Lysosome-Targeted Phosphine-Imine Half-Sandwich Iridium(III) Anticancer Complexes: Synthesis, Characterization, and Biological Activity

Article Cite This: Organometallics XXXX, XXX, XXX−XXX pubs.acs.org/Organometallics Lysosome-Targeted Phosphine-Imine Half-Sandwich Iridium(III) Anticancer Complexes: Synthesis, Characterization, and Biological Activity Yuliang Yang, Lihua Guo,* Zhenzhen Tian, Xingxing Ge, Yuteng Gong, Hongmei Zheng, Shaopeng Shi, and Zhe Liu* Organometallics Downloaded from pubs.acs.org by ALBRIGHT COLG on 04/09/19. For personal use only. Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of Life-Organic Analysis and Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, Department of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China S Supporting Information * ABSTRACT: The synthesis, characterization, and catalytic ability of converting coenzyme NADH to NAD+ and the anticancer activity of half-sandwich iridium(III) complexes with general formula of [(η5-Cpx)Ir(P^N)Cl]PF6 (Cpx: Cp* or biphenyl Cpxbiph derivatives; P^N: various phosphine-imine ligands) were investigated. The crystal structure of the complex Ir4 showed a piano-stool geometry around the iridium(III) center. This type of iridium(III) complexes had suﬃcient stability in aqueous solution. Most of the complexes showed good anticancer activities toward A549 cancer cells, which were higher than the clinical drug cisplatin. In this series, complex Ir8 displayed the highest anticancer activity against A549 cells (IC50 = 4.7 μM), showing an approximately 4.5-fold more potent activity than cisplatin (IC50 = 21.30 μM). The structure−activity relationship study showed that the cytotoxicity of these complexes may be primarily attributed to the coordination between iridium(III) and the coordinating atoms, and the nature of the imine N-substituents may not be a major factor aﬀecting cytotoxicity. Furthermore, this family of complexes causes cell death by cell stress, inducing apoptosis and necrosis, overproduction of reactive oxygen species, and disruption of the mitochondrial membrane potential. Most interestingly, the use of confocal microscopy provides insights into the microscopic mechanism that the typical complex Ir3 can penetrate into A549 cancer cells through a non-energy-dependent pathway and speciﬁcally distribute in lysosomes. ■ INTRODUCTION Cancer is one of the leading causes of death worldwide.1 Platinum-based anticancer drugs are the predecessor of inorganic chemotherapeutic drugs for the treatment of various cancer types.2−4 However, the side eﬀects of platinum drugs, such as nausea, vomiting, losing hair, drug resistance, and substantial side eﬀects aﬀect fast-growing tissues, thereby limiting their clinical usefulness.5−9 Thus, researchers focus on discovering non-platinum metallodrugs with reduced side eﬀects and coordination properties. Anticancer metallodrugs are continuously developed in the ﬁeld of bioorganometallic chemistry depending upon the rational design of novel and sophisticated ligand frameworks to obtain eﬀective chemotherapeutic agents with a superior toxicity proﬁle. Among these heavy metal complexes, bidentate chelating ligands bearing phosphorus and/or nitrogen donor atoms have wide applications, such as homogeneous catalysts10−15 and anticancer drugs.16−23 Modiﬁcations of the bidentate chelating ligands of anticancer metallodrugs, such as alteration of the steric and electronic properties of the P- or Nbound substituents, are eﬀective strategies to tune the chemical and biological properties of the compounds. Many organometallic anticancer complexes bearing symmetrical phosphorus or nitrogen donor atoms in their bidentate chelating ligands have been reported.24−29 For instance, Quirante et al. designed a © XXXX American Chemical Society group of nonplanar cycloplatinated complexes with bidentate phosphine ligand and 2-phenylaniline ligand (Scheme 1, I).24 Scheme 1. Reported Transition-Metal Anticancer Complexes and Our Current Work Received: February 6, 2019 A DOI: 10.1021/acs.organomet.9b00080 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Table 1. Organometallic IrIII Cyclopentadienyl [(η5Cpx)Ir(P^N)Cl]PF6 Complexes Studied in This Work The high antiproliferative activity of such complexes toward four selected cell lines was attributed to a catalytic process, which produces H2O2 as reactive oxygen species (ROS). This oxidant mechanism of action (MoA), which is diﬀerent from that of platinum drugs, is similar to those of half-sandwich iridium(III) complexes.30 Additionally, Marchetti’s group synthesized a set of water-soluble half-sandwich ruthenium(II) complexes with varying α-diimine ligands (Scheme 1, II).25 The toxicological properties of such complexes depend on the nature of the αdiimine N-substituents. Our group developed a family of halfsandwich iridium(III) and ruthenium(II) complexes bearing P,P-chelated ligands (Scheme 1, III).26 These complexes showed anticancer potency superior to that of cisplatin, and the toxicity properties may be related to the redox MoA. These studies motivated us to synthesize half-sandwich iridium(III) complexes containing phosphine-imine (P^N) chelating ligands and investigate their biological activity. Herein, we report the synthesis, characterization, and cytotoxic properties against the A549 cancer cell line and the catalytic transfer hydrogenation activity of half-sandwich iridium(III) complexes as supported by various phosphineimine ligands with a general formula of [(η5-Cpx)Ir(P^N)Cl]PF6. To our knowledge, this is the ﬁrst in vitro evaluation of these novel phosphine-imine half-sandwich iridium(III) complexes for their cytotoxic activity against the A549 cancer cell. The eﬀect of adjusting the coordination environment of the imine moiety on the catalytic ability in converting coenzyme NADH to NAD+ and anticancer activity was discussed. Moreover, to better understand their MoAs, we investigated the chemical and biological behavior and the cellular imaging property of these complexes. ■ RESULTS AND DISCUSSION Synthesis and Characterization. New half-sandwich iridium(III) complexes Ir1−Ir8 were prepared via wellestablished procedures as illustrated in Scheme 2 and Table 1. complex Cpx P^N Ir1 Ir2 Ir3 Ir4 Ir5 Ir6 Ir7 Ir8 Cp* Cp* Cp* Cp* Cp* Cp* Cpxbiph Cpxbiph L1 L2 L3 L4 L5 L6 L4 L6 for complexes Ir1−Ir8 were observed at 1586.9−1617.4 cm−1 in the FT-IR spectra. X-ray Crystal Structures. The molecular structure of complex Ir4 was conﬁrmed by single-crystal X-ray diﬀraction. Crystallographic collection and reﬁnement data are summarized in Tables S1 and S2. As illustrated in Figure 1, complex Ir4 adopts the general pseudo-octahedral three-legged piano-stool conﬁguration, with the phosphine-imine chelating ligand occupying two coordination positions, the pentamethylcyclo- Scheme 2. Synthesis Route of Phosphine-Imine HalfSandwich Iridium(III) Complexes Ir1−Ir8 These complexes were successfully prepared in moderate yields (46−78%) by reacting [(η5-Cp*)IrCl2]2 or [(η5-Cpxbiph)IrCl2]2 with corresponding phosphine-imine ligands L1−L6 in a 1:2 molar ratio at room temperature in CH2Cl2 (Scheme 2). The complexes Ir1−Ir8 were isolated as their PF6− salts and were characterized by multinuclear NMR spectroscopy (Figures S1− S16), Fourier transform infrared (FT-IR) spectroscopy (Figures S17−S24), mass spectrometry (Figures S25−S32), elemental analysis, and X-ray crystallography. The characteristic peaks in the 1H NMR of these complexes were found at δ 8.07−8.79 ppm, which corresponded to the proton of the C(H)N group. Also, the characteristic peaks of the coordinating phosphorus atom of these complexes were found at δ 6.64−12.32 ppm in the 31 P NMR spectra. In addition, the CN stretching vibrations Figure 1. X-ray crystal structure of [(η5-Cp*)Ir(L4)Cl]PF6 (Ir4) with the thermal ellipsoids drawn at the 50% probability level. The hydrogen atoms and PF6− counterions have been omitted for clarity. Selected bond lengths (Å) and angles (deg): Ir−C(centroid) = 1.8722, Ir−P = 2.2822(11), Ir−N = 2.116(4), Ir−Cl = 2.3984(11), P−Ir−N = 87.34(10), P−Ir−Cl = 90.00(4), N−Ir−Cl = 83.88(10). B DOI: 10.1021/acs.organomet.9b00080 Organometallics XXXX, XXX, XXX−XXX Article Organometallics the cytotoxicity was even higher than those previously reported for P^P and N^N-chelated iridium(III) complexes.25,26,37 Within this class, the eﬀect of introducing a biphenyl substituent to the Cp* ring and three types of imine Nsubstituents in the phosphine-imine framework on the cytotoxicity of these iridium(III) complexes was systematically investigated. First, the aromatic and aliphatic rings of the imine moiety determined the anticancer activity of the complexes. For example, complex Ir4 (25.3 μM) bearing a cyclohexyl group in the imine moiety displayed lower cytotoxicity than complex Ir1 (6.5 μM), which contain a phenyl substituent in the same position. Additionally, introducing biphenyl substituents to the Cp* ring optimizes cytotoxicity compared with their parent Cp* complexes (Ir4: 25.3 μM vs Ir7: 5.8 μM; Ir6: 7.9 μM vs Ir8: 4.7 μM). Finally, for the two other classes of imine N-substituents, the introduction of ortho alkyl substituents in the aniline and the length of the methylene chain on imine moiety had little inﬂuence on the cytotoxicity of these complexes. For example, when the ortho alkyl substituents in aniline increased from the hydrogen atom to the isopropyl group (Ir1: 6.5 μM vs Ir2: 7.2 μM vs Ir3: 6.9 μM) or when the number of the methylene group on the imine moiety increased from 0 to 2 (Ir1: 6.5 μM vs Ir5: 6.3 μM vs Ir6: 7.9 μM), the cytotoxicity exhibited an insigniﬁcant change. Overall, the cytotoxicity of these complexes may be primarily attributed to the coordination between iridium(III) and coordinating atoms, and the nature of the imine N-substituents may not be a major factor aﬀecting cytotoxicity. Reaction with NADH. Half-sandwich Ir(III) anticancer complexes may implicate an oxidant MoA through catalytic hydride transfer from NADH to NAD+ because this redox pair is involved in relevant redox signaling pathways within cells.30,38 To evaluate the eﬀect of three diﬀerent types of N-bound substituents on accelerating the oxidation of NADH to NAD+, we monitored the conversion reaction of complexes Ir1, Ir3, Ir4, and Ir6 (1 μM) with NADH (100 μM) in 5% CH3OH/95% H2O by using a UV/vis spectrophotometer (Figures 2a and S46). The turnover numbers (TONs) of complexes Ir1 (19.4), Ir3 (66.3), Ir4 (26.6), and Ir6 (25.4) were calculated based on the diﬀerence in absorbance at 339 nm (Figure 2b). Overall, increasing the steric hindrance of ortho-substituents in the aniline enhanced the catalytic activity (Ir1: 19.4 vs Ir3: 66.3). However, the results for Ir1, Ir4, and Ir6 did not allow the clear determination of the inﬂuence of the diﬀerent types of imine Nsubstituents on catalytic activity. Notably, all tested complexes in this system have more superior catalytic ability in converting coenzyme NADH to NAD+ (up to 16.2-fold) than the structurally similar half-sandwich iridium(III) complexes containing the P^P-chelating ligand under the same test conditions.26 The favorable catalytic potency of these complexes in converting NADH to NAD+ may oﬀer a pathway for generating ROS and may provide eﬃcient oxidant-based therapy. Interaction with Nucleobases. The study on the reaction of metal anticancer drugs with both mode nucleobase 9ethylguanine (9-EtG) and 9-methyladenine (9-MeA) provides mechanistic insights into the rationalization of the observed cytotoxicity of these complexes. To assess the nature of interactions of the new iridium(III) complexes Ir3 and Ir6 (ca. 1 mM) with 2.0 molar equivalent of mode nucleobase 9-EtG or 9-MeA, we monitored their reactions in 80% CD3OD/20% D2O by 1H NMR spectroscopy at 310 K. No additional peaks were observed over a period of 24 h (Figures S47−S50), thereby pentadienyl (Cp*) ring acting as a three-coordinated ligand, and the monodentate ligand (chloride atom) occupying the sixth coordination site. In addition, the metal center is part of a sixmembered (PN)Ir chelate ring system. The Ir−Cl and Ir− Cp*(centroid) distances are 2.3984(11) and 1.8722 Å, respectively. Notably, the Ir−P bond length [Ir−P = 2.2822(11) Å] is longer than Ir−N [Ir−N = 2.116(4) Å]. Stability Studies. Generally, the stability of anticancer metal complexes is a signiﬁcant factor aﬀecting their functions in biological systems. Thus, the solution’s stability in a mixture of 60% dimethyl sulfoxide (DMSO)-d6/40% D2O of complexes Ir1−Ir8 was studied by utilizing 1H NMR spectroscopy at a physiological temperature of 310 K. The presence of DMSO-d6 ensured suﬃcient solubility of the complex. As illustrated in Figures S33−S40, no change was observed in the 1H NMR spectra over 24 h, thereby indicating that these complexes were stable under such conditions. Complexes Ir4 and Ir6 (1.5 mM) were also evaluated for their stability in a 60% DMSO-d6/40% phosphate-buﬀered saline (PBS) (pH ≈ 7.2, PBS is prepared from D2O) buﬀer mixture by utilizing 1H NMR spectroscopy. As shown in Figures S41 and S42, the 1H NMR spectra of complexes Ir4 and Ir6 showed no signals of formation of aqua species, which clearly exhibited that these complexes were stable in PBS buﬀer solution at 310 K for over 24 h. Complexes Ir4 and Ir6 were also monitored over a period of 8 h by ultraviolet/ visible (UV/vis) spectroscopy in 10% DMSO/90% PBS (pH ≈ 7.2) at 298 K to further investigate the stability of these complexes (Figures S43 and S44). The results were consistent with the NMR analysis, thereby indicating their suﬃcient stability in diluted solutions, as is the case of cell culture. Overall, these iridium(III) complexes were stable and remained intact during biological experiments. In Vitro Cytotoxicity. A number of previous research studies have shown that modiﬁcations of the ligand were a contributing factor to toxicological properties of anticancer complexes.31−36 The in vitro cytotoxicity of complexes Ir1−Ir8 and cisplatin to the human lung cancer cell line (A549) was evaluated by colorimetric MTT assay for 24 h. The IC50 values (concentration that produces 50% cell death) are tabulated in Tables 2 and S3 and Figure S45. A majority of complexes were more active than cisplatin to the A549 cells. Notably, complex Ir8, showing the most potent activity against A549 cells, displayed approximately 4.5-fold more potent activity than cisplatin. The P^N-chelated complexes in this system showed high cytotoxicity toward A549 cells. For some of the complexes, Table 2. Inhibition of the Growth of A549 Cancer Cells by Complexes Ir1−Ir8 and Cisplatina complex IC50 (μM) [(η -Cp*)Ir(L1)Cl]PF6 (Ir1) [(η5-Cp*)Ir(L2)Cl]PF6 (Ir2) [(η5-Cp*)Ir(L3)Cl]PF6 (Ir3) [(η5-Cp*)Ir(L4)Cl]PF6 (Ir4) [(η5-Cp*)Ir(L5)Cl]PF6 (Ir5) [(η5-Cp*)Ir(L6)Cl]PF6 (Ir6) [(η5-Cpxbiph)Ir(L4)Cl]PF6 (Ir7) [(η5-Cpxbiph)Ir(L6)Cl]PF6 (Ir8) cisplatin 6.5 ± 1.0 7.2 ± 2.0 6.9 ± 0.6 25.3 ± 0.1 6.3 ± 0.1 7.9 ± 0.9 5.8 ± 0.2 4.7 ± 1.5 21.3 ± 1.7 5 a IC50 values are drug concentrations necessary for 50% inhibition of cell viability. Data are presented as means ± standard deviations (SDs) and cell viability is assessed after 24 h of incubation. C DOI: 10.1021/acs.organomet.9b00080 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Figure 2. (a) UV/vis spectra of the reaction of complex Ir3 (1 μM) with NADH (100 μM) in 5% MeOH/95% H2O (v/v) at 298 K for 8 h. (b) TONs of complexes Ir1, Ir3, Ir4, and Ir6. Figure 3. Apoptosis analysis of A549 cells after 24 h of exposure to complexes Ir3 and Ir6 at 310 K determined by ﬂow cytometry using AV-ﬂuorescein isothiocyanate versus PI staining. Populations for cells in four stages treated by complexes Ir3 and Ir6. Data are quoted as mean ± SD of three replicates. indicating that no reaction with 9-EtG and 9-MeA occurred. Additionally, these reaction mixtures were analyzed by mass spectrometry. The formation of nucleobase adducts was not detected. Hence, potential DNA target sites are not prone to attack from these half-sandwich iridium(III) complexes. Apoptosis Assay. To evaluate whether such complexes lead to cell death through apoptosis or necrosis, we carried out annexin V (AV)/propidium iodide (PI) dual-staining in A549 cells after incubation with complexes Ir3 and Ir6 for 24 h. As shown in Figure 3 and Tables S4 and S5, following incubation with complexes Ir3 and Ir6 at concentrations of 0.5 × IC50, 1 × IC50, 2 × IC50, and 3 × IC50, distinct apoptosis and necrosis were observed. As indicated in the histogram, when A549 cells were treated with test complexes at low concentrations, most of the cells underwent apoptosis. However, when the test complex at high concentrations was used, a dose-related increase in the percentage of necrosis was detected. At the maximum concentration (3 × IC50) of complexes Ir3 and Ir6, 20.73 and 45.35% of cells underwent late apoptosis, respectively, which were higher compared with the control group (9.64%). Additionally, the percentages of cells that underwent necrosis were 77.23 and 47.99% at 3 × IC50, respectively, which were higher compared with the vehicle-treated group (1.85%). These results indicated that cell death occurred because of apoptosis and necrosis. Mitochondrial Membrane Potential. The mitochondria play crucial roles in activating cell apoptosis. The change of mitochondrial membrane potential (MMP) (Δψm) is one of the earliest events in apoptosis. Hence, Δψm should be monitored when cell apoptosis starts. The inﬂuence of iridium(III) complexes on MMP was examined by staining with a lipophilic cationic ﬂuorescent probe, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraeD DOI: 10.1021/acs.organomet.9b00080 Organometallics XXXX, XXX, XXX−XXX Article Organometallics Figure 4. Changes in MMP of A549 cancer cells induced by complexes Ir3 (a) and Ir6 (b). Figure 5. Analysis of ROS levels through ﬂow cytometry after A549 cells were treated with complexes Ir3 (a) and Ir6 (b) at the concentrations of 0.25 × IC50 and 0.5 × IC50 for 24 h and stained with H2DCFDA. Figure 6. Confocal microscopy images of A549 cells co-labeled with Ir3 (10 μM, 1 h) and MTDR (500 nM, 20 min) or LTDR (75 nM, 20 min) at 37 °C. Ir3 (λex: 488 nm, λem: 520 ± 30 nm); MTDR (λex: 644 nm, λem: 700 ± 30 nm); LTDR (λex: 594 nm, λem: 630 ± 30 nm). Scale bar: 20 μm. thylbenzimidazolylcarbocyanine iodide (JC-1). As depicted in Figures 4 and S5, and Tables S6 and S7. A549 cells were treated with complexes Ir3 and Ir6 at 0.25, 0.5, 1.0, and 2.0 equivalents of IC50 for 24 h, followed by staining with JC-1 and analysis by ﬂow cytometry. A substantial dose-related red-to-green color shift was observed, thereby suggesting MMP loss. The percentage of depolarized cells increased from 12.77 to 89.77% and 4.00 to 79.23% for Ir3 and Ir6 at 2 × IC50, respectively. Hence, the loss of MMP may be an apoptotic mediating factor. ROS Determination. The increase of the intracellular ROS level is a well-known MoA.39,40 This family of complexes triggers the formation of intracellular ROS in A549 cancer cells and was evaluated via ﬂow cytometry by utilizing 2′,7′-dichloroﬂuor- escein diacetate (H2DCFDA) staining. Intracellular ROS can promote conversion of nonﬂuorescent H2DCFDA to the ﬂuorescent product 2′,7′-dichloroﬂuorescein (DCF).41 As illustrated in Figures 5 and S52, compared with the control group, the DCF intensity indicated a concentrate-dependent increase upon treatment with complexes Ir3 and Ir6 at concentrations of approximately 0.25 × IC50 value and 0.5 × IC50 value for 24 h, respectively. The increased ROS level for these complexes played an important role in their anticancer activity and was deemed as the MoA in this system. Notably, similar half-sandwich C,N-chelating iridium anticancer complexes were used to generate the ROS by catalytic hydride transfer from the coenzyme NADH to oxygen.30 As a result, the ROS level elevation may be attributed to the conversion of E DOI: 10.1021/acs.organomet.9b00080 Organometallics XXXX, XXX, XXX−XXX Article Organometallics NADH to NAD+ by these complexes, which was also observed in this system. In addition, in the “reaction with NADH” section, Ir6 showed a lower catalytic ability in converting coenzyme NADH to NAD+ than Ir3 (Ir6: 25.4 vs Ir3: 66.3). This result is consistent with Ir6’s lower ROS level elevation compared with Ir3. Therefore, the ROS level elevation is correlated with the conversion of NADH to NAD+ in this system. Intracellular Localization. The cellular localization of anticancer metallodrugs can be visualized by employing confocal microscopy imaging due to their intrinsic emission properties.42,43 To understand which organelles would be targeted by these iridium(III) complexes, we performed colocalization experiments of Ir3 and LysoTracker Deep Red (LTDR) or MitoTracker Deep Red (MTDR) within A549 cells by employing confocal microscopy. As shown in Figure 6, Ir3 eﬃciently accumulates in A549 cells after 1 h of incubation and exhibits an intense and punctate staining pattern as a result of its compartmentalization. Co-localization analysis with the lysosome dye LTDR (75 nM) displays that Ir3 (10 μM) selectively accumulates in lysosomes. The Pearson’s colocalization coeﬃcients obtained for Ir3 with LTDR was 72 ± 2%. Negligible colocalization was observed for Ir3 with the MTDR. These results indicated that Ir3 can speciﬁcally distribute in the lysosomes. Lysosomal Damage. Lysosomes are membrane-bound organelles, which are involved in many physiological processes.44−46 To assess whether lysosomal damage was the pathway of cell death, we utilized acridine orange as a probe to evaluate the dysfunction of lysosomes. Acridine orange is the most reported and commercially available LysoTrackers because it emits red ﬂuorescence in lysosomes and green ﬂuorescence in the cytosol and nuclei.47,48 As illustrated in Figure 7a, A549 cells tosis inhibitor chloroquine). As indicated in Figure S53, no distinct alteration of the luminescent intensity in the three experiment groups were observed compared with the vehicletreated group (37 °C), thereby suggesting that Ir3 entered A549 cells via a non-energy-dependent pathway. Notably, the previously reported half-sandwich iridium(III) complexes with P^P chelating ligands showed diﬀerent cellular uptake mechanisms (an energy-dependent pathway) from these complexes.49 ■ CONCLUSIONS A series of novel half-sandwich iridium(III) complexes containing various phosphine-imine ligands were successfully synthesized and fully characterized. Stability studies revealed that the chemical composition of these complexes in aqueous media and in DMSO solutions did not change in the biological experiments. Most of these complexes displayed high cytotoxicity for the A549 cell line, distinctly surpassing the cisplatin activity in this cell type. Replacing the cyclohexyl group in the imine moiety with a phenyl group enhanced the cytotoxicity. Moreover, the introduction of biphenyl substituents onto the Cp* ring optimized cytotoxicity compared with their parent Cp* complexes. Additionally, the ortho alkyl substituents in aniline and the length of the methylene chain on the imine moiety did not aﬀect the cytotoxicity of the complexes. The cytotoxicity of these complexes may be primarily attributed to the coordination between iridium(III) and coordinating atoms, and the nature of the imine N-substituents may not be a major factor aﬀecting cytotoxicity. Cell death mechanism studies indicated that these complexes caused cell apoptosis and necrosis, generated ROS, and disrupted the MMP. Further mechanism studies through confocal microscopy showed that this class of complexes was taken up via non-energy-dependent mechanisms and mainly gathered in the lysosomes in A549 cancer cells. The disruption of lysosomes was also responsible for cell death. These preliminary results implied that this work may be helpful for the development and design of potential anticancer complexes with mixed P/N-based chelating ligands. ■ EXPERIMENTAL SECTION Chemicals and Reagents. IrCl3·nH2O, 1,2,3,4,5-pentamethylcyclopentadiene (95%), butyllithium solution (1.6 M in hexane), 2,3,4,5-tetramethyl-2-cyclopentenone (95%), 4-bromo-biphenyl, 2diphenylphosphinobenzaldehyde, aniline, 2,6-dimethylaniline, 2,6diisopropylaniline, cyclohexylamine, benzylamine, 2-phenylethylamine, 9-EtG, 9-MeA, and reduced form of nicotinamide-adenine dinucleotid (NADH) were purchased from commercial sources and used without further puriﬁcation. [(η5-Cp*)IrCl2]2 (dimer 1) and [(η5-Cpxbiph)IrCl2]2 (dimer 2) were prepared by a method in the literature.50 The phosphine-imine ligands (L1−L6) were obtained through a Schiﬀ-base condensation reaction previously reported.51−57 Synthesis of the Complexes. General Method. A mixture of the phosphine-imine ligand (0.10 mmol) and [(η5-Cpx)IrCl2]2 (0.05 mmol) was dissolved in CH2Cl2 (20 mL) and stirred at room temperature for 20 h. Then, solid KPF6 (0.60 mmol) was added with stirring. After 2 h, KPF6 was ﬁltered oﬀ. The solvent was removed under reduced pressure. The resulting solid was puriﬁed by recrystallization, providing orange crystals. [(η5-Cp*)Ir(L1)Cl]PF6 (Ir1) yield: 47 mg (54%). 1H NMR (500 MHz, DMSO): δ 8.62 (d, J = 2.4 Hz, 1H, HCN), 7.87−7.80 (m, 3H, Ar-H), 7.72 (ddt, J = 18.3, 15.9, 7.8 Hz, 11H, Ar-H), 7.62 (t, J = 7.6 Hz, 1H, Ar-H), 7.49 (dd, J = 10.6, 7.8 Hz, 1H, Ar-H), 7.41 (t, J = 7.5 Hz, 1H, Ar-H), 7.12 (d, J = 7.7 Hz, 2H, Ar-H), 1.02 (d, J = 2.3 Hz, 15H, Cp*-H). 31 P NMR (202 MHz, DMSO): δ 11.41 (s), −133.65 (s), −137.17 (s), −140.68 (s), −144.19 (s), −147.71 (s), −151.22 (s), −154.73 (s). FT- Figure 7. Confocal luminescence imaging of acridine orange (5 μM) stained A549 cells after diﬀerent treatments. (a) Only acridine orange; (b) acridine orange and complex Ir3 (1 × IC50); (c) acridine orange and complex Ir3 (2 × IC50). λex: 488 nm; λem: 490−530 nm (green channel); 605−645 nm (red channel). Scale bars: 20 μm. alone treated with acridine orange (5 μM) showed red ﬂuorescence in the lysosomes, thereby indicating that the lysosomes of A549 cells were intact under such conditions. When the A549 cells were treated with complex Ir3 at 1 × IC50 and 2 × IC50, the red ﬂuorescence of acridine orange severely decreased in a dose-dependent manner (Figure 7b,c), thereby suggesting that lysosomal integrity was disrupted. Hence, complex Ir3 may induce cell death via the disruption of lysosomes. Cellular Uptake Mechanisms. These complexes crossed the membrane rapidly and preferentially accumulated in the perinuclear region. Therefore, the cellular uptake mechanisms of drug molecules were investigated by confocal microscopy. A549 cells were treated with Ir3 at lower temperature (4 °C), or 37 °C, or upon pretreatment with diﬀerent inhibitors (energy inhibitor carbonyl cyanide m-chlorophenyl hydrazone and the endocyF DOI: 10.1021/acs.organomet.9b00080 Organometallics XXXX, XXX, XXX−XXX Article Organometallics IR (KBr disk, cm−1): ν(CN imine) 1568.9. MALDI-TOF-MS (m/ z): calcd for C35H35ClIrPN, 728.1825; found, 728.1370 [(η5-Cp*)Ir(L1)Cl]+. Elemental analysis calcd (%) for C35H35NClIrP2F6: C, 48.14; H, 4.04; N, 1.60. Found: C, 48.29; H, 4.08; N, 1.65. [(η5-Cp*)Ir(L2)Cl]PF6 (Ir2) yield: 63 mg (70%). 1H NMR (500 MHz, CDCl3): δ 8.07 (d, J = 3.2 Hz, 1H, HCN), 7.72−7.65 (m, 4H, Ar-H), 7.64−7.58 (m, 5H, Ar-H), 7.54 (td, J = 8.0, 2.8 Hz, 2H, Ar-H), 7.28 (d, J = 3.8 Hz, 1H, Ar-H), 7.23 (s, 1H, Ar-H), 7.18 (d, J = 6.9 Hz, 1H, Ar-H), 7.07−7.00 (m, 3H, Ar-H), 2.27 (s, 3H, o-aniline-CH3), 1.37 (s, 3H, o-aniline-CH3), 1.16 (d, J = 2.4 Hz, 15H, Cp*-H). 31P NMR (202 MHz, CDCl3): δ 6.64 (s), −133.94 (s), −137.46 (s), −140.98 (s), −144.50 (s), −148.02 (s), −151.53 (s), −155.05 (s). FT-IR (KBr disk, cm−1): ν(CN imine) 1602.0. MALDI-TOF-MS (m/z): calcd for C37H39ClIrPN, 756.2138; found, 756.1720 [(η5-Cp*)Ir(L2)Cl]+. Elemental analysis calcd (%) for C37H39NClIrP2F6: C, 49.30; H, 4.36; N, 1.55. Found: C, 49.49; H, 4.42; N, 1.58. [(η5-Cp*)Ir(L3)Cl]PF6 (Ir3) yield: 44 mg (46%). 1H NMR (500 MHz, CDCl3): δ 8.07 (d, J = 2.9 Hz, 1H, HCN), 7.68 (tt, J = 14.9, 7.4 Hz, 5H, Ar-H), 7.63−7.59 (m, 1H, Ar-H), 7.51 (dd, J = 6.7, 4.6 Hz, 4H, Ar-H), 7.34−7.27 (m, 3H, Ar-H), 7.24 (d, J = 7.8 Hz, 2H, Ar-H), 7.16− 7.03 (m, 2H, Ar-H), 3.46−3.43 (m, 1H, iPr-CH), 2.19−2.12 (m, 1H, i Pr-CH), 1.38 (d, J = 6.8 Hz, 3H, iPr-CH3), 1.19 (d, J = 2.4 Hz, 15H, Cp*-H), 1.10 (d, J = 6.6 Hz, 3H, iPr-CH3), 1.06 (d, J = 6.7 Hz, 3H, iPrCH3), 0.18 (d, J = 6.6 Hz, 3H, iPr-CH3). 31P NMR (202 MHz, CDCl3): δ 6.89 (s), −133.94 (s), −137.46 (s), −140.98 (s), −144.50 (s), −148.02 (s), −151.54 (s), 155.06 (s). FT-IR (KBr disk, cm−1): ν(C N imine) 1599.0. MALDI-TOF-MS (m/z): calcd for C41H47ClIrPN, 812.2764; found, 812.1550 [(η5-Cp*)Ir(L3)Cl]+. Elemental analysis calcd (%) for C41H47NClIrP2F6: C, 51.43; H, 4.95; N, 1.46. Found: C, 51.38; H, 4.83; N, 1.54. [(η5-Cp*)Ir(L4)Cl]PF6 (Ir4) yield: 58 mg (66%). 1H NMR (500 MHz, DMSO): δ 8.64 (d, J = 2.5 Hz, 1H, HCN), 7.82 (dd, J = 7.3, 3.8 Hz, 1H, Ar-H), 7.75−7.53 (m, 12H, Ar-H), 7.46 (dd, J = 10.0, 8.0 Hz, 1H, Ar-H), 4.03 (t, J = 11.8 Hz, 1H, Cy-H), 1.95 (t, J = 11.7 Hz, 2H, CyH), 1.84 (d, J = 13.3 Hz, 1H, Cy-H), 1.75 (t, J = 12.2 Hz, 2H, Cy-H), 1.68 (d, J = 12.5 Hz, 1H, Cy-H), 1.63−1.53 (m, 1H, Cy-H), 1.53−1.47 (m, 1H, Cy-H), 1.44 (d, J = 14.1 Hz, 1H, Cy-H), 1.40 (d, J = 2.1 Hz, 15H, Cp*-H), 1.23 (dd, J = 25.8, 12.8 Hz, 1H, Cy-H). 31P NMR (202 MHz, DMSO): δ 12.32 (s), −133.65 (s), −137.17 (s), −140.68 (s), −144.19 (s), −147.71 (s), −151.22 (s), −154.73 (s). FT-IR (KBr disk, cm−1): ν(CN imine) 1617.4. MALDI-TOF-MS (m/z): calcd for C35H41ClIrPN, 734.2294; found, 734.1700 [(η5-Cp*)Ir(L4)Cl]+. Elemental analysis calcd (%) for C35H41NClIrP2F6: C, 47.81; H, 4.70; N, 1.59. Found: C, 47.96; H, 4.80; N, 1.53. Crystals of complex Ir4 qualiﬁed for X-ray analysis were obtained by slow diﬀusion of hexane into a concentrated solution of complex Ir4 in dichloromethane. [(η5-Cp*)Ir(L5)Cl]PF6 (Ir5) yield: 53 mg (60%). 1H NMR (500 MHz, CDCl3): δ 8.11 (d, J = 1.7 Hz, 1H, HCN), 7.63 (dd, J = 13.1, 7.3 Hz, 6H, Ar-H), 7.59−7.48 (m, 6H, Ar-H), 7.42 (td, J = 7.8, 2.7 Hz, 2H, Ar-H), 7.30−7.27 (m, 3H, Ar-H), 7.21−7.16 (m, 2H, Ar-H), 5.43 (d, J = 15.4 Hz, 1H, ArCH2N), 5.30 (d, J = 15.4 Hz, 1H, ArCH2N), 1.41 (s, 15H, Cp*-H). 31P NMR (202 MHz, CDCl3): δ 8.02 (s), −133.70 (s), −137.22 (s), −140.74 (s), −144.26 (s), −147.78 (s), −151.30 (s), −154.82 (s). FT-IR (KBr disk, cm−1): ν(CN imine) 1614.7. MALDI-TOF-MS (m/z): calcd for C36H37ClIrPN, 742.1981; found, 742.1977 [(η5-Cp*)Ir(L5)Cl]+. Elemental analysis calcd (%) for C36H37NClIrP2F6: C, 48.73; H, 4.20; N, 1.58. Found: C, 48.63; H, 4.25; N, 1.63. [(η5-Cp*)Ir(L6)Cl]PF6 (Ir6) yield: 70 mg (78%). 1H NMR (500 MHz, DMSO): δ 8.29 (d, J = 1.8 Hz, 1H, HCN), 7.76 (t, J = 7.6 Hz, 1H, Ar-H), 7.73−7.61 (m, 6H, Ar-H), 7.57 (dt, J = 8.9, 3.5 Hz, 6H, ArH), 7.48 (dd, J = 7.3, 3.7 Hz, 1H, Ar-H), 7.29 (dd, J = 11.1, 4.4 Hz, 2H, Ar-H), 7.24 (dd, J = 6.2, 3.9 Hz, 1H, Ar-H), 7.23−7.17 (m, 2H, Ar-H), 4.53−4.41 (m, 1H, ArCH2CH2N), 4.28−4.17 (m, 1H, ArCH2CH2N), 2.71 (ddd, J = 13.4, 8.3, 5.0 Hz, 1H, ArCH2CH2N), 2.58 (dt, J = 13.7, 8.1 Hz, 1H, ArCH2CH2N), 1.38 (s, 15H, Cp*-H). 31P NMR (202 MHz, DMSO): δ 8.09 (s), −133.65 (s), −137.16 (s), −140.68 (s), −144.19 (s), −147.70 (s), −151.22 (s), −154.73 (s). FT-IR (KBr disk, cm−1): ν(CN imine) 1611.9. MALDI-TOF-MS (m/z): calcd for C37H39ClIrPN, 756.2138; found, 756.2055 [(η5-Cp*)Ir(L6)Cl]+. Elemental analysis calcd (%) for C37H39NClIrP2F6: C, 49.30; H, 4.36; N, 1.55. Found: C, 49.11; H, 4.29; N, 1.49. [(η5-Cpxbiph)Ir(L4)Cl]PF6 (Ir7) yield: 63 mg (62%). 1H NMR (500 MHz, DMSO): δ 8.61 (s, 1H, HCN), 7.85−7.80 (m, 3H, Ar-H), 7.75−7.70 (m, 5H, Ar-H), 7.69−7.60 (m, 11H, Ar-H), 7.54−7.49 (m, 3H, Ar-H), 7.43 (t, J = 7.4 Hz, 1H, Ar-H), 3.66 (t, J = 11.7 Hz, 1H, CyH), 2.23 (s, 3H, Cpxbiph-CH3), 1.98 (d, J = 11.7 Hz, 1H, Cy-H), 1.68 (s, 2H, Cy-H), 1.66−1.57 (m, 4H, Cy-H and Cpxbiph-CH3), 1.54 (d, J = 1.7 Hz, 3H, Cpxbiph-CH3), 1.45 (dt, J = 21.1, 7.3 Hz, 1H, Cy-H), 1.30 (d, J = 10.7 Hz, 2H, Cy-H), 1.03−0.93 (m, 2H, Cy-H), 0.30 (s, 3H, CpxbiphCH3), −0.13 (dt, J = 22.1, 11.0 Hz, 1H, Cy-H). 31P NMR (202 MHz, DMSO): δ 12.29 (s), −133.65 (s), −137.16 (s), −140.68 (s), −144.19 (s), −147.70 (s), −151.22 (s), −154.73 (s). FT-IR (KBr disk, cm−1): ν(CN imine) 1616.8. MALDI-TOF-MS (m/z): calcd for C46H47ClIrPN, 872.2764; found, 872.2042 [(η5-Cpxbiph)Ir(L4)Cl]+. Elemental analysis calcd (%) for C46H47NClIrP2F6: C, 54.30; H, 4.66; N, 1.38. Found: C, 54.46; H, 4.69; N, 1.39. [(η5-Cpxbiph)Ir(L6)Cl]PF6 (Ir8) yield: 53 mg (51%). 1H NMR (500 MHz, DMSO): δ 8.79 (d, J = 1.9 Hz, 1H, HCN), 7.94−7.70 (m, 9H, Ar-H), 7.65−7.29 (m, 14H, Ar-H), 7.24−7.11 (m, 3H, Ar-H), 6.96− 6.88 (m, 2H, Ar-H), 4.30 (td, J = 11.6, 5.8 Hz, 1H, ArCH2CH2N), 4.13 (td, J = 11.6, 4.8 Hz, 1H, ArCH2CH2N), 3.20 (td, J = 12.0, 5.2 Hz, 1H, ArCH2CH2N), 2.43 (td, J = 12.0, 4.2 Hz, 1H, ArCH2CH2N), 1.93 (d, J = 1.7 Hz, 3H, Cpxbiph-CH3), 1.70 (d, J = 3.9 Hz, 3H, Cpxbiph-CH3), 1.49 (d, J = 3.0 Hz, 3H, Cpxbiph-CH3), 0.53 (s, 3H, Cpxbiph-CH3). 31P NMR (202 MHz, DMSO): δ 7.08 (s), −133.66 (s), −137.17 (s), −140.68 (s), −144.20 (s), −147.71 (s), −151.22 (s), −154.73 (s). FT-IR (KBr disk, cm−1): ν(CN imine) 1616.8. MALDI-TOF-MS (m/z): calcd for C48H45ClIrPN, 894.2607; found, 894.2693 [(η5-Cpxbiph)Ir(L6)Cl]+. Elemental analysis calcd (%) for C48H45NClIrP2F6: C, 55.46; H, 4.36; N, 1.35. Found: C, 55.60; H, 4.44; N, 1.30. ■ ASSOCIATED CONTENT * Supporting Information S The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.9b00080. Details of the Experimental Section, 1H NMR spectrum, P NMR spectrum, FT-IR spectra, MALDI-TOF-MS, UV/vis spectrum and 1H NMR spectra for stability studies, low-ﬁeld region of the 1H NMR spectra for reactions of complexes Ir3 and Ir6 with nucleobases, analysis of ROS levels, confocal images of A549 cells, and crystallographic data for complex Ir4 (CCDC number: 1877562) (PDF) 31 Accession Codes CCDC 1877562 contains the supplementary crystallographic data for this paper. 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. ■ AUTHOR INFORMATION Corresponding Authors *E-mail: guolihua@qfnu.edu.cn (L.G.). *E-mail: liuzheqd@163.com (Z.L.). ORCID Lihua Guo: 0000-0002-0842-9958 Zhe Liu: 0000-0001-5796-4335 Notes The authors declare no competing ﬁnancial interest. G DOI: 10.1021/acs.organomet.9b00080 Organometallics XXXX, XXX, XXX−XXX Article Organometallics ■ (19) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen, Z.-S. The development of anticancer ruthenium(II) complexes: from single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017, 46, 5771−5804. (20) Zou, T.; Lum, C. T.; Lok, C.-N.; Zhang, J.-J.; Che, C.-M. Chemical biology of anticancer gold(III) and gold(I) complexes. Chem. Soc. Rev. 2015, 44, 8786−8801. 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