| ObjectiveThe occurrence of tumor is a multi-step,multi-stage complex process,which seriously endangers the health of human beings.Recently,the occurrence rate of tumor has risen.The occurrence of tumor is closely related to the abnormal DNA methylation.Therefore,it is highly desirable to design and synthesis of novel DNMT-targeted antitumor drugs.Previously,our research group has prepared two series of non nucleoside DNMT inhibitors of carbazole derivatives with virtual screening method based on DNMT1 composite crystal,and their antitumor activity was also measured.Based on this,we introduced bromine into carbazole 3 site or 3,6 sites and changed the side chains.Thus,a series designed compounds were synthesized and evaluated antitumor activities.Methods1?The design and synthesis of carbazole derivativesIn this work,the key intermediates 3-bromo-9-(oxiran-2-ylmethyl)-9H-carbazole and 3,6-dibromo-9-(oxiran-2-ylmethyl)-9H-carbazole were synthesized from carbazole by bromination,N-alkylation and other steps.And then the intermediates were reacted with a series of amine to prepare the target compounds.We obtained two series of carbazole derivatives:43 compounds with mono molecule carbazole fragment and compound with dimer carbazole fragments,and their structures were confirmed by melting test,TLC,NMR spectroscopy and LC-MS.2?The antitumor activity assays of carbazole derivatives were evaluated in vitro2.1 The inhibitory ratios of the 44 target compounds against DNMT1 were tested by ELISA.2.2 The anti-proliferation effects of target compounds were tested with MTT,which were performed in HCT116,MNK-45,A549 cancer cells,and gemcitabine,paclitaxel,and cisplatin were selected as positive control respectively.According to the inhibition rate,the IC50(50 percent inhibitory concentration)of each tumor cell line was calculated,which was used to evaluate the anti-proliferative effect of NGA on tumor cells in vitro.2.3 HCT116 cell were treated with compound 2 and 12 for 24 h to detect the cell cycle by flow cytometry after PI stainning,while the apoptotic effect on HCT116 cell induced by compounds 2,12,42 were tested after stained with Annexin V-FITC/PI.Result1?44 compounds of containing carbazole structure were synthesized.2?Compound 1,2,12,13,24,25,26,30,31,34,35,37,41,42 can inhibit DNMT1 catalytic activity in a dose-dependence manner.Compounds 2,12,25,26,41,42 half inhibition rate of IC50 is 10,9.6,16,2.7,14,1.2 ?M.MTT assay results show that:1)The IC50 of major compounds were lower than those of positive drugs,and exhibited dose-dependent and time-dependent inhibition of HCT116 and MNK-45.Compound 2 is the best(the IC50 values of 4.12±0.22?5.53±0.20?2.68±0.08 ?M for HCT116,MNK-45 and A549 in 24h);2)Compound 2 and 12 could induce HCT116 cell cycle to arrest in G1 phase in a dose dependent manner;3)Compounds 2,12,and 42 could induce apoptosis of HCT116 cells,and the apoptotic cells increased significantly with the increase of concentration.Conclusion44 compounds of containing carbazole structure were synthesized and characterized.Compounds were evaluated by in vitro antitumor activity screening assays and enzyme activity inhibition assays.The results showed that compounds 2,12 and 42 not only had the better inhibitory potency against three cell lines,but also had a certain inhibitory activity to DNMT1.The work provides solid data for the further research towards new antitumor drugs. |
PDF Full Text Request