Design,Synthesis And Bioevaluation Of Imidazo[1,2-a]Pyrazine Derivatives As Tubulin Polymerization Inhibitors With Potent Anticancer Activities

As a major public health problem worldwide,cancer has become one of the major diseases causing human death,which not only seriously damages human health,but also brings heavy mental pressure and economic burden to patients,society and countries.As an important component of the eukaryotic cytoskeleton,microtubules are heterodimers formed by the assembly of α-tubulin and β-tubulin subunits,and their heads and tails are connected to form long strips of fibrils,and 13 fibrils are arranged longitudinally to form a hollow cylindrical structure.The polymerization kinetics of microtubules is closely related to the formation of cellular mitosis spindles,the maintenance of cell morphology and motility of cells.In addition,microtubules are also involved in the construction of the endothelial cytoskeleton,and the growth of cells and various physiological processes are inseparable from the nutrient supply of peripheral blood vessels.Therefore,the factors that affect the dynamic balance of tubulin polymerization and depolymerization are also bound to affect the blood flow supply of cells,thereby affecting cell growth.Given their vital role in cell life activities,microtubules have become one of the key targets of antitumor drug research.At present,it is known that tubulin has six drug-binding sites,of which microtubule-targeted drugs targeting paclitaxel and vinblastine sites are the most widely used anti-tumor drugs in clinic,but due to its poor water solubility,bone marrow suppression,neurotoxicity,multi-drug resistance,easy to produce hypersensitivity reactions and other shortcomings,the further development of these tubulin inhibitors is limited in the clinic.Therefore,the development of novel tubulin inhibitors with better pharmacokinetic properties is imminent.Compared with other binding sites,the colchicine binding site between the tubulin a and the β subunit has the advantages of small size,simple inhibitor structure,wide variety and high anti-tumor activity,so it has attracted great attention from medicinal chemists.Tubulin polymerization inhibitors that target this site are called tubulin colchicine binding site inhibitors(CBSIs).CBSIs have low toxicity,and they are not substrates of multidrug-resistant efflux pumps,which can overcome the problem of multidrug resistance caused by p-gp,and are still effective against MDR cells.They are water-soluble and do not need chemical solubilization,therefore,CBSIs can avoid the generation of allergic reactions.In addition,CBSIs can also be used as vascular disruptors to destroy blood vessels around tumor cells,and play an anti-tumor cell proliferation,invasion and metastasis effect.In this paper,we designed and synthesized a series of novel imidazole[1,2-a]pyrazine derivatives as potential tubulin inhibitors binding to colchicine-site using bioelectron isosteric principle,ring fusion strategy and structural optimization.These compounds displayed efficient anti-proliferative activities(micromolar to nanomolar)against a panel of cancer cell lines(including HepG-2,HCT-116,A549 and MDA-MB-231 cells).Among them,compound TB-25 exhibited the strongest inhibitory effects against HCT-116 cells with an IC50 of 23 nM.Mechanism studies revealed that TB-25 could inhibit tubulin polymerization in vitro to a certain extent,and destroy the dynamic equilibrium of microtubules in HCT-116 cells.In addition,TB-25 dose-dependently induced G2/M phase cell cycle arrest and apoptosis in HCT-116 cells.Furthermore,TB-25 suppressed HCT-116 cell migration in a concentration-dependent manner.Finally,molecular docking showed that TB-25 occupied colchicine binding site in tubulin in a similar mode of action to CA-4.Collectively,these results suggest that TB-25 represents a promising tubulin polymerization inhibitor deserving further investigation.