Chemical Constituents of Phaius mishmensis

The partitioned n-hexane, CHCl3, and EtOAc extracts from the crude MeOH extract of Phaius mishmensis showed considerable cytotoxicities against the human breast carcinoma (MCF-7), lung carcinoma (NCI-H460), and central nervous system carcinoma (SF-268) cell lines. Four new compounds, phaindole (1), (7′R,8′R)-phaithrene (2), methyl 3-hydroxy-4,5-dimethoxypropiophenone (3), and methyl hematinate (4), as well as 44 known compounds were isolated from the MeOH extract of Phaius mishmensis. The structures of the compounds were determined using spectroscopic methods.

Compound 4 was isolated as a white solid and was confirmed to have the molecular formula C 9 H 12 NO 4 by the HR-FABMS signal at m/z 198.0767. From the 1 H NMR spectrum (Table 1), the structure of 4 was determined to possess a mutually coupled ethylene group at δ 2.61 ppm (H-2 ) and 2.71 ppm (H-1 ), a methyl group at δ 2.00 ppm (4-CH 3 ), and a methoxyl group at δ 3.67 ppm (3 -OCH 3 ). The HMBC correlation of the 3 -OCH 3 with a carbonyl C-3 (δ 172.5 ppm); H-1 , H-2 , and 4-CH 3 with accidently coinciding olefinic carbons C-3 and C-4 (δ 139.7 ppm); as well as a NOE correlation between 4-CH 3 and H-1 indicated a cis CH 3 C=CCH 2 CH 2 COOCH 3 fragment. The remaining two carbonyl carbon signals at δ 171.4 ppm (C-2) and 171.5 ppm (C-5) and a broad NH proton signal at δ 7.52 ppm as well as the HMBC correlations between H-1 and C-2 and 4-CH 3 and C-5 formed an imido -O=CNHC=O-fragment. By combining these two fragments, the structure of 3-(methoxycarbonyl)ethyl-4-methyl-2,5-pyrroledione was thus deduced as methyl hematinate (4). This compound has been obtained from the photooxygenation of biliverdin [4]. However, this is the first time it has been isolated naturally.

Cytotoxicity of Chemical Constituents of P. mishmensis
The partitioned n-hexane, CHCl 3 , and EtOAc extracts from the crude MeOH extract of P. mishmensis showed considerable cytotoxicities against the MCF-7, NCI-H460, and SF-268 cell lines (Table 2). Unfortunately, most of the isolated compounds, except tryptanthrin (13) and phaitanthrin-A (14) [28], did not exhibit significant cytotoxicity against the tested cell lines. This result suggested that the practically insoluble tryptanthrin (13) could disperse in organic layers during extraction.

General
Optical rotations were measured on a Jasco DIP-370 digital polarimeter (JASCO, Tokyo, Japan). CD spectra were determined on a Jasco J-715 spectropolarimeter (JASCO, Tokyo, Japan). UV spectra were recorded on an Agilent 8453 spectrophotometer (Agilent Technologies, m, CA, USA). The IR spectra measured using a Nicolet Magna FT-IR spectrophotometer (Nicolet Instrument, Inc., Madison, WI, USA). The 1 H-, 13 C-, and 2D NMR spectra were recorded on Bruker Avance 300, AMX 400, and Avance-500 FT-NMR spectrometers (Bruker, Karlsruhe, Germany) at room temperature. All chemical shifts (δ) are given in ppm using tetramethylsilane 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 plants of P. mishmensis were collected from Nanto Hsien, Taiwan, in October 2003. The collection was authenticated by Professor Chang-Sheng Kuoh, Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan. A voucher specimen (No. PLW-0304) was deposited in the Herbarium of National Cheng Kung University, Tainan, Taiwan.

Extraction and Isolation
The air-dried P. mishmensis plants (3.5 kg) were extracted with MeOH (7 × 8 L) under reflux. The combined extracts were concentrated under reduced pressure to give a dark brown syrup. The syrup was suspended in H 2 O and then partitioned successively with n-hexane, CHCl 3 , and EtOAc. These concentrated layers were stored in a refrigerator at −20 • C before they were purified.
The concentrated CHCl 3 layer (15 g) was chromatographed on a silica gel column by eluting with a gradient of CHCl 3 and MeOH (20:1, v/v to 100% MeOH) to yield seven fractions. After repeated chromatography on silica gel followed by preparative TLC, fraction 2 gave 2-methoxycarbonylindolin-3-one (7)

Cytotoxicity Assay
The cytotoxicity assay was carried out according to a procedure described previously [29]. Carcinoma cells MCF-7 and SF-268 were maintained in DMEM (Dulbecco's Modified Eagle Medium, Fisher Scientific, HyClone, Logan, UT, USA) and NCI-H460 were maintained in RPMI (Roswell Park Memorial Institute, MP Biomedicals, Inc., Solon, OH, USA) medium supplemented with 10% fetal bovine serum (Biological Industries Inc., Cromwell, CT, USA). Firstly, the MCF-7, NCI-H460, and SF-268 cells were plated at a density of 5 × 10 3 cells per well in 96-well plates overnight and then treated with different concentrations of the isolated compounds. After 48 h, MTS cell proliferation assay kit (Promega, Madison, WI, USA) was added to each well; then, the experiment was performed as the manufacturer recommended (Promega). The absorbance was measured at 490 nm on a MQX200R microplate reader (BioTek, Winooski, VT, USA).