Rare Ophiuroid-Type Steroid 3β,21-, 3β,22-, and 3α,22-Disulfates from the Slime Sea Star Pteraster marsippus and Their Colony-Inhibiting Effects against Human Breast Cancer Cells

Two new steroid 3β,21-disulfates (1, 2) and two new steroid 3β,22- and 3α,22-disulfates (3, 4), along with the previously known monoamine alkaloid tryptamine (5) were found in the ethanolic extract of the Far Eastern slime sea star Pteraster marsippus. Their structures were determined on the basis of detailed analysis of one-dimensional and two-dimensional NMR, HRESIMS, and HRESIMS/MS data. Compounds 1 and 2 have a Δ22-21-sulfoxy-24-norcholestane side chain. Compounds 3 and 4 contain a Δ24(28)-22-sulfoxy-24-methylcholestane side chain, which was first discovered in the polar steroids of starfish and brittle stars. The influence of substances 1–4 on cell viability, colony formation, and growth of human breast cancer T-47D, MCF-7, and MDA-MB-231 cells was investigated. It was shown that compounds 1 and 2 possess significant colony-inhibiting activity against T-47D cells, while compounds 3 and 4 were more effective against MDA-MB-231 cells.


Introduction
Highly oxidized steroid compounds are low-molecular-weight metabolites of sea stars (starfish) that belong to the phylum Echinodermata, class Asteroidea.These polar substances differ from related ones from other marine invertebrates in terms of their significant structural diversity and are subdivided into several structural groups, including polyhydroxysteroids, mono-, di-, and triglycosides of polyhydroxysteroids, steroid alkaloids, steroid conjugates with fatty acids, cyclic steroid triglycosides, and asterosaponins-steroid glycosides with carbohydrate moieties from three to six monosaccharide residues [1][2][3][4].Polar steroid compounds derived from sea stars were shown to exhibit a considerable variety of physiological effects, namely antimicrobial, antiviral, immunomodulatory, neurotrophic, anti-inflammatory, and others [5][6][7][8].In the last two decades, many new reports have appeared on studies of the antitumor activity of polar steroid compounds from starfish as well as on possible molecular mechanisms of action of these compounds [9,10].
Starfish steroids often contain a single sulfate group, which can be located either in the tetracyclic core or side chain, or in the carbohydrate chain of molecule.In contrast to starfish, representatives of another class of echinoderms-ophiuroids or brittle stars (Ophiuroidea)contain the characteristic steroid 3α,21-disulfates with mainly 5β-or ∆ 5 -cholestane cores, which are distinctive features of these animals [11][12][13][14].Notably, such steroid 3β(or α),21disulfates have been found in six starfish species of the Pterasteridae family instead of polar steroid metabolites common in representatives of the class Asteroidea [15].The physiological properties of ophiuroid-like steroid compounds from the sea stars of the Pterasteridae family have been insufficiently studied so far.Previously, these compounds from the starfish P. pulvillus were only shown to exhibit hemolytic activity on mouse erythrocytes [16].Recently, we have investigated the structures of new steroid 3β,21-and 3β,22-disulfates from the sea star Pteraster marsippus that was collected off the coast of the Kuril Islands (Sea of Okhotsk) [15].The mixture of two substances with oxo-group at the C-7 position in the tetracyclic pattern of P. marsippus was determined to exhibit a significant cytotoxic effect against two-and three-dimensional cultures of human breast carcinoma ZR-75-1 cells [15].In the present paper, in continuation of our investigation on the fraction of disulfated steroids from P. marsippus, the isolation and characterization of new disulfate ophiuroid-type steroid compounds 1-4 and the known compound 5 are described.Moreover, the cytotoxic activity and capability of compounds 1-4 to inhibit the viability, colony formation and growth of human breast cancer T-47D, MCF-7, and MDA-MB-231 cells are reported.We did not use normal cells, since the amount of isolated substances was limited (did not exceed 1 mg) and previous work showed that substances of this structural group did not exhibit significant cytotoxicity against human epithelial kidney HEK293 cells at concentrations of up to 100 µM [15].starfish, representatives of another class of echinoderms-ophiuroids or brittle stars (Ophiuroidea)-contain the characteristic steroid 3α,21-disulfates with mainly 5β-o Δ 5 -cholestane cores, which are distinctive features of these animals [11][12][13][14].Notably, such steroid 3β(or α),21-disulfates have been found in six starfish species of the Pterasteridae family instead of polar steroid metabolites common in representatives of the class Aster oidea [15].The physiological properties of ophiuroid-like steroid compounds from the sea stars of the Pterasteridae family have been insufficiently studied so far.Previously these compounds from the starfish P. pulvillus were only shown to exhibit hemolytic ac tivity on mouse erythrocytes [16].Recently, we have investigated the structures of new steroid 3β,21-and 3β,22-disulfates from the sea star Pteraster marsippus that was collected off the coast of the Kuril Islands (Sea of Okhotsk) [15].The mixture of two substances with oxo-group at the C-7 position in the tetracyclic pattern of P. marsippus was deter mined to exhibit a significant cytotoxic effect against two-and three-dimensional cultures of human breast carcinoma ZR-75-1 cells [15].In the present paper, in continuation of ou investigation on the fraction of disulfated steroids from P. marsippus, the isolation and characterization of new disulfate ophiuroid-type steroid compounds 1-4 and the known compound 5 are described.Moreover, the cytotoxic activity and capability of compounds 1-4 to inhibit the viability, colony formation and growth of human breast cancer T-47D MCF-7, and MDA-MB-231 cells are reported.We did not use normal cells, since the amount of isolated substances was limited (did not exceed 1 mg) and previous work showed that substances of this structural group did not exhibit significant cytotoxicity against human epithelial kidney HEK293 cells at concentrations of up to 100 µM [15].
In addition to steroid disulfates 1-4, the non-steroid metabolite tryptamine (5) was found in the sea star P. marsippus.The peak of protonated molecule [M + H] + at m/z 161.1073 in the (+)HRESIMS (Figure S29) exhibited the molecular formula of 5 was C 10 H 12 N 2 .The 13 C-NMR and DEPT spectra indicated the availability of 10 carbons in 5, including 2 methylenes, 5 methines, and 3 carbons unbound to the protons (Figures S31 and S32).The 1 H-1 H COSY and HSQC spectra showed the aromatic proton structural fragment from C-4 to C-7 and bonding of protons from C-2 to C-β (Figures S33 and S34).The main HMBC cross-peaks from H-2 to C-3, C-9; from H-4 to C-3, C-6, and C-8; from H-6 to C-4, C-8; from H-7 to C-5, C-9; from H-α to C-3, C-β; and from H-β to C-2, C-3, C-9, and C-α and the main ROESY correlations from H-4 to H-α, and H-β; and from H-2 to H-α, H-β confirmed the indolamine sceleton in 2 (Figures S35 and S36).As a result, the analysis of the NMR spectra and the HRESIMS led to the identification of compound 2 as tryptamine.The previously known tryptamine, a monoamine alkaloid, was found in animals, humans, and plants, although it was first discovered in starfish.Alkaloids have not often been found in starfish.For instance, tyramine and salsolinol as cations in salts of sulfated steroids and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTSA) were isolated from the sea star Lethasterias nanimensis chelifera [25].In addition, imbricatin [26], ovothiol A [27], and a number of guanidine metabolites [28] were found in some species of starfish.
The lack of high cytotoxicity of compounds 1-4 confirmed their safety, so they were used at non-toxic concentrations of 12.5, 25, and 50 µM for the following assays.The lack of high cytotoxicity of compounds 1-4 confirmed their safety, so they were used at non-toxic concentrations of 12.5, 25, and 50 µM for the following assays.
In addition, a known monoamine alkaloid tryptamine was obtained from P. marsippus in quantities comparable to steroid substances.Tryptamine was discovered in the starfish for the first time.Although some alkaloids were previously isolated from several species of the starfish both in the free form and in the form of salts with sulfated steroids as cations, this finding probably indicates the importance of tryptamine in the physiology of P. marsippus.

Figure 1 .
Figure 1.Chemical structures of steroids 1−4 and compound 5. Steroid 1 had the molecular formula C 26 H 40 O 8 S 2 Na 2 , determined from the peaks of [M − Na] − ion at m/z 567.2073 and [M − 2Na] 2− ion at m/z 272.1090 in the (−)HRESIMS, and from the peak of cationized molecule [M + Na] + at m/z 613.1847 in the (+)HRESIMS.

Figure 3 .
Figure 3. Main ROESY correlations for steroids 1-4.Steroid 3 had the molecular formula C 28 H 44 O 8 S 2 Na 2 , determined from the peaks of [M − Na] − ion at m/z 595.2392 and [M − 2Na] 2− ion at m/z 286.1253 in the (−)HRESIMS, and from the peak of cationized molecule [M + Na] + at m/z 641.2165 in the (+)HRESIMS.It was found that there were two sulfate groups in 3, which followed from the HRESIMS, NMR data, and the (−)HRESIMS/MS spectrum of precursor [M − 2Na] 2− ion at m/z 286.1254, containing of peaks of fragment ions at m/z 475.2904 [M − 2Na − HSO 4 ] − and 96.9610 [HSO 4 ] − (Figure S15).Comparison of the NMR spectroscopic data of steroids 3 and 1 indicated that the signals of protons and carbons belonging to the tetracyclic part of 3 are identical to those of 1, that proved the cholestane nucleus with ∆ 5 -3β-sulfoxy structural fragment in 3 (Figures S16 and S17).At the same time, the chemical shifts of H 3 -21 to C-17, C-20, and C-22; from H-22 C-20, and C-24; from H-23 to C-28; and from H-25 to C-24, C-26, C-27, and C-28 confirmed the position of a sulfoxy group at C-22 and the 24(28)-double bond.

Figure 4 .
Figure 4.The cytotoxic activity of compounds 1-4 against human breast cancer cells.(a) T-47D, (b) MCF-7, and (c) MDA-MB-231 cells were incubated with 1-4 (6.25, 12.5, 25, and 50 µM) or cisplatin (Cis) (6.25, 12.5, 25, and 50 µM) for 72 h.MTS assay was used to evaluate cytotoxicity of compounds.The results are presented as the mean ± SD for triplicate experiments.The asterisk (*) indicates a significant decrease in cell viability of cancer cells treated with different concentrations of