Design,synthesis And Structure-Activity Relationship Of Novel CA-4 Analogues As Tubulin Polymerization Inhibitors

Tubulin is the major component of the cell cytoskeleton and plays an important role in a variety of cell functions,mainly involving in mitosis,cell division and maintenance of cell morphology.Tubulin is considered to be the best anticancer target so far.Combretastatin A-4（CA-4）is a natural product originating from the South Africa shrubs（Combretum caffrum）and one of the simplest and peculiar tubulin inhibitor.Two novel potent tubulin inhibitors from it,CA-4P and AVE8062,have been marketed.However,their structures are still unstable.Isocombretastatin A-4（isoCA-4）is a bioisostere of CA-4,it has a simpler and more stable structure while maintains a strong activity similar to CA-4 in inhibiting tubulin.This paper designed and synthesized two classes,four series of novel tubulin polymerization inhibitors based on the structures of CA-4 and isoCA-4,and anti-tumor cell proliferation activity assay and activity mechanism of two series analogues were completed.The specific findings are as follows:（1）Employing the Mn（OAc）₃-mediated[3+2]cycloaddition reaction and 1,3-dipole[3+2]cycloaddition reaction as key synthetic steps,21 dihydrofuran series analogues of isoCA-4and 17 new dihydroisoxazole series analogues of isoCA-4 were synthesized at first time through six steps.（2）Introducing the Cu（OAc）₂-mediated[3+2]cycloaddition reaction as a key synthetic step,2 dihydrofuran series analogues of CA-4 were synthesized at first time.Based on above synthesis,the synthesis rout of cyclohexen series analogues of CA-4 were completed firstly and optimized.3 analogues were obtained.（3）Two series analogues of isoCA-4,38 compounds,were tested for anti-tumor cell proliferation using the SRB method.16 analogues had IC₅₀ values of less than 10μM for PC-3 cells,of which 6g had the highest cytotoxic activity with an IC₅₀ value of 0.47μM.For He La cells,6 analogues with IC₅₀ values of less than 10μM,5p had the strongest cytotoxic activity with an IC₅₀ value of 2.32μM.（4）The biological activity mechanism of two series analogues of isoCA-4 were completed with 6g as the representative:tubulin polymerization assay in vitro experiments showed that 6g was a dose-dependent tubulin polymerization inhibitor.Immunohistochemistry studies exhibited that this analogue could potent inhibite the assembly of tubulin in PC-3 cells.Cell cycle analysis displayed that both 6g and CA-4 could largely arrest PC-3 cells in G2/M phase.Molecular modeling experiments also showed that the two chiral configurations of 6g efficiently binded to tubulin in similar manners.These results consistently advised that 6g had the same properties as literatures reported tubulin polymerization inhibitors.In summary,using[3+2]cycloaddition reactions,we synthesized the dihydrofuran and dihydroisoxazole series analogues of isoCA-4,and the dihydrofurans and cyclohexene series analogues of CA-4.And,we found that both of two series analogues of isoCA-4 could inhibit the tubulin polymerization,which provided theoretical data for the novel anti-tumor drugs inhibiting tubulin.