Novel Mixed-Ligand Copper(I) Complexes: Role of Diimine Ligands

Article pubs.acs.org/jmc

Novel Mixed-Ligand Copper(I) Complexes: Role of Diimine Ligands on Cytotoxicity and Genotoxicity Valentina Gandin,† Marina Porchia,‡ Francesco Tisato,‡ Alessandro Zanella,‡ Emilia Severin,§ Alessandro Dolmella,† and Cristina Marzano*,† †

Dipartimento di Scienze del Farmaco, Università di Padova, via Marzolo 5, 35131 Padova, Italy IENI-CNR, Corso Stati Uniti 4, 35127 Padova, Italy § Genetics Department, ‘Carol Davila’ University of Medicine and Pharmacy, 24, Kiseleff str., RO-011346 Bucharest, Romania ‡

S Supporting Information *

ABSTRACT: Novel tetrahedral copper(I) mixed-ligand complexes of the type [Cu(X)(N∩N)(PCN)], 3−10, where X = Cl or Br, N∩N = 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen), 5,6-dimethyl1,10-phenanthroline (dmp), and dipyrido-[3,2-d:2′,3′-f ]-quinoxaline (dpq), and PCN = tris-(2-cyanoethyl)phosphine, have been synthetized and characterized by NMR, ESI-MS, and X-ray diffraction on two representative examples, [CuCl(phen)(PCN)]·DMF (5· DMF) and [CuBr(dpq)(PCN)]·2DMF (10·2DMF). Cu(I) complexes were evaluated for their in vitro antitumor properties against a panel of human cancer cell lines, including cisplatin- and multidrugresistant sublines. The most effective complex, [CuCl(dpq)(PCN)] (9), exhibited nanomolar cytotoxicity toward both sensitive and resistant cancer cells, but it significantly inhibited the growth of cultured normal cells. In vitro DNA assays and single cell gel electrophoresis revealed that 9 induced DNA fragmentation resulting in cell apoptosis. In parallel, fluorescence in situ hybridization (FISH) micronucleus assay attested high levels of genotoxicity following treatment of peripheral blood lymphocytes with complex 9, suggesting that the potential risk posed by diimine metal complexes should be carefully reconsidered.

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INTRODUCTION Although highly effective toward a number of solid tumors,1 Pt(II) anticancer drugs (cisplatin and the second and third generation analogues carboplatin and oxaliplatin)2 cause severe toxic effects on normal tissues and induce early appearance of resistance phenomena, even at the beginning of their administration or after the first therapeutic cycles.1,3,4 These drawbacks have stimulated an extensive search and prompted chemists to develop alternative strategies based on different metals with improved pharmacological properties and aimed at different targets.5,6 Actually, there are several metal-based compounds that can attack cancer interfering with biological processes other than DNA replication.7,8 Examples of alternative molecular targets for metal-based drugs include thiol-containing proteins, proteasome, matrix metalloproteases, telomerases, topoisomerases, glutathione-S-transferases, and histone deacetylases. Copper complexes are considered good alternatives to platinum drugs and several families of copper complexes have been studied as potential antitumor agents in recent years.9 Although only little information is available on the molecular basis of their mechanism of action, copper complexes have attracted great attention and provided encouraging perspectives based on modes of action different from that of platinum drugs.10−13 In contrast to exogenous © 2013 American Chemical Society

platinum, copper is found in all living organisms and is a crucial trace element in redox chemistry, growth, and development. However, copper can also be toxic to cells, due to its redox activity and its affinity for binding sites that should be occupied by other metals. Elevated levels of copper have been detected in various types of human cancer cells.11,12 The altered metabolism of cancer cells and the differential response between normal and tumor cells to copper are the basis for the development of copper complexes endowed with antineoplastic characteristics. Diimine copper complexes, in particular those including 2,2′bipyridine (bipy) and 1,10-phenanthroline (phen), have been investigated for their biological activities since the 1970s.14 More recently, detailed studies on the coordination chemistry of copper−phen compounds have revealed that ligation of the first phen to the copper ion is a highly favored process, whereas the association of the second phen is rather disfavored, especially under physiological conditions.15 Two rather different synthetic strategies have been evaluated to increase the stability of diimine copper complexes through (i) the connection of the two phen ligands by a bridging chain, Received: June 26, 2013 Published: August 21, 2013 7416

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thereby enhancing the chelation ability of the resulting polydentate bis-dimine ligand or (ii) the use of the monosubstituted [Cu(phen)] moiety as a building block to which different donor ligands (mainly amino acids or different polydentate chelates) have been coordinated. Examples of the second more investigated option include mixed-ligand complexes registered under the Casiopeinas trademark16 and other variable assemblies.17−20 All these mixed-ligand compounds exhibit similar findings in terms of DNA binding and cleavage, cytotoxic effects, and induction of apoptosis. Overall, it has been demonstrated that physicochemical features, such as planarity, hydrophobicity, and size of the diimine, the nature of the coligand, and the coordination geometry of the metal complex, all played important roles in determining the binding/ intercalating mode of copper complexes to DNA. Cellular processing of DNA damage drives the cell to the activation of apoptosis signal transduction pathways. In recent years, our research on potential cytotoxic copper compounds has been focused on the use of hydrophilic tertiary phosphines as ligands.21,22 The resulting aqueous soluble compounds were proven to be easier to handle during the in vitro tests and, more importantly, showed cytotoxic activity against human tumor cell lines belonging to a variety of tumor types including cisplatin and multidrug resistant phenotypes.22 It has also been found that the cytotoxic activity of these phosphine Cu(I) complexes was correlated with their ability to act as endoplasmic reticulum (ER) stress inducers, likely via proteasome inhibition, thus triggering a nonapoptotic mechanism of programmed cell death (PCD), termed paraptosis.23,24 Among our synthesized phosphine compounds, the mixed, linear [Cu(X)(PCN)] (PCN = tris-(2-cyanoethyl)phosphine, X = Cl, 1; X = Br, 2) showed biological properties consistent with the above-mentioned findings accomplished with encouraging cytotoxic activities.25 The coordination vacancy exhibited by these molecules make them suitable candidates for the introduction of an additional diimine ligand in the coordination sphere of the metal, as already demonstrated by the syntheses of ternary Cu(I) complexes of the type [Cu(X)(diimine)(P)], where X = Br and I, diimine = bipy or phen and P = tris(cyclohexyl)phosphine, that have recently been reported as potential catalysts and photoluminescent devices.26,27 The purpose of this study was to investigate whether the introduction of a diimine coligand in the phosphine copper(I) scaffold led to a synergistic effect and could represent a promising strategy for the development of potent copper-based anticancer agents. Hence, we have synthetized a series of novel [Cu(X)(diimine)(PCN)], namely, [CuCl(bipy)(PCN)] (3), [CuBr(bipy)(PCN)] (4), [CuCl(phen)(PCN)] (5), [CuBr(phen)(PCN)] (6), [CuCl(dmp)(PCN)] (7), [CuBr(dmp)(PCN)] (8), [CuCl(dpq)(PCN)] (9), and [CuBr(dpq)(PCN)] (10) (X = Cl or Br; diimine = 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen), 5,6-dimethyl-1,10-phenanthroline (dmp), and dipyrido-[3,2-d:2′,3′-f ]-quinoxaline (dpq) (Scheme 1). All of these complexes have been characterized both in the solid state and in the solution state by means of conventional physicochemical techniques, including multinuclear NMR spectroscopy and X-ray crystal structure determination for 5·DMF and 10·2DMF. A biological evaluation of the mixed copper(I) complexes 3−10, in comparison with the [Cu(X)(PCN)] complexes 1 and 2 and cisplatin, has been performed. The cytotoxic activity against various human cancer cell lines, including cisplatin-sensitive and

Scheme 1. Ligands and Complexes Utilized in This Work

-resistant cells, was investigated. Growth-inhibitory effects were compared with those induced in human nontransformed cells. Mechanisms underlying copper(I) complex-induced cell death were examined by means of flow cytometric analyses and comet assay, the latter applied for detecting DNA strand breaks. Cytotoxicity data were compared with those resulting from genotoxicity testing. Actually, a major problem associated with conventional chemotherapeutic agents, such as cisplatin, is the induction of genotoxic effects that may lead to an increased risk of secondary malignancies.28−30 Therefore, an important goal in metal-based drug development is the identification of agents able to kill rapidly dividing cells with minimal mutagenic side effects. To date, besides the wide variety of substituted diimine copper complexes that have been proposed, very few studies have attempted to examine their genotoxic potential.31 On these bases, the clastogenic and aneugenic events induced by the most promising derivatives were evaluated by the micronucleus assay combined with the fluorescence in situ hybridization (FISH) technique.

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RESULTS Synthesis and Characterization of Mixed-Ligand Copper(I) Complexes. The mixed-ligand copper(I) complexes were prepared by mixing equimolar amounts of PCN with the pertinent Cu(I) halide salt (CuCl or CuBr) and diimine (bipy, phen, dmp, or dpq) in warm dimethylformamide (DMF). All of the complexes are yellow-orange solids stable in air and in solution for weeks. These mixed-ligand complexes are soluble in DMSO, sparingly soluble in other polar solvents, and insoluble in chlorinated solvents. For example, solubility of representative complexes 1 and 9 in aqueous solution is in both cases S = 6 × 10−4 M, corresponding to ca. 0.18 mg/mL and ca. 0.3 mg/mL, respectively. The copper compounds were characterized by means of microanalysis, ESI(+) mass spectrometry, UV−vis, and multinuclear (1H, 31P) NMR spectroscopies. In addition, the molecular structure of the prototype complexes [CuCl(phen)(PCN)]·DMF (5·DMF) and [CuBr(dpq)(PCN)]·2DMF (10·2DMF) was determined 7417

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Figure 1. ORTEP view of the complexes [CuCl(phen)(PCN)]·DMF (5·DMF, left) and [CuBr(dpq)(PCN)]·2DMF (10·2DMF, right), together with the numbering scheme. Ellipsoids are at the 40% probability level; hydrogen atoms have been omitted for clarity. In the view of 5, the bonds made by the C(20) carbon atom with refined site occupancy of 0.40 have been drawn in white.

by single-crystal X-ray diffraction analysis. ORTEP32 diagrams of 5·DMF and 10·2DMF are outlined in Figure 1, while crystallographic data and selected bond lengths and angles are summarized in Tables 1 and 2, respectively. The overall

Table 2. Selected Bond Lengths (Å) and Angles (deg) for the Complexes [CuCl(phen)(PCN)] (5·DMF) and [CuBr(dpq)(PCN)] (10·2DMF) 5·DMF Cu−Cl Cu−P Cu−N(1) Cu−N(2) N(1)−C(1) N(1)−C(5) N(2)−C(6) N(2)−C(10) P−Ca Cl−Cu−P Cl−Cu−N(1) Cl−Cu−N(2) P−Cu−N(1) P−Cu−N(2) Cu−P−C(13) Cu−P−C(16) Cu−P−C(19) N(1)−Cu−N(2)

Table 1. Crystallographic Data for the Complexes [CuCl(phen)(PCN)]·DMF (5·DMF) and [CuBr(dpq)(PCN)]·2DMF (10·2DMF) empirical formula formula wt wavelength (Å) temp (K) cryst syst cryst size (mm) space group a (Å) b (Å) c (Å) β (deg) vol (Å3) Z (molecules/unit cell) calcd density (Mg m−3) absorp coeff, μ (mm−1) F(000) independent (unique) reflns obsd reflections [I > 2σ(I)] data/params/restraints Goodness-of-fita on F2 final R indices [I > 2σ(I)] largest diff peak and hole (e Å−3)

5·DMF

10·2DMF

C24 H27 N6 O P Cl Cu 545.48 0.71073 294.1(1) monoclinic 0.25 × 0.18 × 0.15 P21/n (No. 14) 8.343(5) 15.605(5) 20.060(5) 95.36(1) 2600(2) 4 1.393 1.032 1128 5642

C29 H34 N9 O2 P Br Cu 715.07 1.54178 150.0(1) monoclinic 0.40 × 0.38 × 0.20 C2/c (No. 15) 39.605(8) 14.234(3) 11.434(2) 96.11(3) 6409(2) 8 1.482 3.238 2928 4889

4833

4137

5642/354/2 1.188 R1b = 0.0657; wR2c = 0.1357 0.880 and −0.501

4889/392/0 1.026 R1b = 0.0320; wR2c = 0.0858 0.729 and −0.281

a

10·2DMF 2.344(2) 2.172(1) 2.073(3) 2.082(3) 1.334(6) 1.350(5) 1.360(5) 1.333(5) 1.837 107.0(1) 105.0(1) 110.4(1) 127.1(1) 124.0(1) 120.9(2) 112.6(2) 120.9(2) 80.4(2)

Cu−Br Cu−P Cu−N(2) Cu−N(1) N(2)−C(12) N(2)−C(13) N(1)−C(14) N(1)−C(1) P−Ca Br−Cu−P Br−Cu−N(2) Br−Cu−N(1) P−Cu−N(2) P−Cu−N(1) Cu−P−C(15) Cu−P−C(18) Cu−P−C(21) N(1)−Cu−N(2)

2.474(1) 2.190(1) 2.072(2) 2.090(2) 1.327(3) 1.361(3) 1.354(3) 1.321(3) 1.838 103.6(1) 117.2(1) 106.9(1) 123.0(1) 124.8(1) 118.0(1) 111.5(1) 117.4(1) 80.4(1)

Mean of three values.

Goodness-of-fit = [∑w(Fo2 − Fc2)2/(Nobsns − Nparams)]1/2, based on all data. bR1 = ∑||Fo| − |Fc||)/∑|Fo|. cwR2 = [∑w(Fo2 − Fc2)2/ ∑w(Fo2)2]1/2. a

arrangement of the complexes looks pretty much alike; a superimposition (Figure 2) of the atoms defining the metal coordination environment in the two molecules (ten atoms in total) by means of the Structure Overlay routine in Mercury33 yields an RMS deviation of 0.15 Å and shows that the clearest

Figure 2. Mercury’s Structure Overlay superimposition of complexes 5 (light gray) and 10 (green).

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Table 3. In Vitro Antitumor Activity IC50 (μM) ± SDa compound [CuCl(PCN)], 1 [CuBr(PCN)], 2 [CuCl(bipy)(PCN)], 3 [CuBr(bipy)(PCN)], 4 [CuCl(phen)(PCN)], 5 [CuBr(phen)(PCN)], 6 [CuCl(dmp)(PCN)], 7 [CuBr(dmp)(PCN)], 8 [CuCl(dpq)(PCN)], 9 [CuBr(dpq)(PCN)], 10 bipy phen dmp dpq PCN cisplatin

MCF-7 6.98 4.51 6.61 6.98 0.31 1.23 0.73 0.25 0.25 0.31 9.53 4.32 2.12 8.32 >100 9.60

± ± ± ± ± ± ± ± ± ± ± ± ± ±

A375 2.06 0.96 1.28 1.36 0.21 0.74 0.11 0.01 0.09 0.12 2.34 1.22 1.21 2.18

± 0.21

6.94 7.28 6.65 8.85 0.35 1.02 0.43 0.56 0.23 0.34 10.36 2.48 3.41 6.63 >100 13.23

± ± ± ± ± ± ± ± ± ± ± ± ± ±

A431 1.01 1.73 1.25 1.34 0.12 0.68 0.16 0.09 0.22 0.16 3.04 1.54 1.18 1.87

± 2.61

7.89 7.99 9.76 9.97 0.43 1.09 0.30 0.19 0.21 0.52 12.65 1.03 1.24 3.04 >100 2.25

± ± ± ± ± ± ± ± ± ± ± ± ± ±

A549 1.99 1.83 1.63 2.35 0.08 0.51 0.13 0.03 0.15 0.22 2.41 0.31 0.10 0.50

± 0.73

3.37 2.74 7.24 7.52 0.68 0.79 0.51 0.32 0.22 0.53 9.76 2.98 3.12 3.54 >100 16.34

± ± ± ± ± ± ± ± ± ± ± ± ± ±

A2780 1.26 1.17 2.35 1.52 0.20 1.23 0.71 0.42 0.17 0.21 1.37 1.12 1.17 1.25

± 0.81

5.89 5.79 5.52 5.87 0.17 0.21 0.43 0.64 0.11 0.31 14.43 3.38 3.15 4.46 >100 3.64

± ± ± ± ± ± ± ± ± ± ± ± ± ±

HCT-15 1.11 1.01 1.73 2.24 0.02 0.14 1.03 0.70 0.04 0.22 2.07 1.12 1.25 2.07

± 0.81

6.37 2.28 5.33 5.62 0.10 0.40 0.42 0.19 0.06 0.07 13.51 2.97 4.36 5.04 >100 13.12

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.17 0.63 2.95 1.25 0.04 0.26 0.33 0.11 0.05 0.12 1.77 1.55 1.65 2.53

± 1.32

Cells ((3−8) × 104·mL−1) were treated for 72 h with increasing concentrations of tested compounds. Cytotoxicity was assessed by MTT test. IC50 values were calculated by a four parameter logistic model (P < 0.05). SD = standard deviation. a

In the solution state, coordination of PCN to the copper(I) ion is assessed by the 31P NMR singlet, which is downfield shifted ca. 13 ppm compared with the signal observed at δ = −25.0 ppm for uncoordinated PCN. Such signal is also broadened (half-height line width of ca. 150 Hz in the complexes compared with the value of 25 Hz observed for uncoordinated PCN) due to the quadrupolar relaxation induced by the 63Cu and 65Cu nuclei (both having I = 3/2). Similar broadening and slight downfield shift are experienced by coordinated methylene proton signals of PCN and aromatic proton signals, the latter indicating coordination of the diimine ligand. Coordination of the halide group no longer occurs in the solution state in acetonitrile, DMSO, or methanol. In fact, the UV−visible and NMR profiles of each pair of [CuX(diimine)(PCN)] complexes (X = Cl or Br) are superimposable indicating that the halide likely becomes the counteranion of a supposed trigonal [Cu(diimine)(PCN)]+ species in solution. Such cation is clearly detected in the ESI(+) mass spectrum of each individual complex, along with additional [Cu(diimine)2]+ and [Cu(PCN)(MeCN)]+ fragment ions due to rearrangements of the trigonal species in the ion trap. Cytotoxicity against Cultured Cancer Cells. The in vitro antitumor activity of tetrahedral [Cu(X)(N∩N)(PCN)] complexes 3−10 and of previously described linear [Cu(X)(PCN)] complexes 1 and 2, as well as of the corresponding uncoordinated ligands (bipy, phen, dmp, dpq, and PCN) was evaluated versus several human cancer cell lines derived from solid tumors by the MTT test. The cytotoxicity parameters, in terms of IC50 (the median growth inhibitory concentration calculated from dose−survival curves) obtained after 72 h exposure, are listed in Table 3. Cell lines representative of lung (A549), colon (HCT-15), ovarian (A2780), breast (MCF-7), and cervical (A431) cancers, along with melanoma (A375), have been included. For comparison purposes, cytotoxicity of cisplatin was assessed under the same experimental conditions. Cancer cell lines included in the screen were endowed with different degrees of sensitivity to cisplatin and showed a consistently good response to treatment with all tested compounds (concentration- and time-dependent

difference is about the orientation of one of the cyanoethyl arms. In both molecules, the copper environment is distorted tetrahedral with a N2PX (X = Cl, Br) donor set. The “bite” angle of the cyclic diimine is 80.4(1)° in both complexes, narrower than ideal 109.5°. As a consequence, also the other angles involving N atoms in the tetrahedron deviate from ideality, up to 127.1(1)° in 5·DMF and 124.8(1)° in 10·2DMF. The metal ion is coplanar with the phen plane in 5·DMF, whereas it stands 0.26 Å off the dpq plane in 10·2DMF. Accordingly, in 5·DMF, the five-membered ring formed by the metal and the diimine upon coordination is perfectly planar, while in 10·2DMF there is a slight deformation toward an envelope conformation. In addition, in both complexes PCN assumes an orientation approaching the energetically more favorable anti arrangement with respect to the Cu−X bond, with dihedral angles of 164.6° and 158.7° in 5·DMF and 10· 2DMF, respectively. Five out of six cyanoethyl residues are also approaching the anti-orientation with only one (in 5·DMF) showing instead a syn−clinal conformation (Figure 2). A search in the CCDC34 database shows that other chemically related mononuclear Cu(I) complexes in which the metal has an N2PX donor set (with the N2 atoms belonging to a cyclic imine) are only about 20 (see Supporting Information and references therein) and that only three complexes including PCN have been structurally characterized so far.35,36 In the series of [CuX(phen)(PCN)] complexes (X = Cl, Br, or I), the change of the halide has little effect on other bonds involving copper. Considering all the complexes grouped in Table S1, Supporting Information, the mean Cu−N distance in chloride, bromide, and iodide complexes is, respectively, 2.095, 2.092, and 2.081 Å, whereas some dependence from the pertinent halide is observed for Cu−P distances, the mean values being 2.180, 2.190, and 2.201 Å for chloride, bromide and iodide, respectively. The latter values compare well with those found in 5·DMF (2.172(1) Å) and in 10·2DMF (2.190(1) Å). A more detailed comparison of metrical data for the series of N2PX−Cu(I) complexes is reported in Table S1 in the Supporting Information. 7419

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Table 4. Cross-Resistance Profiles IC50 [μM] ± SDa compound [CuCl(PCN)], 1 [CuBr(PCN)], 2 [CuCl(bipy)(PCN)], 3 [CuBr(bipy)(PCN)], 4 [CuCl(phen)(PCN)], 5 [CuBr(phen)(PCN)], 6 [CuCl(dmp)(PCN)], 7 [CuBr(dmp)(PCN)], 8 [CuCl(dpq)(PCN)], 9 [CuBr(dpq)(PCN)], 10 cisplatin doxorubicin

2008 5.27 4.62 8.72 9.12 0.49 0.90 0.69 0.32 0.35 0.57 2.22

± ± ± ± ± ± ± ± ± ± ±

2.04 2.51 1.74 1.37 0.09 0.32 0.41 0.12 0.14 0.32 1.03

IC50 [μM] ± SDa b

C13*

RF

± ± ± ± ± ± ± ± ± ± ±

0.7 0.6 1.0 0.9 0.7 1.2 0.5 1.6 0.9 1.2 10.2

3.89 2.65 8.98 8.01 0.36 1.13 0.32 0.51 0.32 0.69 22.77

2.71 2.00 2.42 1.12 0.05 0.29 0.71 0.33 0.02 0.52 2.01

LoVo 7.65 8.25 6.22 7.32 0.04 0.43 0.69 0.15 0.12 0.25

± ± ± ± ± ± ± ± ± ±

2.23 2.00 0.85 1.88 0.13 0.17 0.32 0.13 0.14 0.13

0.54 ± 0.86

LoVo MDR

RFb

± ± ± ± ± ± ± ± ± ±

0.9 0.9 1.2 1.2 1.5 1.2 1.0 1.1 1.8 1.4

6.76 7.77 7.64 8.97 0.06 0.54 0.67 0.17 0.21 0.35

2.16 2.21 2.37 1.26 0.02 0.16 0.34 0.09 0.11 0.21

13.23 ± 1.35

24

Cells (3 × 104·mL−1) were treated for 72 h with increasing concentrations of tested compounds. Cytotoxicity was assessed by MTT test. IC50 values were calculated by a four parameter logistic model (P < 0.05). SD = standard deviation. bRF = IC50 resistant/IC50 sensitive. a

values for all derivatives, calculated on the 2008−C13* cell pairs, were 6−20 times lower than that of cisplatin, attesting the ability of these species to overcome the acquired cisplatin resistance. Tested on LoVo MDR colon cancer cells, suitably selected for their resistance to doxorubicin and thus retaining the MDR phenotype, all derivatives yielded RF values roughly 20 times lower than that obtained with doxorubicin, suggesting that these agents are not potential MDR substrates. Among all mixed-ligand copper(I) complexes, once again complex 9 was found to be the most potent derivative, with IC50 values ranging from 0.12 to 0.35 μM against all sensitive and resistant cancer cells. On the basis of the above data, [CuCl(dpq)(PCN)] (9) was selected for further biological evaluation, and [CuCl(PCN)] (1) was used for comparison purposes. Cytotoxicity against Cultured Nontumor Cells. Cytotoxicity of complexes 1 and 9 was also evaluated against two human nontumor cell lines. In Figure 3, IC50 values calculated for human embryonic kidney HEK293 cells (human noncancerous cells in rapid proliferation) and human colon CCD18Co fibroblasts are summarized. [CuCl(dpq)(PCN)], 9 was the most lethal compound both in HEK293 and CCD-18Co cells with IC50 values in the submicromolar range. Conversely, complex 1 elicited a cytotoxic activity slightly lower (roughly 1.5-fold) than that recorded after cisplatin treatment. The selectivity index value (SI = quotient of the average IC50 toward normal cells divided by the average IC50 for the malignant cells) of 9 was comparable to that calculated with cisplatin, whereas 1 showed a SI roughly 2-fold higher than that obtained with the reference metallodrug. Cellular Uptake. In an attempt to correlate cytotoxic activity with cellular uptake, the copper content was evaluated in 2008 cells treated for 6, 12, 24, and 48 h with equitoxic drug concentrations (IC25 and IC50 values) of copper(I) compounds. The intracellular copper amount was quantified by means of GF-AAS analysis, and the results, expressed as ng metal/mg of cellular proteins, are shown in Figure 4. Treatment of 2008 cells with complex 9 resulted in a marked intracellular time- and dose-dependent copper accumulation that, after only 6 h treatment with IC25 and IC50 doses, was found to be 2.6- and 5.2-fold over the control, respectively (Figure 4B). In contrast, upon treatment of 2008 cells with complex 1 significantly lower intracellular copper levels were achieved.

antiproliferative profiles, data not shown), except the uncoordinated PCN ligand, which proved to be totally ineffective.25 Complexes [CuCl(PCN)] (1) and [CuBr(PCN)] (2) showed a significant in vitro antitumor activity, with IC50 values in the low micromolar range, 1.5 times lower than those recorded with cisplatin. Uncoordinated diimine ligands displayed noteworthy cytotoxic activity as well, as already reported in the literature.37 In detail, bipy showed a cytotoxic profile quite similar to cisplatin, except in A431 cervical and A2780 ovarian cancer cells, where it was about 5-fold less active. Phen, dmp, and dpq displayed an in vitro antitumor activity significantly higher (up to 5 times) than that of cisplatin, mean IC50 values (μM) being 2.86 (4.32−1.03), 2.90 (4.36−1.24), 5.17 (8.32−3.04), and 9.69 (16.34−2.35) for phen, dmp, dpq, and cisplatin, respectively. Complexes 5−10 showed a potent cytotoxic activity, with IC50 in the submicromolar range and markedly lower than that recorded with cisplatin. Phen-containing complexes 5 and 6 showed mean IC50 values from 3- to 8-fold lower than the uncoordinated phen ligand values (0.34, 0.79, and 2.86 μM, respectively). Also with complexes 7 and 8, an increase in cytotoxic potency ranging from about 6 to 8 times was reached over the uncoordinated 5,6-dmp ligand (0.48, 0.35, and 2.90 μM, respectively). A clear-cut enhanced cell-killing effect was gained by coordinating dpq to the [Cu(X)(PCN)] species, since mean IC50 values for complexes 9 and 10 were from 14to 29-fold lower than those calculated with dpq ligand alone (0.18, 0.36, and 5.17 μM, respectively). In particular, compound 9 distinguished itself as the most promising derivative promoting a growth inhibitory effect that exceeded that of cisplatin by a factor ranging from 10 (A431 cervix carcinoma cells) to 164 (HCT-15 colon cancer cells). The antiproliferative activity of mixed-ligand copper(I) complexes was also investigated in two additional cell line pairs, including a cisplatin-resistant cell line (C13* human ovarian adenocarcinoma cells) and a multidrug-resistant (MDR) variant (LoVo MDR human colon adenocarcinoma cells).38 Table 4 shows the cytotoxicity parameters, in terms of IC50 values and of resistance factor (RF), the latter defined as the ratio between IC50 values calculated for the resistant cells and those obtained with the sensitive ones. All copper derivatives exhibited a different cross-resistance profile than cisplatin, possessing a quite similar cytotoxic potency both on cisplatin-sensitive and on cisplatin-resistant cell lines. The RF 7420

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complex 1 caused a 1.3- and 1.6-fold increase of S/M/G2 phase cells after 24 and 48 h treatment, respectively, and a concomitant decrease of cells in G1 phase. No significant change of 2008 cells in sub-G1 phase was observed, suggesting that apoptosis was not the major mechanism of [CuCl(PCN)]induced cell growth inhibition. Because PI cytofluorimetric analysis is not able to discriminate the S and M phases from the G2 phase due to the equal DNA cellular content, the mitotic index (MI) in 2008 cells treated with IC50 doses of copper(I) complexes 1 or 9 for 24 h was calculated (Table 6). Compared with control cells, both copper(I) complexes reduced MI, but complex 9 completely abolished the cell cycle progression to mitosis. Hoechst 33258 Staining. Cell death in 2008 cells treated for 48 h with IC50 doses of complexes 1 or 9 was also studied monitoring cellular morphological changes of Hoechst 33258stained cells by fluorescent microscopic analysis (Figure 5). Compared with control cells, 9-treated cells presented brightly stained nuclei and typical apoptosis morphological features, such as chromatin condensation and fragmentation. In contrast, in cells treated with complex 1, no indication of chromatin fragmentation was detected. Comet Assay. In the newly synthetized mixed-ligand copper(I) complexes, the bidentate diimine coligand is expected to function as a DNA recognition element.20 Therefore, we investigated DNA damage in 2008 cells after treatment with 9 for 6 and 12 h, using the alkaline single cell gel electrophoresis (Comet assay). The results were compared with those obtained after treatment of 2008 cells with equitoxic concentrations of 1. Figure 6A shows the results obtained with 2008 control cells or cells treated with IC50 doses of complexes 1 or 9 for 12 h. The 9-treated cells displayed a statistically significant increase in electrophoretic migration of the DNA fragments, evidenced by well-formed comets, whereas complex 1 did not cause any detectable DNA fragmentation. In Figure 6B, the tail lengths (length of DNA migration), which are directly related to the DNA fragment size, calculated for cells treated for 6 or 12 h with 9 are illustrated. Complex 9 provoked a marked time-dependent increase in comet tail

Figure 3. Cytotoxicity against nontumor cells. Cells (5 × 104) were treated for 72 h with increasing concentrations of the tested compounds. The cytotoxicity was assessed by the MTT test. The IC50 values were calculated by 4-PL (P < 0.05). Insert shows SI values (the quotient of the average IC50 toward normal cells divided by the average IC50 for the malignant cells). Error bars represent standard deviation. *p < 0.05; **p < 0.01 compared with the control.

Cell Cycle Analysis. In order to investigate the effect induced by treatment with copper(I) complexes on cell cycle perturbations and induction of apoptosis, the DNA content of 2008 cells treated with IC50 doses of complexes 1 or 9 for 24 and 48 h was measured by FACS analysis (Table 5). Compared with control cells, the percentages of 2008 cells at different cell cycle phases were modified upon treatment with copper(I) complexes in a time-dependent manner, even if in a different way by 1 and 9. In more detail, after 24 h treatment, complex 9 caused a decrease of cell population in G1 phase (from 64% to 49%) with a concomitant marked increase of the cell population in sub-G1 phase (from 2% to 16%), suggesting the activation of an apoptotic pathway. The sub-G1 shift was further enhanced after 48 h treatment. Conversely, exposure to

Figure 4. Intracellular accumulation of [CuCl(PCN)] (A) and [CuCl(dpq)(PCN)] (B). 2008 cells were incubated with IC25 or IC50 of copper complexes for 6, 12, 24, and 48 h, and copper content was detected by GF-AAS analysis. Error bars indicate the standard deviation. *p < 0.05; **p < 0.01 compared with the control. 7421

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Table 5. Cell Cycle Profilesa cycle phase

Ctr

[CuCl(PCN)] (1), 24 h

[CuCl(dpq)(PCN)], (9), 24 h

[CuCl(PCN)] (1), 48 h

[CuCl(dpq)(PCN)] (9), 48 h

sub-G1 G1 S/G2/M

2.01 ± 0.54 63.87 ± 2.14 33.22 ± 2.04

2.19 ± 0.52 53.07 ± 2.14 43.15 ± 1.97

15.73 ± 1.13 49.09 ± 2.16 31.00 ± 2.71

2.31 ± 0.19 45.05 ± 1.73 52.21 ± 2.50

25.37 ± 2.16 36.12 ± 2.95 35.76 ± 3.16

a Percentage of 2008 cells in different cell cycle phases after 24 and 48 h exposure with IC50 of [CuCl(PCN)], 1 and [CuCl(dpq)(PCN)], 9 vs control untreated cells.

Table 6. Mitotic Indexa compound

MI, %

Ctr [CuCl(PCN)], 1 [CuCl(dpq)(PCN)], 9

16.5 5.2 0.8

indicator that complex 9 caused chromosomal aberrations in human lymphocytes. The relationship between the MN induction and the cytotoxicity for the two copper(I) derivatives is depicted in Figure 7C. Each point was gained by combining data reported in Table 7 and the data about cell viability resulting from MTT test on treated lymphocytes. The data underlined a linear relationship between the yield of chromosomal damage and cytotoxicity for complex 9 (r = 0.93), whereas no linear relationship has been calculated for 1 (r = 0.43). Therefore, at equitoxic doses, 1 and 9 induced a rather different antiproliferative effect that was associated with a high genotoxicity risk only for complex 9. In Vitro DNA Interactions. To gain insight into the molecular interactions of complexes 1 and 9 with DNA, we performed some in vitro experiments using calf thymus (CT)DNA and supercoiled (SC) plasmid pUC19. The binding modes of 1 and 9 to DNA were characterized through electronic absorption titrations and the representative UV−vis spectra in the presence of constant CT-DNA concentration are shown in Figure 8A. Upon increase of the complex/CT-DNA molar ratio (r) from 0.1 to 1, a decrease in the absorption intensities (hypochromism, Δε, 65−93%) of the π−π* absorption bands with red shifts (2−5 nm) was observed for complex 9. Such changes suggested an intercalative binding mode likely due to a strong stacking interaction between the extended planar dpq moiety and the DNA base pairs. Conversely, no modifications in absorption intensity were detected for complex 1 (data not shown), indicating the absence of DNA binding interactions. The competitive DNA binding of complexes 9 and 1 has been monitored via emission intensity of EB intercalated into CT-DNA as a function of copper complex concentration.39 A decrease of EB emission intensity as a function of the increasing amount of complex 9 was detected, whereas no significant variations were recorded after addition of increasing amounts of complex 1. Complex 9 efficiently competed with EB for the intercalative binding sites on DNA and totally replaced it at the highest concentration. KSV (Stern−Volmer constant) was calculated according to the classical Stern−Volmer equation:39

a

2008 cells were treated with IC50 of tested complexes and processed for MI calculation. At least 150 cells were counted for each condition, and MI was calculated as the ratio of the number of dividing cells to the total number of cells multiplied by 100.

lengths indicating that the diimine-containing complex induced massive DNA fragmentation. Micronucleus Assay and FISH Analysis. In order to provide information on the genotoxic potentials of copper(I) derivatives, a cytokinesis-block micronucleus (CBMN) test combined with FISH assay was planned. Human lymphocytes from healthy donors were treated for 4 h at equitoxic conditions of tested compound (IC50, IC25, and IC10 values) and reincubated for additional 40 h with fresh medium. Four hours before harvesting, lymphocytes were treated with the actin polymerization inhibitor cytochalasin B. Mitomycin C (0.12 μM), a clastogenic mutagen, was used as positive control. CBMN frequencies, obtained from replicates of two independent experiments (donors A and B) after treatment with increasing doses of 9 and 1, are summarized in Table 7. Complex 9 induced statistically significant increases of CBMN frequency at all concentrations tested. Notably, a dosedependent increase in lymphocyte CBMNi was detected (Figure 7A) and complex 9, at the highest dose, was found to increase the frequency of CBMN about 4-fold over the control, similarly to mitomycin C. In contrast, there were no differences in the frequency of CBMNi between cells exposed to equitoxic doses of complex 1 and the negative control cells (p > 0.05), thus providing evidence of no genotoxicity of 1 at all used doses. A FISH analysis with a centromeric DNA probe on MN induced by complex 9 was performed to discriminate MN of aneugenic and clastogenic origin. Almost all (85.6%) analyzed CBMNi were signal negative, indicating a predominantly clastogenic effect (an exemplary image of complex 9-induced CBMN is shown in Figure 7B). These observations are a strong

F0/F = 1 + KSVr

Figure 5. Hoechst 33258 staining. 2008 cells were incubated for 48 h with IC50 of [CuCl(PCN)], 1 or [CuCl(dpq)(PCN)], 9 and stained with the fluorescent dye Hoechst 33258. 7422

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Figure 6. Comet assay. 2008 cells were treated for different incubation times (6 or 12 h) with IC50 values of [CuCl(PCN)] (1) or [CuCl(dpq)(PCN)] (9) and then processed for comet assay. (A) Representative images of 2008 cells after 12 h incubation. (B) Comet tail length, calculated from the center of the cell and measured in micrometers with CellF software. The error bars indicate the S.D..

Table 7. Frequencies of Binucleated Lymphocytes with MN (CBMN) Induced by Complexes 1 and 9 treatment

dose

BN lymphocytes, rep. 1/rep. 2

CBMN, rep. 1/rep. 2

CBMN (%), rep. 1/rep. 2

avg CBMN (%) ± SD

Donor A Ctr [CuCl(PCN)], 1 [CuCl(PCN)], 1 [CuCl(PCN)], 1 [CuCl(dpq)(PCN)], 9 [CuCl(dpq)(PCN)], 9 [CuCl(dpq)(PCN)], 9 mitomycin C

IC10 IC10 IC25 IC50 IC10 IC25 IC50 0.12 μM

1978/1965 1981/1973 1996/1984 1967/1971 1893/1923 1873/1931 1899/1915 1965/1896

Ctr [CuCl(PCN)], 1 CuCl(PCN)], 1 CuCl(PCN)], 1 [CuCl(dpq)(PCN)], 9 [CuCl(dpq)(PCN)], 9 [CuCl(dpq)(PCN)], 9 mitomycin C

IC10 IC10 IC25 IC50 IC10 IC25 IC50 0.12 μM

1963/1898 1932/1896 1955/1879 1976/1898 1941/1903 1893/1911 1912/1867 1965/1904

27/25 31/29 30/27 26/27 52/54 97/99 136/138 152/149

1.37/1.27 1.56/1.47 1.50/1.36 1.32/1.37 2.75/2.81 5.17/5.13 7.15/7.21 7.74/7.86

1.32 1.51 1.43 1.35 2.78 5.15 7.18 7.80

± ± ± ± ± ± ± ±

0.07 0.06 0.10 0.04 0.04 0.02 0.04 0.08

20/22 30/27 31/30 25/27 53/56 95/99 138/141 150/146

1.02/1.16 1.55/1.42 1.53/1.59 1.27/1.45 2.73/2.94 5.02/5.18 7.22/7.55 7.74/7.67

1.09 1.49 1.56 1.36 2.84 5.10 7.39 7.71

± ± ± ± ± ± ± ±

0.10 0.09 0.04 0.12 0.15 0.11 0.23 0.05

Donor B

where F0 is the emission intensity in the absence of quencher, F is the emission intensity in the presence of quencher, and r is the concentration ratio of the complex to DNA. The constant was determined from the slope of the diagram F0/F versus r (see Figure 8B). The Ksv value (0.97) quantified a strong emission quenching ability for complex 9, whereas a negligible DNA binding affinity was detected (Ksv value of 0.67) for complex 1. Since 1979, Sigman et al. discovered that the [CuIIphen)2] complex exhibited an efficient DNA cleavage activity.14 On this basis, complexes 1 and 9 were tested for DNA cleaving properties by gel electrophoresis. Cleavage ability was measured from conversion of DNA from the SC form (form I) of plasmid pUC19 DNA to the nicked-circular form (NC, form II). SC pUC19 DNA (1 μg/mL) was incubated with increasing complex concentrations (range 1−25 μM) for 3 h. Figure 8C

shows representative gel electrophoresis patterns resulting from incubation of SC-DNA with 5 and 25 μM of complex 1 or 9. Treatment with complex 9 resulted in a concentrationdependent cleavage of DNA. Even at the lowest concentration (lane 2), complex 9 was able to convert SC-DNA into the NC form, whereas at 25 μM (lane 3), it cleaved DNA completely (absence of the SC conformation). Linearized DNA was not observed under these conditions, suggesting that cleavage occurred randomly. Similar DNAase activity of other diimine copper complexes has been previously described.27 Conversely, complex 1 did not have any DNA cleaving ability, even at the highest concentration (lane 5).

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DISCUSSION Several families of copper-based drugs have been proposed as good alternatives to platinum drugs as potential antitumor 7423

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Figure 7. (A) Dose-dependent frequency of CBMN in cultured lymphocytes after treatment with IC50, IC25, and IC10 of 9 and 1. Average values of Donors A and B; error bars represent SD. (B) Exemplary image (100×) of FISH stained CBMN lymphocytes treated with 9. (C) Relationship between CBMN frequency and cell viability in human lymphocytes. Cells were treated with increasing doses (IC50, IC25, and IC10) of 1 and 9 and then processed for MTT test or MN analysis.

complexes exceeded those of the corresponding ligands by a factor ranging from about 3 to 30 attesting that metal coordination led to an increase of antitumor efficacy. This property was strongly influenced by the nature of the coordinated diimine, as assessed by the analysis of the data achieved by cytotoxicity testing and summarized in Table 3. It is worthy of note that the four uncoordinated diimines showed cytotoxic activities in the 1−13 μM range (bipy < dpq < dmp ≈ phen), values comparable with those shown by cisplatin, while PCN elicited negligible toxicity. Several classes of planar heterocyclic aromatic diimines have been already described as potent cytotoxic agents due to their ability to interact with DNA by aromatic π stacking between base pairs.41 Considering the copper complexes 3−10, there was no significant variation in the cell killing effect ongoing from chloro to bromo analogues, confirming spectroscopic (UV−vis) and spectrometric (ESI-MS) evidence that indicated dissociation of the halide group with formation of charged [Cu(N∩N)(PCN)]+[X]− species in the solution state. Incorporation of bipy in complexes 3 and 4 did not significantly modify the antiproliferative activity of precursor complexes 1 and 2. Conversely, the incorporation of more rigid phen, dmp, and dpq chelates indeed represented the determining factor to enhance the cytotoxic potency of the [Cu(X)(PCN)] moiety by 1−2 orders of magnitude. A more detailed description of structure−activity relationships is reported in the Results section (Cytotoxicity against Cultured Cancer Cells). Remark-

agents due to their selective cytotoxic action on tumor cells. Based on a physiologically relevant, endogenous metal, copper complexes are assumed to be less toxic for normal cells with respect to cancer cells. Moreover, even if still little information is available on the molecular basis of the mode of action of copper complexes, there is a growing body of evidence demonstrating that they possess mechanism(s) of action different from platinum drugs. In a previous study, we have shown that [Cu(X)(PCN)]type complexes 1 and 2 displayed moderate to high in vitro antitumor activity against several human tumor cell lines.25 Two-coordinated linear [Cu(X)(PCN)]-type complexes present coordination vacancies that can be profitably filled by other ligands to afford tetrahedral species, that represent a more common geometry for copper(I) compounds.40 Planning our study, we speculated that the insertion of an additional ligand in the [Cu(X)(PCN)] framework could also improve the biological activity of the resulting complexes. According to the well-established antiproliferative activity elicited by diiminecontaining copper complexes,11,12 we decided to incorporate various bidentate diimine (from semirigid bipy to more rigid phen, dmp, and dpq chelates; Scheme 1) to induce the formation of tetrahedral assemblies. The synthetic approach proved to be feasible, as demonstrated by the easy preparation and characterization of distorted tetrahedral [Cu(X)(N∩N)(PCN)]-type complexes 3−10 (see Scheme 1, and Figures 1 and 2). The cytotoxic activity of all diimine containing 7424

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observations on several classes of phosphine copper(I) complexes.42 From the in vitro antitumor efficacy testing, [CuCl(dpq)(PCN)] (9) proved to be the most cytotoxic complex of the series, displaying an average IC50 of 0.18 μM, up to 2 orders of magnitude lower than that of cisplatin. Complex 9 was hence selected for further biological evaluation in comparison with the diimine-free [CuCl(PCN)] complex 1. The higher cytotoxicity displayed by the more lipophilic complex 9 compared with 1 well correlated with the more efficient cellular uptake of 9, quantified by AAS analysis. For different classes of metal complexes, it has already been demonstrated that the overall lipophilicity of the complex is the strongest structural determinant for uptake efficiency and drug potency.12 Moreover, whereas intracellular accumulation of 9 displayed a linear pattern with respect to time and concentration (Figure 4B), internalization of 1 showed patterns suggesting the involvement of a saturable process. The different uptake profiles over time likely reflect different mechanisms of cellular internalization. Hence, the diimine-containing lipophilic complex 9 might be internalized via passive diffusion, whereas complex 1 might exploit an active transport mechanism resembling that utilized by endogenous copper, through human copper transporter 1 (hCtr1). This observation gains importance in light of hCtr1 overexpression in cancer cells and tissues,43 accounting for the better selectivity of 1 toward cancer cells pointed out by growth inhibition assays performed on noncancerous cells. Instead, passive diffusion mechanisms may induce higher copper internalization (and cytotoxicity) but less cell-type selectivity. Cell cycle and fluorescence microscopic analyses evidenced that the treatment with [CuCl(dpq)(PCN)] (9) resulted in a significant increase of cell population in sub-G1 phase and a massive chromatin condensation, consistent with the triggering of apoptotic cell death. Conversely, complex 1 failed to induce the appearance of apoptotic hallmarks, under any experimental conditions. In 9, the bidentate diimine dpq is expected to function as a DNA recognition element thus conferring to this copper complex DNA binding and cleavage properties.20 Basically DNA damage to cancer cells was evident from Comet assay. Actually, in 2008 ovarian cancer cells treated with 9, a marked time-dependent increase in comet tail lengths has been detected indicating that the diimine-containing complex induced a massive DNA fragmentation. In an attempt to exploit DNA interaction mode, we performed in vitro DNA experiments, using CT-DNA and supercoiled pUC19 plasmid DNA. Besides confirming the ability of complex 9 to cleave DNA, these studies proposed an intercalative binding mode likely due to a strong stacking interaction between the extended planar dpq moiety and the DNA base pairs. Conversely, no significant modifications in DNA properties were detected after incubation with complex 1 suggesting lack of DNA interactions. Taken together, these data confirmed DNA as the major cellular target for diiminecontaining mixed-ligand copper(I) complexes. Moreover, they strengthen the hypothesis that [CuCl(PCN)] cell-killing effect instead relies on mechanism(s) other than DNA damage. DNA lesions resulting from exposure to DNA damaging agents stall DNA replication and collapse replication forks, resulting in cell death. However, if not repaired properly, many of these genomic insults can induce gene mutations or chromosomal alterations. Thus, developing anticancer agents

Figure 8. (A) Spectra of solutions containing CT-DNA (0.14 mM) and increasing concentration of complex 9 in Tris-HCL buffer, pH = 7.3. (B) Stern−Volmer quenching plots of the complexes 1 and 9. KSV values of 0.67 for 1 and 0.97 for 9. (C) Agarose gel electrophoresis patterns of SC pUC19 DNA incubated with complex 1 or 9 in Tris buffer at 37 °C for 3 h. Lane 1, DNA control; lane 2, DNA + 9 (5 μM); lane 3, DNA + 9 (25 μM); lane 4, DNA + 1 (5 μM); lane 5, DNA + 1 (25 μM).

ably, whereas free phen, dmp, and dpq (and also cisplatin) appeared more effective against human cervical carcinoma A431 cells, all diimine containing complexes displayed noticeable specificity against HCT-15 tumor cells with the strongest effectiveness shown by dpq-containing complexes (IC50 values of 0.06 and 0.07 μM for complexes 9 and 10, respectively). The specificity against a human colon carcinoma cell line confirms a distinctive property of most phosphinoCu(I)-type species.24 Moreover, mixed-ligand complexes 3−10, tested against a cisplatin-resistant ovarian carcinoma subline (C13* cells) and multidrug-resistant LoVo human colon-carcinoma subline (LoVo MDR), overcame cisplatin- and MDR-resistance phenomena with RF values 6−20 times lower than those shown by the reference drugs, cisplatin and doxorubicin, respectively. These findings are in agreement with our previous 7425

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then warmed to 60 °C in an oil bath. After 6 h, the mixture was filtered, and the dark-red filtrate was placed in a freezer. Yellow-orange crystals suitable for X-ray diffraction analysis were collected after 7 days. [CuCl(bipy)(PCN)], 3. Anal. Calcd for C19H20ClCuN5P: C 50.90, H 4.50, N 15.62. Found: C 50.73, H 4.48, N 15.34. Yield: 57%. 1H NMR (DMSO-d6, ppm): 2.04 (bs, 6H, P−CH2), 2.73 (m, 6H, NC− CH2), 7.58 (bs, 2H, H5,5′bipy), 8.05 (bs, 2H, H4,4′bipy) 8.48 (m, 2H, H3,3′bipy), 8.79 (bs, 2H, H6,6′bipy). 31P{H} NMR (DMSO-d6, ppm): −12.90 (bs). UV−vis (MeCN; λ, nm): 242, 298. ESI-MS(+) (m/z assignment, % intensity): 412 ([M − Cl]+, 100), 375 ([M − Cl − PCN ]+, 55), 297 ([M − Cl − bipy + MeCN]+, 43), 157 ([bipy + H]+, 44). [CuBr(bipy)(PCN)], 4. Anal. Calcd for C19H20BrCuN5P: C 46.31, H 4.09, N 14.21. Found: C 45.65, H 4.12, N 14.61. Yield: 51%. 1H NMR (DMSO-d6, ppm): 2.06 (m, 6H, P−CH2), 2.73 (m, 6H, NC− CH2), 7.75 (bs, 2H, H5,5′bipy), 8.13 (bs, 2H, H4,4′bipy), 8.53 (m, 2H, H3,3′bipy), 8.83 (bs, 2H, H6,6′bipy). 31P{H} NMR (DMSO-d6, ppm): −12.50 (bs). UV−vis (MeCN; λ, nm): 242, 298. ESI-MS(+) (m/z assignment, % intensity): 412 ([M − Br]+, 50), 375 ([Cu(bipy)2]+, 100), 297 ([M − Br − bipy + MeCN]+, 37). [CuCl(phen)(PCN)]·DMF, 5·DMF. Anal. Calcd for C24H27ClCuN6OP: C 52.84, H 4.99, N 15.41. Found: C 53.01, H 4.92, N 15.26. Yield: 64%. 1H NMR (DMSO-d6, ppm): 2.09 (dt, 2JHH = 7.3 Hz, 2JHP = 7.5 Hz; 6H, P−CH2−), 2.75 (m, NC−CH2−), 2.71 and 2.85 (s + s, 3H + 3H; DMFMe), 7.93 (s, 1H; DMFH), 8.03 (dd, 2 JH3,H2 = 3.3 Hz, 2JH3H4 = 7.5 Hz; 2H, H3,8), 8.18 (s, 2H, H5,6), 8.74 (m, 2JH4H3 = 7.5 Hz; 2H, H4,7), 9.25 (d, 2JH2,H3 = 3.3 Hz; 2H, H2,9). 31 P{H} NMR (DMSO-d6, ppm): −12.5 (bs). UV−vis (MeCN; λ, nm): 227, 269, 286 (sh). ESI-MS(+) (m/z assignment, % intensity): 436 ([M − Cl]+, 45], 298 ([M − Cl − phen + MeCN]+, 38), 181 ([phen + H]+, 100). [CuBr(phen)(PCN)]·DMF, 6·DMF. Anal. Calcd for C24H27BrCuN6OP: C 48.86, H 4.61, N 14.25. Found: C 48.62, H 4.49, N 14.16. Yield: 59%. 1H NMR (DMSO-d6, ppm): 2.10 (dt, 2JHH = 7.3 Hz, 2JHP = 7.5 Hz; 6H, P−CH2-), 2.76 (m, NC−CH2−), 2.71 and 2.85 (s + s, 3H + 3H; DMFMe), 7.94 (s, 1H; DMFH), 8.05 (dd, 2 JH3,H2 = 3.3 Hz, 2JH3H4 = 7.5 Hz; 2H, H3,8), 8.19 (s, 2H, H5,6), 8.75 (m, 2JH4H3 = 7.5 Hz; 2H, H4,7), 9.25 (d, 2JH2,H3 = 3.3 Hz; 2H, H2,9). 31 P{H} NMR (DMSO-d6, ppm): −11.50 (bs). UV−vis (MeCN; λ, nm): 227, 270, 285 (sh). ESI-MS(+) (m/z assignment, % intensity): 436 ([M − Br]+, 17), 298 ([M − Br − phen + MeCN]+, 18), 181 ([phen + H]+, 100). [CuCl(dmp)(PCN)], 7. Anal. Calcd for C23H24ClCuN5P: C 55.20, H 4.83, N 14.00. Found: C 54.86, H 4.76, N 14.28. Yield: 65%. 1H NMR (DMSO-d6, ppm): 2.06 (m, 6H, P−CH2), 2.75 (m, 6H, NC− CH2), 2.79 (s, 6H, 5,6 CH3), 8.03 (bs, 2H, H3,8), 8.85 (m, 2H, H4,7), 9.19 (bs, 2H, H2,9). 31P{H} NMR (DMSO-d6, ppm): −12.3 (bs). UV−vis (MeCN; λ, nm): 232, 277, 298 (sh). ESI-MS(+) (m/z assignment, % intensity): 209 ([dmp + H]+, 100), 464 ([M − Cl]+, 5). [CuBr(dmp)(PCN)], 8. Anal. Calcd for C23H24BrCuN5P: C 50.70, H 4.44, N 12.85. Found: C 50.75, H 4.28, N 12.47. Yield: 63%. 1H NMR (DMSO-d6, ppm): 2.07 (m, 6H, P−CH2), 2.74 (m, 6H, NC− CH2), 2.78 (s, 6H, 5,6 CH3), 8.02 (bs, 2H, H3,8), 8.85 (m, 2H, H4,7), 9.19 (bs, 2H, H2,9). 31P{H} NMR (DMSO-d6, ppm): −12.1 (bs). UV−vis (MeCN; λ, nm): 232, 278, 300 (sh). ESI-MS(+) (m/z assignment, % intensity): 209 ([dmp + H] +, 100), 464 ([M − Br]+, 12). [CuCl(dpq)(PCN)], 9. Anal. Calcd for C23H20ClCuN7P: C 52.67, H 3.84, N 18.70. Found: C 52.75, H 4.01, N 18.47. Yield: 53%. 1H NMR (DMSO-d6, ppm): 2.09 (m, 6H, P−CH2), 2.73 (m, 6H, NC− CH2), 8.18 (bs, 2H, H3,8), 9.26 (s, 2H, H5,6), 9.36 (bs, 2H, H2,9), 9.58 (br. s, 2H, H4,7). 31P{H} NMR (DMSO-d6, ppm): −12.0 (bs). UV−vis (MeCN; λ, nm): 255, 288, 339. ESI-MS(+) (m/z assignment, % intensity): 527 ([M − Cl − PCN + dpq]+, 27), 488 ([M − Cl)]+, 25), 297 ([M − Cl − dpq + MeCN]+, 15), 233 ([dpq + H]+, 100). [CuBr(dpq)(PCN)]·2DMF, 10·2DMF. Anal. Calcd for C29H34BrCuN9O2P: C 48.71, H 4.79, N 17.63. Found: C 48.75, H 4.61, N 17.97. Yield: 59%. 1H NMR (DMSO-d6, ppm): 2.13 (m, 6H,

that kill rapidly dividing cells with minimal potentially deadly side effects of chromosomal alterations and mutagenesis would be highly desirable. Notwithstanding the huge number of [Cu(L1)(N∩N)]+/0-type complexes synthetized and tested so far (L 1 = amino acids or other bidentate chelates), investigations on their potential genotoxicity are still limited.31 Therefore, a further important task of our work was to assess the genotoxic potential of 1 and 9 in human peripheral lymphocytes by CBMN assay. This test is extensively used to evaluate irreversible impacts on genetic toxicity and increased MN frequency predicts the risk of cancer in humans. Actually, CBMN assay is one of the most successful and reliable tests for genotoxic carcinogens according to OECD (Organization for Economic Co-operation and Development).44 Exposure to complex 9 induced statistically significant increases of CBMN frequency, even at the lowest concentration. In contrast, there was no difference in the frequency of CBMN between cells exposed to equitoxic doses of complex 1 and control cells attesting that 1 did not cause potentially detrimental genomic instability. Moreover, a FISH analysis with a centromeric DNA probe allowed recognition of a clastogenic activity for 9, in line with the occurrence of a significant DNA damage. The genotoxicity caused by the diimine-containing complex 9 raises doubts about the usefulness of the general approach of using diimine ligands as efficient DNA targeting probes in the development of potential anticancer copper complexes. In our study, the incorporation of a diimine coligand in the [Cu(X)(PCN)] moieties indeed led to more cytotoxic but, at the same time, to more genotoxic copper complexes. This latter feature, which would give rise to drug resistance or the development of secondary tumors and abnormal reproductive outcomes, has to be seriously taken into account in view of a possible therapeutic use of diimine-containing copper compounds. Therefore, we may suggest that a careful analysis of the genotoxic potential of diimine-containing copper complexes should always flank the in vitro antitumor studies.

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EXPERIMENTAL SECTION

Materials and General Methods. All solvents and commercially available substances were of reagent grade and used without further purification. The Cu(I) precursors CuCl and CuBr and the diimine ligands 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen), and 5,6dimethyl-1,10-phenanthroline (dmp) were purchased from SigmaAldrich. Dipyrido-[3,2-d:2′,3′-f ]-quinoxaline (dpq) was instead prepared according to a published procedure.18 Tris-(2-cyanoethyl)phosphine (PCN) was purchased from Strem Chemical Inc. (Germany). Elemental analyses (C, H, N) were performed on a Carlo Erba 1106 elemental analyzer establishing a >95% purity for all the samples. 1H and 31P NMR spectra were recorded on a Bruker AMX-300 instrument. Chemical shifts, in ppm, for 1H NMR spectra were relative to internal Me4Si. 31P NMR chemical shifts were referenced to an 85% H3PO4 standard. The 31P NMR spectroscopic data were accumulated with 1H decoupling. UV−visible spectra were recorded on a UV500 spectrophotometer (Spectronic-Unicam). Mass spectra were recorded by an electrospray LCQ ThermoFinnigan mass spectrometer. Synthesis of Mixed-Ligand Copper(I) Complexes. [CuCl(PCN)] (1) and [CuBr(PCN)] (2) were prepared according to the literature.25 Copper complexes 3−10 were synthesized with a method similar to that reported by Al-Fayez et al.,35 here detailed for [CuCl(phen)(PCN)], 5. In a two-necked round-bottom flask equipped with a condenser, under stirring, an equimolar amount of copper(I) chloride (99 mg; 1 mmol), PCN (193 mg; 1 mmol), and phen (198 mg; 1 mmol) was dissolved in degassed dimethylformamide (20 mL) at room temperature under a nitrogen atmosphere. The dark mixture was 7426

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P−CH2), 2.76 (m, 6H, NC−CH2), 2.71 and 2.85 (s + s, 6H + 6H; DMFMe), 7.93 (s, 2H; DMFH), 8.20 (dd, 2JH3,H2 = 2.4 Hz, 2JH3H4 = 7.9 Hz; 2H, H3,8), 9.27 (s, 2H, H5,6), 9.37 (d, 2JH3,H2 = 2.5 Hz; 2H, H2,9), 9.58 (d, 2JH4H3 = 7.9 Hz; 2H, H4,7). 31P{H} NMR (DMSO-d6, ppm): −11.9 (bs). UV−vis (MeCN; λ, nm): 255, 289, 339. ESI-MS(+) (m/z assignment, % intensity): 527 ([M − Br − PCN + dpq]+, 65), 488 ([M - Br)]+, 25), 297 ([M − Br − dpq + MeCN]+, 10), 233 ([dpq + H]+, 100). X-ray Diffraction Analyses. Single crystals of the complexes [CuCl(phen)(PCN)]·DMF (5·DMF) and [CuBr(dpq)(PCN)]· 2DMF (10·2DMF), of X-ray quality were grown from a DMF solution at −20 °C. The specimen selected for 5·DMF was mounted on a Philips PW1100 diffractometer, and the specimen selected for 10· 2DMF on an Oxford Diffraction Gemini E diffractometer equipped with a 2K × 2K EOS CCD area detector and sealed-tube Enhance Mo and Cu X-ray sources, under a cold nitrogen stream provided by an Oxford Instruments CryojetXL sample chiller. The impinging radiation was always graphite-monochromated. The diffraction data for 5·DMF were collected with the ω−2θ scan technique at room temperature by using Mo Kα radiation (λ = 0.71073 Å). Data for 10· 2DMF (2856 frames in 58 runs with a step of 1°) were collected by means of ω scans at T = 150.0(1) K using Cu Kα radiation (λ = 1.54178 Å), in a 1024 × 1024 pixel mode and 2 × 2 pixel binning. During both experiments, data were corrected for Lorentz and polarization effects, as well as for absorption. The (empirical) absorption corrections were performed either by means of Φ-scans45 (5·DMF) or by means of a multiscan approach with the scaling algorithm SCALE3 ABSPACK using equivalent reflections (10· 2DMF).46 Unit cell parameters were determined by least-squares refinement of 30 well-centered high-angle reflections (5·DMF) or by least-squares refinement of the angular settings of 18334 strongest reflections chosen from the entire data collection (10·2DMF). Two standard reflections every 150 measurements (5·DMF) or two reference frames were checked every 50 frames (10·2DMF) to ensure crystal stability. No sign of deterioration was detected in either case. The structures were solved by direct methods with SIR9747 (5·DMF) or SHELXTL NT48 (10·2DMF) and refined by standard full-matrix least-squares based on Fo2 with the SHELXL-9748 program. During the refinement of 5, a peak appeared in a position compatible with a second arrangement of one of the cyanoethyl carbons. At the end of the refinement, only the atom was modeled as disordered over two sites, C(20) and C(20A). The two positions were refined with partial occupancies of 0.60 and 0.40, respectively. All non-H atoms of 5 and 10 were allowed to vibrate anisotropically in the last cycles of refinement. Most of H atoms were placed instead in calculated positions and refined as “riding model”; exceptions were the H(24) and the phenanthroline hydrogens in 5, which were located in the Fourier-difference synthesis and were refined isotropically. The Uiso values of hydrogen atoms were calculated from the Ueq of the pertinent carbon atom. Main crystallographic data are listed in Table 1; selected bond lengths and angles are listed in Table 2. Full listings of atomic coordinates, bond lengths and angles, and anisotropic thermal parameters are available as Supporting Information. Experiments with Cultured Human Cells. Cu(I) complexes and the corresponding uncoordinated ligands were dissolved in DMSO just before the experiment, and a calculated amount of drug solution was added to the cell growth medium to a final solvent concentration of 0.5%, which had no detectable effect on cell killing. Cisplatin was dissolved in 0.9% sodium chloride solution. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cisplatin were obtained from Sigma Chemical Co, St. Louis, USA. Cell Cultures. Human lung (A549), breast (MCF-7), colon (HCT15), and ovarian (A2780) carcinoma cell lines along with melanoma (A375) were obtained from American Type Culture Collection (ATCC, Rockville, MD). Human nontumor colon CCD-18Co and embryonic kidney HEK293 cells were obtained from European Collection of Cell Cultures (ECACC, Salisbury, UK). Human ovarian cancer cell lines 2008 and its cisplatin resistant variant, C13*, were kindly provided by Prof. G. Marverti (Dept. of Biomedical Science of Modena University, Italy). Human cervical carcinoma cells A431 were

kindly provided by Prof. F. Zunino (Division of Experimental Oncology B, Istituto Nazionale dei Tumori, Milan, Italy). LoVo human colon-carcinoma cell line and its multidrug-resistant subline (LoVo MDR) were kindly provided by Prof. F. Majone (Department of Biology of Padova University, Italy). Cell lines were maintained in the logarithmic phase at 37 °C in a 5% carbon dioxide atmosphere using the following culture media containing 10% fetal calf serum (Euroclone, Milan, Italy), antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin), and 2 mM L-glutamine: (i) RPMI-1640 medium (Euroclone) for MCF-7, HCT-15, A431, 2780, 2008, and C13* cells; (ii) F-12 HAM’S (Sigma Chemical Co.) for LoVo, LoVo MDR, and A549 cells; (iii) DMEM for A375 cells, and (iv) MEM for CCD-1-Co cells. LoVo MDR culture medium also contained 0.1 μg· mL−1 doxorubicin. For human lymphocytes, whole blood (0.5 mL) of two healthy blood donors (A and B) was incubated in complete RPMI 1640 medium (4.5 mL). Cell division was promoted by the addition of 1% phytohemagglutinin (GIBCO BRL, Paris). The cultures were set up in duplicate and incubated for 24 h in a humidified 5% CO2 atmosphere at 37 °C. MTT Assay. The growth inhibitory effect toward tumor cells was evaluated by means of MTT assay.49 Briefly, (3−8) × 103 cells/well, dependent upon the growth characteristics of the cell line, were seeded in 96-well microplates in growth medium (100 μL). After 24 h, the medium was removed and replaced with fresh media containing the compound to be studied at the appropriate concentration. Triplicate cultures were established for each treatment. After 72 h, each well was treated with 10 μL of a 5 mg/mL MTT saline solution, and following 5 h of incubation, 100 μL of a sodium dodecylsulfate (SDS) solution in HCl 0.01 M was added. After an overnight incubation, cell growth inhibition was detected by measuring the absorbance of each well at 570 nm using a Bio-Rad 680 microplate reader. Mean absorbance for each drug dose was expressed as a percentage of the control untreated well absorbance and plotted vs drug concentration. IC50 values, the drug concentrations that reduce the mean absorbance at 570 nm to 50% of those in the untreated control wells, were calculated by four parameter logistic (4-PL) model. Evaluation was based on means from at least four independent experiments. Cellular Uptake. The 2008 cells (7 × 105) were seeded in 25 cm2 flasks in growth medium (10 mL). After 24 h, the medium was replaced, and the cells were incubated for 6, 12, 24, and 48 h with tested compounds. Cells were washed with PBS and harvested. Samples were subjected to three freezing/thawing cycles at −80 °C and then vigorously vortexed. Aliquots were removed for the determination of protein content by the BioRad protein assay (BioRad). The samples were added with 1 mL of highly pure nitric acid (Cu ≤0.005 μg/kg, TraceSELECT Ultra, Sigma Chemical Co.) and transferred into a microwave Teflon vessel. Subsequently, samples were submitted to standard procedures using a speed wave MWS-3 Berghof instrument (Eningen, Germany). After cooling, each mineralized sample was analyzed for copper amount by using a Varian AA Duo graphite furnace atomic absorption spectrometer (Varian, Palo Alto, CA, USA) at 324 nm. The calibration curve was obtained using known concentrations of standard solutions purchased from Sigma Chemical Co. Flow Cytometric Analysis. Drug-induced cell cycle perturbations and DNA fragmentation were analyzed by flow cytometry after DNA staining with propidium iodide (PI) according to Nicoletti et al.50 Briefly, 2008 cells (5 × 105) were exposed for 24 or 48 h to IC50 doses of tested compounds. One milliliter of a PI solution, containing 50 μg/ mL PI, 0.1% m/v Triton X-100, and 0.01% m/v sodium citrate, was added to cells and then incubated for 25 min at 4 °C in the dark. Percentage of cells in different cell cycle phases was measured by FACScalibur flow cytometer (Becton-Dickinson, CA) using a 550− 600 nm filter. Analysis was performed by Cell Quest Diva software (Becton-Dickinson, CA). Mitotic Index (MI) Determination. MI was determined as previously described.23 Briefly, 105 2008 cells were treated with IC50 of tested complexes. After 24 h incubation, cells were treated with colchicine (10−4 M) for 4 h, washed with cold PBS, and then treated 7427

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with 1% sodium citrate for 12 min at room temperature. Cells were fixed with methanol/acetic acid solution (3:1), spread on clean slides, air-dried, and stained with a 10% Giemsa solution. At least 150 cells were counted for each concentration, and MI was calculated as the ratio of the number of dividing cells to the total number of cells multiplied by 100. Comet Assay. Single-cell gel electrophoresis for detection of DNA damage was performed using the Comet assay reagent kit purchased from Trevigen (Trevigen Inc., Gaithersburg, MD, US) according to the manufacturer’s instructions. Briefly, 2008 (105) cells were seeded in 25 cm2 flasks in growth medium (6 mL). After 24 h, cells were incubated for different exposure times (6 and 12 h) with IC50 of tested compounds. Subsequently, cells were washed twice with cold PBS, harvested, centrifuged, and resuspended at 1 × 105 cell/mL in 1% low melting point agarose (LMPA, Trevigen). Then 50 μL of cells−LMPA mixture was layered onto frosted microscope slides, which were precoated with 1% normal agarose. After the agar had been allowed to set at 4 °C, the slides were immersed in lysis buffer (100 mM Na2EDTA, 2.5 M NaCl, 10 mM Tris pH 10.0, and 1% Triton X-100) for 1 h at 4 °C. The slides were then incubated in an alkaline electrophoresis solution (1 mM EDTA, 300 mM NaOH, pH > 13) at 4 °C for 40 min, followed by electrophoresis (1 V/cm) at 4 °C for 30 min. The slides were washed with neutralization buffer three times before immersion in absolute ethanol for 20 min and air-dried at room temperature. The DNA was stained with SYBR Green (1 μg/mL) for 5 min at 4 °C. A total of 50 comets per slide, randomly captured at the constant depth of the gel, were examined at 40 magnification in a fluorescence microscope (Olympus BX41, Milano, Italy; excitation, 495 nm; emission, 521 nm) connected through a black and white camera to a computer-based image analysis system. Comets were randomly captured at a constant depth of the gel, avoiding the edges of the gel, occasional dead cells, and superimposed comets. DNA damage was measured as tail length (distance of DNA migration from the middle of the body of the nuclear core) using Cell-F software (Olympus). Micronucleus Assay and FISH Analysis. The cytokinesis block micronucleus (CBMN) assay was carried out in human lymphocytes according to Fenech.51,52 Cells were cultured in 6-well plates for 24 h and then exposed to tested compound at increasing concentrations (IC50, IC25, and IC10 values) for 4 h. Following, all cultures were washed with PBS, reincubated for additional 40 h with fresh medium and then supplemented with 3 μg/mL cytochalasin B (Sigma Aldich) for 4 h before harvesting. In total, lymphocyte cultures were incubated for 72 h at 37 °C. Then cells were washed 3 times with PBS and suspended in a hypotonic solution (75 mM KCl) for 3 min at room temperature. Cells were then resuspended in a cold fixative methanol− acetic acid (4:1) solution, smeared on precleaned microscope slides, air-dried, and stained with 5% Giemsa (Sigma Diagnostics) according to standard procedures. Slices were scored at 100× magnification using an Olympus BX41 light microscope (Olympus) equipped with a color Moticam 2000 digital camera and basic image acquisition CellF software (Olympus). MN identification was carried out according to established criteria.51 Per culture, 2 × 1000 cells with well-preserved cytoplasm were examined. The total number of MN scored was recorded, and CBMN frequency was calculated as MN number per 1000 cells for each condition. The distinction between aneugenic and clastogenic effects was achieved by FISH analysis performed using a human pancentromeric probe directly labeled with GFP (Resnova, Roma, Italy). Briefly, the slides were denatured in 70% formamide in 2× SSC at 70 °C for 2 min and dehydrated through a series of ethanol washes (70%, 80%, and 95%). The probe was denatured at 96 °C for 5 min, and 10 μL was applied onto each slide. Slides were then coated with a coverslip, sealed, and hybridized overnight in a moist chamber at 37 °C. Subsequently, slides were washed several times in 10% formamide in 2× SSC and finally in 4× SSC containing 0.05% Tween20 for 10 min. The slides were counterstained with 4′,6-diamino-2-phenylindole (DAPI), air-dried, and mounted in an antifade reagent (90% glycerol in 0.1 M sodium borate, pH 9; 3% propylgallate). The slides were analyzed using 100× magnification under a fluorescence microscope

(Olympus) equipped with a Olympus XM10 monochrome CCD camera and images were acquired and analyzed by CellF software (Olympus). Hoechst 33258 Staining. The 2008 cells were seeded into 8-well tissue-culture slides (BD Falcon, Bedford, MA, USA) at 5 × 104 cells/ well (0.8 cm2). After 24 h, cells were washed twice with PBS, and following 24 or 48 h of treatment with IC50 dose of tested compounds, cells were fixed in 4% freshly prepared, ice-cold paraformaldehyde, postfixed in ethanol, and air-dried. Slides were then stained for 5 min with 10 μg/mL of Hoechst (2′-(4-hydroxyphenyl)-5-(4-methyl-1piperazinyl)-2,5′-bi-1H-benzimidazole trihydrochloride hydrate, Sigma-Aldrich) in PBS before being examined by fluorescence microscope (Olympus). In Vitro DNA Interactions. Electronic Spectral Titration. Absorption titration experiments were performed by maintaining a fixed CT-DNA concentration (0.14 mM) and rising Cu(I) complex concentration (producing reaction mixtures with varied mole ratio r of complex to CT-DNA; r range from 0.1 to 1). Copper(I) compounds were dissolved in a mixed solvent of 1% DMSO and 99% Tris-HCl buffer. The reference solution was the corresponding Tris-HCl buffer solution. The sample solutions were scanned in the range 200−500 nm on a computer-controlled Varian Coulter DU 800 spectrophotometer. EB−DNA Competition Experiments. The ethidium bromide (EB) displacement experiments were performed by titration with aliquots of tested complexes of a Tris buffer solution containing 10 μM DNA and 0.33 μM EB (saturated binding levels). The fluorescence spectrum of the solution was obtained by exciting at 520 nm and measuring the emission spectra at 587 nm. The influence of the addition of compound 9 at increasing concentration to the DNA−EB complex solution has been obtained by recording the variation of fluorescence emission spectra. The quenching efficiency for each complex was evaluated according to the classical Stern−Volmer equation:

F0/F = 1 + KSVr where F0 is the emission intensity in the absence of quencher, F is the emission intensity in the presence of quencher, KSV is the Stern− Volmer constant, and r is the concentration ratio of the complex to DNA. KSV constant was determined from the slope of the diagram F0/ F versus r. pUC19 DNA Cleavage Activity. DNA unwinding and cleavage ability of Cu(I) complexes was evaluated by agarose gel electrophoresis. Samples of DNA plasmid pUC19 (1 μg/μL) were incubated with increasing concentration (range 5−25 μM) of tested complexes in Tris buffer (50 mM Tris, 18 mM NaCl, pH 8.2) at 37 °C for 3 h in the dark. The reaction was quenched by the addition of 3 μL of loading buffer (0.25% bromophenol blue and 30% glycerol), and samples were loaded onto a 1% agarose gel in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.2). The gels were subjected to electrophoresis for 4 h at 50 V, followed by staining with 0.5 μg/mL ethidium bromide overnight. Gel bands were visualized using a UV transilluminator and photographed using an Olympus digital camera. Statistical Analysis. All the values are the means ± SD of not less than three measurements. Multiple comparisons were made by ANOVA followed by Tukey−Kramer multiple comparison test (**P < 0.01; *P < 0.05).

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ASSOCIATED CONTENT

S Supporting Information *

Complete crystallographic data in the form of cumulative CIF file (CCDC for complex 5·DMF (935408) and for complex 10· 2DMF (935409)), extended list of structural parameters in known N2PX−Cu(I) complexes (Table S1), and detailed discussion of metrical parameters. This material is available free of charge via the Internet at http://pubs.acs.org. 7428

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AUTHOR INFORMATION

Corresponding Author

*Fax: +39 049 8275366. Tel: +39 049 8275347. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by University of Padova (Progetto di Ateneo CPDA121973/12). Financial support for the acquisition of the Oxford Diffraction Gemini E diffractometer was provided by the University of Padova through the 2008 Scientific Equipment for Research initiative.

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ABBREVIATIONS

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REFERENCES

bipy, 2,2′-bipyridine; phen, 1,10-phenanthroline; dmp, 5,6dimethyl-1,10-phenanthroline; dpq, dipyrido-[3,2-d:2′,3′-f ]quinoxaline; PCN, tris-(2-cyanoethyl)phosphine; ER, endoplasmic reticulum; PCD, programmed cell death; FISH, fluorescence in situ hybridization; CBMN, cytokinesis-block micronucleus; MI, mitotic index; ET, ethidium bromide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GF-AAS, graphite furnace atomic absorption spectroscopy; FACS, fluorescence activated cell sorting; hCTR1, human copper transporter 1

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