Cyclometalated iridium(III)-guanidinium complexes as mitochondria-targeted anticancer agents

Accepted Manuscript Cyclometalated iridium(III)-guanidinium complexes as mitochondria-targeted anticancer agents Xing-Dong Song, Xia Kong, Shu-Fen He, Jia-Xi Chen, Jing Sun, Bing-Bing Chen, JinWu Zhao, Zong-Wan Mao PII:

S0223-5234(17)30484-1

DOI:

10.1016/j.ejmech.2017.06.038

Reference:

EJMECH 9533

To appear in:

European Journal of Medicinal Chemistry

Received Date: 28 April 2017 Revised Date:

28 May 2017

Accepted Date: 22 June 2017

Please cite this article as: X.-D. Song, X. Kong, S.-F. He, J.-X. Chen, J. Sun, B.-B. Chen, J.-W. Zhao, Z.W. Mao, Cyclometalated iridium(III)-guanidinium complexes as mitochondria-targeted anticancer agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.038. 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.

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Graphical Abstract: :

Cyclometalated iridium(III)-guanidinium complexes as

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mitochondria-targeted anticancer agents

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Xing-Dong Song, Xia Kong, Shu-Fen He, Jia-Xi Chen, Jing Sun, Bing-Bing

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Chen, Jin-Wu Zhao, Zong-Wan Mao

Four cyclometalated iridium(III) complexes containing guanidinium ligands have been synthesized and characterized. These complexes display moderate cytotoxicity by specifically targeting mitochondria and inducing a cascade of apoptotic events

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related to mitochondrial dysfunction.

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Cyclometalated iridium(III)-guanidinium complexes as

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mitochondria-targeted anticancer agents

Xing-Dong Songa†, Xia Kongb†, Shu-Fen Hea, Jia-Xi Chena, Jing Suna*, Bing-Bing

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c

School of Pharmacy, Guangdong Medical University, Dongguan, 523808, China

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a

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Chena, Jin-Wu Zhaoa, Zong-Wan Maoc*

School of Basic Medicine, Guangdong Medical University, Dongguan, 523808, China

MOE Key laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen



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University, Guangzhou, 510275, China

Contributed equally to this work.

* Corresponding authors. Tel.: +86-769-2289-6322; +86-20-8411-3788. E-mail address: [email protected] (J. Sun); [email protected] (Z.-W. Mao).

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Abstract Guanidinium-functionalized molecules are commonly studied for their use as pharmaceutically active compounds and drugs carriers. Herein, four cyclometalated

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iridium(III) complexes containing guanidinium ligands have been synthesized and characterized as potential anticancer agents. These complexes exhibit moderate antitumor activity in HeLa, MCF-7, HepG2, CNE-2, and A549 human tumor cells.

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Interestingly, all complexes showed higher cytotoxicity than cisplatin against a

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cisplatin-resistant cell line A549R, and less cytotoxicity on the nontumorigenic LO2 cells. Intracellular distribution studies suggest that these complexes are selectively localized in the mitochondria. Mechanism studies indicate that these complexes arrested the cell cycle in the G0/G1 phase and can influence mitochondrial integrity,

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pathways.

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inducing cancer cell death through reactive oxygen species (ROS)-dependent

Keywords: Ir(III) complex; Guanidinium; Cytotoxicity; Mitochondria; Apoptosis

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1. Introduction In recent years, the development of multifunctional agents that can realize simultaneous tumor targeting, imaging and treatment has become a major strategy in

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cancer treatment research. As most of the research focuses on the nanoparticles or nanosized drugs [1,2], metal complexes have also stimulated researchers’ interest because of their unique photochemical and photophysical properties and the

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potentiality as anticancer agents [3-5]. Among these metal complexes, organometallic

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iridium complexes have recently emerged as promising alternatives and are expected to overcome the limitations of platinum-based drugs [6-8]. Different from platinum-based drugs, the targets of anticancer Ir(III) complexes include DNA [9,10], proteins [11,12], mitochondria [13,14], and lysosomes [15], yet the mechanisms of

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such complexes on a cellular level remain largely unknown. From an advantageous perspective, Ir(III) complexes have become one of the most attractive classes of phosphorescent heavy metal complexes in bioimaging and biosensing due to their

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high quantum yields, large Stokes shifts, long-lived luminescence, good photostability

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and cell permeability [16]. Additionally, the intense fluorescence of the resulting Ir(III) complexes can facilitate cellular location and anticancer mechanism studies. Mitochondria, known as the energy powerhouse of a cell, represent a key

intracellular signaling hub and are emerging as important determinants of several aspects of cancer development and progression [17]. Mitochondria are indispensable for energy production and, hence, for the survival of eukaryotic cells. Meanwhile, mitochondria are crucial regulators of the intrinsic pathway of apoptosis [18]. For 3

ACCEPTED MANUSCRIPT instance, mutations of the mitochondrial or nuclear DNA that affect components of the mitochondrial respiratory chain result in inefficient ATP production, ROS overproduction, and oxidative damage to mitochondria and other macromolecules.

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Furthermore, multiple hallmarks of cancer cells, including limitless proliferative potential, insensitivity to anti-growth signals, impaired apoptosis, enhanced anabolism and decreased autophagy, have been linked to mitochondrial dysfunctions [19,20].

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Additionally, mitochondria are reservoirs for numbers apoptosis-promoting proteins

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that are essential for apoptosis induction, such as B-cell lymphoma protein 2 (Bcl-2) protein family [18]. Because mitochondrial-targeted compounds represent a promising approach to eradicate chemotherapy-refractory cancer cells, there has been growing interest in developing mitochondria-targeted fluorescent therapeutic agents,

21].

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which can monitor changes in mitochondrial status during the therapeutic process [13,

Guanidine is one of the strongest organic bases, owing to stabilization of the

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guanidinium ion via the Y delocalization effect [22]. The hydrogen bonding acceptor

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and donor abilities of the guanidino group play important roles in supramolecular formation as well as in drug design in the field of medicinal chemistry [23]. Guanidine and its metal complexes exhibit a varied range of chemical, biological, and medicinal applications, and compounds possessing the guanidine functional group have received attention as antimalarial [24,25], antimicrobial [26,27], and antitumor agents [28-30]. Chung and coworkers reported a novel class of guanidine-containing molecules that exhibit selectivity toward mitochondria [31,32]. In this paper, a series 4

ACCEPTED MANUSCRIPT of cyclometalated Ir(III) complexes containing guanidinium groups as ligands, [Ir(N-C)2L](PF6)2

(

L

=

1-(3-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenyl)guanidine cation; N–C =

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benzo[h]quinoline (bzq, 1), 2-(p-tolyl)pyridine (tpy, 2), 2-phenylpyridine (ppy, 3), and 2-(2-thienyl)pyridine (thpy, 4)) were designed, synthesized and characterized (Fig. 1). The in vitro antiproliferative activity of 1−4 was investigated against several cancer

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cell lines as well as a nontumorigenic cell line. To elucidate the possible anticancer

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mechanisms, the impacts of complexes 1 and 2 were evaluated for cellular localization, mitochondrial damage, ROS level, cell cycle distribution and initiation of a series of mitochondria-associated events that leads to cellular apoptosis. Fig. 1

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2. Results and discussion

2.1 Synthesis, characterization and photophysical properties Ligand L was synthesized according to our previous work [33]. Complexes 1–4

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were synthesized by reacting two equivalents of L with the Ir(III) chloro-bridged

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dimer. The synthetic routes of the complexes are elucidated in Scheme S1.These complexes were characterized by ESI-MS, 1H NMR (Fig. S1-S8), and elemental analysis. The UV-vis absorption spectra of 1–4 in phosphate-buffered saline (PBS), CH3CN and CH2Cl2 at 298 K are shown in Fig. 2. The absorption spectra of 1–4 feature an intense absorption band at approximately 260-320 nm and a relatively weak band at approximately 360-420 nm, which are attributed to intraligand (π→π*) transition and metal-to-ligand charge-transfer (MLCT) absorption, respectively. All 5

ACCEPTED MANUSCRIPT complexes exhibited green to yellow emission in PBS, CH3CN, and CH2Cl2 under ambient conditions upon excitation at 405 nm (Fig. S9). Fig. 2

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2.2 In vitro cytotoxicity The in vitro cytotoxic activities of these four Ir(III) complexes were evaluated against human cervical carcinoma cells (HeLa), human breast adenocarcinoma cancer

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cells (MCF-7), human hepatocellular liver carcinoma cells (HepG2), human

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nasopharyngeal carcinoma cells (CNE-2), human lung adenocarcinoma epithelial cells(A549), cisplatin-resistant A549 (A549R) and one nontumorigenic cell line LO2, were determined by MTT assay (Table 1). Based on the IC50 values, all complexes showed moderate antitumor activity to cancer cell lines, including A549R cells, which

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indicates that they can overcome cisplatin resistance. Notably, complex 1 displayed an approximately 4-fold higher anticancer efficacy than cisplatin in killing A549R cells. These complexes in the current study are found to have IC50 values higher to previous

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work [13,14]. Four complexes exhibited differences in their antitumor activities,

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where complex 1 was more active against the HeLa, MCF-7, and A549R tumor cell lines, especially HeLa cells. Thus, this HeLa cell line was used for further investigation regarding the underlying mechanisms. Table 1

2.3 Lipophilicity and cellular uptake properties The lipophilicity (log Po/w) of a compound has a strong influence on its cellular uptake, localization, and cytotoxicity [34,35]. The lipophilicity of complexes 1–4 was 6

ACCEPTED MANUSCRIPT determined by the flask-shaking method. The log Po/w values obtained for the compounds are in the following order: 1 (0.37) > 3 (0.036) > 2 (-0.010) > 4 (-0.23). Complex 1 was much more lipophilic than complexes 2, 3, and 4, owing to the

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presence of two relatively polar auxiliary ligands in the latter complexes. As iridium is an exogenous element, the cellular uptake levels of complexes 1−4 can be quantitatively determined by inductively coupled plasma mass spectrometry

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(ICP-MS) measurements. After incubation with 40 µM complexes for 3.5 h, the

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intracellular iridium contents of compounds were determined to follow the order: 1 (151.73 ng per 106 cells) > 3 (82.25 ng per 106 cells) > 2 (75.72 ng per 106 cells) > 4 (64.34 ng per 106 cells). It has been reported that the greater hydrophobictity results in higher cellular uptake and higher cytotoxicity [36]. The greatest lipophilicity of

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complex 1 exhibited the highest cellular uptake and cytotoxicity. Under the same conditions, the antiproliferative activity of the four complexes in HeLa cells was in the order: 1 > 2 ≈ 3 ≈ 4; thus, 1 and 2 were chosen as the model

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compounds to elucidate the anticancer mechanism. Due to the rich photophysical

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properties of cyclometalated Ir(III) complexes, intracellular distribution can be monitored by fluorescence microscopy. As shown in Fig. S10, complexes 1 and 2 appeared to reside in the cytoplasm after 3.5 h of incubation with HeLa cells and showed green fluorescence. Colocalization analysis of 1 and 2 with the organelle-specific stain MitoTracker® Red CMXRos (MTR) demonstrates that the complexes can specifically target mitochondria (Fig. 3A). Pearson’s colocalization coefficients of 1 and 2 with MTR were determined to be 0.82 and 0.86, respectively. 7

ACCEPTED MANUSCRIPT Meanwhile, minimal colocalization of 1 and 2 with Lyso-tracker Red (LTR) was observed, which indicates that complexes 1 and 2 possess mitochondrial specificity (Fig. 3B).

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Fig. 3 Mechanisms that small molecules follow to penetrate the cell membrane include energy-dependent (e.g., endocytosis and active transport) and energy-independent

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(e.g., facilitated diffusion and passive diffusion) pathways [37,38]. Pretreatment of

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HeLa cells with metabolic inhibitor carbonyl cyanide 3-chloro-phenylhydrazone (CCCP) or at a lower temperature (4 °C) can lead to reduced cellular uptake efficiency (Fig. S11). However, the ability of 1 and 2 to cross the plasma membrane is not affected by the presence of chloroquine, an endocytosis modulator inhibiting the

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acidification of endosomes. The results indicate that complexes 1 and 2 enter HeLa cells possibly through an energy-dependent pathway, (e.g., active transport), and endocytosis is not responsible for the uptake process. The same results were also

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found using other cyclometalated Ir(III) complexes in a previous report [13].

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2.4 Mitochondrial damage

As the bioenergetic center of the cell, dysfunction of mitochondria is closely

connected with apoptosis and several key apoptotic events, such as mitochondrial permeability transition and the reduction of mitochondrial membrane potential (MMP) [36]. In addition, maintenance of the integrity of the mitochondrial membrane plays a vital role in cell survival. Once the membrane integrity of mitochondria is damaged, the pro-death factors are released from mitochondria and initiate cell death signaling 8

ACCEPTED MANUSCRIPT [39]. To investigate whether the accumulation of complexes 1 and 2 in mitochondria could

cause

mitochondrial

dysfunction,

the

cationic

dye

5,5',6,6'-tetrachloro-1,1'-3,3'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) was

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used to detect changes in MMP (∆Ψm) by confocal microscopy and flow cytometry. Upon JC-1 staining, mitochondria depolarization was indicated by a decrease in red fluorescence (JC-1 aggregates) and an increase in green fluorescence (JC-1

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monomers). As shown in Fig. S12, complexes 1 and 2 caused a red-to-green color

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shift in most of the treated cells, indicating the loss of MMP. The changes in MMP were also detected by determining the percentage of red and green fluorescent intensities using flow cytometry. As shown in Fig. 4, after HeLa cells were treated with Ir(III) for 6 h, the cell ratio of mitochondrial depolarization also increased from

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4.4±0.2% (control) to 43.0±2.1% for 1 (40 µM) and to 48.6±1.9% for 2 (120 µM). These results demonstrate that 1 and 2 can impair mitochondrial integrity. Fig. 4

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2.5 ROS detection

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Mitochondria are major sites of cellular ROS production, and mitochondrial dysfunction and elevated intracellular ROS levels are two closely related events in cell death [40]. Furthermore, numerous studies have shown that the loss of mitochondrial function can markedly increase intracellular ROS levels [41]. Considering that complexes 1 and 2 could cause mitochondrial depolarization, their effect on intracellular ROS levels were examined by 2',7'-dichlorodihydrofluorescein dicetate (DCFH-DA) fluorescence assay. Confocal microscopic analysis of 9

ACCEPTED MANUSCRIPT DCFH-DA stained Ir(III) treated cells showed a significant concentration-dependent increase in the intensity of DCF staining compared to the vehicle-treated cells (Fig. S13). This result was further confirmed by flow cytometry (Fig. 5). In comparison

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with the control, 1 (40 µM) and 2 (120 µM) treatment resulted in a 30-fold and 35-fold increase, respectively, in DCF mean fluorescence intensity (MFI). These

Fig. 5

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2.6 Caspase-3/7 activation

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results suggest that ROS play a vital role in Ir(III)-induced cell death.

The activation of caspase-3/7 is generally regarded as one of the most obvious characteristics in the apoptosis stage for many cells [42]. To gain insight into the antiproliferative mechanism of these complexes, a caspase-3/7 activity assay was

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carried out. As shown in Fig. S14, complexes 1 and 2 had only a marginal effect on the activation of caspase-3/7 after a 24 h treatment with HeLa cells. The results suggest that the activation of apoptosis by these Ir(III) complexes was

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caspase-independent.

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2.7 Cell-cycle arrest

Inhibition of the cell cycle is a known target for the treatment of cancer [43]. To

gain further insight into the mechanism of the growth inhibitory effects of these complexes, HeLa cells were treated with complexes 1 and 2 at different concentrations for 24 h for cell cycle analysis via propidium iodide (PI) staining and flow cytometry. As shown in Fig. 6 and S15, compared with the vehicle-treated control, cells treated with complexes 1 and 2 showed an obvious increase in the 10

ACCEPTED MANUSCRIPT proportion of cells in the G0/G1 phase, and a concomitant decrease in the percentage of cells in G2/M and S phases. These results reveal that the effect of complexes 1 and 2 on cell cycle progression is concentration-dependent. Following a 24-h iridium

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treatment, 1 (40 µM) and 2 (120 µM), the percentages of cells in G2/M (Control: 8.26 ± 1.17%; 1: 1.13 ± 1.28%; 2: 2.54 ± 0.17%) and S phase decreased (Control: 35.89 ± 3.25%; 1: 12.26 ± 0.73%; 2: 10.32 ± 0.12%), while there was an accumulation of cells

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in the G0/G1 phase (Control: 55.84 ± 2.08%; 1: 86.61 ± 2.02%; 2: 87.13 ± 0.29%)

arrest in HeLa cells.

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(Table S1). These data indicate that 1 and 2 mainly caused G0/G1 phase cell cycle

Fig. 6

2.8 Induction of apoptosis

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The ability of complexes 1 and 2 to induce apoptosis in HeLa cells was monitored using Hoechst 33342 staining and the Annexin V assay. In general, apoptosis is characterized by distinct morphological features including cell shrinkage, plasma

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membrane blebbing, chromatin condensation, DNA fragmentation and apoptotic

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bodies [44]. HeLa cells were treated with different concentrations of Ir(III) complexes for 24 h and then photographed using a confocal microscopy. As shown in Fig. S16, a clear morphological change was observed as the necrotic cells were uniformly stained blue. Compared to the control cells, the proportion of atypical nuclei increased in a concentration-dependent manner. The percentages of cells showing abnormal nuclei are shown in Fig. 7, and Ir(III) (1, 40 µM; 2, 120 µM) treatment increased the

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ACCEPTED MANUSCRIPT percentage of abnormal nuclei (1, 80.12 ± 3.26%; 2, 69.29 ± 2.63%) compared with the vehicle-treated cells (4.47 ± 0.92%). Fig. 7

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2.9 Western blot analysis It is known that Bcl-2 and Bax are two members of the Bcl-2 protein family that regulate the balance between cell proliferation and apoptosis, in apoptotic cells. The

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anti-apoptotic protein Bcl-2 can block the release of cytochrome c from the

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mitochondria, whereas pro-apoptotic protein, Bax induces the release of cytochrome c and other pro-apoptotic factors from the mitochondria [45]. Decreases in Bcl-2 and cytochrome c levels promote apoptotic cell death by directly activating the mitochondrial apoptotic pathway [46]. As a universal inhibitor of cyclin-CDK

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complexes, P21 blocks the entry of cells at the G0/G1-S phase transition checkpoint induces apoptosis [47]. In addition, activated Caspase-3 is known as an “effector caspase” and serves as an important event in the apoptotic pathway. Our data

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demonstrate that the expression level of Bax was progressively increased when the

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HeLa cells were treated with 1 and 2, while the expression level of Bcl-2 was decreased (Fig. 8). This result indicates that the complexes can up-regulate and down-regulate Bax and Bcl-2. Under the same conditions, a concentration-dependent increase in the expression of protein P21 was also observed. We showed that the two complexes enhanced ROS generation. ROS mediated inactivation of CDKs by oxidation and enhanced expression of P21 can cause cell cycle arrest in G1- and S-phases resulting in reduced cellular proliferation. However, no markedly 12

ACCEPTED MANUSCRIPT suppression of Caspase-3 was seen for either complexes, which further prove that the activation of apoptosis by these Ir(III) complexes was caspase-independent. Fig. 8

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3. Conclusions In conclusion, we have synthesized and characterized four Ir(III) complexes containing guanidinium ligands, which demonstrated antitumor activity to HeLa,

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MCF-7, HepG2, CNE-2, and A549 tumor cells. Interestingly, these complexes

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showed higher antiproliferative activities than cisplatin against cisplatin-resistant A549 cells, indicating that these four complexes can overcome cisplatin resistance. These complexes have similar structures and exert anticancer activity through the same mechanisms. They can be effectively taken into HeLa cells and specifically

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target to mitochondria. Mechanism studies show that complexes 1 and 2 induced a series of events associated with mitochondrial damage in HeLa cells including ROS production, cell cycle change, and apoptosis. Western blot analysis further confirmed

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that these Ir(III) complexes prompted the up-regulation of P21 and then caused

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mitochondrial dysfunction by regulating the pro-apoptotic and anti-apoptotic Bcl-2 family members. In general, our study demonstrates that these Ir(III) complexes have high potential to be utilized as mitochondria-targeted anticancer agents.

4. Experimental Section 4.1 Materials Iridium chloride hydrate and NH4PF6 were purchased from Alfa Aesar (USA); bzq, tpy, ppy, thpy, cisplatin, DMSO, MTT, CCCP, JC-1, DCFH-DA, PI, Hoechst 33342 13

ACCEPTED MANUSCRIPT and Annexin V-FITC Apoptosis Detection Kit were purchased from Sigma Aldrich (USA); MTR and LTR were purchased from Life Technologies (USA). All reagents and solvents were purchased commercially and used without further purification

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unless specifically noted. 4.2 Physical measurement

Microanalysis (C, H, N, and S) was carried out using an Elemental Vario EL CHNS

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analyzer (Germany). ESI-MS spectra were recorded on a Thermo Finnigan LCQ

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DECA XP spectrometer (USA). 1H NMR spectra were recorded on a Bruker Avance 400 spectrometer (Germany) with DMSO-d6 as the solvent and SiMe4 as an internal standard at 400 MHz and room temperature. UV-vis spectra were recorded on a Varian Cary 300 spectrophotometer (USA), and emission spectra were recorded on a

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Perkin-Elmer Lambda-850 spectrophotometer and LS55 spectrofluorophotometer (USA). Cell imaging experiments were carried out on Leica SP8 confocal microscope (Wetzlar Leica, Germany). Flow cytometric analysis was performed using a BD

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FACS CantoTM II flow cytometer (Becton Dickinson, USA).

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4.3 Preparation of ligands and complexes Ligand L [33], the cyclometalated Ir(III) chloro-bridged dimers [Ir(bzq)2Cl]2 [48],

[Ir(tpy)2Cl]2 [49], [Ir(ppy)2Cl]2 [50], and [Ir(thpy)2Cl]2 [37] were prepared according to literature methods.

4.3.1 [Ir(bzq)2(L)](PF6)2 (1) After the addition of [Ir(bzq)2Cl]2 (0.125 mmol, 1 equiv) and ligand L (0.25 mmol, 2 equiv) to 45 mL CH2Cl2/CH3OH (2:1, v/v), the mixture was refluxed for 4 h under 14

ACCEPTED MANUSCRIPT the protection of argon in the dark. Upon cooling, a 6-fold excess of NH4PF6 was added, and the mixture was stirred for another 2 h, then evaporated to dryness under reduced pressure. The crude product was dissolved in CH2Cl2 and purified by column

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chromatography on silica gel eluted with CH2Cl2/CH3OH/NH3·H2O (5:1:0.05, v/v/v). The desired product was further recrystallized from CH2Cl2/diethyl ether. Yield: 0.21 g, 68.4%. Anal. Calcd for C46H33F12N9P2Ir·2H2O (1229.99): C, 44.92; H, 3.03; N,

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10.25. Found: C, 44.88; H, 2.91; N, 10.53. ESI-MS: m/z = 902.20 [M-PF6-]+, 451.40

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[M-2PF6-]2+. 1H NMR (400 MHz, DMSO-d6): δ 15.23 (s, 1H), 10.13 (s, 1H), 9.60 (d, J = 8.3 Hz, 1H), 9.18 (d, J = 8.1 Hz, 1H), 8.53 (d, J = 8.0 Hz, 2H), 8.42 (d, J = 8.0 Hz, 1H), 8.35 (s, 1H), 8.15 (dd, J = 14.9, 4.5 Hz, 2H), 8.01 (d, J = 7.4 Hz, 6H), 7.90 (d, J = 8.7 Hz, 2H), 7.71 (t, J = 7.8 Hz, 2H), 7.66 – 7.57 (m, 6H), 7.45 (d, J = 6.9 Hz, 3H),

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7.24 (t, J = 7.5 Hz, 2H), 6.33 (d, J = 7.0 Hz, 2H). 4.3.2 [Ir(tpy)2(L)](PF6)2 (2)

This complex was synthesized in a manner identical to that described for complex 1,

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except [Ir(tpy)2Cl]2 (0.125 mmol, 1 equiv) was used instead of [Ir(bzq)2Cl]2. Yield:

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0.22 g, 72.7%. Anal. Calcd for C44H37F12N9P2Ir·2H2O (1210.00): C, 43.68; H, 3.42; N, 10.42. Found: C, 43.98; H, 3.31; N, 10.53. ESI-MS: m/z = 882.25 [M-PF6-]+, 441.80 [M-2PF6-]2+. 1H NMR (400 MHz, DMSO-d6): δ 14.59 (s, 1H), 9.89 (s, 1H), 9.20 (s, 2H), 8.30 – 8.14 (m, 7H), 8.10 (d, J = 6.9 Hz, 2H), 7.85 (t, J = 7.8 Hz, 4H), 7.72 (t, J = 7.7 Hz, 1H), 7.55 (s, 4H), 7.46 (t, J = 7.5 Hz, 3H), 6.97 – 6.89 (m, 4H), 6.10 (s, 2H), 2.11 (s, 6H). 4.3.3 [Ir(ppy)2(L)](PF6)2 (3) 15

ACCEPTED MANUSCRIPT This complex was synthesized in a manner identical to that described for complex 1, except [Ir(ppy)2Cl]2 (0.125 mmol, 1 equiv) was used instead of [Ir(bzq)2Cl]2. Yield: 0.20 g, 69.9%. Anal. Calcd for C42H33F12N9P2Ir (1145.92): C, 44.02; H, 2.90; N, 11.00.

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Found: C, 44.14; H, 3.11; N, 10.53. ESI-MS: m/z = 854.20 [M-PF6-]+, 427.25 [M-2PF6-]2+. 1H NMR (400 MHz, DMSO-d6): δ14.91 (s, 1H), 9.98 (s, 1H), 9.45 (s, 1H), 9.21 (s, 1H), 8.35 (d, J = 7.6 Hz, 1H), 8.28 (d, J = 8.1 Hz, 3H), 8.15 (d, J = 26.1

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Hz, 5H), 7.97 (d, J = 7.7 Hz, 2H), 7.89 (t, J = 7.7 Hz, 2H), 7.74 (t, J = 7.9 Hz, 1H),

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7.59 (s, 4H), 7.52 (d, J = 5.6 Hz, 2H), 7.46 (d, J = 8.0 Hz, 1H), 7.08 (t, J = 7.5 Hz, 2H), 7.04 – 6.92 (m, 4H), 6.31 (d, J = 7.5 Hz, 2H). 4.3.4 [Ir(thpy)2(L)](PF6)2 (4)

This complex was synthesized in a manner identical to that described for complex 1,

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except [Ir(thpy)2Cl]2 (0.125 mmol, 1 equiv) was used instead of [Ir(bzq)2Cl]2. Yield: 0.23 g, 76.7%. Anal. Calcd for C38H33F12N9P2S2Ir·2H2O (1198.03): C, 38.13; H, 3.11; N, 10.52; S, 5.35. Found: C, 38.35; H, 3.28; N, 10.36; S, 5.58. ESI-MS: m/z = 865.85

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[M-PF6-]+, 433.20 [M-2PF6-]2+. 1H NMR (400 MHz, DMSO-d6): δ 15.52 (s, 1H),

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10.28 (s, 1H), 9.77 (s, 1H), 9.19 (s, 1H), 8.48 (d, J = 7.7 Hz, 1H), 8.38 (s, 1H), 8.12 (s, 5H), 7.91 – 7.61 (m, 14H), 7.43 (t, J = 6.9 Hz, 4H), 6.81 (dd, J = 9.1, 4.5 Hz, 2H), 6.28 (d, J = 4.5 Hz, 2H).

Acknowledgments

We are grateful to the National Natural Science Foundation of China (21101034), the Science and Technology Plan of Guangdong Province (2016A020217020), the Yong Teachers Training Plan of Guangdong Province (Yq2013086), and the Natural 16

ACCEPTED MANUSCRIPT Science Foundation of Guangdong Medical University (Z2016006).

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ACCEPTED MANUSCRIPT Table 1 IC50 values of tested compounds towards different cell linesa IC50 (µM) Compounds MCF-7

HepG2

CNE-2

A549

A549R

LO2

1

8.96±0.34

13.44±0.53

20.35±0.83

44.99±4.27

13.65±0.63

13.63±2.25

41.67±1.54

2

34.13±3.17

22.87±1.01

58.07±4.97

30.32±0.30

31.08±2.47

18.37±0.96

50.37±3.25

3

35.33±2.52

46.56±0.20

62.77±3.13

44.99±4.27

38.65±2.87

35.36±1.44 107.04±2.66

4

36.42±1.75

39.81±0.44

62.73±2.70

46.67±0.55

32.67±4.34

22.68±1.25

64.31±2.93

Cisplatin

14.19±0.25

12.52±1.03

17.97±0.29

14.71±0.17

12.79±1.13

60.04±3.01

40.00±3.75

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HeLa

IC50 values are drug concentrations necessary for 50% inhibition of cell viability.

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Data are presented as means ± standard deviations obtained in at least three

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independent experiments and trestment period was 48 h.

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Fig. 3. (A) Confocal microscopy images of HeLa cells co-labeled with complexes 1 and 2 (40 µM, 3.5 h ) and MTR (100 nM, 0.5 h). (B) Confocal microscopy images of HeLa cells co-labeled with 1 and 2 (40 µM, 3.5 h ) and LTR (100 nM, 0.5 h). The

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excitation wavelengths were 405 nm for Ir(III) complexes and 552 nm for MTR and

MTR and LTR, respectively.

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LTR. The fluorescence was collected at 570 ± 20 nm for 1 and 2, and 610 ± 20 nm for

Fig. 4. Effects of complexes 1 and 2 on MMP analyzed by JC-1 staining and flowcytometry. HeLa cells were treated with Ir(III) complexes at the indicated concentrations for 6 h. JC-1 was excited at 488 nm and monitored simultaneously at

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530 ± 15 (green) and 590 ± 15 (red).

Fig. 5. Effects of complexes 1 and 2 on ROS generation. HeLa cells incubated with complexes 1 and 2 at 37 °C for 6 h, after which they were labelled with H2DCFDA

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Fig. 6. Effects of complexes 1 and 2 on the distribution of HeLa cells in cell cycle population at different concentrations for 24 h treatment. Fig. 7. Histograms of HeLa cells stained with Annexin V-FITC conjugate after treatment with complexes 1 and 2 at different concentration for 24 h. FITC fluorescence was analyzed by flowcytometry with excitation at 488 nm and emission at 530 ± 20 nm.

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0.6

1 2 3 4

0.5

Abs

0.4 0.3 0.2

0.0 250

300

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450

500

Wavelength/nm 0.8

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1 2 3 4

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1.0 0.8

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0.2

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350 400 450 Wavelength/nm

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300

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550

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Fig.3 (B)

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Fig. 5

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S

G2/M

75

25 0

Control

10 20 40 1 (µM)

40 80 120 2 (µM)

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Fig. 6

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50

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% Cell Number

100

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ACCEPTED MANUSCRIPT Four Ir(III) complexes containing guanidinium ligands were synthesized. Four complexes displayed higher antiproliferative activities than cisplatin against cisplatin-resistant A549 cells. Complexes 1 and 2 mainly located mitochondria in HeLa cancer cells.

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Complexes 1 and 2 increased ROS production and induced cell cycle arrest in G0/G1

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phase.