Inorganica Chimica Acta 487 (2019) 138–144

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Inorganica Chimica Acta

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Research paper

Mixed-ligand copper(II) complexes with tetrazole derivatives and 2,2′- T

bipyridine, 1,10-phenanthroline: Synthesis, structure and cytotoxic activity
Julia A. Ereminaa,b,c, Elizaveta V. Lidera,b, , Denis G. Samsonenkoa,b, Liliya A. Sheludyakovaa,b,
⁎

Alexey S. Berezina, Lyubov S. Klyushovac, Vladimir A. Ostrovskiid, Rostislav E. Trifonovd,e

a
Nikolaev Institute of Inorganic Chemistry SB RAS, 3, Acad. Lavrentiev Ave., 630090 Novosibirsk, Russia
b
Novosibirsk State University, 2, Pirogova Str., 630090 Novosibirsk, Russia
c
Federal Research Center of Fundamental and Translational Medicine, 2/12 Timakova str., 630117 Novosibirsk, Russia
d
Saint Petersburg State Institute of Technology, 26, Moskovsky Prospect, Saint Petersburg 190013, Russia
e
Saint Petersburg State University, 7/9 Universitetskaya nab., Saint Petersburg 199034, Russia

ARTICLE INFO ABSTRACT

Keywords: The [Cu2(2,2′-bipy)2(L1)4] (1), [Cu2(1,10-phen)2(L1)4] (2), [Cu(2,2′-bipy)(L2)2]n (3) and [Cu2(1,10-phen)2(L2)4]
Mixed ligand Cu(II) complexes (4) complexes, where HL1 – 5-phenyltetrazole, and HL2 – 1H-tetrazole, have been synthesized. All complexes
1,10-Phenanthroline have been characterized by elemental analysis, IR, EPR spectroscopy and X-ray diffraction. The complexes
Tetrazole possess distorted tetragonal-pyramidal coordination geometry. Compounds 1, 2, 4 show μ-5-phenyl-tetrazole/
Cytotoxicity
tetrazole bridged dinuclear structures, while compound 3 reveals polymeric structure. The effect of compounds
MCF-7
on viability of the MCF-7 and Hep-2 cell lines was investigated in vitro. The study showed that tetrazole ligands
Hep-2
HL1 and HL2 are non-toxic at tested concentrations (1–50 μM), while 1,10-phen and 2,2′-bipy posses cytotoxicity.
All of the complexes exhibit significant dose-dependent cytotoxic effect and have the potential to act as efficient
cytotoxic drugs. The complexes [Cu(1,10-phen)Cl2] (5) and [Cu(2,2′-bipy)Cl2] (6) have also been obtained to
establish the influence of insertion of tetrazole ligands in compounds on their cytotoxic properties.

1. Introduction formation of reactive oxygen species, interaction with DNA, inhibition

Inorganic, metal-organic and coordination compounds are widely
used in medicine as diagnostic and antitumor agents. Cisplatin and its
derivatives are still considered to be one of the most effective drugs [1],
despite the dose-related side effects during treatment (nephrotoxicity,
neurotoxicity, etc.) [2,3] and inherited or acquired resistance to
therapy in patients [4], which limit clinical use of platinum drugs.
Copper complexes are potentially attractive as antitumor agents, since
antineoplastic drugs based on endogenous metals are expected to ex-
hibit fewer side effects than platinum analogs. In this field copper
complexes have already shown encouraging results [5–7]. Many types
of cancer, such as prostate, breast, colon, lung, brain, are accompanied
by an increased copper content in the tumor and/or a change in the
distribution of copper in the body [8,9]. However, the mechanism of
copper accumulation in tumors, as well as the influence of this process
on their development, have not been studied in detail. As a result,
copper complexes that exert a direct cytotoxic effect are actively stu-
died. Among the various coordination compounds of copper, many
complexes with anticancer activity, due to inhibition of proteasomes,
of topoisomerases and induction of apoptosis [10] have been identified.
Mixed-ligand copper(II) complexes based on 1,10-phenanthroline
and other types of ligands are of interest to scientists [11,12]. Ruiz-
Azuara and colleagues synthesized a series of cytotoxic copper com-
plexes registered under the name Casiopeinas® (Cas) [13]. These
compounds are mixed ligand complexes of copper (II) with the general
formula [Cu(NN)(AA)][NO3], where NN is the donor neutral diimine
(1,10-phenanthroline (1,10-phen), 2,2′-bipyridine (2,2′-bipy) or their
substituted analogues), AA is NO- or OO-donor systems (amino acids or
acetylacetonates). Investigation of the relationship between the struc-
ture and activity of these compounds (QSAR) [14] has shown that the
presence of a central condensed aromatic ring in phenanthroline-con-
taining complexes is necessary to preserve the antiproliferative effect,
and the nature of the second NeO- or OeOedonor ligand has little
effect on biological activity. The most promising complexes from the
Casiopeinas series undergo a pre-clinical trial.
An important factor for conducting intensive research in this field is
the biological activity of the ligands themselves. Tetrazole derivatives
are potential pharmacologically active compounds. Many tetrazole

⁎
Corresponding author.
E-mail address: lisalider@ngs.ru (E.V. Lider).

https://doi.org/10.1016/j.ica.2018.12.011
Received 31 October 2018; Received in revised form 6 December 2018; Accepted 6 December 2018
Available online 07 December 2018
0020-1693/ © 2018 Published by Elsevier B.V.
J.A. Eremina et al.
derivatives have already found their application in medicine as drugs
with antiviral, antifungal and antibacterial activity [15]. However,
tetrazoles and copper complexes with tetrazole derivatives have been
poorly studied as antitumor agents. One of the possible reasons is the
tendency of tetrazoles to form practically insoluble metal-organic co-
ordination polymers, which limits their use in medicine.
Many copper phen/bipy complexes with other co-ligands have been
extensively studied, but there is still a gap to design and study new
ones. We were interested in the evaluation of the biological properties
of complexes that combine pharmacologically active products (tetra-
zole derivatives) and endogenous metals, thereby insights were made
into the cytotoxicity of copper(II) complexes with tetrazole and diimine
ligands. In this work, we have synthesized a series of mixed-ligand
copper(II) complexes of the type [Cu2(diimine)2(tetrazole)4] and [Cu
(diimine)(tetrazole)2]n, where tetrazole is 5-phenyltetrazole (HL1) or
1H-tetrazole (HL2) and diimine is 2,2′-bipyridine or 1,10-phenanthro-
line and studied their cytotoxic activity against MCF-7 (human breast
adenocarcinoma) and Hep-2 (human larynx carcinoma) cell lines.
2. Experimental section
2.1. Reagents and materials
All chemicals used were of reagent grade and were used as pur-
chased without any further purification. Tetrazole ligands HL1 and HL2
(Scheme 1) were provided by Rostislav E. Trifonov group [16].
2.2. Methods and instrumentation
Elemental analysis (C, H, and N) was performed using Euro EA 3000
analyzer. IR absorption spectra were recorded on SCIMITAR FTS 2000
and VERTEX-80 spectrophotometers at 4000–100 cm−1 (in fluoridated
oil – in the region of 4000–1500 cm−1, in Vaseline oil –
1500–400 cm−1).
XRD powder analysis of complexes 1–4 was performed on a
Shimadzu XRD-7000 diffractometer (CuKα radiation, Ni filter, 5–50° 2θ
range, 0.03° 2θ step, room temperature). EPR spectra were recorded on
a Varian E−109 spectrometer in X- and Q-bands at 300 K. The 2,2-
Diphenyl-1-picrylhydrazyl (marked hereafter as DPPH, g = 2.0036)
was used as standard.
2.3. Synthesis
2.3.1. Synthesis of [Cu2(2,2′-bipy)2(L1)4] (1)
The literature procedure [17] for the preparation of this complex
was followed. An aqueous solution (4 ml) of copper acetate (0.020 g,
0.1 mmol) and an ethanol solution (3 ml) of 2,2′-bipyridine (0.016 g,
0.1 mmol) were mixed and the resulting deep blue solution was stirred
for 5 min. To the mixture was added an ethanol solution (3 ml) of HL1
(0.029 g, 0.2 mmol). The reaction mixture was stirred for another 5 min
and it was left for slow evaporation at room temperature. Deep blue
plate-like crystals were obtained after a week.
Yield (0,01 g): 20%. Elemental analysis (%): Calc. for C48H36N20Cu2:
C, 56.5; H, 3.6; N, 27.5. Found: C, 56.7; H, 3.6; N, 27.4.
Scheme 1. 2,2′-Bipyridine, 1,10-phenanthroline, 5-phenyltetrazole (HL1) and tetrazole (HL2) ligands.
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Inorganica Chimica Acta 487 (2019) 138–144
All other complexes were synthesized by following a procedure si-
milar to that of [Cu2(2,2′-bipy)2(L1)4].
2.3.2. Synthesis of [Cu2(1,10-phen)2(L1)4] (2)
Complex [Cu2(1,10-phen)2(L1)4] have been previously synthesized
by M. Saha and et al. using different method (metal mediated [2 + 3]
cycloaddition reaction between copper bound azide polymer and dif-
ferent organonitriles) [18].
An aqueous solution (3 ml) of copper acetate (0.020 g, 0.1 mmol)
and an ethanol solution (2 ml) of 1,10-phenantroline (0.020 g,
0.1 mmol) were mixed and the resulting deep blue solution was stirred
for 5 min. To the mixture was added an ethanol solution (3 ml) of HL1
(0.029 g, 0.2 mmol). During the stirring a solid precipitated from the
solution. Blue colored precipitate was filtered out, washed with ethanol
and water and dried in air. Deep blue crystals were obtained after a
month from mother liquor.
Yield (0,02 g): 38%. Elemental analysis (%): Calc. for C52H36N20Cu2:
C, 58.5; H, 3.4; N, 26.2. Found: C, 57.4; H, 3.1; N, 25.9.
2.3.3. Synthesis of [Cu(2,2′-bipy)(L2)2]n (3)
An aqueous solution (5 ml) of copper acetate (0.040 g, 0.2 mmol)
and an ethanol solution (3 ml) of 2,2′-bipyridine (0.031 g, 0.2 mmol)
were mixed and the resulting deep blue solution was stirred for 5 min.
To the mixture was added an ethanol solution (3 ml) of HL2 (0.028 g,
0.4 mmol). During the stirring a small amount of solid precipitated from
the solution. Blue colored precipitate was filtered out, washed with
ethanol and water and dried in air. Composition of precipitate could not
be established based on the results of elemental analysis. Deep blue
crystals suitable for single-crystal X-ray diffraction were obtained after
a month from mother liquor.
Yield (0,01 г) – 25%.Elemental analysis (%): Calc. for C12H10N10Cu:
C, 40.3; H, 2.8; N, 39.1. Found: C, 39.5; H, 2.9; N, 36.3.
2.3.4. Synthesis of [Cu2(1,10-phen)2(L2)4] (4)
An aqueous solution (5 ml) of copper acetate (0.040 g, 0.2 mmol)
and an ethanol solution (4 ml) of 1,10-phenantroline (0.040 g,
0.2 mmol) were mixed and the resulting deep blue solution was stirred
for 5 min. To the mixture was added an ethanol solution (4 ml) of HL2
(0.028 g, 0.4 mmol). During the stirring a small amount of solid pre-
cipitated from the solution. Blue colored precipitate was filtered out,
washed with ethanol and water and dried in air. Composition of pre-
cipitate could not be established based on the results of elemental
analysis. Deep blue crystals suitable for single-crystal X-ray diffraction
were obtained after a month from mother liquor.
Yield (0,003 г) – 4%. Due to the low yield of the complex elemental
analysis was not performed.
2.3.5. Synthesis of [Cu(2,2′-bipy)Cl2] (5)
An ethanol solution (4 ml) of copper (II) chloride (0.102 g,
0.6 mmol) was added to ethanol solution (3 ml) of 2,2′-bipyridine
(0.047 g, 0.3 mmol) with stirring. Immediately formed precipitate was
filtered out, washed with ethanol and dried in air.
Yield (0,05 g): 58%. Elemental analysis (%): Calc. for
C10H8N2Cl2Cu: C, 41.3; H, 2.8; N, 9.6. Found: C, 40.1; H, 2.9; N, 9.2.
J.A. Eremina et al. Inorganica Chimica Acta 487 (2019) 138–144

Table 1 Positions of hydrogen atoms of organic ligands were calculated geo-

Crystal data and structure refinement for 3 and 4. metrically and refined in the riding model. The crystallographic data
Identification code 3 4 and details of the structure refinements are summarized in Table 1.
CCDC 1841344 and 1841345 contain the supplementary crystal-
Empirical formula C12H10CuN10 C28H20Cu2N20 lographic data for this paper. These data can be obtained free of charge
Formula weight 357.84 763.72
from The Cambridge Crystallographic Data Center at http://www.ccdc.
Crystal system Monoclinic Monoclinic
Space group P21 P21/c
cam.ac.uk/data_request/cif.
a, Å 7.1902(3) 7.8306(5)
b, Å 9.3876(5) 10.9866(8)
2.5. Cytotoxic activity
c, Å 9.9623(4) 16.9857(12)
β, deg. 92.037(4) 98.481(7)
V, Å3 672.02(5) 1445.33(18) Cell viability was evaluated by Hoechst /PI staining by standard
Z 2 2 method as previously described [22]. Human larynx carcinoma cell line
D(calc.), g/cm3 1.768 1.755 (Hep−2) was grown on 96 wells plate in IMDM medium in CO2-in-
μ, mm−1 1.643 1.534
cubator at 37 °C. After 24 h cells were treated with tested compounds
F(0 0 0) 362 772
Crystal size, mm 0.19 × 0.13 × 0.11 0.28 × 0.17 × 0.09 dissolved in ethanol for 48 h at concentrations of 1, 5, 12,5, 25 and
θ range for data 3.44–30.35 3.30–29.49 50 μM. For identification of live and dead cells, treated cells and control
collection, deg. cells were stained with a mixture of fluorescent dyes Hoechst 33,342
Index ranges −9 ≤ h ≤ 10, −7 ≤ h ≤ 10,
(Sigma-Aldrich) and propidium iodide (Invitrogen) for 10 min at 37 °C.
−12 ≤ k ≤ 12, −14 ≤ k ≤ 10,
−14 ≤ l ≤ 13 −22 ≤ l ≤ 19
IN Cell Analyzer 2200 (GE Healthcare, UK) was used to perform at least
Reflections collected/ 3778/2823 6811/3389 four fields per well automatic imaging in bright-field and fluorescence
independent channels. In accordance with the morphological changes of the cell
Rint 0.0162 0.0168 during apoptosis and death the cells were classified by means of IN Cell
Reflections with 2685 2970
Investigator image analysis software as live cells (normal nuclei: non-
I > 2σ(I)
Goodness-of-fit on F2 1.059 1.066 condensed chromatin uniformly dispersed over the entire nucleus),
Final R indices R1 = 0.0264, R1 = 0.0294, apoptotic cells (round cells, bright chromatin that is highly condensed
[I > 2σ(I)] wR2 = 0.0595 wR2 = 0.0701 or fragmented) and dead cells (primarily stained with propidium due to
R indices (all data) R1 = 0.0289, R1 = 0.0361,
impaired permeability: enlarged nuclei with smooth normal structure
wR2 = 0.0609 wR2 = 0.0741
Largest diff. peak/hole, 0.329/−0.312 0.325/−0.462
or slightly condensed nuclei) [22]. All data shown are mean of three
e/Å3 wells. The quantitative data were expressed as the mean ± standard
deviation (SD).

2.3.6. Synthesis of [Cu(1,10-phen)Cl2] (6) 3. Results and discussion

An ethanol solution (4 ml) of copper (II) chloride (0.051 g,
0.3 mmol) was added to ethanol solution (4 ml) of 1,10-phenanthroline
(0.059 g, 0.3 mmol) with stirring. Immediately formed precipitate was
filtered out, washed with ethanol and dried in air.
Yield (0,07 g): 78%. Elemental analysis (%): Calc. for
C12H10N2Cl2Cu: C, 45.8; H, 2.6; N, 8.9. Found: C, 44.4; H, 2.5; N, 8.6.
2.4. X-ray crystallography
Diffraction data for single-crystals 3 and 4 were obtained at 130 K
on an automated Agilent Xcalibur diffractometer equipped with an area
AtlasS2 detector (graphite monochromator, λ(MoKα) = 0.71073 Å, ω-
scans). Integration, absorption correction, and determination of unit
cell parameters were performed using the CrysAlisPro program package
[19]. The structures were solved by dual space algorithm (SHELXT
[20]) and refined by the full-matrix least squares technique (SHELXL
[21]) in the anisotropic approximation (except hydrogen atoms).
3.1. Synthesis
The Cu2+ complexes with ligands HL1 and HL4 were synthesized by
the reaction of water solutions of the Cu(OAc)2·H2O with ethanol so-
lutions of 1,10-phenantroline or 2,2′-bipyridine and ligands according
to the scheme (Schemes 2 and 3).
The syntheses were carried out with stirring and at room tempera-
ture. The complexes were synthesized at the ratio M: 1,10-phen (2,2′-
bipy): L = 1:1:2.
3.2. Description of crystal structures
The complex [Cu2(1,10-phen)2(L2)4] (4) crystallizes in P21/c space
group. Asymmetric unit of 4 contains a copper(II) cation, an o-phe-
nantroline molecule and two tetrazolate anions. Cu(II) cation has tet-
ragonal pyramidal coordination environment of five nitrogen atoms. An

Scheme 2. Synthesis route of the copper complexes with 5-phenyltetrazole.

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J.A. Eremina et al. Inorganica Chimica Acta 487 (2019) 138–144

Scheme 3. Synthesis route of the copper complexes

with 1H-tetrazole.

Fig. 1. The structure of [Cu2(1,10-phen)2(L2)4] (4) complex (ellipsoids of 50%

probability). Hydrogen atoms are omitted.
Fig. 3. Mid-IR spectra of HL2 at 300 K.

o-phenantroline molecule and two tetrazolate anions occupy four co-

ordination cites at the base of the pyramid, another tetrazolate anion
occupies the apical position. Cu–N(base) distances are in the range of
1.9971(16)–2.0415(15) Å. Coordination polyhedron demonstrates tet-
ragonal elongation common for copper(II) complexes, Cu–N(apical)
distance is 2.2741(16) Å. Two Cu(II) cations are joined together via two
bridging tetrazolate anions to form binuclear complex [Cu2(1,10-
phen)2(L2)4] (Fig. 1). In the crystal packing, the binuclear complexes
are situated in special positions 2b with coordinates (½, 0, 0) and (½,
½, ½), one of the systems of inversion centers. There are no specific
interactions between the complexes except van der Waals.
The complex [Cu(2,2′-bipy)(L2)2]n (3) crystallizes in P21 space
group. Asymmetric unit of 3 contains a copper(II) cation, an o-phe-
nantroline molecule and two tetrazolate anions. In the structure 3, the
coordination environment of Cu(II) cation is the same as in structure 4.
Cu–N(base) distances are in the range of 1.981(3)–2.038(3) Å, Cu–N
(apical) distance is 2.205(3) Å. Cu(II) cations are joined together via
bridging tetrazolate anions to form zig-zag polymeric chains (Fig. 2).
The chains are situated on screw axes 21 along b direction. There are no
specific interactions between the chains except van der Waals.
The identity of single crystals of complexes 1–3 and the synthesized
polycrystalline phases was evidenced by powder X-ray diffraction
analysis (Fig. S1-3). The observed X-ray diffraction pattern of com-
pound 4 shows that the complex is amorphous.
3.3. IR spectra
Ligands and complexes are characterized by IR spectroscopy. For
the spectrum of unsubstituted tetrazole HL2 (Fig. 3) and the HL1 ligand,
a broad structured band is observed in the region of 3300–1800 cm−1.
Similar spectroscopic picture (pattern) in the high-frequency region is
typical for compounds with strong intermolecular [23] and in-
tramolecular hydrogen bonds [24,25].
As a result of ligand deprotonation when complexing, the ν(NH) band
disappears. The IR-spectra of compounds exhibit ν(CH) bands of aromatic
rings at 3125–2800 cm−1. For 1–4 in the region 1614–1486 cm−1 the
number and position of bands, that attributable to the ring vibrations,
change in comparison with free ligands spectra (Table 2). This indicates
the coordination of nitrogen atoms to copper atoms. The new bands in the
300–100 cm−1 range are assigned to the ν (Cu–N).

Table 2
Assignment of the IR spectral bands of HL1, HL2 and complexes 1–4.
Compound Assignment, cm−1

HL1 1 2 HL2 3 4

ν (CH) 3125 3111 3086 2966 3123 3125

3044 3080 3067 2921 3063 3062
3036 3046 2926 2926
Rrings + δ(NH) 1614 1603 1593 1603 1601 1605
1564 1570 1584 1521 1575 1579
1486 1540 1520 1493 1517
1496 1499 1493
ν (Cu-N) – 289 271 – 298 –
Fig. 2. Fragment of the polymeric chain in the structure of [Cu(2,2′-bipy)
265 255 285
(L2)2]n (3) (ellipsoids of 50% probability). Hydrogen atoms are omitted.

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J.A. Eremina et al.
Table 3
The g-tensor values of compounds 1 and 2 at 300 K obtained by the EPR
spectroscopy.
Complex g⊥ g||
1 2.077(3) 2.27(1)
2 2.067(2) 2.25(1)
3.4. EPR results
Spectra are described by spin-Hamiltonian
H = g (Hx S x + Hy S y ) + g Hz Sz with S = 1/2 and the axial g-tensor
(values presented in Table 3). The axial symmetry of g-tensors and
unresolved hyperfine structure may point out to the presence of the
weak exchange interaction between paramagnetic centers. The mole-
cules of complexes are dimers according X-ray diffraction data, but
there is no dipole-dipole interaction between paramagnetic centers
despite the small distance between copper ions (R(Cu-Cu) = 3,99 (1),
3,97 (2)).
3.5. In vitro cytotoxicity study
The cytotoxic activity of HL1, HL2, 2,2′-bipyridine, 1,10-phenan-
troline, 1–6, CuCl2 and Cu(OAc)2 on the viability of Hep−2 cells was
examined in the presence of different concentrations of tested com-
pounds dissolved in ethanol (copper(II) salts were dissolved in water).
MCF-7 cell line was also used to evaluate cytotoxicity of complexes 1–4.
The cytotoxicity studies were carried out using dual staining with
Hoechst 33342/propidium iodide (PI) with differentiation of cells into
live and apoptotic. The half maximal inhibitory concentration (IC50)
was defined as drug concentration that reduces the number of living
cells by 50%.
1,10-phenanthroline is a well-known divalent metal chelating
agent, that forms strong complexes with most metal ions. Furthermore,
1,10-phen possesses many biological activities, it is known as DNA in-
tercalating agent, cholinesterase and protease inhibitor [26–28].
Copper complexes with 1,10-phen also have DNA binding and cleaving
ability [29,30] while planar geometry of 1,10-phen facilitates the in-
tercalative interaction with DNA [10,31]. Biological properties of 2,2′-
bipy and its complexes have been little investigated. In this study, 1,10-
phenanthroline and 2,2′-bipyridine were tested under the same condi-
tions as complexes. The IC50 values of 1,10-phen and 2,2′-bipy against
Hep-2 cell line were 12,1 ± 0,2 and 6,9 ± 0,9 µM respectively.
Moreover, diimine ligands exhibit strong induction of apoptosis
(30–40%) in Hep-2 cancer cells in comparison with complexes (Fig. 4C
and D).
Cytotoxicity was not observed at tested concentrations (1–50 μM,
72 h) after exposing cells to water solutions of Cu(OAc)2 and CuCl2.
These results are in agreement with those reported in the literature
[32,33]. Li et al. studied the cell viabilities of different concentrations
of Cu(OAc)2 and CuCl2 against HaCaT (Human adult low Calcium high
Temperature) keratinocytes by MTT assay. The cytotoxic effects of
these copper compounds on HaCaT cells were observed at concentra-
tion > 580 μM after 48 h of incubation.
[Cu(2,2′-bipy)Cl2] and [Cu(1,10-phen)Cl2] complexes were pre-
pared according to the literature procedure [34] and used as a com-
parison. The IC50 values of these two complexes were in the low mi-
cromolar range and comparable to the results reported in the literature
(IC50 < 8 µM for all tested cell lines) [35,36]. Nave et al. have reported
that [Cu(1,10-phen)Cl2] showed potent cytotoxic effect toward dif-
ferent tumor cell lines studied by MTS assay. They also incorporated
complex in long circulating liposomes and showed that it kept the cy-
totoxic properties [37].
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Inorganica Chimica Acta 487 (2019) 138–144
Ligand HL1 showed negligible cytotoxic activity against Hep-2 cell
line. As can be seen in Fig. 4A treatment of Hep-2 cells for 48 h with HL1
initiated death no > 20% of cells after incubation with highest tested
compound concentration (50 µM) (Fig. 4A). The 48-h incubation with
[Cu2(2,2′-bipy)2(L1)4] had stronger effect on Hep-2 cells: 19% and 29%
of dead cells after incubation with 25 and 55 µM of the drug respec-
tively, while the percentage of apoptosis was 26–30%. Exposure of Hep-
2 cells to [Cu2(1,10-phen)2(L1)4] for 48 h was more potent than effect of
[Cu2(2,2′-bipy)2(L1)4] and initiated cell death at a level 96% already at
5 µM concentration of drug (Fig. 4E and F). The cytotoxic effect of the
complex [Cu2(1,10-phen)2(L2)4] with the nontoxic ligand HL2 on Hep-2
cells appears at concentration of 5 μM: the percentage of dead cells
increases to 28%. At the same time, for the complex [Cu(2,2′-bipy)
(L2)2]n activity was observed only against the MCF-7 cell line. More-
over, our investigation revealed that MCF-7 cell line was significantly
more sensitive to 2,2′-bipyridine containing complexes 1, 3 than Hep-2
cell line. High level of apoptosis (30–40%) was observed while treating
MCF-7 cells with [Cu2(1,10-phen)2(L1)4] and Hep-2 cells with
[Cu2(2,2′-bipy)2(L1)4] (Fig. 4B and E).
The half maximal inhibitory concentration (IC50) was calculated
from curves constructed by plotting cell survival (%) versus drug con-
centration (µM). Obtained IC50 values are summarized in Table 4. The
IC50 value of [Cu(2,2′-bipy)Cl2] was 34,7 μM, thus, replacement of
chloride-ion with nontoxic ligand HL1 in [Cu2(2,2′-bipy)2(L1)4] caused
negligible reduction of cytotoxic activity (IC50 = 40,1 μM). Complex
[Cu2(1,10-phen)2(L1)4] displayed the highest potency in killing Hep−2
cells with an IC50 value of 2,7 μM. Compared to the cytotoxicity of [Cu
(1,10-phen)Cl2] (IC50 = 3,03 μM), presence of ligand HL1 in [Cu2(1,10-
phen)2(L1)4] practically did not influence the IC50 value. The same
pattern was observed for mixed-ligand complexes with HL2.
Several trends were revealed from the cytotoxicity screening.
Firstly, cytotoxicity of the resulting compounds was apparently related
to the presence of 1,10-phenanthroline and 2,2′-bipyridine in com-
plexes. The IC50 values of complexes remained approximately at the
same level as IC50 values of toxic ligands (1,10-phen, 2,2′-bipy). Thus,
insertion of HL1 and HL2 had a slight effect on cytotoxic activity of
complexes. However, it should be noted that complexation of copper
acetate(II) with 1,10-phen and nontoxic ligands HL1 and HL2 led to the
formation of complexes with a bit higher cytotoxicity than 1,10-phen.
However, this pattern was not observed for 2,2′-bipy and its complexes.
Secondly, treatment of cells with 1,10-phen and 2,2′-bipy led to fairly
high level of apoptosis (20–40%), while the percentage of apoptotic
cells decreased after exposure to complexes. Cell death was primarily
due to the loss of membrane integrity without apoptosis. Thirdly,
complexes containing 1,10-phenantroline were much more toxic than
complexes containing 2,2′-bipyridine. This reduction of the cytotoxic
activity of 2,2′-bipy-complexes is possibly related to the fact that 1,10-
phenanthroline provides more planarity for its complexes and enables
their stronger intercalative interactions with DNA compared to com-
plexes with 2,2′-bipy [38].
4. Conclusions
A series of mixed-ligand copper(II) complexes with 1,10-phenan-
throline, 2,2′-bipyridine and tetrazole ligands have been synthesized
and fully characterized. All complexes possess cytotoxic activity against
Hep-2 and MCF-7 human cell lines. Cytotoxicity of the compounds was
apparently related to the presence of cytotoxic 1,10-phenanthroline and
2,2′-bipyridine in complexes. Complexes containing 1,10-phenanthro-
line were much more toxic than complexes containing 2,2′-bipyridine.
Our results indicate that apoptosis wasn’t involved in mechanism of
action of tested complexes. Cell death was primarily due to the loss of
membrane integrity without apoptosis. But treatment of cells with 1,10-
phenanthroline and 2,2′-bipyridine led to fairly high level of apoptosis
(20–40%).
J.A. Eremina et al. Inorganica Chimica Acta 487 (2019) 138–144

Fig. 4. Effect of some compounds on the viability of Hep-2 and MCF-7 cells determined by dual staining with Hoechst 33342/propidium iodide: A – HL1 (Hep-2 cell
line), B – [Cu2(1,10-phen)2(L1)4] (2) (MCF−7 cell line), C – 2,2′-bipyridine (Hep-2 cell line), D – 1,10-phenanthroline (Hep-2 cell line), E – [Cu2(2,2′-bipy)2(L1)4] (1)
(Hep−2 cell line), F – [Cu2(1,10-phen)2(L1)4] (2) (Hep-2 cell line).

Table 4 Acknowledgments
Cytotoxic activity (expressed by IC50) of the studied complexes and ligands
against Hep-2 and MCF-7 cell lines. This work was supported by the Russian Science Foundation
Compound IC50 (µM)–Hep-2 IC50 (µM)–MCF-7 (Project № 18-73-00294).
The authors thank Anna P. Zubareva and Valentina V. Ankudovich
Cu(OAc)2 > 50 – for the elemental analysis, Nina I. Alferova for the IR data, and Ilya V.
CuCl2 > 50 –
Korolkov for the X-ray phase analysis data. This work involved the use
HL1 > 50 –
[Cu2(2,2′-bipy)2(L1)4] (1) 40,1 ± 1,7 10,1 ± 5,1 of equipment from the Multi-Access Center “Proteomics” of Federal
[Cu2(1,10-phen)2(L1)4] (2) 2,7 ± 0,2 4,8 ± 0,5 Research Center of Fundamental and Translational Medicine
HL2 > 50 – (Novosibirsk, Russia).
[Cu(2,2′-bipy)(L2)2]n (3) > 50 31,9 ± 9,8
[Cu2(1,10-phen)2(L2)4] (4) 6,1 ± 0,5 3,8 ± 0,1
2,2′-Bipyridine 6,9 ± 0,9 –
1,10-Phenantroline 12,1 ± 0,2 – Appendix A. Supplementary data
[Cu(2,2′-bipy)Cl2] (5) 34,7 ± 0,4 –
[Cu(1,10-phen)Cl2] (6) 3,03 ± 0,39 – Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ica.2018.12.011.

143
J.A. Eremina et al.
References
[1] C. Winter, P. Albers, Testicular germ cell tumors: pathogenesis, diagnosis and
treatment, Nat. Rev. Endocrinol. 7 (2011) 43–53, https://doi.org/10.1038/nrendo.
2010.196.
[2] D.J. Higby, H.J. Wallace Jr., J.F. Holland, Cis-diamminedichloroplatinum (NSC-
119875): a phase I study, PMID: 4586953, Cancer Chemother. Rep. 57 (1973)
459–463.
[3] E. Cvitkovic, J. Spaulding, V. Bethune, J. Martin, W.F. Whitmore, Improvement of
cis-dichlorodiammineplatinum (NSC 119875): therapeutic index in an animal
model, Cancer 39 (1977) 1357–1361, https://doi.org/10.1002/1097-
0142(197704)39:4<1357::AID-CNCR2820390402>3.0.CO;2-C.
[4] B. Köberle, M.T. Tomicic, S. Usanova, B. Kaina, Cisplatin resistance: preclinical
findings and clinical implications, Biochim. Biophys. Acta. 1806 (2010) 172–182,
https://doi.org/10.1016/j.bbcan.2010.07.004.
[5] C. Duncan, A.R. White, Copper complexes as therapeutic agents, Metallomics 4
(2012) 127–138, https://doi.org/10.1039/c2mt00174h.
[6] T. Wang, Z. Guo, Copper in medicine: homeostasis, chelation therapy and antitumor
drug design, Curr. Med. Chem. 13 (2006) 525–537, https://doi.org/10.2174/
092986706776055742.
[7] C. Marzano, M. Pellei, F. Tisato, C. Santini, Copper complexes as anticancer agents,
Anti-Cancer Agents Med. Chem. 9 (2009) 185–211, https://doi.org/10.2174/
187152009787313837.
[8] K.G. Daniel, R.H. Harbach, W.C. Guida, Q.P. Dou, Copper storage diseases: Menkes,
Wilson's, and cancer, Front. Biosci. 9 (2004) 2652–2662, https://doi.org/10.2741/
1424.
[9] S.B. Nayak, V.R. Bhat, D. Upadhyay, S.L. Udupa, Copper and ceruloplasmin status in
serum of prostate and colon cancer patients, Indian J. Physiol. Pharmacol. 47
(2003) 108–110 PMID: 12708132 (http://europepmc.org/abstract/MED/
12708132).
[10] C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Advances in
copper complexes as anticancer agents, Chem Rev. 114 (2014) 815–862, https://
doi.org/10.1021/cr400135x.
[11] F. Mohammadizadeh, S.K. Falahati-pour, A. Rezaei, M. Mohamadi, M.R. Hajizadeh,
M.R. Mirzaei, A. Khoshdel, M.A. Fahmidekhar, M. Mahmoodi, The cytotoxicity
effects of a novel Cu complex on MCF-7 human breast cancerous cells, BioMetals 31
(2018) 233–242, https://doi.org/10.1007/s10534-018-0079-5.
[12] C. Rajarajeswari, M. Ganeshpandian, M. Palaniandavar, A. Riyasdeen,
M.A. Akbarsha, Mixed ligand copper(II) complexes of 1,10-phenanthroline with
tridentate phenolate/pyridyl/(benz)imidazolyl Schiff base ligands: covalent vs non-
covalent DNA binding, DNA cleavage and cytotoxicity, J. Inorg. Biochem. 140
(2014) 255–268, https://doi.org/10.1016/j.jinorgbio.2014.07.016.
[13] L. Ruiz-Azuara, E.M. Bravo-Gomez, Copper compounds in cancer chemotherapy,
Curr. Med. Chem. 17 (2010) 3606–3615, https://doi.org/10.2174/
092986710793213751.
[14] M.E. Bravo-Gómez, J.C. García-Ramos, I. Gracia-Mora, L. Ruiz-Azuara,
Antiproliferative activity and QSAR study of copper(II) mixed chelate [Cu(N–N)
(acetylacetonato)]NO3 and [Cu(N–N)(glycinato)]NO3 complexes (Casiopeínas®), J.
Inorg. Biochem. 103 (2009) 299–309, https://doi.org/10.1016/j.jinorgbio.2008.
10.006.
[15] (a) V.A. Ostrovskii, R.E. Trifonov, E.A. Popova, Medicinal chemistry of tetrazoles,
Russ. Chem. Bull. 61 (2012) 768–780, https://doi.org/10.1007/s11172-012-0108-
4;
(b) E.A. Popova, A.V. Protas, R.E. Trifonov, Tetrazole derivatives as promising
anticancer agents, Anticancer. Agents Med. Chem. 17 (2018) 1856–1868, https://
doi.org/10.2174/1871520617666170327143148.
[16] V.A. Ostrovskii, E.A. Popova, R.E. Trifonov, Developments in tetrazole chemistry
(2009–16), Adv. Heterocyclic. Chem. 123 (2017) 1–62, https://doi.org/10.1016/
bs.aihch.2016.12.003.
[17] Z.-H. Shao, J. Luo, R.-F. Cai, X.-G. Zhou, L.-H. Weng, Z.-X. Chen, Bis(μ-5-phe-
nyltetrazolate-k2N2:N3)bis[(2,2'-bipyridine-k2N, N')bis(5-phenyltetrazolate-kN2)
copper(II)], Acta Crystallogr. 60 (2004) 225–227, https://doi.org/10.1107/
S1600536804001345.
[18] M. Saha, K.M. Vyas, L.M.D.R.S. Martins, N.M.R. Martins, A.J.L. Pombeiro,
S.M. Mobin, D. Bhattacherjee, K.P. Bhabak, S. Mukhopadhyay, Copper(II) tetra-
zolato complexes: role in oxidation catalysis and protein binding, Polyhedron 132
(2017) 53–63, https://doi.org/10.1016/j.poly.2017.04.016.
[19] CrysAlisPro 1.171.38.41. Rigaku Oxford Diffraction. 2015.
[20] G.M. Sheldrick, SHELXT – Integrated space-group and crystal-structure determi-
nation, Acta Crystallogr. A71 (2015) 3–8, https://doi.org/10.1107/
S2053273314026370.
144
Inorganica Chimica Acta 487 (2019) 138–144
[21] G.M. Sheldrick, Crystal structure refinement with SHELXL, Acta Cryst. C 71 (2015)
3–8, https://doi.org/10.1107/S2053229614024218.
[22] A.V. Artem'ev, J.A. Eremina, E.V. Lider, O.V. Antonova, E.V. Vorontsova,
I.Yu. Bagryanskaya, Luminescent Ag(I) scorpionates based on tris(2-pyridyl)phos-
phine oxide: synthesis and cytotoxic activity evaluation, Polyhedron 138 (2017)
218–224, https://doi.org/10.1016/j.poly.2017.09.041.
[23] M. Brela, J. Stare, G. Pirc, M. Sollner-Dolenc, M. Boczar, M.J. Wójcik, J. Mavri, Car-
parrinello simulation of the vibrational spectrum of a medium strong hydrogen
bond by two-dimensional quantization of the nuclear motion: application to 2-
Hydroxy-5-nitrobenzamide, J. Phys. Chem. 116 (2012) 4510–4518, https://doi.
org/10.1021/jp2094559.
[24] L.I. Yudanova, V.A. Logvinenko, L.A. Sheludyakova, N.F. Yudanov,
P.P. Semyannikov, S.I. Kozhemyachenko, I.V. Korol’kov, N.A. Rudina,
A.V. Ishchenko, Maleates of Mn(II), Fe(II), Co(II), and Ni(II) as precursors for
synthesis of metal-polymer composites, Russ. J. Inorg. Chem. 59 (2014) 1180–1186,
https://doi.org/10.1134/S0036023614100222.
[25] M.B. Bushuev, B.A. Selivanov, N.V. Pervukhina, D. Naumov, Sheludyakova, L.A. Yu,
M.I. Rakhmanova, A.Ya. Tikhonov, S.V. Larionov, Zinc(II) complexes with an imi-
dazolylpyridine ligand: luminescence and hydrogen bonding, J. Coord. Chem. 67
(2014) 611–622, https://doi.org/10.1080/00958972.2014.892589.
[26] B. Wermuth, U. Brodbeck, Inhibition of acetylcholinesterase activity by aromatic
chelating agents, Eur. J. Biochem. 35 (1973) 499–506, https://doi.org/10.1111/j.
1432-1033.1973.tb02865.x.
[27] T.A. Day, G.Z. Chen, The metalloprotease inhibitor 1,10-phenanthroline affects
Schistosoma mansoni motor activity, egg laying and viability, Parasitology 116
(1998) 319–325 PMID: 9585934 (https://www.ncbi.nlm.nih.gov/pubmed/
9585934).
[28] A. Gil, M. Melle-Franco, V. Branchadell, M.J. Calhorda, How the intercalation of
phenanthroline affects the structure, energetics, and bond properties of DNA base
pairs: theoretical study applied to adenine-thymine and guanine-cytosine tetramers,
J. Chem. Theory Comput. 11 (2015) 2714–2728, https://doi.org/10.1021/
ct5006104.
[29] P.R. Chetana, R. Rao, S. Saha, R.S. Policegoudra, P. Vijayan, M.S. Aradhya,
Oxidative DNA cleavage, cytotoxicity and antimicrobial studies of L-ornithine
copper (II) complexes //, Polyhedron 48 (2012) 43–50, https://doi.org/10.1016/j.
poly.2012.08.081.
[30] R. Galindo-Murillo, J.C. García-Ramos, L. Ruiz-Azuara, T.E. Cheatham, F. Cortés-
Guzmán, Intercalation processes of copper complexes in DNA, Nucleic Acids Res. 43
(2015) 5364–5376, https://doi.org/10.1093/nar/gkv467.
[31] M. McCann, A.L.S. Santos, B.A. Silva da, M.T.V. Romanos, A.S. Pyrrho,
M. Devereux, K. Kavanagh, I. Fichtnerg, A. Kelletth, In vitro and in vivo studies into
the biological activities of 1,10-phenanthroline, 1,10-phenanthroline-5,6-dione and
its copper(II) and silver(I) complexes, Toxicol. Res. 1 (2012) 47–54, https://doi.
org/10.1039/C2TX00010E.
[32] H. Li, P.Z. Toh, J.Y. Tan, M.T. Zin, C.-Y. Lee, B. Li, M. Leolukman, H. Bao, L. Kang,
Selected biomarkers revealed potential skin toxicity caused by certain copper
compounds, Sci. Rep. 6 (2016) 37664–37675, https://doi.org/10.1038/srep37664.
[33] A. Semisch, J. Ohle, B. Witt, A. Hartwi, Cytotoxicity and genotoxicity of nano – and
microparticulate copper oxide: role of solubility and intracellular bioavailability,
Part Fibre Toxicol. 10 (2014) 11, https://doi.org/10.1186/1743-8977-11-10.
[34] M. Boltz, A. Blanc, G. Laugel, P. Pale, B. Louis, Heterogenization of [Cu(2,2′-
bpy)Cl2] and [Cu(1,10-phen)Cl2] on polyoxometalates: new catalysts for the se-
lective oxidation of tetralin, Chin. J. Catal. 32 (2011) 807, https://doi.org/10.
1016/S1872-2067(10)60239-2.
[35] T. Hara, H. Matsuzaki, T. Nakamura, E. Yoshida, T. Ohkubo, H. Maruyama,
C. Yamamoto, S. Saito, Kaji T., Cytotoxicity of zinc, copper and rhodium complexes
with 1,10-phenanthroline or 2,9-dimethyl-1,10-phenanthroline in cultured vascular
endothelial cells, Fundam. Toxicol. Sci. 3 (2016) 109–113, https://doi.org/10.
2131/fts.3.109.
[36] M.L. Low, C.W. Chan, P.Y. Ng, I.H. Ooi, M.J. Maah, S.M. Chye, K.W. Tan, S.W. Ng,
C.H. Ng, Ternary and binary copper(II) complexes: synthesis, characterization, ROS-
inductive, proteasome inhibitory, and anticancer properties, J. Coord. Chem. 70
(2017) 223–241, https://doi.org/10.1080/00958972.2016.1260711.
[37] M. Nave, R.E. Castro, C.M.P. Rodrigues, A. Casini, G. Soveral, M.M. Gaspar,
Nanoformulations of a potent copper-based aquaporin inhibitor with cytotoxic ef-
fect against cancer cells, Nanomedicine (Lond.) 11 (2016) 1817–1830, https://doi.
org/10.2217/nnm-2016-0086.
[38] A. Meenongwa, R.F. Brissos, C. Soikum, P. Chaveerach, P. Gamez, Y. Trongpaniche,
U. Chaveerach, Effects of N, N-heterocyclic ligands on the in vitro cytotoxicity and
DNA interactions of copper(II) chloride complexes from amidino-O-methylurea li-
gands, New J. Chem. 40 (2016) 5861–5876, https://doi.org/10.1039/C5NJ03439F.