Study On Synthesis And Application Of Acridinium Esters

As a kind of highly efficient chemiluminescence reagent, Acridine esters are commonly used in clinical immunoassays and biological activity determination of enzymes. In this work two series of acridine ester derivatives were designed: the first class is unsubstituted acridine 9-carboxylic acid benzyl ester derivatives and the second is a substituted acridine 9-carboxylic acid phenyl derivative which was intended for the determination of biological activity of paraoxonase in blood. First class of compounds started from diphenylamine which reacted with oxalyl chloride to generate N-phenyl alizarine with yield of 78.6%; then, N-phenyl alizarine reacted with KOH through rearrangement reaction to give acridine 9 -carboxylic acid with yield of 94.1%. Acridine 9-carboxylic acid reacted with thionyl chloride to parepare Acridine 9-carboxylic chlorides, which reacted with methyl-phenol, cresol, p-nitrophenol, and acetoxy phenol to give corresponding acridine 9 -carboxylic acid benzyl ester derivatives (1c, 2c, 3c, 4c). In order to improve the chemiluminescence efficiency and enhance the stability of Acridine esters, the acridine 9-carboxylic acid benzyl ester derivatives reacted with methyl trifluoromethyl sulfonate to obtain target compounds. All the intermediates and target compounds were characterized by 1HNMR and IR. The total yield of each target coumpound was as follows: 1d (29%), 2d (40%), 3d (31%), 4d (22%).In the synthesis of substituted acridine esters, Ullman reaction was used to optimize the preparation of substituted diphenylamine to improve the yield of the reaction process. Ullman reactions were carried out according to the latest literature, using amino acids (L-proline) as ligand and cuprous iodide as catalyst under the condition of microwave heating for 4 hours to give substituted diphenylamine with the yield of 76%. Substituted diphenylamine then reacted with oxalyl chloride at the presence of AlCl3 to give substituted N-phenyl alizarine. Rearrangement reaction of N-phenyl alizarine was carried out in the solution of potassium hydroxide to prepare substituted acridine 9-carboxylic acid; however, the target compound was not obtained. It was concluded that the substituted group influenced the rearrangement reaction.After confirming the structure of target compounds, the fluorescence spectra and chemical luminescence kinetics of each compound were investigated. It was found that acridine ester with electron donating group on its leaving group had stronger CL intensity than that with electron withdrawing group. Meanwhile leaving group also affects the nature of acridine esters's fluorescence spectrum. After obtaining the chemical luminescence property of target compounds synthesized in this work, we tentatively discussed the determination of paraoxonase activity in blood. The biological activity of paraoxonase was characterized by comparing chemiluminescent intensity change before and after adding the serum into acridine ester. It was showed that after adding serum, acridine ester's chemiluminescence intensity was significantly reduced, and the chemiluminescence intensity of all four target compounds was reduced by 90%.