Design And Synthesis Of Fluorescent Probes Targeting Nucleoside Transporter 1 For In Vitro And In Vivo Tumor Diagnosis

Cancer is a growing public health problem in China,missed and delayed diagnosis often leads to lost opportunities to effective treatments,worse clinical outcomes and an increased risk of death.Take liver cancer as an example,survival rates for liver cancer largely depend on the extent of disease at diagnosis.More than 55%of patients with liver cancer in China are in Stage III and IV when diagnosis,while in the United States and Japan,there are are only 15%and 5%patients in the advanced stage,respectively.The average cure rate in the early clinical stage of cancer can reach more than 80%.If cancer can be strangled in the early clinical stage,it will not only have a high cure rate,but also significantly reduce the disease burden of cancer patients.Therefore,the early diagnosis and treatment of cancer is particularly important,and it has always been the focus of scientific research workers.A series of cancer diagnosis technologies and treatment methods have been developed one after another.Fluorescent molecular imaging is an emerging technology.It can be targeted to specific molecules or cells to be abnormal or normal biological processes and visual spatial described on time at the cellular or molecular level by a certain design,and is a non-invasive functional imaging mode.Nucleosides and bases are the basic units of genetic material in organisms and participate in numerous physiological and biochemical processes.Because it is a hydrophilic macromolecule that cannot simply diffuse through biological membranes,a special nucleoside transporter（NT）is needed to help its transport.At present,a large number of studies have shown that nucleoside transporters are highly expressed in many tumor cells and tissues,such as pancreatic cancer,ovarian cancer,lung cancer,breast cancer,and liver cancer.In this paper,we use uridine,which has affinity for nucleoside transporters,as the pharmacophore and two different fluorescent groups:7-amino-4-methylcoumarin（CUM）and 4-chloro-7-Nitrobenzofuroxan（NBD）was combined organically.Two new types of equilibrative nucleoside transporter 1（ENT1）inhibitors with fluorescent properties were designed and synthesized.The obtained molecular probe can theoretically be combined with ENT1 to not only inhibit the growth of tumors,but also can screen and detect cancer due to its fluorescence characteristics,and realize the integration of cancer diagnosis and treatment.All the important intermediates and final products in this subject have been structurally confirmed by ¹H NMR,¹³C NMR and high-resolution mass spectrometry.For the synthesized S1 and S2 fluorescent probes,we first carried out physical and chemical properties detection,and further carried out in vivo and in vitro activity studies.The results showed that:1)The transport absorption of probe S1 and S2 in MDA-MB-231 of human triple negative breast cancer cells with high expression of ENT1 was significantly higher than that in MCF10A of human normal breast epithelial cells with low expression of ENT1.The targeting and specificity of probes against ENT1 were confirmed at the cellular level,and probes S1 and S2 also showed good stability of biological matrix and fluorescence imaging effect.2)The cytotoxicity of probe compounds S1 and S2 on triple-negative breast cancer cells and normal breast epithelial cells were tested.The result showed that the IC50 of S1 and S2MDA-MB-231 cells are 14.51μM and 7.08μM,respectively,but they have no toxic side effects on normal breast epithelial cells.3)Probe S2 and the known nucleoside transporter-specific inhibitor NBMPR（Nitrobenzylmercaptopurine riboside）,showed similar inhibitory effects on gemcitabine’s anti-cancer activity in the same in vitro experiment.The experimental results indirectly verified the inhibition of probe S2 on NT transport channel.4)Due to the longer fluorescence wavelength of probe S2,we performed in vivo imaging experiments on the probe S2 in a nude mouse model of triple-negative breast cancer,and found that S2 has good tumor targeting and fluorescence accumulation effect in vivo.Through the comprehensive analysis of the above results,we believe that the synthesized probes S1 and S2 are fluorescent probes with targeting and specificity to ENT1,and show good biological activities in vivo and in vitro.They are expected to be developed into a kind of time imaging,quantification,high resolution,and a new integrated method of cancer diagnosis and treatment that target specific tumors.tumors.