Design,Synthesis And Biological Evalution Of Triflurormethylated Anion Transproters

Homeostasis of cellular anions provides the premise and basis for maintaining the normal physiological function of cells.Destroying the homeostasis of cellular chloride anions may change the concentration of intracellular chloride,increase lysosomal pH,inhibit lysosomal enzyme activity,disrupt autophagy,and eventually lead to cell death.Therefore,synthetic compounds that are capable of facilitating the transmembrane transport of anions may be developed as a class of potential antitumor drugs.Because they are able to destroy the homeostasis of cellular chloride anions and trigger tumor cell death,anion transporters may provide a new strategy for the design of antitumor drugs.Studies to date have shown that some synthetic anion transporters exbibit promising antitumor activity.Modification of anion transporters with trifluoromethyl groups can not only improve the lipophilicity and hence make it easier for them to enter phospholipid bilayer membranes from water phase,but also change the electron density distribution of the compounds and enhance the affinity of anion transporters toward anions.These two effects may play a crucial role in improving the anion transport and biological activity of anion transporters.To date,trifluoromethylated anion transporters have been reported,and some of them have been demonstrated to exhibit strong anion transport and biological effect.This dissertation consists of three chapters.In Chapter One,the research progress in the application of trifluoromethyl group in anion transporters is reviewed,with the aim to provide some useful guidance for the rational design of effective anion transporters.In Chapter Two,nine trifluoromethylated 1,3-bis(2-benzimidazolyl)benzene derivatives 1-9 were synthesized and their anion transport activity and antitumor activity were systematically studied.Studies by means of 1H NMR titration and ESI MS showed that compounds 1-9 are able to form stable 1:1 complexes with chloride anions.Chloride selective electrode techniques and pH discharge experiments based on EYPC liposomes showed that compounds 1-9 exhibit strong anion transport activity.Structure-activity relationship analysis showed that the transmembrane anion transport activity may be improved by introducing electron-withdrawing substituents,such as NO2,F or CF3 on the central phenyl ring of the 1,3-bis(2-benzimidazolyl)benzene,and/or on the benzimidazolyl subunits.Mechanistic studies based on anion-cation selectivity and U-tube experiments showed that compounds 1-9 may exert anion transport activity most probably through a process of anion exchange via a carrier mechanism.Acridine orange co-staining and Magic Red Cathepsin assays showed that compounds 1-9 are able to alkalize the lysosomes and reduce the activity of lysosomal Cathepsin B enzymes.MQAE results showed that compounds 1-9 can facilitate the influx of chloride anions into cells.The results obtained from MTT assay,flow cytometry and JC-1 co-staining showed that compounds 1-9 exhibit strong cytotoxicity in an apoptotic fashion.In iorder to increase the ability of anion transporters to specifically target.organelles and investgate their distributions within cells,in Chapter Three,we designed compound 12 having a morpholinyl group and coumarin subunit,and its analogue 13 without a morpholinyl group.LysoTrackerTM Deep Red co-staining showed that compound 12 can target lysosomes specifically,whereas compound 13 exhibits lower ability to target lysosomes,suggesting that a morpholinyl group plays a determinant role in improving the ability to target lysosomes.Liposomal and in vitro anion transport based on pH discharge experiments,LysoSensor Green DND-189 co-staining and Magic Red Cathepsin assays showed that compounds 12 and 13 exhibit moderate,but comparable ion transport activity,and compound 12 exhibit stronger capability to alkalinize lysosomes.To further improve the anion transport activity,two trifluoromethylated analogs of compound 12,that is compounds 15 and 16 were synthesized.Both compounds 15 and 16 can target lysosomes specifically.Studies based on pH discharge and chloride selective electrode techniques showed that compared with compound 12,compounds 15 and 16 exhibit significantly improved anion transport activity.In addition,compounds 15 and 16 are able to alkalinize lysosomes more efficiently than compound 12.These results showed that modification with trifluoromethyl groups can improve the anion transport activity of these anion transporters as well as their capacity to alkalinize lysosomes.The biological effects of such coumarin-squaramide conjugates triggered by targeting and alkalizing lysosomes,will be further explored.In summary,two series of anion transporters based on 1,3-bis(2-benzimidazolyl)benzene and morpholine-coumarin-squaramide were synthesized and their biological effects were evaluated in details.The present findings demonstrated that modification of anion transporters with trifluoromethyl groups can significantly improve the anion transport activity and biological activity.These results may provide some useful guidances for the rational design of effective anion transporters.