Nickel–quinolones interaction

Journal of Inorganic Biochemistry 104 (2010) 740–749

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Journal of Inorganic Biochemistry j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i n o r g b i o

Nickel–quinolones interaction Part 3 — Nickel(II) complexes of the antibacterial drug flumequine Kalliopi C. Skyrianou a, Franc Perdih b, Iztok Turel b, Dimitris P. Kessissoglou a, George Psomas a,⁎ a b

Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, P.O. Box 135, GR-54124 Thessaloniki, Greece Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia

a r t i c l e

i n f o

Article history: Received 11 December 2009 Received in revised form 12 March 2010 Accepted 16 March 2010 Available online 24 March 2010 Keywords: Quinolones Flumequine Ni(II) complexes Interaction with calf-thymus DNA Interaction with albumin

a b s t r a c t Nickel(II) complexes with the first-generation quinolone antibacterial agent flumequine in the presence or absence of nitrogen donor heterocyclic ligands (4-benzylpyridine, pyridine, 2,2′-bipyridine or 1,10phenanthroline) have been structurally characterized by physicochemical and spectroscopic techniques. The experimental data suggest that flumequine acts as deprotonated bidentate ligand coordinated to Ni(II) through the carboxylato and ketone oxygen atoms. The crystal structures of bis(4-benzylpyridine)bis (flumequinato)nickel(II) 2, (2,2′-bipyridine)bis(flumequinato)nickel(II) 4 and (1,10-phenanthroline)bis (flumequinato)nickel(II) 5 have been determined by X-ray crystallography and are the first crystal structures of flumequinato complexes reported. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes bind to CT DNA and bis(aqua)bis(flumequinato)nickel(II) exhibits the highest binding constant to CT DNA. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. The cyclic voltammograms of the complexes recorded in DMSO solution and in 1/2 DMSO/buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) solution have shown that in the presence of CT DNA they bind to CT DNA by the intercalative binding mode. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. © 2010 Elsevier Inc. All rights reserved.

1. Introduction The bioinorganic chemistry of nickel [1–3] has been rapidly expanded due to the increasing number of nickel complexes of biological interest reported in the literature. In this context structurally characterized nickel complexes have been able to act as antiepileptic [4], anticonvulsant [5] agents or vitamins [6] or have shown antibacterial [7–9], antifungal [9–11], antimicrobial [12] and anticancer/ antiproliferative [13–16] activity. A principal target of metals in the cancer cells is the coordination with DNA, bound selectively to it through the oxygen of phosphates and/or to heterocyclic nitrogen atoms of DNA bases [17,18]. Therefore, the interaction of Ni(II) complexes with DNA has been mainly dependent on the structure of the ligand exhibiting intercalative behavior [18–23] and/or DNA cleavage ability [24,25]. Flumequine, Hflmq (Fig. 1), is a synthetic first-generation quinolone structurally related to nalidixic and oxolinic acids or ofloxacin [26] and was first prepared by Rinker Laboratory in 1976 [27,28]. Quinolones (or quinolonecarboxylic acids) are a group of antibacterial agents that effectively inhibit DNA replication and are commonly used

⁎ Corresponding author. Tel.: + 30 2310997790; fax: +30 2310997738. E-mail address: [email protected] (G. Psomas). 0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2010.03.007

as treatment for many infections [29–31]. Flumequine is highly effective in treating urinary tract infections. It also is reported to have activity against some Gram-positive and Gram-negative microorganisms [32]. It is used in veterinary medicine for treatment of animal diseases caused by a wide-range of Gram-negative bacteria (Escherichia coli, Salmonella and Pasteurella) [33]. To our knowledge, no crystal structures of flumequine complexes have been reported in the literature yet, although the role of flumequine in the treatment of urinary tract infections is known for the last three decades [28]. Taking into consideration that metal complexes with drugs may exhibit more pronounced biological properties in comparison to the free drugs, we have initiated the study of nickel(II) complexes containing quinolone antimicrobial agents [19–21]. In this context, we report the synthesis, the structural characterization, the electrochemical and the biological properties of the neutral mononuclear nickel(II) complexes with the first-generation quinolone antibacterial drug flumequine with or without nitrogen donor heterocyclic ligand such as 4-benzylpyridine (4bzpy), pyridine (py), 2,2′-bipyridine (bipy) or 1,10-phenanthroline (phen) [Ni(flmq)2(4bzpy)2], 2, [Ni(flmq)2(py)2], 3, [Ni(flmq)2(bipy)], 4, [Ni(flmq)2(phen)], 5, and [(Ni(flmq)2(H2O)2], 1. The crystal structures of complexes 2, 4 and 5 have been determined by X-ray crystallography. The binding properties of the complexes with calf-thymus (CT) DNA have been investigated with UV spectroscopy and cyclic voltammetry. Competitive binding studies

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2.2. Synthesis of the complexes

Fig. 1. Flumequine (Hflmq = 6,7-dihydro-9-fluoro-5-methyl-1-oxo-1H,5H-[ij]quinolizine-2-carboxylic acid).

with ethidium bromide (EB = 3,8-Diamino-5-ethyl-6-phenyl-phenanthridinium bromide) have been performed in order to investigate the existence of a potential intercalation of the complexes to CT DNA. The affinity of Hflmq and complexes 1–5 for human serum albumin (HSA) and bovine serum albumin (BSA) has been investigated with fluorescence spectroscopy. 2. Experimental 2.1. Materials – instrumentation – physical measurements Flumequine, CT DNA, BSA, HSA and EB were purchased from Sigma, NaCl and all solvents were purchased from Merck, trisodium citrate was purchased from Riedel-de Haen and NiCl2· 6H2O, bipy, phen, py, 4bzpy and KOH were purchased from Aldrich Co. All the chemicals and solvents were reagent grade and were used as purchased. Tetraethylammonium perchlorate (= TEAP) was purchased from Carlo Erba and, prior to its use, it was recrystallized twice from ethanol 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 1.89, indicating that the DNA was sufficiently free of protein contamination [34]. The DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M−1 cm−1 [35]. 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 using mercury tetrathiocyanatocobaltate(II) as a calibrant. C, H and N elemental analysis were performed on a Perkin-Elmer 240B elemental analyzer. Molecular conductivity measurements were carried out with a Crison Basic 30 conductometer. Fluorescence spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer. Cyclic voltammetry (CV) studies were performed on an Eco chemie Autolab Electrochemical analyzer. Cyclic voltammetric experiments were carried out in a 30 mL three-electrode 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.

2.2.1. [Ni(flmq)2(H2O)2], 1 Flumequine (0.6 mmol, 157 mg) and KOH (0.6 mmol, 33 mg) dissolved in 15 mL of methanol, were added to a methanolic solution (10 mL) of NiCl2·6H2O (0.3 mmol, 72 mg) and the reaction mixture was refluxed for 1.5 h. The resultant blue solution was left for slow evaporation. After a few days a light blue microcrystalline product was deposited and collected with filtration. Yield: 150 mg, 80%. Anal. Calcd. for [Ni(flmq)2(H2O)2] (C28H24F2N2NiO10) (MW = 645.21): C 52.12, H 3.75, N 4.34; found C 51.89, H 3.98, N 4.45. IR: νmax/cm−1 ν(O–H)w, 3400 (m(medium)); ν(C O)ket, 1625 (vs(very strong)); νasym(CO2), 1592 (vs); νsym(CO2), 1375 (vs); Δ = νasym(CO2) − νsym (CO2): 217 cm−1 (KBr disk); UV–vis: λ/nm (ε/M−1 cm−1) as nujol mull: 760, 655, 460 (sh(shoulder)), 405 (sh), 325, 280; in DMSO: 765 (10), 640 (15), 455 (sh) (15), 398 (sh) (150), 330 (1900), 295 (8600); 10Dq = 13072 cm−1, B = 1258 cm−1. μeff = 3.22 BM. The complex is soluble in DMSO and is non-electrolyte. 2.2.2. [Ni(flmq)2(4bzpy)2]·4MeOH, 2·4MeOH The addition of a methanolic solution (15 mL) of flumequine (0.6 mmol, 157 mg) and KOH (0.6 mmol, 33 mg) to a methanolic solution (10 mL) of NiCl2· 6H2O (0.3 mmol, 72 mg) was followed by the addition of 2 mL of 4-benzylpyridine resulted a dark blue solution that was stirred for 2 h and left for slow evaporation. Dark blue crystals of [Ni(flmq)2(4bzpy)2]·4MeOH, 2·4MeOH suitable for X-ray structure determination, were deposited after a few days. Yield: 200 mg, 65%. Anal. Calcd. for [Ni(flmq)2(4bzpy)2]·4MeOH (C56H60F2N4NiO10) (MW = 1045.79): C 64.32, H 5.78, N 5.36; found C 63.90, H 5.58, N 5.03. IR: νmax/cm−1 ν(C O)ket 1620 (vs); νasym(CO2): 1588 (vs); νsym(CO2): 1375 (vs); Δ = νasym(CO2) − νsym(CO2): 213 cm−1 (KBr disk); UV–vis: λ/nm (ε/M−1 cm−1) as nujol mull: 755, 645, 455 (sh), 400 (sh), 325, 295; in DMSO: 750 (25), 650 (45), 452 (sh) (50), 395(sh) (240), 330 (4800), 290 (12100); 10Dq = 13333 cm−1, B = 1225 cm−1. μeff = 3.31 BM. The complex is soluble in DMSO and DMF and is non-electrolyte. 2.2.3. [Ni(flmq)2(py)2], 3 Complex 3 was prepared in a similar way to 2 with the use of pyridine instead of 4-benzylpyridine. The deposited green microcrystalline solid was collected after a few days. Yield: 150 mg, 70%. Anal. Calcd. for [Ni(flmq)2(py)2] (C38H32F2N4NiO6) (MW = 737.40): C 61.90, H 4.37, N 7.60; found C 61.70, H 4.28, N 7.78. IR: νmax/cm−1 ν(C O)ket 1621(vs); νasym(CO2): 1588 (vs); νsym(CO2): 1397 (vs); Δ = νasym(CO2) − νsym(CO2): 191 cm−1 (KBr disk); UV–vis: λ/nm (ε/ M−1 cm−1) as nujol mull: 760, 645, 460 (sh), 401 (sh), 330, 297; in DMSO: 754 (sh) (16), 640 (28), 452 (sh) (25), 397(sh) (90), 332 (3870), 292 (15700); 10Dq = 13263 cm−1, B = 1241 cm−1. μeff = 2.82 BM. The complex is soluble in DMSO, DMF and ethanol and is non-electrolyte. 2.2.4. [Ni(flmq)2(bipy)]·6.4H2O, 4·6.4H2O Flumequine (0.6 mmol, 157 mg) was dissolved in methanol (15 mL) followed by the addition of KOH (0.6 mmol, 33 mg). The solution was added slowly, and simultaneously with a methanolic solution of bipy (0.3 mmol, 46 mg), to a methanolic solution (10 mL) of NiCl2·6H2O (0.3 mmol, 72 mg) and the resultant solution was left for slow evaporation. Blue crystals of [Ni(flmq)2(bipy)]·6.4H2O, 4·6.4H2O suitable for X-ray structure determination, were deposited after a few days. Yield: 180 mg, 70%. Anal. Calcd. for [Ni(flmq)2(bipy)]·6.4H2O (C38H42.9F2N4NiO12.4) (MW =851.21): C 53.62, H 5.18, N 6.58; found C 53.40, H 5.01, N 6.37. IR: νmax/cm−1; ν(C O)ket: 1620 (vs); νasym(CO2): 1583 (vs); νsym(CO2): 1397 (vs); Δ = νasym(CO2) − νsym(CO2): 186 cm−1 (KBr disk); UV–vis: λ/nm (ε/M−1 cm−1) as nujol mull: 760, 645, 455 (sh), 405 (sh), 333, 298 ; in DMSO: 760 (13), 642 (25), 450 (sh) (40), 401 (sh) (220), 330 (2960), 299 (10000); 10Dq= 13158 cm−1, B =1286 cm−1.

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μeff = 2.87 BM. The complex is soluble in DMSO, DMF, ethanol and CH3CN and is non-electrolyte. 2.2.5. [Ni(flmq)2(phen)]·1.4MeOH·1.1H2O, 5·1.4MeOH·1.1H2O Complex 5 was prepared in a similar way to 4 with the use of phen (0.3 mmol, 54 mg) instead of bipy. Blue crystals of [Ni(flmq)2 (phen)]·1.4MeOH·1.1H2O , 5·1.4MeOH·1.1H2O suitable for X-ray structure determination, were deposited after a week. Yield: 145 mg, 65%. Anal. Calcd. for [Ni(flmq)2(phen)]·1.44MeOH·1.13H2O (C41.44H38.02F2N4NiO8.57) (MW=825.89): C 60.27, H 4.64, N 6.78; found C 60.97, H 4.49, N 6.53. IR: νmax/cm−1 ν(C O)ket 1625 (vs); νasym(CO2): 1594 (vs); νsym(CO2): 1399 (vs); Δ = νasym(CO2) − νsym(CO2): 195 cm−1 (KBr disk); UV–vis: λ/nm (ε/M−1 cm−1) as nujol mull: 755, 650, 450 (sh), 397 (sh), 330, 295; in DMSO: 755 (sh) (14), 652 (21), 455 (sh) (25), 395 (sh) (160), 330 (1780), 295 (15000); 10Dq = 13245 cm−1, B=1217 cm−1. μeff =2.97 BM. The complex is soluble in DMSO, DMF and acetonitrile and is non-electrolyte. 2.3. X-Ray crystal structure determination Single-crystal X-ray diffraction data were collected at 150(2) K with a Nonius Kappa CCD diffractometer with graphite monochromated Mo-Kα radiation (λ = 0.71073 Å). The data were processed by using DENZO [36]. The structure was solved by direct methods implemented in SIR-97 [37] and refined by a full-matrix least-squares procedure based on F2 with SHELXL-97 [38]. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were placed at calculated positions and treated using appropriate riding models unless otherwise noted. Crystallographic data are listed in Table 1. In the crystal structure of 2·4MeOH one flumequine ligand has a disorder at C26, C28 in ratio 0.81:0.19 and it was refined using DFIX instructions; the other flumequine ligand has similar but poorly resolved disorder at C14 and C12, which was refined using SIMU and DELU instructions. Two methanol molecules are disordered over two positions (0.73:0.27 and 0.55:0.45), and DFIX instruction was used for the C55–O9 bond refinement (Fig. S1). Hydrogen atom attached to methanol oxygen, atom O10, was not found in difference Fourier maps, was not placed at calculated position and was not included in the refinement. In the crystal structure of 4·6.43H2O one flumequine ligand has a disorder at C26, C28 in ratio 0.60:0.40 and it was refined using SIMU and DELU instructions. There are five water solvate molecules in the structure, which were refined with full occupation factor (O7–O11; Table 1 Crystallographic data for the complexes.

Formula Fw T Crystal system Space group a= b= c= α= β= γ= Volume Z D(calc) Abs. coef., μ F(000) θ range GOF on F2 R1 wR2 a b c

2·4MeOH

4·6.43H2O

5·1.44MeOH·1.13H2O

C56H60F2N4NiO10 1045.79 150(2) Triclinic P1̄ 10.7040(3) 13.9053(3) 17.3766(4) 101.257(2) 92.600(2) 95.872(2) 2517.64(11) 2 1.380 0.457 1100 3.70–27.42 1.053 0.0494 a 0.1211 a

C38H42.86F2N4NiO12.43 851.21 150(2) Monoclinic P21/n 17.1581(3) 13.4176(2) 17.4587(3) 90 105.7954(10) 90 3867.58(11) 4 1.462 0.581 1777 3.38–27.47 1.028 0.0537 b 0.1442 b

C41.44H38.02F2N4NiO8.57 825.89 150(2) Monoclinic P2/n 11.3491(2) 9.9321(2) 17.0806(4) 90 102.5247(13) 90 1879.51(7) 2 1.459 0.588 858 3.17–27.47 1.045 0.0527 c 0.1409 c

8927 reflections with IN2σ(I). 7407 reflections with IN2σ(I). 3763 reflections with IN2σ(I).

two of them are in disorder), and two water solvate molecules with refined occupation factor 0.90(3) for O12 and 0.53(2) for O13. Hydrogen atoms attached to water oxygen atoms O7 were found in difference Fourier maps and refined as Uiso(H) = 1.2Ueq(O), DFIX instruction was used for O7–H7A bond refinement. Hydrogen atoms on other water oxygen atoms (O8–O13) were not found in difference Fourier maps and were not included in the refinement (Fig. S2). In the crystal structure of 5·1.44MeOH·1.13H2O flumequine ligand has a disorder at C12, C14 in ratio 0.50:0.50 and it was refined using SIMU and DELU instructions. Crystals were isolated as a mixed water/ methanol solvate, with MeOH:H2O ratio 0.72:0.28, DFIX instruction was employed for the C21–O4 bond refinement (Fig. S3). Hydrogen atoms attached to water oxygen atoms O5 and O6 were not found in difference Fourier maps and were not included in the refinement. 2.4. DNA binding studies The interaction of flumequine and complexes 1–5 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 compounds 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. The magnitude of the binding strength of compounds with CT DNA can be estimated through 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 and is given by the ratio of ½DNA versus [DNA] (Insets in Fig. 7), slope to the y intercept in plots ðεA −εf Þ according to the equation [39]: ½DNA ½DNA 1 = + ðεA −εf Þ ðεb −εf Þ Kb ðεb −εf Þ

ð1Þ

where [DNA] is the concentration of DNA in base pairs, εA = Aobsd/ [compound], εf = the extinction coefficient for the free compound and εb = the extinction coefficient for the compound in the fully bound form. The interaction of complexes 1–5 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 DNA in buffer (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). The intercalating effect of flumequine and complexes 1–5 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 Stern–Volmer constant KSV is used to evaluate the quenching efficiency for each compound according to the Eq. (2): Io = 1 + KSV ½Q  I

ð2Þ

where Io and I are the emission intensities in the absence and the presence of the quencher, respectively, [Q] is the concentration of the

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quencher (Hflmq or complexes 1–5). KSV is the Stern–Volmer Io constant and can be obtained by the slope of the diagram vs [Q]. I 2.5. Albumin binding experiments 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 Hflmq or complexes 1–5 as quenchers with increasing concentration [40,41]. Fluorescence spectra were recorded from 300 to 500 nm at an excitation wavelength of 296 nm. The fluorescence spectra of Hflmq and complexes 1–5 in buffer solutions were recorded under the same experimental conditions and exhibited a maximum emission 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. The Stern–Volmer and Schatchard graphs may be used in order to study the interaction of a quencher with serum albumins. According to Stern–Volmer quenching equation [42]: Io = 1 + kq t0 ½Q  = 1 + KSV ½Q ; I

ð3Þ

where Io = the initial tryptophan fluorescence intensity of SA, I = the tryptophan fluorescence intensity of SA after the addition of the quencher, kq = the quenching rate constants of SA, KSV = the dynamic quenching constant, τo = the average lifetime of SA without the quencher, [Q] = the concentration of the quencher respectively, KSV = kqτo and, taking as fluorescence lifetime (τo) of tryptophan in SA at around 10−8 s [42], the dynamic quenching constant (KSV, M−1) Io vs [Q] (Figs. S5(A) can be obtained by the slope of the diagram I and S6(A)), and subsequently the approximate quenching constant (kq, M−1 s−1) may be calculated. Using the Scatchard [43] equation: ΔI Io

½Q 

= nK−K

ΔI Io

ð4Þ

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

ΔI Io

½Q 

versus

ΔI (Figs. S5(B) and S6(B)) and n is given by Io

the ratio of y intercept to the slope [43]. 3. Results and discussion 3.1. Synthesis and spectroscopic study of the complexes The synthesis of the complexes was achieved via the aerobic reaction of flumequine (C13H11FNO-COOH) and KOH with NiCl2· 6H2O in the absence, Eq. (5) for 1, or presence of the corresponding N-donor heterocyclic ligand, e.g. 4-bzpy (C12H11N) for 2 Eq. (6) and phen (C12H8N2) for 5 Eq. (7), according to the equations: NiCl2 ·6H2 O þ 2C13 H11 FNO  COOH þ 2KOH→½NiðC13 H11 FNO  COOÞ2 ðH2 OÞ2  þ2KCl þ 6H2 O

ð5Þ

NiCl2 ·6H2 O þ 2C13 H11 FNO  COOH þ 2KOH þ2C12 H11 N→½NiðC13 H11 FNO  COOÞ2 ðC12 H11 NÞ þ 2KCl þ 8H2 O NiCl2 ·6H2 O þ 2C13 H11 FNO  COOH þ 2KOH þC12 H8 N2 →½NiðC13 H11 FNO  COOHÞ2 ðC12 H8 N2 Þ þ 2KCl þ 8H2 O

ð6Þ

ð7Þ

743

IR spectroscopy has been used in order to confirm the deprotonation and binding mode of flumequine. The observed absorption bands at 3435(br,m) cm−1 , 1718(s) cm−1 and 1270(s) cm−1 attributed to the stretching vibrations ν(H–O), ν(C O)carboxylic and ν(C–O)carboxylic, respectively, of the carboxylic moiety (–COOH) of flumequine, have been replaced in the IR spectra of the complexes by two very strong characteristic bands in the range 1580–1595 cm−1 and 1375–1400 cm−1 assigned as antisymmetric, νasym(C O), and symmetric, νsym(C O), stretching vibrations of the carboxylato group, respectively, while that of ν(H–O) has disappeared. The difference Δ [= νasym(C O) − νsym(C O)], a useful characteristic tool for determining the coordination mode of the carboxylato ligands, gives a value in the range 185–217 cm−1 indicative of a monodentate coordination mode [44]. The vibration ν(C O)ket is slightly shifted from 1618 cm−1 to 1620–1625 cm−1 upon bonding. The overall changes of the IR spectrum suggest that flumequinato ligand is coordinated to the nickel via the ketone oxygen and a carboxylato oxygen [29]. The UV–vis spectra of the complexes recorded as nujol mull and in DMSO solution show similar pattern suggesting that the complexes retain the same structure in solution. In the visible region, three lowintensity bands are observed in the region 750–765 nm (band I), 640– 652 nm (band II) and 450–455 nm (band III) nm assigned to d–d transitions, typical for distorted octahedral Ni2+ complexes [3,20,21]. For octahedral local symmetry, band I can be attributed to a 3A2g → 3T2g transition, band II to a 3A2g → 3T1g transition and band III to a 3 A2g → 3T1g(P) transition. The values of the ratio 10Dq/B (=10.2– 10.9) are within the range expected for octahedral Ni2+ complexes [20,21]. Additionally, an absorption band assigned to the ligand-tometal charge transfer transition for the quinolone ligand [19–21,45,46] appears at around 400 nm. The observed magnetic moment values (μeff = 2.82–3.31 BM) not showing spin-orbit interaction are typical for paramagnetic octahedral Ni(II) complexes with d8 configuration and are close to the spinonly value (=2.83 MB) at room temperature for a magnetically isolated Ni(II) system [3]. 3.2. Crystal structure of [Ni(flmq)2(4bzpy)2]·4MeOH, 2·4MeOH A diagram of 2 is shown in Fig. 2, and selected bond distances and angles are listed in Table 2. The complex is monomeric, the flumequinato ligand behaves as a bidentate deprotonated ligand and is coordinated to nickel ion via the ketone oxygen and a carboxylate oxygen. The nickel atom is six-coordinate and is in octahedral environment of two nitrogen and four oxygen atoms from the two 4-benzylpyridine ligands from the two flumequinato ligands, respectively. The arrangement of the two flumequinato ligands is such that the two ketone oxygen atoms [O(1)–Ni(1)–O(4) = 85.42(6)°] are in a cis arrangement and the two carboxylato oxygen atoms [O(2)–Ni(1)– O(5) = 179.14(6)°] are trans to each other. The Ni–Ocarb distances of oxygen atoms in trans position [Ni(1)–O(2) = 2.030(2) Å, Ni(1)– O(5) = 2.014(2) Å] are shorter than the Ni–Oket [Ni(1)–O(1) =2.063 (2) Å, Ni(1)–O(4) = 2.075(2) Å] occupying cis positions. The Ni–N bond distances [Ni(1)–N(3) = 2.079(2) Å, Ni(1)–N(4) = 2.085(2) Å] are similar to the values reported for other Ni(II) complexes with Ndonor heterocyclic ligands [47,48]. The N4bzpy–Ni–N4bzpy angle [N (3)–Ni(1)–N(4) = 95.44(7)°] shows a cis arrangement of the two 4benzylpyridine ligands. Similar structural arrangements have also been observed in the crystal structure of [Ni(sparfloxacinato)2 (pyridine)2]·6.4H2O [19]. 3.3. Crystal Structure of [Ni(flmq)2(bipy)]· 6.4H2O, 4·6.4H2O A diagram of 4 is shown in Fig. 3, and selected bond distances and angles are listed in Table 3. The complex is mononuclear and the flumequinato ligand behaves as a bidentate deprotonated ligand

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Fig. 2. A drawing of the molecular structure of 2 with only the heteroatoms labeling. Fig. 3. A drawing of the molecular structure of 4 with only the heteroatoms labeling.

coordinated to nickel atom via the ketone oxygen and a carboxylate oxygen. The nickel atom is bound to two bidentate flumequinato ligands and a bidentate 2,2′-bipyridine ligand forming a distorted octahedron with six corners occupied by the two nitrogen atoms of bipy and four oxygen atoms from the flumequinato ligands. The Ni–O distances Ni–Oket [Ni(1)–O(1) = 2.062(2) Å and Ni(1)–O(4) = 2.017(2) Å] and Ni–Ocarb [Ni(1)–O(2) = 2.029(2) Å and Ni(1)–O(5) = 2.041(2) Å] are slightly shorter than the Ni–N [Ni(1)–N(3)= 2.056(2) Å and Ni (1)–N(4)= 2.066(2) Å] distance. The two ketone oxygen atoms [O (1)–Ni(1)–O(4) = 87.78(7)°] and the two carboxylato oxygen atoms [O(2)–Ni–O(5) = 91.15(7)°] lie in cis arrangement. On the contrary, in the crystal structures of the previously reported Ni(II)–quinolone complexes [Ni(oxolinato)2(bipy)] [21] and [Ni(sparfloxacinato)2 (phen)] [20], the two carboxylato oxygen atoms are in trans arrangement. The N(3)–Ni(1)–N(4) angle observed is 79.39(9)° and is similar to reported values of other chelating polycyclic diimines [47,49]. The bipyridine ligand is planar with the nickel atom lying in this plane. The Ni–N bond distances [2.056(2) and 2.066(3) Å] are similar to the values reported for other α-diimine complexes [48]. 3.4. Crystal structure of [Ni(flmq)2(phen)]·1.44MeOH·1.13H2O, 5·1.44MeOH·1.13H2O A diagram of 5 is shown in Fig. 4, and selected bond distances and angles are listed in Table 4. The complex is mononuclear and the

Table 3 Selected bond distances and angles for complex 4·6.43H2O.

Table 2 Selected bond distances and angles for complex 2·4MeOH. Bond

(Å)

Ni(1)–O(1) Ni(1)–O(4) Ni(1)–N(3) O(2)–C(10) O(5)–C(24)

2.063(2) 2.075(2) 2.079(2) 1.263(3) 1.266(3)

Angles

(°)

O(1)–Ni(1)–O(2) O(1)–Ni(1)–O(5) O(1)–Ni(1)–N(4) O(2)–Ni(1)–O(5) O(2)–Ni(1)–N(4) O(4)–Ni(1)–N(3) O(5)–Ni(1)–N(3) N(3)–Ni(1)–N(4)

85.93(6) 93.77(6) 173.77(7) 179.14(6) 89.91(7) 173.40(7) 88.97(7) 95.44(7)

Ni(1)–O(2) Ni(1)–O(5) Ni(1)–N(4) O(3)–C(10) O(6)–C(24)

O(1)–Ni(1)–O(4) O(1)–Ni(1)–N(3) O(2)–Ni(1)–O(4) O(2)–Ni(1)–N(3) O(4)–Ni(1)–O(5) O(4)–Ni(1)–N(4) O(5)–Ni(1)–N(4)

flumequinato ligand behaves as a bidentate deprotonated ligand coordinated to nickel atom via the ketone oxygen and a carboxylate oxygen. The nickel atom is bound to two bidentate flumequinato ligands and a bidentate 1,10-phenanthroline ligand forming a distorted octahedron with six corners occupied by the two nitrogen atoms of phen and four oxygen atoms from the flumequinato ligands. The Ni–O distances Ni– Oket [Ni(1)–O(1) = 2.028(2)] and Ni–Ocarb [Ni(1)–O(2) = 2.010(2)] are shorter than the Ni–N [Ni(1)–N(2) = 2.091(2)] distance. The two carboxylato oxygen atoms [O(2)–Ni(1)–O(2)i = 95.72(10)°] are in a cis arrangement and the two ketone oxygen atoms [O(1)–Ni(1)–O(1)i = 179.89(10)°] are trans [symmetry codes: (i) 1/2 − x, y, 1/2 − z]. This arrangement of the coordinated quinolone oxygen atoms around the metal has been also observed in the crystal structure of [Zn (oxolinato)2(phen)]·2MeOH complex where oxolinic acid is a firstgeneration quinolone, with the ketone oxygen atoms found in trans arrangement [O(3)–Zn–O(3)′ = 178.2(2)°] and the two coordinated carboxylato oxygen atoms lying on cis positions [O(1)–Zn–O(1)′ = 105.3(2)°] of the octahedron [50]. The N(2)–Ni(1)–N(2)′ angle observed is 79.45(11)° and is similar to reported values of other chelating polycyclic diimines [47,49]. The 1,10-phenanthroline ligand is planar with the nickel atom lying in this plane. The Ni–N bond distances are equal to 2.091(2) Å and are similar to the values reported for other α-diimine complexes [48].

2.030(2) 2.014(2) 2.085(2) 1.256(3) 1.249(3)

85.42(6) 89.32(7) 91.77(6) 91.82(7) 87.40(6) 90.09(7) 90.34(7)

Bond

(Å)

Ni(1)–O(1) Ni(1)–O(4) Ni(1) ... O(3) O(1)–C(4) O(3)–C(1) O(5)–C(15)

2.062(2) 2.017(2) 4.068 1.257(3) 1.243(3) 1.257(3)

Angles

(°)

O(1)–Ni(1)–O(2) O(1)–Ni(1)–O(5) O(1)–Ni(1)–N(4) O(2)–Ni(1)–O(5) O(2)–Ni(1)–N(4) O(4)–Ni(1)–N(3) O(5)–Ni(1)–N(3) N(3)–Ni(1)–N(4)

87.22(7) 177.24(6) 92.45(7) 91.15(7) 175.70(8) 173.38(8) 92.87(7) 79.39(9)

Ni(1)–O(2) Ni(1)–O(5) Ni(1) ... O(6) O(2)–C(1) O(4)–C(18) O(6)–C(15)

O(1)–Ni(1)–O(4) O(1)–Ni(1)–N(3) O(2)–Ni(1)–O(4) O(2)–Ni(1)–N(3) O(4)–Ni(1)–O(5) O(4)–Ni(1)–N(4) O(5)–Ni(1)–N(4)

2.029(2) 2.041(2) 4.156 1.268(3) 1.264(3) 1.260(3)

87.78(7) 89.52(7) 89.59(8) 96.32(8) 89.98(7) 94.68(8) 89.34(7)

K.C. Skyrianou et al. / Journal of Inorganic Biochemistry 104 (2010) 740–749

745

Fig. 4. A drawing of the molecular structure of 5 with only the heteroatoms labeling.

For all structures flumequinato ligands form a π–π interaction with flumequinato ligands of neighboring molecules with Cg1···Cg1i distance of 3.550(1) Å and Cg2···Cg3ii distance of 3.508(1) Å [Cg1, Cg2 and Cg3 are the N1/C1–C4/C9, N2/C15–C18/C23 and C18–C23 ring centroids; symmetry codes: (i) 1 − x, 1 − y, 1 − z, (ii) 1 − x, − y, 1 − z] for 2 (Fig. 5), Cg1···Cg2i distance of 3.881(2) Å [Cg1 and Cg2 are the C5–C10 and N1/C2–C5/C10 ring centroids; symmetry codes: (i) −x, −y, −z] for 4 (Fig. S2) and Cg1···Cg1ii distances of 3.708(3) Å [Cg1 is the C4–C9 ring centroid; symmetry codes: (ii) −x, −y, 1 − z] for 5 (Fig. S3). Due to π–π interactions chain frameworks of molecules is formed and the lattice structures are stabilized by a network of hydrogen bonding interactions (Tables S1–S3). 3.5. Interaction with DNA The study of the interaction of quinolones and their complexes with DNA is of great importance since their activity as antibacterial drugs is focused on the inhibition of DNA replication [51] by targeting essential type II bacterial topoisomerases such as DNA gyrase and topoisomerase IV [30]. DNA can provide three distinctive binding sites for quinolone metal complexes (groove binding, electrostatic binding to phosphate group and intercalation) [52–54]. The changes observed in the UV spectra upon titration may give evidence of the existing interaction mode, since a hypochromism due to π → π* stacking interactions may appear in the case of the intercalative binding mode, while red-shift (bathochromism) may be observed when the DNA duplex is stabilized [39]. The UV spectra have been recorded for a constant CT DNA concentration in different [compound]/[DNA] mixing ratios (r). UV spectra of CT DNA in the presence of Hflmq or a complex derived for diverse r values are shown representatively for 2 in Fig. 6. The intensity at λmax = 258 nm is decreased accompanied by a red-shift of the λmax up to 266 nm for all compounds, indicating that the interaction with CT DNA results in the direct formation of a new complex with doublehelical CT DNA. The observed red-shift is an evidence of the

Fig. 5. π–π interactions of molecule 2 with neighboring molecules along b axis.

stabilization of the CT DNA duplex and the hypochromism may be attributed to interaction between the aromatic chromophores (either from flumequinato and/or the N-donor ligands) of the complexes and DNA base pairs [39].

Table 4 Selected bond distances and angles for complex 5·1.44MeOH·1.13H2O. Bond

(Å)

Ni(1)–O(1) Ni(1)–N(2) O(3)–C(1)

2.028(2) 2.091(2) 1.254(3)

Angles

(°)

O(1)–Ni(1)–O(2)′ O(1)–Ni(1)–N(2) O(1)–Ni(1)–N(2)′ O(2)–Ni(1)–O(2)′ N(2)–Ni(1)–N(2)′

90.95(7) 86.01(7) 93.91(7) 95.72(10) 79.45(11)

Ni(1)–O(2) O(2)–C(1)

O(1)–Ni(1)–O(1)′ O(1)–Ni(1)–O(2) O(2)–Ni(1)–N(2) O(2)–Ni(1)–N(2)′

2.010(2) 1.259(3)

179.89(10) 89.13(7) 170.40(7) 92.64(7)

Fig. 6. UV spectra of CT DNA in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the absence or presence of [Ni(flmq)2(4bzpy)2] 2. The arrows show the changes upon increasing amounts of complex.

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K.C. Skyrianou et al. / Journal of Inorganic Biochemistry 104 (2010) 740–749

In Fig. 7, the changes occurred in the spectra of a 10−5 M solution of the compounds during the titration upon addition of CT DNA in diverse r values may be observed. In the UV region, the intense absorption bands observed in the spectra of the complexes are attributed to the intra-ligand transition of the coordinated groups of flumequinato ligands [50]. Any interaction between each complex and CT DNA could perturb its intra-ligand centered spectral transitions [54]. In the UV spectrum of Hflmq, the band centered at 326 nm presents a hypochromism of 15% (Fig. 7(A)) suggesting tight binding to CT DNA while for the band at 340 nm a hyperchromism is present. Both bands are accompanied by a red-shift of 2 nm (up to 328 nm and 342 nm, respectively), suggesting tight binding to CT DNA and stabilization. The hyperchromic effect may suggest binding to CT DNA ascribed to external contact (electrostatic or groove binding) [54]. For 1, both bands centered at 335 nm and 348 nm exhibit initially a hypochromism of 10% (Fig. 7(B)) suggesting tight binding to CT DNA. Further addition of DNA results in a hyperchromism accompanied by a blue-shift of 8 nm for both bands. In the UV spectrum of 2, a hypochromism is present for the band centered at 334 nm (Fig. 7(C)) while the band at 348 nm exhibits a hyperchromism accompanied by a blue-shift of 5 nm. The behavior of complex 3 is similar. For 5, the intensity of the bands centered at 329 nm and 339 nm decreased in

the presence of increasing amounts of CT DNA, while a blue-shift of 3 nm (up to 326 nm) has been observed for the former band (Fig. 7 (D)). Quite similar behavior is observed in the case of complex 4. The results derived from the UV titration experiments suggest that flumequine and its complexes bind to CT DNA [55] although the exact mode of binding cannot be merely proposed by UV spectroscopic titration studies. Nevertheless, the flumequinato ligands may provide an aromatic/planar moiety so that the binding of the complexes involving intercalation between the base pairs of CT DNA cannot be ruled out [50,56]. The calculated Kb values for Hflmq and complexes 1–5 are given in Table 5 and suggest a moderate binding of the compounds to CT DNA [50,56]. The Kb values of complexes 1, 3 and 5 are higher than that of Hflmq (Kb = 3.53 (±0.45) × 105 M−1) suggesting that flumequinato ligand presents higher affinity to CT DNA, when it is coordinated to Ni(II) in the presence of aqua, pyridine or 1,10-phenanthroline ligands while the co-existence of 4-benzylpyridine or 2,2′-bipyridine ligands results in a significant decrease of the affinity for DNA. The Kb values of Hflmq and complexes 1, 3 and 5 are of the same order to the classical intercalator EB binding affinity for CT DNA, (Kb = 1.23(±0.07) × 105 M−1) [57]. The electrochemical investigations of metal–DNA interactions can also provide a useful supplement to spectroscopic methods and yield information about interactions with both the reduced and oxidized form of the metal [58]. The complete scan in the range +1.0 V to

Fig. 7. UV spectra of complex ([Compound] = 1 × 10−5 M) (A) Flumequine (B) [Ni(flmq)2(H2O)2] 1 (C) [Ni(flmq)2(4bzpy)2] 2 and (D) [Ni(flmq)2(phen)] 5, in DMSO solution in the presence of CT DNA at increasing amounts. The arrows show the changes upon increasing amounts of CT DNA. Insets: plots of

½DNA vs [DNA]. ðεA −εf Þ

K.C. Skyrianou et al. / Journal of Inorganic Biochemistry 104 (2010) 740–749 Table 5 The DNA binding constants (Kb) and the Stern–Volmer constants (KSV) of Hflmq and complexes 1–5. Compound

Kb (M−1)

KSV (M−1)

Hflmq [Ni(flmq)2(H2O)2, ] 1 [Ni(flmq)2(4bzpy)2], 2 [Ni(flmq)2(py)2], 3 [Ni(flmq)2(bipy)], 4 [Ni(flmq)2(phen)], 5

3.53(± 0.45) × 105 6.75(± 0.55) × 105 6.81(± 0.40) × 104 6.36(± 0.40) × 105 2.82(± 0.09) × 103 5.38(± 0.20) × 105

1.19(± 0.06) × 106 6.60(± 0.40) × 105 1.54(± 0.04) × 105 1.30(± 0.08) × 105 5.90(± 0.45) × 105 2.55(± 0.08) × 104

−1.0 V of 4 in 0.4 mM DMSO solution consists of a cathodic wave at −520 mV and an anodic wave at −310 mV consisting a quasireversible wave assigned to the couple Ni(II)/Ni(I) [20,21,59]. The rest complexes exhibit similar behavior and the corresponding potentials are given in Table 6. The cyclic voltammograms of the complexes have been also recorded in 0.4 mM 1:2 DMSO:buffer solution and the quasi-reversible redox couple Ni(II)/Ni(I) has been studied upon addition of CT DNA and the corresponding potentials as well as their shifts are given in Table 6. In general, the electrochemical potential of a small molecule will shift positively when it intercalates into DNA double helix, and it will shift in a negative direction in the case of electrostatic interaction with DNA [60]. Additionally, when more potentials than one exist, a positive shift of Ep1 and a negative shift of Ep2 imply that the molecule can bind to DNA by both intercalation and electrostatic interaction [61]. No new redox peaks appeared after the addition of CT DNA to each complex, but the current of all the peaks decreased significantly, suggesting the existence of an interaction between each complex and CT DNA. The decrease in current can be explained in terms of an equilibrium mixture of free and DNA-bound complex to the electrode surface [62]. For increasing amounts of CT DNA, one of the cathodic (Epc) or the anodic (Epa) potentials of complexes shows a positive shift (ΔEpc = (+12) − (+127) mV) (Table 6) suggesting an intercalative mode of binding [20,21,60], while for complexes 1–4 the other potential shifts to more negative values (ΔEpa = (−32) − (−21) mV) showing that the co-existence of electrostatic interaction in these cases cannot be ruled out [20,21,61]. In order to examine the ability of the compounds to displace EB (=3,8-Diamino-5-ethyl-6-phenyl-phenanthridinium bromide) from its EB–DNA complex, a competitive EB binding study has been undertaken with fluorescence experiments [56]. EB is a phenanthridine fluorescence dye and is a typical indicator of intercalation [63], forming soluble complexes with nucleic acids and emitting intense fluorescence in the presence of CT DNA due to the intercalation of the planar phenanthridinium ring between adjacent base pairs on the double helix. The changes observed in the spectra of EB on its binding to CT DNA are often used for the interaction study between DNA and other compounds, such as metal complexes [64]. Flumequine and complexes 1–5 show no fluorescence at room temperature in solution or in the presence of CT DNA, and their binding to DNA cannot be directly predicted through the emission spectra. Therefore, competitive EB binding studies could be undertaken in

747

order to examine the binding of each compound with DNA. EB does not show any appreciable emission in buffer solution due to fluorescence quenching of the free EB by the solvent molecules. Upon addition of Hflmq or complexes 1–5 to a solution containing EB, neither quenching of free EB fluorescence has been observed nor new peaks in the spectra appear. The fluorescence intensity is highly enhanced upon addition of CT DNA, due to its strong intercalation with DNA base pairs. Addition of a second molecule, which may bind to DNA more strongly than EB results in a decrease of the DNA-induced EB emission due to the replacement of EB, and/or electron transfer [65]. The emission spectra of EB bound to CT DNA in the absence and presence of each compound have been recorded for [EB] = 20 μM, [DNA] = 26 μM for increasing amounts of each compound. The addition of Hflmq or complex 1–5 at diverse r values (Fig. 8) results in a significant decrease of the intensity of the emission band of the DNA–EB system at 592 nm (up to 35% of the initial EB–DNA fluorescence intensity for Hflmq, 23% for 1, 21% for 2, 10% for 3, 25% for 4, and 45% for 5) indicating the competition of the complexes with EB in binding to DNA. The observed quenching of DNA–EB fluorescence especially for complexes 1–4 suggests that they can significantly displace EB from the DNA–EB complex and they can probably interact with CT DNA by the intercalative mode [20,66,67]. The Stern–Volmer plots of DNA–EB (Fig. S4) illustrate that the quenching of EB bound to DNA by the compounds is in good agreement (R = 0.99) with the linear Stern–Volmer Eq. (2), which proves that the replacement of EB bound to DNA by each compound results in a decrease of the fluorescence intensity [63]. The high KSV (Table 5) values of complexes 1–5 show that they can displace EB and bind to the DNA [50,56,68]. 3.6. Binding of bovine and human serum albumin Serum albumins are proteins amongst those involved in the transport of metal ions and metal complexes with drugs through the blood stream. The interaction of Hflmq and complexes 1–5 with bovine serum albumin (BSA) has been studied from tryptophan emission-quenching experiments. BSA is the most extensively studied serum albumin, due to its structural homology with human serum albumin (HSA). HSA contains 585 amino acid residues with only one tryptophan located at position 214, while BSA has two tryptophans at positions 134 and 212 along the chain [69,70]. HSA and BSA can bind reversibly to a large number of endogenous and exogenous compounds [71]. BSA and HSA solutions exhibit a strong fluorescence emission with a peak at 343 nm and 351 nm, respectively, due to the tryptophan residues, when excited at 295 nm [40]. The changes in the emission spectra of tryptophan in BSA or HSA are primarily due to change in protein conformation, subunit association, substrate binding or denaturation [69]. Hflmq and complexes 1–5 in buffer solutions exhibit a maximum emission at 365 nm under the same experimental conditions and the SA fluorescence spectra have been corrected before the experimental data processing. Addition of Hflmq or complexes 1–5 to HSA results in fluorescence quenching (up to 45% of the initial fluorescence intensity of HSA for

Table 6 Cathodic and anodic potentials (in mV) for the redox couple Ni(II)/Ni(I) in DMSO and in 1/2 DMSO/buffer solution of the complexes in the absence and presence of CT DNA. a

Complex

Epc

[Ni(flmq)2(H2O)2], 1 [Ni(flmq)2(4bzpy)2], 2 [Ni(flmq)2(py)2], 3 [Ni(flmq)2(bipy)], 4 [Ni(flmq)2(phen)], 5

−620 −520 −525 −520 −515

a b c d

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

Epa

a

−250 −275 −275 −330 −350

Epc(f) b

Epc(b) c

ΔEpc

−678 −845 −808 −712 −746

−699 −833 −783 −693 −746

−21 + 12 + 25 + 19 0

d

Epa(f)

b

−445 −405 −347 −91 −67

Epa(b) −318 −436 −379 −118 −26

c

ΔEpad + 127 −31 −32 −27 + 41

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K.C. Skyrianou et al. / Journal of Inorganic Biochemistry 104 (2010) 740–749 Table 7 The HSA binding constants and parameters (Ksv, kq, K, n) derived for Hflmq and complexes 1–5.

Fig. 8. (A) Plot of EB relative fluorescence intensity at λem = 592 nm (%) vs r (r = [compound]/[DNA]) for Hflmq and complexes 1–5 in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).

Compound

KSV (M−1)

kq (M−1 s−1)

K (M−1)

n

Hflmq [Ni(flmq)2(H2O)2], 1 [Ni(flmq)2(4bzpy)2], 2 [Ni(flmq)2(py)2], 3 [Ni(flmq)2(bipy)], 4 [Ni(flmq)2(phen)], 5

1.00(± 0.17) × 105 2.04(± 0.12) × 104 3.01(± 0.11) × 104 2.36(± 0.16) × 104 4.98(± 0.22) × 104 4.22(± 0.32) × 104

1.00(± 0.17) × 1013 2.04(± 0.12) × 1012 3.01(± 0.11) × 1012 2.36(± 0.16) × 1012 4.98(± 0.22) × 1012 4.22(± 0.32) × 1012

2.37 × 106 1.12 × 106 1.25 × 105 1.72 × 105 1.19 × 105 1.29 × 105

0.67 1.03 0.83 0.44 0.70 0.65

(2.0 × 1010 M−1 s−1) indicating the existence of static quenching mechanism [70]. Using the Scatchard [43] equation (4), the values of K (association binding constant, (M−1)) and n (number of binding sites per albumin) for Hflmq and complexes 1–5 have been calculated from the slope and

Hflmq (Fig. 9(A)), 62% for 1, 39% for 2, 64% for 3, 49% for 4 and 53% for 5, as calculated after the correction of the initial fluorescence spectra) due to possible changes in protein secondary structure of HSA, indicating the binding of Hflmq or each complex to HSA [72]. The Stern–Volmer and Scatchard plots may be used in order to study the interaction of the compounds with serum albumins. The Stern–Volmer plots (Fig. S5(A)) show that the curves have fine linear relationships (r = 0.99) according to the Stern–Volmer quenching Eq. (3) and the calculated values of Ksv and kq (τo ∼ 10−8 s [42]) for the interaction of Hflmq and complexes 1–5 with HSA, as obtained by the slope of the diagram, are given in Table 7 and indicate good HSA binding propensity of the complexes with Hflmq exhibiting the strongest protein-binding ability. The kq values (N1012 M−1 s−1) are higher than diverse kinds of quenchers for biopolymers fluorescence

ΔI Io

versus ½Q  ΔI (Fig. S5(B)) [43] and are given in Table 7. It is obvious that Hflmq Io exhibits the highest K value, suggesting that the coordination of Hflmq to Ni(II) results in a decreased affinity for HSA. On the other hand, the n value of Hflmq increases when flumequine is coordinated to Ni(II) in the absence of the N-donor heterocyclic ligands, while the presence of a N-donor ligand results in a decrease. The quenching provoked by Hflmq or complexes 1–5 to the BSA fluorescence is significant (up to 27% of the initial fluorescence intensity for Hflmq (Fig. 9(B)), 39% for 1, 20% for 2, 30% for 3, 24% for 4 and 20% for 5) indicating that the binding of each compound to BSA quenches the intrinsic fluorescence of the tryptophans in BSA [69]. The Stern–Volmer equation applied for the interaction with BSA graph in Fig. S6(A) shows that the curves have fine linear relationships (r = 0.99) according to Eq. (3). The calculated values of KSV and kq as obtained by the slope of the diagram Hflmq and 1–5 are given in Table 8 and indicate their good BSA binding propensity and complexes 2, 3 and 5 exhibit the strongest protein-binding ability. The kq values (∼1013 M−1 s−1) are higher than diverse kinds of quenchers for biopolymers fluorescence (2.0 × 1010 M−1 s−1) suggesting a static quenching mechanism [70]. From the Scatchard graph (Fig. S6(B)) and equation (4) [43], the association binding constant of each compound has been calculated (Table 8) with complex 1 exhibiting the highest K value. The K values of Hflmq is relatively high and, in general, increases slightly when it is bound to Ni(II) as found for complexes 1–5. The n value of Hflmq increases when flumequine is coordinated to Ni(II) either in the absence or in the presence of the N-donor ligands (Table 8). Comparing the affinity of Hflmq and complexes 1–5 for BSA and HSA (K values), it is obvious (Tables 7 and 8) that Hflmq and complexes 1–4 show higher affinity for HSA than BSA, while complex 5 exhibits higher binding constant for BSA than for HSA.

by the ratio of y intercept to the slope, respectively, in plots

4. Conclusions The synthesis and characterization of the neutral mononuclear nickel(II) complexes with the first-generation quinolone antibacterial Table 8 The BSA binding constants and parameters (Ksv, kq, K, n) derived for Hflmq and complexes 1–5. Compound

Fig. 9. Plot of % relative fluorescence intensity at (A) λem = 352 nm (%) vs r (r = [compound]/[HSA]) and (B) λem = 342 nm (%) vs r (r = [compound]/[BSA]) for Hflmq and complexes 1–5 in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).

Hflmq [Ni(flmq)2(H2O)2], 1 [Ni(flmq)2(4bzpy)2], 2 [Ni(flmq)2(py)2], 3 [Ni(flmq)2(bipy)], 4 [Ni(flmq)2(phen)], 5

KSV (M−1)

kq (M−1 s−1) 4

8.27(± 0.36) × 10 7.25(± 0.30) × 104 1.84(± 0.06) × 105 1.62(± 0.04) × 105 1.24(± 0.06) × 105 1.68(± 0.10) × 105

K (M−1) 12

8.26(± 0.36) × 10 7.25(± 0.30) × 1012 1.84(± 0.06) × 1013 1.62(± 0.04) × 1013 1.24(± 0.06) × 1013 1.68(± 0.10) × 1013

n 4

6.67 × 10 7.12 × 105 6.67 × 104 6.64 × 104 9.34 × 104 8.85 × 104

0.66 0.99 1.34 1.18 1.12 1.23

K.C. Skyrianou et al. / Journal of Inorganic Biochemistry 104 (2010) 740–749

agent flumequine in the absence or presence of a nitrogen donor heterocyclic ligand 4-benzylpyridine, pyridine, 2,2′-bipyridine or 1,10-phenanthroline has been achieved. In these complexes, flumequine is bound to nickel(II) via the ketone oxygen and a carboxylato oxygen. The crystal structures of [Ni(flmq)2(4bzpy)2], [Ni(flmq)2 (bipy)] and [Ni(flmq)2(phen)], determined by X-ray crystallography, are the first reported crystal structures of flumequinato complexes. The diversity of the arrangements of the coordinated quinolones oxygen atoms lying in the coordination sphere may be attributed to the Ν donor ligands. UV spectroscopy and cyclic voltammetry studies have revealed the ability of flumequine and complexes 1–5 to bind to DNA. The binding strength of the complexes with CT DNA calculated with UV spectroscopic titrations have shown that [Ni(flmq)2(H2O)2] exhibits the highest Kb value among the compounds examined. Cyclic voltammetric studies proposed the intercalation as the most possible binding mode to DNA while competitive binding studies with EB have confirmed the ability of the compounds to displace EB from the EB–DNA complex. Flumequine and its complexes show good binding affinity to BSA and HSA proteins giving relatively high binding constants. Especially, Hflmq and complexes 1–4 exhibit higher affinity for HSA than BSA. Acknowledgements Financial support from the Slovenian Research Agency (ARRS) through project P1-0175 is gratefully acknowledged. Appendix A. Supplementary data CCDC 756950–756952 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; or [email protected]). Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinorgbio.2010.03.007. References [1] R.K. Andrews, R.L. Blakeley, B. Zerner, in: H. Sigel, A. Sigel (Eds.), Metal Ions in Biological Systems, vol. 23, Marcel Dekker inc., New York, 1988, pp. 165–284. [2] M.A. Halcrow, G. Christou, Chem. Rev. 94 (1994) 2421–2481. [3] F. Meyer, H. Kozlowski, in: J.A. McCleverty, T.J. Meyer (Eds.), Comprehensive Coordination Chemistry II, vol. 6, Elsevier, 2003, pp. 247–554. [4] P. Bombicz, E. Forizs, J. Madarasz, A. Deak, A. Kalman, Inorg. Chim. Acta 315 (2001) 229–235. [5] G. Morgant, N. Bouhmaida, L. Balde, N.E. Ghermani, J. d'Angelo, Polyhedron 25 (2006) 2229–2235. [6] O.Z. Yesilel, M.S. Soylu, H. Olmez, O. Buyukgungor, Polyhedron 25 (2006) 2985–2992. [7] N.C. Kasuga, K. Sekino, C. Koumo, N. Shimada, M. Ishikawa, K. Nomiya, J. Inorg. Biochem. 84 (2001) 55–65. [8] 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. [9] R. Kurtaran, L.T. Yildirim, A.D. Azaz, H. Namli, O. Atakol, J. Inorg. Biochem. 99 (2005) 1937–1944. [10] 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. [11] E.M. Jouad, G. Larcher, M. Allain, A. Riou, G.M. Bouet, M.A. Khan, X.D. Thanh, J. Inorg. Biochem. 86 (2001) 565–571. [12] W. Luo, X. Meng, X. Sun, F. Xiao, J. Shen, Y. Zhou, G. Cheng, Z. Ji, Inorg. Chem. Commun. 10 (2007) 1351–1354. [13] Z. Afrasiabi, E. Sinn, W. Lin, Y. Ma, C. Campana, S. Padhye, J. Inorg. Biochem. 99 (2005) 1526–1531. [14] M.C. Rodriguez-Arguelles, M. Belicchi Ferrari, F. Bisceglie, C. Pelizzi, G. Pelosi, S. Pinelli, M. Sassi, J. Inorg. Biochem. 98 (2004) 313–321. [15] J. Garcia-Tojal, J.L. Pizarro, A. Garcia-Orad, A.R. Perez-Sanz, M. Ugalde, A. Alvarez Diaz, J.L. Serra, M.I. Arriortua, T. Rojo, J. Inorg. Biochem. 86 (2001) 627–633. [16] 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. [17] A. Juris, V. Balzani, F. Barigelletti, S. Campagna, P. Belser Test, Coord. Chem. Rev. 84 (1988) 85–277. [18] P.J. Cox, G. Psomas, C.A. Bolos, Bioorg. Med. Chem. 17 (17) (2009) 6054–6062. [19] K.C. Skyrianou, C.P. Raptopoulou, V. Psycharis, D.P. Kessissoglou, G. Psomas, Polyhedron 28 (2009) 3265–3271.

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