Unusual Polyhydroxylated Steroids from the Starfish Anthenoides laevigatus, Collected off the Coastal Waters of Vietnam

Four new polyhydroxylated steroids 1–4 were isolated along with two previously known related steroids 5 and 6 from the methanolic extract of the starfish Anthenoides laevigatus collected off the coastal waters of Vietnam. Structures of new compounds were substantially elucidated by one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy and HRESIMS techniques. Heptaol 1 and hexaol 2 contain the common 5α-cholestane skeleton, while hexaol 3 and heptaol 4 have the rare among starfish steroid compounds 5β-cholestane skeleton. Compounds 1, 5, and 6 do not show cytotoxic effects against normal JB6 Cl41 and cancer HT-29 and MDA-MB-231 cells, however they inhibit cell proliferation and colony formation of cancer HT-29 and MDA-MB-231 cells.


Introduction
Polyhydroxylated steroids have been found in diverse marine species of ophiuroids, gorgonians, sponges, and other marine invertebrates. However, the class Asteroidea (also known as starfish or sea stars) is the richest source of these kind of steroids [1][2][3][4][5][6][7]. These substances generally contain from four to nine hydroxyl groups in the steroid nucleus and side chains and are characterized by a wide variety of chemical structures. Polyhydroxylated steroids are present in starfish in both free, sulfated, or glycosylated by one to three monosaccharide residues forms. These compounds usually occur in very complicated mixtures that are often difficult to separate into individual components by chromatographic methods. In addition to the original chemical structure, polyhydroxylated steroids attract attention with a wide diversity of biological effects including neuritogenic, neuroprotective, antiviral, anti-inflammatory, immunomodulatory, and other activities [7,8]. Recently, new knowledge about the cancer preventive activity and toxicity against different human tumor cell lines and molecular mechanisms of action of some starfish steroid compounds has been acquired [9]. Moreover, for the first time, it has been shown that starfish polyhydroxylated compounds demonstrated the radio sensitizing activity that was realized through apoptosis induction by the regulation of anti-and pro-apoptotic protein expression followed by activation of caspases and DNA degradation [10]. On the basis of the data obtained, we assume that the study of polyhydroxylated steroids from the starfish could lead to new promising results on their biological activities.
The search for new metabolites from starfish is a long-term direction of the studies of G.B. Elyakov Pacific Institute of Bioorganic Chemistry which celebrated its 55th anniversary in 2019. Continuing our research on biologically active steroid metabolites from the starfish species inhabiting the Vietnamese sea waters [11][12][13][14][15], herein, we describe the results of our investigation of steroid constituents from the methanolic extract of the starfish Anthenoides laevigatus Liao & A.M. Clark, 1989 (order Valvatida, family Goniasteridae), collected off the coastal waters of the Qui Nhon Province, Vietnam. We have isolated and structurally studied four new polyhydroxylated steroids 1−4, along with two previously known related steroids 5 and 6. Additionally, the action of compounds 1, 5, and 6 on cell viability and proliferation of normal and cancer cells, as well as colony formation of cancer cells in a soft agar clonogenic assay in vitro, have been investigated.
According to the presence of the [M + Na] + sodium adduct ion peak at m/z 491.3341 in the (+)HRESIMS spectrum and the [M -H]deprotonated ion peak at m/z 467.3380 in the (-)HRESIMS spectrum, the molecular formula C 27 H 48 O 6 of compound 3 has been found to be identical to that of 2 ( Figure S16). The detailed comparison of the 1 H-and 13 C-NMR spectra of compounds 3 and 2 has revealed that the proton and carbon resonances belonging to the steroid C/D rings and side chain of 3 are close to those of 2 indicating the 14,15α,16β,26-tetrahydroxy substitution in 3, while the proton and carbon signals of the steroid A/B rings of 3 substantially differed from those of 2 (Tables 1 and 2, Figures S17 and S18). The proton and carbon signals in the NMR spectroscopic data attributable to the A/B rings of 3 showed the presence of angular methyl group CH 3 -19 (δ H 1.13 s and δ C 26.3) and two oxygenated methines CH-3 (δ H 3.99 br. q (J = 2.7) and δ C 67.1) and CH-6 (δ H 3.66 q (J = 2.7) and δ C 74.3). The signal of CH 3 -19 in the 13 C-NMR spectrum of 3 was shifted from δ C 16.1 to 26.3 in comparison with that of 2. This fact strongly testified to cis-A/B ring fusion in 3 [16,20]. The coupling constant J = 2.7 of the broad quartet of H-3 corresponded well to the 3β-hydroxyl group in 5β-cholestane nucleus, and the coupling constant J = 2.7 of the quartet of H-6 indicated the 6β-hydroxyl group in 3 [16]. All the proton and carbon signals associated with the steroid nucleus and side chain were assigned by 2D experiments (Tables 1 and 2, Figure 3, Figures S19). Proton and carbon chemical shifts of the steroid A/B rings of 3 were similar to the corresponding data of (25S)-5β-cholestane-3β,6β,15α,16β,26-pentaol isolated from the starfish L. clathrata [16]. The key ROESY cross-peaks H 3 (Figure 3). As a result, steroid 3 was proved to be the cis-A/B ring fusion isomer of steroid 2 and its structure was established as (25S)-5β-cholestane-3β,6β,14,15α,16β,26-hexaol.  1 and 2, Figures S24). The deshielded shift of the signal of CH 3 -19 at δ C 25.6 in the 13 C-NMR spectrum and the existence of the ROESY cross-peak H 3 -19/H-5 immediately showed a 5β-cholestane skeleton in 4. The proton connectivities from C-1 to C-9 in A/B rings were ascertained using the COSY and HSQC experiments. The 1 H-and 13 C-NMR spectroscopic data, referred to the steroid A/B rings of 4, revealed the proton and carbon chemical shifts of three oxygenated methines, including CH-3 (δ H 3.70 br. q (J = 3.7) and δ C 72.2), CH-4 (δ H 3.66 t (J = 3.7) and δ C 75.3), and CH-6 (δ H 4.00 q (J =3.3) and δ C 73.3). The irradiation of the proton H-5 in the 1D TOCSY experiment gave an enhancing signal of the neighboring proton H-4, that confirmed the presence of an additional hydroxyl group at C-4 in 4 as compared with 3 ( Figure S30). Small values of the coupling constants of the protons H-3, H-4, and H-6 showed the absence of their axially axial interaction with neighboring protons. As a result, the 3β,4α,6β-trihydroxy pattern and the cis-A/B ring fusion were determined. Accordingly, the structure of 4 was established as (25S)-5β-cholestane-3β,4α,6β,14,15α,16β,26-heptaol.

The Effect of Compounds 1, 5, and 6 on Cancer Cells' Viability and Proliferation of Normal and Cancer Cells
In the first step of bioactivity investigations, the cytotoxicity of compounds 1, 5, and 6 was determined by measuring the metabolic activity of normal mouse epidermal JB6 Cl41, human colorectal carcinoma HT-29, and breast cancer MDA-MB-231 cells using MTS reagent. None of the tested compounds inhibited the viability of JB6 Cl41, HT-29, and MDA-MB-231 cells by 50% at concentrations up to 100 µM. The compounds 1, 5, and 6 decreased the cell viability by less than 20% at 100 µM (data not shown).
Next we determined the ability of the investigated compounds to affect cell proliferation of the tested cell lines. JB6 Cl41, HT-29, and MDA-MB-231 cells were treated with compounds 1, 5, and 6 at a non-toxic concentration of 20 µM for 24, 48, and 72 h. All tested compounds inhibited cell growth to a comparable degree (Figure 4). Compounds 1, 5, and 6 decreased proliferation of JB6 Cl41 cells by 20%, 22%, and 24%, respectively; HT-29 cells by 20%, 22%, and 26%, respectively; and MDA-MB-231 cells by 24%, 27%, and 29%, respectively, after 72 h of treatment. Cell viability was estimated using the MTS assay. Data are represented as the mean ± SD as determined from triplicate experiments. A Student's t-test was used to evaluate the data with the following significance levels: *p < 0.05, **p < 0.01, ***p < 0.001.

The Effect of Compounds 1, 5, and 6 on Colony Formation of Cancer Cells
The effect of compounds 1, 5, and 6 on the colony formation of human cancer HT-29 and MDA-MB-231 cells was investigated using the soft agar assay. Compounds 1, 5, and 6 (20 µM) were demonstrated to possess comparable inhibiting activity on colony formation of cancer cells and the decrease in the colony number of HT-29 cells was by 18%, 12%, and 18%, respectively, while MDA-MB-231 cells was by 35%, 30%, and 31%, respectively as compared with non-treated cells (control) ( Figure 5). In the present study it was demonstrated that triple negative human breast cancer cells MDA-MB-231 were more sensitive to the treatment of compounds 1, 5, and 6 than colorectal carcinoma cells HT-29. In summary, compounds 1, 5, and 6 are non-cytototoxic against normal JB6 Cl41 and cancer HT-29 and MDA-MB-231 cell lines at concentrations up to 100 µM, however, they are able to suppress cell proliferation and colony formation of cancer HT-29 and MDA-MB-231 cells.

Extraction and Isolation
The fresh animals (2.2 kg) were chopped into small pieces and extracted four times by MeOH with heating. The MeOH extract was evaporated in vacuo, and the residue (37 g) was dissolved in H 2 O (1.3 L). The H 2 O-soluble fraction was passed in two portions through an Amberlite XAD-2 column (7.5 × 28 cm) and eluted with distilled H 2 O until a negative chloride ion reaction was obtained, followed by elution with EtOH. The combined EtOH eluate was evaporated to give a brownish material (4.4 g). The resulting total fraction was chromatographed on a Si gel column (6.5× 15 cm) using CH 3 Cl-EtOH (stepwise gradient, 3:1→1:2, v/v), EtOH, and EtOH-H 2 O (stepwise gradient, 20:1→9:1, v/v) to give ten main fractions (1−10). Fractions 4 and 5 mainly contained the mixtures of polyhydroxylated steroids based on TLC data on Si gel plates in the eluent system toluene-EtOH (9:5, v/v). HPLC separation of fraction 4 (110 mg) on a Discovery C18 column with 55% aq. EtOH (1.5 mL/min) as an eluent system yielded pure 5 (4.1 mg, t R 48.8 min) and subfraction 4.1 and 4.2 that were further purified on a YMC-Pack Pro C18 column with 78% aq. MeOH (0.9 mL/min) as an eluent system to give pure 2 (0.9 mg, t R 15.3 min), 3 (0.9 mg, t R 14.1 min), and 4 (1.1 mg, t R 10.4 min). HPLC separation of fraction 5 (182 mg) on a Discovery C18 column with 55% aq. EtOH (1.5 mL/min) as an eluent system gave pure 1 (49.6 mg, t R 21.0 min) and 6 (23.6 mg, t R 26.1 min).

MTS Assay
To determine the cytotoxic activity of compounds 1, 5, and 6, JB6 Cl41, HT-29, and MDA-MB-231 cells were seeded at a density of 1.0 × 10 4 cells/200 µL of complete MEM/5% FBS, McCoy's 5A/10% FBS, and DMEM/10% FBS media, respectively, in 96-well plates. After incubation for 24 h, the cells were treated with tested compounds in the range of concentration 5-100 µM, while the control was treated with the complete medium only. Cells were cultured for additional 24 h at 37 • C in 5% CO 2 atmosphere. Subsequently, MTS reagent (20 µL) was added to each well, and the cells were incubated for an additional 3 h at 37 • C in 5% CO 2 . Absorbance was measured at 490/630 nm by a Power Wave XS microplate reader (BioTek, Winooski, VT, USA). All tested samples were carried out in triplicates. Compound's concentration causing 50% of cell viability inhibition (IC 50 ) were calculated.
To analyze the anti-proliferative activity of compounds 1, 5, and 6, the cells (1.0 × 10 4 cells/200 µL) were treated with tested compounds at concentration of 20 µM and incubated for an additional 24, 48, and 72 h at 37 • C in 5% CO 2 . MTS reagent (20 µL) was added to each well, and the cells were incubated for an additional 3 h at 37 • C in 5% CO 2 . Absorbance was measured at 490/630 nm using a microplate reader. All tested samples were analyzed in triplicates.

Soft Agar Assay
Cells (2.4 × 10 4 /mL) were grown in 1 mL of 0.3% Basal Medium Eagle's agar containing 10% FBS. The cells were treated by compounds 1, 5, and 6 at non-toxic concentration of 5, 10, and 20 µM. The cultures were maintained at 37 • C in 5% CO 2 incubator for 2 weeks and the number and size of the colonies were determined using a Motic microscope AE 20 (XiangAn, Xiamen, China) and ImageJ software bundled with 64-bit Java 1.8.0_112 (NIH, Bethesda, Maryland, USA).

Statistical Analysis
Results are expressed as the mean ± standard deviation (SD). Student's T test was used to evaluate the data with the following significance levels: *p < 0.05, **p < 0.01, ***p < 0.001. All assays were performed in at least three independent experiments.

Conclusions
Four new polyhydroxylated steroids were isolated along with two previously known related steroids from the Vietnamese starfish A. laevigatus and their chemical structures were elucidated. Two new compounds have the common 5α-cholestane skeleton, while the other two new compounds have the 5β-cholestane skeleton, which are rare among starfish steroids. Previously, only two steroid compounds with the cis-A/B ring junction were known from two species of the starfish L. clathrata and T. novaecaledoniae. Three of the substances that were isolated from A. laevigatus did not show cytotoxic effects against normal JB6 Cl41 and human colorectal cancer HT-29 and breast cancer MDA-MB-231 cells, however they suppressed cell proliferation and colony formation of cancer HT-29 and MDA-MB-231 cells.