Design,Synthesis,Biological Evaluation And Molecular Modeling Of 2-amino-4-oxo-6-substituted-Pyrrolo[2,3-d] Pyrimidines

In cellular metabolism,folate coenzymes are required in more than 20 interrelated enzymatic reactions,which are necessary to maintain de novo synthesis of the essential building blocks of DNA(i.e.purine nucleoside and pyrimidine nucleoside)as well as the synthesis of certain important amino acids.Therefore,classic antifolates typified by methotrexate(MTX),pemetrexed(PMX),and pralatrexate(PDX)remain important chemotherapeutic agents in the process of various cancers treatment,microbial infections,inflammatory disorders,and autoimmune diseases.In 1949,MTX was first synthesized and to be used alone or in combination with other chemotherapeutic agents to treat breast cancer,osteogenic sarcoma and acute lymphocytic leukemia.PDX came in the mid-1990s and was approved for the treatment of the relapsed or refractory T-cell lymphoma.The RFC-? is often overexpression in cancer cells to ensure enough folate uptake for the vigorous cell division,PDX,is considered to be selective towards cancer cells,which is transported into cells through RFC-?.Both MTX and PDX are potent inhibitors of dihydrofolate reductase(DHFR),which functions as a catalyst for the reduction of dihydrofolate to tetrahydrofolate.The highly basic 2,4-diaminopyrimidine moiety in the pteridine ring of these compounds is protonated and forms an ionic bond with the active site of the DHFR enzyme and accounts,in part,for their high affinity for DHFR.PMX,originally developed as a thymidylate synthase(TS)inhibitor,is a multitargeted agent with a 6-5 fused pyrrolo[2,3-d]pyrimidine nucleus instead of the more common 6-6 fused pteridine or quinazoline ring structure.Preliminary studies revealed that PMX polyglutamates are not only potent inhibitors of TS but also of DHFR and glycinamide ribonucleotide(GAR)formyltransferase(GARFTase)and 5-aminoimidazole-4-carboxamide(AICA)ribonucleotide formyltransferase(AICARFTase)in de novo purine nucleotide biosynthesis.PMX has been approved for the treatment of malignant pleural mesothelioma in combination with cisplatin and recently in nonsmall lung cancer by the FDA.Many types of transformed cancer cells have an elevated dependence on the de novo purine biosynthetic pathway and an impaired purine salvage pathway,while normal cells can rely solely on the salvage pathway for purines.This allows such sensitive cancer cells to be targeted selectively over normal cells by inhibitors of de novo purine biosynthesis,including GARFTase and AICARFTase inhibitors.Lometrexol(LMTX),the first GARFTase inhibitor to enter clinical trials,was synthesized in 1985.LMTX is a poor inhibitor of both DHFR and TS,but a potent inhibitor of GARFTase(Ki=6nM)and a substrate for folypoly-?-glutamate synthetase(FPGS).Patients treated with LMTX in phase-I clinical trials developed severe and cumulative myelosuppression and mucositis.The cumulative toxicity of LMTX is thought to be due in part to its ability to be transported by both the RFC and FR,resulting in increased cellular levels.Targeting only one folate-dependent enzyme,as with most currently available antifolates,are frequently associated with drug resistance.Thus,it is of interest to develop dual or multitargeted antifolates.In our studies,a series of 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines were designed,synthesized and biologically evaluated as potential multitargeted antifolates.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-ol to give ethyl 2-(2-amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)acetate(13).The reduction of intermediate 13 by lithium triethylborohydride and successive mesylation afforded the key mesylate 15.Nucleophilic substitution by mercaptoacetic or mercaptopropionic acid methyl esters,followed by hydrolysis and condensation with pyridinyl-methylamines provided the nonclassical compounds 1-6,whereas condensation with L-glutamic acid diethyl ester hydrochloride and saponification afforded the classical analogs 7-8.Nucleophilic substitution of 14 with ethyl 4-hydroxybenzoate or methyl 4-mercaptobenzoate,followed by hydrolysis,condensation with L-glutamic acid diethyl ester hydrochloride and saponification afforded the classical compounds 9-10.The chemical structures of key intermediates and target products were confirmed by melting point,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 and molecular modeling studies were also applied to investigate the mechanism of action of these compounds.Results:By using the designed synthetic routes,the target compounds 1-10 were obtained successfully and confirmed by HRMS,1H-NMR and 13C-NMR.The synthetic strategies were also optimized.The biological activities of target compounds were evaluated,and their intracellular mechanism and molecular modeling studies were applied as well.Conclusion:All target compounds exhibited inhibitory activities toward KB,SW620 and A549 tumor cell lines.The most potent compounds of this series,8,is a better inhibitor against A549 cells than MTX and PMX.Nucleoside protection assays establish compound 8 a dual inhibitor of TS and AICARFTase.Target compound 8 alternates the cell cycle of SW620 cells with S-phase 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.