Design,Synthesis And Biological Evaluation Of Novel Selective FAK Inhibitors And PROTACs

Focal adhesion kinase（FAK）is a nonreceptor tyrosine kinase consisting of a FERM domain,kinase domain,FAT domain and proline-rich regions.Upon activation by membrane receptors such as integrins,RTKs,GPCRs,and cytokine receptors,FAK initiates downstream signaling cascades,such as PI3K-AKT,ERK,and JNK,to perform various physiological functions.Various tumors overexpress FAK,including neuroblastoma,pancreatic cancer,ovarian cancer,and cervical cancer.FAK contributes to tumor progression by promoting cell motility,survival and proliferation,as well as angiogenesis.It also influences the expression of chemokines and cytokines,and drives the fibrotic and immunosuppressive microenvironment.Overexpression of FAK and high p-FAK have been found in ovarian cancer cases.FAK overexpression correlates with poor overall survival,high-stage tumors,and distant metastasis in ovarian cancer patients.Inhibition of FAK by small molecules decreases tumor growth,angiogenesis,and metastasis in preclinical research,and corresponding clincal trials are undergoing.It suggests that FAK is a potential therapeutic target for the treatment of ovarian cancer.However,unsatisfactory response and safety place restrictions on the clinical applications of FAK inhibitors as a single agent.Thus,there remains a need to develop more FAK-targeting small molecules with better efficacy and safety for the treatment of ovarian cancer.1.Design and synthesis of selective FAK inhibitors for the treatment of ovarian cancerWe applied scaffold hopping strategies and designed novel FAK inhibitors with a7H-pyrrolo[2,3-d]pyrimidine scaffold to retain important H-bond interactions with Cys502 in the hinge region.C2 substitution points to the solvent exposed region,and was fixed as an arylamine with a hydrophilic group.The results of SAR studies indicated that compounds with 1H-benzo[d]imidazole substitution at the C4 position,which have both a methoxy at the ortho-position and a hydrophilic group at the para-position of anilines,were mort potent.Replacement of 1H-benzo[d]imidazole by a 1H-indole,1H-indazol-3-amine or 1-methyl-1H-pyrazole decreased the potency of comound A13–A26.To further improve the inhibitory activity,we designed compounds with 7-hydroxy-2,3-dihydro-1H-inden-1-one,and indone might retain a H-bond interaction with Asp564 of the DFG motif.Derivatives B1–B29 with SAR at C2 position mainly exhibited potent activities against FAK(FAK IC₅₀=1.6～550 n M).Among them,compounds with a methoxy at the ortho-position or a methyl at the meta-position of anilines were mort potent.Methyl or chlorine modification of the indone（B30–B37）was tolerated.SAR of the scaffold showed that 7H-pyrrolo[2,3-d]pyrimidine was a favorable scaffold to retain interactions with FAK.Next,compounds with IC₅₀ values less than 5 n M were selected to evaluate their aqueous solubility and in vitro metabolic stability.Compound B16(FAK IC₅₀=1.9n M),which had good solubility（456μg/m L）and moderate stability in liver microsomes(T_1/2=113 min),was selected for subsequent investigation.Human kinome-wide selectivity profile suggested that B16 was a highly selective and potent FAK inhibitor,which significantly inhibited seven kinases out of 422 kinases at 1μM（>80%inhibition）.The predicted binding mode demonstrated that there were several hydrogen bonds between B16 and FAK,such as 7H-pyrrolo[2,3-d]pyrimidine with Cys502 or Glu500 in the hinge binding region,the carbonyl of indone with Asp564 of the DFG motif,and the aniline with Arg426 or Glu506.B16 inhibited the phosphorylation of FAK and downstream Src and AKT,restrained the migration and invasion of ovarian cancer cells,and diminished the expression of MMP-2 and MMP-9.In a subcutaneous PA-1 xenograft model,administration with 60 mg/kg B16 resulted in significant tumor growth inhibition（TGI=72.5%）.Neither obvious loss of body weight,nor abnormality of vital organs and blood biochemistry was observed.B16,which had potent acvitivities in vitro and in vivo,was a potential candidate for the treatment of ovarian cancer.2.Design,synthesis and biological evaluation of PROTACs targeting FAK degradationAs a multidomain protein,FAK not only displays catalytic activity through kinase domain,but also functions as a scaffold to participate in protein-protein interactions.Kinase-independent functions of FAK are involved in promoting tumor growth,metastasis,and stem cell characteristics.Synchronously repress kinase activities and scaffold functions of FAK is expected to exert better anti-tumor activity.Proteolysis-targeting chimeras（PROTACs）,which are bifunctional molecules that induce the degradation of proteins of interest（POI）by the ubiquitin-proteasome system,are applied to degrade various disease-related proteins and have promising prospects.Degradation induced by PROTACs could completely block the oncogenic activity of FAK.Thus,we designed FAK PROTACs based on a potent and highly selective inhibitor B16,and investigated the anti-tumor activity in vitro and in vivo.The docking model demonstrated that the piperidine motif of B16 was exposed to the solvent,which made it a suitable site for installing a linker.Degraders D1–D12were designed using thalidomide as an E3 ligase CRBN binder with an alkyl or polyethylene glycol linker.Compound D4(FAK IC₅₀=3.5 n M)induced potent FAK degradation in several ovarian cancer cell lines,and the degradation of FAK was time-and dose-dependent.Inhibition of p-FAK Y397 was also induced with the decrease of FAK protein.Rescue experiments indicated that D4 degraded FAK through ubiquitin-proteasome system,which required binding to both FAK and the E3 ligase CRBN.Furthermore,global proteomic profiling showed that D4 was a highly selective FAK degrader,which specifically dowregulated the protein level of FAK in PA-1 cells.D4inhibited colony formation of ovarian cancer cells in a concentration-dependent manner,and induced G₀/G₁ cell cycle arrest.Treatment with D4 for 24 h decreased the phosphorylation of Src in a dose-dependent manner,and suppressed ovarian cancer cell migration and invasion.In a subcutaneous PA-1 xenograft model,administration with 15 mg/kg D4 resulted in tumor growth inhibition and was tolerated.The combination of D4 with cisplatin led to further repression of tumor growth（TGI=80.4%）.Furthermore,D4 induced a considerable decrease in the number of metastatic nodules in an ES-2 mouse xenograft model.Meanwhile,B16 also displayed anti-metastasis activity in vivo.Taken together,we designed and discovered a potent and highly selective FAK inhibitor B16,and further developed a highly selective FAK degrader D4 with in vitro and in vivo activities.B16 and D4 will serve as not only promising therapeutic leads for treatment of ovarian cancer,but also a useful tool box to probe kinase-independent activities of FAK in cancers.