Synthesis, biological evaluation of novel iridium(III) complexes targeting mitochondria toward melanoma B16 cells.

{"full_text": " European Journal of Medicinal Chemistry 247 (2023) 115046\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, biological evaluation of novel iridium(III) complexes targeting\nmitochondria toward melanoma B16 cells\nYuhan Yuan 1, Yuanyuan Zhang 1, Jing Chen, Chunxia Huang, Haimei Liu, Wenlong Li,\nLijuan Liang, Yi Wang, 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: A new ligand 2-(1E,3E,5E,7E)-2,6-dimethyl-8-(2,6,6-trimethylcyclohex-1-yl)octa-1,2,5,7-tetraen-1-yl)-1H-imi\u00ad\nIridium(III) complexes dazo[4,5-f][1,10]phenanthroline (DTOIP) was synthesized and combined with [Ir(ppy)2Cl]2\u22c52H2O (ppy =\nApoptosis deprotonated Hppy: 2-phenylpyridine), [Ir(piq)2Cl]2\u22c52H2O (piq = deprotonated Hpiq: 1-phenylisoquinoline) and\nCell cycle arrest\n [Ir(bzq)2Cl]2\u22c52H2O (bzq = deprotonated Hbzq: benzo[h]quinolone) to form [Ir(ppy)2(DTOIP)](PF6) (Ir1), [Ir\nRNA-sequence\nAntitumor in vivo\n (piq)2(DTOIP)](PF6) (Ir2), and [Ir(bzq)2(DTOIP)](PF6) (Ir3), respectively. The complexes were characterized by\n elemental analysis, high-resolution mass spectrometry (HRMS), 1H NMR and 13C NMR. The antiproliferative\n activity of the complexes toward B16, BEL-7402, Eca-109 and normal LO2 cells was evaluated by 3-(4,5-\n dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Complexes Ir1, Ir2 and Ir3 showed high\n antiproliferative activity against B16 cells with a low IC50 values of 0.4 \u00b1 0.1, 2.0 \u00b1 0.1 and 1.4 \u00b1 0.09 \u03bcM,\n respectively. Three-dimensional (3D) in vitro cell models also demonstrated that the iridium(III) complexes have\n a remarkable cytotoxicity to B16 cells. The experiments of cellular uptake, mitochondrial localization, and\n intracellular distribution of the drugs proved that the three iridium(III) complexes can enter the mitochondria,\n leading to the loss of mitochondrial membrane potential (MMP), decreased glutathione (GSH) levels, causing an\n increase of intracellular ROS content, and DNA damage, finally inducing apoptosis. RNA-sequence and bioin\u00ad\n formatics analyses were used to analyze the differentially expressed genes and enriched biology processes.\n Antitumor in vivo demonstrated that complex Ir1 (5 mg/kg) exhibits a high efficacy to inhibit the tumor growth\n with an inhibitory rate of 71.67%. These results show that the complexes may be potent anticancer candidate\n drugs.\n\n\n\n\n1. Introduction side effects such as myelosuppression and hair loss, these side effects\n limited its clinical application [4]. This stimulated scientists to find\n Cancer is one of the most widespread serious diseases. It is charac\u00ad anticancer compounds of other transition metals as alternative of\nterized by uncontrolled growth of abnormal cells [1]. Due to the un\u00ad cisplatin [5].\ncontrolled cell proliferation, invasion of surrounding and distant tissues In recent years, nonplatinum anticancer drugs have received wide\u00ad\naggressive metastasis, cancer is generally considered as a group of spread attention, among these nonplatinum metal complexes, iridium-\ncomplex and multifaceted diseases [2] Chemotherapy is one of the based metal compounds have been paid a great attention. Similar to\ncommon clinical treatments for cancer, which is usually combined with platinum compounds, the properties of iridium(III) complexes such as\nother methods such as radiotherapy and surgery to prolong cancer pa\u00ad redox potentials, ligand exchange kinetics and coordination numbers\ntient survival. Cisplatin, one of the representations of metal-based were widely studied. Not only in catalysts and optoelectronic sensors\nanticancer drugs, was the first transition metal drug that was used for these transition metal complexes had a widespread application, but also\nchemotherapy. Its mechanisms were interfering with the aspect of DNA in biological and medicinal fields, the anticancer activity of iridium(III)\nreplication, repair, translation, or cell division [3]. Unfortunately, complexes has made a great achievement [6\u201325].\ncisplatin kills both cancer and normal cells, which causes some serious To obtain much information on the anticancer activity of iridium(III)\n\n\n * Corresponding author.\n E-mail address: lyjche@gdpu.edu.cn (Y. Liu).\n 1\n These authors contribute equally to this work.\n\nhttps://doi.org/10.1016/j.ejmech.2022.115046\nReceived 7 October 2022; Received in revised form 12 December 2022; Accepted 21 December 2022\nAvailable online 22 December 2022\n0223-5234/\u00a9 2022 Elsevier Masson SAS. All rights reserved.\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\ncomplexes, in this paper, owing to (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6- ligand, therefore, the NH proton of the imidazole ring is very active and\ntrimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenal including multiple easy to be exchanged between the two imidazole nitrogen atoms in so\u00ad\nconjugate system, these conjugate system may produce a large influence lution [26]. In the 13C NMR spectra, the peaks of 64.8, 56.0, 56.9 and\non the biological activity, hence, we chose (2E,4E,6E,8E)-3,7-dimethyl- 55.9 ppm for DTOIP, Ir1, Ir2 and Ir3 are assigned to the C (u) atoms, the\n9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenal as starting chemical shifts of 35.9, 34.7, 30.8 ppm for DTOIP, 33.9, 32.6, 18.5 ppm\nraw material to synthesize a novel polypyridyl ligand DTOIP (DTOIP = for Ir1, 33.9, 32.7, 18.8 ppm for Ir2, 33.9, 32.6, 18.7 ppm for Ir3 are\n(2-(1E,3E,5E,7E)-2,6-dimethyl-8-(2,6,6-trimethylcyclohexen-1-en-1-yl) attributed to the carbon atoms of C (t), C (r) and C (v), respectively. The\nocta-1,3,5,7-tetraene-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) peaks of 23.6, 23.1, 20.8, 16.3, 14.6 ppm for DTOIP, 21.5, 18.5, 18.7,\nand conjugated with three precursors, [Ir(ppy)2Cl]2\u22c52H2O (ppy = 14.4, 12.6 ppm for Ir1, 21,6, 19.1, 18.8, 14.4, 12.7 ppm for Ir2, 21.5,\ndeprotonated Hppy: 2-phenylpyridine), [Ir(piq)2Cl]2\u22c52H2O (piq = 19.0, 18.7, 14.3, 12.6 ppm for Ir3 are attributed to the carbon atoms of\ndeprotonated Hpiq: 1-phenylisoquinoline) and [Ir(bzq)2Cl]2\u22c52H2O (bzq five methyl groups. Hence, after the ligand DTOIP bonded with the\n= deprotonated Hbzq: benzo[h]quinolone) to synthesize three iridium metal to form complexes, a change of chemical shifts was observed.\n(III) complexes: [Ir(ppy)2(DTOIP)](PF6) (Ir1), [Ir(piq)2(DTOIP)](PF6) The UV\u2013Vis absorption spectra of 20.0 \u03bcM of Ir1, Ir2, Ir3 in ethanol\n(Ir2), [Ir(bzq)2(DTOIP)](PF6) (Ir3) (Scheme 1). The complexes were are presented in Fig. S1a (supporting information). Each complex\ncharacterized by elemental analysis, HRMS, 1H NMR and 13C NMR. The exhibited two bands at wavelength of 257 (\u03b5 = 61,500) and 392 nm (\u03b5 =\ncytotoxicity of the complexes Ir1, Ir2 and Ir3 against cancer B16, BEL- 48,000) for Ir1, 291 (\u03b5 = 68,500) and 385 nm (\u03b5 = 48,500) for Ir2, 253\n7402, Eca-109 and normal LO2 cells was evaluated by 3-(4,5-dime\u00ad (\u03b5 = 71,000) and 395 nm (\u03b5 = 49,500) for Ir3. The bands below 300 nm\nthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Ir1, are assigned to the intraligand (IL) \u03c0\u2013\u03c0* transitions, while the bands at\nIr2 and Ir3 were found to be highly toxic to B16 in vitro. Since mito\u00ad 380\u2013400 nm are attributed to the metal-to-ligand charge transfer tran\u00ad\nchondrial dysfunction is associated with pathological changes in many sition. Fig. S1b (supporting information) shows the luminescence\ncancers, cardiovascular diseases and neurodegenerative disorders, spectra of Ir1 (0.4 \u03bcM), Ir2 (2.0 \u03bcM), and Ir3 (1.4 \u03bcM) in PBS buffer at\nstudies on mitochondria as drug target point are of increasing interest. In room temperature, the maximum emission wavelengths appear at 599\nthis paper, we investigated the effects of the three complexes on tar\u00ad nm for Ir1, 629 nm for Ir2 and 609 nm for Ir3. The stability of the\ngeting mitochondria, impact on mitochondrial and DNA damage, complexes in PBS solution was detected at 0 and 24 h, as shown in\napoptosis mediated by intracellular ROS levels. We exploited RNA- Fig. S1c (supporting information), no change of the peak shape was\nsequence analysis to find the apoptotic pathways. Additionally, the found, which suggests that the complexes are stable. The purity of the\nantitumor activity in vivo was also explored. complexes was investigated by HPLC using methanol and water (Vme\u00ad\n thanol:VH2O = 85:15 for Ir1, 90:10 for Ir2 and 90:10 for Ir3) as mobile\n2. Results and discussion phase, during a period of 30 min, only a peak was observed, indicating\n that the complexes are pure (Fig. S1d) (supporting information), the\n2.1. Synthesis and characterization values of the purity for Ir1, Ir2 and Ir3 are 99.53, 96.85 and 98.28%,\n respectively.\n The ligand DTOIP was prepared through condensation of 1,10-phe\u00ad\nnanthroline-5,6-dione with (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trime\u00ad 2.2. pKa determination\nthylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenal in the presence of NH4Ac\nand ethanol and refluxed for 6 h. After cooling to room temperature, the Charge is an important characteristic of drug molecules, since ioni\u00ad\nbrown precipitate was obtained. The corresponding iridium(III) com\u00ad zation sites determine the pKa at a particular pH. The pKa in turn can\nplexes were synthesized by direct reaction of DTOIP with the appro\u00ad affect many parameters, including solubility, dissolution rate, reaction\npriate precursor complexes in a mixture of methanol/dichloromethane kinetics, formulation, cell permeability, tissue distribution, renal elim\u00ad\n(Scheme 1) and purified by column chromatography. In the HRMS ination, metabolism, protein binding and receptor interactions [27]. The\nspectra, the determined molecular weights of the complexes are pKa value of the compound is a unique parameter specific to the\nconsistent with the expected values. In the 1H NMR spectra, the five chemical structure. According to Manallack, the pKa distribution of\nsingle peaks of chemical shifts less than 3 ppm are assigned to the five drugs is mainly related to the nature of its functional groups, and the\nmethyl groups, while chemical shift of 6.55 (s, 1H) in the ligand is compounds with minimally one charge with a pKa < 4 for acids and\nattributed to the hydrogen at the position of C (h), the peak for the correspondingly a pKa > 10 for bases can\u2019t cross the blood-brain barrier\nproton on nitrogen atom of the imidazole ring was not observed. This by passive diffusion [28]. The transport and distribution of most drugs\nmay be caused by metal coordination inducing electron deficiency in the are affected by passive diffusion, which depends on lipophilicity,\n\n\n\n\n Scheme 1. Synthetic route for the ligand and complexes Ir1, Ir2 and Ir3.\n\n 2\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\nbecause of the need to cross the lipid barrier [29\u201331]. It is well known Table 2\nthat the un-ionized form of drug is usually lipid soluble and diffuses Uptaken amounts of the complexes in the cells and intracellular distribution (ng\nreadily across cell membranes. The pH range of the extracellular envi\u00ad metal/106 cells).\nronment of tumor cells is about 6.7 [32]. The pH values of the complexes complex uptaken amount cytoplasm mitochondria nuclei\n(100 \u03bcM) were determined to be 6.44 for Ir1, 6.52 for Ir2 and 6.25 for Ir1 168.00 \u00b1 4.23 38.00 \u00b1 2.31 3.87 \u00b1 0.64 2.01 \u00b1 0.15\nIr3 dissolved in acetonitrile/water mixtures (v/v, 3:7). The pH was Ir2 51.67 \u00b1 2.35 10.30 \u00b1 1.02 2.11 \u00b1 0.25 1.01 \u00b1 0.04\ntitrated by potentiometric titration with hydrochloric acid (100 mM) to Ir3 102.33 \u00b1 3.55 29.83 \u00b1 2.73 1.74 \u00b1 0.11 1.33 \u00b1 0.21\nreduce the pH to about 2.0. Then KOH solution (60 mM) was slowly\ntitrated into the above solution and the change of pH was recorded. The\n amounts of Ir1, Ir2 and Ir3 are 168.00 \u00b1 4.23, 51.67 \u00b1 2.35 and 102.33\npKa value was obtained by plotting pH versus KOH volume. As shown in\n \u00b1 3.55 ng metal/106 cells, which follows the order of Ir1 > Ir3 > Ir2,\nFig. S2 (supporting information), the pKa values are 6.12 for Ir1, 6.18 for\n this is consistent with those of cytotoxic activity of the complexes\nIr2 and 6.32 for Ir3, respectively, indicating that Ir1, Ir2, Ir3 have low\n against B16 cells. In general, the mitochondria and nuclei are important\nionization in the extracellular environment of tumor cells and can easily\n targets for drug, after the drugs enter the cells, the drugs may accumu\u00ad\nenter the tumor cells.\n late in the cytoplasm, mitochondria and nuclei, hence, the intracellular\n distribution among the cytoplasm, mitochondria and nuclei was deter\u00ad\n2.3. In vitro cytotoxicity determination mined used ICP-MS by isolating pure cellular compartments. After a\n treatment of B16 cells with 20.0 \u03bcM of the complexes for 4 h, the\n The in vitro cytotoxicity of Ir1-Ir3 toward B16, BEL-7402, Eca-109 complexes enter the cell and most of the complexes accumulate in the\nand normal LO2 cell was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5- cytoplasm and to a smaller extent in the mitochondria and nuclei\ndiphenyltetrazolium bromide (MTT) method [33]. The obtained IC50 (Table 2). Moreover, we found that the total amounts of the complexes\nvalues are listed in Table 1, unexpectedly, the ligand DTOIP shows a Ir1, Ir2 and Ir3 distributing in the cytoplasm, mitochondria and nuclei\ncertain degree cytotoxicity, but its cytotoxic activity is lower than those are smaller than those of the cellular uptaken amounts. According to our\nof iridium(III) complexes Ir1, Ir2 and Ir3. Complexes Ir1, Ir2, Ir3 previous work, we discovered that the iridium(III) complexes [Ir\nexhibit high cytotoxic efficacy against the selected cancer cells. In (ppy)2(PEYIP)](PF6) (PEYIP = 2-phenethynyl-1H-imidazo[4,5-f][1,10]\nparticular, the complexes show very high cytotoxic activity toward B16 phenanthroline) [34] and [Ir(piq)2(IPPH)](PF6) (IPPH = (2S,3R,5S,\ncells with a low IC50 value of 0.4 \u00b1 0.1, 2.0 \u00b1 0.1, 1.4 \u00b1 0.09 \u03bcM for Ir1, 6R)-2-(2-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenoxy)-6-(hy\u00ad\nIr2 and Ir3, respectively. Moreover, the cytotoxic activity of the com\u00ad droxymethyl)tetrahydro-2H-pyran-3,4,5-triol) [35] locate at the endo\u00ad\nplexes against B16, BEL-7402 and Eca-109 is higher than those of plasmic reticulum, while complexes [Ir(ppy)2(CPIP)](PF6) (CPIP =\ncisplatin and [Ir(piq)2(NPIP)](PF6) (IC50 = 5.9 \u00b1 0.5 \u03bcM, against B16) 2-(4-chlorophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline) [36] and\n[9], but their cytotoxicity is lower toward BEL-7402 cells than our [Ir(ppy)2(HMNPIP)](PF6) (HMNPIP = 2-(1H-imidazo[4,5-f][1, 10]phe\u00ad\nprevious iridium(III) complex [Ir(ppy)2(dcdppz)](PF6) (dcdppz = 11, nanthroline-3-yl)-6-methoxy-4-nitrophenol) [37] enter the lysosomes.\n12-dichlorodipyrido[3,2-a:2\u2032 ,3\u2032 -c]phenazine) [15]. According to Hence, besides entering the cytoplasm, mitochondria and nuclei, it is\nTable 1, the anticancer activity of the complexes follows the order of Ir1 possible for the complexes to enter the lysosomes and endoplasmic re\u00ad\n> Ir3 > Ir2 toward B16, BEL-7402, Eca-109 and normal LO2 cells. ticulum, which results in a difference between the total cellular uptaken\nBecause the complexes show the highest cytotoxic efficacy against B16 amounts and total amounts of the complexes distributing in the cyto\u00ad\ncell among the selected cancer cells, this cell line was selected for the plasm, mitochondria, and nuclei.\nfollowing cell experiments.\n 2.5. Inhibitory proliferation of iridium(III) complexes in 3D cells culture\n2.4. Cellular uptake and intracellular drug distribution\n The cells are usually cultured as monolayers on a flat surface, and\n The pKa values show that the complexes can enter the cell, the these conditions do not accurately reflect what is happening in vivo\nanticancer efficacy is closely related to the uptaken amount of the because proper tissue structure and cell-cell contact are lost in this two-\ncomplexes by the cells. Therefore, the cell uptake of Ir1, Ir2, and Ir3 by dimensional (2D) systems [38,39]. Three dimensional (3D) cells culture\ntumor cells was qualitatively and quantitatively studied under fluores\u00ad can better simulate the in vivo environment, which plays a very\ncence microscopy and inductively coupled plasma mass spectrometry important role in the research of anti-cancer drugs [40,41]. Qualitative\n(ICP-MS), respectively. As shown in Fig. S3a (supporting information), evaluation of the effect of Ir1-3 on the growth of B16 cells was per\u00ad\nafter B16 cells were exposed to IC50 concentration of the complexes for formed with live/dead staining (Calcein-AM/Hoechst 33342/PI). As\n4 h, the cell nuclei were stained blue with 2-(4-amidinophenyl)-6- shown in Fig. 1, B16 cells treated with IC50 concentration of Ir1, Ir2 and\nindolecarbamidine dihydrochloride (DAPI), the complexes emit green Ir3 for 24 h showed the number of live cells (green fluorescence) stained\nfluorescence, no complete overlap of the green and blue fluorescence by Calcein-AM gradually decreased, and the number of dead cells\nwas discovered, indicating that the complexes can enter the cells and stained with red fluorescence by PI gradually increased compared with\nmainly distribute in the cytoplasm. those of untreated cells. The results indicate that the complexes can\n In addition, to quantitatively explore the cellular uptake levels of Ir1, effectively inhibit the cell proliferation in B16 cells.\nIr2 and Ir3, the accumulation of the three complexes in B16 cells was\ndetermined by ICP-MS. After a 4 h exposure of B16 cells to 20.0 \u03bcM 2.6. Cell cycle arrest determination\ncomplexes, the uptaken amounts are listed in Table 2, the uptaken\n The cell cycle is closely related to the proliferation and development\nTable 1 of tumor cells. Cell cycle arrest is commonly observed in many anti\u00ad\nIC50 values (\u03bcM) of iridium(III) complexes toward selected cancer cells for 48 h. tumor drugs [42,43]. Therefore, the drugs inhibit the proliferation of\n Complexes B16 BEL-7402 Eca-109 LO2 cancer cells through inhibiting the mitotic process of tumor cells. To\n DTOIP 2.6 \u00b1 0.03 35.0 \u00b1 4.1 19.1 \u00b1 3.3 12.7 \u00b1 0.3 evaluate the mechanism of the complexes inhibiting the cell growth, we\n Ir1 0.4 \u00b1 0.1 5.2 \u00b1 0.06 2.5 \u00b1 0.07 7.1 \u00b1 0.2 used flow cytometry to the effects of the complexes on the cell cycle\n Ir2 2.0 \u00b1 0.1 19.2 \u00b1 0.4 4.8 \u00b1 0.4 14.6 \u00b1 0.7 distribution. As shown in Fig. S4 (supporting information), the treat\u00ad\n Ir3 1.4 \u00b1 0.09 6.1 \u00b1 0.03 3.6 \u00b1 0.05 7.5 \u00b1 0.4 ment of B16 cells (a) with IC50 concentration of Ir1 (b), Ir2 (c) and Ir3\n Cisplatin 19.8 \u00b1 2.3 15.7 \u00b1 3.1 11.6 \u00b1 1.1 18.7 \u00b1 2.5\n (d) for 24 h resulted in an increase of 41.65, 40.26 and 41.69% in the cell\n\n 3\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\n\n\n Fig. 1. 3D model of B16 cells stained with calcein AM and PI after a treatment with IC50 concentration of the complexes for 24 h.\n\n\nat G0/G1 phase, accompanied by a corresponding reduction of per\u00ad reference. The green fluorescence intensity was calculated according to\ncentage in the cell at S or G2/M phase. The results indicate that the the following equation:\ncomplexes inhibit the cell growth at the G0/G1 phase. In our previous\nwork, we found that the complexes [Ir(piq)2(NPIP)](PF6) inhibit the cell Igreen fluorescence = Icomplex in the cell+JC-1 \u2013 Icomplex in the cells\ngrowth at G2/M phase against B16 cells [9]. Therefore, we think that the Where Icomplex in the cell+JC-1 stands for the cells were treated with the\ndifferent iridium(III) complexes inhibit the cell growth at the different complexes and then JC-1 was added, whereas Icomplex in the cells stands for\nphase toward the same cancer cells. the cells were treated with the complex (without JC-1).\n As shown in Fig. S5c (supporting information), compared with the\n control, incubation of B16 cells with CCCP, IC50 concentration of Ir1,\n2.7. Mitochondrial depolarization\n Ir2, Ir3 led to a reduction of the ratio (R) of red/green fluorescence.\n These data also confirm that Ir1, Ir2, and Ir3 can cause a decrease in\n Mitochondria play a crucial role in apoptosis, necrosis, autophagy,\n MMP.\nand secretion of calcium ions [44]. The mitochondrial membrane po\u00ad\ntential (MMP, \u0394\u03a8m) is a fundamental characteristic reflecting the\nintegrity of mitochondria [45]. The distribution of the complexes show 2.8. Intracellular reactive oxygen species (ROS) levels\nthat the complexes locate at the mitochondria, we also used microscope\nto observe the colocation of the complexes at the mitochondria. As 2\u2032 ,7\u2032 -dichlorofluorescindiacetate (DCFH-DA) can cross the cell\nshown in Fig. S5a (supporting information), the mitochondria were membrane and is hydrolyzed by intracellular esterase to non-fluorescent\nstained red with Mirotracker Red, while the complexes emit green 2\u2032 ,7\u2032 -dichloro-3,6-fluorandiol (DCFH). In the presence of reactive oxy\u00ad\nfluorescence, the overlap of the red and green fluorescence indicates gen species (ROS), DCFH is oxidized to the highly fluorescent dichlor\u00ad\nthat the complexes locate at the mitochondria. The Pearson\u2019s colocali\u00ad ofluorescein (DCF) [49]. Mitochondrial dysfunction are usually\nzation coefficients (PCC) were calculated by analyzing the red and green accompanied by the change of intracellular reactive oxygen species\nfluorescence intensity (Image pro plus 6.0 software) in 50 cells accord\u00ad (ROS) levels, high levels of ROS can activate a variety of signal pathways\ning to literature [46]. The PCC values are 0.96 for Ir1, 0.97 for Ir2 and which results in apoptosis and induce cell death [50,51]. To detect the\n0.95 for Ir3, which indicated an existence of positive correlation. change of intracellular ROS level, DCFH-DA was used as fluorescent\n The loss of MMP is characterized by an increase in green fluorescence probe. As shown in Fig. S6a (supporting information), the green fluo\u00ad\n(JC-1 monomer) and a decrease in red fluorescence (JC-1 aggregates) rescence of B16 cells treated with IC50 concentration of Ir1, Ir2, and Ir3\n[47,48]. As shown in Fig. S5b (supporting information), incubation of for 24 h increased significantly compared with that in the control, which\nB16 cells with IC50 concentration of Ir1, Ir2 and Ir3 causes a decrease in indicates that Ir1, Ir2, and Ir3 can induce the cells to produce more ROS\nmitochondrial membrane potential, we observed that the red fluores\u00ad and thus mediate apoptosis of B16 cells. To quantitatively compare the\ncence intensity decreased, and the green fluorescence increased effect, the green fluorescence intensity was detected using flow cytom\u00ad\ncompared with the control. Owing to the complexes emitting weak etry (Fig. S6b, supporting information). Due to possible cross-color\ngreen fluorescence, it is possible for the complexes to cause a cross-color interference of weak green fluorescence emitted by the complexes\ninterference with JC-1 monomer. To exclude the disturbance of the with fluorescence probe, in the detection of intracellular ROS level,\ngreen fluorescence emitted by the complexes, we used flow cytometry to complexes were used as reference. The average green fluorescence in\u00ad\nquantitatively determine the red fluorescence (JC-1 aggregates) and tensity increased by 3.20, 3.95 and 5.30 times for Ir1, Ir2 and Ir3\ngreen fluorescence (JC-1 monomers), the complexes were used as compared with that in the control, respectively. These results further\n\n 4\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\nshow that Ir1, Ir2 and Ir3 can increase intracellular ROS levels. were downregulated, and 337 genes were upregulated in B16 cells\n treated with 2IC50 concentration of Ir1 for 24 h compared with the\n control group. Next, we performed Go analysis and found that system\n2.9. Glutathione metabolism\n development, multicellular organism development and intracellular\n signal transduction were all biological processes that were significantly\n Glutathione (GSH) is involved in scavenging reactive oxygen/nitro\u00ad\n enriched in the Ir1-treated cells (Fig. 2c). KEGG pathway enrichment\ngen species (ROS/RNS) and is an antioxidant [52]. Glutathione meta\u00ad\n analysis revealed that Ir1 affected several signaling pathways associated\nbolism intersects with most cell death pathways, such as apoptosis,\n with cancer, including glutathione metabolism, PI3K/AKT and p53\nautophagy, and necrosis [53]. High GSH levels shield cells from the\n signaling pathways (Fig. 2d).\nactivity of chemotherapeutic drugs [54]. Therefore, to potentiate the\nefficacy of drugs causing oxidative stress or avoiding acquired resistance\nto chemotherapy, we can further investigate the effect of the complexes\n 2.11. Activation of endogenous apoptotic pathway\non GSH metabolism. As shown in Fig. S7a (supporting information), we\nfound that the GSH levels of cells treated by 2IC50 concentrations of Ir1,\n External aggression by a chemical compound sensed by the cells\nIr2, and Ir3 for 24 h were significantly lower than those of the control\n causes them to undergo two major forms of death, necrosis, and\ngroup. GSH reacts with lipid ROS and then forms glutathione disulfide\n apoptosis [55]. Potential of Ir1-Ir3 to induce B16 cell death was\n(GSSG), a decrease of ratio of GSH/GSSG is considered to be a significant\n explored by AnnexinV/PI dual-labeling method. As shown in Fig. 3a, the\nindicator of oxidative stress. Hence, we also determined the ratio of\n percentage of early apoptotic cells (Q3) and late apoptotic cells (Q2)\nGSH/GSSG, as shown in Fig. S7b (supporting information), the ratio of\n from 5.20% in the control (I) to 17.25% for Ir1 (II), 15.97% for Ir2 (III)\nGSH/GSSG reduced compared with that in the control after B16 cells\n and 16.07% for Ir3 (IV), respectively. The increase in apoptotic popu\u00ad\nwere exposed to 2IC50 concentrations of Ir1, Ir2, and Ir3 for 24 h,\n lation indicates the ability of complexes Ir1, Ir2, Ir3 to induce apoptosis.\nindicating an enhancement of oxidative stress. The results indicate that\n The apoptotic ability follows the order of Ir1 > Ir3 > Ir2, which is line\nthe complexes can induce a decrease of intracellular GSH levels and an\n with those of cytotoxicity of the complexes toward B16 cells.\nincrease of oxidative stress.\n The RNA-sequencing analysis indicated that the complexes induce\n apoptosis via several signaling pathways. The apoptotic mechanism was\n2.10. RNA-sequencing analysis researched by Western blot analysis. The Bcl-2 protein family de\u00ad\n termines the commitment of cells to apoptosis [56]. The enhanced\n To explore the potential mechanism of action of Ir1 on B16 cells, we expression of Bcl-2 makes it difficult for hematopoietic cells to die and\nused bioinformatics to investigate the key signaling pathways and bio\u00ad promotes the accumulation of lymphocytes, which usually eventually\nlogical processes in which the complex exerts its anti-tumor activity. We leads to cancer death [57,58]. Caspase 3 is a major mediator of apoptosis\nconstructed volcano (Fig. 2a) and heat map (Fig. 2b) to show the dis\u00ad activated during cellular exposure to cytotoxic drugs [59]. Many studies\ntribution and expression of these genes. Fig. 2b shows that 306 genes have shown that caspase 3 promotes stress-induced cancer cell growth,\n\n\n\n\nFig. 2. Bioinformatics identified key pathways and biological processes associated with Ir1 sensitivity in B16 cells. (a) Volcano map of Ir1. (b) Heat map of Ir1 (inset:\nthe number of up or downregulated genes). (c) GO analysis. (d) Kyoto Encyclopedia of Genes and Genomes pathway analyses.\n\n 5\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\n\n\nFig. 3. (a) Apoptosis percentage of B16 (I) induced by IC50 concentration of Ir1 (II), Ir2 (III) and Ir3 (IV) for 24 h, Q2, Q3, Q4 stand for late, early apoptosis and\nliving cells. (b and c) Expression of cleaved PARP, PI3K, AKT, caspase 3, Bcl-2 and Bax in B16 cells exposure to IC50 concentration of Ir1, Ir2 and Ir3 for 24 h.\n\n\ncellular migration, invasiveness, and tumor angiogenesis [60\u201362]. Poly control after a 24 h exposure of B16 cells to IC50 concentration of Ir1, Ir2\nADP-ribose polymerase (PARP) is one of the most important substrates and Ir3. The enhancement of the green fluorescence shows an increase\nof caspase 3, which is associated with DNA repair and gene integrity of \u03b3-H2AX level. In addition, we further investigated the expression of\n[63]. Initiator caspases lead to the processing of executioner caspase 3, \u03b3-H2AX. See from Fig. S8b (supporting information), the complexes can\nsubsequently mediating the apoptotic cascade, including PARP cleavage up-regulate the expression of \u03b3-H2AX. All these results indicate that Ir1,\n[64]. Many studies have shown that PI3K (phosphatidylinositol 3-kina\u00ad Ir2 and Ir3 can remarkably increase the \u03b3-H2AX level, which further\nse)/AKT (protein kinase B) signaling pathway components are demonstrates that the complexes can cause DNA damage, finally lead to\nfrequently altered in human cancers and that the PI3K/AKT pathway apoptosis.\nmay play a decisive role in drug resistance phenotypes. In addition, AKT\nand PI3K have gene amplification in tumor cells [65], hence, we need to\ninhibit the expression of AKT and PI3K in tumor cells. As shown in 2.13. Antitumor activity in vivo\nFig. 3b and c, Bax (Bcl2-associated X) expression increased, PARP\ncleavage occurred, and Bcl-2, caspase 3, PI3K and AKT expression B16 cells with high activity were inoculated subcutaneously in mice\ndecreased in B16 cells treated with IC50 concentration of Ir1, Ir2 and Ir3 and then grew into spherical tumors. To evaluate the antitumor efficacy,\nfor 24 h. Taken together, we deduced that Ir1, Ir2, and Ir3 can regulate mice were injected with saline and Ir1. In addition, the body weights of\nthe expression of apoptosis-related proteins and finally induce cell death mice and the tumor size were also monitored every day. From Fig. 4a,\nthrough the intrinsic apoptotic pathway. and 4d, tumors in the Ir1 (2 mg/kg), Ir1 (3 mg/kg) and Ir1 (5 mg/kg)\n treated groups were significantly diminished compared to those in the\n saline group. Fig. 4b shows the relative tumor volumes in the saline and\n2.12. DNA damage experiment Ir1 (2 mg/kg) treated groups, the tumors of the saline group grew\n rapidly and the relative tumor volumes of the mice in the saline group\n Ataxia-telangiectasia mutated proteins (ATM) plays a critical role in were significantly larger than those in the Ir1 (2 mg/kg) treated group\nDNA damage signaling originating at DNA double-strand breaks (DSBs) on 8th day. Fig. 4c shows that no significant changes were found in the\n[66]. ATM can phosphorylate various targets to promote the cellular body weight of mice in the saline and Ir1 (2 mg/kg) treatment groups\nresponse to damage. An important ATM target in chromatin is the his\u00ad during the treatment of 8 days. In addition, on the 9th day, in the Ir1-\ntone protein H2AX. The phosphorylated form of H2AX, is known as treated group, the drug of Ir1 (2 mg/kg) was not injected into the mice,\n\u03b3-H2AX [67]. The increase of \u03b3-H2AX is a marker of DNA cleavage. As we continue to observe the change of tumor volume and the weight of\nshown in Fig. S8a (supporting information), we can clearly observe that the mice, as shown in Fig. 4b, the relative tumor volume of mice in the\nmore bright green fluorescence was found compared with that in the Ir1 (2 mg/kg) treated group increased rapidly after drug\n\n 6\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\n Fig. 4. The in vivo antitumor activity of Ir1 (2 mg/\n kg), Ir1 (3 mg/kg) and Ir1 (5 mg/kg) toward B16\n xenograft model. (a) Photographs of tumor in the\n vehicle group and Ir1 (2 mg/kg) treatment group. (b)\n Relative tumor volumes and (c) mean weight of mice\n in the vehicle group and Ir1 (2 mg/kg) treatment\n group. (d) Photographs of tumor in the vehicle group\n and Ir1 (3 mg/kg, 5 mg/kg) treatment group. (e)\n Relative tumor volumes and (f) mean weight of mice\n in the vehicle group and Ir1 (3 mg/kg, 5 mg/kg)\n treatment group. (g) H&E staining of lung, liver,\n brain, spleen, kidney, heart, and tumor. (h) \u03b3-H2AX\n staining of tumor sections. (i and j) H-Score and Area\n density in the vehicle, 3 and 5 mg/kg of Ir1.\n\n\n\n\ndiscontinuation, and it was approach to the relative tumor volume of respectively, which also indicated that Ir1 (5 mg/kg) showed a high\nmice in the saline group on the 18th day after drug discontinuation. antitumor efficacy.\nFrom Fig. 4c, it was found that the body weight of mice in the saline To assess the toxic effects of Ir1 in vivo, major organs (lung, liver,\ngroup and Ir1 (2 mg/kg) treated group showed an increasing trend after brain, spleen, kidney, heart) and tumors were stained by H&E staining in\ndrug discontinuation. Fig. 4e shows the relative tumor volumes of mice the saline, Ir1 (3 mg/kg) and Ir1 (5 mg/kg) drug-treated groups to\nin the saline, Ir1 (3 mg/kg) and Ir1 (5 mg/kg) treated groups. The tu\u00ad determine the potential toxicity. As shown in Fig. 4g, compared with the\nmors in the saline group grew rapidly, and the relative tumor volumes of saline-treated group, Ir1-treated groups exhibited no significant damage\nmice in the saline group were significantly larger than those in the Ir1 (3 in the lung, liver, brain, kidney, and heart, only a massive proliferation\nmg/kg) and Ir1 (5 mg/kg) drug-treated groups on the 9th day. Fig. 4f of extramedullary hematopoietic cells within the red marrow was\nshows that no significant changes were found in the body weight of mice observed in the spleen. The above results suggest that Ir1 has a good\nin the saline group, Ir1 (3 mg/kg) and Ir1 (5 mg/kg) drug-treated groups antitumor effect on B16 cells in vivo and is a powerful antitumor\nduring the treatment. Taken together, the above experimental results candidate for B16 cells. Since \u03b3-H2AX is an important marker of DNA\nshowed that Ir1 showed obvious inhibition of tumor growth, indicating damage, \u03b3-H2AX immunostaining can provide more insight into the\nthat Ir1 can exert a high inhibitory effect in vivo, while the effect of Ir1 anti-tumor mechanism. As shown in Fig. 4h, comparing with \u03b3-H2AX in\non the body weight of mice during treatment was close to that of the tumor sites of the saline group, Ir1 (3 mg/kg) and Ir1 (5 mg/kg) treated\nsaline group, indicating that Ir1 exerts anti-tumor efficacy with less groups enhanced the expression of \u03b3-H2AX, indicating that Ir1 may\neffect on the body weight of mice. Ir1 (2 mg/kg), Ir1 (3 mg/kg) and Ir1 cause DNA damage to exert antitumor effects. In addition, tumors from\n(5 mg/kg) achieved an inhibitory rate of 42.57%, 56.14% and 71.67%, mice were stripped and analyzed for immunohistochemical positive area\n\n\n 7\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\nintensity. We assessed the expression of \u03b3-H2AX in tumors by calculating cells), BEL-7402 (human hepatocellular carcinoma) and normal LO2\nthe Histochemistry score (H-score) and area density. As can be seen from (human normal liver) cell lines were obtained from Sun Yat-Sen Uni\u00ad\nFig. 4i and j, both H-Score and area density are higher in the treated versity (Guangzhou). Fluorescent probes and assay kits were purchased\ngroup than those in the control group, and the values in 5 mg/kg group from Beyotime Biotechnology (Shanghai, China). IrCl3\u22c53H2O was ob\u00ad\nare greater than those in the 3 mg/kg group. These data indicate that Ir1 tained from the Kunming Institution of Precious Metals. 2-phenylpyri\u00ad\ncan significantly increase the expression level of \u03b3-H2AX in mouse dine, benzo[h]quinoline, 1-phenylisoquinoline, 1,10-phenanthroline,\ntumors. (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-\n 2,4,6,8-tetraenal, were purchased from Beijing HWRK Chemical Co., Ltd\n3. Conclusions (Beijing). The HRMS spectra were measured by direct injection with\n Waters Xevo G2-XS QTof mass analyzer. 1H NMR and 13C NMR spectra\n In this study, we synthesized a new ligand DTOIP and three novel were recorded on a Varian-500 spectrometer with dimethyl sulfoxide\niridium(III) complexes, Ir1-Ir3, which are highly cytotoxic to B16 cells (DMSO\u2011d6) as solvent and tetramethylsilane (TMS) as an internal stan\u00ad\nin vitro. The complexes can enter the cell in large quantities and can dard at 500 MHz at room temperature. UV\u2013Visible and emission spectra\naccumulate in the mitochondria. Meanwhile, the three complexes were measured in the UV-2550 ultraviolet spectrophotometer and RF-\ninduce B16 cell cycle arrest in G0/G1 phase, which eventually triggers 5301PC fluorescence spectrophotometer (Shimadzu, Japan).\napoptosis. Mechanistic studies have shown that these complexes can\ninduce apoptosis by depolarizing mitochondrial membranes, increasing 4.2. Synthesis of ligand and complexes\nintracellular ROS levels, decreasing intracellular GSH, causing DNA\ndamage. In addition, RNA-sequencing analysis shows that the complexes 4.2.1. Preparation of ligand DTOIP\ncause apoptosis via several pathways including glutathione metabolism, A mixture of 1,10-phenanthroline-5,6-dione (0.42 g, 2 mmol),\nPI3K/AKT and p53 signaling pathways. The Western blot analysis (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)nona-\n(PARP, PI3K, AKT, Caspase 3, Bcl-2 and Bax) confirmed that Ir1, Ir2 and 2,4,6,8-tetraenal (0.57 g, 2 mmol) in ethanol (40 mL) and ammonium\nIr3 complexes induce apoptosis through inhibition of PI3K/AKT acetate (3.08 g, 40 mmol) was refluxed at 78 \u25e6 C for 6 h. After cooling to\nsignaling pathway. The anti-tumor activity of Ir1 in vivo demonstrated room temperature, the yellow precipitate was washed three times with\nthat Ir1 has high anti-tumor activity with few toxic side effects and can ice water, the yellow solid was obtained. Yield: 80%. Anal Calcd for\nimprove the physical status of mice well. In summary, these results C32H34N4: C, 80.98, H, 7.22, N, 11.80%. Found: C, 80.82, H, 7.01, N,\nclearly indicate that these three complexes may induce apoptosis 11.98%. 1H NMR (DMSO\u2011d6, 500 MHz): \u03b4 9.01 (dd, 2H, Ha,a\u2019, J = 1.5, J\nthrough the mitochondria-mediated pathway, inhibition of PI3K/AKT = 4.0 Hz), 8.54 (d, 2H, Hc,c\u2019, J = 8.0 Hz), 7.82-7.79 (m, 2H, Hb,b\u2019), 6.97-\nsignaling pathway and the DNA damage pathway (Fig. 5). This work is 6.92 (m, 1H, Hj), 6.62 (d, 1H, Ho, J = 8.5 Hz), 6.55 (s, 1H, Hh), 6.33-6.25\nhelpful for the understanding the anticancer mechanism of iridium (m, 1H, Hk), 6.22-6.17 (m, 2Hl,n), 2.69 (s, 3H, Hv), 2.20 (s, 3H, Hw),\ncomplexes. 2.02-1.95 (m, 2H, Hr), 1.90 (s, 3H, Hx), 1.70 (s, 3H, Hy), 1.58-1.55 (m,\n 2H, Hs), 1.46-1.43 (m, 2H, Ht), 1.02 (s, 3H, Hz). 13C NMR (DMSO\u2011d6,\n4. Experimental 125 MHz): 152.3 (Ce,e\u2019), 149.7 (Ca,a\u2019), 145.6 (Cg), 143.9 (Ci), 139.4 (Cp),\n 139.2 (Cn), 138.9 (Cm), 133.9 (Cc), 133.3 (Cc\u2019), 132.5 (Cj), 131.5 (Cd),\n4.1. Materials and methods 131.2 (Cd\u2019), 129.7 (Cq), 128.8 (Cl), 127.0 (Co), 125.3 (Ck), 121.9 (Cf,f\u2019),\n 119.7 (Cb), 119.3 (Cb\u2019), 117.3 (Ch), 64.8 (Cu), 35.9 (Ct), 34.7 (Cr), 30.9\n Fetal Bovine Serum (FBS), newborn calf serum (NBCS), as well as the (Cv), 23.6 (Cs), 23.1 (Cy), 20.8 (Cz), 16.3 (Cw), 14.6 (Cx). HRMS (CH3CN)\nculture medium of RPMI (Roswell Park Memorial Institute) 1640 and for C32H34N4: m/z = 473.2803 ([M \u2212 H]\u2013).\nDMEM (Dulbecco\u2019s Modified Eagle Medium) were all purchased from\nGibco company. Ultrapure MilliQ water was used in all experiments. The 4.2.2. Synthesis of complex [Ir(ppy)2(DTOIP)] (PF6) (Ir1)\ncancer B16 (mouse melanoma cells), Eca-109 (human esophageal cancer Ir1 was prepared by refluxing cis-[Ir(ppy)2Cl]2 (0.292 g, 0.25 mmol)\n\n\n\n\n Fig. 5. Apoptotic mechanism of the complexes inducing B16 cells.\n\n 8\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\n[68] and DTOIP (0.171 g, 0.5 mmol) in 45 mL of methanol/dichloro\u00ad (C7,7\u2019), 136.5 (Cc,c\u2019), 133.7 (Cj), 132.2 (Cq), 130.5 (Cl), 130.5 (C12,12\u2019),\nmethane (VCH3OH:VCH2Cl2 = 1:2, v/v) for 6 h under argon. At the end of 129.7 (Co), 129.5 (C4,4\u2019)), 129.3 (C5,5\u2019), 128.5 (C8,8\u2019), 127.2 (C6,6\u2019), 126.9\nthe reaction, an excess of NH4PF6 was added and stirred for 2 h. The (C11,11\u2019), 126.7 (Ck,d,d\u2019), 126.4 (C9,9\u2019), 124.2 (C10,10\u2019), 122.7 (C2,2\u2019,f,f\u2019),\nsolution was filtered and concentrated to give a reddish brown solid. The 120.3 (Cb,b\u2019), 116.8 (Ch), 55.9 (Cu), 33.9 (Ct), 32.6 (Cr), 28.8 (Cv), 21.5\ncrude product was then purified by neutral alumina column chroma\u00ad (Cs), 19.0 (Cy), 18.7 (Cz), 14.3 (Cw), 12.6 (Cx). HRMS (CH3CN, Fig. S11c,\ntography using dichloromethane and acetone as eluents. The yellow supporting information) for C58H50N6PF6Ir: m/z = 1023.3757 ([M \u2212\npowder was obtained. Yield: 78%. Anal Cacld for C54H50N6PF6Ir: C, PF6]+).\n57.90, H, 4.50, N, 7.50%. Found: C, 57.55, H, 4.91, N, 7.86%. 1H NMR\n(DMSO\u2011d6, 500 MHz, Fig. S9a, supporting information): \u03b4 9.04 (d, 2H, 4.3. Purity determination of the complexes\nHa,a\u2019, J = 5.0 Hz), 8.25 (d, 2H, H1,1\u2019, J = 9.0 Hz), 8.11 (d, 2H, Hc,c\u2019, J =\n5.0 Hz), 8.04-8.01 (m, 2H, H8,8\u2019), 7.94 (d, 2H, H7,7\u2019, J = 8.0 Hz), 7.87 (t, The purity of the complexes was analyzed by COSMOSIL 5C18-MS-II\n2H, H8,8\u2019, J = 7.5 Hz), 7.48 (d, 2H, H10,10\u2019, J = 5.5 Hz), 7.06 (t, 4H, H9,9\u2019, column (250 mm \u00d7 10 mm) on 25 \u25e6 C. We used H2O with 0.1% tri\u00ad\n4,4\u2019, J = 7.5 Hz), 6.99-6.93 (m, 4H, H3,3\u2019,2,2\u2019), 6.67-6.43 (m, 2H, Hj,o), fluoroacetic acid (TFA) as mobile phase A and methanol with 0.1% TFA\n6.34-6.18 (m, 4H, Hh,k,l,n), 2.67 (s, 3H, Hv), 2.24 (s, 3H, Hw), 2.07-2.01 as mobile phase B with a flow rate of 3 mL/min. The different elution\n(m, 2H, Hr), 1.90 (s, 3H, Hx), 1.70 (s, 3H, Hy), 1.60-1.56 (m, 2H, Hs), programs eluted the complexes Ir1, Ir2 and Ir3. The elution program of\n1.48-1.43 (m, 2H, Ht), 1.02 (s, 3H, Hz). 13C NMR (DMSO\u2011d6, 125 MHz, Ir1 was H2O (0.1% TFA): MeOH (0.1% TFA) = 15:85, Ir2 was 10:90 and\nFig. S9b, supporting information): 166.9 (Ce,e\u2019), 152.4 (C5), 152.1 (C5\u2019), Ir3 was 10:90, and the detection wavelength was set at 257 nm, 291 nm\n150.6 (Ca), 150.4 (Ca\u2019), 149.1 (C1,1\u2019), 148.2 (C6,6\u2019), 145.4 (Cg), 143.9 and 253 nm.\n(Ci), 139.2 (Cp), 138.7 (Cn), 137.6 (C3), 137.3 (C3\u2019), 137.1 (Cm), 136.5\n(Cc), 136.1 (Cc\u2019), 135.4 (Cj), 132.2 (Cq), 132.1 (Cl), 131.7 (C8), 131.2 4.4. pKa determination\n(C8\u2019), 130.2 (C10,10\u2019), 129.9 (Co), 129.6 (C7), 129.3 (C7\u2019), 128.7 (Ck),\n127.2 (C9,9\u2019), 126.9 (C11,11\u2019), 125.0 (Cd,d\u2019), 123.8 (C4,4\u2019), 123.5 (Cf,f\u2019), The pKa values of the complexes were determined by potentiometric\n122.3 (Cb,b\u2019), 119.9 (Ch), 116.7 (C2,2\u2019), 56.0 (Cu), 33.9 (Ct), 32.6 (Cr), titration using a pH meter (Basic pH Meter PB-10, Sartorius), calibrated\n28.8 (Cv), 21.5 (Cs), 18.7 (Cy), 18.5 (Cz), 14.4 (Cw), 12.6 (Cx). HRMS with standard buffers of pH 4.01, 6.86 and 9.18. The complexes Ir1, Ir2\n(CH3CN, Fig. S9c, supporting information) for C54H50N6PF6Ir: m/z = and Ir3 (1.0 mM) were dissolved in acetonitrile and water (v/v, 3:7),\n975.3721 ([M \u2212 PF6]+). and adjusted pH values to 2 with hydrochloric acid (100 mM). Then,\n titration was then performed with KOH (60 mM).\n4.2.3. Synthesis of complex [Ir(piq)2(DTOIP)] (PF6) (Ir2)\n The complex [Ir(piq)2(DTOIP)](PF6) was prepared in an identical 4.5. Cell culture\nmanner described as Ir1, and cis-[Ir(ppy)2Cl]2 was replaced by cis-[Ir\n(piq)2Cl]2 [68]. Yield: 66%. Anal Calcd for C62H54N6PF6Ir: C, 61.02, H, B16, Eca-109, LO2 were cultured in Dulbecco\u2019s Modified Eagle Me\u00ad\n4.46, N, 6.87%. Found: C, 61.38, H, 4.84, N, 6.43%. 1H NMR (DMSO\u2011d6, dium (DMEM) containing 10% heat-inactivated fetal bovine serum\n500 MHz, Fig. S10a, supporting information): \u03b4 9.02 (d, 4H, Ha,a\u2019,1,1\u2019, J (FBS) and penicillin (100 U/ml)/streptomycin (0.1 mg/ml) (both\n= 7.5 Hz), 8.41 (d, 2H, Hc,c\u2019, J = 8.0 Hz), 8.03-7.89 (m, 6H, Hb,b\u2019,11,11\u2019,4, Gibco). BEL-7402 were cultured in Roswell Park Memorial Institute\n4\u2019), 7.88-7.85 (m, 6H, H2,2\u2019,5,5\u2019,6,6\u2019), 7.43 (d, 2H, H7,7\u2019, J = 6.5 Hz), 7.37 1640 (RPMI 1640) supplemented with 10% FBS, penicillin (100 U/mL)/\n(d, 2H, H14,14\u2019, J = 6.5 Hz), 7.18 (t, 2H, H12,12\u2019, J = 7.0 Hz), 6.96 (t, 2H, streptomycin (0.1 mg/mL).\nH13,13\u2019, J = 7.5 Hz), 6.67-6.44 (m, 2H, Hj,o), 6.34-6.18 (m, 4H, Hh,k,l,n),\n2.67 (s, 3H, Hv), 2.23 (s, 3H, Hw), 2.05-1.98 (m, 2H, Hr), 1.91 (s, 3H, Hx), 4.6. Cytotoxicity assay in vitro\n1.70 (s, 3H, Hy), 1.60-1.51 (m, 2H, Hs), 1.48-1.42 (m, 2H, Ht), 1.02 (s,\n3H, Hz). 13C NMR (DMSO\u2011d6, 125 MHz, Fig. S10b, supporting infor\u00ad Cytotoxicity of Ir1-Ir3 were evaluated against the cancer and normal\nmation): 167.9 (C9,9\u2019), 152.8 (Ce), 152.5 (Ce\u2019), 151.9 (Ca,a\u2019), 148.3 (C10, cell lines using the MTT assay. The various cell lines were seeded in 96-\n10\u2019), 148.3 (C1,1\u2019), 148.2 (Cg), 145.4 (Ci), 143.9 (Cp), 142.1 (Cn), 140.8 well plate at a density of 2000 cells/well overnight. Then, seven\n(Cm), 137.7 (C3), 137.5 (C3\u2019), 137.4 (Cj), 137.3 (C5), 137.2 (C5\u2019), 136.6 different concentrations (100, 50, 25, 12.5, 6.25, 3.125, 1.56 \u03bcM) of Ir1,\n(Cq), 132.1 (Cl), 131.7 (C12,12\u2019), 130.7 (C14), 130.6 (C14\u2019), 129.4 (Cc,c\u2019), Ir2, Ir3 and DTOIP (complexes and ligand were dissolved in DMSO, the\n128.8 (Co), 128.4 (C7,7\u2019), 127.7 (C11), 127.3 (C11\u2019), 127.0 (Ck), 126.5 final concentration of DMSO is 0.05%) were added to the wells indi\u00ad\n(C13,13\u2019), 125.6 (C6,6\u2019), 124.7 (C15,15\u2019), 123.6 (C4,4\u2019), 122.4 (C8,8\u2019), 122.2 vidually, after 48 h of incubation, MTT (9:1, v/v) was added into every\n(Cd,d\u2019), 116.7 (Cf,f\u2019), 114.7 (Cb,b\u2019,h), 111.8 (C2,2\u2019), 56.9 (Cu), 33.9 (Ct), well and incubated at 37 \u25e6 C for 4 h. The purple formazan product was\n32.7 (Cr), 28.9 (Cv), 21,6 (Cs), 19.1 (Cy), 18.8 (Cz), 14.4 (Cw), 12.7 (Cx). dissolved with 100 \u03bcL dimethyl sulfoxide. Finally, the cell viability was\nHRMS (CH3CN, Fig. S10c, supporting information) for C62H54N6PF6Ir: measured at a wavelength of 490 nm using a microplate reader. The\nm/z = 1075.4601 ([M \u2212 PF6]+). mean values were obtained through three independent experiments.\n\n4.2.4. Synthesis of complex [Ir(bzq)2(DTOIP)](PF6) (Ir3) 4.7. Cellular uptake\n Ir3 was synthesized in an identical manner described as Ir1 and cis-\n[Ir(ppy)2Cl]2 was replaced with cis-[Ir(bzq)2Cl]68 2. Yield: 77%. Anal B16 cells were inoculated in 12-well plates at a density of 4 \u00d7 104\nCalcd for C58H50N6PF6Ir: C, 59.63, H, 4.31, N, 7.19%. Found: C, 59.24, cells/well. The cells were treated with IC50 concentration of Ir1, Ir2 and\nH, 4.74, N, 7.55%. 1H NMR (DMSO\u2011d6, 500 MHz, Fig. S11a, supporting Ir3 for 4 h. Then the cells were stained with DAPI (1:100) incubated at\ninformation): \u03b4 9.05 (d, 2H, Ha,a\u2019, J = 8.0 Hz), 8.51 (d, 2H, H1,1\u2019, J = 8.0 37 \u25e6 C for 20 min in dark and observed under ImageXpress R Micro XLS\nHz), 8.10 (d, 2H, Hc,c\u2019, J = 4.5 Hz), 7.99-7.93 (m, 4H, H3,3\u2019,8,8\u2019), 7.92- System (MD company, USA).\n7.87 (m, 4H, H5,5\u2019,6,6\u2019), 7.57 (d, 2H, H10,10\u2019, J = 8.0 Hz), 7.45-7.43 (m, The cell uptake was quantitatively determined by inductively\n2H, H9,9\u2019), 7.22 (t, 2H, Hb,b\u2019, J = 7.0 Hz), 7.07-6.96 (m, 2H, H2,2\u2019) 6.64- coupled plasma-mass spectrometry (ICP-MS, Thermo Fisher Scientific\n6.45 (m, 2H, Hj,o), 6.32-6.17 (m, 4H, Hh,k,l,n), 2.67 (s, 3H, Hv), 2.22 (s, iCAP Qc). B16 cells (5 \u00d7 105 cells/well) were seeded in 6-well plate and\n3H, Hw), 2.04-1.98 (m, 2H, Hr), 1.91 (s, 3H, Hx), 1.70 (s, 3H, Hy), 1.60- incubated with 20.0 \u03bcM of Ir1, Ir2 and Ir3 for 4 h when the cells arrived\n1.55 (m, 2H, Hs), 1.48-1.43 (m, 2H, Ht), 1.03 (s, 3H, Hz). 13C NMR logarithmic phase. Then, washing the adherent cells twice with PBS\n(DMSO\u2011d6, 125 MHz, Fig. S11b, supporting information): 156.4 (Ce,e\u2019), containing 5 mM EDTA. After trypsinization and centrifugation of the\n152.4 (Ca,a\u2019), 148.8 (C1), 148.7 (C1\u2019), 147.2 (C13,13\u2019), 144.4 (Cg), 143.6 suspension at 800 rpm for 5 min, repeating the above processes till the\n(Ci), 141.8 (Cp), 140.3 (Cn), 138.3 (Cm), 137.5 (C3), 137.3 (C3\u2019), 137.1 residual complexes were completely removed. The cells were digested\n\n 9\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\nwith 60% HNO3 at 60 \u25e6 C to completely release the uptaken iridium(III) were washed three times with cool PBS and stained with 5,5\u2032 ,6,6\u2032 -tet\u00ad\ncomplexes from the cells and then a 5 mL solution was obtained by rachloro-1,1\u2032 -3,3\u2032 -tetrethylbenzimidalylcarbocyanine iodide (JC-1), the\nadding Milli-Q water. The uptaken mounts were calculated through the cells were then observed under the ImageXpress R Micro XLS System\nfollowing procedures: (I) determining the intensity (193Ir) of different (MD company, USA) and quantitatively analyzed by flow cytometry\nconcentrations of iridium standard solution, through linear fitting to (Beckman Instruments, NJ). Carbonylcyanide-m-chlorophenyl hydra\u00ad\nobtain a fitting equation (x-axis: concentration of sample; y-axis: in\u00ad zone (CCCP) was used as a positive control.\ntensity). (II) determining the intensity (193Ir) in the sample, then\ncalculate the uptaken amount according to the fitting equation. 4.12. Reactive oxygen species levels\n\n4.8. Intracellular drug distribution B16 cells were placed in confocal Petri dishes at a density of 5 \u00d7 104\n overnight. Then the cells were exposed to IC50 concentration of Ir1, Ir2\n Intracellular distribution of the complexes was tested using induc\u00ad and Ir3 for 24 h, thereafter, the cells were washed three times with cool\ntively coupled plasma mass spectrometry (ICP-MS). B16 cells treated PBS and stained with 2\u2032 ,7\u2032 -dichlorodihydrofluorescein diacetate (DCFH-\nwith 20.0 \u03bcM Ir1, Ir2 and Ir3 for 4 h were collected separately and DA, 1:1000) and observed under the laser confocal microscope (Leica,\nwashed four times with PBS containing EDTA. The number of cells were TCS SP8 SR).\nsame in each sample when detecting the distribution of the complexes in\nthe mitochondria, cytoplasm, nucleus. Then cytoplasm, nucleus and 4.13. Apoptosis assay by Annexin V-FITC/PI staining method\nmitochondria were extracted according to the corresponding organelle\nextraction kit (BestBio, Shanghai). All the samples were digested with When B16 cells in the 6-well plate entered the exponential growth\nnitric acid overnight and detected by ICP-MS. phase, the cells were treated with IC50 concentration of Ir1, Ir2 and Ir3\n for 24 h. After trypsinization and centrifugation, the cells were washed\n4.9. Cytotoxicity of iridium(III) complexes in 3D cells culture twice with PBS. Then, re-suspended in 195 \u03bcL of 1 \u00d7 Annexin V-FITC\n binding buffer. Next, 10 \u03bcL of propidium iodide (PI) and 5 \u03bcL of\n Add 150 \u03bcL of Matrigel matrix\uff088 mg/mL) to the bottom of the Annexin-FITC were sequentially added to each sample, and the cells\nconfocal dish, and then incubated at 37 \u25e6 C for 30 min. A549 cells in the were analyzed by flow cytometer (Beckman Instruments, NJ).\nexponential growth phase were trypsinized to obtain a single-cell sus\u00ad\npension. Approximately 4 \u00d7 104 diluted A549 cells were transferred to 4.14. Cell cycle arrest determination\nMatrigel-coated confocal dishes. Then 200 \u03bcL of the substrate mixture\n(0.8 mg/mL) was gently added to the confocal culture dish to form a gel- Exponentially growing B16 cells were exposed to IC50 concentration\ncell-gel structure and incubated continuously for 6 days. To determine of Ir1, Ir2 and Ir3 for 24 h in the 6-well plates, respectively. The cells\nthe cytotoxicity of iridium(III) complexes, the cells of 3D multicellular were trypsinized and fixed with 75% ethanol overnight at 4 \u25e6 C. Then,\ntumor spheroids cultured with IC50 concentration of Ir1, Ir2 and Ir3 for the cells were washed twice with cold PBS, re-suspended in 190 \u03bcL of\n24 h were stained with Calcein-AM/Hoechst 33342/PI and observed PBS buffer containing 4 \u03bcL of PI (1 mg/mL), 4 \u03bcL of RnaseA (10 mg/mL)\nunder a confocal microscopy (Leica, TCS SP8 SR). and 0.2 \u03bcL of Triton X-100. The cells were analyzed by flow cytometry\n (Beckman Instruments, NJ).\n4.10. RNA sequencing analysis\n 4.15. Glutathione (GSH) measurement\n B16 cells were inoculated in 6-well culture plates (5 \u00d7 105 cells) and\nincubated in a 37 \u25e6 C incubator for 24 h. The growing B16 cells were B16 cells were inoculated in 6-well plates (5 \u00d7 105 cells per well) and\nexposed to Ir1 at a concentration of 2IC50 for 24 h. Adherent cells were treated with 2IC50 concentration of Ir1, Ir2, Ir3 for 24 h. Cells were\nwashed twice using pre-chilled PBS. The appropriate amount of RNA collected by trypsinization and GSH levels were measured by GSH assay\nextraction solution (Servicebio, China) was added, and cells were well kit (Beyotime Biotech, Shanghai, China).\nblown using a pipette to lyse them adequately, followed by isolation of\ntotal RNA using Trizol Reagent (Invitrogen Life Technologies), after 4.16. DNA damage experiment\nwhich the concentration, quality and integrity were determined using a\nNanoDrop spectrophotometer (Thermo Scientific). Sequencing libraries The cells were seeded into 12-well plates overnight, the cells were\nwere generated using the TruSeq RNA Sample Preparation Kit (Illumina, treated with IC50 concentration of Ir1, Ir2, Ir3 for 24 h, Then, the cells\nSan Diego, CA, USA). To select cDNA fragments with a length of pref\u00ad were fixed with 75% ethanol and washed three times with PBS, then\nerably 200 bp, library fragments were purified using the AMpure XP added blocking buffer for immunol staining (Beyotime Biotechnology,\nsystem (Beckman Coulter, Beverly, CA, USA). The library fragments China) to block the cells for 30 min. Primary antibody was added and\nwere then quantified using Agilent high-sensitivity DNA analysis on a incubated overnight at 4 \u25e6 C, then fluorescent secondary antibody and\nBioAnalyst 2100 system (Agilent, Santa Clara, CA, USA). Finally, the Hoechst were added and incubated for 1 h at 37 \u25e6 C. Finally, the cells\nsequencing library was sequenced on a Hiseq platform (Illumina) by were observed under Xpress Micro XLS. Next, we used Western blot to\nShanghai Personal Biotechnology Cp. Ltd. analyze the expression of \u03b3-H2AX in B16 cells.\n\n4.11. Mitochondrial depolarization 4.17. Western blot analysis\n\n Mito tracker Red was used as a fluorescent probe to detect the drug B16 cells were seeded into 6-well plates (5 \u00d7 105 cells per well) for\nentering mitochondria. B16 cells incubated in confocal petri dishes were 24 h and incubated with IC50 concentration of the complexes for 24 h.\ntreated with IC50 concentration of Ir1, Ir2 and Ir3 for 4 h, respectively. Then the cells were harvested in lysis buffer. After sonication, the\nThen the cells were stained with Mito Tracker Red FM (ThermoFisher, samples were centrifuged for 20 min at 13,000 g. The protein concen\u00ad\n100 nM) and photographed under laser confocal microscope (Leica, TCS tration of the supernatant was determined by BCA (bicinchoninic acid)\nSP8 SR). assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis\n Mitochondrial membrane potential (MMP) assay: B16 cells were loaded an equal amount of protein per lane was performed. Gels were\nseeded into 12-well plates (5 \u00d7 105 cells) overnight. The cells were then transferred to poly (vinylidene difluoride) membranes (Millipore)\ntreated with IC50 concentration of Ir1, Ir2 and Ir3 for 24 h. The cells and blocked with 5% non-fat milk in TBST (20 mM Tris-HCl, 150 mM\n\n 10\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n\nNaCl, 0.05% Tween 20, pH 8.0) (Tris = tris(hydroxymethylamino\u00ad Bcl-2 B-cell lymphoma-2\nmethane, Tween = polyoxyethylene monolaurate sorbaitan) buffer for 1 BCA bicinchoninic acid\nh. The membranes were incubated with primary antibodies at 1:1000 bzq benzo[h]quinolone\ndilutions in 5% non-fat milk at 4 \u25e6 C overnight, and after washed four cccp carbonylcyanide-m-chlorophenylhydrazone\ntimes with TBST for a total of 30 min, then the secondary antibodies Calcein-AM calcein-acetoxymethylester\nwere conjugated with horseradish peroxidase at 1:1000 dilution for 1 h DMEM Dulbecco\u2019s Modified Eagle Medium\nat room temperature and washed for four times with TBST. The blots DMSO dimethylsulfoxide\nwere visualized with the Amersham ECL (electrochemiluminescence) DCHF 2\u2032 ,7\u2032 -dichloro-3,6-fluorandiol\nand western blotting detection reagents according to the manufacturer\u2019s DCHF-DA 2\u2032 ,7\u2032 -dichlorodihydrofluorescein diacetate\ninstructions. To assess the presence of a comparable amounts of proteins DCF dichlorofluorescein\nin each lane, the membranes were stripped finally to detect the \u03b2-actin. Eca-109 human esophageal cancer cells\n EDTA ethylene diamine tetraacetic acid\n4.18. Anti-tumor experiment in vivo FBS fetal bovine serum\n FITC fluorescein isothiocyanate\n C57BL/6 mice were provided by Guangdong Medical Laboratory HeLa human cervical cancer cells\nAnimal Center (Guangzhou, China). B16 cells were cultivated in Dul\u00ad ICP-MS inductively coupled plasma mass spectrometry\nbecco\u2019s modified Eagle medium (DMEM, Gibco) containing 10% fetal JC-1 5,5\u2032 ,6,6\u2032 -Tetrachloro-1,1\u2032 ,3,3\u2032 -tetraethyl-imidacarbocyanine\ncalf serum and penicillin (100 U/mL)/streptomycin (0.1 mg/mL) iodide\n(Gibco) at 37 \u25e6 C in 5% CO2. The mice were randomly divided into 3 LO2 human normal hepatocytes\ngroups (n = 5). To obtain the tumor model, B16 cells were subcutane\u00ad MTT 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide\nously injected into the underarm of each mouse. After 10 days, Ir1 (2, 3 MMP mitochondrial membrane potential\nand 5 mg/kg) was administered intraperitoneal (i.p.) daily. The tumor PBS phosphate buffer saline\nsize and the weight of the mice were recorded every day. At the end of piq 1-phenylisoquinoline\nthe administration, all mice were executed. The lung, heart, liver, brain, Hppy 2-phenylpyridine\nkidney, and spleen of mice were dissected and analyzed. The tumors PARP poly ADP-ribose polymerase\nwere excised, and the inhibitory rate was calculated according to the PI3K phosphatidylinositol-3-kinase\nfollowing equation: PI propidium iodide\n PMSF phenylmethylsulfonyl fluoride\n[(W1 \u2013 W2)/W1] \u00d7 100% RNase ribonuclease\n RIPA 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium\n W1 is the average tumor weight in the control group, W2 is the\n deoxycholate\naverage tumor weight in the treated group.\n ROS reactive oxygen species\n RPMI 1640 Roswell Park Memorial Institute 1640\n4.19. Statistical analysis SGC-7901 human gastric adenocarcinoma\n TMS tetramethylsilane\n Data are expressed as mean \u00b1 standard deviation (SD), and each Tris tris(hydroxymethyl)aminomethane\nvalue in this study was obtained from three independent experiments \u03b3-H2AX phosphorylated histone H2AX\nrepeated. *P < 0.05 was considered a statistically significant difference.\n References\nDeclaration of competing interest\n [1] B.J. Aragon-Ching, W.L. Dahut, Anti-angiogenesis approach to genitourinary\n cancer treatment, Update Cancer Therapeut. 3 (2009) 182\u2013188.\n The authors declare that they have no known competing financial [2] C. De Martel, J. Ferlay, S. Franceschi, Global burden of cancers attributable to\ninterests or personal relationships that could have appeared to influence infections in 2008: a review and synthetic analysis, Lancet Oncol. 13 (2012)\nthe work reported in this paper. 607\u2013615.\n [3] J. Hildebrandt, R. Trautwein, D. Kritsch, N. Ha\u0308fner, H. Go\u0308rls, M. D\u00fcrst, I.\n B. Runnebaum, W. Weigand, Synthesis, characterization and biological\nData availability investigation of platinum(II) complexes with asparagusic acid derivatives as\n ligands, Dalton Trans. 48 (2019) 936\u2013944.\n Data will be made available on request. [4] E.S. Antonarakis, A. Emadi, Cancer chemother, Pharmacology 66 (2010) 1\u20139.\n [5] J.J. Cao, Y. Zheng, X.W. Wu, C.P. Tan, M.H. Chen, N. Wu, L.N. Ji, Z.W. Mao,\n Anticancer cyclometalated iridium(III) complexes with planar ligands:\nAcknowledgements mitochondrial DNA damage and metabolism disturbance, J. Med. Chem. 62 (2019)\n 3311\u20133322.\n [6] Z.H. Wang, Z.X. Lv, X.C. Liu, Y.T. Yu, J.Y. Chang, R.X. Dong, C.Y. Li, X.A. Yuan,\n This work was supported by the National Nature Science Foundation Z. Liu, Anticancer application of ferrocene appended configuration-regulated half-\nof China (No 21877018). sandwich iridium(III) pyridine complexes, J. Inorg. Biochem. 237 (2022), 112010.\n [7] J. Masternak, A. Gilewska, B. Barszcz, I. \u0141akomska, K. Kazimierczuk, J. Sitkowski,\n J. Wietrzyk, A. Kamecka, M. Milczarek, Ruthenium(II) and iridium(III) complexes\nAppendix A. Supplementary data as tested materials for new anticancer agents, Materials 13 (2020) 3491.\n [8] H.W. Zhang, X.F. Liao, X.Y. Wu, C.L. Shi, Y.Y. Zhang, Y.H. Yuan, W.L. Li, J.\n Supplementary data to this article can be found online at https://doi. W. Wang, Y.J. Liu, Iridium(III) complexes entrapped in liposomes trigger\n mitochondria-mediated apoptosis and GSDME-mediated pyroptosis, J. Inorg.\norg/10.1016/j.ejmech.2022.115046. Biochem. 228 (2022), 111706.\n [9] Y.H. Yuan, C.L. Shi, X.Y. Wu, W.L. Li, C.X. Huang, L.J. Liang, J. Chen, Y. Wang, Y.\nAbbreviations J. Liu, Synthesis and anticancer activity in vitro and in vivo evaluation of iridium\n (III) complex on mouse melanoma B16 cells, J. Inorg. Biochem. 232 (2022),\n 111820.\nA549 human lung carcinoma [10] L.L. Wang, R.L. Guan, L.N. Xie, X.X. Liao, K. Xiong, T.W. Rees, Y. Chen, L.N. Ji,\nAKT protein kinase B H. Chao, An ER-targeting iridium(III) complex that induces immunogenic cell\n death in non-small-cell lung cancer, Angew Chem. Int. Ed. Engl. 60 (2021)\nB16 mouse melanoma cells 4657\u20134665.\nBEL-7402 human hepatocellular\nBax Bcl-2 associated x protein\n\n 11\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n[11] G. Gupta, S. Cherukommu, G. Srinivas, S.W. Lee, S.H. Mun, BODIPY-based Ru(II) [40] Y. Imamura, T. Mukohara, Y. Shimono, Y. Funakoshi, N. Chayahara, M. Toyoda,\n and Ir(III) organometallic complexes of avobenzone, a sunscreen material: potent N. Kiyota, S. Takao, S. Kono, T. Nakatsura, H. Minami, Comparison of 2D- and 3D-\n anticancer agents, J. Inorg. Biochem. 189 (2018) 17\u201329. culture models as drug-testing platforms in breast cancer, Oncol. Rep. 33 (2015)\n[12] Y.Y. Gu, H.Y. Wen, Y.Y. Zhang, L. Bai, Y. Zhou, H.W. Zhang, L. Tian, J. Hao, Y. 1837\u20131843.\n J. Liu, Studies of anticancer activity in vivo and in vitro behaviors of liposomes [41] B. Weigelt, C.M. Ghajar, M.J. Bissel, The need for complex 3D culture models to\n encapsulated iridium(III) complex, J. Biol. Inorg. Chem. 26 (2021) 109\u2013122. unravel novel pathways and identify accurate biomarkers in breast cancer, Adv.\n[13] W.J. Wang, Y.Y. Ling, Y.M. Zhong, Z.Y. Li, C.P. Tan, Z.W. Mao, Ferroptosis- Drug Deliv. Rev. 69\u201370 (2014) 42\u201351.\n enhanced cancer immunity by a ferrocene-appended iridium(III) diphosphine [42] C. Billecke, S. Finniss, L. Tahash, C. Miller, T. Mikkelsen, N.P. Farrell, O. Bo\u0308gler,\n complex, Angew Chem. Int. Ed. Engl. 61 (2022), e202115247. Polynuclear platinum anticancer drugs are more potent than cisplatin and induce\n[14] G. Gupta, P. Kumair, J.Y. Ryu, J. Lee, S.M. Mobin, C.Y. Lee, Mitochondrial cell cycle arrest in glioma, Neuro Oncol. 8 (2006) 215\u2013226.\n localization of highly fluorescent and photostable BODIPY-based ruthenium(II), [43] H. Wu, L. Chen, F. Zhu, X. Han, L. Sun, K. Chen, The cytotoxicity effect of\n rhodium(III), and iridium(III) metal complexes, Inorg. Chem. 58 (2018) resveratrol: cell cycle arrest and induced apoptosis of breast cancer 4T1 cells,\n 8587\u20138595. Toxins 11 (2019) 731.\n[15] W.G. Chen, X.H. Cai, Q. Sun, X.H. Guo, C.M. Liang, H. Tang, H.M. Huang, H. Luo, [44] S.M. Yoo, Y.K. Jung, A molecular approach to mitophagy and mitochondrial\n L.M. Chen, J.C. Chen, Design and synthesis of aptamer-cyclometalated iridium(III) dynamics, Mol. Cell. 41 (2018) 18\u201326.\n complex conjugate targeting cancer cells, Eur. J. Med. Chem. 236 (2022), 114335. [45] R.C. Scaduto Jr., L.W. Grotyohann, Measurement of mitochondrial membrane\n[16] Y.Y. Zhang, W.D. Fei, H.W. Zhang, Y. Zhou, L. Tian, J. Hao, Y.H. Yuan, W.L. Li, Y. potential using fluorescent rhodamine derivatives, Biophys. J. 76 (1999) 469\u2013477.\n J. Liu, Increasing anticancer effect in vitro and vivo of liposome-encapsulated [46] J. Adler, Quantifying colocalization by correlation: the Pearson correlation\n iridium(III) complexes on BEL-7402 cells, J. Inorg. Biochem. 225 (2021), 111622. coefficient is superior to the mander\u2019s overlap coefficient, I. Parmryd, Cytometry\n[17] J.S. Nam, M.G. Kang, J. Kang, S.Y. Park, S.J.C. Lee, H.T. Kim, J.K. Seo, O.H. Kwon, 77A (2010) 733\u2013742.\n M.H. Lim, H.W. Rhee, T.H. Kwon, J. Am. Chem. Soc. 138 (2016) 10968\u201310977. [47] J. Yang, J.X. Zhao, Q. Cao, L. Hao, D. Zhou, Z. Gan, L.N. Ji, Z.W. Mao,\n[18] C. Fu, Q. Lv, J. Fan, S.M. Wu, M. Lei, X. Zhang, X.R. Li, W. Zhou, Y. Yu, W.S. Ren, C. Simultaneously inducing and tracking cancer cell metabolism repression by\n Z. Zhao, G.J. Liao, Discovery of polypyridyl iridium(III) complexes as potent agents mitochondria-immobilized rhenium(I) complex, ACS Appl. Mater. Interfaces 9\n against resistant Candida albicans, Eur. J. Med. Chem. 233 (2022), 114250. (2017) 13900\u201313912.\n[19] R.P. Paitandi, R.K. Gupta, R.S. Singh, G. Sharma, B. Koch, D.S. Pandey, Eur. J. Med. [48] K. Elefantova, B. Lakatos, J. Kubickova, Z. Sulova, A. Breier, Detection of the\n Chem. 84 (2014) 17\u201329. mitochondrial membrane potential by the cationic dye JC-1 in L1210 cells with\n[20] J.P. Liu, C.Z. Jin, B. Yuan, X.G. Liu, Y. Chen, L.N. Ji, H. Chao, Chem. Commun. 53 massive overexpression of the plasma membrane ABCB1 drug transporter, Int. J.\n (2017) 2052\u20132055. Mol. Sci. 19 (2018) 1985.\n[21] Y.Y. Zhang, Y. Zhou, H.W. Zhang, L. Tian, J. Hao, Y.H. Yuan, W.L. Li, Y.J. Liu, [49] C.P. LeBel, H. Ischiropoulos, S.C. Bondy, Evaluation of the probe 2\u2019,7\u2019-\n DNA-binding and evaluation of anticancer activity in vitro and in vivo of iridium dichlorofluorescin as an indicator of reactive oxygen species formation and\n (III) polypyridyl complexes, J. Inorg. Biochem. 224 (2021), 111580. oxidative stress, Chem. Res. Toxicol. 5 (1992) 227\u2013231.\n[22] B.B. Chen, N.L. Pan, J.X. Liao, M.Y. Huang, D.C. Jiang, J.J. Wang, H.J. Qiu, J. [50] E. Georgieva, D. Ivanova, Z. Zhelev, R. Bakalova, M. Gulubova, I. Aoki,\n X. Chen, L. Li, J. Sun, J. Inorg. Biochem. 219 (2021), 111450. Mitochondrial dysfunction and redox imbalance as a diagnostic marker of \u201cfree\n[23] Y. Liu, B. Liu, C.X. Xu, L. He, Y.C. Wan, L.N. Ji, Z.W. Mao, Mitochondria-targeted radical diseases\u201d, Anticancer Res. 37 (2017) 5373\u20135381.\n phosphorescent cyclometalated iridium(III) complexes: synthesis, characterization, [51] Y. Meng, C.W. Chen, M.M.H. Yung, W. Sun, J. Sun, Z. Li, J. Li, Z. Li, W. Zhou, S.\n and anticancer properties, J. Biol. Inorg. Chem. 25 (2020) 597\u2013607. S. Liu, A.N.Y. Cheung, H.Y.S. Ngan, J.C. Braisted, Y. Kai, W. Peng, A. Tzatsos, Y. Li,\n[24] F.L. Xie, Z.T. Huang, L. Bai, J.W. Zhu, H.H. Xu, Q.Q. Long, Q.F. Guo, Y. Wu, S. Z. Dai, W. Zheng, D.W. Chan, W. Zhu, DUOXA1-mediated ROS production\n H. Liu, Antitumor activity studies of iridium(III) polypyridine complexes-loaded promotes cisplatin resistance by activating ATR-Chk1 pathway in ovarian cancer,\n liposomes against gastric tumor cell in vitro, J. Inorg. Biochem. 225 (2021), Cancer Lett. 428 (2018) 104\u2013116.\n 111603. [52] C. Espinosa-Diez, V. Miguel, D. Mennerich, T. Kietzmann, P. Sa\u0301nchez-Pe\u0301rez,\n[25] G. Ludwig, S. Mijatovic\u0301, I. RanCelovic\u0301, M. Bulatovic\u0301, D. Miljkovic\u0301, D. Maksimovic\u0301- S. Cadenas, S. Lamas, Antioxidant responses and cellular adjustments to oxidative\n Ivanic\u0301, M. Korb, H. Lang, D. Steinborn, G.N.K. Cerovic\u0301, Eur. J. Med. Chem. 69 stress, Redox Biol. 6 (2015) 183\u2013197.\n (2013) 216\u2013222. [53] A.L. Ortega, S. Mena, J.M. Estrela, Glutathione in cancer cell death, Cancers 3\n[26] J.Z. Wu, B.H. Ye, L. Wang, L.N. Ji, J.Y. Zhou, R.H. Li, Bis(2,2\u2032 -bipyridine) (2011) 1285\u20131310.\n ruthenium(II)complexes with imidazo[4,5-f][1,10]-phenanthroline or 2- [54] E. Desideri, F. Ciccarone, M.R. Ciriolo, Targeting glutathione metabolism: partner\n phenylimidazo[4,5-f][1,10]phenanthroline, J. Chem. Soc. Dalton Trans. 8 (1997) in crime in anticancer therapy, Nutrients 11 (2019) 1926.\n 1395\u20131402. [55] Q.Y. Yi, W.Y. Zhang, M. He, F. Du, W.Z. Wang, Y.J. Wang, Y.Y. Gu, L. Bai, Y.J. Liu,\n[27] D.J. Adams, L.R. Morgan, Tumor physiology and charge dynamics of anticancer Anticancer and antibacterial activity in vitro evaluation of iridium(III) polypyridyl\n drugs: implications for camptothecin-based drug development, Curr. Med. Chem. complexes, J. Biol. Inorg. Chem. 24 (2019) 151\u2013169.\n 18 (9) (2011) 1367\u20131372. [56] P.E. Czabotar, G. Lessene, A. Strasser, J.M. Adams, Control of apoptosis by the BCL-\n[28] G.H. Lu, X.S. Miao, L. Dou, Crosstalk of physiological pH and chemical pKa under 2 protein family: implications for physiology and therapy, Nat. Rev. Mol. Cell Biol.\n the umbrella of physiologically based pharmacokinetic modeling of drug 15 (2014) 49\u201363.\n absorption, distribution, metabolism, excretion, and toxicity, Expet Opin. Drug [57] D.L. Vaux, S. Cory, J.M. Adams, Bcl-2 gene promotes haemopoietic cell survival\n Metabol. Toxicol. 17 (2021) 1103\u20131124. and cooperates with c-myc to immortalize pre-B cells, Nature 335 (1988) 440\u2013442.\n[29] A. Avdeef, Physicochemical profiling (solubility, permeability and charge state), [58] T.J. McDonnell, N. Deane, F.M. Platt, G. Nunez, U. Jaeger, J.P. McKearn, S.\n Curr. Top. Med. Chem. 1 (2001) 277\u2013351. J. Korsmeyer, Bcl-2-immunoglobulin transgenic mice demonstrate extended B cell\n[30] E.H. Kerns, L. Di, Multivariate pharmaceutical profiling for drug discovery, Curr. survival and follicular lymphoproliferation, Cell 57 (1989) 79\u201388.\n Top. Med. Chem. 2 (2002) 87\u201398. [59] M. Zhou, X. Liu, Z. Li, Q. Huang, F. Li, C.Y. Li, Caspase-3 regulates the migration,\n[31] H. van de Waterbeemd, D.A. Smith, B.C. Jones, Lipophilicity in PK design: methyl, invasion and metastasis of colon cancer cells, Int. J. Cancer 143 (2018) 921\u2013930.\n ethyl, futile, J. Comput. Aided Mol. Des. 15 (2001) 273\u2013286. [60] X. Feng, Y. Yu, S. He, J. Cheng, Y. Gong, Z. Zhang, X. Yang, B. Xu, X. Liu, C.Y. Li,\n[32] D.T. Manallack, E. Yuriev, D.K. Chalmers, The influence and manipulation of acid/ L. Tian, Q. Huang, Dying glioma cells establish a proangiogenic microenvironment\n base properties in drug discovery, Drug Discov, Today Technol 27 (2018) 41\u201347. through a caspase 3 dependent mechanism, Cancer Lett. 385 (2017) 12\u201320.\n[33] Y. Liu, D.A. Peterson, H. Kimura, D. Schubert, Mechanism of cellular 3-(4,5- [61] M. Mukai, T. Kusama, Y. Hamanaka, T. Koga, H. Endo, M. Tatsuta, M. Inoue, Cross\n dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, talk between apoptosis and invasion signaling in cancer cells through caspase-3\n J. Neurochem. 69 (1997) 581\u2013593. activation, Cancer Res. 65 (2005) 9121\u20139125.\n[34] J.W. Wang, H.M. Liu, X.Y. Wu, C.L. Shi, W.L. Li, Y.H. Yuan, Y.J. Liu, D.G. Xing, [62] K. Lauber, E. Bohn, S.M. Kro\u0308ber, Y.J. Xiao, S.G. Blumenthal, R.K. Lindemann,\n Induction of apoptosis in SGC-7901 cells by iridium(III) complexes via endoplasmic P. Marini, C. Wiedig, A. Zobywalski, S. Baksh, Y. Xu, I.B. Autenrieth, K. Schulze-\n reticulum stress-mitochondrial dysfunction pathway, J. Biol. Inorg. Chem. 27 Osthoff, C. Belka, G. Stuhler, S. Wesselborg, Apoptotic cells induce migration of\n (2022) 455\u2013469. phagocytes via caspase-3-mediated release of a lipid attraction signal, Cell 113\n[35] W.L. Li, C.L. Shi, X.Y. Wu, Y.Y. Zhang, H.M. Liu, X.Z. Wang, C.X. Huang, L.J. Liang, (2003) 717\u2013730.\n Y.J. Liu, Light activation of iridium (III) complexes driving ROS production and [63] S.W. Wu, C.H. Su, Y.C. Ho, R. Huang-Liu, C.C. Tseng, Y.W. Chiang, K.L. Yeh, S.\n DNA damage enhances anticancer activity in A549 cells, J. Inorg. Biochem. 236 S. Lee, W.Y. Chen, C.J. Chen, Y.C. Li, C.Y. Lee, Y.H. Kuan, Genotoxic effects of 1-\n (2022), 111977. nitropyrene in macrophages are mediated through a p53-dependent pathway\n[36] L. Bai, W.D. Fei, Y.Y. Gu, M. He, F. Du, W.Y. Zhang, L.L. Yang, Y.J. Liu, Liposomes involving cytochrome c release, caspase activation, and PARP-1 cleavage,\n encapsulated iridium(III) polypyridyl complexes enhance anticancer activity in Ecotoxicol. Environ. Saf. 213 (2021), 112062.\n vitro and in vivo, J. Inorg. Biochem. 205 (2020), 111014. [64] J. Yuan, A. Najafov, B.F. Py, Roles of caspases in necrotic cell death, Cell 167\n[37] Y.Y. Gu, L. Bai, Y.Y. Zhang, H.W. Zhang, D.G. Xing, L. Tian, Y. Zhou, J. Hao, Y. (2016) 1693\u20131704.\n J. Liu, Liposome as drug delivery system enhance anticancer activity of iridium(III) [65] J.A. Fresno Vara, E. Casado, J. de Castro, P. Cejas, C. Belda-Iniesta, M. Gonza\u0301lez-\n complex, J. Liposome Res. 4 (2021) 432\u2013455. Baro\u0301n, PI3K/Akt signalling pathway and cancer, Cancer Treat Rev. 30 (2004)\n[38] A. Riedl, M. Schlederer, K. Pudelko, M. Stadler, S. Walter, D. Unterleuthner, 193\u2013204.\n C. Unger, N. Kramer, M. Hengstschla\u0308ger, L. Kenner, D. Pfeiffer, G. Krupitza,\n H. Dolznig, Comparison of cancer cells in 2D vs 3D culture reveals differences in\n AKT\u2013mTOR\u2013S6K signaling and drug responses, J. Cell Sci. 130 (2017) 203\u2013218.\n[39] M. Zoetemelk, M. Rausch, D.J. Colin, O. Dormond, P. Nowak-Sliwinska, Short-term\n 3D culture systems of various complexity for treatment optimization of colorectal\n carcinoma, Sci. Rep. 9 (2019) 7103.\n\n\n 12\n\fY. Yuan et al. European Journal of Medicinal Chemistry 247 (2023) 115046\n\n[66] Y. Shiloh, Ataxia-telangiectasia and the Nijmegen breakage syndrome: related [68] S. Sprouse, K.A. King, P.J. Spellane, R.J. Watts, Photophysical effects of metal-\n disorders but genes apart, Annu. Rev. Genet. 31 (1997) 635\u2013662. carbon \u03c3 bonds in ortho-metalated complexes of iridium(III) and rhodium(III),\n[67] M. Goldstein, M.B. Kastan, The DNA damage response: implications for tumor J. Am. Chem. Soc. 106 (1985) 6647\u20136653.\n responses to radiation and chemotherapy, Annu. Rev. Med. 66 (2015) 129\u2013143.\n\n\n\n\n 13\n\f", "pages_extracted": 13, "text_length": 95138}