Low Toxicity β-Cyclodextrin-Caged 4,4 - ACS Publications - American

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Low Toxicity β‑Cyclodextrin-Caged 4,4′-Bipyridinium-bis(siloxane): Synthesis and Evaluation Narcisa Marangoci,† Stelian S. Maier,†,‡ Rodinel Ardeleanu,† Adina Arvinte,† Adrian Fifere,† Anca Roxana Petrovici,† Alina Nicolescu,† Valentin Nastasa,§ Mihai Mares,§ Sorin A. Pasca,§ Ramona F. Moraru,§ Mariana Pinteala,*,† and Anca Chiriac∥ †

Centre of Advanced Research in Bionanoconjugates and Biopolymers, “Petru Poni” Institute of Macromolecular Chemistry of Romanian Academy, Iasi 700487, Romania ‡ Department of Textile and Leather Chemical Engineering, “Gheorghe Asachi” Technical University of Iasi, Iasi 700050, Romania § Faculty of Veterinary Medicine, “Ion Ionescu de la Brad” University of Agricultural Sciences and Veterinary Medicine, Iasi 700489, Romania ∥ Nicolina Medical Center, CMI Dermatology, Iasi 700613, Romania S Supporting Information *

ABSTRACT: The toxicity of viologens can be significantly reduced by including them in tight [2]rotaxane structures alongside β-cyclodextrin, thus turning them into candidates of pharmaceutical interest. Here, we report a synthesis pathway for a benign viologen, by capping a small β-cyclodextrin-caged molecule, the 4,4′-bipyridine, with minimal-length presynthesized axlestopper segments of the propyl-3-pentamethyldisiloxane type. After 90 min from the oral administration to laboratory mice, the product concentration in the bloodstream reaches a value equivalent to 0.634% of the initial dose of 800 mg·kg−1. As compared to the nude viologen having the same structure, which proved to be lethal in doses of 40 mg·kg−1, the product induces reversible morphological changes in the liver, kidney, lung, and cerebellum, up to a dose of 400 mg·kg−1, with higher dosages giving rise to a chronic slow evolution. due to their ability to accomplish DNA strand scission.10 Methyl viologen (Paraquat) has been extensively used as a herbicide,11−13 and numerous works were done to evaluate its action on the human body because of the severe suspicions about its toxicity against mammals,14 induced by its low redox potential.15−17 Hatcher et al.18 have proved the ability of several pesticides, including those from the class of viologen herbicides, to increase the incidence of Parkinson’s disease. Once ingested, 4,4′-bipyridyl viologens (bPy2+) can be enzymatically reduced to form a radical cation (bPy•+) which, in the presence of O2 and/or H2O2, generates highly reactive radicals (e.g., HO•), causing the oxidation of some species of biochemical importance, like cell membrane lipids, proteins, and nucleic acids.8 In this context, scientists are trying to develop new strategies for the treatment of viologen poisoning but also to capitalize its redox potential in pharmacological purposes. Of current interest is the host−guest complexation with cyclodextrins (CDs), which can be evidenced by spectroscopic methods based on the changes of spectra upon complexation.19

1.0. INTRODUCTION Through appropriate chemical architecturing, toxic compounds like viologens can be transformed into pharmacologically active species.1,2 As we will further demonstrate, the toxicity of viologens (1,1′-di(hydroxycarbyl)-4,4′-bipyridinium salts, according IUPAC nomenclature) is significantly diminished by host−guest complexation, which turns them into candidates of pharmaceutical interest. Viologens became interesting to the scientific world in 1933 when Michaelis reported his first study on their electrochemical properties.3 Surprisingly for those early studies, viologens showed the lowest redox potential as compared with any other organic compound, together with a significant degree of redox reversibility. Shortly after that, they became the “parents” of a whole herbicide family, mainly because of their redox potential.4 It has been also demonstrated that electrochemically reduced viologens can further reduce compounds that are not electroactive by themselves.4,5 Recently, viologens were involved in advanced applications such as electrochromic display devices,6 molecular wires in molecular electronic devices,7 and prooxidants in oxidative stress testing.8 Viologens also revealed antibacterial efficiency9 toward Escherichia coli, © 2014 American Chemical Society

Received: November 5, 2013 Published: February 24, 2014 546

dx.doi.org/10.1021/tx400407e | Chem. Res. Toxicol. 2014, 27, 546−557

Chemical Research in Toxicology

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6.05 g (50 mmol) allyl bromide in 10 mL of dry toluene was added dropwise under a nitrogen atmosphere. The mixture was further stirred for 20 h, under the same conditions. The reaction progress was monitored by FTIR via the disappearance of the peak at 2140 cm−1. Finally, the solvents and unreacted allyl bromide were removed by vacuum distillation. The reaction product was a mixture of α- and βaddition isomers (β:α molar ratio of about 8:1, as calculated from the 1 H NMR spectrum). Br-PMDS was obtained as a brown viscous product, in a yield of 81.2% (10.33 g). 1 H NMR (400 MHz, CDCl3) δ ppm: 0.09−0.24 (m, Si−CH3), 0.52−0.62 (m, Si−CH2), 1.42−1.67 (m, Si-CH2−CH2-CH2−Br), 3.39−3.41 (m, Si-CH2−CH2−CH2-Br). 2.4. Preparation of the [2]Rotaxane Structure Consisting of β-CD-Caged Propyl-pentamethyldisiloxane Modified Viologen (β-CD/bPy2+PMDS). The β-CD/bPy2+PMDS complex was prepared by the quaternization of the nitrogen atoms28,30 of bPy with the monofunctional disiloxane bromide (Br-PMDS), in a Br/bPy molar ratio of 2:1 by the following procedure: 1.29 g (1 mmol) of the β-CD/ bPy inclusion complex was dissolved in 5 mL of anhydrous DMF under magnetic stirring, and then 0.53 g (2 mmol) of Br-PMDS was added dropwise. The reaction mixture was heated at 100 °C, for 20 h, under continuous stirring. The final solution was cooled, and the reaction product was precipitated in 50 mL of anhydrous acetone. The precipitate was then filtered, dried, and recrystallized twice from anhydrous DMF in anhydrous acetone. Finally, the precipitate was dried at 50 °C, in a vacuum oven, for 24 h (yield 71%). 1 H NMR (400 MHz, DMSO-d6) δ ppm: 0.05−0.09 (m, 30H, Si− CH3), 0.53−0.54 (m, 4H, Si−CH2), 1.82−1.98 (m, 4H, Si-CH2−CH2CH2−Br), 3.29−3.38 (m, 14H, H-2, H-4), 3.56−3.68 (m, 21H, H-3, H-5, H-6), 4.89−5.07 (br, 11H, H-1 and CH2-N+), 8.65−8.67 (m, 4H, Ar), 9.27−9.30 (m, 4H, Ar). 2.5. Preparation of N,N′-Bis(1-propyl-3-pentamethyldisiloxane)-4,4′-bipyridinium Dibromide (bPy2+PMDS). bPy2+PMDS was prepared in the same way as β-CD/bPy2+PMDS, but the β-CD/ bPy inclusion complex was replaced by bPy (yield 75%). 1H NMR (400 MHz, DMSO) δ ppm: 0.01−0.20 (m, 30H, Si−CH3), 0.65−0.77 (m, 4H, Si−CH2), 1.95−2.04 (m, 4H, Si-CH2−CH2-CH2−Br), 5.01− 5.04 (m, 4H, CH2N+), 8.85 (d, J = 6.5 Hz, 4H, Ar), 9.46 (d, J = 6.5 Hz, 4H, Ar). 2.6. Nuclear Magnetic Resonance (NMR). The NMR spectra have been recorded on a Bruker Avance DRX 400 spectrometer operated at 400.1 MHz for 1H. The proton chemical shifts were reported in ppm, relative to the solvent residual peak as internal standard (DMSO-d6, 2.51 ppm). 2.7. Determination of the Stoichiometry of the β-CD/bPy Complex. β-CD and bPy were dissolved in deuterated water to obtain stock solutions with a concentration of 10 mM. By keeping the total volume constant at 600 μL, nine binary mixtures having β-CD/bPy molar ratios varying from 0 to 1 were prepared and subjected to 1H NMR investigation to record the chemical shifts of H-a protons of bPy and of OH-3 protons of β-CD. OH-3 protons were chosen because they are located within the β-CD cavity, being prone to interact with H-a protons. The Δδ changes of the successively measured values were multiplied by the corresponding molar fractions and plotted against the molar fraction of bPy in the mixtures, to construct a classic Job plot. 2.8. Electrochemical Investigation. Cyclic voltammetric experiments (CV) were performed on an AutoLab PGSTAT 302N electrochemical system (Eco Chemie Utrecht, The Netherlands). A glassy carbon working electrode (0.07 cm2), a Pt wire counter electrode, and an Ag/AgCl reference electrode were used in a singlecompartment cell. The working electrode was polished with 0.05 and 0.3 μm alumina and abundantly rinsed with water prior to electrochemical measurements. The experiments were conducted in 0.1 mol·L−1 NaClO4 as supporting electrolyte (pH 4) prepared with distilled water. All solutions were deoxygenated by nitrogen purging and maintained under an inert atmosphere during the electrochemical experiments. Each CV study was performed by at least 10 repetitive cycles, scanning the potential between 0.7 V to −1.6 V vs Ag/AgCl, in the above-mentioned supporting electrolyte.

Electrochemical techniques of detection and investigation of CD inclusion complexes are also applied when the guest molecules are redox-active.20 β-Cyclodextrin (β-CD) is a macrocycle composed of seven glucopyranose units attached by α-1,4-linkages. It is able to include different guest molecules into its hydrophobic cavity, generating stable inclusion complexes,21−23 but also pseudorotaxane and/or rotaxane structures.24,25 Because of their unique properties (oxidative stability, low toxicity, physiological inertness, antiadhesive properties, etc.), polymethyldisiloxanes are used in a wide range of biomedical applications such as the production of artificial skin, contact lenses, drug-delivery systems, and transdermal therapeutic systems.26 It is presumable that, besides their antislipping effect relative to the β-CD macrocycle, short chain siloxane end-units (like those of 1,1,3,3,3-pentamethyldisiloxane type, PMDS) will synergically contribute to the biostability of the inclusion complex by decreasing its rate of decomposition in the body.27 The objectives of the present work are to synthesize and characterize a 4,4′-bipyridyum derivative of viologen type having significantly reduced toxicity and to validate it by in vivo tests on mice. In this respect, the intrinsic toxicity of the compound was masked by complexation with β-cyclodextrin, and the structural stability of the resulting inclusion complex was ensured by siloxane stopper end-units, forming a tight [2]rotaxane architecture. To prove the toxicity reduction, the in vivo tests were performed using both the nude viologen and the β-CD complexed one, the histopathological echoes of various administered doses on the liver, kidney, lung, and cerebellum being compared.

2.0. EXPERIMENTAL PROCEDURES 2.1. Chemicals and Reagents. 4,4′-Bipyridine (bPy) (Mw = 156.18 Da), allyl bromide (1-bromoprop-2-ene, Mw = 120.98 Da), βcyclodextrin (β-CD) (Mw= 1134.98 Da), pentamethyldisiloxane (PMDS) (Mw = 148.35 Da), tetramethyldisiloxane (TMS), propyl bromide, and all the general purpose chemicals were purchased from Sigma-Aldrich. Aqueous solutions were prepared using double distiled water (ddH2O). 2.2. Preparation of β-CD/4,4′-Bipyridine Inclusion Complex (β-CD/bPy). The inclusion complex was prepared by the coprecipitation method, as described in previous works.21,28 Briefly, 2.27 g (2 mmol) of β-CD was dissolved in 120 mL of ddH2O by shaking at 30 °C for 30 min to obtain a saturated solution. Then, 0.31 g of bPy (2 mmol) was added under continuous stirring for 25 h, at room temperature, until a white precipitate appeared. The white crude product was filtered using Sartorius quantitative grade 389 filter paper (84 g·m−2, particle size retention 8 to 12 μm) and then washed three times with small amounts of ddH2O to remove unreacted products. The final precipitate was dried at 50 °C, in a vacuum oven, for 24 h (yield 73%). The 1H NMR spectrum of the β-CD/bPy complex in DMSO is presented in Figure S1 of Supporting Information. 1 H NMR (400 MHz, DMSO-d6) δ ppm: 3.29−3.38 (m, 14H, H-2, H-4), 3.56−3.70 (m, 21H, H-3, H-5, H-6), 4.45 (t, J = 5.4 Hz, 7H, OH-6), 4.83 (d, J = 3.2 Hz, 7H, H-1), 5.68 (d, J = 2.1 Hz, 7H, OH-2), 5.73 (d, J = 6.9 Hz, 7H, OH-3), 7.84 (d, J = 6.1 Hz, 4H, Ar), 8.74 (d, J = 6.1 Hz, 4H, Ar). 2.3. Preparation of 1-Bromopropyl-3-pentamethyldisiloxane (Br-PMDS). Br-PMDS was prepared as previously reported.29 In a typical procedure, 6.7 g (50 mmol) of PMDS was dissolved in 40 mL of degassed dry toluene. The resulting solution was stirred and heated at 60−70 °C, under nitrogen atmosphere, then 0.1 mL of 2% H2PtCl6 solution in anhydrous isopropanol was added. After 30 min of continuous stirring, when the color of the solution turned to pale brown, the temperature was increased to 80−90 °C, and a solution of 547



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2.9. In Vivo Tests. The maximum tolerated doses (MTD)31 for bPy2+PMDS and β-CD/bPy2+PMDS were determined on gnotobiotic 18-week-old nulliparous female mice (Cantacuzino Institute, Bucharest, Romania). The study was conducted according to the 63/2010/ EU Directive, being performed with the permission of the Ethical Committee of the Institution. The pre-experiment acclimatization of the mice was conducted in identical temperature (22 ± 0.7 °C) and humidity (60 ± 10%) conditions, and the circadian cycle (light/darkness) was established to 12 h each. The animals were kept for 7 days in the laboratory conditions and were daily monitored for possible disease conditions or abnormal behavior. Each experimental group (5 mice/dose level) was placed in polycarbonate cages, where a surface of approximately 300 cm2/mouse was provided. All animals had permanent access to potable water and standard feed with the following composition: 23% proteins, 10% lipids, 50% carbohydrates, 8% raw fibres, and 9% vitamins− minerals premix (Cantacuzino Institute, Bucharest, Romania). The in vivo experiment was conducted on a total number of 60 mice, divided into three groups: the bPy2+PMDS group (n = 25, average weight 25 ± 0.48 g), the β-CD/bPy2+PMDS group (n = 30, average weight 25 ± 0.19 g), and the control group (n = 5, average weight 25 ± 0.54 g). Toxicity tests were performed in identical conditions, using equivalent doses of reference compound (bPy2+PMDS). For the determination of MTD of β-CD/bPy2+PMDS, the first dosage level was considered the one established as MTD for bPy2+PMDS, in order to minimize the number of tested mice. For bPy2+PMDS, five dose levels were assessed (20, 40, 100, 200, and 400 mg·kg−1 body weight), while for β-CD/bPy2+PMDS, six dose levels were tested (corresponding to 40, 100, 200, 400, 800, and 1600 mg· kg−1 body weight). Distilled water (pH 6.5, conductivity 25 μS) was used as vehicle for both products. Each solution was administered orally by gavage, within a volume of 0.6 mL/mouse. When the calculated volume of the solution was higher than 0.6 mL, it was fractionated twice or three times and was administered at 3 h intervals. All mice received a single dose of the tested compound at day zero. For each dosage level, the tested mice were individually monitored for 14 days, as previously described by Gad and Chengelis,32 by taking into consideration the following six variables: the behavioral changes, the body weight dynamics, the potential toxicity signs, the rate of mortality, the gross necropsy, and the histopathological exams. Body weight was determined right before the animals were euthanized, after an interval of seven days for the ones that survived, including the control group. The mortality rate was expressed as a percentage of surviving mice at 7 and 14 days. The dying mice were considered deceased, being euthanized and examined from the morphological and histopathological points of view. The 5 mice control group only received distilled water (0.6 mL/ mouse). At the end of the monitoring period, the mice of the control group were euthanized for collecting the necessary samples for comparative histopathological exams. 2.10. Necropsy and Histopathological Exam. In order to perform the necropsy and to collect samples for histopathology exams, the mice were euthanized with isoflurane gas. The gross macroscopic examination of the tissues and organs was done in comparison with the mice of the control group, and the following parameters were assessed: color, volume, texture, and appearance of the samples surface. After necropsy, the liver, kidney, lung, and brain were retained from each mouse, for histopathological examination. All organs were subsequently processed according to Ruehl-Fehlert et al.’s recommendation.33 Briefly, all samples were placed in 10% buffered formalin and embedded in paraffin using a Leica TP1020 tissue processor (Leica Microsystems GmbH, Germany). Sections of 5 μm thickness were obtained with a SLEE CUT 6062 Microtome (SLEE Medical GmbH, Germany), deparaffinized, and stained by the Masson trichromic technique. Qualitative histology was performed from stained sections using a Leica DM 750 microscope (Leica Microsystems GmbH, Germany), with an attached Leica ICC50 HD digital camera (Leica Microsystems GmbH, Germany). The images were processed with Leica Application Suit Software (LAS), version 4.2.

The histopathological examination was performed on the same organs and under same conditions, for all mice. 2.11. Detection of the β-CD/bPy2+PMDS Inclusion Complex in Mouse Blood. To demonstrate the presence of β-CD/ bPy2+PMDS in the bloodstream, the product was quantitated by HPLC, in triplicate, as described below. One half and one and a half hours after product ingestion, under deep anesthesia, from the body of mice treated with 800 mg of product·kg−1, about 1 mL of blood was collected by cardiac puncture, just before euthanasia. The blood samples were immediately mixed with EDTA as an anticoagulant, in 2 mL Eppendorf tubes, and then were centrifuged to separate blood plasma (4000g, 10 min). Precisely 0.3 mL of the resulted supernatant was introduced into 10 mL centrifuge tubes and mixed with 2 mL of a mixture of 1:4 methyl tert-butyl ether/ethyl acetate to extract lipids which cause poor peak shape and retention shifts. After vortexing, the resulted emulsion was broken by centrifugation (6000g, 15 min), and then the aqueous layer of the supernatant was entirely collected and slowly dried at 40 °C. The dried matter was further dissolved in precisely 0.3 mL of methanol to prepare the analytical samples. The chromatographic analyses were carried out using a Perkin-Elmer HPLC system with a Flexar Quaternary LC Pump, a Flexar LC Autosampler, and a Flexar Refractive Index LC Detector. An EC 250/ 4.6 Nucleosil 50-5 C18 column was used, with methanol as mobile phase and a flow of 0.7 mL·min−1. Calibration was performed starting from solutions containing 5 to 25 mg·mL−1 β-CD/bPy2+PMDS in methanol, on three parallel sample series. The areas of the HPLC peaks were used as ordinates. The slope of the calibration line resulted to be 5.291 (calculated for zero intercept; adjusted R2 = 0.997), and the limits of detection (LOD) and of quantitation (LOQ) were 9.818 and 57.457 μg·mL−1, respectively. 2.12. Chemical Modeling of the Inclusion Process of bPy into the β-CD Cavity. The starting molecular conformation of β-CD and bPy was built using the graphical tool of the HyperChem 8.0 software application.34 The molecular geometries were fully optimized using the PM3 quantum mechanics semiempirical method, under the HyperChem software application. The molecular coordinates were defined by placing the intersection of the XYZ Cartesian coordinate axis in the center of the β-CD cavity so that the glycoside oxygen atoms are considered to be in the XY plane, and the Z axis becomes the cavity axis. The longer dimension of the bPy molecule was placed along the Z axis, and the intermolecular distances were monitored between the center cavity and the dummy (“*”) atom placed in the middle of the covalent bond that links the two heterocycles. Therefore, the plane of the bPy molecule coincides with the YZ plane.35 In a first stage, the favorable angular orientation was sought by placing the guest in the cavity center and by rotating around the Z axis, successively optimizing the structure with the PM3 calculation method at 10° intervals. In a second stage, bPy was moved along the Z axis by keeping the favorable angular orientation constant and optimizing the molecular geometry at 1 Å intervals. The binding energy (in kJ·mol−1) was registered as a function of the distance inside the cavity.

3.0. RESULTS The formation of an inclusion complex between the 4,4′bipyridinium moiety and β-CD is likely to decrease the toxicity of viologens. The caging stability of the viologen reactive segment into β-CD cavity can be provided by designing an adequate architecture of the inclusion complex of the [2]rotaxane type. If the complex must be endowed with pharmacological action, the main structural peculiarities which must be considered in designing the thread axle of [2]rotaxane are (i) a low value of both total length and equivalent diameter to facilitate the transfer across biological barriers and the access to the intimate morphology of target substrata and (ii) an increased backbone flexibility of the stopper end-units to firmly disfavor β-CD slipping-out, in virtue of their multiple thermodynamic possible conformations distinguished by low 548



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Figure 1. Synthesis pathway of the β-CD/bPy2+PMDS complex of the [2]rotaxane type.

Figure 2. PM3 optimized molecular structure of the stable β-CD/bPy inclusion complex: side view (a), front view (b), and the variation of the system energy during the inclusion process (c).

atom of the presynthesized 1-(3-bromopropyl)-pentamethyldisiloxane. The feasibility of the above-mentioned first step of the synthesis was theoretically investigated by modeling the process of bPy inclusion into the hydrophobic cavity of β-CD. Figure 2 depicts the geometry of the stable inclusion product and the variation of the system energy during the intrusion of the host molecule through the wide rim, and its crossing through the βCD cavity. Simulation using the “one set of binding sites”37 method demonstrates that the two precursors geometrically and energetically accommodate and that an axial symmetric assembly results between the 7.05 Å long bPy and the 7.9 Å height β-CD torus, which still exposes the nitrogen atoms to chemical attack. A more than 2-fold level of energy must be exceeded to extract the guest, which allows some mild chemical

energetic thresholds. In this respect, the [2]rotaxane-type product we have synthesized and tested was designed to have a 4,4′-bipyridyum core and two short siloxane chains. The main constraint in developing its molecular structure was the ability to synthesize it via the capping method, after the core precursor (4,4′-bipyridine) was included in the β-CD cavity. 3.1. Synthesis of the β-CD/bPy2+PMDS Inclusion Complex of the [2]Rotaxane Type. According to the IUPAC nomenclature,36 the viologen-based product we have synthesized is a [2]{[1][(1,1′-di(propyl-3-pentamethyldisiloxane)-4,4′-bipyridinium]-rotaxa-[β-cyclodextrin]}. Figure 1 summarizes the synthesis pathway, consisting of two steps: (i) formation of the inclusion complex of β-CD and 4,4′bipyridine (bPy), through coprecipitation, and (ii) quaternization of nitrogen atoms of caged 4,4′-bipyridine, via the bromide 549



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Table 1. Chemical Shifts of bPy and bPy2+PMDS Protons, for Both the Free and the β-CD Hosted Molecules, Recorded in D2O compound

a

β-CD

bPy

β-CD/bPy

bPy2+PMDS

protona

δ (ppm)

H-1 H-2 H-3 H-4 H-5, H-6 H-b H-a

5.06 3.64 3.96 3.57 3.84−3.90

δ (ppm)

δ (ppm)

δ (ppm)

δ (ppm)

7.74 8.62

5.11 3.69 3.94 3.62 3.82−3.92 7.80 8.76

8.57 9.15

5.06 3.65 3.94 3.58 3.83−3.90 8.54 9.11

β-CD/bPy2+ PMDS

Labeling of protons is in Figures 4 and S1, Supporting Information.

Figure 3. ROESY spectrum of the β-CD/bPy inclusion complex.

synthesized 3-bromopropyl-1-pentamethyldisiloxane (BrPMDS). This method was chosen to prevent the reaction between the bPy2+ dication and β-CD, which involves radical cation intermediates.39 To point out the achievement of the expected result of the synthesis, the noncaged viologen (bPy2+PMDS) was also synthesized and was used as the model compound in comparing with the final inclusion complex. The 1H NMR spectrum of β-CD/bPy2+PMDS, presented in Figure 4, confirms the molar ratios of 1:1:2 between the compositional units of the inclusion complex (see also the 1H NMR spectrum description in section 4.0, which quantitatively expresses the ratios between the protons belonging to the three compositional units). An additional confirmation of obtaining the desired inclusion complex was provided by the 2D [1H, 1H] ROESY NMR spectrum presented in Figure 5, which depicts the intermolecular interactions between the guest viologen and the host β-CD (marked by the two squares). The evident shifts, as compared with the spectrum of the β-CD/bPy inclusion complex, are due to the effect of the prolonged chain of the stopper compositional units. A slight asymmetric split of the peaks corresponding to the a and b protons of the caged bPy2+ moiety is evident in the 1H NMR spectrum presented in Figure 4. This fact suggests a

transformation to be further performed on it, provided a highly specific reactant is used. The effective formation of the β-CD/bPy inclusion complex was proven via 1H NMR spectroscopy by analyzing the shifts induced as a result of complexation. Data in Table 1 demonstrate the downfield shifting of all of the protons of caged bPy and of some protons of host β-CD, as compared with those of the corresponding free species, which confirms the inclusion of bPy into the β-CD cavity. The 2D [1H,1H] ROESY NMR supplementary experiment revealed the correlation among H-3 (3.94 ppm) and H-5, H-6 (3.82−3.92 ppm) of host β-CD, and H-a (7.80 ppm), and H-b (8.76 ppm) of guest bPy (see Figure 3 and, in addition, the corresponding 1 H NMR spectrum in Supporting Information Figure S1). A stronger interaction of bPy protons with the H-6 protons (CH2 vicinal to primary hydroxyls of β-CD) indicates the strong retention of the guest at the level of the narrow rim of β-CD. The 1:1 stoichiometry was confirmed by the 1H NMR variant of the Job plot procedure of continuous variation of molar ratio in host−guest mixtures.38 Figure S2 in Supporting Information depicts the results. The second step of the β-CD/bPy2+PMDS complex synthesis was performed by quaternization of nitrogen atoms of the caged-bPy, by stoichiometric reaction with the previously 550



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Figure 4. 1H NMR spectrum of the β-CD/bPy2+PMDS inclusion complex in D2O.

placed β-CDs on the rotaxane axels usually induce larger splitting effects or even distinctive peaks of bPy moiety protons.40 3.2. Electrochemical Characterization of β-CD/ bPy2+PMDS. Viologen toxicity originates in its high ability to abundantly generate reactive oxygen species (ROS), as a consequence of its continuous redox cycling ability, which mainly generates O2•− species.41 The evolving superoxide radicals are aggressive oxidants which progressively denature proteins, break nucleic acids, and peroxidize lipids, thus making local conditions incompatible with life at the cellular level. In vivo, the viologen-mediated ROS generation is an enzymedriven process14 which may be modulated or even inhibited by four mechanisms,8 one of which involves the caging of the bipyridyl moiety in inclusion complexes. The effect of inclusion on the redox capacity of viologens can be measured by cyclic voltametry. Furthermore, this method is able to simulate the cyclic processess of reduction and oxidation, offering valuable information on the stability of the inclusion products. In our study, the voltammograms of the nude bPy2+PMDS viologen were compared with those of the caged one, the β-CD/ bPy2+PMDS inclusion complex, in order to prove lectrochemical activity inhibition as a result of caging. The general electrochemical reactions in which the individual nude viologen molecule can take part are as follows. Figure 5. ROESY spectrum of the β-CD/bPy2+PMDS inclusion complex.

distinct coupling with the host protons due to the neighborhood of the two β-CD rims but not to the siloxane end-segments of bPy2+PMDS, which are symmetrically identical. Asymmetric guest structures and/or asymmetrically

First reduction: vbPy 2 + + e− → vbPy +•

(1)

Second reduction: vbPy +• + e− → vbPy 0

(2)

Outside an inclusion complex, both in solution and (especially) at the surface of an electrochemical electrode, viologen species are involved in several secondary processes of dimerization and comproportionation.42,43 551



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Figure 6. Cyclic voltammogram for oxido-reduction processes of bPy2+PMDS in DMSO and 0.1 M NaClO4.

Figure 7. Electrochemical stability of bPy2+PDMS in DMSO with 0.1 M NaClO4. Inset: comparison of redox peaks for the first and the tenth cycles.

Figure 8. Comparative electrochemical behavior of bPy2+PDMS (dotted lines) and of β-CD/bPy2+PMDS (continuous lines) during the first voltametric cycle (a) and after 10 successive cycles (b). 552



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Dimerization: 2vbPy +• ↔ (vbPy +)2

(3)

Comproportionation: vbPy 2 +• + vbPy 0 → 2vbPy +•

(4)

57.4 μg ·mL−1), it is placed below the limit of quantitation established for the calibration line (LOQ = 9.8 μg mL−1). Therefore, it may be accepted as an extrapolated value but will not be considered significant at a p-level of 0.01 (as the calibration points are). The blood concentration after 90 min is, however, statistically well estimated based on the calibration line at a level of confidence of 99%, confirming the presence of β-CD/bPy2+PMDS in the blood in an amount of about 0.634% of the orally administered dose. To comparatively investigate the effect of viologen caging on its toxicity, an in vivo experiment was conducted on mice exposed to different dose levels of bPy2+PMDS and β-CD/ bPy2+PMDS in unique administration, parallel with a control group. The mice in the bPy2+PMDS group, to which doses of 20 or 40 mg·kg−1 were administered, did not exhibit behavioral changes or other clinical signs of toxicity in comparison to the control group, except for a slight anxiety and inertia in movements. When the necropsy and histopathological exams were performed, no injury that could confirm the toxic status was noticed. Exposing the mice to higher doses of bPy2+PMDS (100 and 200 mg·kg−1) has led to significant behavioral changes: the animals were absent and passive, and all the movements inside the cage were difficult. Their interest in food and water decreased progressively, and the animals became severely dehydrated. The phenomena were more obvious at a dosage level of 400 mg·kg−1, when significant changes in body weight were noticed (from 25 ± 0.48 g on day zero to 21 ± 0.2 g on day 14). These changes were correlated to clinical signs of neurotoxicity (tremor and paraplegia) and were accompanied by depressive behavior (weakness, lethargy, and sedation), bradypnea, cyanosis, hypothermia, piloerection, and eyelids ptosis. The general condition was of emaciation, the animals being unable to feed themselves and to drink water. In the condition of severe dehydration at this dosage level, it was decided to euthanize the animals at the interval of 48−96 h from the beginning of the experiment, all being practically moribund. The high mortality rate (100%) at this dosage level and the short death interval, together with high dehydration, emphasize the high toxicity of bPy2+PMDS. The necropsy of mice from the 400 mg·kg−1 bPy2+PMDS group pointed out changes of the liver consistency, friability, and yellowish color, which are highly suggestive of liver steatosis. From a histological point of view, the liver presented microvesicular steatosis with centrolobular localization (Figure 9a). Within the cytoplasm of hepatocytes, lipids were present as numerous small droplets without fusion tendency (Figure 9b). Nuclei remain centered, in some cases with necrobiotic changes that induce cell death (peripheral chromatin condensations and sharp angle aspect) (Figure 9c). Steatosis appeared also at the renal level and was represented by small triglyceride vacuoles within the cytoplasm of the nephrocytes (Figure 9d). These lesions indicated an acute irreversible toxic phenomenon. Taking into consideration these facts, the maximum tolerated dose (MTD) for bPy2+PMDS was established to be 40 mg·kg−1, as calculated after a 14 day survey. In the case of the β-CD/bPy2+PMDS mice group, at an equivalent dosage level of 40 mg·kg−1, no clinical signs occurred in any of the tested mice. The general status was unmodified, and the animals exhibited a constant interest for water and food. The same unaltered general status was noticed at dosage

Figure 6 depicts a representative first voltammetric scan cycle of bPy2+PDMS dissolved in DMSO containing 0.1 M NaClO4 at a scan rate of 100 mV/s. It displays two cathodic peaks at −0.61 V (peak Ic) corresponding to the first reduction process (eq 1) and at approximately −0.9 V (peak IIc), which corresponds to the second reduction process (eq 2). Both electron uptakes are electrochemically reversible as shown by the anodic peaks positioned at −0.55 V (Ia) and −0.87 V (IIa). The third redox wave, IIIa, at approximately −0.42 V, is presumably due to the formation of π-complex dimers, as a consequence of electrode processes, according to eq 3. This is confirmed by voltammograms in Figure 7, which comprise 10 successive scans at potentials varying between 0.7 V and −1.6 V. The increase of the IIIa peak current, concomitantly with the amplitude decrease of the couple Ia and Ic peaks, is assigned to the dimerization process. The dimeric species, (vbPy+)2, generated by electrode oxidation, appears to be stably adsorbed on the electrode surface, thus explaining the change of the couple peaks IIc and IIa as a characteristic of vbPy+•/vbPy0 transition (eq 2). The inset of Figure 7 comparatively shows the first and the tenth scans, emphasizing the effect of cation radical dimerization (eq 3), which results in adsorption/ deposition of the dimers on the electrode surface. This finding is in agreement with the reported statement that vbPy+•-type species are prone to dimerization in aqueous media.44 The net effect of the inclusion complex generation, as reflected by its electrochemical reactivity, is depicted in Figure 8. The stable inclusion of the bipyridyl moiety of bPy2+PMDS into the β-CD cavity induces a prominent decrease of the current of peaks Ia and Ic and the absence of the redox couple IIa and IIc. The monocation radical viologen (of vbPy+• type) is therefore strongly bound within the hydrophobic cavity of βCD, and no further redox processes can take place. Moreover, Figure 8a proves that the dimerization of the vbPy+• species, mediated by electrode oxidation processes (according to eq 3), does not occur when the bipyridil moiety is included in the cavity of β-CD (peak IIIa is absent). After 10 voltametric cycles (Figure 8b), the stability of the redox peaks becomes obvious. The reduced amplitude of the peaks associated with the inclusion complex, regardless the cycle number, is due to the hindered electronic transfer between the vbPy2+ segment and the potentiostat electrode, caused by cyclodextrin presence. The results are in good agreement with those reported by Yasuda et al.,45 showing that the aqueous solution of an inclusion complex based on small molecular viologen and βciclodextrin is capable of inhibiting the dimerization of viologen derivatives. 3.3. Evaluation of the Toxicological Potential of the βCD/bPy2+PMDS Inclusion Complex. The penetration of the β-CD/bPy2+PMDS inclusion complex in the bloodstream of treated mice was proved by chromatographic analyses of the blood plasma. Blood samples were collected in triplicate at two distinct time intervals, 30 and 90 min, after the administration of product doses of 800 mg·kg−1. Using the corresponding HPLC peak areas, the values of the inclusion complex concentration in mice blood were calculated based on the calibration line equation and were found to be 27.1 ± 7.3 μg· mL−1 at 30 min and 63.4 ± 5.6 μg·mL−1 at 90 min. Even if the first value is above the detection limit of the method (LOD = 553



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(weakness and sedation), change in daily routine of the mice (passivity and inertia), tremor, paraplegia, cyanosis, hypothermia, piloerection, severe dehydration, and emaciation. The average body weight of the mice decreased from 25 ± 0.19 g to 22 ± 0.14 g. The moribund animals were euthanized, and necropsy examination pointed out liver lesions suggestive of steatosis. The histopathological examination of mice treated with 1600 mg·kg−1 β-CD/bPy2+PMDS showed toxicity signs with chronic evolution but with reversible morphological changes, expressed in the liver by rare hepatocytes with large intracytoplasmic lipid vacuoles, which pushed the nucleus toward the cell periphery (Figure 11a). Minor changes in the nuclei of the dystrophic hepatocytes were also present, which was the proof for reversible macrovesicular hepatic steatosis. The majority of hepatocytes had inhomogeneous cytoplasm and were mildly hyperhydrated, without degenerative nuclear changes. At the renal level, a cortex mild edema was noticed, indicating a toxic status with chronic evolution (Figure 11b). In the lungs, perivascular and peribronchiolar interstitial mild fibrosis was identified (Figure 11c and d), while in the cerebellum, necrobiosis of Purkinje neurons was seen: dark cytoplasm, angled in shape, and the nuclei either were missing (cariolysis) or were intensively hematoxilinic with abnormal disposition (Figure 11e and f). On the basis of clinical and histopathological exams of mice treated with β-CD/bPy2+PMDS, MTD was established to 400 mg·kg−1. As compared with the nude viologen, which induced acute, irreversible toxic lesions with lethality in all mice after a dose of 400 mg·kg−1, the inclusion complex administered in the same dose did not modify in a visible manner the health status of the mice, no lethality being registered after 14 days. Higher doses of β-CD/bPy 2+PMDS (800 and 1600 mg·kg−1 ) determined low lethality when compared with that of a 400 mg·kg−1 bPy2+PMDS dose, permitting a quite chronic evolution of the toxic phenomena (tremor and paraplegia, Purkinje neurons degeneration, and lung fibrosis).

Figure 9. Histological examination of samples taken from the liver (a, b, and c), and kidney (d) of mice treated with 400 mg·kg−1 bPy2+PMDS. Masson trichromic stain.

levels of 100 and 200 mg·kg−1, a fact confirmed by the absence of the clinical signs and of the macro- and microscopic lesions. At the dosage level of 400 mg·kg−1 β-CD/bPy2+PMDS, after 14 days of surveillance, small changes of the general status could be noticed, consisting of inconstant passivity and lethargy. The mice though kept their interest in water and food and showed no dehydration signs. The necropsy exam did not emphasize important changes of the monitored organ. The survival rate of the mice treated with β-CD/bPy2+PMDS was significantly higher than those recorded in the bPy2+PMDS group, for both 200 and 400 mg·kg−1 doses (Figure 10), emphasizing the lack of toxicity of the inclusion complex. At 800 mg·kg−1, the deterioration of health status was noticed, which is more obvious at 1600 mg·kg−1. After 48−96 h from the beginning of the experiment, the animals exhibited the same signs as thoe of the bPy2+PMDS group mice at a dosage level of 400 mg·kg−1. Clinically, the toxicity of the β-CD/ bPy2+PMDS product was manifested by a depressive behavior

Figure 10. Survival rates for 200 mg·kg−1 (a) and 400 mg·kg−1 (b) for bPy2+PMDS and β-CD/bPy2+PMDS groups. 554



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bipyridine and the 1-bromopropyl-3-pentamethyldisiloxane. The success of the synthesis relies on providing the caging stability of the first cited precursor, during the capping process. To prove 4,4′-bipyridine caging into β-cyclodextrin cavity and the stoichiometry of the construct, 1H and [1H, 1H] ROESY NMR investigations were done. The structure of the inclusion complex was certified by 1D and 2D NMR techniques, and its electrochemical properties were investigated by cyclic voltammetry in comparison with the nude viologen as the reference molecule. The interaction of the dicationic bipyridil moiety of guest viologen with the βcyclodextrin host causes pronounced changes in cyclic redox behavior, which were evident starting with the first voltammetric cycle and were increased up to the tenth cycle, confirming both the formation and the stability of the inclusion complex. The propensity of nude viologen to dimerize and to get adsorbed at the electrode surface was completely inhibited by bipyridil moiety caging, thus indicating the ability to significantly limit the possible involvement of the inclusion complex in toxicologic mechanisms. The ability of the inclusion complex to reach the bloodstream was confirmed. Ninety minutes after the oral administration of a dose of 800 mg·kg−1, about 0.634% of the product was found in the blood plasma of the laboratory mice. To confirm the lowering of toxicity by firmly caging the bipyridil moiety of the viologen, in vivo tests were performed, involving three mouse batches: (i) those treated with the nude viologen, (ii) those treated with the inclusion complex, and (iii) a control group. As compared with the first batch for which a dose of 40 mg·kg−1 was lethal, the survival of the second one was noted up to a dose of 400 mg·kg−1, but a chronic evolution was noticed up to 1600 mg·kg−1. A histopathologic exam revealed reversible morphological changes at the liver, kidney, lung, and cerebellum level, up to 1600 mg·kg−1 doses of the inclusion complex. As a general conclusion, the [2]rotaxane viologen-containing product that we have synthetized is chemically stable and toxicologically benign. It is presumable that its redox potential could be capitalized for pharmaceutical uses. Globally, our article proves that the molecular architecturing approach consisting of caging of the bipyridil moiety of viologens into the β-cyclodextrin cavity represents a feasible way to produce non- or mildly toxic pharmaceutical candidate species.

Figure 11. Histological examination of samples taken from the liver (a), kidney (b), lung (c and d), and cerebellum (e and f) of mice treated with 1600 mg·kg−1 β-CD/bPy2+PMDS. Masson trichromic stain.

4.0. DISCUSSION The goal of the work was to investigate the possibility of decreasing viologen toxicity while preserving as much as possible their electrochemical activity, aiming to provide a potential bactericidal activity in the context of an increased maximum tolerated dose at the level of as many tissues as possible. In this regard, we describe a method to synthesize an inclusion complex of a viologen species (the N,N′-bis(1-propyl3-pentamethyldisiloxane)-4,4′-bipyridinium dibromide) with βcyclodextrin and aim to prove the significant decrease of the product’s toxicity as compared with that of the nude, uncomplexed viologen. From the chemical point of view, the complex is a [2]rotaxane compound having highly flexible propyl-pentamethyldisiloxane segments as stopper end-units on the thread axle. Both the design principles (low volume but conformational versatile stoppers, supplementarily endowed with carrying activity and biostability-enhancing effect) and the synthesis pathway (capping a small caged molecule, the 4,4′bipyridine, by using minimal-length presynthesized axle-stopper segments) were developed having in view the potential use of the product as a pharmacological vector. Such a vector could valorize the ability of the inclusion complexation process to modulate the redox activity of the bipyridil moiety, rendering controllable the compound’s involvement in the in vivo enzyme-driven oxidation cycles. The simplicity of the inclusion complex synthesis (one step, via quaternization of nitrogen atoms) is due to the careful presynthesis of the precursors: the β-cyclodextrin-caged 4,4′-

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ASSOCIATED CONTENT

S Supporting Information *

H NMR spectrum of the β-CD/bPy inclusion complex, in DMSO, and the Job plot confirming the 1:1 stoichiometry of the β-CD/bPy inclusion complex. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +40-232-217454. E-mail: [email protected]. Funding

This work has been supported by a grant of the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, project: PNII-ID-PCCE-2011-2-0028. The support is very gratefully acknowledged. Notes

The authors declare no competing financial interest. 555



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(19) Paul, B. K., Samanta, A., and Guchhait, N. (2010) Modulation of excited-state intramolecular proton transfer reaction of 1-hydroxy-2naphthaldehyde in different supramolecular assemblies. Langmuir 26, 3214−3224. (20) Bergamini, J. F., Belabbas, M., Jouini, M., Aeiyach, S., Lacroix, J.C., Chane-Ching, K. I., and Lacaze, P. C. (2000) Electrochemical and pH control of the complexation/decomplexation of 4-amino-N,Ndiphenylamine with β-cyclodextrin. J. Electroanal. Chem. 482, 156− 167. (21) Spulber, M., Pinteală, M., Fifere, A., Harabagiu, V., and Simionescu, B. C. (2009) New Therapeutically Agent with Increased Antifungal Activity, in Macrocyclic Chemistry, New Research Developments (Fitzpatrick, D. W., and Ulrich, H. J., Eds.) pp 435−446, Nova Science Publishers Inc., New York. (22) Silion, M., Dascalu, A., Simionescu, B. C., Pinteala, M., and Ungurenasu, C. (2011) Synthesis and anti-HIV activity of βcyclodextrin-C6-sulfate/3-azido-3′-deoxythymidine inclusion complex. J. Polym. Sci., Part A: Polym. Chem. 49, 1730−1733. (23) Uekaji, Y., Jo, A., Urano, A., and Terao, K. (2013) Application of β-Cyclodextrin in Nanomedicinal Foods and Cosmetics, in BioNanotechnology: A Revolution in Food, Biomedical and Health Sciences (Bagchi, D., Bagchi, M., Moriyama, H., and Shahidi, F., Eds.) pp 179− 211, Blackwell Publishing Ltd., Oxford, U.K. (24) Marangoci, N., Farcas, A., Pinteala, M., Harabagiu, V., Simionescu, B. C., Sukhanova, T., Bronnikov, S., Grigoryev, A., Gubanova, G., Perminova, M., and Perichaud, A. (2008) Polyrotaxanes composed of β-cyclodextrin and polydimethylsiloxanes: synthesis, morphology and thermal behavior. High Perform. Polym. 20, 251−266. (25) Marangoci, N., Fifere, A., Farcas, A., Harabagiu, V., Pinteala, M., Simionescu, B. C., and Perichaud, A. (2008) Synthesis and characterization of polyrotaxanes based on cyclodextrins and viologen-modified polydimethylsiloxanes. High Perform. Polym. 20, 553−566. (26) Abbasi, F., Mirzadeh, H., and Katbab, A. A. (2001) Modification of polysiloxane polymers for biomedical applications: a review. Polym. Int. 50, 1279−1287. (27) Andriani, Y., Morrow, I. C., Taran, E., Edwards, G. A., Schiller, T. L., Osman, A. F., Darren, J., and Martin, D. J. (2013) In vitro biostability of poly(dimethyl siloxane/hexamethylene oxide)-based polyurethane/layered silicate nanocomposites. Acta Biomater. 9, 8308−8317. (28) Pinteala, M., Harabagiu, V., Cotzur, C., Holerca, M. N., and Simionescu, B. C. (1994) Functional Polysiloxanes. 2. On the reaction of hydroxypropyl- and aminoalkyl-terminated polydimethylsiloxanes with cyclic anhydrides. Polym. Bull. 32, 173−178. (29) Ogoshi, T., Nishida, Y., Yamagishi, T. A., and Nakamoto, Y. (2010) High yield synthesis of polyrotaxane constructed from pillar[5]arene and viologen polymer and stabilization of its radical cation. Macromolecules 43, 7068−7072. (30) Marangoci, N., Fifere, A., Farcas, A., Harabagiu, V., Pinteala, M., and Simionescu, B. C. (2008) Synthesis and characterization of polyrotaxanes based on cyclodextrins and viologen-modified polydimethylsiloxanes. High Perform. Polym. 20, 553−566. (31) McNaught, A. D., and Wilkinson, A. (1997) Maximum Tolerated Dose (MTD), in Compendium of Chemical Terminology (the “Gold Book”) (McNaught, A. D., and Wilkinson, A., Eds.) 2nd ed., pp 65 and 2068, Blackwell Scientific Publications, Oxford, U.K. (32) Gad, S. C., and Chengelis, P. C. (1998) Systemic Acute Toxicity Testing, in Acute Toxicology Testing (Gad, S. C., and Chengelis, P. C., Eds.) 2nd ed., pp 221−256, Elsevier Inc., Paris. (33) Ruehl-Fehlert, C., Kittel, B., Morawietz, G., Deslex, P., Keenan, C., Mahrt, C. R., Nolte, T., Robinson, M., Stuart, B. P., and Deschl, U. (2003) Revised guides for organ sampling and trimming in rats and mice. Exp. Toxic. Pathol. 55, 91−106. (34) HyperChem Professional 8, Hypercube, Inc., Gainesville, FL. (35) Liu, L., and Guo, Q. X. (2004) Use of quantum chemical methods to study cyclodextrin chemistry. J. Inclusion Phenom. Macrocyclic Chem. 50, 95−103.

ACKNOWLEDGMENTS We thank Dr. Maria Cazacu for allowing us to use the HyperChem 8.0 package.

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ABBREVIATIONS PM3, parametric model 3 REFERENCES

(1) Asaftei, S., and De Clercq, E. (2010) “Viologen” dendrimers as antiviral agents: The effect of charge number and distance. J. Med. Chem. 53, 3480−3488. (2) Lazniewska, J., Janaszewska, A., Miłowska, K., Caminade, A. M., Mignani, S., Katir, N., El Kadib, A., Bryszewska, M., Majoral, J. P., Gabryelak, T., and Klajnert-Maculewicz, B. (2013) Promising lowtoxicity of viologen-phosphorus dendrimers against embryonic mouse hippocampal cells. Molecules 18, 12222−12240. (3) Michaelis, L., and Hill, E. S. (1933) The viologen indicators. J. Gen. Physiol. 16, 859−873. (4) Bird, C. L., and Kuhn, A. T. (1981) Electrochemistry of the viologens. Chem. Soc. Rev. 10, 49−82. (5) Asakura, N., Hiraishi, T., Kamachi, T., and Okura, I. (2001) Lysine linked viologen for substrate of hydrogenase on hydrogen evolution. J. Mol. Catal. A: Chem. 174, 1−5. (6) Pospíšil, L., Fiedler, J., Hromadová, M., Gál, M., Valásě k, M., Pecka, J., and Michl, J. (2006) Search for a one-electron reduction of the cation radical of an “extended viologen” p-phenylene-bis-4,4 ′-(1aryl-2,6-diphenylpyridinium). J. Electrochem. Soc. 153, E179−E183. (7) Hromadová, M., Kolivoska, V., Sokolova, R., Gál, M., Pospíšil, L., and Valásě k, M. (2010) On the adsorption of extended viologens at the electrode | electrolyte interface. Langmuir 26, 17232−17236. (8) Wang, K., Guo, D. S., Zhang, H. Q., Li, D., Zheng, X. L., and Liu, Y. (2009) Highly effective binding of viologens by p-sulfonatocalixarenes for the treatment of viologen poisoning. J. Med. Chem. 52, 6402−6412. (9) Shi, Z., Neoh, K. G., and Kang, E. T. (2005) Antibacterial activity of polymeric substrate with surface grafted viologen moieties. Biomaterials 26, 501−508. (10) Núñez, M. E., Hall, B. D., and Barton, J. K. (1999) Long-range oxidative damage of DNA: effects of distance and sequence. Chem. Biol. 6, 85−97. (11) Mees, G. C. (1960) Experiments on the herbicidal action of 1,1′-ethylene-2,2′-dipyridylium dibromide. Ann. Appl. Biol. 48, 601− 612. (12) Zweig, G., Shavit, N., and Avron, M. (1965) Diquat (1,1ethylene-2,2′-dipyridylium dibromide) in photo-reactions of isolated chloroplasts. Biochim. Biophys. Acta 109, 332−346. (13) Dodge, A. D. (1971) The mode of action of the bipyridylium herbicides, paraquat and diquat. Endeavour 30, 130−135. (14) Dinis-Oliveira, R. J., Duarte, J. A., Sánchez-Navarro, A., Remião, F., Bastos, M. L., and Carvalho, F. (2008) Paraquat poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit. Rev. Toxicol. 38, 13−71. (15) Zielonka, J., Rybak, M., Celińska, J., Adamus, J., Marcinek, A., and Gebicki, J. (2006) Effect of heparin on viologen-stimulated enzymatic NADH depletion. Chem. Res. Toxicol. 19, 668−673. (16) Wallace, M. A., Bailey, S., Fukuto, J. M., Valentine, J. S., and Gralla, E. B. (2005) Induction of phenotypes resembling CuZnsuperoxide dismutase deletion in wild-type yeast cells: an in vivo assay for the role of superoxide in the toxicity of redox-cycling compounds. Chem. Res. Toxicol. 18, 1279−1286. (17) Anderson, F. R., Haris, T. A., Hay, M. P., and Denny, W. A. (2003) Enhanced conversion of DNA radical damage to double strand breaks by 1,2,4-benzotriazine 1,4-dioxides linked to a DNA binder compared to tirapazamine. Chem. Res. Toxicol. 16, 1477−1483. (18) Hatcher, J. M., Pennell, K. D., and Miller, G. W. (2008) Parkinson’s disease and pesticides: a toxicological perspective. Trends Pharmacol. Sci. 29, 322−329. 556



Article

(36) Yerin, A., Wilks, E. S., Moss, G. P., and Harada, A. (2008) Nomenclature for rotaxane and pseudorotaxane. Pure Appl. Chem. 80, 2041−2068. (37) Hembury, G., Rekharsky, M., Nakamura, A., and Inoue, Y. (2000) Direct correlation between complex conformation and chiral discrimination upon inclusion of amino acid derivatives by beta- and gamma-cyclodextrins. Org. Lett. 2, 3257−3260. (38) Djedaini-Pilard, F., Lin, S. Z., Perly, B., and Wouessidjewe, D. (1990) High-field nuclear magnetic resonance techniques for the investigation of a β-cyclodextrin:indomethacin inclusion complex. J. Pharm. Sci. 79, 643−646. (39) Liu, Y., Zhao, Y. L., Zhang, H. Y., and Song, H. B. (2003) Polymeric rotaxane constructed from the inclusion complex of βcyclodextrin and 4,4′- dipyridine by coordination with nickel(II) ions. Angew. Chem., Int. Ed. 42, 3260−3263. (40) Takashima, Y., Yuting, Y., Otsubo, M., Yamaguchi, H., and Harada, A. (2012) Supramolecular hydrogels formed from poly(viologen) cross-linked with cyclodextrin dimers and their physical properties. Beilstein J. Org. Chem. 8, 1594−1600. (41) Jacob, S., and Dietz, K. J. (2009) Systematic Analysis of Superoxide-Dependent Signaling in Plant Cells: Usefulness and Specificity of Methyl Viologen Application, in Plant Stress Biology: From Genomics to Systems Biology (Hirt, H., Ed.) pp 179−196, WileyVCH Verlag GmbH & Co. KGaA, Weinheim, Germany. (42) Tang, X., Schneider, T. W., Walker, J. W., and Buttry, D. A. (1996) Dimerized π-complexes in self-assembled monolayers containing viologens: An origin of unusual wave shapes in the voltammetry of monolayers. Langmuir 12, 5921−5933. (43) John, S. A., Kasahara, H., Okajima, T., Tokuda, K., and Ohsaka, T. (1997) Electrochemical study on monomer-dimer equilibria of a series of monolayers of asymmetric viologens on the electrode surface in the presence of hexafluorophosphate ion. J. Electroanal. Chem. 436, 267−270. (44) Quintela, P. A., Diaz, A., and Kaifer, A. E. (1998) Dimerization of the methylviologen cation radical in anionic micellar and polyelectrolyte solutions. Langmuir 4, 663−667. (45) Yasuda, A., Kondo, H., Itabashi, M., and Seto, J. (1986) Structure changes of viologen+ β-cyclodextrin inclusion complex corresponding to the redox state of viologen. J. Electroanal. Chem. 210, 265−275.

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