Design,Synthesis,and Antitumor Evaluation Of Quinoxalinone Derivatives As Potent BRD4 Inhibitors

As a chromatin reader,bromodomain-containing protein 4（BRD4）is an epigenetic regulatory protein,whose overexpression is closely related to tumors,inflammation and autoimmune diseases.As a result,it has become one of the most effective drug targets.Since the first BRD4 inhibitor（+）-JQ1 was reported,a variety of inhibitors（such as OTX-015,CPI0160 and ABBV-075,etc.）have entered into clinical trials.However,due to the drawbacks such as drug resistance and dose-dependence toxicity,no compound has been successfully marketed.Therefore,research on novel BRD4 inhibitors still has good potential.In this paper,ABBV-075 was used as a template compound.Based on the COMFA model analysis,32 quinoxalinone BRD4 inhibitors were designed and synthesized by classic drug design strategies such as scaffold hopping.Further,the BRD4 inhibitory activity and antiproliferative activity were also evaluated（MV-4-11,PC-3,MCF-7,Hep G2 and HT-29 cells）.Among them,the preferred compound X15 exhibited promising enzyme inhibitory activity with BRD4 BD1 IC₅₀ valuing 82.3 n M and demonstrated pleasurable selective anti-proliferation activity on Hep G2 cells(IC₅₀=1.13±0.07μM).Besides,the toxicity towards normal cell line GES-1 was also low with IC₅₀ valuing57.24±5.46μM.The docking mode with BRD4 BD1 protein manifested that X15 had a tight interaction with KAc pocket and formed favorable interactions with ZA channel and WPF region.In the preliminary mechanism studies,WB experiments showed that X15 had a significant dose-dependent inhibitory effect on the key downstream protein c-Myc of BRD4.Wood healing,transwell and WB experiment demonstrated that X15 could effectively inhibit the migration of Hep G2 cells through regulating the expression of migration-related proteins（such as Snail,E-cadherin,Occludin and MMP-9 proteins）.Colony formation and flow cytometry assay manifested that X15 could inhibit the colony formation,arrest the cell cycle at G₀/G₁and induce apoptosis of Hep G2 cells in a dose-dependent way.In addition,the Swiss ADME forecast showed that X15 conformed to the five principles of Lipinski and had a certain potential of patent medicine.In conclusion,a series of novel quinoxalinone derivatives was designed and synthesized through scaffold hopping strategy,which exhibited pleasurable BRD4inhibitory activity and anti-proliferation activity,providing experience to the BRD4inhibitors in liver cancer.