Synthesis, structure and biological activity of nickel(II) complexes with mefenamato and nitrogen-donor ligands.

Journal of Inorganic Biochemistry 145 (2015) 79–93

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Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio

Synthesis, structure and biological activity of nickel(II) complexes with mefenamato and nitrogen-donor ligands Xanthippi Totta a, Aikaterini A. Papadopoulou a, Antonios G. Hatzidimitriou a, Athanasios Papadopoulos b, George Psomas a,⁎ a b

Laboratory of Inorganic Chemistry, Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece Department of Nutrition and Dietetics, Faculty of Food Technology and Nutrition, Alexandrion Technological Educational Institution, Sindos, Thessaloniki, Greece

a r t i c l e

i n f o

Article history: Received 27 November 2014 Received in revised form 16 January 2015 Accepted 16 January 2015 Available online 26 January 2015 Keywords: Nickel(II) complexes Mefenamic acid Interaction with DNA Antioxidant activity

a b s t r a c t Six novel nickel(II) complexes with the non-steroidal anti-inflammatory drug mefenamic acid (Hmef) with the nitrogen-donor heterocyclic ligands 2,2′-bipyridine (bipy), 2,2′-bipyridylamine (bipyam), 1,10-phenanthroline (phen), 2,2′-dipyridylketone oxime (Hpko) or pyridine (py) and/or the oxygen-donor ligands CH3OH or H2O were synthesized and characterized by physicochemical and spectroscopic techniques. The crystal structures of [Ni(mef-O)2(bipy)(CH3OH)2] (1), [Ni(mef-O)2(phen)(CH3OH)2] (2), [Ni(mef-O,O′)2(bipyam)] (3) and [Ni(mef-O)2(Hpko)2]∙CH3OH (4·CH3OH) were determined by X-ray crystallography. The ability of the complexes to scavenge 1,1-diphenyl-picrylhydrazyl, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and hydroxyl radicals was investigated and the in vitro inhibitory activity against soybean lipoxygenase was evaluated; complexes 3 and 4 were the most active compounds. Spectroscopic (UV and fluorescence), electrochemical (cyclic voltammetry) and physicochemical (viscosity measurements) techniques were employed in order to study the binding mode of the complexes to calf-thymus (CT) DNA and to calculate the corresponding binding constants; for all complexes, intercalation was the most possible mode of DNA-binding. The interaction of the complexes with serum albumins was studied by fluorescence emission spectroscopy and the values of the albumin-binding constants were determined. © 2015 Elsevier Inc. All rights reserved.

1. Introduction For many years, nickel was not considered to play an important biological role. This aspect changed completely in 1975 after the discovery of the presence of Ni in the active center of the enzyme urease [1]. Since then, the biological role of nickel was rapidly expanded because of the determination of more nickel-containing or nickel-dependent enzymes [2,3]. Furthermore, a lot of nickel complexes bearing biological activity have been reported including Ni(II) complexes with antitumor antibiotics [4] and others acting as anticonvulsant [5] or antiepileptic drugs [6] or vitamins [7]. Additionally, a plethora of nickel(II) complexes exhibiting noteworthy potential antibacterial [8–10], antifungal [10, 11], anti-inflammatory [12], anti-leishmania [13], antioxidant [14–16] and antiproliferative activity towards diverse cell lines [17–19] was reported. A significant number of Ni(II) compounds acting as DNAintercalators [20–22] or DNA-cleaving agents [23,24] were also found in the literature. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used agents as anti-inflammatories, analgesics and antipyretics [25]. The purpose of their use is to inhibit the cyclooxygenase-mediated ⁎ Corresponding author. Tel.: +30 2310997790; fax: +30 2310997738. E-mail address: [email protected] (G. Psomas).

http://dx.doi.org/10.1016/j.jinorgbio.2015.01.009 0162-0134/© 2015 Elsevier Inc. All rights reserved.

production of prostaglandins [26]. Additionally, they have shown antiproliferative activity against a series of cancer cell lines via diverse mechanisms including apoptosis [27], synergism with drugs [28], DNA-interaction [29] and free radical scavenging [30]. Apart from their involvement in mechanism leading to proliferation, free radicals play also an important role in the inflammatory process; therefore, their scavenging may often be considered as a first means of evaluating the antioxidant activity of the compounds [31]. Additionally, the ability of the compounds to inhibit lipoxygenase (LOX) may serve indirect evidence of their total antioxidant activity and is a preliminary step towards the anti-inflammatory activity studies [32]. On the basis of characteristic chemical groups, the NSAIDs consist of phenylalkanoic acids, anthranilic acids, oxicams, salicylate derivatives, sulfonamides and furanones with phenylalkanoic acids, anthranilic acids and salicylate derivatives bearing a carboxylic group [25,33]. In regard to the Ni(II) complexes with NSAIDs as ligands, although quite a few of them have been isolated and studied [33–38], the crystal structure of only one Ni(II) complex bearing NSAIDs as ligands has been determined, i.e. [Ni(dicl)(Hdicl)(Hpko)2](dicl), where dicl is the NSAID diclofenac [38]. Mefenamic acid (Hmef, Fig. 1(A)) is a derivative of anthralinic acid and is similar to the NSAIDs tolfenamic and flufenamic acid [25]. As an NSAID, mefenamic acid is used effectively as an analgesic and

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X. Totta et al. / Journal of Inorganic Biochemistry 145 (2015) 79–93

Fig. 1. The syntax formula of (A) mefenamic acid (Hmef), (B) 2,2′-bipyridine (bipy), (C) 1,10-phenanthroline (phen), (D) 2,2′-bipyridylamine (bipyam), (E) 2,2′-dipyridylketone oxime (Hpko) and (F) pyridine (py).

antipyretic agent and its side-effects (headaches, diarrhea and vomiting) are rather mild. The crystal structures of a series of tin(IV) [39], copper(II) [40–42], cobalt(II) [43], zinc(II) [44] and manganese(II) [45] complexes with mefenamato ligands have been found in the literature. Most of the reported metal–mefenamato complexes have shown an enhanced biological profile in comparison to free Hmef. The mefenamato complexes were more active than free Hmef in regard to the DNA- and albumin-binding properties and exhibited pronounced antioxidant activity [42–44]. As a continuation of our recent research concerning metal– mefenamato complexes [33–35], we present herein the synthesis of six novel Ni(II) complexes with mefenamic acid in the presence of the nitrogen-donor ligands 2,2′-bipyridine (bipy), 2,2′-bipyridylamine (bipyam), 1,10-phenanthroline (phen), 2,2′-dipyridylketone oxime (Hpko) or pyridine (py) (Fig. 1) and/or oxygen-donor ligands H2O or methanol (MeOH). The complexes were characterized by physicochemical (elemental analysis, molecular conductivity and room-temperature magnetic measurements), spectroscopic (IR and UV–vis) and electrochemical (cyclic voltammetry) techniques. Additionally, the crystal structures of [Ni(mef-O)2(bipy)(MeOH)2], 1, [Ni(mef-O)2(phen)(MeOH)2], 2, [Ni(mef-O,O′)2(bipyam)], 3 and [Ni(mef-O)2(Hpko)2]·MeOH, 4·MeOH were determined by X-ray crystallography. In order to evaluate the biological impact of the complexes, our studies were also focused on: (i) the investigation of the antioxidant activity by determining the ability of the complexes to scavenge 1,1-diphenyl-picrylhydrazyl (DPPH), hydroxyl radicals (•OH) and 2,2′-azinobis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS+•) radicals and the in vitro inhibitory activity against soybean LOX, (ii) the determination of the binding affinity and mode of the complexes to calf-thymus (CT) DNA directly by UV spectroscopy, cyclic voltammetry, viscosity measurements and indirectly via the ethidium bromide (EB) displacement ability of the complexes from the EB–DNA compound by fluorescence spectroscopy and (iii) the binding affinity of the complexes towards bovine (BSA) and human serum albumin (HSA) by fluorescence spectroscopy.

2. Experimental

were reagent grade and were used as purchased. TEAP was recrystallized twice from ethanol and dried under vacuum, prior to its use. DNA stock solution was prepared by dilution of CT DNA to buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) followed by exhaustive stirring at 4 °C for three days, and kept at 4 °C for no longer than a week. The stock solution of CT DNA gave A260/A280 (ratio of UV absorbance at 260 and 280 nm) in the range 1.85–1.89, indicating that the DNA was sufficiently free of protein contamination [46]. The DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M−1 cm−1 [47]. The infrared (IR) spectra of the compounds were recorded in the range 400–4000 cm−1 on a Nicolet FT-IR 6700 spectrometer with samples prepared as KBr pellets. The UV–visible (UV–vis) spectra of the compounds were recorded as nujol mulls and in solution at concentrations in the range 10− 5–5 × 10−3 M on a Hitachi U-2001 dual beam spectrophotometer. C, H and N elemental analyses were performed on a PerkinElmer 240B elemental analyzer. The molar conductivity measurements were carried out in 1 mM DMSO solution of the complexes with a Crison Basic 30 conductometer. Room temperature magnetic measurements were carried out on a magnetic susceptibility balance of Sherwood Scientific (Cambridge, UK) by the Faraday method using mercury tetrathiocyanatocobaltate(II) as a calibrant. The fluorescence spectra of the compounds were recorded in solution on a Hitachi F7000 fluorescence spectrophotometer. The viscosity experiments were carried out using an ALPHA L Fungilab rotational viscometer equipped with an 18 mL LCP spindle. The cyclic voltammetry studies were performed on an Eco Chemie Autolab Electrochemical analyzer and the 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 vapors. All electrochemical measurements were performed at 25.0 ± 0.2 °C.

2.1. Materials and instrumentation 2.2. Synthesis of the complexes Mefenamic acid, NiCl2·6H2O, bipy, bipyam, Hpko, phen, py, KOH, trisodium citrate, NaCl, CT DNA, BSA, HSA, tetraethylammonium perchlorate (TEAP) and EB were purchased from Sigma-Aldrich Co and all solvents were purchased from Merck. All the chemicals and solvents

2.2.1. Synthesis of [Ni(mef)2(bipy)(MeOH)2], 1 A methanolic solution (10 mL) containing Hmef (0.4 mmol, 97 mg) and KOH (0.4 mmol, 22 mg) was stirred for 1 h at room temperature.


The resultant solution was added simultaneously with a methanolic solution (5 mL) of bipy (0.2 mmol, 61 mg) to a methanolic solution (5 mL) of NiCl2·6H2O (0.2 mmol, 48 mg). The solution was stirred for 30 min and left for slow evaporation at room temperature. Blue crystals of [Ni(mef)2(bipy)(MeOH)2], 1 (80 mg, 70%), suitable for X-ray structure determination, were collected after a week. Anal. Calcd. for [Ni(mef)2(bipy)(MeOH)2] (C42H44N4NiO6) (MW = 759.54): C 66.42, H 5.84, N 7.36%; found C 66.12, H 5.77, N 7.36%. IR (KBr disk): ν max , cm−1; νasym(CO2): 1575 (vs (very strong)); νsym(CO2): 1382 (vs); Δ = νasym(CO2) − νsym(CO2): 193 cm−1; ρ(C–H)bipy: 766 (s (strong)); UV–vis: λ, nm as nujol mull: 990, 625 (sh (shoulder)), 402 (sh), 341 (sh), 301; λ, nm (ε, M−1 cm−1) in DMSO: 995 (10), 620 (20), 397 (sh) (120), 342 (sh) (5800), 298 (19,500); 10 Dq = 10,050 cm−1, B = 745 cm−1, 10 Dq/B = 13.5. μeff = 3.17 BM, at room temperature. The complex is soluble in DMSO, DMF, CHCl3, CH2Cl2 and acetone and is non-electrolyte (ΛM = 20 μS/cm, 1 mM in DMSO). 2.2.2. Synthesis of complexes 2–5 Complexes 2–5 were prepared in a similar way to 1 with the use of phen (0.2 mmol, 36 mg) for 2, bipyam (0.2 mmol, 34 mg) for 3, Hpko (0.4 mmol, 80 mg) for 4 and py (2 mL) for 5 instead of bipy. [Ni(mef)2(phen)(MeOH)2], 2: Blue crystals of [Ni(mef)2(phen) (MeOH)2], 2 (90 mg, 60%), suitable for X-ray structure determination, were collected after ten days. Anal. Calcd. for [Ni(mef)2(phen)(MeOH)2] (C44H44N4NiO6) (MW = 783.57): C 67.45, H 5.66, N 7.15%; found C 67.45, H 5.53, N 7.10%. IR (KBr disk): νmax, cm− 1; νasym(CO2): 1576 (vs); νsym(CO2): 1366 (vs); Δ = 210 cm−1; ρ(C–H)phen: 725 (s); UV– vis: λ, nm as nujol mull: 995, 630 (sh), 402 (sh), 341 (sh), 289; λ, nm (ε, M−1 cm−1) in DMSO: 1005 (8), 635 (15), 405 (sh) (75), 340 (sh) (5900), 294 (17,200); 10 Dq = 9950 cm−1, B = 706 cm−1, 10 Dq/B = 14.1. μ eff = 3.45 BM, at room temperature. The complex is soluble in DMSO, DMF, CHCl3, CH2Cl2 and acetone and is non-electrolyte (ΛM = 12 μS/cm, 1 mM in DMSO). [Ni(mef)2(bipyam)], 3: Blue crystals of [Ni(mef)2(bipyam)], 3 (100 mg, 70%), suitable for X-ray structure determination, were collected after five days. Anal. Calcd. for [Ni(mef)2(bipyam)] (C40H37N5NiO4) (MW = 710.47): C 67.62, H 5.25, N 9.86%; found C 67.22, H 5.38, N 9.66%. IR (KBr disk): νmax, cm− 1; νasym(CO2): 1578 (vs); νsym(CO2): 1405 (vs); Δ = 173 cm−1; ρ(C–H)bipyam: 770 (s); UV–vis: λ, nm as nujol mull: 990, 640 (sh), 405 (sh), 348 (sh), 305; λ, nm (ε, M−1 cm−1) in DMSO: 995 (10), 625 (20), 410 (sh) (60), 340 (sh) (3700), 300 (13,600); 10 Dq = 10,050 cm− 1 , B = 682 cm − 1 , 10 Dq/B = 14.7. μeff = 2.95 BM, at room temperature. The complex is soluble in DMSO and DMF and is non-electrolyte (ΛM = 11 μS/cm, 1 mM in DMSO). [Ni(mef)2(Hpko) 2 ]·MeOH, 4·MeOH: Red–orange crystals of [Ni(mef)2(Hpko)2]·MeOH, 4·MeOH (110 mg, 55%), suitable for Xray structure determination, were collected after ten days. Anal. Calcd. for [Ni(mef)2(Hpko)2]·MeOH (C53H50N8NiO7) (MW = 969.74): C 65.65, H 5.20, N 11.56%; found C 65.93, H 5.04, N 11.17%. IR (KBr disk): νmax, cm−1; νasym(CO2): 1608 (vs); νsym(CO2): 1387 (vs); Δ = 221 cm− 1; ρ(C–H)Hpko: 783 (s); UV–vis: λ, nm as nujol mull: 965, 629, 391 (sh), 349 (sh), 291; λ, nm (ε, M− 1 cm− 1) in DMSO: 970 (15), 620 (35), 385 (sh) (200), 343 (9500), 287 (16,000); 10 Dq = 10,310 cm−1, B = 745 cm−1, 10 Dq/B = 13.8. μeff = 3.22 BM, at room temperature. The complex is soluble in DMSO and DMF and partially soluble in CH3CN and is non-electrolyte (ΛM = 13 μS/cm, 1 mM in DMSO). [Ni(mef)2(py)2(H2O)2], 5: Light blue microcrystalline product of [Ni(mef)2(py)2(H2O)2] (100 mg, 68%) was deposited and collected by filtration after five days. Anal. Calcd. for [Ni(mef)2(py)2(H2O)2] (C40H42N4NiO6) (MW = 733.51): C 65.50, H 5.77, N 7.64%; found C 65.38, H 5.97, N 7.36%. IR (KBr disk): νmax, cm− 1; νasym(CO2): 1577 (vs); νsym(CO2): 1380 (vs); Δ = 197 cm− 1; ρ(C–H)py: 695 (s); UV– vis: λ, nm as nujol mull: 1010, 690, 409 (sh), 345 (sh), 302; λ, nm (ε, M− 1 cm− 1) in DMSO: 1000 (5), 695 (15), 405 (sh) (50), 342 (sh)

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(4500), 298 (12,500); 10 Dq = 10,000 cm − 1 , B = 605 cm − 1 , 10 Dq/B = 16.5. μeff = 3.30 BM, at room temperature. The complex is soluble in DMSO, DMF, CH3CN, CHCl3, CH2Cl2 and acetone and is non-electrolyte (ΛM = 10 μS/cm, 1 mM in DMSO). 2.2.3. Synthesis of [Ni(mef)2(MeOH)4], 6 Complex 6 was prepared by the reaction of a methanolic solution (10 mL) of Hmef (0.4 mmol, 97 mg), which was deprotonated by the addition of KOH (0.4 mmol, 22 mg) and 1 h stirring, and a methanolic solution of (5 mL) NiCl2·6H2O (0.2 mmol, 48 mg). Light green microcrystalline product of [Ni(mef)2(MeOH)4], 5 (90 mg, 75%) was collected with filtration after a month. Anal. Calcd. for [Ni(mef)2(MeOH)4] (C34H44N2NiO8) (MW = 667.44): C 61.19, H 6.65, N 4.20%; found C 60.97, H 6.45, N 4.46%. IR (KBr disk): νmax, cm−1; νasym(CO2): 1578 (vs); νsym(CO2): 1387 (vs); Δ = 191 cm− 1; UV–vis: λ, nm as nujol mull: 990, 690, 405 (sh), 342 (sh), 304; λ, nm (ε, M− 1 cm− 1) in DMSO: 995 (5), 700 (15), 400 (sh) (65), 338 (sh) (5100), 299 (13,500); 10 Dq = 10,050 cm− 1, B = 609 cm−1, 10 Dq/B = 16.5. μ eff = 3.39 BM, at room temperature. The complex is soluble in DMSO, DMF, CHCl3, CH2Cl2 and acetone and is non-electrolyte (ΛM = 18 μS/cm, 1 mM in DMSO). 2.3. X-ray crystallography Crystals of 1–4 were taken from the mother liquor and mounted at room temperature on a Bruker Kappa APEX2 diffractometer equipped with a triumph monochromator using Mo Kα radiation. Unit cell dimensions were determined and refined by using the angular settings of at least 100 high intensity reflections (N 10 σ(I)) in the range 15 b 2θ b 40°. Intensity data were recorded using φ and ω scans. All crystals presented no decay during the data collection. The frames collected for each crystal were integrated with the Bruker SAINT software package [48], using a narrow-frame algorithm. Data were corrected for absorption using the numerical method (SADABS) based on crystal dimensions [49]. All structures were solved using the SUPERFLIP package [50], incorporated in Crystals. Data refinement (full-matrix leastsquares methods on F2) and all subsequent calculations were carried out using the Crystals version 14.40b program package [51]. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were located by difference maps at their expected positions and refined using soft constraints. By the end of the refinement, they were positioned geometrically using riding constraints to bonded atoms. Crystal data as well as details of data collection and structure refinement for the compounds are given in Table 1. Illustrations were drawn by CAMERON [52]. Further details on the crystallographic studies as well as atomic displacement parameters are given as Supporting information in the form of cif files. CCDC 1035954–1035957 contain 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). 2.4. DNA-binding studies 2.4.1. Study with UV spectroscopy The interaction of complexes 1–6 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). The UV spectra of CT DNA have been recorded for a constant DNA concentration in the presence of each compound at diverse [compound]/[DNA] mixing ratios (= r). The binding constant of the complexes with DNA, Kb (in M−1), has been determined by the Wolfe– vs [DNA] using the Shimer equation (Eq. S1) [53] and the plots ðε½DNA A −ε f Þ

UV spectra of the compound recorded for a constant concentration in

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Table 1 Crystallographic data for complexes 1–4.

Formula Fw T(K) Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) Volume (Å3) Z d(calc), Mg m−3 Abs. coef., μ, mm−1 F(000) GOF on F2 Range of h, k, l Reflections with I N 2σ(Ι) R1 wR2 a

1

2

3

4·MeOH

C42H44N4Ni1O6 759.54 295 Orthorhombic Iba2 29.627(2) 7.2318(6) 17.6218(13) 90 90 90 3775.6(3) 4 1.336 0.567 1600 0.999 0 → 36, 0 → 8, −22 → 21 2508 0.0661a 0.1212a

C44H44Ni1N4O6 783.57 295 Orthorhombic Pbcn 16.788(2) 25.611(3) 9.1084(10) 90 90 90 3916.2(4) 4 1.329 0.549 1648 1.003 −21 → 21, −32 → 32, −11 → 11 2381 0.0419a 0.0754a

C40H37N5Ni1O4 710.47 296 Triclinic P −1 11.1189(8) 12.9242(12) 13.8262(12) 70.917(5) 86.365(5) 68.064(5) 1737.82(15) 2 1.358 0.608 744 1.053 −14 → 14, −16 → 16, −17 → 17 4641 0.0659a 0.0842a

C53H50N8Ni1O7 969.74 295 Monoclinic P 21/c 10.3970(9) 22.330(2) 21.184(2) 90 102.091(7) 90 4809.2(5) 4 1.339 0.465 2032 1.000 −13 → 12, −22 → 28, 0 → 26 6744 0.0641a 0.1039a

For reflections with I N 2σ(Ι).

the presence of DNA for diverse r values. Control experiments with DMSO were performed and no changes in the spectra of CT DNA were observed.

calculated according to the linear Stern–Volmer equation (Eq. S2) [54] and the plots IoI vs [Q]. 2.5. Albumin binding experiments

2.4.2. Cyclic voltammetry studies The interaction of complexes 1–6 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 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. 2.4.3. Viscometry 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 DNA ([DNA] = 0.1 mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) was measured in the presence of increasing amounts of the compounds (up to the value of r = 0.26). All measurements were performed at room temperature. The obtained data are presented as (η/η0)1/3 versus r, where η is the viscosity of DNA in the presence of the compound, and η0 is the viscosity of DNA alone in buffer solution.

The protein binding study was performed by tryptophan fluorescence quenching experiments using BSA (3 μM) or 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 1–6 as quencher with increasing concentration. The fluorescence spectra were recorded from 300 to 500 nm at an excitation wavelength of 295 nm [55]. The mefenamato complexes 1–6 in buffer solution did not exhibit any emission bands when the spectra were recorded under the same experimental conditions (i.e. excitation at 295 nm) [42]. The influence of the inner-filter effect [56] on the measurements was evaluated by eq. S3. The Stern–Volmer and Scatchard equations (Eq. S4–S6) [57] and graphs have been used in order to study the interaction of each quencher with serum albumins and calculate the dynamic quenching constant KSV (in M− 1), the approximate quenching constant kq (in M−1 s−1), the association binding constant K (in M−1) and the number of binding sites per albumin n. 2.6. Antioxidant biological assay

2.4.4. EB competitive studies with fluorescence spectroscopy 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 DNA–EB complex. The DNA–EB complex was prepared by adding 20 μM EB and 26 μM CT DNA in buffer (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). The possible intercalating effect of the compounds was studied by adding a certain amount of a solution of the compound step by step into a 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 with excitation wavelength at 540 nm. Complexes 1–6 show no fluorescence at room temperature in solution or in the presence of DNA under the same experimental conditions; therefore, the observed quenching is attributed to the displacement of EB from its EB–DNA complex. The values of the Stern–Volmer constant (K SV, in M− 1 ) have been

In the in vitro assays each experiment was performed at least in triplicate and the standard deviation of absorbance was less than 10% of the mean. 2.6.1. Determination of the reducing activity of the stable radical DPPH To a solution of DPPH (0.1 mM) in absolute ethanol an equal volume of the compounds dissolved in ethanol was added. As control solution ethanol was used. The concentration of the solution of the compounds was 0.1 mM. The absorbance at 517 nm was recorded at room temperature, after 20 and 60 min in order to examine the time-dependence of the radical scavenging activity [31]. The radical scavenging activity of the compounds was expressed as the percentage reduction of the absorbance values of the initial DPPH solution (RA%). Nordihydroguaiaretic acid (NDGA) and butylated hydroxytoluene (BHT) were used as reference compounds.


2.6.2. Competition of the tested compounds with DMSO for hydroxyl radicals The hydroxyl radicals generated by the Fe3+/ascorbic acid system, were detected according to Nash [31], by the determination of formaldehyde produced from the oxidation of DMSO. The reaction mixture contained EDTA (0.1 mM), Fe3 + (167 μM), DMSO (33 mM) in phosphate buffer (50 mM, pH 7.4), the tested compounds (concentration 0.1 mM) and ascorbic acid (10 mM). After 30 min of incubation (37 °C) the reaction was stopped with CCl3COOH (17% w/v) and the absorbance at λ = 412 nm was measured. Trolox was used as an appropriate standard. The competition of the compounds with DMSO for •OH, generated by the Fe3+/ascorbic acid system, expressed as percent inhibition of formaldehyde production, was used for the evaluation of their hydroxyl radical scavenging activity (•OH%). 2.6.3. Assay of radical cation scavenging activity ABTS was dissolved in water to a 2 mM concentration. ABTS radical cation (ABTS+•) was produced by reacting ABTS stock solution with 0.17 mM potassium persulfate and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. Because ABTS and potassium persulfate react stoichiometrically at a ratio of 1:0.5, this will result in incomplete oxidation of the ABTS. Oxidation of the ABTS commenced immediately, but the absorbance was not maximal and stable until more than 6 h had elapsed. The radical was stable in this form for more than 2 days when stored in the dark at room temperature. The ABTS+• solution was diluted with ethanol to an absorbance of 0.70 at 734 nm. After addition of 10 μL of diluted compounds or standards (0.1 mM) in DMSO, the absorbance reading was taken exactly 1 min after initial mixing [31]. The radical scavenging activity of the complexes was expressed as the percentage inhibition of the absorbance of the initial ABTS solution (ABTS%). 6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid (trolox) was used as an appropriate standard. 2.6.4. Soybean lipoxygenase inhibition study in vitro The in vitro study was evaluated as reported previously [31]. The tested compounds dissolved in ethanol were incubated at room temperature with sodium linoleate (0.1 mM) and 0.2 mL of enzyme solution (1/9 × 10−4 w/v in saline). The conversion of sodium linoleate to 13hydroperoxylinoleic acid at 234 nm was recorded and compared with the appropriate standard inhibitor caffeic acid. 3. Results and discussion 3.1. Synthesis and characterization The complexes were prepared in high yield via the aerobic reaction of mefenamic acid, deprotonated by KOH, with NiCl2·6H2O in the presence of the corresponding N,N′-donor heterocyclic ligand (bipy, phen, bipyam of Hpko) in a ratio NiCl2: mef−1: N,N′-donor of 1:2:1, for complexes 1–4, respectively, or in the presence of excess of py or MeOH in a 1:2 NiCl2: mef− 1 ratio for complexes 5 or 6, respectively. The complexes are stable in air, soluble in DMSO and DMF and are nonelectrolytes (for 1 mM DMSO solution, ΛM = 10–20 μS/cm). The complexes were characterized by elemental analysis, IR and UV–vis spectroscopic techniques, magnetic measurements at room temperature and, in the case of 1–4, by X–ray crystallography. Furthermore, the cyclic voltammograms of the complexes in DMSO solution were also recorded. The deprotonation and the binding mode of mefenamic acid were confirmed by examining the IR spectra of the complexes. The absorption band at 3370 (m (medium)) cm−1 which was observed in the IR spectrum of Hmef and was attributed to the ν(O–H) stretching vibration disappeared upon binding to the nickel, inferring thus the deprotonation of the carboxylato group. The bands at 1655 (vs) cm−1 and 1255 (s) cm−1 which were attributed to the stretching vibrations ν(C_O)carboxylic and

83

ν(C–O)carboxylic of the carboxylic moiety (−COOH) of Hmef, respectively, shifted in the IR spectra of complexes 1–6 in the range 1575–1608 cm−1 and 1366–1405 cm−1 and were assigned to the antisymmetric, νasym(CO2), and the symmetric, νsym(CO2), stretching vibration of the carboxylato group, respectively. The most characteristic tool for determining the coordination mode of the carboxylato ligands is the parameter Δ [=νasym(CO2) − νsym(CO2)]. The values of Δ for the complexes are lying in the range 173–221 cm−1; for complex 3 the value of Δ (173 cm−1) rather indicates a bidentate mode of binding while the Δ values of complexes 1, 2, and 4–6 (193–221 cm− 1) are indicative of asymmetrically binding mode of the mefenamato ligands [58]. The UV–vis spectra of the complexes were recorded as nujol mull and in DMSO solution and exhibited similar pattern suggesting that the complexes retain their structure in solution. In the visible region, three low-intensity bands were observed; band I was present in the region 970–1005 nm (ε = 5–15 M−1 cm−1), band II was observed at 620–700 nm (ε = 15–35 M− 1 cm− 1) and band III appeared in the region 385–410 nm (ε = 50–200 M − 1 cm − 1). These three bands may be assigned to d–d transitions and are typical for distorted octahedral Ni2 + complexes. More specifically, for octahedral local symmetry, band I is attributed to a 3A 2g → 3 T2g electronic transition, band II to a 3A 2g → 3 T1g electronic transition and band III to a 3 A2g → 3T1g(P) electronic transition. The values of the ratio 10 Dq/B (= 13.5–16.5) are within the range expected for octahedral Ni 2 + complexes [2,14]. The UV–vis spectra of the complexes were also recorded in the pH range 6–8 in the presence of diverse buffer solutions (150 mM NaCl and 15 mM trisodium citrate at pH values regulated by HCl solution) and there were not any significant changes (shift of the λmax or new peaks). Thus, it may be inferred that the complexes are stable under the experimental conditions where the biological experiments are performed (pH = 7). Additionally, the UV–vis spectra of a DMSO solution (5 × 10−3 M) of the complexes were recorded within the timeframe used for the biological tests (up to 72 h) and no significant changes were observed. Therefore, we may conclude the stability of the compounds in solution during the time, too. Based on the similarity of the UV–vis spectral pattern observed for the complexes in nujol and in DMSO solution (during the time involved in the biological tests), in the presence of the buffer solution (150 mM NaCl and 15 mM trisodium citrate at the pH = 7.0) used in the biological experiments and in the pH range = 6–8 as well as in combination to the non-dissociation in DMSO solution (ΛM ≤ 20 μS/cm, in 1 mM DMSO solution) we may firmly conclude that the compounds keep their integrity in solution. In the cyclic voltammogram of 5 in DMSO solution (Fig. S1), two cathodic waves appear at −525 mV (Epc1) and −1310 mV (Epc2) followed by two anodic waves at −880 mV (Epa2) and −320 mV (Epa1) constituting two quasi-reversible waves. According to the literature, the two quasi-reversible waves could be assigned to redox couples Ni(II)/ Ni(I) (Epc1 and Epa1) and Ni(I)/Ni(0) (Epc2 and Epa2) [59,60]. Complexes 1–4 and 6 exhibit similar electrochemical behavior in DMSO and the corresponding potentials are given in Table 2. As observed from the magnetic measurements, the μeff values (=2.95–3.45 BM) for the complexes are typical for paramagnetic octahedral Ni(II) complexes with d8 configuration and are close to the spin-

Table 2 Cathodic and anodic potentials (in mV) for the redox couples of the complexes in DMSO solution. Complex

Εpc1

Epc2

Epa2

Epa1

[Ni(mef)2(bipy)(MeOH)2], 1 [Ni(mef)2(phen)(MeOH)2], 2 [Ni(mef)2(bipyam)], 3 [Ni(mef)2(Hpko)2], 4 [Ni(mef)2(py)2(H2O)2], 5 [Ni(mef)2(MeOH)4], 6

−480 −505 −505 −530 −525 −525

−1150 −1180 −1250 −1265 −1310 −930

−840 −770 −875 −1030 −880 −730

−355 −320 −340 −250 −320 −315

84


only value μeff (=2.83 BM) at room temperature for a magnetically isolated Ni(II) system [2]. 3.2. Structure of the complexes 3.2.1. Description of the crystal structures of complexes [Ni(mef-O)2 (bipy)(MeOH)2], 1, and [Ni(mef-O)2(phen)(MeOH)2], 2 The crystal structures of complexes [Ni(mef-O)2(bipy)(MeOH)], 1, and [Ni(mef-O)2(phen)(MeOH)], 2, are similar and their characteristic features will be discussed together. A drawing of the molecular structures of the complexes is shown in Fig. 2 and selected bond distances and angles are listed in Table 3. Complexes 1 and 2 are mononuclear and the two mefenamato ligands are deprotonated and coordinated to nickel in a monodentate fashion via oxygen O(1) with the uncoordinated oxygen atoms O(2) forming a strong hydrogen bond with the coordinated methanol ligands (H(214)…O(2) = 1.700 Å in 1 and H(31)…O(2) 1.768 Å in 2). Ni(1) is six-coordinated bearing a NiN2O4 chromophore which is consisted of two carboxylate oxygen atoms (O(1) and O(1)′) of the mefenamato ligands, two methanol oxygen atoms (O(3) and O(3)′) and two nitrogen atoms (N(1) and N(1)′) from bipy and phen for 1 and 2, respectively. The environment around nickel could be rather considered as a distorted octahedron with the methanol oxygens and bipy or phen nitrogens occupying the equatorial positions of the octahedron and the carboxylato oxygens lying trans at the axial positions. Similar structure was also reported for the Co(II)–mefenamato complex [Co(mef)2(bipy)(MeOH)2] [43]. A search in the Cambridge Crystallographic Data Centre (CCDC) database has revealed few mononuclear nickel–carboxylato complexes having similar formulas [Ni(carboxylato-O)2(N,N′-donor)(O-solvent)2]

Fig. 2. A drawing of the molecular structure, the intra-molecular H-bonds and partial (only the heteroatoms) labeling of (A) complex 1 and (B) complex 2.

Table 3 Selected bond distances and angles for complexes 1 and 2. 1

2

Bond distance Ni(1)–O(1) Ni(1)–O(3) Ni(1)–N(1) O(1)–C(1) O(2)–C(1) O(3)–C(21)a or C(22)a

(Å) 2.025(4) 2.042(6) 2.064(7) 1.251(8) 1.245(8) 1.374(8)

(Å) 2.0342(18) 2.065(2) 2.078(2) 1.262(3) 1.226(3) 1.365(4)

Bond angle O(1)–Ni(1)–O(3) O(1)–Ni(1)–O(3)′ O(1)–Ni(1)–O(1)′ O(1)–Ni(1)–N(1) O(1)–Ni(1)–N(1)′ O(1)′–Ni(1)–O(3) O(1)′–Ni(1)–O(3)′ O(1)′–Ni(1)–N(1) O(1)′–Ni(1)–N(1)′ O(3)–Ni(1)–O(3)′ O(3)–Ni(1)–N(1) O(3)–Ni(1)–N(1)′ O(3)′–Ni(1)–N(1) O(3)′–Ni(1)–N(1)′ N(1)–Ni(1)–N(1)′

(°) 88.1(2) 92.1(2) 179.8(4) 93.5(3) 86.4(2) 92.1(2) 88.1(2) 86.4(2) 93.5(3) 90.7(4) 96.2(3) 171.3(3) 171.3(3) 96.2(3) 77.5(4)

(°) 90.74(9) 89.02(8) 179.64(12) 85.13(8) 95.14(8) 89.02(8) 90.74(8) 95.14(8) 85.13(8) 98.60(13) 91.05(9) 168.78(9) 168.78(9) 91.05(9) 79.97(13)

a

C(21) for 1 and C(22) for 2. (′) for 1: 1 − x, −y, z, (′) for 2: 2 − x, y, 1.5 − z

(where N,N′-donor = phen, bipy or its derivatives, and O-solvent = H2O, ethanol or butanol) with complexes 1 and 2. Indeed, only two and three mononuclear Ni(II) analogues of 1 and 2, respectively, were found [61–63]. Both structures are further stabilized by intraligand hydrogen bonds between the secondary amino groups (H(21)) and the coordinated carboxylato oxygens (O(1)) and by the intramolecular hydrogen bonds between the methanol ligand (H(214) or H(31)) and the uncoordinated oxygen atom of the mefenamato ligands (O(2)), (Table S1).

3.2.2. Crystal structure of complex [Ni(mef-O,O′)2(bipyam)], 3 A drawing of the molecular structure of [Ni(mef-O,O′)2(bipyam)], 3 is shown in Fig. 3, and selected bond distances and angles are listed in Table 4. The complex is mononuclear and the mefenamato ligands behave as bidentate chelating ligands in deprotonated mode coordinated to the nickel ion via the carboxylato groups in a rather asymmetric chelating mode (C(1)–O(1) = 1.264(5) Å and C(1)– O(2) = 1.261(5) Å, C(16)–O(3) = 1.278(4) Å and C(16)–O(4) = 1.255(5) Å). Ni(1) is six-coordinated with a NiN2O4 chromophore forming a distorted octahedron around nickel; the six vertices of the octahedron are occupied by four carboxylate oxygen atoms from two mefenamato ligands and two nitrogen atoms from bipyam. The distances around nickel are not equal; the Ni(1)–Nbipyam distances (Ni(1)–N(42) = 2.021(3) Å and Ni(1)–N(38) = 2.030(3) Å) are shorter than the Ni(1)–Omef distances (in the range 2.035(3)–2.211(3) Å). Concerning the search of CCDC database for similar nickel–bipyam complexes, the outcome was rather surprising since there were not found any reported structures of mononuclear nickel complexes of the formula [Ni(carboxylato-O,O′)2(bipyam)]. The structure of the complex is further stabilized by the formation of intraligand hydrogen bonds between the secondary amino groups and the carboxylato oxygen atoms of the mefenamato ligands (N(7)– H(71)⋯O(2) and N(29)–H(291)⋯O(4)) and by the intermolecular hydrogen bonds between the amino group of bipyam and the mefenamato O(3) of the neighboring molecule (N(40)–H(401)⋯O(3)′, Table S1), subsequently forming supramolecular dimers (Fig. 3) around an inversion center which is located in the middle of the Ni(1)…Ni(1)′ distance (=6.680(3) Å).


85

Fig. 3. The molecular structure, partial labeling, intra- and inter-molecular H-bonds of 3.

3.2.3. Structure of cis,cis,trans-[Ni(mef-O)2(Hpko-N,N′)2]·H3OH, 4·CH3OH A drawing of the molecular structure of [Ni(mef-O)2(Hpko-N,N′)2]· CH3OH, 4·CH3OH is shown in Fig. 4 and selected bond distances and angles are listed in Table 5. In complex 4, Ni(II) is six-coordinated and is surrounded by two mefenamato and two Hpko ligands showing a distorted octahedral environment with a NiN4O2 metal center. The bond distances around Ni(1) are rather close (Table 5) with the Ni(1)–O(3) bond distance (= 2.027(3) Å) being the shortest among the bond distances (=2.069(3)–2.084(4) Å). These distances are close to those of previously reported nickel complex with the NSAID diclofenac (= Hdicl) [Ni(dicl)(Hdicl)(Hpko)2](dicl) [38]. The mefenamato ligands are lying in cis positions and behave as deprotonated monodentate ligands coordinated to nickel ion via a carboxylato oxygen. One of the monodentate carboxylate groups of

Table 4 Selected bond distances and angles for complex 3. Bond distance Ni(1)–O(1) Ni(1)–O(2) Ni(1)–O(3) C(1)–O(1) C(1)–O(2) Ni(1)–O(4) Ni(1)–N(38) Ni(1)–N(42) C(16)–O(3) C(16)–O(4)

(Å) 2.035(3) 2.211(3) 2.112(3) 1.264(5) 1.261(5) 2.109(3) 2.030(3) 2.021(3) 1.278(4) 1.255(5)

Bond angle

(°)

O(1)–Ni(1)–O(2) O(1)–Ni(1)–O(3) O(1)–Ni(1)–O(4) O(1)–Ni(1)–N(38) O(1)–Ni(1)–N(42) O(3)–Ni(1)–O(4) O(3)–Ni(1)–N(38) O(3)–Ni(1)–N(42) O(2)–Ni(1)–O(3) O(2)–Ni(1)–O(4) O(2)–Ni(1)–N(38) O(2)–Ni(1)–N(42) O(4)–Ni(1)–N(38) O(4)–Ni(1)–N(42) N(38)–Ni(1)–N(42)

61.64(11) 154.90(11) 97.37(11) 102.35(13) 94.54(12) 61.83(10) 96.95(12) 101.22(12) 101.43(10) 88.10(11) 93.93(12) 156.18(11) 158.59(12) 96.12(12) 90.60(13)

the mefenamato ligands is bound to nickel in symmetric fashion (C(1)–O(1) = C(1)–O(2) = 1.267(4) Å) and the other one in an asymmetric mode (C(16)–O(3) = 1.281(4) Å and C(16)–O(4) = 1.261(4) Å). Weak intraligand interaction through hydrogen bonds between the amine hydrogen atoms and the coordinated oxygen atoms (H(51)∙∙∙O(1) = 1.981 Å and H(535)∙∙∙O(3) = 1.925 Å) contribute to the stabilization of the complex (Table S1). The two Hpko ligands act as bidentate neutral chelating ligands and are bound to nickel via a pyridine nitrogen and the oxime nitrogen while the ketonoxime oxygen remains uncoordinated. This coordination mode of Hpko ligand (1.0110 according to the Harris notation [64]) has been observed in a series of mononuclear metal complexes including [Zn(mefenamato)2(Hpko)2] [44], [Zn(tolfenamato)2(Hpko)2] [65] and [Ni(dicl)(Hdicl)(Hpko)2 ](dicl) [38] as well as in the trinuclear Ni(II) complex [Ni3(shi)2(Hpko)2(py)2] (where H3shi = salicylhydroxamic acid) [66]. According to the CCDC database, there are five more mononuclear Ni(II) complexes of the formula [Ni(carboxylato-O)2(oxime-N,N′)2] [38,67–70]. Furthermore, the ketoxime groups form hydrogen bonds with the adjacent uncoordinated carboxylic oxygen atoms O(2) and O(4) of the mefenamato ligands (H(61)∙∙∙O(2) = 1.664 Å and H(537)∙∙∙O(4) 1.687 Å, Table S1). The lattice of the complex is further stabilized by the formation of a hydrogen bond between the solvate

Fig. 4. The molecular structure and intra-molecular H-bonds of 4·CH3OH with only the heteroatom labeling.

86


a NiN2O4 chromophore for 5 and two oxygen atoms of the mefenamato ligands and four oxygen atoms coming from four methanol ligands forming a NiO6 chromophore in the case of complex 6. The structures of complexes 5 and 6 are similar to those of the previously reported complexes [Cu(mef)2(py)2(MeOH)2] [42,71] and [Co(mef)2(MeOH)4] [43], respectively.

Table 5 Selected bond distances and angles for complex 4. Bond distance

(Å)

Ni(1)–O(1) Ni(1)–O(3) Ni(1)–N(1) C(1)–O(1) C(1)–O(2) N(2)–O(5) C(53)–O(7) Ni(1)–N(2) Ni(1)–N(3) Ni(1)–N(4) C(16)–O(3) C(16)–O(4) N(4)–O(6)

2.069(3) 2.027(3) 2.075(3) 1.267(4) 1.267(4) 1.367(4) 1.446(8) 2.070(3) 2.074(3) 2.084(3) 1.281(4) 1.261(4) 1.366(4)

Bond angle

(ο)

O(1)–Ni(1)–O(3) O(1)–Ni(1)–N(1) O(1)–Ni(1)–N(2) O(1)–Ni(1)–N(3) O(1)–Ni(1)–N(4) N(2)–Ni(1)–N(3) N(2)–Ni(1)–N(4) N(3)–Ni(1)–N(4) O(3)–Ni(1)–N(1) O(3)–Ni(1)–N(2) O(3)–Ni(1)–N(3) O(3)–Ni(1)–N(4) N(1)–Ni(1)–N(2) N(1)–Ni(1)–N(3) N(1)–Ni(1)–N(4)

84.63(11) 91.83(11) 88.50(11) 171.61(11) 98.25(11) 96.70(12) 168.09(12) 77.81(12) 175.84(12) 99.54(11) 88.01(11) 90.89(11) 78.15(12) 95.67(12) 91.77(12)

3.3. Antioxidant capacity of the compounds

methanol molecule and one of the uncoordinated pyridine nitrogen (N(7)) of a dipyridylketoxime ligand (H(71)∙∙∙N(7) = 2.133 Å). 3.2.4. Proposed structures for complexes 5 and 6 Based on the existing IR, UV–vis, magnetic measurements and elemental analysis data, complexes 5 and 6 are neutral mononuclear complexes with the mefenamato ligands behaving as deprotonated monodentate ligands bound to nickel ion via a carboxylate oxygen atom. According to the magnetic data μeff (=3.30–3.39 BM), a square planar geometry around nickel may be ruled out (in such a case a diamagnetic behavior 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) [2]. Additionally, the UV–vis spectra of the complexes suggest a distorted octahedral environment; the vertices of the octahedron are occupied by two oxygen atoms of the mefenamato ligands, two oxygen atoms of two aqua ligands and two nitrogen atoms from the two pyridine ligands giving

The investigation of the antioxidant activity of NSAIDs and their complexes may be a first approach of any potential anti-inflammatory and anticancer activity since NSAIDs are agents related to the inhibition of free radical production acting as radical scavengers [72]. Therefore, the ability of complexes 1–6 to scavenge radicals such as DPPH, ABTS and hydroxyl ones and to inhibit in vitro the soybean LOX activity was determined as a first means to evaluate their antioxidant ability. DPPH scavengers may offer protection against rheumatoid arthritis and inflammation, since DPPH scavenging is closely related to anti-aging and anti-inflammatory activity [31]. Hydroxyl radical (•OH) scavengers may serve as protectors from the reactive oxygen species by activation of the prostaglandins, and the scavenging activity of ABTS cationic radical (ABTS+•) is a marker of the total antioxidant activity [31]. Furthermore, the LOX inhibitors are considered as potential antioxidants or free radical scavengers [32], since LOX are enzymes involved in the transformation of arachidonic acid to leukotrienes and play an important role in several inflammatory and allergic diseases [73]. Additionally, a comparison of the resultant activity to well-known antioxidant agents such as NDGA, BHT, trolox and caffeic acid used as reference compounds (Table 6) revealed its significance. All complexes show similar or higher scavenging activity of all radicals examined (i.e. DPPH, •OH and ABTS+•) than free Hmef (Table 6). The DPPH scavenging activity of the complexes was not time-dependent since no significant changes of the scavenging were observed during 20-min and 60-min measurements. The DPPH scavenging activity of all compounds is of the same magnitude (RA% = 7.42–13.03%) and significant lower than that of the reference compounds NDGA (RA% = 81.02%) and BHT (RA% = 31.30%). The hydroxyl scavenging activity of the complexes (•OH% = 75.28–85.57%) is higher than that of free Hmef and for complexes 2–4 is even higher than the reference compound trolox (•OH% = 82.80%). The ABTS scavenging activity of the compounds (ΑΒΤS% = 89.61–97.23%) is equal to or higher than the reference compound caffeic acid (ΑΒΤS% = 91.80%). All compounds present significant inhibition (IC50 = 25.64–46.71 μM) against soybean lipoxygenase (Table 3), especially when compared to the reference compound caffeic acid (IC50 = 600 μM). Among the complexes, the most active compounds are: (i) complex 5 for DPPH scavenging (RA% = 13.03 (±0.68)%), (ii) complexes 3 and 4 for •OH scavenging (•OH% = 85.57 (±0.80)% and 85.56 (±0.42)%, respectively), (iii) complex 4 for ABTS+• scavenging (ΑΒΤS% = 97.23

Table 6 % DPPH scavenging ability (RA%), % superoxide radical scavenging activity (ABTS%), competition % with DMSO for hydroxyl radical (•OH%), and in vitro inhibition of soybean lipoxygenase (LOX) (IC50, in μM) for complexes 1–6. Compound

RA% (0.1 mM, 20 min)

RA% (0.1 mM, 60 min)

• OH % (0.1 mM)

ΑΒΤS% (0.1 mM)

LOX IC50 (μM)

Hmef [43] [Ni(mef)2(bipy)(MeOH)2], 1 [Ni(mef)2(phen)(MeOH)2], 2 [Ni(mef)2(bipyam)], 3 [Ni(mef)2(Hpko)2], 4 [Ni(mef)2(py)2(H2O)2], 5 [Ni(mef)2(MeOH)4], 6 NDGA BHT Trolox Caffeic acid

5.72 (±0.08) 12.52 (±0.60) 7.42 (±0.78) 12.31 (±0.64) 10.32 (±0.18) 12.56 (±0.82) 10.49 (±0.74) 81.02 (±0.18) 31.30 (±0.10) nta nta

11.74 (±0.20) 10.34 (±0.67) 10.92 (±0.54) 12.42 (±0.24) 12.49 (±0.12) 13.03 (±0.68) 12.38 (±0.52) 82.60 (±0.17) 60.00 (±0.38) nta nta

66.32 (±0.38) 76.09 (±0.14) 83.18 (±0.27) 85.57 (±0.80) 85.56 (±0.42) 75.28 (±0.64) 78.12 (±0.89) nta nta 82.80 (±0.13) nta

92.51 (±0.44) 89.92 (±0.92) 90.03 (±0.33) 93.56 (±0.83) 97.23 (±0.76) 94.76 (±0.12) 89.61 (±0.68) nta nta 91.80 (±0.17) nta

48.52 (±0.88) 46.71 (±0.48) 33.48 (±0.24) 25.64 (±0.38) 35.18 (±0.92) 35.41 (±0.36) 37.67 (±0.05) nta nta nta 600 (±0.3)

a

nt = not tested; Each experiment was performed at least in triplicate SD b ±10%.


A

B

C

D

87

Fig. 5. Comparison of the antioxidant activity of the reported Ni(II), Cu(II), Zn(II) and Co(II)–mefenamato complexes in regard to the average (A) % DPPH scavenging ability (RA%), (B) competition % with DMSO for hydroxyl radical (•OH%), (C) % ABTS radical scavenging activity (ABTS%), and (D) in vitro inhibition of soybean lipoxygenase (LOX) (IC50, in μM).

(±0.76)%) and (iv) complex 3 for LOX inhibitory activity (IC50 = 25.64 (±0.38) μM). In conclusion, the low DPPH scavenging activity of the complexes in combination with their more pronounced hydroxyl and ABTS scavenging activity is evidence of selective activity of the complexes to scavenge hydroxyl and ABTS radicals. This is not unexpected since, in the literature, there are a lot of examples concerning compounds showing selective DPPH scavenging activity [74,75] or others being more active against hydroxyl and ABTS radicals [14,76,77]. A comparison of the antioxidant activity of complexes 1–6 with that of recently reported Co(II) [43], Cu(II) [42] and Zn [44] mefenamato complexes (Table S2) reveals that the scavenging activity of complexes 1–6, especially of DPPH and hydroxyl radicals, is lower than that of their Co(II), Cu(II) and Zn(II) analogues; this is something quite expected, if we consider the biological role and the wider medicinal use of Cu and Zn compounds (Fig. 5). It was unexpected to discover that the Ni(II)– mefenamato complexes are in average better scavengers against ABTS radicals and more active LOX-inhibitors than Co(II), Cu(II) and Zn(II)– mefenamato complexes, although the most active compound is a Cu(II) complex in each case. It should be also noted that in the case of complexes 1–6, the presence of a N,N′-donor ligand results in more active species than [Ni(mef)2(MeOH)4], 6, while in the case of Co(II) [43], Cu(II) [42] and Zn [44] analogues, the complexes [Co(mef)2(MeOH)4], [Cu2(mef)4(H2O)2] and [Zn(mef)2(H2O)4], respectively, are the most active compounds. Furthermore, the ABTS scavenging activity of complex 4 and the LOX inhibitory activity of 3 are in the top-three best results among the metal–mefenamato complexes.

hydrogen-bonding or hydrophobic bonding along major or minor groove of DNA helix) and (iii) cleavage of the DNA helix [78]. In recent studies the potential anticancer and/or anti-inflammatory activity of NSAIDs and their complexes was related to their affinity to DNA [29,79]. Within this context and as continuation of our studies [33,42–44], the interaction of complexes 1–6 with CT DNA is investigated by UV spectroscopy, cyclic voltammetry, DNA viscosity measurements and ethidium bromide displacement examined by fluorescence emission spectroscopy. 3.4.1. DNA-binding studies with UV spectroscopy UV–vis spectroscopy may provide useful information in regard to the interaction mode of complexes with DNA as well as the strength of this binding. Within this context, the UV spectra of a CT DNA solution (1.5–2 × 10−4 M) were recorded in the presence of the complexes at increasing amounts (for different [complex]/[DNA] mixing ratios (=r)) as

3.4. Interaction of complexes 1–6 with calf-thymus DNA On the bases of their structure and the nature of the coordinated ligands, metal complexes may bind to double-stranded DNA via: (i) covalent bonding (with the replacement of one or more labile ligands by a nitrogen base of DNA), (ii) non-covalent interactions (π → π* stacking interaction of the complex and DNA nucleobases resulting to intercalation, development of Coulomb forces between metal complexes and the phosphate groups of DNA leading to electrostatic interactions and groove binding as a result of van der Waals or

Fig. 6. UV spectra of CT DNA (2 × 10−4 M) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the absence or presence of [Ni(mef)2(py)2(H2O)2], 5. The arrow shows the changes upon increasing amounts ((a) → (e)) of the complex.

88


A

DNA results in the direct formation of a new complex with doublestranded CT DNA. Quite similar is the behavior of CT DNA in the presence of the other complexes. In the UV spectra of the complexes 1–6 (1 × 10− 5 M), two intraligand bands appear; band I at 287–300 nm and band II as a shoulder in the region 338–343 nm. The absorbance and the position of these bands may be perturbed upon interaction with CT DNA (addition of the DNA solution up to r′ = [DNA] / [complex] = 1.2). More specifically, in the UV spectra of complex 4, band I appearing at 287 nm and band II at 343 nm exhibit in the presence of increasing amounts of CT DNA a hypochromism of ~ 4% and ~ 40%, respectively (Fig. 7(A) for r ′ = 0–0.8). Additionally, the hypochromism of band I is accompanied by a slight red-shift (~1 nm), while the addition of CT DNA results in an elimination of band II. Similar is the behavior of complexes 1 and 2 in the presence of DNA where band II exhibits a less intense hypochromism (up to 15%) which is accompanied by a 2-nm red-shift (Table 7). In the UV spectra of complexes 3, 5 and 6 in the presence of CT DNA (representatively shown for 6 in Fig. 7(B), for r′ = 0–0.8), band I at ~300 nm exhibits a slight hyperchromism (up to 8.5%) and band II shows a more intense hypochromism (up to 16%); for both bands a slight red-shift (up to 3 nm) appears (Table 7). For all complexes, the observed hypochromism is attributed to π → π stacking interaction between the aromatic chromophore (from mefenamato and/or the N-donor ligands) of the complex and DNA base pairs inferring the possible interaction by an intercalative mode [80]. Although intercalation may be proposed based on the existing spectroscopic data as the most possible mode interaction, it cannot be merely concluded only by UV spectroscopic titration studies and some more experiments including cyclic voltammetry and viscosity measurements were conducted so as to verify the existence of intercalation. In order to evaluate the binding strength of the compounds with CT DNA, the DNA-binding constants (Kb) were calculated by monitoring the changes of the absorbance at the corresponding λmax with increas-

B

Fig. 7. UV spectra of DMSO solution (1 × 10−5 M) of (A) [Ni(mef)2(Hpko)2], 4 and (B) [Ni(mef)2(MeOH)4], 6 in the presence of increasing amounts of CT DNA (r′ = [DNA]/[complex] = 0–0.8). The arrows show the changes upon increasing amounts ((a) → (e)) of CT DNA.

well as the UV spectra of the complexes (1 × 10−5 M) in the presence of CT DNA at increasing amounts. The existence of any interaction between the complex and CT DNA will perturb the band of CT DNA at 258–260 nm or the intra-ligand transition bands of the complexes, respectively, during the titrations; the red-shift shows stabilization of the structure while the blue-shift is evidence of structural destabilization. Furthermore, the intense hypochromism of the transition band, which, in most cases, is accompanied by a bathochromism, is evidence of an intercalative binding mode. The UV spectra of a CT DNA solution in the presence of complex 5 at increasing r values are shown representatively in Fig. 6. The decrease of the absorbance at λmax = 258 nm indicates that the interaction with CT

ing concentrations of CT DNA by the plots

½DNA ðεA −ε f Þ

versus [DNA]

(Fig. S2) using the Wolfe–Shimer equation [53] (Eq. S1). The Kb values of complexes 1–6 (Table 7) are relatively high suggesting strong binding of the complexes to CT DNA and similar to that of the classical intercalator EB (=1.23 (±0.07) × 105 M−1) [81], with complex 6 having the highest Kb value (=2.62 (±0.35) × 105 M−1) among the compounds; with the exception of complex 2, the Kb of the complexes are higher than that of free Hmef suggesting that its coordination to Ni(II) results in an increase of the Kb value. The Kb values of complexes 1–6 are of the same magnitude to those of the metal–NSAID complexes reported so far [42–44]. 3.4.2. Study of the DNA-interaction with cyclic voltammetry Cyclic voltammetry is a commonly used technique that may provide useful information for the mode of interaction of metal ions or metal complexes with DNA [82]. The cyclic voltammograms of each complex in a 1/2 DMSO/buffer solution were recorded upon addition of CT DNA

Table 7 Spectral features of complexes 1–6 in the presence of CT DNA and DNA-binding constants (Kb). Compound

Band (λ, nm) ((ΔA/A0) (%)a, Δλ (nm)b)

Kb (M−1)

Hmef [43] [Ni(mef)2(bipy)(MeOH)2], 1 [Ni(mef)2(phen)(MeOH)2], 2 [Ni(mef)2(bipyam)], 3 [Ni(mef)2(Hpko)2], 4 [Ni(mef)2(py)2(H2O)2], 5 [Ni(mef)2(MeOH)4], 6

324 (+10, −) 298 (−4, +2), 342 (−15, +2) 294 (−3, +2), 340 (−8, +2) 300 (+6.5, +3), 340 (−16, +1) 287 (−4, +1), 343 (−40, elmc) 298 (+8.5, +2), 342 (−15, +3) 299 (+8, +2), 338 (−8.5, +2)

1.05 (±0.02) × 105 1.20 (±0.23) × 105 8.26 (±0.19) × 104 1.46 (±0.33) × 105 1.15 (±0.06) × 106 1.19 (±0.25) × 105 2.62 (±0.35) × 105

a b c

“+” denotes hyperchromism, “−” denotes hypochromism. “+” denotes red-shift, “−” denotes blue-shift. elm = eliminates.


89

(representatively shown for 5 in Fig. S3). A decrease of the current intensity was observed without any new redox peaks suggesting equilibrium between free and DNA-bound complex as evidence of the complex–DNA interaction [82]. The cathodic Epc and anodic Epa potentials of the redox couple Ni(II)/Ni(I) for each complex as well as their shifts upon addition of CT DNA are given in Table 8. Upon addition of CT DNA, the cathodic and the anodic potentials exhibit predominantly a positive shift (ΔEpc/a = (− 5) − (+70) mV). In general, the shift of an electrochemical potential towards a negative direction occurs in the case of an electrostatic interaction with DNA, while a positive shift of the potential takes place in the case of intercalation [60]. Therefore, based on the positive shifts of the cathodic and anodic potentials we may conclude the existence of intercalation as the most possible mode of interaction between the complexes and CT DNA bases [42,43,60]; a conclusion which is in accordance to spectroscopic and viscosity experiments. 3.4.3. Viscosity measurements of DNA solution The viscosity of a DNA solution is sensitive to DNA length changes which may occur in the presence of a compound interacting to DNA. The relation of DNA viscosity (η/η0) and to 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 [83,84]. Therefore, the relative DNA viscosity as a subsequence of DNA length changes is influenced by the presence of a DNA-binder and important information concerning the mode of DNA-binding may be derived. Viscosity measurements were carried out on CT DNA solutions (0.1 mM) upon addition of increasing amounts of complexes 1–6 (up to the value of r = 0.27). It is observed that the relative DNA viscosity increases considerably in the presence of increasing amounts of the complexes (Fig. 8). This behavior indicates that the complexes insert between the DNA bases because of an intercalative binding mode between DNA and each complex. In general, in the case of classic intercalation, the DNA bases will be separated so as to let the intercalating compound enter in between; as a result the DNA helix length will increase leading to enhanced DNA viscosity. When a compound binds to DNA grooves via a partial and/or non-classic intercalation (including electrostatic interaction and groove-binding), the DNA helix will show a bend or a kink accompanied probably by a slight decrease of its effective length; in this case, the viscosity of DNA will be slightly decreased or remain practically unchanged [42–44,84]. 3.4.4. EB-displacement studies Ethidium bromide (EB = 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) is a typical indicator of intercalation to DNA through the planar EB phenanthridine ring since such intercalation results in the appearance of an intense fluorescence emission band at 592 nm (λexcitation = 540 nm) due to the formation of the EB–DNA compound. The addition of a compound able to intercalate to DNA equally or stronger than EB will induce significant quenching of the EB–DNA fluorescence emission [55,85]. Complexes 1–6 do not show any fluorescence emission at room temperature in solution or in the presence of

Table 8 Cathodic and anodic potentials (in mV) for the redox couples of the complexes in DMSO/ buffer solution in the absence or presence of CT DNA. Complex

Epc(f)a

Epc(b)b

ΔΕpcc

Epa(f)a

Epa(b)b

ΔΕpac

[Ni(mef)2(bipy)(MeOH)2], 1 [Ni(mef)2(phen)(MeOH)2], 2 [Ni(mef)2(bipyam)], 3 [Ni(mef)2(Hpko)2], 4 [Ni(mef)2(py)2(H2O)2], 5 [Ni(mef)2(MeOH)4], 6

−745 −730 −702 −670 −715 −718

−750 −731 −705 −670 −715 −701

−5 −1 −3 0 0 +17

−530 −535 −530 −495 −535 −482

−500 −465 −460 −444 −475 −437

+30 +70 +70 +51 +60 +45

a b c

Epc/a in DMSO/buffer in the absence of CT DNA (Epc/a(f)). Epc/a in DMSO/buffer in the presence of CT DNA (Epc/a(b)). ΔEpc/a = Epc/a(b) − Epc/a(f).

Fig. 8. Relative viscosity (η/ηo)1/3 of CT DNA ([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– 6 at increasing amounts (r = [complex] / [DNA]).

CT DNA under the same experimental conditions (λexcitation = 540 nm). Furthermore, the addition of the complexes to a solution of EB does not influence the fluorescence of free EB and does not result in the appearance of new peaks in the spectra. Thus, the changes observed in the fluorescence emission spectra of a solution that contains the EB–DNA compound upon addition of the complexes may be useful to examine the EB-displacing ability of the complexes. The fluorescence emission spectra of pre-treated EB–DNA ([EB] = 20 μM, [DNA] = 26 μM) were recorded in the presence of increasing amounts of each complex 1–6 up to the value of r = 0.22 (Fig. 9(A)). The addition of the complexes results in a significant decrease of the intensity (Fig. 9(B)) of the emission band of the DNA–EB system at 592 nm (the final quenching is up to 70.5–82.5% of the initial EB–DNA fluorescence, Table 9) indicating the strong competition of the complexes with EB in binding to DNA. The observed quenching of DNA–EB fluorescence emission for complexes 1–6 suggests that the complexes can displace EB from the DNA–EB complex, thus revealing the interaction with CT DNA by the intercalative mode [54]. The Stern–Volmer plots (Fig. S4) illustrate that the quenching of the EB–DNA fluorescence emission induced by the complexes 1–6 is in good agreement (R = 0.99) with the linear Stern–Volmer equation (Eq. S2), thus indicating that the observed quenching is a result of the displacement of EB from EB–DNA by each complex [42–44]. The KSV values (Table 9) of the complexes are rather high verifying tight binding to DNA, with complex 1 bearing the highest KSV value (= 3.82 (± 0.08) × 105 M− 1) among the complexes. The KSV values of complexes 1–6 are within the range found for a series of metal complexes with NSAIDs as ligands [33,42–44]. 3.5. Interaction with serum albumins Serum albumins (SAs) are responsible for the transport of ions and drugs to cells and tissues through the bloodstream; therefore, their role is important and they are the most abundant proteins in plasma [55]. Within this context, it is significant to investigate the interaction of biological active compounds (such as complexes 1–6) with SAs, in order to get a preliminary monitoring of their potential transport towards their targets in the body [86]; as a result differentiated biological properties of the compound or novel transport pathways may arise. The solutions of human SA (HSA, having a tryptophan at position 214, i.e. Trp-214) and its homologue bovine SA (BSA, with two tryptophans Trp-134 and Trp-212) exhibit an intense fluorescence emission when excited at 295 nm, with λem,max = 351 nm and 343 nm, respectively, due to the existence of tryptophans [55]. The mefenamato complexes 1–6 did not show any significant fluorescence emission under the

90


A

A

B B

Fig. 9. (A) Fluorescence emission spectra (λexcit = 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 = 0.20). The arrow shows the changes of intensity upon increasing amounts of 1. (B) Plot of EB relative fluorescence emission intensity at λem = 592 nm (%) vs r (r = [complex] / [DNA]) (150 mM NaCl and 15 mM trisodium citrate at pH = 7.0) in the presence of complexes 1–6 (up to 17.5% of the initial EB–DNA fluorescence intensity for 1, 24.5% for 2, 22.5% for 3, 29.5% for 4, 25.5% for 5 and 20% for 6).

same experimental conditions (i.e. λex = 295 nm) [42,43]. The innerfilter effect was also taken into consideration and was calculated with Eq. S3 [56]; it was not found so significant affecting slightly the measurements. The fluorescence emission spectra of HSA and BSA exhibited in the presence of complexes 1–6 a significant decrease of the intensity (Fig. 10) which was much more pronounced in the spectra of BSA (quenching of the SA fluorescence up to ~85% and ~91% in the presence of complex 1 for HSA and BSA, respectively, Fig. 11). The observed quenching in the fluorescence emission spectra of the SAs is attributed to possible changes in tryptophan environment of SA induced by

Table 9 Percentage of EB–DNA fluorescence quenching (ΔI/Io, %) and Stern–Volmer constants (KSV) for complexes 1–6. Compound

EB–DNA fluorescence quenching (%ΔI/Io)

KSV (M−1)

Hmef [42] [Ni(mef)2(bipy)(MeOH)2], 1 [Ni(mef)2(phen)(MeOH)2], 2 [Ni(mef)2(bipyam)], 3 [Ni(mef)2(Hpko)2], 4 [Ni(mef)2(py)2(H2O)2], 5 [Ni(mef)2(MeOH)4], 6

80 82.5 75.5 77.5 70.5 74.5 70.0

1.58 (±0.06) × 105 3.82 (±0.08) × 105 2.70 (±0.08) × 105 2.89 (±0.08) × 105 1.34 (±0.05) × 105 2.16 (±0.09) × 105 1.88 (±0.06) × 105

Fig. 10. Fluorescence emission spectra (λexit = 295 nm) of (A) HSA ([HSA] = 3 μM) and (B) BSA ([BSA] = 3 μM), in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the absence or in the presence of increasing amounts of complex 1 (up to the value of r = [1]/[SA] = 6). The arrows show the changes of intensity upon increasing amounts ((a) → (k)) of 1.

changes in albumin secondary structure, indicating thus the binding of each compound to SA [87]. The values of the quenching constant (kq) for the interaction of complexes 1–6 with both albumins were calculated from the corresponding Stern–Volmer plots (Figs. S5 and S6) using the Stern–Volmer quenching equation (Eq. S4); the results are given in Table 10. The values of kq suggest significant SA quenching ability, they are higher than 1013 M−1 s−1 and indicate the existence of static quenching mechanism [54]. The kq values of the complexes are similar or higher, in most cases, than the those of free Hmef, with 1 exhibiting the highest quenching ability for both albumins (kq(HSA),1 = 3.23 (± 0.13) × 1013 M−1 s−1 and kq(BSA),1 = 5.70 (±0.13) × 1013 M−1 s−1). The values of the kq are within the range found for a series of metal-complexes bearing mefenamato [42–44] and other NSAIDs as ligands [33]. The values of the binding constant (K) of the complexes to both albumins were determined from the corresponding Scatchard plots (Figs. S7 and S8) using the Scatchard equation (Eq. S6) and are given in Table 10. The K values of all complexes 1–6 are relatively high and higher than that of free Hmef; they are of the same magnitude for both albumins with complex 5 exhibiting the highest K value (K(HSA),5 = 3.85 (±0.20) × 105 M−1). Furthermore, the K values of the complexes are of the same magnitude with those calculated for a series of metal complexes with mefenamato [42–44] and other NSAIDs as ligands [33]. In general, the K values of complexes 1–6 are in the range 1.35 × 105–3.85 × 105 M−1 and are considered relatively high showing


A

91

which are considered as the strongest known non-covalent interaction [88], we may conclude the ability of the compounds to get released from the albumins probably upon arrival at their targets [87].

4. Conclusions

B

Fig. 11. (A) Plot of % relative fluorescence emission intensity at λem = 351 nm (%) vs r (r = [complex] / [HSA]) for complexes 1–6 (up to 14.5% of the initial HSA fluorescence for 1, 26.5% for 2, 29.5% for 3, 28% for 4, 42% for 5 and 38% for 6) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). (B) Plot of % relative fluorescence emission intensity at λem = 343 nm (%) vs r (r = [complex]/[BSA]) for complexes 1–6 (up to 9% of the initial BSA fluorescence for 1, 19% for 2, 17% for 3, 26% for 4, 17.5% for 5 and 24.5% for 6) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).

the ability of the compounds to bind to albumins and get transferred by them towards their target cells or tissues. If the K values are compared to the association constant of avidin and diverse ligands (K ≈ 1015 M−1)

The neutral mononuclear nickel(II) complexes with the non-steroidal anti-inflammatory drug mefenamic acid in the presence of the nitrogen-donor heterocyclic ligands 2,2′-bipyridine, 1,10phenanthroline, 2,2′-bipyridylamine, 2,2′-dipyridylketone oxime, pyridine and/or the O-donor ligand methanol or H 2 O were prepared and characterized. The crystal structures of four complexes, namely [Ni(mef) 2 (bipy)(MeOH) 2 ], [Ni(mef) 2 (phen)(MeOH) 2 ], [Ni(mef) 2 (bipyam)] and [Ni(mef) 2 (Hpko) 2 ]∙MeOH, were determined by X-ray crystallography. In the six resultant complexes, the mefenamato ligands are deprotonated and bound to Ni(II) in a bidentate O,O′-chelating mode in complex [Ni(mef)2(bipyam)] and in a monodentate binding mode in the rest complexes. The complexes 1–6 have high quenching ability of BSA and HSA fluorescence presenting tight binding affinity to these proteins as suggested by the relatively high binding constants, which are indicative of their binding to SA and potential of the release arrival at their targets. The complexes were more active than free Hmef in regard to the in vitro scavenging activity of DPPH, hydroxyl and superoxide radicals and inhibition of soybean lipoxygenase, especially acting as hydroxyl- and superoxide-scavengers and significant LOX-inhibitors. Diverse spectroscopic, physicochemical and electrochemical techniques were employed to examine the binding of the complexes to CT DNA. The binding constants of the complexes to CT DNA were calculated by UV spectroscopic titrations with [Ni(mef)2(Hpko)2], 4 having the highest Kb value among the compounds. Measurement of the DNA viscosity and cyclic voltammetry experiments indicated intercalation as the most possible interaction mode with CT DNA, a conclusion which was verified by the EB-displacement ability of the complexes. According to the existing results from the in vitro albumin- and DNAbinding and antioxidant activity studies, complexes 1–6 might be considered interesting for their use as potential nickel metallodrugs. The biological activity of the complexes could be further evaluated by investigating biological properties such as the anti-inflammatory activity and antiproliferative activity in diverse cell lines and/or by proposing the mechanism of action. Alternatively, novel similar Ni(II) complexes could be prepared and characterized by modifying the NSAID or the nitrogen-donor ligand or by inserting mixed-atom (N,O-, O,S-, N,S-) donors as co-ligands.

Table 10 The BSA and HSA binding constants and parameters (KSV, kq, K, n) derived for complexes 1–6. Compound

KSV (M−1)

kq (M−1 s−1)

K (M−1)

n

BSA Hmef [42] [Ni(mef)2(bipy)(MeOH)2], 1 [Ni(mef)2(phen)(MeOH)2], 2 [Ni(mef)2(bipyam)], 3 [Ni(mef)2(Hpko)2], 4 [Ni(mef)2(py)2(H2O)2], 5 [Ni(mef)2(MeOH)4], 6

2.78 (±0.20) × 105 5.70 (±0.13) × 105 2.19 (±0.04) × 105 2.66 (±0.07) × 105 1.56 (±0.04) × 105 2.52 (±0.05) × 105 1.65 (±0.06) × 105

2.78 (±0.20) × 1013 5.70 (±0.13) × 1013 2.19 (±0.04) × 1013 2.66 (±0.07) × 1013 1.56 (±0.14) × 1013 2.52 (±0.05) × 1013 1.65 (±0.06) × 1013

1.35 (±0.22) × 105 3.23 (±0.14) × 105 3.10 (±0.14) × 105 2.33 (±0.11) × 105 1.35 (±0.07) × 105 3.22 (±0.09) × 105 2.11 (±0.10) × 105

1.20 1.08 0.93 1.04 1.05 0.95 0.96

HSA Hmef [42] [Ni(mef)2(bipy)(MeOH)2], 1 [Ni(mef)2(phen)(MeOH)2], 2 [Ni(mef)2(bipyam)], 3 [Ni(mef)2(Hpko)2], 4 [Ni(mef)2(py)2(H2O)2], 5 [Ni(mef)2(MeOH)4], 6

7.13 (±0.34) × 104 3.23 (±0.13) × 105 1.45 (±0.04) × 105 1.20 (±0.04) × 105 1.30 (±0.09) × 105 1.18 (±0.15) × 105 7.81 (±0.48) × 104

7.13 (±0.34) × 1012 3.23 (±0.13) × 1013 1.45 (±0.04) × 1013 1.20 (±0.04) × 1013 1.30 (±0.09) × 1013 1.18 (±0.15) × 1013 7.81 (±0.48) × 1012

1.32 (±0.15) × 105 2.44 (±0.08) × 105 2.23 (±0.14) × 105 2.03 (±0.16) × 105 3.42 (±0.23) × 105 3.85 (±0.20) × 105 3.00 (±0.20) × 105

0.82 1.04 0.91 0.88 0.83 0.66 0.69

92


Abbreviations ABTS BHT bipy bipyam BSA CT DMF DPPH EB Hpko Hmef HSA LOX mef NDGA NSAID phen py s SA sh TEAP trolox vs

2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation butylated hydroxytoluene 2,2′-bipyridine 2,2′-bipyridylamine bovine serum albumin calf-thymus N,N-dimethylformamide 1,1-diphenyl-picrylhydrazyl ethidium bromide, 3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide 2,2′-dipyridylketone oxime mefenamic acid, 2-(2,3-dimethylphenylamino)benzoic acid, N-(2,3-xylyl)anthranilic acid human serum albumin soybean lipoxygenase mefenamato anion nordihydroguaiaretic acid non-steroidal anti-inflammatory drug 1,10-phenanthroline pyridine strong serum albumin shoulder tetraethylammonium perchlorate 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid very strong

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