Functional Upgrading of an Organo-Ir(III) Complex to an Organo-Ir(III) Prodrug as a DNA Damage-Responsive Autophagic Inducer for Hypoxic Lung Cancer Therapy.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Functional Upgrading of an Organo\u2212Ir(III) Complex to an\n Organo\u2212Ir(III) Prodrug as a DNA Damage-Responsive Autophagic\n Inducer for Hypoxic Lung Cancer Therapy\n Meng-Meng Wang,\u00a7 Dong-Ping Deng,\u00a7 An-Min Zhou,\u00a7 Yan Su,* Zheng-Hong Yu,* Hong Ke Liu,\n and Zhi Su*\n Cite This: Inorg. Chem. 2024, 63, 4758\u22124769 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 12:10:41 (UTC).\n\n\n\n\n ABSTRACT: The efficiency of nitrogen mustards (NMs), among\n the first chemotherapeutic agents against cancer, is limited by their\n monotonous mechanism of action (MoA). And tumor hypoxia is a\n significant obstacle in the attenuation of the chemotherapeutic\n efficacy. To repurpose the drug and combat hypoxia, herein, we\n constructed an organo\u2212Ir(III) prodrug, IrCpNM, with the\n composition of a reactive oxygen species (ROS)-inducing moiety\n (Ir\u2212arene fragment)\u2212a hypoxic responsive moiety (azo linker)\u2212a\n DNA-alkylating moiety (nitrogen mustard), and realized DNA\n damage response (DDR)-mediated autophagy for hypoxic lung\n cancer therapy for the first time. Prodrug IrCpNM could\n upregulate the level of catalase (CAT) to catalyze the decomposition of excessive H2O2 to O2 and downregulate the expression\n of the hypoxia-inducible factor (HIF-1\u03b1) to relieve hypoxia. Subsequently, IrCpNM initiates the quadruple synergetic actions under\n hypoxia, as simultaneous ROS promotion and glutathione (GSH) depletion to enhance the redox disbalance and severe oxidative\n and cross-linking DNA damages to trigger the occurrence of DDR-mediated autophagy via the ATM/Chk2 cascade and the\n PIK3CA/PI3K-AKT1-mTOR-RPS6KB1 signaling pathway. In vitro and in vivo experiments have confirmed the greatly\n antiproliferative capacity of IrCpNM against the hypoxic solid tumor. This work demonstrated the effectiveness of the DNA\n damage-responsive organometallic prodrug strategy with the microenvironment targeting system and the rebirth of traditional\n chemotherapeutic agents with a new anticancer mechanism.\n\n\n \u25a0 INTRODUCTION\n The hypoxic microenvironment, as the major feature of the\n overcome the hypoxic environment would still be the best\n choice.11,12\n tumor microenvironment (TME), increased the resistance of Metal\u2212arene complexes, with the common formula of\n cancer cells to the traditional chemotherapeutic treatments and [(Cpx)M(L\u2227L\u2032)Z]0/n+ (Cpx, the arene caps; M, metal ions;\n attenuated the therapeutic effectiveness.1,2 To resolve the L\u2227L\u2032, chelated ligands; Z, the leaving groups), were first\n problem of tumor hypoxia, O2 self-sufficient systems were most reported by Sadler and group as the potential anticancer agents\n desired to supply extra O2, such as the utilization of (Scheme 1a).13,14 Emerging evidence has revealed that the\n overexpressed H2O2 in the TME to produce O2.3\u22125 However, anticancer mechanisms for the oragnometallic complexes were\n even the overexpression of H2O2 was found in hypoxic cancer distinct from classical cis-Pt through DNA damage-mediated\n cells, the manipulation of endogenous enzymes to catalyze the apoptosis.15 The typical Ir\u2212arene complex, Ir(Cp*)(ppy)py\n decomposition of H2O2 to produce O2 to relieve the hypoxic (Cp*, pentamethylcyclopentadienyl; ppy, 2-phenylpyridine;\n condition was still a huge challenge.6,7 On the other hand, py, pyridine), could result in the mitochondria-relevant\n azoreductase was highly expressed in the hypoxic tumors, damages as elevation of reactive oxygen species (ROS) and\n which could reductively cleave the azo bonds.8,9 For example, loss of the mitochondrial membrane potential (MMP).16\n many nanodrugs were designed with an azo-containing system\n as the outer shell for hypoxic cancer treatment, followed by the Received: January 6, 2024\n release of the internal active anticancer agents.10 However, the Revised: February 4, 2024\n nanosystem was usually very complicated with three or four Accepted: February 8, 2024\n independent components, which was a time-consuming work Published: February 26, 2024\n and the exact mechanism was hard to explain. In this case,\n small molecular anticancer agents with the capacity to\n\n \u00a9 2024 American Chemical Society https://doi.org/10.1021/acs.inorgchem.4c00060\n 4758 Inorg. Chem. 2024, 63, 4758\u22124769\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 1. Schematic Illustration of the Anticancer Mechanism for the Multifunctional Prodrug IrCpNM: (a) Reported\nClassical Organo\u2212Iridium Anticancer Agents, (b) Illustration of the Modification of the Small Molecular Prodrug Strategy,\nand (c) Design of IrCpNM and the Activation by Azoreductase\n\n\n\n\nHowever, the underlying molecular mechanism has remained Chk2 cascade (ataxia-telangiectasia-mutated/checkpoint kinase\nunclear. Moreover, new cell death pathways apart from 2) and triggered by the DNA double-strand breaks (DSBs).\napoptosis are urgent to be developed to bypass the flexible The cell death mode for IrCpNM-treated A549 cells was\nTME repellence of tumor cells. The alkylating agents bearing DDR-mediated autophagy, which was first found for the metal-\nthe N(CH2CH2Cl)2 moiety, commonly known as \u2018the nitrogen based anticancer agent and regulated by the PIK3CA/PI3K-\nmustards (NMs)\u2019, are among the early chemotherapeutic AKT1-mTOR-RPS6KB1 signaling pathway. Both in vitro and\nagents against cancer. They exert their biological functions by in vivo biological experiments confirmed the greatly anti-\nforming covalent alkyl linkages due to reaction with proliferative capacity of the complex IrCpNM. This work not\nnucleophilic entities, viz., N7 of guanine in DNA, which led only provided the example of the successful small molecular\nto the impairment of DNA replication and transcription.17 prodrug strategy in metal\u2212arene anticancer agents but also\nHowever, the single component of the Ir\u2212arene complex or demonstrated the effectiveness of the DNA damage-responsive\nnitrogen mustard lost their antiproliferative capacity under organometallic prodrug strategy with the tumor microenviron-\nhypoxia, which suggested that both anticancer mechanisms ment targeting system. Furthermore, this work offered a new\nwere highly dependent on the concentration of oxygen. insight for the rebirth of traditional chemotherapeutic agents\n To minimize the hypoxic influence, the small molecular with a new anticancer mechanism through rational design.\nprodrug strategy was adopted to overcome the limitations of\nsingle-agent therapy,18 which was applied for the first time for\nthe metal\u2212arene anticancer agent (Scheme 1b). The hypoxic\n \u25a0 RESULTS AND DISCUSSION\n Design, Syntheses, and Characterization. Complexes\nazoreductase-responsive azo bond was considered and the new IrCpNM and IrCpNH2 were synthesized according to the\ndouble halberd molecular IrCpNM was synthesized, with the previous literature methods with minor modifications (shown\nconstruction of the ROS-inducing moiety (Ir\u2212arene frag- in Scheme S1).16 In short, the precursor Ir(Cp*)(ppy)Cl\nment)\u2212the hypoxic reductase-responsive moiety (azo linker)\u2212 reacted with the azo-containing ligand or 4-aminopyridine to\nthe DNA-alkylating moiety (nitrogen mustard) (Scheme 1c). form complexes IrCpNM and IrCpNH2, respectively. The\nIrCpNM exhibited superior cytotoxicity to A549 cells under obtained complexes were fully characterized by 1H NMR, 13C\nhypoxia (1% O2) with the IC50 value of 6 \u03bcM and comparative NMR, elemental analysis (EA), ESI-HRMS, and HPLC\nselectivity to cancer cells. The hypoxia has been significantly (Figures S1\u2212S16). The HPLC spectra of IrCpNM were also\nrelieved due to the self-provided O2 from the decomposition of conducted at pH = 7 solution for 24 h, which indicated that\nH2O2 with the upregulated catalase activity and downregulated IrCpNM owned relatively high stability and remained the\nHIF-1\u03b1 after IrCpNM treatment. IrCpNM could accumulate same after 24 h standing in the dark condition (Figure S17).\nin the subcellular mitochondria and nuclei, and it could induce The UV\u2212vis absorption spectra of IrCpNM has indicated that\nthe quadruple synergetic actions, as simultaneous ROS the complex IrCpNM could be stable in the presence of\nelevation and glutathione (GSH) consumption to enhance various biological species, such as cysteine, homocysteine, etc.\nthe redox disbalance and severely oxidative and covalently (Figure S18a\u2212g). However, the obvious interaction between\ncross-linking DNA damages to initiate DNA damage response the complex IrCpNM and the antioxidant glutathione (GSH)\n(DDR)-mediated autophagy. DDR was driven by the ATM/ has been observed, as the absorption intensity of IrCpNM\n 4759 https://doi.org/10.1021/acs.inorgchem.4c00060\n Inorg. Chem. 2024, 63, 4758\u22124769\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. Extracellular reponse to the mimic of azoreductase SDT. (a) LC traces and (b) UV\u2212vis spectra of IrCpNM, IrCpNH2, and IrCpNM (20\n\u03bcM) in the presence of SDT (2 mM, pH 7.4 at 310 K) for 30 min. Gradient: 20% B to 95% B in 25 min, then 80% A for 5 min (A: water with 0.1%\nformic acid, B: CH3OH with 0.1% formic acid).\n\nTable 1. IC50 Values of (\u03bcM) of IrCpNM, IrCpNH2, NM, Mixture, and cis-Pt toward A549, A549R, and HLF Cell Lines after\n48 h Treatment under Normoxia (20% O2) and Hypoxia (1% O2), Respectivelya\n complexes IrCpNM IrCpNH2 NM mixtureb cis-Pt\n normoxia (20% O2)\n A549 1.7 \u00b1 0.2 8.1 \u00b1 0.9 72.4 \u00b1 4.6 9.7 \u00b1 0.5 9.2 \u00b1 0.6\n A549R 8.0 \u00b1 1.5 19.5 \u00b1 3.0 >100 22.3 \u00b1 2.5 38.1 \u00b1 1.5\n HLF 12.7 \u00b1 0.4 43.3 \u00b1 0.3 158 \u00b1 4.6 42.2 \u00b1 2.7 4.9 \u00b1 1.3\n SIc 7.5 5.4 2.2 4.4 0.5\n hypoxia (1% O2)\n A549 5.6 \u00b1 0.4 26.9 \u00b1 1.9 118 \u00b1 14 37.6 \u00b1 0.8 10.7 \u00b1 0.9\n A549R 31.9 \u00b1 2.9 51.6 \u00b1 4.8 >200 56.4 \u00b1 2.7 47.1 \u00b1 2.2\n SIc 2.3 1.6 1.3 1.1 0.5\na\nData are shown as the mean \u00b1 standard deviation (S.D., n = 3). bMixture means the physical mixture of IrCpNH2 and NM with the ratio of 1:1.\nc\nCancer selectivity index (SI) is defined as the ratio of IC50 (HLF) in normoxia/IC50 (A549) in normoxia or hypoxia, respectively.\n\ndropped in a concentration-dependent manner with the dithionite (SDT), as a chemical mimic of azoreductase, was\naddition of GSH (Figure S18h). With the decoration of applied,9 and the reduction process was observed by liquid\nnitrogen mustard, the lipophilicity (log Po/w) of IrCpNM was chromatography mass spectrometry (LC-MS) (Figures 1a and\nelevated to 2.37, compared to 0.04 of the complex IrCpNH2 S21). Complexes IrCpNM and IrCpNH2 owned the retention\n(Table S1), suggesting that IrCpNM could be easier to enter times of 11.43 and 9.75 min with the m/z values of 804.3 and\ncells than IrCpNH2 and would benefit its bioapplication.19 576.3, respectively (Figures 1a and S21). In the presence of\n Interaction with DNA. Both fragments Ir\u2212arene and SDT, the complex IrCpNM was completely disappeared and\nnitrogen mustard were reported with the capacity to interact two new peaks with the retention times of 9.75 and 8.03 min\nwith DNA;16,20 thereby, the binding constants of IrCpNM and were observed with the m/z values of 576.3 and 560.2,\nIrCpNH2 to dsDNA were then calculated from the titration respectively, which could be assigned to the species of\nexperiment,21 as 2.2 \u00d7 104 and 4.2 \u00d7 104, respectively (Figure [Ir(Cp*)(ppy)(4-aminopyridine)] + and [Ir(Cp*)(ppy)-\nS19). Further, an in vitro DNA cleavage experiment was (pyridine)]+. The activation of IrCpNM by SDT was further\ncarried out on the supercoiled pBR322 plasmid using agarose demonstrated by UV\u2212vis spectra, where the pink color of the\ngel (Figure S20). Cisplatin, as the DNA cross-linking agent, IrCpNM solution immediately changed to colorless after the\nexhibited an outstanding capability of binding to pBR322 addition of SDT (Figure 1b). These results suggested that the\nplasmid DNA (Figure S20b,c). The small molecular nitrogen prodrug IrCpNM could be activated by azoreductase inside\nmustard could bind to DNA under the low concentration (50 cancer cells under hypoxia to release the active fragments of\n\u03bcM, Figure S20c), and it damaged the DNA into small species Ir\u2212arene and nitrogen mustard.\nthat smeared all over the gel at a high concentration (100 \u03bcM, Cytotoxicity Assessment. The cytotoxicity of IrCpNM\nFigure S20b). The complex IrCpNM could induce the and IrCpNH2 was then assessed on human nonsmall cell lung\nconformational transition from the supercoiled plasmid to cancer A549 cells, its cisplatin-resistant variant A549R cells,\nopen-circular, as well as the linear plasmid in a concentration- human breast cancer cell lines MCF-7 and MDA-MB-231, and\ndependent manner, while the complex IrCpNH2 indicated no human normal embryo-lung fibroblasts (HLFs) by MTT assay\nobvious phenomenon under the same condition (Figure S20). under both normoxia (20% O2) and hypoxia (1% O2)\nThese results elucidated that the complex IrCpNM was conditions (Tables 1 and S2). Compared to the control\ncapable of inducing DNA damage. sample, the number of A549 cells was obviously decreased and\n Extracellular Response to SDT. Due to the existence of the morphology apparently changed after 24 h exposure of\nthe azo group in IrCpNM, the response to azoreductase has IrCpNM under the hypoxia condition (Figure S22), suggesting\nbeen first evaluated under the hypoxia environment. Sodium that the complex IrCpNM could effectively inhibit the\n 4760 https://doi.org/10.1021/acs.inorgchem.4c00060\n Inorg. Chem. 2024, 63, 4758\u22124769\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 2. Intracellular uptake and distribution of IrCpNM and IrCpNH2 in A549 cells under hypoxia. (a) Cellular uptake (ng/\u03bcg protein) and (b)\nintracellular distribution of Ir in A549 cells after incubation with IrCpNM or IrCpNH2 (10 \u03bcM) for 6 h at 310 K under hypoxia. (c) Ir content of\ngenomic DNA in A549 cells after treatment with IrCpNM and IrCpNH2 (5 \u03bcM) for 24 h at 310 K under hypoxia (n = 3). Error bars: S.D., n = 3.\n\n\n\n\nFigure 3. Oxidative stress and self-supplied O2 in IrCpNM-treated A549 cells under hypoxia. (a) Confocal images and (b) quantitative flow\ncytometry results of the overall ROS level stained with DCF (10 \u03bcM) for 30 min in A549 cells treated with IrCpNM and IrCpNH2 (10 \u03bcM) for 24\nh at 310 K under hypoxia. (c) Confocal images of O2 content pretreatment with RDDP dye (5 \u03bcM) for 4 h in A549 cells then treated with\nIrCpNM (5, 10 \u03bcM) for 24 h at 310 K under hypoxia and (d) quantitative results of RDDP intensity in panel (c) inversely proportional to O2\ncontent. (e) H2O2 content and (f) CAT activity in A549 cells after the indicated complex treatments (5 and 10 \u03bcM for IrCpNM and 10 \u03bcM for\nIrCpNH2, NM and cis-Pt) for 24 h at 310 K under hypoxia. (g) Flow cytometry quantification of the MMP labeled with JC-1 (5 \u03bcg/mL) for 30\nmin in A549 cells treated with IrCpNM and IrCpNH2 (10 \u03bcM) for 24 h at 310 K under hypoxia. Error bars: S.D., n = 3. (h) Representative TEM\nimages of the mitochondrial morphology and autophagic process in A549 cells treated with IrCpNM (10 \u03bcM) for 24 h at 310 K under hypoxia.\nThe orange, green, and pink arrows represented the healthy mitochondria, damaged mitochondria, and autolysosome, respectively.\n\nproliferation of A549 cells. Nitrogen mustard (NM) could be Furthermore, IrCpNM indicated the best antiproliferative\nregarded as nontoxic (IC50 > 110 \u03bcM) under hypoxia. performance to A549 cells, among the tested cancer lines. The\nIrCpNM indicated a much better antiproliferative performance mixture exhibited even less cytotoxicity than that of IrCpNH2,\nthan IrCpNH2 and the mixture to A549 cells under hypoxia, suggesting that free NM hardly contributed to the cytotoxicity\nwhere the IC50 value of IrCpNM (6 \u03bcM) was much lower than and even antagonized IrCpNH2. This phenomenon was also\nthose of IrCpNH2 (27 \u03bcM) and the mixture (38 \u03bcM). found for the mixture of cisplatin with naproxen.22 The\n 4761 https://doi.org/10.1021/acs.inorgchem.4c00060\n Inorg. Chem. 2024, 63, 4758\u22124769\n\fInorganic Chemistry pubs.acs.org/IC Article\n\ncytotoxicity indicated that the linkage of the Ir\u2212arene fragment cells, where the fluorescence of RDDP could be quenched by\nwith NM by the azo group could generate the synergetic effect O2. The red fluorescence intensity has been significantly\nto enhance the anticancer performance. attenuated in a dose-dependent manner after IrCpNM\n Moreover, the complex IrCpNM showed much less treatment, suggesting that the O2 content has been obviously\ncytotoxicity to normal HLF cells than to A549 cells with the elevated in the IrCpNM-treated A549 cells (Figure 3c,d).\nselectivity index (SI) of 2.3/7.5 under the hypoxia/normoxia Furthermore, H2O2 could be the resource of endogenous O2\nconditions, respectively (Table 1), which indicated that and decomposed to O2 and H2O by catalase (CAT);6,28 thus,\nIrCpNM could attenuate the side effects from the treatment the H2O2 content and CAT activity in A549 cells were then\ncomparing to cisplatin (SI value of 0.5).23 Even the IC50 value examined after IrCpNM treatment (Figure 3e\u2212f). The results\nfor IrCpNM to A549 cells under hypoxia (6 \u03bcM) was higher demonstrated that the H2O2 content has been dramatically\nthan that under normoxia (2 \u03bcM). IrCpNM still exhibited a consumed (Figure 3e) and CAT activity has been significantly\npromising anticancer efficiency under hypoxia. According to promoted in a dose-dependent manner after IrCpNM\nprevious reports, the hypoxia-inducible factor (HIF-1\u03b1), as the treatment (Figure 3f), which further confirmed that O2 could\nmarker for the hypoxic condition of cells, would be be self-provided from the decomposition of overexpressed\ndramatically overexpressed under hypoxia.24 Western blotting H2O2 by the elevated CAT activity. This self-provided O2\ndemonstrated that the expression of HIF-1\u03b1 in A549 cells could relieve the hypoxia and attenuate the expression of HIF-\nunder hypoxia was downregulated after IrCpNM treatment, 1\u03b1.\nsuggesting that the hypoxia was effectively relieved (Figure The massive ROS production under hypoxia would trigger a\nS23). series of mitochondrial damages. 29 Mitochondrial Ca 2+\n Intracellular Accumulation and Distribution. The homeostasis was highly related to the permeability of the\nintracellular uptakes and distributions of IrCpNM and mitochondrial membrane, and the Ca2+ overload would\nIrCpNH2 were examined by inductively coupled plasma promote the opening of the mitochondrial permeability\nmass spectrometry (ICP-MS). The cellular Ir content for transition pore (mPTP), eventually leading to the loss of the\nIrCpNM is 0.73 ng/\u03bcg protein, about a 1.3-fold elevation to mitochondrial membrane potential (MMP).30 A free Ca2+\n0.57 ng/\u03bcg protein for IrCpNH2 (Figure 2a and Table S3), fluorescence probe, Fluo-4 AM, was used to monitor the\nwhich is ascribed to the better lipophilicity of IrCpNM.25 In intracellular Ca2+ content, and an obvious green fluorescence\ncontrast to the complex IrCpNH2, an obvious increased was observed in the 10 \u03bcM IrCpNM-treated A549 cells\naccumulation of IrCpNM in the nuclei was observed from 20 (Figure S26). Furthermore, the loss of the MMP was also\nto 35%, which was due to the existence of nitrogen mustard. demonstrated with the observation of CLSM and flow\nThe accumulation in the mitochondria was similar for both cytometry. IrCpNM-treated A549 cells emitted a strong\nIrCpNM (39%) and IrCpNH2 (38%), which suggested that green fluorescence with the mitochondria-selective aggregate\nthe Ir\u2212arene fragment owned a strong mitochondria-targeting dye JC-1 and the depolarized mitochondria jump from 15.3%\ncapacity (Figure 2b and Table S3). Considering the similar (control sample) to 71.6% with 10 \u03bcM IrCpNM (Figures 3g\nDNA-binding affinity, the binding capacities of IrCpNM and and S27). These results suggested that IrCpNM could induce\nIrCpNH2 to genomic DNA of A549 cells were then examined. the mitochondrial Ca2+ overload and loss of the MMP under\nAfter treatments, the genomic DNA in A549 cells was hypoxia.\nextracted and the quantity of binding Ir content was measured The integration of the mitochondria is essential to maintain\nby ICP-MS. About 40% elevation of DNA-binding Ir content the physiological functions,31 and mitochondrial morphology\nwas found for the complex IrCpNM (25.2 pg/\u03bcg) compared was then examined by transmission electron microscopy\nto that of IrCpNH2 (17.7 pg/\u03bcg) (Figure 2c). This suggested (TEM). The mitochondria in the untreated A549 cells\nthat the elevated DNA-binding content of IrCpNM mainly owned a clear-outlined mitochondrial double-layer membrane\nresulted from a higher intracellular uptake. and the cristae structure (Figure 3h, orange arrowheads). In\n ROS Elevation and Mitochondrial Damage. The contrast, vacuolation containing intracellular contents was\nimpact of IrCpNM treatment under hypoxia on the observed after IrCpNM treatment (Figure 3h, pink arrows),\nmitochondria was first evaluated since approximately 40% of and most of the mitochondrial cristae structures were\nIrCpNM accumulated in the mitochondria. Mitochondria is destroyed or disappeared (Figure 3h, green arrowheads),\nthe main site for ROS generation,26 and the overall indicating that the mitochondria were severely damaged and\nintracellular ROS production was probed with 2\u2032,7\u2032-dichloro- autophagic cell death would be initiated.\nfluorescin diacetate (DCF-DA) fluorescence assay by flow Disruption of Redox Homeostasis. Since excessive ROS\ncytometry and confocal laser scanning microscopy (CLSM). A production under hypoxia was observed in IrCpNM-treated\ndramatic concentration-dependent ROS elevation was ob- A549 cells, the redox homeostasis of cells must be disrupted.\nserved in IrCpNM-treated A549 cells, which has been elevated Glutathione (GSH), as the primary endogenous antioxidant in\n4.7-fold compared to the control sample with the concen- cells, could protect cancer cells from oxidative stress to\ntration of 10 \u03bcM (Figures 3a,b and S24). Meanwhile, the perform normal physiological activities. Intracellular GSH\ncomplex IrCpNH2, as expected, showed a concentration- could be oxidized to oxidized glutathione (GSSG) by the\ndependent ROS production under hypoxia (Figure S25), while direct interaction with radicals or by glutathione peroxidases\nthe ROS production in the presence of 10 \u03bcM IrCpNH2 was (GPx) during the reduction of hydrogen peroxide. Meanwhile,\nmuch less than that of IrCpNM (Figures 3a,b and S24). This GSSG would be simultaneously reduced back to GSH by\nimplied that the ROS elevation in IrCpNM-treated A549 cells nicotinamide adenine dinucleotide phosphate (NADPH) with\nwas ascribed to the mitochondrial damage. the catalyst of glutathione reductase (GR) to maintain redox\n To demonstrate the endogenous O2 generation in A549 cells homeostasis, where the NADPH was oxidized to NADP+.4,28\nto relieve the hypoxia, [Ru(dpp)3]Cl2 (RDDP), as the oxygen As expected, after IrCpNM treatment, the contents of GSH\nindicator,27 was incubated with the IrCpNM-treated A549 and GSSG in A549 cells have been simultaneously down- and\n 4762 https://doi.org/10.1021/acs.inorgchem.4c00060\n Inorg. Chem. 2024, 63, 4758\u22124769\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 4. Disruption of GSH homeostasis in IrCpNM-treated A549 cells under hypoxia. (a\u2212d) Concentration of GSH, GSSG, NADP+, and\nNADPH (\u03bcM/\u03bcg protein) and (e, f) GR and GPx activities (mU/mg protein) in A549 cells treated with indicated complexes (5 and 10 \u03bcM for\nIrCpNM and 10 \u03bcM for IrCpNH2, NM and cis-Pt) for 24 h at 310 K under hypoxia. Error bars: S.D., n = 3.\n\nupregulated, respectively (Figure 4a,b). And the expression of \u03b3-H2AX expression of A549 cells in the presence of the\nGPx was also elevated in a concentration-dependent manner, antioxidant ROS inhibitor NAC was further examined (Figure\nwhich promoted the transformation from GSH to GSSG 5d,e). The immunoblotting result indicated that the expression\n(Figure 4f). In addition, even the levels of NADPH and level of \u03b3-H2AX was significantly inhibited by 33% with the\nNADP+ upregulated after IrCpNM treatment, the enzyme addition of NAC, illustrating that DNA damage in the\nactivity of GR has been dramatically inhibited (Figure 4c\u2212e). IrCpNM-treated A549 cells partially resulted from oxidative\nEven the GR activity was suppressed in a concentration- stress. Thus, IrCpNM could simultaneously initiate the cross-\ndependent manner in the presence of IrCpNM; the GSH linking and oxidative DNA damage in A549 cells.\ncontent remains the same. This may be due to the complexity DNA Damage Response-Mediated Autophagy\nof the cellular environment, where the GSH content has been through the ATM/Chk2 Cascade and the PIK3CA/PI3K-\ndetermined by many factors other than GR activity, such as the AKT1-mTOR-RPS6KB1 Pathway. DNA damage could\nlevel of cysteine.32 These results indicated that the disruption trigger various cellular responses, such as DNA repair, cell\nof redox homeostasis was mainly ascribed to the limited cycle arrest, apoptosis, necrosis, and autophagy.37,38 As\nreduction of GSSG to GSH, resulting in excessive ROS reported, DNA damage response (DDR) could be driven by\nproduction. two major pathways, the ataxia-telangiectasia-mutated/check-\n Initiation of DNA Damage. \u03b3-H2AX (the phosphorylated point kinase 2 (ATM/Chk2) and the ATM- and RAD3-related\nform of the histone protein H2AX), as the biomarker of DNA serine/threonine kinase (ATR/Chk1) signaling cascade,\ndouble-strand breaks (DSBs),22,33 was then examined. As is respectively.39,40 The ATM/Chk2 pathway could be activated\nknown, NM and cis-Pt are the alkylating agents of DNA, and \u03b3- by DSBs, whereas ATR/Chk1 signaling was usually activated\nH2AX expression in NM- and cis-Pt-treated A549 cells was by single-strand breaks (SSBs) or bulky DNA lesions. The\nsignificantly elevated as well as the cisplatin-treated cells phosphorylation levels of Chk1 and Chk2 in the IrCpNM-\n(Figure 5b). It is worthy to note that \u03b3-H2AX expression in treated A549 cells were evaluated by immunoblotting assay\nIrCpNM-treated A549 cells was 1.4 and 1.6 times higher (Figure 5f). In contrast to both upregulated expressions in\ncompared to those in NM- and cis-Pt-treated cells, respectively cisplatin-treated A549 cells, only Chk2 phosphorylation has\n(Figure 5b,c), which suggested that IrCpNM could induce been obviously increased by 50 and 98% with different\nmore serious DNA damage than NM. Furthermore, the concentrations of IrCpNM treatment (Figure 5f,g), suggesting\nimmunofluorescence intensity of \u03b3-H2AX foci was also that the major mode of DNA damage was DSBs, which was\ndramatically enhanced in IrCpNM-treated A549 cells, consistent with the observation from \u03b3-H2AX expression and\nsuggesting that IrCpNM treatment strengthened the frequency the comet assay.\nof DNA double-strand breaks (Figure S28).34 In addition, The expression of p53, as a downstream effector of DNA\nalkaline comet assay revealed that the DNA double helix has damage,33,41 has been studied and the results indicated that\nbeen partially denatured and some nuclear DNA has become p53 expression was severely elevated in the IrCpNM-treated\nsingle-stranded after IrCpNM treatment (Figure 5a).35 A549 cells (Figure 5b,c), indicating that DNA damage could\n In order to detect whether DNA damage was also mediated not be repaired and the cell death process could be initiated.\nby oxidative stress (OS) from the excessive ROS production,36 The analysis of cell cycle distribution and dual staining of\n 4763 https://doi.org/10.1021/acs.inorgchem.4c00060\n Inorg. Chem. 2024, 63, 4758\u22124769\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 5. DDR-mediated autophagy in 24 h IrCpNM-treated A549 cells through the ATM/CHK2 pathway under hypoxia. (a) Comet assay of\nA549 cells treated with IrCpNM (10 \u03bcM) for 24 h at 310 K under hypoxia. (b) Expression level of \u03b3-H2AX and p53 in A549 cells treated with\nindicated complexes (5 and 10 \u03bcM for IrCpNM and 10 \u03bcM for IrCpNH2, NM and cis-Pt) for 24 h at 310 K under hypoxia. (c) The corresponding\nprotein contents in panel (b) relative to GAPDH. (d) Expression level of \u03b3-H2AX and LC3 with or without the preincubation of NAC (2.5 mM)\nfor 1 h in A549 cells then treated with or without IrCpNM (10 \u03bcM) for 24 h at 310 K under hypoxia. (e) The corresponding protein contents in\npanel (d) relative to GAPDH. (f) Expression level of proteins involved in DDR-mediated autophagy signaling pathways in A549 cells treated with\nindicated complexes (5 and 10 \u03bcM for IrCpNM and 10 \u03bcM for IrCpNH2, NM and cis-Pt) for 24 h at 310 K under hypoxia. (g) The corresponding\nprotein contents in panel (f) relative to GAPDH. (h) Immunofluorescence staining of LC3 in A549 cells treated with IrCpNM, IrCpNH2, and cis-\nPt (10 \u03bcM) for 24 h at 310 K under hypoxia.\n\nAnnexin V-FITC and PI assay illustrated that IrCpNM could (Figure 5f\u2212h). The conversion from LC3-I (microtubule-\nnot induce cell cycle arrest and trigger apoptosis in A549 cells associated protein 1 light chain 3) to its lipidation form LC3-II,\n(Figure S29a,b). Furthermore, the cell viability was not a reliable marker of autophagy,42 was initiated after IrCpNM\nelevated with the presence of several inhibitors, including z- treatment in a concentration-dependent manner (Figure 5f,g).\nVAD-fmk, necrostatin-1 (Nec-1), or ferrostatin-1 (Fer-1), The apparently increased fluorescence intensity was also\nsuggesting that the cell death did not result from apoptosis, observed in the IrCpNM-treated A549 cells (Figure 5h).\nnecrosis, or ferroptosis after IrCpNM treatment (Figure S30). Furthermore, the p62 expression level was also downregulated\nIn contrast, 3-methyladenine (3-MA) and chloroquine, the in the IrCpNM-treated A549 cells, which would degrade with\nmost-used autophagy inhibitor, could elevate the cell viability, the autophagosomes to complete the autophagic flux (Figure\nwhich suggested that autophagy was the predominant death 5f,g).43 These results confirmed the occurrence of DNA\nmode of the IrCpNM-treated A549 cells (Figure S31). damage response-mediated autophagy in the IrCpNM-treated\n The expression of the autophagy-related protein light chain A549 cells.\nprotein 3 (LC3) was then examined to confirm the occurrence It is well known that the signaling pathway of phosphati-\nof autophagy by immunofluorescence and immunoblotting dylinositol-4,5-bisphosphate 3-kinase, catalytic subunit \u03b1\n 4764 https://doi.org/10.1021/acs.inorgchem.4c00060\n Inorg. Chem. 2024, 63, 4758\u22124769\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 6. Antiproliferation of IrCpNM on the different biological models. (a) 2D A549 monolayer cells after treatment with IrCpNM, IrCpNH2,\nNM, and cis-Pt (10 \u03bcM) for 24 h at 310 K under hypoxia and (b) 3D A549 tumor spheroids after treatment with IrCpNM, IrCpNH2, NM, and cis-\nPt (20 \u03bcM) for 7 days. (c, d) Statistical fluorescence intensity of calcein AM in panels (a) and (b), respectively, and (e) volume of 3D tumor\nspheroids in panel (b). (f, g) Representative photographs and the weight of tumor samples (n = 6) after treatment with saline, IrCpNM, and\nIrCpNH2 (5.0 mg kg\u22121 of body weight) for 18 days, respectively. *p < 0.05, ***p < 0.001, ****p < 0.0005. (h, i) Time-dependent tumor growth\ncurves and the body weight of the mice during 18 days. Error bars: S.D., n = 6. (j) H&E and TUNEL staining assays of tumor tissues at the end of\nthe indicated treatment.\n\n(PIK3CA/PI3K)-v-akt murine thymoma viral oncogene and IrCpNH2 was then evaluated in three biological models, as\nhomologue 1 (AKT1)- mechanistic target of rapamycin the 2D monolayer cells, the 3D multicellular tumor spheroids\n(mTOR)- ribosomal protein S6 kinase, 70 kDa, polypeptide (MCTSs), and the A549 tumor-bearing mice models.45 As\n1 (RPS6KB1) could regulate the occurrence of autophagy in shown in Figure 6a,6c, nearly no green fluorescence was\ncells.44 To further demonstrate the inhibition of the PIK3CA/ observed after 24 h IrCpNM treatment, suggesting that the\nPI3K-AKT1-MTOR-RPS6KB1 pathway to induce autophagy complex IrCpNM killed most of the A549 cells and exhibited\nin the IrCpNM-treated A549 cells, the expression of the the greatest antiproliferative effect toward A549 cells among\nrelated proteins was further examined by Western blotting complexes IrCpNH2, NM, and cisplatin.\n(Figure 5f,g). After treatment with IrCpNM for 24 h, an 3D tumor spheroids of A549 cells were cultured to mimic\nobvious suppression of phosphorylated AKT1 (p-Akt) at the microenvironment of the solid tumor with an average\nser473 and phosphorylated RPS6KB1 (p-p70S6K) at thr389 in diameter of 500 \u03bcm and were treated with complexes IrCpNM\na concentration-dependent manner was observed compared to and IrCpNH2, respectively (Figures 6b,d\u2212e and S32). After 7\nthe untreated A549 cells (Figure 5f,g). Furthermore, the days incubation with IrCpNM, the spheroid volume shrunk to\ninitiation of autophagy was also coordinated with unc-51-like only 10% of the control sample (Figure 6e). In contrast, the\nkinase 1 (ULK1, also known as autophagy-related (ATG)-1), spheroids treated with NM and cisplatin showed very limited\nwhich could be inhibited by mTOR.38 The Western blotting influence on the proliferation of A549 spheroids.\nresults showed that phosphorylated ULK1 (p-ULK1) at ser757 Inspired by the in vitro study, the in vivo antitumor\nwas suppressed in a concentration-dependent manner in performance of IrCpNM and IrCpNH2 in A549 tumor-bearing\nIrCpNM-treated A549 cells (Figure 5f,g). Moreover, in the mice models was further evaluated (Figures 6f\u2212j and S33).\npresence of NAC, autophagy was also inhibited, as the ratio of The tumor-bearing mice were randomly divided into three\nLC3-II/LC3-I was decreased (Figure 5d,e). This suggested groups (6 mice/group) and intratumorally injected with\nthat DNA damage response-mediated autophagy regulated by IrCpNM or IrCpNH2 (5 mg kg\u22121 mice weight) or saline\nthe PIK3CA/PI3K-AKT1-mTOR-RPS6KB1 signaling pathway once every other day for 18 days. The growth of the tumors\nresulted from both the cross-linking and oxidative DNA was significantly inhibited after IrCpNM treatment compared\ndamages, which provided a new anticancer mechanism for the to the control and the IrCpNH2-treated group. The average\nmetal\u2212arene anticancer agents. tumor volumes for the IrCpNM- and IrCpNH2-treated groups\n Biological Evaluation In Vitro and In Vivo. The were 737.1 and 1047.0 mm3, respectively. The control group\nantiproliferative performance of both complexes IrCpNM was characterized with a rapid growth of the tumor volume up\n 4765 https://doi.org/10.1021/acs.inorgchem.4c00060\n Inorg. Chem. 2024, 63, 4758\u22124769\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 7. Proposed mechanism of action for IrCpNM.\n\nto 1498.1 mm3 (Figure 6f,6h). Meanwhile, the tumor weight of\nthe IrCpNM-treated group was the lowest compared to the\n \u25a0 CONCLUSIONS\n In this work, considering the tumor hypoxia microenvironment\nother groups (Figure 6g). In addition, the body weight of the and the monotonous mechanism of exiting drugs, an\nmice treated with IrCpNM or IrCpNH2 showed no obvious azoreductase-responsive multifunctional complex IrCpNM\ndifference to the control group during the treatment, as the was designed and successfully synthesized. The organo\u2212Ir(III)\nsteady growth (Figure 6i), which indicated that IrCpNM prodrug IrCpNM was constructed with the ROS-inducing\nowned potent antitumor activity in vivo and low toxicity to the moiety\u2212the azoreductase-responsive moiety\u2212the DNA alkylat-\nmice. ing moiety, which could initiate the quadruple synergetic\n Hematoxylin and eosin (H&E) staining and terminal actions in A549 cells. The hypoxia has been significantly\ndeoxynucleotidyl transferase dUTP nick-end labeling relieved due to downregulated HIF-1\u03b1 and the self-provided\n(TUNEL) staining assays were also performed to evaluate O2 from the decomposition of H2O2 with upregulated catalase\nthe therapeutic efficacy of the different treatments (Figures 6j activity by IrCpNM. The Ir\u2212arene fragment could promote\nand S33). The images of the H&E and TUNEL-stained tumor the elevation of ROS; the azo bond could be simultaneously\ntissue showed that IrCpNM induced a high level of cell death reduced by azoreductase and GSH to enhance the disbalance\nin the mice (Figure 6j). However, the control group showed no of redox homeostasis, resulting in the occurrence of oxidative\nobvious tumor necrosis. Notably, no apparent lesions of the DNA damage; nitrogen mustard could covalently link to DNA,\nmajor organs were observed in mice treated with IrCpNM or resulting in cross-linking DNA damage. Both oxidative and\nIrCpNH2 (Figure S33), indicating the minimal adverse effects cross-linking DNA damage triggered the DDR by the ATM/\nand the excellent biocompatibility of IrCpNM. Chk2 cascade with the DNA double-strand breaks. DDR-\n Mechanism of Action. Emerging studies have revealed mediated autophagy was subsequently regulated by the\nthat DNA binding does not appear to be the major antitumor PIK3CA/PI3K-AKT1-mTOR-RPS6KB1 signaling pathway,\n which was a new anticancer mechanism for the metal-based\nmechanism of current organo\u2212iridium complexes. Instead,\n anticancer agents. This work not only provided the example of\nincrease of ROS and loss of MMP might significantly\n the successful small molecular prodrug strategy for metal\u2212\ncontribute to their anticancer activities.16 Here, we demon- arene agents to resolve the hypoxic obstacle but also\nstrate that the organo\u2212iridium prodrug, upon triggering the demonstrated the effectiveness of the DNA damage response\noccurrence of ROS storms, can subsequently induce a cascade strategy with the tumor microenvironment targeting system.\nof physiological responses in tumor cells, including redox Moreover, this work offered a new insight for the rebirth of\nimbalance and DNA oxidative damage, ultimately leading to traditional chemotherapeutic agents with a new anticancer\nthe activation of DDR-mediated autophagy for hypoxic lung mechanism through rational design.\ncancer therapy. The overall antitumor mechanism of IrCpNM\nis depicted in Figure 7. Specifically, this prodrug can respond\nto overexpressed azoreductase under hypoxia, thereby\n \u25a0 EXPERIMENTAL SECTION\n Chemicals and Reagents. [Ir(Cp*)2Cl] 2 (Cp* =\nameliorating the hypoxic tumor environment through self- tetramethyl cyclopentadienyl), ppy (2-phenylpyridine), 4-\nprovided O 2 from the decomposition of H 2 O 2 with aminopyridine, and NH4PF6 were purchased from Heowns.\nupregulated catalase activity and downregulated HIF-1\u03b1. N,N-Bis(2-chloroethyl)aniline was obtained from Macklin. All\nConsequently, the Ir\u2212arene and nitrogen mustard fragments commercial chemicals and reagents for syntheses were of\ncan accumulate in the subcellular mitochondria and nuclei, analytical grade and used as received without further\ninducing the quadruple synergetic actions, including simulta- purification. Deuterated solvents for NMR purposes were\nneous ROS promotion and GSH depletion leading to redox obtained from Merck and Cambridge Isotopes.\ndisbalance, and oxidative and cross-linking DNA damages to Synthesis of the Ligand (L). The ligand (E)-N,N-bis(2-\ninitiate DDR-mediated autophagy through the ATM/Chk2 chloroethyl)-4-(pyridin-4-yldiazenyl)aniline (L) was synthe-\ncascade and the PIK3CA/PI3K-AKT1-mTOR-RPS6KB1 path- sized from the coupling of 4-aminopyridine and nitrogen\nway. mustard (NM) in a slightly modified way according to\n 4766 https://doi.org/10.1021/acs.inorgchem.4c00060\n Inorg. Chem. 2024, 63, 4758\u22124769\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nliterature procedures.46 Briefly, to a stirred solution of 4- 119.39, 111.85, 89.81, 8.55. Elemental analysis: calcd (%) for\naminopyridine (1.5 mmol, 141 mg) in a mixture solvent of C26H29F6IrN3P: C, 43.33; H, 4.06; N, 5.83; found: C, 43.51;\nACN/H2O (1:1, v/v) at 273 K under an argon atmosphere, H, 4.21; N, 5.63. ESI-MS (CH3OH): calcd for [IrCpNH2 -\nNaNO2 (1.6 mmol, 110 mg) was added and the solution was PF6\u2212]+ m/z = 576.2, found m/z = 576.3. ESI-HRMS\nstirred for 10 min, after which concentrated HCl (6 mmol, 0.5 (CH3OH): calcd for [IrCpNH2 - PF6\u2212]+ m/z = 576.1991,\nmL) was added and the solution was stirred for another 30 found m/z = 576.1977.\nmin. Subsequently, NM (1 mmol, 218 mg) in ACN was added\ndropwise and the mixture was stirred for 3 h at 273 K, after\nwhich 100 mL of water was added. The mixture was extracted\n \u25a0 ASSOCIATED CONTENT\n * Supporting Information\n s\u0131\nwith DCM (3 \u00d7 50 mL), the organic layers were combined The Supporting Information is available free of charge at\nand dried over with MgSO4, and the solvent was removed in https://pubs.acs.org/doi/10.1021/acs.inorgchem.4c00060.\nvacuo. The crude product was purified by silica gel Materials and experimental details and the NMR, ESI-\nchromatography eluted with petroleum ether/ethyl acetate MS, confocal images, and flow cytometry results for\n(10:1, v/v) to give the pure L. Yield: 150 mg (43%). 1H NMR complexes IrCpNM and IrCpNH2 (PDF)\n(400 MHz, CDCl3) \u03b4 (ppm): 8.96\u22128.59 (m, 2H), 8.08\u22127.85\n(m, 2H), 7.78\u22127.57 (m, 2H), 6.93\u22126.67 (m, 2H), 3.89 (t, J =\n6.9 Hz, 4H), 3.73 (t, J = 6.9 Hz, 4H). 13C NMR (101 MHz,\nCDCl3) \u03b4 (ppm): 157.73, 151.04, 149.70, 144.50, 126.21,\n \u25a0 AUTHOR INFORMATION\n Corresponding Authors\n116.15, 111.64, 53.46, 40.18. ESI-MS (CH3OH): calcd for Yan Su \u2212 Jiangsu Collaborative Innovation Center of\n[C15H16Cl2N4]+ m/z = 323.08, found m/z = 323.10. Biomedical Functional Materials, College of Chemistry and\n Synthesis of Complexes (IrCpNM and IrCpNH2). The Materials Science, Nanjing Normal University, Nanjing\niridium precursor Ir(Cp*)(ppy)Cl was synthesized and 210023, China; Department of Rheumatology and\npurified as previously reported.47 The complexes IrCpNM Immunology, Jinling Hospital, Medical School of Nanjing\nand IrCpNH2 were prepared by the similar method following University, Nanjing 210002, China; Email: suyanahnu@\nthe reference.16 Generally, a solution of the chloride complex 163.com\nIr(Cp*)(ppy)Cl (0.1 mmol, 51.8 mg) and AgNO3 (1 mol Zheng-Hong Yu \u2212 Department of Rheumatology and\nequiv) in MeOH/H2O (1:1, v/v) was refluxed in the dark Immunology, Jinling Hospital, Medical School of Nanjing\nunder an argon atmosphere for 3 h. Then, the precipitate University, Nanjing 210002, China; Email: m_fish@189.cn\n(AgCl) was removed by filtration, and the ligand L or 4- Zhi Su \u2212 Jiangsu Collaborative Innovation Center of\naminopyridine (1 mol equiv) was added to the filtrate. The Biomedical Functional Materials, College of Chemistry and\nreaction mixture was refluxed under an argon atmosphere Materials Science, Nanjing Normal University, Nanjing\novernight, which was then exchanged with NH4PF6 (10 mol 210023, China; orcid.org/0000-0002-1339-0525;\nequiv). Email: zhisu@njnu.edu.cn\n [Ir(Cp*)(ppy)(L)]PF6 (IrCpNM). The crude product was Authors\npurified by silica gel chromatography eluted with n-hexane/ Meng-Meng Wang \u2212 Jiangsu Collaborative Innovation Center\nethyl acetate (10:1, v/v) to give the pure product. The of Biomedical Functional Materials, College of Chemistry and\ncomplex IrCpNM was obtained as a red powder. Yield: 0.056 g Materials Science, Nanjing Normal University, Nanjing\n(59%). 1H NMR (400 MHz, CDCl3) \u03b4 (ppm): 9.15 (d, J = 5.7 210023, China\nHz, 1H), 8.53\u22128.47 (m, 2H), 7.94 (dd, J = 7.6, 1.1 Hz, 1H), Dong-Ping Deng \u2212 Jiangsu Collaborative Innovation Center\n7.81 (td, J = 7.0, 2.8 Hz, 4H), 7.67\u22127.60 (m, 2H), 7.57\u22127.51 of Biomedical Functional Materials, College of Chemistry and\n(m, 2H), 7.41 (td, J = 7.5, 1.3 Hz, 1H), 7.19 (td, J = 7.5, 1.1 Materials Science, Nanjing Normal University, Nanjing\nHz, 1H), 6.76\u22126.69 (m, 2H), 3.87 (t, J = 6.8 Hz, 4H), 3.71 (t, 210023, China\nJ = 6.7 Hz, 4H), 1.65 (s, 15H). 13C NMR (101 MHz, CDCl3) An-Min Zhou \u2212 Jiangsu Collaborative Innovation Center of\n\u03b4 (ppm): 166.76, 160.82, 158.20, 154.19, 152.19, 150.80, Biomedical Functional Materials, College of Chemistry and\n145.35, 144.49, 138.99, 134.81, 132.00, 126.99, 125.30, 124.33, Materials Science, Nanjing Normal University, Nanjing\n123.91, 119.23, 119.17, 111.77, 90.47, 53.41, 40.30, 8.56. 210023, China\nElemental analysis: calcd (%) for C36H39Cl2F6IrN5P: C, 45.52; Hong Ke Liu \u2212 Jiangsu Collaborative Innovation Center of\nH, 4.14; N, 7.37; found: C, 45.66; H, 4.27; N, 7.21. ESI-MS Biomedical Functional Materials, College of Chemistry and\n(CH3OH): calcd for [IrCpNM - PF6\u2212]+ m/z = 804.2, found Materials Science, Nanjing Normal University, Nanjing\nm/z = 804.3. ESI-HRMS (CH3OH): calcd for [IrCpNM - 210023, China\nPF6\u2212]+ m/z = 804.2212, found m/z = 804.2159.\n [Ir(Cp*)(ppy)(4-aminopyridine)]PF6 (IrCpNH2). The Complete contact information is available at:\npure product of IrCpNH2 was obtained by recrystallizing https://pubs.acs.org/10.1021/acs.inorgchem.4c00060\nfrom dichloromethane/diethyl ether. The complex IrCpNH2\nwas obtained as a yellow powder. Yield: 0.061 g (85%). 1H Author Contributions\n \u00a7\nNMR (400 MHz, d6-DMSO) \u03b4 (ppm): 8.94 (dd, J = 5.8, 1.5 M.-M.W., D.-P.D., and A.-M.Z. contributed equally.\nHz, 1H), 8.15 (d, J = 8.1 Hz, 1H), 7.99 (td, J = 7.8, 1.5 Hz, Notes\n1H), 7.89 (dd, J = 7.6, 1.1 Hz, 1H), 7.85 (dd, J = 7.9, 1.4 Hz, The authors declare no competing financial interest.\n1H), 7.65\u22127.56 (m, 2H), 7.49 (ddd, J = 7.3, 5.7, 1.4 Hz, 1H),\n7.34 (td, J = 7.4, 1.4 Hz, 1H), 7.13 (td, J = 7.5, 1.1 Hz, 1H),\n6.78 (s, 2H), 6.34\u22126.23 (m, 2H), 1.56 (s, 15H). 13C NMR\n \u25a0 ACKNOWLEDGMENTS\n The authors appreciate the financial support from the National\n(101 MHz, CDCl3) \u03b4 (ppm): 167.17, 161.34, 154.37, 151.50, Natural Science Foundation of China (NSFC) (grant nos.\n151.17, 145.03, 138.69, 134.61, 131.87, 124.47, 124.36, 123.58, 21977052 and 22277056) and the Distinguished Young\n 4767 https://doi.org/10.1021/acs.inorgchem.4c00060\n Inorg. 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