Design,Synthesis And Bioactivity Of Tubulin Inhibitors Targeting Colchicine Sites

Tubulin inhibitors play an important role in the treatment of malignant tumors.Most of the tubulin inhibitors used in the clinic targeted vinblastine and paclitaxel binding sites.However,these two types of tubulin inhibitors have more toxic and side effects,and most of them come from plants,which are difficult to extract.It is more difficult to synthetize because of their complex chemical structures.Tubulin inhibitors targeting colchicine sites are expected to overcome the above shortcomings due to their unique biological effects and simple chemical synthesis.No targeting colchicine binding site inhibitors are currently on the market.Therefore,it is of great significance to exploit microtubulin inhibitors with novel structures targeting colchicine sites.In this paper,colchicine binding site of microtubules was selected as the target,and chose a variety of structural fragments with anti-tumor activity.Three series of compounds were designed and synthesized based on drug molecular hybridization strategy.HepG2（human liver cancer cell line）,A549（human lung cancer cell line）,U937（human leukemia cell line）and Y79（human retinoblastoma cell line）were selected to evaluate their anti-proliferation activities in vitro by CCK8 assay and SRB assay.The compound 8p with good activity was screened to study the mechanism of antitumor action.Our research work is as follows:1.Based on the fragment of 3,4,5-trimethoxyphenl binding with the pockets of colchicine sites;maintaining the conformation of isoxazole、oxazoline、pyrazoline andα,β-unsaturated ketoxime and potential antitumor activity for aromatic heterocycles like benzothiophene、naphthalene、quinoline、indole et.al,we have designed structures.Three series of compounds were designed and synthesized by reasonable splices of the three pharmacophore subunits.Testing the antiproliferative activity evaluation in vitro for three series of compounds,the results showed that the seriesⅠand seriesⅡcompounds showed very weak antiproliferative activity,and However,the seriesⅢcompounds withα,βunsaturated oxime as the linker showed relatively good anti-proliferation activities on HepG2 and U937 cells.Additionally,in three series of compounds,the activity of indoles better than other heterocycles.2.Based on the good anti-proliferative activity of seriesⅢ,the compound 8p was screened out with the best activity(IC₅₀=65～251n（44）).The compound 8p was selected for preliminary anti-tumor mechanism study.Molecular docking experiments revealed the compound 8p embedded drain pocket of colchicine binding site and came into being dipole interaction with surrounding amino acid residues;Microtubule polymerization and immunofluorescence assay confirmed that 8p played an anti-tumor role by destroying the microtubule network structure and inhibiting microtubule polymerization;Cycle and apoptosis experiments showed that 8p could block cells in G2/M phase and ultimately induce apoptosis,thus exerting anti-tumor effect;Scratch and Transwell assay demonstrated that compound 8p could inhibit the migration and invasion of cancer cells in a dose-dependent manner.In summary,a total of 51 compounds were designed and synthesized in this paper,and the structures of these compounds were confirmed by NMR and HR-MS.At the same time,the compound 8p with strong anti-proliferation effect was screened out by in vitro anti-proliferation activity evaluation experiment for preliminary anti-tumor mechanism study.This is an important complement to tubulin inhibitors that target colchicine sites,and it is of great significance for the subsequent discovery of small-molecule inhibitors with better activity.