Anticancer Activity and Autophagy Involvement of Self-Assembled

Article pubs.acs.org/Organometallics

Anticancer Activity and Autophagy Involvement of Self-Assembled Arene−Ruthenium Metallacycles Abhishek Dubey,†,⊥ Yong Joon Jeong,‡,⊥ Jae Ho Jo,† Sangkook Woo,† Dong Hwan Kim,† Hyunuk Kim,§ Se Chan Kang,*,‡ Peter J. Stang,∥ and Ki-Whan Chi*,† †

Department of Chemistry, University of Ulsan, Ulsan 680-749, Republic of Korea Department of Life Science, Gachon University, Seongnam 461-701, Republic of Korea § Energy Materials Laboratory, Korea Institute of Energy Research, Daejeon 305-343, Republic of Korea ∥ Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States

Downloaded by KTH ROYAL INST OF TECHNOLOGY on September 11, 2015 | http://pubs.acs.org Publication Date (Web): September 11, 2015 | doi: 10.1021/acs.organomet.5b00512

‡

S Supporting Information *

ABSTRACT: A suite of six metallacycles (1−6) was generated via coordination-driven self-assembly using the three dicarboxylate-bridged arene−Ru precursors [Ru2(μ-η4OO∩OO)(η6-p-iPrC6H4Me)2][CF3SO3]2; (OO∩OO = oxalate (A1), 2,5-dihydroxy-1,4-benzoquinonato (dobq) (A2), 5,8-dihydroxy-1,4-naphthoquinonato (donq) (A3); CF3SO3 = triflate) with one of two dipyridyl ligands (L1 and L2). The metallacycles were isolated in excellent yield (86−92%) as triflate salts and characterized by proton (1H) and carbon-13 (13C) nuclear magnetic resonance (NMR) and electrospray ionization−mass spectrometry (ESI-MS) to confirm their structural assignments. Single-crystal X-ray crystal analysis of 1 revealed that two L1 ligands bridged two A1 acceptors to form a rectangular architecture. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to evaluate the in vitro cytotoxicities relative to two chemotherapeutic agents: namely, cisplatin and doxorubicin. Metallacycles 3 and 6 potently inhibited the growth of HCT-15 human colon and AGS human gastric cancer cells. The hollow fiber (HF) assay was performed to investigate the in vivo antitumor activities of metallacycles 3 and 6. Metallacycle 6 was more effective in inhibiting HCT-15 cells than metallacycle 3 in both in vitro and in vivo studies. Moreover, 3 and 6 induced autophagic activity in HCT-15 cells. These results suggested that the autophagic response elicited by metallacycles 3 and 6 could mediate the anticancer effects observed in human colorectal cancer cells.

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INTRODUCTION Autophagy is a form of programmed cell death and is considered one of the most important molecular responses.1 Autophagy has recently been recognized as a mechanism that can induce cell death in various cancer cells and, therefore, may be a potential cancer therapy.2 Autophagy is reported to be induced by the inhibition of antiautophagic proteins such as tissue transglutaminase 2 (TG2), B-cell lymphoma 2 protein (Bcl-2), and protein kinase Cδ (PKCδ).3 This induction would probably lead to autophagic cell death in some apoptosis-resistant cancers such as breast, colon, and pancreatic cancers.3 Drugs that can induce autophagy may activate this cell death mechanism as an alternative when cells fail to undergo apoptosis and, therefore, could serve as potential novel therapies. On the other hand, in some cases, suppression of autophagy may boost the effects of cancer treatments such as in radiotherapy, DNA-damaging agents, and antihormone therapies by hindering cancer cell survival and enhancing cell death.4 Therefore, on the basis of the cellular features, either the suppression or the stimulation of autophagy could be able to provide therapeutic advantages © XXXX American Chemical Society

against cancers. Furthermore, the development of autophagy modulators may extend our understanding of novel cancer therapies and eventually lead to the development of new therapeutic approaches for cancer. Over the past several decades, metallopharmaceuticals have been used as therapeutic agents for the treatment of a variety of diseases.5 The discovery of Pt-based cisplatin drugs6 as antitumor agents motivated the development of numerous Ptbased analogues as potential antitumor agents. Furthermore, most of them have shown promising results, such as carboplatin and oxaliplatin, which are clinically used worldwide.7 However, some drawbacks are associated with these complexes, such as (1) neurotoxicity, nephrotoxicity, and myelotoxcity, (2) limited range of antitumor activity, and (3) potential acquired or intrinsic resistance of some tumors.8 In order to overcome these obstacles and develop safer and more effective remedial agents, intensive efforts have been devoted toward the design and pharmacological evaluation of other metal-based antitumor Received: June 14, 2015

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DOI: 10.1021/acs.organomet.5b00512 Organometallics XXXX, XXX, XXX−XXX

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Organometallics


Scheme 1. Synthesis of Arene−Ru Metallacycles 1−6

agents.9 Recently, Ru complexes, especially with amine and imine ligands, have shown very promising anticancer activity. Additionally, some of these complexes have shown low toxicity and high activity against tumors that are resistant to Pt-based drugs.10 Two of the most promising RuIII complexes, imidazolium trans-[tetrachlorido(1H-imidazole)((S)-dimethyl sulfoxide)ruthenate(III)] (NAMI-A) and indazolium and sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (NKP-1339), have successfully passed phase I clinical trials.11 While small mononuclear Ru complexes are currently showing promising activities, studies have revealed that macromolecules also show selective cellular uptake and retention in cancer cells due to damaged lymphatic drainage.12 Therefore, this result is theoretically very encouraging for the development of new macromolecular anticancer agents. Recent studies have also shown that large drug molecules exert vital, effective, and sizeselective uptake into tumor cells.13 Therefore, large metal-based self-assembled supramolecular coordination complexes (SCCs) may facilitate the design of new drug molecules for biological applications.14,15 The inspiration for using SCCs in biological applications originates from their characteristic properties such as (1) easier fine tuning of dimensions of complexes, (2) ease of selection of metal ions with specific size, coordination geometry, and oxidation state and versatility for biological applications, (3) simple incorporation of essential functional groups on SCCs through pre- or post-self-assembly modifications, and (4) the suitability of internal cavities of SCCs as drug-delivery systems. Recently, Therrien et al. utilized the concept of self-assembly and synthesized various Ru-based self-assembled complexes using a combination of organometallic half-sandwich Ru clips and dipodal or tripodal donors. They demonstrated that Rubased SSCs can act as hosts for encapsulation of photosensitizers and studied their biological significance.16 At the outset of our research program, we sought to develop new arene−Ru supramolecular complexes consisting of arene−Ru acceptors and various pyridyl donors, focusing on potential

anticancer activity and cellular pharmacology,17 Here, we report the synthesis of six new potential [2 + 2] assemblies (1−6), which are oxalate-, 2,5-dioxido-1,4-benzoquinonato (dobq)-, and 5,8-dioxido-1,4-naphthaquinonato (donq)-based O,Obridging ligands, between the Ru centers of acceptors A1− A3, with the two distinct rigid organic donors L1 and L2. These syntheses were followed by structural characterization and anticancer activity assays using both in vitro and in vivo methods. A series of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and hollow fiber (HF) assays were conducted to provide evidence of their anticancer activities. Moreover, we investigated the involvement of autophagy in metallacycle-induced cancer cell death using the monodansylcadaverine (MDC) staining assay.18

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RESULTS AND DISCUSSION The SCCs 1−3 in Scheme 1 were synthesized by stirring 3,6bis(pyridin-4-yl)-1,2,4,5-tetrazine (L1) with [Ru2(μ-η4-C2O4)(MeOH)2(η6-p-iPrC6H4Me)2][CF3SO3]2 (A1), [Ru2(dobq)(M eOH) 2 ( η 6 -p-iPrC 6 H 4 Me) 2 ] [C F 3 SO 3 ] 2 ( A2 ), or [Ru2(donq)(H2O)2(η6-p-iPrC6H4Me)2][CF3SO3]2 (A3) in an equimolar ratio (1/1) in CH3NO2/CH3OH (1/1, v/v) for 24 h at room temperature. The reaction solutions were filtered and concentrated to reduce the volume, and then diethyl ether was added to precipitate the product. In contrast, SCCs 4−6 were self-assembled over the course of 36 h at 40 °C in CH3NO2/ CH3OH (1/1, v/v) solutions containing equimolar amounts of A1−A3 with 2,5-bis(pyridin-4-ylethynyl)furan (L2). The complexes were fully characterized using proton (1H) and carbon-13 (13C) nuclear magnetic resonance (NMR), electrospray ionization−mass spectrometry (ESI-MS), ultraviolet− visible (UV−vis) absorption spectroscopy, and elemental analysis, while the structure of 1 was determined in the solid state using X-ray crystallography. In the 1H NMR spectra of 1−3, the Hα nuclei of the pyridine rings experienced upfield shifts (Δδ 0.2−0.5 ppm) in comparison to L1. In each case, the upfield shifts of the spectra B


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Organometallics

solutions prior to recording their UV−vis absorption spectra (Figure 2). The intense bands at 250 and 350 nm for L1 and


were most likely due to the loss of electron density following coordination of the pyridine N with the Ru(II) centers (Figure 1). However, protons of the capped p-cymene moiety,

Figure 1. Proton (1H) nuclear magnetic resonance (NMR) spectra of L1 (top) and 1 (bottom) recorded in CD3NO2.

benzoquinone protons of 2, and naphthoquinone protons of 3 did not show any significant changes in chemical shift in comparison to A1−A3. In order to elucidate the exact composition of the product, we performed ESI-MS. The ESIMS spectra of the multiply charged ions for 1 at m/z 579.57 [1 − 3CF3SO3]3+ and m/z 397.41 [1 − 4CF3SO3]4+, for 2 at m/z 422.39 [2 − 4CF3SO3]4+, and for 3 at m/z 646.35 [3 − 3CF3SO3]3+ are in agreement with the theoretical isotopic distribution patterns (Figures S8−S14 in the Supporting Information) and confirmed the formation of [2 + 2] selfassembled tetranuclear macrocycles 1−3. Similarly, pyridyl protons of complexes 4−6 also showed significant upfield shifts in comparison to free L2. Protons of the capped p-cymene moiety, benzoquinone protons of 5 (δ 5.76 ppm), and naphthoquinone protons of 6 (δ 7.24 ppm) did not show any significant changes in chemical shift in comparison to A1−A3. The ESI mass spectra of 4−6 showed peaks at m/z 602.38 [4 − 3CF3SO3]3+ for 4, 635.73 [5 − 3CF3SO3]3+ and 439.55 [5 − 4CF3SO3]4+ for 5, and 669.07 [6 − 3CF3SO3]3+ for 6 (Figures S15−S17 in the Supporting Information). The appearance of these peaks and their similarity to the theoretical isotropic distribution patterns confirmed the formation of [2 + 2] self-assembled tetranuclear macrocycles 1−3. Complexes 1−6, their corresponding metal acceptors A1− A3, and donors L1 and L2 were dissolved in methanol

Figure 2. Ultraviolet−visible (UV/vis) spectra of metallarectangles (a) 1−3 and (b) 4−6 and (c) acceptors (A1−A3) and donors (L1 and L2).

L2, respectively, corresponded to intraligand π → π* transitions, which were also present in the spectra of metallacycles 1−6 after self-assembly. High-energy absorption bands were observed for metal acceptors A1−A3 at 250−300 nm, while low-energy absorption bands from ∼400 to 550 nm were observed for the donq spacer of A3. Molecular Structure of 1·(C2H5)2O·CH3OH. The molecular structure of 1 was further determined using X-ray singlecrystal analysis. The vapor diffusion of diethyl ether into a methanol solution of 1 caused the complex 1·(C2H5)2O· CH3OH to crystallize, which was suitable for X-ray diffraction using synchrotron radiation. The crystal structure of 1· (C2H5)2O·CH3OH showed two crystallographically independent tetranuclear metallacycles located at the inversion center. One of the independent tetranuclear metallacycles is described in Figure 3. The tetradentate dicarboxylate ligands bridged two Ru sites vertically, while the dipyridyl donor L1 connected two Ru sites horizontally. Two dipyridyl donors were bent inward toward the center of the rectangle. Therefore, pyrene moieties of the two independent metallacycles exhibited intramolecular π−π interactions with bond lengths of 3.326 and 3.163 Å C


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Table 2. Half-Maximum Inhibitory Concentration (IC50) Values of Acceptor A3, Donors L1 and L2, Metallacycles 1− 6, Cisplatin, And Doxorubicin in AGS and HCT-15 Human Cancer Cell Lines


IC50 (μM)a

Figure 3. (a) X-ray crystal structure of tetranuclear metallacycle 1. Solvent molecules, counteranions, and hydrogen atoms are omitted for clarity (green, Ru; red, O; blue, N; gray, C). (b) Corey−Pauling-Koltun (CPK) model of 1.

sample

AGS

1 2 3 4 5 6 L1 L2 A3 cisplatin doxorubicin

>200 180 ± 12 30 ± 1 >200 105 ± 10 23 ± 1 >200 152 ± 5 125 ± 8 107 ± 2 3±0

HCT-15 >200 >200 29 ± >200 >200 27 ± >200 >200 >200 12 ± 15 ±

1

3

2 1

a

IC50 denotes the drug concentration necessary for 50% inhibition of cell viability. Data are given as mean ± standard error (SE) values.

short-term in vivo pharmacodynamic studies without problems such as immune cell infiltration.19 The high in vitro anticancer activity of complexes 3 and 6 motivated us to perform an HF assay. The semipermeable HFs loaded with HCT-15 cells were implanted in the intraperitoneal (IP) and subcutaneous (SC) compartments of host mice, which were treated with 100 μg (kg bw)−1 day−1 of complexes 3 and 6 for 7 days. The results showed that HCT-15 cell proliferation was inhibited by both complexes 3 and 6 (IP, Figure 4) by 14.2 and 21.8% (p < 0.05

(Figure S18 in the Supporting Information). Table 1 shows the significant bond distances and angles. In Vitro Anticancer Activity. Cell viability and cytotoxicity tests in various cells are mainly used for efficacy screening and cytotoxicity analysis of drug candidates. The in vitro anticancer efficacies of metallacycles 1−6, their respective ligands (L1 and L2), and acceptor A3 were determined by measuring growthinhibitory activity against AGS and HCT-15 human cancer cell lines (gastric and colorectal carcinomas, respectively). These cells were exposed to increasing concentrations of the compounds for 24 h, and then cell viability was evaluated. The results were compared with those of the clinically used anticancer agents cisplatin and doxorubicin (Table 2). The results demonstrated that 1, 4, and donor L1 were inactive (IC50 > 200 μM) against both cancer cell lines. Acceptor A3, donor L2, and metallacycles 2 and 5 showed poor activity, with IC50 values between 105 and 180 and >200 μM against AGS and HCT-15, respectively. Complexes 3 and 6 showed significant activity. The data indicate that 3 and 6 (IC50 30 and 23 μM, respectively) were more potent than cisplatin (IC50 107 μM) against AGS cancer cells and comparable (IC50 29 and 27 μM, respectively) to cisplatin and doxorubicin (IC50 12 and 15 μM, respectively) against HCT-15 cells. In Vivo Anticancer Activity in HF Animal Model. The hollow-fiber (HF) assay was used as an in vivo screening model to provide a quantitative indication of anticancer activity on the basis of the standards of the National Cancer Institute. The HF assay has several advantages over the classical tumor implantation model, including retrieval of tumor cells uncontaminated by host cells for subsequent analysis. Many studies have demonstrated that the HF assay is appropriate for

Figure 4. Antitumor effects of 3 and 6 against the HCT-15 cell line using hollow-fiber (HF) assay in nude mice. Results are given as means ± standard error (SE) values (n = 6). Statistical analysis was performed using one-way analysis of variance (ANOVA) ((*) p < 0.05 and (**) p < 0.01) in comparison with control.

and 0.01 vs control, respectively). Furthermore, complex 6 was more effective in inhibiting HCT-15 cells than complex 3 was in both in vitro and in vivo experiments. Although complex 6 was not as efficient as cisplatin (37.9%), the present study suggests that it manifested antitumor potential against color-

Table 1. Bond Lengths (Å) and Angles (deg) for 1·(C2H5)2O·CH3OH Ru(1A)−N(1A) Ru(1A)−O(2) Ru(2A)−O(3) N(1A)−Ru(1A)−O(2) N(1A)−Ru(1A)−O(1) O(2)−Ru(1A)−O(1)

2.078(8) 2.104(6) 2.149(8) 83.3(3) 84.1(3) 78.7(3)

Ru(1A)−O(1) Ru(2A)−N(6A) Ru(2A)−O(4) N(6A)−Ru(2A)−O(4) N(6A)−Ru(2A)−O(3) O(4)−Ru(2A)−O(3) D

2.116(6) 2.076(9) 2.088(8) 85.2(3) 84.8(3) 79.0(3) DOI: 10.1021/acs.organomet.5b00512 Organometallics XXXX, XXX, XXX−XXX

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Organometallics

low concentrations comparable with those of cisplatin and doxorubicin. In addition, complexes 3 and 6 also exhibited potent antitumor activity against HCT-15 cells in in vivo HF assays. Furthermore, we found that complexes 3 and 6 induced autophagy in HCT-15 cells. These results suggest that the autophagic response elicited by complexes 3 and 6 could mediate the anticancer effects in human colorectal cancer cells. Further studies to elucidate the detailed autophagic mechanism of these complexes are underway.


ectal cancer. However, the responses observed with cells implanted at the SC site were less intense at 7.9, 8.5, and 10.0% for 3, 6, and cisplatin, respectively. These results are probably attributable to less blood-vessel development, which would inhibit delivery as described previously.17d,e Autophagy Activity. Autophagic programmed cell death has been extensively investigated as a tumor suppressor on the basis of its prevention of the accumulation of damaged proteins. Numerous anticancer agents induce cancer cell death by autophagy activation, which is frequently associated with G1 arrest. Complexes 3 and 6 exhibited potent in vitro and in vivo anticancer activities, and therefore, we sought to investigate whether these activities were associated with autophagy activation. Monodansylcadaverine (MDC) staining can be used to detect autophagic vacuoles, which accumulate during the mature autophagy process.20 As shown in Figure 5, the

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EXPERIMENTAL SECTION

General Details. The acceptor clips [Ru 2 (μ-η 4 -C 2 O 4 )(MeOH) 2 (η 6 -p-iPrC 6 H 4 Me) 2 ][CF 3 SO 3 ] 2 (A1), 21 [Ru 2 (dobq)(MeOH)2(η6-p-PriC6H4Me)2][CF3SO3]2 (A2),22 and [Ru2(donq)(H2O)2(η6-p-PriC6H4Me)2][CF3SO3]2 (A3)23 were synthesized under an atmosphere of dry nitrogen using a standard Schlenk technique following previously reported procedures. 3,6-Bis(pyridin-4yl)-1,2,4,5-tetrazine (L1)24 and 2,5-bis(pyridin-4-ylethynyl)furan (L2)25 were synthesized by following previously reported procedures. The solvents were dried and distilled according to standard procedures in the published literature.26 The 1H and 13C NMR spectra were recorded using a Bruker 300 MHz spectrometer, and the chemical shifts (δ) in 1H NMR spectra were reported in ppm relative to tetramethylsilane (Me4Si) as the internal standard (0.0 ppm). Mass spectra were recorded using a Micromass Quattro II triple-quadrupole mass spectrometer using ESI with the MassLynx software suite. Elemental analyses were performed using an Elemental GmbH Vario EL-3 instrument. From a single crystal of 1·(C2H5)2O·CH3OH, the diffraction data were collected at 100 K using an ADSC Quantum 210 CCD diffractometer with synchrotron radiation (λ 0.80000 Å) at the Supramolecular Crystallography Beamline 2D (Pohang Accelerator Laboratory (PAL), Pohang, Korea). The HKL2000 program was used to process and scale the raw data.27 The structures of the compounds were elucidated and confirmed using direct methods, and the refinements were performed with full-matrix least squares on F2 with appropriate software implemented in the SHELXTL program package.28 Methods. Cell Culture and MTT Assay. The cell lines used in this experiment were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The AGS human gastric and HCT-15 human colorectal carcinoma cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium with 5% fetal bovine serum (FBS) at 37 °C under a humidified atmosphere of 5% CO2. Cell viability following treatment with the compounds was evaluated by the MTT assay. Cells were seeded into 96-well plates and incubated at 37 °C, and then cells were stimulated with each compound for the indicated periods of time. Cells were incubated with 10 μL of solution for 4 h at 37 °C and 5% CO2; 100 μL of DMSO was added to each well. The 96-well plates were read by a multireader (Tecan, Switzerland) at 550 nm for absorbance density, and final values were expressed as the ratio of absorbance of treated to untreated cells. The IC50 values for cell growth were determined by fitting the plot of the logarithmic percentage of surviving cells against the logarithm of the drug concentration using a linear regression function. In Vivo Implantation and HF Assay. Polyvinylidene fluoride (PVDF) hollow fibers were purchased from Spectrum Laboratories, Inc., (Houston, TX, USA). HFs with 1.2 and 1 mm outer and inner diameters, respectively, and a molecular weight cutoff point of 500 kDa were flushed with normal growth medium and then filled with HCT15 cells at a density of 5 × 105 cells/mL. Each fiber was subsequently heat-sealed with a hot smooth-jawed needle holder every 1.5 cm along the length and cut into segments with 2 mm tails. The sealed fibers were incubated in a six-well culture plated for 24 h prior to implantation into the mice. For in vivo implantation of HFs, 6-weekold male nu/nu nude mice were obtained from SLC Inc., (Hamamatsu, Japan) and were kept for 7 days prior to experimentation. Mice were provided with sterile standard mouse chow (Purina 5001 Rodent

Figure 5. Relative autophagy activity of complexes 3 and 6 in the HCT-15 cell line. Results are given as means ± standard error (SE) values. Statistical analysis was performed using one-way analysis of variance (ANOVA) ((*) p < 0.05 and (**) p < 0.01) in comparison with control.

number of MDC-labeled autophagic vacuoles markedly increased following treatment with complexes 3 and 6. Furthermore, we observed that both complexes induced a strong concentration-dependent increase in autophagy activity at low concentrations (0−5 μM), while tamoxifen (positive control) increased autophagy at high concentrations (50−100 μM, Figure S19 in the Supporting Information). Specifically, complex 6 induced a greater than 2-fold increase in autophagy activity. However, at higher concentrations of complexes 3 and 6, autophagy activities decreased. These results were paralleled by a decrease in cell proliferation and increase in cell death. During complex 6 induced HCT-15 tumor cell death, a large increase in autophagy activity occurred. This present result suggests that complex 6 may exert potent anticancer activity through autophagic cell death, and further studies are required to determine the precise molecular mechanisms.

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CONCLUSION In summary, six tetranuclear metallacycles of varying sizes were prepared using coordination-driven self-assembly between arene−Ru-based acceptors and dipyridyl donors. The formation of the metallacycles was confirmed using 1H and 13C NMR and ESI-MS spectroscopic techniques, and the molecular structure of metallarectangle 1 was unambiguously confirmed by singlecrystal X-ray diffraction analysis. The cytotoxicity of these metallacycles against AGS and HCT-15 human cancer cell lines was evaluated, with complexes 3 and 6 exhibiting IC50 values at E


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(d, J = 6.2 Hz, 8H), 2.86−3.01 (m, 4H), 2.20 (s, 12H), 1.38 (d, J = 7.0 Hz, 24H); 13C NMR (75 MHz, [D4]methanol) δ 172.5, 163.3, 154.7, 147.3, 132.4, 128.7, 127.9, 103.8, 99.3, 84.0, 83.4, 32.7, 22.8, 18.3; MS (ESI) for 3 (C88H80F12N12O20Ru4S4) 646.35 (found), 646.32 (required) for [3 − 3CF3SO3]3+. Anal. Calcd for 3·H2O: C, 43.96; H, 3.44; N, 6.99. Found: C, 43.88; H, 3.63; N, 6.76. Self-Assembly of Metallacycle 4. 2,5-Bis(pyridin-4-ylethynyl)furan (L2; 2.70 mg, 0.01 mmol) and Ru acceptor A1 (8.56 mg, 0.01 mmol) were placed in a 4 dram vial, followed by the addition of CH3OH and CH3NO2 (1 mL each), and the reaction mixture was stirred at 40 °C for 36 h. The reaction solution was filtered using a cotton plug and concentrated to reduce the volume (0.2 mL), and then diethyl ether (3 mL) was added to precipitate the product. The resulting powder was vacuum-dried to give 4 as a yellow solid (yield 88%). The spectral data are as follows: 1H NMR (300 MHz, [D4]methanol) δ 8.03 (d, J = 6.0 Hz, 8H), 7.53 (d, J = 6.0 Hz, 8H), 6.89 (s, 4H), 5.92 (d, J = 6.1 Hz, 8H), 5.75 (d, J = 6.1 Hz, 8H), 2.74− 2.88 (m, 4H), 2.21 (s, 12H), 1.35 (d, J = 6.7 Hz, 24H); 13C NMR (75 MHz, [D4]methanol) δ 172.5, 153.9, 139.2, 134.8, 128.3, 122.3, 104.0, 99.1, 92.7, 89.1, 83.7, 83.3, 32.7, 22.7, 18.2; MS (ESI) for 4 (C84H76F12N4O22Ru4S4) 602.38 (found), 602.38 (required) for [4 − 3CF3SO3]3+. Anal. Calcd for C84H76F12N4O22Ru4S4 (2254.04): C, 44.76; H, 3.40; N, 2.49. Found: C, 44.58; H, 3.69; N, 2.43. Self-Assembly of Metallacycle 5. 2,5-Bis(pyridin-4-ylethynyl)furan (L2; 2.70 mg, 0.01 mmol) and Ru acceptor A2 (9.06 mg, 0.01 mmol) were placed in a 4 dram vial, followed by the addition of CH3OH and CH3NO2 (1 mL each), and the reaction mixture was stirred at 40 °C for 36 h. The reaction solution was filtered using a cotton plug and concentrated to reduce the volume (0.2 mL), and then diethyl ether (3 mL) was added to precipitate the product. The resulting powder was vacuum-dried to give 5 as a wine red solid (yield 86%). The spectral data are as follows: 1H NMR (300 MHz, [D4]methanol) δ 8.27 (d, J = 6.3 Hz, 8H), 7.49 (d, J = 6.3 Hz, 8H), 6.93 (s, 4H), 6.03 (d, J = 6.2 Hz, 8H), 5.76−5.84 (m, 12H), 2.80−2.95 (m, 4H), 2.19 (s, 12H), 1.35 (d, J = 7.0 Hz, 24H); 13C NMR (75 MHz, [D4]methanol) δ 185.5, 154.2, 139.0, 134.7, 128.4, 121.9, 119.9, 105.5, 103.0, 100.2, 92.2, 88.1, 85.0, 83.5, 32.8, 22.7, 18.3; MS (ESI) for 5 (C92H80F12N4O22Ru4S4) 635.73 (found), 635.72 (required) for [5 − 3CF3SO3]3+ and 439.55 (found), 439.55 (required) for [5 − 4CF3SO3]4+. Self-Assembly of Metallacycle 6. 2,5-Bis(pyridin-4-ylethynyl)furan (L2; 2.70 mg, 0.01 mmol) and Ru acceptor A3 (9.57 mg, 0.01 mmol) were placed in a 4 dram vial, followed by the addition of CH3OH and CH3NO2 (1 mL each), and the reaction mixture was stirred at 40 °C for 36 h. The reaction solution was filtered using a cotton plug and concentrated to reduce the volume (0.2 mL), and then diethyl ether (3 mL) was added to precipitate the product. The resulting powder was vacuum-dried to give 6 as a sea green solid (yield 90%). The spectral data are as follows: 1H NMR (300 MHz, [D4]methanol) δ 8.42 (d, J = 6.6 Hz, 8H), 7.43 (d, J = 6.6 Hz, 8H), 7.24 (s, 8H), 6.92 (s, 4H), 5.84 (d, J = 6.4 Hz, 8H), 5.61 (d, J = 6.4 Hz, 8H), 2.75−2.89 (m, 4H), 2.09 (s, 12H), 1.32 (d, J = 6.8 Hz, 24H); 13 C NMR (75 MHz, [D4]methanol) δ 172.5, 153.3, 138.8, 134.5, 128.2, 121.6, 119.9, 112.8, 105.2, 101.3, 92.4, 87.7, 86.0, 84.2, 32.2, 22.7, 17.5; MS (ESI) for 6 (C100H84F12N4O22Ru4S4) 669.07 (found), 669.07 (required) for [6 − 3CF3SO3]3+; Anal. Calcd for 6·H2O: C, 48.58; H, 3.51; N, 2.27. Found: C, 48.26; H, 3.74; N, 2.39. X-ray Diffraction Data for 1·(C2H5)2O·CH3OH. All of the nonhydrogen atoms were refined anisotropically. Hydrogen atoms were added to their geometrically ideal positions except for the OH groups of methanol and the water molecules. The X-ray diffraction data for 1· (C2H5)2O·CH3OH are as follows: C74H84F12N12O24Ru4S4, Mw = 2286.05, triclinic, P1̅ (No. 2), a = 16.578(3) Å, b = 17.187(3) Å, c = 17.842(4) Å, α = 86.14(3)°, β = 87.59(3)°, γ = 73.73(3)°, V = 4867.6(16) Å3, Z = 2, T = 100 K, μ(synchrotron) = 1.072 mm−1, ρcalcd = 1.560 g cm−3, 28065 reflections measured, 14207 unique (Rint = 0.0153), R1 = 0.1282, wR2 = 0.4233 for 12781 reflections (I > 2σ(I)), R1 = 0.1324, wR2 = 0.4361 (all data), GOF = 2.239, 1292 parameters, and 217 restraints.

Chow, Purina, St. Louis, MO, USA) and water ad libitum. All animals were housed at a temperature of 22 ± 2 °C and humidity of 55 ± 5% under a 12 h light−dark cycle. The mice were anesthetized by IP administration of a mixture of Zoletil 50 (Virbac, Carros, France) and Rompun (Bayer Korea, Seoul, Korea) at a 3/1 ratio (1 mL/kg). The sealed HF segments were implanted SC and IP using an 11 gauge Trocar insert and were stapled. Two days after HF implantation, mice were treated daily for 7 days by oral gavage with 100 μg (kg bw)−1 day−1 of complexes 3 and 6 while cisplatin was administered intravenously at 2 mg/(kg bw) every 2 days. In order to evaluate the numbers of viable cells in the HFs, they were longitudinally dissected in 0.5 mL of ethylenediaminetetraacetic acid (EDTA) and washed with EDTA solution for 3 min, 0.5 mL of trypsin for 5 min, and medium for 3 min. The wash solutions were then centrifuged at 500g for 5 min to collect the cells, and an MTT assay was used to determine the number of viable cells. Labeling of Autophagic Vacuoles with Monodansylcadaverine (MDC). Autophagosomes in the cells were detected with fluorescent monodansylcadaverine (MDC) staining.18 Cells were incubated with 50 μM MDC for 1 h at 37 °C, fixed in 4% paraformaldehyde (15 min), washed twice with phosphate-buffered saline (PBS), and then dissolved in DMSO. The fluorescence was determined with a multiplate reader (excitation and emission wavelengths of 380 and 525 nm, respectively). Self-Assembly of Metallarectangle 1. 3,6-Bis(pyridin-4-yl)1,2,4,5-tetrazine (L1; 2.36 mg, 0.01 mmol) and Ru acceptor A1 (8.56 mg, 0.01 mmol) were placed in a 4 dram vial, followed by the addition of CH3OH and CH3NO2 (1 mL each), and the reaction mixture was stirred at room temperature for 24 h. The reaction solution was filtered using a cotton plug and concentrated to reduce the volume (0.2 mL), and then diethyl ether (3 mL) was added to precipitate the product. The resulting powder was vacuum-dried to give 1 as a brown solid (yield 92%). The spectral data are as follows: 1 H NMR (300 MHz, [D3]nitromethane) δ 8.47 (d, J = 6.0 Hz, 8H), 8.37 (d, J = 6.0 Hz, 8H), 5.96 (d, J = 6.3 Hz, 8H), 5.81 (d, J = 6.3 Hz, 8H), 2.85−2.98 (m, 4H), 2.27 (s, 12H), 1.39 ppm (d, J = 6.9 Hz, 24H); 13C NMR (75 MHz, [D3]nitromethane) δ 166.9, 158.0, 150.0, 136.8, 119.7, 99.1, 93.9, 78.1, 77.7, 27.0, 17.0, 12.9; MS (ESI) for 1 (C72H72F12N12O20Ru4S4) 579.57 (found), 579.57 (required) for [1 − 3CF3SO3]3+ and 397.41 (found), 397.42 (required) for [1 − 4CF3SO3]4+. Anal. Calcd for C72H72F12N12O20Ru4S4 (2185.93): C, 39.56; H, 3.32; N, 7.69. Found: C, 39.23; H, 3.63; N, 7.37. Self-Assembly of Metallarectangle 2. 3,6-Bis(pyridin-4-yl)1,2,4,5-tetrazine (L1; 2.36 mg, 0.01 mmol) and Ru acceptor A2 (9.06 mg, 0.01 mmol) were placed in a 4 dram vial, followed by the addition of CH3OH and CH3NO2 (1 mL each), and the reaction mixture was stirred at room temperature for 24 h. The reaction solution was filtered using a cotton plug and , concentrated to reduce the volume (0.2 mL), and then diethyl ether (3 mL) was added to precipitate the product. The resulting powder was vacuum-dried to give 2 as a wine red solid (yield 90%). The spectral data are as follows: 1 H NMR (300 MHz, [D3]nitromethane) δ 8.66 (d, J = 6.4 Hz, 8H), 8.49 (d, J = 6.4 Hz, 8H), 6.01 (d, J = 6.1 Hz, 8H), 5.75−5.89 (m, 12H), 2.87−3.03 (m, 4H), 2.25 (s, 12H), 1.40 (d, J = 6.9 Hz, 24H); 13 C NMR (75 MHz, [D3]Nitromethane) δ 180.5, 150.1, 119.4, 100.1, 97.3, 94.9, 79.3, 78.1, 27.2, 17.0, 12.9; MS (ESI) for 2 (C80H76F12N12O20Ru4S4) 422.39 (found), 422.44 (required) for [2 − 4CF3SO3]4+. Anal. Calcd for 2·H2O: C, 41.70; H, 3.41; N, 7.30. Found: C, 41.83; H, 3.27; N, 6.90. Self-Assembly of Metallarectangle 3. 3,6-Bis(pyridin-4-yl)1,2,4,5-tetrazine (L1; 2.36 mg, 0.01 mmol) and Ru acceptor A3 (9.57 mg, 0.01 mmol) were placed in a 4 dram vial, followed by the addition of CH3OH and CH3NO2 (1 mL each), and the reaction mixture was stirred at room temperature for 24 h. The reaction solution was filtered using a cotton plug and concentrated to reduce the volume (0.2 mL), and then diethyl ether (3 mL) was added to precipitate the product. The resulting powder was vacuum-dried to give 3 as a sea green solid (yield 91%). The spectral data are as follows: 1 H NMR (300 MHz, [D3]nitromethane) δ 8.81 (d, J = 6.3 Hz, 8H), 8.44 (d, J = 6.3 Hz, 8H), 7.28 (s, 8H), 5.82 (d, J = 6.2 Hz, 8H), 5.63 F


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ASSOCIATED CONTENT

* Supporting Information S


The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.organomet.5b00512. CCDC-993943 (1) also contains supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre at www.ccdc.cam.ac.uk/data_request/cif. NMR and MS details, X-ray crystal structure of metallarectangle 1, and comparative stability test data of metallacycles 3 and 6 in the cell culture medium and DMSO in HCT-15 cells (PDF) X-ray crystal structure and crystallographic data of metallarectangle 1 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail for S.C.K.: [email protected]. *E-mail for K.-W.C.: [email protected]. Author Contributions ⊥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS K.-W.C. gratefully acknowledges the generous financial support of the B asic Science Research p rogram (NRF2013R1A1A2006859) and the Priority Research Center Program (2009-0093818) through the National Research Foundation of Korea and the X-ray diffraction experiments performed at the Pohang Accelerator Laboratory in Korea. P.J.S. thanks the National Institutes of Health (NIH) for financial support (GM-57052). H.K. thanks the Research and Development Program of the Korea Institute of Energy Research (KIER, B5-2513).

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REFERENCES

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