Steroidal Carboxylic Acids from Soft Coral Paraminabea acronocephala

Three new steroidal carboxylic acids, paraminabic acids A–C (1–3) were isolated from a Formosan soft coral Paraminabea acronocephala. The structures of these compounds were established by extensive spectroscopic analysis and chemical methods. Application of the PGME method allowed the establishment of the absolute configurations at C-25 and C-24 for 1 and 2, respectively. Compound 3 showed potent cytotoxicity toward Hep3B, MDA-MB-231, MCF-7, and A-549 cancer cell lines, with IC50 values ranging from 2.05 to 2.83 μg/mL. Compounds 2 and 3 were found to inhibit the accumulation of the pro-inflammatory iNOS protein.


Results and Discussion
The ethanolic extract of the soft coral P. acronocephala was partitioned between EtOAc and H 2 O to afford the EtOAc-soluble fraction. It was then subjected to silica gel column chromatography. The fractions containing steroids were selected for further study, based on characteristic methyl signals in the 1 H NMR spectrum. These fractions were subsequently subjected to a series of chromatographic separations to afford three new steroidal carboxylic acids, paraminabic acids A-C (1-3).
The HRESIMS and 13 C NMR spectroscopic data of paraminabic acid A (1) suggested a molecular formula of C 27 H 38 O 3 , appropriate for nine degrees of unsaturation. The 13 C NMR and DEPT spectroscopic data (Table 1) displayed 27 carbon signals, including 4 methyls, 7 methylenes, 11 methines, and 5 quaternary carbons. A broad O-H stretching absorption in the region of 3400-2600 cm −1 is ascribable to a carboxylic acid, which was evidenced by the carbon resonance at δ C 180.6 (C). The same steroidal nucleus as that of paraminabeolides D and E was deduced for 1 by detailed comparison of their NMR spectroscopic data [1]. The side chain moiety of 1 resembles that of a known steroidal carboxylic acid, (25S)-3-oxocholesta-1,4-dien-26-oic acid [4], isolated from the Indonesian soft coral Minabea sp. However, 1 varied from (25S)-3-oxocholesta-1,4-dien-26-oic acid in the respective side chain. The proton resonances at δ H 5.50 (1H, dt, J = 15.6, 6.4 Hz, H-23) and 5.44 (1H, dd, J = 15. 6,8.8 Hz,, measured in C 5 D 5 N, were due to the presence of a trans C-22/C-23 double bond, which was confirmed by the HMBC correlations from H 3 -21 to C-17, C-20, and C-22. The absolute configuration at C-25 was determined by the application of Kusumi's method (PGME method) [5][6][7]. The chemical shift differences of (S)-PGME amide (1a) and (R)-PGME amide (1b) (Δδ = δ (S) − δ (R) ) were summarized in Figure 2 and established the R configuration at C-25. Paraminabic acid B (2) gave the same molecular formula, C 27 H 38 O 3 , as that of 1, based on the analysis of the HRESIMS and 13 C NMR spectroscopic data ( Table 1). The NMR spectroscopic data of 2 are similar to those of 1, but differences were observed in their side chains. The HMBC correlations from H 3 -21 to C-17, C-20, and C-22 allowed the assignment of a C-22/C-23 double bond. A large coupling constant (15.2 Hz, C 5 D 5 N) between H-22 and H-23 suggested the E geometry of this double bond. The H-23 signal appeared as a doublet of doublets, revealing that the adjacent carbon (C-24) should be a methine. This might be due to the attachment of a methyl group (δ H 1.03, 3H, d, J = 6.4 Hz, H 3 -27) at C-24 (Table 2). This was confirmed by the HMBC correlations from H 3 -27 to C-23, C-24, and C-25 as well as from H 2 -25 to the carboxyl carbon (C-26). The absolute configuration at C-24 of 2 was determined by the application of Kusumi's method developed for chiral β,β-disubstituted propionic acid derivatives [6,7]. The 1 H NMR shift differences (Δδ = δ (R) − δ (S) ) between the diastereomeric (R)-and (S)-PGME amides, 2a and 2b, respectively, are summarized in Figure 2 and establish the 24S configuration for 2. The HRESIMS and 13 C NMR spectroscopic data of paraminabic acid C (3) established a molecular formula of C 27 H 38 O 4 and nine degrees of unsaturation. The IR absorptions at 3419 and 1714 cm −1 suggested the presence of hydroxy and carbonyl groups, respectively. Both 1 H and 13 C NMR spectra of 3 lacked signals of the angular methyl group, which might be replaced by a carboxylic acid according to the carbon signal at δ C 176.8 (C) ( Table 1). The carboxylic acid attached at C-13 was further confirmed by the HMBC correlations from both H 2 -12 and H-17 to C-18. The trans C-22/C-23 double bond was deduced by the HMBC correlations from H 3 -21 to C-17, C-20, and C-22 as well as J value (15.2 Hz) ( Table 2) between H-22 and H-23. In addition, the downfield-shifted quaternary carbon at δ C 69.9 was ascribable to a hydroxy group attached at C-25, which was correlated by H 2 -24, H 3 -26, and H 3 -27 in the HMBC spectrum. The cytotoxicity of compounds 1-3 against HepG2, Hep3B, MDA-MB-231, MCF-7, and A-549 cancer cells was studied and shown in Table 3. Compound 3 showed potent cytotoxicity toward Hep3B, MDA-MB-231, MCF-7, and A-549 cancer cell lines, with IC 50 values ranging from 2.05 to 2.83 μg/mL. We also investigated the inhibition of these compounds toward LPS-induced pro-inflammatory protein (iNOS and COX-2) expression in RAW264.7 macrophage cells by Western blot analysis. At a concentration of 10 μM, compounds 2 and 3 reduced the levels of iNOS to 63.9 ± 6.3% and 53.5 ± 8.6%, respectively; whereas, compound 2 enhanced the expression of COX-2 (130.5 ± 9.8%) in comparison with those of control cells stimulated with LPS only (100% for both iNOS and COX-2). Also, compound 3 could inhibit the expression of iNOS protein but did not induce cytotoxicity in macrophage cells as determined through internal control β-actin expression, as shown in Figure 3. These results indicate that 3 possesses moderate anti-inflammatory activity and potent cytotoxicity, and might be useful for further medicinal study.

General Experimental Procedures
Optical rotations were determined with a JASCO P1020 digital polarimeter. The IR spectra were obtained on a JASCO FT/IR-4100 spectrophotometer. The NMR spectra were recorded on a Bruker AVANCE 300 FT-NMR (or Varian MR-400 NMR) instrument at 300 MHz (or 400 MHz) for 1 H (referenced to TMS for both CDCl 3 and C 5 D 5 N) and 75 MHz (or 100/125 MHz) for 13 C (referenced to δ C 77.0 for CDCl 3 and to internal TMS for C 5 D 5 N). ESIMS were recorded by ESI FT-MS on a Bruker APEX II mass spectrometer. Silica gel 60 (230400 mesh, Merck, Darmstadt, Germany) and LiChroprep RP-18 (Merck, 40-63 μm) were used for column chromatography. Precoated silica gel plates (Kieselgel 60 F254, 0.25 mm, Merck, Darmstadt, Germany) and precoated RP-18 F254S plates (Merck, Darmstadt, Germany) were used for TLC analyses. High-performance liquid chromatography was performed on a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV detector at 210 nm and a semi-preparative reversed-phase column (Hibar Purospher RP-18e, 5 μm, 250 × 10 mm, Merck, Darmstadt, Germany).

Animal Material
The soft coral P. acronocephala was collected by scuba divers, off the western coast of Pingtung county, in May 2009, at a depth of 10 m, and was stored in a freezer until being extracted. This soft coral was identified by Prof. Chang-Fong Dai, Institute of Oceanography, National Taiwan University. A voucher specimen (specimen No. 200905PA) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

Cytotoxicity Testing
Cell lines were purchased from the American Type Culture Collection (ATCC). Compounds were assayed for cytotoxicity against human liver carcinoma (HepG2 and HepG3), human breast carcinoma (MCF-7 and MDA-MB-231), and human lung carcinoma (A-549) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method [8]. Freshly trypsinized cell suspensions were seeded in 96-well microtiter plates at densities of 5000-10,000 cells per well with tested compounds added from DMSO-diluted stock. After 3 days in culture, attached cells were incubated with MTT (0.5 mg/mL, 1 h) and subsequently dissolved in DMSO. The absorbency at 550 nm was then measured using a microplate reader. The IC 50 is the concentration of agent that reduced cell growth by 50% under the experimental conditions.

In Vitro Anti-Inflammatory Assay
Macrophage (RAW264.7) cell was purchased from ATCC. In vitro anti-inflammatory activities of tested compounds were measured by examining the inhibition of lipopolysaccharide (LPS) induced upregulation of iNOS and COX-2 proteins in macrophage cells using Western blotting analysis [9,10].

Conclusions
Our previous investigation on P. acronocephala has successfully discovered marine withanolides with potent anti-inflammatory activity. In this study, we reported three steroidal carboxylic acids, of which 3 exhibited potent cytotoxicity toward Hep3B, MDA-MB-231, MCF-7, and A-549 cancer cell lines. Compound 2, the second member of 27-norergostan-26-oic acid obtained from nature [11,12], was isolated from the soft coral for the first time. Our present investigation demonstrated that the soft coral, P. acronocephala, is a useful source for the discovery of bioactive substances.