Water-Soluble Dinitrosyl Iron Complex (DNIC): a Nitric Oxide Vehicle

Aug 30, 2016 - Synopsis. {Fe(NO)2}9 [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (DNIC 2) is a water-soluble and stable nitric oxide (NO) donor. The investigation...
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Water-Soluble Dinitrosyl Iron Complex (DNIC): a Nitric Oxide Vehicle Triggering Cancer Cell Death via Apoptosis Shou-Cheng Wu,† Chung-Yen Lu,† Yi-Lin Chen,† Feng-Chun Lo,† Ting-Yin Wang,† Yu-Jen Chen,‡ Shyng-Shiou Yuan,§ Wen-Feng Liaw,*,† and Yun-Ming Wang*,‡,∥ †

Department of Chemistry and Frontier Research Center on Fundamental and Applied Sciences of Matters, National Tsing Hua University, Hsinchu 30013, Taiwan ‡ Department of Biological Science and Technology, Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30013, Taiwan § Translational Research Center, Department of Medical Research, and Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan ∥ Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan S Supporting Information *

ABSTRACT: Nitric oxide (NO) is an important cellular signaling molecule that modulates various physiological activities. Angiogenesis-promoting activities of NO-donor drugs have been explored in both experimental and clinical studies. In this study, a structurally well characterized and water-soluble neutral {Fe(NO) 2 } 9 DNIC [(S(CH 2 ) 2 OH)(S(CH 2 ) 2 NH 3 )Fe(NO) 2 ] (DNIC 2) was synthesized to serve as a NO-donor species. The antitumor activity of DNIC 2 was determined by MTT assay, confocal imaging, and Annexin-V/PI staining. The IC50 values of DNIC 2 were 18.8, 42.9, and 38.6 μM for PC-3, SKBR-3, and CRL5866 tumor cells, respectively. Moreover, DNIC 2 promoted apoptotic cell death via activation of apoptosis-associated proteins and inhibition of survival associated proteins. In particular, DNIC 2 treatment suppressed PC-3 tumor growth by 2.34- and 19.3-fold at 7 and 21 days, in comparison with the control group. These results indicate that water-soluble DNIC 2 may serve as a promising drug for cancer therapy.

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death.11,12 For example, tumor cells treated with Roussin’s red salt (RRS, [Fe2S2(NO)4[Na]2]) or Roussin’s black salt (RBS, [NH4][Fe4S3(NO)7]) were more susceptible to γ-radiationinduced cell death.13 Since sodium nitroprusside (SNP) showed a significant toxicity to tumor cells but not normal cells, SNP was adopted as a novel NO-releasing agent for the treatment of hematological malignancy.7 A recent study indicated that NO caused apoptotic cell death in tumor cells through inhibition of mitochondrial respiration and DNA syntheses.14 The most abundant nitric oxide derived cellular adduct, {Fe(NO)2}9 dinitrosyl iron complexes with the characteristic EPR signal g = 2.03, was first discovered in blood vessels.15 In vivo, DNICs act as a natural NO carrier to protect cells from the toxic effects of NO, while conserving its physiological properties at lower concentrations of DNICs.16 Thus, DNIC may be a useful NO carrier that can modulate diverse physiological functions, such as inhibition of platelet aggregation, dilation of vascular smooth muscles via activating

INTRODUCTION Nitric oxide (NO), biosynthesized endogenously from Larginine, oxygen, and NADPH by nitric oxide synthases (NOSs),1 is an important signaling molecule that modulates many physiological functions including smooth muscle relaxation, neuronal transmission, immune system responses and platelet aggregation at physiological levels (nM).2,3 In contrast, NO generated by macrophages and neutrophils is cytotoxic for pathogens and tumor cells at higher levels (μM).4−7 Over the past decades, the fundamental studies and pharmaceutical/clinical applications of this small molecule have attracted a widespread interest across many disciplines. Due to the short lifetime (2−5 s) and high reactivity of NO, pharmacological delivery of a sufficient amount of nitric oxide to biological targets on demand is very difficult.8 Therefore, a number of organic (nitrate/nitrite and S-nitrosothiols) and inorganic compounds (dinitrosyl iron complexes (DNICs) and metal nitrosyl compounds (M-NO)) have been developed to serve as NO-transport and NO-release species in synthetic chemistry.9,10 It was reported that the NO-releasing agent DEANO ([(C2H5)2N(N(O)NO)][Na]) sensitized hypoxic mammalian cells and tumor cells to radiation-induced cell © XXXX American Chemical Society

Received: June 29, 2016

A

DOI: 10.1021/acs.inorgchem.6b01562 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry soluble guanylate cyclase, and enhancement of cardiac resistance to ischemia.2,3 DNICs at higher concentrations, in another way, may act as mobile NO carriers to conduct NOrelated pathological functions, especially in inhibiting carcinogenesis and suppressing the tumor growth via codelivery of nitric oxide and chemotherapeutic agents.10,16 Earlier studies revealed that DNICs could inhibit glutathione reductase and induce heat shock protein (HSP70) expression.17−19 In addition, DNIC-GS were proved to selectively suppress endometriosis in rats20−22 and DNIC-thiosulfate triggered apoptosis in Jurkat cells.23 Moreover, recent studies demonstrated that the decomposition of DNIC with glutathione or thiosulfate by exogenous Fe2+ chelators caused apoptotic death of HeLa cells.24,25 The combinational treatment of DNIC ([S5Fe(NO)2]−) at low concentration and UV-A light induced apoptosis in K562 cells.26 Furthermore, cotreatment of DNIC ([Fe2(C2H5OS)2(NO)4]) and curcumin displayed the synergetic effect on inhibition of B16-F10 cells.27 These approaches signified the activity of DNICs as a potential pharmacological NO vehicle. Dinuclear {Fe(NO)2}9-{Fe(NO)2}9 DNICs (also called Roussin’s red esters (RREs)) contain biological functions similar to those of DNICs. Here, the electronic state of the {Fe(NO)2}n core of DNICs is generally designated by Enemark−Feltham notation.28 In a previous study, we demonstrated that {Fe(NO)2}9 DNICs and RREs are interconvertible via protonation of DNICs and bridged-thiolate cleavage of RREs.29 In addition, NO delivery by DNICs can be modulated by the coordinated ligands. In this study, the structurally well characterized and water-soluble neutral {Fe(NO)2}9 DNIC [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (DNIC 2) was isolated. DNIC 2 containing biocompatible mixed-cysteamine-2-mercaptoethanol-coordinate ligands is expected to modulate NO release. The in vitro cytotoxicity of DNIC 2 was investigated by MTT assay, confocal imaging, and Annexin-V/PI staining in several cancer cell lines, including prostate cancer cells (PC-3), breast cancer cells (SKBR-3), and non-small-cell lung cancer cells (CRL5866). Moreover, the cytotoxic mechanism induced by DNIC 2 was investigated by Western blotting. In particular, the in vivo antitumor efficacy of DNIC 2 was evaluated in nude mice bearing subcutaneous xenografts of PC-3 tumors.

Scheme 1. Synthetic Scheme of DNIC 2

[(Cys)2Fe(NO)2]− displaying IR νNO signals at 1727 (s) and 1772 (s) cm−1 in H2O,10 the aqueous (D2O) IR νNO spectrum of DNIC 2 exhibited the diagnostic νNO stretching frequencies at 1715 (s) and 1765 (s) cm−1 (1685 (s) and 1730 (s) cm−1 (DMSO)) (Table 1). DNIC 2 was stable in THF at ambient Table 1. IR νNO Stretching Frequencies of {Fe(NO)2}9 DNICs: [(C2H5S)2Fe(NO)2]−, DNIC 2, [(Cys)2Fe(NO)2]−, and Peptide-Bound DNICs CnA-DNIC (n = 1−4) IR νNO (cm−1) [(C2H5S)2Fe(NO)2] DNIC 2

−

[(Cys)2Fe(NO)2]− KCACK-DNIC (C1A-DNIC) KCAACK-DNIC (C2A-DNIC) KCAAACK-DNIC (C3A-DNIC) KCAAAACK-DNIC (C4A-DNIC)

1672, 1688, 1715, 1685, 1727, 1722, 1722, 1720, 1721,

1715 1733 1765 1730 1772 1767 1768 1767 1766

(THF) (THF) (D2O) (DMSO) (H2O) (H2O) (H2O) (H2O) (H2O)

ref 21 this work

10 10 10 10 10

temperature and exhibited the isotropic EPR signal gav = 2.029 at 298 K (a rhombic pattern with principal g values of g1 = 2.039, g2 = 2.027 ,and g3 = 2.016 at 77 K (THF) and g1 = 2.014, g2 = 2.029, g3 = 2.042 at 77 K (H2O)) (Figure 1). We noticed

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RESULTS AND DISCUSSION Conversion of Dinuclear {Fe(NO) 2 } 9 -Fe(NO) 2 } 9 [(NO)2Fe(μ-S(CH2)2NH2)]2 (RRE 1) into the Neutral {Fe(NO)2}9 DNIC [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (DNIC 2) Triggered by 2-Mercaptoethanol. In contrast to the steric effect of the nucleophile [C2H5S]− on reduction of Roussin’s red ester (RRE) yielding [(μ-S(CH2)2NH2))Fe(NO)2]2−,30 the direct conversion of dinuclear {Fe(NO) 2 } 9 -Fe(NO) 2 } 9 [(NO)2Fe(μ-S(CH2)2NH2)]2 (RRE 1) to the neutral {Fe(NO) 2}9 DNIC [(S(CH2) 2OH)(S(CH 2)2 NH3)Fe(NO)2] (DNIC 2) was observed when a THF solution of RRE 1 was treated with 2 equiv of 2-mercaptoethanol at ambient temperature (Scheme 1). DNIC 2 was characterized by IR, UV−vis, EPR, and single-crystal X-ray diffraction. In comparison with the IR νNO stretching frequencies at 1672 and 1715 cm−1 exhibited by the complex [(SC2H5)2Fe(NO)2]−,29 the electronic perturbation caused by the nitrogen protonation of the [S(CH2)2NH2]-coordinated ligand of DNIC 2 shifts the IR νNO stretching frequencies to 1688 (s) and 1733 (s) cm−1 (THF). In comparison to the cysteine-containing DNIC

Figure 1. EPR spectrum of DNIC 2 in H2O (black line) at 77 K (g1 = 2.014, g2 = 2.029, and g3 = 2.042) and simulation (red line).

that DNIC 2 is moderately air stable in the solid state but airsensitive in aqueous solution. The synthetic method described above may provide an accessible alternative reaction pathway to synthesize neutral thiolate-containing water-soluble {Fe(NO)2}9 DNICs. One equivalent of 18-crown-6 ether added to a THF solution of DNIC 2 afforded dark brown crystals of DNIC 2·18-crown-6 ether with the protonated pendant amine stabilized by the coordination to crown ether, suitable for single-crystal X-ray diffraction. The single-crystal X-ray structure of DNIC 2 is depicted in Figure 2, and selected bond dimensions are presented in the figure caption. Cellular Uptake of Water-Soluble RRE 1. To probe the possibility of RRE 1 uptake by cells, treatment of RRE 1 with 20 equiv of L-cysteine (Cys), reduced glutathione (GSH), and serum (10%) was examined, respectively. As shown in Figure 3, B

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glutathione, and serum might trigger the transformation of RRE 1 into Cys-/GSH-/BSA-bound DNICs. In the controlled experiments (Figure 4), the intracellular characteristic EPR signal (g = 2.03) was observed in the DNIC 2 treated SKBR-3 cells (50 μM) for 2.5 and 30 min, respectively. In a similar fashion, the intracellular EPR signals (g = 2.03) from the treatment of SKBR-3 cells with RRE 1 (50 μM) for 2.5 and 30 min were also observed, respectively. This result suggests the intact RRE 1 may penetrate the cells, and then intracellular RRE 1 may undergo transformation into Cys-/GSH-/BSA-bound {Fe(NO)2}9 DNICs. The time-course measurements revealed that the EPR signals of intracellular {Fe(NO)2}9 DNICs completely decayed in 3 h. A previous study has proposed that DNICs may serve as NO vehicles to induce NO-mediated cell death. The facile transformation of RRE 1 into Cys-/GSH-bound DNICs in the presence of Lcysteine/reduced glutathione prompted us to investigate tumor cell death induced by DNIC 2 released NO. Cytotoxicity of DNIC 2. To assess the potential anticancer ability of the water-soluble DNIC 2, tumor cells (PC-3, SKBR3, and CRL5866 cells) were exposed to various concentrations of DNIC 2 and then cell survival was measured by MTT assays. As shown in Figure 5A,C,E, treatment of DNIC 2 reduced cell viability in a concentration-dependent manner in all three cancer cell lines. The IC50 values (the concentration of a substance (a particular drug or an inhibitor) for 50% inhibition of a specific biological or biochemical function) of DNIC 2 were 18.8, 42.9, and 38.6 μM for PC-3, SKBR-3, and CRL5866 cells, respectively (Figure 5B,D,F) The IC50 value of PC-3 cells was ∼2-fold lower than those of SKBR-3 and CRL5866 cells after DNIC 2 treatment. Confocal Microscopy Imaging. The penetration and localization of DNIC 2 to tumor cells (PC-3, SKBR-3, and CRL5866) were investigated by confocal microscopy. In these experiments, FA-OMe (5-amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid methyl ester, green fluorescence) was used to detect the nitric oxide released from DNIC 2.31 As shown in Figure 6A, DNIC 2 treated PC-3 cells showed marked green fluorescent signals in cytoplasm, especially in mitochondria (yellow color when merged), whereas only very weak green fluorescent signals appeared when additional NO scavenger was added. Similar results were observed in SKBR-3 and CRL5866 cells upon treatment with DNIC 2 or NO scavenger (Figure 6B,C). Furthermore, the average intracellular nitric oxide concentrations of PC-3, SKBR-3, and CRL5866 cells were about 124, 146, and 152 μM, respectively, which were similar to

Figure 2. ORTEP drawing and labeling scheme of DNIC 2 with thermal ellipsoids drawn at 30% probability. Selected bond lengths (Å) and angles (deg): Fe(1)−N(1) 1.687(3), Fe(1)−N(1A) 1.687(3), Fe(1)−S(1) 2.2793(11), Fe(1)−S(1A) 2.2793(11), N(1)−O(4) 1.180(4), N(1A)−O(4A) 1.180(4); N(1A)−Fe(1)−N(1) 114.9(2), N(1A)−Fe(1)−S(1) 105.38 (10), N(1)−Fe(1)−S(1) 112.85(11), N(1A)−Fe(1)−S(1A) 112.85(11), N(1)−Fe(1)−S(1A) 105.38(10), S(1)−Fe(1)−S(1A) 105.16(6).

Figure 3. EPR spectra derived from reaction of RRE 1 and 20 equiv of L-cysteine (green line), 20 equiv of reduced glutathione (GSH) (blue line), and 10% serum (red line) in PBS, respectively, and standard DNIC [(PhS)2Fe(NO)2]− (100 μM (black line), 200 μM (yellow line)).

the EPR signal at g = 2.03 implicated the quantitative formation of L-cysteine-bound DNIC (93%), glutathione-bound DNIC (63%), and bovine serum albumin bound DNICs (46%), on the basis of the standard paramagnetic [PPN][(C6H5S)2Fe(NO)2]. These results demonstrated that L-cysteine, reduced

Figure 4. EPR spectra derived from SKBR-3 cells treated with (A) RRE 1 or (B) DNIC 2 in PBS for 2.5 min (black line), 30 min (blue line), 60 min (red line), and 180 min (green line), respectively. C

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Figure 5. In vitro cell viability of PC-3 (A), SKBR-3 (C), and CRL5866 (E) cells upon DNIC 2 treatment at different concentrations for 24 h and the IC50 values of PC-3 (18.8 μM, B), SKBR-3 (42.9 μM, D), and CRL5866 (38.6 μM, F) cells treated with DNIC 2.

those of the nitric oxide relative report.32 As a result, these findings demonstrated that DNIC 2 could penetrate and release nitric oxide in tumor cells. Tumor Cell Apoptosis. Exposure of phosphatidylserine at the outer leaflet of the cell membrane, which is conveniently measured by Annexin-V staining, is a hallmark of apoptosis.33 In addition, propidium iodide (PI), which stains for DNA, has been widely used in conjunction with Annexin-V staining for detecting apoptosis.34 To determine the induction of apoptosis by DNIC 2, treated tumor cells were analyzed by the AnnexinV/PI staining method. Figure 7 shows two-dimensional frequency contour plots of green fluorescence (Annexin-VFITC) versus red fluorescence (PI) using flow cytometry. Tumor cells, treated with DNIC 2 for 1 h (18.8 μM for PC-3, 42.9 μM for SKBR-3, and 38.6 μM for CRL5866), displayed both high green and red fluorescence intensities (upper right), representing late apoptotic cells. In contrast, tumor cells without DNIC 2 treatment appeared in subsets with low green and red fluorescence intensities (lower left). After DNIC 2 treatment (18.8 μM for PC-3, 42.9 μM for SKBR-3, and 38.6 μM for CRL5866), the apoptosis levels of PC-3, SKBR-3, and CRL5866 cells rose to 69.1 ± 3.5, 70.5 ± 4.1, and 83.2 ± 2.7%, respectively. These results clearly demonstrated that DNIC 2 treatments promoted apoptosis in tumor cells. SAPK/JNK- and Bcl2-Related Apoptosis Pathways. Oxidative stress and DNA damage are associated with the

activation of the SAPK/JNK pathway, leading to activation of stress-response genes. SAPK/JNK activation is necessary for apoptotic cell death and induction of activating transcription factor 3 (ATF3).35 It has been documented that upregulation of ATF3 is required for cell death by NO-releasing prodrugs in non-small-cell lung cancer (NSCLC) cells.36 To determine whether DNIC 2 treatment, which induces ROS/RNS and stress signaling, was associated with cell death, DNIC 2 treated cells were analyzed by Western blot for markers of apoptosis. As shown in Figure 8A, treatment of tumor cells with DNIC 2 (IC50 dosage for 1 h) induced MKK4 protein and resulted in the activation of apoptosis-associated proteins (JNK, ATF2, cJun, and ATF3) in tumor cells. In addition to the SAPK/JNK pathway, the cell survival associated proteins (P-AKT, P-BAD, Bcl2, and Bcl-xL) were determined by Western blot analysis. Bcl2 was isolated from the chromosomal breakpoint in follicular B-cell lymphoma,37−39 and it has the unique role of prolonging cell survival by inhibiting a variety of apoptotic cell death. To further confirm that DNIC 2 induced apoptotic cell death in tumor cells, Bcl2 associated proteins were analyzed by Western blot (Figure 8B). As expected, expression of survival-associated proteins, P-AKT and P-BAD, was inhibited by DNIC 2 treatments for 1 h (18.8 μM for PC-3, 42.9 μM for SKBR-3, and 38.6 μM for CRL5866). Furthermore, the expression of Bcl2 and Bcl-xL proteins was decreased by the downregulation of P-BAD. These D

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investigate whether DNIC 2 inhibited tumor growth, nude mice bearing subcutaneous PC-3 tumor xenografts were intravenously injected with/without DNIC 2 (0.2 mg/kg) every 3 days for 21 days. The tumor sizes and body weights of the xenografted nude mice between the DNIC 2 treatment group and the control group were compared. As shown in Figure 9A, the tumor growth in nude mice was markedly retarded by DNIC 2 treatment on day 21. The tumor sizes of DNIC 2 treatment group were 2.34- and 19.3-fold smaller than those of the control group on day 7 and day 21, respectively (Figure 9B). Furthermore, the body weight changes of nude mice of these two groups were also examined (Figure 9C). On day 7, DNIC 2 treated and untreated tumor-bearing nude mice lost 5.5 ± 0.9 and 1.4 ± 0.4% of their initial body weights, respectively. On day 21, DNIC 2 treated nude mice were 9.0 ± 0.9% lighter, whereas the body weights of control group mice were only 1.2 ± 1.3% lighter. After statistical analysis, the body weights were not significantly different between both groups of mice. In addition, the blood biochemical parameters and the histology of mouse organs were examined after 21 days. In comparison to the control group, DNIC 2 treated mice did not show significant changes in the body weights (Figure 9), blood biochemical parameters (Table 2), and histology of the prostates, kidneys, livers, hearts, muscles, and spleens (Figure 10). Morphology and Immunohistochemistry Studies. Morphology and immunohistochemistry (IHC) on apoptosisrelated proteins were studied in PC-3 tumor tissue specimens of tumor-bearing nude mice after treatment with DNIC 2 (0.2 mg/kg) every 3 days for 21 days. Sections were stained by H&E for morphology or stained by anticaspase 9, anticaspase 3, antiPARP, anti-MKK4, or anti-ATF3 antibodies for apoptosisrelated proteins. As shown in Figure 11, specific morphological changes, indicative of apoptosis, were identified upon tumor sections of nude mice treated with DNIC 2 (arrows). Furthermore, the IHC analyses showed that the antibodies specific for apoptosis-associated proteins (caspase 9, caspase 3, PARP, MKK4, and ATF3) were selectively stained in the tumor cells (arrows) treated by DNIC 2. Thus, DNIC 2 at IC50 dosage induced apoptosis-associated proteins in PC-3 tumor xenograft tissues. These findings further supported the Western blot analyses (Figure 8) and indicated that DNIC 2 activated apoptosis of PC-3 cells in vivo. Prussian Blue Staining. The localization of DNIC 2 in PC-3 tumor tissue specimens after DNIC 2 treatment was further supported by Prussian blue staining. As shown in Figure 12, conspicuous blue spots (arrows) were present in the PC-3 tumor tissue with DNIC 2 treatment, whereas such blue spots were almost absent in the control group. This may be attributed to the higher permeability of the blood vessels in tumors (EPR effect). The higher permeability led to DNIC 2 being passively accumulated in solid tumors after intravenous administration. This phenomenon was also found in a previous report.40 These results clearly demonstrated the localization of DNIC 2 in tumor tissues in vivo.

Figure 6. Confocal microscopy images of PC-3 (A), SKBR-3 (B), and CRL5866 (C) cells incubated with DNIC 2 (18.8 μM for PC-3, 42.9 μM for SKBR-3, and 38.6 μM for CRL5866, respectively) for 30 min at 37 °C (green, FA-OMe; red, MitoTracker Red CMXRos).

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CONCLUSIONS In summary, a stable and water-soluble NO donor species (DNIC 2) for antitumor application was synthesized. The in vitro and in vivo results demonstrate that DNIC 2 could efficiently inhibit the growth and proliferation of tumor cells and induce tumor cell death via an apoptosis pathway. Thus, DNIC 2 could potentially be developed as an anticancer drug.

results indicated that DNIC 2 treated tumor cells activated apoptosis-associated proteins and inhibited survival-associated proteins, leading to apoptotic death in tumor cells. In Vivo Tumor Growth, Body Weight Changes, Blood Biochemical Parameters, and Histology Studies. To E

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Figure 7. Annexin-V/PI staining of tumor cells (PC-3, SKBR-3, and CRL5866 cells) treated with DNIC 2 (18.8 μM for PC-3, 42.9 μM for SKBR-3, and 38.6 μM for CRL5866, respectively) for 1 h at 37 °C.

Figure 8. Activation of stress signaling pathways (A) and expression of cell survival proteins (B) in tumor cells (PC-3, SKBR-3, and CRL5866 cells) after DNIC 2 treatment (18.8 μM for PC-3, 42.9 μM for SKBR-3, and 38.6 μM for CRL5866, respectively). (C−H) Quantitation of the relative protein levels in tumor cells (PC-3, SKBR-3, and CRL5866 cells) with/without DNIC 2 treatment.

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or in a glovebox (nitrogen gas). Solvents were distilled under nitrogen from the appropriate drying agents (diethyl ether from CaH2, hexane and tetrahydrofuran (THF) from sodium benzophenone) and stored

MATERIALS AND METHODS

Materials. Manipulations, reactions, and transfers of samples were conducted under nitrogen according to standard Schlenk techniques F

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Figure 9. Antitumor effects of DNIC 2 in mice bearing PC-3 tumors: (A) representative images of mice from different groups; (B) average tumor volume change of mice bearing PC-3 tumors after treatment with/without DNIC 2 (0.2 mg/kg) via tail veins every 3 days until day 21; (C) body weight changes of nude mice bearing PC-3 tumors after treatment with/without DNIC 2. Asterisks indicate P < 0.05 as determined by Student’s t test.

Table 2. Biochemical Profiles of the Nude Mice after Treatment with DNIC 2 for 21 Days control DNIC 2

AST

ALT

BUN

creatinine

186.32 ± 26.85 197.54 ± 36.71

67.22 ± 8.36 72.87 ± 11.21

23.75 ± 3.12 20.66 ± 2.43

0.14 ± 0.03 0.15 ± 0.04

Figure 10. Immunohistochemistry analysis of tissue specimens of nude mice after treatment with DNIC 2 (0.2 mg/kg) every 3 days until day 21. Tissue sections from mouse organs were stained by H&E for morphology observation in mice treated with/without DNIC 2. in dried, nitrogen-filled flasks over 4 Å molecular sieves. Nitrogen was purged through these solvents before use. The solvent was transferred to a reaction vessel via a stainless steel cannula under positive pressure of nitrogen. The reagents cysteamine, thiophenol, L-cysteine, 18crown-6 ether (TCI/Alfa/Aldrich/Eskay Engiechem), and O2 (San fu, 99.9%) were used as received, and complex [(NO)2Fe(μ-S(CH2)2NH2)]2 was synthesized on the basis of a literature report.41 Annexin-V/PI staining kit and MitoTracker Red CMXRos were purchased from Life Technologies (Carlsbad, CA, USA). The primary antibodies (mouse anti-JNK, anti-ATF2, anti-c-Jun, anti-ATF3, antiAKT, anti-BAD, anti-Bcl2, anti-Bcl-xL, anticaspase 9, anticaspase 3, anti-PARP, anti-MKK4, and anti-β-actin) and secondary antibody (Rabbit polyclonal antibody to mouse IgG-H&L) were purchased from Abcam (Cambridge, MA, USA). Polyvinylidene difluoride membranes (PVDF), RIPA buffer, Nuclear Fast Red, and Tween-20 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Matrigel was purchased from BD Bioscience (Bedford, MA, USA), and all the

chemicals were used directly without any further purification unless otherwise stated. Methods. IR spectra νNO stretching frequencies were recorded on a PerkinElmer Spectrum One B spectrophotometer with sealed solution cells (Boston, MA, USA). EPR spectra were recorded using a Bruker Biospin E580 X-band spectrometer (Milton, ON, Canada). UV−vis spectra were recorded on an Agilent 8453 UV−vis spectroscopy system (Santa Clara, CA, USA). Analyses of carbon, hydrogen, and nitrogen were obtained with a Heraeus CHN-O-Rapid analyzer (Hanau, Germany). The flow cytometry experiments were carried out using a FACScan flow cytometer (San Jose, CA, USA). The confocal fluorescence studies were performed using a Leica TCS-SP5-X AOBS confocal microscope system (Bensheim, Germany). Preparation of [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (DNIC 2). To a flask containing 2-mercaptoethanol (0.0160 g, 0.205 mmol), was added 20 mL of THF solution of complex [(NO) 2 Fe(μS(CH2)2NH2)]2 (0.0384 g, 0.1 mmol). The reaction solution was stirred under N2 for 10 min at ambient temperature. The resulting G

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Inorganic Chemistry

Figure 11. Immunohistochemistry analysis of PC-3 tumor tissue specimens of nude mice after treatment with DNIC 2 (0.2 mg/kg) every 3 days until day 21. Sections were stained by H&E for morphology observation and incubated with anticaspase 9, anticaspase 3, anti-PARP, anti-MKK4, or anti-ATF3 antibodies for the expression of apoptosis-related proteins (arrows). The scale bar is 100 μm. bovine serum. At 2.5, 30, 60, and 180 min intervals, respectively, the culture medium was collected, transferred to an EPR tube, and frozen in liquid nitrogen. In the meantime, cultured cells were harvested, washed three times with ice-cold phosphate-buffered saline, centrifuged at 1000g for 1 min, and resuspended with PBS (200 μL). The cell suspension was transferred to an EPR tube and immediately frozen with liquid nitrogen. EPR spectra were recorded using a Bruker E580 X-band spectrometer at 77 K. EPR measurement conditions were as follows: microwave frequency, 9.5 GHz; microwave power, 1.191 mW; modulation frequency, 100 kHz; modulation amplitude, 0.5 mT. EPR Spectroscopy. EPR measurements were performed at X band using a Bruker ELEXSYS E580 spectrometer equipped with an ELEXSYS superhigh-sensitivity probehead cavity. At 77 and 298 K, Xband EPR spectra of DNIC 2 (H2O) were obtained with the frequency at 9.66 GHz. The microwave power and modulation amplitude of DNIC 2 were 1.191 mW and 0.5 G at 100.00 kHz, respectively. X-ray Crystallography. Crystallographic data and structure refinement parameters of DNIC 2 are summarized in the Supporting Information. The crystals of DNIC 2 chosen for X-ray diffraction studies were measured at a size of 0.60 × 0.41 × 0.09 mm3. Each crystal was mounted on a glass fiber and quickly coated in epoxy resin. Unit-cell parameters were obtained by least-squares refinement. Diffraction measurements were carried out on a Bruker X8 APEX II CCD diffractometer for DNIC 2 with graphite-monochromated Mo Kα radiation (λ = 0.7107 Å) and between 2.41 and 25.00° for DNIC 2. Least-squares refinement of the positional and anisotropic thermal parameters of all non-hydrogen atoms and fixed hydrogen atoms was based on F2. A semiempirical from equivalent absorption correction was made for DNIC 2. The SHELXTL structure refinement program was employed.42 Cell Culture. All tumor cells (PC-3, SKBR-3, and CRL5866) were cultured in RPMI or DMEM medium and supplemented with 10% fetal bovine serum (FBS) (GIBCO). All cells were cultured in a humidified incubator at 37 °C with 5% CO2. In Vitro Cytotoxicity Assay. All of the three tumor cell lines were used to measure the in vitro cytotoxicity of DNIC 2. Tumor cells were plated in 24-well plates at a density of 1 × 105 cells/well for 24 h, and then DNIC 2 was added at different concentrations (0, 10, 20, 30, 40, 50, 60, 70, or 80 μM) for 24 h. Culture media were removed, and the cells were washed three times with PBS, followed by determination of cell viability using the MTT conversion test. Briefly, MTT (100 μL) solution was added to each well. After incubation for 2 h, each well was

Figure 12. Histological analyses of PC-3 tumor tissue specimens without DNIC 2 treatment (A) and with DNIC 2 (0.2 mg/kg) treatment (B) every 3 days until day 21. Sections were stained with Prussian blue (arrows). The scale bar is 100 μm. yellow-green solution was monitored by FTIR. The IR νNO stretching frequencies shifting from 1749 (s), 1774 (s), and 1808 cm−1 to 1688 (s) and 1733 (s) cm−1 (THF) implicated the formation of the neutral {Fe(NO)2}9 DNIC [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (DNIC 2). The solution was filtered through Celite, and hexane was added slowly to the filtrate to precipitate the yellow-brown oily DNIC 2. One equivalent of 18-crown-6 ether was added to the THF solution of DNIC 2 for crystallization, and crystals (DNIC 2·18-crown-6 ether) suitable for XRD analysis were obtained from the THF solution of DNIC 2 layered with hexane at −20 °C for 2 weeks (yield 0.0191 g, 77%). IR: 1688 (s), 1733 (s) (νNO) cm−1 (THF); 1685 (s), 1730 (s) cm−1 (DMSO); 1720 (s), 1766 (s) cm−1 (10% DMSO + 90% PBS). Absorption spectrum (λmax, nm (ε, M−1 cm−1)): 315 (4481), 368 (4365), 438 (1954) (THF); 342 (5543), 395 (4430) (10% DMSO + 90% PBS). Anal. Calcd for C16H36FeN3O9S2: C, 35.96; H, 6.79; N, 7.86. Found: C, 36.06; H, 6.79; N, 8.02. Detection of Dinitrosyl Iron Complexes (DNICs) Derived from Conversion of {Fe(NO) 2 } 9 -Fe(NO) 2 } 9 [(NO) 2 Fe(μ-S(CH2)2NH2)]2 (RRE 1) and {Fe(NO)2}9 DNIC [(S(CH2)2OH)(S(CH2)2NH3)Fe(NO)2] (DNIC 2) by Electron Paramagnetic Resonance (EPR) Spectroscopy. For each experiment, 3.5 × 106 cells were plated onto a 10 cm cell culture dish and cultured for 12 h. The culture medium was removed, and the cells were washed three times with 1 × PBS. The stock solution of RRE 1 or DNIC 2 (100 or 10 μM, respectively) was added to the cell cultures and incubated for 2.5, 30, 60, or 180 min at 37 °C, respectively. The same treatments were also done in the presence of 20-fold L-cysteine or 10% fetal H

DOI: 10.1021/acs.inorgchem.6b01562 Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

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treated with DMSO (50 μL) for 3−5 min. Absorption at 570 nm was measured on a plate reader. The result was represented as mean ± SD % (n = 4) with the untreated cells (control) as 100% viability. Confocal Microscopy Imaging. All tumor cells were seeded on cover glasses (24 × 24 mm) at a density of 1 × 104 cells/well and allowed to grow for 24 h. Then tumor cells were incubated with 5amino-2-(6-hydroxy-3-oxo-3H-xanth-en-9-yl)benzoic acid methyl ester (FA-OMe)31 for 4 h for nitric oxide detection and DNIC 2 (IC50 dosage) for 30 min at 37 °C, washed three times with PBS, and fixed with 4% formaldehyde solution for 30 min at room temperature. Mitochondria were stained with MitoTracker Red CMXRos (ex/em: 579/599 nm). Cover glasses containing fixed cells were mounted in a mixture of PBS and glycerol (1/1) on a microscope slide, and the cells were observed using a confocal imaging system. Annexin-V/PI Staining Assay. All tumor cells were seeded on cover glasses (24 × 24 mm) at a density of 1 × 105 cells/well and allowed to grow for 24 h. Thereafter, tumor cells were incubated with DNIC 2 (IC50 dosage) for 30 min at 37 °C. Apoptotic cells were determined using an Annexin-V/PI apoptosis staining kit according to the manufacturer’s instructions. Then the cells were collected and resuspended in 500 μL of binding buffer, with 5 μL of Annexin-VFITC and 5 μL of PI added. Finally, the samples were analyzed with the FACScan flow cytometer. Western Blot Assay. All tumor cells were washed twice with cold PBS and lysed on ice with RIPA buffer (10 mM PBS, 1% NP40, 0.1% sodium dodecyl sulfate (SDS), 5 mM EDTA, 0.5% sodium deoxycholate, and 1 mM sodium orthovanadate) with the presence of protease inhibitors, and the protein lysates were quantified. An equal amount of protein lysates (50−80 μg) was separated by SDS polyacrylamide gel, electrotransferred to polyvinylidene fluoride membranes (PVDF), and blocked with 5% nonfat dry milk in pH 7.5 Tris buffer (100 mM NaCl, 50 mM Tris, 0.1% Tween-20). Membranes were immunoblotted against the primary antibodies (mouse anti-JNK, anti-ATF2, anti-c-Jun, anti-ATF3, anti-AKT, antiBAD, anti-Bcl2, anti-Bcl-xL, or anti-β-actin) (1/1000 dilution) for 1 h. After it was probed with the primary antibody, the membrane was washed and incubated with a secondary antibody (rabbit polyclonal antibody to mouse IgG-H&L) (1/5000 dilution) coupled to horseradish peroxidase (HRP) for 1 h. The antibody−antigen complexes were identified with ECL Western blotting detection reagents, and signals were detected by enhanced chemiluminescence. The β-actin was used as an internal control. Animal Model. BALB/cAnN.Cg-Foxn1nu/CrlNarl mice (6−8 weeks old, female) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. Animal experiments were performed in accordance with the institutional guidelines. PC-3 tumor cells in 100 μL PBS (1 × 106 cells) with Matrigel were subcutaneously injected to the flanks of nude mice for in vivo experiments. In Vivo Tumor Growth, Body Weight Changes, and Histology Studies. Nude mice (n = 4) bearing PC-3 tumors were treated with DNIC 2 (0.2 mg/kg) via tail veins every 3 days until day 21. The control groups received saline only. The tumor growth and body weight changes of nude mice were monitored on days 7 and 21. Tumor sizes were determined using caliper measurement. After 21 days, both groups of tumor-bearing mice were sacrificed and the prostate, kidney, liver, heart, muscle, and spleen tissues were fixed in 10% formalin and embedded in paraffin. Sections (6−10 μm) from the representative organs were stained by H&E for histology studies. Morphology, Immunohistochemistry, and Prussian Blue Staining. Tumor-bearing mice were sacrificed, and PC-3 tumors were fixed in 10% formalin and embedded in paraffin. Sections (6−10 μm) from representative tumors were stained by H&E for morphology observation and incubated with anticaspase 9, anticaspase 3, antiPARP, anti-MKK4, or anti-ATF3 antibodies for study of the expression of apoptosis-related proteins. In addition, slides were stained by Nuclear Fast Red for morphology observation and Prussian blue for iron recognition. Then all the sections were imaged and captured with a microscope.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b01562. X-ray crystallographic files in CIF format for structure determinations of DNIC 2 ([(S(CH 2 ) 2 OH)(S(CH2)2NH3)Fe(NO)2]) (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*W.-F.L.: tel, 886-3-5715131 ext. 35663; fax, 886-3-5711082; email, wfl[email protected]. *Y.-M.W.: tel, 886-3-5712121 ext. 56972; fax, 886-3-5729288; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the Ministry of Science and Technology and Ministry of Health and Welfare of the Republic of China for financial support (health and welfare surcharge of tobacco products) under contract nos. MOST 104-2113-M-009-012MY2, MOST 103-2627-M-009-001, and MOHW105-TDU-B212-134005. This research was also particularly supported by “Aim for the Top University Plan” of the National Chiao Tung University and Ministry of Education. The authors thank the core facility of Multiphoton and Confocal Microscope System (MCMS) at National Chiao Tung University, Hsinchu, Taiwan.

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REFERENCES

(1) Marletta, M. A.; Hurshman, A. R.; Rusche, K. M. Curr. Opin. Chem. Biol. 1998, 2, 656−663. (2) Keese, M. A.; Böse, M.; Mülsch, A.; Schirmer, R. H.; Becker, K. Biochem. Pharmacol. 1997, 54, 1307−1313. (3) Manukhina, E. B.; Malyshev, I. Y.; Malenyuk, E. B.; Zenina, T. A.; Podkidyshev, D. A.; Mikoyan, V. D.; Kubrina, L. N.; Vanin, A. F. Bull. Exp. Biol. Med. 1998, 125, 23−25. (4) Brune, B.; Gotz, C.; Messmer, U. K.; Sandau, K.; Hirvonen, M.R.; Lapetina, E. G. J. Biol. Chem. 1997, 272, 7253−7258. (5) Evans, T. J.; Buttery, L. D.; Carpenter, A.; Springall, D. R.; Polak, J. M.; Cohen, J. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 9553−9558. (6) Filep, J. G.; Baron, C.; Lachance, S.; Perreault, C.; Chan, J. S. Blood 1996, 87, 5136−5143. (7) Tsumori, M.; T, J.; Koshimura, K.; Kawaguchi, M.; Murakami, Y.; Kato, Y. Acta Biochim. Polym. 2002, 49, 139−144. (8) Song, Q.; Tan, S.; Zhuang, X.; Guo, Y.; Zhao, Y.; Wu, T.; Ye, Q.; Si, L.; Zhang, Z. Mol. Pharmaceutics 2014, 11, 4118−4129. (9) Gorodetsky, A. A.; Barton, J. K. Langmuir 2006, 22, 7917−7922. (10) Lin, Z.-S.; Lo, F.-C.; Li, C.-H.; Chen, C.-H.; Huang, W.-N.; Hsu, I.-J.; Lee, J.-F.; Horng, J.-C.; Liaw, W.-F. Inorg. Chem. 2011, 50, 10417−10431. (11) Kielbik, M.; Klink, M.; Brzezinska, M.; Szulc, I.; Sulowska, Z. Nitric Oxide 2013, 35, 93−109. (12) Mitchell, J. B.; Wink, D. A.; DeGraff, W.; Gamson, J.; Keefer, L. K.; Krishna, M. C. Cancer Res. 1993, 53, 5845−5848. (13) Bourassa, J.; DeGraff, W.; Kudo, S.; Wink, D. A.; Mitchell, J. B.; Ford, P. C. J. Am. Chem. Soc. 1997, 119, 2853−2860. (14) Ushmorov, A.; Ratter, F.; Lehmann, V.; Droge, W.; Schirrmacher, V.; Umansky, V. Blood 1999, 93, 2342−2352. (15) Vanin, A. F. Biochemistry (Moscow) 1995, 60, 441−447. (16) Suryo Rahmanto, Y.; Kalinowski, D. S.; Lane, D. J. R.; Lok, H. C.; Richardson, V.; Richardson, D. R. J. Biol. Chem. 2012, 287, 6960− 6968.

I

DOI: 10.1021/acs.inorgchem.6b01562 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry (17) Boese, M.; Keese, M. A.; Becker, K.; Busse, R.; Mülsch, A. J. Biol. Chem. 1997, 272, 21767−21773. (18) Malyshev, I. Y.; Malugin, A. V.; Golubeva, L. Y.; Zenina, T. A.; Manukhina, E. B.; Mikoyan, V. D.; Vanin, A. F. FEBS Lett. 1996, 391, 21−23. (19) Malyshev, I. Y.; Zenina, T. A.; Golubeva, L. Y.; Saltykova, V. A.; Manukhina, E. B.; Mikoyan, V. D.; Kubrina, L. N.; Vanin, A. F. Nitric Oxide 1999, 3, 105−113. (20) Burgova, E. N.; Adamyan, L. V.; Tkachev, N. A.; Stepanyan, A. A.; Vanin, A. F. Biophysics 2012, 57, 87−89. (21) Adamyan, L. V.; Burgova, E. N.; Tkachev, N. A.; Mikoyan, V. D.; Stepanyan, A. A.; Sonova, M. M.; Galkin, A. V.; Vanin, A. F. Biophysics 2013, 58, 222−227. (22) Burgova, E. N.; Tkachev, N. A.; Adamyan, L. V.; Mikoyan, V. D.; Paklina, O. V.; Stepanyan, A. A.; Vanin, A. F. Eur. J. Pharmacol. 2014, 727, 140−147. (23) Kleschyov, A. L.; Strand, S.; Schmitt, S.; Gottfried, D.; Skatchkov, M.; Sjakste, N.; Daibera, A.; Umanskyd, V.; Munzela, T. Free Radical Biol. Med. 2006, 40, 1340−1348. (24) Giliano, N. Y.; Konevega, L. V.; Noskin, L. A.; Serezhenkov, V. A.; Poltorakov, A. P.; Vanin, A. F. Nitric Oxide 2011, 24, 151−159. (25) Vanin, A. F. Nitric Oxide 2016, 54, 15−59. (26) Chen, T.-N.; Lo, F.-C.; Tsai, M.-L.; Shih, K.-N.; Chiang, M.-H.; Lee, G.-H.; Liaw, W.-F. Inorg. Chim. Acta 2006, 359, 2525−2533. (27) Shiau, R. J.; Wu, J. Y.; Chiou, S. J.; Wen, Y. D. Planta Med. 2012, 78, 1342−1350. (28) Enemark, J. H.; Feltham, R. D. Coord. Chem. Rev. 1974, 13, 339−406. (29) Lu, T.-T.; Huang, H.-W.; Liaw, W.-F. Inorg. Chem. 2009, 48, 9027−9035. (30) Lu, T.-T.; Tsou, C.-C.; Huang, H.-W.; Hsu, I.-J.; Chen, J.-M.; Kuo, T.-S.; Wang, Y.; Liaw, W.-F. Inorg. Chem. 2008, 47, 6040−6050. (31) Shiue, T.-W.; Chen, Y.-H.; Wu, C.-M.; Singh, G.; Chen, H.-Y.; Hung, C.-H.; Liaw, W.-F.; Wang, Y.-M. Inorg. Chem. 2012, 51, 5400− 5408. (32) Barker, S. L. R; Clark, H. A.; Swallen, S. F.; Kopelman, R. Anal. Chem. 1999, 71, 1767−1772. (33) van Engeland, M.; Nieland, L. J. W.; Ramaekers, F. C. S.; Schutte, B.; Reutelingsperger, C. P. M. Cytometry 1998, 31, 1−9. (34) Riccardi, C.; Nicoletti, I. Nat. Protoc. 2006, 1, 1458−1461. (35) Maciag, A. E.; Nandurdikar, R. S.; Hong, S.-Y.; Chakrapani, H.; Diwan, B.; Morris, N. L.; Shami, P. J.; Shiao, Y.-H.; Anderson, L. M.; Keefer, L. K.; Saavedra, J. E. J. Med. Chem. 2011, 54, 7751−7758. (36) Maciag, A. E.; Chakrapani, H.; Saavedra, J. E.; Morris, N. L.; Holland, R. J.; Kosak, K. M.; Shami, P. J.; Anderson, L. M.; Keefer, L. K. J. Pharmacol. Exp. Ther. 2011, 336, 313−320. (37) Tsujimoto, Y.; Gorham, J.; Cossman, J.; Jaffe, E.; Croce, C. M. Science 1985, 229, 1390−1393. (38) Bakhshi, A.; Jensen, J. P.; Goldman, P.; Wright, J. J.; McBride, O. W.; Epstein, A. L.; Korsmeyer, S. J. Cell 1985, 41, 899−906. (39) Hockenbery, D.; Nunez, G.; Milliman, C.; Schreiber, R. D.; Korsmeyer, S. J. Nature 1990, 348, 334−336. (40) Dreher, M. R.; Liu, W.; Michelich, C. R.; Dewhirst, M. W.; Yuan, F.; Chilkoti, A. J. Natl. Cancer Inst. 2006, 98, 335−344. (41) Lu, C.-Y.; Liaw, W.-F. Inorg. Chem. 2013, 52, 13918−13926. (42) Sheldrick, G. M. SHELXTL, Program for Crystal Structure Determination; Siemens Analytical X-ray Instruments Inc., Madison, WI, 1994.

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DOI: 10.1021/acs.inorgchem.6b01562 Inorg. Chem. XXXX, XXX, XXX−XXX