Design And Synthesis Of COX-2 Targeting Two-photon Fluorescent Probe And Research On The Imaging Performance Of Cancer Cell

Cyclooxygenase-2（COX-2）is an inducible enzyme,and not or lowly expressed in most normal tissues.However,COX-2 has been reported to be overexpressed in various cancers,and its expression level is closely correlated with tumor growth,invasion and metastasis.Therefore,COX-2 has been considered as a promising biomarker for cancer diagnosis.Finding an effective method to track the expression level of COX-2 and distinguish cancer cells at different stages will provide a new direction for cancer detection and staging.Fluorescence imaging technology based on small molecule fluorescence probe has many advantages,such as high sensitivity,rapid non-destructive analysis and real-time detection.Compared with the traditional microimaging technology,the two-photon imaging technology possesses the unique advantages of less light damage,low degree of optical bleaching,good imaging depth,high spatial resolution and signal-to-noise ratio.It is prominent in the visualization of tumor cells and has great potential in the application of clinical noninvasive detection of tumors.In this thesis,a series of COX-2 specific two-photon fluorescent probes based on1,3,5-triazine were designed and synthesized.Their fluorescent properties and live cell imaging were studied systematically.Results in more detail are as follows:（1）With 1,3,5-triazine as the basic structure,3,5-dimethypyrazole as the pull-electron substituent,the fluorescence group was constructed.Linear alkyl diamines with different lengths were selected to link the indomethacin recognition group to the triazine fluorescent group.Two-photon fluorescent probe molecules NTMPY-C2-IMC、NTMPY-C6-IMC and NTMPY-C8-IMC were synthesized.Their structures were characterized by ¹H NMR,¹³C NMR and MS.NTMPY-C2-IMC and NTMPY-C6-IMC were selected to study the imaging performance of living cells with different COX-2 expression levels.Among them,NTMPY-C2-IMC can successfully identify MDA-MB-231 breast cancer cells with high COX-2 expression levels in both single-photon and two-photon excitation modes.However,in MCF-7 breast cancer cells with low COX-2 expression and MDA-MB-231 cells with COX-2 knockout,the fluorescent signals were very weak.（2）Another series of probes NTBTA-C2-IMC、NTBTA-C6-IMC and NTBTA-C8-IMC were synthesized with benzotriazole as the pull-electron substituent to replace 3,5-dimethylpyrazole.The fluorescence principle and photophysical properties are also discussed.Compared with the 3,5-dimethypyrazole substituent probes,these probes possess enlargedπ-conjugated system and more rigid structure,stronger intramolecular charge transfer,redshift of the maximum emission wavelength and decreased fluorescence intensity.NTBTA-C6-IMC was selected to image prostate cancer cells and evaluate the expression level of COX-2,thereby to distinguish prostate cancer cells with different stages of differentiation.In both single-photon and two-photon excitation modes,NTBTA-C6-IMC can recognize COX-2 in PC3 cells and LNCa P cells and distinguish these two cells with different COX-2 expression levels.The recognition effect was found better than that of pyrazol-triazine-indomethacin analogues.