Scholarly article on topic 'ent-Atisane and ent-kaurane diterpenoids from Isodon rosthornii'

ent-Atisane and ent-kaurane diterpenoids from Isodon rosthornii Academic research paper on "Chemical sciences"

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{"Isorosthornins D-G" / Diterpenoids / " Isodon rosthornii "}

Abstract of research paper on Chemical sciences, author of scientific article — Rui Zhan, Xiao-Nian Li, Xue Du, Wei-Guang Wang, Ke Dong, et al.

Abstract A new ent-atisanoid (1) and three new ent-kauranoids (2-4) belonging to different types, along with four known compounds were isolated from Isodon rosthornii. Their structures were established by means of extensive spectroscopic analysis. The absolute configuration of 1 was further determined by X-ray diffraction. Compounds 1 and 5 are the first example of atisane-type diterpenoid from this plant.

Academic research paper on topic "ent-Atisane and ent-kaurane diterpenoids from Isodon rosthornii"

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Fitoterapia

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ent-Atisane and ent-kaurane diterpenoids from Isodon rosthornii☆

Rui Zhan a,b, Xiao-Nian Lia, Xue Du a, Wei-Guang Wang a,b, Ke Dong a, Jia Su a, Yan Lia, Jian-Xin Pu a'*, Han-Dong Sun a'*

a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, Yunnan, PR China

b University of the Chinese Academy of Sciences, Beijing 100039, PR China

ARTICLE INFO ABSTRACT

Article history: A new ent-atisanoid (1) and three new ent-kauranoids (2-4) belonging to different types, Received 7 February 2013 along with four known compounds were isolated from Isodon rosthornii. Their structures Accepted in revised form 14 April 2013 were established by means of extensive spectroscopic analysis. The absolute configuration of Available online 6 May 2013 1 was further determined by X-ray diffraction. Compounds 1 and 5 are the first example of --atisane-type diterpenoid from this plant.

Keywords: © 2013 The Authors. Published by Elsevier B.V. All rights reserved. Isorosthornins D-G Diterpenoids Isodon rosthornii

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1. Introduction

Isodon is a cosmopolitan and important genus of Lamiaceae family. The use of Isodon species in Chinese folk medicine has a long tradition. Investigations proved Isodon species to be a pool of diverse structures of diterpenoids with a range of bioac-tivities, most of which were ent-kauranoids [1]. The discovery of a series of compounds with promising use in anti-inflammatory and anti-tumors, such as eriocalyxin B [2,3], pharicin B [4], oridonin [5], ponicidin [6], and adenanthin [7] further supported their folk use and brought great attention.

Isodon rosthornii, used to treat rheumatism and sore throat in Chinese folk therapy, was distributed in Sichuan and Guizhou provinces of China [8]. Previous studies on this species just led to the isolation of seven ent-kauranoids, some of which showed anti-bacteria activity [8-13]. For the secondary metabolites of the genus Isodon often differ when

☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Corresponding authors. Tel.: +86871 65223251; fax: +86 871 65216343.

E-mail addresses: pujianxin@mail.kib.ac.cn (J.-X. Pu), hdsun@mail.kib.ac.cn (H.-D. Sun).

grows in different ecological environments, in continuation of our effort to find bioactive substances, we explored the plant indigenous to Qionglai City of Sichuan Province, which had even not been studied on the secondary metabolites. From the EtOAc section, two ent-atisanoids, including a new one, isorosthornin D (1), and six ent-kauranoids including a new 11,20-epoxy-ent-kauranoid (2), a new 3,20-epoxy-ent-kauranoid (3) and a new enmein-type diterpenoid (4) were isolated (Fig. 1). The absolute configuration of 1 were further confirmed by X-ray diffraction study. In this paper, we reported the isolation, stucture elucidation and the cytotoxic evaluation.

2. Experimental

2.1. 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 and DRX-500 spectrometers with TMS as internal standard. Unless otherwise specified, chemical shifts (6) were expressed in ppm with reference to the solvent signals. HRESIMS was

0367-326X/$ - see front matter © 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/! 0.1016/j.fitote.2013.04.009

Fig. 1. The structures of compounds 1-8.

performed on an API QSTAR time-of-flight spectrometer. HREIMS was performed on a Waters Auto Spec Premier P776 spectrometer. HSCCC was performed on TBE-300B. Semipreparative HPLC was performed on an Agilent 1100 HPLC with a Zorbax SB-C18 (9.4 mm x 25 cm) column. Column chromatography was performed with silica gel (200-300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, China), Lichroprep RP-18 gel (40-63 |jm, Merck, Darmstadt, Germany) and MCI gel (75-150 |jm, Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC, and spots were visualized by heating Si gel plates sprayed with 5% H2SO4 in EtOH.

2.2. Plant material

The aerial parts of I. rosthornii were collected Qionglai City of Sichuan Province, China, in July 2008, and was identified by Prof. Xi-Wen Li, Kunming Institute of Botany. A voucher specimen (KIB 200809003) has been deposited in the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences.

2.3. Extraction and Isolation

The aerial parts (10 kg) of I. rosthornii were shade dried, powdered, and extracted with the 70% aqueous acetone (20 L) for four times (two days for each time) at room temperature. The resulting extract was evaporated to be concentrated. Then the concentrate without acetone (7 L) was partitioned by EtOAc. The EtOAc soluble portion (485 g) was subjected to column chromatography on a silica gel column (2000 g, 100200 mesh) and eluting with step wise gradient of CHCl3-Me2CO (1:0, 9:1, 8:2, 7:3, 6:4, 1:1) to afford five major fractions (Fi-F5). F2-F4 were first subjected to column chromatography on MCI gel respectively, eluted with CH3OH:H2O 90:10. After that, fraction 2 was purified by repeat flash chromatography on silica gel (200-300 mesh, eluted with petroleum ether-acetone 30:1) to afford compound 6 (10 mg).

Fraction 3 (63 g) was separated by RP-18 gel column chromatography (eluted with CH3OH:H2O 30:70-80:20 gradient) to yield four fractions (C1-C4). C1 was subjected to HSCCC (CHCl3:CH3OH:H2O 4:3:2) and then was rechro-matographed by silica gel, eluted with CHCl3:acetone 30:1 to give compound 1 (25 mg). C2 was purified by HSCCC (CHCl3:MeOH:H2O 4:3:2) and then was separated by semi-preparative HPLC with CH3OH:H2O 50:50 to afford compounds 2 (2 mg), 5 (2 mg), and 8 (5 mg).

Fraction 4 (85 g) was first chromatographed by Rp-18 gel column chromatography with elution of CH3OH:H2O (30:7080:20 gradient) and then was subjected to HSCCC (CHCl3: CH3OH:H2O 4:3:2). After that, silica gel column (eluted with CHCl3:acetone 30:1) and semipreparative HPLC with elution of CH3OH:H2O (50:50) were used to yield compounds 3 (2 mg), 4 (120 mg), and 7 (3 mg).

2.4. Spectroscopic data

Isorosthornin D (1), colorless crystals. [a]21 D = — 24.7 (c = 0.68, MeOH). UV (MeOH): 203 (3.22) nm. IR (KBr): 3427, 2928, 2851,1694,1679,1089,1071,1031 cm-1. NMR: see Tables 1 and 2. HR-ESI-MS: 357.2031 ([M + Na]+, C20H30O4Na; calc. 357.2041).

Isorosthornin E (2), colorless oil. [a]186 D = +41.4 (c = 1.6, MeOH). UV (MeOH): 204 (3.39), 289 (2.84) nm. IR (KBr): 3424, 2924, 1631, 1432, 1032 cm-1. NMR: see Tables 1 and 2. HR-EI-MS: 378.1663 ([M]+, C20H26O7; calc. 378.1679).

Isorosthornin F (3), white powder. [a]263 D = — 97.5 (c = 0.51, MeOH). UV (MeOH): 204 (3.39) nm. IR (KBr): 3421, 2931, 1705, 1663, 1439, 1084, 1048 cm-1. NMR: see

Table 1

13C NMR data of compounds 1-4 (in C5D5N, 6 in ppm, J in Hz)a.

No. 1 2 3 4

1 27.7 (t) 80.0 (d) 27.5 (t) 76.4 (d)

2 37.5 (t) 30.4 (t) 22.5 (t) 24.3 (t)

3 77.7 (d) 41.9 (t) 76.3 (d) 37.6 (t)

4 39.3 (s) 35.6 (s) 38.2 (s) 31.2 (s)

5 52.1 (d) 53.6 (d) 58.3 (d) 55.0 (d)

6 38.5 (t) 75.6 (d) 73.0 (d) 102.0 (d)

7 212.8 (s) 208.9 (s) 210.5 (s) 173.2 (s)

8 59.8 (s) 63.5 (s) 63.8 (s) 59.1 (s)

9 44.3 (d) 51.9 (d) 54.4 (d) 40.9 (d)

10 37.0 (s) 60.2 (s) 54.3 (s) 50.9 (s)

11 28.7 (t) 75.1 (d) 64.3 (d) 19.0 (t)

12 37.3 (d) 39.1 (t) 40.1 (t) 32.7 (t)

13 38.1 (t) 46.7 (d) 48.5 (d) 46.5 (d)

14 66.1 (d) 75.5 (d) 76.8 (d) 74.4 (d)

15 67.1 (d) 73.1 (d) 74.1 (d) 75.5 (d)

16 155.8 (s) 159.0 (s) 156.6 (s) 159.7 (s)

17 108.8 (t) 110.3 (t) 108.0 (t) 109.1 (t)

18 27.8 (q) 36.3 (q) 29.6 (q) 33.1 (q)

19 15.7 (q) 23.2 (q) 24.2 (q) 23.3 (q)

20 14.7 (q) 177.6 (s) 99.6 (d) 73.3 (t)

OMe 54.3

a NMR data of compounds 1, 4 were recorded at 100 MHz; data for compounds 2 and 3 were recorded at 150 MHz. Assignments were made based on DEPT, HSQC, COSY, HMBC, and ROESY experiments.

Tables 1 and 2. HR-EI-MS: 394.2000 ([M]+, C21H30O7; calc. 394.1992).

Isorosthornin G (4), white powder. [a]26.3 D = — 75.3 (c = 1.00, MeOH).UV(MeOH): 202 (3.15), 254 (2.37) nm. IR (l<Br): 3417, 2948,1716,1060 cm-1. NMR: see Tables 1 and 2. HR-EI-MS: 364.1891 ([M]+, C20H28O6; calc. 364.1886).

2.5. The cytotoxicity assay

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 the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (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) [14]. Briefly, 100 |jL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition, both with an initial density of 1 x 105 cells/mL in 100 ^L 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 controls. After the incubation, MTT (100 |ag) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 |iL of 20% SDS-50% DMF after removal of 100 |jL 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 the Reed and Muench's method [15].

2.6. X-ray crystal structure analysis

Colorless crystals of 1 were obtained in CH3OH. Intensity data were collected at 100 K on a Bruker APEX DUO diffrac-tometer equipped with an APEX II CCD, using Cu Ka radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS-97 [16]. Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The H-atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystallographic data (excluding structure factor tables) for the structures reported have been deposited with the Cambridge Crystallographic Data Center as supplementary publications no. CCDC 923358 for 1. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44(0) (1223) 336 033); e-mail: deposit@ ccdc.cam.ac.uk].

Crystallographic data for compound (1): C20H30O4-H2O, M = 352.46, orthorhombic, a = 7.19610(10) A, b = 11.6805(2) A, c = 21.1935(3) A; a = 90.00°, j3 = 90.00°, Y = 90.00°, V = 1781.40(5) A3, T = 100(2) K, space group P212121, Z = 4, 8453 reflections measured, 3070 independent reflections (Rint = 0.0330). The final R1 values were 0.0345 (I > 2o(I)). The final wR(F2) values were 0.0901 (I > 2o(I)). The final Rj values were 0.0345 (all data). The final wR(F2) values were 0.0901 (all data). Flack parameter = — 0.01(14).

3. Results and discussion

The EtOAc extract was subjected to repeated column chromatography to afford eight diterpenoids, including four new ones which were named as isorosthornins D-G (Fig. 1).

Table 2

'H NMR spectroscopic data (in C5D5N, 6 in ppm, J in Hz) of compounds 1-4a.

No. 1 2 3 4

1a 1.81 (m) 4.10 (dd, 10.5, 3.5) 2.52 (overlapped) 4.85 (overlapped)

1b 1.11 (m)

2a 1.55 (m) 2.43 (m) 1.97 (m) 1.84 (m)

2b 0.98 (overlapped) 1.84 (overlapped) 1.80 (dd, 13.7,11.5)

3a 3.77 (dd, 11.0, 4.8) 1.59 (m) 3.40 (m) 1.32 (m)

3b 1.33 (overlapped) 1.25 (m)

5 1.53 (m) 1.84 (overlapped) 1.66 (d, 12.8) 2.26 (s)

6 2.63 (br s) 5.19 (br s) 4.90 (d, 12.8) 5.79 (s)

9 2.53 (m) 4.15 (d, 5.5) 2.80 (br s) 4.02 (dd, 13.0, 6.6)

11a 1.73 (m) 6.04(dd, 7.8, 5.5) 4.87 (overlapped) 2.22 (m)

11b 1.45 (m) 1.65 (m)

12a 2.55 (m) 2.73 (m) 2.54 (overlapped) 2.44 (m)

12b 2.08 (dd, 15.0, 7.8) 2.34 (d, 10.4) 1.86 (overlapped)

13a 2.30 (ddd, 13.0, 9.0, 3.8) 3.04 (d, 7.8) 3.13 (br s) 2.06 (d, 8.0)

13b 1.85 (overlapped)

14 4.79 (dd,9.0, 3.8) 5.14 (br s) 4.84 (br s) 4.87 (overlapped)

15 5.77 (s) 6.51 (s) 6.46 (d, 9.7) 6.39 (s)

17a 5.52 (s) 5.70 (s) 5.66 (s) 5.67 (s)

17b 5.23 (s) 5.32 (s) 5.41 (s) 5.25 (s)

18 1.04 (s) 1.34 (s) 1.12 (s) 1.06 (s)

19 0.97 (s) 1.90 (s) 1.61 (s) 1.02 (s)

20a 1.11 (s) 5.66 (overlapped) 4.77 (d, 8.0)

20b 4.28 (d, 8.0)

OMe 3.46 (s)

a NMR data for compounds 1 and 4 were recorded at 500 MHz, and data of compounds 2 and 3 were recorded at 600 MHz; Assignments were made based on DEPT, HSQC, COSY, HMBC, and ROESY experiments.

The structures of the known compounds were determined by comparing spectroscopic data with literature values and were identified as isorubesin E (5) [17], jianshirubesin D (6) [18], rubescensin W (7) [19], and jianshirubesin E (8) [18].

Compound 1 was obtained as colorless crystals. Its molecular formula was established as C20H30O4 by HR-ESI-MS data (m/z 357.2031 [M + Na]+,calcd. 357.2041), indicating of six degrees of unsaturation. Absorption bands at 3427, 1694, and 1679 cm-1 in the IR spectrum accounted for the presence of OH, C=O, and C=CH2 groups. In the 13C NMR and DEPT spectra, 20 carbon signals were observed: including three methyls, six methylenes (including an olefinic one), six methines (including three oxygenated ones), and five quaternary carbons (including a carbonyl one and an olefinic one). Considering main diterpenoids, ent-kauranoids from Isodon species, compound 1 was first assumed to be a 20-non-oxygenated-ent-kauranoid, while HMBC correlations observed from H-9 (6H 2.53) and H2-17 (6H 5.52 and 5.23) to C-12 (6C 37.3) suggested it a rearranged 16(13 ^ 12)-abeo-ent-kaurane skeleton (ent-atisane skeleton) [20]. With the 1H-1H COSY, HSQC and HMBC spectra, the planar structure of 1 was elucidated. The HMBC correlations from H3-18 (6H 1.04) to C-3 (6C 77.7), from H2-17 to C-15 (6C 67.1), H-9 and H-15 (6H 5.77) to C-14 (6C 66.1) proved the linkage of OH groups to C-3, C-14 and C-15, respectively. The HMBC correlations from H-5 (6H 1.53) and H-9 to C-7 (6C 212.8) permitted the location of the carbonyl carbon at C-7 (Fig. 2). Therefore, 1 was presumed to be 3,14,15-trihydroxy-ent-atis-16-en-7-one.

The ROESY correlations helped assign the relative configuration of 1. Correlations of H-3/H-1j3/H-5j3, H-14/H3-20/ H-13a and H-15/H-13j3 confirmed H-3, H-14 and H-15 to be j3, a, and a-oriented, respectively (Fig. 2). To determine the absolute configuration of 1, a single crystal of 1 was analyzed by X-ray crystallography. Bearing on four oxygen atoms in the molecular, the final refinement on Cu Ka data resulted in a Flack parameter of - 0.01 (14), allowing an unambiguous assignment of the complete absolute configuration of 1 as shown in its formula (Fig. 3) [21]. All chiral centers, C-3, C-5, C-8, C-9, C-10, C-12, C-14 and C-15 were determined as R, S, S, R, S, R, R and R, respectively. Therefore, compound 1 was determined to be 3a,14j3,15j3-trihydroxy-ent-atis-16-en-7-one, named as isorothornin D (1).

Fig. 3. X-ray crystal structure of 1.

The molecular formula of 2 was determined as C20H26O7 on the basis of the HREIMS data. Analysis of the spectroscopic data of 2 revealed it a 11,20-epoxy-ent-kauranoid, with extreme similarity to 6 [18]. The only difference was an additional OH substituted at C-14 in 2 which was deduced by the HMBC correlations from H-14 (ÔH 5.14) to C-9 (ÔC 51.9), C-12 (ÔC 39.1), and C-16 (ÔC 159.0). The HMBC correlations from H2-3 (ÔH 1.59 and 1.33), H-5 (ÔH 1.84), and H-9 (ÔH4.15) toC-1 (ÔC80.0), from H-6 (ÔH 5.19) to C-5 (ÔC 53.6) and C-8 (ÔC 63.5), and from H-15 (ôH 6.51) to C-16 and C-17 (ôC 110.3) confirmed the connections of other three OH groups to C-1, C-6, and C-15, respectively, the same as those in 6. The HMBC correlations from H-5, H-9, and H-15to the ketone carbonyl (ÔC 208.9), from H-1 (ÔH 4.10), H-5, and H-9 to the lactone carbonyl (ôC 177.6) permitted the location of the ketone and lactone carbonyls at C-7 and C-20, respectively.

The ROESY experiment verified that the relative configuration of H-1, H-6, H-11, and H-15 of compound 2 were j3-, a-, j3-, a-oriented, respectively, identical to compound 6 (Fig. 4) [18]. The correlations ofH-14/H-12a verified H-14 to be a-oriented. Thus, compound 2 was elucidated

ROESY: H r ^ H

Fig. 4. Key ROESY correlations of 2.

as 1a,6j3,14j3,15j3-tetrahydroxy-11a,20-olide-ent-kaur-16-en-7-one, named as isorothornin E (2).

The HREIMS of compound 3 suggested the molecular formula of C21H30O7, indicative of seven degrees of unsatu-ration.The absorption bands at 3421,1705, and 1663 cm-1 in its IR spectrum revealed the existence of OH, C=O, and C=C groups. In its 13C NMRand DEPT spectra (Table 1), 21 carbon signals were observed, which were assigned to three methyls (including an oxygenated one), four methylenes (including an olefinic one), nine methines (including six oxygenated ones), and five quaternary carbons (including a carbonyl one and an olefinic one). These data suggested 3 to be a 20-oxygenated ent-kauranoid which was further proved by the 2D NMR spectra. The HMBC correlations from H-3 (SH 3.40) to C-20 (6C 99.6) confirmed 3 to be a 3,20-epoxy-ent-kauranoid; correlations from OMe (SH 3.46) to C-20 permitted the connection of OMe to C-20 directly; correlations from H-6 (SH 4.90) to C-4 (SC 38.2) and C-5 (SC 58.3), from H-11 (SH 4.87) to C-13 (6C 48.5) and C-8 (6C 63.8), from H-14 (SH 4.84) to C-9 (6C 54.4) and C-16 (6C 156.6), and from H2-17 (Sh 5.66 and 5.41) to C-15 (SC 74.1) verified the linkage of OH groups to C-6, C-11, C-14 and C-15, respectively; correlations from H-5 (SH 1.66) and H-15 (SH 6.46) to C-7 (6C 210.5) permitted the location of the carbonyl carbon at C-7 (Fig. 5). Consequently, compound 3 was assigned as 6,11,14,15-tetrahydroxy-20-methoxy-3,20-epoxy-ent-kaur-16-en-7-one.

The relative configuration of 3 was derived by the ROESY spectrum. The correlations of H-3/H-13/H-5), H-6/H3-19a/ H-20, H-11/H-12a/H-OMe, H-14/H-20, and H-15/H-13a confirmed H-3, H-6, H-11, H-14 and H-15 to be )-, a-, a-, a-, and a-oriented, respectively. The R* configuration of C-20 was determined by the correlations from H-20 to H3-19a and H-2a. As a result, 3 was characterized as 20(R*)-63,113,14), 15)3- tetrahydroxy- 20-methoxy-3a,20- epoxy- ent-kaur-16-en-7-one, named as isorothornin F (3).

Compound 4 was obtained as white amorphous powders. The HREIMS of 4 exhibited a [M]+ peak at 364.1891, which suggested the molecular formula of C20H28O6, indicating of seven degrees of unsaturation. Its IR spectrum had absorption bands at 3417, 1716 and 1060 cm-1, accounting for the presence of OH, C=O, and C=C groups. In its 13C NMR and DEPT spectra (Table 1), 20 carbon signals were observed, which were assigned to two methyls, six methylenes (including an olefinic one and an oxygenated one), seven methines (including four oxygenated ones), and five quaternary carbons (including a carbonyl one and an olefinic). These data indicated that 4 was a 6,7-seco-1,7-olide-ent-kauranoid, similar to epinodosinol [22,23]. The only difference verified by the 1H-1H COSY, HSQC, and HMBC spectra was the connection of OH to C-14 instead of the one substituted at C-11 in epinodosinol. The ROESY spectrum supported the same relative configurations of H-1, H-6 and H-15 with epinodosinol. The configuration of H-14 was determined to be 3-oriented by the ROESY correlation of H-14/H-13). Hence, 4 was elucidated as 63,14a,15a-trihydroxy-6,7-seco-6,20-epoxy-1a,7-olide-ent-kaur-16-ene, named as isorothornin G (4).

To the best of our knowledge, compounds 1 and 5 are the first example of atisane-type diterpenoids from this plant. Furthermore, those sub-types of ent-kauranoids, 11,20-epoxy-ent-kauranoid (2 and 6-8), 3,20-epoxy-ent-kauranoid (3) and enmein-type ent-kauranoid (4) were also isolated from Isodon rosthornii for the first time.

Considering the folk use of the Isodon genus, these isolates except 2 and 3 were tested for in vitro cytotoxicity against A-549, HL-60, MCF-7, SMMC-7721, and SW-480 human cancer cell lines using the MTT method [14]. Compound 6 showed significant inhibitory activity against HL-60 cell line (Table 3).

Acknowledgment

This project was supported financially by the National Natural Science Foundation of China (No. 81172939 to J.-X. Pu), the Major State Basic Research Development Program of China (No. 2009CB522300), the reservation-talent project of Yunnan Province (2011CI043 to J.-X. Pu), the Science and

Table 3

IC50 values (|iM) of diterpenoids from I. rosthornii for human tumor cell lines.

'H-'H COSY: H■

Compounda HL-60 SMMC-7721 A-549 MCF-7 SW480

6 5.00 >40 30.11 16.22 22.62

DDPb 2.0 16.2 17.5 17.8 12.8

Paclitaxelb <0.008 < 0.008 1.36 < 0.008 0.04

■H HMBC : H'

Fig. 5.1H-1H COSY, selected HMBC of 3.

a Other selected ones not listed in the table were inactive (IC50 > 40 |iM) for all cell lines.

DDP (cisplatin) and paclitaxel were used as positive controls.

Technology Program of Yunan Province (nos. 2008IF010 and 2008CD162), and the Independent Research Program of the Chinese Academy of Sciences (No. KSCX2-EW-J-24 to J.-X. Pu).

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