Interactions of ruthenium(II) compounds with sulfasalazine and N,N′-heterocyclic ligands with proteins

Accepted Manuscript Interactions of ruthenium(II) compounds with sulfasalazine and N,N'-heterocyclic ligands with proteins Ariane Carla Campos de Melo, Jaime Murilo Salvo Vidal Pereira Santana, Kelen Jorge Rodrigues da Costa Nunes, Mayra de Amorim Marques, Guilherme Augusto Piedade de Oliveira, Adolfo Henrique Moraes, Elene Cristina PereiraMaia PII: DOI: Reference:

S0020-1693(17)30986-6 http://dx.doi.org/10.1016/j.ica.2017.08.037 ICA 17831

To appear in:

Inorganica Chimica Acta

Received Date: Revised Date: Accepted Date:

23 June 2017 16 August 2017 18 August 2017

Please cite this article as: A.C.C. de Melo, J.M.S. Santana, K.J.R. Nunes, M. de Amorim Marques, G.A.P. de Oliveira, A.H. Moraes, E.C. Pereira-Maia, Interactions of ruthenium(II) compounds with sulfasalazine and N,N'heterocyclic ligands with proteins, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica. 2017.08.037

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Interactions of ruthenium(II) compounds with sulfasalazine and N,N'-heterocyclic ligands with proteins

Ariane Carla Campos de Meloa, Jaime Murilo Salvo Vidal Pereira Santanaa, Kelen Jorge Rodrigues da Costa Nunesa, Mayra de Amorim Marquesb, Guilherme Augusto Piedade de Oliveira b, Adolfo Henrique Moraesa, Elene Cristina Pereira-Maiaa*

a

Department of Chemistry, Universidade Federal de Minas Gerais, 31270901, Belo

Horizonte, MG, Brazil b

Institute of Medical Biochemistry Leopoldo de Meis, Universidade Federal do Rio de

Janeiro, 21941590, Rio de Janeiro, RJ - Brazil

* Author to whom correspondence should be addressed [email protected] Tel.: +55-31-3409-5727; Fax: +55-31-3409-5700.

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Abstract Two complexes of Ru(II) possessing the general formula [Ru(ssz)2L](PF6) were prepared, in which ssz is sulfasalazine and L is either 2,2′-bipyridine (1) or 1,10-phenanthroline (2). The complexes were characterized by elemental and conductivity analyses, UV-Vis, vibrational, NMR and electrospray ionization mass spectroscopies. The geometry around ruthenium(II) is distorted octahedral. The metal ion is bound to the carboxylate of ssz in a bidentate way and to two heterocyclic nitrogens of bpy or phen. Spectrofluorimetric measurements indicate that complexes bind to BSA with affinity constants ranging from 104 to 105 M-1. Both compounds are significantly cytotoxic in a chronic myelogenous leukemia cell line in a concentration dependent manner. UV-light exposure for 5 min increases the cytotoxicity of 1 by three times and that of 2 by ten times. In addition, upon UV light irradiation, the complexes are able to damage the bovine serum albumin molecule, at concentrations that do not affect BSA in dark conditions. It is worth to note that the complexes also bind to Abl-SH3 protein, which is responsible for the development of chronic myelogenous leukemia. Interactions with proteins seem to be significant for the cytotoxic and photocytotoxic activity of these complexes.

Keywords: ruthenium; sulfasalazine; photocytotoxic activity; protein interactions; chronic myelogenous leukemia; Abl-SH3 domain

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1. Introduction cis-Diamminedichloroplatinum(II), or cisplatin, has become one of the most important chemotherapeutic agents for the treatment of a wide spectrum of solid tumors. Unfortunately, toxic effects and the development of resistance to cisplatin make the search for new agents a necessity [1]. Ruthenium possesses some favorable characteristics that render its complexes promising antitumoral agents, such as ligand exchange kinetics similar to platinum, preferential accumulation in tumor tissues, and low toxicity [2,3]. Three ruthenium compounds entered clinical trials for cancer treatment: the indazolium(HIn) trans[tetrachlorobis(1H-indazole)ruthenate(III)] and its sodium analogue (IT-139), and the imidazolium(HIm) trans-[tetrachloro(dimethylsulfoxide)(1H-imidazole)ruthenate(III)] (NAMI-A) [4,5]. Despite the structural similarities, the former induces apoptosis in primary tumors whereas the later decreases the formation of metastasis [6,7]. Although the mechanism of action of ruthenium compounds is not completely clear, some reports emphasize the role of DNA binding [8] and others point to the role of the interactions with proteins [9]. Interactions with extracellular matrix components, cell surface, blood proteins and other cytosolic targets may be responsible for the anticancer and antimetastatic effect of different ruthenium compounds [9,10,11]. It has also been suggested that the formation of adducts between NAMI-A and human serum albumin (HSA) are likely to be responsible for the anti-metastatic action of this Ru(III) compound [7,12]. The structure of the complex [RuCl5(ind)]2- bound to HSA revealed the presence of two mol of metal complex per protein, at subdomains IB and IIA. In the IB subdomain, His-146 replaces one chlorine atom of [RuCl5(ind)]2- whereas in the IIA subdomain, [RuCl5(ind)]2- binds to Lys-199 and His-242 [13]. Several ruthenium compounds were developed aiming at treat cancer [2,3,13-16]. Moreover, Ru(II)-polypyridyl complexes have received great attention in the development of metal-based photodynamic therapy agents [17-22]. Photodynamic therapy (PDT) is a method

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of cancer treatment, which uses a photoactive drug and light to induce cell death through an oxygen-dependent mechanism. This process generates reactive oxygen species that attack biomolecules such as proteins, DNA, and lipid. Ruthenium(II) complexes with polypyridyl ligands induce apoptosis in cancer cells by a ROS-mediated mitochondrial pathway [23,24]. Upon irradiation, oligonucleotide-conjugates containing [Ru(phen)3]2+ cleaved a singlestranded DNA representing the bcr-abl gene [25]. The bcr-abl gene, absent in normal cells, encodes for a tyrosine kinase protein, Abl, implicated in the pathogenesis of chronic myelogenous leukemia (CML) [26]. We have been exploring metal coordination to antibiotic molecules, in an attempt to obtain more active agents [27]. Sulfasalazine is used to treat inflammatory bowel diseases and rheumatoid arthritis [28]. It also inhibits lymphoma growth [29] and intensifies temozolomide cytotoxicity in human glioblastoma cells by inhibition of the xc- cystine transporter [30]. In this work, we describe the synthesis and characterization of two heteroleptic complexes of ruthenium(II) containing sulfasalazine (ssz) and α-α-diimines as ligands. The new complexes are photocytotoxic in CML cells. Upon light irradiation complexes damage bovine serum albumin. In addition, the complexes bind to the SH3 domain of cABl tyrosine kinase, which is responsible for the development of chronic myelogenous leukemia.

2. Experimental 2.1 Material and Instruments 2-hydroxy-5-[(E)-2-{4-[(pyridin-2-yl)sulfamoyl]phenyl}diazen-1-yl]benzoic acid (ssz), 2,2′-bipyridine (bpy), 1,10-phenanthroline (phen), and RuCl3.nH2O were purchased from Sigma Co. (St. Louis, MO, USA). All other chemicals were reagent-grade and were used without further purification.

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Elemental analyses were performed in a Perkin–Elmer 2400 CHN analyzer. Conductivity studies were carried out with a Digimed DM 31 conductivity meter using a cell of constant 1.130 cm-1, spectroscopic grade nitromethane (Merck) (ΛM = 8.20 S cm2 mol−1) and tetramethylammonium bromide (ΛM=79.12 S cm2 mol−1) as a standard. Infrared spectra were recorded over the region 400–4000 cm−1 with a Perkin–Elmer 283 B spectrometer. The samples were examined in KBr pellets. NMR spectra were obtained in a Bruker Avance DRX 400 spectrometer with tetramethylsilane as an internal standard using dmso-d6. Full scan mass spectra were obtained in a MicroTOF LC Bruker Daltonics spectrometer equipped with an electrospray source operating in positive ion mode. Samples were dissolved in acetone/acetonitrile (1/1) and were injected in the apparatus by direct infusion. A Cary 100 Varian spectrometer was used for UV-vis absorption measurements. Fluorescence spectra were recorded in a Shimadzu RF5301PC spectrophotometer. A stock solution of the complexes (1 × 10-2 mol L-1) was prepared in acetonitrile and further diluted in HEPES buffer (20 mmol L-1), pH 7.2. Light irradiation at 365 nm was achieved by using a Spectroline Model CX-20 - ultraviolet fluorescence analysis cabinet. 2.2 Synthesis of [Ru(bpy)2ssz](PF6) and [Ru(phen)2ssz](PF6) The precursors, cis-Ru(bpy)2Cl2 and cis-Ru(phen)2 Cl2, were prepared according to a literature method [31]. The complexes were synthesized using a general procedure, in which the precursor (0.40 mmol, 0.21 g of cis-Ru(bpy)2Cl2 or 0.40 mmol, 0.23 g of cisRu(phen)2Cl2) and ssz (0.60 mmol, 0.239 g) were mixed in 40.0 mL of an ethylene glycol/water solution (7:1, v/v). The mixture was refluxed for 6h, while the solution turned from purple to red. Afterwards, 40.0 mL of water was added and the mixture was filtered to remove solid impurities. Subsequently 12.27 mmol (2.31 g) of KPF6 was added to the filtrate, which leads to the formation of a red solid. The complexes were separated by filtration,

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washed thoroughly with water, ethanol and ether diethyl and dried. Furthermore, complexes were purified by alumina chromatography, using acetone/ ethyl acetate (1:10, v/v) as eluent. Complex 1: Yield 68 %. IR (KBr): νmax = 3430, 3078, 1630, 1590, 1528, 1484, 1456, 1392, 1294, 1138, 1086 cm−1. ΛM = 90.86 S cm2 mol−1 in nitromethane. Electronic spectrum (aqueous solution): λmax = 498 nm (ε = 6882 M−1 cm−1). Anal. Calc. For [Ru(C10H8N2)2 C18H13N4O5S](PF6) (956.10 g mol−1): C 47.70, H 3.16, N 11.76, found C 47.35, H 3.22, N 11.78. Complex 2: Yield 68 %. IR (KBr): νmax= 3410, 3080, 1630, 1590, 1528, 1482, 1456, 1390, 1290, 1138, 1084 cm−1. ΛM = 93.00 S cm2 mol−1 in nitromethane. λmax = 495 nm (ε = 6333 M−1 cm−1). Anal. calc. for [Ru(C12H8N2)2C18H13N4O5S](PF6) (1004.10 g mol−1): C 50.21, H 3.01, N 11.15, found C 50.15, H 3.20, N 10.94. 2.3 Cells, culture and drug sensitivity assays The K562 cell line was purchased from the Rio de Janeiro Cell Bank (number CR083 of the RJCB collection). This cell line was established from pleural effusion of a 53 year-old female with chronic myelogenous leukemia in terminal blast crisis. Cells were cultured in RPMI 1640 (Sigma Chemical Co.) medium supplemented with 10% fetal calf serum (CULTILAB, São Paulo, Brazil) at 37 °C in a humidified 5% CO2 atmosphere. Cultures grow exponentially from 105 cells mL-1 to about 8 × 105 cells mL-1 in three days. Cell viability was checked by Trypan Blue exclusion. The cell number was determined by Coulter counter analysis. For photocytotoxicity assessment, 1 × 10 5 cells mL-1 were cultured in the dark for 4 h in the absence and the presence of a range of concentrations of tested compounds. Subsequently, cells were washed three times with ice-cold phosphate-buffered saline (PBS) to eliminate the culture medium. After replacement of the culture medium with PBS, the cells were photoirradiated with UV-A light (365 nm, 610 µW cm-2) for 5 min in a Spectroline

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Model CX-20 - ultraviolet fluorescence analysis cabinet. After irradiation, PBS was replaced with RPMI 1640 medium supplemented with 10% fetal calf serum, and incubation was continued for a further 72 h in the dark. The sensitivity to compound was then evaluated by the concentration that inhibits cellular growth by 50%, IC50. The cytotoxicity assessments were also performed in the dark following the same protocol but without irradiation. Stock solutions of the compounds were prepared in acetonitrile. 2.4 Interactions with BSA and Abl-SH3 domain The interaction of complexes 1 and 2 with bovine serum albumin (BSA) and Abl-SH3 protein was studied by fluorescence spectroscopy. Protein was dissolved in 20 mmol L-1 HEPES buffer at pH 7.2. A stock solution of each complex in acetonitrile at the concentration of 1.0 × 10 -2 mol L-1 was diluted to 5.0 × 10-4 mol L-1 in HEPES buffer (pH 7.2). To a solution containing 1.0 × 10-6 mol L-1 of protein, increasing concentrations of the tested complexes were added and the emission spectra registered after excitation at 280 nm. Complex concentrations ranged from 0 to 16 × 10-6 mol L-1. All measurements were performed in triplicate. 2.5 BSA photodamage Aqueous solutions containing 1.0 × 10 -6 mol L-1 of complexes and 1.0 × 10 -6 mol L-1 BSA (pH 7.2) were irradiated with UV-A light (365 nm, 610 µW cm-2) for 30 min. Afterwards, the protein was analyzed by emission fluorescence, with excitation wavelength equal to 280 nm.

3. Results and discussion The complexes [Ru(bpy)2(ssz)](PF6), 1, and [Ru(phen)2(ssz)](PF6), 2, were synthesized by the straightforward reaction of cis-[Ru(bpy)2Cl2] or cis-[Ru(phen)2Cl2] with

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ssz, as portrayed in scheme 1. Ssz acts as a bidentate ligand replacing two chlorides from the precursor.

N

N N Ru

S N H O

Cl + Cl

N

0.6 mmoL

N

N HN O S O

O ethylene glicol/water N

N

12 mmol KPF6 OH

HO

0.4 mmoL

N

O

N

N

N O

N

O N

N

N N Ru

Cl +

N

N HN O S O

S N H O 0.6 mmoL

ethylene glicol/water N

N

12 mmol KPF6 OH

0.4 mmoL

HO

O

Cl

N

(PF6)

Ru

HO

O

N

N N

N O

(PF6)

Ru N

O N

HO

Scheme 1. Reaction scheme showing the preparation of complexes 1 and 2.

3.1 Characterization of the complexes The molar conductivity values of 10−3 M solutions of both complexes in nitromethane at 25°C were in the range reported for 1:1 electrolytes, from 60 to 115 S cm2 mol−1 [32].

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The modifications observed in the infrared spectra of complexes 1 and 2 as compared to that of free ssz indicate the participation of the carboxylic oxygen in metal coordination. A strong absorption at 1676 cm-1 in the spectrum of free ssz (Fig. S1) assigned to carboxyl stretching ν(C=O) is not present in the spectra of complexes (Fig S2 and S3). In the complexes, the assymmetric stretching vibration of carboxylate appears at 1590 cm-1 and the symmetric one at 1456 cm-1. The difference between νas(COO-) and νs(COO-), 134 cm-1, is indicative of the coordination of carboxylate as a chelating ligand [33]. In the 1H-NMR spectra of complex 1 and 2, the presence of 27 aromatic hydrogens is in accordance with the stoichiometry of 1ssz: 1Ru: 2L, in which L = bpy or phen. In the range δ 6.4−9.0 the signals of aromatic protons from ssz and the N-heterocyclic ligand (bpy or phen) appear overlapped. A broad signal in the region 10-16 ppm in the free ligand, corresponding to two protons, was assigned to the sulfonamidic and the carboxylic hydrogens (Fig S4). In complexes 1 and 2 this broad band integrates to one proton, due to the deprotonation of the carboxylic oxygen (Fig S5 and S6). The electronic spectra of complexes 1 and 2 with their respective ligands are shown in Fig. 1. The ligands bpy, phen, and ssz exhibit bands centered at 280 nm, 264 nm, and 359 nm respectively, due to intraligand π-π* and n- π* transitions, which undergo bathochromic shifts in the complexes. New bands around 500 nm appear in the spectra of complexes due to metalto-ligand charge transfer transitions (MLCT, d-π). The UV-vis spectra of complexes did not change with time, up to 24 h, attesting their stability in aqueous solution.

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Fig 1. Electronic spectra of aqueous solutions of complexes 1 and 2 at 3.0 × 10-5 mol L-1.

The presence of the complexes in solution was confirmed by ESI-MS studies (Fig S7 and S8). The ESI-MS spectrum of complex 1 in positive mode gives a main peak at m/z 811.101 assigned to [Ru(C10H8N2)2 C18H13N4O5S]+ and that of complex 2 at m/z 859.103 assigned to the species [Ru(C12H8N2)2C18H13N4O5S]+. The isotopic distribution for the proposed species was calculated with the program LabSolutions/LCMSSolutions (2010) and there is a good accordance with the experimental spectra.

3.2 Cytotoxic and photocytotoxic effect

Both complexes were able to inhibit the growth of myelogenous leukemia cells in a concentration dependent manner. In order to investigate the influence of light on cytotoxicity of the complexes, cells were exposed to UV-A light for 5 min after being incubated with compounds for 4 h. Both complexes exhibit higher cytotoxicity under UV-light exposure in comparison to their dark activity. The IC50 values obtained in the dark and after irradiation are shown in Table 1. It is noteworthy that complex 2 exhibits a photocytotoxic index, IC50 dark / IC50 irradiated, of 10. Free sulfasalazine is not cytotoxic in the experimental conditions.

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Table 1. Photocytotoxicity of compounds 1 and 2

compound

-1

IC50 (µmol L-1)

photocytotoxic index

IC50 (µmol L ) dark irradiated 24.72 ± 0.30

8.90 ± 0.16

2.78

2

8.90 ± 0.20

0.92 ± 0.01

9.67

ssz

> 50

-

-

1

IC50 is the concentration required to inhibit 50% of cell growth, after 4 h of incubation, in dark conditions or after 5 min of UV-A light exposure. The values are the mean of triplicate determinations.

3.3 BSA binding studies Spectrophotometric studies indicated that the complexes did not interact with CTDNA. Therefore, their interactions with model proteins were studied. The binding stability of a compound to serum albumin is important to estimate its usefulness as therapeutic agent. The effective dose levels for a drug rely on the level of unbound drug in the circulation because strong binding to serum albumin prevents the binding to the pharmacological target. In order to evaluate the interaction of complexes 1 and 2 with bovine serum albumin (BSA) the binding constants were determined with the help of fluorescence spectroscopy. A representative titration of BSA with the complexes can be seen in Fig. 2. Addition of complexes causes a quenching in the fluorescence emission centered at 340 nm, when excited at 280 nm, indicating that the complexes interact with the protein. Complexes alone are not fluorescent in the same conditions.

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Fig 2 Interaction of complexes 1 and 2 with bovine serum albumin. Fluorescence emission spectra of BSA (1.0 × 10 -6 M, excitation = 280 nm) in the presence of increasing complex concentrations. Complex-to-BSA molar ratios are a = 0, b = 1.66, c = 3.31, d = 4.95, e = 6.57, f = 8.19, g = 9.80, h = 11.40, i = 12.90, j = 14.50, l = 16.10. Inset: log (F0-F) / F] versus log [Q].

The relative fluorescence intensity is directly proportional to the concentration of the quencher, which is the complex in the present case. Fluorescence data were analyzed with the help of the Stern–Volmer equation, F0/F = 1 + kqτ0 [Q] = 1 + Ksv in which F0 and F are the fluorescence intensities in the absence and presence of complex respectively, [Q] is the concentration of complex, Ksv the Stern–Volmer quenching constant, kq the quenching rate constant and τ0 the average lifetime of the fluorophore in the excited state, usually 10-8 s for a biomacromolecule [34]. The Stern-Volmer (Ksv) and the binding constant (Kb) values obtained for the complexes are listed in Table 2. In a dynamic quenching, the maximum scattering collision quenching constant of various quenchers is 2.0 × 10 10 L mol-1 s-1 [35]. As the calculated kq values are much greater than that, the quenching of BSA fluorescence by complexes 1 and 2 is probably due to a static quenching mechanism.

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The binding constant, Kb, was calculated accordingly to the equation log [(F0−F)/F] = log Kb + n log [Q] in which [Q] is the concentration of complex, n is the number of binding sites, F0 and F are the fluorescence intensity in the absence and in the presence of quencher, and n is the number of binding sites. A plot of log [(F0−F)/F] versus log [Q] gives straight line, whose slope gives the value of Kb and the intercept the value of n (inset Fig. 2). In both cases, the number of binding sites is approximately one, which indicates that there is only binding site in BSA for the studied complexes. These values are comparable to those reported for other ruthenium complexes with BSA [34, 36, 37]. The binding constant of a compound to serum albumin should be high enough to guarantee its transportation and distribution but low enough to ensure that the compound will be released to reach its pharmacological target. Therefore, the binding constants of complexes 1 and 2 are within an optimum range [38].

Table 2 Stern-Volmer constants (Ksv), quenching rate constant (kq), binding constant (Kb), number of binding sites (n) and linear regression determination coefficients (R2) for the interaction of complexes 1 and 2 with BSA. Complex

Ksv (L mol-1)

Kq (L mol-1 s-1)

Kb (L mol-1)

n

R2

1

1.69 x 10 5

1.63 x 10 13

2.80 x 104

0.89

0.9588

2

1.09 x 10 5

1.09 x 10 13

8.26 x 104

1.00

0.9942

3.4 Photodamage of BSA by the ruthenium complexes We have firstly checked that the addition of 1.0 × 10 -6 M of each complex to a solution of 1.0 × 10 -6 M BSA did not modify its emission spectrum in dark conditions. In addition, 30 min of UV-A light exposure did not affect BSA spectrum in the absence of complexes.

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The intensity of BSA fluorescence around 340 nm, assigned to tryptophan residues, decreases by photo-irradiation in the presence of complexes. Figure 3 shows the fluorescence emission spectra of solutions containing 1.0 × 10-6 M of BSA in the absence or presence of 1.0 × 10 -6 M of the complexes after UV-A irradiation for 30 min. The concentration of damaged BSA is determined as follows: [damaged BSA] = (F0 – F/F0) × [BSA]0 in which F and F0 are the fluorescence intensities of BSA with or without the treatment and [BSA]0 is the initial concentration of BSA (1.0 × 10 -6 M) [39]. The concentration of damaged BSA was 5.23 × 10-8 and 3.79 × 10-7 M, for complexes 1 and 2, respectively.

Fig 3. Photodegradation of BSA by the synthesized complexes. Emission spectra of solutions containing 1.0 × 10-6 M of BSA in the absence or presence of 1.0 × 10-6 M of complexes 1 and 2 after irradiation for 30 min at λ=365 nm. λexc = 280 nm, pH 7.2.

Upon light irradiation, a sensitizer molecule can be excited to a triplet state and oxidize biomacromolecules by two distinct mechanisms: type I, when the excited sensitizer transfer electrons to oxygen or oxygen-containing species with the generation of reactive oxygen species or type II, when the energy transfer to the ground state 3O2 results in the

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generation of highly reactive singlet oxygen (1O2) [40]. The quenching in the fluorescence of tryptophan residues caused by low concentrations of the ruthenium compounds was not observed without previous exposure to light irradiation. Amino acids are the primary target of an oxidative attack on proteins [41]. Therefore, the pronounced effect of complex 2 can be explained by the amino acid oxidation through a photosensitized reaction.

3.5 Abl-SH3 domain binding The interaction of complexes 1 and 2 with Abl-SH3 was also studied by fluorescence spectrometry. The binding constants determined for complexes 1 and 2 were, respectively, 4.16 × 104 and 4.02 × 10 5 M-1. Representative titrations of Abl-SH3 with the complexes can be seen in Fig. 4.

Fig 4 Interaction of complexes 1 and 2 with domain Abl-SH3. Fluorescence emission spectra of Abl-SH3 (1.0 × 10-6 M, λexcitation = 280 nm) in the presence of increasing complex concentrations. Complex-to-BSA molar ratios are a = 0, b = 2.00, c = 3.96, d = 5.92, e = 7.87, f = 9.80, g = 11.7, h = 13.61, i = 15.50, j = 17.37, l = 19.20. Inset: log (F0-F) / F] versus log [Q].

4. Conclusions

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Two new heteroleptic complexes of ruthenium(II), [Ru(bpy)2ssz](PF6) and [Ru(phen)2ssz](PF6), have been synthesized and fully characterized. Both compounds are able to inhibit CML cellular growth in a concentration-dependent manner and the substitution of phen for bpy increases the activity. UV-light irradiation greatly enhances their cytotoxic activity, which places these complexes as candidates for photodynamic therapy. Photodynamic therapy is a process that involves the production of reactive oxygen species, which can attack biological macromolecules such as proteins, DNA, or lipids. The ability of the ruthenium complexes to damage BSA upon light irradiation suggests that protein oxidation can be involved in the mechanism of action. Nevertheless, the occurrence of DNA photodamage cannot be discarded. In addition, the complexes interact with the SH3 domain of the Abl tyrosine kinase protein, which is responsible for the development of CML.

Supplementary material Infrared, 1H NMR and ESI-MS spectra of the complexes are presented as supplementary material (Fig. S1 to S8).

Acknowledgements We thank CNPq, FAPEMIG, CAPES, and INCT-Catálise for financial support.

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20

25

1000 dark irradiated

Intensity a.u

-1

IC50 (µ mol L )

20 15 10

750 500 250

5 0 1

Compound

2

0 330 360 390 420 450 480 510 Wavelenght (nm)

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Heteroleptic complexes of ruthenium(II) with sulfasalazine and diimines were prepared The complexes inhibit the growth of chronic myelogenous leukemia cells Exposure to UV-A irradiation increases the cytotoxicity by 3 and 10 times The complexes are able to bind and photodegrade bovine serum albumin The complexes interact with the SH3 domain of the Abl tyrosine kinase protein