Design,Synthesis And Biological Activity Of Novel Pyrimidine Thymidylate Synthase Inhibitors

Objective:Inhibiting cell nucleotide metabolism to promote apoptosis is an important direction for cancer treatment.Seeking high-efficiency and low-toxicity chemotherapy drugs to prolong patient survival is the main goal of contemporary drug development.Thymidylate synthase?TS?,as a key rate-limiting enzyme in the initiation and repair of cellular DNA synthesis,is highly expressed at the site of vigorous cell replication and directly affects cell proliferation and metabolism.When TS activity is inhibited,it directly affects the cell cycle process,prevents DNA repair,and eventually induces apoptosis.TS has obvious expression differences in cancerous tissue and adjacent tissues,making it one of the classic targets for the development of chemotherapy drugs.Traditional TS inhibitors have many side effects,so it is of great medical significance to develop TS inhibitors with new structure and high efficiency and low toxicity.Methods:This research is mainly divided into three parts:1.Extract and combine the key pharmacophores of two types of TS inhibitors,which are currently most widely used in clinical research,to design and synthesize a series of new TS inhibitors,namely A-series of compounds.Through MTT assay and drug selectivity index?SI?evaluation,potential compounds were obtained and their anticancer mechanisms were further verified.2.Further targeted structural optimization,a series of new structure TS inhibitors with multiple effects?B-series compounds?,were designed and synthesized.Through in vitro TS assay and MTT assay,and structure-activity relationship studies,finally,compounds with anti-tumor proliferation ability were obtained.Further studies on molecular docking were conducted to study the possible modes of action of compound in TS?IJUJ?active pockets.The anti-tumor cell proliferation ability of the compound was further verified by flow cytometry,and its regulation on the proliferation or apoptotic signal pathway was verified by western blot analysis.Then,through transwell and cancer cell scratch experiments,explore the role of novel TS inhibitors in inhibiting the migration of cancer cells after targeted transformation,and verify the regulation of other signaling pathways at the protein level.In vitro angiogenesis and in vivo experiments,verify the compound's anti-cancer proliferation ability.3.BasedontheoriginaldesignofN-phenyl-?2,4-dihydroxypyrimidine-5-sulfonamido?benzoylhydrazide,and 1,3,4-oxadiazole instead of the formylhydrazine structure,synthesis of TS inhibitors with new structures,namely C-series compounds.Carry out in vitro TS assay and MTT assay,and structure-activity relationship research.Colony formation experiments,flow cytometry,and mitochondrial membrane potential determination experiments were used to verify the antitumor cell proliferation ability of C-series compounds in vitro.And its influence on related signal pathways was analyzed and verified by western blot.In vitro angiogenesis experiments and chicken embryo allantoic membrane experiments verify the inhibitory effect of compounds on angiogenesis.Finally,through xenograft tumor experiments and lung carcinoma in situ mouse model tests,the effects on tumor proliferation inhibition and the effects on survival cycle and toxic effect were evaluated.Results:1.A series of 42 compounds with novel structures were designed and synthesized as TS inhibitors based on the binding principle.The results of TS enzyme activity assay showed that a few compounds were more enzymatic inhibitory than PTX.However,the results of cell proliferation assays showed that most of the compounds had good anti-proliferative activity against A549,OVCAR-3,SGC7901 and MDA-MB-231cells.The IC₅₀ value of compound 10l on A549 cells was 1.26?M,which was better than pemetrexed(PTX,IC₅₀=3.31?M).In addition,the selectivity index?SI was 16.71?of compound 10l was higher than PTX.Flow cytometry analysis showed that compound 10l?apoptosis rate of 39.4%?could induce apoptosis of A549 cells and effectively inhibit tumor cell proliferation.Further western blot analysis showed that compound 10l could induce intrinsic apoptosis by regulating the activation of caspase-3,increasing the expression of cleaved caspase-3 and reducing the ratio of bcl-2/bax.2.Based on the structure of the A series of lead compounds,18 N-phenyl-?2,4-dihydroxypyrimidin-5-sulfonamido?phenylurea derivatives of the B series were further designed and synthesized as TS inhibitors.Biological evaluation results show that the target compound can significantly inhibit TS enzyme activity in vitro,and most compounds have excellent antitumor activity against six kind of cancer cell lines.Especially compared with PTX?SI was 7.71?,compound L14e?SI is 19.79?has excellent antitumor ability and safety on A549 cells(IC₅₀=0.67?M)and H460 cells(IC₅₀=0.81?M).Further research suggests that compound L14e could cause G1/S phase arrest and then induce intrinsic apoptosis.The results of tanwells experiment,western blot analysis,and in vitro angiogenesis experiments proved that compound L14e could inhibit the activation of EGFR signaling pathway,and finally achieved the purpose of inhibiting cancer cell migration and angiogenesis in cancer tissue.In addition,in vivo pharmacological evaluation showed that compound L14e had significant antitumor activity in A549 cell tumor-bearing mice,with a tumor suppression rate of 84.86,much higher than PTX?TGI was 63.42?.At the same time,HE staining and serum biochemical analysis showed that compound L14e was less toxic than PTX and had less damage to major organs.3.In vitro biological evaluation revealed that C-series N-?3-?5-phenyl-1,3,4-oxadiazol-2-yl?phenyl?-2,4-dihydroxypyrimidine-5-sulfonamido derivatives all have significant inhibition of TS enzyme activity and excellent antitumor activity against five cancer cell lines.In particular,compound 7f not only had a high drug selectivity index?SI was 21.33?,but also has excellent cell proliferation inhibitory effect on eight kind of NSCLC cells.The results of flow cytometry analysis showed that compound 7f not only had the ability to inhibit A549 cell cycle progression,but also could change the mitochondrial membrane potential and induce apoptosis.In-depth research shows that compound 7f could induce mitochondrial pathway apoptosis by up-regulating the expression of wild-type P53 in A549 and PC-9 cells without affecting the expression of mutant P53.At the same time,compound 7f could inhibit angiogenesis both in vivo and in vitro.In in vivo studies,compared to PTX,compound 7f significantly inhibited tumor growth in A549 cell xenograft mice and had a higher tumor suppression rate?87.36?.Moreover,compound 7f can prolong the survival of LLC implanted mice?lung carcinoma in situ?more effectively than PTX.Conclusion:Based on understanding the structure and function of TS,the structure analysis and advantages and disadvantages of existing TS inhibitors were summarized in this study.A series of new TS inhibitors were designed and synthesized based on the principle of chemical combination to obtain new TS inhibitors with high efficiency and low toxicity.With this new structure as a template,the structure was optimized in 2 times.Through in vitro and in vivo biological evaluations,compounds L14e and 7f,which were better than PTX in SI and antitumor ability,were finally obtained.At the same time,compounds L14e and 7f also had excellent anti-angiogenic effects.These two structures of TS inhibitors provide a new reference for the development of chemotherapeutics for NSCLC.