Ni(II) complexes with non-steroidal anti-inflammatory drug diclofenac: Structure and interaction with DNA and albumins

Accepted Manuscript Ni(II) complexes with non-steroidal anti-inflammatory drug diclofenac: Struc‐ ture and interaction with DNA and albumins Myrto Kyropoulou, Catherine P. Raptopoulou, Vassilis Psycharis, G. Psomas PII: DOI: Reference:

S0277-5387(13)00428-2 http://dx.doi.org/10.1016/j.poly.2013.05.043 POLY 10162

To appear in:

Polyhedron

Received Date: Accepted Date:

11 April 2013 22 May 2013

Please cite this article as: M. Kyropoulou, C.P. Raptopoulou, V. Psycharis, G. Psomas, Ni(II) complexes with nonsteroidal anti-inflammatory drug diclofenac: Structure and interaction with DNA and albumins, Polyhedron (2013), doi: http://dx.doi.org/10.1016/j.poly.2013.05.043

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ni(II) complexes with non-steroidal anti-inflammatory drug diclofenac: Structure and interaction with DNA and albumins Myrto Kyropoulou1, Catherine P. Raptopoulou2, Vassilis Psycharis2, G. Psomas1,* 1

Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of

Thessaloniki, GR-54124 Thessaloniki, Greece. 2

Institute of Advanced Materials, Physicochemical Processes, Nanotechnology & Microsystems,

Department of Materials Science, NCSR “Demokritos”, GR-15310 Aghia Paraskevi Attikis, Greece. Abstract The interaction of nickel(II) with the non-steroidal anti-inflammatory drug sodium diclofenac (Nadicl) in the presence of the N,N'-donor heterocyclic ligands 2,2'-dipyridylketone oxime (Hpko), 2,2'-bipyridine (bipy) or 1,10-phenanthroline (phen) leads to the formation of mononuclear Ni(II) complexes. The crystal structure of [Ni(dicl)(Hdicl)(Hpko)2](dicl)·CH3OH·0.6H2O (1·CH3OH·0.6H2O) has been determined by X-ray crystallography. The interaction of the complexes with human or bovine serum albumins has been studied by fluorescence spectroscopy revealing their good binding affinity to the albumins with high binding constant values. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes can bind to CT DNA with [Ni(dicl)(Hdicl)(Hpko)2](dicl) exhibiting the highest binding constant to CT DNA. Complex 1 can bind to CT DNA via intercalation as concluded by studying its cyclic voltammograms in the presence of CT DNA solution and by DNA solution viscosity measurements, while for complexes [Ni(dicl)2(bipy)] (2) and [Ni(dicl)2(phen)] (3) a non-classic intercalative mode has been concluded. Competitive studies of the complexes with ethidium bromide (EB) have shown their moderate to significant ability can displace the DNA-bound EB suggesting a competition with EB. Keywords: nickel complexes; non-steroidal anti-inflammatory drugs; diclofenac; interaction with DNA; interaction with serum albumins 1. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most frequently used medical drugs as analgesics, anti-inflammatories and antipyretics [1]. NSAIDs act by inhibiting the *

Tel.:+30+2310997790; Fax:+30+2310997738; E-mail: [email protected] 1

production of prostaglandins which is mediated by cyclo-oxygenase (COX) [2]. NSAIDs present antitumorigenic properties attributed to COX-independent mechanisms [3,4], to apoptosis via an activation of caspases [5] or via an unknown molecular mechanism where free radical may be involved [6]. Thus, the interaction of NSAIDs and their complexes with DNA should be considered of great importance and further evaluated as a means to the potential anticancer activity since few relevant reports on the interaction of NSAIDs and their complexes with DNA have been published so far [7,8]. The chemical classes of NSAIDs comprise salicylate derivatives, phenylalkanoic acids, oxicams, anthranilic acids, sulfonamides and furanones [9]. Sodium diclofenac (=Nadicl, Scheme 1) is a potent NSAID of the phenylalkanoic acids’ group exhibiting favourable anti-inflammatory, analgesic and antipyretic properties [10]. It is mainly used in painful and inflammation conditions like rheumatoid arthritis, spondilytis, and osteoarthritis. This drug inhibits COX activity in vitro with no significant effect on phospholipase A2 or on lipoxygenase enzymes [11]. In the literature, the crystal structures of three copper(II) [12-14], a cadmium(II) [15] and a tin(IV) [16] complexes with diclofenac ligands have been found. The discovery in 1975 that urease is a nickel enzyme [17] was the first evidence of the important role of nickel in biological systems. Since then, the role of Ni has been largely expanded, not only due to the determination and the significant increase of the number of nickel-dependent or nickel-containing enzymes [18,19], but also because of the plethora of reported nickel complexes showing biological activity. Nickel complexes have been reported to act as anticonvulsant [20], antiepileptic [21] agents or vitamins [22]; they have also presented antibacterial [23,24], antifungal [24,25], antimicrobial [26,27], antioxidant [28] and antiproliferative/anticancer [29,30] activities. Furthermore, the interaction of Ni(II) complexes with biomolecules such as serum albumins [27,3133] or DNA [31-36] has been recently studied revealing that the binding to these molecules and its mode is mainly dependent on the structure of the ligand [37]. A thorough survey of the literature concerning Ni(II) complexes with NSAIDs has revealed few reports [38-41] describing the isolation of such complexes without the presentation of any crystal structure. Given the increasing biological interest in metal complexes and especially nickel compounds as well the significance of NSAIDs in medicine, we report the synthesis, the structural and spectroscopic characterization and the biological properties of nickel(II) complexes with the NSAID sodium diclofenac in the presence of a N,N'-donor heterocyclic ligand such as 2,2'dipyridylketone oxime (=Hpko), 2,2'-bipyridine (=bipy) or 1,10-phenanthroline (=phen). The interaction of Ni(II) with Nadicl in the presence of Hpko leads to the formation of complex [Ni(dicl)(Hdicl)(Hpko)2](dicl)·CH3OH·0.6H2O (=1·CH3OH·0.6H2O) that has been structurally characterized by X-ray crystallography, while in the presence of bipy or phen complexes 2

[Ni(dicl)2(bipy)] (=2) or [Ni(dicl)2(phen)] (=3) have been isolated, respectively. The complexes have been characterized with physicochemical and spectroscopic techniques and their electrochemical behavior has been also investigated. In order to investigate the possibility of existence of any potential biological activity of complexes 1-3, the study of the biological properties of the complexes has been focused on (i) their binding properties with CT DNA investigated by UV spectroscopy, cyclic voltammetry and viscosity measurements, (ii) the ability to displace ethidium bromide (=EB) from the EB-DNA complex as a means to clarify the existence of a potential intercalation of the complexes to CT DNA in competition to the classical DNA-intercalator EB performed by fluorescence spectroscopy and (iii) the affinity of the complexes to bovine (=BSA) and human serum albumin (=HSA) - binding to these proteins involved in the transport of metal ions and metal-drug complexes through the blood stream may reveal lower or enhanced biological properties of the original drug, or new paths for drug transportation [42] - investigated by fluorescence spectroscopy. 2. Experimental 2.1 Materials - Instrumentation - Physical measurements The chemical reagents NiCl2⋅6H2O, sodium diclofenac, phen, bipy, Hpko, KOH, CT DNA, BSA, HSA, EB, TEAP, NaCl and trisodium citrate were purchased from Sigma-Aldrich Co and all solvents were purchased from Merck. All chemicals and solvents were reagent grade and were used as purchased without any further purification. TEAP was recrystallized twice from ethanol, prior to its use, and dried under vacuum. DNA stock solution was prepared by dilution of CT DNA to buffer (containing 15 mM trisodium citrate and 150 mM NaCl at pH 7.0) followed by exhaustive stirring for three days, and kept at 4°C for no longer than a week. The stock solution of CT DNA gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) of 1.87, indicating that the DNA was sufficiently free of protein contamination. The DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M-1cm-1 [12]. Infrared (IR) spectra (400-4000 cm-1) were recorded on a Nicolet FT-IR 6700 spectrometer with samples prepared as KBr disk. UV-visible (UV-vis) spectra were recorded as nujol mulls and in solution at concentrations in the range 10-5 - 10-3 M on a Hitachi U-2001 dual beam spectrophotometer. Room temperature magnetic measurements were carried out by the Faraday method. C, H and N elemental analysis were performed on a Perkin-Elmer 240B elemental analyzer. Molar conductivity measurements were carried out with a Crison Basic 30 conductometer. Fluorescence spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer. 3

Cyclic voltammetry studies were performed on an Eco chemie Autolab Electrochemical analyzer. Cyclic voltammetry experiments were carried out in a 30 mL three-electrode electrolytic cell. The working electrode was platinum disk, a separate Pt single-sheet electrode was used as the counter electrode and a Ag/AgCl electrode saturated with KCl was used as the reference electrode. The cyclic voltammograms of the complexes were recorded in 0.4 mM DMSO solutions and in 0.4 mM 1/2 DMSO/buffer solutions at ν = 100 mV s-1 where TEAP and the buffer solution were the supporting electrolytes, respectively. Oxygen was removed by purging the solutions with pure nitrogen which had been previously saturated with solvent vapours. All electrochemical measurements were performed at 25.0 ± 0.2°C. 2.2. Synthesis of the complexes 2.2.1 [Ni(dicl)(Hdicl)(Hpko)2](dicl)·CH3OH·0.6H2O, 1·CH3OH·0.6H2O A methanolic solution (10 mL) of Nadicl (126 mg, 0.4 mmol) and a methanolic solution (5 mL) of Hpko (79 mg, 0.4 mmol) were added simultaneously and dropwise in a methanolic solution (10 mL) of NiCl2·6H2O (47 mg, 0.2 mmol). The resultant solution was stirred for 5 min and left for slow evaporation. Light-green crystals of [Ni(dicl)(Hdicl)(Hpko)2](dicl)·CH3OH·0.6H2O, 1·CH3OH·0.6H2O, suitable for X-ray structure determination were collected after a couple of days. Yield: 180 mg, 65 %. Anal. Calcd. for [Ni(dicl)(Hdicl)(Hpko)2](dicl)·CH3OH·0.6H2O (C65H54.2Cl6N9NiO9.6) (MW = 1386.38): C 56.31, H 3.94, N 9.09; found C 56.25, H 3.79, N 8.85. IR (KBr disk): sym(CO2):

max,

cm-1; (C=O)carboxylic: 1708 (very strong (vs));

asym(CO2):

1591 (vs);

1387 (vs); (C-O)carboxylic: 1281 (s); ∆ = νasym(CO2) - νsym(CO2) = 204 cm-1; UV-vis: λ,

nm (ε, M-1 cm-1) as nujol mull: 975, 575, 398 (sh), 350, 304(sh); in DMSO: 960 (13), 590 (160), 405(sh) (375), 346 (1500), 307 (1900); 10Dq = 10415 cm-1, B = 693 cm-1. µeff = 2.87 BM. The complex is soluble in DMSO (ΛM = 20 mho cm2 mol-1, in 1 mM DMSO) and partially soluble in DMF, ethanol and chloroform. 2.2.2 [Ni(dicl)2(B)] (B = bipy for 2, phen for 3) Complexes 2 and 3 have been prepared in a similar way to complex 1 with the use of the corresponding N,N’-donor ligand (B), bipy (0.2 mmol, 31 mg) for 2 and phen (0.2 mmol, 36 mg) for 3. Pale green microcrystalline product of [Ni(dicl)2(bipy)] and [Ni(dicl)2(phen)], respectively, was collected after a few days. Data for 2: Yield: 120 mg, 75 %. Anal. Calcd. for [Ni(dicl)2(bipy)] (C40H28Cl4N4NiO4) (MW = 805.19): C 56.69, H 3.51, N 6.96; found C 56.51, H 3.38, N 6.77. IR (KBr disk): asym(CO2):

1576 (vs);

sym(CO2):

max,

cm-1;

1423 (vs); ∆ = 153 cm-1; UV-vis: λ, nm (ε, M-1 cm-1) as nujol 4

mull: 1010, 605, 390 (sh)), 315(sh), 295; in DMSO: 995 (15), 609 (25), 395(sh) (160), 310(sh) (1800), 296 (6500); 10Dq = 10050 cm-1, B = 772 cm-1. µeff = 3.01 BM. The complex is soluble in ethanol and DMSO (ΛM = 8 mho cm2 mol-1, in 1 mM DMSO) and partially soluble in DMF. Data for 3: Yield: 115 mg, 70 %. Anal. Calcd. for [Ni(dicl)2(phen)] (C40H28Cl4N4NiO4) (MW = 829.21): C 57.94, H 3.40, N 6.76; found C 58.25, H 3.49, N 6.95. IR (KBr disk): asym(CO2):

1575 (vs);

sym(CO2):

max,

cm-1;

1425 (vs); ∆ = 150 cm-1; UV-vis: λ, nm (ε, M-1 cm-1) as nujol

mull: 1005, 601, 391 (sh), 291; in DMSO: 985 (10), 595 (20), 397(sh) (240), 294 (sh) (8500); 10Dq = 10151 cm-1, B = 769 cm-1. µeff = 2.96 BM. The complex is soluble in DMSO (ΛM = 6.5 mho cm2 mol-1, in 1 mM DMSO) and partially soluble in DMF, ethanol, acetonitrile, dichloromethane and chloroform. 2.3 DNA- and albumin-binding studies The interaction of complexes 1-3 with CT DNA has been studied with UV spectroscopy in order to investigate the possible binding modes to CT DNA and to calculate the binding constants to CT DNA (Kb). In UV titration experiments, the spectra of CT DNA in the presence of each compound have been recorded for a constant CT DNA concentration in diverse [compound] / [CT DNA] mixing ratios (r). The binding constants, Kb, of the compounds with CT DNA have been determined using the UV spectra of the compound recorded for a constant concentration in the absence or presence of CT DNA for diverse r values. Control experiments with DMSO were performed and no changes in the spectra of CT DNA were observed. Viscosity experiments were carried out using an ALPHA L Fungilab rotational viscometer equipped with an 18 mL LCP spindle and the measurements were performed at 100 rpm. The viscosity of a DNA solution has been measured in the presence of increasing amounts of complexes 1-3. The relation between the relative solution viscosity (η/η0) and DNA length (L/L0) is given by the equation L/L0 = (η/η0)1/3, where L0 denotes the apparent molecular length in the absence of the compound. The obtained data are presented as (η/η0)1/3 versus r, where η is the viscosity of DNA in the presence of complex, and η0 is the viscosity of DNA alone in buffer solution. The interaction of complexes 1-3 with CT DNA has been also investigated by monitoring the changes observed in the cyclic voltammogram of a 0.40 mM 1:2 DMSO:buffer solution of complex upon addition of CT DNA at diverse r values. The buffer was also used as the supporting electrolyte and the cyclic voltammograms were recorded at ν = 100 mV s-1. The competitive studies of each compound with EB have been investigated with fluorescence spectroscopy in order to examine whether the compound can displace EB from its CT DNA-EB complex. The CT DNA-EB complex was prepared by adding 20 µM EB and 26 µM CT 5

DNA in buffer (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). The intercalating effect of complexes 1-3 with the DNA-EB complex was studied by adding a certain amount of a solution of the compound step by step into the solution of the DNA-EB complex. The influence of the addition of each compound to the DNA-EB complex solution has been obtained by recording the variation of fluorescence emission spectra. The protein binding study was performed by tryptophan fluorescence quenching experiments using bovine (BSA, 3 µM) or human serum albumin (HSA, 3 µM) in buffer (containing 15 mM trisodium citrate and 150 mM NaCl at pH 7.0). The quenching of the emission intensity of tryptophan residues of BSA at 343 nm or HSA at 351 nm was monitored using complexes 1-3 as quenchers with increasing concentration [43]. Fluorescence spectra were recorded in the range 300-500 nm at an excitation wavelength of 296 nm. The fluorescence spectra of the complexes were recorded under the same experimental conditions and a maximum emission appeared at 365 nm. Therefore, the quantitative studies of the serum albumin fluorescence spectra were performed after their correction by subtracting the spectra of the compounds. 2.4 X-ray structure determination A crystal with approximate dimensions 0.15 × 0.26 × 0.39 mm was taken from the mother liquor and immediately cooled to -113°C. Diffraction measurements were made on a Rigaku RAXIS SPIDER Image Plate diffractometer using graphite monochromated Cu Kα radiation. Data collection (ω-scans) and processing (cell refinement, data reduction and Empirical absorption correction) were performed using the CrystalClear program package [44]. Important crystallographic data are listed in Table 1. The structure was solved by direct methods using SHELXS-97 [45] and refined by full-matrix least-squares techniques on F2 with SHELXL-97 [46]. Further experimental crystallographic details for 1·CH3OH·0.6H2O: 2θmax = 130 °; reflections collected/unique/used, 42119/10462 [Rint = 0.0618]/10462; 824 parameters refined; (∆/σ)max = 0.001; (∆ρ)max/(∆ρ)min = 0.997/-0.655 e/Å3; R1/wR2 (for all data), 0.0866/0.1823. All hydrogen atoms were introduced at calculated positions as riding on bonded atoms. All non-hydrogen atoms were refined anisotropically, except of the water solvate molecules which were refined isotropically with occupation factors fixed to 0.30, and no hydrogen atoms included in the refinement. 3. Results and discussion 3.1 Synthesis and spectroscopic study The synthesis of the complexes has been achieved in high yield via the aerobic reaction of NiCl2·6H2O with Nadicl in the presence of the corresponding N-donor ligand (Hpko, bipy, phen). 6

The resultant complexes are soluble mainly in DMSO and partially soluble in other organic solvents (DMF, ethanol and chloroform). In the IR spectrum of Nadicl, the bands at 1575(s) cm-1 and 1399(s) cm-1 [12] attributed to antisymmetric, νasym(C=O), and symmetric, νsym(C=O), stretching vibrations, respectively, have shifted in the IR spectra of the complexes to 1575-1591 cm-1 and 1387-1425 cm-1, respectively. More specifically, in the spectrum of 1 the bands atttibuted to νasym(C=O) and νsym(C=O) are located at 1591 cm-1 and 1387 cm-1, respectively, and the difference ∆ (= νasym(C=O) - νsym(C=O), a useful characteristic tool for determining the coordination mode of the carboxylato ligands [43]) has a value of 204 cm-1 which suggests a monodentate binding mode for the carboxylato groups of diclofenac. Additionally, the existence of bands appearing at 1708 (vs) cm-1 and 1281 (s (strong)) cm-1 which can be attributed to the stretching vibrations ν(C=O)carboxylic and ν(C-O)carboxylic of the carboxylic moiety (-COOH), respectively, may provide evidence of the co-existence of a neutral diclofenac as Hdicl in the complex. In the case of complexes 2 and 3, only the νasym(C=O) and νsym(C=O) stretching vibrations are observed at 1575-1576 cm-1 and 1423-1425 cm-1, respectively, providing a ∆ value within the range 150-153 cm-1 that is indicative of a bidentate chelating coordination mode [47] for the diclofenac carboxylato groups. The UV-vis spectra of the complexes have been recorded as nujol mull and in dmso solution and have shows similar pattern suggesting that the complexes retain their structure in solution. In the visible region, three low-intensity bands are observed in the region 960-995 nm (band I), 590609 nm (band II) and 395-405 nm (band III) nm assigned to d-d transitions, typical for distorted octahedral Ni2+ complexes [19,27,28]. For octahedral local symmetry, band I is attributed to a 3

A2gÆ3T2g electronic transition, band II to a 3A2gÆ3T1g electronic transition and band III to a

3

A2gÆ3T1g(P) electronic transition. The values of the ratio 10Dq/B (=13.0-15.0) are within the

range expected for octahedral Ni2+ complexes [27,28]. The fact that the complexes either dissociate partially (complex 1) or are non-electrolytes (complexes 2 and 3) in DMSO solution (ΛM = 6.5-20 mho cm2 mol-1, in 1 mM DMSO) and they have the same UV-Vis spectral pattern in nujol and in DMSO solution as well as in the presence of the buffer solution used in the biological experiments (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) suggests that the compounds keep their integrity in solution [48]. The observed from the magnetic measurements values of µeff (= 2.87-3.01 BM) for the complexes are typical for paramagnetic octahedral Ni(II) complexes with d8 configuration and are close to the spin-only value µeff (= 2.83 BM) at room temperature for a magnetically isolated Ni(II) system [19,27,28]. 3.2 Structure of the complexes 7

3.2.1 Crystal structure of [Ni(dicl)(Hdicl)(Hpko)2](dicl)·CH3OH·0.6H2O Compound 1 is a mononuclear cationic complex of Ni (Figure 1) where the cationic unit [Ni(dicl)(Hdicl)(Hpko)2]+ is neutralized by an anion of deprotonated diclofenac. The molecular structure of the cation [Ni(dicl)(Hdicl)(Hpko)2]+ as well as two lattice dicl- ligands appears in Figure 1, and selected bond distances and angles are given in Table 2. It should be noted that the crystal structure of 1 is the first crystal structure reported of a Ni(II) complex with NSAID as a ligand. In the cationic unit of 1, the nickel atom, Ni(1), is six-coordinate lying in a distorted octahedral environment formed by four nitrogens of two Hpko ligands and two oxygens of two diclofenac ligands. The bond distances around the nickel atom are close but not equal, with the coordinated carboxylate oxygens (Ni(1)-O(21) = 2.037(2) Å and Ni(1)-O(31) = 2.045(2) Å) lying closer to Ni than the Hpko nitrogens (Ni(1)-N(1) = 2.065(3)Å, Ni(1)-N(2) = 2.085(3) Å, Ni(1)N(11) = 2.070(3) Å and Ni(1)-N(12) = 2.069(3)Å). The two coordinate diclofenac ligands are lying in cis positions and are found in different modes; one diclofenac ligand is deprotonated and is bound to Ni(1) via carboxylate oxygen O(21) in a symmetric fashion (C(41)-O(21) =1.267(4) Å and C(41)=O(22) = 1.259(4) Å); the second diclofenac is a neutral ligand and is monodentately bound to nickel in asymmetric fashion (C(61)O(31) = 1.272(4) Å and C(61)-O(32)= 1.248(4) Å) via O(31) where the hydrogen atom is also located. The Hpko ligands act as bidentate neutral chelators and are coordinated to nickel via the pyridine nitrogen and the oxime nitrogen with ketonoxime oxygen and the second pyridine nitrogen remaining unbound. This coordination mode of Hpko ligand (1.0110 according to the Harris notation [49]) has been observed in a series of mononuclear metal complexes [50] and in the trinuclear Ni(II) complex [Ni3(shi)2(Hpko)2(py)2] (where H3shi = salicylhydroxamic acid and py = pyridine) [23]. The two coordinated Hpko and the two diclofenac ligands are held together through hydrogen bonds and weak - stacking interactions. In particular the intra-molecular O(1)···O(32) and O(11)···O(22) hydrogen bonds (Table 3) are developed between the protonated ketonoxime oxygen atom of Hpko and one of the carboxylato oxygen atoms of diclofenac. The - stacking interactions are developed between the coordinated pyridine ring of Hpko and one of the phenyl rings of diclofenac; specifically - stacking is developed between the pyridine ring defined by atoms N(1)-C(5) of Hpko and the phenyl ring defined by atoms C(43)-C(48) of diclofenac (the average interplanar distance between the two rings which form an angle of 9.7o is 3.985 Å) and 8

between the ring defined by N(11)-C(25) and C(63)-C(68) (the average interplanar distance between the two rings which form an angle of 11.8o is 3.858 Å). Intra-molecular hydrogen bonds are also developed between the NH group of the diclofenac ligands and their carboxylato oxygen atoms (Table 3). The lattice diclofenac anion reveals all the possible weak interactions which is possible to develop with neighobouring molecules resulting in an interesting supramolecular chain structure (Figure 2). Two lattice diclofenac anions are linked to two complex cations through hydrogen bonds developed between the carboxylato oxygen atoms O(31) and O(42) (Table 2) and chalogen bond interactions between Cl(5) and the ketonoxime oxygens O(11) (Cl(5)···O(11) = 3.063Å) thus forming {[Ni(dicl)(Hdicl)(Hpko)2](dicl)}2 tetramers. Both lattice diclofenac anions participating in the tetramer formation interact weakly through - stacking interactions developed between the chloro-substituted phenyl rings defined by atoms C(89)-C(94) belonging to centrosymmetrically related anions of lattice diclofenac (the average interplanar distance between the two parallel rings is 3.560 Å) of neighboring tetramer which results in the formation of chains parallel to the a crystallograpfic axis. MeOH solvate molecules through hydrogen bond O(1m)···O(21) (Table 3) are linked to the neutral coordinated diclofenac molecule. The chalogen bond value of 3.063 Å (with N(12)-O(11)···Cl(5) = 155.1°) mentioned above is close to the short range of values taken by this type of bonds [51-54]. This type of weak interactions of diclofenac molecule has been also discussed in relation to its interactions with small proteins [55] such as lactoferrin [56], transthyretin [57] and COX-2 [58]. 3.2.2 Proposed structures for complexes 2 and 3. Based on IR, UV-Vis and magnetic measurements data, complexes 2 and 3 are neutral mononuclear complexes with the diclofenac ligands behaving as deprotonated bidentate ligands bound to nickel ion via the carboxylate oxygen atoms in a chelating mode. The UV-Vis spectra suggest a distorted octahedral environment. The octahedron is formed by four oxygen atoms of the diclofenac ligands and two nitrogen atoms provided by a 2,2′-bipyridine or a 1,10-phenanthroline ligand. Based on the magnetic data µeff (= 2.96-3.01 BM), a square planar geometry may be ruled out - in such a case a diamagnetic behaviour for a d8 electronic configuration system should be expected- while based on the close to spin-only µeff values, a tetrahedral coordination environment may be also excluded - a spin-orbit coupling increasing the µeff value for Ni(II) complexes is predicted [18,19]. Similar structures have been reported for a series of Cu(II) complexes with carboxylate NSAIDs as ligands [12,48,59]. 9

3.3 Binding of the complexes to serum albumin proteins Serum albumin (SA) is the most abundant protein in plasma. SA is mainly involved in the transport of metal ions, drugs and their metal complexes through the blood stream [42]. Human serum albumin (HSA) has a tryptophan located at position 214, while bovine serum albumin (BSA), the most extensively studied serum albumin due to its structural homology with HSA, has two tryptophans at positions 134 and 212 [60]. BSA and HSA solutions exhibit an intense fluorescence emission with λem,max = 343 nm and 351 nm, respectively, due to the tryptophan residues, when excited at 295 nm [43]. Complexes 1-3 exhibit a maximum emission at 365 nm under the same experimental conditions. Therefore, the quantitative studies of the BSA or HSA fluorescence emission spectra were performed after their correction by subtracting the spectra of the compounds Addition of complexes 1-3 to a SA solution results in moderate to significant quenching of BSA fluorescence at λ = 343 nm (Figure 3(A)) (quenching up to 93% of the initial fluorescence intensity for 2) and to a less pronounced quenching of the HSA fluorescence at λ = 351 nm (Figure 3(B)) (quenching up to 73% of the initial fluorescence intensity for 2). The observed quenching may be due to possible changes in protein secondary structure leading to changes in tryptophan environment of BSA or HSA, and thus indicating the binding of each complex to the albumins [61]. The Stern-Volmer and Scatchard equations and graphs may be used in order to study the interaction of a quencher with serum albumins. From Stern-Volmer quenching equation [43]: Io = 1 + k q t 0 [Q] = 1 + K SV [Q] I

(eq. 1),

where Io = the initial tryptophan fluorescence intensity of SA, I = the tryptophan fluorescence intensity of SA after the addition of the quencher (i.e. complexes 1-3), kq = the quenching rate constants of SA, KSV = the dynamic quenching constant, τo = the average lifetime of SA without the quencher and [Q] = the concentration of the quencher, the dynamic quenching constant (KSV, M-1) can be obtained by the slope of the diagram

Io vs [Q] (figures S1 and S2). From the equation KSV I

= kqτo and taking as fluorescence lifetime (τo) of tryptophan in SA at ~10-8 s [62], the approximate quenching constant (kq, M-1 s-1) may also be calculated. The calculated values of Ksv and kq for the interaction of the complexes with BSA and HSA are given in Table 4 and suggest good binding propensity of the complexes with 2 exhibiting the highest SA quenching ability (kq(BSA) = 7.35(±0.56) x 1013 M-1s-1 and kq(HSA) = 1.26(±0.13) x 1013 M-1s-1). The kq values (>1012 M-1s-1) of all complexes are higher than diverse kinds of quenchers for biopolymers fluorescence (~ 2.0 x 1010 M1 -1

s ) indicating the existence of static quenching mechanism [60]. 10

From the Scatchard equation [61]: ∆I

Io = nK K ∆I [Q] Io

(eq. 2)

where n is the number of binding sites per albumin and K is the association binding constant, K (M−1) may be calculated from the slope in plots

∆I

Io versus ∆I (Figures S3 and S4) and n is given [Q] Io

by the ratio of y intercept to the slope [61]. The results concerning the K and n values are given in Table 4, showing complex 2 exhibits the highest K value for BSA and complex 3 for HSA. Additionally, the n value of Nadicl decreases for BSA in the cases of the complexes and increases for HSA when coordinated to Ni(II). In conclusion, the complexes exhibit higher K values for HSA than for BSA. Furthermore, the quenching of SA fluorescence provoked by complexes 1-3 is more pronounced than their Cu(II) analogues [12] and, subsequently, the corresponding quenching constants (kq) of 1-3 are higher than those reported for Cu(II) diclofenac complexes for both albumins. Additionally, complexes 2-3 exhibit the highest association binding constant values among the metal diclofenac complexes reported. In general, the binding constant of a compound to a protein such as an albumin should be at an optimum range; (a) high enough to allow binding and possible transfer by the protein and (b) not too high to get released upon arrival at its target. Bearing that in mind, the K values of all complexes 1-3 may be considered to be within such a range; high enough (2.54x104 - 1.31x106 M-1) to allow the binding of the complexes to SAs and also significantly below the association constant of the one of strongest known non-covalent bonds for the interaction between avidin and ligands (K ≈ 1015 M-1), suggesting a possible release from the serum albumin to the target cells [61]. 3.4 Interaction of the complexes with calf-thymus DNA The potential anticancer and the anti-inflammatory activity of the NSAIDs and their complexes are often related to their ability to interact with DNA [7,8]. Within this context, the number of such studies so far is considered limited; complexes of oxicams can bind to DNA via intercalation [8], while the interaction of DNA with a series copper(II) complexes with phenylalkanoic acids naproxen and diclofenac, the anthralinic acid derivatives mefenamic acid flufenamic acid and the salicylate derivative diflunisal as well as with cobalt(II) naproxenato or mefenamato complexes has been recently reported [12,48,59,63-67]. In general, the binding of transition metal complexes to DNA takes place via a covalent (a labile ligand of the complex is replaced by a nitrogen base of DNA, e.g. guanine N7) and/or a noncovalent (intercalation, electrostatic or groove binding) interaction [12,48]. 11

3.4.1 DNA-binding study with UV spectroscopy The UV spectra have been recorded for a constant CT DNA concentration in different [complex]/[DNA] mixing ratios (r). UV spectra of CT DNA in the presence of a complex derived for diverse r values (up to 0.3) are shown representatively for 3 in Figure 4. Complexes 1-3 exhibit similar behavior upon their addition on CT DNA solution. The decrease of the intensity at λmax = 258 nm is accompanied with a red-shift of the λmax up to 261 nm for all compounds indicating that the interaction with CT DNA results in the direct formation of a new complex with double-helical CT DNA [68]. The observed hypochromism may be attributed to π Æ π* stacking interaction between the aromatic chromophore of the complexes and DNA base pairs consistent with the intercalative binding mode [69], while the red-shift (bathochromism) may be considered an evidence of stabilization of CT DNA duplex [70]. In the UV region of the spectra of the complexes (Figure 5), the intense absorption bands observed in the spectra of the complexes can be attributed to the intraligand transition of the coordinated groups of the NSAID ligands [12,63-67]. The existence and the possible mode of interaction between each complex and CT DNA may be revealed by the changes in the intraligand centered spectral transitions of complexes 1-3 upon addition of CT DNA solution in diverse r values. In the UV spectrum of 1 (Figure 5(A)), the bands centered at 350 nm (band I) and 306 nm (band II) exhibit in the presence of increasing amounts of CT DNA (up to 1/r = 2) a significant hypochromism up to 40% and 25%, respectively. The observed hypochromic effect may suggest tight binding to CT DNA probably by intercalation. In the UV spectrum of 2 (Figure 5(B)), a significant hyperchromism of the band at 295 nm is observed suggesting tight binding probably by external interaction. For complex 3, a slight hyperchromism may be also observed. Although the exact mode of binding cannot be merely proposed by UV spectroscopic titration studies, the results collected from the UV titration experiments suggest that all compounds can bind to CT DNA [71]. The observed hypochromic effect may be considered as first evidence of tight binding to CT DNA probably by intercalation for 1, while the significant hyperchromism observed for 2 may suggest that the possibility of intercalation between 2 and DNA should be ruled out [72]. The magnitude of the binding strength of compounds with CT DNA may be estimated through the calculation of the binding constant Kb, which can be obtained by monitoring the changes in the absorbance at the corresponding λmax with increasing concentrations of CT DNA. Kb 12

is given by the ratio of slope to the y intercept in plots

[DNA] versus [DNA] (Figure S5), (εA − εf )

according to the equation [69]:

1 [DNA] [DNA] + = (ε A ε f ) (ε b ε f ) K b (ε b ε f )

(eq. 3)

where [DNA] is the concentration of DNA in base pairs, εA = Aobsd/[complex], εf = the extinction coefficient for the free complex and εb = the extinction coefficient for the complex in the fully bound form. The values of Kb for the complexes, as calculated by eq. 3 and the plots in Figure S5, are given in Table 5.

The Kb values are moderate to high suggesting a strong binding of the compounds to CT DNA [12,48]. It is quite obvious that the coordination of diclofenac to Ni(II) results in a increase of the Kb value. Complex 1 exhibits the highest Kb value (3.63(±0.12)x105 M-1) among the compounds which is higher than that of the classical intercalator EB (Kb = 1.23(±0.07) x 105 M-1) [12,73]. A comparison to the corresponding Cu(II) diclofenac complexes will reveal that, in most cases, 1-3 exhibit higher Kb values than their Cu(II) analogues [12]. 3.4.2 Study of the DNA-binding with cyclic voltammetry

The investigation of metal-DNA interaction with electrochemical techniques such as cyclic voltammetry is considered as a complement to spectroscopic methods and it can provided useful information for both the reduced and oxidized form of the metal. The redox couple for each complex in 1/2 DMSO/buffer solution studied upon addition of CT DNA (up to 1/r = 2) as well as the shifts of the cathodic Epc and anodic Epa potentials attributed to the redox couple Ni(II)/Ni(I) are given in Table 6.

Complexes 1-3 exhibit the same electrochemical behavior upon addition of CT DNA (Table 6). No new redox peaks appeared after the addition of CT DNA to each complex; nevertheless, the observed decrease in current intensity, attributed to an equilibrium mixture of free and DNA-bound complex to the electrode surface, may suggest the existence of an interaction between each complex and CT DNA [12,48,74]. For increasing amounts of CT DNA, the cathodic potentials (Epc) exhibit a shift (∆Epc = (-10) - (+15) mV) and the anodic (Epa) potentials a positive shift (∆Epa = (+15) - (+85) mV); representatively, the cyclic voltammograms of complex 3 in the absence and presence of CT DNA are shown in Figure 6. 13

The electrochemical potential usually presents a positive shift, upon intercalation of the metal ion or complex to DNA, while, in the case of electrostatic interactions with DNA, the potential will shift to a negative direction. Additionally, in the case of more than one potentials, the positive shift of Ep1 accompanied by a negative shift of Ep2 may suggest that the binding of the molecule to DNA by both intercalation and electrostatic interaction [74,75]. Therefore, from the experimental data the existence of intercalation between the complexes and CT DNA bases may be suggested while in the case of complexes 2 and 3, the existence of electrostatic interactions may be also proposed [12,48,74]. 3.4.3 DNA-binding study with viscosity measurements

The measurement of the viscosity of DNA solution upon addition of a compound may provide significant aid to clarify the interaction mode of a compound with DNA, since it is sensitive to DNA length changes [12,63-65]. Viscosity measurements were carried out on CT DNA solutions (0.1 mM) upon addition of increasing amounts of the complexes (up to the value of r = 0.24). The relative viscosity of DNA solution exhibits a significant increase upon addition of complex 1 and remains practically unchanged upon addition of complexes 2 and 3 (Figure 7).

In the case of classic intercalative binding mode, the insertion of the compound in between the DNA base pairs results in an increase in the separation of base pairs at intercalation sites in order to host the bound compound; thus, the increase of the length of the DNA helix will be obvious through an increase of DNA viscosity, the magnitude of which is usually in accordance to the strength of the interaction. Furthermore, the binding of a compound to DNA grooves via a partial or non-classic intercalation (i.e. electrostatic interaction or external groove-binding) may provoke a bend or kink in the DNA helix and subsequently a shortening of its effective length; as a result, the viscosity of the DNA solution may show a slight decrease or may remain unchanged [63-65,76]. In conclusion, the behaviour of the DNA viscosity observed upon addition of the complexes may be considered an evidence of the existence of an intercalative binding mode between DNA and complex 1, and a non-classic intercalation between DNA and complexes 2 and 3 [63-65]; a conclusion which is in accordance to that derived from UV spectroscopic and electrochemical studies. 3.4.4 Competitive studies with ethidium bromide

Ethidium bromide (EB = 3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide) is a typical indicator of intercalation because it emits intense fluorescence in the presence of CT DNA as a result of strong intercalation of the planar EB phenanthridine ring between adjacent base pairs 14

on the double helix [77]. The changes observed in the fluorescence emission spectra of a solution containing the EB-DNA compound may be used to study the interaction between DNA and other compounds, such as metal complexes, since the addition of a compound that is capable to intercalate to DNA equally or more strongly than EB may result in a quenching of the EB-DNA fluorescence emission [78]. The emission spectra of EB bound to CT DNA in the absence and presence of each complex have been recorded for [EB] = 20 µM, [DNA] = 26 µM for increasing amounts of the complex (up to the value of r = 0.15) where the existence of EB is obvious at λem,max ~ 602 nm. The addition of complexes 1-3 at diverse r values results in a moderate to significant decrease of the intensity of the emission band of the DNA-EB system at 592 nm (the final fluorescence is up to 32-34% of the initial EB-DNA fluorescence intensity for all complexes) indicating the existence of a moderate competition of the complexes with EB in binding to DNA (Figure 8). The observed quenching of DNA-EB fluorescence from the complexes suggests that the complexes have a moderate ability to displace EB from the DNA-EB compound, and their interaction with CT DNA by the intercalative mode cannot be totally ruled out [48,63-65,74] although such strong evidence from the viscosity measurements only for complex 1.

The Stern-Volmer constant, KSV (in M-1), may be used to evaluate the quenching ability of each complex according to the equation (eq. 4): Io = 1 + K SV [Q ] I

(eq. 4)

where Io and I are the emission intensities in the absence and the presence of the quencher, respectively, [Q] is the concentration of the quencher (complexes 1-3). KSV is be obtained by the slope of the diagram

Io vs [Q] in Stern-Volmer plots of DNA-EB. The experimental data (Figure I

S6) indicate that the quenching of EB bound to DNA provoked by complexes 1-3 is in good agreement (R = 0.99) with the linear Stern-Volmer equation (eq. 4). The relatively high KSV (Table 7) values of complexes 1-3 show that they can bind tightly to the DNA [48,63-67] with complexes 2 and 3 exhibiting higher KSV values than free sodium diclofenac.

In general, the quenching of EB-DNA fluorescence provoked by complexes 1-3 is similar or higher than that of the corresponding Cu(II) diclofenac complexes and the KSV values of 2 and 3 especially are higher than their Cu(II) analogues [12]. 4. Conclusions

15

The synthesis and characterization of the mononuclear nickel(II) complex with the nonsteroidal anti-inflammatory drug sodium diclofenac in the presence of the N,N'-donor heterocyclic ligands 2,2'-dipyridylketone oxime, 2,2'-bipyridine or 1,10-phenanthroline has been achieved. The presence of Hpko results in a cationic nickel(II) complex of the formula [Ni(dicl)(Hdicl)(Hpko)2](dicl)·CH3OH·0.6H2O which is the first reported crystal structure of Ni(II) complex with a NSAID as ligand determined by X-ray Crystallography. In this complex, the coordinate monodentate diclofenac ligands are via bound a carboxylato oxygen atom lying in a deprotonated or a neutral mode, while the cationic unit [Ni(dicl)(Hdicl)(Hpko)2]+ is further neutralized by a diclofenac anion. The complexes show good quenching ability of the BSA and HSA fluorescence and tight binding affinity to these proteins giving relatively high binding constants. Their behavior towards serum albumins is more pronounced than their Cu(II) analogues previously studied [12]. UV spectroscopy studies have revealed the ability of the complexes to bind to CT DNA with [Ni(dicl)(Hdicl)(Hpko)2](dicl) exhibiting the highest Kb value among the complexes examined, which is higher than the Kb value of EB. Competitive binding studies with EB have revealed a moderate to significant ability of the complexes to displace the typical intercalator EB from the EBCT DNA complex. DNA viscosity measurements in combination with cyclic voltammetry studies have revealed an intercalative binding mode of complex 1 to CT DNA and the existence of a nonclassic intercalation between DNA and complexes 2 and 3. Furthermore, the DNA-binding properties of complexes 1-3 are more pronounced than their copper(II) analogues. 5. Abbreviations

bipy

2,2’-bipyridine

BSA

bovine serum albumin

COX

cyclo-oxygenase

CT

calf-thymus

DMF

N,N-dimethylformamide

EB

ethidium bromide, 3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide

HSA

human serum albumin

Nadicl

sodium diclofenac, sodium 2-(2,6-dichlorophenylamino)phenylacetate)

NSAID

non-steroidal anti-inflammatory drug

phen

1,10-phenathroline

s

strong

SA

serum albumin 16

sh

shoulder

TEAP

tetraethylammonium perchlorate

vs

very strong

∆

νasym(CO2) - νsym(CO2)

Appendix A. Supplementary data

CCDC-931523 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+44) 1223-336-033; or [email protected]). Supplementary data associated with this article can be found, in the online version, at doi: ……….

17

References

[1]

C.P. Duffy, C.J. Elliott, R.A. O'Connor, M.M. Heenan, S. Coyle, I.M. Cleary, K. Kavanagh,

S. Verhaegen, C.M. O'Loughlin, R. NicAmhlaoibh, M. Clynes, Eur. J. Cancer 34 (1998) 12501259. [2]

A.R. Amin, P. Vyas, M. Attur, J. Leszczynskapiziak, I.R. Patel, G. Weissmann, S.B.

Abramson, Proc. Natl. Acad. Sci. 92 (1995) 7926-7930. [3]

W.J. Wechter, E.D. Murray, D. Kantoci, D.D. Quiggle, D.D. Leipold, K.M. Gibson, J.D.

McCracken, Life Sci. 66 (2000) 745-753. [4]

M.L. Smith, G. Hawcroft, M.A. Hull, Eur. J. Cancer 36 (2000) 664-674.

[5]

L. Klampfer, J. Cammenga, H.G. Wisniewski, S.D. Nimer, Blood 93 (1999) 2386-2394.

[6]

A. Inoue, S. Muranaka, H. Fujita, T. Kanno, H. Tamai, K. Utsumi, Free Radic. Biol. Med.

37 (2004) 1290-1299. [7]

T. Zhang, T. Otevrel, Z.Q. Gao, Z.P. Gao, S.M. Ehrlich, J.Z. Fields, B.M. Boman, Cancer

Research 61 (2001) 8664-8667. [8]

S. Roy, R. Banerjee, M. Sarkar, J. Inorg. Biochem. 100 (2006) 1320-1331.

[9]

J.E. Weder, C.T. Dillon, T.W. Hambley, B.J. Kennedy, P.A. Lay, J.R. Biffin, H.L. Regtop,

N.M. Davies, Coord. Chem. Rev. 232 (2002) 95-126. [10]

S.B. Etcheverry, D.A. Barrio, A.M. Cortizo, P.A.M. Williams, J. Inorg. Biochem. 88 (2002)

94-100. [11]

J. Sharma, A.K. Singla, S. Dhawan, Int. J. Pharm. 260 (2003) 217-227.

[12]

F. Dimiza, F. Perdih, V. Tangoulis, I. Turel, D.P. Kessissoglou, G. Psomas, J. Inorg.

Biochem. 105 (2011) 476-489. [13]

D. Kovala-Demertzi, A. Theodorou, M.A. Demertzis, C.P. Raptopoulou, A. Terzis, J. Inorg.

Biochem. 65 (1997) 151-157. [14]

C. Castellari, G. Feroci, S. Ottani, Acta Cryst. Sect. C 55 (1999) 907-910.

[15]

D. Kovala-Demertzi, D. Mentzafos, A. Terzis, Polyhedron 12 (1993) 1361-1370.

[16]

N. Kourkoumelis, M.A. Demertzis, D. Kovala-Demertzi, A. Koutsodimou, A. Moukarika,

Spectrochimica Acta Part A 60 (2004) 2253-2259. [17]

N.E. Dixon, C. Gazzola, R.L. Blakeley, B. Zerner, J. Am. Chem. Soc. 97 (1975) 4131-4133.

[18]

F. Meyer, H. Kozlowski, in: J.A. McCleverty, T.J. Meyer (Eds.), Comprehensive

Coordination Chemistry II, vol. 6, Elsevier, 2003, pp. 247-554. [19]

R.K. Andrews, R.L;Blakeley, B. Zerner, B. in: H. Sigel, A. Sigel (Eds.), Metal Ions in

Biological Systems, vol. 23, Marcel Dekker Inc., New York, 1988, pp. 165-284. 18

[20]

G. Morgant, N. Bouhmaida, L. Balde, N.E. Ghermani, J. d'Angelo, Polyhedron 25 (2006)

2229-2235. [21]

P. Bombicz, E. Forizs, J.Madarasz, A. Deak, A. Kalman, Inorg. Chim. Acta 315 (2001) 229-

235. [22]

O.Z. Yesilel, M.S. Soylu, H. Olmez, O. Buyukgungor, Polyhedron 25 (2006) 2985-2992.

[23]

M. Alexiou, I. Tsivikas, C. Dendrinou-Samara, A.A. Pantazaki, P. Trikalitis, N. Lalioti,

D.A. Kyriakidis, D.P. Kessissoglou, J. Inorg. Biochem. 93 (2003) 256-264. [24]

R. Kurtaran, L.T. Yildirim, A.D. Azaz, H. Namli, O. Atakol, J. Inorg. Biochem. 99 (2005)

1937-1944. [25]

R. del Campo, J.J. Criado, E. Garcia, M.R. Hermosa, A. Jimenez-Sanchez, J.L. Manzano, E.

Monte, E. Rodriguez-Fernandez, F. Sanz, J. Inorg. Biochem. 89 (2002) 74-82. [26]

W. Luo, X. Meng, X. Sun, F. Xiao, J. Shen, Y. Zhou, G. Cheng, Z. Ji, Inorg. Chem.

Commun. 10 (2007) 1351-1354. [27]

K.C. Skyrianou, E.K. Efthimiadou, V. Psycharis, A. Terzis, D.P. Kessissoglou, G. Psomas,

J. Inorg. Biochem. 103 (2009) 1617-1625. [28]

K.C. Skyrianou, F. Perdih, A.N. Papadopoulos, I. Turel, D.P. Kessissoglou, G. Psomas, J.

Inorg. Biochem. 105 (2011) 1273-1285. [29]

Z. Afrasiabi, E. Sinn, W. Lin, Y. Ma, C. Campana, S. Padhye, J. Inorg. Biochem. 99 (2005)

1526-1531. [30]

A. Buschini, S. Pinelli, C. Pellakani, F. Giordani, F. Belicchi, F. Bisceglie,M. Giannetto, G.

Pelosi, P. Tarasconi, J. Inorg. Biochem. 103 (2009) 666-677. [31]

K.C. Skyrianou, F. Perdih, I. Turel, D.P. Kessissoglou, G. Psomas, J. Inorg. Biochem. 104

(2010) 161-170. [32]

K.C. Skyrianou, F. Perdih, I. Turel, D.P. Kessissoglou, G. Psomas, J. Inorg. Biochem. 104

(2010) 740-749. [33]

K.C. Skyrianou, V. Psycharis, C.P. Raptopoulou, D.P. Kessissoglou, G. Psomas, J. Inorg.

Biochem. 105(2011) 63-74. [34]

P.J. Cox, G. Psomas, C.A. Bolos, Bioorg. Med. Chem. 17 (2009) 6054-6062.

[35]

Y. Jin, M.A. Lewis, N.H. Gokhale, E.C. Long, J.A. Cowan, J. Am. Chem. Soc. 129 (2007)

8353-8361. [36]

G. Barone, N. Cambino, A. Ruggirello, A. Silvestri, A. Terenzi, V.T. Liveri, J. Inorg.

Biochem. 103 (2009) 731-737. [37]

G. Psomas, D.P. Kessissoglou, Dalton Trans. 42 (2013) 6252-6276.

[38]

D. Kovala-Demertzi, S.K. Hadjikakou, M.A. Demertzis, Y. Deligiannakis, J. Inorg.

Biochem. 69 (1998) 223-229. 19

[39]

D. Kovala-Demertzi, J. Inorg. Biochem. 79 (2000) 153-157.

[40]

Z.H. Chohan, M.S. Iqbal, H.S. Iqbal, A. Scozzafava, C.T. Supuran, J. Enz. Inhib. Med.

Chem. 17 (2002) 87-91. [41]

D. Kovala-Demertzi, D. Hadjipavlou-Litina, A. Primikiri, M. Staninska, C. Kotoglou, M.A.

Demertzis, Chem. & Biodiver. 6 (2009) 948-960. [42]

C. Tan, J. Liu, H. Li, W. Zheng, S. Shi, L. Chen, L. Ji, J. Inorg. Biochem. 102 (2008) 347-

358. [43]

J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 3nd ed., Springer, New York, 2006.

[44]

Rigaku/MSC (2005). CrystalClear. Rigaku/MSC Inc., The Woodlands, Texas, USA.

[45]

Sheldrick, G.M., SHELXS-97: Structure Solving Program, University of Göttingen,

Germany, 1997. [46]

Sheldrick, G.M., SHELXL-97: Crystal Structure Refinement Program, University of

Göttingen, Germany, 1997. [47]

K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, Part

B: Applications in Coordination, Organometallic, and Bioinorganic Chemistry, 6th ed., Wiley, New Jersey, 2009. [48]

F. Dimiza, S. Fountoulaki, A.N. Papadopoulos, C.A. Kontogiorgis, V. Tangoulis, C.P.

Raptopoulou, V. Psycharis, A. Terzis, D.P. Kessissoglou, G. Psomas, Dalton Trans. 40 (2011) 8555-8568. [49]

R.A. Coxall, S.G. Harris, D.K. Henderson, S. Parsons, P.A. Tasker, R.E.P. Winpenny, J.

Chem. Soc., Dalton Trans. (2000) 2349-2356. [50]

C.J. Milios, T.C. Stamatatos, S.P. Perlepes, Polyhedron 25 (2006) 134-194.

[51]

I. Mossakowska, W. Grazyna, Acta Cryst. C63 (2007) o123-o125.

[52]

M. Kubicki, P. Wagner, Acta Cryst. C63 (2007) o454-o457

[53]

F.H. Allen, Acta Cryst. B58 (2002) 380-388.

[54]

M. Fourmigue, Curr. Opin. Sol. State & Mat. Sci. 13 (2009) 36-45.

[55]

E. Parisini, P. Metrangolo, T. Pilati, G. Resnati, G. Terraneo Chem. Soc. Rev. 40 (2011)

2267-2278. [56]

R. Mir, N. Singh, G. Vikram, R.P. Kumar, M. Sinha, A. Bhushan, P. Kaur, A. Srinivasan, S.

Sharma, T.P. Singh, Biophys. J. 97 (2009) 3178-3186. [57]

T. Klabunde, H.M. Petrassi, V.B. Oza, P. Raman, J.W. Kelly, J.C. Sacchettini, Nat. Struct.

Molec. Biol. 7 (2000) 312-321. [58]

S.W. Rowlinson, J.R. Kiefer, J.J. Prusakiewicz, J.L. Pawlitz, K.R. Kozak, A.S. Kalgutkar,

W.C. Stallings, R.G. Kurumbail, L.J. Marnett, J. Biol. Chem. 278 (2003) 45763-45769. 20

[59]

S. Fountoulaki, F. Perdih, I. Turel, D.P. Kessissoglou, G. Psomas, J. Inorg. Biochem. 105

(2011) 1645-1655. [60]

Y. Wang, H. Zhang, G. Zhang, W. Tao, S. Tang, J. Luminescence 126 (2007) 211-218.

[61]

V. Rajendiran, R. Karthik, M. Palaniandavar, H. Stoeckli-Evans, V.S. Periasamy, M.A.

Akbarsha, B.S. Srinag, H. Krishnamurthy, Inorg. Chem. 46 (2007) 8208-8221. [62]

J.R. Lakowicz, G. Weber, Biochemistry 12 (1973) 4161-4170.

[63]

F. Dimiza, A.N. Papadopoulos, V. Tangoulis, V. Psycharis, C.P. Raptopoulou, D.P.

Kessissoglou, G. Psomas, Dalton Trans. 39 (2010) 4517-4528. [64]

F. Dimiza, A.N. Papadopoulos, V. Tangoulis, V. Psycharis, C.P. Raptopoulou, D.P.

Kessissoglou, G. Psomas, J. Inorg. Biochem. 107 (2012) 54-64. [65]

S. Tsiliou, L.-A. Kefala, F. Perdih, I. Turel, D.P. Kessissoglou, G. Psomas, Eur. J. Med.

Chem. 48 (2012) 132-142. [66]

A. Tarushi, X. Totta, C. Raptopoulou, V. Psycharis, G. Psomas, D.P. Kessissoglou, Dalton

Trans. 41 (2012) 7082-7091. [67]

C. Tolia, A.N. Papadopoulos, C.P. Raptopoulou, V. Psycharis, C. Garino, L. Salassa, G.

Psomas, J. Inorg. Biochem. 123 (2013) 53-65. [68]

Q. Zhang, J. Liu, H. Chao, G. Xue, L. Ji, J. Inorg. Biochem. 83 (2001) 49-55.

[69]

A.M. Pyle, J.P. Rehmann, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am.

Chem. Soc. 111 (1989) 3053-3063. [70]

E.C. Long, J.K. Barton, Acc. Chem. Res. 23 (1990) 271-273.

[71]

A. Jancso, L. Nagy, E. Moldrheim E. Sletten, J. Chem. Soc. Dalton Trans. (1999) 1587-

1594. [72]

G. Pratviel, J. Bernadou, B. Meunier, Adv. Inorg. Chem. 45 (1998) 251-262.

[73]

A. Dimitrakopoulou, C. Dendrinou-Samara, A.A. Pantazaki, M. Alexiou, E. Nordlander,

D.P. Kessissoglou, J. Inorg. Biochem. 102 (2008) 618-628. [74]

G. Psomas, J. Inorg. Biochem. 102 (2008) 1798-1811.

[75]

M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989) 8901-8911.

[76]

J.L. Garcia-Gimenez, M. Gonzalez-Alvarez, M. Liu-Gonzalez, B. Macias, J. Borras, G.

Alzuet, J. Inorg. Biochem. 103 (2009) 923-934. [77]

W.D. Wilson, L. Ratmeyer, M. Zhao, L. Strekowski, D. Boykin, Biochemistry 32 (1993)

4098-4104. [78]

S. Dhar, M. Nethaji, A.R. Chakravarty, J. Inorg. Biochem. 99 (2005) 805-812.

21

Figure and Scheme captions Scheme 1. Sodium diclofenac (=Nadicl). Figure 1. A drawing of the molecular structure of the cationic unit [Ni(dicl)(Hdicl)(Hpko)2]+ and

two lattice ligands of 1 with selective atoms labeling. Intramolecular hydrogen bonds, intermolecular hydrogen bonds and halogen bonds are shown in dark green, yellow and light green colours respectively. Figure 2. A part of the chain extended parallel to the a-axis of {[Ni(dicl)(Hdicl)(Hpko)2](dicl)}2

tetramers formed in the structure of 1. The complex cations are coloured in blue and the lattice diclofenac anions are coloured in red. The intra-tetramer hydrogen bond and chalogen interactions are shown with yellow and light green dashed lines, respectively. The inter-tetramer π-π stacking interactions are shown with dark green dashed lines. Figure 3. (A) Plot of % relative BSA fluorescence intensity at λem = 342 nm (%) vs r (r =

[complex]/[BSA]) for complexes 1-3 (46% of the initial fluorescence intensity for for 1, 7% for 2 and 27% for 3) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). (B) Plot of % relative HSA fluorescence intensity at λem = 351 nm (%) vs r (r = [complex]/[HSA]) for complexes 1-3 (67% of the initial fluorescence intensity for 1, 27% for 2 and 40% for 3) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). Figure 4. UV spectra of CT DNA (1.35x10-4 M) in buffer solution (150 mM NaCl and 15 mM

trisodium citrate at pH 7.0) in the absence and presence of [Ni(dicl)2(phen)], 3 (up to r = 0.3). The arrow shows the changes upon increasing amounts of complex. Figure 5. UV spectra of a DMSO solution (2x10-5 M) of (A) [Ni(dicl)(Hdicl)(Hpko)2](dicl), 1 and

(B) [Ni(dicl)2(bipy)], 2 in the presence of CT DNA at increasing amounts (up to 1/r = 2). The arrows show the changes upon increasing amounts of CT DNA. Figure 6. Cyclic voltammogram of 0.4 mM 1/2 DMSO/buffer (containing 150 mM NaCl and 15

mM trisodium citrate at pH=7.0) solution of [Ni(dicl)2(phen)], 3, in the absence or presence of CT DNA (1/r = 2). The arrows show the changes upon addition of CT DNA ([DNA] = 0.2 mM). Scan rate = 100 mV s-1. Supporting electrolyte = buffer solution. Figure 7. Relative viscosity (η/ηo)1/3 of CT DNA (0.1 mM) in buffer solution (150 mM NaCl and

15 mM trisodium citrate at pH 7.0) in the presence of complexes 1-3 at increasing amounts (r) up to r = 0.24. Figure 8. (A) Emission spectra (λexit = 540 nm) for EB-DNA ([EB] = 20 µM, [DNA] = 26 µM) in

buffer solution in the absence and presence of increasing amounts of complex 1 (up to the value of r = 0.15). The arrow shows the changes of intensity upon increasing amounts of 1. (B) Plot of EB22

DNA relative fluorescence intensity (%I/Io) at λem = 592 nm vs r (r = [compound]/[DNA]) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the presence of complexes 1-3 (quenching up to 32% of the initial EB-DNA fluorescence for 1, 34% for 2 and 32% for 3).

23

O Cl

H

H2C

N

Cl Scheme 1

24

C

ONa

Figure 1

25

Figure 2

26

BSA fluorescence (%)

(A) 100

80 60 40 20 0

1

0

2

1

3

2

3

4

5

6

7

6

7

r=[complex]/[BSA]

HSA fluorescence (%)

(B) 100

80 60 40 20 0

1

0

1

2

3

2

3

4

5

r=[complex]/[HSA]

Figure 3

27

1.2 1.0

A

0.8 0.6 0.4 0.2 0.0 240

260

280

λ (nm)

Figure 4

28

300

320

(A) 1.4

1.2 1.0

A

0.8 0.6 0.4 0.2 0.0 280

320

360

400

440

λ (nm) (B)

1.4 1.2 1.0

A

0.8 0.6 0.4 0.2 0.0

280

300

320

λ (nm)

Figure 5

29

340

360

380

-150

I (µA)

-100 -50 0 50 100 -200

3 3 + DNA -400

-600

E (mV)

Figure 6

30

-800

-1000

1.3

1 2 3

(η / η ο )

1/3

1.2

1.1

1.0 0.00

0.05

0.10

0.15

r= [compound]/[DNA]

Figure 7

31

0.20

0.25

(A)

2500 2000

I

1500 1000 500 0 560

580

600

620

640

660

680

700

λ (nm)

EB-DNA fluorescence (I/Io%)

(B)

100 80 60 40 20 1

0 0.00

0.02

2

0.04

3

0.06

0.08

0.10

r = [Complex]/[DNA]

Figure 8

32

0.12

0.14

Tables Table 1. Crystallographic data for complex 1·CH3OH·0.6H2O. 1·CH3OH·0.6H2O

Formula

C65H54.20Cl6N9NiO9.60

Fw

1386.38

T (Κ)

160(2)

Crystal system

monoclinic

Space group

P21/n

a (Å)

13.7540(2)

b (Å)

26.1971(5)

c (Å)

17.8373(3)

α (ο )

90.00

β (ο )

102.867(1)

ο

γ( )

90.00 3

a

Volume (Å )

6265.66(18)

Ζ

4

D(calc), Mg m–3

1.470

Abs. coef., µ, mm–1

3.355

Reflections with I>2σ(I)

7448

GOF on F2

1.110

R1=

0.0667 a

wR2=

0.1649 a

w=1⁄[σ2(Fo2)+(αP)2+bP] and P = [max (Fo2,0)+2Fc2]/3,

R1 = Σ(|Fo|-|Fc|)/Σ(|Fo|) and wR2 = {Σ[w(Fo2-Fc2)2]/Σ[w(Fo2)2]}1/2.

33

Table 2. Selected bond distances and angles for complex 1. Bond distances

(Å)

Bond distances

(Å)

Ni(1)-O(21)

2.037(2)

Ni(1)-O(31)

2.045(2)

Ni(1)-N(1)

2.065(3)

Ni(1)-N(2)

2.085(3)

Ni(1)-N(11)

2.070(3)

Ni(1)-N(12)

2.069(3)

O(21)-C(41)

1.267(4)

O(22)-C(41)

1.259(4)

O(31)-C(61)

1.272(4)

O(32)-C(61)

1.248(4)

O(1)-N(2)

1.367(3)

O(11)-N(12)

1.364(3)

(°)

Bond angles

(°)

Bond angles

O(21)-Ni(1)-O(31)

85.1(1)

O(31)-Ni(1)-N(1)

175.6(1)

O(21)-Ni(1)-N(1)

90.5(1)

O(31)-Ni(1)-N(2)

102.2(1)

O(21)-Ni(1)-N(2)

87.2(1)

O(31)-Ni(1)-N(11)

88.0(1)

O(21)-Ni(1)-N(11)

173.1(1)

O(31)-Ni(1)-N(12)

86.5(1)

O(21)-Ni(1)-N(12)

101.8(1)

N(2)-Ni(1)-N(11)

94.1(1)

N(1)-Ni(1)-N(2)

77.7(1)

N(2)-Ni(1)-N(12)

168.1(1)

N(1)-Ni(1)-N(11)

96.4(1)

N(11)-Ni(1)-N(12)

78.0(1)

N(1)-Ni(1)-N(12)

94.2(1)

34

Table 3. Hydrogen bonds in the structure of 1. Interaction

D···A (Å)

H···A (Å)

D-H···A (o)

Symmetry operation

O(1)-H(1o)···O(32)

2.593

1.764

168.8

x, y, z

O(11)-H(11o)···O(22)

2.519

1.688

169.3

x, y, z

O(31)-H(31o)···O(42)

2.590

1.863

143.8

x, y, z

N(21)-H(21n)···O(22)

2.969

2.313

131.4

x, y, z

N(31)-H(31n)···O(32)

2.908

2.253

131.2

x, y, z

N(41)-H(41n)···O(41)

3.362

2.537

156.4

x, y, z

O(1m)-H(1m)···O(21)

2.795

1.958

173.7

0.5+x, 0.5-y, 0.5+z

35

Table 4. The SA constants and parameters derived for complexes 1-3.

BSA Compound

Νadicl [12]

KSV (M-1)

kq (M-1s-1) 4

8.11(±0.34)x10

4

Κ (M-1)

8.11(±0.34)x10

12

4.80(±0.39)x10

12

n

5

3.55 x10

2.21(±0.23)x10

1.61 5

0.66

[Ni(dicl)(Hdicl)(Hpko)2](dicl), 1

4.80(±0.39)x10

[Ni(dicl)2(bipy)], 2

7.35(±0.56)x105

7.35(±0.56)x1013

1.31(±0.11)x106

0.97

[Ni(dicl)2(phen)], 3

1.52(±0.08)x105

1.52(±0.08)x1013

1.72(±0.13)x105

0.98

Compound

KSV (M-1)

kq (M-1s-1)

Κ (M-1)

n

Νadicl [12]

1.81(±0.17)x104

1.81(±0.17)x1012

1.63x105

0.32

[Ni(dicl)(Hdicl)(Hpko)2](dicl), 1

2.75(±0.10)x104

2.75(±0.10)x1012

2.54(±0.27)x104

1.04

[Ni(dicl)2(bipy)], 2

1.26(±0.13)x105

1.26(±0.13)x1013

3.60(±0.41)x105

0.83

[Ni(dicl)2(phen)], 3

4

12

5

0.63

HSA

7.38(±0.14)x10

36

7.38(±0.14)x10

6.87(±0.42)x10

Table 5. The DNA binding constants (Kb) for complexes 1-3. Complexes

Kb (M-1)

Νa-diclofenac [12]

3.16x104

[Ni(dicl)(Hdicl)(Hpko)2](dicl), 1

3.63(±0.12)x105

[Ni(dicl)2(bipy)], 2

2.21(±0.08)x105

[Ni(dicl)2(phen)], 3

3.67(±0.17)x104

37

Table 6. Cathodic and anodic potentials (in mV) for the redox couple Ni(II)/Ni(I) in 1/2

dmso/buffer solution of complexes 1-3 in the absence and presence of CT DNA. Complex

Epc(f)

a

Epc(b)

b

∆Epc c

Epa(f)

a

Epa(b)

b

∆Epa c

[Ni(dicl)(Hdicl)(Hpko)2](dicl),1

-785

-770

+15

-510

-425

+85

[Ni(dicl)2(bipy)], 2

-750

-760

-10

-445

-430

+15

[Ni(dicl)2(phen)], 3

-745

-755

-10

-480

-455

+25

a

Epc/a(f) in dmso/buffer in the absence of CT DNA

b

Epc/a(b) in dmso/buffer in the presence of CT DNA

c

∆Epc/a = Epc/a(b) - Epc/a(f)

38

Table 7. The Stern-Volmer constants (KSV) of EB-DNA fluorescence for complexes 1-3. Compound

Ksv (M-1)

Νadicl [12]

2.47x105

[Ni(dicl)(Hdicl)(Hpko)2](dicl), 1

1.40(±0.03)x105

[Ni(dicl)2(bipy)], 2

9.43(±0.20)x105

[Ni(dicl)2(phen)], 3

1.07(±0.04)x106

39

2 sodium diclof enac + Ni2+ + 2 Hpko Antiinflammatorydrug

Ni2+

DNA- intercalator

Synopsis

The mononuclear nickel(II) complexes of the non-steroidal anti-inflammatory drug diclofenac in the presence of N,N’-donor ligands exhibit pronounced binding behaviour towards DNA and serum albumins compared to the free drug.

Graphical abstract

40