Design,Synthesis And Bioimaging Study Of A Novel Pyrimidine Antitumor Fluorescent Agent

The treatment of cancer remains a major challenge in the field of medicine.Pyrimidine small molecules have excellent antitumor activity due to their unique structure.However,the disadvantages of large toxic side effects,poor optical properties,and low oral availability also limit the application of these drugs.Due to its non-invasive,high specificity,high sensitivity,and the ability to obtain higher resolution advantages at the cellular level,fluorescence imaging technology has become an important research tool in the biomedical field,enabling in situ real-time monitoring of target molecules.The purpose of this study is to improve the optical properties of pyrimidine compounds through structural modification and the introduction of fluorescent groups,and to attempt to use pharmaceutical polymer excipients to coat fluorescent agents,improve their swelling and bioavailability,and apply them to biological fluorescence imaging.In this paper,the strategy of introducing boron fluoride or curcumin into the pyrimidine mother nucleus is used to improve its fluorescence emission characteristics;Through structural modification,pharmacodynamic groups such as alkynyl,benzene,and methoxy groups are introduced into the side chain,and the carbon chain length of the side chain is changed;Improving the ability of compounds to penetrate biofilms by adjusting their lipid water partition coefficient;The compound is prepared in the form of a hydrochloride or sulfate,and the resulting fluorescent agent is coated with a pharmaceutical excipient Eudragit^?to improve the targeting and stability of the fluorescent agent and increase bioavailability.The obtained compounds are characterized by nuclear magnetic resonance,infrared spectroscopy,mass spectrometry,and other means to ensure structural accuracy.The optical properties of the fluorescent agent were measured using UV visible absorption spectroscopy and photoluminescence spectroscopy;The new type of anti tumor fluorescent agent was thoroughly analyzed through cytotoxicity experiments,transmission scanning electron microscopy,in vitro and in vivo imaging,and drug release in vitro and in vivo.The details are as follows:（1）Design,synthesis,and photophysical properties of Boron-Fluoride pyrimidine derivatives as antitumor fluorescent agents（Series 1）.Firstly,using ethyl 4-chloro-2-methylthiopyrimidine-5-carboxylate as the starting material（SC）,an intermediate with terminal alkynes was obtained through nucleophilic substitution.Secondly,methylthio side chains were oxidized with a strong oxidant（m-chloroperoxybenzoic acid）,ketone carbonyls were introduced onto the sulfur atoms,and the benzene ring was introduced through a condensation reaction.Finally,four different anti tumor fluorescent agents（PD-3a,PD-3b,PD-3c,and PD-3d）were obtained by introducing boron trifluoride ether into the fluorophore.In the testing of optical properties,the maximum emission wavelengths of the four fluorescent agents are between 566-569 nm.Through the MTT test,the IC₅₀ values of the four compounds achieved the expected results.In particular,the IC₅₀ value of compound PD-3d in He La cells was 19.24±5.88μM,slightly higher than 5-fluorouracil.Flow cytometry analysis showed that these four compounds have the ability to induce tumor cell apoptosis,and the proportion of compound PD-3d inducing early apoptosis of Hela cells was31.3%.At the same time,molecular docking results show that hydrogen bonds,π-πbonds,and T-πbonds are the main forces.This provides new ideas for tumor treatment and provides potential for rapid identification of intracellular drug concentrations.（2）Design and bioactivity study of Eudragit^?encapsulated p H-Sensitive enteric/gastric soluble fluorescent agents（Series 2）.Pyrimidine and curcumin drugs were successfully combined through a simple reaction step and encapsulated with different types of Eudragit^?（intestinal/gastric solubility and permeability）.In order to observe the optical properties of the complex in the gastrointestinal microenvironment,UV and fluorescence tests were conducted in different p H buffer solutions.The results show that the maximum emission wavelength of compound PD-5 is concentrated between 520-600 nm.With the increase of p H value,the maximum emission wavelength of the complex PD-5-L gradually shifts red,while the maximum emission wavelength of PD-5-E shifts blue.TEM results showed that compound PD-5 was uniformly wrapped by four polymer materials,forming spherical particles with uniform and moderate size,and could be absorbed by cells.Cell imaging results showed that the complex could enter the nucleus and partially distribute in the cytoplasm.In addition,by simulating the binding between compounds and proteins through molecular docking,the results show that hydrogen bonds are the main force.The purpose of this study is to provide new ideas for the development of novel anti-tumor composite fluorophores and novel gastrointestinal targeted drug delivery（3）Based on Eudragit^?acrylic resin coated curcumin pyrimidine multi salt strategy:for biological activity,in vivo and in vitro imaging,and drug distribution research（Series 3）.Use three different types of Eudragit^?（Gastric solubility,enteric solubility,and permeability）Coated with two salts under mild reaction conditions to obtain six nano polymeric complexes.In order to simulate the environment in the gastrointestinal tract,nanoparticles were characterized by UV vis spectroscopy and fluorescence spectroscopy in PBS buffer with different p H values.The TEM experimental results showed that the potential drug was uniformly dispersed in the drug carrier material,with a particle size between 100-500 nm.Confocal microscope imaging experiments were conducted in different channels of HCT-116 and LO2 cells.The results showed that the composite had good biocompatibility and imaging performance.The calculation of Pearson’s coefficient indicates that some complexes can also enter the nucleus.Cell proliferative toxicity test（CCK8）and TEM experiments of cell uptake localization showed that the complexes had positive anti-tumor activity and low cytotoxicity,and the distribution of the complexes in the nucleus and cytoplasm was observed.In vivo imaging experiments,strong fluorescence intensities of compounds were detected in subcutaneous tumor tissue of mice.The release of six compounds has the potential for slow and controlled release and can be well targeted to the stomach of mice.Therefore,curcumin derivatives in salt form are ideal potential antitumor drugs,Eudragit^?is an excellent drug carrier.