Design,Synthesis,Antitumor Activity Research On KDR And Hsp90 Inhibitors And Specific Modification Of Oligopeptide Primary Amino Groups By Dihydrooxazoloquinoline Quaternary Ammonium Salt

Malignant tumors have always been one of the important factors threatening human health.Molecularly targeted therapy and immunotherapy,hormonal therapy and cytotoxic chemotherapy constitute the main means of modern cancer drug therapy.As a form of molecular medicine,molecularly targeted therapy blocks the growth of tumor cells by interfering with specific targeted molecules required for tumorigenesis and tumor growth,rather than by simply interfering with all rapidly dividing cells as with conventional chemotherapy.Therefore,molecularly targeted therapy has specific anti-tumor effect,less damage to normal tissues,and significantly reduced toxicity?kidney function damage and myelography suppression etc.?.At present,molecularly targeted therapy mostly uses monoclonal antibody drugs and small molecule drugs?mainly tyrosine kinase inhibitors?in clinical practice.KDR?vascular endothelial growth factor receptor-2?belongs to the family of protein tyrosine kinases and is highly expressed in many malignant tumors.Developing KDR inhibitors provides a good way to discover new molecularly targeted anti-tumor drugs.The biology,pharmacology and medicinal chemistry of Hsp90 are also very active in drug discovery and clinical development.Hsp90 has received increasing attention as a potential target for anti-tumor.This dissertation consists of four chapters.The first chapter is about the research progress of KDR and Hsp90 inhibitors in recent years including the KDR and Hsp90inhibitors on the market and in clinical stages.Based on molecular docking-based virtual screening and pharmacophore analysis of existing KDR inhibitors,in the second chapter,we designed and synthesized 79 new?-ketoamide compounds and tested inhibitory activity against KDR.We analyzed structure-activity relationship of the?-ketoamide compounds.35 compounds had higher KDR inhibition rates than the positive control drug Sorafenib?inhibition rate was 82%?at a concentration of 0.1?M.The inhibition rates of compounds 2-10c and 2-28o for KDR at the concentration of0.1?M were 92.6%and 83.5%?the inhibition rates of Sorafenib and Regorafenib were 82%and 85.1%?.The inhibition rates of 2-10c for PDGFR-?^D842V and C-Kit^D816H at the concentration of 1?M were 90.6%and 98.1%?the inhibition rates of the positive control drug Sunitinib were 87.9%and 96.4%?.Moreover both 2-10c and2-28o have good metabolic stability in human and rat liver microsomes,and moderate metabolic stability in mouse liver microsomes,which has in-depth research value.In addition to having good KDR inhibitory activity?inhibition rate of 2-28a for KDR was 92.9%at 0.1?M?,the inhibition rates of 2-28a for C-Kit^D816H,C-Kit^D816V and C-Kit^D816Y were 40.8%,62.1%and 65.2%at the concentration of 0.1?M,respectively.Further cell assays showed that compound 2-28a inhibited the proliferation of mouse mast cell carcinoma P815(C-Kit^D816Y)and human mast cell carcinoma HMC1.2(C-Kit^D816V)cells with IC₅₀ values of 1.19±0.18 and 4.67±0.45?M(IC₅₀ values of positive control drug Ponatinib values were 1.415±0.45 and 1.23±0.17?M).Compound 2-28a had moderate metabolic stability in human and rat liver microsomes and low metabolic stability in mouse liver microsomes.Compound 2-12a?inhibition rate of 2-12a for KDR was 93.4%at 0.1?M?,a multi-target kinase inhibitor,can inhibit PDGFR-?^D842V,PDGFR-?^V561D and C-Kit^D816H and the inhibition rates of2-12a for these three mutant kinases at a concentration of 1?M were 92.2%,97.6%and 83.1%,respectively.2-12a had poor metabolic stability in human liver microsomes,moderate metabolic stability in rat liver microsomes,and good metabolic stability in mouse liver microsomes.In the third chapter,we described the design,synthesis and antitumor activity of Hsp90 inhibitors.We synthesized 24 Hsp90 inhibitors and find three compounds with higher anti-tumor activity than the lead compound X66.We divided the 1,3,5-triazine compounds into three fragments,and examined the effects of different substituents on the activity of the compounds,and summarized the structure-activity relationship.Classical Hsp90 inhibitors can induce significant heat shock responses,resulting in up-regulation of heat shock protein HSPs?Hsp72 and Hsp27?with anti-apoptotic and tumorigenic effects.Most synthesized compounds?eg 3-7a,3-7b,3-7c?will not raise Hsp72.Compounds 3-7a,3-7b,3-7c were able to induce significant autophagy by pharmacological activity tests and showed potent antitumor activity in SK-BR-3 with high expression of HER2 and A549 cell line with high expression of EGFR(IC₅₀=0.6¹.6?M).The most active compound 3-7b(IC₅₀=0.1⁰.4?M),which binds to the N-terminal domain of Hsp90 in a manner different from the traditional Hsp90inhibitor?GM?,can also induce degradation of the client protein,but not increase the expression of Hsp72.The ability to bind to Hsp90?KD=0.219?M?,Hsp90 inhibitory activity and antitumor activity(IC₅₀=0.1⁰.4?M)of 3-7b compared to lead compound X66(KD=5.3?M;IC₅₀=6.1¹0.8?M)had a significant improvement.In addition,compound 3-7b showed in vivo anti-tumor activity against EGFR-positive A549 lung cancer xenografts.It laid the foundation for further design and development of new Hsp90 anti-tumor drugs.Peptides have unique pharmacological activities and good target specificity and are one of the main sources of designing new drugs.Compared with traditional small molecule drugs,it has better safety,tolerance and effectiveness.However,peptide drugs themselves have some shortcomings,such as low cell membrane permeability,metabolic instability,poor oral bioavailability,and short circulating half-life.Therefore,the use of chemical modification strategies to improve the druggability of peptides has greatly promoted the development of peptide drugs.In general,N-terminal chemical modification strategies for peptides include acetylation,alkylation,oximation,quinonylation,aromatization,and so on.Although these chemical modification strategies have been widely used,there are still some limitations,such as high reaction temperature,use of toxic solvents and metal reagents,poor reaction selectivity or poor compatibility with other sensitive groups.In the fourth chapter of this dissertation,a novel method about site-selective N-quinolinylation of primary amino groups for complex drug molecules and oligopeptides by novel dihydrooxazo[3,2-a]quinoline quaternary ammonium salt was developed.The process is carried out under mild reaction conditions without any metal reagents and has good compatibility with other reactive groups such as secondary amino groups,amides,alcoholic hydroxyl groups,phenolic hydroxyl groups,disulfide bonds and cyano groups.Therefore,this new method can be widely used in the fields of medicinal chemistry and chemical biology to promote the development of peptide drugs.