Synthesis And Biological Activities Of Genistein Chemically Modified Compounds

Genistein(5,7,4'-trihydroxyisoflavone) is a ubiquitous phytoestrogen isoflavone in nature with a wide variety of biological activities,including chemoprevention of breast and prostate cancers,cardiovascular disease and post-menopausal ailments.However,genistein has not been applied widely in clinical therapy as drugs because of its weak bioavailability resulting from its poor lipophilicity and hydrophilicity,and its first pass effect.In this paper,we focused on the chemical modification using genistein as a lead-compound,the antioxdative activities of novel genistein derivatives,the interactions of genistein derivatives with bovine serum albumin(BSA) and calf thymus DNA(ctDNA),and also the antitumor activities of genistein lactosides.To improve the solubility,enhance the targeting property,and block the first pass effect of genistein,lactose and ferulic acid were selected to conduct the glycosylation and esterification reactions with genistein respectively.The first regiospecific synthesis of genistein 4'-O-β-lactoside,genistein 7-O-β-lactoside and genistein 7,4'-di-O-β-lactoside from unprotected genistein under phase-transfer-catalyzed conditions were presented.Two new genistein esterified derivatives, genistein 7-acetylferulic acid ester and genistein 7,4'-di-acetylferulic acid ester were first synthesized based on Schotten-Bamann reaction.The structures of target compounds were characterized by IR,MS,¹H NMR and ¹³C NMR spectra.The antioxidative and free radical scavenging activities of genistein and its chemically modified compounds were assessed by using four methods,namely the anti-DPPH free radical,Fenton reaction with 1,10-phenanthroline-Fe²⁺ spectro-photometry, pyrogallol autoxidation assays and protective effects on hydroxyl radicals mediated DNA damage.The results showed that the abilities of the glycoslated and esterified derivatives of genistein in scavenging DPPH and·OH radicals were improved,and the free radical scavenging activities of esterified derivatives were stronger than those of genistein lactosides.Esterified derivatives of genistein were effective in scavenging·O₂^-.In contrast,the inhibiting rates of genistein lactosides on·O₂^- were much lower than that of genistein aglycone and even had negative effects because of their self-oxidation.Genistein and its chemically modified compounds also possess great abilities to protect DNA from being destroyed by hydroxyl radicals,these abilities were not only related to their abilities in scavenging·OH radicals,but also influenced by other factors,such as their interactions with DNA.Under the imitated physiological conditions,the interactions of genistein and its chemically modified compounds with bovine serum albumin(BSA) were investigated using fluorescence quenching technique and UV-vis absorption spectroscopy.It was observed that all of them could effectively quench the intrinsic fluorescence of BSA. The binding of BSA with genistein and its lactosides can be associated with the static fluorescence quenching mechanisms together with the inner filter effects of the compounds.The fluorescence quenching processes of BSA quenched by genistein esterified derivatives were multi-mechanisms coexistent,and were governed by static quenching mechanisms only at low concentrations of the compounds.The binding processes of tested compounds with BSA were both spontaneous.The binding forces of genistein esterified derivatives with BSA were much stronger than those of genistcin lactosides.From the thermodynamic parameters,the primary binding patterns between genistein and its two mono-substituted lactosides and BSA were interpreted as hydrophobic interactions,the binding of genistein 7-acetylferulic acid ester to BSA involved electrostatic interactions,whereas the binding of two genistein di-substituted derivatives to BSA involved hydrogen bonds and Van der Waals forces. The binding average distances between genistein and its derivatives and BSA were determined based on the F(o|¨)rster theory and the results indicated that the energy transfer from BSA to the compounds occurred with high possibility.Besides,the interactions of BSA with other compounds did not change the conformation of BSA except for genistein and genistein 7-O-β-lactoside via synchronous fluorescence spectrometry.The structural model of genistein 7-O-β-lactoside and calf thymus DNA(ctDNA) complex was set up for the first time,and the molecular dynamics simulation was applied to study the interactions between the compound and ctDNA.The interactions of genistein and its chemically modified compounds with ctDNA in Tris-HCl buffer were also studied using UV-vis spectroscopy,fluorescence spectroscopy and viscosity measurements.The results indicated that genistein and its lactosides could interact with DNA through groove binding.Compared with genistein 7,4'-di-acetylferulic acid ester,there was a relatively strong interaction between genistein 7-acetylferulic acid ester and ctDNA.The binding constants of genistein 7-acetylferulic acid ester and genistein 7,4'-di-acetylferulic acid ester with DNA at 293 K were 2.81×10⁶ L·mol ^-1 and 1.19×10⁴ L·mol^-1,and the binding modes were evaluated to be intercalative and groove binding,respectively.The antitumor activities of genistein and its lactosides were estimated by fluorescence screening test using ethidium bromide(EB) as a fluorescence probe.In addition,comparative antitumor activities of genistein 4'-O-β-lactoside and genistein 7-O-β-lactoside were tested by MTT method.The results of the proposed fluorescence method are in agreement with those of the MTT method.The inhibitory ratios of the two genistein mono-substituted lactosides against two kinds of tumor cells,SMMC-7721 and MCF-7,were higher than those of genistein.