Capilloquinol: A Novel Farnesyl Quinol from the Dongsha Atoll Soft Coral Sinularia capillosa

Capilloquinol (1), possessing an unprecedented farnesyl quinoid skeleton, was isolated from the Dongsha Atoll soft coral Sinularia capillosa. The structure of capilloquinol was elucidated by extensive analysis of spectroscopic data. The cytotoxicity and antiviral activity against human cytomegalovirus of 1 was evaluated in vitro.


Scheme 1. Possible biogenetic pathway for 1.
Capilloquinol (1) was evaluated for cytotoxicity against P-388, A-459, and HT-29 cancer cell lines as well as antiviral activity against human cytomegalovirus. Metabolite 1 displayed cytotoxicity against P-388, with an ED 50 of 3.8 μg/mL. With the exception of the above finding, 1 did not show cytotoxic against A-459, and HT-29 cancer cell lines, nor was it active against human cytomegalovirus (HCMV).

General Experimental Procedures
Optical rotations were determined with a JASCO P1020 digital polarimeter. Ultraviolet (UV) and infrared (IR) spectra were obtained on a JASCO V-650 and JASCO FT/IR-4100 spectrophotometers, respectively. The NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR spectrometer at 500 MHz for 1 H and 125 MHz for 13 C, respectively. Chemical shifts are expressed in δ (ppm) referring to the solvent peaks δ H 3.30 and δ C 49.0 for CD 3 OD, respectively, and coupling constants are expressed in Hz. ESI-MS were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Silica gel 60 (Merck, Germany, 230-400 mesh) and LiChroprep RP-18 (Merck, 40-63 μm) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F 254 , 0.25 mm) and precoated RP-18 F 254s plates (Merck) were used for thin-layer chromatography (TLC) analysis.

Animal Material
The soft coral S. capillosa was collected by hand using SCUBA along the coast reefs offshore from the Dongsha Atoll in April 2007, at a depth of 8-10 m, and was stored in a freezer at −20 °C for two months until extraction. Identification was kindly verified by Prof. Chang-Feng Dai, Institute of Oceanography, National Taiwan University, Taiwan. A voucher specimen (TS-06) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Taiwan.

Extraction and Isolation
The acetone extract of S. capillosa was concentrated to a brown gum, which was partitioned with EtOAc and H 2 O. The EtOAc-soluble residue (60 g) was subjected to Si 60 CC using n-hexane-EtOAc mixtures of increasing polarity for elution, to yield 40 fractions. Fraction 14 (0.8 g) was applied to a RP-18 gravity column to obtain a mixture (72 mg) that was further purified by RP-18 HPLC eluted with MeOH-H 2 O (9:1) to yield 1 (2.0 mg).
The frozen soft coral (2 kg) was chopped into small pieces and extracted exhaustively by maceration with fresh acetone for 24 h at room temperature. The quantity of solvent used for each extraction (2 L) was at least three times the amount of the soft coral material used. The acetone extracts were filtered and concentrated under vacuum to yield a brownish oily residue, which was subsequently partitioned between EtOAc and H 2 O. The resulting EtOAc-soluble residue (60 g) was subjected to column chromatography on silica gel using n-hexane with increasing amounts of EtOAc, and finally 100% MeOH as elution, to fractionate roughly 40 fractions on the basis of the 1 H NMR spectroscopic data and TLC analyses. Fraction 14 (0.8 g) eluted with n-hexane-EtOAc (2:1) was subjected to was applied to a RP-18 gravity column to obtain a mixture (72 mg) that was further purified by RP-18 HPLC eluted with MeOH-H 2 O (9:1) to yield 1 (2.0 mg).

Cytotoxicity Assay
Cytotoxicity was determined against P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells using a modification of the MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method. The provision of the P-388 cell line was provided by J. M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection. The experimental details of this assay were carried out according to a previously described procedure [17][18][19].

Anti-HCMV Assay
To determine the effects of natural product upon HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural product. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of 24-well dish. Antiviral activity was expressed as IC 50 (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after 7 days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [20].