Hypoxia-Targeting Organometallic Ru(II)–Arene Complexes with

Jun 28, 2018 - Synopsis. As hypoxia is an important factor to limit chemotherapeutic efficacy in tumors, we herein report a few of ruthenium(II)−are...
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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

Hypoxia-Targeting Organometallic Ru(II)−Arene Complexes with Enhanced Anticancer Activity in Hypoxic Cancer Cells Jian Zhao,†,‡ Wanchun Li,† Shaohua Gou,*,†,‡ Shuang Li,† Shengqiu Lin,† Qianhui Wei,† and Gang Xu*,†,‡ †

Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China

‡

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S Supporting Information *

ABSTRACT: As hypoxia is an important factor to limit chemotherapeutic efficacy in tumors, we herein report three ruthenium(II)−arene complexes containing a hypoxia inducible factor-1α inhibitor (YC-1), which endow the organometallic complexes with potential for hypoxia targeting. In vitro tests showed the resulting complexes had higher anticancer activities in hypoxia than in normoxia against the tested cancer cell lines. Western blot analysis revealed that complexes 1−3 blocked HIF1α protein accumulation under hypoxic conditions. Moreover, these complexes displayed much less cytotoxicity toward the normal human umbilical vein endothelial cell line (HUVEC), indicating that complexes 1−3 may be selectively cytotoxic for human cancer cell lines. These findings proved that ligation with YC-1 endowed these organometallic ruthenium(II) complexes with potential for hypoxia targeting in addition to enhancing their anticancer activities.

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INTRODUCTION The therapeutic values of metal-based agents have gained increasing interest since the discovery of cisplatin in 1965.1 Platinum(II) drugs, particularly cisplatin, carboplatin, and oxaliplatin, are now widely used in clinical practice,2−4 which exhibit their activities by covalently binding to DNA and forming stable DNA adducts with guanines and adenine bases.5,6 However, their irreversible binding to DNA and the lack of the selectivity have resulted in severe side-effects.7 Therefore, ruthenium complexes with relatively lower toxicity were considered to be an attractive alternative to platinum drugs.8−16 So far two ruthenium(III) compounds, [IndH][trans-Ru(Ind)2Cl4] (KP1019, Ind = indazole) and [ImH][trans-Ru(DMSO)(Im)Cl4] (NAMIA, Im = imidazole), have entered clinical trials,17,18 especially KP1019 and its sodium salt KP1339 (Figure 1), which have successfully finished a phase I clinical trial with promising activity.19,20 Besides, organometallic ruthenium(II)−arene complexes also show great potential for cancer therapy because the coordination ligands in arene−ruthenium(II) complexes can provide more opportunity to modulate their pharmacological properties such as cellular accumulations and kinetic reactivities.21−29 For examples, [(C6H5Ph)Ru(en)Cl][PF6] (RM175, en = ethylenediamine) and [(piPrC6H4Me)Ru(pta)Cl2] (RAPTA-C, pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]-decane) (Figure 1) are currently at an advanced preclinical stage.19,30−32 Hypoxia, caused by the imbalance between oxygen supply and consumption, is an important hallmark of cancer progression.33−36 The reduced oxygen levels in tumor tissues © XXXX American Chemical Society

Figure 1. Representative anticancer ruthenium(II) complexes.

result in the stabilization and accumulation of hypoxia inducible factor-1α (HIF-1α) protein,37,38 and the resultant HIF-1α can dimerize with its β subunit to form a transcription factor that binds to hypoxia response elements (HREs) and activates a number of genes involved in vascular epidermal growth factor (VEGF) and erythropoietin (EPO).39 Thus, HIF-1α plays an essential role in tumorigenesis by regulating the expression of various genes associated with tumor metabolism, angiogenesis, metastasis, proliferation, and differReceived: April 18, 2018

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DOI: 10.1021/acs.inorgchem.8b01070 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry Scheme 1. Preparation of Ligand L1 and Complexes 1−3a

a

Reagents and conditions: (a) KOH, DMF, I2, rt, 4 h; (b) benzyl bromide, t-BuOK, THF, rt, 4 h; (c) (5-formylfuran-2-yl)boronic acid, Pd(PPh3)4, Na2CO3, DMF, 90 °C, 12 h; (d) NaBH4, MeOH, rt, 2 h; (e) succinic anhydride, Et3N, DMF, 4 h; (f) 5-amino-1,10-phenanthroline, HATU, Et3N, DMF, 60 °C, 12 h; (g) [(arene)RuCl2]2, DCM, rt, 4 h.

entiation.39 Overexpression of HIF-1α was detected in more than 90% of colon, lung, and prostate cancers, but no expression was detected in their corresponding normal tissues.40 In addition, HIF-1α overexpression was found to be associated with resistance to treatment and poor patient prognosis in the hypoxic region around solid tumors.38 Consequently, HIF-1α becomes a compelling drug target for the treatment of tumor angiogenesis and cancer.41−44 1-Benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole (YC-1) has been reported as an effective HIF-1α inhibitor by stimulation of factor inhibiting HIF (FIH)-dependent p300 dissociation from HIF-1α,45−47 which can enhance chemosensitivity of chemotherapy drugs like cisplatin and sorafenib.48,49 Therefore, we designed and prepared three organometallic ruthenium(II) compounds bearing a YC-1 moiety, which are expected to target the hypoxic tumor microenvironment and exhibit their anticancer activity through multiple mechanisms so as to improve the therapeutic efficacy of chemotherapeutic drugs.

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et al.54 Thus, the hydrolysis behavior of complexes 1−3 was studied using UV−vis spectroscopy. It was observed that the hydrolysis was accompanied by a gradual increase of the absorption band around 275 nm, which was chosen for kinetic calculation (Figure 2). The experimental data fitted well to a monoexponential function, and the hydrolysis rate constants (k) and half-times (t1/2) of complexes 1−3 were shown in Table 1. Obviously, the hydrolysis rate of complex 3 is the fastest (t1/2 = 13.8 min) as compared with complexes 1 and 2. The relative order of the hydrolysis rates of these complexes is 3 > 1 > 2, which is due to the fact that increased electron density at Ru(II) from the aromatic ligand with more alkyl groups can improve the Cl− lability and facilitate the hydrolysis reaction.55,56 The study indicated that the arene groups play an important role in controlling the hydrolysis rates of the arene− Ru(II) complexes, which may further affect their biological activities. In Vitro Cytotoxicity. The cytotoxic activities of complexes 1−3 and ligand L1 have been investigated against two human cancer cell lines (HCT-116 and A549 cancer cells) and human umbilical vein endothelial cell line (HUVEC) under normoxic or hypoxic conditions together with cisplatin as a positive agent. Each complex was measured at eight different concentrations against the tested cell lines, and the corresponding IC50 values (dose required to inhibit 50% cellular growth) were determined from dose−survival curves (Figure S12). According to the IC50 values (Table 2), ligand L1 exhibited little cytotoxicity under normoxia, while complexes 1−3 showed dose-dependent inhibition against the tested cell lines with IC50 values ranging from 22.8 to 76.3 μM. Notably, complex 3 (22.8 ± 1.2 μM) showed comparable cytotoxicity to that of cisplatin (19.9 ± 0.9 μM) in HCT-116 cells, which may partly attribute to its increased hydrolysis rate. Moreover, all complexes exhibited markedly improved cytotoxic activity

RESULTS AND DISCUSSION

Synthesis and Characterization. Complexes 1−3 were prepared by following the procedure shown in Scheme 1 in which YC-1 was obtained as described previously.50 The resulting arene−ruthenium(II) complexes were characterized by elemental analysis and 1H and 13C NMR spectra along with ESI−MS spectrometry (Figures S1−S11). All the spectral data were compatible with the proposed molecular structures of complexes 1−3. Hydrolysis Studies. Hydrolysis of the organometallic Ru(II)−arene complexes with the type of [(6η-arene)Ru(NN)Cl]+ (NN = chelating diamine ligand) is believed as a key activation step before they bind to the biomolecules,51−53 and a relationship between the aquation rate for arene-−Ru(II) complexes and cytotoxicity was proposed by Wang and Sadler B

DOI: 10.1021/acs.inorgchem.8b01070 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Figure 2. Time-dependent UV−vis spectra for the aquation of 0.05 mM complexes 1 (a), 2 (c), and 3 (e) (95% H2O/5% MeOH). Absorbance− time trace (275 nm) and monoexponential fit obtained for the hydrolysis of complexes 1 (b), 2 (d), and 3 (f) at 310 K.

under hypoxia against HCT-116 and A549 cells with HF (hypoxia factor: the toxicity under normoxia vs hypoxia) values range from 1.8 to 3.8, indicating that YC-1 can enhance the antiproliferative activity of the Ru(II)−arene complexes against hypoxic cancer cells. Additionally, significant morphological changes to the HCT-116 cells were observed when they were treated with complex 3 at different concentrations (12.5, 50, and 100 μM) under hypoxia (Figure S13). In contrast,

Table 1. Hydrolysis Rate Constants (k) and Half-Times (t1/2) of Complexes 1−3 (95% H2O/5% MeOH) at 310 K complex

1

2

3

−4

5.03 ± 0.23 23.0

3.89 ± 0.19 29.7

8.39 ± 0.29 13.8

−1

k (10 s ) t1/2 (min)

Table 2. Cytotoxicity of the Compounds against HCT-116 and A549 Cancer Cells under Normoxic and Hypoxic Conditions IC50 values (μM) HCT-116 compound 1 2 3 L1 cisplatin

normoxia 65.7 57.5 22.8 112.9 19.9

± ± ± ± ±

0.9** 2.5** 1.2* 8.6** 0.9

a

A549b d

hypoxia

HF

± ± ± ± ±

3.8 3.2 1.8 2.1 1.0

17.5 17.9 12.7 53.7 19.0

1.4 0.8 0.6** 4.5** 1.3

normoxia 76.3 65.5 36.3 147.1 20.9

± ± ± ± ±

3.2** 4.8** 1.6** 8.7** 1.5

HUVECc d

hypoxia

HF

± ± ± ± ±

2.4 2.7 2.3 2.3 1.0

31.7 24.7 15.7 63.8 21.7

2.5** 0.9* 0.6** 2.3** 1.3

normoxia 129.1 92.6 153.2 128.9 16.6

± ± ± ± ±

10.6** 5.7** 9.8** 11.9** 1.5

a

Human colorectal cancer cell line. bHuman nonsmall-cell lung cancer cell line. cHuman umbilical vein endothelial cell line. Data are expressed as the mean (±SD) for three independent experiments. dHF (hypoxia factor): the toxicity under normoxia vs hypoxia. *p < 0.05 and **p < 0.01 compared with the value of cisplatin. C

DOI: 10.1021/acs.inorgchem.8b01070 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry cisplatin showed similar cytotoxicity under both normoxic and hypoxic conditions against the tested cell lines. Notably under hypoxia, the IC50 values of complexes 1−3 were lower than that of cisplatin against HCT-116 cells, implying that these complexes were more active than cisplatin in hypoxic HCT116 cells. Overall, complexes 1−3 showed selective and markedly improved cytotoxic activity against hypoxic cancer cells, proving that the introduction of YC-1 to the Ru(II)− arene complexes is an effective way to enhance anticancer activity in hypoxic cancer cells. Cellular Accumulation. The intracellular ruthenium accumulation of complexes 1−3 in HCT-116 cells was studied to investigate the possible relationship between cellular accumulation and cytotoxicity. As shown in Figure 3 (Table

Figure 4. (a) HCT-116 cells treated with complexes 1−3 under hypoxia for 12 h were examined for the expression of HIF-1α proteins using Western blot analysis. (b) Densitometric analysis of the expression of apoptosis-regulated proteins normalized with GAPDH. The relative expression of each protein was represented by the density of the protein band/density of GAPDH band. The data are representative of three independent experiments. *p < 0.05 and **p < 0.01 compared with the value of control.

staining and flow cytometry assay, with cisplatin as positive control. As demonstrated in Figures 5 and 6, HCT-116 cells exhibited numerous apoptotic cells with condensed or Figure 3. Intracellular accumulation of complexes 1−3 in HCT-116 cells after 24 h of incubation under normoxic and hypoxic conditions. Data are expressed as the mean (±SD) for three independent experiments. **p < 0.01.

S1), the relative order of the cellular accumulation of complexes 1−3 in normoxia is 3 > 1 > 2, which is in accordance with the hydrophobicity of the arene ligands (hexamethylbenzene > cymene > benzene). Thus, the higher accumulation of complex 3 may be another reason for its superior cytotoxicity to complexes 1 and 2. While under hypoxia, the ruthenium contents of complexes 1−3 in HCT116 cells dramatically increased, and all these complexes had much higher Ru amount than they did under normoxia, indicating their ability to target hypoxic cancer cells. The results showed that the introduction of YC-1 can promote the cellular accumulation of the Ru(II)−arene complexes in hypoxic cancer cells and lead to more potent anticancer activity for these compounds. Western Blot. In order to determine the effect of these complexes on the expression of HIF-1α under hypoxic conditions, Western blot analysis was applied to detect the HIF-1α protein expression in HCT-116 cells. As shown in Figure 4, the expression of HIF-1α decreased after treatment with complexes 1−3 and L1 that all blocked HIF-1α protein accumulation in a dose-dependent manner. This study implied that our metal complexes could effectively inhibit HIF-1α protein accumulation under hypoxic conditions in HCT-116 cells. Apoptosis Studies. Apoptotic analysis of complexes 1−3 and L1 against HCT-116 cells was performed under both normoxic and hypoxic conditions by the Hoechst 33342 DNA

Figure 5. Cell morphological observation for cell apoptosis induction on the HCT-116 cells treated with complexes 1−3, L1, and cisplatin at 30 μM for 48 h, respectively: (a) normoxia and (b) hypoxia. Cells were stained by Hoechst 33342 (size bar = 20 μm). D

DOI: 10.1021/acs.inorgchem.8b01070 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

complexes containing a hypoxia inducible factor-1α inhibitor (YC-1) were first designed and synthesized. In vitro tests showed the resulting complexes had higher anticancer activities in hypoxia than in normoxia against the tested cancer cell lines, especially complex 3, with more alkyl groups than complexes 1 and 2, exhibiting superior cytotoxicity to cisplatin under a hypoxic condition. Significantly, these complexes are much less cytotoxic than cisplatin against human umbilical vein endothelial cells (HUVEC), suggesting that they have selectivity for cancer cells over normal cells. Both kinetic study on the hydrolysis and cellular accumulation tests indicated that the enhancing cytotoxicity of these complexes under either normoxia or hypoxia matched their hydrolysis rates and cellular accumulations in HCT-116 cancer cells positively. Moreover, complex 3 was the most active to induce apoptosis among the tested compounds under hypoxia, particularly in early stage apoptosis. Western blot analysis revealed that complexes 1−3 as well as the ligand containing a YC-1 moiety blocked HIF-1α protein accumulation in a dosedependent manner. These findings proved that ligation with YC-1 endowed these organometallic ruthenium(II) complexes with potential for hypoxia targeting in addition to enhancing their anticancer activities. Consequently, our study provides a useful way for further development of ruthenium(II) anticancer agents with selective identification of cancer cells and enhanced antitumor activity under hypoxia.

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EXPERIMENTAL SECTION

Materials and Measurements. All chemicals and solvents were of analytical reagent grade and used without further purification. [Ru(η6-arene)Cl2]2 (arene = cymene, benzene, and hexamethylbenzene) and YC-2 were prepared according to previous reports.57,34 1H and 13C NMR spectra were measured on Bruker Avance III-HD 600 MHz spectrometer. Mass spectra were recorded an Agilent Q-TOF 6540 spectrometer. Elemental analysis of C, H, and N used a Vario MICRO CHNOS elemental analyzer (Elementar). Hydrolysis study was performed on a Shimadzu UV2600 instrument equipped with a thermostatically controlled cell holder. Cancer cells were obtained from Jiangsu KeyGEN BioTECH company (China). Cell apoptosis experiments were measured by flow cytometry (FAC Scan, Becton Dickenson) and analyzed by Cell Quest software. Synthesis of YC-2. To a solution of YC-1 (0.84 g, 2.76 mM) dissolved in DMF (50 mL) were added succinic anhydride (0.55 g, 5.54 mM) and TEA (0.56g, 5.53 mM). The reaction mixture was refluxed for 4 h, and then DMF was removed under reduced pressure. The product was purified by column chromatography (silica gel, methanol/dichloromethane 1:9) to give YC-2. Yield: 1.04 g (93.3%). White solid. Anal. Calcd (%) for C23H20N2O5: C 68.31, H 4.98, N 6.93. Found: C 68.20, H 4.91, N 6.79; 1H NMR (600 MHz, DMSO) δ 2.50−2.52 (m, 2H), 2.57−2.59 (m, 2H), 5.19 (s, 2H), 5.73 (s, 2H), 6.71−6.72 (d, 1H, J = 3.4 Hz), 7.02−7.03 (d, 1H, J = 3.4 Hz), 7.24− 7.28 (m, 4H), 7.30−7.33 (m, 2H), 7.45−7.47 (m, 1H), 7.76−7.77 (d, 1H, J = 8.5 Hz), 8.10−8.12 (d, 1H, J = 8.2 Hz), 12.24 (s, 1H) ppm. Synthesis of L1. YC-2 (0.50 g, 1.24 mM), HATU (0.51 g, 1.34 mM), TEA (0.14g, 1.40 mM), and DMF (7.5 mL) were added into a 25 mL round-bottom flask. The mixture was stirred at room temperature for 2 h. Then 5-amino-1,10-phenanthroline (0.20 g, 1.02 mM) was added, and the resulting mixture was stirred at 60 °C for 12 h. The solvent was then removed by evaporation under reduced pressure. Column chromatography (eluent 30:1 DCM/methanol) gave L1. Yield: 0.32 g (54.0%). Light yellow solid. Anal. Calcd (%) for C35H27N5O4: C 72.28, H 4.68, N 12.04. Found: C 72.20, H 4.81, N 11.96; 1H NMR (600 MHz, DMSO) δ 2.77−2.79 (m, 2H), 2.88− 2.90 (m, 2H), 5.24 (s, 2H), 5.70 (s, 2H), 6.74−6.75 (d, 1H, J = 3.4 Hz), 7.01−7.02 (d, 1H, J = 3.3 Hz), 7.18−7.26 (m, 4H), 7.29−7.31 (m, 2H), 7.39−7.42 (m, 1H), 7.72−7.74 (d, 1H, J = 8.2 Hz), 7.75− 7.77 (q, 1H, J = 4.4 Hz), 7.84−7.86 (q, 1H, J = 4.4 Hz), 8.08−8.09

Figure 6. Flow cytometry analysis for apoptosis of HCT-116 cells induced by complexes 1−3, L1, and cisplatin at the concentration of 30 μM for 48 h: (a) normoxia and (b) hypoxia.

fragmented nuclei cells, while nuclei of the control cells retained the regular round contours, exhibiting that the tested compounds induced more apoptotic cells than the control group. Moreover, under a hypoxic condition, cells treated with complex 3 were more condensed and brighter than those in other groups (Figure 5), suggesting the significant cell apoptosis induction of complex 3 on HCT-116 cells. The results were further verified by flow cytometry assay. Under normoxia, the apoptotic rates of HCT-116 cells treated with complexes 1−3 increased as compared with that of the untreated cells (Figure 6). The apoptotic rate of complex 3 (44.88%) was compatible to that of cisplatin (46.87%), and the relative order of inducing apoptosis against HCT-116 cells is cisplatin (46.87%) ≈ 3 (44.88%) > 1 (35.87%) ≈ 2 (32.54%) > L1 (19.69%), which is in line with the result of the cytotoxicity assay to some extent. Once hypoxia was conducted, both the complexes and their ligand L1 induced an improved incidence of early to late stage apoptosis in HCT116 cells compared with that in normoxia. Notably, complex 3 had a higher apoptotic rate than cisplatin, proving that it had advantages over cisplatin to induce cell apoptosis under a hypoxic condition. This further verified the considerable antiproliferative effect of complex 3 in hypoxic HCT-116 cells.

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CONCLUSION In view of hypoxia as an important factor to limit chemotherapeutic efficacy in clinical practice, we purposed to develop organometallic ruthenium(II)−arene complexes targeting the hypoxic tumor microenvironment so as to enhance their anticancer activity. In this study, three ruthenium(II)−arene E

DOI: 10.1021/acs.inorgchem.8b01070 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

MTT Assay. Cytotoxicity of complexes 1−3, ligand L1, and cisplatin against HCT-116, A549, and HUVEC cells was determined by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Cells (5000−10000 per well) with better vitality were seeded in 96-well plates. The compounds were dissolved by DMF (water for cisplatin) and diluted with medium to various concentrations (the final concentration of DMF was less than 0.4%). After being incubated under a normoxic condition (20% O2, 5% CO2, and 75% N2) or hypoxic condition (1% O2, 5% CO2, and 94% N2) at 37 °C for 72 h, cells were stained with MTT (5 mg/mL) for another 5 h, and then the medium was thrown away and replaced by 150 mL of DMSO. The inhibition of cell growth induced by the tested complexes was detected by measuring the absorbance of each well at 570/630 nm using enzyme labeling instrument. The IC50 values were calculated by SPSS software after three parallel experiments. Cellular Accumulation. HCT-116 cells were seeded in six-well plates at a density of 106 cells/well. After overnight incubation, 50 μM complexes 1−3 were added, respectively. After incubation under a normoxic condition (20% O2, 5% CO2, and 75% N2) or hypoxic condition (1% O2, 5% CO2, and 94% N2) at 37 °C for 24 h, cells were collected and washed three times with ice-cold PBS, then centrifuged at 1000 rpm for 10 min and resuspended in 1 mL of PBS. A volume of 100 μL was taken out to determine the cell density. Then the remaining cells were digested by HNO3 (200 μL, 65%) at 65 °C for 4 h. The Ru level in cells was measured by ICP−MS after three parallel experiments. Apoptosis Assessment by Hoechst 33342 Staining. HCT-116 cells were seeded in 24-well plates at 1 × 105 cells/well and incubated overnight. Cells were incubated with 30 μM of the tested compounds under a normoxic condition (20% O2, 5% CO2, and 75% N2) or hypoxic condition (1% O2, 5% CO2, and 94% N2) at 37 °C for 48 h. Then the cells were rinsed twice in PBS and stained with Hoechst 33342 fluorescent dye for 10 min in the dark at 37 °C. Cell apoptosis was examined under the fluorescence microscope with excitation wavelength of 330−380 nm, and data were collected from three independent experiments. Apoptosis Analysis by Flow Cytometry. HCT-116 cells were grown in a six-well plate at a density of 2 × 105 cells/well and cultured overnight. The tested complexes were added, which were diluted to a concentration of 30 μM. After incubation under a normoxic condition (20% O2, 5% CO2, and 75% N2) or hypoxic condition (1% O2, 5% CO2, and 94% N2) for 48 h, cells were collected by centrifugation (5 min, 25 °C, 2000 rpm). Then, the cells were washed twice with cold water and resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). The cells were stained with 5 μL of Annexin V- FITC and then with 5 μL of propidium iodide (20 μg/ mL) for 15 min in the dark at room temperature. The fluorescence of cells was detected by an annexin V-FITC apoptosis detection kit (Roche) according to the manufacturer’s protocol, and cells were quantified by system software (Cell Quest; BD Biosciences). Western Blot. HCT-116 cells were grown in a six-well plate at a density of 2 × 105 cells/well and cultured until the cell density reached 80%. The solutions of complexes 1−3 and L1 were diluted down in media to give the required concentration (0.1 , 1 , or 5 μM) for addition to the cells, and the cells were cultured under a normoxic condition (20% O2, 5% CO2, and 75% N2) or hypoxic condition (1% O2, 5% CO2, and 94% N2) for 12 h at 37 °C. HCT-116 cells were lysed in cell lysis buffer and collected by centrifugation at 13 000 rpm for 20 min at 4 °C. Proteins from cell lysates were separated by 8− 12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidine difluoride (PVDF) membrane (Amersham Biosciences). The membrane was blocked with PBST containing 5% nonfat dry milk for 1 h and further incubated with monoclonal antihuman HIF-1α antibody (Santa Cruz Biotechnology, USA) overnight at 4 °C under gentle shaking. After that, the membrane was incubated with the secondary antibody (1:2000) for 1 h at RT (25 °C). Protein blots were detected with chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). GAPDH was used as loading control.

(d, 1H, J = 8.2 Hz), 8.18 (s, 1H), 8.45−8.47 (1H, d-d, J = 8.1, 1.4 Hz), 8.69−8.70 (d, 1H, J = 7.4 Hz), 9.01 (m, 1H), 9.03−9.04 (1H, dd, J = 4.3, 1.6 Hz), 9.12−9.13 (1H, d-d, J = 4.2, 1.5 Hz), 10.27 (m, 1H) ppm. General Procedure for Synthesis of Complexes 1−3. A solution of L1 (0.29 g, 0.50 mM) in CH2Cl2 (10 mL) was added to a suspension of [Ru(arene)Cl2]2 (0.21 mM) in CH2Cl2 (15 mL) dropwise. The mixture was stirred at room temperature for 8 h. Then the reaction mixture was concentrated to 3 mL. The crude product was separated, washed with Et2O (3 × 10 mL) and cold methanol (3 × 10 mL), and dried in a vacuum-dryer. Complex 1. Yield: 0.23 g (61.3%). Yellow-green powder. Anal. Calcd (%) for C45H41Cl2N5O4Ru: C 60.88, H 4.65, N 7.89. Found: C 60.73, H 4.74, N 7.66. ESI−MS: m/z [M − Cl]+ = 852.2. 1H NMR (600 MHz, DMSO) δ 0.89−0.91 (t, 6H, J = 6.6 Hz), 2.17 (s, 3H), 2.59−2.64 (m, 1H), 2.76−2.78 (m, 2H), 2.95−2.97 (m, 2H), 5.23 (s, 2H), 5.71 (s, 2H), 6.12−6.15 (m, 2H), 6.36−6.38 (t, 2H,, J = 6.4 Hz), 6.73−6.74 (d, 1H, J = 3.4 Hz), 7.02−7.03 (d, 1H, J = 3.4 Hz), 7.19−7.26 (m, 4H), 7.30−7.32 (m, 2H), 7.39−7.42 (m, 1H), 7.73− 7.75 (d, 1H, J = 8.5 Hz), 8.06−8.11 (m, 2H), 8.18−8.21 (m, 1H), 8.48 (s, 1H), 8.79−8.80 (d, 1H, J = 8.0 Hz), 9.27−9.28 (d, 1H, J = 8.0 Hz), 9.89 (m, 1H), 10.02 (m, 1H), 11.12 (m, 1H) ppm; 13C NMR (150 MHz, DMSO-d6) δ 18.69, 22.12, 22.14, 29.17, 30.86, 31.23, 52.44, 58.31, 84.23, 84.39, 86.42, 86.57, 103.23, 104.48, 108.41, 110.75, 113.28, 120.73, 121.50, 122.14, 126.05, 126.45, 126.85, 127.37, 127.73, 128.05, 129.08, 130.12, 133.95, 135.48, 135.65, 137.76, 138.50, 140.77, 143.18, 145.87, 149.06, 149.59, 155.35, 156.60, 172.02, 172.61 ppm. Complex 2. Yield: 0.21 g (59.6%). Yellow-green powder. Anal. Calcd (%) for C41H33Cl2N5O4Ru: C 59.21, H 4.00, N 8.42. Found: C 59.03, H 4.14, N 8.20. ESI−MS: m/z [M − Cl]+ = 796.2. 1H NMR (600 MHz, DMSO) δ 2.82−2.84 (m, 2H), 3.04 (m, 2H), 5.28 (s, 2H), 5.76 (s, 2H), 6.40 (s, 6H), 6.79−6.80 (d, 1H, J = 3.1 Hz), 7.06− 7.07 (d, 1H, J = 3.1 Hz), 7.22−7.25 (d, 1H, J = 7.5 Hz), 7.29−7.38 (m, 5H), 7.44−7.46 (m, 1H), 7.78−7.80 (d, 1H, J = 8.5 Hz), 8.11 (m, 1H), 8.13−8.15 (d, 1H, J = 8.2 Hz), 8.23 (m, 1H), 8.50 (s, 1H), 8.82−8.83 (d, 1H, J = 8.0 Hz), 9.30−9.31 (d, 1H, J = 8.2 Hz), 10.03 (m, 1H), 10.17 (m, 1H), 11.18 (m, 1H) ppm; 13C NMR (150 MHz, DMSO-d6) δ 29.19, 31.32, 52.41, 58.29, 87.11, 108.38. 110.71, 113.29, 120.69, 121.49, 122.11, 125.85, 126.56, 126.65, 127.35, 127.73, 128.04, 129.07, 130.12, 133.92, 135.46, 135.77, 137.76, 138.46, 140.73, 143.33, 146.02, 149.06, 149.57, 155.56, 156.82, 172.03, 172.61 ppm. Complex 3. Yield: 0.25 g (65.2%). Yellow-green powder. Anal. Calcd (%) for C47H45Cl2N5O4Ru: C 61.64, H 4.95, N 7.65. Found: C 61.38, H 5.11, N 7.53. ESI−MS: m/z [M − Cl]+ = 880.3. 1H NMR (600 MHz, DMSO) δ 2.10 (s, 18H), 2.75−2.77 (m, 2H), 2.98−3.00 (m, 2H), 5.21 (s, 2H), 5.69 (s, 2H), 6.72−6.73 (d, 1H, J = 2.9 Hz), 7.00−7.01 (d, 1H, J = 2.9 Hz), 7.18−7.25 (m, 4H), 7.28−7.31 (m, 2H), 7.38−7.40 (m, 1H), 7.72−7.73 (d, 1H, J = 8.4 Hz), 8.08−8.09 (m, 2H), 8.16 (m, 1H), 8.45 (s, 1H), 8.73−8.75 (d, 1H, J = 7.8 Hz), 9.24−9.27 (m, 2H), 9.37−9.38 (m, 1H), 11.16 (m, 1H) ppm; 13C NMR (150 MHz, DMSO-d6) δ 15.75, 29.19, 31.23, 52.42, 58.29, 95.81, 108.41. 110.75, 113.28, 120.71, 121.50, 122.14, 126.24, 126.27, 127.06, 127.36, 127.73, 128.05, 129.07, 129.87, 134.05, 135.36, 135.46, 137.75, 138.05, 140.74, 143.23, 145.92, 149.04, 149.58, 153.05, 154.24, 172.06, 172.62 ppm. Hydrolysis Studies. Hydrolysis of complexes 1−3 was recorded on a Shimadzu UV2600 instrument equipped with a thermostatically controlled cell holder. The UV−vis spectra were recorded by scanning from 185 to 600 nm every 5 min at 37 °C, and 275 nm was selected for the kinetic study. The time-dependent absorbance was fitted using Origin 9.0 to give the first order rate constant k. Cell Culture. HCT-116 (human colorectal cancer cell line) and A549 (human nonsmall cell lung cancer cell line) were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS). All media were also supplemented with 100 mg/mL of penicillin and 100 mg/ mL of streptomycin. F

DOI: 10.1021/acs.inorgchem.8b01070 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry Statistical Analysis. Differences among samples were calculated with the two-tailed Student’s t-test using an independent samples ttest in SPSS 16.0. Differences among groups were considered statistically significant at P < 0.05.

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single molecule compounds to nanomaterials. Chem. Soc. Rev. 2017, 46, 5771−5804. (12) Hartinger, C. G.; Zorbas-Seifried, S.; Jakupec, M. A.; Kynast, B.; Zorbas, H.; Keppler, B. K. From bench to bedside-preclinical and early clinical development of the anticancer agent indazolium trans[tetrachlorobis (1H-indazole) ruthenate (III)](KP1019 or FFC14A). J. Inorg. Biochem. 2006, 100, 891−904. (13) Han Ang, W.; Dyson, P. J. Classical and non-classical ruthenium-based anticancer drugs: Towards targeted chemotherapy. Eur. J. Inorg. Chem. 2006, 20, 4003−4018. (14) Zhao, J.; Zhang, D.; Hua, W.; Li, W.; Xu, G.; Gou, S. Anticancer Activity of Bifunctional Organometallic Ru (II) Arene Complexes Containing a 7-Hydroxycoumarin Group. Organometallics 2018, 37, 441−447. (15) Tian, M.; Li, J.; Zhang, S.; Guo, L.; He, X.; Kong, D.; Zhang, H.; Liu, Z. Half-sandwich ruthenium (ii) complexes containing N̂ Nchelated imino-pyridyl ligands that are selectively toxic to cancer cells. Chem. Commun. 2017, 53, 12810−12813. (16) Li, J.; Tian, M.; Tian, Z.; Zhang, S.; Yan, C.; Shao, C.; Liu, Z. Half-Sandwich Iridium (III) and Ruthenium (II) Complexes Containing P̂ P-Chelating Ligands: A New Class of Potent Anticancer Agents with Unusual Redox Features. Inorg. Chem. 2018, 57, 1705− 1716. (17) Hartinger, C. G.; Jakupec, M. A.; Zorbas-Seifried, S.; Groessl, M.; Egger, A.; Berger, W.; Zorbas, H.; Dyson, P. J.; Keppler, B. K. KP1019, a new redox-active anticancer agent-preclinical development and results of a clinical phase I study in tumor patients. Chem. Biodiversity 2008, 5, 2140−2155. (18) Bergamo, A.; Sava, G. Ruthenium anticancer compounds: myths and realities of the emerging metal-based drugs. Dalton Trans. 2011, 40, 7817−7823. (19) Riedl, C. A.; Flocke, L. S.; Hejl, M.; Roller, A.; Klose, M. H.; Jakupec, M. A.; Kandioller, W.; Keppler, B. K. Introducing the 4Phenyl-1, 2, 3-Triazole Moiety as a Versatile Scaffold for the Development of Cytotoxic Ruthenium (II) and Osmium (II) Arene Cyclometalates. Inorg. Chem. 2017, 56, 528−541. (20) Trondl, R.; Heffeter, P.; Kowol, C. R.; Jakupec, M. A.; Berger, W.; Keppler, B. K. NKP-1339, the first ruthenium-based anticancer drug on the edge to clinical application. Chem. Sci. 2014, 5, 2925− 2932. (21) Guerriero, A.; Oberhauser, W.; Riedel, T.; Peruzzini, M.; Dyson, P. J.; Gonsalvi, L. New Class of Half-Sandwich Ruthenium (II) Arene Complexes Bearing the Water-Soluble CAP Ligand as an in Vitro Anticancer Agent. Inorg. Chem. 2017, 56, 5514−5518. (22) Wang, F.; Chen, H.; Parsons, S.; Oswald, I. D.; Davidson, J. E.; Sadler, P. J. Kinetics of Aquation and Anation of Ruthenium (II) Arene Anticancer Complexes, Acidity and X-ray Structures of Aqua Adducts. Chem. - Eur. J. 2003, 9, 5810−5820. (23) Batchelor, L. K.; Paunescu, E.; Soudani, M.; Scopelliti, R.; Dyson, P. J. Influence of the Linker Length on the Cytotoxicity of Homobinuclear Ruthenium (II) and Gold (I) Complexes. Inorg. Chem. 2017, 56, 9617−9633. (24) Ma, L.; Ma, R.; Wang, Z.; Yiu, S.-M.; Zhu, G. Heterodinuclear Pt (IV)-Ru (II) anticancer prodrugs to combat both drug resistance and tumor metastasis. Chem. Commun. 2016, 52, 10735−10738. (25) Prior, T. J.; Savoie, H.; Boyle, R. W.; Murray, B. S. pHDependent Modulation of Reactivity in Ruthenium (II) Organometallics. Organometallics 2018, 37, 294−297. (26) Caruso, F.; Pettinari, R.; Rossi, M.; Monti, E.; Gariboldi, M. B.; Marchetti, F.; Pettinari, C.; Caruso, A.; Ramani, M. V.; Subbaraju, G. V. The in vitro antitumor activity of arene-ruthenium (II) curcuminoid complexes improves when decreasing curcumin polarity. J. Inorg. Biochem. 2016, 162, 44−51. (27) Ma, L.; Lin, X.; Li, C.; Xu, Z.; Chan, C.-Y.; Tse, M.-K.; Shi, P.; Zhu, G. A cancer cell-selective and low-toxic bifunctional heterodinuclear Pt(IV)-Ru(II) anticancer prodrug. Inorg. Chem. 2018, 57, 2917−2924.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01070. 1 H and 13C NMR and ESI−MS spectra of complexes 1− 3, dose-dependent cell viability curves, and cellular accumulation data (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Shaohua Gou: 0000-0003-0284-5480 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the National Natural Science Foundation of China (Grant No. 21601034) and Jiangsu Province Natural Science Foundation (Grant No. BK20160664) for financial aids to this work. We also thank the Fundamental Research Funds for the Central Universities (Project 2242016K30020 and 2242017K41025) and Priority Academic Program Development of Jiangsu Higher Education Institutions for the construction of fundamental facilities are also appreciated.

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REFERENCES

(1) Rosenberg, B.; Van Camp, L.; Krigas, T. Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode. Nature 1965, 205, 698−699. (2) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Trans. 2010, 39, 8113−8127. (3) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The Next generation of platinum drugs: targeted Pt (II) agents, nanoparticle delivery, and Pt (IV) prodrugs. Chem. Rev. 2016, 116, 3436−3486. (4) Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7, 573−584. (5) Todd, R. C.; Lippard, S. J. Inhibition of transcription by platinum antitumor compounds. Metallomics 2009, 1, 280−291. (6) Zhao, J.; Wang, D.; Xu, G.; Gou, S. Improve the anticancer potency of the platinum (II) complexes through functionalized leaving group. J. Inorg. Biochem. 2017, 175, 20−28. (7) Galanski, M.; Keppler, B. K. Searching for the magic bullet: anticancer platinum drugs which can be accumulated or activated in the tumor tissue. Anti-Cancer Agents Med. Chem. 2007, 7, 55−73. (8) Yan, Y. K.; Melchart, M.; Habtemariam, A.; Sadler, P. J. Organometallic chemistry, biology and medicine: ruthenium arene anticancer complexes. Chem. Commun. 2005, 0, 4764−4776. (9) Meier-Menches, S. M.; Gerner, C.; Berger, W.; Hartinger, C. G.; Keppler, B. K. Structure-activity relationships for ruthenium and osmium anticancer agents-towards clinical development. Chem. Soc. Rev. 2018, 47, 909−928. (10) Alessio, E.; Mestroni, G.; Bergamo, A.; Sava, G. Ruthenium antimetastatic agents. Curr. Top. Med. Chem. 2004, 4, 1525−1535. (11) Zeng, L.; Gupta, P.; Chen, Y.; Wang, E.; Ji, L.; Chao, H.; Chen, Z.-S. The development of anticancer ruthenium (II) complexes: from G

DOI: 10.1021/acs.inorgchem.8b01070 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

transcription 3 (STAT3) in hepatocellular carcinoma. Mol. Cancer 2014, 13, 7. (49) Lau, C. K.; Yang, Z. F.; Lam, S. P.; Lam, C. T.; Ngai, P.; Tam, K. H.; Poon, R. T.-P.; Fan, S.-T. Inhibition of Stat3 activity by YC-1 enhances chemo-sensitivity in hepatocellular carcinoma. Cancer Biol. Ther. 2007, 6, 1900−1907. (50) Xiao, J.; Jin, C.; Liu, Z.; Guo, S.; Zhang, X.; Zhou, X.; Wu, X. The design, synthesis, and biological evaluation of novel YC-1 derivatives as potent anti-hepatic fibrosis agents. Org. Biomol. Chem. 2015, 13, 7257−7264. (51) Zheng, W.; Luo, Q.; Lin, Y.; Zhao, Y.; Wang, X.; Du, Z.; Hao, X.; Yu, Y.; Lv, S.; Ji, L. Complexation with organometallic ruthenium pharmacophores enhances the ability of 4-anilinoquinazolines inducing apoptosis. Chem. Commun. 2013, 49, 10224−10226. (52) Du, J.; Zhang, E.; Zhao, Y.; Zheng, W.; Zhang, Y.; Lin, Y.; Wang, Z.; Luo, Q.; Wu, K.; Wang, F. Discovery of a dual-targeting organometallic ruthenium complex with high activity inducing early stage apoptosis of cancer cells. Metallomics 2015, 7, 1573−1583. (53) Liu, Z.; Habtemariam, A.; Pizarro, A. M.; Fletcher, S. A.; Kisova, A.; Vrana, O.; Salassa, L.; Bruijnincx, P. C. A.; Clarkson, G. J.; Brabec, V.; Sadler, P. J. Organometallic Half-sandwich Iridium Anticancer Complexes. J. Med. Chem. 2011, 54, 3011−3026. (54) Wang, F.; Habtemariam, A.; van der Geer, E. P.; Fernandez, R.; Melchart, M.; Deeth, R. J.; Aird, R.; Guichard, S.; Fabbiani, F. P.; Lozano-Casal, P. Controlling ligand substitution reactions of organometallic complexes: tuning cancer cell cytotoxicity. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 18269−18274. (55) Dougan, S. J.; Melchart, M.; Habtemariam, A.; Parsons, S.; Sadler, P. J. Phenylazo-pyridine and Phenylazo-pyrazole Chlorido Ruthenium(II) Arene Complexes: Arene Loss, Aquation, and Cancer Cell Cytotoxicity. Inorg. Chem. 2006, 45, 10882−10894. (56) Bugarcic, T.; Habtemariam, A.; Deeth, R. J.; Fabbiani, F. P. A.; Parsons, S.; Sadler, P. J. Ruthenium(II) arene anticancer complexes with redox-active diamine ligands. Inorg. Chem. 2009, 48, 9444−9453. (57) Bennett, M. A.; Smith, A. K. Arene ruthenium(II) complexes formed by dehydrogenation of cyclohexadienes with ruthenium(III) trichloride. J. Chem. Soc., Dalton Trans. 1974, 2, 233−241.

(28) Paunescu, E.; Soudani, M.; Martin, P.; Scopelliti, R.; Lo Bello, M.; Dyson, P. J. Organometallic Glutathione S-Transferase Inhibitors. Organometallics 2017, 36, 3313−3321. (29) Zhang, Y.; Zheng, W.; Luo, Q.; Zhao, Y.; Zhang, E.; Liu, S.; Wang, F. Dual-targeting organometallic ruthenium(II) anticancer complexes bearing EGFR-inhibiting 4-anilinoquinazoline ligands. Dalton Trans 2015, 44, 13100−13111. (30) Guichard, S.; Else, R.; Reid, E.; Zeitlin, B.; Aird, R.; Muir, M.; Dodds, M.; Fiebig, H.; Sadler, P.; Jodrell, D. Anti-tumour activity in non-small cell lung cancer models and toxicity profiles for novel ruthenium (II) based organo-metallic compounds. Biochem. Pharmacol. 2006, 71, 408−415. (31) Scolaro, C.; Bergamo, A.; Brescacin, L.; Delfino, R.; Cocchietto, M.; Laurenczy, G.; Geldbach, T. J.; Sava, G.; Dyson, P. J. In vitro and in vivo evaluation of ruthenium(II)-arene PTA complexes. J. Med. Chem. 2005, 48, 4161−4171. (32) Murray, B. S.; Babak, M. V.; Hartinger, C. G.; Dyson, P. J. The development of RAPTA compounds for the treatment of tumors. Coord. Chem. Rev. 2016, 306, 86−114. (33) Moyer, M. W. Targeting hypoxia brings breath of fresh air to cancer therapy. Nat. Med. 2012, 18, 636−637. (34) Xu, Z.; Zhao, J.; Gou, S.; Xu, G. Novel hypoxia-targeting Pt (IV) prodrugs. Chem. Commun. 2017, 53, 3749−3752. (35) Wilson, W. R.; Hay, M. P. Targeting hypoxia in cancer therapy. Nat. Rev. Cancer 2011, 11, 393−410. (36) Purkait, K.; Karmakar, S.; Bhattacharyya, S.; Chatterjee, S.; Dey, S. K.; Mukherjee, A. A hypoxia efficient imidazole-based Ru(II) arene anticancer agent resistant to deactivation by glutathione. Dalton Trans. 2015, 44, 5969−5973. (37) Ryan, H. E.; Lo, J.; Johnson, R. S. HIF-1α is required for solid tumor formation and embryonic vascularization. EMBO J. 1998, 17, 3005−3015. (38) Bos, R.; van der Groep, P.; Greijer, A. E.; Shvarts, A.; Meijer, S.; Pinedo, H. M.; Semenza, G. L.; van Diest, P. J.; van der Wall, E. Levels of hypoxia-inducible factor-1α independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer 2003, 97, 1573−1581. (39) Harris, A. L. Hypoxia - a key regulatory factor in tumour growth. Nat. Rev. Cancer 2002, 2, 38−47. (40) Semenza, G. L. Expression of hypoxia-inducible factor 1: mechanisms and consequences. Biochem. Pharmacol. 2000, 59, 47−53. (41) Xia, Y.; Choi, H.-K.; Lee, K. Recent advances in hypoxiainducible factor (HIF)-1 inhibitors. Eur. J. Med. Chem. 2012, 49, 24− 40. (42) Semenza, G. L. Targeting HIF-1 for cancer therapy. Nat. Rev. Cancer 2003, 3, 721−732. (43) Wang, J.; Kou, J.; Hou, X.; Zhao, Z.; Chao, H. A ruthenium(II) anthraquinone complex as the theranostic agent combining hypoxia imaging and HIF-1α inhibition. Inorg. Chim. Acta 2017, 454, 176− 183. (44) Lv, W.; Zhang, Z.; Zhang, K. Y.; Yang, H.; Liu, S.; Xu, A.; Guo, S.; Zhao, Q.; Huang, W. A Mitochondria-targeted Photosensitizer Showing Improved Photodynamic Therapy Effects Under Hypoxia. Angew. Chem., Int. Ed. 2016, 55, 9947−9951. (45) Kim, H.-L.; Yeo, E.-J.; Chun, Y.-S.; Park, J.-W. A domain responsible for HIF-1α degradation by YC-1, a novel anticancer agent. Int. J. Oncol. 2006, 29, 255−260. (46) Shin, D. H.; Kim, J.-H.; Jung, Y.-J.; Kim, K.-E.; Jeong, J. M.; Chun, Y.-S.; Park, J.-W. Preclinical evaluation of YC-1, a HIF inhibitor, for the prevention of tumor spreading. Cancer Lett. 2007, 255, 107−116. (47) Chen, C.-J.; Hsu, M.-H.; Huang, L.-J.; Yamori, T.; Chung, J.-G.; Lee, F.-Y.; Teng, C.-M.; Kuo, S.-C. Anticancer mechanisms of YC-1 in human lung cancer cell line, NCI-H226. Biochem. Pharmacol. 2008, 75, 360−368. (48) Kong, J.; Kong, F.; Gao, J.; Zhang, Q.; Dong, S.; Gu, F.; Ke, S.; Pan, B.; Shen, Q.; Sun, H. YC-1 enhances the anti-tumor activity of sorafenib through inhibition of signal transducer and activator of H

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