Nimesulide Silver Metallodrugs, Containing the Mitochondriotropic

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Nimesulide Silver Metallodrugs, Containing the Mitochondriotropic, Triaryl Derivatives of Pnictogen; Anticancer Activity against Human Breast Cancer Cells Christina N. Banti,*,† Constantina Papatriantafyllopoulou,‡ Maria Manoli,‡ Anastasios J. Tasiopoulos,‡ and Sotiris K. Hadjikakou*,† †

Inorganic and Analytical Chemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece Department of Chemistry, University of Cyprus, 1678 Nicosia, Cyprus

‡

S Supporting Information *

ABSTRACT: Novel silver(I) metallo-drugs of the nonsteroidal anti-inflammatory drug nimesulide (nim) and the mitochondriotropic triaryl derivatives of pnictogen ligands (tpE, E = P (tpp, tptp, or totp), As (tpAs), Sb (tpSb)) with the formulas {[Ag(nim) (tpp)2]DMF} (1), [Ag(nim) (tptp)2] (2), [Ag(nim) (totp)] (3), [Ag(nim) (tpAs)2] (4), and [Ag(nim) (tpSb)3] (5) ((tpp = triphenyphosphine, tptp = tri(p-tolyl)phosphine, totp = tri(otolyl)phosphine, tpAs = triphenylarsine, tpSb = triphenylantimony, and DMF = dimethylformamide) were synthesized and characterized by melting point, vibrational spectroscopy (mid-Fourier transform IR), 1H NMR, UV−visible spectroscopic techniques, and X-ray crystallography. The in vitro cytotoxic activity of 1−5 against human breast adenocarcinoma cancer cell lines: MCF-7 (estrogen receptor (ER) positive) and MDA-MB-231 (ER negative) was determined. The genotoxicity on normal human fetal lung fibroblast cells (MRC-5) caused by 1−5 was evaluated by fluorescence microscopy. The absence of micronucleus in MRC-5 cells confirms the in vitro non toxicity behavior of the compounds. Because of the morphology of the cells, an apoptotic pathway was concluded for the cell death. The apoptotic pathway, especially though the mitochondrion damage, was confirmed by DNA fragmentation, cell cycle arrest, and permeabilization of the mitochondrial membrane tests. The molecular mechanism of action of 1−5 was further studied by (i) the binding affinity of 1−5 toward the calf thymus (CT) DNA, (ii) the inhibitory activity of 1−5 against lipoxygenase (an enzyme that oxidizes polyunsaturated fatty acids to leukotrienes or prostaglandins), and (iii) the catalytic activity of 1−5 on the oxidation of linoleic acid (an acid that partakes in membrane fluidity, membrane enzyme activities, etc.) to hyperoxolinoleic acid by oxygen.

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INTRODUCTION Breast cancer is among the most widespread cancer types in the world, nowadays. It is estimated that millions of women alive have breast cancer diagnosed within the last five years, and the incidence rates are increasing.1,2 This malignancy is hormonedependent, since estrogen receptors (ERs) are expressed, which probably act as both growth and survival factors.3 New strategies in its chemotherapy involve metallotherapeutics, which interact with ERs. Human breast adenocarcinoma cells positive to ERs (MCF-7) and negative to ERs (MDA MB 231) are cell lines used as models for breast cancer.3 Human clinical trials, epidemiological evidence, and laboratory bioassays strongly support the protective role of nonsteroidal anti-inflammatory drugs (NSAIDs) in the incidence of mammary cancer, tumor burden, and tumor volume.4 Especially, the NSAIDs are beneficial to hormone steroid-dependent malignancies.2,3,5 Nimesulide has been proven to suppress key enzymes for the biosynthesis of estrogen in breast cancer cells,4 and it increases the apoptotic rate.6 Furthermore, nimesulide derivatives were more active © XXXX American Chemical Society

against breast cancer than the parent drug, and they induced apoptosis into cells through the mitochondrion.7 Mitochondrial targeted compounds are promising for the development of novel chemotherapeutic drugs. This is because mitochondria are the powerhouse of cancer cells, and therefore they are used as targets for the development of new compounds that lead cells to death. The permeabilization of the mitochondrial membrane unleashes apoptosis activators, which are located in mitochondria.8 Conjugation of mitochondriotropic ligands with specific metallotherapeutics may be used for the delivery of the drugs in to the mitochodria. For example, the mitochondrial membrane dilutes the triphenylphosphonium (tppH+) due to its high lipophilicity and its stable cationic charge.8 Lipoxyganase (LOX), an enzyme mainly distributed to mitochondrion, catalyzes the oxidation of arachidonic acid to leukotrienes or linoleic acid to prostaglandins. LOX inhibition induces apoptosis, since mitochondrial Received: May 21, 2016

A

DOI: 10.1021/acs.inorgchem.6b01241 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry dysfunction is associated with the reduced capacity for fatty acid oxidation.9 Therefore, LOX inhibition study is relevant to mitochondrial damage in cancer cells. Linoleic acid is also one of the main inner mitochondrial membrane components10 that is enzymatically oxidized upon releasing from membrane.11 Thus, the 13-S-hydroxyoctadecadienoic acid (13-S-HODE), a linoleic acid metabolite, affects adversely mitochondrial function, and it causes mitochondrial degradation.12 Moreover, silver ions cause accumulation of Ag2Se in mammals, and therefore silver(I) complexes are considered as TrxR (thioredoxin reductase) inhibitors.13,14 Silver N-heterocyclic carbene complexes oxidize the thioredoxin system, leading to the dimerization of peroxiredoxin 3. This demonstrates the ability of silver(I) carbene complexes to reach the mitochondrion.15 The complex [Na4(ImPrSO3)2]AgCl (NaHImPrSO3 = sodium 3,3′-(1H-imidazole-3-ium-1,3-diyl) dipropane-1-sulfonate) exhibits significant in vitro antiproliferative activity, which is correlated with its strong ability to inhibit thioredoxin reductase and through this to the activation of an apoptotic pathway.16 Moreover, the Ag(I) compounds of norharmane (9H-pyrido[3,4-b]indole; Hnor) with general formula [Ag(Hnor)2](anion) (anion = ClO4−, NO3−, and BF4−) were in vitro tested for their antiproliferative activity. Cell growth inhibitory effects were shown by these complexes against two different cancer cell lines, with the perhlorate to be the strongest one, having comparable activity to the cisplatin.17 Silver(I) compounds with the NSAIDs (aspirin, naproxen, or salicylic acid), however, exhibit significant antiproliferative activity against the breast adenocarcinoma cells (MCF-7 or MDA MB 231 cell lines), and they are, in vitro, less potent against noncancerous (normal) cells. Moreover, silver(I) compounds found to interact with nuclear DNA, LOX, and mitochondrion, causing cell apoptosis through the mitochondrial signaling pathway. The low toxicity against humans of silver ions and its compounds enable its use in the development of new metallotherapeutics.3,18−22 Recently, the carbene−silver acetate analogue (1-methyl-3-(p-cyanobenzyl)benzimidazole-2ylidene)silver(I)acetate (SBC1) was in vitro tested against human neuroblastoma cells, human colon carcinoma cells, and prostate cancer cells. SBC1 was proven to bind DNA strongly. SBC1 was also in vivo tested in mice showing a borderline toxicity.23 In the course of our studies for the development of new efficient metallodrugs for the breast cancer chemotherapy,2,3,18−30 we designed and synthesized five new metallodrugs of the NSAID nimesulide (nim) and the mitochondriotropic triaryl derivatives of pnictogens as ligands: triphenyphosphine (tpp), tri(p-tolyl)phosphine (tptp), tri(o-tolyl)phosphine (totp), triphenylarsine (tpAs), and triphenylantimony (tpSb) (Scheme 1). The aim of this work is to link together nimesulide (that suppresses key enzymes in breast cancer cells and increases apoptosis), silver(I), and a mitochondriotropic ligand (for mitochondria-specific drug delivery). The formulas of the compounds are {[Ag(nim) (tpp)2]DMF} (1), [Ag(nim) (tptp)2] (2), [Ag(nim) (totp)] (3), [Ag(nim) (tpAs)2] (4), and [Ag(nim) (tpSb)3] (5) (where DMF = dimethylformamide). Compounds 1−5 were characterized by their melting points, IR, 1H NMR, UV−vis spectra, and single-crystal X-ray diffraction (XRD) patterns. Berners-Price et.al31 have shown that the increasing of the lipophilicity led to the improvement of the activity of silver−phosphine complexes toward cancer cells. Thus, apart from their mitochondriotropic behavior, the use of nim and tpE (E = P (tpp, tptp or totp), As (tpAs), Sb

Scheme 1

(tpSb)) for the preparation of silver(I) metallodrugs aims in the increasing of the lipophylicity with simultaneous retention of the water solubility. The tpE residues of 1−5 are expected to retain the high solubility in organic media, while the position and the kind of the polar groups (O2N- and CH3SO2- groups) of nim might lead to an increasing of water solubility. Additionally, despite their toxicity, organophosphorus, organoarsenic, and organoantimonials are already used as drug constituents (e.g., in antiarthritic drug auronofine, etc). Although the crystal structure of the ligand nim is already known, its refinement was completed here once again, to conclude on the changes it undergoes upon its coordination to silver(I) ions and for the clarification of the exact polymorph type, among the three different ones isolated up to now, that is present in our experiments.32−34 The nim, tpE, and 1−5 were tested for their in vitro antiproliferative activity against breast adenocarcinoma cells, MCF-7 (ER positive), and MDA MB 231 (ER negative) cell lines, in vitro. The genotoxicity on normal human fetal lung fibroblast cells (MRC-5) caused by 1−5 was evaluated by fluorescence microscopy. The apoptotic pathway of 1−5 was tested by DNA fragmentation, cell cycle arrest, and permeabilization of the mitochondrial membrane. The molecular mechanism is elucidated by the mean of their interaction with the intracellular components: (i) calf thymus (CT) DNA, (ii) LOX, (iii) catalytic oxidation of the linoleic acid.

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RESULTS AND DISCUSSION General Aspects. Crystals of 1−5 were grown by slow evaporation of DMF solutions. Complexes 1−5 were formed from the reaction of triaryl derivatives of pnictogen (tpp, tptp, totp, tpAs, and tpSb) with the product between the AgNO3 and nimesulide, which was previously treated with equimolar amount of KOH (Chart 1). The formulas of the compounds were defined by spectroscopic methods, while their structures were determined by single-crystal XRD analysis. The crystals of 1−5 are airstable at room temperature. The silver compounds should be stored in darkness. Solid-State Studies. Crystal and Molecular Structure of {[Ag(nim) (tpp)2]dmf} (1), [Ag(nim) (tptp)2] (2), [Ag(nim) (totp)] (3), [Ag(nim) (tpAs)2] (4), and [Ag(nim) (tpSb)3] (5). Although the structure of nim is already known, (i) the modification in the crystallization processes of the drug followed here and (ii) the existence of at least three different polymorphs of the drug (Polymorph A: WINWUL: C2/c; a = 33.657(3), b = 5.1305(3), c = 16.0816(10) Å, β = 92.368(8)ο; R B


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Inorganic Chemistry Chart 1

= 4.01%, WINWUL02: C2/c; a = 33.231(2), b = 5.0720(4), c = 15.8736(12) Å, β = 92.712(2)ο; R = 3.4%, Polymorph B: ACICAQ: P21/c; a = 14.522(1), b = 10.439(1), c = 10.523(1) Å, β = 109.460(4)ο; R = 4.13%. Polymorph C: WINWUL01: Pca21; a = 16.1268(19), b = 5.0411(6), c = 32.761(4) Å; R = 4.01%)33,34 prompt us to the completion of new data set of the X-ray diffraction measurements and rerefinement. The crystallographic parameters of nim used for the synthesis of 1−5 were: space group: C2/c; a = 33.241(5), b = 5.068(5) c = 15.874(5), Å, β = 92.689(5)ο; R = 4.41%, which classify it within those of Polymorph A type. Molecular diagrams of nim and 1−5 along with their selected bond distances and angles are shown in Figures 1−6.

Figure 2. ORTEP diagram together with the numbering scheme of 1 compound. Selected bond lengths (Å) and angles [deg]: Ag1−P1 = 2.4203(13), Ag1−P2 = 2.4348(13), Ag1−N2 = 2.292(3), Ag1··· O3(ether) = 2.740, Ag1···O5(sulfonyl) = 2.888, P1−Ag1−P2 = 127.86(3), P1−Ag1−N2 = 120.20(7), P2−Ag1−N2 = 111.51(7).

Two pnictogen atoms (E = P or As) from tpp, tptp, and tpAs and one nitrogen from a deprotonated nimesulide are binding to the Ag(I) ion forming a trigonal planar geometry around silver(I) ion in the case of 1, 2, and 4, respectively. Thus, since the Ag, E, E, and N atoms are coplanar, the Ag···O(ether) (etheric oxygen of nim) distance (Ag1−O3(ether) = 2.740 Å (1), Ag1− O1(ether) = 2.806 Å (2), and Ag1−O5(ether) = 2.694 Å (4)) can not be considered as a bonding interaction, although it is shorter than the sum of the Ag−O van der Waals radii, which lie between 3.65 and 4.08 Å.35 Otherwise, the silver atom should be higher from the E, E, N plane. A solvent molecule (DMF) is cocrystallized in the crystal lattice of 1. One phosphorus atom from totp and one nitrogen from a deprotonated nimesulide form linear geometry around silver ion in 3. However, in this case the short Ag1−O5(ether) bond length (2.626(3) Å) should be taken into account turning the geometry to T shape. The existence of this Ag1−O5(ether) interaction is further supported by the deviation from the linearity of the P−Ag−N (P1−Ag1−N2 = 175.84(8)°). Three antimony atoms from tpSb and one nitrogen from the

Figure 1. ORTEP diagram together with the numbering of nim. Selected bond lengths (Å) and angles [deg]: S1−O1 = 1.428(2), S1− O2 = 1.430(2), S1−N1 = 1.644(3), N1−C2 = 1.407(4), N2−C5 = 1.457(4), O1−S1−O2 = 119.21(12), C7−O5−C8 = 118.31(19), S1− N1−C2 = 124.24(17), O3−N2−C5 = 118.1(2), O4−N2−C5 = 119.0(2). C


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Inorganic Chemistry

Figure 3. ORTEP diagram together with the numbering scheme of 2 compound. Selected bond lengths (Å) and angles [deg]: Ag1−P1 = 2.4109(14), Ag1−P2 = 2.4403(14), Ag1−N1 = 2.291(3), Ag1··· O1(ether) = 2.806, Ag1···O2(sulfonyl) = 2.953, P1−Ag1−P2 = 131.22(3), P1−Ag1−N1 = 118.34(6), P2−Ag1−N1 = 110.24(6).

Figure 5. ORTEP diagram together with the numbering scheme of 4 compound. Selected bond lengths (Å) and angles [deg]: Ag1−As1 = 2.5350(4), Ag1−As2 = 2.5051(5), Ag1−N1 = 2.268(3), Ag1−O5(ether) = 2.694, Ag1···O2(sulfonyl) = 2.974, As1−Ag1−As2 = 124.60(2), As1− Ag1−N1 = 111.29(8), As2−Ag1−N1 = 123.08(8).

deprotonated nimesulide are binding to the Ag(I) ion forming a tetrahedron around silver(I) in 5. The corresponding Ag1··· O3(ether) distance in 5 is 3.732 Å. In conclusion the ligand nim coordinates through N atom in monodentate manner in 1, 2, 4, and 5, while in case of 3 it chelates the silver(I) through N and O(ether). Steric Effect of Arylphosphines. The angle of the cone that encloses the phosphine ligand and the metal ion in the vertex position is known as cone angle.22,36 The cone angle is a measure of the size of a ligand, and it depends to the M−P

bond distance and consequently of the type of the metal ions. The coordinates of the atoms of 1−3 as theses were measured from their X-ray analysis were used in the cone angles determination.36 The values for 1−3 are 155.2° (1), 154.1° (2), and 195.9° (3; Figure S1). The cone angles found in 1−3 are greater than those reported for tpp (145°),37 tptp (146°),38 and totp (183°)39 (Table 1) due to the steric demands caused by nim.

Figure 4. ORTEP diagram together with the numbering scheme of 3 compound. Selected bond lengths (Å) and angles [deg]: Ag1−P1 = 2.3736(15), Ag1−N2 = 2.179(3), Ag1−O5(ether) = 2.626(3), Ag1···O1(sulfonyl) = 2.903, P1−Ag1−O5 = 115.79(6), P1−Ag1−N2 = 175.84(8), O5− Ag1−N2 = 67.98(10). D


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Inorganic Chemistry

vs(SO2), respectively, are observed at 1384, 963 (1), 1282, 967 (2), 1297, 962 (3), 1284, 963 (4), and 1272, 971 (5) cm−1 in the spectra of 1−5. The bands at 521−500 cm−1 in the spectra of free phosphines are attributed to the v(C−P) vibrations (Figures S8−S10). These bands are observed at 512 (1), 512 (2), and 521 (3) cm−1 in the spectra of 1−3. The bands at 471 and 454 cm−1 are attributed to the v(C−As) vibrations in 4 and to the v(C−Sb) vibration in 5, respectively (Figures S11 and S12). The presence of v(C−E) (E = P, As, Sb) vibration bands in the IR spectra of the compounds confirm the coordination of these ligands (Ar3E). Thus, IR spectra show the consistency between the powders and their structures, which were solved from XRD patterns. Solution Studies. The stability of compounds 1−5 in dimethyl sulfoxide (DMSO) solution was tested by UV−vis (Figures S13−S17) and 1H NMR (Figures S18−S22) spectra for a period of 48 h in DMSO-d6 solutions. The period of at least 48 h for the stability of the compounds was chosen, since the biological experiments require 48 h of incubation of the cell cultures with the compounds. No changes were observed between the initial UV and 1H NMR spectra of freshly prepared solutions and the corresponding spectra when measured after 24 and 48 h confirming the retention of the structures in solution. The negligible changes that are observed in the electronic absorption spectra of 1 and 4 (Figures S13 and S16) are due to the slight dissociation of the Ag−E (E = P (tpp) or As (tpAs)) bonds after a period of 48 h.31 Moreover, the assignments of the 1H NMR spectra of 1−5 were based on their crystal structure data, while the presence of the resonance signals for the H nucleus in the expected chemical shifts due to their orientation in their crystal structures support the retention of the structures of 1−5 in solution. Unless protected from ligand exchange reactions, the instability of Ag(I) complexes at physiological concentrations of Cl− ions is also known. Thus, the stability of 1−5 in the buffer solution containing 15 mM trisodium citrate and 150 mM NaCl at pH = 7, which is used for the binding studies toward CT DNA, was checked (Figure S23). No differences were observed between the initial spectrum of the complexes in buffer solution and the one after 10 min. This suggests the stability of the Ag(I) complexes 1−5 for the experimental time scale (0.5 min; Figures S23). 1 H Nuclear Magnetic Resonance Studies. The absence of H(11N) resonance signal in the 1H NMR spectra of 1−5

Figure 6. ORTEP diagram together with the numbering scheme of 5 compound. Selected bond lengths (Å) and angles [deg]: Ag1−Sb1 = 2.7309(12), Ag1−Sb2 = 2.7321(11), Ag1−Sb3 = 2.7313(11), Ag1− N1 = 2.317(6), Ag1···O2(sulfonyl) = 3.244, Sb1−Ag1−Sb2 = 111.31(2), Sb1−Ag1−Sb3 = 117.12(2), Sb2−Ag1−Sb3 = 105.73(2), Sb1−Ag1− N1 = 103.94(14), Sb2−Ag1−N1 = 113.73(14), Sb3−Ag1−N1 = 105.11(11).

Vibrational Spectroscopy. The ν(C−N) vibration band in the mid-IR spectra of 1−5 is observed at 963 (1), 966 (2), 966 (3), 963 (4), and 980 cm−1 (5; Figures S2−S6) and at 978 cm−1 in free nim (Figure S7). The strong shift that undergoes the ν(C−N) vibration band indicates the coordination of nim through N donor atom toward silver(I) cation.40 The vas(NO2) and vs(NO2) vibration bands at 1519 and 1318 cm−1, respectively, in the spectrum of the nim, are observed at 1487, 1285 (1), 1487, 1221 (2), 1503, 1206 (3), 1487, 1214 (4), and 1478, 1214 (5) cm−1, respectively (Figures S2−S6). The vibration bands at 1338 and at 976 cm−1 in the IR spectrum of nim, which are assigned to the vas(SO2) and

Table 1. Chemical Shifts (ppm) of the Resonance Signals Observed in 1H NMR Spectra of the Ligands and Complexes 1−5 in DMSO-d6 nimesulide

Ar3E

10.12 ppm (H(11N)), 8.01−8.0 ppm (H(8,8′C)), 7.50−7.42 ppm (H(7,7′C)) and 7.15−7.12 ppm (H(3C)), 3.16 ppm (H(10C) of the methyl group)

1 2

7.85−7.80 ppm (H(8,8′C)), 6.68−6.65 ppm (H(3C)), 2.57 ppm (H(10C)) 7.85−7.81 ppm (H(8,8′C) of nim), 6.70−6.67 ppm (H(3C)of nim), 2.61 (H(10C))

3

7.89−7.83 ppm (H(8,8′C)), 6.67−6.63 ppm (H(3C)), 2.78 ppm (H(10C))

4 5

7.91−7.86 ppm (H(8,8′C)), 6.87−6.84 ppm (H(3C)), 2.80 ppm (H(10C)) 7.87−7.82 (H(8,8′C)), 6.92−6.89 ppm (H(3C)), 2.73 ppm (H(10C)) E

tpp: 7.36−7.19 ppm (aromatic protons) tptp: 7.46−7.03 ppm (aromatic protons), 2.26−2.32 ppm (H(CH3-)) totp: 7.28−6.55 ppm (aromatic protons), 2.27 ppm (H(CH3-)) tpAs: 7.37−7.22 ppm (aromatic protons) tpSb: 7.39−7.31 ppm (aromatic protons) 7.19−6.98 ppm (aromatic protons) 7.15−6.99 ppm (aromatic protons), 2.28 ppm of H(CH3-) 7.20−7.02 ppm (aromatic protons), 2.27 ppm of H(CH3-) 7.40−7.15 ppm (aromatic protons) 7.40−7.34 ppm (aromatic protons) DOI: 10.1021/acs.inorgchem.6b01241 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Table 2. Metal Complexes with Non-Antiinflammatory Drugs against Two Adenocarcinoma Breast Cell Lines MCF-7 (Estrogen Positive) MDA-MB 231 (Estrogen Negative) and One Normal Human Fetal Lung Fibroblast Cell (MRC-5 Cells) IC50 (μM)a complexes [Ag(tpp)2(nim)] (1) [Ag(tptp)2(nim)] (2) [Ag(totp) (nim)] (3) [Ag(tpAs)2(nim)] (4) [Ag(tpSb)3(nim)] (5) nim tpp tptp totp tpAs tpSb {[Ag(tpp)3(asp)](dmf)} (6) Ag(tpSb)3(asp) (7) {[Ag(tpp)3(napr)](H2O)} (8) [Ag(tptp)2(napr)] (9) [Ag(tpp)2(salH)] (10) [Ag(tptp)2(salH)] (11) [Ag(tmtp)2(salH)] (12) Ag(tpSb)3(salH) (13) cisplatin

cone angle (deg)

MCF-7

2.3 ± 4.4 ± 3.3 ± 5.2 ± 4.7 ± >30 >30 >30 >30 >30 >30

148.2

1.0 ± 0.1 3.8 ± 0.2 2.7 ± 0.1 4.9 ± 0.3 4.6 ± 0.1 >30 28.9 ± 1.4 27.4 ± 1.7 23.9 ± 0.8 27.2 ± 1.2 >30 2.3 ± 0.3 5.9 ± 0.4 0.7 ± 0.1

14.9 ± 0.9

149.5

155.2 154.1 195.9

158.1 157.8 159.2 169.2

2.2 2.3 1.7 5.9 3.2 6.8

± ± ± ± ± ±

0.2 0.3 0.3 0.8 0.1 0.3

TPI MCF-7

TPI MDA-MB 231

Kb (1 × 104) M−1

LOX (IC50 μM)

2.9 ± 0.1 6.6 ± 0.1 3.1 ± 0.1 6.0 ± 0.1 4.0 ± 0.1 >30 >30 20.9 ± 0.9 25.2 ± 0.5 19.4 ± 0.7 >30 2.9 ± 0.1

2.9 1.7 1.1 1.2 0.9

1.3 1.5 0.9 1.2 0.9

20.0 ± 0.0 86.2 ± 27.0 12.5 ± 4.9 4.0 ± 0.5 8.9 ± 2.0

12.2 5.8 10.8 19.8 11.0 >30

b b b b b b

11.0 ± 2.8

7.2

19,20

4.8 ± 0.8

0.8

32.8 ± 8.5

5.1

3 21

4.7 ± 1.8 13.3 ± 6.5 7.2 ± 1.1 5.3 ± 0.8

>30 2.3 >30 >30

MDA-MB 231 0.1 0.1 0.1 0.1 0.4

7.3 ± 0.2 8.2

MRC-5

1.3

3.1 ± 0.3

1.3

5.0 ± 0.1 6.3

1.6 0.9

0.3

0.7 0.8

65.9

ref

21 19,20 22 22 3 19,26

The concentration which cause 50% of cell viability; Kb = binding constants toward CT DNA, LOX (IC50 μM) = inhibition οf lipoxygenase in IC50 values (μM). bThis work; TPI is defined as the IC50 value against normal cell lines divided by the IC50 value against cancer cell lines of the same or similar tissue are also determined), aspH = aspirin, nap = naproxen, salH2 = salicylic acid, tpSb = triphenyl antimony(III), tptp = tri(ptolyl)phosphine, tmtp = tri(m-tolyl)phosphine. a

(Figures S18−S22), which is observed at 10.12 ppm in the spectrum of nim (Figure S24), indicates the coordination of the drug to the silver ion through N donor atom. The strong shift observed in the signal of H(3C; Scheme 1) in 3 is due to the bonding interaction of the etheric oxygen of nim with the silver (see X-ray structures). The assignment of the spectra of 1−5 is summarized in Table 1, and they are compared with the corresponding ones of triarylpnictogens (Ar3E; E = P, Figures S25−S27; E = As or Sb, Figures S28 and S29) Biological Tests. Antiproliferative Activity. Metallodrugs 1−5 were tested for their in vitro antiproliferative activity against two human breast adenocarcinoma cell lines, MCF-7 (ER positive) and MDA-MB-231 (ER negative), by the means of sulforhodamine B (SRB) assay after 48 h of incubation. Sulforhodamine B (SRB) assay is used for drug-toxicity testing against cancerous or noncancerous cell lines. SRB assay relies on the ability of the dye to bind protein components of the cells that have been fixed to the culture plates. SRB advantages include high level of sensitivity and staining independent of cell metabolic activity.41 To ascertain the influence of the ERs in the mechanism of action of 1−5 the MCF-7 and MDA-MB-231 cell lines were chosen.2,3 The IC50 values are summarized in Table 2. The IC50 values of 1−5 lie between 1.0 and 4.6 μM (activity order: 1 > 3 > 2 > 5 > 4) against MCF-7 cells, while against the MDA-MB 231, between 2.3 and 5.2 μM with the same activity order. The activity of 1 against MCF-7 is 2.3-fold higher than the corresponding one against MDA-MB-231 cells. Generally, the breast cancer MCF-7 cells (positive to ER) are more sensitive to 1−3 in contrast to the MDA-MB-231 cells (negative to ER). Compounds 4 and 5 behave similarly against both cell lines. The ligands used (nimesulide, triarylphosphines,

triphenylarsine, and triphenylstibine), however, exhibit significantly lower activity than their complexes (Table 2). The selectivity of 1−3 against MCF-7 might be due to the involvement of ERs in their mechanism of action. This assumption is further supported by the sensitivity that is also exerted by MCF-7 than MDA-MB-231 cells for the triarylphosphine containing silver-NSAIDs drugs of aspirin and salicylic acid (Table 2). Compound 1 exhibits the strongest activity against MCF-7 cells (IC50 = 1.0 μM), which is the second-strongest activity among the NSAID-metal drugs studied in our group (Table 2). However, the {[Ag(tpp)3(napr)](H2O)} (nap = naproxen) is the most potent (IC50 = 0.7 μM). Complexes 1−3 of triarylphosphine exhibit stronger activity than cisplatin, which rises up to 7 times (1) against MCF-7 cells and 3 times (1) against MDA-MB-231 cells (Table 2). Among arsenic or antimony-containing compounds, both exhibit congener activities with cisplatin. The mixed ligands silver(I) complexes with NSAIDs and triarylphosphines of low cone angle values exhibit higher bioactivity (low IC50 values) against MCF-7 cells (Figure 7).22 This indicates that the steric demands of triarylphosphines affect the cell viability of MCF7 cells. The high value of the cone angle measured in 3 (196 deg) due to the almost linear orientation of the P−Ag−N (175.84(8) deg) in contrast to the tetrahedral or trigonal geometries observed in the rest of mixed ligand silver complexes of NSAIDs and triarylphosphines led to the outlier behavior of this compound. The toxicity of 1−5 is evaluated against normal human fetal lung fibroblast cells (MRC-5 cells). The IC50 values of 1−5 lie between 2.9 and 6.6 μM (higher toxicity order: 1 > 3 > 4 > 5 > F


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Inorganic Chemistry

cells (2.9 (1), 1.7 (2), 1.1 (3), 1.2 (4), and 0.9 (5)) classify 1−4 into potent antitumor agents against MCF-7 cells, while the corresponding TPI values against MDA-MB-231 cells (1.3 (1), 1.5 (2), 0.9 (3), 1.2 (4), and 0.9 (5)) classify 1−2 and 4 into potent antitumor agents against MDA-MB-231 cells. The higher TPI value against MCF-7 cells is observed for 1 (TPI order: 1 > 2 > 4 > 3 > 5) indicating its stronger antiproliferative than toxic activity. Also, 2 shows the highest TPI value against MDA-MB-231 cells (TPI order: 2 > 1 > 4 > 3 = 5). The lower TPI value against both cell lines found for the triarylstipinecontaining compound 5 is due to its high toxicity. For comparison the corresponding TPI values exhibited by cisplatin against MCF-7 and MDA-MB-231 cells are quoted as 0.9 and 0.8, respectively (Table 2). These are indicative of the antitumor than toxic behavior of the metallodrugs 1−5, especially of 1 and 2 against both cell lines studied. It is noteworthy to mention the significantly high TPI value (∼3) measured for 1 against MCF-7 cells. Although a high activity of AgNO3 water solutions against MCF-7 (3.3 ± 0.2 μM) was observed19 (Table 2), its use in medicine is prevented because of its high water solubility and its low lipophylicity. These lead to a consuming of large amount of silver ions by precipitation of silver(I) chloride complexes

Figure 7. pIC50 (= −log(IC50) values vs cone ange of triaryl phosphines.

2). The therapeutic potency index (TPI),3,26 which is defined as the IC50 value against normal cells toward the corresponding IC50 value against cancer cells of similar tissue, are also determined, lies between 0.9 and 2.9 against both cell lines. TPI values greater than 1 (IC50 against normal cells > IC50 against tumor cells) indicate selectivity of the agent against cancerous than normal cells. The higher TPI value the better the metallotherapeutic agent is. The TPI values against MCF-7

Figure 8. Representative pictures with micronucleus formed in MRC-5 cells treated with IC50 values of 1−2 (A) and 3−5 (B) for a period of 48 h; arrow indicates micronucleus in MRC-5 cells. G


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Inorganic Chemistry

Cell Morphology Study. To conclude on the type of the cell death, cell morphological observations were performed. These observations reveal important characteristics of apoptotic or necrotic cells. Morphological changes appear when light microscopy is used on the MCF-7 cells treated by 1−5 at their IC50 values for 48 h, which show apoptosis for the cell death. Both control and treated cells with DMSO showed normal morphologies (Figure 9). However, upon treatment

within few hours from their application. On the one hand, complexes of triarylphosphines 1−3 overcome this disadvantage due to their stability in water. The lower activity of 4−5 than 1−3, on the other hand, might be due to their lower water solubility. In conclusion the triarylphosphine-containing metallodrugs 1−3 with trigonal or T-shape geometries around the silver(I) ions exhibit (i) higher activity than the chemotherapeutic drug cisplatin, (ii) selectivity on tumor cells positive to ERs, and (iii) low toxicity against normal cells. Arsenic or antimonycontaining metallodrugs, however, show (i) lower antiproliferative activity, (ii) higher toxicity, and (iii) no selectivity. Evaluation of Genotoxicity by Micronucleus Assay. The presence of micronuclei (MN) is a biomarker of mutagenic, genotoxic, or teratogenic agent influence.42 In the presence of exogenous factors (such as chemical agents), the MN is formed during the metaphase−anaphase transition of the mitosis. The MN appears as small membrane-bound DNA fragments in the cytoplasm of interphase cells.43 The MN is unable to be incorporated to daughter cells.42 The micronucleus assay has been widely used in monitoring genetic damage in different tissue and cell types.44 To avoid the screening of compounds on animals for their toxicological effects, human cell lines in culture are used due to their high sensitivity.45 To ascertain the genotoxicity caused by 1−5 against MRC-5 normal cells (see above), the possible induction of micronucleus frequencies was checked when MRC-5 cells are treated by the complexes at the concentration of IC50. The micronucleus frequencies in the MRC-5 cell culture without treatment are 0.8% and 1.0% after treatment with DMSO. A meaningless MN (0.1%) was detected when 2 was used, while when 1, 3, and 5 were used it became: 1.0 (1), 1.8 (3), and 1.0% (5; Figure 8A and Table 3). These values are comparable Table 3. Percentage of Micronucleus of 1−5 after the Treatment of MRC-5 with 1−5 at the Concentrations of IC50 Values percentage of micronucleus (%) control DMSO 1 2 3 4 5 cisplatin

0.8 1.0 1.0 0.1 1.8 2.8 1.0 1.6

Figure 9. Changes in the morphology of MCF-7 cells incubated with 1−5, cisplatin, and DMSO for 48 h as examined by phase contrast microscopy.

or less than that caused by cisplatin (1.6% at the concentration of 26 μM; Figure 8B and Table 3) indicating no mutagenic, genotoxic, or teratogenic activity of 1, 3, and 5. However, in case of 4, 2.8% of MN were observed in MRC-5 cells. Mechanism of Action. The mechanism of action of 1−5 against MCF-7 was clarified by the means of (a) cell morphology studies, (b) DNA fragmentation, (c) cell cycle arrest, and (d) permeabilization of the mitochondrial membrane test. The activity of metallodrugs was studied only against MCF-7, since this cell line is generally more sensitive to 1−5. The molecular mechanism of action of 1−5 was further studied by their (i) binding affinity toward the CT DNA, (ii) inhibitory activity against LOX, and their (iii) catalytic activity on the oxidation of linoleic acid to hyperoxolinoleic acid by oxygen.

with 1−5, the MCF-7 cells were shrunk and rounded, the cells contact was lost, and they were detached from the plate in contrast to the untreated cells. These findings clearly indicated that 1−5 reduce the cell viability of MCF-7 cells by inducing apoptosis.20,21,46 The MCF-7 cells culture under incubation with cisplatin, a known apoptotic agent, is also shown in Figure 9. The similarities in morphologies of cell cultures incubated with 1−5 with those observed when cisplatin is used suggest apoptotic cell death of MCF-7. DNA Fragmentation Study. To verify the apoptotic type of MCF-7 cell death when they are treated with 1−5, DNA fragmentation study was performed. MCF-7 cells were H


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Inorganic Chemistry

and the resveratrol induce S-phase arrest in different types of human cancer cells.51 The percentage of cells in the G2/M phase when they are treated with 2 is increased to 35.6%, while the nontreated cells (control) show 21.6% cells in the G2/M phase (Figure 11A). Therefore, compound 2 accumulates cells in the G2/M phase of the cell cycle by delaying or inhibiting cell cycle progression at the G2/M phase.52 The cell cycle arrest of cisplatin was also studied against MCF-7 cells at the concentration of 6.8 μM (IC50). The control cells are spread in 4.2% sub-G1 phase, 54.3% in G0/G1, 23.1% in S, and 17.8% in G2/M phase. Incubated MCF-7 cells with cisplatin show an increase in the number of apoptotic cells through sub-G1 phase (9.1%, toward 4.2% of the non -treated cells). Cell cycle arrest in S phase is 36.4% of the cells toward 23.1% of nontreated and in the G2/M phase is 22.1% toward 17.8% of the untreated cells (Figure 11B). This is expected, since cisplatin is known to cause cell arrest at S and G2/M phases depending on both p53 status and drug concentration.53 In conclusion 1−5 are causing cell cycle arrest either in at G2/ M (2) or S (1, 3−5) phases similarly to cisplatin. Detection of the Loss of the Mitochondrial Membrane Permeabilization. The mitochondrion participates in the apoptosis regulation.54 The opening of the mitochondrial permeability leads to a characteristic type of cell death, while it is often considered as the “point of no return” toward the apoptosis.55 One of the decisive events of the apoptotic process is the opening of the mitochondrial permeability transition pore, causing an increase in the permeability of the outer mitochondrial membrane and the release of soluble proteins such as cytochrome c.54 The release of cytochrome c from mitochondria is considered to be a primary activator of the caspase cascade.56 It has been known that the induction of mitochondrial membrane permeabilization (MMP) in tumor cells constitutes the goal of anticancer chemotherapy.55 The assay is optimized for the detection of the loss of the MMP in cells. To evaluate the mitochondrial modifications in MCF-7 cells, the cells were exposed in 1−5 at their IC50 values. The cationic hydrophobic mitochondrial potential dye accumulates in normal mitochondria, while in the treated cells, MMP collapse results in decreased fluorescence, indicating apoptosis and the release of cytochrome c in the cytosol. The percent of decrease of fluorescence observed is 15.7% (1), 35.6% (2), 25.0% (3), 28.8% (4), and 37.3% (5). Moreover, the fluorescence of MCF7 cells treated by 100 μM of cisplatin is decreased by 55.6%.57 These values show (i) the relative loss of MMP of MCF-7 cells upon treatment with 1−5 and (ii) the release of cytochrome c, which consequently results in the activation of cell apoptosis. The higher activity is caused by metallodrugs 5 and 2 (activity order: 5 > 2 > 4 > 3 > 1). Therefore, the apoptotic way of cell death when they are treated with metallodrugs 1−5 is also concluded by the loss of the MMP studies in accordance with (a) the cell morphology, (b) DNA fragmentation, and (c) cell cycle arrest experiments. Study of the Peroxidation of Linoleic Acid by the Enzyme Lipoxygenase in the Presence of 1−5. LOX is mainly distributed to mitochondrion, and its inhibition induces apoptosis, since it is associated with the mitochondrial dysfunction.9 The influence of metallodrugs on the catalytic oxidation of linoleic acid by the enzyme LOX was studied in a concentration range from 2 to 30 μM. The degree of LOX activity (A, %) in the presence of 1−5 was calculated according to the method described previously (Table 2).3,19,58 Metal-

incubated with 1−5 or cisplatin at their IC50 values (Figure 10). The 1−5 and cisplatin caused DNA laddering, which is a typical

Figure 10. DNA electrophoresis of MCF-7 cells incubated with 1−5 and cisplatin for 48 h. (A) control; (B) incubated with 1; (C) incubated with 2; (D) incubated with 3; (E) incubated with 4; (F) incubated with 5, and (G) incubated with cisplatin.

pattern of internucleosomal fragmentation. The similarity in the behavior of 1−5 with cisplatin toward MCF-7 cells clearly suggests the apoptotic activity of the complexes, in contrast to the untreated cells, where no fragmentation is observed. Cell Cycle. One of the hallmarks for apoptosis is the internucleosomal DNA fragmentation. Thus, DNA fragments of apoptotic cells can be identified by their sub-G1 peak on DNA frequency histograms.47 The detection of the sub-G1 peak in DNA flow cytometric profiles indicates the presence of apoptotic cells.48 To determine the possible effect of 1−5 on the progression of cell cycle, a flow cytometric analysis was performed to quantify the percentage of MCF-7 cells that underwent apoptosis giving a sub-G1 peak.49 For this purpose, exponentially growing MCF-7 cells were treated with 1−5 and cisplatin in their IC50 values for 48 h, stained with propidium iodide, and the amount of DNA was analyzed by flow cytometry. Figure 11 illustrates the pronounced effects on the cell cycle caused from the compounds 1−5 or cisplatin as the number of cells versus DNA content in sub-G1, G0/G1, S, and G2/M phases. The FACs data of cell cycle studies are given in the Supporting Information (Figures S30−S32). The control cells (not treated cells) are spread in 3.1% sub-G1 phase, 52.9% in G0/G1, 21.1% in S, and 21.6% in G2/M phase. The five stages of the cell cycle include (i) the G1 phase, a period during which the synthesis of enzymes needed for DNA replication is occurred; (ii) the S phase, a period of DNA replication; (iii) the G2 phase, where the cell continues to grow and produce new proteins; (iv) the M phase, where the cell divides into two daughter cells; and (v) the quiescent G0 phase, the last stage of the cycle, where the cell remains. After incubation of the MCF7 cells with 1−5, a significant increase in the number of apoptotic cells in sub-G1 phase (4.6% (1), 5.6% (2), 5.4% (3), 8.8% (4), and 7.1% (5)) was observed toward the control group (3.1%). The percentage of cells in S phase, however, was increased to 25.2% (1), 27.1% (3), 32.9% (4), and 33.7% (5) only when they were treated with 1 and 3−5. The percentage of the nontreated cells in S phase is 21.1%. Thus, compounds 1 and 3−5 suppress cell proliferation by inhibiting DNA synthesis and inducing S-phase cell cycle arrest.50 Many anticancer agents such as the mitomycin C, the hydroxyurea, I


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Inorganic Chemistry

Figure 11. Effects of 1−5 (A) and cisplatin (B) on cell cycle against MCF-7 cells. The relative number of cells within each cell cycle was determined by flow cytometry. Number of cells in sub-G1, G0/G1, S, and G2/M phase are indicated.

The Influence of 1−5 upon the Peroxidation of Linoleic Acid. The linoleic acid is oxidized enzymatically upon release from inner mitochondrial membrane.10,11 To measure the influence of 1−5 against mitochondrion, their interaction with linoleic acid is studied. Peroxidation of the linoleic acid leads into deconstruction of the mitochondrial membrane and through this to its dysfunction. This peroxidation of linoleic acid goes through free radicals mechanism.58 Compounds that catalyze the peroxidation of linoleic acid (the substrate of the enzyme LOX) to hyperoxo linoleic acid also inhibit the enzyme LOX with the same manner.58 This is because the LOX inhibition proceeds through free radicals mechanism, similarly

lodrug 2 shows the highest inhibitory activity against LOX (IC50 = 5.8 μM). Complexes 1−5 show higher LOX inhibitory activity than cisplatin (IC50 = 65.9 μM19,58) or nimesulide (>30 μM). Thus, the presence of the mitochondriotropic ligands such as phosphines (1−3) enhances their LOX inhibitory activity. We have shown that inhibition of LOX, caused by metallodrugs, is connected with their antiproliferative activity.21 This is also followed by 1−3, since they strongly inhibit LOX, bind CT DNA, and exhibit antiproliferative activity. Thus, the higher antiproliferative activity observed for 2 may also be attributed to its higher LOX inhibitory activity (Table 2). J


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Inorganic Chemistry

an aromatic chromophore and the base pairs of DNA suggesting the intercalative mode of binding.26 The hyperchromic effect of 22% (4) and 55% (5), respectively, on the other hand, suggests binding of the drugs to CT DNA either by external contact or by uncoiling of the helix structure of DNA caused by the complexes.20 The binding constants (Kb) of 1−5 toward CT DNA were evaluated by monitoring the changes in absorbance of the UV spectra of metallodrugs ([1, 3, 5] = 25 μM. [2] = 50 μM, [4] = 20 μM) at 450−440 nm, in the case of 1 and 400−410 nm (2− 5), with increasing concentration of CT DNA (Figure S34). Kb is obtained from the ratio of the slope to the y-intercept in plots [DNA]/(εa − εf) versus [DNA].20 Compound 2 exhibits the highest DNA binding constant (Kb = (86.2 ± 27.0) × 104 M−1); binding constant order: 2 > 1 > 3 > 5 > 4; Table 2. The high binding constants values (Kb) of NSAIDs-metallodrugs toward CT DNA are accompanied by low IC50 values (high activity) against breast cancer cells (Table 2). High cytotoxic activity of copper(II) and zinc(II) metallodrugs with high binding constancies toward DNA has already been reported.59 b. Fluorescence Spectroscopic Studies. The DNA−complex binding properties can also be studied by fluorescent spectroscopy, since a hypochromism is recorded in the UV spectra of the DNA−complex solutions of variable proportions of 1−3. Ethidium bromide (EtBr) emits intense fluorescent light in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs. Since EtBr intercalates DNA in the minor groove, its displacement by a compound is suggestive of an intercalative or minor groove binding.26,60 Thus, the intensity of fluorescence emission of the DNA solutions that were pretreated with EtBr and that contain complexes were recorded. The fluorescence emission at 605 nm of the solutions of CT DNA (26 μM), which contain EtBr (2.3 μM) in the presence or absence of 1−3 (0−250 μM), were measured after excitation with radiation at 527 nm (Figure S35). Application of the equations reported elsewhere61 results in the apparent binding constants (Kapp). The fluorescence quenching spectra show that the emission band at 605 nm undergoes a hypochromism by 16% (1), 23% (2), and 22% (3) upon increasing of the concentration of 1−3 as compared with the initial fluorescence intensity of the solutions without the complexes 1−3. The displacement of the EtBr molecules from DNA binding sites from 1−3 is clearly indicated by the decrease in the fluorescence intensity. The Kapp values calculated for the complexes 1−3 are 2.2 × 104 (1), 3.2× 104 (2), and 3.2× 104 M−1 (3), supporting intercalative or minor groove binding of 1−3 toward DNA. In conclusion the interaction of complexes 1−3 with CT DNA, which has been verified earlier with UV−vis absorption studies (see above), is further supported by fluorescence spectroscopy of the EtBr− DNA emission spectra in the absence and presence of the metallodrugs 1−3. Therefore, 2 exhibits the higher binding affinity for CT DNA, the higher LOX inhibitory activity, and the higher catalytic activity in linoleic acid oxidation among metallodrugs 1−5 tested.

with the promotion of the peroxidation of linoleic acid. Thus, the free radicals that promote the oxidation of linoleic acid inhibit the LOX activity. The influence of 1−5 upon the peroxidation of linoleic acid solution (0.3 mM) is evaluated by recording the absorbance of hyperoxolineleic acid formed at 234 nm (ε = 25 000 (M−1 cm−1)28). Only 2 catalyzes the peroxidation of the linoleic acid (Figure 12) at the

Figure 12. Accumulation level of hyperoxolynolieic acid (λmax = 234 nm) at 25 °C in the absence of 2 or ciplatin and in the presence at the concentrations of 0.6 μM (ratio 1/500) and 1.4 μM (ratio 1/250).

concentrations of 0.4 and 1.6 μM of 2 at 25 °C. The initial slope (Vo) of the oxidation reaction of free linoleic acid at 25 °C is 0.66 × 10−6 s−1. The corresponding Vo values of this reaction in the presence of various concentrations of 2 (0.6 and 1.4 μM) are 303 × 10−6 s−1 and 51.7 × 10−6 s−1, respectively. Cisplatin also catalyzes the peroxidation of linoleic acid. The corresponding Vo values of this reaction in the presence of 0.6 and 1.4 μM of cisplatin are 0.8 × 10−6 s−1 and 103 × 10−6 s−1, respectively (Figure 12). Therefore, both 2 and cisplatin act similarly. The catalytic peroxidation of linoleic acid that has been caused by 2 maybe causes a damage of the cell or mitochondrion membrane, since it removes an essential component such as linoleic acid from it. This is also supported by the strong loss of the mitochondrial membrane permeabilization and the high LOX inhibitory activity caused by 2. DNA Binding Studies. a. Ultraviolet−Visible Absorption Spectroscopic Studies. To rationalize the mechanism of action of the metallodrugs 1−5 against MCF-7 cells, their interaction with CT DNA was examined by UV−visible absorption spectroscopy. There are three binding modes with DNA: (i) electrostatic interaction with the negatively charged nucleic sugar−phosphates, which are along the external DNA double helix and do not possess selectivity, (ii) binding interaction with the grooves of DNA double helix, and (iii) intercalation between the stacked base pairs of native DNA.3 The configuration of the double-helix structure of DNA due to interaction with metallodrugs can be assigned either by hypochromism or by hyperchromism.20 Thus, hypochromism is attributed to intercalated or electrostatic binding mode, while hyperchromism is assigned to the breakage of hydrogen bonds, which stabilized the secondary structure of DNA.20 A slight decrease in the absorption intensity (at λmax = 258 nm) is observed upon the presence and absence of 1−3 at various r values (r = [complex]/[DNA], [DNA] = 5 × 10−5 M for 1 and 3 and 1 × 10−4 M for 2) suggesting intercalation or electrostatic binding between DNA and 1−3 (Figure S33). The percent of hypochromicity is 21 (1), 4 (2), and 8 (3) % (Figure S33). In the case of 2, the hypochromicity is accompanied by a blue shifting of 2 nm, indicating strong stacking interaction between

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CONCLUSIONS Nowadays, mitochondria-targeted compounds are promising entities for chemotherapy.8 Nimesulide, a known NSAID, and its derivatives exhibit significant activity against breast cancer cells by inducing apoptosis through mitochondrion.6 This prompted us to design, synthesize, and fully characterize five new nimesulide metallodrugs of silver(I) that contain the K


Inorganic Chemistry

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auxiliary mitochondriotropic triaryl derivatives of pnictogen ligands of the general formula [Ag(nim) (Ar3E)n] (n = 1 (3); n = 2 (1, 2, 4); n = 3 (5); E = P, As, Sb). The rationale of this work is to link together nimesulide (which suppresses key enzymes in breast cancer cells and increases apoptosis), silver(I), and a mitochondriotropic ligand (for mitochondriaspecific drug delivery). The geometry around silver(I) ion is either trigonal planar (1, 2, 4), tetrahedral (5), or T-shaped (3). Their mechanism against breast cancer cell lines was studied. Metallodrugs 1−5 show higher in vitro antiproliferative activity against human breast adenocarcinoma cell lines MCF-7 and MDA-MB-231 than cisplatin (a chemotherapeutic drug in clinical use; Table 2). The triarylphosphine-containing metallodrugs 1−3 of tpp, tptp, and totp exhibit selectivity on tumor cells positive to ERs and low toxicity against normal cells. On the contrary arsenic- or antimony-containing metallodrugs show lower antiproliferative activity and no selectivity. This sensitivity of MCF-7 to 1−3 might be due to the involvement of ERs in their mechanism of action, as a subsequence of the suppressing of the key enzymes involved in the biosynthesis of estrogen by nimesulide.4 The TPI values against MCF-7 or MDA-MB 231 cells are higher than unity in case of 1 and 2 indicating stronger antiproliferative than toxic activity. Compounds 1−5 were evaluated for their genotoxicity against MRC-5 cells. Only 2 causes no genetic damage to normal cells, indicating no mutagenic, genotoxic, or teratogenic effect (Figure 8 and Table 3). The apoptotic type of cell death of MCF-7 cells after treatment with 1−5 was concluded by their morphology (Figure 9), DNA fragmentation (Figure 10), and cell cycle arrest (Figure 11). The metallodrugs 1 and 3−5 suppress cell proliferation by inhibiting DNA synthesis and inducing S-phase cell cycle arrest, while 2 accumulates cells in the G2/M phase of the cell cycle by delaying or inhibiting cell cycle progression at the G2/M phase. A similar mechanism is also proposed for cisplatin.53 To rationalize the apoptotic cell death the MMP was investigated. The mitochondrion takes part in one of the regulation pathways of apoptosis.54 Since nim is known to induce apoptosis through mitochondrion, this pathway was studied for 1−5. Apart from this, a synergistic outcome is expected due to the presence of the mitochondriotropic pnictogen ligands in 1−5. Metallodrugs 1−5 cause the loss of MMP and the release of cytochrome c, which consequently results in the activation of apoptosis. Metallodrug 2 exhibits the strongest LOX (a mitochondrial enzyme) inhibitory activity among 1−5, which is greater than the corresponding one of cisplatin. Only 2 catalyzes the peroxidation of the linoleic acid (a component of mitochondrial membrane) causing serious damage of the cell or mitochondrion membrane, by removing an essential component such as linoleic acid from it. This is further supported by the loss of the mitochondrial membrane permeabilization and the high LOX inhibitory activity. Metallodrug 2 also exhibits the highest DNA binding affinity among 1−5. In conclusion, the lipophilic, mitochondrion-targeted compound 2, with the O2N- and CH3SO2- polar groups, exhibits selectivity for breast cancer cells positive to ERs causing their death by activation of apoptosis through mitochondrial dysfunction. Thus, 2 is proved to be a promising tool for the development of chemotherapy.

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EXPERIMENTAL SECTION

Materials and Instruments. All solvents used were of reagent grade, while triphenylphosphine, tri(p-tolyl)phosphine, tri(o-tolyl)phosphine, triphenyl arsine, and triphenyl antimony (Sigma-Aldrich, Merck) were used without further purification. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, glutamine, and trypsin were purchased from Gibco, Glasgow, U.K. Phosphate buffer saline (PBS), CT DNA, ethidium bromide, propidium iodide, Triton X-100, agarose, ethylenediaminetetraacetic acid (EDTA), RNase A, and Proteinase K were purchased from Sigma-Aldrich. Dimethyl sulfoxide and boric acid were from Riedel-de Haen. Melting points were measured in open tubes with a Stuart Scientific apparatus and are uncorrected. IR spectra in the region of 4000−370 cm−1 were obtained from KBr discs, with a PerkinElmer Spectrum GX Fourier transform IR spectrophotometer. The 1H NMR spectra were recorded on a Bruker AC 250 MHz FT-NMR instrument in DMSO-d6 solution. A UV-1600 PC series spectro-photometer of VWR was used to obtain electronic absorption spectra. FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA) was obtained for the cell cycle. Synthesis and Crystallization of 1−5. A suspension of 0.5 mmol nimesulide (0.185 g) in water (8 cm3) was treated with 0.5 cm3 KOH 1 N, and the resulting clear solution was added dropwise to a stirred solution of 0.5 mmol silver nitrate (0.085 g) in water (5 cm3) at room temperature. A yellow powder precipitated immediately, which was filtered off and dried under vacuum over calcium chloride. The resulting powder was dissolved in DMF (10 mL), and 0.262 g (1 mmol) of triphenylphosphine (1), 0.304 g (1 mmol) of tri(ptolyl)phosphine (2), 0.152 g (0.5 mmol) of tri(o-tolyl)phosphine (3), 0.306 g (1 mmol) of triphenylarsine (4), or 0.529 g (1.5 mmol) of triphenyl antimony (5) was added. The mixture was stirred for 30 min, and the yellow solution was kept in darkness. Yellowish crystals of 1−5 suitable for X-ray analysis were grown from slow evaporation of the solution after 2 d. 1. Yellow crystal. Yield: 0.055 g; mp 193−198 °C. Elemental analysis found: C: 61.50; H: 4.92, N: 4.23; S: 3.55%; calculated for C52H47AgN3O6P2S: C: 61.73; H: 4.68; N: 4.15; S: 3.17%. IR (cm−1), (KBr): 1680 s, 1581 m, 1487 vs, 1434 vs, 1384 m, 1285 vs, 1111 m, 1081 s, 963 s, 512 s; 1H NMR (ppm) in DMSO-d6: 2.57 (s, H(10C) of nimesulide), 7.87−7.82 (q, H(8,8′C) of nimesulide), 6.61−6.58 (d, H(3C) of nimesulide), 7.47−6.94 (m, aromatic)); UV−vis (DMSO): λ = 260 nm (log ε = 4.52), λ = 435 nm (log ε = 4.20) 2. Yellow crystal. Yield: 0.067 g; mp 179−183 °C. Elemental analysis found: C: 64.33; H: 5.61, N: 2.78; S: 3.34%; calculated for C55H53AgN2O5P2S: C: 64.52; H: 5.21; N: 2.74; S: 3.13%. IR (cm−1), (KBr):1580 s, 1487 vs, 1282 vs, 1221 s, 1187 m, 1080 vs, 967 s, 630 m, 512 vs; 1H NMR (ppm) in DMSO-d6: 2.61 (s, H(10C) of nimesulide), 7.85−7.81 (q, H(8,8′C) of nimesulide), 6.66−6.32 (d, H(3C) of nimesulide), 7.45−6.96 (m, aromatic), 2.28 (s, H(CH3) of tptp); UV− vis (DMSO): λ = 260 nm (log ε = 4.58), λ = 435 nm (log ε = 4.38). 3. Yellow crystal. Yield: 0.070 g; mp 218−225 °C. Elemental analysis found: C: 56.30; H: 3.81, N: 3.78; S: 4.50%; calculated for C34H32AgN2O5PS: C: 56.76; H: 4.48; N: 3.89; S: 4.46%. IR (cm−1), (KBr): 1583 vs, 1503 vs, 1381 m, 1297 m, 1206 s, 962 vs, 816 m, 754 s, 690 s, 521 s; 1H NMR (ppm) in DMSO-d6: 2.78 (s, H(10C) of nimesulide), 7.89−7.83 (q, H(8, 8′C) of nimesulide), 6.67−6.63 (d, H(3C) of nimesulide), 7.54−7.00 (m, aromatic), 2.89 (s, H(CH3) of totp); UV−vis (DMSO): λ = 260 nm (log ε = 4.32), λ = 435 nm (log ε = 4.37). 4. Yellow crystal. Yield: 0.169 g; mp 165−167 °C. Elemental analysis found: C: 57.30; H: 4.11, N: 2.88; S: 3.55%; calculated for C49H41AgAs2N2O5S: C: 57.27; H: 4.02; N: 2.73; S: 3.12%. IR (cm−1), (KBr): 1580 s, 1487 vs, 1435 vs, 1284 vs, 1214 s, 1080 s, 963 s, 805 m, 471 s; 1H NMR (ppm) in DMSO-d6: 2.80 (s, H(10C) of nimesulide), 7.91−7.86 (q, H(8,8′C) of nimesulide), 6.87−6.84 (d, H(3C) of nimesulide), 7.54−6.99 (m, aromatic); UV−vis (DMSO): λ = 260 nm (log ε = 4.34), λ = 435 nm (log ε = 4.35). 5. Yellow crystal. Yield: 0.159 g; mp 127−131 °C. Elemental analysis found: C: 54.30; H: 3.61, N: 1.98; S: 2.21%; calculated for C67H56AgN2O5SSb3: C: 54.58; H: 3.83; N: 1.90; S: 2.17%. IR (cm−1), L


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Inorganic Chemistry (KBr): 1577 s, 1478 vs, 1431 vs, 1333 s, 1272 s, 1215 m, 971 m, 454 s, 527 m; 1H NMR (ppm) in DMSO-d6: 2.73 (s, H(10C) of nimesulide), 7.87−7.82 (q, H(8,8′C) of nimesulide), 6.92−6.89 (d, H(3C) of nimesulide), 7.46−6.98 (m, aromatic); UV−vis (DMSO): λ = 260 nm (log ε = 4.49), λ = 435 nm (log ε = 4.25). X-ray Structure Determination. Intensity data for the crystals of 1−5 and nimesulide were collected on an Oxford Diffraction CCD instrument, using graphite monochromated Mo radiation (λ = 0.710 73 Å). Cell parameters were determined by least-squares refinement of the diffraction data from 25 reflections. All data were corrected for Lorentz-polarization effects and absorption.62 The structures were solved with direct methods with SHELXS9763 and refined by full-matrix least-squares procedures on F 2 with SHELXL97.64 All non-hydrogen atoms were refined anisotropically; hydrogen atoms were located at calculated positions and refined via the “riding model” with isotropic thermal parameters fixed at 1.2 (1.3 for CH3 groups) times the Ueq value of the appropriate carrier atom. nim. C13H12N2O5S, MW = 308.32, monoclinic, space group C2/c, a = 33.241(5), b = 5.068(5), c = 15.874(5) Å, α = 90, β = 92.689(5), γ = 90°, V = 2671(3) Å3, Z = 8, T = 100 K, ρ (calc) = 1.533 g cm−3, μ = 0.3 mm−1, F(000) = 1280. 4397 reflections measured, 2334 unique (Rint = 0.043). The final R1 = 0.0441 (for 1980 reflections with I > 2s(I)) and wR(F2) = 0.1297 (all data) S = 0.91. 1. C49H41AgN2O5P2S, C3H6NO, MW = 1011.81, triclinic, space group P1̅, a = 12.627(5), b = 13.245(5), c = 14.592(5) Å, α = 97.969(5), β = 97.130(5), γ = 92.879(5)°, V = 2392.5(15) Å3, Z = 2, T = 100 K, ρ (calc) = 1.405 g cm−3, μ = 0.6 mm−1, F(000) = 1042. 16 013 reflections measured, 8410 unique (Rint = 0.032). The final R1 = 0.0406 (for 7195 reflections with I > 2s(I)) and wR(F2) = 0.1069 (all data) S = 1.05. 2. C55H53AgN2O5P2S, MW = 1023.87, triclinic, space group P1,̅ a = 10.626(5), b = 10.821(5), c = 22.212(5) Å, α = 101.258(5), β = 91.047(5), γ = 99.428(5)°, V = 2467.7(17) Å3, Z = 2, T = 100 K, ρ (calc) = 1.378 g cm−3, μ = 0.6 mm−1, F(000) = 1060. 16 767 reflections measured, 8737 unique (Rint = 0.037). The final R1 = 0.0397 (for 7404 reflections with I > 2s(I)) and wR(F2) = 0.0998 (all data) S = 1.03. 3. C34H32AgN2O5PS, MW = 719.53, triclinic, space group P1̅, a = 9.460(5), b = 11.154(5), c = 16.521(5) Å, α = 94.375(5), β = 102.621(5), γ = 111.303(5)°, V = 1561.5(12) Å3, Z = 2, T = 100 K, ρ (calc) = 1.530 g cm−3, μ = 0.8 mm−1, F(000) = 736. 10 593 reflections measured, 5491 unique (Rint = 0.037). The final R1 = 0.0366 (for 4785 reflections with I > 2s(I)) and wR(F2) = 0.0890 (all data) S = 1.05. 4. C49H41AgAs2N2O5S, MW = 1027.62, monoclinic, space group P21/n, a = 15.4416(4), b = 14.2826(4), c = 19.7348(6) Å, α = 90, β = 96.343(3), γ = 90°, V = 4325.8(2) Å3, Z = 4, T = 100 K, ρ (calc) = 1.578 g cm−3, μ = 6.3 mm−1, F(000) = 2072. 27 159 reflections measured, 7671 unique (Rint = 0.052). The final R1 = 0.0381 (for 6549 reflections with I > 2s(I)) and wR(F2) = 0.1297 (all data) S = 1.06. 5. C67H56AgN2O5SSb3, MW = 1474.36, triclinic, space group P1̅, a = 14.284(5), b = 14.492(5), c = 14.643(5) Å, α = 86.056(5), β = 78.750(5), γ = 78.750(5)°, V = 2926.3(18) Å3, Z = 2, T = 100 K, ρ (calc) = 1.673 g cm−3, μ = 1.8 mm−1, F(000) = 1456. 22 829 reflections measured, 10 285 unique (Rint = 0.057). The final R1 = 0.0462 (for 7804 reflections with I > 2s(I)) and wR(F2) = 0.1222 (all data) S = 1.03. Additional crystallographic information is available in the Supporting Information. Steric Effect of Arylphosphines. The cone angle values for 1−5 were determined using the software algorithm denoted in ref 36. Biological Tests. Solvents Used. The biological experiments including assessment of the cell viability with SRB assay, cell morphology, DNA fragmentation, cell cycle, micronucleus, and the permebalization of mitochondrial membrane were performed in DMSO/DMEM solutions 0.01−0.3% v/v DMSO in DMEM for the complexes 1−5. Stock solutions of complexes 1−5 (0.01 M) in DMSO were freshly prepared and diluted with cell culture medium to the desired concentration. For DNA binding studies, the experiments

performed in DMSO/buffer solutions (0.000 25−0.025% v/v DMSO). For LOX inhibitor activation and the peroxidation of linoleic acid, the experiments were performed in DMSO/buffer solutions (0.0002− 0.004% v/v DMSO) for the complexes 1−5. Doubly distilled (dd) water was used to dilute cisplatin. This water solution of cisplatin in dd water (0.005 M) was used in all biological experiments. Sulforhodamine B Assay. Stock solutions of the complexes 1−5 (0.01M) in DMSO were freshly prepared and diluted with cell culture medium to the desired concentration (2−10 μM). For the experiments, cells were plated (100 μL per well) in 96-well flatbottom microplates at various cell inoculation densities (MCF-7, MDA-MB-231, and MRC-5:6000, 6000, and 2000 cells/well, respectively). Cells were left for 24 h at 37 °C to resume exponential growth and stabilization and afterward exposed to tested agents for 48 h by the addition of an equal volume (100 μL) of either complete culture medium (control wells) or twice the final drug concentrations diluted in complete culture medium (test wells). Drug cytotoxicity was measured by means of a SRB colorimetric assay giving the percent of the survival cells toward the control (untreated cells) absorbance. The SRB assay was performed as previously described.25 The culture medium was aspirated before fixation, and 50 μL of 10% cold trichloroacetic acid (TCA) was gently added to the wells. Microplates were left for 30 min at 4 °C, washed five times with deionized water, and left to dry at room temperature for at least 24 h. Subsequently, 70 μL of 0.4% (w/v) sulforhodamine B (Sigma) in 1% acetic acid solution was added to each well and left at room temperature for 20 min. SRB was removed, and the plates were washed five times with 1% acetic acid before air drying. Bound SRB was solubilized with 200 μL of 10 mM unbuffered Tris-base solution. Absorbance was read in a 96-well plate reader at 540 nm. Results are expressed in terms of IC50 values, which is the concentration of drug required to inhibit cell growth by 50% compared to control, after 48 h of incubation of the complexes toward cell lines.30 Calculation of Results. For each tested compound a dose−effect curve was drawn. Sextuplicate determinations gave a coefficient of variation (CV) (standard deviation/mean %) in the range of 1−5%, resulting in standard error (SE) that was very low in all cases. The data showing inhibition of cellular growth are expressed as the fraction of cells that remains unaffected (fu; survival fraction, SF), which is derived from the following equation: fu = ODx/ODc (where ODx and ODc represent the test and the control optical density, respectively). Drug potency was expressed in terms of IC50 values (50% inhibitory concentration) calculated from the plotted dose−effect curves (through least-squares regression analysis). A significant difference was presumed to exist when p ≤ 0.05 (Student’s t test). Micronucleus. A micronucleus test was performed in accordance with the previously reported method.65,66 MRC-5 cells were seeded (at a density of 2 × 104 cells/well) in glass coverslips, which were afterward placed in six-well plates with 3 mL of cell culture medium and incubates for 24 h. MRC-5 cells were exposed with 1−5 in IC50 values for a period of 48 h. After the exposure of 1−5, the coverslips were washed three times with PBS and with a hypotonic solution (75 mM KCl) for 10 min at room temperature. The hypotonized cells were fixed by at least three changes of 1/3 acetic acid/methanol (v/v). The coverslips were also washed with cold methanol containing 1% acetic acid. The coverslips were stained with acridine orange (5 μg/ mL) for 15 min at 37 °C. After, the coverslips rinsed three times with PBS to remove any excess acridine orange stain. The number of micronucleated cells per 1000 cells was determined. Cell Morphology. MCF-7 cells morphology of was observed under an inverse microscope, after incubation of MCF-7 cells by 1−5 complexes, for 48 h.20,21 DNA Fragmentation. The MCF-7 cells were incubated with complexes 1−5 and ciplatin for 48 h, as reported previously.20,21 MCF7 cells were seeded at a density of 3 × 105 cells/well in 10 mm plates at 37 °C for 48 h. The cultures media were transferred to a test tube with the attached cells, which were washed twice with PBS and detached with a silicone spatula. The test tube was centrifuged at 3000 rpm for 10 min, and the supernatant was removed. The cell pellets were resuspended in 1 mL of PBS, transferred to a microtube, and M


Article

Inorganic Chemistry

then derived from the equation Vo = −(A1b1 + A2b2), from the diagram absorbance versus time (s).28,58 DNA Binding Studies. Ultraviolet−Visible Studies. DNA stock solution was prepared by dilution of CT DNA to buffer (containing 15 mM trisodium citrate and 150 mM NaCl at pH = 7). The stock solution of CT DNA gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) of 1.89, indicating that the DNA was sufficiently free of protein contamination. The DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M−1 cm−1. For the titration experiments, UV spectra of CT DNA in buffer solution in the absence and presence of 1−5 at r values of 0, 0.02, 0.05, 0.07, 0.09, 0.10, 0.11, and 0.13; (r = [complex]/[DNA], [DNA] = 5 × 10−5 M) were recorded. For binding constants Kb value determinations, UV spectra of complexes 1−5 in absence and presence of CT DNA at r values 1, 0.5, 0.25, 0.17, 0.125, and 0.1 (r = [complex]/ [DNA], [complex] = 30 μM (in the case of 1 complex), 50 μM (in the case of 2 complex), 25 μM (in the case of 3 and 5 complexes), and 20 μM (in the case of 2 complex), [CT DNA] = 10−100 μM) were also recorded. This study was performed as described previously.20,21 Fluorescence Studies. The fluorescence spectroscopy method using EtBr was employed to determine the relative DNA binding properties of complexes 1−3 into CT DNA. The emission spectra at 605 nm of EtBr (2.3 μM) solutions that contain CT DNA (26 μM) in the absence or presence of various concentrations of complexes 1−3 (0, 25, 50, 100, 150, 200, and 250 μM) were recorded upon their excitation at 527 nm.26

centrifuged at 3000 rpm for 15 min, and the supernatant was removed again. In microtube 100 μL of lysis buffer, consisting of 0.5% Triton X100, 0.01 M Tris-HCl pH = 7.4, and 0.01 M EDTA pH = 8, was added and was incubated on ice for 30 min. The lysate was centrifuged at 6000 rpm for 90 min. The supernatant was then transferred to a microtube and incubated with 2 μL of RNase A (10 mg mL−1) for 2 h at 37 °C and then with 2.5 μL of Proteinase K (20 mg mL−1) for 2 h at 37 °C. DNA from the supernatant was precipitated overnight at −20 °C via the addition of 20 μL of 5 M NaCl and 120 μL of isopropyl alcohol. The microtube was centrifuged at 6000 rpm for 90 min, airdried, and resuspended in 20 μL of TE (Tris/EDTA) buffer solution, at pH = 7.4, consisting of 0.01 M Tris-HCl, pH = 7.4, and 0.001 M EDTA, pH = 8. A loading buffer, which contained 50% glycerol, 0.1% bromophenol blue, and 0.001 M EDTA, pH = 8, was then added to the samples at a ratio of 1:10 (v/v). Electrophoresis was performed on 1.6% agarose gel in 0.5×TBE (Tris/Borate/EDTA) buffer solution (from 5×TBE stock buffer solution consisting of 4M Tris, 0.02 M EDTA pH= 8, 0.4M Boric acid) which it is also contains 1 μg ml−1 ethidium bromide, for 120 min at 40 V. The DNA was visualized under ultraviolet light. The λ HindIII DNA Ladder was used as ladder. Cell Cycle. MCF-7 cells were seeded at a density of 1 × 105 cells/ well in six-well plates at 37 °C for 24 h. Cells were treated with 1−5 at the indicated IC50 values for 48 h. Afterward, the cells were trypsinized and washed twice with PBS and separated by centrifugation. With the addition of 1 mL of cold 70% ethanol, the cells were incubated overnight at −20 °C. For analysis, the cells were centrifuged and transferred into PBS, incubated with RNase (0.2 mg/mL) and propidium iodide (0.05 mg/mL) for 40 min at 310 K, and then analyzed by flow cytometry using a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA). For each sample, 10 000 events were recorded. The resulting DNA histograms were drawn and quantified using the FlowJo software (version FlowJo X 10.0.7r2).51 Detection of the Loss of the Mitochondrial Membrane Permeabilization. MCF-7 cells were treated with 1−5 at IC50 values. After 48 h of incubation period of 1−5, the cell medium was removed, and the Dye Loading Solution was added. The cells were incubated in 5% CO2 at 37 °C for 30 min. Afterward, 50 μL of Assay Buffer B is added of each well and incubated for 30 min. The fluorescence intensity is measured at λex = 540 and λem = 590 nm. The MMP assay kit used was purchased from sigma Aldrich “Mitochondria Membrane Potential Kit for Microplate Readers, MAK147”. Study of the Peroxidation of Linoleic Acid by the Enzyme Lipoxygenase in the Presence of Complexes. These studies were performed as previously reported.20,21,58 Boric acid 0.2 M was used as buffer solution at pH = 9. The solution of the substrate (linoleic acid) was prepared as below: 0.05 mL of linoleic acid was dissolved in 0.05 cm3 95% ethanol in a volumetric flask. The appropriate volume of H2O was gradually added in the flask. The prepared solution (5 cm3) was added to 30 cm3 of borate buffer. The enzyme (LOX) solution was prepared in ice cold bath from a solution of 10 000 U of enzyme per milliliter. An amount of 500 U per 3 cm3 of reaction mixture is used in every experiment. A unit of LOX causes an increase in absorption at 234 nm equal to 0.001 per minute. The enzyme activity was monitored by UV analysis. Enzyme solution (0.05 cm3) was added to a cuvette containing 2 cm3 of linoleic acid solution and the appropriate amounts of buffer and the solution of the inhibitors 1−5. The activity of the enzyme was determined by monitoring the increase in the absorption caused by the oxidation of linoleic acid at 234 nm (ε = 25 000 M−1 cm−1). The concentration of the substrate was kept constant (0.3 mM), while the amounts of buffer and inhibitor solutions varied according to the inhibitors concentration (2−30 μM). The Influence of 1−5 upon the Peroxidation of Linoleic Acid. A stock solution of 1−5 in DMSO 0.001 M was prepared. Three solutions of 1−5 (0.6 and 1.4 μM) were then formed by dilution of the stock solution using buffer boric acid at 0.2 M, pH 9, while the concentration of the linoleic acid was kept at 0.3 mM (linoleic acid/ catalyst molar ratios is 1/500 and 1/250). The measurements were done at 234 nm. The initial rate (Vo) was calculated by the equation Id = C + A1 exp(−b1t) + A2 exp(−b2t), and the initial reaction slope is

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b01241. Crystallographic data (excluding structure factors) for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication with CCDC Nos. 1442668 (nim), 1442665 (1), 1442664 (2), 1442663 (3), 1442667 (4), 1442666 (5). Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax: (+44) 1223−336−033; e-mail: deposit@ ccdc.cam.ac.uk). Cone angles of the complexes; mid-IR spectra; UV− visible spectra; 1H NMR spectra; the FACs data; UV spectra of CT DNA; A/Ao versus [complex] plots; CT DNA titration spectra; graphical plot of [DNA]/(εα − εf) versus [DNA]; emission spectra of EB bound to DNA in the presence of complexes (PDF) X-ray crystallographic data (CIF) X-ray crystallographic data (CIF) X-ray crystallographic data (CIF) X-ray crystallographic data (CIF) X-ray crystallographic data (CIF) X-ray crystallographic data (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. (C.N.B.) *E-mail: [email protected]. Phone: 30-26510-08374. Fax: 3026510-08786. (S.K.H.) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.K.H. acknowledges the Oncology Department of Novartis Hellas S.A.C.I. for the financial support (Project No. 81939). N


Article

Inorganic Chemistry

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Nimesulide was provided from Help pharmaceutical, which is acknowledged. C.N.B. and S.K.H. would like to thank the Unit of Bioactivity Testing of Xenobiotics, of the Univ. of Ioannina, for providing access to the facilities. C.N.B. and S.K.H. would like to thank the Atherothrombosis Research Centre of the Univ. of Ioannina for providing access to the flow cytometer. The COST Action CM1105 “Functional metal complexes that bind to biomolecules” is acknowledged for the stimulating discussions. C.N.B. and S.K.H. acknowledge the National Scholarships Foundation of Greece (IKY) for the postdoctoral research fellowship of excellence program IKY-Siemens (Project No. 22957)

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