Mitochondrial-targeted iridium(III) complexes suppress tumor growth through inducting immunogenic cell death to activate immune response.

{"full_text": " European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\n Contents lists available at ScienceDirect\n\n\n European Journal of Medicinal Chemistry\n journal homepage: www.elsevier.com/locate/ejmech\n\n\nResearch paper\n\nMitochondrial-targeted iridium(III) complexes suppress tumor growth\nthrough inducting immunogenic cell death to activate immune response\nShuanghui Tang a , Yueyao Ding b, Ziyan Zhang b, Yan Yang c,*, Chunxia Huang a , Lin Zhou a ,\nShuang Tian a , Hui Yin b,** , Yunjun Liu a,***\na\n School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China\nb\n Department of Microbiology and Immunology, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China\nc\n Department of Pharmacy, The Affiliated Guangdong Second Provincial General Hospital of Jinan University, Guangzhou, 510317, 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: A new ligand, 2-(2-hydroxyl-4-methyl)phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (IPMP), and [Ir\nIridium(III) complexes (ppy)2(IPMP)]PF6 (7a), [Ir(bzq)2(IPMP)]PF6 (7b), and [Ir(piq)2(IPMP)]PF6 (7c) have been prepared and char\u00ad\nMitochondrial targeting acterized by HRMS, NMR spectra. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)\nFerroptosis\n assays revealed that 7b exhibited excellent activity (IC50 = 4.5 \u00b1 0.4 \u03bcM), while 7a and 7c showed good\nAntitumor in vivo\nImmune response\n cytotoxicity (IC50 = 8.5 \u00b1 0.9 \u03bcM and 8.9 \u00b1 2.2 \u03bcM) against non-small cell lung cancer A549 cells. The ex\u00ad\n periments of cellular uptake and mitochondrial localization demonstrate that these new iridium(III) complexes\n are readily taken up by A549 cells and accumulate in the mitochondria and damage the structure of the mito\u00ad\n chondria, which results in the loss of mitochondrial membrane potential (MMP), elevated lipid peroxidation, as\n well as DNA damage, the inhibition of microtubule polymerization, hindrance of the cell cycle in the G0/G1\n phase, and release of cytochrome c, collectively leading to apoptosis. Furthermore, upregulation of Beclin-1,\n overexpression of NF-\u03baB and downregulation of GPX4 protein were observed, which resulted in the activation\n of autophagy, pyroptosis and ferroptosis, respectively. In the C57BL/6 mouse model, the 7b demonstrated\n promising in vivo antitumor efficacy, with a tumor inhibitory rate of 66.9 %. Additionally, the complexes induce\n an immunogenic cell death to activate immune response, further enhance CD8+ T cells and efficiently inhibit\n tumor growth. Collectively, we consider that the complexes may be utilized as potential candidate agents for the\n treatment of A549 cancer.\n\n\n\n\n1. Introduction Anticancer chemotherapeutic agents based on platinum (Pt) such as\n cisplatin (approval by the FDA in 1978) are widely used, but their severe\n Cancers are prevalent and pose a serious threat to human health. side effects and resistance have limited their clinical application [8\u201311].\nLung cancer has one of the highest incidence rates of any malignancy Therefore, there is a critical need for the development of a\nworldwide, exceeding those of breast, prostate, and colorectal cancers. broad-spectrum, high-potency, and low-toxicity metal-based anticancer\nFurthermore, it is the leading cause of cancer-related mortality [1]. Lung drug. Recently, the coordination of iridium(III) has emerged as a\ncancer can be classified into two primary categories: small cell lung promising approach in the design of antitumor drugs [12]. In compari\u00ad\ncancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC consti\u00ad son to the planar Pt(II) complexes, the unique structural multiplicity and\ntutes the majority (up to 85 % of total cases) and exhibits a 5-year versatility conferred by the octahedral coordination of Ir(III) have been\nsurvival rate of just 15.9 % [2]. At present, chemotherapy is one of shown to enhance the feasibility of biomolecular targeting. In addition,\nthe most widely used and effective strategies for NSCLC treatment Ir(III) complexes possess luminescent imaging and therapeutic functions\ncompared to other approaches, such as surgery, molecularly targeted that will be beneficial for the visualization of anti-cancer mechanisms\ntherapies, and immunotherapies [3\u20137]. [13,14]. Some Ir(III) complexes have been found to exhibit high\n\n\n * Corresponding author.\n ** Corresponding author.\n *** Corresponding author.\n E-mail addresses: yany@gd2h.org.cn (Y. Yang), huiyin0103@gdpu.edu.cn (H. Yin), lyjche@gdpu.edu.cn (Y. Liu).\n\nhttps://doi.org/10.1016/j.ejmech.2025.117926\nReceived 16 April 2025; Received in revised form 29 June 2025; Accepted 30 June 2025\nAvailable online 1 July 2025\n0223-5234/\u00a9 2025 Elsevier Masson SAS. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\nselectivity for various cancer cell lines and effectively to target mito\u00ad phenanthroline (IPMP) and its three Ir(III) complexes: [Ir(ppy)2(IPMP)]\nchondria [15\u201317], leading to mitochondrial dysfunction and the inhi\u00ad PF6 (7a), [Ir(bzq)2(IPMP)]PF6 (7b), and [Ir(piq)2(IPMP)]PF6 (7c)\nbition of cancer cell proliferation. In recent years, researchers (Scheme 1). The cytotoxicity of 7a, 7b and 7c against cancerous (A549,\ndiscovered that a lot of metal complexes including Ir(III), Cu(I), Rh(III), LA795) and normal (NIH3T3) cells was evaluated using the 3-(4,\nRu(II) show excellent anticancer efficiency [18\u201326]. Liang et al. re\u00ad 5-dimethylthiazole)-2,5-diphenyltetrazolium bromide (MTT) assay. The\nported that iridium(III) complexes accumulated the mitochondrial inner cellular apoptosis, cell substructure localization and antitumor molec\u00ad\nmembrane of HeLa cells with high selectivity and exhibited a high ular mechanisms of 7a, 7b and 7c on A549 cells were investigated in\nantineoplastic activity [27]. Ortyl reported that iridium(III) complexes detail. Finally, the antitumor in vivo efficacy of 7b was evaluated in the\ncan produce ROS upon light activation and act as photosensitizers for subcutaneous A549 tumor-bearing nude mouse model and the anti\u00ad\nphotodynamic therapy (PDT) to cause cell death [28]. In addition, tumor mechanism was explored.\nactivation of immune response to increase CD8+ T cells has been paid a\ngreat attention, Liu et al. found that a Ru(II) complex causes an immu\u00ad 2. Results and discussion\nnogenic cell death, which activated an immune response to enhance\nCD8+ T cells, further induced an increment of TNF-\u03b1, IFN-\u03b3 and a 2.1. Synthesis and characterization of 7a, 7b and 7c\ndecrease of IL-10 amount and efficiently prevent tumor growth [29].\nYang et al. reported that an Ir(III) complex directly kill liver cancer cells IPMP was obtained by refluxing 1,10-phenanthroline-5,6-dione,\nthrough inducing necroptosis and activating the necroptosis-related NH4Ac and 2-hydroxyl-4-methylbenzaldehyde in glacial acetic acid.\nimmune response [30]. Liang et al. found that the Ru(III) complexes 7a, 7b and 7c were gained by the reaction of IPMP with [Ir\ntrigger Gasdermin E-mediated pyroptosis and an immune response to (ppy)2Cl]2\u22c5H2O, [Ir(bzq)2Cl]2\u22c5H2O, or [Ir(piq)2Cl]2\u22c5H2O in a mixed\ninduce cell death [31]. To obtain more information on anticancer ac\u00ad solvent of methanol and dichloromethane. These compounds were pu\u00ad\ntivity and mechanism of Ir(III) complexes, we prepared a new ligand rified through the neutral column chromatography to gain the desired\n2-(2-hydroxyl-4-methyl)phenyl-1H-imidazo[4,5-f][1,10] 7a, 7b and 7c. These complexes were characterized by HRMS, NMR. The\n\n\n\n\nScheme 1. Synthetic route for ligand IPMP and complexes 7a, 7b, 7c.\nSynthetic conditions, reagents, and solvent: (I) NH4Ac, HAc, reflux for 2 h at 130 \u25e6 C; (II) [Ir(ppy)2Cl]2, 15 mL CH3OH, 30 mL CH2Cl2, reflux for 6 h at 40 \u25e6 C under Ar;\n(III) [Ir(bzq)2Cl]2, 15 mL CH3OH, 30 mL CH2Cl2, reflux for 6 h at 40 \u25e6 C under Ar; (IV) [Ir(piq)2Cl]2, 15 mL CH3OH, 30 mL CH2Cl2, reflux for 6 h at 40 \u25e6 C under Ar.\n\n 2\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\nresults from HRMS show that the calculated values are consistent with order of 7b < 7a < 7c, which aligns with those of cytotoxicity of 7b > 7a\nthe determined molecular weights. 1H NMR spectra show that the peaks > 7c. Similar results can be found in other iridium(III) complexes [19,\nof 2.33, 2.35, 2.30 and 2.31 ppm for IPMP, 7a, 7b and 7c are assigned to 34]. Due to these complexes exhibiting high cytotoxic activity towards\nthe proton of \u2013CH3. The signal of the proton of the hydroxyl group (-OH) A549 cells, this cell line was selected for the subsequent experiments.\nin the benzene ring was not found. Likely due to a rapid interchange Inhibition of tumor proliferation and metastasis represents a crucial\nbetween the two nitrogen atoms within the imidazole ring, the signal of aspect of cancer treatment [35]. To investigate the inhibitory effects of\nthe proton in the imidazole ring was not also observed. In the 13C NMR 7a, 7b and 7c on the proliferative and migratory abilities of A549 cells,\nspectra, the signals of 21.60, 21.66, 21.53 and 21.62 ppm for IPMP, 7a, cell colony and wound healing assays were performed. As shown in\n7b and 7c are attributed to the \u2013CH3. Fig. S3a (SI), the number of A549 cell in 7a, 7b and 7c-treated groups\n To investigate the stability of 7a, 7b and 7c in PBS solution, the significantly reduced compared with that in the control, indicating that\nUV\u2013Vis absorption spectra of 7a, 7b and 7c at 0 h and 24 h were these Ir-complexes effectively inhibit the cell colony. Additionally,\nrecorded. As illustrated in Fig. S1a (SI), the complexes (20 \u03bcM) exhibited A549 cells were co-cultured with IC50 concentration of 7a, 7b and 7c for\nan absorption at wavelengths of 256 nm (\u03b5 = 24300) for 7a, 250 nm (\u03b5 = 24 h to assess the efficacy of these compounds on inhibiting wound\n26500) and 345 nm (\u03b5 = 17400) for 7b, 297 nm (\u03b5 = 14050) and 362 nm healing. Fig. S3b (SI) showed that 7a, 7b and 7c can significantly sup\u00ad\n(\u03b5 = 12900) for 7c. At room temperature, in PBS solution, the peak shape press the cell migration. The scratch condition was quantitatively\nat 0 h and 24 h remained unchanged, indicating that these complexes are analyzed using Image J software, as shown in Fig. S3c (SI), compared to\nstable. Additionally, we used fluorescence spectra to examine the sta\u00ad the control group with a healing degree of 0.50, the healing degrees in\nbility of the complexes in PBS solution at 0 and 24 h, see from Fig. S1b the 7a, 7b and 7c scratches are 0.27, 0.32 and 0.28, respectively, which\n(SI), no change in the peak shape also indicates that the complexes are further suggests that 7a, 7b and 7c can effectively block cell migration.\nstable in PBS solution. Additionally, FAK (focal adhesion kinase) protein assumes a pivotal role,\n Using methanol and water as mobile phase, the purity of 7a, 7b and which can control the adhesion, migration, invasion, and proliferation\n7c was examined by HPLC (Lc25, C18 column, flow rate 3 mL/min, of cells. Its overexpression in a wide variety of cancers can lead to a poor\nRehen Technology, China). A main peak was uncovered, showing that cancer prognosis [36]. As illustrated in Fig. S3d (SI), the expression of\n7a, 7b, 7c are pure (>95 %) (Fig. S2 and SI). intracellular FAK protein was found to be decreased after 20 h of\n exposure to 7a, 7b and 7c, providing substantial evidence that 7a, 7b and\n 7c are capable of inhibiting the migration and proliferation in A549\n2.2. In vitro cytotoxic activity, cell colony and wound healing studies cells.\n Angiogenesis is an important pathological feature of tumorigenesis,\n The complexes were dissolved in DMSO and diluted with PBS to and inhibition of angiogenesis is an effectively method for tumor ther\u00ad\nobtain different concentration of 7a, 7b and 7c. To evaluate the anti\u00ad apy. As depicted in Fig. S3e (I) (SI), we found a reduction of the number\ncancer activity of these complexes in vitro, the antiproliferative activ\u00ad of tumor vasculature after a 6 h treatment of A549 cells with 4.5 \u03bcM of\nities of 7a, 7b and 7c toward A549, LA795 and normal cell NIH3T3 were 7b, indicating that 7b can prevent the angiogenesis. In addition, VEGFA\ninvestigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte\u00ad (vascular endothelial growth factor A) is a highly specific pro-vascular\ntrazolium bromide (MTT) method [32]. The IC50 values for 7a, 7b and 7c endothelial growth factor, which promotes increasing vascular perme\u00ad\nare presented in Table 1. In detail, all the complexes exhibit excellent ability, extracellular matrix degeneration, vascular endothelial cell\ntoxicity against the A549 cells with IC50 values of 8.5 \u00b1 0.9 \u03bcM for 7a, migration, proliferation, and angiogenesis, hence, inhibition of the\n4.5 \u00b1 0.4 \u03bcM for 7b, and 8.9 \u00b1 2.2 \u03bcM for 7c. 7b shows higher anti\u00ad expression of VEGFA is an important manner to prevent the tumor cell\ncancer activity than cisplatin on A549 and LA795 cells, their cytotoxic proliferation, observing from Fig. S3e (II) (SI), 7a-7c can inhibit the\nactivity is comparable to that of liposome-entrapped complex [Ir expression of VEGFA, which shows that 7a, 7b and 7c block the tumor\n(ppy)2(BAPIP)](PF6) (IC50 = 4.9 \u00b1 1.0 \u03bcM) [18]. We also discovered angiogenesis, further prevent the cancer proliferation. Taken together,\nthat the complexes reveal moderate cytotoxic activity on normal the complexes can efficiently block cell proliferation and migration.\nNIH3T3 cells. Additionally, we also determined the IC50 value of ligand\nIPMP against A549 cells, we found that IPMP shows moderate cytotoxic\nactivity toward A549 cells, furthermore, the cytotoxic activity enhances 2.3. Cellular uptake, co-localization, change of mitochondrial membrane,\nafter the ligand was formed complexes. The anticancer efficiency follows release of cytochrome c and apoptosis\nthe order of 7b > 7a > 7c on A549 and LA795 cells. This can be\nexplained by the energy gap (\u0394E) between the lowest unoccupied orbital The initial step in the anti-cancer effect of a drug is its capability of\n(LUMO) and the highest occupied orbital (HOMO). Geometry optimi\u00ad crossing the cellular membrane. Thus, the cellular uptake was explored.\nzation of compounds 7a-7c with orbital calculations was performed As illustrated in Fig. S4 (SI), while a 12 h of co-incubation of A549 cells\nusing Gaussian 09 package [33] at the level of B3LYP density functional with 8.5 \u03bcM of 7a, 4.5 \u03bcM of 7b and 8.9 \u03bcM of 7c, we found that Hoechst\nin conjunction with the 6-31G(d) basis sets for C, H, N, O atoms, 33342 stained the cell nuclei blue, 7a, 7b, and 7c emitted green fluo\u00ad\nLanL2DZ for Ir atom. Generally, the small \u0394E value leads to high anti\u00ad rescence. The envelopment of the blue by the green indicated that 7a,\ncancer efficiency. As depicted in Fig. 1 and Table 1, the electronic clouds 7b, and 7c are able to enter the cancer cell to cumulate in the cytoplasm.\nmainly focus on phenanthroline in the LUMO orbitals, whereas the Mitochondria represent a pivotal cellular organelle in eukaryotic\nelectronic clouds in HOMO orbitals mainly focus on the ancillary ligands cells, with a pivotal role in both the induction and control of apoptosis.\nppy, bzq and piq. The \u0394E values of ELUMO \u2013 EHOMO or ES1 \u2013 ET1 follow the The mitochondria-specific red fluorescently labeled probe\n\nTable 1\nIC50 values (\u03bcM) of IPMP, 7a, 7b, 7c toward cancer cells for 48 h and the energy of LUMO, HOMO, S1, T1 (eV).\n complex A549 LA795 NIH3T3 LUMO HOMO \u0394EL-H S1 T1 \u0394ES1-T1\n\n IPMP 11.8 \u00b1 2.1 \u2013 \u2013 \u200b \u200b \u200b \u200b \u200b \u200b\n 7a 8.5 \u00b1 0.9 13.1 \u00b1 3.0 12.5 \u00b1 2.3 \u2212 4.6055 \u2212 7.5383 2.9328 2.5506 1.8953 0.6553\n 7b 4.5 \u00b1 0.4 8.3 \u00b1 0.3 12.0 \u00b1 0.5 \u2212 4.5818 \u2212 7.4268 2.8450 2.5315 1.9157 0.6159\n 7c 8.9 \u00b1 2.2 43.2 \u00b1 2.2 14.7 \u00b1 2.7 \u2212 4.4806 \u2212 7.4319 2.9513 2.4328 1.4079 1.0249\n cisplatin 6.6 \u00b1 0.7 19.0 \u00b1 0.5 18.3 \u00b1 2.4 \u200b \u200b \u200b \u200b \u200b \u200b\n\nData for cisplatin from Ref [23,37].\n\n 3\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\n\n\n Fig. 1. The energy gap between LUMO and HOMO (eV) of 7a, 7b and 7c.\n\n\nMitoTracker\u00ae Deep Red FM (ThermoFisher, 100 nM) was employed to potential (MMP), JC-1 (5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetraethyl-imida\u00ad\ninvestigate the co-localization of 7a, 7b and 7c in the mitochondria. carbocyanine), a fluorescent probe, was utilized. JC-1 is a specific\nFig. S5a (SI) depicts in the control condition, wherein mitochondria marker of membrane potential, as aggregates JC-1 emits red fluores\u00ad\nwere stained red. Following a 4 h treatment period, the merge (yellow) cence, which is because the cell membrane potential is at a high po\u00ad\nof red and green fluorescence (emitted by the complexes) suggests that tential, and damaged cells by depolarizing MMP, as monomer JC-1 is\n7a, 7b, 7c locate at the mitochondria. In our recent work, we found that broken down (green). As Seen from Fig. S5b (SI), the control group\nthe complexes enter the cancer cells to accumulate in the cytoplasm, and exhibits intense red fluorescence, whereas A549 cells exhibit a notable\nfinally the complexes focus on the mitochondria, Consequently, the green fluorescence after 20 h of treatment with the IC50 concentration of\naccumulation of the complexes in the organelles is a dynamical process 7a, 7b and 7c. The results indicate that the MMP decreased following a\n[29]. The ICP-MS experiments have been performed and we found that treatment of A549 cells with 7a, 7b and 7c. The use of a cyto c antibody\nthe amounts of 7b distributing in the mitochondria and cell nuclei are in conjunction with specific immunolabeling revealed that A549 cells\n10.3030 (\u00b11.04) and 0.7284 (\u00b10.11) ng/106 cells after a treatment of co-incubated with 7a, 7b and 7c for 20 h exhibited a more pronounced\n24 h of A549 cells with 10 \u03bcM of 7b, hence, most of the complex dis\u00ad green fluorescence in comparison to the control group (Fig. S5c and SI).\ntributes at the mitochondria. This indicates that the cells can release a significant amount of cyto c\n Afterwards, we examined the impact of the complex on the mito\u00ad molecules from their mitochondria.\nchondrial structure through transmission electron microscopy (TEM). As 7a, 7b and 7c co-localized at the mitochondria, disrupt the mito\u00ad\nseen from Fig. 2a, we observed the changes in the mitochondrial chondrial structure and function and induce a release of cyto c from the\nmorphology in A549 cells. In the blank group, the mitochondrial mitochondria, which can activate caspase 3, finally cause cell apoptosis.\nmorphology was well preserved, the inner and outer membranes were To ascertain the impact of 7a, 7b, and 7c on apoptosis in A549 cells, the\nintact, and the cristae were obvious. In contrast, the mitochondrial apoptotic percentage was examined. As illustrated in Fig. 2b, the control\nstructure in the 7c-treated group was significantly altered. Vacuolar group (I) exhibited an early and late apoptotic percentage of 5.92 %.\nstructures were formed in the mitochondrial matrix, and cristae were Following the treatment of A549 cells with 7a (II), 7b (III), and 7c (IV)\nirregularly structured and appeared to be broken or fused, indicating over a period of 20 h, the early and late apoptotic percentage of A549\nloss of contents and almost complete impairment of function. cells was found to be 40.1 %, 43.21 % and 45.1 %, respectively, the\n A normal mitochondrial membrane potential is important for safe\u00ad apoptotic percentage increases by 34.18 % for 7a, 37.29 % for 7b and\nguard of the functions of mitochondrial oxidative phosphorylation and 39.18 % for 7c. These results clearly show that 7a, 7b and 7c can effi\u00ad\nadenosine 5\u2032-triphosphate (ATP) production. The impairment of mito\u00ad ciently cause apoptosis.\nchondrial function is manifested by increased mitochondrial membrane The mitochondrial damage-mediated intrinsic pathway has been\npermeability, decreased membrane potential, the release of cytochrome demonstrated to induce apoptosis in A549 cells [39,40]. A series of\nc (cyto c), and ultimately apoptosis [38]. To further evaluate the impact proteins associated with the intrinsic pathway to induce apoptosis was\nof 7a, 7b and 7c on the changes in the mitochondrial membrane examined for the expression levels by western blotting. On the one hand,\n\n\n 4\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\n\n\nFig. 2. (a) Mitochondrial structure damage while A549 cells were treated with 2 \u00d7 IC50 concentration of 7c for 24 h, (b) apoptotic percentage after the treatment of\nA549 cells (I) with IC50 concentration of 7a (II), 7b (III) and 7c (IV) for 20 h, (c) expression of PARP, caspase 3, Bcl-2, Bax, PI3K, AKT, mTOR and p-mTOR proteins,\n(d) TGF-\u03b2 and p53 signaling pathways while A549 cells were treated with IC50 concentration of 7a, 7b and 7c for 24 h.\n\n\nprotein molecules containing the BH3 structure play a pivotal role in PI3K/AKT/mTOR signaling pathway.\nregulating the intrinsic pathway, including BID, Bax, and Bcl-2 [41,42]. In addition, we explored the TGF-\u03b2 and p53 signaling pathways. As\nOn the other hand, the initiation of apoptosis results in the degradation depicted in Fig. 2d, the complexes downregulated the expression of TGF-\nof caspase 3, an apoptotic execution factor, which is employed to cleave \u03b2, activated SMAD pathway, downregulated the expression of SMAD2,\nthe cellular substrate poly ADP-ribose polymerase (PARP) [43,44]. As SMAD3, p-SMAD2, p-SMAD3 and SMAD4, we also discovered that the\nshown in Fig. 2c, a reduction in the expression level of caspase 3 was complexes increased the expression of p53 protein. Taken together, the\nobserved following the co-incubation of A549 cells with 7a, 7b and 7c complexes cause cell death through TGF-\u03b2/SMAD and p53 signaling\nfor 24 h, indicating that caspase 3 was activated. Additionally, the pathways.\nexpression level of PARP was downregulated and cleavage occurs,\nindicating that PARP was cleaved and activated by caspase 3. At the\nsame time, it was also observed that the expression of the anti-apoptotic 2.4. DNA damage, tubulin polymerization, cell cycle arrest and\nBcl-2 was downregulated, while the expression of pro-apoptotic Bax was autophagy\nupregulated. The results demonstrated that 7a, 7b, and 7c induced\napoptosis through the intrinsic pathway, which is mediated by mito\u00ad DNA damage can impede DNA replication and induce cell cycle ar\u00ad\nchondrial damage. Additionally, 7a, 7b and 7c inhibit PI3K, AKT, mTOR rest through discrete mechanisms. When a double-strand DNA break\nand p-mTOR. Therefore, 7a-7c also trigger apoptosis through blocking occurs, the core histones H2AX is phosphorylated to generate \u03b3H2AX,\n which is an important marker for detecting the DNA damage [45,46]. As\n\n 5\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\nillustrated in Fig. 3a, the control group displays a weak green fluores\u00ad in numerous biological processes. They are essential for transport of\ncence, while the cells treated with 7a, 7b, and 7c exhibit distinct green substances and the transduction of signals, and they also maintain the\nfluorescence following co-incubation with the \u03b3H2AX antibody. Conse\u00ad morphological structure of cells [49]. Mutations and mis-regulation of\nquently, it can be surmised that 7a, 7b, and 7c can induce DNA microtubule structure result in errors in cell cycle division, which in turn\ndouble-strand breaks. leads to cell cycle arrest. The microtubule morphology remained unal\u00ad\n The processes of replication and division that occur during the cell tered in the control cells, exhibiting a perfectly ordered and tightly\ncycle are accompanied by significant alterations to the cytoskeleton and packed structure. However, following the administration of 7a, 7b and\ncell morphology [47,48]. Microtubule structures play an important role 7c for a period of 20 h, the cell microtubule structure exhibited a\n\n\n\n\nFig. 3. (a) DNA-damage via determination of \u03b3-H2AX, (b) The \u03b1-tubulin measurement, (c) cell cycle distribution, control (I), 7a (II), 7b (III), 7c (IV), (d) expression of\ncell cycle-related proteins, (e) autophagy, while A549 cells were incubated with IC50 concentration of 7a, 7b and 7c for 20 h.\n\n 6\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\ndispersed configuration (Fig. 3b). The results demonstrate that 7a, 7b formation of autophagic particle or autophagic vesicle. To investigate\nand 7c are capable of impeding cell cycle progression and inducing cell the ability of 7a, 7b and 7c cause autophagy, we used mono\u00ad\ndeath by disrupting the normal microtubule structure of cells. dansylcadaverine (MDC) as a probe to examine the autophagic effect. As\n To elucidate the mechanism of 7a, 7b and 7c inhibiting cell prolif\u00ad observed from Fig. S6 (SI), after a treatment of A549 cells with 8.5 \u03bcM of\neration, an assay of the cell cycle arrest was performed. As seen from 7a, 4.5 \u03bcM of 7b and 8.9 \u03bcM of 7c for 20 h, the autophagic vesicles were\nFig. 3c, we uncovered a large increase of 13.52 % for 7a (II), 16.81 % for discovered in the complexes-treated group in comparison to the control.\n7b (III), and 13.55 % for 7c (IV) in the G0/G1 phase in comparison to the It is well known that the upregulation of Beclin-1 and downregulation of\ncontrol, meanwhile, we also observed a reduction in the S and G2/M p62 proteins are a mark of autophagic occurrence. As shown in Fig. 3e,\nphases. These results indicate that 7a, 7b and 7c block cell proliferation 7a-7c upregulated Beclin-1 and downregulated the expression of p62\nduring the G0/G1 phase. protein, which showcases that 7a, 7b and 7c are able to cause\n Cell cycle assays have demonstrated that 7a, 7b and 7c induce A549 autophagy.\ncell cycle arrest in G0/G1 phases. Additionally, it has been established\nthat the progression of G1 phases cells necessitates integrin-dependent 2.5. RNA-sequence and signaling pathways\nsignaling, particularly cyclin D1 induction and positive regulation of\ncyclin-dependent kinases (CDKs) [50,51]. p21 is a protein kinase in\u00ad To further explore the mechanism of the complex on the inhibition of\nhibitor that plays a crucial role in regulating cell cycle progression. By tumor cell proliferation and cell death in A549 cells, RNA-seq was per\u00ad\ninhibiting CDK4, CDK6, and cyclin D, as well as CDK2 and cyclin E, p21 formed. Bioinformatics was used to further study and verify the effect of\npromotes the temporary arrest of cells at the G1/S and G2/M transitions 7b on the gene level of A549 cells. As illustrated in Fig. 4, through the\n[52,53]. The results from Fig. 3d demonstrated that A549 cells exposed volcano map and the heat map, we can see a significant difference in\nto 7a, 7b and 7c exhibited a significant reduction in the expression of gene expression between the control and 7b treatment groups. Among\nCDK6, CDK4 and cyclin D1, while the expression of p21 was signifi\u00ad them, there were 53 up-regulated genes and 85 down-regulated genes.\ncantly upregulated, which indicates that the cells were blocked in the G1 Then by KEGG enrichment analysis, we found several pathways\nphase and unable to progress to the S phase, and resulted in an increase including p53, TNF and TGF-\u03b2 signaling pathways. Moreover, GO\nin the proportion of cells in the G0/G1 phase, which was consistent with enrichment analysis discovered differential gene involvement inner\nthe findings from the cell cycle analysis. mitochondrial membrane protein complex, mitochondrial respirasome,\n Autophagy plays a key role in regulating cellular processes, main\u00ad response to oxygen levels, cell population proliferation, regulation of\ntaining tissue integrity and determining the course of cellular demise. cell migration, ATP biosynthetic process. In summary, RNA-seq results\nPromoting autophagy represents a potential avenue for the treatment of showed that iridium(III) metal complex 7b prevents cell proliferation\ntumors. Autophagy is a process that swallows its own cytoplasmic pro\u00ad through p53, TNF and TGF-\u03b2 signaling pathways, which aligns with the\nteins or organelles and encapsulates them into vesicles, where they results from cell apoptosis.\ncombine with lysosomes to form autophagic lysosomes that degrade\ntheir encapsulated contents. Autophagy plays a key role in maintaining\ncellular homeostasis [54]. Incidence of autophagy can result in a\n\n\n\n\n Fig. 4. RNA-sequence analysis in A549 cells exposed to 9.0 \u03bcM of 7b for 24 h, (a) heatmap, (b) volcano, (c) GO enrichment, (d) KEGG enrichment.\n\n 7\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\n2.6. Pyroptosis studies together, it can conclude that apoptosis and pyroptosis are the main\n mode to trigger cell death.\n Pyroptosis is a type of programmed cell death, one of several forms of To further affirm the above results, we explored the expression of\nprogrammed cell death. Pyroptosis represents a promising therapeutic caspase 1 for pyroptosis, p62 for autophagy, caspase 3 for apoptosis. As\ntarget for overcoming tumor drug resistance and triggering anti-tumor illustrated in Fig. 6c\u2013e, the downregulation of caspase 1 and caspase 3\nimmune responses [55,56]. Mitochondrial damage has been demon\u00ad was blocked compared with 7c alone. These results further demonstrate\nstrated to enhance the onset of cellular pyroptosis and to amplify that the cell death is mainly caused by apoptosis and pyroptosis.\ndownstream inflammatory responses. To investigate the effect of 7a, 7b\nand 7c on pyroptosis, a morphological examination of cells was con\u00ad\nducted. As shown in Fig. 5a, A549 cells were co-incubated with 7a, 7b, 2.8. Acute toxicity and antitumor in vivo\nand 7c for 20 h, we uncovered the formation of bubbles (red triangles),\nwhich is caused because of the disruption of cell membranes and cell The maximum tolerated dose (MTD) of an antineoplastic drug in vivo\nswelling, which is driven by the difference in osmotic pressure between can be initially assessed by observing the behavior and body weight\nthe interior of the cell and the external environment of the cell. On the changes of the animals after administration. The in vitro experiments\nother hand, LDH (lactate dehydrogenase) release is a direct indicator of indicated that 7b exhibited the highest anticancer activity among these\npyroptosis, when the integrity of the cell membrane is disrupted, LDH is complexes, therefore, it was selected for further investigation in vivo.\nreleased from the cell, resulting in an increase in the extracellular con\u00ad Healthy Kunming mice were randomly assigned to four groups: control,\ntent of LDH [57]. As illustrated in Fig. 5b, A549 cells were treated with 12.5 mg/kg, 25 mg/kg and 50 mg/kg of 7b, each received a continuous\n7a, 7b, or 7c, the LDH content was increased, indicating the rupture of administration of 7b over a 10-day period. Fig. 7a illustrates the weight\nA549 cell membrane. These results suggest that 7a, 7b, and 7c are change of mice. The weight change trend in the 12.5 mg/kg adminis\u00ad\ncapable of inducing pyroptosis. tration group and the control group mice is comparable, exhibiting a\n Pyroptosis can be defined as an inflammatory cell death process. The slow growth pattern. The weight of mice in the 25 mg/kg administration\ninvolvement of NF-\u03baB protein is a necessary component, as it has a group exhibited a slight decline from the 2nd to 5th day, followed by a\nsubstantial influence on the inflammatory cascade. The key function of gradual increase from the 6th day onwards. In contrast, the body weight\nNF-\u03baB proteins is to activate the pro-inflammatory entity caspase 1. The of mice in the 50 mg/kg dosing group exhibited a sharp decline during\nactivation of caspase 1 initiates the cleavage of GSDMD-F to GSDMD-N, the initial seven days of dosing, followed by a period of relatively stable\nwhich results in the perforation of the cell membrane, cellular rupture, weight. On the 9th day of this period, one mouse in this group died.\nand subsequent release of cellular contents. As illustrated in Fig. 5c, the Furthermore, an examination of the behavior and hair status of mice in\nexpression of NF-\u03baB protein was found to be elevated, while caspase 1 each group throughout the experiment revealed that the hair of mice in\nexpression demonstrated a notable reduction, GSDMD-F was transferred both the control group and the 12.5 mg/kg administration group was\ninto GSDMD-N. Taken together, we consider that 7a, 7b, and 7c can smooth and neat, and their behavior was active. The mice in the 25 mg/\ninduce pyroptosis. kg administration group exhibited slight messiness in their fur, accom\u00ad\n panied by a small amount of fecal contamination. Additionally, their\n overall behavior was reduced, and they displayed a tendency to curl up\n2.7. Main death mode studies in a corner. In the 50 mg/kg dosing group, the hair of the mice exhibited\n a messy and dull appearance, and secretions adhered to the mouth, nose,\n In this article, we found that 7a-7c cause cell death through and vagina. Furthermore, the mice displayed aberrant behavior,\napoptosis, pyroptosis, autophagy. To validate the main mode to cause including a reduction in overall activity and a tendency to lie in a prone\ncell death, we applied inhibitors Z-VAD-FMK (20 \u03bcM), VX765 (0.8 \u03bcM) position. The results indicate that the LD50 value of 7b against healthy\nand 3-MA (20 \u03bcM) for apoptosis, pyroptosis and autophagy to explore Kunming mice is 25 mg/kg.\nthe main manner. In the first, we investigated the change of cell The LD50 value, as determined by the 10-day acute toxicity test, is\nviability. As shown in Fig. 6a, addition of Z-VAD-FMK and VX765 led to 25 mg/kg for 7b. In the antitumor in vivo assay, we selected about 1/\nan increment in the cell viability of 27.3 % and 19.7 % in comparison to 14LD50 and 1/9LD50 values as dosing groups. A xenogeneic A549 lung\n7c alone, while 3-MA only increased by 2.2 %, indicating that the main cancer model was constructed in BALB/c nude mice. The mice were\nmanners of causing cell death are apoptosis and pyroptosis. Also, we randomly divided into three groups: control group, low dosing group\nexamined the early and late apoptotic percentage. As depicted in Fig. 6b, (1.8 mg/kg), and high dosing group (2.7 mg/kg). We administered an\nthe total apoptotic percentage in the early and late decreased by 10.61 intraperitoneal injection of 7b into nude mice for seven consecutive\n%, indicating that apoptosis is main manner to induce cell death. Taken days. During this period, we monitored and recorded the mice\u2019s daily\n\n\n\n\nFig. 5. Pyroptosis assay: (a) cell morphology, (b) LDH release (positive: LDH release reagent), (c) expression of NF-\u03baB, caspase 1 and GSDMD while a 20 h-treatment\nof A549 cells with IC50 concentration of 7a, 7b and 7c.\n\n 8\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\n\n\nFig. 6. (a) cell viability assay of A549 cells exposure to IC50 concentration of 7c for 36 h, (b) apoptosis assay of A549 cells exposure to IC50 concentration of 7c for 12\nh, (I) control, (II) Z-VAD-FMK, (III) 7c, (IV) 7c + Z-VAD-FMK, (c\u2013e) expression of caspase 1, p62 and caspase 3 while A549 cells were exposed to IC50 concentration of\n7c for 24 h.\n\n\nbody weight and tumor size. After 7 days, following the euthanasia of on the three groups of tumors, with the markers of DNA damage and cell\nnude mice and the subsequent collection of the tumor and its principal proliferation-related antigen Ki67 being studied, respectively. As illus\u00ad\norgans. As illustrated in Fig. 7b, the administration group exhibited a trated in Fig. 7h, in the assay of \u03b3H2AX, the positive rate in the control,\nsignificantly inhibited tumor growth compared to the control group. 1.8 mg/kg and 2.7 mg/kg groups are 0.51 %, 2.61 % and 13.70 %. This\nFig. 7c illustrates that there was no statistically significant difference in suggests that the complex can cause apoptosis. While in the assay of\nbody weight between the control and drug-treated groups. Fig. 7d re\u00ad Ki67, the positive rates are 18.96 %, 7.52 % and 5.84 % in the control,\nveals that the relative tumor volume in the control group exhibited a 1.8 mg/kg and 2.7 mg/kg groups, a decrease of the positive rate in\u00ad\nrapid increase. In contrast, the growth trend of the low-dose group was dicates that the complex can prevent tumor proliferation. The results\nslower than that in the control group. Furthermore, the growth rate in indicated that 7b could induce apoptosis and inhibit tumor proliferation.\nthe high-dose group was notably slower and significantly lower than In conclusion, these findings demonstrate the potent anti-tumor efficacy\nthat in the control group and the low-dose group. The mean tumor of 7b in vitro and in vivo, as well as its favorable safety profile in key\nweight (Fig. 7e) for the three groups was 0.88 g, 0.65 g, and 0.29 g, organs. Therefore, it can be proposed that 7b may be a suitable drug\nrespectively. The tumor inhibitory rates in the 1.8 mg/kg, 2.7 mg/kg candidate for the treatment of non-small cell lung cancer.\nadministration group were 26.1 % and 66.9 %. In the Fig. 7f, the weights\nof heart in the control, 1.8 mg/kg and 2.7 mg/kg are 0.1155 \u00b1 0.02, 2.9. Mechanism studies of inhibiting tumor growth\n0.1009 \u00b1 0.009 and 0.1109 \u00b1 0.008 g, respectively. Therefore, the\nshape and weight of heart have no obvious change. These findings 2.9.1. Immunogenic cell death assay\ndemonstrated that 7b exhibited high anti-tumor efficacy with no Immunogenic cell death (ICD) is a form of regulated cell death that\napparent adverse effects on mice. activates tumor-specific immune responses in the presence of normal\n H&E staining and pathological analysis were performed on key or\u00ad immune function. Tumor cells expose the endoplasmic reticulum pro\u00ad\ngans and tumor. The results are presented in Fig. 7g. There were no teins calreticulin (CRT) and heat shock proteins (HSP) to the cell\ndiscernible abnormalities in the heart, liver, spleen, lung, and kidney in membrane during the pre-apoptotic phase, while releasing the nuclear\nthe administration group compared with the control group. The lung of protein high mobility group box-1 (HMGB1) extracellularly, and these\nnude mice treated with 7b exhibited a small number of granulocytes, signaling factors promote the uptake of dying cells by macrophages\nhemorrhage of bronchioles and peripheral alveoli, and an irregular [58]. Inducing tumor cells to undergo immunogenic cell death to release\narrangement of their epithelial cells. The combined experimental data damage-associated molecular patterns has become a key component in\nindicated that 7b exhibited anti-tumor activity in vivo and did not result antitumor therapy [59].\nin any significant toxic effects on various organs. Surface-exposed CRT, HMGB1 and HSP70 were analyzed by smart\n Subsequently, immunohistochemistry (ICH) analysis was conducted cell imaging to explore whether 7a, 7b and 7c could induce ICD in A549\n\n 9\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\n\n\nFig. 7. (a) The relative weight of mice in 10 treated days during the treatment with different concentration of 7b (12.5, 25 and 50 mg/kg), (b) shape of tumor, (c)\nMean weight of mice, (d) relative tumor volume, (e) mean weight of tumor, (f) heart of mice, (g) H&E staining assay of heart, liver, spleen, lung, kidney, brain, and\ntumor, (h) Immunohistochemical analysis of \u03b3H2AX and Ki67 in tumor tissues of xenograft nude mice.\n\n\n\n\n 10\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\ncells. As major markers of ICD, the adenosine triphosphate (ATP) of cytokines IL-10, IFN-\u03b3 and TNF-\u03b1 was measured using ELISA method.\nsecretion, calreticulin (CRT) exposure on the membrane, and high The results shown in Fig. 8a revealed that the content of IL-10 was\nmobility group box 1 (HMGB1) release from nuclei content was exam\u00ad significantly lower than those of the blank group, while the contents of\nined. As shown in Figs. S7a, b, c and d (SI), A549 cells were exposed to IFN-\u03b3 and TNF-\u03b1 were higher than those in the control group. The results\nIC50 concentration of 7a-7c for 24 h, the ATP content increased, the demonstrated that 7b and 7c could further enhance the antitumor effect\ngreen fluorescence of CRT, HMGB1 and HSP70 on the surface of the drug by reducing the content of IL-10 and increasing IFN-\u03b3 and TNF-\u03b1.\ngroup was significantly enhanced compared with the blank group. This GM1, an important component of lipid rafts, is involved in T cell\nsuggests that the complexes can elicit the release of the damage- maturation, activation, and the formation of immune synapses. Lipid\nassociated molecules CRT, HSP70, and HMGB1 from A549 cells, acti\u00ad rafts are microstructural domains in cell membranes enriched with\nvate tumor-specific immune responses, and show promise for inducing sphingolipids and cholesterol that enrich for signaling molecules and\nimmunogenic cell death in A549 cells. Meanwhile, we also used Western promote T-cell receptor (TCR) activation. The high expression of GM1 in\nblot to confirm the upregulation of the expression of CRT, HSP70 and CD8+ T cells and its enrichment in lipid rafts allow it to play an\nMHGB1 (Fig. S7d and SI), the Western blot results were consistent with important role in regulating signaling and function of CD8+ T cells [60].\nthe results obtained from the Smart Cell Imager, further confirming that We measured CD8+ T cells and GM1 by immunostaining as shown in the\n7a, 7b and 7c may induce immunogenic cell death. Fig. 8b found that the green fluorescence of the fits 7b and 7c was\n significantly enhanced compared to the control, indicating an increase\n2.9.2. Activation of the immune response in CD8+ T cells. We also determined the percentage of CD44 and CD62L\n IFN-\u03b3, TNF-\u03b1 and IL-10, as an important cytokine involved in the cells in peripheral blood mononuclear cells and spleen by measuring the\nimmune response, play a key role in combating tumorigenesis and percentage of CD44 and CD62L cells in peripheral blood mononuclear\nprogression. Firstly, LLC (mouse lung cancer) cells were injected into the cells and spleen. It was observed from the Fig. 8c and d that compounds\nblack C57BL/6J mice, after 7 days, the blood was obtained, the content 7b and 7c were able to increase the number of CD44 and CD62L cells in\n\n\n\n\nFig. 8. Determination of lymphocyte from the tumor tissue, (a) the content of TNF-\u03b1, IFN-\u03b3 and IL-10, (b) GM1 and CD8+ T cells, memory-T cells from the PBMC (c)\nand spleen (d). (e) H&E staining assay of heart, liver, spleen, lung, kidney, brain, and tumor.\n\n 11\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\nPBMC and spleen. 0.25 mmol) [61] for 7a, cis-[Ir(bzq)2Cl]2 (0.30 g, 0.25 mmol) [61] for\n Tissue organs of mice in each group were stained with H&E to detect 7b, cis-[Ir(piq)2Cl]2 (0.32 g, 0.25 mmol) [61] for 7c were dissolved in 45\npathological damage at the end of the experiment. As shown in Fig. 8e, mL CH3OH/CH2Cl2 (CH3OH:CH2Cl2 = 1:2, v/v) and refluxed at 40 \u25e6 C for\nno significant damage was discovered in the heart, liver, spleen, kidney, 6 h. Once the solution has reached room temperature, the excess NH4PF6\nand brain in all groups. However, a slight thickening of the alveoli is aqueous solution was added into the solution and stirred for 1.5 h. The\nseen in the lung tissue, and the tumor tissue shows cellular permutation solvent was removed through vacuum decompression, resulting in the\ndisorganization, nuclear consolidation, and hemorrhage. These suggest formation of a yellow powder. The obtained crude product was sepa\u00ad\nthat 7b and 7c does not only cause significant damage in mice but also rated by neutral alumina column chromatography,\ndisrupt tumor cell function and cause tumor necrosis. dichloromethane-acetonitrile (v/v, 4:1) was used as the eluent. The\n orange-yellow band fractions were collected, concentrated, and dried.\n3. Conclusions Finally, the yellow powder (7a), orange-red powder (7b), and dark red\n powder (7c) were obtained.\n In this study, a new ligand, IPMP, and three new compounds, [Ir 7a: 84 % yield. HRMS (CH3CN, Fig. S8 and SI): Calcd for C42H30Ir\u00ad\n(ppy)2(IPMP)]PF6 (7a), [Ir(bzq)2(IPMP)]PF6 (7b), and [Ir(piq)2(IPMP)] N6OPF6: m/z = 827.2110 ([M \u2212 PF6]+), found m/z = 827.2120 ([M \u2212\nPF6 (7c), were synthesized and characterized. The experimental results PF6]+). 1H NMR (DMSO\u2011d6, 500 MHz, Fig. S9 and SI): The signals of the\nindicated that the three complexes exhibited the most pronounced proton of hydroxyl group in the benzene ring and the proton in the\ninhibitory effect on the proliferation of the A549 cell line. In vitro results imidazole ring were not observed. \u03b4 9.26 (d, 2H, J = 8.5 Hz), 8.26 (d,\nhave demonstrated that complexes can target the mitochondria, disrupt 2H, J = 8.0 Hz), 8.16\u20138.13 (m, 3H), 8.09\u20138.06 (m, 2H), 7.95 (d, 2H, J =\nmitochondrial function, and thereby cause a reduction of mitochondrial 7.5 Hz), 7.87 (t, 2H, J = 7.5 Hz), 7.50 (d, 2H, J = 6.0 Hz), 7.06 (t, 2H, J\nmembrane potential. These complexes exhibit remarkable efficacy in the = 7.5 Hz), 7.00\u20136.92 (m, 6H), 6.29 (d, 2H, J = 7.5 Hz), 2.35 (s, 3H). 13C\ninhibition of tumor cell proliferation and migration, as well as in the NMR (125 MHz, DMSO\u2011d6, Fig. S10 and SI) \u03b4 172.33, 167.38, 157.66,\ninhibition of tumor cell growth at the G0/G1 phase. Additionally, the 150.85, 149.66, 148.93, 144.57, 144.52, 142.79, 139.18, 132.98,\ncomplexes can cause apoptosis, autophagy and pyroptosis, while 131.71, 130.75, 127.53, 127.17, 125.56, 124.33, 122.86, 120.98,\napoptosis and pyroptosis are two main manners to trigger cell demise. 120.46, 118.05, 21.66.\nInterestingly, 7b shows high antitumor efficiency with an inhibitory rate 7b: 86 % yield. HRMS (CH3CN, Fig. S11 and SI): Calcd. for\nof 66.9 %. The mechanism studies illustrate that the complexes induce C46H30IrN6OPF6: m/z = 875.2110 ([M \u2212 PF6]+), found m/z = 875.2120\nimmunogenic cell death to activate immune response to increase CD8+ T ([M \u2212 PF6]+). 1H NMR (DMSO\u2011d6, 500 MHz, Fig. S12 and SI): The sig\u00ad\ncell and increase the content of cytokines IFN-\u03b3 and TNF-\u03b1, reduce the nals of the proton of hydroxyl group in the benzene ring and the proton\ncontent IL-10, which further kill tumor cells. The findings from in vitro in the imidazole ring were not observed. \u03b4 9.02 (d, 2H, J = 8.5 Hz), 8.50\nexperiments demonstrate that the complexes elicit cell death in A549 (d, 2H, J = 8.0 Hz), 8.18 (d, 1H, J = 8.0 Hz), 7.99\u20137.94 (m, 6H), 7.87 (d,\ncells through four distinct pathways (I) The complexes cause a mito\u00ad 2H, J = 8.5 Hz), 7.82\u20137.79 (m, 2H), 7.57 (d, 2H, J = 8.0 Hz), 7.44 (q,\nchondrial dysfunction, release cyto c, and activate caspase 3, regulate 2H), 7.22 (t, 2H, J = 7.5 Hz), 6.77\u20136.73 (m, 2H), 6.33 (d, 2H, J = 7.5\nthe expression of Bcl-2 family proteins and inducing apoptosis; (II) the Hz), 2.30 (s, 3H). 13C NMR (DMSO\u2011d6, 125 MHz, Fig. S13 and SI):\ncomplexes cause damage to cellular DNA and microtubules, and block 172.48, 157.93, 157.05, 148.99, 148.52, 146.91, 143.53, 140.92,\ncellular division at the G0/G1 phase, thereby promoting apoptosis; (III) 137.86, 134.19, 132.15, 130.16, 129.94, 127.13, 126.92, 126.26,\nthe complexes induce autophagy via inhibiting PI3K/AKT/mTOR 124.65, 123.21, 120.61, 119.81, 117.21, 21.53.\nsignaling pathway; (IV) the complexes regulate NF-\u03baB proteins, thereby 7c: 81 % yield. HRMS (CH3CN, Fig. S14 and SI): Calcd. for\nactivate caspase 1, which induces cellular pyroptosis. Consequently, this C50H34IrN6OPF6: m/z = 927.2523 ([M \u2212 PF6]+), found m/z = 927.2556\nwork will prove beneficial to the future development of anti-tumor ([M \u2212 PF6]+). 1H NMR (DMSO\u2011d6, 400 MHz, Fig. S15 and SI): The sig\u00ad\nmechanisms and the design of new Ir(III) complexes. nals of the proton of hydroxyl group in the benzene ring and the proton\n in the imidazole ring were not observed. \u03b4 9.11 (q, 2H), 9.02 (d, 2H, J =\n4. Experimental 8.0 Hz), 8.40 (d, 2H, J = 8.0 Hz), 8.20 (d, 1H, J = 7.6 Hz), 8.00 (d, 2H, J\n = 9.0 Hz), 7.94\u20137.84 (m, 8H), 7.44 (d, 2H, J = 6.4 Hz), 7.38 (d, 2H, J =\n4.1. Synthesis and characterization 6.8 Hz), 7.16 (t, 2H, J = 8.0 Hz), 6.96 (t, 2H, J = 7.6 Hz), 6.81\u20136.78 (m,\n 2H), 6.32 (d, 2H, J = 7.2 Hz), 2.31 (s, 3H). 13C NMR (DMSO\u2011d6, 125\n4.1.1. Synthesis of ligand IPMP MHz, Fig. S16 and SI): 172.50, 168.38, 157.88, 154.93, 145.85, 143.39,\n To a solution of 1,10-phenanthroline-5,6-dione (2 mmol, 0.42 g) and 141.07, 136.92, 132.45, 132.20, 131.07, 130.97, 129.81, 128.16,\n2-hydroxyl-4-methylbenzaldehyde (2 mmol, 0.28 g) in glacial acetic 126.99, 126.91, 126.63, 126.04, 125.65, 122.68, 122.64, 120.15,\nacid (30 mL) was added ammonium acetate (40 mmol, 3.1 g) in a three- 117.46, 21.62.\nneck flask. The mixture was stirred at 130 \u25e6 C and refluxed for 2 h. After\ncooling the reaction to room temperature, the solution was neutralized 4.2. In vitro cytotoxicity test\nwith concentrated ammonia water, and the resulting precipitate was\nwashed with double-distilled water. The yellow precipitate was The cytotoxicity in vitro of 7a, 7b and 7c on the selected cancer cells\ncollected by suction filtration, and the IPMP ligand was obtained after was tested by 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bro\u00ad\ndrying in a vacuum drying oven. The yield was 89 %. The molecular mide (MTT) method [28]. The cancer cells were seeded in 96-well plates\nformula of IPMP is C20H14N4O. 1H NMR (DMSO\u2011d6, 500 MHz): The at a density of 1.0 \u00d7 105 cells per well and cultured for 24 h. The\nsignals of the protons in the hydroxyl group (-OH) and in the imidazole different concentration of 7a, 7b and 7c were added into the wells to\nring were not found. \u03b4 9.01 (d, 2H, J = 7.5 Hz), 8.85 (d, 2H, J = 7.5 Hz), treat the cancer cells for 48 h, followed by adding 10 \u03bcL of MTT and 90\n8.00 (d, 1H, J = 8.0 Hz), 7.81\u20137.79 (m, 2H), 6.86 (d, 2H, J = 7.5 Hz), \u03bcL of medium. The plate was then incubated in an incubator at 37 \u25e6 C for\n2.33 (s, 3H). 13C NMR (DMSO\u2011d6, 125 MHz): 173.01, 167.29, 157.43, 4 h. After incubation, discarding the culture medium, adding 100 \u03bcL of\n150.67, 149.49, 148.87, 144.41, 139.20, 132.86, 131.66, 130.78, DMSO into the plate and shake for 6 min using a multimode microplate\n125.46, 124.23, 122.99, 120.37, 21.60. HRMS (CH3OH): Calcd for reader to determine the absorbance (490 nm). The test was repeated\nC20H14N4O: m/z = 326.1168, Found: m/z = 325.1049 ([M \u2212 H]-). three times independently and the mean value was calculated.\n\n4.1.2. Synthesis of 7a, 7b, and 7c\n Under argon, IPMP (0.16 g, 0.5 mmol) and cis-[Ir(ppy)2Cl]2 (0.27 g,\n\n 12\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\n4.3. Western blotting experiments the mice are administered with the drug in a seven-dose regimen. The\n tumor size and mice weight were recorded throughout the experiment.\n The Western blot was carried out by the following six procedures: (I) Finally, the tumors were peeled off and the brain, heart, liver, spleen,\nA549 cells were seeded evenly in 6-well plates and incubated overnight, and lung tissues were collected, and the slices were stained with he\u00ad\nwhen the adherent cells grow to 80 %, the cells were together incubated matoxylin-eosin (H&E).\nwith IC50 concentration of 7a, 7b and 7c at 37 \u25e6 C for 20 h. (II) After\nincubation, wash the cells three times with ice-cold PBS, then add the\ncell lysates to the 6-well plate, scrape the cells in the 6-well plate and 4.6. Assay of immunofluorescence staining\nplace a 1.5 mL tube, and continue lysis for 10 min. At the end of the lysis,\nthe cells are placed in a cryogenic centrifuge and the supernatant is LLC (mouse lung cancer) cells were injected the black C57BL/6J\ntaken as the target total protein. (III) The total protein content was mice, we observed the tumor model after 7 days. Then 2.7 mg/kg 7b was\nmeasured using the BCA Quantification Kit and electrophoresed on an intraperitoneally injected the mice for continuous 7 days. The tumors\nSDS-SPAGE gel for 3 h to separate the same amount of denatured pro\u00ad were excised, then the tumors were taken into the embedding box,\ntein. A pre-stained colored protein marker was used as an internal dehydrated for 15 h, embedded in paraffin, and gain the tumor sections.\nreference. (IV) The gel is then split, transferred to a polyvinylidene The sections were baked overnight, dewaxed according to the proced\u00ad\nfluoride membrane, and blocked with 5 % nonfat dry milk in TBST ure, and put into citrate antigen repair solution to repair the sections.\nbuffer solution for 2 h. (IV) The membrane is hybridized with primary After cooling, the sections were closed with a drop of goat serum\nantibody (1:5000 dilution) overnight at 4 \u25e6 C. (V) Then the membrane is blocking solution for 1 h, then incubated with primary antibody over\u00ad\nfurther hybridized with horseradish peroxidase-conjugated secondary night. Next day, the secondary antibody was incubated for 1 h, then\nantibody (1:5000 dilution) for 2 h. (VI) Each protein was visualized Hoechst 33342 was incubated for 5 min in the dark, and anti-fluorescent\nusing an enhanced chemiluminescence (ECL) assay kit according to the bursting agent was added to close the sections. Ultimately, the sections\nmanufacturer\u2019s instructions. Finally, the protein bands were visualized were observed under fluorescence microscope.\nand analyzed in a FluorChem E instrument.\n\n4.4. Acute toxicity test 4.7. Data analysis\n\n Healthy mice from the Guangdong Medical Laboratory Animal Statistical significance was assessed with t-tests and *P < 0.05 is\nCenter (Guangzhou, China) were randomly divided into four groups. significant.\nEach group consisted of half male and half female mice, and the mice Note: The experimental procedure for material and methods, cell\nwere fed in the Animal Experiment Center of Guangdong Pharmaceu\u00ad culture, cellular uptake, cell scratch experiment, cell cloning experi\u00ad\ntical University. The mice were housed in a temperature-controlled ment, mitochondrial damage, mitochondrial localization and mito\u00ad\nenvironment (25 \u25e6 C) with a relative humidity of 40\u201350 %. The feeding chondrial membrane potential, apoptosis assay, cell cycle arrest studies,\nconditions and experimental methods were conducted in accordance \u03b3H2AX detection, \u03b1-tubulin assay, cytochrome c release, autophagy,\nwith the relevant regulations and principles of the Animal Center of lactate dehydrogenase (LDH) release and ATP determination can be\nGuangdong Pharmaceutical University and approved by the Animal discovered in the Supporting Information.\nEthics Committee of Guangdong Pharmaceutical University. Following\nthe safety quarantine period, the four groups of mice were designated as\n CRediT authorship contribution statement\nthe blank control group and the experimental group (7b at concentra\u00ad\ntions of 0, 12.5, 25, and 50 mg/kg, respectively). The body weight of\n Shuanghui Tang: Writing \u2013 original draft, Methodology, Investiga\u00ad\neach mouse was recorded at the same time each day, and 7b of each\n tion. Yueyao Ding: Investigation, Formal analysis, Data curation. Ziyan\nconcentration gradient was injected intraperitoneally. The mouse hair\n Zhang: Investigation, Data curation. Yan Yang: Writing \u2013 original draft,\nand its activity status were monitored and recorded. Following a 10-day\n Methodology, Conceptualization. Chunxia Huang: Investigation,\nperiod of continuous administration, the mice were injected with 150\n Formal analysis. Lin Zhou: Formal analysis, Data curation. Shuang\nmg/kg sodium isoambarbital and subsequently euthanized via cervical\n Tian: Software, Formal analysis. Hui Yin: Writing \u2013 review & editing,\ndislocation following anesthesia. The maximum tolerated dose of mice\n Funding acquisition, Conceptualization. Yunjun Liu: Supervision,\nwas ultimately determined based on the drug concentration, weight,\n Project administration, Funding acquisition.\nhair, and behavioral characteristics of the mice.\n\n4.5. In vivo antitumor activity\n Declaration of competing interest\n Given the evident in vitro toxicity of 7b on A549 cells, the in vivo\n Authors declare no competing interests exist.\nanti-tumor effect of 7b was investigated using the A549 xenograft tumor\nmodel. The BALB/c nude mice were obtained from the Guangdong\nMedical Laboratory Animal Center (Guangzhou, China) and were Acknowledgements\nmaintained in the Animal Experiment Center of Guangdong Pharma\u00ad\nceutical University (25 \u25e6 C, relative humidity 40\u201350 %) in a specific This research was supported by the National Natural Science Foun\u00ad\npathogen-free (SPF) environment. All experimental procedures were dation of China (Nos. 21877018, 82171700).\napproved by the Animal Ethics Committee of Guangdong Pharmaceu\u00ad\ntical University. A549 cells exhibiting robust growth characteristics Appendix A. Supplementary data\nwere directly inoculated beneath the armpits of nude mice. Following a\nseven-day incubation period, small cauliflower-like tumors emerged at Supplementary data to this article can be found online at https://doi.\nthe site of tumor inoculation in the mice. The mice were randomly org/10.1016/j.ejmech.2025.117926.\ndivided into three groups, with six mice in each group and half male and\nhalf female, respectively. The groups were designated as the blank Data availability\ncontrol group, the low dose group (1.8 mg/kg), and the high dose group\n(2.7 mg/kg). Once the tumor reaches approximately 5 mm in diameter, Data will be made available on request.\n\n 13\n\fS. Tang et al. European Journal of Medicinal Chemistry 297 (2025) 117926\n\n\nReferences [28] P. Szymaszek, M. Tyszka-Czochara, J. Ortyl, Iridium(III) complexes as novel\n theranostic small molecules for medical diagnostics, precise imaging at a single cell\n level and targeted anticancer therapy, Eur. J. Med. Chem. 276 (2024) 116648.\n [1] F. Bray, M. Laversanne, H. Sung, J. Ferlay, R.L. 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