A ruthenium nitrosyl cyclam complex with appended anthracenyl fluorophore

Polyhedron 173 (2019) 114117

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A ruthenium nitrosyl cyclam complex with appended anthracenyl fluorophore Rodrigo Gibaut de Souza Góis a,1, Elisangela Fabiana Boffo a, José Carlos Toledo Júnior b, Karla Furtado Andriani c, Giovanni Finoto Caramori c, Anderson de Jesus Gomes d, Fabio Gorzoni Doro a,e,⇑ a

Departamento de Química Geral e Inorgânica, Instituto de Química, Universidade Federal da Bahia, Salvador, BA, Brazil Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brazil Departamento de Química, Universidade Federal de Santa Catarina, Campus Universitário Trindade, Florianópolis, SC, Brazil d Universidade de Brasília, Campus Ceilândia, Brasília, DF, Brazil e Universidade Federal do Triângulo Mineiro, Campus Universitário de Iturama, Av. Rio Paranaíba, 1251, CEP 38280-000 Iturama, MG, Brazil b c

a r t i c l e

i n f o

Article history: Received 14 March 2019 Accepted 15 August 2019 Available online 23 August 2019 Keywords: Ruthenium Nitric oxide Cyclam Fluorescence NO-release

a b s t r a c t The complex trans-[Ru(NO)(H2O)(L)](ClO4)3 (L = (1-anthracen-9-ylmethyl)-1,4,8,11-tetraazacyclotetradecane) (RuNOL) was synthesized and characterized, and its photophysics and reactivity were investigated. The FTIR spectrum displays a mNO band at 1840 cm
1, indicating a nitrosonium character. 1H and 13 C signals in 1D and 2D NMR spectra were assigned and are consistent with the trans configuration. The UV–Vis spectrum displays maximal absorption bands at 341 nm (log e 3.74), 363 nm (log e 3.91), 378 nm (log e 3.97) and 397 nm (log e 3.91). Coordination of the [RuNO] moiety to the L ligand strongly quenches the anthracenyl fluorescence, and thus RuNOL exhibits only very weak emission at 390, 418, 440 and 472 nm. Electrochemical studies indicate that cathodic peak potentials at +1.10 V(Ic),
0.1 V(IIc) and
0.4 V(IIIc) (vs Ag/AgCl) are related to An+/An, {RuNO}6/7 and {RuNO}7/8 reduction processes, respectively. The pKa of coordinated water was estimated as 2.8 ± 0.2 by DPV. The emission of the pendant fluorophore increases upon electrochemical or chemical reduction of RuNOL, and the resulting switch-ON fluorescence is possibly due to NO release from the target complex. Spectroscopic titrations with DNA resulted in hypochromism and red shifts and allowed estimation of binding constants of 1.9  103 (L) and 1.8  103 (RuNOL). Molecular docking showed that RuNOL complex has a great affinity with DNA. The RuNOL complex showed low cytotoxicity toward MCF-7 and NIH-3T3 and was ineffective in healthy HUVEC and A7r5 cell lines in the range of concentration of 1  10
7–1  10
4 mol L
1. Ó 2019 Elsevier Ltd. All rights reserved.

1. Introduction Nitric oxide (nitrogen monoxide, NO) plays multiple roles under both physiological and pathological conditions [1]. The effects of NO in biological media are influenced by its concentration, site of production and lifetime [2,3]. The outstanding properties of nitric oxide have stimulated the development of several types of NO-carriers [4]. Among them, metal nitrosyl (or nitrite) complexes with Mn [5], Fe [6] and Ru [7,8] have drawn considerable attention since they can be fine-tuned to deliver NO controllably and locally using different activation strategies. ⇑ Corresponding author at: Universidade Federal do Triângulo Mineiro, Campus Universitário de Iturama, Av. Rio Paranaíba, 1251, CEP 38280-000 Iturama, MG, Brazil. E-mail address: [email protected] (F.G. Doro). 1 Present address: Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, campus do Pici, Fortaleza, CE, Brazil. https://doi.org/10.1016/j.poly.2019.114117 0277-5387/Ó 2019 Elsevier Ltd. All rights reserved.

Control and monitoring of the release of NO from NO-donor metal complexes are crucial for understanding its chemical and biological functions. Light-triggered NO-donors have been successfully used for controlled delivery of nitric oxide to biological targets [9]. Ford and co-workers designed several metal complexes capable of releasing NO upon irradiation by visible light [9]. The side attachment of fluorescein dye derivatives to Roussin’s red salt [10] or pendant fluorophores such as dansyl or anthracenyl in chromium cyclam nitrite complexes can also be used to deliver NO upon photoactivation [11]. Following a different approach, Mascharak and co-workers investigated the direct attachment of dyes such as fluorescein methyl ester [12] or resofurin [13] directly to a {RuNO}6 core. This approach resulted not only in NO release by visible light photoactivation of these complexes, but also in a photoinduced turn-ON fluorescence leading to ‘‘trackable” NO-donors [13]. The Ru–NO bond in ruthenium nitrosyls can lead to significant electron delocalization over the metal–nitrosyl system, which can

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complicate assignment of formal oxidation states. Enemark and Feltham [14] proposed a simplifying notation, {RuNO}n, where n is the number of d-electrons in Ru center plus p* NO electrons. Thus, [RuII–NO+], [RuIII–NO] and [RuIV–NO
] are all represented by {RuNO}6. Since experimental and theoretical evidence support the [RuII–NO+] canonical form in [Ru(NO)L(mac)]n+ and other {RuNO}6 nitrosyl complexes [15–18], we will use both Enemark and Feltham notation or individual canonical forms as appropriate throughout this manuscript. The ruthenium tetraamine and tetraazamacrocycle nitrosyl complexes, [Ru(NO)L(N4)]q (N = NH3 or N4 = tetraazamacrocycle), have shown suitable properties for application as NO-carriers [19–21]. These complexes allow the fine tuning of properties of coordinated NO by judicious choice of ligands around the [RuNO] moiety, [7,19,21] resulting in controlled NO release upon chemical reduction or photoactivation [7,22,23]. For instance, the exposure of trans-[Ru(NO)(NH3)4L]3+ (L = P(OEt)3 or pyridine) complexes to a mitochondrial suspension suggested that these compounds are reduced by mitochondrial NADH, releasing NO [24,25]. Ruthenium tetraammine nitrosyl complexes also show a broad range of NOdependent biological effects, for example in the control of blood pressure and parasitic infections, as well as in tumor cell death in different models of cancer [19,26,27]. Our group has explored the properties of ruthenium nitrosyl complexes with macrocyclic ligands such as cyclam (1,4,8,11tetraazacyclotetradecane) and cyclen (1,4,7,10-tetraazacyclododecane) [20,28]. The trans-[Ru(NO)Cl(cyclam)](PF6)2 complex can release NO slowly (k–NO = 6.1  10
4 s
1) as a result of the reduction and substitution of NO by H2O [15], and it has very low toxicity towards V79 cell lines (IC50 >3000 lmol L
1) [29]. The chemical versatility of cyclam allows its functionalization by appending selected groups to N or C atoms of the macrocycle [30,31], providing a simple means to tailoring the chemical properties of the complexes. Our previous works showed that fac-[Ru(NO) Cl2(j3N4,N8,N11(1-carboxypropyl)cyclam)]Cl (1-carboxypropyl) cyclam = (1-(carboxypropyl)-1,4,8,11-tetrazacyclotetradecane)) exhibited the j3 coordination mode for the N-appended macrocycle, which in turn influences its structure, electronic properties, and reactivity [32,33]. For instance, NO release after reduction (kobs-NO = 2.1 s
1) was found to be about four orders of magnitude larger than for trans-[Ru(NO)Cl(cyclam)](PF6)2 [33]. The photophysical properties of cyclam metal complexes bearing anthracenyl or dansyl fluorophores N-appended to the macrocyclic ring are sensitive to the metal center and its oxidation state [34,35] as well as to axial ligands such as NO [36]. Conceivably, changes to the {RuNO}6 center in a ruthenium nitrosyl tetraazamacrocyclic complex with an appended fluorophore could result in a fluorescence response. Combining the features above, we designed a ruthenium nitrosyl cyclam complex with an N-appended anthracenyl fluorophore, trans-[Ru(NO)(H2O)(L)](ClO4)3 (L = (1-anthracen9-ylmethyl)-1,4,8,11-tetraazacyclotetradecane) (RuNOL, Fig. 1). In this work we report the synthesis, characterization and relevant chemical and electrochemical reactivities and photophysical properties of this complex.

2. Materials and methods 2.1. Chemicals Cyclam was purchased from Strem Chemicals. Ruthenium nitrosyl chloride hydrate, Ru(NO)Cl3xH2O and 9-(chloromethyl) anthracene were purchased from Sigma-Aldrich. The ligand (1anthracen-9-ylmethyl)-1,4,8,11-tetraazacyclotetradecane) (L) was synthesized according to a previously published procedure [37].

23

21 20

19

17 18

NO N 5

6

16

19´

17´ 18´

1

Ru

N

11

8

9

20´

14

N

N

7

22'

15

2

3

4

21´

22

12 13

10

OH2

Fig. 1. Chemical structure of RuNOL complex. Atom numbering used to identify the nuclei in NMR discussion is shown. Hydrogen atoms numbering follow those indicated for carbon and nitrogen.

N,N-Dimethylformamide (DMF) was dried over CaSO4, distilled under reduced pressure and stored with 4 Å molecular sieves. All other solvents and reagents were of analytical grade and used as supplied or purified when necessary, according to standard methods [38]. All manipulations of air-sensitive compounds were carried out following conventional techniques [39]. Trypsin, antibiotic–antimycotic solution (Ab/Am), RPMI-1640 (modified), Dulbecco’s Modified Eagle Medium (DMEM), penicillin, streptomycin, and L-glutamine, gentamicin, were acquired from Sigma Chemical Company (St. Louis, MO, USA). Fetal bovine serum was from (GIBCO). Deionized water was used throughout.

2.2. Synthesis of trans-[Ru(NO)(H2O)(L)](ClO4)3 Ru(NO)Cl3xH2O (144 mg, 6.1  10
4 mol) was dissolved in 10 mL of argon-degassed dry DMF in a three-necked round-bottom flask. To this solution, 223 mg (5.7  10
4 mol) of L dissolved in 10 mL of argon-degassed dry DMF was added dropwise. The resulting mixture was heated to 90 °C with continuous argon bubbling for 1 h. After filtration of the reaction mixture through a glass frit, the solvent was eliminated under reduced pressure. The reaction product was dissolved in 50 mL of water, and H2O:HClO4 (1:1 v/ v) solution was added dropwise. The greenish solid formed was filtered off and discarded, and the resulting yellow solution was concentrated under vacuum to ca. 20 mL. After cooling overnight in the refrigerator, 244 mg of crude product was collected by filtration. Under stirring, this solid was dissolved in a minimum amount of aqueous HClO4 solution (pH 1) at 60 °C. After one hour, an excess of AgNO3 was added and a white solid formed instantly. The resulting mixture was kept in the dark under stirring at 40 °C for 12 h and then filtered over a pad of celite to remove the fine AgCl formed. Addition of LiClO4 and concentration of the solution afforded the trans-[Ru(NO)(H2O)(L)](ClO4)3 complex, which was isolated as a fine yellow powder. Yield 38% (185 mg, 2.2  10
4 mol). Caution: Although no detonations occurred in any of our preparations, perchlorate salts should be handled with care. Anal. Calc. for C25H38N5O15RuCl3 (for 1 H2O) (856.02 g mol
1): C, 35.08; H, 4.47; N 8.18. Found: C, 36.58; H, 4.61; N, 8.72. HR-ESI-MS positive mode in MeOH:H2O 1:1 (4500 V, direct introduction, 180 °C): m/z (%) 520.1614 (100%) [RuIINO+(L)2
]+ (mtheor = 520.1650).

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2.3. Physical measurements C, H and N elemental analyses were performed on a PerkinElmer 240B elemental analyzer. Electron Spray Ionization Tandem Mass Spectrometry (ESI-MS/MS) measurements were performed using a Bruker Daltonics Esquire 3000 Plus mass spectrometer. High-Resolution Electron Spray Ionization Mass Spectrometry (HR-ESI-MS) measurements were performed using a Bruker Daltonics Micro-Tof mass spectrometer. Both analyses were conducted in positive ion ionization mode and samples were diluted in a CH3OH:H2O (1:1) mixture. UV–Vis spectra were recorded using a Varian Cary50 spectrophotometer with a 1-cm length quartz cell. Fourier Transformed Infrared (FTIR) spectra were obtained in a BOMEM MB-102 equipment using KBr pellets. Nuclear Magnetic Resonance (NMR) experiments were conducted at LABAREMN (Laboratório Baiano de Ressonância Magnética Nuclear) and spectra were recorded on a Varian Inova 500 instrument equipped with a 5 mm probe at 300 K with frequencies of 500 MHz for hydrogen and 125 MHz for carbon nuclei. Standard pulse sequences were used for two-dimensional (2D) correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC). Samples (15 mg) were dissolved in CD3CN (0.7 mL). The 1H and 13 C chemical shifts (d) are given relative to the signal of CD3CN. Differential pulse voltammetry (DPV) measurements were performed on a Princeton Applied Research (PAR) 273A model potentiostat/galvanostat. These experiments were performed using a conventional three-electrode system. Glassy carbon was used as the working electrode for CV and DPV measurements. A platinum wire and Ag/AgCl electrodes were used as a counter-electrode and reference electrode, respectively. The influence of chloride concentration on peak potentials was investigated at 0, 1.0 and 4.0 mol L
1. The acidity of the coordinated water molecule (pKa) was estimated by running DPV measurements after each addition of NaOH solution to a 10
3 mol L
1 of RuNOL in CF3COOH/CF3COONa (pH 1.0, I = 0.1 mol L
1) solution. The pH range studied was from 1.0 to 4.5. Chronocoulometry experiments were performed on an AutoLab PGSTAT 30, and a graphite rod (60  12 mm) or plate (22  22 mm) was used as working electrode. NO detection was performed using an inNO Nitric Oxide Measuring System from Innovative Instruments Inc. This apparatus detects NO directly by means of an amperometric technique. All electrochemical measurements were conducted in Argon de-aerated aqueous solutions. Throughout this work, all the reported potentials are referred to Ag/AgCl, 3 mol L
1 KCl (0.21 V versus NHE), unless otherwise stated. 2.4. Fluorescence spectroscopy Fluorescence emission spectra were recorded on a Shimadzu fluorescence spectrophotometer RF-5301PC model using a quartz cell with 1.0 cm optical path. Samples of RuNOL (or L) in CF3COOH (pH 1.0) (30 lmol L
1) were excited at 370 nm (slit 5) and fluorescence intensities were recorded from 380 to 550 nm. Fluorescence turn-ON measurements of RuNOL were obtained from chronocoulometry experiments, collecting samples at different charge values, or by addition of Eu2+ solution (1, 5, 10 and 25) to a fully argon-degassed solution of the complex. In the latter case, fluorescence emission was monitored by recording spectra at 30 s time intervals (kem at 390 nm). 2.5. DNA-interaction experiments Stock solutions of fish sperm DNA were prepared by dissolving 20 mg in 10 mL of 50 mmol L
1 NaCl/5 mmol L
1 Tris–HCl buffer (pH 7.4) with stirring overnight at 8 °C. The ratio of UV absorbance

at 260 and 280 nm, A260/A280, gave values of 1.8–1.9, indicating that the DNA was sufficiently free of protein [40]. DNA concentrations were determined by using an attenuation coefficient of 6600 mol
1 L cm
1 at 260 nm [40] and expressed in terms of base pairs. Stock solutions were stored at 6 °C and used after no more than 5 days. For studies on the effect of salt concentration on the UV–Vis absorption spectra of the complex-DNA adduct (see below), solutions of DNA were prepared without NaCl. Interaction studies of RuNOL (or L) with DNA were performed by spectroscopic titration. To a solution of RuNOL or L (10 lmol L
1) in 3 mL of Tris–HCl buffer (pH 7.4) prepared without any addition of organic solvent, fixed amounts of DNA were added to each cuvette to eliminate the absorbance of DNA itself. Both solutions were mixed carefully five times with the aid of a 1 mL micropipette and allowed to equilibrate for 5 min before absorption spectra were recorded. The binding constants (Kb) were estimated from the spectroscopic titration data using Eq. (1) [41]:

½DNA

ea
ef


¼

½DNA

eb
ef


þ

1 Kb

eb
ef




ð1Þ

where [DNA] is the concentration of DNA in base pairs, ea is the apparent absorption coefficient and eb and ef are the attenuation coefficients of fully bound and free compounds, respectively. A plot of [DNA]/(ea
ef) versus [DNA] will give a slope of 1/(ea
ef) and intercept of 1/Kb(eb
ef). Kb is calculated by the ratio of the slope to the intercept. The effect of NaCl concentration on the UV–Vis absorption spectra of complex–DNA adduct was studied as follows. The RuNOL complex in Tris–HCl buffer (pH 7.4) solution was previously saturated (fully bound) with DNA, then the concentration of NaCl in solution was progressively increased by addition of fixed amounts of 4.0 mol L
1 NaCl solution. After each addition, the solutions were mixed carefully five times with the aid of a 1 mL micropipette and allowed to equilibrate for 5 min before absorption spectra were recorded. 2.6. DFT calculations and molecular docking In order to take into account the physiological pH in molecular docking studies, the geometry optimization and vibrational frequencies calculations were performed for trans-[Ru(NO)(OH) (L)]2+ with ORCA v.3.0.2 [42] package by employing the model B3LYP-D3 [43–45] + ECP[Ru] [43–45]/def2-SVP [46]. No symmetry constraints were employed during the geometry optimization procedure. The Zeroth Order Relativistic Approximation was used to account the relativistic effects of Ru [47,48]. The absence of imaginary eigenvalues in the Hessian matrix confirmed that the structures reported herein corresponds to as local minima in on the potential energy surface. Only data for trans-III configuration of trans-[Ru(NO)(OH)(L)]2+ are reported since this configuration was found to be the most stable configuration among trans ruthenium nitrosyl tetraazamacrocycle complexes [17]. To investigate the interaction of trans-[Ru(NO)(OH)(L)]2+ with DNA the molecular docking approach was performed with AutoDock Vina v.1.1.2 [49]. We also performed the molecular docking of L (as protonated form, L2+) to elucidate the role of ruthenium ion on the complex interaction with DNA. The +2 charge on L is due to the pKa of amine groups in the mono-N-substituted cyclam [50]. For docking runs the trans-[Ru(NO)(OH)(L)]2+ and L2+ optimized structures were both treated as a flexible ligands to allow the inclusion of torsional angles and thus, the chose receptor was a beta-DNA dodecamer duplex with 1.9 Å of resolution (PDB code: 1BNA) [51]. Both ligands and receptor were prepared with AutoDock Tools package v.1.5.6 [52] in which the crystallized water was removed from the crystal structure of DNA. The nonpolar hydrogens were treated with extended-atom representation and

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R.G. de Souza Góis et al. / Polyhedron 173 (2019) 114117

only polar ones were accounted (pH 7.4) for both ligands and receptor. The active site around DNA was fixed in a grid box of 60:60:100 points along x, y and z axes, respectively, and the configuration search was performed with AutoDock Vina v.1.1.2 with the default configurations, except exhaustiveness in which was set to 10. The docked figures as well as the post-docking analyses were obtained using Chimera v.1.11 [53]. 2.7. In vitro evaluation The cell lines used in this study were obtained from the American Type Culture Collection (ATCC, Bethesda, Maryland): Michigan Cancer Foundation-7 (MCF-7; ATCCÒ HTB-22TM) – breast cancer cell; Fibroblast cell line (NIH-3T3; ATCCÒ CRL-1658TM); Human Umbilical Vein Endothelial Cells (HUVEC; ATCCÒ CRL-1730TM); A7r5 rat aortic smooth muscle (A7r5; ATCCÒ CRL-1444TM). The cell viability of RuNOL was evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tetrazolium reduction assay [54,55]. The four cell lines (MCF-7, NIH-3T3, HUVEC, and A7r5) were tested. After ten passages the cells were employed in passages 12–25, during which time they exhibited stable morphological characteristics by light microscopy. The cell lines MCF-7 and NIH-3T3 were cultured in RPMI 1640 medium supplemented with 10% FBS and antibiotics (50 IU mL
1 penicillin, and 50 lg/mL streptomycin), at 37 °C, 5% CO2 in the air. HUVEC and A7r5 cells were cultured in DMEM medium containing 10% FBS, 150 lg/mL penicillin, 150 lg/mL streptomycin, 300 lg/mL neomycin, and 250 lg/mL gentamycin [56]. For comparison purposes, the cytotoxicity of RuNOL was evaluated under the same experimental conditions. The percentage of cell viability was calculated with respect to control cells that were incubated without the drug, as described in previous studies [19,55]. The IC50 was determined from the dose–response curve for each cell line. All experiments were performed in triplicate. 2.8. Statistical analysis Statistical analysis was performed using Graph Pad Prism, version 6.0. The significance level of difference between the experimental groups and controls was assessed using two-way ANOVA and the Post hoc Tukey test. The results are presented as the mean ± standard deviation (SD). Statistical significance was considered when P < 0.05 (each experiment, n = 3). 3. Results and discussion 3.1. Synthesis The synthesis of the RuNOL complex was performed with readily available precursors (L and RuNOCl3xH2O) in dry DMF. This method resulted in a shorter preparation time (1 h) than other methods that use precursors such as K2[Ru(NO)Cl5] or RuNOCl3xH2O and the macrocycle in ethanol or methanol, which usually take from 8 to 18 h of reflux [15,32]. Notably, the FTIR spectrum of the crude product displayed two bands in the region of coordinated NO (1888 and 1840 cm
1), and after the acid hydrolysis with an excess of AgNO3 only the band at 1840 cm
1 was observed. 3.2. Vibrational, mass and magnetic nuclear resonance spectroscopy The FTIR spectrum of RuNOL (Fig. S1) shows bands of mNH (3140–2840 cm
1) and of dCH and mCH of the macrocycle in the regions of 1480–1250 cm
1 and 960–740 cm
1. A sharp band of medium intensity at 620 cm
1 can be assigned to CAH bending in the anthracenyl fluorophore. The characteristic triple-degener-


1 ate strong band of ClO
. The mNO band 4 is centered at 1114 cm
1 at 1840 cm appears in the range generally associated with nitrosyl metal complexes and is consistent with a nitrosonium character (NO+) in the {RuNO}6 moiety [20,57]. The mNO band of RuNOL is close to that observed for trans-[Ru(NO)(OH)(cyclam)](PF6)2 (1830 cm
1), [16] but it is shifted to lower energy from that observed for trans-[Ru(NO)Cl(cyclam)](ClO4)2 (1875 cm
1) [15]. Consistent with the presence of coordinated water, and with results reported for the analogous trans-[Ru(NO)(H2O)(NH3)4]Cl3 [58], bands at 3437 cm
1 (broad, mOH), 1622 cm
1 (dOH) and 941 cm
1 (weak, mRu–OH2) were observed. Mass spectroscopy techniques have been applied for characterization of ruthenium nitrosyl complexes [32,59,60]. The structure and fragmentation chemistry of the gaseous RuNOL was investigated by the ESI-MS/MS technique and spectra are presented in the Supplementary material (Fig. S2). For a solution of RuNOL in H2O:MeOH (1:1), a predominant cluster of isotopologues ranging from m/z 635.1 to 642.0 was observed. This isotopic pattern characterizes the presence of the Ru atom and is consistent with the C25H35N5O6RuCl (m/z 638.1) composition, which could be + described as a [RuII(NO+)(OH
)L,ClO
4 ] ion pair. The conversion of aqua to hydroxo ligand is expected due to the solvent used and the estimated pKa of coordinated water (see Section 3.4). The other clusters of lower m/z correspond to a series of fragmentations involving the loss H2O, NO and even the anthracenyl pendant arm, as revealed by the ESI-MS/MS data. The ESI-MS/MS for collisioninduced dissociation of a selected isotopologue of mainly m/z 638.1 shows that the gaseous cationic complex fragments initially by loss of either H2O, generating the species of composition + C25H33N5O5RuCl (m/z 620.2), or H2O, ClO
4 and H , generating the species of composition C25H32N5ORu (m/z 520.2). Note that the latter species was the same as that detected in HR-ESI-MS spectra, [RuIINO+(L)2
]+ (m/z 520.1614). The collision-induced dissociation of [RuIINO+(L)2
]+ results in loss of the anthracenyl pendant arm (m/z 330.4) and NO, generating [RuIII(L)2
]+ (m/z 300.2), which was also observed in ESI-MS/MS of fac-[Ru(NO)Cl2(j3N4,N8, N11(1-carboxypropil)cyclam)]+ [32] under similar conditions. Proposed gas phase fragmentation patterns for RuNOL are presented in Fig. S3. 1D and 2D NMR techniques are particularly useful to analyze structural differences promoted by appending N-substituents to cyclam in ruthenium nitrosyl complexes such as trans-[Ru(NO) Cl(1-pramcyclam)]3+ (1-pramcyclam = 1-(3-propylammonium)1,4,8,11-tetraazacyclotetradecane) [23], fac-[Ru(NO)Cl2(j3N4, N8,N11(1-carboxypropil)cyclam)]+ [32], and [Ru(NO)(N-(2methylpyridyl)cyclam)]3+ (N-(2-methylpyridyl)cyclam) = N-(2methylpyridyl)-1,4,8,11-tetraazacyclotetradecane) [61], compared to trans-[Ru(NO)Cl(cyclam)]2+ [15]. The RuNOL complex was characterized by 1D and 2D (1H, 13C, COSY, HMBC, HSQC) NMR spectroscopy techniques to gain structural information. Assignments of 1H and 13C were made by comparing the chemical shift values in the spectra of our complex to those of the free ligand and related species [23,61], and (mainly) on the correlations observed in the 2D spectra. The 1H and 13C NMR spectra of RuNOL are consistent with the presence of the 1-anthracen-9-ylmethyl group bound to the cyclam ring. The HSQC spectrum (Fig. S4) confirms the presence of only one major isomer. The 1H NMR signals at 8.45, 7.66, 7.58, 8.16 and 8.73 ppm, referred to as H18,180 , H19,190 , H20,200 , H21,210 and H23, respectively (see Fig. 1), can be assuredly assigned to the anthracenyl fluorophore. The broad 1H NMR signals in the 4.75–5.00 ppm and 5.90–6.10 ppm ranges can be assigned to the NH hydrogens of cyclam (Fig. S5). These two signals show a 2:1 ratio between their integrals. The pattern of NH in the 1H NMR spectra of the metal complex with cyclam and analogous ligands correlates with the

R.G. de Souza Góis et al. / Polyhedron 173 (2019) 114117

geometrical configuration and with the presence of N-appended groups [20], as can be seem in trans-[Ru(NO)Cl(1-pramcyclam)]3+ (2:1) [23], fac-[Ru(NO)Cl2(j3N4,N8,N11(1-carboxypropil)cyclam)]+ (1:1:1) [32], cis-[Ru(NO)(N-(2-methylpyridyl)cyclam)]3+ (1:1:1) [61] and trans-[Ru(NO)Cl(cyclam)]2+ (2:2) [15]. Thus, the ratio between the two NH integrals in 1H NMR of RuNOL is consistent with the trans configuration in a mono-N-substituted cyclam ruthenium nitrosyl complex. The HSQC spectrum was used to identify the geminal pairs of CH2 hydrogens, assigning them to the corresponding 13C signals (Fig. S4). The COSY spectrum (Fig. S6) showed correlations that allowed determination of the correct signal numbering according to the proposed structure (Fig. 1). The doublet hydrogen signal in the 5.40–5.60 ppm range, assigned to hydrogens in C15, exhibited heteronuclear correlation in HMBC with the 2;14;16;17 carbon nuclei (Fig. 2). Since C2 and C14 signals are in the aliphatic region, they were assigned to carbons in the macrocycle ring neighboring N1. The carbons C16 and C17 are in the aromatic region, indicating that C15 (–CH2–) is the group responsible for the link between the anthracenyl fluorophore and cyclam macrocycle. This is also supported by COSY correlations between hydrogens in C15 with those in C2 and C14. Following the scalar connectivities in the COSY spectrum, a sequential assignment of the 1H NMR spectrum was performed (Table 1). In the 13C NMR spectrum of RuNOL (Fig. S7), eight carbon signals in the aromatic region are observed due to the anthracenyl group, in which six pairs of carbon atoms are chemically equivalent, as also detected in the free ligand. The 13C NMR spectrum also displays 10 signals in the 25–60 ppm range assigned to the (–CH2–) aliphatic carbons of the macrocyclic ligand instead of six, as observed for trans-[Ru(NO)(OH)(cyclam)]2+ [16]. This is a consequence of the rigidity of the macrocycle combined with the lack of symmetry elements, which in turn results in inequivalence of the aliphatic cyclam carbons in RuNOL. Taken together, these results along with those of electrochemical experiments (see Section 3.4) provide suitable information on the structure and composition of the complex under investigation. 3.3. Electronic absorption and emission spectra The electronic absorption spectrum of RuNOL in CF3COOH (pH 1) is shown in Fig. S8 and the main absorption bands are listed in Table 2 along with those of L for comparison purposes. The elec-

Fig. 2. 2D [1H,

13

5

tronic spectrum of RuNOL is largely dominated by the absorption bands of the anthracenyl group. The most intense band at 258 nm has been attributed to a p–p* transition of symmetry type 1 1 B2u A1g [62] and the quartet at 340–420 nm region is typical of anthracene vibrationally resolved bands. Coordination of the {RuNO}6 moiety to L leads to a consistent (5 nm) red shift, indicating a ground state interaction between the anthracenyl fluorophore and {RuNO}6 core. Similar red shifts have been observed for related octahedral Co(III) [63] and Cr(III) dichlorido complexes [64] with analogous fluorophore-functionalized cyclam ligands. The fluorescence emission spectrum of L (kex 370 nm) displays the expected quartet emission of anthracene. Under the same conditions, RuNOL showed very weak and broad emissions at 472, 440, 418, 390 nm characteristic of the p–p* fluorescence from the anthracenyl group [64] (Table 2, Fig. 3). Notably, the analogous complex trans-[Ru(NO)Cl(cyclam)]2+ did not show fluorescence emission. In comparison with L, macrocycle complexation with {RuNO}6 dramatically quenched (>99%) the singlet state of anthracene. This clearly shows that emission of the anthracenyl group is influenced by the {RuNO}6 center. Fluorescence quenching of cyclam with appended anthracene was observed upon coordination of metal ions such as Cr(III), Co(III), Ni(II), and Cu(II) [37,64,65]. The quenching of fluorophore emission has been attributed mainly to an energy transfer (ET) process to metal-centered states. At least in Co(III) and Cr(III) complexes, the contribution of electron transfer (eT) processes were not ruled out [64,65]. Fabbrizzi and coworkers [35] proposed that the ET process in Ni(II) complexes with anthracenyl pendant arm could be favored by the –CH2– linker. The flexibility of this group may allow folding and occasional intramolecular van der Waals contact between the metal-containing subunit and the anthracene fragment. The same mechanisms are expected to be responsible for fluorescence quenching of L upon coordination of the {RuNO}6 center. 3.4. Electrochemistry and chemical reactivity The monoelectronic electrochemical reduction assigned to the {RuNO}6/7 core (formally NO+/0) in [Ru(NO)L(N4)]q+ (N4 = tetraazamacrocycle or N = NH3) complexes results in NO dissociation with variable aquation rates [7,15,20]. For these ruthenium nitrosyls, the {RuNO}6/7 reduction potential has been shown to be accessible to important biological reducers [19,24]. Electrochemical studies of L and RuNOL were performed mainly by differential

C] HMBC NMR spectrum of RuNOL in CD3CN solution.

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Table 1 H, 13C, COSY, HMBC NMR data in CD3CN solution for RuNOL.

1

1

13

– 1.98/2.69 2.81/3.5 4.90, br(s) 3.06/3.14 1.53/2.70 3.10/3.19 6.02, br(s) 3.06/3.52 2.81/3.51 4.90, br(s) 3.47/3.91 2.01/2.40 2.54/2.82 5.48 (d, 15.0 Hz)/5.56 (d, 15.0 Hz) – – 8.45 br(s) 7.66 (t) 7.58 (t) 8.16 (d) – 8.73 (s)

– 56.0 53.5 – 50.5 29.2 53.1 – 53.4 56.8 – 54.4 29.7 59.3 53.2 134.1 123.5 124.8 128.0 126.5 130.5 132.3 132.1

H d(multiplicity)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17,170 18,180 19,190 20,200 21,210 22,220 23

C

1

H–1H COSY

1

H–13C HMBC

– 13;15 4 3 6 5 8 7,9,10 8 8;11;12 9;10;12 10;12;14 2 12;15 2;14 – – 19 18;20 19;21 20;23 – 21

– 3;5;14 – – 2;3;6 – – – – 12 – – – 12;13;15 2;14;16;17 – – – 16;21 23 16;19;23 – 16;17;21

Abbreviations: d – doublet, s – singlet, t – triplet, br – broad.

Table 2 Main absorption, emission and redox parameters of L and RuNOL.

a b c

Compound

Absorptiona kmax (nm) (log e)

Emissiona kmax (nm)

Potential (Ep) (V vs Ag/AgCl)

L

337 353 370 390

(3.77) (3.99) (4.07) (3.98)

470 436 414 390

+1.20 (Ic)b +1.10 (Ia)b

RuNOL

341 363 378 397

(3.74) (3.91) (3.98) (3.91)

472 440 418 390

+1.10 (Ic)c +1.06 (Ia)c
0.10 (IIc)c
0.40 (IIIc)c +0.10 (IVa)c

In 0.1 mol L
1 CF3COOH solution. In KCl/HCl (I = 0.1 mol L
1, pH 1) In CF3COONa/CF3COOH (I = 0.1 mol L
1, pH 1) solution.

Fig. 3. Fluorescence emission spectra of L and RuNOL in CF3COOH solution (pH 1).

pulse voltammetry in aqueous solution at pH 1. The main peak potentials are collected on Table 2, and voltammograms are provided in the Supplementary material (Fig. S9 and S10). An investigation of the influence of pH and chloride concentration on the peak potentials of RuNOL was also performed.

In the range of
1.00 V to 1.50 V, the differential pulse voltammograms of L (Fig. S9) display only one reduction peak (Ic, +1.20 V) with the corresponding oxidation peak (Ia, +1.06 V). Considering that the Ep of anthracene is +0.99 V (An+/An versus Ag, AgClO4 0.001 mol L
1 in acetonitrile) [66], Ia/Ic is probably due to the redox (An+/An) process centered on the pendant anthracenyl fluorophore. In the same potential range, the cathodic scan of RuNOL displays two well-defined reduction peaks at +1.10 V(Ic),
0.10 V(IIc) and one broad and poorly resolved peak at
0.40 V(IIIc) (Fig. S10). Since the corresponding oxidation peak Ia (+1.05 V) also appears in the anodic scan of complex, Ia/Ic was assigned to the An+/An redox process in RuNOL. The peak IIc appears in the range of potentials associated with the {RuNO}6/7 redox process of ruthenium nitrosyl macrocyclic complexes [7,20] but it is positively shifted (0.2 V) compared to those of trans-[Ru(NO)Cl(cyclam)]2+ and trans-[Ru(NO)Cl(1-pramcyclam)]3+ [15,23]. Importantly, the corresponding peak in the anodic branch (IIa) is observed only in the lower range of potentials (
0.35 V to +1.3 V) (Fig. 4, left). The peak potential of IIc was also found to be dependent on pH in the range of 1.0–4.5 and on the concentration of Cl
ions at pH 1. With the increase of pH, the current intensity of peak IIc decreases while a new peak appears at more negative values (Fig. 4, right). With the decrease of pH back to 1 by careful addition of trifluoroacetic acid, the peak IIc reappears at the same potential. These results are consistent with the deprotonation of coordinated water and formation of the trans hydroxo derivative. The increase of chloride concentration from 0 to 1.0 mol L
1 at , pH 1, also results in changes in the peak potential of IIc, which shifts from
0.10 V to
0.20 V. Increasing the concentration of Cl
up to 4.0 mol L
1 resulted in no further changes (Fig. S11). It is well known that different ligands trans to NO influence the reduction potential of the nitrosyl ligand (formally NO+/NO0), as has been demonstrated for a series of trans-[Ru(NO)L(NH3)4]q+ complexes [7,21]. Notably, for trans-[Ru(NO)Cl(cyclam)]2+ no influence of pH was observed on the {RuNO}6/7 reduction potential in the range of 1.0–6.0, nor of chloride concentration [15]. On the other hand, this potential has been shown to be dependent on pH and Cl
concentration in the trans-[Ru(NO) (OH2)(NH3)4]2+ complex [58]. Thus, these results suggest not only the substitution of labile coordinated water by chloride ion, resulting

R.G. de Souza Góis et al. / Polyhedron 173 (2019) 114117

7

Fig. 4. Left: differential pulse voltammogram of RuNOL in CF3COOH/CF3COONa (pH 1.0, I = 0.1 mol L
1) at 25 °C, scan rate 50 mV s
1. Right: dependence of current (I) and potential (E) on pH in differential pulse voltammograms (cathodic scan) of RuNOL in CF3COOH/CF3COONa (initial pH 1.0, I = 0.1 mol L
1), at 25 °C, scan rate 50 mV s
1.

in the formation of a chlorido complex in solution, but also supports the assignment of peak IIc to the {RuNO}6/7 reduction process for RuNOL. Accordingly, the peak IIIc is possibly related to the {RuNO}7/8 reduction process [15,67]. The peak IVa, which is also pH-dependent, is consistent with the RuIII/II process in the diaquo complex formed by NO dissociation after electrochemical reduction of RuNOL. These assignments agree with the results of controlled-potential electrolysis (at pH 1 without Cl
), which shows a decrease of the peak current of IIc and an increase in IVa (Fig. S12). The propositions above regarding chemical and electrochemical reactivity are summarized in Scheme 1. Since aqua and hydroxo forms of RuNOL showed different peak potential values and the current is proportional to the concentration of each form, DPV measurements at different pH values allowed us to estimate the pKa of the coordinated water in RuNOL as 2.8 ± 0.2 using an Ipc  pH plot (Fig. S13) [68]. The estimated pKa is close to those reported for trans-[Ru(NO)(OH2)(cyclam)]3+ (pKa 3.0) [15], trans-[Ru(NO)(OH2)(NH3)4]3+ (pKa 3.1 ± 0.1) [58] and mer-[RuNO(NH3)3(NO2)(H2O)]2+ (pKa 2.4 ± 0.2), in which NO+ is trans to the H2O ligand [69]. The high acidity of coordinated water trans to NO+ has been related to the hardening of the Ru(II) fragment, due to the strong p-acceptor character of the nitrosonium ligand [58]. As stated previously, the fluorescence emission in metal complexes with tetraazamacrocycles with N-appended fluorophores has been shown to be highly sensitive to the nature and oxidation state of the metal center [34,35], and thus the same would be expected in the {RuNO}6 core as well. To investigate electrochemical reduction of RuNOL and its relationship to the photophysical properties of the anthracenyl group, fluorescence spectra were run with samples taken at different applied charge values during

chronocoulometry experiments (Fig. 5, left). As can be observed, there is an increase in fluorescence RuNOL as the applied charge increases. A similar experiment was performed positioning the NO-sensor right along the side of the graphite rod working electrode to investigate the relationship of fluorescence increase and NO release. As soon as the electrochemical cell was turned on (around 200 s), an increase of NO-sensor current was registered (Fig. 5, right). Notably, no current increase for the NO-sensor was observed for the CF3COONa/CF3COOH solution only, or up to an applied potential equal to
0.20 V in RuNOL solution. Chemical reduction of RuNOL at pH 1 with Eu2+ (EuIII/II =
0.55 V versus Ag–AgCl–NaCl (satd)) [70], a well-known monoelectronic reducing agent for [Ru(NO)L(N4)]q+ complexes [15,71], was also monitored by fluorescence spectroscopy. In agreement with the electrochemical experiments, the addition of Eu2+ to a degassed solution of RuNOL resulted in a substantial increase of fluorescence emission (Fig. 6). The increase in fluorescence emission is time-dependent and reaches a maximum after 600 s at the experimental conditions of Fig. 6. A similar pattern was observed when a stoichiometric amount or 5- and 10-fold excess of Eu+2 relative to RuNOL were used, except that the time to reach the plateau decreased slightly as the Eu2+ concentration increased. Since Eu2+ one-electron reduction of trans-[Ru(NO)Cl(cyclam)]2+ and trans-[Ru(NO)(NH3)4L]q+ complexes results in NO dissociation [15,71], it seems reasonable to assume a similar behavior for RuNOL. Thus, the NO dissociation and formation of the diaqua complex seems to be the origin of fluorescence increase. These findings are interesting since the increase in fluorescence emission could be, at least in principle, used to monitor NO release from RuNOL dynamically.

Scheme 1.

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R.G. de Souza Góis et al. / Polyhedron 173 (2019) 114117

Fig. 5. Left: changes in fluorescence emission spectra of samples collected during a chronocoulometry experiment (Eapp
0.20 V) (kex = 370 nm). Right: chronoamperogram of NO-sensor during chronocoulometry experiment (Eapp
0.20 V) of RuNOL. Both experiments were performed in CF3COONa/CF3COOH solution (I = 0.1 mol L
1, pH 1), at 25 °C.

Fig. 6. Left: changes in fluorescence emission spectra after addition of Eu2+ solution (25 excess) to 27 lmol L
1 of RuNOL in CF3COOH solution (pH 1) (kex = 370 nm). Right: time course for fluorescence increase (kem = 390 nm).

3.5. DNA-interaction studies and molecular docking The interaction of metal complexes with DNA has attracted considerable attention [72]. The DNA interaction with complexes of Cu [73], Pt [74], and Ru [75] has been studied by means of UV–Vis absorption titration with increasing amounts of DNA. For instance, the interaction of ruthenium(II) quinonediimine complexes of cyclam with DNA have been investigated [75]. Analysis of the DNA binding properties showed that cis-[Ru(phi)(cyclam)]2+ (phi = 9,10-phenanthroquinonediimine) can intercalate with this host with a binding constant of 5.0  104 mol
1 L (at 20 °C). Such ability was attributed to the spatial geometry of this complex and the large aromatic planar surface of the quinodiimine derivative, which provides the p–p interactions essential to the intercalation process [75]. The absorption spectra of L and RuNOL in the presence of increasing concentrations of DNA are shown in Fig. 7. As Fig. 7 shows, increasing DNA concentration results in significant changes in the absorption spectra of L and the RuNOL complex. A decrease in the anthracene absorption bands is seen, which results in hypochromism at the absorption maxima of about 54% for both species. Bathochromic shifts (Dkmax) of 4 nm and 6 nm were observed for L and for RuNOL, respectively. For ionic anthracenyl derivatives such as 9-anthrylmethylammonium chloride, the red shifts have been attributed to a decrease in the energy gap of HOMO–LUMO orbitals of the chromophore, resulting from the DNA–probe interaction [76]. Absorption spectra of L and

RuNOL also show isosbestic points at 312 and 392 nm and 310 and 422 nm, respectively, and these are consistent with conversion of the free to the bound type of chromophore. The similarities in spectral changes observed for both species and the large planar aromatic surface of the pendant anthracenyl group suggest that intercalation of the functional group may play a role in the DNA– RuNOL interaction. Table 3 summarizes the relevant results of this study. The absorbance data were used to estimate the DNA binding constants (Kb) (Table 3) by the half-reciprocal method using Eq. (1). The binding plots of [DNA]/(ea
ef) versus [DNA] (Fig. 7) are linear and resulted in apparent binding constants of 1.9  103 mol
1 L for L and 1.8  103 mol
1 L for RuNOL, indicating a moderate interaction with DNA when compared to those observed for ionic 9- and 10-substituted anthracene derivatives reported by Kummar and coworkers (104 mol
1 L) [76]. To gain further insight on the type of interaction between the ruthenium nitrosyl and DNA, we investigated the effect of NaCl concentration on the absorption spectra of the DNA–RuNOL adduct by spectroscopic titration. The absorption spectra recorded with increasing amounts of NaCl showed a discrete hyperchromism of 22% at 380 nm and a small hypsochromic shift of 4 nm (Fig. S14). It has been demonstrated that addition of anionic surfactants or salts such as NaCl destabilizes the DNA–anthracenyl intercalation complex [77]. These results have been rationalized as a consequence of charge neutralization of the polyanionic DNA phosphate backbone due to interactions with cations, which in turn reduce

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Fig. 7. Absorption titration of L (5.3  10
5 mol L
1) and RuNOL (8.0  10
5 mol L
1) in 50 mmol L
1 NaCl/5 mmol L
1 Tris-HCl buffer solution (pH 7.4) with increasing concentration of fish sperm DNA at room temperature, and plots of the [DNA]/(ea
ef) vs [DNA] (in base pairs).

Table 3 Selected data from UV–Vis absorption titration of L and RuNOL with DNA.a Compound

L

RuNOL

a

Absorption maxima (nm)

Isosbestic points (nm)

Free

Bound

334 350 368 388

338 354 372 392

312 392

340(sh) 360 378 398

346(sh) 368 384 402

310 412

Dkmax (nm)

% hypochromism

Kb (mol
1 L)

4

54

1.9  103

6

54

1.8  103

In Tris-HCl buffer (50 mmol L
1 NaCl/5 mmol L
1, pH 7.4).

repulsion between the double strands, leading to close packing and consequently less space for an existing intercalator [77]. Thus, an increase in NaCl concentration should result in opposite behavior compared to the increase in DNA in the guest solution, i.e., increase of absorption and hypsochromic shift of the intercalator, as was observed for RuNOL. From the results above, it seems likely that the interaction of RuNOL with DNA is assisted by intercalation of the pendant anthracenyl group. Despite the evidence of intercalation, intermolecular hydrogen bonding and electrostatic interactions of the positively charged nitrosyl complex with DNA cannot be ruled out. Molecular docking provided further insights on the interactions of RuNOL with DNA. As shown on Table 4 the molecular docking results suggest that the interaction of trans-[Ru(NO)(OH)(L)]2+ with DNA is stabilized by a factor up to 1.0 kcal/mol in comparison with L2+/DNA interaction, taking into account the most stable configuration. When the ruthenium cation is included, it not only enhances the magnitude of the binding affinity but also promotes a break of the DNA domains specificity. For example, the binding affinity for the first

configuration is
6.9 kcal/mol (
8.3 kcal/mol) for L2+ (RuNOL) interaction with DNA whereas the interaction occurs between G2A–C23B/C3A–G22B (C3A–G22B/G4A–C21B) base pair domains, Figs. 8 and 9. Besides, the small values of rmsd, for the three most stable configurations for L2+/DNA is less than 3 Å and confirm that the related configurations present similar conformations. Conversely, for trans-[Ru(NO)(OH)(L)]2+/DNA interactions, the second most stable configuration occurs at T7A–A18B and C9A–G16B base pairs domains with distinct conformation from 1, as shown the larger values of rmsd, upper to 3 Å. However, as show Table 4 for the remains configurations the interaction between trans-[Ru (NO)(OH)(L)]2+ and DNA occurs at a different domains than the observed for the three first ones, although near G4A–C21B base pairs. In addition, our initial molecular docking analysis showed that the intermolecular hydrogen bonds play a minor role for trans[Ru(NO)(OH)(L)]2+/DNA and L2+/DNA stabilizations, within the only one hydrogen bond found for the former interaction (configuration 1) and occurring between the oxygen atom of NO ligand with the

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Table 4 Molecular docking analysis for trans-[Ru(NO)(OH)(L)]2+ (RuNOL) and L2+ (L) as ligands with DNA as the receptor, modeled at flexible/rigid regime. Affinity is given in kcal.mol
1, the rmsd distance from configuration 1 is given in Å (lower bond/upper bond). Ligands

Configurations

Binding Affinity

rmsd distance from 1

Site

RuNOL

1 2 3 4 5 6 7 8 9


8.3
7.8
7.8
7.7
7.7
7.7
7.6
7.6
7.6

0 13.56 4.39 2.65 13.47 4.39 13.03 4.30 13.47

C3A–G22B/G4A–C21B T7A–A18B/C9A–G16B C3A–G22B/G4A–C21B C3A–G22B/G4A–C21B A5A–T20B/T7A–A18B G2A–C23B/G4A–C21B A5A–T20B/T7A–A18B G2A–C23B/G4A–C21B G4A–C21B/A6A–T19B

L

1 2 3 4 5 6 7 8 9


6.9
6.8
6.6
6.6
6.5
6.5
6.4
6.4
6.2

0 2.99 0.37 2.53 2.33 3.34 4.81 3.90 5.37

G2A–C23B/C3A–G22B

Fig. 8. Docking graphics for the first three stable configurations of L2+/DNA interaction.

hydrogen atom from guanine (4A). According to Fig. 9 is expected that trans-[Ru(NO)(OH)(L)]2+ complex should intercalate with beta-DNA dodecamer duplex in reason of the presence of anthracenyl group and in agreement with DNA titration results. However, different modes complex/DNA could also be associated but rather seen in molecular docking analysis, being necessary additional insights provided by other useful analysis such as reactive molecular dynamics. Nevertheless, as showed elsewhere [78], the interaction between charged ruthenium complexes with DNA is modulated mainly by the electrostatic contribution, and thus we also expected the same pattern occurs for these investigated systems. 3.6. In vitro evaluation There is a broad range of metal complexes that have received considerable attention for medical treatment of diseases, such as cancer [19,54,55], cardiovascular problems [79,80], arthritis [81], and parasitosis [82,83]. Metallic-based therapies have been used for decades in anticancer protocols despite their limitations, such as the resistance of certain types of cancers or side effects. Follow-

ing these leads, we have recently characterized the biological activities of a new class of metallodrug candidates containing a ruthenium center [27,55,84,85]. The focus of our group is the evaluation of ruthenium-based complexes as a biological tool [7,19,20,27]. This choice is based mainly on their stability and kinetic inertness, and lower toxicity compared to platinum compounds such as cisplatin [86,87]. The cytotoxic properties of RuNOL were evaluated using four cell lines containing fibroblast (NIH3T3), breast cancer (MCF-7), endothelial cell (HUVEC) and aortic smooth muscle (A7r5), and the results are displayed in Fig. 10. The toxicity of the RuNOL complex (1  10
7–1  10
4 mol L
1) toward the HUVEC and A7r5 cell lines is quite low, with cell survival consistently greater than 95% or similar to control (i.e., cells not treated with the complex). However, the MCF-7 and NIH-3T3 cells were sensitive to the cytotoxic effects of RuNOL. As shown in Fig. 10, our results revealed that RuNOL decreased the percentage of viable cells of fibroblast and murine cancer cells (NIH-3T3 and MCF-7) by approximately 45% in concentration-dependent manners, with an IC50 value of 4.5  10
4 mol L
1, which is in the range of results from studies using similar compounds [19].

R.G. de Souza Góis et al. / Polyhedron 173 (2019) 114117

11

Fig. 9. Docking graphics for the first three stable configurations of trans-[Ru(NO)(OH)(L)]2+/DNA interaction.

fact, this behavior could be a potential benefit of the use of RuNOL over other antitumoral drugs. 4. Conclusions

Fig. 10. MTT assay-measured cell viability of NIH-3T3, MCF-7, HUVEC, and A7r5. *P < 0.05, **P < 0.01 and ***P < 0.001 (each experiment, n = 3). Cells were treated with the indicated concentrations of RuNOL for two hours.

Studies conducted in our group showed that trans-[Ru(NO) Cl(cyclam)]2+ exhibits low toxicity as judged from cytotoxicity experiments in cultures of vascular smooth muscle cells (VSMC) (IC50 >1.5 mM) [85] and V79 cells (IC50 >3.0 mM) [29]. This complex also showed in vitro inhibition of proliferation/migration of VSMC [85] and in vivo blood pressure reduction in hypertensive Wistar rats [29]. These results have been attributed to the NO release from the compound, following reduction of the complex in the cell environment [19,29,85,88]. This attribution agrees with the biological activity related to NO release from other ruthenium nitrosyls [89,90]. Analyzing the data from MTT assays in NIH-3T3 and MCF-7 cell lines, it seems that the complex had lower values of cell death in the range of most antitumoral drugs (submicromolar to micromolar) range [91]. However, the increased doses used up to 450 lM displayed better action for the complex, increasing the NO released. The use of this type of complex could be an additional feature in cancer therapy, in particular in future I.V. administration, since as displayed in Fig. 10 the vasculature system main cells (endothelial and smooth muscle) were not affected or damaged. In

In summary, we have described the synthesis, chemical properties, DNA interaction, and toxicity of a nitrosyl ruthenium complex containing a cyclam N-appended anthracenyl fluorophore. Altogether, experimental results support the structure and composition of the proposed complex. The pendant anthracenyl group provided a suitable approach to tune the chemical and photophysical properties while maintaining the trans configuration in the RuNOL complex. The planar fluorophore also assisted DNA interaction. The one-electron reduction, which triggers NO aquation, is accompanied by a significant increase of the anthracenyl fluorescence. This increase turns RuNOL complex into a potential tool to monitor NO delivery directly from fluorescence changes and may paves the way to tracking exogenous NO release from the complex in cells and tissues. Thus, the selective N-functionalization of the cyclam ligand proved to be a successful strategy to improve the chemistry and potential applications of ruthenium nitrosyl complexes with tetraazamacrocyclic ligands and will be further explored using other pendant fluorophores. Acknowledgments R. G. S. Góis gives thanks to UFBA and CNPq for undergraduate and masters scholarships. E. F. Boffo gives thanks to FAPESB and CNPq. K. F. Andriani to CNPq (grant number 141700/2013-0 and number 302408/2014-2). A. J. Gomes gives thanks to FAPDF (grant number 193.000.890/2015) and CNPq (grant number 303540/2016-8). F. G. Doro gives thanks to FAPESB (grant number PPP 0039/2010) and CNPq (grant number 402687/2016-7) and to Professor Roberto Santana da Silva and Dr. Juliana Biazotto for help with NO measurements. Professor Elia Tfouni is also thanked for his friendship and scientific collaboration over more than a decade. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.poly.2019.114117.

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