| BACKGROUDIn medicine,cancer refers to the malignant tumor originated from epithelial tissue,which is due to that the body affacted by a variety of physical,chemical and other tumorigenic factors under the action of local tissue cells loose their normal regulation of growth and lead to abnormal proliferation and differentiation of the formation of the mass.The growth of these lumps is not regulated by the body's physiology,but destroys the normal tissues and organs,which determines that once they are formed,they will not stop growing with the elimination of etiology.After years of epidemiological studies,experimental and clinical observations,people found more than 80%of malignant tumors are related to environmental factors.Various environmental and genetic carcinogenic factors may cause non-lethal DNA damage in a synergistic or sequential manner to activate proto-oncogenes or(and)inactivate tumor suppressor genes,coupled with apoptosis-regulating genes and(or)DNA repair gene changes,so that cells transformed.The transformed cells can be polyclonal first,and after a long multi-stage evolution process,one of the clones unrestrictedly amplifiesrelatively,and by additional mutation,forms subclones with different characteristics selectively,fanally obtains infiltration and migration ability,thus forming malignant tumor.Therefore,tumor is a genetic diseaseessentially.So far,more than 40 pathogenic genes have been discovered,and their encoded protein products include growth factors,protein kinases,GTP-binding proteins and so on.Many oncoproteins and growth factor receptors are protein kinases,and transformed cells show higher levels of protein phosphorylation than normal cells.Under the control of protein kinases,these phosphorylated proteins act as downstream effector molecules and,by amplifying the signaling pathway,lead to aberrant transcription of certain key genes and abnormal cell proliferation,which resulting cancer.Despite advances in medicine,how many cancers take the lives of millions of patients every year and even the so-called "miraculous pill" can only prolong patients'lives by an average of a few weeks.Moreover,the existing drugs are generally only for one kind of tumors,if the cancer changes too much,the seemingly useful drugs will not work anymore.Even if some drugs eliminate the cancer temporarily,some highly-mutated cells may be in a latent state,the disease will recur.At present,poor treatment,severe side effects and multiple drug-acquired tolerance are common problems in clinical treatment of cancer.The research and development of strong specificity,therefore,it is a great challenge for the pharmaceutical industry to develop anti-tumor drugs with strong specificity and small edge effect.Disorders in the cell cycle are considered to be important markers of human cancer,and the changes in the components of their regulatory organs are closely related to the occurrence of most tumors.Each stage of the cell cycle is prepared for the latter phase in order to achieve the purpose of obtaining two daughter cells with the same genetic material.Before cells entering the next period have to be "checked",which called the checkpoint.The purpose of checkpoint is to arrest the cell cycle when cells encounter environmental stress or DNA damage,and thereforeplays a key role in maintaining genes stability,which can lead to tumor formation if impaired in these checkpoints.Among the several checkpoints(G1/S?S/G2?G2/4?M/G0)in the cell cycle,G1/S and G2/M are the most important.However,tumor cells generally have defects in the G1/S checkpoint,and tumor cells tend to selectively remain in the G2/M phase after DNA damage.Therefore,the G2/M checkpoint becomes an attractive target for tumor therapy.When the tumor cells arrest in the G2/M phasefor a long time,they can inhibit tumor growth,on the other hand,the tumor cells can also accumulate DNA damage and trigger apoptosis,thus achieving the purpose of treating the tumor.In addition,apoptosis is the autonomic,orderd death of cells controlled by genes,the study found that abnormal apoptosis is an important cause of cancer in recent years,The inactivation of some genes which induce apoptosis,such as Bax,and overexpression of apoptotic genes,such as Bcl-2,play an important role in the occurrence of malignant tumors,andthe damage of apoptosis pathway is the main cause of tumor resistance to treatment.For example,80%of human hepatoma cells overexpress XIAP protein to inhibit the activation of caspases,more than 50%of the tumor cells in anti-apoptotic protein Bcl-2 expression increased,these mechanisms directly or indirectly promote the occurrence of tumors and development of tumor.Abnormal proliferation caused by disruption of the cell cycle regulatory mechanisms is the most essential biological characteristic of all cancer cells.Cyclin-dependent kinases(CDKs)as a crucial cycle regulatory proteases,which are closely linked to cell cycle or transcriptional regulation.And the extremely active of CDKs is frequently detected in lung cancer,colon cancer,breast cancer,and malignant lymphoma.So specific CDKs inhibitors which can disrupt the cancer cell cycle and induce its apoptosis are effective for the control and cure of cancer,the development of CDKs inhibitors,especially highly selective CDKs,has become a very popular field in the research of anti-tumor drugs.OBJECTIVEWith our previous design synthesis of small molecules of ATP competitive CDKs inhibitors C-4 for lead compound,by modifying the structure modification,we design synthesis of a series of 2-amino pyrimidine compounds(series I),and study the antitumor activity of these compounds in vitro,preliminarily elucidate its mechanism to provide some reference for the discovery and research of selective CDKs inhibitors.METHODS AND RESULTS1.Synthesis of the target compoundsBased on our previously reported compound C-4,possessing significant antiproliferative activity,this paper designed and synthesised ten novel target compounds by a two-step nucleophilic substitution reaction using 2,4-dichloropyrimidine as the raw material.2.MTT method was uesd to detect cell activityDifferent concentrations of series I compounds were incubated with colon cancer cell line HCT 116,cervical cancer cell line Hela,hepatoma cell line Hep G2 and human prostate cancer cell line PC-3 for 48 hours respectively.MTT assay showed that most of the compounds of series I inhibited the growth of the above four tumor cells significantly.In particular,the most promising compound I-7 displayed the IC50 values of 0.4365 ± 2.56 ?M,1.846 ± 0.42 ?M,2.28 ± 0.99 ?M,46.02 ± 0.60?M for above four cells,respectively.3.Flow cytometry was uesd to determine cell cycle distribution and apoptosisAfter treatment of HCT 116 and Hela with different concentrations of compound?-7,we found that ?-7 could change the proportion of HCT 116 and Hela in the cell distribution of each cycle and arrest the cell growth in G2/M phase obviously,moreover,?-7 also induced apoptosis in a dose-dependent manner.4.Western blotting was uesd to determine G2/M and apoptosis correlative proteinsWe investigated the effect of compound ?-7 on HCT 116 and Hela related protein,and found that compound ?-7 reduced the expression of p-CDK1,CyclinB1,CDK2,p21wafl/Cip1 and anti-apoptotic protein Bcl-2 with the increase of thej concentration and the extension of the action time,but basically did not change the expression of pro-apoptotic protein Bax,while increasing the expression of Cleaved PARP and p53 protein.5.Statistical analysisData analysis and mapping are based on the software of GraghPad Prism 5.0(data analysis and mapping software).CONCLUSION1.Ten new structure of 2,4-diaminopyrimidine compounds were designed and synthesised and all target compounds were characterized by nuclear magnetic resonance hydrogen spectrum(1H-NMR),carbon-13 nuclear magnetic resonance spectroscopy(13C-NMR),EI low resolution mass spectrometry(ESI-MS),and elemental analysis or high resolution mass spectrometer(HRMS).2.compound ?-7 significantly inhibits the activity of human colon cancer cell line HCT 116,human cervical cancer cell line Hela,human prostate cancer cell line PC-3 and human hepatoma cell line Hep G2,with IC50 values of 0.4-46 ?M3.compound ?-7 induces G2/M cell cycle arrest by inhibiting CDK1/CyclinB1 complex.4.compound ?-7 can down-regulate the expression of anti-apoptotic protein Bcl-2 and cause the downstream PARP protein to be activated,thereby inducing cell apoptosis.5.The function of ?-7 in inducing cell cycle arrest and apoptosis in HCT 116 and Hela is related to the regulation of the key factors p21 waf1/Cip1 and p53 protein. |
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