Design,Synthesis And Antitumor Activity Of Antitubulin Agents Targeting The Colchicine Binding Site

Tubulin inhibitors acting on paclitaxel or vinblastine sites are widely used as antimitotic agents in clinical practice at present.However,their disadvantages,such as large molecular weight,complex chemical structure,poor water solubility,certain neurotoxicity and myelosuppression toxicity,limit their further application in clinical practice.Tubulin inhibitors targeting colchicine sites can largely overcome the above shortcomings and indirectly kill cancer cells by disrupting cancer angiogenesis.Because the targeted endothelial cells have better genetic stability than cancer cells,they are less likely to develop drug resistance.However,only tubulin inhibitors targeting the colchicine site have entered the clinical stage,and there are no FDAapproved drugs on the market.Therefore,there is an urgent need to develop novel chemically diverse,safe and effective tubulin inhibitors targeting colchicine sites with both anti-angiogenesis and anti-tumor properties.In this paper,natural products 2-methoxy estradiol and chalcone,with a broad range of biological activities,was selected as the basic skeleton.By means of chemical modification and drug design principle such as isostere and hybridization etc.,our research focuses on the incorporation of different new groups on natural products,aimed at increasing the novelty and diversity of natural product structure,as well as always endowed them with new or improved biological activities and laid the foundation for the research and development of new tubulin inhibitor.A total of 95 bioactive small molecules that belong to three series were designed and synthesized.All these compounds were evaluated for their half of tumor cell proliferation inhibition rate(IC50 value)after 72h action aginst six cell lines of MCF-7(human breast cancer cells),MGC-803(human gastric cancer cells),HepG2(human liver cancer cells),Hela(human cervical cancer cells),A549(non-small cell lung cancer)and U937(human leukemia cells)by using SRB or CCK-8 method.Among them,four active compounds with the best anti-tumor activity were selected for the in-depth study of the antitumor mechanism of action.Immunofluorescence assay,in vitro microtubule polymerization assay,radioactive ligand binding assay and molecular docking assay were used to detect the binding mode and anti-proliferation activity of the compounds to the tubulin sites.The effects of the compounds on tumor cell cycle,apoptosis,mitochondrial membrane potential and reactive oxygen species were determined by flow cytometry.Western blotting assay was used to detect the effects of compounds on the expression of Cyclin B1,P21,pCdc2 and apoptotic proteins(Caspase-3/-7/-9,PARP)in tumor cells.Tubule formation assay of human umbilical vein endothelial cells(HUVEC)in vitro was used to detect the antiangiogenic activity of the compounds.Wound healing and Transwell tests were performed to determine whether the compound could inhibit HUVEC and tumor cell migration and invasion.Green fluorescent protein(GFP)labeled transgenic zebrafish Tg(Flk:EGFP)embryos was used to observe the effects of compounds on angiogenesis in vivo;Tumor cells labeled with red protein(CMTPX)were injected into the yolk of Tg(Flk:EGFP)embryos of transgenic zebrfish to observe the inhibitory effects of the compounds on tumor formation and metastasis.(1)A series of 27 novel C3,C17 double modified and C17 modified 2ME2 derivatives were designed and synthesized.Among them,compounds 9i showed a broad spectrum and most potent proliferation inhibitory activity(IC50=0.063～0.139μM),9i displayed inhibitory activity against MCF-7cells with IC50 value of 0.069μM,compared with positive control CA-4 and 2ME2(IC50=0.01 μM and 3.901 μM respectively).Further mechanism studies showed that compound 9i could specifically bind to the colchicine site of tubulin,inhibit microtubule polymerization and interfere with the normal formation of mitogenic spindles(in vitro IC50=4.87μM for 9i and IC50=6.6μM for colchicine).MCF-7 cells were blocked in the G2/M phase by regulating the expression of Cyclin B1,Cdc2,P21 and other cell cycle-related proteins.PARP is cleaved through the activation of Caspase family protein cascade,and apoptosis is eventually induced in MCF-7 cells.In vivo and in vitro experiments in zebrafish showed that 9i could significantly inhibit the formation of proliferative tubeles,the migration and invasion of HUVECs,as well as the migration and invasion of MCF-7 cells and the growth and metastasis of tumor cells in zebrafish.These results suggest that 9i may have significant effects on tumor proliferation,metastasis and angiogenesis.In addition,9i has good physical and chemical properties and metabolic stability.(2)In series 2 the parent structure of chalcone was modified,methyl and fluorine atoms were introduced to limit the conformation of chalcone,and a total of 21 compounds was synthesized.After preliminary bioactivity evaluation,we found two with good bioactivity,compounds 3m and 6c,wherein the IC50 values of compound 6c against HeLa,U937 and MGC-803 were 0.025,0.025 and 0.057 μM respectively.By binding to colchicine sites on tubulin,3m and 6c interfered with tubulin assembly to different degrees and damaged the formation of microtubule network structure,resulting in cell damage(in vitro IC50=12.23 μM).3m and 6c selectively arrested cell cycle of MCF-7 and MGC-803 at G2/M phase respectively.Compared with 6c,compound 3m increased the expression of Cyclin B1 in a concentration-dependent manner and down-regulated the expression of P21 and p-Cdc2.Abnormal degradation of Cyclin B1 might be caused by the deceased activity of Cdc2 and the blocked spindle formation.In addition,3m induces apoptosis of MCF-7 cells by lowering mitochondrial membrane potential,inducing reactive oxygen species(ROS)production and activating Caspase cascade.The mechanism of apoptosis induced by 6c was the same as that induced by 3m.Finally,both in vitro and in vivo zebrafish experiments showed that 3m and 6c had significant inhibitory effects on angiogenesis,HUVEC and tumor cell migration and invasion.These results indicate that 3m and 6c have significant effects on tumor proliferation,metastasis and angiogenesis.(3)In series 3 the pharmacophore model of HDAC inhibitor was introduced on the estradiol skeleton,and 47 HDAC and tubulin dual target inhibitors was designed and synthesized,in which 19 compounds were screened for their inhibitory activity less than 5μM against 6 cancer cell lines tested.Compound S-18 exhibited the best and broad-spectrum anti-proliferation activity in vitro(ICso=0.485～2.538μM),showed moderate HDAC2 selectivity against HDAC1/2/6/10 with IC50 values of 0.06μM and 0.12μM,respectively.Studies have shown that S-18 can destroy the ordered fibrous tubular microtubule structure,arrest the growth of A549 cells in the G2/M phase by inducing mitochondrial membrane potential reduction and reactive oxygen species(ROS)production,and activating Caspase cascade reaction.In addition,zebrafish experiments in vitro and in vivo showed that S-18 had significant inhibitory effects on angiogenesis,migration and invasion of vascular endothelial cells and tumor cells.These results suggest that S-18 can not only inhibit tumor angiogenesis by targeting tumor blood vessels,but also inhibit the migration and invasion of A549 cells and exert anti-tumor activity.To sum up,this study preliminarily elucidates that compounds 9i,3m,6c,S-18 have two main mechanisms of killing tumor cells:on one hand,they inhibit tubulin polymerization by targeting on tubulin to block spindle formation,thus blocking cells in G2/M phase,unable to complete normal mitosis,and eventually apoptosis;On the other hand,by inhibiting the proliferation,migration and invasion of vascular endothelial cells and the process of vascular formation of endothelial cells,the supply of tumor nutrients and oxygen is cut off,thus interfering the proliferation,invasion and metastasis of tumor cells.In conclusion,these three compounds have dual effects of anti-angiogenesis and cytotoxicity on tumor cells.Using a molecule that has both cytotoxic and antiangiogenic activity,rather than using a combination of multiple drugs,can expect fewer mechanism-independent adverse reactions.These three compounds designed and synthesized by us have strong inhibitory activities against angiogenesis and have inhibitory effects on multiple stages and the whole process of angiogenesis.These characteristics enhance the practical significance of further research on them.