Cytotoxic Polyhydroxysteroidal Glycosides from Starfish Culcita novaeguineae

Four new polyhydroxysteroidal glycosides—culcinosides A–D (1, 2, 4, and 7)—along with three known compounds—echinasteroside C (3), linckoside F (5), and linckoside L3 (6)—were isolated from the ethanol extract of starfish Culcita novaeguineae collected from the Xisha Islands of the South China Sea. The structures of new compounds were elucidated through extensive spectroscopic studies and chemical evidence, especially two-dimensional (2D) NMR techniques. The cytotoxicity of the new compounds against human glioblastoma cell lines U87, U251, and SHG44 were evaluated.


Introduction
The starfish is a sea animal belonging to Asteroidea: Echinodermata that is distributed worldwide; there are approximately 1900 species grouped into 370 genera [1]. Steroidal glycosides are the predominant metabolites of starfish, and are responsible for their general toxicity. Based on structural characteristics, they have been subdivided into three groups: asterosaponins, polyhydroxysteroidal glycosides, and cyclic steroidal glycosides [2]. Polyhydroxysteroidal glycosides are abundant in the metabolism of starfish; more than 500 have been identified in total [3]. In general, polyhydroxysteroidal glycosides consist of an oxygenated steroidal aglycone with more than three hydroxy groups, and one or two (rarely three) monosaccharide residues attached to the steroidal nucleus or side chain. Polyhydroxysteroidal glycosides have been reported to show a broad spectrum of biological activities, including hemolytic, cytotoxic, immunoregulatory, anti-bacterial, neuritogenic, anti-inflammatory, and anti-biofouling effects [4][5][6][7][8][9][10][11][12]. Culcita novaeguineae is plentiful in the South China Sea; it is used as a folk medicine for the treatment of rheumatism, and as a tonic in China [13]. The chemical constituent investigation of this starfish has led to the isolation of several polyhydroxysteroid glycosides and asterosaponins by scientists around the world [14,15]. The previous work that our team carried out on Culcita novaeguineae led to the isolation of a series of novel asterosaponins. Some of these have shown significant cytotoxicity against several human cancer cell lines, such as asterosaponin 1, through suppressing the proliferation of human glioblastoma cell line U87 with an IC 50 of 4.3 µg/mL [2,6,[16][17][18][19]. However, no polyhydroxysteroidal glycosides, as a large class of bioactive metabolites of starfish, were found in our previous work. Therefore, as part of a continuous search for bioactive steroidal glycosides from starfish, we aimed for the polyhydroxysteroidal glycosides in Culcita novaeguineae. Herein, we report the isolation, structural elucidation, and biological activity screening of four new polyhydroxysteroidal glycosides, culcinosides A-D (1, 2, 4, and 7), together with three report the isolation, structural elucidation, and biological activity screening of four new polyhydroxysteroidal glycosides, culcinosides A-D (1, 2, 4, and 7), together with three known compounds, which were identified as echinasteroside C (3), linckoside F (5), and linckoside L3 (6), through a comparison of the physical and spectra data with literature values (Figure 1) [10,12,20].

Structure Elucidation
Culcinoside A (1) was isolated as a colorless powder, and was positive for the Liebermann-Burchard and Molisch tests, which indicated that it might be a steroidal glycoside. . A comprehensive analysis and comparison of the above data with that of co-isolated linckoside L3 (6) indicated that they may share the same steroidal aglycone and side chain (Table 1). A difference was recognized in ring D, where a secondary alcohol (δC 83.1, δH 4.04) in compound 6 was replaced by one methylene (δH 2.15 and 2.27, δC 42.3, CH2-16) of compound 1. Furthermore, this substitution was witnessed by the upfield shift of C-15 (δC 69.5, δH 4.90) in compound 1 when compared with that of 6 (δC 80.6, δH 4.17). The position of the double bond was determined by

Structure Elucidation
Culcinoside A (1) was isolated as a colorless powder, and was positive for the Liebermann-Burchard and Molisch tests, which indicated that it might be a steroidal glycoside. The molecular formula of compound 1 was determined as C 34  , and two oxygenated quaternary carbon (δ C 76.0, δ C 76.2). A comprehensive analysis and comparison of the above data with that of co-isolated linckoside L3 (6) indicated that they may share the same steroidal aglycone and side chain ( Table 1). A difference was recognized in ring D, where a secondary alcohol (δ C 83.1, δ H 4.04) in compound 6 was replaced by one methylene (δ H 2.15 and 2.27, δ C 42.3, CH 2 -16) of compound 1. Furthermore, this substitution was witnessed by the upfield shift of C-15 (δ C 69.5, δ H 4.90) in compound 1 when compared with that of 6 (δ C 80.6, δ H 4.17). The position of the double bond was determined by HMBC correlations for H-4 to C-2, C-6, and C-10; H-3 and H-5 to C-4 and C-5; and H-19 to C-5. This connection was further confirmed by the 1 H-1 H COSY correlations for H-3 to H-4. The assignment of the NMR signals associated with the aglycone moiety and side chain (Table 1) was derived from HSQC, 1 H-1 H COSY, HMBC, and TOCSY experiments. The normally occurring 2-O-methyl-β-D-xylopyranose (2-OMe-Xyl) moiety in the polyhydroxysteroidal glycosides was detected in compound 1 by analyzing the one-dimensional (1D) and two-dimensional (2D) NMR spectra, and by comparing the monosaccharide signals with those co-isolated known compounds and the literature values [21][22][23]. This was further confirmed by the demethylation and acid hydrolysis of compound 1 with 2 M trifluoroacetic acid, followed by derivatization with 1-(trimethylsily)-imidazole, gas chromatography (GC) analysis sequentially, and a comparison with the corresponding derivatives of a standard monosaccharide [6,8]. The attachment of the monosaccharide to C-3 was confirmed by HMBC correlation for H-3 to C-1 (δ C 104.8) of the Xyl, and H-1 (δ H 4.84) of the Xyl to C-3 of the aglycone ( Figure 2). The NOESY correlations of H-3 to H-1b, H-1b to H-9, H-6 to H-7b, and H-7b to H-9, and the lack of correlation between H-1 and H-6 indicated the α-orientation of H-3 and H-6 ( Figure 3). The β-orientation of the hydroxy at C-15 was determined by the NOESY correlation of H-16b to H-15 and H-17, and the lack of correlation between H-14 and H-15. The 20R configuration was deduced from the NOESY correlation of H-20 to H-18, and the large coupling constant of H-17 (J = 9.1) [10]. It has been reported that small but important differences in the signals for the HC(28), H'C(28), and C(28) atoms in the NMR spectra of the synthetic 24R and 24S epimers of 24-hydroxymethyl-24-hydroxycholesterol (24R: ∆δ H = 0.04, δ C = 66.0; 24S: ∆δ H = 0.06, δ C = 66.3) [12]. The NMR spectroscopic data of compound 1 at CH 2 -28 (∆δ H = 0.04, δ C = 66.1) coincided with that of the 24R epimer. Thus, the absolute configuration of C-24 was determined as R. Therefore, the structure of compound 1 was established as    [11,20]. The chemical shift of the anomeric proton at δH 4.84 (J = 7.6) associated with the anomeric carbon at δC 104.8 in the HSQC spectrum and the carbon signals at δC 61.2, 67.5, 71.6, 78.16, and 85.6 suggested the presence of the 2-O-methyl-β-D-xylopyranose monosaccharide residue, and this was further confirmed by acid hydrolysis followed by GC analysis. The connection of the monosaccharide to C-3 was deduced from the HMBC correlation for H-1′ of Xyl to C-3 of the aglycone (Figure 2). All of the H and C signals of compound 2 were assigned by the 2D NMR spectra, including HSQC, 1 H-1 H COSY, and , and one quaternary oxygenated carbon C-8 (δ C 76.0). All of the above data were similar to the known compounds echinasteroside C (3) and linckoside A, which possessed the same ∆ 4 -3β,6β,8,15α,16β-pentahydroxycholestane aglycone [11,20]. The chemical shift of the anomeric proton at δ H 4.84 (J = 7.6) associated with the anomeric carbon at δ C 104.8 in the HSQC spectrum and the carbon signals at δ C 61.2, 67.5, 71.6, 78.16, and 85.6 suggested the presence of the 2-O-methyl-β-D-xylopyranose monosaccharide residue, and this was further confirmed by acid hydrolysis followed by GC analysis. The connection of the monosaccharide to C-3 was deduced from the HMBC correlation for H-1 of Xyl to C-3 of the aglycone (Figure 2). All of the H and C signals of compound 2 were assigned by the 2D NMR spectra, including HSQC, 1 H-1 H COSY, and HMBC (Table 1). The structure of the side chain for compound 2 was elucidated on the basis of 2D NMR spectra. The tetrasubstituted double bond at C-24 and C-25, and the C-28 hydroxymethyl were determined by HMBC correlations for H-23 (2H) to C-24, C-25 and C-28; H-26 and H-27 to C-24 and C-25; and H-28 (2H) to C-23, C-24, and C-25. The 3β, 6β, 15α, and 16β orientations for the aglycone of 2 were confirmed by the cross-peaks of the NOESY spectrum (  Culcinoside C (4), a colorless powder, was positive for the Liebermann-Burchard and Molisch tests, which indicated that it might be a steroidal glycoside. The HRESIMS at m/z 663.3696 [M + Na] + (calculated for C34H56O11Na, 663.3720) indicated that the molecular formula of 4 was C34H56O11. The 1 H, 13 1, 2, 4, and 7).

General Experimental Procedures
Optical rotations were measured with a Perkin-Elmer 343 polarimeter (German Perkin-Elmer Corporation, Boelingen, Germany). 1D and 2D NMR spectra were recorded on a Bruker AVANCE III 500 and 800 MHz spectrometer with TMS (Tetramethylsilane) as the internal standard. ESIMS and HRESIMS were carried out on a Micromass Quattro mass spectrometer (Waters, Shanghai, China). HPLC was carried out on a Dionex P680 liquid chromatograph (Dionex, Germering, Germany) equipped with a UV 170 UV/Vis detector using a YMC-Pack C18 column (20 × 250 mm i.d., 5 µm, YMC Co., Ltd., Kyoto, Japan) and monitored at 206 nm, 225 nm, 275 nm, and 300 nm, simultaneously. GC was performed on a Finnigan Voyager apparatus using an l-Chirasil-Val column

Animal Material
The starfish were collected from the South China Sea (Xisha Islands, Sansha, Hainan Province, China) in August 2015. The organisms were identified as Culcita novaeguineae by Dr. Ning Xiao (Institute of Oceanology, Chinese Academy of Science, Qingdao, China). A voucher specimen (No. HX201508) was deposited in the Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University (Xi'an, China).

Demethylation and Acid Hydrolysis of the New Compounds
The new compounds (each 1.5 mg) were mixed with 1 mL of dry dichloromethane and 0.01 mL of boron tribromide at −80 • C for 30 min, and then stood overnight at 10 • C under anhydrous conditions. The solvent and reagent were evaporated to dryness in vacuo at room temperature. The demethylated derivative of the new compound was heated with 1.0 mL of trifluoroacetic acid (TFA) at 120 • C for 2 h. The reaction mixture was evaporated in vacuo, and the residue was partitioned between CH 2 Cl 2 and H 2 O. The aqueous phase was concentrated and dissolved in 1-(trimethylsilyl)imidazole and anhydrous pyridine (0.1 mL). Then, the solution was stirred at 60 • C for 5 min, and dried with a stream of N 2 . The residue was partitioned between CH 2 Cl 2 and H 2 O. The CH 2 Cl 2 layer was analyzed by GC with an initial temperature of 100 • C for 1 min, and then temperature programmed to 180 • C at a rate of 5 • C/min. The peak of the derivative of the sample was detected at 11.25 and 12.46 min for compound 1, 11.25 and 12.45 min for compound 2, 11.23 and 12.45 for compound 4, and 11.24 and 12.26 min for compound 7. The retention time of the authentic samples after being treated simultaneously with 1-(trimethylsily)imidazole in pyridine were 11.23 and 12.44 min (D-xylose), and 11.34 and 12.40 min (L-xylose), respectively [6].

Assays for In Vitro Cytotoxicity
The cytotoxicity of new compounds 1, 2, 4, and 7 against human glioblastoma cell lines U87, U251, and SHG44 were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay method in vitro. All of the cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL benzyl penicillin, and 100 U/mL streptomycin at 37 • C in a humidified atmosphere with 5% CO 2 . The logarithmic phase cells were seeded on 96-well plates at a concentration of 4 × 10 3 cell/mL, and incubated with various concentrations (100 µM, 80 µM, 60 µM, 40 µM, 20 µM, 10 µM, 1 µM, and 0.25 µM in medium containing less than 0.1% DMSO) of test compounds in triple wells for 48 h, and doxorubicin was used as the positive control. Next, 20 µL MTT (5 mg/mL) was added to each well, and incubated for another 4 h. The water-insoluble dark blue formazan crystals formed during MTT cleavage in actively metabolizing cells were dissolved in DMSO. The optical density of each well was measured with a Bio-Rad 680 microplate reader at 570 nm. Cytotoxicity was expressed as the concentration of drug inhibiting cell growth by 50% (IC 50 ).
Supplementary Materials: The NMR and HRESIMS data of the new compounds and the 13 C-NMR data of the known compounds are available online at www.mdpi.com/1660-3397/16/3/92/s1.