Design,Synthesis And Biological Evaluation Of Novel TRK Inhibitors

The three members of the tropomyosin receptor kinase（TRK）family,namely TRKA（encode by NTRK1）,TRKB（NTRK2）and TRKC（NTRK3）,belong to receptor tyrosine kinases（RTKs）,which are transmembrane proteins.NTRK gene fusion,point mutation,and in-frame deletions produce a ligandindependent constitutively activated protein with carcinogenic potential.Due to the structural or functional loss of the extracellular domain,some long-tested means such as antibody therapy are ineffective for treating these genetic transforms,while the small-molecule inhibitors have become a promising approach.There are several TRK inhibitors in clinical trials,two of which,Larotrectinib and Entrectinib,have got gratifying progress.In basket trials,TRK inhibitors（such as Larotrectinib and Entrectinib）have remarkable and durable antitumor activity in patients with diverse cancers harbouring NTRK oncogenic fusions.These successes have led to more interests in small molecule inhibitors against TRK.The compound designs of this study were divided into two parts.The first one was refer to the research ideas of Entrectinib,through the analysis of the results of computer simulation docking of PHA-E429 with TRKA,and meanwhile drew lessons from Entrectinib’s optimization ideas and the ring fusion strategy in conventional drug chemical structure optimization experience,and then a series of 10 compounds of 3-amino-1H-pyrazolo-[3,4-b]pyridine-5-formamide derivatives were designed and synthesized as class I to investigate whether different 5-site carbonyl benzene rings structures had differentπ-πinteractions with proteins and the differences in the presence of 3-site different adipose amines at the hydrophilic point.The second one was adopt a design strategy based on molecular similarity.Based on the structural analysis of the macrocyclic inhibitors Selitrectinib and Repotrectinib,this study retained the common nucleus in TRK inhibitors,pyrazolo[1,5-a]pyrimidine,also retained the atoms number of 13-membered macrocyclic,mainly to explore the impact of increase of ring on the pyrimidine side of the pyrazolo[1,5-a]pyrimidine nucleus and different Linkers on the activity.Through these discussions,this study designed and synthesized 10 new 13-members-macrocyclic compounds as class II TRK inhibitor.In this study,a total of 20 compounds with novel structure were designed and synthesized,and the obtained compounds were confirmed by mass spectrometry,¹H-NMR and ¹³C-NMR.In this study,in vitro kinase inhibition tests were performed on the synthesized compounds,and the inhibition rate value of 500 nM was used as the measurement standard.These tests showed that:Class I compounds generally have a poor level of inhibitory activity against TRKA,and the ALK inhibitory activity test based on the consideration of multi-target inhibitors had also achieved unsatisfactory results;Class II compounds generally had a good inhibitory activity on TRKA.The comparison between II-09 and II-08 showed that whether the ring is increased or not had little effect on the activity.Among the different ring increase forms,the inhibitory activity of II-07 was poorer than that of other compounds,which indicated that when the six-membered ring was extended on the pyrimidine side,it had an adverse effect when there was a methyl substitution on the carbon which was adjacent to the oxygen atom.In II-04 and II-10,seven-membered ring was constructed on the pyrimidine side,but these compounds was active.II-05 used the same Linkers as Repotrectinib,as expected,it showed excellent inhibitory activity.The fluorine substituted position of the compounds we designed were different from those of Selitrectinib or Repotrectinib on the other aryl groups,but most of the compounds still showed good inhibitory activity,and it was speculated that the change of fluorine position did not have significant effect on the activity.