Accepted Manuscript Synthesis and antioxidant activities of transition metal complexes based 3-hydroxysalicylaldehyde-S-methylthiosemicarbazone Tülay Bal-Demirci, Musa Şahin, Esin Kondakç ı, Mustafa Özyürek, Bahri Ülküseven, Reşat Apak PII: DOI: Reference:

S1386-1425(14)01585-6 http://dx.doi.org/10.1016/j.saa.2014.10.088 SAA 12903

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

23 September 2014 12 October 2014 23 October 2014

Please cite this article as: T. Bal-Demirci, M. Şahin, E. Kondakç ı, M. Özyürek, B. Ülküseven, R. Apak, Synthesis and antioxidant activities of transition metal complexes based 3-hydroxysalicylaldehyde-Smethylthiosemicarbazone, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.10.088

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Synthesis and antioxidant activities of transition metal complexes based 3hydroxysalicylaldehyde-S-methylthiosemicarbazone Tülay Bal-Demircia*, Musa Şahina, Esin Kondakçıb, Mustafa Özyürekb, Bahri Ülküseven a and Reşat Apakb a, *

Department of Chemistry, Inorganic Chemistry Section, Istanbul University, 34320, Avcilar,

Istanbul, Turkey, Corresponding author.E-mail: [email protected], Tel.: +90 2124737070. b

Department of Chemistry, Analytical Chemistry Section, Istanbul University, 34320, Avcilar,

Istanbul, Turkey

Abstract The nickel(II), iron(III), oxovanadium(IV) complexes of the 3-hydroxysalicylidene-S-methylthiosemicarbazone(L)

were

obtained

from

the

3-hydroxysalicyldehyde-S-

methylthiosemicarbazone with the R1-substituted-salicylaldehyde (R1 : H, 3-OH) in the presence of Ni(II), Fe(III), VO(IV) as template ion. The ligand and its complexes were characterized by elemental analysis, electronic, UV/Vis., 1H-NMR, EPR and IR studies. The free ligand and its metal complexes have been tested for in vitro antioxidant capacity by reduction of copper(II) neocuproine (Cu(II)-Nc) using the CUPRAC method. The ligand exhibited more potent in vitro antioxidant capacity than its complexes. The obtained trolox equivalent antioxidant capacity (TEAC) value of the iron(III) complex (TEACCUPRAC = 3.27) was higher than those of other complexes. Furthermore, the antioxidant activity of the free ligand and its complexes were determined by in vitro methods measuring the scavenging activity of reactive oxygen species (ROS) including hydroxyl radical ( OH), superoxide anion radical (O2· -), and hydrogen peroxide (H2O2), showing that especially the V(IV) and Fe(III) complexes had significant scavenging activity for ROS. Keywords: Thiosemicarbazone, nickel, iron, vanadium, antioxidant

Introductions Thiosemicarbazones form an important class in chemistry and pharmacology. Thiosemicarbazones and their metal complexes have biological activities as antiviral [1-3], antibacterial [4-6], antitumor [7-11], anticancerogenic [12-17] and insulin mimetic-antidiabetic properties [18, 19] and are used as pesticide [20,

1

21] and fungicide [22, 23] in agriculture, as analytical reagent [24-27] and catalyzer [28, 29] in process chemistry as well. The importance of these compounds is that thiosemicarbazones have more than one donor atom and their metal complexes show variable behaviors depending on the type and position of substituent on thiosemicarbazone, type and charge of metal atom [30-34]. Therefore, we have studied synthesis of thiosemicarbazones and their metal complexes and have investigated their biological activities. In earlier papers, we saw that nickel(II) and iron(III) complexes of S-methylthiosemicarbazones have efficient cytotoxic activities for leukemia cells and may show remarkable therapeutic drug potential due to their cytotoxicities at 1–5 µM against K562 cells [16, 17]. Free radicals are highly reactive compounds and free radicals at high concentrations can damage all components of cells such as DNA, proteins, cell membranes. Antioxidants are crucial chemicals to retard autoxidation and to neutralize free radicals associated with the development of cancer and other health problems. The combination of certain foods, natural and synthetic rubbers, and gasolines with oxygen in air at room temperature cause to undesirable status such as rancidity in foods, loss of elasticity in rubbers and formation of gums in gasolines. The potential value of antioxidants has prompted scientists to search for the cooperative effects of compounds for improving antioxidant activity and cytotoxicity. To protect biomolecules against the attack of ROS and to suppress the resultant oxidative damage, various natural and synthetic ROS scavengers and antioxidants have been developed and studied. Among them, thiosemicarbazones and their metal complexes have been evaluated for their free radical scavenging activity [35-37]. Recognizing the importance of thiosemicarbazone metal complexes as antioxidants, we have synthesized and characterized thiosemicarbazone derivative, 3-hydroxy salicylidene-S-methyl-thiosemicarbazone (L) (Scheme 1) and its Ni(II), Fe(III) and VO(IV) complexes. The Schiff base and its metal complexes were characterized by elemental analyses, IR, 1 H NMR, ESR spectral analysis, and molar conductance studies. The in vitro antioxidant capacities as well as ROS scavenging activities of these compounds were investigated systematically. We know that thiosemicarbazones have cytotoxic activities and are selective for leukemia cells, K562, in previous studies [16, 17]. Therefore, we prepared the nickel and iron complexes (1a, 1b, 2a, 2b) which have cytotoxic activities and synthesized new oxovanadium(IV) complexes (1c, 2c) of those and investigated antioxidant capacities of them in this paper (Scheme 1). The antioxidant capacities of the compounds were determined by Normal CUPRAC (CUPric Reducing Antioxidant Capacity) assay [38], Hydroxyl radical scavenging (HRS) activity [39], Hydrogen peroxide scavenging (HPS) activity [40], Superoxide anion radical scavenging activity (SRSA) [41, 42] methods.

2

X

OH

R1: H

b

+NiCl2/FeCl3/VOSO4 c

4

1 d

c

4

+

N S

CH3

O

NH2

N

d

s

1a, 1b, 1c

OH

1

r

N

N N

S OH

q

M

OH

b

p O

O

R1

H3C L

X

OH

R1: 3-OH

b

+NiCl2/FeCl3/VOSO4

HO

q

O

O M

4

1

c d

N N

r

N S

s CH3

2a, 2b, 2c 1a: (M: Ni), (X : -); 1b: (M: Fe), (X: Cl); 1c: (M: V), (X: =O); 2a: (M: Ni), (X : -); 2b: (M: Fe), (X: Cl); 2c: (M: V), (X: =O) Scheme 1:

The formation of the complexes [MLX].

Experimental Section General Procedures All chemicals were of reagent grade and used as commercially purchased without further purification. The elemental analyses were performed on a Thermo Finnigan Flash EA 1112 Series Elemental Analyzer and Varian Spectra–220/FS Atomic Absorption spectrometer. UV-Vis spectra were obtained with the aid of an ATI-Unicam UV-Visible Spectrometer UV2 Series. Infrared spectra of the compounds were recorded on KBr pellets with a Mattson 1000 FT-IR spectrometer. 1H-NMR spectra were recorded on Bruker AVANCE- 500 model spectrometer. Magnetic measurements were carried out at room temperature by the Gouy technique with a MK I model device obtained from Sherwood Scientific. The molar conductivities of the compounds were measured in 10−3 M DMSO solution at 25±1°C using a digital WPA CMD 750 conductivity meter. The ESI-MS analyses were carried out in positive and negative ion modes using a Thermo Finnigan LCQ Advantage MAX LC/MS/MS. The mobile phase consisted of MeOH. Hypersil Betabasic-8 (5 µ, 100 mm×4.6 mm) column was used at a flow rate of 0.3 ml/min at 25°C. ESI-MS inlet conditions; in the positive ion mode: heated capillary temp., 200°C; sheath gas flow rate, 40 units; capillary voltage, (−20)-(−45) V and tube lens offset, 20 V; in the negative ion mode: heated capillary temp., 270–290°C; sheath gas flow rate, 40 units; capillary voltage, 20 V and tube lens offset, 20 V. Spectrophotometric antioxidant capacity and activity measurements of trolox and synthesized compounds were performed by using a Perkin Elmer Lambda 35 UV-Vis spectrophotometer using a pair of matched quartz cuvettes of 1 cm thickness. The pH measurements were made with the aid of a HI 221 Calibration Check Microprocessor pH-meter using a glass electrode.

3

Synthesis

of

N1 -3-hydroxysalicylidene-S-methyl-thiosemicarbazone

(L):

The

3-hydroxysalicylidene-S-methyl-

thiosemicarbazone was synthesized from the 3-hydroxy-salicylaldehyde with methyl iodide in equimolar ratios as in the reported procedure [43]. The color, yield (%), m.p. (°C), R f value (stationary/mobile phase), elemental analysis, UV-visible (λmax, nm, in DMF), IR (KBr, cm−1) and 1H-NMR (DMSO-d6, 25°C, δ ppm) data of L were given as follows: L: dirty yellow, 175-176 °C, 84 %, 0.2632 (CHCl3/20CHCl3 +1MeOH), Anal.Calc. for C9H11N3O2S (225 g): C, 48.00; H, 4.89; N,18.66; S, 14.22, Found: C, 48.25; H, 4.82; N, 18.59; S, 14.18%. UV–Vis spectrum in DMF [ λmax (nm), ε (dm3 cm-1 mol-1)] : 243, 315. FT-IR(KBr, cm−1): νa(NH)3472, νs(NH) 3349, ν(OH) 3218, δ(NH) 1620, ν(C=N1), ν(N 2=C) 1618, 1582, ν(C-O) 1162, 1139. 1H-NMR (DMSO-d6, 25 ◦C, ppm): δ 11.586, 10.692(cis/trans ratio: 5/2, s, 1H, OH), δ 9.050, 8.915(cis/trans ratio: 3/7, s, 1H, R(OH)), δ 8.410, 8.285 (syn/anti ratio:3/7, s, 1H, CH=N1), δ 6.877(s, 2H, NH2 ), 6.969-6.832(d-d, J:7.81, J:1.46, 1H, d), 6.8086.771(d-d, J:7.812, J:1.46, 1H, b), 6.696(t, 1H, J:7.812, c), 2.442, 2.381(cis/trans ratio:5/2, s, 3H, S-CH3). Synthesis of the nickel (II), iron(III) and oxovanadium(IV) complexes: The nickel(II) and iron(III) complexes of N1-3hydroxysalicylidene-N 4-R1-salicylidenethiosemicarbazidato chelate (1a, 1b, 2a, 2b) were prepared in the same way as that in previous study [17]. N 1-3-Hydroxysalicylidene-N4 -salicylidene-S-methyl-thiosemicarbazidato-oxovanadium(IV)

1c

was

synthesized

from

3-

hydroxysalicylaldehyde-S-methylthiosemicarbazone (1g, 1mol) and salicylaldehyde (0.47 ml, 1mol) in the presence of oxovanadium(IV)sulphate

(1.12

g,

1

mol)

in ethylalcohol.

2c

was

obtained

from

3-hydroxysalicylaldehyde-S-

methylthiosemicarbazone with 3-hydroxysalicylaldehyde and VOSO4 using the same method. The color, yield (%), m.p. (°C), Rf value (stationary/mobile phase), elemental analysis, UV-visible (λmax nm, in DMF), IR (KBr, cm−1), 1H-NMR (DMSO-d6, 25°C, δ ppm), ESI-MS data of 1a-c and 2a-c were given as follows: 1a: Claret; 268-269 0C; 52%; 0.16; Anal. Calc. for C16H13N3O3SNi (386,05 g): C, 49.78; H, 3.39; N,10.88; S, 8.31; Ni, 15.20, Found: C, 49.81; H, 3.36; N, 10.85; S, 8.33; Ni, 15.19% ; Ω (in 10-3M DMSO, ohm-1 cm2 mol-1): 5.2. FT-IR(KBr, cm-1): ν(OH) 3426, ν(C=N) 1612, 1593, 1582 ν(C-O) 1166, 1146, 1127. UV–Vis spectrum in DMF[ λmax (nm), ε (dm3 cm-1 mol-1) ]: 260 ( 16460), 300 (8270), 328 (7720), 401(10020), 483sh (2490), 549sh (1760), 816(62).

1

H-NMR(DMSO-d6, 250C, ppm): 8.85, 8.43

(cis/trans ratio: 2/9, s, 1H, R1(OH)), 8.59 (d, J:4.4, 1H, CH=N1 ), 8.31 (d, J: 6.34, 1H, CH=N4), 7.01 (d, J: 8.78, 1H, b), 7.49 (ddd, J: 6.83, J:1.95, 1H, c), 7.78 (dd, J: 8.3, J:1.95 1H, d), 6.81 (dd, J: 7.32, J:1.47 1H, p), 6.74 (t, J: 7.81, 1H, q), 6.51 (t, J: 7.81, 1H, r), 7.03 (dd, J: 8.3, J:1.46 1H, s), 2.73 (s, 3H, S-CH3). 1b: Bright black; > 390 0C; 9%; µeff (B.M.) 5.88; Anal. Calc. for C16H13N3O3SFeCl (418,66 g): C, 45.90; H, 3.13; N,10.04; S, 7.66; Fe, 13.34, Found: C, 45.88; H, 3.12; N, 10.07; S, 7.66; Fe, 13.33% ; Ω (in 10-3 M DMSO, ohm-1 cm2 mol-1 ): 19.52. FTIR(KBr, cm-1): ν(OH) 3438, ν(C=N) 1607, 1593, 1576, ν(C-O) 1169, 1153,1130. UV–Vis spectrum in DMF [ λmax (nm), ε (dm3 cm-1 mol-1)]: 261 (15000), 297 (18140), 354sh (13605), 459sh (5258), 529sh (2390). m/z (+c ESI-MS, %relative abundance): 383 [MCl] (100.00), 384 [M-Cl+H] (20.39), 385 [M-Cl+2H] (7.59), 386 [M-Cl+3H] (8.77), 399 [M-Cl+H+CH3] (12.02), 415 [M-3H] (33.50), 766 [2M-2Cl] (8.61), 787 [2M-SCH3 ] (24.42), 788 [2M-SCH3 +H] (11.59), 803 [2M-Cl+2H] (18,90), 1185 [3M-2Cl+2H] (9.52),1203 [3M-3Cl+3H2O] (7.63), 1550 [4M-4Cl+H2O] (8.42); m/z (-c ESI-MS, %relative abundance):416 [M-2H] (2.48), 434 [M+H+CH3] (6.20), 449 [MH+CH3+OH] (100.00), 450 [M+CH3 +OH] (39.86), 465 [M+SCH 3] (60.75), 466 [M+SCH3+H] (17.32), 467 [M+SCH3+2H] (44.03), 468 [M+SCH3+3H] (10.32), 469 [M+SCH3 +4H] (12.70), 483 [M+SCH3+H2O] (13.76), 860 [2M+Na] (3.52). 1c: Dark brown; > 390 0C; 62%; µeff (B.M.) 1.59; Anal. Calc. for C 16H 13N 3O4SV (394.30 g): C, 48.74; H, 3.32; N,10.66; S, 8.13; O, 16.23, Found: C, 48.56; H, 3.36; N, 10.69; S, 8.09; O, 16.20% ; Ω (in 10-3M DMSO, ohm-1 cm2 mol-1 ): 10.32. FT-IR(KBr, cm1

): ν (OH) 3415, ν (C=N) 1600, 1561, ν (C-O) 1146,1123, ν (V=O) 976, ν (V-O) 475, ν (V-N) 515. m/z (+c ESI-MS, %relative

abundance): 394 [M] (100.00), 395 [M+H] (28.12), 396 [M+2H] (10.20), 397 [M+H] (8.34), 618 [M+L -H] (4.49), 788 [2M] (6.18) 2a: Claret; 326(decomp) 0C; 68%; 0.12; Anal. Calc. for C 16H 13N 3O4SNi (402,05 g): C, 47.80; H, 3.26; N,10.45; S, 7.98; Ni, 14.60, Found: C, 47.82; H, 3.24; N, 10.47; S, 7.98; Ni, 14.59% ; Ω (in 10-3M DMSO, ohm-1 cm2 mol-1): 8.3. FT-IR(KBr, cm-1): ν(OH) 3422, 3407, ν(C=N) 1612, 1597, 1582, ν(C-O) 1168, 1150. UV–Vis spectrum in DMF [ λmax (nm), ε (dm3 cm-1 mol-1)]: 261 ( 15940), 315 (10090), 400 (10250), 524(1770), 549sh (1710), 819(67). 1H-NMR(DMSO-d 6, 250C, ppm): 8.58, 8.38 (s, 2H, R1(OH)),

4

8.64 (s, 1H, CH=N1), 8.37 (s, 1H, CH=N 4), 6.90 (dd, J: 7.32, J:1.46, 1H, b), 6.59 (ddd, J:8.3, 1H, c), 7.23 (dd, J: 8.78, J:1.46 1H, d), 6.80 (dd, J: 7.32, J:1.46, 1H, q), 6.53 (t, J: 7.81, 1H, r), 7.03 (dd, J: 8.3, J:1.47 1H, s), 2.73 (s, 3H, S-CH3). 2b: Bright black; > 390 0C; 26%; µeff (B.M.) 5.88; Anal. Calc. for C16H13N3O4SFeCl (434,65 g): C, 44.21; H, 3.01; N,9.67; S, 7.38; Fe, 12.85, Found: C, 44.22; H, 2.99; N, 9.65; S, 7.38; Fe, 12.87% ; Ω (in 10-3 M DMSO, ohm-1 cm2 mol-1): 16.56. FT-IR (KBr, cm-1): ν(OH) 3430, ν(C=N) 1615, 1597, 1584, ν(C-O) 1161, 1130. UV–Vis spectrum in DMF [ λmax (nm), ε (dm3 cm-1 mol1

)]: 252 (17720), 325(18660), 360sh (14980), 447sh (6020), 546

sh

(830). m/z (+c ESI-MS, %relative abundance): 399 [M-Cl]

(100.00), 400 [M-Cl+H] (27.95), 401 [M-Cl+2H] (9.30), 430 [M-4H] (13.76), 431 [M-2H] (51.10), 432 [M-H] (21.35), 433 [M-H] (6.68), 436 [M+H] (7.12), 437 [M+2H] (15.42), 438 [M+3H] (11.10), 798 [2M-2Cl] (8.14), 1197 [3M-3Cl] (5.15); m/z (-c ESIMS, %relative abundance): 433 [M-2H] (4.15), 434 [M-H] (6.71), 435 [M] (100.00), 436 [M+H] (24.15), 437 [M+2H] (20.41). 2c: Dark brown; > 390 0C; 74%; µeff (B.M.) 1.62; Anal. Calc. for C 16H 13N 3O5SV (410.30 g): C, 46.84; H, 3.19; N,10.24; S, 7.82; O, 19.50, Found: C, 46.92; H, 3.21; N, 10.19; S, 7.80; O, 19.55% ; Ω (in 10-3M DMSO, ohm-1 cm2 mol-1 ): 12.46. FT-IR(KBr, cm1

): ν (OH) 3400, 3376, ν (C=N) 1615, 1584, 1553, ν (C-O) 1146, ν (V=O) 976, ν (V-O) 469, ν (V-N) 515. m/z (+c ESI-MS,

%relative abundance): 410 [M] (100.00), 411 [M+H] (48.24), 412 [M+2H] (18.30), 413 [M+3H] (22.55), 414 [M+4H] (22.55), 688.7 [2M-2VO+2H] (7.09), 739 [2M-V+2H] (8.40).

Antioxidant capacity and activity Normal CUPRAC (CUPric Reducing Antioxidant Capacity) assay: The CUPRAC method, as described by Apak et al. [38], is based on the reduction of a cupric neocuproine complex (Cu(II)-Nc) by antioxidants to the yellow-orange colored cuprous chelate (Cu(I)-Nc). To a test tube were added 1 mL of 10 mM CuCl2.2H2 O, 1 mL of 7.5 mM Nc, 1 mL of 1.0 M pH 7 NH4Ac buffer solution, x mL antioxidant sample solution and (1.1 - x) mL H2O in this order. The mixture in a total volume of 4.1 mL was incubated for 30 min, and the absorbance at 450 nm was recorded against a reagent blank. The calibration curves (absorbance vs concentration graphs) of each compound were constructed under the described conditions, and their trolox equivalent antioxidant capacities (TEAC coefficients, found as the ratio of the molar absorptivity of each compound to that of trolox in the CUPRAC method) were calculated. All determinations were carried out at least three times and in triplicate at each separate concentration of the samples in order to generate consistent data with statistical errors. Hydroxyl radical scavenging (HRS) activity: The hydroxyl radicals (·OH) in aqueous media were generated through the Fenton system and spectrophotometrically determined - via hydroxylation of a salicylate probe - by the modified CUPRAC method [39]. To a test tube were added 1.5 mL of phosphate buffer (pH 7.0), 0.5 mL of 10 mM sodium salicylate (probe material), 0.25 mL of 20 mM Na2 -EDTA, 0.25 mL of 20 mM FeCl2 solution, (2-x) mL H 2O, (x) mL scavenger sample solution at a concentration of 5 x 10-4 M (all complexes), and 0.5 mL of 10 mM H2O 2 rapidly in this order. The mixture in a total volume of 5 mL was incubated for 10 min in a water bath kept at 37 °C. At the end of this period, the reaction was stopped with adding 0.5 mL of 268 U mL-1 catalase solution, and vortexed for 30 s. To 0.5 mL of the incubation solution, the modified CUPRAC method was applied. The absorbance at 450 nm of the final solution at 4.5 mL total volume was recorded 5 min later against a reagent blank. The inhibition ratio of scavengers (%) was calculated. At least three determinations were performed. Hydrogen peroxide scavenging (HPS) activity: The ability of the synthesized complexes to scavenge hydrogen peroxide was determined according to the method of Özyürek et al. [40]. To a test tube were added 0.7 mL of phosphate buffer (pH 7.4), 0.4 mL of 1 mM H2O2, 0.4 mL of 0.1 mM CuCl2.2H2O in this order (hydrogen peroxide incubation solution, used as reference). To other two test tubes were added 0.5 mL of phosphate buffer (pH 7.4), 0.4 mL of 1.0 mM H 2O2 , 0.2 mL scavenger sample solution, and 0.4 mL of 0.1 mM CuCl2.2H2O solution rapidly in this order (scavenger solutions-I and -II, identical at this stage). The mixtures in a total volume of 1.5 mL were incubated for 30 min in a water bath kept at 37 °C. At the end of this period, to reference and scavenger solution-I was added 0.4 mL H2O and to scavenger solution-II was added 0.4 mL of 268 U mL-1 catalase solution (the

5

contents of scavenger solution-I and -II became different at this stage), and vortexed for 30 s. To 1.0 mL of the final incubation solutions, the HPS–CUPRAC method was applied. The absorbance at 450 nm of the final solution at 5.0 mL total volume was recorded 30 min later against a reagent blank. The inhibition ratio of the tested compound (%) was calculated. Three determinations were carried out in order to generate consistent data with statistical errors. Superoxide anion radical scavenging activity: The commonly used indirect reference method of determining O2·- is the reduction of NBT [41] to the insoluble blue formazan. SRSA (superoxide radical scavenging activity) was evaluated by spectrophotometric measurement of formazan formed from NBT reduction by O 2·- . To a test tube was added (2.5–x) mL of DMSO, (x) mL (usually, x=0.5) of scavenger solution at a concentration of 5 x 10-4 M, 2.0 mL of 468 µM NADH, 1.0 mL of 300 µM NBT, in this order. DMSO was used as solvent for completely solubilizing the formazan compounds formed in the original NBT assay [42]. The reaction was started by adding 1.0 mL of 60 µM PMS solution to the incubation mixture. The mixture in a total volume of 6.5 mL was incubated for 5 min in a water bath kept at 25 °C, and the absorbance was read at 560 nm against DMSO. Decreased absorbance of the incubation reaction mixture indicated increased SRSA. The inhibition ratio of scavengers (%) was calculated. Results were expressed as the mean ± standard deviation (SD) of triplicate determinations.

Results and discussion Synthesis and spectral studies The nickel(II), iron(III) and oxovanadium(IV) complexes of N1-3-Hydroxysalicylidene-N4R1-salicylidene-S-methyl-thiosemicarbazidato-

chelates

were

obtained

from

3-

hydroxysalicylaldehyde-S-methylthiosemicarbazone and R1-salicylaldehyde in the presence of metal ions (Ni(II), Fe(III) and VO(IV)) in ethanol. The nickel (II) complexes with red color, the iron(III) complexes with black color and the oxovanadium(IV) complexes with dark brown color are stable and are very much soluble in polar aprotic solvents such as DMF and DMSO. The µeff values of nickel(II) complexes 1a, 2a show diamagnetic form that is attributed to square-planar structure. The magnetic susceptibility value of iron(III) complexes 1b, 2b (5.88 BM) is assigned to five unpaired electrons of high-spin iron(III). Magnetic measurement results of oxovanadium(IV) complexes 1c, 2c as 1.59 and 1.62 BM, respectively, also indicate paramagnetic structure corresponding to V(IV) state. The molar conductivity measurements of complexes show that nickel is in non-electrolytic structure, and that oxovanadium(IV) complexes are more conductive than nickel(II). The iron complexes have relatively high conductance values (19.52 and 16.52) because the chlorine atom coordinates to iron(III). The band observed at 243 nm in the UV-vis spectrum of ligand is attributed to π →π* transition belonging to substituted benzene group. Also, π →π* and n →π* transitions belonging to imine group are located in the same region. A π-p conjugation occurs because oxygen lone electron pair on -OH group enters into conjugation with the π bond. As a result of this, n →π* absorptions shift to red and the band is observed around 315 nm. In the UV-vis spectra of the 6

complexes, it was seen λ1(max) at 252-261 nm, λ2( max) at 297-360 nm, λ3(max) at 400 nm (for nickel complexes 1a, 2a), 447-459 nm (for iron complexes, 1b, 2b), λ4(max) at 524-549 nm range, λ5( max) at 816-819 nm range (for 1a, 2a). In comparison of the spectra of complexes with that of ligand L, λ1(max) and λ2(max) are assigned to intra-ligand π →π transitions, λ3(

max)

is attributed to charge

transfer transitions. While the weak transitions in the 524-549 nm range and 816-819 nm range of the spectra of nickel complexes 1a, 2a belong to weak d-d transition, these bands are masked by strong λ3(max) absorption in the iron complexes and cannot be clearly distinguished. The

νa(NH),

νs(NH)

and

δ(NH2)

bands

of

3-hydroxysalicylaldehyde-S-methyl-

thiosemicarbazone ligand (L) that were monitored at 3472, 3349 and 1620 cm-1, respectively, in the ligand spectra, are absent due to chelation in the IR spectra of the complexes. The band at 3218 cm-1 in the IR spectrum of L is attributed to OH stretching band in the 2-position of salicylaldehyde. This band disappears in the spectra of the complexes 1a-c, 2a-c after the coordination of the ligand to the metal ion. This result confirms that phenolic oxygen of L coordinates to metal atom by losing hydrogen atom. The band of 3-OH lies between 3438 and 3376 cm-1 and is recorded as a broadened band in the spectra of the complexes. In addition, the IR spectra of the oxovanadium(IV) complexes 1c, 2c show the bands in the regions 976, 475–469 and 515 cm−1 belonging to ν(V=O), ν(V-O) and ν(V-N), respectively [44].

L has several isomers [43] because the structure and isomerism can be clearly seen in the 1

H-NMR spectra of the L. The hydroxyl group on the 2-position of benzene ring was recorded as

two signals at 11.586 and 10.692 ppm in 5:2 isomer ratio, whereas substituted-hydroxyl group on the 3-position of benzene ring was recorded as two signals at 9.050 and 8.915 ppm in 3:7 isomer ratio. In the 1H-NMR spectra of the nickel(II) complexes, the bands that are attributed to hydroxyl group on 2-position were absent due to coordination to a metal atom by losing a proton. However, substituted-hydroxyl group(R1) was monitored at 8.85 and 8.43 ppm in 2:9 isomer ratios in 1a spectra, at 8.58 and 8.38 ppm in 2a spectra. The syn/anti isomerism occurs at 8.410 and 8.285 ppm in 2:7 ratios because of rotation around CH=N1 was not seen due to hindered rotation in the complex spectra 2a, 2b. Nevertheless, the occurred new CH=N4 band after complexation was recorded next to CH=N1 imine group at 8.59 and 8.31 ppm for 1a, at 8.64 and 8.37 ppm for 2a. The band assigned to NH2 group at 6.877 ppm was not seen in the spectra of nickel complexes 1a,

2a because of the coordination to aldehyde group. The mass spectra of the iron and vanadyl chelates 1b, 1c, 2b, 2c gave 100% relative abundances, the [M-Cl] (383), [M] (394), [M-Cl] (399), and [M] (410) peaks, respectively. The determined molecular peaks prove the [FeLCl] and [VOL] compositions. 7

On the basis of vibrational analysis, we suggest binding of the ligands to the nickel(II), iron(III) and oxovanadium(IV) ion through the azomethines and the deprotonated hydroxylic oxygens of thiosemicarbazone ligand.

Antioxidant Studies Antioxidant capacity of synthesized ligand and complexes measured by the CUPRAC method Thiosemicarbazones have many bioactivities such as antibacterial, antifungal and anticancer activities. The activity of these compounds is strongly dependent upon the nature of the hetero atomic ring and the position of attachment of thiosemicarbazone group to the ring as well as the form of thiosemicarbazone moiety [45, 46]. There were few papers about the antioxidant capacity and free radical scavenging activity of thiosemicarbazones [47]. Synthesized thiosemicarbazone compounds were assayed using the normal (at room temperature) and incubated (at 50 °C) CUPRAC methods [39] against trolox as the standard reference compound. Incubated measurements were performed to speed up the oxidative conversion of the tested possible antioxidant compounds with the CUPRAC reagent within 30 min protocol time of the assay. The linear calibration equations of these compounds (as absorbance in a 1 cm cell vs molar concentration) gave the molar absorption coefficient (ε) as the slope. The CUPRAC molar absorption coefficient of the tested antioxidant divided by that of trolox under the same conditions gave the trolox equivalent antioxidant capacity (TEAC), or TEAC coefficient of that compound tested for antioxidant power (Table 1). The TEAC coefficients of the Ni(II) complexes were 0.20 and 0.47, as 1a and 2a, respectively. The TEAC coefficients of the Fe(III) complexes were 0.88 and 3.27, as 1b and 2b, respectively. For Fe(III) and Ni(II) complexes, the more -OH groups there were in the ring, the larger was the TEAC value, because electron transfer-based antioxidant capacity is usually associated with electron donation from the phenolic hydroxyl groups. In the literature, the antioxidant capacity generally increases with increasing number of -OH groups [48]. Amongst the compounds screened for antioxidant capacity, L and 2b showed good antioxidant capacity. As we compared the synthesized ligand and complexes with trolox (as reference), the TEAC coefficients of L and 2b were higher than trolox (TEACtrolox = 1.0). The remaining compounds (1a, 1b, 1c, 2a and 2c) showed less antioxidant capacity with lower TEAC values.

8

The electron donation ability from the phenolic hydroxyl groups (and thus electron transfer-based antioxidant capacity) decreases with transition metal ion complexation because of the participation of these electron-donating sites to metal coordination. The presence of –NH group in the thiosemicarbazone moiety of the uncomplexed ligand also contributes to the overall antioxidant capacity largely supplied by phenolic –OH groups, because it was shown to donate an H-atom to DPPH· radical, a well-known antioxidant capacity measurement reagent, i.e. after H donation, the molecule can stabilize by resonance hybridization [49]. After the parent ligand, the only compound showing substantial CUPRAC antioxidant capacity is the Fe(III) complex 2b (Table 1). Compared to the analogic Fe(III) compound 1b having one free phenolic –OH, the dramatic increase of TEAC seen in 2b comes from the second –OH (Table 1). Fe(III) from hard electron-donating sites (O, N-chelate) is significantly stabilized [50] so that phenolic –OH groups, aside from the coordinated Fe(III), can easily donate their electrons to increase the TEAC values.

Table 1. The TEAC (trolox equivalent antioxidant capacity) coefficients of the samples with respect to the CUPRAC assay.

Sample

TEACnormal TEACinc

OH OH

L

NH2

N

4.89

5.30

0.20

0.23

0.88

0.67

0.90

0.81

N S

CH3

OH

O

1a

O Ni

N

N N S

CH3

OH

1b

Cl

O

O

Fe N

N N S

CH3

OH

1c

O

O

O

V N

N N S

CH3

9

OH

HO

O

O

2a

Ni N

0.47

0.54

3.27

3.87

0.52

0.58

N N S

CH3

OH HO

2b

Cl

O

O

Fe N

N N S

OH

2c

CH3

HO

O

O

O

V N

N N S

CH3

Trolox εN : 1.67 x 10 4 L mol-1 cm-1 εI : 1.86 x 104 L mol-1 cm-1

Hydroxyl radical scavenging (HRS) activity Hydroxyl radical (•OH), which can easily react with amino acids, DNA and membrane components, showed the strongest chemical reactivity among ROS. Hydroxyl radicals, generated by reaction of iron-EDTA complex with H2O2, attack salicylate to form products that, upon incubating in solutions under acid conditions, yield a yellow colored product with Cu(II)neocuproine. Added radical scavengers compete with salicylate for the •OH produced, and diminish chromophore formation from Cu(II)–neocuproine. Above-mentioned model was used to measure HRS activities of trolox and synthesized thiosemicarbazone complexes as % inhibition ratio [40]. The order of HRS activity of the synthesized compounds in terms of % inhibition ratio was: 2c ≥ 1c ≥ 2a > 1a > 1b > 2b > L. The HRS activities of the synthesized compounds were less than that of trolox (the HRS activity of trolox is 81.90%). The reason that almost all compounds displayed similar (or less divergent) HRS values in between 50-65% (Table 2) is because •OH is the most rapidly reacting ROS (also having the highest reduction potential) capable of reacting with almost all organic substances −irrespective of their electron-donating functional groups− with second-order rate constants of 108-109 M-1s-1.

Hydrogen peroxide scavenging (HPS) activity

10

Hydrogen peroxide (H2O2), a biologically relevant, non-radical oxidizing species, may be formed in tissues through oxidative processes. The HPS activity of synthesized complexes were determined without interference by directly measuring the concentration of H2O2 using the HPS– CUPRAC method at 450 nm in the presence of a Cu(II) salt (since H2O2 is relatively stable, and not scavenged unless transition metal compounds are present as catalysts) [41]. The percentage inhibition ratio ranged between 7.83–90.14% (Table 2). The order of HPS activity of the synthesized compounds in terms of % inhibition ratio was: 1c > 2c > 2b > 1b > 1a > 2a > L. The HPS activities of the synthesized compounds, except for the 1a, 2a and L, were higher than trolox (17.0%). The highest HPS activity was displayed by two compounds, i.e. 1c, 2c, both being V(IV) complexes. The extraordinarily high HPS ability of V(IV) complexes, irrespective of the free phenolic -OH groups of all compounds tested, most probably arises from the V(IV)-V(V) oxidation by hydrogen peroxide [51]. H2O2, a relatively innocuous compound by itself, strongly reacts with the lower oxidation states of transition metal ions, participating in Fenton-type reactions, thereby becoming rapidly scavenged. Since the other metal complexes contained transition metal ions already in their higher oxidation states (such as Fe(III)), they did not exhibit high HPS values (Table 2).

Superoxide anion radical scavenging activity Superoxide anion (O2· -) derived from dissolved oxygen by PMS–NADH coupling reaction reduces NBT in this system. In this method, O2· - reduces the yellow dye (NBT2+) to produce the blue formazan which is measured spectrophotometrically at 560 nm. Antioxidants are able to inhibit formazan formation [42]. The decrease of absorbance at 560 nm with antioxidants indicates the consumption of O2· - in the reaction mixture. The percentage inhibition of O2· - by 5 x 10-4 M concentrations of the synthesized compounds were found as between 8.00–98.95% (Table 2). The order of superoxide anion radical scavenging (SRSA) activity of the synthesized compounds in terms of % inhibition ratio was: 1c ≥ 2c > 2b > 1b > L > 1a ≥ 2a. The SRSA values of the 1b, 1c,

2b and 2c were higher than trolox (36.3%), as 98.68, 88.04, 83.90 and 98.95%, respectively. SRSA mainly arises from the reaction affinity of the superoxide anion radical with redox-active transition metal ions and their complexes. The low SRSA of the compounds 1a, 2a (Table 2) is probably due to the redox stability of Ni(II) oxidation state in the complexes. It is noteworthy that both Fe(III) complexes 1b, 2b and V(IV) complexes 1c, 2c displayed relatively high SRSA values (Table 2) because of their affinity toward O2· - to indulge into redox reactions. O2· - may act as an oxidizing agent toward most biologically important molecules, and yet become a reducing agent 11

for certain compounds (such as NBT, used in its colorimetric determination). Thus the complexes of both Fe(III) in its higher oxidation state and of V(IV) in its lower oxidation state were good scavengers of superoxide anion radicals (Table 2). Fenton-like chemistry as a suitable property for SRSA may still be possible with complexed Fe(III) (assuming an outer-sphere process) if the complex is in the right redox potential [50].

Table 2. Hydrogen peroxide, superoxide radical and hydroxyl radical scavenging activities of the samples, as % inhibition ratio.

% Inhibition ratio Scavenger

Hydrogen peroxide scavenging activity

Superoxide radical scavenging activity

Hydroxyl radical scavenging activity

L

7.83 ± 0.32

16.12 ± 0,74

49.64 ± 0.82

1a

16.79 ± 1.23

8.19 ± 0.35

61.66 ± 3.04

1b

17.92 ± 0.90

83.90 ± 3.67

56.57 ± 2.29

1c

90.14 ± 1.46

98.95 ± 0.01

63.89 ± 0.68

2a

10.93 ± 0.09

8.00 ± 0.31

63.42 ± 3.01

2b

18.85 ± 0.34

88.04 ± 2.18

50.66 ± 0.44

2c

81.23 ± 2.57

98.68 ± 0.10

64.96 ± 2.87

Initial concentrations: 5 x 10 -4 M

Conclusion We synthesized the 3-hyroxysalicylaldehyde-S-methylthiosemicarbazone and its nickel(II), iron(III) and oxovanadium(IV) complexes of and investigated antioxidant activities. We saw that the compounds have high antioxidant values. The antioxidant properties of the ligand and complexes have also been tested using CUPRAC method in which ligand L and complex 2b exhibited better antioxidant capacity than the other complexes. Moreover, V(IV) and Fe(III) complexes of the ligand exhibited high ROS scavenging properties (depending on the type of ROS) compared to the parent ligand and trolox (as reference).

12

Acknowledgement This work was partially supported by TUBITAK (Project Number: SBAG-109S188) and by the Scientific Research Projects Coordination Unit of Istanbul University (Project Number: 22391YADOP).

References

[1] C.Jr. Shipman, S.H. John Smith, C. Drach, D.L. Klayman, Antimicrob Agents Chemother. 19(4) (1981) 682-685. [2] C. Prins, S.G. Cresawn, R.C. Condit, J. Biol. Chem. 279(43) (2004) 44858-71. [3] G. Pelosi, F. Bisceglie, F. Bignami, P. Ronzi, P. Schiavone, M.C. Re, C. Casoli, E. Pilotti, J. Med. Chem. 53 (24) (2010) 8765-8769. [4]

N.C. Kasuga, K. Sekino, C. Koumo, N. Shimada, M. Ishikawa, K. Nomiya, J. Inorg. Biochem. 2001, 84(1-2)

(2001) 55-65. [5] M.C. Rodríguez-Argüelles, P. Tourón-Touceda, R. Cao, A.M. García-Deibe, P. Pelagatti, C. Pelizzi, F. Zani, J. Inorg. Biochem. 103(1) (2009) 35-42. [6] V.E. Ivanov, N.G. Tikhomirova, A.B. Tomchin, N.V. Razukrantova, Pharm. Chem. J. 23(5) (1989) 413-5. [7] Z. Afrasiabi, E. Sinn, W. Lin, Y. Ma, C. Campana, S. Padhye, J. Inorg. Biochem. 99(7) (2005) 1526-31. [8] A.P. Da Silva, M.V. Martini, C.M. De Oliveira, S. Cunha, J.E. De Carvalho, A.L. Ruiz, C.C. Da Silva, Eur. J. Med. Chem. 45(7) (2010) 2987-93. [9] M. Mohan, M. Kumar, A. Kumar, P.H. Madhuranath, N.K. Jha, J. Inorg. Biochem. 32(4) (1988) 239-49. [10] Z. Kovacevic, S. Chikhani, D. B. Lovejoy, D.R. Richardson, Mol. Pharmacol. 80(4) (2011) 598-609. [11] J. Yuan, D.B. Lovejoy, D.R. Richardson, Blood, 104 (2004) 1450-8. [12] P. Quach, E. Gutierrez, M.T. Basha, D.S. Kalinowski, P.C. Sharpe, D.B. Lovejoy, P.V. Bernhardt, P.J. Jansson, D.R. Richardson, Mol. Pharmacol. 82 (2012) 105-14. [13] D.S. Kalinowski, P. Quach, D.R. Richardson, Future. Med. Chem. 1(6) (2009) 1143-51. [14] A.C. Sartorelli, L. Tai-Shun, Japan patent 128230, (1994). [15] F.A. French and E.J.Jr. Blanz, Cancer Res. 26 (1966) 1638-40. [16] T. Bal, B. Atasever, Z. Solakoğlu, S. Erdem-Kuruca, B. Ülküseven, Eur. J. Med. Chem. 42(2) (2007) 161-167. [17] B. Atasever, B. Ülküseven, T. Bal-Demirci, S. Erdem-Kuruca, Z. Solakoğlu, Invest. New Drugs 28 (2010) 421432. [18] R.Yanardag, T. Bal Demirci, B. Ülküseven, S. Bolkent, S. Tunalı, Ş. Bolkent, Eur. J Med. Chem. 44 (2009) 818826. [19] I.C. Mendes, L.M. Botion, A.V.M. Ferreira, E.E. Castellano, H. Beraldo. Inorg. Chim. Acta 362 (2009) 414– 420. [20] X. Yan, W. Zhen, L. Yun, D. Wei, X. Jing, L. Pei, Y. Xinling, Chin. J. Org. Chem. 32(07) (2012) 1278-83. [21] L-E.K. Pedersen, A. Svendsen, P.D. Klemmensen, Pestic. Sci. 15 (1984) 462-70. [22] R.W. Addor, G. Lamb, U.S. Patent 3,824,317, (1974). [23] Dat P. Le.; US. Patent 4,696,938, (1987). [24] K.M.M.S. Prakash, L.D. Prabhakar, D.V. Reddy, Analyst 111 (1986) 1301-6. [25] M.P. Martinez, M. Valcarcel, F. Pino, Anal. Chim. Acta 81(1) (1976) 157-65.

13

[26] K.H. Reddy, N.B.L. Prasad, T.S. Reddy, Talanta, 59 (2003) 425-33. [27] A.P. Kumar, P.R. Reddy, V.K. Reddy, J. Korean Chem. Soc. 51(4) (2007) 331-8. [28] G. Xie, P. Chellan, J. Mao, K. Chibale, G.S. Smith, Adv. Synth. Catal. 352(10) (2010) 1641-7. [29] T. Mahamo, M.M. Mogorosi, J.R. Moss, S.F. Mapolie, C.J. Slootweg, K. Lammertsma, G.S. Smith, J. Organomet. Chem. 703 (2012) 34-42. [30] T. Bal-Demirci,, Polyhedron, 27(1) (2008) 440-446. [31] B. Ulkuseven, T. Bal-Demirci, M. Akkurt, S.P. Yalcin, O. Buyukgungor, Polyhedron, 27(18) (2008) 3646-3652. [32] M. Sahin, A. Koca, N. Ozdemir, M. Dincer, O. Buyukgungor, T. Bal-Demirci, B. Ulkuseven, Dalton Trans. 39(42), (2010) 10228-10237. [33] T. Bal-Demirci, M. Akkurt, S.P.Yalcin, O buyukgungor, Trans. Met. Chem. 35(1) (2010) 95-102. [34] Ş. Güveli, A. Koca, N. Özdemir, T. Bal-Demirci, B. Ülküseven, New J. Chem. (2014) DOI: 10.1039/C4NJ00556B. [35] D. S. Raja, N. S. P. Bhuvanesh, K. Natarajan, Eur. J. Med. Chem. 46 (2011) 4584-94. [36] P. Mariana, D. Bruno, T. Jeanette, B. Lucia, G. Mercedes, C. Hugo, O. A. Claudio, N. Ester, G. Dinorah, O. Lucia, Eur. J. Med. Chem. 44 (2009) 4937-43. [37] T. Bal-Demirci, M. Sahin, M. Ozyurek, E. Kondakci, B. Ulkuseven, Spectrochimica Acta Part A 126 (2014) 317-323. [38] R. Apak, K. Güçlü, M. Özyürek, S.E. Karademir, J. Agric. Food Chem. 52 (2004) 7970-7981. [39] M. Özyürek, B. Bektaşoğlu, K. Güçlü, R. Apak, Anal. Chim. Acta 616 (2008) 196-206. [40] M. Özyürek, B. Bektaşoğlu, K. Güçlü, N. Güngör, R. Apak, J. Food Comp. Anal. 23 (2010) 689-698. [41] H. Yu, X. Liu, R. Xing, S. Liu, C. Li, P. Li, Bioorg. Med. Chem. Lett. 15 (2005) 2659-2664. [42] B. Bekdeşer, M. Özyürek, K. Güçlü, R. Apak, Anal. Chem. 83(14), (2011) 5652-5662 [43] C. Yamazaki, Can. J. Chem. 53(4) (1975) 610–620. [44] V.B. Arion, V.Ch. Kravtsov, R. Goddard, E. Bill, J.I. Gradinaru, N.V. Gerbeleu, V. Levitschi, H. Vezin, Y.A. Simonov, J. Lipkowski, V.K. Bel’skii, Inorg. Chim. Acta 317 (2001) 33-44. [45] Z. Zhong, R. Xing, P. Li, G. Mo, Int. J. Biol. Macromol. 47 (2010) 93-7. [46] B. Prathima, Y. S. Rao, S. A. Reddy, Y.P. Reddy, A.V. Reddy, Spectrochim. Acta Part A, 77 (2010) 248-52. [47] S. Ghosh, A.K. Misra, G. Bhatia, M.M. Khan, A.K. Khanna, Bioorg. Med. Chem. Lett. 19 (2009) 386-89. [48] A. Puglisi, J. Spencer, V. Oliveri, G. Vecchio, X. Kong, J. Clarke, J. Milton, Dalton Trans. 41 (2012) 2877-83. [49] D.T. Nguyen, T.H. Le, T.T.T. Bui, Eur. J. Med. Chem. 60 (2013) 199-207. [50] Y. Yu, D.S. Kalinowski, Z Kovacevic, A.R. Siafakas, P.J. Jansson, C. Stefani, D.B. Lovejoy, P.C. Sharpe, P.V. Bernhardt, D.R. Richardson, J. Med. Chem. 52(17) (2009) 5271-94. [51] M. Mohammadi, R. Yazdanparast, Exp. Toxicol. Pathol. 62 (2010) 533-8.

14

HIGHLIGHT •

We synthesized Ni(II),Fe(III),VO(IV) complexes of thiosemicarbazone and characterized

•

The compounds were tested for in vitro antioxidant capacity using the CUPRAC method.

•

It was measured the scavenging activities of reactive oxygen species (ROS), also.

•

The ligand exhibited more potent in vitro antioxidant capacity than its complexes.

•

V(IV) and Fe(III) complexes had significant scavenging activity for ROS

15

GRAPHICAL ABSTRACT

NiCl2 / FeCl3 / VOSO4

OH OH

N

+

NH2

X

HO

R

R O

O

OH

M N N

O

N

N S

S CH3

16

CH3