Three New Phytoecdysteroids Containing a Furan Ring from the Roots of Achyranthes bidentata Bl

Three new phytoecdysteroid compounds, named niuxixinsterone A (1), B (2) and C (3) with acetal functions in the side-chain were isolated from Achyranthes bidentata Bl. The structures were established as (20R,22R,24S)-20-O,22-O-(5′-hydroxymethyl)-furfurylidene-2β,3β,14α,25-tetrahydroxy-5β-ergost-7-en-6-one (1), (20R,22R)-20-O,22-O-(5′-hydroxymethyl)-furfurylidene-2β,3β,25-trihydroxy-14β-methyl-18-nor-5β-cholesta-7,12-dien-6-one (2) and (20R,22R,25R)-20-O,22-O-(5′-hydroxymethyl)-furfurylidene-2β, 3β,5β,14α,26-pentahydroxycholest-7-en-6-one (3) by means of spectroscopic evidence.


Introduction
Achyranthes bidentata Bl., a member of Amaranthaceae, is an erect perennial herbaceous plant widely distributed and grown in hilly districts of India, China, Japan and Java. The roots of A. bidentata named "Niuxi" in Chinese, is an important medicinal herbal and documented in the Chinese Pharmacopeia. It is usually prescribed by practitioners of Traditional Chinese Medicine (TCM) as a tonic, emmenagogue, antiarthritic, diuretic, and antifertility agent to nourish the liver and kidneys, strengthen bones and muscles, and invigorate circulation [1]. Modern pharmacological studies have
In this paper, we describe the isolation and structure elucidation of three new phytoecdysteroids with acetal functions [15] (Figure 1) isolated from the EtOH extracts of A. bidentata. In previous experiments, we discovered ecdysterone, inokosterone and serfurosterone A [16]. The three new phytoecdysteroids had a similar structure to serfurosterone A, and we presumed they might have similar pharmacological activity. The structure determination of the phytoecdysteroids from A. bidentata could establish a basis for further pharmacological experiments. Here, we describe the isolation and structure elucidation of three new phytoecdysteroid compounds on the basis of the spectroscopic analysis, including 1D, 2D-NMR techniques and HRESIMS.

Results and Discussion
Compound 1 was obtained as a white amorphous powder and had a [M+Na] + ion peak at m/z 625.3359 in the HRESIMS, corresponding to a molecular formula of C 34 H 50 O 9 . The UV spectrum was consistent with presence of a 7-en-6-one chromophore in an ecdysteroid, with a maximum value at 248 nm. By analyzing the 1 H-and 13 C-NMR spectroscopic data, it was determined that 1 was an ecdysone analog with a furan ring-containing substituent.
Compound 2, a white amorphous powder, was assigned a molecular formula of C 33 H 46 O 8 by HRESIMS, which exhibited a [M+Na] + ion peak at m/z 593.3091. The UV spectrum was consistent with presence of the ecdysteroid chromophore as above. Extensive NMR analysis of the parent nucleus indicated that compounds 2 and 1 were structurally different in terms of the presence of additional one olefin signal and the loss of one oxyquaternary carbon signal.
Compound 3, a white amorphous powder, was established by HRESIMS (m/z 627.3150 [M+Na] + ) as C 33 H 48 O 10 with a maximal UV absorption at 247 nm. Comparison of the 13 C-NMR spectroscopic data of the tetracyclic ring system between 3 and 1 indicated that the major difference was a methine at δ C 51.4 (C-5) in 1 being replaced by an oxyquaternary at δ C 80.0 in 3. The HMBC correlations between H-7 and H 3 -19 with the signal at δ C 80.0, respectively, which established the presence of an OH substituent at C-5. The 5β-OH configuration was indicated by the upfield resonance for the CH 3 -19 at δ C 17.1 [20].
In compound 3, three methyl singlets at δ H 1.12, 0.99 and 1.38 were reasonably attributed to the methyl groups at C-18, C-19 and C-21 by a HMBC experiment. Additionally, the methyl doublet δ H 1.04 is correlated in HMBC to C-23, C-24 and C-26, which is only in agreement with the presence of this methyl group at C-25. Comparison of 13 C-NMR chemical shifts of C-23 ～ 27 in 3 with palythoalone B [17] and 25R-inokosterone [21] indicated a 25R configuration.
Many plants have been found to be rich in ecdysteroids [22]. A multiplicity of ecdysteroids have been isolated from members of the Amaranthaceae [23][24][25][26][27]. The common structural features are characteristic of the ecdysteroids: ∆7-6-keto grouping in ring B; cis-junction of A/B rings; hydroxyl groups in positions 1, 2, 3, 5, 11, and 14 of the steroidal core; and the side chain usually containing an (R)-C22-group. In most cases, phytoecdysteroids are isolated in a free state, although many their derivatives (ethers, esters, and glycosides) have also been found.
However, the distribution of ecdysteroids with a furan ring in the side-chain in natural sources is very limited. They have been found so far only in the plant Serratula wolffii [16]. The new isolates 1-3 constitute a series of ecdysteroids containing a furan ring in the side-chain, all of them with an acetal group at C-1′, which have not been reported previously from A. bidentata. It is difficult to decide whether the three ecdysteroids are originally present in the plant or if it is artifacts formed during root drying, but we consider that these compounds are genuine compounds as they can be characterized by their UPLC-MS retention time and fragmentation pattern in ethanol extracts of fresh root. It is well know that 20,22-condensation reaction can take place under certain conditions [28], so compounds 1-3 are new endogenous compounds.

General
The melting points (uncorrected) were measured on a Kofler micromelting point apparatus. Optical rotations were measured with a PE-241 digital polarimeter. IR spectra were recorded on an IR-47 spectrometer. The NMR spectra were recorded on Bruker DPX 400 (400 MHz for 1 H-NMR and 100 MHz for 13 C-NMR), respectively. Chemical shifts are given as δ values with reference to tetramethylsilane (TMS) as an internal standard, and coupling constants are given in Hz. The HRESIMS analyses were conducted on IonSpec Ultima 7.0T FTICR. Preparative HPLC (Waters, Delta 600-2487) was performed on Pegasil ODS II (5 μm, 10 × 250 mm, Senshu Pak, Japan). Macroporous absorption resin (D101 Crosslinked Polystyrene, Nan Kai, Tian Jin, China) was employed for column chromatography. Silica gel (100-200 mesh) for column chromatography and silica gel H for TLC were obtained from Qingdao Marine Chemical Factory, Qingdao, Shandong Province, China. ODS-A (120 A, 50 μm) was obtained from YMC Co.

Plant Material
The