Synthesis and mitochondria-localized iridium (III) complexes induce cell death through pyroptosis and ferroptosis pathways.

{"full_text": " European Journal of Medicinal Chemistry 268 (2024) 116295\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\nSynthesis and mitochondria-localized iridium (III) complexes induce cell\ndeath through pyroptosis and ferroptosis pathways\nHuiyan Hu , Fan Zhang , Zhujun Sheng , Shuang Tian , Gechang Li , Shuanghui Tang , Yajie Niu ,\nJiawan Yang , Yunjun Liu *\nSchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, 510006, PR China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: This paper introduces a new ligand, 4,6-dichloro-5-(1H-imidazo [4,5-f]phenanthroline-2-yl)pyrimidin-2-amine\nIridium(III) complexes (DPPA), and its corresponding new iridium(III) complexes: [Ir(ppy)2(DPPA)](PF6) (2a) (where ppy represents\nFerroptosis deprotonated 2-phenylpyridine), [Ir(bzq)2(DPPA)](PF6) (2b) (with bzq indicating deprotonated benzo[h]quin\u00ad\nRNA-Sequence\n oline), and [Ir(piq)2(DPPA)](PF6) (2c) (piq denoting deprotonated 1-phenylisoquinoline). The cytotoxic effects of\nPyroptosis\nAntitumor in vivo\n both DPPA and 2a, 2b, and 2c were evaluated against human lung carcinoma A549, melanoma B16, colorectal\n cancer HCT116, human hepatocellular carcinoma HepG2 cancer cell lines, as well as the non-cancerous LO2 cell\n line using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. While DPPA\n exhibited moderate anticancer activity toward A549, B16, HCT116 and HepG2 cells, complexes 2a, 2b, and 2c\n displayed remarkable efficacy against A549, B16, and HCT116 cells. The cell colonies and wound healing were\n investigated. Moreover, various aspects of the anticancer mechanisms were explored. The cell cycle analyses\n revealed that the complexes block cell proliferation of A549 cells during the S phase. Complex 2c induce an early\n apoptosis, while 2a and 2b cause a late apoptosis. The interaction of 2a, 2b and 2c with endoplasmic reticulum\n and mitochondria was identified, leading to elevated ROS and Ca2+ amounts. This resulted in a reduced mito\u00ad\n chondrial membrane potential, mitochondrial permeability transition pore opening, and an increase of cyto\u00ad\n chrome c. Also, ferroptosis was investigated through measurements of intracellular glutathione (GSH),\n malondialdehyde (MDA), and recombinant glutathione peroxidase (GPX4) protein expression. The pyroptosis\n was explored via cell morphology, release of lactate dehydrogenase (LDH) and expression of pyroptosis-related\n proteins. RNA sequencing was applied to examine the signaling pathways. Western blot analyses illuminated that\n the complexes regulate the expression of Bcl-2 family proteins. Additionally, an in vivo antitumor study\n demonstrated that complex 2c exhibited a remarkable inhibitory rate of 58.58% in restraining tumor growth. In\n summary, the findings collectively suggest that the iridium(III) complexes induce cell death via ferroptosis,\n apoptosis by a ROS-mediated mitochondrial dysfunction pathway and GSDMD-mediated pyroptosis.\n\n\n\n\n1. Introduction fluorescence lifetime, and minimal cytotoxicity, have emerged as\n promising candidates in areas like bioimaging, cellular fluorescent\n Carcinogenesis is believed to arise from DNA alterations within cells, probes, and agents for visualizing biological targets [5\u20138]. Certain\ncoupled with the inhibition of growth suppressors. These events lead to iridium (III) complexes exhibit targeted therapeutic properties, specif\u00ad\nuncontrollable cell growth, migration to a remote organization, and ically through their ability to accumulate within tumor cells and target\nultimately result in an aggressive metastasis [1\u20133]. While cisplatin has mitochondria. This localization triggers an overload of reactive oxygen\ndemonstrated successful anticancer applications, the clinical utility of species and causes changes of mitochondrial membrane potential, ulti\u00ad\ncisplatin is curtailed by side-effects [4], which stimulates scientists to mately culminating in mitochondrial dysfunction and consequent cell\nfind other metallic drugs to replace cisplatin. Iridium (III) complexes, death [9\u201311]. Recent years have witnessed substantial attention\nrenowned for their favorable attributes such as photostability, high directed towards the potential of iridium (III) complexes, revealing a\nquantum yield, effective cell membrane permeability, extended repertoire of intriguing anticancer activities [12\u201323]. For instance,\n\n\n * Corresponding author.\n E-mail address: lyjche@gdpu.edu.cn (Y. Liu).\n\nhttps://doi.org/10.1016/j.ejmech.2024.116295\nReceived 16 January 2024; Received in revised form 26 February 2024; Accepted 28 February 2024\nAvailable online 29 February 2024\n0223-5234/\u00a9 2024 Elsevier Masson SAS. All rights reserved.\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\nZhang et al. demonstrated that [Ir(PPY)2(DCDPPZ)](PF6) interacts with ppm) at the imidazole ring was discovered, whereas in the spectra of 2a,\nDNA through minor-groove intercalation, thereby triggering apoptosis 2b and 2c, this signal was not observed. Owing to coordination of ligand\nin SGC-7901 cells via dysfunction of mitochondria [10]. Similarly, [Ir with metal atom, induction of electron deficiency resulted in a very\n(PPY)2(TFPIP)](PF6), when encapsulated in liposomes, triggers both active of NH proton in the imidazole ring. In PBS solution, the UV\u2013Vis\nROS-mediated apoptosis and GSDME-mediated pyroptosis [24]. and luminescence spectra of 2a, 2b and 2c was determined. As illus\u00ad\nNotably, Sobczak-The\u0301pot reported that a half-sandwich iridium (III) trated in Fig. S1a (SI), 2a, 2b, 2c (20.0 \u03bcM) exhibit a maximum absor\u00ad\ncomplex, IrBDP, efficiently interacts on the mitochondria and the bance at 266 (\u03b5 = 29350), 263 (\u03b5 = 28600) and 293 nm (\u03b5 = 31950). The\nendoplasmic reticulum, leading to cell death [25]. In our previous work, luminescence of 2a (\u03bbex = 310 nm), 2b (\u03bbex = 305 nm) and 2c (\u03bbex = 300\nwe found the iridium (III) complexes or liposomes entrapped iridium nm) is depicted in Fig. S1b (SI), the maximum for 2a, 2b, 2c appears at\n(III) complexes coordinated by the polypyridyl ligands containing one or 629, 619 and 609 nm, respectively.\ntwo chloride atoms or \u2013NH2 show high anticancer efficiency against In PBS solution, the stability of 2a, 2b and 2c was examined by\nA549 cells [13,26,27]. In light of pursuit of enhanced insight into UV\u2013Vis spectra, See from Fig. S2 (SI), at 0 and 48 h, no change in the\nanticancer activities and mechanisms, this study introduces a novel peak shape was observed, which indicated that the complexes are stable\npolypyridyl ligand simultaneously containing two chloride atoms and in PBS solution in all the cell experiments.\none \u2013NH2: 4,6-dichloro-5-(1H-imidazo [4,5-f]phenanthroline-2-yl) CH3OH and H2O as a mobile phase, the purity of 2a, 2b and 2c was\npyrimidin-2-amine (DPPA). Subsequently, complexes were synthe\u00ad measured by HPLC. As depicted in Fig. S3 (SI), the chromatograms\nsized: [Ir(ppy)2(DPPA)](PF6) (2a) (where ppy denotes deprotonated exhibited a dominant peak during the 30-min analysis period, with\n2-phenylpyridine), [Ir(bzq)2(DPPA)](PF6) (2b) (with bzq representing purities of 95.05% for 2a, 96.02% for 2b and 95.67% for 2c.\ndeprotonated benzo[h]quinoline), and [Ir(piq)2(DPPA)](PF6) (2c) (piq In all the cell experiments, complexes 2a, 2b and 2c were dissolved in\nindicating deprotonated 1-phenylisoquinoline) (Scheme 1). Detailed DMSO (final DMSO concentration <0.05%), the complexes were diluted\ninvestigations for 2a, 2b and 2c were conducted to evaluate the anti\u00ad to obtain a series of different concentration of the complexes. During the\ncancer effects in vitro and in vivo. process of dilution, no precipitate was found.\n\n2. Results and discussion\n Table 1\n Selected bond lengths [\u00c5] and bond angles [\u25e6 ].\n2.1. Syntheses and characterization\n Ir\u2013N1 2.070(5) Ir\u2013N2 2.069(5)\n The ligand 4,6-dichloro-5-(1H-imidazo[4,5-f]phenanthroline-2-yl) Ir\u2013N3 2.151(5) Ir\u2013N4 2.153(4)\npyrimidin-2-amine (DPPA) was prepared through refluxing Ir\u2013C15 2.025(5) Ir\u2013C30 2.028(5)\n N1\u2013Ir\u2013N3 88.05(19) N1\u2013Ir\u2013N4 99.14(19)\nphenanthroline-5,6-diamine with 2-amino-4,6-dichloropyrimidine-5-\n N2\u2013Ir\u2013N1 171.36(19) N2\u2013Ir\u2013N3 98.57(19)\ncaraldehyde in ethanol. 2a, 2b, 2c were synthesized by the reaction of N2\u2013Ir\u2013N4 87.79(18) N3\u2013Ir\u2013N4 77.26(18)\n[Ir(ppy)2Cl]2\u22c5H2O, [Ir(bzq)2Cl]2\u22c5H2O or [Ir(piq)2Cl]2\u22c5H2O with DPPA in C15\u2013Ir\u2013N1 79.8(2) C15\u2013Ir\u2013N2 94.1(2)\nCH2Cl2 and CH3OH. The complexes were characterized by 1H NMR, 13C C15\u2013Ir\u2013N3 94.4(2) C15\u2013Ir\u2013N4 171.6(2)\nNMR and HRMS spectra. In the HRMS spectra of the complexes, the C15\u2013Ir\u2013C30 91.4(2) C30\u2013Ir\u2013N1 94.8(2)\n C30\u2013Ir\u2013N2 79.1(2) C30\u2013Ir\u2013N3 174.00(19)\ndetermined molecular weights are line with those of calculated values.\n C30\u2013Ir\u2013N4 97.0(2)\nIn the 1HNMR spectra of ligand DPPA, the single signal of proton (14.1\n\n\n\n\n Scheme 1. Synthetic route of DAPP and its complexes 2a, 2b, 2c.\n\n 2\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\n The selected bond lengths and angles are listed in Table 1, the ORTEP Table 2\ndiagram for 2c is depicted in Fig. 1, in the crystal structure, no anion PF\u22126 IC50 values (\u03bcM) of DPPA and 2a, 2b, 2c towards the selected cancer cells for 48\nwas uncovered due to exit of H atom from the imidazole ring, similar h.\nresults can be discovered in our previous work [28]. While in the 19F Complexes A549 B16 HCT116 HepG2 LO2 SI\nNMR spectra of 2c, six fluorine atoms were observed, which further DPPA 17.8 \u00b1 17.6 \u00b1 18.8 \u00b1 26.5 \u00b1 43.7 \u00b1 2.5\nvalidates that the leaving of H atom in the imidazole ring in the crystal 1.3 1.2 1.8 4.3 4.4\nstructure of 2c without anion. The Ir atom, a distorted octahedral ge\u00ad 2a 5.6 \u00b1 8.4 \u00b1 10.9 \u00b1 15.0 \u00b1 54.8 \u00b1 9.8\nometry, is coordinated by ligand DPPA and two ancillary ligands piq. 0.1 0.8 0.2 1.5 1.9\n 2b 3.9 \u00b1 4.4 \u00b1 3.7 \u00b1 0.1 11.4 \u00b1 32.4 \u00b1 8.3\nThe mean bond lengths of Ir\u2013N and Ir\u2013C are 2.111 and 2.027 \u00c5.\n 0.3 1.1 0.7 1.4\n 2c 2.2 \u00b1 2.5 \u00b1 2.5 \u00b1 0.1 3.1 \u00b1 0.9 35.0 \u00b1 15.9\n2.2. IC50 values determination 0.1 0.7 0.5\n Cisplatin 6.1 \u00b1 28.8 \u00b1 15.3 \u00b1 8.8 \u00b1 1.2 18.4 \u00b1 3.0\n The assessment of cellular cytotoxicity was undertaken utilizing the 0.4 3.3 1.2 3.2\n\nMTT assay (MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte\u00ad SI Selectivity index of DPPA, 2a, 2b and 2c of IC50 values of LO2 cell versus A549\ntrazolium bromide) [29] to investigate the impact of DPPA and 2a, 2b, cell.\nand 2c on cell proliferation. Selected cancer cell lines were exposed to\nvarying concentrations (1.56\u2013100 \u03bcM) of ligand DPPA, as well as 2a, 2b, 2.3. Cell colonies and wound healing studies\nand 2c for a duration of 48 h, with the resulting IC50 values summarized\nin Table 2. Notably, 2a, 2b, and 2c exhibited potent cytotoxic activity Malignant tumor cells grow rapidly and infiltratively, invading sur\u00ad\nagainst A549, B16, and HCT116 cells. Furthermore, the complexes rounding tissues and forming indistinct borders. Hence, it is urgent to\ndemonstrated a notably heightened cytotoxic effect compared to the find new drugs to inhibit cancer cell proliferation and migration. To\nligand DPPA on the selected cancer cell lines under identical conditions, explore the impact of 2a, 2b and 2c on cell migration and growth, we\nsuggesting a substantial enhancement of cytotoxic activity upon DPPA carried out the cell colonies and wound healing experiments. As\nto form complexes 2a, 2b and 2c. Also, Table 2 illustrates that the depicted in Fig. S4 (SI), A549 cells were subjected to treatment with 2a,\ncytotoxicity of the complexes on A549, B16, HCT116, HepG2 follow the 2b, and 2c for 24 h, resulting in a discernible reduction of viable cells.\nsequence of 2c > 2b > 2a. This can be explained by the lipid water Fig. S5 (SI) further demonstrated an evident increase in wound width\npartition coefficient. The lipid water partition coefficient of 2a, 2b and relative to the control, indicative of the complexes\u2019 pronounced efficacy\n2c were measured to be 0.2585, 0.2721 and 0.3609, respectively. in hindering both cell colony formation and migration.\nConsequently, the cytotoxic activity of 2a, 2b and 2c is line with their\nlipid water partition coefficient. The cytotoxic potential of the com\u00ad\n 2.4. Cellular uptake, co-location, mitochondrial permeability, and the\nplexes towards A549, B16, and HCT116 cells was found to surpass that\n change of mitochondrial membrane potential\nof cisplatin, and their cytotoxic activity is higher than iridium(III)\ncomplex Ir(ppy)2(BAPIP[Ir(ppy)2(BAPIP)](PF6) (BAPIP = 5-bro\u00ad\n A fundamental prerequisite for a drug to exert an anti-cancer effect is\nmo-2-amino-2\u2019-(phenyl-1H-imidazo[4,5-f][1,10]phenanthroline and\n its ability to traverse the cellular membrane. Consequently, we initiated\ncomparable with liposome-encapsulated iridium (III) complex (IC50 =\n our investigation by probing the cellular uptake. As demonstrated in\n4.9 \u00b1 1.0 \u03bcM for A549, 5.7 \u00b1 0.3 \u03bcM for B16) against A549 and B16\n Fig. S6a (SI), following co-incubation of A549 cells with 2a, 2b, and 2c\n[30]. The selectivity indexes of 2a, 2b and 2c of IC50 values of LO2 cell\n for 12 h, Hoechst 33342 stained cell nuclei blue, while 2a, 2b and 2c\nversus A549 cell are 9.8, 8.3 and 15.9, respectively. The high SI values\n exhibited green fluorescence. Merging the images revealed an envel\u00ad\nindicated that 2a, 2b and 2c have choice on the cancer and non-cancer\n opment of the blue nuclei by the green fluorescence, strongly indicating\ncells. Given that 2a, 2b, and 2c exhibited the highest cytotoxic efficacy\n that 2a, 2b and 2c effectively penetrated the cell and accumulated\non A549 cells, this cell line was chosen for subsequent anticancer ac\u00ad\n within the cytoplasm. Given the significance of the endoplasmic retic\u00ad\ntivity studies.\n ulum as a hub for cellular synthesis beyond nucleic acids, encompassing\n proteins, lipids, and sugars, and the pivotal role of mitochondria in\n furnishing energy for various cellular activities, we proceeded to\n investigate whether 2a, 2b and 2c localized within the endoplasmic\n reticulum or mitochondria.\n We have quantitatively examined the cellular uptake, the distribu\u00ad\n tion of the complexes in the mitochondria, cell nuclei and cytoplasm\n using inductively coupled plasma-mass spectrometry (ICP-MS). As listed\n in Table 3, A549 cells were incubated with 10.0 \u03bcM of 2a, 2b and 2c for\n 4 h, all the uptaken amounts of the complexes 2a, 2b and 2c are 73.47 \u00b1\n 2.24, 138.45 \u00b1 4.23 and 99.82 \u00b1 3.53 ng/106 cells, while the uptaken\n amounts distributed in the mitochondria are 55.60 \u00b1 2.03 for 2a, 74.01\n \u00b1 2.74 for 2b and 75.81 \u00b1 3.12 ng/106 cells for 2c, hence, most com\u00ad\n plexes uptaken by the cells distribute in the mitochondria and cyto\u00ad\n plasm, few amounts of the complexes enter the cell nuclei. The cellular\n uptaken amounts follow the order 2b > 2c > 2a, this is not line with\n\n Table 3\n The uptaken amount and the distribution of the complexes in the cells (ng/106\n cells).\n complexes uptaken amount mitochondria nuclei cytoplasm\n\n 2a 73.47 \u00b1 2.24 55.60 \u00b1 2.03 1.07 \u00b1 0.03 6.79 \u00b1 0.24\n 2b 138.45 \u00b1 4.23 74.01 \u00b1 2.74 1.33 \u00b1 0.04 36.46 \u00b1 1.35\nFig. 1. X-ray crystal structure of 2c set at a 50% probability. The H atoms, two\n 2c 99.82 \u00b1 3.53 75.81 \u00b1 3.12 1.32 \u00b1 0.02 21.48 \u00b1 1.22\nwater and two methanol molecules are omitted for clarity.\n\n 3\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\nthose of cytotoxicity of 2c > 2b > 2a, consequently, we consider that the apoptotic pathway [31]. In our examination of intracellular calcium\ncytotoxic activity is not only closely related to the cellular uptaken concentration, we employed Fluo-3 AM as a fluorescence probe. Fluo-3\namount, but also the nature of the complexes. AM is a widely used indicator for Ca2+ levels, upon entering the cells, it\n As depicted in Figs. S6b and S6c (SI), treatment of A549 cells with 2a, undergoes cleavage by endogenous esterase to generate Fluo-3, which\n2b and 2c for 4 h exhibited red staining with ER Tracker Red or Mito\u00ad readily binds with Ca2+ to emit green fluorescence. As illustrated in\nTracker Red, respectively. The co-localization of the red and green Fig. S8a (SI), co-incubation of A549 cells with 2a, 2b, and 2c for 24 h led\nfluorescence signified that 2a, 2b and 2c indeed localized within endo\u00ad to a noticeable increase in green fluorescence, indicating an augmen\u00ad\nplasmic reticulum and mitochondria. This interaction with the mito\u00ad tation in intracellular Ca2+ levels. Quantitative analysis (Figs. S8b and\nchondria, in turn, elicits the opening of the mitochondrial membrane SI) further corroborated this finding, demonstrating a 1.99-fold increase\npermeability transition pore. To further discern the influence of 2a, 2b for 2a, 1.98-fold increase for 2b, and 2.08-fold increase for 2c in com\u00ad\nand 2c on this transition pore, A549 cells were subjected to incubation parison to the control. Collectively, our results strongly suggest that the\nwith lonomycin (positive control) and 2a, 2b and 2c for 24 h. As revealed complexes have the capacity to elevate intracellular Ca2+ content.\nin Fig. S6d (SI), the green fluorescence was attenuated in comparison to\nthe control or the negative group, suggesting that the complexes prompt 2.7. Release of cytochrome C\nthe opening of the mitochondrial permeability transition pore, culmi\u00ad\nnating in mitochondrial dysfunction and a subsequent reduction in Cytochrome C (cyto C) serves as an essential electron transporter\nmitochondrial membrane potential. This was corroborated by the find\u00ad within the mitochondrial respiratory chain. Upon the initiation of\nings in Fig. S6e (SI), where, under normal conditions, JC-1 emitted vivid apoptosis, specifically through the intrinsic pathway, alterations in\nred fluorescence, but upon treatment with cccp (a positive control) or mitochondrial membrane permeability result in the release of cyto C\n2a, 2b, and 2c for 24 h, the emission shifted to green fluorescence, into the cytoplasm [32,33]. This translocation of cytochrome C is a\nindicative of decreased mitochondrial membrane potential. To account pivotal event in activating the mitochondrial signaling pathway during\nfor potential interference of the green fluorescence emitted by 2a, 2b apoptosis. Illustrated in Fig. S9 (SI), the control cells displayed a weak\nand 2c, we used 2a, 2b and 2c as references to eliminating the impact of green fluorescence signal, suggesting minimal cytochrome C release.\n2a, 2b and 2c on the green fluorescence intensity. Illustrated in Fig. S6f However, when A549 cells were subjected to 2a, 2b, and 2c for 24 h, a\n(SI), substantiated that the treatment of A549 cells with cccp or 2a, 2b, conspicuous increase in green fluorescence was observed. This height\u00ad\nand 2c caused a reduction in the ratio of red to green fluorescence ened fluorescence is indicative of a notable release of cyto C from the\nrelative to the control. Collectively, our observations affirm the com\u00ad mitochondria into the cytoplasm, substantiating the activation of the\nplexes\u2019 cellular entry and cytoplasmic accumulation, their interaction intrinsic apoptotic pathway induced by the complexes.\nwith the endoplasmic reticulum and mitochondria, resulting in\ncompromised mitochondrial function, the open of the mitochondrial 2.8. Impact of the complexes on RNA-sequence\npermeability transition pore, and ultimately, a decline in mitochondrial\nmembrane potential. RNA was extracted after A549 cells were incubated with 2c for 24 h.\n Total RNA was analyzed by RNA-Sequence to further explore the\n2.5. Intracellular ROS determination cellular signaling pathway by which 2c hindering the cell proliferation.\n As shown in Fig. 2a (heat map) and 2b (volcano map), we found that 2c\n Reactive oxygen species (ROS) and various apoptotic inducers are regulated 151 genes expression, in which 61 and 90 genes were raised\nknown to initiate diverse forms of apoptosis, often accompanied by and downgraded, respectively. As shown in Fig. 2c, the KEGG enrich\u00ad\ndisruptions in mitochondrial function such as depolarisation and altered ment shows that 2c causes cell death through several signaling pathways\nmembrane potential. Given that the complexes induce an open of the including calcium ion and ferroptosis. The GO enrichment analysis\nmitochondrial permeability transition pore along with a reduction in found that biological process, cellular process, and molecular function\nmitochondrial membrane potential, it is reasonable to expect conse\u00ad were enriched (Fig. 2d).\nquential changes in the concentration of reactive oxygen species. To\ngauge intracellular ROS levels, we employed the 2\u2032,7\u2032-dichlorodihydro- 2.9. Cell cycle distribution, apoptosis, and expression of proteins\nfluorescein diacetate (DCFH-DA) as non-fluorescent cell-permeant\nprobe. Upon internalization, hydrolysis, and oxidation by a variety of The observed impact of the complexes on cell cytotoxicity, colony\nROS, DCHF-DA is transformed into 2\u2032,7\u2032-dichlorofluorescein (DCF), formation, and wound healing collectively validates their efficacy in\nemitting characteristic green fluorescence. As illustrated in Fig. S7a (SI), restraining cell proliferation and migration. To unravel the potential\nminimal green fluorescence was discovered in the control group, mechanism responsible for the inhibition of cell growth, a meticulous\nwhereas upon incubation of A549 cells with Rosup (as a positive con\u00ad examination of cell cycle distribution was conducted. The results in\ntrol), 2a, 2b, and 2c for 24 h, an enhancement of green fluorescence Fig. 3a elucidate a noteworthy increase of 4.5% for 2a (II), 8.1% for 2b\nintensity was uncovered. This starkly indicates that 2a, 2b and 2c (III), and 8.7% for 2c (IV) in the S phase, accompanied by corresponding\nsignificantly elevate intracellular ROS amounts. To preclude the po\u00ad reductions in the G0/G1 and G2/M phases when contrasted with the\ntential influence of the complexes\u2019 green fluorescence emission, we used control (I). This signifies that the complexes exert their inhibitory effect\ncomplexes as references to quantitatively measure the green fluores\u00ad primarily during S phase.\ncence intensity. As depicted in Fig. S7b (SI), the green fluorescence in\u00ad Given that hindrance of cell proliferation often entails cell death or\ntensity increased by 1.89 \u00b1 0.32, 1.42 \u00b1 0.18, 1.47 \u00b1 0.22, and 1.21 \u00b1 apoptosis, a pivotal programmed process, the impact of 2a, 2b and 2c on\n0.14 times for Rosup, 2a, 2b, and 2c, respectively, compared to the apoptosis was further examined using acridine orange. See from Fig. 3b,\ncontrol. The effect on ROS amounts followed the sequence 2b > 2a > 2c, the living and apoptotic cells were stained green, we observed the\nwhich diverges from the order of cytotoxic activity of 2a, 2b and 2c apoptotic features of apoptotic cells such as nuclear shrinkage, chro\u00ad\ntowards A549 cells. matin condensation. In addition, the apoptotic percentage was deter\u00ad\n mined with flow cytometry, cisplatin was used as a positive control. As\n2.6. Intracellular Ca2+ concentration presented in Fig. 3c, treatment of A549 cells (I) with 2 \u00d7 IC50 of 2a (IV),\n 2b (VII), 2c (X) and cisplatin (XII) for 24 h resulted in a substantial rise of\n Elevation in intracellular calcium ion levels is known to trigger the 4.56%, 10.59%, 9.19% and 3.38%, respectively, in early apoptotic cells\nopen of the mitochondrial permeability transition pore, ultimately when compared to the control. However, A549 cells were incubated\nleading to the release of cytochrome C and initiation of the intrinsic with 2 \u00d7 IC50 of 2a (IV), 2b (VII), 2c (X) and cisplatin (XII) for 48 h\n\n 4\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\n\n\n Fig. 2. RNA-sequence determination (a) Heap map, (b) volcano map, (c) and (d) KEGG and GO enrichment.\n\n\n(Fig. 3d), the apoptotic percentage increased by 13.24% for 2a, 14.84% suppressing it by preventing the depletion of glutathione (GSH), ulti\u00ad\nfor 2b and 79.64% for cisplatin in the late apoptosis, while 2c caused an mately thwarting ferroptosis [36,37]. The depletion of GSH occurs\nincrease of 67.98% in the early apoptosis. Therefore, we conclude that through its reaction with lipid ROS, resulting in the production of\n0.5 \u00d7 IC50 or IC50 concentration of 2a, 2b and 2c show low or no glutathione disulfide (GSSG). As displayed in Fig. 4a, a discernible\napoptotic effect, 48h-treatement displayed a high apoptotic efficacy reduction in GSH content is observed in A549 cells treated with com\u00ad\nthan that of 24h-treatment, 2a, 2b and cisplatin induce a late apoptosis, plexes 2a, 2b, and 2c when compared to the control, illustrating the\nwhile 2c causes an early apoptosis. The late apoptotic effect of 2a and 2b effective reduction of GSH content by the complexes.\nis lower than that of cisplatin, the early apoptotic efficacy of 2c is higher Lipid peroxidation, as evidenced by malondialdehyde (MDA) levels,\nthan cisplatin after a treatment of 48 h. was subsequently assessed to further explore the influence of 2a, 2b, and\n The expression of Bax, PARP (poly ADP-ribose polymerase), caspase 2c on cellular lipid peroxidation. As demonstrated in Fig. 4b, treatment\n3 and Bcl-2 was investigated by Western blot. It\u2019s noteworthy that the of A549 cells with 2a, 2b and 2c for 24 h led to a notable increase in MDA\ncleavage of PARP serves as a hallmark of apoptosis initiated by caspase levels when contrasted with the control, signifying their impact on lipid\nactivity. Additionally, Bcl-2 family proteins, encompassing pro- peroxidation. Inhibition of GPX4 activity is recognized to prompt\napoptotic Bax and anti-apoptotic Bcl-2, are pivotal in the orchestration intracellular lipid peroxidation, a pivotal marker of ferroptosis. This is\nof cellular apoptosis [34,35]. This intricate interplay becomes apparent corroborated in Fig. 4c, where the expression of GPX4 was down\u00ad\nin Fig. 3e, wherein co-incubation of A549 cells with 2a, 2b, and 2c for 24 regulated in response to treatment of A549 cells with 2a, 2b, and 2c.\nh leads to a downregulation of PARP, caspase 3, and Bcl-2, alongside an Assessment of lipid peroxidation was further facilitated using 4,4-\nincrease in Bax protein expression. Taken together, we consider that 2a, difluoro-5-(4-phenyl-1,3-butadenyl)-4-bora-3a,4a-diaza-sindacene-3-\n2b and 2c orchestrate apoptosis through caspase 3 activation, PARP undecanoic acid (C11-BODIPY) as a fluorescent marker. This fluorescent\ncleavage, and modulation of Bcl-2 family protein expression. marker dichotomously represents low and high degrees of lipid perox\u00ad\n idation through red and green fluorescence, respectively [38,39]. As\n presented in Fig. 4d and e, the control group displayed red fluorescence,\n2.10. Complexes inducing ferroptosis indicative of a low degree of lipid peroxidation, whereas the groups\n treated with 2a, 2b, and 2c exhibited bright green fluorescence, signi\u00ad\n Distinguished by its distinct iron-dependent nature and different fying a high degree of lipid peroxidation. Collectively, these results\nfrom autophagy, necrosis, and apoptosis, ferroptosis is an emerging form illuminate that 2a, 2b, and 2c induce lipid peroxidation and conse\u00ad\nof cell death. The increase in ROS is intricately linked with both quently invoke ferroptosis, providing a comprehensive understanding of\napoptosis and ferroptosis. Central to the regulation of lipid peroxidation the mechanisms underpinning their cellular impact. These results show\nis glutathione peroxidase 4 (GPX4), which plays a pivotal role in\n\n 5\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\n\n\nFig. 3. (a) cell cycle distribution, (b) morphological apoptosis, (c) and (d) apoptosis after A549 cells (I) were exposed to 0.5 \u00d7 IC50, IC50 and 2 \u00d7 IC50 concentrations\nof 2a (II-IV), 2b (V-VII), 2c (VIII-X) and IC50 and 2 \u00d7 IC50 concentrations of cisplatin (XI-XII) for 24 or 48 h.\n\n\n\n 6\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\n\n\nFig. 4. (a) GSH, (b) MDA concentration determination, (c) the expression of GPX4, (d) lipid peroxidation detection while A549 cells were stained with C11-\nBODIPY581/591, (e) Ratio of red versus green fluorescence.\n\n\nthat the complexes can cause ferroptosis. subsequent release of cellular contents, culminating in an inflammatory\n response. As shown in Fig. 5c, the complexes cleave GSDMD-F into\n GSDMD-N, downregulate caspase 1, upregulate the expression of NF-kB\n2.11. Induction of pyroptosis protein. Collectively, 2a, 2b and 2c can trigger pyroptosis.\n\n Programmed cell death is an orchestrated process triggered by spe\u00ad\ncific signals or stimuli, ensuring the preservation of the internal cellular 2.12. Antitumor effect in vivo\nenvironment. Among the diverse forms of programmed cell death,\ncellular pyroptosis is notable. To probe the impact of the complexes on Given that 2c exhibited the most potent anticancer efficacy on A549\npyroptosis, an examination of cell morphology was carried out. In cells, thus, 2c was chosen for further in vivo antitumor investigations.\nFig. 5a, co-incubation of A549 cells with 2a, 2b, and 2c for 28 h revealed The initial step was to determine the LD50 value through acute toxicity\ncharacteristic membrane bubbles on the plasma membrane. This phe\u00ad assessment. As depicted in Fig. 6a and b, the administration of 60, 90,\nnomenon arises due to the breach of the cell membrane and subsequent and 120 mg/kg doses of 2c to Kunming mice led to noticeable reductions\ncellular swelling driven by osmotic pressure disparities between the cell in body weight and instances of mice mortality. However, no significant\ninterior and its surroundings, signifying the ability of 2a, 2b, and 2c to alterations in body weight and no mouse deaths were uncovered in the\ninduce pyroptosis. 30 or 40 mg/kg doses of 2c. Therefore, LD50 value was 64 mg/kg after\n Lactate dehydrogenase (LDH) release serves as a direct indicator of 14 days of treatment.\npyroptosis. As illustrated in Fig. 5b, treatment of A549 cells with 2a, 2b, For the subsequent in vivo antitumor study, athymic BALB/c nude\nor 2c resulted in elevated LDH content compared to the control, mice were allocated into three groups (n = 6): control, 3.2, and 5.0 mg/\nproviding further evidence of the pyroptotic effect induced by these kg of 2c. The mice were treated with the respective doses of 2c, and their\ncomplexes. body weights and tumor volumes were monitored daily. Upon comple\u00ad\n The molecular underpinnings of cellular pyroptosis encompass two tion of the 8-day treatment, the nude mice were euthanized, then the\nkey pathways, one of which is reliant on the classical caspase 1 pathway. tumors were collected. Fig. 6c, d, and 6e illustrate that the tumor size\nIn the context of innate immune response, pathogenic invasion triggers and weight in the 2c groups are notably diminished compared to the\nthe formation of inflammasome complexes that activate caspase 1. control group. The tumor inhibitory rate reached 34.04% and 58.58%\nActivated caspase 1 serves a dual role: it cleaves Gasdermin D to yield a for the 3.2 mg/kg and 5.0 mg/kg of 2c, respectively (Fig. 6f). The\npeptide harboring the N-terminal reactive domain of Gasdermin D, inhibitory rate of 2c is lower than liposome-entrapped iridium(III)\ninducing cell membrane perforation, cellular rupture, and the complex [Ir(ppy)2(DAPIP)](PF6) (inhibitory rate of 70.4%) [30].\n\n\n\n\n Fig. 5. Pyroptosis assay (a) cell morphology, (b) LDH release, (c) expression of GSDMD, caspase 1, NF-kB.\n\n 7\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\n\n\nFig. 6. In vivo antitumor (a) mean body weight of kunming mice, (b) survival rate of Kunming mice, (c, d and e) Photographs of tumor size and weight from\ntreatment and vehicle groups, (f) Inhibitory rate of 3.2 mg/kg and 5.0 mg/kg of 2c, (g) Body weight of the nude mice and (h) relative tumor volumes after the tumors\nwere treated with 2c for 8 days, (i) Histopathological sections using H&E staining.\n\n\nImportantly, as demonstrated in Fig. 6g, in the body weights of the mice, 3. Conclusions\nthere were no discernible alterations between the control and 2c groups.\nThe relative tumor volumes displayed a decrease in the 2c groups In conclusion, this study successfully synthesised and comprehen\u00ad\ncomparison to the control (Fig. 6h). Additionally, histological exami\u00ad sively characterised three iridium (III) complexes, namely 2a, 2b, and\nnation of various tissues including heart, liver, lung, spleen, kidney, and 2c. The complexes enter the cells and mainly distribute in the mito\u00ad\nbrain using Hematoxylin-Eosin (H&E) staining revealed no significant chondria and cytoplasm. Notably, these complexes demonstrated sig\u00ad\ndeviations in heart, spleen, kidney, brain and liver tissue, only observing nificant cytotoxic activity against A549, B16, and HCT116 cancer cells.\na mild edema and the presence of bruising in the lungs (Fig. 6i). Taken Moreover, they effectively hindered cell colonies and cell migration.\ntogether, these findings underscore the potent antitumor efficacy of 2c Further exploration of their impact on cell cycle distribution indicated\nin vivo and its favorable safety profile in key organs. that the complexes exerted their anti-proliferative effects by arresting\n A549 cells at the S phase. Additionally, 2a, 2b and 2c were found to elicit\n intricate changes within the cells. Specifically, they heightened\n\n\n 8\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\nintracellular amount of ROS, triggered a reduction in mitochondrial meticulously dissolved in 28 mL CH2Cl2 and 14 mL CH3OH and refluxed\nmembrane potential and induced augmented intracellular calcium ion at 40 \u25e6 C for a period of 6 h under an argon atmosphere. Then the solution\ncontent, culminating in the release of cytochrome c. RNA-sequence an\u00ad was allowed to cool, subsequently, saturated aqueous NH4PF6 was\nalyses underscored the comprehensive impact of complex 2c on gene added and stirred for 2 h, yielding a distinctive yellow precipitate. The\nexpression, revealing a significant modulation of 61 upregulated and 90 obtained crude product underwent purification via neutral alumina\ndownregulated genes. KEGG enrichment analysis shed light on the column chromatography, utilizing CH2Cl2-acetone (v/v, 5:1) as the\npathways including calcium ions and ferroptosis as pivotal players in the eluting agent, the yellow band fraction was collected, finally the yellow\ninduction of cell death. The downregulation of GPX4 expression, a powder was attained through removing the solvent.\nreduction of glutathione (GSH) concentration, an elevation in malon\u00ad 2a, yield: 80%. 1H NMR (DMSO\u2011d6, 500 MHz) (Figs. S10 and SI): \u03b4\ndialdehyde (MDA) levels and C11-BODIPY581/591 dyed experiments 9.01 (d, J = 8.0 Hz, 2H), 8.24 (d, J = 8.0 Hz, 2H), 8.12\u20138.09 (m, 1H),\nshow that the complexes can induce ferroptosis. The finding of charac\u00ad 8.00 (d, J = 5.0 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H), 7.91\u20137.84 (m, 4H),\nteristic membrane bubbles, cleavage of GSDMD-F into GSDMD-N, 7.72 (s, 1H), 7.51 (d, J = 6.5 Hz, 2H), 7.04 (t, J = 7.5 Hz, 2H), 7.00\u20136.92\ndownregulation of caspase 1, upregulation of the expression of NF-kB (m, 4H), 6.31 (d, J = 7.5 Hz, 2H). 13C NMR (DMSO\u2011d6, 125 MHz)\nprotein and LDH release indicate that the complexes can cause pyrop\u00ad (Figs. S11 and SI): 172.66, 167.43, 167.33, 162.05, 161.58, 151.68,\ntosis. The in vivo antitumor assessments further accentuated the po\u00ad 151.05, 149.64, 149.48, 148.33, 146.69, 144.55, 144.22, 143.42,\ntential of complex 2c, showcasing remarkable antitumor efficacy 139.07, 138.94, 132.59, 132.13, 131.76, 130.62, 127.32, 126.46,\n(58.58%) without adverse effects on critical organs. Complex 2c cause 125.46, 124.26, 122.76, 122.62, 120.35. IR (KBr, cm\u2212 1): 3406, 2974,\nan early apoptosis, 2a and 2b induce a late apoptosis. The collective 1639, 1477, 1420, 1350, 1267, 1163, 1064, 1031, 820, 757, 557. 19F\nevidence suggests that the complexes induce cell death via apoptosis, NMR (DMSO\u2011d6, 500 MHz): 69.38 (3F), \u2212 70.90 (3F). HRMS (CH3CN):\nferroptosis and GSDMD-mediated pyroptosis. This work provides valu\u00ad C39H25N9Cl2IrPF6: m/z = 882.12145 [(M \u2212 PF6)+].\nable insights for both understanding the anticancer properties of these 2b, yield: 77%. 1H NMR (DMSO\u2011d6, 500 MHz) (Figs. S12 and SI): \u03b4\niridium (III) complexes and guiding the design and synthesis of novel 8.98 (d, J = 7.5 Hz, 2H), 8.50 (d, J = 7.0 Hz, 2H), 8.08 (t, J = 4.0, 1H),\niridium-based anticancer agents. 8.01\u20137.97 (m 6H), 7.87 (d, J = 9.0 Hz, 2H), 7.81\u20137.79 (m, 2H), 7.71 (s,\n 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.45\u20137.42 (m, 2H), 7.21 (t, J = 7.5 Hz,\n4. Experimental 2H), 6.34 (d, J = 7.5 Hz, 2H). 13C NMR (DMSO\u2011d6, 125 MHz) (Figs. S13\n and SI): 172.59, 167.99, 162.05, 161.58, 157.01, 156.88, 149.19,\n4.1. Materials and method 148.49, 147.87, 147.17, 143.86, 140.93, 140.87, 137.93, 137.82,\n 134.19, 132.59, 132.17, 130.15, 129.92, 129.06, 127.31, 127.11,\n Starting with the acquisition of essential reagents, IrCl3\u22c53H2O was 126.46, 124.66, 123.22, 120.76, 120.62. IR (KBr, cm\u2212 1): 3407, 3032,\nprocured from J&K Scientific, while Aladdin supplied 1,10-phenanthro\u00ad 1635, 1445, 1404, 1328, 1137, 1070, 1049, 851, 753, 557. 19F NMR\nline and DMSO. Additionally, Equation Biotechnology Co., Ltd. provided (DMSO\u2011d6, 500 MHz): 69.38 (3F), \u2212 70.90 (3F). HRMS (CH3CN):\n4\u2032,6-diamidino-2-phenylindole (DAPI). Complementary reagents for the C43H25N9Cl2IrPF6: m/z = 930.12256 [(M \u2212 PF6)+].\nstudy encompassed 1-phenylpyridine (ppy), 1-phenylisoquinoline (piq), 2c, yield: 75%. 1H NMR (DMSO\u2011d6, 500 MHz) (Figs. S14 and SI): \u03b4\nand benzo[h]quinolone (bzq), which were acquired from Beijing HWRK 9.00 (d, J = 7.5 Hz, 4H), 8.39 (d, J = 8.0 Hz, 2H), 7.99 (dd, J = 2.0, J =\nChem Co., Ltd. The iridium dimeric precursors cis-[Ir(ppy)2Cl]2, [Ir 7.5 Hz, 4H), 7.88\u20137.84 (m, 8H), 7.43\u20137.39 (m, 4H), 7.16 (t, J = 7.5 Hz,\n(bzq)2Cl]2 and [Ir(piq)2Cl]2 were synthesized according to the methods 2H), 6.94 (t, J = 7.5 Hz, 2H), 6.32 (d, J = 8.0 Hz, 2H). 13C NMR\ndescribed as the literature [40]. (DMSO\u2011d6, 125 MHz) (Figs. S15 and SI): 172.54, 168.32, 162.19,\n With dimethyl sulfoxide as the solvent and tetramethylsilane (TMS) 161.56, 154.90, 154.41, 145.86, 141.43, 141.26, 136.95, 132.74,\nas an internal standard, NMR spectra were conducted utilizing a Varian- 132.42, 132.19, 131.04, 130.95, 130.43, 129.78, 128.15, 126.89,\n500 spectrometer (500 MHz). Luminescence and UV\u2013Vis spectra mea\u00ad 126.01, 122.78, 122.66. IR (KBr, cm\u2212 1): 3410, 2992, 1636, 1504, 1455,\nsurements were also executed. The molecular weights of the complexes 1404, 1385, 1271, 1149, 1133, 1080, 1048, 845, 756, 556. 19F NMR\nwere meticulously determined employing High Resolution Mass Spec\u00ad (DMSO\u2011d6, 500 MHz): 69.39 (3F), 70.90 (3F). HRMS (CH3CN):\ntrometry (HRMS). C47H29N9Cl2IrPF6: m/z = 982.15472 [(M \u2212 PF6)+].\n\n4.2. Synthesis of DPPA and 2a, 2b and 2c 4.3. Detection of purity of 2a, 2b and 2c\n\n4.2.1. Preparation of DPPA The purity of 2a, 2b and 2c was carried out with COSMOSIL 5C18-MS-\n A solution consisting of 0.198 g of 2-amino-4,6-dichloropyrimidine- II column (250 mm \u00d7 10 mm) at 25 \u25e6 C. Mobile phase A consisted of 0.1%\n5-carbaldehyde (1.0 mmol) and 0.21 g of 1,10-phenanthroline-5,6-dione trifluoroacetic acid (TFA) with H2O, mobile phase B was made of 0.1%\n(1.0 mmol) was meticulously dissolved in 25 mL CH3CH2OH and TFA with methanol, the flow rate reached 3 mL/min. The volume ratios\nrefluxed at 80 \u25e6 C for a duration of 24 h. Upon completion of the reflux of VCH3OH:VH2O were 90:10 for 2a, 95:5 for 2b, 95:5 for 2c, the detected\nprocess, and following cooling to ambient temperature, the solvent was wavelengths were 254 nm for 2a, 210 nm for 2b and 2c.\ncarefully removed. A brown-colored powder was subsequently obtained.\nThe resulting yield amounted to 82%. Anal. Cacld for C17H9Cl2N7: C, 4.4. Determination and refinement of crystal structure 2c\n53.42, H, 2.37, N, 25.65%. Found: C, 53.18, H, 2.51, N, 25.80%. 1HNMR\n(DMSO\u2011d6, 500 MHz): 14.1 (s, 1H), 9.05 (d, J = 5.5 Hz, 1H), 8.88 (d, J = Suitable for X-ray diffraction analysis, single crystal of 2c, was grown\n8.5 Hz, 1H), 8.76 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 7.87\u20137.76 (m, 2H), via slow evaporation of 2c methanol solution (CCDC 2293711) and\n6.48 (s, 2H). 13C NMR (DMSO\u2011d6, 125 MHz): 162.66, 161.71, 148.73, Table 4 listed the crystal data for 2c. A SuperNova, Dual, Cu at home/\n148.52, 147.84, 144.37, 144.01, 143.98, 135.76, 130.29, 130.12, near, AtlasS2 diffractometer in the range 3.654 < \u03b8 < 73.003\u25e6 with CuK\u03b1\n126.48, 124.57, 124.11, 123.89, 120.30, 119.64. HRMS (CH3OH): m/z radiation (\u03bb = 1.54184 \u00c5) at 150 K was used to measure the crystal\n= 381.9126 ([M \u2212 1]). structure of 2c. Direct method was applied to solve the structure of 2c to\n obtain the position of all non-H atoms. All calculation was achieved\n4.2.2. Synthesis of 2a, 2b and 2c applying the SHELXTL-97 system [41].\n A combination of cis-[Ir(ppy)2Cl]2 (0.215 g, 0.20 mmol) [40] for 2a,\n[Ir(bzq)2Cl]2 (0.234 g, 0.20 mmol) [40] for 2b, [Ir(piq)2Cl]2 (0.255 g,\n0.20 mmol) [40] for 2c and DPPA (0.152 g, 0.40 mmol) was\n\n 9\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\nTable 4 (10% fetal bovine serum, FBS) overnight. The cells were treated with\nCrystal data of 2c\u22c52H2O\u22c52CH3OH. IC50 concentrations of 2a, 2b and 2c for 24 h. The cells were washed\n C49H40Cl2N9Ir twice with PBS and separated using trypsin-EDTA solution. The\n collected cells were centrifuged to eliminate the supernatant and\n Mr 1082.09\n T [K] 150(2) washed twice with cold PBS, and fixed with 75% ethanol overnight in a\n \u03bb [\u00c5] 1.54184 refrigerator. Next day, the cells were centrifuged to eliminate the su\u00ad\n Crystal system Monoclinic pernatant and washed twice with cold PBS. A mixture of RNAse (30 \u03bcL,\n Space group P21/n 10 mg mL\u2212 1) and propidium iodide (30 \u03bcL, 1.0 mg mL\u2212 1), Tritonx-100\n a [\u00c5] 10.0777(2)\n b [\u00c5] 12.7179(3)\n (10 \u03bcL) and PBS were added to the cells and incubated at 37 \u25e6 C for 30\n c [\u00c5] 18.3379(5) min, the cells were analyzed using a FACS Calibur flow cytometer.\n \u03b1 [\u25e6 ] 81.834(2)\n \u03b2 [\u25e6 ] 80.622(2) 4.9. Examination of cell morphology and lactate dehydrogenase (LDH)\n \u03b3 [\u25e6 ] 72.910(2)\n content\n V [\u00c53] 2205.43(2)\n Z 2\n Dc [ g\u22c5m\u2212 3] 1.629 Seeded in a 6-well plate overnight, A549 cells were then treated with\n Crystal size [mm] 0.21 \u00d7 0.19 \u00d7 0.12 2a, 2b and 2c for 28 h. Afterwards, the cell morphology was carefully\n \u03b8 range for data collection[\u25e6 ] 3.654 to 73.003 observed under an inverted light microscope.\n Limiting indices, hkl \u2212 8 \u2264 h \u2264 12, \u2212 13 \u2264 k \u2264 15, \u2212 20 \u2264 l \u2264 22\n Reflections collected 16070\n For LDH release studies, cells cultured in a 96-well plate were co-\n Independent reflections (Rint) 8480 incubated with 2a, 2b and 2c for a period of 24 h. After this treat\u00ad\n Goodness-of-fit on F2 1.015 ment, LDH release solution was added to the control group for an hour.\n R1/wR2 [I > 2\u03c3(I)][a] 0.0528/0.1203 Thereafter, the supernatant was transferred to new wells and mixed with\n R1/wR2 (all data) [a] 0.0639/0.1262\n LDH detection working solution at 25 \u25e6 C for 30 min, with precautions\n Largest diff. peak and hole [e \u00c5\u2212 3] 2.040 and \u2212 1.457\n taken to avoid light. The absorbance was measured at 490 nm.\n\n4.5. Cell viability and IC50 values determination 4.10. Acute toxicity\n\n Cytotoxic activity assessment against cell lines of human lung car\u00ad Given the relatively low IC50 value of the complex 2c on A549 cells,\ncinoma A549, melanoma B16, colorectal cancer HCT116, human he\u00ad 2c was selected for in vivo antitumor experiments. A half of male and\npatocellular carcinoma HepG2 and non-cancer cell LO2 was conducted female Kunming mice (KM mice) of 5\u20136 weeks of age, with body weight\nusing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide range of 20\u201330 g, were selected and kept in SPF barrier environment.\n(MTT) method [29]. In this approach, cells were seeded into 96-well Then they were divided into 6 groups (n = 6). The mice were fasted for\nmicroassay culture plates at a density of 5 \u00d7 103 cells per well and 12 h before administration, the control group was injected with saline\nallowed to grow overnight at 37 \u25e6 C within a 5% CO2 incubator. intraperitoneally, different concentration (30, 40, 60, 90, 120 mg/kg) of\nFollowing the initial incubation, varying concentrations of the com\u00ad 2c was injected in the 2c-treated groups, the mice were continuously\nplexes and ligands were introduced into the wells. The control wells observed for 14 days, and the weights of the Kunming mice were\nwere supplemented with 100 \u03bcL of culture medium. The plates were measured daily. On the 14th day, the mice were executed. The survival\nsubsequently co-incubated for 48 h. After the incubation period, a stock rate (%) = (Nnumber of surviving mice/Ntotal number of mice tested) \u00d7 100%. The\nsolution of MTT dye (25 \u03bcL, 4 mg mL\u2212 1) was introduced into each well. experimental data recorded daily were compiled and analyzed on Excel\nFollowing an additional 4-h treatment, MTT formazan crystals were software, and finally the LD50 value of complex 2c was gained by SPSS\ndissolved by a solution (100 \u03bcL) of DMSO (50%) and sodium dodecyl software.\nsulfate (20%). A microplate spectrophotometer was applied to measure\nthe optical density at 490 nm. 4.11. Antitumor activity in vivo\n\n4.6. Cellular uptake studies Complex 2c emerged as the candidate for in vivo antitumor evalu\u00ad\n ation, all the experimental procedures were authorized by Institutional\n Seeded in a 12-well plate of A549 at a density of 1.5 \u00d7 105 cells per Animal Center of Guangdong Pharmaceutical University. Guangdong\nwell overnight, 2a, 2b and 2c were added in the above cells for 12 h at Provincial Laboratory Animal Center provides BALB/c nude mice (3\u20134\n37 \u25e6 C. Then, the plate was washed three times with PBS buffers to weeks). The mice were injected with a total of A549 (6 \u00d7 106/\u03bcL),\neliminate the residual complexes, and the cells were further dyed with subsequently distributing them into randomized groups: the control\nHoechst 33324 for 20 min. After that, the cells were washed twice with group and the positive group. Intraperitoneal injections of 2c (3.2 or 5.0\nice-cold PBS, the residual Hoechst was eliminated, ultimately, the cells mg/kg) were administered daily. The mice\u2019s body weights and the di\u00ad\nwere photographed. ameters of tumors were documented daily. The mice were humanely\n euthanized on the 8th day, and the tumors were meticulously collected\n4.7. Cellular apoptosis studies and weighed.\n\n Seeded in the six-well plates overnight, A549 cells were incubated 4.12. Data analysis\nwith IC50 concentrations of 2a, 2b and 2c for 24 h, then A549 cells were\nharvested, washed three times with PBS. The cells were dyed with 50 \u03bcg All data was obtained as means \u00b1 standard error and the difference\nmL\u2212 1 PI (propidium iodide) and 1 mg mL\u2212 1 Annexin V-FITC in PBS on was evaluated using student\u2019s-test. Differences are significant at *P <\nice in the dark for 15 min. Finally, the cells were analyzed using flow 0.05, **P < 0.01, ***P < 0.001. At least three independent experiments\ncytometry. were performed.\n Note: The procedures for cell culture, lipid water partition coeffi\u00ad\n4.8. Cell cycle arrest assay cient, wound healing experiment, cell colonies formation, co-location,\n intracellular ROS concentration determination, GSH content determi\u00ad\n In this experimental approach, 2 \u00d7 105 A549 cells were meticulously nation, intracellular MDA concentration, change of the mitochondrial\nseeded in individual wells of 6-well plates and cultured in a medium membrane potential, intracellular Ca2+ levels determination, release of\n\n 10\n\fH. Hu et al. European Journal of Medicinal Chemistry 268 (2024) 116295\n\n\ncytochrome c, RNA-sequence assay, protein expression assay can be [14] L.L. Wang, R.L. Guan, L.N. Xie, X.X. Liao, K. Xiong, T.W. Rees, Y. Chen, L.N. Ji,\n H. Chao, An ER-targeting iridium(III) complex that induces immunogenic cell\nfound in the supporting information.\n death in non-small-cell lung cancer, Angew. Chem., Int. Ed. Engl. 60 (2021)\n 4657\u20134665.\nCRediT authorship contribution statement [15] Y. Wang, Y.Z. Li, J. Chen, H.M. Liu, Y. Zhou, C.X. Huang, L.J. Liang, Y.J. Liu, X.\n Z. Wang, Anticancer effect evaluation of iridium(III) complexes targeting\n mitochondria and endoplasmic reticulum, J. Inorg. Biochem. 239 (2023) 112054.\n Huiyan Hu: Writing \u2013 original draft, Methodology, Investigation. [16] G. Gupta, S. Cherukommu, G. Srinivas, S.W. Lee, S.H. Mun, BODIPY-based Ru(II)\nFan Zhang: Formal analysis, Data curation. Zhujun Sheng: Software. and Ir(III) organometallic complexes of avobenzone, a sunscreen material: potent\n anticancer agents, J. Inorg. Biochem. 189 (2018) 17\u201329.\nShuang Tian: Data curation. Gechang Li: Formal analysis. Shuanghui\n [17] J. Chen, H.M. Liu, Y.C. Chen, H.Y. Hu, C.X. Huang, Y. Wang, L.J. Liang, Y.J. Liu,\nTang: Software. Yajie Niu: Formal analysis. Jiawan Yang: Visualiza\u00ad Iridium(III) complexes inhibit the proliferation and migration of BEL-7402 cells\ntion, Software. 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