Synthesis And Biological Activity Of Indole-2-ones As BRD4(D1)Selective Inhibitors

Bromodomain protein 4（BRD4）,a member of the BET family,plays a role in regulating gene transcription and cellular processes via binding to the acetylated lysine residues on the tail of histones,and recruiting the transcription factors related with chromatin regulation proteins.Thus,targeting BRD4 protein is an effective strategy for the treatment of various diseases（especially malignant tumors and chronic inflammation）.Currently,a variety of BRD4 inhibitors have been discovered,some of which have been included in different stages of human clinical trials,such as I-BET762,OTX-015,and ABBV-075.However,pan-BRD4 inhibitors usually show adverse effects in clinical trials,such as dose limiting toxicity and thrombocytopenia.Those inhibitors that are selective for the D1 or D2 domain of BRD4 have important clinical value,such as GSK340 and LT052,which can provide better specificity and help people to study the pharmacological effects of BRD4 and distinguish the biological characteristics of each domain,which can also improve the clinical effect and reduction of toxic and side effects.In previous work,we had found that indole-2-ones had potent BRD4 inhibitory activity,unfortunately,they lacked selectivity for different domains.In order to seek for BRD4 inhibitors with higher selectivity to D1 domain,and study its anti-tumor activity and mechanism,we synthesized a series of 5-substituted,5,6-substituted and 5,7-substituted Indole-2-ones based on studying the structural differences between the D1and D2 domains of BC Loop of BRD4.And the preliminary studies on the biological activities and their structure-activity relationship had been carried out.Specifically,using different substituted indolinones or 4-nitrophenylacetic acid as raw materials,thirty-two indolin-2-one compounds were synthesized through nitration,aldol condensation,reduction,Suzuki coupling and acylation reactions.Their inhibitory activities against BRD4（D1）and BRD4（D2）were determined by time-resolved fluorescence analysis（TR-FRET）,and most of the compounds showed strong D1enzymatic inhibitory activity(IC₅₀<100 n M).Among them,compound 28c expressed the highest domains selectivity to D1 for 22.1 times,although its BRD4（D1/D2）IC₅₀was 38 n M and 838 n M,it expressed poor anti-proliferative activity against cancer cells,which might be due to too strong structural rigidity and poor transmembrane ability.Compound 29a against BRD4（D1/D2）with the IC₅₀ values of 41 n M and 313 n M,the selectivity of D1 domain was 7.6 times more than D2 domain.In the cytotoxicity experiment,compound 29a displayed potent activity with the IC₅₀ values of 4.64±0.30μM,0.78±0.03μM and 5.57±1.03μM against three cancer cells lines（HL-60,MV4-11 and HT-29）for 72 hours,respectively.Especially,29a had low toxicity(IC₅₀=91.80±1.11μM)against normal GES-1 cells for 72 hours.In docking model,the indolinone nucleus of compound 29a could form hydrogen bonds with the KAc recognition region,while the methoxyphenyl and N-methyl piperazine groups occupied the WPF region and the BC Loop respectively in the docking study,which could form a strong interaction with the BRD4（D1）protein.In the study of the anti-proliferative mechanism,we found that compound 29a blocked MV4-11 cell cycle in G0/G1 phase and induced cell apoptosis in dose-dependent manner.In addition,compound 29a inhibited the expression of proto-oncogene c-Myc in tumor cells.Furthermore,we evaluated the drug-like properties of 29a,which conformed to the five principles and had well drug-like properties.In conclusion,the research in this thesis showed that 5,6-disubstituted indol-2-ones displayed effective BRD4（D1）inhibitory activity and strong anti-tumor proliferation activity,providing experience for the study of BRD4（D1）selective inhibitors.