Antidepressant Abietane Diterpenoids from ... - ACS Publications

Feb 8, 2013 - ... and Peking Union Medical College, Beijing 100050, People's Republic of China ...... Use of Zanzibar copal (Hymenaea verrucosa Gaertn...
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Antidepressant Abietane Diterpenoids from Chinese Eaglewood Lin Yang,†,‡,§ Lirui Qiao,† Chengxue Ji,† Dan Xie,† Ning-Bo Gong,† Yang Lu,† Jianjun Zhang,† Jungui Dai,*,† and Shunxing Guo*,‡ †

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 ‡ Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, People’s Republic of China § College of Life and Environmental Sciences, Minzu University of China, Beijing 100081, People’s Republic of China S Supporting Information *

ABSTRACT: Ten new abietane diterpenoids, aquilarabietic acids A−J (1−10), and a new podocarpane diterpenoid, aquilarabietic acid K (11), were isolated from the petroleum ether and ethanol extracts of Chinese eaglewood. Among them, 3, 9, and 10 are artifacts. Their structures were established on the basis of data from extensive spectroscopic and X-ray diffraction analyses. Bioassay results indicated that 1 at 10 μM demonstrated remarkable antidepressant activity in vitro by inhibiting norepinephrine reuptake in rat brain synaptosomes by 81.4% and with an IC50 value of 9.1 × 10−7 M.

C

proton (δH 4.13, t, J = 2.8 Hz, H1-7), and an olefinic proton (δH 5.71, d, J = 1.2 Hz, H1-14). The 13C NMR and DEPT data (Table 2) showed 20 carbon resonances including four methyl, six methylene, three methine (one oxygenated), and four quaternary carbons (two oxygenated), a trisubstituted double bond (δC 138.3, s, C-8; 137.2, d, C-14), and a carbonyl carbon (δC 182.5, s, C-18). Therefore, the data indicated that one carbonyl group, one olefinic moiety, and a tricyclic unit were present to explain the detected five degrees of unsaturation, and 1 was determined to be an abietic acid-type diterpenoid18 with three hydroxy groups. The molecular structure of 1 with the three hydroxy groups located at C-7, C-9, and C-13 and the C8−C-14 double bond was determined via the combined 1H−1H COSY, HSQC, and HMBC spectra (Figure 2). Both the lack of NOE correlations between H-5/H-7 and the small coupling constant for H-7 (t, J = 2.8 Hz) indicated an α-orientation for the 7-OH, which was confirmed by the single-crystal X-ray diffraction analysis (Figure 3). To prove the above assignment and determine the orientation of the 13-OH in 1, a singlecrystal X-ray diffraction pattern was obtained via the anomalous scattering of Mo Kα radiation. An ORTEP drawing with the indicated atom-numbering scheme is shown in Figure 3 and demonstrates the α-orientation of the 13-OH. Thus, the structure of 1 was identified as 7α,9α,13α-trihydroxyabiet8(14)-en-18-oic acid. Aquilarabietic acid B (2) possessed the same molecular formula as compound 1 according to HRESIMS analysis. The

hinese eaglewood (“Chenxiang” in Chinese), the resinous wood from the tree Aquilaria sinensis Gilg (Thymelaeaceae), is a Chinese crude drug used as a traditional sedative, analgesic, and digestive medicine in China.1 Chenxiang is distributed in Southeast Asia and used as medicine and incense in many Asian countries.2 Previous phytochemical investigations of eaglewood have resulted in the identification of sesquiterpenes3−9 and chromone derivatives.10−16 Our previous studies on the chemical constituents of Chinese eaglewood led to the isolation of several 2-(2-phenylethyl)chromones.17 By continuing the studies on the bioactive chemical constituents of Chinese eaglewood via bioassay-guided fractionation through the in vitro inhibition of serotonin and norepinephrine reuptake in rat brain synaptosomes, 10 new abietane diterpenoids, aquilarabietic acids A−J (1−10, Figure 1), and a new podocarpane diterpenoid, aquilarabietic acid K (11, Figure 1), were obtained. Among them, 3, 9, and 10 are artifacts. Herein, we describe the isolation and structural elucidation of compounds 1−11 and their in vitro antidepressant activity.

■

RESULTS AND DISCUSSION Aquilarabietic acid A (1) was crystallized from acetone as colorless needles. The molecular formula of 1 was determined to be C20H32O5 on the basis of its HRESIMS, which indicated five degrees of unsaturation. The IR spectrum showed absorption peaks at 3236 cm−1 (hydroxy group) and 1731 cm−1 (carbonyl group). The 1H NMR data for 1 (Table 1) contained two methyl singlets (δH 1.16, s, H3-19; 0.83, s, H320), an isopropyl group (δH 0.87, d, J = 6.8 Hz, H3-16; 0.86, d, J = 6.4 Hz, H3-17; 1.69, m, H1-15), an oxygenated methine © 2013 American Chemical Society and American Society of Pharmacognosy

Received: October 5, 2012 Published: February 8, 2013 216

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Figure 1. Structures of compounds 1−11. 1

H and 13C NMR spectroscopic data (Tables 1 and 2) of 2 were similar to those of 1. The primary difference was the presence of an oxygenated methine at δC 70.0 (C-12) in 2 instead of the oxygenated quaternary carbon at δC 77.4 (C-9) in 1, which indicates the absence of the hydroxy group at C-9 in 2. The key HMBC correlations of both H-14 (δH 5.47) and H-15 (δH 1.74) to C-12 (δC 70.0) supported the presence of the hydroxy group at C-12 in 2. The α-orientation of the 12-OH was established using the NOE correlations between H-12/H15 and H-20 (Figure 4). Thus, 2 was determined to be 7α,12α,13α-trihydroxyabiet-8(14)-en-18-oic acid. Aquilarabietic acid C (3) was obtained as pale yellow, block crystals, and its molecular formula was determined to be C23H36O5 by HRESIMS (m/z 391.2499 [M − H]−, calcd for C23H35O5, 391.2485), which indicated six degrees of unsaturation. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) of 3 were similar to those of 2 except for the addition of an acetonide moiety [δH 1.34, s, H3-1′ and 1.33, s, H3-2′; δC 28.5 (q, C-1′), 27.2 (q, C-2′), and 106.9 (s, C-3′)], which indicated that 3 was an acetonide derivative of 2. Thus, 3 was determined to be 7α,12α,13α-trihydroxyabiet-8(14)-en-18-oic acid acetonide, which might be an artifact because it was not detected in the crude extract (Supporting Information, pp S60, 61), and acetone was used as eluent during chromatography. The molecular formula of aquilarabietic acid D (4) was C20H32O5 on the basis of HRESIMS analysis. It contained three hydroxy groups and a double bond, according to the NMR (Tables 1 and 2) and DEPT data. The key HMBC correlations between H-6 (δH 4.04)/C-5 (δC 51.9)/C-7 (δC 131.5), H-14 (δH 3.75)/C-8 (δC 141.2)/C-13 (δC 75.3), and H-7 (δH 5.49)/ C-5 (δC 51.9)/C-14 (δC 76.4) indicated that the three hydroxy groups were located at C-6, C-13, and C-14 and that the double bond was at C-7(8). The α-orientations of the 6- and 14-OHs were determined by the NOE correlations of H-6/H-19/H-20 and H-14/H-15, respectively. Thus, the structure of 4 was determined to be 6α,13α,14α-trihydroxyabiet-7-en-18-oic acid. Aquilarabietic acid E (5) was isolated as a pale yellow, amorphous solid, and its molecular formula was determined to be C20H30O6 by HRESIMS, which indicated six degrees of unsaturation. The IR spectrum indicated the presence of hydroxy group(s) (3520 cm−1) and an α,β-unsaturated carbonyl group (1648 cm−1). The 1H and 13C NMR data (Tables 1 and 2) showed two tertiary methyl groups (δH 1.21,

s, H3-19; 0.86, s, H3-20), a hydroxyisopropyl group [δH 1.17 and 1.33 (s, H3-16 and H3-17); δC 24.1 (q), 25.4 (q), and 76.8 (s)], six methylene groups, two methines (one oxygenated), four quaternary carbons (two oxygenated), an α,β-unsaturated carbonyl [δC 131.4 (s, C-8), 171.2 (s, C-9), and 201.6 (s, C-7)], and a carbonyl carbon [δC 181.5, s, C-18]. The molecular structure of 5 was determined via the combined HMQC and HMBC data in comparison to the NMR data of 4, which indicated that 5 had an abietic acid skeleton bearing three hydroxy groups at C-13, C-14, and C-15 and an α,βunsaturated carbonyl moiety at C-7−8(9). Thus, 5 was determined to be 13α,14α,15-trihydroxy-7-oxoabiet-8-en-18oic acid. Aquilarabietic acid F (6) had the molecular formula C20H28O3, as determined by HRESIMS (m/z 317.2124 [M + H]+). The IR absorption at 1776 cm−1 indicated a γbutyrolactone group. Compound 6 had the same molecular structure as 13β,14β-epoxyabiet-7-en-19,6β-olide19 based on its HSQC and HMBC data. The different NMR data [δC 72.8, δH 4.87 (1H, dd, J = 5.0, 2.0 Hz) for the above compound; δC 74.9, δH 4.79 (1H, d, J = 11.2 Hz) for 6] at C-6 suggest that these two compounds may be stereoisomers. To prove the above assignment and determine the absolute configuration of 6, a single-crystal X-ray diffraction pattern was obtained via the anomalous scattering of Cu Kα radiation. An ORTEP drawing with the indicated atom-numbering scheme is shown in Figure 5 and demonstrates a configuration of 4R,5S,6R,9R,10R,13S,14S for 6. Thus, the structure of 6 was determined to be 13β,14β-epoxyabiet-7-en-18,6α-olide. Aquilarabietic acid G (7) had the same molecular formula, C20H32O5 (m/z 375.2132 [M + Na]+), as compounds 1, 2, and 4. The three hydroxy groups were assigned to the same locations as 2 by HMBC analysis. The differences in chemical shifts in the 1H and 13C NMR data (Tables 1 and 2) indicated that 2 and 7 might be stereoisomers. The β-orientation of the 12-OH group and α-isopropyl group in 7 were established via an NOE experiment (Figure 3) by the H-12/H-9/H-15 correlations; the β-orientation of H-7 was assigned on the basis of the absence of any H-5/H-7 correlation and the broad singlet for H-7. Thus, compound 7 was defined as 7α,12β,13βtrihydroxyabiet-8(14)-en-18-oic acid. Aquilarabietic acid H (8) was assigned the molecular formula C20H26O3 on the basis of its positive HRESIMS, which yielded 217

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218

2′

1.09, s 0.75, s

0.90, d (6.6)

0.88, d (7.2)

1.74, mc

5.47, s

1.62, mc 3.82, dd (6.0, 3.0)

1.33b, s

1.22, s 0.77, s 1.34b, s

0.93, d (6.6)

0.95, d (7.2)

1.82, mc

5.46, s

1.46, mc 4.13, s

4

mc mc mc s

1.30, s 0.87, s

0.83, d (6.0)

0.88, d (6.0)

1.80, mc

1.42, 1.69, 1.53, 3.75,

1.50, mc

2.10, m

5.49, br s

4.04, dd (10.4, 1.6)

1.57, m 2.17, br d (10.4)

c

1.47, mc 1.66, mc

1.78, m 1.14, ddd (12.8, 12.8, 4.8) 1.54, mc

c

c

mc mc mc br s

1.21, s 0.86, s

1.17, s

1.33, s

2.36, 1.81, 1.78, 4.49,

2.38, mc

2.11, br d (14.8)

2.53, mc

1.59, m 2.49, mc

c

1.60, mc 1.75, mc

1.76, mc

1.90, m 1.38, mc

5 c

c

0.96, d (7.2) 0.95, d (7.2) 1.24, s 0.86, s

1.60, mc

1.68, m 1.62, mc 3.16, s

c

1.34, mc (2H)

1.76, m

6.22, s

c

1.64, m 1.88, d (10.8) 4.79, d (11.2)

2.04, m

c

1.68, mc (2H)

1.84, m 1.12, mc

6

7

1.12, s 0.78, s

0.90, d (7.2)

0.80, d (7.2)

2.06, sept (7.2)

5.55, s

1.59, mc 3.62, dd (12.0, 4.2)

2.29, br dd (8.4, 8.4) 1.67, mc

4.08, br s

1.39, br d (13.8)

1.68, mc

c

1.54, m 2.34, br d (11.4)

1.83, m

c

1.66, m 1.15, ddd (13.2, 13.2, 6.0) 1.53, mc (2H)

c

1.21, s 1.11, s

2.07, s

5.29, s; 4.97, s

7.38, d (2.0)

7.31, dd (8.4, 2.0)

7.19, d (8.4)

4.67, br d (4.0)

2.07, ddd (13.6, 13.6, 4.8) 1.61, br d (14.0)

1.88, dddd (12.8, 12.8, 12.8, 4.4) 1.61, br d (14.0) 2.48, dd (12.8, 2.0)

2.31, br d (13.2) 1.44, ddd (12.8, 12.8, 3.6) 1.76, mc (2H)

8

1.21, s 1.11, s 2.99, s

1.44, s

1.44, s

7.29, s

7.23, d (0.8)

7.23, d (0.8)

4.66, dd (4.8, 1.2)

2.08, ddd (13.6, 13.6, 4.4) 1.61, br d (14.0)

1.90, dddd (13.2, 13.2, 13.2, 4.0) 1.60, br d (14.0) 2.48, dd (13.2, 2.0)

1.76, mc (2H)

2.32, br d (13.2) 1.43, mc

9

1.28, 0.84, 3.68, 3.45, 1.25,

s s q (7.2) q (7.2) t (7.2)

1.08, d (6.6)

2.41, sept (6.6) 1.11, d (6.6)

5.85, s

1.86, mc 3.91, br s

2.16, dd (12.0, 4.2) 2.06, mc

5.49, t (2.4)

1.16, mc

2.36, br d (13.8) 2.02, mc

1.86, m (2H)

c

1.62, mc (2H)

1.88, m 1.21, mc

c

10

1.26, s 1.12, s

1.56, mc 2.60, mc 2.22, mc 2.62, mc; 2.41, mc

2.15, mc

2.57, mc 1.62, mc

2.48, mc

2.53, mc

1.79, mc 2.22, mc

1.66, mc 1.84, mc

1.69, mc

1.90, mc 1.20, mc

11

Data were measured in methanol-d4 at 600 MHz for both 2 and 7 and 400 MHz for 1, 4, 5, 8, and 9 and in CDCl3 at 600 MHz for both 3 and 10, 400 MHz for 6, and 300 MHz for 11. bValues can be interchanged. cMultiplicity not determined due to overlapping signals.

a

0.86, d (6.4)

17

1.16, s 0.83, s

0.87, d (6.8)

16

19 20 1′

1.69, mc

15

14

12

mc mc mc d, (1.2)

1.71, mc

11

1.42, 1.85, 1.65, 5.71,

2.43, m

2.33, br dd (6.6, 6.6) 1.77, mc 2.11, m

4.29, br s

4.07, t (3.0)

1.56, mc

168, m 2.50, dd (13.2, 2.4) 1.76, mc

c

1.53, mc 1.85, mc

1.59, mc

1.66, m 1.20, mc

c

3

4.13, t (2.8)

1.51, mc

1.46, mc

1.83, ddd, (12.6, 12.6, 4.8) 1.48, mc 2.47, dd (13.2, 3.0)

1.60, mc

c

1.52, m 2.87, dd (13.2, 2.8) 1.66, mc

1.78, m

2

1.61, m 1.17, ddd (12.6, 12.6, 4.8) 1.51, mc (2H)

c

7 8 9

6

5

3

1.53, mc (2H)

2

c

1.82, m 1.32, mc

c

1

1

no.

Table 1. 1H NMR Spectroscopic Data for Compounds 1−11 (J in Hz)a

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a

219

31.7, 18.9, 37.8, 47.9, 35.9, 33.2, 74.5, 138.3, 77.4, 42.9, 27.6, 30.9, 73.7, 137.2, 34.9, 16.9, 17.2, 182.5, 17.4, 18.1,

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′

CH2 CH2 CH2 qC CH CH2 CH qC qC qC CH2 CH2 qC CH CH CH3 CH3 qC CH3 CH3

37.8, 18.2, 36.6, 46.6, 41.6, 31.1, 72.4, 139.5, 41.4, 36.9, 23.4, 74.7, 81.8, 128.4, 34.2, 17.6, 17.9, 182.1, 16.7, 14.5, 28.5, 27.2, 106.9,

39.4, 19.7, 38.7, 49.2, 43.9, 33.6, 73.6, 142.0, 44.3, 39.7, 26.7, 70.0, 74.3, 129.8, 35.5, 17.7, 16.7, 186.9, 18.5, 14.8,

CH2 CH2 CH2 qC CH CH2 CH qC CH qC CH2 CH qC CH CH CH3 CH3 qC CH3 CH3

3

2 CH2 CH2 CH2 qC CH CH2 CH qC CH qC CH2 CH qC CH CH CH3 CH3 qC CH3 CH3 CH3 CH3 qC

40.1, 18.9, 39.8, 45.1, 51.9, 67.9, 131.5, 141.2, 48.4, 37.2, 20.1, 29.6, 75.3, 76.4, 33.9, 16.5, 16.5, 184.6, 17.9, 16.4,

4 CH2 CH2 CH2 qC CH CH CH qC CH qC CH2 CH2 qC CH CH CH3 CH3 qC CH3 CH3

35.6, 19.0, 37.7, 47.8, 46.2, 38.5, 201.6, 131.4, 171.2, 40.6, 23.4, 22.9, 73.8, 67.1, 76.8, 24.1, 25.4, 181.5, 17.1, 18.2,

5 CH2 CH2 CH2 qC CH CH2 qC qC qC qC CH2 CH2 qC CH qC CH3 CH3 qC CH3 CH3

38.1, 19.2, 24.3, 41.5, 56.6, 74.9, 126.5, 139.8, 52.4, 35.6, 17.4, 24.7, 64.4, 60.8, 33.9, 17.7, 18.2, 180.8, 15.2, 14.4,

6 CH2 CH2 CH2 qC CH CH CH qC CH qC CH2 CH2 qC CH CH CH3 CH3 qC CH3 CH3

39.4, 19.1, 38.1, 47.9, 43.0, 33.2, 72.9, 146.2, 48.4, 38.8, 26.7, 70.1, 74.1, 126.7, 33.8, 18.2, 16.7, 182.5, 17.5, 14.7,

7 CH2 CH2 CH2 qC CH CH2 CH qC CH qC CH2 CH qC CH CH CH3 CH3 qC CH3 CH3

38.9, 19.6, 37.6, 48.3, 40.9, 32.2, 68.4, 137.1, 149.9, 38.6, 124.9, 126.2, 139.9, 128.6, 144.4, 111.9, 21.9, 182.1, 17.1, 24.6,

8 CH2 CH2 CH2 qC CH CH2 CH qC qC qC CH CH qC CH qC CH2 CH3 qC CH3 CH3

38.9, 19.7, 37.6, 48.2, 40.9, 32.4, 68.4, 137.1, 149.4, 38.6, 125.1, 126.7, 144.1, 129.0, 78.1, 28.2, 28.1, 182.1, 17.1, 24.7, 50.9,

9 CH2 CH2 CH2 qC CH CH2 CH qC qC qC CH CH qC CH qC CH2 CH2 qC CH3 CH3 CH3

37.9, 18.0, 37.0, 46.1, 43.8, 25.7, 123.7, 134.6, 44.7, 33.9, 25.7, 73.9, 142.2, 126.0, 32.6, 21.6, 22.0, 182.5, 16.7, 14.4, 64.3, 15.8,

CH2 CH2 CH2 qC CH CH2 CH qC CH qC CH2 CH qC CH CH CH3 CH3 qC CH3 CH3 CH2 CH3

10

Data were measured in methanol-d4 at 150 MHz for both 2 and 7 and 100 MHz for 1, 4, 5, 8, and 9 and in CDCl3 at 150 MHz for both 3 and 10 and 100 MHz for 6 and 11.

1

no.

Table 2. 13C NMR Spectroscopic Data for Compounds 1−11a CH2 CH2 CH2 qC CH CH2 qC CH CH qC CH2 CH2 qC CH2

181.7, qC 16.0, CH3 13.8, CH3

37.8, 17.7, 36.8, 46.8, 48.4, 40.6, 208.3, 49.7, 54.3, 36.5, 25.5, 40.7, 210.4, 40.8,

11

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an m/z of 337.1764 [M + Na]+ for eight degrees of unsaturation. The molecular structure of 8 was determined by combining the 1H−1H COSY, HSQC, and HMBC data, which were similar to those of 7β-hydroxy-15-endehydroabietic acid,20 except for a smaller coupling constant (δH 4.67, br d, J = 4.0 Hz, H-7) observed for 8. The NOE correlations of H-7/H19/H-20 also supported the presumed 7α-OH orientation. Accordingly, the structure of 8 was defined to be 7αhydroxyabieta-8,11,13,15-tetraen-18-oic acid. Aquilarabietic acid I (9) possessed the molecular formula C21H30O4 according to the HRESIMS (m/z 369.2037 [M + Na]+, calcd for C21H30NaO4, 369.2036). The 1H and 13C NMR data of 9 (Tables 1 and 2) were similar to those of 7α,15dihydroxy-15-dehydroabietic acid21 except for the presence of a methoxy group instead of the hydroxy group. The HMBC correlations between the methoxy protons (δH 2.99) and C-15 (δC 78.1) indicated that the methoxy group was located at C15. Therefore, 9 was determined to be 7α-hydroxyabieta-15methoxy-8,11,13-trien-18-oic acid, which may be an artifact because it was not detected in the crude extract (Supporting Information, pp S60, 62), and MeOH was used as solvent during purification. Aquilarabietic acid J (10) had the molecular formula C22H34O3 on the basis of its HRTOFMS (m/z 345.2454 [M − H]+). The 1H and 13C NMR data of 10 (Tables 1 and 2) were similar to those of 12α-hydroxyabietic acid22 except for the presence of an ethoxy moiety instead of the hydroxy group. HMBC correlations between H-12 (δH 3.91, br s) and C-1′ (δC 64.3) supported this deduction. Accordingly, 10 was determined to be 12α-ethoxyabieta-7,13-dien-18-oic acid, a presumed artifact of 12α-hydroxyabieta-7,13-dien-18-oic acid because it was not detected in the crude extract (Supporting Information, pp S60, 63), and EtOH was used as solvent during purification. Aquilarabietic acid K (11) was assigned the molecular formula C17H24O4 based on its HRESIMS (m/z 607.3211 [2M + Na]+, calcd for C34H48O8Na, 607.3241). Its IR spectrum indicated the presence of carbonyl groups (1711, 1684 cm−1). The 1H and 13C NMR data of 11 (Tables 1 and 2) were similar to those of 8(14)-podocarpen-7,13-dion-18-oic acid23 except for the absence of the double bond at C-8(14). This agreed with the HMBC correlations of both H-5 (δH 2.22) and H-6 (δH 2.48) to C-7 (δC 208.3) and both H-12 (δH 2.60) and H-14 (δH 2.41) to C-13 (δC 210.4). Accordingly, 11 was determined to be 7,13-dioxopodocarpan-18-oic acid. Serotonin and norepinephrine reuptake inhibitors may be potential antidepressants. The in vitro antidepressant activities of the 11 compounds were preliminarily evaluated using rat brain synaptosomes by inhibiting both [3H]-5-HT and [3H]NE reuptake24 using duloxetine as a positive control. The results showed that compounds 1, 8, and 9 remarkably exhibited in vitro antidepressant activity at 10 μM, inhibiting [3H]-NE reuptake in the rat brain synaptosomes by 81.4%, 73.8%, and 70.4%, respectively (Table 3), and the IC50 of compound 1 was determined to be 9.1 × 10−7 M. In addition, compound 2 showed potent in vitro antidepressant activity, having inhibited [3H]-5-HT reuptake in the rat brain synaptosomes by 63.8% at 10 μM, and an IC50 value of 3.2 × 10−6 M. Interestingly, when comparing the structures of 1, 8, and 9 to the other eight compounds, it could be hypothesized that the activities of 1, 8, and 9 may be attributed to the αorientation of their C-7 hydroxy and the substitution of the hydroxy group at C-9 or aromatization of ring C. Undoubtedly,

Figure 2. Key HMBC (→) and 1H−1H COSY (−) correlations for compounds 1 and 7.

Figure 3. ORTEP diagram of compound 1.

Figure 4. Key NOE correlations for compounds 2 and 7.

Figure 5. ORTEP diagram of compound 6.

220

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Table 3. Inhibition of [3H]-5-HT and [3H]-NE Reuptake in Rat Brain Synaptosomes by 1−11

a

compound (10 μM)

inhibition of [3H]-5-HT reuptake (%)

inhibition of [3H]-NE reuptake (%)

duloxetine 1 2 3 4 5 6 7 8 9 10 11

89.5 42.5 63.8 (3.2 × 10−6 Ma) 54.9 31.6 52.7 51.1 36.9 38.0 38.1 51.1 32.5

81.4 81.8 (9.1 × 10−7 Ma) 39.1 37.6 37.2 0 8.1 34.4 73.8 70.4 13.4 18.9

semipreparative reversed-phase HPLC with MeOH−H2O (9:11, v/v) to afford 5 (7 mg) and 7 (5 mg). Fraction D5 (3.5 g) was subjected to silica gel (100 g) column chromatography eluted with CHCl3−MeOH (from 99:1 to 1:1, v/v) to yield fractions D5a−D5e. Fraction D5e was subjected to semipreparative reversed-phase HPLC with MeOH−H2O (3:2, v/v) to yield 4 (10 mg). Fraction D6 (4.8 g) was subjected to silica gel (152 g) column chromatography eluted with CHCl3−MeOH (from 99:1 to 1:1, v/v) to yield fractions D 6a −D 6d . The recrystallization of fraction D6c in acetone yielded 1 (45 mg). Fraction E (29.0 g) was subjected to silica gel (350 g) column chromatography and eluted with petroleum ether−acetone (9:1−1:2, v/v) to yield fractions E1−E6. Fraction E1 (250 mg) was purified over semipreparative reversed-phase HPLC with MeOH−H2O (4:1, v/v) to afford 3 (20 mg). Fraction E5 (2.5 g) was subjected to column chromatography over silica gel (40 g), eluted with petroleum ether− acetone (3:1−1:2, v/v), and chromatographed further over an RP-18 column eluted with a MeOH−H2O gradient system (30−100%, v/v) to afford compound 2 (32 mg). The petroleum ether extract (212 g) was subjected to silica gel (200−300 mesh, 1.8 kg) column chromatography eluted with petroleum ether−EtOAc (1:0−0:1, v/v) to afford fractions I−P. Fraction K (16.0 g) was subjected to column chromatography over silica gel (300 g) eluted with petroleum ether−EtOAc (1:0−1:5, v/v) to give fractions K1−K5. Fraction K4 (2.4 g) was subjected to column chromatography over silica gel (50 g) eluted with petroleum ether− acetone (249:1−1:2, v/v) and further purified via semipreparative reversed-phase HPLC with MeOH−H2O (39:11, v/v) to afford 6 (15 mg). Fraction K5 (0.9 g) was subjected to semipreparative reversedphase HPLC with MeOH−H2O (39:11, v/v) to yield 10 (129 mg). Aquilarabietic acid A (1): colorless needles (acetone); mp 216− 218 °C; [α]20D −10.2 (c 0.3, MeOH); IR νmax 3477, 3419, 3236, 2942, 2571, 1731, 1459, 1382, 1237, 1026, 971, 895, 849 cm−1; for 1H (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) data, see Tables 1 and 2, respectively; positive HRESIMS m/z 375.2161 [M + Na]+ (calcd for C20H32O5Na, 375.2147). Aquilarabietic acid B (2): white power; [α]20D −80.6 (c 0.2, MeOH); IR νmax 3353, 2937, 2876, 1655, 1556, 1468, 1390, 1312, 1172, 1033, 1003, 976, 947, 886 cm−1; for 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data, see Tables 1 and 2; negative HRESIMS m/z 351.2168 [M − H]− (calcd for C20H31O5, 351.2172). Aquilarabietic acid C (3): pale yellow, block crystals (acetone); mp 186−188 °C; [α]20D −17.8 (c 0.5, MeOH); IR νmax 3441, 3191, 2934, 2874, 1670, 1454, 1380, 1220, 1041, 1012, 976, 947, 846 cm−1; for 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) data, see Tables 1 and 2; negative HRESIMS m/z 391.2499 [M − H]− (calcd for C23H35O5, 391.2485). Aquilarabietic acid D (4): orange, amorphous solid; [α]20D +12.4 (c 0.1, MeOH); IR (KBr) νmax 3417, 2937, 2874, 1701, 1464, 1387, 1240, 1182, 1156, 1030, 999, 941, 875 cm−1; for 1H (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) data, see Tables 1 and 2; positive HRESIMS m/z 375.2158 [M + Na]+ (calcd for C20H32O5Na, 375.2147). Aquilarabietic acid E (5): pale yellow, amorphous solid; [α]20D +41.8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 243 (3.86) nm; IR νmax 3520, 3278, 2974, 2938, 2874, 2609, 1694, 1649, 1624, 1454, 1410, 1372, 1263, 1241, 1175, 1118, 1005, 973, 948, 844 cm−1; for 1H (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) data, see Tables 1 and 2; positive HRESIMS m/z 367.2096 [M + H]+ (calcd for C20H31O6, 367.2115). Aquilarabietic acid F (6): colorless, block crystals [CHCl3− acetone−MeOH (1:1:1)]; mp 178−180 °C; [α]20D +24.7 (c 0.2, acetone); IR νmax 3526, 2962, 2938, 2873, 1776, 1468, 1385, 1074, 941 cm−1; for 1H (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; positive HRESIMS m/z 317.2124 [M + H]+ (calcd for C20H29O3, 317.2111). Aquilarabietic acid G (7): pale yellow, block crystals (MeOH); mp 198−199 °C; [α]20D −23.0 (c 0.1, MeOH); IR νmax 3623, 3340, 2943, 2657, 1694, 1558, 1463, 1376, 1284, 1255, 1073, 1021, 971, 876, 797 cm−1; for 1H (methanol-d4, 600 MHz) and 13C NMR (methanol-d4,

IC50 value.

further and more extensive investigation is necessary to validate this hypothesis.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting point measurements were conducted using an XT4A MP apparatus (uncorrected). The optical rotations were measured using a Perkin-Elmer model 341LC polarimeter. All UV spectra were recorded with a Cary 300 spectrophotometer. The IR spectra were measured using a Nicolet 5700 FT-IR microscope spectrometer. The NMR spectra were recorded using a Varian MP-300 or 400 or VNS-600 spectrometer. The EI mass spectra were measured using an AutoSpec Ultima-TOF mass spectrometer (Micromass, UK). The HRESIMS spectra were measured using a Q-trap ESI mass spectrometer. Semipreparative HPLC was conducted using a Shimadzu LC-6AD instrument with a Shimadzu RID-10A detector and a Grace Adsorbosphere XL C18 column (250 mm × 10 mm, i.d. 5 μm). Column chromatography was performed using either silica gel (200−300 mesh; Qingdao Marine Chemical Inc., China) and RP-18 gel (Chromatorex, 40−75 μm; Fuji Silysia Chemical Ltd., Japan) or a Grace Allsphere silica column (250 mm × 10 mm, i.d., 5 μm). Fractions were monitored by TLC, and the spots were visualized by illuminating with an ultraviolet lamp and spraying with 5% H2SO4 in EtOH, followed by heating at 105 °C. Plant Material. Chinese eaglewood was purchased from an herbal medicine market in Wenchang County, Hainan Province, People’s Republic of China, in January 2009 and was identified by one of the authors (S.G.). A voucher specimen (No. 2009-01-AS) was deposited at the herbarium of the Institute of Medicinal Plant Development, Chinese Academy of Medicinal Sciences and Peking Union Medical College. Extraction and Isolation. Chinese eaglewood (7.5 kg, dry weight) was first crushed and extracted (3 × 8 h) with petroleum ether (15 L, 60−90 °C) under reflux to yield a residue (258 g) after solvent removal at reduced pressure. The residual material was extracted by refluxing (3 × 8 h) in EtOH (15 L) to yield a brown powder (935 g) after removal of solvent under reduced pressure. The EtOH extract (783 g) was subjected to silica gel (200−300 mesh, 1.9 kg) column chromatography eluted successively with petroleum ether (60−90 °C), petroleum ether−EtOAc (4:1, 1:1, 1:2, 1:5, v/v), EtOAc, acetone, and MeOH to afford fractions A−H. Silica gel (792 g) column chromatography of fraction D (43.8 g) with petroleum ether− EtOAc (1:2, v/v) resulted in subfractions D1−D6. Fraction D4 (19.0 g) was subjected to silica gel (82 g) column chromatography and eluted with petroleum ether−EtOAc (from 1:1 to 1:3, v/v) to yield fractions D4a−D4e. Compound 9 (23 mg) was isolated from fraction D4a by repeated column chromatography over silica gel eluted with petroleum ether−EtOAc (from 2:1 to 1:2, v/v). Fraction D4b was subjected to semipreparative normal-phase HPLC with n-hexane−EtOAc (3:1, v/v) to afford 11 (7 mg) and 8 (48 mg). Fraction D4d was also subjected to 221

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150 MHz) data, see Tables 1 and 2; ESIMS m/z 375 [M + Na]+; positive HRESIMS m/z 375.2132 [M + Na]+ (calcd for C20H32O5Na, 375.2147). Aquilarabietic acid H (8): colorless needles (MeOH); mp 105−106 °C; [α]20D −1.2 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 210 (4.52), 248 (4.23) nm; IR (KBr) νmax 3322, 3084, 2938, 2868, 2630, 1697, 1626, 1558, 1497, 1454, 1381, 1227, 1181, 1124, 1044, 957, 887, 830 cm−1; for 1H (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) data, see Tables 1 and 2; positive HRESIMS m/z 337.1751 [M + Na]+ (calcd for C20H26O3Na, 337.1774). Aquilarabietic acid I (9): white, amorphous solid; [α]20D +5.9 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 204 (4.37), 214 (4.14) nm; IR νmax 3293, 3124, 2974, 2931, 2616, 1720, 1498, 1463, 1369, 1332, 1252, 1183, 1149, 1051, 959, 916, 873, 833 cm−1; for 1H (methanol-d4, 400 MHz) and 13C NMR (methanol-d4, 100 MHz) data, see Tables 1 and 2; positive HRESIMS m/z 369.2037 [M + Na]+ (calcd for C21H30O4Na, 369.2036). Aquilarabietic acid J (10): colorless oil; [α]20D −24.1 (c 0.6, acetone); UV (MeOH) λmax (log ε) 203 (4.81), 242 (4.50) nm; IR νmax 2929, 2870, 1695, 1461, 1385, 1274, 1186, 1151, 1076, 949, 903, 867, 799 cm−1; for 1H (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) data, see Tables 1 and 2; negative HRTOFMS m/z 345.2454 [M − H]− (calcd for C22H33O3, 345.2421). Aquilarabietic acid K (11): pale yellow needles (MeOH); mp 188− 189 °C; [α]20D −14.3 (c 0.1, MeOH); IR (KBr) νmax 3149, 2956, 2926, 2870, 1712, 1684, 1477, 1461, 1449, 1331, 1224, 1201, 1107, 1090, 1001, 977, 825 cm−1; for 1H (CDCl3, 300 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2; positive HRESIMS m/z 607.3211 [2 M + Na]+ (calcd for C34H48O8Na, 607.3241). X-ray Crystallographic Analysis. The crystallographic data for 1 were collected on an AFC10HF+CCD diffractometer using graphitemonochromated Mo Kα radiation (λ = 0.71073 Å). The crystallographic data for 6 were collected on a Rigaku MicroMax 002 diffractometer using graphite-monochromated Cu Kα radiation (λ = 1.54187 Å). The structures of 1 and 6 were solved using direct methods (SHELXL-97) and expanded using difference Fourier techniques (DIRDIF99). Crystal Data for 1. Aquilarabietic acid A (1) was crystallized from acetone to yield colorless needles. A single crystal of dimensions 0.43 × 0.27 × 0.17 mm3 was used for the X-ray measurements. Crystal data: C20H32O5, M = 352.22, space group P212121, a = 7.3445(13) Å, b = 12.413(2) Å, and c = 19.978(4) Å, V = 1821.3(6) Å3, Z = 4, Dcalcd = 1.285 mg/m3, R1 = 0.0354, wR2 = 0.0817, and S = 1.002. Crystal Data for 6. Aquilarabietic acid F (6) was crystallized from CHCl3−acetone−MeOH (1:1:1) to yield colorless block crystals. A single crystal of dimensions 0.11 × 0.17 × 0.39 mm3 was used for the X-ray measurements. Crystal data: C20H28O3, M = 316.44, space group P212121, a = 11.102(4) Å, b = 11.339(5) Å, and c = 13.543(4) Å, V = 1705.0(9) Å3, Z = 4, Dcalcd = 1.233 mg/m3, R1 = 0.0470, wR2 = 0.1294, and S = 1.078. The supplementary crystallographic data for compounds 1 and 6 have been deposited at the Cambridge Crystallographic Data Centre under the reference numbers CCDC 903692 and 903693. Copies of the data can be obtained, free of charge, by applying to the Director at CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, fax: +44 1223 336033 or e-mail: [email protected]. Assessment of the in Vitro Antidepressant Activity. The antidepressant activities of the 11 compounds were preliminarily evaluated using rat brain synaptosomes via the inhibited [3H]-5-HT and [3H]-NE reuptake method.24 Duloxetine was used as a positive control.

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Article

AUTHOR INFORMATION

Corresponding Author

*Tel: 86-10-62829619. Fax: 86-10-62896288. E-mail: [email protected] (S.G.). Tel: 86-10-63165195. Fax: 86-10-63017757. E-mail: [email protected] (J.D.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science & Technology Major Project “Key New Drug Creation and Manufacturing”, China (No. 2012ZX09301002-001-005); the State Ethnic Affairs Commission (Grant No. 09ZY014); and “985 Project” of Minzu University of China (MUC985-9).

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

1 H/13C NMR, 2D NMR, NOE, IR, UV, and HRMS spectra for all compounds (1−11), single-crystal X-ray crystallographic data for compounds 1 and 6, and HPLC-DAD/MS chromatograms of compounds 3, 9, and 10 and the extracts are available free of charge via the Internet at http://pubs.acs.org.

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