Synthesis, characterization, and biological evaluation of new tetrazole-based platinum(II) and palladium(II) chlorido complexes - Potent cisplatin analogues and their trans isomers

Article abstract of DOI:10.1016/j.jinorgbio.2012.12.001

Two series of tetrazole-containing platinum(II) and palladium(II) chlorido complexes, trans-[ML₂Cl₂] (M = Pt, Pd) and cis-[PtL ₂Cl₂]·nH₂O (n = 0, 1), where L is 1- or 2-substituted 5-aminotetrazole, h

Full text of DOI:10.1016/j.jinorgbio.2012.12.001

Journal of Inorganic Biochemistry 120 (2013) 44–53
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis, characterization, and biological evaluation of new tetrazole-based
platinum(II) and palladium(II) chlorido complexes — Potent cisplatin analogues and
their trans isomers
b
b
c
Tatiyana V. Serebryanskaya a, , Tatiana Yung , Alexey A. Bogdanov , Andrei Shchebet ,
Steven A. Johnsen c,d, Alexander S. Lyakhov a, Ludmila S. Ivashkevich a, Zhanna A. Ibrahimava e,
Tatiyana S. Garbuzenco e, Tatiyana S. Kolesnikova e, Natalya I. Melnova e,
Pavel N. Gaponik a, Oleg A. Ivashkevich a
⁎
a
Research Institute for Physical Chemical Problems, Belarusian State University, Leningradskaya 14, 220030, Minsk, Belarus
Department of Molecular Biophysics, Faculty of Physics, St. Petersburg State University, Ulyanovskaya 1, 198504 St. Petersburg, Russian Federation
Department of Molecular Oncology, Göttingen Center for Molecular Biosciences, University of Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany
Center of Experimental Medicine, Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany
b
c
d
e
Scientific and Production Republican Unitary Enterprise “LOTIOS”, Z. Byaduli 10, 220034 Minsk, Belarus
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 July 2012
Received in revised form 21 October 2012
Accepted 5 December 2012
Available online 13 December 2012
Two series of tetrazole-containing platinum(II) and palladium(II) chlorido complexes, trans-[ML2Cl2] (M=Pt,
Pd) and cis-[PtL2Cl2]·nH2O (n=0, 1), where L is 1- or 2-substituted 5-aminotetrazole, have been synthesized
and thoroughly characterized. Configuration of platinum(II) complexes obtained from the reaction of
5-aminotetrazoles with K2PtCl4 has been found to vary depending on the nature of tetrazole derivatives and
reaction conditions. According to in vitro cytotoxic evaluation, only platinum complexes display noticeable
antiproliferative effect, and their cytotoxicity depends strongly on their geometry and hydrophobicity of the
carrier ligands. The most promising complexes are cis-[Pt(1-apt)2Cl2]·H2O and cis-[Pt(2-abt)2Cl2]·H2O, where
1-apt is 5-amino-1-phenyltetrazole and 2-abt is 5-amino-2-tert-butyltetrazole. In comparison with cisplatin, they
show comparable cytotoxic potency against cisplatin-sensitive human cancer cell lines, cis-[Pt(2-abt)2Cl2]·H2O
performing substantially higher activity against cisplatin-resistant cell lines. Cell cycle studies in H1299 cell line
indicated that cis-[Pt(2-abt)2Cl2]·H2O induced apoptosis launched from G2 accumulations. The DNA interaction
with cis-[Pt(1-apt)2Cl2]·H2O was followed by UV spectroscopy, circular dichroism, hydrodynamic and electro-
phoretic mobility studies. Both cis-[Pt(1-apt)2Cl2]·H2O and cis-[Pt(2-abt)2Cl2]·H2O complexes appeared to be
significantly less toxic than cisplatin in mice, while only compound cis-[Pt(1-apt)2Cl2]·H2O displayed noticeable
efficacy in vivo.
Keywords:
5-Aminotetrazoles
Platinum(II) complexes
Palladium(II) complexes
Cisplatin analogues
DNA interaction
Antitumor activity
© 2012 Elsevier Inc. All rights reserved.
1. Introduction
[20–24], quinoline and isoquinoline [6]. Biological activity of platinum
complexes with polynitrogen heterocycles, particularly tetrazoles and
Platinum complexes with nitrogen-containing heterocycles are of
great interest as potent antitumor agents. The spatial hindrance
caused by these ligands has been shown to enhance activity of both
cis and trans platinum complexes [1–4]. Moreover, an application of
N-containing heterocycles appeared to be efficient in designing active
palladium-based compounds [5].
A great number of cis platinum complexes as well as their trans
analogues with different heterocycles have been investigated up to
date. Most of the studies in this area were concerned with the
complexes of pyridine or its derivatives [6–15]. Among other consid-
ered heterocycles were imidazoles [15–19], thiazole [6], pyrazoles
triazoles, is considerably less studied. For example, Bekhit et al. reported
the antitumor activity of platinum(II) complexes of tetrazolo[1,5-a]
quinolines [25]. More recently, two tetrazolato-bridged binuclear
platinum(II) complexes were shown to be considerably more active
than their triazolato- and pyrazolato-analogues when studied against
H460 human NSCLC cell line, and one of them appeared to be non-
cross-resistant with cisplatin in vitro and low-toxic in vivo [26]. It should
be noted that the ability of tetrazole moiety to decrease toxicity of
modified medicines is well known and, owing to this fact, it is widely
used as bioisoster of different biologically active groups [27,28]. Due
to variety of possible substituents, N-mono- and C,N-disubstituted
tetrazoles are promising carrier ligands for designing new potential
antitumor agents that are able to overcome the most common draw-
backs of convenient platinum drugs such as high toxicity, low solubility
⁎
Corresponding author. Tel.: +375 17 2095198; fax: +375 17 2264696.
E-mail address: serebryanskaya.t@gmail.com (T.V. Serebryanskaya).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.12.001

T.V. Serebryanskaya et al. / Journal of Inorganic Biochemistry 120 (2013) 44–53
45
2 equiv of the nitrogen-containing nucleophiles is considered to be a
common method to prepare cisplatin analogues [30]. In spite of this
fact, potassium tetrachloridoplatinate was found to react with 2 equiv
of 5-aminotetrazoles 1a, 1b, and 1d to give trans-isomeric products or
inseparable mixtures of both isomers, and only for tetrazole 1c
this method afforded to obtain cis-isomeric product. Therefore, cis
platinum(II) chlorido complexes with substituted 5-aminotetrazoles
were prepared by direct reaction of 2 equiv of the tetrazole derivative
(1a–1d) with K2PtCl4 in 1 M aqueous solution of HCl. Cis platinum
complexes of 5-aminotetrazoles 1a, 1b, and 1d were isolated as
monohydrates. It is noteworthy that formation of trans platinum
complexes or mixtures of cis and trans isomers under the reaction of
K2PtCl4 with nitrogen-containing nucleophiles has been previously
described for pyrazoles [31–38], 2-nitroimidazoles [39,40], 3- and
4-acetylpyridines [41], 1- and 2-adamantylamines [42], anilines [43]
etc. Addition of HCl to the reaction mixture was earlier used to improve
the yield and purity of pyrazole-based cisplatin analogues [31–35].
Although no explanation was suggested in those papers to clarify how
the presence of HCl in solution could promote formation of cis-
isomeric products, it is likely that an excess of chloride ions in the reac-
tion mixture leads to a decrease in solubility of cis platinum complexes,
so that it accelerates their precipitation and prevents the process of
isomerization in solution.
Platinum(II) complexes trans-[PtL2Cl2] (L=1b–1d) were synthesized
by reaction of tetrazole L with platinum(II) chloride (PtCl2) in toluene/
THF mixture. Alternatively, complexes trans-[PtL2Cl2] (L=1a, 1d) were
obtained by reaction of K2PtCl4 with free 5-aminotetrazoles in neutral
aqueous media, as mentioned above, or by cis/trans isomerization
occurring under recrystallization of corresponding cis forms from hot
acetonitrile. It is noteworthy that cis/trans isomerization under similar
conditions, e.g. under heating in organic solvents, has been previously
reported for cis platinum(II) chlorido complexes of 5-nitroimidazoles
[40,44], pyrazoles [31–35], and cyclobutylamine [45]. The similarities in
solution behavior of platinum(II) complexes of pyrazoles, nitroimidazoles
and N-substituted tetrazoles may be caused by low basicity of these
heterocycles, as suggested earlier [39], or by spatial hindrance they
induce in the structure of cis-isomeric compound [40].
Fig. 1. Structures of 1- and 2-substituted 5-aminotetrazoles employed as ligands.
and tumor resistance. Despite that, at the beginning of our study, there
were no examples in the literature dealing with antitumor activity of
cisplatin analogues containing N-substituted tetrazoles as carrier
ligands. Moreover, synthesis and structure of platinum complexes with
these ligands were insufficiently explored to carry out their cytotoxic
studies.
Recently, trans palladium(II) and platinum(II) chlorido complexes
with 5-amino-2-tert-butyltetrazole have been synthesized and structur-
ally characterized [29]. The platinum complex has been shown to
possess promising cytotoxic activity against HeLa tumor cells, the IC50
for this compound being average between cisplatin and carboplatin.
The present study was initiated to extend the knowledge on the reactiv-
ity of tetrazole-containing ligands towards platinum and palladium salts
and biological activity of the tetrazole-based cisplatin analogues and
their trans isomers. In the present paper, we report the synthesis of
twelve platinum(II) and palladium(II) chlorido complexes with 1-
and 2-substituted 5-aminotetrazoles 1a–1d (Fig. 1). Physico-chemical
properties of the complexes were thoroughly investigated, and their
antitumor activity in vitro and in vivo was characterized.
2. Results and discussion
2.1. Synthesis and characterization
All complexes were characterized by elemental analysis, APCI
The complexes trans-[PdL2Cl2] were prepared by the reaction of
tetrazoles 1a–1d with palladium(II) chloride (PdCl2) dissolved in 1 M
HCl as described previously [29].
General procedures for the synthesis of the cis and trans platinum(II)
chlorido complexes with 5-aminotetrazoles 1a–1d are depicted in
Fig. 2. The reaction of potassium tetrachloridoplatinate (K2PtCl4) with
mass spectrometry, and IR spectroscopy in the range 4000–50 cm−1
.
Both cis and trans platinum(II) complexes were also studied using 1H
and 13C NMR spectroscopy, and their purity was ascertained by HPLC.
Under APCI conditions, the fragmentation of palladium and platinum
complexes, dissolved in CH3CN, proceeded through the formation
of ionic species [ML2(CH3CN)Cl]+, where M=Pd, Pt and L=5-
aminotetrazole 1a–1d, in agreement with previous results for other
platinum complexes with N-heterocyclic ligands [9,46,47].
The assignment of the bands in the IR spectra was based on previous
works on vibrational spectra of N-substituted 5-aminotetrazoles [48]
and their silver(I) [49], platinum(II), and palladium(II) complexes [29]
as well as platinum(II) complexes with other N-containing heterocycles
[50]. The spectra of cis platinum compounds showed more bands in
comparison with their trans isomers (Table S1). Noteworthy, not only
the bands assigned to coordination unit's stretching vibrations ν(Pt–N)
{250–300 сm−1} and ν(Pt–Cl) {320–345 cm−1}, but also the bands cor-
responding to stretching-deformation ν(С–NH2)+δ(NH2), deformation
δ(N–H), rocking ρ(NH2) {1100–1150 cm−1} and mixed ν(Pt–N)+γtz
{450–500 сm−1} vibrations were found to split or broaden in the
spectra of cis forms. The appearance of additional bands in the mid-
IR spectra of cis isomers can be caused by their lower symmetry, in
agreement with previous data on splitting of the δ(N–H) bands in
the vibrational spectra of cisplatin [51] and cis isomeric cobalt(III)
ethylendiamine complexes [52–54].
In the 1H NMR spectra of platinum complexes, the most character-
istic NH2 peaks undergo large downfield shifts with respect to those
of the free 5-aminotetrazoles 1a–1d (Table 1). This can be explained
by an electron density withdraw after coordination binding resulting
Fig. 2. Synthesis of platinum(II) and palladium(II) chlorido complexes with 5-
aminotetrazoles 1a–1d.

46
T.V. Serebryanskaya et al. / Journal of Inorganic Biochemistry 120 (2013) 44–53
Table 1
Table 2
1H and 13C shifts in the NMR spectra of cis and trans platinum(II) chlorido complexes
with 5-aminotetrazoles 1a–1d.
Selected bond lengths (Å) in the crystal structure of
trans-[Pd(1-amt)2Cl2].
Compound
1H NMR
13C NMR
N1–N2
N2–N3
N3–N4
N1–C5
N4–C5
C5–Namino
Pd–N4
1.362 (3)
1.282 (3)
1.358 (3)
1.343 (3)
1.334 (3)
1.327 (3)
2.009 (2)
2.009 (2)
2.2839 (12)
2.2970 (12)
⁎
δ, ppm
Δδ
δ, ppm
Δδ
trans-[Pt(1-amt)2Cl2]
cis-[Pt(1-amt)2Cl2]·H2O
trans-[Pt(1-apt)2Cl2]
cis-[Pt(1-apt)2Cl2]·H2O
trans-[Pt(2-amt)2Cl2]
cis-[Pt(2-amt)2Cl2]
6.32
6.40
6.49
6.59
5.73
5.83
5.73
5.79
1.1
152.53
154.88
153.9
154.3
165.8
165.8
165.2
165.6
−4.04
−0.92
−1.9
−1.5
−1.6
−1.6
−2.1
−1.7
1.18
1.02
1.12
0.96
1.06
0.95
1.01
Pd–Cl
trans-[Pt(2-abt)2Cl2]
cis-[Pt(2-abt)2Cl2]·H2O
⁎
Δδ (ppm)=δcomplex −δligand
.
bonds lie in a narrow range of 1.334(3)–1.362(3) Å. The tetrazole ring
is planar within 0.0020 (17) Å. In the complex molecule, the planes of
the tetrazole rings are inclined at ca 59° with respect to the coordination
plane. H atoms of the amino groups are involved in hydrogen bonds
(Table S2) with Cl1 and N2 atoms of neighboring complex molecule,
forming a three-dimensional network. Crystal packing of trans-[Pd(1-
amt)2Cl2] is shown in Fig. S1.
in an increase in the N–H acid character of 5-aminogroup. On the
contrary, coordination leads to the upfield shift of C(5) signals in com-
parison with the 13C NMR spectra of free 5-aminotetrazoles. However,
signals of NH2 protons and C(5) carbons were found to be more
deshielded in the spectra of cis isomers than in those of their trans con-
geners, in consistence with previous results for platinum complexes
with other N-containing heterocycles [31–34,55–59].
2.3. Lipophilicity
The geometry of the complexes was assigned on the basis of IR spec-
troscopy and X-ray data, and supported by HPLC and NMR spectroscopy.
The lipophilicity of tetrazole-based platinum complexes was evalu-
ated as log P values using the shake-flask method as described earlier
[66]. Weighted amounts of complexes were partitioned between an
aqueous and 1-octanol phases. Platinum concentrations in the aqueous
phase were measured by atomic absorption spectrometry. Based on
these results, the partition coefficients were calculated (Table 3).
All complexes turned out to be more lipophilic than cisplatin and
carboplatin, but only complexes based on the tetrazole derivatives
with large organic substituents like tert-butyl or phenyl possess positive
log P values. Lipophilicity of the trans complexes is higher than that of
their cis isomers.
2.2. Description of crystal structures
Earlier [29], we determined molecular and crystal structures of
trans-[Pd(2-abt)2Cl2] (monoclinic and triclinic crystal forms) and
trans-[Pt(2-abt)2Cl2]. The structure of cis-[Pt(2-amt)2Cl2] was solved
from powder X-ray diffraction data [60]. All attempts to prepare sin-
gle crystals of cis-isomeric products failed due to their easy isomeriza-
tion under recrystallization. It should be noted that, before our study
was started, only a few crystal structures of palladium(II) chlorido com-
plexes with neutral N-substituted tetrazoles were available in the liter-
ature [61–64]. For platinum analogues, crystal data were limited only to
platinum(IV) chlorido complex with 1-methyltetrazole [65]. So,
cis-[Pt(2-amt)2Cl2] was the first cis platinum(II) complex with neutral
N-substituted tetrazole which crystal structure was established by
X-ray analysis. Moreover, determination of its structure let us to
confirm the assignment of synthesized platinum complexes to cis and
trans isomers.
Here we report molecular and crystal structure of trans-[Pd(1-
amt)2Cl2]. The complex crystallizes in the monoclinic space group C2/c,
with four formula units per unit cell. The compound presents a molecular
complex (Fig. 3). Complex molecule reveals C2 symmetry, with the Pd,
Cl1, and Cl2 atoms being on two-fold axis. Coordination unit PdCl2N2 is
slightly distorted square, with all atoms lying in the plane. The tetrazole
ring bond lengths are in expected ranges (Table 2). The shortest bond is
formally double bond N(2)–N(3) of 1.282(3) Å. Other tetrazole ring
2.4. In vitro cytotoxic activity
Antiproliferative activity of N-substituted 5-aminotetrazoles and
their platinum(II) and palladium(II) complexes in vitro against human
cervical carcinoma cell line (HeLa) was assessed by trypan blue test.
Cisplatin and carboplatin were added to the test as positive control.
5-Aminotetrazoles 1a–1d and their palladium(II) chlorido complexes
appeared to be inactive against HeLa cell line, with IC50 values exceeding
100 μM. Cytotoxic effects of platinum complexes are given in Table 3. As
seen, cis platinum complexes are more cytotoxic than their trans conge-
ners. Moreover, cytotoxicity of tetrazole-containing complexes non-
linearly depends on their lypophilicity, so that cis-[Pt(2-abt)2Cl2] with
log P value close to 0 showed the highest cytotoxicity.
Cytotoxicity of the most active tetrazole-based complexes cis-[Pt(1-
apt)2Cl2]·H2O, cis-[Pt(2-abt)2Cl2]·H2O, and trans-[Pt(2-abt)2Cl2] was
tested against human cancer cell lines with different sensitivity to
Cl1
Table 3
Lipophilicity and cytotoxic activity against HeLa cell line (trypan blue assay) of
tetrazole-containing platinum(II) chlorido complexes in comparison with cisplatin
and carboplatin.
N3
N2
N4
Pd1
Compound
Log P
IC50, μM
C6
trans-[Pt(1-amt)2Cl2]
cis-[Pt(1-amt)2Cl2]·H2O
trans-[Pt(2-amt)2Cl2]
cis-[Pt(2-amt)2Cl2]
trans-[Pt(2-abt)2Cl2]
cis-[Pt(2-abt)2Cl2]·H2O
trans-[Pt(1-apt)2Cl2]
cis-[Pt(1-apt)2Cl2]·H2O
Cisplatin
−0.94
−1.15
−1.17
−1.01
0.40
−0.05
1.06
0.39
>50
40
45
C5
N1
33
N7
5.6 [29]
0.9
50
1.3
0.7
Cl2
−2.27 [67]
−1.63 [67]
Fig. 3. Complex molecule of trans-[Pd(1-amt)2Cl2], with atom labeling scheme for the
asymmetric unit and displacement ellipsoids at the 50% probability level.
Carboplatin
12.5

T.V. Serebryanskaya et al. / Journal of Inorganic Biochemistry 120 (2013) 44–53
47
Table 4
Cytotoxic activity of tetrazole-containing platinum(II) chlorido complexes against
cisplatin-sensitive and cisplatin-resistant cell lines complexes in comparison with
cisplatin.
Compound
IC50, μМ
HeLa
RD
U2OS H1299 А2780 А2780cisR
(RF)
cis-[Pt(1-apt)2Cl2]·H2O 15.9 0.2 10.1 0.4 17.1
64.2
5.4
64.8
21.8
n.d.
8.4
n.d.
6.2
n.d.
15.2 (1.8)
n.d.
cis-[Pt(2-abt)2Cl2]·H2O
trans-[Pt(2-abt)2Cl2]
Cisplatin
8.4 0.2 19.5 0.4 n.d.a
58.6 0.3 20.2 0.2 27.4
7.9 0.2 20.7 0.2 3.6
37.1 (6.0)
a
n.d. — not determined.
cisplatin and measured by МТТ- and MTS-based assays. According to
Table 4, cis-[Pt(1-apt)2Cl2]·H2O and cis-[Pt(2-abt)2Cl2]·H2O demonstrate
cytotoxicity against cisplatin-sensitive cell lines comparable to cisplatin,
while cis-[PtCl2(2-abt)2]·H2O markedly outperforms cisplatin against
cisplatin-resistant cells (see Fig. S2). Complex trans-[Pt(2-abt)2Cl2]
shows significantly lower activity.
Fig. 5. CD spectra of CT DNA (0.003%) in the presence of different amounts of
cis-[Pt(1-apt)2Cl2]·H2O.
2.5. Biophysical studies
with small molecules, so the changes in CD spectra are commonly used
to discriminate between different modes of coordinative binding of
platinum drugs to DNA [74,75].
The following techniques have been used to probe the changes,
induced by complex cis-[Pt(1-apt)2Cl2]·H2O in DNA structure: UV
absorption and circular dichroism (CD) spectroscopy, hydrodynamic
methods (low-gradient viscometry and dynamic birefringence), and
gel electrophoresis.
The CD spectral changes of the CT DNA upon binding with
cis-[Pt(1-apt)2Cl2]·H2O under different experimental conditions are
shown in Figs. 5 and 6. With increase of platinum content, the gradual
increase in the positive band intensity is observed followed by the sub-
sequent hypochromism of this band (Fig. 5). These specific changes,
namely initial stabilization of base stacking at low drug concentration
and its following destruction with growth of drug amounts, are a
distinctive feature of the coordinative mode of DNA binding peculiar
to cisplatin and other cis platinum complexes [73,75]. This allows to
conclude that binding mode of cis-[Pt(1-apt)2Cl2]·H2O to DNA is quite
close to that of cisplatin. The negative band of the CD spectrum shows
gradual decrease in the intensity with growth of the tetrazole-based
complex concentration in the solution, indicating loss of the DNA helical
structure.
2.5.1. UV–vis titration with calf thymus DNA
The electronic absorption spectroscopy in the UV–vis range is the
useful technique to investigate the interactions of metal compounds
with bio-molecules, including DNA. The absorption spectra of CT DNA
in the presence of different amounts of complex cis-[Pt(1-apt)2Cl2]·H2O
are shown in Fig. 4. Addition of this complex to a DNA solution results in
hyperchromism and bathochromic shift of a band at 257 nm compared
to blank DNA, which is generally accepted to be consistent with coordi-
nation binding of platinum compounds to DNA [68–71].
2.5.2. Circular dichroism with calf thymus DNA
To specify the coordination site of the tetrazole-based complex to
DNA, the CD experiments at different values of acidity of DNA solutions
were performed. It is known that DNA protonation on N7 position of
guanine occurs at pH lower than 4.7 and leads to certain changes in
the CD spectrum, namely to appearance of second positive maximum
CD spectroscopy is a common technique to probe DNA conforma-
tional changes in solution [72]. A solution of the most abundant B-form
of DNA exhibits a positive band (275 nm) due to base stacking and a
negative band (245 nm) due to right-handed helicity of DNA [72,73].
The CD signals are quite sensitive to the mode of DNA interactions
Fig. 4. UV–vis absorption spectra of CT DNA (0.003%) in the presence of different
amounts of cis-[Pt(1-apt)2Cl2]·H2O (ri is a molar ratio of platinum complex to pair of
nucleotide phosphates).
Fig. 6. CD spectra of CT DNA (0.004%) for different amounts of cis-[Pt(1-apt)2Cl2]·H2O
at different pH values: 1 — ri =0, pH 6.2; 2 — ri =0, pH 4.3; 3 — ri =0.17, pH 6.04;
4 — ri =0.17, pH 4.4; 5 — ri =1, pH 5.4; 6 — ri =1, pH 4.4.

48
T.V. Serebryanskaya et al. / Journal of Inorganic Biochemistry 120 (2013) 44–53
mobility of the covalently closed circular (ccc) and open (oc) forms
of pFL44s plasmid DNA. A view of gel, obtained for this complex and
cisplatin, is shown in Fig. 7. Cisplatin binding to plasmid DNA causes
unwinding of ccc and oc forms of plasmid DNA which results in retar-
dation of ccc form and simultaneous increase in mobility of oc form, in
agreement with previous reports [72,80].
Similarly to cisplatin, a rise of content of cis-[Pt(1-apt)2Cl2]·H2O in
plasmid DNA solutions reduces electrophoretic mobility of ccc form
(Fig. 7). This indicates that both compounds cause similar changes
in tertiary structure of superhelical plasmid. However, no changes in
electrophoretic mobility of oc form were observed for DNA samples
incubated with cis-[Pt(1-apt)2Cl2]·H2O. This result may be due to
lower affinity of the tetrazole-containing complex to open circular form
of plasmid DNA in comparison with cisplatin.
Fig. 7. Agarose gel electrophoresis of pFL44s plasmid DNA after 24 hour incubation
with cis-[Pt(1-apt)2Cl2]·H2O (1 ri=2.4, 2 ri=1.6, 3 ri=1.2, 4 ri=0.8, 5 ri=0.4) and
cisplatin (cis-DDP, ri=1.3).
2.6. Cell cycle analysis
The analysis of cell cycle phase distribution was performed on
H1299 cell line treated with cisplatin or cis-[Pt(2-abt)2Cl2]·H2O in
equitoxic concentrations corresponding to IC80 (60 μM and 14 μM
respectively) for 12 h. Cells were fixed, stained with propidium iodide
and analyzed on flow cytometer. As seen from Fig. 8, high concentration
of cisplatin causes distinct block of cell cycle in G1 phase which is in
accordance with previously published data [81]. cis-[Pt(2-abt)2Cl2]·H2O
blocks cells in G2 phase, this effect being typically observed in cells treat-
ed with low concentrations of cisplatin. However, massive death of
cis-[Pt(2-abt)2Cl2]·H2O-treated cells is already detected at this point
indicating that although cells are still able to replicate DNA, they also
become more prone to apoptosis. This could be due to lower reparability
of cis-[Pt(2-abt)2Cl2]·H2O-induced lesions. Alternatively, G2 cells can be
just arrested while the cells trying to replicate the DNA undergo apopto-
sis immediately. Timecourse studies on synchronized cells are required
to clarify the mechanism of cis-[Pt(2-abt)2Cl2]·H2O cytotoxicity.
at about 255 nm [76–78]. Fig. 6 displays CD spectra of CT DNA in the
absence and in the presence of cis-[Pt(1-apt)2Cl2]·H2O at different pH
values. As seen, at pH lower than 4.7, binding of the complex to CT
DNA prevents its protonation, confirming that N7 position of guanine
is the main site of the coordination of cis-[Pt(1-apt)2Cl2]·H2O to DNA,
similarly to cisplatin.
2.5.3. Hydrodynamic studies
Hydrodynamic measurements sensitive to changes of hydrodynamic
volume of macromolecules are used as the least-ambiguous methods
to probe a binding mode of small molecules to DNA in solutions in the
absence of crystal data [71,79]. To elucidate the binding mode of cis-
[Pt(1-apt)2Cl2]·H2O to DNA, relative viscosity and relative birefringence
measurements were carried out at fixed DNA concentration by varying
the concentration of the complex. The results obtained were compared
to those of cisplatin and cis-[Pt(en)Cl2] (en is 1,2-ethylendiamine) and
shown in Figs. S3 and S4. As seen from Fig. S3, upon increasing the
amounts of the complex cis-[Pt(1-apt)2Cl2]·H2O, the relative viscosity
of CT DNA decreases up to 25% indicating the reduction of the macro-
molecules' volume similarly to the behavior of cisplatin and cis-[Pt(en)
Cl2]. The increase of platinum complexes concentration also leads to
the significant decline in relative birefringence of CT DNA solution
(Fig. S4), the effect being more pronounced for cis-[Pt(1-apt)2Cl2]·H2O
and cis-[Pt(en)Cl2], which can be explained by similar changes in tertiary
structure of DNA macromolecules upon binding with these complexes.
2.7. In vivo studies in mice bearing Erlich ascyte carcinomas
On the basis of the in vitro studies, cis-[Pt(1-apt)2Cl2]·H2O and
cis-[Pt(2-abt)2Cl2]·H2O were selected for in vivo experiments. Acute
toxicity of cisplatin and its tetrazole-based analogues were checked on
8- to 10-week-old white mongrel mice of both sexes. The complexes, at
different concentrations, were injected iv daily for 5 days or once as a sin-
gle dose, and animal weight and survival were evaluated. Toxicity studies
showed that both cis-[Pt(1-apt)2Cl2]·H2O and cis-[Pt(2-abt)2Cl2]·H2O
were nontoxic at 50 mg/kg as a single dose or 10 mg/kg per day, while
for cisplatin a maximal nontoxic dose was 2.5 mg/kg per day.
2.5.4. Electrophoretic mobility studies
The influence of cis-[Pt(1-apt)2Cl2]·H2O on the tertiary structure
of DNA was assessed by its ability to modify the electrophoretic
The antitumor efficacy of the compounds was evaluated in mice
bearing Erlich ascyte carcinoma as detailed in the Experimental section.
Briefly, mice were injected ip with 103 Erlich ascyte carcinoma cells. To
Fig. 8. Cell cycle distribution of H1299 cells treated with 60 μM of cisplatin and 14 μM of cis-[Pt(2-abt)2Cl2]·H2O for 12 h.

T.V. Serebryanskaya et al. / Journal of Inorganic Biochemistry 120 (2013) 44–53
49
Table 5
Effect of platinum complexes on the survival time in tumor-induced mice.
3.2. Instruments
1H and 13C NMR spectra were recorded on a Brucker Avance 400
spectrometer at 298 K using acetonitrile-d3 as a solvent and TMS as
an internal standard. IR spectra of the powder samples were measured
on a Nicolet Thermo Avatar 330 spectrometer using Smart Diffuse
Reflection accessory in the range of 4000–400 cm−1. Far-IR spectra of
the powdered samples were measured on a Bruker Vertex 70 in the
range of 400–50 cm−1. Single-crystal X-ray data were collected on a
Nicolet R3m diffractometer using graphite monochromated Mo Kα ra-
diation (λ=0.71073 Ǻ) at room temperature. CHN elemental analysis
was performed using a VARIO EL-elemental analyzer. Determination
of Pt and Pd content was performed using ICP AES technique on a Varian
Liberty II spectrometer.
HPLC of platinum complexes was performed on an Agilent 1200
chromatograph using a Zorbax-NH2 column eluted with H2O/MeCN
(10:90) at 30 °С. APCI-MS spectra were obtained by preparing the
samples in CH3CN and infusing into the Acсela mass spectrometer
equipped with LCQ-Fleet detector. The mass spectra were recorded
with a scan range of m/z 200–1000 for positive ions.
Compound
Dose, mg/kg
Median survival time (day)
ILS (%)
Experiment 1
Control
Cisplatin
–
20.8 1.0
23.0 2.2
7.8 0.6
–
1.25
3.75
1.25
3.75
7.50
10.0
10.6
–
a
a
cis-[Pt(1-apt)2Cl2]·Н2О
14.4 2.5
–
18.2 0.9
28.8 1.6
–
a
32.0
4.6
22.8 1.5
Experiment 2
Control
Cisplatin
–
26.1 1.4
23.5 3.3
23.3 1.0
24.8 1.3
–
–
–
–
2.5
5.0
7.5
cis-[Pt(2-abt)2Cl2]·Н2О
a
Statistically significant.
determine the therapeutic efficacy, cisplatin (1.25–3.75 mg/kg) or cis-
[Pt(1-apt)2Cl2]·H2O or cis-[Pt(2-abt)2Cl2]·H2O (1.25–10.0 mg/kg) were
injected iv. Treatment began on day 3 after tumor inoculation and was re-
peated daily for 5 days. The non-treated animals served as the control.
Each group was composed of 5 or 9 mice. The median survival and %
ILS (percentage increase in life span) of treated (T) over control (C) an-
imals [(T−100/C)−100] were calculated. As seen from Table 5, cis-
[Pt(1-apt)2Cl2]·H2O appeared to be more effective in vivo than
cisplatin. Cisplatin increased the life span of the mice by 10.6%, cis-
[Pt(1-apt)2Cl2]·H2O by 32% (Fig. 9), while cis-[Pt(2-abt)2Cl2]·H2O, de-
spite its high in vitro cytotoxic activity, showed no efficacy in vivo.
3.3. Log P determination
Log P values of these compounds were determined using a shake-
flask method [66]. Aqueous sodium chloride (0.9% w/v) and 1-octanol
(99.5%, Sigma-Aldrich) phases were saturated for 1 week. In case of
1-mat and 2-mat derivatives weighed amounts of platinum complexes
(1–2 mg) were dissolved in the aqueous phase (10 mL) and an equal
volume of saturated 1-octanol was added. Alternatively, weighed
amounts of 1-pat and 2-tbat platinum complexes (1–2 mg) were
dissolved in 10 mL of 1-octanol and an equal volume of aqueous
phase was added. The resulting solutions were mixed for 1 h. Samples
were filtered and platinum content of the aqueous phases determined
by atomic absorption spectroscopy (k=265.9 nm) using Carl Zeiss 30
AAS apparatus. The experiments were performed in triplicate. Platinum
content of 1-octanol phase was calculated as a difference between
concentrations before and after mixing of the phases. The logarithm of
the ratio of platinum concentrations in the organic and aqueous phases
was calculated to determine log P values reported in Table 3.
3. Experimental section
3.1. Materials
Potassium
tetrachloridoplatinate(II),
platinum(II)
chloride,
palladium(II) chloride dihydrate and 5-aminotetrazole were purchased
from commercial sources and used without further purification. 5-
Amino-1-methyltetrazole [82], 5-amino-2-methyltetrazole [82], 5-
amino-1-phehyltetrazole [83], and 5-amino-2-tert-butyltetrazole [84]
were synthesized as described earlier.
3.4. X-ray structure determinations
Experimental details of X-ray structure analysis for compounds
cis-[Pt(2-amt)2Cl2] [60], trans-[Pt(2-abt)2Cl2] [29], and trans-[Pd(2-
abt)2Cl2] [29] were given elsewhere. Crystal data, data collection
and refinement details for trans-[Pd(1-amt)2Cl2] are summarized in
Table S3. X-Ray data were collected on a Nicolet R3m diffractometer
using graphite monochromated Mo Kα radiation (λ=0.71073 Ǻ) at
room temperature. The structure was solved by direct methods
(SIR2004 [85]) and refined by full-matrix least-squares technique on
F2 (SHELXL [86]). All non-H atoms were refined anisotropically. H
atoms of the amino group were determined from Fourier map and
refined with constraints on N–H bond length. H atoms of the methyl
group were placed in calculated positions and refined in the “riding
model” approximation, with Uiso(H)=1.5Uiso(C). Molecular graphics
was performed with the programs PLATON [87] and ORTEP-3 for
Windows [88].
3.5. Synthesis and characterization of the platinum(II) complexes
For the synthesis of cis-[Pt(1-amt)2Cl2]·H2O, K2PtCl4 (415 mg,
1 mmol) was dissolved in 10 mL of 1 M HCl aqueous solution. A solu-
tion of 1a (198 mg, 2 mmol) in 5 mL of distilled water was added, and
the mixture was stirred for 24 h at room temperature. The yellow pre-
cipitate was collected by filtration and washed with 20 mL of distilled
water. Yield: 440 mg (91%). Found: C, 9.7; H, 2.3; N, 28.7; Pt, 40.0. Calc.
Fig. 9. In vivo therapeutic activity of cis-[Pt(1-apt)2Cl2]·Н2О (7.5 mg/kg, triangles,
dotted line) and cisplatin (1.25 mg/kg, circles, dashed line) compared to that of untreated
group (squares, solid line) of mice with EAC. The treatment is described in Experimental
section.

50
T.V. Serebryanskaya et al. / Journal of Inorganic Biochemistry 120 (2013) 44–53
for C4H12N10Cl2OPt: C, 9.95; H, 2.5; N, 29.05; Pt, 40.4%. 1H NMR
(400 MHz; CD3CN; δ/ppm): 6.4 (2H, s, NH2); 3.75 (3H, s, CH3). 13C
NMR (100 MHz; CD3CN; δ/ppm): 154.9 (C(5)); 34.15 (CH3). APCI-MS
(m/z): 470 ([Pt(1-amt)2(CH3CN)Сl]+).
(3H, s, CH3). 13C NMR (100 MHz; CD3CN; δ/ppm): 165.8 (C(5));
42.01 (CH3). APCI-MS (m/z): 469 ([Pt(2-amt)2(CH3CN)Сl]+).
trans-[Pt(2-abt)2Cl2] (triclinic crystal form) was obtained as described
earlier [29].
For the synthesis of cis-[Pt(1-apt)2Cl2]·H2O, K2PtCl4 (415 mg,
1 mmol) was dissolved in 10 mL of 1 M HCl aqueous solution. A solu-
tion of 1b (322 mg, 2 mmol) in 10 mL of ethanol was added, and the
mixture was stirred for 24 h at room temperature. The yellow precip-
itate was collected by filtration and washed with 20 mL of distilled
water. Yield: 550 mg (92%). Found: C, 27.5; H, 2.3; N, 22.9; Pt, 31.7.
Calc. for C14H16Cl2N10OPt: C, 27.7; H, 2.6; N, 23.1; Pt, 31.2%. 1H NMR
(400 MHz; CD3CN; δ/ppm): 6.59 (2H, s, NH2); 7.65–7.55 (5H, m, Ph).
13C NMR (100 MHz; CD3CN; δ/ppm): 154.3 (C(5)); 133.35, 132.0,
131.3, 126.1 (Ph). APCI-MS (m/z): 593 ([Pt(1-apt)2(CH3CN)Сl]+).
cis-[Pt(2-amt)2Cl2] was synthesized as described for cis-[Pt(1-
amt)2Cl2]·H2O starting from 5-aminotetrazole 1c. Yield: 390 mg (84%).
Found: C, 10.2; H, 2.0; N, 30.0; Pt, 42.2. Calc. for C4H10N10Cl2Pt: C,
10.35; H, 2.2; N, 30.2; Pt, 42.0%. 1H NMR (400 MHz; CD3CN; δ/ppm):
5.83 (2H, s, NH2); 4.10 (3 H, s, CH3). 13C NMR (100 MHz; CD3CN;
δ/ppm): 165.8 (C(5)); 41.86 (CH3). APCI-MS (m/z): 469 ([Pt(2-
amt)2(CH3CN)Сl]+).
cis-[Pt(2-abt)2Cl2]·H2O was synthesized by the procedure described
for cis-[Pt(1-apt)2Cl2]·H2O using 5-aminotetrazole 1d as a ligand. Yield:
205 mg (36%). Found: C, 21.4; H, 4.1; N, 24.55; Pt, 34.5. Calc. for C10H24
Cl2N10OPt: C, 21.2; H, 4.3; N, 24.7; Pt, 34.4%. 1H NMR (400 MHz; CD3CN;
δ/ppm): 5.79 (2H, s, NH2); 1.56 (9H, s, tBu). 13C NMR (100 MHz;
CD3CN; δ/ppm): 165.6 (C(5)); 67.08 (C(CH3)3); 28.72 (CH3). APCI-MS
(m/z): 554 ([Pt(2-abt)2(CH3CN)Сl]+).
For the synthesis of trans-[Pt(1-amt)2Cl2], K2PtCl4 (415 mg,
1 mmol) was dissolved in 10 mL of distilled water. A solution of 1a
(198 mg, 2 mmol) in 10 mL of distilled water was added, and the
mixture was stirred for 24 h at room temperature. The yellow precip-
itate was collected by filtration, washed with 20 mL of distilled water,
dried on air and dissolved in hot acetonitrile. After 4 days, the
light-yellow crystalline product was filtered off and dried on air.
Yield: 380 mg (86%). Found: C, 10.2; H, 2.05; N, 30.0; Pt, 41.7. Calc.
for C4H10N10Cl2Pt: C, 10.35; H, 2.2; N, 30.2; Pt, 42.0%. 1H NMR
(400 MHz; CD3CN; δ/ppm): 6.32 (2H, s, NH2); 3.78 (3H, s, CH3). 13C
NMR (100 MHz; CD3CN; δ/ppm): 152.53 (C(5)); 34.11 (CH3). APCI-
MS (m/z): 470 ([Pt(1-amt)2(CH3CN)Сl]+).
trans-[Pt(2-abt)2Cl2] (monoclinic form) was obtained as described
for trans-[Pt(1-amt)2Cl2] using ethanol to dissolve 1d. Yield: 350 mg
(64%). Found: C, 21.8; H, 3.95; N, 25.4; Pt, 35.7. Calc. for C10H22Cl2N10Pt:
C, 21.9; H, 4.05; N, 25.6; Pt, 35.6%. 1H NMR (400 MHz; CD3CN; δ/ppm):
5.73 (2H, s, NH2); 1.66 (9H, s, tBu). 13C NMR (100 MHz; CD3CN;
δ/ppm): 165.2 (C(5)); 67.26 (C(CH3)3); 28.78 (CH3). APCI-MS (m/z):
554 ([Pt(2-abt)2(CH3CN)Сl]+).
3.6. Synthesis and characterization of the palladium(II) complexes
For the synthesis of trans-[Pd(1-amt)2Cl2], PdCl2·2H2O (214 mg,
1 mmol) was dissolved in 10 mL of hot 1 M HCl aqueous solution
and the resulting solution was allowed to cool to room temperature.
A solution of 1a (198 mg, 2 mmol) in 10 mL of distilled water was
added, and the mixture was stirred 2 h at room temperature. The yel-
low precipitate was collected by filtration and washed with 20 mL of
distilled water. Single crystals of trans-[Pd(1-amt)2Cl2] suitable for
X-ray analysis were obtained by slow evaporation of the filtrate at am-
bient temperature. Yield: 365 mg (97%). Found: C, 12.6; H, 2.55; N, 37.1;
Pd, 28.2. Calc. for C4H10N10Cl2Pd: C, 12.8; H, 2.7; N, 37.3; Pd, 28.5%.
APCI-MS (m/z): 382 ([Pd(1-amt)2(CH3CN)Сl]+).
trans-[Pd(2-amt)2Cl2] was synthesized as described for trans-
[Pd(1-amt)2Cl2] starting from 5-aminotetrazole 1c. Yield: 375 mg
(99%). Found: C, 12.6; H, 2.5; N, 37.1; Pd, 28.6. Calc. for C4H10N10Cl2Pd:
C, 12.8; H, 2.7; N, 37.3; Pd, 28.5%. APCI-MS (m/z): 382 ([Pd(2-
amt)2(CH3CN)Сl]+).
trans-[Pd(1-apt)2Cl2] was synthesized by analogy with trans-[Pd(1-
amt)2Cl2] using ethanol to dissolve 1b. Yield: 435 mg (87%). Found: C,
33.55; H, 2.7; N, 27.9; Pd, 21.4. Calc. for C14H14Cl2N10Pd: C, 33.65; H,
2.8; N, 28.0; Pd, 21.4%. APCI-MS (m/z): 506 ([Pd(1-apt)2(CH3CN)Сl]+).
trans-[Pd(2-abt)2Cl2] was synthesized as described earlier [29]. Yield:
94%. Found: C, 26.0; H, 4.7; N, 30.2; Pd, 23.0. Calc. for C10H22N10Cl2Pd: C,
26.13; H, 4.82; N, 30.47; Pd, 23.15%. 1H NMR (400 MHz; CD3CN; δ/ppm):
5.30 (2H, s, NH2); 1.67 (9H, s, C(CH3)3). 13C NMR (100 MHz; CD3CN; δ/
ppm): 164.4 (C(5)); 65.25 (C(CH3)3); 28.76 (C(CH3)3). APCI-MS (m/z):
466 ([Pd(2-abt)2(CH3CN)Сl]+).
3.7. In vitro cytotoxicity
3.7.1. Trypan-blue assay
Tests were performed at N.N. Alexandrov National Cancer Centre of
Belarus, as described earlier [29]. Stock 10−2 M solutions of test com-
pounds were freshly prepared in dimethylsulfoxide (DMSO), and then
dilutions from 10−4 to 10−6 M in distilled water were made.
3.7.2. MTT assay
МТT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
was purchased from “Sigma”, USA. Stock 10−2 M solutions of test plat-
inum compounds were freshly prepared in DMSO, then 10−3 dilutions
in distilled water were made.
For the synthesis of trans-[Pt(1-apt)2Cl2], a solution of 1b (322 mg,
2 mmol) in 10 mL of THF was added to a suspension of PtСl2 (266 mg,
1 mmol) in 10 mL of toluene. The reaction mixture was stirred for 2 h
at 40–50 °C, and the resulting yellow solution was cooled to 0 °C. A
small amount of light-yellow amorphous product was obtained from
reaction mixture and washed with cold THF. Yield: 52 mg (9%).
Found: C, 28.4; H, 2.3; N, 23.7; Pt, 32.8. Calc. for C14H14Cl2N10Pt: C,
28.6; H, 2.4; N, 23.8; Pt, 33.2%. 1H NMR (400 MHz; CD3CN; δ/ppm):
6.49 (2H, s, NH2); 7.66–7,59 (5 H, m, Ph). 13C NMR (100 MHz;
CD3CN; δ/ppm): 153.9 (C(5)); 133.15, 131.9, 131.2, 126.0 (Ph).
APCI-MS (m/z): 594 ([Pt(1-apt)2(CH3CN)Сl]+).
The human cervical carcinoma cell line (HeLa) and human rhab-
domyosarcoma cell line (RD) were cultured in RPMI-1640 medium
(“Serva”, Germany) supplemented with 10% FBS, 1 mM sodium pyru-
vate, 2 mM L-glutamine, 100 u/mL penicillin, and 100 μg/mL streptomy-
cin (“Sigma”, USA) at 37 °C in an atmosphere of 95% of air and 5% CO2.
Cells were seeded in 96-well sterile plates at a density of 103 cells/well
in 100 μL of medium and were incubated for 24 h. The platinum
compounds were added to final concentrations from 0 to 250 μM in a
volume of 100 μL/well. 48 h later the MTT solution containing 150 mМ
phosphate buffer (рН 7.2) and 150 mМ saline was added into each
well to final concentration 5 mg/mL, and the plate was incubated at
37 °C in a humidified 5% CO2 atmosphere for 4 h. At the end of the incu-
bation period the medium was removed and the formazan product was
dissolved in 150 μL of DMSO. The cell viability was evaluated by mea-
surement of the absorbance at 450 nm, using an absorbance reader
“Biotek EL Χ 800G”. All experiments were made in triplicate. IC50 values
(compound concentration that produces 50% of cell growth inhibition)
were calculated using BioDataFit 1.02.
For the synthesis of trans-[Pt(2-amt)2Cl2],
a solution of 1c
(198 mg, 2 mmol) in 10 mL of THF was added to a suspension of
PtСl2 (266 mg, 1 mmol) in 10 mL of toluene. The reaction mixture
was stirred for 2 h at 40–50 °C and then cooled to rt. The resulting
light-yellow crystalline precipitate was filtered and recrystallized
from acetonitrile. Yield: 345 mg (74%). Found: C, 10.2; H, 2.0; N,
30.0; Pt, 41.6. Calc. for C4H10N10Cl2Pt: C, 10.35; H, 2.2; N, 30.2; Pt,
42.0%. 1H NMR (400 MHz; CD3CN; δ/ppm): 5.73 (2H, s, NH2); 4.23

T.V. Serebryanskaya et al. / Journal of Inorganic Biochemistry 120 (2013) 44–53
51
3.7.3. MTS assay
were added to each sample. The electrophoresis of these mixtures was
performed in agarose gel (0.8% in TAE buffer) for 3 h at 5 V/cm. Then,
the DNA was dyed with ethidium bromide solution in the same buffer.
A sample of blank pFL44s plasmid DNA was used as control. The DNA
bands were visualized with a Vilber Lourmat transilluminatior and
fixed with digital camera.
Cell death was evaluated by using a system based on the
tetrazolium compound MTS ([3-(4,5-dimethyl-2-thiazolyl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)]-2H-tetrazolium], inner salt)
which is reduced by living cells to yield a soluble formazan product that
can be detected colorimetrically. МТS (CellTiter 96® AQueous One Solu-
tion) was purchased from “Promega”, USA. Stock 10−2 M solutions of
test platinum compounds were freshly prepared in DMSO, then 10−3
dilutions in distilled water were made.
3.9. Cell cycle analysis
The human cervical carcinoma cell line (HeLa), human osteosarcoma
cell line (U2OS), human non-small-cell lung adenocarcinoma cell line
(H1299), and human ovarian tumor cell lines (A2780/A2780cisR)
were cultured in DMEM medium (Gibco) supplemented with 10% FBS,
1 mM sodium pyruvate, 100 u/mL penicillin, and 100 μg/mL streptomy-
cin (“Gentaur”, Germany) at 37 °C in an atmosphere of 95% of air and 5%
CO2. Cells were seeded in 96-well sterile plates at a density of 103 cells/
well in 50 μL of medium and were incubated for 3–4 h. The test
compounds were added to final concentrations from 0 to 200 μM in a
volume of 50 μL/well. 48 h later 20 μL of MTS solution was pipetted
into each well and the plate was incubated at 37 °C in a humidified 5%
CO2 atmosphere for 1 h. The cell viability was evaluated by measure-
ment of the absorbance at 490 nm, using an Absorbance Reader
BIOTEC EL800-PC. All experiments were made in triplicate. IC50 values
(compound concentration that produces 50% of cell growth inhibition)
were calculated using GraphPadPrism 5.
H1299 cells were treated with 60 μM of cisplatin or 14 μM of
cis-[Pt(2-abt)2Cl2]·H2O for 12 h. After trypsinization, cells were
washed with PBS and fixed with 70% ethanol overnight at 4 °C. The
next day ethanol was removed and fixed cells were resuspended in
PBS containing 40 μg/mL of propidium iodide and 0.5 μg/mL RNAse A.
After incubation for 3 h at 4 °C, cells were analyzed on GUAVA PCA-96
flow cytometer.
3.10. In vivo evaluation of toxicity and antitumor efficacy
Cancer cell lines Ehrlich ascytic carcinoma (EAC) were obtained
from Institute of Pharmacology and Biochemistry of NAS of Belarus.
The cells were maintained as ascites tumor in mice by intraperitoneal
inoculation of 106 viable cells and then used to induce cancer in
animal model (white mongrel mice).
Acute toxicity and antitumor efficacy of the tetrazole-containing
platinum complex were checked on white mongrel mice and compared
to cisplatin. Mice of both sexes, in good condition, weighing 18 to 22 g,
without signs of infection, and 8 to 10 weeks old, were included in the
experiments. For acute toxicity assessment, the tetrazole-containing
complexes and cisplatin, at different concentrations, were injected iv
in the tail vain as a single dose or daily for 5 days in a volume 0.2 mL.
For antitumor efficacy evaluation, mice were injected ip with 106
Erlich ascyte carcinoma cells. The viability of these cells was >90%
by trypan blue exclusion. Then, the tetrazole-containing complexes
and cisplatin, at different concentrations (Table 5), were injected iv
in the tail vain daily once for 5 days in a volume 0.2 mL. Treatment
began on day 3 after tumor inoculation, and animal weight was measured
in the day of inoculation and then daily for 5 days. The non-treated ani-
mals served as the control. Each group was composed of five (Experiment
1) or nine (Experiment 2) mice. The median survival time and % ILS
(percentage increase in life span) of treated (T) over control (C) animals,
(T−100/C)−100, were calculated and given in Table 5.
3.8. Biophysical assays
Interaction of complex cis-[Pt(1-apt)2Cl2]·H2O with calf thymus
(CT) DNA (“Sigma”) was studied using UV–vis absorption and circular
dichroism (CD) spectroscopy, low-gradient viscometry, dynamic
birefringence, and gel-electrophoresis. Molecular weight of CT DNA
(107 g/mol) was found from the magnitude of intrinsic viscosity in
0.15 M NaCl. Due to low water solubility of cis-[Pt(1-apt)2Cl2]·H2O,
it was dissolved in an aqueous solution containing 1% DMSO. The
stock solutions of cis-[Pt(1-pat)2Cl2]·H2O (10−5 M) were prepared
immediately before use. All systems contain 5 mM NaCl and 1% DMSO.
Measurements were performed at 21 °C. Details of hydrodynamic mea-
surements are given in Supplementary data.
3.8.1. Spectral study
Concentration of CT DNA in solutions was determined from the
difference in the absorbance of hydrolyzed solutions in the presence
of 4% HClO4 at wavelengths λ=270 and 290 nm [89]. The nativeness
of DNA was judged from the value of hyperchromic effect upon dena-
turation of the macromolecule. The final samples were prepared by
mixing the equal volumes of CT DNA solution and platinum complex
solution at different ri value (ri is defined as the molar ratio of platinum
complex to pair of nucleotide phosphates).
UV–vis absorption spectra in the range 200–600 nm were recorded
on a Specord UV–vis spectrophotometer using quartz cuvette (1 сm
path length). The spectra of the complex were subtracted from the
spectra of the complex-DNA adducts by computer.
Circular dichroism (CD) spectra in the range 220–340 nm were
recorded with a Jobin–Yvon Mark IV dichrograph and are given in
the units of extinction M−1 cm−1. For the experiment with different
pH values, the complexes of CT DNA with cis-[Pt(1-pat)2Cl2]·H2O
where incubated at 4 °C for 24 h; then the pH value of the solutions
2, 4 and 6 was changed by adding a small amount of HCl, and CD spectra
were taken. The ri values are given for the final solutions.
4. Conclusion
In the present study, we have shown that tetrazole-containing
platinum(II) complexes are a potent platform for design of new
promising anticancer agents, capable of overcoming cisplatin resis-
tance. Series of platinum(II) and palladium(II) chlorido complexes
with C,N-substituted tetrazoles were prepared and characterized. The
geometry of their coordination units was unambiguously proved by
spectral studies, HPLC and X-ray analysis.
The ligands and the complexes were evaluated for their cytotoxic ac-
tivity. Cytotoxicity of tetrazole-containing platinum complexes was found
to be strongly dependent on their lipophilicity and coordination unit ge-
ometry. The most active cis platinum compounds cis-[Pt(1-apt)2Cl2]·H2O
and cis-[Pt(2-abt)2Cl2]·H2O showed antiproliferative activity comparable
to that of cisplatin. It is noteworthy that cis-[Pt(2-abt)2Cl2]·H2O exhibits
lower resistance factors than cisplatin when tested against cisplatin-
resistant cell line. This fact shows that tetrazole-containing complexes
are able to partially overcome the cisplatin resistance developed by the
A2780R cancer cell line.
3.8.2. Electrophoretic mobility study
pFL44s Plasmid DNA 0.0025% was incubated in the presence of
cis-[Pt(1-apt)2Cl2]·H2O or cisplatin for 24 h at 4 °C, after this the
loading buffer (50% glycerol, 0.25% bromophenol blue, 0.25% xylene
cyanol) and TAE buffer (0.04 M Tris-acetate, 1 mM EDTA, pH 7.4)
To probe differences in mechanisms of cytotoxic action of cisplatin
and its tetrazole-containing analogues, DNA binding and cell cycle stud-
ies were performed. The biophysical studies showed that tetrazole-
containing platinum complexes behaved mostly like cisplatin as far as

52
T.V. Serebryanskaya et al. / Journal of Inorganic Biochemistry 120 (2013) 44–53
interaction with DNA is concerned. On the basis of UV and CD spectral
studies and hydrodynamic measurements, DNA was shown to be the
most plausible cellular target of tetrazole-based cisplatin analogues,
though a distinct dissimilarity with cisplatin behavior was observed
from gel-electrophoresis experiments suggesting the slightly different
mode of DNA binding. The results of cell cycle study also testified to
the different mechanism of cis-[Pt(2-abt)2Cl2]·H2O-induced cell death
as compared with cisplatin. These differences may contribute to the
lower acute toxicity of tetrazole-containing compounds in vivo in com-
parison with cisplatin as well as to the higher in vitro activity of cis-
[Pt(2-abt)2Cl2]·H2O against the cisplatin-resistant cell line.
In view of the promising potential as anticancer agents of this
series of compounds, further studies will be carried out to unveil
structure–activity relationships for this kind of complexes and to
improve stability, solubility, and pharmacokinetic properties of the
leading compounds.
Appendix A. Supplementary data
CCDC 861349 contain the supplementary crystallographic data for
trans-[Pd(1-amt)2Cl2] and may be obtained free of charge at www.
ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge Crystallo-
graphic Data Centre, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax:
int. code +44(0)1223/336-033, e-mail: deposit@chemcrys.cam.ac.uk).
The results of IR spectroscopy, crystallographic data for trans-
[Pd(1-amt)2Cl2], experimental details of hydrodynamic measurements,
Fig. S1 (crystal packing of trans-[Pd(1-amt)2Cl2]), Fig. S2 (growth
inhibition of H1299, A2780 and A2780CisR cell lines), Fig. S3 (relative
viscosity of CT DNA solution in the presence of Pt complexes), and
Fig. S4 (relative birefringence of CT DNA solution in the presence of Pt
complexes) are given in Supplementary data.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jinorgbio.2012.12.001.
References
5. Abbreviations
[1] J. Holford, S.Y. Sharp, B.A. Murrer, M. Abrams, L.R. Kelland, Br. J. Cancer 77 (1998)
366–373.
[2] F.I. Raynaud, F.E. Boxall, P.M. Goddard, Clin. Cancer Res. 3 (1997) 2063–2074.
[3] M. Coluccia, G. Natile, Anticancer Agents Med. Chem. 7 (2007) 111–123.
[4] U. Kalinowska-Lis, J. Ochocki, K. Matlawska-Wasowska, Coord. Chem. Rev. 252
(2007) 1328–1345.
1-amt
5-amino-1-methyltetrazole
1-apt
2-abt
2-amt
A2780
5-amino-1-phehyltetrazole
5-amino-2-tert-butyltetrazole
5-amino-2-methyltetrazole
Human ovarian tumor cell line
[5] J. Kuduk-Jaworska, A. Puszko, M. Kubiak, M. Pełczynska, J. Inorg. Biochem. 98
(2004) 1447–1456.
[6] M.V. Beusichem, N. Farrell, Inorg. Chem. 31 (1992) 634–639.
[7] G. McGowan, S. Parsons, P.J. Sadler, Chem. Eur. J. 11 (2005) 4396–4404.
[8] F.J. Ramos-Lima, O. Vrana, A.G. Quiroga, C. Navarro-Ranninger, A. Halamikova, H.
Rybnıckova, L. Hejmalova, V. Brabec, J. Med. Chem. 49 (2006) 2640–2651.
[9] S. Roy, J.A. Westmaas, F. Buda, J. Reedijk, J. Inorg. Biochem. 103 (2009)
1278–1287.
[10] G.M. Rakic, S. Grguric-Sipka, G.N. Kalucerovic, S. Gomez-Ruiz, S.K. Bjelogrlic, S.S.
Radulovic, Z.Lj Tesic, Eur. J. Med. Chem. 44 (2009) 1921–1925.
[11] G. McGowan, S. Parsons, P.J. Sadler, Inorg. Chem. 44 (2005) 7459–7467.
[12] A. Martinez, J. Lorenzo, M.J. Prieto, M. Font-Bardia, X. Solans, F.X. Avilés, V.
Moreno, Bioorg. Med. Chem. 15 (2007) 969–979.
[13] S.M. Aris, D.A. Gewirtz, J.J. Ryan, K.M. Knott, N.P. Farrell, Biochem. Pharmacol. 73
(2007) 1749–1757.
[14] R.M. Medina, J. Rodriguez, A.G. Quiroga, F.J. Ramos-Lima, V. Moneo, A. Carnero, C.
Navarro-Ranninger, M.J. Macazaga, Chem. Biodivers 5 (2008) 2090–2100.
[15] F. Huq, H. Daghriri, Q.J. Yu, P. Beale, K. Fisher, Eur. J. Med. Chem. 39 (2004)
691–697.
A2780cisR cisplatin-resistant human ovarian tumor cell line
AAS atomic absorption spectrometry
APCI-MS atmospheric pressure chemical ionization mass spectrometry
ccc form covalently closed circular form
CD
CT DNA calf thymus DNA
DMEM Dulbecco's Modified Eagle Medium
DMSO
EAC
EDTA
en
circular dichroism
dimethyl sulfoxide
Ehrlich ascytic carcinoma
ethylenediaminetetraacetate
1,2-ethylendiamine
FBS
fetal bovine serum
H1299 human non-small-cell lung adenocarcinoma cell line
[16] E. Pantoja, A. Gallipoli, S. van Zutphen, D.M. Tooke, A.L. Spek, C. Navarro-
Ranninger, J. Reedijk, Inorg. Chim. Acta 359 (2006) 4335–4342.
[17] E. Pantoja, A. Gallipoli, S. van Zutphen, S. Komeda, D. Reddy, D. Jaganyi, M. Lutz,
D.M. Tooke, A.L. Spek, C. Navarro-Ranninger, J. Reedijk, J. Inorg. Biochem. 100
(2006) 1955–1964.
HeLa
HPLC
IC50
human cervical carcinoma cell line
high-performance liquid chromatography
50% inhibitory concentration
IC80
80% inhibitory concentration
[18] S. van Rijt, S. van Zutphen, H. den Dulk, J. Brouwer, J. Reedijk, Inorg. Chim. Acta
359 (2006) 4125–4129.
ICP AES inductively coupled plasma atomic emission spectrometry
[19] F. Gumus, G. Eren, L. Acık, A. Celebi, F. Ozturk, S. Yılmaz, R.I. Sagkan, S. Gur, A.
Ozkul, A. Elmalı, Y. Elerman, J. Med. Chem. 52 (2009) 1345–1357.
[20] F.K. Keter, S. Kanyanda, S.S.L. Lyantagaye, J. Darkwa, D.J.G. Rees, M. Meyer, Cancer
Chemother. Pharmacol. 63 (2008) 127–138.
[21] E. Budzisz, U. Krajewska, M. Rozalski, A. Szulawska, M. Czyz, B. Nawrot, Eur. J.
Pharmacol. 502 (2004) 59–65.
[22] C. Mock, I. Puscasu, M.J. Rauterkus, G. Tallen, J.E.A. Wolff, B. Krebs, Inorg. Chim.
Acta 319 (2001) 109–116.
[23] V. Montoya, J. Pons, X. Solans, M. Font-Bardia, J. Ros, Inorg. Chim. Acta 359 (2006)
25–34.
ILS
IR
log P
MTS
increase in life span
infra red
partition coefficient
[3-(4,5-dimethyl-2-thiazolyl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)]-2H-tetrazolium]
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide)
МТT
oc form open circular form
PBS
RD
RPMI medium Roswell Park Memorial Institute medium
TAE buffer Tris-acetate-EDTA buffer
THF
[24] M. Miernicka, A. Szulawska, M. Czyz, I.-P. Lorenz, P. Mayer, B. Karwowski, E.
Budzisz, J. Inorg. Biochem. 102 (2008) 157–165.
phosphate-buffered saline
human rhabdomyosarcoma cell line
[25] A.A. Bekhit, O.A. El-Sayed, T.A.K. Al-Allaf, H.Y. Aboul-Enein, M. Kunhi, S.M. Pulicat,
K. Al-Hussain, F. Al-Khodairy, J. Arif, Eur. J. Med. Chem. 39 (2004) 499–505.
[26] S. Komeda, Y.-L. Lin, M.A. Chikuma, ChemMedChem 6 (2011) 987–990.
[27] R.N. Butler, in: A.R. Katritzky, C.W. Rees (Eds.), Comprehensive Heterocyclic
Chemistry, Tetrazoles, vol. 5, Pergamon Press, Oxford, 1984, pp. 834–837.
[28] R.N. Butler, in: A.R. Katritzky, C.W. Rees, E.F.V. Scriven (Eds.), Comprehensive
Heterocyclic Chemistry II, Tetrazoles, vol. 4, Pergamon Press, Oxford, 1996,
pp. 672–675.
tetrahydrofuran
tetramethylsilane
human osteosarcoma cell line
TMS
U2OS
[29] S.V. Voitekhovich, Т.V. Serebryanskaya, A.S. Lyakhov, P.N. Gaponik, O.A. Ivashkevich,
Polyhedron 28 (2009) 3614–3620.
[30] D.M. Roundhill, in: G. Wilkinson, R.D. Gillard, J.A. McCleverty (Eds.), Сomprehensive
Сoordination Сhemistry, vol. 5, Pergamon Press, Oxford, New York, Beijing, Frankfurt,
Sao Paulo, Sydney, Tokyo, Toronto, 1987, pp. 351–531.
Acknowledgments
[31] C.G. van Kralingen, J.K. de Ridder, J. Reedijk, Trans. Met. Chem. 5 (1981) 73–77.
[32] A.V. Khripun, S.I. Selivanov, V.Yu. Kukushkin, M. Haukka, Inorg. Chim. Acta 359
(2006) 320–326.
[33] K. Sakai, Y. Tomita, T. Ue, K. Goshima, M. Ohminato, T. Tsubomura, K. Matsumoto,
K. Ohmura, K. Kawakami, Inorg. Chim. Acta 297 (2000) 64–71.
We are grateful to Dr. Alexander V. Baranovsky (Institute of
Bioorganic Chemistry, National Academy of Science of Belarus) for
recording NMR spectra and the interpretation.

T.V. Serebryanskaya et al. / Journal of Inorganic Biochemistry 120 (2013) 44–53
53
[34] A. Boixassa, J. Pons, X. Solans, M. Font-Bardia, J. Ros, Inorg. Chim. Acta 355 (2003)
254–263.
[63] Z.G. Aliev, T.K. Goncharov, B.M. Grachev, S.V. Kurmaz, V.P. Roschupkin, Koord.
Khim. 17 (1991) 1101–1105.
[35] A.V. Khripun, M. Haukka, V.Yu. Kukushkin, Russ. Chem. Bull. (2006) 247–255.
[36] E. Budzisz, M. Malecka, B. Nawrot, Tetrahedron 60 (2004) 1749–1759.
[37] M.A. Cinellu, S. Stoccoro, G. Minghetti, A.L. Bandini, G. Banditelli, B. Bovio, J.
Organomet. Chem. 372 (1989) 311–325.
[64] D.J. Chadwick, R.A.W. Johnstone, P.J. Price, M.M. Harding, Acta Crystallogr. C44
(1988) 367–369.
[65] A.V. Khripun, V.Yu. Kukushkin, G.I. Koldobskii, M. Haukka, Inorg. Chem. Commun.
10 (2007) 250–254.
[38] V. Montoya, J. Pons, X. Solans, M. Font-Bardia, J. Ros, Inorg. Chim. Acta 358 (2005)
2312–2318.
[66] D. Garmann, M. Galanski, G. Weber, G.V. Kalayda, B.K. Keppler, U. Jaehde, J. Inorg.
Biochem. 105 (2011) 709–717.
[39] N. Farrell, T.M. Gomes Carneiro, F.W.B. Einstein, T. Jones, K.A. Skov, Inorg. Chim.
Acta 92 (1984) 61–66.
[67] J.A. Platts, S.P. Oldfield, M.M. Reif, A. Palmucci, E. Gabano, D. Osella, J. Inorg.
Biochem. 100 (2006) 1199–1207.
[40] J.R. Bales, A. Mazid, P.J. Sadler, A. Aggarwal, R. Kuroda, S. Neidle, D.W. Gilmour, B.J.
Peart, C.A. Ramsden, J. Chem. Soc., Dalton Trans. (1985) 795–802.
[41] G.M. Rakic, S. Grguric-Sipka, G. Kalucerovic, S. Go mez-Ruiz, S. Bjelogrlic, S.S.
Radulovic, Z.Lj Tesic, Eur. J. Med. Chem. 44 (2009) 1921–1925.
[42] F.D. Rochon, J.M. Doyon, I.S. Butler, Inorg. Chem. 32 (1993) 2717–2723.
[43] P.-C. Kong, F.D. Rochon, Inorg. Chim. Acta 61 (1982) 269–271.
[44] J.R. Bales, C.J. Coulson, D.W. Gilmour, A. Mazid, S. Neidle, B.J. Peart, R. Kuroda, C.A.
Ramsden, P.J. Sadler, J. Chem. Soc. Chem. Commun. (1983) 432–433.
[45] C.J.L. Lock, M. Zvagulis, Inorg. Chem. 20 (1981) 1817–1823.
[46] P.K. Basu, A. González, C. López, M. Font-Bardía, T. Calvet, J. Organomet. Chem.
694 (2009) 3633–3642.
[68] P. Horasek, J. Drobnik, Biochim. Biophys. Acta 254 (1971) 341–347.
[69] J.-P. Macquet, T. Theophanides, Inorg. Chim. Acta 18 (1976) 189–194.
[70] J.-P. Macquet, J.-L. Butour, Biochimie 60 (1978) 901–914.
[71] V. Brabec, V. Kleinwächter, J.-L. Butour, N.P. Johnson, Biophys. Chem. 35 (1990)
129–141.
[72] A.D. Richards, A. Rodger, Chem. Soc. Rev. 36 (2007) 471–483.
[73] S. Roy, J.A. Westmaas, K.D. Hagen, G.P. van Wezel, J. Reedijk, J. Inorg. Biochem. 103
(2009) 1288–1297.
[74] A.M. Tamburro, L. Celotti, D. Furlan, V. Guantieri, Chem. Biol. Interact. 16 (1977)
1–11.
[75] J.-P. Macquet, J.-L. Butour, Eur. J. Biochem. 83 (1978) 375–387.
[76] N.A. Kas'ianenko, S.F. Bartoshevich, E.V. Frisman, Mol. Biol. 19 (1985) 1386–1393.
[77] Y. Courtois, P. Fromageot, W. Guschlbauer, Eur. J. Biochem. 6 (1968) 493–501.
[78] N.A. Kasyanenko, S.A. Prokhorova, E.F. Haya Enriquez, S.S. Sudakova, E.V. Frisman,
S.A. Dyachenko, N.A. Smorygo, B.A. Ivin, Colloids Surf., A 148 (1999) 121.
[79] J.-L. Butour, J.-P. Macquet, Biochim. Biophys. Acta 653 (1981) 305–315.
[80] M.V. Keck, S.J. Lippard, J. Am. Chem. Soc. 114 (1992) 3386–3390.
[81] E. Roubalová, V. Kvardová, R. Hrstka, Š. Bořilová, E. Michalová, L. Dubská, P.
Müller, P. Sova, B. Vojtěšek, Invest. New Drugs 28 (2010) 445–453.
[82] A.H. Ronald, G.F. William, J. Am. Chem. Soc. 76 (1954) 923–926.
[83] S.V. Voitekhovich, P.N. Gaponik, A.S. Lyakhov, O.A. Ivashkevich, Tetrahedron 64
(2008) 8721–8725.
[84] A.N. Vorobyov, P.N. Gaponik, P.T. Petrov, O.A. Ivashkevich, Synthesis (2006)
1307–1312.
[85] M.C. Burla, R. Caliandro, M. Camalli, B. Carrozzini, G.L. Cascarano, L. De Caro, C.
Giacovazzo, G. Polidori, R. Spagna, J. Appl. Crystallogr. 38 (2005) 381–388.
[86] G.M. Sheldrick, Acta Crystallogr. A64 (2008) 112–122.
[87] A.L. Spek, Acta Crystallogr. D65 (2009) 148–155.
[47] T. Tian, I. Mutikainen, G.P. van Wezel, N. Aliaga-Alcalde, J. Reedijk, J. Inorg.
Biochem. 103 (2009) 1221–1227.
[48] A. Bigotto, R. Klingendrath, Spectrochim. Acta 46A (1990) 1683–1692.
[49] K. Karaghiosoff, T.M. Klapötke, C. Miro Sabate, Chem. Eur. J. 15 (2009) 1164–1176.
[50] C. Engelter, A.T. Hutton, D.A. Thornton, J. Mol. Struct. 44 (1978) 23–30.
[51] G.M. Barrow, R.H. Krueger, F. Basolo, J. Inorg. Nucl. Chem. 2 (1956) 340–344.
[52] J.P. Faust, J.V. Quagliano, J. Am. Chem. Soc. 76 (1954) 5346–5349.
[53] M.M. Chamberlain, J.C. Bailar, J. Am. Chem. Soc. 81 (1959) 6412–6415.
[54] M.L. Morris, D.H. Busch, J. Am. Chem. Soc. 82 (1960) 1521–1524.
[55] C.G. van Kralingen, J.K. de Ridder, J. Reedijk, Inorg. Chim. Acta 36 (1979) 69–77.
[56] C.G. van Kralingen, J. Reedijk, Inorg. Chim. Acta 30 (1978) 171–177.
[57] P.-C. Kong, F.D. Rochon, Can. J. Chem. 56 (1978) 441–445.
[58] C. Tessier, F.D. Rochon, Inorg. Chim. Acta 322 (2001) 37–46.
[59] F.M. Macdonald, P.J. Sadler, Polyhedron 10 (1991) 1443–1448.
[60] L.S. Ivashkevich, Т.V. Serebryanskaya, A.S. Lyakhov, P.N. Gaponik, Acta Crystallogr.
C67 (2011) m195–m198.
[61] H. Gallardo, I.M. Begnini, A. Neves, I. Vencato, J. Braz. Chem. Soc. 11 (2000)
274–280.
[62] Z.G. Aliev, T.K. Goncharov, B.M. Grachev, V.P. Roschupkin, Koord. Khim. 16 (1990)
570–573.
[88] L.J. Farrugia, J. Appl. Crystallogr. 30 (1997) 565–566.
[89] A.S. Spirin, Biochemistry 23 (1958) 656–662.