Yellow Pigments, Fomitellanols A and B, and Drimane Sesquiterpenoids, Cryptoporic Acids P and Q, from Fomitella fraxinea and Their Inhibitory Activity against COX and 5-LO

Yellow pigments, fomitellanols A (1a) and B (2a), and drimane-type sesquiterpenoid ethers of isocitric acid, cryptoporic acids P (3) and Q (4), have been isolated fromthe fruiting bodies of Fomitellafraxinea (Polyporaceae). Their structures were established by a combination of extensive NMR spectroscopy and/or X-ray crystallographic analyses, and their biological activity against COX-1, COX-2, and 5-LO was investigated.


Introduction
In the course of our research aimed at the discovery of biologically active compounds from fungi, we previously studied the chemical constituents of four genera belonging to the Polyporaceae: Laetiporus versisporus [1], Laetiporus sulphureus var. miniatus [2], Elfvingia applanata [3], Fomitopsis pinicola [4], and Daedalea dickisii [5]. We subsequently initiated an investigation of Fomitella fraxinea (FR) Imaz. belonging to the same family. This fungus grows on dead trees in broad-leaved forests and is widely distributed in Japan [6,7]. Previous phytochemical studies on this OPEN ACCESS fungus have led to the discovery of lanostane triterpenes, fomitellic acids A-D and their inhibitory activity against calf DNA polymerase  and rat DNA polymerase  [8], and a mannofucogalactan, fomitellan A, with a mitogenic effect [9]. The fruiting bodies of F. fraxinea were extracted with 70% isopropanol and the extract, after concentration, was dissolved in ethyl acetate. Fractionation of the EtOAc-soluble portion led to the isolation and characterization of four new compounds, which were designated as fomitellanols A (1a), B (2a) and cryptoporic acids P (3), and Q (4) (Figure 1), along with two known compounds, cryptoporic acids B (5) [10,11] and N (6) [12] (Figure 2). We describe here the isolation and structure elucidation of 1a, 2a, 3, and 4 by extensive NMR and/or X-ray experiments, and the inhibitory activities of 1a, 3, 4, and 6 against COX and 5-LO are also described.

General
Optical rotations were taken on a Jasco DIP-1030 polarimeter. UV spectra were recorded on a Shimadzu UV-1650PC, IR spectra were recorded on a Jasco FT/IR-5300, CD spectra were recorded on a Jasco J-725 and NMR spectra on a Varian Unity 600 spectrometer in C 5 D 5 N using TMS as an internal standard. NMR experiments included COSY, DEPT, HMQC, HMBC and ROESY. Coupling constants (J values) are given in Hz. The FABMS was measured on a JEOL JMS-700 mass spectrometer. Column chromatography was carried out on silica gel (230-400 mesh; Merck). Analytical TLC was performed on precoated Merck F 254 silica gel plates and visualized by spraying with 30% H 2 SO 4 . HPLC was carried out on a Jasco PU-1580 pump equipped with a Jasco UV-970 detector and a Wakopack C30-5 column (5 m, 20 mm i.d. × 250 mm, Wako).

Materials
The fruit bodies were collected in Tokushima, Japan, in autumn 2008. A voucher specimen (TB 3085) is deposited in the Herbarium of Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan. Material was identified by Dr. T. Hattori, the researcher of Forestry and Forest Products Research Institute.

p-Bromobenzoylation of 1a
To a solution of 1a (15 mg) in pyridine (2 mL) were added p-bromobenzoyl chloride (15 mg) and 4-dimethylaminopyridine (2 mg). The reaction mixture was stirred at room temperature for 24 h and then concentrated in vacuo to give a residue, which was purified by HPLC (ODS, 85% MeOH) to afford 1b (5 mg) as yellow needles from diethyl ether-EtOH; mp 170-172 °C. X-ray crystallographic analysis confirmed the structure of 1b (absolute configuration; ORTEP diagram, Figure 4).

X-ray Crystallographic Data for 1b
Single crystals of 1b, obtained by slow evaporation of MeOH, were selected, fitted onto a glass fiber, and measured at −173°C with a Bruker Apex II ultra diffractometer using Mo K radiation. Data correction and reduction were performed with the crystallographic package Apex II. The structures were solved by direct methods using SHELXS-97) and refined using full matrix least-squares based on Compounds 5 (10 mg) and 6 (10 mg) were acetylated overnight with Ac 2 O and pyridine (2 mL of each), respectively. The usual work-up afforded the acetates of 6 and 5, which were identified by comparisons of their NMR data and optical rotations, as 3 and 4, respectively.

COX-1 and COX-2-Catalyzed Prostaglandin Biosynthesis Assay in Vitro
Experiments were performed according to Futaki et al. [14] with modification. In brief, 200 U of COX-1 or COX-2 enzyme was suspended in 0.1 M Tris-HCl buffer (PH 7.5) containing hematin (1 M) and phenol (2 mM), as co-factors. The reaction medium was preincubated with sample for 2 min at 37 C, and 51.4 M of [1-14 C] arachidonic acid (Sigma, St. Louis, MO, USA) was added and incubated for 2 min at 37 C. To terminate the reaction and extract PGE 2 , 400 L of n-hexane/EtOAc (2:1, V/V) was added to the reaction mixture and the preparation was centrifuged at 2,000 rpm for 1 min. The organic solvent phase was discarded. The extraction procedure was repeated twice, then 50 L EtOH was added to the aqueous phase, and the preparation was at 2,000 rpm for 1 min. The amount of PGE 2 was measured by radioimmunoassay using a liquid scintillation counter. COX-1 (EC1.14.99.1, isolated from ram seminal vesicles; Cayman Chemical Company, Ann Arbor, MI, USA) and COX-2 (isolated from sheep placenta, purity 70%; Cayman Chemical Company) were used.

Conclusions
Two yellow pigments, fomitellanols A (1a) and B (2a) and drimane sesquiterpenoid ethers of isocitric acid, cryptoporic acids P (3) and Q (4), were isolated from the fruiting bodies of Fomitella fraxinea (Polyporaceae) together with two known compounds, cryptoporic acids B (5) and N (6). The structures of 1a, 2a, 3, and 4 was determined by extensive spectroscopic analysis, and the absolute configuration was determined by X-ray analysis and/or the chemical transformations from 5 and 6, respectively. Additionally, the known compounds, 5 and 6 are reported for the first time from Fomitella fraxinea. Compounds 1a and 2a were isolated as the 1-isopropoxy derivatives of 1 and 2, respectively. They might possibly be artifacts derived from the reaction of the corresponding hemiacetals, with the isopropanol used in the extraction. Compounds 1a, 3, 4, and 6 were tested for anti-inflammatory activities against COX-1, COX-2 and 5-LO in in vitro assays. It was assumed that compound 1 was most likely a dual inhibitor of the pathway involved in arachidonic acid metabolism in this study, although its activity is not apparent for COX-2 at the present. The result provides a potential explanation for the use of this fungus as herbal medicine in the treatment of inflammation, and it is potentially useful for developing new anti-inflammatory agents.