Design,Synthesis And Antitumor Activity Evaluation Of Olmutinib Derivatives

With the increasing number of people died of cancer every year,cancer has become one of the major threats to human health.With the development of molecular biology,people have a deeper understanding of the occurrence and development mechanism of cancer.There is an abnormal expression of the Epidermal Growth Factor Receptor（EGFR）signaling pathway in a variety of tumor tissues,which is closely related to tumor invasion and metastasis.The development of small molecular inhibitors targeting EGFR has become a potential strategy for the treatment of cancer,especially non-small cell lung cancer（NSCLC）.This paper reviews the EGFR tyrosine kinase inhibitors（TKIs）of small molecule receptors,and summarizes,analyzes and optimizes the synthesis route of Olmutinib,the third generation EGFR inhibitor that marketed in Korea.In addition,in combination with the results of structure-activity relationship studies of Olmutinib and the other third EGFR TKIs,the lead compound Olmutinib was structurally modified.The structure of pyrimidine amino group and acrylamide was preserved,and the molecular U-shaped scaffold was maintained.Through the introduction of N-methylpyrazole and anisidine like side chains,the influence of the type of amino side chain near the solvent area on the activity of the compounds was investigated;the methoxy group on the anisidine group was replaced by cyano group and the nitrogen atom was introduced into the benzene ring of the anisidine structure to explore the electrophilic and repellent properties of the side chain and the influence of the change of electron cloud density of the benzene ring on the activity of the compounds;through the introduction of different length of flexible chain at the end of Acrylamide,the reaction activity between acrylamide warhead and CYS-797 is changed.In addition,according to the results of molecular docking,the cavity of thiophene pyrimidine parent nucleus has a large transformation range,and the parent nucleus is designed to be replaced by thiapyran-pyrimidine in the later stage to observe its influence.At last,we designed ten series of 65 Olmutinib derivatives based on two different parent nuclear scaffolds.The synthesis route of Olmutinib was summarized and analyzed,and its synthesis was optimized to some extent.The total yield of the optimized route was17.7%higher than that of the patented route.It simplified the post-treatment,reduced the cost.The synthesis of the target compounds was based on the synthesis route of Olmutinib.Starting from methyl 3-aminothiophene-2-carboxylate and dimethyl 3,3’-thiodipropionate,the parent nuclei of thiophene-pyrimidine and thiapyran-pyrimidine were prepared by cyclization and condensation,and then the key intermediates A4 and B5 were obtained by chlorination and nucleophilic substitution,which obtained the intermediates A5a-A5e and B6a-B6e by nucleophilic substitution reaction with different amines.A5a-A5e and B6a-b6e were reduced by hydrazine hydrate,and then the reduced products were amidated with different kinds of small molecular acids.Finally,the target compound X1-X65 was obtained.The structures of all the target compounds were confirmed by ¹H NMR,and some of them were confirmed by ¹³C NMR and TOF MS.All of the target compounds were tested for cell activity,and some of the compounds with outstanding activity were further studied,including concentration dependent test,kinase activity screening and evaluation,AO/Hoechst3358immunofluorescence staining experiment,cell cycle apoptosis test,and molecular docking simulation.Compared with the lead compound Olmutinib,most of the target compounds got lower activity on human normal cells LO2,indicating that the target compounds had lower side effects in vitro.The compounds showed better inhibitory activity on mutant lung cancer cell line H1975 and wild type lung cancer cell line A549 than other cells,indicating that the compounds mainly played a role by blocking the EGFR signal pathway.Further screening of kinase activity confirmed this conclusion.The inhibition rate of X27,the best compound of thiophene-pyrimidine series,on EGFR^T790M/L858R was as high as 103.6%（1μM）,the IC50 was as low as tens of nanomoles,and the late apoptosis of cancer cells was induced in a dose-dependent manner.The IC50 of the optimal compound X41 of thiapyran-pyrimidine series on EGFR^T790M/L858R was 132.1n M,and it could induce the late apoptosis of cancer cells in a dose-dependent manner,and block the cancer cells in G2/M phase.Based on the in vitro activity evaluation,physical parameter analysis and molecular docking results,the structure-activity relationship of the target compounds was summarized as follows:（1）the compounds were embedded in the protein kinase domain in the U-shape,and the pyrimidine amino group and Michael receptor acrylamide warhead interacted with the amino acid residues MET-793 and ARG-841in the cavity,all of which were pharmacodynamic groups.（2）The activity of acrylamide warhead was the best when there was no side chain at the tail end,and the activity of the side chain at the tail end was decreased when there were branch chains or fluorine atom.Compounds containing fluorine atom in the side chain had an increased inhibitory effect on LO2.Fluorine atom could change the distribution range of the electron cloud of the compound,weaken the ability of the compound to interact with amino acid residues,and also reduce the selectivity of the compound to cancer cells.（3）The target compounds with electron absorbing or electron donating groups inserted into the benzene ring in the side chain pointing to the solvent region,had moderate or even excellent antiproliferative activity to H1975 and A549 cells.When there was nitrogen atom on the side chain benzene ring,the electron cloud density of the benzene ring decreased,resulting in a slight decrease in activity.（4）The substitution of thiapyran-pyrimidine for thiophene-pyrimidine had no significant effect on the activity.（5）The parameters of Clog P and TPSA were closely related to the acrylamide warhead and the side chain in the solvent zone.In conclusion,in this paper,the synthesis and optimization of Olmutinib were carried out.65 novel Olmutinib derivatives based on two different parent nuclear structures were obtained through the structural transformation of Olmutinib,and their structure-activity relationship and mechanism of action were preliminarily analyzed and discussed,which provided optimization and transformation ideas for the study of EGFR inhibitors,and pointed out the direction for later research.