Design,Synthesis,and Biological Evaluation Of 3-aminoindazole Derivatives As Highly Selective Covalent Inhibitors Of FGFR4

Fibroblast growth factor receptor 4（FGFR4）is a member of the FGFR family,can bind to a variety of fibroblast growth factors（FGFs）and participate in the regulation of cell proliferation,differentiation and migration,lipid metabolism,bile acid biosynthesis and glucose metabolism.The FGFR4 isoform specifically utilizes endocrine FGF19 as its intracellular ligand and has been highlighted as an oncogenic driver in a subset of solid tumors,especially HCC,breast cancer and rhabdomyosarcoma,making FGFR4 an attractive target for further research.Great efforts have been made to develop FGFR4 inhibitors.Erdafitinib（2019）,pemigatinib（2020）,and infigratinib（2021）that have been approved by the FDA are all pan-FGFR inhibitors.Among them,only erdafitinib with balanced activity across FGFR1-4 has been approved by the FDA for the treatment of metastatic urothelial carcinoma.Pemigatinib and infigratinib target FGFR1/2/3 while displaying a relatively weak potency against FGFR4.So far,no selective FGFR4 inhibitor has been approved,and more effective FGFR4 selective inhibitors are urgently needed in clinical practice.Moreover,mutations in the kinase domain of FGFR4,especially at the gatekeeper residue（Val550）,have been demonstrated as a major concern for the development of clinically acquired resistance in patients receiving first-generation FGFR4-targeted therapies.Therefore,there is an urgent need to develop next-generation selective FGFR4 inhibitors with a different chemical scaffold that can effectively overcome mutation acquired resistance.Based on the co-crystal structures of reported inhibitors and FGFR4,structure-based rational drug design strategy was used to design and synthesize a series of 3-aminoindazole derivatives as highly selective covalent inhibitors of wild-type and gatekeeper mutant variants of FGFR4.One representative compound 2-2v exhibited excellent potency against FGFR4^WT,FGFR4^V550L,and FGFR4^V550M with nanomolar activity in both the biochemical and cellular assays,while sparing FGFR1/2/3.Compound 2-2v exhibited excellent potency against FGFR4,FGFR4^V550L and FGFR4^V550Mwith IC₅₀values of 0.5,0.25 and 1.6 n M,respectively.Compound 2-2v also strongly suppressed proliferation of Ba/F3 cells transfected with FGFR4^WT,FGFR4^V550Land FGFR4^V550M with IC₅₀ values of 3.5,4.7 and 8.1 n M,respectively.Moreover,2-2v exhibited potent antiproliferative activity against FGFR4-dependent MDA-MB-453,Hep3B,Huh-7 and JHH-7 cancer cells with IC₅₀ values of 34,29,72and 54 n M,respectively.Kinome-wide screen against 66 kinases possessing available cysteines in the hinge region further confirms compound 2-2v’s target specificity at a concentration of 1.0μM.In Ba/F3-FGFR4^WT and Ba/F3-FGFR4^V550L,compound 2-2v almost completely inhibited the phosphorylation of FGFR4 and its downstream proteins FRS2 and MAPK at a concentration of 30 n M.Co-crystal complexes of compound 2-2v with FGFR4^WT,FGFR4^V550L,FGFR4^V550M reveal that it is covalently bound to the FGFR4 protein in a“U-shaped”conformation.While compound 2-2v demonstrated modest in vivo antitumor efficacy in nude mice bearing the Huh-7xenograft model（the tumor growth inhibition values were 25.9 and 46.9%at dosages of 20 and 40 mg/kg for 17 continuous days,respectively）which may be due to its unfavorable pharmacokinetic properties or fast resynthesis rate of FGFR4 in Huh7cell line.Therefore,a series of compounds were obtained by optimizing acrylamide“warhead”of 2-2v.Preliminary kinase activity results showed that compound 2-2ze could exhibit strong inhibitory activity against FGFR4 kinase.More detailed studies on PD and PK studies of these compounds are ongoing.In this study,a series of novel 3-aminoindazole derivatives as highly selective covalent inhibitors of FGFR4^WTand gatekeeper mutants FGFR4^V550L/M were successfully designed and synthesized using structure-based rational drug design strategy,it provides a promising new starting point for future drug discovery combating FGFR4 gatekeeper mediated resistance in HCC patients.