Potent Half-Sandwich 16-/18-Electron Iridium(III) and Ruthenium(II) Anticancer Complexes with Readily Available Amine-Imine Ligands.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Potent Half-Sandwich 16-/18-Electron Iridium(III) and Ruthenium(II)\n Anticancer Complexes with Readily Available Amine\u2212Imine Ligands\n Lihua Guo,* Pengwei Li, Jiaxing Li, Yuwen Gong, Xiaoyuan Li, Tingjun Wen, Xinxin Wu, Xinyi Yang,\n and Zhe Liu*\n Cite This: Inorg. Chem. 2023, 62, 21379\u221221395 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ABSTRACT: The synthesis and biological evaluation of stable 16-\n electron half-sandwich complexes have remained scarce. We herein\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:21:40 (UTC).\n\n\n\n\n present the different coordination modes (16-electron or 18-\n electron) between half-sandwich iridium(III) complexes and\n ruthenium(II) complexes derived from the same amine\u2212imine\n ligands chelating hybrid sp3-N/sp2-N donors. The 16-electron\n iridium(III) and 18-electron ruthenium(II) complexes with differ-\n ent counteranions were obtained and identified by various\n techniques. The promising cytotoxicity of these complexes against\n A549 lung cancer cells, cisplatin-resistant A549/DPP cells, cervical\n carcinoma HeLa cells, and human hepatocellular liver carcinoma HepG2 cells was observed with IC50 values ranging from 5.4 to 16.3\n \u03bcM. Moreover, these complexes showed a certain selectivity (selectivity index: 2.1\u22123.7) toward A549 cells and BEAS-2B normal\n cells. The variation of metal center, counteranion, 16/18-electron coordination mode, and ligand substituents showed little influence\n on the cytotoxicity and selectivity of these complexes. The mechanism of action study showed that these complexes could target\n mitochondria, induce the depolarization of the mitochondrial membrane, and promote the generation of intracellular reactive oxygen\n species (ROS). Further, the induction of cell apoptosis and the perturbation of the cell cycle in the G0/G1 phase were also observed\n for these complexes. Overall, it seems that the redox mechanism dominated the anticancer efficacy of these complexes.\n\n\n 1. INTRODUCTION complexes using different bidentate XY chelating ligands.\n Chemotherapy has been the primary approach to cancer Notably, XY has mostly been chosen as an N,N donor among\n treatment, despite significant advancements over the past 50 these complexes. One of the important N,N-chelating arene\u2212\n years. Platinum-based drugs, which mainly comprised cisplatin, ruthenium(II) anticancer complexes was RM175 containing an\n carboplatin, and oxaliplatin, have been well-studied in treating ethylenediamine (en) ligand (Scheme 1, I), which has\n various tumors.1,2 However, these anticancer drugs lack achieved success in both in vitro and in vivo cytotoxic\n selectivity, have many serious side effects, and can lead to assessments.21 Moreover, RM175 did not reveal any cross-\n drug resistance.3\u22126 As a result, many research efforts have resistance against cisplatin-resistant A2780cis cells, suggesting\n a distinctive mode of anticancer action.22,23 Sadler and co-\n focused on developing alternative anticancer complexes with\n workers have shown that iridium(III) complexes bearing N,N-\n high selectivity and novel mechanisms of action (MoAs) to\n chelating bipyridine (bpy) were approximately twice as potent\n reduce side effects and overcome drug resistance.7\u221212 Various\n than cisplatin against A2780 cancer cell line (Scheme 1, II).\n complexes of platinum group metals, including iridium,\n The enhanced anticancer efficiency of these complexes was\n rhodium, ruthenium, and osmium, have been developed for\n due to their increased hydrophobicity and DNA-binding\n this purpose. In particular, azole-based ruthenium complexes,\n activities.24 The Os(II) complex FY-26 with the asymmetric\n NAMI-A and KP1019, have entered clinical trials.13,14\n N,N-chelating donor has a novel mechanism of action centered\n In the last decade, platinum group-based half-sandwich\n on inducing dysfunctional mitochondria and increasing\n anticancer complexes of the formula [(\u03b75-Cpx)/(\u03b76-arene)M-\n reactive oxygen species (ROS) levels, thereby allowing it\n (XY)Z]0/+ (XY = bidentate chelating ligand; Z = monodentate\n labile ligand, mostly chosen as Cl\u2212; Cpx = substituted\n cyclopentadienyl; M = iridium, rhodium, ruthenium, and Received: October 4, 2023\n osmium) have provided a versatile and simple platform for Revised: November 27, 2023\n developing new organometallic anticancer drugs due to their Accepted: November 28, 2023\n structural diversity and unique MoAs.15\u221220 The majority of Published: December 14, 2023\n these studies have been directed toward the synthesis and\n investigation of 18-electron cationic and neutral anticancer\n\n \u00a9 2023 American Chemical Society https://doi.org/10.1021/acs.inorgchem.3c03471\n 21379 Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 1. Reported Organometallic N,N-chelating Half-Sandwich Platinum Group Metal Complexes and Our Current Work\n\n\n\n\npossible to combat cancer cell drug resistance and exert outcome.37 Mitochondrial-targeting drugs usually exhibit two\nselective activity toward cancer cells and normal cells (Scheme main characteristics: high positive charge and strong hydro-\n1, III).25\u221227 Our group has also been working to develop a phobicity.38 Since cancer cells have a higher mitochondria\nseries of platinum group-based half-sandwich complexes with membrane potential compared to normal cells,39 the hydro-\nN,N-chelating ligand systems. For example, the pyridyl-imine phobic cations tend to accumulate more in the mitochondria\nhalf-sandwich iridium(III) and ruthenium(II) complexes were of cancer cells. Furthermore, drugs with strong hydrophobic\nable to convert nicotinamide adenine dinucleotide (NADH) to properties can increase their affinity with mitochondrial\nNAD+, increase ROS levels, disrupt mitochondrial membranes membranes, leading to disruption of normal metabolic\n(causing mitochondrial dysfunction), and show promising homeostasis and dysregulation of intracellular ROS levels.40\u221242\ncytotoxicity toward A549 and HeLa cells (Scheme 1, IV and It should be noted that the coordination fashion of the\nV).28\u221230 Due to the constant oxidative stress caused by high above-mentioned N,N-chelating complexes was sp3-N/sp3-N\nlevels of ROS generation, cancer cells are highly sensitive to amine\u2212metal (H2N \u2192 metal, Scheme 1, I) or sp2-N/sp2-N\nchanges in the cell\u2019s redox state.31 Some selected pyridyl-imine imine\u2212metal (C\ufffdN \u2192 metal, Scheme 1, II\u2212VII). We were\nruthenium(II) complexes displayed anticancer selectivity interested in extending our research to hybrid sp3-N/sp2-N\ntoward A549 cells over BEAS-2B cells (Scheme 1, V).29,30 In chelating complexes. However, our preliminary attempt to\naddition, a set of iridium(III) and ruthenium(II) complexes obtain these complexes using sp3-N/sp2-N pyridyl-amine\nwith \u03b1-diimine N,N-chelating ligands were also prepared. ligands generated a mixture of pyridyl-imine (sp2-N/sp2-N)\nThese complexes can induce cell apoptosis through ROS- and pyridyl-amine (sp3-N/sp2-N) complexes due to their easily\nmediated signaling and display no cross-resistance with oxidizable nature, which made it difficult to investigate the\ncisplatin (Scheme 1, VI and VII).32\u221234 anticancer efficiency of these sp3-N/sp2-N-chelating complexes\n Mitochondria are closely associated with a variety of cellular (Scheme 1, VIII and IX).43 Herein, based on the [NH2, N]\nprocesses, such as energy generation, apoptotic induction, and Schiff base ligands, we prepared a series of amine\u2212imine (sp3-\nredox signaling.32,35,36 Cancer cells show varying degrees of N/sp2-N) half-sandwich iridium(III) and ruthenium(II)\nmitochondrial dysfunction, such as altered energy metabolism, complexes (Scheme 2). It was surprising that this simple and\nhigher mitochondrial membrane potential (MMP), and readily accessible Schiff base, which showed the hybrid sp3-N/\nincreased oxidative stress. These characteristics offer chances sp2-N donor, has not yet been used in synthesizing platinum\nto target cancer cell mitochondria for an optimal therapeutic group metal-based half-sandwich complexes. The different\n 21380 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 2. Synthesis of Amine\u2212Imine Ligands (a) and the Corresponding Half-Sandwich Iridium(III) and Ruthenium(II)\nComplexes with Cl\u2212 (b) and PF6\u2212 (c) as Counteranions\n\n\n\n\ncoordination modes between iridium(III) (16-electron, five- (v/v, ca. 1/1) at room temperature yielded 16-electron [NH,\ncoordinated, without monodentate labile Cl\u2212) and ruthenium- N]-coordinated iridium(III) complexes Ir1\u2212Ir2 and 18-\n(II) (18-electron, six-coordinated) complexes were observed. electron [NH2, N]-coordinated ruthenium(II) complexes\nNotably, the synthesis of stable 16-electron platinum group Ru1\u2212Ru2 as Cl\u2212 salts (Scheme 2b), Cl\u2212 as the counteranion\nmetal-based half-sandwich complexes and their biological with the formula of [(\u03b75-Cpx)Ir(NH, N)Cl]Cl and ([(\u03b76-p-\nevaluation have been rarely reported.44 All of the complexes cymene)Ru(NH2, N)Cl]Cl). Likewise, treatment of D1\u2212D3\nin this system were highly active toward several cancer cell with ligands L1\u2212L5 and an excess of ammonium salt NH4PF6\nlines, showed the ability to circumvent platinum resistance, and led to the formation of 16-electron iridium(III) complexes\nexhibited certain selectivity toward cancer cells over normal Ir3\u2212Ir8 and 18-electron ruthenium(II) complexes Ru3\u2212Ru7\ncells. In particular, their possible MoAs in vitro, including as the PF6\u2212 salts in 46\u221257% isolated yields (Scheme 2c). Thus,\nmitochondria-targeting, ROS production, and cell apoptosis, NH4PF6 served only as a reagent for anion exchange in this\nwere determined. reaction. All of these new complexes were air-stable and highly\n soluble in commonly used solvents like acetonitrile, dimethyl\n2. RESULTS AND DISCUSSION sulfoxide (DMSO), methanol and dichloromethane but poorly\n 2.1. Synthesis and Characterizations. The amine\u2212 soluble in aqueous solution. Therefore, nontoxic amounts of\nimine Schiff base ligands L1\u2212L5 can be easily prepared from a DMSO were used in the subsequent biological experiments to\nsimple single-step p-toluenesulfonic acid (TsOH)-catalyzed assist dissolution. The purity and identity of these complexes\nreaction of 2-aminobenzaldehyde with anilines in 67\u221274% have been determined through spectroscopic analysis (1H,\n 13\nisolated yields (Scheme 2a). Ligands L2\u2212L5 were previously C{1H} NMR, and mass spectra) and CHN elemental\nknown.45,46 The identity of ligand L1 was also verified by 1H analysis. The 1H NMR spectra of 16-electron iridium(III)\nNMR (Figure S1), 13C{1H} NMR (Figure S2), and mass complexes showed two characteristic peaks corresponding to\nspectrometry (Figure S35). This type of amine\u2212imine ligand the proton of the NH group (chemical shift: 11.80\u221215.31\nprovides an easy-to-handle, cheap, and versatile platform for ppm) and the CH\ufffdN group (chemical shift: 8.68\u22129.32 ppm).\nthe development of platinum group metal-based half-sandwich However, the signal for the NH group of Ir3 was not observed.\ncomplexes chelating hybrid sp3-N/sp2-N donors. In the case of 18-electron ruthenium(II) complexes, the\n The chloro-bridged bimetallic iridium(III) precursors D1 characteristic peak corresponding to CH\ufffdN was at 8.30\u22128.55\n([(\u03b75-Cp*)IrCl2]2), D2 ([(\u03b75-Cpph)IrCl2]2), and ruthenium- ppm, which was similar to the 16-electron iridium(III)\n(II) precursor D3 ([(\u03b76-p-cymene)RuCl2]2) were prepared complexes. The signals of each proton of the NH2 group in\naccording to the literature.44,47 Reactions of D1 and D3 with Ru1 and Ru2 are displayed separately. One peak correspond-\nthe corresponding amine\u2212imine ligands in CH3OH/CH2Cl2 ing to the NH2 group was at 10.13 and 10.47 ppm, while the\n 21381 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. X-ray crystal structures of complexes Ir1 (a), Ir2 (b), Ir3 (c), Ir4 (d), Ir6 (e), Ir8 (f), and Ru4 (g) with the thermal ellipsoids drawn at\nthe 50% probability level. The hydrogen atoms in all of these complexes and PF6\u2212 in Ir3, Ir4, Ir6, Ir8, and Ru4 have been omitted for clarity.\n\nother peak was at 4.95\u22125.01 ppm. However, only one peak, ruthenium(II) complexes. The reaction of 18-electron complex\ncorresponding to one of the exchangeable NH2 protons, was Ru3 with a slight excess of deprotonating agent NaOAc in the\nobserved in the 1H NMR spectra of Ru3\u2212Ru7. In the 13C{1H} mixture of dichloromethane and methanol at room temper-\nNMR spectra, the characteristic peaks of these complexes were ature afforded 16-electron complex Ru8 in 95% isolated yield\nat 160.44\u2212164.29 ppm (Figures S4, S6, S8, S10, S12, S14, S16, (Scheme 3). The molecular structure of Ru8 was determined\nS18, S20, S22, S24, S26, S28, S30 and S32), which were\nassigned to the imine carbon of the CH\ufffdN group. Moreover, Scheme 3. Synthesis of 16-Electron Ru8 in the Presence of\nsingle-crystal structures were also obtained for some typical Base NaOAc\ncomplexes.\n The molecular structure of Ir1\u2212Ir4, Ir6, Ir8, and Ru4 was\nconfirmed using single-crystal X-ray diffraction analysis, as\nshown in Figure 1a\u2212g and Tables S1\u2212S2. The iridium(III)\ncomplexes Ir1\u2212Ir4, Ir6, and Ir8 had an unsaturated 16-\nelectron geometry without coordination of chlorine atoms to\nthe metal center, while the ruthenium(II) complex Ru4\nadopted an 18-electron six-coordinated \u201cthree-legged piano-\nstool\u201d geometry with Cl\u2212 as the monodentate labile ligand.\nThe Ir\u2212N1 bonds (1.924 to 1.996 \u00c5) in these 16-electron by 1H, 13C{1H} NMR (Figures S33 and S34), elemental\ncomplexes were shorter than the Ir\u2212N2 bond distances analysis, mass spectrum (Figures S51), and X-ray crystallog-\n(1.985\u22122.068 \u00c5). In contrast, the Ru\u2212N1 bond (metal\u2212 raphy (Figure 2). A frequency shift of 13.28 ppm was observed\namine bond: 2.144 \u00c5) in the 18-electron complex Ru4 was in the 1H NMR spectra of Ru8 due to the proton of the NH\nlonger than that of Ru\u2212N2 bond distance (metal\u2212imine bond: group. Ru8 adopted a 16-electron five-coordinated geometry\n2.114 \u00c5). This difference in bond lengths may originate from and no monodentate labile group Cl\u2212 was bound to the metal\nthe different coordination modes between the iridium(III) and center (Figure 2). In contrast to the 18-electron six-\nruthenium(II) complexes. The distances of the C\u2212N1 single coordinated ruthenium(II) complex Ru4, the Ru\u2212N1 bond\nbond (1.343\u22121.445 \u00c5) on the six-membered chelating ring of (metal\u2212amine bond: 1.943 \u00c5) in the 16-electron complex Ru8\nthese complexes were slightly longer than those of the C\ufffdN2 was shorter than the Ru\u2212N2 bond distance (metal\u2212imine\nbond (1.292\u22121.404 \u00c5) on the six-membered chelating ring. bond: 2.014 \u00c5). However, this result was consistent with the\nThe variation of counteranions from Cl\u2212 to PF6\u2212 had little 16-electron iridium(III) complexes in this system, further\nimpact on the bond distances and angles of these complexes supporting the key role of coordination mode in determining\n(Ir1 vs Ir3; Ir2 vs Ir6). the metal\u2212nitrogen bond lengths.\n Further, our interest in 16-electron five-coordinated 2.2. Absorption and Emission Spectroscopy. The UV\u2212\nruthenium(II) complexes prompted us to perform the vis absorption spectra of Ir1\u2212Ir8 (Figure 3a) and Ru1\u2212Ru8\ndeprotonation reaction of these 18-electron amine\u2212imine (Figure 3b) were recorded at 37 \u00b0C in methanol solutions. The\n 21382 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n nm, respectively. These absorption bands that fall below 320\n nm were associated with \u03c0\u2212\u03c0* ligand-centered spin-allowed\n transitions. Additionally, the absorption bands between 320\n and 490 nm can be attributed to spin-allowed charge transfer\n from metal to ligand. Notably, the absorption behavior of these\n complexes was similar to that of earlier reported half-sandwich\n iridium(III) and ruthenium(II) complexes.48\u221251\n Upon excitation at \u03bbex = 398 nm, Ir1\u2212Ir8 (Figure 3c) and\n Ru1\u2212Ru8 (Figure 3d) exhibited emission maxima (\u03bbem)\n ranging from 465 to 477 nm (Ir1: 471 nm, Ir2: 476 nm, Ir3:\n 477 nm, Ir4: 472 nm, Ir5: 470 nm, Ir6: 465 nm, Ir7: 472 nm,\n Ir8: 475 nm, Ru1: 472 nm, Ru2: 471 nm, Ru3: 472 nm, Ru4:\n 465 nm, Ru5: 468 nm, Ru6: 468 nm, Ru7: 470 nm, Ru8: 472\n nm) at 37 \u00b0C in methanol solutions. Overall, the emission\nFigure 2. X-ray crystal structures of complex Ru8 with the thermal spectra of these complexes were insensitive to the change in\nellipsoids drawn at the 50% probability level. The hydrogen atoms metal ions, counteranion (Cl\u2212 or PF6\u2212), coordination mode\nand PF6\u2212 have been omitted for clarity. (16- or 18-electron), and ligand substituents. The relative\n emission quantum yields (\u03a6) of complexes Ir3, Ru3, and Ru8,\niridium(III) complexes Ir1\u2212Ir8 exhibited a relatively sharp measured using fluorescein as the standard, were relatively low\nband with a maximum in the range of 265\u2212294 nm. In in ethanol solutions. Specifically, Ir3 had a quantum yield of\naddition, two broad and less intense bands with maxima at 0.148, Ru3 had a quantum yield of 0.084, and Ru8 had a\napproximately 320 and 406 nm were observed. Similarly, the quantum yield of 0.065. The average lifetimes of Ir3, Ru3, and\nruthenium(II) complexes Ru1\u2212Ru8 displayed a sharp band Ru8 were 2.85, 1.91, and 1.97 ns, respectively (Figure S52),\nwith a maximum at 285\u2212320 nm, as well as two very weak and suggesting that these complexes were fluorescent. It should be\nbroad bands with a maximum at approximately 370 and 490 noted that the similar weakly fluorescent behavior has also\n\n\n\n\nFigure 3. (a) UV\u2212visible absorbance spectra of complexes Ir1\u2212Ir8 (20 \u03bcM) in methanol solutions at 37 \u00b0C. (b) The UV\u2212visible absorbance\nspectra of complexes Ru1\u2212Ru8 (20 \u03bcM) in methanol solutions at 37 \u00b0C. (c) Normalized emission spectra of complexes Ir1\u2212Ir8 (20 \u03bcM) in\nmethanol at 37 \u00b0C (Ir1\u2212Ir8: \u03bbex = 398 nm). (d) Normalized emission spectra of complexes Ru1\u2212Ru8 (20 \u03bcM) in methanol at 37 \u00b0C (Ru1\u2212Ru8:\n\u03bbex = 398 nm).\n\n 21383 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 1. IC50 Values of Complexes Ir1\u2212Ir8 and Ru1\u2212Ru8 Tested toward Cancer and Normal Cell Lines and Comparison with\nCisplatin\n IC50 (\u03bcM)\n complexes A549 A549/DDP HeLa HepG2 BEAS-2B SIa\n Ir1 11.17 \u00b1 0.06 13.06 \u00b1 0.41 10.31 \u00b1 0.25 16.29 \u00b1 0.09 23.36 \u00b1 0.24 2.1\n Ir2 11.01 \u00b1 0.22 12.98 \u00b1 0.16 10.48 \u00b1 0.13 15.35 \u00b1 0.16 23.04 \u00b1 0.17 2.1\n Ir3 10.75 \u00b1 0.14 12.23 \u00b1 0.13 9.89 \u00b1 0.37 14.83 \u00b1 0.06 22.66 \u00b1 0.21 2.1\n Ir4 10.01 \u00b1 0.16 11.21 \u00b1 0.19 9.74 \u00b1 0.32 14.27 \u00b1 0.15 22.89 \u00b1 0.06 2.3\n Ir5 8.61 \u00b1 0.05 11.31 \u00b1 0.25 9.02 \u00b1 0.18 12.16 \u00b1 0.31 21.32 \u00b1 0.25 2.5\n Ir6 8.34 \u00b1 0.16 10.37 \u00b1 0.11 8.85 \u00b1 0.06 11.98 \u00b1 0.24 21.84 \u00b1 0.32 2.6\n Ir7 6.32 \u00b1 0.09 8.64 \u00b1 0.09 7.15 \u00b1 0.03 9.88 \u00b1 0.32 20.25 \u00b1 0.54 3.2\n Ir8 5.41 \u00b1 0.19 7.23 \u00b1 0.06 6.26 \u00b1 0.08 9.29 \u00b1 0.14 20.17 \u00b1 0.34 3.7\n Ru1 8.29 \u00b1 0.10 9.94 \u00b1 0.07 8.94 \u00b1 0.31 11.34 \u00b1 0.18 22.65 \u00b1 0.31 2.7\n Ru2 8.65 \u00b1 0.06 9.52 \u00b1 0.03 8.65 \u00b1 0.10 11.87 \u00b1 0.25 22.18 \u00b1 0.09 2.6\n Ru3 7.41 \u00b1 0.05 8.23 \u00b1 0.14 7.98 \u00b1 0.13 10.36 \u00b1 0.26 21.17 \u00b1 0.08 2.9\n Ru4 6.91 \u00b1 0.24 8.14 \u00b1 0.22 6.88 \u00b1 0.06 9.59 \u00b1 0.09 21.51 \u00b1 0.22 3.1\n Ru5 6.64 \u00b1 0.07 8.31 \u00b1 0.15 6.25 \u00b1 0.31 9.29 \u00b1 0.06 20.98 \u00b1 0.11 3.2\n Ru6 6.52 \u00b1 0.03 8.25 \u00b1 0.19 6.56 \u00b1 0.13 10.67 \u00b1 0.29 21.89 \u00b1 0.17 3.4\n Ru7 6.26 \u00b1 0.06 8.47 \u00b1 0.08 6.05 \u00b1 0.22 11.78 \u00b1 0.30 21.78 \u00b1 0.36 3.5\n Ru8 9.12 \u00b1 0.15 10.16 \u00b1 0.12 9.89 \u00b1 0.13 12.98 \u00b1 0.33 22.18 \u00b1 0.28 2.4\n Cisplatin 23.96 \u00b1 0.23 >100 7.55 \u00b1 0.06 22.7 \u00b1 0.58 28.27 \u00b1 0.28 1.2\na\n SI: selectivity index represents the IC50 ratio of BEAS-2B normal cells to A549 cancer cells.\n\nbeen observed in some other reported half-sandwich iridium- ployed to proceed with further anticancer studies under\n(III) and ruthenium(II) complexes.43,44,49 Probably, the aqueous conditions.\nexploration of MoAs through bioimaging can be facilitated Some rarely reported half-sandwich 16-electron iridium(III)\nby the photoluminescence characteristic of these complexes. and ruthenium(II) complexes have been shown to react with\n 2.3. Solution Stability and Reactivity. To investigate the two-electron donors to form the stable 18-electron com-\nstability of the complexes in an aqueous solution, Ir3, Ir4, Ru1, pounds.57,58 When PPh3, CH3CN, or CO was added into an\nRu3, Ru5, and Ru8 were monitored at 37 \u00b0C in 85% DMSO- NMR tube containing a CDCl3 solution of 16-electron\nd6/15% phosphate-buffered saline (PBS) (v/v, pH \u2248 7.2, complexes Ir6, Ir7, and Ru8, no additional peaks in the 1H\nprepared from D2O) solutions by 1H NMR (Figures S53\u2212 NMR spectra of Ir6, Ir7, and Ru8 were detected over a period\nS58). No additional peaks were observed in the 1H NMR of 24 h (Figures S61\u2212S65), indicating that the 16-electron\nspectra for 24 h, and the assignment of protons was completely iridium(III) and ruthenium(II) complexes in this system\nin agreement with their molecular structures, suggesting that exhibited inert reactivity toward two-electron donors, such as\nno decomposition or ligand dissociation occurred and these PPh3, CH3CN, and CO. The inert reactivity of these\ncomplexes had sufficient stability under test conditions. complexes was also consistent with their above-mentioned\n The stability of these complexes had also been estimated at stable nature in aqueous solutions.\n37 \u00b0C in 20% DMSO/80% PBS (v/v, pH \u2248 7.2, prepared from 2.4. Cytotoxicity. With cisplatin as the standard control,\nH2O) using UV\u2212vis spectroscopy at different time intervals the cytotoxicity of Ir1\u2212Ir8 and Ru1\u2212Ru8 toward A549 lung\nover a period of 8 h. Negligible or minor changes were cancer cells, cisplatin-resistant A549/DPP cells, cervical\n carcinoma HeLa cells, human hepatocellular liver carcinoma\nobserved in the absorption spectra of these complexes (Figures\n HepG2 cells, and noncancerous BEAS-2B cells was evaluated\nS59 and S60), evidencing their stability in diluted solutions\n using MTT assay (Table 1). Neither the bimetallic precursors\nwith a high content of water, which was also in agreement with\n nor the free amine\u2212imine ligands displayed cytotoxicity against\nthe NMR analysis. However, the absorption intensity in the A549 and HeLa cancer cells (IC50 > 100 \u03bcM) (Table S3).\nspectra of 18-electron complexes Ru1 and Ru2 changed, while However, all of the amine\u2212imine complexes Ir1\u2212Ir8 and\nno obvious shift for the absorption bands was observed, Ru1\u2212Ru8 showed strong cytotoxicity toward A549, A549/\nsuggesting the hydrolysis of metal\u2212Cl bond (Cl\u2212/H2O DDP, HeLa, and HepG2 cells with IC50 values in a narrow\nexchange) in Ru1 and Ru2, which were consistent with range of 5.41\u221211.17, 7.23\u221213.06, 6.05\u221210.48, and 9.29\u221216.29\nsome reported ruthenium(II) complexes containing the \u03bcM, respectively, which were comparable to or even better\nmonodentate labile chloride ligand.52\u221254 Notably, the hydrol- than commercial cisplatin. As a result, the anticancer efficiency\nysis of metal\u2212Cl bond generally represented an activation step of these complexes arose from the chelation of the free ligands\nfor a large number of reported anticancer complexes.55,56 Half- with the iridium(III) or ruthenium(II) ion. Specifically, these\nlives (t1/2) and hydrolysis rate constants (k) of Ru1 (t1/2 = 129 complexes were approximately 2\u22124 times more potent than\nmin, k = 0.00538 min\u22121) and Ru2 (t1/2 = 176 min, k = 0.00394 cisplatin toward A549 cancer cells (5.41\u221211.17 vs 23.96 \u03bcM).\nmin\u22121) were calculated by fitting the absorption difference to Interestingly, all of these iridium(III) and ruthenium(II)\npseudo-first-order kinetics. Hence, the complexes in this complexes also showed high cytotoxicity against cisplatin-\nsystem were fairly stable or underwent a relatively slow resistant A549/DPP cells with IC50 values in the range 7.23\u2212\nhydrolysis rate in comparison with the reported half-sandwich 13.06 \u03bcM, suggesting the different MoAs of these complexes\niridium(III) and ruthenium(II) complexes chelating N,N with cisplatin. The trend in potency was maintained across the\ndonors.24,54,56 Consequently, these complexes can be em- different cell lines, indicating that the MoAs were not\n 21384 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 4. (a) Effects of temperatures (37 or 4 \u00b0C), chloroquine (50 \u03bcM), and CCCP (50 \u03bcM) on cellular uptake of Ir3 (2 \u03bcM). Scale bar: 20 \u03bcm,\n\u03bbex = 405 nm, \u03bbem = 430\u2212490 nm. (b) Effects of temperatures (37 or 4 \u00b0C), chloroquine (50 \u03bcM), and CCCP (50 \u03bcM) on cellular uptake of Ru3\n(2 \u03bcM). Scale bar: 20 \u03bcm, \u03bbex = 405 nm, \u03bbem = 430\u2212490 nm.\n\ndependent on the cell type. Notably, the variation of metal changes in their surroundings. When small-molecule com-\ncenter (Ir vs Ru), counteranion (Cl\u2212 vs PF6\u2212), coordination plexes bind to these residues, fluorescence emission can be\nmode (16-electron vs 18-electron) and ligand substitution suppressed. The increase in the concentration of the complexes\nshowed little impact on the cytotoxicity of these complexes. caused a decrease and red shift in the absorption peak at 229\nHowever, the introduction of the extended lipophilic phenyl nm, which may be due to the effect of inducing \u03b1-helix\nsubstituents on the \u03b75-Cpx ring of the iridium(III) complexes perturbations and the impact of polar solvents.59\u221264 In\nled to the increase of the cytotoxicity toward all of the cell lines addition, a progressive increase without any shift was observed\n(Ir3 vs Ir8), which was in agreement with the increasing trend in the absorption peak of BSA at 276 nm for these complexes,\nof the previously reported N,N-chelating half-sandwich suggesting a kind of tiny variation of microenvironment of\niridium(III) complexes bearing bpy or \u03b1-diimine ligands.24,34 aromatic amino acid residues (Tyr and Trp) in BSA.65,66 The\nMoreover, these complexes showed a certain selectivity toward fluorescence intensity of BSA showed a regular decline at 353\nA549 over BEAS-2B cells with the values of selectivity index nm with an increase in the concentration of Ir3, Ir4, Ru4, and\nranging from 2.1 to 3.7. However, all of the iridium(III) and Ru8, suggesting that these complexes interacted with BSA\nruthenium(II) complexes in this system were still active toward through a static quenching mode.67\nnoncancerous BEAS-2B cells. Synchronous fluorescence spectrometry is a valuable tool for\n 2.5. Interaction with Nucleobases. The potential understanding the conformational changes that occur in BSA\nbinding to the DNA model nucleobase 9-methyladenine (9- after adding complexes. When the wavelength interval remains\nMeA) of Ir4, Ru1, Ru2, Ru3, Ru5, and Ru8 was evaluated stable at either 15 or 60 nm, synchronized fluorescence could\nusing 1H NMR spectroscopy in a solution of 85% DMSO-d6/ reveal the characteristic information on Trp or Tyr residues in\n15% D2O (Figures S66\u2212S71). No coordination reaction BSA. The emission wavelength of Trp decreased at 276 nm\noccurred between 9-MeA and these complexes over a period (\u0394\u03bb = 60 nm) with a red shift of 4 nm, while the emission\nof 24 h. In addition, the formation of nucleobase adducts was wavelength of Tyr decreased at 287 nm (\u0394\u03bb = 15 nm) with a\nalso not detected via mass spectrometry. Therefore, DNA minor red shift of 1 nm (Figure S73). These observations\nbinding may not be the primary MoAs for these amine\u2212imine suggested that Ir3, Ir4, Ru4, and Ru8 primarily affected the\niridium(III) and ruthenium(II) complexes, which was further Trp microregion\u2019s conformation when binding to BSA.\nsupported by their low colocalization efficiency in the nucleus 2.7. Cellular Uptake Pathway. Since these complexes\n(see Section 2.8). displayed fluorescence characteristics, laser confocal micros-\n 2.6. Protein Binding Studies. It is important to copy was used to determine how these complexes entered the\nunderstand the interactions between anticancer agents and cells. According to the observation of confocal microscopy\nproteins in cells. Serum albumin (SA) is a major protein in images for the selected 16-electron Ir3 and 18-electron Ru3 at\nblood plasma that helps transport and metabolize complexes. \u03bbex = 405 nm at 37 \u00b0C, it seemed that Ir3 and Ru3 were able to\nIn this study, bovine serum albumin (BSA) was used as a effectively enter A549 cells after 1 h of incubation, as shown by\nsubstitute for human serum albumin (HSA) because it is the presence of punctate green fluorescence in the cytoplasm\nstructurally similar and easier to obtain. The binding potency (Figure 4a,b). It is well-known that small-molecule drugs can\nof complexes Ir3, Ir4, Ru4, and Ru8 with BSA was evaluated penetrate cells through either the energy-dependent or energy-\nby UV\u2212vis absorption and fluorescence spectra (Figure S72). independent pathway.68 In comparison with the control group\nBoth the reference and sample cuvettes were treated with the incubated at 37 \u00b0C, the fluorescence intensity of A549 cells\ncorresponding complexes to eliminate self-absorption. The decreased significantly when incubated with Ir3 or Ru3 at a\nfluorescence characteristic of BSA is usually due to two protein low temperature (4 \u00b0C) or pretreated with carbonyl cyanide 3-\nresidues called tyrosine (Tyr) and tryptophan (Trp). These chloro-phenylhydrazone (CCCP, a metabolic inhibitor). These\nresidues contain aromatic amino acids that are sensitive to results indicated that the cellular uptake mechanism for Ir3\n 21385 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 5. Determination of intercellular localization of Ir3 (a) and Ru3 (b) by confocal microscopy. A549 cells were incubated with Ir3 and Ru3\n(2 \u03bcM) for 1 h at 37 \u00b0C and then coincubated with DAPI (1 \u03bcg/mL), MTDR (500 nM), or LTDR (75 nM) for 1 h, respectively. Ir3 and Ru3, \u03bbex\n= 405 nm, \u03bbem = 460\u2212520 nm; DAPI, \u03bbex = 345 nm, \u03bbem = 410\u2212455 nm; MTDR, \u03bbex = 644 nm, \u03bbem = 660\u2212720 nm; LTDR, \u03bbex = 594 nm, \u03bbem =\n600\u2212660 nm. Scale bar: 20 \u03bcm. The green, red, and blue fluorescence represent Ir3 and Ru3, mitochondria or lysosome, and nucleus, respectively.\n\nand Ru3 was energy-dependent. Further, there was no Hence, the high hydrophobicity of these complexes may also\nsignificant difference in the intracellular fluorescence intensity contribute to the mitochondria-targeting.\nbetween A549 cells treated with the endocytosis inhibitor 2.9. Mitochondrial Membrane Depolarization. Since\nchloroquine and the untreated cells, indicating that endocytosis these complexes could selectively accumulate in the\nwas not responsible for the uptake pathway of Ir3 and Ru3. mitochondria, the possible influence of these complexes on\n 2.8. Cellular Localization. In order to evaluate the the functional status of mitochondria was also investigated.\npossible cellular target of these complexes, intracellular The maintenance of the mitochondrial membrane potential\nlocalization analysis in different organelles was also measured (MMP, \u25b3\u03c8m) is essential for mitochondrial integrity and\nby using confocal microscopy (Figure 5a,b). 4,6-Diamino-2- bioenergetics function. The loss of MMP is often regarded as\nphenyl indole (DAPI), Mito Tracker Red CM-H2XRos an early event in the mitochondrion-mediated apoptosis\n(MTDR), and LysoTracker Red DND-99 (LTDR) were pathway. Thus, analysis of \u25b3\u03c8m in A549 cancer cells was\nemployed as the nucleus and mitochondrial and lysosome performed after exposure to Ir3 or Ru3 at concentrations of\nprobes, respectively. The A549 cells were dual-stained with 0.5, 1, and 2 \u00d7 IC50. The changes in MMP can be detected by\nthese organelle-specific probes and Ir3 or Ru3. After treatment JC-1 staining using flow cytometry. A decrease in the ratio of\nfor 1 h, distinct green fluorescence in the cytoplasm was the red to green fluorescence intensity can be observed when\ndetected, suggesting that Ir3 and Ru3 effectively penetrated mitochondrial depolarization occurred. The MMP in A549\nA549 cells. Ir3 and Ru3 showed a negligible degree of merging cells displayed a significant decrease compared with the\nwith DAPI or LTDR. The Pearson correlation coefficient untreated cells. The concentration-dependent mitochondrial\n(PCC) values for DAPI and LTDR were both very low (Ir3: dysfunction in A549 cells was also observed for both Ir3 and\nPCC = 0 for DAPI and PCC = 0.15 for LTDR; Ru3: PCC = 0 Ru3. When the concentration of Ir3 and Ru3 was increased\n from 0.5 \u00d7 IC50 to 2 \u00d7 IC50, the percentage of A549 cells\nfor DAPI and PCC = 0.09 for LTDR), which suggested that\n suffering mitochondrial membrane depolarization showed an\nthese complexes were not effectively localized in the nucleus\n increase from 34.47 and 13.93% to 57.17 and 42.16%,\nand lysosome. However, these complexes can effectively\n respectively (Figure 6a,b). These results were consistent with\naccumulate in mitochondria with high PCC values (Ir3:\n the aforementioned mitochondria-targeting behavior of Ir3\nPCC = 0.85; Ru3: PCC = 0.88). These results indicated that\n and Ru3. Therefore, these complexes could exert their\nIr3 and Ru3 had a selective localization in the mitochondria, anticancer actions by targeting mitochondria and inducing\nand the cytotoxicity of these complexes may be caused by mitochondrial dysfunction.\nmitochondria-mediated cell death. Since cancer cells contained 2.10. Cellular ROS Determination. The production of\nmore mitochondria than normal cells, they were much more intracellular reactive oxygen species (ROS) is strongly\nsensitive to the disruption of mitochondria than normal cells, associated with mitochondria.69\u221271 Previous studies have\nwhich may lead to certain anticancer selectivity (SI: 2.1\u22123.7) shown that dysfunctional mitochondria are unable to\nof the complexes in this system. Notably, the high positive \u03b6- effectively regulate ROS production, resulting in elevated\npotential of Ir3 (46.58 \u00b1 0.19) and Ru3 (50.93 \u00b1 0.32) was oxidative stress in cancer cells.26,72 It has been reported that\nobserved (Figure S74), which could contribute to targeting anticancer complexes that produce high levels of ROS can\nmitochondria with negative charges on the surface after disturb the redox balance in cells, thus leading to cell apoptosis\nentering the cytosol. Since the mitochondria-targeting was and damage.70,73,74 Therefore, we also became interested in\nlikely associated with the hydrophobicity of these complexes, investigating the effect of these complexes on intracellular ROS\nthe distribution coefficient, i.e., log P value of the selected levels in A549 and cisplatin-resistant A549/DPP cancer cells.\ncomplexes Ir3, Ru3, and Ru8, was also measured using the The total ROS levels induced by the typical complexes Ir3 and\nshake-flask method. These complexes showed almost equal Ru3 at the concentrations of 0.5, 1, and 2 \u00d7 IC50 were\nlog P values ranging from 1.23 to 1.49 (Ir3: 1.49; Ru3: 1.23; measured by fluorescence microscopy after staining with the\nRu8: 1.33), suggesting high hydrophobicity. The small change probe DCFH-DA (Figures 7a\u2212c, S75, and S76). Compared to\nin the hydrophobicity of these complexes was also consistent the control cells, the treated A549 cells with complexes Ir3 and\nwith their similar cytotoxicity toward various cancer cells. Ru3 showed a concentration-dependent increase in intra-\n 21386 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n (Figure S77). The decrease in the absorption intensity of the\n NADH band (339 nm) can be used to determine the\n conversion of NADH to NAD+. The calculated turnover\n numbers (TONs) of Ir3 (5.47), Ru3 (9.81), and Ru8 (6.43)\n were quite similar, which was also in agreement with their\n small change in the ROS generation and cytotoxicity. Thus, the\n catalytic conversion of NADH to NAD+ for these amine\u2212\n imine iridium(III) and ruthenium(II) complexes may also be\n one of the mechanism actions for the generation of ROS.\n 2.11. Apoptosis. One potential goal of anticancer agents is\n to target cancer cells and trigger their death through the\n apoptosis pathway. It has been found that the induction of\n apoptosis was closely related to the disruption of redox\n balance.78,79 As a result, Ir3 and Ru3 were also selected to\n verify whether these complexes can induce cell apoptosis by\n using the dual-staining annexin V/PI assay. A549 or A549/\n DPP cells were incubated with Ir3 or Ru3 at the concentration\n of 0.25, 0.5, and 1 \u00d7 IC50 for 48 h and then detected by flow\n cytometry (Figure 8a\u2212c). In comparison to the control group,\n an increase in the percentage of both early and late apoptotic\n cells was observed for complexes Ir3 and Ru3. When Ir3 and\n Ru3 were at 1 \u00d7 IC50 concentration, a total of 52.5% (31.7%\n early apoptosis and 20.8% late apoptosis) and 29.7% (9.60%\n early apoptosis and 20.1% late apoptosis) of A549 cells\n underwent apoptosis, respectively (Figure 8a,b). In addition,\n the late apoptotic cell populations increased in a concen-\n tration-dependent manner for Ru3, although the increase of\nFigure 6. Changes in the mitochondrial membrane potential of A549 the early apoptotic cell populations was insignificant when the\ncancer cells induced by Ir3 (a) and Ru3 (b). concentrations increased from 0.5 to 1 \u00d7 IC50. Moreover, Ir3\n showed a concentration-dependent increase in both early and\ncellular ROS levels (Figure 7a,b), which indicated that ROS late apoptotic cell populations. A similar trend was also\ngeneration played a key role in the induction of cell death for observed in the cisplatin-resistant A549/DDP cells treated\nthese complexes. Notably, the ROS levels in cisplatin-resistant with Ir3, which was basically identical to that in A549 cells\nA549/DPP cells treated with Ir3 also increased in a (Figure 8c). These observations were correlated with the\nconcentration-dependent manner (Figure 7c). Moreover, the above-mentioned loss of MMP and ROS production of Ir3 and\nfluorescence intensity in A549 cells, which is proportional to Ru3 toward A549 and A549/DPP cells and indicated that\nthe ROS levels, was comparable to that in A549/DDP cells these iridium(III) and ruthenium(II) complexes can induce\nunder the same concentrations of Ir3. Thus, the overcoming of cell death via the apoptotic pathway.\ncisplatin resistance for these complexes can be attributed to the 2.12. Cell Cycle Arrest. The cell cycle arrest may be\nredox mechanism arising from ROS overproduction. affected by apoptotic signals and was also associated with the\n Many reported half-sandwich iridium(III) and ruthenium- acceleration of cell apoptosis. Some transition metal-based\n(II) complexes can produce ROS by the catalytic oxidation of anticancer complexes were reported to induce cell apoptosis by\nnicotinamide adenine dinucleotide (NADH) to NAD+.28,75\u221277 blocking the cell cycle.80,81 The effects of Ir3 and Ru3 on cell\nAs a result, the reaction of Ir3, Ru3, and Ru8 (1 equiv) with cycle arrest in A549 cancer cells were also explored by using\nNADH (ca. 100 equiv) in 10% MeOH/90% H2O (v/v) was flow cytometry (Figures 9, S78, and S79). Treatment of A549\nmonitored within 8 h by UV\u2212vis spectroscopy at 25 \u00b0C cells with Ir3 or Ru3 at the concentrations of 0.25 \u00d7 IC50 and\n\n\n\n\nFigure 7. Analysis of ROS levels by fluorescence microscopy after A549 cells were treated with Ir3 (a) or Ru3 (b) for 24 h at 37 \u00b0C and A549/\nDDP cells were treated with Ir3 (c) for 24 h at 37 \u00b0C and stained with DCFH-DA. Data are quoted as mean \u00b1 standard deviation (SD) of three\nreplicates. p-Values were calculated after a test against the negative control data, *p < 0.05.\n\n 21387 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 8. (a) Apoptosis analysis and the corresponding histograms of apoptosis analysis for A549 cells after 48 h of exposure to Ir3 at 37 \u00b0C. (b)\nApoptosis analysis and the corresponding histograms of apoptosis analysis for A549 cells after 48 h of exposure to Ru3 at 37 \u00b0C. (c) Apoptosis\nanalysis and the corresponding histograms of apoptosis analysis for A549/DDP cells after 48 h of exposure to Ir3 at 37 \u00b0C. Determined by flow\ncytometry using annexin V-FITC vs PI staining. Data are quoted as mean \u00b1 SD of three replicates. p-Values were calculated after a test against the\nnegative control data, *p < 0.05.\n\n\n\n\nFigure 9. Flow cytometry data for the cell cycle distribution of A549 cancer cells exposed to Ir3 (a) and Ru3 (b) for 24 h. The concentrations used\nwere 0.25 \u00d7 IC50 and 0.5 \u00d7 IC50. Cell staining for flow cytometry was carried out using PI/RNase. Data are quoted as mean \u00b1 SD of three\nreplicates. p-Values were calculated after a test against the negative control data, *p < 0.05.\n\n0.5 \u00d7 IC50 for 24 h led to the concentration-dependent can cause malignant cells to move away from the original\nincrease of cell population in the G0/G1 phase, along with a tumor site and travel to other organs.82,83 Cell migration,\ngradual decrease of cell population in S and G2/M phases. invasion, and metastasis were closely related to the degradation\nWhen Ir3 and Ru3 were at the concentration of 0.5 \u00d7 IC50, the of extracellular matrix and different cell adhesion mole-\nproportion of A549 cells in the G0/G1 phase increased by 12.5 cules.84,85 To evaluate the inhibitory effect of Ir3 and Ru3\nand 10.7%, respectively, compared to the untreated group on A549 cancer cell migration, a wound-healing assay was\n(Figures 9, S78, and S79). As a result, both Ir3 and Ru3 conducted (Figure 10a,b). Compared to the control group\ninduced cell cycle perturbation and arrested the cell cycle in (40.5 and 42.5%), the wound closure rate (WCR) of A549\nthe G0/G1 phase. cells incubated with Ir3 and Ru3 decreased significantly to 8.1\n 2.13. Inhibition of Cell Migration. Preventing the spread and 9.0% at 0.5 \u00d7 IC50, respectively. In addition, both Ir3 and\nof cancer cells is highly desired but remains a significant Ru3 exhibited a concentration-dependent pattern for the WCR\nchallenge in cancer treatment. A reduction in surface adhesion of A549 cells. These results suggested that the amine\u2212imine\n 21388 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 10. (a) Wound-healing assay and histogram analysis for A549 cells treated with Ir3 for 24 h. (b) Wound-healing assay and histogram\nanalysis for A549 cells treated with Ru3 for 24 h. Typical images were taken at 0 and 24 h. The widths of the wounds are indicated with lines (\u03bcm).\nScale bar: 100 \u03bcm. Wound closure rate = (R0 \u2212 R1)/R0 \u00d7 100%.\n\ncomplexes in this system showed the ability to impede the 16/18-electron coordination mode, and ligand substitution.\nmigration of A549 cancer cells. Microscopic studies ascertained that these complexes entered\n A549 cells through an energy-dependent pathway and\n3. CONCLUSIONS accumulated mainly in mitochondria. The detection of\n disruption in mitochondrial membrane potential and over-\nThe anticancer efficacy of 16-electron five-coordinated half- production of ROS levels suggested that these complexes may\nsandwich iridium(III) and ruthenium(II) complexes has been induce cell apoptosis (including both early- and late-stage\nless studied compared to the corresponding 18-electron six- apoptosis) via the mitochondrial pathway. Additionally, the\ncoordinated complexes. In this study, a simple and readily cell cycle arrest in the G0/G1 phase and the suppression of the\naccessible amine\u2212imine ligand was used to successfully prepare cell migration were also observed in A549 cells treated by these\na series of potent half-sandwich iridium(III) and ruthenium(II) complexes. This type of half-sandwich amine\u2212imine complex\nanticancer complexes. Unsaturated 16-electron iridium(III) may represent a potent platform for the development of redox-\nand 18-electron ruthenium(II) complexes were formed when based anticancer complexes.\nthe same ligand was used to react with the corresponding\nchloro-bridged bimetallic metal precursors. Further deproto- 4. EXPERIMENTAL SECTION\nnation of the 18-electron ruthenium(II) complex Ru3 by the General considerations and the synthetic procedure for ligand L1 are\nbase NaOAc afforded isolatable 16-electron ruthenium(II) shown in the Supporting Information. The ligands L2\u2212L5 were\ncomplex Ru8. The different coordination modes of these prepared using literature methods.45,46 The bimetallic precursors D1\u2212\ncomplexes were confirmed by a single-crystal X-ray diffraction D3 were prepared according to the previously reported proce-\nanalysis. All of the obtained 16-electron complexes were fairly dures.44,47 The detailed description of biological experiments is also\nstable in aqueous solution and did not react with two-electron shown in the Supporting Information.\ndonors, such as CH3CN, PPh3, and CO, to form 18-electron 4.1. Synthesis of Complexes. 4.1.1. Synthesis of iridium(III)\n and ruthenium(II) complexes with Cl\u2212 as counteranion. The\nadducts. The weak fluorescence for these complexes was complexes Ir1\u2212Ir2 and Ru1\u2212Ru2 were synthesized by the reaction of\nverified by spectroscopic studies. All of the 16-electron and 18- bimetallic metal precursors and ligands (molar ratio, 1:2) in CH2Cl2/\nelecton complexes displayed potent cytotoxicity with the IC50 CH3OH (v/v, ca. 1:1) for 6 h at 25 \u00b0C. The solvent was removed\nvalues lower or comparable to cisplatin against A549 cells, under vacuum and the residue redissolved in CH2Cl2. Subsequently,\nHeLa cells, and HepG2 cells. In particular, high cytotoxicity n-hexane was added to the solution to produce a precipitate, which\nagainst cisplatin-resistant A549/DDP cells was also observed was subsequently filtered and washed with n-hexane. The complexes\nfor these 16-electron and 18-electron complexes, suggesting were subjected to vacuum drying. At room temperature, n-hexane was\n slowly diffused into a CH2Cl2 solution to obtain single crystals of Ir1\nthat they were not cross-resistant with cisplatin. These and Ir2.\ncomplexes also displayed a certain selectivity toward A549 Ir1. (57 mg, Yield 46%). 1H NMR (500 MHz, CDCl3) \u03b4 14.73 (s,\ncells and BEAS-2B normal cells, although the selectivity index 1H, NH), 9.10 (d, J = 7.9 Hz, 1H), 8.87 (s, 1H, CH\ufffdN), 7.69 (t, J =\nwas not high. The anticancer activity and selectivity of these 7.2 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 8.0 Hz, 1H), 7.30\ncomplexes were insensitive to the metal center, counteranion, (d, J = 7.4 Hz, 1H), 7.16 (d, J = 8.3 Hz, 2H), 1.79 (s, 15H, Cp*\u2212\n\n 21389 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\n CH(CH3)2), 2.42 (s, 3H, Aryl\u2212CH3), 2.00 (s, 3H, arene\u2212CH3), 0.91\nCH3), 1.42 (s, 9H, C(CH3)3). 13C NMR (126 MHz, DMSO-d6) \u03b4 (d, J = 6.9 Hz, 6H, CH(CH3)2). 13C NMR (126 MHz, DMSO-d6) \u03b4\n161.18 (CH\ufffdN), 156.11, 150.78, 149.87, 135.68, 135.15, 125.86, 162.76 (CH\ufffdN), 149.51, 147.92, 142.08, 127.97, 127.71, 125.85,\n123.53, 119.81, 118.99, 114.44, 94.18, 92.2, 90.36, 86.61, 82.05, 55.38, 125.35, 125.32, 124.14, 120.57, 116.90, 114.66, 106.35, 100.05, 86.34,\n34.83 (C(CH3)3), 31.73 (C(CH3)3), 10.13 (Cp*\u2212CH3), 9.43 (Cp*\u2212 85.48, 63.32, 31.45 (CH3), 31.09 (C(CH3)3), 29.94 (CH(CH3)2),\nCH3), 9.27 (Cp*\u2212CH3), 8.84 (Cp*\u2212CH3), 8.01 (Cp*\u2212CH3). ESI- 21.48 (CH(CH3)2), 17.85 (arene\u2212CH3). ESI-MS (m/z): calcd for\nMS (m/z): calcd for C27H34IrN2 579.23512, found 579.23330, [M\u2212 C24H27RuN2 445.12177, found 445.12113 [M\u2212Cl\u2212HCl]+. Elemental\nCl]+. Elemental analysis: calcd for C27H34IrN2Cl: C, 52.80; H, 5.58; analysis: calcd for C24H28RuCl2N2: C, 55.81; H, 5.46; N, 5.42, found:\nN, 4.56, found: C, 53.12; H, 5.31; N, 4.31 C, 56.12; H, 5.33; N, 5.52.\n 4.1.2. Synthesis of Iridium(III) and Ruthenium(II) Complexes with\n PF6\u2212 As the Counteranion. The complexes Ir3\u2212Ir8 and Ru3\u2212Ru7\n were synthesized by the reaction of bimetallic metal precursors,\n ligands, and NH4PF6 (molar ratio, 1:2:5) in CH2Cl2/CH3OH (v/v,\n ca. 1:1) for 6 h at 25 \u00b0C. The solvent was removed under vacuum and\n the residue redissolved in CH2Cl2, filtered, and concentrated.\n Subsequently, n-hexane was added to the solution to produce a\n precipitate, which was subsequently filtered and washed with n-\n hexane. The complexes were subjected to vacuum drying. At room\n temperature, n-hexane was slowly diffused into the CH2Cl2 solution to\n obtain single crystals of Ir3, Ir4, Ir6, Ir8, and Ru4.\n Ir2. (58 mg, Yield 48%). 1H NMR (500 MHz, CDCl3) \u03b4 15.31 (s,\n1H, NH), 8.84 (s, 1H, CH\ufffdN), 8.62 (s, 1H), 7.70 (s, 1H), 7.45 (d, J\n= 7.2 Hz, 1H), 7.30 (s, 1H), 7.09 (s, 2H), 2.45 (s, 3H, CH3), 1.98 (s,\n6H, CH3), 1.72 (s, 15H, Cp*\u2212CH3). 13C NMR (126 MHz, CDCl3) \u03b4\n160.44 (CH\ufffdN), 143.13, 137.25, 137.11, 136.60, 135.68, 135.19,\n130.12, 129.89, 129.31, 128.15, 121.18, 116.38, 93.88, 90.96, 85.40,\n20.64 (CH3), 17.89 (CH3), 9.60 (Cp*\u2212CH3), 8.49 (Cp*\u2212CH3), 8.44\n(Cp*\u2212CH3). ESI-MS (m/z): calcd for C26H32IrN2 565.21947, found\n565.21745 [M\u2212Cl]+. Elemental analysis: calcd for C26H32IrN2Cl: C,\n52.03; H, 5.37; N, 4.67, found: C, 52.42; H, 5.16; N, 4.33\n Ir3. (74 mg, Yield 51%). 1H NMR (500 MHz, DMSO-d6) \u03b4 9.32 (s,\n 1H, CH\ufffdN), 7.93 (d, 1H), 7.87 (d, 1H), 7.77 (t, J = 7.6 Hz, 1H),\n 7.62 (d, 2H), 7.31 (m, 3H), 1.60 (s, 15H, Cp*\u2212CH3), 1.36 (s, 9H,\n C(CH3)3). 13C NMR (126 MHz, DMSO-d6) \u03b4 160.71 (CH\ufffdN),\n 155.61, 150.34, 149.32, 135.32, 135.26, 134.79, 134.64, 125.37,\n 119.38, 118.49, 118.34, 113.96, 93.71, 34.34 (C(CH3)3), 31.24\n (C(CH3)3), 30.93 (C(CH3)3), 22.04 (C(CH3)3), 13.94 (Cp*\u2212CH3),\n 8.74 (Cp*\u2212CH3), 8.20 (Cp*\u2212CH3), 8.02 (Cp*\u2212CH3). ESI-MS (m/\n z): calcd for C27H34IrN2 579.23512, found 579.23510, [M\u2212PF6]+.\n Elemental analysis: calcd for C27H34IrN2PF6: C, 44.81; H, 4.74; N,\n Ru1. (54 mg, Yield 48%). 1H NMR (500 MHz, CDCl3) \u03b4 10.13 (s, 3.87, found: C, 45.07; H, 4.61; N, 3.71.\n1H, NH2), 8.62 (d, J = 7.8 Hz, 1H), 8.30 (s, 1H, CH\ufffdN), 7.65 (d, J\n= 8.3 Hz, 2H), 7.57 (t, J = 7.5 Hz, 1H), 7.49 (d, J = 7.4 Hz, 1H),\n7.42\u22127.36 (m, 2H), 7.34 (d, J = 7.4 Hz, 1H), 5.81 (s, 1H), 5.45 (d,\n2H), 5.20 (s, 1H), 4.95 (s, 1H, NH2), 2.57 (m, 1H, arene\u2212\nCH(CH3)2), 1.93 (s, 3H, arene\u2212CH3), 1.29 (s, 9H,C(CH3)3), 0.86\n(d, J = 6.7 Hz, 3H, arene\u2212CH(CH3)2), 0.80 (d, J = 6.6 Hz, 3H,\narene\u2212CH(CH3)2). 13C NMR (126 MHz, DMSO-d6) \u03b4 162.76\n(CH\ufffdN), 149.52, 149.71, 144.29, 142.08, 134.27, 127.97, 127.70,\n125.84, 125.31, 122.69, 120.56, 116.89, 116.28, 106.35, 100.04, 86.34,\n85.48, 31.44 (C(CH3)3), 31.24 (C(CH3)3), 31.20 (C(CH3)3), 31.09\n(C(CH3)3), 29.93 (CH(CH3)2), 21.47 (CH(CH3)2), 17.84 (arene\u2212\nCH3). ESI-MS (m/z): calcd for C27H33RuN2 487.16872, found\n487.16773 [M\u2212Cl\u2212HCl] + . Elemental analysis: calcd for Ir4. (74 mg, Yield 54%).1H NMR (500 MHz, CDCl3) \u03b4 11.97 (s,\nC27H34RuCl2N2: C, 58.06; H, 6.14; N, 5.02, found: C, 58.35; H, 1H, NH), 8.90 (s, 1H, CH\ufffdN), 8.00 (d, J = 8.7 Hz, 1H), 7.76 (t, J =\n5.97; N, 4.90. 7.7 Hz, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 7.6 Hz, 2H), 7.36\n Ru2. (56 mg, Yield 54%). 1H NMR (500 MHz, CDCl3) \u03b4 10.47 (s, (m, 1H), 7.11 (d, J = 7.5 Hz, 2H), 2.53 (s, 3H, CH3), 1.66 (s, 15H,\n1H, NH2), 8.71 (d, J = 7.9 Hz, 1H), 8.41 (s, 1H, CH\ufffdN), 7.71 (d, J Cp*\u2212CH3). 13C NMR (126 MHz, DMSO-d6) \u03b4 160.88 (CH\ufffdN),\n= 8.0 Hz, 2H), 7.65 (t, J = 7.6 Hz, 1H), 7.61 (d, J = 7.4 Hz, 1H), 7.44 130.16, 129.30, 129.03, 123.34, 117.31, 93.67, 92.09, 63.41, 20.47\n(t, J = 7.4 Hz, 1H), 7.26 (d, J = 7.9 Hz, 2H), 5.97 (s, 1H), 5.55 (s, (CH3), 8.84 (Cp*\u2212CH3), 8.22 (Cp*\u2212CH3). ESI-MS (m/z): calcd for\n1H), 5.40 (s, 1H), 5.26 (s, 1H), 5.01 (s, 1H, NH2), 2.63 (m, 1H, C24H28IrN2 537.18817 found 537.19247, [M\u2212PF6]+. Elemental\n\n 21390 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nanalysis: calcd for C24H28IrN2PF6: C, 42.29; H, 4.14; N, 4.11, found:\nC, 42.52; H, 4.01; N, 3.98.\n\n\n\n\n C(CH3)3). 13C NMR (126 MHz, DMSO-d6) \u03b4 161.42 (CH\ufffdN),\n 155.57, 150.43, 149.42, 135.54, 134.88, 134.86, 130.43, 130.33,\n 128.98, 128.32, 125.31, 123.15, 119.85, 118.48, 114.12, 100.62, 99.71,\n Ir5. (80 mg, Yield 57%). 1H NMR (500 MHz, CDCl3) \u03b4 11.80 (s, 91.55, 49.89 (C(CH3)3), 34.32 (C(CH3)3), 31.20 (C(CH3)3), 9.46\n1H, NH), 8.82 (s, 1H, CH\ufffdN), 7.86 (d, J = 6.4 Hz, 1H), 7.66 (m, (Cp ph \u2212CH 3 ), 8.36 (Cp ph \u2212CH 3 ). ESI-MS (m/z): calcd for\n3H), 7.33 (d, J = 6.9 Hz, 2H), 7.04 (s, 2H), 3.89 (s, 3H, OCH3), 1.56 C32H36IrN2 641.25077, found 641.25086 [M\u2212PF6]+. Elemental\n(s, 15H, Cp*\u2212CH3). 13C NMR (126 MHz, DMSO-d6) \u03b4 161.24 analysis: calcd for C32H36IrN2PF6: C, 48.91; H, 4.62; N, 3.56,\n(CH\ufffdN), 158.36, 151.75, 149.31, 135.28, 134.74, 124.58, 119.33, found: C, 49.02; H, 4.58; N, 3.26.\n118.42, 113.95, 113.62, 93.70, 55.66, 22.04 (OCH3), 13.94 (Cp*\u2212\nCH3), 8.91 (Cp*\u2212CH3). ESI-MS (m/z): calcd for C24H28OIrN2\n553.18309, found 553.18330, [M\u2212PF6]+. Elemental analysis: calcd for\nC24H28OIrN2PF6: C, 41.32; H, 4.05; N, 4.02, found: C, 41.76; H,\n3.88; N, 3.73.\n\n\n\n\n Ru3. (71 mg, Yield 53%). 1H NMR (500 MHz, CDCl3) \u03b4 8.42 (s,\n 1H, CH\ufffdN), 7.98 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 7.2 Hz, 3H), 7.57\n (s, 1H), 7.49 (d, 3H), 5.59 (s, 1H), 5.46 (s, 1H), 5.38 (d, 2H), 5.23\n (s, 1H, NH2), 2.77 (m, 1H, CH(CH3)2), 1.95 (s, 3H, arene\u2212CH3),\n 1.39 (s, 9H, C(CH3)3), 0.97 (s, 6H, CH(CH3)2). 13C NMR (126\n MHz, DMSO-d6) \u03b4 163.34 (CH\ufffdN), 135.03, 129.29, 128.49, 125.88,\n Ir6. (70 mg, Yield 49%). 1H NMR (500 MHz, CDCl3) \u03b4 12.06 (s, 123.13, 116.86, 115.19, 100.56, 97.04, 86.84, 85.98, 83.07, 63.83,\n1H, NH), 8.68 (s, 1H, CH\ufffdN), 8.04 (m, 1H), 7.78 (m, 1H), 7.56 34.87 (C(CH3)3), 31.59 (C(CH3)3), 30.45 (C(CH3)3), 24.46\n(m, 1H), 7.34 (d, J = 3.8 Hz, 1H), 7.13 (s, 2H), 2.46 (s, 3H, CH3), (CH(CH3)2), 22.54 (CH(CH3)2), 21.96 (CH(CH3)2), 14.44\n2.00 (s, 6H, CH3), 1.62 (s, 15H, Cp*\u2212CH3). 13C NMR (126 MHz, (arene\u2212CH3). ESI-MS (m/z): calcd for C27H33RuN2 487.16872,\nCDCl3) \u03b4 161.22 (CH\ufffdN), 150.39, 137.49, 136.43, 133.74, 129.60, found 487.16869, [M\u2212PF 6 \u2212HCl] + ; calcd for C 27 H 34 RuClN 2\n129.33, 120.41, 120.05, 114.83, 94.07, 20.83 (CH3), 17.94 (CH3), 523.14540, found 523.14390, [M\u2212PF6]+. Elemental analysis: calcd\n8.92 (Cp*\u2212CH3). ESI-MS (m/z): calcd for C26H32IrN2 565.21947, for C27H34RuClN2PF6: C, 48.54; H, 5.13; N, 4.19, found: C, 48.78; H,\nfound 565.21838, [M\u2212PF 6 ] + . Elemental analysis: calcd for 4.97; N, 4.02.\nC26H32IrN2PF6: C, 44.00; H, 4.54; N, 3.95, found: C, 44.34; H,\n4.32; N, 3.67.\n\n\n\n\n Ru4. (69 mg, Yield 55%). 1H NMR (500 MHz, CDCl3) \u03b4 8.40 (s,\n 1H, CH\ufffdN), 7.92 (d, J = 7.2 Hz, 1H), 7.73\u22127.62 (m, 4H), 7.48 (t, J\n = 7.4 Hz, 1H), 7.27 (s, 2H), 5.32 (m, 4H), 5.22 (s, 1H, NH2), 2.56\u2212\n 2.48 (m, 1H, CH(CH3)2), 2.41 (s, 3H, Aryl\u2212CH3), 1.90 (s, 3H,\n Ir7. (80 mg, Yield 53%). 1H NMR (500 MHz, CDCl3) \u03b4 12.94 (s, arene\u2212CH3), 0.91 (d, 6H, CH(CH3)2). 13C NMR (126 MHz,\n1H, NH), 8.77 (s, 1H, CH\ufffdN), 8.21 (m, 1H), 7.79 (t, J = 7.7 Hz, DMSO-d6) \u03b4 162.82 (CH\ufffdN), 153.48, 139.69, 134.35, 129.14,\n1H), 7.46 (d, J = 7.6 Hz, 1H), 7.43\u22127.37 (m, 3H), 7.33 (d, J = 7.7 123.09, 121.46, 96.48, 86.37, 85.51, 85.00, 84.04, 81.66, 80.08, 30.08\nHz, 1H), 3.73 (m, J = 7.0 Hz, 2H, CH(CH3)2), 1.65 (s, 15H, Cp*\u2212 (CH3), 21.49 (CH(CH3)2), 20.59 (CH(CH3)2), 17.86 (arene\u2212CH3).\nCH3), 1.38 (d, J = 6.8 Hz, 6H, CH(CH3)2), 0.96 (d, J = 6.6 Hz, 6H, ESI-MS (m/z): calcd for C24H27RuN2 445.12177, found 445.12189,\nCH(CH3)2). 13C NMR (126 MHz, DMSO-d6) \u03b4 163.92 (CH\ufffdN), [M\u2212PF 6\u2212HCl] +; calcd for C24H 28RuClN 2 481.09845, found\n152.59, 149.76, 140.39, 135.74, 134.74, 128.09, 123.53, 119.60, 481.09703, [M\u2212PF 6 ] + . Elemental analysis: calcd for\n118.18, 113.47, 93.87, 27.40 (CH(CH3)2), 26.03 (CH(CH3)2), 22.55 C24H28RuClN2PF6: C, 46.05; H, 4.51; N, 4.48, found: C, 46.37; H,\n(CH(CH3)2), 8.71 (Cp*\u2212CH3). ESI-MS (m/z): calcd for C29H38IrN2 4.14; N, 4.29.\n607.26642, found 607.26467, [M\u2212PF6]+. Elemental analysis: calcd for Ru5. (60 mg, Yield 47%). 1H NMR (500 MHz, CDCl3) \u03b4 8.54 (s,\nC29H38IrN2PF6: C, 46.33; H, 5.09; N, 3.73, found: C, 46.53; H, 4.87; 1H, CH\ufffdN), 7.93 (s, 1H), 7.70\u22127.57 (m, 4H), 7.45 (t, J = 7.3 Hz,\nN, 3.51. 1H), 7.05 (s, 2H), 5.53 (m, 4H), 5.44 (s, 1H, NH2), 3.91 (s, 3H,\n Ir8. (80 mg, Yield 51%). 1H NMR (500 MHz, CDCl3) \u03b4 11.97 (s, OCH3), 2.54\u22122.42 (m, 1H, CH(CH3)2), 1.97 (s, 3H, arene\u2212CH3),\n1H, NH), 8.96 (s, 1H, CH\ufffdN), 7.68 (s, 3H), 7.38 (m, 7H), 7.17 (s, 1.01 (s, 6H, CH(CH3)2). 13C NMR (126 MHz, DMSO-d6) \u03b4 162.62\n3H), 1.73 (s, 6H, Cpph\u2212CH3), 1.49 (s, 6H, Cpph\u2212CH3), 1.35 (s, 9H, (CH\ufffdN), 134.68, 129.28, 128.42, 125.10, 115.34, 114.21, 86.84,\n\n 21391 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n hexane. The complexes were subjected to vacuum drying. At room\n temperature, n-hexane was slowly diffused into a CH2Cl2 solution to\n obtain a single crystal of complex Ru8.\n\n\n\n\n85.98, 55.98, 31.42 (OCH3), 30.56 (CH(CH3)2), 24.45 (CH(CH3)2),\n22.53 (CH(CH3)2), 14.43 (arene\u2212CH3). ESI-MS (m/z): calcd for\nC24H27ORuN2 461.11669, found 461.11581, [M\u2212PF6\u2212HCl]+; calcd\nfor C24H28ORuClN2 497.09337, found 497.09254, [M\u2212PF6]+.\nElemental analysis: calcd for C24H28ORuClN2PF6: C, 44.90; H,\n4.40; N, 4.36, found: C, 45.12; H, 4.17; N, 4.15. Ru8. (60 mg, Yield 95%). 1H NMR (500 MHz, CDCl3) \u03b4 13.28 (s,\n 1H, NH), 8.75 (s, 1H, CH\ufffdN), 8.04 (d, J = 8.6 Hz, 1H), 7.65 (d, J =\n 8.3 Hz, 2H), 7.60 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.39\n (m, 3H), 5.72 (d, J = 6.2 Hz, 2H), 5.59 (d, J = 6.2 Hz, 2H), 2.04 (s,\n 3H, arene\u2212CH3), 1.48 (s, 9H, C(CH3)3), 1.11 (d, J = 6.9 Hz, 6H,\n CH(CH3)2). 13C NMR (126 MHz, DMSO-d6) \u03b4 163.35 (CH\ufffdN),\n 158.17, 151.99, 135.94, 129.28, 126.55, 125.91, 123.82, 119.90,\n 114.11, 100.75, 88.11, 84.44, 55.36, 34.88 (CH(CH3)2), 33.46\n (CH(CH3)2), 31.68 (CH(CH3)2), 30.56 (CH(CH3)2), 24.45 (CH-\n (CH3)2), 22.53 (CH(CH3)2), 18.63 (CH(CH3)2), 14.43 (arene\u2212\n CH3). ESI-MS (m/z): calcd for C27H33RuN2 487.16872, found\n Ru6. (60 mg, Yield 46%). 1H NMR (500 MHz, CDCl3) \u03b4 13.45 (s, 487.16846, [M\u2212PF6]+. Elemental analysis: calcd for C27H33RuN2PF6:\n1H, NH2), 8.52 (s, 1H, CH\ufffdN), 8.11 (d, J = 8.6 Hz, 1H), 7.65 (t, J = C, 51.34; H, 5.27; N, 4.44, found: C, 51.68; H, 5.02; N, 4.17.\n7.2 Hz, 1H), 7.51 (m, 2H), 7.17 (s, 2H), 5.64 (d, 2H), 5.51 (d, J = 6.3\nHz, 2H), 2.49 (s, 3H, arene\u2212CH3), 2.38 (m, 1H, CH(CH3)2), 2.04\n(s, 6H, Aryl\u2212CH3), 2.02 (s, 3H, Aryl\u2212CH3), 1.15 (d, J = 6.9 Hz, 6H,\nCH(CH3)2). 13C NMR (126 MHz, CDCl3) \u03b4 162.44 (CH\ufffdN),\n \u25a0\n *\n ASSOCIATED CONTENT\n s\u0131 Supporting Information\n155.49, 151.05, 137.18, 135.44, 133.84, 129.36, 129.28, 121.59, The Supporting Information is available free of charge at\n120.38, 113.98, 105.09, 94.60, 82.50, 31.85 (CH3), 23.41 (CH-\n https://pubs.acs.org/doi/10.1021/acs.inorgchem.3c03471.\n(CH3)2), 20.98 (CH(CH3)2), 19.62 (CH(CH3)2), 18.16 (arene\u2212\nCH3). ESI-MS (m/z): calcd for C26H31RuN2 473.15307, found Additional experimental details and methods, 1H, 13C-\n473.15195, [M\u2212PF 6 \u2212HCl] + . Elemental analysis: calcd for {1H} NMR spectra, and ESI-MS spectra for all\nC26H32RuClN2PF6: C, 47.75; H, 4.93; N, 4.28, found: C, 48.06; H, compounds (Figures S1\u2212S79 and Tables S1\u2212S8)\n4.75; N, 4.11. (PDF)\n Accession Codes\n CCDC 2296029 (Ir1), 2296030 (Ir2), 2296031 (Ir3),\n 2296033 (Ir4), 2296034 (Ir6), 2296037 (Ir8), 2296039\n (Ru4) and 2296042 (Ru8), contain the supplementary\n crystallographic data for this paper. These data can be\n obtained free of charge via www.ccdc.cam.ac.uk/data_\n request/cif, or by emailing data_request@ccdc.cam.ac.uk, or\n by contacting The Cambridge Crystallographic Data Centre,\n 12 Union Road, Cambridge CB2 1EZ, UK; fax: + 441223\n Ru7. (67 mg, Yield 48%). 1H NMR (500 MHz, CDCl3) \u03b4 13.67 (s,\n1H, NH2), 8.55 (s, 1H, CH\ufffdN), 8.17 (d, J = 8.7 Hz, 1H), 7.72\u22127.65\n 336033.\n(m, 1H), 7.52\u22127.49 (m, 1H), 7.46\u22127.40 (m, 4H), 5.64 (d, J = 6.5 Hz,\n2H), 5.60 (d, J = 6.3 Hz, 2H), 3.72 (m, 2H, CH(CH3)2), 2.65 (m,\n1H, arene\u2212CH(CH3)2), 2.02 (s, 3H, arene\u2212CH3), 1.43 (d, J = 6.8\n \u25a0 AUTHOR INFORMATION\n Corresponding Authors\nHz, 6H, CH(CH3)2), 1.15 (d, J = 6.9 Hz, 6H, arene\u2212CH(CH3)2),\n0.95 (d, J = 6.7 Hz, 6H, CH(CH3)2). 13C NMR (126 MHz, DMSO- Lihua Guo \u2212 Key Laboratory of Life-Organic Analysis of\nd6) \u03b4 164.29 (CH\ufffdN), 154.78, 150.03, 140.21, 135.10, 134.99, Shandong Province, Key Laboratory of Green Natural\n120.70, 123.63, 120.87, 84.69, 81.85, 30.50 (CH(CH3)2), 27.98 Products and Pharmaceutical Intermediates in Colleges and\n(CH(CH3)2), 26.14 (CH(CH3)2), 23.33 (CH(CH3)2), 22.92 (CH- Universities of Shandong Province, School of Chemistry and\n(CH3)2), 22.54 (CH(CH3)2), 18.59 (arene\u2212CH3). ESI-MS (m/z): Chemical Engineering, Qufu Normal University, Qufu\ncalcd for C29H37RuN2 515.20002, found 515.20023, [M\u2212PF6\u2212HCl]+. 273165, P. R. China; orcid.org/0000-0002-0842-9958;\nElemental analysis: calcd for C29H38RuClN2PF6: C, 50.04; H, 5.50; N, Email: guolihua@qfnu.edu.cn\n4.02, found: C, 50.24; H, 5.26; N, 3.85. Zhe Liu \u2212 Key Laboratory of Life-Organic Analysis of\n 4.1.3. Synthesis of 16-Electron Ruthenium(II) Complex Ru8. The Shandong Province, Key Laboratory of Green Natural\ncomplex Ru8 was synthesized by the reaction of Ru3 with NaOAc\n(molar ratio: approximately 1:1.5) in CH2Cl2/CH3OH (v/v, ca. 1:1) Products and Pharmaceutical Intermediates in Colleges and\nfor 6 h at 25 \u00b0C. The solvent was removed under vacuum and the Universities of Shandong Province, School of Chemistry and\nresidue redissolved in CH2Cl2, filtered, and concentrated. Sub- Chemical Engineering, Qufu Normal University, Qufu\nsequently, n-hexane was added to the solution to produce a 273165, P. R. China; orcid.org/0000-0001-5796-4335;\nprecipitate, which was subsequently filtered and washed with n- Email: liuzheqd@163.com\n 21392 https://doi.org/10.1021/acs.inorgchem.3c03471\n Inorg. Chem. 2023, 62, 21379\u221221395\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nAuthors (2) Florea, A.-M.; Bu\u0308sselberg, D. Cisplatin as an anti-tumor drug:\n Pengwei Li \u2212 Key Laboratory of Life-Organic Analysis of cellular mechanisms of activity, drug resistance and induced side\n Shandong Province, Key Laboratory of Green Natural effects. Cancers 2011, 3, 1351\u22121371.\n (3) Peng, K.; Liang, B.-B.; Liu, W.; Mao, Z.-W. What blocks more\n Products and Pharmaceutical Intermediates in Colleges and\n anticancer platinum complexes from experiment to clinic: Major\n Universities of Shandong Province, School of Chemistry and problems and potential strategies from drug design perspectives.\n Chemical Engineering, Qufu Normal University, Qufu Coord. Chem. Rev. 2021, 449, No. 214210.\n 273165, P. R. China (4) Ho, G. Y.; Woodward, N.; Coward, J. I. Cisplatin versus\n Jiaxing Li \u2212 Key Laboratory of Life-Organic Analysis of carboplatin: comparative review of therapeutic management in solid\n Shandong Province, Key Laboratory of Green Natural malignancies. Crit. Rev. Oncol. Hematol. 2016, 102, 37\u221246.\n Products and Pharmaceutical Intermediates in Colleges and (5) Cepeda, V.; Fuertes, A. M.; Castilla, J.; Alonso, C.; Quevedo, C.;\n Universities of Shandong Province, School of Chemistry and P\u00e9rez, M. J. Biochemical mechanisms of cisplatin cytotoxicity. Anti-\n Chemical Engineering, Qufu Normal University, Qufu Cancer Agents Med. Chem. 2007, 7, 3\u221218.\n 273165, P. R. China (6) Giaccone, G.; Herbst, R. S.; Manegold, C.; Scagliotti, G.; Rosell,\n Yuwen Gong \u2212 Key Laboratory of Life-Organic Analysis of R.; Miller, V.; Natale, R. B.; Schiller, J. H.; Pawel, Jv.; Pluzanska, A.;\n Gatzemeier, U.; Grous, J.; Ochs, J. S.; Averbuch, S. D.; Wolf, M. K.;\n Shandong Province, Key Laboratory of Green Natural\n Rennie, P.; Fandi, A.; Johnson, D. H. Gefitinib in combination with\n Products and Pharmaceutical Intermediates in Colleges and gemcitabine and cisplatin in advanced non\u2212small-cell lung cancer: a\n Universities of Shandong Province, School of Chemistry and phase III trial\ufffdINTACT 1. J. Clin. Oncol. 2004, 22, 777\u2212784.\n Chemical Engineering, Qufu Normal University, Qufu (7) Allison, M.; Caram\u00e9s-M\u00e9ndez, P.; Hofmann, B. J.; Pask, C. M.;\n 273165, P. R. China Phillips, R. M.; Lord, R. M.; McGowan, P. C. Cytotoxicity of\n Xiaoyuan Li \u2212 Key Laboratory of Life-Organic Analysis of ruthenium(II) arene complexes containing functionalized ferrocenyl\n Shandong Province, Key Laboratory of Green Natural \u03b2-diketonate ligands. Organometallics 2023, 42, 1869\u22121881.\n Products and Pharmaceutical Intermediates in Colleges and (8) Dorairaj, D. P.; Haribabu, J.; Dharmasivam, M.; Malekshah, R.\n Universities of Shandong Province, School of Chemistry and E.; Mohamed Subarkhan, M. K.; Echeverria, C.; Karvembu, R. Ru(II)-\n Chemical Engineering, Qufu Normal University, Qufu p-cymene complexes of furoylthiourea ligands for anticancer\n 273165, P. R. China applications against breast cancer cells. Inorg. Chem. 2023, 62,\n 11761\u221211774.\n Tingjun Wen \u2212 Key Laboratory of Life-Organic Analysis of\n (9) Muhammad, N.; Sadia, N.; Zhu, C.; Luo, C.; Guo, Z.; Wang, X.\n Shandong Province, Key Laboratory of Green Natural Biotin-tagged platinum(IV) complexes as targeted cytostatic agents\n Products and Pharmaceutical Intermediates in Colleges and against breast cancer cells. Chem. Commun. 2017, 53, 9971\u22129974.\n Universities of Shandong Province, School of Chemistry and (10) Xiong, X.; Liu, L.-Y.; Mao, Z.-W.; Zou, T. Approaches towards\n Chemical Engineering, Qufu Normal University, Qufu understanding the mechanism-of-action of metallodrugs. Coord.\n 273165, P. R. China Chem. Rev. 2022, 453, No. 214311.\n Xinxin Wu \u2212 Key Laboratory of Life-Organic Analysis of (11) Karges, J.; Xiong, K.; Blacque, O.; Chao, H.; Gasser, G. Highly\n Shandong Province, Key Laboratory of Green Natural cytotoxic copper(II) terpyridine complexes as anticancer drug\n Products and Pharmaceutical Intermediates in Colleges and candidates. Inorg. Chim. Acta 2021, 516, No. 120137.\n Universities of Shandong Province, School of Chemistry and (12) Qi, Y. Y.; Gan, Q.; Liu, Y. X.; Xiong, Y. H.; Mao, Z. W.; Le, X.\n Chemical Engineering, Qufu Normal University, Qufu Y. Two new Cu(II) dipeptide complexes based on 5-methyl-2-(2\u2032-\n pyridyl)benzimidazole as potential antimicrobial and anticancer\n 273165, P. R. China\n drugs: Special exploration of their possible anticancer mechanism.\n Xinyi Yang \u2212 Key Laboratory of Life-Organic Analysis of Eur. J. Med. Chem. 2018, 154, 220\u2212232.\n Shandong Province, Key Laboratory of Green Natural (13) Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl,\n Products and Pharmaceutical Intermediates in Colleges and M.; Egger, A.; Berger, W.; Zorbas, H.; Dyson, P. J.; Keppler, B. K.\n Universities of Shandong Province, School of Chemistry and KP1019, A new redox-active anticancer agent \u2212 preclinical develop-\n Chemical Engineering, Qufu Normal University, Qufu ment and results of a clinical phase I study in tumor patients. Chem.\n 273165, P. R. China Biodiversity 2008, 5, 2140\u22122155.\n (14) Rademaker-Lakhai, J. M.; van den Bongard, D.; Pluim, D.;\nComplete contact information is available at:\n Beijnen, J. H.; Schellens, J. H. M. A phase I and pharmacological study\nhttps://pubs.acs.org/10.1021/acs.inorgchem.3c03471 with imidazolium-trans-DMSO-imidazole-tetrachlororuthenate, a\n novel ruthenium anticancer agent. Clin. Cancer Res. 2004, 10,\nNotes 3717\u22123727.\nThe authors declare no competing financial interest. (15) Gupta, G.; Kumari, P.; Ryu, J. Y.; Lee, J.; Mobin, S. M.; Lee, C.\n Y. Mitochondrial localization of highly fluorescent and photostable\n\n\u25a0 ACKNOWLEDGMENTS\nThe authors thank the Natural Science Foundation of\n BODIPY-based ruthenium(II), rhodium(III), and iridium(III) metal\n complexes. Inorg. Chem. 2019, 58, 8587\u22128595.\n (16) Romero-Canel\u00f3n, I.; Sadler, P. J. Next-generation metal\nShandong Province (ZR2022MB038), the Young Talents anticancer complexes: multitargeting via redox modulation. Inorg.\n Chem. 2013, 52, 12276\u221212291.\nInvitation Program of Shandong Provincial Colleges and\n (17) Maji, M.; Acharya, S.; Bhattacharya, I.; Gupta, A.; Mukherjee,\nUniversities, and the Taishan Scholars Program for support A. Effect of an imidazole-containing schiff base of an aromatic\nand Li Yankai from Shiyanjia Lab (www.shiyanjia.com) for the sulfonamide on the cytotoxic efficacy of N,N-coordinated half-\nsingle-crystal XRD data analysis. sandwich ruthenium(II) p-cymene complexes. Inorg. 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