Philinopgenin A, B, and C, Three New Triterpenoid Aglycones from the Sea Cucumber Pentacta quadrangulasis

Three new triterpenoid aglycones named Philinopgenin A (1), B (2), and C (3) were isolated from the acid hydrolysate of the crude glycoside mixture prepared from the whole sea cucumber Pentacta quadrangulasis Lesson. The corresponding structures were determined as 16β-acetoxyholosta-8(9), 24(25)-diene-3β-ol (1), 20, 25-epoxy-lanosta-9(11)-ene-3β-ol 18(16)–lactone (2) and 16β-acetoxyholosta-9(11), 24(25)-diene-3β-ol (3), respectively, on the basis of spectral evidence.


Introduction
Investigations of triterpene glycosides in sea cucumbers (Holothuroidea, Echinodermata) have a long history. In the search for new biologically active substances from marine organisms, we have been investigating antitumour constituents in the sea cucumber Pentacta quadrangulasis Lesson, which is widely distributed throughout the South China Sea, especially in the area near Guangdong. Before examining the components of this sea cucumber to elucidate the structure of the biologically active glycosides, we investigated the aglycones. We report herein the isolation, purification, and structural elucidation of three new aglycones named Philinopgenin A (1), B (2), and C (3) from the acid hydrolysate of the crude glycoside fraction.
The molecular formula of Philinopgenin B (2) was determined as C 30 H 46 O 4 (m/z 470) by pseudomolecular ion peaks at m/z 471 ([M+H] + ) and 493 ([M+Na] + ) in the HRESI-MS (positiveion mode). The IR spectrum showed absorptions due to hydroxyl (3446cm -1 ) and γ-lactone (1770cm -1 ) groups. The 13 C-NMR and DEPT spectrum exhibited 30 carbon signals (7×CH 3 6×CH , 8×C). The 1 H-and 13 C-NMR spectra of compound 2 (Table 1) showed some similarities to those of compound 1 and to those of the aglycone moieties of cucumarioside G 2 [3]. The main difference from compound 1 being the replacement of the 16 OAc-substitutuent (δ C 75.0, δ H 5.63) by one oxygen-bearing methine group (δ C 79.2, δ H 4.77) forming the 18(16)-lactone with the C-18 carbonyl group, in agreement with the chemical shifts for C 15 and C 17 , and the replacement of the side chain by a 2,2-dimethyl-6-pyranyl moiety. The quaternary carbons at δ C 73.0 and 71.8 suggested that the side chain is in a pyran form. The correct assignments for the side chain are based the fact that the carbon peaks at δ C 73.0 (C-20) and 28.
The molecular formula of Philinopgenin C (3) was determined as C 32 H 48 O 5 (m/z 512) by pseudomolecular ion peaks at m/z 535 ([M+Na] + ) in the HRESI-MS (positive-ion mode). Compound 3 shows spectral features (Table 1) similar to those of compound 1, except for the signals due to the carbon atoms at the B/C-ring junction. These findings suggest that compound 3 is an olefinic function regioisomer of compound 1. The two olefinic carbon signals at δ=130.2 (s) and 135.6 (s) in the 13 C-NMR spectrum of compound 3 indicate that compound 3 is the ∆ 8(9) isomer of 1. Thus, compound 3 can be identified as 16β-acetoxyholosta-8(9), 24(25)-diene-3β-ol.
About 40 genuine and artifact triterpenoid aglycones have been obtained by acidic or enzymatic hydrolysis of crude sea cucumber glycosides [4]. The newly obtained aglycones, described in this paper, differ from previously reported ones in their substitution patterns. It should be noted, however, that compounds 1, 2, and 3 may be artifacts arising from the extraction treatment, because the coexistence of minute amounts of other not identified regioisomers was noted in the acid hydrolysate of the glycoside mixture, and signals due to a trisubstituted olefin group (∆ 7(8) ) were observed in the NMR spectra of the parent oligoglycoside sulfates of this sea cucumber.

Acknowledgments
This work was supported by the State Foundation for High-Projects Grant "863" from the Ministry of Science and Technology, P. R. China awarded to Y-H Yi.

General
Melting points were determined with a XT5-XMT micro melting point apparatus and are uncorrected. IR spectrum was measured on a BRUKER VECTOR-22 spectrophotometer. The ESI-MS (positive and negative ion modes) was obtained on Micromass Quattro mass spectrometer. 1 H-NMR and 13 C-NMR spectra were recorded in CDCl 3 (at 100 MHz and 400 MHz for 13 C-and 1 H-NMR, respectively) with an Inova-400 spectrometer. Chemical shifts are given in δ values with tetramethylsilane (TMS) as internal standard in 1 H-NMR. Silica gel (Yantai, 200-300 mesh) was used for column chromatography. Pre-coated silica gel plates (Yantai, HSG, 0.15-0.20mm) were used for analytical TLC. HPLC was carried out with Zorbax 300 SB-C18 reverse phase.columns (9.4×250mm, Zorbax).

Extraction and Purification
Air-dried body walls of the sea cucumber Pentacta quadrangulasis (10kg) were extracted with 50% EtOH (50 L). The EtOH was evaporated in vacuo to give a crude extract (23.1g), which was partitioned between water (15 L) and chloroform (15 L). The water layer was extracted with nbutanol (15 L) and the organic layer was evaporated in vacuo to give the n-butanol extract (16.3g). A portion of this extract (6.0g) was heated at reflux in aqueous 15% H 2 SO 4 (2000 mL) and the cooled reaction mixture was then extracted with three portions of chloroform (800 mL, 600 mL and 600 mL), the combined chloroform extracts were washed with water (300 mL), dried over Na 2 SO 4 and concentrated to give an aglycone mixture (1.890g). This mixture was separated by flash chromatography on silica gel (3x40cm column, eluent: 4:1 n-hexane/EtOAc, flow rate: 1.5 mL/min) with detection of eluates by TLC (SiO 2 , 2:1 n-hexane/EtOAc; SCRC reagent) to yield a main fraction (600.9 mg, v R : 80mL-160mL