Design,Synthesis,Biological Evaluation Of Pyrazole Derivatives Containing Acetamide Bond As Potential BRAF^V600EInhibitors

Due to environmental deterioration and lifestyle changes,malignant tumors have become the main cause of human death in the new century with difficult treatment and low recovery rate.It is easy to relapse and metastasis.There are many targets associated with the development of malignant tumor,and the BRAFV600E mutation is closely related to the occurrence,invasion and metastasis of malignant melanoma,thyroid cancer,colon cancer and ovarian cancer.Therefore,BRAFV600E has gradually become a hot target for the research of anti-cancer drugs,and the development of small molecule BRAFV600E inhibitors has become the research focus of anticancer drugs.At present,the drug resistance,relapse and side effects of small molecule BRAFV600E inhibitors have become increasingly prominent.Therefore,it is of great significance to design new and more effective inhibitorsAiming at the target BRAFV600E,studying the binding site of receptor protein BRAFV600E stereostructure and the known structure activity relationship of endogenous ligands extracted from BRAF eutectic,24 pyrazole derivatives containing acetamide bonds were designed and synthesized by computer aided drug design method.The structures of all compounds were confirmed by 'H NMR,13C NMR and ESI-MS methods.Subsequently,the target compounds had anti-tumor proliferation in vitro experiment,BRAFV600E inhibitory activities experiment,induced apoptosis experiment,cell cycle arrest and cell membrane potential experiment to evaluate the antitumor activities of 24 compounds,of which 5r showed high anti-tumor cell proliferation activity(the IC50 value of A375 was 0.96±0.10?M)and BRAFV600E inhibitory activity(IC50=0.10 ± 0.01?M)than positive control vemurafenib(A375 cells IC50=1.05 ± 0.01?M,BRAFV600E IC50=0.04±0.004?M).In addition,compound 5r can induce apoptosis of A375 cells,induce irreversible programmed cell death in A375 cells through destruction of mitochondrial transmembrane potential,and block the A375 cell cycle in G1 phase.The molecular docking model showed that the compound 5r could be tightly bound to the active site of BRAFV600E(PDB encode:4xv9).Based on the above experimental results,the 24 compounds,represented by the compound 5r,are expected to develop new therapeutic agents.The construction of the 3D-QSAR model also provided more valuable theoretical guidance for the research of such inhibitorsLicorice is a traditional Chinese herbal medicine which contains many functional ingredients and has extensive pharmacological activity.At present,glycyrrhizic acid is the main target in the development of licorice,and other ingredients contained in waste residue are abandoned,resulting in a great waste of resources.Glabridin is found in Glycyrrhiza glabra L.It has been known as "whitening gold" because of its safe and significant melanin inhibition effect.At the same time,it has significant effect in anti-oxidation,anti-tumor and other aspects.It has a broad application prospects in medicine,food,cosmetics and other areas.However,due to the complex material components,rare Glabridin content,high cost of production and difficult to chemical synthesis,the research progress of the industrial production of Glabridin is slow,so far there is no complete production process route in the factory,and it has seriously affected the progress of its new drug design.Glabridin is found that its applications scope and effects are affected by its poor water solubilityFirstly,we extracted and purified the main components of licorice residue.A total of 6 compounds including Glabridin were obtained.All the compounds were analyzed by 1H NMR,ESI-MS and 13CNMR,and their chemical structures were finally determined.Secondly,we optimized the production conditions of Glabridin,and designed the process route.The raw material was licorice residue,setting the methanol content of the extraction solution,extracting time,extracting temperature and extraction times four variables,using orthogonal experimental method,using the extraction rate of extract and the content of flavonoids as the standard.Finally,the 70%-80%methanol content,extraction time 1h,extraction temperature 100?,and 2 extraction times are the best conditions.Polyamide chromatography column was selected to purify Glabridin.The licorice residue was extracted with 80%methanol.The extraction solution was diluted to 60%-70%methanol content with the first polyamide column to separate and purify.The effluent was diluted to the content of 30%-40%methanol with the second polyamide column to separate and purify,and then second polyamide column was eluted with 40%-50%methanol.The elution was separated and purified by third polyamide column.The polyamide column was eluted with 80%-90%methanol and the eluent was dried.The purity of Glabridin was 40%,and the purity of 98%was obtained by adding the 20%-30%methanol water recrystallization.Finally,the yield of 40%Glabridin was 4%-5%,and the yield of 98%Glabridin was 0.5%-1%.Finally,the water solubility was improved by structural modification.First,methyl bromide(1 mmol),Glabridin(0.1 mmol)and K2CO3(0.2 mmol)were added together into acetone solution,and was stirred for 8-1 Oh at 60?.The water and ethyl acetate were extracted after the reaction was stopped,and the silica gel column with ethyl acetate:petroleum(1:10,1:6,1:4)gradient elution to separate and purify the product.Then,the white oily solid was obtained.Adding a proper amount of methanol to dissolve the the product,adding a large amount of water,KOH(0.6 mmol),45?-60?,reaction for 2-3 hours.At the end of the reaction,methanol was evaporated.Water was added with acid(1 mol/L diluted hydrochloric acid)to reduce the solution to weak acid.Then,the solution was extracted by ethyl acetate,and the dried organic layer was the desired product.