Design and anticancer behaviour of cationic/neutral half-sandwich iridium(III) imidazole-phenanthroline/phenanthrene complexes.

{"full_text": " Journal of Inorganic Biochemistry 257 (2024) 112612\n\n\n Contents lists available at ScienceDirect\n\n\n Journal of Inorganic Biochemistry\n journal homepage: www.elsevier.com/locate/jinorgbio\n\n\n\n\nDesign and anticancer behaviour of cationic/neutral half-sandwich iridium\n(III) imidazole-phenanthroline/phenanthrene complexes\nAo Lv a, Guangxiao Li a, Pei Zhang b, Rui Tao a, Xiaoshuang Li a, Xueyan Ren a, Peixuan Li a,\nXicheng Liu a, *, Xiang-Ai Yuan a, Zhe Liu a, *\na\n Key Laboratory of Life-Organic Analysis of Shandong Province, Institute of Anticancer Agents Development and Theranostic Application, School of Chemistry and\nChemical Engineering, Qufu Normal University, Qufu 273165, Shandong, China\nb\n College of Life Sciences, Qufu Normal University, Qufu 273165, Shandong, 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: Considerable attention has been devoted to the exploration of organometallic iridium(III) (IrIII) complexes for\nIridium(III) complex their potential as metallic anticancer drugs. In this study, twelve half-sandwich IrIII imidazole-phenanthroline/\nImidazole-phenanthroline phenanthrene complexes were prepared and characterized. Complexes exhibited promising in-vitro anti-\nImidazole-phenanthrene\n proliferative activity, and some are obviously superior to cisplatin towards A549 cells. These complexes\nAnticancer\n possessed suitable fluorescence, and a non-energy-dependent uptake pathway was identified, subsequently\nApoptosis\nLysosomal targeted leading to their accumulation in the lysosome and the lysosomal damage. Additionally, complexes could inhibit\n the cell cycle (G1-phase) and catalyze intracellular NADH oxidation, thus substantiating the elevation of intra\u00ad\n cellular reactive oxygen species (ROS) level, which confirming the oxidative mechanism. Western blotting\n further confirmed that complexes could induce A549 cell apoptosis through the lysosomal-mitochondrial anti\u00ad\n cancer pathway, which was inconsistent with cisplatin. In summary, these complexes offer fresh concepts for the\n development of organometallic non\u2011platinum anticancer drugs.\n\n\n\n\n1. Introduction [8]. Compared to cyclometallic complexes, half-sandwich IrIII com\u00ad\n plexes exhibited superior anticancer activity, and their fluorescence\n Today, cancer is one of the most important diseases endangering properties could also be improved by regulating the peripheral ligands,\nhuman life and health. Cisplatin, a clinically utilized metal-based anti\u00ad which providing the convenient condition for the ascertainment of their\ncancer drug, offers a fresh concept for the creation of metallic anticancer anticancer mechanism [9]. In contrast to platinum-based drugs, half-\nmedications [1,2]. While widely used in clinic, platinum-based drugs sandwich IrIII complexes have been reported to predominantly accu\u00ad\nexhibit the disadvantages of drug resistance, serious toxicity (including mulate in intracellular mitochondria or lysosomes, which facilitating\nrenal toxicity, gastrointestinal toxicity, ototoxicity and neurotoxicity) their active participation in intracellular REDOX reactions, then pro\u00ad\nand low biological selectivity, which limiting the long cycle and large moting the buildup of intracellular ROS and the induction of cell\ndose use in clinical practice [3,4]. Therefore, non\u2011platinum anticancer apoptosis [10]. Half-sandwich IrIII complexes are commonly described\nagents, which showing different mechanism from cisplatin, including as [(\u03b75-CpX)Ir(L)(Z)2]n+ or [(\u03b75-CpX)Ir(L^L)Z]n+, and the screening of\niridium(III) (IrIII) anticancer complexes, have received extensive atten\u00ad peripheral ligands (CpX (cyclopentadiene derivatives), L/L^L (mono\u00ad\ntion as the substitutes for platinum drugs [5\u20137]. dentate or bidentate ligand), or Z (leaving group, usually chlorine)) can\n IrIII anticancer complexes are typically classified into two distinct effectively regulate the anticancer activity of these complexes\nstructural categories: Half-sandwich and cyclometallic IrIII complexes [7,11\u201316]. Now, the activity regulation is mainly focusing on the\n\n\n Abbreviations: Cp*, pentamethyl cyclopentadiene; Cpxph, phenyltetramethyl cyclopentadiene; MTT, (3-(4,5-dimethylmercaptan-2-yl)-2,5-diphenylmethyl bro\u00ad\nmide); IC50, 50% inhibiting concentration; HOMO, the highest occupied molecular orbital; LUMO, the lowest unoccupied molecular orbital; CCCP, carbonyl cyanide\nm-chlorophenyl hydrazine; LTDR, lysotracker deep red, lysosome probe; MTDR, mitotracker deep red, mitochondrial probe; PCC, Pearson co-localization coefficient;\nPI, propidium iodide; ROS, reactive oxygen species; MMP, mitochondrial membrane potential; JC-1, (5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-tetraethylbenzimidazolyl iodo\u00ad\ncarbocyanine); NADH, nicotinamide adenine dinucleotide; TON, turnover number; WCR, wound closure rate.\n * Corresponding authors.\n E-mail addresses: chemlxc@163.com (X. Liu), liuzheqd@163.com (Z. Liu).\n\nhttps://doi.org/10.1016/j.jinorgbio.2024.112612\nReceived 16 February 2024; Received in revised form 17 April 2024; Accepted 13 May 2024\nAvailable online 15 May 2024\n0162-0134/\u00a9 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fA. Lv et al. Journal of Inorganic Biochemistry 257 (2024) 112612\n\n\nscreening of monodentate/bidentate ligands, especially for bidentate 2. Results and discussion\nligands, including N^N (e.g., bipyridine, phenanthroline), N^C (e.g.,\nphenylpyridine, \u03b2-carboline [17]), N^O (e.g., acylhydrazone, picolinic 2.1. Synthesis and structural characterization\nacid, amino acid), N^S (e.g., thiocarbazone, 2-pyridinecarbothioamide\n[18]), O^O (e.g., \u03b2-diketonato [19]), etc., which showing the better ac\u00ad The imidazole-based pro-ligands (L1-L6) were synthesized using a\ntivity. Among them, the most studied were N^N and N^C ligands. Sadler usual four components assembling of 1,10-phenolin-5,6-dione/acenaph\u00ad\nreported series of organoiridium bipyridine (N^N)/phenylpyridine (N^C) thenedione, ammonium acetate, 4-methylaniline and the corresponding\ncomplexes for the first time, which showed the greater potency than benzaldehyde (-F/-H/-OCH3; Scheme 1), and the pure products were\ncisplatin in targeting a variety of cancer cells, including colon cancer, achieved through column chromatography (petroleum ether: ethyl ac\u00ad\nleukemia, prostate cancer, breast cancer, and melanoma [20]. etate = 15:1). Target complexes were prepared through the reaction of\n The composition of many natural and semi-synthetic opioids basic IrIII precursors (([(\u03b75-Cp*)Ir(\u03bc-Cl)Cl]2, Dimer 1; [(\u03b75-Cpxph)Ir(\u03bc-Cl)\nincluding morphine, codeine, and naloxone, consisting of planar aro\u00ad Cl]2, Dimer 2) and twice the proportion of imidazole chelating ligands.\nmatic molecules, is commonly occurring in nature. Over the past two Phenanthrene-based complexes (Ir7-Ir12, [(\u03b75-CpX)Ir(C^N)Cl]) were\ndecades, imidazo-phenanthroline/phenanthrene or derivatives had obtained by adding sodium acetate as an acid binding agent, and\ngarnered escalating interest in the fields of molecular switches, photo\u00ad phenanthroline-based one were precipitated into hexafluorophosphate\ncatalyses, luminescent sensing, photodynamic therapy (PDT), and (Ir1-Ir6, [(\u03b75-CpX)Ir(N^N)Cl]PF6). Characterization was carried out\nanticancer drugs, which contributing to their strong binding with tran\u00ad using NMR and MS spectra. In the cyclopentadiene (\u03b75-CpX) ligand, the\nsition metals, p-delocalized molecular skeleton, pH dependence and hydrogen atoms of methyl were observed in the range of 1.5\u20132.0 ppm,\nstructural controllability for substituents (imidazole ring) to easily and those on the planar imidazo-phenanthroline/phenanthrene were in\nadjust their optical and bioactivity characteristics [21\u201325]. Therefore, the range of 7.5\u20139.5 ppm [32,33]. The results of MS were consistent with\nimidazo-phenanthroline/phenanthrene derivatives were extensively the theoretical calculations ([M-PF6]+ for Ir1-Ir6; [M-Cl]+ for Ir7-Ir12).\nused in the synthesis of various transition metallic anticancer com\u00ad Typically, the anticancer activity of half-sandwich IrIII complexes is\nplexes, including IrIII [26], RuII [27], ZnII [28], and RhI [29]. Among typically attributed to hydrolysis (the broken of Ir\u2013Cl bond), and Ir-H2O\nthem, the investigation of IrIII-based complexes has harvested significant exhibiting the higher activity in comparison to the corresponding ha\u00ad\nattention due to their good photophysical properties, exceptional lides [34]. Hydrolysis is a major step to exert activity for these com\u00ad\nchemical stability, and promising anticancer activity. Long-term lyso\u00ad plexes. The hydrolysis characteristics of these complexes were\nsome tracking was achieved via endocytic trafficking of a self- determined in a solution of DMSO 20%/H2O 80% (v/v) using ultraviolet-\nassembling IrIII imidazo-phenanthroline complex, as demonstrated by visible (UV\u2013Vis) spectroscopy over a duration of 8 h, Fig. S5. However,\nChao et al. [23] Guo et al. prepared two benzothiophenyliso-quinoline- these complexes exhibit the different properties from the classical half-\nderived IrIII complexes, which showed the first instance of ferroptosis sandwich IrIII complexes, which are relatively stable and do not undergo\ncaused by photosensitive IrIII complexes [30]. Chao et al. obtained a hydrolysis. Furthermore, the stability of Ir2 in solution was identified\nmitochondrial-targeting IrIII imidazo-phenanthroline prodrug, which through the 1H NMR spectra, Fig. S6. As shown, in both the pure DMSO\nwas responsive to the tumor microenvironment and able to release two- solvent and the mixed 80% DMSO\u2011d6/20% D2O (v/v) solution, Ir2 still\nphoton photosensitizers for PDT and a glutathione scavenger [22]. Of exhibits good stability after 24 h of incubation. This conclusion implies\ncourse, these reports were primarily on cyclometallic IrIII complexes, that these complexes possess a distinct anticancer mechanism in com\u00ad\nwith less emphasis on half-sandwich IrIII complexes. parison to the traditional IrIII complex, and this stability also provides a\n In this study, six imidazo-phenanthroline/phenanthrene pro-ligands convenient condition for the further investigation of biological\nwere prepared, then reacted with basic IrIII precursors and obtained properties.\ntwelve ionic/neutral half-sandwich IrIII imidazole complexes (Ir1-Ir12,\nChart 1) in this study. The potential of these complexes inhibiting cancer\ncell proliferation was initially evaluated in vitro using the MTT (3-(4,5-\nDimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Addi\u00ad\ntionally, the representative complex (Ir6) was selected as a model to\nevaluate anti-migration, intracellular imaging, anticancer mechanism,\nand anticancer channel of these complexes [31].\n\n\n\n\nChart 1. Structures of half-sandwich iridium(III) imidazole-phenanthroline/ Scheme 1. Design of imidazole proligands (L1-L6) and target complexes\nphenanthrene complexes. (Ir1-Ir12).\n\n 2\n\fA. Lv et al. Journal of Inorganic Biochemistry 257 (2024) 112612\n\n\n2.2. In-vitro proliferation and migration inhibition\n\n A549 cell line (lung epithelial carcinoma, the most common cancer\nof in the developed and developing regions) was incubated with these\ncomplexes for a duration of 24 h and estimated through MTT assay in\nthis study [35]. Cisplatin, commonly utilized in clinical settings, was\nemployed as a reference to distinguish their in-vitro anti-proliferative\nactivity, and the IC50 values (the concentration at which there is a\n50% inhibition of cell growth, a crucial parameter for assessing bioac\u00ad\ntivity) were listed in Table 1. These complexes exhibit promising anti-\nproliferative activity, especially for the \u03b75-Cpxph-based one, all of\nwhich possess superior activity to cisplatin under the same conditions.\nHowever, the substituents on imidazole ligands (F/H/OCH3, which\nrepresent the different absorption and electron donor properties) exhibit\nthe weaker effect on the activity, including variations in coordination\nmodes of imidazole ligands (N^N or C^N). Among these complexes, Ir6,\nL3-based complex exhibits the superior activity, which is even twice as\ngood as cisplatin. Even after 72 h of treatment, Ir6 continues to perform\nwell, including the A549/DDP (cisplatin-resistance) cells, Table S1.\nHowever, Ir6 shows the worse potential for other cancer cells, including\nHepG2, Hela, HT29 and HCT116 cell lines, Table S2. Cytotoxic effect on\nBEAS-2B cells (human normal bronchial epithelial cells) were also\ndetermined. The selectivity index (SI) for Ir6 is 3.00, calculated as the\nratio of the IC50 value of BEAS-2B cells to that of A549 cells, however,\nthat for cisplatin is 1.80. This result indicates that Ir6 has less cytotox\u00ad\nicity and better anti-proliferative activity than cisplatin under the same\nconditions, then possessing favorable anticancer potency.\n The bioactivity of organometallic complexes hinges upon a crucial\nindicator: Energy gap between the highest occupied molecular orbital\n(HOMO) and the lowest unoccupied molecular orbital (LUMO).\nOrganometallic complex generally shows a better reactivity when the\nHOMO and LUMO orbital energy range is smaller [36\u201338]. Then, the\norbital energy ranges of Ir5, Ir6, Ir11, and Ir12 were determined\nthrough a calculation through density functional theory (DFT) using the\nDFT/B3LYP-D3/6-31G(d, p)(C, H, N, Cl)/LANL2DZ (Ir) level in this Fig. 1. Electron cloud distribution of frontier orbitals (HOMO and LUMO) and\n the energy levels of Ir5, Ir6, Ir11 and Ir12.\nstudy [39]. As shown in Fig. 1, the HOMO orbital electron cloud is\nmainly distributed on the central iridium ion and imidazole ligand,\nhowever, that for LUMO is mainly on Ir ion and Cp ligand. \u03b75-Cpxph-\nbased complexes (Ir6/Ir12) show the lower orbital energy level differ\u00ad\nence than that of the corresponding \u03b75-Cp*-based one (Ir5/Ir11). This\nfinding aligns with the observed trend of in-vitro anti-proliferative ac\u00ad\ntivity. (Table 1).\n The process of cancer cell metastasis involves the release of cancer\ncells from the primary cancerous sites and their subsequent migration to\nremote tissues through the lymphatic or circulatory system, resulting in\nthe development of secondary tumor lesions [40]. Inhibiting tumor\nmetastasis is an essential component of advanced tumor treatment\n[41\u201343]. Therefore, wound healing assay was employed to preliminarily\n\nTable 1\nIC50 values and selectivity index of Ir1-Ir12 and cisplatin towards A549 and\nBEAS-2B cells after incubation of 24 h.\n Complexes IC50 (\u03bcM) SI\n (BEAS-2B/A549)\n A549 cell BEAS-2B cell\n\n Ir1 >100 >100 /\n Ir2 18.15 \u00b1 0.44 38.02 \u00b1 0.33 2.09\n Ir3 58.05 \u00b1 0.40 71.99 \u00b1 0.31 1.24\n Ir4 15.73 \u00b1 0.29 27.11 \u00b1 0.69 0.13\n Ir5 >100 97.16 \u00b1 0.42 /\n Ir6 11.49 \u00b1 0.27 34.52 \u00b1 0.18 3.00\n Ir7 22.93 \u00b1 0.34 19.19 \u00b1 0.23 0.84\n Ir8 15.15 \u00b1 0.62 26.03 \u00b1 0.58 1.71\n Ir9 66.37 \u00b1 0.16 70.22 \u00b1 0.36 1.05\n Ir10 15.54 \u00b1 0.31 24.33 \u00b1 0.24 1.57\n Ir11 97.41 \u00b1 0.14 >100 / Fig. 2. (a) Photographs of wound healing against A549 cells following the\n Ir12 19.34 \u00b1 0.52 28.02 \u00b1 0.32 1.45\n treatment of Ir6. Scale bar: 200 \u03bcM; (b) Bar chart of WCR values after the\n Cisplatin 21.32 \u00b1 1.71 38.43 \u00b1 0.62 1.80\n treatment of Ir6.\n\n 3\n\fA. Lv et al. Journal of Inorganic Biochemistry 257 (2024) 112612\n\n\nevaluate the anti-metastasis potential of these complexes towards A549 mitochondria, which indicating that lysosomes are the main target lo\u00ad\ncells in this study. As Fig. 2 demonstrated, R0 and R1 represent the cations in A549 cells. Interestingly, the presence of Ir6 does not induce\nbreadth of \u201cwounds\u201d (the areas free of A549 cells) after the treatment of A549 cell death immediately, which opens up the possibility of studying\nIr6 (specially selected complex, which demonstrating the best activity the changes of lysosome morphology timely. Once lysosome injury, the\namong these complexes) for 0 h and 24 h. The values of wound closure subsequent release of enzymes into the cytoplasm can trigger cell\nrate (WCR), calculated by WCR = (R0-R1)/R0 \u00d7 100%, exhibit a dose- apoptosis or necrosis [47\u201350]. Typically, lysosome integrity can be\ndependent reduction after the treatment of Ir6. The value of WCR is determined through acridine orange (AO, an acid fluorescent probe),\nonly half that of the control after the incubation of Ir6 at 0.75 \u00d7 IC50. which demonstrates red fluorescence emission within the lysosome and\nAbove all, including promising anti-proliferative properties, Ir6 also green fluorescence emission within the cytoplasm. Therefore, A549 cells\npossesses the anti-migratory capability. Therefore, Ir6 is selected as a were treated with Ir6 (1.0 \u00d7 IC50) over times, then stained with AO to\nrepresentative to investigate the underlying anticancer mechanism of assess the lysosomal integrity. As shown in Fig. 3c, in the control group,\nthese complexes. a significant amount of red fluorescence is observed, indicating the\n intact lysosomes. However, after the treatment of Ir6, the loss of red\n emission becomes readily discernible with the naked eyes, which indi\u00ad\n2.3. Cellular localization analysis\n cating the damage of lysosomal integrity and subsequent cell death of\n A549 cells.\n Drugs with good intracellular absorption routes are able to enter cells\neasily and show physiological effects. For organometallic complexes,\nenergy-dependent mode (e.g., active transport and endocytosis) and 2.4. Apoptosis assay\nnon-energy-dependent mode (e.g., passive transport and free diffusion)\nare two main cellular uptake mechanism [44,45]. Ir6 shows a strong Cell apoptosis is a highly regulated process in which cells undergo\nabsorption at 320 nm and a weak absorption observed at 410 nm programmed cell death to maintain tissue homeostasis, and this process\nattributing to the \u03c0-\u03c0* transition and the metal-ligand charge transfer is regulated and accompanied by distinct cell morphological alterations\ntransition, respectively, then accompanied by an emission at 486 nm [51]. Metallic complexes typically exert their anticancer effects through\n(Fig. S7). Therefore, A549 cells were pretreated with chloroquine the induction of apoptosis [52]. Then the effect of Ir6 on A549 cell\n(endocytosis modulator) or carbonyl cyanid-m-chlorophenylhydrazone apoptosis was detected by a dual probe of membranin V-FITC/ Propyl\n(CCCP, metabolism inhibitor) for 2 h, then subsequently incubated with iodide (PI) after the treatment for 24 h. As shown in Fig. 4a and\nIr6 at 310 K/277 K to investigate the uptake mode, Fig. 3a. As shown, Table S3, Ir6 can significantly promote A549 cell apoptosis in a\nthe smooth entry into A549 cells under all conditions confirms that Ir6 concentration-dependent mode. When Ir6 increasing from 1.0 \u00d7 IC50 to\nfollows a non-energy-dependent cellular uptake mode. 3.0 \u00d7 IC50, there is a 22.8% increase in the proportion of apoptotic cells\n As shown in Fig. 3b, using the laser confocal microscopy, the intra\u00ad (including early and late apoptosis), however, the rate of survival is\ncellular co-localization of Ir6 was detected based on its suitable emission 87.5% in the control group. Compared with the control, the early\nwithout any interference with MitoTracker Deep Red (MTDR) and apoptosis proportion showed a 46.45% increase, which confirming the\nLysoTracker Deep Red (LTDR), the conventional mitochondrial and anticancer activity of Ir6 by inducing early apoptosis.\nlysosomal probe, respectively [46]. Evidently, Ir6 could accumulate in The essential characteristic for tumor is that cell cycle regulation is\nthe intracellular lysosomes of A549 cells following a Pearson co- out of control and cell proliferation is infinite [53,54]. Therefore, it\nlocalization coefficient (PCC) of 0.70, however, that is only 0.20 for makes sense to develop anticancer drugs that concentrating on the in\u00ad\n hibition of cell cycle progression. Cell cycle, e.g., G1 phase (cells expe\u00ad\n rience growth and prepare for DNA replication), S phase (DNA synthesis\n occurs) and G2 phase (cells prepare for division), refers to one cell di\u00ad\n vision to the next, ultimately leading to the formation of daughter cells\n [55,56]. When the cell cycle is disrupted or inhibited, it can elicit\n various cellular responses, including apoptosis [51]. As shown in Fig. 4b\n and Table S4, the proportion of A549 cells is mainly concentrated in G1\n phase, which increases by 11.63% after the incubation of Ir6 from 0.5 \u00d7\n IC50 to 2.0 \u00d7 IC50, then showing a dose-dependent increase. This\n disruption of cell cycle is a potential mechanism through which Ir6\n exerting its effects on A549 cells.\n Reactive oxygen species (ROS) are generated as inherent byproducts\n during cellular metabolism, particularly through aerobic respiration in\n the mitochondria. If excessively elevated, they can induce oxidative\n stress and inflict damage on various cellular components, including\n membrane proteins. High levels of intracellular ROS can initiate a\n cascade of events, including disrupting cell signaling pathways and\n inducing apoptosis [57,58]. In this study, the intracellular ROS levels\n were assessed after the treatment of Ir6 for 24 h. To detect ROS levels, a\n fluorescent probe called DCFH-DA (2,7-dichlorodihydrofluorescein\n diacetate) was used, which would be oxidized by ROS and result is the\n formation of the fluorescent DCF (2,7-dichlorofluorescein), Fig. 5a. ROS\n levels of the experimental group are significantly improved compared to\n those in the control, although the increase is not significant with the\n increase of Ir6. A significant 2.62-fold increase is found for the intra\u00ad\nFig. 3. (a) Photomicrographs of A549 cells after treated with CCCP/chloro\u00ad cellular ROS level after the treatment of Ir6 at 2.0 \u00d7 IC50 in comparison\nquine and Ir6; (b) Photomicrographs of A549 cells after incubation of MTDR/ to the control.\nLTDR and Ir6. Ir6, MTDR and LTDR was excited at 405, 644 and 594 nm, then Mitochondria could convert cellular energy and serve as reservoirs\ncollected at 500 \u00b1 70, 690 \u00b1 30 and 630 \u00b1 30 nm, respectively; (c) Photo\u00ad for storing electrochemical potential energy within cell membranes. An\nmicrographs of A549 cells after the incubation of Ir6 and AO. Scale bar: 20 \u03bcm. uneven distribution of protons or other ions across the inner membrane\n\n 4\n\fA. Lv et al. Journal of Inorganic Biochemistry 257 (2024) 112612\n\n\n\n\nFig. 4. (a) Statistical analysis and histograms of apoptotic A549 cells after treated with Ir6 and dyed with Annexin VFITC/PI; (b) Cell cycle distribution ratio and\nhistograms of A549 cells after treated with Ir6 and dyed with PI/RNase. Data were quoted as mean \u00b1 SD of three replicates.\n\n\ncould lead to the decline of mitochondrial membrane potential (MMP), and Cyt-C upregulate. Based on these results, it can be inferred that Ir6\nthen accompanied by an improvement of intracellular ROS level [59]. can interact with LAMP-1 protein, leading to extensive lysosomal\nHence, to investigate the state of MMP, A549 cells were exposed to Ir6 membrane permeability, releasing cathepsin CB into the cytoplasm\nand subsequently labeled with JC-1 (an efficient fluorescent probe, originally presenting in lysosomes, inducing the change of MMP, then\nwhich emitting red or green fluorescence depending on its existence in finally releasing pro-apoptotic factor Cyt-C and leading to apoptosis. The\nmonomeric or aggregated forms, then corresponding to an increase or results obtained from the downregulation of LAMP-1 and upregulation\ndecrease of intracellular MMP, respectively), Fig. 5b and Table S5. As of CB and Cyt-C strongly support the hypothesis that Ir6 follows a\nshown, the proportion of mitochondrial membrane depolarized cells lysosomal-mitochondrial apoptotic pathway.\n(highlighted by green fluorescence) increases by 70.41% with the in\u00ad\ncrease of Ir6 (0.5 \u00d7 IC50 to 3.0 \u00d7 IC50), then showing a dose-dependent 3. Conclusion\nimprovement. Furthermore, NADH, the reduced form of nicotinamide\nadenine dinucleotide, is essential as a regulatory marker for the energy In this study, a total of twelve cationic/neutral half-sandwich IrIII\nproduction process within mitochondria. Assessing the redox state of imidazole complexes were synthesized and characterized. These com\u00ad\nNADH serves as a valuable parameter for characterizing the mitochon\u00ad plexes showed favorable stability in the biological test environment,\ndrial activity in vivo [60]. Typically, organometallic complexes could exhibiting distinct characteristics compared to the traditional half-\nusually accelerate the oxidation of NADH, then inducing the improve\u00ad sandwich IrIII complexes. Complexes had good anti-proliferative and\nment of intracellular ROS levels [61]. Therefore, NADH was hatched anti-migration ability towards A549 cancer cells, especially for \u03b75-Cpxph-\nwith Ir6 in a solution of 20% CH3OH and 80% H2O (v/v), then moni\u00ad based one. Ir6, L3-based complex, possessed the best activity among\ntored by UV\u2013Vis spectra to detect the time-dependent interaction be\u00ad these complexes, which was even twice as good as cisplatin against A549\ntween them. As shown in Fig. 5c, the absorbance at 259 nm (NAD+, the cells. Meanwhile, Ir6 showed the better activity towards A549/DDP cells\noxidation state of NADH after losing hydrogen) and 339 nm (NADH) and the less cytotoxicity, then being a potential substitute for cisplatin.\nincreases and decreases after the incubation. These changes are conve\u00ad Ir6 entered into A549 cells through a non-energy-dependent mode,\nniently detected through a turnover number (TON, 60.1), which further specifically targeting intracellular lysosomes and causing the disruption\nascertaining the favorable biocatalysis property of Ir6. Above all, of lysosomal integrity. Moreover, Ir6 exhibited the ability of acceler\u00ad\ndespite of primarily accumulating in lysosomes without significant ating NADH oxidation, leading to a reduction in MMP and improvement\nmitochondrial targeting, Ir6 can cause the lysosomal damage, resulting of intracellular ROS levels, disrupting the cell cycle at the G1 phase, and\nin the hydrolases entering the cytoplasm, then acting on mitochondria ultimately inducing apoptosis. Western blotting further confirmed Ir6\nand reducing MMP, increasing the intracellular ROS level, and pre\u00ad following a lysosomal-mitochondrial anticancer pathway and showing a\nsenting a lysosomal-mitochondria apoptotic channel. different anticancer mechanism from cisplatin. However, most of the\n To further understand the effect of lysosomes and mitochondria on objective complexes are highly toxic in comparison to cisplatin and\nIr6-induced apoptosis, the expression levels of lysosomal membrane show the worse activity towards cancer cells other than A549 cells.\nprotein (LAMP-1), cytochrome C oxidase (Cyt\u2013C), and cathepsin (CB) Additionally, the modification of peripheral ligands shows little effect\nwere determined using Western blotting, Fig. 6 [62,63]. As shown, the on the activity, which also requiring further adjustment to improve.\nexpression of LAMP-1 significantly downregulates, indicating a decrease Above all, half-sandwich IrIII imidazole complex may be a potential\nin lysosomal integrity. On the other hand, the expression levels of CB substitute for platinum-based drugs and warrant further study.\n\n\n 5\n\fA. Lv et al. Journal of Inorganic Biochemistry 257 (2024) 112612\n\n\n\n\nFig. 5. (a) Intracellular ROS levels after the treatment of Ir6 for 24 h; (b) The proportion of JC-1 aggregates and JC-1 monomers because of Ir6; (c) UV\u2013Vis spectra of\nNADH (100 \u03bcM) in 20% MeOH/80% H2O (v/v) inducing by Ir6 (1.0 \u03bcM) over 8 h.\n\n\n\n\nFig. 6. (a) Protein expression (LAMP-1, CB, and Cyt C) after the treatment of Ir6 at a gradient concentration. To ensure equal protein loading, \u03b2-Actin was used as a\nloading control; (b) Histograms depicted the alterations in protein expression across a range of concentrations.\n\n\n\n\n 6\n\fA. Lv et al. Journal of Inorganic Biochemistry 257 (2024) 112612\n\n\n4. Experimental section = 8.2 Hz, 1H), 8.91 (d, J = 5.1 Hz, 1H), 8.77 (d, J = 5.3 Hz, 1H),\n 7.93\u20137.89 (m, 1H), 7.76\u20137.72 (m, 1H), 7.66 (d, J = 7.9 Hz, 3H), 7.55 (d,\n4.1. General information J = 5.4 Hz, 4H), 7.45 (d, J = 7.7 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 6.85\n (d, J = 8.8 Hz, 3H), 3.81 (s, 3H, OCH3), 2.58 (s, 3H, CH3), 1.88 (s, 3H,\n All synthesized materials and organic solvents were purchased from Cp-CH3), 1.81 (d, J = 5.3 Hz, 6H, Cp-CH3), 1.75 (s, 3H, Cp-CH3). ESI-MS\nRhea Biotechnology Co. LTD and used without further treatment. The (m/z): Calcd for C42H37ON4ClF6PIr, 986.1927; Found 841.2221, [M-\nfundamental iridium precursors ([(\u03b75-Cp*)Ir(\u03bc-Cl)Cl]2, Dimer 1; [(\u03b75- PF6]+. Elem. anal. Calcd for C42H37N4F6POClIr: C, 51.14; H, 3.78; N,\nCpxph)Ir(\u03bc-Cl)Cl]2, Dimer 2) were prepared following the literature 5.68%; Found C, 51.32; H, 3.83; N, 5.55%.\nprocedures [20]. Imidazole-phenanthroline/ phenanthrene pro-ligands\n(L1-L6) were prepared according to the reported methods [64,65]. 4.3. Synthesis of half-sandwich IrIII imidazole complexes (Ir7-Ir12)\n\n4.2. Synthesis of half-sandwich IrIII imidazole complexes (Ir1-Ir6) Under a nitrogen atmosphere, a mixture of basic iridium precursors\n (Dimer1/2, 0.10 mmol), twice the proportion of the imidazole- phen\u00ad\n Under a nitrogen atmosphere, a mixture of basic iridium precursors anthrene ligands (L4-L6, 0.20 mmol) and sodium acetate (0.40 mmol)\n(Dimer1/2, 0.10 mmol) and twice the proportion of the imidazole- was dissolved in methanol (30 mL) and refluxed for 12 h. Then, the\nphenanthroline ligands (L1-L3, 0.20 mmol) was dissolved in methanol solvent was removed under vacuum, and 3 mL dichloromethane was\n(30 mL) and stirred at room temperature for 24 h. Then, ammonium added. After filtration, yellow products (Ir7-Ir12) were obtained by\nhexafluorophosphate (0.80 mmol) was added and reacted for another 6 adding n-hexane (30 mL) to facilitate diffusion. The NMR and MS\nh. The solvent was removed under vacuum, and 3 mL dichloromethane spectra were depicted in Figs. S3-S4 The data were as follows:\nwas added. After filtration, yellow products (Ir1-Ir6) were obtained by Ir7: Yield 85.7 mg (56.1%). 1H NMR (500 MHz, CDCl3) \u03b4 9.02 (d, J =\nadding n-hexane (30 mL) to facilitate diffusion. The NMR and MS 8.9 Hz, 1H), 8.74 (t, J = 9.1 Hz, 2H), 7.91 (t, J = 7.2 Hz, 1H), 7.74 (t, J =\nspectra were depicted in Figs. S3-S4 The data were as follows: 7.7 Hz, 1H), 7.60 (q, J = 7.2 Hz, 3H), 7.52 (d, J = 7.9 Hz, 1H), 7.47 (d, J\n Ir1: Yield 101.8 mg (55.8%). 1H NMR (500 MHz, DMSO) \u03b4 9.37 (dd, = 7.9 Hz, 1H), 7.41 (dd, J = 8.6, 2.5 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H),\nJ = 8.1, 7.0 Hz, 2H), 9.26 (d, J = 5.2 Hz, 1H), 8.31 (dd, J = 8.2, 5.3 Hz, 7.17 (d, J = 8.2 Hz, 1H), 6.52 (td, J = 8.6, 2.5 Hz, 1H), 6.32 (dd, J = 8.7,\n1H), 7.99 (dd, J = 8.6, 5.3 Hz, 1H), 7.73\u20137.66 (m, 3H), 7.64 (dd, J = 8.4, 5.3 Hz, 1H), 2.68 (s, 3H, CH3), 1.60 (s, 15H, Cp-CH3). ESI-MS (m/z):\n5.1 Hz, 2H), 7.57 (d, J = 8.3 Hz, 2H), 7.30 (t, J = 8.9 Hz, 2H), 2.52 (d, J Calcd for C38H33N2ClFIr, 764.1946; Found 729.2283, [M-Cl]+. Elem.\n= 6.4 Hz, 3H, CH3), 1.51 (s, 15H, Cp-CH3). ESI-MS (m/z): Calcd for anal. Calcd for C38H33N2ClFIr: C, 59.71; H, 4.35; N, 3.67%; Found C,\nC36H32N4ClF7PIr, 912.1571; Found 767.1982, [M-PF6]+. Elem. anal. 59.92; H, 4.46; N, 3.55%.\nCalcd for C36H32N4F7PClIr: C, 47.40; H, 3.54; N, 6.14%; Found C, 47.63; Ir8: Yield 91.2 mg (55.2%). 1H NMR (500 MHz, CDCl3) \u03b4 8.76 (d, J =\nH, 3.61; N, 6.02%. 7.9 Hz, 1H), 8.67 (d, J = 8.4 Hz, 1H), 8.61 (d, J = 8.2 Hz, 1H), 7.64 (t, J\n Ir2: Yield 105.8 mg (54.3%). 1H NMR (500 MHz, CDCl3) \u03b4 9.24 (d, J = 7.5 Hz, 1H), 7.58\u20137.53 (m, 1H), 7.49 (dd, J = 8.1, 5.7 Hz, 3H),\n= 8.2 Hz, 1H), 8.84 (d, J = 5.1 Hz, 1H), 8.70 (d, J = 5.3 Hz, 1H), 7.84 7.46\u20137.38 (m, 2H), 7.28 (q, J = 8.1 Hz, 5H), 7.19 (dd, J = 13.7, 6.6 Hz,\n(dd, J = 8.2, 5.4 Hz, 1H), 7.67 (dd, J = 8.6, 5.2 Hz, 1H), 7.61 (dd, J = 2H), 7.14 (d, J = 8.3 Hz, 1H), 6.90 (t, J = 8.4 Hz, 2H), 2.45 (s, 3H, CH3),\n7.3, 4.1 Hz, 3H), 7.55 (dd, J = 8.7, 5.3 Hz, 2H), 7.51\u20137.46 (m, 5H), 7.38 1.83 (s, 6H, Cp-CH3), 1.73 (m, J = 2.6 Hz, 6H, Cp-CH3). ESI-MS (m/z):\n(d, J = 7.6 Hz, 1H), 7.25 (d, J = 8.6 Hz, 1H), 6.97 (t, J = 8.6 Hz, 2H), Calcd for C43H35N2ClFIr, 826.2102; Found 791.2401, [M-Cl]+. Elem.\n2.51 (s, 3H, CH3), 1.82 (s, 3H, Cp-CH3), 1.76 (d, J = 6.4 Hz, 6H, Cp-CH3), anal. Calcd for C43H35N2ClFIr: C, 62.49; H, 4.27; N, 3.39%; Found C,\n1.66 (s, 3H, Cp-CH3). ESI-MS (m/z): Calcd for C41H34N4ClF7PIr, 62.64; H, 4.36; N, 3.22%.\n974.1727; Found 829.2066, [M-PF6]+. Elem. anal. Calcd for Ir9: Yield 81.8 mg (54.8%). 1H NMR (500 MHz, CDCl3) \u03b4 9.26 (d, J =\nC41H34N4F7PClIr: C, 50.54; H, 3.52; N, 5.75%; Found C, 50.62; H, 3.59; 8.0 Hz, 1H), 9.09 (d, J = 8.0 Hz, 1H), 8.01 (t, J = 7.5 Hz, 1H), 7.93 (t, J\nN, 7.66%. = 7.5 Hz, 1H), 7.53 (d, J = 7.7 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.33 (t,\n Ir3: Yield 91.6 mg (51.2%). 1H NMR (500 MHz, CDCl3) \u03b4 9.27 (d, J = J = 7.6 Hz, 2H), 7.18 (dd, J = 13.6, 5.9 Hz, 4H), 6.87\u20136.79 (m, 2H), 2.48\n8.0 Hz, 1H), 9.10 (d, J = 8.0 Hz, 1H), 8.02 (t, J = 7.5 Hz, 1H), 7.54 (d, J (s, 3H, CH3), 1.66 (s, 15H, Cp-CH3). ESI-MS (m/z): Calcd for\n= 7.7 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.20 C38H34N2ClIr, 746.2040; Found 711.2318, [M-Cl]+. Elem. anal. Calcd\n(dd, J = 13.6, 5.9 Hz, 4H), 6.85\u20136.80 (m, 2H), 2.48 (s, 3H, CH3), 1.60 (s, for C38H34N2ClIr: C, 61.15; H, 4.59; N, 3.75%; Found C, 61.44; H, 4.67;\n15H, Cp-CH3). ESI-MS (m/z): Calcd for C36H33N4ClF6PIr, 894.1665; N, 3.63%.\nFound 749.2047, [M-PF6]+. Elem. anal. Calcd for C36H33N4F6PClIr: C, Ir10: Yield 91.2 mg (56.4%). 1H NMR (500 MHz, CDCl3) \u03b4 9.05 (d, J\n48.35; H, 3.72; N, 6.26%; Found C, 48.66; H, 3.81; N, 6.18%. = 8.2 Hz, 1H), 8.73 (d, J = 8.2 Hz, 1H), 8.71\u20138.68 (q, J = 15.3, 8.4 Hz,\n Ir4: Yield 102.5 mg (53.6%). 1H NMR (500 MHz, DMSO) \u03b4 9.36 (d, J 2H),8.16\u20138.07 (dd, J = 15.3, 8.4 Hz, 3H), 7.91 (t, J = 7.2 Hz, 1H), 7.74\n= 8.2 Hz, 1H), 9.08 (d, J = 6.2 Hz, 1H), 8.97 (d, J = 5.2 Hz, 1H), 8.25 (t, J = 7.7 Hz, 1H), 7.60 (q, J = 7.2 Hz, 3H), 7.52 (d, J = 7.9 Hz, 1H),\n(dd, J = 8.2, 5.3 Hz, 1H), 7.94 (dd, J = 8.6, 5.3 Hz, 1H), 7.73 (dd, J = 7.47 (d, J = 7.9 Hz, 1H), 7.41 (dd, J = 8.6, 2.5 Hz, 1H), 7.33 (t, J = 7.7\n8.1, 2.3 Hz, 1H), 7.63 (dd, J = 11.0, 5.5 Hz, 5H), 7.54 (dd, J = 14.2, 7.7 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 6.84\u20136.80 (m, 3H), 6.36 (d, J = 7.8 Hz,\nHz, 5H), 7.50 (d, J = 6.3 Hz, 2H), 2.50 (d, J = 2.7 Hz, 3H, CH3), 1.83 (s, 1H), 2.68 (s, 3H, CH3), 1.82 (s, 3H, Cp-CH3), 1.77\u20131.75 (m, 6H, Cp-CH3),\n6H, Cp-CH3), 1.73 (m, J = 2.6 Hz, 6H, Cp-CH3). ESI-MS (m/z): Calcd for 1.66 (s, 3H, Cp-CH3). ESI-MS (m/z): Calcd for C43H36N2ClIr, 808.2196;\nC41H35N4ClF6PIr, 956.1821; Found 811.2179, [M-PF6]+. Elem. anal. Found 773.2501, [M-Cl]+. Elem. anal. Calcd for C43H36N2ClIr: C, 63.88;\nCalcd for C41H35N4F6PClIr: C, 51.49; H, 3.69; N, 5.86%; Found C, 51.75; H, 4.49; N, 3.47%; Found C, 64.11; H, 4.61; N, 3.38%.\nH, 3.77; N, 5.75%. Ir11: Yield 78.4 mg (54.9%). 1H NMR (500 MHz, CDCl3) \u03b4 9.33 (d, J\n Ir5: Yield 99.6 mg (53.9%). 1H NMR (500 MHz, CDCl3) \u03b4 9.32 (d, J = = 8.2 Hz, 1H), 9.08 (dd, J = 10.8, 5.2 Hz, 2H), 8.08 (dd, J = 8.2, 5.3 Hz,\n8.2 Hz, 1H), 9.10 (d, J = 5.3 Hz, 1H), 9.07 (d, J = 5.1 Hz, 1H), 8.09 (dd, 1H), 7.85 (dd, J = 8.5, 5.1 Hz, 2H), 7.66 (d, J = 8.5 Hz, 1H), 7.55 (dd, J\nJ = 8.2, 5.3 Hz, 1H), 7.85 (dd, J = 8.6, 5.3 Hz, 1H), 7.65 (d, J = 8.6 Hz, = 16.6, 6.6 Hz, 4H), 7.45 (d, J = 8.2 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H),\n1H), 7.57 (d, J = 8.7 Hz, 2H), 7.52 (dd, J = 8.1, 6.1 Hz, 2H), 7.46 (d, J = 6.87 (d, J = 8.8 Hz, 2H), 3.82 (s, 3H, OCH3), 2.59 (s, 3H, CH3), 1.76 (s,\n8.1 Hz, 1H), 7.31 (d, J = 9.9 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 3.82 (s, 15H, Cp-CH3). ESI-MS (m/z): Calcd for C39H36ON2ClIr, 714.2446;\n3H, OCH3), 2.59 (s, 3H, CH3), 1.75 (s, 15H, Cp-CH3). ESI-MS (m/z): Found 776.2122, [M-Cl]+. Elem. anal. Calcd for C39H36ON2ClIr: C,\nCalcd for C37H35N4OF6PClIr, 924.1770; Found 779.2182, [M-PF6]+. 60.33; H, 4.67; N, 3.61%; Found C, 60.56; H, 4.78; N, 3.50%.\nElem. anal. Calcd for C37H35ON4ClF6PIr: C, 48.08; H, 3.82; N, 6.06%; Ir12: Yield 93.4 mg (55.7%). 1H NMR (500 MHz, CDCl3) \u03b4 9.30 (d, J\nFound C, 48.31; H, 3.91; N, 5.96%. = 8.2 Hz, 1H), 8.91 (d, J = 5.1 Hz, 1H), 8.77 (d, J = 5.3 Hz, 1H),\n Ir6: Yield 103.0 mg (52.2%). 1H NMR (500 MHz, CDCl3) \u03b4 9.30 (d, J 7.93\u20137.89 (m, 1H), 7.75\u20137.72 (m, 1H), 7.66 (d, J = 7.9 Hz, 3H),\n\n 7\n\fA. Lv et al. Journal of Inorganic Biochemistry 257 (2024) 112612\n\n\n7.58\u20137.54 (m, 5H), 7.45 (d, J = 7.7 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), activity of some half-sandwich cyclometalated Rh (III) and Ir (III) complexes,\n Organometallics 34 (2015) 4491\u20134506.\n6.85 (d, J = 8.8 Hz, 3H), 3.81 (s, 3H, OCH3), 2.58 (s, 3H, CH3), 1.83 (s,\n [12] J. Ruiz, C. Vicente, C. Haro, D. Bautista, Novel bis-C, N-cyclometalated iridium (III)\n6H, Cp-CH3), 1.73 (m, 6H, Cp-CH3). ESI-MS (m/z): Calcd for thiosemicarbazide antitumor complexes: interactions with human serum albumin\nC44H38ON2ClIr, 838.2302; Found 803.2613, [M-Cl]+. Elem. anal. Calcd and DNA, and inhibition of cathepsin B, Inorg. Chem. 52 (2013) 974\u2013982.\nfor C44H38ON2ClIr: C, 63.03; H, 4.57; N, 3.34%; Found C, 53.1; H, 3.55; [13] Z. Liu, A. Habtemariam, A.M. Pizarro, S.A. Fletcher, A. Kisova, O. Vrana, L. Salassa,\n P.C.A. Bruijnincx, G.J. Clarkson, V. Brabec, P.J. Sadler, Organometallic half-\nN, 3.37%. sandwich iridium anticancer complexes, J. Med. Chem. 54 (2011) 3011\u20133026.\n [14] K. Lo, K. Zhang, Iridium(III) complexes as therapeutic and bioimaging reagents for\nCRediT authorship contribution statement cellular applications, RSC Adv. 2 (2012) 12069\u201312083.\n [15] S.J. Lucas, R.M. Lord, R.L. Wilson, R.M. Phillips, V. Sridharan, P.C. 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