Isolation and Synthesis of a Bioactive Benzenoid Derivative from the Fruiting Bodies of Antrodia camphorata

A new enynyl-benzenoid, antrocamphin O (1,4,7-dimethoxy-5-methyl-6-(3′-methylbut-3-en-1-ynyl)benzo[d][1,3]dioxide), and the known benzenoids antrocamphin A and 7-dimethoxy-5-methyl-1,3-benzodioxole, were isolated from the fruiting bodies of Antrodia camphorata (Taiwanofungus camphoratus). The structure of antrocamphin O was unambiguously assigned by the analysis of spectral data (including 1D and 2D NMR, high-resolution MS, IR, and UV) and total synthesis. Compound 1 was prepared through the Sonogashira reaction of 5-iodo-4,7-dimethoxy-6-methylbenzene and 2-methylbut-1-en-3-yne as the key step. The benzenoids were tested for cytotoxicity against the HT29, HTC15, DLD-1, and COLO 205 colon cancer cell lines, andactivities are reported herein.


Introduction
Antrodia camphorata (Taiwanofungus camphoratus), also known as "Niu-chang-chih," belongs to the family Polyporaceae in the Basidiomycetes. Designated an endangered species in Taiwan, the fungus is a rare and unique mushroom used in folk medicine and as a health food. To date, over 200 constituent compounds, consisting of small molecules (terpenoids, benzenoids, lignans, benzoquinone derivatives, and succinic and maleic derivatives) and macromolecules (polysaccharides, nucleic acids, and proteins), have been reported [1][2][3]. Preparations from the fruiting bodies have been used for the prevention or treatment of numerous maladies, including liver diseases, food and drug intoxication, diarrhea, abdominal pain, hypertension, itchy skin, and tumorigenic diseases [4]. Previous research has shown the anti-cancer and anti-inflammatory effects of the triterpenoids derived from A. camphorate [3,5]. The major chemical constituents, the enynyl-benzenoids, are the key components responsible for anti-inflammatory activity [6,7]. Because the growth rate of A. camphorata in both the wild and under cultivation is very slow, the fruiting bodies are rare and expensive. Therefore, synthesis of the active compounds is very important. In this paper, we isolated and elucidated the structure of a new active compound, the enynyl-benzenoid antrocamphin O (1), and the known benzenoids, antrocamphin A (2) [8] and 4,7-dimethoxy-5-methyl-1,3-benzodioxole (3) [9], from the fruiting bodies of A. camphorata. We also describe the total synthesis of 1. The benzenoids were tested for cytotoxicity against the HT29, HTC15, DLD-1, and COLO 205 colon cancer cell lines, and their activities are reported herein.

General Methods
IR spectra were recorded on a JASCO 4100 FT-IR spectrometer. NMR data were recorded on a Bruker DRX-500 SB 500 MHz instrument (Bruker, Rheinstetten, Germany) with CDCl 3 as solvent and TMS as internal standard. ESIMS and HRESIMS data were acquired using an ABI API 4000Q Trap and ABI XL Q-TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA), respectively. Semi-preparative HPLC was performed on a Hitachi L-7110 HPLC with a refractive index detector (Thermo Separation Products, Sunnyvale, CA, USA). A Phenomenex Luna silica column (5 μm, 10 × 250 mm, 3 mL·min −1 flow rate) was used for normal-phase separations. Silica gel (Merck, Geduran ® Si 60 0.063-0.2 mm) was used for column chromatography. All solvents were either ACS or HPLC grade and were obtained from JT Baker (Phillipsburg, NJ, USA). 5-FU was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Plant Material
The fruiting bodies of Antrodia camphorata were collected in Taoyuan, Taiwan, in the summer of 2009, and kindly provided to our laboratory by the Cosmos Biotech. Company (Longtan, Taiwan). The sample was authenticated by Tien-Wang Chiu (general manager of Cosmos Biotech. Company), and a voucher specimen (AC2009-1) was deposited at the School of Pharmacy, Taipei Medical University, Taipei, Taiwan.

Cell Culture
Human colon cancer cells (HCT15, HT29, DLD-1, and COLO 205) were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37 °C in a CO 2 incubator. Cells grew to 90% confluence, were harvested with trypsin/EDTA, and sub-cultured in a new tissue culture flask after removing trypsin and EDTA. The cancer cells used in this study had passage numbers of less than ten.

Cytotoxicity Assay
Colon cancer cells (1 × 10 5 cells) were seeded into 96-well plates and cultured overnight, allowing cell attachment. This was followed by treatment with compounds 1, 2, and 3 for 48 h at various concentrations (from 0.001 to 100 μM), as indicated, and 5-FU as a positive control. At the end of the experiment, the cytotoxicities of the compounds were examined by the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test. MTT solution (400 mg/mL) was added into the cultured wells and incubated for 1 h. The medium was then aspirated, and the dark blue crystal product was extracted with DMSO. Colorimetric changes were read on a microtiter plate reader with a 570 nm filter and a reference wavelength of 430 nm.

Statistical Analysis
All data were expressed as the mean value ± S.E.M. Comparisons were subjected to the Student's two-tailed t test. Significance was accepted at p < 0.05.

Conclusions
In conclusion, we have achieved the first total synthesis of the rare new natural product, antrocamphin O (1), using the Sonogashira reaction as the key step. The intermediate 3, from the fruiting bodies of A. camphorata, was also successfully synthesized during the process (Scheme 1).
Whereas enynyl-benzenoids are considered the key components responsible for the anti-inflammatory activities demonstrated by these fungi, this paper offers the first report of the inhibition of COLO 205 cancer cells by such compounds too [13].