Design,Synthesis,Biological Evaluation And Molecular Modeling Of Novel Classical And Non-Classical Folic Acid Antagonists

It has long been the focus of medicinal chemists to discover antitumor-agents through interfering with the process of folate metabolism.In human folate cycle,only tetrahydrofolate has physiological functions.Dihydrofolic-acid cannot be used,unless it is reduced to tetrahydrofolate by dihydrofolate reductase?DHFR?.Tetrahydrofolate provides active one-carbon units that allows thymidylate synthase?TS?to catalyze the reductive methylation of 2'-deoxyuri-dine-5'-monophosphate?d UMP?to 2'-deoxy-thymidineglycoside-5'-monophosp-hate?d TMP?,which is the only de novo pathway for the synthesis of d TMP,a key precursor for nucleic acid synthesis.Tetrahydro-folate is also involved in the generation and metabolism of many other bioprecursors.During the rapid growth period of tumor cells,if DHFR and TS are inhibited,tumor cells will be dead due tolack of thymine.Therefore,DHFR and TS have been considered as major targets for cancer chemothe-rapy.Methotrexate?MTX?and pemetrexed?PMX?are widely used antitumor-agents in clinics.Their main intracellular targets are DHFR and TS,respectively.The chemical structure of MTX is similar to that of folic acid.MTX blocks the reduction of dihydrofolate to tetrahydrofolate by competitive inhibition of DHFR and reduces one carbon unit donor in cancer cells,which results in inhibition of DNA,RNA and protein synthesis,eventually leading to cancer cell death.MTX is in the form of polyglutamate intracellularlly,which makes it long-term presence in certain cells and results in accumulate toxicity.DHFR gene mutation leads to decreased affinity for MTX,resulting in drug resistance.Therefore,the discovery of novel antitumor agentsis mainly focused on multitargeted antifolates to improve selectivity and to overcome drug resistance.Using compound A previously reported by our group as a lead compou-nd,a series of 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines were designed and synthesized as antifolates and potential antitumor agents.Their antiproliferative activity,mechanism of action and molecular simulation were also studied.Objective:The study is aimed to establish the synthetic method of a series of 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines,to optimize the reaction conditions and to obtain the target compounds.It is also aimed to evaluate the biological activities of these compounds and to reveal their mechanism of action through in vitro metabolic assays and molecular modeling studies.Methods:The synthesis started from the cyclization of ethyl 4-chloro-3-oxobutanoate with 2,6-diaminopyridin-4-oxopyrimidinel to give Ethyl 2-?2-amino-4-oxo-3,7-dihydro-1H-pyrrolo[2,3-d]pyrimidine?acetate?15?.The redu-ction of intermediate 15 by lithium triethylborohydride and successive mesyl-ation afforded the key mesylate 17.Nucleophilic substitution reaction with sodium azide via intermediate 17 gave the key intermediate 2-amino-6-?2-azi-doethyl?-3,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-4-one?18?.Nucleophilic substitution reaction of sodium azide with intermediate 17 gave compound2-amino-6-?2-azidoethy?-3,7-dihydro-1H-pyrrolo[2,3-d]pyrimidin-4-one?18?.Reduction of the azido group of 18 to an amino group using Pd/C and a hydr-ogen system gave compound 19.Under the treatment of HOBt/EDCI,compo-und 19 was condensed with orthometa or para phthalic acid monomethyl ester respectively to give intermediates 29,21 and 25.Compounds 29,21 and 25were condensed with 2-,3-,or 4-aminomethylpyridine respectively under the treatment of HOBt/EDCI to obtain the target compounds 1-9.Intermediates29,21 or 25 underwent condensation reaction with diethyl L-glutamate hydr-ochloride catalyzed by N-methylmorpholine?NMM?and 2-Chloro-4,6-dime-thoxy-1,3,5-triazine?CDMT?.Targetcompounds 10,11 and 12 were obtained after hydrolysis and acidification.The chemical structures of key intermedia-tes and target products were confirmed by HRMS,¹H-NMR and ¹³C-NMR.Tumor cell lines such as KB,SW620 and A549 were selected to test the antiproliferative activities of target compounds with MTX and PMX as positive controls.Nucleoside protection assays,dihydrofolate reduct-ase assay and molecular modeling studies have also been used to study the mechanism of action of these compounds.Results:The target compounds 1-12 were successfully synthesized according to the designed routes.The structures of important intermediates and target compounds were confirmed by MS,¹H-NMR and ¹³C-NMR.The synthetic strategies were optimized.In vitro antitumor activity test,mechanism of action studies and molecular simulation of compound 1-9 were also completed.Conclusion:All target compounds exhibited inhibitory activities toward KB,SW620 and A549 tumor cell lines.The most potent compounds of this series 1-3 were better inhibitors against KB cells than MTX and PMX.Nucleoside protection assay and dihydrofolate reductase assay indicated compounds 1,2 and 3 were DHFR inhibitors.Target compound 1,2,3alternates the cell cycle of KB cells with Sphase accumulation and induces apoptosis,leading to cell death.The docking results are consistent with the results of our in vitro metabolic assays,which provide supports for the design of this series of compounds.