Synthesis Of Fused 3-amino-1,2,4-triazole Compounds And Evaluation Of Anti-idiopathic Pulmonary Fibrosis Activity

Background and Objective:Idiopathic pulmonary fibrosis?IPF?is a type of chronic progressive disease that is most common in idiopathic interstitial pneumonia?IIPs?and is characterized by chronic,progressive,fibrotic interstitial lung disease of unknown cause^[1].Patients with IPF are usually diagnosed with a poor prognosis.There are few treatments for IPF,and the efficacy is poor,and the side effects are large.Therefore,it is urgent to develop new drugs to treat IPF.Studies have shown that the epithelial mesenchymal transition?EMT?process and the production of IPF have a great relationship;In the process of EMT in epithelial cells,the cells undergo morphological changes,appearing as fusiform,the cell outline becomes blurred,the expression of the marker proteins in the cells is down-regulated?such as E-Cad protein?,and the expression of marker proteins in interstitial cells up-regulation?eg?-SMA protein?^[2].The EMT process is mainly initiated by the action of transforming growth factor-??TGF-??,thus blocking the transmission of TGF-?₁ signal in lung tissue cells,which can prevent the occurrence of EMT process,and it is expected to achieve prevention and treatment of IPF^[3].Reference to existing TGF-?₁ inhibitor structures and related research support^[4],we plan to synthesize a series of fused 3-amino-1,2,4-triazole compounds.Summarizing and referring to the synthesis method of the predecessors,the reaction is carried out under mild conditions by continuous optimization,and the product is obtained in a higher yield.A549 cells were first used to evaluate the cytotoxicity of the compounds,and an in vitro model of pulmonary fibrosis was constructed to detect the reversal activity of the series of compounds on the EMT process.Methods:1.Compound synthesis:Adding isothiocyanate to aryl hydrazine at room temperature?r.t.?with ethanol as solvent,and then performing I₂ mediated oxidative desulfurization/cyclization under basic conditions?potassium carbonate?to produce corresponding condensation 3-amino-1,2,4-triazole.2.Compound toxicity test on A549 cells:A549 Cell growth inhibition was detected by MTT assay after treatment with 25,50,100,200,400?mol/L compound or positive control?SB-431542?for 72 h.3.Compound detection of EMT reversal activity:The cells were treated with 10ng/ml TGF-?₁,10?mol·l-1 compound or positive control?SB-431542?for 48 h,and the E-Cad protein and?-SMA protein were detected by immunofluorescence and Western Blot.Express the situation.Results:1.The fused 3-amino-1,2,4-triazole compound was successfully synthesized,and the product yield was between 41%and 97%.The synthesis method is simple in operation,mild in condition,high in yield,good in adaptability,and can be conveniently synthesized on a gram scale scale;the key catalytic oxidant iodine element is cheap,easy to obtain,and green and non-polluting.2.The toxic effects of the compounds and the positive control on A549 cells were small,and the CC₅₀ values were all greater than 300?M.The cytotoxicity of compounds 1a,1d,1e,1k,1n,1r,1t,1u,1z and 1aa was small,and the CC₅₀ values are greater than 1000?M;compound 1l and 1y have slightly greater cytotoxicity,CC₅₀ values are 302?M and 387?M;the remaining compounds 1g,1h,1s and 1v have CC₅₀ values between 400?M and 1000?M.The positive control compound has a CC₅₀ value of 450?M.3.Six compounds with better activity were screened by immunofluorescence experiments,which were 1d,1g,1n,1r,1y and 1aa.Western Blot assay showed that two compounds,1g and 1r,significantly up-regulated the expression of E-Cad protein and down-regulated the expression of?-SMA protein in the comparison model group and the positive group.The activity was similar to that of the positive control compound.According to the structure-activity relationship analysis,it is known that the 3-amino group-bonded aryl compound 1d has activity in which the benzene ring has a fluorine atom and 1g has activity in which a cyano group is substituted,wherein 1g of the activity is more prominent;the 3rd amino group is bonded to an aliphatic hydrocarbon group.Both 1n and 1r have better activity,and the cyclohexyl activity of 1r is better.When the nitrogen atom is substituted on the fused ring,the 1z activity of adding thiomethyl group is poor,while the activity of 1y and 1aa is better.Conclusion:1.A preferred method for synthesizing fused 3-amino-1,2,4-triazole compounds is given.2.The synthesized compounds have some activity in reversing the EMT process,suggesting that there may be a role in the treatment of IPF.