Synthesis Of 2-substituted-4-arylaminopyrazolo[1,5-a][1,3,5]triazines And Preliminary Study On Their Antitumor Activity

Cancer has seriously affected the health and living quality of people.With the emergence and development of "targeted therapy",small-molecule targeted therapies have become a hot research area in the treatment of cancer.Researchers have developed some anti-cancer drugs based on the characteristics of tyrosine kinase,which brings great hope for cancer treatment.The third generation of TKIs solved the drug resistance and toxic side effects of the first and second generations of inhibitors,and achieved good results in clinical practice.Unfortunately,the new drug resistance of the third generation TKIs has recently been discovered in clinical practice,thus design new anticancer drugs are ungently needed.It has been found that pyrazolo[1,5-a][1,3,5]triazine,a heterocyclic compound containing four nitrogen atoms,has strong biological activity,and has shown great potential in the treatment of diseases such as parkinson's syndrome and anxiety.We used pyrazolo[1,5-a][1,3,5]triazine as core structure,designed and synthesized a series of pyrazolo[1,5-a][1,3,5]triazine derivatives.The structures of the target compounds were characterized by NMR,IR and HRMS.Finally,a preliminary antiproliferative test of the synthesized compounds was performed against human tumor cell.The main work of this paper are as follows:(1)The relationship between protein tyrosine kinase and cancer,and the hopes and disappointments brought by small-molecule tyrosine kinase inhibitors in the treatment of cancer were briefly described.A N-containing heterocyclic pyrazolo[1,5-?][1,3,5]triazine with good biological activity was proposed as the core structure,and a virtual compound library was obtained by assigning multiple structural units to different positions of the core.Using EGFR as the target protein,the library compounds were scored by Sybyl software to select 32 compounds as target compounds.(2)Based on the summary of the literature,we selected an economical,green,simple synthetic route to synthesize the target compound.Firstly,the core structure,1H-3H-2-thioxo-pyrazolo[1,5-?][1,3,5]triazin-4-one?was synthesized by the reaction of 2H-3-aminopyrazole and ethyl isothiocyanate in dichloromethane at room temperature,and after moving solvent,and sequently cyclization under basic conditions.The process of two steps are simple,and the yields are more than 90%.Secondly,4-(3-chloropropyl)morpholine and 1-(3-chloropropyl)-4-methylpiperazine were respectively synthesized from morpholine,N-methylpiperazine through nucleophilic substitution with 1-bromo-3-chloropropane.4-(2-chloroethyl)morpholine hydrochloride,1-(2-chloroethyl)-4-methylpiperazine hydrochloride,1-(2-chloroethyl)piperazine hydrochloride and 2-chloro-N,N-diethylethan-l-amine hydrochloride were synthesized from morpholine,N-methylpiperazine etc through nucleophilic substitution with bromoethanol,and then reaction with SOCl2,with the yields of 70%-81%.Then these synthesized compounds reacted respectively with 1H-3H-2-thioxo-pyrazolo[1,5-a][1,3,5]triazin-4-one to give six 2-substituted-3H-pyrazolo[1,5-?][1,3,5]triazin-4-one in 83%-95%yields.Finally,2-substituted-3H-pyrazolo[1,5-a][1,3,5]triazin-4-one was chlorinated with POCl3,and sequently the nucleophilic substitution of 2-substituted-4-Cl-pyrazolo[1,5-a][1,3,5]triazines with six aromatic amines,including 3-chloro-4-fluoroaniline,3-ethynylaniline,3-chloro-4-(3-fluorobenzy loxy)aniline,3-chloro-4-(pyridin-2-ylmethoxy)aniline,3-chloro-4-(3-(trifluoromethyl)phenoxy)aniline and 2-methoxy-4-fluoro-5-nitroaniline,were carried out,respectively.The substitution reactions using Et3N as acid-binding agent and THF as solvent.Thus,32 compounds,2-substituted-4-arylaminopyrazolo[1,5-a][1,3,5]triazines were successfully prepared in 42-71%yields.(3)With the clinical anticancer drugs lapatinib and gefitinib as positive controls,the antiproliferative activities of these new compounds were evaluated by MTT assay on four human tumor cells,including colon cancer cells SW480,epidermal squamous cell carcinoma A431,non-small cell lung cancer cells A549 and NCI-H1975.The results showed that most of the compounds have inhibitory effect on the proliferation of the four cancer cells line.Especially the compounds F1,F5,F6,F9,F12,F13,F18 and F31 showed potent inhibition of proliferation on four tumor cell lines.And the IC50 against the four cells are respectively 2.82-6.27 ?M,2.86-7.63 ?M,2.23-4.79?M,2.34-5.08 ?M.The antiproliferative activities of these compounds are significantly better than that of the positive controls lapatinib and gefitinib.