Effective copper(II) and nickel(II) complexes with N3O and ON3 thiosemicarbazidato ligands. Synthesis, structural analysis and in vitro cytotoxity on melanoma B16F10 cells

Article abstract of DOI:10.1016/j.ica.2019.119347

Novel copper(II) and nickel(II) complexes were newly synthesized by template condensation using 2-amino-3,5-dibromo- or 2-hydroxy-5-bromo-benzaldehyde S-propyl thiosemicarbazones as starting materials. The complexes with asymmetric donor atom set were ide

Full text of DOI:10.1016/j.ica.2019.119347

Journal Pre-proofs
Effective copper(II) and nickel(II) complexes with N3O and ON3 thiosemicar-
bazidato ligands. Synthesis, structural analysis and in vitro cytotoxity on mel-
anoma B16F10 cells
Musa Şahin, Songül Eğlence-Bakır, Merve Alpay, Selin Alpay, Recep
Özmerdivenli, Bahri Ülküseven
PII:
S0020-1693(19)31070-9
https://doi.org/10.1016/j.ica.2019.119347
ICA 119347
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
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2 December 2019
5 December 2019
Please cite this article as: M. Şahin, S. Eğlence-Bakır, M. Alpay, S. Alpay, R. Özmerdivenli, B. Ülküseven,
Effective copper(II) and nickel(II) complexes with N3O and ON3 thiosemicarbazidato ligands. Synthesis,
structural analysis and in vitro cytotoxity on melanoma B16F10 cells, Inorganica Chimica Acta (2019), doi:
https://doi.org/10.1016/j.ica.2019.119347
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Effective copper(II) and nickel(II) complexes with N3O and ON3
thiosemicarbazidato ligands. Synthesis, structural analysis and in vitro
cytotoxity on melanoma B16F10 cells
Musa Şahina,*,musahin@istanbul.edu.tr, Songül Eğlence-Bakıra, Merve Alpayb, Selin
Uluçayc, Recep Özmerdivenlid, Bahri Ülküsevenc
aFaculty of Science, Department of Chemistry, Istanbul University, Istanbul, Turkey
bMD Anderson Cancer Center, Department of Cancer Systems Imaging, Houston, USA
cFaculty of Engineering, Department of Chemistry, Istanbul University-Cerrahpaşa, Istanbul,
Turkey
dFaculty of Medicine, Department of Physiology, Adnan Menderes University, Aydın, Turkey
*Corresponding author.
Graphical abstract
Highlights
Synthesis and structural characterization of N3O and ON3-type copper(II) and nickel(II)
complexes.
Cytotoxicity properties of the complexes on melanoma B16F10 and fibroblast BJ ATCC
CRL-2522 cell lines.
Abstract
Novel copper(II) and nickel(II) complexes were newly synthesized by template condensation
using
2-amino-3,5-dibromo-
or
2-hydroxy-5-bromo-benzaldehyde
S-propyl
thiosemicarbazones as starting materials. The complexes with asymmetric donor atom set
1
were identified through use of analytical and spectroscopic methods (IR, H NMR, ESI-MS

and X-ray crystallography). Cytotoxicity potents of the N3O and ON3-type complexes were
determined on melanoma B16F10 and fibroblast BJ ATCC CRL-2522 cell lines. The cell
lines were exposed to the complexes in the concentration range of 6-500 µM for 24 h. The
results indicated selective effect of the nickel and copper complexes in favor of healthy cells.
Namely, relative low concentrations of the test molecules resulted in cytotoxic effects on
malignant cells while higher concentrations were required for normal cell death. The nickel
complexes were more effective than the copper complexes and a nickel complex (1a) showed
selectively cytotoxcity by IC50 value of 1,978 μg/ml on B16F10 cells compared to the 5,464
μg/ml value of CRL-2522 cells.
Keywords: Copper(II) and Nickel(II); Thiosemicarbazone; X-ray structure; Cytotoxicity;
Melanoma
1. Introduction
Biologically active synthetic molecules have been one of the priorities of chemistry
researches and the design of molecules are performed to obtain the biomimetic properties are
desired. Schiff bases are popular compounds in drug investigations due to their nitrogen-rich
conjugated structures. In recent years, metal complexes of Schiff bases have been examined
more frequently, especially copper (II), iron(III), nickel (II) or oxo-metal ions such as
vanadyl, molybdenyl centered ones [1-6].
Researches have been revealed a wide range of biological activity depending on the metal ion
and the substituents of the ligand. The iron(III) complexes of N-salicylidene-lysine and
hydroxynaphthylidene-arginine as netropsin mimics have DNA binding and photonuclease
activity [7]. Some copper(II) complexes of N2O-Schiff bases have been reported as anti-
cancer effective chemicals [8,9]. However, copper(II) complexes with N2O2 ligands are also
known with their antimicrobial [10-12] and antifungal properties, and even some copper(II)
complexes have shown better antifungal activity compared to the free ligand [13].
Oxovanadium(IV) complexes with salen-type Schiff bases were widely examined as anti-
cancer and anti-diabetic drugs [14]. A platinum(II) complex of salphen-type Schiff base
ligand exhibits in vitro cytotoxicities against seven human tumor cells in quite low IC50 values
via apoptosis mechanism [15]. Examination for the biological properties of palladium

complexes of ON, ONS, and ONN-chelating Schiff bases, particularly thiosemicarbazones,
have been determined some antimicrobial [16], antitumor [17,18], and antiviral [19] activities.
After publication of the selective cytotoxic effects of some iron(III) centric N2O2 donor
thiosemicarbazidato complexes on leukemia cells [20], the studies related to metal complexes
of such tetradenate thiosemicarbazones increased. The oxovanadium(II) complex with a
N1,N4-diarylidene-S-methylthiosemicarbazone having in vivo insulinomimetic effect [21], a
nickel(II) complex showing in vitro cytotoxity on HL60 leukemia cells [22], and the
vanadium(VI) complexes having antioxidant properties [23] are some of biologically effective
complexes with N2O2-thiosemicarbazidato ligand.
Melanoma is the most dangerous skin cancer that results from the malignant growth of color
cells called "melanocytes" in the top layer of our skin. The worldwide incidence is 3 in
100,000 and 1 person per hour dies from melanoma [24]. As it is in the world, the frequency
of seeing it in Turkey is increasing [25]. Melanoma; starts and grows rapidly on a stain or
blemish over a benign or normal spot, with a dark brown or black color, 3-4 mm wide,
rounded, with a regular streak or bump resembling the edges [26]. Melanoma treatment is
possible if the cancer does not exceed the upper layer in the early period, the tumor is
surgically removed and 100 percent healing is achieved. When melanoma progress, newly
designed compounds can be achieved for a novel treatment [27].
So far most studies have tended to focus on substituted 2-hydroxy-aldehydes as carbonyl
compounds to obtain template complexes of thiosemicarbazones [28, 29, 30]. Herein, we
presented new copper(II) and nickel(II) complexes having asymmetric donor sets, ON3 or
N3O, derivated by using a 2-amino-aldehyde in addition to a 2-hydroxy-aldehyde (Fig. 1). By
study, the thiosemicarbazone-based nickel(II) and copper(II) complexes were fully
characterized and determined antiproliferation properties on melanoma B16F10 and normal
epidermal CRL-2522 cell lines.
2. Experimental
2.1. Materials and physical measurements

The elemental analyses were determined by a Thermo-Finnigan Flash EA 1112 Series
Elementary Analyzer. Infrared spectra of complexes were recorded on an Agilent Carry 630
1
FTIR-ATR spectrophotometer in the range of 4000–600 cm-1 at ambient temperature. H-
NMR spectra were recorded on a Bruker Avance-500 model spectrometer relative to SiMe4
using deuterated chloroform as solvent at 25 ± 2 °C. The mass spectra were recorded on a
Thermo Finnigan LCQ Advantage MAX system using ion-trop mass analyzer for ESI source.
The X-ray intensity data of 1a were measured on a Bruker D8 VENTURE system equipped
with a multilayer monochromator and a Mo Kα Sealed tube (λ = 0.71073 Å) and for 1b data
were measured on a Rigaku RAXIS RAPID imaging plate area detector with graphite
monochromated Mo Kα radiation. Details of the data collection conditions and the parameters
of refinement process were given in Table 1. The single crystal in dimensions of 0.010 ×
0.030 × 0.500 mm for 1a and 0.80 × 0.10 × 0.10 mm for 1b were obtained directly from
ethanol/chloroform solution.
2.2. Synthesis
The complexes with ON3 chelating thiosemicarbazidato ligands were synthesized by using S-
propyl-N1-2-hydroxy-5-bromobenzylidene thiosemicarbazone and S-propyl-N1-2-hydroxy-3-
methoxybenzylidene thiosemicarbazone. The complexes with N3O chelating
thiosemicarbazidato ligands were synthesized by using S-propyl-N1-2-amino-3,5-
dibromobenzylidene thiosemicarbazone.
2.2.1. Synthesis of S-propyl-N1-2-amino-3,5-dibromobenzylidene thiosemicarbazone
The thiosemicarbazide (5 mmol, 0.45 g) and 1-bromopropane (5 mmol, 0.45 mL) were mixed
in 20 mL ethanol. The mixture was refluxed for 3 h. A solution of 2-amino-3,5-dibromo
benzaldehyde (5 mmol, 1.39 g) in ethanol/chloroform mixture (1:1, 10 mL) was then added.
The reaction mixture was refluxed for 4 h, and the clear solution was cooled to room
temperature. An aqueous solution of NaHCO3 (5%, w/v) was added to the reaction mixture
for neutralization to pH 7. The resulting yellow precipitate, S-propyl-N1-2-amino-3,5-
dibromobenzylidene thiosemicarbazone, was filtered off, washed with water and diethyl ether,
and dried over P2O5 in vacuo.

The colors, m.p. (°C), yields (%), elemental analyzes, IR (cm−1) and 1H NMR (CDCl3, 25 °C,
ppm) data of the thiosemicarbazones are given as follows:
0
S-propyl-N1-2-hydroxy-5-bromobenzylidene thiosemicarbazone: Light yellow, 138 C,
90%, Anal. Found (Calc.) for C11H14BrN3OS (316.22 g/mol) C, 42.01 (41.78); H, 4.89 (4.46);
N, 13.33 (13.29); S, 10.65 (10.14)%. IR: υ(OH) 3277, υs(NH2) 3476, υas(NH2) 3420, υ(C=N)
1605, υ(C-O) 1150, υ(C-H) 2929, 2968. 1H NMR: 11.55, 11.34 (s, 1H, OH), 8.28, 8.15 (s, 1H,
i:2/1 CH=N1), 7.30-6.79 (m, 3H, aromatics), 5.00 (s, 2H, NH2), 2.99, 2.84 (t, 2H, i:2/1,
J:7.32, S-CH2-), 1.68 (m, 2H, -CH2-), 1.01, 0.96 (t, 3H, i:2/1, J:7.32, CH3-).
S-propyl-N1-2-hydroxy-3-methoxybenzylidene thiosemicarbazone: Yellow, 267 0C, 82%,
Anal. Found (Calc.) for C12H17N3O2S (267.35 g/mol) C, 53.95 (53.91); H, 6.71(6.41); N,
15.72 (15.72); S, 12.32 (11.99)%. IR υ(OH) 3220, υs(NH2) 3416, υas(NH2) 3308, υ(C=N)
1600, υ(C-O) 1120, υ(C-H) 2952, 2929, 2871. 1H NMR: 11.68 (s, 1H, OH), 8.37, 8.25 (s, 1H,
i:2/1 CH=N1), 6.85-6.75 (m, 3H, aromatics), 5.02 (s, 2H, NH2), 3.85 (s, 3H, -OCH3), 3.00,
2.87 (t, 2H, i:2/1, J:7.32, J:6.83, S-CH2-), 1.68 (m, 2H, -CH2-), 0.98 (m, 3H, CH3-).
0
S-propyl-N1-2-amino-3,5-dibromobenzylidene thiosemicarbazone: Yellow, 155 C, 66%,
Anal. Found (Calc.) for C11H14Br2N4S (394.13 g/mol) C, 33.50 (33.52); H, 3.60 (3.58); N,
14.20 (14.22); S, 8.00 (8.14)%. IR: υs(NH2) 3469, υas(NH2) 3249, υs(NH2) 3395, υas(NH2)
3275 υ(C=N) 1605, υ(C-O) 1128, υ(C-H) 2948, 2929, 2856. 1H NMR: 8.36, 8.24 (s, 1H, i:1/1
CH=N1), 7.58-7.49 (m, 2H, aromatics), 7.18-6.75 (s, t, 4H, NH2), 2.98, 2.92 (t, 2H, i:1/1,
J:7.32, J:6.83, S-CH2-), 1.62 (m, 2H, -CH2-), 0.98 (t, 3H, J:7.32, CH3-).
2.2.2. Synthesis of N1-2-hydroxy-5-bromobenzylidene-N4-2-amino-3,5-dibromobenzylidene-S-
propylthiosemicarbazidato Nickel(II) (1a)
NiCl2.6H2O (0.23g, 1 mmol) was dissolved in absolute ethanol (20 ml). A solution of S-
propyl-N1-2-hydroxy-5-bromobenzylidene thiosemicarbazone (0.31 g, 1 mmol) and 2-amino-
3,5-dibromo benzaldehyde (0.27 g, 1 mmol) in ethanol/chloroform mixture (1:1, 20 ml) was
added drop wise to the metal solution with vigorous stirring. The red colored reaction mixture
was stirred at 40 oC for 2 h and than allowed to stand at room temperature for a few days. The
brown product was collected by filtration and washed with cold chloroform. The powder
crystals were dried over P2O5 under vacuum.

The other nickel (2a) and copper (2b) complexes were synthesized using the same procedures
described above.
1
The colors, m.p. (°C), yields (%), elemental analyzes, IR (cm−1), H NMR (for 1a and 2a)
and ESI-MS (for 1b and 2b) data of the complexes are given as follows:
0
1a: Brown, 166 C; 38 %, Anal. Found (Calc.) for C18H15Br3N4NiOS (633.81 g/mol): C,
34.24 (34.11); H, 2.16 (2.39); N, 8.56 (8.84); S, 4.51 (5.06) %. IR: υ(NH) 3326, υ(C=N1)
1
1597, υ(C=N4) 1571, υ(C-O) 1149, υ(C-H) 2957, 2890. H-NMR: 8.06 (s, 1H, CH=N1), 7.92
(s, 1H, CH=N4), 7.45-7.10 m (5H, aromatics), 7.10 (m, 1H, Ar-NH), 3.31 (m, 2H, S-C1H2),
1.84 (m, 2H, -C2H2), 1.10 (t, 3H, -C3H3).
0
1b: Redish Brown, 229 C, 58 %; Anal. Found (Calc.) for C19H18Br2N4CuO2S (589.79
g/mol): C, 38.52 (38.69); H, 2.80 (3.03); N, 9.47 (9.50); S, 5.28 (5.44) %. IR: υ(NH) 3328,
υ(C=N1) 1593, υ(C=N4) 1571, υ(C-O) 1120, υ(C-H) 2948, 2920, 2901. m/z (+c ESI-MS):
590.0 ([M+H]+, 100%).
0
2a: Brown, ˃ 300 C; 42 %, Anal. Found (Calc.) for C18H15Br3N4NiOS (633.81 g/mol): C,
34.15 (34.11); H, 2.49 (2.39); N, 8.63 (8.84); S, 5.24 (5.06) %. IR: υ(NH) 3338, υ(C=N1)
1
1575, υ(C=N4) 1524, υ(C-O) 1127, υ(C-H) 2965, 2967. H-NMR: 8.25 (s, 1H, CH=N1), 8.22
(s, 1H, CH=N4), 7.60-7.08 m (5H, aromatics), 5.54 (s, 1H, Ar-NH), 3.32 (t, 2H, S- C1H2),
1.84 (m, 2H, -C2H2), 1.10 (t, 3H, -C3H3).
2b: Redish brown, 210 0C; 53 %, Anal. Found (Calc.) for C19H18Br2N4CuO2S (589.79 g/mol):
C, 38.84 (38.69); H, 2.92 (3.03); N, 9.55 (9.50); S, 5.26 (5.44) %. IR: υ(NH) 3349, υ(C=N1)
1612, υ(C=N4) 1582, υ(C-O) 1153, υ(C-H) 2961, 2929. m/z (+c ESI-MS): 590.0 ([M+H]+,
100%).
2.3. Cell Culture
The melanoma cell line B16F10 was purchased from Istanbul Institute. B16F10 cells and
CRL-2522 were maintained in DMEM (Dulbecco's Modified Eagle Medium) with and
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Irvine Scientific, Santa Ana,
California, USA) containing 20% Fetal bovine serum (Irvine Scientific, Santa Ana,
California, USA). Aganist contamination, using 50 µg/mL gentamycin sulfate solution (Irvine

Scientific, Santa Ana, California, USA). Cells were cultured in 25-75 cm2 flasks (BD Falcon,
Rockville, MD, USA) in a humidified 5% CO2 air at 37 °C. The medium was changed related
to confluence rapidity. Cells were subcultured 1:4 ratio with Trypsin-EDTA solution (Irvine
Scientific, Santa Ana, California, USA) before designed-molecules application.
2.4. Cell Proliferation/Viability Assay
Based on cell proliferation, have counted by trypan blue assay [31]. Additionally, melanoma
and fibroblast cells were seeded in 96-well plates for MTT viability assay. In each well of 96,
1x105 cells/well density of in 100 μL of culture medium were managed. The concentrations
were 500 µg/ml to 0 during application on cells. Stock solutions were prepared 1 mg/ml, then
diluted equally before treatment. The cells were incubated 24 h preparation of enable
attachment. Dead or unattached cells were removed by washing out with PBS (Phosphate
Buffered Saline). (The Quick Cell Proliferation Assay Kit, Biovision, San Francisco, USA).
Assays were performed according to the document. The cells were briefly seeded in 96-well
in control well and diluted concentration of different molecules administration as a final
volume 100 μL medium. Following 1 h incubation, absorbance values were measured at 590
nm in a spectrophotometric reader. The results were presented as % cell viability. Each
experiment was done in triplicate.
3. Results and discussion
3.1. Spectral data
The formation of the complexes can be clearly concluded from the disappearing of OH and
NH2 streching vibrations and the formation of a new azomethine absorbtion band (C=N4) with
1571 cm1 besides C=N1 group for 1a and 1b complexes. The new band assigned to the
streching vibration of -NH related to the anilino group was recorded at 3326 cm1 for 1a and
3328 cm1 for 1b. Similarly, for complexes, 2a and 2b, obtained by using 2-amino-3,5-
dibromo-S-propyl thiosemicarbazone, the disappearance of amidic NH2 band and the
appearance of anilinic NH band at 3338 cm1 (for 2a) and 3349 cm1 (for 2b) are the infrared
data proved the complex formation. New azomethine bands, ν(C=N4), were observed at 1524
cm1 for 2a and 1582 cm1 for 2b.
1
When H-NMR spectra of the nickel complexes were examined, the ON3 ligand structure of
1a is confirmed by disappearance of proton signal belonging to phenolic OH and observation
of one proton signal for NH group originated from 2-amino-3,5 dibromo-aldehyde. Two

proton signals no observed after the formation of 2a were from the 2-amino- and 2-hydroxy
groups of the aldehydes condensed to the N1 and N4 nitrogens, respectively.
The mass spectra analysis results of the copper complexes are given in the experimental
sections. The mass data exhibited the protonated molecular ion peak m/z [M+H]+: 590.0
(100%) for each of 1b and 2b which are agreement with the molecular formula equal to
589.79 g/mol.
3.2. Crystalography
The structural views of the complexes are presented in Figures 2 and 3. The images with cell
axes and the hydrogen bond interactions in the crystal packing are given in Figures 4 and 5.
Selected bond distances and angles are listed in the Table 2. X-ray crystallography studies
have revealed that nickel complex (1a) and copper complex (1b) belong to the triclinic and
orthorhombic crystal system, respectively (Table 1).
Table 3 shows the data about the hydrogen bonding in the crystal pack for 1a and 1b
compounds. The unit cell of 1a consist of two unit of nickel complexes in which complex
units form centrosymmetric dimeric structures by C1-H4∙∙∙Br2 hydrogen bond interaction
with distances 2.80 Å along the crystallographic c* axis (Fig. 4). In the packing diagram of 1b
complex along b axis, the molecules form non-centrosymmetric dimers held together by C10-
H9∙∙∙O2 and C7-H3∙∙∙O2 hydrogen bond interactions with distances 2.70 Å and 2.52 Å,
respectively (Fig. 5).
The coordination geometries of the copper complexes may be described as slightly distorted
square plane being a phenolic oxygen, two imin nitrogen and an anilino nitrogen in corners.
According to the angles of diagonal axes, O1-Ni-N1 (178.9º), N3-Ni-N4 (176.6º), O1-Cu-N3
(173.3º), and N1-Cu-N4 (172.5º), the tetragonal distortion of the copper complexes are higher
(Fig.6). The bond lengths between nickel center and donor atoms are shorter than in copper
complexes as a consequence of stronger binding of the ligand backbone to nickel ion. As we
already know from the studies so far, the template effect of nickel(II) with d8 electronic
structure is more powerful compared to copper(II) to form a square planar complex [32, 33].

3.3.Antiproliferation activity
The effects of the test samples on the antiproliferation of skin cancer cells (B16F10) were
investigated by comparing to fibroblast BJ ATCC CRL-2522 cells with concentration-
dependent manner. The relationships of cell viability-concentration were shown in Figure 7
and 8. Half-maximal inhibitory concentration (IC50) values were determined after 24 h
incubation (Table 4). All complexes were dissolved in DMSO before treatment and only
DMSO solution using a positive control.
When the graphs in figures 7 and 8 are examined depending on cell viability, it is seen that the
concentrations higher than 12.65 µg/ml of all complexes cause proliferation effect. The
proliferation is more pronounced in B16F10 fibroblast cells. Actually, all of the complexes
have sufficient activity below 12.65 µg/ml. The proliferation is more pronounced in the
fibroblast cells. Essentially, the complexes have sufficient activity below 12.65 µg/ml. The
nickel complexes showing relative low antiproliferation on B16F10 cells at higher
concentrations may be considered to be more advantageous for treatment.

Morphological alterations observed in melanoma cells treated with these complexes supported
the MTT results. The morphological image captured by Olympos inverted microscope IX83
and showed that each complexes stimulated cell membrane damage at different levels. Some
unfounded cell structures thought to be caused by effect pathways of the test samples were
also monitored (Fig. 9 ). The 24 h exposure with the complexes at 500 µM to 6 µM diluted
doses resulted in varying extents of melanoma cells. Treatment using copper complexes at
over doses of 250 µM resulted in a remarkable increase cell proliferation (Fig. 10).
The image differences indicate that the complexes have some different anti-proliferative
activities on cancer cells compared to the epidermal cells. These chemical structures are
effective anticancer agents on tissue culture and they cause to cell damage at the nuclear level
which turn leads to arrest division of the cell and further more degenerative change and finally
death of the cell.
4. Conclusion
New nickel(II) and copper(II) complexes with ON3 and N3O chelating thiosemicarbazidato
ligands were synthesized and characterized by using analytical and spectroscopic methods.
The structures were confirmed by X-ray diffraction analysis. Antiproliferation potantials of
the chelate complexes on melanoma B16F10 cells were examined by comparing the healthy
CRL-2522 ones.
MTT results showed that the nickel complexes were relatively more effective on melanoma.
In particular, complex 1a showed a selective cytotoxicity with an IC50 value of 1.978 µg/ml
on B16F10 melanoma cells versus 5.464 µg/ml in CRL-2522 epidermal cells. All complexes
have proliferation effect on CRL-2522 at concentrations higher than 12.65 µg/ml. On
melonama, the copper complexes exhibited proliferation at concentrations higher than 62.5
µg/ml while the nickel complexes have antiproliferative effect at each concentration.

Observation of morphological alterations suggests that the concentration of the copper
complexes in tumor cells and tissue should be high enough to induce stimulation of the
mechanism of apoptosis. According to our findings, tumors can be treated with the copper
complexes that are not toxic to cells but which can bind to tumor cellular with cytotoxic
selectivity. In accordance cell viability data, the nickel complexes 1a and 2a were determined
to be the more effective and copper complexes 1b and 2b were the less toxic.
In accordance cell viability data, the nickel complexes 1a and 2a were determined to be the
more effective and copper complexes 1b and 2b were the less toxic. However, by observation
of morphological alterations suggests that the concentration of the copper complexes in tumor
cells and tissue should be high enough to induce stimulation of the mechanism of apoptosis.
According to our findings, tumors can be treated with the copper complexes at lower than
62.5 µg/ml concentration that are not toxic to cells but which can bind to tumor cellular with
cytotoxic selectivity. In this reason, the copper complexes can be used as a model to obtain
the drug active ingredient.
Acknowledgement
This work was supported by the Scientific Research Projects Coordination Unit of Istanbul
University (Project Number: 22391).
Appendix A. Supplementary material
CCDC 1842525 (for 1a) and CCDC 1842526 (for 1b) contain the supplementary
crystallographic data for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac. uk/data_request/cif.
References

[1] S. A. Hosseini-Yazdi, A. Mirzaahmadi, A. A. Khandar, V. Eigner, M. Dušek, M.
Mahdavi, S. Soltani, F. Lotfipour, J. White, Polyhedron, 124 (2017) 156-165.
[2] B. Kaya, O. Şahin, M. Bener, B. Ülküseven, Journal of Molecular Structure, 1167 (2018)
16-22.
[3] Y. Kaya, A. Erçağ, A. Koca, Journal of Molecular Structure, 1102 (2015) 117-126.
[4] T. Bal-Demirci, M. Şahin, M. Özyürek, E. Kondakçı, B. Ülküseven, Spectrochimica Acta
Part A: Molecular and Biomolecular Spectroscopy, 126 (2014) 317-323.
[5] A. Akbari, H. Ghateazadeh, R. Takjoo, B. Sadeghi-Nejad, M. Mehrvar, J. T.
Mague, Journal of Molecular Structure, 1181 (2019) 287-294.
[6] S. Eğlence, M. Şahin, M. Özyürek, R. Apak, B. Ülküseven, Inorganica Chimica Acta, 469
(2018) 495-502.
[7] M. S. A. Begum, S. Saha, M. Nethaji, A. R. Chakravarty, Journal of Inorganic Biochem.
104 (2010) 477–484.
[8] X. Qiao, Z.-Y. Ma, C.-Z. Xie, F. Xue, Y.-W. Zhang, J.-Y. Xu, Z.-Y. Qiang, J.-S. Lou, G.-
J. Chen, S.-P. Yan, Journal of Inorg Biochem. 105 (2010) 728-37.
[9] K. Hu, F. Li, Z. Zhang, F. Liang, New J. Chem. 41 (2017) 2062-2072.
[10] E. Ispir, S. Toroglu, A. Kayraldiz, Trans. Met. Chem., 33 (2008) 953-960.
[11] M. V. Angelusiu, S. F. Barbuceanu, C. Draghici, G. L. Almajan, Eur. J. Med. Chem., 45
(2010) 2055-2062.
[12] P. J. K. Inba, B. Annaraj, S. Thalamuthu, M. A. Neelakantan, Bioinorganic Chemistry
and Applications 2013 (2013) 1- 11.
[13] K. B. Gudasi, M. S. Patil, R. S. Vadavi, R. V. Shenoy, S. A. Patil, M. Nethaji, Trans.
Met. Chem., 31 (2006) 580-585.
[14] K. Kustin, J. Costa Pessoa, D. C. Crans, American Chemical Society, Washington DC
(2007) 340 –351.
[15] Y. Peng, H. Zhong, Z.-F. Chen, Y.-C. Liu, G.-H. Zhang, Q.-P. Qin, H. Liang, Chem. and
Pharm. Bull. 62 (2014) 221-228.
[16] S. A. Khan, M. Yusuf, European Journal of Medicinal Chem. 44 (2009) 2270-2274.
[17] S. Padhye, Z. Afrasiabi, E. Sinn, J. Fok, K. Mehta, N. Rath, Inorg. Chem. 44 (2005)
1154-1156.
[18] A.Garoufis, S.K.Hadjikakou, N.Hadjiliadis, Coordination Chemistry Reviews, 253
(2009) 1384-1397
[19] P. Genova, T.Varadinovaa A.I. Matesanz, D. Marinova, P. Souza, Toxicology and
Applied Pharmacology 197 (2004) 107– 112.

[20] T. Bal, B. Atasever, Z. Solakoğlu, S. Erdem-Kuruca, B. Ülküseven, European Journal of
Medicinal Chem. 42 (2007) 161-167.
[21] R. Yanardağ, T. Bal-Demirci, B. Ülküseven, S. Bolkent, S. Tunalı, Ş. Bolkent, European
Journal of Medicinal Chem. 44 (2009) 818-826.
[22] B. Kaya, B. Atasever-Arslan, Z. Kalkan, H. Gür, B. Ülküseven, General Physiology and
Biophysics 35 (2016) 451–458.
[23] T. Bal-Demirci, M. Şahin, M. Özyürek, E. Kondakçı, B. Ülküseven, Spectrochimica
Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 317–323.
[24] A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, and M. J. Thun, Cancer Journal for
Clinicians 57 (2007) 43–66.
[25] International Agency for Research on Cancer. "Global cancer observatory." World
Health Organization.(2018) http://gco. iarc. fr. Accessed 8.
[26] B. R. Smoller, Modern Pathology 19 (2006) 34–40.
[27] P. Quatresooz, G. E. Pierard, C. Pierard-Franchimont, International Journal of Molecular
Medicine 24 (2009) 295–301.
[28] S. Duman, İ. Kızılcıklı, B. Ülküseven, Phosphorus, Sulfur, Silicon Relat. Elem. 190
(2015) 342–351.
[29] P. Kumaria , T. S. Lobanaa, R. J. Butcher, A. Castineiras, M. Zeller, Inorganica Chimica
Acta 482 (2018) 268–274.
[30] R. Takjoo, A. Hashemzadeh, H.A. Rudbari, F. Nicolo, J. Coord. Chem. 66 (2013) 345-
357.
[31] W. Strober, Trypan blur exclusion test of cell viability. Current protocols in immunology
(1997) 21.1: A. 3B. 1-A. 3B. 2.
[32] M. D. Revenco, O. V. Palamarciuc, P. N. Bourosh, J. Lipkowski, M. Gdaniec, Y. A.
Simonov, R. Clérac, Inorganica Chimica Acta 368 (2011) 157–164.
[33] M. Guricova, M. Pizl, Z. Smekal, L. Nadherny, J. Cejka, V. Eigner, I. Hoskovcova,
Inorganica Chimica Acta 477 (2018) 248–256.
Fig. 1. The metal chelates. For ON3 is R1, R2, M: H, Br, Ni (1a); OCH3, H, Cu (1b),
For N3O is R1, R2, M: H, Br, Ni (2a); OCH3, H, Cu (2b)

Fig. 2. ORTEP diagram of 1a showing the labelling scheme of all atoms with 50% probability
level.
Fig. 3. ORTEP diagram of 1b showing the labelling scheme of all atoms with 50% probability
level.
Fig. 4. Packing and hydrogen bonding interaction of 1a along c* view with unit cell
Fig. 5. Packing and hydrogen bonding interactions of 1b along b view with unit cell
Fig. 6. Comparison of some bond angles and lengths of 1a and 1b complexes
Fig.7. Cell viability ratios obtained by MTT assay for the nickel complexes
Fig. 8. Cell viability ratios obtained by MTT assay for the copper complexes
Fig. 9. Morphological alterations on melanoma cells after treatment with nickel complex 1a
Fig. 10. Morphological alterations on melanoma cells after treatment with copper complex 1b
Table 1. Crystal data and structure refinement for 1a and 1b
1a
1b
CCDC deposit number
Empirical formula
1842525
1842526
C18H15Br3N4NiOS
brown, rod
C19H18Br2N4CuO2S
black, chunk
Crystal colour, habit

Crystal size (mm)
Formula weight (g/mol)
Temperature (K)
Wavelength (Å)
Crystal system
0.010 x 0.030 x 0.500 0.80 x 0.10 x 0.10
633.84
589.79
302(2)
293(5)
0.71073
0.71073
Triclinic
orthorhombic
Pbca
Space group
P – 1
Cell dimensions
a = 7.082(6) Å
b = 12.394(10) Å
c = 13.144(10) Å
α = 107.93(2)°
ß = 101.94(2)°
γ = 93.81(2)°
1063.4(15)
2
a = 18.8339(3) Å
b = 8.73970(10) Å
c = 25.4167(3) Å
α = 89°
ß = 89°
γ = 90°
Cell volume (Å3)
Cell Formula units (Z)
Dcal (g/cm−3)
4183.66(10)
8
1.980
1.873
Absorption coefficient (mm−1)
F (000)
6.664
5.000
616
2328
h, k, l ranges
−8/7, −14/14, −15/14
-22/22, -9/10, -30/30
Reflections collected
6847
158576
Independent reflections
3715 [R(int) = 0.1593] 4205 [R(int) = 0.052]
Data/restraints/parameters
Goodness-of-fit (GOF) on F
Final R indices [I>2σ(I)]
3715 / 268 / 255
1.006
4205/3657/280
0.830
R = 0.126, wR = 0.286 R = 0.112, wR = 0.098
Largest diff. peak and hole 2.927 and -0.927
(e−/Å−3)
1.31 and -1.03
Table 2. Selected bond distances (Å) and angles (°) for 1a and 1b
1a
1b
1a
distances(Å) Atoms
1b
distances(Å) atoms
atoms
angle (°) atoms
angle (°)
173.3(3)
172.5(4)
93.3(3)
O1- Ni1
N3- Ni1
N1- Ni1
N4- Ni1
1.86(1)
O1- Cu1
N1- Cu1
N3- Cu1
N4- Cu1
1.904(7)
1.942(9)
1.954(8)
1.918(8)
O1- Ni- N1
N3- Ni- N4
O1- Ni- N4
O1- Ni- N3
178.9(8) O1- Cu1- N3
176.6(9) N1- Cu1- N4
1.89(2)
1.86(2)
1.85(2)
86.6(9)
96.0(8)
O1- Cu1- N4
O1- Cu1- N1
93.1(3)

N4-C15
O1-C8
S1-C4
N3-N2
N3-C5
N1-C12
1.30(4)
1.30(3)
1.76(2)
1.42(2)
1.31(3)
1.38(4)
S1-C9
1.74(1)
1.35(1)
1.31(1)
1.39(1)
1.32(1)
1.32(1)
N4- Ni- N1
N1- Ni- N3 82.9(9)
94.5(9)
N1- Cu1- N3
N3- Cu1- N4
80.8(4)
93.0(3)
N4-C15
O1-C1
N1-N2
N1-C8
N3-C13
Table 3. Hydrogen bond parameters (Å and o) of complex 1a and 1b
D—H····A
1a
D—H
0.96
H····A
2.80
D····A
3.72(4)
D—H····A
158
C1-H4C···Br2
1b
C10-H9···O2
C7-H3····O2
0.95(3)
0.94(2)
2.70(2)
2.52(2)
3.46(2)
3.29(2)
138(2)
138(1)
Table 4. IC50 values of the molecules for B16F10 and CRL2522 cell lines
Cell Lines
IC50 (µg/ml)
1a
1b
2a
2b
B16F10
CRL2522
1,978
5,464
3,919
2,673
3,594
5,883
4,205
4,373