marine drugs

: Two new steroid 3 β ,21-disulfates ( 1 , 2 ) and two new steroid 3 β ,22-and 3 α ,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 HRES-IMS/MS data. Compounds 1 and 2 have a ∆ 22 -21-sulfoxy-24-norcholestane side chain. Compounds 3 and 4 contain a ∆ 24(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
Sea cucumbers are marine invertebrates belonging to the class Holothuroidea of the phylum Echinodermata.They have wide distributions in all the oceans from shallow waters to significant depths.Sea cucumbers are mainly detritophages, collecting food from superficial sediments through their tentacles.The representatives of the order Dendrochirotida, including Cucumaria djakonovi, are filtrators, i.e., they filter solid particles disperged in sea water also using their branched tentacles.For this reason, sea cucumbers have great importance for the ecosystem, providing a sort of cleaning service by recycling and decomposing the substrate.Nowadays, a growing number of marine organisms are being chemically investigated in the search for new biomolecules with pharmacological potential.Sea cucumbers are among them because they produce specific metabolites, triterpene glycosides, which are uncommon in the animal kingdom.Investigations of the triterpene glycosides of diverse representatives of the class Holothuroidea have a long history, beginning from structural studies of the artifact genines obtained as a result of acid hydrolysis, followed by the structure establishment of native compounds [1,2].The continuation of these researches resulted in the appearance of the modern approaches of the separation of complex mixtures of native metabolites giving individual glycosides, including minor ones, possessing unique structural features [3,4].All these broaden the fundamental knowledge about the chemical diversity of natural products in general and of triterpenoids and their derivatives in particular.Actually, each new research study of the glycosidic composition of previously uninvestigated species or reinvestigation of species that were previously studied, but using modern techniques and equipment, leads to the finding of novel compounds [4,5].Fairly recently, the metabolomic studies of the glycosidic profiles of diverse species of holothuroids appeared, providing very useful data on the chemical diversity of glycosides, their distribution, and their content inside the different internal parts of animals, as well as their ecological signal functions [6][7][8][9][10].The interest of researchers in glycosides has been increased by their diverse and unique biological activity, including anticancer activity against different types of tumors, and immunomodulatory properties [11][12][13].Hence, some of these substances isolated from C. djakonovi demonstrated a promising action against human breast cancer cells, especially against the most aggressive triple-negative MDA-MB-231 cell line, suppressing cells' viability, inhibiting colony formation and growth, and inhibiting the migration of cells [5].These data indicate that glycosides could be used in the target therapy of breast cancer.An additional direction of scientific interest in these metabolites, based on the use of them as chemotaxonomic markers, allows for defining the systematic position and resolving taxonomic issues by analysis of the structural features of glycosides.Particularly, on the one hand, the isolation of novel compounds from C. djakonovi corroborated the apartness of this species from other closely related Cucumaria species; on the other hand, the presence of the same compounds in different species of the genus Cucumaria confirmed the specificity of glycosides at the genus level [5,14].The analysis of the structure-activity relationships of these metabolites shows the significance of the different parts of molecules for the demonstration of bioactivity and their influence on the mechanisms of action of glycosides [15].The accumulation of such data provides the opportunity to predict glycosides' bioactivity.So, the calculations of the quantitative structure-activity relationships (QSAR) for the glycosides from C. djakonovi are one of the first steps in this prospective direction.As data are accumulated, the combinatorial libraries of glycosides are formed and expanded, and, on the basis of the structural biogenetic rows of aglycones and the carbohydrate chains of glycosides, the pathways of biosynthesis could be traced [4], revealing the sequence of enzymes acting during the formation of glycosides, which is necessary for the regulation of the biosynthesis of these promising compounds in the future.
As a continuation of the study of the glycosides of C. djakonovi collected in Avacha Gulf, the isolation, structure elucidation, and biological activity testing of a series of new triterpene glycosides, djakonoviosides C 1 (1), D 1 (2), E 1 (3), and F 1 (4), and several known compounds were reported.The chemical structures of 1-4 were elucidated by the analyses of the 1 H, 13 C NMR, 1D TOCSY, and 2D NMR ( 1 H, 1 H COSY, HMBC, HSQC, ROESY) spectra as well as the HR-ESI mass spectra.All the original spectra are displayed in Figures S1-S57 in the Supplementary Materials.The hemolytic activity against human erythrocytes and the cytotoxicity against human breast cancer cell lines MCF-7, T-47D, and triple-negative MDA-MB-231, as well as the non-tumor mammary epithelial cell line MCF-10A, were studied.QSAR analysis was conducted for the series of 20 triterpene glycosides found in C. djakonovi.
As data are accumulated, the combinatorial libraries of glycosides are formed and expanded, and, on the basis of the structural biogenetic rows of aglycones and the carbohydrate chains of glycosides, the pathways of biosynthesis could be traced [4], revealing the sequence of enzymes acting during the formation of glycosides, which is necessary for the regulation of the biosynthesis of these promising compounds in the future.
As a continuation of the study of the glycosides of C. djakonovi collected in Avacha Gulf, the isolation, structure elucidation, and biological activity testing of a series of new triterpene glycosides, djakonoviosides C1 (1), D1 (2), E1 (3), and F1 (4), and several known compounds were reported.The chemical structures of 1-4 were elucidated by the analyses of the 1 H, 13 C NMR, 1D TOCSY, and 2D NMR ( 1 H, 1 H COSY, HMBC, HSQC, ROESY) spectra as well as the HR-ESI mass spectra.All the original spectra are displayed in Figures S1-S57 in the Supplementary Materials.The hemolytic activity against human erythrocytes and the cytotoxicity against human breast cancer cell lines MCF-7, T-47D, and triple-negative MDA-MB-231, as well as the non-tumor mammary epithelial cell line MCF-10A, were studied.QSAR analysis was conducted for the series of 20 triterpene glycosides found in C. djakonovi.

Structure Elucidation of Glycosides
The crude glycosidic sum of Cucumaria djakonovi (1.379 g) was obtained after the hydrophobic chromatography of the concentrated ethanolic extract on a Polychrom-1 column (powdered Teflon, Biolar, Latvia).Then, it was chromatographed on Si gel columns (CC) with the stepped gradient of the eluents system of CHCl       S1 and S2 The orientation of this substituent was confirmed by the NOE correlation between H-16 and H-32 as well as by the value of the coupling constant (J 16/17 = 8.7 Hz) [16].
Table 1. 13 C and 1 H NMR chemical shifts and HMBC and ROESY correlations of the aglycone moiety of djakonovioside C 1 (1).Extensive analysis of the 1 H, 13 C NMR, and HSQC spectra of the carbohydrate moiety of djakonovioside C 1 (1) (Table 2; Figures S1-S7) indicated the presence of the pentasaccharide chain with β-glycosidic bonds because five doublets of the anomeric protons at δ H 4.73-5.25 (J = 6.8-8.0Hz) and the signals of the corresponding anomeric carbons at δ C 102.4-105.6 were observed.Analysis of the 1 H, 1 H COSY, and 1D TOCSY spectra of each monosaccharide residue started from the anomeric proton, followed by ROESY and HSQC correlations analyses, allowed to determine the monosaccharide composition and the positions of glycosidic bonds.Hence, it was found that the second sugar in the chain is the xylose residue (Xyl2) that is linked to the Xyl1 residue at C-2.The second sugar unit (Xyl2) is bound with another two monosaccharides, being a branchpoint of the chain.The third monosaccharide unit-Glc3-was attached to C-4 Xyl2, and the additional xylose residue (Xyl5) was attached to C-2 Xyl2, causing glycosylation effects (δ C-4 Xyl2 77.9 and δ C-2 Xyl2 82.6).The analysis of the NMR spectra showed that 3-O-methylglucose (MeGlc4) was a terminal monosaccharide unit linked to C-3 Glc3.The positions of the glycosidic linkages were corroborated by the ROESY and HMBC correlations between the H-1 Xyl1 and H-3 (C-3) of the aglycone, H-1 Xyl2 and H-2 (C-2) Xyl1, H-1 Glc3 and H-4 (C-4) Xyl2, H-1 MeGlc4 and H-3 (C-3) Glc3, and H-1 Xyl5 and H-2 (C-2) Xyl2 (Table 2).The single sulfate group was attached to the C-4 Xyl1, causing an α-shifting effect of its signal to δ C 76.1, instead of the δ C ~70 observed in non-sulfated compounds.S16).The aglycone of djakonovioside D 1 (2) (Table 3, Figures S9-S15) was structurally close to that of 1, only differing by the position of the double bond in the side chain.So, all the signals of the polycyclic nuclei in the NMR spectra of 2 and 1 were almost coincident.The signals of the side chain were assigned by the analysis of the NMR spectra of 2: an isolated spin system formed by the protons from H-22 to H-24 was found in the 1 H, 1 H COSY spectrum, and the characteristic signals in the 13 C and 1 H NMR spectra at δ C 123.9 (C-24), δ H 5.00 (m, H-24) and 132.1 (C-25) corresponded to the 24(25)-double bond.Its position was confirmed by the correlations H-26(27)/C: 24, 25, 27 (26) in the HMBC spectrum.Extensive analysis of the 1 H, 13 C NMR, 1 H, 1 H COSY, 1D TOCSY, HSQC, and ROESY spectra of the carbohydrate part of djakonovioside D 1 (2) and okhotoside A 2 -1 (5) (Tables 4 and S3; Figures S9-S15 and S33-S38) revealed the presence of the same monosulphated pentasaccharide chains.The typical signals for quinovose residue were absent, while three signals characteristic for the hydroxymethylene groups of glucopyranose residues at δ C 61.4, 61.1, and 61.7 were observed.Further analysis of the sugar composition and sequence, as well as the glycosidic bond positions, showed that the second and third units in the chain were glucose residues (Glc2 and Glc3); the 3-O-methylglucose (MeGlc4) and xylose (Xyl5) residues were terminal, forming a carbohydrate chain branched by C-2 Glc2.A sulphate group was attached to C-4 Xyl1 (δ C 76.1), as in djakonovioside C 1 (1).The fragment ion peaks in the (−)ESI-MS/MS of 2 (Figure S16) were observed at m/z The molecular formula of djakonovioside E 1 (3) was determined as C 56 H 85 O 33 S 3 Na 3 from the [M 3Na − Na] − ion peak at m/z 1427.3942 (calc. 1427.3936), the [M 3Na − 2Na] 2− ion peak at m/z 702.2037 (calc.702.2022), and the three-charged ion [M 3Na − 3Na] 3− at m/z 460.4732 (calc.460.4717) in the (−)HR-ESI-MS (Figure S24) that confirmed the presence of three sulfate groups.In the 1 H and the 13 C NMR spectra of the carbohydrate chain of djakonovioside E 1 (3) (Table 5, Figures S17-S23), the four doublets of the anomeric protons at δ H 4.68-5.09(J = 7.3-8.5Hz) and the signals of the corresponding anomeric carbons at δ C 103.8-104.7 were indicative of a tetrasaccharide chain with β-glycosidic bonds between sugars.The first monosaccharide connected to the C-3 of the aglycone was a xylose (Xyl1) sulfated by C-4 (deduced from the deshielding of this signal to δ C 76.1).The subsequent analysis of the 1D TOCSY, 1 H, 1 H COSY, HSQC, and ROESY spectra revealed that the second residue was a glucose, which was linked to C-2 Xyl1.The third sugar-glucose (Glc3)-was attached to the typical position-C-4 Glc2 (cross-peak H-1 Glc3/H-4 Glc2 in the ROESY spectrum)-and was sulphated by C-6 (deshielding of the signal of C-6 Glc3 to δ C 67.4).Terminal 3-O-methylglucose residue was bound to C-3 Glc3, that was deduced from the corresponding NOE correlation (Table 5) and also contained a sulphate group because the signal of C-6 MeGlc4 was observed at δ C 67.0.Hence, the tetrasaccharide chain of 3 was new, having glucose as the second unit and bearing three sulfate groups.
The fragment ion peaks in the (−)ESI-MS/MS of 3 (Figure S24) were observed as a result of the fragmentation of the [M 3Na − Na] − ion at m/z 1427.5, giving ions at m/z 1367.4 The molecular formula of djakonovioside F 1 (4) was determined as C 59 H 93 O 34 S 3 Na 3 from the [M 3Na − Na] − ion peak at m/z 1487.4467(calc.1487.4511), the [M 3Na − 2Na] 2− ion peak at m/z 732.2320 (calc.732.2310), and the [M 3Na − 3Na] 3− ion peak at m/z 480.4926 (calc.480.4909) in the (−)HR-ESI-MS (Figure S32).Noticeably, for the isotope composition of the pseudomolecular ion of 4, the predominance of the [M 3Na − Na + 2] − ion peak is inherent.A normal isotope distribution was observed for two-and three-charged pseudomolecular ions.This is obviously explained by the chemical structure of the side chain.The protons of the methylene group CH 2 -23 adjacent to the 22-oxo group and the 24(25)-double bond were easily exchanged to deuterium when the glycoside was dissolved in the mixture C 5 D 5 N/D 2 O for the NMR spectra acquisition.The signal of CH 2 -23 could not be accumulated in the 13 C NMR spectrum of 4 for the same reason.Therefore, the spectra were repeatedly acquired in C 5 D 5 N/H 2 O, which resulted in the appearance of the signal at δ C 37.0 that corresponded to the proton's signal at δ H 3.61 (m, H-23) in the HSQC spectrum.
The aglycone of djakonovioside F 1 (4) (Table 6; Figures S25-S30) did not contain a γ-lactone ring (deduced from the absence of the characteristic signal in the 13 C NMR spectrum at δ C ~180 (C-18)) being of the non-holostane type.Actually, the signals of CH 3 -18 were observed at δ C 24.5 and δ H 1.29 (s).The signals in the downfield region of the 13     S17-S23.
Isokoreoside A (9) (Tables S7-S8; Figures S52-S57) was first isolated as a desulfated derivative from the fraction of the trisulfated glycosides of C. conicospermium [19], which was separated into individual compounds after the procedure of solvolytic desulfation.In native form, this compound was later obtained from C. frondosa [17].
Cucumarioside A 3 -2 (8) was isolated as native glycoside from C. djakonovi for the first time.The structure of 8 was determined earlier on the basis of its desulfated derivative, obtained the same way as 9 from Cucumaria conicospermium [19].So, the 2D NMR spectra and the assignments of the signals of 8 are provided first (Tables 8 and 9, Figures S45-S51).

Biologic Activity of the Glycosides
The cytotoxic activity of the compounds isolated from C. djakonovi was studied against three types of human breast cancer cells (MCF-7, T-47D, and triple negative MDA-MB-231), as well as the non-tumor mammary epithelial cell line MCF-10A.Djakonovioside A 1 [5] and cisplatin were used as the positive controls.Cytotoxic activity against all the selected cell lines was assessed using the MTT method (Table 10).Djakonovioside F 1 (4), okhotoside A 2 -1 (5), and cucumarioside A 2 -5 (6) showed strong hemolytic activity against human erythrocytes, with ED 50 0.51 ± 0.01, 1.53 ± 0.14, and 1.63 ± 0.13 µM, respectively.The hemolytic activity of djakonoviosides C 1 (1) and D 1 (2) was slightly lower but significant and close to each other.Djakonovioside D 1 (2), cucumarioside A 3 -2 (8), and isokoreoside A (9) demonstrated moderate hemolytic activity but were not active against all human tumor cell lines.Frondoside A 2 -3 (7) and koreoside A (10) did not show hemolytic or cytotoxic activity in the concentration range up to 50 µM.
To study antiproliferative properties, three most active glycosides were selected: djakonoviosides C 1 (1) and E 1 (3) and cucumarioside A 2 -5 (6).The prolonged incubation of cells for 48 and 72 h with glycosides 1 and 6 did not increase their EC 50 ; but, more importantly, the glycosides did not lose cytotoxicity over time.Only djakonovioside E 1 (3) showed an antiproliferative effect when incubated with MCF-7 cells for 48 h, demonstrating approximately a two-fold increase in EC 50 (0.78 ± 0.32 µM) (Figure 2a).
The clonogenic (or colony formation) assay is a standard in vitro cell survival assay based on the ability of a single cell to grow into a colony.In the human body, this uncontrolled growth of tumor cells leads to the formation of metastases.To study the effect of selected glycosides on the formation and growth of tumor cell colonies, a range of nontoxic concentrations was used against the MDA-MB-231 cell line (Figure 3a).Additionally, colonies of MCF-7 lines were exposed to the action of non-toxic doses of djakonovioside E 1 (3) (Figure 3b).For all tested compounds, a dose-dependent effect of inhibiting colony growth was observed.Cucumarioside A 2 -5 (6) at a concentration of 1 µM demonstrated the greatest inhibitory effect on the formation and growth of colonies: 44.32 ± 0.77% of the control.The inhibitory effects of djakonovioside C 1 (1) at concentrations of 2 and 1 µM were the same-approximately 25% compared to the control.Djakonovioside E 1 (3) blocked the formation of colonies of both MDA-MB-231 and MCF-7 to the same extent: at a concentration of 2 µM in relation to MDA-MB-231 cells to 35.68 ± 2.00% of the control and in relation to MCF-7 cells to 30.73 ± 0.49% of the control (Figure 3a,b).
Scratch analysis is used to study the effects of compounds with potential antitumor activity on cell motility and cell-cell interactions.In the control group of the MDA-MB-231 cell line, the scratch was overgrown within 24 h, while in the control group of the MCF-7 cell line, this happened after 72 h (Figure 4e).All the selected glycosides in the concentration range below their EC 50 inhibited the migration of breast cancer MDA-MB-231 and MCF-7 cells in a dose-dependent manner.The greatest effect, about 85% as compared to the control, was observed for cucumarioside A 2 -5 (6) at a concentration of 1 µM after 24 h of incubation with MDA-MB-231 cells (Figure 4b).Images of CFDA SE fluorescently labeled MCF-7 cells incubated with djakonovioside E 1 (3) demonstrate the reliable blockage of the migration of cells of the MCF-7 line under the glycoside action (Figure 4e).
The cytotoxic activity of djakonovioside E1 (3) was maximal in the series against the MCF-7 and MDA-MB-231 cell lines with a half-maximal inhibitory concentration of 1.52 ± 0.14 µM (Figure 2a) and 2.19 ± 0.17 µM (Table 10), respectively.Cucumarioside A2-5 (6) was the most active compound from the series in relation to T-47D cells (IC50 5.81 ± 0.86 µM (Figure 2b).Djakonovioside C1 (1) and cucumarioside A2-5 (6) demonstrated a pronounced effects against the MDA-MB-231 cell line (IC50 of 7.67 ± 0.32 and 2.58 ± 0.1 µM, respectively) (Figure 2c,d).To study antiproliferative properties, three most active glycosides were selected: djakonoviosides C1 (1) and E1 (3) and cucumarioside A2-5 (6).The prolonged incubation  of cells for 48 and 72 h with glycosides 1 and 6 did not increase their EC50; but, more importantly, the glycosides did not lose cytotoxicity over time.Only djakonovioside E1 (3) showed an antiproliferative effect when incubated with MСF-7 cells for 48 h, demonstrating approximately a two-fold increase in EC50 (0.78 ± 0.32 µM) (Figure 2a).The clonogenic (or colony formation) assay is a standard in vitro cell survival assay based on the ability of a single cell to grow into a colony.In the human body, this uncontrolled growth of tumor cells leads to the formation of metastases.To study the effect of selected glycosides on the formation and growth of tumor cell colonies, a range of non-toxic concentrations was used against the MDA-MB-231 cell line (Figure 3a).Additionally, colonies of MCF-7 lines were exposed to the action of non-toxic doses of djakonovioside E1 (3) (Figure 3b).For all tested compounds, a dose-dependent effect of inhibiting colony growth was observed.Cucumarioside A2-5 (6) at a concentration of 1 µM demonstrated the greatest inhibitory effect on the formation and growth of colonies: 44.32 ± 0.77% of the control.The inhibitory effects of djakonovioside C1 ( 1  Scratch analysis is used to study the effects of compounds with potential antitumor activity on cell motility and cell-cell interactions.In the control group of the MDA-MB-231 cell line, the scratch was overgrown within 24 h, while in the control group of the MCF-7 cell line, this happened after 72 h (Figure 4e).All the selected glycosides in the concentration range below their EC50 inhibited the migration of breast cancer MDA-MB-231 and MCF-7 cells in a dose-dependent manner.The greatest effect, about 85% as compared to the control, was observed for cucumarioside A2-5 ( 6) at a concentration of 1 µM after 24 h of incubation with MDA-MB-231 cells (Figure 4b).Images of CFDA SE fluorescently labeled MCF-7 cells incubated with djakonovioside E1 (3) demonstrate the reliable blockage of the migration of cells of the MCF-7 line under the glycoside action (Figure 4e).

Correlational Analysis and QSAR Model
The quantitative structure-activity relationship (QSAR) approach was applied to analyze the correlation between the hemolytic activity values and structures of all the glycosides isolated from C. djakonovi.Three-dimensional models of the glycosides were built, protonated at pH 7.4, and subjected to energy minimization.A conformational search was performed with MOE 2020.0901CCG software [22], and the dominant glycoside conformations were selected for further analysis.A set of various 2D and 3D descriptors (379 in total) responsible for physicochemical properties, as well as energy values and topological indexes numerically expressing the geometric properties of molecular structures, was calculated and analyzed using the QuaSAR-Descriptor tool of the MOE 2020.0901CCG software [22].
Noticeably, the descriptors choice has a fundamental significance, since no "almighty descriptor set" modeling all the activities and properties has been found yet.So, the selection of a suitable descriptor set for each activity and type of analyzed compounds is needed.So, in addition to the descriptors characterizing the physicochemical properties of the molecules (polarizability, refractive index, surface charge distribution, dipole

Correlational Analysis and QSAR Model
The quantitative structure-activity relationship (QSAR) approach was applied to analyze the correlation between the hemolytic activity values and structures of all the glycosides isolated from C. djakonovi.Three-dimensional models of the glycosides were built, protonated at pH 7.4, and subjected to energy minimization.A conformational search was performed with MOE 2020.0901CCG software [22], and the dominant glycoside conformations were selected for further analysis.A set of various 2D and 3D descriptors (379 in total) responsible for physicochemical properties, as well as energy values and topological indexes numerically expressing the geometric properties of molecular structures, was calculated and analyzed using the QuaSAR-Descriptor tool of the MOE 2020.0901CCG software [22].
Noticeably, the descriptors choice has a fundamental significance, since no "almighty descriptor set" modeling all the activities and properties has been found yet.So, the selection of a suitable descriptor set for each activity and type of analyzed compounds is needed.So, in addition to the descriptors characterizing the physicochemical properties of the molecules (polarizability, refractive index, surface charge distribution, dipole moment, hydrogen bonds' potential strength (donors and acceptors) [23], hydrophobic volume, surface area, atomic valence connectivity index, etc.), following descriptors as the presence/absence of 18(20)-lactone and the side chain, carbohydrate chain branching, and nature of the second sugar residue (glucose, quinovose, and xylose), the sulfate groups' number and positions were added to the descriptors set provided by the MOE-QuaSAR-Descriptor software (2020.0901CCG).The correlational analysis revealed the direct positive correlation between the hemolytic activities of the tested compounds in vitro and such descriptors as the molecular refractivity, log of the octanol/water partition coefficient [24], and partial charges distribution on the van der Waals surface area.In contrast, the diameter of the molecule, principal moment of inertia describing the different aspects of molecular shape, VDW surface area (Å 2 ), molecular VDW volume (Å 3 ), lowest hydrophobic energy, and approximation of the sum of the VDW surface areas of hydrophobic atoms (Å 2 ) were found to negatively correlate.
The analysis of the principal components (PCA) decreased the number of descriptors, leaving only those having a substantial contribution, and resulted in the division of the glycosides into two groups (Figure 5), which indicated the right way of the descriptor's choice.The linear QSAR model was built with the QuaSAR-Model tool of the MOE 2020.0901CCG software [22] using these descriptors.The model fits well with the experimental data on the hemolytic activities of glycosides with a correlation coefficient r 2 = 0.94702 and RMSE = 0.05234 (Figure S58).The model was cross-validated with r 2 cros = 0.82341 and RMSE cros = 0.24613.The QSAR model includes 148 terms, 58 from those that have the biggest contribution; however, the reduction in the number of descriptors to the latter value resulted in the quality deterioration of the correlation model.All these data indicate the extremely complex nature of the relationships between the structure of glycosides and their membranolytic action, with the multiple negligible effects of plenty of descriptors causing a considerable effect in combination with each other.
Mar. Drugs 2023, 21, x FOR PEER REVIEW 18 of 28 moment, hydrogen bonds' potential strength (donors and acceptors) [23], hydrophobic volume, surface area, atomic valence connectivity index, etc.), following descriptors as the presence/absence of 18(20)-lactone and the side chain, carbohydrate chain branching, and nature of the second sugar residue (glucose, quinovose, and xylose), the sulfate groups' number and positions were added to the descriptors set provided by the MOE-QuaSAR-Descriptor software (2020.0901CCG).The correlational analysis revealed the direct positive correlation between the hemolytic activities of the tested compounds in vitro and such descriptors as the molecular refractivity, log of the octanol/water partition coefficient [24], and partial charges distribution on the van der Waals surface area.In contrast, the diameter of the molecule, principal moment of inertia describing the different aspects of molecular shape, VDW surface area (Å 2 ), molecular VDW volume (Å 3 ), lowest hydrophobic energy, and approximation of the sum of the VDW surface areas of hydrophobic atoms (Å 2 ) were found to negatively correlate.The analysis of the principal components (PCA) decreased the number of descriptors, leaving only those having a substantial contribution, and resulted in the division of the glycosides into two groups (Figure 5), which indicated the right way of the descriptor's choice.The linear QSAR model was built with the QuaSAR-Model tool of the MOE 2020.0901CCG software [22] using these descriptors.The model fits well with the experimental data on the hemolytic activities of glycosides with a correlation coefficient r 2 = 0.94702 and RMSE = 0.05234 (Figure S58).The model was cross-validated with r 2 cros = 0.82341 and RMSEcros = 0.24613.The QSAR model includes 148 terms, 58 from those that have the biggest contribution; however, the reduction in the number of descriptors to the latter value resulted in the quality deterioration of the correlation model.All these data indicate the extremely complex nature of the relationships between the structure of glycosides and their membranolytic action, with the multiple negligible effects of plenty of descriptors causing a considerable effect in combination with each other.
Four reported glycosides (4 and 8-10) contained non-holostane aglycones without a lactone ring, including a new one in djakonovioside F 1 (4).Noticeably, djakonovioside F 1 (4) is the only compound from this row having a normal non-shortened side chain.The other three aglycones are hexa-nor-lanostane derivatives.Djakonovioside F 1 (4), cucumarioside A 3 -2 (8), and koreoside A (10) formed a biogenetic row reflecting the formation in the process of the biosynthesis of nor-lanostane derivatives through the stage of C-22 oxidation and following the oxidative cleavage of the 20( 22)-covalent bond.This leads to the elimination of a side chain.The same way is realized during the biosynthesis of steroid hormones.Remarkably, the set of trisulfated pentaosides of C. djakonovi (djakonovioside F 1 (4), isokoreoside A (9), and koreoside A ( 10)) complements and resembles the analogical set found in C. frondosa [17]: three pairs of isomers differed by the positions of the double bonds in the lanostane nuclei had a differently substituted C-22 or a shortened side chain.Compound 4, having a carbonyl group at C-22, can be considered as a missing link in the row of aglycones of the C. frondosa glycosides that fills the gap between the 22-hydroxylated (22-O-acetylated) and hexa-nor-lanostane derivatives.
Interestingly, isokoreoside A ( 9) is the single glycoside from C. djakonovi having a 9(11)-double bond.This indicates that two oxidosqualenecyclases (OSCs) (parkeol syntase and 9βH-lanosta-7,24-diene-3β-ol syntase) are operating in the biosynthesis and forming different types of polycyclic nuclei of the glycosides of C. djakonovi.However, the parkeol syntase seems to be preferably engaged in the biosynthesis of free 14α-methyl sterols with a 9(11)-double bond, while the 9βH-lanosta-7,24-diene-3β-ol synthase's action leads to triterpene aglycones' formation.Recent investigations into sea cucumber glycosides showed that some species contain mainly one type of aglycones with a certain position of intranuclear double bond, but the others accumulate glycosides with different positions for the double bonds in the polyclic system [4].However, even when glycosides preferably contain aglycones with one position of the intranuclear double bond, for example, ∆ 7 (8) aglycones in E. fraudatrix [36] and S. horrens [37] and ∆ 9 (11) -aglycones in A. japonicus [38], the genes of at least two OSCs are expressed, albeit with different efficiencies.In C. djakonovi, the situation seems to be different: the level of expression of parkeol syntase and its activity is obviously on the high level; however, the major part of biosynthesized parkeol is subsequently used for the formation of 14α-methylsterols with a 9(11)-double bond.Such parkeol-derived sterols are very typical for sea cucumbers belonging to the order Dendrochirotida, including the family Cucumariidae [39][40][41][42][43].
Generally, five new aglycones were found in the glycosides isolated from C. djakonovi [5].Noticeably, the new aglycones are mainly inherent for monosulfated compounds, while, among trisulfated compounds, only one glycoside having a novel aglycone was discovered.Eight types of carbohydrate chains comprised the glycosides of C. djakonovi.Two sugar chains, know earlier from the glycosides of C. okhotensis [18], C. japonica [34], and C. frondosa [44,45], are the parts of the djakonoviosides of groups A and B (monosulphated tetra-and pentaosides, with second quinovose and third xylose residues).Among the last series of glycosides from C. djakonovi, two types of carbohydrate moieties were found for the first time, while four were known earlier.The monosulfated pentasaccharide chain of djakonovioside C 1 (1), with xylose as the second unit, was new.Such a structural feature is very rare for holothurious glycosides.It was only found in two of several hundred compounds known from representatives of the order Dendrochirotida [46,47].Additionally, the trisulfated tetrasaccharide chain of djkonovioside E 1 (3), with glucose as the second unit, was revealed first.The oligosaccharide parts of djakonoviosides D 1 (2) and F 1 (4) and known compounds 5-10 are also characteristic for other representatives of the Cucumaria genus: C. okhotensis [18], C. conicospermium [19], C. frondosa [17,20], C. japonica [48], and C. koreaensis [21].These data confirm the possibility of using glycosides as chemotaxonomic markers.Belonging to C. djakonovi to the genus Cucumaria is undoubtfully evidenced by the presence of the same glycosides, characteristic to the other species of the genus.The supposition that all Cucumaria species share the same mono-, di-, and trisulfated pentasaccharide branched chains and species-specific aglycones is corroborated by the structures of C. djakonovi glycosides [14].However, the set of sugar chains biosynthesized by sea cucumbers of the Cucumaria genus is broadened with novel oligosaccharide moieties.
Biogenetic analysis of sugar chain structures of C. djakonovi glycosides showed that each type of carbohydrate chain is formed by a single way, including glycosylation with a certain monosaccharide residue or sulfation reactions.The branching of pathways occurs at the stage of glycosylation of the monoxylosides with additional xylose (forming the djakonovioside C 1 (1) chain), quinovose, or glucose residues (Figure 6).Subsequently the pathway of the biosynthesis of quinovose-containing glycosides divides depending on the type of third monosaccharide (xylose or glucose), which glycosylates quinovose residue by C-4.At this stage, tetrasaccharide chains of the djakonoviosides of group A are formed.Next, branching with xylose leads to the djakonoviosides of group B. In the case of glycosides having glucose as the third sugar, the tetrasaccharide precursors are obviously initially subjected to glycosylation, forming the pentasaccharide chains of cucumarioside A 2 -5 (6) and frondoside A 2 -3 (7), followed by further sulfation leading to disulfated cucumarioside A 3 -2 (8) and trisulfated djakonovioside F 1 (4), isokoreoside A (9), and koreoside A (10).
moieties were found for the first time, while four were known earlier.The monosulfated pentasaccharide chain of djakonovioside C1 (1), with xylose as the second unit, was new.Such a structural feature is very rare for holothurious glycosides.It was only found in two of several hundred compounds known from representatives of the order Dendrochirotida [46,47].Additionally, the trisulfated tetrasaccharide chain of djkonovioside E1 (3), with glucose as the second unit, was revealed first.The oligosaccharide parts of djakonoviosides D1 (2) and F1 (4) and known compounds 5-10 are also characteristic for other representatives of the Cucumaria genus: C. okhotensis [18], C. conicospermium [19], C. frondosa [17,20], C. japonica [48], and C. koreaensis [21].These data confirm the possibility of using glycosides as chemotaxonomic markers.Belonging to C. djakonovi to the genus Cucumaria is undoubtfully evidenced by the presence of the same glycosides, characteristic to the other species of the genus.The supposition that all Cucumaria species share the same mono-, di-, and trisulfated pentasaccharide branched chains and speciesspecific aglycones is corroborated by the structures of C. djakonovi glycosides [14].However, the set of sugar chains biosynthesized by sea cucumbers of the Cucumaria genus is broadened with novel oligosaccharide moieties.
Biogenetic analysis of sugar chain structures of C. djakonovi glycosides showed that each type of carbohydrate chain is formed by a single way, including glycosylation with a certain monosaccharide residue or sulfation reactions.The branching of pathways occurs at the stage of glycosylation of the monoxylosides with additional xylose (forming the djakonovioside C1 (1) chain), quinovose, or glucose residues (Figure 6).Subsequently the pathway of the biosynthesis of quinovose-containing glycosides divides depending on the type of third monosaccharide (xylose or glucose), which glycosylates quinovose residue by C-4.At this stage, tetrasaccharide chains of the djakonoviosides of group A are formed.Next, branching with xylose leads to the djakonoviosides of group B. In the case of glycosides having glucose as the third sugar, the tetrasaccharide precursors are obviously initially subjected to glycosylation, forming the pentasaccharide chains of cucumarioside A2-5 ( 6) and frondoside A2-3 (7), followed by further sulfation leading to disulfated cucumarioside A3-2 (8) and trisulfated djakonovioside F1 (4), isokoreoside A (9), and koreoside A (10).The third direction of sugar chains' biosynthesis is realized when the glucose glycosylates monoxylosides.The next steps of the chain's elongation are glycosylation with glucose and 3-O-methylglucose (third and fourth residues).Then, two pathways are possible: the two stages of sulfation resulting in the chain of djakonovioside F 1 (4) or the glycosylation of the C-2 position of Glc2 leading to the chain of djakonovioside D 1 (2).
So, the biosynthesis of the carbohydrate moieties of C. djakonovi glycosides looks rather strictly directed, in comparison with that of the aglycones, exhibiting the time shifts of some stages that is typical for the mosaic type of biosynthesis.The mosaicism of the biosynthesis of the glycosides of C. djakonovi clearly appeared at the level of the whole molecules as the combination of different sugar moieties with the same aglycones, due to the parallel and independent biosynthesis of these parts of molecules.There are some examples: cucumariosides A 2 -5 (6) and A 0 -1 and okhotoside A 1 -1 [5], as well as djakonoviosides C 1 (1) and E 1 (3) and okhotoside A 2 -1 (5), share the same aglycone.
It is interesting that hexa-nor-lanostane glycosides, structurally similar to sex (steroid) hormones, are mainly trisulfated or rarely disulfated (the biosynthetic precursors of trisulfated) compounds in the representatives of the genus Cucumaria [17,19,21], including C. djakonovi.These data, along with the metabolite profiling of a representative of the family Sclerodactylidae-the sea cucumber Eupentacta fraudatrix [10], where nor-lanostane derivatives were quantitatively predominant in the gonads-probably indicate that the role of such metabolites is different from that of the other glycosides, which provide the chemical defense of sea cucumbers.Actually, it is known that glycosides also synchronize the oocyte maturation in the holothurious population.So, a high level of sulfation makes them more polar and hydrophilic and facilitates their release into the surrounding sea water.Such an exchange with chemical signals provides the simultaneous readiness to spawn all the animals in the population.

Tendencies of Biologic Activity of the Glycosides: Observed and Calculated Structure Activity Relationships (SAR and QSAR)
The observed structure-activity relationships based on the hemolytic activity demonstrated that the most active compounds, okhotoside A 2 -1 (5) and cucumarioside A 2 -5 (6), from the tested series have holostane-type aglycones and monosulphated pentasaccharide chains.The high hemolytic effect of djakonovioside F 1 (4) was unexpected due to the absence of a lactone in 4 and the presence of three sulfate groups.The presence of a 22-keto group in the side chain of 4 in close proximity to the 20-OH group obviously resulted in the intramolecular hydrogen bond formation leading to a spatial structure similar to the lactone ring that resulted in the increase of the activity.Djakonovioside C 1 (1), having the same aglycone as okhotoside A 2 -1 (5), showed slightly lower activity due to the presence of xylose as the second residue of the sugar chain.Djakonovioside D 1 (2), being isomeric to 5 by the double bond position in the side chain, also was slightly less hemolytic.The absence of the activity of frondoside A 2 -3 ( 7) is easily explained by the presence of a hydroxy group in its side chain [15].The low hemolytic activity of cucumarioside A 3 -2 (8) and isokoreoside A ( 9) is determined by the presence of non-holostane aglycones with shortened side chains; the same features led to the complete loss of the activity of koreoside A (10).The partial compensation of the hemolytic action of 8 and 9, in comparison with compound 10, was presumably realized due to the presence of two sulfate groups instead of three in 8 and the 9(11)-position of the intranuclear double bond in 9. Erythrocytes were, as usual, more sensitive to the membranolytic action of glycosides than cancer cells.
Quantitative structure-activity relationships were calculated on the basis of correlational analysis of the physicochemical properties and structural features of the glycosidic molecules and their membranolytic activity and revealed the extremely complex nature of such relationships.The characteristics of molecules related with charged groups, such as polarization under the influence of the electric fields of neighboring ions, surface charge distribution, and impact of hydrophobic/hydrophilic areas and some others considerably influence the membranotropic activity of glycosides.The discovered dependence of the activity upon such chemical peculiarities as the number and positions of double bonds, single bond chain (aglycone side chain) length, availability of 18(20)-lactone, branching and monosaccharide composition of the carbohydrate chain, and positions and numbers of sulfate groups logically follows from the above physical characteristics.More importantly, the QSAR results are consistent with the observed structure-activity relationships.Actually, the availability of a normal non-shortened side chain is urgently needed for the glycoside to be active (i.e., hexa-nor-lanostane compounds 8-10 are almost not active).The presence of 18 (20)-lactone also provides significant activity, as illustrated not only by C. djakonovi glycosides [5] but also is a common tendency [15].The negative correlation of the molecular volume and shape with the hemolytic activity is confirmed by the observation that tetraosides with linear carbohydrate chains showed stronger effects than the corresponding pentaosides [5,15].The calculations showed that the number of sulfate groups has an ambiguous effect on the activity of the tested glycosides.In this case, the calculations are also backed with observations: disulfated cucumarioside A 3 -2 (8) is more active than trisulfated koreoside A (10).The presence of a third sulfate group, unlike the second one, is not conducive to the membranotropic properties of the analyzed glycosides.It should be noted that the influence of sulfate groups on the membranolytic action of the triterpene glycosides depends on the architecture of their carbohydrate chains and the positions of attachment of these functional groups.Hence, increasing the numbers of sulfates in the glycosides with tetrasaccaride and pentasaccharide chains branched by the C-2 of quinovose leads to the activity decreasing [15], which is in good accordance with the observations: trisulfated tetraoside djakonovioside E 1 (3) is weakly hemolytic.So, the complicated and ambiguous character of structure-activity relationships is related to the diverse impact on the membranolytic action of certain structural elements and their combinations in the glycosidic molecules.
Regarding the cytotoxicity of the tested compounds against human breast cancer cells, the selective action of djakonovioside E 1 (3) against the ER-positive MCF-7 cell line and the triple-negative MDA-MB-231 cell line, which fail to express receptors to sex hormones and have no approved targeted therapeutics, was the most important finding, especially amid the absence of a toxic effect in relation to normal mammary epithelial cells (MCF-10A) and low hemolytic activity.The MDA-MB-231 (triple-negative breast cancer) cell line was the most sensitive to cytotoxic action, while the MCF-7 cell line was the most resistant.
The action of djakonoviosides C 1 (1), E 1 (3), and cucumarioside A 2 -5 (6), which are the most active against cancer cells, was more deeply studied.It was shown that these compounds did not lose cytotoxicity over time, and djakonovioside E 1 (3) demonstrated an antiproliferative effect.The compounds were able to inhibit colony formation and growth in selected cell lines, with cucumarioside A 2 -5 (6) demonstrating the greatest inhibitory effect.The same glycoside was the strongest inhibitor of tumor cell migration.The other glycosides also reliably suppressed cell motility.Such properties of the studied glycosides corroborate their potential to be used as anticancer agents.

Animals and Cells
The specimens of sea cucumber Cucumaria djakonovi (family Cucumariidae; order Dendrochirotida) were gathered by scuba diving from a depth of 14-15 m near Starichkov's Island (Avacha Gulf) in July 2007.The taxonomic identification of the animals was performed by Stepanov V.G.Voucher specimen is kept in the Pacific Institute of Geography, Kamchatka Branch, Petropavlovsk-Kamchatsky, Russia.

Wound Scratch Migration Assay
Attached to special migration plate plastic bottom MCF-7 or MDA-MB-231 cells were separated by a silicone insert (Culture-insert 2 Well 24, ibiTreat).After removing an insert, gap between the cells was 500 ± 50 µm.Cell debris and floating cells were deleted by washing twice with PBS; 10 µM of 5 mM initial solution of CFDA SE ((5,6)carboxyfluorescein succinimidyl ester) (LumiTrace CFDA SE kit, Lumiprobe, Moscow, Russia) in DMSO was dissolved in PBS and added to cells for 5 min at 37 • C; after washing twice with PBS, the fresh culture medium was added.Then, cells were treated with various concentrations of glycosides or culture medium only (vehicle control) and left for 24 and 72 h.Cell migration into the wound area was observed under a fluorescence microscope (MIB-2-FL, LOMO, Russia) with objective 10× magnification.

Building a QSAR Model
QSAR model for the set of 20 glycosides was built using QuaSAR-Descriptor and QuaSAR-Model tools of MOE 2020.0901software [22].The procedure involved the following steps: a charge calculation and structure optimization, glycosides conformational search, descriptors calculation, correlational analysis, principal component analysis (PCA), removing the descriptors collinear with another descriptor (unnecessary descriptors), building a QSAR model and the model's cross-validation, removing the descriptors not contributing to the model, and model checking by making a graph showing the correlation between the model-predicted value and the experimental activity value expressed as pED 50 .

Conclusions
As result of thorough research on the glycosidic composition of the sea cucumber Cucumaria djakonovi, 11 new djakonoviosides and 9 known glycosides, found earlier in other representatives of the Cucumaria genus, were isolated in general.Twelve different aglycones were the parts of the found compounds, and five of them were new ones, including a unique one having a 23,16-hemiketal fragment.Nine types of carbohydrate chains composed the glycosides of C. djakonovi.Two types of sugar moieties were revealed first, including those with xylose or glucose residue in the second position of the chain.
The pathways of biosynthesis of the aglycones and carbohydrate moieties of C. djakonovi's glycosides were proposed, showing the regularities characteristic of the mosaic type of biosynthesis.
It was shown that, on the one hand, the unique species-specific composition of the glycosides is inherent for each species of the genus Cucumaria, and, on the other hand, the presence of some common glycosides for all representatives of the genus is typical.Some of the glycosides from C. djakonovi display promising anti-breast-cancer effects expressed as the inhibition of cells' viability, functioning, and motility-the aspects of carcinogenesis occurring in the organism.
Quantitative structure-activity relationships analysis confirmed the complicated, tricky character of these correlations because of the influence on the activity of a large number of properties and peculiarities of the molecules that act in combination altogether.Importantly, the calculated results of QSAR are in good accordance with the observed SAR, concluded on the basis of the experimental data.All these indicate that the application of this instrument for the prediction or modeling of the biologic activity of sea cucumbers' triterpene glycosides is rather prospective and useful.

3 )a
Recorded at 125.67 MHz in C 5 D 5 N/D 2 O. b Recorded at 500.12 MHz in C 5 D 5 N/D 2 O. c Bold-interglycosidic positions.d Italics-sulfate positions.Multiplicity by 1D TOCSY.The original spectra of 4 are provided in Figures S25-S31 .
Mar. Drugs 2023, 21, x FOR PEER REVIEW 16 of 28 ) at concentrations of 2 and 1 µM were the same-approximately 25% compared to the control.Djakonovioside E1 (3) blocked the formation of colonies of both MDA-MB-231 and MCF-7 to the same extent: at a concentration of 2 µM in relation to MDA-MB-231 cells to 35.68 ± 2.00% of the control and in relation to MCF-7 cells to 30.73 ± 0.49% of the control (Figure 3a,b).

Figure 5 .
Figure 5. Three-dimensional plot of hemolytic activity (pED50) depending on the principal components' values (PCA1-PCA3) calculated for 20 glycosides.The glycosides that demonstrated hemolytic activity with ED50 ≤ 10 µM were outlined as active and are marked in red, while the rest are marked in violet.

Figure 5 .
Figure 5. Three-dimensional plot of hemolytic activity (pED 50 ) depending on the principal components' values (PCA1-PCA3) calculated for 20 glycosides.The glycosides that demonstrated hemolytic activity with ED 50 ≤ 10 µM were outlined as active and are marked in red, while the rest are marked in violet.

Figure 6 .
Figure 6.The scheme of biosynthesis of carbohydrate chains of the glycosides of C. djakonovi.

Figure 6 .
Figure 6.The scheme of biosynthesis of carbohydrate chains of the glycosides of C. djakonovi.

mult. a δ H mult. (J in Hz) b,c,d HMBC ROESY
Recorded at 125.67 MHz in C 5 D 5 N/D 2 O. b Recorded at 500.12 MHz in C 5 D 5 N/D 2 O. c Bold-interglycosidic positions.d Italics-sulfate positions.Multiplicity by 1D TOCSY.The original spectra of 2 are provided in Figures S9-S15. a

Table 5 .
Cont.Recorded at 125.67 MHz in C 5 D 5 N/D 2 O. b Recorded at 500.12 MHz in C 5 D 5 N/D 2 O. c Bold-interglycosidic positions.d Italics-sulfate positions.Multiplicity by 1D TOCSY.The original spectra of 3 are provided in Figures a

Table 6 .
Cont.Recorded at 125.67 MHz in C 5 D 5 N/D 2 O. b Recorded at 500.12 MHz in C 5 D 5 N/D 2 O. * Recorded at 176.04 MHz in C 5 D 5 N/H 2 O.The original spectra of 4 are provided in Figures S25-S30. a

Table 8 .
Cont.Recorded at 125.67 MHz in C 5 D 5 N/D 2 O. b Recorded at 500.12 MHz in C 5 D 5 N/D 2 O.The original spectra of 8 are provided in Figures S45-S51. a

Table 9 .
Cont.Recorded at 125.67 MHz in C 5 D 5 N/D 2 O. b Recorded at 500.12 MHz in C 5 D 5 N/D 2 O. c Bold-interglycosidic positions.d Italics-sulfate positions.Multiplicity by 1D TOCSY.The original spectra of 8 are provided in Figures S45-S51. a

Table 11 .
Tumor cell selectivity index (SI; a ratio of IC 50 calculated for healthy and cancer cells) of tested glycosides.