Design, Synthesis And Biological Evaluation Of Pyrrolo [2,3-d] Pyrimidines

As an important class of anti-metabolic drugs,antifolates play an important role in chemotherapy as antitumor,anti-bacterial,antifungal and anti-inflammatory agents.Several drugs such as methotrexate,pemetrexed,raltitrexedandpralatrexatehavebeenapprovedasantitumor chemotherapeutic agents.These drugs have been widely used clinically.Among them,pemetrexed completed the goal of 2.594 billion dollars in the global market.The intracellular targets of antifolates mainly include dihydrofolate reductase?DHFR?and thymidylate synthase?TS?.For example,methotrexate and pralatrexate are typical DHFR inhibitors,whereas pemetrexed and raltitrexed predominantly inhibit TS.DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolic acid in cells,while TS is the essential enzyme that catalytizes the transfer of deoxyuridine monophosphate?dUMP?to thymidine monophosphate?dTMP?.Inhibition of DHFR and TS causes lack of reduced folic acid or"thymineless state"leading to cell death,as cells can not complete the exact DNA replication.In addition to DHFR and TS,there are two enzymes that play a key role in de novo purine biosynthesis,and they are?-glycinamide ribonucleotide formyltransferase?GARFTase?and5-aminoimidazole-4-carboxamidenucleoside?5-aminoimidazole-4-carboxamideribonucleotideformyltransferase,AICARFTase?.They can also serve as targets of antifolates.The intracellular targets of most antifolates including methotrexate,pralatrexate and raltitrexed are relatively simple.They mainly inhibit DHFR and?or?TS.Tumor cells can easily produce resistance by mutation or overexpress after a period of clinical use.Pemetrexed was identified as the first multi-target antifolates.The major targets of pemetrexed include TS,DHFR,GARFTase and AICARFTase.Although pemetrexed is not easy to get resistance compared to other single-target antifolates,it still causes serious side effects due to lack of selectivity toward tumor cells.In previous work,were portedaseries of 6-substituted pyrrolo[2,3-d]pyrimidines.These compounds can be selectively transported in cells by protone coupled folate transporter?PCFT?but not the reduced folate carriers?RFCs?,resulting in selectivity to tumor cells.Compound 1a inhibited KB cells and IGROV1 cells with IC₅₀0 of 3.22 nM and 122.7 nM respectively.The intracellular target of 1a is GARFTase,which is important in purine biosynthesis.In this study,a series of new compounds were designed and synthesized by using compound 1a as a lead compound,which has transport selectivity and multiple intracellular targets.The aim of this study is to maintain the existing transport selectivity and to generate multi-target inhibition through structure modification of the lead compound.Objective:To establish a synthetic method of pyrrolo[2,3-d]pyrimidines,complete the synthesis of the target compounds,optimize the reaction conditions,and to screen the target compounds in vitro for anti-tumor biological activity.Methods:Commercially available 2,4-diamino-6-hydroxypyrimidine?11?and ethyl-4-chloroacetoacetate?12?were condensed in sodium acetate water solution to form the key intermediate 13,which was then hydrolyzed under basic condition to give acid 14.Compound 14 was condensed with methyl aminobenzoate,methyl p-methylbenzoate,4-??methylamino?methyl?benzoate methyland5-aminofuran-2-carboxylate,respectively,followedby saponification to produce carboxylic acids 17a-17d.Final condensation with L-glutamic acid diethyl ester hydrochloride,followed by saponification produced target compounds 1-4.Ester 13 was reduced to alcohol 22 by lithium triethylborohydride,and then converted to mesylate 23 under the treatment of methanesulfonyl chloride.Mesylate 23 reacted with sodium azide to afford the key intermediate 24.A“click reaction”of intermediate 24 with different terminal alkynes,followed by saponification produced carboxylic acids27a-27c.Target compounds 5-7 were synthesized by condensation of carboxylic acids 27a-27c with L-glutamate diethyl ester hydrochloride,followed by hydrolyzation under basic condition.Following the same method,target compounds 8-10 were successfully obtained by coupling of carboxylic acids 27a with different aminomethylpyridines.The structures of key intermediates and target products were confirmed by HRMS,¹H-NMR and¹³C-NMR.KB cells?oral epidermoid carcinoma?,MCF7 cells?human breast cancer?and SW620 cells?colon cancer?were selected to evaluate antiproliferative activities of target compounds with MTX and PMX as positive controls.Nucleotide protection assay?adenosine and thymidine?/AICA?5-aminoimidazole-4-carboxamide?were also carried out to determine the cellular targets of synthesized compounds.Results:The target compounds 1-10 were successfully synthesized according to the designed synthetic route.The structures of the important intermediates and target compounds were confirmed by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy.The in vitro antitumor activity of compounds 1-7 and their mechanism of actionwere studied.Conclusions:Target compounds showed significantly potent antitumor activity.Compound 1 had an IC₅₀0 of 7.5 nM against KB cells which is more potent than that of the standard MTX and PMX.Compound 1 was also active against SW620 cells and MCF7 cells with IC₅₀0 of 0.98?M and 1.13?M,respectively.