Five New Biphenanthrenes from Cremastra appendiculata

Five new biphenanthrenes, cremaphenanthrenes A–E (1–5), along with six known ones, were isolated from the ethanolic extract of the tubers of Cremastra appendiculata (D. Don) Makino (Orchidaceae). Their structures were elucidated on the basis of extensive spectroscopic analyses. All the compounds obtained were tested in vitro for cytotoxic activities against colon (HCT-116), cervix (Hela), and breast (MDA-MB-231) cancer cell lines. They all showed moderate or weak cytotoxicities to the above cancer cell lines.


Introduction
Phenanthrenes are a rather uncommon class of aromatic metabolites, which are presumably formed by oxidative coupling of the aromatic rings of stilbene precursors, and have been reported in higher plants, mainly in the Orchidaceae family [1]. The phenanthrenes isolated thus far may be classified into three major groups: monophenanthrenes, biphenanthrenes, and triphenanthrenes, and most natural phenanthrenes occur in monomeric form [1]. To date, less than one hundred biphenanthrenes and two triphenanthrenes have been isolated from the Orchidaceae family [2][3][4][5][6][7][8][9][10][11][12][13]. Biphenanthrenes have been reported to possess various biological activities including cytotoxicity, antimicrobial, spasmolytic, anti-inflammatory, antiplatelet aggregation, and antiallergic activities [1]. Currently, the cytotoxic activities of biphenanthrenes have attracted much interest, and biphenanthrenes may potentially be served as novel class of antitumor candidate [4].
The tuber of Cremastra appendiculata (D. Don) Makino (Orchidaceae) is one main source of "Shancigu", which is a famous traditional Chinese medicine with a long history for treating cancers [14]. About forty phenanthrenes, including ten biphenanthrenes, have been isolated from this title plant in previous phytochemical investigations [2,3,13,15,16]. The in vitro cytotoxic activities of some biphenanthrenes are better than the corresponding monomeric phenanthrenes [1,2]. In a previous article, eight new benzylphenanthrenes, and a few known compounds, were reported from this herb [13,15,16]. Our continuing investigations on the constituents of this plant have led to the isolation of five new biphenanthrenes, together with six known ones ( Figure 1). In this paper, we report the structural identification of five previously unreported biphenanthrenes, namely cremaphenanthrenes A-E (1)(2)(3)(4)(5), as well as their cytotoxic activities.

Results and Discussion
The tubers of C. appendiculata were extracted with 95% EtOH to yield a dark brown residue, which was suspended in distilled water and partitioned successively with petroleum ether (PE), ethyl acetate (EtOAc), and n-butyl alcohol (n-BuOH). The PE and EtOAc partitions were chromatographed over silica gel, Sephadex LH-20 and ODS columns, and semi-preparative HPLC to obtain eleven biphenanthrenes (1-11) (Figure 1). Their structures were elucidated based on extensive spectroscopic analyses (Supplementary Materials).
Compounds 6 and 7 (Tables 3 and 4, Figure 2) were obtained as brown amorphous powders. The molecular formula was deduced as C 30 H 24 O 6 according to their HR-ESI-MS. Their IR, UV, and 1D-NMR spectra were similar with those of 4 and 5, but their molecular weights were fourteen mass units less than those of 4 and 5. All of the above information indicated the character of biphenanthreneether of 6 and 7. Compared with 5, 6 had a hydroxyl located at C-7 instead of a methoxyl in the same position of 5, which was confirmed by the HMBC correlations from H-5 to C-7. Thus, the structure of 6 was elucidated as 2,7,7 -trihydroxy-4,4 -dimethoxy-9 ,10 -dihydro-1,2 -biphenanthreneether. It was a known compound with the name blestrin C [12]. In comparation with 4, 7 also possessed a hydroxyl located at C-7 instead of a methoxyl in the corresponding position of 4, the HMBC correlations from H-5 to C-7 further confirmed the deduction. Therefore, the structure of 7 was defined as 2,7,7 -trihydroxy-4,5 -dimethoxy-9 ,10 -dihydro-1,2 -biphenanthreneether, which was a known compound having the name blestrin D [12]. The assignments of 1 H-, 13 C-NMR data of blestrin C (6) and D (7) in the literature were partly different with ours [12]. In this paper, we assigned 1D NMR data of the two compounds based on the comprehensive analyses of 1 H-NMR, 13 C-NMR, HSQC, HMBC, and NOESY spectra.

Cytotoxicity Assay
The cytotoxicities of 1-11 were evaluated by the MTT method [2], using paclitaxel as a positive control. Their cytotoxicities against human colon (HCT-116), cervix (Hela), and breast (MDA-MB-231) cancer cell lines were determined. The results (Table 5) indicated that 1-11 showed moderate or weak cytotoxicities to the tested cancer cell lines. Among them, compounds 10 and 11 showed moderate cytotoxicities to all the above cancer cell lines with IC 50 values range of (12.13 ± 0.38)-(17.43 ± 3.07) µmol/L. They are all hexasubstituted phenanthrene-phenanthrene dimer with hydroxyl and methoxyl located at C-2, C-2 , C-4, C-4 , C-7, and C-7 . Compound 8 was a phenanthrene-9,10-dihydrophenanthrene dimer with the identical substitution pattern to 10. However, 8 only had weak cytotoxicities against the tested three cancer cell lines. Compounds 3 and 8 had the same phenanthrene and 9,10-dihydrophenanthrene moieties with the different linkage positions (1-1 or 3-1 connections, respectively), while the cytotoxicities of 3 were better than those of 8, which indicated that the linkage position of phenanthrene and 9,10-dihydrophenanthrene moieties affected the activity. All of the above results suggested that the cytotoxicities of biphenanthrenes might be associated with the following factors: the number of substituted hydroxyl and methoxyl, the hydroxyl and methoxyl location, the linkage position of two phenanthrene moieties, and the type of moiety (phenanthrene or 9,10-dihydrophenanthrene).

Plant Material
The tubers of C. appendiculata were collected in Yunnan province in June 2011. The plant materials were identified by one of the authors (Prof. P.F. Tu), and a voucher specimen (No. DJL20110628) was deposited at the Herbarium of Peking University Modern Research Center for Traditional Chinese Medicine.