Isolation and Cytotoxicity Evaluation of the Chemical Constituents from Cephalantheropsis gracilis

Cephalantheropsis gracilis afforded five new compounds: cephalanthrin-A (1), cephalanthrin-B (2), cephathrene-A (3), cephathrene-B (4), methyl 2-(aminocarbonyl)phenylcarbamate (5), and 52 known compounds. The structures of the new compounds were determined by spectroscopic analysis. Among the compounds isolated, tryptanthrin (6), phaitanthrin A (7), cephalinone D (19), and flavanthrin (30) showed significant cytotoxicity against MCF-7, NCI-H460, and SF-268 cell lines.


Introduction
The genus Cephalantheropsis (also known as Cephalanceropsis) belongs to the Orchidaceae family and is comprised of eight species distributed in Southeast Asia. The plant, Cephalantheropsis gracilis (Lindl.) Shiu-Ying Hu var. gracilis, is an orchid native to Taiwan and grows in forests at altitudes of 500-1500 m throughout the island [1]. The crude methanol extract of C. gracilis showed significant cytotoxicity against human breast carcinoma (MCF-7), lung carcinoma (NCI-H460), and central nervous system carcinoma (SF-268) cell lines in our preliminary screening. In earlier papers, the isolation of indole alkaloids was reported from C. gracilis, but they are unlikely to be responsible for such anticancer activity [2,3]. In the course of continuing the search for bioactive molecules from C. gracilis, two new quinazolines, cephalanthrin-A (1) and cephalanthrin-B (2), two new dihydrophenanthrenes, cephathrene-A (3) and cephathrene-B (4), and a methyl 2-(aminocarbonyl)phenylcarbamate (5) [4] (Figure 1) as well as 52 known compounds were obtained and identified from a methanol extract (in addition to common long-chain fatty acids, chlorophylls, and steroids). Herein, we describe the structural elucidation of these new compounds and the cytotoxic properties of all compounds identified toward several human cancer cell lines.
Due to the small specific rotations of compounds 1 ([α]D +8.0°) and 2 ([α]D +3.0°), we suspected they might be not optically pure compounds. The configuration of compounds 1 and 2 has not been determined due to the isolation of insufficient amounts of these materials. However, we adopted the similar structure of phaitanthrin A (7) as a model. First, our attempts to synthesize a pair of diastereomeric esters by acylating 7 with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride [(+)-MTPACl] [6], even with acetyl chloride, were unsuccessful. This most likely is due to steric inhibition at the tertiary alcohol, which is present in the tryptanthrin skeleton. We then tried to analyze the C-6 chemical shift behaviors using the chiral shift reagents, tris[3-trifluoroacetyl-D-and

General
Optical rotations were measured on a Jasco DIP-370 digital polarimeter (JASCO, Tokyo, Japan). UV spectra were recorded on an Agilent 8453 spectrophotometer (Agilent Technologies, Palo Alto, CA, USA). IR spectra were recorded on a Nicolet Magna FT-IR spectrophotometer (Nicolet Instrument, Inc., Madison, WI, USA). NMR spectra were recorded on a Bruker Avance 300 (Bruker, Karlsruhe, Germany) and AMX 400 spectrometers (Bruker, Karlsruhe, Germany), and all chemical shifts are given in ppm using tetramethylsilane (δ 0.00) as an internal standard. Mass spectra were obtained on a VG 70-250S spectrometer by a direct inlet system (Micromass Corp., Manchester, UK).

Plant Material
Whole Cephalantheropsis gracilis plants were collected from Pingtung Hsien, Taiwan, in December 2004, as authenticated by Chang-Sheng Kuoh, Department of Biology, National Cheng Kung University, Tainan, Taiwan. A voucher specimen (No: PLW-0401) was deposited in the Herbarium of National Cheng Kung University, Tainan, Taiwan.

Extraction and Isolation
The dried aerial parts of C. gracilis (2.4 kg) were extracted with MeOH (8 L) under reflux 8 times. The combined extracts were concentrated under reduced pressure to produce a dark brown syrup. The syrup was then suspended in H2O and then partitioned with hexane, CHCl3, and EtOAc, successively. The concentrated hexane layer (47 g) was chromatographed on a silica gel column by eluting with a gradient of hexane-Me2CO (10:1 to pure Me2CO) to give six fractions. Fraction 3 was chromatographed on silica gel by elution with hexane-i-Pr2O (1:3 to pure i-Pr2O) to give 48 (9.6 mg). The EtOAc extract (20 g) was subjected to column chromatography using Cosmosil 75 C18 and eluted with a gradient of H2O-MeOH (from pure H2O to pure MeOH) to give nine fractions. Fraction 2 was subjected to further chromatography on a Cosmosil 75 C18 column eluting with a gradient of H2O-MeOH (from pure H2O to pure MeOH) to give 21 (3.6 mg) and 47 (12.5 mg). Fraction 3 was chromatographed on a silica gel column eluting with a gradient of i-Pr2O-MeOH (15:1 to pure MeOH) to give 22 (2.2 mg). Fraction 4 was subjected to repeated chromatography on a silica gel column eluting with a gradient of i-Pr2O-MeOH (10:1 to pure MeOH) to give 1 (1.4 mg), 16 (3.5 mg), 36 (12.6 mg), and 41 (44.5 mg). Fraction 5 was chromatographed on a silica gel column eluting with a gradient of i-Pr2O-MeOH (20:1 to pure MeOH) to give 8 (9.8 mg), 15 (22.2 mg), and 38 (2.9 mg). Fraction 6 was further purified over silica gel eluting with a gradient of CHCl3-MeOH (15:1 to pure MeOH) to give 20 (20.9 mg). Fraction 7 was further chromatographed on a silica gel column eluting with a gradient of i-Pr2O-MeOH (8:1 to pure MeOH) to give 30 (4.0 mg).

Cytotoxicity Assay
The cytotoxicity assay was carried out according to the procedure described in the literature [17].