Cadmium(II) complexes of 5-bromo-salicylaldehyde and α-diimines: Synthesis, structure and interaction with calf-thymus DNA and albumins

Polyhedron 107 (2016) 136–147

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Cadmium(II) complexes of 5-bromo-salicylaldehyde and a-diimines: Synthesis, structure and interaction with calf-thymus DNA and albumins Ariadni Zianna a, Maja Šumar Ristovic´ a,b, George Psomas a, Antonis Hatzidimitriou a, Evdoxia Coutouli-Argyropoulou c, Maria Lalia-Kantouri a,⇑ a b c

Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece Faculty of Chemistry, University of Belgrade, Studenski trg 12-16, Belgrade, Serbia Department of Organic Chemistry and Biochemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece

a r t i c l e

i n f o

Article history: Received 5 November 2015 Accepted 14 January 2016 Available online 22 January 2016 Keywords: Cadmium(II) complexes Crystal structure Interaction with calf-thymus DNA Competitive studies with ethidium bromide Interaction with serum albumins

a b s t r a c t Five Cd(II) complexes, four dinuclear (1–4) and a mononuclear one (5), with the anion of 5-Br-salicylaldehyde (5-Br-salo
), were synthesized and characterized as [Cd(5-Br-salo)2(CH3OH)]2 (1), [Cd(5-Br-salo)2 (bipy)]2 (2), [Cd(5-Br-salo)2(phen)]2 (3), [Cd(5-Br-salo)(neoc)(NO3)]2 (4) and [Cd(5-Br-salo)2(dpamH)] (5). The complexes were characterized by spectroscopy (IR, UV–vis, 1H NMR), elemental analysis and molar conductivity measurements. The structures for three complexes (2, 3 and 5) were determined by X-ray crystallography providing different coordination modes of the 5-Br-salicylaldehydo ligand. The complexes bind to DNA mainly by intercalation, as concluded by the viscosity measurements and the EB-displacement studies from the EB–DNA complex and present high DNA-binding constants (Kb), as calculated by the Wolfe–Shimer equation and plots. The interaction of the complexes with serum albumins was studied by fluorescence emission spectroscopy and the determined binding constants exhibit relative high values. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction It is well known that cadmium is a highly toxic metal and is considered to be a carcinogen by the International Agency of Research on Cancer [1], but the mechanism of its action remains largely unknown [2]. Cadmium can interact with DNA and may act as a carcinogen through epigenetic and/or other mechanisms such as mitogenic effects on gene expression, inhibition of DNA repair and inhibition of apoptosis [3]. Apart from that major drawback, research has shown that there are cadmium complexes possessing antimicrobial [4–6], as well as antifungal properties [7]. Furthermore, studies have been reported in regard to the cytotoxic activity of Cd(II) complexes [8,9]. It has also been shown that Abbreviations: 5-Br-saloH, 5-bromo-salicylaldehyde; bipy, 2,20 -bipyridine; BSA, bovine serum albumin; CT, calf-thymus; DMF, N,N-dimethylformamide; dpamH, 2,20 -dipyridylamine; EB, ethidium bromide, 3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide; HSA, human serum albumin; K, SA-binding constant; Kb, DNAbinding constant; kq, approximate quenching constant; KSV, Stern–Volmer constant; m, medium; neoc, 2,9-dimethyl-1,10-phenanthroline; phen, 1,10-phenanthroline; r, [complex]/[DNA] mixing ratio; r0 , [DNA]/[complex] mixing ratio; s, strong; saloH, salicylaldehyde, 2-hydroxy-benzaldehyde; sm, strong-to-medium; sh, shoulder; w, weak; D, m4
m1. ⇑ Corresponding author. Tel./fax: +30 2310 997844. E-mail address: [email protected] (M. Lalia-Kantouri). http://dx.doi.org/10.1016/j.poly.2016.01.020 0277-5387/Ó 2016 Elsevier Ltd. All rights reserved.

cadmium may enhance the activity in several zinc containing metalloenzymes, but its potential as a zinc analogue is relatively unexplored [10,11]. The interaction of transition metal complexes with DNA is a crucial field of scientific interest, mainly because of their versatile applications in cancer research and molecular biology [12,13]. Transition metal complexes provide two advantages as DNA-binding agents [14]. Due to their well-defined geometries, they may be used for reversible recognition of nucleic acids and often possess electrochemical or photophysical properties, enhancing the functionality of the binding agent [15]. The interaction of cadmium complexes with DNA has also been investigated [16–20]. 2-hydroxy-benzaldehyde (salicylaldehyde) and its derivatives can coordinate strongly to metal ions. They coordinate mainly in a bidentate mode with transition metals in the mono-anionic form, acquiring variant geometries, such as square–planar [21], square– pyramidal [22] and octahedral [23]. From a biological aspect, salicylaldehyde derivatives have shown antimicrobial properties [24,25] and have been proposed for the removal of low concentration cadmium(II) from aqueous media [26]. During the past years, we have initiated in our laboratory the synthesis and characterization of transition metal complexes coordinated to substituted salicylaldehyde and benzophenone ligands

A. Zianna et al. / Polyhedron 107 (2016) 136–147

[27–34]. Especially, we have focused on the synthesis of metal complexes with salicylaldehydes and their interaction with DNA. More specifically, a series of zinc(II), copper(II) and cadmium(II) complexes with salicylaldehydes or benzophenones were studied in regard to their binding affinity to calf-thymus (CT) DNA [19,20,31–34], and cobalt(II) analogues with the nitrogen-donor 2,20 -dipyridylamine (dpamH) as co-ligand have exhibited noteworthy anticancer activity [30]. As a continuation of our research, we have synthesized and characterized novel cadmium(II) complexes with the ligand HL = 5-bromo-2-hydroxy-benzaldehyde (5bromo-salicylaldehyde, abbreviated as 5-Br-saloH) in the absence [Cd(5-Br-salo)2(CH3OH)]2 (1), or presence (2–5) of the a-diimines 2,20 -dipyridine (bipy), dpamH, 1,10-phenanthroline (phen) and 2,9-dimethyl-1,10-phenanthroline (neoc) (Scheme 1). The obtained neutral compounds have different structures according to the a-diimine used (Scheme 2). Their characterization was achieved by physicochemical measurements and spectroscopic methods (IR, UV–vis, 1H NMR) and formulated as [Cd(5-Br-salo)2 (bipy)]2 (2), [Cd(5-Br-salo)2(phen)]2 (3), [Cd(5-Br-salo)(neoc)(NO3)]2 (4) and [Cd(5-Br-salo)2(dpamH)] (5). The crystal structures for complexes 2, 3 and 5 were determined by single-crystal X-ray crystallography. The ability of the complexes to bind to CT DNA was investigated by (i) UV–vis spectroscopic titration studies and the binding constants Kb to CT DNA have been determined, (ii) measurements of the viscosity of DNA solution in the presence of increasing amounts of the compounds and (iii) competitive binding studies with the classic intercalator ethidium bromide (EB) performed by fluorescence spectroscopy in order to assess the ability of the compounds to displace EB from the EB–DNA complex. Finally, the affinity of complexes 1–5 for human serum albumin (HSA) and bovine serum albumin (BSA) was also evaluated and the corresponding binding constants were determined.

2. Experimental 2.1. Materials The a-diimines (bipy, dpamH, phen and neoc), 5-Br-saloH, Cd (NO3)24H2O, CH3ONa, trisodium citrate, NaCl, CT DNA and EB were obtained as reagent grade from Sigma–Aldrich Co and used as received. Solvents for the preparation and physical measurements

H Br

5

6

4 3

(A) 5'

6

6'

2

OH

4'

4 N

N

O

5

5'

3'

3

6'

6

N

6

5

7

3 9

N

N

2

5 4

8

(E)

H3C

3 9

N

N

2

of ‘‘extra pure” grade were obtained from Chemlab without further purification. The 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 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 [35]. The DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using e = 6600 M
1cm
1 [36]. 2.2. Instrumentation – physical measurements The infrared (IR) spectra (400–4000 cm
1) were recorded on a Nicolet FT-IR 6700 spectrometer with samples prepared as KBr pellets. The UV–vis spectra were recorded as Nujol mulls and in DMSO solutions at concentrations in the range 10
5–10
3 M on a Hitachi U-2001 dual beam spectrophotometer. 1H NMR spectra were recorded at 300 MHz on a Bruker AVANCEIII 300 spectrometer using DMSO-d6 as solvent. C, H and N elemental analyses were performed on a PerkinElmer 240B elemental microanalyzer. Molecular conductivity measurements of 1 mM DMSO solution of the complexes were carried out with a Crison Basic 30 conductometer. Fluorescence spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer. 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.

2.3. Synthesis of the complexes 2.3.1. Synthesis of [Cd(5-Br-salo)2(CH3OH)]2, (1) Complex 1 was prepared according to the published procedures [33,34] by the addition of a methanolic solution (15 mL) of 5-BrsaloH (1 mmol, 201 mg) deprotonated with CH3ONa (1 mmol, 54 mg) to a methanolic solution (10 mL) of Cd(NO3)24H2O (0.5 mmol, 154 mg) at room temperature. The reaction mixture was stirred for 2 h and then turned into yellow. The solution was filtered off and left for slow evaporation. After a few days, yellowish microcrystalline solid was collected with filtration and was airdried. Yield 51.0%, 140 mg, analyzed as [Cd(5-Br-salo)2(CH3OH)]2, (C30H24Br4Cd2O10) (MW = 1088.95): C, 33.09; H, 2.22; Found: C, 33.02; H, 2.20. IR spectrum (KBr): selected peaks in cm
1: 3440 (medium,(m)) m(OAH) of coordinated methanol, 1636 (strong, (s)) v(C@O), 1296 (strong-to-medium (sm)) m(CAO?Cd), 508 (m) m(CdAO); UV–vis: as Nujol mull, k/nm: 330, 425; in DMSO, k/nm (e/M
1 cm
1): 257(12 370), 325(shoulder (sh)), 422(7800); molar conductivity in 1 mM DMSO = 13 lS/cm. 1H NMR spectrum in DMSO-d6 (d/ppm): 9.64 (2H, s, H7 5-Br-salo), 7.41 (2H, d, J = 2.8 Hz H6 5-Br-salo), 7.23 (2H, dd, J = 9.2, 2.8 Hz, H4 5-Br-salo), 6.53 (2H, J = 9.2 Hz, H3 5-Br-salo).

3

N

7

4

8

4

6 7 N H

(C)

(B)

(D)

C

5

4' 3'

7

1

137

CH3

Scheme 1. The syntax formula of (A) 5-Br-saloH, (B) bipy, (C) dpamH, (D) phen and (E) neoc with the H atoms numbering.

2.3.2. Synthesis of the cadmium mixed-ligand complexes (2–5) The complexes 2–5 were prepared according to the published procedure [19]. The reaction of a methanolic solution of Cd(NO3)2 4H2O with of 5-Br-saloH in methanol (deprotonated by sodium methoxide) and an a-diimine (bipy, phen, neoc, dpamH), led to the preparation of mixed-ligand cadmium complexes (compounds 2–5) with different formulae and coordination modes of the ligand 5-Br-salicylaldehyde. Alternatively, from the reaction, of [Cd(5-Brsalo)2(MeOH)]2 (1) with the corresponding a-diimine, only the complexes [Cd(5-Br-salo)2(bipy)]2 (2) and [Cd(5-Br-salo)2(phen)]2 (3) were formed.

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Br

Br

Br H

Br

H

C

C O

O

O

H C

O

O

O

CH3OH

N

O

Cd

Cd

H

C

O

N Cd

Cd

CH3OH

N

O O

O

C

H

C

H

O

O

O

N

O

O

C

C H

H Br

(A)

Br

(B)

Br

Br

Br

C

N

O

O N

H

Br

H

CH 3

C

O

O

N

O

O

Cd

CH3

O

O

(C)

O

N

C

H 3C

H

H

N

Br

N

O

N

O

(D)

NH

Cd

O

Cd O

CH 3

N

O

C

Br

Scheme 2. Molecular structure representation of the prepared cadmium complexes (1–5) (A) [Cd(5-Br-salo)2(CH3OH)]2 (1), (B) [Cd(5-Br-salo)2(N-N)]2 (N-N = bipy for 2 and phen for 3), (C) [Cd(5-Br-salo)(neoc)(NO3)]2 (4) and (D) [Cd(5-Br-salo)2(dpamH)] (5).

2.3.2.1. Synthesis of [Cd(5-Br-salo)2(bipy)]2, (2). Complex 2 was prepared by the addition of a methanolic solution (15 mL) of 5-BrsaloH (1 mmol, 201 mg) deprotonated with CH3ONa (1 mmol, 54 mg) to a methanolic solution (10 mL) of Cd(NO3)24H2O (0.5 mmol, 154 mg) and bipyridine (0.5 mmol, 78 mg). The reaction mixture was stirred for two hours at 50 °C and left for slow evaporation. The formed yellow crystals were suitable for X-ray structure determination, yield 53.0%, 175 mg, and analyzed as [Cd(5-Br-salo)2(bipy)]2, (C48H32Br4Cd2N4O8) (MW = 1337.22): C, 43.11; H, 2.41; N, 4.19; Found: C, 43.02; H, 2.39; N, 4.12%. IR spectrum (KBr): selected peaks in cm
1: 1658 and 1629(s) m(C@O), 1581(s) m(C@N), 1303(sm) m(CAO?Cd), 836(m) and 729(sm) q (CAH)pyridyl, 539 (m) m(CdAO), 492 (m) m(CdAN); UV–vis: as Nujol mull, k/nm: 272, 344, 420; in DMSO, k/nm (e/M
1 cm
1): 270 (13 580), 339 (4000), 415 (780); molar conductivity in 1 mM DMSO = 15 lS/cm. 1H NMR spectrum in DMSO-d6 (d/ppm): 9.87 (2H, s, H7 5-Br-salo), 7.48 (2H, d, J = 2.8 Hz, H6 5-Br-salo), 7.32 (2H, dd, J = 9.0, 2.8 Hz, H4 5-Br-salo), 6.68 (2H, d, J = 9.0 Hz, H3 5Br-salo) and 8.70 (2H, d, J = 4.2 Hz, H3 and H3-bipy), 8.48 (2H, d, J = 7.2 Hz, H6 and H6-bipy), 8.08 (2H, t, J = 7.2 Hz, H5 and H5-bipy), 7.59 (2H, dd, J = 7.2, 4.4 Hz, H4 and H4-bipy). 2.3.2.2. Synthesis of [Cd(5-Br-salo)2(phen)]2, (3). Yellow crystals suitable for X-ray structure determination, yield 55.0%, 190 mg, analyzed as [Cd(5-Br-salo)2(phen)]2, (C52H32Br4Cd2N4O8) (MW = 1385.26): C, 45.09; H, 2.33; N, 4.04; Found: C, 45.10; H, 2.33; N, 4.02%. IR spectrum (KBr): selected peaks in cm
1: 1662 (s) and 1629(s) m(C@O), 1582(sm) m(C@N), 1302(sm) m(CAO? Cd), 845(m), 770(m) and 729(sm) d(CAH)phen, 526(m) m(CdAO); 489(m) m(CdAN); UV–vis: as Nujol mull, k/nm: 268,310, 425; in

DMSO, k/nm (e/M
1 cm
1): 265 (13 370), 300 (sh), 422 (7100); molar conductivity in 1 mM DMSO = 14 lS/cm. 1H NMR spectrum in DMSO-d6 (d/ppm):9.74 (2H, s, H7 5-Br-salo), 7.30 (2H, d, J = 2.9 Hz, H6 5-Br-salo), 7.10 (2H, dd, J = 9.2, 2.9 Hz, H4 5-Br-salo), 6.45 (2H, d, J = 9.2 Hz, H3 5-Br-salo) and 9,03 (2H, d, J = 4.4 Hz, H2 and H9-phen), 8.79 (2H, d, J = 8.1 Hz, H4 and H7 -phen), 8.19 (2H, s, H5 and H6-phen), 8.02 (2H, dd J = 8.1, 4.4 Hz, H3 and H8-phen). 2.3.2.3. Synthesis of [Cd(5-Br-salo)(neoc)(NO3)]2, (4). Yellow microcrystalline solid, yield 50.0%, 145 mg, analyzed as [Cd(5-Br-salo) (neoc)(NO3)]2, (C42H32Br2Cd2N6O10) (MW = 1165.38): C, 43.39; H, 2.77; N, 7.21; Found: C, 43.44; H, 2.85; N, 7.15%; IR spectrum (KBr): selected peaks in cm
1: 1644(s) m(C@O), 1589(m) m(C@N), 1315(s) m(CAO?Cd), 1445(s) and 1298(s) (ONO2), 844(m), 770 (m) and 729(sm) d(CAH)neoc, 551(m) m(CdAO); 479(m) m(CdAN); UV–vis: as Nujol mulls, k/nm: 273 330, 410; in DMSO, k/nm (e/M
1 cm
1): 270(14 690), 329 (sh), 407 (7300); molar conductivity in 1 mM DMSO = 20 lS/cm. 1H NMR spectrum in DMSO-d6 (d/ppm):9.47 (1H, s, H7 5-Br-salo), 7.45 (1H, d, J = 2.8 Hz, H6 5-Brsalo), 7.28 (1H, dd, J = 9.2, 2.8 Hz H4 5-Br-salo), 6.52 (1H, d, J = 9.2 Hz, H3 5-Br-salo) and 8.60 (2H, br, H4- and H7-neoc), 8.05 (2H, s, H5- and H6-neoc), 7.86 (2H, d, J = 7.3 Hz, H3- and H8-neoc), 3.00 (6H, s, CH3-neoc). 2.3.2.4. Synthesis of [Cd(5-Br-salo)2(dpamH)] (5). Yellow crystals suitable for X-ray structure determination, yield 59.0%, 200 mg, analyzed as [Cd(5-Br-salo)2(dpamH)], (C24H17Br2CdN3O4) (MW = 683.62): C, 42.16; H, 2.51; N, 6.14; Found: C, 42.31; H, 2.59; N, 6.23%. IR spectrum (KBr): selected peaks in cm
1: 3309, 3252 and 3192 (weak, (w)) m(NAH)dpamH, 1634(s) d(NAH)dpamH,

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1626(s) m(C@O), 1580(m) m(C@N), 1318(sm) m(CAO?Cd), 846(m), 770(m), 741(m) and 727(m) d(CAH)dpamH, 529 (m) m(CdAO); 491 (m) m(CdAN); UV–vis: as Nujol mulls, k/nm: 270, 317, 422; in DMSO, k/nm (e/M
1 cm
1): 269 (17 600), 316 (3200), 421(9400); molar conductivity in 1 mM DMSO = 11 lS/cm. 1H NMR spectrum in DMSO-d6 (d/ppm):9.63 (2H, s, H7 5-Br-salo), 7.40 (2H, d, J = 2.9 Hz, H6 5-Br-salo), 7.20 (2H, dd, J = 9.2, 2.9 Hz, H4 5-Br-salo), 6.51 (2H, d, J = 9.2 Hz, H3 5-Br-salo) and 9.53 (1H, br, NH dpamH), 8.20 (2H, d, J = 4.7 Hz, H3 and H3-dpamH), 7.71–7.60 (4H, m, H5, H5, H6, H6-dpamH), 6.84 (2H, dd as t, J = 6.0 Hz, H4 and H4-dpamH). 2.4. X-ray crystal structure determination Single crystals of [Cd(5-Br-salo)2(bipy)]2 (2), [Cd(5-Br-salo)2 (phen)]2 (3) and [Cd(5-Br-salo)2(dpamH)] (5), suitable for crystal structure analysis were obtained by slow evaporation of their mother liquids at room temperature. The crystals were mounted at room temperature on a Bruker Kappa APEX2 diffractometer equipped with a triumph monochromator using Mo Ka radiation. Unit cell dimensions were determined and refined by using the angular settings of at least 200 high intensity reflections (>10r(I)) in the range 11 < 2h < 36°. Intensity data were recorded using / and x scans. All crystals presented no decay during the data collection. The frames collected for each crystal were integrated with the Bruker SAINT Software package [37], using a narrow-frame algorithm. Data were corrected for absorption using the numerical method (SADABS) based on crystal dimensions [38]. All the structures were solved using the SUPERFLIP package [39], incorporated in Crystals. Data refinement (full-matrix least-squares methods on F2) and all subsequent calculations were carried out using the Crystals version 14.40b program package [40]. All nonhydrogen 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. Illustrations with 50% ellipsoids probability were drawn by CAMERON [41]. Crystallographic data for complexes 2, 3 and 5 are presented in Table 1. 2.5. DNA-binding studies In order to study the interaction of CT DNA with the compounds, the compounds were initially dissolved in DMSO (1 mM). Mixing of such solutions with the aqueous buffer DNA solutions used in the studies never exceeded 5% DMSO (v/v) in the final solution, which was needed due to low aqueous solubility of most compounds. All studies were performed at room temperature. The interaction of the free 5-Br-saloH with CT DNA has been recently reported [31]. Study with UV spectroscopy: The interaction of 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. Control experiments with 5% DMSO were performed and no changes in the spectra of CT DNA were observed. The UV spectra of CT DNA in the presence of each compound were recorded for a constant CT DNA concentration in diverse mixing ratios (r = [compound]/[DNA]). The DNA-binding constants of the compounds, Kb (in M
1), were determined by the Wolfe–Shimer equation [42] (Eq. (S1)) and the plots [DNA]/(eA
ef) versus [DNA] using the UV spectra of the compounds recorded, for a constant concentration in the presence of DNA for diverse r values. Viscometry: 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 compounds 1–5 up to the value of r = 0.45. All measurements were

performed at room temperature. The obtained data are presented as (g/g0)1/3 versus r, where g is the viscosity of DNA in the presence of the compound, and g0 is the viscosity of DNA alone in buffer solution. 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 lM EB and 26 lM CT DNA in buffer (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). The possible intercalating effect of the compounds (5-BrsaloH and complexes 1–5) was studied by adding a certain amount of a solution of the compound step by step into a solution of the pre-treated 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. The compounds do not show any fluorescence emission 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 (KSV, in M
1) were calculated according to the linear Stern– Volmer equation [43] (Eq. (S2)) and the plots Io/I versus [Q]. 2.6. Interaction with albumins The albumin binding study was performed by tryptophan fluorescence quenching experiments using BSA (3 lM) or HSA (3 lM) 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 1–5 as quencher with increasing concentration. The fluorescence spectra were recorded from 300 to 500 nm at an excitation wavelength of 295 nm [44]. Most of compounds 1–5 exhibited under the same experimental conditions (i.e. excitation at 295 nm) a low-intensity emission band in the region 395–415 nm. Therefore, the quantitative studies of the serum albumin (SA) fluorescence spectra were performed after their correction by subtracting the spectra of the compounds. The influence of the inner-filter effect [45] on the measurements was evaluated by Eq. (S3). The Stern–Volmer and Scatchard equations (Eqs. (S4)– (S6)) [46] and graphs were used in order to study the interaction of each quencher with SAs and calculate the dynamic quenching constant KSV (in M
1), the approximate quenching constant kq (in M
1 s
1), the SA-binding constant K (in M
1) and the number of binding sites per albumin (n) (Tables S1 and S2). The interaction of 5-Br-saloH with HSA and BSA has been recently reported [33]. 3. Results and discussion 3.1. Synthesis-general considerations of the complexes The reaction of Cd(NO3)24H2O with 5-Br-saloH in methanol (deprotonated by sodium methoxide) afforded solid microcrystalline compound in good yield, according to reaction 1.

2CdðNO3 Þ2  4H2 O þ 4ð5-Br-saloHÞ þ 4CH3 ONa ! ½Cdð5-Br-saloÞ2 ðCH3 OHÞ2 þ 4NaNO3 þ 2CH3 OH þ 8H2 O ðreaction1Þ The obtained cadmium(II) complex [Cd(5-Br-salo)2(CH3OH)]2 (1), is neutral and possesses 1:2 metal-to-ligand composition, as it is indicated from elemental analyses. It is soluble in MeOH (slightly), DMF and DMSO, but not in CH3CN, CH3COCH3, CH2Cl2, EtOH, H2O and Et2O.

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Table 1 Crystallographic data for complexes 2, 3 and 5.

Empirical formula Molecular mass Crystal system T (K) Radiation type k (Å) Space group Unit cell dimensions a (Å) b (Å) c (Å) a (°) b (°) c (°) V (Å3) Z l (mm
1) Crystal density, Dx (g cm
3) Crystal size (mm) h range for data collection (°)/completeness (%) Range of h, k, l Measured reflections/independent reflections/Rint Parameters Goodness-of-fit on F2 (GOF) Final R indices R1, wR2 [I > 2r(I)] R1, wR2 (all data) Largest difference peak/hole (e Å
3)

2 [Cd(5-Br-salo)2(bipy)]2

3 [Cd(5-Br-salo)2(phen)]2

5 [Cd(5-Br-salo)2(dpamH)]

C48H32Br4Cd2N4O8 1337.22 monoclinic 295 Mo Ka 0.71073 P121/c1

C52H32Br4Cd2N4O8 1385.26 triclinic 295 Mo Ka 0.71073
P1

C24H17Br2Cd1N3O4 683.62 triclinic 295 Mo Ka 0.71073
P1

11.2697(3) 20.7743(7) 10.2901(3) 90 100.657(2) 90 2367.57(12) 2 4.331 1.876 0.14  0.18  0.42 1.839–30.107/99.2
15?15,
29?29,
14?14 54 251/6932/0.051 298 1.000

9.7965(6) 11.1603(7) 11.6491(6) 99.560(4) 91.046(4) 104.588(4) 1213.03(13) 1 4.230 1.896 0.27  0.34  0.39 1.776–26.929/99.6
12?12,
14?14,
14?14 16 921/5240/0.050 316 1.000

7.9608(9) 11.0982(13) 13.5866(14) 96.304(5) 97.457(6) 90.460(5) 1182.7(2) 2 4.338 1.919 0.10  0.15  0.41 2.258–28.363/93.1
10?10,
14?14,
18?18 19 432/5513/0.042 307 1.000

0.0421, 0.0647 0.0684, 0.0730 1.18/
0.93

0.0425, 0.0792 0.0812, 0.0875 0.81/
0.93

0.0502, 0.0748 0.1320, 0.1110 2.21/
1.49

The reaction of a methanolic solution of the Cd(NO3)24H2O with 5-Br-saloH in methanol (deprotonated by sodium methoxide) and with an a-diimine (bipy, phen, neoc, dpamH) led to the preparation of complexes 2–5, respectively, with different formulae and coordination modes of the ligand 5-Br-salicylaldehyde, depending on the a-diimine used, as indicated in reactions (2)–(4).

3.2. Spectroscopy (IR, UV–vis and 1H NMR)

2CdðNO3 Þ2  4H2 O þ 4ð5-Br-saloHÞ þ 4CH3 ONa þ 2a-diimineðbipy or phenÞ ! ½Cdð5-Br-saloÞ2 ða-diimineÞ2 þ 4NaNO3 þ 4CH3 OH þ 8H2 O

ðreaction2Þ

2CdðNO3 Þ2  4H2 O þ 2ð5-Br-saloHÞ þ 2CH3 ONa þ 2neoc ! ½Cdð5-Br-saloÞðneocÞðNO3 Þ2 þ 2NaNO3 þ 2CH3 OH þ 8H2 O ðreaction3Þ CdðNO3 Þ2  4H2 O þ 2ð5-Br-saloHÞ þ 2CH3 ONa þ dpamH ! ½Cdð5-Br-saloÞ2 ðdpamHÞ þ 2NaNO3 þ 2CH3 OH þ 4H2 O ðreaction4Þ Alternatively, complexes [Cd(5-Br-salo)2(bipy)]2 (2) and [Cd (5Cl-salo)2(phen)]2 (3) were also isolated from the reaction of [Cd(5Br-salo)2(MeOH)]2 (1) with the corresponding a-diimine (bipy, phen), as indicated in reaction 5.

½Cdð5-Cl-saloÞ2 ðMeOHÞ2 þ a-diimineðbipy or phenÞ ! ½Cdð5-Cl-saloÞ2 ða-diimineÞ2

oxygen atom in all complexes, but in a bidentate and/or tridentate manner in different complexes, as it will be clarified by spectroscopy and X-ray crystallography. The crystal structure for the compounds 2, 3 and 5 was verified by single-crystal X-ray diffraction analysis.

ðreaction5Þ

The synthesized cadmium(II) complexes are neutral and soluble in DMF and DMSO, but insoluble in most organic solvents and H2O. Evidence of the coordination mode of the ligands in the novel cadmium compounds is also arisen from the interpretation of the IR, UV and 1H NMR data of the 5-bromo-salicylaldehyde ligand, the a-diimines and the complexes. In these compounds, the a-diimine is coordinated bidentately through the heterocyclic nitrogen atoms, while 5-bromo-salicylaldehyde behaves as monoanionic ligand, being coordinated to cadmium ion through the phenolato

IR spectroscopy confirmed the deprotonation of 5-Br-saloH and its binding mode as well as the binding modes of the a-diimines. In the IR spectra of the complexes, the peaks of the stretching and bending vibrational modes of the phenolic OH around 3200 cm
1 and 1400 cm
1, respectively, disappeared indicating the deprotonation of the salicylaldehyde [47,48]. Additionally, the peak originating from the CAO stretching vibration at 1276 cm
1, exhibited in IR spectra of the complexes positive shifts towards 1300 cm
1 denoting coordination through the deprotonated phenolic oxygen. The peak at 1660 cm
1, attributable to the aldehyde bond m (HC@O) of the free 5-Br-saloH, shifted towards lower wavenumber (1630 cm
1) in the complexes, denoting, thus, the bidentate mono-anionic character of the ligand [49]. This is valid for complexes 1, 4 and 5. However, for complexes 2 and 3 with bipy and phen, respectively, apart from the peak at 1630 cm
1, there is an additional peak at 1660 cm
1, which means the co-existence of non-coordinated aldehyde oxygen of salicylaldehyde ligand in the complexes. The intense bands at 1590 cm
1 attributed to the stretching vibrations m(C@N)aromatic are also present in the mixed-ligand complexes, denoting the coordination through the nitrogen atoms of the a-diimine ligands. In the IR spectrum of the compound 4, the presence of coordinated nitrato ligands is also demonstrated due to the presence of two strong bands located at 1445 and 1298 cm
1, assigned to the m4 and m1 vibrations modes of the nitrate group, respectively, (C2V symmetry, coordinated nitrate group). The magnitude of the splitting D, where D = m4
m1, is 147 cm
1 and it is typical of monodentate (MAOANO2) bonding of nitrato ligands [50].

A. Zianna et al. / Polyhedron 107 (2016) 136–147

141

Fig. 1. 1H NMR spectrum of complex (A) [Cd(5-Br-salo)(neoc)(NO3)]2 (4) and (B) [Cd(5-Br-salo)2(dpamH)] (5) in DMSO-d6.

The UV spectra of the complexes have been recorded as Nujol mull and in DMSO solution and are similar. In addition, in order to explore the stability of complexes in buffer solution, the UV spectra in the series of pH (pH range 6–8, since the biological experiments are performed at pH = 7) with the use of diverse buffer solutions (150 mM NaCl and 15 mM trisodium citrate at pH values regulated by HCl solution) were also recorded. Significant changes (shift of the kmax or new peaks) were not observed in

the spectra, indicating that complexes 1–5 keep their integrity in the pH range 6–8 [20,51]. 1 H NMR spectroscopy was also used to confirm the deprotonation of the salicylaldehyde and the stability of the complexes in solution. The deprotonation of the phenolic hydrogen can be easily deduced from the absence of the AOH signal, which is obvious to the 1H NMR spectra of the free 5-Br-saloH, appearing as single peak at d = 11 ppm. All the expected sets of signals related to the

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presence of the ligands in the corresponding compounds are present; four signals for the aromatic protons of 5-Br-salo
and four ones of the a-diimine ligands, as shown, representatively, for complexes 4 and 5 in Fig. 1. From the number of protons appeared in the 1H NMR spectra of the complexes, we can conclude that the ratio 5-Br-salo to a-diimine is 2:1 in complexes 2, 3 and 5, and 1:1 in complex 4. All signals are slightly shifted as expected upon binding to cadmium ion. The 1H NMR spectra of the title complexes give the protons, attributable to the aldehydo group at d = 9.87–9.47 ppm and the benzene protons of the 5-Br-salo
at d = 7.48–6.45 ppm, while the protons due to the a-diimines at d = 9.03–6.84 ppm. The absence of additional set of signals related to dissociated ligands suggests that all complexes remain intact in solution [19,20,52–54]. The fact that the complexes are non-electrolytes in DMSO solution (KM = 11–30 mho cm2 mol
1, in 1 mM DMSO solution) having the same UV spectral pattern in Nujol, in DMSO solution and in the presence of the buffer solution and their 1H NMR spectra confirm no dissociation, suggests that the compounds are stable and keep their integrity in solution [20,52,55]. 3.3. Description of the structures for complexes 2, 3 and 5 The molecular structures of three complexes (2, 3 and 5) with the atom numbering scheme are depicted in Figs. 2 and 3, respectively, and selected bond distances and angles are cited in Table 2. In general, the 5-X-salo
ligands may be coordinated to metal ions via three different modes (Scheme 3): (a) in a bidentate chelating mode, through the phenolic oxygen and the aldehyde oxygen (Scheme 3(I)) as found in a series of mononuclear Co(II) [56–58], Zn(II) [59] and Cr(III) [60] complexes, (b) in a mixture of bridging mode with the phenolic oxygen being bidentately bound to both metal ions (M1 and M2) forming a monoatomic bridge and the aldehyde oxygen bound monodentately to M1 forming a chelate ring (Scheme 3(II)), as reported in a series of homodimetallic (Cu(II)) [61,62] or heterodimetallic (Al-Rh) [63] complexes, and (c) in a bridging mode, with the phenolic oxygen forming a monoatomic (O,O)-bridge bound to two metal ions (M1 and M2), and the aldehyde oxygen remaining non-coordinated (Scheme 3(III)).

Fig. 3. Molecular structure of [Cd(5-Br-salo)2(dpamH)] (5) with the displacement ellipsoids shown at the 50% probability level. The hydrogen atoms are omitted for clarity.

3.3.1. Description of the structures of [Cd(5-Br-salo)2(bipy)]2, (2), and [Cd(5-Br-salo)2(phen)]2, (3) The complexes [Cd(5-Br-salo)2(bipy)]2 (2) and [Cd(5-Br-salo)2 (phen)]2 (3) are both dinuclear and have similar structures (Fig. 2). In these complexes, the four deprotonated 5-Br-salo
ligands are coordinated to cadmium ions, in two different coordination modes. Two of them are coordinated to a cadmium ion in a bidentate chelating mode, through the phenolic oxygen O(3) and the aldehyde oxygen O(4) forming a six-membered chelate ring (i.e. being in mode I in Scheme 3), while the other two ligands serve as bidentate bridging ligands being bound to two cadmium ions via the phenolic oxygen atom O(1) and having the aldehyde oxygen O(2) non-coordinated (as in mode III in Scheme 3). This kind of coordination mode is novel, since there has not been found

Fig. 2. Molecular structures of (A) [Cd(5-Br-salo)2(bipy)]2 (2) and (B) [Cd(5-Br-salo)2(phen)]2 (3) with the displacement ellipsoids shown at the 50% probability level. The hydrogen atoms are omitted for clarity.

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A. Zianna et al. / Polyhedron 107 (2016) 136–147 Table 2 Selected bond distances (Å) and angles (°) for complexes 2, 3 and 5. Bond distance (Å)

2

3

5

3.5075(5) 2.286(3) 2.278(3) 2.184(3) 2.300(3) 2.376(4) 2.321(4)

3.4925(7) 2.380(4) 2.233(4) 2.191(4) 2.320(5) 2.360(5) 2.342(5)

— 2.239(4) 2.344(4) 2.227(3) 2.313(4) 2.292(4) 2.255(4)

2

3

Bond angle (°)

5

O1 —Cd1—O1 O1i—Cd1—O3 O1—Cd1—O3 O1i—Cd1—O4 O1—Cd1—O4 O3—Cd1—O4 O1i—Cd1—N1 O1—Cd1—N1 O3—Cd1—N1 O4—Cd1—N1 O1i—Cd1—N2 O1—Cd1—N2 O3—Cd1—N2 O4—Cd1—N2 N1—Cd1—N2 Cd1i—O1—Cd1

79.55(11) 103.61(12) 95.71(11) 95.27(11) 174.14(12) 82.79(11) 164.31(12) 101.31(12) 91.93(13) 84.42(13) 93.89(11) 91.01(12) 162.11(13) 92.07(12) 70.46(13) 100.45(11)

81.64(16) 91.95(15) 109.22(16) 168.63(15) 92.64(17) 80.61(16) 92.84(16) 157.17(15) 93.02(18) 96.11(19) 86.99(16) 86.69(16) 163.75(18) 102.56(17) 70.85(18) 98.36(16)

O1—Cd1—O2 O1—Cd1—O3 O2—Cd1—O3 O1—Cd1—O4 O2—Cd1—O4 O3—Cd1—O4 O1—Cd1—N1 O2—Cd1—N1 O3—Cd1—N1 O4—Cd1—N1 O1—Cd1—N2 O2—Cd1—N2 O3—Cd1—N2 O4—Cd1—N2 N1—Cd1—N2

77.47(15) 164.71(14) 89.39(14) 90.26(13) 81.43(15) 79.95(13) 95.16(14) 172.32(14) 98.20(13) 100.93(13) 95.61(14) 95.67(15) 93.41(14) 172.75(14) 82.81(14)

Symmetry

(i) 1
x, 1
y, 2
z

(i) 2
x, 1
y,
z

Cd(1). . .Cd(1) Cd1—O1 Cd1—O1 Cd1—O3 Cd1—O4 Cd1—N1 Cd1—N2

i

Bond angle (°) i

H X

C

O

X

C

O

C O

X

M1

M

O (I)

H

H

O (II)

O

M1 M2

M2

(III)

Scheme 3. Coordination modes of 5-X-salo
ligands (X = AH, ACl, ABr, ANO2, ACH3).

in the literature any compound with coordination mode III. Each cadmium ion is six-coordinated having a distorted octahedral geometry with a CdO4N2 chromophore; the coordination sphere of each cadmium is completed by two nitrogen atoms (N(1) and N(2) of an a-diimine, forming a five-membered chelate ring with Cd. The distance between the two cadmium ions Cd(1). . . Cd(1)0 = 3.5085 Å for 2 and 3.493 Å for 3 and is typical for dinuclear cadmium complexes [64,65]. 3.3.2. Description of the structure of [Cd(5-Br-salo)2dpamH)] (5) In the mononuclear complex [Cd(5-Br-salo)2(dpamH)] (5) (Fig. 3), one dpamH ligand is coordinated in a bidentate manner (through its two pyridine nitrogen atoms) as a neutral ligand forming a six-membered ring with the cadmium ion, while the deprotonated 5-Br-salo ligands are coordinated to the cadmium ion in bidentate chelating mode (through the deprotonated phenolic oxygen and the carbonyl oxygen), also forming a six-membered chelate. The cadmium ion is six-coordinated having a distorted octahedral geometry with a CdO4N2 chromophore. The structure is further stabilized by hydrogen-bonds of the type Nbipyam-H. . .Oi(Ph) between two neighboring molecules of complex [Cd(5-Br-salo)2(dpamH)], i.e. N3—H31. . .O1i with H31. . . O1i = 2.0320 Å, N3. . . O1i = 2.841(8) Å and N3—H31. . .O1i = 156.51(14)° (Fig. S1). 3.4. Interaction with calf-thymus DNA Metal complexes can bind with CT DNA covalently and/or noncovalently. In covalent interactions, at least one labile ligand of the

complexes is replaced by a nitrogen atom of a DNA-base such as guanine N7 atom. Non-covalent binding can be classified into three types: (i) intercalation of the metal complex into the DNA helix via p–p stacking, (ii) external binding (electrostatic) on the outside of the helix and (iii) groove-binding (van der Walls interactions and hydrogen bonding) [66,67].

3.4.1. DNA-binding studies by UV absorption spectroscopy UV spectroscopy is a useful tool to investigate the existence and the binding strength of the interaction between a compound and CT DNA, since any type of interaction may perturb the band of CT DNA at 258–260 nm or the intra-ligand transition bands of the complex during the titrations. Bathochromism is evidence of stabilization of the structure; when it is accompanied by hypochromism, due to p–p stacking interactions, a conclusion concerning the DNA-interaction via intercalation may be deduced [68]. Therefore, the UV spectra of a CT DNA solution were recorded in the presence of complexes 1–5 at increasing amounts (for different [compound]/[DNA] mixing ratios = r) as well as the UV spectra of the compounds in the presence of CT DNA at increasing amounts (r0 = [DNA]/[compound] mixing ratio). The UV spectra of DNA solution (1.1–1.5  10
4 M) were recorded in the presence of complexes 1–5 at increasing r values (up to 0.3) (representatively shown for 3 in Fig. 4(A)). The UV spectra of the DNA solution in the presence of complexes 1–5 are quite similar exhibiting slight hyperchromism, which may be considered as evidence of the formation of a new adduct of the compound

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A. Zianna et al. / Polyhedron 107 (2016) 136–147

(A) 1.2

1.6 1.5 (1/3)

1.0

(η/η0)

A

0.8 0.6 0.4

(B)

25 0

260

27 0

280

λ (nm)

2 90

300

3 10

0.9 0.0

320

0.1

0.2

0.3

0.4

r = [Compound]/[DNA] Fig. 5. Relative viscosity (g/g0)1/3 of CT DNA (0.1 mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the presence of 5-Br-saloH and complexes 1–5, at increasing amounts (r = 0–0.35).

0.4

[DNA] (Fig. S2) using the Wolfe–Shimer equation [42]. The Kb constants of complexes 1–5 (Table 3) are relatively high suggesting strong binding of the complexes to CT DNA and similar to or higher than that of the classical intercalator EB (=1.23(±0.07)  105 M
1) [69], with 5 having the highest Kb constant (=3.83(±0.04)  106 M
1) among the complexes. The Kb constants of complexes 1–5 are higher than those of analogous Zn complexes with 5-Brsalo
ligands reported [31] and significantly higher than a series of Cd complexes found in the literature [16–19,20].

0.3

A

1.2

1.0

0.5

0.2 0.1 0.0

1.3

5-Br-saloH (1) (2) (3) (4) (5)

1.1

0.2 0.0

1.4

300

350

400

450

500

λ (nm) Fig. 4. (A) UV spectra of CT DNA (1.2  10
4 M) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the absence or presence of complex 3. The arrow shows the changes upon increasing amounts of the complex. (B) UV spectra of DMSO solution of complex 2 (2  10
5 M) in the presence of increasing amounts of CT DNA (r0 = 0–0.9). The arrows show the changes upon increasing amounts of CT DNA.

with double-helical DNA resulting in the stabilization of the DNA duplex [68]. In the UV–vis spectra of the complexes two discrete bands may be observed; band I in the range 315–338 nm and band II at 407– 421 nm. Upon addition of CT DNA, these bands are perturbed (representatively shown for complex 2 in Fig. 4(B)). More specifically, band I shows a moderate hypochromism up to 20% while band II exhibits a much more pronounced hyperchromism (Table 3). The position of kmax presents a slight red- or blue-shift. The obtained data may suggest the binding of the complexes to CT DNA, but safe conclusions concerning the interaction mode between the complexes and CT DNA cannot arise from the existing UV spectroscopic studies and viscosity measurements should be employed in order to clarify the DNA-binding mode. The magnitude of the binding strength a compound with CT DNA may be evaluated through the calculation of the Kb constants. The Kb constants were obtained by the plots [DNA]/(eA
ef) versus

3.4.2. DNA-binding study with viscosity measurements The viscosity of DNA is sensitive to DNA length changes when interacting with a compound since the relative solution viscosity (g/g0) and relative DNA length (L/L0) are related by the equation (L/L0) = (g/g0)1/3 [70–71]. The interaction via intercalation (insertion of the compound in between the DNA-base pairs) will result in an increase of the separation distance of DNA-base pairs lying at intercalation sites in order to host the bound compound, causing an increase of the length of the DNA helix and inducing subsequently an increase of DNA viscosity, the magnitude of which is usually in accordance to the strength of the interaction. In the case of binding to DNA grooves via a partial or non-classic intercalation (i.e. electrostatic interaction or external groove-binding), a bend or kink in the DNA helix may happen leading to a slight shortening of its effective length, and the change in viscosity of the DNA solution is less pronounced or there is no change at all [70–72]. This characteristic can be proven helpful in clarifying the interaction with DNA. The viscosity measurements were carried out on CT DNA solutions (0.1 mM) upon addition of increasing amounts of the compounds (up to the value of r = 0.35) at room temperature. The relative DNA-viscosity exhibited an increase upon addition of the compounds 1–5 (Fig. 5). The observed behavior of the DNA

Table 3 UV–vis spectral features of the UV spectra of 5-Br-saloH and complexes 1–5 upon addition of DNA. UV band (k in nm) (percentage of hyperchromism or hypochromism (DA/A0, %), blue-/red-shift of the kmax (Dk(nm)) and the corresponding DNA binding constants (Kb). Complex 5-Br-saloH [31] [Cd(5-Br-salo)2(CH3OH)]2, 1 [Cd(5-Br-salo)2(bipy)]2, 2 [Cd(5-Br-salo)2(phen)]2, 3 [Cd(5-Br-salo)(neoc)(NO3)]2, 4 [Cd(5-Br-salo)2(dpamH)], 5 a b c

‘‘+” denotes hyperchromism and ‘‘
” denotes hypochromism. ‘‘+” denotes bathochromism and ‘‘
” denotes hypochromism. ‘‘++” denotes extreme hyperchromism.

Kb (M
1)

Band (DA/A0a, Dkb) c

336 (
51, +2); 423 (++ ,
5) 332 (
10, +1); 418 (+35, 0) 338 (
15, +8); 415 (+55,
1) 330(sh) (
15, 0); 421 (+30,
2) 329 (
20, 0); 407 (++, +2) 316 (
2, 0); 421 (+40,
2)

7.13(±0.22)  105 7.62(±0.15)  105 4.23(±0.10)  105 1.36(±0.08)  106 1.33(±0.06)  105 3.83(±0.04)  106

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A. Zianna et al. / Polyhedron 107 (2016) 136–147

viscosity may be considered evidence of the existence of an intercalative binding mode to DNA.

3.4.3. EB-displacement studies A solution containing the EB–DNA compound shows an intense fluorescence emission band at 592 nm (with kexcitation = 540 nm) as a result of the intercalation of the planar EB phenanthridine ring in-between adjacent DNA-base pairs; for this reason, EB is considered a typical indicator of intercalation to DNA. If a compound which can intercalate to DNA equally or stronger than EB is added into this solution, a significant quenching of the EB–DNA fluorescence emission may be induced. Complexes 1–5 do not show any fluorescence emission at room temperature in solution or in the presence of CT DNA or EB under the same experimental conditions (kexcitation = 540 nm). Therefore, the changes observed in the fluorescence emission spectra of the EB–DNA solution, when complexes 1–5 are added, may be useful to examine the complexes’ EB-displacing ability as an indirect evidence of their intercalating ability [73]. The fluorescence emission spectra of pre-treated EB–DNA ([EB] = 20 lM, [DNA] = 26 lM) were recorded in the presence of increasing amounts of each complex 1–5 up to the value of r = 0.45 (representatively shown for complex 3 in Fig. 6(A)). The addition of the complexes resulted in a significant quenching of the fluorescence emission band of the DNA–EB compound at 592 nm which was up to 79.5% of the initial EB–DNA fluorescence (Fig. 6(B) and Table 4), indicating the significant ability of the complexes to

(A) 6000

Table 4 Percentage of EB–DNA fluorescence quenching (DI/Io, %) and Stern–Volmer constants (KSV, M
1) for 5-Br-saloH and its Cd(II) complexes 1–5. Compound

DI/Io (%)

KSV (M
1)

5-Br-saloH [31] [Cd(5-Br-salo)2(CH3OH)]2, (1) [Cd(5-Br-salo)2(bipy)]2, (2) [Cd(5-Br-salo)2(phen)]2, (3) [Cd(5-Br-salo)(neoc)(NO3)]2, (4) [Cd(5-Br-salo)2(dpamH)], (5)

79.5 72.2 53.0 75.1 63.9 63.6

3.01(±0.13)  105 8.28(±0.23)  104 1.91(±0.04)  105 9.93(±0.31)  105 4.05(±0.11)  105 1.28(±0.06)  105

displace EB for the EB-compound and revealing, thus, indirectly the interaction with CT DNA by the intercalative mode [43]. The Stern–Volmer plots (Fig. S3) illustrate that the EB–DNA fluorescence emission quenching is in good agreement (R = 0.99) with the linear Stern–Volmer equation (Eq. (S2)); thus, the observed quenching may be attributed to the displacement of EB from EB– DNA by each complex [29,30]. The KSV constants (Table 4) of the complexes were calculated by the Stern–Volmer equation using the Stern–Volmer plots (Fig. S3) are relatively high suggesting tight binding to DNA, with complex 3 having the highest KSV constant (=9.93(±0.31)  105 M
1) among the complexes. The KSV constants of complexes 1–5 are higher than those of their Zn analogues [31]. 3.5. Interaction with serum albumins Serum albumins are the major soluble protein constituents of the circulatory system. They have many physiological functions

(A)

100

5000

80

I/Io (%)

I

4000 3000

60

2000

40

1000

20

0 550

600

650

700

0

750

(nm)

(B)

100 5-Br-saloH (1) (2) (3) (4) (5)

80 60

20 0

0.3

0.4

3

0.5

r = [Compound]/[DNA] Fig. 6. (A) Fluorescence emission spectra (kexcitation = 540 nm) for EB–DNA ([EB] = 20 lM, [DNA] = 26 lM) in buffer solution in the absence and presence of increasing amounts of complex 3 (up to r = 0.05). The arrow shows the changes of intensity upon increasing amounts of 3. (B) Plot of EB–DNA relative fluorescence emission intensity at kemission = 592 nm (%) vs r (r = [complex]/[DNA]) (150 mM NaCl and 15 mM trisodium citrate at pH = 7.0) in the presence of complexes 1–5 (up to 27.8% of the initial EB–DNA fluorescence intensity for 1, 47.0% for 2, 24.9% for 3, 36.1% for 4 and 36.4% for 5).

4

5

6

7

5-Br-saloH (2) (4)

(1) (3) (5)

60

20

0.2

2

100

40

0.1

1

r=[Complex]/[HSA]

40

0 0.0

0

(1) (3) (5)

80

I/Io (%)

EB-DNA fluorescence (%)

(B)

5-Br-saloH (2) (4)

0

1

2

3

4

5

6

7

r=[Complex]/[BSA] Fig. 7. (A) Plot of HSA % relative fluorescence intensity at kem = 351 nm (%) vs r (r = [complex]/[HSA]) for 5-Br-saloH and complexes 1–5 (up to 79% of the initial HSA fluorescence for 5-Br-saloH, 77% for 1,55% for 2, 35% for 3, 33.5% for 4, 51% for 5) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). (B) Plot of BSA % relative fluorescence intensity at kem = 342 nm (%) vs r (r = [complex]/[BSA]) for 5-Br-saloH and complexes 1–5 (up to 53% of the initial BSA fluorescence for 5Br-saloH, 54% 1, 40% for 2, 8% for 3, 19.5% for 4, 36% for 5) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).

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A. Zianna et al. / Polyhedron 107 (2016) 136–147

Table 5 The HSA- and BSA-quenching and binding constants derived for the free 5-Br-saloH and its complexes 1–5. Compound 5-Br-saloH [33] [Cd(5-Br-salo)2(CH3OH)]2, (1) [Cd(5-Br-salo)2(bipy)]2, (2) [Cd(5-Br-salo)2(phen)]2, (3) [Cd(5-Br-salo)(neoc)(NO3)]2, (4) [Cd(5-Br-salo)2(dpamH)], (5)

kq(HSA) (M
1 s
1)

K(HSA) (M
1) 11

2.31(±0.32)  10 1.47(±0.12)  1012 4.11(±0.26)  1012 9.72(±0.29)  1012 1.03(±0.46)  1013 5.40(±0.21)  1012

[74] and the most important one is to transport drugs and other bioactive small molecules through the blood-stream [74,75]. BSA has been one of the most extensively albumins studied, especially because of its structural homology with HSA [75]. BSA is constituted of three homologous domains (I, II, III) and has two tryptophans, Trp-134 and Trp-212, embedded in the first subdomain IB and subdomain IIA, respectively. HSA is a globular protein composed of 585 aminoacid residues in three homologous a-helices domains (I–III) bearing only one tryptophan located at position 214 along the chain, in subdomain IIA [76,77]. Solutions of BSA and HSA exhibit an intense fluorescence emission band with kem,max = 342 nm and 351 nm, respectively, when excited at 295 nm [45] because of the tryptophan residues. Most of complexes 1–5 in buffer solutions exhibited a maximum emission in the region 395–415 nm under the same experimental conditions and the SA fluorescence emission spectra were corrected before the calculations processing. The inner-filter effect was also taken into consideration and was calculated with Eq. (S3); it was not found so significant affecting slightly the measurements [45,46]. The addition of complexes 1–5 to a SA solution resulted in a low to moderate quenching of the SA fluorescence emission band (i.e. at kem = 351 nm for HSA and at kem = 342 nm for BSA) which is in the case of BSA was more pronounced (Fig. 7). The observed SA quenching in the presence of a compound may be attributed to changes in tryptophan environment of SA as a result of possible changes in SA secondary structure due to binding of the compound to SA [77]. The kq constants for the compounds were calculated from the corresponding Stern–Volmer plots (Figs. S4 and S5) using the Stern–Volmer quenching equation (Eqs. (S4) and (S5)) and they are cited in Table 5. In almost all cases, the quenching ability of complexes 1–5 is higher than that of free 5-Br-saloH. Complex 4 exhibits the highest kq constant for HSA (kq(HSA),4 = 1.03(±0.46)  1013 M
1 s
1) and complex 3 for BSA (kq(BSA),3 = 6.16(±0.34)  1013 M
1 s
1) and are similar to that of other cadmium complexes [16]. The kq constants are higher than 1010 M
1 s
1 indicating, thus, the existence of static quenching mechanism [43]. The K constants of the compounds to both the SAs were calculated from the corresponding Scatchard plots (Figs. S6 and S7) using the Scatchard equation (Eq. (S6)) and they are given in Table 5. The K constants of the complexes are similar to that of free 5-Br-saloH, with complexes 2 and 3 exhibiting the highest association constants for HSA (K(HSA),2 = 1.74(±0.15)  105 M
1) and BSA (K(BSA),3 = 2.42(±0.12)  105 M
1), respectively, among the complexes. The K constants of the compounds are in the range 4.07  104– 1.43  106 M
1, indicating their rather tight binding to SA in order to get transported either towards their potential targets or away from Cd-poisoned organs or tissues during a potential detoxification process; the compounds may get released when arriving at the potential targets or the excretion site, if removed from a poisoned area, since their non-covalent binding to the SA is less tight than the strongest known non-covalent (irreversible) bonds attributed to the interaction of diverse ligands to the protein avidin with K value = 1015 M
1 [78,79].

kq(BSA) (M
1 s
1) 6

1.43(±0.09)  10 1.06(±0.08)  105 1.74(±0.15)  105 1.60(±0.07)  105 1.35(±0.12)  105 9.10(±0.43)  104

K(BSA) (M
1) 12

4.63(±0.31)  10 3.40(±0.21)  1012 8.82(±0.35)  1012 6.16(±0.34)  1013 2.46(±0.13)  1013 9.37(±0.29)  1012

1.43(±0.08)  105 4.07(±0.32)  104 5.85(±0.35)  104 2.42(±0.12)  105 1.17(±0.09)  105 1.10(±0.06)  105

4. Conclusions Five cadmium complexes with 5-bromo-salicylaldehyde in the absence or presence of a a-diimine (bipy, phen, neoc or dpamH) were synthesized and characterized. The reaction of Cd(NO3)2 4H2O and deprotonated 5-Br-salicylaldehyde resulted in the formation of complex [Cd(5-Br-salo)2(CH3OH)]2 (1), while the addition of the a-diimine led to the formation of complexes [Cd (5-Br-salo)2(bipy)]2 (2), [Cd(5-Br-salo)2(phen)]2 (3), [Cd(5-Br-salo) (neoc)(NO3)]2 (4) and [Cd(5-Br-salo)2(dpamH)] (5). The structures of three complexes (2, 3 and 5) were determined by X-ray crystallography, revealing combinations of different coordination modes of the deprotonated ligand 5-Br-salicylaldehyde to the cadmium ion, i.e. bidentate/tridentate chelating/bridging ligation. The ability of the complexes to bind to CT DNA was estimated by UV spectroscopic studies. The DNA-binding constants were calculated so as to estimate the magnitude of the binding of the complexes to CT DNA. All complexes exhibit relatively high binding constants, with complex 5 possessing the highest Kb constant among the complexes (3.83(±0.04)  106 M
1). An intercalative binding mode of the complexes to DNA was mainly supported by viscosity measurements, a conclusion which was further enforced by the ability of the complexes to displace the classical intercalator EB from its EB–DNA compound. Complexes 1–5 were also studied in regard to their affinity to human and bovine serum albumins. The complexes exhibited similar SA-fluorescence quenching ability and similar SA-binding affinity with free 5-Br-saloH as estimated through the values of the quenching and the SA-binding constants, respectively. Complexes can bind reversibly to the albumins as concluded by the values of their association constants. The present results may be considered encouraging for future studies concerning the Cd complexes with possible modifications of the structure, the substituted salicylaldehyde or the a-diimine ligands.

Acknowledgment This work was funded by program of ‘‘Aristeia 2014”, Aristotle University of Thessaloniki, Greece.

Appendix A. Supplementary data CCDC 1054640–1054642 contains the supplementary crystallographic data for compounds 2, 3 and 5. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving. html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail: [email protected]. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.poly.2016.01.020.

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