Synthesis And Biological Activity Of Novel Pyrazolopyridine Tubulin Destabilizers

Microtubules are globular proteins composed of?-and?-tubulin heterodimers,and alternate cycles of polymerization and depolymerization that occur in a dynamic equilibrium state.Microtubules play a vital role in cellular processes,including the movement of organelles,intracellular transport,the formation and maintenance of cell shapes,and cell division.As its important role in cellular processes,drugs that target tubulin have become the focus of research in recent years.Drugs that interfere with microtubule polymerization are tubulin destabilizers,and drugs that inhibit microtubule depolymerization are tubulin stabilizers.Combretastatin A-4?CA-4?is a tubulin destabilizer that binds to the colchicine site,but it has been disabled in clinical use for its poor water solubility and adverse reactions.In order to find more potent antitumor drugs,we designed and synthesized a series of new pyrazolopyridin-type tubulin destabilizers based on the basic skeleton of CA-4,and evaluated their biological activity.First,we use 2-chloro-6-methoxypyridine as a raw material,perform an electrophilic substitution reaction using NBS,then undergoes a nucleophilic addition under the action of n-butyl lithium to eliminate the reaction to provide the intermediate 2-chloro-6-methoxynicotinaldehyde.The target compound 11a-e was then obtained by condensation reaction,electrophilic substitution reaction,and Buchwald-Hartwig coupling reaction.The target compounds 12a and 12c were produced by nucleophilic substitution reaction with different intermediates.Finally,the target compound 13g-p were prepared by deprotection under the condition of concentrated HCl-ethanol.We evaluated the cytotoxicity of 17 target compounds on cancer cells HCT-116,A549,HeLa,MCF-7 and HepG2 by MTT experiments,and the toxic effect of normal cells WI-38.Compounds 13g and 13k were found to have good inhibitory activity on cancer cells and low toxicity to normal cells.The IC₅₀ values of 13g and 13k on five cancer cells were in the range of 0.067?6.00?M and 0.012?5.79?M,respectively.We performed experiments on both compounds to inhibit tubulin polymerization,and found that 13g showed stronger anti-tubulin polymerization activity,however 13k showed weaker anti-tubulin polymerization.Molecular simulation docking with Schrodinger software revealed that 13g had a good binding to amino acid residues such as Cys 241 and Leu 255 at the colchicine site,and 13g inhibited tubulin polymerization by binding to the colchicine site.So we conducted mechanism studies of the two compounds separately.Compound 13g changed the morphology of tubulin in cells,and arrested G₂/M phase by inhibition of the expression of cyclins Cdc 2,Cdc25c and Cyclin B1 in MCF-7 cells,and affected colony formation.It also inhibited HUVEC cell migration and tubule formation,thereby acting as an antiangiogenic agent.With treatment of 13k,HeLa cells were blocked in the G₂/M phase by reducing the expression of cyclin Cdc 2,Cdc25c and Cyclin B1,and further,through the mitochondrial pathway,the anti-apoptotic proteins Bcl-xl,Bcl-2 and pro-apoptotic protein Bad were regulated to induce tumor cell apoptosis.In summary,a series of new pyrazolopyridine CA-4 analogues were designed and synthesized,we initially screened out two compounds 13g and 13k with better activities.Through their preliminary biological activity evaluation and mechanism research,it was proved that 13g and 13k are potential tubulin destabilizing agents.