Djakonoviosides A, A1, A2, B1–B4 — Triterpene Monosulfated Tetra- and Pentaosides from the Sea Cucumber Cucumaria djakonovi: The First Finding of a Hemiketal Fragment in the Aglycones; Activity against Human Breast Cancer Cell Lines

Seven new monosulfated triterpene glycosides, djakonoviosides A (1), A1 (2), A2 (3), and B1–B4 (4–7), along with three known glycosides found earlier in the other Cucumaria species, namely okhotoside A1-1, cucumarioside A0-1, and frondoside D, have been isolated from the far eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida). The structures were established on the basis of extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds of groups A and B differ from each other in their carbohydrate chains, namely monosulfated tetrasaccharide chains are inherent to group A and pentasaccharide chains with one sulfate group, branched by C-2 Qui2, are characteristic of group B. The aglycones of djakonoviosides A2 (3), B2 (5), and B4 (7) are characterized by a unique structural feature, a 23,16-hemiketal fragment found first in the sea cucumbers’ glycosides. The biosynthetic pathway of its formation is discussed. The set of aglycones of C. djakonovi glycosides was species specific because of the presence of new aglycones. At the same time, the finding in C. djakonovi of the known glycosides isolated earlier from the other species of Cucumaria, as well as the set of carbohydrate chains characteristic of the glycosides of all investigated representatives of the genus Cucumaria, demonstrated the significance of these glycosides as chemotaxonomic markers. The membranolytic actions of compounds 1–7 and known glycosides okhotoside A1-1, cucumarioside A0-1, and frondoside D, isolated from C. djakonovi against human cell lines, including erythrocytes and breast cancer cells (MCF-7, T-47D, and triple negative MDA-MB-231), as well as leukemia HL-60 and the embryonic kidney HEK-293 cell line, have been studied. Okhotoside A1-1 was the most active compound from the series because of the presence of a tetrasaccharide linear chain and holostane aglycone with a 7(8)-double bond and 16β-O-acetoxy group, cucumarioside A0-1, having the same aglycone, was slightly less active because of the presence of branching xylose residue at C-2 Qui2. Generally, the activity of the djakonoviosides of group A was higher than that of the djakonoviosides of group B containing the same aglycones, indicating the significance of a linear chain containing four monosaccharide residues for the demonstration of membranolytic action by the glycosides. All the compounds containing hemiketal fragments, djakonovioside A2 (3), B2 (5), and B4 (7), were almost inactive. The most aggressive triple-negative MDA-MB-231 breast cancer cell line was the most sensitive to the glycosides action when compared with the other cancer cells. Okhotoside A1-1 and cucumarioside A0-1 demonstrated promising effects against MDA-MB-231 cells, significantly inhibiting the migration, as well as the formation and growth, of colonies.


Introduction
Marine invertebrates belonging to the class Holothuroidea are named the "pearls of the sea" because some of them present valuable sea food and almost all, if not all, produce triterpene glycosides, demonstrating various biologic activities. Despite the fact that the number of sea cucumber species whose glycosidic compositions have been studied is steadily growing, the majority of them are still unexplored because of their inaccessibility or the complexity of isolating the individual compounds from the extracts obtained from the producer organisms. Every new investigation of the glycosidic composition of unstudied species of sea cucumbers or the reinvestigation of the glycosidic composition of the species studied earlier, using the modern methods of isolation of individual substances from the multicomponent mixtures, resulted in the finding of dozens of new structural variants of the glycosides, which have unique features [1][2][3][4][5]. The early studies of some representatives of the genus Cucumaria showed the species specificity of glycosidic composition that allowed the use of these compounds for resolving the taxonomic challenges arising because of the high phenotypical polymorphism within one species of Cucumaria, probably because of a long evolutionary history [6]. The comparing of the glycosides from some Cucumaria species revealed they are characterized by significant structural variability of the aglycones [6][7][8][9][10][11][12][13][14], while they shared the same carbohydrate chain structures, including predominant mono-, di-, and trisulfated pentaosides with the branching xylose unit attached to the second monosaccaride (Qui). Tetrasaccharide monosulfated sugar chains are also present in the glycosides of several Cucumaria species [11,15], but these compounds are not so abundant, as a rule.
It is known that the glycosides from Cucumaria possess cytotoxic, proapoptotic, and immunomodulatory properties. The immunomodulatory preparation Cumaside, created on the basis of cucumarioside A 2 -2 isolated from the sea cucumber Cucumaria japonica, also demonstrating the anticancer action [16]. Nowadays, triterpene glycosides from sea cucumbers attract the attention of scientists worldwide, being potential antitumor agents demonstrating cytotoxic and antiproliferative action against different human cancer cells in vitro, initiating apoptosis, inhibiting tubules formation, adhesion, migration, invasion, and the angiogenesis of cancer cells [17][18][19][20][21][22][23]. Breast cancer (BC) is the mostly widespread (more than 2.2 million of cases were registered in 2020) and leading cause of death from oncologic diseases for women. The treatment for breast cancer is personalized because it depends on the disease's stage and the molecular-biological type of the cancer, which is determined by the presence in tumor cells of the receptors for estrogen, progesterone, or human epidermal growth factor (HER2) [24]. The described biochemical features of BC give an advantage for the development of a target therapy against each molecular type of BC. Since searching for suppressors of breast cancer cells is an important scientific and medical task, the investigation of cytotoxic action against human breast cancer cell lines MCF-7, T-47D, and triple-negative MDA-MB-231 was undertaken.

Structure Elucidation of the Glycosides
The crude glycosidic sum of Cucumaria djakonovi (13.79 g) was obtained after the hydrophobic chromatography of the concentrated ethanolic extract on a Polychrom-1 column (powdered Teflon, Biolar, Latvia). Its initial separation was achieved using the chromatography on Si gel columns (CC) with the stepped gradient of the eluents system  Three known triterpene glycosides were found in the glycosidic fraction of C. djakonovi. The structures of the known compounds were examined by the analysis of NMR Three known triterpene glycosides were found in the glycosidic fraction of C. djakonovi. The structures of the known compounds were examined by the analysis of NMR and MS data followed by comparison with the literature data that led to identification of okhotoside A 1 -1 isolated first from C. okhotensis [10], cucumarioside A 0 -1 from C. japonica [25], and frondoside D from C. frondosa [26] ( Figure S65).
The sugar configurations in glycosides 1-7 were assigned as D on the basis of the analogy with all other known triterpene glycosides from sea cucumber.
Extensive analysis of the 1 H, 1 H COSY, 1D TOCSY, HSQC, and ROESY spectra of the carbohydrate moieties of compounds 1-3 (Table 1, Tables S1 and S2; Figures S1-S7, S10-S16 and S18-S24) indicated that the identical monosulfated tetrasaccharide chains are characteristic of these glycosides. The monosaccharide composition was determined as two xylose (Xyl1 and Xyl3), one quinovose (Qui2), and 3-O-methylglucose (MeGlc4). The positions of the glycosidic linkages established by the ROESY and HMBC correlations corresponded to the linear tetrasaccharide chain with β-glycosidic bonds: Xyl1 bonded to C-3 of the aglycone, Qui2 linked to C-2 Xyl1, Xyl3-to C-4 Qui2, and terminal MeGlc4-to C-3 Xyl3 (Table 1). The presence of a sulfate group was established by distinctive values of δ C of C-4 Xyl1 observed at δ C 75.9 and C-5 Xyl1 observed at δ C 64.1 (α-and β-shifting effects of sulfate group) and corroborated by the MS data of each glycoside. Such a structure of sugar moiety is common for the glycosides from the sea cucumbers of different taxa. The glycosides, bearing this carbohydrate chain and isolated from C. djakonovi, were named djakonoviosides of group A.  Figure S8). The spectra of the aglycone part of 1 (  (20)-lactone at δ C 179.7 (C-18) and δ C 85.3 (C-20), 7(8)-double bond at δ C 120.2 (C-7) and δ C 145.7 (C-8), and O-acetyl group δ C 169.5 (OCOCH 3 ) and δ C 21.3 (OCOCH 3 ), attached to C-16 (δ C 75.2) in the polycyclic nucleus. The position of this functionality was corroborated by the cross-peaks between H-15 and C-16 and the methyl group of acetoxy substituent and C-16 in the HMBC spectrum of 1. Common for the glycosides, the β-orientation of the 16-acetoxy group in djakonovioside A (1) was confirmed by ROE correlation H-16/H-32. The protons of the side chain (H-22-H-27) formed an isolated spin system in the COSY spectrum, showing that the signal of H-23 was shifted downfield to δ H 4.08. A corresponding carbon signal deduced by the HSQC spectrum was observed at δ C 65.9, indicating the presence of a hydroxyl group at this position. The known frondoside D [26], isolated from C. djakonovi along with new compounds, has the same aglycone as djakonovioside A (1). The (23S) configuration was suggested for frondoside D based on the comparison of the δ C of C-22-C-24 with those for stichlorogenol-the aglycone of the glycosides from Stichopus chloronotus-the stereochemistry of which had been established by X-ray crystallography [27]. To assign the configuration of the C-23 chiral center in compound 1, the modified Mosher's method was applied [28]. The aglycone moieties of djakonoviosides A 1 (2) and B 1 (4) were identical to each other, which was deduced from the coincidence of their NMR spectra (Table 3 and Table  S3, Figures S10-S15 and S26-S31). The signals corresponding to the polycyclic system of the aglycone part of 2 (Table 3, Figures S10-S15) were close to those of 1, indicating their identity. The signal of C-22 deduced from the common lanostane derivative HMBC correlations H-21/C-22 was observed at δ C 78.3. The corresponding proton signal (H-22) was observed at δ H 5.71 as a singlet. This proton was correlated in the HMBC spectrum with the signal of a quaternary carbon at δ C 213.4 (C-23), corresponding to the oxo group. Therefore, a 22-hydroxy-23-oxo fragment was supposed to be present in the side chain of 2. The coupling patterns of H-22 (s) and H-24 (dd), as well as the MS data of 2, confirmed the supposition.
The attempt to assign a C-22 configuration in compound 2 using the modified Mosher's method failed because the 22-O-MTPA esters were not formed. However, the analysis of the biogenetic background resulted in the assignment of a 22S configuration in djakonovioside A 1 (2). Earlier, the absolute R configuration of the C-22 chiral center was elucidated by Mosher's method in the glycosides of the holostane type isolated from the sea cucumber Cladolabes schmeltzii [29]. The same 22R configuration was established in the non-holostane aglycone of frondoside C by comparing its NMR data with those of model isomeric derivatives by C-22 [30]. Cucumarioside H 8 , isolated from Eupentacta fraudatrix and having a 16,22-epoxy fragment in the aglycone, is also characterized by the 22R configuration, which was established based on ROE correlation H-16/H-22 [31]. Hence, all these data suggested the same configuration, but in the case of 2 and 4 were designated as 22S (because of the changing of substituent seniority in comparison with earlier known glycosides). The configuration of the C-22 chiral center should be the same because of the significance of this stereocenter for the enzymatic cleavage of the side chain in the process of the biosynthesis of the nor-lanostane-type aglycones [32]. Moreover, the presence of ROE correlation H-16/H-22 in the spectrum of 4 ( Figure S30) confirmed this supposition.  2 Table 4); because of the vicinity of quaternary carbons C-20 and C-23, the latter signal was observed at δ C 96.2. The values of the chemical shifts indicated the presence of a hydroxy group at C-22 along with the hemiketal fragment formed by C-23 and C-16 in the aglycone of 3. The HMBC correlation between H-16 and C-23 observed for 3 confirmed this supposition. Only this structure of the aglycone of 3 corresponded to the chemical formula deduced from HR-ESI-MS data. The configuration of C-22 in 3 is the same as suggested for 2, which was deduced based on biogenetic background. The configuration of the C-23 chiral center was proposed on the basis of the observed ROE correlations and the evaluation of interatomic distances in the MM2-optimized models of the aglycones of 3 having αor β-orientation of the hydroxyl group at C-23 ( Figure 2). The distances between H-22 and H 2 -24 in the 23α-OH model were much less than in the 23β-OH model, indicating the probability of the observation of ROE correlations H-22/H-24 only in the case of 23α-OH. Taking into account the fact that the referred cross-peaks were observed in the ROESY spectra of 3 and 7 (Tables 4 and S7), the configuration of C-23 was assigned as R. 27 24. The configuration of C-22 in 3 is the same as suggested for 2, which was deduced based on biogenetic background. The configuration of the C-23 chiral center was proposed on the basis of the observed ROE correlations and the evaluation of interatomic distances in the MM2-optimized models of the aglycones of 3 having α-or β-orientation of the hydroxyl group at C-23 ( Figure 2). The distances between H-22 and H2-24 in the 23α-OH model were much less than in the 23β-OH model, indicating the probability of the observation of ROE correlations H-22/H-24 only in the case of 23α-OH. Taking into account the fact that the referred cross-peaks were observed in the ROESY spectra of 3 and 7 (Tables 4  and S7), the configuration of C-23 was assigned as R. For the additional corroboration of the aglycone structure in djakonoviosides A2 (3) and B4 (7), the latter compound, which was isolated in sufficient amount, was acetylated For the additional corroboration of the aglycone structure in djakonoviosides A 2 (3) and B 4 (7), the latter compound, which was isolated in sufficient amount, was acetylated to give the derivative of 7a (Figure 3 Figure S64) and corresponded to the peracetylated derivative with 11 acetoxy groups. The signals in the 13 C NMR spectrum of the aglycone moiety of 7a (Table S8, Figures S59-S63) corresponded to the holostane-type polycyclic nucleus (18(20)-lactone signals at δ C 178.4 (C-18) and 77.4 (C-20)) with a 7(8)-double bond (δ C 120.7 (C-7) and 145.6 (C-8)). The signal of C-16 was observed at δ C 77.2 being deshielded in comparison with that of the native compound 7 (δ C-16 70.2). The signals of an additional double bond were observed at δ C 143.2 (C-23), δ C 120.6 (C-24), and δ The derivative 7b has 11 acetoxy groups and one hydroxyl group that is obviously 23-OH because the tertiary hydroxyl is usually not exposed to acetylation. Thus, the structures of 7a and 7b corroborated the presence of a 23,16-hemiketal fragment in the aglycones of djakonoviosides A 2 (3) and B 4 (7). The triterpene nucleus with a hemiketal fragment was found for the first time among the diversity of known sea cucumber aglycones.
peak at m/z 1789.6109 [MNa+Na] (C80H111O40SNa), as well as compound 7b correspondin to molecular formula C80H113O41SNa by the ion peak at m/z 1807.6210 [MNa+Na] + in the (+ HR-ESI-MS. The derivative 7b has 11 acetoxy groups and one hydroxyl group that is ob viously 23-OH because the tertiary hydroxyl is usually not exposed to acetylation. Thus the structures of 7a and 7b corroborated the presence of a 23,16-hemiketal fragment in th aglycones of djakonoviosides A2 (3) and B4 (7). The triterpene nucleus with a hemiketa fragment was found for the first time among the diversity of known sea cucumber agly cones. These data indicate that djakonovioside A2 (3) Extensive analysis of the 1 H, 1 H COSY, 1D TOCSY, HSQC, and ROESY spectra of th carbohydrate parts of djakonoviosides B1-B4 (4-7) (Tables 5 and S4-S6; Figures S26-S32 S34-S40, S42-S48, and S50-S56) indicated they contain, identical to each other, monosul phated pentasaccharide chains branched at C-2 Qui2. The monosaccharide compositio was determined as three xylose (Xyl1, Xyl3, and Xyl5) residues, one quinovose (Qui2) res idue, and one 3-O-methylglucose (MeGlc4) residue. The comparison of the 13 C NMR These data indicate that djakonovioside Extensive analysis of the 1 H, 1 H COSY, 1D TOCSY, HSQC, and ROESY spectra of the carbohydrate parts of djakonoviosides B 1 -B 4 (4-7) (Tables 5 and S4-S6; Figures S26-S32, S34-S40, S42-S48 and S50-S56) indicated they contain, identical to each other, monosulphated pentasaccharide chains branched at C-2 Qui2. The monosaccharide composition was determined as three xylose (Xyl1, Xyl3, and Xyl5) residues, one quinovose (Qui2) residue, and one 3-O-methylglucose (MeGlc4) residue. The comparison of the 13 C NMR spectra of 4 and 1 showed the similarity of the signals of Xyl1, Xyl3, and MeGlc4, while the signal of C-2 Qui was deshielded to δ C 83.1 because of the effect of glycosylation, as well as five additional signals corresponding to xylose unit that were observed in the spectrum of 4. The positions of the glycosidic linkages established by the ROESY and HMBC correlations were the same as in the djakonoviosides of group A with additional correlation corresponding to the β-(1→2) glycosidic bond between C-1 Xyl5 and C-2 Qui2 (Table 5). Such a structure of a carbohydrate chain is characteristic of the cucumariosides of the A 0 group found first in Cucumaria japonica [25]. The glycosides, bearing this sugar chain and isolated from C. djakonovi, were named the djakonoviosides of group B.
The molecular formula of djakonovioside B 1 (4) was determined to be C 60 H 93 O 31 SNa from the [M Na -Na] −  These data indicate that djakonovioside The molecular formula of djakonovioside B 2 (5) was determined to be C 58 H 91 O 29 SNa from the [M Na -Na] − ion peak at m/z 1283.5371 (calc. 1283.5372) in the (−)HR-ESI-MS ( Figure S41). The signals of the polycyclic system in the aglycone moiety of djakonovioside B 2 (5) were close to those in djakonovioside A 2 (3) (Tables 4 and 6 (Table 6, Figures S34-S39). Hence, the aglycone of djakonovioside B 2 (5) differed from that of djakonovioside A 2 (3) by the absence of the 22-OH group and was also found for the first time in the holothurious glycosides.     Figure S49). The aglycone moiety of 6-those signals were deduced from the extensive analysis of the NMR spectra (Table 7, Figures S42-S48)-was found to be of the lanostane type, having 18(16)-lactone (from the signals of C-16 at δ C 80.0 and C-18 at δ C 182.8, as well as from the distinctive signals of H-16 at δ H 5.10 and H-17 at δ H 2.81, both being singlets [29], and the shielded signal of C-20 (δ C 72.8), compared to the same signals in the spectra of compounds 1-5 (at δ C ∼ 82)). The 16β-O configuration was confirmed by the absence of coupling constant J 17/16 and by the ROE correlation H-16/H-21 in the spectra of 6. The common lanostane derivative 20S configuration was corroborated by the cross-peaks H-17/H-21 and H-21/H-12 observed in the ROESY spectrum of djakonovioside B 3 (6). The hydroxyl group was attached to C-23 (δ C 66.5) in the side chain of 6, as in djakonovioside A (1) and frondoside D, which was deduced from the analyses of the COSY (H-22

Biosynthetic Pathways of the Aglycones of the Glycosides from C. djakonovi
It is known that holostane-type aglycones are biosynthesized via the hydroxylation of C-20 in a triterpene precursor followed by C-18 oxidation, resulting in the formation of 18(20)-lactone. When the hydroxyl groups are simultaneously present at C-16 and C-20 of the 18-carboxylated derivative, the formation of 18(16)-lactone occurred [32]. This process takes place during the biosynthesis of djakonovioside B 3 (6). The hydroxylation with subsequent acetylation of C-16 preceding the oxidation of C-18 prevents the formation of 18(16)-lactone and leads to the synthesis of holostane-type aglycones in djakonoviosides A (1), A 1 (2), B 1 (4), okhotoside A 1 -1, cucumarioside A 0 -1, and frondoside D.
Taking into account the fact that the glycosides are the products of a mosaic type of biosynthesis, different biosynthetic stages can be shifted in time or change places in the sequence of the enzymatic oxidative reactions. Presumably, this occurred in the processes of formation of the aglycones with hemiketal fragments (compounds 3, 5, and 7). Their precursor probably contains 16-hydroxyl, 18(20)-lactone, and obviously, a highly oxidized side chain with the 23-oxo group that is inherent for the aglycones of okhotoside A 1 -1, cucumarioside A 0 -1, djakonoviosides A 1 (2), and B 1 (4). The obtained data indicate that different aglycones of C. djakonovi glycosides are exposed to the action of the same monooxygenase during their biosynthesis. The intramolecular attack of the hydrogen of the hydroxy group at C-16 to the 23-oxo group leads to the cyclization of a 23,16-hemiketal fragment. The biogenetic network formed as a result of the biosynthesis of the aglycones found in the glycosides isolated from C. djakonovi is illustrated in Figure 4.  In the process of the biosynthesis of compounds 3, 5, and 7, the formation of pyranose hemiketal fragments is realized as quite similar to the formation of the pyranose forms of sugars as a result of ring-chain tautomerism-a non-enzymatic intramolecular reaction occurred in an open-chain isomer, which produces more stable cyclic compounds. In our case, a similar process may lead to the formation of compounds 3, 5, and 7 with alphapyranose fragments, as shown in Figure 2. It is considered that the acetate groups are introduced through intermediate hydroxy derivatives at catalysis by O-acetyltransferases. The probable reason for the conversion into 23,16-hemiketals characteristic of C. djakonovi is the retardation of O-acetyltransferase action on an intermediate 16-hydroxylated derivative.

Bioactivity of the Glycosides and Structure-Activity Relationships
Cytotoxic activity of djakonoviosides A-B4 (1-7), as well as of known glycosides isolated from C. djakonovi ( Figure S65), against erythrocytes and human breast cancer cell lines (MCF-7, T-47D, and triple negative MDA-MB-231), as well as leukemia HL-60 and embryonic kidney HEK-293, has been studied. Chitonoidoside L [33] and cisplatin were used as the positive controls ( Table 8). The activity of the glycosides against MCF-7, T-47D, MDA-MB-231, and HEK293 cells were examined by MTT assay and against HL-60 by MTS assay.  In the process of the biosynthesis of compounds 3, 5, and 7, the formation of pyranose hemiketal fragments is realized as quite similar to the formation of the pyranose forms of sugars as a result of ring-chain tautomerism-a non-enzymatic intramolecular reaction occurred in an open-chain isomer, which produces more stable cyclic compounds. In our case, a similar process may lead to the formation of compounds 3, 5, and 7 with alphapyranose fragments, as shown in Figure 2. It is considered that the acetate groups are introduced through intermediate hydroxy derivatives at catalysis by O-acetyltransferases. The probable reason for the conversion into 23,16-hemiketals characteristic of C. djakonovi is the retardation of O-acetyltransferase action on an intermediate 16-hydroxylated derivative.

Bioactivity of the Glycosides and Structure-Activity Relationships
Cytotoxic activity of djakonoviosides A-B 4 (1-7), as well as of known glycosides isolated from C. djakonovi ( Figure S65), against erythrocytes and human breast cancer cell lines (MCF-7, T-47D, and triple negative MDA-MB-231), as well as leukemia HL-60 and embryonic kidney HEK-293, has been studied. Chitonoidoside L [33] and cisplatin were used as the positive controls ( Table 8). The activity of the glycosides against MCF-7, T-47D, MDA-MB-231, and HEK293 cells were examined by MTT assay and against HL-60 by MTS assay. Six glycosides from ten tested demonstrated potent hemolytic action, while the rest of the compounds were only moderately hemolytic because of the presence of hydroxy-groups in the side chains along with an additional 23,16-hemiketal cycle, making the aglycone more rigid (compounds 3, 5, and 7), or non-holostane aglycone and hydroxy group in the side chain (glycoside 6). The cancer cells were, as usual, less sensitive to the membranolytic action of the glycosides than of the erythrocytes. It is noticeable that compounds 1 and 2 demonstrated rather high cytotoxic activity despite both bearing the hydroxyl groups in the side chain, whose activity-decreasing action is obviously compensated for by the presence of holostane aglycones and tetrasaccharide linear monosulfated chains [4,34].
Some patterns of the structure-activity relationships were deduced from the analysis of the cytotoxic actions of the tested glycosides against cancer cell lines. Okhotoside A 1 -1 was the most active compound in the series because of the presence of a tetrasaccharide linear chain and holostane aglycone with a 7(8)-double bond and 16β-O-acetoxy group [11] without any hydroxyl groups. Cucumarioside A 0 -1 [25], having the same aglycone as okhotoside A 1 -1 ( Figure S65), was slightly less active because of the presence of a branching xylose residue at C-2 Qui, while the activity of frondoside D [26], characterized by the same pentasaccharide sugar chain, was twofold decreased compared with cucumarioside A 0 -1 because of the presence of the 23-OH group in the aglycone. The possible decrease in the cytotoxicity level of djakonovioside A 1 (2), featuring the aglycone identical to frondoside D, was compensated for by the presence of a tetrasaccharide chain identical to that of okhotoside A 1 -1. Generally, the activity of the djakonoviosides of group A was higher than that of the djakonoviosides of group B containing the same aglycones, indicating the significance of a linear sugar chain containing four monosaccharide residues for the demonstration of membranolytic action by the glycosides. All the compounds containing hemiketal fragments, djakonovioside A 2 (3), B 2 (5), and B 4 (7), were inactive against cancer cells, as was djakonovioside B 3 (6), having non-holostane aglycone and a hydroxyl group in the side chain. All these patterns are in good agreement with the structure-activity relationships established earlier for the triterpene glycosides of sea cucumbers [34].
As regards the sensitivity of cancer cell lines to glycoside action, HL-60 and MDA-MB-231 (triple-negative breast cancer) were exposed to cytotoxic action to the greatest extent, while the MCF-7 cell line was more resilient.
For further investigation of glycoside action on colony formation and migration, the triple-negative breast cancer MDA-MB-231 cells were used, and the most active okhotoside A 1 -1 and cucumarioside A 0 -1, as well as djakonoviosides A (1) and A 1 (2), were selected. The glycosides did not lose cytotoxicity over time, and their effects increased after 48 and 72 h of incubation with the MDA-MB-231 cells ( Figure 5). For example, cucumarioside A 0 -1 demonstrated an almost twofold increase in the activity (EC 50 6.04 2.45 and 2.19 µM) after 48 and 72 h of exposition ( Figure 5C).
Clonogenic analysis is commonly used to study the influence of cytotoxic compounds on the survival and division of the cells and their ability to form colonies. The investigation of the action of selected glycosides in non-cytotoxic concentrations on the formation and growth of colonies of MDA-MB-231 cells demonstrated that the maximum inhibitory effect (70.76 ± 0.13% of the control) was observed for okhotoside A 1 -1 at a concentration of 0.5 µM (Figure 6). Cucumarioside A 0 -1 significantly inhibited the growth of colonies at all concentrations studied; the maximal blockage of the formation and growth of colonies by 43.54 ± 6.07% of the control was observed at a concentration of 0.5 µM. Djakonovosides A (1) and A 1 (2) showed a dose-dependent effect: a statistically significant inhibition of colony growth by 41.39 ± 3.16% and 19.24 ± 0.25% of the control was observed for 1 and 2, respectively, at the maximum concentration of 2 µM.
Migration of tumor cells plays a crucial role in the process of metastases growth, so the search for substances capable of inhibiting this process is very important. The ability of the glycosides to inhibit migration of MDA-MB-231 cells was tested in vitro by wound scratch migration assay. In the control group, MDA-MB-231 cells completely close migration to the wound area at 24 h ( Figure 7A). The statistically significant effects of the glycosides on migration were also observed after 24 h of incubation. Clonogenic analysis is commonly used to study the influence of cytotoxic compounds on the survival and division of the cells and their ability to form colonies. The investigation of the action of selected glycosides in non-cytotoxic concentrations on the formation and growth of colonies of MDA-MB-231 cells demonstrated that the maximum inhibitory effect (70.76 ± 0.13% of the control) was observed for okhotoside A1-1 at a concentration of 0.5 µM ( Figure 6). Cucumarioside A0-1 significantly inhibited the growth of colonies at all concentrations studied; the maximal blockage of the formation and growth of colonies by 43.54 ± 6.07% of the control was observed at a concentration of 0.5 µM. Djakonovosides A (1) and A1 (2) showed a dose-dependent effect: a statistically significant inhibition of colony growth by 41.39 ± 3.16% and 19.24 ± 0.25% of the control was observed for 1 and 2, respectively, at the maximum concentration of 2 µM. Migration of tumor cells plays a crucial role in the process of metastases growth, so the search for substances capable of inhibiting this process is very important. The ability  Clonogenic analysis is commonly used to study the influence of cytotoxic compounds on the survival and division of the cells and their ability to form colonies. The investigation of the action of selected glycosides in non-cytotoxic concentrations on the formation and growth of colonies of MDA-MB-231 cells demonstrated that the maximum inhibitory effect (70.76 ± 0.13% of the control) was observed for okhotoside A1-1 at a concentration of 0.5 µM ( Figure 6). Cucumarioside A0-1 significantly inhibited the growth of colonies at all concentrations studied; the maximal blockage of the formation and growth of colonies by 43.54 ± 6.07% of the control was observed at a concentration of 0.5 µM. Djakonovosides A (1) and A1 (2) showed a dose-dependent effect: a statistically significant inhibition of colony growth by 41.39 ± 3.16% and 19.24 ± 0.25% of the control was observed for 1 and 2, respectively, at the maximum concentration of 2 µM.  Compound 1 showed a maximum blockage of the migration by 79.92 ± 0.27% in relation to control at a concentration of 1 µM ( Figure 7A,B). A dose-dependent inhibitory effect was observed for 2 and okhotoside A 1 -1 ( Figure 7C,E). Djakonovioside A 1 (2) inhibited the migration of tumor cells by 79.52 ± 9.12% as compared to control at a concentration of 2 µM after 24 h incubation, while okhotoside A 1 -1 inhibited the migration by 74.76 ± 4.41%, already at the minimum studied concentration of 0.05 µM. Cucumarioside A 0 -1 strongly affected cell migration at all concentrations with approximately equal intensity ( Figure 7D). Thus, at the maximum concentration of 0.5 µM, the blocking of migration was 84.62 ± 2.89% in comparison with control, and at the minimum concentration of 0.05 µM, this value was 83.61 ± 0.72%. scratch migration assay. In the control group, MDA-MB-231 cells completely close migration to the wound area at 24 h ( Figure 7A). The statistically significant effects of the glycosides on migration were also observed after 24 h of incubation. Compound 1 showed a maximum blockage of the migration by 79.92 ± 0.27% in relation to control at a concentration of 1 µM ( Figure 7A,B). A dose-dependent inhibitory effect was observed for 2 and okhotoside A1-1 ( Figure 7C,E). Djakonovioside A1 (2) inhibited the migration of tumor cells by 79.52 ± 9.12% as compared to control at a concentration of 2 µM after 24 h incubation, while okhotoside A1-1 inhibited the migration by 74.76 ± 4.41%, already at the minimum studied concentration of 0.05 µM. Cucumarioside A0-1 strongly affected cell migration at all concentrations with approximately equal intensity ( Figure 7D). Thus, at the maximum concentration of 0.5 µM, the blocking of migration was 84.62 ± 2.89% in comparison with control, and at the minimum concentration of 0.05 µM, this value was 83.61 ± 0.72%.
Generally, some of the investigated glycosides demonstrated encouraging action against breast cancer cells, suppressing their viability and inhibiting the formation and growth of colonies and the ability of the cells to migrate of the most aggressive triplenegative MDA-MB-231 line of breast cancer.

Animals and Cells
The specimens of sea cucumber Cucumaria djakonovi (family Cucumariidae; order Dendrochirotida) were collected in the Avacha Gulf near Starichkov's Island in

Extraction and Isolation
The body walls and tentacles of the sea cucumbers were minced and extracted twice with refluxing 70% EtOH. The dry weight of raw material after extraction was 663.5 g. The combined extracts were concentrated to dryness in vacuum, dissolved in H2O, and chromatographed on a Polychrom-1 column (powdered Teflon, Biolar, Latvia). The first elution of the inorganic salts and impurities with H 2 O, followed by the elution of glycosides with 55% acetone, produced 1379 mg of a crude glycoside fraction. Its initial separation was achieved by using the chromatography on Si gel columns (CC) with the stepped gradient of the system of eluents CHCl 3 /EtOH/H 2 O in ratios 100:50:4, 100:75:10, 100:100:17, and 100:125:25 resulting in the obtaining of five fractions. Fractions I and II were repeatedly subjected to CC with the system of eluents CHCl 3 /EtOH/H 2 O (100:75:10), each for subsequent purification that resulted in obtaining subfractions 1 (244 mg) and 2 (640 mg), respectively. The HPLC of subfraction 1 on a reversed-phase column, Supelco Ascentis RP-Amide (

Acetylation of Djakonovioside B 4 (7)
Glycoside 7 (4 mg) was dissolved in 1 mL of a mixture of absolute pyridine and acetyl anhydride (1:1) and kept at room temperature for 12 h. The mixture was evaporated in vacuo (at 60 • C) to obtain acetylated derivative 7a and dissolved in C 5 D 5 N for NMR spectra registration. Derivative 7b was obtained by the same procedure, excluding the heating. All the substances were tested in concentrations from 0.1 µM to 50 µM. Cisplatin was used as positive control. The cell suspension (180 µL) and solutions (20 µL) of tested glycosides in different concentrations were injected in wells of 96-well plates (MCF-7, T-47D, MDA-MB-231, and HEK293-7 × 103 cells/well) and incubated at 37 • C for 24 h in an atmosphere with 5% CO 2 . Glycosides 1 and 2, cucumarioside A 0 -1, and okhotoside A 1 -1 at concentrations of 1.25-10.0 µM were incubated with MDA-MB-231 cells at 37 • C for 24, 48, and 72 h in an atmosphere with 5% CO 2 . After incubation, the glycosides with medium were replaced by 100 µL of fresh medium. Then, 10 µL of MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA) stock solution (5 mg/mL) was added to each well, followed by incubation of the microplate for 4 h. After this procedure, 100 µL of SDS-HCl solution (1 g SDS/10 mL d-H2O/17 µL 6 N HCl) was added to each well and incubated for 18 h. The absorbance of the converted dye formazan was determined with a Multiskan FC microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm. Cytotoxic activity of the tested glycosides was calculated as a concentration that caused 50% cell metabolic activity inhibition (IC50). The experiments were conducted in triplicate; p < 0.05.

Cytotoxic Activity (MTS Assay) (for HL-60)
The cells of the HL-60 line (10 × 10 3 /200 µL) were placed in 96-well plates at 37 • C for 24 h in a 5% CO 2 incubator and, then, treated with tested glycosides and cisplatin as positive control at concentrations between 0.1 and 50 µM for an additional 24 h of incubation. Then, the cells were incubated with 10 µL MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) for 4 h, and the absorbance in each well was determined at 490/630 nm with a PHERA star FS plate reader (BMG Labtech, Ortenberg, Germany). The experiments were conducted in triplicate. The results were presented as the percentage of inhibition that produced a reduction in absorbance after tested glycosides treatment compared to the nontreated cells (negative control); p < 0.01.

Hemolytic Activity
Erythrocytes were obtained from human blood (AB(IV) Rh+) by centrifugation with phosphate-buffered saline (PBS) (pH 7.4) at 4 • C for 5 min by 450 g on a LABOFUGE 400R centrifuge (Heraeus, Hanau, Germany) three times. Then, the erythrocytes residue was resuspended in ice cold phosphate saline buffer (pH 7.4) to a final optical density of 1.5 at 700 nm and kept on ice. For the hemolytic assay, 180 µL of erythrocyte suspension was mixed with 20 µL of test compound solution, as well as control, chitonoidoside L [33], in V-bottom 96-well plates. After 1 h of incubation at 37 • C, the plates were exposed to centrifugation for 10 min at 900 g in a laboratory LMC-3000 centrifuge (Biosan, Riga, Latvia). Then, 100 µL of supernatant was carefully decanted and transferred into new flat plates. The values of the lysis of the erythrocytes were found by measuring the concentration of hemoglobin in the supernatant with a Multiskan FC microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA); λ = 570 nm. The effective dose causing 50% hemolysis of erythrocytes (ED50) was calculated with a SigmaPlot 10.0 computer program. All the experiments were carried out in triple repetitions; p < 0.01.

Colony Formation Assay
The influence of glycosides on the proliferation of MDA-MB-231 cells was analyzed by clonogenic assay [35]. Briefly, MDA-MB-231 cells were cultured on 6-well plates at a density of 1 × 10 3 cells per well in control media (MEM media, 10% FBS, 10,000 U/mL of penicillin and 10,000 µg/mL of streptomycin) or in media supplemented with different concentrations of glycosides. Cells were incubated for one week at 37 • C with 5% CO 2 until the cells in the control plates formed colonies that were visible to the eye and were of a substantial size (at least 50 cells per colony). For fixation and staining, the media were removed and the cells were washed twice with PBS. The colonies were fixed with methanol for 25 min, then washed with PBS and stained with 0.5% crystal violet solution for 25 min at room temperature. The plates were then washed with water and air dried.

Wound Scratch Migration Assay
To analyze the influence of the tested compounds on tumor cell migration, MDA-MB-231 cells attached to the plate's plastic bottom were separated by a silicone insert from special migration plates (Culture-insert 2 Well 24, ibiTreat); then, the insert was removed, leaving a gap of 500 ± 50 µm (according to the manufacturer's data) between the cells. The cells were washed twice by PBS to remove cell debris and floating cells and loaded with a fluorescent probe, a (5,6)-carboxyfluorescein succinimidyl ester (CFDA SE) dye (LumiTrace CFDA SE kit, Lumiprobe, Moscow, Russia). The initial solution of CFDA SE at a concentration of 5 mM in DMSO was dissolved in PBS to prepare a 10 µM solution and was added to the cells for 5 min at 37 • C; then, the cells were washed twice with PBS, and fresh culture medium was added. After that, the cells were treated with various concentrations of glycosides and left for 8 and 24 h. Cells treated with culture medium only were used as control. Cell migration into the wound area was then observed under a fluorescence microscope (MIB-2-FL, LOMO, Saint Peterburg, Russia) with an objective ×10 magnification.

Conclusions
At the first stage of investigation of the glycosidic composition of the sea cucumber Cucumaria djakonovi, two fewer polar fractions containing monosulfated tetra-and pentaosides have been studied that resulted in the isolation of seven new djakonoviosides, A-B 4 (1-7), and three known glycosides found earlier in other representatives of the Cucumaria genus. The analysis of the structural peculiarities of isolated compounds revealed five different aglycones, four of them found for the first time, and two types of carbohydrate chains common for the glycosides of the Cucumaria species. Therefore, the trend that was disclosed earlier concerning the species specificity of the set of aglycones, as well as the genus-specific raw carbohydrate chains of the glycosides of the sea cucumbers belonging to genus Cucumaria [6], was confirmed by the structures of compounds from C. djakonovi. Unprecedented structural features of the glycosides 3, 5, and 7 consist of the presence of a pyranose 23,16-hemiketal cycle, formed similarly to the appearance of pyranose forms of sugars. Probably, it is connected with the retardation of acylation of a free hydroxyl at C-16 by the corresponding O-acetyltransferases in Cucumaria djakonovi.
Noticeably, the known okhotoside A 1 -1 and cucumarioside A 0 -1 isolated earlier from C. djakonovi demonstrated promising effects against the most aggressive triple-negative MDA-MB-231 cell line of breast cancer, significantly inhibiting the formation and growth of colonies and the migration of cells.  Institutional Review Board Statement: Ethical review and approval were waived for this study due to absence of humans and laboratory animals in the experiments.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.