Triphenylphosphine-Modified Iridium^III, Rhodium^III, and Ruthenium^II Complexes to Achieve Enhanced Anticancer Selectivity by Targeting Mitochondria.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Triphenylphosphine-Modified IridiumIII, RhodiumIII, and RutheniumII\n Complexes to Achieve Enhanced Anticancer Selectivity by Targeting\n Mitochondria\n Zhe Liu,* Hanxiu Fu, Heqian Dong, Kangning Lai, Zhihao Yang, Chunyan Fan, Yuting Luo,\n Wenting Qin, and Lihua Guo*\n Cite This: Inorg. Chem. 2024, 63, 24736\u221224753 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 11:28:25 (UTC).\n\n\n\n\n ABSTRACT: The incorporation of an organelle-targeting moiety\n into compounds has proven to be an effective strategy in the\n development of targeted anticancer drugs. We herein report the\n synthesis, characterization, and biological evaluation of novel\n triphenylphosphine-modified half-sandwich iridiumIII, rhodiumIII,\n and rutheniumII complexes. The primary goal was to enhance\n anticancer selectivity through mitochondrial targeting. All these\n triphenylphosphine-modified complexes exhibited promising cyto-\n toxicity in the micromolar range (5.13\u221223.22) against A549 and\n HeLa cancer cell lines, surpassing the activity of comparative\n complexes that lack the triphenylphosphine moiety. Noteworthy is\n their good selectivity toward cancer cells compared to normal\n BEAS-2B cells, underscored by selectivity index ranging from 7.3 to\n >19.5. Mechanistically, these complexes primarily target mitochondria rather than interacting with DNA. The targeting of\n mitochondria and triggering mitochondrial dysfunction were confirmed using both confocal microscopy and flow cytometry. Their\n ability to depolarize mitochondrial membrane potential (MMP) and enhance reactive oxygen species (ROS) was observed, thereby\n leading to intrinsic apoptotic pathways. Moreover, these complexes lead to cell cycle arrest in the G2/M phase and demonstrated\n antimigration effects, significantly inhibiting the migration of A549 cells in wound-healing assays.\n\n\n 1. INTRODUCTION generating reactive oxygen species (ROS), disrupting mito-\n Cancer remains a leading cause of mortality and a significant chondrial membrane potential (MMP), storing calcium ions,\n barrier to an increasing global life expectancy. Despite various and facilitating apoptosis mediated by mitochondrial path-\n available treatments, chemotherapy continues to be a crucial ways.20,21 Mitochondrial targeting represents a strategic focus\n and indispensable option for cancer management. Platinum- on organelle-specific intervention.22 Triphenylphosphine is a\n based medications, including cisplatin, carboplatin, and commonly used mitochondria-targeting lipophilic cation,\n oxaliplatin, are well-known for their efficacy in treating diverse composed of a positively charged phosphonium ion and\n tumors.1,2 However, these anticancer agents often suffer from a three phenyl rings that enhance its lipophilicity.23 The phenyl\n lack of selectivity, significant adverse effects, and the potential rings are spatially positioned to shield the phosphorus atom\n for developing resistance.3\u22125 Consequently, identifying new from dissolution. Additionally, the positive charge on the\n targets for anticancer drug action and developing novel phosphorus atom is distributed across the three phenyl rings,\n anticancer agents remain central research priorities in cancer creating a delocalized positive charge that facilitates the\n therapy.6\u22128 Organelle-targeted antitumor drugs can effectively passage of triphenylphosphine through the lipid bilayer,\n address these problems and have become one of the hotspots allowing it to specifically target the interior of mitochon-\n in current antitumor drug research.9\u221216 Notably, considering dria.24\u221226 The first organic small molecule of its kind, methyl-\n that numerous conditions, including cancer, diabetes, neuro-\n degenerative diseases, and ischemia\u2212reperfusion injury, are\n linked to mitochondrial dysfunction, there has been an Received: September 19, 2024\n increasing focus on targeting mitochondria in drug develop- Revised: November 13, 2024\n ment over the past two decades.17\u221219 Accepted: December 2, 2024\n Mitochondria are crucial organelles that are primarily Published: December 16, 2024\n responsible for energy production within cells. Additionally,\n they play roles in various other cellular processes, including\n\n \u00a9 2024 American Chemical Society https://doi.org/10.1021/acs.inorgchem.4c03975\n 24736 Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 1. Known Triphenylphosphine-containing Compounds, Half-Sandwich Complexes, and Our Current Work\n\n\n\n\ntriphenylphosphonium (TPP) cation, selectively accumulates preclinical and clinical trials, positioning them as potential drug\nin mammalian cell mitochondria in response to higher candidates.40,41 However, most of these complexes continue to\nmembrane potentials (Scheme 1, I).27,28 The delivery of suffer from unclear targets and mechanisms of actions (MoAs),\ndrugs to mitochondria was optimized by directly attaching coupled with limited selectivity between normal and cancerous\nbioactive molecules to TPP using alkyl chains or other covalent cells.42,43 Our group is committed to advancing the develop-\nbonds, refining their application in mitochondrial biology ment of organometallic complexes of iridiumIII, rhodiumIII, and\n(Scheme 1, II).29\u221231 rutheniumII with N^N chelating ligands.44\u221248 Notably, some of\n In particular, TPP and its derivatives have also been the imine\u2212pyridyl (Scheme 1, VI) and imine\u2212amine (Scheme\nemployed, although remain scarce, as building blocks or 1, VII and VIII) complexes have shown promising cytotoxic\nchelating ligands to develop metal-based agents for anticancer effects and displayed anticancer selectivity against A549 cancer\nor antimicrobial applications.31\u221234 For example, when cisplatin cells relative to BEAS-2B normal cells, mediated by a ROS-\nis conjugated with TPP (Scheme 1, III and IV), it based redox mechanism.48\u221250 These encouraging results have\npreferentially targets mitochondria rather than nuclear DNA, inspired us to explore new platinum group metal-based\nintegrating into the mitochondrial genome to influence cellular anticancer complexes through the coupling with the\nfunctions.33,34 This strategy also effectively overcame cisplatin triphenylphosphonium (PPh3+) moiety, potentially combining\nresistance.33 Generally, the mitochondrion-targeting system, the advantages of mitochondria-targeting ability of\ndriven by triphenylphosphine and leveraging mitochondrial PPh3+moiety and specific properties of half-sandwich metal\nmembrane potential, is designed to direct drugs preferentially complexes (Scheme 1). Herein, we synthesized a series of\nto the mitochondria of cancer cells. The hyperpolarization of triphenylphosphine-modified half-sandwich iridiumIII, rhodiu-\nboth the cancer cell membrane and mitochondrial membranes mIII, and rutheniumII complexes, specifically designed to target\nenables selective drug accumulation at these sites. This strategy mitochondria. This targeting ability appears to enhance the\nnot only increases the direct cytotoxic effects on cancer cells generation of reactive oxygen species (ROS), disrupt\nbut also minimizes potential toxicity to normal cells.35 mitochondrial membrane potential (MMP), and trigger\n In recent years, half-sandwich organometallic complexes, apoptosis in cancer cells, leading to selective cytotoxic effects\n on A549 cancer cells compared with normal BEAS-2B cells.\nparticularly those based on iridiumIII, rhodiumIII, rutheniumII\nand osmiumII with the structural type [(\u03b76-arene)/(\u03b75-\nCp*)M(XY)Cl]0/+ (where Cp* represents C5(CH3)5 and XY 2. RESULTS AND DISCUSSION\ndenotes bidentate chelating ligands), have garnered significant 2.1. Synthesis and Characterizations. The chloro-\ninterest in cancer research (Scheme 1, V).36\u221239 bridged bimetallic iridiumIII precursor D1 ([(\u03b75-Cp*)IrCl2]2),\n These complexes are celebrated for their modifiable rhodiumIII precursor D2 ([(\u03b75-Cp*)RhCl2]2), and rutheniumII\nstructure, which allows for a rich diversity of molecular precursor D3 ([(\u03b76-p-cymene)RuCl2]2) were synthesized\nstructures and biological activities. Unlike traditional platinum- following established methods in the literature.51\u221253 The-\nbased drugs, these metal complexes follow distinct mechanisms phenanthroline-based N^N chelating ligand L1 was synthesized\nof action, potentially overcoming platinum resistance and in a moderate yield by the reaction of (4-(4-formylphenoxy)-\nreducing toxicity. Notably, the rutheniumII complexes NAMI-A butyl) triphenylphosphonium bromide with 1,10-phenanthro-\nand KP1019 have demonstrated promising outcomes in both line-5,6-dione and the excess ammonium acetate using a\n 24737 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 2. Synthesis of Ligands L1 (a), Synthesis of Half-Sandwich IridiumIII, RhodiumIII, and RutheniumII Complexes (b),\nand the Detailed Structures of Ir1, Ir2, Rh1, Rh2, Ru1, and Ru2 (c)\n\n\n\n\nmodified procedure (Scheme 2a). L2, which did not contain moiety and the PF6\u2212 counteranion produce a singlet and a\ntriphenylphosphine moiety, was also prepared following septet at approximately \u03b420 and \u2212140 ppm, respectively\nliterature method.32 The triphenylphosphine-modified com- (Figure 1). Conversely, only a septet, corresponding to the\nplexes Ir1, Rh1, and Ru1 were synthesized in 59\u221271% yields PF6\u2212 counteranion, was observed in complexes Ir2, Rh2, and\nby reacting the metal precursors D1, D2, or D3 with the Ru2 without triphenylphosphine moiety (Figures S9, S14, and\ncorresponding ligands in a solution of CH2Cl2 and CH3OH S21). This result further validates the successful synthesis of\n(v/v = 1:1) (Scheme 2b). For comparison, complexes Ir2, the targeted complexes. Particularly, the variation of the metal\nRh2, and Ru2, which lack the triphenylphosphine moiety, were center (Ir1 vs Rh1 vs Ru1) has minimal impact on the\nsimilarly prepared (Scheme 2b). The formation of these chemical shifts in the 31P NMR spectra. Unfortunately, despite\ncomplexes were fully confirmed by 1H, 13C, and 31P NMR numerous attempts, we have been unable to successfully\n(Figures S4\u2212S21), elemental analysis and mass spectroscopy crystallize these metal complexes into single crystals.\n(positive mode: Figures S22\u2212S27 and negative mode: Figures 2.2. Absorption and Emission Spectroscopy. UV\u2212\nS28\u2212S30). In the 1H NMR spectra of these complexes, molar visible (UV\u2212vis) absorption spectra for these iridiumIII,\nequivalents of bound Cp*/arene per mole of ligand were rhodiumIII, and rutheniumII complexes were recorded in\ndetected, indicating coordination between the ligands and the methanol at 37 \u00b0C (Figure 2a). A prominent absorption\nmetal ions. 31P NMR analysis distinctly shows that in the peak was noted between 270 and 298 nm across these\ncomplexes Ir1, Rh1, and Ru1 the triphenylphosphine (PPh3+) complexes. Further, broader and less intense absorption peaks\n 24738 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. 31P NMR spectrum of phosphorus complexes Ir1, Rh1, and Ru1.\n\n\n\n\nFigure 2. (a) UV\u2212visible absorbance spectra of Ir1, Ir2, Rh1, Rh2, Ru1, and Ru2 (20 \u03bcM) in MeOH solutions at 37 \u00b0C. (b) Normalized emission\nspectra of Ir1, Ir2, Rh1, Rh2, Ru1, and Ru2 (20 \u03bcM) in MeOH at 37 \u00b0C.\n\naround approximately 310 and 410 nm were also detected. The between 511 and 513 nm (Ir1: 512 nm, Ir2: 513 nm, Rh1: 512\npeaks observed below 300 nm are indicative of ligand-centered nm, Rh2: 511 nm, Ru1: 512 nm, and Ru2: 513 nm) at 37 \u00b0C\n\u03c0\u2212\u03c0* transitions, while those between 300 and 450 nm are in methanol (Figure 2b). Consistent emission patterns across\ntypically associated with metal-to-ligand charge transfer or d\u2212d these spectra also suggest a minimal influence of the metal\ntransitions, which is consistent with those reported in other center and ligand variations on the emission bands. The\nhalf-sandwich iridiumIII, rhodiumIII, and rutheniumII com- emission quantum yields of Ir1, Rh1, and Ru1 are notably low\nplexes.54\u221256 The absorption spectra for these complexes show in methanol solutions (Ir1: 0.12%, Rh1: 0.27%, and Ru1:\ncomparable features, indicating that changes in the metal ion 0.24%), with fluorescein used as the reference standard.\nand ligand substitution have little effect on their spectral Additionally, the average lifetimes of Ir1, Rh1, and Ru1 were\nabsorption bands. recorded at 5.14, 5.50, and 4.75 ns respectively (Figure S31),\n When excited at \u03bbex = 286\u2212290 nm, the complexes Ir1, Ir2, indicating their fluorescent properties. Notably, this weak\nRh1, Rh2, Ru1, and Ru2 exhibited emission peaks (\u03bbem) photoluminescence is consistent with observations in other\n 24739 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nreported half-sandwich iridiumIII, rhodiumIII, and rutheniumII complexes in the acidic environment characteristic of cancer\ncomplexes.46,56,57 However, the photoluminescent character- cells, we prepared a buffered solution using 10% DMSO/90%\nistics of these complexes provide a potential method for PBS (v/v), adjusting the pH to approximately 5.5 with an\nobserving their cellular localization and accumulation, which acetic acid/sodium acetate buffer, and conducted stability tests.\ncould offer detailed insights into their MoAs. The results showed that this pH adjustment did not\n 2.3. Solution Stability. To evaluate the stability of Ir1, significantly impact the hydrolysis rate of the complexes\nIr2, Rh1, Rh2, Ru1, and Ru2 in aqueous solutions, hydrolysis (Figure S39, Table 1). Overall, the potential stability of these\nexperiments were conducted in a solution of 80% DMSO-d6/ complexes for further investigation of their anticancer activity\n20% PBS (pH \u2248 7.4, made with D2O) (v/v) at 37 \u00b0C, using under aqueous conditions has been confirmed.\n1\n H NMR analysis. Given their limited solubility at high water 2.4. Cytotoxicity. Using cisplatin as a reference, the\ncontent, a higher proportion of DMSO-d6 was used to cytotoxicity of complexes Ir1, Ir2, Rh1, Rh2, Ru1, and Ru2\nmaintain a sufficient complex concentration for reliable toward A549 lung cancer cells, cervical carcinoma HeLa cells,\nspectroscopic measurement. Over a 24 h period, no additional and noncancerous BEAS-2B cells was assessed via MTT assay\npeaks were detected in the 1H NMR spectra, and the proton (Table 2). The precursors D1\u2212D3 and ligands L1\u2212L2 showed\nassignments remained consistent with their established\nmolecular structures (Figures S32\u221237). This indicates that Table 2. IC50 Values of Ligands, Precursors, and Complexes\nthe complexes did not undergo decomposition or ligand Tested Toward Cancer and Normal Cell Lines and Their\ndissociation, demonstrating their stability under the tested low- Comparison with Cisplatin\nwater content conditions.\n IC50 (\u03bcM)\n The stability of the complexes Ir1, Ir2, Rh1, Rh2, Ru1, and\nRu2 was also evaluated at 37 \u00b0C in a solution of 10% DMSO/ Complexes A549 HeLa BEAS-2B SIa\n90% PBS (v/v, pH \u2248 7.4, prepared with H2O, high water L1 >100 >100 >100 -\ncontent solution) using UV\u2212vis spectroscopy across various L2 >100 >100 >100 -\ntime intervals over a 24-h period. While no significant shifts in D1 >100 >100 >100 -\nthe absorption bands were noted, there were changes in the D2 >100 >100 >100 -\nabsorption intensity of these complexes (Figure S38). This D3 >100 >100 >100 -\nbehavior suggests hydrolysis of the metal-Cl bond (Cl\u2212/H2O Ir1 5.13 \u00b1 0.25 12.78 \u00b1 0.12 >100 >19.5\nexchange), aligning with observations from other reported half- Ir2 46.28 \u00b1 0.24 28.34 \u00b1 0.18 51.21 \u00b1 0.14 1.1\nsandwich iridiumIII, rhodiumIII, and rutheniumII complexes that Ru1 6.25 \u00b1 0.18 12.79 \u00b1 0.20 82.34 \u00b1 0.22 13.2\nfeature monodentate labile chloride ligands.48,58 This outcome Ru2 43.72 \u00b1 0.19 31.89 \u00b1 0.13 46.28 \u00b1 0.11 1.1\nthrough UV\u2212vis analysis appears to be inconsistent with the Rh1 12.50 \u00b1 0.23 23.22 \u00b1 0.17 91.41 \u00b1 0.09 7.3\nNMR results. However, in line with the trends noted in earlier Rh2 33.85 \u00b1 0.17 32.29 \u00b1 0.06 46.28 \u00b1 0.24 1.4\nstudies of half-sandwich metal complexes,59,60 these complexes Cisplatin 24.89 \u00b1 0.21 8.39 \u00b1 0.08 29.21 \u00b1 0.22 1.2\nare likely to undergo Cl\u2212/H2O exchange more readily in a\n SI: The selectivity index represents the IC50 ratio of BEAS-2B\ndiluted solutions with high water content, akin to typical cell normal cells to A549 cancer cells. Data are quoted as mean \u00b1\nculture conditions. Significantly, the hydrolysis of the metal\u2212 standard deviation (SD) of three replicates. Statistical analysis using t\nCl bond often acts as an activation step for many anticancer test indicated a significant difference in IC50 values between A549 and\ncomplexes.59 The half-lives (t1/2) and hydrolysis rate constants BEAS-2B cells for Ir1, Ir2, Ru1, Ru2, Rh1 and Rh2 (*p < 0.05).\n(k) were calculated for these complexes by fitting the changes\nin absorption to pseudo-first-order kinetics (Table 1). Overall, no obvious cytotoxicity toward A549 and HeLa cancer cells\n (IC50 > 100 \u03bcM). In contrast, all tested complexes exhibited\nTable 1. The Half-Life and Hydrolysis Rate of IridiumIII, effective cytotoxicity toward A549 and HeLa cancer cells, with\nRhodiumIII, and RutheniumII in Different Solvent Systems IC50 values in a range of 5.13\u221246.28 \u03bcM, surpassing or\n 10% DMSO/90% PBS 10% DMSO/90% PBS\n matching the efficacy of cisplatin. Thus, this anticancer\n pH \u2248 7.4 pH \u2248 5.5 effectiveness is ascribed to the chelation between the free\n Complexes t1/2 (min) k (min\u22121) t1/2 (min) k (min\u22121)\n ligands and the iridiumIII, rhodiumIII, and rutheniumII ion. Ir1,\n Rh1, and Ru1 featuring the triphenylphosphine moiety,\n Ir1 120.75 0.00574 149.06 0.00462\n exhibited significantly enhanced potency (3 to 9 times greater)\n Ir2 116.10 0.00597 139.19 0.00498\n against A549 cells compared to their counterparts Ir2, Rh2,\n Rh1 200.33 0.00346 276.15 0.00251\n and Ru2 with IC50 values of 5.13\u221212.50 \u03bcM versus 33.85\u2212\n Rh2 133.55 0.00519 152.01 0.00456\n 46.28 \u03bcM. A similar trend was also observed in Hela cells,\n Ru1 155.41 0.00446 141.17 0.00491\n where, although the increase in cytotoxicity was less\n Ru2 117.08 0.00592 121.81 0.00569\n pronounced than in A549 cells, the potency still reached 1.5\n to 3 times higher (12.78\u221223.22 \u03bcM versus 28.34\u221232.29 \u03bcM).\nthese complexes underwent relatively slow hydrolysis, with Basically, under the same ligand conditions, variations in the\nhalf-lives ranging from 116.1 to 200.3 min, demonstrating a metal center had minimal impact on the cytotoxicity exhibited\nslower rate of hydrolysis compared to some reported half- by these complexes toward the same cancer cell lines,\nsandwich iridiumIII, rhodiumIII, and rutheniumII complexes suggesting that the ligand environment predominantly dictates\nwith N,N-donor ligands.48,61 The hydrolysis rates for these their anticancer activity. A distinctive feature of these\ncomplexes follow a decreasing order: Ir2 > Ir1, Rh2 > Rh1, triphenylphosphine-modified complexes is their enhanced\nand Ru2 > Ru1, indicating that the introduction of the selectivity between cancerous and normal cells. Ir1 was\ntriphenylphosphine moiety into these complexes effectively inactive (IC50 > 100 \u03bcM) toward noncancerous BEAS-2B\nreduces chloride loss. Additionally, to assess the stability of the cells and showed a selectivity index (SI) of >19.5 toward A549\n 24740 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 3. (a) 1H NMR spectrum of complex Ir1 (1 mM) and NADH (5 mM) in CD3OD and D2O (v/v 4:1) at 37 \u00b0C after 15 min. Peaks labeled\n(red solid triangle) and (black solid circle) correspond to the formed Ir\u2212H complex and chlorido complex Ir1. (b) UV\u2212vis spectra of the reaction\nof NADH (100 \u03bcM) with Ir1, Rh1, and Ru1 and positive control (Cp*Ir(phpy)Cl) (1 \u03bcM) in 10% MeOH/90% H2O (v/v) at 25 \u00b0C for 8 h.\n\nand BEAS-2B cells. Moreover, complexes Rh1 and Ru1 twice as high as that of Ir1, Rh1, and Ru1, indicating that\nexhibited minimal cytotoxicity toward BEAS-2B cells, with lipophilicity may not be the only factor influencing their\nIC50 values that were 7.3 and 13.2 times higher, respectively, cytotoxicity and selectivity. This discrepancy points to the\nthan those observed in A549 cancer cells. Conversely, the potential role of redox-based mechanisms, as supported by our\ncomplexes Ir2, Rh2, and Ru2 without a triphenylphosphine observations in subsequent biological experiments (catalytic\nmoiety showed no significant selectivity between cancer and oxidation of NADH to NAD+, mitochondria targeting,\nnormal cells. Thus, it seemed that the introduction of the mitochondrial dysfunction, and ROS generation).\ntriphenylphosphine moiety in this system led to both the 2.5. DNA and Protein Binding Results. Given that the\nenhanced cytotoxicity and improved selectivity of these cytotoxic effects of many anticancer complexes are related to\ncomplexes. DNA binding, the capability of complexes Ir1, Rh1, and Ru1\n The highly lipophilic nature of triphenylphosphine led us to to bind with the model nucleobase 9-methyladenine (9-MeA)\nexplore whether the cytotoxicity of these complexes is was initially assessed using 1H NMR spectroscopy in a solution\ninfluenced by their lipophilicity. Consequently, the octanol/ of 80% DMSO-d6/20% D2O. Over 24 h, no interactions were\nwater partition coefficients (log P) for these complexes were detected between the complexes and 9-MeA under the\nmeasured by using the shake-flask method. The resulting log P experimental conditions (Figures S40\u2212S42). Additionally,\nvalues displayed the following trend: Ir1 (1.55) > Ir2 (0.75), mass spectrometry analysis confirmed the absence of any\nRh1 (0.97) > Rh2 (0.61), and Ru1 (1.28) > Ru2 (0.68). In nucleobase adducts. The interactions of complexes Ir1, Rh1,\ncancer cells, a consistent trend was observed, indicating that and Ru1 with CT-DNA were subsequently examined by using\nthe cytotoxicity of these complexes correlated to their UV\u2212vis absorption spectroscopy (Figure S43). The experi-\nlipophilicity was observed. However, in normal BEAS-2B ments involved adding increasing concentrations of CT-DNA\ncells, the cytotoxicity of Ir2, Rh2, and Ru2 was approximately (0\u221243.6 \u03bcM) to solutions containing 60 \u03bcM of each complex.\n 24741 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 4. Determination of intercellular localization of Ir1 (a), Ir2 (b), Rh1 (c), Rh2 (d), Ru1 (e), Ru2 (f) by confocal microscopy. A549 cells\nwere incubated with Ir1, Rh1, and Ru1 (2 \u03bcM) for 1 h at 37 \u00b0C, then coincubated with DAPI (1 \u03bcg/mL), MTDR (500 nM) or LTDR (75 nM)\nfor 1 h, respectively. Scale bar: 20 \u03bcm. The green, red, and blue fluorescence represent Ir1, Rh1, and Ru1, mitochondria, lysosome, and nucleus,\nrespectively.\n\nThis increase in CT-DNA concentration resulted in hyper- and many therapeutic drugs.68 Consequently, exploring the\nchromism and a slight red shift at the absorption peak, typically reactions of anticancer metallodrugs with proteins is essential\nindicative of noncovalent electrostatic binding.62,63 Addition- for understanding their toxicity, biodistribution, and mecha-\nally, the Benesi\u2212Hildebrand equation was applied to nisms of action within this novel class of anticancer agents. The\ndetermine the intrinsic equilibrium binding constants (Kb) of binding affinity of complexes Ir1, Rh1, and Ru1 with BSA was\nthese complexes with CT-DNA. The determined Kb values assessed using UV\u2212vis absorption and fluorescence spectros-\nranged from 5.88 \u00d7 103 to 1.60 \u00d7 104 M\u22121 (Ir1: Kb = 1.02 \u00d7 copy (Figures S44 and S46). To reduce self-absorption, both\n104, Rh1: Kb = 1.60 \u00d7 104 M\u22121, Ru1: Kb = 5.88 \u00d7 103 M\u22121). the reference and sample cuvettes were treated with the\nThese values were notably lower than those of previously respective complexes. BSA\u2019s fluorescence is primarily derived\nreported half-sandwich complexes (Kb > 105 M\u22121),64\u221266 from tyrosine (Tyr) and tryptophan (Trp), two aromatic\nsuggesting a relatively weak interaction with CT-DNA.\n amino acids that are sensitive to environmental changes. When\nFurthermore, the binding affinity values (Kb) of Ir1, Rh1,\n small-molecule complexes interact with these residues, a\nand Ru1 also did not correlate with their cytotoxicity toward\n reduction in the fluorescence emission is typically observed.\ncancer cells, indicating that DNA binding might not be the\npredominant mechanism of action for these half-sandwich As the concentrations of Ir1, Rh1, and Ru1 increased, a\ncomplexes. This speculation is further reinforced by the decrease and red shift in the absorption peak at 233 nm were\nobserved low colocalization efficiency of these complexes observed (Figure S44a\u2212c), likely due to \u03b1-helix disruptions\nwithin the nucleus (see Section 2.7 below). and the effects of polar solvents.69 Moreover, a consistent\n Bovine serum albumin (BSA) serves as an economical model increase with no shift was noted in BSA\u2019s absorption peak at\nfor investigating the interactions between anticancer agents 279 nm for these complexes, indicating subtle changes in the\nand blood plasma proteins due to its plentiful presence in surrounding environment of the aromatic amino acid residues\nplasma, exceptional binding properties, and similarity to in BSA (Tyr and Trp).70 Additionally, the fluorescence\nhuman serum albumin (HSA).67 BSA is known to bind a intensity of BSA consistently decreased at 353 nm as the\nwide range of substrates, including hormones, metal cations, concentration of the complexes increased (Figure S44d\u2212f),\n 24742 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nsuggesting that the interaction with BSA occurs through a DND-99 (LTDR) for lysosomes, were used.79 A549 cells were\nstatic quenching mechanism.71,72 simultaneously stained with organelle-specific probes and the\n Synchronous fluorescence spectrometry proves essential for corresponding complexes. Following a 1 h treatment, distinct\nexamining the conformational changes in BSA upon green fluorescence was observed in the cytoplasm, indicating\ninteraction with the complexes. Maintaining a fixed wavelength effective penetration of these complexes into A549 cells. All of\ninterval of either 15 or 60 nm enables the detection of specific these complexes exhibited minimal colocalization with DAPI\nchanges in the Trp or Tyr residues of BSA. Specifically, the and LTDR, evidenced by low Pearson correlation coefficient\nemission wavelength for Trp decreased at 273 nm (\u0394\u03bb = 60 (PCC) values (PCC = 0.01\u22120.03 for DAPI and PCC = 0.11\u2212\nnm) with a 5 nm red shift, while the emission for Tyr showed a 0.34 for LTDR). Thus, these complexes did not localize\ndecrease at 288 nm (\u0394\u03bb = 15 nm) with a minor red shift of 2 effectively within the nucleus or lysosomes. However, the\nnm (Figure S45). These results suggest that complexes Ir1, complexes Ir1, Rh1, and Ru1, which contain the triphenyl-\nRh1, and Ru1 predominantly influence the conformation of phosphine moiety, effectively accumulate in mitochondria, as\nthe Trp microregion when they interact with BSA. indicated by high PCC values (Figure 4a,c, and e, Ir1: PCC =\n 2.6. Catalytic Hydride Transfer. Nicotinamide adenine 0.95; Rh1: PCC = 0.94; Ru1: PCC = 0.94). In contrast, Ir2,\ndinucleotide (NADH) and its oxidized counterpart, NAD+, are Rh2, and Ru2, lacking the triphenylphosphine moiety, showed\ncrucial for cellular homeostasis. The NADH/NAD+ ratio and a negligible degree of merging with mitochondria (Figure 4b, d\nNAD+ concentration are integral to various intracellular redox and f, Ir2: PCC = 0.23; Rh2: PCC = 0.24 Ru2: PCC = 0.23).\nprocesses. Cancer cells typically undergo oxidative stress due to The comparison of colocalization coefficients reveal that\nelevated levels of oxidizing species generated by their active incorporating triphenylphosphine significantly enhances the\nmetabolism.73 Consequently, they are more sensitive to complexes\u2019 targeting of mitochondria, suggesting that the\nfluctuations in the NADH/NAD+ ratio and NAD+ concen- cytotoxicity of these complexes might be attributed to\ntrations than normal cells. Manipulating the NADH/NAD+ mitochondria-mediated cell death. Given that cancer cells\nbalance with catalytic anticancer complexes could enhance possess more mitochondria than normal cells, they are more\nselectivity toward cancer cells over normal cells. The use of susceptible to mitochondrial disruption. This differential\nhydride transfer catalysis in cancer drug design has been sensitivity may contribute to the anticancer selectivity of\nextensively explored recently.74 In light of these considerations, these complexes with a triphenylphosphine moiety. Notably,\nwe examined the interactions of Ir1, Rh1, and Ru1 with the complexes Ir1, Rh1, and Ru1 demonstrated significantly\nNADH via 1H NMR spectroscopy. NADH (5 equiv) was higher positive zeta potentials\ufffd44.99 \u00b1 0.19, 50.95 \u00b1 0.22\nadded to the solutions of Ir1 (Figure 3a), Rh1 (Figure S46), and 20.56 \u00b1 0.32 respectively (Figure S48), compared to the\nand Ru1 (Figure S47), each at a concentration of 1 mM, in a negative zeta potentials of Ir2, Rh2, and Ru2\ufffd\u22126.27 \u00b1 0.12,\nmixture of CD3OD and D2O (4:1 v/v). This led to the \u221213.83 \u00b1 0.21, and \u22126.61 \u00b1 0.16 respectively (Figure S49).\nappearance of new NMR peaks at 9.0, 9.3, and 9.6 ppm, This characteristic could enhance their targeting of mitochon-\ncorresponding to the oxidized form of NAD+ (Figure 3a). dria, which possess negatively charged surfaces, upon entry\nHowever, after 12 h, no peaks associated with Ir\u2212H, Rh\u2212H, into the cytosol. The higher positive zeta potentials of Ir1,\nand Ru\u2212H hydrides were observed, likely due to the instability Rh1, and Ru1 may facilitate their accumulation in the\nof these hydride adducts.75 Subsequently, we assessed the mitochondria of cancer cells, which typically exhibit higher\ninteraction of Ir1, Rh1, Ru1, and Cp*Ir(phpy)Cl (phpy = mitochondrial membrane potentials than those of normal\nphenylpyridine, as a positive control)76 with NADH at a 1:100 cells,80 potentially improving their efficacy as targeted\nmolar ratio in a solution of 10% MeOH/90% H2O (v/v) using anticancer agents. Moreover, as aforementioned, Ir1, Rh1,\nUV\u2212vis spectroscopy (Figure 3b). Compared to the untreated and Ru1 showed much higher log P values than Ir2, Rh2, and\ncontrol group without metal complex catalysts, the presence of Ru2 which may improve their mitochondrial targeting ability.\nIr1, Rh1, Ru1, and Cp*Ir(phpy)Cl catalysts led to a noticeable Generally, anticancer complexes with high lipophilicity can\ndecrease in the intensity of the NADH absorption band at 339 disrupt normal metabolic homeostasis and intracellular ROS\nnm over an 8 h period, confirming that the observed effects levels by increasing their interaction with mitochondrial\nwere indeed attributable to catalytic action rather than external membranes.81\nfactors. The turnover numbers (TON) were calculated as The biodistribution of Ir1 in different subcellular compart-\n40.07 for Ir1, 38.90 for Rh1, 28.30 for Ru1, and 14.15 for ments of A549 and BEAS-2B cells was quantitatively analyzed\nCp*Ir(phpy)Cl. The significantly higher TON values for Ir1, using ICP-MS.82\u221284 After 48 h of exposure to Ir1, the iridium\nRh1, and Ru1 compared to those of Cp*Ir(phpy)Cl content in the mitochondria, cytoplasm (without mitochon-\ndemonstrate that the catalytic efficiency of these new dria), and nucleus fractions isolated from A549 and BEAS-2B\ncomplexes exceeds that of Cp*Ir(phpy)Cl. Notably, previous cells was measured (Figure 5). The results revealed that the\nstudies have demonstrated that the catalytic oxidation of majority of iridium was localized and accumulated in the\nNADH to NAD+ increases ROS levels, thereby providing an mitochondrial fraction of both A549 and BEAS-2B cells.\noxidative mechanism of action.77,78 Consequently, the ability Furthermore, Ir1 showed a higher accumulation in the\nof these complexes to produce ROS in both cancer and normal mitochondria of A549 cells compared to normal cells. These\ncells was further explored in follow-up experiments (Section observations are consistent with the observations from the\n2.9). aforementioned cellular localization experiments, further\n 2.7. Cellular Localization and Cellular Uptake Path- elucidating the mitochondrial targeting behavior of these\nway. Confocal microscopy was used for intracellular local- complexes.\nization analysis to determine the potential targets of these Laser confocal microscopy was also utilized to investigate\ncomplexes (Figure 4a\u2212f). Probes, such as 4,6-diamino-2- how complexes Ir1, Rh1, and Ru1 enter cells. Confocal\nphenyl indole (DAPI) for the nucleus, Mito Tracker Red CM- microscopy images captured at \u03bbex = 405 nm and 37 \u00b0C\nH2XRos (MTDR) for mitochondria, and Lyso Tracker Red revealed that after 1 h of incubation, Ir1, Rh1, and Ru1\n 24743 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n is a widely utilized fluorescent probe that is ideal for detecting\n MMP due to its color-changing properties. In a high\n membrane potential environment, JC-1 aggregates in the\n mitochondrial matrix, forming J-aggregates that emit red\n fluorescence. Conversely, when the membrane potential is\n low, JC-1 exists as a monomer and emits green fluorescence,\n allowing for the easy detection of decreases in membrane\n potential through the shift from red to green fluorescence.87,88\n The results are expressed as the red/green fluorescence\n intensity ratio. In addition, we used CCCP (carbonyl cyanide\n m-chlorophenyl hydrazone) as a positive control. CCCP is a\n well-established mitochondrial uncoupler that disrupts MMP\nFigure 5. Subcellular distribution of iridium in A549 and BEAS-2B by allowing protons to flow across the mitochondrial\ncells after incubation with Ir1(10 mM) for 1 h. The amount of membrane, thus, reducing the membrane potential. A549\niridium in mitochondrial, cytoplasmic, and nuclear fractions is\nmeasured by ICP-MS. Data were collected at least 3 times\n cells were exposed to Ir1 and Ir2 at concentrations of 0.25, 0.5,\nindependently. and 1.0 \u00d7 IC50 (Figure 7). Ir1 caused a substantial decrease in\n MMP in A549 cancer cells relative to untreated cells.\n Specifically, as the concentration of Ir1 increased from 0.25\nsuccessfully penetrated A549 cells, as evidenced by speckled \u00d7 IC50 to 1 \u00d7 IC50, the percentage of cells with mitochondrial\ngreen fluorescence within the cytoplasm (Figure 6). It is widely\n membrane depolarization rose by 58.7%, from 26.4% to 85.1%\nrecognized that small-molecule drugs can enter cells via either\n (Figure 7a). For comparison, treatment with Ir2 in A549 cells\nenergy-independent or energy-dependent pathways.81 Com-\n and Ir1 in BEAS-2B normal cells resulted in only minor\npared to the control group maintained at 37 \u00b0C, the\n changes in mitochondrial membrane depolarization, at 11.6%\nfluorescence intensity in A549 cells incubated with Ir1, Rh1,\nor Ru1 at a low temperature (4 \u00b0C) or pretreated with and 17.9%, respectively (Figure 7b,c). These findings align\ncarbonyl cyanide 3-chlorophenylhydrazone (CCCP, a meta- closely with the cytotoxicity and anticancer selectivity observed\nbolic inhibitor) decreased significantly. This suggests that the in these complexes. Specifically, complex Ir1 demonstrated\nuptake of Ir1, Rh1, and Ru1 is energy-dependent. Additionally, higher cytotoxicity and selective toxicity toward A549 cancer\nno significant differences were observed when comparing A549 cells compared to BEAS-2B normal cells. Moreover, Ir1 also\ncells treated with the endocytosis inhibitor chloroquine to exhibited greater cytotoxicity against both A549 and Hela\nuntreated cells, suggesting that endocytosis is not the primary cancer cells compared to that of Ir2. Therefore, the\nuptake pathway for these complexes. incorporation of the triphenylphosphine moiety in these\n 2.8. Mitochondrial Membrane Depolarization. Due to complexes may enhance their anticancer efficacy through the\nthe selective accumulation of these complexes in mitochondria, mitochondrial pathway, specifically by targeting mitochondria\ntheir potential effects on mitochondrial functionality were also and disrupting their normal function.\nexplored. The mitochondrial membrane potential (MMP, 2.9. Cellular ROS Determination. The generation of\n\u0394\u03c8m) represents the electrical gradient essential for cellular intracellular ROS is closely linked to mitochondrial function.89\nfunctions, with a voltage difference across the mitochondrial Numerous studies have demonstrated that impaired mitochon-\nmembrane.85 Mitochondrial damage and the loss of this dria fail to regulate ROS production efficiently, leading to\nmembrane potential are key early events in the initiation of the increased oxidative stress in cancer cells.90 Since cancer cells\napoptotic cascade. These disruptions initiate a series of typically endure heightened oxidative stress compared to\nbiochemical changes within the mitochondrial membrane normal cells, any additional increase in ROS levels induced by\nthat lead to apoptosis.86 We employed the JC-1 probe and anticancer complexes tends to have a less pronounced effect on\nflow cytometry to assess changes in the MMP of A549 cells the redox balance in normal cells, potentially underpinning the\nand BEAS-2B cells incubated with complexes Ir1 and Ir2. JC-1 selectivity of these agents.91,92 Motivated by observations that\n\n\n\n\nFigure 6. Effects of temperatures (37 or 4 \u00b0C), chloroquine (50 \u03bcM) and CCCP (50 \u03bcM) on cellular uptake of Ir1 (2 \u03bcM) (a), Ru1 (2 \u03bcM) (b),\nand Rh1 (2 \u03bcM) (c). Scale bar: 20 \u03bcm.\n\n 24744 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 7. (a,d) Changes in the mitochondrial membrane potential of A549 cancer cells induced by Ir1. (b,e) Changes in the mitochondrial\nmembrane potential of A549 cancer cells induced by Ir2. (c,f) Changes in the mitochondrial membrane potential of BEAS-2B cells induced by Ir1.\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 after a test against the untreated control data, *p < 0.05, **p < 0.01.\n\n\n\n\nFigure 8. Analysis of ROS levels by fluorescence microscope after A549 cells were treated with Ir1 (a,d) and Ir2 (b,e) for 24 h at 37 \u00b0C. Analysis of\nROS levels by fluorescence microscope after BESA-2B cells were treated with Ir1 (c,f) for 24 h at 37 \u00b0C. Stained with DCFH-DA. ROSup was used\nas a positive control to induce reactive oxygen species (ROS) production. Data are quoted as mean \u00b1 SD of three replicates. p-Values were\ncalculated after a test against the untreated control data, *p < 0.05, **p < 0.01.\n\nsome half-sandwich complexes, which are capable of either to NAD+, also elevate ROS levels in cancer cells,44 we\ntargeting mitochondria or promoting the oxidation of NADH measured ROS levels in A549 cells and BEAS-2B cells exposed\n 24745 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 9. (a) Apoptosis analysis for A549 cells after treatment with complex Ir1 at the concentrations of 0.25 \u00d7 IC50, 0.5 \u00d7 IC50, and 1 \u00d7 IC50 for\n48 h. (b) Apoptosis analysis for BEAS-2B cells after treatment with complex Ir1 at the concentrations of 0.25 \u00d7 IC50, 0.5 \u00d7 IC50, and 1 \u00d7 IC50 for\n48 h. (c) Histograms of apoptosis analysis for A549 and BEAS-2B cells after treatment with complex Ir1 at the concentrations of 0.25 \u00d7 IC50, 0.5 \u00d7\nIC50, and 1 \u00d7 IC50 for 48 h. Positive control was treated with cisplatin (Figures S52 and S53). Data are quoted as the mean \u00b1 standard deviation\n(SD) of three replicates. p-Values were calculated after a test against the untreated control data, *p < 0.05, **p < 0.01.\n\n\n\n\nFigure 10. Flow cytometry data for cell cycle distribution of A549 cancer cells exposed to Ir1 (a), Rh1 (b), Ru1 (c), and cisplatin (d) for 24 h.\nConcentrations used were 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\nstandard deviation (SD) of three replicates. p-Values were calculated after a test against the untreated control data, *p < 0.05, **p < 0.01.\n\nto various concentrations of complexes Ir1 and Ir2 (Figure 8). concentrations, and no obvious concentration-dependent\nThe measurements were performed by using a fluorescence increase was observed. Furthermore, only minimal changes in\nmicroscope with DCFH-DA as the probe. This assay assesses ROS levels were detected in BEAS-2B cells despite increasing\noverall oxidative stress rather than identifying specific ROS. In concentrations of Ir1. These results are consistent with the\ncomparison to the untreated control cells, there is a noticeable demonstrated cytotoxicity and anticancer selectivity of the\nconcentration-dependent rise in fluorescence intensity. The complexes in this system. Notably, several previously reported\nlevel of ROS (36.4%) at the concentration of 2 \u00d7 IC50 was on half-sandwich complexes have been shown to generate ROS\npar with the positive control treated with ROSup, indicating through catalytic hydride transfer from NADH to O2.61,76\nelevated ROS levels, was observed in A549 cells treated with Given that we have observed the catalytic transfer of NADH to\ncomplex Ir1, suggesting that these complexes may disrupt the NAD+ with these complexes in this system, it is plausible that\nintracellular redox through the generation of ROS. However, the induction of ROS could also be related to the oxidation of\nthe fluorescence intensity in A549 cells, indicative of ROS NADH to NAD+. However, since targeting mitochondria and\nlevels, was lower for Ir2 compared to Ir1 at the same disrupting mitochondrial membrane potential (MMP) also\n 24746 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 11. (a) Wound-healing assay for A549 cells treated with Ir1 for 24 h. (b) Histograms analysis for A549 cells treated with Ir1 for 24 h;\ntypical images were taken at 0 and 24 h. (c) Wound-healing assay for A549 cells treated with cisplatin for 24 h. (d) Histograms analysis for A549\ncells treated with cisplatin for 24 h; typical images were taken at 0 and 24 h. The widths of the wounds are indicated with lines (\u03bcm). Scale bar: 500\n\u03bcm. Wound Closure Rate: (R0 \u2212 R1)/R0 \u00d7 100%. Data are quoted as mean \u00b1 standard deviation (SD) of three replicates. p-Values were calculated\nafter a test against the untreated control data, *p < 0.05, **p < 0.01.\n\nimpairs the effective regulation of ROS production, mitochon- conditions, only a marginal increase in early apoptotic cells was\ndria-mediated ROS overproduction cannot be excluded. observed, ranging from 3.9% to 5.4% (Figure 9b,c).\nRegardless, the elevated levels of ROS can be considered one Specifically, at a 1 \u00d7 IC50 concentration of Ir1, early apoptosis\nof MoAs for these complexes. occurred in just 5.4% of BEAS-2B cells, a minimal change from\n 2.10. Apoptosis. Previous reports indicate that anticancer that in the untreated group. This selective induction of\ncomplexes generating high ROS levels can disrupt cellular apoptosis aligns with the observed low cytotoxicity of Ir1\nredox balance, thereby inducing apoptosis and cellular toward BEAS-2B normal cells, suggesting that these complexes\ndamage.93,94 The apoptosis-inducing mechanism of cell death preferentially target cancer cells while sparing normal cells.\nwas investigated by using the annexin V/PI assay. A549 cells Active caspase-3 is a key effector in multiple apoptotic\nwere treated with Ir1, Rh1, Ru1, and cisplatin as a positive pathways, making it an effective marker for detecting apoptotic\ncontrol at concentrations of 0.25, 0.5, and 1 \u00d7 IC50 for 48 h, cells via flow cytometry.95 After treatment with Ir1 at 0.5\u00d7\nand analyzed via flow cytometry (Figure 9). A concentration- IC50 and 1\u00d7 IC50 concentrations for 24 h, 19.5% and 30.9% of\ndependent rise in early apoptotic cell populations was observed active caspase 3-positive A549 cells were determined,\nfor these complexes (Figures 9a,b, S50\u2212S53). To validate the respectively. These percentages were significantly higher than\nassay\u2019s reliability, cisplatin was used as a comparison, as it is the 9.77% observed in untreated cells (Figure S54), further\nknown to induce apoptosis in cancer cells. For example, when indicating that these complexes induce cell death primarily\nA549 cells were treated with Ir1, Rh1, Ru1, and cisplatin at a through the apoptotic pathway.\nconcentration of 1 \u00d7 IC50, the percentages of cells undergoing 2.11. Cell-Cycle Arrest. Cell cycle arrest, often triggered\nearly apoptosis were 57.4%, 96.1%, 51.7%, and 16.1%, by apoptotic signals, is closely linked to the acceleration of\nrespectively. Notably, cisplatin induces both early and late apoptosis. Many anticancer complexes induce apoptosis by\napoptosis in A549 and BEAS-2B cells (Figures S52 and S53), impeding the cell cycle.96 The impact of Ir1, Rh1, and Ru1 on\nreflecting its dual-phase apoptotic effect. In contrast, the cell cycle arrest in A549 cancer cells was investigated by using\ncomplexes in this system primarily trigger early apoptosis, flow cytometry. Treatment with these complexes at concen-\nsuggesting a mechanistic difference. However, the results for trations of 0.25 \u00d7 IC50 and 0.5 \u00d7 IC50 for 24 h resulted in a\ncisplatin confirmed the effectiveness of the experimental setup, concentration-dependent rise in the cell population at the G2/\nthereby strengthening the reliability of the observed apoptotic M phase, along with a decrease in the S and G0/G1 phases\neffects induced by Ir1, Rh1, and Ru1. In contrast, when BEAS- (Figures 10a\u2212c and S55\u2212S57). Specifically, at a concentration\n2B normal cells were treated with Ir1 under the same of 0.5 \u00d7 IC50, the proportion of A549 cells in the G2/M phase\n 24747 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nincreased by 32.26%, 5.55%, and 2.39% for Ir1, Rh1, and Ru1, (4-formylphenyl)butyl) triphenylphosphine bromide (0.518 g, 1.0\nrespectively, compared to the untreated group. Cisplatin was mmol), ammonium acetate (1.734 g, 22.5 mmol), and ethanol (30\nused as a comparison for cell cycle analysis, primarily causing mL) was refluxed at 85 \u00b0C for 24 h. After reaction solution was\ncell cycle arrest in the G0/G1 and S phases (Figures 10d and cooled, added KPF6 saturation solution to give a brown precipitate.\n Filter, wash with water, and dry to obtain a brown powder. Yield,\nS58). This arrest pattern differs from that induced by the\n 0.318 g (45%). This compound is previously known. 1H NMR\ncomplexes in this system, indicating distinct mechanisms of (DMSO-d6, 500 MHz) \u03b4 13.66 (s, 1H), 9.03 (d, J = 3.9 Hz, 2H), 8.96\nbiological action between these two metal-based compounds. (d, 1H), 8.91 (d, 1H), 8.24 (d, 2H), 7.92 (t, 3H), 7.87 (m, 14H), 7.13\nConsequently, Ir1, Rh1, and Ru1 were able to induce cell cycle (d, 2H), 4.16 (t, 2H, OCH2), 3.78 (m, 2H, CH2CH2), 2.03 (m, 2H,\nperturbations, leading to arrest in the G2/M phase. Notably, CH2CH2), 1.82 (m, 2H, PCH2). 13C NMR (DMSO-d6, 126 MHz): \u03b4\ndespite differences in the metal centers (iridiumIII, rhodiumIII, 160.15 (C-O), 151.25 (C\ufffdN), 148.03, 135.43, 134.03, 130.77,\nand rutheniumII), all complexes consistently induced early 130.27, 128.28 (C-N), 123.86 (C-NH), 123.07, 119.30, 119.06,\napoptosis, indicating that early apoptosis induction was not 118.62, 118.38), 115.42, 66.65 (OCH2), 29.58 (OCH2CH2), 20.03\ninfluenced by the type of metal center. (PCH2), 18.97 (PCH2CH2). 31P NMR (202 MHz, DMSO-d6): \u03b4\n 2.12. Inhibition of Cell Migration. Tumor metastasis 24.08, \u2212137.16, \u2212140.67, \u2212144.18, \u2212147.70, \u2212151.21.\nrepresents a grave aspect of malignancy, and repeated cell\nmigration can lead to dire outcomes, often underlying the\nintractability of tumors.97 Understanding how antitumor drugs\nblock metastasis is essential for developing new treatments that\nenhance the body\u2019s antimetastatic capabilities. Ir1 was selected\nfor further metastasis studies due to its high cytotoxicity and\nsuperior selectivity. A wound-healing assay was conducted to\ninvestigate the effects of Ir1 on inhibiting the migration of 4.2. Synthesis of Complexes. 4.2.1. General Procedures.\nA549 cancer cells (Figure 11a,b). The results showed a Synthesis of iridiumIII, rhodiumIII, and ruthenium II complexes: A\nsignificant reduction in the wound closure rate (WCR) for cells mixture of bimetallic precursors (D1\u2212D3) (1 equiv), ligands (L1\u2212\ntreated with complex Ir1 at 0.5 \u00d7 IC50, decreasing from L2) (2 equiv), and NH4PF6 (2 equiv) in CH2Cl2/CH3OH (v/v,\n45.98% in the untreated control to 15.24%. For comparison, approximately 1:1) was stirred at ambient temperature for 24 h.\nthe WCR of cells treated with cisplatin, used as a positive Afterward, the solvents were evaporated by using a rotary evaporator.\ncontrol at 0.5 \u00d7 IC50, decreased from 34.36% in the control The resulting solid was dissolved in CH2Cl2 and filtered. The filtrate\ngroup to 18.53% (Figures 11c,d). Additionally, Ir1 demon- was subsequently concentrated, and the residue was recrystallized\nstrated a dose-dependent decrease in the WCR of A549 cancer from CH2Cl2 and hexane to yield a pale yellow powder.\ncells, indicating that complex Ir1 was effective at curtailing the\nin vitro migration of A549 cancer cells.\n\n3. CONCLUSIONS\nIn summary, this study successfully synthesized and charac-\nterized novel triphenylphosphine-modified half-sandwich\niridiumIII, rhodiumIII, and rutheniumII complexes. These\ncomplexes exhibit potent cytotoxicity against A549 and HeLa\ncancer cell lines, notably surpassing that of comparative Ir1: (69 mg, Yield 54%). 1H NMR (DMSO-d6, 500 MHz) \u03b4 14.24\ncomplexes lacking a triphenylphosphine moiety. Most (s,1H, NH), 9.32 (s, 2H), 9.24 (d, 2H), 8.27 (d, 4H), 7.92 (t, 3H),\n 7.88 (m, 13H), 7.18 (d, 2H), 4.18 (t, 2H, OCH2), 3.70 (m, 2H\nimportantly, they also demonstrate improved selectivity toward CH2CH2), 1.99 (m, 2H, CH2CH2), 1.78 (d, 2H, PCH2), 1.73 (s, 15H,\ncancer cells over normal BEAS-2B cells with high selectivity Cp*-CH3). 13C NMR (DMSO-d6, 126 MHz) \u03b4 160.75 (C-O),\nindex. The enhanced anticancer efficacy of these complexes is 153.47, 150.36 (C\ufffdN), 144.32, 135.43 (P-Ph3), 134.07 (P-Ph3),\nattributed primarily to their ability to target mitochondria and 133.22, 130.77 (P-Ph3), 128.77 (C-N), 127.83 (C-NH), 122.34,\ndisrupt mitochondrial function, as confirmed through confocal 119.30, 118.62, 115.62, 89.62, 66.87 (OCH2), 29.58 (OCH2CH2),\nmicroscopy and flow cytometry. These complexes effectively 20.11 (p-CH2), 18.95 (p-CH2CH2), 8.68 (Cp*-CH3). ESI-MS (m/z):\ndepolarize mitochondrial membrane potential, increase ROS calcd for C51H48ClIrN4OP 991.2884, found 991.2811 [M\u2212H\u2212\nproduction, and trigger intrinsic apoptosis pathways. Addi- 2PF6]+. 31P NMR (202 MHz, DMSO-d6): \u03b4 24.07, \u2212133.65,\ntionally, they induce cell cycle arrest at the G2/M phase and \u2212137.17, \u2212140.68, \u2212144.19, \u2212147.71, \u2212151.22, \u2212154.73. Elemental\npresent significant potential to prevent metastasis. Overall, the analysis: calcd for C51H49ClIrN4OP3F12: C, 47.76; H, 3.85; N, 4.37,\nuse of triphenylphosphine-modified complexes to target found: C, 48.01; H, 3.67; N, 4.21.\nmitochondria presents a potential approach for developing\nanticancer agents with enhanced efficacy and reduced side\neffects.\n\n4. EXPERIMENTAL SECTION\nL2 was synthesized following methods from the literature.98 D1\u2212D3\nwere obtained using previously reported procedures51,53 General Ir2: (65 mg, Yield 70%). 1H NMR (DMSO-d6, 500 MHz) \u03b4 14.23\nconsiderations and detailed experimental procedures for biological (s, 1H, NH), 9.32 (s, 2H), 9.25 (s, 2H), 8.35 (m, 4H), 7.23 (d, 2H),\nassays are provided in the Supporting Information. 4.12 (t, 2H, OCH2), 1.78 (d, 2H, CH2CH2), 1.78 (m, 15H, Cp*\u2212\n 4.1. Synthesis of Ligands. L1: (4-(4-formylphenyl)butyl) CH3), 1.49 (m, 2H, CH2CH2), 0.98 (t, 3H, CH2CH3). 13C NMR\ntriphenylphosphine bromide were synthesized by reference method. (DMSO-d6, 126 MHz) \u03b4 161.10 (C-O), 153.67, 150.63 (C\ufffdN),\nA mixture of 1,10-phenanthroline-5,6-dione (0.210 g, 1.0 mmol), (4- 150.39, 145.45, 144.29, 133.78, 133.24, 128.83 (C-N), 127.80 (C-\n\n 24748 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nNH), 115.60, 89.60, 68.02 (OCH2), 31.16 (OCH2CH2), 19.20\n(CH2CH3), 14.21 (CH2CH3), 8.70 (Cp*-CH3), 8.18 (Cp*-CH3).\nESI-MS (m/z): calcd for C33H35ClIrN4O 731.2129, found 731.2124\n[M\u2212PF6]+. Elemental analysis: calcd for:C33H35ClIrN4OPF6: C,\n45.23; H, 4.03; N, 6.39, found: C, 45.52; H, 3.86; N, 6.12.\n\n Ru2: (57 mg Yield 68%). 1H NMR (DMSO-d6, 500 MHz) \u03b4 14.25\n (s, 1H, NH), 9.86 (d, 2H), 9.24 (d, 2H), 8.26 (d, 2H), 8.25 (d, 2H),\n 7.21 (d, 2H), 6.34 (d, 2H), 6.12 (d, 2H), 4.11 (t, 2H), 2.62 (m, 1H,\n CH(CH3)2), 2.21 (s, 3H, CCH3), 1.76 (m, 2H), 1.49 (m, 2H), 0.98\n (t, 3H, CH2CH3), 0.91 (d, 6H, CH(CH3)2). 13C NMR (DMSO-d6,\n 126 MHz) \u03b4 160.99 (C-O), 154.04, 153.42 (C\ufffdN), 143.43, 132.68,\n 128.65, 126.64 (C-N), 122.09 (C-NH), 115.55, 104.42, 103.4, 86.83,\n 86.66, 85.97, 84.45, 67.97 (OCH2), 31.18 (CH(CH3)2), 30.89 (CH2),\n Rh1: (68 mg Yield 58%). 1H NMR (DMSO-d6, 500 MHz) \u03b4 9.33 22.31 (CH(CH3)2), 19.20 (arene-CH3), 18.72 (CH2CH3), 14.17\n(m, 2H), 9.24 (d, 2H), 8.24 (m, 3H), 7.92 (m, 3H), 7.86 (m, 13H), (CH2CH3).ESI-MS (m/z): calcd for C33H34ClRuN4O 639.1465,\n7.18 (d, 2H), 4.18 (t, 2H, OCH2), 3.71 (m, 2H, CH2CH2), 1.99 (m, found 639.1464, [M\u2212PF 6 ] + . Elemental analysis: calcd for\n2H, CH2CH2), 1.83 (m, 2H, PCH2), 1.76 (d, 15H, Cp*-CH3). 13C C33H34ClRuN4OPF6: C, 50.55; H, 4.37; N, 7.51. found: C, 50.76;\nNMR (DMSO-d6, 126 MHz) \u03b4 160.76 (C-O), 153.19 (C\ufffdN), H, 4.21; N, 7.34.\n150.66, 143.12, 135.42, 134.07, 133.01, 130.75, 128.69, 127.43,\n122.25, 119.30, 118.62, 115.64, 99.29, 97.44, 66.84 (OCH2), 29.73\n(OCH2CH2), 20.53 (p-CH2CH2), 20.13 (P-CH2CH2), 8.99 (Cp*-\nCH3). 31P NMR (202 MHz, DMSO-d6): \u03b4 24.07, \u2212137.17, \u2212140.68,\n \u25a0 ASSOCIATED CONTENT\n * Supporting Information\n s\u0131\n\n\u2212144.19, \u2212147.71, \u2212151.22. ESI-MS (m/z): calcd for The Supporting Information is available free of charge at\nC51H48ClRhN4OP 901.2309, found 901.2297, [M\u2212H\u22122PF6]+. https://pubs.acs.org/doi/10.1021/acs.inorgchem.4c03975.\nElemental analysis: calcd for C51H49ClRhN4OP3F12: C, 51.34; H, Additional experimental details and methods, 1H, 31P,\n4.14; N, 4.70, found:C, 51.59; H, 3.92; N, 4.46\n and 13C{1H} NMR spectra, and ESI-MS spectra for all\n compounds (PDF)\n\n \u25a0 AUTHOR INFORMATION\n Corresponding Authors\n Zhe Liu \u2212 Key Laboratory of Life-Organic Analysis of\n Rh2: (60 mg Yield 71%). 1H NMR (DMSO-d6, 500 MHz) \u03b4 9.30 Shandong Province, Key Laboratory of Green Natural\n(d, 2H), 9.26 (d, 2H), 8.32 (m, 4H), 7.20 (d, 2H), 4.10 (t, 2H, Products and Pharmaceutical Intermediates in Colleges and\nOCH2), 1.82 (m, 2H, CH2CH2), 1.77 (s, 15H, Cp*-CH3), 1.49 (m, Universities of Shandong Province, School of Chemistry and\n2H, CH2CH2), 0.98 (t, 3H, CH2CH3). 13C NMR (DMSO-d6, 126 Chemical Engineering, Qufu Normal University, Qufu\nMHz) \u03b4 160.85 (C-O), 154.53, 148.28 (C\ufffdN), 129.22, 126.29 (C- 273165, P. R. China; orcid.org/0000-0001-5796-4335;\nNH), 121.99, 114.86, 96.82, 67.85 (OCH2), 31.23 (CH2CH2CH2), Email: liuzheqd@163.com\n19.22 (CH2CH3), 13.85 (CH2CH3), 9.10 (Cp*-CH3). ESI-MS (m/z): Lihua Guo \u2212 Key Laboratory of Life-Organic Analysis of\ncalcd for C33H35ClIrN4O 641.1788, found 605.1780 [M\u2212PF6]+. Shandong Province, Key Laboratory of Green Natural\nElemental analysis: calcd for C33H35ClIrN4OPF6: C, 50.36; H, 4.48;\nN, 7.12. found: C, 50.52; H, 4.26; N, 6.96. 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; orcid.org/0000-0002-0842-9958;\n Email: guolihua@qfnu.edu.cn\n Authors\n Hanxiu Fu \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 Ru1: (56 mg Yield 48%). 1H NMR (DMSO-d6, 500 MHz) \u03b4 14.16\n Universities of Shandong Province, School of Chemistry and\n(s, 1H, NH), 9.86 (d, 2H), 9.20 (d, 2H), 8.24 (d, 3H), 8.19 (s, 1H), Chemical Engineering, Qufu Normal University, Qufu\n7.92 (t, 3H), 7.87 (m, 13H), 7.17 (d, 2H), 6.34 (d, 2H), 6.11 (d, 2H), 273165, P. R. China\n4.17 (t, 2H), 3.74 (m, 2H), 2.65 (m, 1H, CH(CH3)2), 2.21 (s, 3H, Heqian Dong \u2212 Key Laboratory of Life-Organic Analysis of\nCCH3), 2.02 (m, 2H), 1.77 (m, 2H), 0.90 (d, 6H, CH(CH3)2). 13C Shandong Province, Key Laboratory of Green Natural\nNMR (DMSO-d6, 126 MHz) \u03b4 160.74 (C-O), 154.16, 153.35 (C\ufffd Products and Pharmaceutical Intermediates in Colleges and\nN), 143.46, 135.44 (P-Ph3), 134.03, 132.67, 130.78 (P-Ph3), 128.66, Universities of Shandong Province, School of Chemistry and\n126.68, 122.31 (C-NH), 119.31 (C-N), 118.62, 115.62, 104.44, Chemical Engineering, Qufu Normal University, Qufu\n103.47, 86.76, 85.98, 84.46, 66.80 (OCH2), 30.89 (CH(CH3)2), 22.09 273165, P. R. China\n(CH(CH3)2), 20.51 (OCH2CH2), 20.10 (arene-CH3), 18.96 (p-\n Kangning Lai \u2212 Key Laboratory of Life-Organic Analysis of\nCH2CH2), 18.73 (P-CH2CH2). 31P NMR (202 MHz, DMSO-d6): \u03b4\n24.08, \u2212133.65, \u2212137.16, \u2212140.68, \u2212144.19, \u2212147.70, \u2212151.22. ESI- Shandong Province, Key Laboratory of Green Natural\nMS(m/z):calcd for C 51 H 48 ClRuN 4 OP 2 F 6 1045.1940, found Products and Pharmaceutical Intermediates in Colleges and\n1045.1916, [M-PF6]+. Elemental analysis: calcd for Universities of Shandong Province, School of Chemistry and\nC51H48ClRuN4OP3F12: C, 51.46; H, 4.06; N, 4.71, found: C, 51.59; Chemical Engineering, Qufu Normal University, Qufu\nH, 3.91; N, 4.44. 273165, P. R. China\n 24749 https://doi.org/10.1021/acs.inorgchem.4c03975\n Inorg. Chem. 2024, 63, 24736\u221224753\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n Zhihao Yang \u2212 Key Laboratory of Life-Organic Analysis of (9) Singh, D. Organelle Targeted Drug Delivery: Key Challenges,\n Shandong Province, Key Laboratory of Green Natural Recent Advancementsand Therapeutic Implications. Endocr. Metab.\n Products and Pharmaceutical Intermediates in Colleges and Immune. 2024, 24, 1480\u22121487.\n Universities of Shandong Province, School of Chemistry and (10) Sahu, P.; Agarwal, S.; Tomar, S.; Vyas, S.; Kashaw, S. K.;\n Chemical Engineering, Qufu Normal University, Qufu Rajoriya, V. Multifunctional Nanoparticles for Organelle-Specific\n 273165, P. R. China Targeted Drug Delivery in Cancer Therapy. Curr. Nanomed. 2022, 12,\n Chunyan Fan \u2212 Key Laboratory of Life-Organic Analysis of 191\u2212203.\n (11) Shao, X.; Meng, C.; Song, W.; Zhang, T.; Chen, Q. Subcellular\n Shandong Province, Key Laboratory of Green Natural\n Visualization: Organelle-Specific Targeted Drug Delivery and\n Products and Pharmaceutical Intermediates in Colleges and Discovery. Adv. Drug Delivery Rev. 2023, 199, 114977\u2212114977.\n Universities of Shandong Province, School of Chemistry and (12) Li, W.; Li, T.; Pan, Y.; Li, S.; Xu, G.; Zhang, Z.; Liang, H.; Yang,\n Chemical Engineering, Qufu Normal University, Qufu F. Designing a Mitochondria-Targeted Theranostic Cyclometalated\n 273165, P. R. China Iridium(III) Complex: Overcoming Cisplatin Resistance and\n Yuting Luo \u2212 Key Laboratory of Life-Organic Analysis of Inhibiting Tumor Metastasis through Necroptosis and Immune\n Shandong Province, Key Laboratory of Green Natural Response. J. Med. Chem. 2024, 67, 3843\u22123859.\n Products and Pharmaceutical Intermediates in Colleges and (13) Ma, X.; Lin, N.; Yang, Q.; Liu, P.; Ding, H.; Xu, M.; Ren, F.;\n Universities of Shandong Province, School of Chemistry and Shen, Z. Y.; Hu, K.; Meng, S.; Chen, H. Biodegradable copper-iodide\n Chemical Engineering, Qufu Normal University, Qufu clusters modulate mitochondrial function and suppress tumor growth\n 273165, P. R. China under ultralow-dose X-ray irradiation. Nat. Commun. 2024, 15 (1),\n Wenting Qin \u2212 Key Laboratory of Life-Organic Analysis of 8092.\n Shandong Province, Key Laboratory of Green Natural (14) Wang, B.; Hu, S.; Teng, Y.; Chen, J.; Wang, H.; Xu, Y. Z.;\n Products and Pharmaceutical Intermediates in Colleges and Wang, K. Y.; Xu, J. G.; Cheng, Y. Z.; Gao, X. Current advance of\n Universities of Shandong Province, School of Chemistry and nanotechnology in diagnosis and treatment for malignant tumors.\n Chemical Engineering, Qufu Normal University, Qufu Signal Transduction Targeted Ther. 2024, 9 (1), 200.\n 273165, P. R. China (15) Yang, Y.; Zou, X.; Sun, Y.; Chen, F.; Zhao, J.; Gou, S.\n Naphthalene Diimide-Functionalized Half-Sandwich Ru(II) Com-\nComplete contact information is available at: plexes as Mitochondria-Targeted Anticancer and Antimetastatic\nhttps://pubs.acs.org/10.1021/acs.inorgchem.4c03975 Agents. Inorg. Chem. 2023, 62, 9649\u22129660.\n (16) Gupta, A.; Pandey, A. K.; Mondal, T.; Bhattacharya, J.; Sasmal,\nNotes P. K. Multifunctional Iridium(III)\u2212Platinum(IV) Conjugates as\nThe authors declare no competing financial interest. Potent Anticancer Theranostic Agents. J. Med. Chem. 2023, 66,\n 8687\u22128704.\n\n\u25a0 ACKNOWLEDGMENTS\nThe authors thank the Taishan Scholars Program, the Natural\n (17) Singh, D. A sojourn on mitochondria targeted drug delivery\n systems for cancer: Strategies, clinical and future prospects.\n Mitochondrion 2024, 74, 101826.\nScience Foundation of Shandong Province (ZR2022MB038), (18) Murphy, M. P.; Hartley, R. C. Mitochondria as a therapeutic\nthe Young Talents Invitation Program of Shandong Provincial target for common pathologies. Nat. Rev. Drug Discovery 2018, 17,\nColleges and Universities for support, and the Shiyanjia Lab 865\u2212886.\n(https://www.shiyanjia.com) for the mass spectrum test. 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