Antitumor Effects of Ir(III)-2H-Indazole Complexes for Triple Negative Breast Cancer

pubs.acs.org/IC Article Antitumor Eﬀects of Ir(III)‑2H‑Indazole Complexes for Triple Negative Breast Cancer Rajeeva Lochana Panchangam, Ramdas Nishanth Rao, Musuvathi Motilal Balamurali, Tejashri B. Hingamire, Dhanasekaran Shanmugam, Venkatraman Manickam, and Kaushik Chanda* Downloaded via TSINGHUA UNIV on November 5, 2022 at 09:01:51 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. Cite This: Inorg. Chem. 2021, 60, 17593−17607 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: In this work, we have synthesized a series of novel C,Ncyclometalated 2H-indazole-ruthenium(II) and -iridium(III) complexes with varying substituents (H, CH3, isopropyl, and CF3) in the R4 position of the phenyl ring of the 2H-indazole chelating ligand. All of the complexes were characterized by 1H, 13C, high-resolution mass spectrometry, and elemental analysis. The methyl-substituted 2H-indazole-Ir(III) complex was further characterized by single-crystal X-ray analysis. The cytotoxic activity of new ruthenium(II) and iridium(III) compounds has been evaluated in a panel of triple negative breast cancer (TNBC) cell lines (MDA-MB-231 and MDA-MB-468) and colon cancer cell line HCT-116 to investigate their structure−activity relationships. Most of these new complexes have shown appreciable activity, comparable to or signiﬁcantly better than that of cisplatin in TNBC cell lines. R4 substitution of the phenyl ring of the 2Hindazole ligand with methyl and isopropyl substituents showed increased potency in ruthenium(II) and iridium(III) complexes compared to that of their parent compounds in all cell lines. These novel transition metal-based complexes exhibited high speciﬁcity toward cancer cells by inducing alterations in the metabolism and proliferation of cancer cells. In general, iridium complexes are more active than the corresponding ruthenium complexes. The new Ir(III)-2H-indazole complex with an isopropyl substituent induced mitochondrial damage by generating large amounts of reactive oxygen species (ROS), which triggered mitochondrionmediated apoptosis in TNBC cell line MDA-MB-468. Moreover, this complex also induced G2/M phase cell cycle arrest and inhibited cellular migration of TNBC cells. Our ﬁndings reveal the key roles of the novel C−N-cyclometalated 2H-indazole-Ir(III) complex to speciﬁcally induce toxicity in cancer cell lines through contributing eﬀects of ROS-induced mitochondrial disruption along with chromosomal and mitochondrial DNA target inhibition. lung cancer.10 In the most prevalent triple negative breast cancer (TNBC), the cells do not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2), which are generally present in other therapeutically responsive breast cancer subtypes.11 Moreover, the developed drug resistance caused by impaired hormonal HER-2 targeted therapy makes it very diﬃcult to treat. For these reasons, though only 15−20% of breast cancers are of the TNBC type, it is considered as the most aggressive and highly metastatic and has a poor prognosis with increased risk of reoccurrence and low survival rates. However, chemotherapy remains a key therapeutic option for treating early and advanced stages of TNBC. Women with TNBC are 1. INTRODUCTION The discovery of cisplatin as an anticancer drug by Rosenberg et al. in 1969 revolutionized cancer chemotherapy and extended the range of commonly applied chemotherapeutics purely from organic drugs to cyclometalated drugs.1,2 Gandin et al. demonstrated the anticancer potential of the Pt(IV) derivative of cisplatin, with two axial ligands such as aspirin, ibuprofen, or dichloroacetate, or phenylbutyrate against human cancer cell lines with 100-fold more potency than cisplatin.3 This further inspired medicinal chemists to look beyond for other possible and alternate chemotherapeutics that included metal complexes with Ru, Os, Ir, Fe, and Rh metal centers4−8 speciﬁcally to overcome the limitations associated with platinum chemotherapy.9 The design of cyclometalated halfsandwich metal complexes was based on the ability of hydrophobic arene ligands to facilitate free diﬀusion through the cell membrane along with additional coordinating sites for bidentate ligands, thus allowing the biological and pharmacological activities to be eﬀectively tuned. Breast cancer is the world’s second most common lethal cancer for women after © 2021 American Chemical Society Received: July 21, 2021 Published: November 12, 2021 17593 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC reported to harbor mutations in the BRCA gene, which makes possible therapeutics that speciﬁcally inhibit DNA targets,12,13 as evidenced with platinum complexes like cisplatin and carboplatin that are known to directly inhibit DNA. On the contrary, the development of severe side eﬀects and drug resistance in TNBC patients made these compounds ineﬃcient treatment options that paved the way for the development of other metal-based chemotherapeutics. Furthermore, this led to the development of the in vivo antimetastatic activity of ruthenium complexes14,15 and less toxic ruthenium(II) and iridium(III)16 complexes as angiogenesis inhibitors with their selective cytotoxicity. Cyclometalated Ir(III) complexes have been extensively studied for their anticancer activities, contrastenhancing applications in cell imaging, and peptide labeling.17 Recently, Komarnicka et al. studied the anticancer potential of half-sandwich Ir(III) complexes with phosphine derivatives of ﬂuoroquinolones along with interaction among DNA, albumin, and apo-transferrin.18 Targeting apoptosis, in particular mitochondrial targets (mutations that hamper its metabolism), 19 will be the primary aim in developing any anticancerous therapeutic strategies. Other possibilities for tuning mitochondrial functions include ceasing cancer cell proliferation and inducing cell death.20,21 The recently developed mitochondrion-targeting chemotherapeutic molecules serve as promising candidates for targeting cancer cells.22 Likewise, the novel synthesis and use of small heterocyclic molecules as potential chelating ligands for cyclometalation have also been well recognized in recent years. The currently prevalent rational design approaches serve as a tool for the generation of promising metallodrugs with improved anticancer activity.23 The design concept for the currently synthesized 2H-indazole-bearing Ru and Ir complexes originated from the recognition of the biological role of the core 2H-indazole moiety in having antitumor24 and modulatory activity versus estrogen receptors25 and in theranostic applications.26 Interestingly, the 2H-indazole ligands form the core in many best selling drugs such as pazopanib and niraparib, having the ability to target drug resistant, epidermal growth factor receptor (EGFR)-overexpressing tumors.27 Here we have designed the 2H-indazole C,N-cyclometalated ruthenium(II) and iridium(III) complexes with the hypothesis that altering substituents on the metal-coordinated ligands can inﬂuence the activity of the complex. Therefore, the phenyl ring of the ligand is substituted with both electron-donating and electron-withdrawing groups that favor the structure− activity relationship (SAR) of the complexes. In this work, we have investigated the cytotoxicity, apoptosis, cell cycle arrest, and inhibition of cell migration in a panel of triple negative breast cancer cell lines. We further demonstrate that these new complexes represent promising therapeutic agents for modulating triple negative breast cancer and could serve as an eﬃcient strategy for modifying the existing metallo-drug cisplatin. Article overall yield of 78−95%. Inspired by the introduction of C,Ncyclometalated complexes with heterocyclic ligands,28 we attempted further synthesis of new cyclometalated 2H-indazole ruthenium(II) and iridium(III) complexes as depicted in Scheme 1, as anticancer agents on aggressive triple negative breast cancer cells. Scheme 1. Synthesis of 2H-Indazole-Containing Ruthenium(II) and Iridium(III) Complexes For the synthesis of ruthenium complexes 4a−d, 2Hindazole ligands 1a−d were treated with p-cymene ruthenium(II) [(p-cymene)RuCl2]2 and sodium acetate in dichloromethane at room temperature under a N2 atmosphere for 20 h to aﬀord the corresponding complexes in 70−75% yields. Similarly, iridium complexes 5a−d were synthesized using a similar method starting from the corresponding [(η5-C5Me5)IrCl2]2 in 80−85% yields. Structures of ruthenium complexes 4a−d were unequivocally conﬁrmed from the 1H NMR spectra by the disappearance of one aromatic proton and the introduction of four doublets at 5.1−5.9 ppm, where as for half-sandwich iridium(III) complexes 5a−d, the appearance of a singlet at 1.7 ppm for 15 protons corresponding to pentamethylcyclopentadienyl (C5Me5) moiety in 1H NMR spectra conﬁrmed the complexes. The structures of other synthesized complexes were also established by spectroscopic and analytical methods, including 1H NMR, 13C NMR, and ESI-MS techniques (Supporting Information). Compound 5b was also characterized by X-ray diﬀraction from the crystal obtained by slow diﬀusion of hexane into a saturated solution of 5b in CH2Cl2/EA. The structure and numbering are shown in Figure 1. Crystallographic data along with selected bond lengths and angles are listed in Tables S3−S9. The pentamethylcyclopentadienyl group displays the common πbonded η5-coordination mode, whereas the 2-(p-tolyl)-2Hindazole assumes a bidentate-chelated coordination mode (C,N); the two rings of the 2H-indaozle and phenyl moieties are coplanar. An in-depth investigation of the structural characteristics for all of the reported complexes (4a−d and 5a−d) was carried out through computational methods using the B3LYP functional and DEF2-TZVPP basis set for ligands and atoms and SDD for Ir and Ru. The computed results were compared with the corresponding X-ray crystallographic data (Table S10). The electronic properties of all of the complexes were studied in diﬀerent solvents of varying polarity and protic 2. RESULTS AND DISCUSSION 2.1. Synthesis and Characterization of the Novel C−N Cyclometalated Complexes. The substituted 2H-indazole ligands 1 were synthesized via a one-pot synthetic transformation using Cu2O rhombic dodecahedra as the nanocatalyst.24 The ligands were substituted with electron-donating moieties (methyl, isopropyl, etc.) and a triﬂuoromethyl group as the electron-withdrawing moiety. All ligands reported here were synthesized by a cyclo-condensation reaction with an 17594 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Article Figure 1. Single-crystal ORTEP diagram of compound 5b (50% thermal ellipsoids). Selected bond lengths (angstroms) and angles (degrees) for 5b: Ir−C19, 2.052(5); Ir−N1, 2.081(4); Ir−Cl, 2.408(11); Ir−Cp*(centroid), 1.81(2); C19−Ir−N1, 77.2(18); N1−Ir−Cl, 85.49(11); C19−Ir− Cl, 88.67(13); Cp*(centroid)−Ir−Cl, 92.6(13); Cp*(centroid)−Ir−N1, 143.6(19); Cp*(centroid)−Ir−C19, 139.1(2). CCDC number 2056445. Figure 2. Computed (B3LYP/def2TZVPP) ESP surface maps drawn onto a total electron density surface (isovalue of 0.0004) and scaled between the electrostatic potential of ±3.948 ×10−2 au for iridium complexes (5c) and ±4.118 ×10−2 au for ruthenium complexes (4b). Red-colored surfaces are more positive electrostatic potential, and blue-colored surfaces more negative electrostatic potential. extinction coeﬃcient values of ∼200 M−1 cm−1. Moreover, the values of the molar extinction coeﬃcient, as given in Table S1, indicate that the observed transitions are of a π−π* nature. The ﬂuorescence spectra of complexes 4a−d and 5a−d in diﬀerent solvents as mentioned above were recorded in the range of 330−550 nm and at an excitation wavelength of 300 nm. Unlike the absorption spectra, a prominent shift was observed in the emission band maxima in diﬀerent solvents with diﬀerent polarities and protic natures (Figure S2). The results indicate the inﬂuence of dielectric parameters on stabilizing the various electronic levels of the complexes. An increase in the quantum yield was observed with an increase in the polarity of the solvents, and the values are listed in Table S1. The full width at half-maximum (fwhm) revealed that the observed band corresponds to a single species in the excited state in all of the solvents except for CH2Cl2. This could be due to the formation of additional species whose electronic transitions overlap with each other in the excited state. The same was also revealed from the excitation spectrum, where diﬀerent band maxima were observed when spectra were recorded at diﬀerent emission wavelengths (data not shown), indicating that the emission band in CH2Cl2 has two diﬀerent originating species. To investigate the stability of synthesized complexes 4a−d and 5a−d in aqueous medium, the absorption spectra were recorded in water with 5% DMSO at diﬀerent time intervals (0 nature to investigate their applications as potential optical probes, as well as in photodynamic therapy for cancer treatments. Stock solutions (5 mM) of 4a−d and 5a−d for the study were prepared in neat methanol, and further dilutions to achieve the working concentration of ∼5 μM in the respective solvent were carried out after evaporating the residual methanol. The electronic spectra of complexes 4a−d and 5a−d could not be recorded in water due to their poor solubility, and hence, the spectra had to be recorded in neat water with saturated aqueous solutions of the complexes. All other experiments in aqueous medium were performed in the presence of 5% DMSO to solubilize the complexes. The ultraviolet−visible absorption spectra in various solvents like CH2Cl2 DMF, ethyl acetate, methanol, and water in the range of 220−450 nm were recorded to investigate their solvatochromic eﬀect, and the results are depicted in Figure S2. The long wavelength absorption band of ruthenium complexes 4a−d was centered at ∼280 and 330 nm, and that of iridium complexes 5a−d at ∼280 nm. The spectra of ruthenium complexes were relatively more structured than those of iridium complexes, indicating the inﬂuence of solvents on perturbing various electronic levels to increase degeneracy. The short wavelength absorption band corresponds to the intraligand transitions, while the transitions that correspond to the long wavelength absorption band centered ∼330 nm are caused by the d−d transition, as evidenced by the low molar 17595 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Article Table 1. Cytotoxicities of Synthesized Compounds against Diﬀerent Cell Lines [IC50 (micromolar)]a compound MDA-MB-231 (SI)b MDA-MB-468 (SI)b HCT-116 (SI)b HEK 293 1a 1b 1c 1d 4a 4b 4c 4d 5a 5b 5c 5d cisplatin 74.03 ± 2.78 (1.35) 29.23 ± 1.52 (3.42) 19.29 ± 0.41 (3.98) >100 (∼1) 37.14 ± 1.95 (1.04) 1.36 ± 0.74 (25.58) 4.97 ± 0.56 (6.65) 11.12 ± 0.20 (1.61) 3.33 ± 0.09 (7.19) 2.77 ± 0.06 (8.27) 1.19 ± 0.05 (15.6) 5.07 ± 0.11 (3.39) 4.95 ± 0.05 65.43 ± 3.75 (1.52) 22 ± 1.46 (4.54) 18.94 ± 0.66 (4.03) 71.1 ± 0.79 (1.34) 22.96 ± 0.25 (1.68) 1.69 ± 0.25 (20.57) 4.20 ± 0.07 (7.86) 5.15 ± 0.11 (3.47) 0.95 ± 0.03 (25.2) 0.75 ± 0.06 (30.56) 0.52 ± 0.02 (35.71) 4.13 ± 0.08 (4.17) 2.27 ± 0.03 76.26 ± 1.20 (1.31) 63.08 ± 0.72 (1.58) 22.65 ± 0.84 (3.37) >100 (∼1) 40.3 ± 1.19 (∼1) 1.3 ± 0.003 (26.75) 6.03 ± 0.10 (5.48) 9.43 ± 0.19 (1.89) 3.47 ± 0.11 (6.90) 1.59 ± 0.04 (14.41) 1.36 ± 0.01 (13.65) 5.4 ± 0.09 (3.19) 1.89 ± 0.06 >100 >100 76.49 ± 1.48 95.92 ± 2.52 38.53 ± 1.04 34.78 ± 0.49 33.03 ± 1.02 17.85 ± 0.40 23.95 ± 0.28 22.92 ± 0.02 18.57 ± 0.05 17.23 ± 0.12 9.89 ± 0.05 Cells were incubated with the indicated compounds for 48 h. Data are presented as the means ± standard deviations, and cell viability was assessed after incubation for 48 h. bSelectivity Index. a 2.3. Cellular Kinetics and Uptake Characteristics. The synthesis and characterization of the cellular uptake properties of metal complexes were investigated in 2H-indazole iridium(III) (5a−d) complexes, and their potential was compared with that of their ruthenium(II) counterparts. Parameters like lipophilicity, nature of the substituent, and molecular size play crucial roles in determining the cellular uptake ability or the drug likelihood of synthesized complexes. To estimate the lipophilicity of the complexes, the conventional shake ﬂask method was employed to follow their partition coeﬃcients in n-octanol/water (Po/w).29 It was also revealed in the stability studies described above that iridium complexes are not hydrolyzed under aqueous and physiological conditions. In addition, the cellular uptake eﬃcacies of 4b and 5c were determined in MDA-MB-468 cell lines using inductively coupled plasma mass spectrometry (ICP-MS) as shown in Figure S7. The result has revealed a disproportionate concentration dependence in cellular accumulation. In the case of iridium(III) complexes, the uptake potentials of 5c per 106 cells were observed to be 0.186 μg at 3 μM and 0.274 μg at 10 μM, while in the case of ruthenium(II) complexes, 4b revealed potentials of 0.061 μg at 5 μM and 0.0791 μg at 10 μM. Moreover, the uptake kinetics was also assessed by the observed time-dependent increase in cytotoxicity by following the MTT proliferation studies. 2.4. In Vitro Cytotoxicity. Generally, low cytotoxicity toward noncancerous cells and speciﬁc killing of malignant cells are prerequisites for an ideal antineoplastic drug. Assessing quantitatively the mitochondrial integrity through a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay serves as the eﬀective strategy while screening for the cellular proliferative index in a multitude of cytotoxic ligands. With the multiwall format and with a change in the treatment intervals, the variation in cancer cell proliferation can be eﬃciently quantiﬁed using the MTT assay. In this study, the in vitro cytotoxicity of ruthenium(II) (4a−d) and iridium(III) (5a−d) was evaluated by the MTT assay along with cisplatin as a positive metallo-drug against MDA-MB-231, MDA-MB468 (TNBC cells), and HCT-116 (human colon cancer cell) cells and compared with the cytotoxicity against noncancerous HEK 293 (human embryonic kidney) cells. After treatment for 48 h, all of the complexes exhibit high cytotoxicity with favorable IC50 values, which is even better than those of cisplatin against all human cancer cell lines tested (Table 1). It and 24 h) in the presence and absence of 150 mM NaCl (Figure S3). We could see that the stabilities of iridium complexes (5a−d) were quite high under the conditions described above, while in the case of ruthenium complexes (4a−d), a hypochromic shift in the absorption band was observed, indicating their relatively poor stability in an aqueous environment. To ascertain these observations, the 1H NMR spectrum of complex 5c in a 10% DMSO/D2O solvent mixture was recorded and no signiﬁcant changes in the spectral pattern were observed even after 48 h, suggesting that the M−L bonds from the complexes are intact (Figure S5). The stability of all of the complexes was investigated in aqueous medium in the presence and absence of chloride ions. In addition, to further mimic the cellular and physiological conditions the stability was analyzed in the presence of 1 mM GSH and 150 mM NaCl. The relevant results are shown in Figure S4. It could be observed that the iridium complexes exhibited higher stability in the presence and absence of NaCl and GSH. It was also revealed that these complexes possess the ability to react with intracellular nucleophiles and are proposed as potential candidates for various therapeutic applications. Therefore, further investigations of anticancer potentials were limited to iridium complexes. 2.2. Electrostatic Potential Surface. To understand the inﬂuence of peripheral substitution at the C,N-cyclometalated ligand on the activity of complexes, quantum chemical calculations were performed on C−N cyclometalated ruthenium(II) (4a−d) and iridium(III) complexes (5a−d) featuring H, methyl, and isopropyl (electron-donating groups) and triﬂuoromethyl (electron-withdrawing group) groups at position 4 of the phenyl ring. Electrostatic potential (ESP) surfaces for all of the substitution patterns are shown in Figure S6. The localized natural charge on the metal atom is higher for 5c and 4b (0.1716 and 0.3060, respectively) than for all of the reported complexes. Also, mapping of the ESP for complexes 5c and 4b as shown in Figure 2 displays lower electron density at the phenyl ring in comparison to that of the N-donor 2Hindazole ring system, which could be probably due to the peripheral substitution in the phenyl ring. In addition, the observed electron density on the indazole moiety is more prominent for the iridium complex than for the ruthenium complexes. 17596 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Article Figure 3. Complex 5c induced morphological changes and inhibited colony formation ability in TNBC cells. (A) Images representing morphological changes induced by complex 5c at the indicated concentrations after treatment for 24 h. (B) (a) Representative images of a clonogenic survival assay of MDA-MB-468 cells and (b and c) histograms representing the colony number and colony forming eﬃciency, respectively, of MDA-MB-468 cells. The ﬁgures depicted are representative of three independent experiments. Results are represented as means ± standard deviations (SD), and asterisks and double daggers denote values signiﬁcantly diﬀerent from those of the control and cisplatin respectively, */‡p < 0.05. **/‡‡p < 0.01. ****/‡‡‡‡p < 0.0001. treated cells appeared to be round with subsequent detachment and cell death. The same was observed in cells treated with 5a (2.3 μM), 5b (3.5 μM), and 5d (8.2 μM) for 24 h, as shown in Figure S8A. Similar morphological changes were also observed in the case of 4b, and the same is depicted in Figure S9. The observations were more pronounced with increasing concentrations of 5c. The results were compared with those of vehicle control-treated cells that stayed healthy and did not exhibit cell death. Aggressive TNBC cells undergo uncontrolled cell division and thus have the ability to produce colonies even when plated at a lower cell density. Usually, this colony forming ability could be related to their proliferative index and is monitored to check the mitotic ability of cancer cells after treatment with test compounds or ionizing radiation.30 MDA-MB-468 cells treated with 5c (1 and 3 μM) for 24 h showed a signiﬁcant reduction in colony number and in the size of the colonies in a concentration-dependent manner compared to the values of the control and cisplatintreated cells as shown in Figure 3B. The highest concentration (3 μM) almost abolished the ability of TNBC cells to form colonies. Thus, it can be inferred that Ir(III) complex 5c exerted both cytotoxic and cytostatic was revealed that the cytotoxic activities of iridium(III) (5a− d) complexes were higher than those of ruthenium(II) (4a−d) complexes. In particular, complex 5c exhibited signiﬁcantly lower IC50 values against MDA-MB-231 (1.19 ± 0.05 μM), MDA-MB-468 (0.52 ± 0.026 μM), and HCT-116 (1.36 ± 0.01 μM) cells. Also, the cytotoxocity results clearly revealed the speciﬁcity of 5c for cancer cells over noncancerous cells. This highlights the speciﬁcity of these metal drug complexes toward fast-growing cancer cell lines rather than the normal noncancerous cells. The iridium(III) (5a−d) complexes showed good cytotoxicity against cancer cells compared to that of cisplatin. Among the synthesized complexes, iridium(III) (5a−d) complexes exhibited prominent antitumor activity. The data in Table 1 revealed 5c to be the most eﬀective with consistent cytotoxicity against the MDA-MB-468 cell line. To further conﬁrm the cytotoxicity of Ir(III) complexes (5a−d) and Ru(II) complex 4b against MDA-MB-468 cells, we have investigated the drug-induced morphological changes under a phase-contrast microscope. As shown in Figure 3A, in comparison with cisplatin, the cells treated with 5c (1 and 3 μM) exhibited signiﬁcant alterations in their morphology. The 17597 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Article resistance to chemotherapeutic agents by overriding apoptosis and become unresponsive to treatment modalities.36,37 Compared to cisplatin, in MTT and other assays, iridium complexes were highly eﬀective in inducing cell death. Thus, it is possible that iridium(III) complex 5c might eﬀectively induce apoptosis in the tumor cell, which could be an eﬀective strategy for targeting TNBC. The ﬂipped exposure of phosphatidylserine (PS) to the outer side of the cell membrane is a general marker for apoptosis. To determine whether cell death induced by 5c on MDA-MB-468 cell lines occurs via apoptosis or necrosis, we carried out quantitative analysis of phosphatidylserine externalization by ﬂow cytometry using Annexin V as an apoptosis marker. The diﬀerent cell populations gated and tracked by this method were Q1-LL Live [Annexin V−/PI−), Q1-LR Early apoptosis (Annexin V+/ PI−), Q1-UR Late apoptosis (Annexin V+/PI+), and Q1-UL Necrotic (Annexin V−/PI+). Compared to the control and cisplatin (5 μM) treatment groups, there is a striking increase in the percentage of early and late apoptotic cell populations in 5c-treated groups (Figure 5A). Also there is a signiﬁcant concentration-dependent increase in positive apoptotic populations with 5c-treated cells (Figure 5B). At the lower concentration of 5c of 1 μM, ∼18.75% and ∼16.20% of the cells were found to be in early and late apoptotic stages, respectively. Signiﬁcantly, on the contrary, treatment with 3 μM complex 5c shows 7.49% and 76.03% of the cells were found to be in early and late apoptotic stages, respectively. A small fraction of the population (4.30% and 2.54%) was found to be of necrotic cells in the two concentrations of Ir conjugates applied. In comparison, cells treated with 5 μM cisplatin showed 10.77% early apoptotic cells, 13.15% late apoptotic cells, and 3.98% necrotic cells. Thus, the cytotoxic eﬀects seen with Ir-complexed 2H-indazoles were very similar to or in some cases more eﬃcient than those of the platinum drugs. A small proportion of necrotic cells (4.30% and 2.54% at 1 and 5 μM, respectively) were seen in cells treated with 5c. In comparison, cisplatin-treated cells showed 10.77%, 13.5%, and 3.98% early apoptotic, late apoptotic, and necrotic cells, respectively. Mitochondria are said to be the energy reservoir of the cells and the site of ATP synthesis. Cancer cells have the ability to beneﬁcially alter cellular energetics by altering mitochondrial metabolism. This aberrant energetic process inﬂuences the growth, division, tumor progression, and even drug response and thus makes mitochondria potential targets for anticancer drug discovery.34,38,39 It has been reported that metal-based anticancer drug cisplatin exerts its eﬀects by targeting nuclear DNA and can provoke drug resistance by interfering with DNA repair mechanisms.34 However, other metal complexes, with distinct modes of action compared to that of cisplatin, could overcome this possibly by targeting other molecules.40 Interestingly, mitochondrial DNA (mtDNA) is highly vulnerable when treated with transition metals and could become an eﬀective target due to the lack of a repair mechanism and histone protection.19,34 It is well-known that the mitochondrial damage and loss of the mitochondrial membrane potential (ΔΨm) are said to be early events in the mitochondrion-mediated apoptosis pathway and considered to be a hallmark for apoptosis. The cells with a decreased ΔΨm can irreversibly enter apoptosis by causing the release of cytochrome c and activating caspases.38 Previous reports suggested that Ir(III) complexes can evoke ROS generation and also reduce the ΔΨm to exert pro-apoptotic properties activities. While the improved eﬃcacy of platinum drugs was explored,31 an attempt to use novel C−N cyclometalated 2Hindazole iridium(III) complexes could enhance the availability of ligand-conjugated metallo-drug options for the treatment of TNBC cells. 2.4.1. Determination of Intracellular Reactive Oxygen Species (ROS). With respect to metal drugs, the generation and upregulation of intracellular ROS play an important role while initiating apoptosis through both intrinsic and extrinsic apoptotic pathways.32,33 An increased level of mitochondrial ROS can damage mtDNA and thereby induce the mitochondrion-mediated intrinsic apoptotic pathway in cancer cells.34 ROS-based therapeutics are well recognized both in classical chemotherapy and with the recent targeted approaches.35 To investigate the possibility of ROS generation upon treatment of MDA-MB-468 cells with iridium(III) complex 5c, we used the cell-permeable, ROS speciﬁc dye DCFDA (2′,7′dichloroﬂuorescein diacetate). The DCFDA dye will be converted to nonﬂuorescent DCFH by cellular esterases, which is then subsequently oxidized to highly ﬂuorescent DCF in the presence of ROS. As shown in Figure 4, only the Figure 4. Complex 5c induced generation of ROS in TNBC cells. The generation of subcellular ROS was analyzed by ﬂuorescence microscopy with DCFDA staining. The generated ROS levels were higher in the complex 5c (1 and 3 μM) and cisplatin (5 μM) treatment groups than in the untreated control. This ﬁgure is representative of three independent experiments (scale bar, 100 μm). background ﬂuorescence was observed in control untreated cells while in cells treated with 5c (1 and 3 μM), the ﬂuorescence was visibly enhanced, indicating the increase in the level of ROS induced by 5c in MDA-MB-468 cells. Similar results were seen for cells treated with cisplatin (5 μM). Eﬀectively, the treatment with 5c substantially induced generation of ROS, which lead to oxidative stress and eventually the death of MDA-MB-468 cells. We have also analyzed other iridium(III) complexes such as 5a (2.3 μM), 5b (3.5 μM), and 5d (8.2 μM) for their ability to increase the level of ROS as depicted in Figure S8B in the TNBC cell line and found that all of the iridium(III) complexes are capable of increasing the level of ROS when used at their IC 50 concentrations. 2.4.2. Apoptotic Study. Escaping apoptosis is one of the important properties of cancer cells, which leads to uncontrolled cell proliferation and the development of resistance to various therapies. TNBC cells frequently develop 17598 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Article Figure 5. Complex 5c induced apoptosis in triple negative breast cancer (TNBC) cells. (A) Flow cytometry analysis of apoptosis by Annexin V FITC and PI. MDA-MB-468 cells treated with control, 5c (1 and 3 μM), and cisplatin (5 μM) for 24 h. The histograms represent the distribution of cells in live, early apoptosis, late apoptosis, and necrotic phases. (B) Bar diagram representing the percentage of apoptotic cells. Results are represented as mean ± SD, and asterisks and double daggers denote values signiﬁcantly diﬀerent from those of the control and cisplatin, respectively. */‡p < 0.05. ****/‡‡‡‡p < 0.0001. ΔΨm, and these changes are more prominent than those of cisplatin-treated cells. On the contrary, untreated cells emitted red ﬂuorescence indicative of healthy mitochondria, thereby revealing the prominent role played by the mitochondrial apoptosis during iridium(III)-conjugated 2H-indazole toxicity. 2.4.3. Enhanced Activation of Caspase 3 by Ir(III) Complex 5c. When the mitochondrial ΔΨm is disrupted, the release of cytochrome c triggers a cascade of events that eventually activates caspases and leads to apoptotic cell death. During apoptosis, cells undergo distinct morphological changes like cell shrinkage, chromatin condensation, nuclear fragmentation, outer membrane blebbing, and changes in the cytoskeletal dynamics that are mediated by active execution caspases. The activation of caspase 3 represents an early event of apoptosis, which plays an important role in the subsequent execution of apoptosis by cleaving key structural proteins associated with nuclear, mitochondrial, and cytoskeletal integrity, thus leading to apoptotic cell death.46,47 To conﬁrm the involvement of caspase activation, and thereby the mitochondrial route of intrinsic apoptosis in complex 5cinduced cell death, we next examined the activation of caspase 3/7 by using CellEvent Caspase-3/7 Green ReadyProbes. This probe has the inhibitory DEVD peptide bound to the ﬂuorochrome, thus preventing it from binding to DNA in the healthy cells. However, upon activation of caspase 3/7 in apoptotic cells, activation-linked cleavage of the DEVD peptide in turn releases the ﬂuorochrome, which readily enters the nucleus and emits green ﬂuorescence. In this study, the MDAMB-468 cells treated with complex 5c (1 and 3 μM) and cisplatin (5 μM) for 24 h showed bright green ﬂuorescence, which indicates the activation status of caspase 3/7 as in Figure 7. The absence of any ﬂuorescence in the untreated cells conﬁrms the complex 5c speciﬁc, caspase-mediated cellular apoptosis in TNBC cells. Caspase 3/7 activation was also simultaneously.22,34,41−44 Thus, to reveal the involvement of the mitochondrial apoptotic pathway exerted by 5c and the associated change in ΔΨm, TNBC cells were subjected to JC-1 staining-based characterization. This cationic dye selectively accumulates as J-aggregates that emit red ﬂuorescence in healthy cells and remain in the monomeric form, which emits green ﬂuorescence, in apoptotic cells due to the decreased membrane potential.45 From Figure 6A, it is clear that cells treated with 5c showed concentration-dependent changes in Figure 6. Complex 5c induced mitochondrion-mediated apoptosis in triple negative breast cancer (TNBC) cells. Fluorescence microscopy images of mitochondrial membrane potential changes analyzed by JC1 staining. MDA-MB-468 cells showed a decreased ΔΨm after being treated with complex 5c (1 and 3 μM) compared with those of untreated cells (Control) and cells treated with cisplatin (5 μM) for 24 h. The ﬁgures are representative of three independent experiments (scale bar, 100 μm). 17599 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Article Figure 7. Complex 5c induced caspase 3/7 activation in triple negative breast cancer (TNBC) cells. Fluorescence microscopy images of caspase 3/ 7 activation in MDA-MB-468 cells treated with complex 5c for 24 h. The cells treated with complex 5c (1 and 3 μM) and cisplatin (5 μM) showed bright green ﬂuorescence, which indicates the binding of free nucleic acid dye to the DNA upon clevage of inhibitory DEVD peptide by activated eﬀector caspases 3/7. Where as in untreated (Control) cells there was no ﬂuorescence was observed. The ﬁgures are representative of three independent experiments (scale bar, 125 μm). Figure 8. Complex 5c induced nuclear fragmentation in triple negative breast cancer cells. (A) Representative images of DAPI staining of cells treated with complex 5c (1 and 3 μM) and cisplatin (5 μM) that showed an altered nuclear morphology and nuclear fragmentation indicated with white arrows compared to control cells. Representative images shown from three or more independent experiments. (B) Bar diagram representing the quantiﬁcation of DNA fragmentation by the diphenylamine (DPA) method. Results are represented as means ± SD. Asterisks and double daggers denote values signiﬁcantly diﬀerent from those of the control and cisplatin, respectively. */‡p < 0.05. ****/‡‡‡‡p < 0.0001 (scale bar, 100 μm). Figure 9. Complex 5c induced cell membrane blebbing in triple negative breast cancer cells. Scanning electron microscopy images of the formation of membrane blebs in MDA-MB-468 cells treated with complex 5c for 24 h. Control cells show a smooth cell surface, whereas the cells treated with complex 5c (1 and 3 μM) and cisplatin (5 μM) show an altered morphology with the formation of cell membrane blebs. White arrows indicate cellular blebbing. Representative images shown from three independent experiments (magniﬁcation, 15.00K×). To further verify and visually conﬁrm the complex 5cinduced apoptotic cell death, w also studied the eﬀect of complex 5c on the nuclear morphology of MDA-MB-468 cells using DAPI staining (Figure 8A). The cells incubated with complex 5c (1 and 3 μM) for 24 h showed changes in nuclear observed in the cells treated with iridium(III) complexes 5a (2.3 μM), 5b (3.5 μM), and 5d (8.2 μM) at their respective IC50 concentrations for 24 h as shown in Figure S8C, which indicated the apoptotic induction ability of the iridium(III) complexes in TNBC cells. 17600 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Article Figure 10. Complex 5c induced changes in the expression levels of pro- and antiapoptotic proteins. Real time PCR analysis of (a) Bax, (b) Bad, and (c) Bcl-2 mRNA expression in MDA-MB-468 cells treated with complex 5c (1 and 3 μM) and cisplatin (5 μM) for 24 h and the control. The mRNA levels are normalized to internal control GAPDH and displayed as the fold change. The results are expressed as means ± SD. Asterisks and double daggers denote values signiﬁcantly diﬀerent from those of the control and cisplatin, respectively. **/‡‡p < 0.01. ***/‡‡‡p < 0.001. ****/‡‡‡‡p < 0.0001. ns indicates nonsigniﬁcant data. Figure 11. Complex 5c induced changes in the actin cytoskeleton structure in TNBC cells. Confocal microscopy images of phalloidin staining show changes in the actin cytoskeleton in MDA-MB-468 cells treated with complex 5c (1 and 3 μM) and cisplatin (5 μM) for 24 h and control cells. Complex 5c induced F-actin remodeling and morphological changes in MDA-MB-468 cells. Representative images shown from three or more independent experiments (scale bar, 40 μm). using the diphenylamine (DPA) method (Figure 8B). The results indicate that the percentage of fragmented DNA signiﬁcantly increases in a concentration-dependent manner in cells treated with complex 5c (1 and 3 μM) compared to those of the control and cells treated with cisplatin (5 μM). This indicates that the extent of apoptosis induced by Ir(III) complex 5c is high compared to that of the standard anticancer drug cisplatin. morphology, typically with more condensed and fragmented nuclei compared with cells treated with cisplatin (5 μM). However, the untreated control cells possessed healthy nuclear staining. A similar nuclear morphology and similar condensation were also observed in the cells treated with iridium(III) complexes 5a (2.3 μM), 5b (3.5 μM), and 5d (8.2 μM) at their respective IC50 concentrations for 24 h as shown in Figure S8D. The apoptotic eﬀect of complex 5c on MDA-MB-468 cell lines was also quantitatively conﬁrmed 17601 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Article Figure 12. Complex 5c induced G2/M phase cell cycle arrest in TMBC cells. (A) Representative results of cell cycle analysis from a ﬂow cytometer assay in MDA-MB-468 cells treated with complex 5c (1 and 3 μM) and cisplatin (5 μM) for 24 h and the control. (B) Bar diagram representing the G0/G1, S, and G2/M phase proportions of MDA-MB-468 cells. Data shown are representative of three independent experiments. Results are represented as means ± SD. Asterisks and double daggers denote values signiﬁcantly diﬀerent from those of the control and cisplatin, respectively. */‡p < 0.05. **/‡‡p < 0.01. ***/‡‡‡p < 0.001. ****/‡‡‡‡p < 0.0001. ns indicates nonsigniﬁcant data. Figure 13. Complex 5c attenuated the cellular migration ability of triple negative breast cancer cells. MDA-MB-468 cells were treated with diﬀerent concentrations of complex 5c, and cell migration was examined by a wound healing assay and a transwell migration assay. (A) (a) Representative microscopic images of wound healing in which the dotted lines represent the borders of the wound. (b) Bar diagram representing wound closure percentages. The open wound area was measured by ImageJ software, and the percentage of wound closure was calculated and is represented in bar diagrams. (B) (a) Representative images of a transwell migration assay. (b) Bar diagrams representing the number of migrating cells per ﬁeld. Representative images are shown from three or more independent experiments. Results are represented as means ± SD. Asterisks and double daggers denote values signiﬁcantly diﬀerent from those of the control and cisplatin, respectively. **/‡‡p < 0.01. ***/‡‡‡p < 0.001. ****/‡‡‡‡p < 0.0001. ns indicates nonsigniﬁcant data. cisplatin-treated cells. This result again visually indicates that complex 5c is capable of causing apoptotic cell death in TNBC cells. The cell membrane blebs were also observed in the cells treated with iridium(III) complexes 5a (2.3 μM), 5b (3.5 μM), and 5d (8.2 μM) at their respective IC50 concentrations for 24 h as shown in Figure S8E. The induction of apoptosis by Ir(III) complex 5c is evident from the increased mRNA levels of pro-apoptotic genes Bax and Bad and the reduced level of expression of antiapoptotic Cell membrane blebbing is one of the hallmarks of apoptosis. To visualize cell membrane blebbing induced by complex 5c in MDA-MB-468 cells, we next performed scanning electron microscopy. From Figure 9, it is evident that the cells treated with diﬀerent concentrations of complex 5c have undergone changes in the cell surface characterized by the formation of unique apoptotic membrane blebs. In contrast, the control cells appeared to be normal without the membrane blebs. Cell membrane blebs were also seen in 17602 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Article 3. CONCLUSIONS We have developed a successful divergent synthesis of novel C,N-cyclometalated 2H-indazole ruthenium(II) and iridium(III) complexes of the types [(η6-p-cymene)RuCl(-N,C-2Hindazole)] and [(η5-C5Me5)IrCl(-N,C-2H-indazole)] with various substituents (H, Me, isopropyl, and CF3) in the R4 position of the phenyl ring of the 2H-indazole ligand of ruthenium (4a−d) and iridium complexes (5a−d). The structures of all complexes were unequivocally conﬁrmed by 1 H NMR, 13C NMR, HRMS, and elemental analyses. In addition, complex 5b was conﬁrmed by single-crystal X-ray diﬀraction analysis. The cytotoxic activity of the new ruthenium(II) and iridium(III) complexes has been evaluated in human cancer cell lines such as TNBC cell lines MDA-MB231 and MDA-MB-468 and on colon cancer cell line HCT116. Almost all new complexes have shown appreciable activity, comparable to or signiﬁcantly better than that of cisplatin in TNBC cell lines. Increased potency was observed in Ir(III) complex 5c when the R4 substituent of the phenyl ring of the 2H-indazole ligand is an isopropyl group as compared to the parent compounds in all cell lines. Moreover, Ir(III) complex 5c induced mitochondrial damage by generating large amounts of ROS, which triggered mitochondrion-mediated apoptosis in TNBC cell line MDA-MB-468. Moreover, complex 5c also induced G2/M phase cell cycle arrest and inhibited cellular migration of TNBC cells. As reported recently for planar ligand-derived cyclometalated Ir(III) complexes by Cao et al.,34 with our ﬁnding involving signiﬁcant induction of ROS and mitochondrial disruption, it is possible that in addition to chromosomal DNA, targets like mtDNA damage might also play a key role in C,Ncyclometalated 2H-indazole Ir(III) complex-mediated toxicity. The Ir(III) complex reported herein could eﬀectively target TNBC cells with high speciﬁcity. In general, this work provides strong evidence that Ir(III) complex 5c could be further studied to explore the fullest anticancer potential as such or with few modiﬁcations. Also, it could be utilized as a potential mitochondrial targeting anticancer agent. gene Bcl-2 mRNA. From these results, it is evident that Ir(III) complex 5c primarily induced mitochondrion-mediated apoptosis in MDA-MB-468 cells as shown in Figure 10. 2.5. Changes in Cytoskeletal Dynamics and Consequences. The changes in the cytoskeletal network in MDAMB-468 cells treated with Ir(III) complex 5c were observed by staining with F-actin using Alexa Fluoro 488 Phalloidin. The incubation of MDA-MB-468 cells with 1 and 3 μM complex 5c strongly modiﬁed the structure of the actin cytoskeleton when compared to that seen in control and cisplatin-treated cells. These results suggest the cytoskeletal changes from collapsed actin networks are associated typically with apoptosis and are triggered by complex 5c as shown in Figure 11. 2.5.1. Cell Cycle Arrest. Application of chemotherapeutic drugs usually arrests the cell cycle process and mediates cancer cell death due to insuﬃcient DNA repair. To investigate whether Ir(III) complex 5c modulates any cell cycle phase of MDA-MB-468 cells, we tracked cell cycle phases P2 (G0/G1 phase), P3 (S-phase), and P4 (G2/M phase) in the treated population by ﬂow cytometry after propidium iodide (PI) staining (Figure 12). The results indicate that the cells treated with diﬀerent concentrations of complex 5c (1 and 3 μM) and cisplatin (5 μM) showed increased proportions of cells in G2/ M phases when compared to control cells. In the control group, the proportions of cells present in each phase are 63.13%, 17.45%, and 18.90% in phases P2−P4, respectively. In the case of cells treated with complex 5c, at 1 μM 52.19%, 17.25%, and 27.53% were in phases P2−P4, respectively, while at 3 μM 53.48%, 14.14%, and 30.66% were in phases P2−P4, respectively. This shows the signiﬁcant concentration-dependent increase in arrested cells in the G2/M phase of the cell cycle upon treatment with complex 5c. Cells treated with 5 μM cisplatin showed 61.16%, 12.61%, and 25.64% in phases P2− P4, respectively. This indicated that complex 5c induced G2/ M phase cell cycle arrest in MDA-MB-468 cells in a manner similar to that of cisplatin. Modulation of cell cycle arrest at the G2/M phase through enhancing the arrest or abrogating the arrest has been used to improve the cytotoxicity of therapeutic agents.48 Interestingly, the enhanced G2/M arrest by 2Hindazole-ligated iridium(III) in MDA-MB-468 cells supports the view of G2/M arrest followed with the enhanced cytotoxicity by 2H-indazole conjugates. 2.5.2. Cell Migration. The process of metastasis involves spreading of the cancer cells from the primary tumor site to secondary sites, which is eﬀected through cellular migration and invasion within circulatory systems and in the tissue matrix. We investigated the eﬀect of Ir(III) complex 5c on the cell migratory ability of MDA-MB-468 cells in vitro by performing a wound healing migration assay and a transwell migration assay. As shown in Figure 13A, complex 5c inhibited the migratory potential of MDA-MB-468 cells, which is evident from the concentration-dependent decreased rate of wound closure compared to that of the control cells. Also, inhibition of wound closure is comparatively eﬀective compared to than the positive control cisplatin treatment. The transwell migration assay in Figure 13B suggests that the number of migrated cells was signiﬁcantly decreased in cells treated with complex 5c compared to that of the untreated control and that of the cisplatin treatment group. This indicates that complex 5c is capable of inhibiting cellular migration of TNBC cells in vitro, and thus, it can alter the metastatic potential of this aggressive breast cancer cell type. 4. EXPERIMENTAL SECTION 4.1. Chemistry. 4.1.1. General Methods. Unless otherwise indicated, all common reagents and solvents were used as obtained from commercial suppliers without further puriﬁcation. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker DRX400 spectrometer. Chemical shifts are reported in parts per million relative to the internal solvent peak. Coupling constants, J, are given in hertz. Multiplicities of peaks are given as d (doublet), m (multiplet), s (singlet), and t (triplet). Mass spectra were recorded on a PerkinElmer Calrus 600 GC-MS spectrometer. Elemental analyses were carried out using a PerkinElmer 2400 Series II instrument. Highresolution mass spectra (HRMS) were recorded in ESI mode using a Thermo Exactive LC-MS mass spectrometer. Ultraviolet−visible spectroscopy was carried out on a UV-2550 instrument (Shimadzu Corp., Kyoto, Japan). Solvents were dried by the usual methods. [(η6p-Cymene)RuCl2]2 and [(η5-C5Me5)IrCl2]2 were obtained from Sigma-Aldrich (Bangalore, India). The syntheses of metal complexes 4a−d and 5a−d were carried out using the method reported in the literature with a slight modiﬁcation.28 4.1.2. General Procedure for the Synthesis of Substituted 2HIndazole Ru(II) Complexes 4a−d. In a round-bottom ﬂask equipped with a magnetic bar under a nitrogen atmosphere, 2-phenyl-2Hindazole (1 mmol) 1a was dissolved in a freshly distilled dichloromethane solution (5 mL). Sodium acetate (1.2 mmol) was added to the ﬂask at room temperature while its contents were being constantly stirred followed by the addition of [(η6-p-cymene)RuCl2]2 (0.5 17603 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC mmol) 2. The reaction mixture was stirred at room temperature for 20 h, and the progress of the reaction was monitored by TLC. After the complex had formed, diethyl ether (10 mL) was added to the mixture. The reaction mixture was stirred for 10 min to precipitate the product. The crystalline product was ﬁltered through a fritted funnel and dried well. Yellow-colored ruthenium complex 4a was obtained in good yield (72%): Rf = 0.25 (40% EtOAc/n-hexane); 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 8.23 (d, J = 7.2 Hz, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.47−7.43 (m, 2H), 7.20−7.16 (m, 2H), 7.05 (dd, J = 6.8, 1.6 Hz, 1H), 5.93 (d, J = 5.8 Hz, 1H), 5.74 (d, J = 5.8 Hz, 1H), 5.41 (d, J = 5.8 Hz, 1H), 5.17 (d, J = 5.8 Hz, 1H), 2.28−2.23 (m, 1H), 2.11 (s, 3H), 0.88 (d, J = 6.90 Hz, 3H), 0.72 (d, J = 6.90 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 148.5, 141.1, 140.5, 128.0, 127.2, 123.4, 122.8, 121.5, 118.4, 115.9, 113.1, 102.3, 99.1, 89.5, 89.3, 82.4, 81.0, 30.7, 22.5, 21.7, 18.9; MS (ESI, MS) 429 (M − Cl)+; HRMS (ESI) calcd for C23H23N2Ru m/z 429.0905, found m/z 429.0903. Anal. Calcd for 4a (C23H23ClN2Ru): C, 59.54; H, 5.00; N, 6.04. Found: C, 59.51; H, 5.02; N, 6.08. Compound 4b was synthesized using a procedure similar to that used for the synthesis of compound 4a: yield 73%; light yellow solid; Rf = 0.26 (40% EtOAc/n-hexane); 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1H), 8.03 (s, 1H), 7.96 (d, J = 8.7 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.44−7.40 (m, 1H), 7.33 (d, J = 7.9 Hz, 1H), 7.19−7.16 (m, 1H), 6.86 (d, J = 7.8 Hz, 1H), 5.92 (d, J = 5.8 Hz, 1H), 5.73 (d, J = 5.8 Hz, 1H), 5.44 (d, J = 5.8 Hz, 1H), 5.16 (d, J = 5.8 Hz, 1H), 2.42 (s, 3H), 2.27−2.22 (m, 1H), 2.10 (s, 3H), 0.87 (d, J = 6.9 Hz, 3H), 0.72 (d, J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 148.4, 141.0, 128.9, 136.7, 130.1, 127.8, 121.4, 120.9, 118.0, 115.9, 112.7, 102.2, 98.9, 89.3, 82.7, 80.8, 30.7, 22.5, 21.7, 21.5, 19.0; MS (ESI, MS) 443 (M − Cl)+; HRMS (ESI) calcd for C24H25N2Ru m/z 443.1061, found m/z 443.1090. Anal. Calcd for 4b (C24H25ClN2Ru): C, 60.31; H, 5.27; N, 5.86. Found: C, 60.33; H, 5.18; N, 5.84. Compound 4c was synthesized using a procedure similar to that used for the synthesis of compound 4a: yield 75%; pale yellow solid; Rf = 0.27 (40% EtOAc/n-hexane); 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 8.01 (s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 6.8 Hz, 1H), 7.36−7.32 (m, 1H), 7.14−7.10 (m, 1H), 6.89 (d, J = 8.0 Hz, 1H), 5.83 (d, J = 5.8 Hz, 1H), 5.69 (d, J = 5.8 Hz, 1H), 5.29 (d, J = 5.8 Hz, 1H), 5.09 (d, J = 5.8 Hz, 1H), 2.97− 2.90 (m, 1H), 2.26−2.19 (m, 1H), 2.03 (s, 3H), 1.29−1.26 (m, 6H), 0.85 (d, J = 6.90 Hz, 3H), 0.68 (d, J = 6.90 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 147.4, 146. 138.2, 137.7, 127.4, 126.8, 121.6, 120.3, 116.9, 114.9, 111.7, 101.1, 98.4, 88.7, 88.0, 80.9, 79.9, 33.2, 29.7, 28.6, 23.6, 22.9, 21.5, 20.6; MS (ESI, MS) 471 (M − Cl)+; HRMS (ESI) calcd for C26H29N2Ru m/z 471.1374, found m/z 471.1341. Anal. Calcd for 4c (C26H29ClN2Ru): C, 61.71; H, 5.78; N, 5.54. Found: C, 61.69; H, 5.58; N, 5.49. Compound 4d was synthesized using a procedure similar to that used for the synthesis of compound 4a: yield 70%; pale green solid; Rf = 0.31 (40% EtOAc/n-hexane); 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 2H), 7.87 (d, J = 8.92 Hz, 1H), 7.51 (d, J = 8.72 Hz, 1H), 7.41− 7.39 (m, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.14−7.07 (m, 2H), 5.89 (d, J = 5.8 Hz, 1H), 5.68 (d, J = 5.8 Hz, 1H), 5.40 (d, J = 5.8 Hz, 1H), 5.15 (d, J = 5.8 Hz, 1H), 2.19−2.12 (m, 1H), 2.07 (s, 3H), 0.79 (d, J = 7.0 Hz, 3H), 0.64 (d, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 164.4, 148.8, 136.8, 128.7, 123.2, 123.0, 121.9, 120.9, 119.7, 115.8, 112.7, 103.3, 99.6, 89.6, 89.4, 83.0, 81.0, 30.7, 22.5, 21.7, 19.0; MS (ESI, MS) 495 (M − Cl)+; HRMS (ESI) calcd for C24H21N2ClF3Ru m/z 531.0383, found m/z 531.0377. Anal. Calcd for 4d (C24H22ClF3N2Ru): C, 54.19; H, 4.17; N, 5.27. Found: C, 54.19; H, 3.92; N, 5.18. In a round-bottom ﬂask equipped with a magnetic bar under a nitrogen atmosphere, 2-phenyl-2H-indazole 1a (1 mmol) was dissolved in a freshly distilled dichloromethane solution (5 mL). Sodium acetate (1.2 mmol) was added to the ﬂask at room temperature while its contents were being constantly stirred followed by the addition of [(η5-C5Me5)IrCl2]2 3 (0.5 mmol). The reaction mixture was stirred at room temperature for 20 h, and the progress of the reaction was monitored by TLC. After the complex had formed, dichloromethane was distilled under reduced pressure and diethyl Article ether (10 mL) was added to the mixture. The reaction mixture was stirred for 10 min to precipitate the product. The crystalline product was ﬁltered through a fritted funnel and dried well. Orange-colored iridium complex 5a was obtained in good yield (82%): Rf = 0.26 (40% EtOAc/n-hexane); 1H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.72−7.66 (m, 2H), 7.52 (d, J = 8.0 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.20−7.16 (m, 2H), 7.07 (dd, J = 8.0, 1.2 Hz, 1H), 1.76 (s, 15H); 13C NMR (100 MHz, CDCl3) δ 147.7, 146.9, 136.9, 128.4, 128.3, 122.9, 122.7, 121.6, 119.1, 115.2, 112.4, 88.5, 9.6; MS (ESI, MS) 521 (M − Cl)+; HRMS (ESI) calcd for C23H25ClIrN2 m/z 557.1336, found m/z 557.1349. Anal. Calcd for 5a (C23H24ClIrN2): C, 49.67; H, 4.35; N, 5.04. Found: C, 49.72; H, 4.26; N, 5.04. Compound 5b was synthesized using a procedure similar to that used for the synthesis of compound 5a: yield 82%; yellow solid; Rf = 0.30 (40% EtOAc/n-hexane); 1H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 7.89 (d, J = 7.5 Hz, 1H), 7.72−7.67 (m, 2H), 7.52 (d, J = 7.8 Hz, 1H), 7.37 (t, J = 6.84 Hz, 1H), 7.18 (s, 1H), 7.07 (dd, J = 7.4, 1.2 Hz, 1H), 1.77 (s, 15H) 1.55 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 147.4, 146.4, 145.7, 136.8, 136.5, 134.1, 127.1, 122.7, 121.5, 120.5, 120.0, 117.6, 114.0, 111.1, 87.3, 20.4, 8.6; MS (ESI, MS) 535 (M − Cl)+; HRMS (ESI) calcd for C24H26IrN2 m/z 535.1719, found m/z 535.1699. Anal. Calcd for 5b (C24H26ClIrN2): C, 50.56; H, 4.60; N, 4.91. Found: C, 50.68; H, 4.58; N, 4.89. Compound 5c was synthesized using a procedure similar to that used for the synthesis of compound 5a: yield 85%; pale yellow solid; Rf = 0.34 (40% EtOAc/n-hexane); 1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.72 (d, J = 7.2 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.29− 7.27 (m, 2H), 7.19−7.15 (m, 1H), 7.05 (dd, J = 7.6, 1.2 Hz, 1H), 3.02−2.95 (m, 1H), 1.4 (s, 15H), 1.30 (d, J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 148.4, 147.5, 146.7, 140.3, 135.1, 128.1, 122.5, 121.5, 118.6, 115.1, 112.1, 88.4, 34.1, 24.7, 23.7, 9.6; MS (ESI, MS) 564 (M − Cl)+; HRMS (ESI) calcd for C26H31IrN2 m/z 564.2116, found m/z 564.2114. Anal. Calcd for 5c (C26H30ClIrN2): C, 52.20; H, 5.06; N, 4.68. Found: C, 52.26; H, 5.02; N, 4.42. Compound 5d was synthesized using a procedure similar to that used for the synthesis of compound 5a: yield 80%; pale green solid; Rf = 0.29 (40% EtOAc/n-hexane); 1H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 8.13 (s, 1H), 7.69 (t, J = 9 Hz, 2H), 7.55 (d, J = 8.3 Hz, 1H), 7.40 (t, J = 6.88 Hz, 1H), 7.31 (d, J = 8.44 Hz, 1H), 7.21 (t, J = 7.72 Hz, 1H), 1.77 (s, 15H); 13C NMR (100 MHz, CDCl3) δ 154.5, 148.0, 146.5, 144.6, 131.2, 129.5, 126.6, 120.7, 120.0, 118.4, 117.6, 112.6, 108.5, 86.4, 7.1; MS (EI, MS) 589 (M − Cl)+; HRMS (ESI) calcd for C24H23F3IrN2 m/z 589.1437, found m/z 589.1425. Anal. Calcd for 5d (C24H23ClF3IrN2): C, 46.19; H, 3.71; N, 4.49. Found: C, 46.23; H, 3.83; N, 4.69. 4.2. Biology. 4.2.1. General Methods. MTT, dimethyl sulfoxide (DMSO), diphenylamine (DPA), 4% paraformaldehyde, glutaraldehyde, methanol, and Triton X-100 were purchased from Himedia (Mumbai, India). Cisplatin, DCFDA, and TRI Reagent were purchased from Sigma-Aldrich (Bangalore, India). Propidium iodide, JC-1 stain, DAPI, Hoechst 33342, the dead cell apoptosis kit with Annexin V FITC and PI for ﬂow cytometry, CellEvent Caspase-3/7 Green ReadyProbes Reagent, and Alexa Fluoro 488 Phalloidin were purchased from ThermoFisher Scientiﬁc (Bangalore, India). The PrimeScript RT reagent Kit and SYBR Premix Ex Taq were purchased form DSS Takara Bio India Pvt. Ltd. (Bangalore, India). All of the primers were purchased from Euroﬁns (Bangalore, India). All of the compounds tested were freshly prepared and dissolved in DMSO. The ﬁnal concentration of DMSO was maintained at 0.25% in all of the experiments. 4.2.2. Cell Culture and Reagents. Human MDA-MB-468 and MDA-MB-231 TNBC cells, human colon cancer cell line HCT-116, and human HEK 293 embryonic kidney cells were obtained from the National Center for Cell Sciences (NCCS, Pune, India). Cells were routinely maintained at 37 °C and 5% CO2 in DMEM (Dulbecco’s modiﬁed Eagle’s medium, Himedia), complemented with 10% FBS (fetal bovine serum, Himedia), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Himedia). 17604 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. Chem. 2021, 60, 17593−17607 Inorganic Chemistry pubs.acs.org/IC Tejashri B. Hingamire − Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune 411008, India; Academy of Scientiﬁc and Innovative Research (AcSIR), Ghaziabad 201002, India Dhanasekaran Shanmugam − Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune 411008, India; Academy of Scientiﬁc and Innovative Research (AcSIR), Ghaziabad 201002, India Venkatraman Manickam − Department of Biosciences, School of Biosciences and Technology, Vellore Institute of Technology, Vellore 632014, India 4.2.3. Cell Viability Assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) (Himedia) assay was performed to check the cell viability as described previously.14 In brief, MDAMB-468, MDA-MB-231, HCT-116, and HEK 293 cell lines were seeded at a density of 1 × 104 cells/well in 96-well plates. Cells were exposed to diﬀerent concentrations of 4a−d, 5a−d, and cisplatin for 48 h. After incubation, 25 μL of an MTT (5 mg/mL in 1× PBS) solution was added to each well and incubated for 4 h at 37 °C. The resulting formazan crystals were then solubilized with 100 μL of DMSO, and the color intensity of formazan was measured at 490 nm using a microplate reader (BioTek-ELx800). The results were compared with those of the untreated control, and each analysis point was assessed in triplicate. The cisplatin was used as a positive control, and DMSO was used as the vehicle control for cell viability inhibition. 4.2.4. Computational Details. Quantum chemical calculations were performed with the Gaussian software. All geometry optimizations were performed using the B3LYP functional with the LanL2 basis set. From these geometries, all reported data were obtained by means of vertical excitations using the same functional along with the more polarized def2-TZVPP basis set for all atoms except for iridium and ruthenium, for which the SDD basis set was used. ■ Complete contact information is available at: https://pubs.acs.org/10.1021/acs.inorgchem.1c02193 Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS The authors thank the Chancellor and Vice Chancellor of VIT for providing the opportunity to carry out this study. R.N.R., R.L.P., V.M., M.M.B., and K.C. thank the management of this university for providing seed money as a research grant. K.C. thanks ICMR, Government of India, for funding through Grant 45/03/2019-BIO/BMS. The authors also thank the Central Instrumentation Facility of VIT for recording the spectra and the Sophisticated Analytical Instrumentation Facility (SAIF), Gauhati University, for use of the singlecrystal X-ray diﬀractometer. ASSOCIATED CONTENT * Supporting Information sı The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c02193. Copies of 1H NMR, 13CNMR, and HRMS spectra of complexes 4a−d and 5a−d, absorption and ﬂuorescence spectra of complexes in diﬀerent solvents, stability study of complexes 4a−d and 5a−d, ESP maps of 4a, 4c, 4d, 5a, 5b, and 5d, ICP-MS analysis of complexes 4b and 5c in the MDA-MB-468 cell line and complexes 4b, 5a, 5b, and 5d that induced morphological changes in MDAMB-468 cells after 24 h, single-crystal X-ray data of compound 5b, and comparison of XRD- and DFTderived structural details for 5b (PDF) ■ REFERENCES (1) Rosenberg, B.; VanCamp, L.; Trosko, J. E.; Mansour, V. H. 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These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. ■ Article AUTHOR INFORMATION Corresponding Author Kaushik Chanda − Department of Chemistry, School of Advanced Science, Vellore Institute of Technology, Vellore 632014, India; orcid.org/0000-0002-7555-9322; Email: chandakaushik1@gmail.com Authors Rajeeva Lochana Panchangam − Department of Biosciences, School of Biosciences and Technology, Vellore Institute of Technology, Vellore 632014, India Ramdas Nishanth Rao − Department of Chemistry, School of Advanced Science, Vellore Institute of Technology, Vellore 632014, India Musuvathi Motilal Balamurali − Chemistry Division, School of Advanced Sciences, Vellore Institute of Technology, Chennai 600127, India; orcid.org/0000-0001-79489950 17605 https://doi.org/10.1021/acs.inorgchem.1c02193 Inorg. 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