Copper(II) complexes of N3O tripodal ligands appended with pyrene and polyamine groups: Anti-proliferative and nuclease activities

Accepted Manuscript Copper(II) complexes of N3O tripodal ligands appended with pyrene and polyamine groups: Anti-proliferative and nuclease activities

Doti Serre, Sule Erbek, Nathalie Berthet, Xavier Ronot, Véronique Martel-Frachet, Fabrice Thomas PII: DOI: Reference:

S0162-0134(17)30595-0 doi:10.1016/j.jinorgbio.2017.11.006 JIB 10366

To appear in:

Journal of Inorganic Biochemistry

Received date: Revised date: Accepted date:

17 August 2017 28 October 2017 4 November 2017

Please cite this article as: Doti Serre, Sule Erbek, Nathalie Berthet, Xavier Ronot, Véronique Martel-Frachet, Fabrice Thomas , Copper(II) complexes of N3O tripodal ligands appended with pyrene and polyamine groups: Anti-proliferative and nuclease activities. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jib(2017), doi:10.1016/j.jinorgbio.2017.11.006

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ACCEPTED MANUSCRIPT Copper(II) complexes of N3O tripodal ligands appended with pyrene and polyamine groups: Anti-proliferative and nuclease activities

Doti Serre,a Sule Erbek,b Nathalie Berthet,a Xavier Ronot,b Véronique Martel-Frachet,b and Fabrice

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Thomasa*

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a. Département de Chimie Moléculaire, Université Grenoble Alpes, UMR-5250 CNRS UGA, CS 40700, 38058 Grenoble Cedex 9, France. Email : [email protected].

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b. EPHE, PSL Research University, IAB, INSERM UGA U1209 - CNRS UMR 5309, 38700 La Tronche, France

ACCEPTED MANUSCRIPT Research highlights: - N3O tripodal ligands were appended by DNA targeting groups. - The copper complexes interact tightly with DNA. - The complexes exhibit micromolar nuclease activity.

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- The best nucleases show IC50 lower than cis-platin.

ACCEPTED MANUSCRIPT Abstract: A series of tripodal ligands based on the 2-tert-butyl-4-R-6-phenol was synthesized, where R = aldehyde (HL1), R = putrescine-pyrene (HL2) and R = putrescine (HL3). A dinucleating ligand wherein a putrescine group connects two tripodal moieties was also prepared (H2L4). The corresponding copper complexes (1, 2, 3, and 4, respectively) were prepared and characterized. We determined the phenol’s pKas in the range 2.47-3.93. The DNA binding constants were determined at

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6×106, 5.5×105 and 2.7×106 for 2, 3 and 4, respectively. The complexes display a metal-centered reduction wave at Epc,red = -0.45 to -0.5 V vs. saturated calomel electrode, as well as a ligand-centered

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oxidation wave above 0.57 V at pH 7. In the presence of ascorbate they promote an efficient cleavage

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of DNA, with for example a concentration required to cleave 50% of supercoiled DNA of 1.7 M for 2. The nuclease activity is affected by the nature of the R group: putrescine-pyrene ≈ bis-ligating >

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putrescine > aldehyde. The species responsible for strand scission is the hydroxyl radical. The cytotoxicity of the complexes was evaluated on bladder cancer cell lines sensitive or resistant to cis-

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platin. The IC50 of complexes 2 and 4 span over a short range (1.3 – 2 M) for the two cell lines. They are lower than those of the other complexes (3.1-9.7 M) and cis-platin. The most active compounds

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block the cell cycle at the G1 phase and promote apoptosis.

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Keywords: Copper; nuclease; DNA; tripodal ligand; phenol; anti-tumour.

Abbreviations: Boc: tert-ButylOxyCarbonyl; CT: Charge Transfer; CT DNA: Calf Thymus DeoxyriboNucleic Acid; CV: Cyclic Voltammetry; DFT: Density Functional theory; DMEM: Dulbecco's Modified Eagle's Medium; DMF: N,N-dimethylformamide; DMSO: dimethylsulfoxide; DNA: DeoxyriboNucleic Acid; EDTA: Ethylene Diamine Tetraacetic Acid; EI/DCI: Electron Ionization / Desorption Chemical Ionization; EPR: Electron Paramagnetic Resonance; ESI-MS:

ACCEPTED MANUSCRIPT ElectroSpray Ionization Mass Spectrometry; EtBr: Ethidium Bromide; FCS: Fetal Calf Serum; FITC: Fluorescein IsoThioCyanate; HR-MS: High Resolution Mass Spectrometry; L DNA: Linear DNA; MTT: 3-(4,5-DiMethylThiazol-2-yl)-2,5-diphenylTetrazolium bromide; NC DNA: Nicked Circular DNA; NMR: Nuclear Magnetic Resonance; NTO: Natural Transition Orbital; PBS: Phosphate Buffered Saline; X174 RF1: X174 Replicative Form 1; PI: Propidium Iodide; Q-TOF : Quadrupole

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Time-Of-Flight; RF: Resistance Factor; RPMI: Roswell Park Memorial Institute; SC DNA: Supercoiled DNA; SCE: Saturated Calomel Electrode; TBE: Tris - Boric acid - Ethylene Diamine

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Tetraacetic Acid; TD-DFT: Time-Dependent Density Functional theory; UV-Vis: UV-Visible.

ACCEPTED MANUSCRIPT 1. Introduction Inorganic complexes provide a versatile platform for the design of anti-cancer agents.[1-17] Perhaps the most representative example is cis-platin (and its most recent derivatives oxaliplatin and carboplatin), which is one of the most potent anti-tumour drugs.[2, 6-9] Its biological activity results from the formation of platinum-DNA adducts inside the nucleus, which hamper cellular processes and

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ultimately lead to cellular apoptosis.[2, 6-9] Despite cis-platin being one of the most efficient anti-cancer agents, severe side-effects (nephrotoxicity, emetogenesis and neurotoxicity)[18, 19] and drug resistance limit its clinical use. In contrast to platinum, copper is an essential bio-element.[23] It is involved

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[20-22]

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in several natural processes such as dioxygen transport, oxidation catalysis and signalling. By following the logic that biologically relevant metal ions may be less toxic than non-biological ones,

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several groups have designed new families of anti-cancer agents based on copper complexes.[10-12, 15-17] These investigations are also bolstered by the fact that correlations have been recently established

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between the serum copper levels and the tumor incidence, malignant progression [24-27] as well as the response to treatment in some human cancers. [28] In addition, copper was found to be an essential

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cofactor for the tumour angiogenesis process. [29]

Generally, the copper complexes that exhibit an interesting anti-proliferation activity also show a nuclease activity. [10-12, 14, 16, 30] The in vitro DNA cleavage reaction proceeds according to two main

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mechanisms, which usually depend on the presence or absence of exogenous agents. In the absence of exogenous agents, it is a hydrolytic pathway which is favoured, [31-42] although some examples of

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oxidative cleavage have been reported.[43-54] In the hydrolytic pathway the nucleophilic attack at the DNA phosphates is facilitated by metal coordination. This is instead an oxidative pathway that is observed in the presence of a reductant or oxidant.[55-77] Typically, the copper(II) is reduced to copper(I), which reacts with dioxygen to form toxic reactive oxygen species (O22-, O2•- and/or OH•) responsible for DNA strand scission. We recently reported a first generation of copper nucleases based on N3O tripodal ligands bearing a sterically hindered phenolate moiety, whose archetype is HL0 (scheme 1). We showed that the

ACCEPTED MANUSCRIPT complexes exhibit promising anti-proliferative activity (more active than cis-platin) against the bladder tumor cell lines RT112 and RT112CP (cis-platin resistant).[78]

Scheme 1

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We herein report a second generation of compounds, which incorporate either a putrescine or a putrescine-pyrene arm (Scheme 1). This ligand design allowed us to improve both the anti-

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proliferative activity against cancer cell lines and the nuclease activity of the complexes. The choice of

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these groups was motivated by the fact that i) putrescine is a natural polyamine whose targets at physiological pH (when the amines are protonated) are nucleic acids, [79] ii) polyamine analogues

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exhibit an antitumor activity in several cell lines (including breast and lung)[80-82] and iii) the pyrene is a planar and hydrophobic polyaromatic molecule, which can interact with DNA through -stacking

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(intercalation between the base pairs).[83] We report in this article the preparation and characterization

2. Materials and methods

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of the copper complexes, as well as the evaluation of their anti-proliferative and nuclease activities.

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2.1 General methodologies

All chemicals were of reagent grade and were used without purification. NMR spectra were recorded

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on a Bruker AM 300 (1H at 300 MHz) spectrometer. Chemical shifts are quoted relative to tetramethylsilane (TMS). Mass spectra were recorded on a Thermofinningen (EI/DCI) or a Bruker Esquire ESI-MS apparatus. For pKa determinations UV/Vis spectra were recorded on a Cary Varian 50 spectrophotometer equipped with a Hellma immersion probe (1.000 cm path length). The temperature in the cell was controlled using a Lauda M3 circulating bath and the pH was monitored using a Methrom 716 DMS Titrino apparatus. A least square fit of the titration data was realized with the SPECFIT software. The fluorescence spectra were recorded on a Cary Eclipse spectrometer. Xband EPR spectra were recorded on a Bruker EMX Plus spectrometer equipped with a Bruker nitrogen

ACCEPTED MANUSCRIPT flow cryostat and a high sensitivity cavity. Spectra were simulated using the Bruker SIMFONIA software. Electrochemical measurements were carried out using a CHI 620 potentiostat. Experiments were performed in a standard three-electrode cell under argon atmosphere. A glassy carbon disc electrode (3 mm diameter), which was polished with 1 mm diamond paste, was used as the working electrode. The auxiliary electrode is a platinum wire, while a SCE was used as reference.

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2.2 Synthesis

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HL1, HL2, 3-tert-butyl-4-hydroxy-benzaldehyde and N1-(pyren-1-ylmethyl)butane-1,4-diamine were

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prepared according to literature procedures.[84-86] For HL3boc, HL3 and H2L4 (see below) the numbering used for the assignment of the NMR resonances is depicted in ESI.

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HL3boc. The ligand (3-tert-Butyl-4-hydroxy-5-benzaldehyde) (HL1, 300 mg, 0.74 mmol) and tertbutyl-N-(4-aminobutyl)carbamate (140 mg, 0,74 mmol) were dissolved in MeOH (30 mL) and the

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reaction mixture was stirred for overnight at room temperature. Sodium borohydride (84 mg, 2.22 mmol) was then added by small fractions during 3h and the reaction was further stirred for 2h. The

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reaction mixture was extracted with AcOEt, washed with water and brine and dried over anhydrous

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Na2SO4. The solution was evaporated under vacuum to afford yellow-brown oil. Column chromatography on silica gel with CH2Cl2/MeOH (95/5) with a gradient of triethylamine (0 - 5%) as

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the eluent afforded HL3boc as a colorless oil in a 57 % yield. NMR 1H (400 MHz, CDCl3) : δ (ppm) = 1.42 (s, 9H, t-Bu); 1.45 (s, 9H, t-Bu); 1.52 (m, 4H, f and g); 2.58 (s, 3H, Me-pyr); 2.62 (t, 2H, e or h);

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3.10 (m, 2H, e or h); 3.65 (s, 2H, a); 3.77 (s, 2H, b); 3.82 (s, 2H, d); 3.83 (s, 2H, c); 6.88 (d, 1H, 4JH-H = 1,9 Hz, H1 or H2); 6,99 (d, 1H, 3JH-H = 7,6 Hz, H9); 7.09 (m, 2H, H7 and H1 or H2); 7.12 (ddd, 1H, 3

JH-H = 7.5 Hz, 3JH-H = 5.0 Hz, 4JH-H = 1.3 Hz, H4); 7.38 (d, 1H, d, 3JH-H = 7.5 Hz, H6); 7.49 (t, 3JH-H =

7.6 Hz, H8); 7.60 (td, 1H, 3JH-H = 7.5 Hz, 4JH-H = 1.8 Hz, H5); 8.51 (dd, 3JH-H = 5.0 Hz, 4JH-H = 1.3 Hz, H3); 10.81 (s, 1H, OH phenol). NMR 13C (Q.DEPT, 400 MHz, CDCl3) : δ (ppm) = 24.27 (CH3, Mepyr); 27.35; 28.05 (CH2); 28.58; 29,70 (CH3, t-Bu phenol and Boc); 34.92 (C, t-Bu phenol); 40.60 (C, t-Bu Boc); 46.28; 49.12; 54.05; 57.74; 59.45; 59.59 (CH2); 120.20; 121.81; 122.23 (CHaro); 123.03 (Caro); 123.61; 126.44; 128.05 (CHaro); 129.29; 136.61 (Caro); 136.69; 136.94; 149.00 (CHaro); 155.63;

ACCEPTED MANUSCRIPT 156.18; 157.50; 158.14; 158.67 (Caro). C34H49N5O3 • CH2Cl2 • 2CH3OH: calcd. C, 60.30; H, 8.39; N, 9.25; found: C 59.98; H 8.39; N 9.52%. IR (ATR): ν (cm-1) = 3281 (broad), 2951, 2860, 2815, 1698, 1594, 1524, 1473, 1454, 1444, 1384, 1369, 1242, 1201, 1173, 1087, 1043, 989, 872, 755. HR-MS (QTOF): m/z, 576.3900; Calcd: 576.3914 for [M+H]+. HL3. The ligand HL3boc ( 202 mg, mg, 0.35 mmol) is dissolved in ethanol (20 mL) saturated with

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gaseous HCl beforehand. After one hour stirring at room temperature the solvent is evaporated under vacuum, affording HL3 as a white hydrochloride salt in a quantitative yield. NMR 1H (400 MHz,

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D2O) : δ (ppm) = 1.29 (s, 9H, t-Bu); 1.78 (m, 4H, f and g); 2.81 (s, 3H, Me-pyr); 3.07 (m, 4H, e and

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h); 4.01 (s, 2H, b); 4.12 (s, 2H, a); 4.39 (s, 2H, d); 4.41 (s, 2H, c); 7.16 (s, 1H, H2); 7.21 (s, 1H, H1); 7.77 (d, 1H, 3JH-H = 7.9 Hz, H7); 7.89 (m, 2H, H4 and H9); 7.97 (d, 1H, 3JH-H = 7.9 Hz, H6); 8.37 (t, 1H, JH-H = 7,9 Hz, H8); 8.46 (t, 1H, 3JH-H = 7.9 Hz, H5); 8.68 (d, 1H, 3JH-H = 5.6 Hz, H3). RMN 13C

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3

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(Q.DEPT, 400 MHz, D2O) : δ (ppm) = 19.00 (CH3, Me-pyr); 22.71; 24.05 (CH2); 28.93 (CH3, t-Bu); 34.11 (C, t-Bu); 38.82; 46.16; 50.47; 57.36; 57.43; 57.83 (CH2); 122.57; 124.59 (Caro); 124.95 ; 126.20 ; 127.14 ; 127.54 ; 129.38 ; 130.29 (CHaro); 138.99 (Caro); 141.33 ; 146.49 ; 146.70 (CHaro);

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150.79 ; 151.90 ; 154.35 ; 154.66 (Caro). C29H49Cl4N5O3 • 4HCl • 2H2O: calcd. C 52.97; H 7.51; N

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10.65%; found: C 52.94; H 7.85; N 10.55%. IR (ATR): ν (cm-1) = 3392 (broad), 2993, 2955, 2819,1650, 1597, 1477, 1458, 1429, 1366, 1309, 1242, 1207. MS (ESI): m/z (positive mode) 476.4

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[M+H]+; m/z (negative mode) 474.3 [M-H]-. H2L4. 3-tert-Butyl-4-hydroxy-5-benzaldehyde (HL1, 500 mg, 1.24 mmol) and 1,4-butane diamine (55

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mg, 0.62 mmol) were dissolved in MeOH (30 mL). Then, 6 molar equivalents of NaBH4 (141 mg, 3.72 mmol) were added in small fractions over a period of 3h under stirring at room temperature. The reaction was further stirred for 2h. The solvent was evaporated under vacuum and the reaction mixture was extracted with AcOEt, washed with water and brine and dried over anhydrous Na2SO4. The solution was evaporated under vacuum to afford yellow-brown oil that is sensitive to carbonatation. Column chromatography on silica gel with CH2Cl2/MeOH (95/5) with a gradient of triethylamine (0 5%) as the eluent afforded HL4 as a white powder in a 52 % yield. NMR 1H (400 MHz, CDCl3) : δ (ppm) = 1.44 (s, 18H, t-Bu); 1.57 (m, 4H, f); 2.57 (s, 6H, Me-pyr); 2.64 (m, 4H, e); 3.64 (s, 4H, a);

ACCEPTED MANUSCRIPT 3.76 (s, 4H, b); 3.81 (s, 4H, c); 3.82 (s, 4H, d); 6.86 (d, 2H, 4JH-H = 1.8 Hz, H2); 6.99 (d, 2H, 3JH-H = 7.6 Hz, H7); 7.10 (m, 6H, H1-H4-H9); 7.37 (d, 2H, 3JH-H = 7.7 Hz, H6); 7.49 (t, 2H, 3JH-H = 7.6 Hz, H8); 7.59 (td, 2H, 3JH-H = 7.7 Hz, 4JH-H = 1.8 Hz, H5); 8.50 (dd, 3JH-H = 4.6 Hz, 4JH-H = 0.7 Hz, H3); 10.84 (s, 1H, OHphenol). RMN 13C (Q.DEPT, 400 MHz, CDCl3) : δ (ppm) = 23.22 (CH3, Me-pyr); 27.97 (CH2 f); 29.71 (CH3, t-Bu); 34,93 (C, t-Bu); 49.22; 53.78 ; 57.72; 59.45 ; 59.59 (CH2); 120.21 ; 121.82 ; 122.23

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(CHaro); 123.11 (Caro); 123.63; 126.52; 128.16; 136.68; 136.94; 149.00 (CHaro); 155.73; 157.49; 158.13; 158.64 (Caro). C54H70N8O2 • H2CO3 • 0.5H2O: calcd. C 70.71; H 7.88; N 11.99%; found: C

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70.00; H 7.75; N 12.14%. IR (ATR): ν (cm-1) = 3400 (broad), 2949, 1594, 1578, 1457, 1432, 1366,

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1296, 1242, 1207, 1191, 1147, 1090, 1001. HR-MS (Q-TOF): m/z, 863.5674; Calcd: 863.5700 for [M+H]+.

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Copper complexes. The copper complex 1 was isolated as single crystals, as previously described,[84] and used under this form for the spectroscopic and biological studies. Complex 2 were prepared in situ

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by mixing equimolar amounts of CuCl2 • 2 H2O, triethylamine and the ligand in DMF, as reported in literature.[84] Complexes 3 and 4 were prepared in a similar way, except that two molar equivalent of

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copper were used in the case of 4. The concentrated DMF solutions of the complexes were diluted in

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2.3 DFT calculations

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water in order to get a final medium of composition (DMF:H2O) (10:90).

All density functional theory (DFT) calculations were performed with the Gaussian suite (release

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9.0)[87] by using the B3LYP functional[88-90] in combination with the 6-31g* basis set for all the atoms. After geometry optimization a frequency calculation was systematically run in order to ensure that the optimized structures correspond to real energy minima and not saddle points. The relative energies are the sum of electronic and thermal free energies at 298 K. The spectroscopic properties were calculated by time dependent DFT[91] at the same level of theory than geometry optimization. The solvent was taken in account through a polarized continuum model (PCM)[92] and the 30 lowest energy excitations were calculated. 2.4 Determination of the DNA binding constants

ACCEPTED MANUSCRIPT The DNA binding constants were obtained from titration of solutions of the complexes with CT DNA. DNA (type I, fibers, from Sigma Aldrich) was first purified by four successive extractions with the phenol-CHCl3-isoamyl alcohol 25-24-1 mixture. Then 0.1 volume (with respect to the aqueous phase) of a 3 M sodium acetate solution and 2 volumes of ethanol were added and the solution was stored overnight at 253 K. The precipitated DNA was solubilised and analyzed by UV-Vis to ensure that the

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A260/A280 ratio was ≥ 1.8. For the direct determination of the binding constants by fluorescence the complex (1 M) was mixed with various concentrations of CT DNA (5 - 200 M in base pair) in a

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buffered H2O:DMF 90:10 solution containing [Tris-HCl] = 0.05 M, [NaCl] = 0.02 M at pH 7. After

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the equilibrium was reached (10 min) the fluorescence spectrum was recorded at 298 K. The DNA binding constants K were deduced from a Scatchard–Von Hippel plot. Typically, the ratio r/c was

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plotted against r to give a straight line whose slope is K. r corresponds to the number of bound molecules per site, while c represents the concentration of free drug. The concentration of bound

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molecules was obtained from the expression C0 × (f0-f)/(f0-fb), where f0 is the fluorescence of the free drug, f the fluorescence at any DNA concentration, fb the fluorescence of the drug bound to DNA and

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C0 the total concentration of drug. c was deduced from the mass balance. In order to avoid

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inaccuracies inherent to the determination of the concentration of free and bound molecules at the end and beginning, respectively, of the titration we removed the two firsts and two lasts points. Each binding constant was obtained from a duplicate experiment. For the competition experiment both the

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DNA and ethidium bromide (EtBr) concentrations were kept constant at 20 M and increasing

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concentrations of complex were added (2 - 50 M). The fluorescence was measured with an excitation wavelength of 517 nm, which corresponds to the excitation maximum for EtBr. The data were treated as detailed in the text, by using a binding constant for EtBr of 4.9 105 M-1.[93] 2.5 Procedure for DNA cleavage experiments X174 RF1 DNA was purchased from Fermentas and was stored at -20°C. The typical reaction mixture, containing double-stranded DNA and the copper complexes in a 10 mM phosphate solution (pH 7.2) with 10% (vol) DMF, was incubated at 37°C for the required time, with or without additives. After the incubation period, the reaction was quenched at -20°C, followed by the addition of loading

ACCEPTED MANUSCRIPT buffer (6x loading dye solution, Fermentas). The reaction mixture was loaded on a 0.8 % agarose gel in Tris-Boric acid-EDTA (TBE) buffer (pH 8), (0.5 x TBE) and electrophoresis was performed at 70 V for 2h30 - 3h. After DNA migration, the gels were stained by incubation for 10 min with a 1g/ml EtBr solution then washed with distilled water. The gels were visualised and the fluorescence quantified using Imager Typhoon 9400 and Image Quant Software. The cleavage efficiency was

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measured by determining the ability of the complex to convert the supercoiled DNA (SC) to nicked

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circular form (NC) and linear form (L).

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2.6 Cell culture

Human bladder cancer cell line RT112 and VERO cells (derived from the kidney of a healthy green

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monkey) were purchased from Cell Lines Service (Eppelheim, Germany). Cisplatin resistant RT112 cells (RT112-CP) were kindly provided by B. Köberle (Institute of Toxicology, Clinical Centre of

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University of Mainz, Mainz, Germany). RT112 and RT112-CP cells were cultured in RPMI 1640 medium and VERO cells in DMEM. These media are supplemented with 10% (v/v) fetal calf serum

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(FCS) and 2 mM glutamine (Invitrogen Life Technologies, Paisley, UK). Cells were maintained at

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37°C in a 5% CO2-humidified atmosphere and tested to ensure freedom from mycoplasma contamination.

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2.7 Cell proliferation assay

Inhibition of cell proliferation by copper complexes was measured by a MTT (3-(4,5-Dimethylthiazol-

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2-yl)-2,5-diphenyltetrazolium bromide) assay. RT112 and RT112-CP cells were seeded into 96-well plates (5 x 103 cells / well) in 100 µl of culture medium. After 24 h, cells were treated with cisplatin (Sigma-Aldrich, Lyon France) or complexes at various concentrations. In parallel, a control with DMF (vehicle alone) at the same dilutions was done. Following incubation for 48 h, 10 µl of a MTT (Euromedex, Mundolsheim, France) stock solution in PBS at 5 mg/ml was added in each well and the plates were incubated at 37 °C for 3 h. Plates were then centrifugated 5 min at 1500 rpm before the medium was discarded and replaced with DMF (100 µl/well) to solubilize water-insoluble purple formazan crystals. After 15 min under shaking, absorbance was measured on an ELISA reader (Tecan,

ACCEPTED MANUSCRIPT Männedorf, Switzerland) at a test wavelength of 570 nm and a reference wavelength of 650 nm. Absorbance obtained by cells treated with the same dilution of the vehicle alone (DMF) was rated as 100% of cell survival. Each data point is the average triplicates of three independent experiments. 2.8 Cell cycle analysis

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RT112 cells were cultured in 6-wells plates (2 x 105 cells/well) for 24 h before treatment with DMF or complexes for 24 h or 48 h. Trypsinized and floating cells were then pooled, fixed with 70% ethanol,

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washed with PBS and stained with 20 µg/ml propidium iodide (PI) in the presence of 0.5 mg/ml of

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RNAse (Sigma-Aldrich, Lyon France). Data acquisitions were performed with the Accuri C6 cytometer (BD Biosciences, Le Pont de Claix, France) equipped with a 488 nm laser Argon and PI

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fluorescence was collected with a 585±44 nm filter. Parameters from 2x104 cells were acquired using the Cell Quest Pro software (Becton Dickinson). The percentage of cell cycle distribution in the G1, S

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and G2+M phases was determined using FCS Express 5 software (De Novo Sofware, Los Angeles, CA).

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2.9 Apoptosis detection

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RT112 and RT112-CP cells were treated with DMF or complexes for 24 h or 48 h. Trypsinized and floating cells were then pooled and labeled with FITC-coupled Annexin V (Miltenyi Biotec, Paris,

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France) and PI following the manufacturer's instructions. Data acquisitions were performed with the Accuri C6 cytometer. Parameters from 2x104 cells were acquired using the C-Flow software. For each

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condition, fractions of live cells (Annexin V negative / PI negative), early apoptotic cells (Annexin V positive / PI negative) and late apoptotic cells (Annexin V positive / PI positive cells) were quantified with FCS Express 5 software (De Novo Software). The experiments were repeated three times independently.

3. Results and discussion 3.1 Synthesis of the ligands

ACCEPTED MANUSCRIPT The ligand synthesis is based on the preparation of an aldehyde precursor and a reductive amination. We recently reported the synthesis of the ligands HL1 and HL2.[84] Reductive amination of HL1 by the pyrene-putrescine conjugate PyrPt[86] affords the ligand HL2.[84] Reductive amination of HL1 by the tert-butyl-N-(4-aminobutyl)carbamate affords the ligand HL3boc, which was deprotected under acidic conditions to give HL3. The reaction of 1,4-butane-diamine with two equivalents of HL1 in the

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presence of NaBH4 affords the binucleating ligand H2L4.

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3.2 Formation of the complexes followed by mass spectrometry and UV-Vis spectroscopy Equimolar mixtures of the ligands HL1-3 and copper chloride were prepared in DMF and diluted in

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water for characterization by mass spectrometry (ESI-MS). For all the mixtures we obtained clean spectra with a single dominant peak corresponding to the expected copper complex. Indeed, a

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prominent peak is observed at m/z = 465.16 (monocation) in the case of HL1, which corresponds to [(L1)- + Cu2+]. Regarding HL2 the main peak is observed at m/z = 787.08 and assigned to [(HL2) + Cl-

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+ Cu2+]. For HL3 the dominating peak is observed at m/z = 269.13 and corresponds to a dication. It is

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formulated as [(HL3) + Cu2+]. Finally, the dinucleating ligand H2L4 gives, in the presence of two molar equivalents of copper, a prominent peak at m/z = 1095.31 (monocation), together with a peak of weaker intensity at m/z = 1059.32, which are assigned to [(H2L4) + 3 Cl- + 2 Cu2+] and [(HL4)- + 2 Cl-

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+ 2 Cu2+], respectively. Thus, ESI-MS confirms the formation of complexes with the expected (1:1)

Figure 1

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and (2:1) stoichiometries (M:L) for HL1-3 and H2L4, respectively.

The addition of copper chloride to a DMF solution of the ligands HL1-3 and H2L4 in the presence of NEt3 (to ensure phenol deprotonation) induces a colour change of the solution from pale yellow to violet, which is characterized by the development of a new band at around 470 nm (see below). Consistent with the ESI-MS data its intensity is maximal at one molar equivalent added for HL1-3,

ACCEPTED MANUSCRIPT disclosing the formation of complexes with a 1:1 stoichiometry (complexes denoted 1, 2 and 3, respectively, scheme 2).[94] In the case of H2L4 two molar equivalents or copper must be added in order to reach the plateau, confirming the dinucleating properties of this ligand, which forms the bimetallic

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complex denoted 4 (scheme 2).

Scheme 2

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In order to gain insight into the structure and the behaviour of the complexes in aqueous medium we

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investigated their spectroscopic properties as a function of the pH (Figure 2).[24] For solubility reasons (the complexes are not soluble in neat water) we formed the 1:1 (1-3) or 2:1 (4) M:L complexes in situ

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in a concentrated DMF solution, and next diluted them in water until reaching a water:DMF ratio of 90:10. At pH = 7 the UV-Vis spectra of all the complexes are dominated by a band at ca. 470 nm

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(Table 1), which corresponds to the phenolate-to-copper charge transfer (CT) transition.[78, 84, 85, 95-98][99] This assignment is corroborated by the blue shift of the max on going from 1 (electron withdrawing

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CHO para substituent) to 2-4 (electron donating alkyl para substituent). A low intensity shoulder is in

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general observed at lower energy, which is assigned to the copper(II) d-d transitions (Table 1). The intensity of the CT band is twice as large in complex 4 in comparison to complex 1, in agreement with

Figure 2

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the chelation of two metal centers.

Upon decreasing the pH from 7 to 2 the charge transfer transition decreases in intensity, consistent with the protonation of the phenolate moiety. The evolution of the spectra as a function of the pH was fitted to give the pKa values: 2.47±0.01, 3.93±0.02, 3.77±0.01, for 1, 2 and 3, respectively (scheme 3).

Scheme 3

ACCEPTED MANUSCRIPT For complex 4 the titration data were refined by using a model involving two protons, giving a -log2 value of 7.97±0.03. For every complex the pKa values are much lower than 7, indicating that the complexes exist mainly under their deprotonated form under physiological conditions. These values deserve few comments. Firstly, complex 1 shows the smallest pKa value, in line with the electron withdrawing effect of the aldehyde para substituent. Secondly, the pKa values are much lower than

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those reported for complexes featuring axial phenolates.[96-98] The easier deprotonation in our case reflects a stronger Cu-O bond, suggesting an equatorial positioning of the phenolate.[84, 96-98] The

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formation of a 5-membered chelate ring with the -methylpyridine in equatorial position is indeed

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strongly unfavored for steric reasons (steric clash due to the methyl substituent). This enforces an axial positioning of the -methylpyridine, and subsequently an equatorial coordination of the phenolate.

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This hypothesis is fully supported by the crystal structure of 1.[84]

Table 1

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3.3 EPR spectroscopy

The EPR spectra of complexes 1, 2, 3 and 4 at 100 K display the typical pattern of mononuclear copper(II) compounds. The (S = ½) signal is axial and hyperfine splitted, as a result of the interaction

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of the spin with the copper nuclear spin (ICu = 3/2). From simulation of the spectra we obtained the spin Hamiltonian parameters listed in Table 2. The ordering of g is indicative with a dx2-dy2 ground spin

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state for the copper ion, consistent with a square planar or square pyramidal geometry.

Figure 3 At pH = 7 the spectra of 2, 3 and the boc protected 3(Boc) are superimposable, supporting a similar metal ion geometry (Figure 3). This subsequently rules out the coordination by the terminal amine in the case of 3. The EPR spectrum of 4 exhibits the same shape than that of 3, but its intensity is twice as large. It can therefore be concluded that the two copper centers are magnetically uncoupled. This

ACCEPTED MANUSCRIPT idea is reinforced by the absence of MS = 2 resonance at around g = 4, as well as similarities in the linewidth for both 3 and 4. We interpret this result by a high flexibility of the putrescine arm and the functionalization in para position, which both do not favour a spatial proximity of the metal ions. The EPR spectra recorded at pH = pKa are broader than those taken at pH = 7, pKa+1 and pKa-1. The broadening is substantial in the parallel region, consistent with the presence of an equimolar mixture

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of the copper-phenol and copper-phenolate complexes in solution. Examination of the spectra in the

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low field region at pH = pKa+1 than at pKa-1 reveals substantial changes in the hyperfine coupling constant A//, congruent with a change in the copper ion geometry. An empirical relationship based on

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the g///A// ratio has been often used to estimate the tetragonal distortions of the copper ion.[100] Whatever is the pH the g///A// ratio is 126-133 cm within the present series (Table 2), indicative of

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weak tetragonal distortions. It is worth noting that the g///A// ratio is smaller at pH = pKa-1 than at

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pKa+1 (Table 2). Thus the distortions are less marked at low pH, presumably due to the weak coordinating ability of the phenol, which does not impose significant distortion in the coordination

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polyhedron.

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Interestingly, the spin Hamiltonian parameters for the phenolate complexes slightly differ between the saline (0.1 M NaCl) and Tris-HCl buffers (0.05 M containing 0.02 M of NaCl). We take this as evidence that an exchangeable ligand completes the coordination sphere of the copper center, in

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agreement with the structures depicted in Scheme 3. In order to gain insight on the nature of this exchangeable group we prepared the four complexes by mixing the ligand and the appropriate amount

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of CuCl2 2 H2O in DMF:H2O (10:90) without supporting electrolyte and added to the resulting solutions either NaCl or silver trifluoromethane sulfonate in excess. In this latter case precipitation of AgCl is observed, indicating removal of chloride from the solution. The EPR spectra without additive are very similar to those taken with silver trifluoromethane sulfonate, but they differ from those recorded in the NaCl 0.1 M medium. The main difference concerns the g// value, which is systematically lower in the NaCl 0.1 M medium (g// = 0.005). We take this behaviour as evidence of a change in the coordination polyhedron, which is assigned to the binding of Cl-. The increase in g// can indeed be interpreted in terms of an increased delocalization of the spin over the exogenous

ACCEPTED MANUSCRIPT chloride ligand, in agreement with DFT calculations (Mulliken spin population of 0.686 vs. 0.691 for the phenolate complexes with chloride and water equatorially bound, respectively). Thus, these data support binding of a chloride molecule at the equatorial position of the copper in an electrolytic

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medium containing a large amount of NaCl, such as that used for spectroscopic titrations.

Table 2

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3.4 DFT calculations

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We investigated the structure and spectroscopic properties of the copper complexes by DFT and TDDFT calculations (see ESI). In order to minimize the computational cost we designed an archetype

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denoted HLarch wherein the alkylammonium chain in para position of the phenol was replaced by a

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methyl substituent, which exerts similar electronic effects (Figure 4). The geometry of the copper complexes under their protonated “phenol” ([(HLarch)Cu(X)]y) and deprotonated “phenolate” [(Larch)Cu(X)]y-1) forms was optimized by taking in account the solvent (water) and by considering

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both a water molecule (X = H2O, Figure 4) and chloride ion (X = Cl) as exogenous ligand. With the

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aim of gaining insight on the positioning of the phenolic moiety we used as initial guess structures wherein this group is arbitrary located either in equatorial or axial environment. For

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[(HLarch)Cu(H2O)]2+ the isomer showing the phenol bound in axial position lies 7.2 kcal/mol below the one that features an equatorially bound phenol. For [(Larch)Cu(H2O)]+ (deprotonated) the

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difference in energy between the isomers is 6.2 kcal/mol, but this is now the structure showing an equatorially bound phenolate that is lower in energy. We next considered complexes featuring an exogenous chloride ion instead of the water molecule since such structures might form in the saline buffer medium. Both the axial positioning of the phenol and the equatorial positioning of the phenolate are again favoured, but the energy differences between the conformers are smaller (2.9 and 0.7 kcal/mol for the phenol and phenolate forms, respectively).

ACCEPTED MANUSCRIPT Figure 4 The absorption spectrum of the complexes was then computed by TD-DFT methods. We considered only the most favoured isomers in our calculations. For [(HLarch)Cu(H2O)]2+ an electronic excitation is predicted at 635 nm, which corresponds to the band detected at 680-690 nm in the visible spectrum of 2-4. The oscillator strength is rather small (fosc = 0.0017), in line with the small molar extinction

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coefficient observed experimentally. Consistent with its low intensity its assignment is dxz or dyz  dx2-y2 transition. When [(HLarch)Cu(Cl)]+ is considered instead of [(HLarch)Cu(H2O)]2+ the dxz or dyz 

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dx2-y2 transition is predicted at lower energy, 652 nm (fosc = 0.0012), in better agreement with the

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experimental spectrum. We next examined the deprotonated complex [(Larch)Cu(H2O)]+ (equatorial phenolate) with a coordinated water molecule. An intense electronic excitation is computed at 576 nm

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(fosc = 0.0454), which is assigned to the experimental band at 457-461 nm. The oscillator strength is

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much larger than in the protonated complex, in agreement with experiment. The NTO analysis discloses a different origin for this band, which is phenolate-to-copper charge transfer transition. Interestingly, calculations predict a shift of this band towards 547 nm when the chloride ion is

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considered as exogenous ligand instead of the water molecule, without significant change of oscillator

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strength (fosc = 0.0433). This predicted max again better fits with the experimental values, supporting

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the binding of a chloride ion in electrolytic media.

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3.5 Electrochemical behaviour of the complexes The electrochemical behaviour of the complexes (Figure 5, Table 3) has been investigated by cyclic voltammetry (CV) in a water: DMF 90:10 medium. Before commenting on the CV of the putrescine appended complexes, we will first discuss the behaviour of the archetype 1 (Figure 4a). The CV curve of 1 displays a cathodic peak at Epc,red = -0.46 V vs. SCE, which corresponds to the reduction of the copper(II) to copper(I). On the reverse scan an oxidation peak is observed at Epa,red = -0.12 V. This peak is absent from the anodic scan, when the initial potential is set at -0.2 V. Thus, Epa,red at Epc,red are found as a pair of semi-reversible peaks, which are attributed to the copper(II)/copper(I) redox couple.

ACCEPTED MANUSCRIPT The large Ep value (Ep = 0.34 V) attests that a chemical reaction is coupled to the reduction, which we assign to rearrangement and/or protonation of the phenolate.[84] Upon scanning towards a more positive potential an irreversible oxidation wave is detected at Epa,ox = 0.96 V (Table 3). It is assigned to the oxidation of the phenolate into a phenoxyl radical.[48, 78, 84, 85, 95-97, 101] The irreversibility of the wave is indicative of a low stability of the radical species, which is not unexpected owing to the

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electron withdrawing properties of the CHO substituent.

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Figure 5

We next examined the electrochemical behaviour of the conjugate PyrPt (Figure 5b). No clear

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reduction wave could be discerned when scanning down to -1.4 V, while an anodic signal could be observed at 0.86 V, followed by ill-defined broad oxidation wave above 1 V. The oxidation peaks

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arises from the irreversible oxidation of the amines[102] followed by the pyrene group.[103]

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Table 3

The CV curves of 2 and 3 are depicted in Figure 5c-d, while that of 4 is shown in ESI. The

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copper(II)/copper(I) redox couple in 2, 3, and 4 is observed as a pair of well separated peaks Epa,red and Epc,red, similarly to 1. This again supports a rearrangement of the coordination sphere associated to the

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electron transfer. The substituent effect on the Cu(II)/Cu(I) redox couple, if any, is difficult to appreciate due to broadening of the cathodic peak (Epc,red). The characteristic features of the PyrPt substituent are observable above 0.8 V in the CV of 2, in addition to a shoulder in the range 0.5-0.7 V, which is assigned to the phenoxyl/phenolate redox couple. This latter wave is observed at a lower potential in 2, 3 and 4 when compared to 1, consistent with the electron donating properties of the alkyl para substituent.[101]

ACCEPTED MANUSCRIPT 3.6 Fluorescence study The pyrene moiety is known to form excimers in solution, which are responsible for its intense fluorescence at around 470 nm. Upon addition of DNA to 2 the fluorescence at 468 nm progressively decreases. This behaviour is consistent with the intercalation of the pyrene moiety in DNA. The spectral changes were fitted by using a Scatchard – Von Hippel model (Figure 6),[104] giving a K value

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of 2.6 × 106 M-1 (n = 2, Table 4). The K value is three orders of magnitude larger than that of the copper complex of HL0,[78] pointing out the beneficial influence of the pyrene-putrescine chain. It is

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slightly smaller than that reported for PyrPt (1.1 × 107 M-1).[86] However, this difference likely arises

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from the higher salinity of the medium in our case (0.07 M versus 0.0097 M in Collette’s work), which disfavours ionic interactions between the ammoniums and the sugar-phosphate backbone of

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DNA. Finally, the K values of 2 is much higher than that of the classical intercalator ethidium bromide

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(EtBr) and putrescine,[105] showing the synergetic effect of the polyamine chain and the tripodal unit.

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Figure 6

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Table 4

In order to shed some light on the role of the pyrene and the tripodal unit in the strength of the

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interaction with DNA we conducted competition experiments between EtBr and 2-4. [106] This

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methodology has been successfully used to measure the binding constant of putrescine to DNA,[107] despite the fact that this polyamine is not an intercalator. A plot of the fluorescence intensity as a function of the [complex]/[EtBr] ratio is shown in Figure 7 for 2 (those of PyrPt, 3 and 4 are shown in ESI). We calculated the concentration of the complex that induces a quenching of 50 % of fluorescence. The following equation:

gives access to the apparent binding constants Kapp. By using the reported values KEtBr = 6.9 × 105 M-1 [93]

and [EtBr] = 20 M we calculated the Kapp values reported in Table 4. The Kapp of 2, 3 and 4 are

ACCEPTED MANUSCRIPT 6 × 106, 5.5 × 105 M-1 and 2.7 × 106 M-1, respectively. We also determined the binding constant of PyrPt under our experimental conditions, which gave 1.7 × 106 M-1. It must be underlined that the agreement between the K determined by the direct method for 2 and the Kapp from competition experiment is quite reasonable. When the Kapp of 2 is compared to that of 3 it immediately comes that the pyrene moiety enhances the

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binding constant by ca. one order of magnitude. In addition, when the Kapp of 4 is compared to those of 2 and 3 one can conclude that an extra tripodal unit is a better anchor than the terminal amine, but it is

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not as much as efficient as a pyrene moiety. In line with these results, the apparent binding constant of

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PyrPt is four times smaller than that of 2, again showing that the copper-tripodal unit enhances the

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binding to DNA.

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Figure 6

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3.7 DNA cleavage

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The nuclease activity of the copper(II) complexes has been investigated by gel electrophoresis, by using a plasmidic X174 RF1 supercoiled DNA, in a water: DMF 90:10 phosphate buffer (10 mM) at pH 7.2. Upon cleavage, the covalently closed circular supercoiled (SC) plasmidic DNA is converted

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into the nicked circular (NC) form (single strand breakage) and then to the linear (L) form, which

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corresponds to a double strand breakage.

Figure 8 The nuclease activity was first investigated in the absence of reductant, after 1h of incubation at 37°C by using concentrations of complexes ranging between 3 and 50 M. The results are summarized in Figure 8. No significant cleavage activity was observed for complex 1 at 50 M. The gel electrophoresis shows the disappearance of the SC form between 10 and 30 M (full disappearance at

ACCEPTED MANUSCRIPT 50 M) for complex 2 (Figure 8b) and between 30 and 50 M for complex 4 (Figure 8d), without concomitant apparition of the typical pattern for single or double strand cleavage. The absence of nicked circular or linear form in the gel has prompted us to repeat the experiment over a sharper range of complex concentrations. Between 10 and 30 M (complex 2, Figure 8f) or between 20 and 50 M (complex 4, ESI), i. e. before disappearance of the band, the supercoiled plasmid DNA clearly

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experiences a delay in the migration. This strongly suggests that the binding of the complexes promotes condensation of the DNA. It is indeed known that the polyamine chains, protonated under

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physiological conditions, can interact strongly with the phosphate groups of DNA. In some cases it

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leads to the aggregation of DNA,[108-111] which manifests by a delay in the migration, as experimentally observed for 2 and 4. Regarding 3 (Figure 8c) and especially in the case of 3(Boc) (Figure 8e) the

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effect is less pronounced, consistent with their weaker affinity with DNA.

Figure 9

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We next examined the nuclease activity of the complexes in the presence of two reductants,

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mercaptoethanol and ascorbic acid, after one hour of reaction at 37°C (Table 5).[112] As shown in Figure 9, all the complexes exhibit a higher nuclease activity in the presence of a reductant than

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without it. As the concentrations used to promote cleavage are lower in this experiment, the putative DNA condensation generally does not hamper the observation of the NC form. A common observation

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is that the cleavage activity is higher when the reductant is ascorbic acid than mercaptoethanol. We interpret this result by the fact that mercaptoethanol could potentially bind to the copper(I) (soft Sdonor), interfering with the reaction. We will therefore limit our discussion to the results obtained with ascorbic acid in the following section. Complex 2 is the most active nuclease, which cleaves 50 % DNA at the concentration of 1.7 M. Complex 1, which bears neither a pyrene, nor a putrescine chain is the least active compound (45 M for 50% of cleavage), while complex 3 is intermediate (14 M). Thus, the nuclease activity is correlated to the nature of the R substituent according to: pyreneputrescine > putrescine > CHO. This trend reveals that the affinity of the complex for DNA is the

ACCEPTED MANUSCRIPT main determinant for the nuclease activity of the compounds. Interestingly, at a concentration of 30 M, complex 3 also promotes very efficiently the double strand cleavage of DNA. At this concentration the cleavage produced an equimolar mixture of NC and L forms. Regarding the dicopper complex 4, gel electrophoresis displays the total disappearance of the SC form at concentrations in complex higher than 20 M, without appearance of bands characteristic of the NC and L forms. This

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peculiar behaviour is in line with that observed in the absence of reductant, hampering the determination of precise IC50 values. It is worth noting that both the NC and L forms could be

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observed with ratio of 67:33 in the case of 4 at a concentration of 15 M, while no more SC form was

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present. For comparison the SC form fully disappeared at a complex concentration in the range 5-10

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M in the case of 2. It is therefore clear that complex 4 is also a powerful nuclease.

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Table 5

In order to gain insight into both the binding mode and the reaction mechanism we conducted

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experiments in the presence of various scavengers and exogenous agents. The results obtained for 1, 2

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and 3 (at concentrations inducing significant cleavage) are depicted in Figure 10. By using a concentration of 2 of 8 M, it is observed that 75 % of the plasmid was converted into its NC form

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after 1 hour of reaction. The addition of superoxide dismutase (lane 4), catalase (lane 7) or NaN3 (lane 5) did not affect the yield of cleavage, showing that neither superoxide, nor hydrogen peroxide or

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singlet dioxygen is involved in the reaction. When CH3CH2OH (lane 2) or DMSO (lane 3, OH• scavengers) are added to the medium, the ratio of NC form drops to 63 and 42 %, respectively, indicative of the involvement of the hydroxyl radical in the cleavage reaction. Thus, it can be proposed that the copper(II) ion is reduced by ascorbic acid into copper(I), which reacts with molecular dioxygen to form the freely diffusible hydroxyl radical responsible for DNA strand scission.[10,15] The essential role of the copper in this mechanism could be confirmed by the inhibitory effect of EDTA (lane 6, 32 % NC form), which competes with the ligands for metal chelation.

ACCEPTED MANUSCRIPT Figure 10 The salt NaCl (350 M) has a marginal effect on the DNA cleavage (lane 9), while the addition of a minor groove binder (Hoechst 33258, lane 8) significantly alters the activity of 2 (48 % of NC form). These results indicate that 2 preferentially binds to the minor groove of DNA. They are consistent with the observation made by Ramirez et al. that free putrescine interacts primarily with the minor groove

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of duplex DNA[113] and suggest that the polyamine chain of 2 has the sufficient degree of freedom to

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interact with DNA in a similar way than the free putrescine. We can therefore propose a binding model wherein the putrescine chain interacts with the sugar-phosphate backbone in the minor groove,

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while the pyrene moiety is intercalated into base pairs. Consistent with this assumption, the Hoechst 33258 agent, which dramatically alters the nuclease activity of 2 has only a minor effect on the activity

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of 1 (46 % versus 40 % of NC form after 1 hour incubation, by using a complex concentration of 50

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3.8 Anti-proliferative activity

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M).

These promising nuclease activities of the complexes in vitro encouraged us to investigate their activity in cellulo. The standard treatment for muscle-invasive and metastatic bladder cancer is a

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neoadjuvant cis-platin-based combination chemotherapy followed by radical cystectomy. However, chemoresistance against cis-platin and its toxicity limit its use to patients with advanced bladder

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cancer. So, we evaluated the cytotoxicity of copper complexes on bladder cancer cell lines sensitive (RT112) or resistant to cis-platin (RT112-CP). Cells were incubated with various concentrations of the complexes for 48 h and proliferation was then monitored by a MTT assay. The obtained IC50 values are summarized in Table 6.

Table 6

ACCEPTED MANUSCRIPT Except for 1, the anti-proliferative activity of the complexes seemed to be directly correlated with their nuclease activity and their affinity towards DNA. Complex 2, which presented the highest cleavage activity was 5 times more active than 3, which appeared as less efficient nuclease. Complex 4 was the most active compound, with an IC50 of about 1 µM. Interestingly, complexes 2 and 4 presented the same cytotoxic activity against the cis-platin-resistant cells RT112-CP and were about 20 times more

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active than cis-platin against this cell line. Remarkably, all compounds appended by at least a putrescine chain were more efficient than cis-platin against RT112 cells and more importantly, all the

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complexes (irrespective of the phenolate para substituent) overcome the resistance observed with cis-

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platin on RT112-CP cells. As shown in Table 6, the RT112-CP cells are 2.6 times more resistant to cis-platin than the RT112 parental cell line, as expressed by the resistance factor (RF). On the other

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hand, the RF for copper complexes ranged between 0.6 and 1.8.

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The cytotoxicity of the complexes was also evaluated on the VERO normal non-tumor cell line. All the complexes were 1.5-3.6 fold less active against VERO cells compared to tumoral cell lines. It is particularly noteworthy that complex 4, the most active compound, is about 2.5 times less cytotoxic

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3.9 Apoptosis induction

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against VERO cells than on RT112 or RT112-CP cell lines.

In order to determine whether the anti-proliferative activity of the complexes results from apoptosis

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induction, we performed an annexin V/propidium iodide (PI) double staining, followed by flow cytometry analysis after incubating the cells with 1, 2 and 4, i.e. the compounds that show the lowest IC50. Based on the results of the MTT assay, RT112 cells were treated with the IC50 or 1.5 fold the IC50 concentration for 24 or 48 h. As shown on Figure 11, the fraction of apoptotic cells was dramatically increased in a dose and time dependent manner after incubation with all the investigated complexes. After a 48 h treatment with the IC50 concentration, the percentage of apoptotic cells were 42% for 1 and 2 and 43% for 4, compared to 12% for the control condition without treatment. These results

ACCEPTED MANUSCRIPT clearly indicate that the apoptotic pathway is implicated in the anti-proliferative activity of the complexes.

Figure 11

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3.10 Cell cycle arrest

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To further understand the mechanisms underlying proliferation inhibition and apoptosis induction, we evaluated the effect of the complexes on cell cycle progression. RT112 cells were treated with IC50

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concentrations of molecules 1, 2 and 4. After 24 h, the cell cycle phase distribution of treated cells was

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determined by flow cytometry. Our results showed that 2 and 4 caused a significant accumulation of RT112 cells at the G1 phase with a concomitant decrease of the number of cells in S and G2+M

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phases (Figure 12). The percentage of cells at the G1 phase after incubation with complexes 2 and 4 were 72% and 73%, respectively, compared to 51% in the non-treated cells. Thus, both 2 and 4 appeared as powerful blockers of the cell cycle, in line with their strong inhibitory effect on RT112

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cells proliferation.

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4. Conclusions

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Figure 12

In summary, we herein report an original functionalization of two tripodal ligands with putrescine and putrescine-pyrene chains. The copper-phenolate complexes were prepared and characterized. The binding constants towards duplex DNA are greatly enhanced (up to 3 orders of magnitude) in comparison to previously described copper-tripodal complexes that do not harbour these chains. This new generation of compounds shows a high nuclease activity in the presence of ascorbic acid, with a remarkable IC50 of 1.7 M for 2. Furthermore, the most active nucleases are also very potent antiproliferative agents towards RT112 and RT112-cis-platin resistant tumour cell lines. Our complexes

ACCEPTED MANUSCRIPT present a higher activity against tumoral cells compared to normal cells, which is particularly important for limiting the toxicity against healthy tissues and side effects. They block the cell cycle at the G1 phase, inducing apoptosis, which could be the consequence of the activation of DNA damage checkpoint. These new copper complexes are promising molecules, since they had a much greater anti-proliferative activity than cis-platin, a conventional agent used for the treatment of metastatic

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bladder cancer.

ACCEPTED MANUSCRIPT Aknowledgements The authors wish to acknowledge the LabEx ARCANE (ANR-11-LABX-0003-01), the EPHE, the University Grenoble Alpes (AGIR grant), as well as the Region Rhone-Alpes (ARC Santé) for

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financial supports.

ACCEPTED MANUSCRIPT References

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[1] Z. Guo, P.J. Sadler, Medicinal Inorganic Chemistry, Adv. Inorg. Chem., 49 (1999) 183-306. [2] B. Lippert, Cisplatin, Chemistry and Biochemistry of a Leading Anticancer Drug, Wiley-VCH, Weinheim, 1999. [3] T. Storr, K.H. Thompson, C. Orvig, Design of targeting ligands in medicinal inorganic chemistry, Chem. Soc. Rev., 35 (2006) 534-544. [4] L. Ronconi, P.J. Sadler, Using coordination chemistry to design new medicines, Coord. Chem. Rev., 251 (2007) 1633-1648. [5] J. Reedijk, Medicinal Applications of Metal Complexes Binding to Biological Macromolecules, Macromolecular Symposia, 270 (2008) 193-201. [6] J. Reedijk, Improved understanding in platinium antitumour chemistry, Chem. Commun., (1996) 801-806. [7] D. Lebwohl, R. Canetta, Clinical development of platinum complexes in cancer therapy: an historical perspective and an update, Eur. J. Cancer, 34 (1998) 1522-1534. [8] D. Wang, S.J. Lippard, Cellular processing of platinum anticancer drugs, Nat Rev Drug Discov, 4 (2005) 307-320. [9] W. Xiaoyong, Fresh Platinum Complexes with Promising Antitumor Activity, Anti-Cancer Agents Med. Chem., 10 (2010) 396-411. [10] M. Cristina, P. Maura, T. Francesco, S. Carlo, Copper Complexes as Anticancer Agents, AntiCancer Agents Med. Chem., 9 (2009) 185-211. [11] T. Saverio, M. Luciano, Copper Compounds in Anticancer Strategies, Curr. Med. Chem., 16 (2009) 1325-1348. [12] F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Copper in diseases and treatments, and copper-based anticancer strategies, Med. Res. Rev., 30 (2010) 708-749. [13] C. Duncan, A.R. White, Copper complexes as therapeutic agents, Metallomics, 4 (2012) 127-138. [14] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Advances in Copper Complexes as Anticancer Agents, Chem. Rev., 114 (2014) 815-862. [15] W. Wong, Copper for breast cancer metastasis, Science, 344 (2014) 1238. [16] R. Galindo-Murillo, J.C. García-Ramos, L. Ruiz-Azuara, I.I.I.T.E. Cheatham, F. Cortés-Guzmán, Intercalation processes of copper complexes in DNA, Nucleic Acids Res., 43 (2015) 5364-5376. [17] K. Garber, Targeting copper to treat breast cancer, Science, 349 (2015) 128-129. [18] D. Screnci, M.J. McKeage, Platinum neurotoxicity: clinical profiles, experimental models and neuroprotective approaches, J. Inorg. Biochem., 77 (1999) 105-110. [19] A. Rajeswaran, A. Trojan, B. Burnand, M. Giannelli, Efficacy and side effects of cisplatin- and carboplatin-based doublet chemotherapeutic regimens versus non-platinum-based doublet chemotherapeutic regimens as first line treatment of metastatic non-small cell lung carcinoma: A systematic review of randomized controlled trials, Lung Cancer, 59 (2008) 1-11. [20] L.R. Kelland, S.Y. Sharp, C.F. O’Neill, F.I. Raynaud, P.J. Beale, I.R. Judson, Mini-review: discovery and development of platinum complexes designed to circumvent cisplatin resistance, J. Inorg. Biochem., 77 (1999) 111-115. [21] M.A. Fuertes, C. Alonso, J.M. Pérez, Biochemical Modulation of Cisplatin Mechanisms of Action:  Enhancement of Antitumor Activity and Circumvention of Drug Resistance, Chem. Rev., 103 (2003) 645-662. [22] Z.H. Siddik, Cisplatin: mode of cytotoxic action and molecular basis of resistance, Oncogene, 22 (2003) 7265-7279. [23] M.C. Linder, Biochemistry of Copper, Plenum press, New York, 1991. [24] A. Chan, F. Wong, M. Arumanayagam, Serum Ultrafiltrable Copper, Total Copper and Caeruloplasmin Concentrations in Gynaecological Carcinomas, Ann. Clin. Biochem., 30 (1993) 545549. [25] M. Zowczak, M. Iskra, L. Torliński, S. Cofta, Analysis of serum copper and zinc concentrations in cancer patients, Biol. Trace Elem. Res., 82 (2001) 1.

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ACCEPTED MANUSCRIPT Figure captions Figure 1 Electrospray mass spectra of the in situ generated copper complexes (a) 1; (b) 2; (c) 3; (d) 4. The complexes were obtained by mixing the ligand with the appropriate amount of copper(II) chloride in DMF and further dilution with water at pH 7 for analysis. Figure 2 pH-dependence of the electronic spectrum of 2 in a H2O: DMF 90: 10 solution. [NaCl] = 0.1

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Figure 3 X-Band EPR spectra in a water: DMF 90:10 medium at pH 7. (a) 1; (b) 2; (c) 3; (d) simulation by using the parameters given in Table 2 for 2; (e) 4. [Tris-HCl] = 0.05 M, [NaCl] = 0.02

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M; T, 100 K; microwave frequency, 9.44 GHz; microwave power, 4 mW; mod. Freq.,100 KHz; mod. Amp. 0.4 mT.

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Figure 4 Geometry optimized structures for the archetypes: (a) [(HLarch)Cu(H2O)]2+ with an axial phenol; (b) [(HLarch)Cu(H2O)]2+ with an equatorial phenol; (c) [(Larch)Cu(H2O)]+ with an axial

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phenolate (as initial guess); (d) [(Larch)Cu(H2O)]+ with an equatorial phenolate.

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Figure 5 Cyclic voltammetry curves in a water:DMF 90:10 medium at pH 7. (a) 1; (b) PyrPt; (c) 2; (d) 3. [Tris-HCl] = 0.05 M, [NaCl] = 0.02 M; T, 298 K; scan rate, 0.1 V sec-1; The potentials are given

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versus the SCE reference.

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Figure 6 Scatchard – Von Hippel plot for the binding of complex 2 to CT DNA (from fluorescence data). Water: DMF 90:10 medium,[Tris-HCl] = 0.05 M, [NaCl] = 0.02 M; T, 298 K. Figure 7 Variation in the ethidium bromide (EtBr) fluorescence upon addition of complex 2; (a) Fluorescence spectra, the arrow indicates changes upon addition of 0-50 M of complex 2 in the presence of 20 M EtBr. Water: DMF 90:10 medium at pH 7; [Tris-HCl] = 0.05 M, [NaCl] = 0.02 M; T, 298 K;exc = 527 nm. (b) Plot of the fluorescence intensity as a function of the [complex]/[EtBr] ratio.

ACCEPTED MANUSCRIPT Figure 8 Agarose gel electrophoresis patterns of the cleavage reaction of X174 supercoiled DNA (20 M base pairs) mediated by the copper complexes in a phosphate buffer 10 mM pH 7.2 (+ 10 % DMF) at 37°C for 1 h, in the absence of reductant. Abbreviations: NC: nicked circular, SC: supercoiled. The concentrations of the complexes indicated on the top are in M. (a) 1; (b) 2; (c) 3; (d) 4; (e) 3(Boc); (f) Expended view of the region 10-30 M in the case of 2.

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Figure 9 Agarose gel electrophoresis patterns of the cleavage of X174 supercoiled DNA (20 M

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base pairs) mediated by the copper complexes in a phosphate buffer 10 mM pH 7.2 (+ 10 % DMF) at 37°C for 1 h in the presence of reductant. [ascorbate] = 0.8 mM. Abbreviations: NC: nicked circular,

SC

SC: supercoiled, L: linear. The concentrations of the complexes indicated on the top are in M. (a) 2;

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(b) 3; (c) 4.

Figure 10 Agarose gel electrophoresis patterns of supercoiled X174 DNA (20 M base pairs)

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incubated with the copper complexes in a phosphate buffer 10 mM pH 7.2 (+ 10 % DMF) at 37°C for 1 h; [ascorbate] = 0.8 mM; (a) 1, 50 M; (b) 2, 8 M; (c) 3, 30 M. Lane 0, DNA control; lane 1,

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DNA + complex (without scavenger); lanes 2-10, DNA + complex in the presence of agents: lane 2,

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ethanol; lane 3, DMSO (2 L); lane 4, Superoxide Dismutase (0.5 unit); lane 5, NaN3 (100 M); lane 6, EDTA (10 mM); lane 7, Catalase (0.1 unit); lane 8, Hoechst 33258 (100 M); lane 9, NaCl (350

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M).

Figure 11 Flow cytometry analysis after 48 h treatment with the copper complexes. Colour code:

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Green, late apoptosis; red, early apoptosis; blue, viable cells (necrosis corresponds to less than 0.3 % of the cells). (a) Control; (b) 2, 2.5 M; (c) 2, 3.75 M; (d) 4, 2 M; (e) 4, 3 M; (f) 1, 5 M; (g) 1, 7.5 M. Figure 12 Cell distributions after 24h treatment of RT112 cells with the copper complexes at a concentration corresponding to the IC50. (a) Control without complex; (b) treatment with 1; (c) treatment with 2; (d) treatment with 4. Colour code: Green, phases G2, M; Red, phase S; Blue, phases G0, G1.

ACCEPTED MANUSCRIPT Tables:

Complex pKa

phenolate formb

phenol formc

max

max



d

-d



1

2.47±0.01

481

790

-

2

3.93±0.02

460

1020

680

33

3

3.77±0.01

461

780

690

35

4

7.97±0.03e 457

2000

684

172

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Table 1. pKa values and electronic spectra of the copper complexesa

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a

SC

Fitted with the SPECFIT software. Data obtained in a H2O:DMF 90:10 solution, [NaCl] = 0.1 M. The pH is adjusted by addition of HClO4 or NaOH. The number in bracket corresponds to the standard deviation in the pKa value for the equilibrium depicted in Scheme 3. b

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Phenolate-to-copper charge transfer transition. A broad shoulder is observed at lower energy (ca. 700 nm) with a much lower intensity, which corresponds to the d-d transitions (not indicated in the table). Copper d-d transition.

d

At the lowest pH investigated (1.8) the phenolate-to-copper charge transfer was still observable.

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c

The value indicated corresponds to the -logβ2 value associated to the equilibrium: [(H2L4)Cu2]4+ H+ + [(L4)Cu2]2+. The fit was not improved by considering two stepwise protonation steps.

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e

2

ACCEPTED MANUSCRIPT

4

g/A e

2.053 2.250

1.4

17.0

126

2.056 2.261

2.0

17.0

127

3.60 d

2.055 2.262

1.4

16.4

132

7b

2.051 2.248

1.4

17.0

126

c

2.055 2.262

1.8

17.1

126

4.99 d

2.051 2.258

1.5

16.2

133

7b

2.053 2.247

1.7

17.0

126

2.78 c

2.055 2.264

1.8

16.9

128

4.78 d

2.054 2.260

1.4

16.4

132

7b

2.053 2.247

1.7

17.0

126

3.07 c

2.054 2.262

1.8

17.1

126

5.06 d

2.052 2.258

1.5

16.2

2.97

3

A//

c

1.62

2

A_|_

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7b

g//

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1

g_|_

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Complex pH

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Table 2. Spin Hamiltonian parameters for the copper complexesa

133

In H2O:DMF 90:10 frozen solution. A for Cu (I = 3/2) in mT.

b

[Tris-HCl] = 0.05 M, [NaCl] = 0.02 M.

c

[NaCl] = 0.1 M. The pH is adjusted by addition of HClO4 to the value corresponding to ca. pKa-1.

d

[NaCl] = 0.1 M. The pH is adjusted by addition of NaOH to the value corresponding to ca. pKa+1.

e

ratio expressed in cm.

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a

ACCEPTED MANUSCRIPT Table 3. Electrochemical properties of the compounds at pH 7 a Epc,red

Epa,red

Epa,ox

1

-0.46

-0.12

0.96

2

-0.5br

0.07

0.57, 0.69,0.8b

3

-0.46

-0.17

0.68,0.9b

4

-0.5br

0.08

0.57, 0.68, 0.85b

PyrPt

-

-

0.86, 1.0b

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Complex

a

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Ill-defined.

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b

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In a H2O:DMF 90:10 solution, [Tris-HCl] = 0.05 M, [NaCl] = 0.02 M. Reference, SCE; T, 298 K; Br: broad.

ACCEPTED MANUSCRIPT Table 4. Binding constants at pH 7 Compound

K (M-1)

Method

Cu-N3O tripod

a

Direct

Kapp (M-1)

Ref

2.0×10

3

[78]

2.6×10

6

This work b

Direct

3

Competition Competition

6×106 c 5.5×105 c

This work b This work b

4

Competition

2.7×106

This work b

Putrescine

Mobility

1.0×105 c

PyrPt

Competition Direct 1.1×107

[101] [86] d

1.7×106 c

EtBr

Competition Direct 6.9×105

This work b [87]

1.0×105

SC

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[99]

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2

Cu-N3O tripod: copper(II) complex of the 2-tert-butyl-4-methoxy-6-phenol.

b

In a buffered H2O:DMF 90:10 solution, [Tris-HCl] = 0.05 M, [NaCl] = 0.02 M, T = 298 K, pH = 7.

c

Calculated by using a K value for EtBr of 4.9 105 (reference 87).

d

10 mM sodium cacodylate buffer, pH 7.

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a

ACCEPTED MANUSCRIPT Table 5. Nuclease activity at pH 7.2 a Complex

Mercaptan Ascorbic acid

1

> 50

45

2

14

1.7

3

32

14

4

b

-b

-

Expressed as the concentration of complex (in M) that produces 50 % of cleavage of X174 supercoiled DNA. [DNA] = 20 M base pairs; T = 37°C; t = 1 h; [reductant] =0.8 mM; phosphate buffer 10 mM; pH = 7.2; water:DMF (90:10).

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a

b

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SC

The gel electrophoresis displays only the disappearance of the SC form, due to significant fragmentation or condensation of DNA (see the text). The IC50 value cannot be determined under these conditions.

ACCEPTED MANUSCRIPT Table 6. Antiproliferative activities of the complexesa

VERO c

RT112

RT112-CP

Cis-platin

ND

9.1 ± 1.2

23.8 ± 1.2

2.6

1

11.1 ± 1.5

3.1 ± 0.3

5.5 ± 0.2

1.8

2

3.1 ± 1.0

2.0 ± 0.2

1.5 ± 0.1

0.75

3

14.7 ± 1.4

9.7 ± 1.1

9.7 ± 0.6

1.0

4

3.2 ± 0.6

1.4 ± 0.3

1.3 ± 0.1

0.9

The concentration resulting in 50% loss of cell viability relative to untreated cells (IC50) was

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a

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RFb

IC50 (µM)

Complexes

SC

determined from dose-response curves. Results represent means ± SD of three independent experiments. RF is the relative ratio of IC50 in both the cis-platin-resistant and the sensitive cell lines. RF is the relative ratio of IC50 in both the cis-platin-resistant and the sensitive cell lines.

c

Not determined.

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b

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D

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Schemes and figures:

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Scheme 1 Formula of the organic compounds of interest in this study.

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Scheme 2 in situ generated complexes.

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Scheme 3 General structures and protonation equilibrium of the complexes (Ex: H2O or Cl-).

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2x106

3x107

(b) +

1x106

5x105

+

2x107

1x107

0 458 460 462 464 466 468 470 472 474

0 780

785

m/z

SC

6x106

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Intensity (a. u.)

+

8x106

2x105

4x106 2x106

0 266

268

270

272

0 1085

1090

1095

1100

1105

1110

m/z

D

m/z

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Intensity (a. u.)

4x10

5

(d)

2+

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1x107

(c) 6x10

795

m/z

8x105

5

790

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Intensity (a. u.)

Intensity (a. u.)

(a)

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Figure 1 Electrospray mass spectra of the in situ generated copper complexes (a) 1; (b) 2; (c) 3; (d) 4. The complexes were obtained by mixing the ligand with the appropriate amount of copper(II) chloride

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in DMF and further dilution with water at pH 7 for analysis.

ACCEPTED MANUSCRIPT

1.4 1.2 1.0

A

0.8 0.6

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0.4 0.2 0.0 500

600

700

800

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400

SC

 / nm

Figure 2 pH-dependence of the electronic spectrum of 2 in a H2O: DMF 90: 10 solution. [NaCl] = 0.1

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M, T = 298 K, l = cm. The pH varies from 2.56 to 6.94 and the arrow indicates the spectral change

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D

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upon increasing the pH.

ACCEPTED MANUSCRIPT

(a) (b) (c) d'' dB

280

300

320

340

360

SC

B / mT

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260

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(d) (e)

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Figure 3 X-Band EPR spectra in a water: DMF 90:10 medium at pH 7. (a) 1; (b) 2; (c) 3; (d) simulation by using the parameters given in Table 2 for 2; (e) 4. [Tris-HCl] = 0.05 M, [NaCl] = 0.02

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M; T, 100 K; microwave frequency, 9.44 GHz; microwave power, 4 mW; mod. Freq.,100 KHz; mod.

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Amp. 0.4 mT.

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SC

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ACCEPTED MANUSCRIPT

Figure 4 Formula of the archetype [(HLarch)Cu(H2O)]2+ and geometry optimized structures for the archetypes: (a) [(HLarch)Cu(H2O)]2+ with an axial phenol; (b) [(HLarch)Cu(H2O)]2+ with an equatorial

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equatorial phenolate.

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phenol; (c) [(Larch)Cu(H2O)]+ with an axial phenolate (as initial guess); (d) [(Larch)Cu(H2O)]+ with an

ACCEPTED MANUSCRIPT

(a) (b)

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I/A (c)

-1.0

-0.5

0.0

0.5

1.0

SC

-1.5

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(d)

E/V

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Figure 5 Cyclic voltammetry curves in a water:DMF 90:10 medium at pH 7. (a) 1; (b) PyrPt; (c) 2; (d)

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3. [Tris-HCl] = 0.05 M, [NaCl] = 0.02 M; T, 298 K; scan rate, 0.1 V sec-1; The potentials are given

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CE

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D

versus the SCE reference.

ACCEPTED MANUSCRIPT

36000

32000

r/c

28000

24000

0.010

0.012

0.014

PT

20000

0.016

SC

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r

Figure 6 Scatchard – Von Hippel plot for the binding of complex 2 to CT DNA (from fluorescence

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CE

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D

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data). Water: DMF 90:10 medium, [Tris-HCl] = 0.05 M, [NaCl] = 0.02 M; T, 298 K.

450

600

(a)

500

Fluorescence / a.u.

Fluorescence (a. u.)

ACCEPTED MANUSCRIPT

400 300 200 100

(b) 400 350 300 250

600

650

700

750

0.0

800

 (nm)

0.2

0.4

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200 0 550

0.6

0.8

1.0

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[complex]/[EtBr]

Figure 7 Variation in the ethidium bromide (EtBr) fluorescence upon addition of complex 2; (a)

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Fluorescence spectra, the arrow indicates changes upon addition of 0-50 M of complex 2 in the

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presence of 20 M EtBr. Water: DMF 90:10 medium at pH 7; [Tris-HCl] = 0.05 M, [NaCl] = 0.02 M; T, 298 K;exc = 527 nm. (b) Plot of the fluorescence intensity as a function of the [complex]/[EtBr]

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D

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ratio.

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SC

RI

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ACCEPTED MANUSCRIPT

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Figure 8 Agarose gel electrophoresis patterns of the cleavage reaction of X174 supercoiled DNA (20 M base pairs) mediated by the copper complexes in a phosphate buffer 10 mM pH 7.2 (+ 10 %

D

DMF) at 37°C for 1 h, in the absence of reductant. Abbreviations: NC: nicked circular, SC:

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supercoiled. The concentrations of the complexes indicated on the top are in M. (a) 1; (b) 2; (c) 3; (d)

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4; (e) 3(Boc); (f) Expended view of the region 10-30 M in the case of 2.

SC

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ACCEPTED MANUSCRIPT

Figure 9 Agarose gel electrophoresis patterns of the cleavage of X174 supercoiled DNA (20 M

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base pairs) mediated by the copper complexes in a phosphate buffer 10 mM pH 7.2 (+ 10 % DMF) at 37°C for 1 h in the presence of reductant. [ascorbate] = 0.8 mM. Abbreviations: NC: nicked circular,

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SC: supercoiled, L: linear. The concentrations of the complexes indicated on the top are in M. (a) 2;

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(b) 3; (c) 4.

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ACCEPTED MANUSCRIPT

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Figure 10 Agarose gel electrophoresis patterns of supercoiled X174 DNA (20 M base pairs) incubated with the copper complexes in a phosphate buffer 10 mM pH 7.2 (+ 10 % DMF) at 37°C for

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1 h; [ascorbate] = 0.8 mM; (a) 1, 50 M; (b) 2, 8 M; (c) 3, 30 M. Lane 0, DNA control; lane 1, DNA + complex (without scavenger); lanes 2-10, DNA + complex in the presence of agents: lane 2,

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ethanol; lane 3, DMSO (2 L); lane 4, Superoxide Dismutase (0.5 unit); lane 5, NaN3 (100 M); lane

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6, EDTA (10 mM); lane 7, Catalase (0.1 unit); lane 8, Hoechst 33258 (100 M); lane 9, NaCl (350

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M).

ACCEPTED MANUSCRIPT

4% 8% 11% 20% 15% 19% 15%

80

31%

27%

28%

60

46%

25%

57% 40

88% 59%

58%

57%

20

23%

PT

%

32%

35%

44%

(f) (g)

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(a) (b) (c) (d) (e)

SC

0

RI

100

Figure 11 Flow cytometry analysis after 48 h treatment with the copper complexes. Colour code:

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Green, late apoptosis; red, early apoptosis; blue, viable cells (necrosis corresponds to less than 0.3 % of the cells). (a) Control; (b) 2, 2.5 M; (c) 2, 3.75 M; (d) 4, 2 M; (e) 4, 3 M; (f) 1, 5 M; (g) 1,

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7.5 M.

ACCEPTED MANUSCRIPT

100

25% 80 60

24%

15%

15%

13%

12%

72%

73%

(c)

(d)

29%

23%

40

51%

47%

(a)

(b)

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20

PT

%

NU

SC

0

Figure 12 Cell distributions after 24h treatment of RT112 cells with the copper complexes at a

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concentration corresponding to the IC50. (a) Control without complex; (b) treatment with 1; (c) treatment with 2; (d) treatment with 4. Colour code: Green, phases G2, M; Red, phase S; Blue, phases

AC

CE

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D

G0, G1.

ACCEPTED MANUSCRIPT

Copper(II) complexes of N3O tripodal ligands appended with pyrene and polyamine groups: Antiproliferative and nuclease activities Doti Serre, Sule Erbek, Nathalie Berthet, Christian Philouze, Xavier Ronot, Véronique Martel-Frachet and Fabrice Thomas*

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The pyrene and polyamine groups enhance the affinity of the copper complexes towards DNA. The complexes exhibit a high nuclease activity in the presence of reductant, and show a much greater anti-proliferative activity than cis-platin towards bladder cancer cells.

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Graphical abstract