Design,Synthesis And Bioactivity Evaluation Of Antitumor Agents Targeting BRAF Kinase

Dysfunction of signaling pathways in cells is one of the important causes of major diseases such as malignant tumors.MAPK is closely related to the occurrence and development of diseases.MAPK is one of the most important signaling pathways in cells,including multiple parallel branches,which jointly regulate cell growth,differentiation,migration and apoptosis.The most detailed studied in the MAPK is the ERK pathway(RAS/RAF/MEK/ERK cascade),and its mutation and abnormal activation often cause the occurrence of tumor,such as the melanoma.The RAF is the most active and key member of the ERK pathway,in which BRAF kinase is the most mutagenic subtype of kinases.The mutation rate of BRAF in malignant tumors is about 8%(90%of the mutations is BRAFV600E mutation),and the mutation rate in melanoma cells is about 80%.BRAFV600E is one of the hot targets for developing targeted inhibitors of tumors,especially the melanoma.Several BRAF inhibitors have been approved for clinical treatment of tumors,such as dabrafenib,trametinib and vemurafenib.Although these drugs significantly improve the overall and progression free survival of the cancer patients,the recurrence and drug resistance are frequently reported in clinical use.It is an urgent need to develop inhibitors with novel backbones that can reduce relapse and drug resistanceThe general structure of BRAFV600E inhibitors contains a hydrogen bond donor,which could interact with the hydrophobic pockets of the target protein.The hydrogen bond donor is often a heterocyclic compound such as pyrazole,imidazole,pyridine,or a substituted aromatic group.Pyrazole,one of five membered aromatic heterocyclic compounds,has active chemical properties and many biological activities,such as anti-tumor,anti-bacterial,anti-inflammatory.Based on this knowledge and combined with the active fragments reported in the literature and the research results of our group on BRAF inhibitors,we exploited a new scaffold with pyrazole structural units by virtual screening.Subsequently,we obtained the lead compound by scaffold growth and then obtained a series of compounds by deriving it.Using virtual screening,the derivatives were ranked according to the docking score for the selecting of the best hit(compound 4m),whose binding models were further verified by molecular dynamics simulation.The results showed that compound 4m was closely linked to the active site of BRAF kinase through two hydrogen bonds and some weak interaction forces and the docking model of compound 4m and BRAF protein has high stability.All compounds of 4a-4t were synthesized and the structures were characterized by 1H NMR,13C NMR and EI-MS.Subsequently,we evaluated the activity of all the compounds synthesized and studied the structure-activity relationship of the compounds 4a-4t based on the experimental results.The experimental results suggested that most of these compounds had moderate anti-tumor activity.Among these compounds,compound 4m showed the better BRAFV600E kinase inhibitory activity(IC50 value of 0.09±0.01μM)and anti-proliferation activity(the IC50 value of 1.08±0.07μM).Besides,in A375 cells,compound 4m could induce the cell apoptosis and cell cycle arrest in a dose-dependent and time-dependent manner.In addition,cytotoxicity tests to 293T cells showed that most compounds had low toxicity and safety.The purpose of this project is to provide an important guid for the following structure optimization and the development of a new BRAF inhibitor,which is more efficient and low toxic,through the docking scoring,bioactivity assessment and structure-activity relationship analysis.