Bioactive Abietane and ent-Kaurane Diterpenoids ... - ACS Publications

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Bioactive Abietane and ent-Kaurane Diterpenoids from Isodon tenuifolius Jian-Hong Yang,†,‡ Xue Du,† Fei He,† Hai-Bo Zhang,† Xiao-Nian Li,† Jia Su,† Yan Li,† Jian-Xin Pu,*,† and Han-Dong Sun† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China. S Supporting Information *

ABSTRACT: Three new abietane diterpenoids, isoabietenins A−C (1−3), and 13 new ent-kauranoids, tenuifolins A−M (4− 16), along with four known compounds (17−20), were isolated from the aerial parts of Isodon tenuifolius. The structures of the new metabolites were established on the basis of detailed spectroscopic analysis. The absolute configurations of 1, 15, and 16 were confirmed by singlecrystal X-ray diffraction. Selected compounds were evaluated for their cytotoxicity against a small panel of human tumor cell lines, and some compounds showed inhibitory effects. Furthermore, several isolates exhibited inhibitory activity against nitric oxide production in LPS-activated RAW264.7 macrophages.

cancer cell lines and their inhibitory activity against LPSinduced NO production in RAW264.7 macrophages.

Isodon (formally Rabdosia) includes about 150 species and is one of the largest genera of the plant family Lamiaceae. It has attracted considerable attention as a rich source of diverse diterpenoids, including ent-kauranes, abietanes, labdanes, pimaranes, isopimaranes, gibberellanes, and clerodanes, of which several have interesting biological properties, such as anti-inflammatory, antibacterial, and antitumor activities.1−4 The use of Isodon species in Chinese popular folk medicine has a long tradition. For example, in 1977, a standardized extract of Isodon rubescens (Hemsl.) H. Hara was successfully developed in mainland China into a drug product, used in treating sore throats and inflammation.5 Isodon tenuifolius (W. W. Smith) Kudo, a small shrub, is distributed mainly in the northwest of Yunnan Province and the southwest of Sichuan Province6 and is a folk medicine used to treat influenza, dysentery, jaundice, snakebite, and a variety of types of inflammation.7 Previous phytochemical investigations of this plant have shown the presence of six ent-kauranoids.8,9 In the present investigation, three new abietane diterpenoids, isoabietenins A−C (1−3), and 13 new ent-kauranoids, tenuifolins A−M (4−16), along with four known compounds (17−20) have been isolated from I. tenuifolius. Among the new compounds obtained, compound 1, characterized by the presence of a rare five-membered-ring structure, is the first example of an abietane diterpenoid with an epoxide ring between rings A and C. Compound 14, in contrast, features an unusual five-membered ring through an ether bond between C11 and C-16. In this study, the isolation and structure elucidation of the diterpenoid constituents of I. tenuifolius are described as well as the cytotoxicity evaluation against several © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A 70% aqueous acetone extract of the air-dried and powdered aerial parts of I. tenuifolius was partitioned between EtOAc and H2O. The EtOAc layer was subjected to repeated column chromatography and HPLC to afford three new abietane diterpenoids, isoabietenins A−C (1−3), and 13 new entkauranoids, tenuifolins A−M (4−16), along with four known substances, adenanthin C (17),10 adenanthin B (18),10 adenanthin E (19),11 and inflexarabdonin E (20).12 Isoabietenin A (1) was obtained as colorless needles and gave the molecular formula C20H28O3, as deduced from its positive-ion HRESIMS ([M + Na]+ m/z 339.1929). Its IR spectrum indicated absorption bands for hydroxy groups (3406 cm−1) and an aromatic ring (1483, 1460 cm−1). The 1H and 13 C NMR spectra showed 20 carbon signals (Tables 1 and 2), including those for two quaternary methyls, a hydroxymethyl, an isopropyl, and an aromatic ring. These signals are characteristic of an abieta-8,11,13-triene derivative. Analysis of the 2D-NMR spectroscopic data and comparison with those of pomiferin A13 led to the conclusion that C-7 and C-18 are each substituted by a hydroxy group and that a five-membered ring through an oxygen atom occurred between C-1 and C-11, from the HMBC correlations from H-1 (δH 4.54, dd, J = 11.2, 6.7 Hz) to C-11 (δC 157.8, s). The observed ROESY correlations Received: November 5, 2012 Published: January 17, 2013 256

dx.doi.org/10.1021/np300772e | J. Nat. Prod. 2013, 76, 256−264

Journal of Natural Products

Article

the hydroxy methyl group is α-oriented (Figure 1). Singlecrystal X-ray diffraction analysis using the anomalous scattering of Cu Kα radiation yielded a Flack14 parameter of 0.25(19) (CCDC 907612), confirming the structural assignment made (Figure 2). Thus, the structure of 1 was determined as 7α,18dihydroxy-1α,11-epoxyabieta-8,11,13-triene, and this compound has been given the trivial name isoabietenin A. Isoabietenin B (2) was isolated as colorless needles and yielded a pseudomolecular ion peak in the positive-ion HRESIMS at m/z 359.2190 [M + Na]+, indicative of the molecular formula C20H32O4. Identifiable from the NMR spectroscopic data for 2 were resonances consistent with a tetrasubstituted double bond (δC 142.7 s and 150.6, s) and a carbonyl carbon atom (δC 200.5, s) (Table 1). In the absence of any other sp or sp2 carbon, the gross structure of 2 must be tricyclic. This information, along with the HMBC correlations traced from four methyls, suggested the presence of an abietane diterpenoid. Observation of the HMBC correlations from H-7 (δH 4.30, d, J = 4.1 Hz) to C-5, C-8 (δC 150.6, s), C-9 (δC 142.7, s), and C-14, from H-12, H-14, H-15, H3-16, and H3-17 to C-13 (δC 75.8, s), and from H2-18 (δH 3.73, 3.51, 2H, each d, J = 10.5 Hz) to C-3, C-4, C-5, and CH3-19 permitted the assignment of the tetrasubstituted double bond at C-8 and C-9 and the three hydroxy groups at C-7, C-13, and C-18, respectively. The carbonyl carbon was assigned to C-11 by the correlations from H-12 and H-14 to C-11 (δC 200.5 s) in the HMBC spectrum. The ROESY correlations of H-7/H-14β

from H-1 and H-7 to H3-20, and from H2-18 to H-3α, H-5α, and H-6α, indicated that H-1 and H-7 are both β-oriented and Table 1. 13C NMR Data of 1−16 in C5D5N, δ in ppm position

1a

2b

3c

4d

5d

6d

7d

8d

9d

10d

11d

12d

13d

14d

15d

16d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CH3O-17 AcO-1

93.3 26.0 30.9 36.1 40.6 32.1 66.3 139.6 134.7 44.4 157.8 105.4 150.1 114.9 35.4 24.6 24.6 70.8 21.3 23.1

36.0 19.0 35.9 38.5 41.2 28.9 69.7 150.6 142.7 37.9 200.5 51.3 75.8 40.5 38.7 17.4 17.0 71.9 17.9 19.4

79.3 31.5 74.7 36.8 54.8 204.7 74.7 72.2 58.3 40.1 71.8 28.2 25.6 34.4 153.8 195.1 133.8 28.7 21.9 15.0

38.3 28.0 77.8 38.6 54.8 18.8 37.3 50.7 64.1 39.5 65.1 41.8 37.8 34.7 209.2 151.8 111.1 28.8 17.9 16.4

33.7 26.5 75.0 38.2 48.9 20.0 36.6 45.5 56.5 37.9 65.5 42.9 40.2 39.8 82.9 159.6 105.7 29.5 22.5 17.7

78.8 32.8 74.7 38.4 44.3 36.5 210.0 57.7 54.3 43.0 68.2 40.8 38.4 36.3 77.6 151.9 108.2 28.2 21.5 13.1

80.5 33.8 75.4 37.7 37.8 27.2 77.7 47.8 49.3 43.0 70.5 40.5 38.9 35.1 83.0 157.0 105.0 29.0 22.6 14.5

33.8 26.0 74.9 37.3 59.1 212.4 55.1 50.7 56.4 44.0 65.2 42.4 39.8 36.5 82.2 158.0 106.6 28.1 22.6 19.0

80.0 33.1 75.5 37.0 57.7 211.6 55.6 50.8 56.4 48.5 65.8 42.7 39.5 37.5 82.3 157.9 106.4 27.5 22.7 15.6

75.2 37.5 76.1 37.5 58.1 212.2 54.9 50.3 54.0 49.5 71.5 39.1 38.8 36.7 82.6 157.0 105.6 27.7 22.7 15.4

76.9 31.5 75.8 37.2 58.8 210.4 51.9 51.3 63.7 47.1 209.6 52.6 40.2 35.9 81.2 154.6 109.3 27.6 22.3 17.8

74.1 36.9 76.1 37.4 58.5 211.8 50.7 55.0 60.4 51.0 70.9 37.6 37.0 37.0 205.2 150.2 113.2 27.6 22.5 15.9

33.0 22.4 77.9 36.1 60.2 211.3 50.1 55.0 64.7 45.2 71.1 76.6 45.4 31.1 206.5 146.7 115.6 27.1 21.9 18.3

78.4 31.8 75.8 37.0 59.8 209.7 50.5 49.1 53.5 45.7 78.2 38.2 40.2 38.6 78.7 86.5 62.9 27.8 22.7 16.0

76.0 37.1 75.5 37.2 50.3 211.3 82.1 55.4 56.5 50.8 71.2 36.8 33.9 33.2 223.0 54.9 72.7 27.5 23.1 15.5 58.5

170.5, 20.7

170.5 22.0

79.7 32.6 75.4 37.1 49.6 210.9 81.9 54.8 54.3 49.3 68.5 31.5 32.4 34.3 218.1 56.5 70.2 27.3 22.9 16.1 58.6 170.2 22.0

169.8 21.4

169.1 21.1

169.8 21.2

169.9 21.5

169.7 21.1

170.2 21.5

AcO-3 AcO-7 AcO-11 AcO-15

a

170.2 20.9 170.1 21.3 169.7 20.5

170.3 21.4 170.6 22.1

169.4 21.5

169.6 21.8

169.5 21.4 171.4 20.9

Recorded in (CD3)2CO, 100 MHz. bRecorded at 125 MHz. cRecorded in DMSO, 150 MHz. dRecorded at 100 MHz. 257



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Table 2. 1H NMR Data of 1−8 in C5D5N, δ in ppm (J in Hz) 1a

position 1α 1β 2α 2β 3α 3β 5 6α 6β 7α 7β 9 11 12α 12β 13 14α 14β 15 16 17a 17b 18a 18b 19 20 AcO-1 AcO-7 AcO-11 AcO-15 HO-3 HO-8 HO-11 HO-15 a

2b

4.54, dd (11.2, 6.7) 1.59, m 1.78, m 1.83, m 1.12, m 1.61, overlap 1.62, overlap 2.50, m

1.09, 3.00, 1.54, 1.78, 1.78, 1.48, 2.31, 2.16, 1.83,

overlap d (12.0) overlap overlap overlap overlap d (12.8) d (13.5) overlap

5.09, dd (8.8, 2.3)

4.30, d (4.1)

6.39, s

2.77, s 2.77, s

6.83, s 2.83, m 1.18, d (6.9) 1.18, d (6.9)

3.36, 2.68, 1.83, 1.07, 1.01,

d (18.1) d (18.1) overlap d (6.8) d (6.8)

3.24, 3.17, 0.93, 1.26,

3.73, 3.51, 0.94, 1.47,

d (10.5) d (10.5) s s

d (10.4) d (10.4) s s

3c 5.30, m 1.70, m 1.87, m 3.20, brs 3.59, s

4d

5b

1.90, 1.19, 1.82, 1.87,

overlap overlap m m m brd (14.0) m overlap m brd (15.6) s s overlap m brs overlap m

2.12, 1.77, 1.77, 2.02, 3.61,

m overlap overlap overlap brs

1.82, 1.55, 1.34, 1.09, 2.02, 2.19, 4.21, 2.12, 2.00, 2.65, 2.00, 1.46, 4.04,

brd (12.1) m m m overlap s brs overlap overlap brs overlap d (12.9) d (9.9)

6b

7d

5.86, overlap

5.85, dd (10.7, 4.8)

2.01, m 2.25, m 3.66, brs

2.13, m 2.19, overlap 3.72, brs

2.38, brd (12.9) 2.72, dd (14.5, 12.9) 2.60, d (14.5)

1.76, 2.86, 1.60, 3.82,

m d (11.8) m brs

2.57, 5.89, 1.99, 1.99, 2.51, 1.71, 1.15, 4.46,

s s m m d (3.2) d (12.0) m s

8b 2.33, 1.83, 2.04, 1.78, 3.50,

m m m m brs

5.20, 2.23, 5.26, 1.72, 1.85, 3.13, 1.75, 1.77,

s s brs overlap m m overlap overlap

3.35, 0.88, 1.60, 1.33, 1.35, 2.38, 1.79, 4.24, 2.23, 2.06, 3.01, 2.22, 1.43,

9.47, 6.22, 6.20, 1.09,

s s s s

6.01, s 5.24, s 1.15, s

5.37, s 5.17, s 1.16, s

5.12, s 5.02, s 1.05, s

5.25, s 4.96, s 1.19, s

5.35, s 5.15, s 1.33, s

1.01, 1.07, 1.97, 2.18, 1.94,

s s s s s

1.00, s 1.00, s

0.88, s 1.00, s

0.90, s 1.37, s 1.84, s

0.93, s 1.28, s 2.19, s

1.46, s 1.06, s

1.91, s 2.20, s 6.65, s

1.87, s

5.00, s 5.61, s

5.64, d (4.2)

2.89, 5.84, 2.22, 1.96, 2.58, 1.82, 1.74, 6.52,

s overlap m m brs d (13.8) m s

3.29, s

2.04, 3.14, 2.71, 4.27, 2.04, 1.88, 2.60, 1.97, 1.20, 4.02,

d (12.2) d (12.2) s brs overlap m brs d (12.9) brd (12.9) d (10.0)

6.04, d (3.4)

6.26, d (4.1) 5.75, d (9.9)

6.52, d (3.2) 5.87, d (10.0)

Recorded in (CD3)2CO, 400 MHz. bRecorded at 500 MHz. cRecorded in DMSO, 600 MHz. dRecorded at 400 MHz.

Figure 1. Key HMBC and ROESY correlations of 1.

and H-12α, H-14α/H-15, and H-12β/H3-17 indicated the configurations of H-7 and HO-13 are both β-oriented. Therefore, isoabietenin B was determined as 7α,13β,18trihydroxyabieta-8(9)-en-11-one. Isoabietenin C (3) gave the molecular formula C26H36O10, as established from the positive-ion HRESIMS at m/z 531.2215 [M + Na]+, indicating nine degrees of unsaturation. Except for three acetoxy groups, 20 carbon atoms found in the 13C NMR and DEPT spectra consisted of a carbonyl carbon, an aldehydic carbon, an olefinic quaternary carbon, an olefinic methylene carbon, seven methine carbons, of which four are oxygenated, three methylene carbons, three quaternary carbons with one

Figure 2. X-ray crystal structure of 1.

oxygenated carbon, and three methyl carbons, which were again consistent with a skeleton of an abietane diterpenoid. The carbonyl carbon was assigned as C-6 from the long-range correlations of H-5 and H-7 with C-6 (δC 204.7, s) in the HMBC spectrum. On the basis of the analysis of the HMBC 258



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C-5 (Δ 5.9 ppm) in 5, caused by the γ-steric compression effect between HO-3β and H-1β and H-5β, confirmed that H-3 is αoriented. Therefore, tenuifolin B (5) was elucidated as 3β,11β,15β-trihydroxy-ent-kaur-16-ene. The molecular formulas of tenuifolin C (6) and tenuifolin D (7) were determined as C26H36O8 and C24H36O7, respectively, according to their HRESIMS. Comparison of the NMR data with those of 18 indicated that the acetoxy group at C-7 in the latter compound is replaced by a carbonyl group in 6, and the hydroxy group at C-15 is acetylated. These assignments were verified by the HMBC correlations of H-5, H-9, and H-14 with C-7 (δC 210.0, s) and of H-15 (δH 6.52, s) with the acetyl carbonyl (δC 170.6, s). Comparison of the NMR data of 7 with those of 18 revealed that the only difference was that an acetoxy group at C-7 in 18 is reduced to a hydroxy group at the same position in 7. Moreover, the correlations observed in the ROESY spectrum indicated that the orientations of the substituents in 6 and 7 are the same as those of 18. Thus, 6 and 7 were characterized as 3β-hydroxy-1α,11β,15β-triacetoxyent-kaur-16-en-7-one and 3β,7β,15β-trihydroxy-1α,11β-diacetoxy-ent-kaur-16-ene, respectively. Tenuifolin E (8) was obtained as an amorphous powder. Its molecular formula, C20H30O4, was determined by the [M + Na]+ ion peak at m/z 357.2051 (calcd 357.2041). The 1H and 13 C NMR data of 8 were similar to those of 5, except for the absence of signals for an sp3 methylene and evidence for a carbonyl group. The HMBC correlations observed from H-5, H-9, and H-15 to C-7, and H-5 and H-7 to C-6 (δC 212.4, s), indicated that the carbonyl is located at C-6 in 8. Tenuifolins F (9) and G (10) were assigned the same molecular formula, C22H32O6, as determined by HRESIMS (m/z 415.2094 [M + Na]+ and 415.2097 [M + Na]+, respectively). Detailed comparison of the NMR data of 9 with those of 8 indicated that these two compounds are closely comparable. The only significant difference observed was that the sp3 methylene for C-1 in 8 is replaced by an acetoxy group (δC 80.0, d) in 9. The main difference found between 9 and 10 concerned the substituent groups located at C-1 and C-11. Comparison of the spectroscopic data of 10 with those of 9 revealed both substances to be quite similar structurally, except that the location of the acetyl group at C-1 in 9 was changed to C-11 in 10. The HMBC and 1H−1H COSY spectra were used to support the above deductions. The ROESY correlations of H1/H-5β and H-9β indicated that H-1 is β-oriented in 9 and 10. The relative configurations of 8−10 were assigned as being the same as those of 5 from the similar carbon and proton chemical shifts and the ROESY correlations observed. Thus, compounds 8−10 were determined structurally as 3β,11β,15β-trihydroxyent-kaur-16-en-6-one, 3β,11β,15β-trihydroxy-1α-acetoxy-entkaur-16-en-6-one, and 1α,3β,15β-trihydroxy-11β-acetoxy-entkaur-16-en-6-one, respectively. Tenuifolin H (11) was assigned the molecular formula C22H30O6 from its HRESIMS and NMR data. Comparison of the spectroscopic data of 11 with those of 9 (Tables 1 and 3) showed similarities except for the hydroxy group at C-11 in 9 being replaced by a carbonyl group (δC 209.6, s) in 11. This allowed the relative configurations of HO-3 and HO-15 to be determined with a β-orientation, and AcO-1 as α-oriented; so 11 was assigned as 3β,15β-dihydroxy-1α-acetoxy-ent-kaur-16ene-6,11-dione. The molecular formula of tenuifolin I (12) was determined to be C22H30O6 from the HRESIMS. Comparison of the NMR data of 12 with those of 10 led to the deduction that the only

correlations of H-13 with C-15 (δC 153.8, s), C-16 (δC 195.1, d), and C-17 (δC 133.8, t) and of H2-17 (δH 6.20, 6.22, 2H, each s) with C-15 and C-16, the olefinic bond conjugated with the aldehydic group could be located at C-13. The remaining oxygenated functionalities of 3 were established accordingly, with AcO-1β, HO-3α, AcO-7α, HO-8α, and AcO-11α deduced from the HMBC correlations of H-1/C-3, C-5, C-9, C-10, and an acetyl carbonyl, of H-3/C-1 and C-5, of H-7/C-5, C-9, and an acetyl carbonyl, and of H-11/C-8, C-10, C-13, and an acetyl carbonyl, and also the ROESY correlations of H-1α/H-5α and H-9α, of H-3β/H3-18 and H3-19, of HO-3α/H-5α, and of H7β/H-11β and H3-20β. Consequently, 3 (isoabietenin C) was established as 3α,8α-dihydroxy-1β,7α,11α-triacetoxy-6,16-dioxoabiata-15-ene. Tenuifolin A (4) was assigned as C20H30O3, as deduced from the HRESIMS (m/z 341.2100 [M + Na]+) and in accordance with its NMR data. The 13C NMR and DEPT spectra were used to resolve the 20 carbon signals as three methyls, seven methylenes (including one sp2 methylene), five methines (of which two were oxygenated), and five quaternary carbons (including one sp2 carbon and one carbonyl), which was consistent with a skeleton of an ent-kaur-16-en-15-one.15 Two oxymethine protons at δH 3.35 (1H, m) and 4.24 (1H, s), observed in the 1H NMR spectrum (Table 2), could be located at C-3 and C-11, respectively, as determined by the HMBC correlations from H-3 to C-5, C-18, and C-19 and from H-11 to C-8, C-10, and C-13. The 1H−1H COSY correlations of H21/H2-2/H-3, H-5/H2-6/H2-7, and H-9/H-11/H2-12/H-13/H214 were used to establish the spin systems of -CH2(C-1)CH2(C-2)-CH(C-3)-, -CH(C-5)-CH2(C-6)-CH2(C-7)-, and -CH(C-9)-CH(C-11)-CH2(C-12)-CH(C-13)-CH2(C-14)-, respectively. These features indicated the structure of tenuifolin A (4) as 3α,11β-dihydroxy-ent-kaur-16-en-15-one. Tenuifolin B (5) was assigned the molecular formula C20H32O3, by HRESIMS (m/z 343.2244 [M + Na]+). Inspection of the 1D- and 2D-NMR spectra led to the conclusion that this compound is also an ent-kaurane diterpenoid with a similar structure to that of 4. The carbonyl signal for C-15 in 4 was replaced by signals for an oxymethine (δH 4.04, d, J = 9.9 Hz; δC 82.9, d) of 5. In the 13C NMR spectrum, the upfield shift for C-9 from δC 64.1 in 4 to δC 56.5 in 5, caused by the γ-steric compression effect between HO-15β and H-9β, supported H-15 as being α-oriented. The βorientation of HO-15 was determined by the ROESY correlations of H-15α with H-7α and H-14β. Further analysis of the ROESY spectrum revealed that H-3 of 5 showed NOEs with H3-18 and H3-19 (Figure 3) instead of correlating with H5β and H-9β, indicating the β-orientation of HO-3. In contrast to the 13C NMR data of 4, the downfield shift of C-19 (Δ 4.6 ppm) and the relatively upfield shifts of C-1 (Δ 4.6 ppm) and

Figure 3. Key COSY, HMBC, and ROESY correlations of 5. 259



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Table 3. 1H NMR Data of 9−16 in C5D5N, δ in ppm (J in Hz) 9a

position

a

1α 1β 2α 2β 3 5 7α 7β 9 11 12α 12β 13 14α 14β 15 16 17a

10a

11b

12a

13a

6.12, dd (11.3, 4.7)

4.89, m

6.05, dd (11.4, 5.0)

4.85, m

2.24, m 2.08, overlap 3.56, s 3.41, s 2.10, d (11.6) 3.19, d (11.6) 2.95, s 4.88, d (0.4) 2.12, overlap 1.99, overlap 2.59, d (3.2) 1.92, d (12.1) 1.23, dd (12.1, 3.2) 4.02, d (9.8)

2.38, m 2.33, m 3.62, brs 3.36, s 2.36, d (12.1) 3.24, d (12.1) 2.86, s 7.10, d (4.3) 2.24, m 1.91, overlap 2.52, brs 1.97, d (12.7) 1.22, brd (12.7) 4.00, d (10.2)

1. 94, m 1.47, m 3.55, s 3.27, s 2.20, d (13.6) 3.09, d (13.6) 3.60, s

2.31, 2.28, 3.59, 3.41, 2.10, 3.51, 2.65, 7.03, 2.10, 2.10, 2.89, 2.46, 1.46,

5.30, s

5.32, s

5.39, s

6.01, s

6.18, s

17b

5.12, s

5.03, s

5.11, s

5.23, s

5.41, s

18 19 20 CH3O-17 AcO-1 AcO-3 AcO-11 AcO-15 HO-1 HO-3 HO-7 HO-11 HO-12 HO-15

1.27, s 1.49, s 1.33, s

1.34, s 1.53, s 1.39, s

1.28, s 1.48, s 1.47, s

1.34, s 1.50, s 1.43, s

1.10, s 1.42, s 1.62, s

1.97, s

2.50, d (12.6) 2.74, dd (12.6, 2.4) 2.90, d (2.4) 2.52, d (10.2) 1.38, brd (10.2) 4.12, s

m m brs s overlap d (12.8) s d (2.3) overlap overlap d (3.3) d (12.2) overlap

1.88, 1.88, 1.87, 1.60, 4.69, 3.02, 2.20, 3.63, 2.62, 4.49, 4.59,

overlap overlap overlap overlap s s d (13.8) d (13.8) s s brs

3.38, brs 3.21, d (12.0) 1.46, brd (12.0)

1.99, s

14b

15a

16a

5.92, dd (11.2, 4.7)

4.94, m

5.87, m

2.30, 1.98, 3.55, 3.30, 2.16, 2.90, 3.06, 4.64, 2.10, 2.04, 2.85, 2.04, 1.34, 4.98,

2.38, 2.28, 3.65, 4.65, 4.25,

m m brs s s

2.36, 2.09, 3.58, 4.67, 4.19,

3.07, 6.80, 2.13, 1.94, 2.50, 2.15, 2.11,

s d (4.6) overlap m s overlap overlap

2.86, s 5.86, d (4.7) 2.08, m 2.08, m 2.68, brs 2.17, overlap 1.68, brd (12.0)

m m s s d (14.0) d (14.0) s s d (11.4) overlap m d (11.4) m s

4.02, dd (19.7, 11.5) 4.02, dd (19.7, 11.5) 1.25, s 1.48, s 1.28, s

m m s s s

2.93, m 3.59, m

3.01, m 3.92, m

3.49, m

3.66, m

1.36, 1.53, 1.43, 3.17,

1.31, 1.48, 1.39, 3.28, 2.17,

s s s s

1.98, s

s s s s s

1.88, s 1.91, s

1.80, s

1.83, s

1.86, s

6.33, d (4.5) 6.48, s 6.67, s

6.76, s 6.65, s

1.91, s 6.69, s

6.51, d (4.8) 6.45, s

6.66, s

6.49, d (4.7) 6.42, d (3.1)

6.49, s 6.02, d (9.8)

6.60, s 7.28, s 4.47, d (10.2)

Recorded at 400 MHz. bRecorded at 500 MHz. 13

difference structurally is that the hydroxy group at C-15 in 10 is replaced by a ketone group in 12. The α-orientation of HO-1 was suggested from the ROESY correlations of H-1 observed with H-5β and H-9β. The relative configurations of both HO-3 and AcO-11 were determined as β-oriented. Thus, the structure of 12 was established as 1α,3β-dihydroxy-11β-acetoxy-ent-kaur16-ene-6,15-dione. Tenuifolin J (13) was found by HRESIMS to possess the molecular formula C22H30O6, the same as that of 11 and 12. Its NMR data suggested 13 to be an ent-kauranoid with three oxygenated carbons, an exocyclic double bond, and two ketone groups. Analysis of the 2D-NMR data and comparison with 12 suggested that the two ketone groups are located at C-6 and C15, and the exocyclic double bond is at C-16 and C-17, the same as in 12. The C-11 and C-12 positions were found to be each substituted by a hydroxy group, with C-3 acetylated, on the basis of the HMBC correlations. Moreover, from the ROESY correlations of H-11 with H3-20α, and H-12 with H-17, HO-11 was deduced to be β-oriented, and HO-12 α-oriented. Therefore, compound 13 was concluded to be 11β,12αdihydroxy-3β-acetoxy-ent-kaur-16-ene-6,15-dione. Tenuifolin K (14) gave the molecular formula C24H34O8, as established from the positive-ion HRESIMS at m/z 473.2157 [M + Na]+, indicating eight degrees of unsaturation. Its 1H and

C NMR spectra were found to be similar to those of 9. However, close comparison of the 13C NMR spectroscopic data of these two compounds revealed that the exocyclic doublebond signals (δC 157.9, s, and δC 106.4, t) in 9 were changed to an oxymethylene and an oxygenated quaternary carbon (δC 62.9, t, and δC 86.5, s) in 14. In a HMBC experiment, a correlation was observed between H-11 (δH 4.64, 1H, s) and C16 (δC 86.5, s), which indicated the occurrence of a fivemembered ring in 14 through an ether bond between C-11 and C-16. This was confirmed by the downfield chemical shift of C11 from δC 65.8 in 9 to δC 78.2 in 14. On the basis of the NOESY correlations of H-3/H3-18 and H3-19, H-11/H3-20, and H-15/H2-7 and H-14β, compound 14 (tenuifolin K) was elucidated as 3β,17-dihydroxy-1α,15β-diacetoxy-11β,16-epoxyent-kaur-6-one. Tenuifolin L (15) was assigned the molecular formula C23H34O8 from the positive-ion HRESIMS. Comparison of the NMR data of 15 with those of 12 revealed that the two compounds resemble each other structurally, with the main differences being that the exomethylene group in 12 is replaced by a methine proton (δC 54.9, d, C-16) and a methoxymethyl group (δC 58.5, s, OMe, and δC 72.7, t, C-17) in 15. The downfield shift of C-15 (Δ 17.8 ppm) in 15, caused by the absence of the conjugated system, was consistent with these 260



Article

inferences. The α-orientation of the methoxymethyl group was established by the ROESY correlations of H-17/H-14β and H16/H-12β. X-ray crystallographic analysis using a crystal of 15 and Cu Kα radiation (CCDC 907613) was used to confirm the structure deduced (Figure 4). Thus, the structure of 15 was assigned as 16(S)-1α,3β,7β-trihydroxy-17-methoxy-11β-acetoxy-ent-kaur-6,15-dione.

Table 4. Cytotoxic Activities of Diterpenoids from Isodon tenuifolius against Tumor Cell Linesa compound

HL-60

SMMC-7721

A-549

MCF-7

SW-480

3 4 12 13 16 17 20 cisplatin paclitaxol

4.7 4.7 0.8 3.8 4.4 1.8 1.7 2.6 10 7.4 1.9 6.3 >10 3.2 2.2 >10 10 >10 4.5 >10 >10 >10 6.8 >10 10 >10 3.0 >10 >10 4.8 4.8 >10 10 >10 4.3 >10 >10 5.1 4.6 >10 0.15

a Results are expressed as IC50 values in μM. Cell lines: HL-60, acute leukemia; SMMC-7721, hepatie cancer; A-549, lung cancer; MCF-7, breast cancer; SW-480, colon cancer. Compounds 1, 2, 5−11, 15, 18, and 19 were inactive for all cell lines (IC50 > 10 μM).

Inflammation is a systemic response aimed to decrease the toxicity of harmful agents and to repair damaged tissue. A key feature of the inflammatory response is the activation of phagocytic cells involved in the host defense. Nitric oxide, the free radical produced by the inducible NO synthase (iNOS) isoform, is an essential component of the host innate immune and inflammatory response to a variety of pathogens. All isolates except compound 14 were tested for their inhibitory activity against NO production in LPS-stimulated RAW264.7 cells. Compounds 12, 17, and 20 showed strong inhibitory activity against NO production (Table 5). The viability of


Tenuifolin M (16) gave a pseudomolecular ion peak at m/z 503.2247 [M + Na]+, consistent with the molecular formula C25H36O9. Comparison of their 1D- and 2D-NMR data indicated that 16 and 15 are almost identical, except for an additional acetoxy group in 16. Another difference was the strong ROESY correlation of H-17/H-14β and H-16/H-12β for 15 and of H-17/H-12β and H-16/H-14β in 16. Thus, the βorientation of the methoxymethyl group could be proposed for 16, which was confirmed by X-ray analysis (Figure 5). Thus, 16 was assigned as 16(R)-3β,7β-dihydroxy-17-methoxy-1α,11βdiacetoxy-ent-kaur-6,15-dione.

Table 5. Inhibitory Effects of the Diterpenoids from Isodon tenuifolius on LPS-Activated NO Production in RAW264.7 Cellsa

a

compd

IC50 (μM)

compd

IC50 (μM)

1 2 3 4 5 6 7 8 9 10

>25 >25 10.8 6.9 >25 >25 >25 >25 >25 >25

11 12 13 15 16 17 18 19 20 MG-132

>25 5.8 8.8 >25 >25 3.8 >25 >25 5.7 0.15

Each value represents the mean ± SEM (n = 3).

RAW264.7 cells was determined by the MTS assay.17 At the highest concentration, none of the compounds tested showed any obvious cytotoxicity toward RAW264.7 cells.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D- and 2D-NMR spectra were recorded on Bruker AM-400, DRX-500, and Avance III 600 spectrometers with TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. Mass spectra were performed on an API QSTAR time-of-flight spectrometer. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 25 cm) column. Column chromatography was performed with silica gel (200−300 mesh, Qingdao Marine Chemical, Inc., Qingdao,


All diterpenoids isolated, except for 14 due to sample limitations, were assayed for their cytotoxicity against five human tumor cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480), using the MTT method.16 Among these, compounds 3, 4, 12, 13, 16, 17, and 20 showed some cytotoxic potency, while compounds 17 and 20 exhibited activity (IC50 < 10 μM) for all five cell lines used (Table 4). 261



Article

People’s Republic of China) and MCI gel (75−150 μM, Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC, and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. Plant Material. The aerial parts of I. tenuifolius were collected in Shangri-la County of Yunnan Province, People’s Republic of China, in August 2008, and identified by Prof. Xi-Wen Li, Kunming Institute of Botany. A voucher specimen (KIB 20080810) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and Isolation. The air-dried and powdered aerial parts of I. tenuifolia (10 kg) were extracted with 70% aqueous Me2CO (40 L × 3) at room temperature and concentrated in vacuo to yield a residue, which was partitioned between H2O and EtOAc. The EtOAc extract (500 g) was chromatographed over a column containing MCI CHP 20P gel (90% CH3OH−H2O). The 90% CH3OH fraction (430 g) was subjected to silica gel (200−300 mesh) column chromatography (CC), eluting with a CHCl3−Me2CO gradient system (1:0, 9:1, 8:2, 7:3, 6:4, 1:1, 0:1), to afford fractions A−G. Fraction B (2.7 g) was chromatographed over silica gel (petroleum ether−Me2CO, 20:1−2:1 gradient) to give three subfractions, B1−B3. Fraction B2 (150 mg) was purified by RP-18 chromatography (80% CH3OH−H2O) to give 6 (6 mg) and 17 (13 mg). Fraction C (30 g) was subjected to CC on silica gel (200−300 mesh), eluted with CHCl3−Me2CO (50:1−2:1 gradient), to obtain five fractions, C1−C5. Compounds 1 (4 mg), 4 (3 mg), and 18 (25 mg) were precipitated from fraction C1. After repeated CC (silica gel, petroleum ether−Me2CO, 15:1−2:1 gradient), fraction C2 afforded 14 (2 mg). Fraction C3 was separated by RP-18 (50−100% MeOH− H2O), then by HPLC with 55% CH3CN−H2O, to obtain compounds 9 (3 mg), 10 (3 mg), 13 (11 mg), 16 (47 mg), and 20 (16 mg). Fraction D (29 g) was separated by passage over RP-18 (30−80% MeOH−H2O) into fractions D1−D6. After repeated CC (silica gel, CHCl3−Me2CO, 30:1−1:1), fraction D1 afforded 15 (4 mg). Fraction D2 was purified by HPLC with 45% CH3CN−H2O to yield 8 (7 mg), 11 (21 mg), and 12 (12 mg). Compound 3 (4 mg) crystallized from fraction D3, while compounds 5 (3 mg) and 7 (7 mg) were obtained from fraction D4 by silica gel CC, by elution with CHCl3−MeOH (20:1−2:1 gradient). Fraction E (46 g) was separated by RP-18 CC (30−80% MeOH− H2O) into fractions E1−E6. Compound 2 (6 mg) crystallized from fraction E3, and the mother liquor was passed through a silica gel column, eluted with CHCl3−MeOH, 15:1−2:1, to yield 19 (98 mg). Isoabietenin A (1): colorless crystals; [α]17D −72.6 (c 0.13, acetone); UV (MeOH) λmax (log ε) 287 (3.22), 207 (4.20) nm; IR (KBr) νmax 3406, 2958, 2871, 1483, 1460 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 339 (100) [M + Na]+; positive-ion HRESIMS m/z 339.1929 [M + Na]+ (calcd for C20H28O3Na, 339.1936). Isoabietenin B (2): white solid; [α]18D +94.8 (c 0.13, acetone); UV (MeOH) λmax (log ε) 240 (3.49) nm; IR (KBr) νmax 3460, 2921, 1653 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 359 (100) [M + Na]+; positive-ion HRESIMS m/z 359.2190 [M + Na]+ (calcd for C20H32O4Na, 359.2198). Isoabietenin C (3): white solid; [α]17D −18.5 (c 0.15, acetone); UV (MeOH) λmax (log ε) 289 (2.88), 216 (3.58) nm; IR (KBr) νmax 3497, 2937, 1736, 1692, 1249, 1234 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 531 (100) [M + Na]+; positive-ion HRESIMS m/z 531.2215 [M + Na]+ (calcd for C26H36O10Na, 531.2206). Tenuifolin A (4): white solid; [α]18D −70.8 (c 0.12, acetone); UV (MeOH) λmax (log ε) 237 (3.14), 205 (3.15) nm; IR (KBr) νmax 3433, 2931, 1639 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESIMS m/z 341 (100) [M + Na]+; positive-ion HRESIMS m/z 341.2100 [M + Na]+ (calcd for C20H30O3Na, 341.2092). Tenuifolin B (5): white solid; [α]15D −71.7 (c 0.16, acetone); UV (MeOH) λmax (log ε) 207 (3.50) nm; IR (KBr) νmax 3470, 3385, 3299, 2931, 2899, 2850, 1432 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 343 (100) [M + Na]+; positive-ion

HRESIMS m/z 343.2244 [M + Na]+ (calcd for C20H32O3Na, 343.2249). Tenuifolin C (6): white solid; [α]20D −14.1 (c 0.15, CHCl3−MeOH, 1:1); UV (MeOH) λmax (log ε) 204 (3.63) nm; IR (KBr) νmax 3575, 2937, 1728, 1257, 1243 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 499 (100) [M + Na]+; positive-ion HRESIMS m/z 499.2316 [M + Na]+ (calcd for C26H36O8Na, 499.2307). Tenuifolin D (7): white solid; [α]18D −9.9 (c 0.16, acetone); UV (MeOH) λmax (log ε) 204 (3.47) nm; IR (KBr) νmax 3441, 2936, 1738, 1251 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion FABMS m/z 437 (100) [M + H]+; positive-ion HRESIMS m/z 459.2369 [M + Na]+ (calcd for C24H36O7Na, 459.2358). Tenuifolin E (8): white solid; [α]14D −44.7 (c 0.13, acetone); UV (MeOH) λmax (log ε) 207 (3.51) nm; IR (KBr) νmax 3379, 2933, 1713, 1639, 1045 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positiveion FABMS m/z 335 (100) [M + H]+; positive-ion HRESIMS m/z 357.2051 [M + Na]+ (calcd for C20H30O4Na, 357.2041). Tenuifolin F (9): white solid; [α]17D −40.0 (c 0.14, acetone); UV (MeOH) λmax (log ε) 207 (3.52) nm; IR (KBr) νmax 3482, 3366, 2927, 2881, 1713, 1263 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive-ion ESIMS m/z 415 (100) [M + Na]+; positive-ion HRESIMS m/z 415.2094 [M + Na]+ (calcd for C22H32O6Na, 415.2096). Tenuifolin G (10): white solid; [α]15D −38.8 (c 0.15, acetone); UV (MeOH) λmax (log ε) 206 (3.53) nm; IR (KBr) νmax 3451, 2984, 2930, 2876, 1715, 1237 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive-ion ESIMS m/z 415 (100) [M + Na]+; positive-ion HRESIMS m/z 415.2097 [M + Na]+ (calcd for C22H32O6Na, 415.2096). Tenuifolin H (11): white solid; [α]18D +38.4 (c 0.15, acetone); UV (MeOH) λmax (log ε) 203 (3.44) nm; IR (KBr) νmax 3443, 2924, 1711, 1246 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive-ion ESIMS m/z 413 (100) [M + Na]+; positive-ion HRESIMS m/z 413.1947 [M + Na]+ (calcd for C22H30O6Na, 413.1940). Tenuifolin I (12): white solid; [α]18D −103.5 (c 0.15, acetone); UV (MeOH) λmax (log ε) 237 (3.57) nm; IR (KBr) νmax 3499, 3446, 2880, 1718, 1264 cm−1; 1H and 13C NMR data, see Tables 1 and 3; negativeion ESIMS m/z 425 (100) [M + Cl]−; negative-ion HRESIMS m/z 425.1734 [M + Cl]− (calcd for C22H30O6Na, 425.1730). Tenuifolin J (13): white solid; [α]17D −34.3 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 238 (3.36), 201 (3.32) nm; IR (KBr) νmax 3439, 2931, 1715, 1644, 1248 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive-ion ESIMS m/z 413 (100) [M + Na]+; positive-ion HRESIMS m/z 413.1950 [M + Na]+ (calcd for C22H30O6Na, 413.1940). Tenuifolin K (14): white solid; [α]17D −28.9 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 202 (3.23) nm; IR (KBr) νmax 3439, 1716, 1629, 1242 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive-ion ESIMS m/z 473 (100) [M + Na]+; positive-ion HRESIMS m/z 473.2157 [M + Na]+ (calcd for C24H34O8Na, 473.2151). Tenuifolin L (15): colorless crystals; [α]18D −37.8 (c 0.17, acetone); UV (MeOH) λmax (log ε) 205 (3.09) nm; IR (KBr) νmax 3481, 3375, 2956, 1714, 1253, 1076 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive-ion ESIMS m/z 461 (100) [M + Na]+; positive-ion HRESIMS m/z 461.2143 [M + Na]+ (calcd for C23H34O8Na, 461.2151). Tenuifolin M (16): colorless crystals; [α]16D −44.3 (c 0.12, acetone); UV (MeOH) λmax (log ε) 206 (2.98) nm; IR (KBr) νmax 3464, 3447, 2940, 1732, 1239 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive-ion ESIMS m/z 503 (100) [M + Na]+; positive-ion HRESIMS m/z 503.2247 [M + Na]+ (calcd for C25H36O9Na, 503.2257). X-ray Crystal Structure Analysis. The intensity data for isoabietenin A (1), tenuifolin L (15), and tenuifolin M (16) were collected on a Bruker APEX DUO diffractometer using graphitemonochromated Cu Kα radiation. The structures of these compounds were solved by direct methods (SHELXS-97),18 expanded using difference Fourier techniques, and refined by the program and fullmatrix least-squares calculations. The non-hydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions. Crystallographic data for the structures of isoabietenin A 262



Article

Determination of Cytotoxic Effects. The cytotoxicity of the test compounds was evaluated using a MTS assay.17 Briefly, RAW264.7 cells, 2 × 105 cells/well, were seeded in 96-well plates. After 24 h incubation, cells were treated with or without the test compounds at given concentrations for 18 h. Then, MTS was added to each well and the plates were kept for 4 h. Test compounds were dissolved in DMSO, and absorbance was read at 490 nm. Cytotoxicity was calculated by cell viability of cells without compounds as 100%.

(1), tenuifolin L (15), and tenuifolin M (16) have been deposited in the Cambridge Crystallographic Data Centre database (deposition numbers CCDC 907612, 907613, and 907614). Copies of the data can be obtained free of charge from the CCDC at www.ccdc.cam.ac.uk. Crystal data of isoabietenin A (1): C20H28O3, M = 316.42, monoclinic, a = 10.2652(3) Å, b = 8.4425(3) Å, c = 19.5390(6) Å, α = 90.00°, β = 96.6360(10)°, γ = 90.00°, V = 1681.98(9) Å3, T = 100(2) K, space group P21, Z = 4, μ(Cu Kα) = 0.649 mm−1, 12 894 reflections measured, 5434 independent reflections (Rint = 0.0460). The final R1 values were 0.0533 (I > 2σ(I)). The final wR(F2) values were 0.1531 (I > 2σ(I)). The final R1 values were 0.0537 (all data). The final wR(F2) values were 0.1542 (all data). The goodness of fit on F2 was 1.057. Flack14 parameter = 0.25(19). The Hooft parameter is 0.10(7) for 2134 Bijvoet pairs.19,20 Crystal data of tenuifolin L (15): C23H34O8, M = 438.50, monoclinic, a = 25.4016(6) Å, b = 6.6022(2) Å, c = 14.2850(3) Å, α = 90.00°, β = 116.2140(10)°, γ = 90.00°, V = 2149.29(9) Å3, T = 100(2) K, space group C2, Z = 4, μ(Cu Kα) = 0.842 mm−1, 15 718 reflections measured, 3696 independent reflections (Rint = 0.0736). The final R1 values were 0.0414 (I > 2σ(I)). The final wR(F2) values were 0.1059 (I > 2σ(I)). The final R1 values were 0.0415 (all data). The final wR(F2) values were 0.1063 (all data). The goodness of fit on F2 was 1.045. Flack14 parameter = 0.18(13). Crystal data of tenuifolin M (16): C25H36O9, M = 480.54, orthorhombic, a = 8.9493(2) Å, b = 15.1680(4) Å, c = 17.9800(4) Å, α = β = γ = 90.00°, V = 2440.66(10) Å3, T = 100(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.821 mm−1, 11 285 reflections measured, 4235 independent reflections (Rint = 0.0300). The final R1 values were 0.0342 (I > 2σ(I)). The final wR(F2) values were 0.0914 (I > 2σ(I)). The final R1 values were 0.0345 (all data). The final wR(F2) values were 0.0918 (all data). The goodness of fit on F2 was 1.077. Flack14 parameter = 0.00(13). Cytotoxicity Assays. The following human tumor cell lines were used: HL-60, SMMC-7721, A-549, MCF-7, and SW-480, which were obtained from ATCC (Manassas, VA, USA). All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA).17 Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before test compound addition, while suspended cells were seeded just before test compound addition, both with an initial density of 1 × 105 cells/mL in 100 μL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive control. After the incubation, MTT (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS−50% DMF after removal of 100 μL of medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench’s method.16 Nitric Oxide Production in RAW 264.7 Macrophages. Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 105 cells/well) containing RPMI 1640 medium (Hyclone) with 10% FBS under a humidified atmosphere of 5% CO2 at 37 °C. After 24 h preincubation, cells were treated with serial dilutions of the compounds at the maximum concentration of 25 μM in the presence of 1 μg/mL LPS for 18 h. Each compound was dissolved in DMSO and further diluted in medium to produce different concentrations. NO production in each well was assessed by adding 100 μL of Griess reagent (reagents A and B, respectively, Sigma) to 100 μL of each supernant from LPS (Sigma)-treated or LPS- and compound-treated cells in triplicate. After 5 min incubation, the absorbance was measured at 570 nm with a 2104 Envision multilabel plate reader (Perkin-Elmer Life Sciences, Inc., Boston, MA, USA). MG-132 was used as a positive control.21

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ASSOCIATED CONTENT

S Supporting Information *

This material (1H, 13C NMR, DEPT, HSQC, HMBC, NOESY, HRESIMS, UV, and IR spectra of compounds 1, 5, and 14; 1H, 13 C NMR, DEPT, and HRESIMS spectra of compounds 2−4, 6−13, 15, and 16; X-ray data of compounds 1, 15, and 16) is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (86) 871-5223616. Fax: (86) 871-5216343. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported financially by the NSFC-Joint Foundation of Yunnan Province (No. U0832602 to H.-D.S.), the National Natural Science Foundation of China (No. 81172939 to J.-X.P.), the Major State Basic Research Development Program of China (No. 2009CB522300), the Reservation-Talent Project of Yunnan Province (2011CI043 to J.-X.P.), and the Major Direction Projection Foundation of CAS Intellectual Innovation Project (No. KSCX2-EW-J-24 to J.-X.P.).

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REFERENCES

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