Studies On The Synthesis And Biological Activities Of The Derivatives Of Esculetin

Objective:A series of esculetin derivatives were designed and synthesized,and their anti-HBV and anti-tumor activities were screened and studied.Methods:（1）Synthesis of esculetin derivatives:Sixteen of C-7 and C-6,7 derivatives were synthesized by etherification reaction and twenty esculetin derivatives with C-7 amine side chains were synthesized by etherification and nucleophilic substitution.Three C-4substituted derivatives were synthesized by Pechmann condensation reaction and three C-8 substituted amine derivatives were synthesized by Mannich reaction.（2）Screening of anti-HBV activity of derivatives in vitro:Using Hep G2.2.15 cell line as model,the toxicity of each compound to Hep G2.2.15 cell line was detected by MTT assay.Hep G2.2.15 cells were treated with derivative samples of different concentrations,and the supernatant was collected after continuous culture for 9 days and the secretion of hepatitis B surface antigen（HBs Ag）and e antigen（HBe Ag）in the supernatant was detected by enzymy-linked immunosorbent assay（ELISA）.（3）Screening of antitumor activity of derivatives in vitro:Human colon cancer cells（LOVO）,human liver cancer cells（Hep G2）,human cervical cancer cells（He La）,human lung cancer cells（NCI-H460）and human ductal cancer cells（T47D）were used as cell models and the antitumor activity of each derivative was detected by MTT assay.The effect of the compounds on tumor cell apoptosis was determined by flow cytometry.Results:（1）42 esculetin derivatives were synthesized and their structures were identified by ¹H NMR,¹³C NMR and ESI-MS.（2）In the anti-HBV experiment in vitro,the inhibitory effects of the compounds on HBs Ag and HBe Ag were detected by enzyme-linked immunoassay.The compounds a7 and a8showed anti-HBV activity,the two compounds significantly inhibit the secretion of both antigens.Their IC₅₀ are 254.17±9.08μM（HBs Ag）,220.00±15.10μM（HBe Ag）and 198.27±4.13μM（HBs Ag）,284.14±43.27μM（HBe Ag）,respectively.Compound c9 showed the best inhibitory effect on the single antigens HBe Ag(IC₅₀=25.87±3.70μM,SI=8.03)and compound c20 showed the best inhibitory effect on the single antigens HBs Ag(IC₅₀=21.37±4.50μM,SI>20.32),both of their activity were higher than that of the positive drug lamivudine(IC_{50HBs Ag}=334.10±38.52μM,SI_{HBs Ag}=3.07,IC_{50HBe Ag}=478.34±12.10μM,SI_{HBe Ag}=2.15).In addition,MTT results showed that the toxicity to Hep G2.2.15 cells was less than that of esculetin.（3）The antitumor activities of derivatives were detected by MTT assay.The results showed that compound a3 exhibited excellent antitumor activity against five kinds of tumor cells,with IC₅₀ values of 23.41±0.19（Lovo）,16.26±0.19（Hep G2）,28.02±0.25（Hela）,38.24±0.20（NCI-H460）and 39.26±0.07（T47D）μM,respectively.The results showed that compound a3could promote the apoptosis of Hep G2 cells,and the apoptosis rate was concentration dependent.Conclusions:In all synthetic esculetin derivatives,some etherified derivatives with C-7 substituted groups have anti-HBV activity and individual etherified derivatives at 6 and 7 sites showed a wide range of antitumor activities.It was suggested that the introduction of side chain could improve the anti-HBV activity of the compound while Hydrocarbon chains introduced by etherification also play a role in increasing the antitumor activity.