Sesquiterpenes and Alkaloids from the Roots of Alangium

Jun 4, 2013 - antiviral activity against Coxsackie virus B3 with IC50 values of 1.4−15.4 μM. Compounds 2−4, 7, and 9−13 showed antioxidant acti...
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Sesquiterpenes and Alkaloids from the Roots of Alangium chinense Yan Zhang, Yun-Bao Liu, Yong Li, Shuang-Gang Ma, Li Li, Jing Qu, Dan Zhang, Xiao-Guang Chen, Jian-Dong Jiang, and Shi-Shan Yu* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Four new sesquiterpenes (1−4), four new alkaloids (5a, 6a, 6b, and 7), and nine known compounds (5b and 8−15) were isolated from an ethanolic extract of roots of Alangium chinense. The structure of 1 was confirmed by X-ray crystallography. The configurations of 5 and 6 were assigned by chiral HPLC analysis and CD spectra. Compounds 3, 4, 8−13, and 15 exhibited antiviral activity against Coxsackie virus B3 with IC50 values of 1.4−15.4 μM. Compounds 2−4, 7, and 9−13 showed antioxidant activities against Fe2+cysteine-induced rat liver microsomal lipid peroxidation, with IC50 values of 3.8−45.7 μM. Compound 5b displayed neuritis inhibitory activity against microglial inflammation factor, with an IC50 value of 6.7 μM. None of the compounds exhibited detectable cytotoxic activity toward any of five tumor cell lines (A549, Be-17402, BGC-823, HCT-8, and A2780) in the MTT assay.

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lants of the genus Alangium are distributed in the tropics and subtropical area of the Eastern Hemisphere, nine species of which are known to occur in South China.1 Alangium chinense (Lour.) Harms (Alangiaceae) is a deciduous shrub common in China, and the roots, flowers, and leaves of this plant have historically been used in traditional Chinese medicine.2 Previous phytochemical investigations have demonstrated that this plant contains alkaloids3 and phenolic glycosides.4,5 As part of a program to study bioactive substances from medicinal plants, an ethanolic extract of dried roots of A. chinense was investigated. We describe herein the isolation, structure elucidation, and biological activities of four new sesquiterpenes (1−4), four new alkaloids (5a, 6a, 6b, and 7), and nine known compounds, (2S)-N-hydroxybenzylanabasine (5b),6 4,5-dimethoxycanthin-6-one (8),7 lacinilene C (9),8 7hydroxycadalene (10),9 2,7-dihydroxycadalene (11),10 mansonone E (12),11 mansonone H (13),11 mansonone C (14),12 and (1S,4R)-7,8-dihydroxycalamenene (15),13 from the ethanolic extract.

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RESULTS AND DISCUSSION Compound 1 had the molecular formula C15H22O3, as established by HRESIMS [m/z 251.1639 [M + H]+ (calcd for 251.1642)], with 5 degrees of unsaturation. The IR spectrum showed OH (3370 cm−1) and α,β-unsaturated carbonyl (1664 cm−1) absorptions. The 13C NMR and DEPT spectra of compound 1 revealed the presence of an α,βunsaturated carbonyl group (δC 122.6, 160.9, and 199.4), two sp3 quaternary carbons (δC 75.5 and 81.4), four sp3 methines (δC 32.1, 40.4, 55.9, and 82.6), two methylenes (δC 21.0 and 33.5), and four methyls (δC 31.1, 25.8, 17.5, and 10.8) in the structure. The NMR data (Tables 1 and 2) implied that the structure of compound 1 was similar to the known compound dihydroisoperezinone, except for the absence of a double bond © XXXX American Chemical Society and American Society of Pharmacognosy

at C-3.14 1H−1H COSY and HSQC analyses revealed two isolated spin systems: (a) C(5)H−C(6)H2−C(7)H2−C(8)H− C(15)H3 and (b) C(4)H−C(3)H−C(14)H3. HMBC correlations (Figure S1) from H3-15/H-5 to C-9 and from H2-6 to C10, together with the spin system (a) deduced above, led to the assignment of ring A. The HMBC correlations (Figure S1) from H-4 to C-9/C-2 and from H-1 to C-3/C-10, as well as the spin system (b) C(4)H−C(3)H−C(14)H3 and the α,βReceived: January 24, 2013

A

dx.doi.org/10.1021/np4000747 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Data of Compounds 1−4 and 7a 1b

pos. 1 2 3 4 5 6

2c

3d

4e

7f

5.88 s 8.60 s

7 8 11 12 13 14 15 −OCH3

2.74 4.05 2.33 2.06 1.67 1.85 1.49 2.66

m d (3.0) brd (7.2) m m m m m

1.27 1.03 1.25 1.12

s s d (7.2) d (6.6)

5.84 s

6.72 s

6.85 s

7.47 s

7.74 s

7.89 s

7.00 3.24 1.17 1.19 1.28 2.16 2.83

7.71 3.60 1.27 1.27 10.5 2.27

8.30 3.71 1.33 1.33

s m d (7.0) d (7.0) s s s

s m d (7.0) d (7.0) s s

s m d (7.0) d (7.0)

8.05 d (8.0) 7.58 d (8.0)

2.50 s 2.83 s 2.97 s

2.35 s 4.03 s

a

600 MHz for 1 and 500 MHz for 2−4 and 7. Proton coupling constants (J) in Hz were given in parentheses. bIn CDCl3. cIn DMSO-d6. dIn methanol-d4. eIn acetone-d6. fIn pyridine-d5.

Table 2. 13C NMR Data of Compounds 1−4 and 7a pos.

1b

2c

3d

4e

1 2 3 3a 4 5 6 6a 7 8 9 10 11 12 13 14 15 −OCH3

122.6 199.4 40.4

81.3 200.7 116.8

110.7 165.7 112.1

102.8 164.5 112.7

82.6 55.9 21.0

162.8 128.3 123.6

158.3 126.9 125.8

154.6 126.3 124.3

33.5 32.1 160.9 75.5 81.4 31.1 25.8 10.8 17.5

157.8 112.4 143.3 121.4 28.3 22.2 21.9 31.3 15.8 52.8

158.5 103.3 135.5 122.0 30.2 23.4 23.4 194.0 16.7

157.2 109.6 133.8 122.3 30.3 23.4 23.4 173.3 16.9 52.4

10R (Figure 1). Therefore, compound 1 was named 3S,4R,5S,8R,10R-tetrahydroperezinone.

7f 143.9 126.9 132.5 130.6 134.9 146.6 124.7 181.9 151.5 123.0 150.2 120.4 15.6 10.7 24.1

a 150 MHz for 1 and 125 MHz for 2, 3, 4, and 7. bIn CDCl3. cIn DMSO-d6. dIn methanol-d4. eIn acetone-d6. fIn pyridine-d5.

Figure 1. ORTEP drawing of the X-ray crystal structure of 1.

Compound 2 had the molecular formula C16H20O3, by HRESIMS at m/z 283.1309 [M + Na]+ (calcd for 283.1305, C16H20O3Na), with 7 degrees of unsaturation. The IR spectrum showed absorptions typical of OH (3243 cm−1), α,βunsaturated carbonyl (1658 cm−1), and aromatic ring (1605 and 1571 cm−1) moieties. The 1H NMR spectrum of compound 2 (Table 1) revealed signals of an OCH3 (δH 2.83, 3H, s), four methyls [δH 1.17 (3H, d, J = 7.0 Hz), 1.19 (3H, d, J = 7.0 Hz), 1.28 (3H, s), 2.16 (3H, s)], three olefinic protons [δH 5.84 (1H, s), 7.47 (1H, s), 7.00 (1H, s)], and one methine proton (δH 3.24, 1H, m). The 13C NMR (Table 2) and DEPT spectra of compound 2 contained 16 carbon signals, including one OCH3 (δC 52.8), four CH3 (δC 22.2, 21.9, 31.3, and 15.8), four methine carbons (δC 128.3, 116.8, 112.4, and 28.3), and seven quaternary carbons (δC 200.7, 162.8, 157.8, 143.3, 123.6, 121.4, and 81.3). The NMR data resembled those of lacinilene C, which had no OCH3 at C-1.8 By comparison of the NMR data of lacinilene C with those of compound 2, the

unsaturated carbonyl unit deduced above, allowed for the assignment of ring B, which fused with ring A through C-9 and C-10. Additionally, the HMBC correlations (Figure S1) from H3-12/H3-13 to C-5 and from H2-6 to C-11 suggested that C11 was connected to C-5 of ring A. Identification of the connection between C-4 and C-11 through an oxygen atom to form ring C was initially hampered by the lack of an HMBC correlation from H-4 to C-11. However, this unit was established unambiguously by an X-ray experiment. The relative configuration of compound 1 was determined by NOESY and NOE experiments. Correlations of H-4 with H-3/ H-5/H-14/H-12 and H-1 with H-15 determined the relative configuration of 1 to be that shown in Figure S2. X-ray data confirmed the relative configuration of compound 1. In addition, the value of the Flack parameter (0.1(2)) determined by the X-ray analysis allowed for assignment of the absolute configurations of the chiral carbons to be 3S, 4R, 5S, 8R, and B

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Table 3. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data of Compounds 5 and 6 in CDCl3

most distinctive difference in compound 2 was the presence of an OCH3. The HMBC correlation (Figure S1) from the methoxy group (δH 2.83, 1H, s) to C-1 (δC 81.3) indicated that the OCH3 was attached to C-1. Further analysis of the 1H−1H COSY, HSQC, and HMBC data verified the structural similarities and differences between compound 2 and lacinilene C, leading to the proposed structure of 1-methoxylacinilene C. The only chiral carbon (C-1) in the structure of compound 2 was determined to have an S absolute configuration based on a positive Cotton effect at 322.5 nm (Δε +0.20, the n → π* transition of the α,β-unsaturated ketone) in the CD spectrum.15 Consequently, the structure of 2 was (1S)-1-methoxylacinilene C. Compound 3 had the molecular formula C15H16O3, with 8 degrees of unsaturation. The IR spectrum contained OH (3258 cm−1), conjugated aldehyde (1620 cm−1), and aromatic ring (1577 cm−1) absorptions. The 1H NMR spectrum of compound 3 (Table 1) showed resonances of one aldehyde group (δH 10.5, 1H, s), three aromatic protons [δH 7.74 (1H, s), 7.71 (1H, s), and 6.72 (1H, s)], three methyls [δH 2.27 (3H, s) and 1.27 (6H, d, J = 7.0 Hz)], and one methine (δH 3.60, 1H, m). The 13C NMR (Table 2) and DEPT spectra of 3 contained 15 carbon signals, including one aldehyde (δC 194.0), three methyl (δC 16.7, 23.4, and 23.4), three aromatic methine (δC 126.9, 112.1, 103.3, and 30.2), one sp3 methine (δC 30.2), and seven quaternary carbons (δC 165.7, 158.5, 158.3, 135.5, 125.8, 122.0, and 110.7). The NMR data closely resembled those of 2,7-dihydroxycadalene.16 The most distinctive difference was the presence of the aldehyde group in compound 3. HMBC correlations (Figure S1) from the aldehyde proton (H14) to C-1 and C-2 revealed that the aldehyde carbon (C-14) was attached at C-1. Further analysis of the 1H−1H COSY, HSQC, and HMBC data verified the structural similarities and differences between compound 3 and 2,7-dihydroxycadalene, leading to the proposed structure for compound 3 as shown. Compound 4 (C16H18O4) showed IR absorptions for OH (3421 cm−1), carbonyl (1643 cm−1), and aromatic ring (1603 and 1540 cm−1) groups. The 1H and 13C NMR data of compound 4 (Tables 1 and 2) were similar to those of compound 3. The differences were the absence of the aldehyde group (δH 10.5, 1H; δC 194.0) and the presence of one carbonyl group (δC 173.3) and a methoxyl group (δC 52.4, δH 4.03) in compound 4. The HMBC correlation (Figure S1) from the methoxy signal (δH 4.03) to the carbonyl carbon at C14 suggested that the structure of compound 4 was the methyl ester of 14-carbonyl-2,7-dihydroxycadalene. Analysis of the 1 H−1H COSY, HSQC, and HMBC data verified the structural similarities and differences between compound 4 and 3,7dihydroxycadalene, leading to the proposed structure for 4 as shown. Compound 5 had the molecular formula C17H20N2O, with 9 degrees of unsaturation. The IR spectrum showed absorptions for OH (3039 cm−1) and aromatic ring (1590 and 1482 cm−1) groups. The 13C NMR spectrum of compound 5 (Table 3) showed 17 carbon signals, including 11 aromatic carbons, five methylene carbons, and one methine carbon. 1H−1H COSY and HSQC analyses revealed four isolated spin systems [(a) C(2)H−C(3)H2−C(4)H2−C(5)H2−C(6)H2, (b) C(4′)H− C(5′)H−C(6′)H, (c) C(3″)H−C(4″)H−C(5″)H−C(6″)H, and (d) C(7″)H2]. HMBC correlations (Figure S1) from H2 to C-6 and from H-6 to C-2 indicated that C-2 and C-6 were connected through a nitrogen atom to form a 6H-piperidine ring (A). HMBC correlations (Figure S1) from H-2′ to C-6′

5 pos.

δC

2 3 4

67.0 25.6 24.6

5

35.8

6

53.5

2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 7″

149.5 138.1 134.8 124.1 149.3 157.2 121.2 128.4 119.2 128.5 115.9 58.8

6

δH (J in Hz) 3.21 1.75 1.47 1.86 1.83 1.86 3.21 2.09 8.54

m m m m m m m m brs

7.78 d (6.5) 7.29 dd (5.0, 8.0) 8.50 dd (1.5, 5.0)

6.82 6.69 7.10 6.76 3.98 3.03

d (7.5) t (7.5) t (7.5) d (7.5) d (14.0) d (14.0)

δC 90.9 25.2 40.6 20.1 48.6 148.4 140.5 134.4 123.3 148.2 152.6 118.8 127.2 120.5 127.7 116.3 49.9

δH (J in Hz) 1.78 2.03 1.69 1.82 1.69 2.90 2.81 8.78

m d (14.0) m m m m d (11.0) s

7.81 d (7.5) 7.19 dd (5.0, 8.0) 8.45 dd (1.5, 5.0)

6.79 6.80 7.12 6.97 3.97 3.38

overlap overlap m d (8.0) d (17.0) d (17.0)

and from H-6′ to C-2′ implied the presence of a piperidine ring (B)-containing spin system (b). HMBC correlations (Figure S1) from H-2 to C-2′/C-3′/C-4′ and from H-2′/H-4′ to C-2 suggested that ring B was connected to ring A through a C2− C3′ bond. HMBC correlations (Figure S1) from H-6″ to C-1″/ C-2″ and from H-3″ to C-1″/C-2″ indicated the presence of a 1,2-disubstituted benzene group (ring C), which bears spin system (c). HMBC correlations (Figure S1) from H-7″ to C1″/C-2″/C-3″ and from H-7″ to C-2/C-6 showed that C-7″ was connected to C-2″ of ring C and the nitrogen atom of ring A. Thus, the planar structure of compound 5 was the same as the known compound N-hydroxybenzylanabasine.6 Compound 5 contained only one chiral center at C-2 and was confirmed to be a racemic mixture due to its low specific rotation (+0.1) and the absence of distinct Cotton effects in the CD spectrum. Chiral HPLC (Figure S3) was performed to afford the enantiomers (5a and 5b). The CD spectra of the resolved enantiomers (5a and 5b) were recorded (Figures S46, S47). The CD spectrum of 5a displayed a positive Cotton effect at 278.5 nm (Δε +3.49) corresponding to the 1Lb band of the pyridine chromophore. According to the benzene sector and benzene chirality rules for pyridylethylamines,17 the 2R configuration was assigned to 5a. Therefore, the structure of 5a was established, and it was named (2R)-N-hydroxybenzylanabasine. Similarly, 5b was established as (2S)-N-hydroxybenzylanabasine according to the negative Cotton effect at 279 (Δε −9.22). Compound 5a is a new compound, and 5b is a new natural product.6 The molecular formula of compound 6, C17H20N2O2, was established by HRESIMS and was larger than compound 5 by 16 mass units. Compound 6 exhibited OH (3040 cm−1) and aromatic ring (1585 and 1486 cm−1) absorptions in the IR spectrum. The 13C NMR data of compound 6 (Table 3) revealed marked similarities to compound 5, except that the carbon signal for C-2 was shifted downfield to δC 90.9 (Table 3). The absence of one of the methine signals in the 13C NMR C

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Table 4. Antiviral Activity of Compounds 3, 4, 8−13, and 15a toward Coxsakie Virus B3 IC50 (μM)

a

3

4

8

9

10

11

12

13

15

RBVb

1.4

11.3

7.4

15.4

5.3

10.1

4.7

3.1

7.4

222.2

Compounds 2, 5−7, and 14 all had IC50 >20 μM. bPositive control (ribavirin).

Table 5. Antioxidant Activity of Compounds 2−4, 7, and 9−13a IC50 (μM)

a

2

3

4

7

9

10

11

12

13

vitamin Eb

23.8

14.7

4.9

3.9

39.8

4.3

3.8

45.7

17.8

54.2

Compounds 8, 14, and 15 were inactive. bPositive control.

sites of unsaturation, suggesting the presence of one remaining ring in the structure, and indicated that the carbonyl carbon (C7) of the α,β-unsaturated carbonyl unit was connected to C-6a to form another ring. The OH at C-8 was inferred from the molecular formula and the chemical shift of the quaternary carbon at C-8. The structure was further supported by the enhancement between H3-14 and H-5, as well as that between H3-12 and H-4/H-2, which were observed in the NOE difference spectrum of compound 7. Thus, 7 was established as 8-hydroxy-3,6,9-trimethyl-7H-benzo[de]quinolin-7-one. The known alkaloid (8) and seven known sesquiterpenes (9−15) were identified based on their spectroscopic profiles (NMR, UV, MS, and CD) and comparison to published data. Four different assays (in vitro anti-Coxsackie virus B3 activity assay, antioxidant assay, inhibitory effects on nitric oxide production in LPS-activated microglia, and cytotoxicity assay) were carried out to evaluate the bioactivities of the isolated compounds. Nine compounds (3, 4, 8−13, and 15) exhibited antiviral activities against Coxsackie virus B3, as shown in Table 4. Infections with Coxsackie virus B are reported to be associated with the development of myocarditis, pancreatitis, meningitis, and encephalitis. Therefore, the reported data could add new knowledge for the development of chemical entities able to inhibit Coxsackie virus. Compounds 2−4, 7, and 9−13 showed antioxidant activities against Fe2+-cysteine-induced rat liver microsomal lipid peroxidation (Table 5). Compound 5b displayed neuritis inhibitory activity against microglial inflammation factor, with an IC50 value of 6.7 μM. However, its isomer, compound 5a, showed weak activity (>20 μM) in the same assay, indicating that chirality played an important role in neuritis inhibition. None of the compounds (1−15) were cytotoxic in the MTT assay.

spectrum of compound 6, in comparison with compound 5, indicated that the hydrogen of this methine group was replaced by an OH (absence of the 1H signal) in compound 6. Analysis of the 1H−1H COSY, HSQC, and HMBC data verified the structural similarities and differences between compounds 6 and 5, leading to the proposed structure of compound 6. Compound 6 was also confirmed to be a racemic mixture by its low specific rotation and the absence of distinct Cotton effects in the CD spectrum. Chiral HPLC and the CD spectra of the resolved enantiomers (6a and 6b) confirmed that 6 was a racemic mixture. On the basis of the benzene sector and benzene chirality rules for pyridylethylamines,17 a negative Cotton effect at 278.5 nm (Δε −0.61), corresponding to the 1 Lb band of the pyridine chromophore, assigned the 2S configuration to 6a and the 2R configuration to 6b. Therefore, the structure of 6a was established, and it was named (2S)-2hydroxy-N-hydroxybenzylanabasine. Compound 6b was named (2R)-2-hydroxy-N-hydroxybenzylanabasine. Both 6a and 6b are new compounds. Compound 7 was obtained as a red, amorphous powder. Its molecular formula was established as C15H14NO2 by HRESIMS at m/z 240.1021 [M + H]+ (calcd for 240.1019), with 10 degrees of unsaturation. The IR spectrum showed absorptions for OH (3359 cm−1), α,β-unsaturated carbonyl (1649 cm−1), and aromatic ring (1615 and 1572 cm−1) groups. The 1H NMR spectrum of compound 7 showed three aromatic protons at δH 8.60 (1H, s, H-2), 8.05 (1H, d, J = 8.0 Hz, H-4), and 7.58 (1H, d, J = 8.0 Hz, H-5) in the downfield region and methyl signals at δH 2.50, 2.83, and 2.97 (each 3H, s, Me-12, 13, and 14). The 1 H−1H COSY and HSQC data showed the presence of one isolated spin system [C(4)H−C(5)H]. The 13C NMR and DEPT spectra of compound 7 contained 15 carbon signals, including three methyl carbons (δC 10.7, 15.6, 24.1), three aromatic methine carbons (δC 130.6, 134.9, 143.9), eight aromatic quaternary carbons (δC 120.4, 123.0, 124.7, 126.9, 132.5, 146.6, 150.2, 151.5), and one quaternary carbonyl group (δC 181.9). All of the protons were assigned to the corresponding carbons by HSQC experiments. On the basis of the HMBC correlations (Figure S1) from H3-14 to C-5, C-6, and C-6a, from H-5 to C-3a, C-6a, and C-14, and from H-4 to C-3, C-3a, C-11, and C-6a, as well as the HMBC correlations from H3-12 to C-2, C-3, and C-3a and from H-2 to C-3, C-3a, C-12, and C-10, an isoquinoline moiety was established, with methyl groups attached to C-6 and C-3. HMBC correlations (Figure S1) from H3-13 [2.83 (3H, s)] to C-8, C-9, and C-10 suggested that the β carbon (C-9) of the α,β-unsaturated carbonyl unit was connected at C-10. The isoquinoline moiety and α,β-unsaturated carbonyl group accounted for 9 of the 10

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an XY5B micromelting point apparatus and were uncorrected. Optical rotations were recorded on a JASCO P-2000 automatic digital polarimeter. UV spectra were measured on a JASCO V650 spectrophotometer. CD spectra were recorded on a JASCO J815 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR spectrometer. NMR spectra were recorded on INOVA-500 and SX-600 spectrometers. ESIMS spectra were measured on an Agilent 1100 Series LC/MSD ion trap mass spectrometer. HRESIMS data were recorded on an Agilent Technologies 6250 Accurate-Mass QTOF LC/MS spectrometer. EIMS and HREIMS data were recorded on an AutoSpec Ultima-TOF MS spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with an SPD-10A detector, using a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Sephadex LH-20 (Amersham Pharmacia Biotech AB, Sweden), ODS (45−70 μm, Merck), and silica gel (200−300 mesh, Qingdao Marine D

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νmax 3370, 2975, 1664, 1457, 1364, 1219, 1050, 999, 864 cm−1; 1H NMR (600 MHz, CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3) data, see Table 2; ESIMS m/z 523 [2 M + Na]+, 273 [M + Na]+; HRESIMS m/z 251.1639 [M + H]+ (calcd for C15H23O3, 251.1642). X-ray Crystallographic Analysis of Compound 1 (ref 18). Crystallization from CH2Cl2−CH3OH (1:1) using the vapor diffusion method yielded colorless crystals of compound 1. A crystal (0.13 mm × 0.21 mm × 0.36 mm) was separated from the sample and mounted on a glass fiber. Data were collected using a Rigaku MicroMax 002 CCD detector with a graphite monochromator and Cu Kα radiation, λ = 0.71073 Å at 173(2) K. Crystal data: C15H21O3, M = 249.33, space group orthorhombic, P212121; the unit cell dimensions were determined to be a = 9.003(3) Å, b = 8.280(5) Å, and c = 18.182(5) Å; V = 1355.4(10) Å3, Z = 4, Dcalcd = 1.222 mg/m3, F(000) = 540. The 878 measurements yielded 2558 independent reflections after the equivalent data were averaged and Lorentz and polarization corrections were applied. The structure was solved by direct methods using the SHELXS-97 program, expanded using Fourier techniques, and refined by the program SHELXL-97 and full-matrix least-squares calculations. The final refinement gave R1 = 0.0455 and wR2 = 0.1240. (1S)-1-Methoxylacinilene C (2): white, amorphous powder; [α]20D 22 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 207 (4.30), 233 (4.27), 347 (3.87) nm; CD (MeOH) 250.5 (Δε +1.18), 322.5 (Δε +0.20) nm; IR (KBr) νmax 3243, 2970, 1658, 1605, 1571, 1460, 1381, 1314, 1263, 1137, 1104, 1026, 891, 715, 542 cm−1; 1H NMR (500 MHz, DMSO-d6) data, see Table 1; 13C NMR (125 MHz, DMSO-d6) data, see Table 2; ESIMS m/z 283 [M + Na]+, 259 [M − H]−; HRESIMS m/z 283.1309 [M + Na]+ (calcd for C16H20NaO3, 283.1305). Compound 3: brown needles (CH2Cl2−CH3OH); mp 153−154 °C; UV (MeOH) λmax (log ε) 231 (4.40), 358 (3.73) nm; IR (KBr) νmax 3258, 2962, 1620, 1577, 1544, 1465, 1368, 1248, 1204, 1134, 874 cm−1; 1H NMR (500 MHz, CD3OD) data, see Table 1; 13C NMR (125 MHz, CD3OD) data, see Table 2; ESIMS m/z 245 [M + H]+, 243 [M − H]−; HRESIMS m/z 245.1177 [M + H]+ (calcd for C15H17O3, 245.1172). Compound 4: brown needles (CH2Cl2−CH3OH); mp 117−118 °C; UV (MeOH) λmax (log ε) 230 (4.43), 334 (3.70) nm; IR (KBr) νmax 3421, 2954, 1643, 1603, 1540, 1468, 1433, 1364, 1251, 1201, 1133, 949, 884, 842, 813, 662 cm−1; 1H NMR (500 MHz, acetone-d6) data, see Table 1; 13C NMR (125 MHz, acetone-d6) data, see Table 2; EIMS m/z 274; HREIMS m/z 274.1198 (calcd for C16H18O4, 274.1205). N-Hydroxybenzylanabasine (5, racemic mixture): colorless oil; [α]20D +0.1 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 207 (3.96), 264 (3.38) nm; IR (KBr) νmax 3039, 2935, 2815, 1590, 1482, 1254, 1089, 986, 803, 757, 716 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Table 3; ESIMS m/z 269 [M + H]+, 267 [M − H]−; HRESIMS m/z 269.1645 [M + H]+ (calcd for C17H21N2O, 269.1648). (2R)-N-Hydroxybenzylanabasine (5a): colorless oil; [α]20D +3.6 (c 0.03, MeOH); CD (MeOH) 228.5 (Δε +8.20), 278.5 (Δε +3.49) nm. (2S)-N-Hydroxybenzylanabasine (5b): colorless oil; [α]20D 3.6 (c 0.03, MeOH); CD (MeOH) 229.5 (Δε −23.1), 279 (Δε −9.22) nm. 2-Hydroxy-N-hydroxybenzylanabasine (6, racemic mixture): colorless oil; [α]20D −0.3 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 207 (3.98), 264 (3.40) nm; IR (KBr) νmax 3040, 2940, 2859, 1585, 1486, 1454, 1303, 1237, 1117, 1030, 948, 907, 756, 716 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Table 3; ESIMS m/z 283 [M − H]−; HRESIMS m/z 285.1600 [M + H]+ (calcd for C17H21N2O2, 285.1598). (2S)-2-Hydroxy-N-hydroxybenzylanabasine (6a): colorless oil; [α]20D −4.3 (c 0.03, MeOH); CD (MeOH) 231 (Δε −1.98), 278.5 (Δε −0.61) nm. (2R)-2-Hydroxy-N-hydroxybenzylanabasine (6b): colorless oil; [α]20D +4.3 (c 0.03, MeOH); CD (MeOH) 228.5 (Δε +0.83), 245.5 (Δε +0.48), 279 (Δε +0.45) nm. 8-Hydroxy-3,6,9-trimethyl-7H-benzo[de]quinolin-7-one (7): red, amorphous powder; UV (MeOH) λmax (log ε) 210 (4.42), 346 (3.60), 449 (3.28) nm; IR (KBr) νmax 3359, 1649, 1615, 1572, 1450, 1391,

Chemical Inc. China) were used for column chromatography (CC). TLC was conducted with glass precoated with silica gel GF254 (Qingdao Marine Chemical Inc., China). Plant Material. The roots of A. chinense were collected from Guangxi Province, China, in July 2009 and identified by Prof. Peng-Fei Tu (Peking University School of Pharmaceutical Sciences). A voucher specimen (ID-S-2356) has been deposited in the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences, People’s Republic of China. Extraction and Isolation. Air-dried, powdered roots of A. chinense (10 kg) were macerated for 3 h with 40 L of aqueous 95% EtOH and refluxed for 6 h (40 L × 3). The filtrate was concentrated under reduced pressure, and the residue (500 g) was suspended in H2O and then successively partitioned with petroleum ether, EtOAc, and nBuOH. The petroleum ether extract (60 g) was subjected to a silica gel column (200−300 mesh, 1 kg) and eluted sequentially with petroleum ether containing increasing amounts of acetone (1:0, 50:1, 30:1, 20:1, 10:1,5:1, 3:1, and 0:1), to yield nine fractions, S1−S9. Fraction S3 (2.3 g) was chromatographed over a Sephadex LH-20 column with CH2Cl2−MeOH (5:1) and purified via silica gel CC (petroleum ether−EtOAc, 15:1) to yield compounds 10 (17 mg), 14 (25 mg), and 15 (12 mg). Fraction S7 (2.81 g) was chromatographed over Sephadex LH-20 with CH2Cl2−MeOH (5:1) and further purified by preparative HPLC [CH3CN−H2O (55:45)] to yield compounds 3 (8 mg) and 4 (10 mg). Fraction S8 (2.1 g) was chromatographed over a Sephadex LH-20 with CH2Cl2−MeOH (5:1) and further purified by preparative HPLC [CH3CN−H2O (45:55)] to yield compound 11 (10 mg). Air-dried, powdered roots of A. chinense (200 kg) were macerated for 12 h with 800 L of aqueous 95% EtOH and refluxed for 6 h (800 L × 3). After removal of the solvent under vacuum, the resultant residue (8 kg) was suspended in acidic H2O (100 L) and acidified to pH 2 with HCl to afford acidic H2O-soluble and acidic H2O-insoluble fractions. The acidic mixture was then filtered and partitioned with petroleum ether. The acidic H2O phase was basified to pH 10 with NaOH and then partitioned with CHCl3 to yield the CHCl3 extract (90 g). The alkaline H2O phase was then acidified to pH 7 with HCl and partitioned with n-BuOH to yield an n-BuOH extract (210 g). The crude CHCl3 extract (90 g) was fractionated using a basified silica gel column (pH 8−9, 200−300 mesh, 1.6 kg), eluting with petroleum ether containing increasing amounts of EtOAc (1:0, 50:1, 20:1, 10:1, 5:1, 1:1), and then eluted with CH2Cl2−MeOH (10:1 to 0:100) to afford eight fractions (A−H). Fraction B (0.39 g) was chromatographed over Sephadex LH-20 with CH2Cl2−MeOH (5:1) repeatedly to yield 7 (20 mg). Fraction C (1.2 g) was fractionated via an ODS column (45−70 μm, 400 g) eluting with a gradient of MeOH (5− 100%) in 0.03% TFA−H2O to yield four major fractions (C1−C4). Fraction C1 (32 mg) was purified via a silica gel column (petroleum ether−acetone−diethylamine, 10:1:0.1) to yield compounds 5 (20 mg) and 6 (2 mg). Compound 5 was isolated using a chiral analytical column (90% n-hexane−10% 2-propanol, 1 mL/min) to yield compounds 5a (1 mg) and 5b (1 mg). Compound 6 was isolated using a chiral analytical column (85% n-hexane−15% 2-propanol, 1 mL/min) to yield compounds 6a (0.5 mg) and 6b (0.5 mg). Fraction C4 (30 mg) was further purified by reversed-phase preparative HPLC (CH3CN−H2O−TFA, 42.5:57.5:0.03) to afford compounds 8 (6 mg) and 12 (2 mg). Fraction E (7.0 g) was fractionated via an ODS column (45−70 μm, 400 g) by eluting with a gradient of MeOH (5− 100%) in H2O to yield six major fractions (E1−E6). Fraction E6 (1.6 g) was chromatographed over a Sephadex LH-20 column with CH2Cl2−MeOH (5:1) and was further purified by reversed-phase preparative HPLC (MeOH−H2O−TFA, 50:50:0.03) to afford compounds 2 (2 mg), 9 (5 mg), and 13 (4 mg). Fraction E4 (1.1 g) was chromatographed over a Sephadex LH-20 column with CH2Cl2−MeOH (5:1) and was further purified by reversed-phase preparative HPLC (MeOH−H2O−TFA, 40:60:0.03) to afford compound 1 (4 mg). (3S,4R,5S,8R,10R)-Tetrahydroperezinone (1): white needles (CH2Cl2−CH3OH); mp 146−147 °C; [α]20D −73.9 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 228 (4.19) nm; CD (MeOH) 221 (Δε +2.16), 246.5 (Δε −13.6), 324.5 (Δε +3.54) nm. IR (KBr) E

dx.doi.org/10.1021/np4000747 | J. Nat. Prod. XXXX, XXX, XXX−XXX

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1325, 1265, 1210, 1137, 1082, 809, 776, 642 cm−1; 1H NMR (500 MHz, C5D5N) data, see Table 1; 13C NMR (125 MHz, C5D5N) data, see Table 2; ESIMS m/z 240 [M + H]+; HRESIMS m/z 240.1021 [M + H]+ (calcd for C15H14NO2, 240.1019). In Vitro Anti-Coxsackie Virus B3 Activity Assay. Confluent Vero cells grown in 96-well microplates were infected with 100 median tissue culture infective doses (100TCID50) of Cox B3 virus. After 1 h of adsorption at 37 °C, the monolayers were washed with phosphatebuffered saline (PBS) and incubated at 37 °C in maintenance media (MEM plus 2% fetal bovine serum (FBS)) with or without different concentrations of test compounds. The viral cytopathic effect (CPE) was observed when the viral control group reached 4+, and the antiviral activity of the tested compounds was determined by Reed and Muench analyses.19 Cytotoxicity. Vero cells were plated into a 96-well plate. After 24 h, the monolayer cells were incubated in the presence of various concentrations of test compounds. After 48 h of culture at 37 °C and 5% CO2 in a carbon dioxide incubator, the cells were monitored by CPE. The median toxic concentration (TC50) was calculated by Reed and Muench analyses.20 Inhibitory Effects on Nitric Oxide Production in LPSActivated Microglia. The BV2 cell line, a microglia cell line, was purchased from the Cell Culture Centre at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. LPS (from Escherichia coli 055:B5) was obtained from Sigma-Aldrich. After preincubation for 24 h in a 96-well plate, the cells were treated with various concentrations of isolated compounds and then stimulated with 300 ng/mL LPS for 24 h. The production of NO was determined by assaying the concentration of nitrite in the culture supernatant. Briefly, 100 μL of culture supernatant was incubated with 100 μL of Griess reagent (a 1:1 mixture of 1% sulfanilamide in 5% H3PO4 and 0.1% N-(1-naphthyl)ethylenediamine in distilled water) at room temperature for 20 min. Sodium nitrite was used to generate a standard curve. The OD value of the samples at 550 nm was measured. Untreated culture cells were used as a negative control. Curcumin was used as the positive control (IC50 3.1 μM).21 Antioxidant Assay. Antioxidant assays were performed according to reported procedures.22 Vitamin E was selected as the positive control. The activities were determined by measuring the content of malondialdehyde (MDA), a compound produced during microsomal lipid peroxidation induced by Fe2+-cysteine. MDA was detected using the thiobarbituric acid (TBA) method. Briefly, 1.0 mg of microsomal protein in 1 mL of 0.1 M PBS buffer (pH 7.4) was incubated with 0.2 μM cysteine and the test samples at 37 °C for 15 min. Lipid peroxidation was initiated by the addition of 0.05 mM FeSO4. After incubation, 1 mL of 20% trichloroacetic acid was added to terminate the reaction. The mixture was centrifuged for 10 min at 3000 rpm. The supernatant was removed and reacted with 0.67% TBA for 10 min at 100 °C. After cooling, the MDA was quantified by UV/vis (absorbance at 532 nm), from which the inhibition rate (IR) was calculated as IR [%] = 100% At/(AP − Ac) × 100, where Ap, At, and Ac refer to the absorbance of Fe2+-cysteine, test compound, and control (solvent only), respectively.

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ACKNOWLEDGMENTS This project was supported by the National Science and Technology Project of China (No. 2009ZX09311-004) and the Natural Science Foundation of China (No. 201072234). We are grateful to the Department of Instrumental Analysis, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, for measuring the IR, UV, NMR, and MS spectra.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

IR, MS, and 1D and 2D NMR spectra for compounds 1−7; UV and CD spectra for compounds 1, 2, 5, and 6. HPLC profile of compound 5. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-63165324. Fax: +86-10-63017757. Notes

The authors declare no competing financial interest. F

dx.doi.org/10.1021/np4000747 | J. Nat. Prod. XXXX, XXX, XXX−XXX