Synthesis And Anti-cervical Cancer Activity Of Halogenated Methoxy Chalcone Derivatives

Objective:(1)The design and synthesis of halogenated methoxy chalcone derivatives.(2)The anti cervical cancer activity of the synthesized target compounds was studied in vitro,and the target compounds with better activity were screened.(3)We will screen better active compounds and MDM2 protein for docking experiments,and explore the binding mode and binding sites of target compounds to MDM2 protein.Methods:(1)The Claisen-Schmidt reaction principle was adopted,in which the target compound 1 was synthesized by acid method,alkali method and microwave method.Halogenated Methoxy Chalcone synthesis by KOH/EtOH method and grinding method;The acid method uses boric acid as catalyst and ethylene glycol as solvent;The alkali method uses KOH as the catalyst,and the anhydrous ethanol is used as the reaction solvent;Microwave method adopted neutral Al2O3 as reaction medium,KOH as catalyst and microwave as reaction condition.Grinding method using neutral Al2O3 as the reaction medium,KOH as a catalyst,grinding as a reaction condition;The melting point of the compound was measured by using a microscope melting point meter.The structure of the compound was confirmed by 1H-NMR and 13C-NMR.(2)In vitro cell model of cervical cancer HeLa,SiHa and hamster ovary CHO cells.The synthesis of compounds as drugs,cisplatin as positive control drug,inhibition of proliferation by MTT method to detect target compounds on cervical cancer cells and hamster ovary.(3)The cells with better activity were screened for apoptosis.The cells after 24 h were stained with BD(FITC Annexin/PI)apoptosis kit and detected by upflow cell analyzer.(4)The target compounds with better activity were screened and molecular docking experiment was carried out with the AutoDock Vina 1.1.2 software and the MDM2 protein.Results:(1)Synthesis of 18 compounds,and the yield is between 37～88%(2)In the concentration range of 10 to 50μg/mL,each compound had a significant inhibitory effect on HeLa and SiHa cells of cervical cancer,and the inhibitory effect of compounds on CHO proliferation in cervical cancer cells and normal hamster ovary cells was analyzed.Among them,4-chloro-2’,4’-dimethoxychalcon(Compound 3),3,4,5-trimethoxy-2’,4’-dimethoxychalcon(Compound 7)and 3,4,5 trimethoxy-3’-hydroxy-4’-methoxy chalcone(Compound 15)showed higher activity.The compound 3,7 and 15 in HeLa cells for 48 h The IC50 values were 11.30,9.34,and 8.12 μg/mL,respectively,and the activity was stronger than that of the lead compound ILG IC50(25.36 μg/mL)(P<0.05).The IC50 values of each compound on SiHa cells at 72 h were 10.78.10.21,and 3.98 μg/mL,respectively,demonstrating significant anti-cervical cancer activity in vitro.The IC50 values of the three compounds against normal CHO cells of hamster ovary for 48 h were 25.34.10.44.and 10.43 μg/mL.respectively,similar to the toxicity of lead compound ILG,indicating that compounds 3,7.and 15 had low toxicity to normal cells.(3)The compounds 3,7,15 have better apoptosis induction effect on cervical cancer SiHa cells.The apoptosis rate was 49%when the concentration of compound 3 was 25 μg/mL,and the apoptosis rate was 45.1%when the concentration of compound 7 was 25 μg/mL and the concentration of compound 15 was 25 μg/mL role of 48,the proportion of apoptosis reached 41.2%.Three compounds demonstrated well-induced apoptosis.Cell cycle experiments of compound 3 against SiHa have shown that these compounds block the proliferation of cervical cancer cells in G2/M phase.(4)The molecular docking results showed that the binding free energy of compound 3 to MDM2 protein was-7.2 kcal/mol,that of compound 7 to MDM2 protein was-6.2 kcal/mol,that of compound 15 to MDM2 protein was-6.3 kcal/mol,The results showed that all three compounds could bind well with MDM2 protein,of which compound 3 and MDM2 protein were most closely associated.Compound 3 after docking conformation shows its 4-Chlorphenyl can occupy the D site of them,and the original crystal structure in 7HC 6-chloro indole ring part can be overlapped,and 2,4-dimethoxyacetophenone toward the B site,and surrounding amino acid residues to form hydrogen bonds,internal 4-Chlorphenyl are deep pockets of activity,the formation of strong interactions between hydrophobic.Conclusion:Through the synthesis and activity studies of 18 derivatives,it was shown that the halogenated and methoxylated modification of the lead compound ILG can significantly increase the activity of anti-cervical cancer,while the toxicity to normal cells is relatively low.Among them,compound 3,The inhibitory activity of compound 7 and 15 on the two kinds of cervical cancer cells and the promotion of apoptosis were the most significant,and compound 3 could block the proliferation of cervical cancer SiHa cells in the G2/M cycle.The selected three derivatives could be used as In-depth research and development of potential drug candidates.The results of molecular pairing showed that compound 3 and the proposed target MDM2 protein can form a stable complex and have a high target match.