Design,Synthesis And Biological Evaluation Of 3,5-Diarylpyridine Derivatives As Inhibitors Of Tubulin Polymerization

Objective:Combretastatin A-4（CA-4）,a natural compound isolated from the South African shrub Combretum caffrum,has strong anti-mitotic and anti-angiogenic effects.Due to its outstanding antitumor activity and simple structure,CA-4 has received much attention as a lead structure to investigate new antitumor drugs.The purpose of this study is to design new and effective anti-tumor compounds targeting tubulin sites.Methods:Synthesis of target compounds:We synthesized 3-bromo-5-iodopyridine using3,5-dibromo-pyridine as the starting material,followed by a reaction with 3,4,5-trimethoxyphenylboronic acid,Pd（PPh₃）₄,and K₂CO₃ in 1,4-dioxane/water to afford 3-bromo-5-（3,4,5-trimethoxyphenyl）pyridine,followed by reaction with different substituted phenylboronic acids,Pd（PPh₃）₄,and K₂CO₃ in 1,4-dioxane/water to generate 21 target compounds.Mass spectrometry,nuclear magnetic resonance hydrogen spectroscopy and nuclear magnetic resonance carbon spectroscopy were used to verify the accuracy of the structure of the compounds.In vitro evaluation:anti-tumor proliferation tests were performed on three tumor cell lines,namely,human gastric cancer cell line SGC-7901,human breast cancer cell line MCF-7,and human cervical cancer cell line He La,respectively,using the MTT method to screen the best active compounds,and then microtubule polymerization experiments were performed in vitro using an in vitro microtubule polymerization kit,and the immunofluorescence assay was used to visually reflect the effect of the target compounds on The inhibition of microtubule polymerization was visualized by immunofluorescence assay,and the ability of the target compounds to inhibit cell cycle and induce apoptosis was tested by flow cytometry,and finally the binding of the target compounds to microtubule proteins was verified by using the molecular docking software Discovery Studio 3.0.Results:21 diarylpyridine analogues of CA-4 were designed and synthesized.Mass spectrum,nuclear magnetic resonance hydrogen spectrum and carbon spectrum showed the accuracy of the structure of the compounds.MTT method showed that some of the compounds showed moderate to strong antiproliferative activity,and the IC₅₀ value of the most active compound 13t to three tumor cells was 0.19-0.33μM.The microtubule polymerization experiment in vitro showed that compound 13t could significantly inhibit microtubule polymerization.The immunofluorescence experiment showed that compound13t destroyed the stability of microtubule.The cell cycle experiment and cell apoptosis experiment showed that compound 13t could inhibit the mitosis of tumor cells at G2/M phase,thus inducing tumor cell apoptosis.The molecular docking experiment showed that,13t may exert its biological activity by binding to colchicine sites on tubulin.Conclusion:We synthesized a series of diarylpyridines by introducing pyridine fragments into the CA-4 framework to replace the double bonds between A and B rings,and evaluated their anti-proliferation activity and inhibition of tubulin polymerization.Some target compounds showed moderate to strong antiproliferative activity,with IC₅₀ values at the micromolar level.Among all synthetic compounds,the existence of indole group is the main factor affecting the biological activity.Through further biological experiments,including inhibition of microtubule polymerization in vitro,immunofluorescence,cell cycle analysis and apoptosis analysis,the mechanism of action was studied;The results showed that the effects of these compounds on microtubules and cells were similar to those of CA-4,which suggested that the binding target might be colchicine.Our work not only expanded the exploration of homeopathic double bond modification of tubulin inhibitor CA-4,but also provided a group of CA-4 analogues with moderate to strong antiproliferative activity.