Systematic Investigation of Coordination Chemistry in Iridium(III) and Ruthenium(II) Complexes Derived from Pyridyl-Amine Ligands and Their Anticancer Evaluation.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Systematic Investigation of Coordination Chemistry in Iridium(III)\n and Ruthenium(II) Complexes Derived from Pyridyl\u2212Amine Ligands\n and Their Anticancer Evaluation\n Lihua Guo,* Zhihao Yang, Heqian Dong, Kangning Lai, Hanxiu Fu, Yuwen Gong, Susu Li, Mingbo Yue,\n and Zhe Liu*\n Cite This: Inorg. Chem. 2025, 64, 10379\u221210401 Read Online\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:27:11 (UTC).\n\n\n\n\n ABSTRACT: A systematic investigation of the coordination chemistry\n of iridium(III) and ruthenium(II) complexes synthesized from pyridyl\u2212\n amine ligands was performed, focusing on how ligand steric hindrance\n and metal centers affect oxidation behavior, coordination modes, and\n biological activities. The study revealed that steric hindrance at the\n ligand\u2019s bridge carbon strongly influenced both oxidation behavior and\n coordination modes. Smaller substituents (e.g., H and Me) facilitated\n oxidation to form pyridyl\u2212imine species under adventitious oxygen,\n whereas bulky substituents (e.g., i-Bu and mesityl) suppressed oxidation,\n yielding stable pyridyl\u2212amine or 16-electron pyridyl\u2212amido complexes.\n Moreover, iridium(III) complexes were more prone to oxidation than\n the corresponding ruthenium(II) complexes under similar conditions.\n The aqueous stability of the newly synthesized complexes was\n confirmed. Cytotoxicity assays demonstrated that most of the complexes exhibited notable anticancer potency against A549,\n HeLa and cisplatin-resistant A549/DDP cancer cells. Mechanistic studies suggested a redox-driven pathway involving the catalytic\n oxidation of NADH to NAD+, the elevation of ROS levels and depolarization of the mitochondrial membrane. Notably, pyridyl\u2212\n amine complexes induced apoptosis, while 16-electron pyridyl\u2212amido complexes did not, though both caused S phase cell cycle\n arrest. Additionally these complexes can inhibit A549 cell migration, suggesting their potential to reduce cancer metastasis.\n\n\n 1. INTRODUCTION ruthenium(II) complexes selectively targeted lysosomes and\n Platinum-based anticancer drugs have been highly effective in demonstrated high selectivity for A549 cancer cells over BEAS-\n treating various tumors.1\u22126 Nonetheless, issues such as side 2B normal cells.42,43 Additionally, iridium(III) and ruthenium-\n effects and drug resistance have driven the search for (II) complexes featuring \u03b1-diimine N,N-chelating ligands were\n alternative metal-based therapies. Among these, half-sandwich synthesized (Scheme 1, IV and V). These complexes overcome\n organometallic complexes of platinum group metals (e.g., cisplatin resistance by initiating apoptosis through ROS-related\n iridium, ruthenium, osmium and rhodium) with piano-stool mechanisms.44\u221246\n geometries have gained significant attention as potential The aforementioned N,N-chelating complexes can be\n anticancer agents.7\u221213 These complexes have shown significant categorized into amine (sp3-N/sp3-N)-metal (Scheme 1, I)\n anticancer potential, operating through mechanisms of action and imine (sp2-N/sp2-N)-metal (Scheme 1, II\u2212V) coordina-\n (MoAs) that differ from those of platinum-based drugs. tion modes. With this basis, we further investigated hybrid sp3-\n Research has primarily centered on exploring and investigating N/sp2-N chelating complexes. It was observed that sp2-N/sp3-\n the 18-electron neutral and cationic complexes containing N pyridyl\u2212amine complexes (Scheme 1, VI) could undergo\n diverse bidentate XY chelating ligands.13,14 Among these, N,N- oxidation to form pyridyl\u2212imine iridium(III) complexes\n donor ligands have been predominantly utilized.15\u221239 A (Scheme 1, VII) in the presence of trace molecular oxygen.47\n notable example is RM175, an arene\u2212ruthenium(II) complex\n with an ethylenediamine (en) ligand (Scheme 1, I), which has\n demonstrated considerable cytotoxicity in vitro and in vivo.40 Received: December 31, 2024\n Our group has observed that cationic half-sandwich pyridyl\u2212 Revised: March 18, 2025\n imine iridium(III) and ruthenium(II) complexes generate Accepted: May 13, 2025\n substantial reactive oxygen species (ROS), disrupt mitochon- Published: May 17, 2025\n drial membranes, and exhibit significant anticancer effects\n against A549 cancer cells (Scheme 1, II and III).41\u221243 Certain\n\n \u00a9 2025 American Chemical Society https://doi.org/10.1021/acs.inorgchem.4c05599\n 10379 Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 1. Reported Organometallic N,N-Chelating Half-Sandwich Platinum-Group Metal Complexes, Along with the Hybrid\nCoordination Modes Investigated in This Work\n\n\n\n\nScheme 2. Synthesis of Ligands L1\u2212L6\n\n\n\n\nFurthermore, by varying reaction conditions (e.g., nitrogen vs We found that increasing the steric bulk of substituents on the\nadventitious oxygen atmospheres) and ligand structures, we bridge carbon linking the pyridyl and amine moieties\nsuccessfully isolated three distinct types of complexes: pyridyl\u2212 effectively suppressed oxidation. Notably, introducing a highly\namine iridium(III) complexes, their oxidized pyridyl\u2212imine\ncounterparts, and pyridyl\u2212amido complexes. This preliminary sterically demanding group resulted in the direct formation of a\nobservation of oxygen- and ligand-dependent structural stable pyridyl\u2212amido 16-electron complex without oxidation\ndiversity prompted us to conduct a systematic investigation (Scheme 1). Moreover, the coordination chemistry of these\nof the coordination chemistry of iridium(III) complexes based complexes was also significantly influenced by the metal center\non pyridyl\u2212amine ligands. Additionally, we are intrigued by (iridium(III) vs ruthenium(II)). In particular, the cytotoxicity\nhow a different metal center, such as ruthenium(II), might\ninfluence coordination behavior. Thus, our study was also and potential MoAs of these pyridyl\u2212amine and 16-electron\nextended to systematically investigate the coordination pyridyl\u2212amido iridium(III) and ruthenium(II) complexes\nchemistry of the corresponding ruthenium(II) complexes. were also systematically examined in vitro.\n 10380 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 3. Coordination Structures and Oxidation Tendencies of iridium(III) Complexes with Varying Steric Ligands\nSynthesized in This System\n\n\n\n\n2. RESULTS AND DISCUSSION pyridyl\u2212imine iridium(III) complexes with 64.0\u221271.5%\n yields.47 These results indicate that both Ir1amine and Ir2amine\n 2.1. Synthesis of Ligands. Pyridine\u2212amine ligands L1\u2212\n undergo oxidation within a short reaction time, highlighting\nL6 were obtained in moderate yields through the reduction of\n their susceptibility to oxidation (oxidation-prone, Scheme 3).\npyridine\u2212imine compounds using different reduction agents,\n Most interestingly, replacing H or CH3 with a bulky i-Bu group\nsuch as LiAlH4, AlMe3, AlEt3, Al(i-Bu)3 and 2,4,6-Me3Ph\n on the bridge carbon inhibited oxidation, yielding a pyridyl\u2212\n(Mesityl)-MgBr (Scheme 2). Thus, the steric bulk of bridge\n amido 16-electron complex (Scheme 3, Ir4amido) with a clean\ncarbon substituents between the pyridyl and amine groups can isolated yield.47 The formation of Ir4amido likely occurred in\nbe adjusted using various reduction agents. Ligands L1, L2, L4 situ via deprotonation of the pyridyl\u2212amine complex and was\nand L5 were previously known.47,48 The identity and purity of stabilized by the steric hindrance provided by the bulky i-Bu\nnew ligands L3 and L6 were also verified by 1H NMR, 13C group. These initial results suggest that the steric hindrance\nNMR (Figures S1\u2212S4), and mass spectrometry (Figures S26 from substituents on the bridge carbon greatly impacts the\nand S27). In the 1H NMR spectra, the characteristic peaks of coordination mode of iridium(III) complexes derived from\nthese ligands appeared at 4.08\u22124.34 ppm, corresponding to the pyridyl\u2212amine ligands. This observation motivated us to\nN\u2212H proton. systematically investigate these effects. To illustrate the impact\n 2.2. Synthesis of Iridium(III) and Ruthenium(II) of steric hindrance more comprehensively, we depicted the\nComplexes. Our previous study demonstrated that the structures of Ir1amine, Ir2amine and Ir4amido from our previous\nreactions of pyridine\u2212amine ligands L1 or L2 with [(\u03b75- work,47 alongside the newly synthesized complexes Ir3amine,\nCp*)IrCl2]2 (D1) (Cp* = C5(CH3)5) in methanol for 2 h, Ir5amido, and Ir6amido in this study (Scheme 3).\nunder trace amounts of adventitious oxygen (with no nitrogen Under the same reaction conditions (methanol, 24 h,\ndegassing, and the oxygen present was simply that mixed into adventitious oxygen), the ethyl-substituted ligand (moderate\nthe system from the ambient air), produced a mixture of steric hindrance) afforded the pyridyl\u2212amine complex Ir3amine\npyridyl\u2212amine complexes (Scheme 3, Ir1amine or Ir2amine) and (Scheme 4a and Figures S5, S6 and S28) in a 62.8% isolated\ntheir corresponding oxidation products, namely pyridyl\u2212imine yield with no immediate oxidation. However, prolonged\ncomplexes (Scheme 1, VII).47 The acidic N\u2212H proton of storage (14 days in solution) resulted in gradual oxidation to\npyridyl\u2212amine ligands was considered to facilitate the form Ir3imine (Scheme 4a), indicating that the ethyl group\ngeneration of oxidized pyridyl\u2212imine complexes.47,49\u221251 partially retards, but does not completely prevent oxidation.\nExtending the reaction time from 2 to 24 h yielded oxidized Building on our earlier observation that introducing a sterically\n 10381 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 4. Synthesis of Pyridyl\u2212Amine Complex Ir3amine, Pyridyl\u2212Imine Complex Ir3imine and Pyridyl\u2212Amido Complexes\nIr5amido, Ir6amido and Ir7amido\n\n\n\n\nbulky i-Bu group prevented oxidation and led to a stable 16- crucial influence of steric effects on stabilizing complexes and\nelectron complex (Ir4amido),47 we further employed a mesityl- modulating their oxidation behavior.\nsubstituted ligand with even greater steric hindrance. This We were also interested in systematically investigating the\napproach resulted in the exclusive formation of a 16-electron coordination modes of pyridyl\u2212amine-based complexes when\npyridyl\u2212amido complex (Ir5amido) in 58.5% yield, with no the metal center was switched to ruthenium(II) (Scheme 5).\noxidation observed (Scheme 4b). It is important to note that The reaction of ligand L1 with the dimer [(\u03b76-p-cymene)-\nthe presence of a \u03b2-hydrogen (i.e., the hydrogen attached to RuCl2]2 (D2) in methanol for 2 h produced the pyridyl\u2212amine\nthe bridge carbon adjacent to the metal center) in ligands L1\u2212 complex Ru1amine in a 61.7% yield (Scheme 6a, Figures S14,\nL5 is a prerequisite for oxidation to occur. To address this S15 and S33). Additionally, extending the reaction time from 2\nissue, we synthesized ligand L6 (Scheme 2), which features a to 24 h yielded a mixture of pyridyl\u2212amine and the\nquaternary carbon on the bridge substituted with Me and Et corresponding oxidized pyridyl\u2212imine complex (Scheme 6b),\ngroups, thereby lacking any \u03b2-hydrogen. Reaction of L6 with with a molar ratio of 3:1 as determined using 1H NMR\n spectroscopy (Figure S39). When the reaction time was further\nD1 yielded a stable 16-electron pyridyl\u2212amido complex\n extended to 96 h, only the oxidized pyridyl\u2212imine complex\nIr6amido, rather than the pyridyl\u2212amine complex (Scheme\n was obtained (Scheme 6c and Figure S39). These results\n4c). These observations demonstrate that the simultaneous\n indicates that prolonged reaction time promotes oxidation of\nintroduction of bulky substituents on the bridge carbon, such Ru1amine under conditions of adventitious oxygen.\nas Me and Et in Ir6amido significantly enhances steric When using the methyl-substituted ligand L2, reactions\nhindrance, which effectively prevents oxidation and results in conducted for 2 or 24 h produced only pyridyl\u2212amine\nthe generation of stable 16-electron pyridyl\u2212amido complexes. complexes (yielding Ru2amine at 61.4%, Scheme 7a and Figure\nTo confirm the 16-electron structure of Ir6amido, we used a S40). However, extending the reaction time to 96 h resulted in\nsimilar ligand L7, which also features a quaternary carbon on a 1:1 mixture of pyridyl\u2212amine and pyridyl\u2212imine complexes\nthe bridge with two methyl substituents. Reacting L7 with D1 (Scheme 7b and Figure S40).\nyielded Ir7amido (Scheme 4d), and single crystals were Compared to the unsubstituted ligand L1, the methyl\nsuccessfully obtained. The 16-electron structure of Ir7amido substituent increases steric hindrance, making the complex\nwas confirmed through single-crystal analysis (see Section 2.4: more resistant to oxidation. Moreover, for both L1 and L2,\nX-ray Crystallography). These observations emphasized the using dichloromethane instead of methanol\ufffdeven up to 96\n 10382 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 5. Coordination Structures and Oxidation Tendencies of ruthenium(II) Complexes with Varying Steric Ligands\nSynthesized in This System\n\n\n\n\nScheme 6. Results of the Reaction between D2 and L1 in the Presence of Small Amounts of Adventitious Molecular Oxygen\nunder the Reaction Time of (a) 2, (b) 24 or (c) 96 h in CH3OH\n\n\n\n\n 10383 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 7. Results of the Reaction between D2 and L2 in the Presence of Small Amounts of Adventitious Molecular Oxygen\nunder the Reaction Time of (a) 2, 24 or (b) 96 h in CH3OH and Synthesis of Pyridyl\u2212Amine Complexes Ru1amine and Ru2amine\nin the Presence of Small Amounts of Adventitious Molecular Oxygen under the Reaction Time of 2, 24, or 96 h in the Non-\nPolar Solvent CH2Cl2 (c)\n\n\n\n\nh\ufffdresults exclusively in pyridyl\u2212amine complexes (Scheme 2.3. Characterization and Reactivity of Iridium(III)\n7c, Figures S41 and S42). This is likely because the nonpolar and Ruthenium(II) Complexes. Comprehensive spectro-\ndichloromethane inhibits deprotonation, reducing the N\u2212H scopic and analytical methods including 1H and 13C NMR\nacidity and thereby preventing oxidation. spectroscopy (Figures S5\u2212S25), mass spectrometry (Figures\n Using the bulky ethyl-substituted ligand L3, no oxidation S28\u2212S38), and elemental analysis have been employed to\nwas observed in either methanol or dichloromethane, even characterize all of these new complexes. For example, 1H NMR\nafter 96 h (Scheme 8a, Figures S43 and S44). The pyridyl\u2212 spectroscopy of Ir3amine revealed two distinct signals\namine complex Ru3amine was isolated in a 61.1% yield after 24 corresponding to the N\u2212H proton at 7.12 ppm and the\nh in methanol. Similarly, increasing the steric hindrance further CH\u2212N proton at 5.36 ppm (Figure S5), confirming the amine-\nto the i-Bu group (L4) also prevented oxidation. However, metal coordination via an sp3-N donor. The 1H NMR spectra\nunlike the i-Bu-substituted iridium(III) complex Ir4amido of representative ruthenium complex Ru1amine also displayed\n(which forms a 16-electron structure), the product Ru4amine two distinct peaks: one for the N\u2212H proton at 6.80 ppm and\nremained a highly oxidation-resistant pyridyl\u2212amine complex another for the CH2\u2212N protons at 4.40 and 4.59 ppm (Figure\n(Scheme 8b), showing no oxidation even after 14 days in S14). In the case of Ru1amine, the CH2 group signals appeared\nmethanol, and was isolated in a 68.6% yield. When the mesityl- separately, attributed to the two diastereomeric protons,\nsubstituted ligand L5 (with exceptionally high steric consistent with previously reported pyridyl\u2212amine iridium(II)\nhindrance) was reacted with D2, a 16-electron pyridyl\u2212 complexes.47 The solution stability of Ir3amine and Ru3amine\namido complex Ru5amido was formed (Scheme 8c). Likewise, was verified using 1H NMR spectroscopy, with no changes\nusing ligand L6\ufffdwhich features a quaternary bridge carbon observed over a period of 96 h (Figures S46 and S47),\n(substituted with Me and Et) and lacks a \u03b2-hydrogen\ufffdyielded demonstrating its good stability.\na 16-electron pyridyl\u2212amido complex Ru6amido (Scheme 8d), 1\n H and 13C NMR spectra for the 16-electron pyridyl\u2212amido\nconsistent with the behavior observed in the iridium(III) complexes (Ir5amido, Ir6amido, Ru5amido and Ru6amido) displayed\nsystem. Additionally, we conducted a comparison experiment high stability over extended periods (e.g., 24 h in CDCl3).\nin which molecular oxygen was deliberately introduced into the Previous studies revealed that 16-electron half-sandwich\nreaction system by bubbling oxygen through the reaction iridium(III) complexes interact with various two-electron\nmixture to increase the oxygen concentration and fully saturate donors to yield stable 18-electron complexes.52,53 However,\nthe system, while maintaining the same reaction conditions. adding PPh3, CH3CN, or CO to an NMR solution of Ir5amido\nSpecifically, for Ru3amine and Ru4amine, despite the introduction and Ru5amido in CDCl3 (for CH3CN and CO) or DMSO-d6\nof a higher oxygen concentration, no oxidation occurred, and (for PPh3) showed no changes in 1H NMR peaks after 24 h\nthe reaction products remained exclusively pyridyl\u2212amine (Figures S48\u2212S53). This indicates that Ir5amido and Ru5amido\ncomplexes (Figure S45), similar to those obtained under trace remained unreactive toward these two-electron donors under\noxygen conditions. This indicates that increasing the oxygen the tested conditions. These results further confirm the\nconcentration did not alter the reaction outcome. exceptional stability of 16-electron pyridyl\u2212amido iridium(III)\n 10384 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 8. Synthesis of Pyridyl\u2212Amine Complex Ru3amine, Ru4amine, Pyridyl\u2212Amido Complexes Ru5amido, Ru6amido. Synthesis\nof Pyridyl\u2212Amine Complex Ru3amine in the Presence of Small Amounts of Adventitious Molecular Oxygen under the Reaction\nTime of 2, 24, or 96 h in CH3OH or CH2Cl2\n\n\n\n\nand ruthenium(II) complexes in this system. Additionally, a Cl\u2212 ion was clearly identified at the metal center (Figure 1a\nnoticeable color difference was observed: while the 18-electron and Table S1). In contrast, crystals of the oxidized product\npyridyl\u2212amine complexes (e.g., Ir3amine, Ru3amine) typically Ir3imine, obtained after 14 days in solution, displayed a planar\nexhibit orange or yellow hues, the 16-electron pyridyl\u2212amido five-membered metallacycle. The planar structure arises from\ncomplexes display deeper red (e.g., Ir5amido and Ir6amido) or the coplanarity of the newly formed C\ufffdN double bond (C6\u2212\ndeep purple (e.g., Ru5amido and Ru6amido) colors (Figure S54), N2 bond length of 1.266(10) \u00c5) and the adjacent pyridyl\nfurther corroborating the differences in their coordination moiety (Figure 1b and Table S2). Additionally, Ir5amido\nmodes. displayed a five-coordinated piano-stool structure, lacking a\n 2.4. X-ray Crystallography. A dedicated X-ray crystallo- Cl\u2212 leaving group at the metal center (16-electron). A\ngraphic study was carried out to unambiguously determine the nonplanar five-membered metallacycle was identified, with a\ncoordination geometries and structural parameters of selected C6\u2212N2 bond length of 1.465(12) \u00c5 (Figure 1c and Table S3),\ncomplexes (Figure 1 and Table S1\u2212S8). For the iridium(III) aligning with a single C\u2212N bond. Furthermore, although\nseries, single-crystal X-ray diffraction analysis of Ir3amine multiple attempts to crystallize the 16-electron Ir6amido were\nconfirmed the formation of a nonplanar five-membered unsuccessful, the use of a similar bridge carbon structure (also\nmetallacycle involving the Ir center and the ligand\u2019s sp3-N a quaternary carbon) with two methyl substituents on the\ndonor atoms. The observed C6\u2212N2 bond length of 1.490(10) ligand (L7), enabled the isolation and crystallographic\n\u00c5 is characteristic of a single C\u2212N bond, and a coordinated determination of 16-electron Ir7amido (Scheme 4d, Figure 1d\n 10385 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. X-ray crystal structures of complexes Ir3amine (a), Ir3imine (b), Ir5amido (c), Ir7amido (d), Ru1amine (e), Ru2amine (f), Ru4amine (g) and\nRu5amido (h) with the thermal ellipsoids drawn at the 50% probability level. The hydrogen atoms in all of these complexes and PF6\u2212 in Ir3amine,\nIr3imine, Ir5amido, Ir7amido, Ru1amine, Ru2amine, Ru4amine and Ru5amido have been omitted for clarity.\n\nand Table S4), also confirming the formation of a 16-electron standard conditions. However, the introduction of steric\npyridyl\u2212amido structure of analogue Ir6amido. For the hindrance with substituents like i-Bu or mesityl leads to the\nruthenium(II) complexes, X-ray data obtained for Ru1amine, generation of stable pyridyl\u2212amido 16-electron complexes\nRu2amine and Ru4amine revealed a nonplanar five-membered (e.g., Ir4amido, Ir5amido), where oxidation is effectively sup-\nmetallacycle with a C6\u2212N2 bond length of 1.500(3), 1512(3) pressed. In contrast, ruthenium(II) complexes exhibit higher\nand 1.469(5) \u00c5 (Figure 1e\u2212g and Tables S5\u2212S7), respectively, intrinsic resistance to oxidation. Even with smaller substituents\nconsistent with a single C\u2212N bond, supporting the amine- like H or Me, oxidation proceeds more slowly and requires\nmetal coordination mode. However, Ru5amido displayed a 16- extended reaction times or specific conditions to form oxidized\nelectron five-coordinated piano-stool structure, without a Cl\u2212 products. For bulkier substituents such as Et, i-Bu, or mesityl,\nleaving group at the metal center. Similarly, a nonplanar five- oxidation is entirely suppressed, resulting in stable pyridyl\u2212\nmembered metallacycle was noted in Ru5amido (Figure 1h and amine (Ru4amine), or pyridyl\u2212amido complexes (Ru5amido).\nTable S8), with a C6\u2212N2 bond length of 1.523(13) \u00c5, The observed differences can be attributed to the inherent\nindicating a single C\u2212N bond. electronic properties of the two metals. Iridium, as a heavier\n 2.5. Differences between Coordination Chemistry of group 9 metal, exhibits stronger \u03c0-backbonding capabilities,\nIridium(III) and Ruthenium(II) Complexes. Steric hin- which stabilize low-valent or electron-deficient states like the\ndrance is crucial in influencing the oxidation behavior of both 16-electron configuration. This greater propensity for electron\niridium(III) and ruthenium(II) complexes. Larger substituents deficiency makes iridium(III) complexes more susceptible to\non the bridge carbon effectively suppress oxidation and oxidation when steric protection is insufficient. Ruthenium, as\nstabilize the 16-electron species. Specifically, in both iridium- a group 8 metal, has weaker \u03c0-backbonding interactions with\n(III) and ruthenium(II) systems, smaller substituents like H ligands, favoring more electron-rich 18-electron configurations.\nand Me on the bridge carbon result in complexes that are more This electronic preference enhances the stability of ruthenium-\nsusceptible to oxidation. By contrast, introducing bulkier (II) complexes against oxidation, even in cases with less steric\nsubstituents such as Et, i-Bu, or mesityl significantly increases hindrance.\nsteric hindrance, which prevents oxidation and leads to the 2.6. Proposed Mechanism for the Oxidative Dehy-\nformation of stable oxidation-resistant pyridyl\u2212amine com- drogenation of Pyridyl\u2212Amine Complexes. Previous\nplexes or pyridyl\u2212amido 16-electron complexes. Iridium(III) studies have demonstrated that the N\u2212H proton in pyridyl\u2212\ncomplexes are inherently more prone to oxidation. When amine ligands can facilitate the oxidation of pyridyl\u2212amine\nsmaller substituents are present, oxidation occurs rapidly under complexes to pyridyl\u2212imine complexes upon exposure to\n 10386 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nadventitious oxygen.51 Building on Go\u0301mez et al.\u2019s reported (Figure S61). The stability of 16-electron pyridyl\u2212amido\nmechanism for amine to imine oxidation,49 the proposed complexes, such as those formed with bulky substituents (i-Bu,\noxidation mechanism of MIII/IIamine to MIII/IIimine in this mesityl, or disubstituted groups), further supports the\nsystem is illustrated in Figure 2. The production of H2O2 proposed oxidation mechanism (Figure 2). These pyridyl\u2212\n amido complexes may be generated in situ through\n deprotonation of the pyridyl\u2212amine complexes and exhibit\n significant stability, which likely prevents further oxidation by\n molecular oxygen. Additionally, the nonpolar nature of\n dichloromethane (CH2Cl2) appears to hinder the deprotona-\n tion of pyridyl\u2212amine complexes by reducing the N\u2212H\n proton\u2019s acidity. This reduced acidity may suppress or\n completely prevent the generation of oxidized pyridyl\u2212imine\n complexes.\n 2.7. Aqueous Stability. Evaluating the stability of metal\n complexes in aqueous and biological conditions is essential in\n drug development. It has been previously reported that half-\nFigure 2. Proposed oxidation mechanism of pyridyl\u2212amine to\npyridyl\u2212imine complexes, with hydrogen peroxide detected in the\n sandwich iridium(III) and ruthenium(II) pyridyl\u2212imine\nRu1amine solution after 24 h. complexes are prone to hydrolysis.41,42 Specifically, pyridyl\u2212\n imine iridium(III) complexes, synthesized by reacting\n pyridine\u2212imine ligands with dimer precursor [(\u03b75-Cp*)-\nduring the oxidation reaction in the preparation of Ru1amine MCl2]2, were found to undergo M\u2212Cl hydrolysis (Cl\u2212/H2O\nand Ru2amine was confirmed using Quantofix peroxide test exchange) in aqueous media.41 Hydrolysis is often seen as an\nstrips, which turned light blue (Scheme 6b,c, Scheme 7b, activation process, making the aqua complex (M\u2212OH2) more\nFigures 2, S55b,c, and S57c). In contrast, for reactions where reactive than the chloride-bound (M\u2212Cl) form in some metal-\nno oxidation occurred, no H2O2 was detected using the same based anticancer complexes.38,55 In our previous work, we\ntest strips (Figures S55a, S56, S57a,b, S58, S59 and S60). The demonstrated that Ir1amine and Ir2amine, when introduced into\npresence of H2O2 was also determined by iodometry.54 aqueous media, could undergo simultaneous oxidation and\nReaction solutions collected at various time points were M\u2212Cl bond hydrolysis.47 In contrast, the 16-electron pyridyl\u2212\nmixed with 2 mL of a 0.1 M acetic acid solution in methanol amido complex Ir4amido exhibited significantly greater stability\nand 2 mL of a 0.4 M potassium iodide (KI) solution in under similar conditions. UV\u2212vis spectroscopy indicated only\nmethanol, then allowed to stand for 30 min. Under acidic minor changes in its spectral profile, and 1H NMR analysis\nconditions, H2O2 reacts with iodide ions (I\u2212) to form triiodide confirmed that most of the parent complex remained intact.47\nions (I3\u2212), which exhibit strong absorption near 355 nm. The The stability of the newly synthesized complexes Ir3amine,\nexperimental data indicated that a trace amount of hydrogen Ir3imine, Ir5amido, Ir6amido, Ru1amine, Ru2amine, Ru3amine,\nperoxide was generated during the oxidative transformation of Ru4amine, Ru5amido and Ru6amido was also evaluated at 37 \u00b0C\nthe complex, with its concentration progressively increasing as in a solution of 20% DMSO/80% PBS (v/v, obtained from\nthe reaction time lengthened and the oxidation level intensified H2O, pH \u2248 7.4, high water content solution) using UV\u2212vis\n\n\n\n\nFigure 3. UV\u2212vis spectra of Ir3amine, Ir3imine, Ir5amido, Ru3amine, and Ru5amido over 24 h at 37 \u00b0C in 20% DMSO/80% PBS (v/v): (a) Ir3amine; (b)\nIr3imine; (c) Ir5amido; (d) Ru3amine; (e) Ru5amido.\n\n 10387 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nspectroscopy over 24 h at different time intervals (Figures 3 and S66). By fitting the rate constants obtained at these three\nand S62). Changes in the absorption intensity, but without temperatures (25 \u00b0C, 31 and 37 \u00b0C) to the Arrhenius\nsignificant shifts in the absorption bands, were observed for the equation, we calculated the activation energies (Ea, kJ/mol) for\npyridyl\u2212imine Ir3imine and the pyridyl\u2212amine Ir3amine, the hydrolysis of each complex (Table 2): Ir3amine (45.87 kJ/\nRu1amine, Ru2amine, Ru3amine, and Ru4amine (Figures 3 and mol), Ir3imine (48.98 kJ/mol), Ru1amine (51.63 kJ/mol),\nS62). These results indicated that the complexes underwent Ru2amine (44.59 kJ/mol), Ru3amine (36.21 kJ/mol) and\nhydrolysis, while the newly synthesized pyridyl\u2212amine Ru4amine (41.68 kJ/mol). However, there is no consistent\ncomplexes did not undergo oxidation, likely due to their trend in activation energies as influenced by either the metal\nresistance to oxidation under the test conditions. By fitting the center or the ligand modifications.\nabsorption changes to pseudo-first-order kinetics, we deter- As discussed earlier, the 16-electron pyridyl\u2212amido\nmined the half-lives (t1/2) and hydrolysis rate constants (k) for complexes demonstrated stability in CDCl3 solution and\nthese complexes (Table 1). Overall, both the pyridyl\u2212imine remained unreactive with two-electron donors like CH3CN,\n PPh3 and CO. However, minor changes in the UV\u2212vis\nTable 1. Half-Life and Hydrolysis Rate of Ir3amine, Ir3imine, absorption intensity of Ir5amido, Ir6amido, Ru5amido, and\nRu1amine, Ru2amine, Ru3amine and Ru4amine at Varying pH Ru6amido were observed after 24 h in a high-water-content\nLevels solution (20% DMSO/80% PBS, v/v, obtained from H2O, pH\n \u2248 7.4) at 37 \u00b0C (Figures 3c,e and S62a,e). These changes\n 20% DMSO/80% PBS 20% DMSO/80% PBS\n pH \u2248 7.4 pH \u2248 5.5 suggest that H2O likely coordinates to the 16-electron parent\n complexes, leading to the partial formation of 18-electron\n complexes t1/2 (min) k (min\u22121) t1/2 (min) k (min\u22121)\n amine\n aquated species (e.g., Ir\u2212OH2) in aqueous media. Under pH\n Ir3 222.86 0.00311 235.32 0.00294 5.5 conditions (20% DMSO/80% PBS, v/v, buffered to pH 5.5\n Ir3imine 203.82 0.00340 220.46 0.00314 with acetic acid/sodium acetate), similar changes were also\n Ru1amine 179.56 0.00386 199.79 0.00346 observed compared to those at pH 7.4 (Figure S64). To ensure\n Ru2amine 211.35 0.00328 248.16 0.00279 sufficient complex concentration for reliable spectroscopic\n Ru3amine 231.82 0.00299 252.96 0.00274 measurements, the stability of Ir3amine, Ir3imine, Ir5amido,\n Ru4amine 225.04 0.00308 238.58 0.00290 Ir6amido, Ru1amine, Ru2amine, Ru3amine, Ru4amine, Ru5amido and\n Ru6amido was also evaluated under conditions with a higher\nand pyridyl\u2212amine complexes displayed relatively slow proportion of DMSO-d6 and lower proportion of water (80%\nhydrolysis, with half-lives ranging from 179.6 to 231.8 min. DMSO-d6/20% PBS, v/v, obtained from D2O, pH \u2248 7.4) at 37\nThis indicates a slower rate of hydrolysis compared to other \u00b0C. 1H NMR spectroscopy was used to monitor the complexes\nreported half-sandwich Ir(III) and Ru(II) complexes with N,N- over a 24 h period. For Ir3amine, Ru1amine, Ru3amine, and\ndonor ligands.56,57 Notably, the hydrolysis rates of the Ru4amine, a new set of peaks corresponding to the aqua\npyridyl\u2212imine and pyridyl\u2212amine complexes in this system complex was observed (Figures S67, S71 S73 and S74),\nwere fairly consistent and appeared to be largely independent indicating the hydrolysis of the Mt\u2212Cl bond, However, the\nof the metal center or modifications to the ligands. Moreover, extent of hydrolysis remained low (Ir3amine: 13%, Ru1amine:\nwe also evaluated the complexes\u2019 stability under acidic 16%, Ru3amine: 17%, Ru4amine: 21%) within 24 h. In contrast,\nconditions that mimic the cancer cell microenvironment. A the other complexes exhibited spectra with no new peaks, and\nsolution of 20% DMSO/80% PBS (v/v) was buffered to pH all proton assignments were consistent with their known\n5.5 using acetic acid and sodium acetate, and stability tests molecular structures (Figures S69, S70, S73 and S75\u2212S76).\nwere also performed at 37 \u00b0C. The results revealed that the This indicates that no decomposition or ligand dissociation\nacidic environment did not notably alter the hydrolysis rates of occurred during the test, confirming the stability of these\nthe complexes (Table 1 and Figure S63). Additionally, rate complexes under reduced water content conditions. Overall,\nconstants and half-lives were measured at two additional the potential of these complexes for further anticancer activity\ntemperatures (25 and 31 \u00b0C, pH \u2248 7.4, Table 2, Figures S65 studies in aqueous conditions has been established.\n 2.8. Cytotoxicity. The cytotoxicity of the newly synthe-\nTable 2. Hydrolysis Data for Ir3amine, Ir3imine, Ru1amine, sized complexes, using cisplatin as a standard control, was\nRu2amine, Ru3amine and Ru4amine at Various Temperatures evaluated against lung cancer A549 cells, cervical carcinoma\n HeLa cells, cisplatin-resistant A549/DDP cells, and non-\n k (min\u22121) t1/2(min)\n cancerous BEAS-2B cells using the MTT assay (Table 3).\n Metal precursors D1, D2, and ligands L1-L6 showed no\n complexes 25 \u00b0C 31 \u00b0C 37 \u00b0C Ea (kJ/mol) cytotoxicity against these cells (IC50 > 100 \u03bcM). However,\n amine\n Ir3 0.00235 0.00286 0.00311 45.87 Ir3amine, Ir3imine, Ir5amido, Ir6amido, Ru3amine, Ru4amine, Ru5amido\n 294.89 242.31 222.86 and Ru6amido exhibited potent cytotoxic effects against A549\n Ir3imine 0.00257 0.00305 0.00340 48.98 and HeLa cells, with IC50 values ranging from 17.28 to 27.54\n 269.65 227.21 203.82 \u03bcM and 15.24 to 29.25 \u03bcM, respectively, which are on par with\n Ru1amine 0.00295 0.00323 0.00386 51.63 or even surpass commercial cisplatin. This suggests that the\n 234.92 214.44 179.56 anticancer activity of these complexes is primarily due to the\n Ru2amine 0.00264 0.00275 0.00328 44.59 interaction between the free ligands and the iridium(III) or\n 262.50 252.00 211.35 ruthenium(II) ions. It should be noted that Ru1amine showed\n Ru3amine 0.00242 0.00271 0.00299 36.21 no activity against all three cancer cell lines, while Ru2amine\n 286.36 255.70 231.82 exhibited activity only against HeLa cells. However, no clear\n Ru4amine 0.00238 0.00286 0.00308 41.68 structure\u2212activity relationship was observed between the steric\n 291.18 242.31 225.04 bulk of the ligand substituents and the anticancer activity.\n 10388 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 3. Cytotoxicity of the Ligands, Precursors and Complexes in Cancer and Normal Cell Lines after 48 h of Incubation\n IC50 (\u03bcM)\n compounds A549 HeLa A549/DDP BEAS-2B SIa\n L1 >100 >100 >100 >100\n L2 >100 >100 >100 >100\n L3 >100 >100 >100 >100\n L4 >100 >100 >100 >100\n L5 >100 >100 >100 >100\n L6 >100 >100 >100 >100\n D1 >100 >100 >100 >100\n D2 >100 >100 >100 >100\n Ir3amine 27.54 \u00b1 0.24 22.65 \u00b1 0.27 65.47 \u00b1 0.12 50.28 \u00b1 0.13 2.38\n Ir3imine 25.44 \u00b1 0.26 29.25 \u00b1 0.17 71.27 \u00b1 0.34 63.41 \u00b1 0.34 2.80\n Ir5amido 18.75 \u00b1 0.18 19.95 \u00b1 0.21 23.58 \u00b1 0.30 27.39 \u00b1 0.34 1.26\n Ir6amido 20.12 \u00b1 0.57 21.25 \u00b1 0.41 26.27 \u00b1 0.34 29.41 \u00b1 0.64 1.31\n Ru1amine >100 >100 >100 >100\n Ru2amine >100 70.32 \u00b1 0.25 >100 >100\n Ru3amine 21.02 \u00b1 0.21 28.24 \u00b1 0.09 60.45 \u00b1 0.24 41.75 \u00b1 0.22 2.87\n Ru4amine 23.12 \u00b1 0.14 29.21 \u00b1 0.09 63.12 \u00b1 0.17 53.72 \u00b1 0.19 2.73\n Ru5amido 19.02 \u00b1 0.21 18.24 \u00b1 0.19 24.63 \u00b1 0.47 31.75 \u00b1 0.36 1.29\n Ru6amido 17.28 \u00b1 0.09 15.24 \u00b1 0.16 22.74 \u00b1 0.29 28.64 \u00b1 0.15 1.31\n Cisplatin 24.89 \u00b1 0.21 8.39 \u00b1 0.08 38.14 \u00b1 1.01 29.21 \u00b1 0.22 1.20\na\n SI: The selectivity index is the ratio of IC50 values for BEAS-2B normal cells to A549 cancer cells.\n\nInterestingly, most of these iridium(III) and ruthenium(II) However, no clear correlation between cellular uptake and\ncomplexes exhibited significant cytotoxicity against cisplatin- cytotoxicity was observed. Aside from Ru1amine, which showed\nresistant A549/DDP cells, with IC50 values ranging from 22.74 no activity, the other complexes displayed similar cytotoxicity\nto 26.27 \u03bcM, suggesting their mechanisms of action differ from against A549 cells. Therefore, differences in cellular accumu-\ncisplatin. The 16-electron pyridyl\u2212amido complexes Ir5amido, lation do not explain the variations in cytotoxicity among these\nIr6amido, Ru5 amido and Ru6amido, exhibited significantly complexes.\nenhanced potency (ca. 3 times greater) against A549/DDP 2.9. DNA and Protein Binding Results. The potential\ncells compared to pyrydyl\u2212imine complex Ir3imine and interactions of the representative complexes Ir3amine, Ir5amido,\npyridyl\u2212amine complexes Ir3amine, Ru3amine and Ru4amine Ru3amine, Ru5amido and adriamycin (as a positive control) with\nwith IC50 values of 22.74\u221226.27 \u03bcM versus 60.45\u221271.27 Calf Thymus DNA (CT-DNA) were examined through UV\u2212\n\u03bcM. In addition, MTT assay was conducted on noncancerous vis absorption spectroscopy (Figure S77), considering that\nBEAS-2B cells, but no significant selectivity (SI: 1.26\u22122.87) DNA binding often contributes to the cytotoxic activity of\nwas observed between cancer and normal cells. The IC50 values metal-based anticancer complexes. In these experiments,\nof these complexes (27.39\u221263.41 \u03bcM) were similar to or lower solutions containing 60 \u03bcM of each complex were mixed\nthan that of cisplatin (29.21 \u03bcM). with increasing concentrations of CT-DNA (0\u221243.6 \u03bcM).\n Notably, lung cancer is a leading cause of mortality This resulted in hyperchromism and a slight red shift at the\nworldwide, with high fatality rates in both developed and absorption peak, characteristic of noncovalent electrostatic\ndeveloping countries. Given that current treatments, such as binding.41,58 The intrinsic equilibrium binding constants (Kb)\ncisplatin, have not demonstrated ideal efficacy in the treatment were calculated using the Benesi\u2212Hildebrand equation and\nof lung cancer, we thus selected A549 lung cancer cells as the found to range from 1.19 \u00d7 104 to 2.42 \u00d7 104 M\u22121 (Figure\nmodel for the subsequent biological studies. Moreover, four S78: Ir3amine: Kb = 2.42 \u00d7 104 M\u22121, Ir5amido: Kb = 2.04 \u00d7 104\nrepresentative complexes\ufffdIr3amine, Ir5amido, Ru3amine, and M\u22121, Ru3amine: Kb = 1.19 \u00d7 104 M\u22121, Ru5amido: Kb = 1.24 \u00d7 104\nRu5amido\ufffdwere selected for further biological evaluation (see M\u22121). These values were notably lower than those for\nSections 2.15 below) since this selection allowed us to adriamycin (11.2 \u00d7 104 M\u22121) and for similar half-sandwich\nsystematically compare the effects of different metal centers complexes in previous studies (Kb > 105 M\u22121),29,59,60\n(Ir3amine vs Ru3amine; Ir5amido vs Ru5amido) and coordination suggesting relatively weak CT-DNA interactions. Furthermore,\nmodes (16-electron vs 18-electron, Ir3amine vs Ir5amido; the Kb did not correlate with the cytotoxicity of these\nRu3amine vs Ru5amido). complexes, indicating that DNA binding is unlikely to be the\n The total cellular accumulation of complexes Ir3amine, main MoA for these half-sandwich complexes.\nIr5amido, Ru1amine, Ru3amine, and Ru5amido was quantified in It is crucial to understand how anticancer agents interact\nA549 cancer cells treated with each complex (5 \u03bcM) for 48 h with cellular proteins, as these proteins are pivotal in the\nby ICP\u2212MS to determine if there was a correlation between transport and metabolism of drugs. Serum albumin (SA), a\ncellular uptake levels of the complexes and cytotoxicity. The major blood plasma protein, is known for its ability to bind and\nintracellular metal accumulation (ng/\u03bcg protein) in A549 cells transport metal-based complexes. Bovine serum albumin\nwas determined tobe Ir5amido (0.598) > Ir3amine (0.514) > (BSA) was selected as a substitute for human serum albumin\nRu5amido (0.391) > Ru1amine (0.346) \u2248 Ru3amine (0.341), (HSA) in this study due to its similar structure and easier\nindicating that iridium(III) complexes are generally more accessibility. The interactions between Ir3amine, Ir5amido,\ninternalized than the corresponding ruthenium(II) complexes. Ru3amine, Ru5amido, fluorescein (as a positive control) and\n 10389 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 4. Docking model of complexes (a) Ir3amine and (c) Ru5amido positioned in the hydrophobic cavity of BSA (PDB ID: 4OR0). Interaction\ndetails between complexes (b) Ir3amine and (d) Ru5amido with the polypeptide chains.\n\nBSA were investigated through UV\u2212vis absorption and Ru3amine, and Ru5amido interact with BSA, causing subtle\nfluorescence spectroscopy (Figure S79a\u2212e). To eliminate alterations in the structure of its aromatic residues, an effect\npotential self-absorption effects, both the reference and sample similar to that of the positive control fluorescein.\ncuvettes were pretreated with the respective complexes. The Molecular docking studies of the selected representative 18-\nfluorescence behavior of BSA arises from the aromatic amino electron Ir3amine and 16-electron Ru5amido complexes were\nacids tyrosine (Tyr) and tryptophan (Trp), which respond to carried out using the AutoDock suite and Q-SiteFinder, which\nchanges in their environment. Binding of small molecules to are designed to identify potential binding sites and assess\nthese residues can lead to a decrease in fluorescence intensity. ligand\u2212protein interactions. The crystal structure of bovine\nAs the complexes and fluorescein concentration increased, the serum albumin (BSA) (PDB ID: 4OR0) was retrieved from\nabsorption peak of BSA at 229 nm decreased and shifted the Protein Data Bank. Initially, Q-SiteFinder was used to\nslightly, likely due to \u03b1-helix changes and the effect of polar pinpoint probable binding regions within BSA. Docking\nsolvents.61\u221265 Additionally, the absorption peak at 276 nm simulations were then conducted using AutoDock, where\nexhibited a gradual increase without a shift, indicating subtle iridium and ruthenium parameters, absent from AutoDock\u2019s\nchanges in the microenvironment surrounding the aromatic default force field, were manually added to the parameter\nresidues (Tyr and Trp).66,67 The fluorescence intensity of BSA library. Flexible residues within a 6 \u00c5 radius of the binding\nat 353 nm steadily decreased with increasing complex or pocket were chosen to enable adaptive docking. The docking\nfluorescein concentration (Figure S79f\u2212j), indicating inter- results showed several significant interactions between Ir3amine,\nactions through a static quenching mechanism.68 Synchronous Ru5amido and BSA, including electrostatic, hydrogen bonding,\nfluorescence spectroscopy is an effective method for examining and \u03c0-cation interactions (Figure 4). Specifically, the 18-\nthe conformational alterations in BSA caused by interactions electron pyridyl\u2212amine complex Ir3amine formed a salt bridge\nwith the complexes. By maintaining a fixed wavelength interval with LYS221 and ARG217, along with a \u03c0-cation interaction\nof 15 or 60 nm, this method can reveal specific alterations in with TRP213. Additionally, a \u201cnonconventional\u201d hydrogen\nthe microenvironments of Tyr and Trp residues.69,70 At \u0394\u03bb = bond with PRO446 was observed. In the case of the 16-\n60 nm, the emission of Trp shifted to 276 nm with a 3 nm red electron pyridyl\u2212amido complex Ru5amido, \u03c0-cation interac-\nshift. Similarly, at \u0394\u03bb = 15 nm, Tyr exhibited a decrease in tions were noted with LYS294 and ARG217. Several\nintensity at 287 nm, accompanied by a 2 nm red shift (Figure \u201cnonconventional\u201d hydrogen bonds were also identified,\nS80), which points to changes in its microenvironment and involving residues like TYR340 and ASP450. These results\nconformation. These results imply that Ir3amine, Ir5amido, provide deeper insight into the binding mechanisms of Ir3amine\n 10390 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 5. UV\u2212vis spectra of the reaction between NADH (100 \u03bcM) and (a) Ir3amine, (b) Ir5amido, (c) Ru3amine and (d) Ru5amido (1 \u03bcM) in 10%\nMeOH/90% H2O (v/v) at 25 \u00b0C for 8 h.\n\n\n\n\nFigure 6. Fluorescence microscopy analysis of ROS levels in A549 cells after 4 h treatment with (a) Ir3amine, (b) Ir5amido, (c) Ru3amine, and (d)\nRu5amido at 37 \u00b0C. Cells were stained with DCFH-DA, and ROSup served as a positive control for ROS induction. p-values were calculated\ncompared to the untreated control (*p < 0.05, **p < 0.01).\n\nand Ru5amido with BSA, offering valuable information on their part (NAD+) are crucial for regulating cellular balance and\npotential effects on transport, stability, and bioavailability. maintaining homeostasis. The NADH/NAD+ ratio and NAD+\n 2.10. Catalytic Hydride Transfer. Coenzymes nicotina- levels are integral to various intracellular redox reactions.71 In\nmide adenine dinucleotide (NADH) and its oxidized counter- recent years, the use of hydride transfer catalysis in anticancer\n 10391 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 7. Changes in mitochondrial membrane potential of A549 cancer cells induced by (a) Ir3amine, (b) Ir5amido, (c) Ru3amine, and (d) Ru5amido.\nCCCP (Carbonyl cyanide m-chlorophenyl hydrazone) was used as a positive control to induce mitochondrial membrane depolarization. Data are\nquoted as mean \u00b1 SD of three replicates. p-values were calculated compared to the untreated control (*p < 0.05, **p < 0.01).\n\ndrug design has attracted considerable interest.72 The Sadler NADH. The capacity of these complexes to disrupt the\ngroup demonstrated that NADH can transfer a hydride to aqua NADH/NAD+ balance may trigger ROS production and\niridium(III) cyclopentadienyl complexes, generating ROS such activate an oxidative mechanism, prompting further explora-\nas H2O2, which acts as part of the oxidant MoA.73\u221275 Similarly, tion of their potential ROS generation in cancer cells.\nour group revealed that half-sandwich pyridine\u2212imine iridium- 2.11. Cellular ROS Determination. Pyridyl\u2212imine\n(III) and ruthenium(II) complexes with sp2-N/sp2-N coordi- iridium(III) and ruthenium(II) complexes were found to\nnation can facilitate the catalytic conversion of NADH to significantly elevate ROS levels in A549 cells.41\u221243,76 This\nNAD+. This process elevates ROS levels, triggers oxidative prompted us to explore the impact of switching the\nstress, and plays a role in promoting cell death (Scheme 1, II coordination mode from pyridyl\u2212imine to pyridyl\u2212amine or\nand III).41\u221243 Along this line, we also explored the catalytic pyridyl\u2212amido complexes on ROS generation. ROS levels\nhydride transfer activity of new hybrid sp2-N/sp3-N pyridyl\u2212 induced by the representative complexes Ir3amine, Ir5amido,\namine and pyridyl\u2212amido complexes (Ir3amine, Ir5amido, Ru3amine and Ru5amido at the concentrations of 0.5, 1.0, and 2.0\nRu3amine, and Ru5amido) in this system. The interactions of \u00d7 IC50 were determined using DCFH-DA staining and\nthese complexes with NADH at a molar ratio of 1:100 were fluorescence microscopy (Figures 6 and S83). A549 cells\nmonitored in a solution comprising 10% methanol and 90% treated with these complexes exhibited a noticeable, concen-\nwater (v/v) over an 8 h period using UV\u2212vis spectroscopy tration-dependent increase in fluorescence intensity, indicating\n(Figure 5). A decrease in the NADH absorption peak at 339 higher ROS levels compared to control cells (Figures 6a\u2212d\nnm was observed, indicating the occurrence of catalytic and S83). Thus, these complexes could potentially disturb the\nactivity. Turnover numbers (TONs) were calculated as intracellular redox balance by generating ROS. Previous studies\nfollows: 16.40 for Ir3amine, 21.54 for Ir5amido, 21.90 for have shown that many half-sandwich complexes with different\nRu3amine, and 18.84 for Ru5amido. These values align with chelating ligands can generate ROS through catalytic hydride\nthose observed for earlier pyridine\u2212imine complexes.41,43 To transfer from NADH to O2.77\u221279 Similarly, in this system, the\nverify NADH oxidation, separate HPLC and ESI-MS experi- conversion of NADH to NAD+, as noted in Section 2.10, was\nments were also conducted (Figures S81, 82). HPLC analysis also observed for these complexes, may contribute to ROS\nof NADH and NAD+ standards produced distinct retention generation. Overall, these results indicate that the anticancer\ntimes. After incubating NADH with complex Ir3amine at 25 \u00b0C effects of these complexes can be attributed to the elevated\nfor 24 h, the HPLC profile showed a diminished NADH peak ROS levels.\nand an emerging NAD+ peak, indicating that Ir3amine catalyzed 2.12. Mitochondrial Membrane Depolarization. Given\nthe oxidation of NADH to NAD+. Analysis of the reaction that these complexes can induce excessive reactive oxygen\nmixture by ESI-MS also revealed a NAD+ signal at m/z 664.1 species (ROS) and mitochondria are vital regulators of ROS\n([M + H]+, Figure S82), further corroborating the oxidation of production, we investigated whether they affect mitochondrial\n 10392 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 8. Cell cycle distribution of A549 cells treated with (a) Ir3amine, (b) Ir5amido, (c) Ru3amine, and (d) Ru5amido for 24 h at 0.25 \u00d7 IC50 and 0.5\n\u00d7 IC50 concentrations was analyzed by flow cytometry using PI/RNase staining. Data are shown as mean \u00b1 SD from three replicates, with p-values\ncalculated against the untreated control group (*p < 0.05, **p < 0.01).\n\n\n\n\nFigure 9. Apoptosis analysis and corresponding histograms for A549 cells treated with (a) Ir3amine, (b) Ir5amido, (c) Ru3amine, and (d) Ru5amido for\n48 h at 37 \u00b0C. p-values were determined relative to the untreated control group (*p < 0.05, **p < 0.01).\n\n\n 10393 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 10. Protein expression levels analyzed by Western blot after 24 h treatment with (a) Ir3amine and (c) Ru5amido at concentrations of 0.5, 1.0,\nand 2.0 \u00d7 IC50. Histograms showing the changes of Bcl-2/Bax ratio levels at various (b) Ir3amine and (d) Ru5amido concentrations. Data represent\nthe mean \u00b1 SD from three independent experiments. Statistical significance compared to the control: *p < 0.05, **p < 0.01.\n\nfunction. The mitochondrial membrane potential (MMP, of 0.25, 0.5, and 1.0 \u00d7 IC50, and apoptosis was analyzed via\n\u25b3\u03c8m) is crucial for maintaining mitochondrial integrity and flow cytometry (Figures 9 and S89\u2212S92). Compared to the\nbioenergetics, and its loss (depolarization) is a key event in cell control group, both Ir3amine and Ru3amine significantly\ndeath pathways. A549 cancer cells were treated with Ir3amine, increased the percentage of apoptotic cells, with notable\nIr5amido, Ru3amine, and Ru5amido at 0.5, 1.0, and 2.0 \u00d7 IC50, and differences in early and late apoptosis. At 1.0 \u00d7 IC50, Ir3amine\ntheir effects on MMP were evaluated by JC-1 staining and flow induced apoptosis in 79.1% of A549 cells, with 17.9%\ncytometry (Figures 7 and S84). Depolarization is indicated by attributed to early apoptosis and 61.2% to late apoptosis\na decrease in the red-to-green fluorescence ratio. The results (Figure 9a). The late apoptotic population increased with\nshowed a concentration-dependent mitochondrial dysfunction: concentration, while the early apoptotic population showed\nas the drug concentration rose from 0.5 \u00d7 IC50 to 2 \u00d7 IC50, the minimal changes between 0.5 and 1.0 \u00d7 IC50. For Ru3amine, a\npercentage of cells with depolarized mitochondria increased total of 31.1% of cells underwent apoptosis at 1.0 \u00d7 IC50,\nfrom 37.5%, 35.2%, 22.8%, and 28.7% to 82.2%, 86.3%, 70.5%, consisting of 22.0% early and 9.1% late apoptosis (Figure 9c).\nand 84.0%, respectively. Hence, these complexes may exert Unlike Ir3amine, Ru3amine exhibited a concentration-dependent\ntheir anticancer effects by inducing mitochondrial dysfunction. increase in the early apoptotic population as the concentration\n 2.13. Cell-Cycle Arrest. The inhibition of cancer cell rose from 0.5 to 1.0 \u00d7 IC50. In contrast, Ir5amido and Ru5amido\ngrowth by anticancer drugs may arise from inducing cell cycle showed minimal changes in apoptotic cell populations across\narrest, apoptosis, or a combination of these processes.80,81 the tested concentrations, with results comparable to the\nThus, flow cytometric analysis was conducted to investigate control group and no clear concentration-dependent increase\nwhether the inhibition of cell growth was due to cell cycle observed (Figure 9b,d). These results suggest that the pyridyl\u2212\narrest. Treatment with these complexes Ir3amine, Ir5amido, amine complexes are capable of inducing apoptosis, whereas\nRu3amine and Ru5amido at concentrations of 0.25 \u00d7 IC50 and 0.5 the pyridyl\u2212amido complexes did not show any obvious\n\u00d7 IC50 for 24 h resulted in a concentration-dependent increase apoptotic effect. Both types of complexes exhibit similar\nin the S-phase cell population, with a corresponding reduction cytotoxicity, yet the pyridyl\u2212amido complexes did not show\nin the G2/M and G0/G1 phases. At 0.5 \u00d7 IC50, the percentage obvious apoptotic effects. This discrepancy may suggest that\nof A549 cells in the S phase was elevated by 13.00% (Ir3amine), apoptosis is not the primary mode of cell death for the\n17.70% (Ir5amido), 18.25% (Ru3amine), and 5.55% (Ru5amido) pyridyl\u2212amido complexes in A549 cells. Possible reasons\ncompared to the untreated control group (Figures 8 and S85\u2212 include the differences in the mechanisms of action, like cell\nS88). These results demonstrate that both the pyridyl\u2212amine cycle arrest, which do not necessarily trigger classical apoptotic\nand pyridyl\u2212amido complexes primarily induce growth markers.\ninhibition through S-phase cell cycle arrest, regardless of Within the Bcl-2 protein family, which plays a key role in\ntheir coordination mode. apoptosis, the pro-apoptotic regulator Bax helps maintain the\n 2.14. Apoptosis. Anticancer complexes producing high balance between cell survival and programmed death by\nROS levels can disturb the cellular redox balance, a process downregulating Bcl-2. In this study, Western blot analysis was\nstrongly linked to apoptosis induction and cellular damage.82,83 carried out to quantify Bax and Bcl-2 levels, aiming to\nTo determine the apoptosis-inducing mechanism of cell death, determine whether both pyridyl\u2212amine and pyridyl\u2212amido\nan annexin V/PI assay was conducted. A549 cells were treated complexes modulate apoptotic pathways (Figure 10). Notably,\nwith Ir3amine, Ir5amido, Ru3amine, and Ru5amido at concentrations 18-electron pyridyl\u2212amine complex Ir3amine increased the Bcl-\n 10394 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 11. (a) Wound-healing assay showing A549 cells treated with Ir3amine for 24 h. (b\u2212e) Histograms of A549 cells treated with Ir3amine,\nIr5amido, Ru3amine, and Ru5amido for 24 h. Representative images were taken at 0 and 24 h, with wound widths indicated by lines (\u03bcm). Scale bar:\n200 \u03bcm. Wound closure rate was calculated as (R0 \u2212 R1)/R0 \u00d7 100%. Data are presented as mean \u00b1 SD from three replicates, with p-values\ncompared to the untreated control group (*p < 0.05, **p < 0.01).\n\n2/Bax ratio in A549 cells, suggesting that this effect contributes plexes. Ruthenium(II) complexes exhibited higher resistance\nsignificantly to apoptosis, whereas 16-electron pyridyl\u2212amido to oxidation compared to iridium(III) complexes, with\ncomplex Ru5amido did not significantly alter the Bcl-2/Bax moderate steric hindrance favoring stable pyridyl\u2212amine\nratio, further indicating a nonapoptotic mechanism of cell complexes and extreme steric hindrance enabling the\ndeath. These observations align with the results of the formation of pyridyl\u2212amido 16-electron complexes. The\napoptosis analysis described above (Figure 9). aqueous stability of these newly synthesized complexes was\n 2.15. Inhibition of Cell Migration. Inhibiting cancer cell confirmed. Most of the complexes demonstrated anticancer\nmigration is a key challenge in cancer therapy, as decreased potency against A549 lung cancer, HeLa cervical cancer, and\nsurface adhesion allows malignant cells to detach from the cisplatin-resistant A549/DDP cells. Interaction studies re-\nprimary tumor and spread to other parts of the body.84,85 Cell vealed weak binding affinities with CT-DNA, suggesting that\nmigration, metastasis and invasion are closely associated with DNA binding is not the primary mechanism of their anticancer\nthe breakdown of the extracellular matrix and the activity of potency. The further mechanistic investigations suggested a\ncell adhesion molecules.86,87 To assess the impact of Ir3amine, potential redox-driven pathway, where the catalytic conversion\nIr5amido, Ru3amine, and Ru5amido on A549 cancer cell migration, of NADH to NAD+, elevated ROS levels and mitochondrial\na wound-healing experiment was performed (Figures 11 and dysfunction may contribute to their anticancer activity.\nS93\u221295). At 0.5 \u00d7 IC50, the wound closure rates (WCR) for Pyridyl\u2212amine complexes exhibited the ability to trigger\ncells treated with Ir3amine, Ir5amido, Ru3amine, and Ru5amido were apoptosis, whereas 16-electron pyridyl\u2212amido complexes did\nsignificantly reduced to 10.78%, 11.12%, 8.10% and 11.90% not show a comparable apoptotic response. However, both\nrespectively, compared to the control group values of 41.76%, types of complexes caused S-phase cell cycle arrest. In\n39.98%, 44.38% and 43.76%. Furthermore, all four complexes particular, wound-healing assays demonstrated that these\nexhibited a concentration-dependent inhibitory effect on cell complexes effectively inhibited A549 cell migration, indicating\nmigration. These results demonstrate that both pyridyl\u2212amine their potential to suppress cancer metastasis.\nand pyridyl\u2212amido complexes in this system effectively\nsuppress the migration of A549 cancer cells in vitro. 4. EXPERIMENTAL SECTION\n Ligands L1, L2, L4, and L5 were synthesized following established\n3. CONCLUSIONS methods.47,48 The preparation of bimetallic precursors D1 and D2\n adhered to previously reported protocols.88,89 Further experimental\nThis study systematically investigated the ligand- and metal-\n details, encompassing general methodologies and descriptions of\ndependent coordination chemistry of iridium(III) and biological assays, are presented in the Supporting Information. No\nruthenium(II) complexes synthesized from pyridyl\u2212amine uncommon hazards are noted.\nligands. Ligand steric hindrance and metal centers were 4.1. Synthesis of Ligands.\nfound to significantly influence their oxidation behavior and\ncoordination modes. Smaller substituents, such as H and Me,\nfacilitated oxidation to pyridyl\u2212imine complexes under\nadventitious oxygen conditions, whereas bulkier substituents,\nsuch as i-Bu and mesityl, suppressed oxidation, stabilizing\neither pyridyl\u2212amine or 16-electron pyridyl\u2212amido com-\n 10395 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n 4.1.1. L3. A solution of 2,6-diisopropyl-N-(pyridin-2-ylmethylene)- MHz, CDCl3): \u03b4 162.99, 151.39, 141.30, 141.06, 139.71, 139.21,\naniline (5.32 g, 20.00 mmol) in 100 mL of toluene was prepared, and 88.96 (C5Me5), 72.12 (N\u2212CH), 28.45 (i-Pr-CH), 27.69 (i-Pr-CH),\ntriethylaluminum (30.00 mmol) was carefully added dropwise under 27.62 (CH2CH3), 26.70 (i-Pr-CH3), 26.02 (i-Pr-CH3), 25.68 (i-Pr-\nnitrogen. The mixture was then heated at reflux for 12 h. After CH3), 24.49 (i-Pr-CH3), 9.96 (CH2CH3), 8.60 (Cp*-CH3). ESI-MS\ncooling, the reaction was quenched by adding 1 M NaOH solution (m/z): calcd for C30H43CIrN2, 659.2744; found, 659.2714, [M-PF6]+.\nwhile keeping it in an ice bath. The organic phase was washed with Elemental analysis: calcd for C30H43ClF6IrN2P: C, 44.80; H, 5.39; N,\nbrine twice, then dried and filtered, and the solvent evaporated under 3.48. Found: C, 44.92; H, 5.37; N, 3.49.\nreduced pressure. The product was recrystallized from hexane to\nafford white crystalline product. Yield: 3.84 g (64.9%).1H NMR (500\nMHz, CDCl3): \u03b4 8.63 (d, 1H), 7.44 (m, 1H), 7.23 (m, 1H), 7.15 (m,\n1H), 7.09 (m, 1H), 6.96 (m, 1H), 6.86 (d, 1H), 4.08 (s, 1H, NH),\n3.88 (m, 1H, CHCH2CH3), 3.23 (m, 2H, i-Pr\u2212CH), 2.00 (m, 2H,\nCH2CH3), 1.21 (d, 6H, i-Pr\u2212CH3), 1.02 (d, 6H, i-Pr\u2212CH3), 0.78 (t,\n3H, CH2CH3). 13C NMR (126 MHz, CDCl3): \u03b4 162.02, 149.87,\n141.86, 141.79, 135.86, 123.42, 123.28, 122.94, 122.08, 67.51 (NH\u2212\nCH), 28.53(CH2CH3), 27.55 (i-Pr-CH), 24.25 (i-Pr-CH3), 24.18 (i-\nPr-CH3), 11.15 (CH2CH3). ESI-MS (m/z): calcd for C20H29N2, 4.2.2. Ir3imine. First, pyridyl\u2212amine Ir3amine was synthesized by the\n297.2331; found, 297.2345, [M + H]. general method, followed by the formation of the pyridyl\u2212imine\n complex Ir3imine via gradual oxidation (14 days) during the cultivation\n of single crystal. Red flaky crystals. Yield 46.4 mg (57.8%). 1H NMR\n (500 MHz, CDCl3): \u03b4 8.91 (d, 1H), 8.21 (d, 2H), 7.96 (m, 1H), 7.33\n (m, 3H), 3.44 (m, i-Pr\u2212CH), 3.03 (m, 1H, i-Pr\u2212CH), 2.63 (m, 1H,\n CH2), 2.49 (m, 1H, CH2), 1.44 (s, 15H, Cp*\u2212CH3), 1.37 (d, 3H, i-\n Pr\u2212CH3), 1.27 (d, 3H, CH2CH3), 1.22 (d, 3H, i-Pr\u2212CH3), 1.15 (d,\n 4.1.2. L6. A solution of 2,6-diisopropyl-N-(1-(2-pyridin)- 3H, i-Pr\u2212CH3), 0.95 (d, 3H, i-Pr\u2212CH3). 13C NMR (126 MHz,\nethylidene)benzenamine (5.60 g, 20.00 mmol) in 100 mL of toluene CDCl3): \u03b4 171.67 (C\ufffdN), 152.24, 151.05, 148.12, 144.41, 142.66,\nwas prepared, and triethylaluminum (30.00 mmol) was carefully 139.57, 129.55, 129.18, 124.74, 124.05, 99.99, 91.03 (C5Me5), 27.82\nadded dropwise under nitrogen. The mixture was then heated at reflux (i-Pr-CH3), 27.61 (i-Pr-CH), 27.58 (CH2CH3), 26.57 (i-Pr-CH3),\nfor 12 h. After cooling, the reaction was quenched by adding 1 M 26.00 (i-Pr-CH3), 23.72 (i-Pr-CH3), 21.63 (i-Pr-CH3), 13.75\nNaOH solution while keeping it in an ice bath. The organic phase was (CH2CH3), 8.51 (Cp*-CH3). ESI-MS (m/z): calcd for C30H41ClIrN2,\nwashed with brine twice, then dried and filtered, and the solvent 657.2588; found, 657.2576, [M-PF6]+. Elemental analysis: calcd for\nevaporated under reduced pressure, yielding a clear, colorless oil as C30H41ClF6IrN2P: C, 44.91; H, 5.15; N, 3.49. Found: C, 44.81; H,\nthe final product. Yield: 3.56 g (57.4%). 1H NMR (500 MHz, 5.17; N, 3.48.\nCDCl3): \u03b4 8.62 (s, 1H), 7.59 (m, 1H), 7.44 (d, 1H), 7.23 (t, 1H),\n7.11 (m, 3H), 4.34 (s, 1H, NH), 3.10 (m, 2H, i-Pr\u2212CH), 2.16 (m,\n1H, CH2CH3), 1.94 (m, 1H, CH2CH3), 1.23 (s, 3H, CH3), 1.11 (d,\n6H, i-Pr\u2212CH3), 1.00 (d, 6H, i-Pr\u2212CH3), 0.68 (t, 3H, CH2CH3). 13C\nNMR (126 MHz, CDCl3): \u03b4 166.76, 148.22, 146.64, 141.07, 135.94,\n124.39, 122.94, 121.21, 120.52, 61.82 (NH\u2212C), 38.56 (CH2), 28.25\n(i-Pr-CH), 24.13 (i-Pr-CH3), 23.91 (i-Pr-CH3), 22.69 (CH3), 8.97\n(CH2CH3). ESI-MS (m/z): calcd for C21H31N2, 311.2487; found,\n311.2491, [M + H].\n 4.2. Synthesis of Complexes. General method: A mixture of\nD1/D2 (0.05 mmol, 1 equiv), pyridyl\u2212amine ligands (0.10 mmol, 2 4.2.3. Ir5amido. Red powder. Yield 50.2 mg (58.5%). 1H NMR (500\nequiv), and NH4PF6 (0.20 mmol, 4 equiv) was prepared in methanol. MHz, CDCl3): \u03b4 9.19 (d, 1H), 7.84 (m, 2H), 7.20 (m, 2H), 7.10 (d,\nThe solution was stirred at room temperature for 24 h to ensure 1H), 7.01 (d, 1H), 6.68 (s, 1H), 6.54 (s, 1H), 4.90 (s, 1H, CH\u2212N),\ncomplete reaction. Afterward, the solvent was removed under reduced 3.11 (m, 1H, i-Pr\u2212CH), 2.76 (m, 1H, i-Pr\u2212CH), 2.14 (s, 3H, Ar\u2212\npressure, leaving a crude residue. This residue was dissolved in CH3), 1.69 (s, 15H, Cp*-CH3), 1.65 (s, 3H, Ar\u2212CH3), 1.40 (s, 3H,\ndichloromethane, filtered through a Celite pad, and the filtrate was Ar\u2212CH3), 1.31 (d, 3H, i-Pr\u2212CH3), 1.28 (d, 3H, i-Pr\u2212CH3), 1.22 (d,\ntreated with an excess of n-hexane to precipitate. The solid product 3H, i-Pr\u2212CH3), \u22120.15 (d, 3H, i-Pr\u2212CH3). 13C NMR (126 MHz,\nwas collected, thoroughly washed with n-hexane, and dried under\n CDCl3): \u03b4 169.75, 150.47, 150.15, 144.21, 143.71, 139.31, 139.11,\nvacuum to obtain the final complexes. (Ir1amine, Ir2amine and Ir4amido\n 138.95, 138.87, 132.84, 129.98, 128.81, 127.17, 125.64, 124.67,\nhave been reported in our previous work47).\n 124.08, 122.34, 89.66 (C5Me5), 86.21 (CH), 27.31 (Ar\u2212CH3), 27.19\n (Ar\u2212CH3), 26.16 (i-Pr-CH), 23.81 (i-Pr-CH), 21.34 (i-Pr-CH3),\n 20.62 (i-Pr-CH3), 18.64 (i-Pr-CH3), 9.46 (Cp*-CH3). ESI-MS (m/z):\n calcd for C37H48IrN2, 713.3447; found, 713.3437, [M-PF6]+.\n Elemental analysis: calcd for C37H48F6IrN2P: C, 51.80; H, 5.64; N,\n 3.27. Found: C, 51.95; H, 5.62; N, 3.26.\n\n\n\n 4.2.1. Ir3amine. Orange powder. Yield 50.5 mg (62.8%). 1H NMR\n(500 MHz, CDCl3): \u03b4 8.64 (d, 1H), 8.01 (t, 1H), 7.60 (t, 1H), 7.44\n(d, 1H), 7.36 (m, 1H), 7.29 (m, 2H), 7.12 (m, 1H, NH), 5.36 (m,\n1H, CH\u2212N), 3.50 (m, 1H, i-Pr\u2212CH3), 2.86 (m, 1H, i-Pr\u2212CH), 2.08\n(m, 1H, CH2), 1.99 (m, 1H, CH2), 1.44 (d, 3H, i-Pr\u2212CH3), 1.39 (s,\n15H, Cp*\u2212CH3), 1.37 (s, 3H, i-Pr\u2212CH3), 1.31 (d, 3H, i-Pr\u2212CH3),\n1.26 (d, 3H, i-Pr\u2212CH3), 0.86 (t, 3H, CH2CH3). 13C NMR (126\n\n 10396 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n 4.2.4. Ir6amido. Red powder. Yield 47.5 mg (60.7%). 1H NMR (500 4.2.7. Ru2amine. Yellow powder. Yield 42.9 mg (61.4%). 1H NMR\nMHz, CDCl3): \u03b4 9.10 (d, 1H), 8.05 (t, 1H), 7.78 (t, 1H), 7.58 (d, (500 MHz, CDCl3): \u03b4 9.17 (d, 1H), 7.97 (t, 1H), 7.63 (t, 1H), 7.42\n1H), 7.25 (d, 1H), 7.16 (t, Hz, 2H), 3.04 (m, 1H, i-Pr\u2212CH), 2.51 (m, (m, 1H), 7.37 (m, 2H), 7.30 (m, 1H), 6.18 (m, 1H, CH\u2212N), 5.79 (d,\n1H, i-Pr\u2212CH), 1.93 (m, 1H, CH2CH3), 1.68 (m, 1H, CH2CH3), 1.60 1H), 5.54 (d, 1H), 5.26 (d, 1H), 5.19 (m, 2H, Ar p-cymene\n(s, 15H, Cp*\u2212CH3), 1.48 (d, 3H, i-Pr\u2212CH3), 1.35 (s, 3H, CH3), 1.27 (1H)+NH (1H)), 3.39 (m, 2H, i-Pr\u2212CH), 2.64 (m, 1H, i-Pr\u2212CH),\n(d, 3H, i-Pr\u2212CH3), 1.13 (d, 3H, i-Pr\u2212CH3), 0.96 (d, 3H, i-Pr\u2212CH3), 2.09 (s, 3H, Ar\u2212CH3), 1.63 (d, 3H, CHCH3), 1.46 (d, 3H, i-Pr\u2212\n0.28 (t, 3H, CH2CH3). 13C NMR (126 MHz, CDCl3): \u03b4 169.90, CH3), 1.38 (d, 3H, i-Pr\u2212CH3), 1.35 (d, 3H, i-Pr\u2212CH3), 1.22 (d, 3H,\n151.32, 148.71, 146.16, 143.45, 139.75, 126.94, 125.92, 124.69, i-Pr\u2212CH3), 1.17 (d, 3H, i-Pr\u2212CH3), 0.91 (d, 3H, i-Pr\u2212CH3). 13C\n123.97, 120.35, 89.27 (C5Me5), 85.18 (N\u2212C), 39.71 (CH2), 29.03 NMR (126 MHz, CDCl3): \u03b4 162.06, 155.02, 141.44, 141.05, 140.25,\n(CH3), 28.16 (i-Pr-CH), 27.43 (i-Pr-CH), 25.99 (i-Pr-CH3), 25.87 (i- 140.09, 128.15, 126.86, 126.49, 125.63, 122.76, 103.70, 100.20, 85.26,\nPr-CH3), 24.55 (i-Pr-CH3), 23.19 (i-Pr-CH3), 9.88(Cp*-CH3), 9.75 85.12, 82.84, 82.02, 66.26 (CH), 30.23 (Ar-i-Pr-CH), 29.17 (i-Pr-\n(CH2CH3). ESI-MS (m/z): calcd for C31H45IrN2, 637.3134; found, CH), 28.32 (i-Pr-CH), 26.20 (i-Pr-CH3), 25.45 (i-Pr-CH3), 25.29 (i-\n637.3144, [M-PF6]+. Elemental analysis: calcd for C31H44F6IrN2P: C, Pr-CH3), 24.53 (i-Pr-CH3), 22.32 (Ar-i-Pr-CH3), 21.95 (Ar-i-Pr-\n47.62; H, 5.67; N, 3.58. Found: C, 47.52; H, 5.69; N, 3.57. CH3), 20.47 (Ar-CH3), 17.73 (CHCH3). ESI-MS (m/z): calcd for\n C29H40ClF6N2Ru, 553.1924; found, 553.1896, [M-PF6]+. Elemental\n analysis: calcd for C29H40ClF6N2PRu: C, 49.89; H, 5.78; N, 4.01.\n Found: C, 50.01; H, 5.76; N, 4.02.\n\n\n\n\n 4.2.5. Ir7amido. Red powder. Yield 40.3 mg (57.7%). 1H NMR (500\nMHz, CDCl3): \u03b4 9.02 (d, 1H), 8.04 (t, 1H), 7.75 (m, 1H), 7.63 (d,\n1H), 7.26 (d, 2H), 6.84 (d, 2H), 2.46 (s, 3H, Ar\u2212CH3), 1.57 (s, 15H,\nCp*\u2212CH3), 1.46 (s, 6H, CH3). Due to the limited solubility of this\ncomplex, 13C NMR spectroscopy was not performed. ESI-MS (m/z): 4.2.8. Ru3amine. Yellow powder. Yield 43.5 mg (61.1%). 1H NMR\ncalcd for C25H32IrN2, 553.2195; found, 553.2179, [M-PF6]+. (500 MHz, CDCl3): \u03b4 9.21 (s, 1H), 7.97 (s, 1H), 7.66 (s, 1H), 7.39\nElemental analysis: calcd for C25H32F6IrN2P: C, 43.04; H, 4.62; N, (m, 2H), 7.31 (d, 1H), 6.41 (m, 1H, CH\u2212N), 5.66 (d, 1H), 5.50 (d,\n4.02. Found: C, 43.16; H, 4.60; N, 4.03. 1H), 5.28 (d, 1H), 5.20 (m, 1H, NH), 5.08 (s, 1H), 3.44 (m, 1H, i-\n Pr\u2212CH), 3.31 (m, 1H, i-Pr\u2212CH), 2.68 (m, 1H, i-Pr\u2212CH), 2.06 (s,\n 3H, Ar\u2212CH3), 1.77 (m, 2H, CH2), 1.60 (d, 3H, i-Pr\u2212CH3), 1.40 (d,\n 3H, i-Pr\u2212CH3), 1.38 (d, 3H, i-Pr\u2212CH3), 1.21 (d, 3H, i-Pr\u2212CH3),\n 1.19 (d, 3H, i-Pr\u2212CH3), 0.97 (d, 3H, i-Pr\u2212CH3), 0.72 (t, 3H,\n CH2CH3). 13C NMR (126 MHz, CDCl3): \u03b4 160.79, 155.27, 141.44,\n 140.88, 140.78, 139.56, 128.04, 127.01, 126.42, 125.89, 122.98,\n 104.55, 100.62, 85.05, 83.86, 83.02, 82.37, 70.90 (CH), 30.26 (Ar-i-\n Pr-CH), 29.25 (i-Pr-CH), 28.36 (i-Pr-CH), 26.48 (CH2CH3), 25.68\n (i-Pr-CH3), 25.05 (i-Pr-CH3), 24.96 (i-Pr-CH3), 22.62 (i-Pr-CH3),\n 4.2.6. Ru1amine. Yellow powder. Using the general method, a 21.93 (Ar-i-Pr-CH3), 17.86 (Ar-i-Pr-CH3), 8.76 (Ar-CH3), 1.02\ncombination of pyridyl\u2212imine and pyridyl\u2212amine complexes was (CH2CH3). ESI-MS (m/z): calcd for C30H42ClN2Ru, 567.2080;\ninitially obtained. To isolate the pure pyridyl\u2212amine complex found, 567.2059, [M-PF 6 ] + . Elemental analysis: calcd for\nRu1amine, the reaction duration was reduced to 2 h, ensuring minimal C30H42ClF6N2PRu: C, 50.60; H, 5.94; N, 3.93. Found: C, 50.71; H,\noxidation and yielding the desired product without contamination 5.92; N, 3.94.\nfrom the oxidized pyridyl\u2212imine species. Yield 42.2 mg (61.7%). 1H\nNMR (500 MHz, CDCl3): \u03b4 9.36 (d, 1H), 7.92 (t, 1H), 7.71 (t, 1H),\n7.38 (m, 2H), 7.28 (d, 2H), 6.80 (m, 1H, NH), 5.85 (d, 1H), 5.58 (d,\n1H), 5.10 (q, 2H), 4.59 (m, 1H, CH2), 4.40 (m, 1H, CH2), 3.40 (m,\n1H, i-Pr\u2212CH), 2.87 (m, 1H, i-Pr\u2212CH), 2.74 (m, 1H, i-Pr\u2212CH), 1.99\n(s, 3H, Ar\u2212CH3), 1.52 (d, 3H, i-Pr\u2212CH3), 1.44 (d, 3H, i-Pr\u2212CH3),\n1.33 (d, 3H, i-Pr\u2212CH3), 1.23 (d, 3H, i-Pr\u2212CH3), 1.21 (m, 6H, i-Pr\u2212\nCH3). 13C NMR (126 MHz, CDCl3): \u03b4 159.71, 154.52, 142.38,\n139.88, 139.03, 138.07, 127.30, 126.26, 125.82, 123.22, 120.07,\n104.35, 98.67, 83.25, 83.11, 82.87, 80.72, 59.95 (CH2), 29.76 (Ar-i-Pr-\nCH), 27.82 (i-Pr-CH), 27.26 (i-Pr-CH), 25.07 (i-Pr-CH3), 24.82 (i- 4.2.9. Ru4amine. Yellow powder. Yield 50.8 mg (68.6%). 1H NMR\nPr-CH3), 24.51 (i-Pr-CH3), 21.58 (i-Pr-CH3), 21.11 (Ar-i-Pr-CH3), (500 MHz, CDCl3): \u03b4 9.40 (d, 1H), 7.97 (t, 1H), 7.73 (t, 1H), 7.38\n21.07 (Ar-i-Pr-CH3), 16.28 (Ar-CH3). ESI-MS (m/z): calcd for (d, 2H), 7.32 (m, 1H), 7.20 (d, 1H), 6.28 (d, 1H, CH\u2212N), 5.66 (d,\nC28H38ClN2Ru, 539.1767; found, 539.1685, [M-PF6]+. Elemental 1H), 5.45 (d, 1H), 5.21 (d, 1H), 5.14 (d, 1H), 4.63 (m, 1H,\nanalysis: calcd for C28H38ClF6N2PRu: C, 49.16; H, 5.60; N, 4.09. CH2CH(CH3)2), 3.36 (m, 1H, i-Pr\u2212CH), 2.88 (m, 1H, i-Pr\u2212CH),\nFound: C, 49.25; H, 5.58; N, 4.08. 2.71 (m, 1H, i-Pr\u2212CH), 1.96 (s, 3H, Ar\u2212CH3), 1.79 (m, 2H, CH2),\n 1.53 (d, 3H, i-Pr\u2212CH3), 1.46 (d, 3H, i-Pr\u2212CH3), 1.38 (d, 3H, i-Pr\u2212\n CH3), 1.35 (m, 6H, CH2CH(CH3)2), 1.18 (d, 3H, i-Pr\u2212CH3), 0.75\n (d, 3H, i-Pr\u2212CH3), 0.72 (d, 3H, i-Pr\u2212CH3). 13C NMR (126 MHz,\n CDCl3): \u03b4 164.96, 155.37, 143.93, 139.87, 139.81, 139.22, 128.10,\n 127.62, 126.31, 125.36, 121.87, 106.74, 99.54, 84.64, 83.80, 82.52,\n 81.87, 71.25 (CH), 49.91 (CH2), 30.88 (Ar-i-Pr-CH), 28.97 (i-Pr-\n CH), 28.19 (i-Pr-CH), 26.89 (CH2CH(CH3)2), 25.81 (i-Pr-CH3),\n 25.43 (CH2CH(CH3)2), 25.02 (i-Pr-CH3), 23.37 (i-Pr-CH3), 22.50\n (i-Pr-CH3), 21.81 (Ar-i-Pr-CH3), 21.72 (Ar-i-Pr-CH3), 16.98 (Ar-\n\n 10397 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nCH3). ESI-MS (m/z): calcd for C32H46N2Ru, 559.2626; found, ment data (Figures S1\u2212S95, and Tables S1\u2212S16)\n559.2659, [M-HCl-PF 6 ] + . Elemental analysis: calcd for (PDF)\nC32H47ClF6N2PRu: C, 51.92; H, 6.26; N, 3.78. Found: C, 51.80; H,\n6.28; N, 3.77. Accession Codes\n CCDC 2396390 (Ir3amine), 2396393 (Ir3imine), 2396396\n (Ir5amido), 2409367 (Ir7amido), 2396392 (Ru1amine), 2396391\n (Ru2amine), 2396395 (Ru4amine) and 2396394 (Ru5amido)\n contain the supplementary crystallographic data for this\n paper. These data can be obtained free of charge via www.\n ccdc.cam.ac.uk/data_request/cif, or by emailing data_\n request@ccdc.cam.ac.uk, or by contacting The Cambridge\n Crystallographic Data Centre, 12 Union Road, Cambridge\n CB2 1EZ, UK; fax: +44 1223 336033.\n 4.2.10. Ru5amido. Purple powder. Yield 46.7 mg (60.9%). 1H NMR\n(500 MHz, CDCl3): \u03b4 10.07 (d, 1H), 7.71 (dt, 2H), 7.21 (d, 1H),\n7.02 (d, 1H), 6.82 (d, 1H), 6.63 (s, 1H, CH\u2212N), 6.52 (s, 1H), 6.02\n \u25a0 AUTHOR INFORMATION\n Corresponding Authors\n(d, 1H), 5.79 (d, 1H), 5.52 (s, 2H), 4.75 (s, 1H), 3.01 (m, 2H, i-Pr\u2212 Lihua Guo \u2212 Key Laboratory of Life-Organic Analysis of\nCH), 2.70 (m, 1H, i-Pr\u2212CH), 2.20 (s, 3H, Ar\u2212CH3), 2.12 (s, 3H, Shandong Province, Key Laboratory of Green Natural\nAr\u2212CH3), 1.58 (s, 3H, Ar\u2212CH3), 1.38 (d, 3H, i-Pr\u2212CH3), 1.33 (d, Products and Pharmaceutical Intermediates in Colleges and\n3H, i-Pr\u2212CH3), 1.31 (d, 6H, i-Pr\u2212CH3), 1.28 (m, 6H, i-Pr\u2212CH3 Universities of Shandong Province, School of Chemistry and\n(3H)+Ar\u2212CH3 (3H)), \u22120.08 (d, 3H, i-Pr\u2212CH3). 13C NMR (126 Chemical Engineering, Qufu Normal University, Qufu\nMHz, CDCl3): \u03b4 167.69, 154.13, 142.40, 142.35, 139.04, 138.91, 273165, P. R. China; orcid.org/0000-0002-0842-9958;\n138.85, 138.70, 132.46, 129.96, 127.74, 127.22, 124.77, 124.63,\n124.30, 121.62, 101.36, 92.27, 86.38, 84.34, 83.52, 82.27, 81.34 (CH), Email: guolihua@qfnu.edu.cn\n31.44 (Ar-i-Pr-CH), 27.91 (i-Pr-CH), 27.89 (i-Pr-CH), 27.49 (i-Pr- Zhe Liu \u2212 Key Laboratory of Life-Organic Analysis of\nCH3), 27.36 (i-Pr-CH3), 25.50 (i-Pr-CH3), 23.42 (i-Pr-CH3), 23.40 Shandong Province, Key Laboratory of Green Natural\n(Ar-i-Pr-CH3), 23.03 (Ar-i-Pr-CH3), 21.44 (Ar\u2212CH3), 20.58 (Ar- Products and Pharmaceutical Intermediates in Colleges and\nCH3), 19.81 (Ar\u2212CH3), 18.74 (Ar\u2212CH3). ESI-MS (m/z): calcd for Universities of Shandong Province, School of Chemistry and\nC37H47N2Ru, 621.2783; found, 621.2872, [M-PF6]+. Elemental Chemical Engineering, Qufu Normal University, Qufu\nanalysis: calcd for C37H47F6N2PRu: C, 58.03; H, 6.19; N, 3.66. 273165, P. R. China; orcid.org/0000-0001-5796-4335;\nFound: C, 58.15; H, 6.16; N, 3.67. Email: liuzheqd@163.com\n Authors\n Zhihao Yang \u2212 Key Laboratory of Life-Organic Analysis of\n Shandong Province, Key Laboratory of Green Natural\n Products and Pharmaceutical Intermediates in Colleges and\n Universities of Shandong Province, School of Chemistry and\n Chemical Engineering, Qufu Normal University, Qufu\n 273165, P. R. China\n Heqian Dong \u2212 Key Laboratory of Life-Organic Analysis of\n 4.2.11. Ru6amido. Purple powder. Yield 44.5 mg (64.5%).1H NMR Shandong Province, Key Laboratory of Green Natural\n(500 MHz, CDCl3): \u03b4 9.62 (d, 1H), 7.65 (t, 1H), 7.49 (t, 1H), 6.98 Products and Pharmaceutical Intermediates in Colleges and\n(d, 1H), 6.93 (m, 1H), 5.58 (d, 1H), 5.52 (d, 1H), 5.31 (d, 1H), 5.29 Universities of Shandong Province, School of Chemistry and\n(d, 1H), 3.19 (m, 1H, i-Pr\u2212CH), 2.66 (m, 1H, i-Pr\u2212CH), 2.05 (m, Chemical Engineering, Qufu Normal University, Qufu\n1H, i-Pr\u2212CH), 1.42 (m, 1H, CH2CH3), 1.35 (m, 6H, i-Pr\u2212CH3),\n1.27 (m, 1H, CH2CH3), 1.09 (s, 3H, Ar\u2212CH3), 1.07 (d, 6H, i-Pr\u2212\n 273165, P. R. China\nCH3), 1.00 (s, 3H, CH3), 0.97 (d, 3H, i-Pr\u2212CH3), 0.86 (d, 3H, i-Pr\u2212 Kangning Lai \u2212 Key Laboratory of Life-Organic Analysis of\nCH3), 0.01 (t, 3H, CH2CH3). 13C NMR (126 MHz, CDCl3): \u03b4 Shandong Province, Key Laboratory of Green Natural\n167.57, 155.02, 151.73, 144.69, 141.74, 139.09, 127.05, 124.99, Products and Pharmaceutical Intermediates in Colleges and\n124.69, 124.57, 120.22, 104.98, 93.29, 86.17, 83.84, 81.79, 79.03, Universities of Shandong Province, School of Chemistry and\n78.99 (N\u2212C), 39.49 (Ar- i-Pr-CH), 31.32 (CH2), 28.75 (CH3), 27.71 Chemical Engineering, Qufu Normal University, Qufu\n(i-Pr-CH), 26.95 (i-Pr-CH), 26.89 (i-Pr-CH3), 26.51 (i-Pr-CH3), 273165, P. R. China\n24.33 (i-Pr-CH3), 24.05 (i-Pr-CH3), 23.14 (Ar-i-Pr-CH3), 22.78 (Ar-i- Hanxiu Fu \u2212 Key Laboratory of Life-Organic Analysis of\nPr-CH3), 17.60 (Ar-CH3), 9.70 (CH2CH3). ESI-MS (m/z): calcd for Shandong Province, Key Laboratory of Green Natural\nC31H43N2Ru, 545.2470; found, 545.2479, [M-PF6]+. Elemental\nanalysis: calcd for C31H43F6N2PRu: C, 53.98; H, 6.28; N, 4.06.\n Products and Pharmaceutical Intermediates in Colleges and\nFound: C, 53.89; H, 6.30; N, 4.05. Universities of Shandong Province, School of Chemistry and\n Chemical Engineering, Qufu Normal University, Qufu\n\n\u25a0\n*\n ASSOCIATED CONTENT\ns\u0131 Supporting Information\n 273165, P. R. China\n Yuwen Gong \u2212 Key Laboratory of Life-Organic Analysis of\n Shandong Province, Key Laboratory of Green Natural\nThe Supporting Information is available free of charge at Products and Pharmaceutical Intermediates in Colleges and\nhttps://pubs.acs.org/doi/10.1021/acs.inorgchem.4c05599. Universities of Shandong Province, School of Chemistry and\n Chemical Engineering, Qufu Normal University, Qufu\n Additional experimental details and methods, 1H and 273165, P. R. China\n 13\n C {1H}NMR spectra, and ESI-MS spectra for all Susu Li \u2212 Key Laboratory of Life-Organic Analysis of\n compounds, experimental images and biological experi- Shandong Province, Key Laboratory of Green Natural\n 10398 https://doi.org/10.1021/acs.inorgchem.4c05599\n Inorg. Chem. 2025, 64, 10379\u221210401\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n Products and Pharmaceutical Intermediates in Colleges and acylthiourea ligands in half-sandwich Ru(II) complexes and their\n Universities of Shandong Province, School of Chemistry and cytotoxic evaluation. Inorg. Chem. 2020, 59, 5072\u22125085.\n Chemical Engineering, Qufu Normal University, Qufu (14) Habtemariam, A.; Melchart, M.; Fern\u00e1ndez, R.; Parsons, S.;\n 273165, P. R. China Oswald, I. D. H.; Parkin, A.; Fabbiani, F. P. A.; Davidson, J. E.;\n Dawson, A.; Aird, R. E.; Jodrell, D. I.; Sadler, P. J. Structure-activity\n Mingbo Yue \u2212 Key Laboratory of Life-Organic Analysis of\n relationships for cytotoxic ruthenium(II) arene complexes containing\n Shandong Province, Key Laboratory of Green Natural N,N-, N,O-, and O,O-chelating ligands. J. Med. Chem. 2006, 49,\n Products and Pharmaceutical Intermediates in Colleges and 6858\u22126868.\n Universities of Shandong Province, School of Chemistry and (15) Cepeda, V.; Fuertes, M. A.; Castilla, J.; Alonso, C.; Quevedo,\n Chemical Engineering, Qufu Normal University, Qufu C.; Perez, J. M. Biochemical mechanisms of cisplatin cytotoxicity.\n 273165, P. R. China; orcid.org/0000-0002-8534-7606 Anti-Cancer Agents Med. Chem. 2007, 7, 3\u221218.\nComplete contact information is available at: (16) Giaccone, G.; Herbst, R. S.; Manegold, C.; Scagliotti, G.;\n Rosell, R.; Miller, V.; Natale, R. B.; Schiller, J. H.; von Pawel, J.;\nhttps://pubs.acs.org/10.1021/acs.inorgchem.4c05599\n Pluzanska, A.; et al. Gefitinib in combination with gemcitabine and\n cisplatin in advanced non-small-cell lung cancer: a phase III trial\u2013\nNotes INTACT 1. J. Clin. Oncol. 2004, 22, 777\u2212784.\nThe authors declare no competing financial interest. (17) Li, Y.; Tan, C. P.; Zhang, W.; He, L.; Ji, L. N.; Mao, Z. W.\n\n\u25a0 ACKNOWLEDGMENTS\nWe gratefully acknowledge the support provided by the\n Phosphorescent iridium(III)-bis-N-heterocyclic carbene complexes as\n mitochondria-targeted theranostic and photodynamic anticancer\n agents. Biomaterials 2015, 39, 95\u2212104.\n (18) Su, X. X.; Wang, W. J.; Cao, Q.; Zhang, H.; Liu, B.; Ling, Y. Y.;\nTaishan Scholars Program and the Young Talents Invitation Zhou, X. T.; Mao, Z. W. A carbonic anhydrase IX (CAIX)-anchored\nProgram of Shandong Provincial Colleges and Universities. We rhenium(I) photosensitizer evokes pyroptosis for enhanced anti-\nalso extend our thanks to Shiyanjia Lab (www.shiyanjia.com) tumor immunity. Angew. Chem., Int. 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