Design,synthesis And Antitumor Activity Study Of Novel Pan-FGFR Inhibitors And Selective FGFR4 Inhibitors

Over the past two decades,the rapid development of receptor tyrosine kinase（RTKs）inhibitors for anti-cancer targeted therapy has significantly improved the quality of life in some patients with advanced malignancies and prolonged their survival period.However,these small-molecule targeted drugs still have some problems including low clinical response rate and drug resistance.The fibroblast growth factor receptors（FGFRs）are an important family members of RTKs.FGFR aberrations caused by gene amplification,mutation and fusion are identified in a variety of human solid cancers.Although the combination of tumor molecular classification and molecular targeted therapy has improved the clinical response rate of some FGFR inhibitors and promoted their clinical progress,the current FGFR inhibitors still face the following challenges:（1）there are so many FGFR subtypes classified by gene amplification,mutation and fusion in cancer,that the current FGFR inhibitions cannot meet the clinical needs for all FGFR-associated tumors;（2）a part of FGFR inhibitors exhibit serious side effects,such as hyperphosphatemia and eye diseases,due to poor target selectivity;（3）acquired resistance to FGFR inhibitors has been found in clinical trials,the most frequent resistant mutations appear at the gatekeeper amino acid in FGFR kinase domain.Therefore,it is of great clinical benefits to develop novel highly selective and highly bioactive FGFR inhibitors to supplement structural diversification or to overcome drug-resistant mutations.Part Ⅰ.Design,synthesis and anti-gastric cancer study of novel pyrido[1,2-a]pyrimidinone pan FGFR inhibitorsThrough analyzing the respective binding mode of the marketed drug Erdafitinib and the reported compound 34 with FGFR1,we utilized the scaffold hopping strategy to design a series of pyrido[1,2-a]pyrimidinone derivatives as novel FGFR inhibitors.The structure-activity relationships（SARs）study of more than one hundred synthesized compounds showed that:（1）increasing the aromaticity of R₁ group adjacent to pyrido[1,2-a]pyrimidinone was helpful to enhance the activity,and the pyrazole ring at this position was the dominant fragment.Incorporation of some substituents linked with the nitrogen of pyrazole was acceptable for the activity;（2）introduction of a hydrophilic group or a basic center linked to the nitrogen atom of pyrazole can maintain the activity against FGFR1 and increase the aqueous solubility;（3）introduction of a alkyl chain at R₄position is helpful to enhance the activity against FGFR1,and most of the obtained compounds maintain the high activity against FGFR4,and show a certain selectivity for FGFR4.Through the systematic SARs study,we obtained seven compounds,57i,61b,61f,63c,63d,67c and 67d,which can not only retain good enzymatic activity and cellular activity(IC₅₀<10 n M)but also improve their aqueous solubility compared with the lead Erdafitinib.Among them,compound57i exhibited excellent enzymatic activity(FGFR1-4,IC₅₀=0.57,2.0,0.80 and1.4 n M),cellular activity(SNU-16 IC₅₀=3.3 n M,KATOIII IC₅₀=1.1 n M),improved aqueous solubility（321μg/m L）and acceptable molecular weight（462.6g/mol）.Therefore,57i was chosen as the ideal compound for further antitumor mechanism study and in vitro and in vivo pharmacodynamics evaluation.In the kinases inhibition profile,only 9 hits including FGFR1-4 exhibited>90%inhibition at a concentration of 1μM of 57i,which indicating its good kinase selectivity.The thermal shift assay was also used to verify 57i and FGFR interaction,and the results displayed that the addition of 57i increased the thermal stability of FGFR2 in SNU-16 cells.Western blot assays in SNU-16 and KATOIII cells showed that 57i could target FGFR2 and inhibited the auto-phosphorylation of FGFR2,phosphorylation of FRS2,AKT and ERK1/2 at low nanomolar concentration.To elucidate the interaction mode of 57i with FGFR,we conducted a molecular docking analysis using a reported crystal structure of FGFR1.The result revealed that 57i had a similar biding mode with Erdafitinib and maintained the two key hydrogen bonds formed by the pyrido[1,2-a]pyrimidinone core and methoxy group with FGFR1 Ala564 and Asp641,respectively.On the contrary,the methyl pyrazole ring of 57i extending into solvent region made a small rotation with respect to the pyrido[1,2-a]pyrimidinone core.Such slight rotation did not affect the interaction of methyl pyrazole ring with amino acid Leu484,which resulted in improved enzymatic potency when compared with the lead 34.Meanwhile,this rotation disrupted the planarity of compound 57i,which may have been a factor its higher aqueous solubility when compared with Erdafitinib.MTT and cell counting experiments showed that 57i strongly inhibited the growth of gastric cancer cells SNU-16 and KATOIII in a dose-and time-dependent manner.After cells were treated with 57i for 48 h,the cell cycle of SNU-16 and KATOIII cells was arrested in G0/G1 phase,and the cell cycle related protein level of CDK2,CDK4,CDK6,Cyclin D and Cyclin E decreased markedly.Compound57i also could induce cell apoptosis in a dose-and time-dependent manner,and the proportion of late apoptotic cells was significantly higher than that of early apoptotic cells.The pro-apoptotic proteins cleaved caspase-3 and cleaved PARP increased significantly when the concentration of 57i exceeded 5 n M.Based on the good performance of 57i in vitro activity,we further investigated its pharmacokinetics and pharmacodynamics in vivo.The oral bioavailability of57i in rats was 14.94%after a single administration of 30 mg/kg.The retention time of drug concentration in the body beyond the effective concentration was more than 20 h,which indicating a low frequency of drug delivery.In a SNU-16xenograft model,57i was orally administered at a dose of 15 mg/kg once daily.Four weeks later,the tumor growth inhibition（TGI）was 67.3%.When the dose increased to 30 mg/kg/day,the TGI could reach 104.6%and the tumor growth was inhibited completely.During the treatment period,the body weight of mice did not change significantly.After the administration,there was no obvious change in the hematopoietic parameters and thological changes of main organs of the mice in57i treated group,indicating that the mice were tolerated with the dosage,and the compounds were therapeutically safe.By immunohistochemical analysis of tumor in vivo,we found that the number of p-FGFR2 and Ki-67 positive staining cells in 57i treated tumor tissues decreased significantly,while the number of cleaved caspase-3 positive staining cells increased significantly,indicating that 57i regulated FGFR2 phosphorylation,inhibited cell proliferation and caused apoptosis of SNU-16 gastric cancer cells in vivo.In conclusion,through reasonable molecular design and systematic SARs study,a new compound 57i bearing the pyrido[1,2-a]pyrimidinone corew as discovered as a novel and potent FGFR inhibitor.Its good antitumor activity in vivo and in vitro and improved aqueous solubility made it possible for further pre-clinical development.Part Ⅱ.Design,synthesis and antitumor study of a novel series of N-（2-（1H-indazole-3-yl）phenyl）acrylamide FGFR4 inhibitorsTo obtain highly active and selective FGFR inhibitors which can overcome the resistant“gatekeeper”mutation of FGFR4,through analyzing the binding model of the current FGFR inhibitors with FGFR,we found that the indazole FGFR inhibitors have the potential to overcome the resistant mutation of“gatekeeper”amino acid.Then we introduced the 2-acrylamide-phenyl group into the 3-position of indazole to generate a novel type of FGFR4 covalent inhibitor,which can selectively bind FGFR4.Through the SARs study of the synthesized 3,6-disubstited（80-Series）and 3,5-disubstituted（90-series）indazole derivatives,we found:（1）The introduction of 2-acrylamide-phenyl at the 3-position of indazole can maintain the high activity and selectivity against FGFR4;（2）Incorporation of some substituents on the benzene ring bearing acrylamide was acceptable for the activity;（3）The 1-（3,5-dichloropyridin-4-yl）ethoxy group substituted at 5-position of indazole is beneficial for activity,and the R configuration of chiral carbon is the active conformation.Among the two series of compounds,the optimal compound 80i in the 80-series derivatives exhibited the highest enzymatic activity against FGFR4 with an IC₅₀of 8.0 n M,but showed low inhibitory activity against Ba F3/ETV6-FGFR4-V550L cells with an IC₅₀ of 11μM,which was equivalent to that of BLU554(IC₅₀=6.6μM).In the 90-series derivatives,the active compound 90e not only showed good FGFR4 activity(IC₅₀=5.7 n M)and antiproliferative potency against Huh7 and Hep3B cells(IC₅₀=28 and 17 n M,respectively),but also strongly inhibited the growth of resistant Ba F3/ETV6-FGFR4-V550L cells with an IC₅₀ of 8.3 n M,which was better than that of BLU554(IC₅₀=6.7μM)and LY2874455(IC₅₀=32n M).Partial kinases inhibition profile showed that 90e exhibited good selectivity for FGFR4.In view of that the selective FGFR4 inhibitor 90e is capable of overcoming the gatekeeper mutations of FGFR4,it is worthy for further evaluation as next-generation selective FGFR4 inhibitors.