Synthesis Of Topoisomerase ? Inhibitors And Their Antitumor Mechanism

DNA topoisomerase(topo),an enzyme responsible for resolving the topological structure of DNA,plays an important role in some basic life activities of cell,such as replication,transcription,recombination,repair,chromatin reconstitution,M phase chromosome concentration and separation,and so forth.As Topo poisons are more favorable and widely used in clinic in the treatment of various neoplastic diseases.However,long-term usage of Topo II poisons is associated with serious side effects,including multi-drug resistance(MDR),secondary malignancy and cardiotoxicity,which have limited the therapeutic outcomes.Additionally,long-term use of Topo II poisons(e.g.Etoposide)is often accompanied by severe DNA damage or are easily excluded from the cells by the drug efflux pump,thus leading to reduced concentration of intracellular drugs and decreased drug sensitivity.Even worse,due to its toxic profile,Topo II poisons often cause an intracellular Topo II levels,resulting in a decrease of drug sensitivity to Topo II inhibitors.Many naturally occurring acridone derivatives are reported to display promising antitumor,antibacterial,antiparasitic,and anti-inflammatory activities,and also exhibit P-gp inhibitory potency by working as MRP family protein inhibitors.Inspired by these bioactivities of acridone derivatives,we firstly conducted nitro reaction at potision 5 of 2,6-dichlorobenzoic acid.The secord step took us long time to synthesize,finally Buchwald-Hartwig reaction was used to obtain compound C.Then FC acylation was conducted to obtain compound D.E series compound were synthezied through a nucleophilic attack process,and F series compounds were synthesized through reduction action.The synthesized compounds were evaluated their anti-proliferative activity to tumor cells.Three cell lines were selected(HCT116,SW480,HeLa)and obtained IC50 values of these compounds.Among them,some compounds performed promising anti-tumor activity(IC50<10 ?M:E24?E25?E27?E29?E30?E31?E32?E33?F8.Then those compound were screened for topoisomerase II activity though gel electrophoresisa and obtained potent Topo ? inhibitor:E24?E25?E29-E33.Among them,the most potent compound E33 was submitted to study the mechanism of action.It resulted that E33 could inhibit Topo II mediate-DNA relaxation assay and KDNA decantenation assay,indicating E33 could inhibit Topo II in vitro.And E33 reduced significantly cell viability and performed more potent activity than Etoposide;E33 could apparently inhibit the colony formation of tumor cells;E33 inhibited cell woud-healing rate of tumor cells;Interestingly,we found that E33 could not induce apparent cell cycle arrest and apoptosis but we used doule thymidine block to prove E33 can induce G2/M arrest in HCT116;To expore the reason of cell cycle arrest.at the same time,considering the role of Topo II in cell cycle,chromosomes spreading assay was conducted.It resulted that E33 could inhibit the condensation of chromosome thus leading to G2/M arrest.In order to evaluate wether E33 exert anti-tumor activity through inhibiting celluar Topo II,Topo-DNA cleavage complex trapping assay was conducted.We found that E33 could inhibit cellular Topo II but did not induce Topo II degradation.We also found that E33 did not induce DNA damage in tumor cells,and this result explained why E33 did not induce Topo II degradation These results indicated E33 performed potent anti-tumor activity and E33 was differ from classic Topo II posion.Futher researches were needed to evaluate the bioactivities of E33.