Phytoecdysteroids from the Roots of Achyranthes bidentata Blume

Two new phytoecdysteroids, (25S)-20,22-O-(R-ethylidene)inokosterone (1) and 20,22-O-(R-3-methoxycarbonyl)propylidene-20-hydroxyecdysone (2), together with six known phytoecdysteroids 3–8 were isolated from the roots of Achyranthes bidentata Blume. The new structures were established on the basis of spectroscopic studies and chemical evidences. The absolute configuration at C-25 in the structure of known compound 3 was determined by chemical and spectroscopic means.

Compound 3, showing the molecular formula C 28 H 44 O 7 , was deduced to be the same ecdysteroid compound inokosterone-20,22-acetonide from the roots of Leuzea carthamoides recently reported in the literature [22], by comparison of its spectroscopic data (Tables 1 and 2) with reported values.However, at that time the authors had yet not clarified the absolute configuration at C-25 in the structure.In order to determine the stereochemistry at C-25 in the structure of 3, a similar acid hydrolysis like that performed for 1 was conducted, and the free ecdysteroid that was released from 3 was confirmed to be (25S)-inokosterone (8) on the basis of a co-TLC elution test and NMR analyses, suggesting that the absolute configuration at C-25 in 3 should also be S configuration.Therefore, the complete structure of 3 was determined as (25S)-inokosterone-20,22-acetonide.
Among the eight isolated compounds, 1 and 2 are two new phytoecdysteroids, each characterized by having an acetal group in the molecule.In particular, 2 is so far the first example of phytoecdysteroid acetalated at the side chain with a 4-oxobutanoic acid unit.Compounds 3-5 were found in Achyranthes bidentata roots for the first time.

General
Optical rotations were measured on a Perkin-Elmer 341 polarimeter with MeOH as solvent.UV spectra were recorded in MeOH on a Perkin-Elmer Lambda 35 UV-Vis spectrophotometer.IR spectra (KBr) were taken on a Bruker Tensor 27 spectrophotometer in cm −1 .NMR spectra were recorded in C 5 D 5 N and CD 3 OD on a Bruker DRX-400 instrument using the residual solvent peak as reference.
ESIMS were collected on an MDS SCIEX API 2000 LC/MS/MS instrument.HRESIMS data were obtained on a Water Q-TOF Premier mass spectrometer and HREIMS data were obtained on a Finigan MAT 95XP mass spectrometer.Preparative HPLC was conducted using a CXTH P3000 HPLC pump and a UV3000 UV-Vis Detector with a Fuji-C18 column (10 µm-100 A).For column chromatography (CC), silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), YMC ODS-A (50 μm, YMC Co. Ltd., Kyoto, Japan) and Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden) were used.Fractions were monitored by TLC, and spots were visualized by heating the silica gel plates sprayed with 10% H 2 SO 4 in ethanol.

Plant Materials
Roots of A. bidentata Blume were purchased from Anguo Professional Market for Chinese Materia Medica, in April 2010, and were collected in Anguo County, Hebei Province, China.Plants were authenticated by Fu-Wu Xing (South China Botanical Garden, Chinese Academy of Sciences), and a voucher specimen (No. 20100408A) was deposited in the Laboratory of Phytochemistry of South China Botanical Garden, Chinese Academy of Sciences.