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To overcome the acquired resistance and the significant side-effects of the reported drugs, four new ruthenium(II) complexes with alkynyl (Ru1, Ru2, Ru3, Ru4) were designed and synthesized. Ru1, Ru2, Ru3 and Ru4 were characterized by ESI-MS, 1H NMR, 1H–1H COSY NMR and elemental analysis. Compared with Ru2, Ru3, Ru4 and cisplatin, the anti-tumor experiments in vitro and vivo confirmed that Ru1 could most effectively inhibit tumor growth. In the experiments of safety evaluation in vivo, Ru1 could avoid any detectable side-effects compared with cisplatin. DNA binding experiments and cell cycle experiments showed that Ru1 exhibited the strongest DNA binding ability and interfered with the cell cycle by inserting DNA to inhibit tumor growth. The study demonstrated that Ru1 had the potential to be an exciting new drug candidate for glioblastoma treatment. [ABSTRACT FROM AUTHOR]