Mixed valence copper(I,II) binuclear complexes with unexpected structure: Synthesis, biological properties and anticancer activity

Article abstract of DOI:10.1021/jm500154f

We have synthesized and characterized a panel of new binuclear mixed valence Cu(I,II) complexes containing substituted 2-alkylthio-5-arylmethylene- 4H-imidazolin-4-ones with unusual structure. These complexes are shown to be cytotoxic for various cell lin

Full text of DOI:10.1021/jm500154f

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Brief Article
Mixed valance copper (I,II) binuclear complexes with unexpected
structure: synthesis, biological properties and anticancer activity.
Alexander G. Majouga, Maria I. Zvereva, Maria P. Rubtsova, Dmitry A. Skvortsov, Andrei V.
Mironov, Dulat M. Azhibek, Olga O. Krasnovskaya, Vasily M. Gerasimov, Anna V. Udina, Nikolay
I. Vorozhtsov, Elena K. Beloglazkina, Leonid A. Agron, Larisa V. Mikhina, Alla V. Tretyakova,
Nikolay Vasil'evich Zyk, Nikolay S. Zefirov, Alexander V. Kabanov, and Olga A. Dontsova
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 20 Jun 2014
Downloaded from http://pubs.acs.org on June 25, 2014
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Mixed valance copper (I,II) binuclear complexes with unexpected
structure: synthesis, biological properties and anticancer activity.
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Alexander G. Majouga†‡ *, Maria I. Zvereva†₫‡*, Maria P. Rubtsova†₫‡, Dmitry A. Skvortsov†,
┼
Аndrei V. Mironov†, Dulat M. Azhibek†˩, Olga O. Krasnovskaya†, VasilyM. Gerasimov†, Anna V.
Udina, Nikolay I. Vorozhtsov†, Elena K. Beloglazkina†, Leonid Agron†, Larisa V. Mikhina₸,Alla V.
Tretyakova₸, Nikolay V. Zyk†, Nikolay S. Zefirov†, Alexander V. Kabanov†ǁ and Olga A. Dontsoꢀ
va†₫.
†Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1/3, 119991, Moscow, Russia.
₫Belozersky Institute, Lomonosov Moscow State University, Leninskie Gory, 1/40, 119992, Moscow, Russia.
ǁUniversity of North Carolina at Chapel Hill, Genetic Medicine Building, room 1094, Campus Box 7362, Chapel Hill, NC
27599ꢀ7362, Center for Nanotechnology in Drug Delivery and Division of Molecular Pharmaceutics, Eshelman School of
Pharmacy.
₸ Federal StateꢀFinanced Institution "Research Center for Toxicology and Hygienic Regulation of Biopreparations" of Fedꢀ
eral MedicoꢀBiological Agency,102A, Lenin str., Serpukhov, Moscow Region, 142253, Russia.
┼
National University of Science and Technology MISiS, Leninsky Ave, 4, 119049, Moscow, Russian Federation.
˩ Skolkovo Institute of Science and Technology, ul. Novaya, d.100, Skolkovo, 143025, Russian Federation.
KEYWORDS mixed valence copper complex, thiohydantoin derivatives, nuclear localization, TUNEL, polymerase inhibiꢀ
tion, telomerase inhibition, anticancer activity.
ABSTRACT:We have synthesized and characterized a panel of new binuclear mixed valance Cu(I, II) complexes containing subꢀ
stituted 2ꢀalkylthioꢀ5ꢀarylmethyleneꢀ4Hꢀimidazolinꢀ4ꢀones with unusual structure. These complexes are shown to be cytotoxic for
various cell lines. We have found that these compounds did not intercalate DNA, inhibited number of polymerases (telomerase
predominantly), accumulated in the cell nucleus and caused DNA degradation. Preliminary studies revealed that lead compound
inhibited human breast adenocarcinoma growth in mice model.
Metalꢀbased anticancer drugs play an important role in chemoꢀ
therapy. The discovery of cisplatin resulted in tremendous
growth in research associated with inorganic metal complexes
for cancer therapy.1 Therefore, in recent years there has been
a rapid expansion in research and development of novel metalꢀ
based anticancer drugs to improve clinical effectiveness, to
reduce general toxicity and to broaden the spectrum of activiꢀ
ty. The variety of metal ion functions in biology has stimulatꢀ
ed the development of new metallodrugs other than Pt drugs
with the aim to obtain compounds with alternative mechaꢀ
nisms of action. Among nonꢀPt compounds, copper complexes
are potentially attractive. It has been established that the propꢀ
erties of copperꢀcoordinated compounds are largely deterꢀ
mined by the nature of ligands and donor atoms bound to the
metal ion. Cytotoxic activity of copperꢀcontaining complexes
is based either on induction of cell apoptosis or enzyme inhibiꢀ
tion.2,3 Among the diversity of copper complexes most reꢀ
searcher's attention attracts copper bis(thiosemicarbazone)
complexes4 and casiopeinas,5,6 copperꢀbased coordinated comꢀ
plexes with generic structure of [Cu(N–N)(O–N)]+ or [Cu(N–
N)(O–O)]+]. Copper bis(thiosemicarbazone) complexesare
used in application of radioactive copper isotopes in radioꢀ
pharmaceuticals. The use of coordination compounds as an
imaging agent are currently in clinical trials.7,8 It was shown
that these complexes exhibit anticancer activity by inhibition
of DNA and RNA synthesis and disruption of ATP producꢀ
tion, and surprisingly none of them function through ROS
generation4. In contrast to the first type complexes, the generaꢀ
tion of ROS is a major factor of the high toxicity and antiꢀ
cancer activity of casiopeinas.9,10
In an attempt to receive more insight and determine the action
mechanism and antitumor activity of copper complexes, we
have synthesized and characterized new binuclear mixed valꢀ
ance Cu(I,II) complexes containing substituted 2ꢀalkylthioꢀ5ꢀ
arylmethyleneꢀ4Hꢀimidazolinꢀ4ꢀones. These complexes were
shown to be cytotoxic for various cell lines. We have shown
that in experiments in vitro these compounds inhibit Human
telomerase, HIV reverse transcriptase, T7 RNA polymerase,
calf DNA polymeraseβ, Taq DNA polymerase to different
extend, do not intercalate DNA and do not cause significant
DNA cleavage. However, these complexes accumulate in the
nucleus within the cell and cause rapid chromosomal DNA
degradation which results in the cell death. Preliminary studies
revealed that lead compound inhibits human breast adenocarꢀ
cinoma growth in mice model.
As it was previously described initial 5ꢀarylmethylene substiꢀ
tuted thiohydantoines were prepared by Knoevenagel condenꢀ
sation reaction between heteroaromatic aldehydes and the
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thiohydantoin.11 The key step in the formation of the thiohyꢀ
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pound, the triangle coordination of Cu(I) atom is usually alꢀ
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dantoin backbone was achieved by a Sꢀ alkylation reactions in
the basic conditions (Scheme 1).
most flat, unequal angels, which is atypical for such type of
copper coordination.Cl11ꢀCu1ꢀN12 angle is 151.9o while the
other angels Cl11ꢀCu1ꢀN11and N11ꢀCu1ꢀN12are 109.8oand
98.0orespectably.
Scheme 1. Synthesis of 2ꢀalkylthioꢀ5ꢀarylmethyleneꢀ4Hꢀ
imidazolinꢀ4ꢀone ligands L1ꢀL6.
We have studied electrochemical behavior of complexes 2,
3 and 4 by means of cyclic voltammetry and rotating disk
electrode techniques. Under the same conditions oneꢀelectron
and twoꢀelectron reduction waves are observed during the
reverse scan and assigned to the Cu2+ to Cu1+and Cu1+ to
Cu0(respectably) which are quasi reversible in nature (see Taꢀ
ble S5,Supporting Information).
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The anticancer activities of 1-6 against the MCFꢀ7 (human
breast adenocarcinoma), SiHa (human cervical squamous cell
carcinoma) and HEK293 (human embryonic kidney) cell lines
have been tested with a help of 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ
2,5ꢀdiphenyltetrazolium bromide (MTT) assay.13 The comparꢀ
ison of these data with the widely used cisplatin (literature and
our data) and doxorubicine under identical conditions by using
MTT assay is shown in Table 1.
Coordination of copper ions was essential for cytotoxicity of
novel compounds. Indeed, IC50 values for free ligands were
much higher than the values for corresponding complexes
(Table 1). For cytotoxicity not only does the coordination of
copper ions play an important role, but also does the nature of
ring substituents. The most active complexes (2,3) contained
allyl or phenyl substituents. Complexes with aliphatic substitꢀ
uents, placed at the same positions in the ring (4,5,6), were
less active. For further biological studies complex 2 was choꢀ
sen, because in comparison to complex 3, it had a perfect acꢀ
tivity and better solubility in biological media. The cytotoxiciꢀ
ty for the complex 2 was tested in broadened panel of human
cancer cell lines which included MDAꢀMBꢀ231, HepG2, PC3
cell lines that represent human breast adenocarcinoma, human
hepatocellular carcinoma and human prostate adenocarcinoma
(see Table S6, Supporting Information). In all of the cases
complex 2 showed similar toxicity.
Reagents and conditions: (a) Br(CH2)2Br, K2CO3, DMF, rt.
Presence of azido group allows us to modify complexes
with fluorescent dyes via the well known clickꢀchemistry apꢀ
proaches.
The Cu(I,II) complexes were prepared by direct reaction of
ligands dissolved in dichloromethane with CuCl2·2H2O in
methanol. According to the Xꢀray data received during the
study, absolutely unexpectedly, we revealed a novel type of
mixed valence copper (I,II) complexes (Figure 1).
Figure 1. Copper complexes based on 2ꢀalkylthioꢀ5ꢀ
arylmethyleneꢀ4Hꢀimidazolinꢀ4ꢀones.
Table 1. The level of cytotoxicity measured in vitro by vari-
ous complexes.
№
IC50, µM
MCFꢀ7
3.7±1.6
15.9±1.4
2.1±0.8
11.3±2.6
7.4±1.4
>100
IC50, µM
SiHa
3.0±0.2
61.4±25.5
2.2±0.7
>100
IC50, µM
HEK293
2.5±0.4
>100
2.3±0.9
53.0±4.4
25.3±1.2
>100
2
L2
3
L3
4
L4
Figure 2. Xꢀray molecular structures of Cu (I, II) complex
2. Solid thermal ellipsoids are reported at the 30% probability
level. C (black), N (blue), Cl (green), O (red), S (yellow), Cu
(brown).
3.9±2.3
>100
6
L6
Dox
Cisplatin
13.4±3.8
12.0±10
2.1±0.8
64.13±3.9a
>30b
8.5±0.4
54.8±10.5
2.0±0.8
ꢀ
12.7±3.7
37.3±15.9
1.1±0.1
12,4±3,9
The crystal structures of 2, 4, 5 and 6 were determined by
Xꢀray crystallography (see Figures 2, S1, TablesS1ꢀ4, Supꢀ
porting Information). All synthesized complexes have the
same geometry and metal ion surroundings (Figure 2). Organꢀ
ic ligand consists of two equivalent parts, each part is linked
with copper atoms. Even though both part of molecule are
identical (symmetrical), during the interaction with copper
they become very different in structure. The main difference is
in the coordination of copper atoms: one atom is three coordiꢀ
nated, while the other atom is four coordinated. The calculaꢀ
tion of oxidation state of copper atoms by bond valence sum
a14; bꢀin our experiments;
To test the complex 2 influence on the HEK293 cells morꢀ
phology, they were treated with complex 2 (2.6 µM) for 3
hours. Significant changes such as contraction, loss of adherꢀ
ence and rounded shape were detected (see Figure S2, Supꢀ
porting Information). Next, we have tested an uptake of the
copper complex by the cell. Inductively coupled plasma mass
spectrometry detected the 10times enrichment of Cu2+ in cells
compared to the natural copper cell level after incubation with
3µM of copper complex 2 (see Table S7, Supporting Inforꢀ
mation). Thus, after the cells were treated with complex
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method gives 2.00 for Cu(II) and 1.54 for Cu(I). The coorꢀ
dination of both copper atoms is highly asymmetric. The deꢀ
formation of copper coordination polyhedron is common, but
usually, the deformation is not too strong. In the titled comꢀ
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Figure 4. DNA damage occurs in HEK293 cells after complex 2
2,copper ions accumulated inside of the cell. We used fluoresꢀ
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treatment according to flow cytometry analysis. HEK293 cells
without treatment (A) and after treatment with complex 2 (B) and
ligand L2 (C) were analyzed by PIꢀstaining followed by flow
cytometry (upper panel). At the lower panel forward and side
scattering for the same samples are shown.
cent microscopy to investigate whether the copper ions enter
the cell as a part of copper containing complex2. Since comꢀ
plexes 1-4 have low fluorescence level and are insufficient for
microscopy, we used complexes 5-6 bearing azido group to
conjugate them with diSulfoꢀCy5 alkyne (Cyandye LLC)
fluorophore using clickꢀchemistry approach15 after the cells
were fixed for microscopy. Series of fluorescent microscopy
images (Figure 3) showed intracellular localization of complex
5 conjugate with its predominant presence in the nucleus. In
contrast to complex 5, localization of ligand L5 marked with
the same fluorescent dye under the same conditions was also
detected inside the cell, but spread over the entire cell volume.
To prove that complex 2 causes significant DNA destruction
TUNEL16 that allows to detect DNA breaks was performed.
All cells demonstrated TUNEL signal after treatment with
complex 2 (Figure5) thus confirming that complex 2 causes
DNA cleavage followed by massive DNA fragmentation by
cellular nucleases that provide free DNA 3’ꢀend necessary for
induction of TUNEL signal.
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Figure5. DNA damage occurs in HEK293 cells after complex
2 treatment according to TUNEL assay. HEK293 cells without
treatment (A) and after treatment with 6.5 µM complex 2 (B) and
16 µM ligand L2 (C) were analyzed by TUNEL assay.
DNA damage can occur due to the formation of reactive oxꢀ
ygen species upon cell treatment with copper complexes. To
investigate whether ROS are essential for copper complexes
induced cell death, ability of complex 2 to stimulate the apꢀ
pearance of intracellular ROS was assessed by flow cytometry
with the fluorescent CMꢀH2DCFDA probe17 in HEK293 cells
(see Figure S3, Supporting Information). ROS value increased
after incubation with 2.6 µM of complex 2 and 16 µM of ligꢀ
and L2. The level of ROS production was increased similarly
for cells treated with complex 2 or ligand L2 or H2O2in comꢀ
parison with untreated HEK293 cells. Although similar levels
of ROS production was detected both for complex 2 and ligꢀ
and L2, DNA damage happened only in the case of complex 2
but not ligand L2 (see Figure S3, Supporting Information).
These data suggest that formation of ROS didn't play essential
role in DNA fragmentation activity of the desired complexes.
We have shown that complex 2 causes DNA damage within
the cell nucleus not due to ROS induction. To understand the
mechanism of this process we decided to test whether this
complex can directly interact with DNA and cleave it.
There are several types of interaction between DNA and
small molecules: formation of a covalent bond, intercalation,
the interaction with the bases or sugarꢀphosphate backbone.18
To test whether complex 2 can bind DNA as intercalatꢀ
ing agent or minor groove binder we used fluorescenceꢀbased
ethidium bromide displacement protocol.4 If the substance
competes with ethidium bromide for DNA binding and cause
ethidium bromide dissociation from preformed DNA adduct
that results in ethidium bromide fluorescence emission
quenching. We monitored such quenching from either ligand
L2 or corresponding copper complex 2. Both substances did
not cause detectable quenching thus indicating that these comꢀ
pounds did not bind DNA (see FigureS4, Supporting Inforꢀ
mation). Majority of substances interacting with DNA by difꢀ
ferent mechanisms affect duplex stability and thus their bindꢀ
ing could be detected by the influence on dsDNA oligonucleoꢀ
tide thermal denaturation. Melting curves for both DNA duꢀ
plex and DNAꢀRNA heteroduplex did not show any difference
Figure 3. The cellular uptake properties of the complex 5. (A)
HEK293 cells after complex 5 uptake were stained with DAPI
and conjugated with diSulfoꢀCy5 alkyne; (B) HEK293 cells after
ligand L5 uptake were stained with DAPI and conjugated with
diSulfoꢀCy5 alkyne
.
To investigate changes in DNA integrity after complex acꢀ
cumulation in cellular nucleus we performed PI DNA staining
followed by flow cytometry (Figure 4). We observed the sigꢀ
nificant decrease in amount of PIꢀpositive cells in the presence
of complex 2 that provided an evidence that the number of
long DNA fragments capable for PI binding was reduced. The
analysis of forward and side scattering distribution of these
samples revealed the dramatic changes in morphology of cells
after treatment with complex 2. In contrast, L2 did not affect
the HEK293 cells morphology and distribution of PIꢀpositive
cells. These data allow us to propose that complex 2 targets
copperꢀions to cellular nucleus that causes the significant
DNA damage.
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250
in presence or absence of complex 2 (See Figures S5, S6,
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Supporting Information) thus providing additional evidence
that complex 2 does not interact with DNA duplex or DNAꢀ
RNA heteroduplex. Number of organic ligands and metal
complexes can interact with noncanonical forms of DNA, like
Gꢀquadruplexes.19ꢀ21 Complex 2 has its own fluorescence, sufꢀ
ficient for measuring interaction with Gꢀquadruplexes. We
studied complex 2 fluorescence in the presence of 0.2ꢀ4 fold
excess of singleꢀstranded or quadruplex DNA and didn't obꢀ
serve any changes (See Figures S7, S8, Supporting Inforꢀ
mation), that proves the absence of the strong complexꢀDNA
binding. We checked whether the complex 2 cleaves DNA in
vitro. The conversion of supercoiled DNA into relaxed DNA
as indicator of the cuts/nicks in DNA induced by compound
was used. For that purpose pUC18 plasmid DNA was incubatꢀ
ed with increasing concentrations of complex 2 as described4
and resulted DNA molecules were analyzed by agarose gel
electrophoresis. The results are shown in Figure 6. Accumulaꢀ
tion of relaxed plasmid DNA form was observed starting from
10 µM of complex 2. It means that at this concentration one
phosphorꢀester bond of DNA per 3500bp is cleaved.
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Telomerase
HIV reverse
T7 RNA
Calf DNA
Taq DNA
transcriptase
polymerase
polimerase β
polymerase
Figure 7.Telomerase(1), HIV reverse transcriptase (2), T7 RNA
polymerase (3), calf thymus DNA polymerase
polymerase (5) inhibition data for complex 2.
β (4), Taq DNA
All other polymerases tested were also inhibited by complex
2 but with lower efficiency. However, these results showed
that indeed the variety of DNA operating enzymes and possiꢀ
bly DNA binding proteins can bind complex 2 and may stimuꢀ
late copper ion release near DNA in the cell nucleus. Such
release can provide the appearance of shortꢀliving hydroxyl
radicals at direct proximity to DNA that cause DNA damage.
It should be mentioned that we have detected significant
level of telomerase inhibition by complex 2. Telomerase is a
very attractive molecular target for developing therapeutic
anticancer agents due to difference in the performance of acꢀ
tive enzyme in normal and cancerous tissues (for review
see22). We used two independent approaches to investigate
telomerase inhibition by complex 2: TRAP assay23 and direct
telomerase assay24 (See FiguresS9, 10, 15, Supporting Inforꢀ
mation). Complex 2 inhibited telomerase with IC50 6.5±4 ꢁM
in TRAP assay, but ligand L2 showed lower telomeraseꢀ
inhibitory activity that proves that copper ions are essential for
inhibition process. Complex 2 can possibly inhibit telomerase
by binding to catalytic core or/and siteꢀdirected cleavage of
telomerase RNA or telomerase elongation product by hydroxꢀ
yl radicals upon copper ions release after complex interaction
with the enzyme. The fact that complex 2 was inhibiting HIV
reverse transcriptase to similar extend suggests that this reaꢀ
gent can bind to conserved reverse transcriptase catalytic core.
Despite complex 2 inhibits telomerase activity in vitro we
cannot exclude that complex 2 disrupts indirectly shelterin
complex and induce telomereꢀdependent DNAꢀdamage similar
to situation observed for SWNTs25. Thus, in spite complex 2
itself cannot be considered as a potential telomerase specific
anticancer drug due to significant DNA damage in cell nucleꢀ
us, it provides a chemical platform for searching for telomerꢀ
ase inhibitors with different ions and/or substituents that may
have telomerase inhibiting activity without DNA damaging
due to copper release.
Figure 6.Test of DNA cleavage in vitro by complex 2. A. Agaꢀ
rose gel electrophoresis of pUC18 plasmid DNA treated with 0ꢀ
200 µM complex 2 or 0ꢀ200 µM CuCl2. “0” –DNA incubated in
the same buffer conditions without treatment, “M” – “1 kb+”
DNA marker.
Within the cell, the DNA was significantly destroyed into
fragments after cell treatment with the complex, but in soluꢀ
tion, we could detect only limited DNA degradation after
longer incubation with higher concentration of the reagent.
Such a difference, allows us to hypothesize that there is a partꢀ
ner in the cell that can interact with the complex and stimulate
DNA damage by release of copper ions at direct proximity to
DNA followed by massive DNA degradation by nucleases.
Based on that we have decided to test if this reagent can interꢀ
act with DNA binding proteins. For that purpose, we tested
whether complex 2 could inhibit such important DNA operatꢀ
ing enzymes as DNA or RNA polymerases. Effect of complex
2 on different polymerase under enzyme reaction conditions
were tested by standard assays (See Figures 7, S9ꢀS15, Supꢀ
porting Information). The strongest inhibitory effect was obꢀ
served for human telomerase. It's important to mention that
complex 2 was stable during an incubation for a long time in a
buffer used for experimental procedures (See Figure S16,
Supporting Information).Complex 2 inhibit HIV reverse tranꢀ
scriptase, which has similar catalytic core, at similar level
(Figure 7).
It should be mentioned that complex 2 inhibit different polꢀ
ymerases. All these enzymes share the possibility to interact
with nucleic acids. If we compare the structure of complex 2
with the structure of nucleic acid chain we could notice that
the position of aromatic rings and the distance between them
resemble that in DNA chain (See FigureS17, Supporting Inꢀ
formation). That allows us to hypothesize that complex 2 can
interact with the variety of DNA binding proteins and induce
DNA damage by copper release and subsequent DNA degraꢀ
dation in the cell nucleus. Complex 2 is a mixed valance copꢀ
per (I,II) complex. It is known that Cu(I) catalyzes the producꢀ
tion of hydroxyl radical from endogenous hydrogen peroxide
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after release from complex that is highly damaging for cellular
solvent. Chemical shifts are given in parts per million (ppm) (δ
relative to residual solvent peak for H).Elemental analysis
1
lipids, proteins and especially for nucleic acids.26, 27
1
2
3
4
5
6
7
8
9
Complex 2 was active in antiproliferative assay with differꢀ
ent cancer cell cultures. To test whether this reagent can have
antitumor activity in vivo we used mouse breast adenocarciꢀ
noma model. MTD for C57Bl mice for complex 2 in 10%
DMSO was 24 mg/kg correspondingly. Five intraperitoneal
injection of complex 2 at MTD to animals did not lead to
deaths during the course and during the observation period ꢀ
14 days, the mice body weight loss did not exceed 10%.
Breast adenocarcinoma 755was inoculated to mice lines
C57BL/6 (female), treatment began 48 hours after vaccination.
Complex 2 (14 and 12 mg/kg/d) was intraperitoneally injected
with intervals of 24 hours for 5 days. Statistical comparison
was made between control and treated groups (10 mice in each
group). A pꢀvalue <0.05 was considered to be statistically
significant. Indicator of tumor growth inhibition for mice with
a course of the test substance at a dose of 12 mg/kg, was
46.1% on the seventh day after the end of treatment and 36.1%
ꢀ on the fourteenth day after the end of treatment. For a dose of
24 mg/kg it was 73.5% on the seventh day after the treatment,
and 59,5% ꢀ on the fourteenth day after the end of treatment.
These data show that the complex 2 demonstrates antitumor
activity in vivo (See Table S8, Supporting Information).
was performed on a vario MICRO cube elemental analyzer,
Elementar Analysen systeme GmbH, Hanau Germany. IR
spectra were recorded on a Varian 800 FTꢀIR Scimitar series.
All compounds exhibited >95% purity according to elemental
analysis (within ±0.4% of the calculated value).
Materials. Initial 2ꢀthioxoꢀ5ꢀ(pyridineꢀ2ꢀylmethylene)ꢀ3,5ꢀ
dihydroꢀ4Hꢀimidazoleꢀ4ꢀones were prepared previously by
Beloglazkina28.
General method for the synthesis of L1-L6.Selected 2ꢀ
thioxoꢀ5ꢀ(pyridineꢀ2ꢀylmethylene)ꢀ3,5ꢀdihydroꢀ4Hꢀimidazoleꢀ
4ꢀon (2 equivalents), α,ωꢀdibromoalkane (1 equivalent) and
solid potassium carbonate (3 equivalents) were suspended in
DMF (10 ml) at 00С. The mixture was stirred for two hours at
RT. Water (50 ml) was added and resulting precipitate was
filtered, washed with water, ethanol and ether.
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(5Z,
5’Z)ꢀ1,2ꢀbisꢀ[3ꢀpropylꢀ5ꢀ(pyridineꢀ2ꢀylmethylene)ꢀ3,5ꢀ
Brightꢀ
dihydroꢀ4Hꢀ4ꢀoxoimidazolꢀ2ꢀyl)ꢀthio]ꢀethan(L1):
yellow solid (82%); mp 152ºС. 1H NMR (400 MHz, CDCl3) δ
= 8.69(d, J=8.0 Hz, 1H, HαꢀPy,), 8.65 (d, J=4.7 Hz, 1Н, Hβ’ꢀ
Py,), 7.63 (td, J1=7.4Hz, J2=2.0 Hz, 1Н, HβꢀPy,), 7.19 (dd,
J1=7.5 Hz, J2=0.9 Hz, 1Н, HγꢀPy,), 7.12 (s, 1H, CH=), 7.12 (s,
1H, CH=), 3.93 (s, 2H, SCH2), 3.60 (t, J=7.5 Hz, 2H, CH2N),
1.72 (m, 2Н, CH2), 0.96 (t, J=7.5 Hz, 3H, CH3)ppm. IR(KBr):
υ =1705(C=O), 1675(C=N), 1640(C=C) cmꢀ1. Elemental analꢀ
ysis calculated (%) for C26H28N6O2S2: C 59.98, H 5.42, N
16.14. Found C 59.89, H 5.48, N 15.97.
CONCLUSIONS
As a result of our research a number of novel nitrogen conꢀ
taining organic ligands were synthesized. It is interesting to
note, that for the first time formation of the mixed valance
copper (I,II) complexes from Cu(II) precursor and imidazolinꢀ
4ꢀones was observed. The characterization has been based on
singleꢀcrystal XꢀRay analysis, elemental analysis and cyclic
voltamperometry. Copper containing complexes were cytotoxꢀ
ic for variety of cancer cell lines. In contrast to majority of
other known metalꢀcontaining drugs these compounds were
shown to be accumulated at the cell nucleus and the presence
of copper ions as a complex constituent was essential for both
accumulation in the cell nucleus and rapid DNA fragmentation
accompanied with DNA 3’ꢀend generation as a result of nuꢀ
clease cleavage. Copper containing complexes were able to
inhibit number of polymerases that allowed us to hypothesize
that interaction of copper containing complexes with the proꢀ
teins in the vicinity of DNA may result in copper ions release
that cause DNA damage followed by rapid DNA degradation
by cellular nucleases. Experiments with mice models showed
that complex 2 is less toxic than wellꢀknown drug cisplatin28
and have pronounced antitumor effect. Novel class of metal
complexes,based on 2ꢀthioxoimidazolꢀ2ꢀone heterocyclic core
could be utilized as a smart platform for construction of antiꢀ
cancer chemotherapy agents. Ability to penetrate cell memꢀ
brane, localize in cell nuclear and inhibit of polymerase opens
access to development of novel nonꢀcopper metal complexes
with less toxicity.
(5Z,5’Z)ꢀ1,2ꢀbisꢀ[3ꢀ(propenꢀ2ꢀyl)ꢀ5ꢀ(pyridineꢀ2ꢀylmethylene)ꢀ
3,5ꢀdihydroꢀ4Hꢀ4ꢀoxoimidazolꢀ2ꢀyl)ꢀthio]ꢀethan(L2):Brightꢀ
yellow solid (92%);mp 187ºС. 1H NMR (400 MHz, CDCl3) δ
8.65(m, 2H, HαꢀPy + Hβ’ꢀPy,), 7.62 (t, J=7.5 Hz, 1Н, HβꢀPy,),
7.19 (m, 1Н, HγꢀPy,), 7.14 (s, 1H, CH=), 7.12 (s, 1H, CH=), 5,
82 (m, 1Н, СН=), 5.23 (m., 2Н, СН2=), 4.23 (m, 2Н, CH2N)
3.89 (s, 2H, SꢀCH2) ppm. IR (KBr): υ =1720(C=O),
1680(C=N), 1640(C=C)cmꢀ1. Elemental analysis calculated
(%) for C26H26N6O2S2: C 60.44, H 4.68, N 16.27. Found C
60.14, H 4.48, N 16.03.
(5Z,
5’Z)ꢀ1,2ꢀbisꢀ[3ꢀphenylꢀ5ꢀ(pyridineꢀ2ꢀylmethylene)ꢀ3,5ꢀ
dihydroꢀ4Hꢀ4ꢀoxoimidazolꢀ2ꢀyl)ꢀthio]ꢀethan(L3):Yellow solid
(73%); mp 259ºС. 1H NMR (400 MHz, CDCl3) δ8.75(d,J=7.9
Hz, 1H,HαꢀPy,), 8.66(d, J=4.0 Hz,1Н, Hβ’ꢀPy), 7.81(td, J1=7.3
Hz, J2=2.3 Hz,1Н, HβꢀPy), 7.42(m, 3Н, НꢀPh), 7.29(m, 2Н, Нꢀ
Ph), 7.11(td, J1=7.5 Hz, J2=1.0 Hz,1H, HγꢀPy), 7.18(s, 1Н,
СН=), 3.11(t, J=7.5 Hz, 2Н, SCH2) ppm. IR (KBr): υ =
1710(C=O), 1670(C=N), 1640(C=C)cmꢀ1. Elemental analysis
calculated (%) for С32Н24N6S2O2:С 65.31, Н 4.08, N 14.29.
Found С 65.28, Н 4.10, N 14.11.
(5Z, 5’Z)ꢀ1,2ꢀbisꢀ[3ꢀcyclopropylꢀ5ꢀ(pyridineꢀ2ꢀylmethylene)ꢀ
3,5ꢀdihydroꢀ4Hꢀ4ꢀoxoimidazolꢀ2ꢀyl)ꢀthio]ꢀethan(L4): Brightꢀ
yellow solid (59%); mp 200ºС. 1H NMR (400 MHz, CDCl3) δ
8.67 (m, 2H, Hα’Hβ’ꢀPy) 7.67 (t, J = 7.00 Hz, 1Н, HβꢀPy), 7.22
(bs, 1Н, HγꢀPy), 7.08 (s, 1Н,CH=), 3.87 (s, 2H, СН2S), 2.66
(m, 1Н, СН(СН2)2), 1.04 (m, 4Н,СН(СН2)2). IR (KBr): υ =
1710 (C=O), 1670 (C=N), 1640 (C=C) cmꢀ1. Elemental analyꢀ
sis calculated (%) for C26H26N6O2S2: C 60.44, H 4.68, N
16.27. Found C 60.23, H 4.57, N 16.01.
EXPERIMENTAL SECTION
Chemistry. All solvents and chemicals were used as purꢀ
chased without further purification. The progress of all reacꢀ
tions was monitored on Silufol precoated silica gel plates
(with fluorescence indicator UV254) using ethyl acetate/nꢀ
hexane as solvent system. Melting points (mp) were taken in
open capillaries on a Stuart melting point apparatus SMP11
and are uncorrected. Proton (1H) NMR spectra were recorded
on a Bruker Avance 400 (400.13 MHz for 1H) using CDCl3 as
(5Z,5’Z)ꢀ1,2ꢀbisꢀ[3ꢀ(2ꢀazidoethyl)ꢀ5ꢀ(pyridineꢀ2ꢀylmethylene)ꢀ
3,5ꢀdihydroꢀ4Hꢀ4ꢀoxoimidazolꢀ2ꢀyl)ꢀthio]ꢀethan (L5): Yellow
1
solid (48%); mp 150ºС. H NMR (400 MHz, CDCl3) δ 8.66
(m, 2H, Hα, Нβ’ꢀPy), 7.65(td, J1 = 7.63 Hz, J2 = 1.76 Hz, 1H,
HϒꢀPy), 7.21 (m, 1H, HβꢀPy ), 7.15 (s, 1H, CH=), 3.95 (s, 2H
,СН2S ), 3.83 (t, J=5.87 Hz, 2H, NCH2CH2 N3), 3.61 (t,
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Page 6 of 8
J=5.67 Hz, 2H, NCH2 CH2N3).IR (KBr, cmꢀ1): υ = 2080 (N3
),1680 (С=O), 1630 (С=С), 1600 (С=N).Elemental analysis
calculated (%) for C24H22N12O2S2: C 50.16, H 3.86, N 29.25.
Found C 50.37, H 3.90, N 29.07.
and the International Associated Laboratory LIAꢀNUCPROT
1
2
3
4
(CNRSꢀRussia). We thank Cyandye LLC for providing diSulfoꢀ
Cy5 alkyne and Rostock group. The authors are thank Russian
Ministry of Science and Education (11.G34.31.0004, K1ꢀ2014ꢀ
022).
Synthesis of (5Z, 5’Z)ꢀ1,2ꢀbisꢀ[3ꢀ(3ꢀazidopropyl)ꢀ5ꢀ(pyridineꢀ
2ꢀylmethylene)ꢀ3,5ꢀdihydroꢀ4Hꢀ4ꢀoxoimidazolꢀ2ꢀyl)ꢀthio]ꢀ
5
1
ABBREVIATIONS USED
ethan(L6): Yellow solid (51%); mp 145ºС. H NMR (400
6
7
8
9
MHz, CDCl3) δ = 8.77 (d, J = 5.48, 1H, HꢀαPy), 8.66 (d, J =
8.02, 1H, Hβ'ꢀPy), 7.80(t, J= 6.46 Hz, 1H, HϒꢀPy), 7.32 (t, J=
5.38 Hz, 1H, HβꢀPy), 7.15 (s, 2H, CH=), 4.02 (s,2H, СН2S),
3.75 (t, J = 6.85 Hz, 2H, CH2N3), 3.43 (t, J = 6.26 Hz, 2H,
NCH2), 1.97 (m, 4H, NCH2 CH2CH2N3). IR (KBr, cmꢀ1): υ =
2092 (N3), 1730 (С=O, C=N), 1660(С=C).Elemental Analysis
calculated (%) for C26H26N12O2S2: C 51.81, H 4.35, N 27.89.
Found C 51.48, H 4.35, N 27.75.
General method for the synthesis of complexes 1-6.To a
solution of 0.068 mmol of selected ligand L1-L6 in 2 ml of
CH2Cl2 (placed in a glass tube with approximately 1 cm diamꢀ
eter) pure EtOH (1 ml) was slowly added, in order to form a
diphasic system. Then a solution of equivalent amount of
CuCl2·2H2O (0.136 mmol) in 2–3 ml of EtOH was added
down the tube side, without mixing the ligand and salt soluꢀ
tions. Thereafter the tube was covered and kept for crystal
solid precipitation. The solid was filtered and dried in air.
1.Brown crystals (45%); mp> 340 °C. Elemental analysis
calculated (%)for C26H28N6O2S2 *CuCl2*CuCl: C 41.41, H
3.74, N 11.14. Found C 41.30, H 3.77, N 11.05.
DAPI,
TUNEL, TdTꢀmediated dUTP Nick End Labeling; CMꢀ
H2DCFDA, 5ꢀ(andꢀ6)ꢀchloromethylꢀ2',7'ꢀdichlorodihydroꢀ
2ꢀ(4ꢀamidinophenyl)ꢀ1Hꢀindoleꢀ6ꢀcarboxamidine);
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fluorescein diacetate, acetyl ester; TRAP, telomeric repeat
amplification protocol; PI, propidiumiodide; SWNTs, single
walled carbon nanotubes.
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2. Brown crystals (40%); mp>340 °C. Elemental analysis
calculated (%)for C26H24N6O2S2* CuCl2*CuCl: C 41.63, H
3.23, N 11.15. Found: C 41.38, H 3.30, N, 10.97.
3.Brown crystals (55%); mp> 340 °C. Elemental analysis
calculated (%)for C32H24N6O2S2*CuCl2*CuCl: C 46.75, H
2.94, N 10.22. Found: C 46.62, H 2.99, N 10.27.
4. Brown crystals (38%); mp 200 °C. Elemental analysis calꢀ
culated (%)forC26H24N6O2S2 *CuCl2*CuCl:
C 41.63, H
3.23, N 11.15 . Found: C 41.39, H 3.30, N 11.02.
5.Brown crystals (35%); mp> 340 °C. Elemental analysis calꢀ
culated (%)for C24H22N12O2S2 *CuCl2*CuCl:
35.67, H2.74, N 20.80 . Found: C 35.73, H 2.56, N20.50.
C
6.Brown crystals (30%); mp> 340 °C. Anal. Elemental analyꢀ
sis calculated (%)for С26H26N12O2S2Cu2Сl3: С 37.35, Н 3.13,
N 20.10. Found: С 37.24, Н 3.29, N 20.02.
ASSOCIATED CONTENT
Supporting Information. Synthesis details, structural data, bioꢀ
chemical in vitro and in vivo data, materials and methods, abbreꢀ
viations supplied as Supporting Information. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* Phone: +7 495 9394020. Fax: +7 495 9328846. Eꢀmail: majouꢀ
ga@org.chem.msu.ru, zvereva@genebee.msu.ru
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Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manuꢀ
script. / ‡These authors contributed equally.
ACKNOWLEDGMENTS
The study was supported by Russian Foundation for Basic Reꢀ
search (12ꢀ03ꢀ33148, 12ꢀ04ꢀ00988ꢀа, 14ꢀ04ꢀ01092ꢀa); PNR 5.13;
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Cu
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In vivo anticancer activity
DNA damage in nucleus
Nuclear localization
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