Design,Synthesis And Biological Activities Of Benzofurans And Pyrazolopyridines As Tubulin Polymerization Inhibitors

Microtubules are hollow tubular structures in which?-and?-tubulins are joined end to end.They maintain cell morphology,material transport and signal transmission by maintaining the dynamic balance of polymerization and depolymerization.In addition,they are components of centrosome and spindle.So microtubules become an important target in cancer treatment.Combretastatin A-4?CA-4?is a tubulin polymerization inhibitor that blinds to the colchicine site.However,its poor water solubility and bioavailability hindered its development as an anti-tumor drug.In order to find novel inhibitors of tubulin polymerization with good anti-tumor activities,two CA-4 derivatives bearing benzo[b]furan and pyrazolopyridine were designed and synthesized,and their biological activities were also evaluated.First,we designed a series of hybrids of 3,4,5-trimethoxybenzene and benzofuran with triazole as bridge linker.First,3,4,5-trimethoxybenzidine was prepared from3,4,5-trimethoxybenzaldehyde by Corey–Fuchs reaction.At the same time,o-hydroxybenzonitrilewasreactedwith ethylbromoacetatetoyield3-aminobenzofuran-2-carboxylic acid ethyl ester,which was then subjected to azide reactions,ester hydrolysis reactions,amidations or esterification reactions to provide3-N₃-benzofuran derivatives.Finally,Click reactions between 3-N₃-benzofuran derivatives with 3,4,5-trimethoxyphenylacetylene gave the target compounds 13a-i and 14a-i.The proliferation inhibitory activities of these 18 compounds on HCT-116,HeLa,HepG-2 and A549 cell lines were detected by MTT assay.The results showed that 14g was the best potent compound with the IC₅₀ values in range of 0.57?5.7?M for the four cancer cells.Bioactivity experiments such as flow cytometry and western blotting showed that compound 14g effectively inhibited tubulin polymerization,destroyed intracellular microtubule network,and arrested cells in the G2/M phase by regulating the expression of cyclic related proteins such as cyclin B1,Cdc25C and Cdc2.Further studies exhibited that 14g induced A549 cell apoptosis by regulating the expression of apoptosis-related proteins such as Bcl-2 and Bcl-xl and decreasing the level of mitochondrial membrane potential.Wound healing assay revealed that14g prevented the migration of A549 cells.Molecular simulation docking in the Schrodinger software found 14g had a good combination with Cys241 and Val181 at the colchicine site.These results indicated that 14g displays its antitumor activity by inhibiting tubulin polymerization.In addition,we have conducted a preliminary study on the synthesis methods and biologicalactivitiesofpyrazolopyridinederivatives.Specifically,3-bromo-2-chloro-6-methoxypyridineasrawmaterialreactedwith3,4,5-trimethoxybenzene under n-butyllithium to obtain hydroxy intermediate,which was then subjected to PCC oxidation and hydration.After that,cyclization reaction with N₂H₄·H₂O and the halogenated alkane substitution reactions were carried out to obtain the target compounds 8 and 9a-c.The MTT assay showed that 8 had the best activity,and the IC₅₀ value for HeLa cells was 0.26±0.12?M.The structure-activity relationship discussion and molecular simulation docking revealed that hydrogen bonding at the 1-NH on the pyrazole of 8 and Thr179 at the colchicine site was critical for activity.In summary,we have designed and synthesized two kinds of benzofuran and pyrazolopyridine analogues of CA-4.Bioactivity experiments showed that benzofuran derivative 14g was a potent inhibitor of tubulin polymerization;pyrazolopyridine derivative 8 had good selectivity for HeLa cells.The compounds with the pyrazolopyridine ring need further structural optimization.Our results provided sufficient evidences to find novel drugs as tubulin polymerization inhibitors.