Chlorination Modification Of Chalcone Derivative Isoliquiritigenin And Its Anti-Cervical Cancer Activity And Mechanism

Objective:?1?A series of chlorinated chalcone derivatives were synthesized and characterized by the structural design and modification of the chalcone derivative isoglycyrrhizin.?2?To study the proliferation inhibitory activity of isoglycyrrhizin and chlorinated chalcone derivatives on human cervical cancer cells and normal cells in vitro,and to analyze the structure-activity relationship to select effective low-toxic target compounds.?3?To study the effect of target compounds on the pro-apoptotic effect and cell cycle of human cervical cancer cells,and to determine the expression level of apoptosis-related gene mRNA.?4?Calculate the free energy of the binding protein of the target compound and the human cervical cancer cell proliferation-inhibiting pathway,and determine the content of the cancer pathway protein by experiments.Methods:?1?using acetophenone and benzaldehyde derivatives as raw materials,using the classical Claisen-Schmidt reaction principle,using a reaction under normal temperature and microwave catalysis,using KOH as a catalyst and anhydrous ethanol as a reaction solvent for chlorination.The synthesis of ketone derivatives were mainly purified by water recrystallization,and the structure was characterized by ¹H-NMR and ¹³C-NMR.?2?Human cervical cancer cells HeLa,SiHa and Chinese hamster normal ovary cell CHO were used as test cells,and the target compound was synthesized as a reagent.Cisplatin was used as a positive control drug.The target compound was determined by MTT method for HeLa,SiHa and The proliferation inhibition rate of CHO cells was calculated and the IC₅₀ value was calculated.?3?Flow cytometry was used to determine the effect of target compounds with low toxicity on the pro-apoptotic effect and cell cycle of HeLa and SiHa cells.Fluorescence quantitative RT-PCR was used to determine the apoptosis-promoting compounds.Effects of related genes CDK-4,Bcl-2,ALDH1 A1,OTC-4,UHRF1,BIRC7,TP53 and BIRC5 mRNA expression levels.?4?Using computer-assisted molecular docking technology to molecularly dock the effective low-toxic compounds with the?-tubulin 1A chain and MDM2 protein A chain,calculate the binding free energy and analyze the binding mode,and determine the effective low toxicity compound by Elisa method.The content of?-tubulin and MDM2 protein in the cells after SiHa cells were treated for 24 h.Results:?1?78 chlorochalcone derivatives were synthesized and identified as target compounds by structural characterization.In the anhydrous ethanol or alcohol-water mixed system,the appearance of the compound is different in the form of crystals,mainly needle crystals.and the yield was between 20.5and 98.7%.The normal temperature reaction time was 4¹2 h,the microwave liquid reaction time was 10¹80 s.?2?The isoglycyrrhizin's IC₅₀ values for HeLa,SiHa and CHO cells for 24 h was 66.63,75.48 and 57.98?g/mL,compound 3 was 5.17,25.54 and31.77?g/mL,compound 5 was 22.58,20.19 and 58.36?g/mL,compound 6 was 58.54,24.32,and 69.36?g/mL,compound 46 was 13.28,5.02,and 63.66?g/mL,compound 47was 7.67,4.31,and>100?g/mL,and compound 49 was 23.14,9.98,and>100?g/mL.?3?In the apoptosis and cycle experiments,the duration of action was 24 h.At 75?g/mL,the apoptotic rate of isoglycyrrhizin to SiHa and HeLa cells was 58.1%and 23.3%,and that of compound 5 was 82.2%and 61.8%,Compound 6 was 73.1%and 87.5%,and Compound 49 was 60.7%and 60.7%.Isoglycyrrhizin increased the G₂/M phase of SiHa cells by 28.15%at 10?g/mL,increased the S phase of SiHa cells by 13.12%at 100?g/mL,and increased the S phase of HeLa cells by 30.47%at 25?g/mL.Compound 5increased the SiHa cells by 43.9%and 10.64%at S and G₂/M at 100?g/mL,and increased by 34.04%and 4.78%for HeLa cells in S and G₂/M phases.Compound 6increased 15.48%and 4.08%for SiHa cells at G₀/G₁ and G₂/M at 100?g/mL,and15.38%for HeLa cells at S phase.?4?In the quantitative RT-PCR experiment,the expression levels of seven apoptosis-related genes were up-regulated in the presence of ALDH1 A1 at the 10,25 and 75?g/mL start to lower.At the 25 and 75?g/mL of compound 5,the 8 target genes showed a downward trend.Compound 49 generally showed a significant downward trend.?5?In the molecular docking experiment,the binding free energy of isoglycyrrhizin to?-tubulin 1A chain and MDM2 A chain was-8.3and-7.2 kcal/mol,and compound 3 was-8.8 and-7.7 kcal/mol,Compound 5 was-8.7and-7.9 kcal/mol.?6?At 100?g/mL,the content of?-tubulin was 98.15 pg/ml after compound 49 was applied to SiHa cells for 24 h,which was significantly higher than that of blank group and isoglycyrrhizin?P<0.05?.The content of MDM2 protein in SiHa cells treated with compound 5 was 5.35 ng/ml lower than that in the blank group and isoglycyrrhizin,and there was statistical difference?P<0.05?.Conclusion:78 kinds of chlorochalcone derivatives were obtained by structural modification of isoglycyrrhizin.The microwave liquid phase reaction was suitable for the synthesis of compounds which are difficult to react at room temperature and have a long reaction time.The reaction at room temperature is suitable for a large number of reactions.The synthesis of the compound precipitated by the microparticles.The water recrystallization method simplifies the purification and separation process of the chlorinated chalcone derivatives,improves the yield,and is environmentally friendly.In the study of the proliferation inhibitory activity against SiHa,HeLa and CHO cells,a series of effective low-toxic chalcone derivatives?compared with isoglycyrrhizin?were obtained,in which compounds5,6 and 49 promoted SiHa and HeLa cells.The apoptotic effect was significant.Compounds 5,6,8 and 49 significantly inhibited the S phase arrest of SiHa and HeLa cells in the cycle experiment.The expression levels of 8 apoptotic gene mRNA of compounds 5 and 49 were significantly lower than those of isoglycyrrhizin?P<0.05?.The molecular docking results showed that the binding free energy of compounds 3 and 5 to?-tubulin 1A chain and MDM2 A chain was lower than that of isoglycyrrhizin.At 100?g/mL,the content of?-tubulin was higher than that of other compounds after compound49 was applied to SiHa cells for 24 h?P<0.05?.MDM2 protein was observed after compound 5 was applied to SiHa cells for 24 h.The content was lower than other compounds and isoglycyrrhizin,and there was a statistical difference?P<0.05?.