) 7.15, d (8.0) 3.83, s

s s s d (10.0)

1.46, s 13.25, s

(δC 117.2) suggested the position of this group at C-2 and C-3. The positions of the remaining functional groups were determined by additional 2D NMR spectra, and the structure of compound 10 (cudratrixanthone U) was confirmed from its HRESIMS data. According to their HRESIMS data, compounds 11 and 12 were found to share the same molecular formula of C30H30O7. Detailed analysis of the 1H and 13C NMR spectroscopic data (Table 2) suggested that 11 and 12 possess 1,3,6,7tetraoxygenated xanthone skeletons, with both possessing a 1,1-dimethylallyl group, a p-substituted benzyl group, and a prenyl group. Differences between 11 and 12 involved the respective substituent patterns of their aromatic rings. In 11, the HMBC cross-peaks of H-11/C-1 (δC 160.3) and C-3 (δC 160.8), Me-19 and Me-20/C-4 (δC 110.0), and H-23/C-8a (δC 111.6) indicated the positions of the 1,1-dimethylallyl group, the p-substituted benzyl group, and the prenyl group to be at C2, C-4, and C-8, respectively. In contrast to 11, the HMBC cross-peaks of Me-12 and Me-13/C-2 (δC 114.2), H-16/C-3 (δC 161.4) and C-4a (δC 154.4), and H-23/C-6 (δC 150.7) in 12 suggested that the 1,1-dimethylallyl group, the p-substituted benzyl group, and the prenyl group are attached at C-2, C-4, F



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Table 4. 1H and 13C NMR Spectroscopic Data for Compounds 17−21 in Acetone-d6 17 position

δC, type


δC, type


1 2

115.4, 152.1,

C C

157.5,

CH

3

138.5,

C

111.5,

CH

4

152.6,

C

182.6,

5 6 7

123.8, 126.9, 37.1,

C CH CH2

160.5, 110.3, 164.2,

8

76.1,

CH

9 10

148.9, 110.7,

C CH2

4.82, s

157.5, 106.3,

11

18.0,

CH3

4.75, s 1.75, s

29.6,

CH2

12

201.5,

C

76.3,

CH

13

140.5,

C

148.2,

C

14

116.5,

CH

110.4,

CH2

4.93, s

15 16

158.2, 119.7,

C CH

18.3,

CH3

4.76, s 1.83, s

17

130.3,

CH

18

121.3,

CH

7.10, s 2.83, dd (13.5, 5.0) 2.64, dd (13.0, 8.0) 4.19, dd (8.0, 5.0)

δC, type 122.2, 162.8,

C C

103.2,

CH

C

163.0,

C

C C C

105.0, 132.2, 171.3,

C CH C

34.6,

CH2

132.5, 130.5,

C CH

115.9,

CH

156.5,

C

115.9,

CH

130.5,

CH

8.04, d (5.5) 6.21, d (5.5)


δC, type 116.3, 162.2,

C C

108.3,

CH

129.7,

CH

7.52, s

105.2, 162.3, 171.7,

C C C

3.79, s

28.0,

CH2

132.7, 130.4,

C CH

115.5,

CH

156.1,

C

115.5,

CH

130.4,

CH

6.45, s


6.55, d (8.5) 7.59, d (8.5)

116.2, 159.9,

C C

121.3,

C

130.0,

CH

105.2, 160.9, 171.8,

C C C

δH, mult (J in Hz)

7.46, s

35.1,

CH2

131.8, 130.5,

C CH

116.0,

CH

156.3,

C

116.0,

CH

130.5,

CH

28.1, 132.0,

CH2 C

3.98, s

130.2,

CH

115.7,

CH

7.09, d (8.0) 6.68, d (8.5)

19 20

156.6, 115.7,

C CH

21

130.2,

CH

52.4,

CH3

OH OMe

60.6,

CH3

7.17, d (2.5)

95.3,

CH

6.42, s

C C

3.05, dd (14.0, 3.0) 2.91, dd (14.0, 8.0) 4.42, dd (8.0, 3.0)

7.06, d (8.0) 6.74, d (8.0)

6.74, d (8.0) 7.06, d (8.0)

3.90, s

δC, type

7.15, d (8.0) 6.67, d (8.5)

6.67, d (8.5) 7.15, d (8.0)

7.11, d (8.0) 7.38, t (8.0) 7.16, d (8.0)

12.24, s 4.04, s

13.24, s 52.3,

CH3

10.78, s 3.86, s

52.4,

CH3

11.30, s 3.89, s

3.89, s

7.02, d (8.5) 6.75, d (8.5)

6.75, d (8.5) 7.02, d (8.5)

6.68, d (8.5) 7.09, d (8.0) 11.22, s 3.86, s

(1H, s, H-4‴a), 4.77 (1H, s, H-4‴b), 4.38 (1H, dd, J = 8.0, 3.0 Hz, H-2‴), 2.97 (1H, dd, J = 14.5, 3.5 Hz, H-1‴a), 2.83 (1H, dd, J = 14.5, 8.0 Hz, H-1‴b), and 1.82 (3H, s, Me-5‴) was observed. The 13C NMR spectroscopic data (Table 3) revealed the presence of 25 carbon signals, including three methyl carbons at δC 28.3 (C-4″ and C-5″) and 18.4 (C-5‴), three methylene carbons at δC 110.4 (C-4‴), 42.7 (C-3), and 29.4 (C-1‴), seven methine carbons at δC 128.7 (C-2″), 126.0 (C6′), 122.6 (C-1″), 104.2 (C-3′), 96.4 (C-8), 76.6 (C-2‴), and 75.1 (C-2), a carbonyl carbon at δC 197.8 (C-4), six oxygenated tertiary carbons at δC 166.6 (C-7), 162.9 (C-9), 162.8 (C-5), 156.1 (C-4′), 155.2 (C-2′), and 77.1 (C-3″), and five quaternary carbons at δC 148.3 (C-3‴), 118.8 (C-1′), 114.8 (C-5′), 106.8 (C-6), and 103.0 (C-10). The HMBC crosspeaks of H-1″ and H-2″/C-5′ (δC 114.8) and of H-1‴b/C-5 (δC 162.8) and C-7 (δC 166.6) suggested that the 2,2-

and C-5, respectively. Further analysis of the COSY, HSQC, and HMBC spectroscopic data supported the structures proposed for compounds 11 (cudratrixanthone V) and 12 (cudratrixanthone W) as shown. Compound 13 was obtained as a brown, amorphous solid with a molecular formula of C25H26O7, consistent with 13 degrees of unsaturation, as evidenced by HRESIMS analysis. The 1H NMR spectroscopic data (Table 3) showed characteristic signals of a 5-hydroxyflavanone moiety at δH 12.69 (1H, s, OH-5), 5.68 (1H, dd, J = 13.5, 3.0 Hz, H-2), 3.16 (1H, dd, J = 17.0, 13.5 Hz, H-3a), and 2.75 (1H, dd, J = 17.0, 3.0 Hz, H-3b) as well as three aromatic methine signals at δH 7.19 (1H, s, H6′), 6.37 (1H, s, H-3′), and 6.00 (1H, s, H-8). Additionally, a 2,2-dimethylpyran group at δH 6.37 (1H, d, J = 9.5 Hz, H-1″), 5.58 (1H, d, J = 9.5 Hz, H-2″), and 1.40 (6H, s, Me-4″ and Me5″) as well as a 2-hydroxy-3-methylbut-3-enyl group at δH 4.93 G



Article

NMR analysis (Figure 1). Therefore, the new compound 16 (cudraflavone H) was assigned as shown. Compound 17, isolated as a yellow, amorphous solid, exhibited a molecular formula of C19H20O6, suggesting 10 degrees of unsaturation, as deduced by HRESIMS analysis. The 1 H NMR spectroscopic data (Table 4) gave evidence of a chelated hydroxy group at δH 12.24 (1H, s, OH-2), five aromatic methine signals at δH 7.38 (1H, t, J = 8.0 Hz, H-17), 7.17 (1H, d, J = 2.5 Hz, H-14), 7.16 (1H, d, J = 8.0 Hz, H-18), 7.11 (1H, d, J = 8.0 Hz, H-16), and 7.10 (1H, s, H-6), a 2hydroxy-3-methylbut-3-enyl group at δH 4.82 (1H, s, H-10a), 4.75 (1H, s, H-10b), 4.19 (1H, dd, J = 8.0, 5.0 Hz, H-8), 2.83 (1H, dd, J = 13.5, 5.0 Hz, H-7a), 2.64 (1H, dd, J = 13.0, 8.0 Hz, H-7b), and 1.75 (3H, s, Me-11), and a methoxy group signal at δH 4.04 (3H, s, OMe-4). The 13C NMR spectroscopic data (Table 4) showed 19 carbon resonances assigned to a methoxy carbon at δC 60.6 (OMe-4), a methyl carbon at δC 18.0 (C-11), two methylene carbons at δC 110.7 (C-10) and 37.1 (C-7), six methine carbons at δC 130.3 (C-17), 126.9 (C-6), 121.3 (C18), 119.7 (C-16), 116.5 (C-14), and 76.1 (C-8), a carbonyl carbon at δC 201.5 (C-12), four oxygenated tertiary carbons at δC 158.2 (C-15), 152.1 (C-2), 152.6 (C-4), and 138.5 (C-3), and four quaternary carbons at δC 148.9 (C-9), 140.5 (C-13), 123.8 (C-5), and 115.4 (C-1). These 1D NMR data were closely comparable to those of cudracuspiphenone A,17 which contains a benzophenone carbon framework with a C5 group. The only difference was that a prenyl group is replaced by the 2-hydroxy-3-methylbut-3-enyl group. Additional 2D NMR data, including HMBC, suggested the structure of compound 17 (cudraphenone E) (Figure 1). Compound 18, isolated as a yellow, amorphous solid, gave an elemental formula of C14H14O5 as determined by HRESIMS analysis, indicating eight degrees of unsaturation. The 1H NMR data (Table 4) showed the characteristic signals of a 5hydroxychromone moiety at δH 13.24 (1H, s, OH-5), 8.04 (1H, d, J = 5.5 Hz, H-2), and 6.21 (1H, d, J = 5.5 Hz, H-3). Moreover, an aromatic proton signal at δH 6.42 (1H, s, H-8) and the signals indicating the occurrence of a 2-hydroxy-3methylbut-3-enyl group at δH 4.93 (1H, s, H-14a), 4.76 (1H, s, H-14b), 4.42 (1H, dd, J = 8.0, 3.0 Hz, H-12), 3.05 (1H, dd, J = 14.0, 3.0 Hz, H-11a), 2.91 (1H, d, J = 14.0, 8.0 Hz, H-11b), and 1.83 (3H, s, Me-15) were observed. The 13C NMR data (Table 4) exhibited 14 carbon signals, consisting of one methyl carbon at δC 18.3 (C-15), two methylene carbons at δC 110.4 (C-14) and 29.6 (C-11), four methine carbons at δC 157.5 (C-2), 111.5 (C-3), 95.3 (C-8), and 76.3 (C-12), a carbonyl carbon at δC 182.6 (C-4), three oxygenated tertiary carbons at δC 164.2 (C7), 160.5 (C-5), and 157.5 (C-9), and three quaternary carbons at δC 148.2 (C-13), 110.3 (C-6), and 106.3 (C-10). These data were closely comparable with those of 5,7-dihydroxy-6prenylchromone, except that a prenyl group was replaced by the 2-hydroxy-3-methylbut-3-enyl group,18 as evidenced by the 2D NMR studies (Figure 1). Consequently, the new compound 18 (cudrachromone A) was assigned as shown. Compound 19, isolated as a yellow, amorphous solid, showed a molecular formula of C15H14O5, as deduced by HRESIMS analysis. The 1H NMR spectroscopic data (Table 4) exhibited a sharp hydroxy group signal at δH 10.78 (1H, s, OH4), six aromatic methine signals at δH 7.52 (1H, s, H-6), 7.06 (2H, d, J = 8.0 Hz, H-10 and H-14), 6.74 (2H, d, J = 8.0 Hz, H11 and H-13), and 6.45 (1H, s, H-3), a methylene signal at δH 3.79 (2H, s, H-8), and a methyl ester signal at δH 3.86 (3H, s, COOMe). The 13C NMR spectroscopic data (Table 4) showed

dimethylpyran group is fused at C-4′ and C-5′ and that the 2hydroxy-3-methylbut-3-enyl group is attached at C-6, respectively. Furthermore, the positions of the remaining substituents were deduced by additional 2D NMR experiments (Figure 1). Consequently, compound 13 (cudraflavanone G) was assigned as shown. The absolute configuration of 13 at C-2 was determined by the ECD spectroscopic technique. The positive Cotton effect (CE) for the n → π* transition at 342 nm (Δε +5.5) and the negative CE for the π → π* transition at 290 nm (Δε −30.4) indicated a 2S configuration, which was consistent with previous reports.15 However, the absolute configuration of C-2‴ was not determined, despite comparing the experimental and calculated ECD spectra, because both predicted ECD curves of the 2‴R and 2‴S conformers exhibited almost the same patterns (Figure S67, Supporting Information). The molecular formulas of compounds 14 and 15 were determined to be C25H26O7 based on HRESIMS analysis. The 1 H and 13 C NMR spectroscopic data of 14 and 15 demonstrated almost the same signals and indicated that they possess a 5-hydroxyflavanone framework with two C5 groups similar to those of 13, except for the replacement of a 2hydroxy-3-methylbut-3-enyl group by a 2-(1-hydroxy-1methylethyl)dihydrofuran group. Additional 2D NMR data such as HMBC suggested the entire structures of 14 and 15. The absolute configurations at C-2 were assigned as R and S for 14 and 15, respectively. While the ECD spectrum of 14 exhibited a negative CE at 337 nm (Δε −1.4) and a positive CE at 295 nm (Δε +6.0), the ECD spectrum of 15 showed a positive CE at 335 nm (Δε +2.6) and a negative CE at 297 nm (Δε −5.1).15 However, the absolute configurations of C-2‴ were not determined, as was the case for 13 (Figure S67, Supporting Information). Accordingly, the structures of compounds 14 and 15 were elucidated as shown, and they have been given the trivial names of (2R)- and (2S)cudraflavanones H, respectively. Compound 16, obtained as a yellow, amorphous solid, was assigned a molecular formula of C25H22O7, corresponding to 15 degrees of unsaturation, as determined via HRESIMS analysis. The 1H NMR data (Table 3) showed the presence of a hydroxy group at δH 13.25 (1H, s, OH-2), a characteristic signal of a 5hydroxyflavone framework. In addition, four aromatic methine signals were observed at δH 7.15 (1H, d, J = 8.0 Hz, H-6′), 6.55 (1H, d, J = 2.0 Hz, H-3′), 6.47 (1H, dd, J = 8.5, 2.5 Hz, H-5′), and 6.32 (1H, s, H-8). Furthermore, a 2-oxo-3-methylbut-3enyl group at δH 6.07 (1H, s, H-4″a), 5.80 (1H, s, H-4″b), 3.83 (2H, s, H-1″), and 1.81 (3H, s, Me-5″) and a 2,2-dimethylpyran group at δH 6.67 (1H, d, J = 10.0 Hz, H-1‴), 5.75 (1H, d, J = 10.0 Hz, H-2‴), and 1.46 (6H, s, Me-4‴ and Me-5‴) were observed. The 13C NMR data (Table 3) suggested the presence of 25 carbon resonances assigned to three methyl carbons at δC 28.3 (C-4‴ and C-5‴) and 17.8 (C-5″), two methylene carbons at δC 125.0 (C-4″) and 35.3 (C-1″), six methine carbons at δC 132.1 (C-6′), 129.3 (C-2‴), 115.9 (C-1‴), 108.2 (C-5′), 103.9 (C-3′), and 95.5 (C-8), two carbonyl carbons at δC 198.2 (C2″) and 182.6 (C-4), seven oxygenated tertiary carbons at δC 163.6 (C-2), 161.9 (C-4′), 160.2 (C-7), 158.4 (C-9), 157.4 (C2′), 156.8 (C-5), and 78.7 (C-3‴), and five quaternary carbons at δC 145.0 (C-3″), 117.6 (C-3), 112.1 (C-1′), 105.7 (C-6), and 105.3 (C-10). Analysis of the 1D NMR data suggested the structural similarity of compound 16 to cudraflavone B,16 and the only difference was the replacement of a prenyl group by the 2-oxo-3-methylbut-3-enyl group, as evidenced by the 2D H



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15 carbon resonances assigned to a methylene carbon at δC 34.6 (C-8), six methine carbons at δC 132.2 (C-6), 130.5 (C-10 and C-14), 115.9 (C-11 and C-13), and 103.2 (C-3), the methyl ester carbon at δC 171.3 (C-7) and 52.2 (COOMe), three oxygenated tertiary carbons at δC 163.0 (C-4), 162.8 (C2), and 156.5 (C-12), and three quaternary carbons at δC 132.5 (C-9), 122.2 (C-1), and 105.0 (C-5). The positions of all substituents were determined by detailed analysis of the 2D NMR spectra, including HMBC (Figure 1). Accordingly, the new compound 19 (cudraphenol A) was assigned as shown. The molecular formula of compound 20 was determined to be C15H14O5, as deduced by HRESIMS analysis. The 1H and 13 C NMR data (Table 4) were similar to those of 19, except that the position of the hydroxy group in 20 was transposed from C-4 to C-6, as evidenced by the aromatic proton signals at δH 7.59 (1H, d, J = 8.5 Hz, H-4) and 6.55 (1H, d, J = 8.5 Hz, H-3) as well as the HMBC cross-peaks of OH-6/C-1 (δC 116.3), C-5 (δC 105.2), and C-6 (δC 162.3) and H-8/C-6. Consequently, the new compound 20 (cudraphenol B) was determined as shown. Compound 21 was assigned a molecular formula of C22H20O6, as determined by HRESIMS analysis. The 1H and 13 C NMR spectroscopic data (Table 4) were comparable to those of 20, except for the presence of a p-substituted benzyl group at C-3, as evidenced by the HMBC cross-peaks of H-15/ C-2 (δC 159.9) and C-3 (δC 121.3). Further analysis of the COSY, HSQC, and HMBC spectroscopic data supported the structure of the new compound 21 (cudraphenol C) as shown. Additionally, 54 previously reported compounds were identified as dicycloeuchrestaflavanone B (22),19 cudraflavone A (23),16 5,7-dihydroxychromone (24),20 isoimperatorin (25),21 xanthyletin (26),22 4-hydroxybenzaldehyde (27),23 glutinol (28),24 achilleol A (29),25 oleic acid (30),26 demethylsuberosin (31),27 5-dehydroxybavachinone A (32),28 imperatorin (33),21 isoencecalin (34),29 2,4-dihydroxymethylbenzoate (35),30 4-(methoxymethyl)phenol (36),31 α-amyrin (37),32 gentisein,33 toxyloxanthone B,34 2-deprenylrheediaxanthone B,35 4′-O-demethylcrotaramin,36 brosimine B,37 pinocembrin,38 euchrestaflavanone C,39 4′-hydroxyisolonchocarpin,40 naringenin,41 6-prenylnaringenin,42 tomentosanol D,43 dalenin,44 parvisoflavone A,45 alpinumisoflavone,12 8-hydroxygenistein,46 laburnetin,47 biochanin A,48 erythrinins B, C, and G,11,49 cudracuspiphenone A,17 eriosematin A,50 decursinol angelate,51 cis-3′,4′-diisovalerylkhellactone,52 7-hydroxycoumarin,53 8-methoxypsoralen,54 bergapten,55 hyuganin C,56 4hydroxy-3-(4-hydroxybenzyl)benzyl methyl ether,57 2,4-bis(4hydroxybenzyl)phenol,58 4-hydroxymethylbenzoate,59 vanillin,60 3-methoxycarbonylindole,61 4-hydroxybenzalacetone,62 lanosterol,63 γ-hexadecanolactone,64 hexadecanoic acid,65 and 9,12-octadecadienoic acid,66 by comparing their spectroscopic data with published literature values. All isolated compounds were evaluated for their neuroprotective effects against 6-OHDA-induced cell death, with curcumin used as a positive control (EC50: 6.2 μM), and compounds 22−30 exhibited activities with EC50 values of 1.9− 30.2 μM (Table 5). Moreover, the 75 isolated compounds as well as 34 previously reported xanthones were evaluated in two other neurodegenerative disease models, as summarized in Table 6. Consequently, compounds 6, 31, and cudratricusxanthone J showed potent or moderate neuroprotective effects on MPP+-induced neurotoxicity with EC50 values of 0.2−10.3 μM, which were comparable to that of betulinic acid when used as a positive control (EC50: 4.3 μM). 6-OHDA and MPP+ generate

Table 5. Neuroprotective Effects of Isolated Compounds against 6-OHDA-Induced Cell Death EC50 (μM)a compound

6-OHDA

22 23 24 25 26 27 28 29 30 curcumin

9.1 15.5 1.9 9.2 8.0 30.2 12.9 7.2 9.5 6.2

a The EC50 is defined as the concentration affording half-maximal protection and is expressed in each case as the mean of triplicate determinations.

Table 6. Neuroprotective Effects of Isolated Compounds against MPP+- and OGD-Induced Cell Death EC50 (μM)a compound

MPP+

OGD

1 6 23 25 29 31 32 33 34 35 36 37 cudratricusxanthone J cudratrixanthone D cudratrixanthone E cudratrixanthone H cudratrixanthone I cudratrixanthone J cudratrixanthone K cudratrixanthone M cudratrixanthone N 3-O-methylcudratrixanthone G cudraxanthone D 3-O-demethylcudraxanthone B gerontoxanthone C positive control

>50.0 10.3 >50.0 >50.0 >50.0 0.2 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 8.1 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 >50.0 4.3b

12.2 >50.0 13.5 3.5 6.5 8.6 8.8 8.4 22.3 35.5 7.9 13.3 >50.0 13.2 28.5 34.9 23.6 11.2 8.9 6.0 3.5 2.9 4.8 33.3 17.2 3.0c

a The EC50 is defined as the concentration affording half-maximal protection and is expressed in each case as the mean of triplicate determinations. bBetulinic acid. cCarnosine.

excessive reactive oxygen species and elicit dopaminergic neuronal cell death. Furthermore, compounds 1, 23, 25, 29, and 31−37, cudratrixanthones D, E, H−K, M, and N, 3-Omethylcudratrixanthone G, cudraxanthone D, 3-O-demethylcudraxanthone B, and gerontoxanthone C exhibited neuroprotective activities against OGD-induced cell death with EC50 values of 2.9−35.5 μM when using carnosine as a positive control (EC50: 3.0 μM). OGD provides conditions that I



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mixtures of n-hexane/CHCl3 (10:1 to 0:1) to give oleic acid (30, 15.4 mg). Fr 4.4.7.2 (1.1 g) was chromatographed on a silica gel MPLC column with CHCl3/MeOH mixtures (1:0 to 1:1) to yield four fractions (Fr 4.4.7.2.1 to Fr 4.4.7.2.4). Fr 4.4.7.2.3 (120.0 mg) was subjected to a C18 RP silica gel column with MeOH/H2O mixtures (9:1 to 1:0) to give five fractions, with purification by RP-HPLC with MeOH/H2O mixtures (4:1 to 9:1) to yield cudraflavone A (23, 11.0 mg). Fr 4.4.7.4 (350.0 mg) was fractionated on a C18 RP silica gel column with MeOH/H2O mixtures (9:1 to 1:0) to afford five fractions, with purification by RP-HPLC with MeOH/H2O mixtures (7:3 to 9:1), to give dicycloeuchrestaflavanone B (22, 1.8 mg), 2,4dihydroxymethylbenzoate (35, 4.2 mg), 4′-O-demethylcrotaramin (2.9 mg), and hexadecanoic acid (16.7 mg). Fr 4.4.7.5 (200.0 mg) was subjected to a C18 RP silica gel column with MeOH/H2O mixtures (9:1 to 1:0) to give five fractions, followed by purification with RPHPLC using MeOH/H2O mixtures (17:3 to 9:1), to afford 9,12octadecadienoic acid (4.5 mg). Fr 4.4.7.6 (280.0 mg) was chromatographed on a C18 RP silica gel column with MeOH/H2O (9:1 to 1:0) mixtures to yield six fractions (Fr 4.4.7.6.1 to Fr 4.4.7.6.6). Fr 4.4.7.6.1 (43.0 mg) was purified by RP HPLC with MeOH/H2O mixtures (17:3 to 9:1) to afford cudraphenol A (19, 4.3 mg), cudraphenol B (20, 2.9 mg), cudraphenol C (21, 1.7 mg), and 4-hydroxymethylbenzoate (5.3 mg). Fr 4.4.4.8 (150.0 mg) was chromatographed on a silica gel column to afford γ-hexadecanolactone (120.0 mg). Fr 4.4.7.7 (124.0 mg) was subjected to passage over a C18 RP silica gel column with MeOH/H2O mixtures (4:1 to 1:0) to give seven fractions and then purification by RP-HPLC with MeOH/H2O (7:3) to yield 4(methoxymethyl)phenol (36, 9.6 mg). Fr 4.4.8 (418.8 mg) was separated on a silica gel column with CHCl3/MeOH mixtures (100:1 to 10:1) to yield 13 fractions (Fr 4.4.8.1 to Fr 4.4.8.13). Fr 4.5 (7.2 g) was fractionated on a silica gel column with mixtures of CHCl3/ MeOH (1:0 to 0:1) to yield seven fractions (Fr 4.5.1 to Fr 4.5.7). Fr 4.5.2 (4.3 g) was chromatographed on a C18 RP silica gel column with MeOH/H2O mixtures (8:2 to 1:0) to yield 15 fractions (Fr 4.5.2.1 to Fr 4.5.2.15). Fr 4.5.2.1 (99.5 mg) was purified by RP-HPLC with MeOH/H2O mixtures (4:1 to 1:0) to afford 4-hydroxybenzaldehyde (27, 3.0 mg), vanillin (2.0 mg), and 3-methoxycarbonylindole (1.3 mg), and the remaining impure fraction obtained was further separated on a silica gel column with n-hexane/EtOAc (5:1) to give 4-hydroxy-3(4-hydroxybenzyl)benzyl methyl ether (1.8 mg) and 2,4-bis(4hydroxybenzyl)phenol (1.3 mg). Fr 4.5.2.2 (37.1 mg) was separated by RP-HPLC with mixtures of MeOH/H2O (1:1 to 1:17) to give imperatorin (33, 2.2 mg), xanthyletin (26, 3.5 mg), demethylsuberosin (31, 2.9 mg), brosimine B (2.0 mg), and pinocembrin (1.0 mg). Fr 4.5.2.3 (270.0 mg) was separated on a silica gel MPLC column with mixtures of n-hexane/EtOAc (5:1 to 1:1) to afford eight fractions (Fr 4.5.2.3.1 to Fr 4.5.2.3.8). Fr 4.5.2.3.2 (39.0 mg) and Fr 4.5.2.3.3 (39.0 mg) were separated by RP-HPLC with mixtures of MeOH/H2O (3:2 to 4:1) to give decursinol angelate (15.6 mg) and parvisoflavone A (1.3 mg), respectively. Fr 4.5.2.3.4 (32.7 mg) was separated by RPHPLC with mixtures of MeOH/H2O (7:3 to 19:1) to obtain cudraflavanone G (13, 7.4 mg). Fr 4.5.2.4 (382.0 mg) was separated on a silica gel MPLC column with n-hexane/EtOAc mixtures (5:1 to 1:1) to give seven fractions (Fr 4.5.2.4.1 to Fr 4.5.2.4.7). Fr 4.5.2.4.6 (16.0 mg) was purified by RP-HPLC with MeOH/H2O mixtures (7:3 to 9:1) to yield (2R)-cudraflavanone H (14, 1.5 mg) and (2S)cudraflavanone H (15, 4.6 mg). Fr 4.5.2.5 (69.4 mg) was separated by RP-HPLC with MeOH/H2O mixtures (7:3 to 1:0) to give euchrestaflavanone C (6.9 mg). Fr 4.5.2.6 (420 mg) was separated on a silica gel MPLC column with mixtures of CHCl3/MeOH (100:1 to 1:1) to yield six fractions (Fr 4.5.2.6.1 to Fr 4.5.2.6.6). Fr 4.5.2.6.2 (230.0 mg) was fractionated on a silica gel column with CHCl3/ acetone (10:1) to give seven fractions, followed by RP-HPLC purification with MeOH/H2O mixtures (7:3 to 1:0) to afford 4′hydroxyisolonchocarpin (1.0 mg) and alpinumisoflavone (2.3 mg). Fr 4.5.2.7 (185.0 mg) was separated using a silica gel column with mixtures of CHCl3/acetone (50:1 to 1:1) to give seven fractions, followed by purification with RP HPLC using MeOH/H2O mixtures (7:3 to 1:0) to obtain cis-3′,4′-diisovalerylkhellactone (1.8 mg). Fr 4.5.3 (270.6 mg) was chromatographed on a C18 RP silica gel column

produce neuronal damage in diverse cell types during cerebral ischemia.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-2000 (JASCO, Tokyo, Japan). UV spectra were obtained by an Optizen POP UV−vis spectrophotometer (Mecasys, Daejeon, Korea). IR spectra were obtained with a Varian 640 FT-IR spectrometer (Varian, Palo Alto, CA, USA). ECD spectra were obtained with a JASCO J-1100 spectropolarimeter. NMR spectra were recorded on a Varian 500 MHz NMR spectrometer with tetramethylsilane as an internal standard. ESITOFMS data were acquired on a Waters Q-TOF micromass spectrometer. MPLC was performed using a Biotage Isolera One system with a SNAP cartridge KP-Sil 100 g (Biotage AB, Uppsala, Sweden). HPLC was performed with a Waters system comprising a 515 pump and a 2996 PDA detector with a YMC J’sphere ODS-H80 column (4 μm, 250 × 10 mm i.d., YMC, Kyoto, Japan) and a YMC Pack ODS-A column (5 μm, 250 × 20 mm i.d.). Column chromatography was carried out using silica gel (230−400 mesh, Merck, Darmstadt, Germany), C18 reversed-phase (RP) silica gel (12 μm, YMC), and Sephadex LH-20 gel (18−111 μm, GE Healthcare AB, Stockholm, Sweden). TLC was performed using plates precoated with silica gel (0.25 mm, Merck). 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 6hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium (MPP+), streptomycin, carnosine, and betulinic acid (≥97.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Curcumin (≥98.5%) was purchased from Enzo Life Science (Farmingdale, NY, USA). Plant Material. The root bark from C. tricuspidata was collected by the Korea Forest Research Institute, Southern Forest Research Center, Jinju, Korea, in September 2008 and authenticated by Dr. Hak Ju Lee. A voucher specimen (accession number KH1-4-090814) was deposited at the Department of Biosystems and Biotechnology, Korea University, Seoul, Korea. Extraction and Isolation. The dried root bark of C. tricuspidata (13.0 kg) was ground and extracted with MeOH (48, 20, and 18 L) at room temperature, and the combined extract was evaporated under reduced pressure. The dark brown extract (702.1 g) was suspended in H2O and partitioned sequentially with n-hexane and EtOAc to give an EtOAc-soluble extract (213.0 g) and a hexane-soluble residue (69.2 g). The EtOAc extract was subjected to passage over a silica gel column with CHCl3/MeOH mixtures (1:0 to 1:1) to yield seven fractions (Fr 1 to Fr 7). Fr 4 (36.0 g) was further fractionated on a silica gel column with mixtures of n-hexane/EtOAc (30:1 to 0:1) to afford eight fractions (Fr 4.1 to Fr 4.8). Fr 4.3 (3.4 g) was precipitated with MeOH, and the MeOH-soluble layer was further chromatographed on a silica gel column with n-hexane/EtOAc mixtures (40:1 to 1:1) to yield seven fractions (Fr 4.3.1 to Fr 4.3.7). Fr 4.3.3 (900.0 mg) was purified using a Sephadex LH-20 column with CHCl3/MeOH (2:1) and finally purified by RP-HPLC with MeOH/H2O (1:0) to give αamyrin (37, 7.0 mg) and lanosterol (5.3 mg). Fr 4.3.2 (81.5 mg) was applied to a C18 RP silica gel column with MeOH/H2O mixtures (9:1 to 1:0) to give eight fractions (Fr 4.3.2.1 to Fr 4.3.2.8). Fr 4.3.2.1 (11.0 mg) was purified by RP-HPLC with MeOH/H2O (3:1) to afford isoencecalin (34, 4.8 mg). Fr 4.3.2.7 (10.0 mg) was subjected to passage over a silica gel column with n-hexane/CHCl3 (1:1) to yield achilleol A (29, 7.4 mg). Fr 4.3.2.8 (15.0 mg) was separated by silica gel column chromatography with n-hexane/CHCl3 mixtures (2:1 to 1:1) to give glutinol (28, 8.0 mg). Fr 4.4 (4.8 g) was applied to a silica gel column with n-hexane/CHCl3/MeOH mixtures (1:10:0 to 0:5:1) to give 10 fractions (Fr 4.4.1 to Fr 4.4.10). Fr 4.4.6 (620.0 mg) was precipitated with MeOH, and the MeOH-soluble layer was further chromatographed on a silica gel column with CHCl3/MeOH (100:1) to give five fractions, with purification by RP-HPLC with MeOH/H2O mixtures (9:1 to 1:0) to yield isoimperatorin (25, 3.3 mg). Fr 4.4.7 (2.5 g) was fractionated on a silica gel MPLC column with CHCl3/ acetone mixtures (1:0 to 0:1) to yield eight fractions (Fr 4.4.7.1 to Fr 4.4.7.8). Fr 4.4.7.1 (69.0 mg) was subjected to a silica gel column with J



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with MeOH/H2O mixtures (3:2 to 1:0) to give 10 fractions and then further separated by RP-HPLC with MeOH/H2O mixtures (7:3 to 1:0) to afford 16-methoxycudratrixanthone M (4, 4.9 mg) and cudratrixanthone R (6, 2.5 mg). Fr 4.5.4 (175.5 mg) was fractionated on a C18 RP silica gel column with MeOH/H2O mixtures (7:3 to 1:0) to yield six fractions followed by RP-HPLC with MeOH/H2O mixtures (3:2 to 1:0) to afford cudratrixanthone Q (2, 6.3 mg). Fr 4.5.5 (1.1 g) was subjected to passage over a C18 RP silica gel column with MeOH/H2O mixtures (7:3 to 1:0) to obtain eight fractions (Fr 4.5.5.1 to Fr 4.5.5.8). Fr 4.5.5.1 (16.0 mg) and Fr 4.5.5.3 (28.6 mg) were separated by RP-HPLC with MeOH/H2O mixtures (1:4 to 1:0) to afford 7-O-demethylcudratrixanthone C (8, 3.4 mg), cudraphenone E (17, 2.5 mg), and cudracuspiphenone A (3.0 mg), respectively. Fr 4.5.5.4 (60.0 mg), Fr 4.5.5.5 (80.0 mg), and Fr 4.5.5.6 (54.0 mg) were purified by RP-HPLC with mixtures of MeOH/H2O (1:4 to 1:0) to obtain cudratrixanthone P (1, 2.7 mg), cudratrixanthone T (9, 62.0 mg), and cudratrixanthone S (7, 2.9 mg), respectively. Fr 4.5.5.7 (133.5 mg) was separated by RP-HPLC with MeOH/H2O mixtures (1:4 to 1:0) to give cudratrixanthone U (10, 62.0 mg). Fr 4.5.5.8 (620.0 mg) was chromatographed on a Sephadex LH-20 column with CHCl3/MeOH (1:1) to yield five fractions and then further purified by RP-HPLC with MeOH/H2O mixtures (1:1 to 1:0) to give cudratrixanthone V (11, 5.6 mg) and cudratrixanthone W (12, 9.6 mg). Fr 4.5.6 (500.0 mg) was fractionated on a Sephadex LH-20 column with CHCl3/MeOH (1:1) to give seven fractions, followed by purification with RP-HPLC using MeOH/H2O mixtures (1:1 to 1:0), to obtain 16-hydroxycudratrixanthone M (5, 6.1 mg) and 16hydroxycudratrixanthone Q (3, 5.2 mg). Fr 4.6 (8.1 g) was subjected to a C18 RP silica gel column with MeOH/H2O mixtures (3:2 to 1:0) to yield 26 fractions (Fr 4.6.1 to Fr 4.6.26). Fr 4.6.2 (42.9 mg) and Fr 4.6.3 (93.3 mg) were purified by RP-HPLC with MeOH/H2O mixtures (1:4 to 3:2) to afford 5,7-dihydroxychromone (24, 17.6 mg), naringenin (7.6 mg), 8-hydroxygenistein (1.6 mg), 7-hydroxycoumarin (6.6 mg), 8-methoxypsoralen (3.3 mg), and 4-hydroxybenzalacetone (2.8 mg). Fr 4.6.4 (78.3 mg) was purified by RP-HPLC with MeOH/ H2O mixtures (1:4 to 3:2) to obtain cudrachromone A (18, 2.8 mg), gentisein (2.6 mg), tomentosanol D (13.6 mg), and bergapten (2.0 mg). Fr 4.6.5 (153.3 mg) was separated by RP-HPLC with MeOH/ H2O mixtures (2:3 to 13:7) to give 5-dehydroxybavachinone A (32, 5.7 mg) and laburnetin (74.6 mg). Fr 4.6.8 (130.2 mg) was purified by RP-HPLC with MeOH/H2O mixtures (2:3 to 3:2) to afford eriosematin A (2.0 mg). Fr 4.6.10 (731.0 mg) was fractionated on a silica gel column with mixtures of CHCl3/acetone (30:1 to 5:1), followed by purification with RP-HPLC using MeOH/H2O mixtures (1:4 to 1:0), to obtain toxyloxanthone B (1.0 mg), 2-deprenylrheediaxanthone B (4.7 mg), 6-prenylnaringenin (4.6 mg), erythrinin B (115.7 mg), erythrinin C (2.1 mg), erythrinin G (2.6 mg), and hyuganin C (3.3 mg). Fr 4.6.10 (731.0 mg) was chromatographed on a silica gel column with mixtures of CHCl3/acetone (10:1 to 1:1), followed by purification with RP-HPLC using MeOH/H2O mixtures (1:4 to 1:0), to afford cudraflavone H (16, 2.1 mg) and dalenin (2.8 mg). Cudratrixanthone P (1): yellow, amorphous solid; [α]25D +13.7 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 244 (4.1), 262 (4.2), 320 (3.9), 378 (3.7) nm; IR νmax (ATR) 3244, 2926, 1626, 1467, 1407, 1276, 1188 cm−1; ECD (c 0.5 mM, MeCN) Δε −1.6 (203), −0.4 (216), −0.4 (224), −0.3 (234), −1.7 (251), +0.3 (285); 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 1; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-4, C-9a; H-5/C-4b, C-6, C-7, C-8a; Me-12/C-4, C-11, C-13, C-14; Me-13/C-4, C-11, C-12, C14; H-16/C-7, C-8, C-18; Me-19/C-17, C-18, C-20; Me-20/C-17, C18, C-19; ESIMS (negative) m/z 411 [M − H]−; ESIMS (positive) m/ z 413 [M + H]+; HRESIMS m/z 411.1449 [M − H]− (calcd for C23H23O7, 411.1444). Cudratrixanthone Q (2): yellow, amorphous solid; [α]25D +17.3 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 244 (4.2), 260 (4.2), 326 (4.0), 369 (3.8) nm; IR νmax (ATR) 3401, 2972, 1599, 1476, 1336, 1299, 1183 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 1; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/C-4b, C-6, C-7, C-9; Me-12/C-2, C-11, C-13, C-14;

Me-13/C-2, C-11, C-12, C-14; H-14/C-11, C-12, C-13; H-15/C-11, C-14; H-16/C-4b, C-5, C-6, C-17, C-18; H-17/C-5, C-16, C-18, C-19; Me-19/C-17, C-18, C-20; Me-20/C-17, C-18, C-19; ESIMS (negative) m/z 411 [M − H]−; ESIMS (positive) m/z 413 [M + H]+; HRESIMS m/z 411.1439 [M − H]− (calcd for C23H23O7, 411.1444). 16-Hydroxycudratrixanthone Q (3): yellow, amorphous solid; [α]25D +22.2 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 244 (4.2), 260 (4.2), 323 (4.0), 364 (3.8) nm; IR νmax (ATR) 3275, 2970, 1599, 1473, 1328, 1295, 1188 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/C-4b, C-6, C-7, C-9; Me-12/C-11, C13, C-14, Me-13/C-2, C-11, C-12, C-14; H-14/C-11, C-12, C-13; H15/C-11, C-14; H-16/C-4b, C-5, C-6, C-17, C-18; H-17/C-5, C-16, C-18, C-19; Me-19/C-17, C-18, C-20; Me-20/C-17, C-18, C-19; ESIMS (negative) m/z 427 [M − H]−; ESIMS (positive) m/z 429 [M + H]+; HRESIMS m/z 427.1391 [M − H]− (calcd for C23H23O8, 427.1393). 16-Methoxycudratrixanthone M (4): yellow, amorphous solid; [α]25D +21.2 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 245 (4.1), 259 (4.1), 322 (3.8), 361 (3.7) nm; IR νmax (ATR) 3378, 2927, 1657, 1614, 1472, 1291, 1185 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 1; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/C-4b, C-6, C-7, C-9; Me-12/C-2, C11, C-13, C-14; Me-13/C-2, C-11, C-12, C-14; H-15/C-11, C-14; H16/C-5, C-6; Me-19/C-17, C-18, C-20; Me-20/C-17, C-18, C-19; OMe-16/C-16; ESIMS (negative) m/z 441 [M − H]−; ESIMS (positive) m/z 443 [M + H]+; HRESIMS m/z 441.1565 [M − H]− (calcd for C24H25O8, 441.1549). 16-Hydroxycudratrixanthone M (5): yellow, amorphous solid; [α]25D +7.6 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 246 (4.3), 259 (4.3), 323 (4.1), 361 (3.9) nm; IR νmax (ATR) 3302, 2931, 1656, 1479, 1303, 1204, 1140 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/C-4b, C-6, C-7, C-9; Me-12/C-2, C11, C-13, C-14; Me-13/C-2, C-11, C-12, C-14; H-15/C-11, C-14; H17/C-6, C-16; Me-19/C-17, C-18, C-20; Me-20/C-17, C-18, C-19; ESIMS (negative) m/z 427 [M − H]−; ESIMS (positive) m/z 429 [M + H]+; HRESIMS m/z 427.1377 [M − H]− (calcd for C23H23O8, 427.1393). Cudratrixanthone R (6): yellow, amorphous solid; UV (MeOH) λmax (log ε) 241 (4.2), 259 (4.1), 335 (4.1) nm; IR νmax (ATR) 3243, 2974, 1584, 1443, 1399, 1273, 1202 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 1; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-1, C-3, C-4, C-9a; H-5/C-4b, C-6, C-7, C-8a; Me12/C-4, C-11, C-13, C-14; Me-13/C-4, C-11, C-12, C-14; H-14/C-12, C-13; H-15/C-11; H-16/C-7, C-8, C-17; Me-19/C-17, C-18, C-20; Me-20/C-17, C-18, C-19; OMe-3/C-3; ESIMS (negative) m/z 423 [M − H]−; ESIMS (positive) m/z 425 [M + H]+; HRESIMS m/z 423.1451 [M − H]− (calcd for C24H23O7, 423.1451). Cudratrixanthone S (7): yellow, amorphous solid; UV (MeOH) λmax (log ε) 248 (4.2), 263 (4.3), 316 (3.9), 369 (3.6) nm; IR νmax (ATR) 3397, 2926, 1601, 1471, 1358, 1290, 1195 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/ C-4b, C-6, C-7, C-9; Me-12/C-2, C-11, C-13, C-14; Me-13/C-2, C-11, C-12, C-14; H-15/C-11, C-14; H-16/C-5, C-6, C-17; H-19/C-17, C18, C-20; Me-20/C-17, C-18, C-19; ESIMS (negative) m/z 409 [M − H]−; ESIMS (positive) m/z 411 [M + H]+; HRESIMS m/z 409.1282 [M − H]− (calcd for C23H21O7, 409.1287). 7-O-Demethylcudratrixanthone C (8): yellow, amorphous solid; UV (MeOH) λmax (log ε) 242 (4.1), 261 (4.1), 321 (3.9), 358 (3.7) nm; IR νmax (ATR) 3389, 2926, 1643, 1462, 1409, 1276, 1181 cm−1; 13 C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 1; HMBC correlations OH-1/C-1, C-2, C-9a; H-2/C-1, C-3, C-4, C-9a; H-5/C-6, C-7, C-8a; Me-12/C-4, C-11, C-13, C-14; Me-13/C-4, C-11, C-12, C-14; H-16/C-7, C-8, C-8a, C-17; H-19/C-17; Me-20/C-17, C18, C-19; ESIMS (negative) m/z 409 [M − H]−; ESIMS (positive) m/ z 411 [M + H]+; HRESIMS m/z 409.1274 [M − H]− (calcd for C23H21O7, 409.1287). K



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ESIMS (positive) m/z 439 [M + H]+; HRESIMS m/z 437.1599 [M − H]− (calcd for C25H25O7, 437.1600). (2S)-Cudraflavanone H (15): brown, amorphous solid; [α]25D −62.4 (c 0.01, MeOH); UV (MeOH) λmax (log ε) 219 (4.5), 295 (4.2) nm; IR νmax (ATR) 3369, 2920, 1658, 1619, 1460, 1205, 1141 cm−1; ECD (c 0.1 mM, MeCN) Δε +24.0 (208), −5.1 (231), +0.2 (256), −5.1 (297), +2.6 (335); 13C and 1H NMR (500 and 125 MHz, CDCl3); 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 3; HMBC correlations H-3/C-2, C-4; OH-5/C-5, C-6, C-10; H-8/C6, C-7, C-9, C-10; H-3′/C-1′, C-4′, C-5′; H-6′/C-2, C-4′, C-1″; H-1″/ C-4′, C-3″; H-2″/C-5′, C-3″; Me-4″/C-2″, C-3″, C-5″; Me-5″/C-2″, C-3″, C-4″; H-1‴/ C-6, C-2‴, C-3‴; Me-4‴/C-2‴, C-3‴, C-5‴; Me5‴/C-2‴, C-3‴, C-4‴; ESIMS (negative) m/z 437 [M − H]−; ESIMS (positive) m/z 439 [M + H]+; HRESIMS m/z 437.1602 [M − H]− (calcd for C25H25O7, 437.1600). Cudraflavone H (16): yellow, amorphous solid; UV (MeOH) λmax (log ε) 219 (4.4), 280 (4.3), 326 (3.9) nm; IR νmax (ATR) 3254, 2924, 1655, 1616, 1464, 1303, 1176 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 3; HMBC correlations H-8/C-6, C-7, C9, C-10; H-3′/C-1′, C-2′, C-4′, C-5′; H-5′/C-1′, C-3′; H-6′/C-2, C-2′, C-4′; H-1″/C-2, C-3, C-4; H-4″/C-3″, C-5″; H-1‴/C-5, C-7, C-3‴; H-2‴/C-6, C-3‴; Me-4‴/C-2‴, C-3‴, C-5‴; Me-5‴/C-2‴, C-3‴, C4‴; ESIMS (negative) m/z 433 [M − H]−; ESIMS (positive) m/z 435 [M + H]+; HRESIMS m/z 433.1289 [M − H]− (calcd for C25H21O7, 433.1287). Cudraphenone E (17): yellow, amorphous solid; [α]25D +7.0 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 211 (4.3), 300 (4.0) nm; IR νmax (ATR) 3316, 2924, 1623, 1443, 1314, 1267, 1201 cm−1; ECD (c 0.5 mM, MeCN) Δε +3.2 (204), −6.4 (215), +11.2 (224), −0.4 (234), +2.0 (242); 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 4; HMBC correlations OH-2/C-1, C-2, C-3, H-6/C-2, C-4, C-7, C-12; H-7/C-5, C-6, C-8, C-9; H-8/C-5, C-9, C-10, C-11; H-10/ C-8, C-11; H-11/C-8, C-9, C-10; H-14/C-18; H-16/C-14, C-18; H17/C-13, C-15; H-18/C-12, C-14, C-16; OMe-4/C-4; ESIMS (negative) m/z 343 [M − H]−; ESIMS (positive) m/z 345 [M + H]+; HRESIMS m/z 343.1176 [M − H]− (calcd for C19H19O6, 343.1182). Cudrachromone A (18): yellow, amorphous solid; [α]25D +8.3 (c 0.04, MeOH); UV (MeOH) λmax (log ε) 211 (4.3), 261 (4.1), 294 (3.8) nm; IR νmax (ATR) 3248, 2927, 1647, 1460, 1305, 1178 cm−1; ECD (c 0.5 mM, MeCN) Δε −1.1 (208), +1.6 (223), +1.0 (238), +2.0 (249), −3.7 (289), +0.7 (327); 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 4; HMBC correlations H-2/C-3, C-4, C-9; H3/C-2, C-10; H-8/C-6, C-7, C-9, C-10; H-11/C-5, C-6, C-7, C-12; H14/C-12, C-15; H-15/C-12, C-13, C-14; ESIMS (negative) m/z 261 [M − H]−; ESIMS (positive) m/z 263 [M + H]+; HRESIMS m/z 261.0775 [M − H]− (calcd for C14H13O5, 261.0763). Cudraphenol A (19): pale yellow, amorphous solid; UV (MeOH) λmax (log ε) 229 (4.1), 262 (3.8), 306 (3.5) nm; IR νmax (ATR) 3248, 2924, 1663, 1511, 1436, 1353, 1271, 1179 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 4; HMBC correlations H3/C-1, C-4, C-5; OH-4/C-3, C-4, C-5; H-6/C-2, C-7, C-8; H-8/C-1, C-2, C-4, C-6, C-10, C-14; H-10/C-8, C-12, C-14; H-11/C-9, C-12, C-13; H-13/C-9, C-11, C-12; H-14/C-8, C-10, C-12; OMe-7/C-7; ESIMS (negative) m/z 273 [M − H]−; ESIMS (positive) m/z 275 [M + H]+; HRESIMS m/z 273.0760 [M − H]− (calcd for C15H13O5, 273.0763). Cudraphenol B (20): pale yellow, amorphous solid; UV (MeOH) λmax (log ε) 227 (4.2), 266 (3.9), 301 (3.6) nm; IR νmax (ATR) 3135, 2925, 1674, 1510, 1437, 1277, 1203, 1142 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 4; HMBC correlations H3/C-1, C-2, C-5; H-4/C-6, C-7; OH-6/C-1, C-5, C-6; H-8/C-1, C-2, C-6, C-9, C-10, C-14; H-10/C-8, C-12, C-14; H-11/C-9, C-12, C-13; H-13/C-9, C-11, C-12; H-14/C-8, C-10, C-12; OMe-7/C-7; ESIMS (negative) m/z 273 [M − H]−; ESIMS (positive) m/z 275 [M + H]+; HRESIMS m/z 273.0763 [M − H]− (calcd for C15H13O5, 273.0763). Cudraphenol C (21): pale yellow, amorphous solid; UV (MeOH) λmax (log ε) 227 (4.4), 267 (3.9), 308 (3.6) nm; IR νmax (ATR) 3298, 2923, 1611, 1510, 1440, 1375, 1239, 1072 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 4; HMBC correlations H-

Cudratrixanthone T (9): yellow, amorphous solid; [α]25D +8.2 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 243 (4.3), 259 (4.3), 323 (4.1), 369 (3.9) nm; IR νmax (ATR) 3393, 2924, 1608, 1464, 1296, 1194 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 1; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C4a, C-9a; H-8/C-4b, C-6, C-7, C-9; Me-12/C-2, C-11, C-13, C-14; Me-13/C-2, C-11, C-12, C-14; H-14/C-11, C-12, C-13; H-15/C-11, C-14; H-16/C-4b, C-5, C-6, C-17, C-18; H-19/C-17, C-18, C-20; Me20/C-17, C-18, C-19; ESIMS (negative) m/z 411 [M − H]−; ESIMS (positive) m/z 413 [M + H]+; HRESIMS m/z 411.1441 [M − H]− (calcd for C23H23O7, 411.1444). Cudratrixanthone U (10): yellow, amorphous solid; [α]25D +10.9 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 245 (4.3), 259 (4.3), 323 (4.1), 364 (3.9) nm; IR νmax (ATR) 3215, 2926, 1656, 1480, 1291, 1232, 1159 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-4/C-2, C-3, C-4a, C-9a; H-8/C-4b, C-6, C-7, C-8a, C-9; Me-12/C-2, C-11, C-13, C-14; Me-13/C-2, C-11, C-12, C-14; H-14/C-12, C-13; H-15/C-11, C-14; H-16/C-4b, C-5, C-6, C-17, C-18; H-17/C-5, C-19; H-19/C-17, C-18; Me-20/C-17, C-18, C-19; ESIMS (negative) m/z 411 [M − H]−; ESIMS (positive) m/z 413 [M + H]+; HRESIMS m/z 411.1459 [M − H]− (calcd for C23H23O7, 411.1444). Cudratrixanthone V (11): yellow, amorphous solid; UV (MeOH) λmax (log ε) 246 (4.2), 263 (4.2), 320 (4.0), 369 (3.7) nm; IR νmax (ATR) 3397, 2927, 1615, 1460, 1285, 1194 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-5/C-7, C-8a; H-11/C-1, C-2, C-3, C-12, C13; H-13/C-11, C-15, C-17; H-14/C-12, C-15, C-16; H-16/C-12, C14, C-15; H-17/C-11, C-13, C-15; Me-19/C-4, C-18, C-20, C-21; Me20/C-4, C-18, C-19, C-21; H-21/C18, C-19, C-20; H-22/C-18, C-21; H-23/C-6, C-8, C-8a, C-24, C-25; H-24/C-26, C-27; Me-26/C-24, C25, C-27; Me-27/C-24, C-25, C-26; ESIMS (negative) m/z 501 [M − H]−; ESIMS (positive) m/z 503 [M + H]+; HRESIMS m/z 501.1919 [M − H]− (calcd for C30H29O7, 501.1913). Cudratrixanthone W (12): yellow, amorphous solid; UV (MeOH) λmax (log ε) 230 (4.3), 241 (4.2), 263 (4.3), 323 (4.0), 374 (3.8) nm; IR νmax (ATR) 3385, 2928, 1613, 1463, 1298, 1199 cm−1; 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 2; HMBC correlations OH-1/C-1, C-2, C-9a; H-8/C-4b, C-6, C-9; Me-12/C-2, C-11, C-13, C-14; Me-13/C-2, C-11, C-12, C-14; H-15/C-11; H-16/ C-3, C-4, C-4a, C-17, C-18; H-18/C-20, C-21; H-19/C-17, C-20, C22; H-21/C-19, C-20; H-22/C-18, C-20; H-23/C-4b, C-5, C-6, C-24, C-25; Me-26/C-24, C-25, C-27; Me-27/C-24, C-25, C-26; ESIMS (negative) m/z 501 [M − H]−; ESIMS (positive) m/z 503 [M + H]+; HRESIMS m/z 501.1921 [M − H]− (calcd for C30H29O7, 501.1913). Cudraflavanone G (13): brown, amorphous solid; [α]25D −79.6 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 223 (4.5), 292 (4.1) nm; IR νmax (ATR) 3242, 2972, 1634, 1499, 1303, 1155 cm−1; ECD (c 0.1 mM, MeCN) Δε +20.2 (207), −2.9 (237), +0.6 (254), −8.6 (291), +2.7 (340); 13C and 1H NMR (500 and 125 MHz, CDCl3); 13C and 1 H NMR (500 and 125 MHz, acetone-d6), see Table 3; HMBC correlations H-3/C-2, C-4, C-1′; OH-5/C-5, C-6, C-10; H-8/C-6, C-7, C-9, C-10; H-3′/C-1′, C-4′, C-5′; H-6′/C-2, C-2′, C-1″; H-1″/C-3″; H-2″/C-5′, C-3″; Me-4″/C-2″, C-5″; Me-5″/C-2″, C-4″; H-1‴/C-5, C-6, C-7, C-2‴; H-4‴/C-2‴, C-3‴, C-5‴; Me-5‴/C-2‴, C-3‴, C-4‴; ESIMS (negative) m/z 437 [M − H]−; ESIMS (positive) m/z 439 [M + H]+; HRESIMS m/z 437.1606 [M − H]− (calcd for C25H25O7, 437.1600). (2R)-Cudraflavanone H (14): brown, amorphous solid; [α]25D −199.2 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 216 (4.6), 295 (4.3) nm; IR νmax (ATR) 3308, 2921, 1659, 1619, 1459, 1192, 1140 cm−1; ECD (c 0.1 mM, MeCN) Δε −11.4 (205), +1.9 (229), −1.0 (255), +6.1 (296), −1.4 (337); 13C and 1H NMR (500 and 125 MHz, CDCl3); 13C and 1H NMR (500 and 125 MHz, acetone-d6), see Table 3; HMBC correlations H-3/C-2, C-4; OH-5/C-5, C-6, C-10; H-8/C6, C-7, C-9, C-10; H-3′/C-1′, C-4′, C-5′; H-6′/C-2, C-4′, C-1″; H-1″/ C-4′, C-3″; H-2″/C-5′, C-3″; Me-4″/C-2″, C-3″, C-5″; Me-5″/C-2″, C-3″, C-4″; H-1‴/ C-6, C-7, C-2‴, C-3‴; Me-4‴/C-2‴, C-3‴, C-5‴; Me-5‴/C-2‴, C-3‴, C-4‴; ESIMS (negative) m/z 437 [M − H]−; L



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4/C-2, C-7, C-15; H-8/C-1, C-2, C-6, C-9; OH-6/C-1, C-5, C-6; H10/C-8, C-12, C-14; H-11/C-9, C-12, C-13; H-13/C-9, C-11, C-12; H-14/C-8, C-10, C-12; H-15/C-2, C-3, C-16, C-17, C-21; H-17/C-15, C-19, C-21; H-18/C-16, C-19, C-20; H-20/C-16, C-18, C-19; H-21/ C-15, C-17, C-19; OMe-7/C-7; ESIMS (negative) m/z 379 [M − H]−; ESIMS (positive) m/z 381 [M + H]+; HRESIMS m/z 379.1182 [M − H]− (calcd for C22H19O6, 379.1182). Computational Methods. Conformational searches were performed under the MMFF molecular mechanics force field in Spartan’14 software,13 and the selected conformers were optimized with DFT calculations at the B3LYP/6-31+G(d,p) level using the Gaussian 09 package.14 TDDFT ECD calculations of the optimized conformers were performed at the CAM-B3LYP/TZVP level with a CPCM solvent model in MeCN. The calculated ECD spectra were simulated with a half bandwidth of 0.2−0.3 eV, and ECD curves were generated by SpecDis 1.64 software.67 The ECD spectra were weighted by Boltzmann distribution after UV correction. Cell Culture. The SH-SY5Y human neuroblastoma cell line (ATCC No. CRL-2266) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich) and 10% heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT, USA). Cells were maintained at 37 °C in a saturated humidity atmosphere containing 95% air and 5% CO2. Determination of Cell Viability against 6-OHDA. SH-SY5Y cells were plated at a density of 2 × 104 cells/200 μL/well in 96-well plates for 24 h. The cells were treated with various concentrations of samples and 6-OHDA (100 μM) for an additional 24 h. Cell viability was determined by treatment with MTT dissolved in phosphatebuffered saline (PBS) (0.5 mg/mL) at 37 °C for 4 h. The PBS was carefully removed, and formazan crystals were dissolved with DMSO. The absorbance of this solution was measured at 540 nm using a SpectraMaxM5 microplate reader (Molecular Devices, Sunnyvale, CA, USA). Determination of Cell Viability against MPP+. SH-SY5Y cells were plated at a density of 2 × 104 cells/200 μL/well in 96-well plates for 24 h and then treated with MPP+ (2 mM) for 48 h. Cell viability was determined by treatment with MTT dissolved in PBS (0.5 mg/ mL) at 37 °C for 4 h. The PBS was carefully removed, and formazan crystals were dissolved with DMSO. Absorbance of this solution was measured at 540 nm using a SpectraMaxM5 microplate reader. To prevent exposure of MPP+, which is a neurotoxin, it was handled in a laboratory fume hood wearing N95 dust masks and gloves. Determination of Cell Viability against OGD. SH-SY5Y cells were plated in DMEM with 10% FBS and glucose at a density of 2 × 104 cells/200 μL/well in 96-well plates for 24 h, and the medium was then changed to serum-free and glucose-free RPMI. Cells were treated with various concentrations of samples or vehicle. After treatment with a sample, cells were incubated in a modular incubator chamber-101 (Billups-Rothenberg, Del Mar, CA, USA) containing a mixture of 95% N2 and 5% CO2 at 37% for 16 h to create a hypoxic environment. After establishing the hypoxia chamber, the medium was replaced with RPMI including 10% FBS and glucose, and cells were incubated under normoxic conditions in humidified 95% air and 5% CO2 at 37 °C for 24 h in order to reoxygenate the cells and restore glucose levels. In the normoxic control group, cells were cultured with DMEM including 10% FBS and glucose under normal conditions. Cell viability was determined by treatment with MTT dissolved in PBS (0.5 mg/mL) at 37 °C for 4 h. The PBS was carefully removed, and formazan crystals were dissolved with DMSO. Cell viability was determined by measuring absorbance at 540 nm with a SpectraMaxM5 microplate reader.

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H and 13C NMR and MS spectra of new compounds as well as 1H NMR data of known compounds (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +82-2-880-2473. Fax: +82-2-888-9122. E-mail: mars@ snu.ac.kr (W. Mar). *Tel: +82-2-3290-3017. Fax: +82-2-953-0737. E-mail: [email protected] (D. Lee). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant from the National Research Foundation of Korea (NRF2015R1D1A1A01060321), BK21 Plus program in 2015 through NRF funded by the Ministry of Education of Korea, and the Institute of Life Science and Natural Resources, Korea University.

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REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00204. M



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Chemical Constituents Isolated from the Root Bark of Cudrania

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