Design,Synthesis,and Biological Evaluation Of 3,5-dual Substituted Aminopyridine Derivatives As ROS1 Kinase Inhibitors

Receptor tyrosine kinases(RTKs)are critical players in cellular communication network and function.Proto-oncogene tyrosine-protein kinase ROS(ROS1)is one of the last two remaining orphan the human receptor tyrosine kinases.The ligands and mechanism of activation of human ROS1 kinase have not been fully identified in different body tissues so far.Accumulating evidence advocates that ROS1 has become a potential drug discovery target.Crizotinib,which has been granted accelerated approval for the treatment of ROS1 rearranged NSCLC in March 2016.Despite rapid and impressive initial effects,a common concern of many patients is the invariably development of resistance.In those case,a secondary mutation G2032R in CD74-ROS1,have been most frequently detected.Hence,it’s absolutely important for the medicinal chemistry researchers to further explore and produce selective and potent ROS1 inhibitors as therapeutic strategy for different human malignancies.Based on the co-crystal structure of ROS 1 complexed with crizotinib,as well as our previous studies,we consider that the 2-aminobenzyloxypridine acts as a molecule’s skeleton of ROS 1 inhibitors.To design,synthesis,biological evaluation,and preliminarily structure-activity relationships analysis were performed for series compounds according to Crizotinib by comprehensively using the methods of structure-based design,chemical synthesis and evaluations of biological activity.We found seven novel compounds which showed significant potency and strong targeting in the enzymic and cellular level.The study lay the foundation for developing ROS1 inhibitors with clinical application value.The primary contents are listed as follows:1.Based on the co-crystal structure of ROS1 complexed with crizotinib as well as our previous studies,Inspired by the "DFG-in" conformation,we would like to design the selective and potent ROS kinase inhibitors.2.Starting materials and other reagents were purchased from commercial vendors,we designed and synthesized three series of the new aminopyridine derivatives.Starting materials,via reduction,acidation,hydrolyzation,Mitsunobu reaction,Suzuki-Miyaura coupling reaction,bromination and other reactions to synthesize three series of the new aminopyridine derivatives(9a-9j,18a-18z,19a-19z and 20a-20s).The chemical structures of the new synthetic compounds were characterized by ESI-MS,1H NMR and 13C NMR.3.To our surprise,we found that a majority of compounds were active against ROS1 but inactive against ALK and c-MET.A total of 27 new synthetic compounds showed good selectivity against ROS1 kinase.The percent inhibition values range of-13～37%of the enzymatic activity of c-MET kinase was observed,percent inhibition values range of 3～53%against ALK and the percentage of inhibition ranging from 61%～101%against ROS 1.4.Based on the results anti-proliferative results,we could find that all of the selected compounds had a lower cytotoxic activies against HaCaT cell line than Crizotinib and showed little potency against A549 cell line.the enzymatic and cellular activities were consistent for some new compounds,compound 18r,19k,19v,20a,20d,201 and 20m showed no potency against H3122 cell line(IC50>100 μM)and displayed little activities against A549 cell line,but had high activities against HCC78 cell line Particularly,compounds 19k,19v and 20m showed high potency and selectivity against ROS1 kinase and HCC78 cell line.The cellular and enzymatic potency were inconsistent for some other compounds,compounds 9e,9j,19q and 20k displayed little activities toward ROS1-addicted HCC78 cell line,we supposed that the different sensitivity to ROS1 fusion genes induced by synthetic compounds led to different response in HCC78 cell line or eventually developed resistance to compounds.Interestingly,the compounds,19o,20f,20i,20n displayed inferior activities against ROS1 kinase,but they exhibited good antiproliferative activities against HCC78 cell lines.It suggested that the compounds might induce cell apoptosis,or accelerate cell metabolism,or have some other mechanisms.5.Based on the enzymic activity(ALK,ROS1 and c-MET),the primarily structure-activity relationships of the novel synthetic compounds were analyzed.With regard to the pyrazole NH substitution,It had no distinct difference when the related substituted groups such as methyl,ethyl,and propyl groups.The 3,5-dimethylisoxazol analogs turned out to show an obvious increase selectivity against ROS1 because of the decrease potency of ALK kinase.1-methyl-1H-pyrazol-4-yl,1-ethyl-1H-pyrazol-4-yl,and 1-propyl-1H-pyrazol-4-yl had a similar potency against ROS1 kinase,then followed by 1-methyl-1H-pyrazol-5-yl,thiophene,1-methyl pyrrole,3,5-dimethylisoxazole.ROS1 inhibitory activities of ortho-benzyloxyaminopyridine derivatives displayed similar to meta benzyloxyaminopyridine derivatives,but it led to a slight decrease in potency against ALK,Novel compounds showed no marked impact on activity ROS1 kinase whether the property of substitutions phenyl ring of the aminopyridine was big or not,but it displayed significant increase against ROS1 kinase.Electron donating groups on the phenyl group of the benzyloxyaminopyridine compounds in favor of the activity increased,among them,4-Me was the optimal group,then followed by 3-Me,4-OMe,4-F,2,4-di-F,3,4-di-OMe,4-OEt,4-CF3,4-OCF3.