Astataricusones A–D and Astataricusol A, Five New Anti-HBV Shionane-Type Triterpenes from Aster tataricus L. f.

Five new shionane-type triterpenes, astataricusones A–D (compounds 1–4) and astataricusol A (5), together with one known shionane-type triterpene 6 were obtained from the roots and rhizomes of Aster tataricus L. f. Their structures were elucidated on the basis of spectroscopic data, mainly NMR and MS data. The absolute configurations of 1 and 4 was determined by single crystal X-ray diffraction and CD analysis. Compound 2 showed inhibitory activity on HBsAg secretion with an IC50 value of 23.5 μM, while 2 and 6 showed inhibitory activities on HBeAg secretion with IC50 values of 18.6 and 40.5 μM, and cytotoxicity on HepG 2.2.15 cells with CC50 values of 172.4 and 137.7 μM, respectively. Compounds 2 and 6 also exhibited inhibitory activities on HBV DNA replication with IC50 values of 2.7 and 30.7 μM, respectively.


Introduction
Shionane-type triterpenoids possessing a unique all six-membered tetracyclic skeleton and 3-oxo-4-monomethyl moieties have been isolated only from Compositae plants [1]. The first studies on these compounds were done in the 1960s by Ourisson and Takahashi [2,3]. The stereostructures of rings A-D were determined by total synthesis in the 1970s by Ireland [4,5], but only six ones were reported from natural resources until now [6]. It has been reported that shionane-type triterpenes are the main constituents of Aster tataricus L. f. and have been shown to possess antitussive and expectorant activities [1,7,8]. In 2010 three new anti-HBV shionane-type triterpenes have been reported from A. tataricus by us [9]. As a continuation of our work on A. tataricus [9][10][11][12][13], we conducted further phytochemical studies on its roots and rhizomes. This has now led to the isolation of other five new shionane-type triterpenes, astataricusones A-D 1-4 and astataricusol A (5), and a known one, epishionol (6) (Figure 1). Herein we describe their isolation and structural elucidation, as well as their anti-HBV activity.
Compound 4 was obtained as a white powder. Its molecular formula was determined as C 30 H 52 O 3 by HREIMS at m/z 460.3922 [M] + (calcd. for 460.3916). Comparing the NMR data with 1 indicated that 4 had a different side chain. The structure of 4 was established as 21,22-dihydroxy-shion-3-one based on the 1D and 2D NMR data. The stereochemistry at C-21 were amenable to CD analysis with dimolybdenum tetracetate. Acyclic 1,2-diols can form a complex with Mo 2 (AcO) 4 and the complex produces a significant induced CD spectrum (ICD). According to the rule proposed by Snatzke [18,19], the diagnostic band at around 310 nm has the same sign of the O-C-C-O dihedral angle in the favored conformation in the 1,2-diol moiety. So assignment of the absolute configuration of the chiral centers in the 1,2-diol moiety could be given based on the sign of the band at arond 310 nm of the ICD spectrum. In particular an S-monosubstituted glycol gives rise to a positive Cotton effect at around 310 nm. Thus, the positive sign observed at 315 nm in the mixture ICD spectrum of 4 and Mo 2 (OAc) 4 ( Figure 4) allowed to assign the S-configuration to C-21 in 4. Therefore, the structure of 4 was established as (4R,5S,8S,9S,10S,13S,14R,17S,21S)-21,22-dihydroxy-shion-3-one, and it was named astataricusone D. Compound 5 was assigned the molecular formula C 30 H 52 O 2 by its HREIMS data at m/z = 444.3973 [M] + (calcd. 444.3967). Comparing the NMR data with 6 [14,15] and 1, 5 had the same cyclic skeleton as 6 and the same side chain as 1, which was supported by the HMBC correlations between H-21 (δ H 3.99, 1H, t, J = 6.2 Hz) and C-29 (δ C 17.3), C-20 (δ C 29.8), C-19 (δ C 38.9), C-30 (δ C 111.2), and H-30 (δ H 4.92, 1H, s) and C-21 (δ C 77.0). The configuration of 5 was established to be identical to 1 based on the similar ROESY correlations and 6 based on the similar CD data. Therefore, the structure of 5 was established as (3S,4R,5S,8S,9S,10S,13S,14R,17S,21S)-shion-22(30)-en-3,21-diol, and the compound was named astataricusol A.
Compounds 1-6 were tested for anti-HBV activity in HBV antigen secretion and DNA replication of HepG 2.2.15 cells using the literature methods [20][21][22]. Results indicated that 2 showed inhibitory activity on HBsAg secretion with an IC 50 value of 23.5 μM, while 2 and 6 showed inhibitory activities on HBeAg secretion with IC 50 values of 18.6 and 40.5 μM, and cytotoxicity on HepG 2.2.15 cells with CC 50 values of 172.4 and 137.7 μM, respectively; 2 and 6 exhibited inhibitory activities on HBV DNA replication with IC 50 values of 2.7 and 30.7 μM.

General
Melting points were determined on a TECH X-4 micro melting point apparatus without correction. Optical rotations were measured with a Horiba SEPA-300 polarimeter (Horiba, Kyoto, Japan). UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer (Shimadzu, Kyoto, Japan). IR spectra were obtained by a Tenor 27 spectrophotometer (Bruker, Karlsruhe, Germany) using KBr pellets. CD spectra were recorded with an Applied Photophysics Chirascan spectrometer (Applied Photophysics Ltd., London, UK). 1D and 2D NMR spectra were run on Bruker DRX-500 or AM-400 spectrometers (Bruker, Karlsruhe, Germany) with TMS as internal standard; coupling constants were expressed in Hertz and chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Mass spectra were recorded on a VG Autospec-3000 spectrometer (VG, Manchester, UK) or an API QSTAR Pulsar TOF spectrometer (AB-MDS Sciex, Concord, ON, Canada).

Plant Material
The roots and rhizomes of Aster tataricus L. f. was commercially purchased from the Yunnan Lv-Sheng Pharmaceutical Co. Ltd. (Kunming, China) and identified by Prof. Xi-Wen Li at Kunming Institute of Botany (voucher No. 200704).

In Vitro Anti-HBV Assay
The in vitro anti-HBV activity of compounds 1-6 were analyzed by the secretion of HBV antigen and DNA replication from the HepG 2.2.15 cell line. The toxicity of the compounds was evaluated by the sulforhodamine B method. DMSO alone was used as a solvent control. All the compounds were tested for their anti-HBV activity with the highest concentration of 20 g/mL. Lamivudine (3TC) was used as the positive control.

Cell Line and Cell Culture
The widely used HepG 2.2.15 cell line was applied for the assay of anti-HBV activity. In this study, the HepG 2.2.15 cell lines, which were stably transfected with the HBV genome, were a gift from Prof. Yong-Tang Zheng, Kunming Institute of Zoology, Chinese Academy of Sciences and cultured in RPMI−1640 (Gibco, Carlsbad, CA, USA) medium supplemented with 10% FBS (Haoyang Biological Manufacture Co., Ltd. Tianjin, China) and 400 μg/mL G148 (Calbiochem, San Diego, CA, USA). All cultures were maintained at 37 °C in a moist atmosphere containing 5% CO 2 .

Analysis of Secreted HBV Antigens
Inhibitory activity of compounds 1-6 on the secretion of HBV antigens in HepG 2.2.15 cells was evaluated with an ELISA method (Kehua Bio-engineering Co., Ltd, Shanghai, China). The procedures were performed according to that described in the previous literature [20,21] with some modifications. Cells were seeded in 96-well microplates at a density of 5 × 10 4 cells/mL and cultured at 37 °C, 5% CO 2 for 24 h. Different concentrations of compounds were added in the wells. The medium was replaced every 3 days with fresh medium and compounds. After cultured for 12 days, the supernatants were collected, and the levels of HBsAg and HBeAg in the supernatants were evaluated according to the manufacturer's instructions. The absorbance was measured at 450/630 nm using a microplate reader (SpectraMax 190, Molecular Devices, San Francisco, CA, USA).

Assay for HBV DNA Replication
Inhibitory activity of compounds 2 and 6 on HBV DNA replication in HepG 2.2.15 cells was examined by a fluorescence quantitative PCR kit (Shanghai Fuxing Bio-engineering Co., Ltd, Shanghai, China). After the cells were treated with or without the compounds for 12 days, the HBV DNA level in HepG2.2.15 cells was evaluated according to the manufacturer's instruction with a Real-Time PCR Systems (ABI7500, Applied Biosystems, Foster city, CA, USA).

Cytotoxicity Assay
Cytotoxicity of compounds 1-6 on HepG 2.2.15 cells was tested by a sulforhodamine B method [22] (SRB, Sigma, St. Louis, MO, USA). Firstly, HepG 2.2.15 cells were seeded in a 96-well microplate for 24 h. Compounds, dissolved in DMSO and diluted with the medium, were placed in each well and incubated for another 72 h at 37 °C. Then cells were fixed with 50% ice-cold trichloroacetic acid at 4 °C for 1 h and stained with 0.4% SRB in 1% acetic acid solution for 15 min. After removal of excessive dye, SRB was resuspended in 10 mM Tris buffer, and the absorbance was measured at 560 nm with the microplate reader above-mentioned.

X-ray Crystallographic Analysis
The intensity data for astataricusone A (1) was collected on a Bruker APEX DUO diffractometer using graphitemonochromated CuKα radiation. Its structure was solved by direct methods (SHELXS97), expanded using difference Founier techniques, and refined by the program and full-matrix least-squares calculations. The nonhydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions. Crystallographic data for the structure of 1 has been deposited in the Cambridge Crystallographic Data Centre (deposition number CCDC 926578). Copies of the data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk.

Determination of Absolute Configuration of the 21,22-Diol Group in 4
According to the procedure [18], the first CD spectrum (CD 1 ) of the 1:1.2 mixture of 4:Mo 2 (OAc) 4 at the concentration of 0.95 mg/mL in DMSO was recorded after mixed immediately. Then the second CD spectrum (CD 2 ) was recorded at 30 min after mixed. Finally the induced CD (ICD) of 4 was calculated by the formula: ICD = CD 2 − CD 1 .

Conclusions
Five new shionane-type triterpenes 1-5, named astataricusones A-D (compound 1-4) and astataricusol A (5), were isolated from Aster tataricus, together with a known one 6. According to previous investigations on Aster species, we have evaluated the inhibitory activities of all compounds against the secretion of HBV antigen and DNA replication from the HepG 2.2.15 cell line. Compounds 2 and 6 showed significant anti-HBV activity. The results prove the potential for the use of this plant as a herbal medicine in the treatment of HBV.