Design,Synthesis And Biological Evaluation Of Novel BRD4 Inhibitors

Epigenetics is a concept corresponding to genetics.Epigenetics refers to changes in gene expression levels that are not caused by changes in gene sequence,including DNA methylation,histone acetylation,non-histone acetylation,non-coding RNA regulation,and chromatin conformational changes.Among them,potential antitumor drugs targeting DNA methylation and histone modification are the focus of current researches,and it is widely accepted that epigenetic drugs will be one of the important cornerstones of the cancer treatment.Bromodomain is a kind of conserved protein domain that can specifically recognize histone acetylated lysine,which exists in a variety of epigenetic regulatory proteins.At present,61 kinds of BRD have been found in the human.According to the difference of proteins’ functions in which they are located,these domains are subdivided into 8 families,of which bromodomain and extra-terminal domain(BET)proteins belong to the second family and was most thoroughly studied among BRD proteins.The BET family consists of BRD2,BRD3,BRD4,and BRDT,and BRD4 was selected as the research object in this paper.The N-terminal of BRD4 contains two bromodomains(BD1 and BD2)with 95% sequence identity in the binding pocket of KAc.However,their biological roles are markedly different.Furthermore,although the drug-like properties of BRD4 inhibitors have been verified preliminarily in clinical trials for the treatment of cancers and cardiovascular diseases,only a few inhibitors fulfill the selectivity between two homologous bromodomains of BRD4 and mechanism-based toxicity appeared in clinical trials of some pan-BRD4 inhibitors,impeding more extensive clinical application.Hence the acquisition of novel BRD4 inhibitors with excellent selectivity is imperative.The research contents of this paper include the following three aspects:(1)The theophylline core was utilized as the structural basis and 7-benzyl theophylline derivatives were designed as novel BRD4 inhibitors by molecular docking.Then,our target compounds with moderate inhibitory potency were synthesized.IC50 values ranged from 35 μM to 50 μM.In the further process of optimization,we found that the1,7-dibenzyl substituted theophylline derivatives possessed obvious selectivity for BRD4-BD1 and significant structural differences from the known BRD4 inhibitors.Therein,compound 16 d showed the highest selectivity for two bromodomains of BRD4,the inhibition of BD1(IC50 = 2.51 μM)was 20 times greater than that of BD2.Furthermore,the potential binding modes of 1,7-dibenzyl substituted theophylline derivatives with BRD4 were explored by molecular docking studies,showing that residue Asn140,Trp81,Leu92 and Ile146 in the binding pocket of BRD4-BD1 made an important contribution on inhibitory activity and Ile146 might be the key for 1,7-dibenzyl substituted theophylline derivatives to fulfill selectivity of BRD4-BD1.In the cancer cell proliferative inhibition assay,compound 16 d exhibited moderate antiproliferation potency on leukemia cell line MV4-11 with inhibition rate of 63.17% and a weak inhibitory effect on BRD4 insensitive cell lines and the inhibition rates were between 24.48% and 33.89% at a concentration of 10 μM.In addition,the synthesis process of 1,7-dibenzyltheophylline derivatives was optimized and the optimal reaction conditions were determined.(2)Commercially available tyrosine kinase inhibitors were conducted to perform molecular docking studies.The results showed that Nilotinib displayed the best docking conformation with BRD4-BD1 and possessed the potential to be a dual target inhibitor of BRD4/kinase.Therefore,the activity of Nilotinib and its analogs were evaluated.The molecular fragment of Nilotinib,3-(4-methyl-1H-imidazole-1-yl)-5-trifluoromethylaniline,was found to exhibit a certain inhibitory effect against BRD4 and the inhibitory rate against BRD4-BD1 was 40.70% at a concentration of 10 μM.In order to obtain compounds with better activities,a series of its derivatives were synthesized.Among them,compounds 22 g and 22 h showed higher inhibitory activities with inhibition rates of 47.28% and 60.10%,respectively.Furthermore,the possible binding modes of compounds were also researched.(3)In view of the excellent anti-inflammatory effects of andrographolide and BRD4 inhibitors,and molecular docking studies suggested that the diterpene lactone ring might be a privileged fragment to simulate KAc.Therefore,the inhibitory potency of andrographolide and andrographolide bisulfite was tested,and the results showed that andrographolide bisulfite showed weak inhibitory activity against BRD4 with inhibition rate of 32.55% at 10 μM.In conclusion,through the design,synthesis and activity screening of theophylline derivatives,3-(4-methyl-1H-imidazole-1-yl)-5-trifluoromethylaniline derivatives and andrographolide derivatives,it was found that 1,7-dibenzyl substituted theophylline derivative 16 d possessed obvious selective inhibition of BRD4-BD1.The results of this study enrich the structural types of BRD4 inhibitors and provide a valuable research basis for the development of drugs targeting BRD4.