Investigating novel tubulin polymerization inhibitors: design, synthesis, LC/MS cellular permeability, in silico studies, and in vitro assessment

In this study, chalcones 5, and 6 and pyrazolines 7, and 8 were designed and synthesized as combrestatin A-4 (CA-4) analogues. The anticancer effect of the synthesized compounds 5-8 was assessed against a panel of cancer cell lines at 10 mu M. Results revealed that the 3-benzyloxy chalcone 5 exhibited the highest GI % (81.43%) against all the cancer cell lines, and recorded the highest anticancer activity against HuH-7 liver cancer cell line (IC50 = 5.59 mu M). The effect of 5-8 on the microtubules network was visualized via immunofluroescence detection. The 3-benzyloxy chalcone 5, and the 4-phenethyl chalcone 6 revealed microtubules destabilizing effects as CA-4, however, the pyrazolines 7, and 8 showed microtubules stabilizing effects similar to that of paclitaxel. Moreover, it caused cell cycle arrest at G2/M phases as well as early and late apoptosis and necrosis induction in HuH-7 cells as recorded by flow cytometry. The ADME properties of the synthesized compounds 5-8 were investigated and their in vitro cellular permeability was also determined. The 3-benzyloxy chalcone 5 exhibited acceptable drug likeness properties and passed the Lipinski, Ghose, Veber and Egan rules filters, and revealed a good cellular permeability (41%) according to the LC-MS/MS permeability assay. Finally, molecular docking and dynamic studies were performed to investigate the binding modes of 5-8. It was revealed that the 3-benzyloxy chalcone 5 exhibit a stable binding to the tubulin via multiple interactions with the key amino acids at the colchicine binding site.Graphical abstractChalcone 5 revealed mean GI50 values 1.59-25.10 mu M and a microtubules destabilizing agent.