Design,Synthesis And Biological Activity Evaluation Of Aminopyridine Containing Spiro Derivatives As EGFR/HER2 Inhibitors

The increasing incidence rate and mortality rate of cancer had caused widespread concern in the society.Epidermal growth factor receptor（EGFR or HER2）overexpression is one of the main causes of cancer.In tumor cells,overexpression of EGFR/HER2 binds to ligand factors and activates downstream signaling pathways,leading to cancer cell proliferation,angiogenesis,tumor invasion and metastasis and other physiological processes.EGFR/HER2 inhibitors can significantly inhibit the activation of related pathways and slow down the above changes to play an anti-tumor role.Neratinib,as an EGFR/HER2 inhibitor,will reduce the expression of phosphorylation and carcinogenic signals of receptors.It was approved for listing in 2017,and has obvious therapeutic effects on non-small cell lung cancer,colon cancer and breast cancer.In our previous work,we designed and synthesized a series of indole spiro compounds,which showed good antitumor effects,In this project,a novel EGFR/HER2double target inhibitor was designed and synthesized by using neuratinib as the lead compound and introduction of indole spiro ring fragment:（1）5-bromo-2-chloropyrimidin-4-amine derivatives（intermediates3a-3d）were synthesized by modifying 3-chloro-4-（pyridine-2-methoxy）aniline,the pharmacophore of the lead compound neratinib.3-chloro-4-（pyridine-2-methoxy）aniline derivatives（2a-2d）were synthesized by Hofmann alkylation of 2-chloro-4-aminophenol derivatives,and then 2,4-dichloro-5-bromopyrimidine was introduced to obtain intermediate 3a-3d（Structural segment I）;（2）The 5’-methoxy-1’,2’-dihydrospiro[cyclopropane]-1,3’-indole]-6’-amine（Intermediate 8）was synthesized by alkylation using 5-methoxy-2,3-dihydro-1H-indole-2-one as the starting material by alkylation,nitration and reduction.The 5-bromo-N²-（5’-methoxyspiro[cyclopropane-1,3’-indole]-6’-yl）pyrimidin-2,4-diamine derivatives（Intermediates 9a-9d）were synthesized by combining intermediate 8（Structural fragment II）and intermediate 3a-3d（Structural fragment I）.The 5-nitrile-N²-（5’-methoxyspiro[cyclopropane-1,3’-indole]-6’-yl）pyrimidin-2,4-diamine derivatives（intermediates 10a-10d）were synthesized by the reaction of compounds 9a-9d with zinc cyanide in the solvent of N,N-dimethylformamide and water using tris（dibenzylacetone）dipalladium（PD₂（DBA）₃）and 1,1’-ferrocene bis（diphenylphosphine）（DPPF）as catalysts.Finally,novel compounds A1-A7 containing 5’-methoxyspiro[cyclopropane-1,3’-dihydroindole]and 4-（（4-（benzyloxy）phenyl）amino）pyrimidine-5-carbonitrile were obtained by acylation of 10a-10d.（3）The 3,4-dihydrospiro[benzo[b][1,4]oxazine-2,1’-cyclopropane]was introduced to replace spiro[cyclopropane-1,3’-dihydroindole]in a series,and new compounds containing 7-methoxy-3,4-dihydrospiro[benzo[b][1,4]oxazine-2,1’-cyclopropane]and 4-（（4-（benzyloxy）phenyl）amino）pyrimidine-5-carbonitrile B1-B4 were obtained.The starting materials of these compounds are 1-fluoro-5-methoxy-2,4-dinitrobenzene and 1-hydroxycyclopropane-1-carboxylic acid methyl ester.The 7-methoxy-3,4-dihydrospiro[1,4-benzoxazin-2,1’-cyclopropane]-6-amine （Intermediate 14）was synthesized by Williamson synthesis and two-step reduction reaction,which was used as structural fragment III.The5-bromo-N²-（7-methoxy-3,4-dihydrospiro[benzo[b][1,4]oxazine-2,1’-cycl opropane]-6-yl）pyrimidin-2,4-diamine derivatives（Intermediate 15a-15d）were prepared by one reaction with the 3a-3d（structura fragment I）.Then,5-nitrile-N²-（7-methoxy-3,4-dihydrospiro[benzo[b][1,4]oxazine-2,1’-cycl opropane]-6-yl）pyrimidine-2,4-diamine derivatives（Intermediates16a-16d）were obtained by substitution reaction.After acylation,the target products B1-B4 were obtained by separation.The structures of intermediates and end products were confirmed by MS and ¹H-NMR,and the purity of that was determined by liquid phase method.（4）The inhibition of EGFR/HER2 protein kinase activity of A1-A7and B1-B4 was studied by near scintillation counting method with neratinib as positive control.The results showed that the residual activity of EGFR-wt kinase ranged from 68%to 97%and that of HER2 ranged from 87%to 100%at the drug concentration of 0.5μM.In which A5 was the most prominent,and the residual activity of EGFR-wt kinase was68%,HER2 was 90%.Compared with the kinase residual activity in the presence of A1（92%）/B1（86%）,A2（97%）/B2（76%）,A3（95%）/B3（79%）and A6（94%）/B4（85%）,it was found that 3,4-dihydrospiro[benzo[b][1,4]oxazine-2,1’-cyclopropane]had better inhibitory effect on EGFR-wt than spiro[cyclopropane-1,3’-dihydroindole].In conclusion,11 novel spiro substituted aminopyrimidine EGFR/HER-2 double target inhibitors and 31 intermediates were designed and synthesized,and the inhibition experiments of A1-A7,B1-B4 on EGFR-wt and HER2 kinase were carried out.The preliminary experimental data showed that the new aminopyrimidine compounds had limited inhibitory effect on the two protein kinases compared with the control neratinib activity,but A4 and A5 could provide reference for the subsequent structural transformation.