iNOS Inhibitory Activity of Sesquiterpenoids and a ... - ACS Publications

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iNOS Inhibitory Activity of Sesquiterpenoids and a Monoterpenoid from the Rhizomes of Curcuma wenyujin Guo-Ping Yin,†,‡,∥ Liang-Chun Li,⊥,∥ Qing-Zhe Zhang,†,‡ Yue-Wei An,†,‡ Jing-Jing Zhu,*,†,‡ Zhi-Min Wang,*,†,‡ Gui-Xin Chou,§ and Zheng-tao Wang§ †

Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, People’s Republic of China National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicines, Beijing 100700, People’s Republic of China ⊥ School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang 621010, People’s Republic of China § The MOE Key Laboratory for Standardization of Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Eleven new sesquiterpenoids, wenyujinins A−K (1−11), and a new monoterpenoid, wenyujinin L (12), were isolated from the rhizomes of Curcuma wenyujin. Their structures and relative configurations were elucidated using 1D and 2D NMR, X-ray crystallographic analysis, and HRESIMS data. The absolute configurations of 1, 2, 3, 4, 6, 8, 9, and 10 were determined by comparison of the experimental and calculated ECD spectra. The absolute configuration of 5 was determined from the ECD data of the [Rh2(OCOCF3)4] complex, whereas those of 7 and 12 were determined from the ECD spectra of the compounds alone. Compounds 7 and 7a strongly inhibited the induction of NO production by LPS, with IC50 values of 7.6 and 8.5 μM, respectively. Compounds 6 and 10 moderately inhibited NO production with IC50 values of 47.7 and 48.6 μM, respectively. terpenoid were isolated, and their inducible nitric oxide synthase (iNOS) inhibitory activities were determined.

Curcuma wenyujin Y. H. Chen et C. Ling, a member of the Zingiberaceae family, a plant used in traditional medicine, is distributed throughout Southeast China. C. wenyujin is widely used to treat blood stasis, relieve jaundice, stimulate menstrual discharge, and relieve pain.1 Recent pharmacological evidence indicates that extracts of Curcuma plants exhibit a broad range of biological activities, including anti-inflammatory, analgesic, antioxidant, and antitumor effects.2−7 Traditionally the steamed and nonsteamed rhizomes of C. wenyujin have been clinically used in two herbal medicines, Wen-E-Zhu (WEZ) and PianJiang-Huang (PJH).1 PJH has antimicrobial and anti-inflammatory properties and has been used to alleviate the pain associated with arthrosis and skin infections. WEZ is used to treat cancer, viral infections, and gynecological diseases. Previous phytochemical investigations of the rhizomes of C. wenyujin have described the presence of sesquiterpenoids.8−12 Whereas much research has been performed on WEZ,13−15 little chemical information is available on PJH. During WEZ processing, a large quantity of the water-soluble constituents may be lost when boiling in water. However, water-soluble constituents are believed to be present in greater amounts in PJH than in WEZ. Here, a systematic phytochemical study of the compounds in PJH that are retained in aqueous extracts is reported. Eleven new sesquiterpenoids and a new mono© XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Wenyujinin A (1) was obtained as white needles. The molecular formula was determined as C15H24O3 based on the HRESIMS (m/z 275.1611 [M + Na]+, calcd for 275.1623) and 13 C NMR data. The UV spectrum showed an absorption maximum at 252 nm (MeOH, log ε 3.73), indicating the presence of an α,β-unsaturated carbonyl moiety. The 1H NMR data (Table 1) indicated the presence of four methyl groups [δH 1.04 (3H, d, 6.6), 1.23 (3H, s), 1.91 (3H, s), 2.08 (3H, s)]. The 13C NMR (Table 3) and HSQC data showed 15 carbon resonances, including four methyl (δC 17.1, 23.9, 24.4, 32.9), four methylene (δC 23.6, 28.9, 38.3, 50.9), two methine (δC 55.5, 44.8), one carbonyl (δC 202.6), two olefinic quaternary (δC 130.1, 140.9), and two oxygenated tertiary carbons (δC 73.7, 82.2). The 1H−1H COSY (Figure 1) correlations revealed one spin system corresponding to the C-1−C-2−C-3−C-4−C14 moiety. The HMBC correlations (Figure 1) from Me-14 to C-3/C-4/C-5 confirmed that the Me-14 was linked to C-4. The Received: December 5, 2013

A

dx.doi.org/10.1021/np400984c | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H NMR Data of Compounds 1−7 (600 MHz; J in Hz) position 1 2

1a

2a

2c

2.18, t (10.2) a, 1.85, m b, 1.37, m a, 1.94, m b, 1.37, m 1.89, m

2.12, m α, 1.94, m β, 1.38, m a, 1.88, m b, 1.24, m 2.14, m

1.95, m α, 1.68, m β, 1.43, m a, 1.78, m b, 1.17, m 2.00, m

12 13

a, 2.61, d (15.0) b, 2.46, d (15.0) a, 3.15, d (15.0) b, 2.46, d (15.0) 2.08, s 1.91, s

α, 2.76, d (14.5) β, 2.06, d (14.5) β, 3.20, d (16.5) α, 2.43, d (16.5) 2.11, s 1.92, s

α, 2.55, d (14.2) β, 2.05, d (14.2) β, 3.20, d (16.3) α, 2.19, d (16.3) 1.96, s 1.82, s

14

1.04, d (6.6)

1.02, d (6.5)

15

1.23, s

1.20, s

3 4 5 6

9

OH

3b

4b

5b

2.22, m α, 1.67, m β, 1.61, m a, 1.77, m b, 1.64, m

3.08, m a, 1.78, m b, 1.72, m α, 1.83, m β, 1.72, m

2.65, m a, 1.90, m b, 1.70, m a, 1.74, m b, 1.70, m

2.00, m a, 2.35, brd (13.6) b, 2.02, m

2.13, m a, 2.13, m

2.24, m a, 2.55, brd (11.9) b, 2.21, m

a, 4.76, s

b, 1.98, dd (15.3, 10.9) a, 4.85, s

b, 4.74, s

b, 4.72, s

1.73, s 1.63, s

1.77, s 1.64, s

1.84, s 1.79, s

0.93, d (6.8)

1.22, s

1.31, s

1.21, s

1.03, s

1.75, s

1.80, s

2.10, s

2.27, s

6b

6d

3.01, t (9.0) a, 2.07, m b, 2.07, m a, 1.88, m b, 1.44, m 2.59, m

3.13, t (9.0) a, 2.25, m b, 2.00, m a, 1.83, m b, 1.57, m 2.81, m

7b α, 2.14, m β, 1.81, m α, 1.90, m β, 1.77, m 2.18, d (8.8) β, 2.95, brd, 16.9

6.66, s

7.20, s

α, 2.68, ddd (16.9, 9.1, 2.1) β, 2.37, d (14.4) α, 2.35, d (14.4)

7.23, s 2.18, d (1.2) 1.08, d (6.6) 4.27, d (15.6) 4.21, d (15.6)

7.28, s 2.19, d (1.2) 1.42, d (6.6) 4.68, d (16.2) 4.54, d (16.2)

1.80, d (2.0) 1.18, s 1.40, s

4.09, s (OH5) 4.91, s (OH10)

a Recorded in CDCl3. bRecorded in methanol-d4. cRecorded in DMSO-d6. dRecorded in pyridine-d5. The 1H NMR chemical shift of Me-12 vs Me-13 of 1 cannot be differentiated.

HMBC correlations from Me-13 to C-7/C-11/C-12 and Me-12 to C-7/C-11/C-13 suggested the presence of an isobutylene moiety. The HMBC correlations from Me-15 to C-1/C-9/C-10 confirmed the linkage of C-15 to C-10. Additionally, the HMBC spectrum showed correlations from H-6a to C-5/C-7/ C-8/C-11 and H-9b to C-1/C-7/C-8/C-10. The planar structure of 1 was thus identified as a guaiane-type sesquiterpenoid. The structure and relative configuration of 1 were confirmed unequivocally by X-ray crystallography (Figure 2). The absolute configuration of 1 was determined as (1R,4S,5S,10S) by comparison of calculated electronic circular dichroism (ECD) and optical rotation (OR) values to experimental data (see Supporting Information).16−18 Wenyujinin B (2) was obtained as white needles from ethyl acetate. The molecular formula was determined as C15H24O3 based on the HRESIMS (m/z 275.1614 [M + Na]+, calcd for 275.1623) and 13C NMR data. The UV, 1H NMR, and 13C NMR data (Tables 1 and 3) were similar to those of 1. The fact that 2 possessed the same planar structure as 1 was supported by the HMBC correlations from Me-12 to C-7/C-11/C-13, Me-13 to C-7/C-11/C-12, Me-14 to C-3/C-4/C-5, Me-15 to C-1/C-9/C-10, H-6α to C-1/C-4/C-5/C-7/C-8/C-11, H-9b to C-1/C-7/C-8/C-10/C-15, H-1 to C-2/C-5/C-9/C-10, OH-5 to C-1/C-4/C-5/C-6, and OH-10 to C-1/C-9/C-10/C-15. The 1 H−1H COSY correlations revealed the spin system of the C1−C-2−C-3−C-4−C-14 moiety. The relative configuration of 2 was determined from the NOESY spectrum. The correlations of H-1 with Me-15/H-4/OH-5, H-6β with H-2β/H-9β, H-2β with H-9β, and H-6α with Me-13 suggested that H-1, H-4, OH5, and Me-15 were α-oriented, whereas Me-14 was β-oriented. The absolute configuration of 2 was determined to be

(1S,4S,5R,10S) by comparison of calculated ECD and OR values to experimental data (see Supporting Information).16−18 Wenyujinin C (3) was obtained as light yellow oil. The molecular formula was determined as C15H24O3 from the HRESIMS (m/z 275.1633 [M + Na]+, calcd for C15H24O3Na, 275.1623) and 13C NMR data. The UV spectrum showed an absorption maximum at 236 nm (MeOH, log ε 4.03), indicating the presence of an α,β-unsaturated carbonyl moiety. The 1H NMR data (Table 1) indicated four methyl groups [δH 1.22 (3H, s), 1.63 (3H, s), 1.73 (3H, s), 1.75 (3H, s)] and two olefinic protons [δH 4.76 (1H, s), 4.74 (1H, s)]. The 13C NMR (Table 3), DEPT, and HSQC data showed 15 carbon B



Article

Table 2. 1H NMR Data of Compounds 7a and 8−12 (600 MHz; J in Hz) 7ab

position 1 2

3

a, 2.10, ddd (14.0, 11.2, 7.3) b, 1.65, ddd (14.0, 9.0, 5.0) a, 1.89, m b, 1.84, m

8b

8c

2.63, d (8.8)

6

a, 2.95, brd (16.7) b, 2.83, ddd (16.7, 8.9, 1.8)

7 9

a, 2.29, d (13.8)

15 OH

a

10b

11a

1.89, m 1.65, m

4.41, brd (7.0) a, 2.31, tdd (14.2, 7.0, 3.9) b,1.90, brd (14.2)

α, 2.15, m

1.73, m a, 1.71, m

β,1.93, m

β,1.75, m

b, 1.48, m

α, 2.06, dd (13.5, 10.9) β, 1.82, m

α, 1.93, dd (13.5, 10.9) β, 1.67, m

a, 1.92, m

a 1.85, m

b, 1.43, m 1.92, m

b, 1.49, m 1.91, m

2.92, dd (10.8, 9.0) β, 2.35, m

2.76, dd (10.8, 9.0) β, 2.18, m

α, 1.57, dd (14.4, 10.8) 2.76, dd (8.2, 1.9) 6.10, s

α, 1.39, dd (14.4, 10.8) 2.65, dd (8.2, 1.9) 5.99, s

b, 2.18, d (13.8) 10 11 12 13 14

9d

α, 2.30, m

4 5

9a

1.79, d (1.8) 1.18, s

1.04, s 1.43, s 1.02, s

0.93, s 1.32, s 0.87, s

1.41, s

2.13, d (1.0)

2.01, d (1.0) 4.74, s (OH-4)

a, 2.60, d (15.5) b, 2.33, d (15.5)

a, 2.61, d (14.9) b, 2.52, d (14.9)

a, 2.01, d (13.7) b, 1.94, m

a, 2.34, d (13.7) b, 2.30, d (13.7)

1.87, s 1.68, s 0.96, d (6.7) 1.13, s 2.95, s (OH-8) 2.66, s (OH-10)

2.01, s 1.58, s 1.03, d (6.7) 1.29, s 4.92, s

12b

12a

5.48, d (9.8) 4.78, td (11.0, 4.2)

2.31, m

2.34, m

α, 1.72, td (14.0, 3.9) β, 1.53, brd (14.0)

α, 2.62, dd (12.0, 4.2) β, 1.36, m

a, 1.94, m

a, 1.95, m

3.51, dd (13.4, 3.6) α, 2.90, m

3.81, s

b, 1.81, m a, 2.31, m b, 1.65, m 3.11, td (9.6, 6.5)

b, 1.81, m a, 2.32, m b, 1.62, m 3.07, td (9.6, 6.5)

5.79, s

5.79, s

a, 2.31, m

a, 2.38, m

b, 2.18, m

b, 2.01, m

2.00, d (0.9) 1.24, s

1.95, d (0.9) 1.28, s

β, 2.66, dd (14.4, 3.6)

5.69, s

a, 3.78, d (16.5) b, 3.72, d (16.5)

2.12, s 1.93, s 1.79, s

7.10, s 2.12, d (0.7) 1.66, s

1.19, s

1.35, s

4.77, s

Recorded in CDCl3. bRecorded in methanol-d4. cRecorded in DMSO-d6. dRecorded in pyridine-d5.

Wenyujinin D (4) was determined to have the same molecular formula as 3 from the HRESIMS (m/z 275.1622 [M + Na]+, calcd for C15H24O3Na, 275.1623) and 13C NMR data. The UV, 1H NMR, and 13C NMR data (Tables 1 and 3) were similar to those of 3. However, comparison of the 1H and 13 C NMR data revealed that the H-1 resonance of 4 was shifted from δH 2.22 to 3.08 and that the C-15 resonance of 4 was shifted from δC 17.0 to 22.6. Thus, 4 had the same planar structure as 3, but the orientation of the C-1 substituent was different. The HSQC and HMBC spectra confirmed this conclusion. The NOESY correlations of H-1 with H-5, Me-14 with H-3α, and Me-13 with H-6b and the absence of correlations of Me-14 with H-1/H-5 suggested that H-1 and H-5 were β-oriented and that Me-14 was α-oriented. The absolute configuration of 4 was determined to be (1S,4S,5S) by comparison of calculated ECD (Figure 4) and OR values to experimental data.16−18 Wenyujinin E (5) was obtained as a colorless oil. The molecular formula of 5 was determined as C15H24O3 from the HRESIMS (m/z 275.1606 [M + Na]+, calcd for C15H24O3Na 275.1623) and 13C NMR data. The 1H NMR data (Table 1) of 5 showed five methyl groups [δH 1.21 (3H, s), 1.84 (3H, s), 1.79 (3H, s), 2.10 (3H, s), 2.27 (3H, s)]. The 13C NMR (Table 3), DEPT, and HSQC data indicated 15 carbon resonances, including five methyl (δC 20.5, 21.3, 21.4, 27.0, 29.3), three methylene (δC 24.8, 29.3, 39.2), two methine (δC 50.7, 54.9), two carbonyl (δC 206.9, 211.7), one oxygenated tertiary (δC 79.8), and two olefinic quaternary carbons (δC 135.9, 137.8). In

resonances, including four methyl (δC 17.0, 17.8, 21.2, 22.1), four methylene (δC 25.5, 28.1,40.1, 110.7), two methine (δC 52.0, 52.1), one carbonyl (δC 181.0), one oxygenated tertiary (δC 78.9), and three olefinic quaternary carbons (δC 125.2, 134.4, 147.0). The HMBC spectrum showed correlations from Me-12 to C-7/C-8/C-11/C-13, Me-13 to C-7/C-8/C-11/C-12, Me-14 to C-3/C-4/C-5, Me-15 to C-1/C-9/C-10, H-5 to C-1/ C-4/C-6/C-7/C-10, H-6a to C-1/C-4/C-5/C-7/C-8/C-11, H9a to C-1/C-10/C-15, and H-1 to C-2/C-3/C-5/C-6/C-9/C10/C-15. These spectroscopic data were similar to those of gajutsulactone A.19 The primary difference between the spectra of these two compounds was that the chemical shift of C-8 in 3 was deshielded from δC 167.2 to 181.0. The indices of hydrogen deficiency revealed that the lactone moiety in gajutsulactone A was replaced by a carboxyl moiety in 3. The relative configuration of 3 was inferred from the NOESY spectrum. The correlations of H-1 with Me-14, H-5 with H-2β, and Me-13 with H-6a, and the absence of correlations of H-5 with H-1/Me-14, indicated the α-orientation of H-1 and Me-14 and the β-orientation of H-5. The absolute configuration of 3 was determined using TDDFT calculations.16−18 The calculated ECD spectrum for 3 was similar to the experimental spectrum (Figure 3). Furthermore, the OR was calculated at the PBE1PBE/6-311++G(2d,p) level. The computed OR value of 3 was −41, also similar to the experimental value of −30. Thus, based on both the ECD and OR values, the absolute configuration of 3 was determined to be (1R,4S,5S). C


a

55.5 23.6 28.9 44.8 82.2 38.3 130.1 202.6 50.9 73.7 149.0 23.9 24.4 17.1 32.9

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

58.7 23.5 27.5 45.1 81.4 28.7 129.2 202.4 50.6 74.3 149.6 23.9 23.8 13.1 32.6

2a

3b 52.0 25.5 40.1 78.9 52.1 28.1 134.4 181.0 110.7 147.0 125.2 21.2 17.8 22.1 17.0

2c

58.3 22.5 27.1 44.4 80.8 27.9 130.1 202.3 50.6 72.9 145.5 23.3 23.1 13.0 31.7

48.8 24.9 38.2 81.8 50.2 26.3 135.5 179.7 109.7 146.8 125.5 21.5 18.6 24.1 22.6

4b 54.9 24.8 39.2 79.8 50.7 29.3 135.9 206.9 29.3 211.7 137.8 21.4 20.5 21.3 27.0

5b

6d 48.8 24.2 30.1 39.9 84.1 196.1 119.6 158.5 113.3 147.5 122.9 138.7 9.8 15.6 64.4

6b 48.0 23.8 29.2 39.2 83.6 195.6 119.2 157.9 113.8 144.5 122.7 138.7 8.4 13.9 64.2

96.0 26.5 37.7 79.9 48.7 18.9 160.0 106.3 45.8 78.1 117.4 173.0 6.3 23.4 21.5

7b 94.8 26.9 38.2 79.3 48.9 19.6 160.9 105.5 45.5 75.4 117.3 172.7 6.3 24.4 26.3

7ab 82.6 34.4 39.2 75.3 52.5 17.6 56.3 203.2 131.2 165.2 73.4 28.6 28.3 24.2 22.7

8b 82.6 35.0 40.0 74.9 52.8 18.1 56.3 201.8 131.4 164.5 73.3 30.0 29.7 25.8 23.8

8c 55.4 25.8 29.4 38.9 86.4 36.0 134.9 102.0 45.9 72.6 129.3 22.6 19.2 12.3 28.4

9a

10b 73.2 20.9 27.4 68.7 82.0 31.5 131.2 200.7 126.4 145.7 146.4 22.3 22.4 19.9 25.9

9d 56.7 27.0 31.2 40.5 86.2 37.8 138.0 103.9 48.7 72.6 124.7 23.5 20.3 13.9 31.2

134.9 66.6 46.8 63.6 66.2 191.5 122.3 156.5 41.8 132.0 123.3 138.3 10.2 16.7 16.4

11a

Recorded in CDCl3. bRecorded in methanol-d4. cRecorded in DMSO-d6. dRecorded in pyridine-d5. The 13C NMR chemical shift of C-12 vs C-13 of 1 cannot be differentiated.

1a

position

Table 3. 13C NMR Data of Compounds 1−12 (150 MHz) 50.4 82.0 38.4 28.0 42.4 164.5 124.6 198.8 36.6 23.5 24.0

12a

49.9 81.0 37.5 27.7 42.4 166.8 123.5 200.4 35.9 22.2 22.2

12b

Journal of Natural Products Article

Figure 1. Key HMBC and 1H−1H COSY correlations of compounds 1, 6, 10, and 12.

Figure 2. ORTEP drawing of compound 1.

Figure 3. Calculated and experimental ECD spectra of compound 3 in MeOH.

the HMBC spectrum, strong correlations from Me-9 to C-7/C8, Me-12 to C-7/C-11/C-13, and Me-13 to C-7/C-11/C-12 as well as weak long-range correlations from Me-12/Me-13 to C6/C-8 suggested that the Me-12−C-11 (Me-13)−C-7−C-8−C9 moiety was an α,β-unsaturated carbonyl moiety. The HMBC correlations from Me-14 to C-3/C-4/C-5 and Me-15 to C-1/C10 suggested the presence of Me-14−C-4(C-3)−C-5 and Me15−C-10−C-1 moieties. The HMBC correlations from H-6a to

D



Article



C-1/C-4/C-5/C-7/C-8/C-11 and H-1 to C-2/C-3/C-4/C-5/ C-6/C-10/C-15 suggested the presence of C-5−C-6−C-7 and C-1−C-2−C-5 moieties. Through the analysis of these data, the planar structure of 5 was determined to be that of a secoguaiane sesquiterpenoid. The NOESY correlations of Me-14 with H-5/H-1 and H-5 with Me-12 and the absence of correlations of H-1 with H-6a/H-6b and Me-14 with H-6a/H6b indicated that Me-14, H-1, and H-5 were α-oriented. An Sconfiguration for C-4 was supported by the positive Cotton effect at 350 nm in the Rh2 (OCOCF3)4-induced ECD spectrum, based on the empirical bulkiness rule proposed by Snatzke.20,21 Thus, the absolute configuration of 5 was assigned as (1R,4S,5R). Wenyujinin F (6) was obtained as a light yellow oil. The molecular formula of 6, C15H18O4, was determined from the HRESIMS (m/z 285.1102 [M + Na]+, calcd for C15H18O4Na 285.1103) and 13C NMR data. The 1H NMR data (Table 1) showed two methyl groups [δH 1.42 (3H, d, 6.6 Hz), 2.19 (3H, d, 1.2 Hz)], two olefinic protons [δH 7.20 (1H, s), 7.28 (1H, s)], and two methylene protons [δH 4.68 (1H, d, 16.2 Hz), 4.54 (1H, d, 16.2 Hz)]. The 13C NMR (Table 3) and HSQC data indicated 15 carbon resonances, including two methyl (δC 9.8, 15.6), three methylene (δC 24.2, 30.1, 64.4), four methine (δC 39.9, 48.8, 113.3, 138.7), one carbonyl (δC 196.1), three olefinic quaternary (δC 119.6, 122.9, 147.5), and two oxygenated tertiary carbons (δC 84.1, 158.5). The NMR spectra of 6 were similar to those of zedoarol;22 the primary difference was the presence of the allylic alcohol group in 6. This deduction was supported by the HMBC correlations (Figure 1) from Me-13 to C-7/C-11/C-12, Me-14 to C-3/C-4/C-5, H-1 to C-2/C-5/ C-6/C-9/C-10, H-4 to C-3/C-5/C-6/C-14, H-9 to C-1/C-7/ C-8/C-10/C-15, and H-12 to C-7/C-8/C-11/C-13. The NOESY correlations of H-1 with H-4 and the absence of correlations of H-1 with Me-14 indicated the β-orientation of Me-14 and the α-orientation of H-1 and H-4. The pyridineinduced solvent shifts23,24 of H-2β (δ pyridine − δ methanol = 0.18 ppm), H-3β (0.13 ppm), and Me-14 (0.34 ppm) supported the β-orientation of the OH group at C-5. The absolute configuration of 6 was determined to be (1S,4S,5S) based on the calculated and experimental ECD data (Figure 5).16−18 Wenyujinin G (7) was obtained as a colorless oil. The molecular formula of 7 was determined as C15H20O5 on the basis of HRESIMS (m/z 303.1216 [M + Na]+, calcd for C15H20O5Na 303.1208) and 13C NMR data. The 1H NMR data (Table 1) showed resonances corresponding to three methyl groups [δH 1.80 (3H, d, 2.0 Hz), 1.18 (3H, s), 1.40 (3H, s)]. The 13C NMR (Table 3) and HSQC data showed 15 carbon resonances, including three methyl (δC 6.3, 21.5, 23.4), four methylene (δC 18.9, 26.5, 37.7, 45.8), one methine (δC 48.7),

one carbonyl (δC 173.0), two olefinic quaternary (δC 117.4, 160.0), three oxygenated tertiary (δC 78.1, 79.9, 96.0), and a dioxygenated secondary carbon (δC 106.3). The 1H NMR and 13 C NMR spectra were similar to those of phaeocaulisin A (7a), which had been isolated previously from Curcuma phaeocaulis.25 Compound 7 was proposed to have the same planar structure as 7a. This deduction was supported by the HMBC correlations from Me-13 to C-7/C-11/C-12, Me-14 to C-3/C-4/C-5, Me15 to C-1/C-9/C-10, H-5 to C-1/C-4/C-6/C-7/C-10/C-14, and H-9a to C-1/C-7/C-8/C10/C-15. The NOESY correlations of H-5 with Me-14/Me-15, H-2α with Me-15, H-3α with Me-14, and H-6α with H-9α suggested α-orientations of H-5, Me-14, and Me-15 and a β-orientation of the oxygen bridge. The absolute configuration of 7 was determined using the empirical rule for α,β-unsaturated-γ-lactones in the ECD spectrum. The characteristic Cotton effect at 230 nm (Δε +9.1) and 255 nm (Δε −3.2) indicated that the absolute configuration of C-8 was S. Thus, the absolute configuration of 7 was determined to be (1R,4S,5S,8S,10S).25 Wenyujinin H (8) was isolated as a colorless oil, and its molecular formula was determined as C15H22O3 from the HRESIMS (m/z 251.1650 [M + H]+, calcd for C15H23O3 251.1647) and 13C NMR data. The UV spectrum showed an absorption maximum at 234 nm (MeOH, log ε 3.92), indicating the presence of an α,β-unsaturated carbonyl moiety. The 1H NMR data (Table 2) showed four methyl groups [δH 0.87 (3H, s), 0.93 (3H, s), 1.32 (3H, s), 2.01(3H, d, 1.0 Hz)]. The 13C NMR (Table 3) and HSQC data indicated 15 carbon resonances, including four methyl (δC 23.8, 25.8, 29.7, 30.0), three methylene (δC 18.1, 35.0, 40.0), three methine (δC 52.8, 56.3, 131.4), one carbonyl (δC 201.8), one olefinic quaternary (δC 164.5), and three oxygenated tertiary carbons (δC 73.3, 74.9, 82.6). The HMBC spectrum showed correlations from Me-14 to C-3/C-4/C-5, Me-12 to C-7/C-11/C-13, Me-13 to C-7/C-11/C12, Me-15 to C-1/C-9/C-10, and OH-4 to C-3/C4/C-5/C-14. These data suggested that 8 was a guaiane sesquiterpenoid. The 13C NMR data were similar to those of epiprocurcumenol,26 except that the resonance of C-1 at δC 46.4 in epiprocurcumenol was deshielded to δC 82.6 and that the resonances of C-7 at δC 134.5 and C-11 at δC 140.8 in epiprocurcumenol were shielded to δC 56.3 and 73.3, respectively. Analysis of the chemical shifts and indices of hydrogen deficiency indicated that the C-7−C-11 olefinic bond in epiprocurcumenol was replaced with an oxygen bridge connecting C-1 and C-11 in 8. The NOESY correlations (Figure 6) of H-5 with Me-13/H-2β/H-6β, Me-15 with H-3α/ H-2α/Me-14, H-7 with H-6β/Me-12/Me-13, and H-6α with Me-14 indicated that H-5 and the oxygen bridge were βE



Article

with Me-15/H-4 and Me-13 with H-6a and the absence of correlations of H-1 with Me-14 and Me-15 with H-6a/H-6b indicated an α-orientation of Me-14 and β-orientations of H-1, Me-15, and the oxygen bridge. The absolute configuration of 9 was determined to be (1R,4R,5S,8R,10R) based on the calculated and experimental ECD data (Figure 8).16−18

Figure 6. Key NOESY correlations of compounds 8, 10, and 11.


oriented and that Me-14 was α-oriented. The absolute configuration of 8 was determined to be (1R,4S,5R,7R) based on the calculated and experimental ECD data (Figure 7).16−18

Wenyujinin J (10) was obtained as a colorless oil and had a molecular formula of C15H22O3 based on the HRESIMS (m/z 251.1632 [M + H]+, calcd for C15H23O3 251.1647) and 13C NMR data. The UV spectrum showed an absorption maximum at 254 nm (MeOH, log ε 3.54), indicating the presence of an α,β-unsaturated carbonyl moiety. The 1H NMR data (Table 2) of 10 showed four methyl groups [δH 1.19 (3H, s), 1.79 (3H, s), 1.93 (3H, s), 2.12 (3H, s)] and one olefinic proton [δH 5.69 (1H, s)]. The 13C NMR (Table 3) and HSQC data indicated 15 carbon resonances, including four methyl (δC 22.3, 22.4, 19.9, 25.9), three methylene (δC 20.9, 27.4, 31.5), three methine (δC 73.2, 82.0, 126.4), one carbonyl (δC 200.7), one oxygenated tertiary (δC 68.7), and three olefinic quaternary carbons (δC 146.4, 145.7, 131.2). In the HMBC spectrum (Figure 1), correlations from Me-14 to C-1/C-9/C-10 and Me15 to C-3/C-4/C-5 suggested the presence of Me-14−C-10(C1)−C-9 and Me-15−C-4(C-3)−C-5 moieties. The HMBC correlations from Me-12 to C-7/C-11/C-13 and Me-13 to C-7/ C-11/C12 suggested the presence of the isobutylene moiety. Comparison of the NMR data with literature values suggested that the planar structure of 10 was consistent with that of a germacrane sesquiterpenoid. The HMBC correlations from H1 to C-2/C-3/C-5/C-9/C-10 and H-5 to C-1/C-3/C-4/C-6/ C-7 indicated that the oxygen bridge connected C-1 and C-5. The NOESY correlations (Figure 6) of H-5 with Me-15/H-6β, H-6α with H-3α, and Me-13 with H-6β and the absence of correlations of Me-15 with H-1 indicated that Me-15, H-1, and H-5 were α-oriented and that the oxygen bridge was β-oriented. The absolute configuration of 10 was determined to be (1R,4S,5R) based on the calculated and experimental ECD data (Figure 9).16−18 Wenyujinin K (11) was obtained as a colorless oil. The molecular formula was determined as C15H18O4 from the HRESIMS (m/z 285.1109 [M + Na]+, calcd for C15H18O4Na 285.1103) and 13C NMR data. The 1H NMR data (Table 2) showed three methyl groups [δH 1.35 (3H, s), 1.66 (3H, s), 2.12 (3H, d, 0.7 Hz)]. The 13C NMR (Table 3) and HSQC data indicated 15 carbon resonances, including three methyl (δC 10.2, 16.4, 16.7), two methylene (δC 41.8, 46.8), four methine (δC 66.2, 66.6, 134.9, 138.3), one carbonyl (δC 191.5), two oxygenated tertiary (δC 63.6, 156.5), and three olefinic


Wenyujinin I (9) was obtained as a colorless oil. Its molecular formula was determined as C15H24O3 based on the HRESIMS (m/z 275.1620 [M + Na]+, calcd for C15H24O3Na 275.1623) and 13C NMR data. The 1H NMR (Table 2) data indicated the presence of four methyl groups [δH 1.03 (3H, d, 6.7 Hz), 1.29 (3H, s), 1.58 (3H, s), 2.01 (3H, s)]. The 13C NMR (Table 3) and HSQC data showed 15 carbon resonances, including four methyl (δC 13.9, 20.3, 23.5, 31.2), four methylene (δC 27.0, 31.2, 37.8, 48.7), two methine (δC 40.5, 56.7), two olefinic quaternary (δC 124.7, 138.0), two oxygenated tertiary carbons (δC 72.6, 86.2), and a dioxygenated secondary carbon (δC 103.9). The 13C NMR spectrum of 9 was similar to that of neocurcumenol.19 The major difference between the 13C NMR data of 9 and those of neocurcumenol was that the chemical shifts of C-1 and C-10 in 9 were shifted from δC 137.6 to 56.7 and from δC 123.5 to 72.6, respectively. Analysis of the indices of hydrogen deficiency and the chemical shifts showed the presence of a hydroxy group (C-10) in 9 instead of the double bond (C-1 and C-10) in neocurcumenol. This deduction was supported by the HMBC correlations from Me-14 to C-3/C-4/C-5, Me-15 to C-1/C-9/C-10, Me-12 to C7/C-8/C-11/C-13, Me-13 to C-7/C-8/C-11/C-12, H-9a to C1/C-7/C-8/C-10/C-15, H-6a to C-1/C-4/C-5/C-7/C-8/C-11, and OH-8 to C-7/C-8/C-9. The NOESY correlations of H-1 F



Article

α,β-unsaturated cyclohexanone moiety, indicated that the absolute configuration of 12 was (1R,2R,5R).29 The effects of compounds 1−12 regarding the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)activated RAW 264.7 mouse peritoneal macrophage cells were evaluated. Compounds 7 and 7a showed strong inhibition, 6 and 10 exhibited moderate inhibition, while the other compounds showed weak iNOS inhibitory activity (Table 4) Table 4. Inhibitory Effects of Compounds 1−12 on NO Production Induced by LPS in Macrophages Figure 9. Calculated and experimental ECD spectra of compound 10 in MeOH.

quaternary carbons (δC 122.3, 123.3, 132.0). The 1H and 13C NMR spectra of 11 were similar to those of zederone.27,28 However, the chemical shift of δC 66.6 for C-2 in 11 was different from its shift of δC 24.6 in zederone. Analysis of the indices of hydrogen deficiency and the chemical shifts indicated that C-2 carried a hydroxy substituent in 11. This deduction was supported by the HMBC correlations from Me-13 to C-7/ C-11/C-12, Me-15 to C-3/C-4/C-5, Me-14 to C-1/C-9/C-10, H-12 to C-7/C-8/C-11/C-13, H-9a to C-1/C-7/C-8/C-10/C14, H-3a to C-1/C-2/C-4/C-5/C-15, and H-5 to C-3/C-4/C-6. The NOESY correlations (Figure 6) of H-3β with H-1/H-5, H1 with H-5, and H-2 with Me-14/Me-15 indicated that H-5 was β-oriented and that H-2 and Me-15 were α-oriented. Thus, 11 could be derived from zederone by hydroxylation at C-2. The hypothesis was consistent with the similar OR values of these two compounds.27,28 The absolute configuration of 11 was hence tentatively determined to be (2R,4S,5R). Wenyujinin L (12) was isolated as a colorless oil. Its molecular formula was determined as C11H16O2 from the HRESIMS (m/z 203.1042 [M + Na]+, calcd for C11H16O2Na, 203.1048) and 13C NMR data. The UV spectrum showed an absorption maximum at 238 nm (MeOH, log ε 3.87), indicating the presence of an α,β-unsaturated carbonyl moiety. The 1H NMR data (Table 2) showed two methyl groups [δH 1.24 (3H, s), 2.00 (3H, d, 0.9 Hz)] and one olefinic proton [δH 5.79 (1H, s)]. The 13C NMR (Table 3), DEPT, and HSQC data indicated 11 carbon resonances, including two methyl (δC 22.2, 22.2), three methylene (δC 27.7, 35.9, 37.5), three methine (δC 42.4, 49.9, 123.5), one carbonyl (δC 200.4), one oxygenated tertiary (δC 81.0), and one olefinic quaternary carbon (δC 166.8). A C-5−C-4−C-3 moiety was indicated by the 1H−1H COSY data (Figure 1). The HMBC correlations (Figure 1) from Me-11 to C-1/C-2/C-3 and Me-10 to C-5/C6/C-7/C-8, as well as analysis of the proton shifts, implied the existence of two partial structures, i.e., the Me-11−C-2(C-3)− C-1 and Me-10−C-6(C-5)−C-7−C-8 moieties. In addition, the HMBC spectrum showed correlations from H-5 to C-1/C-3/ C-4/C-6/C-7/C-8/C-10 and H-9a to C-1/C-2/C-5/C-7/C-8. Analysis of the HMBC correlations and indices of hydrogen deficiency indicated the presence of two cyclic moieties linked at C-1 and C-5. The relative configuration of 12 was determined from the NOESY spectrum. The correlation of H-1 with H-5 and the absence of correlations of Me-11 with H1/H-5 suggested that H-1 and H-5 were β-oriented and that Me-11 was α-oriented. The absolute configuration was confirmed using the ECD spectrum. According to the empirical rule for α,β-unsaturated cyclohexanones, the negative Cotton effect at 326 nm (Δε −0.5), based on the n−π* transition of

compound

IC50a (μM)

1 2 3 4 5 6 7 7a 8 9 10 11 12 hydrocortisoneb

89.2 92.4 68.4 66.5 78.7 47.7 7.6 8.5 79.8 87.6 48.6 78.2 77.9 51.4

a

Inhibitory effects of compounds 1−12 against LPS-induced NO production in RAW 264.7 macrophages. bPositive control.

compared to the positive control. The cytotoxicity of these compounds against RAW 264.7 cells was also evaluated using the MTT assay.30 None of the compounds showed significant cytotoxicity at the effective concentrations for inhibition of NO production.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with an Autopol V Plus polarimeter. UV spectra were recorded on a Shimadzu UV2550 spectrophotometer. ECD spectra were measured on a JASCO J-815 spectropolarimeter. ESIMS spectra were obtained on an Agilent 1200 Series LC/MSD ion-trap mass spectrometer. HRESIMS spectra were recorded on an Agilent Technologies 6520 accurate mass Q-TOF LC/MS spectrometer and an Agilent 1100 series LC/MSD ion-trap mass spectrometer. NMR spectra were recorded at 600 MHz for 1H and 150 MHz for 13C on a Bruker AV-600 spectrometer in CDCl3 with TMS as an internal standard. Analytical HPLC was performed on an Agilent 1100/1260 Infinity system with Dikma C18 (5 μm, 4.6 × 250 mm) and Apollo C18 (5 μm, 4.6 × 250 mm) columns. Semipreparative HPLC was performed on a Varian Cary-100/BIO UV−vis spectrophotometer with Cosmosil C18 (5 μm, 28 × 250 mm) and Kromasil C18 (5 μm, 10 × 250 mm) columns. Column chromatography was performed on silica gel (100−200, 200−300, and 300−400 mesh, Qingdao Marine Chemical Inc., Qingdao, China), ODS (50 μm, YMC, Japan), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), and HPD-100 macroporous resin (Beijing Credit Technology Co., LTD, Beijing, China). GF254 plates (Qingdao Marine Chemical Inc., Qingdao, China) and reversed-phase silica gel plates (Merck, USA) were used for TLC. Fractions were monitored by TLC, and the spots were visualized by heating the silica gel plates after spraying with 10% H2SO4 in EtOH. Plant Material. The dried rhizomes of C. wenyujin were collected from Ruian, Zhejiang Province, P. R. China, in April 2009 and identified by one of the authors (Z.-M.W.). A voucher specimen (No. PJH-0904) has been deposited in the Herbarium of the Institute of G



Article 13

Chinese Materia Medica, China Academy of Chinese Medical Sciences. Extraction and Isolation. The dried rhizomes of C. wenyujin (25.0 kg) were extracted with boiling water (3 × 10 L; 2 h each) and filtered. The combined filtrates were subjected to HPD-100 macroporous resin and eluted with water, 30% EtOH, 60% EtOH, and 95% EtOH to yield three fractions, A−C. The water fraction was discarded. The 30% EtOH fraction (A, 297.4 g) was subjected to silica gel column chromatography (CC) eluting with a mixture of petroleum ether (60− 90 °C) and acetone (5:1 to 0:1) with increasing polarity to yield 14 fractions, A1−A14, based on TLC. Fr. A2 (0.3 g) was further fractionated with repeated CC on a silica gel column eluting with petroleum ether (60−90 °C) and EtOAc (20:1 to 0:1) with increasing polarity to yield seven subfractions, A2.1−A2.7, based on TLC. Fr. A2.6 (104.6 mg) was separated by preparative HPLC with MeCN− H2O (12:88) to yield compound 9 (12.8 mg). Fr. A4 (2.6 g) was further fractionated with repeated CC on silica gel eluting with CHCl3 and MeOH (100:1 to 0:1) to give nine subfractions, A4.1−A4.9. Fr. A4.3 (0.7 g) was subjected to preparative HPLC using MeCN−H2O (35:65) to yield six subfractions, A4.3.1−A4.3.6. Fr. A4.3.2 (42.9 mg) was further fractionated by preparative HPLC using MeCN−H2O (25:75) to yield compound 5 (14.7 mg). Fr. A4.4 (0.8 g) was separated using preparative HPLC with MeCN−H2O (20:80 to 50:50) to yield five subfractions, A4.4.1−A4.4.5. Fr. A4.4.2 (72.7 mg) was separated by preparative HPLC with MeCN−H2O (13:87) to yield compounds 12 (6.0 mg) and 8 (6.5 mg). Fr. A4.4.5 (0.5 g) was separated on an ODS silica gel column eluting with MeOH−H2O (5:5 to 1:0) to give five subfractions, A4.4.5.1−A.4.4.5.5. Fr. A4.4.5.5 (46.0 mg) was further fractionated using preparative HPLC with MeCN− H2O (20:80) to give compound 10 (3.9 mg). Fr. A4.8 (80.0 mg) was separated on an ODS silica gel column eluting with MeOH−H2O (5:5 to 1:0) to give compounds 3 (6.28 mg) and 4 (4.7 mg). Fr. A5 (12.5 g) was further fractionated with repeated CC on silica gel eluting with petroleum ether (60−90 °C) and acetone (8:1 to 0:1) with increasing polarity to yield six subfractions, A5.1−A5.6, based on TLC. Fr. A5.5 (2.9 g) was separated on an ODS silica gel column eluting with MeOH−H2O (2:8 to 1:0) to give seven subfractions, A5.5.1−A5.5.7. Fr. A5.3.5 (0.5 g) was separated by preparative HPLC with MeOH− H2O (30:70) to yield compounds 7 (3.2 mg) and 7a (5.5 mg). Fr. B12 (8.5 g) was further fractionated by repeated CC on silica gel (petroleum ether−EtOAc, 9:1 to 7:3) to give eight subfractions, B12.1−B12.8. Fr. B12.5 (50 mg) was further separated on silica gel (petroleum ether−EtOAc, 99:1 to 80:20) to yield compounds 1 (15 mg) and 2 (10 mg). Fr. B12.6 (0.3 g) was further separated by HPLC with MeOH−H2O (50:50) to yield compounds 11 (2.8 mg) and 6 (5.0 mg). Wenyujinin A (1): white needles (MeOH); [α]20D −50 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 252 (3.73) nm; ECD (MeOH) λmax (Δε) 200 (+4.97), 255 (−2.13), 314 (−0.87) nm; 1H NMR and 13 C NMR data, see Tables 1 and 3; HRESIMS m/z 275.1611 [M + Na]+ (calcd for C15H24O3Na, 275.1623). Wenyujinin B (2): white needles (MeOH); [α]20D +200 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 252 (3.79) nm; ECD (MeOH) λmax (Δε) 200 (−6.69), 257 (+6.04), 312 (+1.80) nm; 1H NMR and 13 C NMR data, see Tables 1 and 3; HRESIMS m/z 275.1614 [M + Na]+ (calcd for C15H24O3Na, 275.1623). Wenyujinin C (3): light yellow oil (MeOH); [α]20D −30 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 236 (4.03) nm; ECD (MeOH) λmax (Δε) 208 (+3.19), 231 (−0.67) nm; 1H NMR and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 275.1633 [M + Na]+ (calcd for C15H24O3Na 275.1623). Wenyujinin D (4): light yellow oil (MeOH); [α]20D +40 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 236 (4.05) nm; ECD (MeOH) λmax (Δε) 200 (+4.65), 235 (−1.09) nm; 1H NMR and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 275.1622 [M + Na]+ (calcd for C15H24O3Na 275.1623). Wenyujinin E (5): colorless oil (MeOH); [α]20D −47 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 247 (3.62) nm; ECD (MeOH) λmax (Δε) 244(−1.68), 289 (+0.65), 305 (+0.69) nm; 1H NMR and

C NMR data, see Tables 1 and 3; HRESIMS m/z 275.1606 [M + Na]+ (calcd for C15H24O3Na 275.1623). Wenyujinin F (6): light yellow oil (MeOH); [α]20D +80 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 222 (4.14), 332 (3.78) nm; ECD (MeOH) λmax (Δε) 220 (+8.54), 331 (−1.52), 371 (+0.71) nm; 1H NMR and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 285.1102 [M + Na]+ (calcd for C15H18O4Na 285.1103). Wenyujinin G (7): colorless oil (MeOH); [α]20D +50 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 212 (3.69) nm; ECD (MeOH) λmax (Δε) 230 (+9.09), 255 (−3.25); 1H NMR and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 303.1216 [M + Na]+ (calcd for C15H20O5Na 303.1208). Phaeocaulisin A (7a): UV, [α], MS, 1H NMR (DMSO-d6), and 13C NMR (DMSO-d6) data, see Supporting Information. 1H NMR (methanol-d4) and 13C NMR (methanol-d4) data, see Tables 2 and 3. Wenyujinin H (8): colorless oil (MeOH); [α]20D +100 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 234 (3.92) nm; ECD (MeOH) λmax (Δε) 253 (+10.09), 327 (+2.23) nm; 1H NMR and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 251.1650 [M + H]+ (calcd for C15H23O3 251.1647). Wenyujinin I (9): colorless oil (MeOH); [α]20D +200 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 204 (3.63) nm; ECD (MeOH) λmax (Δε) 200 (+14.10) nm; 1H NMR and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 275.1620 [M + Na]+ (calcd for C15H24O3Na 275.1623). Wenyujinin J (10): colorless oil (MeOH); [α]20D +36 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 254 (3.54) nm; ECD (MeOH) λmax (Δε) 322 (+0.79), 260 (+2.66), 222 (−2.67) nm; 1H NMR and 13 C NMR data, see Tables 2 and 3; HRESIMS m/z: 251.1632 [M + H]+ (calcd for C15H23O3 251.1647). Wenyujinin K (11): colorless oil (MeOH); [α]20D +120 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 (+3.96), 280 (+3.38) nm; 1H NMR and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 285.1109 [M + Na]+ (calcd for C15H18O4Na 285.1103). Wenyujinin L (12): colorless oil (MeOH); [α]20D −10 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 238 (3.87) nm; ECD (MeOH) λmax (Δε) 209 (−6.80), 326 (−0.50) nm; 1H NMR and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 203.1042 [M + Na]+ (calcd for C11H16O2Na 203.1048). X-ray Crystallographic Analysis of Wenyujinin A (1). After crystallization from MeOH−H2O (1:1) using the vapor diffusion method, colorless crystals were obtained for 1. A crystal (0.21 × 0.22 × 0.61 mm) was used for the X-ray measurements. Intensity data were collected on a Rigaku MicroMax 002+ diffractometer with Cu Kα radiation using the ω and κ scan technique to a maximum 2θ value of 144.84° (295 K). The structure was solved by direct methods using SHELXS-97. All non-hydrogen atoms were refined anisotropically using the least-squares method, and all hydrogen atoms were positioned by geometric calculations and difference Fourier overlapping calculation. Flack parameter = 0.0(4). Crystallographic data for 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 975362). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]). Crystallographic data for 1: C15H24O3, M = 252.35, monoclinic, P21, a = 10.369(7) Å, b = 5.883(9) Å, c = 12.350(8) Å, β = 102.51(1)°, V = 735(11) Å3, Z = 2, Dcalcd = 1.140 g/cm3, 2145 reflections independent, 1878 reflections observed (|F|2 ≥ 2σ|F|2), R1 = 0.0538, wR2 = 0.1478, S = 1.064. Computational Methods for ECD Spectra. First, conformational studies were performed using Amber and the MMFF94S force field, respectively. The geometries with a relative energy from 0 to 5.5 kcal/mol were selected for calculation at the B3LYP/6-31G(d)// B3LYP/3-21G(d) level to obtain global minima. Conformers within an energy range of 3 kcal/mol from the global minima were subjected to geometrical optimization (DFT/B3LYP/6-31G(d)) in the gas phase, followed by calculation of vibrational modes to confirm these minima. H



Article

After the conformational search, 19 conformations with the lowest energy were found for 3; 15 for 4; one for 1, 6, 8, and 9; and two for 2 and 10. ECD spectra of the above conformations were calculated at the B3LYP/6-311+G(d,p) level in MeOH (SCRF/IEFPCM). OR values were calculated at the PBE1PBE/6-311++G(2d,p) level. The calculated ECDs/OR were weighted prior to comparison with the experimental results. All of the DFT calculations reported in this study were performed with the Gaussian 03 package. iNOS Inhibitory Activity Bioassay. Inhibition of lipopolysaccharide-induced nitric oxide production in RAW 264.7 mouse macrophage cells: cells were evaluated using 96-well plates (1 × 105 cells/well) and allowed to adhere for 2 h at 37 °C in 5% CO2 in air. Next, the cells were treated with 1 μg/mL LPS for 24 h with or without test compound (5 μg/mL). DMSO was used as the solvent. Test compounds were tested at a final concentration of 0.2% (v/v) in cell-culture supernatant. NO production was determined from the accumulation of nitrite in the culture supernatant using Griess reagent.31,32

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(11) Lou, Y.; Zhao, F.; He, H.; Peng, K. F.; Chen, L. X.; Qiu, F. Chem. Biodiversity 2010, 7, 1245−1253. (12) Wang, D.; Huang, W.; Shi, Q.; Hong, C. T.; Cheng, Y. Y.; Ma, Z. Z.; Qu, H. B. Nat. Prod. Commun. 2008, 3, 861−864. (13) Lou, Y.; Zhao, F.; He, H.; Peng, K. F.; Zhou, X. H.; Chen, L. X.; Qiu, F. J. Asian Nat. Prod. Res. 2009, 11, 737−747. (14) An, Y. W.; Hu, G.; Yin, G. P.; Zhu, J. J.; Zhang, Q. W.; Wang, Z. M.; Peng, J.; Fan, B. J. Chromatogr. Sci. 2013, DOI: 10.1093/chromsci/ bmt149. (15) Hu, D.; Ma, N.; Lou, Y.; Qu, G. X.; Qiu, F. J. Shenyang Pharm. Univ. 2008, 25, 188−190. (16) (a) Harada, N.; Nakanishi, K.; Berova, N. In Comprehensive Chiroptical Spectroscopy: Applications in Stereochemical Analysis of Synthetic Compounds, Natural Products, and Biomolecules; Berova, N., Polavarapu, P. L., Nakanishi, K., Woody, R. W., Eds.; Wiley: NJ, USA, 2012; Vol. 2, p 115. (b) Stephens, P. J.; Pan, J. J. J. Org. Chem. 2007, 72, 7641−7649. (17) Louzao, I.; Seco, J. M.; Quinoa, E.; Riguera, R. Chem. Commun. 2010, 46, 7903−7095. (18) Zhang, J.; Li, L. C.; Wang, K. L.; Liao, X. J.; Deng, Z.; Xu, S. H. Bioorg. Med. Chem. Lett. 2013, 23, 1079−1082. (19) Matsuda, H.; Morikawa, T.; Toguchida, I.; Ninomiya, K.; Yoshikawa, M. Heterocycles 2001, 55, 841−846. (20) Gerards, M.; Snatzke, G. Tetrahedron: Asymmetry 1990, 1, 221− 236. (21) Frelek, J.; Szczepek, W. Tetrahedron: Asymmetry 1999, 10, 1507−1520. (22) Shiobara, Y.; Asakawa, Y.; Kodama, M.; Takemoto, T. Phytochemistry 1986, 25, 1351−1353. (23) Takano, I.; Yasudam, I.; Takeya, K.; Itokawa, H. Phytochemistry 1995, 40, 1197−1200. (24) Demarco, P. V.; Farkas, E.; Doddrell, D.; Mylari, B. L.; Wenkert, E. J. Am. Chem. Soc. 1968, 90, 5480−5486. (25) Liu, Y.; Ma, J. H.; Zhao, Q.; Liao, C. R.; Ding, L. Q.; Chen, L. X.; Zhao, F. J. Nat. Prod. 2013, 76, 1150−1156. (26) Kuroyanagi, M.; Ueno, A.; Koyama, K.; Natori, S. Chem. Pham. Bull. 1990, 38, 55−58. (27) Shibuya, H.; Hamamoto, Y.; Cai, Y.; Kitagawa, I. Chem. Pharm. Bull. 1987, 35, 924−927. (28) Phan, M. G.; Phan, T. S. J. Chem. (Vietnam) 2000, 38, 96−99. (29) Snatzke, G. Tetrahedron 1965, 21, 413−419. (30) Johansson, M.; Kopcke, B.; Anke, H.; Sterner, O. J. Antibiot. 2002, 55, 104−106. (31) Dirsch, V. M.; Stuppner, H.; Vollmar, A. M. Planta Med. 1998, 64, 423−426. (32) Li, J.; Zhao, F.; Li, M. Z.; Chen, L. X.; Qiu, F. J. Nat. Prod. 2010, 73, 1667−1671.

ASSOCIATED CONTENT

S Supporting Information *

Crystallographic data of 1; MS, 1D NMR, 2D NMR, and ECD spectra for compounds 1−12 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(J.-J. Zhu) Tel: 86-10-64014411. Fax: 86-10-64013996. Email: [email protected]. *(Z.-M. Wang) Tel: 86-1084014128. Fax: 86-10-64013996. Email: [email protected]. Author Contributions ∥

G.-P. Yin and L.-C. Li contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the National Natural Science Foundation of China (Nos. 30901961, 81373960) and the Fundamental Research Funds for the Central Public Welfare Research Institutes (Nos. ZZ20092015, ZZ070833) for their financial support of this work. The support of the Supercomputing Center, CNIC, CAS, for computer time is acknowledged.

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REFERENCES

(1) The State Pharmacopoeia Commission of PRC. Pharmacopoeia of the People’s Republic of China; China Medical Science and Technology Press: Beijing, China, 2010; Vol. 1, pp 285−286. (2) Tohda, C.; Nakayama, N.; Hatanaka, F.; Komatsu, K. Evid.-Based. Complementary Altern. Med. 2006, 3, 255−260. (3) Endo, K.; Kanno, E.; Oshima, Y. Phytochemistry 1990, 29, 797− 799. (4) Yu, Z. Q.; Schmaltz, R. M.; Bozeman, T. C.; Paul, R.; Rishel, M. J.; Tsosie, K. S.; Hecht, S. M. J. Am. Chem. Soc. 2013, 135, 2883−2886. (5) Zhang, P.; Huang, W.; Song, Z. H.; Zhang, M.; Cheng, L.; Cheng, Y. Y.; Qu, H. B.; Ma, Z. J. Phytochem. Lett. 2008, 1, 103−106. (6) Pank, S. Y.; Kim, D. S. H. L. J. Nat. Prod. 2002, 65, 1227−1231. (7) Syu, W. J.; Shen, C. C.; Don, M. J.; Ou, J. C.; Lee, G. H.; Sun, C. M. J. Nat. Prod. 1998, 61, 1531−1534. (8) Ma, Z. J.; Meng, Z. K.; Zhang, P. Fitoterapia 2009, 80, 374−376. (9) Wang, L. X.; Deng, Z. W.; Huang, K. X.; Lin, W. H. Chin. J. Chin. Mater. Med. 2008, 33, 785−788. (10) Lou, Y.; He, H.; Wei, X. C.; Li, X. G.; Chen, L. X.; Qiu, F. J. Shenyang Pharm. Univ. 2010, 27, 195−199. I


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