Mitochondria Localized Anticancer Iridium(III) Prodrugs for Targeted Delivery of Myeloid Cell Leukemia-1 (Mcl-1) Inhibitors and Cytotoxic Iridium(III) Complex.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Mitochondria Localized Anticancer Iridium(III) Prodrugs for\n Targeted Delivery of Myeloid Cell Leukemia\u20111 (Mcl-1) Inhibitors and\n Cytotoxic Iridium(III) Complex\n Tejal Dixit, Monika Negi, and V. Venkatesh*\n Cite This: Inorg. Chem. 2024, 63, 24709\u221224723 Read Online\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ABSTRACT: Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic\n oncoprotein overexpressed in several malignancies and acts as one\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:29:28 (UTC).\n\n\n\n\n of the promising therapeutic targets for cancer. Even though there\n are several small molecule based Mcl-1 inhibitors reported, the\n delivery of Mcl-1 inhibitor at the target site is quite challenging. In\n this regard, we developed a series of mitochondria targeting\n luminescent cyclometalated iridium(III) prodrugs bearing Mcl-1\n inhibitors via ester linkage due to the presence of Mcl-1 protein in\n the outer mitochondrial membrane. Among the synthesized\n prodrugs, IrThpy@L2 was found to exhibit the potent cytotoxicity\n (IC50 = 30.93 nM) against HCT116 cell line when compared with\n bare Mcl-1 inhibitors (IC50 > 100 \u03bcM). Mechanistic studies further\n revealed that IrThpy@L2 quickly gets internalized inside the\n mitochondria of HCT116 cells and undergoes activation in the presence of overexpressed esterase which leads to the release of two\n cytotoxic species i.e. Mcl-1 inhibitors (I-2) and cytotoxic iridium(III) complex (IrThpy@OH). The improved cytotoxicity of\n IrThpy@L2 is due to the mitochondria targeting ability of iridium(III) prodrug, subsequent esterase activated release of I-2 to\n inhibit Mcl-1 protein and IrThpy@OH to generate reactive oxygen species (ROS). After prodrug activation, the released cytotoxic\n species cause mitochondrial membrane depolarization, activate a cascade of mitochondria-mediated cell death events, and arrest the\n cell cycle in S-phase which leads to apoptosis. The potent anticancer activity of IrThpy@L2 was further evident from the drastic\n morphological changes, size reduction in the solid tumor mimicking 3D multicellular tumor spheroids (MCTS) of HCT116.\n\n\n \u25a0 INTRODUCTION\n Protein\u2212protein interactions (PPIs) play a pivotal role in\n development of small molecule inhibitors to target these PPIs.5\n However, it is difficult to target these PPIs as their binding\n sites are usually wide and shallow,6 making it difficult for the\n several cellular processes like signal transduction, enzymatic\n small molecules to interact.7,8 Therefore, it is a great challenge\n activity, structural organization, gene regulation, protein\n to design small molecules that can inhibit these PPIs with high\n folding, and quality control, etc., enabling cells to carry out\n selectivity and good binding affinity.\n their functions and respond to environmental cues.1\u22124 These\n Antiapoptotic proteins play significant role for the survival\n interactions occur when two or more proteins bind together\n and progression of tumor by engaging cross talk with pro-\n via specific binding sites, which help to regulate cellular\n apoptotic proteins, which makes them appealing targets in\n processes such as cell growth, differentiation, and apoptosis.\n several cancer therapies.9\u221211 Mcl-1 is one such critical\n Dysregulation or disruption of normal protein\u2212protein\n antiapoptotic protein that belongs to the Bcl-2 family of\n interactions can lead to various molecular and cellular changes\n proteins that is generally overexpressed in various cancers like\n like activation of oncogenic signaling pathways, dysregulation\n estrogen receptor-positive breast cancer, melanoma, leukemia,\n of apoptosis and cell cycle control, disruption of tumor nonsmall cell lung cancer, ovarian, liver, colorectal, and human\n suppressor functions, activation of angiogenesis and metastasis cervical cancers, etc.12\u221214 In cancer cells, the overexpression of\n that promote tumor growth and progression.5 Understanding\n the aberrant protein\u2212protein interactions involved in tumori-\n genesis is crucial for developing targeted therapies that can Received: September 18, 2024\n selectively disrupt or modulate these interactions to inhibit Revised: November 6, 2024\n tumor growth. Accepted: December 2, 2024\n Researchers are actively studying these interactions to Published: December 12, 2024\n identify potential therapeutic targets and develop strategies\n to interfere with their associations. One such strategy is the\n\n \u00a9 2024 American Chemical Society https://doi.org/10.1021/acs.inorgchem.4c03950\n 24709 Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. (A) Synthetic scheme for the preparation of Ir(III) prodrugs. (B) Molecular structure of all six synthesized iridium(III) prodrugs.\n\nMcl-1 can contribute to tumor growth, resistance to radio- through which it exerts its activity, but until now, no Mcl-1\ntherapy, chemotherapy, and reduced patient survival rates.15,16 inhibitor has been approved for clinical applications.22,23 Some\nIts upregulation allows cancer cells to evade programmed cell of them have entered clinical trials (AZD5991, AMG-176,\ndeath, thereby promoting their survival and contributing to NCT03218683, and NCT03797261), but unfortunately got\ntumor progression.17,18 Due to its crucial role in cancer cell terminated due to their severe side effects.24 There are several\nsurvival, targeting Mcl-1 protein has become an intense area of drawbacks associated with small molecule based Mcl-1\nresearch in anticancer therapies.19,20 Researchers are actively inhibitors such as low bioavailability, low membrane\ndeveloping Mcl-1 inhibitors which could bind Mcl-1 protein permeability, and off-target bindings, etc.25 Recent studies\neffectively and with high degree of selectivity.21 In this have shown that most of the Mcl-1 inhibitors lack proper\ndirection, several small molecule inhibitors have been mitochondrial membrane penetration, and exhibit poor\ndeveloped to target the Mcl-1 protein and inhibit the PPIs anticancer activity.26 Therefore, not only structural modifica-\n 24710 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\ntion to enhance the binding affinity with Mcl-1 protein but also\nmore attention is required for the proper organelle targeting\n \u25a0 RESULTS AND DISCUSSION\n Design, Synthesis and Characterization. Herein, Mcl-1\nability of Mcl-1 inhibitors. Over the past decade, there has inhibitor appended cyclometalated Ir(III) prodrugs were\nbeen a great leap in the discovery of small molecule-based prepared in two steps. First, The \u03bc-chlorido-bridged dimers\ninhibitors. Several small molecules such as benzothiophene-2- [(ppy)2-Ir-\u03bc-Cl]2, [(bzq)2-Ir-\u03bc-Cl]2 and [(thpy)2-Ir-\u03bc-Cl]2\ncarboxylic acid, indole-2-carboxylic acid, benzofuran-2-carbox- were synthesized from IrCl3.xH2O using the procedure\nylic acid, and their derivatives were identified as potent Mcl-1 reported by us previously.87 The esterification of Mcl-1\ninhibitors.27 But, delivering them in the mitochondria is of inhibitors (5-chloroindole-2-carboxylic acid (I-1) and 3-\nparamount importance for eliciting potent anticancer activity.28 chlorobenzo[b]thiophene-2-carboxylic acid (I-2) with 4,4\u2032-\n After the success of Cisplatin in the anticancer arena, various Bis(hydroxymethyl)2,2\u2032-bipyridine was carried out to prepare\nother metal-based anticancer complexes have captivated the L1 and L2 respectively (N\u2227N ligands).88 Ir(III) prodrugs were\nattention of researchers over the past few decades.29\u221241 The synthesized using [(ppy)2-Ir-\u03bc-Cl]2, [(bzq)2-Ir-\u03bc-Cl]2 and\nanticancer activity of iridium(III) complexes have been [(thpy)2-Ir-\u03bc-Cl]2 along with L1 and L2 as mentioned in\nextensively studied because of their diverse mechanism of (Figure 1A, S1). All the synthesized complexes were further\naction, kinetic stability in biological systems which can be characterized by 1H & 13C NMR, High-resolution mass\nconsidered further as potential alternative for conventional spectrometry (HRMS) (Figure S2\u2212S34), and the structure\nplatinum-based anticancer drugs to overcome the side effects of IrPpy@L2 was confirmed from single crystal XRD.\nand drawbacks of platinum resistance.42\u221247 Cyclometalated Single-Crystal X-Ray Diffraction Studies. The single crystal\niridium(III) complexes have demonstrated significant anti- of IrPpy@L2 was grown by using a slow evaporation method\ncancer activity against a variety of cancer types, including both in a DCM-toluene (1:1, v/v) solvent mixture. The crystal\nsolid tumors and leukemic cells, as they penetrate the cancer structure of IrPpy@L2 is shown (Figure 2A). IrPpy@L2\ncells and target various subcellular organelles.48\u221253 They can\ninduce cancer cell death through multiple modes of action,\ndifferent from those of conventional platinum drugs, including\napoptosis (programmed cell death), autophagy (self-degrada-\ntion of cellular components), inhibition of protein\u2212protein\ninteractions, and disruption of cellular signaling pathways.54\u221266\nFurthermore, these complexes have opulent photophysical\nproperties,67 including high quantum yields,68,69 good photo-\nstability,70,71 long emission lifetimes,72,73 large Stokes shifts,74\ntwo-photon absorption (TPA), and high photobleaching\nresistance75 making them effective candidates for cancer\nimaging and diagnostics.76\u221280\n Considering all of the above aspects, we have designed and\nsynthesized mitochondria targeting cyclometalated iridium-\n(III) complexes bearing Mcl-1 inhibitors as prodrugs. The\npositive charge on the cyclometalated iridium(III) prodrugs\nfavors its mitochondria-targeting ability which is also\nstrategically a prominent target in several cancer therapies.\nThis approach facilitates the better uptake of prodrugs81 due to\nimproved lipophilicity, imparts mitochondria localizing abil-\nity,82 and mitochondrial esterase mediated delivery of Mcl-1\ninhibitors at the target site.83\u221285\n In this work, six positively charged cyclometalated iridium- Figure 2. (A) X-ray crystal structure of IrPpy@L2 at a 50% thermal\n ellipsoids probability level (hydrogen atoms were omitted for clarity).\n(III) prodrugs IrPpy@L1, IrPpy@L2, IrBzq@L1, IrBzq@L2, (B, C) Absorption and emission spectra of designed iridium(III)\nIrThpy@L1, IrThpy@L2 (L1 and L2 = 2,2\u2032-bipyridine-4,4\u2032- prodrugs (10 \u03bcM) performed in DMSO/PBS (v/v, 0.4:99.6) at room\ndiester derivatives of 5-chloroindole-2-carboxylic acid (I-1), temperature.\nand 3-chlorobenzo[b]thiophene-2-carboxylic acid (I-2) re-\nspectively) were synthesized (Figure 1B). Complexes IrPpy@\n crystallized in the monoclinic P 1 21/c 1 space group. The\nOH, IrBzq@OH, and IrThpy@OH which lack the inhibitor details of single crystal data refinement parameters are\npart and are already known for their cytotoxicity were also summarized in (Table S1). The selected bond lengths and\nsynthesized as reference compounds.86 IrThpy@L2 was found bond angles for IrPpy@L2 are provided in (Table S2).\nto be the most potent complex and showed excellent Photophysical Properties. The absorption and emission\ncytotoxicity against the HCT116 cell line. Fluorescence studies properties of iridium(III) prodrugs were studied in DMSO/\nand HRMS studies were conducted to investigate esterase PBS (v/v, 0.4:99.6) (Figure 2B, C) and DMSO (Figure S35A,\nmediated activation of prodrug IrThpy@L2 in vitro. The B) at room temperature. In all the iridium(III) prodrugs, the\nphotophysical properties, cellular uptake studies, subcellular higher energy intense absorption bands that lie in the UV\u2212vis\nlocalization, in vitro cytotoxicity, mitochondrial outer mem- region of <350 nm could be attributed to the mixed spin-\nbrane permeabilization (MOMP), ROS generation, cell cycle allowed ligand-centered (LC) transition (\u03c0\u2212\u03c0*) for C\u2227N and\narrest, and bioimaging studies were carried out to understand N\u2227N ligands.89,90 The less intense absorption bands falling\nthe mode of action of IrThpy@L2. between low energy region of UV\u2212vis i.e. 350\u2212450 nm\n 24711 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n(DMSO) and 400\u2212600 nm (DMSO/PBS, 0.4%) is due to higher accumulation of IrThpy@L2 could be attributed to its\nmixed singlet and triplet metal-to-ligand charge transfer higher lipophilicity compared to others as the water-octanol\n(1MLCT and 3MLCT) transitions.91 All six synthesized partition coefficient value (log Po/w) follows the order\ncomplexes display bright fluorescence emission maximum IrThpy@L2 > IrThpy@L1 > IrPpy@L2 > IrBzq@L2 >\nbetween 580 and 620 nm after exciting at 405 nm. Notably, IrPpy@L1 > IrBzq@L1 (Table S3). The uptake of iridium(III)\nIrThpy@L2 has a bathochromic shifted (red-shifted) emission prodrugs in HCT116 cells correlates well with their\nmaxima at 620 nm as compared to the rest of the prodrugs, lipophilicity.\nsuggesting that the combination of thpy ligand with L2 causes In-Vitro Cytotoxicity. The cytotoxicity of all six synthe-\nthis shift. All of the prodrugs possess a large Stokes shift of sized iridium(III) prodrugs, their corresponding reference\nabout 200 nm. Emission quantum yields of all the iridium(III) complexes, inhibitors, and Cisplatin was studied against HeLa\nprodrugs range from 0.007 to 0.065 in aerated DMSO at room (Human Cervical Carcinoma), HepG2 (Human Hepatocel-\ntemperature (Table S3). Moreover, the emission quantum lular Carcinoma), HCT116 (Human Colorectal Carcinoma),\nyield of all the synthesized Ir(III) complexes were compared A549 (Human Pulmonary Carcinoma), and human normal\nwith the respective reference complexes in DMSO/PBS (0.4%) liver cells (WRL-68) with the help of the MTT (3-(4,5-\nat room temperature (Table S3). Notably, they were found to dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide)\n assay. The half-maximum inhibitory concentration (IC50)\nbe 2.4 to 4.5 times higher than their corresponding reference\n values after 48 h of iridium(III) prodrugs treatment are listed\ncomplexes; the observed difference may be due to the presence\n in (Table 1) and (Figure 3B,C,D,E). All the iridium(III)\nof substituted N\u2227N ligands. Overall, the synthesized prodrugs prodrugs showed good antiproliferative activity against all the\nhold good photophysical properties which could be utilized to tested cancer cell lines, especially against HCT116 cell line\nunderstand their subcellular localization. with the IC50 values lying between 0.03 to 7.06 \u03bcM. Among all\n Lipophilicity and Cellular Uptake. Inductively coupled six iridium(III) prodrugs, IrThpy@L2 has shown the best\nplasma mass spectrometry (ICP-MS) was utilized to activity against HCT116 cancer cell line. The IC50 value of\nquantitatively estimate the cellular uptake of the iridium(III) IrThpy@L2 against the HCT116 cell line is 600-fold and 200-\nprodrugs. HCT116 cells were incubated with 5 \u03bcM iridium- fold less than that of Cisplatin and its corresponding reference\n(III) prodrugs for 1 h. It was observed that the accumulation of complex (IrThpy@OH) respectively. These results suggest\niridium inside cells in nM/7 \u00d7 104cells is highest for IrThpy@ that IrThpy@L2 has the substantial capability to induce\nL2 compared to others (Figure 3A) and (Table S3). This cytotoxicity against HCT116 cell line whereas both the Mcl-1\n inhibitors, I-1 and I-2 were found to be nontoxic even at higher\n concentrations. This could be attributed to their lower\n lipophilicity and off-target bindings. As the HCT116 cell line\n contains ample amount of esterase and overexpressed Mcl-1\n protein,92\u221295 therefore, the hydrolysis of IrThpy@L2 is feasible\n and further releases cytotoxic species which lead to the\n generation of ROS and Mcl-1 inhibition that resulted in\n nanomolar toxicity. All of the complexes were found to be\n more selective for the human cancer cell line as compared to\n the human normal cell (WRL-68) (Table 1) and (Table S4).\n The selectivity of the complexes is mainly due to the prodrug\n strategy developed in this approach, which results in higher\n IC50 values for normal cells. Due to aberrant metabolic\n activities and altered mitochondrial functions, cancer cells\n produce elevated levels of esterase and Mcl-1 protein when\n compared with normal cells.76,83,96\u2212102 Therefore, cancer cells\n provide an ideal environment to activate prodrug and exhibit\n nanomolar toxicity. These results thus confirmed our strategy\n of appending Mcl-1 inhibitors to the iridium scaffold for\n simultaneously inhibiting Mcl-1 protein and elevating ROS for\n inducing mitochondria mediated cell death selectively in\n cancer cells.\n Most notably, the cytotoxicity of iridium(III) prodrugs\n correlates well with their cellular uptake and lipophilicity.\n IrThpy@L2 exhibited potent cytotoxicity with highest uptake\n against HCT116 cell line; therefore, it has been considered as a\n lead molecule to perform further biological studies.\n In-Vitro Activation of IrThpy@L2 Prodrug. In all of the\n designed iridium(III) prodrugs, Mcl-1 inhibitors are attached\nFigure 3. (A) The cellular uptake of iridium(III) prodrugs in\n via ester linkage. Therefore, they undergo hydrolysis in the\nHCT116 cells after exposure to IrPpy@L1, IrPpy@L2, IrBzq@L1,\nIrBzq@L2, IrThpy@L1, and IrThpy@L2 for 1 h (5 \u03bcM each). (ICP- presence of mitochondrial esterase after reaching the\nMS was used to determine the concentration of iridium). (B, C, D, E) mitochondria. Herein, porcine liver esterase (PLE) was used\nCell viability assay was performed for IrThpy@L2, IrThpy@OH, and to study the hydrolysis of ester bonds in IrThpy@L2 as\nI-2 against HeLa, HepG2, HCT116, and A549 cells after 48 h depicted in (Figure 4D). To validate the esterase mediated\ntreatment. release of Mcl-1 inhibitors in vitro, HRMS and fluorescence\n 24712 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 1. IC50 (\u03bcM) of the Synthesized Complexes, Inhibitors, and Cisplatin Against HCT116, HepG2, HeLa, A549, and WRL-\n68 Cell Lines Expressed as Mean \u00b1 Standard Deviation (n = 3) at 48 h\n Compounds IC50 (\u03bcM)\n HCT116 HepG2 HeLa A549 WRL-68\n IrPpy@L1 4.22 \u00b1 0.91 6.05 \u00b1 1.10 7.28 \u00b1 1.00 10.83 \u00b1 1.57 >50\n IrPpy@L2 0.41 \u00b1 0.23 1.29 \u00b1 0.14 2.05 \u00b1 0.84 8.99 \u00b1 3.33 49.38 \u00b1 2.82\n IrBzq@L1 7.06 \u00b1 3.31 9.84 \u00b1 1.83 10.51 \u00b1 1.49 15.65 \u00b1 1.38 >50\n IrBzq@L2 2.25 \u00b1 1.92 3.43 \u00b1 1.18 4.86 \u00b1 2.42 9.00 \u00b1 1.07 >50\n IrThpy@L1 1.94 \u00b1 0.72 2.15 \u00b1 1.52 4.08 \u00b1 2.33 6.18 \u00b1 1.69 44.17 \u00b1 1.97\n IrThpy@L2 0.03 \u00b1 0.01 0.05 \u00b1 0.01 0.97 \u00b1 0.75 2.93 \u00b1 1.55 39.18 \u00b1 0.62\n IrPpy@OH 7.94 \u00b1 1.76 9.31 \u00b1 1.96 11.39 \u00b1 1.08 22.91 \u00b1 0.97 30.54 \u00b1 0.33\n IrBzq@OH 11.52 \u00b1 1.36 16.17 \u00b1 1.06 17.68 \u00b1 0.57 23.62 \u00b1 1.64 35.5 \u00b1 1.35\n IrThpy@OH 6.27 \u00b1 2.07 7.66 \u00b1 1.18 8.67 \u00b1 2.16 17.82 \u00b1 1.01 23.22 \u00b1 0.48\n Cisplatin 18.01 \u00b1 2.18 18.28 \u00b1 2.33 16.87 \u00b1 0.43 25.87 \u00b1 0.88 22.33 \u00b1 0.35\n I-1 >100 >100 >100 >100 >100\n I-2 >100 >100 >100 >100 >100\n\n\n\n\nFigure 4. (A) Time-dependent and (B) concentration-dependent changes in fluorescence intensity of IrThpy@L2 (10 \u03bcM) after treatment with\nporcine liver esterase (PLE) at room temperature (\u03bbexc= 405 nm). (C) HRMS spectra of IrThpy@L2 (10 \u03bcM) in the absence of PLE (top) and in\nthe presence of PLE (0.2 U/mL) after being incubated for 70 min at room temperature (bottom). (D) Schematic diagram representing the esterase\nmediated release of IrThpy@OH and I-2 from IrThpy@L2.\n\nspectroscopy studies were performed. The time-dependent After that, concentration-dependent fluorescence emission\nfluorescence emission intensity of IrThpy@L2 was measured at spectra were also recorded in which IrThpy@L2 was treated\n10 min time interval after treatment with PLE (Figure 4A). with different concentrations of PLE with the same time\nThere was a gradual decrement in the fluorescence intensity intervals (Figure 4B). With the increase in PLE concentration,\nsuggesting that IrThpy@L2 gets hydrolyzed into IrThpy@OH the fluorescence emission intensity decreased, which again\nand I-2 by PLE as the fluorescence intensity of IrThpy@L2 is indicates the hydrolysis of ester bonds to yield cytotoxic\napproximately 8 times greater than that of IrThpy@OH species, i.e. IrThpy@OH and I-2 (Figure 4D). Additionally,\n(Figure S35C). A control experiment was conducted to IrThpy@L2 was incubated with PLE in PBS for 70 min. The\nexamine the stability of IrThpy@L2 in PBS in the absence of HRMS spectra were recorded before and after the PLE\nPLE (Figure S35D). There was no noticeable change in the treatment. After PLE treatment with IrThpy@L2, it showed\nemission intensity of IrThpy@L2 in DMSO/PBS (0.4%) m/z peaks designated to the intact complex as well as complex\nbuffer solution even after 70 min, suggesting that the complex IrThpy@OH (Figure 4C) which further confirms the\nis stable in PBS buffer and gets activated only in the presence enzymatic hydrolysis of the lead complex to release the\nof esterase. cytotoxic species.\n 24713 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n Cellular Localization Studies. The subcellular local- the red region was utilized to examine its subcellular\nization of IrThpy@L2 in HCT116 cells was studied by using localization. To perform this study, HCT116 cells were treated\nfluorescence microscopy. The fluorescence emission property with IrThpy@L2 for 30 min and then incubated separately\nof IrThpy@L2 was utilized to study its uptake in HCT116 with organelle-specific staining dyes such as Mitotracker\ncells; bright red fluorescence emanating from the cells Green, Lysotracker Green, and 4\u2032,6-diamidino-2-phenylindole\nrepresents significant uptake of IrThpy@L2 in HCT116 cells (DAPI). Fluorescence images were further visualized to\n(Figure 5A). Also, the fluorescence emission of IrThpy@L2 in evaluate the colocalization studies. The colocalization results\n suggested that the green fluorescence of Mitotracker Green\n overlapped to a greater extent with the red fluorescence of\n IrThpy@L2 in the merged channel (appeared as yellow) with\n Pearson\u2019s correlation coefficient of 0.8 (Figure 5B). On the\n contrary, IrThpy@L2 exhibits poor colocalization for lysosome\n and nucleus with Pearson\u2019s correlation coefficient of 0.3 and\n 0.2, respectively (Figure 5C, D). These results revealed the\n maximum accumulation of IrThpy@L2 inside mitochondria\n rather than lysosomes and nuclei.\n Mechanism of Cell Death Investigation. The cytotox-\n icity result of IrThpy@L2 and prodrug activation studies\n encouraged us to investigate ROS production and further its\n implication in the induction of the cell death process. ROS\n generation by IrThpy@L2 inside cells was determined using\n the ROS probe, i.e., 2,7-dichlorodihydrofluorescein diacetate\n (DCFH-DA). Basically, a nonfluorescent cell-permeable dye\n DCFH-DA gets oxidized to form dichlorofluorescein (DCF) in\n the presence of ROS, which shows bright green fluores-\n cence.103 HCT116 cells were treated with IrThpy@L2 and\n stained with DCFH-DA. H2O2-treated cells were used as a\n positive control. The results revealed that untreated cells\n exhibited a weak fluorescence emission. Green emission was\n observed from a few cells for the positive control. Cells treated\nFigure 5. (A) The cellular uptake images of IrThpy@L2 after 1 h with IrThpy@L2 showed the highest green fluorescence for\nincubation with the HCT116 cell line. Magnification: 40x, scale bar: DCF (Figure 6A). ROS concentration is directly proportional\n20 \u03bcm. Intracellular localization of IrThpy@L2 in HCT116 cells to the fluorescence intensity of DCF (\u03bbem = 535 nm). So, the\ncostained with organelle targeting dye, i.e. Mitotracker Green (ex/em: fluorescence emission of DCF was quantified using a\n490/516 nm) (B), Lysotracker Green (ex/em: 504/511 nm) (C) and\nDAPI (ex/em: 364/454 nm) (D). Magnification: 60x, scale bar: 10\n microplate reader. The result demonstrated that there is\n\u03bcm. about a 7-fold increase in emission intensity of DCF in the\n presence of IrThpy@L2 when compared with control cells,\n\n\n\n\nFigure 6. (A) Determination of ROS generation by DCFH-DA in HCT116 cells before and after treatment with IrThpy@L2 and H2O2 treated\ncells as a positive control. Magnification: 40x. scale bar: 20 \u03bcm. (B) Corresponding statistical analysis of the generation of ROS in HCT116 cells.\nOne-way ANOVA with Turkey multiple comparison test was used to measure the p values, ***p < 0.001, and data expressed as mean \u00b1 SD (n =\n2). (C) Western blot analysis quantifying different protein expressions using a densitometric plot. (D) Effect of IrThpy@L2 on MMP analyzed by\nflow cytometry using JC-1 dye.\n\n 24714 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nconfirming the elevated ROS levels (Figure 6B). Therefore, positive control Cisplatin exhibits bright fluorescence due to its\nthese results clearly signify that the activation of IrThpy@L2 in DNA targeting ability. The greater extent of nuclear\nthe presence of mitochondrial esterase releases ROS generator deformation and disintegration observed for IrThpy@L2\nIrThpy@OH86 which further induces ROS-mediated apopto- demonstrated that cells were undergoing DNA damage,\nsis. ultimately leading to apoptosis (Figure 8A).\n To further confirm the activation of prodrug IrThpy@L2, The lowering of MMP and nuclear fragmentation further\nWestern blot analysis was conducted to quantify the expression encouraged us to analyze the cell cycle progression at 6 h post-\nof antiapoptotic Mcl-1 protein and proapoptotic Bax protein. treatment with IrThpy@L2 in HCT116 cells. Propidium\nThe results indicated the downregulation of Mcl-1 and iodide (PI) staining is utilized to evaluate the cell cycle\nupregulation of Bax, which further attests to the induction of distribution by using flow cytometry analysis. It shows the\nthe apoptosis that proceeds via inhibition of Mcl-1 protein distribution of cells in distinct phases of the cell cycle (G0/G1,\n(Figure 6C). Overall, our experimental results validated the S, Sub G1, and G2/M) for IrThpy@L2, Cisplatin as a positive\nesterase mediated activation of prodrug IrThpy@L2 and control and untreated cells (Figure 8B). The results indicated\nsubsequent release of Mcl-1 inhibitor15 and IrThpy@OH that IrThpy@L2 is arresting the cell cycle in the S phase\nwhich induce mitochondrial outer membrane permeabilization compared with untreated cells and Cisplatin. In addition, along\n(MOMP) resulting in mitochondria-mediated apoptosis by with a significant reduction in the G0/G1 phase, there was an\nROS generation and Mcl-1 inhibition (Figure 7). increment in cell population in the Sub G1 phase for IrThpy@\n L2 treated cells. These results demonstrate that complex\n IrThpy@L2 induced cell death in HCT116 after the disruption\n of cell cycle progression.\n Flow cytometry analysis with Annexin V- fluorescein\n isothiocyanate (Annexin V-FITC) and propidium iodide (PI)\n was conducted to determine the extent of cell apoptosis and\n necrosis in HCT116 cells. For cells incubated with IrThpy@\n L2, 7.3% and 25.5% of the cell populations were in the early\n and late apoptosis stages, respectively (Figure 8C). Whereas at\n a similar concentration of Cisplatin, only 12.1% and 9.4% of\n cells were in the early and late apoptosis stages, respectively.\n These results confirmed that the synthesized prodrug could\n induce cell apoptosis more effectively than Cisplatin.\n To further confirm the cytotoxic activity of the prodrug,\n fluorescein diacetate (FDA) and PI costaining assay were\n performed for IrThpy@L2 treated HCT116 cells (Figure 8D).\n FDA (permeable dye) is hydrolyzed by cellular esterase to\nFigure 7. Schematic diagram represents the plausible mechanism of show intense green fluorescence for live cells. PI (non-\naction of synthesized iridium(III) prodrugs. permeable dye) emits red fluorescence for dead cells after\n binding to DNA.106 The fluorescence microscopic images of\n The induction of cellular apoptosis through mitochondrial HCT116 treated with 5 \u03bcM IrThpy@L2 after 6 h showed a\ndysfunction was evaluated for IrThpy@L2 by measuring the decrement in green fluorescence compared to untreated cells.\nmitochondrial membrane potential (MMP). Mitochondrial No green fluorescence is observed after 8h of incubation with\ndysfunction is visualized using 5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032- IrThpy@L2. On the other hand, the proportion of cells stained\ntetraethylimidacarbocyanine iodide (JC-1). For a healthy with PI was significantly higher in the case of IrThpy@L2\npolarized mitochondrion, JC-1 emits red fluorescence when treated cells when correlated with the untreated control group.\nit exists in an aggregated form. On the other hand, JC-1 shows Thus, these results confirmed that IrThpy@L2 has the\ngreen fluorescence when it exists as a monomer in the case of potential to induce cell death in HCT116 cells.\ndepolarized mitochondria with low MMP.104 3D Multicellular Tumor Spheroids Studies. 3D Multi-\n HCT116 cells treated with IrThpy@L2 showed green cellular tumor spheroids were found to depict the most\nfluorescence (Figure S37), representing the loss of mitochon- promising 3D in vitro model used to study where the invasive\ndrial integrity. Moreover, red fluorescence is seen for untreated nature of cancer is much related to the in vivo model.107\nHCT116 cells depicting high MMP due to the JC-1 aggregates. Moreover, MCTS studies are considered as the perfect in vitro\nThe flow cytometry analysis further showed a higher model to represent the therapeutic potential of the drug. The\npercentage of IrThpy@L2 treated cells present in the images were taken through an inverted microscope and\nmonomeric region (58.1%) as compared to the untreated subjected to morphological analysis.\ncells (25.5%) which suggests that IrThpy@L2 causes a When spheroids were treated with IrThpy@L2, the loss in\nsignificant loss in the mitochondrial membrane potential of the solidity of MCTS and disintegration was witnessed,\nHCT116 cells (Figure 6D). compared with the untreated (control) MCTS (Figure 9A).\n Nuclear deformation and fragmentation studies were The compact perimeter in the case of untreated spheroids was\nperformed using nuclear staining dye, DAPI, to investigate destroyed for IrThpy@L2 treated spheroids after 6 days, which\nthe cellular DNA damage process using fluorescence indicated the cytotoxic potential of IrThpy@L2 against\nmicroscopy.105 IrThpy@L2 treated cells show intense HCT116 MCTS. Furthermore, to confirm the cell death\nfluorescence for DAPI due to condensed chromatin, thus activity in MCTS, fluorescence microscopic analysis of live/\nrepresenting the DNA damage process. Untreated HCT116 dead cell staining was performed using FDA (for live cells) and\ncells showed no signs of nuclear fragmentation. However, PI (for dead cells). The images were visualized in the green\n 24715 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 8. (A) Nuclear fragmentation of HCT116 cells treated with IrThpy@L2 followed by DAPI staining. Magnification: 40x, scale bar: 20 \u03bcm.\n(Arrow represents condensation of DNA inside the nucleus). (B) Histogram profile of HCT116 in various stages of the cell cycle with stacked bar\ngraph represents the cell percentage in each phase of the cell cycle after 6 h of treatment with IrThpy@L2 at 5 \u03bcM concentration. The subG1 cell\npopulation represents apoptotic/necrotic cells. (C) Quantification of apoptotic HCT116 cells using flow cytometric analysis data of cells stained\nwith Annexin V-FITC and PI. (D) FDA/PI costaining for HCT116 cells for untreated cells and IrThpy@L2 treated cells after 6 and 8 h. (Green\nfluorescence depicts viable cells, and red fluorescence indicates dead cells). Magnification: 40x, scale bar: 20 \u03bcm.\n\n\n\n\nFigure 9. (A) Morphology changes in HCT116 MCTS after incubation with IrThpy@L2 (5 \u03bcM) and Cisplatin (50 \u03bcM). (B) Fluorescence\nmicroscope images of live/dead cells in HCT116 MCTS after FDA/PI staining. Magnification: 10\u00d7, scale bar: 100 \u03bcm.\n\nchannel (for FDA) and red channel (for PI) after 48 h of IrThpy@L2 is capable of effectively inducing apoptosis in the\ntreatment with IrThpy@L2. Fewer cells stained in green MCTS.\nchannels were observed for IrThpy@L2 treated cells compared\nwith untreated control groups (Figure 9B). In addition to this,\ncells stained with PI in the red channel were remarkably higher\n \u25a0 CONCLUSIONS\n In summary, we synthesized six cyclometalated iridium(III)\nfor IrThpy@L2 (5 \u03bcM) treated MCTS. Similar observation prodrugs bearing Mcl-1 inhibitors. Among them, IrThpy@L2\nwas noted for positive control Cisplatin (50 \u03bcM) treated exhibited excellent cytotoxicity against the HCT116 cancer cell\nMCTS at higher concentrations. These results confirmed that line, which was approximately 600 times more potent than\n 24716 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nCisplatin. Most notably, IrThpy@L2 was found to be more was prepared according to a previous reported literature.88 After that,\nselective towards cancer cells as compared to normal human Cs2CO3 (5.84 mmol, 4 equiv) was suspended in dry DMF at 0 \u00b0C,\nhepatic cells (WRL-68). Uptake studies suggest that IrThpy@ then I-1/I-2 (2.92 mmol, 2 equiv) were added at the same\nL2 effectively internalizes in the HCT116 cell line and temperature. Then 5 (1.46 mmol) was added in portions, and the\n reaction mixture was shifted to room temperature (30 \u00b0C). The\nsubstantially localizes in the mitochondria. Furthermore, PLE reaction mixture was further allowed to stir at room temperature for\nmediated activation of IrThpy@L2 was evident from 12 h. After completion of the reaction, the product precipitated out\nfluorescence emission and HRMS studies. Western blotting was washed with water and further used for complex synthesis without\nanalysis was performed to understand the mitochondrial further purification.\nesterase mediated activation of IrThpy@L2. The generation Ligand L1. Off-white solid (Yield 97%). 1H NMR (500 MHz,\nof ROS, MMP depolarization, downregulation of Mcl-1 DMSO-d6) \u03b4 12.25 (s, 2H), 8.71 (d, J = 5.0 Hz, 2H), 8.47 (s, 2H),\nprotein, and upregulation of Bax protein further attest the 7.77 (d, J = 2.1 Hz, 2H), 7.57 (dd, J = 5.0, 1.4 Hz, 2H), 7.49 (dd, J =\nactivation of IrThpy@L2 in the HCT116 cell line due to the 8.8, 0.6 Hz, 2H), 7.31\u22127.27 (m, 4H), 5.55 (s, 4H); 13C NMR (126\noverexpression of mitochondrial esterase. Furthermore, the MHz, DMSO-d6) \u03b4 160.71, 155.25, 149.71, 146.54, 136.00, 128.06,\n 127.75, 125.18, 124.89, 122.51, 121.28, 118.67, 114.40, 108.10, 64.55.\nactivity of IrThpy@L2 was also tested against 3D multicellular\n HRMS (ESI): m/z calculated for [M + H]+: 571.0862. Found:\ntumor spheroids (MCTS) to mimic the solid tumor micro- 571.0962.\nenvironment. Our work signifies that incorporation of Mcl-1 Ligand L2. Off-white solid (Yield 95%). 1H NMR (500 MHz,\ninhibitors into the iridium(III) complex not only improves its DMSO-d6) 8.74 (d, J = 5.0 Hz, 2H), 8.54 (s, 2H), 8.17 (d, J = 8.1 Hz,\nanticancer activity but also imparts targeting ability. This 2H), 8.01 (d, J = 7.7 Hz, 2H), 7.66 (m, J = 26.1, 8.1, 7.1, 1.1 Hz, 4H),\nstrategy can be further exploited to design targeted multifunc- 7.57 (d, J = 3.5 Hz, 2H), 5.59 (s, 4H); 13C NMR (126 MHz, DMSO-\ntional prodrugs by conjugating metal complexes with other d6) \u03b4 160.18, 155.27, 149.78, 146.06, 138.12, 136.26, 129.08, 126.60,\nbioactive molecules/drugs to open new avenues for futuristic 126.42, 125.25, 123.76, 123.51, 122.48, 118.74, 65.55. HRMS (ESI):\nanticancer therapies. m/z calculated for [M + H] +: 605.0085. Found: 605.0177.\n General Procedure for the Synthesis of Iridium(III) Prodrugs. As\n\n\u25a0 EXPERIMENTAL SECTION\n General Materials and Methods. All starting materials, reagents,\n shown in Figure 1A, IrPpy@L1, IrPpy@L2, IrBzq@L1, IrBzq@L2,\n IrThpy@L1, and IrThpy@L2 were prepared by adding a solution of\n appropriate Ir(III) chloro-bridged dimer in DCM (10 mL) into the\nand solvents were purchased from commercial vendors and used solution of ligand L1/L2 (2.1 equiv) in DMF (5 mL) and the reaction\nwithout any further purification. Column chromatography was mixture was stirred for 12 h at 70 \u00b0C under the nitrogen atmosphere,\nperformed using silica gel (100\u2212200 mesh). Iridium(III)chloride followed by anion exchange with saturated NH4PF6 solution in\nhydrate, MitoTracker Green FM, LysoTracker Green DND-26, (3- methanol (5 mL) for 2 h. The desired product was achieved after\n(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), purification by silica gel column chromatography (5:95 v/v\nJC-1 assay kit, FITC Annexin V/Dead Cell Apoptosis Kit and Fetal Methanol/DCM).\nbovine serum (FBS) were purchased from Thermo Fisher Scientific. General Procedure for the Synthesis of Reference Complexes.\n2-Phenylpyridine, Porcine liver esterase (PLE), 2\u2032,7\u2032-dichlorofluor- Complexes IrPpy@OH, IrBzq@OH, IrThpy@OH were synthesized\nescein diacetate (DCFH-DA), 4\u2032,6-diamidino-2-phenylindole (DAPI) using similar strategy as mentioned above with a slight modification in\nand Fluorescein diacetate (FDA) were purchased from Sigma-Aldrich. the synthetic procedure by using a methanolic solution of 4 in place of\nDulbecco\u2019s modified Eagle\u2019s medium (DMEM), Roswell Park L1/L2. Briefly, a solution of appropriate Ir(III) chloro-bridged dimer\nMemorial Institute medium 1640 (RPMI 1640) and cell-lysis RIPA in DCM (10 mL) was added into the methanolic solution of ligand 4\nbuffer were purchased from HIMEDIA. 4,4\u2032-Dimethyl-2,2\u2032-bipyridyl, (2.1 equiv) (10 mL) and the reaction mixture was stirred for 12 h at\n5-Chloroindole-2-carboxylic acid were purchased from TCI. Anti- 60 \u00b0C under the nitrogen atmosphere, followed by anion exchange\nbodies Mcl-1, Bax for Western blot were purchased from Cloud-Clone with saturated NH4PF6 solution in methanol (5 mL) for 2 h. The\nCorp. and \u03b2-actin was purchased from Bio-Rad. Propidium iodide was desired product was achieved after purification via silica gel column\npurchased from SRL. Benzo[h]quinoline, 2-(Thiophen-2-yl)pyridine chromatography in CH2Cl2/CH3OH.\nand 3-Chlorobenzo[b]thiophene-2-carboxylic acid were purchased Complex IrPpy@L1. Bright yellow solid (Yield 45%). 1H NMR\nfrom BLD Pharma India. Deuterated solvents, such as DMSO-d6 (500 MHz, DMSO-d6) \u03b4 12.20 (s, 2H), 8.98 (s, 2H), 8.26 (d, J = 8.2\n(dimethyl sulfoxide) and CDCl3 (chloroform), were purchased from Hz, 2H), 7.95\u22127.86 (m, 8H), 7.68 (d, J = 5.1 Hz, 4H), 7.46 (d, J =\nEurisotop. All the experiments were performed using deionized water 8.8 Hz, 2H), 7.33\u22127.23 (m, 4H), 7.17 (t, J = 6.2 Hz, 2H), 7.03 (t, J =\nwherever required. The rest of the chemicals were all analytical grade 7.4 Hz, 2H), 6.91 (t, J = 7.5 Hz, 2H), 6.20 (d, J = 7.5 Hz, 2H), 5.62\nand obtained from commercial sources. (s, 4H); 13C NMR (126 MHz, DMSO-d6) \u03b4 166.89, 160.49, 155.24,\n The cyclometalated iridium(III) chloro-bridged dimers [(ppy)2-Ir- 150.37, 150.09, 149.15, 149.02, 143.87, 138.95, 136.02, 131.14,\n\u03bc-Cl]2, [(bzq)2-Ir-\u03bc-Cl]2, and [(thpy)2-Ir-\u03bc-Cl]2 were prepared 130.37, 127.83, 127.70, 126.69, 125.29, 125.18, 124.95, 124.04,\naccording to the previous literature methods.87 All of the synthesized 123.11, 122.43, 121.20, 120.17, 114.35, 108.50, 63.98. HRMS (ESI):\ncomplexes were characterized by 1H NMR, 13C NMR, and HRMS m/z calculated for [M-PF6] +: 1071.1804. Found: 1071.1806.\nspectrometry. All the complexes were dissolved in DMSO before Complex IrPpy@L2. Bright yellow solid (Yield 47%). 1H NMR\nperforming the biological experiments and the concentration of (500 MHz, DMSO-d6) \u03b4 8.93 (s, 2H), 8.28 (d, J = 8.2 Hz, 2H), 8.08\nDMSO was 0.4% vol in DMEM with 10% FBS. (d, J = 8.2 Hz, 2H), 7.97\u22127.91 (m, 8H), 7.82 (d, J = 4.8 Hz, 2H),\n General Instrumentation. Nuclear magnetic resonance spectra 7.64 (ddd, J = 30.4, 15.8, 6.5 Hz, 6H), 7.18 (t, J = 6.1 Hz, 2H), 7.03\n(NMR) were recorded by a JEOL-500 MHz spectrometer (1H NMR: (t, J = 7.5 Hz, 2H), 6.91 (td, J = 7.6, 1.0 Hz, 2H), 6.19 (d, J = 7.3 Hz,\n500 MHz, 13C NMR: 126 MHz) using DMSO-d6 and CDCl3 as the 2H), 5.68 (s, 4H); 13C NMR (126 MHz, DMSO-d6) \u03b4 166.81,\ndeuterated solvent. High-resolution mass spectra (HRMS) were 159.97, 155.21, 150.21, 150.17, 149.02, 148.72, 143.81, 138.87,\nrecorded in Agilent 6545 LC/Q-TOF mass spectrometer. UV\u2212visible 138.08, 136.08, 131.07, 130.28, 129.01, 126.73, 126.48, 126.28,\nabsorption spectra were recorded on Shimadzu UV-2600 UV/vis/ 125.12, 125.00, 123.98, 123.53, 123.34, 122.78, 122.35, 120.10, 64.76.\nNIR spectrophotometer. The emission spectra were recorded using HRMS (ESI): m/z calculated for [M-PF6] +: 1105.1028. Found:\nHoriba Fluoromax spectrophotometer. The MTT and quantification 1105.1030.\nof DCFH-DA assays were performed with the help of SYNERGY H1 Complex IrBzq@L1. Yellow solid (Yield 41%). 1H NMR (500\nmicroplate reader. MHz, DMSO-d6) \u03b4 12.18 (s, 2H), 9.03 (s, 2H), 8.59 (d, J = 7.5 Hz,\n General Synthetic Procedures for Ligands (L1 and L2) and 2H), 8.18\u22128.14 (m, 2H), 7.98 (d, J = 8.9 Hz, 2H), 7.88 (dd, J = 14.2,\nIr(III) Complexes. Ligands L1 and L2. As shown in Figure S1(e), 2-5 7.3 Hz, 4H), 7.78 (d, J = 5.2 Hz, 2H), 7.67 (d, J = 1.8 Hz, 2H), 7.62\n\n 24717 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n(dd, J = 8.1, 5.5 Hz, 2H), 7.56 (d, J = 7.9 Hz, 2H), 7.44 (d, J = 8.8 Hz, diffractometer (Mo K\u03b1 \u03bb = 0.71073). The total exposure time was\n2H), 7.30\u22127.25 (m, 4H), 7.19 (t, J = 7.6 Hz, 2H), 6.24 (d, J = 7.1 Hz, 4.92 h. The frames were integrated with the Bruker SAINT software\n2H), 5.61 (s, 4H); 13C NMR (126 MHz, DMSO-d6) \u03b4 161.71, package using a narrow-frame algorithm. The integration of the data\n157.58, 156.86, 151.86, 150.40, 150.04, 148.43, 141.43, 139.05, using a monoclinic unit cell yielded a total of 227513 reflections to a\n137.24, 135.09, 131.07, 130.85, 129.75, 129.04, 128.91, 128.07, maximum \u03b8 angle of 26.43\u00b0 (0.80 \u00c5 resolution), of which 12237 were\n127.93, 126.51, 126.16, 125.55, 124.37, 124.18, 122.42, 121.66, independent (average redundancy 18.592, completeness = 99.3%, Rint\n115.59, 109.69, 64.37. HRMS (ESI): m/z calculated for [M-PF6] +: = 5.88%, Rsig = 3.45%) and 10661 (87.12%) were greater than 2\u03c3(F2).\n1119.1804. Found: 1119.1842. The final cell constants of a = 14.2102(14) \u00c5, b = 30.898(3) \u00c5, c =\n Complex IrBzq@L2. Yellow solid (Yield 40%). 1H NMR (500 14.9071(15) \u00c5, \u03b1 = 90\u00b0, \u03b2 = 113.649(3)\u00b0, \u03b3 = 90\u00b0, volume =\nMHz, DMSO-d6) \u03b4 8.97 (s, 2H), 8.59 (dd, J = 8.1, 0.9 Hz, 2H), 8.17 5995.5(10) \u00c53, are based upon the refinement of the XYZ-centroids\n(dd, J = 5.4, 1.0 Hz, 2H), 8.06 (d, J = 8.1 Hz, 2H), 7.98 (d, J = 8.9 Hz, of 9733 reflections above 20 \u03c3(I) with 4.954\u00b0 < 2\u03b8 < 50.38\u00b0. Data\n2H), 7.92\u22127.88 (m, 6H), 7.71 (d, J = 5.6 Hz, 2H), 7.66\u22127.54 (m, were corrected for absorption effects using the Multi-Scan method\n8H), 7.18 (t, J = 7.6 Hz, 2H), 6.22 (d, J = 7.0 Hz, 2H), 5.67 (s, 4H); (TWINABS). The ratio of minimum to maximum apparent\n13\n C NMR (126 MHz, DMSO-d6) \u03b4 160.01, 156.31, 155.62, 150.84, transmission was 0.609. The calculated minimum and maximum\n148.85, 148.77, 147.06, 140.17, 138.11, 137.78, 136.10, 133.81, transmission coefficients (based on crystal size) are 0.6090 and\n129.81, 129.59, 129.07, 128.47, 126.81, 126.78, 126.52, 126.33, 0.9220. The structure was solved and refined using the Bruker\n125.01, 124.29, 123.58, 123.39, 122.94, 122.78, 120.40, 64.79. HRMS SHELXTL Software Package, using space group P 1 21/c 1, with Z =\n(ESI): m/z calculated for [M-PF6] +: 1153.1028. Found: 1153.1049. 4 for the formula unit, C52H34Cl2IrN4O4S2. The final anisotropic full-\n Complex IrThpy@L1. Brown solid (Yield 49%). 1H NMR (500 matrix least-squares refinement on F2 with 614 variables converged at\nMHz, DMSO-d6) \u03b4 12.20 (s, 2H), 8.98 (s, 2H), 7.93 (dd, J = 5.8, 1.6 R1 = 6.56% for the observed data and wR2 = 15.69% for all data. The\nHz, 2H), 7.87 (d, J = 5.7 Hz, 2H), 7.82 (ddd, J = 8.2, 7.4, 1.4 Hz, goodness-of-fit was 1.121. The largest peak in the final difference\n2H), 7.77 (ddd, J = 8.2, 1.5, 0.8 Hz, 2H), 7.70 (d, J = 2.1 Hz, 2H), electron density synthesis was 2.324 e\u2212/\u00c53 and the largest hole was\n7.67 (d, J = 4.7 Hz, 2H), 7.61\u22127.58 (m, 2H), 7.48\u22127.45 (m, 2H), \u22121.301 e\u2212/\u00c53 with an RMS deviation of 0.135 e\u2212/\u00c53. On the basis of\n7.33 (dd, J = 2.2, 0.9 Hz, 2H), 7.28 (dd, J = 8.8, 2.1 Hz, 2H), 6.98 the final model, the calculated density was 1.225 g/cm3 and F(000),\n(ddd, J = 7.4, 5.9, 1.6 Hz, 2H), 6.19 (d, J = 4.7 Hz, 2H), 5.64 (s, 4H); 2196 e\u2212. Crystal data, data collection parameters, and structure\n13\n C NMR (126 MHz, DMSO-d6) \u03b4 163.14, 160.64, 155.54, 152.51, refinement details are given in Table S1. Selected bond distances and\n150.86, 149.71, 149.43, 139.66, 136.41, 136.18, 131.26, 130.51, angles are given in Table S2. CCDC 2310258 (IrPpy@L2).\n127.98, 127.84, 126.96, 125.44, 125.09, 123.23, 121.36, 121.29, Hydrolysis of IrThpy@L2 by PLE in Vitro. Time and\n118.59, 114.53, 108.64, 64.11. HRMS (ESI): m/z calculated for [M- Concentration Dependent Emission Spectra. IrThpy@L2 (10\nPF6] +: 1083.0933. Found: 1083.0964. \u03bcM) was freshly prepared in 3 mL of PBS, and then a suspension\n Complex IrThpy@L2. Brown solid (Yield 44%). 1H NMR (500 of porcine liver esterase (PLE) (0.2 U/mL) in PBS was added. Time\nMHz, DMSO-d6) \u03b4 8.92 (s, 2H), 8.10\u22128.07 (m, 2H), 7.93\u22127.90 (m, dependent emission spectra were recorded after every 10 min.\n2H), 7.89 (d, J = 5.7 Hz, 2H), 7.86\u22127.83 (m, 2H), 7.82\u22127.80 (m, Similarly, concentration dependent emission spectra were recorded by\n2H), 7.77 (ddd, J = 8.2, 1.4, 0.8 Hz, 2H), 7.67\u22127.64 (m, 4H), 7.62\u2212 changing the concentration of PLE from 0.2 to 0.8 U/mL after 10 min\n7.58 (m, 4H), 6.98 (ddd, J = 7.4, 5.9, 1.5 Hz, 2H), 6.19\u22126.16 (m, incubation.\n2H), 5.69 (s, 4H); 13C NMR (126 MHz, DMSO-d6) \u03b4 163.34, HRMS. IrThpy@L2 (10 \u03bcM) was freshly prepared in 3 mL of PBS,\n160.46, 155.79, 152.54, 151.20, 149.95, 149.27, 139.89, 138.47, and then a suspension of porcine liver esterase (PLE) (0.2 U/mL) in\n136.63, 136.48, 131.41, 130.72, 129.48, 127.20, 126.99, 126.74, PBS was added. After the mixture was incubated at 298 K for 70 min,\n125.36, 123.91, 123.77, 123.05, 121.51, 118.78, 65.18. HRMS (ESI): acetone (ice cold) was added to quench the hydrolysis. The sample\nm/z calculated for [M-PF6] +: 1117.0156. Found: 1117.0159. was centrifuged, and the supernatant was collected and analyzed by\n Complex IrPpy@OH. Bright yellow solid (Yield 81%). 1H NMR HRMS.\n(500 MHz, CDCl3) \u03b4 9.04 (s, 2H), 7.89 (d, J = 8.2 Hz, 2H), 7.79\u2212 Determination of Lipophilicity. The lipophilicity of the\n7.65 (m, 8H), 7.50 (d, J = 5.5 Hz, 2H), 7.37 (d, J = 5.5 Hz, 2H), complexes was measured in an octanol\u2212water system using the\n7.03\u22126.99 (m, 4H), 6.90 (t, J = 7.4 Hz, 2H), 6.29 (d, J = 7.6 Hz, 2H), shake flask method. Solutions of IrPpy@L1, IrPpy@L2, IrBzq@L1,\n4.91 (s, 4H); 13C NMR (126 MHz, DMSO-d6) \u03b4 163.04, 156.22, IrBzq@L2, IrThpy@L1, and IrThpy@L2 were prepared in an\n155.23, 152.67, 149.98, 149.31, 139.33, 136.14, 131.57, 130.95, aqueous phase at a concentration of 10 \u03bcM. Equal volumes of the\n130.41, 128.70, 125.80, 121.74, 121.02, 118.31, 61.28. HRMS (ESI): solution and 1-octanol saturated with H2O were mixed and placed in\nm/z calculated for [M-PF6] +: 717.1841. Found: 717.1861. an orbital shaker overnight at 500 rpm. The samples were separated\n Complex IrBzq@OH. Yellow solid (Yield 85%). 1H NMR (500 into 2 phases after centrifugation at 8000 rpm for 10 min. Then the\nMHz, DMSO-d6) \u03b4 8.78 (s, 2H), 8.58 (d, J = 7.9 Hz, 2H), 8.09 (d, J = concentrations of the complexes in both phases were determined by\n5.1 Hz, 2H), 7.98 (d, J = 8.8 Hz, 2H), 7.89 (d, J = 8.9 Hz, 2H), 7.76 UV\u2212vis. absorbance. The lipo-hydro partition coefficient Log Po/w\n(d, J = 5.7 Hz, 2H), 7.61 (dd, J = 8.0, 5.5 Hz, 2H), 7.54 (dd, J = 12.4, were calculated using the following formula:\n6.7 Hz, 4H), 7.18 (t, J = 7.6 Hz, 2H), 6.22 (d, J = 7.1 Hz, 2H), 5.78 Po/w = Co/Cw = Ao/Aw (A stands for absorbance)\n(t, J = 5.5 Hz, 2H), 4.74 (d, J = 5.4 Hz, 4H); 13C NMR (126 MHz, Cell Lines and Culture. Human hepatic cells (WRL-68), HeLa\nDMSO-d6) \u03b4 166.96, 156.07, 155.14, 150.75, 149.43, 148.83, 143.89, (human cervical cancer), HCT116 (human colon cancer), A549\n138.81, 131.19, 130.32, 128.99, 128.30, 125.71, 125.15, 123.96, (human pulmonary carcinoma), and HepG2 (human liver cancer) cell\n122.30, 121.80, 120.09, 61.35. HRMS (ESI): m/z calculated for [M- lines were purchased from the National Centre for Cell Science\nPF6] +: 765.1841. Found: 765.1844. (NCCS), Pune, India. Dulbecco\u2019s modified eagle medium (DMEM)\n Complex IrThpy@OH. Brown solid (Yield 80%). 1H NMR (500 with fetal bovine serum (FBS, 10%), penicillin G (100 units/mL),\nMHz, DMSO-d6) \u03b4 8.73 (s, 2H), 7.81 (t, J = 7.6 Hz, 2H), 7.78\u22127.74 sodium bicarbonate (2.2 g/L), and streptomycin (100 mg/mL) is\n(m, 4H), 7.68\u22127.64 (m, 4H), 7.53 (d, J = 5.6 Hz, 2H), 6.96 (t, J = 6.5 used to grow the cells. Cells were cultured at approximately 80%\nHz, 2H), 6.18 (d, J = 4.7 Hz, 2H), 5.81 (t, J = 5.5 Hz, 2H), 4.77 (d, J confluence and kept in a humified incubator at 37 \u00b0C with 5% CO2 in\n= 5.3 Hz, 4H); 13C NMR (126 MHz, DMSO-d6) \u03b4 163.22, 156.37, cell culture flasks (tissue cultured) with different sizes depending on\n155.41, 152.85, 150.17, 149.49, 139.51, 136.31, 131.13, 130.59, the type of experiments to be performed.\n125.98, 121.90, 121.19, 118.49, 61.46. HRMS (ESI): m/z calculated ICP-MS Analysis for Cellular Uptake Studies. The cellular\nfor [M-PF6] +: 729.0970. Found: 729.0974. accumulation of iridium(III) prodrugs in HCT116 cells was analyzed\n Crystallographic Structure Determination. A specimen of by ICP-MS. The concentration of iridium for each sample was\nC52H34Cl2IrN4O4S2, approximate dimensions 0.034 \u00d7 0.106 \u00d7 0.227 determined using an Agilent 8900 ICP-MS Triple Quad. Briefly, 7 \u00d7\nmm, was used for the X-ray crystallographic analysis, and the 104 HCT116 cells were seeded onto a tissue-cultured 12-well plate for\nmeasurements were performed on a Bruker APEX-II CCD 24 h. Then, cells were treated with 5 \u03bcM Ir(III) prodrugs for 1 h.\n\n 24718 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nAfter the incubation, cells were washed with PBS, trypsinized, and for 10\u221215 min inside incubator. Then the cells were washed with PBS\ncollected. Cells were then digested with 68% of HNO3 (200 \u03bcL) for and incubated with DAPI (1 \u03bcg/mL) for 30 min in the dark. After\n24 h at room temperature. The sample solutions were further diluted that, cells were washed twice with PBS. DAPI-stained images of cells\nto a final volume of 10 mL with distilled water and then proceeded for were obtained with the help of a fluorescence microscope under a\nICP-MS. blue channel.\n Fluorescence Imaging. For cellular uptake imaging studies, 7 \u00d7 JC-1 Staining. HCT116 cells with a density of 7 \u00d7 104 cells per\n104 cells were grown over the coverslip in a 6-well plate for 24 h in well were seeded onto six-well plates and allowed to grow for 24 h.\nmedia with 10% FBS. The cells were treated with 5 \u03bcM of IrThpy@ Cells were treated with IrThpy@L2 (5 \u03bcM) for 4 h. After this period,\nL2 for 1 h. After completion of the incubation time, cells were washed cells were washed with PBS and stained with JC-1 (5 \u03bcg/mL) for 30\ncarefully with PBS twice to remove excess compound and then min under dark conditions. Cells were thoroughly washed with PBS\ncoverslips were mounted over the glass slides for fluorescence\n twice, and images were captured by keeping the exposure time\nimaging. All of the imaging experiments were performed using an\n constant.\nOlympus fluorescence microscope under different channels. Images\n JC-1 Flow Cytometric Analysis. HCT116 cells were seeded onto\nwere analyzed by using Fiji ImageJ software.\n Intracellular Localization. HCT116 cells (7 \u00d7 104 cells per well) a six-well plate with a density of 1 \u00d7 105 cells per well for 24 h and\nwere seeded onto 6 well plates and incubated with IrThpy@L2 (5 then treated with 5 \u03bcM of IrThpy@L2 for 4 h. Post treatment, cells\n\u03bcM) for 30 min in DMEM media. After this, cells were washed with were trypsinized and carefully harvested to make cell pellets by\nPBS and incubated with Lysotracker Green (300 nM), Mitotracker centrifugation. Each cell pellet was resuspended in 0.2 mL working\nGreen (300 nM), and DAPI (1 \u03bcg/mL) separately for 30 min in the solution of JC-1 dye (5 \u03bcg/mL) and incubated for 30 min. Then, cells\ndark inside the incubator. Cells were washed thoroughly with PBS, were washed carefully with PBS twice and resuspended in PBS, and\nand images were taken with the help of a fluorescence microscope. immediately flow cytometric analysis was performed using BD FACS\nThe images obtained were visualized with Fiji ImageJ software. The instrument, and data was analyzed by BD FACSDiva v8 software.\nvalue of Pearson\u2019s correlation coefficient was determined using the Western Blotting. HCT116 cells were seeded into six-well plates\nColoc2 plugin with a scatter plot. and incubated under a 5% CO2 incubator for 24 h. After that, the cells\n Cytotoxicity Assay. The cell viability or MTT assay was were treated with the control and IrThpy@L2 (1 \u03bcM) for 12 h. Then,\nperformed using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium cells were harvested after trypsinization and lysed in the RIPA lysis\nbromide (MTT) dye. The cytotoxicity was examined in the HCT116, buffer. The lysates of all groups obtained were centrifuged at 12000\nHeLa, WRL-68, A549, and HepG2 cell lines. Briefly, 1 \u00d7 104 cells per rpm at 4 \u00b0C for 10 min, and the supernatant was collected. Protein\nwell were cultured in 96 well plates and grown for 24 h in complete samples were separated on SDS-PAGE gel and onto PVDF\nmedia in a humidified incubator with 5% CO2. The stock solutions of membranes. The membranes were blocked with 5% freshly prepared\niridium(III) prodrugs (12 mM) and inhibitors (12 mM) were skim milk for 1 h at room temperature and then incubated with\nprepared in DMSO and further diluted with media (with 10% FBS) primary antibody and secondary antibody, respectively. The targeted\nso that the final concentration of DMSO in all the wells was lower proteins were detected with a chemiluminescent substrate. Gel Doc\nthan 0.5%. Cells were further treated with different concentrations (5,\n was used to visualize the band intensity, which was further quantified\n15, 30, 50 \u03bcM) of prodrugs and inhibitors for 48 h in media. FDA-\n using ImageJ software.\napproved drug, i.e., Cisplatin, was used as a positive control. After\n Cell Cycle Assay. Using PI staining, analysis of various stages of\nincubation, cells were carefully washed with PBS to remove the media.\nCells were loaded with a freshly prepared MTT solution (5 mg/mL) the cell cycle were analyzed for IrThpy@L2. In brief, HCT116 cells (1\nfor 4 h at 37 \u00b0C in the incubator. After 4 h, the formazan crystals (for \u00d7 105 cells per well) were incubated with IrThpy@L2 (5 \u03bcM) and\nactive cells) were dissolved in 150 \u03bcL of DMSO. Finally, the Cisplatin (50 \u03bcM) for 6 h. After that, the treated cells were washed\nabsorbance was measured at 570 nm using a microplate reader. The with PBS, trypsinized, and harvested by centrifugation. The pellets\ncell viability was determined by comparing the absorbance of drug- were carefully washed with ice-cold PBS, fixed with 70% ethanol, and\ntreated cells to that of the cells alone. All experiments were examined incubated for 2 h at 4 \u00b0C. After that, 100 \u03bcL of RNase (100 \u03bcg/mL)\nin three independent tests (n = 3) and represented as the mean \u00b1 and PI (50 \u03bcg/mL) were simultaneously added, and flow cytometry\nstandard deviation. was performed.\n ROS Generation in HCT116 Cell Line. Intracellular ROS levels Annexin V-FITC/PI Assay. HCT116 cells with a density of 1 \u00d7\nwere detected and quantified using DCFH-DA. Briefly, 7 \u00d7 104 105 cells per well were seeded in 6 well plates and then incubated with\nHCT116 cells per well were seeded in 6 well plates for 24 h in media. 5 \u03bcM of IrThpy@L2 for 6 h. Cells were carefully washed with PBS,\nH2O2 (working concentration= 50 \u03bcM) was taken as a positive trypsinized, and harvested through centrifugation. Cells were washed\ncontrol. The cells were treated with 5 \u03bcM of IrThpy@L2 and twice with ice-cold PBS and resuspended in 100 \u03bcL of 1x binding\nincubated for 2 h at 37 \u00b0C in a humidified incubator with 5% CO2. buffer. Then, 5 \u03bcL of Annexin V-FITC and 5 \u03bcL of PI solution were\nCells were carefully washed with 1 mL of PBS and then subjected to added, and samples were incubated for 15 min in the dark. Finally,\nDCFH-DA (20 \u03bcM) staining at 37 \u00b0C for 20 min under dark flow cytometric analysis was performed.\ncondition. Cells were then washed with PBS twice, and images were FDA/PI Costaining. HCT116 cells were grown onto a six-well\ntaken under green channels with the help of a fluorescence plate with a density of 7 \u00d7 104 cells per well for 24 h. The cells were\nmicroscope. All of the images were obtained at the same exposure treated with 5 \u03bcM IrThpy@L2 for 6 and 8 h. After this, cells were\ntime. After this, cells were trypsinized, collected by centrifugation, and carefully washed with PBS and stained with FDA (8 \u03bcg/mL) and PI\nfurther dissolved in PBS. The fluorescence intensity of DCF in each\n (2 \u03bcg/mL) for 5 min in the dark. Then, cells were washed slowly with\ngroup was quantified using a microplate reader with excitation and\n PBS twice, and images were captured using a fluorescence\nemission wavelengths of 485 and 535 nm, respectively. The results\nwere plotted as a fold increase in ROS generation by H2O2 and microscope.\nIrThpy@L2 correlated with the relative fluorescence of DCF with Studies in 3D Spheroid. HCT116 cells with a density of 2 \u00d7 104\nthree independent experiments with n = 2 replicates. cells per well were seeded in 96 U-shaped (ultralow attachment) well\n Nuclear Fragmentation Assay. For DAPI staining, 7 \u00d7 104 plates and grown to assemble as spheroids for 72 h. Spheroids were\nHCT116 cells were seeded onto six-well plates and grown for 24 h in then incubated with 5 \u03bcM IrThpy@L2 and 50 \u03bcM Cisplatin for 48 h.\na complete media. Cells were further treated with 5 \u03bcM IrThpy@L2 The integrity of the spheroids was observed for 6 days using an\nfor 6 h. Cisplatin (50 \u03bcM) was taken as positive control. Cells were inverted microscope. Simultaneously, treated spheroids were stained\ncarefully washed with PBS and then fixed with 4% freshly prepared using FDA/PI solution for 30 min in the dark at 37 \u00b0C. After that,\nparaformaldehyde and kept inside the incubator for 10\u221215 min. Cells samples were washed with PBS, and at last, images were captured\nwere washed again with PBS and permeabilized by 0.1% TritonX100 using a fluorescence microscope.\n\n 24719 https://doi.org/10.1021/acs.inorgchem.4c03950\n Inorg. Chem. 2024, 63, 24709\u221224723\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\u25a0\n*\n ASSOCIATED CONTENT\ns\u0131 Supporting Information\n (2) Lampe, J. N. Advances in the understanding of protein-protein\n interactions in drug metabolizing enzymes through the use of\n biophysical techniques. Front. Pharmacol. 2017, 8, 521.\nThe Supporting Information is available free of charge at (3) Rao, V. S.; Srinivas, K.; Sujini, G. N.; Kumar, G. N. S. Protein-\nhttps://pubs.acs.org/doi/10.1021/acs.inorgchem.4c03950. protein interaction detection: Methods and analysis. Int. J. Proteomics.\n NMR and HRMS spectra of L1, L2, IrPpy@L1, IrPpy@ 2014, 2014, 147648.\n L2, IrBzq@L1, IrBzq@L2, IrThpy@L1, and IrThpy@ (4) Michnick, S. W. Exploring protein interactions by interaction-\n L2. Crystallographic details of IrPpy@L2 with bond induced folding of proteins from complementary peptide fragments.\n length and bond angles (PDF) Curr. Opin. Struct. Biol. 2001, 11, 472\u2212477.\n (5) Deng, H.; Huang, M.; Liu, H.; Zhang, H.; Liu, L.; Gao, B.; Li, X.;\nAccession Codes Li, J.; Niu, Q.; Zhang, Z.; Luan, S.; Zhang, J.; Jing, Y.; Liu, D.; Zhao,\nDeposition Number 2310258 contains the supplementary L. Development of a series of novel Mcl-1 inhibitors bearing an indole\ncrystallographic data for this paper. These data can be obtained carboxylic acid moiety. Bioorg. Chem. 2022, 127, 106018.\nfree of charge via the joint Cambridge Crystallographic Data (6) Deng, H.; Han, Y.; Liu, L.; Zhang, H.; Liu, D.; Wen, J.; Huang,\n M.; Zhao, L. Targeting myeloid leukemia-1 in cancer therapy:\nCentre (CCDC) and Fachinformationszentrum Karlsruhe\n advances and directions. J. Med. Chem. 2024, 67, 5963\u22125998.\nAccess Structures service. (7) Chen, L.; Fletcher, S. Mcl-1 inhibitors: A patent review. Expert\n\n\u25a0 AUTHOR INFORMATION\nCorresponding Author\n Opin. Ther. Pat. 2017, 27, 163\u2212178.\n (8) Song, T.; Wang, Z.; Ji, F.; Feng, Y.; Fan, Y.; Chai, G.; Li, X.; Li,\n Z.; Zhang, Z. Deactivation of Mcl-1 by dual-function small-molecule\n V. Venkatesh \u2212 Department of Chemistry, Indian Institute of inhibitors targeting the Bcl-2 homology 3 domain and facilitating Mcl-\n Technology Roorkee, Roorkee, Uttarakhand 247667, India; 1 ubiquitination. Angew. Chem., Int. Ed. 2016, 55, 14250\u221214256.\n orcid.org/0000-0001-9520-6842; Email: venkatesh.v@ (9) Clifton, M. C.; Dranow, D. M.; Leed, A.; Fulroth, B.; Fairman, J.\n cy.iitr.ac.in W.; Abendroth, J.; Atkins, K. A.; Wallace, E.; Fan, D.; Xu, G.; Ni, Z. J.;\n Daniels, D.; Drie, J. V.; Wei, G.; Burgin, A. B.; Golub, T. R.; Hubbard,\nAuthors B. K.; Serrano-Wu, M. H. A maltose-binding protein fusion construct\n Tejal Dixit \u2212 Department of Chemistry, Indian Institute of yields a robust crystallography platform for MCL1. PLoS One 2015,\n Technology Roorkee, Roorkee, Uttarakhand 247667, India 10, No. 0125010.\n (10) Negi, A.; Murphy, P. V. Development of Mcl-1 inhibitors for\n Monika Negi \u2212 Department of Chemistry, Indian Institute of\n cancer therapy. Eur. J. Med. Chem. 2021, 210, 113038.\n Technology Roorkee, Roorkee, Uttarakhand 247667, India (11) Maeda, Y.; Takahashi, H.; Nakai, N.; Yanagita, T.; Ando, N.;\nComplete contact information is available at: Okubo, T.; Saito, K.; Shiga, K.; Hirokawa, T.; Hara, M.; Ishiguro, H.;\nhttps://pubs.acs.org/10.1021/acs.inorgchem.4c03950 Matsuo, Y.; Takiguchi, S. Apigenin induces apoptosis by suppressing\n Bcl-xl and Mcl-1 simultaneously via signal transducer and activator of\nAuthor Contributions transcription 3 signaling in colon cancer. Int. J. Oncol. 2018, 52,\nV.V. and T.D. conceptualized the study. T.D. synthesized and 1661\u22121673.\ncharacterized the complexes, and T.D. and M.N. performed all (12) Friberg, A.; Vigil, D.; Zhao, B.; Daniels, R. N.; Burke, J. P.;\nthe biological studies. T.D. performed and analyzed the single Garcia-Barrantes, P. M.; Camper, D.; Chauder, B. A.; Lee, T.;\n Olejniczak, E. T.; Fesik, S. W. Discovery of potent myeloid cell\ncrystal XRD analysis of the synthesized complex. The\n leukemia 1 (Mcl-1) inhibitors using fragment-based methods and\nmanuscript was written through the contributions of all structure-based design. J. Med. Chem. 2013, 56, 15\u221230.\nauthors. All authors have approved the final version of the (13) Harrison, L. R. E.; Micha, D.; Brandenburg, M.; Simpson, K. L.;\nmanuscript. Morrow, C. J.; Denneny, O.; Hodgkinson, C.; Yunus, Z.; Dempsey,\nNotes C.; Roberts, D.; Blackhall, F.; Makin, G.; Dive, C. Hypoxic human\nThe authors declare no competing financial interest. cancer cells are sensitized to BH-3 mimetic\u2212induced apoptosis via\n downregulation of the Bcl-2 protein Mcl-1. J. Clin. Invest. 2011, 121,\n\u25a0 ACKNOWLEDGMENTS\nV.V. sincerely acknowledges Faculty Initiation Grant (FIG),\n 1075\u22121087.\n (14) Zhang, Z.; Wu, G.; Xie, F.; Song, T.; Chang, X. 3-\n thiomorpholin-8-oxo-8h-acenaphtho[1,2-b]pyrrole-9-carbonitrile\nIIT Roorkee, India for the funding support. T.D and M.N (S1) based molecules as potent, dual inhibitors of B-cell lymphoma 2\nacknowledge University Grants Commission (UGC) and IIT (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1): Structure-\nRoorkee respectively for their fellowship. The authors are based design and structure\u2212activity relationship studies. J. Med. Chem.\ngrateful to Dr. B.V.V.S. Pavan Kumar for providing access to 2011, 54, 1101\u22121105.\nthe fluorescence microscope facility. The authors are also (15) Zhang, S.; Zhong, X.; Yuan, H.; Guo, Y.; Song, D.; Qi, F.; Zhu,\nthankful to Mr. Prathapa S.J. from Bruker for assisting in Z.; Wang, X.; Guo, Z. Interfering in apoptosis and DNA repair of\nsolving the crystal structure, Dr. Reena Kumari and Mr. cancer cells to conquer cisplatin resistance by platinum(IV) prodrugs.\nKartikay Tyagi for their valuable suggestions while performing Chem. Sci. 2020, 11, 3829\u22123835.\nbiological studies and Mr. Ramesh Kumar (M.Sc. Student) for (16) Drennen, B.; Goodis, C. C.; Bowen, N.; Yu, W.; Vickers, G.;\nassisting T.D. in ligand synthesis. We acknowledge the Wilder, P. T.; MacKerell, A. D.; Fletcher, S. Scaffold hopping from\n indoles to indazoles yields dual MCL-1/BCL-2 inhibitors from MCL-\nInstitute Instrumentation Centre (IIC), IIT Roorkee for\n 1 selective leads. RSC Med. 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Chem. 2024, 63, 24709\u221224723\n\f", "pages_extracted": 15, "text_length": 124868}