Isolation, chemical modification and biological evaluation of steroids from the starfish acanthaster planci

From the starfish Acanthaster planci was isolated 14 compounds.

The structure of these compounds was determined by mass spectrometry,

nuclear magnetic resonance spectroscopy and other physicochemical

methods. The isolated and identified compounds include: planciside A

(AP1); planciside B (AP2); planciside C (AP3); planciside D (AP4); (3-Osulfothornasterol A (AP5); 5-ergost-7-en-3-ol (AP6); cholesterol (AP7);

astaxanthin (AP8); thymin (AP9); uracil (AP10); acanthaglycoside G

(AP11); pentareguloside G (AP12); acanthaglycoside A (AP13); and

maculoside (AP14)

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titute of Organic Biochemistry - Russian Federal Academy of Sciences - Vladivostok. Migration-cell Wound-healing assay The test substances (at concentration 10 μM) were added into the medium that was prepared a wound on MDA-MB-231 mammary carcinoma cell and check after 48 hours. The closure of the lesion area was measured and determined the percentage of distance traveled by the cell. The test was done at the Pacific Institute of Organic Biochemistry - Russian Federal Academy of Sciences – Vladivostok. CHAPTER 3. EXPERIMENT 3.1. Isolation of compounds from starfish Acanthaster planci This section details how to isolate compounds from A. planci starfish. The separation of compounds was summarized in the diagram in Figure 3.1. Figure 3.1. Diagram of isolated compounds from starfish A.planci 5 3.2. Physical constants and spectrum data of isolated compounds 3.3. Summary of cholesterol derivatives AP7 compound was identified as cholesterol, selected as the start material to convert into polyhydroxysteroid and hydroximinosteroid derivatives. 3.3.1. Synthesis of polyhydroxyl derivatives of cholesterol 3.3.2. Synthesis of hydroximinosteroid derivatives from cholesterol 3.4. Biological activity of isolated compounds and synthetic derivatives 3.4.1. Biological activity of polar steroid compounds isolated from starfish Acanthaster planci 3.4.2. Cytotoxic activity of cholesterol derived derivatives The hydroxysteroid compounds (15c-21c), hydroximinosteroid compounds (23c, 25c, 29c, 31c) and intermediate compounds (22c, 24c, 26c-28c, 30c) were evaluated for cytotoxic activity on three cancer cell lines as Hep G2, HeLa and T98G. CHAPTER 4. RESULTS AND DISCUSSION This chapter presents the results of research on isolating, metabolizing and determining the structure of compounds, results of cytotoxic activity, tumor suppression activity on soft agar and inhibitory activity metastases of the tested compounds. 4.1. Study on chemical composition of starfish Acanthaster planci This section details the results of the structure determination of 14 compounds isolated from A. planci, including 4 new compounds and 8 known compounds. Table 4.21: Summary table of substances isolated from starfish A.planci AP1. Planciside A (new compound) AP2. Planciside B (new compound) 6 AP3. Planciside C (new compound) AP4. Planciside D AP5. 3-O-sulfothornasterol A AP6. 5-ergost-7-en-3-ol AP7. Cholesterol AP8. Astaxanthin AP9. Thymin AP10. Uracil AP11. Acanthasglycoside G (new compound) AP12. Pentareguloside G AP13. Acanthasglycoside A AP14. Maculoside 7 * The following shows the spectral analysis and chemical structure determination of a new glycoside steroid called Acanthaglycoside G (AP11). • AP11 compound: Acanthaglycoside G (new compound) AP11 compound was isolated as solid, amorphous. The molecular formula of AP11 was determined to be C51H81O26SNa (M = 1164) from the [M+Na]+ sodium adduct ion peak at m/z 1187,4533 (theoretical calculations for molecular formula is C51H81Na2O26S = 1187,4532) in the (+) HR-ESI-MS spectrum and the [M–Na]− ion peak at m/z 1141,4735 (theoretical calculations for molecular formula is C51H81O26S = 1141,4737) in the (-) HR-ESI-MS, where M is the molecular mass of the intact sodium salt. In addition, the (–) HR-ESI- MS/MS spectrum of the [M–Na]− ion at m/z 1141 exhibited fragment ion peak at m/z 995 [(M–Na)–C6H10O4]−, 849 [(M–Na)–2xC6H10O4]−, 703 [(M–Na)– 3xC6H10O4]−, 557 [(M–Na)–4xC6H10O4]−, 411 [(M–Na)–5xC6H10O4]−, corresponding to the successive losses of one, two, three, four, and five 6- deoxyhexose units, respectively, and 393 [(M–Na)–4 x C6H10O4 –C6H12O5]−, corresponding to the loss of an oligosaccharide chain in AP11. The MS spectroscopic data confirmed the presence of five 6-deoxyhexoses in the carbohydrate moiety of AP11. 437.1941 1+ 526.3922 1+ 605.2223 2+ 774.6342 1187.4533 3+ 1269.4566 1+ 1367.4546 +MS, 0.8-1.4min #44-83 0 1 2 3 4 5x10 Intens. 400 500 600 700 800 900 1000 1100 1200 1300 m/z 424.1754 497.2043 570.2335 2- 849.3573 995.4151 1141.4735 1- 1163.4545 1127.4573 977.4046 -MS, 1.3-1.8min #76-100 0 1 2 3 4 6x10 Intens. 400 500 600 700 800 900 1000 1100 1200 m/z Fig. 4.21: (+) HR-ESI-MS spectrum of AP11 Fig. 4.22: (-) HR-ESI-MS spectrum of AP11 The 1H-NMR spectrum of AP11 showed signals of 3 methyl groups, characterized by steroid nucleus including 2 methyl groups CH3-18 (s, 0.58 ppm) and CH3-19 (s, 0.94 ppm); 1 signal in CH3-21 weaker field (s, 2.08 ppm). 8 There is also the signal of a proton olefin at δH 5.24 (brt, J 4.2 Hz); an oxymetin group linked to a sulfate group at δH 4.87 (m) ppm; an oxymetin group is directly linked to a carbohydrate chain at the CH-6 position with δH 3.78 (m). In the weak field region, five doublet signals of five proton anomers of 5 monosaccharide units at δH 4.82 (1H, d, J = 7.6 Hz), 4.84 (1H, d, J = 7.9 Hz), 4.97 (1H, d, J = 6.8 Hz), 5.03 (1H, d, J = 7.7 Hz), 5.27 (1H, d, J = 7.7 Hz). Fig. 4.23: 1H-NMR spectrum of AP11 Fig. 4.24: 13C-NMR spectrum of AP11 The 13C-NMR spectrum of AP11 showed the presence of 51 carbon atoms, including: eight CH3 groups, seven CH2 groups, thirty two CH groups and four quater carbons. The presence of olefin carbon was triple substituted in the molecule was determined at δC 116.4/145.8 ppm. Five carbon CH signals at δC 102,3; 103,6; 104,9; 104,9; 106,9 ppm were identified as carbon anomers of the five sugar units. In addition, in the region of 60 - 90 ppm, there are resonance signals of 23 carbon atoms directly linked to the oxygen atom, including 22 carbon oximetin with 2 carbon of aglycon fraction at δC 77,4 (C-3) bind to sulfate group; δC 80,1 (C-6) linked to carbohydrate chain; and 20 carbons of sugar molecules at δC 73,8; 91,0; 74,4; 71,7; 82,3; 75,1; 85,7; 71,4; 84,3; 77,5; 75,7; 72,8; 73,7; 74,9; 72,3; 71,8; 76,2; 77,5; 75,5; 73,4 ppm; and a carbon of C=O group at δC 208,0 ppm. The sequences of proton at C-1 to C-8, C-11 to C-12, and C-8 to C-17 were ascertained from the COSY and HSQC experiments. The HMBC 9 correlation H3-18/C-12, C-13, C-14, C-17; H3-19/C-1, C-5, C-9, C-10; and H3- 21/C-17, C-20 supported the whole structure of the steroidal aglycon. The key ROESY correlations, such as H-5/H-3α, H-7α; H-14/H-12α, H-17; H3-18/H-8, H-15β, H-16β; and H3-19/H-2β, H-4β, H-6β, H-8 confirmed 5α/8β/10β/13β/14α/17α steroidal nucleus and 3β, 6α relative configurations of O-bearing substitutions in AP11. In HMBC spectrum, there is interaction of proton anomer H-1 of Qui1 unit at δH 4,82 ppm with C-6 at δC 80,1 ppm of aglycon, and in ROESY spectrum there is interaction of proton anome H-1 of Qui1 at δH 4,82 ppm with H-6 at δH 3,78 ppm of aglycon. This proves that the position linked of oligosaccharide chain to aglycon is C-6 position. Fig. 4.25: 1D TOCSY spectrum of AP11 1D TOCSY experiments with the irradiation of anomeric proton indicated the resonaces H-1 – H-6 of four quinovose units and H-1 – H-4 of one fucose units, whereas the irradiation of the resonance of the corresponding methyl group resulted in a signal for H-5 of the fucose unit. The positions of the interglycosidic linkages and the attachment of the oligosacchride moiety to the steroidal aglycon at C-6 were elucidated from long-range correlations in the ROESY abd HMBC spectra. The key cross-peaks between H-1 of Quip1 and H- 6 (C-6) of aglycon, H-1 of Quip2 and H-3 (C-3) of Quip1, H-1 of Quip3 and H-4 (C-4) of Quip2, H-1 of Fucp and H-2 (C-2) of Quip3, H-1 of Quip4 and H-2 (C-2) of Quip2 were detected. Therefore, it is possible to infer the position of the link between sugar units and between oligosaccharide moiety to the steroidal 10 aglycon is Fuc(1→2)-Qui3(1→4)-[Qui4(1→2)]-Qui2(1→3)-Qui1-Aglycon. Based on the signals on the 1D-, 2D-NMR spectrum, we can determine the carbon and proton values of oligosaccharide moiety (see Table 4.9). Fig. 4.26: Chemical structure of AP11 Fig. 4.27: Key interactions in the HMBC and ROESY spectrum of AP11 The configuration of the sugar units in AP11 was demonstrated by the method of Leontein et al, the results of the GC spectral analysis showed that all of sugar units of AP11 was D-configuration. On the basis of the above mentioned data, the structure of AP11 was established to be sodium 6α-O-{β-D-fucopyranosyl-(1→2)-β-D-quinovopyranosyl- (1→4)-[β-D-quinovopyranosyl-(1→2)]-β-D-quinovo-pyranosyl-(1→3)-β-D- quinovopyranosyl}-6α-hydroxy-5α-pregn-9(11)-en-20-one-3β-yl sulfate, and named Acanthaglycoside G. Acanthaglycoside G is rare asterosaponin, the carbohydrate moiety of which includes only β-D-fucopyranosyl and β-D-quinovopyranosyl units. Thus, Acanthaglycoside G (AP11) is the first new substance isolated from nature. 11 Table 4.8: Aglycon spectrum data of AP11 C Cac Hab (JHz) ROESY (H→H) HMBC (H→C) 1β 1α 35,8 1,63 m 1,38 m H-11, H3-19 H-3, H-11 2α 2β 29,3 2,81 (brd, J 13,6 Hz) 1,89 (brq, J 12,5 Hz) H3-19 3 77,4 4,87 m H-1, H-5 4α 4β 30,6 3,45 (brd, J 12,6Hz) 1,70 m H3-19 5 49,1 1,48 m H-3, H-7 6 80,1 3,78 m H3-19 7β 7α 41,3 2,66 m 1,28 m H-5 8 35,4 2,06 m H3-18, H3-19 9 146,0 ‒ 10 35,8 ‒ 11 115,8 5,24 (brt, J 4,2 Hz) H-1 12 40,4 2,14 brs H-14, H-17 13 42,3 ‒ 14 53,5 1,33 m H-12, H-17 15α 15β 25,3 1,76 m 1,20 m H3-18 16β 16α 23,0 2,34 (brq, J 10,9 Hz) 1,61 m H3-18 17 63,2 2,51 (t, J 8,7 Hz) H-12, H-14, H3-21 C-13, C-18 18 12,9 0,58 s H-8, H-15, H-16 C-12, C-13, C-14, C-17 19 19,0 0,94 s H-1, H-2, H-4, H-6, H-8 C-1, C-5, C-9, C-10 20 208,0 ‒ 21 30,8 2,08 s H-17 C-17, C-20 a C5D5N, b 500,13 MHz, c 125,75 MHz Table 4.9: Oligosaccharide NMR spectrum data of AP11 C δCac δHab (JHz) ROESY HMBC (H→C) Qui 1 1 104,9 4,82 (d, J 7,6 Hz) H-6 of aglycon; H-3, H-5 Qui1 C-6 of aglycon 2 73,8d 3,97 (t, J 8,3 Hz) C-1, C-3 Qui1 12 3 91,0 3,77 (t, J 8,6 Hz) H-1 Qui1, H-1 Qui2 C-1 Qui 2 4 74,4 3,55 (t, J 9,0 Hz) H-6 Qui1 C-3, C-6 Qui1 5 71,7d 3,69 m H-1 Qui1 6 18,2 1,60 (d, J 6,0 Hz) H-4 Qui1 C-4, C-5 Qui1 Qui 2 1 103.6 4,97 (d, J 6,8 Hz) H-3, H-5 Qui2; H-3 Qui1 C-3 Qui1 2 82.3 4,09 (t, J 7,6 Hz) H-1 Qui4 3 75.1 4,12 (t, J 8,7 Hz) H-1 Qui2 C-2 Qui2 4 85.7 3,56 (t, J 8,7 Hz) H-6 Qui2, H-1 Qui3 C-3 Qui2 5 71.4 3,90 m H-1 Qui2 6 18,0 1,73 (d, J 6,1 Hz) H-4 Qui2, H-1 Qui3 C-4, C-5 Qui2 Qui 3 1 102,3 4,84 (d, J 7,9 Hz) H-3, H-5 Qui3; H-4, H- 6 Qui2 C-4 Qui2 2 84,3 4,00 (t, J 8,3 Hz) H-1 Fuc C-3 Qui3, C-1 Fuc 3 77,5 4,13 (t, J 9,3 Hz) H-1, H-5 Qui3 C-2, C-4 Qui3 4 75,7 3,62 (t, J 8,9 Hz) H-6 Qui3 C-5, C-6 Qui3 5 72,8 3,71 m H-1, H-3 Qui3 C-3 Qui3 6 17,7d 1,48 (d, J 6,0 Hz) H-4 Qui3 C-4, C-5 Qui3 Fuc 1 106,9 5,03 (d, J 7,7 Hz) H-3, H-5 Fuc; H-2 Qui3 C-2 Qui3 2 73,7d 4,41 (dd, J 8,6; 9,5 Hz) C-1, C-3 Fuc 3 74,9 4,06 (dd, J 3,6; 9,5 Hz) H-1, H-5 Fuc 4 72,3 3,99 (d, J 4,0 Hz) H-5, H-6 Fuc C-3 Fuc 5 71,8d 3,78 (q, J 6,5 Hz) H-3, H-4 Fuc C-1, C-4 Fuc 6 16,9 1,49 (d, J 6,2 Hz) H-4 Fuc C-4, C-5 Fuc Qui 4 1 104,9 5,27 (d, J 7,7 Hz) H-3, H-5 Qui4; H-2 Qui2 C-2 Qui2 2 76,2 4,04 (t, J 8,8 Hz) 3 77,5 4,12 (t, J 8,8 Hz) H-1 Qui4 4 75,5 4,01 (t, J 8,7 Hz) H-6 Qui4 5 73,4 3,70 m H-1 Qui4 6 17,8d 1,79 (d, J 6,1 Hz) H-4 Qui4 C-4, C-5 Qui 4 a C5D5N, b 500,13 MHz, c 125,75 MHz, d Assignments may be interchanged 13 4.2. Chemical metabolism of cholesterol Polyhydroxysteroid, hydroximinosteroid compounds with cholesterol- like branched blood are evaluated as potential compounds with toxic activity on many cancer cell lines. Therefore, cholesterol has been chosen as the first raw material for metabolic reactions to produce hydroxyl and oxidized products. 4.2.1. Metabolism of polyhydroxysteroid derivatives Cholesterol (AP7) is hydroxylated to form many -OH groups around the C5/C6 double connection. The hydroxylation agents used are mainly oxidizing agents and are capable of producing diol-type products. Seven hydroxysteroid compounds (15c-21c) were synthesized from cholesterol according to the diagram 4.1 below. Although synthetic compounds are known substances, the content of this study provides effective synthesis methods for active substances, only through one reaction step. These methods can also be used on other steroid compounds from starfish to synthesize polyhydroxysteroid derivatives. Diagram 4.1. Synthesis of polyhydroxysteroid derivatives from cholesterol Reagents and reaction conditions: (i): BH3.THF, H2O2, NaOH, 0oC, 1h (15c: 71%, 16c: 9%); (ii): SeO2, dioxane, H2O, 80oC, 80h (17c: 50%, 18c: 2,0%, 19c: 3,5%); (iii): 4% OsO4/H2O, NMO, reflux, 48h (20c: 74%); (iv): 1. HCOOH 88%, THF/H2O2, 12h, 2. KOH 3% in MeOH (21c: 75%). 14 4.2.2. Metabolism of hydroximinosteroid derivatives Recent studies have shown that the position of the oxime groups and the branch circuit types at the C-17 position of the cholestane-like steroid framework is more active than other types of branch circuits. Therefore, cholesterol was chosen as the first raw material for the synthesis of hydroximinosteroid derivatives. Products with an oxime group at C-3 position; or C-3 and C-6; and epoxy ring at position C-4,5. Four hydroximinosteroid (23c, 25c, 29c, 31c) including 2 new substances (29c, 30c) and 7 intermediates (15c, 22c, 24c, 26c, 27c, 28c, 30c) including 1 new substance (30c) has been summarized according to Figure 4.2 below. Diagram 4.2. Synthesis of hydroximinosteroid derivatives from cholesterol Reagents and reaction conditions: (i): PCC/CH2Cl2, rt, 48h (22c: 80,0 %, 24c: 82,0 %); (ii): BH3.THF/H2O2, NaOH, 0oC, 1h (15c: 80,0 %); (iii): CeCl3.7H2O/NaBH4, CH2Cl2&MeOH (1:1), rt, 1h (26c: 89,0 %); (iv): 1. m- CPBA/CH2Cl2, 2. Dess-Martin/CH2Cl2, 0oC (27c: 12,3 %, 28c: 14,5 %); (v): NH2OH.HCl/Pyridine, 24h (23c: 85,0 %, 25c: 81,0 %, 29c: 19,3 %, 30c: 13,4 %, 31c: 22,5 %). 15 From cholesterol, it is metabolized in different directions to create ketone-mediated derivatives (C = O) at C-3 and C-6 positions; in the C-4,5 position there is a double bond (22c), no double bonds (24c) or an epoxy ring (27c). Finally, these ketones are transformed into hydroximinosteroid products (> C = N-OH), respectively (23c, 25c, 29c, 31c) by hydroxylamine hydrochloride (NH2OH.HCl) in pyridine by tissue method. described by Javier. In particular, when performing chemical oxidation 27c (with epoxy ring at position C-4,5 and ketone group at C-3), two products were obtained, one of which was oxidized (29c) and one product that is not oxidized but opens the epoxy ring in position C-4,5 (30c). The products have been successfully oxidized due to the apparent change in the chemical shift of the carbonyl group within δC 195-210 ppm to the oxime group at δC 155-160 ppm. Combining modern physico-chemical methods and nuclear magnetic resonance spectroscopy structures of all proven products. 4.3. Results of bioactive test 4.3.1. The activity of steroidal glycoside compounds isolated from starfish Acanthaster planci a. Biological activity of polyhydroxysteroid glycoside compounds AP1 was tested for cytotoxic activity on three HCT-116, T-47D and RPMI- 7951 cell lines. The result of AP1 is likely to be cytotoxic on HCT-116 and RPMI- 7951 cell lines with IC50 values of 36 and 58 µM, compared to cisplatin-positive control. Both AP1 and cisplatin were non-toxic on T-47D cell lines (see table 4.23). Table 4.23: In vitro cytotoxic activity of compound AP1 Cell lines (IC50 µM) AP1 Cisplatin HCT-116 36 75 T-47D >150 > 150 RPMI-7951 58 43 16 AP1 inhibited the proliferation of T-47D cell line after 72 hours was 35%, the RPMI-7951 cell line after 48 hours was 27% (Figure 4.54 B, C). While cisplatin almost completely inhibits the growth of T-47D and RPMI- 7951 cell lines (Figure 4.54 B, C). A B C Control AP1, 15 μM Cisplatin, 15 μM Fig. 4.54: Effects of AP1 on the proliferation of HCT-116, T-47D and RPMI-7951 cell lines at a concentration of 15 μM b. Biological activity of asterosaponin compounds AP13 and AP14 compounds have the potential to cause toxic effects on HT-29 rectal cancer cells; MDA-MB-231 mammary carcinoma cells, but not toxic RPMI-7951 malignant melanoma cells at concentrations above 150 µM. The value of IC50 concentration for each cell line showed in Table 4.24. Table 4.24: Cytotoxic activity and affecting tumor formation on soft agar of asterosaponin compounds AP11-AP14 Com. RPMI-7951 HT-29 MDA-MB-231 IC50, µM IF50, µM IC50, µM IF50, µM IC50, µM IF50, µM AP11 >150 >15 >150 >15 >150 >15 AP12 >150 >15 >150 >15 >150 >15 AP13 >150 15 109 11 30 13 AP14 >150 14 90 7 24 8 IC50: the concentration of compounds that caused a 50% reduction in cell viability of human cancer cells; IF50: the concentration of compounds that caused a 50% reduction in colonies formation of human cancer cells 17 AP13 and AP14 compounds effectively inhibit tumor formation of HT-29 and MDA-MB-231 cell lines, and are less effective with RPMI-7951 cell lines. IF50 values corresponding to cell lines showed in Table 4.24. AP13 and AP14 compounds at a concentration of 10 µM can prevent the movement of MDA-MB-231 cells at 26% and 45%, respectively, compared to the control after 48 hours of incubation (Figure 4.55). Meanwhile, compounds AP11 and AP12 cannot stop the movement of these cells. Fig. 4.55: Effects of asterosaponins AP11-AP14 on MDA-MB-231 breast adenocarcinoma migration in humans 4.3.2. Biological activity of cholesterol derivatives a. Biological activity of polyhydroxysteroid derivatives Three substances 16c, 18c, 21c show Hep G2 cytotoxic activity with IC50 value of 11,69; 11,89 and 6,87 μM. Only 21c exhibited T98G cytotoxic activity with IC50 = 2,28 μM, compared with the positive control Paclitaxel (see Table 4.25). Table 4.25: Biological activity of substances 15c-21c on Hep G2 and T98G cell lines Com. IC50 µM Com. IC50 µM Hep G2 T98G Hep G2 T98G 15c >100 >100 19c >100 >100 16c 11,59 >100 20c >100 >100 17c >100 >100 21c 6,87 2,28 18c 11,89 >100 Paclitaxela 0,040 0,023 a positive control Two pairs of substances 15c and 16c; 20c and 21c differ in the OH group configuration at C-6 positions (6α-OH in substances 15c and 20c; 6β- 18 OH in substances 16c and 21c). While 16c has toxic activity on Hep G2 cell lines and 21c has toxic activity on both Hep G2 and T98G cell lines, substances 15c and 20c do not show activity on these tests. Thus, the configuration of the OH group at C-6 of this type of structure may be one of the factors determining their cytotoxic activity. b. Biological activity of hydroximinosteroid derivatives and intermediate compounds Three derivatives 3,6-dihydroximino (23c, 25c, 31c) have stronger cytotoxic activity than 3-hydroximino-6α-hydroxy (29c) on 3 test cell lines. The 3,6-dihydroximino 25c has no double bonds at position C4/5, which can cause toxic inactivation on HepG2 and HeLa cell lines compared with Δ4- 3,6-dihydroximino 23c. While 31c has two oxime groups at position C-3, C- 6 and epoxy ring at position C-4,5 with selective cytotoxic activity on T98G cell line (IC50 = 2,9 μM ) 29c has an oxime group at C-3 and epoxy rings at C-4/5 but does not have this activity. Table 4.26: Cytotoxic activity of substances 22c- 31c on Hep G2, HeLa, T98G cell lines Com. IC50 (µM) Com. IC50 (µM) HepG2 HeLa T98G HepG2 HeLa T98G 22c >100 >100 >100 27c 41,8 72,4 >100 23c 42,4 68,6 70,3 28c >100 74,6 >100 24c >100 >100 >100 29c >100 >100 >100 25c >100 >100 69,8 30c >100 >100 18,5 26c >100 >100 >100 31c >100 >100 2,9 Paclitaxel 0,040 0,031 0,023 Paclitaxela 0,040 0,031 0,023 a positive control Three intermediates (27c, 28c, 30c) with the presence of elemental oxygen at C-4 or C-5 have moderately toxic activity on at least one cell line while the other compounds not active. As such, these steroids have double bonds at the C4/5 position or are linked to the oxygen element at the C-4 position, C-5 may have a positive effect on cytotoxic activity on cancer cell lines tested. 19 CONCLUSIONS 1. From the starfish Acanthaster planci was isolated 14 compounds. The structure of these compounds was determined by mass spectrometry, nuclear magnetic resonance spectroscopy and other physicochemical methods. The isolated and identified compounds include: planciside A (AP1); planciside B (AP2); planciside C (AP3); planciside D (AP4); (3-O- sulfothornasterol A (AP5); 5-ergost-7-en-3-ol (AP6); cholesterol (AP7); astaxanthin (AP8); thymin (AP9); uracil (AP10); acanthaglycoside G (AP11); pentareguloside G (AP12); acanthaglycoside A (AP13); and maculoside (AP14). Among them, four steroidal glycoside are discovered for the first time from nature, including three polyhydroxysteroidal glycosides, namely planciside A (AP1); planciside B (AP2); planciside C (AP3); and an asterosaponin, acanthaglycoside G (AP11). In addition, the compound pentareguloside G (AP12) found the first time in Acanthaster planci starfish in Vietnam. 2. The chemical modification of cholesterol, staring material isolated from this starfish, leads to obtain seventeen derivatives including 7 polyhydroxysteroid derivatives (15c-21c), 4 hydroximinosteroid derivatives (23c, 25c, 29c, 31c) and 6 intermediate derivatives (22c, 24c, 26c, 27c, 28c, 30c), namely: cholestane-3β,6α-diol (15c); cholestane-3β,6β-diol (16c); cholestan-5-ene-3β,4β-diol (17c); cholestan-5-ene-3β,7β-diol (18c); cholestan-5-ene-3β,4β,7β-triol (19c); cholestane-3β,5α,6α-triol (20c); cholestane-3β,5α,6β-triol (21c); cholest-4-ene-3,6-dione (22c); (3E,6E)- dihydroximinocholest-4-ene (23c); cholestane-3,6-dione (24c); (3E,6E)- dihydroximinocholestane (25c); cholest-4-ene-3β,6α-diol (26c); 6-hydroxy- 4,5-epoxycholestane-3-one (27c); 4α,5α-epoxycholestane-3,6-dione (28c); 4α,5α-epoxy-6-hydroxycholestane-3-oxime (29c); 4α,5α,6α-trihydroxy- cholestane-3-one (30c); and 4α,5α-epoxycholestane-3,6-dioxime (31c). 20 In particular, 4α,5α-epoxy-6-hydroxy cholestan-3-oxime (29c); 4α, 5α,6α-trihydroxy-cholestane-3-one (30c); 4α,5α-epoxycholestane-3,6- dioxime (31c) are new substances. 3. Have investigated cytotoxic activity and evaluated the effects of compounds AP1, AP11, AP12, AP13, AP14 to colony formation on soft agar of human cancer cell lines. The results showed that: - Compound AP1 exhibited a moderate cytotoxicity against human colon cancer (HCT-116) and human melanoma (RPMI-7951) cell lines with IC50 = 36 µM and 58 µM, respectively. AP1 compound inhibited cell proliferation of HCT-116, T-47D, and RPMI-7951 cancer cell lines, but had no effect on colony formation of these cells. - Compounds AP13 and AP14 at the same doses moderate inhibited cell viability of human colorectal carcinoma HT-29 and human breast adenocarcinoma MDA-MB-231 cell lines, but not RPMI-7951 cells. IC50 of AP13 and AP14 were 109 and 90 µM in HT-29 cells; and 30 and 24 µM in MDA-MB-231 cells, respectively. Compounds AP13 and AP14 effectively inhibited colony formation of colorectal HT-29 and breast cancer MDA-MB- 231 cells and less melanoma RPMI-7951 cells. The concentrations which caused 50% inhibition of colonies number of AP13 and AP14 were 11 and 7 µM in HT-29 cells, 13 and 8 µM in MDA-MB-231 cells, and 15 and 14 µM in RPMI-7951 cells, respectively. 4. The compounds AP11-AP14 were determined effectively on migration of MDA-MB-231 cells which are human breast adenocarcinoma cells with high metastatic potential by method of in vitro wound healing assay. The results showed that: AP13 and AP14 at concentration 10 were able to prevent migration of MDA-MB-231 cells by 26% and 45%, respectively, compared to control after 48 h of cells incubation. 5. Cytotoxicity of 07 prepared polyhydroxysteroid derivatives (15c- 21c), 04 hydroximinosteroid derivatives (23c, 25c, 29c, 31c) and 06 their 21 intermediate derivatives (22c, 24c, 26c, 27c, 28c, 30c) against three human cancer cell lines including hepatocellular carcinoma (Hep-G2), cervical cancer (HeLa) and glioblastoma (T98G) were studied. The results showed: - Five compounds (16c, 18c, 21c, 23c, 27c) exhibited cytotoxicity activity against Hep-G2 cell lines with values of IC50 = 11,59; 11,89; 6,87; 42,40; and 41,80 µM, respectively. - Three compounds (23c, 27c, 28c) inhibited cytotoxicity activity against HeLa cell lines with values of IC50 = 68,6; 72,4 and 74,6 µM, respectivel

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