Design,Synthesis And Evaluation Of Anti-tumor Activity Of 1H-Indazole VEGFR-2 Inhibitors And Degraders

Vascular epidermal growth factor receptor(VEGFR-2)is an important tyrosine transmembrane protein,which is closely related to tumor angiogenesis,lymph node metastasis and tumor stem cell invasion,and is one of the important targets for antitumor drug research.Sorafenib is the first VEGFR-2 inhibitor to be marketed,but its efficacy is limited due to drug resistance and low oral bioavailability in clinical medication.PROTAC-targeted protein degradation technology is the earliest and most widely studied biotechnology for degradation mechanism and E3 ubiquitin ligase ligands,while VEGFR-2-PROTAC degraders are rarely researched.In this dissertation,VEGFR-2 was used as the research target,and the design,synthesis and in vitro antitumor activity studies of 1H-indazole VEGFR-2 inhibitors and VEGFR-2-PROTAC degraders were carried out,and in vivo drug-like property and mechanism of the active compounds were also evaluated,respectively.In the first part,based on the structure of VEGFR-2 inhibitor Sorafenib,a6-(1H-pyrazole-4-yl)-1H-indazol-3-amine scaffold was designed through a three-dimensional conformation matching-based scaffold hopping design strategy,and the structural modification and optimization of its solvent-exposed area,linker and hydrophobic pocket were carried out.Specifically,using 2-fluoro-4-cyano-bromoben-zene as raw material,26 novel 1H-indazole VEGFR-2 inhibitors were synthesized through ring construction,Buchwald-Hartwig and Suzuki coupling reaction.The results of biological activity evaluation showed that most of the target compounds had good in vitro kinase inhibition and cytotoxic activity.Especially,the representative compound W13 possessed potent VEGFR-2 inhibition(IC₅₀:1.6 n M)and revealed a moderate antiproliferative activity in HUVEC cells(IC₅₀:7.15±2.26?M),and the selectivity for tumor HGC-27 cells(IC₅₀:0.36±0.11?M)was over500-fold higher than normal GES-1 cells indicating lowest toxicity(IC₅₀:187.46±10.13?M).Furthermore,studies on the mechanism of cellular displayed that W13inhibited the migration and invasion of HGC-27 cells by modulating the expression of MMP-9 and E-cadherin proteins in a concentration-dependent manner,and induced apoptosis by increasing intracellular ROS and regulating the expression of apoptotic proteins,Bax and Bcl-x L.Moreover,W13 inhibited cell survival by reducing the phosphorylation of VEGFR-2 in HGC-27 cells,thereby blocking the PI3K-Akt-m TOR signaling pathway,and also exhibited good anti-angiogenic activity in HUVEC tubule formation assays.Moreover,the drug-like property evaluation showed that compound W13 had sui?metabolic half-life for human and mouse liver microsomes,and had no obvious inhibitory effect on CYP3A4,an important subtype of CYP450.The acute toxicity experiment in mice represented that the LD₅₀ of W13 was greater than 3000 mg/kg,and there was no obvious changes in tissue and morphological characteristics in the heart,liver,spleen,lung,kidney.Additionally,pharmacokinetic evaluation experiments in rats represented that the oral bioavailability of W13 was 64.5%.These results demonstrated that W13 displayed a good safety and druggability.In the second part,with the support of PROTAC-targeted protein degradation technology,the Lys residue region on the surface of VEGFR-2 protein was regionally classified by computer-aided drug design.Therefore,relyed on the protein structure-based rational drug design strategy,compound W13 and its derivatives W27and W28 were used as warheads,and VH032 was used as VHL E3 ligase ligand inhibitor,and the two ligands were linked via amidation reaction with an alkanedioic acid or substituted piperazine side chain of different carbon numbers.A total of 25VEGFR-2-PROTAC degraders in 6 series were rationally designed and synthesized,and evaluated its in vitro cytotoxic activity and VEGFR-2 protein degradation activity in HGC-27 cells.Among them,the representative compound P7 exhibited preferable antitumor activity against HGC-27 cells(IC₅₀:2.03±0.17?M),moderate antiproliferative activity in HUVEC cells(IC₅₀:7.07±1.97?M),and less toxic to human normal HEK293T and GES-1 cells in vitro(IC₅₀:14.47±1.68?M and 19.97±0.14?M),and better degradation activity of VEGFR-2 protein in HGC-27 cells(DC₅₀:0.084±0.04?M,D_max:73.7%).Moreover,the mechanism of VEGFR-2 degradation showed that P7 could rapidly and effectively degrade VEGFR-2 protein in a time-dependent manner,and the long-term degradation effect of HGC-27 cells was maintained within24 h after being treated with fresh medium for 24 h.Additionally,P7 degraded VEGFR-2 protein by the formation of ternary complex and the ubiquitin-proteasome(UPS)pathway.Moreover,P7 significantly shortened the half-life of VEGFR-2protein synthesis and had no effect on the expression of VEGFR-2 m RNA during its translation and transcription,proving that P7 only degraded the existing VEGFR-2protein in cells.Furthermore,cell mechanism studies displayed that P7 could inhibit the colony formation,cell migration and invasion of HGC-27 cells in a time-and concentration-dependent manner,and obviously induced HGC-27 cells apoptosis and G₂/M phase cycle arrest.In addition,P7 exhibited better VEGFR-2 protein degradation activity in HUVEC cells(DC₅₀:0.51±0.10?M,Dmax:76.6%).In conclusion,a series of indazole VEGFR-2 inhibitors were discovered through a three-dimensional conformation matching-based scaffold hopping design strategy,especially W13 displayed potent anti-gastric cancer activity,safety and druggability,and further developed it into a VEGFR-2-PROTAC degrader,in which P7 effectively degraded VEGFR-2 protein through the UPS pathway by forming a ternary complex in vitro.Therefore,the research results of this dissertation provide a new proposal for the design and development of VEGFR-2 inhibitors and degraders,and a new strategy and experimental accumulation for the discovery of anti-gastric cancer drugs.