The solid was purified by column chromatography using dichloromethaneCethyl acetate (9:1) as the eluent. mechanistic studies showed that complex 4 induced A549R cell apoptosis via inhibition of thioredoxin reductase (TrxR), elevated intracellular ROS levels, mitochondrial dysfunction and cell cycle arrest, making it an outstanding candidate for overcoming cisplatin resistance. Cisplatin is an effective antitumor agent that acts on DNA and is largely employed as the first metal-based therapeutic in the clinic against a wide spectrum of solid tumors1,2. However, drug resistance to cisplatin limits its applications and represents a continuing challenge3. Drug resistance mainly arises from different 7-Epi-10-oxo-docetaxel cellular adaptations, including reduced SIRT5 cellular drug concentration, increased rates of drug damage repair and drug deactivation4. Theoretically, there is a need for an effective anticancer drug that exhibits increased cellular uptake in tumor cells and is able to maintain sufficient drug concentrations to kill cancer cells5,6. Compared with platinum agents, some of the new transition metal complexes breakdown less easily, which is an important property for the delivery of drugs to locations where they are needed 7-Epi-10-oxo-docetaxel to fight cancers in the body7,8. Worldwide efforts to develop alternative organometallic drug designs that are distinct from cisplatin and have different targets have been directed toward overcoming this issue9,10,11,12,13,14. Due to their octahedral geometry, ruthenium complexes are widely utilized to construct highly effective anticancer agents with high selectivity and fewer (and less severe) side effects compared to platinum drugs15. Ruthenium complexes have been investigated for use as DNA topoisomerase inhibitors16, TrxR inhibitors17, antimicrobial agents18, molecular probes19, and anticancer agents20. Gratifyingly, three ruthenium-based chemotherapeutics are currently in clinical trials. Some ruthenium complexes have been proven to be mitochondria-targeting anticancer drug candidates21, which often induce redox reactions inside cancer cells resulting in an increase in reactive oxygen species (ROS)22. Some studies have observed reduced mitochondrial accumulation of cisplatin in cisplatin-resistant cells23; in contrast, ruthenium-based drugs have been found to have different subcellular distributions and no decrease in the amount of ruthenium was observed in cisplatin-resistant cells24. 7-Epi-10-oxo-docetaxel Moreover, complexes with mitochondria-targeting functionality have been created as efficient anticancer drugs that are immune to cisplatin resistance25,26. Therefore, mitochondria-targeting Ru(II) complexes are potential strong candidates for combating cisplatin-resistant tumor cells. Fluorine substituents have become a common and important drug component. They enhance the lipophilicity and biological activity of drug compounds27,28, and their introduction has been facilitated by the development of safe and selective fluorinating compounds29. Accordingly, the design of drug-like heterocyclic organic small molecules with trifluoromethyl groups that chelate ruthenium has generated promising anticancer drug candidates30. In addition, 2-phenylimidazo[4,5-f][1,10]phenanthroline (PIP) and its derivatives are widely used in medicinal chemistry. Ru(phen)2(PIP)2+ is a famous mitochondria-targeting Ru(II) complex31. As shown in scheme 1, a PIP ligand modified by the incorporation of a trifluoromethyl group into the benzene ring is a core component of our design. Often, 1,10-phenanthroline (phen) is directly used as a bis-chelating ligand to build Ru(II) polypyridyl complexes. The C-N coordination site of the 7,8-benzoquinoline (bq) ligand cyclometalates ruthenium, which can decrease the positive charge of the Ru metal center and increase cellular uptake32,33,34. The hydrogen (H) atom of the NH-functionality in PIP was substituted by a tert-butyl-benzene group to increase lipophilicity. The trifluoromethyl functionality was installed into the PIP ligand as a functional ligand to improve not only the bioavailabilities and membrane permeabilities of the complexes but also the interactions of the Ru complexes with biomolecules. Therefore, we synthesized four Ru(II) complexes with similar structures but distinctly different biological activities to verify that ruthenium cyclometalation in combination with trifluoromethyl and PIP ligands is a simple but competitive method to develop novel metallodrugs for the treatment of cancer. In this work, we studied the changes in biological activity and physicochemical properties resulting from structural modifications of the four Ru(II) complexes (Fig. 1). Complex 4 successfully exhibited potent cytotoxicity that was higher than cisplatin and the other three Ru(II) complexes against all of the screen cancer cell lines. We established 3D multicellular tumor spheroids based on A549R cells, and used this model to investigate the activity of complex 4 toward multidrug-resistant (A549R) tumor cells. The cellular uptake and localization of complex 4 in A549R cells were studied. Furthermore, we investigated the mechanism of complex 4-induced A549R cell apoptosis. The results show.