Design,Synthesis,and Biological Evaluation Of 9-Aryl-5H-Pyrido[4,3-b] Indole Derivatives

Microtubule is the main component of eukaryotic cytoskeleton,which plays an important role in maintaining cell morphology,mitosis,and signal transduction,and is an important target for antitumor drug research and development.Many tubulin inhibitors have significant clinical effects in tumor chemotherapy,but there are also some limitations.Therefore,the research and development of new and efficient tubulin inhibitors is of great significance for tumor treatment.In this paper,a series of novel tubulin inhibitors were designed by replacing the thienopyrrolizinone fragment of NSC 676693 with a more stable carbazole-concentrated heterocycle based on the bioelectronic equivalence principle and computer-aided drug design,and were prepared by a simple and efficient synthetic route and microwave-assisted means.The structures of the compounds were confirmed by mass spectrometry,nuclear magnetic resonance hydrogen spectroscopy,and nuclear magnetic resonance carbon spectroscopy.The in vitro antitumor activity of all target compounds was examined against three tumor cell lines,namely human cervical cancer He La cells,human gastric cancer SGC-7901 cells and human breast cancer MCF-7 cells,using MTT assay,and the conformational relationships were investigated in depth to screen out compounds with better anti-proliferative activity.The antitumor mechanism of action was then investigated by further biological experiments,including in vitro tubulin polymerization inhibition assay,immunofluorescence staining assay,apoptosis,and cell cycle analysis.Finally,molecular docking experiments were performed to simulate the binding mode between the target compounds and tubulin.Twenty-one novel 9-aryl-5H-pyrido[4,3-b]indole derivatives were designed and synthesized.The results of in vitro antitumor activity tests showed that most of the compounds showed moderate antiproliferative activity against tumor cells with IC₅₀ values at the micromolar level.Among them,compound 7k with 3,4,5-trimethoxyphenyl as A-ring and 5H-pyrido[4,3-b]indole as B-ring showed the strongest activity against He La cells with an IC₅₀ value of 8.7±1.3μM.Tubulin polymerization inhibition assay showed that compound 7k was a tubulin polymerization inhibitor and was able to inhibit tubulin polymerization at the protein level in vitro.Immunofluorescence staining assays further demonstrated that 7k could effectively inhibit tubulin polymerization and disrupt the microtubule backbone at the cellular level.Cell cycle and apoptosis assays showed that 7k could block the cell cycle in G2/M phase,interfere with the mitotic process of tumor cells,and eventually cause apoptosis.In addition,molecular docking studies showed that 7k exerts its biological activity by binding to the colchicine site of tubulin.In this study,a series of potential and novel tubulin polymerization inhibitors 9-aryl-5H-pyrido[4,3-b]indoles were synthesized by replacing the B ring of NSC 676693 with a fused heterocycle of carbazole.This study enriches the structural types of tubulin polymerization inhibitors and provides a new strategy for the design of antitumor drugs targeting tubulin.