Designing novel tridentate iridium(III) complexes comprising functionalized benzothiazole ligands to improve anticancer activity by targeting mitochondria.

{"full_text": " i An update to this article is included at the end\n\n\n\n\n Bioorganic Chemistry 161 (2025) 108507\n\n\n Contents lists available at ScienceDirect\n\n\n Bioorganic Chemistry\n journal homepage: www.elsevier.com/locate/bioorg\n\n\n\n\nDesigning novel tridentate iridium(III) complexes comprising\nfunctionalized benzothiazole ligands to improve anticancer activity by\ntargeting mitochondria\u2606\nQin Zhou a,c , Xiao-Bin Zhang a , An-Li Liu a,c , Zhi-Gang Niu a,c,*, Gao-Nan Li a,c,* ,\nFa-Biao Yu b,c\na\n Key Laboratory of Electrochemical Energy Storage and Light Energy Conversion Matreials of Haikou City, Key Laboratory of Electrochemical Energy Storage and Energy\nConversion of Hainan Province, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, China\nb\n Key Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Haikou Trauma, Key Laboratory of Hainan Trauma and Disaster Rescue, The First\nAffiliated Hospital, Hainan Medical University, Haikou 571199, China\nc\n Engineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, Key Laboratory of Hainan Functional Materials and Molecular Imaging, College\nof Emergency and Trauma, Hainan Medical University, Haikou 571199, 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: In recent years, organo\u2011iridium anticancer agents have shown promising antitumor activity toward cancer cells.\nIridium (III) complex In this paper, two benzothiazole-based tridentate ligands, 2,2\u2032-(5-(tert-butyl)-1,3-phenylene)bis(benzo[d]thia\u00ad\nTridentate ligands zole) (L1) and 2,2\u2032-(5-(methyl)-1,3-phenylene)bis(benzo[d]thiazole) (L2), have been designed and synthesized,\nBenzothiazole\n and then combined with 2,2\u2032-bipyridine (bipy) and 1,10-phenanthroline (phen) ancillary ligands to form a series\nAnticancer\nMitochondria\n of novel [Ir(N^C^N)(N^N)Cl]+-type iridium(III) complexes (Ir1-Ir4). The phosphorescence properties of these\n complexes facilitate the visualization of their subcellular localization and interactions with other biomolecules.\n Among them, complex Ir2 has the best cytotoxicity activity toward A549 cells and its antitumor activity was\n further evaluated. Laser confocal assay reveals that Ir2 followed an energy-dependent cellular uptake mechanism\n and specifically accumulates in mitochondria (Pearson colocalization coefficient: 0.89). The anticancer mecha\u00ad\n nism has been explored through apoptosis, cell cycle arrest, western blotting (WB), reactive oxygen species (ROS)\n levels and mitochondrial membrane potential (MMP) changes. The antitumor activity in vivo confirms that Ir2\n could effectively inhibit tumor growth with an inhibitory rate of 71.60 %, which is superior to cisplatin. To the\n best of our knowledge, Ir2 is a rare example of [Ir(N^C^N)(N^N)Cl]+-type complexes as potential anticancer\n agents.\n\n\n\n\n1. Introduction and serious side effects [9,10]. Therefore, a large of efforts have been\n made to explore different anticancer metal complexes as alternative of\n Cancer is a malignant disease threatening human health and life due cisplatin, involving iridium [11], rhenium [12], gallium [13], gold [14],\nto its excessive proliferation, easily dispersed and transported charac\u00ad ruthenium [15], copper [16], rhodium [17] and more. Among them,\nteristics [1\u20133]. Chemotherapy based on metal-organic medications is organometallic Ir(III) complexes as potential anticancer agents have\none of the most prevalent methods in cancer treatment [4,5]. At present, been studied extensively in recent years, because of their rich photo\u00ad\nplatinum drugs represented by cisplatin have proven to be effective physical properties, good biocompatibility and excellent anticancer ac\u00ad\nchemotherapeutic agents against various types of cancers [6\u20138]. With tivities [18\u201322].\nthe widespread use of cisplatin in clinical practice, however, some Based on different ligands, iridium(III) anticancer complexes are\ndrawbacks are gradually found, such as drug resistance, high toxicity mainly classified into four types: cyclopentadienyl [23\u201325],\n\n\n This article is part of a Special issue entitled: \u2018Targeted cancer therapy\u2019 published in Bioorganic Chemistry.\n \u2606\n\n * Corresponding authors at: Key Laboratory of Electrochemical Energy Storage and Light Energy Conversion Matreials of Haikou City, Key Laboratory of Elec\u00ad\ntrochemical Energy Storage and Energy Conversion of Hainan Province, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158,\nChina.\n E-mail addresses: niuzhigang1982@126.com (Z.-G. Niu), ligaonan2008@163.com (G.-N. Li).\n\nhttps://doi.org/10.1016/j.bioorg.2025.108507\nReceived 8 March 2025; Received in revised form 11 April 2025; Accepted 21 April 2025\nAvailable online 22 April 2025\n0045-2068/\u00a9 2025 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fQ. Zhou et al. Bioorganic Chemistry 161 (2025) 108507\n\n\ncyclometalated [26\u201328], arene [29] and carbene [30\u201332]. By compari\u00ad high lipophilicity, which help to enhance the biocompatibility of com\u00ad\nson, cyclometalated Ir(III) complexes possess easier adjustment in plexes through facilitating the cellular uptake and accumulation\nstructure owing to great diversity of coordinating ligands. As such, these [50\u201352]. Based on above considerations, we introduced functionalized\ncomplexes can be precisely structurally modulated through introducing benzothiazole group onto the tridentate ligand to improve anticancer\nbiomolecular functional groups on ligands, with the aim of effectively activity of corresponding Ir(III) complexes, and thus designed two novel\nregulating their cytotoxic profiles and anticancer activities [33]. Lu et al. ligands 2,2\u2032-(5-(tert-butyl)-1,3-phenylene)bis(benzo[d]thiazole) (L1)\nreported two sulfur-coordinated organoiridium(III) complexes (pbtIrSS and 2,2\u2032-(5-(methyl)-1,3-phenylene)bis(benzo[d]thiazole) (L2), and\nand ppyIrSS) with C,N and S,S (dithione) chelating ligands, which then combined bipy and phen ancillary ligands to obtain a series of new\ninhibit breast cancer tumorigenesis and metastasis by blocking Wnt/ type [Ir(N^C^N)(N^N)Cl]+ complexes (Ir1-Ir4, Scheme 1). The structure\n\u03b2-catenin signaling cascade [34]. Mao et al. developed two iridium(III)- of complex Ir1 was studied by X-ray crystallography (Fig. 1). Their\ntripenylamine photosensitizers (IrC and IrF), representing the first photophysical properties and cytotoxic profiles were investigated sys\u00ad\nintegration of pyroptosis and ferroptosis hybrid cell death induction tematically. It was determined that complex Ir2 had good fluorescence\nthrough disrupting redox balance and inducing photo-driven DNA and imaging capability and remarkable cytotoxicity to A549 cells. Subse\u00ad\nKEAP1 cascade damage [35]. Chao et al. reported an iridium(III) com\u00ad quently, the anticancer mechanism in vitro and anticancer effects in vivo\nplex containing the derivate bis(2-chloroethyl)-azane, which localized of Ir2 were discussed and evaluated, respectively. As a result, our\nin the ER and induced ICD in non-small-cell lung cancer [36]. findings demonstrated that complex Ir2 could induce the apoptosis of\n So far, numerous bis/tri-cyclometalated Ir(III) complexes with A549 cells by activating mitochondria apoptotic pathway of ROS and\nbidentate ligands have been designed and prepared, and structure- MMP regulated dysfunction. Furthermore, antitumor assays in nude\nactivity relationships have also been established [37\u201339]. Neverthe\u00ad mice showed Ir2 could significantly prevent tumor growth in vivo,\nless, it is worth mentioning that the exploration of tridentate Ir(III) indicating its potential as novel anticancer drugs.\ncomplexes is an area that remains relatively scarce. Though there have\nbeen a few recent reports of these complexes for biological applications, 2. Results and discussion\nthe cases are only limited to [Ir(N^N^N)(C^N)Cl]+ [40,41], [Ir(N^N^N)\n(C^N^C)]+ [42], [Ir(N^N^N)2]3+ [43] and dinuclear Ir(III) complexes 2.1. Synthesis and characterization\nwith tridentate ligands [44,45]. It is thus to be motivated to develop new\ntypes of tridentate ligand incorporating special functional group to As shown in Scheme 1, the cyclometalated N^C^N ligands (L1 and L2)\napply in the area of iridium-based anticancer therapeutic agents. were prepared in good yields from ring condensation reactions between\n As well known, benzothiazole contains a benzene ring fused to a 2-aminothiophenol and 5-tert-butylisophthalic acid/5-\nthiazole ring, displaying remarkable biological properties, especially methylisophthalic acid. Next, the two ligands reacted with IrCl3\u20273H2O\nhigh anticancer activity [46,47]. In the past three decades, a great deal to generate the corresponding chloro-bridged dimers, and then N^N li\u00ad\nof research groups have been devoted to develop effective anticancer gands (bipy or phen) and silver trifluoromethyl sulfonate (Ag(CF3SO3))\ndrugs with benzothiazole derivatives [48,49]. In addition, conventional were added into the reaction system to form the target Ir(III) complexes\n2,2\u2032-bipyridine (bipy) and 1,10-phenanthroline (phen) ligands possess in moderate yields [53]. The structures of these compounds and\n\n\n\n\n Scheme 1. The synthetic routes of complexes Ir1-Ir4.\n\n 2\n\fQ. Zhou et al. Bioorganic Chemistry 161 (2025) 108507\n\n\n are around 78\u25e6 while the trans N-Ir-N, C-Ir-N and Cl-Ir-N angles are in\n the range of 157.68(18)\u25e6 -175.2(2)\u25e6 .\n\n\n 2.2. Photophysical properties\n\n The UV\u2013vis and emission spectra of Ir1-Ir4 in CH2Cl2 at 298 K are\n depicted in Fig. 2 and the corresponding data are given in Table S3. As\n shown in Fig. 2a, all complexes exhibit a strong absorption band\n (220\u2013350 nm) and a weak absorption band (350\u2013500 nm), which are\n attributed to spin-allowed ligand-centered (1LC) \u03c0-\u03c0* transitions and an\n admixture of metal-to-ligand/intraligand/ligand-to-ligand charge\n transfer (MLCT/ILCT/LLCT) transitions, respectively [56,57]. Through\n the DFT calculation results, the lower-lying absorption of all Ir(III)\n complexes are associated with HOMO\u2192LUMO+1 transitions (Table S4).\n From Table S5, the HOMOs mainly reside on the Ir (29.87\u201330.43 %, Ir1-\n Ir4) and N^C^N ligands (60.29\u201361.62 %, Ir1-Ir4), while the LUMO+1 s\n are mainly located on the N^C^N ligands (97.44 %, Ir1; 66.60 %, Ir2;\n 97.29 %, Ir3; 69.94 %, Ir4) and N^N ligands (31.42 %, Ir2; 28.00 %,\n Ir4). Therefore, the lowest energy bands mainly arise from MLCT/ILCT\n transitions (d\u03c0Ir/N^C^N \u2192 \u03c0*N^C^N, Ir1 and Ir3) and MLCT/ILCT/LLCT\n transitions (d\u03c0Ir/N^C^N \u2192 \u03c0*N^C^N/N^N, Ir2 and Ir4), further supporting the\n above description. There are no distinct differences on the absorption\nFig. 1. Crystal structure of complex Ir1 (CCDC: 2327299). For clarity, edge of complexes Ir1-Ir4, in line with the approximate energy gaps\nhydrogen atoms and the counter anion are omitted. based on the DFT calculations (Fig. 3). It can be also found that iridium\n complexes with the same N^N ligand, Ir1/Ir3 and Ir2/Ir4, have similar\n UV spectral profiles. The observation clearly demonstrates that the\ncomplexes were characterized by 1H NMR (Figs. S6-S11), 13C NMR\n N^C^N ligand modifications (t-Bu and CH3) have a negligible impact on\n(Figs. S12-S15), high-resolution mass spectrometry (HRMS) (Figs. S16-\n the absorption spectra.\nS21) and elemental analysis, as well as Ir1 by X-ray single structure\n Upon photoexcitation, complexes Ir1-Ir4 showed yellow-green\nanalysis (Fig. 1 and Tables S1-S2).\n phosphorescence (photos in Scheme S1 and CIE data in Table S3) with\n Single crystals of complex Ir1 were obtained by slowly evaporating\n emission maximum peaking at \u2248540 nm (Fig. 2b). The vibronic-\nfrom the mixed dichloromethane and methanol solution at room tem\u00ad\n structured emission bands reveal that their emissive excited states\nperature. The molecular structure with the cationic part is shown in\n originate from the combined 3MLCT and 3LC characteristics [58,59]. In\nFig. 1. The crystallographic data and selected bond lengths/angles\n those analogues, the HOMO-1 s involve Ir metal and the N^C^N ligand,\naround the iridium center are listed in Tables S1-S2. The Ir(III) center\n while the LUMO+1 s primarily reside on the N^C^N ligand (Ir1 and Ir3)\npresents a distorted octahedral coordination geometry with N1/N2/C17\n or the N^C^N/N^N ligands (Ir2 and Ir4). So, the lowest-lying triplet states\natoms of L1 ligand, N3/N4 atoms of bipy ligand and monodentate\n (T1) based on HOMO-1 \u2192 LUMO+1 orbital transition exhibit the mixed\nchloride ligand. The N1/N2/C17 atoms are in a meridional arrange\u00ad\n characteristics of MLCT/ILCT for Ir1 and Ir3 or MLCT/ILCT/LLCT for\nment, and nearly coplanar with the N3 atom with the average deviation\n Ir2 and Ir4 (Table S4). The calculated results agree well with the\nfrom a least-squares plane of 0.0427 \u00c5. The chloride ligand is in a po\u00ad\n structured emission profiles, as well as their low-energy absorption\nsition trans to the N4 atom and the length of the Ir\u2013Cl bond (2.3481(15)\n transitions. Besides, the difference in peak wavelength turns evidently\n\u00c5) is much longer than those of other coordination bonds, as previously\n small among the four complexes, implying that the changes of main and\nreported iridium analogues [54,55]. Moreover, Ir\u2013Cl bond is almost\n ancillary ligands seem to not affect their emission spectra. The phos\u00ad\nperpendicular to the N1/N2/C17/N3 plane with bond angles of 88.87\n phorescence relative quantum yields (\u03a6em) of Ir1-Ir4 in dichloro\u00ad\n(14)-93.73(13)\u25e6 . The chelating C-Ir-N (N^C^N) and N-Ir-N (N^N) angles\n methane solutions were measured to be 15.1\u201354.6 %, with lifetimes in\n\n\n\n\n Fig. 2. UV\u2013vis absorption (a) and emission (b) spectra of complexes Ir1-Ir4 in CH2Cl2 solution at room temperature.\n\n 3\n\fQ. Zhou et al. Bioorganic Chemistry 161 (2025) 108507\n\n\n\n\n Fig. 3. Electronic levels and surface distributions of selected frontier molecular orbitals involved in crucial electronic excitations of complexes Ir1-Ir4.\n\n\nthe range 0.48 to 1.15 \u03bcs (Table S1). The quantum yields of Ir1-Ir2 are effect. Overall, their cytotoxicity follows the order: Ir1/Ir2 > Ir3/Ir4 >\nless than the others, reflecting in the higher knr and lower kr values, as a cisplatin, further confirming the necessity of high lipophilicity for\nconsequence of the relatively strong vibrational quenching effect caused improving anticancer activity [24]. In detail, complexes Ir1/Ir2\nby the presence of the two t-Bu groups on the N^C^N ligand. (0.74\u20133.29 \u03bcM) offer the lower IC50 values than their counterparts Ir3/\n Due to the overall photophysical properties of Ir1-Ir4, their cellular Ir4 (1.14\u20135.06 \u03bcM), probably owing to the effect of the t-Bu groups.\naccumulation, uptake and distribution could be more easily monitored Furthermore, the IC50 values of Ir2 against Hela, A549, MGC-803 cells\nby laser scanning confocal microscopy. Thereby, these complexes were are about 1.5-fold lower than those of Ir1, related with the nature of\ntaken forward for anticancer testing. ancillary ligands. And the cytotoxic activity of Ir2 on three above cancer\n cells are about 3.7, 9.0 and 9.0-fold higher than that of cisplatin,\n respectively. Considering the cisplatin-resistance and universality, the\n2.3. Detection of lipophilicity and cytotoxicity\n better candidate was finally assigned as A549 cells [62\u201364]. Besides,\n these benzothiazole-based iridium(III) complexes also exhibit high\n Before proceeding with anticancer studies, the lipophilicity of\n selectivity between cancer cells and non-cancer cells. In particular, Ir2\niridium complexes Ir1-Ir4 were first tested by the partition coefficient in\n reveals the largest selectivity index of 16.7 toward A549 and BEAS-2B\nn-octanol/water system (logPo/w) that reflect their ability to pass\n cells, far surpassing that of cisplatin (SI:1.6). Thus, we selected Ir2\nthrough the cell membrane [60,61]. Using the classical shake-flask\n and A549 cells for further investigation into the underlying mechanisms,\ntechnique, the logPo/w values are determined as the order of Ir2\n since Ir2 was not only more cytotoxic to A549 cells, but also less cyto\u00ad\n(2.25) > Ir1 (2.22) > Ir4 (2.04) > Ir3 (1.91). As can be seen, complex\n toxic to normal cells.\nIr2 exhibits the highest lipophilicity that facilitates its uptake by cancer\ncells, suggesting the complex would possess good cytotoxicity activity.\n The in vitro cytotoxicity of the four complexes against Hela, RKO, 2.4. Solution stability studies\nA549, MGC-803 cancer cells and BEAS-2B non-cancer cells were\nmeasured by using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe\u00ad In order to study the solution stability of complexes Ir1-Ir4, the 1H\nnyltetrazolium bromide (MTT) assay and compared to cisplatin as NMR spectra in DMSO solution were recorded at different time intervals\nreference (Table 1). It\u2019s remarkable that all investigated complexes are (5 min and 24 h) (Figs. S1-S4). There are no significant changes in these\nmore active than cisplatin against four types of cancer cells, which spectral patterns, suggesting that the metal\u2212 ligand bonds in complexes\nsuggests that benzothiazole unit in this system has favorable anticancer remain intact that support their potential as anticancer drug candidates.\n To further evaluate the feasibility for therapeutic application, the sta\u00ad\n bility of representative complex Ir2 was conducted using emission\nTable 1\nIC50 (\u03bcM) values of Ir1-Ir4 and cisplatin toward different cell lines after 24 h of spectra in PBS:DMSO (99:1) solution at room temperature at different\nexposure. PH values (3\u2212 10) and times (0\u2013180 min), respectively (Fig. S5). As\n shown, the luminescence intensity was almost unchanged, indicating\n Complex Hela RKO A549 MGC- BEAS-2B SI\n 803\n that Ir2 would have good biological stability under acidic/alkaline and\n normal cell conditions.\n Ir1 2.95 \u00b1 0.94 \u00b1 3.29 \u00b1 1.13 \u00b1 46.22 \u00b1 14.0\n 0.08 0.35 0.26 0.11 0.81\n Ir2 1.87 \u00b1 1.98 \u00b1 2.23 \u00b1 0.74 \u00b1 37.3 \u00b1 16.7 2.5. Cellular uptake mechanism and cellular localization\n 0.25 0.21 0.08 0.07 1.24\n Ir3 4.08 \u00b1 2.9 \u00b1 3.77 \u00b1 1.9 \u00b1 41.85 \u00b1 11.1\n 0.18 0.07 0.52 0.17 0.44\n Because the cellular uptake mechanism is an important factor when\n Ir4 1.14 \u00b1 5.06 \u00b1 3.89 \u00b1 1.7 \u00b1 39.5 \u00b1 10.2 evaluating Ir2 as an antitumor drug, to ascertain this, four sequent of\n 0.08 0.92 0.20 0.14 0.85 experiments were designed utilizing laser confocal microscopy based on\n Cisplatin 6.92 \u00b1 9.90 \u00b1 20.01 \u00b1 6.70 \u00b1 32.01 \u00b1 1.6 the inherent luminescence property of the iridium(III) complex. A549\n 0.33 0.28 1.96 0.80 0.63\n tumor cells were firstly incubated with Ir2 at the normal temperature of\nSelectivity index (SI): IC50 ratio of BEAS-2B normal cells to A549 cancer cells. the human body (37 \u25e6 C) and a low temperature (4 \u25e6 C), and then co-\n\n 4\n\fQ. Zhou et al. Bioorganic Chemistry 161 (2025) 108507\n\n\ncultured with carbonyl cyanide m-chlorophenylhydrazone (CCCP, initiation of apoptosis in A549 cells.\nmetabolic inhibitor) and chloroquine (endocytic inhibitor), respectively.\nFig. 4a shows that the cellular uptake are apparently suppressed when at 2.8. Western blotting assay\n4 \u25e6 C or CCCP pretreated, while that with chloroquine treatment remains\nalmost unchanged, indicating an energy-dependent cellular uptake To understand the mechanism of Ir2-induced antitumor effects,\nmechanism and not an endocytosis pathway. Western blot analysis was utilized to identify mitochondrial apoptosis\n To further explore the target location of Ir2 in subcellular organelles, pathways in A549 cells by detecting the level of Caspase-3 and Bcl-2\nthe status of mitochondria and lysosome in Ir2-treated A549 cells were family proteins (Fig. 6). The activation of Caspase-3 represents an\ninvestigated using Mito Tracker Deep Red (MTDR, mitochondrial red early event of apoptosis, which cleaves key structural proteins and ul\u00ad\nfluorescent dye) and Lysol Tracker Deep Red (LTDR, lysosomal red timately leads to apoptotic cell death [67,68]. Bcl-2 is in the outer\nfluorescent dye). As shown in Fig. 4b, green and red fluorescence orig\u00ad membrane of mitochondria, whose over expression can inhibit\ninate from Ir2 and mitochondrial/lysosomal dye, respectively. The apoptosis. Bax is a core member of the Bcl-2 family proteins, which can\nmerge of the green and red fluorescence displays that Ir2 mainly accu\u00ad form a heterodimer with Bcl-2 to make apoptosis more likely to occur\nmulates in the mitochondria and slightly distributes in the lysosome [69]. In comparison to the control, the expression levels of Caspase-3\nwith a Pearson co-localization coefficient (PCC) of 0.89 and lower 0.41, and pro-apoptotic protein Bax are increased while the anti-apoptosis\nrespectively. These results demonstrate that Ir2 could specially map to protein Bcl-2 are decreased. This confirms that Ir2 could promote\nmitochondria of A549 cells. apoptosis of A549 cells through the classical mitochondrial pathway,\n proving the above co-localization experiment in the meantime.\n2.6. Apoptosis assay\n 2.9. Evaluation of reactive oxygen species (ROS) generation\n Apoptosis has been described as a \"suicide\" program in various cells,\nwhich is an orderly death regulated by genes [24]. It has been reported In general, the overproduction of ROS induced by transition metal\nthat some transition metal complexes based on Pt(II), Ru(II), Os(II), and complexes could cause mitochondria mediated dysfunction and then\nIr(III) can cause apoptosis and show anticancer activity [65,66]. Thus, lead to apoptosis [70,71]. In order to investigate the efficacy of Ir2\nthe ability of Ir2 to induce apoptosis in A549 cells double-stained with inducing ROS production in A549 cells, 2\u2032,7\u2032-dichlorofluorescein diac\u00ad\nAnnexin V-FITC/propidium iodide (PI) was detected by flow cytometry. etate (DCFH-DA) probe was used to observe changes of intracellular ROS\nWhen the concentrations of Ir2 are 0.5, 1.0 and 2.0 \u00d7 IC50, the per\u00ad level using confocal imaging and flow cytometry. DCFH-DA can be hy\u00ad\ncentages of early and late apoptotic cells increase in a concentration- drolyzed by esterase to non-fluorescent DCFH, and then be oxidized to\ndependent manner (Fig. 5a), demonstrating Ir2 could induce apoptosis generate highly fluorescent DCF by cellular ROS [72]. From Fig. 7a, it\nof A549 cells to achieve the anticancer effect. can be seen that the fluorescence in control cells is weak, whereas the\n fluorescence intensity in cells treated with Ir2 gradually increase in a\n2.7. Cellular cycle arrest concentration-dependent manner. Next, the ROS levels were further\n established by flow cytometry (Fig. 7b). The ROS levels increased by 1.3\n The effect of Ir2 on cell cycle progression was also investigated using times when the concentration of Ir2 was from 0.5 \u00d7 IC50 to 2.0 \u00d7 IC50.\nflow cytometric analysis (Fig. 5b). As shown, Ir2 could effectively block Together, these results suggest that Ir2 could effectively induce intra\u00ad\nthe A549 cell cycle in G0/G1 phase when compared with the control. At cellular ROS production and promote apoptosis.\na gradient concentration (0.5\u20132.0 \u00d7 IC50), the percentage of cells in G0/\nG1 phase increases, accompanied by a corresponding reduction in the S 2.10. Mitochondrial membrane potential (MMP) changes\nand G2/M phases. The concentration-dependent results suggest that Ir2\ncould induce G0/G1 phase cell cycle arrest and further cause a gradual Most transition metal complexes can induce the loss of MMP and\n\n\n\n\nFig. 4. (a) Confocal images of A549 cells treated with Ir2 at 37 \u25e6 C or 4 \u25e6 C, and then incubated with CCCP or chloroquine at 37 \u25e6 C. (b) Colocalization images of Ir2\nwith MTDR and LTDR in A549 cells. Ir2: \u03bbex = 445 nm, \u03bbem = 520 \u00b1 30 nm; MTDR: \u03bbex = 561 nm, \u03bbem = 600 \u00b1 30 nm; LTDR: \u03bbex = 594 nm, \u03bbem = 630 \u00b1 30 nm.\nScale bar: 50 \u03bcm.\n\n 5\n\fQ. Zhou et al. Bioorganic Chemistry 161 (2025) 108507\n\n\n\n\n Fig. 5. Apoptosis assay (a) and cell cycle analysis (b) of A549 cells treated with Ir2 at a gradient IC50 concentration.\n\n\n 2.11. In vivo antitumor and safety analysis\n\n After elucidating the apoptosis-inducing mechanism of Ir2 in vitro, its\n antitumor efficacy and biosafety in vivo were evaluated. The BALB/c\n nude mice were subcutaneously implanted with A549 cells to obtain\n xenograft tumors, and then injected intratumorally with saline/DMSO,\n cisplatin, and Ir2, respectively. The body weight and tumor volume of\n the mice in each group were recorded every two days throughout the\n entire treatment period. After 14 days of treatment, the tumors were\n excised for photographing and weighing. From Figs. 10a-10b, tumors\n treated in Ir2 group are significantly diminished compared with those in\n the cisplatin group and control group. Concretely, the tumor weights of\n the three groups are 1.54 \u00b1 0.38 g, 3.78 \u00b1 0.34 g and 6.63 \u00b1 0.57 g,\n respectively (Fig. 9c and Table S6). Besides, the growth rates of the\n tumor volume and final volume in Ir2 group are obviously smaller than\n those of the other two groups (Fig. 9 and Table S7). Furthermore, the\n tumor inhibition rates of the cisplatin and Ir2 groups are 27.25 % and\nFig. 6. Western blot analysis of Caspase-3 and Bcl-2 family proteins after\n 71.60 %, respectively, confirming that Ir2 has excellent antitumor ef\u00ad\ntreatment of Ir2 at 1.0 \u00d7 IC50 concentration.\n ficacy. During the follow-up period, the mean body weight of mice in\n three test groups remained within the normal range (Fig. 9e and\nactivate mitochondria-related apoptotic pathways [71,73]. Therefore, Table S8), indicating Ir2 has low systemic toxicity and high biocom\u00ad\nthe changes of MMP in Ir2-treated cells were detected using the 5,5\u2032,6,6\u2032- patibility [75,76].\ntetrachloro-1,1\u2032,3,3\u2032-tetraethylbenzimidazolylcarbocyanine iodide (JC- At the end of treatment, the pathological tissues of the tumors in each\n1) fluorescent probe. In high mitochondrial membrane potential, JC-1 group were examined by hematoxylin-eosin (H&E) staining (Fig. 10a). It\naggregates emit red fluorescence; in contrast, when the mitochondria is clearly that Ir2-treated group decreases more tumor cell density than\nare damaged and the membrane potential decreases, JC-1 monomers the control and cisplatin groups, suggesting that Ir2 could induce a\nemit green fluorescence [74]. As seen in Fig. 8a, the negative control serious death of tumor cells. In addition, the cells of major organs (heart,\ngroup emits strong red fluorescence, while the cells treated with Ir2 emit liver, spleen, lung and kidney) in the control and Ir2 groups were also\ngreen fluorescence concentration-dependently, especially the positive stained by H&E (Fig. 10b) and no abnormalities or signs of inflammation\ncontrol group of CCCP emits strong green fluorescence. Flow cytometry were observed in any organs for the Ir2 group. Taken together, the\nanalysis was also performed to quantify MMP in Ir2-treated cells and above experimental results imply that Ir2 has high antitumor efficacy\nFig. 8b illustrates the experiment results. Compared with the negative and good vivo safety, thus could be a tumor-targeting candidate drug for\ncontrol (14.5 %), the percentage of mitochondrial membrane depolar\u00ad A549 cancer.\nized cells increases by 31.7 % at 2.0 \u00d7 IC50, and exhibits a dose-\ndependent trend. Altogether, the above results confirms Ir2 could\ninduce mitochondrial dysfunction in A549 cells and eventually lead to\napoptosis.\n\n\n\n\n 6\n\fQ. Zhou et al. Bioorganic Chemistry 161 (2025) 108507\n\n\n\n\nFig. 7. (a) Intracellular ROS production in A549 cells treated with Ir2 at a gradient IC50 concentration and stained with DCFH-DA. DCF: \u03bbex = 488 nm, \u03bbem = 540 \u00b1\n20 nm. Scale bar: 50 \u03bcm. (b) Intracellular ROS levels in A549 cells after treatment of Ir2 at a gradient IC50 concentration; Histograms of relative ROS levels, and the\ndata are the mean of three replicate experiments \u00b1 SD.\n\n\n\n\nFig. 8. (a) Changes in the MMP of A549 cells treated with Ir2 at a gradient IC50 concentration and stained with JC-1. (b) Histogram of relative MMP changes, and the\ndata are the mean of three replicate experiments \u00b1 SD.\n\n\n3. Experimental section reaction mixture was cooled to room temperature, methanol (20 ml) was\n added to precipitate a white solid and collected to give the desired\n3.1. Synthesis of ligands product L1 as a white solid (1.30 g, 72.2 %). 1H NMR (400 MHz, CDCl3) \u03b4\n 8.55 (t, J = 1.6 Hz, 1H), 8.28 (d, J = 1.6 Hz, 2H), 8.13 (d, J = 8.1 Hz,\n3.1.1. 2,2\u2032-(5-(tert-butyl)-1,3-phenylene)bis(benzo[d]thiazole) (L1) 2H), 7.95 (d, J = 8.0 Hz, 2H), 7.55\u20137.51 (m, 2H), 7.44\u20137.40 (m, 2H),\n A mixture of 5-tert-butylisophthalic acid (1.00 g, 4.50 mmol), 2-ami\u00ad 1.50 (s, 9H). HRMS: m/z 423.0956 [M + Na]+ (calcd 423.1068).\nnothiophenol (1.24 g, 9.90 mmol), triphenyl phosphite (2.79 g, 9.00\nmmol) and tetrabutylammonium bromide (2.9 g, 9.00 mmol) was 3.1.2. 2,2\u2032-(5-(methyl)-1,3-phenylene)bis(benzo[d]thiazole) (L2)\nheated to 120 \u25e6 C and stirred for 2 h in a nitrogen atmosphere. After the L2 was synthesized according to the method described in L1 (white\n\n\n 7\n\fQ. Zhou et al. Bioorganic Chemistry 161 (2025) 108507\n\n\n\n\nFig. 9. Photographs of tumor in vivo (a), tumor sizes (b) and tumor weight (c) in control and cisplatin/Ir2-treated groups (3.0 mg/kg) at the end of treatment. The\ntumor volume (d) and changes in body weight (e) of each group throughout the follow-up period. The data are the mean of three replicate experiments \u00b1 SD.\n\n\n\n\nFig. 10. H&E staining of tumor tissues in three groups (a, scale bar: 20 \u03bcm) and major organs of mice in two groups (b, scale bar: 100 \u03bcm) at the end of treatment.\n\n\nsolid, yield: 76.9 %). 1H NMR (400 MHz, CDCl3) \u03b4 8.56 (d, J = 0.4 Hz, 3.2. Synthesis of Ir(III) complexes\n1H), 8.12 (dd, J = 8.1, 0.4 Hz, 2H), 8.07 (dd, J = 1.6, 0.7 Hz, 2H), 7.95\n(dd, J = 8.0, 0.6 Hz, 2H), 7.55\u20137.51 (m, 2H), 7.44\u20137.40 (m, 2H), 2.56 (s, IrCl3\u22c53H2O (1.0 eq.) and L1/L2 (1.0 eq.) were dissolved in a mixture\n3H). HRMS: m/z 381.0487 [M + Na]+ (calcd 381.0598). of 2-ethoxyethanol and water (v:v = 2:1), and then heated at 120 \u25e6 C for\n\n 8\n\fQ. Zhou et al. Bioorganic Chemistry 161 (2025) 108507\n\n\n8 h under nitrogen atmosphere. After cooling to room temperature, the mitochondrial membrane potential and regulating the expression of\niridium dimer was filtered and dried, which was used in next step Caspase-3 and Bcl-2 family proteins. Another study shows that Ir2 could\nwithout further purification. A mixture of the iridium dimer, 2,2\u2032- induce cell cycle arrest in the G0/G1 phase, inhibit the growth of cancer\nbipyridine/1,10-phenanthroline and silver trifluoromethyl sulfonate cells and eventually trigger apoptosis. Most notably, Ir2 displays a\nwere dissolved in toluene and heated at 110 \u25e6 C for 12 h under nitrogen higher anticancer efficacy compared to cisplatin in a mouse tumor\natmosphere. After the reaction was completed, the solvent was removed model. Overall, our results pave a way for designing new tridentate-type\nin vacuo, then dichloromethane and water was added. The separated iridium(III) complexes as effective drugs for cancer treatment.\norganic phase was washed with brine, dried over Na2SO4, filtered, and\nconcentrated in vacuo. The residue was purified by flash column chro\u00ad CRediT authorship contribution statement\nmatography (DCM: MeOH = 100: 1\u201320: 1) to give complexes Ir1-Ir4.\n Ir1 (yellow-green solid, yield: 25.4 %). 1H NMR (400 MHz, CDCl3) \u03b4 Qin Zhou: Writing \u2013 original draft, Validation, Investigation, Formal\n10.43 (d, J = 4.7 Hz, 1H), 9.36 (d, J = 8.5 Hz, 1H), 8.95 (d, J = 7.8 Hz, analysis, Data curation. Xiao-Bin Zhang: Validation, Investigation,\n1H), 8.77 (t, J = 8.0 Hz, 1H), 8.32\u20138.22 (m, 1H), 8.08 (s, 2H), 7.91 (t, J Formal analysis, Data curation. An-Li Liu: Investigation, Formal anal\u00ad\n= 7.9 Hz, 1H), 7.85 (d, J = 7.9 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), ysis. Zhi-Gang Niu: Writing \u2013 review & editing, Supervision, Project\n7.23\u20137.17 (m, 2H), 7.18\u20137.06 (m, 2H), 6.23 (d, J = 8.5 Hz, 2H), 1.62 (s, administration, Funding acquisition, Conceptualization. Gao-Nan Li:\n9H). 13C NMR (101 MHz, DMSO\u2011d6) \u03b4 179.75, 169.05, 157.42, 156.31, Writing \u2013 review & editing, Supervision, Project administration, Fund\u00ad\n153.97, 150.27, 148.85, 148.25, 141.92, 139.86, 137.72, 131.24, ing acquisition, Conceptualization. Fa-Biao Yu: Supervision, Software,\n129.54, 129.06, 128.34, 126.21, 125.56, 125.16, 124.86, 116.84, 31.54. Resources, Funding acquisition, Data curation.\nHRMS: m/z 783.0972 (calcd 783.0995). Anal. Calcd for C35H27ClF3Ir\u00ad\nN4O3S3: C, 45.08; H, 2.92; N, 6.01; found: C, 45.10; H, 2.89; N, 6.04.\n Ir2 (yellow-green solid, yield: 20.6 %). 1H NMR (400 MHz, CDCl3) \u03b4 Declaration of competing interest\n10.72 (d, J = 5.0 Hz, 1H), 9.27 (d, J = 8.3 Hz, 1H), 8.66 (dd, J = 8.3, 5.0\nHz, 1H), 8.54 (d, J = 7.0 Hz, 1H), 8.46 (d, J = 8.9 Hz, 1H), 8.24 (d, J = The authors declare that they have no known competing financial\n9.0 Hz, 1H), 8.14 (s, 2H), 7.81\u20137.77 (m, 2H), 7.69 (dd, J = 8.2, 5.5 Hz, interests or personal relationships that could have appeared to influence\n1H), 7.60 (d, J = 4.3 Hz, 1H), 7.32\u20137.26 (m, 2H), 7.00 (t, J = 7.9 Hz, the work reported in this paper.\n2H), 6.02 (d, J = 8.4 Hz, 2H), 1.65 (s, 9H). 13C NMR (101 MHz,\nDMSO\u2011d6) \u03b4 179.72, 168.21, 154.89, 151.48, 148.93, 148.32, 148.06, Acknowledgments\n146.71, 140.81, 139.21, 137.88, 131.17, 130.65, 128.52, 128.49,\n128.12, 127.83, 127.13, 126.07, 125.16, 124.72, 116.69, 31.57. HRMS: This work was supported by the National Natural Science Foundation\nm/z 807.0969 (calcd 807.0995). Anal. Calcd for C37H27ClF3IrN4O3S3: C, of China (22061016, 22261016, 22264013), the Natural Science\n46.46; H, 2.85; N, 5.86; found: C, 46.48; H, 2.88; N, 5.87. Foundation of Hainan Province (225RC746, 823MS042) and the Grants\n Ir3 (yellow-green solid, yield: 23.4 %). 1H NMR (400 MHz, CDCl3) \u03b4 for the Innovation Center of Academician Sun Shigang\u2019s Team in Hainan\n10.42 (d, J = 4.4 Hz, 1H), 9.35 (d, J = 8.2 Hz, 1H), 8.93 (d, J = 8.2 Hz, Province.\n1H), 8.77 (t, J = 8.0 Hz, 1H), 8.31\u20138.20 (m, 1H), 7.95\u20137.87 (m, 3H),\n7.84 (d, J = 8.0 Hz, 2H), 7.40 (t, J = 7.3 Hz, 2H), 7.20 (t, J = 7.9 Hz, 3H), Appendix A. Supplementary data\n7.16\u20137.04 (m, 1H), 6.22 (d, J = 8.5 Hz, 2H), 2.76 (s, 2H). 13C NMR (101\nMHz, DMSO\u2011d6) \u03b4 179.45, 168.92, 157.47, 156.34, 153.96, 150.29, Supplementary data to this article can be found online at https://doi.\n148.90, 141.98, 139.93, 137.85, 134.69, 131.25, 129.58, 129.04, org/10.1016/j.bioorg.2025.108507.\n128.51, 128.41, 126.28, 125.63, 124.94, 116.87, 31.59. HRMS: m/z\n741.0505 (calcd 741.0525). Anal. Calcd for C32H21ClF3IrN4O3S3: C, Data availability\n43.17; H, 2.38; N, 6.29; found: C, 43.20; H, 2.42; N, 6.30.\n Ir4 (yellow-green solid, yield: 28.4 %). 1H NMR (400 MHz, CDCl3) \u03b4 Data will be made available on request.\n10.71 (d, J = 5.0 Hz, 1H), 9.25 (d, J = 8.3 Hz, 1H), 8.66 (dd, J = 8.2, 5.0\nHz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.45 (d, J = 8.9 Hz, 1H), 8.23 (d, J =\n References\n9.0 Hz, 1H), 7.97 (s, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.72\u20137.60 (m, 2H),\n7.30 (d, J = 7.2 Hz, 2H), 6.99 (t, J = 8.4 Hz, 2H), 6.01 (d, J = 8.3 Hz, [1] U. Das, B. 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Chem.\n 155 (2025) 108148, https://doi.org/10.1016/j.bioorg.2025.108148.\n\n\n\n\n 11\n\f Update\n Bioorganic Chemistry\n Volume 164, Issue , September 2025, Page\n\n\nDOI: https://doi.org/10.1016/j.bioorg.2025.108810\n\f Bioorganic Chemistry 164 (2025) 108810\n\n\n Contents lists available at ScienceDirect\n\n\n Bioorganic Chemistry\n journal homepage: www.elsevier.com/locate/bioorg\n\n\nCorrigendum\n\nCorrigendum to \u201cDesigning novel tridentate iridium(III) complexes\ncomprising functionalized benzothiazole ligands to improve anticancer\nactivity by targeting mitochondria\u201d [Bioorg. Chem. 161 (2025) 108507]\nQin Zhou a,c , Xiao-Bin Zhang a , An-Li Liu a,c , Zhi-Gang Niu a,c,*, Gao-Nan Li a,c,* , Fa-Biao Yu b,c\na\n Key Laboratory of Electrochemical Energy Storage and Light Energy Conversion Matreials of Haikou City, Key Laboratory of Electrochemical Energy Storage and Energy\nConversion of Hainan Province, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, China\nb\n Key Laboratory of Emergency and Trauma, Ministry of Education, Key Laboratory of Haikou Trauma, Key Laboratory of Hainan Trauma and Disaster Rescue, The First\nAffiliated Hospital, Hainan Medical University, Haikou 571199, China\nc\n Engineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, Key Laboratory of Hainan Functional Materials and Molecular Imaging, College\nof Emergency and Trauma, Hainan Medical University, Haikou 571199, China\n\n\n\n We regret that there are Chinese characters in the Fig. 10, it may mistake could be corrected in next available issue.\ncause inconvenience to readers from other countries. So I hope the\n\n\n\n\n DOI of original article: https://doi.org/10.1016/j.bioorg.2025.108507.\n * Corresponding authors at: Key Laboratory of Electrochemical Energy Storage and Light Energy Conversion Matreials of Haikou City, Key Laboratory of Elec\u00ad\ntrochemical Energy Storage and Energy Conversion of Hainan Province, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158,\nChina.\n E-mail addresses: niuzhigang1982@126.com (Z.-G. Niu), ligaonan2008@163.com (G.-N. Li).\n\nhttps://doi.org/10.1016/j.bioorg.2025.108810\n\nAvailable online 5 August 2025\n0045-2068/\u00a9 2025 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fQ. Zhou et al. Bioorganic Chemistry 164 (2025) 108810\n\n\n\n\n appreciate greatly any of your help.\n We would like to apologise for any inconvenience caused. And we\n\n\n\n\n 2\n\f", "pages_extracted": 14, "text_length": 78195}