Synthesis, characterization and cytotoxic activity of binuclear copper(II)-complexes with some S-isoalkyl derivatives of thiosalicylic acid. Crystal structure of the binuclear copper(II)-complex with S-isopropyl derivative of thiosalicylic acid

Article abstract of DOI:10.1016/j.jinorgbio.2020.111078

Isoalkyl (isoalkyl = isopropyl-(L1), isobutyl-(L2) and isoamyl-(L3)) derivatives of thiosalicylic acid (TSA) were prepared by alkylation of TSA with corresponding isoalkyl-chlorides in the alkaline water-ethanol solution. The new free copper(II)-complexes with corresponding S-isoalkyl derivatives of TSA (C1-copper(II)-complex with S-isopropyl derivative of thiosalicylic acid, C2-copper(II)-complex with S-isobutyl derivative of thiosalicylic acid and C3-copper(II)-complex with S-isoamyl derivative of thiosalicylic acid) have been synthesized by direct reaction of copper(II)-nitrate with ligand precursor and then characterized by microanalysis, infrared spectra (IR) and EPR (electron paramagnetic resonance) spectra. The spectroscopically predicted structure of the obtained binuclear copper(II)-complex with S-isopropyl derivative of thiosalicylic acid was confirmed by X-ray analysis. Single crystals suitable for X-ray measurements were obtained by slow crystallization from a water solution. Newly synthesized precursors S-isoalkyl derivatives of thiosalicylic acid and corresponding copper(II)-complexes moderately reduced viability of human and murine lung cancer cells, they showed similar cytotoxic effect on human colorectal cancer cells as cisplatin and lower cytotoxic effect than cisplatin toward normal fibroblasts, evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric technique. All new complexes exhibited apoptotic effect toward lung cancer cells, stronger than cisplatin, whereas only C3 induced significant apoptosis of colorectal cancer cells. Complex C1 showed significant antiproliferative effect against murine lung cancer cells, LLC1, while C2 reduced expression of Ki67 in human colorectal cancer cells. All tested complexes induced cell cycle arrest of HCT116 cells in G2/M phase.

Full text of DOI:10.1016/j.jinorgbio.2020.111078

Journal Pre-proof
Synthesis, characterization and cytotoxic activity of binuclear
copper(II)-complexes with some S-isoalkyl derivatives of
thiosalicylic acid. Crystal structure of the binuclear copper(II)-
complex with S-isopropyl derivative of thiosalicylic acid
Jelena Dimitrijević, Aleksandar N. Arsenijević, Marija Z.
Milovanović, Nebojša N. Arsenijević, Jelena Z. Milovanović,
Ana S. Stanković, Andriana M. Bukonjić, Dušan Lj. Tomović,
Zoran R. Ratković, Ivan Potočňák, Erika Samoľová, Gordana P.
Radić
PII:
S0162-0134(20)30106-9
DOI:
https://doi.org/10.1016/j.jinorgbio.2020.111078
JIB 111078
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
28 January 2020
24 March 2020
24 March 2020
Please cite this article as: J. Dimitrijević, A.N. Arsenijević, M.Z. Milovanović, et al.,
Synthesis, characterization and cytotoxic activity of binuclear copper(II)-complexes with
some S-isoalkyl derivatives of thiosalicylic acid. Crystal structure of the binuclear
copper(II)-complex with S-isopropyl derivative of thiosalicylic acid, Journal of Inorganic
Biochemistry (2018), https://doi.org/10.1016/j.jinorgbio.2020.111078
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Synthesis, characterization and cytotoxic activity of binuclear
copper(II)-complexes with some S-isoalkyl derivatives of thiosalicylic
acid. Crystal structure of the binuclear copper(II)-complex with
S-isopropyl derivative of thiosalicylic acid
Jelena Dimitrijevića, Aleksandar N. Arsenijevića, Marija Z. Milovanovića, Nebojša N.
Arsenijevića, Jelena Z. Milovanovića,b*, Ana S. Stankovićc, Andriana M. Bukonjićc, Dušan Lj.
Tomovićc, Zoran R. Ratkovićd, Ivan Potočňáke, Erika Samoľováf, Gordana P. Radićc†
a Center for Molecular Medicine and Stem Cell Research, Faculty of Medical Sciences, University of Kragujevac, Svetozara
Markovića 69, 34000 Kragujevac, Republic of Serbia;
b Department of Histology and Embriology, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovića 69,
34000 Kragujevac, Republic of Serbia;
c Department of Phamacy, Faculty of Medical Sciences, University of Kragujevac, Svetozara Markovića 69, 34000
Kragujevac, Republic of Serbia;
dDepartment of Chemistry, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac,
Republic of Serbia;
eP.J. Safárik, University in Košice, Institute of Chemistry, Moyzesova 11, SK-041 54 Košice, Slovakia
fInstitute of Physics of the Czech Academy of Sciences, Na Slovance 2, CZ-182 21 Praha 8, Czech Republic
Abstract
Isoalkyl (isoalkyl = isopropyl-(L1), isobutyl-(L2) and isoamyl-(L3)) derivatives of
thiosalicylic acid (TSA) were prepared by alkylation of TSA with corresponding isoalkyl-
* Corresponding author, Tel.: +38134306800; +38134306800
e-mail: jelenamilovanovic205@gmail.com (J.Z. Milovanović)
† Corresponding author, Tel.: +38134306800; +38134306800
e-mail address: vasic_gordana@yahoo.com (G.P. Radić)
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chlorides in the alkaline water-ethanol solution. The new free copper(II)-complexes with
corresponding S-isoalkyl derivatives of TSA (C1-copper(II)-complex with S-isopropyl
derivative of thiosalicylic acid, C2-copper(II)-complex with S-isobutyl derivative of
thiosalicylic acid and C3-copper(II)-complex with S-isoamyl derivative of thiosalicylic acid)
have been synthesized by direct reaction of copper(II)-nitrate with ligand precursor and then
characterized by microanalysis, infrared spectra (IR) and EPR (electron paramagnetic
resonance) spectra. The spectroscopically predicted structure of the obtained binuclear
copper(II)-complex with S-isopropyl derivative of thiosalicylic acid was confirmed by X-ray
analysis. Single crystals suitable for X-ray measurements were obtained by slow
crystallization from a water solution. Newly synthesized precursors S-isoalkyl derivatives of
thiosalicylic acid and corresponding copper(II)-complexes moderately reduced viability of
human and murine lung cancer cells, they showed similar cytotoxic effect on human
colorectal cancer cells as cisplatin and lower cytotoxic effect than cisplatin toward normal
fibroblasts, evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) colorimetric technique. All new complexes exhibited apoptotic effect toward lung
cancer cells, stronger than cisplatin, whereas only C3 induced significant apoptosis of
colorectal cancer cells. Complex C1 showed significant antiproliferative effect against murine
lung cancer cells, LLC1, while C2 reduced expression of Ki67 in human colorectal cancer
cells. All tested complexes induced cell cycle arrest of HCT116 cells in G2/M phase.
Keywords: S,O-ligands; Copper(II)-complexes; Crystal structure; Biological activity
1. Introduction
Intensive research on the cytotoxic activity of inorganic and organometallic
compounds began after the start of global clinical application of the cis-
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diamminedichloroplatinum(II)-complex (cisplatin) [1-6]. The discovery of new complex
compounds with different ions of transition metals was primarily due to the unfavorable
pharmacological profile of cisplatin (non-selective effect, a wide range of adverse effects, the
development of resistance, etc.) [4-6]. An increasing number of research groups has been
focused on the design and synthesis of copper(II)-complexes capable of interacting with
biomolecules producing a pharmacological action [7-13].
Multidentate ligands with O-, N- or S- donor atoms are a fundamental building unit
for this approach due to their capacity to bridge between central metal atoms in order to form
binuclear or polynuclear complex structures [14,15]. The series of 2-supstituted benzoic acids
are important ligands [16]. 2-Mercaptobenzoic acid shows a variety of possibilities for
coordination providing both hard and soft donor centers. It can bind to hard metal ions
through the oxygen atom from carboxylate group and to soft metal ions through the sulfur
atom from thiol group [15,17-20].
Derivatives of aromatic compounds, such as ligands, are known to indicate biological
activities of interest, especially those related to the thioether functional group, which are
being found in some pharmaceutically active compounds [7,20]. Antitumor effects of
acetylsalicylic acid have been reported and it was partially attributed to apoptosis induction
by regulating the activity of the PTEN (phosphatase and tensin homolog)/AKT
(serine/threonine kinase Akt)/NF-κB (nuclear factor kappa-light-chain-enhancer of activated
B cells)/survivin signalling pathway [21]. The ability of derivatives of thiosalicylic acid to
build complexes with different geometries, together with many metal ions, has been the
subject of numerous investigations [14,18].
The aim of this study is to investigate biological activity of the newly synthesized
S-isoalkyl derivatives of thiosalicylic acid and the corresponding copper(II)-complexes. The
composition and structure of S-isoalkyl derivatives of thiosalicylic acid was assumed on the
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basis of microanalysis, IR (infrared spectra) and NMR (nuclear magnetic resonance)
spectroscopy. The composition and structure of synthesized complexes were assumed on the
basis of microanalysis, IR spectroscopy, EPR (electron paramagnetic resonance) spectra,
magnetic measurements, and molar conductance. Cytotoxic assays in vitro of the three
ligands, S-isoalkyl derivatives of thiosalicylic acid (isoalkyl = isopropyl-(L1), isobutyl-(L2)
and isoamyl-(L3)) as well as corresponding copper(II)-complexes (C1-copper(II)-complex
with S-isopropyl derivative of thiosalicylic acid, C2-copper(II)-complex with S-isobutyl
derivative of thiosalicylic acid and C3-copper(II)-complex with S-isoamyl derivative of
thiosalicylic acid) have been studied for the first time. In addition, this paper outlines the new
real crystal structure of the copper(II)-complex with isopropyl derivative of thiosalicylic acid
(C1). All of obtained compounds were tested by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) colorimetric technique on human and murine lung carcinoma
cells. Antitumor activities of obtained compounds were tested on human and murine lung
carcinoma and human colorectal carcinoma cells.
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 (Potassium bromide) pellet technique were employed.
The magnetic measurements of synthesized complexes were performed at 294 K by
the Evans’ method using a MSB-MK1 balance (Sherwood Scientific Ltd.) with
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Hg[Co(SCN)4] (mercury(II)-tetrathiocyanatocobaltate(II)) as a calibrant; diamagnetic
corrections were calculated from Pascal’s constants.
The molar conductance values of all complexes in DMSO (dimethyl sulfoxide)
measured at 10-3 M concentration were in the range of 8–17 S·cm-2·mol-1, indicating that all
complexes behaved as non-electrolytes.
The 9.8 GHz EPR spectra were recorded at room temperature on a Bruker Elexsys II
540 EPR spectrometer under the following conditions: microwave power 6.325 mW,
modulation amplitude 5 G, modulation frequency 100 kHz, and conversion time 240 ms. The
samples (30 μL of 0.5 mM aqueous solution, in deionized 18 MΩ water) were drawn into
gaspermeable Teflon tubes (Zeus industries, Raritan, NJ). The spectra were recorded and
analyzed using the Bruker Xepr software.
2.2. Syntheses
2.2.1. General procedure for the synthesis of S-isoalkyl thiosalicylic acids (L1)–(L3)
The thioacid ligands L1–L3 were prepared by alkylation of thiosalicylic acid by
means of the corresponding isoalkyl chlorides in alkaline water-ethanol solution [22].
Thiosalicylic acid (1 mmol) was added to a 100 cm3 round bottom flask containing 50 cm3
of 30% solution of ethanol in water and then stirred. A solution of NaOH
(2 mmol in 5 cm3 of water) was added to the acid suspension, whereupon the solution became
clear. The corresponding isoalkyl chlorides (2 mmol) was dissolved in 5 cm3 of ethanol and
transferred to the stirred solution. The resulting mixture was kept overnight at 60°C. The
reaction mixture was transferred into a beaker and ethanol was evaporated off on a water bath.
Diluted hydrochloric acid (2 mol/dm3) was added to the resulting water solution and
S-isoalkyl thiosalicylic acid was precipitated as a white powder. The released acid was
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filtered and washed with plenty of distilled water. The product was dried under vacuum
overnight. Yield: 85–95%.
S-isoropyl-thiosalicylic acid (L1): M.p. 117°C, white powder. Anal. Calc. for
S-ipr-thiosal (S-isoropyl-thiosalicylic acid) = C10H12O2S (Mr = 196.27): C, 61.20; H, 6.16; S,
16.34. Found: C, 60.89; H, 5.85; S, 16.70.
IR (KBr, cm-1): 3788, 3699, 3660, 3052, 2979, 2968, 2929, 2866, 2813, 2641, 2554,
1951, 1925, 1677, 1588, 1561, 1461, 1445, 1404, 1381, 1365, 1326, 1310, 1281, 1270, 1257,
1236, 1171, 1150, 1110, 1061, 1052, 1040, 919, 810, 798, 739, 703, 690, 653, 553, 527, 515,
482, 459 (Figure S1).
1H NMR (200 MHz, CDCl3, d ppm): 1.39 (d, 6.6Hz, 6H), 3.46-3.59 (m, 1H), 7.21-
13
7.29 (m, 1H), 7.41-7.54 (m, 2H), 8.15 (dd, J = 7.8Hz, J = 1.4Hz, 1H). C NMR (50 MHz,
CDCl3, d ppm): 22.7, 36.8, 125.1, 128.3, 129.1, 132.6, 132.8, 139.8, 170.4.
S-isobutyl-thiosalicylic acid (L2): M.p. 104°C, white powder. Anal. Calc. for S-ibu-
thiosal (S-isobutyl-thiosalicylic acid) = C11H14O2S (Mr = 210.29): C, 62.83; H, 6.71; S, 15.25.
Found: C, 62.48; H, 6.31; S, 15.53.
IR (KBr, cm-1): 3067, 2963, 2926, 2869, 2721, 2649, 2560, 1950, 1805, 1679, 1587,
1559, 1463, 1429, 1412, 1383, 1366, 1314, 1290, 1271, 1252, 1168, 1152, 1109, 1060, 1046,
978, 914, 809, 738, 704, 689, 651, 553, 526, 515, 491, 459 (Figure S2).
1H NMR (200 MHz, CDCl3, d ppm): 1.11 (d, 6.6Hz, 6H), 1.90-2.10 (m, 1H), 2.82 (d,
6.8Hz, 2H), 7.15-7.23 (m, 1H), 7.32-7.36 (m, 1H), 7.44-7.53 (m, 1H), 8.13 (dd, J = 8Hz, J =
1.6Hz, 1H). 13C NMR (50 MHz, CDCl3, d ppm): 22.5, 27.8, 41.4, 123.9, 126.1, 126.6,
132.5, 132.9, 143, 171.2
S-isoamyl-thiosalicylic acid (L3): M.p. 89°C, white powder. Anal. Calc. for S-iam-
thiosal (S-isoamyl-thiosalicylic acid) = C12H16O2S (Mr = 224.32): C, 64.25; H, 7.19; S, 14.29.
Found: C, 63.87; H, 6.98; S, 14.50.
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IR (KBr, cm-1): 3778, 3661, 3059, 2954, 2923, 2868, 2851, 2650, 2554, 1943, 1808,
1683, 1586, 1561, 1465, 1432, 1412, 1385, 1367, 1350, 1314, 1303, 1284, 1269, 1255, 1170,
1149, 1118, 1061, 1046, 954, 907, 809, 785, 749, 737, 703, 651, 559, 527, 491, 480, 459
(Figure S3).
1H NMR (200 MHz, CDCl3, d ppm): 0.97 (d, 6.4Hz, 6H), 1.58-1.71 (m, 2H), 1.74-
1.90 (m, 1H), 2.91-2.98 (m, 2H), 7.15-7.23 (m, 1H), 7.33-7.37 (m, 1H), 7.45-7.54 (m, 1H),
13
8.14 (dd, J = 7.8Hz, J = 1.6Hz, 1H). C NMR (50 MHz, CDCl3, d ppm): 22.3, 27.8, 30.3,
37.1, 123.8, 125.8, 126.3, 132.5, 133, 143.1, 171.4.
2.2.2. Preparation of copper(II)-complex with S-isopropyl derivative of thiosalicylic acid,
[Cu2(S-ipr-thiosal)4(H2O)2]·2H2O (C1)
Copper(II)-nitrate trihydrate (0.1000 g, 0.4139 mmol) was dissolved in water (10.0
mL) on a steam bath, and S-isopropyl derivatives of thiosalicylic acid (L1), (0.1623 g, 0.8278
mmol) were added. The mixture was heated for 3 h, during which a solution of lithium
hydroxide (LiOH, 0.0198 g, 0.8278 mmol in water 10.0 mL) was added in small portions. The
solution was then filtered and evaporated to small volume. The blue-green precipitate was
separated by filtration, washed with cold water and air-dried. Yield: 0.3248 g (83.15 %).
Anal. Calc. for [Cu2(S-ipr-thiosal)4(H2O)2]·2H2O = Cu2C40H52O12S4 (Mr = 980.16): C, 49.01;
H, 5.35; S, 13.09. Found: C, 49.33; H, 5.19; S, 13.34. Molar conductivity 8.59 S·cm-2·mol-1.
μ(294 K) = 1.79 μB.
IR (KBr, cm-1): 3789, 3699, 3432, 3054, 2964, 2924, 2865, 2624, 1677, 1615, 1587,
1555, 1510, 1459, 1436, 1402, 1366, 1310, 1282, 1257, 1235, 1167, 1152, 1113, 1065, 1052,
1041, 938, 876, 847, 810, 797, 751, 720, 698, 661, 624, 553, 525, 500, 459 (Figure S4).
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2.2.3. Preparation of copper(II)-complex with S-isobutyl derivative of thiosalicylic acid,
[Cu2(S-ibu-thiosal)4(H2O)2] (C2)
Copper(II)-nitrate trihydrate (0.1000 g, 0.4139 mmol) was dissolved in water (10.0
mL) on a steam bath, and S-isobutyl derivatives of thiosalicylic acid (L2), (0.1739 g, 0.8278
mmol) was added. The mixture was heated for 3 h, during which a solution of LiOH (0.0198
g, 0.8278 mmol in water 10.0 mL) was added in small portions. The solution was then filtered
and evaporated to small volume. The blue-green precipitate was separated by filtration,
washed with cold water and air-dried. Yield: 0.3311 g (79.99 %). Anal. Calc. for [Cu2(S-ibu-
thiosal)4(H2O)2] = Cu2C44H56O10S4 (Mr = 1000.26): C, 52.83; H, 5.64; S, 12.82. Found: C,
52.57; H, 5.33; S, 13.01. Molar conductivity 9.9 S·cm-2·mol-1. μ(294 K) = 1.81 μB.
IR (KBr, cm-1): 3778, 3444, 3060, 2954, 2925, 2868, 2650, 2196, 1684, 1606, 1561,
1465, 1434, 1400, 1367, 1314, 1282, 1269, 1255, 1197, 1168, 1150, 1114, 1062, 1046, 956,
917, 847, 809, 786, 740, 721, 696, 652, 625, 556, 539, 526, 515 (Figure S5).
2.2.4. Preparation of copper(II)-complex with S-isoamyl derivative of thiosalicylic acid,
[Cu2(S-iam-thiosal)4(H2O)2] (C3)
Copper(II)-nitrate trihydrate (0.1000 g, 0.4139 mmol) was dissolved in water (10.0
mL) on a steam bath, and S-isoamyl derivatives of thiosalicylic acid (L3), (0,1855 g, 0.8278
mmol ) was added. The mixture was heated for 3 h, during which a solution of LiOH (0.0198
g, 0.8278 mmol in water 10.0 mL) was added in small portions. The solution was then filtered
and evaporated to small volume. The blue-green precipitate was separated by filtration,
washed with cold water and air-dried. Yield: 0.3569 g (81.62 %). Anal. Calc. for [Cu2(S-iam-
thiosal)4(H2O)2] = Cu2C48H64O10S4 (Mr = 1056.37): C, 54.58; H, 6.11; S, 12.40. Found: C,
54.73; H, 6.33; S, 12.14. Molar conductivity 16.92 S·cm-2·mol-1. μ(294 K) = 1.85μB.
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IR (KBr, cm-1): 3790, 3433, 3054, 2956, 2926, 2868, 2184, 1593, 1550, 1462, 1436,
1401, 1385, 1281, 1258, 1239, 1157, 1112, 1062, 1044, 957, 846, 805, 786, 743, 717, 695,
657, 616, 603, 551, 526, 513, 499 (Figure S6).
2.3. Single crystal X-ray crystallography
A summary of the crystal data and structure refinement for C1 is presented in Table 1.
The data collection was carried out on SuperNova diffractometer from Rigaku OD equipped
with Atlas2 CCD detector. CrysAlisPro was used for data collection and also for cell
refinement, data reduction and absorption correction [23]. The structure was solved by
SHELXT [24] and subsequent Fourier syntheses using SHELXL [25], implemented in
WinGX program suit [26]. Anisotropic displacement parameters were refined for all non-H
atoms. The carbon bonded H atoms were placed in calculated positions and refined riding on
their parent C atoms. H atoms of water molecules involved in hydrogen bonds were found in
a Fourier difference map and refined by riding model. A geometric analysis was performed
using SHELXL. DIAMOND [27] was used for molecular graphics.
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Table 1: Crystal data and structure refinement of C1
C1
Empirical formula
Formula weight
T [K]
C40H52Cu2O12S4
980.13
95(2)
λ [Å]
1.54184
Crystal system
Space group
Triclinic
P-1
Unit cell dimensions [Å, °]
a = 9.5846(2)
b = 10.6895(2)
c = 11.4601(2)
1099.12(4)
1; 1.481
 = 85.1550(10)
 = 87.6090(10)
 = 69.977(2)
Volume [Å3]
Z; calculated density [g·cm-3]
Absorption coefficient [mm-1]
F(000)
3.466
510
Crystal shape, color
Crystal size [mm3]
 range for data collection [°]
Index ranges
prism, light blue
0.103 x 0.078 x 0.039
3.871 – 74.545
-11  h  11, -13  k  13, -14  l  14
Reflections collected / independent 30364 / 4448 [R(int) = 0.0259]
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I>2(I)]
R indices (all data)
4448 / 0 / 262
1.058
R1 = 0.0303, wR2 = 0.0765
R1 = 0.0330, wR2 = 0.0795
Largest diff. peak and hole [e. Å-3] 1.122 and -0.614
2.4. In vitro cytotoxicity studies
2.4.1. Cell culture
A549 human lung adenocarcinoma and LLC1 mouse lung cancer, HCT116 human
colorectal carcinoma, and MRC-5 human normal fibroblasts cell lines were purchased from
American Type Culture Collection. These cells were routinely grown in complete DMEM
(Dulbecco’s Modified Eagle Medium) medium (Sigma Aldrich) in a 5% CO2 (carbon
dioxide) incubator with standard conditions. In all experiments only cell suspensions with
>95% viable cells were used. Trypan blue was used to determine the number of viable tumor
cells.
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2.4.2. MTT assay
The cytotoxic effects of the three copper(II)-complexes and on A549, LLC1, HCT116,
and MRC-5 cells were determined using MTT colorimetric technique [28]. A549, HCT116,
and MRC5 cells were seeded on 96-well plates at a density of 5·103 cells/100 μl (per well) in
complete DMEM growth medium and were allowed to adhere by incubation at 37°C
overnight. After 24 hours, the culture medium were replaced and each well received 100μl of
different compounds, which had been serially diluted two-fold in medium to concentrations
ranging from 1000 to 7.8 μM. LLC1 cells grow in suspension and 5·103 cells were placed in
individual wells and 100 µL of either copper complexes or cisplatin in concentrations ranging
from 1000 to 7.8 μM concentrations were added [29]. In the presence of copper complexes
cells were incubated under standard conditions (37°C/5% CO2) for 72 hours. Upon incubation
the medium was removed, MTT solution (5mg/ml in PBS (phosphate buffered saline), 20 μl)
was added to each well and the 96-multiplates were incubated for additional 4 hours. Using
microplate multimode detector Zenyth 3100 the optical density of each well was determined
at 595 nm. The percentage of cell viability was determined by comparison with untreated
controls according to formula: % of viable cells = (E-B)/(S-B) x 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. The experiments, performed in triplicates, were repeated
three times.
2.4.3. Annexin V Propidium Iodide double staining assay
Phosphatidylserine, normally found on the internal part of the plasma membrane
becomes translocated to the external portion of the membrane at the onset of apoptosis and
becomes available to bind to the FITC (fluorescein isothiocyanate) annexin V. Propidium
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iodide binds cellular DNA (Deoxyribonucleic acid) in cells where the cell membrane has been
significantly compromised [30]. After 24 hours of treatment with tested compound at
concentration 62.5 μM, LLC1 and HCT116 cells were collected, washed in PBS and
resuspended in ice cold binding buffer [10x binding buffer: 0.1 M Hepes/NaOH (sodium
hydroxide) (pH 7.4), 1.4 M NaCl (sodium chloride), 25 mM CaCl2 (calcium chloride)].
AnnexinV-FITC (BD Pharmingen, San Diego, California, USA) and propidium iodide
(Sigma Aldrich) were added to each sample and incubated in the dark for 15 min. The
percentage of dead cells were determined by FACS (fluorescence-activated cell sorting)
Calibur flow cytometer (BD Biosciences, San Jose, USA) and the data were analyzed using
FlowJo (Tree Star).
2.4.4. Flow cytometric analysis of Ki67 expression
LLC1 and HCT116 cells treated with either copper(II)-complexes or cisplatin (62.5
μM) for 24 hours, were fixed and permeabilized with permeabilization buffer (BD
Bioscience) and incubated with Ki67 specific antibody conjugated with fluorescein
isothiocyanate (11-5698-82, eBioscience, San Diego, USA) for 30 minutes. Flow cytometry
was conducted on FACS Calibur flow cytometer (BD Biosciences, San Jose, USA) and the
data were analyzed using FlowJo (Tree Star).
2.4.5. Cell cycle analysis
HCT116 cells were allowed to grow until a confluency of 70–80% was achieved in culture
plates. They were exposed to copper(II)-complexes or cisplatin (62.5 μM) for 12 hours and
the cell cycle analysis was performed with Vybrant® DyeCycle™ Ruby stain (Thermo Fisher
Scientific, Inc. USA) according to manufacturer's instructions. After the treatment, HCT116
cells were stained with Vybrant DyeCycle Ruby and analyzed by FACS Calibur flow
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Cytometer (BD Biosciences, San Jose, USA). The cell cycle distribution was analyzed using
FlowJo software.
2.4.6. Statistics
Statistical analyses were performed using SPSS 20.0 for Windows software (SPSS Inc.,
Chicago, IL). The results were analyzed using the Student’s t-test. The difference was
considered signifcant when p < 0.05.
3. Results and discussion
3.1. Synthesis and chemical characterization
S-isoalkyl (R = isopropyl-(L1), isobutyl-(L2), isoamyl-(L3)) derivatives of thiosalicylic
acid were prepared [22] by alkylation of thiosalicylic acid by means of the corresponding
isoalkyl chlorides in alkaline water-ethanol solution (Scheme 1). The corresponding
[Cu2(S-R-thiosal)4(H2O)2] complexes were obtained by direct reaction of Cu(NO3)2
(copper(II)-nitrate) with S-isoalkyl derivatives of thiosalicylic acid (molar ratio 1:2) in water
solution with equimolar amount of LiOH (Scheme 2).
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Scheme 1. Synthesis method of S-isoalkyl derivates of thiosalicylic acid
R = isopropyl-(L1), isobutyl-(L2), isoamyl-(L3)
Scheme 2. Synthesis of copper(II)-complex with S-isoalkyl derivates of thiosalicylic acid
R = isopropyl-(C1), isobutyl-(C2), isoamyl-(C3)
For infrared spectra of complexes, KBr pellet technique was used. This way a formation of
the complexes was confirmed. Based on the asymmetric valence vibrations of the carboxyl
groups, a conformation of the coordination of copper(II)-ion with S-isoalkyl derivatives of
thiosalicylic acid was obtained. Based on the previous results of structurally similar ligands,
binuclear copper(II)-complexes were expected to be obtained [31]. The asymmetric stretching
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frequencies of the carboxylate moieties were established as criteria for distinguishing between
protonated carboxylate groups (1700-1750 cm-1) and coordinated carboxylate groups (1600-
1700 cm-1) [32].
The lack of absorption between 1700 and 1750 cm-1 in IR spectra of complexes
suggested the coordination was to happen through its carboxyl groups. Ligand’s carboxylate
assymetric stretching bands (1677 cm-1 for L1, 1679 cm-1 for L2, 1683 cm-1 for L3) are
positioned lower than it was expected (1700-1750 cm-1) [33,34]. The reason could be the
ortho positioned R-S groups. These bands in corresponding complexes, C1-C3, are in the
expected range (1600-1650 cm-1) [35] (1615 and 1587 cm-1 for C1, 1606 cm-1 and 1561 cm-1
for C2, 1593 and 1550 cm-1 for C3), indicating their deprotonation and coordination in
complexes [33,34]. Table 2 shows that the strong sharp single symmetric stretching bands of
the coordinated carboxylic groups of the S-alkyl derivatives of thiosalicylic acid are in the
expected range (about 1400 cm-1) [34,36]. The geometry of copper(II)-complexes with S-
isoalkyl derivatives of thiosalycilic acid could only be predicted based on the results of IR
spectra, and it was expected to be the same as the coordination with S-alkyl derivatives of
thiosalycilic acid [22]. The geometry of corresponding complexes was confirmed by results
of X-ray analysis.
Table 2. The most important infrared bands (cm-1) of the investigated compounds.
Compound
S-ipr-thiosal
[Cu2(S-ipr-thiosal)4(H2O)2]·2H2O
S-ibu-thiosal
[Cu2(S-ibu-thiosal)4(H2O)2]
S-iam-thiosal
-S-R
-COO- (as)
1677
1615 (s), 1587 (s)
1679
1606 (s), 1561 (s)
1683
1593 (s), 1550 (s)
-COO- (sim)
1404
690 (m)
698 (m)
689 (m)
696 (m)
703 (m)
695 (m)
1402
1412
1400
1412
[Cu2(S-iam-thiosal)4(H2O)2]
s-strong, m-medium
1401
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Figure S7 shows room temperature of EPR spectra of the investigated copper(II)-
complexes in 1% DMSO/H2O (aqua) (v/v). All spectra are nearly isotropic (g = 2.18, H =
140 G), without resolved hyperfine coupling (S = 1/2, I = 3/2), indicating the same copper(II)-
ion coordination in all three complexes. Similar spectra have been reported for hydrated
binuclear copper compounds, with copper(II)-ions coordinated by oxygen ligands [37]. The
forbidden half-field transition (Ms = ±2) was not detected, indicating that the copper(II)-ions
are not magnetically coupled [38].
3.2. Structural description of the complex [Cu2(S-ipr-thiosal)4(H2O)2]2H2O (C1)
X-ray structural analysis shows that complex C1 crystallizes in the triclinic system
with the space group P-1. Its molecular structure and atomic numbering scheme are shown in
Figure 1. The structure of C1 consists of the neutral paddle-wheel type centrosymmetric
dimer molecule [Cu2(S-ipr-thiosal)4(H2O)2] and two molecules of solvated water. In the
dimer, two Cu(II) atoms are bridged via two pairs of bidentate carboxylate anions thus
forming a square base of four oxygen atoms (Table 3) around each copper. Each carboxylate
coordinates unsymmetrically to the central atoms with one Cu–O distance shorter by ca. 0.2 Å
than the other; average Cu–O distance is 1.961(13) Å. The apical positions of the square
pyramidal copper coordination polyhedra ( = 0.005) are occupied by the oxygen atoms
(O1w) of water molecules with slightly elongated bond lengths relatively to equatorial bonds
(Cu1–O1w = 2.194(2) Å). Due to the possible rotation around the single C1–C2 and C11–C12
bonds in both carboxylates, the dihedral angles between the planes of phenyl rings and
corresponding carboxyle groups are 5.6(1) and 11.0(1)°, respectively. These angles are
significantly smaller than the observed ones in two closely related Cu(II)-complexes with S-
methyl and S-ethyl derivatives of thiosalicylic acid, which we have recently published
[31,39]. As can be seen from the trans O–Cu–O angles in C1, which are not linear, the copper
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atoms are displaced from the respective basal planes towards the apical oxygen atoms by
0.187(1) Å. There are 41 crystal structures of paddle-wheel type [Cu2(ar-COO)4(H2O)2]
(ar-COO are aromatic carboxylic acids) dimer molecules gathered in Cambridge Structural
Database [40]. The comparison of bond lengths and angles in C1 with respective data in CSD
structures shows that the bond lengths and angles in C1 are normal. The CuCu separation
(2.6031(5) Å) in C1 within the dimer, being significantly shorter than the sum of the van der
Waals radii of Cu and indicating a weak CuCu interaction, is also typical since the CuCu
separation in 41 CSD structures is between 2.579 and 2.665 Å with the average value of
2.63(2) Å.
Figure 1. Molecular structure with atom numbering scheme of C1. Displacement ellipsoids
are drawn at 50% probability, hydrogen bonds are shown as red dashed lines. Symmetry code:
i = -x + 1, -y + 1, -z + 1.
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Table 3. Selected bond lengths (Å) and angles (°) for C1.
Cu1–O1
Cu1–O2i
Cu1–O3
Cu1–O4i
Cu1–O1w
Cu1–Cu1i
S1–C3
1.9545(14)
1.9766(14)
1.9473(14)
1.9650(14)
2.1935(16)
2.6031(5)
1.768(2)
1.830(2)
1.7737(19)
1.828(2)
O3–Cu1–O1
O3–Cu1–O4i
O1–Cu1–O4i
O3–Cu1–O2i
O1–Cu1–O2i
O4i –Cu1–O2i
O3–Cu1–O1w
O1–Cu1–O1w
O4i–Cu1–O1w
O2i–Cu1–O1w
90.25(7)
169.14(6)
88.65(6)
90.55(7)
168.84(6)
88.47(6)
98.83(6)
99.62(6)
92.01(6)
91.26(6)
S1–C8
S2–C13
S2–C18
O1–C1
1.253(2)
O2–C1
1.268(2)
O3–C11
O4–C11
1.260(2)
1.259(2)
Symmetry transformation used to generate equivalent atoms: i = -x + 1, -y + 1, -z + 1.
The crystal structure of C1 is completed by a crystalline water molecule being
hydrogen bonded (Table 4). It serves as a donor to S2 and O2 atoms from two different
carboxylic acids of one dimeric unit, and as an acceptor of the two coordinated water
molecules, one of them being from the neighboring dimeric unit. Due to these hydrogen
bonds the dimeric units are tied to form infinite chains along the a axis (Figure 2).
Neighbouring chains are held together by van der Waals forces and form a parquet motif
(Figure 3).
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Figure 2. A chain parallel with the a axis in the crystal structure of C1 showing hydrogen
bonds (red dashed lines). Hydrogen atoms not involved in hydrogen bonds are omitted for
clarity.
Table 4. Hydrogen bonds for C1 [Å and °].
d(D–H)
0.81
0.92
1.04
1.06
<(DHA)
144.4
139.0
154.1
154.9
D–HA
d(HA)
2.11
2.14
1.86
2.25
d(DA)
2.811(3)
2.893(3)
2.836(2)
3.241(2)
O1w–H1O1O2wi
O1w–H2O1O2wi
O2w–H2O2O2
O2w–H1O2S2
Symmetry transformations used to generate equivalent atoms: i = -x + 1, -y + 1, -z + 1; ii = x + 1, y, z.
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Figure 3. A view along the a axis showing the arrangement of neighboring chains in the
structure of C1. Hydrogen bonds within the chains are shown as red dashed lines.
3.3. In vitro cytotoxic activity
3.3.1. Copper(II)-complexes showed lower cytotoxic activity against normal fibroblasts than
cisplatin
Ligands, S-isoalkyl derivatives of thiosalicylic acid reduced viability of human lung
cancer cells only in the highest concentrations, 500 and 1000μM (Figure 4A). Cytotoxic effect
of isoalkyl derivatives of thiosalicylic acid toward mouse lung cancer cells was stronger, they
reduced viability of these cells in concentration 32.5 μM (Figure 4B). All tested copper(II)-
complexes showed dose dependant cytotoxic effect toward A549 and LLC1 cells, higher in
comparison with ligands, but lower in comparison with cisplatin (Figure 4A and 4B).
However, cytotoxic activity of all three copper(II)-complexes toward human colorectal
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carcinoma cells, HCT116, was very similar to cisplatin activity (Figure 4C). Complexes C2
and C3 in medium concentrations from 15.625 to 62.5 showed higher cytotoxicity on
HCT116 cells in comparison with lung cancer cell lines, A549 and LLC1. C3 showed the
highest cytotoxicity toward both A549 and LLC1 cells, while C1 exhibited the lowest effect.
Importantly, copper(II)-complexes had significantly lower cytotoxic effect toward normal
human fibroblasts in comparison with cisplatin in all tested concentrations (Figure 4D).
Cytotoxicity of tested complexes being lower than cisplatin toward MRC-5 fibroblasts in the
highest concentrations, significantly reducing viability of tumor cells, A594 and HCT116,
indicates possible selective cytotoxicity of these new complexes toward tumor cell lines.
3.3.2. Copper(II)-complexes induced apoptosis of tumor cells
The potential of tested complexes to induce apoptotic death was determined by flow
cytometric analysis of treated cells stained with Annexin V FITC and Propidium Iodide. The
results showed that all copper(II)-complexes induced apoptosis in LLC1 cells (Fig 5).
Significantly higher percentage of LLC1 cells treated with C1 (p<0.001) and C2 (p<0.005)
for 24 hours was in early apoptosis in comparison with untreated cells (Fig 5A). Also,
significantly higher percentage of LLC1 cells treated with C1 (p<0.05), C2 (p<0.05), and C3
(p<0.005) for 24 hours was in late apoptosis in comparison with untretaed cells. The
percentage of LLC1 cells treated with copper(II)-complexes in early apoptosis is higher than
the percentage of these cells after cisplatin treatment (Fig 5A). As shown in Fig 5A and 5B
the majority of LLC1 cells after 24 h treatment with copper(II)-complexes and cisplatin were
in late apoptosis. Importantly, the percentages of early and late apoptotic LLC1 cells treated
with copper(II)-complexes are higher in comparison with cisplatin treated LLC1 cells (Fig 5A
and 5B). In agreement with both well known low efficiency of cisplatin on colorectal cancer
and rather weak clinical responses even when used in combination [41,42], the minimal
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percentage of HCT116 cells underwent apoptosis after the cisplatin treatement (Fig 5C).
However C3 copper(II)-complex induced significant apoptosis of HCT116 cells (Fig 5C and
5D). Significantly higher percentage of early (p<0.01) and late (p<0.05) apoptotic HCT116
cells were found after the treatement with C3 (62.5 μM) in comparison with cisplatin treated
cells (Fig 5C). The highest proapototic effect of C3 on tumor cells (Fig. 5) is in line with the
highest reduction of all tested cells by C3 (Fig. 1) evaluated by MTT assay.
3.3.3. C2 complex showed antiproliferative effect against lung cancer cells while C1 against
colorectal cancer cells
Ki67 is a DNA-binding, proliferation-associated protein expressed in proliferating but
not in quiescent cells. Anti-proliferative effects of testing molecules can be evaluated by
assessment of Ki67 expression level in treated cells [43]. The percentage of Ki67 positive
LLC1 treated with all copper(II)-complexes and cisplatin was lower compared to untreated
cells (Fig 6A). As presented in Fig 6A the percentage of Ki67 positive LLC1 cells treated
with C2 (62.5 μM) was significantly lower compared to cisplatin treated cells (Fig 6A). Also,
significantly lower expression of Ki67 was found in cisplatin, C1, and C2 treated cells
compared to untreated LLC1 cells (Fig 6B). The same analysis showed different effects of
tested complexes on HCT116 colorectal cancer cells (Fig 6C and 6D). Among tested new
complexes only C1 showed antiproliferative effect toward HCT116. The percentage of Ki67
positive HCT116 cells treated with C1 was significantly lower (p<0.05) compared to
untretaed cells (Fig 6C). The percentage of Ki67 was found to be lower in cells treated with
C1 as well as in cisplatin treated cells (Fig 6D).
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3.3.4. Copper(II) complexes induce G2/M cell cycle arrest
We further investigated whether new copper(II) complexes affect cell cycle. Hence,
the cell cycle profile of HCT116 cells was determined after exposure to copper(II) complexes
or cisplatin for 12 hours. All three tested complexes (62.5 μM) and cisplatin treatment
significantly increased the percentage of cells in G2/M phase in comparison with untreated
cells (Fig 7). Furthermore, the percentage of cells in S and G0/G1 phases was lower after
copper(II) complexes and cisplatin treatment (Fig 7). C3 had the highest effect on cell cycle
distribution of HCT116 cells, by means of inducing significant arrest in G2/M phase and by
decreasing the percentage of cells in S phase (Fig 7). The obtained data indicate that
copper(II) complexes inhibit cell proliferation through arrest of cell cycle progression in the
G2/M phase and subsequent induction of apoptosis in HCT116 cells. The strongest effect of
C3 on cell cycle disturbance is in line with the highest apoptotic effect (Fig 5C) of this
complex on HCT116 cells.
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A
Cu1
C1
C2
CCu33
Cisplatin
L1
L2
L3
Cisplatin
A549
A549
Cu2
120
100
80
60
40
20
0
120
100
80
60
40
20
0
7,8 15,625 31,25 62,5 125 250 500 1000
7,8 15,625 31,25 62,5 125 250 500 1000
concentration (μΜ)
concentration (μΜ)
B
L1
Cu1
C1
LLC1
LLC1
120
100
80
60
40
20
0
120
C2
L2
Cu2
C3
L3
100
80
60
40
20
0
Cu3
Cisplatin
Cisplatin
7,8 15,625 31,25 62,5 125 250 500 1000
7,8 15,625 31,25 62,5 125 250 500 1000
concentration (μΜ)
concentration (μΜ)
D
C
Cu1
C1
Cu
C2
C3
Cu
cisplatin
Cu
C1
HCT116
MRC-5
Cu
C2
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Cu
C3
cisplatin
7,8 15,625 31,25 62,5 125 250 500 1000
7,8 15,625 31,25 62,5 125 250 500 1000
contentrations (µM)
concentration (µM)
Figure 4: Dose-dependent cytotoxicity of copper(II)-complexes against human and
murine lung cancer cells. Graphs of (A) human (A549) and (B) murine (LLC1) lung cancer,
(C) human colorectal (HCT116), and (D) human normal fibroblasts (MRC-5) cell survival
after 72h growth in the presence of copper(II)-complexes determined by MTT assay. All data
are presented as mean values from three independent experiments performed in triplicates.
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A
B
untreated
cisplatin
Рeаaнrаlyаaпpоoптpоtoзаsis
80
Касна апоптоза
eate apoptosis
**
60
40
20
0
*
*
*
C1
C2
C3
**
***
untreated cisplatin
C1
C2
C3
Annexin V
D
untreated
cisplatin
нетретиране
цисплатина
C
early apoptosis
рана апоптоза
касна апоптоза
eate apoptosis
12
**
10
8
6
*
Cu1
C1
C2
Cu2
C3
Cu3
4
2
0
untreated cisplatin
Cu1
C1
Cu2
C2
Cu3
C3
untreated cisplatin
Annexin V
Figure 5: Copper(II)-complexes induce apoptosis of lung cancer cells, while C3 induces
apoptosis of colorectal cancer cells. Apoptosis of untreated, cisplatin, and copper(II)-
complexes 24h treated (62.5 μM) LLC1 and HCT116 cells was evaluated by flow cytometry
using Annexin V (FITC) and PI double staining. (A) The data are presented as means + SD of
a three independent experiments, * p<0.05, **p<0.005, *** p<0.001 indicate difference
between treated and untreated cells (B) Representative dot plots illustrate the population of
viable (AnnV- PI-), early apoptotic (Ann V+ PI-), late apoptotic (AnnV+ PI+) and necrotic
(AnnV- PI+) LLC1 cells.
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80
60
40
20
0
A
*
Cu1
Cu2
C2
Cu3
C3
нетретиране цисплатина
untretaed
cisplatin C1
B
untreated
cisplatin
C1
C2
C3
Ki67
FSC
Ki67
C
70
*
60
50
40
30
20
10
0
untreated
cisplatin
Cu1
Cu2
C2
Cu3
C3
untreated cisplatin
C1
D
нетретиране
untreated
цисплcаisтpиlнatаin
CCu11
CCu22
CCu33
Ki67
FSC
Figure 6: Copper(II)-complexes attenuate expression of Ki67 in tumor cells. The
percentage of Ki-67 positive LLC1 (A) and HCT116 (C) cells exposed to copper(II)-
complexes or cisplatin (62.5 μM) for 24h and determined by flow cytometry is presented as
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the mean + SD from three independent experiments. The data were analyzed with Student’s t-
test: *p<0.05. Representative histograms of Ki67 expression (mean fluorescence intensity)
and gating strategy presenting exclusion of cell debris and clumps in forward/side scatter plot
in LLC1 (B) and HCT116 (D) cells.
G1
S
G2
80
**
70
60
50
40
30
20
10
0
*
*
*
*
untreated cisplatin
C1
Cu1
C2
Cu2
C3
Cu3
нетретиране цисплатина
untretaed cisplatin
Figure 7: Copper(II) complexes induce G2/M cell cycle arrest in HCT116 cells. HCT116
cells cycle was analyzed by flow cytometry. The results are expressed as the percentage of
cells in different phases of the cell cycle. The data are presented as the mean ± SD, *p<0.05,
**p<0.01 significantly different from untreated cells.
4. Conclusion
In the reaction of copper(II)-nitrate tryhidrate with S-isoalkyl derivatives of
thiosalicylic acids (isoalkyl = isopropyl-(L1), isobutyl-(L2) and isoamyl-(L3)) three new
binuclear copper(II)-complexes were formed. The structure of complexes was predicted
based on microanalysis, infrared, and EPR spectra, magnetic measurements and molar
conductivity. The real geometry of obtained binuclear copper(II)-complexes were confirmed
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by X-ray analysis for copper(II)-complex with S-isopropyl derivative of thiosalicylic acid,
[Cu2(S-ipr-thiosal)4(H2O)2]2H2O (C1). C1 manifested the strongest cytotoxic and apoptotic
effect, while C2 exhibited the highest anti-prolferative activity. The complexes showed tumor
dependant cytotoxic activity, with significantly lower cytotoxic activity toward normal
fibroblasts than cisplatin. All complexes induced G2/M cell cycle arrest and apoptotic death
in tumor cells. C3 had the highest cytotoxic and apopototic effect, while C1 and C2
manifested the highest antiprolferative activity. The significant apoptotic effect of C3, the
induction of G2/M cell cycle arrest and lower activity toward normal cells indicate that this
complex might be of interest for further analysis of its in vivo antitumor effects.
Acknowledgment
This work was financially supported by the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Projects 172016, 172034, 175069),
bilateral project with PR China (06/2018) and the Faculty of Medical Sciences, University of
Kragujevac (MP02/19). The financial supports of Slovak grant agencies VEGA (project
1/0148/19) and APVV (project APVV-18-0016) are gratefully acknowledged. The
crystallographic measurements used instruments of the ASTRA lab established within the
Operation program Prague Competitiveness – project CZ.2.16/3.1.00/24510. We thank prof.
Ana Popović-Bijelić and prof. Miloš Mojović (EPR Lab, Faculty of Physical Chemistry,
University of Belgrade) for acquiring and analyzing the EPR spectra.
Appendix A. Supplementary data
CCDC 1977864 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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