Design,Synthesis And Biological Application Of Bromodomain-containing Protein 4 Inhibitors

As an epigenetic reader,bromodomain-containing protein 4（BRD4）specifically recognizes and binds to acetylated-lysine in histones to regulate the process of gene transcription.Dysfunction of BRD4 is closely related to a variety of human diseases,so BRD4 has been considered as a promising drug target.In the past decade,a series of small molecule inhibitors of BRD4 have been reported which bind to BRD4 by mimicking acetyl-lysine.And some of them exhibited excellent cell proliferation inhibitory effects and antitumor activities.So far,multiple BRD4 small molecule inhibitors（such as RVX-208 and CPI-0610）have entered clinical trials,and most of them are used for malignant solid tumors and leukemia.However,the occurrence of side effects,insufficient clinical efficacy and primary and acquired resistance limited clinical application of some inhibitors.With the emergence of various new strategies,we think there remains new scope and direction for development and application of BRD4 inhibitiors.Based on candidate 51 previously reported by our laboratory,through in-depth analysis of it’s binding mode with BRD4,we mainly explored the structure-activity relationship of the structure located at the solvent region,and designed and synthesized a series of inhibitors.Molecular binding activity,cellular activity and in vitro metabolic stabilities were comprehensively evaluated.Then compound 69 with good metabolic stability of liver microsomes and compound 70 with the best cellular activity were selected for further evaluation.Compound 69 showed a moderate plasma exposure,and its toxicity was comparable to compound 51.Although the toxicity of compound 70 is weaker than compound 51,its pharmacokinetic properties are still not ideal.Based on the current results,we preliminarily identified compounds 69 and 70 as the potential backups of compound 51,and more comprehensive biological evaluation will be carried out.Autosomal dominant polycystic kidney disease（ADPKD）is a systemic disease characterized by progressive enlargement of bilateral kidney cysts,leading to kidney dysfunction.It was reported that BRD4 delays cyst growth in ADPKD,so the development of selective BRD4 inhibitors has great clinical value.According to previous efforts of our laboratory on the discovery of BET and kinase dual-target inhibitors 71,we obtained compound 79 with good selectivity for BET family members and BD1 domain through rational drug design.At a concentration of 1μM,compound79 had little inhibition on the tested 58 kinase.And compound 79 exhibited good pharmacokinetic properties in the mice model,which dosed at 10 mg/kg showed a good plasma exposure with the AUC_0-∞value of 2770 ng·h·m L^-1 and a moderate half-life with the T_1/2 value of 2.35 h.In vitro 3D-MDCK cyst model showed potent inhibitory effect.In a pkd1 ^flox/flox:Ksp-Cre mouse model,compound 79 exhibited high safety and good therapeutic effect,which significantly slowed cyst growth,by subcutaneous injection at a dosage of 3 mg·kg^-1daily.Mechanistic study also confirmed that inhibition of BRD4 with compound 79 could regulate cystic renal epithelial cell proliferation through c-Myc mediated p21 and Rb signaling pathways.In summary,in the first part of this study,we designed and synthesized a series of compounds based on candidate 51,two of which can be further evaluated and developed.In the second part of this study,a class of selective BRD4 inhibitors was synthesized,one of which was applied to ADPKD research in order to explore its potential clinical efficacy.