Modification Of Sugar Chains In Steroidal Saponins And Structure-anticancer Activity Relationship

Steroidal saponins, a class of naturally occuring glycosides, are widely found in terrestrial plants and some marine organisms. Some of the steroidal saponins exhibit moderated to good cardiovascular, antitumor, antifungal, and immunostimulant activities. Both the carbohydrate and aglycon are structurally essential for their bioactivities. Some results suggested that the biological activities of steroidal saponins are strongly dependent on the carbohydrate residues attached to the aglycon. The nature, number, and order of attachment of the sugars in the oligosaccharide associated with the steroidal saponins are crucial for the biological activities. To better define the role of the carbohydrate groups of steroidal saponins and research the relationship of structures and activities of carbohydrate chains, a program was started in our research group to modify the sugar chains in steroidal saponins. The antiproliferative activities against tumor cells of a series of structurally modified compounds were examined using a microculture tetrazolium (MTT) assay.α-Chaconine,α-solanine,α-solamargine andα-solasonine were isolated from potato(Solanum tuberosum L.) sprouts and nigrum(S. nigrum L.)fruits.Two steroidal saponins dioscin and gracillin were ioslation from the rhizomes of Dioscorea nipponica Makino. These products were purified by silica gel column chromatography and characterized by TLC, HPLC and 13C NMR spectra.The selective sulfation to the 6-hydroxy groups of chaconine,solamargine and solanine were carried out in a strategy by the use of protective groups. The 6-hydroxyl groups of the sugar chains in glycoalkaloids were first selectively protected with 4,4'-dimethoxytftyl (DMT-Cl), and then the other hydroxyl groups were acetylated.After the protective group DMTr was removed by using 0.5% TFA in dichloromethane, the free 6-hydroxyl groups were sulfated by chlorosnlfonic acid pyridine to give 6-O-sulfated products.Finally, the acetyl groups were removed to give 6-O-sulfated solamargine in good yields.13C NMR spectra confirmed that glycoalkaloids were sulfated at O-6 of the carbohydrate moiety.Selective 6-OH sulfated of unprotected chaconine with chlorosulfonic acid-pyridine method afforded 6-O-sulfated chaconine in 36% yields. The synthetic route in this strategy need only one procedure. Compared with what is discussed above, this method is more simple and has some advantages. The synthesis of per-O-acetyled chaconine was carried out using a large excess of acetic anhydride as the reagent and solvent and the catalysts employed pyridine to give the peracetyled product in 91% yields.Glycosylation of the 6'-OH of chaconine and solamargine with 2,3,4,6-tetra-O-acetylβ-D-glucopyranosyl bromide using Koenigs-knorr method under the promotion of Ag2O afforded the 6'-O-glycosylated products in 41%-53% yields.Diosgenylβ-D-galacopyranosyl-(1→4)-β-D-glucopyranoside was concisely synthesized from diosgenin in 85% yields. The process was carried out using per-O-acetyledβ-D-galacopyranosyl-(1→4)-β-D-glucopyranosyl trichloroacetimdates as glycosyl donors and diosgenin as glycosyl acceptors in the presence of a catalytic amount of BF3·Et2O. Followed by deprotection, gave the target compound.Diosgenylβ-D-galacopyranosyl-(1→4)-β-D-glucopyranosyl(1→2)-β-D-glucopy ranoside were synthesized efficiently. The synthesis was started from trillin (diosgenylβ-D-glucopyranoside ). Treatment of trillin with benzoic aldehyde and ZnCl2 afforded 4',6'-O-benzylidene trillin in 91% yields. And then the 3'-OH of 4',6'-O-benzylidene trillin was selectively protected with pivaloyl chloride in pyridine at 0℃to 3'-O-pivaloyl-4',6'-O-benzylidene trillin. BF3·Et2O-promoted glycosylation of the free 2'-OH of 3'-O- pivaloyl-4',6'-O-benzylidene trillin with per-O-acetyled lactose trichloroacetimdates gave the 2-O-glycosylated product. Subsequent the protecting groups were removed to give diosgenylβ-D-galacopyranosyl-(1→4) -β-D-glucopyranosyl(1→2)-β-D-glucopyranoside in 85% yields.Selectively acidic hydrolysis of steroidal saponins were reported. Acidic hydrolysis products ofα-solamargine,α-solasonine and dioscin were prepared and characterized by TLC and 13C NMR spectrum. TLC showed that partial acid hydrolysis ofα-solamargine can produce four products: two isomeric disaccharideβ1- andβ₂-solamargine, the monosaccharideγ-solamargine and the aglycon solasodine. Partial acidic hydrolysis ofα-solasonine can produce three products. Our efforts to isolateβ₁-solasonine were unsuccessful. In addition, the yield ofγ-solasonine is quite low, compared toβ₂-solasonine and solasodine, this could be caused either by the rapid hydrolysis of theγ-solasonine or by direct cleavage ofβ-solasonine. Acid-catalyzed partial hydrolysis of carbohydrate groups of dioscin obtained four products. The optimal hydrolytic condition for preparation ofγ-dioscin (trillin) was at 90℃with 2%H₂SO₄-MeOH -H₂O (2:90:10, v/v/v) in a reaction period of 75 min. For preparation ofβ-dioscin, the best condition was at 75℃using 5%H2SO4 -MeOH-H₂O(5:90:10, v/v/v) for 75min.The antiproliferative activities against HCT-8 tumor cells of a series of structurally modified glycoalkaloids were examined using a MTT assay. The IC50 of four glycoalkaloids chaconine, solanine, solamargine and solasonine were 7.12,10.9,10.63, 16.04μM, respectively. These results showed that four glycoalkaloids were active in the assay; the effects of the four glycoalkaloids were concentration dependent in the range of 1⁴0μM. Three 6-O-sulfated glycoalkaloids 6-O-sulfated chaconine, 6,6'-di-O-sulfated solanine and 6-O-sulfated solamargine shown no cytotoxic activity against HCT-8 cells (IC50 >40μM).β1-,β2-,γ-Solamargine andβ2- ,γ-solasonine exhibited low activity against HCT-8 cells. For the aglycon solasodine, the inhibition of HCT-8 cells was 38.1% at low concentration of 7.5μM. Structure-activity relationship studies indicated that the 6-OH group of the sugar molecules seem to be an important factor related to antiandrogenic activity.Four steroidal saponins and their derivatives in vitro antitumour activities were assayed using L929,Hela and H7402 cell lines. The results have shown that solamargine and dioscin were highly cytotoxic toward several human tumor cell lines, whereas solasonine and gracillin seemed to be less activities. The results indicated that the synthetic steroidal saponin diosgenylβ-D-galacopyranosyl-(1→4)β-D–gluco- pyranoside exhibited high activity against the L929 cells in contrast to the low activity against the Hela and H7402 cells. Diosgenylβ-D-galacopyranosyl-(1→4)-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside was inactive at the 30μM levels. The activities of the four hydrolysis products of dioscin were less than that of the parent steroidal saponin. Structure–activity relationship studies indicated that not only the nature and number, but also the order of attachment of the sugars in the oligosaccharide associated with the steroidal saponins, were important in the antiproliferative properties.