Half-sandwich Iridium^IIIN-heterocyclic carbene antitumor complexes and biological applications.

Accepted Manuscript Half-sandwich IridiumIII N-heterocyclic carbene antitumor complexes and biological applications Yali Han, Zhenzhen Tian, Shumiao Zhang, Xicheng Liu, Juanjuan Li, Yanru Li, Yi Liu, Min Gao, Zhe Liu PII: DOI: Reference: S0162-0134(18)30458-6 doi:10.1016/j.jinorgbio.2018.09.009 JIB 10564 To appear in: Journal of Inorganic Biochemistry Received date: Revised date: Accepted date: 31 July 2018 10 September 2018 11 September 2018 Please cite this article as: Yali Han, Zhenzhen Tian, Shumiao Zhang, Xicheng Liu, Juanjuan Li, Yanru Li, Yi Liu, Min Gao, Zhe Liu , Half-sandwich IridiumIII Nheterocyclic carbene antitumor complexes and biological applications. Jib (2018), doi:10.1016/j.jinorgbio.2018.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Half-sandwich IridiumIII N-heterocyclic Carbene Antitumor Complexes and Biological Applications Yali Han, Zhenzhen Tian, Shumiao Zhang, Xicheng Liu*, Juanjuan Li, Yanru Li, Yi Liu, Min Gao, Zhe Liu* Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of PT Life-Organic Analysis and Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, School of Chemistry and Chemical Engineering, Qufu Normal University, Qufu RI 273165, China. *Corresponding author (Email): chemlxc@163.com (X.C. Liu); liuzheqd@163.com (Z. Liu) SC Abstract Series of half-sandwich IrIII N-heterocyclic carbene (NHC) antitumor complexes NU [(η5-Cp*)Ir(C^C)Cl] have been synthesized and characterized (Cp* is pentamethyl cyclopentadienyl, and C^C are four NHC chelating ligands containing phenyl rings at MA different positions). IrIII complexes showed potent antitumor activity with IC50 values ranged from 3.9 to 11.8 µM against A549 cells by the MTT assay. Complexes can D catalyze the conversion of the coenzyme NADH to NAD+ and induce the production PT E of reactive oxygen species (ROS), and bonding to BSA by static quenching mode. Complexes can arrest the cell cycle in G1 or S phase and reduce the mitochondrial membrane potential. Confocal microscopy test show complexes could target the CE lysosome and mitochondria in cells with the Pearson’s colocalization coefficient of 0.82 and 0.21 after 12 h, respectively, and followed by an energy-dependent cellular AC uptake mechanism. Key words: Half-sandwich; IridiumIII complex; N-heterocyclic carbene; Antitumor 1. Introduction Cancer is threatening people's health, and the research on anticancer drugs has caused widespread concern. Anticancer drugs mainly include natural drugs and chemical synthetic drugs [1, 2], among these, metal-centered platinum antitumor drugs are most widely used in clinical practice. Although cisplatin and its derivatives have achieved great success in clinical applications, the development of which is hindered by many drawbacks, e.g., serious side-effects and easily acquired drug 1 ACCEPTED MANUSCRIPT resistance [3-6]. Even so, the successful clinical application of platinum drugs has promoted the research process of other metal antitumor drugs [7]. Recently, non-platinum-based compounds such as iridium complexes are attracting more and more attention [8, 9], and which have significant potential to become alternatives to platinum-based metal anticancer agents [10, 11]. PT Iridium antitumor complexes mainly include two types: half-sandwich and cyclometalated iridium complexes. Due to the higher antitumor activity, RI half-sandwich IrIII complexes have attracted considerable attention. The general form of half-sandwich IrIII complexes can be expressed as [(Cpx)Ir(L^L)Z], Cpx represents SC the electron-rich cyclopentadienyl group and its derivatives, Z is the leaving group, and L^L is chelating ligand. Previous study showed the introduction of electron-rich NU Cp* in IrIII complexes can increase the stability of ligand binding to metal iridium [12-14]. And also, the type and the size of chelating ligands could obviously influence MA the targeted sites, the lipid solubility and even antitumor activity for half-sandwich IrIII complexes. D N-heterocyclic carbene (NHC) metal complexes have been extensively used in PT E various fields, especially as potential anticancer agents [15-17]. Previous study showed cyclic double carbene ligands IrIII complexes directly activate the mitochondrial energy production system of cancer cells, generating reactive oxygen CE species (ROS) and activating mitochondria-dependent cell death signaling pathways [18-20]. However, the study of half-sandwich IrIII NHC complex applied to antitumor AC field was rare [21, 22]. In this study, four half-sandwich IrIII NHC complexes of the type [(Cp*)Ir(C^C)Cl] (Complexes 1-4, Scheme 1) were synthesized and characterized. Cp* is pentamethylcyclopentadienyl, C^C are four diverse NHC chelating ligands containing phenyl rings at different positions [23]. As-synthesized IrIII complexes displayed a favorable antitumor activity against A549 lung cancer cells (IC50: 3.9-11.8 μm) than cisplatin (21.3 μm) under the same conditions. Complexes were able to bind with bovine serum albumin (BSA) and oxidize NADH (nicotinamide adenine dinucleotide) to NAD+, which inducing the production of reactive oxygen species (ROS) [24, 25]. Complexes can arrest the cell cycle and 2 ACCEPTED MANUSCRIPT induce apoptosis. Confocal microscopy test show complexes could target the lysosome and mitochondria in cells with the Pearson’s colocalization coefficient of 0.82 and 0.21 after 12 h, respectively, and followed by an energy-dependent cellular uptake mechanism [26-28]. The results suggest that half-sandwich IrIII NHC D MA NU SC RI PT complexes are hopeful for development as new antitumor agents. Scheme 1. The selcted NHC chelating ligands (L1-L4) and the synthetic process PT E of IrIII NHC complexes (1-4). 2. Results and Discussion CE 2.1 Synthesis and Characterization Half-sandwich IrIII NHC complexes of the type [(Cp*)Ir(C^C)Cl] were synthesized AC in dichloromethane at ambient temperature using silver oxide (Ag2O) catalyzed reaction. NHC chelating ligands were obtained by the reaction of the corresponding imidazole and iodine hydrocarbon. All of IrIII NHC complexes were newly synthesized complexes and achieved in good yields. The synthetic processes are shown in Scheme 1. IrIII NHC complexes and the intermediates were marked by nuclear magnetic resonance spectrum (1H NMR), mass spectroscopy (MS) and elemental analysis. Complexes were non-hygroscopic and highly soluble in common organic solvents such as dichloromethane, chloroform, dimethyl sulfoxide, partially dissolved in methanol, insoluble in ether, hexane and petroleum ether. 3 ACCEPTED MANUSCRIPT 2.2 Cytotoxicity Test The antitumor activity of IrIII NHC complexes against A549 human lung cancer cells was determined by the MTT assay [29-31]. As shown in Table 1, all complexes showed better antitumor activity than cisplatin (widely used in clinical practice), with the IC50 values ranged from 3.9 to 11.8 μM. Compared with complex 1, complexes PT 2-4 have the better antitumor activity, which is mainly due to the enhanced lipid solubility caused by the introduction of more phenyl to NHC chelating ligands. The RI log P (partition coefficient in oil/water) for complexes 1 and 4 were determined by inductively coupled plasma mass spectrometry (ICP-MS), the values were -1.12 and SC -0.57, respectively. The data show that the increase of the benzene ring to NHC chelating ligands can effectively increase the lipid solubility of the complex. However, NU the position and number of benzene rings have little change in the activity of complexes 2-4. In order to further study the antitumor mechanism of IrIII NHC MA complexes, complexes 2 and 3 were selected as representatives, and further studied. D Table 1. IC50 values of complexes 1-4 and cisplatin against A549 cells determined by MTT PT E assay after 24 h. Complex [(η -C5Me5)Ir(L1)Cl] (1) [(η5-C5Me5)Ir(L2)Cl] (2) [(η5-C5Me5)Ir(L3)Cl] (3) [(η5-C5Me5)Ir(L4)Cl] (4) Cisplatin IC50 (µM) 11.8±1.2 5.9±0.4 4.6±0.2 3.9±0.7 21.3±1.7 AC CE 5 2.3 Reaction with NADH NADH and NAD+ are indispensable substances in various bio-catalytic reactions. [32]. NADH can contribute hydride to transition metal-based complex and promote the production of ROS (H2O2), thus serving as a pathway for the oxidation mechanism [33]. Interaction between IrIII NHC complexes and NADH was determined by ultraviolet-visible (UV-Vis) spectrum. The maximum absorbance of NADH and NAD+ can be determined in 339 and 259 nm, respectively. As shown in Fig. 1A (Fig. 4 ACCEPTED MANUSCRIPT S1, ESI), with the increase of complexes, the absorption of 339 nm showed a significantly reduced and an increase in 259 nm which further confirmed the catalytic activity of IrIII NHC complexes [33]. The turn over numbers (TONs) values of complexes 2 and 3 were shown in Fig. 2B. As shown, complex 3 had the bigger TONs value, which was correspond with the result of MTT assay. The bigger of the TONs NU SC RI PT value, the better of antitumor activity. MA Fig. 1 (A) Reaction of complex 3 (1.0 μM) and NADH (100 μM) in a 60% MeOH/40% H2O (v/v) PT E 2.4 Protein interaction D mixed solution was monitored by UV-Vis at 298 K over 8 h. (B) The TONs of complexes 2 and 3. Serum albumin (SA) has the highest abundance in plasma and excellent binding properties, so it is typically used in the delivery of drugs in the blood system [34-37]. CE As a kind of transport protein, bovine serum albumin (BSA) has the advantages of cheap, stable, easy to purify and similar to human serum albumin (HSA) [38], which AC was chose as a model to study the interaction between complexes and proteins. The UV-Vis absorption spectrum of BSA in the presence of complexes 2 and 3 are shown in Fig. 2 and Fig. S2, ESI. The internal filtration effect can be eliminated by UV-Vis and aim at further fluorescence intensity studies. With the increase of complexes, the maximum absorption at 228 nm decreased, which is mainly due to the induced perturbation of BSA because of IrIII compounds. [39-41]. Obvious red-shift was found at 228 nm attribute to the effect of the polar solvent (water). However, there is no obvious shift in 278 nm, which indicated the microenvironment of the three aromatic acid residues in BSA (Trp, Tyr and Phe) was changed because of IrIII 5 ACCEPTED MANUSCRIPT RI PT NHC complexes [42]. SC Fig. 2 (A) Complex 3 was increased from 0 μM to 10 μM, UV-Vis spectra of BSA in Tris-HCl/NaCl buffer solution (pH=7.2). Arrows indicate the direction of change in absorbance as NU the concentration of the complex increases. Inset: Wavelength absorbance changes from 200 to 320 nm. (B) The complex 3 was increased from 0 μM to 10 μM, and the fluorescence spectrum of MA BSA was changed (0.5 μM, λex=280 nm, λem=343 nm). D The fluorescence emission spectra of BSA with different concentrations of PT E complexes 2 and 3 at 298 K are presented in Fig. 2B and Fig. S2B, ESI. As shown, fluorescence intensity of BSA quenched obviously with the increase of complexes. The quenching rate constant Kq and Stern–Volmer quenching constant Ksv were CE calculated by classical Stern-Volmer equation (Fig. S3, ESI), and the binding constant Kb and binding site number n of complexes were obtianed by the Scatchard equation AC (Fig. S4, ESI) [43]. As shown in Table 3, the values of Kq for complexes 2 and 3 were 3.92×1012 and 3.21×1012 M-1 s-1, which are about two orders of magnitude higher than that of a pure dynamic quenching mechanism (2.0×1010 M-1 s-1) [44]. These indicated that the interaction between IrIII NHC complexes and BSA have been followed by a static quenching mechanism dominates. The binding site number (n) was almost the same (~1), but complex 3 having a slightly larger binding constant (Kb) values than complex 2, which was consistent with the conclusion that complex 3 had the higher antitumor activity. 6 ACCEPTED MANUSCRIPT Table 3. Quenching parameters and binding parameters for the interaction of the complex 2 and complex 3 with BSA. Complex 2 3 Ksv (104M-1) 3.92±0.73 3.21±0.29 Kq (1012 M-1s-1) 3.92 3.21 Kb (104 M-1) 2.06 2.55 n 1.29 1.14 PT Information on tyrosine residues and tryptophan residues in the BSA microenvironment can be displayed at the same fluorescence spectra Δλ = 15 nm and RI Δλ = 60 nm [45]. As shown in Figs. S5-S6, ESI, the fluorescence intensity at 291 nm and 285 nm (Δλ = 15 nm and 60 nm), which are the characteristic of the synchronous SC emission spectra for tyrosine and tryptophan residues of BSA, reduced with the NU increase of compounds 2 and 3. At Δλ = 15 nm, a slight blue shift of 2 nm was observed in the complex 3 (Fig. S6), and a slight red shift of 3 nm was observed for MA complex 2 at Δλ = 60 nm. The above results clearly shows that IrIII NHC complexes could act on BSA by affecting its microenvironment. 2.5 Cell cycle analysis D As shown in Fig. 3 (Fig S7, Table S1 and S2, ESI), the cell cycle arrest for A549 PT E cells exposure to complexes 2 and 3 with the concentrations of 0.25, 0.5 and 1.0×IC50 for 24 h have been determined by flow cytometry. At a concentration of 2.0×IC 50, the percentages of cells in the Sub-G1 phase increased from 58.1% to 64.7% when CE exposure to complex 2, which indicating cells failed to synthesize RNA and protein normally, or affecting the energy supply to the next stage of the cell [31, 35]. For AC complex 3, the percentages of cells in the S phase increased 7.8%, which indicating complex 3 may block the synthesis of DNA and histone, or some DNA replication-related enzymes [35]. Compared with the untreated control, the cell cycle was disturbed in sub-G1 and S phase for complexes 2 and 3, respectively. The results indicate that IrIII NHC complexes can disturb the cell growth cycle progression, and achieving the objective of apoptosis. 7 PT ACCEPTED MANUSCRIPT Fig. 3 Histogram data of cell cycle distribution of A549 cancer cells of complexes 2 and 3 after 24 RI h. Tests were performed using 0.25, 0.5, 1.0, and 2.0 times of the IC50 equivalent concentration. SC The data is taken as mean ± SD of three measurements. Cell staining for flow cytometry was NU carried out using PI/RNase. 2.6 Induction of apoptosis MA To determine whether cell function decline was associated with apoptosis, A549 cells were treated with complexes 2 and 3 at 1.0, 2.0, and 3.0 equivalents IC50 for 24 hours. After staining with Annexin, data were measured by flow cytometry [46]. As D shown in Fig. 4 and Tables S3- S4, ESI, the population of the early and late apoptotic PT E phase had a significant increase with the value ranged from 1.0% and 4.3% to 2.1% and 29.8% after 24 h, respectively, when the concentration changed from 1.0×IC50 to CE 3.0×IC50 for complex 2. For complex 3 (Fig. 4), about 39.0% of the A549 cells were undergoing apoptosis, including 32.0% of cells in the late apoptosis, while 95.8% of AC untreated cells are still alive under the same conditions. 8 MA NU SC RI PT ACCEPTED MANUSCRIPT D Fig. 4 (A) Apoptosis analysis of A549 cancer cells after 24 h of exposure to complexes 2 and 3 at PT E 310 K determined by flow cytometry with Annexin V-FITC vs PI staining. (B) Histogram showing populations for A549 cells in four stages treated by complexes 2 and 3. Data are quoted as mean ± CE SD of three replicates. 2.7 Induction of ROS AC The accumulation of ROS produced in mitochondrial will lead to apoptosis, which can be used to explore the function mechanism of antitumor agents. By means of flow cytometry, the level of ROS was determined in A549 cells for complexes 2 and 3 after 24 h. As shown in Fig. 5 and Tables S5-S6, ESI, compared with the control, the level of ROS increased by 1.3 and 1.4 times for complex 2 in the concentration of 0.25×IC50 and 0.50×IC50, respectively. For complex 3, the level of ROS only increased by 1.1 times with the concentration of 0.5×IC50. The results indicate that IrIII NHC complexes could induce the production of ROS, which provide a basis that complexes could affect the mitochondria and lead to apoptosis. 9 PT ACCEPTED MANUSCRIPT RI Fig. 5 A549 cancer cells were incubated with complexes 2 (A) and 3 (B) at concentrations of SC 0.25×IC50 and 0.5×IC50 to induce ROS production. Taking the ROS level of negative control as 2.8 Mitochondrial membrane potential NU the standard to make the histogram. MA Mitochondrial dysfunction may be involved in the cause of cell death [47-49]. The extent of mitochondrial dysfunction can be assessed by the loss of the mitochondrial membrane potential (MMP). As shown in Fig. 6, Fig. S8 and Tables S7-S8, ESI, the D loss of MMP induced by complexes 2 and 3 was assessed by detecting the JC-1 dye PT E (the decrease in red fluorescence and increased green fluorescence) using flow cytometry. At the indicated concentrations, a significant concentration-dependent CE increase was found with a remarkable loss of MMP, e.g. from 34.3% and 7.8% (control) to 60.1% and 30.6% (2.0× IC50) for complexes 2 and 3, respectively, which AC proving IrIII NHC complexes could act on mitochondria and induce apoptosis. 10 CE PT E D MA NU SC RI PT ACCEPTED MANUSCRIPT Fig. 6 (A) At concentration of 0.25×IC50, 0.5×IC50, 1.0×IC50 and 2.0×IC50, the loss of MMP AC induced by complex 2 using JC-1 dye. The red aggregates and green monomers are gated. (B) Histograms for the MMP treated with different concentrations of complex 2. The above data is the average ± SD of three measurements repeated. 2.9 Cell imaging and cellular uptake Subcellular localization of complexes 2 and 3 can be easily determined by laser confocal microscopy in A549 cells on account of their intrinsic luminescence. The Lyso Tracker Red DND-99 (LTRD) and Mito Tracker Deep Red (MTDR) were used as lysosomes and mitochondria fluorescence probes, respectively. As shown in Fig. 7 11 ACCEPTED MANUSCRIPT (Fig. S9, ESI), selected representative complex 3 could effectively target lysosomes with the Pearson coefficients of 0.75, 0.88, and 0.82 when incubated for 1, 6 and 12 h, respectively. In addition, complex 3 could also target mitochondria with the Pearson coefficients of 0.21, 0.17, and 0.23 after 1, 6 and 12 h, respectively, although it was not apparent. And also, the complex did not immediately lead to abnormal cell death, PT E D MA NU SC RI PT enabling us to track changes in lysosome morphology in real time [6]. CE Fig. 7 Confocal microscopic images of A549 cells co-labeled with complex 3 (10 μM, 1h, 6h, 12h) and LTRD (75 nM, 1h). Complex 3 and LTRD were excited at 488 nm and 594 nm, respectively. AC Complex 3 collects fluorescence in 549-651nm and LTRD collects fluorescence in 493-630nm. Scale bar: 20 μm. Lysosomal permeability is often triggered by the disruption of the lysosomal integrity [50]. To investigate whether lysosomal damage induced by complex 3 was accompanied by lysosome targeting specificity. Lysosomal integrity of A549 cells were evaluated by acridine orange (AO) staining after treatment with 3 for 6 h with the concentration of 1.0 × IC50 and 3.0 × IC50 [51, 52]. AO exhibits red fluorescence when accumulated in lysosome and green fluorescence when bounding to RNAs in 12 ACCEPTED MANUSCRIPT the nuclei or cytosol. As shown in Fig. 8, compared with control, red fluorescence basically disappears in lysosome when exposed to complex 3 (1.0 × IC50) after 6 h, PT E D MA NU SC RI PT and obvious lysosomal damage was found at 3.0 × IC50. Fig. 8 AO-loaded A549 cells (a) as a control group, no drug was added to the cells. (b) After addition of complex 3 (1.0 × IC50) for 6 h, AO (5 μM) was added for 15 min after laser confocal CE detection. (c) After addition of complex 3 (3.0 × IC50) for 6 h, AO (5 μM) was added for 15 min after laser confocal detection. Complex 3 was excited at 488 nm and collected at a wavelength of AC 493-630 nm. Scale bar: 20 μm. Drug molecules can enter cells with different transport mechanisms, including energy-dependent mechanisms (such as endocytosis and active transport) and energy-independent mechanisms (such as diffusion-promoting and passive diffusion) [53]. Therefore, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and chloroquine were used as energy inhibitor and endocytic inhibitor to study the mechanism of cellular uptake, respectively. The cells incubated at 277 K and 310 K for 2 h and then pretreatment with CCCP and chloroquine. As shown in Fig. 9, the uptake of cells 13 ACCEPTED MANUSCRIPT reduced efficiency after incubated at 273 K, which suggested that cellular uptake of PT E D MA NU SC RI PT complex 3 was followed by an energy-dependent mechanism. Fig. 9 Effect of incubation temperature (37 ºC and 4 ºC), metabolic inhibitor (CCCP, 50 μM) and CE chloroquine (50 μM) on cellular uptake of 3 (10 μM, 30 min) measured by confocal microscopy. AC Complex 3 was excited at 488 nm and emission was collected at 493-630 nm. Scale bar: 20 µm. 3. Conclusions In this study, four new half-sandwich IrIII NHC complexes were synthesized with simple synthetic procedures. All complexes showed favorable antiproliferative activity. Complex 4 showed the best activity towards A549 lung cancer cells, which were five times higher than the clinical antitumor drug cisplatin. Complexes can effectively bind with BSA, catalyzing NADH to NAD+ and inducing ROS, which will disturb the cell growth cycle and lead to apoptosis. Complexes can specifically target lysosomes and mitochondria, entering cells through energy-dependent mechanisms, 14 ACCEPTED MANUSCRIPT and destroying the integrity of lysosomes. Above all, half-sandwich IrIII NHC complexes could be a promising candidate for further evaluation as antitumor drugs. 4. Experimental section 1H-imidazole, 1H-benzimidazole, 1-benzylbenzimidazole, 1-benzyl-3-methylimidazoliumiodide 1-diphenylmethylimidazole, (L1), 1-(diphenylmethyl) PT -1H-benzimidazole and the dimer [Cp*IrCl2]2 were prepared according to literature procedures [54]. All other reagents are used as supplied by the commercial supplier. RI Nitrogen was used as the drying and filling gas. Synthesis of L2-L4. SC 1-benzylbenzimidazole (2.5 g, 12 mmol) was dissolved in 10 mL of acetonitrile, and then iodomethane (1.1 mL, 18 mmol) were added. The mixture was refluxed NU overnight. After rotary evaporation, yellow oil product was washed with diethyl ether, pure yellow salt was obtained. Yield: 2.2 g (86%). 1H NMR (500 MHz, CDCl3) δ MA 11.23 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.65 (s, 1H), 7.60 (s, 2H), 7.53 (d, J = 6.4 Hz, 2H), 7.39 (d, J = 7.3 Hz, 3H), 5.83 (s, 2H), 4.28 (s, 3H). D L3 and L4 were synthesized using the same method, the representation data is as PT E follows: L3: Yield: 79%. 1H NMR (500 MHz, CDCl3) δ 9.67 (s, 1H), 7.43 (s, 2H), 7.41 (s, 5H), 7.33 – 7.30 (m, 5H), 7.08 (s, 1H), 4.08 (s, 3H). CE L4: Yield: 74%. 1H NMR (500 MHz, CDCl3) δ 10.21 (s, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.45 (s, 10H), 7.29 (s, 1H), 7.20 (s, AC 1H), 4.31 (s, 3H). Synthesis of the complexes 1–4. In a round bottom flask, silver oxide (2.4 eq) and chelating ligands (L1-L4) (2.0 eq) were added to a solution of dichloromethane (CH2Cl2). After 8 h, the mixture was filtered through Celite and washed with CH2Cl2. The combined filtrates were added dropwise to a solution of CH2Cl2 containing [Cp*IrCl2]2 (1.0 eq). The mixture was stirred at room temperature for another 8h and filtered through Celite. The solvent was removed in vacuum， and the product was recrystallized from CH2Cl2 / n-hexane to give a pure yellow product. Detailed characterization data of complexes 1–4 were 15 ACCEPTED MANUSCRIPT as follows: [(η5-C5Me5)Ir(L1)Cl] (1): Yield: 79%. 1H NMR (500 MHz, CDCl3) δ 7.62 (d, J = 7.5 Hz, 1H), 6.97 (dd, J = 12.9, 7.0 Hz, 2H), 6.94 (d, J = 2.0 Hz, 1H), 6.90 (d, J = 1.9 Hz, 1H), 6.81 (t, J = 7.3 Hz, 1H), 4.86 (d, J = 13.8 Hz, 1H), 4.65 (d, J = 13.9 Hz, 1H), 3.93 (s, 3H), 1.68 (s, 15H). Elemental analysis: Found: C, 50.49; H, 5.21; N, 5.64%, calcd for C21H26ClIrN2: C, 50.58; H, 5.26; N, 5.62%. ESI-MS (m/z): calcd for PT C21H26IrN2: 498.67 [M-Cl]+; found 499.42. RI [(η5-C5Me5)Ir(L2)Cl] (2): Yield: 72%. 1H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 6.7 Hz, 1H), SC 7.23 (d, J = 7.4 Hz, 1H), 7.08 – 7.05 (m, 1H), 6.92 (t, J = 7.4 Hz, 1H), 6.81 (t, J = 7.0 Hz, 1H), 5.16 (d, J = 14.2 Hz, 1H), 5.03 (d, J = 14.1 Hz, 1H), 4.04 (s, 3H), 1.82 (s, NU 15H). Elemental analysis: Found: C, 50.78; H, 5.10; N, 5.09%, calcd for C25H28ClIrN2: C, 50.72; H, 5.14; N, 5.11%. ESI-MS (m/z): calcd for C25H28IrN2: 548.73 [M-Cl]+; MA found 549.33. [(η5-C5Me5)Ir(L3)Cl] (3): Yield: 74%. 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 7.6 D Hz, 1H), 7.51 (d, J = 6.7 Hz, 3H), 7.44 (s, 2H), 6.84 (t, J = 7.1 Hz, 1H), 6.80 (s, 1H), PT E 6.61 (t, J = 7.4 Hz, 1H), 6.38 (d, J = 8.7 Hz, 2H), 5.98 (s, 1H), 3.90 (s, 3H), 1.81 (s, 15H). Elemental analysis: Found: C, 52.49; H, 5.23; N, 4.35%, calcd for C27H30ClIrN2: found 575.33. CE C, 53.06; H, 5.11; N, 4.28%. ESI-MS (m/z): calcd for C27H30IrN2: 574.77 [M-Cl]+; [(η5-C5Me5)Ir(L4)Cl] (4): Yield: 68%. 1H NMR (500 MHz, CDCl3) δ 7.95 (d, J = 7.7 AC Hz, 1H), 7.53 (s, 1H), 7.46 (d, J = 6.5 Hz, 4H), 7.09 – 7.04 (m, 2H), 6.84 (t, J = 7.5 Hz, 1H), 6.70 (t, J = 7.4 Hz, 1H), 6.61 (t, J = 7.5 Hz, 1H), 6.46 (d, J = 7.7 Hz, 2H), 5.56 (d, J = 8.6 Hz, 1H), 4.11 (s, 3H), 1.77 (s, 15H). Elemental analysis: Found: C, 59.51; H, 5.18; N, 4.45%, calcd for C31H32ClIrN2: C, 59.59; H, 5.16; N, 4.48%. ESI-MS (m/z): calcd for C31H32IrN2: 624.57 [M-Cl]+; found 625.33. Acknowledgments We thank the University Research Development Program of Shandong Province (J18KA082), the National Natural Science Foundation of China (Grant No. 21671118) and the Taishan Scholars Program for support. 16 ACCEPTED MANUSCRIPT References [1] L. Galluzzi, L. Senovilla, I. Vitale, J. Michels, I. Martins, O. Kepp, M. Castedo, G. Kroemer, Oncogene 31 (2011)1869-1873. [2] C.-H. Leung, H.-J. Zhong, D.S.-H. Chan, D.-L. Ma, Coord. Chem. 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