Triphenylphosphine-modified cyclometalated iridium^III complexes as mitochondria-targeting anticancer agents with enhanced selectivity.

{"full_text": " Bioorganic Chemistry 155 (2025) 108148\n\n\n Contents lists available at ScienceDirect\n\n\n Bioorganic Chemistry\n journal homepage: www.elsevier.com/locate/bioorg\n\n\n\n\nTriphenylphosphine-modified cyclometalated iridiumIII complexes as\nmitochondria-targeting anticancer agents with enhanced selectivity\nHanxiu Fu , Shuli Wang , Yuwen Gong , Heqian Dong , Kangning Lai , Zhihao Yang ,\nChunyan Fan , Zhe Liu 1,*, Lihua Guo *,2\nKey Laboratory of Life-Organic Analysis of Shandong Province, Key Laboratory of Green Natural Products and Pharmaceutical Intermediates in Colleges and Universities\nof Shandong Province, School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165 PR China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: This study presents the development and evaluation of triphenylphosphine-modified cyclometalated iridiumIII\nCyclometalated complexes complexes as selective anticancer agents targeting mitochondria. By leveraging the mitochondrial localization\nIridium capability of the triphenylphosphine group, these complexes displayed promising cytotoxicity in the micromolar\nTriphenylphosphonium\n range (3.12\u20137.24 \u03bcM) against A549 and HeLa cancer cells, these complexes exhibit significantly higher activity\nCytotoxicity\n compared to their unmodified counterparts lacking the triphenylphosphine moiety. Moreover, they demonstrate\nTarget mitochondria\n improved specificity for cancer cells over normal cells, achieving selectivity index in the range of 5.46\u201314.83.\n Mechanistic studies confirmed that these complexes selectively target mitochondria rather than DNA, as shown\n by confocal microscopy and flow cytometry, where they accumulate to induce mitochondrial dysfunction. This\n disruption leads to mitochondrial membrane depolarization (MMP), elevated reactive oxygen species (ROS)\n levels, and activation of intrinsic apoptosis pathways. Furthermore, the complexes induce cell cycle arrest at the\n G2/M phase and suppress the migration of A549 cells.\n\n\n\n\n1. Introduction categorized into two structural classes: half-sandwich iridium complexes\n (Fig. 1, I) and cyclometalated iridium complexes (Fig. 1, II). Among\n Currently, significant progress has been made in the fields of tumor these metal complexes, photoluminescent cyclometalated iridiumIII\ndiagnosis and treatment, with the emergence of novel therapies such as compounds are particularly notable for their strong anticancer proper\u00ad\ntargeted therapy, immunotherapy, and gene therapy, further advancing ties, which are mediated by strategies like targeting specific subcellular\ncancer treatment [1,2]. However, challenges remain in the realm of organelles and disrupting protein function [16\u201321]. Simultaneously,\ncancer therapy, including issues related to the systemic toxicity of small their exceptional optical characteristics, such as high quantum yields,\nmolecule drugs and the problem of drug resistance [3\u20135]. The devel\u00ad significant Stokes shifts, adjustable emission wavelengths, superior\nopment of multifunctional therapeutic agents has opened new possi\u00ad photostability, and extended luminescence lifetimes, have established\nbilities for cancer diagnosis and treatment, particularly with the focus on them as important assets in biological imaging and chemical sensing.\nanticancer drugs that integrate imaging and therapeutic functions These applications encompass tracking organelle activities and\nwithin a single molecular framework [6\u201310]. Nevertheless, as of now, analyzing intracellular chemical species [22\u201326]. By contrast, half-\nthe development and research of multifunctional therapeutic agents still sandwich iridium complexes generally lack these favorable photo\u00ad\nface several scientific challenges [11,12]. physical properties, limiting their use in such applications. Therefore,\n In the past decades, iridium complexes have shown remarkable cyclometalated iridiumIII complexes hold great promise as innovative\nanticancer properties and are emerging as a promising area of research multifunctional theranostic agents, seamlessly integrating imaging ca\u00ad\nin the advancement of metal-containing therapeutic agents for cancer pabilities with anticancer activity into a single platform [16,27\u201329].\ntreatment [13\u201315]. Iridium anticancer complexes can be mainly Mitochondria, a central hub for cell signaling, are vital organelles\n\n\n\n * Corresponding authors.\n E-mail addresses: liuzheqd@163.com (Z. Liu), guolihua@qfnu.edu.cn (L. Guo).\n 1\n ORCID: 0000-0001-5796-4335.\n 2\n ORCID: 0000-0002-0842-9958.\n\nhttps://doi.org/10.1016/j.bioorg.2025.108148\nReceived 18 November 2024; Received in revised form 29 December 2024; Accepted 6 January 2025\nAvailable online 7 January 2025\n0045-2068/\u00a9 2025 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\nresponsible for energy generation and are essential in regulating bio\u00ad previous studies have demonstrated that attaching specific groups, such\nlogical processes, including apoptosis, calcium ion balance, and cellular as chloromethyl on bipyridine (bpy) in cyclometalated iridiumIII com\u00ad\nmetabolism [26,30,31]. Dysfunction of mitochondria is closely associ\u00ad plexes, can facilitate mitochondrial targeting by reacting with thiol\nated with various diseases, including cancer and neurodegenerative groups in mitochondrial proteins via nucleophilic substitution (Fig. 1,\ndisorders [32]. Tumor cells and normal cells exhibit significant in both VII) [16]. Inspired by these encouraging results, we sought to develop\nthe structure and function of their mitochondria. Due to extensive mu\u00ad new anticancer complexes based on platinum group metals, by coupling\ntations, tumor cells have a more fragile redox balance and genomic with the triphenylphosphonium (PPh3+) moiety, aiming to combine the\ninstability, making them more susceptible to mitochondrial damage advantages of mitochondria-targeting ability of PPh3+ moiety and\n[33]. Mitochondrial-targeted compounds have emerged as a promising photoluminescent properties of cyclometalated iridiumIII complexes\nstrategy for combating chemotherapy-resistant cancer cells, driving (Scheme 1). Herein, we synthesized a series of triphenylphosphine-\nincreased interest in the development of luminescent therapeutic agents modified photoluminescent cyclometalated iridiumIII complexes spe\u00ad\ndesigned to target mitochondria [34]. Triphenylphosphine (TPP) is a cifically designed for mitochondrial targeting. This targeted design en\u00ad\nwidely utilized lipophilic cationic ligand that targets mitochondria [35]. hances mitochondrial accumulation and induces apoptosis in cancer\nIts structure features a positively charged phosphorus atom bonded to cells, resulting in high anticancer activity and improved selectivity for\nthree benzene rings, giving it high lipid solubility. The arrangement of cancer cells over normal cells. Additionally, the photoluminescent\nthe phenyl groups shields the phosphorus from solubilization. Addi\u00ad properties of these complexes enable real-time tracking within cells,\ntionally, the positive charge on the phosphorus can be delocalized across offering valuable capabilities for biological imaging. This multifunc\u00ad\nthe three benzene rings, which, along with the negative membrane po\u00ad tional approach effectively addresses key limitations of existing iridium-\ntential of mitochondria, enhances TPP\u2019s ability to traverse lipid bilayers based anticancer agents, such as limited selectivity, unclear targeting,\nand selectively accumulate within mitochondria [35,36]. and ambiguous mechanisms of action.\n The methyl-triphenylphosphonium (TPP) cation, as the first organic\nsmall molecule of its type, demonstrates selective accumulation in 2. Results and discussion\nmammalian cell mitochondria, driven by their high membrane potential\n(Fig. 1, III) [37]. Mitochondrial drug delivery has been enhanced by 2.1. Synthesis and characterizations\ntethering bioactive molecules to TPP via alkyl chains or other covalent\nlinkages, broadening its utility in mitochondrial research (Fig. 1, IV) Cyclometalated iridiumIII precursors D1\u2013D5 were synthesized using\n[38\u201340]. Despite limited applications, TPP and its derivatives have been reported methods [44,45]. The phenanthroline-based N^N chelating\nexplored as scaffolds or chelating ligands in designing metal-based ligand L1 was prepared with a moderate yield through the reaction of (4-\nagents with anticancer and antimicrobial properties [40\u201343]. For (4-formylphenoxy)butyl) triphenylphosphonium bromide with\ninstance, conjugation of cisplatin with TPP (Fig. 1, V and VI) redirects its phenanthroline-dione and excess NH4OAc, following a modified proto\u00ad\naction toward mitochondria rather than nuclear DNA, enabling inte\u00ad col (Scheme 1a). L2, which did not contain triphenylphosphine moiety,\ngration into the mitochondrial genome and modulation of cellular pro\u00ad was also prepared following literature method [41]. The\ncesses [42,43]. Thus, this mitochondrial targeting strategy has been triphenylphosphine-modified complexes Ir1\u2013Ir5 were synthesized in\nshown to effectively overcome cisplatin resistance [42]. Notably, 43\u201361 % yields by reacting the metal precursors D1\u2013D5 with the\n\n\n\n\n Fig. 1. The cyclometalated complexes and half-sandwich complexes, known triphenylphosphine-containing compounds and our current work.\n\n 2\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\n Scheme 1. Preparation of ligands L1 (a), synthesis of cyclometalated iridiumIII complexes (b), and the detailed structures of Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 (c).\n\n\ncorresponding ligands in a solution of CH2Cl2 and CH3OH (v/v = 1:1) elemental analysis and mass spectroscopy (Figs. S33\u2013S42 in Supple\u00ad\n(Scheme 1b). For comparison, the corresponding complexes Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 mentary materials). 1H NMR spectra showed that molar equivalents of\nwhich lack the triphenylphosphine moiety, were similarly prepared bound arene per mole of ligand were detected in these complexes,\n(Scheme 1b). The detailed structures of Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 are also indicating coordination between the ligands and the metal ions. 31P\ndepicted in Scheme 1c. The formation of the desired complexes was NMR analysis distinctly shows that in the complexes Ir1\u2013Ir5, the tri\u00ad\nthoroughly confirmed using phenylphosphine (PPh3+) moiety and the PF6\u00a1 counteranion produce a\n 1\n H, 13C, 31P and 19F NMR (Figs. S1\u2013S32 in Supplementary materials), singlet and a septet at approximately \u03b424 ppm and \u2212 140 ppm,\n\n\n 3\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\nrespectively (Figs. 2, S3, S6, S9, S12 and S16). Conversely, only a septet, Upon excitation at a wavelength of \u03bbex = 388 nm, the Ir1\u2013Ir5 and\ncorresponding to the PF6\u00a1 counteranion, was observed in the complexes Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 complexes exhibit emission wavelengths (\u03bbem) that span from\nIr1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 without triphenylphosphine moiety (Figs. 2, S19, S22, S25, S28 540 nm to 611 nm (Fig. 4b: Ir1 (598 nm), Ir2 (603 nm), Ir3 (609 nm),\nand S32). The 19F NMR analysis clearly showed that the fluoro com\u00ad Ir4 (611 nm), Ir5 (600 nm), Ir1\u02b9\u02b9 (601 nm), Ir2\u02b9\u02b9 (550 nm), Ir3\u02b9\u02b9 (540 nm),\nplexes Ir4 and Ir4\u02b9\u02b9 display two doublets at approximately \u03b4 \u2212 107 ppm Ir4\u02b9\u02b9 (599 nm), and Ir5\u02b9\u02b9 (595 nm)) when measured in methanol at 37 \u25e6 C.\ncorresponding to the fluorine substituents on the phenylpyridine(ppy) Consistent with their absorption spectra, substitutions on the ligands\nligand, while the PF6\u00a1 counteranion produces two singlets at around \u03b4 have no obvious impact on the emission spectra. The relative emission\n\u2212 70 ppm (Figs. S13 and S29). quantum yields (\u03a6) of the iridiumIII complexes in an ethanol solution,\n Multiple attempts to grow single crystals of the triphenylphosphine- determined using fluorescein as a standard, range narrowly from 0.390\ncontaining complexes were unsuccessful. However, Single crystals of the to 0.527 (Ir1: 0.491, Ir2: 0.489, Ir3: 0.527, Ir4: 0.512, Ir5: 0.432, Ir1\u02b9\u02b9:\ntriphenylphosphine-free control complex Ir4\u02b9\u02b9 were successfully pre\u00ad 0.453, Ir2\u02b9\u02b9: 0.390, Ir3\u02b9\u02b9: 0.417, Ir4\u02b9\u02b9: 0.464, Ir5\u02b9\u02b9: 0.437), demonstrating a\npared by allowing n-hexane to slowly diffuse into its CH2Cl2 solution. lack of sensitivity to changes in the complex structure. Furthermore, the\nThe structure of Ir4\u02b9\u02b9 was then determined and validated using X-ray average fluorescence lifetimes of the Ir1\u2013Ir5 complexes were also\ncrystallographic analysis (Fig. 3 and Table S1). In the Ir4\u02b9\u02b9 complex, the measured, with values ranging from 219.52 to 319.19 ns (Fig. S43, Ir1:\ncationic iridiumIII center shows coordination with the N,N-chelating 258.68 ns, Ir2: 219.52 ns, Ir3: 250.08 ns, Ir4: 241.19 ns, Ir5: 319.19 ns),\nligand, resulting in a distorted octahedral configuration around the indicating that these complexes exhibit fluorescence. The photo\u00ad\nmetal center. The coordination around the metal center in the two luminescent properties of these complexes facilitate bio-imaging anal\u00ad\nphenylpyridine (ppy) ligands exhibits a mutually cis C,C and trans N,N ysis, enabling exploration of their mechanisms of action. Thus, these\nconfiguration. The Ir\u2013N bond lengths in 1,10-phenanthroline-based complexes are anticipated to serve as versatile theranostic platforms,\nligand (Ir1\u2013N1:2.119 \u00c5 and Ir1\u2013N2: 2.132 \u00c5) are slightly longer combining anticancer activities with imaging functionalities in a single\ncompared to those in the ppy ligands (Ir1\u2013N5: 2.041 \u00c5 and Ir1\u2013N6: compound.\n2.049 \u00c5).\n\n 2.3. Solution stability\n2.2. Absorption and emission spectroscopy\n The stability of Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 was evaluated in a containing of\n The UV\u2013vis spectra of Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 in methanol solutions at 20 % DMSO and 80 % PBS (obtained using H2O, pH \u2248 7.4,) at 37 \u25e6 C over\n37 \u25e6 C are presented in Fig. 4a. These iridiumIII complexes all show strong a 24-hour period using UV\u2013vis spectroscopy. The absorption spectra\nabsorption bands in the high-energy region (less than 350 nm), which showed minimal changes, indicating that these iridiumIII complexes\nare associated with spin-allowed \u03c0-\u03c0* electronic transitions of the li\u00ad were stable in dilute aqueous solutions (Fig. S44). Additional stability\ngands. Broad and featureless absorption bands in the 350\u2013450 nm range tests were performed using 1H NMR spectroscopy under similar condi\u00ad\nare attributed to metal-to-ligand charge transfer transitions (MLCT) tions (80 % DMSO\u2011d6 and 20 % PBS, pH \u2248 7.4, obtained using D2O) at\n[46,47]. Additionally, lower-energy bands appearing in the visible re\u00ad 37 \u25e6 C. The 1H NMR spectra revealed no new peaks for Ir1\u2013Ir5 and Ir1\u02b9\u02b9-\ngion (>450 nm) are ascribed to a combination of singlet and triplet Ir5\u02b9\u02b9 over the same time period (Figs. S45-S54), confirming that the\nMLCT transitions (1MLCT and 3MLCT). The absorption characteristics of complexes retained their structural integrity without dissociation.\nthese complexes resemble those of cyclometalated iridiumIII complexes Overall, their stability was consistent with that of previously reported N,\nreported in earlier studies [44,48\u201350]. N-chelating cyclometalated iridiumIII complexes, [49,52] supporting\n\n\n\n\n Fig. 2. 31P NMR spectrum of the phosphorus complexes Ir1 and Ir1\u02b9\u02b9.\n\n 4\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\nFig. 3. X-ray crystal structure of complex Ir4\u02b9\u02b9, with thermal ellipsoids shown at 50% probability. For clarity, hydrogen atoms and the anion have been excluded from\nthe illustration.\n\n\n\n\nFig. 4. (a) UV/vis absorption spectra of s Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 (20 \u03bcM) in methanol at 37 \u25e6 C. (b) Normalized fluorescence emission spectra of Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9\n(20 \u03bcM) under the same conditions.\n\n\ntheir suitability for further studies on anticancer activity in aqueous enhances their cytotoxicity towards A549 and HeLa cells compared to\nenvironments. the non-triphenylphosphine analogs Ir1\u02b9\u02b9-Ir5\u02b9\u02b9 (Ir1\u2013Ir5: 3.12\u20139.23 \u00b5M)\n vs. (Ir1\u02b9\u02b9-Ir5\u02b9\u02b9: 12.58\u201382.34 \u00b5M). This modification in Ir1\u2013Ir5 also im\u00ad\n proves selectivity, as evidenced by selectivity index ranging from 5.46 to\n2.4. Cytotoxicity 14.83 when comparing A549 cancer cells to BEAS-2B normal cells. The\n enhanced cytotoxicity and selectivity are likely due to the mitochondria-\n The cytotoxicity of complexes Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 was evaluated on targeting capability conferred by the triphenylphosphine group, which\nlung cancer A 549 cells), cervical cancer HeLa cells, and normal lung allows the complexes to accumulate more effectively in cancer cell\nepithelial BEAS-2B cells using the MTT assay, with cisplatin serving as a mitochondria, thereby increasing their anticancer efficacy. Given the\nreference compound (Table 1). In comparison, the cyclometalated pre\u00ad high lipophilicity of the triphenylphosphine group, we examined\ncursors D1-D5 and ligands L1 and L2 displayed minimal cytotoxicity, whether the cytotoxicity of these complexes is influenced by their lip\u00ad\nwith IC50 values exceeding 100 \u00b5M for both A549 and HeLa cancer cells ophilicity. To explore this, the octanol/water partition coefficients (log\n(Table S2). Conversely, complexes Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 demonstrated P) were determined using the shake-flask method, revealing a distinct\nnotable cytotoxic effects on A549 and HeLa cells, with IC50 values trend in the resulting values: Ir1 (1.35) > Ir1\u02b9\u02b9 (0.68), Ir2 (1.07) > Ir2\u02b9\u02b9\nranging between 3.12\u201353.96 \u00b5M and 3.72\u201382.34 \u00b5M, respectively, (0.63), Ir3 (1.22) > Ir3\u02b9\u02b9 (0.70), Ir4 (0.98) > Ir4\u02b9\u02b9 (0.71), and Ir5 (0.83) >\nindicating that their cytotoxicity may arise from the coordination of Ir5\u02b9\u02b9 (0.66), indicating that triphenylphosphine-containing complexes\nmetal ions with the ligands. Some of these complexes exhibited cytotoxic are significantly more lipophilic. Nevertheless, comparisons among\neffects comparable to or even exceeding those of cisplatin, The incor\u00ad complexes with or without the triphenylphosphine group revealed that\nporation of a triphenylphosphine group in complexes Ir1\u2013Ir5 notably\n\n 5\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\nTable 1 available, was used as a model protein. The UV\u2013visible absorption\nIC50 Values (\u00b5M) of Complexes Ir1\u2013Ir5 and Ir1\u02b9\u02b9-Ir5\u02b9\u02b9 for Cancer and Normal Cell spectra for complexes Ir1\u2013Ir5 (Fig. S62) revealed a significant reduction\nLines, Using Cisplatin as a Control. in absorbance at 225 nm alongside a red shift, suggesting that the\n Complexes IC50 (\u00b5M) complexes disrupt the \u03b1-helix structure of BSA and alter the polarity of\n A549 HeLa BEAS-2B SIa\n the surrounding environment upon binding [51,55,56]. In the presence\n of BSA, a pronounced peak at 275 nm was observed, suggesting modi\u00ad\n Ir1 3.12 \u00b1 0.02 4.71 \u00b1 0.26 46.28 \u00b1 0.16 14.83\n fications in the microenvironment of aromatic amino acids, particularly\n Ir2 4.21 \u00b1 0.16 5.89 \u00b1 0.14 46.21 \u00b1 0.22 10.97\n Ir3 5.29 \u00b1 0.21 5.32 \u00b1 0.17 36.18 \u00b1 0.16 6.83 tryptophan (Trp) and tyrosine (Tyr) [56,57]. Furthermore, when the\n Ir4 7.26 \u00b1 0.09 3.72 \u00b1 0.19 43.47 \u00b1 0.15 5.99 concentration of the complexes increased, the fluorescence intensity of\n Ir5 9.23 \u00b1 0.019 7.24 \u00b1 0.19 50.45 \u00b1 0.04 5.46 BSA at 353 nm showed a significant reduction, indicative of a static\n Ir1\u02b9\u02b9 49.47 \u00b1 0.27 45.81 \u00b1 0.08 102.69 \u00b1 0.13 2.08 quenching mechanism (Fig. S63) [57]. Synchronous fluorescence spec\u00ad\n Ir2\u02b9\u02b9 53.96 \u00b1 0.21 16.52 \u00b1 0.27 91.41 \u00b1 0.26 1.69\n Ir3\u02b9\u02b9 36.41 \u00b1 0.12 82.34 \u00b1 0.07 57.28 \u00b1 0.19 1.57\n troscopy provided further insights into the structural modifications of\n Ir4\u02b9\u02b9 25.75 \u00b1 0.10 36.84 \u00b1 0.12 43.71 \u00b1 0.30 1.70 BSA. At \u0394\u03bb = 15 nm, the spectrum revealed features of Tyr residues,\n Ir5\u02b9\u02b9 48.25 \u00b1 0.15 12.58 \u00b1 0.16 53.47 \u00b1 0.17 1.11 while at \u0394\u03bb = 60 nm, it corresponded to Trp residues. Binding of com\u00ad\n Cisplatin 24.06 \u00b1 0.08 7.41 \u00b1 0.17 38.11 \u00b1 0.28 1.58 plexes Ir1\u2013Ir5 caused a slight red shift (1\u20132 nm) in the emission wave\u00ad\n a\n SI: The selectivity index is calculated as the IC50 ratio of BEAS-2B normal lengths of both Tyr and Trp (Figs. S64 and S65), indicating alterations in\ncells to A549 cancer cells. Values are presented as the mean \u00b1 standard devia\u00ad their microenvironment and conformation [57,58]. These observations\ntion (SD) from three independent experiments. The IC50 values for Ir1\u2013Ir5 and suggest that the complexes interact with BSA by inducing subtle struc\u00ad\nIr1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 showed a statistically significant difference between A549 and BEAS-2B tural changes in its aromatic residues.\ncells (p < 0.05). Molecular docking studies on the Ir1 complex were conducted using\n the AutoDock suite and Q-SiteFinder, tools designed to identify potential\nwhile some correlation exists between log P values and cytotoxicity, it is binding sites and evaluate ligand interactions with proteins. The bovine\nnot consistent across all complexes, particularly in normal cells. This serum albumin (BSA) crystal structure (PDB ID: 4OR0) was obtained\nsuggests that lipophilicity alone may not fully explain the differences in from the Protein Data Bank. Initially, Q-SiteFinder was employed to\ncytotoxicity and selectivity. identify likely binding regions within BSA. Subsequently, docking sim\u00ad\n ulations were performed using AutoDock. As AutoDock\u2019s default force\n2.5. DNA binding studies field does not include parameters for iridium, these were manually\n incorporated into the parameter library. Flexible residues within a 6 \u00c5\n To investigate the potential DNA binding affinity of the anticancer radius of the identified binding pocket were selected to allow adaptive\ncomplexes, we first used 1H NMR spectroscopy in an 80 % DMSO\u2011d6 / docking. The docking results revealed several key interactions between\n20 % D2O solution to examine interactions between the representative Ir1 and BSA, including electrostatic and hydrogen-bonding interactions\ncomplexes Ir1\u2013Ir5 and the model nucleobase 9-methyladenine (9-MeA) (Fig. 5). Notably, salt bridges were formed with ARG217 and ARG198,\n(Figs. S55\u201359). The 1H NMR spectra showed no significant changes over while a \u03c0-cation interaction was observed with ARG217. Additionally,\ntime, suggesting that these complexes did not engage in coordination several \u201cnon-conventional\u201d hydrogen bonds were identified, involving\nreactions with 9-MeA during the 24-hour observation period. Mass residues such as ASP450, ARG194, LEU480, GLU478, GLU353, and\nspectrometry analysis also revealed no detection of nucleobase adducts LEU346. These results further demonstrate the interaction between Ir1\n(Fig. S60). Additionally, we monitored the interaction of calf thymus and BSA, providing valuable insights into the binding mechanisms and\nDNA (CT-DNA) with complexes Ir1\u2013Ir5 by UV\u2013visible absorption potential implications for transport, stability, and bioavailability.\nspectroscopy (Fig. S61). CT-DNA is DNA extracted from calf thymus and\nis commonly used in molecular biology and drug research, especially in 2.7. Cellular localization and cellular uptake pathway\nexperiments involving the interaction of DNA with drugs or other mol\u00ad\necules. Keeping the concentration of the complexes constant while To investigate the potential cellular targets of these complexes, their\nprogressively increasing the CT-DNA concentration, hyperchromicity intracellular localization within various organelles was analyzed using\nand an approximate 5 nm blue shift were observed in the spectra of the confocal microscopy, leveraging their fluorescent properties (Fig. 6). To\ncomplexes, indicating that their interaction with CT-DNA likely occurs visualize specific organelles, MTDR (MitoTracker Red CM-H2XRos),\nvia a noncovalent electrostatic binding mechanism [51,52]. Using the LTDR (LysoTracker Red DND-99), and DAPI (4,6-diamino-2-phenyl\u00ad\nBenesi-Hildebrand equation, we determined the binding constants (Kb) indole) were used as probes for the mitochondria, lysosomes and nu\u00ad\nfor these complexes. The results revealed that the Kb values were 1.38 \u00d7 cleus, respectively [59,60]. A549 cells were co-stained with Ir1\u2013Ir5,\n104 M\u2212 1 for Ir1, 2.83 \u00d7 104 M\u2212 1 for Ir2, 3.37 \u00d7 104 M\u2212 1 for Ir3, 5.66 \u00d7 Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9, and these probes. After 1 h of treatment, clear green fluores\u00ad\n103 M\u2212 1 for Ir4, and 7.11 \u00d7 103 M\u2212 1 for Ir5. These values are signifi\u00ad cence was detected in the cytoplasm, confirming effective cellular up\u00ad\ncantly lower than those reported for other DNA-binding complexes (Kb take of both Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9. None of the complexes exhibited\n> 105 M\u2212 1) [20,53,54], suggesting that these complexes exhibit rela\u00ad notable colocalization with DAPI or LTDR, as reflected by the low\ntively weak binding affinity to CT-DNA. Furthermore, the absence of Pearson correlation coefficients (PCC: 0\u20130.03 for DAPI and 0.14\u20130.29\ncoordination reactions with 9-MeA and the low nuclear co-localization for LTDR), indicating minimal localization in the nucleus or lysosomes.\nefficiency observed in subsequent tests (see section \u201ccellular localiza\u00ad In contrast, complexes Ir1\u2013Ir5 displayed strong accumulation in mito\u00ad\ntion\u201d below) indicate that DNA binding is unlikely to play a significant chondria, evidenced by high PCC values. (Ir1: 0.90; Ir2: 0.92; Ir3: 0.88;\nrole in the biological activity of these complexes. Ir4: 0.89; Ir5: 0.90). Conversely, Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 showed limited mitochondrial\n colocalization, indicated by low PCC values (Ir1\u02b9\u02b9: 0.32; Ir2\u02b9\u02b9: 0.38; Ir3\u02b9\u02b9:\n2.6. Albumin binding studies 0.30; Ir4\u02b9\u02b9: 0.39; Ir5\u02b9\u02b9: 0.21). These comparisons clearly demonstrate that\n the introduction of the triphenylphosphine group significantly enhances\n Human serum albumin (HSA) is an essential protein found in the the complexes\u2019 mitochondrial targeting ability. The cytotoxic effects of\nbloodstream, responsible for transporting and metabolizing various these complexes are likely attributed to mitochondria-mediated cell\nbioactive substances. Understanding its interaction with anticancer death. Given that cancer cells possess a higher mitochondrial content\ndrugs is essential for elucidating their transport, distribution, meta\u00ad than normal cells, they exhibit greater sensitivity to mitochondrial\nbolism, and therapeutic effects [52]. Herein, bovine serum albumin disruption. This heightened vulnerability may contribute to the\n(BSA), which shares a similar structure with HSA and is more readily enhanced anticancer selectivity (SI: 5.46\u201314.84) observed for the\n\n 6\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\nFig. 5. (a) Docking model of complex Ir1 positioned in the hydrophobic cavity of BSA (PDB ID: 4OR0). (b) Interaction details between complex Ir1 and the\npolypeptide chains.\n\n\ntriphenylphosphine-based complexes in this study. Notably, the com\u00ad complexes Ir1 and Ir1\u02b9\u02b9. JC-1, known for its color-shifting properties, is\nplexes Ir1\u2013Ir5 demonstrated significantly higher positive zeta potentials ideal for detecting MMP variations. Under high MMP conditions, JC-1\nof Ir1 (52.14 \u00b1 0.18), Ir2 (54.67 \u00b1 0.23), Ir3 (41.58 \u00b1 0.32), Ir4 (40.45 aggregates in the mitochondrial matrix as J-aggregates, emitting red\n\u00b1 0.26), and Ir5 (36.55 \u00b1 0.15) (Fig. S68), compared to the negative fluorescence. In contrast, at low MMP levels, JC-1 remains as monomers,\nzeta potentials of Ir1\u02b9\u02b9 (\u2212 15.56 \u00b1 0.21), Ir2\u02b9\u02b9 (\u2212 13.79 \u00b1 0.23) Ir3\u02b9\u02b9 producing green fluorescence. The red-to-green fluorescence shift pro\u00ad\n(\u2212 11.06 \u00b1 0.32), Ir4\u02b9\u02b9 (\u2212 16.27 \u00b1 0.26), and Ir5\u02b9\u02b9 (\u2212 19.63 \u00b1 0.15) vides a straightforward way to monitor reductions in MMP. A549 cells\nrespectively (Fig. S69). This property could improve mitochondrial were treated with Ir1 and Ir1\u02b9\u02b9 at concentrations of 0.5 and 1.0, and Ir1\ntargeting, as mitochondria have negatively charged surfaces within the significantly reduced MMP in these cells compared to the untreated\ncytosol. The elevated positive zeta potentials of Ir1\u2013Ir5 may facilitate controls. As Ir1 concentration of increased from 0 to 2 \u00d7 IC50, the\ntheir preferential accumulation in cancer cell mitochondria, which proportion of cells showing mitochondrial membrane depolarization\ntypically have higher membrane potentials than those in normal cells significantly rose by 60.1 %, from 20.1 % to 85.1 % (Fig. 8a, d), closely\n[61], potentially enhancing their effectiveness as targeted anticancer matching the depolarization level of the positive control (67.2 %). In\nagents. Furthermore, the markedly higher log P values of Ir1\u2013Ir5 contrast, Ir1 treatment in BEAS-2B normal cells and Ir1\u02b9\u02b9 treatment in\ncompared to Ir1\u02b9\u02b9-Ir5\u02b9\u02b9, as discussed in section on cytotoxicity, may A549 cells resulted in minimal depolarization, measuring 15.6 % and\nfurther support their mitochondrial localization. Generally, highly 25.2 %, respectively (Fig. 8b, e and 8c, f). These findings align with the\nlipophilic anticancer complexes can disrupt cellular metabolic balance observed cytotoxicity and anticancer selectivity, with Ir1 exhibiting\nand increase intracellular ROS levels by intensifying interactions with greater specificity for A549 cancer cells compared to BEAS-2B normal\nmitochondrial membranes [62]. Cellular uptake of small molecule cells. Furthermore, Ir1 demonstrated stronger cytotoxic effects against\ncomplexes can occur through energy-dependent or energy-independent both A549 and HeLa cancer cells than Ir1\u02b9\u02b9. This suggests that the in\u00ad\nmechanisms [59]. Laser confocal microscopy was used to examine the clusion of the triphenylphosphine moiety enhances the anticancer po\u00ad\ncellular uptake of complexes Ir1 and Ir1\u02b9\u02b9 in A549 cells. Following 1 h of tency of the complexes by specifically targeting mitochondria and\nincubation, clear green fluorescence was observed in the cytoplasm, disrupting their function.\nconfirming successful penetration (Fig. 7). However, at 4 \u25e6 C or with the\nmetabolic inhibitor CCCP, fluorescence intensity dropped significantly,\nindicating that the uptake of both triphenylphosphine-containing and 2.9. Cellular ROS determination\nnon-triphenylphosphine complexes relies on energy. In contrast, treat\u00ad\nment with the endocytosis inhibitor chloroquine did not alter intracel\u00ad Many studies have demonstrated that impaired mitochondria lose\nlular fluorescence levels compared to the 37 \u25e6 C control, ruling out their ability to regulate ROS production efficiently, leading to height\u00ad\nendocytosis as the primary uptake mechanism for Ir1 and Ir1\u02b9\u02b9. ened oxidative stress within cancer cells [67,68] Given that cancer cells\n generally experience higher oxidative stress than normal cells, further\n ROS elevation induced by anticancer complexes is less likely to disrupt\n2.8. Mitochondrial membrane depolarization the oxidative equilibrium in normal cells, potentially contributing to the\n selectivity of these agents [68\u201370]. The impact of varying concentra\u00ad\n Due to the preferential accumulation of these cyclometalated com\u00ad tions (0.5, 1.0, and 2.0 \u00d7 IC50) of Ir1 and Ir1\u02b9\u02b9 complexes on ROS levels in\nplexes in mitochondria, their effects on mitochondrial function were A549 cancer cells and BEAS-2B normal cells was evaluated using the\nfurther investigated. The mitochondrial membrane potential (MMP, fluorescent probe DCFH-DA and observed under a fluorescence micro\u00ad\n\u25b3\u03c8m), an essential electrical gradient for cellular activity, is crucial for scope (Fig. 9). It is important to note that this assay measures overall\nmitochondrial integrity. The loss of MMP is a critical early step in oxidative stress rather than quantifying specific ROS. A549 cells treated\ninitiating apoptosis, triggering a series of biochemical changes within with complex Ir1 showed a marked, concentration-dependent increase\nthe mitochondrial membrane that ultimately result in cell death in fluorescence intensity, correlating directly with elevated ROS levels\n[47,63\u201366]. To assess changes in MMP, flow cytometry with the JC-1 compared to untreated controls (Fig. 9a, d). This trend highlights the\nfluorescent probe was used on A549 and BEAS-2B cells incubated with ability of these complexes to disrupt cellular redox balance by\n\n 7\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\nFig. 6. Confocal microscopy analysis of the intracellular localization of Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9. A549 cells were treated with Ir1\u2013Ir5 and Ir1\u02b9\u02b9\u2013Ir5\u02b9\u02b9 (2 \u03bcM) for 1 h at\n37 \u25e6 C, followed by co-staining with DAPI (1 \u03bcg/mL), MTDR (500 nM), or LTDR (75 nM) for another hour. Scale bar: 20 \u03bcm. Green fluorescence indicates Ir1\u2013Ir5 and\nIr1\u02b9\u02b9\u2013Ir5\u02b9\u02b9, red represents mitochondria or lysosomes, and blue corresponds to the nucleus. (For interpretation of the references to color in this figure legend, the reader\nis referred to the web version of this article.)\n\n\n\n\n 8\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\nFig. 7. Influence of temperature (37 \u25e6 C or 4 \u25e6 C), chloroquine (50 \u03bcM), and CCCP (50 \u03bcM) on the uptake of Ir1 (2 \u03bcM) (a) and Ir1\u02b9\u02b9 (2 \u03bcM) (b) by cells. Scale bar: 20\n\u03bcm, \u03bbex = 405 nm, \u03bbem = 430\u2013490 nm.\n\n\n\n\nFig. 8. (a, d) Effects of Ir1 on the MMP in A549 cancer cells. (b, e) Effects of Ir1 on the MMP in BEAS-2B cells. (c, f) Effects of Ir1\u02b9\u02b9 on the MMP in A549 cancer cells.\nCCCP (Carbonyl cyanide m-chlorophenyl hydrazone) served as a positive control for inducing mitochondrial membrane depolarization. Data are presented as mean\n\u00b1 SD from three replicates. Statistical significance compared to the untreated control: *p < 0.05, **p < 0.01.\n\n\npromoting ROS generation [51,71,72]. However, the changes of ROS 2.10. Apoptosis\nlevels in BEAS-2B cells following an increase in Ir1 concentration were\nnegligible (Fig. 9b, e). At the same concentration, the fluorescence in\u00ad Anticancer complexes with high ROS generation can disrupt the\ntensity of Ir1\u02b9\u02b9 in A549 cells, representing ROS levels, was notably lower cellular redox balance, inducing apoptosis and cellular damage [73].\ncompared to Ir1 (Fig. 9a vs. Fig. 9c). This observation aligns with the The mechanism of apoptosis-induced cell death was evaluated with the\nanticancer activity and selectivity of the iridiumIII complexes, as Ir1 annexin V/PI assay. A549 cancer cells were treated with Ir1 and Ir1\u02b9\u02b9 at\nexhibited selective cytotoxicity toward A549 cancer cells over BEAS-2B 0.25, 0.5, and 1 \u00d7 IC50 for 48 h and analyzed by flow cytometry\nnormal cells. Collectively, these findings suggest that increased ROS (Fig. 10). Both complexes exhibited a concentration-dependent rise in\nproduction is a key mechanism driving the action of these complexes. the late apoptotic cell population. Specifically, 88.28 % of A549 cells\n treated with Ir1 at 1 \u00d7 IC50 underwent late apoptosis (Fig. 10a). The\n\n 9\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\nFig. 9. Fluorescence microscopy analysis of ROS levels in A549 cells treated with Ir1 (a, d) and Ir1\u02b9\u02b9 (c, f) and in BEAS-2B cells treated with Ir1 (b, e) for 24 h at 37 \u25e6 C.\nCells were stained with DCFH-DA, and ROSup served as a positive control for ROS induction. Data are presented as mean \u00b1 SD from three replicates. Statistical\nsignificance compared to untreated controls: *p < 0.05, **p < 0.01.\n\n\nimpact of Ir1 on BEAS-2B normal cells was evaluated under the same population was observed, alongside a decrease in the s and G0/G1 phases\nconditions (Fig. 10b). At 1 \u00d7 IC50, only 11.75 % of cells were in the late (Figs. 12a, b, S66, and S67). At 0.5 \u00d7 IC50, the G2/M phase population\napoptotic stage, showing minimal change compared to the untreated increased by 10.16 % for Ir1 and 3.67 % for Ir1\u02b9\u02b9 compared to untreated\ncontrol. In contrast, Ir1\u02b9\u02b9 exhibited a more pronounced concentration- controls. These results demonstrate that both complexes, with or\ndependent effect on A549 cells, with late apoptotic populations without the triphenylphosphine moiety, induce G2/M phase arrest.\nincreasing from 12.1 % to 14.28 % and then to 16.08 % as the con\u00ad\ncentration rose (Fig. 10c). These results are consistent with the low 2.12. Inhibition of cell migration\ncytotoxicity of Ir1 in normal cells and confirm that these complexes\ninduce apoptosis as the primary mode of cell death. Reduced surface adhesion can facilitate the migration of cancer cells\n Among the various genes that regulate apoptosis, the Bcl-2 protein from the original tumor to other organs, making the prevention of\nfamily, including B cell CLL/Bcl-2, has received significant scientific metastasis a critical challenge in cancer therapy [77,78]. Cell migration,\ninterest. Bax, a key pro-apoptotic protein, is instrumental in maintaining metastasis and invasion are heavily influenced by extracellular matrix\nthe balance between cell survival and programmed death. Upon stim\u00ad degradation and changes in cell adhesion molecules. To investigate the\nulation, Bax downregulates Bcl-2 expression, shifting the balance to\u00ad effect of the representative complex Ir1 on the migration of A549 cancer\nward apoptosis [74,75]. In this study, Western blotting was employed to cells, a wound-healing assay was conducted, as shown in Fig. 13.\nquantify Bax and Bcl-2 protein levels to confirm the involvement of the Treatment with 0.5 \u00d7 IC50 of Ir1 reduced the wound closure rate (WCR)\nmitochondrial apoptosis pathway. Fig. 11 demonstrates that as Ir1 in A549 cells to 12.87 %, compared to 41.51 % in the control group. Ir1\nconcentration increases, Bcl-2 levels significantly decrease, while Bax also demonstrated a dose-dependent reduction in WCR in A549 cancer\nlevels rise correspondingly. These changes indicate that Ir1 promotes cells. These observations suggest that these cyclometalated iridiumIII\napoptosis in A549 cells through the mitochondrial pathway. This complexes effectively inhibit A549 cell migration in vitro.\nconclusion aligns with the findings of the co-localization analysis.\n 3. Conclusions\n2.11. Cell cycle arrest\n In this work, we developed and prepared a set of triphenylphosphine-\n Cell cycle arrest, often triggered by apoptotic signals, plays a critical modified cyclometalated iridiumIII complexes designed to selectively\nrole in facilitating apoptosis. Many anticancer complexes exert their target mitochondria in cancer cells. The inclusion of the triphenyl\u00ad\neffects by disrupting the cell cycle [76]. To evaluate this, we used flow phosphine group significantly enhanced mitochondrial localization and\ncytometry to analyze the impact of Ir1 (containing a triphenylphosphine cytotoxicity in cancer cells while maintaining selectivity over normal\nmoiety) and Ir1\u02b9\u02b9 (without the triphenylphosphine moiety) on cell cycle cells. Through comprehensive cellular analyses, these complexes\nprogression in A549 cancer cells. After 24 h of treatment at 0.25 \u00d7 IC50 demonstrated the ability to effectively depolarize mitochondrial mem\u00ad\nand 0.5 \u00d7 IC50, a concentration-dependent rise in the G2/M phase brane potential, increase ROS production and trigger intrinsic apoptosis.\n\n 10\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\nFig. 10. Cell apoptosis analysis by flow cytometry. (a) A549 cells untreated (control) or treated with various concentrations of Ir1 for 48 h. (b) BEAS-2B cells\nuntreated (control) or treated with Ir1 for 48 h. (c) A549 cells untreated (control) or treated with Ir1\u02b9\u02b9 at different concentrations for 48 h. (d) Apoptosis histograms for\nA549 and BEAS-2B cells treated with Ir1 and Ir1\u02b9\u02b9 at 0.25 \u00d7 IC50, 0.5 \u00d7 IC50, and 1 \u00d7 IC50 for 48 h. Data are presented as mean \u00b1 SD from three replicates. Statistical\nsignificance compared to the control: *p < 0.05, **p < 0.01.\n\n\nMoreover, the promotion of G2/M phase cell cycle arrest and inhibition which were synthesized following adapted protocols from the literature\nof cell migration were also observed for these complexes, indicating [79,80]. The synthesis of metal precursors D1\u2013D5 was carried out using\nmultiple pathways for their anticancer action. These observations previously reported methodologies [18,44]. Further descriptions of the\nhighlight the role of the triphenylphosphine moiety in augmenting both biological assays and experimental protocols are also provided in the\nthe anticancer efficacy and selective mitochondrial targeting of these supplementary materials for reference.\ncomplexes. Moreover, these triphenylphosphine-modified iridiumIII\ncomplexes integrate imaging capabilities with anticancer effects, pre\u00ad\n 4.1. Synthesis of complexes\nsenting a promising approach for developing multifunctional thera\u00ad\nnostic agents.\n General Procedure: The reaction involved mixing cyclometalated\n iridium(III) precursors (1 eq.), ligands (2 eq.), and an excess of NH4PF6\n4. Experimental section\n (2 eq.) in a CH2Cl2/CH3OH solution at a 1:1 ratio (v/v). The mixture was\n stirred at room temperature for 24 h to ensure reaction completion.\n The Supplementary materials include comprehensive details,\n Afterward, the solvents were evaporated under reduced pressure to yield\ncovering general procedures and the preparation of ligands L1 and L2,\n a crude solid. This solid was dissolved in CH2Cl2, filtered to eliminate\n\n 11\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n Ph3), 128.73 (C\u2013N), 127.48 (C-NH), 125.53, 124.30, 122.84, 120.44,\n 119.30, 118.61, 115.64, 66.82 (OCH2), 20.46 (O-CH2CH2), 20.06(P-\n CH2), 18.95 (P-CH2CH2). ESI-MS (m/z): calcd for C63H49IrN6OP\n 1129.3329, found: 1129.3446 [M\u2212 H\u2212 2PF6]+. 31P NMR (202 MHz,\n DMSO\u2011d6): \u03b4 \u2212 151.22, \u2212 147.71, \u2212 144.20, \u2212 140.68, \u2212 137.17, 24.06.\n Elemental analysis: calcd for C63H50IrN6OP3F12: C, 53.28; H, 3.55; N,\n 5.92. found: C, 53.53; H, 3.37; N, 6.71.\n Ir2: isolated yield 61 mg (43 %).1H NMR (500 MHz, DMSO\u2011d6) \u03b4\n 9.22 (d, 2H), 8.30 (d, 2H), 8.23 (d, 2H), 8.18 (d, 1H), 8.11 (m, 2H), 7.98\n (m, 4H), 7.90 (m, 7H), 7.83 (m, 10H), 7.48 (d, 2H), 7.19 (d, 2H), 6.98\n (m,3H), 6.12 (s, 2H), 4.19 (t, OCH2,2H), 3.76 (m, OCH2CH2,2H), 2.13 (s,\n 6H, Ph-CH3), 2.04 (m, 2H, P-CH2CH2), 1.79 (m, 2H,P-CH2CH2). 13C\n NMR (126 MHz, DMSO\u2011d6) \u03b4 167.41, 160.75 (C\u2013O), 153.28, 151.22\n (C\u2013\u2013N), 149.41, 148.76, 144.40, 141.89, 140.10, 139.00, 135.49 (P-\n Ph3), 134.04, 132.50, 132.21, 130.76 (P-Ph3), 128.73 (C\u2013N), 127.45\n (C-NH), 125.48, 123.80, 122.35, 120.04, 119.30, 118.62, 115.62, 66.81\n (OCH2), 31.41(O-CH2CH2), 22.52 (P-CH2), 22.03 (Ph-CH3), 14.42 (P-\n CH2CH2). 31P NMR (202 MHz, DMSO\u2011d6): \u03b4 24.07, \u2212 151.21, \u2212 147.70,\n \u2212 144.19, \u2212 140.67, \u2212 137.16. ESI-MS (m/z): calcd for C65H53IrN6OP\n 1157.3642, found:1157.3772, [M\u2212 H\u2212 2PF6]+. Elemental analysis: calcd\n for: C65H54IrN6O P3F12: C, 53.91; H, 3.76; N, 5.80. found: C, 54.02; H,\n 3.56; N, 6.62.\n Ir3: isolated yield 71 mg (48 %).1H NMR (500 MHz, DMSO\u2011d6) \u03b4\n 9.20 (d, 2H), 8.28 (d, 2H), 8.23 (d, 1H), 8.15 (m, 3H), 7.92 (t, 5H), 7.87\n (m, 16H), 7.41 (d, 2H), 7.17 (d, 2H), 6.89 (t, 2H), 6.70 (d, 2H), 5.74 (d,\n 2H), 4.18 (t, 2H, OCH2CH2), 3.71 (m, 2H, OCH2CH2), 3.59 (s, 6H,\n OCH3), 2.00 (m, 6.5 Hz, 2H, P-CH2CH2), 1.78 (m, 2H, P-CH2CH2). 13C\n NMR (126 MHz, DMSO\u2011d6) \u03b4 167.20, 160.99 (C\u2013O), 153.29, 153.06\n (C\u2013\u2013N), 149.24, 148.81, 144.38, 138.85, 137.30, 135.41 (P-Ph3),\nFig. 11. (a) Protein expression levels analyzed by Western blot after 24 h 134.01, 130.74 (P-Ph3), 128.79, 127.62 (C\u2013N), 127.30 (C-NH), 122.90,\ntreatment with Ir1 at concentrations of 0.5, 1.0, and 2.0 \u00d7 IC50. (b) Histograms\n 122.38, 119.58, 119.27, 118.59, 117.19, 115.56, 107.73, 66.75 (OCH2),\nshowing protein expression levels at various Ir1 concentrations. Data represent\n 54.91 (OCH3), 29.53 (O-CH2CH2), 18.92 (P-CH2), 14.40 (P-CH2CH2).\nthe mean \u00b1 SD from three independent experiments. Statistical significance 31\n P NMR (202 MHz, DMSO\u2011d6): \u03b4 \u2212 151.21, \u2212 147.70, \u2212 144.19,\ncompared to the control: *p < 0.05, **p < 0.01.\n \u2212 140.67, \u2212 137.16, 24.07. ESI-MS (m/z): calcd for, C65H53IrN6O3P\n 1189.3541, found 1189.3678, [M\u2212 H\u2212 2PF6]+. Elemental analysis: calcd\nimpurities, and then was concentrated. Finally, the product was purified\n for: C65H54IrN6O3P3F12:C, 52.74; H, 3.68; N, 5.68. found: C, 52.92; H,\nthrough recrystallization using CH2Cl2 and n-hexane, yielding a light\n 3.51; N, 5.50.\nyellow solid.\n Ir4: isolated yield 78 mg (53 %).1H NMR (500 MHz, DMSO\u2011d6) \u03b4\n Ir1: isolated yield 69 mg (49 %). 1H NMR (500 MHz, DMSO\u2011d6) \u03b4\n 9.24 (d, 2H), 8.33 (m, 3H), 8.24 (d, 2H), 8.11 (m, 2H), 7.99 (t, 2H), 7.92\n9.19 (d, 2H), 8.27 (m, 4H), 8.16 (s, 2H), 8.09 (s, 2H), 7.97 (d, 2H), 7.94\n (t, 3H), 7.87 (m, 15H), 7.57 (d, 2H), 7.18 (d, 2H), 7.10 (m, 4H), 5.73 (d,\n(m, 5H), 7.81 (m 12H), 7.51 (s, 2H), 7.17 (d, 2H), 7.08 (t, 2H), 7.02 (m,\n 2H), 4.18 (t, 2H, OCH2CH2), 3.75 (m, 2H, OCH2CH2), 2.03 (m, 2H, P-\n4H), 6.31 (d, 2H), 4.17 (t, 2H, OCH2), 3.75 (m, 2H, OCH2CH2), 2.03 (m,\n CH2CH2), 1.77 (m, 2H, P-CH2CH2). 13C NMR (126 MHz, DMSO\u2011d6) \u03b4\n2H P-CH2CH2), 1.81 (m, 2H, P-CH2CH2). 13C NMR (126 MHz, DMSO\u2011d6)\n 163.29, 162.32, 160.81 (C\u2013O), 155.02, 153.39 (C\u2013 \u2013N), 150.34, 149.33,\n\u03b4 167.38, 160.77 (C\u2013O), 153.28, 150.86 (C\u2013 \u2013N), 149.60, 148.80,\n 144.09, 140.39, 135.48 (P-Ph3), 134.05, 133.30, 130.76 (P-Ph3),\n144.47, 139.16, 135.61 (P-Ph3), 134.04, 132.64, 131.72, 130.76 (P-\n 128.84, 128.29 (C\u2013N), 127.78 (C-NH), 124.90, 123.79, 122.24, 119.31,\n\n\n\n\nFig. 12. Flow cytometry results showing the cell cycle distribution of A549 cancer cells treated with Ir1 (a) and Ir1\u02b9\u02b9 (b) at concentrations of 0.25 \u00d7 IC50 and 0.5 \u00d7\nIC50. Cells were stained using PI/RNase prior to analysis. Data represent the mean \u00b1 standard deviation (SD) from three independent experiments. Statistical sig\u00ad\nnificance was determined in comparison to untreated controls, with *p < 0.05 and **p < 0.01.\n\n 12\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\nFig. 13. (a) Wound-healing assay of A549 cells after 24 h treatment with Ir1. (b) Histogram analysis of wound closure rates after 24 h treatment. Representative\nimages were taken at 0 h and 24 h, with wound widths marked (\u03bcm). Scale bar: 100 \u03bcm. Wound closure rate was calculated as (R0 \u2212 R1)/R0 \u00d7 100 %. Data are\npresented as mean \u00b1 SD from three replicates. Statistical significance compared to the control: *p < 0.05, **p < 0.01.\n\n\n\n\n 13\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\n118.62, 115.63, 113.87, 99.58, 66.80 (OCH2), 31.41 (O-CH2CH2), 22.52 (s ,1H, NH), 10.51 (s, 1H), 9.22 (s, 1H), 8.69 (d, 2H), 8.11 (s, 2H), 7.87\n(P-CH2), 14.40 (P-CH2CH2). 31P NMR (202 MHz, DMSO\u2011d6): \u03b4 \u2212 151.22, (d, 2H), 7.75 (s, 2H), 7.70 (m, 4H), 7.31 (d, 2H), 7.04 (d, 2H), 6.91 (d,\n\u2212 147.71, \u2212 144.19, \u2212 140.68, \u2212 137.17, 24.06. 19F NMR (471 MHz, 2H), 6.78 (s, 2H), 6.21 (s, 2H), 4.03 (t, 2H, OCH2), 2.18 (s, 6H, Ph-CH3),\nDMSO\u2011d6): \u03b4 \u2212 69.41 (s), \u2212 70.92 (s), \u2212 106.61 (d), \u2212 108.81 (d). ESI-MS 1.83 (m, 2H, OCH2CH2), 1.51 (m, 2H, CH2CH3), 0.99 (t, 3H, 2CH3) 13C\n(m/z): calcd for C63H49IrN6OPF2 1201.3031, found: 601.1616 NMR (126 MHz, DMSO\u2011d6) \u03b4 167.42, 160.94, 153.23, 151.23, 149.42,\n[(M\u2212 2PF6)/2]+. Elemental analysis: calcd for C63H50IrN6OP3F14: C, 148.69, 144.34, 141.88, 140.09, 139.01, 132.47, 128.64 (C\u2013N),\n51.96; H, 3.32; N, 5.77. found: C, 52.13; H, 3.07; N, 5.53. 127.35, 125.47, 123.80, 122.04 (C-NH), 120.04, 115.45, 67.93 (OCH2),\n Ir5: isolated yield 64 mg (47 %).1H NMR (500 MHz, DMSO\u2011d6) \u03b4 31.15 (OCH2CH2), 22.03 (C-CH3), 19.20 (CH2CH3), 14.18 (CH2CH3).\n 31\n9.18 (d, 2H), 8.87 (d, 2H), 8.28 (m, 3H), 8.13 (m, 2H), 7.92 (m, 4H), P NMR (202 MHz, DMSO\u2011d6): \u03b4 \u2212 151.22, \u2212 147.71, \u2212 144.19,\n7.87 (m, 15H), 7.17 (d, 2H), 7.09 (m, 3H), 6.91 (m, 2H), 6.60 (m, 2H), \u2212 140.68, \u2212 137.17. ESI-MS (m/z): calcd for C47H40IrN6O 897.2891,\n6.30 (m, 2H), 4.17 (m, 2H, OCH2CH2), 3.78 (m, 2H, OCH2CH2), 2.04 (m, found: 897.3020 [M\u2212 PF6]+. Elemental analysis: calcd for C47H40Ir\u00ad\n2H, P-CH2CH2), 1.77 (m,2H, P-CH2CH2). 13C NMR (126 MHz, DMSO\u2011d6) N6OPF6: C, 54.17; H, 3.87; N, 8.06 found: C, 54.33; H, 3.69; N, 7.94.\n\u03b4 160.79 (C\u2013O), 153.11 (C\u2013 \u2013N), 149.17, 145.19, 143.63, 139.44, Ir3\u02b9\u02b9: isolated yield 67 mg (59 %).1H NMR (500 MHz, DMSO\u2011d6) \u03b4\n135.64 (P-Ph3), 134.05, 133.18, 132.73, 130.78 (P-Ph3), 129.08, 15.43 (s, 1H, NH)), 10.49 (s, 2H), 9.24 (s, 1H), 8.69 (d, 2H), 8.15 (s, 3H),\n128.75, 127.34 (C\u2013N), 126.99 (C-NH), 123.55, 122.19, 119.30, 118.62, 7.78 (d, 2H), 7.77 (m, 2H), 7.68 (d, 2H), 7.63 (t, 2H), 7.06 (d, 2H), 6.71\n115.62, 112.58, 108.89, 66.81 (OCH2), 31.41 (O-CH2CH2), 22.52 (P- (s, 2H), 6.67 (d, 2H), 5.93 (s, 2H), 4.03 (t, 2H, OCH2), 3.66 (s, 6H,\nCH2), 14.42 (P-CH2CH2). 31P NMR (202 MHz, DMSO\u2011d6): \u03b4 \u2212 154.75. OCH3), 1.81 (m, 2H, OCH2CH2), 1.54 (m, 2H, CH2CH3), 0.99 (t, 3H,\n\u2212 147.70, \u2212 144.19, \u2212 140.68, \u2212 137.68, \u2212 133.65, 24.07. ESI-MS (m/z): CH3).13C NMR (126 MHz, DMSO\u2011d6) \u03b4 167.22, 160.98, 160.63, 153.28,\ncalcd for C59H46IrN8OP 1107.3234, found: 1107.3236 and 554.1661 149.36, 138.83, 137.34, 128.87 (C\u2013N), 127.20, 122.95, 122.25 (C-NH),\n[M\u2212 H\u2212 2PF6]+. Elemental analysis: calcd for C59H47IrN8OP3F12: C, 119.57, 117.22, 115.05, 107.64, 67.79 (OCH2), 54.93 (OCH3), 31.15\n50.72; H, 3.39; N, 8.02. found: C, 50.94; H, 3.55; N,8.21. (OCH2CH2), 19.21 (CH2CH3), 14.18 (CH2CH3). 31P NMR (202 MHz,\n Ir1\u02b9\u02b9: isolated yield 65 mg (61 %).1H NMR (500 MHz, DMSO\u2011d6) \u03b4 DMSO\u2011d6): \u03b4 \u2212 154.74\u2013\u2013151.23, 147.72, \u2212 144.20, \u2212 140.68, \u2212 137.17,\n14.53 (s ,1H, NH), 9.38 (s, 1H), 9.18 (s, 1H), 8.32 (d, 2H), 8.27 (d, 2H), \u2212 133.69. ESI-MS (m/z): calcd for C47H40IrN6O3 929.2791, found:\n8.15 (d, 2H), 8.07 (s, 2H), 7.96 (d, 2H), 7.88 (t, 2H), 7.51 (d, 2H), 7.19 929.2904 [M\u2212 PF6]+. Elemental analysis: calcd for C47H40IrN6O3PF6: C,\n(d, 2H), 7.07 (t, 2H), 7.02 (m, 4H), 6.30 (d, 2H), 4.10 (t, 2H, OCH2), 1.80 52.56; H, 3.75; N, 7.82. found: C, 52.83; H, 3.57; N, 7.64.\n(m, 2H, OCH2CH2), 1.47 (m, 2H, CH2CH3), 0.97 (t, CH3). 13C NMR (126 Ir4\u02b9\u02b9: isolated yield 58 mg (51 %).1H NMR (500 MHz, DMSO\u2011d6) \u03b4\nMHz, DMSO\u2011d6) \u03b4 167.37, 160.75 (C\u2013O), 153.36, 151.02, 149.68, 14.73 (s, 1H, NH)), 9.52 (s, 1H), 9.21 (s, 1H), 8.36 (d, 2H), 8.31 (d, 2H),\n144.51, 139.13, 131.70, 130.70, 128.90 (C\u2013N), 125.52, 124.31, 122.78 8.22 (s, 3H), 8.21 (m, 3H), 7.99 (t, 2H), 7.58 (d, 2H), 7.18 (d, 2H), 7.12\n(C-NH), 122.27, 120.41, 115.22, 67.86 (OCH2), 31.15 (OCH2CH2), (m, 4H), 5.73 (m, 2H), 4.10 (t, 2H, OCH2), 1.80 (m, 2H, OCH2CH2), 1.48\n19.21 (CH2CH3), 14.19 (CH2CH3). ESI-MS (m/z): calcd for C45H36IrN6O (m, 2H, CH2CH3), 0.97 (t, 3H, CH3). 13C NMR (126 MHz, DMSO\u2011d6) \u03b4\n869.2580, found: 869.2700 [M\u2212 PF6]+. 31P NMR (202 MHz, DMSO\u2011d6): 163.30, 162.28, 160.74, 160.17, 155.13, 153.45, 150.45, 140.37,\n\u03b4-151.21, \u2212 147.70, \u2212 144.18, \u2212 140.67, \u2212 137.16. Elemental analysis: 128.92 (C\u2013N), 128.32, 124.96, 123.74, 122.21 (C-NH), 115.15, 113.87,\ncalcd for C45H36IrN6OPF6: C, 53.30; H, 3.58; N, 8.29. found: C, 53.53; H, 99.52, 67.84 (OCH2), 31.41 (OCH2CH2), 22.51 (CH2CH3), 14.40\n3.37; N, 8.01. (CH2CH3). 31P NMR (202 MHz, DMSO\u2011d6): \u03b4 \u2212 151.21. \u2212 147.70,\n Ir2\u02b9\u02b9: isolated yield 65 mg (55 %).1H NMR (CDCl3, 500 MHz) \u03b4 15.43 \u2212 144.19, \u2212 140.67, \u2212 137.16. 19F NMR (471 MHz, DMSO\u2011d6): \u03b4 \u2212 69.41\n\n\n\n\n 14\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\n(s), \u2212 70.92 (s), \u2212 106.60 (d), \u2212 108.81 (d). ESI-MS (m/z): calcd for NMR (202 MHz, DMSO\u2011d6): \u03b4 \u2212 151.22, \u2212 147.71, \u2212 144.19, \u2212 140.68,\nC45H32IrN6OF4 941.2203 found: 941.2394 [M\u2212 PF6]+. Elemental anal\u00ad \u2212 137.13. ESI-MS (m/z): calcd for C41H34IrN8O 847.2485\nysis: calcd for C45H32IrN6OPF10: C, 51.48; H, 3.26; N, 8.00. found: C, found:847.2476 [M\u2212 PF6]+. Elemental analysis: calcd for C41H34Ir\u00ad\n51.63; H, 3.07; N, 7.71. N8OPF6: C, 49.64; H, 3.45; N, 11.30. found: C, 49.83; H, 3.67; N, 11.41.\n Ir5\u02b9\u02b9: isolated yield 56 mg (55 %). 1H NMR (500 MHz, DMSO\u2011d6) \u03b4\n8.87 (d , 2H), 8.33 (d, 2H), 8.27 (s, 2H), 8.05 (m, 2H), 7.73 (d, 2H), 7.18 CRediT authorship contribution statement\n(d, 3H), 7.13 (m, 4H), 6.90 (t, 2H), 6.60 (t, 2H), 6.30 (d, 2H), 4.09 (t, 2H,\nOCH2), 1.75 (m, 2H OCH2CH2), 1.48 (m, 2H, CH2CH3), 0.97 (t, 3H, Hanxiu Fu: Writing \u2013 original draft, Investigation, Formal analysis.\nCH3). 13C NMR (126 MHz, DMSO\u2011d6) \u03b4 143.65, 139.48, 133.53, 133.01, Shuli Wang: Investigation. Yuwen Gong: Investigation. Heqian Dong:\n129.37 (C\u2013N), 128.99, 126.84, 123.49 (C-NH), 115.28, 112.58, 108.88, Investigation. Kangning Lai: Investigation. Zhihao Yang: Investiga\u00ad\n67.86 (OCH2), 31.16 (OCH2CH2), 19.21 (CH2CH3), 14.19 (CH2CH3). 31P tion. Chunyan Fan: Investigation. Zhe Liu: Supervision, Funding\n\n 15\n\fH. Fu et al. Bioorganic Chemistry 155 (2025) 108148\n\n\n\n\nacquisition. Lihua Guo: Writing \u2013 review & editing, Supervision, Project [8] A. Zamora, G. Vigueras, V. Rodr\u00edguez, M.D. Santana, J. Ruiz, Cyclometalated\n iridium(III) luminescent complexes in therapy and phototherapy, Coord. Chem.\nadministration, Conceptualization.\n Rev. 360 (2018) 34\u201376, https://doi.org/10.1016/j.ccr.2018.01.010.\n [9] C.P. Tan, Y.M. Zhong, L.N. Ji, Z.W. Mao, Phosphorescent metal complexes as\nDeclaration of competing interest theranostic anticancer agents: combining imaging and therapy in a single\n molecule, Chem. Sci. 12 (2021) 2357\u20132367, https://doi.org/10.1039/d0sc06885.\n [10] L. Filippi, A. Chiaravalloti, O. Schillaci, R. Cianni, O. Bagni, Theranostic\n The authors declare that they have no known competing financial approaches in nuclear medicine: current status and future prospects, Expert. Rev.\ninterests or personal relationships that could have appeared to influence Med. Devices 17 (2020) 331\u2013343, https://doi.org/10.1080/\nthe work reported in this paper. 17434440.2020.1741348.\n [11] H. Chen, W. Zhang, G. Zhu, J. Xie, X. Chen, Rethinking cancer nanotheranostics,\n Nat. Rev. Mater. 2 (2017) 17024, https://doi.org/10.1038/natrevmats.2017.24.\nAcknowledgments [12] X.D. Zhang, J. Chen, Y. Min, G.B. Park, X. Shen, S.S. Song, Y.M. Sun, H. Wang,\n W. Long, J. Xie, K. Gao, L. Zhang, S. Fan, F. Fan, U. Jeong, Metabolizable Bi2Se3\n nanoplates: biodistribution, toxicity, and uses for cancer radiation therapy and\n We gratefully acknowledge the support from the Taishan Scholars imaging, Adv. Funct. Mater. 24 (2014) 1718\u20131729, https://doi.org/10.1002/\nProgram, the Natural Science Foundation of Shandong Province adfm.201302312.\n(ZR2022MB038), and the Young Talents Invitation Program of Shan\u00ad [13] Z. Liu, P.J. Sadler, Organoiridium complexes: anticancer agents and catalysts, Acc.\n Chem. Res. 47 (2014) 1174\u20131185, https://doi.org/10.1021/ar400266c.\ndong Provincial Colleges and Universities. We also thank Shiyanjia Lab [14] D.L. Ma, C. Wu, K.J. Wu, C.H. Leung, Iridium(III) complexes targeting apoptotic\n(www.shiyanjia.com) for assisting with the single-crystal XRD data cell death in cancer cells, Molecules 24 (2019) 2739, https://doi.org/10.3390/\nanalysis. molecules24152739.\n [15] L.C. Lee, K.K. Leung, K.K. Lo, Recent development of luminescent rhenium(I)\n tricarbonyl polypyridine complexes as cellular imaging reagents, anticancer drugs,\nAppendix A. Supplementary data and antibacterial agents, Dalton. Trans. 46 (2017) 16357\u201316380, https://doi.org/\n 10.1039/C7DT03465B.\n Supplementary materials includes detailed experimental procedures, [16] J.J. Cao, C.P. Tan, M.H. Chen, N. Wu, D.Y. Yao, X.G. Liu, L.N. Ji, Z.W. Mao,\n Targeting cancer cell metabolism with mitochondria-immobilized phosphorescent\nFigures S1\u2013S69, and Tables S1\u2013S7. Crystallographic data for Ir4\u02b9\u02b9 (CCDC cyclometalated iridium(III) complexes, Chem. Sci. 8 (2017) 631\u2013640, https://doi.\n2384027) are provided as supplementary material. The CIF and check\u00ad org/10.1039/C6SC02901A.\nCIF files for Ir4\u02b9\u02b9 are available for free download from the Cambridge [17] W. Lv, Z. Zhang, K.Y. Zhang, H. Yang, S. Liu, A. Xu, S. Guo, Q. Zhao, W. Huang,\n A mitochondria-targeted photosensitizer showing improved photodynamic therapy\nCrystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/c effects under hypoxia, Angew. Chem. Int. Ed. Engl. 55 (2016) 9947\u20139951, https://\nif. Supplementary data to this article can be found online at https doi.org/10.1002/anie.201604130.\n://doi.org/10.1016/j.bioorg.2025.108148. [18] Y. Chen, T.W. Rees, L. Ji, H. Chao, Mitochondrial dynamics tracking with iridium\n (III) complexes, Curr. Opin. Chem. 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