Design,Synthesis,and Biological Evaluation Of Coumarin Derivatives As BRD4 Inhibitors

Bromodomain-containing protein 4（BRD4）,acting as a transcriptional and epigenetic reader,plays a considerable role in various biological processes including transcription.The aberrant expression of BRD4 is currently regarded as one of the most likely cause of many diseases such as cancer.Although a large number of potent bromodomain inhibitors have been discovered,none of them has been marketed.Coumarins and their derivatives have long been known for their biological activity and druggability,which also have rarely been used in BRD4 inhibitors.Therefore,it’s a promising way to design new coumarin-based BRD4 inhibitors.In the preliminary work,25 BRD4 inhibitors with coumarin skeleton(BRD4 IC₅₀=40-200 n M)were obtained by scaffold hopping.In this thesis,the structure-activity relationship and cytotoxic activity were first evaluated.Among them,compound SW18endowed with respectable inhibitory activity(BRD4-BD1 IC₅₀=99 n M,BRD4-BD2IC₅₀=82 n M,MCF-7 IC₅₀=2.01±0.47μM,HGC-27 IC₅₀=7.67±1.00μM)and had been proved to induce apoptosis and to inhibit colony formation,cell cycle,and migration of MCF-7.Furthermore,it can downregulate the formulation of proto-oncogene c-Myc.However,the poor safety window(HEK293T IC₅₀=7.32±3.03μM,GES-1 IC₅₀=14.97±2.30μM)and pharmacokinetics(t_1/2=2.96 h,F=3.79%)suggested it needs further structural optimization.Then,31 coumarin derivatives with certain BRD4（BD1）selectivity were designed and synthesized by molecular assembly based on compound SW18.Among them,compound 29j was outstanding for its activity and selectivity(BRD4-BD1 IC₅₀=113n M,BRD4-BD2 IC₅₀=2639 n M,with 23.35 times over BD2),which also had a much greater safety window in cellular level(HGC-27 IC₅₀=5.91±1.81μM,GES-1 IC₅₀=45.47±0.52μM).The docking result revealed that the carbonyl of coumarin could form hydrogen bonding with Asn140 in the KAc pocket,and the o-methoxybenzene could extend into the WPF shelf and generateπ-πstacking interaction.In addition,the piperazine could reach into the BC Loop and form hydrogen bonding with the BD1-specific Asp144 that drives a better BRD4（BD1）inhibitory of compound 29j.Further studies showed that compound 29j could dose-dependently down-regulat the proto-oncogene c-Myc and increas the level of the oncogene p21.Furthermore,it also had better druggability in the prediction of Swiss ADME.Thus,compound 29j is a potential compound for in-depth study.In summary,we first investigated the antitumor mechanism and pharmacokinetics of pan-BRD4 inhibitors with coumarin skeleton and found that they were poor in safety window and pharmacokinetics.Subsequently,a series of BRD4（BD1）selective inhibitors with coumarin derivatives were designed,synthesized by molecular assembly,which unfolded effective selecting activity of BRD4（BD1）and preferable antitumor activity,providing a basis for the development of selective inhibitors of BRD4（BD1）in the treatment of cancer.