Preliminary Study On The Semi-Synthesis And Anti-Inflammatory And Anti-Human Triple Negative Breast Cancer Activities In Vitro Of Glavonoid 3-O Glycosides From Hesperetin

Objective:Two flavonoid glycosides neamed Nervilifordin-F and Nervilifordin-G with excellent anti-inflammatory activity(IC₅₀=15.15μM,30.82μM,respectively)and low cytotoxicity(EC₅₀>40μM)were isolated from Nervilia fordii（Hance）Schltr..However,the paucity of this natural product from plant sources led us to conduct chemical semi-synthesis of its derivatives from hesperetin,and further investigate their anti-inflammatory and anti-human triple negative breast cancer activities in vitro,which would provide a reference for synthesising of its derivatives,investigating more biological activities and discovering drugs or lead compounds of flavonoids in the future,and which would futher promote the utilization of the potential medicinal value of flavonoids.Methods:A series of novel flavonoids and derivatives were synthesized by methoxylation,oxidative dehydrogenation,benzylation,modified peroxyacetone method,glycoside condensation reaction,debenzylation and deacetylation.Their structures were characterized by ESI-MS,¹HNMR and ¹³C NMR,and their anti-inflammatory activity were evaluated for their inhibitory effects on the nitric oxide（NO）,tumor necrosis factor（TNF-α）and interleukin-6（IL-6）production in LPS-induced RAW264.7 mouse macrophages,and their effects on cell viability were detected by CCK8 assay.Besides,their cytotoxicity on human inflammatory triple-negative breast cancer cells line SUM 149 were tested by MST assay,which would help evaluate their anti-human triple-negative breast cancer activity.Results:（1）Thirty two flavonoids and derivatives（1a-1m,2a,1-18）,confirmed by ESI-MS,¹H NMR and ¹³C NMR,were successfully synthesized,including fifteen natural flavonoids（1a-1d,1f,1g,1j-1l、2a,13-16）and seventeen new flavonoids（1e,1h,1i,1-12,17,18）.（2）Compounds 1c,1g,1i,1j,1l,1-18 showed different degrees of inhibitory effects on NO production induce by LPS macrophages RAW264.7.Compounds 1,7,9,17 showed relatively strong inhibitory activity with IC₅₀values of 46.32±1.29μM,47.23±1.05μM,35.71±1.06μM,45.88±3.60μM,respectively,and also inhibited the production of inflammatory factors such as TNF-αand IL-6,which exhibited certain anti-inflammatory activity in vitro.While other compounds were inactive with inhibition rate<25%at the highest concentration tested（60μM）and haven’t statistical significance at 15,30,60μM concentrations（P>0.05）.（3）Compounds 1b,1f,1h,1j-1m,1,3 showed different degrees of cytotoxicity with IC₅₀ values of≤16.61μM on human inflammatory triple-negative breast cancer cell line SUM 149,and compounds 1i was more cytotoxicity with IC₅₀ values of 1.38±0.31μM;while other compounds were inactive with inhibition rate<50%at the highest concentration tested（20μM）.Conclusion:（1）Thirty two flavonoids（1a-1m,2a,1-18）were successfully synthesized,and their structures wrer confirmed by ESI-MS,¹H NMR and ¹³C NMR,including seventeen new flavonoids.（2）Compounds 1c,1g,1i,1j,1l,1-18 showed different degrees of inhibitory effects on NO production induce by LPS in macrophages RAW264.7.（3）Compounds 1b,1f,1h,1j-1m,1,3 showed different degrees of cytotoxicity on human inflammatory triple-negative breast cancer cell line SUM149.（4）The fators,glycosyl at 3-OH and glycosyl acetylation,increase anti-inflammatory activity,and glucose is more active than galactose.2,3 double bond,3ˊ-OH and proper fat-soluble modification at 7-OH,which can increase cytotoxicity angainst triple-negative breast cancer cells;while the fators,3-OH,5-OH and glycosylated at 3-OH,decrease cytotoxicity.