New Trisulfated Steroids from the Vietnamese Marine Sponge Halichondria vansoesti and Their PSA Expression and Glucose Uptake Inhibitory Activities

Seven new unusual polysulfated steroids—topsentiasterol sulfate G (1), topsentiasterol sulfate I (2), topsentiasterol sulfate H (3), bromotopsentiasterol sulfate D (4), dichlorotopsentiasterol sulfate D (8), bromochlorotopsentiasterol sulfate D (9), and 4β-hydroxyhalistanol sulfate C (10), as well as three previously described—topsentiasterol sulfate D (7), chlorotopsentiasterol sulfate D (5) and iodotopsentiasterol sulfate D (6) have been isolated from the marine sponge Halichondria vansoesti. Structures of these compounds were determined by detailed analysis of 1D- and 2D-NMR and HRESIMS data, as well as chemical transformations. The effects of the compounds on human prostate cancer cells PC-3 and 22Rv1 were investigated.

In addition to unusual structural features, trisulfated steroids possess promising biological properties [19]. In fact, a broad range of activities has been described to trisulfate steroids such as antibacterial [1,15,20], antifungal [15,16,21], antiviral (including anti-HIV and anti-HSV effects) [5,12,13,22,23], antiparasitic [21], and antiplatelet activities [24]. In addition, the inhibition of different enzymes [6,11,18], promotion of angiogenesis [25], and antitumor activity against various tumor cell lines [7,15,17,26] have been described. Thus, the search for new trisulfated steroids from sponges, including the analyses of their chemical structures and physiological properties, continues to be a promising area of research. Hopefully, this may lead to the development of a new generation of drugs for a broad spectrum of diseases.
In the course of our ongoing interest in new biologically active secondary metabolites of marine invertebrates, Halichondria vansoesti sponge, collected in Vietnamese waters during the 49th scientific cruise aboard the R/V 'Academic Oparin', was investigated. As a result, ten trisulfated steroids 1-10 were isolated (Figure 1). Using NMR spectroscopy, including 1 H, 13 C, HSQC, COSY, HMBC, and NOESY, as well as high-resolution mass spectrometry and chemical transformations, 1-4 and 8-10 were identified as new, unusual analogues of topsentiasterol sulfates and halistanol sulfates. Compounds 5-7 were previously known as chlorotopsentiasterol sulfate D, iodotopsentiasterol sulfates D [18], and topsentiasterol sulfate D, respectively [15]. Herein, we report the isolation, structural elucidation, proposed biosynthetic pathways, and the study of the biological activities of the isolated compounds. functionality is combined with a C-9(11)-double bond and a methyl group at C-14. One more subgroup includes topsentiasterol sulfates A-E from the sponge Topsentia sp. [15], Sch 575867 from the deep-water sponge belonging to the family Astroscleridae [16], spheciosterol sulfates from the sponge Spheciospongia sp. [17], as well as chloro-and iodotopsentiasterol sulfates D, isolated from the sponge Topsentia sp. in our laboratory [18]. These compounds contain a common Δ 9(11) -unsaturated, 4β-hydroxy-14α-methyl, 2β,3α,6α-trisulfated steroid nucleus.
In addition to unusual structural features, trisulfated steroids possess promising biological properties [19]. In fact, a broad range of activities has been described to trisulfate steroids such as antibacterial [1,15,20], antifungal [15,16,21], antiviral (including anti-HIV and anti-HSV effects) [5,12,13,22,23], antiparasitic [21], and antiplatelet activities [24]. In addition, the inhibition of different enzymes [6,11,18], promotion of angiogenesis [25], and antitumor activity against various tumor cell lines [7,15,17,26] have been described. Thus, the search for new trisulfated steroids from sponges, including the analyses of their chemical structures and physiological properties, continues to be a promising area of research. Hopefully, this may lead to the development of a new generation of drugs for a broad spectrum of diseases.
In the course of our ongoing interest in new biologically active secondary metabolites of marine invertebrates, Halichondria vansoesti sponge, collected in Vietnamese waters during the 49 th scientific cruise aboard the R/V 'Academic Oparin', was investigated. As a result, ten trisulfated steroids 1-10 were isolated (Figure 1). Using NMR spectroscopy, including 1 H, 13 C, HSQC, COSY, HMBC, and NOESY, as well as high-resolution mass spectrometry and chemical transformations, 1-4 and 8-10 were identified as new, unusual analogues of topsentiasterol sulfates and halistanol sulfates. Compounds 5-7 were previously known as chlorotopsentiasterol sulfate D, iodotopsentiasterol sulfates D [18], and topsentiasterol sulfate D, respectively [15]. Herein, we report the isolation, structural elucidation, proposed biosynthetic pathways, and the study of the biological activities of the isolated compounds.

Results and Discussion
Concentrated EtOH extract of the sponge Halichondria vansoesti was partitioned between aqueous EtOH and n-hexane. The aqueous EtOH-soluble materials were further applied on a reversed-phase column chromatography (YMC-gel) and eluted successively with H ), respectively, were indicated in the (−)-HRESIMS of 1 ( Figure S2).
The data of 1D-and 2D-NMR spectra of 1 (Tables 1 and 2   Further analysis of the 1D-and 2D-NMR data of 1, and the comparison of its NMR data with those in the literature revealed that 1 contains a ∆ 9(11) -4β-hydroxy-14α-methyl-2β,3α,6α-trisulfated steroid core ( Figure 2, substructure I) and the same side chain, containing C-20 (21) to C-24 (29) (Figure 2, substructure III), as that found in the previously described topsentiasterol sulfates A-E [14], Sch 575867 [16], spheciosterol sulfates A-C [17], and chloro-and iodotopsentiasterol sulfates D (5,6) [18]. The 1 H and 13 C NMR spectra of 1 (Tables 1 and 2, Figures S3 and S4) were almost identical to those of topsentiasterol sulfate C [15]. The only exceptions were the signals of the protons linked to C-27 and C-28, which were shifted to lower frequencies (δ H 6.85/δ C 139.4, δ H 3.92/δ C 48.3) in the spectrum of 1. Moreover, the HRESIMS ( Figure S2) data showed that the molecular mass of 1 was 1 amu less than that of topsentiasterol sulfate C. Based on the above data, and in combination with 2D NMR data ( Figures S5-S7), the presence of the 1,5-dihydro-2H-pyrrol-2-one portion in the terminal part of the side chain of 1 ( Figure 2, substructure III) was proposed. To the best of our knowledge, this is the first report on the 1,5-dihydro-2H-pyrrol-2-one moiety found in polysulfated steroids from sponges. Comparison of the NOESY ( Figure S8) data of the steroid nucleus of 1 with those of topsentiasterol sulfate C and the related analogs [15][16][17][18] suggests that all the stereogenic centers of these compounds have the same relative configurations. Key NOESY correlations of the steroid core of 1 are shown in Figure 3. Thus, 1 is a new analogue of the topsentiasterol sulfate C [15], containing a unique structural element with a nitrogen atom in the side chain. Therefore, it was named topsentiasterol sulfate G.
Mar. Drugs 2019, 17, x 5 of 15 of 1 are shown in Figure 3. Thus, 1 is a new analogue of the topsentiasterol sulfate C [15], containing a unique structural element with a nitrogen atom in the side chain. Therefore, it was named topsentiasterol sulfate G.  Detailed studies of the 1D-and 2D-NMR spectra of 1-4, 8 and 9 (including the determination of the relative configuration of stereogenic centers using NOESY data, Tables 1 and 2, Figure 3) were performed. The generated data were compared to those of the known analogs, such as topsentiasterol sulfates A-E [15], Sch 575867 [16], spheciosterol sulfates A-C [17], chlorotopsentiasterol sulfate D (5), and iodotopsentiasterol sulfate D (6) [18]. Indeed, signals of the steroid nuclei in these compounds and in the isolated polysulfated steroids were almost identical. Therefore, it was proposed that 1-4, 8 and 9 have the same Δ 9(11) -4β-hydroxy-14α-methyl-2β,3α,6α-trisulfated steroid nucleus, with a variation of the side chain.  Figure 3. Thus, 1 is a new analogue of the topsentiasterol sulfate C [15], containing a unique structural element with a nitrogen atom in the side chain. Therefore, it was named topsentiasterol sulfate G.  Detailed studies of the 1D-and 2D-NMR spectra of 1-4, 8 and 9 (including the determination of the relative configuration of stereogenic centers using NOESY data, Tables 1 and 2, Figure 3) were performed. The generated data were compared to those of the known analogs, such as topsentiasterol sulfates A-E [15], Sch 575867 [16], spheciosterol sulfates A-C [17], chlorotopsentiasterol sulfate D (5), and iodotopsentiasterol sulfate D (6) [18]. Indeed, signals of the steroid nuclei in these compounds and in the isolated polysulfated steroids were almost identical. Therefore, it was proposed that 1-4, 8 and 9 have the same Δ 9(11) -4β-hydroxy-14α-methyl-2β,3α,6α-trisulfated steroid nucleus, with a variation of the side chain. Detailed studies of the 1D-and 2D-NMR spectra of 1-4, 8 and 9 (including the determination of the relative configuration of stereogenic centers using NOESY data, Tables 1 and 2, Figure 3) were performed. The generated data were compared to those of the known analogs, such as topsentiasterol sulfates A-E [15], Sch 575867 [16], spheciosterol sulfates A-C [17], chlorotopsentiasterol sulfate D (5), and iodotopsentiasterol sulfate D (6) [18]. Indeed, signals of the steroid nuclei in these compounds and in the isolated polysulfated steroids were almost identical. Therefore, it was proposed that 1-4, 8 and 9 have the same ∆ 9(11) -4β-hydroxy-14α-methyl-2β,3α,6α-trisulfated steroid nucleus, with a variation of the side chain.
The molecular formula of 2, C 32 H 47 Na 3  The 1 H and 13 C NMR data (CD 3 OD, Tables 1 and 2, Figures S10 and S11) of the side chain of 2 resemble those of topsentiasterol sulfate A [15], except for the presence of the methyl group at δ H 1.24 t, J = 7.1/δ C 16.1 (C-32) and a methylene group at δ H 3.74, 3.85/δ C 67.1 (C-31). Further analyses of the 2D-NMR spectral data, including COSY and HMBC spectra (Figures S12 and S14), revealed the following correlations: H-32/H-31, H-31/C-32, H-31/C-28, H-28/C-31 ( Figure 2, substructure IV). In addition, HRESIMS spectrum showed that the molecular weight of 2 is 28 amu more than that of topsentiasterol sulfate A [15]. Based on these data, 2 was elucidated as the ethyl ester of topsentiasterol sulfate A [15]. Since 2 has not been previously reported, it was named topsentiasterol sulfate I.
The  Tables 1 and 2, Figures S17 and S18) exhibited the signals of two carboxyl carbon (δ C 166.8 and 171.9) and a trisubstituted double bond (δ H 5.92/δ C 125.9, 157.9). The HMBC spectrum of 3, recorded in DMSO-d 6 ( Figure S21), displayed correlations from H-29 to C-26 and H-27 to C-28. Based on these data and mass spectrometry data, the presence of 2-substituted maleic acid in the side chain of 3 was suggested ( Figure 2, substructure V). The Z-configuration of the double bond in this fragment was established using NOESY experiment, in which a correlation from H-29 to H-27 was observed ( Figure S22).
To confirm the structure of 3, a methylation with diazomethane was carried out. The structure of the resulting product 3a was clarified using 2D-NMR and HRESIMS. Cross peaks from OMe-26 to C-26 and OMe-28 to C-28 were observed in the HMBC spectrum (  Figure S23). These data revealed that a dimethyl maleate was at the terminal of the side chain of the methylated derivative, as in the previously described topsensterol A, a polyhydroxylated steroid from the sponge Topsentia sp. [27]. Additionally, the desulfation reaction of 3 with trifluoroacetic acid was carried out. The structure of the obtained product (11) was established from the analysis of the HRESIMS data ( Figure 4, Figure S24). The molecular formula of 2, C32H47Na3O16S3, was established from the [M3Na − Na] − ion peak at m/z 829.1834 in the (-)-HRESIMS. In addition, the peaks at m/z 403.0980 and 261.0694 in the (-)-HRESIMS of 2 were observed, corresponding to the doubly-and triply-charged ions ([M3Na − 2Na] 2− and [M3Na − 3Na] 3− , respectively) ( Figure S9).
The To confirm the structure of 3, a methylation with diazomethane was carried out. The structure of the resulting product 3a was clarified using 2D-NMR and HRESIMS. Cross peaks from OMe-26 to C-26 and OMe-28 to C-28 were observed in the HMBC spectrum ( Figure 2 Figure S23). These data revealed that a dimethyl maleate was at the terminal of the side chain of the methylated derivative, as in the previously described topsensterol A, a polyhydroxylated steroid from the sponge Topsentia sp. [27]. Additionally, the desulfation reaction of 3 with trifluoroacetic acid was carried out. The structure of the obtained product (11) was established from the analysis of the HRESIMS data ( Figure 4, Figure S24).
Thus, 3 is a new analogue of polysulfated steroids from sponges, with a 2-substituted maleic acid in the terminal part of the side chain. Compound 4 was isolated as an inseparable mixture with the previously reported chlorotopsentiasterol sulfate D (5) and iodotopsentiasterol sulfate D (6) [18] (2:7:1). Detailed analysis of the HRESIMS (Figure S25), 1D-and 2D-NMR spectra (Tables 1,2, Figures S26-S31) of the mixture, as well as the comparison of these data with those for the previously described compounds [15][16][17][18], led to the identification of the structure 4. The molecular formula of 4 was determined as C30H42BrNa3O14S3 from the (-)-HRESIMS whose peaks of singly-, doubly-, and triply-charged ions Thus, 3 is a new analogue of polysulfated steroids from sponges, with a 2-substituted maleic acid in the terminal part of the side chain.
Compound 4 was isolated as an inseparable mixture with the previously reported chlorotopsentiasterol sulfate D (5) and iodotopsentiasterol sulfate D (6) [18] (2:7:1). Detailed analysis of the HRESIMS (Figure S25), 1D-and 2D-NMR spectra (Tables 1 and 2, Figures S26-S31) of the mixture, as well as the comparison of these data with those for the previously described compounds [15][16][17][18], led to the identification of the structure 4. The molecular formula of 4 was determined as C 30 Figure S26) displayed the higher frequency-shifted three pairs of doublets corresponding to H-27 and H-28 of bromo-, chloro-, and iodo-of 3-substituted furans. Comparison of the chemical shift values of these signals to those of chloro-and iodotopsentiasterol sulfates D from the literature [18] allowed us to assign the proton signals at δ H 6.39 (H-27) and 7.48 (H-28) to 4 ( Table 1). Using the integration of the signals corresponding to H-27 and H-28 in the 1 H spectra of the mixture containing 4, 5, and 6 showed that the mixture contains about 20% bromotopsentiasterol sulfate D (4) and 70% and 10% of chloroand iodo-derivatives (5,6) [18], respectively. Additional interpretation of the COSY, HSQC, and HMBC data confirmed that 4 is composed of the substructures I and VI (Figure 2, Figures S28-S30).
New trisulfated steroids, dichlorotopsentiasterol sulfate D (8) and bromochlorotopsentiasterol sulfate D (9), were isolated as an inseparable mixture. Attempts to separate 8 and 9 using repetitive HPLC failed, however, based on HRESIMS and 1D-and 2D-NMR data, it was estimated as a 9:  [28][29][30][31], and two-headed sphingolipids [32]. Moreover, triply-charged ions [M 3Na − 3Na] 3− in the spectra of both compounds were also observed (m/z 263.7026 and 278.3516, respectively) ( Figure S32). Intensities of the isotope peaks calculated for 8 confirm the proposed molecular formula C 30 [18]. The main differences between the NMR spectra of these compounds were the singlet of H-27 at δ H 6.31 for 8 and δ H 6.44 for 9 (integrating these signals, a ratio of 8 to 9 was established as 9:1), instead of two characteristic doublets at δ H 6.39 and 7.36, corresponding to H-27 and H-28 in the 1 H NMR spectrum of monochlorinated compound 5 [18]. Analysis of the COSY, HSQC, and HMBC spectrum confirmed the substructures I and VII (Figure 2, Figures S35-S37) in 8.
To determine the positions of the halogen atoms in 9, we have carried out careful analysis of the 1 H NMR and COSY spectra of the mixture of 4 and 5 (  (26-chloro) in the COSY spectrum. Therefore, in the case of a bulkier bromine substituent at C-26 the chemical shifts of H-24 and H-29 were observed in a higher field. Taking into attention, that the COSY spectra of 8 + 9 (Table 1, Figures S35 and S37a) showed only one cross-peak δ H 2.55 (H-24)/δ H 1.15 (H-29) similar to the cross-peak in the spectrum of 4, the position of the chlorine atom at C-26 in 9 was established. Based on this data and the HRESIMS data (see above), structure 9 was assigned to the bromochlorotopsentiasterol sulfate D. Nevertheless, the localization of Cl-at C-26 and Br at C-28 in 9 need to be further confirmed.  Figure S38). Detailed analysis of the 1 H and 13 C NMR, COSY, HSQC, HMBC, and NOESY spectra of 10 (CD 3 OD, Tables 1 and 2, Figure 2, substructures II and VIII, Figures S39-S44) and a comparison of its 1 H and 13 C chemical shift values with those reported in the literature for the previously described trisulfated steroids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18], indicated that 10 is a previously unreported 4β-hydroxy derivative of halistanol sulfate C [3], which was named 4β-hydroxyhalistanol sulfate C.
Interestingly, unlike all the previously described trisulfated steroids containing 4β-hydroxy group [15][16][17][18], 10 does not contain a C-9/C-11-double bond and the α-methyl group at C-14. Thus, 10 is the first member of a new structural subgroup of trisulfated steroids from sponges.
Interestingly, unlike all the previously described trisulfated steroids containing 4β-hydroxy group [15][16][17][18], 10 does not contain a C-9/C-11-double bond and the α-methyl group at C-14. Thus, 10 is the first member of a new structural subgroup of trisulfated steroids from sponges.
The biosynthesis of unusual side chains of trisulfated steroids, such as 1-9, could be hypothesized to originate from codisterol (12) ( Figure S45) [33]. This process could proceed via the C-27 alkylation of 12, followed by the proton loss and several reactions such as amination or hydratation of double bonds accompanied with cyclization, oxidation, hydrolysis, and halogenation, which would result in the formation of 1-6, 8, and 9 (Scheme 1).

Scheme 1. Proposed biogenesis of the side chains in 1-9.
The biological activities of 3, 7, and 10, as well as of the mixtures of 4 + 5 + 6 and 8 + 9 were investigated using human prostate cancer cells PC-3 and 22Rv1. PC-3 cells are known to be androgenindependent as they do not express the androgen receptor (AR(-)). 22Rv1 expresses both the androgen receptor (AR(+)), and the androgen receptor splice variant 7 (AR-V7(+)), the expression of AR-V7 mediates the resistant of this cell line to androgen-deprivation therapy [34,35]. PSA is a downstream target gene of the androgen receptor (AR) pathway. Thus, suppression of PSA expression may indicate the inhibition of AR-signaling. AR-signaling is essential for the growth and survival of a significant number of prostate cancer cell types. In fact, downregulation of AR signaling mediated by androgen withdrawal is the standard first-line therapy for advanced human prostate cancer [36]. The isolated compounds and the mixtures were found to inhibit the expression of PSA (prostate-specific antigen) in human drug-resistant 22Rv1 cells ( Figure 5A). Compound 3 and the mixture of 4 + 5 + 6, suppressed PSA expression at a concentration as low as 10 µM ( Figure 5A). Note, the IC50s for all the isolated compounds determined with the MTT assay in PC-3 and 22Rv1 cells were >100 µM, which could be due to the androgen-independent nature of these particular prostate cancer cell lines. The biological activities of 3, 7, and 10, as well as of the mixtures of 4 + 5 + 6 and 8 + 9 were investigated using human prostate cancer cells PC-3 and 22Rv1. PC-3 cells are known to be androgen-independent as they do not express the androgen receptor (AR(-)). 22Rv1 expresses both the androgen receptor (AR(+)), and the androgen receptor splice variant 7 (AR-V7(+)), the expression of AR-V7 mediates the resistant of this cell line to androgen-deprivation therapy [34,35]. PSA is a downstream target gene of the androgen receptor (AR) pathway. Thus, suppression of PSA expression may indicate the inhibition of AR-signaling. AR-signaling is essential for the growth and survival of a significant number of prostate cancer cell types. In fact, downregulation of AR signaling mediated by androgen withdrawal is the standard first-line therapy for advanced human prostate cancer [36]. The isolated compounds and the mixtures were found to inhibit the expression of PSA (prostate-specific antigen) in human drug-resistant 22Rv1 cells ( Figure 5A). Compound 3 and the mixture of 4 + 5 + 6, suppressed PSA expression at a concentration as low as 10 µM ( Figure 5A). Note, the IC 50 s for all the isolated compounds determined with the MTT assay in PC-3 and 22Rv1 cells were >100 µM, which could be due to the androgen-independent nature of these particular prostate cancer cell lines. Effect on glucose uptake. PC-3 cells were seeded in the 96-well plate, treated with the test compounds for 24 h in FBS-and glucose-free media, incubated with 2-NBDG, and then the fluorescence was measured. Apigenin (50 µM) was used as a positive control (Apig). Cells treated with vehicle (DMSO) were used as a control (Con). The glucose uptake was normalized to the cell viability, measured by the MTS test. Significant difference from the control is shown as follows: * p < 0.05 (Student's t-test).
Additionally, 3 and 7, as well as the mixtures of 4 + 5 + 6 and 8 + 9 suppressed glucose uptake in 22Rv1 cells ( Figure 5B), whereas 10 did not exhibit this effect (data not shown). Cancer cells are characterized by increased glucose consumption, which is related to their rapid growth and metabolism [37]. Inhibition of glucose uptake either by nutrient deprivation or inhibitors, may suppress cancer cells proliferation and/or sensitize cancer cells to standard therapies. Moreover, recent studies suggested a possible crosstalk between glycolysis and AR-signaling [38]. However, cytotoxic effects and proliferation inhibition were observed only at high concentrations of the isolated compounds (data not shown). Nevertheless, due to the promising activity on AR-receptor signaling and glucose uptake, 3 and 7, as well as the mixtures of 4 + 5 + 6 and 8 + 9 may serve as starting compounds for a development of novel prostate cancer drugs. To the best of our knowledge, this is the very first report on the ability of marine-derived steroid compounds to suppress the PSA expression/androgen receptor signaling, as well as glucose uptake in cancer cells.

General Procedures
Optical rotations were measured using a Perkin-Elmer 343 polarimeter. The 1 H-and 13 C NMR spectra were recorded on an Avance III-700 spectrometer at 700 and 175 MHz, respectively. Chemical shifts were referenced to the corresponding residual solvent signal (δH 3.30/δC 49.60 in CD3OD). The HRESIMS spectra were recorded on a Bruker maXis Impact II mass spectrometer (Bruker, Germany). Low-pressure column liquid chromatography was performed using YMC-GEL ODS-A. HPLC was performed using an Agilent Series 1100 Instrument equipped with a differential refractometer RIDDE14901810 and an YMC-ODS-A (250 × 10 mm) column. Sorbfil Si gel plates (4.5 × 6.0 cm, 5-17 µm, Sorbpolimer, Krasnodar, Russia) were used for thin-layer chromatography. MTT reduction was measured using the F200PRO reader (TECAN, Männedorf, Switzerland). Additionally, 3 and 7, as well as the mixtures of 4 + 5 + 6 and 8 + 9 suppressed glucose uptake in 22Rv1 cells ( Figure 5B), whereas 10 did not exhibit this effect (data not shown). Cancer cells are characterized by increased glucose consumption, which is related to their rapid growth and metabolism [37]. Inhibition of glucose uptake either by nutrient deprivation or inhibitors, may suppress cancer cells proliferation and/or sensitize cancer cells to standard therapies. Moreover, recent studies suggested a possible crosstalk between glycolysis and AR-signaling [38]. However, cytotoxic effects and proliferation inhibition were observed only at high concentrations of the isolated compounds (data not shown). Nevertheless, due to the promising activity on AR-receptor signaling and glucose uptake, 3 and 7, as well as the mixtures of 4 + 5 + 6 and 8 + 9 may serve as starting compounds for a development of novel prostate cancer drugs. To the best of our knowledge, this is the very first report on the ability of marine-derived steroid compounds to suppress the PSA expression/androgen receptor signaling, as well as glucose uptake in cancer cells.

General Procedures
Optical rotations were measured using a Perkin-Elmer 343 polarimeter. The 1 H-and 13 C NMR spectra were recorded on an Avance III-700 spectrometer at 700 and 175 MHz, respectively. Chemical shifts were referenced to the corresponding residual solvent signal (δ H 3.30/δ C 49.60 in CD 3 OD). The HRESIMS spectra were recorded on a Bruker maXis Impact II mass spectrometer (Bruker, Germany). Low-pressure column liquid chromatography was performed using YMC-GEL ODS-A. HPLC was performed using an Agilent Series 1100 Instrument equipped with a differential refractometer RIDDE14901810 and an YMC-ODS-A (250 × 10 mm) column. Sorbfil Si gel plates (4.5 × 6.0 cm, 5-17 µm, Sorbpolimer, Krasnodar, Russia) were used for thin-layer chromatography. MTT reduction was measured using the F200PRO reader (TECAN, Männedorf, Switzerland).

Animal Material
The sponge Halichondria vansoesti (order Suberitida, family Halichondriidae; Figure S46) was collected at a depth of 5-12 m by hand via scuba diving during the 49th scientific cruise on board R/V "Academik Oparin", in the period from 10 November, 2016 to 3 January, 2017, in the South China Sea (the territorial waters of Vietnam, 12 • 34 02 N; 109 • 24 26 E). The sponge material was identified by Grebnev B.B. A voucher specimen is kept under the registration number N 049-232 in the marine invertebrate collection of the G. B. Elyakov Pacific Institute of Bioorganic Chemistry (Vladivostok, Russia).

Extraction and Isolation
The sample of the sponge Halichondria vansoesti was immediately frozen after collection and kept at −20 • C. The biological materials (dry weight 13.7 g) were chopped into small pieces and extracted with EtOH (200 mL × 3). The combined EtOH solution was concentrated to obtain the crude ethanol extract (7.9 g), which was partitioned between n-hexane and aqueous EtOH (9:4

Methylation of 3
Compound 3 (0.5 mg) was converted to methyl ester 3a by treatment with an excess (1.5 mL) of a saturated solution of diazomethane in diethyl ether. The obtained derivative 3a was analyzed using 2D-NMR and HRESIMS (Figure 2, substructure Va, Figure S23).

Desulfation of 3
Compound 3 (<0.1 mg) was dissolved in 500 µL H 2 O and 16 µL of concentrated TFA was added and kept at 100 • C for 4 h. The reaction mixture was concentrated and purified by CC on YMC-GEL (1.5 × 2 cm) and eluted successively with H 2 O→EtOH. The subfraction eluted with EtOH was concentrated to give 11. The obtained derivative was analyzed using HRESIMS ( Figure 4, Figure S24).

Cell Lines and Culture Conditions
22Rv1 and PC-3 cell lines (human prostate cancer cell lines) were purchased from ATCC. Cells were cultured in monolayer in 10% FBS/RPMI media according to the manufacture's protocols and were regularly checked for mycoplasma contamination.

In Vitro MTT-and MTS-Based Drug Sensitivity Assay
The in vitro cytotoxic activity of the isolated compounds was evaluated by the MTT assay (performed as described previously [39]). For glucose uptake assay (3.7.5. Glucose uptake assay, see below) the viability was determined using the MTS assay (performed as described previously [40]). Treatment time was 48 h.

Glucose Uptake Assay
The examination of the effect of the compounds on glucose uptake was carried out using PC-3 cells and the Glucose Uptake Cell-Based Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA) and normalized to the cell viability measured using the MTS test [40]. 12,000 cells per well were seeded in two 96-well plates in 100 µL of media per well, incubated overnight, and treated with the drugs in 100 µL of FBS-free and glucose-free RPMI media per well for 24 h. For glucose uptake measurements, 10 µL of the 2-NBDG solution in FBS-free and glucose-free RPMI media (glucose uptake measurements, final 2-NBDG concentration in the wells was 50 µg/mL) of the vehicle (for cell viability measurements) was added to each well. After 6 h of incubation, the cells were washed twice with PBS (200 µL/well). Next, for the evaluation of glucose uptake, 100 µL of PBS was added to each well. The fluorescence was measured using Infinite F200PRO reader (TECAN, Männedorf, Switzerland). For cell viability measurements, the 100 µL of culture media containing MTS reagent was added to each well, and cell viability was measured using an Infinite F200PRO reader according to the manufacture's protocol.

Statistical Analysis
All assays were repeated at least three times. Results are expressed as the mean ± standard deviation (SD). Student's t-test was used to estimate the significance: * p < 0.05.

Conclusions
Ten polysulfated steroids 1-10 were isolated from the Vietnamese marine sponge Halichondria vansoesti. The structures of seven previously unreported compounds (1-4 and 8-10) were established by 1D-and 2D-NMR spectroscopy, HRESIMS, and chemical transformations . Compounds 1-4, 8, and  9 are new analogues of topsentiasterol sulfates. The characteristic ∆ 9(11) -4β-hydroxy-14α-methyl-2β, 3α, 6α-trisulfated steroid nucleus and unusual side chains, not previously described in trisulfated steroids from sponges, were found in the structures of these compounds. Compound 10 is a new analogue of halistanol sulfate, containing a 4β-hydroxy-2β, 3α, 6α-trisulfated steroid nucleus and this is the first report of this structure in sponge polar steroids. We proposed hypothetical pathways for the biosynthesis of the side chains in new topsentiasterol sulfates. Some of the isolated trisulfated steroids were able to suppress PSA expression and glucose uptake in human prostate cancer cells and thus may serve as starting compounds for the development of novel prostate cancer drugs.