Cytotoxicity of copper(II)-complexes with some S-alkyl derivatives of thiosalicylic acid. Crystal structure of the binuclear copper(II)-complex with S-ethyl derivative of thiosalicylic acid

Article abstract of DOI:10.1016/j.molstruc.2016.03.058

The spectroscopically predicted structure of the obtained copper(II)-complex with S-ethyl derivative of thiosalicylic acid was confirmed by X-ray structural study and compared to previously reported crystal structure of the Cu complex with S-methyl deriva

Full text of DOI:10.1016/j.molstruc.2016.03.058

Accepted Manuscript
Cytotoxicity of copper(II)-complexes with some S-alkyl derivatives of thiosalicylic
acid. Crystal structure of the binuclear copper(II)--complex with S-ethyl derivative of
thiosalicylic acid
Miloš V. Nikolić, Marina Ž. Mijajlović, Verica V. Jevtić, Zoran R. Ratković, Slađana B.
Novaković, Goran A. Bogdanović, Jelena Milovanović, Aleksandar Arsenijević, Bojana
Stojanović, Srećko R. Trifunović, Gordana P. Radić
PII:
S0022-2860(16)30254-X
10.1016/j.molstruc.2016.03.058
MOLSTR 22369
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 17 December 2015
Revised Date: 16 March 2016
Accepted Date: 18 March 2016
Please cite this article as: M.V. Nikolić, M.Ž. Mijajlović, V.V. Jevtić, Z.R. Ratković, S.B. Novaković, G.A.
Bogdanović, J. Milovanović, A. Arsenijević, B. Stojanović, S.R. Trifunović, G.P. Radić, Cytotoxicity of
copper(II)-complexes with some S-alkyl derivatives of thiosalicylic acid. Crystal structure of the binuclear
copper(II)––complex with S-ethyl derivative of thiosalicylic acid, Journal of Molecular Structure (2016),
doi: 10.1016/j.molstruc.2016.03.058.
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ACCEPTED MANUSCRIPT
Copper(II)-complexes were obtained by the direct reaction of copper(II)-nitrate with the S-alkyl
derivatives of thiosalicylic acid (alkyl = benzyl (L1), methyl (L2), ethyl (L3), propyl (L4), butyl
(
L5)). The spectroscopically predicted structure of the obtained copper(II)-complex with
S-ethyl derivative of thiosalicylic acid was confirmed by X-ray analysis. The crystal structure is
built out of centrosymmetric, dinuclear complex molecules where the pairs of Cu(II) are bridged
by four S-ethyl-thiosalicylate ligands forming the paddle-wheel type structure. In the crystal
packing the molecules are connected into a chain by two pairs of the O1w−H…O1 hydrogen
bonds which involve axially coordinated water molecule as a donor and two carbonyl oxygen
atoms as acceptors. The analysis of cancer cell viability showed that all the tested complexes had
low cytotoxic effect on murine colon carcinoma cell lines, but several times higher cytotoxicity
on normal human colon carcinoma cells.

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Cytotoxicity of copper(II)-complexes with some S-alkyl derivatives of
thiosalicylic acid. Crystal structure of the binuclear copper(II)-
-complex with S-ethyl derivative of thiosalicylic acid
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Miloš V. Nikolić , Marina Ž. Mijajlović , Verica V. Jevtić , Zoran R. Ratković , Slađana B.
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Novaković , Goran A. Bogdanović , Jelena Milovanović , Aleksandar Arsenijević , Bojana
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Stojanović , Srećko R. Trifunović , Gordana P. Radić
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Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovića 69, 34000 Kragujevac, Republic of Serbia;
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Department of Chemistry, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac,
Republic of Serbia;
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Vinča Institute of Nuclear Sciences, Laboratory of Theoretical Physics and Condensed Matter Physics, University of
Belgrade, PO Box 522, 11001 Belgrade, Republic of Serbia;
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Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Svetozara
Markovića 69, 34000 Kragujevac, Republic of Serbia;
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Abstract
The spectroscopically predicted structure of the obtained copper(II)-complex with
S-ethyl derivative of thiosalicylic acid was confirmed by X-ray structural study and compared
to previously reported crystal structure of the Cu complex with S-methyl derivative. Single
crystals suitable for X-ray measurements were obtained by slow crystallization from a water
solution. Cytotoxic effects of S-alkyl (R = benzyl (L1), methyl (L2), ethyl (L3), propyl (L4)
and butyl (L5)) derivatives of thiosalicylic acid and the corresponding binuclear copper(II)-
-complexes on murine colon carcinoma cell lines, CT26 and CT26.CL25 and human colon
carcinoma cell line HCT-116 were reported here. The analysis of cancer cell viability showed
*
Corresponding author, Tel.: +38134306800; +38134306800
e-mail address: vasic_gordana@yahoo.com (G.P. Radić)
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that all the tested complexes had low cytotoxic effect on murine colon carcinoma cell lines,
but several times higher cytotoxicity on normal human colon carcinoma cells.
Keywords: binuclear copper(II)-complexes; crystal structure; cytotoxicity
1. Introduction
The success of cisplatin in the treatment of various types of neoplasms has placed the
coordination chemistry of metal-based drugs in the frontline in the cancer chemotherapy
[1-3]. Although cisplatin is widely used as an anticancer drug, the treatment is still dose-
-limited by side effects, narrow therapeutic window and acquired resistance. These problems
in the therapy have increased the interest in developing alternative strategies based on
different metals with improved pharmacological properties. Over the years a large number of
complexes containing platinum or non-platinum metals have been studied as potential cancer
chemotherapeutic agents. Scientists synthesized and characterized various metal complexes,
where the nature of both the metal and the ligand were varied. Investigations in this field are
focused on the use of biologically active complexes, such as copper coordination compounds
[4,5].
Copper-based complexes have been investigated on the assumption that endogenous
metals may be less toxic for normal cells compared to cancer cells. However, copper can also
be toxic due to its redox activity and affinity for binding sites that should be occupied by
other metals. The exact role of copper in cancer is not yet fully understood, but it is possible
to be involved via oxidation processes and the production of ROS (ROS = Reactive oxygen
species) and its involvement in angiogenic processes [6-8].
Numerous copper(II)-complexes exhibiting cytotoxic activity have been reviewed so
far. Structurally, these compounds usually include a variety of aromatic molecules as ligands,
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which can be coordinated through N, S and O donor atoms [3]. The combination of copper(II)
salts and specific copper chelators have been shown to suppress proliferation of different
types of human cancer cells [1]. A great variety of copper complexes including different
classes of ligands, such as derivatives of thiosemicarbazones, imidazole, purine, pyrrolidine
and pyridine have been proposed as potential anticancer agents [9-11]. Copper(II)-complexes
with ligands from the group of NSAIDs (NSAIDs = Nonsteroidal anti-inflammatory drugs)
and other therapeutic ligands were also found to be cytotoxic [12].
Thiosalicylic acid and its derivatives are used for metal determination [13,14], as
modificators for graphite paste electrodes [15], as photoinitiators for free radical
polymerization [16], in cosmetics [17], in inflammatory, allergic and respiratory diseases
treatments [18], as well as Ras-tumor growth inhibitors [19]. In our previously published
paper we investigated the antimicrobial activity of some S-alkyl derivatives of thiosalicylic
acids and the corresponding copper(II)-complexes. We concluded that all the tested
compounds showed moderate or selective antimicrobial activity and low antifungal activity.
The copper(II)-complexes showed similar or higher activity than the corresponding S-alkyl
derivatives of thiosalicylic acid [20].
The complex forming ability of thiosalicylic acid with copper(II) have been previously
recorded. Subrahmanyan et. al. reported the formation of a blue complex containing
copper(II) with thiosalicylic acid in which the metal ion occupied only one of two potential
sites of the ligand: the –SH group [21]. Anjaneyulu et. al. reported the formation of a light
green complex containing copper(II) with thiosalicylic acid and acetylacetone as bidentate
ligands [22]. Then, Ferrer et. al. reported synthesis and characterization of dimeric copper(II)-
-complex with thiosalicylic acid and pyridine [23]. Mawgoud et. al. reported synthesis of
copper(II) ternary complexes with thiosalicylic acid and amino acids as ligands [24].
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Idriss et. al. investigated spectrophotometrically the complex formation between copper(II)-
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-mercaptonic acid and thiosalicylic acid [25].
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The aim of this paper is to research the in vitro cytotoxic effects of the earlier
synthesized S-alkyl derivatives of thiosalicylic acid and the corresponding copper(II)-
-complexes. In this study, we report the cytotoxicity of five ligands precursors, S-alkyl
derivatives of thiosalicylic acid (L1-L5), and their corresponding five copper(II)-complexes
(C1-C5) on murine colon carcinoma cell lines, CT26 and CT26.CL25 and human colon
carcinoma cell line HCT-116. We show here that our new copper(II)-complexes have low
cytotoxic effect on murine colon carcinoma cell lines, but several times higher cytotoxicity on
normal human colon carcinoma cells. In addition, we also report in this paper the new crystal
structure of the Cu complex with ethyl derivative of thiosalicylic acid (C3) in order to
compare the fine structural differences and the effects of intermolecular interactions with
respect to previously reported Cu(II) complex with the methyl derivative of the same ligand
(C2).
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2. Experimental
2.1. Materials and measurements
The reagents were obtained commercially and used without further purification.
Elemental analyses were done on a Vario III CHNOS Elemental Analyzer, Elemental
Analysensysteme GmbH. For infrared spectra a Perkin-Elmer FTIR 31725-X
spectrophotometer and KBr pellet technique were employed.
2.2. Syntheses
2.2.1. General procedure for the synthesis of S-alkyl derivatives of thiosalicylic acid
(L1)–(L5)
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The S-alkyl derivatives of thiosalicylic acid ligands (alkyl = benzyl (L1), methyl
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(L2), ethyl (L3), propyl (L4), butyl (L5)) were prepared [26] by alkylation of thiosalicylic
acid by means of the corresponding alkyl halides in alkaline water-ethanol solution
(Scheme 1).
2.2.2. General procedure for the synthesis of copper(II)-complexes with S-alkyl derivatives of
thiosalicylic acid, (C1)–(C5)
Copper(II)-nitrate trihydrate (0.1000 g, 0.4139 mmol) was dissolved in 10.0 mL of
water on a steam bath and S-alkyl thiosalicylate (alkyl = benzyl (L1), methyl (L2), ethyl (L3),
propyl (L4), butyl (L5)) was added. The reaction mixtures were heated for 3 h and during this
period 10.0 mL of LiOH aqueous solution (0.0348 g, 0.8278 mmol) was added in small
portions and the solutions were filtered and evaporated to a small volume. The blue
precipitates of copper(II)-complexes were separated by filtration, washed with cold water and
air-dried [20]. Single crystals of [Cu (S-et-thiosal) (H O) ], (C3) suitable for X-ray
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measurements were obtained by slow crystallization from a water solution by evaporation.
2.3. Single crystal X-ray crystallography
Single-crystal diffraction data for [Cu (S-et-thiosal) (H O) ] (C3) were collected at
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room temperature on an Agilent Gemini S diffractometer equipped with Cu Kα radiation
(λ = 1.5418 Å). Data reduction and empirical absorption corrections were performed with
CrysAlisPro [27]. Crystal structure was solved by direct methods, using SIR2002 [28] and
refined using SHELXL program [29]. The hydrogen atoms attached to C atoms were placed
at geometrically idealized positions with C–H distances fixed to 0.93, 0.97 and 0.96 Å from
phenyl, methylene and methyl C atoms, respectively. The isotropic displacement parameters
were set equal to 1.2U and 1.5U of the parent C atoms. The hydrogen atoms of the apical
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eq
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water ligands were located in difference Fourier map and refined isotropically. The
crystallographic data are listed in Table 1. The PARST [30] and PLATON [31] programs
were used to perform geometrical calculation and the program ORTEP [32] was employed for
molecular graphics.
2.4. In vitro cytotoxicity studies
2.4.1. Preparation of drug solutions
The complexes were dissolved in 10 % dimethylsulfoxide (DMSO) in distilled water
at a concentration of 10 mM and filtered through a 0.22 mm Millipore filter. These stock
solutions were diluted in culture medium immediately before use. MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide was dissolved (5 mg/mL) in a
phosphate buffer saline having a pH of 7.2, and filtered through the 0.22 mm Millipore filter
before use. All reagents were purchased from Sigma Chemicals.
2.4.2. Cell culture
CT26 and CT26.CL25 cells were purchased from the American Type Culture
Collection (ATCC, Manassas, USA). HCT-116 cells were kindly provided by Dr Danijela
Vignjević (Institute Curie, Paris, France). The cells were maintained in DMEM (Sigma
Aldrich, Munich, Germany) supplemented with 10 % fetal bovine serum (FBS, Sigma
Aldrich, Munich, Germany), penicillin (100 IU/mL), streptomycin (100 ꢀg/mL) in a
humidified atmosphere of 95 % air/5 % CO at 37 °C. Subconfluent monolayers, in log
2
growth phase, were harvested by brief treatment with 0.25 % trypsin and 0.02 % EDTA in
phosphatebuffered saline (PBS, PAA Laboratories GmbH) and washed three times in serum-
-free PBS. The number of viable cells was determined by trypan blue exclusion.
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2.4.3. Cytotoxicity assays
The effects of the tested compounds on cell viability were determined using MTT
colorimetric technique. CT26, CT26.CL25, and HCT116 cells were diluted with growth
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medium to 5·10 cells/mL and aliquots (5·10 cells/100 mL) were placed in individual wells
in 96-multiplates. The next day the medium was exchanged with 100 ꢀL of different
compounds, which had been serially diluted 2-fold in the medium to concentrations ranging
from 1000 ꢀM to 7.8 ꢀM in growth medium. Each compound was tested in triplicate. The
cells were incubated at 37 °C in a 5 % CO for 72 h. After incubation the supernatant was
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removed and 15 % MTT solution (5 mg/mL in PBS, 10 ꢀL) in DMEM medium without FBS
was added to each well. After an additional 4 h of incubation at 37 °C in a 5 % CO , the
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medium with MTT was removed and DMSO (150 ꢀL) with glycine buffer (20 ꢀL) was added
to dissolve the crystals. The plates were shaken for 10 min. The optical density of each well
was determined at 595 nm using microplate Zenyth 3100 Multimode detector. The percentage
of cytotoxicity was calculated using the formula: % cytotoxicity = 100-((E-B)/(S-B)·100),
where B is for background of medium alone, S is for total viability/spontaneous death of
untreated target cells, and E is for experimental well. Each of the tested complexes was
evaluated for cytotoxicity in three separate experiments.
2.4.4. Apoptosis Assay
For the detection of apoptosis, cells were plated at T25 culture flasks and allowed to
grow overnight. After the cells reached subconfluency, medium was replaced with tested
complexes (15 ꢀM). Exposed cells were placed at 37 °C in a 5 % CO incubator for 24h.
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(10x binding buffer: 0,1 M Hepes/NaOH (pH 7,4), 1,4 M NaCl, 25 mM CaCl ) at
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concentration 1·10 / mL. Annexin FITC and propidium iodide (PI) were added to the 100 ꢀL
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of cell suspension and incubated for 15 min at room temperature (25 °C) in the dark. After
incubation 400 ꢀL of 1x binding buffer was added to each tube and stained cells were
analyzed within 1 hour using FACS Calibur (BD, San Jose, USA) and Flow Jo software
(Tri Star). Since, Annexin V FITC staining precedes the loss of membrane integrity that
accompanies the later stage identified by PI, Annexin FITC positive, PI negative indicates
early apoptosis, while viable cells are Annexin V FITC negative, PI negative. Cells that are in
late apoptosis, or already dead are both Annexin V FITC and PI positive.
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3. Results and discussion
3.1. Synthesis and chemical characterization
Copper(II)-complexes
with
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derivatives
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thiosalicylic
acid,
[Cu (S-alkyl-thiosal) (H O) ], were obtained by the direct reaction of copper(II)-nitrate with
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the S-alkyl derivatives of thiosalicylic acid (in a molar ratio of 1:2) in water solution (Scheme
2), as described earlier [20]. Based on the content of the complex and the shape of their
infrared spectra, it can not be concluded with certainty how S-alkyl derivatives of thiosalicylic
acid are coordinated to copper(II)-ion. The proper molecular structure was obtained on the
basis of X-ray analysis of [Cu (S-met-thiosal) (H O) ] [20] and [Cu (S-et-thiosal) (H O) ]
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complexes. Single crystals suitable for X-ray measurements were obtained by slow
crystallization from water solution. Based on crystal structures of the
[Cu (S-met-thiosal) (H O) ] and [Cu (S-et-thiosal) (H O) ] complexes, it can be assumed that
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the other complexes have the binuclear molecular structure.
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3.2. Structural description of the complex [Cu (S-et-thiosal) (H O) ], (C3)
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The single-crystal X-ray analysis showed that C3 crystallizes in the triclinic P-1 space
group. The crystal structure is built out of centrosymmetric, dinuclear complex molecules
where the pairs of Cu(II) are bridged by four S-ethyl-thiosalicylate ligands forming the
paddle-wheel type structure (Fig. 1). The Cu…Cu distance of 2.611(1) is similar to 2.614 Å
found in dimeric copper(II)-acetate [33]. In C3 the Cu(II) is placed in a distorted square-
pyramidal environment (Table 2), formed by oxygen atoms of carboxylate ligands in the basal
plane, and water ligand in the apical position. Copper(II)-ion is displaced from the plane of
the square base for 0.195(1) Å. The geometrical features of C3 could be closely compared
with those reported for Cu(II)-complex with S-methyl-thiosalicylate derivative, C2 [20]
containing two crystallographicaly independent molecules in the asymmetric unit. The Cu−O
coordination bond lengths in C3 are within the ranges observed in the independent molecules
of structure C2. Similarly to the previous structure, each of thiosalicylate ligands (A and B,
Fig. 1) shows unsymmetrical coordination to Cu(II)-ion. Thus, for two ligands of C3 the
Cu−O1 is longer than Cu−O2 bond for 0.028 Å in average (Table 2), while in C2 the
analogue coordination bonds in two independent molecules differ for 0.011 and 0.025 Å in
average. The water oxygen atom in C3 is placed at somewhat shorter distance from Cu(II)-ion
[2.135(1) Å] in comparison to C2 [2.146 Å in average]. Due to the free rotation around the
single C1−C2 bonds in both ligands [1.502(2) and 1.502(3) Å in A and B, respectively], the
dihedral angles between the planes of phenyl ring C2/C7 and carboxyle group are 16.0(2) and
16.6(2)° in A and B, respectively. For complex C2, with two independent molecules, the
variation in these dihedral angles is more pronounced and it ranges from 5.8(2) to 24.3(2)°. It
should be noticed that S-ethyl moieties in both ligands of C3 are nearly coplanar with the
phenyl rings with maximal deviation of terminal C9 atoms of 0.142(5) and 0.154(6) Å in A
and B ligands, respectively.
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In the crystal packing the molecules of C3 are connected into a chain by two pairs of
the O1w−H…O1 hydrogen bonds which involve axially coordinated water molecule as a
donor and two carbonyl oxygen atoms as acceptors (Table 3, Fig. S1). The chain of molecules
involving similar donor and acceptor groups, which is also found in compound C2, appears to
be rather common structural motif of paddle-wheel Cu(II)-complexes heaving the water
molecule as axial ligand. In our previous paper [20] we analyzed the crystal structures of 26
similar [Cu (ArCOO) (H O) ] (ArCOO is any aromatic carboxylate ligand) extracted from
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Cambridge Structural Database [34] in order to understand the factors influencing the
deformation of the corresponding Cu C O paddle-wheel cores. We found that 16 structures
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form similar chain of molecules via O1w−H…O interactions between the water and carboxyle
ligands. In such arrangement the deformation of paddle-wheel cores manly arises i) from the
engagement of the carboxyle O atoms as acceptors in hydrogen bonds and also ii) from the
tendency of the closely adjacent and substituted phenyl rings to avoid the sterical hindrance,
particularly if their rotation is partly reduced. The Cu C O core in C3 is also noticeable
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distorted, as two Cu1−O1−C1 angles (118° in average) involving O1 acceptors of
O1w−H…O1 are significantly smaller in comparison to angles Cu1−O2−C2 (128° in
average).
In the chain the O1w−H…O1 hydrogen bonds are assisted by the pairs of weaker
O1w-H…S1 and C9−H…O2 interactions which also engage the acceptor and donor sites
from the phenyl S-ethyl substituents (Fig. S1). It can be suggested that these interactions
partly reduce the rotation of the ortho-substiuted phenyl rings with respect to the
corresponding carboxyl groups. Apart from the pair of C9−H…O2 interactions no additional
significant contacts can be found for the O2 acceptors. In the chain, the closest distance
between the Cu(II) ions of the linked molecules is 5.117(1) Å, similarly to the distance found
for C2 (5.122 (2) Å). The neighboring chains of C3 are further connected by pairs of
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C−H…π interactions involving the phenyl rings from both aromatic ligands (Table 3). Fig. S2
and Fig. S3 display the fragments of this two-dimensional molecule arrangement as viewed
down a and c axis, respectively.
3.3. In vitro cytotoxic activity
The MTT assay for cell viabilty shows that five newly synthesized precursor S-alkyl
derivatives of thiosalicylic acid, and their corresponding copper(II)-complexes after 72 hours
exhibit relevant cytotoxic activity on human colon carcinoma cells, HCT-116 (Fig. 2), with
significantly lower activity on murine colon carcinoma cell lines especially for concentrations
from 31,25 to 250 µM. The cytotoxic effect was dose-dependent: the decrease of
concentration of the tested complexes was followed by marked increase of tumor cell
viability. Cytotoxic effect of all tested ligands and complexes was stronger on HCT116 cell
line compared to CT26 and CT26.CL25 cell lines. Copper(II)-complexes showed similar,
slightly lower, cytotoxicity on human HCT116 cells compared with cisplatin. However
cytotoxic effect of new copper(II)-complexes from concentrations 31,25 to 250 µM on murine
cell lines CT26 and CT26.CL25 was significantly lower compared to the activity of cisplatin.
There was almost no differences in the cytotoxicity of ligands and complexes on two murine
cell lines CT26 and CT26.CL25.
Among the tested ligand precursors the ligands with shorter alkyl side chain, L1, L2,
and L3 showed higher cytotoxic effect on CT26 and CT26.CL25 tumor cells (Fig. 2)
compared to the ligands with longer alkyl side chains, L4 and L5. Ligands L4 and L5 showed
an important cytotoxic effect only at the higher concentrations (from 500 ꢀM and
1000 ꢀM). Such difference was not observed on HCT116 cell line. However, there was no
striking difference in cytotoxic effect of all five tested complexes on all three tumor cell lines
(Fig. 2).
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All the tested complexes had lower cytotoxic effect on tumor cells cells after their
exposure to complexes for 72 hours when compared to cisplatin (Table 4). The analysis of
IC50 values showed that copper(II)-complexes had significantly stronger antiproliferative
effect on HCT116 cells compared to CT26 and CT26.CL25 cells (Table 4). Complexes C1,
C2, and C3 had lower cytotoxic effect towards CT26 and CT26.CL25 cells compared with
C4 and C5 (Table 4). However, the greater difference in cytotoxicity on these two cell lines
between the ligand and the corresponding complex was observed for complexes with longer
side chains C4 and C5. Copper(II)-complex C1 and C5 had almost the same cytotoxicity
towards HCT116 cells (IC = 60.17 ꢀM vs 59.32 ꢀM ). Other three complexes had slightly
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lower cytotoxic effect on HCT116 cells, with the lowest effect of complex C4 (IC = 91.51
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ꢀM) (Table 4).
Flow cytometry analysis was used to determine the potential for induction of apoptotic
death of tested copper complexes. The percentage of apoptotic CT26, CT26.CL25, and
HCT116 cells after tretament with copper complexes was determined by flow cytometric
detection of Annexin V+ Propidium Iodide+ cells. Our results indicate that tested complexes
after 24 hours induce apoptotic death of CT26, CT26.CL25, and HCT116 cells, higher in
comparison with corresponding ligands (Fig. 3). Mostly similar cytotoxic effects of all tested
complexes and cisplatin (concentration 15 µM) on all three cell lines was detected. This
finding is in correlation with the results of cytotoxic effects of complexes with the same
concentration (15 µM) after 72 hours exposure measured by MTT assay.
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307
308
309
310
311
312
313
314
315
4. Conclusion
Synthesized S-alkyl derivatives of thiosalicylic acid, and their corresponding
copper(II)-complexes demonstrated relevant cytotoxic activity on human colon carcinoma
cells, HCT-116, with significantly lower activity on murine colon carcinoma cell lines.
Cytotoxic effect of all the tested compounds was stronger on HCT116 cell line compared to
CT26 and CT26.CL25 cell lines. Copper(II)-complexes showed slightly lower cytotoxicity on
human HCT116 cells compared to cisplatin. Cytotoxic effect of new copper(II)-complexes on
murine cell lines CT26 and CT26.CL25 was significantly lower compared to activity of
cisplatin.
The spectroscopically predicted structure of the obtained copper(II)-complex with
S-ethyl derivative of thiosalicylic acid was confirmed by X-ray analysis. The crystal structure
is built out of centrosymmetric, dinuclear complex molecules where the pairs of Cu(II) are
bridged by four S-ethyl-thiosalicylate ligands forming the paddle-wheel type structure. In the
crystal packing the molecules are connected into a chain by two pairs of the O1w−H…O1
hydrogen bonds which involve axially coordinated water molecule as a donor and two
carbonyl oxygen atoms as acceptors.
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322
323
324
Acknowledgment
This work was financially supported by the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Projects 172016, 172034, 172035,
175069 and 175103) and Faculty of Medical Sciences for grant MP 2014/01.
Appendix A. Supplementary data
CCDC 1430504 contains the supplementary crystallographic data for this paper. These
data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
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from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
UK; fax: (+44) 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk.
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Table 1. Crystallographic data for C3.
Empirical formula
Formula weight
C H CuO S
18 20 5 2
444.00
Green, prism
0.36 x 0.18 x 0.11
293(2)
1.5418
Color, crystal shape
3
Crystal size (mm )
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
Triclinic
P1−
7.5102(3)
10.8709(5)
12.1557(6)
83.578(4)
89.304(3)
76.314(4)
958.09(8)
2
1.539
3.875
3.66 to 72.64
8944
b (Å)
c (Å)
α
β
γ
(°)
(°)
(°) 3
V (Å )
Z
3
Dcalc (Mg/m )
ꢀ
θ
-1
(mm )
range for data collection (°)
Reflections collected
Independent reflections, Rint
3804, 0.0185
99.9
Full-matrix least-squares on F
3804 / 0 / 245
1.049
Completeness (%) to
Refinement method
θ = 67°
2
Data / restraints / parameters
2
Goodness-of-fit on F
Final R /wR indices [I >2
σ
(I)]
0.0273 / 0.0748
0.0292 / 0.0764
0.313 /−0.279
1
2
Final R /wR indices (all data)
1
2
-3
Largest diff. peak and hole (e Å )

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Table 2. Selected bond lengths (Å) and angles (°) of C3.
i
Cu1−Cu1
Cu1−O1a
Cu1−O2a
2.611(1)
1.988(1)
1.955(1)
1.982(1)
1.958(1)
2.135(1)
1.260(2)
1.255(2)
1.255(2)
1.261(2)
1.761(2)
1.812(2)
1.763(2)
1.815(2)
O1a−Cu1−O1b
O1a−Cu1−O2b
86.18(6)
92.44(5)
91.29(6)
87.84(5)
99.05(6)
97.18(6)
92.42(6)
94.15(6)
168.48(5)
168.66(5)
124.31(16)
115.94(17)
124.36(15)
116.57(15)
i
i
O1b−Cu1−O2a
i
i
Cu1−O1b
Cu1−O2b
Cu1−O1w
O1a−C1a
O2a−C1a
O1b−C1b
O2b−C1b
C3a−S1a
C8a−S1a
C3b−S1b
C8b−S1b
O2a −Cu1−O2b
O1a−Cu1−O1w
O1b−Cu1−O1w
i
O2a −Cu1−O1w
i
O2b −Cu1−O1w
i
O1a−Cu1−O2a
i
O1b−Cu1−O2b
C1a−C2a−C3a
C1a−C2a−C7a
C1b−C2b−C3b
C1b−C2b−C7b
Symmetry codes: (i) −x, −y, −z+1.

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Table 3. Geometrical parameters (Å,°) of hydrogen bonding and weak intermolecular
interactions in C3.
D−H…A
O1w–H1w…O1b
O1w–H2w…O1a
O1w–H1w...S1b
O1w–H2w...S1a
O9a–H9a1…O2a
O9b–H9b3…O2b
C4b–H4b...Cg1
D−H
0.68(3)
0.69(3)
0.68(3)
0.69(3)
0.96
D…A
3.058(2)
3.127(2)
3.412(2)
3.487(2)
3.457(3)
3.404(3)
3.803(2)
H…A
D−H…A
ii
2.50(3) 140(3)
2.50(3) 151(3)
2.77(3) 155(3)
2.88(3) 147(3)
2.56
2.50
3.12
ii
ii
ii
iii
155
157
131
iii
0.96
0.93
iv
Symmetry codes: (ii) -x+1,-y,-z+1 ; (iii) x+1,+y,+z; (iv) 1-x, -1-y, 1-z.
Cg1 refers to centroid of C2a/C7a ring.

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Table 4. IC50 (µM)* for the 72 h of action of the investigated compounds, ligand precursors and
copper(II)-complexes on CT26, CT26.CL25, and HCT-116 cell lines, determined by MTT assay.
Compound
C1
C2
C3
C4
C5
L1
L2
L3
L4
L5
Cisplatin
CT26
CT26.CL25
307,21 ± 44,87
280,23 ± 79,81
286,29 ± 52,19
375,55 ± 25,31
360,l9 ± 18,08
328,79 ± 13,74
427,17 ± 32,27
513,27 ± 79,50
859,56± 67,93
584,53± 40,47
21,21 ± 8,19
HCT116
60,17 ± 11,01
67,21 ± 10,62
75,34 ± 16,53
91,59 ± 15,21
59,32 ± 9,83
137,01 ± 13,74
143,14 ± 17,14
171,24 ± 14,23
130,89± 19,13
82,65± 11,07
25,41 ± 7,14
295,65 ± 36,64
279,16 ± 71,11
291,92 ± 61,24
407,58 ± 38,47
402,29 ± 41,33
374,46 ± 26,75
331,84 ± 64,46
349,14 ± 51,19
848,83± 63,05
585,70± 38,58
9,45 ± 3,06
*mean values ± standard deviation from four independent experiments

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O
OH
O
ONa
O
OH
C
C
C
SH
R-X + 2 NaOH
S
S
Ethanol/H O
R
2
H+
R
+
R= Benzyl (L1), methyl (L2), ethyl (L3), propyl (L4), butyl (L5)
Scheme 1. The preparation of the benzyl (L1), methyl (L2), ethyl (L3), propyl (L4) and butyl
L5) derivatives of thiosalicylic acid
(

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Scheme 2. The preparation of the binuclear copper(II)-complex with S-alkyl derivatives of
thiosalicylic acid

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Figure 1. Crystal structure and the atom numbering scheme of molecule C3. Displacement
ellipsoids are drawn at the 30%.
Figure 2. Representative graphs of CT26, CT26.CL25, and HCT-116 cell survival after 72 h cell
growth in the presence of copper(II)-complexes and ligand precursors. Each point represents a
mean value and standard deviation of 3 experiments with 3 replicates per dose.
Figure 3. Representative results of apoptotic assay of CT26, CT26.CL25, and HCT-116 cells treated
with 15 µM of cisplatin, copper(II) complexes and ligand precursors for 24 hours.

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Fig. 1. Crystal structure and the atom numbering scheme of molecule C3. Displacement
ellipsoids are drawn at the 30%

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Fig. 2. Representative graphs of CT26, CT26.CL25, and HCT-116 cell survival after 72 h cell
growth in the presence of copper(II) complexes and ligand precursors. Each point represents a
mean value and standard deviation of 3 experiments with 3 replicates per dose

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Fig. 3. Representative results of apoptotic assay of CT26, CT26.CL25, and HCT-116 cells
treated with 15 μM of cisplatin, copper(II)-complexes and ligand precursors for 24 hours

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•
•
•
Crystal structure of [Cu (S-et-thiosal) (H O) ]
2 4 2 2
In vitro cytotoxic effects of copper(II)-complexes were determined by MTT test
apoptotic assay of cisplatin, copper(II)-complexes and ligand