Design,synthesis And Evaluation Of Anti-breast Cancer Activity Of Pyrazole [1,5-a] Pyrimidine Derivatives As C-met And PARP1 Inhibitors

ObjectiveThe incidence rate and mortality rate of malignant tumor have increased rapidly,and it has become one of the serious threats to human life and health.Breast cancer is one of the most dangerous types of cancer.The patients are mainly women.At present,the treatment and pharmaceutical research of breast cancer has become a research hotspot.The c-Met is a protein product encoded by proto oncogene MET,which has tyrosine protein kinase activity and its ligand is hepatocyte growth factor（HGF）.PARP1 is the key enzyme in DNA damage repair process,which is mainly involved in the base excision repair（BER）process after DNA single strand breaks.BRCA1/2 genes are mainly involved in the process of double stranded DNA homologous recombination repair（HRR）.BER and HRR are the main mechanisms to maintain genome stability in vivo,and they play a synergistic role.BRCA mutations increase the risk of multiple cancers,including breast cancer.Because the HRR process in breast cancer patients with BRCA germline mutations has been lost,the repair of damaged DNA only depends on BER process.PARP inhibitors can inactivate the BER process by inhibiting the activity of PARP enzyme,which can cause tumor cell death.PARP1 is a key enzyme in DNA damage repair process,which is mainly involved in the base excision repair（BER）process after DNA single strand breaks.BRCA1/2gene is mainly involved in the process of double stranded DNA homologous recombination repair（HRR）.BER and HRR are the main mechanisms to maintain genome stability in vivo,and they play a synergistic role.BRCA mutations increase the risk of multiple cancers,including breast cancer.Because the HRR process in breast cancer patients with BRCA germline mutations has been lost,the repair of damaged DNA only depends on BER process.PARP inhibitors can inactivate the BER process by inhibiting the activity of PARP enzyme,which can cause tumor cell death.There is interaction between c-Met kinase and PARP1.C-met kinase can bind to tyr907 amino acid residues on PARP1 catalytic domain,and then phosphorylate PARP1.Phosphorylation of PARP1 can increase the activity of PARP1 and decrease the binding with PARP inhibitors,which makes cancer cells resistant to PARP inhibition.If we can develop kinase inhibitors targeting c-Met and PARP1,it will be hopeful to eliminate the resistance of PARP1 to its inhibitors,so as to enhance the efficacy of breast cancer.Based on the structure of Cabozantinib,BMS-777607 and PARP1 inhibitor Olaparib,the project will be designed a series of pyrazole[1,5-a]pyrimidine derivatives as c-Met and PARP1 inhibitors by using skeleton transition,splicing principle,bioelectronic and other methods.We will start the anti-breast cancer activity experiment,HTRF tyrosine kinase inhibition experiment,PARP1 enzyme inhibition experiment and structure-activity relationship research to lay the foundation for further research.MethodsA series of pyrazole[1,5-a]pyrimidine derivatives,numbered GMZC-L1～L28,were synthesized by designing a synthetic route with easy raw materials,mild reaction conditions,low cost and simple operation safely.The structures were confirmed by HRMS,¹H NMR and ¹³C NMR.The antitumor activities of the target compounds on MDA-MB-231 cell,MCF-7 cell,HCC1937 cell and Hep G2 cell were measured by CCK-8 method in vitro.The preliminary structure-activity relationship of pyrazole[1,5-a]pyrimidine derivatives was summarized by analyzing the data of anti-tumor cell proliferation in vitro.The effects of target compounds on the clonogenic ability of breast cancer cells were investigated by plate clonogenesis experiment.The effects of target compounds on the migration ability of breast cancer cells were explored by scratch experiment.The effects of target compounds on the cycle and apoptosis of breast cancer cells were explored by cell cycle and apoptosis experiment.HTRF tyrosine kinase experiment was carried out to determine the inhibitory activity of the target compounds on the selected tyrosine kinase.The binding patterns of these compounds to target proteins were studied by molecular docking technology to evaluate the binding ability of these compounds to target proteins.Results1.A series of pyrazole[1,5-a]pyrimidine derivatives,numbered GMZC-L1～L28,were synthesized successfully and their structures were confirmed by HRMS,¹H NMR and ¹³C-NMR.Through Sci Finder search,28 compounds were confirmed to be new compounds.2.In general,the target compounds showed inhibitory activity on MDA-MB-231,MCF-7,HCC1937 and Hep G2 cells.The target compounds showed stronger selectivity to MDA-MB-231 cells.Compounds GMZC-L25,GMZC-L26,GMZC-L27 and GMZC-L28 showed excellent inhibitory activity on four kinds of tumor cells.Their inhibitory activities were better than the positive drugs 5-Fluorouracil and Cabozantinib or equivalent.The IC₅₀ of compound GMZC-L28 to MDA-MB-231,MCF-7,HCC1937and Hep G2 cells were 2.627±0.073,6.233±1.088,5.559±0.317 and 6.902±0.434μmol·L^-1respectively.The inhibitory activity of compound GMZC-L28 is more prominent and it is expected to be used as a candidate anti-tumor compound in the next step of toxicology and pharmacology research.3.The structure-activity relationships between the target compounds and the four tumor cells were similar.It shows that the introduction of p-methoxyphenyl into the5-position R₁ of the parent nucleus was better than that of methyl.The addition of methyl group to 6-position R₂ of the parent nucleus is beneficial to the increase of activity.It is helpful to maintain antitumor activity to introduce fluorine atoms into the middle structure substituted for the 2-position R₃ of phenoxy and the 4-positions on the benzene ring at the end of the side chain at the same time.The antitumor effect of the compounds is not good if only the fluorine atom is introduced into R₃or R₄ site.The introduction of 4-CH₃,4-OCH₃ and 2-CH₃into the position R₄ of the benzene ring at the end of the side chain could not effectively improve the antitumor activity of the compounds.The addition of methyl,ethyl or isopropyl group to position R₅ of pyrimidine diketone ring has little effect on the activity,and the antitumor activity can be maintained.4.Cell morphology observation showed that after administration of GMZC-L28 to MDA-MB-231 cells,it could cause cell shrinkage and rounding,nuclear pyknosis and nucleolysis,and the adherence ability decreased.Plate clonogenic assay showed that GMZC-L28 could inhibit the clonogenic ability of MDA-MB-231 cells in a concentration-dependent manner.Scratch assay confirmed that GMZC-L28 could decrease the migration ability of MDA-MB-231 cells in a concentration-dependent manner.Cell cycle experiments showed that after 48 h of GMZC-L28 acting on MDA-MB-231 cells,it could inhibit the transformation of cell cycle from S phase to G2/M phase,thereby arresting the cell cycle in S phase.Apoptosis experiments showed that GMZC-L28 could induce apoptosis of MDA-MB-231 cells after 48 h of treatment and the higher the drug concentration,the higher the apoptotic rate.5.The result of HTRF tyrosine kinase experiment indicates that the inhibition activity of Cabozantinib on c-Met kinase is significant,and the inhibition rates at the concentrations of 0.5μmol·L^-1 and 25μmol·L^-1are 86.88%and 97.83%,respectively.In contrast,the target compound has a certain inhibitory effect on c-Met kinase,and the inhibitory activity is worse than Cabozantinib.In the experiments of other kinases,we found that several target compounds have moderate inhibitory activities on MER,LCK,FLT3 and ALK kinases.In the further study,it is necessary to consider MER,LCK,FLT3 and ALK kinases.as potential target enzymes to modify the structure of the target compounds,which is expected to obtain multi-target tyrosine kinase inhibitors.6.The results of molecular docking showed that the binding mode of GMZC-L28with c-Met kinase or PARP1 enzyme was different from t the positive drugs cabozantinib or olaparib.This also explains that the inhibitory activity of the compound to c-met kinase is quite different from Cabozantinib.However,GMZC-L28 has a relatively high overall scoring function and the highest consistency evaluation function,indicating that g GMZC-L28 can effectively combine with c-met kinase and PARP1enzyme.ConclusionBased on the structural framework of Cabozantinib,BMS-777607 and PARP1inhibitor olaparib,28 new pyrazol[1,5-a]pyrimidines were successfully synthesized by structural modification.The target compounds showed inhibitory activities on MDA-MB-231,MCF-7,HCC1937 and Hep G2 cells.GMZC-L25,GMZC-L26,GMZC-L27 and GMZC-L28 showed excellent inhibitory activities on four tumor cells,and the inhibitory activities were close to or better than the positive drugs5-Fluorouracil and Cabozantinib.The compound GMZC-L28 was selected to act on MDA-MB-231 cells,and it have antitumor effects on MDA-MB-231 cells in many aspects,including inducing morphological changes,inhibiting the clonal formation of cancer cells,inhibiting the migration of cancer cells,blocking cell cycle in S phase and promoting apoptosis of cancer cells and so on.HTRF tyrosine kinase experiment showed that the target compound had a certain inhibitory effect on c-Met kinase.In the experiments of other kinases,it was found that several target compounds had moderate inhibitory activities on MER,LCK,FLT3 and ALK kinases.The results of molecular docking showed that the binding mode of GMZC-L28 with c-Met kinase or PARP1enzyme was different from the positive drugs Cabozantinib or Olaparib.Docking function scores showed that GMZC-L28 could effectively bind to c-met kinase or PARP1 enzyme.GMZC-L28 can be used as a candidate compound for toxicology,pharmacology,pharmacodynamics and pharmaceutics research,providing basis and basis for further research due to its significant anti-breast cancer activity.