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European Journal of Medicinal Chemistry xxx (2010) 1e8

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

New ruthenium(II) complexes with N-alkylphenothiazines: Synthesis, structure,in vivo activity as free radical scavengers and in vitro cytotoxicity

Milena Krsti�c a, Sofija P. Sovilj b,*, Sanja Grguri�c-�Sipka b, Ivana Radosavljevi�c Evans c, Sun�cica Borozan a,Juan Francisco Santibanez d, Jelena Koci�c d

a Faculty of Veterinary Medicine, University of Belgrade, Bulevar oslobodjenja 18, 11000 Belgrade, Serbiab Faculty of Chemistry, University of Belgrade, P.O. Box 158, 11001 Belgrade, SerbiacDepartment of Chemistry, Durham University, Durham DH1 3LE, UKd Institute for Medical Research, P.O. Box 102, 11129 Belgrade, Serbia

a r t i c l e i n f o

Article history:Received 27 November 2009Received in revised form6 May 2010Accepted 7 May 2010Available online xxx

Keywords:Ruthenium complexesN-alkylphenothiazinesSingle crystal X-ray diffractionAntioxidant enzymesCytotoxicity

* Corresponding author. Tel.: þ381 11 3336742; faxE-mail address: ssovilj@chem.bg.ac.rs (S.P. Sovilj).

0223-5234/$ e see front matter � 2010 Elsevier Masdoi:10.1016/j.ejmech.2010.05.013

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a b s t r a c t

Three new complexes of the general formula L[RuCl3(DMSO)3] (1e3), where L ¼ chlorpromazinehydrochloride, trifluoroperazine dihydrochloride or thioridazine hydrochloride, were prepared andcharacterized by elemental analysis and spectroscopic methods (FT-IR, UVeVis, 1H NMR and 13C NMR). Inaddition, the crystal structure of the complex 2 containing trifluoroperazine dihydrochloride was solvedby single crystal X-ray diffraction. The complex crystallizes in the monoclinic system, space group P21/n,with a ¼ 10.4935(7) �A, b ¼ 18.6836(12) �A, c ¼ 19.9250(13) �A, b ¼ 98.448(2)�, V ¼ 3864.0(4) �A3. Thestructure was refined to the agreement factors of R ¼ 4.79%, Rw ¼ 11.23%. The effect of three differentdoses (0.4, 4.5 and 90.4 mM/kg bw) of complex 2 on superoxide dismutase (SOD) and catalase (CAT)activity was investigated under physiological conditions. Influence on nitrite production (NO2

�) and thelevel of erythrocytes malondialdehyde (MDA) in rats blood was also evaluated. Complex 2 did not affectthe CAT enzyme activity in vivo and did not cause the hydroxyl radicals production. In the 0.4 and 4.5 mM/kg bw doses it showed almost the same or lower SOD activity and nitrite levels, while the dose of90.4 mM/kg bw significantly increased these parameters. Finally, the cytotoxicity of complexes wereassayed in four human carcinoma cell lines MCF-7, MDA-MB-453 (breast carcinoma), SW-480 (colonadenocarcinoma) and IM9 (myeloma multiple cells). Antiproliferative activity in vitro with low IC50

during 48 h of treatment was observed.� 2010 Elsevier Masson SAS. All rights reserved.

1. Introduction

Ruthenium complexes are used in many different fields ofchemistry, particularly bioinorganic [1e10] and in medicine [11,12].Their properties can be altered by the choice of the ligand, whichmeans that complexes potentially have multiple applications[13e16] and the ability to mimic iron binding to biomolecules[11,17]. Many biological properties have been attributed to ruthe-nium complexes including antioxidant activity [18] and cytotoxicity[19,20].

The field of metallodrugs is expanding rapidly, particularly inrelation to the search for new anticancer drugs. Studies of the cells,cell metabolism and enzyme activity are fundamental to theseinvestigations. Reactive oxygen species (ROS) and reactive nitrogenspecies (RNS) are closely linked tomany degenerative diseases such

: þ381 11 2184330.

son SAS. All rights reserved.

et al., New ruthenium(II) com0.1016/j.ejmech.2010.05.013

as Alzheimer’s disease, Parkinson’s, neuronal and cardiac myocytedeath as well as cancer [21]. Living organisms have developedspecific defenses against ROS, such as superoxide dismutase (SOD),glutathione peroxidase (GPx) and catalase (CAT). SOD dismutatessuperoxide radicals (O2�

�) to form hydrogen peroxide, which isturned into water by CAT and GPx, thereby preventing the forma-tion of hydroxyl radicals [22]. Superoxide radicals can also reactwith nitric oxide radicals (NO�) and participate in the formation ofreactive nitrogen species such as peroxynitrite (ONOO�). Perox-ynitrite can be protonated and dissociated into nitrogen dioxideand hydroxyl radical (OH�) which can cause cell damage by lipidperoxidation. Malondialdehyde (MDA) is formed as one of productsof peroxidation of polyunsaturated fatty acids (PUFA’s) by OH�

[23e25].Previous investigations of antioxidant enzyme activities of

ruthenium complexes demonstrated a slight induction of super-oxide dismutase and catalase in the liver tissue of rats [26], but alsoan inhibition of succinate dehydrogenase (SDH) and cytochromeoxidase (COX) activities in brain, heart, skeletal muscle, liver and

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kidney of rats [27]. Ruthenium(II) and ruthenium(III) complexesexhibit antioxidative enzyme activities (SOD, CAT) and might beresponsible for the complex-induced cytotoxicity [28].

Early uses of phenothiazine and its N-alkyl derivatives were inthe aniline dye industry [29]. Since then they have been extensivelyapplied in medicine and veterinary practice as antipsychotic,antiemetical and antihistaminic drugs [30e33]. Recently, theirderivatives have been found to possess antimicrobial effects andhave found application as pharmaceuticals for prion diseases[34,35]. These compounds also exhibit strong cytotoxic and anti-proliferative activity against leukemic cells [36] and anti-proliferative activity on other cancer lines [37]. The cytotoxic effectof phenothiazines can be increased when they are applied withother drugs [38]. Only a few studies of transition metal complexeswith phenothiazine have been reported so far [39e46]. Thesecomplexes are mononuclear, with ligands coordinated in a mono-dentate fashion through heterocyclic sulphur [39,44,45] or throughheterocyclic nitrogen [46].

The rationale for this work was to combine the pharmacologicalproperties exhibited by ruthenium complexes and by phenothia-zines. We report the synthesis and characterization of three newcomplexes 1e3 of Ru(II) with N-alkylphenothiazines (chlorproma-zine hydrochloride (CP.HCl), trifluoroperazine dihydrochloride(TF.2HCl) or thioridazine hydrochloride (TR.HCl), respectively) intheir hydrochloride salt form (Scheme 1). Crystal structure ofcomplex 2 was solved by single crystal X-ray diffraction methodsand this appears to be the first structure determination of TF.2HClreported in the literature. Our findings provide valuable insight notonly into the structure of complex 2, but also into its ability to act asa free radical scavenger (nitrogen(II) oxide, superoxide anion andhydroxyl radicals), and its protective action against lipid perox-idation in vivo under physiological conditions. In addition,complexes 1e3were tested for invitro cytotoxic activity against fourhuman cell lines MCF-7 and MDA-MB-453 (breast carcinoma),SW-480 (colon adenocarcinoma) and IM9 (myeloma multiple celllines).

2. Results and discussion

2.1. Synthesis and characterization of complexes

The complexes L[RuCl3(DMSO)3] (1e3) (L ¼ chlorpromazinehydrochloride (CP.HCl), trifluoroperazine dihydrochloride (TF.2HCl)or thioridazine hydrochloride (TR.HCl)) were synthesized by thereaction of the starting complex [RuCl2(DMSO)4] and the corre-sponding ligands in a molar ratio of 1:1.6 in absolute ethanol. Othermolar ratios attempted gave lower product yields. The complexesare soluble in water and in common organic solvents (methanol,ethanol, chloroform, dimethyl sulfoxide). The molar conductivityvalues obtained for 1 � 10�3 mol dm�3 solution of the 1e3complexes in ethanol [lM ¼ 26.9; 26.5; 47.0 S cm2 mol�1,

Scheme 1. The structures with the atom numbering of the ligands: a) Chlorpromazine hchloride, b) Trifluoroperazine dihydrochloride (TF.2HCl), 10-[3-(4-Methyl-1-piperazinyl)pro(TR.HCl), 10-[2-(1-methyl-2-piperidinyl)ethyl]-2-methylthio phenothiazine hydrochloride.

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respectively] fall into the range anticipated for a 1:1 electrolyte[47]. All obtained complexes exhibit similar absorption spectra,which indicate that the central ion and ligands are bonded ina similar mode. Phenothiazines exibit a band at 306e312 nm due tothe intraligand n / p* transition [38]. In the spectra of thecomplexes this maximum remains unchanged in a shape, but shiftsto longer wavelengths (317e324 nm), suggesting that the addi-tional ligands are included in the formation of the correspondingcomplexes. The unchanged intensity as well as position of thesepeaks recorded on freshly prepared samples and 48 h later, indi-cates similar stability of these compounds during this period [48].

The significant regions of the IR spectra of all complexes are verysimilar and there are several regions of considerable interest. Astrong band of the starting complex [RuCl2(DMSO)4] at 1097.2 cm�1

arising from n(SO) vibration is shifted to slightly lower values in thecomplexes 1e3. The absence of a band at 921 cm�1 of n(SO) (O-bonded) in the new complexes indicates only S-bonding of DMSOligands in the prepared complexes. In addition, the same shifts of d(CSO) vibration confirm the RueS bond. A higher wavenumber forthe na(CS) vibrations in 1e3 with respect to the starting complexmay explain changes in the bond strength between carbon atomand sulphur [49]. The 2300e2600 cm�1 region in the spectra of thefree N-alkylphenothiazine ligands exhibits a strong broad bandassociated with the interaction of the exocyclic quaternaryammonium ion, R3NHþ with the chloride ion [42]. This band hasshifted to higher wavenumber in the spectra of the complexesindicating a change in the strength of the hydrogen bondingbetween the nitrogen atom and the chloride ion. This is consistentwith the fact that the protonated exocyclic amine nitrogen ishydrogen bonded to Cl� which is in turn coordinated to theruthenium. This arrangement has been demonstrated crystallo-graphically for complex 2. A presence of the aromatic ring isconfirmed by bands at 1550e1600 cm�1 (n(CH)ar) and1000e1100 cm�1 (d(CH)ar), as well as a shift at 800 cm�1 (p(CH)).The small positive shifts of 10 cm�1 in heterocyclic n(CSC) modes incomplexes indicate only conformational changes, but not thecoordination of S-atom from N-alkylphenothiazines.

Comparison of the 1H NMR spectra of the free ligands with thoseof the corresponding complexes shows that some of the resonancesshift upon complexation. Chemical shifts at 3.30e3.60 ppm of thecomplexes correspond to the DMSO-protons and indicate the S-bond to the ruthenium [50]. Moreover, the broad singlet whichoccurs far downfield at 10e11.5 ppm in the free ligands, attribut-able to the heterocyclic protons of R3NHþ, exhibits downfield shiftsin all complexes, indicating a change in this proton environment.This is evidenced by the crystal structure, with Cl� and DMSO in theRu coordination sphere and the protonated nitrogen atoms ofligands (for complex 2). It is therefore reasonable to predict thesimilar binding mode in complexes 1 and 3. The aromatic hetero-cyclic ring protons from phenothiazine give resonances in the rangeof 6.80e7.27 ppm. The protons of free ligands exhibit almost the

ydrochloride (CP.HCl), 10-[3-(Dimethylamino)propyl]-2-chloro phenothiazine hydro-pyl]-2-trifluoromethyl phenothiazine dihydrochloride, c) Thioridazine hydrochloride

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same chemical shifts in complexes indicating the outer sphereposition of alkylphenotiazines [40]. The 13C NMR spectra of freeligands and their complexes are very similar. The carbon atoms thatsuffer a greater influence upon complexation are those near the Rucoordination sphere. The 13C NMR data for the complexes and thecorresponding ligands are listed in Table 1. The atom numbering isshown in Scheme 1. A noticeable feature of the spectrum ofcomplex 1 is a higher field shift of carbon atoms from the N-alkylpart of CP.H. Also, the spectrum of complex 3 shows the same high-field shifts of piperidinyl part of molecule. Finally, signals of ligandC-atoms of complex 2 exhibit slight upfield shifts in piperazinylpart of molecules.

Fig. 1. Asymmetric unit of (TF.H2)[RuCl3(DMSO)3]Cl$C2H5OH (2) with the crystallo-graphic numbering scheme; atomic displacement parameters are drawn at a 70%probability level.

2.2. X-ray crystallography of (TF.H2)[RuCl3(DMSO)3]Cl$C2H5OH (2)

The complex (TF.H2)[RuCl3(DMSO)3]Cl$C2H5OH (2) crystallizesin themonoclinic space group P21/n. The molecular structure of thecomplex with the crystallographic numbering scheme is given inFig. 1. The Crystallographic Information File has been deposited aselectronic supplementary information.

It should be noted that single crystal X-ray diffraction stronglysuggests a crystallised solvent content of one ethanol molecule performula unit. This is in contrast to the CHN analysis carried out onbulk sample, where the found composition corresponds moreclosely to half a molecule of ethanol. An attempt was made tomodify the structural model to reflect this composition; howeverthis precluded the refinement from converging and led to unrea-sonable shapes of atomic displacement parameters on the ethanolmolecule. It is possible that the crystallised ethanol content in thesample isn’t homogeneous or constant and that the single crystalselected for X-ray diffraction isn’t fully representative of the bulk inthis respect.

The coordination sphere around Ru(II) consists of three DMSOmolecules and three chloride ions in a slightly distorted octahedralgeometry, with bond angles ranging from 87.29(4)� to 94.11(4)�. Ru(II) e Cl bonds range from 2.432(1) to 2.456(1) �A, and Ru(II) e Sbonds from 2.260(1) to 2.264(1) �A. These values are in excellentagreement with those found for the [RuCl3(DMSO)3]� structuralunit in the [RR’NHOH] [RuCl3(DMSO)3] (R ¼ R0 ¼ H; R¼Me, R0 ¼ H;R¼ R0 ¼ Et) series of complexes [51]. The three SeO bond lengths in

Table 113C NMR chemical shiftsa (in ppm) of the complexes 1e3 and the correspondingligands.

Complex 1 CP.HCl Complex 2 TF.2HCl Complex 3 TR.HCl

C(10a) 146.10 146.02 C(10a) 145.31 143.26 C(10a) 145.50 145.40C(9a) 143.80 143.66 C(9a) 143.42 143.26 C(9a) 144.04 144.04C(2) 133.61 133.66 C(4a,5a) 128.07 128.20 C(2) 138.47 138.43C(4) 128.34 128.31 C(1) 125.52 128.20 C(5a) 127.87 127.82C(8) 127.94 127.89 C(3) 124.07 124.19 C(4a) 127.62 127.76C(6) 127.78 127.89 C(2) 120.06 120.19 C(3) 126.51 127.58C(5a) 125.96 125.87 C(9,4,6) 116.35 116.38 C(1) 125.18 126.38C(4a) 124.65 124.59 C(8,7) 112.23 112.29 C(4) 123.43 123.34C(7) 123.70 123.74 C(21) 54.56 54.84 C(9) 123.05 122.80C(3) 123.12 123.16 C(11,13) 49.71 49.65 C(6) 121.21 121.12C(9) 116.33 116.42 C(15,19) 48.03 48.29 C(8) 116.49 116.38C(1) 116.29 116.33 C(16,18) 44.41 44.35 C(7) 115.28 114.47C(13) 56.55 55.64 C(20) 43.32 42.95 C(19) 64.33 63.56C(11) 44.74 44.28 C(12) 21.23 21.05 C(17) 57.30 56.77C(15) 44.52 42.88 C(13) 44.35 43.02C(12) 21.92 21.59 C(11) 43.35 40.91

C(20) 27.95 28.37C(12) 27.40 27.42C(16) 22.49 22.98C(14) 22.18 22.30C(15) 16.13 16.04

a For the assignment of the various atoms see Scheme 1.

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coordinated DMSO molecules in complex 2 are between 1.487(3)and 1.492(3) �A.

Despite being frequently used in biomedical and neuroscienceresearch, trifluoroperazine does not appear to have been crystal-lographically characterized before and our work seems to be thefirst report of its crystal structure [52]. In complex 2, protonatedtrifluoroperazine ligand is in the outer coordination sphere. In thepiperazinyl ring, the NeC bonds lengths are 1.413(5) and 1.415(3)�Aand the CeNeC angle is 116.0(3)�. The NeC bond lengths in thephenothiazine ring are longer and range from 1.497(6) �A to 1.505(5) �A, while the CeNeC angles are smaller at 110.5(3)�, SeC bondlengths are 1.755(5) and 1.775(4)�A and the C(38)-S(37)-C(71) angleis 97.3(2)�. This geometry represents a significant distortion due tobonding of the phenothiazine fragment in trifluoroperazine relative

Fig. 2. Hydrogen bonding interactions in (TF.H2)[RuCl3(DMSO)3]Cl$C2H5OH (2).

plexes with N-alkylphenothiazines: Synthesis, structure,..., European

Fig. 3. Activity of SOD: activity of SOD on native PAGE (a), total activity of SOD (b), activity of isoenzyme SOD1 (c) and activity of isoenzyme SOD2 (d); I e control group; II e ratstreated i.p. with complex 2 at dose of 0.4 mM/kg bw; III e rats treated i.p. with complex 2 at dose of 4.5 mM/kg bw; IV e rats treated i.p. with complex 2 at dose of 90.4 mM/kg bw.

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to the structure of the phenothiazine molecule itself, in which thecorresponding angles are larger (124.4� and 100.9� for the CeNeCand the CeSeC angle, respectively) [53].

The connectivity between the protonated trifluoroperazine andthe [RuCl3(DMSO)3]� building blocks in the crystal structure of (2)is provided by hydrogen bonding. Each trifluoroperazine formsa NeH.Cl hydrogen bond to a [RuCl3(DMSO)3]� unit ‘above’ itspiperazinyl ring and a NeH.O hydrogen bond toa [RuCl3(DMSO)3]� unit ‘below’ it (Fig. 2). The donoreacceptordistances in these interactions are 3.149(7) �A for the NeH.Clhydrogen bond and 2.975(7) �A for NeH.O.

2.3. Biological assays of complex 2

Complex 2, given intraperitoneally to the rats, did not show anyevidence of latency response. The change in the SOD activities inthe erythrocytes is presented in Fig. 3. An increase of the total SODactivities in dose of 0.4 and 4.5 mM/kg bw, as well as in activities ofisoenzymes SOD1 and SOD2 (Fig. 3aed) is observed. The total SODactivities in blood samples of rats treated with 90.4 mM/kg bw ofcomplex 2 increases by 73% relative to the control group (Fig. 3a, b),

Fig. 4. CAT activities: I e control group; II e rats treated i.p. with complex 2 at dose of0.4 mM/kg bw; III e rats treated i.p. with complex 2 at dose of 4.5 mM/kg bw; IV e ratstreated i.p. with complex 2 at dose of 90.4 mM/kg bw.

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while the SOD1 and SOD2 isoenzyme activity increase by 70 and150%, respectively (Fig. 3c, d) compared to the control group.

The CAT activity in all three doses is slightly lower than in thecontrol group (Fig. 4). These results are in accordance with the SODactivities and demonstrate that complex 2 did not producehydroxyl radicals under physiological conditions.

The decrease of nitrite level (p < 0.05) in dose of 4.5 mM/kg bwsuggests a possible interaction between the complex and nitrogen(II)oxide [26]. Complex 2 is obviously capable of scavenging NO� radicalsin this dose. Similar nitrogen monoxide scavenging capability hasbeen observed in other ruthenium complexes [54]. This significantdifference (p < 0.001) between the control group and the dose of90.4 mM/kg bwsuggests that the rutheniumcomplex in a higher doseeither induces productionofNO� or cannot reactwith it (Fig. 5). In thisdosemorenitrogenmonoxide isproduced than canbescavengedandas a consequence the NO2

� level increases. This type of study may beuseful in providing understanding of specific uptake mechanisms ofruthenium complexes into cells and confirms the affinity of thesecompounds to scavenge NO� in appropriate doses.

Cells of the immune system produce both the superoxide anionand nitric oxide radical during oxidative stress under inflammatory

Fig. 5. Content of NO2�: I e control group; II e rats treated i.p. with complex 2 at dose

of 0.4 mM/kg bw; III e rats treated i.p. with complex 2 at dose of 4.5 mM/kg bw; IV e

rats treated i.p. with complex 2 at dose of 90.4 mM/kg bw.

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Fig. 6. Levels of erythrocytes MDA: I e control group; II e rats treated i.p. withcomplex 2 at dose of 0.4 mM/kg bw; III e rats treated i.p. with complex 2 at dose of4.5 mM/kg bw; IV e rats treated i.p. with complex 2 at dose of 90.4 mM/kg bw.

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processes. Under these conditions, these species may react togetherto produce significant amounts of more reactive species, such as theperoxynitrite anion. The product of peroxynitrite overproduction isthe hydroxyl radical, which causes tissue damage during

Fig. 7. Cytotoxicity curves of complexes 1e3 on MCF-7 (a) and MDA-MB-453 (b) (breast cardifferent cell lines were treated with complexes 1e3 at different concentrations duringindependent experiments are shown.

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pathological processes. As very reactive molecules, they can reactwith and damage cellular components such as membrane lipids[55,56]. Complex 2 in doses lower than 4.5 mM/kg bw is proved asa potent scavenger of NO� under physiological conditions and hasa protective role against cell damages.

Finally, malondialdehyde as an indicator of lipid peroxidationexhibits some differences between the control group and all doses,with a significant difference observed between the control groupand the third dose of 90.4 mM/kg bw (Fig. 6). The major pathway forNO� metabolism is oxidation to nitrite and nitrate with productionof hydroxyl radicals, and this could explain the higher level of MDA.

2.4. Cytotoxic activity of complexes

The cytotoxicity of the complexes 1e3, free phenothiazines aswell as the starting complex [RuCl2(DMSO)4], were studied in a fourhuman cancer cell lines: MCF-7 and MDA-453 (breast cancer);SW-480 (colon adenocarcinoma) and IM9 (myelomamultiple) overa period of 48 h of incubation. The results are analyzed by cellviability curves (Fig. 7) and expressed as IC50 (Table 2).

The starting complex [RuCl2(DMSO)4], did not show any activityon selected cell lines in concentration under 25 mM. Free pheno-thiazines have low cytotoxic activities against selected cell lines,except TR.HCl which exhibits a considerably higher cytotoxicity

cinoma), SW-480 (c) (colon carcinoma) and IM9 (d) (myeloma multiple cell lines). The48 h. Cell viability was determined by MTT assay. Representative curves from three

plexes with N-alkylphenothiazines: Synthesis, structure,..., European

Table 2Inhibition of cell viability (IC50� SD, mM) of the complexes and free ligands onMCF-7 and MDA-MB-453 (breast carcinoma), SW-480 (colon carcinoma) and IM9(myeloma multiple cell lines).a

Compound Cell lines

MCF-7 MDA-MB-453 SW-480 IM9

Complex 1 >25 8.5 � 0.4 22.0 � 1.1 3.00 � 0.1Complex 2 12.7 � 1.5 10.6 � 0.5 11.2 � 0.3 9.8 � 2.5Complex 3 >25 >25 >25 5.50 � 0.2CP.HCl >25 25.0 � 0.5 >25 >25TF.2HCl 20.2 � 0.7 18.5 � 0.4 17.5 � 1.0 20.5 � 0.8TR.HCl 20.3 � 1.7 9.6 � 0.5 10.2 � 0.6 10.4 � 1.0[RuCl2(DMSO)4] >25 >25 >25 >25

a Cytotoxicity as IC50 for each cell line is in average of three independent exper-iments after 48 h of treatment.

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against MDA-MB-432, SW-480 and IM9 cell lines. In case of MCF-7cell lines, TF.2HCl and TR.HCl showed similar activities (IC50 are20.2 and 20.3 mM, respectively), while CP.HCl had less sensitivityagainst these cells (IC50 > 25 mM). Complex 3 was inactive againstthe mentioned all cell lines at doses ranging from 5 to 25 mM. MCF-7 cell line was more sensitive (IC50 of 12.7 � 1.5 mM) to complex 2than to the complexes 1 and 3. Also, cytotoxicity of this compoundis better than rutheniumeindazolium complex (KP 1019) with anIC50 179 � 18 mM in the same cell line [57].

In MDA-MB-453 cell lines, at low concentrations of allcomplexes (w5 mM) a similar cell growth inhibition was observed,while above 5 mM the curves began to separate. Complex 1 has thelowest value of cell inhibition (IC50¼ 8.5 � 0.4 mM) against the cellline MDA-MB-453, but at concentration of 15 mM the inhibition ofcells is about 80%. Complex 2 induced a more rapid inhibition inMDA-MB-453 and SW-480 than in other two cell lines. At relativelyhigh concentrations (15e25 mM) in the breast cancer cell line MDA-MB-453 and colon adenocarcinoma SW-480 the inhibition of cellviability was the same, achieving an almost complete cell death.This compound has a better cytotoxic activity against SW-480 celllines than rutheniumetriazole complexes [5] and induces cancercell apoptosis in lower concentration (Fig. 7c). Therefore, complex 2is over 200%more active against SW-480 cell lines than complexes 1

Table 3Crystallographic data for (TF.H2)[RuCl3(DMSO)3]Cl$C2H5OH (2).

Formula C29H50Cl4F3N3O4RuS4Mr 932.87Colour YellowCrystal system MonoclinicSpace group P21/na, �A 10.4935(7)b, �A 18.6836(12)c, �A 19.9250(13)b, deg 98.448(2)V, �A3 3864.0(4)Z 4Dx, g cm�3 1.603F (000) 1920.0abs.koef.(Mo Ka), cm�1 0.951Crystal size, mm 0.06 � 0.10 � 0.22Diffractometer Bruker SMART 6000Temperature, K 120Radiant (l, �A) Mo K/a (0.71073)Monochromator GraphiteScan type (u-2q)Scan speed, deg/min 10q range, deg (1.502e34.777)Total number of reflections 71.945

Number of reflectionwith I > 3.0/s (I) 8133Rb, % 4.79Rwb , % 11.23

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and 3. Myeloma multiple cell line IM9 is more sensitive to the alltested complexes, but complex 1 inhibited 50% of cells only in theconcentration of 3 mM. Themost efficient compoundwas complex 2,which induced almost total cell mortality at 25 mM,while other twocomplexes inhibited about 80% of IM9 cells. The IC50 values suggesta variation of cell sensitivity in the cell lines studied and increase inthe following order: MCF-7 < SW-480 < MDA-MB-453 < IM9.

3. Conclusion

The present study demonstrates simple synthetic routes tothree new Ru(II) complexes with N-alkylphenothiazine ligands. Thestructure of complex 2, determined by single crystal X-ray diffrac-tion, contains three DMSO molecules and three chlorine ions in theRu coordination sphere and N-alkylphenothiazine out of them.Single crystals of complexes 1 and 3 could not be grown to enablecrystallographic studies, but spectroscopic results obtained forthese complexes suggest structures similar to the complex 2. Bio-logical assays provide clear evidence that complex 2, under phys-iological conditions can act as a scavenger of NO� radicals in lowerdoses of 0.4 and 4.5 mM/kg bw, and as a scavenger of free radicalssuch as superoxide anion and hydroxyl radicals. Complex 2 influ-ences the SOD and CAT activities only in dose of 90.4 mM/kg bw.Complex 2, as non-toxic compound under physiological conditions,could provide potential therapeutic benefits in lower doses (0.4 and4.5 mM/kg bw), in disorders where these reactive species areinvolved. The investigation of antitumor properties of complexes1e3 in four human cell lines (MCF-7, MDA-MB-453, SW-480 andIM9) showed a dose-dependence and variation by cell types. Themost active compound is complex 1, which demonstrates a higheractivity against MCF-7 and IM9, but it is less effective against othercell lines and did not cause complete cell death. Complex 2 is themost sensitive against human breast cancer cell line (MDA-MB-453) and human colon adenocarcinoma cell line (SW-480) thanother two in low concentration and induced almost total cell deathat 25 mM during 48 h of treatment. Also, complex 2 is the onlycompound in our investigations with cytotoxic activities against alltumour cell lines. The selective cytotoxicity of these complexes,especially complex 2 against the cancer cells suggests their greatpotential for development as anticancer drugs.

4. Experimental

4.1. Materials and methods

High-purity grade commercial RuCl3$xH2O (Acros) and ligands(chlorpromazine hydrochloride (CP.HCl), trifluoroperazine dihy-drochloride (TF.2HCl) and thioridazine hydrochloride (TR.HCl))(Aldrich) were used without previous purification, while for thebiological assays the used chemicals were of reagent grade (Merck).The complex [RuCl2(DMSO)4] was obtained by a route described inthe literature [49].

Elemental analyses were carried out on Elemental Vario EL IIImicroanalysers. Molar conductivity of ethanol solutions(1 � 10�3 mol dm�3) was measured at 20 �C by a Jenway-4009conductometer. Electronic spectra of the complex solutions(1.50e2.25$10�4 mol dm�3) were recorded on a GBC UVevisibleCintra 6 spectrometer. Infrared spectra of the ligands and theircomplexes were recorded on a Nicolet 6700 FT-IR spectrometer byATR technique. 1H and 13C NMR spectra were ran on a VarianGemini 2000 spectrometer operating in the Fourier transformmode (200 and 50 MHz) in CDCl3 at room temperature.

Enzyme activities were measured on CECIL CE 2021 UV/VISspectrophotometer, while electrophoresis was performed ona vertical device Mini Ve Hoffer, LKB 2117, Bromma, Uppsala,

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Sweden. The band intensities of the isoenzymes of SOD wereestimated using Scion Image Beta 4.02 software [58]. Nitrite ion(NO2

�) levels were analyzed on a microplate reader (Plate reader,Mod. A1, Nubenco Enterprises, INC) at a wavelength of 545 nm.

4.2. General procedure for the synthesis of complexes

Appropriate amounts of N-alkylphenothiazines were added inone portion (67 mg, 0.19 mmol of chlorpromazine hydrochloride;91 mg, 0.19 mmol of trifluoroperazine dihydrochloride and 77 mg,0.19 mmol of thioridazine hydrochloride) to a solution of[RuCl2(DMSO)4] complex (60 mg, 0.12 mmol) in absolute ethanol(15 cm3) to obtain complexes 1e3. The reaction mixtures wereheated on a silicon bath at 65 �C for 5 h. Upon cooling the mixtureto room temperature yellow precipitates were vacuum filtered off,washed with a small amount of acetone and dried in vacuo (1 and2). The complex 3 was isolated after the filtrate evaporation ofreaction mixture and adding of 10 cm3 of acetone. The yellowproduct was precipitated and filtered off, washed with smallportion of acetone and dried in vacuo. The complexes obtained asanalytically pure samples.

4.2.1. (CP.H)[RuCl3(DMSO)3]$C2H5OH (1)Yield: 55 mg, 56.74%. Anal. Calcd. for (C25H44Cl4N2S4O4Ru) (Mr

807.78) (%): C, 37.17; H, 5.49; N, 3.47; S, 15.88. Found: C, 36.88; H,5.10; N, 3.62; S, 15.59. Selected IR absorption bands: n(R3NHþ)2362.4 cm�1, n(CH)ar 1562.8 cm�1, d(CH)ar 1097.8, 1021.7 cm�1, n(SO) 1097.8 cm�1, p(CH) 804.2 cm�1, n(CSC) 754.4 cm�1, na(CS)679.9 cm�1, d(CSO) 425.6 cm�1 1H NMR (200 MHz, CDCl3):d (ppm) ¼ 1.24 (t, 3H, CH3CH2OH); 2.34e2.4 (m, 2H, CH2CH2CH2N(CH3)2); 2.76 (s, 6H, N(CH3)2); 3.13 (m, 2H, CH2CH2CH2N(CH3)2);3.35 (m, 2H, CH3CH2OH); 3.40e3.48 (m, 6H, (CH3)2SO); 4.01 (t,J ¼ 6 Hz, 2H, CH2CH2CH2N(CH3)2); 6.87e7.27 (m, 7H, phenothia-zine); 8.50 (br.s., 1H).

4.2.2. (TF.H2)[RuCl3(DMSO)3]Cl$0.5C2H5OH (2)Yield: 61 mg, 55.87%. Anal. Calcd. for (C28H47Cl4N3F3S4O3.5Ru)

(Mr 909.84) (%): C, 36.96; H, 5.21; N, 4.62; S, 14.10. Found: C, 36.65;H, 5.24; N, 4.50; S, 14.00. Selected IR absorption bands: n(R3NHþ)2357 cm�1, n(CH)ar 1601.6 cm�1, d(CH)ar 1098, 1022 cm�1, n(SO)1098 cm�1, p(CH) 815.5 cm�1, n(CSC) 762.7 cm�1, na(CS) 722,681 cm�1, d(CSO) 424.2 cm�1 1H NMR (200 MHz, CDCl3):d (ppm) ¼ 1.24 (t, 3H, CH3CH2OH); 2.36 (m, 2H, CH2CH2CH2); 2.89(s, 3H, NCH3); 3.29 (m, 2H, CH2N-piperarazine); 3.37 (m, 2H,CH3CH2OH); 3.44 (m, 6H, (CH3)2SO); 3.74 (q, J ¼ 6.8 Hz, 8H,piperazine); 4.14 (m, 2H, CH2N-phenothiazine); 6.93e7.27 (m, 7H,phenothiazine); 10.3 (br.s., 1H); 11.2 (br.s., 1H).

4.2.3. (TR.H)[RuCl3(DMSO)3]$0.5C2H5OH (3)Yield: 41 mg, 40.84%. Anal. Calcd. for (C28H48Cl3N2S5O3.5Ru) (Mr

836.46) (%): C, 40.21; H, 5.78; N, 3.35; S, 19.17. Found: C, 40.18; H,5.65; N, 3.49; S, 18.95. Selected IR absorption bands: n(R3NHþ)2445 cm�1, n(CH)ar 1589.1, 1566.3 cm�1, d(CH)ar 1018.6 cm�1, n(SO)1094.6 cm�1, p(CH) 800.5 cm�1, n(CSC) 755.3 cm�1, na(CS) 719.4,679.2 cm�1, d(CSO) 426.6 cm�1 1H NMR (200 MHz, CDCl3):d (ppm) ¼ 1.21 (t, 3H, CH3CH2OH); 1.30 (m, 2H, NCH2CH2); 2.50 (m,6H, piperidine); 2.72 (m, 3H, SCH3); 2.92 (m, 3H, NCH3); 3.22e3.50(m, 3H, piperidine; 2H, CH3CH2OH); 3.60 (m, 6H, (CH3)2SO); 4.10 (m,2H, NCH2CH2); 6.81e7.27 (m, 7H, phenothiazine); 8.30 (br.s., 1H).

4.3. Single crystal X-ray diffraction

Single crystals of (TF.H2)[RuCl3(DMSO)3]Cl$C2H5OH (2) suitablefor X-ray diffraction were obtained directly from the reactionmixture after addition of a small amount of acetone by slow

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evaporation at room temperature. A yellow crystal of approximatedimensions of 0.06 � 0.10 � 0.22 mm3 was selected for theexperiment. Data were collected on a Bruker AXS SMART 6000diffractometer using Mo Ka radiation, equipped with a CCDdetector and an Oxford Cryosystems N2 crysotream, at a tempera-ture of 120 K. A full sphere of datawas recorded with a framewidthof 0.3� and a counting time of 30 s per frame. A multiscanabsorption correction [59] was applied to the raw data and theresulting Rint was 4.0%. Frames were integrated using the programSAINT Plus [60]. The structure was solved by direct methods usingSIR-92 software [61] and refined using the Oxford CRYSTALS suite[62]. The positions and atomic displacement parameters of all non-hydrogen atoms were refined; hydrogen atoms were placedgeometrically and treated using a riding model. A summary of thecrystallographic data is given in Table 3.

4.4. Biological assays

The experiments were carried out on adult male Wistar rats(200e300 g) housed in stainless steel cages with wired floors andwith free access to food and water, in a room under controlledconditions (12 h lightedark cycles, temperature 22 � 2 �C). Ratswere randomly assigned to an experimental or a control group. Allexperiments were done according to our institutional guidelinesfor animal research and principles of the European Convention forthe Protection of Vertebrate Animals Used for Experimental andOther (Official Daily N. L 358/1-358/6, 18, December 1986).

Wistar rats were divided into four groups of eight animals:control (group I), and three groups treated i.p. with complex 2dissolved in water, at doses of 0.4 mM/kg bw (group II), 4.5 mM/kgbw (group III) and 90.4 mM/kg bw (group IV). Blood samples(6e8 cm3) were obtained via aorta abdominalis puncture (from allrats, including the control group, 24 h after treatment) andcollected in tubes containing Na-citrate (3.8%w/v) as anticoagulant.Erythrocytes were separated by centrifuge (3000 rpm) and washedin saline solution three times, and the enzyme activities weredetermined immediately.

4.5. Erythrocyte enzyme activities

The CAT activity was determined as described in the literature[63]. One unit of CATactivity was defined as the activity required fordegradation of 1 mmol H2O2 for 60 s at 25 �C and pH 7.0. Activitywas expressed as U/gHb. The SOD activity was determined usingCalbichem’s Superoxide Assay Kit which utilizes 5,6,6a,11b-tetra-hydro-3,9,10-trihydroxybenzo[c]fluorine, recording at 525 nm. SODactivity was expressed as U/gHb. The isoenzymes of SOD (SOD1 andSOD2) were separated on discontinuous polyacrylamide gels [64].The density of each band was estimated with respect to the totalarea/g Hb. LPO in erythrocytes was quantified by measuring theformation of thiobarbituric acid reactive substances (TBARS) [65]and expressed in nM MDA/g Hb. The total Hb content wasmeasured by cyanmethemoglobin method [66]. ELISA method byGriess Reagent [67] was used to analyze nitrite ion (NO2

�) levels.Obtained results were expressed as mM/L.

Data are expressed as the means � SD. Statistical significancewas tested by the one-way Anova, followed by Dunnett’s t-test. Theminimum level of statistical significance was set to p < 0.05.

4.6. Cell culture

Human breast carcinoma cell lines (MCF-7, ATCC/HTB-22; MDA-MB-453, ATCC/HTB-131) and human colon adenocarcinoma cellline (SW-480, ATCC/CCL-228) were maintained in Ham’s F12:DMEM (1:1) (Sigma Chemicals Co, USA) supplemented with 10%

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heat-inactivated fetal bovine serum (FBS, PAA GmbH, Austria).Human myeloma multiple cell line IM9 (ATCC/CCL 159) wascultured in RPMI-1640 (Sigma Chemicals Co, USA) supplementedwith 10% FBS. Cell culture media were supplemented with peni-cillin (100 U/mL), streptomycin (200 mg/mL), HEPES (10 mM) and L-glutamine (2 mM). The cells were grown at 37 �C in 5% CO2 andhumidified air atmosphere.

4.7. Cytotoxicity assay

Cytotoxicity of the investigated compounds was determinedusing MTT assay [68]. Cells (5 � 104) were seeded in 96-well cellculture plates in culture medium and grown over a period of 24 h.Stock solutions of investigated compounds were made in sterilePBS at concentrations of 5 mM. Compounds were diluted withnutrient medium to desired final concentrations and tested atdoses ranging from 5 to 25 mM. Nutrient media without cells wereused as a blank. All samples were done in triplicate.

For the treatment, cells were incubated with different concen-trations of compounds for 48 h at 200 mL final volume. After theincubation period, 20 mL of MTT solution (5 mg/mL in phosphatebuffer solution (PBS), pH 7.2) were added to each sample. Sampleswere incubated over a period of 4 h at 37 �C. To dissolve Formazancrystals in MCF-7, MDA-MB-453 and SW-480 cell cultures themedia were discarded and Formazan crystals were dissolved with100 mL of DMSO:isopropanol (2:3). For IM9 cell line 100 mL of 30%sodium dodecyl sulphate in 0.03 M HCl were added in each well.Absorbance at 530 nm was recorded on an enzyme-linked immu-nosorbent assay reader.

Cytotoxic curves, as well as IC50 (mM) concentrations (defined asconcentrationofdrugthatdecreases the cell viabilityby50%comparedto non-treated control cells), were plotted by Excel software.

Acknowledgements

This work was supported by the Ministry of Science and Tech-nological Development of the Republic of Serbia (Projects No.142028 and 143018).

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