Synthesis,Characterization And Bioactivity Evaluation Of Fluorescent Alkylphosphocholine Analogues

Alkylphosphocholine is a class of synthetic phospholipid drugs developed from2-lysophosphatidylcholine,which has various pharmacological activity such as anti-tumor,anti-parasitic,antibacterial and immune regulation etc.Different from most of the traditional anticancer chemotherapeutics,these compounds act on cell membranes rather than nucleic acids.They can interfere with lipid metabolism and some important signal pathways such as PI3K/AKT pathway and MAPK/ERK pathway etc.Small-molecule fluorescent probes are widely used to analyze biological samples,monitor environmental condition,diagnose disease and track drug metabolism thanks to their small size,strong specificity and high sensitivity.Among them,organic fluorescent dyes have the advantages of good chemical stability,large molar absorption coefficient and high fluorescence quantum yield.Thus,fluorescent probes for specific detection of analytes and efficient imaging in tumor cells and living lesions can be obtained based on rational design.Theranostics which combine biological activity and imaging capabilities have broad application prospects in the early diagnosis and targeted treatment of diseases.Tumor theranostics can not only provide diagnostic information,such as the location,distribution and size of the tumor,but also perform in-situ treatment and monitor the distribution of the drug.In addition,small-molecule theranostics are powerful tools to reveal the mechanism of activity and target of drugs.In this thesis,based on the antitumor activity of alkylphophoscholine and the fluorescent properties of organic fluorescent dyes,a series of fluorescent alkylphosphocholine analogues have been designed and synthesized.Alkylphosphocholine analogue I contains BODIPY as the fluorescent group with ether bond as linking group,while analogue II has coumarin as the fluorescent group with ester bond or amide bond as linking group.Then we investigated the fluorescence properties of the synthesized fluorescent alkylphosphocholine analogues,such as maximum absorption wavelength,maximum emission wavelength and fluorescence quantum yield.We also monitored the cell uptake of BODIPY analogue Ie and coumarin analogue II-2e via confocal microscopy.The results showed that these analogues with good fluorescent properties can quickly enter into tumor cells.The enhancement of the intracellular fluorescence intensity with the extension of co-incubation indicated that the uptake of compounds by tumor cells were in a time-dependent manner.In addition,we found that compound II-2e mainly accumulated in the nucleus when incubated for 24 hours,which suggested that the target of compound II-2e may be located in the nucleus.Then we evaluated the antiproliferation activity of the synthesized compounds in different cancer cells.The structure-activity relationship study showed that compound II-2e maintained comparable antitumor activity of miltefosine.It is worth noting that the antitumor activity was significantly affected by the length of hydrophobic alkyl chain and the linking group between alkylphosphocholine and the fluorescent gorup.Flow cytometry test showed that compound II-2e could induce apoptosis in a dose-dependent manner.Further study displayed that II-2e was able to down-regulate the expression of heat shock protein HSP27 and its client protein Akt,and up-regulate the expression of tumor suppressor p53.In conclusion,we have designed and synthesized a series of fluorescent alkylphosphocholine analogues,which not only exhibited antiproliferative activity against various cancer cells,but also could establish fluorescent imaging in tumor cell.Among them,analogue II is the first alkyl phosphocholine fluorescent probe having coumarin as the fluorescent group.These alkylphosphocholine analogues therefore have the potential to be developed as tumor-targeted diagnostic and therapeutic reagents in the future.