Novel Copper-Containing Cytotoxic Agents Based on 2-Thioxoimidazolones

Article abstract of DOI:10.1021/acs.jmedchem.0c01196

A series of 73 ligands and 73 of their Cu+2 and Cu+1 copper complexes with different geometries, oxidation states of the metal, and redox activities were synthesized and characterized. The aim of the study was to establish the structure-activity relations

Full text of DOI:10.1021/acs.jmedchem.0c01196

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Article
NOVEL COPPER-CONTAINING CYTOTOXIC
AGENTS BASED ON 2-THIOXOIMIDAZOLONES
Olga Krasnovskaya, Dmitry A Guk, Alexey Naumov, Vita N. Nikitina, Alevtina Semkina, Kseniya
Yu. Vlasova, Vadim Pokrovsky, Oksana O Ryabaya, Saida Karshieva, Dmitry A Skvortsov, Irina
Zhirkina, Radik Shafikov, Petr Gorelkin, Alexander Vaneev, Alexander Erofeev, Dmitrii Mazur,
Viktor Aleksandrovich Tafeenko, Vladimir I. Pergushov, Mikhail Melnikov, Mikhail A. Soldatov, Victor
V. Shapovalov, Alexander V. Soldatov, Roman Akasov, Vasily Gerasimov, Dmitry A Sakharov,
Anna A. Moiseeva, Nikolay Vasil'evich Zyk, Elena K. Beloglazkina, and Alexander G. Majouga
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01196 • Publication Date (Web): 28 Sep 2020
Downloaded from pubs.acs.org on September 28, 2020
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Soldatov, Mikhail; Southern Federal University, The Smart Materials
Research Institute
Shapovalov, Victor; Uznyj federal'nyj universitet, Department of Physics
Soldatov, Alexander; Uznyj federal'nyj universitet,
Akasov, Roman; National University of Science and Technology MISIS; I
M Sechenov First Moscow State Medical University
Gerasimov, Vasily; Dmitry Mendeleev University of Chemical Technology
of Russia
Sakharov, Dmitry; Dmitry Mendeleev University of Chemical Technology
of Russia
Moiseeva, Anna; Lomonosov Moscow State University Department of
Chemistry, Chemistry
Zyk, Nikolay; Lomonosov Moscow State University Department of
Chemistry, Chemistry
Beloglazkina, Elena; Lomonosov Moscow State University, Chemistry
Majouga, Alexander; Lomonosov Moscow State University, Chemistry;
Dmitry Mendeleev University of Chemical Technology of Russia; National
University of Science and Technology MISIS
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NOVEL COPPER-CONTAINING CYTOTOXIC AGENTS
BASED ON 2-THIOXOIMIDAZOLONES
1,2
*
2
2
2
Olga O. Krasnovskaya ‡, Dmitry A. Guk ‡, Alexey E. Naumov , Vita N. Nikitina , Alevtina S.
3,4
2
5,6
5
5
Semkina , Kseniya Yu. Vlasova , Vadim Pokrovsky , Oksana O. Ryabaya , Saida S. Karshieva ,
2
,7
2
2
1
Dmitry A. Skvortsov , Irina V. Zhirkina , Radik R. Shafikov , Petr V. Gorelkin , Alexander N.
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Vaneev , Alexander S. Erofeev , Dmitrii M. Mazur , Viktor A. Tafeenko , Vladimir I. Pergushov ,
2
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Mikhail Ya. Melnikov , Mikhail A. Soldatov , Victor V. Shapovalov , Alexander V. Soldatov ,
1,9
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Roman. A. Akasov , Vasily M. Gerasimov , Dmitry A. Sakharov , Anna A. Moiseeva , Nikolay V.
Zyk , Elena K. Beloglazkina and Alexander G. Majouga
2
2
1,2,10
1National University of Science and Technology MISIS, Department of Materials Science of Semiconductors and
Dielectrics, Leninskiy prospect 4, Moscow 101000, Russia
2Lomonosov Moscow State University, Chemistry Department, Leninskie Gory, 1/3, Moscow, 119991, Russia
3Pirogov Russian National Research Medical University, Department of Medical Nanobiotechnologies,
Ostrovityanova 1, Moscow 117997, Russia
4Serbsky National Medical Research Center for Psychiatry and Narcology, Department of Basic and Applied
Neurobiology, Kropotkinskiy 23, Moscow 119991, Russia
5N.N. Blokhin National Medical Research Center of Oncology оf the Ministry of Health of the Russian Federation,
Kashirskoe highway 23, Moscow 115478, Russia
6
People's Friendship University, Moscow, Russia, Miklukho-Maklaya 6, 117198, Moscow, Russia,
7
Higher School of Economics, department of biology and biotechnologies, Myasnitskaya 13, Moscow 101000,
Russia
8The Smart Materials Research Institute Southern Federal University Sladkova, 178/24 Rostov-on-Don, 344090,
Russia
9
I.M. Sechenov First Moscow State Medical University, Trubetskaya 8-2, Moscow 119991, Russia
10Mendeleev University of Chemical Technology of Russia, Miusskaya sq. 9, Moscow 125047, Russia
ABSTRACT: A series of 73 ligands and 73 of their Cu+2 and Cu+1 copper complexes with different geometries, oxidation
states of the metal, and redox activities were synthesized and characterized. The aim of the study was to establish the
structure-activity relationship within a series of analogues with different substituents at the N(3) position, which govern
+2
+1
the redox potentials of the Cu /Cu redox couples, ROS generation ability, and intracellular accumulation. Possible
cytotoxicity mechanisms, such as DNA damage, DNA intercalation, telomerase inhibition, and apoptosis induction have
been investigated. ROS formation in MCF-7 cells and 3D spheroids was proven using Pt-nanoelectrode. Drug
accumulation and ROS formation at 40-60 µm spheroid depths was found to be the key factor for the drug efficacy in the
+2
+1
3D tumor model, governed by the Cu /Cu redox potential. A nontoxic in vivo single dose evaluation for two binuclear
+
1
+2
mixed-valence Cu /Cu redox-active coordination compounds, 72k and 61k, was evaluated.
INTRODUCTION
diagnosis of malignant pathologies, including head and
Organometallic complexes are one of the most
promising types of anticancer drugs. Monovalent and
divalent copper complexes have been investigated in the
last few years as less toxic and cheaper alternatives to
1
5
neck cancer , as well as the hallmark of Alzheimer
16-18
disease, amyloid-β (Aβ).
The redox properties of
copper ions, their ability for intracellular reduction, their
selective accumulation in hypoxic areas, and their
stability in blood flow means copper-containing
therapeutic agents can be used not only for therapeutic
1
,2
+1
platinum complexes. Two copper redox states (Cu and
+
2
Cu ), different possible geometries around the metal
center (tetrahedral, square-planar and octahedral) and a
variety of cytotoxicity mechanisms (proteasome activity
1
9
purposes but also as diagnostic and theranostic agents.
2
3,4
Due to the higher biocompatibility of copper-containing
anticancer drugs in comparison with classical platinum
agents, Cu-based therapy are supposed to be well
tolerated and does not induce substantial body weight
inhibition, telomerase inhibition, formation of reactive
oxygen species (ROS), DNA degradation,6 DNA
intercalation,
5
7
8
and
paraptosis )
make
copper
coordination compounds especially promising antitumor
therapeutic agents.
2
0
loss. Several clinical trials of various redox-active
disulfiram and copper gluconate combinations are
Copper-containing coordination compounds are not
2
1-23
9
currently ongoing.
only effective antitumor agents, but are also effective as
1
0
11
12
A key factor for drug toxicity is having a high cellular
antimicrobial,
anti-tuberculosis,
anti-malarial,
24
antifugal and anti-inflammatory drugs. 64Сu-labeled
1
3
14
uptake. Drug uptake is influenced by such factors as
steric mobility, the oxidation state of the metal cation, the
geometry of the coordination compound, ligand
coordination compounds are promising positron
emission tomography PET imaging agents for the
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Scheme 1. Synthetic Routes to Type 1-3 coordination compounds.
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lipophilicity, and redox activity.25 Cu+1 coordination
compounds show higher cytotoxicity and cell
5(4H)-ones) (Type 3) were prepared according to
1
13
Scheme 1. The H and C NMR and mass spectrometry
data were consistent with the proposed structures of the
ligands (Tables S1-S3, Figures S46-S118). Purity of
targeted compounds was confirmed by HPLC (Tables S4-
S15, Figures S1-S8). The type 3 ligands were all cis-
isomers with magnetically equivalent protons in the
ethylene spacer, except for the ortho-fluoro-substituted
ligands 60, 66 and ligand 65. The Cu , Сu complexes
were prepared via solvent layering technique with a
copper chloride dihydrate: ligands in a 1:1 (1:2) molar
ratio in butanol-1/chloroform system. A series of 73
copper 2-complexes were synthesized and characterized.
The color of the complexes obtained differs from bright
red to black, depending on the copper oxidation state. By
varying the nature of the organic motifs and the donor
atom set, we developed three types of copper
coordination compounds with different geometries: Type
+
2
26
accumulation than Сu
analogues.
Typically,
monovalent Сu+1 coordination compounds are
2
7
+1
phosphine-based due to their high stability , other Cu
coordination compounds are poorly described.
Intracellular redox processes determine cellular
viability, where a disturbance in redox homeostasis may
affect cell death.28 The biological activity of the copper
+2
+1
coordination compound is mostly affected by its
+2
+1
intracellular redox activity, therefore the Cu /Cu redox
potential is one of the most important physicochemical
parameters upon which the effectiveness of the copper-
based drug depends. The relationship between the redox
potentials and both the coordination geometry and
nature of the donor atoms has long been a subject of
2
9
interest in copper chemistry, especially because copper-
containing antitumor drug efficiency correlates with the
aforementioned physicochemical parameters.
+
1.5
+1.5
1 are binuclear Cu /Cu
coordination compounds,
In this work, we set the task of stabilizing the unstable
Cu+1 state by varying the donor atoms and ligand
substituent nature in organic core, as well as the
geometry and the redox activity of the coordination
compounds. We conducted a large-scale study of how the
initial ligand structure influences the copper oxidation
state and the mechanism of cytotoxicity.
with distorted trigonal bipyramidal geometry, and one
bridging chlorine atom located at the same distance from
both metal atoms; Type 2 are mononuclear Cu
+
2
coordination compounds with tetrahedral geometry; and
binuclear type 3 coordination compounds have various
oxidation states and geometries of the copper ions:
+
2
+2
+2
+1
+1
+1
Cu /Cu , Cu /Cu , Cu /Cu . UV-Vis data and stability
studies of coordination compounds are presented at
Figures S14-S24, Table S17.
Recently, we have proven that copper 2-
thioxoimidazolone complexes exhibit antitumor activity
both in vitro and in vivo. The binuclear mixed-valence
Organic motif – copper oxidation state. We assumed
that the presence of strong electron acceptors (R) in
organic core stabilize copper in the monovalent state.
Among the three types of coordination compounds (Table
1), the influence of a substituent in the third position of
the 2-thioxoimidazolidin-4-one ring on the copper
oxidation state is the strongest in type 3, where all three
+
1
+2
Cu /Cu complex showed a telomerase inhibition ability
along with the highest in vivo tumor CA-755 growth
3
0
inhibition. In this work, we have greatly expanded this
series, having synthesized three types of coordination
compounds based on 2-thioxoimidazolones, which are
different in geometry, the oxidation state of copper ions,
the redox behavior and a mechanism of cytotoxic activity.
+
2
+2
+2
+1
+1
+1
possible subtypes (Cu /Cu , Cu /Cu , Cu /Cu ) are
present. Large electron-withdrawing substituents
effectively stabilize Cu (70k-73k). Electron-donating
RESULTS AND DISCUSSION
+1
+2
groups stabilize Cu (52-57k). 58k-69k are mixed-
valence copper coordination compounds. Butyl alcohol is
assumed to be a reducing agent
Synthesis and Characterization. Three types of
ligands based on 2-thioxoimidazolidin-4-ones (Type 1),
-(methylthio)-1H-imidazol-5(4H)-ones (Type 2) and
,2'-(ethane-1,2-diylbis(sulfanediyl))bis-1H-imidazol-
2
2
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35k
41k
46k
72k
3
k
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Figure 1. ORTEP representations of the X-ray structure X-ray crystal structure for 3k, 35k, 41k, 46k, and 72k.
Figure 2. X-ray Absorption Near Edge Structure spectra of Type 3 coordination compounds.
for the copper ion, since the complexation reaction in
acetonitrile does not lead to the formation of monovalent
copper. The structures of the five coordination
compounds in the solid state were established using X-ray
crystallography, the copper oxidation state was
determined via X-ray absorption near edge structure
by two nitrogen atoms and a sulfur atom, which leads to
the binuclear coordination compound formation with a
Cu – Cu distance of 2.519 Å plus one bridging chlorine
atom equidistant from both metal centers (2.346 Å and
2.409 Å).
In Type 2 coordination compounds (P21/n), copper is
coordinated through the two N atoms, forming a
tetrahedral geometry complex. Coordination compound
72k is a binuclear Cu /Cu compound with trigonal-
planar coordination geometry, with Cu-Cl bounding 2.151
Å, Cu-N bounding 1.95 Å and 1.97 Å. (P2
XANES spectroscopy. X-ray absorption near edge
structure spectroscopic studies gives information on the
local chemical environment of the absorbing atom in
solids, liquids or gases. At the K (or L3,2-) edges of a
selected atom, strong resonances appear below the
ionization potential, which are assigned to the transitions
of the 1s (or 2p) electron to unoccupied molecular
3
1
(
XANES) in solid samples, cyclic voltammetry and
electron paramagnetic resonance (EPR) spectra were
used for the DMF solutions of the coordination
compounds.
Crystallography. Five metal complexes 3k (Type 1),
5k, 41k, 46k (Type 2), and 72k (Type 3) were
characterized using single-crystal X-ray crystallography.
Their thermal ellipsoid plots and atom numberings are
shown in Figure 1, and the selected bond lengths are
given in Tables 1, S18. Additionally, the X-ray data for 53k
and 54k have been described previously. In the Type 1
coordination compounds (Aba2), copper is coordinated
+
1
+1
1
1
2 2).
3
3
2
Table 1. Selected bond lengths (Å) for coordination compounds 3k, 35k, 41k, 46k, 72k
3
k
35k
41k
2.229(2)
2.216(2)
2.029(5)
46k
72k
2.154(6)
–
1.99(2)
1.90(1)
–
Cu1 – Cl1
Cu1 – Cl2
Cu1 – N1
2.346(5)
–
2.214(1)
2.222(1)
2.038(3)
1.981(3)
2.198(2)
2.231(1)
2.029(4)
1.964(4)
2.05(1)
1.94(1)
2.519(4)
2.291(5)
2.409(5)
1.65(1)
1.68(1)
Cu1 – N2
2.025(4)
Cu1 – Cu1A
Cu1 – S1A
Cl1 – Cu1A
S1 – C10
–
–
–
–
–
–
–
–
–
1.709(3)
–
–
1.707(5)
–
–
1.708(5)
–
1.67(3)
1.70(3)
S1A – C10A
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orbitals. An analysis of these resonances gives
Table 2. Various types of coordination compounds
obtained.
3
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1
1
1
1
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1
1
1
1
1
2
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6
structure.3
3
information
about
the
electronic
Furthermore, in the XANES spectra, the mean free run
length of the photoelectron, due to its small kinetic energy
Type 1
Type 2
Type 3
(
Ec<50–100 eV), provides a high contribution to multiple
scattering events of the photoelectron allowing to probe
the three-dimensional structure (3D) around the
absorbing atom. The position of the absorption edge not
only depends on the charge (oxidation state/valence) of
the absorbing atom but also on the local atomic structure
3
4
0
1
2
3
4
5
6
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1
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3
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0
1
2
3
4
5
6
7
8
9
0
(bond lengths and angles, coordination numbers,
symmetry, etc.).
The XANES spectra of Type 3 coordination compounds
are presented in Figure 2. Based on an intensity at 8984
eV, we can conclude that the coordination compounds
+
1
+1
+1
+1
7
1k (Cu /Cu ) and 73k (Cu /Cu ) have similar local
№
X
№
X
№
X
Y1
Cl
Y2
Cl
atomic and electronic structures for the copper atoms
+2
+1
(
Figure 2, A). 61k (Cu /Cu ), in comparison with
52
k
1
2
3
4
k
k
k
k
4-F
3-F
2-F
4-Br
3-Br
2-Br
30k
31k
32k
33k
34k
35k
36k
37k
38k
39k
40k
41k
42k
43k
44k
45k
4-F
4-OMe
4-OEt
3,4-OMe
4-tBu
2,4,6-Me
2-tBu
4-F
samples 71k and 72k (Figure 2, B), shows an increase in
the “shoulder” height at 8984 eV and a drop in the
maximum absorption, which implies a similar ligand
environment and different geometries of copper atoms in
5
k
3
3-F
54
k
2-F
5
k
5
4-Br
3-Br
2-Br
7
7
2k and 61k, while the X-ray absorption data of 71k and
2k confirms similar local atomic structures. The X-ray
56
k
5k
absorption spectra of compounds 59k, 58k and 61k
confirm similar local atomic structures of the
coordination compounds and demonstrate an increase in
the “shoulder” height at 8984 eV and a drop in the
maximum absorption when compared with 71k. The
5
k
7
6
k
2
F
-Br, 4-
2-Br, 4-
F
58
k
7k
8k
5
k
6
k
9
4-Cl
4-Cl
3-F
+
2
+2
Cu /Cu compound 52k demonstrates a structural
difference from the mixed-valence 58k, 61k and
monovalent 71k, which indicates a different geometry
and a higher Cu oxidation state (Figure 2, C). The X-ray
absorption data of the difluoro-substituted coordination
compounds 66k and 67k suggests that the complexes are
mixed-valenсe and have different local atomic structures.
Considering the NMR spectra, compound 66k is a trans-
isomer, and 67k has a cis-configuration. The oxidation
states of the cations are also determined from the
reference samples. The XANES spectra of compounds 3k,
0
9
k
3-Cl
3-Cl
2-F
61
k
10k
1k
12k
3k
14k
5k
2-Cl
2-Cl
3-Br
6
k
6
k
2
1
4-OMe
4-OEt
4 -OMe
4-OEt
2-Br
3
2-Cl
3,
4-
3,
OMe
4-
64
k
65
k
66
k
67
k
2-OMe 4-
Cl, 5-Me
2-Br, 4-F
1
OMe
4-tBu
4-tBu
Cl
-
1
2-tBu
2-tBu
2,5-F
1
4k (Type 1), 43k (Type 2) and the XANES spectra of
16k
17k
4-NO
2
2-OMe
-Cl, 5-
3,5-F
4
Type 3 coordination compounds compared with the
2-Me, 3-Cl
2
-Me,
68
k
reference samples of copper oxides CuO, Cu
in the SI (Figures S25-S26).
2
O are given
46k
47k
3-Cl, 4-F
4-NEt2
Cyclic voltammetry. To assess the influence of the
redox properties on the biological activity, as well as to
confirm the oxidation state of copper ions in Type 3
coordination compounds, cyclic voltammetry (CV) and
voltammetry with a rotating disk electrode (RDE) were
used. Due to the low water solubility of the complexes,
2-OMe,
2-Me, 3-
Cl
69
k
18k
4-SMe
4
-Cl, 5-
Me
3
F
-Cl, 4-
2,4,6
Me
-
1
9k
48k
DMF was chosen as the solvent, with 0.1 M Bu
4
NClO as
4
the supporting electrolyte.
20k
2-Me,
49k
50k
51k
4-SMe
2,5-F
3,5-F
70
k
71
k
72
k
73
k
3-Cl
3
2
-Cl
,4,6-
All coordination compounds of Type 1 demonstrated
2
2
2
2
1k
2k
3k
4k
4-Br
quasi-reversible Cu+1.5/Cu+1.5 → Cu /Cu reduction at
+1
+1
Me
-
-
+
1.5
+1.5
potentials Epc ~ 0.00 - 0.18V, and Cu /Cu
Cu /Cu oxidation at potentials Epa ~ 0.34 - 0.58 V
→
4-SMe
4-Cl
+
2
+2
4-J
3-Cl, 4-F
(Figure S27, Table S19). RDE voltammograms prove the
oxidation and reduction process take place in Type 1
coordination compounds (Figure S28).
All coordination compounds of Type 2 showed quasi-
2-Me,
4-NEt
2
2
5k
2,6-Cl
+2
reversible cathodic peaks corresponding to the Cu
→
+
1
Cu reduction at potentials Epc ~ 0.32 - 0.41 V in the
cathodic region (Figure S29, Table S20). The RDE
voltammogram proves that only the oxidation process
occurs in the coordination compound of Type 2 (Figure
S30).
2,4,6-
Cl
2
2
2
2
6k
7k
8k
9k
2,6-Br
2,5-F
3,5-F
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. EPR spectra of Type 3 coordination compounds.
Figure 4. Cyclic voltammograms of Type 3 coordination compounds.
All coordination compounds of Type 3 showed similar
voltammograms, regardless of the oxidation state of
copper (Figures 4, S31, Table S21). Two reversible waves
were observed at the E range 0.3-0.6 mV vs Ag/AgCl,
which could be ascribed to the one-electron oxidation of
cations in the coordination compound based on their
different magnetic susceptibilities. To exclude the
spontaneous oxidation of copper cations, which was
observed in electrochemical experiments, EPR
measurements were made in degassed solutions in the
absence of oxygen. The EPR spectra for coordination
+
1
+2
35
Cu to Cu and chlorine oxidation, respectively.
Recently, Schneider et al. reported a mixed-valence
+
2
+2
+2
+2
compounds 53k (Cu /Cu ), 56k (Cu /Cu ), 58k
+
2
+1
+2
+1
+2
+1
+1
pyridylamide-based complex, and the oxidation of Cu ,
(Cu /Cu ), 59k (Cu /Cu ), 70k (Cu /Cu ), and 72k
(Cu /Cu ) were recorded. In the DMF solution for 70k
there is an admixture of Cu (Figure 3), whereas for
paramagnetic Cu the cations are absent in the degassed
+
1
36
+1
+1
Cu centers was irreversible. In our case, both
reversible oxidation and reduction are observed.
RDE voltammogram allows for the identification of
+
2
+
2
+2
+1
different redox transition currents Cu /Cu (Figure
DMF solution of the coordination compound 72k
+1
+1
+1
+1
S32): for Cu /Cu complexes (71k) only the oxidation
(Cu /Cu ). Despite the fact that the coordination
+1
+2
+1
+1
current is observed (anodic process Cu → Cu ), for
Cu /Cu (52k) complexes only the reduction current is
observed (cathodic reaction Cu → Cu ), for mixed-
valence complexes Cu /Cu (61k) the voltammogram
exhibits both anodic and cathodic limiting currents. Most
complexes of Type 3 are mixed-valence, which implies the
high stability of the resulting Cu /Cu redox system.
Despite the fact that 72k in a solid form is a Cu /Cu
compounds 70k and 72k are monovalent Cu /Cu when
in solid form, as soon as they appear in solution,
spontaneous oxidation occurs with the formation of a
+2
+2
+2
+1
+
1
+2
+2
+1
mixed-valence complex Cu /Cu . The coordination
+
2
+2
compounds 53k and 56k are paramagnetic (Cu /Cu ),
and mixed-valance coordination compounds 58k and
59k have an intermediate position between them due to
the presence of one paramagnetic Cu and one
diamagnetic Cu centers (Figure 3). Additionally, for
+
2
+1
+
1
+1
+2
+
1
complex, which was confirmed by the XANES and X-ray
data (Figures 1, 2), the resulting rotating ring-disk
electrode voltammogram confirms the presence of two
+2
+2
coordination compounds 56k (Cu /Cu ) and 59k
+
1
+2
(Cu /Cu ), the axial and rhombic symmetry of metal
copper
cations
at
different
centers was evaluated (Figure S33).
oxidation states (Figure S32).
instantaneous oxidation of one of the copper cations Cu
Apparently, the
Biological Assays. In vitro cytotoxicity, cellular
accumulation/distribution, protein binding, DNA
intercalation, nuclease activity, apoptosis test, telomerase
inhibition, reactive oxygen species formation in the 2D
cell monolayer and a 3D spheroid model of the copper
complexes (all three types) were studied to clarify the
cytotoxicity mechanism of the synthesized coordination
compounds.
+1
occurs in solution.
EPR spectroscopy. The EPR spectroscopy is an
invaluable method for characterizing the ligand
environment and oxidation states of paramagnetic metal
3
7
complexes. EPR spectroscopy enables unambiguously
+
1
+2
distinguishing Cu (d10, S = 1) /Cu (d9, S = 1/2) metal
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Table 3. Cytotoxicity data of the coordination compounds.
IC50, μM
HEK-
93
МCF-7
3.4±0.4
2.8±0.6
9.2±8.1
9.6±2.9
2.8±0.4
2.9±0.3
9.5±0.8
1.9±0.4
1.4±0.1
2.5±0.5
2.3±0.2
6.8±0.6
5.2±1.0
6.8±0.6
5.1±0.2
1.5±0.2
~15
A-549
4.4±0.3
4.7±0.3
5.2±0.6
24±6.5
3.0±0.5
4.2±1.2
8.3±1.8
5.7±0.6
1.7±0.1
3.3±0.7
3.8±1.4
7.7±0.5
7.7±0.9
6.7±1.5
8.1±2.3
2.3±0.4
~11
Va-13
HEK-293
МCF-7
A-549
Va-13
HEK-293
МCF-7
A-549
Va-13
2
1
2
3
4
k
k
k
k
1.9±0.1
1.6±0.2
2.1±0.2
1.3±0.5
2.4±0.4
0.9±0.1
6.0±2.5
2.3±0.3
1.1±0.1
0.8±0.1
1.6±0.1
2.6±0.4
5.1±2.4
6.3±0.3
4.1±0.2
0.07±0.0
-
2.6±0.2
2.6±0.2
2.5±0.1
9.2±1.8
2.7±0.5
0.9±0.1
2.25±0.1
1.3±0.2
1.9±0.3
>
5
5
5
4k.
5k
9.35±8.23
3±1
7.04±1.05
55±20
17.08±2.07
102±50
5.84±1.95
10±2.8
24k
28k
29k
30k
31k
32k
33k
34k
35k
36k
37k
38k
39k
40k
41k
15.3±1.8
18.1±2.2
~10
31.4±4
~14
17.7±1.4
~16
-
6k.
~14
~12
~54
~17
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-
~9
~15
~17
57k
58k
59k
60k
61k
62k
63k
64k
65k
66k
67k
68k
69k
70k
71k
72k
73k
6.48±1.17
19.4±17.87
31.66±3.7
18.17±4.4
5k
11±9
3.6±0.5
3.6±0.2
2.9±0.2
4.9±0.6
4.6±0.6
1.8±0.5
5±1
~10
1.5±0.7
4.0±0.5
4.3±0.4
3.3±0.3
9.4±0.7
9.0±0.7
6.8±3.0
6.6±1.4
5.4±2
8.1±2.5
~3
6
7
k
k
7±1
1.8±0.2
2.4±0.2
5.1±0.4
5.2±0.8
4.4±0.1
9.5±0.7
8.5±0.7
6.6±0.4
9.6±5.3
9.8±0.9
9.8±0.9
0.8±0.0
2.6±0.4
0.5±0.1
9.2±0.7
5.1±0.2
12.11±1.18
1.24±0.20
1.6±0.6
1.0±2.8
~8
1.7±0.1
1±0.4
1.1±0.1
2.2±0.8
5.7±0.6
3.5±0.2
10.24±3.14
0.73±0.10
1±2
8k
0.3±0.1
9
1
k
1.7±0.3
8.2±1.3
8.1±2.3
18.68±1.32
2.24±0.20
1.3±4.7
1.4±0.9
~23
0k
4.1±0.2
1
0
.02±
2.04±
1.85±
0.91±
11k
1.7±0.3
0.2±0
2.87±1.81
.47
0.54
0.54
0.23
1.05±
1.93±
1.19±
1
1
2k
3k
~5
0.49±0.10
0
.40
0.40
0.30
3.8±1
-
~12
~54
~48
-
15k
4.3±0.3
3.5±0.2
2.3±0.3
1.6±0.1
2.6±0.3
2.3±0.2
4.2±0.2
4±1
1.4±0.2
1.7±0.3
2.4±0.1
-
2.8±1.2
~12
1
1
6
1.6±0.1
3.8±1.4
4.6±0.6
-
6k
2.1±0.3
-
~12
~54
~48
17k
46k
47k
48k
49k
50k
-
-
-
-
-
~19
~3
~3
~9
~3
~`14
~11
~12
~2
~16
~13
~14
~3
-
~6
~11
~8
1.3±0.2
1
1
8k
9k
58±4
-
95±32
~13
45±14
~57
24±7
~31
~4
1.5±0.5
0.5±0.0
-
2.0±0.4
0.6±0.0
~6
1.7±0.5
1.3±0.2
~11
0.8±0.1
~7
20k
-
~3
~8
2
2
1k
2k
-
~55
~75
~66
~10
~14
-
~65
~3
~3
Dox
0.01
0.06
0.37±0.19
0.2±0.04
52k.
3.38±0.55
10.8±3.7
5.61±1.21
19.7±2.4
7.89±0.38
3.03±0.07
4.2±
9.7±
2
3k
2.9±0.9
10.7±2.1
CDDP
0.50±0.05
0.62±0.15
10.96±1.42
5.48±1.44
1
.0
2.9
5
3k
>50
21.1±2.7
+
2
+2
In vitro Anticancer Activity. МТТ data for the
(except Cu /Cu coordination compounds 52k and
53k), while demonstrating cytoplasm accumulation.
Among the
coordination compounds are presented in Table 3.
Additionally, for a number of coordination compounds, a
fluorescent proteins detection assay was used. The
Type 3 coordination compounds, it is clearly seen that
+
2
+2
cytotoxicity was measured by the live-imaging of MCF-7
Cu /Cu (52k and 53k) coordination compounds are
Katushka cells.38 All coordination compounds had a
+1
+1
+2
+1
low-penetrating, while Cu /Cu , Cu /Cu (72k and
61k) show
better cellular accumulation, and as a result are stronger
cytotoxic agents (Tables 4, S23).
DNA damage/DNA intercalation. DNA is
therapeutic target for most metal-based agents, where
nuclease activity is one of the most common antitumor
activities for copper-containing anticancer drugs.
have tested the DNA cleavage ability of all 3 types of
coordination compounds (Tables 4, S24, Figure S34).
None of the Type 1 complexes showed nuclease activity,
while the representatives of Type 2 proved to be chemical
nucleases. The geometry of Type 1 coordination
compounds probably does not allow them to exhibit
nuclease activity, while the small size of Type 2
coordination compounds allows effective DNA
interaction. The lead compound 61k (type 3) showed a
weak nuclease activity, meaning it is not the main
mechanism of cytotoxic action. The lead compound 72k
showed no nuclease activity at all. Additionally, DNA
intercalation was studied by classic competitive binding
higher toxicity than the original ligands, however, several
ligands (4 - 7, 10, and 16) showed comparable
cytotoxicity (Table S22). Most of the compounds 1k - 73k
have cytotoxicity comparable to cisplatin. The mixed-
a
+
2
+1
valence Cu /Cu coordination compound 61k and
+
2
+1
Cu /Cu 72k can be considered as lead compounds.
3
9-41
Binuclear coordination
We
+2
+2
compounds Cu /Cu are generally less toxic than the
mixed-valence and monovalent analogs. A change in the
substituent’s nature and its position has a strong effect on
the cytotoxic activity; our task was to establish the nature
of this influence and the cytotoxicity mechanism.
Intracellular
accumulation/distribution.
An
intracellular accumulation/distribution of all three types
of coordination compounds was studied using the atomic
absorption spectroscopy (AAS) technique.
The Type 1 coordination compounds are the most
penetrating compounds, with high intracellular
accumulation for metal-containing drugs (~40%),
accumulating in the nuclei and mitochondria. Type 2 and
4
2
3
coordination compounds are also high-penetrating
assays with ethidium bromide. Most coordination
ACS Paragon Plus Environment

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Page 8 of 34
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
k
8k
11k
12k
16k
a)
b)
3
0k
36k
41k
44k 52k
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
53k
65k
61k
71k
72k
Figure 5. Apoptosis analysis of MCF-7 cells by Annexin V-FITC/PI staining after treatment with coordination compounds.
5
compounds can intercalate DNA with Kapp ~ 10 M, and
the values obtained correlate with those obtained for
coordination compounds of copper with anticancer
activity. Expectedly, organic ligands do not intercalate
DNA, except for cytotoxic ligand 16 (Table S25, Figures
S35-S37). However, a lack of correlation between the
ability to intercalate DNA and cytotoxicity makes the DNA
intercalation ability noncritical for the cytotoxicity of
studied ligands and coordination compounds.
BSA binding. BSA is a protein of choice by many
biochemists for studying drug-protein interaction and its
contribution to the distribution and metabolism of the
drug through different organs of the human body.44 We
have monitored quenching in the fluorescence properties
of BSA in the presence of chosen ligands and coordination
compounds. It is clear that the fluorescence intensity of
BSA
S26) suggested significant binding of the selected ligand
and complexes to BSA.
4
6
Telomerase inhibition. Since the previously described
4
3
+2
+1
mixed-valence Cu /Cu
2-thioxoimidazolone-based
coordination compound, which was an effective
3
0
antitumor drug in vivo, acted as a telomerase inhibitor,
the study of the ability of coordination compounds to
inhibit telomerase was of particular interest to us. Since
an increased telomerase activity in tumor cells has been
47
repeatedly proven, copper coordination compounds as
48
telomerase inhibitors is of interest.
Among the
representatives of all three types, coordination
compounds capable of inhibiting the telomerase enzyme
were found (1k, 36k and 40k). However, the lead
compound 61k showed the highest inhibition rate
(Tables 4, S27).
Cell death (apoptosis/necrosis). Induction of the
apoptosis by the complexes in the MCF-7 cancer cell line
was analyzed with the Annexin V-FITC/PI double staining
progressively decreases with the binding of copper
complexes (Figures S38-S40). Binding constants with
albumin were measured by the fluorescence emission
49
assay. The results are presented in the dot plots in
Figure 5. The level of phosphatidylserine quantified the
treatment of the MCF-7 cells with the complexes for 24
4
5
7
spectrum of BSA. The calculated values (Kb~10 ) (Table
Table 4. Investigation of the intracellular accumulation and various mechanisms of the antiproliferative
activity of coordination compounds.
Intracellular
accumulation*
DNA damage
(Nuclease
activity)**
DNA
Apoptosis****
Telomerase
intercalation***
inhibition*****
Type 1
Compound 6k, 8k, 16k
tested
5k, 8k, 10k, 1k, 2k, 4k-8k, 10k, 2k, 8k, 11k, 12k, 1k, 5k, 8k, 10k,
16k
12k, 16k
16k
16k
Cu+1.5/Cu+1.5
5
Result
~40%
No
K
b
~10 , except 2k, Early apoptosis
1k
– average
5
k
inhibition rate
Type 2
Compound 30k, 36k
tested
30k, 36k, 39k, 29k, 30k, 35k, 30k, 36k, 41k, 30k, 36k, 39k,
40k
40k
44k
40k
Cu+2
Result
~25%
Average
K
4
b
~105,
0k
except Late
36k, 40k
average
-
apoptosis
inhibition rate
+
2
+2
Type 3
Compound 52k, 53k (Cu /Cu ) 61k, 65k, 71k, 53k, 59k, 71k, 53k, 61k, 65k, 61k, 65k, 71k,
tested
57k,
61k,
65k 72k
72k
71k, 72k
72k
+2
+2
+1
+2
Cu /Cu
(Cu /Cu )
+1
+1
7
2k (Cu /Cu )
~6%; ~20%; ~24%
Result
Weak,
2k
K
b
~105,
no 59k
except Late
Apoptosis,
61k
inhibition rate
–
high
7
–
nuclease
activity
71k
necrosis
4T1 breast cancer cells, 24 hours of incubation; ** pUC18 plasmid DNA ***calf timus DNA+ EB competitive binding method;
*** MCF-7 breast cancer cells, 24 hours of incubation; ***** TRAP method
– 56% of
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Page 9 of 34
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a)
+2
+1
b)
+2
+2
Cu /Cu
Cu /Cu
60
МCF-7 A-549
60
МCF-7
A-549
5
4
3
0
0
0
5
4
3
2
0
0
0
0
+
2
+1
Cu /Cu
+
1
+1
CuI/Cu
20
Cu /Cu
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
0
10
0
+2
+1
Epc mV
Epc Cu /Cu , mV
Copper reducing facilitation
The increase of an electronic withdrawal of organic core
Figure 6. A) The effect of the electron-donor properties of the ligand core on the oxidation state of copper and cytotoxicity of coordination
compound. The higher the electron withdrawal of the ligand, the higher the copper reduction potential, the more stable the monovalent state
of the copper cation, and the higher the cytotoxicity. B) Dependence of the Cu /Cu redox potential on cytotoxicity within each subtype.
The higher the redox Cu /Cu potential, the easier the reduction reaction, and the higher the cytotoxicity of the coordination compound.
+
2
+1
+
2
+1
hours, exhibiting results as viable, early apoptosis, late
apoptosis and necrosis, and their percentages (Table
S28). All the complexes of Type 1 exhibited early
apoptosis; Type 2 and 3 exhibited late apoptosis. Thus,
these results confirm that copper complexes induce
apoptosis instead of necrosis in MCF-7 cancer cells.
Redox potential as a key factor of drug efficiency.
intracellular reduction of a copper-containing drug, along
with the inability of the
Cu - based drug to leave the cell. Recently, Anjum et
al. reported eight bis(thiosemicarbazone)-based copper
coordination compounds, and established some
structure-activity ratios. Electron-donating effects of
the disubstitutions on the diamine backbone leads to a
lowering in the Cu /Cu redox potential, and the authors
suggested that cytotoxicity could be controlled by the
redox potential. Based on this, we also suggested a
cytotoxic efficacy to be directly dependent on the
+
1
53
54
The ability of the organic сore to affect the Cu+2/Cu redox
potential and the drug accumulation was repeatedly
+1
+2
+1
5
0-53
64
proven by the Cu-ATSM series.
Radiolabeled Cu,
6
2
Cu and 60Cu coordination compounds based on
+
2
+1
thiosemicarbazone were used as a hypoxia-sensitive PET-
imaging agent, capable of accumulation in hypoxic
regions. Dearling et al.
proposed a mechanism of trapping and selectivity-
based around the redox behavior of the
Cu /Cu couple. In an attempt to further elucidate this
proposed mechanism, Blower et al. conducted extensive
structure–activity relationship (SAR) studies on a series
of thirteen similar CuATSM complexes. They found that
hypoxia selectivity strongly correlated with both the
electrochemical potential Cu /Cu .
In all three types of coordination compounds, the
redox behavior of copper cations was influenced by a
substituent in the aromatic ring. The dependence сan be
observed when the ortho- and para-halogen-substituted
coordination compounds of all three types are compared
(Table 5). An increase in the size and mesomeric donation
ability of the para-substituent leads to an increase in Epа
(Cu → Cu ) and a decrease in Epс (Cu → Cu ). The
influence of ortho substituents does not coincide with the
influence of para-substituents, and in some cases is
directly opposite, where an increase in the electron
5
0
+
2
+1
51
52
+1
+2
+2
+1
+2
+1
Cu /Cu reduction potential and the alkyl substitution
pattern on the backbone of the ligand. The sensitivity of
Cu-ATSM to hypoxia is achieved by irreversible anaerobic
+
1
withdrawing ability leads to an increase in Epа (Cu
Cu ) and a decrease in Epс (Cu → Cu ) (Table 4).
→
+
2
+2
+1
Table 5. The effect of the substituent in the aromatic ring of the ligand on the redox behavior of copper cations
in the coordination compound. (Epa - electron donation, the lower Epa, then the easier the redox-reaction; Epc
- Electron acceptation, the higher Epc, then the easier redox-reaction)
Para Type Epa
1
IC50, μM
(MCF-7)
Type Epc
2
IC50, μM
(MCF-7)
Type 3
Epa
Epc
IC50, μM
(MCF-
4-F
1k
0.34V
3.4±0.
30k
0.40/0.55V 1.8±0.5
58k 0.54/0.41 0.41/0.54 Mixed-
6.6±1.4
4
valence
4-Cl
-Br
8k
4k
0.45V
1.9±0.4 37k
0.36/0.58V ~5
72k 0.54/0.40
71k 0.55/0.45
-
-
Monovalent 0.6±0.0
+
1
+1
Cu /Cu
4
0.59/0.44V 9.6±2.9 33k
0.32/0.56V 4.4±0.1
2.0±0.4
Orto
Epc Cu+2 → Cu+1
Epa Cu+1→ Cu+2
Epc Cu+2 → Cu+1
0.41/0.12 0.12/0.41
0.56/0.41 0.41/0.56
2-F
3k
0.55V
9.2±2.1 32k
0.12/0,41V 5.2±0.8 60k
0.41/0.56V 2.6±0.3 62k
0.44/0.55V 8.5±0.7 63k
2.6±0.4
9.2±0.7
5.1±0.2
Mixed-
2-Cl
10k
6k
0.53/0.42V 2.5±0.5 39k
0.52/0.44V 2.9±0.3 35k
valence
+
2
+1
2-Br
0.55/0.44 0.44/0.55 Cu /Cu
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6
ROS level detection
Cell Rox Deep Red assay
ROS level detection
Single-cell electrochemical measurement in
MCF-7 cells using Pt-nanoetectrode
2.5
2
2
1.5
1
1.5
1
0
1
2
3
4
5
6
7
8
9
0
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1
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8
9
0
0.5
0
0
.5
0
2
k 6k 8k11k16k 30k36k40k41k44k 52k53k61k65k71k72k
2
k
8k 11k16k12k30k36k 41k44k
53k61k65k71k72k
Figure 7. ROS level quantification in MCF-7 cells by single cell H O level measurement by Pt-nanoelectrode and Cell
2
2
Rox Deep Red fluorescent assay.
0
0
.7
.6
20 µm
2
4
0 µm
0 µm
0.5
.4
0.3
0
60 µm
0 µm
8
0
0
.2
.1
0
7
2k
61k
71k
Figure 8. Electrochemical measurement of the ROS level at various depths of an MCF-7 spheroid preincubated with coordination
compounds.
The charge state of the binuclear coordination
compounds of Type 1 Cu+1.5/Cu makes them different
from Type 2 and Type 3 coordination compounds in redox
behavior. Type 1 coordination compounds are hardly
thereby decreasing the antiproliferative activity and
intracellular accumulation of the coordination compound.
Acceptor substituents in the benzene ring increase the
+1.5
+
2
+1
Epc (Cu → Cu ) redox potential, facilitating the copper
+
1.5
+1.5
+1
+1
reduced (Cu /Cu → Cu /Cu ) in the potential range
reduction reaction up to the formation of stable solid
+
1.5
+1.5
+1
+1
0
.01 – 0.18 V (Epc) and oxidized (Cu /Cu
→
monovalent Cu /Cu complexes, such as 71k, 72k.
The effect of the substituent on the ligand сore is best
seen by its electron withdrawal, that is, the ease of
reduction. The cyclic voltammetry study of ligands 1-73
(Tables S19-S21) revealed that donor ligands reduce the
electron-withdrawing properties of the ligand, while the
acceptor substituents, on the contrary, increase. The
higher the potential of the first electron addition step, the
stronger the electron withdrawal of the ligand. An
increase in the electron withdrawal ability of the ligand
causes an increase in the copper reduction potential,
which contributes to the stabilization of the monovalent
state and increases the cytotoxicity (Figure 6, A).
+2
+2
Cu /Cu ) in the potential range 0.32-0.59 V (Epa). These
coordination compounds should be prone not to the
intracellular reduction, but instead to the electron
+
1
donation. The lower the oxidation potential Epa (Cu
→
+
2
Cu ), the easier the donation of the electron, and the
increased Epa is suggested to negatively affect the
cytotoxicity (Tables 5, 3, S19).
+
2
Type 2 coordination compounds of Cu are capable of
a higher reduction to Cu than the Type 1 Epc (Cu+2
+
1
→
+
1
Cu ) potential range 0.33-0,42 V (Table S20). Despite the
noticeable influence of the ligand environment on the
redox potential, this type of coordination compound did
not show a noticeable correlation between the redox
potential and cytotoxicity. This type contains a labile
thiomethyl group, and the formation of a coordination
compound with reaction products of ligand hydrolysis
can take place in the solution, as described for the
disulfiram-based copper antitumor agent.2
+
2
+1
The lower the potential of the Cu /Cu , the easier it is
to donate electrons. The lower the potential Epc (Cu
Cu ), the worse the antiproliferative activity of the
coordination compound, due to the high stability of the
Cu coordination compound. This dependence can be
explained by the low redox activity of the coordination
compounds, and the inability to undergo reduction within
the cell, which leads to its rapid leaching from the
intracellular environment. The electron-withdrawing
+
2
→
+1
+2
+
2
Type 3 coordination compounds are capable of Cu
→
→
+
1
+2
+2
Cu reduction in the potential range Epc (Cu /Cu
+
1
+1
Cu /Cu ) 0.31-0,44 V, depending on the nature of the
substituent in the N3 position (Table S21). Large electron-
donor alkyl substituents in the benzene ring lower the
Epc (Cu → Cu ), making the reduction of copper
difficult, both in the complex itself and inside the cell,
+2
+1
organic сore increases the reduction Cu /Cu potential,
+
1
providing the high stability of the Cu coordination
compound, and the high redox activity facilitates an
intracellular reduction, which leads to prolonged
+
2
+1
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Table 6. 3D cytotoxicity (MCF-7 spheroid).
1
2
3
4
5
6k
8k
16
30k
41k
42
44k
52k
53k
61k
71k
72k
k
k
4
4
4.3±
.5
96.1±3. 60.0± 26.3±8. 22.1±5.0
4.8
18.2± 40.5±1
6.9
34.9±12.
4
28.4±1. 15.8±4. 17.6±5. 16.7±4.
0
6
0
4
2
8
6
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
intracellular retention and a high cytotoxicity (Figure 6,
B).
Despite the fact that this type shows the lowest redox
potentials (E ½ ~0.44 V), the presence of stable divalent
copper does not contribute to the generation of ROS.
Obviously, the ligand structure determines both the
electrochemical behavior and the ability of coordination
compounds to generate ROS. 30k (E1/2 = 0.48 V) is a more
active ROS generator in comparison with 40k (E1/2 = 0.41
V). Most likely, the more difficulty the coordination
compound has in being reduced inside the cell, forming a
monovalent copper complex, the worse its ability to
generate ROS is. However, for Type 2 there is no apparent
correlation between the ability to generate ROS and
cytotoxicity, which makes the ability to generate ROS a
possibility, but not the main mechanism of action of these
Type 2 coordination compounds.
Type 3 coordination compounds were found to be the
most active ROS-generators. 61k and 72k are active
mixed-valence ROS generators. A higher redox potential
of 71k (E1/2 = 0.51 V) in comparison with 72k (E1/2 = 0.46
V) explains the worse electron-donating ability, and, as a
result, a small number of reactive oxygen species are
formed. Thus, the difference in redox potentials leads to
different behaviors within the cell.
ROS
generation
ability.
Copper-containing
coordination compounds attract wide attention not only
due to the biogenicity of copper ions but also due to the
high redox activity.55 ROS-mediated nuclease activity,
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
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6
7
8
9
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2
3
4
5
6
7
8
9
0
56
5
7
58
mitochondrial membrane depolarization, apoptosis
–
all these well-described cytotoxic mechanisms are
generally the consequence of the reactive oxygen species
formation, which, in turn, is the consequence of redox
+2
+1
59
Cu → Cu transitions. The redox cycle involving
+2
copper coordination compounds is based on the Cu to
+1
Cu reduction by intracellular thiols, such as glutathione
(GSH), under oxygen-containing conditions. Most
+
2
coordination compounds of Cu after entering the cell
quickly form glutathione adducts, which leads to the
+
1
formation of a coordination compound of Cu and
•
+1
glutathione radical GS . In the presence of oxygen, the Cu
complex is capable of generating a superoxide anion,
which can induce the formation of reactive oxygen
species in a fenton-like reaction:59
+
2
+2
+
[
Cu L]+ GSH > [(GS)Cu L] + H
+
2
•
+1
[
(GS)Cu L] > GS + [Cu L]
Cu L] + O
The differences in the ligand structure affect the
+
1
•-
+2
+2
+1
[
2
> O
2
+ [Cu L]
Cu /Cu redox potential, which apparently affects the
ability of the coordination compounds to generate ROS
inside the cell. The presence of copper in both oxidation
We have tested the representatives of each type of the
coordination compounds for their ROS formation ability.
To detect the ROS formation in MCF-7 tumor cells, we first
used CellRox Deep Red fluorescent probe for all sorts of
oxidant species (Figure S41). To detect ROS formation in
single tumor cells, we used electrochemical sensor
systems for ROS detection, previously described by
+2
+1
states Cu /Cu in one coordination compound promotes
the generation of ROS inside the cell. The electrochemical
+2
+1
system containing Cu /Cu can thus be called a redox
buffer that maintains a constant equilibrium between
oxidized and reduced forms of copper.
Based on RDE voltammograms (Figure S26), the
equilibrium potential (I = 0) for mixed-valence Type 3
coordination compounds is ~ 0.5 V, which coincides with
6
1
Erofeev et al. To estimate the amount of ROS and to
show the concentration dependence of the current at the
platinized electrode, we calibrated the anodic response
+
2
+1
the half-wave potential of the Cu /Cu couple for these
complexes obtained from CVs. Since the limiting
cathodic and anodic currents are equal (as for 72k and
with H
2
2
O (Figure S42). We measured the ROS level in
separate MCF-7 cells using a 60 nm – diameter Pt
nanoelectrode for the electrochemical detection of
hydrogen peroxide in single cells (Figure S43). A typical
intracellular voltammogram is presented in Figure S44.
A comparison of the results of the ROS level detection
in the MCF-7 tumor cells quantified by two different
methods are presented in Figure 7. The results obtained
with the use of a nanoelectrode make it possible to more
accurately estimate the number of reactive oxygen
species in single cells after incubation with coordination
compounds.
+2
+1
6
1k), the ratio of Cu and Cu is ≈ 1. If the potential
slightly shifts due to the ROS production in the presence
of oxygen, the oxidation of Cu occurs, the system tends
to restore the equilibrium between Cu and Cu , which
+1
+2
+1
+1
+2
facilitates the regeneration of Cu via reduction of Cu .
The mixed-valence coordination compounds seem to act
as a redox buffer and maintain the equilibrium potential
in which two copper cations of different valences coexist.
The results obtained by the two ROS-detection methods
are well correlated, the difference in the obtained data
may be explained by the different sensitivities of the
fluorescent dye and the nanoelectrode, as well as by the
As seen from the data obtained, structurally similar
coordination compounds are capable of generating ROS
differently. In Type 1 coordination compounds, the most
active ROS-generator was 16k. Apparently, a relatively
higher specificity of the nanoelectrode. The H
2
2
O -specific
electrode is a direct and reliable method for ROS
measuring, while fluorescence peroxide detectors are
susceptible to nonspecific oxidation and false-positive
low redox potential
(compared with other
representatives of this group) (Epa = 0,37 V) determines
a high electron-donating ability, and, as a result, a high
ability to form ROS. For Type1, the ROS generation ability
is similar to Type 2, but for the highest of all three types
of potentials (E ½ ~ 0.55 V), the lowest electron–donating
ability reduces the ability to generate ROS.
The ability of Type 2 coordination compounds to form
ROS inside the cell is also heterogeneous within the
group. The lowest ability to generate ROS was expected.
62-64
reactions.
ROS detection at different depths of the MCF-7
spheroid. 3D spheroid was chosen as an alternative
model to look inside an antitumor drug mechanism of
action. The ROS level was detected at different depths of
MCF-7 spheroids preincubated with coordination
compounds. The measurements were conducted using a
Pt-nanoelectrode, capable of measuring the inside of the
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6
spheroid. Cyclic voltammograms were recorded as the
electrode deepened for every 10 μm inside the spheroid
Figures 8, S45, Table S29). 61k proved to be the most
spectra, XANES, EPR spectroscopy, and cyclic
voltammetry.
detailed relationships
(
A
structure−activity
effective ROS-generating agent at different depths of the
MCF-7 spheroid, where mixed-valence 72k is slightly
inferior to it due to having less redox activity. 71k
expectedly showed less redox activity due to the high
investigation enabled us to suppose the mechanism of
cytotoxic action for each type, as well as the factors
affecting any antiproliferative activity.
+
1.5
+1.5
Type 1 binuclear Cu /Cu coordination compounds
showed high 2D cytotoxicity and high intracellular
accumulation, the ability to cause early apoptosis, the
inability for intracellular reduction, and moderate
cytotoxicity in the 3D model of the MCF-7 spheroid.
+1
+1
stability of the monovalent Cu /Cu complex.
D-Cell Culture/Tumor Spheroids model (MTS
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
assay). The therapeutic efficacy of the drugs in the 3D
spheroid model was evaluated (Table 6, Figure S46).
Type 1 coordination compounds showed a moderate
efficacy in the 3D model. Previously, Type 1 coordination
compounds proved to be highly penetrating (according to
cellular accumulation/distribution experiments) and
highly cytotoxic (MTT assay). However, experiments on a
+
2
Type 2 mononuclear Cu coordination compounds
showed ROS-independent nuclease activity and late
apoptosis induction in MCF-7 cells and confirmed their
effectiveness in the 3D model MCF-7 spheroid. The data
once again confirms that the redox activity of copper
cations and the ability to form ROS is an important, but
not required mechanism of the cytotoxic action of copper
coordination compounds.
3
D spheroid model have shown a low efficiency. This fact
once again shows the difference between 2D cell
experiments and tumor models.
Type 2 coordination compounds showed a high
efficiency, along with the inability to generate ROS in the
spheroids, confirming the fact that this type of
coordination compound exhibits a redox-independent
mechanism of cytotoxic action.
Type 3 coordination compounds have been shown to
be highly effective in the 3D model. 61k and 72k were
found to be effective redox-active antitumor agents,
showing great cytotoxic ability in MCF-7 spheroids, which
confirmed their potential efficiency in solid tumors
therapy.
Type 3 binuclear coordination compounds showed an
+
2
+1
evident correlation of Epc(Cu → Cu ) and an oxidation
state of copper with the structure of the initial ligand.
Electron-withdrawing substituents in position N3 of the
thiohydantoine cycle increased the electron withdrawal
of the ligand, which could be observed by an increase in
the reduction potential in voltammograms. An increase in
the electron withdrawal properties of the organic core
+
2
+1
facilitated the redox transition Cu Cu , up to the
complete stabilization of the coordination compound
with a monovalent state. The valence state of copper
cations in the coordination compounds was confirmed by
the cyclic voltammetry with a rotating disk electrode, EPR
and XANES data.
Nontoxic (NOAEL) single dose evaluation in vivo. All
animal experiments were performed in accordance with
Russian law and were approved by the Ethics Committee
of the N.N. Blokhin Cancer Research Center. Various doses
of 61k and 72k (with 3.5 mg/kg as the highest for 61k
and 4.69 mg/kg as the highest for 72k) were
administered intravenously (i.v.) to groups of mice, four
animals each. Substances were injected immediately after
preparation of the solution. 0.2 ml of solvent were used
for i.v. administration in the control group of animals. The
animals were placed under observation for 1 hour after
administration, then every 2 hours for 12 hours and then
a daily monitoring period for 21 days. Behavioral toxicity
signs and mortality were recorded. Mice treated with 3.5
mg/kg of 61k or 4.69 mg/kg of 72k died immediately
after injection (four of four dead). Mice treated with 2.4
and 2.7 mg/kg of 61k or 2.5 and 3.4 mg/kg 72k had
seizures 20–30 seconds after injection, shallow
respiration and then weakness. These seizures lasted for
2–3 minutes (eight of eight mice), but the animals
appeared to fully recover 5–10 minutes later and there
were no lethal effects during 3 weeks of observation. After
i.v. administration of 0.79 mg/kg of 61k or 1.1 mg/kg 72k
only short-term local convulsions were observed in three
of the four mice with complete recovery in 1–2 minutes.
These single doses were considered to be tolerable doses
with minimum side effects suitable for efficacy
experiments.
It was also shown that the lower the potential Epc
Cu+2Cu+1, the more stable the Cu+2 complex and the
lower the antiproliferative activity of the coordination
compound. We suggest that this is due to the rapid
leaching of the redox-inactive complex Cu+2 from the cell.
Type
3
binuclear mixed-valenсe coordination
compounds 61k and 72k are redox active ROS generators
and cause apoptosis, while not exhibiting nuclease
activity and the ability to intercalate DNA. The redox-
buffer system of mixed-valence Cu+2/Cu+1 in one
compound provides high cytotoxicity, both in the 2D
monolayer and the 3D tumor spheroid model due to the
high ROS-generating ability. The ability of coordination
compounds to generate ROS was proven by the classical
method with a fluorescent dye, and with the help of a
nanoelectrode, capable of single-cell measurements in
single MCF-7 cells.
Additionally, ROS accumulation at different MCF-7
spheroid depths was proven using Pt-nanoelectrode, an
excellent model for drug penetration investigation
possessing the main futures of human solid tumors,
including structural organization and hypoxia. Both 61k
and 72k generate ROS up 100 µm deep in the spheroid,
and 3D cytotoxicity confirmed their efficacy.
In vivo maximum tolerated doses of the coordination
compounds 72k and 61k were determined.
CONCLUSIONS
This work is a large-scale study of the cytotoxic
properties of copper coordination compounds. The aim of
the study was to answer the following question: what
affects the cellular accumulation and cytotoxicity of
copper coordination compounds? The following SAR
were found an increase in the electron-withdrawing
properties of the ligand stabilizes the copper in the
In summary, 73 ligands and 73 complexes based on 2-
thioxoimidazolidin-4-ones (Type 1), 2-(methylthio)-1H-
imidazol-5(4H)-ones (Type 2) and 2,2'-(ethane-1,2-
diylbis(sulfanediyl))bis-1H-imidazol-5(4H)-ones) (Type
1
13
3
) were synthesized and characterized by H and C NMR
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Journal of Medicinal Chemistry
1
2
monovalent state, which promotes ROS generation. The
presence of monovalent copper and a high redox
2 mm) disks were used as the working electrodes, 0.05 M
Bu NClO solution in CH CN or DMF served as the supporting
electrolyte, and Ag/AgCl/KCl(satur.) was used as the reference
electrode. The potential scan rates were 200 mV s. Samples were
dissolved in the predeaerated solvent.
XANES spectra. The spectra were measured using a Rigaku R-
XAS Looper (Japan) X-ray spectrometer equipped with an X-ray
tube as the radiation source (cathode and anode material-
tungsten). All measurements were made in the geometry of
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
4
4
3
+2
+1
potential Cu /Cu contributes to the intracellular
accumulation of the drug and, as a result, its cytotoxic
activity. The high drug efficiency is a consequence of the
combination of physicochemical properties, which can be
tuned by varying the ligand environment and the nature
of donor atoms.
"
passing". As an incident radiation intensity detector, an argon-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
General. Unless otherwise noted, all reagents were purchased
from Sigma Aldrich (Merck) and used without further purification.
NMR spectra were recorded on a Brucker-Avance 400 MHz
filled ionization chamber (300 mbar) was used, and
a
scintillation detector was used to detect the transmitted
radiation. The size of the X-ray beam on the sample was 10x3
2
mm . For each of the samples, the spectrum was measured in 10
spectrometer (USA) in DMSO-d
6
and CDCl
3
with TMS as an internal
passes with subsequent averaging for reference samples in 4
passes. The approximate time for the measurement of each
sample was 15 hours. The measurements of the XAS spectra
behind the K-edge of copper were conducted using a crystal-
monochromator Ge (440) (second-order reflection of the Ge
(220) crystal) in the 8775 - 9500 eV range. The energy resolution
was 0.7 eV. Copper compounds, containing Cu ions with different
standard. HRMS spectra were obtained with Thermo Scientific
Orbitrap Elite (USA) mass spectrometer, ionization: ESI mod with
±3.5 kV, capillary temperature 275ºC. Mass spectra were recorded
with an Orbitrap analyzer, resolution: 480000. Internal standards:
DMSO and diisooctyl phthalate (m/z 157.03515 and 413.26623) in
positive mode and dodecyl sulfate (m/z 265.14790) in negative
mode. Uv-Vis spectra were recorded on Hitachi U2900 (Japan)
spectrophotometer. MALDI Bruker spectra were recorded on
Autoflex II (USA). Elemental analysis was performed on
ELEMENTAR Vario MICRO Cube (Germany).
Purity of organic ligands. The purities of compounds 1-73
were determined by HPLC. HPLC analysis was carried out on a
HPLC system Ultimate 3000 (Thermo Fischer Scientific, USA)
with TSQ Endura triple quadrupole mass spectrometer (Thermo
Fischer Scientific, USA) equipped with a Column XBridge Peptide
BEH C18, 50x2.1 mm, particle size 5 μm. HPLC profiles were
recorded by an UV detector at 260 nm.
charge states: Cu(foil), Cu
reference samples.
2
O, CuO, and CuCl
2
were used as
EPR spectra. were recorded on
a
Varian E-3 EPR
Spectrometer (USA) at the boiling point of liquid nitrogen (77.4
K)
Cytotoxicity in monolayer culture (MTT assay). Human
3
adenocarcinoma MCF-7 cells (5ꢀ×ꢀ10 ) were seeded on a 96-well
plate and incubated overnight. A freshly prepared stock solution
of copper complexes in DMSO (1.0 mg/mL) were diluted in the
cell culture media and added to the cells (0.5% DMSO as the
maximum concentration). After 72 hours, MTT (3-(4,5-
The purity of the compounds 1-73 was confirmed to be ≥95%.
For compounds 1-11, 13-52, 54, 58, 60 - 67, 69, 70.
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
solution was added to the cells to the final concentration of 0.5
Phase A water + 0.1% formic acid, Phase B - acetonitrile + 0.1%
formic acid. Elution mode: 0 to 0.5 minutes 5% Phase B, 0.5 to 12.5
minutes Phase B gradient 5 to 100%, 12.5 to 15 minutes 100% Phase
B, 15 to 17 minutes Phase B gradient 100 to 5%, 17 - 20 minutes 5%
Phase B. Flow rate 300 μl/min, column temperature 30ºC, injection
volume 10 μl.
6
7
mg/ml, according to the standard protocol. After 2 hours of
incubation, the MTT solution was discarded and 140 µl of DMSO
was added. The plates were shaken (60 rpm) to solubilize the
formazan. The absorbance was measured using a microplate
reader (VICTOR X5 Plate Reader, PerkinElmer, USA) at a
wavelength of 565 nm in order to measure the formazan
concentration. IC50 values were estimated from the dose-
response graph (GraphPad Software, Inc). All measurements
were performed in three independent replicates to average the
obtained data.
For compounds 12, 53, 55-57, 59, 68, 69, 71-73.
Phase A water + 0.1% formic acid, Phase B - acetonitrile + 0.1%
formic acid. Elution mode: 0 to 0.5 minutes 50% Phase B, 0.5 to 12.5
minutes Phase B gradient 50 to 100%, 12.5 to 15 minutes 100%
Phase B, 15 to 17 minutes Phase B gradient 100 to 50%, 17 - 20
minutes 50% of Phase B. Flow 300 μl / min, column temperature 30
C, injection volume 10 μl.
MS parameters are the same for all samples: Full Scan Q1, 100 to
1000 Yes, positive ion registration, 3.5 kV, Q1 resolution 0.7 Da,
Chrom Filter 1 second. Sheath gas 40 arb, Aux gas 12 arb, Sweep gas
Fluorescent proteins detection assay. Cytotoxicity was
measured by the live-imaging of MCF7_Katushka cells in
3
8
accordance with Kalinina et al. using Typhoon FLA scanner (GE
Healthcare, USA). Cells were seeded in a 96-well plate (3,000 per
well) in an F12 medium. After 24 hours, the studied substances
diluted in a culture medium were added to cells. The cells were
1
arb, capillary temperature 330ºC, evaporator temperature 300ºC.
Crystallography. The X-ray diffraction study of a single crystal of
incubated for 72 hours at 37°С in 5% СО
atmosphere and analyzed via a high-resolution scanner at r.t.
less than 30 minutes without CO ). The obtained plate images
2
humidified
coordination compounds was conducted on a STOE StadiVari Pilatus
100K diffractometer, Cu-Kα radiation (1.54186 Å), from a GeniX3D
Cu HF generator with a microfocus X-ray tube and a Xenocs
FOX3DHF multilayer thin-film ellipsoidal monochromator (France).
Data collection and processing of the recorded diffraction peaks
were performed using the X-Area 1.67 software package (STOE & Cie
GmbH, Darmstadt, Germany, 2013). The intensities of the reflections
on the frames obtained from the 2D detector were scaled using the
LANA program (incorporated in the X-Area package), upon which
diffraction data processing minimizes the intensity differences of
symmetrically equivalent reflections (multiscan method).
(
2
were processed using ImageJ software.
Cytotoxicity in tumor spheroids (MTS assay). Tumor
spheroid formation was performed using a liquid overlay
technique with agarose-coated plates, as described earlier.6
Briefly, 1.5% wt of agarose in PBS (pH = 7.4) was heated on
water bath for 15 minutes. Then, 50 μL of melted agarose was
added to each well of a flat-bottom 96-well plate under sterile
conditions. Plates with agarose were cooled down to r.t. for 15
minutes. The cells were seeded on agarose-coated plates (10,000
cells/well, 100 μL of media per well) and incubated at 37°C and
8,69
The structural data were deposited with the Cambridge
Crystallographic Data Center (CCDC 1874574).
The structure was solved by the direct method implemented in
the SHELXS-97 software package.65 The positional and thermal
parameters of nonhydrogen atoms were refined in the full matrix
anisotropic approximation. The positions of hydrogen atoms at
heteroatoms (N, O) were determined from the Fourier syntheses and
refined freely. The positions of the hydrogen atoms at the carbon
atoms were calculated and refined in the isotropic approximation
using a riding model. Graphic images of a molecule in the crystal
were made using the DIAMOND program.66
Cyclic voltammetry. experiments were conducted using an
IPC Pro M potentiostat (Russia) with cyclic voltammetry (CV)
and rotating disk electrode (RDE) techniques. Glass-carbon (d =
5
2
% CO for 72 hours to generate spheroids. The formation of
tumor spheroids was then observed using an inverted light
microscope. Then, the solutions (1-50 µM) of the tested drugs
were added to spheroids for 72 hours. The cytotoxicity was
evaluated using
a colorimetric MTS assay based on the
NAD(P)H-dependent dehydrogenase activity, which resulted in
the reduction of the MTS tetrazolium compound and generation
of the soluble formazan product in the cell culture media. The
formazan formation was measured at 490ꢀnm using a Varioskan
Flash reader (Thermo Scientific, USA). The IC50 values were
estimated using the GraphPad Prism Software.
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Journal of Medicinal Chemistry
Page 14 of 34
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Intracellular accumulation/distribution.
Cell preparation/incubation. The mice 4T1 breast cancer cells
were maintained in Roswell Park Memorial Institute medium
RPMI-1640) containing 10% fetal bovine serum (FBS), 2 mM L-
for the spectrofluorimetric studies were diluted with a buffer
solution. Ethidium bromide was added to a buffer solution
containing 2 μM DNA to a concentration of 2 μM. The variable
volumes of the test compound in the buffer were added to the
resulting solution to obtain a series of samples containing DNA (1
µM), Ethidium bromide (1 µM) and a test compound (0 to 0.9 µM
with an increment of 0.1 µM); pure buffer was used as a the control.
The solutions were shaken and left for 15 minutes. The interaction
between the DNA and coordination compounds (ligands) were
evaluated via fluorescence quenching experiments. The DNA
solution (2 mM) and EtBr (2 mM) were prepared in 1 M Tris-HCl
(pH = 7.0). The spectra of the fluorescence quenching experiments
were recorded at r.t. with a DNA excitation wavelength at 502 nm
(the emission wavelength at 530-730 nm; step width 1 nm) by
keeping the concentration of the DNA constant (1 mM) while
varying the concentration of the complexes (ligands) 1-10 mM.
BSA binding. Competitive binding of ligands and coordination
compounds to BSA was assessed on Agilent Cary Eclipse
Fluorescence Spectrophotometer (USA) All operations with BSA
were performed in phosphate buffer (pH = 7.2) with 5% addition of
DMSO. All solutions were used within an hour after preparation.
Equal volumes of the solutions of the studied compounds and pure
phosphate buffer were added to the BSA solution to obtain mixtures
containing the final concentrations of 1 μM BSA and 0, 1, 2, 3, 4, 5,
6, 7, 8, 9 μM of the test compound respectively. The solutions were
shaken and left for 15 minutes at r.t. to establish equilibrium. The
fluorescence spectra were recorded upon excitation at a wavelength
of λ = 280 nm in the spectral range of wavelengths 500 - 700 nm.
Fluorescence spectra were recorded on an Agilent Cary Eclipse
Fluorescence Spectrophotometer without temperature control. The
concentration stability constant was calculated using the Sketchard
(
glutamine, 100 U/ml penicillin, 100 lg/ml streptomycin, and 0.25
2
lg/ml amphotericin B, using 25 cm cell culture flasks (Corning) in
2
a humidified atmosphere (95% air/5% CO ) at 37ºC. The cells were
periodically screened for mycoplasma contamination. Exponentially
growing cells were harvested by trypsinisation and the resulting
2
single cell suspension was seeded in 25 cm cell culture flasks at a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
concentration of 5×10 cells/flask. After 48 hours at 37ºC, when the
cell culture was 80% confluent, the medium of the subconfluent cells
was replaced with 4 ml of a fresh medium containing the desired
drug compound at an appropriate concentration in three replicates.
The flasks were incubated at 37ºC in 5% CO
hours. Then, the cells were washed three times with a saline solution
0.9% NaCl), and 1.0 ml of trypsin–EDTA (0.05%) was added to
2
atmosphere for 24
(
each flask to detach the cells. A complete growth medium (6.0 ml)
was added and the cells were aspirated again by gently pipetting. The
suspended cells in the growth medium were centrifuged at 800 g
4ºC) for 5 minutes. After discarding the supernatant, the cells were
dispersed in 500 μl of PBS and the cells were counted using a TC20
automated cell counter (Biorad).
Subcellular fractionation procedure. Samples of the 4T1 cells
were subjected to three freezing/thawing cycles at -80ºC, treated
with 200 µl of PBS supplemented with 0.1 M NaOH and 0.05%
Tween-20, and vigorously vortexed to lyse the cells. The
homogenate was centrifuged at 600 g for 15 minutes at 4ºC to isolate
the nuclear fraction. Then, the supernatant was carefully transferred
to a new tube and centrifuged at 15000 g for 15 minutes at 4ºC to
isolate the mitochondria fraction. The supernatant was a cytosol
fraction, which was carefully transferred to another tube. All three
fractions were treated with highly pure nitric acid (100 µl for nuclear
and mitochondria fractions, and 300 µl for cytosol fraction) for at
least 48 hours until complete dissolution.
(
7
1
equation:
퐼0 − 퐼
log10
= log10 퐾푎푝푝(퐵푆퐴) + 푛 ∗ log10 퐶(푋)
퐼
where I0 is the fluorescence intensity of a pure BSA solution; I is
the intensity at a concentration C (X) of substance X; n is the number
of binding sites,
AAS assay. Copper accumulation in cell organelles was measured
using an atomic absorption spectrometer (AAS) with KVANT-Z-
ETA electrothermal atomization (KORTEK, Russia). 100 μl of the
Telomerase inhibition. The telomerase assay was performed
with quantitative real-time (RTQ) PCR detection instead of gel
3
sample were mixed with 500 μl of a 5% pure HNO qualification
7
0
modification. For this, 18 μl of TRAP mixtures were prepared,
containing TRAP buffer [20 mM of Hepes-N-
solution in 1.5 ml Eppendorf tubes, followed by thorough mixing and
10 minutes of incubation at a temperature of 60ºC in a water bath.
Then, the contents of the tubes were again mixed and centrifuged for
Hydroxyethylpiperazine-N-ethane-sulfonate (Hepes-KOH)]; 1.5
mM MgCl2; 63 mM KCl; 1 mM EGTA; 0.1 mg/ml of bovine serum
albumin; 0.005% v/v polyoxyethylene (20) sorbitan monolaurate; 20
1
0 minutes at 14500 rpm to separate any possible sediment (no
precipitation was observed for any of the samples). 5% extrapure
HNO solution was prepared by diluting a 65% HNO solution
μM
dNTP;
and
0.5
μM
TS
oligonucleotide
3
3
(
AATCCGTCGAGCAGAGTT). Then, 1 μl of inhibitor solution
(Merck KGaA, Germany, Cu ≤ 0.010 ppm) with DI water. The
prepared sample was thoroughly mixed immediately prior to
measuring the copper content. Resonance copper line λ = 324.8 nm
was used for measurements with a preliminary calibration with five
solutions (0 μg/l, 5 μg/l, 10 μg/l, 50 μg/l, and 100 μg/l) based on the
multielement standard ICP-MS-68 in three replicates. Solution “A”
and refinement of the calibration before measuring each sample (“0”
is the signal value when measuring double distillate).
was added in the sample probes. Water-DMSO solution was added
in the control probes instead of the inhibitor. Then, 1 μl of cellular
extract 2000/μl cells was added to the sample and the control probes
and reaction mixtures were incubated for 30 minutes at 25°C. At the
second stage, an equal volume of the inhibitor solution was added to
the control probes. Then, two units of Taq DNA polymerase
(
Fermentas), up to 0.5 μM of ACX oligonucleotide
(
GCGCGGCTTACCCTTACCCTTACCCTTACCCTAACC), and
DNA degradation. DNA degradatin was detected after agarose
gel electrophoresis (Helicon, Russia) by CFX Gel Imager (Biorad,
USA). The mixtures (15 µl), containing 150 ng pUC18 plasmid
up to 0.17x Sybr I were added in all probes. RTQ-PCR was
performed according to the following scheme: 35 s (94°C), 35 s
(
50°C), 90 s (72°C) (35 cycles, Thermal cycler CFX96, Biorad,
DNA,
20
mM
Hepes-KOH
(4-(2-hydroxyethyl)-1-
USA). The quantity of the telomerase repeats in comparison to the
control without telomerase inhibition was calculated by the formula
piperazineethanesulfonic acid with KOH) pH = 7.8, and 2-200 µM
of complexes were prepared. The mixtures were incubated at 37˚C
for 4 hours and analyzed in 1% agarose gel. The gel densitometry
was estimated with ImageQuant software.
1
/(2^(Cq[assay with inhibitor addition to telomerase reaction] -
Cq[assay with inhibitor addition to the PCR step only])).
Apoptosis/necrosis Annexin V–FITC assay. MCF-7 cells
DNA intercalation. Competitive binding of ligands and
coordination compounds to human DNA intercalated with ethidium
bromide was assessed on a Microplate Reader Clariostar (BMG
LABTECH, Germany), with scan mode Ethidium homodimer-1, or
Agilent Cary Eclipse Fluorescence Spectrophotometer (USA) All
operations with human DNA were performed in a Tris-HCl buffer
solution (pH = 7.2) supplemented with 5% DMSO. The solutions
were used within 30 minutes after preparation. The DNA purity was
determined by the ratio of optical absorption values at wavelengths
λ = 260 nm and λ = 280 nm (A260/A280). The ratio did not exceed
5
(
5×10 ) were seeded on a 6-well plate and incubated overnight.
Then, the cells were treated with drugs at concentrations equal to
IC50 values for 24ꢀhours. Then, the cells were detached with trypsin-
EDTA and washed with PBS (pH = 7.4). Apoptosis and necrosis
were quantified via combined staining of Annexin V-FITC
(
Molecular probes, Waltham, MA, USA) and propidium iodide (PI)
for 15ꢀmin at r.t. and in the dark. The FITC and PI fluorescence were
measured on NovoCyte 2000R flow cytometer (ACEA
a
Biosciences, USA), and the percentages of apoptotic and necrotic
cells were analyzed using NovoExpress v.1.2.4 software. A
minimum of 50,000 cells per sample was acquired and analyzed;
acquisition was performed by setting the forward scatter (FSC) and
side scatter (SSC) on the dot plot to differentiate the population of
1
.8, which means that the DNA was not bound to any protein. The
DNA concentration was determined by the magnitude of the optical
-
1
-1
absorption (λ = 260 nm, ε = 6600 M cm ). The initial solutions were
prepared by dissolving the test compounds in DMSO. The solutions
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Page 15 of 34
Journal of Medicinal Chemistry
1
2
cells from cellular debris (FSC-A vs SSC-A) and doublets (FSC-H
mg/ml for 72k and 61k, respectively. The stock solution was
3
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vs FSC-W).
adjusted to the desired concentration immediately prior to the
experiments using Haemodes-N.
ROS level detection in MCF-7 cells preincubated with
coordination compounds.
Fluorescent-based ROS measurement. Evaluation of the
cytoplasmic ROS was performed using a CellROX Deep Red
fluorescent probe (Molecular Probes/Thermo) according to the
Nontoxic (NOAEL) single dose evaluation in mice. C57Bl6
male mice (six weeks of age, 20–24 g) were obtained from the
N.N. Blokhin Cancer Research Center. The mice were housed
individually and kept at r.t. (20–22ꢁ°C), with a relative humidity
of 45–65% and natural lighting. Various doses of test substances
(3.5 mg/kg and 4.69 mg/kg as the highest for 61k and 72k,
respectively) were administered intravenously (0.2 ml in total),
and each experimental group included four mice. The i.v.
administration of 0.2 ml free DMSO-Haemodes-N solvent was
used for a control group. The animals were placed under
3
manufacturer protocol. In brief, the MCF-7 cells (5ꢀ×ꢀ10 ) were
seeded on a 96-well plate and incubated overnight. Then, the
cells were incubated with 2.5ꢀµM of CellROX Deep Red in DMEM
for 30ꢀminutes and washed in PBS three times. After that, the
medium was replaced with 100ꢀμL of drug solutions for 1 hour.
The cell nuclei were additionally stained by 50ꢀμM of Hoechst
33258 dye solution for 15ꢀminutes. Optical images and CellROX
Deep Red fluorescence intensity data of the MCF-7 cells were
acquired using InCell Analyzer 6000 and the In Cell Analyzer
Workstation software v.3.7.3 (GE Healthcare, USA). The ROS
level was calculated as a ratio of the CellROX Deep Red
fluorescence to the Hoechst 33258 fluorescence to normalize the
signal to the cell amount in each image.
0
1
2
3
4
5
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7
8
9
0
observation for
1 hour after administration. Then, their
conditions were checked every 2 hours for 12 hours followed by
a daily monitoring period for 21 days. Behavioral toxicity signs
and mortality were recorded.
Syntheses of Compounds 1−15, 17-24, 28, 29
Ethyl isothiocyanоacetate was synthesized according to the
described procedure.72
Pt-nanoelectrode experiments For the production of platinum
electrodes, quartz billets with internal and external diameters of
The synthesis procedure was used in accordance with
Kuznetsova et al.72 Ethyl isothiocyanоacetate (1 eq) was added
0.9 mm and 1.2 mm, respectively, were used. These blanks were
dropwise to a vigorously stirred solution of aniline in ~10 ml of
diethyl ether. The mixture was stirred until the white solid
precipitate formed, the solvent was evaporated under reduced
pressure. The resulted powder was dissolved in ~30 ml
ofethanol, 1.1 eq. of solid KOH was added to form a clear red
solution. Then, 1.1 eq. of 2-pyridinecarbaldehyde was added
dropwise and the mixture was stirred for 3 hours at r.t. 1 M HCl
was then added until pH = 7, the precipitate formed was filtered
off, washed with water, ethanol, diethyl ether and air-dried. The
final products were purified by column chromatography on
pulled out on a laser puffer (Sutter, USA), resulting in two
nanocapillaries with a diameter of the order of 100-500 nm.
Pyrolytic carbon was precipitated on the obtained
nanocapillaries with thermal decomposition of the propane-
butane mixture in an oxygen-free environment. We used an
electrochemical method of creating cavities in the carbon
nanoelectrodes with subsequent deposition of platinum. Etching
was performed in a solution containing 0.1 mM NaOH and 10
mM KCl. The nanoscale electrode and reference electrode were
immersed in the solution; аn alternating voltage of
a
2 2
silica gel, eluent: CH Cl : MeOH (10: 1). The products were
isolated as a yellow powders.
symmetrical V-shape with an amplitude of 1.5 – 2 V was applied
to the reference electrode. After soaking in a KCl/NaOH solution,
the treated blanks were transferred to a hexachloroplatinic acid
solution for further electrochemical deposition of platinum at
the tip of the capillary. Both electrodes were placed in a solution
with
nanoelectrode by applying
voltage with an amplitude of 0.8 V.
The potential difference
H
2
PtCl
6
and platinum was deposited on an etched
a
symmetrical sawtooth shape
between
the
platinum
microelectrode and the reference electrode was recorded by a
patch-clamp Model 2400 amplifier ("A-M Systems", USA). The
transfer and recording of the measurements to a computer was
conducted using a ADC-DAC converter USB-6211 ("National
3-(4-fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (1). From 200 mg (1.45
mmol) of 4-fluoroaniline, by the reaction with 210 mg (1.45
mmol) of ethyl isothiocyanatoacetate, followed by addition of
171 mg (1.6 mmol) of pyridine-2-carbaldehyde and 90 mg (1.6
mmol) of solid KOH 1 was obtained as a light yellow powder.
Yield: 347 mg (80%) after column chromatography. 1H NMR
instruments", USA) and the program WinWCP.
A
micromanipulator PatchStar ("Scientifica", Great Britain) was
used to feed the nanosensor. All manipulations were made on
the table of an inverted microscope (Nikon, Japan).
Pt-nanoelectrode calibration. The concentration of ROS
(400 MHZ, DMSO-d
J=4.89 Hz, Нα-Ру), 7.89 (td, 1Н, J
6
, δ, ppm): 11.87 (brs, 1Н, NH), 8.76 (d, 1Н,
=7.83 Hz, J =1.76 Hz, Нβ-Рy),
(
hydrogen peroxide) was evaluated at a potential of +800 mV vs.
Ag/AgCl. Under the given conditions, the platinum catalyzed
reaction 2H ↔ 2H O + O occurs, and since the superoxide
1
2
7.75 (d, 1Н, J=7.83 Hz, Нβ’-Рy), 7.47-7.30 (m, 5Н, Нγ-Ру, Нα, Нα’,
13
2
O
2
2
2
6
Нβ, Нβ’-Рh), 6.79 (s, 1Н, -СН=). C NMR (101 MHz, DMSO-d , δ,
radical is rapidly converted in the solution into hydrogen
peroxide, the total concentration of peroxide determines the
general background of the oxidation processes. Prior to the
measurements, each platinum electrode was calibrated using a
series of standard H O solutions (Figure S25, calibration curve).
2 2
The levels of peroxides in the samples were determined based
ppm) 178.2, 161.1, 153.6, 150.3, 137.9, 131.5, 127.1, 123.8,
-
116.3, 109.0. HRMS (neg.) calculated C15
H
9
FN
3
OS : 298.0451 (1 -
-
-
H) , found 298.0452 (1 - H) . HPLC purity 96.48%.
3-(3-fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (2). From 200 mg (1.45
mmol) of 3-fluoroaniline, by the reaction with 210 mg (1.45
mmol) of ethyl isothiocyanatoacetate, followed by addition of
171 mg (1.59 mmol) of pyridine-2-carbaldehyde and 90 mg
(1.59 mmol) of solid KOH 2 was obtained as a light yellow
on the calibration curve.
DMSO formulation for intravenous administration. The
solutions for i.v. administration were prepared using DMSO
enriched with auxiliary agents (DMSO: Tween 20: F127 94: 5: 1
by weight). The compositions included DMSO (Ph.Eur 5.0,
USP29-NF24), 10% by volume; a nonionic surfactant Tween 20
(Ph. Eur. 8.5, USP29-NF24), 0.5% by volume; and a block
copolymer Pluronic F-127 (Ph. Eu. 7.0, USP26-NF21), 1% by
volume. We dissolved 1 mg 72k or 61k in 300 μl or 540 μl of
DMSO-based solvent, respectively. The resulting solution was
slowly added dropwise by stirring in 2100 μl of Haemodes-N (FS
powder. Yield: 364 mg (84%) after column chromatography. 1
NMR (400 MHz, DMSO-d , δ, ppm): 12.71 (brs, 1H, NH), 8.78 (d,
1H, J=4.89 Hz, Hα-Py), 8.63 (m, 1H, Hγ-Py), 7.91 (td, 1H, J =7.82
Hz, J =1.96 Hz, Hβ-Py), 7.79 (m, 1H, Hβ'-Py), 7.57 (m, 2H, Hα, Hβ-
Ph), 7.33 (m, 3H, Hβ ', Hγ-Ph, Hα’-Ph), 6.82 (s, 1H, -CH=). C NMR
(101 MHz, DMSO-d , δ, ppm) 177.8, 163.6, 153.5, 150.4, 137.9,
130.9, 130.2, 127.2, 125.7, 123.8, 116.7, 109.2. HRMS (neg.)
H
6
1
2
1
3
6
-
-
calculated C15
H
9
FN
3
OS : 298.0451 (2 - H) , found 298.0449 (2 -
-
4
2-2620-97, Ph. Eur.8.0), an isotonic agent with a stabilizing,
H) . HPLC purity 95.03%.
anti-aggregation, detoxification, and plasma substituting effect.
3-(2-fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (3). From 200 mg (1.45
The resulting stock solutions were 0.416 mg/ml and 0.379
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 34
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mmol) of 2-fluoroaniline, by the reaction with 210 mg (1.45
mmol) of ethyl isothiocyanatoacetate, followed by addition of
171 mg (1.59 mmol) of pyridine-2-carbaldehyde and 90 mg
3-(4-chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (8). From 200 mg (1.57
mmol) of 4-chloroaniline, by the reaction with 228 mg (1.57
mmol) of ethyl isothiocyanatoacetate, followed by addition of
185 mg (1.72 mmol) of pyridine-2-carbaldehyde and 97 mg
(1.72 mmol) of solid KOH 8 was obtained as a white-yellow
(
1.59 mmol) of solid KOH, 3 was obtained as a yellow powder.
1
Yield: 325 mg (75%) after column chromatography. H NMR
(
400 MHz, DMSO-d
J=4.89 Hz, Hα-Py), 7.90 (td, 1H, J
.77 (m, 1H, H β'-Py), 7.53 (m, 2H, Hβ, Hβ'-Ph), 7.35 – 7.40 (m,
6
, δ, ppm): 12.06 (brs, 1H, NH), 8.76 (d, 1H,
=7.83 Hz, J =1.76 Hz, Hβ-Py),
2
powder. Yield: 401 mg (81%) after column chromatography. 1
NMR (400 MHz, DMSO-d , δ, ppm): 8.78 (d, 1H, J=4.34 Hz, Hα-
Py), 8.58 (m, 1H, Hβ-Py) 8.45 (m, 1H, Hβ'-Py), 7.45 – 7.34 (m, 5H,
H
1
7
6
1
3
3H, Hγ-Py, Hα, Hγ-Ph), 6.85 (s, 1H, -CH=). C NMR (101 MHz,
DMSO-d , δ, ppm) 177.3, 163.1, 153.3, 150.4, 145.1, 137.9, 133.4,
32.1, 127.3, 123.9, 116.9, 110.0. HRMS (neg.) calculated
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
13
J=7.63 Hz, Hγ-Py, Hα, Hα’, Hβ, Hβ'-Ph), 6.83 (s, 1H, -CH=).
C
1
C
NMR (101 MHz, DMSO-d , δ, ppm) 177.3, 163.2, 153.3, 150.3,
6
-
-
-
15
9 3
H FN OS : 298.0451 (3 - H) , found 298.0453 (3 - H) . HPLC
137.9, 132.8, 132.3, 131.9, 131.2, 130.3, 128.6, 127.3, 123.9,
purity 95.32%.
-
1
-
,
16.6, 109.9. HRMS (neg.) calculated C15
H
9
ClN
3
OS : 316.0125 (8
(Z)-3-(4-bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
-
-
-
H isotopic) , 314.0155 (8 - H) , found 316.0125 (8 - H isotopic)
thioxotetrahydro-4H-imidazol-4-one (4). From 200 mg (1.16
mmol) of 4-bromoaniline, by the reaction with 168 mg (1.16
mmol) of ethyl isothiocyanatoacetate, followed by addition of
-
314.0158 (8 - H) . HPLC purity 97.98%
3
-(3-chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (9). From 200 mg (1.57
mmol) the 4-chloroaniline, by the reaction with 228 mg (1.57
mmol) of ethyl isothiocyanatoacetate, followed by addition of
85 mg (1.72 mmol) of pyridine-2-carbaldehyde and 97 mg
1.72 mmol) of solid KOH 9 was obtained as a white-yellow
1
37 mg (1.28 mmol) of pyridine-2-carbaldehyde and 72 mg
(1.28 mmol) of solid KOH 4 was obtained as a yellow powder.
1
Yield: 330 mg (79%) after column chromatography. H NMR
(400 MHz, DMSO-d , δ, ppm): 8.76 (d, 1H, J=4.30 Hz, Hα-Py), 7.89
td, 1H, J =7.83 Hz, J =1.96 Hz, Hβ-Py), 7.76 (d, 1H, J=7.83 Hz,
Hβ'-Py), 7.45 (m, 2H, Hβ, Hβ'-Ph), 7.32 – 7.36 (m, 3H, Hγ-Py, Hα,
Hα '-Ph), 6.78 (s, 1H, -CH=). 13C NMR (101 MHz, DMSO-d
, δ,
ppm) 177.9, 163.6, 153.5, 150.3, 137.9, 132.4, 131.5, 127.2,
1
(
6
(
1
2
1
powder. Yield: 401 mg (81%) after column chromatography.
H
NMR (400 MHz, CDCl
.91 (m, 1Н, Нβ-Рy), 7.78 (d, 1H, J=7.82 Hz, Нβ’-Рy), 7.58 (m, 1Н,
Нα’-Рh), 7.55 (m, 1Н, Нα, Нβ -Рh), 7.41 (m, 4Н, Нγ-Ру, Hγ-Ph),
3
, δ, ppm): 8.78 (d, 1Н, J=3.91 Hz, Нα-Ру),
6
7
+
1
3
23.8, 122.6, 109.1. HRMS (pos.) calculated C15H11BrN OS :
1
3
6
1
1
6
.81 (s, 1Н, -СН=). C NMR (101 MHz, DMSO-d , δ, ppm) 177.4,
+
+
361.9806 (4 + H isotopic) , 359.9801 (4 + H) , found 361.9778,
65.5, 149.9, 137.6, 134.47, 132.9, 130.5, 128.9, 127.9, 126.8,
+
359.9804 (4 + H) . HPLC purity 96.72%.
-
24.9, 123.4. HRMS (neg.) calculated C15
H
9
ClN
3
OS : 314.0125 (9
3
-(3-bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (5). From the 200 mg
1.16 mmol) of 3-bromoaniline by the reaction with 168 mg
-
-
-
- H isotopic) , 314.0155 (9 - H) , found 314.0129 (9 - H) ,
-
3
16.0160 (9 - H) . HPLC purity 95.22%.
(
3
-(2-chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
(1.16 mmol) of ethyl isothiocyanatoacetate, followed by addition
of 137 mg (1.28 mmol) of pyridine-2-carbaldehyde and 72 mg
thioxotetrahydro-4H-imidazol-4-one (10). From 200 mg
1.57 mmol) of 2-chloroaniline, by the reaction with 228 mg
1.57 mmol) of ethyl isothiocyanatoacetate, followed by addition
of 185 mg (1.72 mmol) of pyridine-2-carbaldehyde and 97 mg
1.72 mmol) of solid KOH 10 was obtained as a white-yellow
(
(
(
1.28 mmol) of solid KOH, 5 was obtained as a yellow powder.
1
Yield: 321 mg (77%) after column chromatography. H NMR
(
400 MHz, DMSO-d
6
, δ, ppm): 8.77 (d, 1H, J=4.37 Hz, Hα-Py), 7.91
(
(
td, 1H, J =7.79 Hz, J
1
2
=1.78 Hz, Hβ-Py), 7.72 – 7.78 (m, 3H, Hγ-Py,
1
powder. Yield: 376 mg (76%) after column chromatography.
NMR (400 MHz, DMSO-d
Py), 7.89 (td, 1H, J =7.91 Hz, J
Py), 7.47 – 7.43 (m, 2H, Hβ, Hβ'-Ph), 7.37 – 7.32 (m, 3H, Hγ-Py,
H
Hα, Hα'-Ph), 7.41 (m, 3H, Hβ'-Py, Hβ-Ph, Hγ-Ph), 6.81 (s, 1H, -
CH=). C NMR (101 MHz, DMSO-d
6
, δ, ppm): 8.76 (d, 1H, J=4.73 Hz, Hα-
=1.94 Hz, Hβ-Py), 7.76 (m, 1H, Hβ'-
1
3
6
, δ, ppm) 169.9, 155.8, 154.9,
1
2
1
50.3, 137.8, 135.4, 132.0, 131.1, 128.6, 126.9, 121.4, 109.2.
+
HRMS (pos.) calculated
isotopic) 359.9801 (5 + H) , found 361.9779 (5 + H) 359.9804
C
15
H11BrN
3
OS : 361.9806 (5
+
H
1
3
Hα, Hγ-Ph), 6.78 (s, 1H, -CH=). C NMR (101 MHz, DMSO-d
6
, δ,
+
+
+
ppm) 177.4, 163.3, 153.3, 150.4, 137.9, 132.3, 132.0, 129.9,
+
(5 + H) . HPLC purity 97.39%.
1
27.3, 125.5, 123.9, 120.8, 120.0, 116.9, 110.0. HRMS (neg.)
3-(2-bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
-
-
calculated C15
H
9
ClN
3
OS : 316.0125 (10 - H isotopic) , 314.0155
thioxotetrahydro-4H-imidazol-4-one (6). From 200 mg (1.16
mmol) of 2-bromoaniline, by the reaction with 168 mg (1.16
mmol) of ethyl isothiocyanatoacetate, followed by addition of
-
-
-
(
10 - H) , found 314.0159 (10 - H) 316.0126 (10 - H isotopic) .
HPLC purity 99.20%.
3-(4-methoxyphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (11). From 200 mg
1.63 mmol) of 4-methoxyaniline by the reaction with 236 mg
1.63 mmol) of ethyl isothiocyanatoacetate, followed by addition
of 191 mg (1.79 mmol) of pyridine-2-carbaldehyde and 100 mg
(1.79 mmol) of solid KOH 11 was obtained as a yellow powder.
Yield: 466 mg (92%) after column chromatography. 1H NMR
137 mg (1.28 mmol) of pyridine-2-carbaldehyde and 72 mg
(
1.28 mmol) of solid KOH 6 was obtained as a yellow powder.
(
(
1
Yield: 225 mg (54%) after column chromatography. H NMR
(400 MHz, DMSO-d
td, 1H, J =8.03 Hz, J
Hβ'-Py), 7.31 (m, 3H, Hγ-Py, Hα, Hγ-Ph), 6.78 (s, 1H, -CH=).
6
, δ, ppm): 8.86 (d, 1H, J=4.69 Hz, Hα-Py), 7.99
(
1
2
=1.66 Hz, Hβ-Py), 7.65 (m, 3H, Hβ, Hβ'-Ph,
1
3
C
NMR (101 MHz, DMSO-d
6
, δ) 177.3, 163.1, 153.3, 150.4, 137.9,
(
400 MHz, DMSO-d
(td, 1H, J =6.91 Hz, J
Hβ’-Py), 7.25 (dd, 1H, J
Hα, Hα'-Ph), 6.87 (m, 2H, Hβ, Hβ'-Ph), 6.83 (s, 1H, -CH=), 3.90 (s,
6
, δ, ppm): 8.46 (d, 1H, J=3.74 Hz, Hα-Py), 7.73
=1.73 Hz, Hβ-Py), 7.39 (d, 1H, J=6.81 Hz,
=7.52 Hz, J =2.02 Hz, Hγ-Py), 7.17 (m, 2H,
1
33.4, 132.9, 132.38, 132.37, 132.0, 130.0, 129.2, 127.3, 123.9,
1
2
+
109.8. HRMS (pos.) calculated C15
H11BrN
3
OS : 361.9786 (6 + H
1
2
+
+
+
isotopic) , 359.9806 (6 + H) , found 361.9782 (6 + H isotopic) ,
+
13
359.9812 (6 + H) . HPLC purity 95.06%.
3 6
3H, p-OCH ). C NMR (101 MHz, DMSO-d , δ, ppm) 178.2, 163.6,
3-(2-bromo-4-fluorophenyl)-5-((Z)-2-
pyridylmethylene)-2-thioxoimidazolidin-4-one (7). From
159.4, 153.3, 149.9, 137.6, 130.0, 129.9, 126.7, 125.5, 123.3,
-
114.1, 108.4, 55.5. HRMS (neg.) calculated
310.0650 (11 - H) , found 310.0654 (11 - H) . HPLC purity
97.31%
C
16
H
12
N
3
O
2
S :
-
-
2
00 mg of (1.06 mmol) 2-bromo-4-fluoroaniline by the reaction
with 154 mg (1.06 mmol) of ethyl isothiocyanatoacetate,
followed by addition of 125 mg (1.17 mmol) of pyridine-2-
carbaldehyde and 66 mg (1.17 mmol) of solid KOH 7 was
obtained as light yellow powder. Yield: 248 mg (62%) after
3-(4-ethoxyphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (12). From 200 mg
(1.46 mmol) of 4-ethoxyaniline, by the reaction with 212 mg
(1.46 mmol) of ethyl isothiocyanatoacetate, followed by addition
of 172 mg (1.60 mmol) of pyridine-2-carbaldehyde and 90 mg
column chromatography. 1H NMR (400 MHZ, CDCl
.69 (d, 1Н, J=3.69 Hz, Нα-Ру), 7.76 (td, 1Н, J =7.74, J
Нβ-Рy), 7.43 (m, 3Н, Hβ-Ph, Нβ’-Рh, Нβ’-Рy), 7.26 (m, 2Н, Нγ-Ру,
Нα-Рh), 6.63 (s, 1Н, -СН=). 13C NMR (101 MHz, CDCl
, δ, ppm)
176.4, 162.7, 159.1, 153.2, 149.6, 137.4, 131.9, 130.1, 128.1,
, δ, ppm):
2
=1.83 Hz,
3
8
1
(1.60 mmol) of solid KOH 12 was obtained as a yellow powder.
Yield: 419 mg (89%) after column chromatography. 1H NMR
3
(400 MHz, DMSO-d
(td, 1H, J =7.70 Hz, J
Hβ'-Py), 7.38 (m, 1H, Hγ-Py), 7.26 (m, 2H, Hα, Hα'-Ph), 7.01 (m,
2
2H, Hβ, Hβ-Ph), 6.76 (s, 1H, -CH=), 4.05 (q, 2H, J=6.97 Hz, OCH -
6
, δ, ppm): 8.75 (d, 1H, J=4.40 Hz, Hα-Py), 7.89
1
26.7, 124.4, 123.3, 120.8, 120.5, 108.6. HRMS (pos.) calculated
1
2
=1.83 Hz, Hβ-Py), 7.75 (d, 1H, J=7.89 Hz,
+
+
+
C
15 3
H10BrFN OS : 379.9687 (7 + H isotopic) , 377.9707 (7 + H) ,
+
+
found 379.9718 (7 + H isotopic) , 377.9706 (7 + H) . HPLC purity
1
3
98.91%
3 6
), 1.32 (t, 3H, J=6.97 Hz, -CH ). C NMR (101 MHz, DMSO-d , δ,
ACS Paragon Plus Environment

Page 17 of 34
Journal of Medicinal Chemistry
1
2
ppm) 178.6, 163.9, 159.1, 153.7, 150.3, 137.9, 130.4, 130.3,
3-(2-methoxy-4-chloro-5-methylphenyl)-5-((Z)-pyridin-
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
27.1, 125.8, 123.7, 114.9, 108.8, 63.8, 15.1. HRMS (neg.)
2-ylmethylene)-2-thioxoimidazolidin-4-one (18). From 200
mg (1.17 mmol) of 2-methoxy-4-chloro-5-methyl aniline,) by the
-
-
calculated C17
-
H
14
O
2
N
3
S (12 - H) : 324.0807, found 324.0812 (12
-
H) . HPLC purity 95.90%
reaction
with
170
mg
(1.17
mmol)
of
ethyl
3
-(3,4-dimethoxyphenyl)-5-((Z)-2-pyridylmethylidene)-
isothiocyanatoacetate, followed by addition of 138 mg (1.30
mmol) of pyridine-2-carbaldehyde and 72 mg (1.30 mmol) of
solid KOH 18 was obtained as light yellow powder. Yield: 253 mg
2
-thioxotetrahydro-4H-imidazol-4-one (13). From 200 mg
(1.31 mmol) of 3,4-dimethoxyaniline, by the reaction with 190
1
mg (1.31 mmol) of ethyl isothiocyanatoacetate, followed by
addition of 154 mg (1.44 mmol) of pyridine-2-carbaldehyde and
3
(60%) after column chromatography. H NMR (400 MHZ, CDCl ,
δ, ppm): 8.67 (m, 1Н, Нα-Ру), 7.74 (m, 1Н, Нβ-Рy), 7.42 (m, 1Н,
8
0 mg (1.44 mmol) of solid KOH 13 was obtained as a yellow
Нβ’-Рy), 7.25 (m, 1Н, Нγ-Ру), 7.11 (s, 1H, Hβ-Ph), 7.04 (s, 1H, Hα’-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
13
powder. Yield: 322 mg (72%) after column chromatography.
NMR (400 MHz, CDCl
.77 (td, 1H, J =7.70 Hz, J
Hz, Hβ’-Py), 7.29 (dd, 1H, J
2H, Hα, Hβ-Ph), 6.85 (m, 1H, Hα'-Ph), 6.60 (s, 1H, -CH=), 3.91 (s,
H
Ph), 6.60 (s, 1Н, -СН=), 3.77 (s, 3H, -OCH
NMR (101 MHz, CDCl
137.3, 136.6, 132.1, 130.6, 128.6, 126.5, 123.0, 119.7, 113.5, 107.9, 56.3,
3 3
), 2,33 (s, 3H, -CH ). C
3
, δ, ppm): 8.69 (d, 1H, J=4.04 Hz, Hα-Py),
=1.65 Hz, Hβ-Py), 7.44 (d, 1H, J=7.89
=7.52 Hz, J =2.02 Hz, Hγ-Py), 6.96 (m,
3
, δ, ppm) 177.8, 163.5, 153.9, 153.6, 149.6,
7
1
2
+
+
1
2
19.2. HRMS (pos.) calculated C17
H
15ClN
3
O
2
S : 360.0569 (18 + H) ,
+
found 360.0603 (18 + H) . HPLC purity 95.14%
1
3
3-(3-chloro-4-fluorophenyl)-5-((Z)-pyridin-2-
3
H, p-OCH
δ, ppm) 178.6, 163.9, 153.6, 150.3, 149.5, 149.0, 137.9, 130.3,
27.1, 126.0, 123.7, 121.7, 113.0, 111.8, 108.7, 56.1, 56.0. HRMS
3 3 6
), 3.88 s, 3H, m-OCH ). C NMR (101 MHz, DMSO-d ,
ylmethylene)-2-thioxoimidazolidin-4-one (19). From 200
mg (1.37 mmol) of 3-chloro-4-fluoro aniline, by the reaction with
199 mg (1.37 mmol) of ethyl isothiocyanatoacetate, followed by
addition of 161 mg (1.51 mmol) of pyridine-2-carbaldehyde and
85 mg (1.51 mmol) of solid KOH 19 was obtained as light yellow
1
-
-
(
3
neg.) calculated
C
17
H
14
O
3
N
3
S : 340.0756 (13
40.0760 (13 - H) . HPLC purity 97.03%
3-(4-t-butylphenyl)-5-((Z) -2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (14). From 200 mg
- H) , found
-
powder. Yield: 366 mg (80%) after column chromatography. 1
NMR (400 MHZ, DMSO-d , δ, ppm): 11.91 (br.s., 1Н, NH), 8.76 (d,
1Н, J=4.70 Hz, Нα-Ру), 7.92 (td, 1Н, J =7.43 Hz, J =1.96 Hz, Нβ-
H
(1.34 mmol) of 4-tertbutylaniline, by the reaction with 194 mg
6
(
1.34 mmol) of ethyl isothiocyanatoacetate, followed by addition
1
2
of 158 mg (1.48 mmol) of pyridine-2-carbaldehyde and 83 mg
Рy), 7.75 (m, 2Н, Нα, Нα’ -Рh), 7.58 (m, 1Н, Нβ -Рh), 7.51 (m, 1Н,
(
1.48 mmol) of solid KOH 14 was obtained as a yellow powder.
Нγ-Ру), 7.40 (m, 1Н, Нβ’-Ру), 6.80 (с, 1Н, -СН=). HRMS (neg.)
1
calculated C H ClFN OS
-
: 332.0066(19 - H)
-
, 334.0031 (19 - H
Yield: 402 mg (89%) after column chromatography. H NMR
15
8
3
-
-
-
(400 MHz, CDCl
3
, δ, ppm): 8.72 (d, 1H, J=5.38 Hz, Hα-Py), 7.84
isotopic) , found 332.0065 (19 - H) , 334.0035 (19 - H isotopic) .
HPLC purity 96.63%
(
m, 1H, Hβ-Py), 7.53 (m, 3H, Hβ, Hβ'-Ph, Hβ'-Py), 7.33 (m, 3H, Hγ-
1
3
3-(2-methyl-3-chlorophenyl)-5-((Z)-pyridin-2-
Py, Hα, Hα'-Ph), 6.62 (s, 1H, -CH=), 1.36 (m, 9H, t-Bu). C NMR
101 MHz, DMSO-d , δ, ppm) 179.0, 164.5, 153.1, 149.9, 147.9,
137.6, 132.0, 129.7, 129.2, 126.9, 126.8, 123.4, 108.9, 35.7, 31.5.
(
6
ylmethylene)-2-thioxoimidazolidin-4-one (20). From 200
mg (1.41 mmol) of 2-methyl-3-chloro aniline, by the reaction
with 204 mg (1.41 mmol) of ethyl isothiocyanatoacetate,
followed by addition of 167 mg (1.56 mmol) pyridine-2-
carbaldehyde and 87 mg (1.56 mmol) of solid KOH 20 was
obtained as light yellow powder. Yield: 414 mg (89%) after
+
+
HRMS (pos.) calculated C19
H
20
N
3
OS : 338.1327 (14 + H) , found
38.1322 (14 + H) . HPLC purity 98.83%
-(2-t-butyl)phenyl)-5-((Z)-pyridin-2-ylmethylene)-2-
thioxoimidazolidin-4-one (15). From 200 mg (1.34 mmol) of
-(tert-butyl)aniline, by the reaction with 194 mg (1.34 mmol)
+
3
3
1
2
column chromatography. H NMR (400 MHZ, CDCl
12.23 (br.s, 1Н, NH), 8.76 (d, 1Н, J=4.70 Hz, Нα-Ру), 7.86 (td, 1Н,
=7.73 Hz, J =1.76 Hz, Нβ-Рy), 7.51 (t, 2Н, J=8.22 Hz, Нβ, Нγ -Рh),
3
, δ, ppm):
of ethyl isothiocyanatoacetate, followed by addition of 158 mg
(1.48 mmol) of pyridine-2-carbaldehyde and 83 mg (1.48 mmol)
of solid KOH 15 was obtained as light yellow powder. Yield: 361
mg (80%) after column chromatography. 1H NMR (400 MHZ,
J
1
2
7.40 (m, 1Н, Нγ-Ру), 7.27 (m, 1Н, Нβ’-Рy), 7.14 (d, 1Н, J=8.22 Hz,
Нα-Рh), 6.62 (s, 1Н, -СН=), 2.20 (s, 3Н, -СH3). 13C NMR (101 MHz,
CDCl
3
, δ, ppm): 11.65 (brs, 1Н, NH), 8.71 (d, 1Н, J=3.91 Hz, Нα-
6
DMSO-d , δ, ppm) 177.1, 163.2, 153.0, 149.9, 137.6, 134.9, 133.7,
Ру), 7.77 (m, 1Н, Нβ-Рy), 7.65 (m, 1Н, Нβ’-Рy), 7.46 (m, 2Н, Нγ-
Ру, Hα’-Ph), 7.27 (m, 2H, Нβ’-Ph, Нγ-Ph),7.00 (m, 1H, Hβ-Ph),
6.63 (s, 1Н, -СН=), 1.38 (s, 9H, t-Bu). 1 C NMR (101 MHz, CDCl
130.4, 129.0, 127.8, 126.8, 123.5, 109.3, 15.0. HRMS (neg.)
-
-
calculated C16
isotopic) , found 328.0317 (20 - H)
HPLC purity 95.12%
3-(2,4,6-methylphenyl)-5-((Z)-pyridin-2-ylmethylene)-
H
11ClN
3
OS : 328.0311 (20 - H) , 330.0282 (20 - H
3
-
-
, 330.0283 (20 - H isotopic) .
-
3
,
δ, ppm) 179.0, 164.8, 153.6, 149.6, 148.6, 137.2, 131.5, 130.5,
1
30.3, 130.0, 129.5, 127.2, 126.5, 123.0, 107.8, 36.1, 31.8.HRMS
+
+
2-thioxoimidazolidin-4-one (21). From 200 mg (1.48 mmol)
of 2,4,6-methylaniline, by the reaction with 215 mg (1.48 mmol)
of ethyl isothiocyanatoacetate, followed by addition of 175 mg
(1.63 mmol) of pyridine-2-carbaldehyde and 91 mg (1.63 mmol)
of solid KOH 21 was obtained as light yellow powder. Yield: 430
(
pos.) calculated
C
+
19
H
20
N
3
OS : 338.1327 (15
38.1359 (15 + H) . HPLC purity 96.97%
3-(2-methyl-4-diethylaminophenyl)-5-((Z)-2-
+ H) , found
3
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-
one (17). To a solution of 200 mg (0.93 mmol) of 2-methyl-4-
diethylaminoaniline hydrochloride in 10 ml of DI water 40 mg
1
mg (90%) after column chromatography. H NMR (400 MHZ,
(1.00 mmol) of sodium hydroxide was added. After extraction
CDCl
=8.22 Hz, J
7.41 (td, 1Н, J
Рh), 6.60 (s, 1Н, -СН=), 2.32 (s, 3Н, -СH
NMR (101 MHz, CDCl , δ, ppm) 176.8, 163.1, 152.9, 149.9, 142.7,
38.9, 137.6, 136.2, 129.6, 128.9, 126.9, 123.5, 109.6, 20.7, 17.3.
3
, δ, ppm): 8.76 (d, 1Н, J=4.70 Hz, Нα-Ру), 7.92 (td, 1Н,
=1.57 Hz, Нβ-Рy), 7.53 (d, 1Н, J=8.22 Hz, Нβ’-Ру),
=7.43 Hz, J =1.96 Hz, Нγ-Ру), 7.02 (s, 2Н, Нβ, Нβ’-
J
1
2
with diethyl ether (3×10 ml) the organic layers were combined
1
2
and dried over anhydrous sodium sulfate. Then, 135 mg (0.93
mmol) of ethyl isothiocyanоacetate was added dropwise to a
vigorously stirred solution of 2-methyl-4-diethylaminoaniline in
diethyl ether. The mixture was stirred overnight at r.t., the
solvent was evaporated under reduced pressure. The resulting
oil was dissolved in 10 ml of ethanol, 56 mg (1.00 mmol) of solid
KOH was added. After a clear red solution was formed, 107 mg
1
3
3
), 2.10 (s, 6Н, 2х-СH
3
).
C
3
1
-
-
HRMS (neg.) calculated
18 16 3
C H N OS : 322.1014 (21 - H) ,
-
-
323.1048 (21 - H isotopic) , found 322.1019 (21 - H) , 323.1052
-
(21 - H isotopic) . HPLC purity 98.34%
3
-(4-methylthiophenyl)-5-((Z)-pyridin-2-ylmethylene)-
(
1.00 mmol) of pyridine-2-carbaldehyde were added dropwise
2
-thioxoimidazolidin-4-one (22). From 200 mg (1.44 mmol)
and mixture was stirred for 3 hours at r.t. The solution was then
acidified with 1 M HCl until pH = 7. The precipitate formed was
filtered off, washed with water, ethanol, diethyl ether and air-
of 4- methylthioaniline, by the reaction with 209 mg (1.44 mmol)
of ethyl isothiocyanatoacetate, followed by addition of 169 mg
(1.58 mmol) of pyridine-2-carbaldehyde and 89 mg (1.58 mmol)
of solid KOH 22 was obtained as light yellow powder. Yield: 259
dried. 17 was obtained as a light yellow powder. Yield: 276 mg
1
(
81%) after column chromatography. H NMR (400 MHz, CDCl
3
,
1
mg (55%) after column chromatography. H NMR (400 MHZ,
δ, ppm): 11.61 (brs, 1H, NH), 8.70 (d, 1H, J=3.91 Hz, Hα-Py), 7.76
CDCl
Ру), 7.80 (td, 1Н, J
=7.83 Hz, J =1.96 Hz, Нγ-Ру), 7.37 (m, 2Н, Нβ’-Py), 7.29 (m, 3Н,
Нα, Нα’-Рh, Нβ’-Ph), 6.63 (s, 1Н, -СН=), 2.50 (s, 3Н, -SСH
NMR (101 MHz, DMSO-d , δ, ppm) 177.8, 163.4, 153.2, 149.9,
3
, δ, ppm): 11.79 (br.s., 1Н, NH), 8.71 (d, 1Н, J=4.70 Hz, Нα-
(t, 1H, J=9.78 Hz, Hβ-Py), 7.41 (d, 1H, J=7.82 Hz, Hβ'-Py), 7.39 (m,
1
=7.73 Hz, J =1.76 Hz, Нβ-Рy), 7.47 (td, 1Н,
2
1H, Hγ-Py), 6.98 (m, 1H, Hβ-Ph), 6.63 (s, 1H, -CH=), 6.57 (m, 2H,
J
1
2
Hα, Hβ'-Ph), 3.38 (q, 4H, J=6.84 Hz, -CH -), 1.19 (t, 6H, J=6.85 Hz,
2
1
3
3
).
C
+
+
-
CH
3
). HRMS (pos.) calculated C20
H
23
N
4
OS : 367.1587 (17 + H) ,
6
+
found 367.1591 (17 + H) . HPLC purity 95.22%
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Journal of Medicinal Chemistry
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8 2 3
(neg.) calculated C15H F N -
OS-: 316.0362 (29 H)-, found
316.0360 (29 - H) . HPLC purity 97.50%
Syntheses of compounds 16, 25, 26, and 27
1
39.6, 137.6, 131.3, 129.9, 129.2, 126.8, 125.8, 123,4, 108.6, 14.6.
-
-
-
HRMS (neg.) calculated
327.0455 (22 - H isotopic) , found 326.0427 (22 - H) , 327.0459
C
-
16
H
12
N
3
OS
2
:
326.0422 (22
-
H) ,
-
-
(22 - H isotopic) . HPLC purity 95.78%
General Protocol (16, 25, 26, 27). In a round bottom flask,
1000 mg of aniline was dissolved in 1 ml of ionic liquid (BMIM-
X) and heated to 100°C. Then isothiocyanate ethyl acetate
(equimolar amount) was slowly added to the solution and
stirred for several hours (TLC control). After the completion of
the reaction, the mixture was diluted with 10 ml of ethanol, 1.1
equivalent of pyridine-2-carbaldehyde and 1.1 equivalent of
solid KOH were added. The reaction was stirred for several
hours at r.t., 1 M HCl was added until pH = 7, the precipitate
formed was filtered off, washed with water, ethanol, diethyl
ether and air-dried. Final products purified by column
3
-
(4-iodophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (23). From 200 mg
0.91 mmol) of 4-iodoaniline, by the reaction with 146 mg (0.91
(
mmol) of ethyl isothiocyanatoacetate, followed by addition of
107 mg (1.00 mmol) of pyridine-2-carbaldehyde and 56 mg
(
1.00 mmol) of solid KOH 23 was obtained as dark yellow
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
powder. Yield: 174 mg (47%) after column chromatography.
H
NMR (400 MHz, DMSO-d
6
, δ, ppm): 11.89 (brs, 1H, NH), 8.75 (d,
1H, J=4.70 Hz, Hα-Py), 7.90 – 7.86 (m, 3H, Hβ-Py, Hβ, Hβ'-Ph),
7.75 (d, 1H, J=8.61 Hz, Hβ'-Py), 7.38 (m, 1H, Hγ-Py), 7.22 – 7.20
chromatography on silica gel, eluent: CH
2
Cl
2
:MeOH. The
(
d, 2H, J=8.22 Hz, Hα, Hα'-Ph), 6.78 (s, 1H, -CH=). 13C NMR (101
MHz, DMSO-d , δ, ppm) 177.5, 164.5, 163.2, 157.8, 153.1, 149.9,
137.6, 131.9, 131.1, 126.8, 123.4, 114.6, 108.8. HRMS (neg.)
products were isolated as yellow
a
powders.
6
-
-
calculated C15
-
H
9
IN
3
OS : 405.9511 (23 - H) , found 405.9510 (23
-
H) . HPLC purity 97.11%
3
-(2-methyl-4-diethylaminophenyl)-5-((Z)-2-
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-
one (24). Sodium hydroxide 40 mg (1.00 mmol) was added to a
solution of 200 mg (0.93 mmol) of 2-methyl-4-
diethylaminoaniline hydrochloride in 10 ml of DI water. After
extraction with diethyl ether (3 × 10 ml) the organic layers were
combined and dried over anhydrous sodium sulfate. Then, 135
mg (0.93 mmol) of ethyl isothiocyanоacetate was added
(Z)-3-(4-nitrophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one (16). In a round bottom
flask, 200 mg (1.45 mmol) of 4-nitroaniline was dissolved in 1
ml of BMIM-N(CN) , and heated to 100°C .Then, 210 mg (1.45
2
dropwise to
a vigorously stirred solution of 2-methyl-4-
mmol) of ethyl isothiocyanоacetate was added dropwise. The
mixture was stirred for 48 hours at 100°C (TLC control). After
the completion of the reaction, the mixture was diluted by 10 ml
of ethanol, 89 mg (1.59 mmol) of solid KOH and 171 mg (1.59
mmol) of pyridine-2-carbaldehyde was added. The mixture was
stirred for 6 hours at r.t. After acidification with 1 M HCl until
pH = 7, the precipitate formed was filtered off, washed with
ethanol, water, diethyl ether and air-dried. 16 was obtained as a
yellow powder. Yield: 137 mg, (29%) after three-time
diethylaminoaniline in diethyl ether. The mixture was stirred
overnight at r.t., the solvent was evaporated under reduced
pressure. Then, 10 ml of EtOH and 56 mg (1.00 mmol) of solid
KOH were added to the resulting oil, followed by addition of 107
mg (1.00 mmol) of pyridine-2-carbaldehyde. The mixture was
stirred for 3 hours at r.t. The solution was acidified with 1 M HCl
until pH = 7. The precipitate formed was filtered off, washed with
water, ethanol, diethyl ether and air-dried. 24 was obtained as
light yellow powder. Yield: 276 mg (81%). 1H NMR (400 MHz,
purification by column chromatography (CH
2
Cl
2
: MeOH 10:1;
CDCl
Py), 7.76 (t, 1H, J
Py), 7.39 (m, 1H, Hγ-Py,), 6.98 (m, 1H, Hβ-Ph), 6.63 (s, 1H, -CH=),
6.57 (m, 2H, Hα, Hβ'-Ph), 3.38 (q, 4H, J=6.84 Hz, -CH -), 1.19 (t,
, δ, ppm) 171.7,
3
, δ, ppm): 11.61 (brs, 1H, NH), 8.70 (d, 1H, J=3.91 Hz, Hα-
1
1
5:1; 20:1). H NMR (400 MHz, DMSO-d
6
, δ, ppm): 8.81 (m, 1H,
1
=9.78 Hz, Hβ-Py), 7.41 (d, 1H, J=7.82 Hz, Hβ'-
Hα-Py), 8.40 (m, 3H, Нα, Hβ, Hβ’-Ph), 7.93 (m, 2H, Нβ, Нβ’-Рy),
.78 (m, 1Н, Нα’-Рh), 7.43 (m, 1H, Hγ-Py), 6.86 (s, 1H, -CH=). 13
NMR (101 MHz, DMSO-d , δ, ppm) 176.9, 163.0, 158.9, 149.9,
147.3, 146.7, 137.6, 130.3, 129.8, 127.6, 126.8, 124.1, 123.5.
7
C
2
6
3 3
6H, J=6.85 Hz -CH ). 13C NMR (101 MHz, CDCl
1
66.5, 158.2, 149.9, 136.7, 135.1, 130.0, 117.9, 113.5, 112.3,
-
-
HRMS (neg.) calculated C15
H
9
N
4
O
3
S : 325.0395 (16 - H) , found
109.6, 109.1, 108.5, 103.2, 95.6, 43.7, 17.9, 12.6. HRMS (pos.)
-
+
3
3
9
25.0397 (16
-
H) . HRMS (pos.) calculated
27.0552 (16 + H) , found 327.0544 (16 + H) . HPLC purity
C
15
H
9
N
4
O
3
S
+
+
calculated C20
+
H
23
N
4
OS : 367.1587 (24 + H) , found 367.1591 (24
+
+
+
H) . HPLC purity 96.91%
7.67%
3-(2,5-difluorophenyl)-5-((Z)-pyridin-2-ylmethylene)-2-
3
-(2,6-dichlorophenyl)-5-((Z)-pyridin-2-ylmethylene)-2-
thioxoimidazolidin-4-one (28). From 200 mg (1.55 mmol) of
,5-difluoro aniline, by the reaction with 225 mg (1.55 mmol) of
ethyl isothiocyanatoacetate, followed by addition of 183 mg
1.71 mmol) of pyridine-2-carbaldehyde and 96 mg (1.71 mmol)
thioxoimidazolidin-4-one (25). In a round bottom flask, 1000
mg (6.17 mmol) of 2,6-dichloroaniline was dissolved in 1 ml of
BMIM-BF and heated to 100°C. Then, 1.89 g (6.17 mmol) of
4
2
(
ethyl isothiocyanоacetate was added dropwise. The mixture was
stirred for 48 hours at 100°C (TLC control). After the completion
of the reaction, the mixture was diluted with 10 ml of EtOH and
726 mg (6.79 mmol) of pyridine-2-carbaldehyde and 380 mg
(6.79 mmol) of KOH were added. The mixture was stirred for 2
hours at r.t., acidified with 1 M HCl until pH = 7. The precipitate
formed was filtered off, washed with ethanol, water, diethyl
ether and air-dried. 25 was obtained as a light yellow powder.
of solid KOH 28 was obtained as light yellow powder. Yield: 371
mg (75%) after column chromatography. 1H NMR (400 MHz,
3
CDCl , δ, ppm): 12.06 (br.s, 1Н, NH), 8.74 (d, 1Н, J=3.91 Hz, Нα-
Ру), 7.85 (t, 1Н, J=7.83 Hz, Нβ-Рy), 7.51 (d, 1Н, J=7.83 Hz, Нβ’-
Ру), 7.36 (m, 1Н, Нγ-Ру), 7.23 (m, 2Н, Нγ, Нβ’-Рh), 7.15 (m, 1Н,
Нα-Ph), 6.65 (s, 1Н, -СН=). 174.6, 166.9, 162.7, 158.1, 156.4,
152.8, 149.9, 137.6, 129.4, 128.2, 126.9, 123.6, 118.7, 117.6,
109.9. HRMS (neg.) calculated C15
found 316.0361 (28 - H) . HPLC purity 98.02%
-
-
H
8
F
2
N
3
OS : 316.0362 (28 - H) ,
1
Yield: 108 mg (5%) after column chromatography. H NMR (400
-
MHz, CDCl
3
, δ, ppm): 11.83 (br.s., 1Н, NH), 8.72 (d, 1Н, J=3.91 Hz,
3
-(3,5-difluorophenyl)-5-((Z)-pyridin-2-ylmethylene)-2-
thioxoimidazolidin-4-one (29). From 200 mg (1.55 mmol) of
,5-difluoro aniline, by the reaction with 225 mg (1.71 mmol) of
Нα-Ру), 7.80 (td, 1Н, J
1
=7.83 Hz, J = 1.96 Hz, Нγ-Рy), 7.52 (d, 2Н,
2
J=6.85 Hz, Нβ’, Нβ-Рh), 7.48 (m, 2Н, Нβ’-Ру, Нγ-Ph), 7.32 (m, 1Н,
3
+
2 3
Нβ-Ру), 6.67 (s, Н, -CH=). HRMS (pos.) calculated C15H10Cl N OS
3
ethyl isothiocyanatoacetate, followed by addition of 183 mg
(1.71 mmol) of pyridine-2-carbaldehyde and 96 mg (1.71 mmol)
of solid KOH 29 was obtained as light yellow powder. Yield: 240
mg (49%) after column chromatography. 1H NMR (400 MHz,
+
+
49.9922 (25 + H) , found 349.9904 (25 + H) . HPLC purity
9
5.94%
3-(2,4,6-trichlorophenyl)-5-((Z)-pyridin-2-ylmethylene)-
2-thioxoimidazolidin-4-one (26). In a round bottom flask,
CDCl
=7.79 Hz, J
7.35 (dd, 1Н, J
Рh), 6.94 (m, 1Н, Нγ -Рh), 6.54 (s, 1Н, -СН=). C NMR (101 MHz,
DMSO-d , δ, ppm) 177.0, 168.1, 162.9, 160.8, 149.9, 145.6, 137.6,
29.7, 126.8, 125.9, 123.5, 119.8, 113.4, 113.1, 109.0.HRMS
3
, δ, ppm): 8.73 (d, 1Н, J=3.85 Hz, Нα-Ру), 7.84 (td, 1Н,
=1.65 Hz, Нβ-Рy), 7.50 (d, 1Н, J=7.70 Hz, Нβ’-Ру),
= 7.34 Hz, J = 5.32 Hz, Нγ-Ру), 7.02 (m, 2Н, Нα,Нα’-
1000 mg (5.10 mmol) of 2,4,6-trichloroaniline was dissolved in
J
1
2
1
ml of BMIM-BF and heated to 100°C. Then, 740 mg (5.10
4
1
2
mmol) of ethyl isothiocyanоacetate was added dropwise. The
mixture was stirred for 48 hours at 100°C (TLC control). After
the completion of the reaction, the mixture was diluted with 10
ml of ethanol, 600 mg (5.61 mmol) of pyridine-2-carbaldehyde
1
3
6
1
ACS Paragon Plus Environment

Page 19 of 34
Journal of Medicinal Chemistry
1
2
and 314 mg (5.61 mmol) of KOH were added. The reaction was
7.44 (m, 1Н, Нβ-Рy), 7.24 - 7.21 (m, 2H, Нα, Нα’-Рh), 7.16 – 7.09
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
stirred for 2 hours at r.t., acidified with 1 M HCl until pH = 7. The
precipitate formed was filtered off, washed with ethanol, water,
diethyl ether and air-dried. 26 was obtained as a light yellow
(m, 3Н, Нβ, Нγ-Рh, -СН=), 2.72 (s, 3H, -SCH
DMSO-d , δ, ppm) 168.0, 161.2, 153.1, 150.4, 140.3, 137.0, 134.2,
131.6, 126.9, 124.5, 122.8, 116.8, 115.6, 100.3, 87.9, 13.5.
HRMS (pos.) calculated C16H FN OS : 314.0758 (31 + H)
314.0761 (31 + H) . HPLC purity 98.34%
3
). C NMR (101 MHz,
6
powder. Yield: 100 mg (6%). after column chromatography. 1
H
+
+
, found
13
3
+
NMR (400 MHz, CDCl
3
, δ, ppm): 11.91 (br.s., 1Н, NH), 8.72 (d, 1Н,
J=3.91 Hz, Нα-Ру), 7.81 (m, 1Н, Нβ-Рy), 7.52 (s, 2H, Hβ, Hβ’-Ph)
5-(Z)-3-(2-fluorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one (32). From 100 mg (0.33
mmol) of 3-(2-fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 3 as a result of reaction
with 110 mg (0.77 mmol) of methyl iodide, in the presence of 22
mg (0.39 mmol) of solid KOH, 32 was obtained as a white-yellow
7
.46 (d, 1Н, J=7.83 Hz, Нβ’-Рy), 7.31 (m, 1Н, Нγ-Ру), 6.67 (s, Н, -
+
+
CH=). HRMS (pos.) calculated C15
found 383.9514 (26 + H) . HPLC purity 96.00%
-(2,6-dibromophenyl)-5-((Z)-pyridin-2-ylmethylene)-2-
H
9
Cl
3
N
3
O
S
383.9532 (26 + H) ,
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
thioxoimidazolidin-4-one (27). In a round-bottom flask, 1000
mg (5.10 mmol) of 2,4-dibromaniline was dissolved in 1 ml of
powder. Yield: 32 mg (31%) after column chromatography. 1
H
BMIM-BF
4
and heated to 100°C. Then, 1.578 mg (3.99 mmol) of
3
NMR (400 MHz, CDCl , δ, ppm): 8.89 (d, 1H, J=7.82 Hz, Hα-Py),
ethyl isothiocyanоacetate was added dropwise. The mixture was
stirred for 48 hours at 100°C (TLC control). After the completion
of the reaction, the mixture was diluted with 10 ml of ethanol,
8.76 (d, 1H, J= 3.91 Hz, Hβ’-Py), 7.91 (t, 1H, J=7.82 Hz, Hy-Py),
7
.50 (m, 1H, Hα-Ph), 7.38 -7.25 (m, 5H, Hβ, Hβ ', Hγ-Ph, Hβ-Py -
+
CH=), 2.76 (s, 3H, -SCH
3
). HRMS (pos.) calculated C16
H13FN
3
OS :
4
69 mg (4.39 mmol) of pyridine-2-carbaldehyde and 246 mg
+
+
314.0758 (32 + H) , found 314.0763 (32 + H) . HPLC purity
(4.39 mmol) of solid KOH were added. The mixture was stirred
9
8.20%
-(Z)-3-(4-bromophenyl)-2-(methylthio)-5-(pyridin-2-
for 2 hours at r.t., acidified with 1 M HCl until pH = 7. The
precipitate formed was filtered off, washed with ethanol, water,
diethyl ether and air-dried. 27 was obtained as a light yellow
5
ylmethylidene)-1H-imidazol-4-one (33). From 100 mg (0.28
mmol) of 3-(4-bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 4 as a result of reaction
with 93 mg (0.65 mmol) of methyl iodide, in the presence of 19
mg (0.33 mmol) of solid KOH, 33 was obtained as a white-yellow
powder. Yield: 90 mg (5%) after column chromatography. 1
H
3
NMR (400 MHz, CDCl , δ, ppm): 12.05 (br.s, 1Н, NH), 8.74 (d, 1Н,
J=4.89 Hz, Нα-Ру), 7.93 (t, 1Н, J=7.82 Hz, Нβ-Рy), 7.71 (d, 1Н,
J=7.33 Hz, Нβ’-Рy), 7.56 (d, 2Н, J=7.92 Hz, Нβ, Нβ’-Рh), 7.41 (m,
1Н, Нγ-Ру), 7.27 (m, 1Н, Нγ-Рh), 6.60 (s, Н, -CH=). HRMS (pos.)
powder. Yield: 42 mg (40%) after column chromatography. 1
NMR (400 MHz, CDCl , δ, ppm): 8.80 (d, 1Н, J=8.22 Hz, Нα-Ру),
.68 (d, 1Н, J=4.70 Hz, Нβ’-Рy), 7.76 (dt, 1Н, J =7.63 Hz, J =1.37
Hz, Нγ-Рy), 7.61 (m, 2H, Нβ-Ру, -CH=), 7.25 (m, 2Н, Нβ, Нβ’ -Рh),
H
3
+
+
calculated C15
(
H
10Br
2
N
3
O
S
439.8891 (27 + H) , found 439.8909
8
1
2
+
27 + H) . HPLC purity 97.38%
Syntheses of compounds 30-51
General Protocol. 1.2 eq. of solid KOH was added to a solution
1
3
7
.20 (m, 2Н, Нα, Нα’-Рh), 2.72 (s, 3H, -SCH
DMSO-d , δ, ppm) 171.9, 167.6, 152.9, 150.1, 139.8, 136.8, 132.3,
31.5, 129.3, 126.6, 123.9, 123.0, 122.5, 12.9.HRMS (pos.)
3
). C NMR (101 MHz,
6
of
thioxotetrahydro-4H-imidazol-4-one in H
2 ml per 100 mg of compound). After a clear red solution was
of
the
3-aryl-5-((Z)
-2-pyridylmethylidene)-2-
1
2
O/EtOH 1:1 mixture
+
+
3
calculated C16H13BrN OS 373.9958 (33 + H) , found 373.9963
(
+
(33 + H) . HPLC purity 95.83%
formed, methyl iodide (2.3 eq.) was added dropwise and the
mixture was stirred for ten minutes. The mixture was placed in
a freezer for 1h., the precipitate formed was filtered off, washed
with an aqueous 1 M KOH, water, ice-cold ethanol, diethyl ether
and air-dried. The products were isolated as a light yellow
powders after purification by column chromatography, eluent
5-(Z)-3-(3-bromophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one (34). From 100 mg (0.28
mmol) of 3-(3-bromophenyl)-5-((Z) -2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 5 as a result of reaction
with 93 mg (0.65 mmol) of methyl iodide, in the presence of 19
mg (0.33 mmol) of solid KOH, 34 was obtained as a white-yellow
2 2
CH Cl : MeOH 20:1.
powder. Yield: 52 mg (50%) after column chromatography. 1
NMR (400 MHz, CDCl , δ, ppm): 8.80 (d, 1Н, J=7.83 Hz, Нα-Ру),
H
3
8.68 (d, 1Н, J=5.67 Hz, Нβ’-Рy), 7.76 (t, 1Н, J=8.23 Hz, Нγ-Рy),
7
.58 (m, 1H, Нα-Рh), 7.52 (s, 1H, -CH=), 7.36 (m, 1Н, Нβ-Рy), 7.30
(m, 1Н, Нα’-Рh), 7.29 – 7.20 (m, 2Н, Нβ, Нγ-Рh), 2.72 (s, 3H, -
13
3 6
SCH ). C NMR (101 MHz, DMSO-d , δ, ppm) 177.5, 167.5, 150.1,
146.7, 139.7, 136.8, 133.5, 132.3, 131.5, 129.3, 126.6, 123.9,
+
1
3
9
3
22.5, 119.1, 110.8, 12.9. HRMS (pos.) calculated C16H13BrN OS
+
+
73.9958 (34 + H) , found 373.9962 (34 + H) . HPLC purity
6.16%
5
-(Z)-3-(4-fluorophenyl)-2-(methylthio)-5-(pyridin-2-
5
-(Z)-3-(2-bromophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one (30). From 100 mg (0.33
mmol) of 3-(4-fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 1 as a result of reaction
with 110 mg (0.77 mmol) of methyl iodide, in the presence of 22
mg (0.39 mmol) of solid KOH, 30 was obtained as a white-yellow
ylmethylidene)-1H-imidazol-4-one (35). From 100 mg (0.28
mmol) of 3-(2-bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 6 as a result of reaction
with 93 mg (0.65 mmol) of methyl iodide, in the presence of 19
mg (0.33 mmol) of solid KOH, 35 was obtained as a white-yellow
powder. Yield: 39 mg (38%) after column chromatography. 1
NMR (400 MHz, CDCl , δ, ppm): 8.92 (d, 1Н, J=8.27 Hz, Нα-Ру),
H
powder. Yield: 30 mg (29%) after column chromatography. 1
H
3
NMR (400 MHz, CDCl
.69 (d, 1H, J=4.70 Hz, Hβ’-Py), 7.74 (m, 2H, Hγ-Py, Hα-Ph), 7.45
m, 1H, Hβ-Py), 7.34 (m, 2H, Hβ, Hβ'-Ph), 7.21 (m, 2H, Hγ-Ph, -
3
, δ, ppm): 8.82 (d, 1H, J=9.00 Hz, Hα-Py),
8.81 (d, 1Н, J=5.09 Hz, Нβ’-Рy), 8.04 (t, 1Н, J=8.42 Hz, Нγ-Рy),
8
7
.48 (m, 1H, Нβ-Ру,), 7.30 (m, 2Н, Нβ, Нβ’ -Рh), 7.19 (m, 2Н, Нα,
(
1
3
3
Нα’-Рh), 7.12 (s, 1Н, -СН=), 2.76 (s, 3H, -SCH ). C NMR (101
1
3
3 6
CH=), 2.71 (s, 3H, -SCH ). C NMR (101 MHz, DMSO-d , δ, ppm)
MHz, DMSO-d
6
, δ, ppm) 181.6, 168.4, 165.0, 161.4, 153.5, 150.4,
168.0, 153.2, 150.5, 140.1, 137.1, 133.9, 131.9, 131.9, 131.8,
1
40.4, 137.0, 130.6, 128.9, 126.8, 124.0, 122.6, 116.8,
1
29.6, 126.9, 124.2, 123.2, 123.0, 122.9, 13.3. HRMS (pos.)
+
1
3
3.43.HRMS (pos.) calculated for C16H13FN OS : 314.0758 (30 +
H) , found 314.0761 (30 + H) . HPLC purity 98.95%
+
+
calculated C16
H13BrN
3
OS 373.9958 (35 + H) , found 373.9967
+
+
+
(35 + H) . HPLC purity 97.01%
5
-(Z)-3-(3-fluorophenyl)-2-(methylthio)-5-(pyridin-2-
5
-(Z)-3-(2-bromo-4-fluorophenyl)-2-(methylthio)-5-
pyridin-2-ylmethylene)-1H-imidazol-5(4H)-one (36). From
00 mg (0.27 mmol) of (Z)-3-(2-bromo-4-fluorophenyl)-5-
pyridin-2-ylmethylene)-2-thioxoimidazolidin-4-one as
ylmethylidene)-1H-imidazol-4-one (31). From 100 mg (0.33
mmol) of 3-(3-fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 2 as a result of reaction
with 110 mg (0.77 mmol) of methyl iodide, in the presence of 22
mg (0.39 mmol) of solid KOH, 31 was obtained as a white-yellow
(
1
(
7
a
result of reaction with 90 mg (0.63 mmol) methyl iodide, in the
presence of 18 mg (0.33 mmol) of solid KOH, 36 was obtained as
powder. Yield: 43 mg (42%) after column chromatography. 1
NMR (400 MHz, CDCl , δ, ppm): 8.81 (d, 1Н, J=8.03 Hz, Нα-Ру),
.68 (d, 1Н, J=3.91 Hz, Нβ’-Рy), 7.76 (t, 1Н, J=8.02 Hz, Нγ-Рy),
H
a white-yellow powder. Yield: 47 mg (45%) after column
3
1
chromatography. H NMR (400 MHz, CDCl
3
, δ, ppm): 8.85 (d, 1Н,
8
J=7.83 Hz, Нα-Ру), 8.72 (d, 1Н, J=3.91 Hz, Нβ’-Рy), 7.84 (m, 1Н,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 34
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Нγ-Рy), 7.45 (m, 2H, Нβ-Ру, Нβ-Рh), 7.30 (m, 1Н, Нβ’-Рh), 7.22
165.2, 159.6, 153.6, 150.1, 140.5, 136.6, 129.3, 126.8, 124.8,
122.6, 115.5, 63.9, 14.9, 13.34. HRMS (pos.) calculated
13
(m, 2Н, Нα-Рh, -СН=), 2.75 (s, 3H, -SCH
3
). C NMR (101 MHz,
+
+
+
CDCl
3
, δ, ppm) 167.9, 159.0, 156.5, 153.0, 149.3, 139.9, 136.8,
C H N O S 340.1115 (41 + H) , found 340.1116 (41 + H) .
18 18 3 2
1
31.0, 128.4, 127.2, 124.4, 123.5, 120.9, 120.7, 116.5, 13.3. HRMS
HPLC purity 99.30%
+
+
(pos.) calculated C16
H
12BrFN
3
OS : 393.9843 (36 + H isotopic) ,
5-(Z)-3-(3,4-dimethoxyphenyl)-2-(methylthio)-5-
(pyridin-2-ylmethylidene)-1H-imidazol-4-one (42). From
100 mg (0.29 mmol) of 3-(3,4-dimethoxyphenyl)-5-((Z)-2-
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one 13
as a result of reaction with 95 mg (0.67 mmol) of methyl iodide,
in the presence of 19.5 mg (0.35 mmol) of solid KOH, 42 was
obtained as a white-yellow powder. Yield: 78 mg (76%) after
+
+
391.9863 (36 + H) , found 393.9879 (36 + H isotopic) ,
+
391.9902 (36 + H) . HPLC purity 96.00%
5
-(Z)-3-(4-chlorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one (37). From 100 mg (0.32
mmol) of 3-(4-chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 8 as a result of reaction
with 103 mg (0.72 mmol) of methyl iodide, in the presence of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
column chromatography. 1H NMR (400 MHz, CDCl
3
, δ, ppm):
.90 (m, 2H, J=7.83 Hz, Hα, Hβ-Py), 8.08 (m, 1H, Hγ-Py), 7.51 (m,
H, Нβ-Рy), 6.97 – 6.81 (m, 4H, Hα, Hα', Hβ –Ph, -CH=), 3.93 (s,
H, p-OCH ), 3.90 (s, 3H, m-OCH ), 2.78 (s, 3H, -SCH ). HRMS
21.5 mg (0.38 mmol) of solid KOH, 37 was obtained as a white-
8
1
3
yellow powder. Yield: 50 mg (47%) after column
1
chromatography. H NMR (400 MHz, CDCl
3
, δ, ppm): 8.82 (d, 1H,
=4.89 Hz, Hβ’-Py), 7.80 (t, 1H,
J=6.85 Hz, Hγ-Py), 7.48 (m, 2H, Hα, Hα'-Ph), 7.42 – 7.30 (m, 3H,
3
3
3
J=7.83 Hz, Hα-Py), 8.72 (d, 1H, J
1
+
H)+, found
356.1064 (42
(pos.) calculated
C
+
18
H
18
N
3
O
2
S
+
356.1065 (42 + H) . HPLC purity 95.02%
Hβ, Hβ '-Ph, Hβ-Py,), 7.22 (s, 1H, -CH=), 2.74 (s, 3H, -SCH
3
+
). HRMS
5-(Z)-3-(4-t-butylphenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one (43). From 100 mg (0.30
mmol) of 3-(4-tert-butylphenyl)-5-((Z)-2-pyridylmethylidene)-
2-thioxotetrahydro-4H-imidazol-4-one 14 as a result of reaction
with 98 mg (0.69 mmol) of methyl iodide, in the presence of 20
mg (0.36 mmol) of solid KOH, 43 was obtained as a white-yellow
+
(
pos.) calculated C16
H13ClN
3
OS 330.0463 (37 + H) , found
+
330.0464 (37 + H) . HPLC purity 96.27%
5
-(Z)-3-(3-chlorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one (38). From 100 mg (0.32
mmol) of 3-(4-chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 9 as a result of reaction
with 104 mg (0.73 mmol) of methyl iodide, in the presence of 21
mg (0.38 mmol) of solid KOH, 38 was obtained as a white-yellow
powder. Yield: 96 mg (91%) after column chromatography. 1
NMR (400 MHz, CDCl , δ, ppm): 8.84 (m, 1Н, Нα-Ру), 8.69 (d, 1Н,
J=4.89 Hz, Нβ’-Рy), 7.78 (m, 1Н, Нγ-Рy), 7.50 (m, 2Н, Нβ, Нβ’ -Рh),
7.22 – 7.21 (m, 4Н, Нβ-Рy, Нα, Нα’-Рh, -СН=), 2.72 (s, 3H, -SCH ),
22 3
1.64 (s, 9H, -tBu). HRMS (pos.) calculated C20H N OS 352.1479
H
3
powder. Yield: 33 mg (31%) after column chromatography. 1
NMR (400 MHz, CDCl , δ, ppm): 8.86 (d, 1Н, J=7.83 Hz, Нα-Ру),
8.73 (d, 1Н, J=3.91 Hz, Нβ’-Рy), 7.85 (t, 1Н, J=7.83 Hz, Нγ-Рy),
.44 (m, 2Н, Нα, Нα’-Рh), 7.38 (m, 1H, Нβ-Рy), 7.28 (m, 3H, Hβ,
H
3
+
3
+
+
(43 + H) , found 352.1479 (43 + H) . HPLC purity 97.14%
5-(Z)-3-(2-t-butylphenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylene)-1H-imidazol-5(4H)-one (44). From 100 mg
(0.30 mmol) of (Z)-3-(2-(tert-butyl)phenyl)-5-(pyridin-2-
7
Hγ-Ph, -СН=), 3.05 (s, 3Н, -SCH
C
3
+
). HRMS (pos.) calculated
OS : 330.0463 (38 + H) , found 330.0454 (38 + H) .
+
+
16
H
13ClN
3
HPLC purity 96.12%
ylmethylene)-2-thioxoimidazolidin-4-one 15 as a result of
reaction with 100 mg (0.70 mmol) of methyl iodide, in the
presence of 20 mg (0.36 mmol) of solid KOH, 44 was obtained as
5
-(Z)-3-(2-chlorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one (39). From 100 mg (0.32
mmol) of 3-(2-chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 10 as a result of reaction
with 103 mg (0.72 mmol) of methyl iodide, in the presence of
a white-yellow powder. Yield: 42 mg (40%) after column
1
chromatography. H NMR (400 MHz, CDCl
3
, δ, ppm): 8.85 (d, 1Н,
J=7.98 Hz, Нα-Ру), 8.73 (m, 1Н, Нβ-Рy), 7.80 (m, 1Н, Нγ-Рy), 7.64
(m, 1H, Нβ’-Ру,), 7.7 (m, 1Н, Нβ’ -Рh), 7.32 (m, 1Н, Нγ-Рh), 7.25
21.5 mg (0.38 mmol) of solid KOH, 39 was obtained as a white-
yellow powder. Yield: 52 mg (49%) after column
(m, 1Н, Нα-Рh),7.21 (s, 1H, -СН=),7.04-7.00 (m, 1H, Нβ –Рh), 2.72
1
13
chromatography. H NMR (400 MHz, CDCl
3
, δ, ppm): 8.82 (d, 1H,
3 3
(s, 3H, -SCH ), 1.37 (s, 9H, -tBu). C NMR (101 MHz, CDCl , δ,
ppm169.1, 153.8, 149.9, 149.3, 140.3, 136.1, 131.5, 130.2, 129.7,
J=7.83 Hz, Hα-Py), 8.69 (d, 1H, J=3.91 Hz, Hβ’-Py), 7.76 (t, 1H,
J=7.43 Hz, Hy-Py), 7.56 (m, 1H, Hα-Ph), 7.46 – 7.32 (m, 4H, Hβ-
1
29.5, 127.2, 127.1, 127.0, 123.9, 123.1, 36.1, 31.8, 13.2. HRMS
+
+
(
pos.) calculated
C
+
20
H
22
N
3
OS : 352.1479 (44
52.1515 (44 + H) . HPLC purity 96.37%
-(Z)-3-(2-methoxy-4-chloro-5-methylphenyl)-2-
(methylthio)-5-(pyridin-2-ylmethylene)imidazolidin-4-one
45). From 100 mg (0.28 mmol) of (Z)-3-(2-methoxy-4-chloro-
-methylphenyl)-5-(pyridin-2-ylmethylene)-2-
thioxoimidazolidin-4-one 18 as a result of reaction with 92 mg
0.65 mmol)of methyl iodide, in the presence of 19 mg (0.34
+ H) , found
Py, Hγ, Hβ, Hβ '-Ph), 7.20 (s, 1H, -CH=), 2.71 (s, 3H, -SCH
3
+
). HRMS
3
+
(
pos.) calculated C16
H13ClN
3
OS 330.0463 (39 + H) , found
5
+
330.0468 (39 + H) . HPLC purity 97.20%
5
-(Z)-3-(4-methoxyphenyl)-2-(methylthio)-5-(pyridin-2-
(
5
ylmethylidene)-1H-imidazol-4-one (40). From 100 mg (0.32
mmol) of 3-(4-methoxyphenyl)-5-((Z)-2-pyridylmethylidene)-
2
-thioxotetrahydro-4H-imidazol-4-one 11 as a result of reaction
(
with 105 mg (0.74 mmol) of methyl iodide, in the presence of 21
mmol) of solid KOH, 45 was obtained as a white-yellow powder.
mg (0.38 mmol) of solid KOH, 40 was obtained as a white-yellow
1
Yield: 49 mg (47%) after column chromatography. H NMR (400
powder. Yield: 63 mg (61%) after column chromatography. 1
H
MHz, CDCl
3
, δ, ppm): 8.84 (d, 1Н, J=7.98 Hz, Нα-Ру), 8.69 (d, 1Н,
=7.76 Hz, J =1.55 Hz, Нγ-Рy),
=4.82 Hz, J =1.11 Hz, Нβ-Ру), 7.18 (s,
H, Hβ-Ph), 7.10 (s, 1H, Hα’-Ph), 7.04 (s, 1Н, -СН=), 3.79 (s, 3H, -
NMR (400 MHz, CDCl
8.76 (d, 1H, J=4.45 Hz, Hβ’-Py), 7.93 (t, 1H, J= 8.42 Hz, Hγ-Py),
.27 (m, 4H, Hα, Hα', Hβ, Hβ'-Ph), 6.96 (m, 2H, Hβ-Py, -CH=), 2.72
3
, δ, ppm): 8.87 (d, 1H, J=8.11 Hz, Hα-Py),
J=3.99 Hz, Нβ’-Рy), 7.77 (td, 1Н, J
1
2
7.22 (ddd, 1H, J
1
=7.37 Hz, J
2
3
7
1
1
3
3 6
(s, 3H, -SCH ). C NMR (101 MHz, DMSO-d , δ, ppm) 169.0,
13
OCH
3
), 2.70 (s, 3H, S-CH
3
).2.33 (s, 3H, Ph-CH
3
). C NMR (101
1
1
C
65.1, 160.3, 153.6, 150.4, 140.6, 137.0, 129.6, 126.8, 125.0,
MHz, CDCl
3
, δ, ppm) 168.9, 167.9, 154.0, 153.8, 149.9, 140.2,
24.8, 122.4, 115.2, 56.0, 13.38. HRMS (pos.) calculated
1
1
+
36.6, 136.0, 131.6, 128.6, 126.9, 123.8, 123.0, 119.0, 113.4, 56.2,
+
+
+
17
H N O S 326.0958 (40 + H) , found 326.0963 (40 + H) .
16 3 2
+
9.1, 13.1.HRMS (pos.) calculated C18H17ClN O S : 376.0696 (45
3 2
HPLC purity 96.05%
-(Z)-3-(4-ethoxyphenyl)-2-(methylthio)-5-(pyridin-2-
+
+
H isotopic) , 374.0725 (45 + H) , found 376.0727 (45 + H
5
+
+
isotopic) , 374.0760 (45 + H) . HPLC purity 95.66%
ylmethylidene)-1H-imidazol-4-one (41). From 100 mg (0.31
mmol) of 3-(4-ethoxyphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 12 as a result of reaction
with 101 mg (0.71 mmol) of methyl iodide, in the presence of 21
mg (0.37 mmol) of solid KOH, 41 was obtained as a white-yellow
5-(Z)-3-(3-chloro-4-fluorophenyl)-2-(methylthio)-5-
(pyridin-2-ylmethylene)-1H-imidazol-5(4H)-one (46). From
1
00 mg (0.30 mmol) of (Z)-3-(3-chloro-4-fluorophenyl)-5-
(pyridin-2-ylmethylene)-2-thioxoimidazolidin-4-one 19 as
a
result of reaction with 98 mg (0.69 mmol) of methyl iodide, in
the presence of 20 mg (0.36 mmol) of solid KOH, 46 was
powder. Yield: 48 mg (46%) after column chromatography. 1
NMR (400 MHz, CDCl , δ, ppm): 8.86 (d, 1H, J=7.83 Hz, Hα-Py),
H
3
obtained as a white-yellow powder. Yield: 44 mg (42%) after
8.77 (m, 1H, J= 4.45 Hz, Hβ-Py), 7.92 (t, 1H, J= 8.31 Hz, Hγ-Py),
1
column chromatography. H NMR (400 MHz, CDCl
3
, δ, ppm): 8.87
7
.34 (m, 2H, Hβ, Hβ '-Ph), 7.24 – 7.19 (m, 4H, Hβ'-Py, Hα, Hα'-Ph,
(
1
d, 1Н, J=7.83 Hz, Нα-Ру), 8.75 (d, 1Н, J=4.89 Hz, Нβ’-Рy), 7.93 (t,
Н, J=6.85 Hz, Нγ-Рy), 7.44 (dd, 2H, J =6.85 Hz, J =1.96 Hz Нα,
Нα’-Рh), 7.39 (m, 1H, Нβ -Рh), 7.31-7.23 (m, 2H, Нβ-Ру, -СН=),
-
CH=), 4.06 (q, 2H, J=6.99 Hz, -CH
2
-), 2.72 (s, 3H, -SCH
3
), 1.42 (t,
1
2
3H, J=6.97 Hz, -CH
3
). 13C NMR (101 MHz, DMSO-d
6
, δ, ppm) 168.8,
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Journal of Medicinal Chemistry
1
2
+
+
2
.76 (s, 3Н, -SCH
3
). HRMS (pos.) calculated C16
H
12ClFN
3
OS+:
found 332.0671 (51 + H) , 333.0705 (51 + H isotopic) . HPLC
purity 97.09%
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
348.0374 (46 + H) , 350.0344 (46 + H isotopic) , found
+
+
348.0369 (46 + H) , 350.0338 (46 + H isotopic) . HPLC purity
7.19%
-(Z)-3-(2-methyl-3-chlorohenyl)-2-(methylthio)-5-
pyridin-2-ylmethylene)-1H-imidazol-5(4H)-one (47). From
00 mg (0.30 mmol) of (Z)-3-(2-methyl-3-chlorohenyl)-5-
pyridin-2-ylmethylene)-2-thioxoimidazolidin-4-one 20 as
Syntheses of compounds 52-73
9
General protocol.
pyridylmethylene)-2-thioxotetrahydro-4H-imidazol-4-one (2
eq.) and dry K CO (3 eq.) was dissolved in dimethylformamide
(2 ml per 200 mg of the mixture). The mixture was cooled to 0°C,
1,2-dibromoethane (1 eq.) was added dropwise. The mixture
was stirred overnight at r.t., and then was quenched with 50 ml
of iced water. The precipitate formed was filtered off, washed
with 1 M KOH, water, diethyl ester and air-dried. The final
A
mixture of 3-aryl-5-((Z)-2-
5
(
1
2
3
(
a
result of reaction with 98 mg (0.69 mmol) of methyl iodide, in
the presence of 20 mg (0.36 mmol) of solid KOH, 47 was
obtained as a white-yellow powder. Yield: 62 mg (60%) after
column chromatography. H NMR (400 MHz, CDCl
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
, δ, ppm): 8.95
products were purified by column chromatography (CH
MeOH 20:1) and isolated as yellowish or white powders.
2 2
Cl :
(d, 1Н, J=7.82 Hz, Нα-Ру), 8.83 (d, 1Н, J=4.89 Hz, Нβ’-Рy), 8.05 (t,
1Н, J=7.82 Hz, Нγ-Рy), 7.50 (m, 2H, -Рh), 7.25 (m, 1H, -Рh), 7.32
(m, 1H, Нβ-Ру), 7.13 (s, 1Н, -СН=), 6.99 (m, 2Н, Нβ, Нβ’-Рh), 2.77
(s, 3Н, -SCH
3
+
), 2.24 (s, 3H, -SCH
OS : 344.0624 (47 + H) , 346.0595 (47 + H isotopic) ,
3
). HRMS (pos.) calculated
+
+
C
17
H
15ClN
3
+
+
found 344.0619 (47 + H) , 346.0589 (47 + H isotopic) . HPLC
purity 96.07%
5
-(Z)-3-(2,4,6-trimethylphenyl)-2-(methylthio)-5-
(pyridin-2-ylmethylene)-1H-imidazol-5(4H)-one (48). From
00 mg (0.30 mmol) of (Z)-3-(2,4,6-trimethylphenyl)-5-
pyridin-2-ylmethylene)-2-thioxoimidazolidin-4-one 21 as
1
(
(
4Z,
pyridylmethylidene)-1-(4-methoxyphenyl)-2,3-dihydro-
H-imidazol-5-one) (52). From 100 mg (0.32 mmol) of 3-(4-
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
a
result of reaction with 101 mg (0.71 mmol) of methyl iodide, in
the presence of 21 mg (0.37 mmol) of solid KOH, 48 was
obtained as a white-yellow powder. Yield: 52 mg (50%) after
column chromatography. H NMR (400 MHz, CDCl
4
methoxyphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 11 as by reaction with 30
mg (0.16 mmol) of 1,2-dibromoethane, in the presence of 66 mg
1
3
, δ, ppm): 8.87
(d, 1Н, J=7.83 Hz, Нα-Ру), 8.76 (d, 1Н, J=4.89 Hz, Нβ’-Рy), 7.90 (t,
(0.48 mmol) of solid K
2
CO
3
, 52 was obtained as a yellow powder.
1
6
2
3
Н, J=7.83 Hz, Нγ-Рy), 7.34 (m, 1H, Нβ-Ру), 7.25 (s, 1Н, -СН=),
.99 (m, 2Н, Нβ, Нβ’-Рh), 2.73 (s, 3Н, -SCH ), 2.33 (s, 3H, -SCH ),
.14 (s, 6H, 2x-CH ). HRMS (pos.) calculated C19
1
Yield: 71 mg (68%) after column chromatography. H NMR (400
3
, δ, ppm): 8.69 (m, 4Н, Нα, Нα’-Рh), 7.58 (m, 2Н, Нβ-
Рy), 7.25 – 7.17 (m, 8Н, Нβ, Нβ’-Рh, Нβ’ -Рy -СН=), 7.01 (m, 4Н,
3
3
MHz, CDCl
+
3
H
20
N
3
OS :
+
+
38.1327 (48 + H) , 339.1361 (48 + H) , found 338.1322 (48 +
Нγ, Нα-Рy), 3.87 (s, 6Н, p-OCH
3
), 3.83 (s, 4Н, -CH
2
CH
2
-). HRMS
+
+
+
H) , 339.1354 (48 + H) . HPLC purity 96.80%
-(Z)-3-(4-methylthiophenyl)-2-(methylthio)-5-(pyridin-
-ylmethylene)-1H-imidazol-5(4H)-one (49). From 100 mg
0.30 mmol) of (Z)-3-(4-methylthiophenyl)-5-(pyridin-2-
ylmethylene)-2-thioxoimidazolidin-4-one 22 as result of
+
+
(
C
pos.) calculated
C
34
H
29
N
6
O
4
S
2
649.1687 (52
H) ,
5
2+
2+
+
34
H
29
N
6
O
4
S
2
325.0880 (52 + 2H) , found 649.1688 (52 + H) ,
325.0882 (52 + 2H) . HPLC purity 95.26%
2
(
2
+
(
4Z, 4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
a
pyridylmethylidene)-1-(4-ethoxyphenyl)-2,3-dihydro-4H-
imidazol-5-one) (53). From 100 mg (0.31 mmol) of 3-(4-
ethoxyphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 12 by reaction with 29 mg
(0.15 mmol) of 1,2-dibromoethane, in the presence of 64 mg
reaction with 101 mg (0.71 mmol) of methyl iodide, in the
presence of 21 mg (0.37 mmol) of solid KOH, 49 was obtained as
a white-yellow powder. Yield: 64 mg (61%) after column
1
chromatography. H NMR (400 MHz, CDCl
3
, δ, ppm): 8.87 (d, 1Н,
=4.89 Hz, Нβ’-Рy), 7.89 (t, 1Н,
J=6.85 Hz, Нγ-Рy), 7.33 (m, 3H, Нβ, Нβ’-Рh, Нβ-Ру), 7.24 (m, 3H,
J=7.83 Hz, Нα-Ру), 8.74 (d, 1Н, J
1
(0.46 mmol) of solid K
2
CO
3
, 53 was obtained as a light yellow
1
powder. Yield: 75.5 mg (72%) after column chromatography. H
NMR (400 MHz, CDCl , δ, ppm): 8.70 (m, 2H, Hα-Py), 8.64 (d, 2H,
3
J= 3.91 Hz, Hβ’-Py), 7.58 (m, 4H, J=7.83Hz, Hα, Hα’-Ph), 7.25-7.17
(m, 4H, Нβ, Нβ’-Ph), 7.08 (m, 2H, Нβ-Рy), 6.99 (m, 4H, -CH=, Hγ-
-), 3.82 (s, 4H, -CH
). HRMS (pos.) calculated C36
339.1036 (53 + 2H) , found 677.1998
53 + H) , 339.1038 (53 + 2H) . HPLC purity 99.87%
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(3,4-dimethoxyphenyl)-2,3-
dihydro-4H-imidazol-5-one) (54). From 100 mg (0.29 mmol)
of 3-(3,4-dimethoxyphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 13 by reaction with 27 mg
Нα, Нα’-Рh, -СН=), 2.74 (s, 3Н, -SCH
HRMS (pos.) calculated C17
3
), 2.52 (s, 3H, Ph-SCH
: 342.0735 (49 + H) ,
3
).
+
+
H
16
+
N
3
OS
2
+
343.0768 (49 + H isotopic) , found 342.0730 (49 + H) ,
+
343.0762 (49 + H isotopic) . HPLC purity 96.63%
Py), 4.09 (m, 4H, -CH
2
2
CH
2
-), 1.46 (m, 6H,
5
-(Z)-3-(2,5-difluorophenyl)-2-(methylthio)-5-(pyridin-
+
J=6.85 Hz, CH
3
H
33
2+
O
4
N
6
S
2
677.2000
2
-ylmethylene)-1H-imidazol-5(4H)-one (50). From 100 mg
+
2+
(
53 + H) , C36
H
34
O
4
N
6
S
2
(0.30 mmol) of (Z)-3-(2,5-difluorophenyl)-5-(pyridin-2-
+
2+
(
ylmethylene)-2-thioxoimidazolidin-4-one 28 as result of
a
(4Z,
reaction with 104 mg (0.73 mmol) of methyl iodide, in the
presence of 22 mg (0.38 mmol) of solid KOH, 50 was obtained as
a white-yellow powder. Yield: 37 mg (35%) after column
chromatography. H NMR (400 MHz, CDCl , δ, ppm): 8.87 (d, 1Н,
3
J=7.83 Hz, Нα-Ру), 8.74 (d, 1Н, J=4.89 Hz, Нβ’-Рy), 7.84 (t, 1Н,
J=7.82 Hz, Нγ-Рy), 7.31-7.17 (m, 3H, Нα, H β’, Hγ-Рh), 7.09 (m,
1
(
0.14 mmol) of 1,2-dibromoethane, in the presence of 60 mg
(0.43 mmol) of solid K
2
CO
3
was obtained 54 as a white-yellow
3
1H, Нβ-Ру), 5.30 (s, 1Н, -СН=), 2.77 (s, 3Н, -SCH ). HRMS (pos.)
1
powder. Yield: 75 mg (73%) after column chromatography.
NMR (400 MHz, CDCl
Hβ'-Py), 7.59 (m, 2H, Hα’-Ph), 7.20 (s, 4H, Hβ-Ph), 6.97-6.80 (м,
H, Hα, Hβ, Hγ-Py), 6.80 (s, 2H, -CH=), 3.95 (s, 6H, p-ОCH ), 3.90
с, 6H, m-ОCH ), 3.84 (s, 4H, -CH CH
- ). 13C NMR (101 MHz,
H
+
+
calculated C16
H
12
F
2
N
3
OS : 332.0669 (50 + H) , 333.0703 (50 + H
3
, δ, ppm): 8.71 (m, 2H, Hα-Ph), 8.66 (m, 2H,
+
+
isotopic) , found 332.0665 (50 + H) , 333.0698 (50 + H
isotopic) . HPLC purity 96.24%
+
6
3
5-(Z)-3-(3,5-difluorophenyl)-2-(methylthio)-5-(pyridin-
(
3
2
2
2
(
-ylmethylene)-1H-imidazol-5(4H)-one (51). From 100 mg
0.30 mmol) of (Z)-3-(3,5-difluorophenyl)-5-(pyridin-2-
ylmethylene)-2-thioxoimidazolidin-4-one 29 as result of
DMSO-d
6
(54 + H) , C36
(
6
, δ, ppm) 149.3, 136.9, 128.6, 127.0, 123.4, 115.3, 106.9,
3.9, 15.3, 14.7. HRMS (pos.) calculated C36
355.0990 (54 + 2H) , found 709.1896
54 + H) , 355.0987 (54 + 2H)2+. HPLC purity 96.78%
+
H
33
2+
O
6
N
6
S
2
709.1903
a
+
2+
H
33
O
6
N
6
S
2
reaction with 104 mg (0.73 mmol) of methyl iodide, in the
presence of 22 mg (0.38 mmol) of solid KOH, 51 was obtained as
a white-yellow powder. Yield: 41 mg (39%) after column
chromatography. H NMR (400 MHz, CDCl
J=7.83 Hz, Нα-Ру), 8.81 (d, 1Н, J=4.89 Hz, Нβ’-Рy), 8.05 (d, 1Н,
J=6.85 Hz, Нγ-Рy), 7.35 (m, 1Н, Нβ-Ру), 6.97 (m, 3H, -Рh), 5.31
+
(
4Z,
pyridylmethylidene)-1-(4-tertbutylphenyl)-2,3-dihydro-
H-imidazol-5-one) (55). From 100 mg (0.30 mmol) of 3-(4-
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
1
3
, δ, ppm): 8.94 (d, 1Н,
4
tert-butylphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 14 by reaction with 28 mg
0.15 mmol) of 1,2-dibromoethane, in the presence of 62 mg
(m, 1H, -СН=), 2.81 (s, 3Н, -SCH
3
). HRMS (pos.) calculated
(
+
+
+
C
16
H
12
F
2
N
3
OS : 332.0669 (51 + H) , 333.0703 (51 + H isotopic) ,
(0.45 mmol) of solid K
2
CO
3
, 55 was obtained as a white powder.
1
Yield: 93 mg (89%) after column chromatography. H NMR (400
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 34
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
MHz, CDCl
3
, δ, ppm): 8.69 (m, 4H, Hα, Hα'-Ph), 7.61 (m, 2H, Hα-
Hβ, Hβ'-Ph), 7.33 (m, 2H, Hγ-Py), 7.23 (m, 4H, Hβ, Hβ '-Py), 7.17
Py), 7.53 (m, 6H, Hβ, Hβ'-Ph), 7.24 – 7.19 (m, 8H, Hγ, Hβ, Hβ '-Py,
-CH =), 3.86 (s, 4Н, -CH
calculated C40
(s, 2H, -CH=),3.93 (s, 4H, -CH
2
CH
2
-). HRMS (pos.) calculated
+
+
+
2
CH
2
-), 1.36 (s, 18H, p-tBu). HRMS (pos.)
C
32
H
23
F
2
N
6
O
2
S
2
+
3
625.1287 (60 + H) , C17H13FN OS 326.0763
+
+
2+
+
H
41
N
6
O
2
+
S
2
701.2727 (55 + H) , C40
H
41
N
6
O
2
S
2
(1/2 60 + CH
CH
)+. HPLC purity 96.10%
(4Z, 4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
2
) , found 625.1295 (60 + H) , 326.0761 (1/2 60 +
2
+
3
+
51.1400 (55 + 2H) , found 701.2730 (55 + H) , 351.1401 (55
2
2H)2+. HPLC purity 95.27%
(4Z,
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(3-bromophenyl)-2,3-dihydro-4H-
imidazol-5-one) (61). From 100 mg (0.28 mmol) of 3-(3-
bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 5 by reaction with 26 mg
(0.14 mmol) of 1,2-dibromoethane, in the presence of 58 mg
pyridylmethylidene)-1-(2,4,6-trimethylphenyl)-2,3-
dihydro-4H-imidazol-5-one) (56). From 100 mg (0.31 mmol)
of 3-(2,4,6-trimethylphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 21 by reaction with 30 mg
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(
0.16 mmol) of 1,2-dibromoethane, in the presence of 66 mg
0.48 mmol) of solid K CO , 56 was obtained as a yellow powder.
(0.42 mmol) of solid K
powder. Yield: 68 mg (65%) after column chromatography.
2
CO
3
, 61 was obtained as a white-yellow
1
2
3
H
1
Yield: 72 mg (69%) after column chromatography. H NMR (400
MHz, DMSO-d , δ, ppm): 8.15 (m, 4Н, Нβ’, Нα-Рy), 7.07 (t, 2Н,
J=7.43 Hz, Нγ-Рy), 6.69 (m, 2Н, Нβ-Рy), 6.63 (s, 2H, -СН=), 6.43
NMR (400 MHz, DMSO-d
6
, δ, ppm): 8.68 (m, 2Н, Нα-Рh), 8.58 (m,
6
2Н, Нα’-Рh) 7.73 (m, 4Н, Нβ, Нγ-Рh), 7.59 (m, 2Н, Нγ-Рy), 7.45
(m, 2Н, Нα-Рy), 7.44 (m, 4Н, Нβ, Нβ’ -Рy), 6.74 (s, 2Н, -CH=), 3.82
+
(s, 4Н, -Рh), 3.29 (m, 4Н, -CH
2
CH
2
-), 1.78 (s, 6H, -CH
3
+
), 1.54 (s,
673.2414
2 2 2 6 2 2
(s, 4Н, -CH CH -). HRMS (pos.) calculated C32H23Br N O S
+
2+
2
373.9869 (61 + 2H)2+,
) , found 746.9673 (61 +
1
2H, 2x-CH
3
). HRMS (pos.) calculated C38
H
37
N
6
O
2
S
2
746.9665 (61 + H) , C32
H
23Br
2
N
6
O
2
S
+
+
+
+
2
(56 + H) , found 673.2426 (56 + H) . HPLC purity 95.61%
C
17
H
13BrN
3
OS 385.9963 (1/2 61 + CH
H)+, 373.9879 (61 + 2H)2+, 385.9961 (1/2 61 + CH
+
2
(
4Z,4'Z)-2,2'-(ethane-1,2-diylbis(sulfanediyl))bis-4-
(pyridin-2-ylmethylene)-(1-(2-t-butylphenyl)-2,3-dihydro-
H-imidazol-5-one) (57). From 100 mg (0.30 mmol) of (Z)-3-
2-(tert-butyl)phenyl)-5-(pyridin-2-ylmethylene)-2-
) . HPLC
purity 98.01%
4
(
(4Z,
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(2-bromophenyl)-2,3-dihydro-4H-
imidazol-5-one) (62). From 100 mg (0.28 mmol) of 3-(2-
bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 6 as a result of reaction
with 26 mg (0.14 mmol) of 1,2-dibromoethane, in the presence
thioxoimidazolidin-4-one 15, by reaction with 28 mg (0.15
mmol) of 1,2-dibromoethane, in the presence of 62 mg (0.45
mmol) of solid K
2
CO
3
, 57 was obtained as a bright yellow
1
powder. Yield: 49 mg (46%) after column chromatography.
NMR (400 MHz, CDCl
m, 4H, Hβ-Py, Hγ-Py), 7.44 (m, 2H. Hα-Py), 7.26 (m, 2H, Hβ-Ph),
H
3
, δ, ppm): 8.68 (m, 4H, Hα-Ph, Hβ’-Py), 7.62
2 3
of 58 mg (0.42 mmol) of solid K CO , 62 was obtained as a white-
(
7
yellow powder. Yield: 74 mg (71%) after column
1
.23 (m, 2H, Hβ'-Ph), 7.16 (m, 2H, Hγ-Ph), 6.96 (m, 2H, =СH-),
chromatography. H NMR (400 MHz, CDCl
3
, δ, ppm): 8.69 (d, 2Н,
1
3
3.84 (s, 4H, -CH
2
CH
2
-). C NMR (101 MHz, CDCl
3
, δ, ppm) 169.7,
J= 8.22 Hz, Нα-Рh), 8.61 (m, 2Н, Нγ-Рy), 7.74 (m, 2Н, Нβ-Рh), 7.56
(m, 2Н, Нα-Рy), 7.40 (m, 6Н, Нβ, Нβ’ –Рy, Hγ-Рh), 7.16 (m, 4Н,
167.4, 153.4, 150.0, 149.2, 139.9, 136.9, 131.5, 130.4, 129.5,
127.3, 127.8, 123.3, 77.00, 36.1, 31.8, 30.1. HRMS (pos.)
calculated C40
Hβ’-Ph, -CH=), 3.83 (s, 4Н, -CH
2
CH
2
-). HRMS (pos.) calculated
+
+
2+
+
+
2+
H
41
N
6
O
2
+
S
2
701.2727 (57 + H) , C40
H
41
N
6
O
2
S
2
C
32
H
23Br
2
N
6
O
2
S
2
32 2 6 2 2
746.9665 (62 + H) , C H23Br N O S
2
+
2+
+
+
3
+
51.1400 (57 + 2H) , found 701.2783 (57 + H) , 351.1435 (57
2H) . HPLC purity 96.19%
373.9869 (62 + 2H) , C17
found 746.9677 (62 + H) , 373.9873 (62 + 2H) , 385.9961 (1/2
H13BrN
3
OS 385.9963 (1/2 62 + CH
2
) ,
2
+
+
2+
+
(4Z, 4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
62 + CH
2
) . HPLC purity 96.28%
pyridylmethylidene)-1-(4-fluorophenyl)-2,3-dihydro-4H-
imidazol-5-one) (58). From 100 mg (0.33 mmol) of 3-(4-
fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
(4Z,
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(2-chlorophenyl)-2,3-dihydro-4H-
imidazol-5-one) (63). From 100 mg (0.32 mmol) of 3-(2-
chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 10 by reaction with 30 mg
(0.16 mmol) of 1,2-dibromoethane in the presence of 66 mg
thioxotetrahydro-4H-imidazol-4-one 1, by reaction with 31 mg
(0.16 mmol) of 1,2-dibromoethane, in the presence of 68 mg
(
0.495 mmol) of solid K CO , 58 was obtained as a bright yellow
2
3
1
powder. Yield: 79 mg (77%) after column chromatography.
NMR (400 MHz, DMSO-d , δ, ppm): 8.66 (m, 4H, Hα, Hα'-Ph), 7.59
m, 2H, Hα-Py), 7.33 – 7.27 (m, 6H, Hβ, Hβ'-Ph, -CH=), 7.22 – 7.19
H
(0.48 mmol) of solid K
Yield: 67 mg (64%) after column chromatography. H NMR (400
2
CO
3
, 63 was obtained as a yellow powder.
1
6
(
(
6
MHz, DMSO-d , δ, ppm): 8.73 (d, 2Н, J=7.83 Hz, Нα-Рh), 7.61 (m,
4Н, Нβ, Нβ’-Рy), 7.56 (m, 2Н, Hγ-Py), 7.39 (m, 2H, Нα-Рy), 7.41-
7.26 (m, 6Н, Нγ, Нβ, Нβ’-Рh), 6.76 (m, 2Н, -CH=), 3.88 (s, 4Н, -
m, 6H, Hγ, Hβ, Hβ '-Py), 3.85 (s, 4H, -CH
2
CH
2
+
-). HRMS (pos.)
+
+
calculated C32
H
23
F
2
N
6
O
2
2
S
2
+
3
625.1287 (58 + H) , C17H13FN OS
) , found 625.1295 (58 + H) , 326.0762
+
+
2
3
26.0763 (1/2 58 + CH
CH
2
CH
2
-). HRMS (pos.) calculated C32
H23Cl
2
N
6
O
2
S
657.0696
)+. HPLC purity 95.46%
+
2+
2+
(1/2 58 + CH
2
(63 + H) , C32
H
24Cl
2
N
6
O
2
S
2
329.0384 (63 + 2H) , found
657.0697 (63 + H) , 329.0382 (63 + 2H)2+. HPLC purity 96.76%
(4Z,4'Z)-2,2'-(ethane-1,2-diylbis(sulfanediyl))bis-4-
(pyridin-2-ylmethylene)-(1-(2-methoxy-4-chloro-5-
methylphenyl)-2,3-dihydro-4H-imidazol-5-one) (64). From
100 mg (0.28 mmol) of (Z)-3-(2-methoxy-4-chloro-5-
methylphenyl)-5-(pyridin-2-ylmethylene)-2-
+
(4Z,
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(3-fluorophenyl)-2,3-dihydro-4H-
imidazol-5-one) (59). From 100 mg (0.33 mmol) of 3-(3-
fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 2 by reaction with 31 mg
(0.16 mmol) of 1,2-dibromoethane, in the presence of 68 mg
(0.49 mmol) of solid K
2
CO
3
, 59 was obtained as a yellow powder.
thioxoimidazolidin-4-one 18, by reaction with 26 mg (0.14
mmol) of 1,2-dibromoethane, in the presence of 58 mg (0.42
1
Yield: 43 mg (42%) after column chromatography. H NMR (400
MHz, CDCl
Py), 7.50 (m, 6H, Hβ, Hγ-Ph, Hγ-Py), 7.16 (m, 2H, -CH=), 7.11 (m,
3
, δ, ppm): 8.68 (m, 4H, Hα, Hα'-Ph), 8.11 (m, 2H, Hα-
mmol) of solid K
powder. Yield: 79 mg (75%) after column chromatography.
NMR (400 MHz, CDCl
2
CO
3
, 64 was obtained as a bright yellow
1
H
4H, Hβ, Hβ '-Py), 4.03 (s, 4H, -CH
2
CH
2
+
-). HRMS (pos.) calculated
3
, δ, ppm): 8.67 (s, 2H, Hα-Ph), 8.66 (d, 2H,
+
+
C
32
H
23
F
2
N
6
O
2
S
2
2
625.1287 (59 + H) , C17
) , found 625.1294 (59 + H) , 326.0761 (1/2 59 +
H13FN
3
OS 326.0763
J=7.83 Hz, Hβ’-Py), 7.59 (m, 2H, Hβ-Py), 7.24 (m, H, Hγ-Py), 7.11
+
+
(
1/2 59 + CH
CH
)+. HPLC purity 98.59%
4Z, 4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
(s, 2H, Hβ-Ph), 7.07 (d, 2H, J=7.83 Hz, Hα-Py), 7.02 (s, 2H, -CH=)
13
2
3.81 (s, 4H, -CH
NMR (101 MHz, CDCl
144.9, 140.2, 136.9, 131.6, 128.8, 127.0, 123.4, 123.0, 118.7, 113.4, 56.2,
2 2 3 3
CH -), 2.32 (s, 6H, -OCH ), 2.15 (s, 6H, -CH ). C
(
3
, δ, ppm) 168.3, 167.0, 153.9, 152.7, 149.2,
pyridylmethylidene)-1-(2-fluorophenyl)-2,3-dihydro-4H-
imidazol-5-one) (60). From 100 mg (0.33 mmol) of 3-(2-
fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 3 as a result of reaction
with 31 mg (0.16 mmol) of 1,2-dibromoethane, in the presence
+
30.1, 19.1. HRMS (pos.) calculated C36
H
32Cl
2 6 4 2
N O S 747.1191 (64
+
+
+ H isotopic) , 745.1220 (64 + H) , 374.0632 (64 + 2H
isotopic) , 373.0347 (64 + 2H) , found 747.1253 (64 + H
isotopic) , 745.1287 (64 + H) , 374.0664 (64 + 2H isotopic)
2
+
2+
+
+
2+
,
2
+
of 68 mg (0.49 mmol) of solid K
2
CO
3
, 60 was obtained as a white-
373.00682 (64 + 2H) . HPLC purity 95.16%
yellow powder. Yield: 74 mg (72%) after column
(4Z,4'Z)-2,2'-(ethane-1,2-diylbis(sulfanediyl))bis(1-(2-
bromo-4-fluorophenyl)-4-(pyridin-2-ylmethylene)-1H-
1
chromatography. H NMR (400 MHz, CDCl
3
, δ, ppm): 8.74 (m, 2H,
Hα-Ph), 8.65 (m, 2H, Hγ-Ph),7.73 (m, 2H, Hα-Py), 7.47 (m, 4H,
imidazol-5(4H)-one) (65). From 100 mg (0.27 mmol) of (Z)-3-
ACS Paragon Plus Environment

Page 23 of 34
Journal of Medicinal Chemistry
1
2
(2-bromo-4-fluorophenyl)-5-(pyridin-2-ylmethylene)-2-
(4Z,
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
thioxoimidazolidin-4-one 7, by reaction with 25 mg (0.13 mmol)
of 1,2-dibromoethane, , in the presence of 56 mg (0.40 mmol) of
pyridylmethylidene)-1-(3-chlorophenyl)-2,3-dihydro-4H-
imidazol-5-one) (70). From 100 mg (0.32 mmol) of 3-(3-
chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 9 by reaction with 30 mg
(0.16 mmol) of 1,2-dibromoethane, in the presence of 66 mg
solid K
2
CO
3
, 65 was obtained as a bright yellow powder. Yield:
1
79 mg (75%) after column chromatography. H NMR (400 MHz,
3
CDCl , δ, ppm): 8.74 (m, 2H, Hα-Ph),8.65 (d, 2H, J=7.83 Hz, Hβ’-
Py),7.73 (m, 2H, Hβ-Py), 7.49-7.43 (m, 2H, Hβ’-Ph), 7.32 (m, 4H,
(0.48 mmol) of solid K
Yield: 73 mg (69%) after column chromatography. H NMR (400
MHz, DMSO-d , δ, ppm): 8.72 (d, 2H, J=7.83 Hz, Нα-Рy), 8.58 (d,
2 3
CO , 70 was obtained as a white powder.
1
Hγ-Py. Hβ-Ph), 7.23 (m, 2H, Hα-Py), 7.17 (s, 2H, -CH=), 3.90 (m,
1
3
4H, -CH
2
CH
2
-). C NMR (101 MHz, DMSO-d
6
, δ, ppm) 167.6,
6
1
1
C
66.4, 159.0, 156.4, 149.4, 140.0, 137.7, 132.6, 129.3, 127.2,
2H, J=4.89 Hz, Нβ’-Рy), 7.61 (m, 8H, Нα’-Рh, Нα-Рh, Нβ-Ph, Нγ-
Py), 7.46 (d, 2H, J=6.85 Hz, Нγ-Рh), 7.29 (m, 2H, Hβ-Py), 6.76 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
24.5, 120.8, 119.3, 40.0, 30.3. HRMS (pos.) calculated
2
+
2+
32
H22Br
2
F
2
+
N
6
O
2
S
2
: 391.9775 (65 + 2H isotopic) , 390.9785
2H, -CH=), 3.89 (s, 4H, -CH
2
CH
2
-). HRMS (pos.) calculated
2
2+
+
+
+
(65 + 2H) , found 391.9798 (65 + 2H isotopic) , 390.9807 (65
C
32
H23Cl
2
N
6
O
2
S
2
657.0696 (70 + H) , found 657.0696 (70 + H) .
2+
+
2H) . HPLC purity 96.27%
HPLC purity 95.81%
(4Z,4'Z)-2,2'-(ethane-1,2-diylbis(sulfanediyl))bis(1-(2,5-
(4Z,
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
difluorophenyl)-4-(pyridin-2-ylmethylene)-1H-imidazol-
(4H)-one) (66). From 100 mg (0.32 mmol) of (Z)-3-(2,5-
pyridylmethylidene)-1-(4-bromophenyl)-2,3-dihydro-4H-
imidazol-5-one) (71). From 100 mg (0.28 mmol) of 3-(4-
bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 4 by reaction with 26 mg
(0.14 mmol) of 1,2-dibromoethane, in the presence of 58 mg
5
difluorophenyl)-5-(pyridin-2-ylmethylene)-2-
thioxoimidazolidin-4-one 28, by reaction with 30 mg (0.16
mmol)of 1,2-dibromoethane, in the presence of 66 mg (0.48
mmol) of solid K
powder. Yield: 46 mg (44%) after column chromatography.
NMR (400 MHz, DMSO-d
2
CO
3
, 66 was obtained as a bright yellow
(0.42 mmol) of solid K
powder. Yield: 73 mg (70%) after column chromatography.
NMR (400 MHz, DMSO-d
2
CO
3
71 was obtained as a bright yellow
1
1
H
H
6
, δ, ppm): 8.69 (d, 2Н, J=7.82 Hz, Нβ-
6
, δ, ppm): 8.68 (m, 2H, Hα-Ph), 8.56 (m,
Рy), 8.59 (m, 2Н, Нα-Рy), 7.68-7.54 (m, 8Н, Нβ-Рy, Нγ-Рy, Нα’-Рh,
Нγ-Рh), 7.31 (m, 2Н, Нβ-Рh), 6.79 (s, 2H, -СН=), 3.91 (s, 4Н, -
2H, Hα'-Ph) 7.76 – 7.73 (м, 4Н, Нβ, Нβ’-Рh), 7.61 (m, 2H, Hα-Py),
7.41 -7.39 (m, 4H, Hβ, Hβ '-Py), 7.27 (m, 2H, Hγ-Py), 6.75 (s, 2H,
+
13
CH
2
CH
2
-). HRMS (pos.) calculated C32
H
21
F
4
N
6
O
2
S
2
661.1104 (66
2 2 6
-CH=), 3.85 (m, 4H, -CH CH -). C NMR (101 MHz, DMSO-d , δ,
+
+
+
+ H) , 662.1137 (66 + H isotopic) , found 661.1113 (66 + H) ,
62.1129 (66 + H isotopic) . HPLC purity 95.96%
ppm) 161.7, 158.6, 148.0, 141.3, 136.4, 123.6, 118.9, 96.9, 39.4.
+
+
+
6
HRMS (pos.) calculated C32
H
23Br
2
N
6
O
+
2
S
2
746.9665 (71 + H) ,
2
+
2+
+
(
4Z,4'Z)-2,2'-ethane-1,2-diylbis(sulfanediyl))bis(1-(3,5-
difluorophenyl)-4-(pyridin-2-ylmethylene)-1H-imidazol-
(4H)-one) (67). From 100 mg (0.32 mmol) of (Z)-3-(3,5-
C
32
H
23Br
2
N
6
O
2
S
2
373.9869 (71
2H)
,
C
17
H
13BrN
3
OS
+
+
385.9963 (1/2 71 + CH
2
) , found 746.9674 (71 + H) , 373.9879
) . HPLC purity 98.51%
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
(71 + 2H)2+, 385.9962 (1/2 71 + CH
(4Z,
+
5
2
difluorophenyl)-5-(pyridin-2-ylmethylene)-2-
thioxoimidazolidin-4-one 7, by reaction with 30 mg (0.6 mmol)
of 1,2-dibromoethane, in the presence of 66 mg (0.48 mmol) of
pyridylmethylidene)-1-(4-chlorophenyl)-2,3-dihydro-4H-
imidazol-5-one) (72). From 100 mg (0.32 mmol) of 3-(4-
chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 8 by reaction with 30 mg
(0.16 mmol) of 1,2-dibromoethane, in the presence of 66 mg
solid K
2
CO
3
, 67 was obtained as a bright yellow powder. Yield:
1
65 mg (62%) after column chromatography. H NMR (400 MHz,
6
DMSO-d , δ, ppm): 8.71 (d, 2Н, J=7.83 Hz, Нα-Рy), 8.56 (d, 2Н,
J=3.91 Hz, Нβ’-Рy), 7.65 (t, 2Н, J=6.85 Hz, Нγ-Рy), 7.50 (t, 2Н,
J=9.78 Hz, Нγ-Рh), 7.37 (d, 4Н, J=7.83 Hz, Нα’,Нα-Рh,), 7.28 (m,
(0.48 mmol) of solid K
Yield: 77 mg (73%) after column chromatography. H NMR (400
2
CO
3
, 72 was obtained as a white powder.
1
2
Н, Нβ-Рy), 6.77 (s, 2H, -СН=), 3.89 (s, 4Н, -CH
2
CH
2
-). HRMS
6
MHz, DMSO-d , δ, ppm): 8.66 (m, 4H, Hα, Hα'-Ph), 7.59 (m, 2H,
+
+
(
pos.) calculated C32
H
21
F
4
N
6
O
2
S
2
661.1104 (67 + H) , 662.1137
Hα-Py), 7.33-7.31 (m, 4H, Hβ, Hβ'-Ph,), 7.22 (m, 2H, -CH=), 7.19
+
+
13
(67 + H isotopic) , found 661.1096 (67 + H) , 662.1122 (67 + H
(m, 6H, Hγ, Hβ, Hβ '-Py), 3.85 (s, 4H, -CH
MHz, DMSO-d
128.8, 123.5, 111.3, 40.9. HRMS (pos.) calculated
2 2
CH -). C NMR (101
+
isotopic) . HPLC purity 96.66%
6
, δ, ppm) 173.0, 164.9, 150.1, 139.4, 136.4, 129.9,
(4Z,4'Z)-2,2'-(ethane-1,2-diylbis(sulfanediyl))bis(1-(2-
methyl-3-chlorophenyl)-4-(pyridin-2-ylmethylene)-1H-
imidazol-5(4H)-one) (68). From 100 mg (0.30 mmol) of (Z)-3-
+
H)+,
2+
C
32
H23Cl
2
N
6
O
2
S
2
657.0696 (72
+
C
32
+
H23Cl
2
N
6
O
2
S
2
2
+
329.0384 (72 + 2H) , found 657.0670 (72 + H) , 329.0381 (72
+ 2H)2+. HPLC purity 98.32%
(
2-methyl-3-chlorophenyl)-5-(pyridin-2-ylmethylene)-2-
thioxoimidazolidin-4-one 20, by reaction with 28 mg (0.15
(4Z,
4'Z)-2,2'-(ethane-1,2-diyldisulfanediyl)bis(4-(2-
mmol) of 1,2-dibromoethane, in the presence of 63 mg (0.45
pyridylmethylidene)-1-(3-chloro-4-fluorophenyl)-2,3-
dihydro-4H-imidazol-5-one) (73). From 100 mg (0.32 mmol)
of 3-(3-chloro-4-fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 19 by reaction with 30 mg
(0.16 mmol) of 1,2-dibromoethane, in the presence of 66 mg
mmol) of solid K
2
CO
3
, 68 was obtained as a bright yellow
1
powder. Yield: 72 mg (70%) after column chromatography.
NMR (400 MHz, DMSO-d
Рy), 8.63 (m, 2Н, Нβ-Рy), 7.67-7.57 (m, 4Н, Нβ’-Рy, Нβ-Рh), 7.42-
H
6
, δ, ppm): 8.71 (d, 2Н, J=7.82 Hz, Нα-
7
4
6
6
9
.31 (m, 6Н, Нβ’-Рh, Нγ-Рy, -CH=), 6.82 (m, 2H, Hα-Ph), 3.88 (m,
(0.48 mmol) of solid K
Yield: 77 mg (73%) after column chromatography. H NMR (400
MHz, DMSO-d , δ, ppm): 8.71 (d, 2Н, J=7.83 Hz, Нα’-Рh), 8.57 (m,
2 3
CO , 73 was obtained as a white powder.
+
1
Н, -CH
2
CH
2
-). HRMS (pos.) calculated
C
34
H
27Cl
2
+
N
6
O
2
S
2
+
85.1014 (68 + H) , 687.0984 (68 + H isotopic) , found
85.1055 (68 + H) , 687.1027 (68 + H isotopic) . HPLC purity
6
+
+
2Н, Нα-Рh), 7.86 (m, 2Н, Нγ-Рy), 7.65 (m, 4Н, Нβ, Hβ’-Рy), 7.57
5.83%
(m, 2Н, Нα-Рy), 7.29 (m, 2H, Нβ-Рh), 6.79 (m, 2H, -СН=), 3.89 (s,
+
(4Z,4'Z)-2,2'-(ethane-1,2-diylbis(sulfanediyl))bis(1-(4-
4Н, -CH
2
CH
2
-). HRMS (pos.) calculated
C
32
H
23Cl
2
+
N
6
O
2
S
2
+
methylthiophenyl)-4-(pyridin-2-ylmethylene)-1H-
imidazol-5(4H)-one) (69). From 100 mg (0.32 mmol) of (Z)-3-
(4-methylthiophenyl)-5-(pyridin-2-ylmethylene)-2-
thioxoimidazolidin-4-one 22, by reaction with 30 mg (0.16
mmol) of 1,2-dibromoethane, in the presence of 64 mg (0.47
693.0513 (73 + H) , 695.0483 (73 + H isotopic) , found
693.0536 (73 + H) , 695.0484 (73 + H isotopic) . HPLC purity
+
+
96.97%
Synthesis of coordination compounds 1k-73k
mmol) of solid K
2
CO
3
, 69 was obtained as a bright yellow
General Protocol. 0.1 ml of butanol-1 was carefully added to
a solution of 15 mg of the corresponding ligand in 1.5 ml of
dichloromethane* to achieve the separated layers. A solution of
1
powder. Yield: 65 mg (63%) after column chromatography.
NMR (400 MHz, DMSO-d
Рy), 8.60 (m, 2Н, Нβ-Рy), 7.67 (t, 2Н, J=7.31 Hz, Нγ-Рy), 7.41-7.29
m, 10Н, Нα’-Рh, Нα-Рh, Нβ’-Рh, Нβ-Рh, Нβ’-Py), 6.77 (s, 2H, -
H
6
, δ, ppm): 8.72 (d, 2Н, J=7.31 Hz, Нα-
2 2
copper (II) chloride dihydrate CuCl *2H O in 1.5 ml of butanol-1
(
is then carefully added to form a two-phase system. The tightly
closed reaction mixture was left for 2 - 7 days in the dark, until
crystallization occurred. If a clear solution without signs of
crystallization or precipitation was formed, crystallization was
the activated by ether diffusion. An open vial with a solution of
СН=), 3.87 (s, 4Н, -CH
2
CH
2
-). HRMS (pos.) calculated
+
+
+
C
34
H
29
N
6
O
2
S
4
681.1230 (69 + H) , 682.1263 (69 + H isotopic) ,
+
+
found 681.1231 (69 + H) , 682.1271 (69 + H isotopic) . HPLC
purity 97.44%
the coordination compound was placed in
a larger vial
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 34
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
containing a small amount of diethyl ether, tightly closed and left
to stand for 24 hours in the dark. The precipitate was separated
by decanting, washed with a small amount of dichloromethane
and then with diethyl ether until the washing solvent became
colorless. The final products were obtained as dark crystalline
powders after drying in air.
C
30
H
18Cu
2
ClBr
2 6 2 2
N O S : C 40.90, H 2.06, N 9.54, found C 41.13, H
1.98, N 8.71.
Preparation of the coordination compound of 3-(2-
bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 6 with copper (II)
chloride dihydrate (6k). 6k was obtained as a black crystalline
precipitate from 15 mg (42 μmol) of 3-(2-bromophenyl)-5-(((Z)-
2-pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one
6 and 7 mg (42 μmol) copper(II) chloride dehydrate. Yield: 6.0
mg (32%). MALDI: m/z = 781 (6 - H) Cu 32%, 844 (6 - H) Cu
100%. HRMS (pos.) calculated 843.7866 (C30
found 843.8512 (C30
*
In the case of compounds 66 and 67, dissolution in
dichloromethane was impossible due to the low solubility and
the delamination was replaced by boiling with copper chloride
in butanol-1 (see procedures 66k and 67k).
Preparation of the coordination compound of 3-(4-
fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 1 with copper (II)
chloride dihydrate (1k). 1k was obtained as a black crystalline
precipitate from 15 mg (50 μmol) 3-(4-fluorophenyl)-5-(((Z)-2-
+
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
+
H
18Cu
2
Br
2
N
6
O
2
S
2
) ,
+
-1
H18Cu
2
Br
2
N
6
O
2
S
2
) , UV-vis (λ, nm/ε, l mol
-
1
cm ): 432/12400. Elemental analysis: calculated (%) for
ClBr : C 40.90, H 2.06, N 9.54, found C 41.02, H
1.98, N 9.91.
C
30
H18Cu
2
2 6 2 2
N O S
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one
1
Preparation of the coordination compound of 3-(2-
bromo-4-fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 7 with copper (II)
chloride dihydrate (7k). 7k was obtained as a black crystalline
precipitate from 15 mg (42 μmol) 3-(2-bromo-4-fluorophenyl)-
5-(((Z)-2-pyridylmethylidene)-2-thioxotetrahydro-4H-
imidazol-4-one 7 and 7 mg (42 μmol) of copper (II) chloride
dihydrate. Yield: 7.6 mg (42%). UV-vis (λ, nm/ε, l mol cm ):
435/16500.
(С30
and 8.5 mg (50 µmol) of copper (II) chloride dihydrate. Yield: 6
+
+
2 2
Cu
mg (32%). MALDI: m/z = 659 (1 - H)
1
2
Cu 32%, 722 (1 - H)
+
2 2 6 2 2
00%. HRMS (pos.) calculated 721.9493 (C30H18Cu F N O S ) ,
+
-1
found 721.9498 (C30
H18Cu
2
F
2
N
6
O
2
S
2
) , UV-vis (λ, nm/ε, l mol
cm ): 432/23834. Elemental analysis: calculated (%) for
-
1
C
2
30
H
18Cu
2
ClF
.36, N 10.78.
Preparation of the coordination compound of 3-(3-
2
N
6
O
2
S
2
: C 47.46, H 2.39, N 11.07, found C 47.42, H
-
1
-1
HRMS
(pos.)
calculated
found
439.8816
439.8806
)2
+
fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
H
H
16Br
16Br
2
Cu
Cu
2
2
F
F
2
2
N
6
O
2
S
2
2+
thioxotetrahydro-4H-imidazol-4-one 2 with copper (II)
chloride dihydrate (2k). 2k was obtained as a black crystalline
precipitate from 15 mg (50 μmol) of 3-(3-fluorophenyl)-5-(((Z)-
(С30
C
H 1.84, N 8.90.
2
N
N
6
O
2
S
2
)
2
. Elemental analysis: calculated (%) for
: C 39.29, H 1.76, N 9.17, found C 39.63,
30
H16Br
2
Cu
2
ClF
2
6
O
2
S
2
2
-pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one
Preparation of the coordination compound of 3-(4-
chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 8 with copper (II)
chloride dihydrate (8k). 8k was obtained as a black
crystalline precipitate from 15 mg (47 μmol) of 3-(4-
chlorophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 8 and 8 mg (47 μmol) of
copper (II) chloride dihydrate. Yield: 6.0 mg (32%). MALDI: m/z
= 692 (8 - H) Cu 13%, 755 (8 - H) Cu 100%. HRMS (pos.)
calculated 753.8902 (C30
(C30
and 8.5 mg (50 μmol) of copper (II) chloride dihydrate. Yield:
+
5.8 mg (31%). MALDI: m/z = 659 (2 - H)
2
Cu 38%, 720 (2 -
+
H)
(C30
2
Cu
H
2
100%.
HRMS
(pos.)
calculated
721.9493
+
+
18Cu
2
F
2
N
6
O
2
S
2
) , found 721.9512 (C30
H18Cu
2
F
2
N
6
O
2
S
2
) ,
-
1
-1
UV-vis (λ, nm/ε, l mol cm ): 432/22967. Elemental analysis:
calculated (%) for C30 ClF (2k + H O) C 46.36, H
.59, N 10.81, found C 47.74, H 2.36, N 10.85.
Preparation of the coordination compound of 3-(2-
H20Cu
2
2
N
6
O
2
S
2
2
2
+
+
2
2
2
+
fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
H
18Cu
2
Cl
2
N
6
O
2
S
2
) , found 753.8904
+
-1
-1
thioxotetrahydro-4H-imidazol-4-one 3 with copper (II)
chloride dihydrate (3k). 3k was obtained as a black crystalline
precipitate from 15 mg (50 μmol) of 3-(2-fluorophenyl)-5-(((Z)-
H
18Cu
2
Cl
2
N
6
O
2
S
2
) , UV-vis (λ, nm/ε, l mol cm ): 432/9683.
Elemental analysis: calculated (%) for C30
45.49, H 2.29, N 10.61, found C 45.19, H 2.35, N 10.59.
2 3 6 2 2
H18Cu Cl N O S C
2-pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one
Preparation of the coordination compound of 3-(3-
chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 9 with copper (II)
chloride dihydrate (9k). 9k was obtained as a black crystalline
precipitate from 15 mg (47 μmol) of 3-(3-chlorophenyl)-5-(((Z)-
2-pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one
9 and 8 mg (47 μmol) of copper (II) chloride dihydrate. Yield:
11.0 mg (50%). MALDI: m/z = 692 (9 - H)
H)
(C30
found
3
and 8.5 mg (50 μmol) of copper (II) chloride dihydrate. Yield:
+
6.1 mg (33%). MALDI: m/z = 659 (3 - H)
2
Cu 28%, 724 (3 -
+
H)
2
Cu
2
100%.
HRMS
(pos.)
calculated
721.9493
+
(C
30
H
18Cu
2
F
2
N
6
O
2
S
2
, found 721.9495 (C30
H
18Cu
2
F
2
N
6
O
2
S
2
) , UV-
-
1
-1
vis (λ, nm/ε, l mol cm ): 432/38497. Elemental analysis:
calculated (%) for C30 ClF C 47.46, H 2.39, N 11.07,
found C 47.85, H 2.41, N 11.00.
H18Cu
2
2 6 2 2
N O S
+
2
Cu 15%, 755 (9 -
+
Preparation of the coordination compound of 3-(4-
bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 4 with copper (II)
chloride dihydrate (4k). 4k was obtained as a black crystalline
precipitate from 15 mg (42 μmol) of 3-(4-bromophenyl)-5-(((Z)-
2
Cu
2
100%.
Cl
753.8901
HRMS
) , 755.8884 (C30
(C30H18Cu Cl N O S ) ,
isotopic) Elemental analysis: calculated
(pos.)
calculated
+
2 6 2 2
Cl N O S
753.8902
isotopic),
755.8878
+
H
18Cu
2
2
N
6
O
2
S
2
H
18Cu
2
+
2 2 6 2 2
+
(C30
H
18Cu
2
Cl
2
N
6
O
2
S
2
(%) for C30
45.34, H 2.30, N 10.38.
Preparation of the coordination compound of 3-(2-
chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 10 with copper (II)
chloride dihydrate (10k). 10k was obtained as a black
crystalline precipitate from 15 mg (47 μmol) of 3-(2-
chlorophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 10 and 8 mg (47 μmol) of
copper (II) chloride dihydrate. Yield: 5.5 mg (30%). MALDI: m/z
= 693 (10 - H)
calculated 753.8902 (C30
H18Cu Cl N O S C 45.49, H 2.29, N 10.61, found C
2 3 6 2 2
2-pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one
4 and 7 mg (42 μmol) of copper (II) chloride dihydrate. Yield: 6.1
+
+
2 2
Cu
mg (33%). MALDI: m/z = 781 (4 - H)
2
Cu 32%, 844 (4 - H)
+
1
00%. HRMS (pos.) calculated 843.7866 (C30 Br
H
18Cu
2
2
N
6
O
2
S
2
) ,
+
-1
found 843.6904 (C30
H18Cu
2
Br
2
N
6
O
2
S
2
) , UV-vis (λ, nm/ε, l mol
-
1
cm ): 432/6533. Elemental analysis: calculated (%) for
ClBr S C 40.90, H 2.06, N 9.54, found C 40.61, H
2.02, N 9.82.
C
30
H18Cu
2
2 6 2
N O
Preparation of the coordination compound of 3-(3-
bromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 5 with copper (II)
chloride dihydrate (5k). 5k was obtained as a black crystalline
precipitate from 15 mg (42 μmol) of 3-(3-bromophenyl)-5-(((Z)-
2-pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one
+
+
2
Cu 32%, 756 (10 - H)
2
Cu
2
100%. HRMS (pos.)
+
H18Cu
2
Cl
2
N
6
O
2
S
2
) , found 753.8901
+
-1
-1
(C30
H
18Cu
2
Cl
2
N
6
O
2
S
2
) , UV-vis (λ, nm/ε,
l
mol
cm ):
432/15417. Elemental analysis: calculated (%) for
Cl C 45.49, H 2.29, N 10.61, found C 45.40, H
2.38, N 10.52.
C
30
H18Cu
2
3 6 2 2
N O S
5
and 7 mg (42 μmol) of copper (II) chloride dihydrate. Yield: 5.9
+
+
mg (32%). MALDI: m/z = 781 (5 - H)
2
Cu 18%, 846 (5 - H)
Br
) , UV-vis (λ, nm/ε, l mol
2
Cu
2
+
Preparation of the coordination compound of 3-(4-
methoxyphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 11 with copper (II)
100%. HRMS (pos.) calculated 843.7866 (C30
H
18Cu
2
2
N
6
O
2
S
2
) ,
+
-1
found 843.7813 (C30
cm ): 432/7183. Elemental analysis: calculated (%) for
H18Cu
2
Br
2
N
6
O
2
S
2
-
1
chloride dihydrate (11k). 11k was obtained as
a black
crystalline precipitate from 15 mg (44 μmol) of 3-(4-
ACS Paragon Plus Environment

Page 25 of 34
Journal of Medicinal Chemistry
1
2
methoxyphenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 11 and 7.5 mg (44 μmol)
one 17 with copper (II) chloride dihydrate (17k). 17k was
obtained as a black crystalline precipitate from 15 mg (41 μmol)
of 3-(2-methyl, 4-diethylaminophenyl)-5-(((Z)-2-
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one 17
and 6.9 mg (41 μmol) of copper(II) chloride dehydrate. Yield: 6.2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of copper (II) chloride dihydrate. Yield: 5.0 mg (29%). MALDI:
+
+
m/z = 685 (11 - H)
2
Cu 32%, 747 (11 - H)
2
Cu
2
100%. HRMS
+
(
pos.) calculated 745.9892 (C32
H24Cu
2
N
6
O
4
S
2
-1
) , found 745.9814
+
-1
-
+
2
(C
32
H24Cu
2
N
6
O
4
S
2
) , UV-vis (λ, nm/ε, l mol cm ): 441/42859.
mg (35%). MALDI: m/z = 333 (17-H) 98%, 856 (17 - H)
+
2 8 2 2
100%. HRMS (pos.) calculated 856.1464 (C40H42Cu N O S ) ,
2
Cu
Elemental analysis: calculated (%) for C32
9.07, H 3.09, N 10.73, found C 49.43, H 3.22, N 10.61.
Preparation of the coordination compound of 3-(4-
H24Cu
2
ClN
6
O
4
S
2
C
+
-1
-
4
found 856.1484 (C40
H42Cu
2
N
8
O
2
S
2
) , UV-vis (λ, nm/ε, l mol cm
1
): 432/25240. Elemental analysis: calculated (%) for
ClN C 53.77, H 4.74, N 12.54, found C 53.43, H
4.88, N 12.17.
ethoxyphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 12 with copper (II)
chloride dihydrate (12k). 12k was obtained as
crystalline precipitate from 15 mg (47 μmol) of 3-(4-
C
40
H42Cu
2
8 2 2
O S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
black
Preparation of the coordination compound of 3-(2-
methoxy-4-chloro-5-methylphenyl)-5-((Z)-2-
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-
one 18 with copper (II) chloride dihydrate (18k). 18k was
obtained as a black crystalline precipitate from 15 mg (47 μmol)
ethoxyphenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 12 and 8.1 mg (47 μmol) of
copper (II) chloride dihydrate. Yield: 6.0 mg (35%). MALDI: m/z
+
+
=
711 (12 - H)
2
Cu 64%, 776 (12 - H)
2
Cu
2
100%. HRMS (pos.)
of
3-(2-methoxy-4-chloro-5-methylphenyl)-5-(((Z)-2-
+
calculated 775.0284 (C34
H29Cu
2
N
6
O
4
S
2
) , found 775.0219
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one 18
and 8 mg (47 μmol) of copper (II) chloride dihydrate. Yield: 5.5
+
-1
-1
(C
34
H29Cu
2
N
6
O
4
S
2
) , UV-vis (λ, nm/ε, l mol cm ): 432/11250.
+
Elemental analysis: calculated (%) for C35
H32Cu
2
ClN
6
O
5
S
2
(12k +
mg (30%). MALDI: m/z = 693 (18 - H)
2
Cu 32%, 756 (18 -
+
CH OH) C 49.85, H 3.82, N 9.97, found C 49.98, H 3.39, N 10.02.
3
H)
(C30
2
Cu
2
100%.
HRMS
(pos.)
calculated
753.8902
+
+
Preparation of the coordination compound of 3-(3,4-
methoxyphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 13 with copper (II)
chloride dihydrate (13k). 13k was obtained as
crystalline precipitate from 15 mg (46 μmol) of 3-(3,4-
methoxyphenyl)-5-(((Z)-2-pyridylmethylidene)-2-
H18Cu
2
Cl
2
N
6
O
2
S
2
) , found 753.8901 (C30
H
18Cu
2
Cl
2
N
6
O
2
S
2
) .
-
1
-1
UV-vis (λ, nm/ε, l mol cm ): 436/34300. Elemental analysis:
calculated (%) for C30 Cl C 46.40, H 2.98, N 9.55,
found C 46.31, H 2.93, N 9.32.
H18Cu
2
3 6 2 2
N O S
a black
Preparation of the coordination compound of 3-(3-
chloro-4-fluorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 19 with copper (II)
thioxotetrahydro-4H-imidazol-4-one 13 and 8.0 mg (46 μmol) of
copper (II) chloride dihydrate. Yield: 7.0 mg (39%). MALDI: m/z
chloride dihydrate (19k). 19k was obtained as
a black
+
+
=
743 (13 - H)
2
Cu 50%, 806 (13 - H)
2
Cu
2
100%. HRMS (pos.)
crystalline precipitate from 15 mg (45 μmol) of 3-(3-chloro-4-
fluorophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 18 and 7.6 mg (45 μmol) of
copper (II) chloride dihydrate. Yield: 9 mg (45%). HRMS (pos.)
+
calculated 806.0104 (C34
H
28Cu N O S ) , found 806.0100
2 6 6 2
+
-1
-1
(C
34
H28Cu
2
N
6
O
6
S
2
) , UV-vis (λ, nm/ε, l mol cm ): 432/33055.
Elemental analysis: calculated (%) for C34 ClN
8.42, H 3.35, N 9.97, found C 48.51, H 3.47, N 9.99.
Preparation of the coordination compound of 3-(4-t-
H28Cu
2
6
O
6
S
2
C
+
4
calculated 789.8713 (C30
H
16Cu
2
Cl
2
F
2
N
6
O
2
S
2
) , found 789.8615
+
(C30
H
16Cu
16Cu
2
Cl
Cl
2.12, N 10.05
2
F
2
N
6
O
2
S
2
) . Elemental analysis: calculated (%) for
F N O S C 43.51, H 1.95, N 10.15, found C 43.90, H
3 2 6 2 2
butylphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 14 with copper (II)
chloride dihydrate (14k). 14k was obtained as
crystalline precipitate from 15 mg (44 μmol) 3-(4-
tretbutylphenyl)-5-(((Z)-2-pyridylmethylidene)-2-
C
30
H
2
a
black
Preparation of the coordination compound of 3-(2-
methyl-3-chlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 20 with copper (II)
chloride dihydrate (20k). 20k was obtained as a black
crystalline precipitate from 15 mg (47 μmol) of 3-(2methyl-3-
thioxotetrahydro-4H-imidazol-4-one 14 and 7.5 mg (44 μmol)
copper (II) chloride dihydrate. Yield: 10.0 mg (59%). MALDI:
+
+
m/z = 735 (14 - H) Cu 46%, 800 (14 - H) Cu 100%. HRMS
2
2
2
chlorophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 20 and 8 mg (47 μmol) of
+
(
pos.) calculated 737.1788 (C38
H38CuN
6
O
2
S
2
-1
) , found 737.1795
+
-1
(C38
H38CuN
6
O
2
S
2
) , UV-vis (λ, nm/ε, l mol cm ): 439/24853.
copper (II) chloride dihydrate. Yield: 14 mg (61%). HRMS (pos.)
+
Elemental analysis: calculated (%) for C38
O) C 50.29, H 4.89, N 9.26, found C 50.64, H 4.22, N 9.36.
Preparation of the coordination compound of 3-(2-t-
H44Cu
2
ClN
6
O
2
S
2
(14k +
calculated
781.9215
(C32
isotopic),
H
22Cl
2
Cu
2
N
found
6
O
2
S
2
) ,
783.9197
781.9179
isotopic).
+
4H
2
(C32
(C32
H
H
22Cl
22Cl
2
Cu
Cu
2
N
N
6
O
O
2
2
S
S
2
)+, 783.9213 (C32
H
22Cl
Cu
N
O
S
+
2
2
2
6
2
2
2
6
2
butylphenyl)-5-((Z)-2-pyridylmethylidene)-2-
Elemental analysis: calculated (%) for C36
2 3 6 2 2
H32Cu Cl N O S (20k
thioxotetrahydro-4H-imidazol-4-one 15 with copper (II)
chloride dihydrate (15k). 15k was obtained as a black
crystalline precipitate from 15 mg (44 μmol) of 3-(2-
tertbutylphenyl)-5-(((Z)-2-pyridylmethylidene)-2-
+ Et O) C 48.35, H 3.61, N 9.40, found C 48.38, H 3.87, N 9.37.
2
Preparation of the coordination compound of 3-(2,4,6-
trimethylphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 21 with copper (II)
chloride dihydrate (21k). 21k was obtained as a black
crystalline precipitate from 15 mg (46 μmol) of 3-(2,4,6-
trimethylphenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 21 and 7.8 mg (46 μmol)
of copper (II) chloride dihydrate. Yield: 13 mg (59%). HRMS
thioxotetrahydro-4H-imidazol-4-one 15 and 7.5 mg (44 μmol) of
copper (II) chloride dihydrate. Yield: 11 mg (50%) HRMS
2+
calculated 399.0449 (C38
H
38CuN
6
O
2
S
2
)
,
found 399.0461
2
+
-1
-1
(C
38
H38CuN
6
O
2
S
2
)
. UV-vis (λ, nm/ε, l mol cm ): 434/28800.
Elemental analysis: calculated (%) for C38
4.63, H 4.34, N 10.06, found C 54.25, H 4.30, N 10.34.
Preparation of the coordination compound of 3-(4-
H38Cu
2
ClN
6
O
2
S
2
C
+
5
(pos.) calculated 770.0620 (C36
H
32Cu
2
N
6
O
2
S
2
) , 772.0602
+
(C36
(C36
H
H
32Cu
32Cu
2
N
N
6
O
O
2
S
S
2
isotopic),
) , 772.0604 (C36
found
770.0624
isotopic).
ClN O S C
6 2 2
+
+
nitrophenyl)-5-((Z)-2-pyridylmethylidene)-2-
2
6
2
2
H
32Cu
2
N
6
O
2
S
2
thioxotetrahydro-4H-imidazol-4-one 16 with copper (II)
chloride dihydrate (16k). 16k was obtained as a black
crystalline precipitate from 15 mg (46 μmol) of 3-(4-
nitrophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 16 and 7.8 mg (46 μmol) of
copper (II) chloride dihydrate. Yield: 6.0 mg (35%). MALDI: m/z
Elemental analysis: calculated (%) for C36
H
32Cu
2
53.56, H 4.00, N 10.41, found C 53.40, H 3.97, N 10.30.
Preparation of the coordination compound of 3-(4-
methylthiophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 22 with copper (II)
chloride dihydrate (22k). 22k was obtained as
a black
+
-1
-1
= 778 (16 - H)
2
Cu
32/10583. Elemental analysis: calculated (%) for
ClN C 44.31, H 2.23, N 13.78, found C 44.30, H
.22, N 13.80.
Preparation of the coordination compound of 3-(2-
2
100%. UV-vis (λ, nm/ε, l mol cm ):
crystalline precipitate from 15 mg (46 μmol) of 3-(4-
methylthiophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 22 and 7.8 mg (46 μmol) of
4
C
2
30
H18Cu
2
8
O
6
S
2
copper (II) chloride dihydrate. Yield: 10 mg (45%). HRMS (pos.)
+
calculated
777.9436
(C32
H24Cu
2
N
found
6
O
2
S
4
) ,
779.9418
777.9442
+
methyl-4-diethylaminophenyl)-5-((Z)-2-
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-
(C32
H24Cu
2
N
N
6
6
O
O
2
S
4
isotopic),
+
2 6 2 4
779.9406 (C32H24Cu N O S isotopic).
+
(C32
H24Cu
2
2
S
4
) ,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 34
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Elemental analysis: calculated (%) for C32
O): C 45.14, H 3.31, N 9.87, found C 44.83, H 2.95, N 9.96.
Preparation of the coordination compound of 3-(4-
iodophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 23 with copper (II)
H
28Cu
2
ClN
6
O
4
S
4
(22k +
C
30
H
16Cu
2
ClF
4 6 2 2
N O S C 45.31, H 2.03, N 10.50, found C 45.38, H
2H
2
2.13, N 10.41.
Preparation of the coordination compound of 3-(3,5-
difluorophenylphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 29 with copper (II)
chloride dihydrate (23k). 23k was obtained as
a
black
chloride dihydrate (29k). 29k was obtained as a black
crystalline precipitate from 15 mg (16.5 μmol) of 3-(4-
iodophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
crystalline precipitate from 15 mg (46 μmol) of 3-(3,5-
difluorophenylphenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 29 and 7.8 mg (46 μmol) of
copper (II) chloride dihydrate. Yield: 10 mg (47%). HRMS
calculated 757.9299 (C30
(C30
C
2.12, N 10.18.
Preparation of the coordination compound 5-(Z)-3-(4-
fluorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 30 with copper (II)
chloride dihydrate (30k). 30k was obtained as a black
crystalline precipitate from 15 mg (50 μmol) of 5-(Z)-3-(4-
fluorophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
imidazol-4-one 30 and 8.5 mg (50 μmol) of copper (II) chloride
dihydrate. Yield: 6.0 mg (32%). MALDI: m/z = 314 (30 + H)
100%, 376 30 Cu 70%, 690 (30)
calculated 423.0114 (C16 13CuFN
thioxotetrahydro-4H-imidazol-4-one 23 and 3 mg (16.5 μmol) of
copper (II) chloride dihydrate. Yield: 6.0 mg (32%). MALDI: m/z
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
+
=
877 (23 - H)
2
Cu 6%, 940 (23 - H)
2
Cu
2
100%. HRMS (pos.)
H
16Cu
2
F
4
N
6
O
2
S
2
) , found 757.9289
+
+
calculated 937.7614 (C30
H
18Cu
2
I
2
N
6
O
2
S
2
) , found 937.7643
H
16Cu
2
F
ClF
4
N
6
O
2
S
2
) . Elemental analysis: calculated (%) for
+
-1
-1
(C
30
H18Cu
2
I
2
N
6
O
2
S
2
) , UV-vis (λ, nm/ε, l mol cm ): 438/19622.
30
H16Cu
2
4 6 2 2
N O S C 45.31, H 2.03, N 10.57, found C 45.32, H
Elemental analysis: calculated (%) for C30
2 2 6 2 2
H18Cu ClI N O S C
36.96, H 1.86, N 8.62, found C 36.76, H 1.70, N 8.49.
Preparation of the coordination compound of 3-(2-
methyl-4-diethylaminophenyl)-5-((Z)-2-
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-
one 24with copper (II) chloride dihydrate (24k). 24k was
obtained as a black crystalline precipitate from 15 mg (41 μmol)
of
3-(2-methyl,
4-diethylaminophenyl)-5-(((Z)-2-
+
pyridylmethylidene)-2-thioxotetrahydro-4H-imidazol-4-one 17
and 6.9 mg (41 μmol) of copper (II) chloride dihydrate. Yield: 6.2
+
+
2
Cu 100%. HRMS (pos.)
+
+
mg (35%). MALDI: m/z = 856 (24 - H)
calculated 856.1464 (C40
2
Cu
2
100%. HRMS (pos.)
H
3
OS*HCOOH) , found 423.0111
+
+
-1
-1
H42Cu
2
N
8
O
2
S
2
) , found 856.1484
(C16
H
13CuFN
392/20417. Elemental analysis: calculated (%) for
13CuCl FN OS C 42.91, H 2.70, N 9.38, found C 42.94, H 2.67,
N 9.22.
3
OS*HCOOH) , UV-vis (λ, nm/ε, l mol cm ):
+
-1
-1
(C
40
H42Cu
2
N
8
O
2
S
2
) , UV-vis (λ, nm/ε, l mol cm ): 432/25240.
Elemental analysis: calculated (%) for C40 ClN
3.77, H 4.74, N 12.54, found C 54.01, H 5.02, N 12.90.
Preparation of the coordination compound of 3-(2,6-
H42Cu
2
8
O
2
S
2
C
C
16
H
2
3
5
Preparation of the coordination compound 5-(Z)-3-(3-
fluorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 31 with copper (II)
dichlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 25 with copper (II)
chloride dihydrate (25k). 25k was obtained as
a
black
chloride dihydrate (31k). 31k was obtained as a black
crystalline precipitate from 15 mg (43 μmol) of 3-(2,6-
dichlorophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 25 and 7.3 mg (43 μmol)
crystalline precipitate from 15 mg (50 μmol) of 5-(Z)-3-(3-
fluorophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
imidazol-4-one 31 and 8.5 mg (50 μmol) of copper (II) chloride
+
copper (II) chloride dihydrate. Yield: 5 mg (27%). HRMS (pos.)
dihydrate. Yield: 5.8 mg (32%). MALDI: m/z = 313 (31) 20%,
+
+
+
calculated 858.7781 (C30
H
16Cl
5
Cu
2
N
6
O
2
S
2
) , found 858.8301
376 31Cu 68%, 690 (31)
423.0114 13CuFN OS*HCOOH) ,
13CuFN
2
Cu 100%. HRMS (pos.) calculated
+
+
(C
30
H16Cl
5
Cu
2
N
6
O
2
S
2
) . Elemental analysis: calculated (%) for
(C16
H
3
+
found
423.0074
mol-1 cm ):
-1
C
30
H16Cl
5
Cu
2
N
6
O
2
S
2
C 41.85, H 1.87, N 9.76, found C 42.05, H 1.79,
(C16
H
3
OS*HCOOH) , UV-vis (λ, nm/ε,
l
N 9.68.
392/13583. Elemental analysis: calculated (%) for C16
Cl FN OS C 42.91, H 2.70, N 9.38, found C 42.86, H 2.69, N 9.31.
Preparation of the coordination compound 5-(Z)-3-(2-
fluorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 32 with copper (II)
H13Cu
Preparation of the coordination compound of 3-(2,4,6-
trichlorophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 26 with copper (II)
2
3
chloride dihydrate (26k). 26k was obtained as
a
black
crystalline precipitate from 15 mg (39 μmol) of 3-(2,4,6-
trichlorophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
chloride dihydrate (32k). 32k was obtained as a black
crystalline precipitate from 15 mg (50 μmol) of 5-(Z)-3-(2-
fluorophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
imidazol-4-one 32 and 8.5 mg (50 μmol) of copper (II) chloride
dihydrate. Yield: 6.1 mg (33%). MALDI: m/z = 374 (32)Cu 11%,
692 (32)
(C16
(C16
thioxotetrahydro-4H-imidazol-4-one 26 and 6.6 mg (39 μmol)
of copper (II) chloride dihydrate. Yield: 6 mg (33%). HRMS (pos.)
+
+
calculated 891.7313 (C30
H
16Cl
6
Cu
2
N
8
O
6
S
2
) , found 891.7231
+
+
(C
30
H16Cl
6
Cu
2
N
8
O
6
S
2
) . Elemental analysis: calculated (%) for
2
Cu 100%. HRMS (pos.) calculated
423.0114
+
C
30
H16Cu
2
Cl
7
N
8
O
6
S
2
C 38.75, H 1.52, N 9.04, found C 38.70, H 1.43,
H
H
13CuFN
13CuFN
3
OS*HCOOH) ,
OS*HCOOH) , UV-vis (λ, nm/ε,
found
423.0109
+
mol-1 cm ):
-1
N 9.06.
3
l
Preparation of the coordination compound of 3-(2,6-
dibromophenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 27 with copper (II)
392/14883. Elemental analysis: calculated (%) for
13CuCl FN OS C 42.91, H 2.70, N 9.38, found C 42.95, H 2.90,
N 9.41.
C
16
H
2
3
chloride dihydrate (27k). 27k was obtained as
a
black
Preparation of the coordination compound 5-(Z)-3-(4-
bromophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 33 with copper (II)
chloride dihydrate (33k). 33k was obtained as a black
crystalline precipitate from 15 mg (42 μmol) of 5-(Z)-3-(4-
bromophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
imidazol-4-one 33 and 7 mg (42 μmol) of copper (II) chloride
3
dihydrate. Yield: 6.1 mg (33%). MALDI: m/z = 390 (33 + CH )
100%, 478 (33)CuCl 12%, 814 (33)
calculated 482.9314 (C16 13CuBrN
482.9303 (C16 13CuBrN
crystalline precipitate from 15 mg (34 μmol) of 3-(2,6-
dibromophenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 27 and 5.8 mg (34 μmol) of
copper (II) chloride dihydrate. Yield: 9 mg (51%). HRMS (pos.)
+
calculated 1001.6061 (C30
H
16Br
4
Cu
2
N
8
O
6
S
2
) , found 1001.6035
+
(C30
H
16Cu
16Br
4
Cu
ClBr
.69, N 8.20.
Preparation of the coordination compound of 3-(2,5-
2
N
8
O
6
S
2
) . Elemental analysis: calculated (%) for
+
C
30
H
2
4
N
8
O
6
S
2
C 34.69, H 1.55, N 8.09, found C 34.80, H
+
+
1
2
Cu 100%. HRMS (pos.)
3
+
H
OS*HCOOH) , found
OS*HCOOH) , UV-vis (λ, nm/ε, l mol
+
-1
difluorophenylphenyl)-5-((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 28 with copper (II)
H
3
-1
cm ): 393/12157. Elemental analysis: calculated (%) for
13CuCl BrN OS C 37.78, H 2.38, N 8.26, found C 37.85, H 2.39,
N 8.31.
chloride dihydrate (28k). 28k was obtained as
a
black
C
16
H
2
3
crystalline precipitate from 15 mg (45 μmol) of 3-(2,5-
difluorophenylphenyl)-5-(((Z)-2-pyridylmethylidene)-2-
thioxotetrahydro-4H-imidazol-4-one 28 and 7.6 mg (45 μmol) of
Preparation of the coordination compound 5-(Z)-3-(3-
bromophenyl)-2-(methylthio)-5-(pyridin-2-
copper (II) chloride dihydrate. Yield: 5 mg (24%). HRMS
ylmethylidene)-1H-imidazol-4-one 34 with copper (II)
chloride dihydrate (34k). 34k was obtained as a black
crystalline precipitate from 15 mg (42 μmol) of 5-(Z)-3-(3-
+
calculated 757.9299 (C30
H
16Cu
2
F
4
N
6
O
2
S
2
) , found 757.9292
+
(C
30
H16Cu
2
F
4
N
6
O
2
S
2
) . Elemental analysis: calculated (%) for
ACS Paragon Plus Environment

Page 27 of 34
Journal of Medicinal Chemistry
1
2
bromophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
imidazol-4-one 34 and 7 mg (42 μmol) of copper (II) chloride
imidazol-4-one 39 and 8 mg (47 μmol) of copper (II) chloride
dihydrate. Yield: 5.5 mg (30%). MALDI: m/z = 330 (39 + H)
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
dihydrate. Yield: 5.9 mg (32%). MALDI: m/z = 375 (34 + H)
+
+
3
7%, 392 (39)Cu 100%, 814 (39)
calculated 438.9814 14CuClN
438.9815 (C16 14CuClN OS*HCOOH) , UV-vis (λ, nm/ε, l mol
2
Cu 73%. HRMS (pos.)
+
8
0%, 438 (34) Cu+ 32%, 815 (34)
2
Cu 100%. HRMS (pos.)
+
(C16
H
3
OS*HCOOH) ,
found
+
calculated 482.9314 (C16
82.9310 (C16 13CuBrN
cm ): 392/11917. Elemental analysis: calculated (%) for
13CuCl BrN OS C 37.78, H 2.38, N 8.26, found C 37.75, H 2.29,
N 8.27.
H
13CuBrN
3
OS*HCOOH) , found
+
-1
+
-1
H
3
4
H
3
OS*HCOOH) , UV-vis (λ, nm/ε, l mol
-
1
cm-1): 382/17298. Elemental analysis: calculated (%) for
13CuCl OS C 41.39, H 2.61, N 9.05, found C 41.35, H 2.59,
N 9.01.
C
16
H
2
3
C
16
H
3 3
N
Preparation of the coordination compound 5-(Z)-3-(2-
bromophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 35 with copper (II)
Preparation of the coordination compound 5-(Z)-3-(4-
methoxyphenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 40 with copper (II)
chloride dihydrate (40k). 40k was obtained as a black
crystalline precipitate from 15 mg (44 μmol) of 5-(Z)-3-(4-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chloride dihydrate (35k). 35k was obtained as
a
black
crystalline precipitate from 15 mg (42 μmol) of 5-(Z)-3-(2-
bromophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
methoxyphenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-
1
H-imidazol-4-one 40 and 7.5 mg (44 μmol) of copper (II)
imidazol-4-one 35 and 7 mg (42 μmol) of copper (II) chloride
chloride dihydrate. Yield: 5.0 mg (29%). MALDI: m/z = 326 (40
+
OMe)
+
dihydrate. Yield: 6.0 mg (32%). MALDI: m/z = 375 (35 + H)
+
+
+
H) 100%, 388 (40)Cu 47%, 423 (40)CuCl , 649 (40-
34%, 475 (35)CuCl+ 18%, 814 (35)
Cu 100%. HRMS (pos.)
OS*HCOOH) , found
+
2
+
2
Cu
51%.
HRMS
(pos.)
calculated
found
S*HCOOH) , UV-vis (λ, nm/ε, l
mol-1 cm ):
435.0314
+
calculated 482.9314 (C16
H13CuBrN
3
+
(C
17
H
H
17CuN
17CuN
3
3
O
O
2
S*HCOOH) ,
435.0316
+
-1
4
82.9307 (C16 13CuBrN
H
3
OS*HCOOH) , UV-vis (λ, nm/ε, l mol
+
-1
(C17
2
-
1
cm ): 392/5950. Elemental analysis: calculated (%) for
13CuCl BrN OS C 37.78, H 2.38, N 8.26, found C 37.79, H 2.36,
N 8.30.
392/13400. Elemental analysis: calculated (%) for
17CuCl S C 44.40, H 3.29, N 9.14, found C 44.44, H 3.32,
N 9.15.
C
16
H
2
3
C
17
H
2 3 2
N O
Preparation of the coordination compound 5-(Z)-3-(2-
bromo-4-fluorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 36 with copper (II)
chloride dihydrate (36k). 36k was obtained as a black
Preparation of the coordination compound 5-(Z)-3-(4-
ethoxyphenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 41 with copper (II)
chloride dihydrate (41k). 41k was obtained as
a black
crystalline precipitate from 15 mg (47 μmol) of 5-(Z)-3-(4-
ethoxyphenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-
crystalline precipitate from 15 mg (42 μmol) of 5-(Z)-3-(2-
bromophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
imidazol-4-one 36 and copper (II) chloride dihydrate 7 mg (42 μ
mol). Yield: 5 mg (25%) HRMS (pos.) calculated 523.8458
1H-imidazol-4-one 41 and 8.1 mg (47 μmol) of copper (II)
chloride dihydrate. Yield: 6.0 mg (35%). MALDI: m/z = 342 (41
+
+
+
+ H)
50%, 441 (41)CuCl
32%, 747 (41)
2
Cu
100%. HRMS (pos.)
+
+
+
(C
16
H11BrCl
2
CuFN
3
OS) , found 523.8463 (C16
H
11BrCl
2
CuFN
3
OS) .
calculated 449.0471 (C18
H
19CuN
3
O
2
S*HCOOH) , found 449.0471
+
-1
-1
UV-vis (λ, nm/ε, l mol-1 cm ): 386/37800. Elemental analysis:
-1
(C18
H
19CuN
3
O
2
S*HCOOH) , UV-vis (λ, nm/ε,
l
mol cm ):
392/13233. Elemental analysis: calculated (%) for
calculated (%) for C16
2 3
H11 CuCl BrFN OS C 36.49, H 2.11, N 7.98,
C
18
H
19CuCl S C 45.62, H 3.62, N 8.87, found C 45.44, H 3.50,
2 3 2
N O
found C 36.55, H 2.24, N 7.96.
Preparation of the coordination compound 5-(Z)-3-(4-
chlorophenyl)-2-(methylthio)-5-(pyridin-2-
N 8.75.
Preparation of the coordination compound 5-(Z)-3-(3,4-
dimethoxyphenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 42 with copper (II)
ylmethylidene)-1H-imidazol-4-one 37 with copper (II)
chloride dihydrate (37k). 37k was obtained as
a black
chloride dihydrate (42k). 42k was obtained as
a black
crystalline precipitate from 15 mg (47 μmol) of 5-(Z)-3-(4-
chlorophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
imidazol-4-one 37 and 8 mg (47 μmol) of copper (II) chloride
crystalline precipitate from 15 mg (46 μmol) of 5-(Z)-3-(3,4-
dimethoxyphenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 42 and 8.0 mg (46 μmol) of
copper (II) chloride dihydrate. Yield: 7.0 mg (39%). MALDI: m/z
+
dihydrate. Yield: 6.0 mg (32%). MALDI: m/z = 330 (37 + H)
=
7
356 (42 + H) 100%, 418 (42)Cu 22%, 453 (42)CuCl+ 36%,
+
+
+
+
1
00%, 392 (37) Cu 82%, 814 (37)
calculated 438.9814 14CuClN
38.9811 (C16 13CuClN
2
Cu 100%. HRMS (pos.)
+
09 (42-OMe)
2
Cu 49%. HRMS (pos.) calculated 465.0420
+
(C16
H
3
OS*HCOOH) ,
found
+
(C
18
H19CuN
19CuN
3
O
O
3
S*HCOOH) ,
S*HCOOH) , UV-vis (λ, nm/ε,
found
465.0411
+
-1
4
H
3
OS*HCOOH) , UV-vis (λ, nm/ε, l mol
+
mol-1 cm ):
-1
(C18
H
3
3
l
-
1
cm ): 382/18034. Elemental analysis: calculated (%) for
13CuCl OS C 41.39, H 2.61, N 9.05, found C 41.44, H 2.67,
N 9.11.
368/149756. Elemental analysis: calculated (%) for
C18H19CuCl N O S C 44.13, H 3.50, N 8.58, found C 44.22, H 3.60,
C
16
H
3 3
N
2
3
3
N 8.62.
Preparation of the coordination compound 5-(Z)-3-(3-
chlorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 38 with copper (II)
Preparation of the coordination compound 5-(Z)-3-(4-t-
butylphenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-
1H-imidazol-4-one 43 with copper (II) chloride dihydrate
(43k). 43k was obtained as a black crystalline precipitate from
15 mg (44 μmol) of 5-(Z)-3-(4-t-buthylphenyl)-2-(methylthio)-
chloride dihydrate (38k). 38k was obtained as
a black
crystalline precipitate from 15 mg (45 μmol) of 5-(Z)-3-(4-
chlorophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
imidazol-4-one 38 and 7.6 mg (45 μmol) of copper (II) chloride
5-(pyridin-2-ylmethylidene)-1H-imidazol-4-one 43 and 7.5 mg
(
44 μmol) ща copper (II) chloride dihydrate. Yield: 10.0 mg
+
+
(59%). MALDI: m/z = 353 (43 + H) 53%, 669 ((43-tBu)-Me)Cu
dihydrate. Yield: 11 mg (50%). HRMS (pos.) calculated 437.9735
+
+Me)Cu+ 27%.
3
27%, 685 (43-tBu)
2
CuCl 100%, 701 (31-tBu)
2
+
(C
16
H13CuClN
3
OS*HCOOH) ,
found
437.9739
+
HRMS (pos.) calculated 461.0834 (C20
H
22CuN
OS*HCOOH) ,
, UV-vis (λ, nm/ε, l
mol cm ): 366/13487. Elemental analysis: calculated (%) for
21CuCl OS C 49.44, H 4.36, N 8.65, found C 49.56, H 4.39,
N 8.72.
OS*HCOOH)+, UV-vis (λ, nm/ε,
mol-1 cm ):
-1
found 461.0833 (C20H22CuN OS*HCOOH)
+
(C
16
H13CuClN
3
l
3
-
1
-1
3
C
92/14567. Elemental analysis: calculated (%) for
13CuCl OS C 41.39, H 2.61, N 9.05, found C 41.31, H 2.58,
C
20
H
2 3
N
16
H
3 3
N
N 9.01.
Preparation of the coordination compound 5-(Z)-3-(2-t-
butylphenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-
Preparation of the coordination compound 5-(Z)-3-(2-
chlorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 39 with copper (II)
1
H-imidazol-4-one 44 with copper (II) chloride dihydrate
(44k). 44k was obtained as a black crystalline precipitate from
chloride dihydrate (39k). 39k was obtained as
a black
1
5
5 mg (44 μmol) of 5-(Z)-3-(4-t-buthylphenyl)-2-(methylthio)-
-(pyridin-2-ylmethylidene)-1H-imidazol-4-one 44 and 7.5 mg
crystalline precipitate from 15 mg (47 μmol) of 5-(Z)-3-(2-
chlorophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
(44 μmol) of copper (II) chloride dihydrate. Yield: 9.5 mg (46%).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 34
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
-1
-1
HRMS (pos.) calculated 415.0775 (C20
15.0769 (C20 22CuN
OS)+, UV-vis (λ, nm/ε,
392/11350. Elemental analysis: calculated (%) for
21CuCl OS C 49.44, H 4.36, N 8.65, found C 49.43, H 4.28,
N 8.65.
H
22CuN
3
OS)+, found
mol cm ):
(C16
H
11CuF
11CuCl
N 9.01.
2
N
2 2 3
3
OS) , UV-vis (λ, nm/ε, l mol cm ): 392/11583.
-1
-1
4
H
3
l
C
16
H
F N OS C 41.26, H 2.38, N 9.02, found C 41.30, H 2.41,
C
20
H
2
N
3
Preparation of the coordination compound 5-(Z)-3-(3,5-
difluorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 51 with copper (II)
chloride dihydrate (51k). 51k was obtained as
crystalline precipitate from 15 mg (45 μmol) of 5-(Z)-3-(3,5-
difluorophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-
1H-imidazol-4-one 51 and 7.6 mg (45 μmol) of copper (II)
Preparation of the coordination compound 5-(Z)-3-(2-
methoxy-4-chloro-5-methylphenyl)-2-(methylthio)-5-
a black
(
pyridin-2-ylmethylidene)-1H-imidazol-4-one 45 with
copper (II) chloride dihydrate (45k). 45k was obtained as a
black crystalline precipitate from 15 mg (47 μmol) of 5-(Z)-3-(4-
chlorophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-1H-
imidazol-4-one 45 and 8 mg (47 μmol) of copper (II) chloride
dihydrate. Yield: 5.0 mg (27%). HRMS (pos.) calculated 481.9998
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
chloride dihydrate. Yield: 10 mg (47%). HRMS calculated
+
393.9882
(C16
H11CuF
2
N
3
OS) ,
found
393.9872
+
(C16
H
11CuF
11CuCl
N 9.40.
2 3
N OS) . Elemental analysis: calculated (%) for
+
(C
18
H16CuN
3
O
2
S + HCOOH) , found 481.9989 (C18
H16CuN
3
O
2
S +
C
16
H
2 2 3
F N OS C 41.26, H 2.38, N 9.02, found C 41.33, H 2.34,
+
-1
-1
HCOOH) , UV-vis (λ, nm/ε, l mol cm ): 392/9467. Elemental
analysis: calculated (%) for C18
.27, found C 42.57, H 3.20, N 8.33.
Preparation of the coordination compound 5-(Z)-3-(3-
H16CuCl
3
N
3
O
2
S C 42.53, H 3.17, N
Preparation of the coordination compound (4Z,4'Z)-2,2'-
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
8
pyridylmethylidene)-1-(4-methoxyphenyl)-2,3-dihydro-
4H-imidazol-5-one) 52 with copper (II) chloride dihydrate
(52k). 52k was obtained as a black crystalline precipitate from
15 mg (22 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-diyl
disulfandiyl)bis(4-(2-pyridylmethylidene)-1-(4-
methoxyphenyl)-2,3-dihydro-4H-imidazole-5-one) 52 as
result of reaction with 7.5 mg (44 μmol) copper (II) chloride
chloro-4-fluorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 46 with copper (II)
chloride dihydrate (46k). 46k was obtained as
a black
crystalline precipitate from 15 mg (44 μmol) of 5-(Z)-3-(3-
chloro-4-fluorophenyl)-2-(methylthio)-5-(pyridin-2-
a
ylmethylidene)-1H-imidazol-4-one 46 and 7.5 mg (44 μmol)
copper (II) chloride dihydrate. Yield: 9.5 mg (46%). HRMS (pos.)
+
dihydrate. Yield: 5.1 mg (30%). MALDI: m/z = 712 52Cu 100%,
+
+
calculated 409.9591 (C16
H
11ClCuFN
3
OS) , found 409.9590
775 52Cu
(C34
2
22%. HRMS (pos.) calculated 711.0909
+
-1
-1
+
+
2
(C16
H11ClCuFN
3
OS) , UV-vis (λ, nm/ε, l mol cm ): 392/12217.
H
28
O
4
N
6
CuS
2
) , found 711.0910 (C34
H
28
O
4
N
6
CuS
) , UV-vis
-1
Elemental analysis: calculated (%) for C17
2H O + CH OH): C 37.10, H 3.48, N 7.64, found C 36.50, H 3.38,
N 7.76
H19CuCl
3
FN
3
O
4
S (46k
(λ, nm/ε,
calculated (%) for C34
found C 44.61, H 3.15, N 9.19.
l
mol-1 cm ): 395/31517. Elemental analysis:
+
2
3
2 4 6 4 2
H28Cu Cl N O S C 44.50, H 3.08, N 9.16,
Preparation of the coordination compound 5-(Z)-3-(2-
methyl-3-chlorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 47 with copper (II)
Preparation of the coordination compound (4Z,4'Z)-2,2'-
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(4-ethoxyphenyl)-2,3-dihydro-4H-
imidazol-5-one) 53 with copper (II) chloride dihydrate
(53k). 53k was obtained as a black crystalline precipitate from
15 mg (23.5 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-
diyldisulfandiyl)bis(4-(2-pyridylmethylidene)-1-(4-
ethoxyphenyl)-2,3-dihydro-4H-imidazole-5-one) 53 as a result
of reaction with 8.1 mg (47 μmol) of copper (II) chloride
chloride dihydrate (47k). 47k was obtained as
a black
crystalline precipitate from 15 mg (44 μmol) of 5-(Z)-3-(2-
methyl-3-chlorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 47 and 7.4 mg (44 μmol) of
copper (II) chloride dihydrate. Yield: 9.5 mg (46%). HRMS
+
calculated 405.9842 (C17
H
14ClCuN
3
OS) , found 405.9836
+
+
(
C
17
H
14ClCuN
14CuCl
N 8.81.
3
OS) . Elemental analysis: calculated (%) for
OS: C 42.69, H 2.95, N 8.79, found C 42.70, H 2.91,
dihydrate. Yield: 6.3 mg (37%). MALDI: m/z = 739 53Cu 100%,
+
C
17
H
3
N
3
801 53Cu
(C36
2
23%. HRMS (pos.) calculated
738.1222
) , UV-vis
+
+
H
32
O
4
N
6
CuS
2
) , found 739.1221 (C36
H
32
O
4
N
6
CuS
2
-1
Preparation of the coordination compound 5-(Z)-3-
(2,4,6-methylphenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 48 with copper (II)
(λ, nm/ε,
calculated (%) for C36
found C 45.75, H 3.45, N 8.93.
l
mol-1 cm ): 395/34383. Elemental analysis:
H
2 4 6 4 2
32Cu Cl N O S C 45.72, H 3.41, N 8.89,
chloride dihydrate (48k). 48k was obtained as
a
black
Preparation of the coordination compound (4Z,4'Z)-2,2'-
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
crystalline precipitate from 15 mg (45 μmol) 5-(Z)-3-(2,4,6-
methylphenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-
pyridylmethylidene)-1-(3,4-dimethoxyphenyl)-2,3-
dihydro-4H-imidazol-5-one) 54 with copper (II) chloride
dihydrate (54k). 54k was obtained as a black crystalline
precipitate from 15 mg (23 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-
diyldisulfandiyl)bis(4-(2-pyridylmethylidene)-1-(3,4-
dimethoxyphenyl)-2,3-dihydro-4H-imidazole-5-one) 54 as
result of reaction with 8.0 mg (46 μmol) of copper (II) chloride
dihydrate. Yield: 5.1 mg (30%). MALDI: m/z = 772 54 Cu 100%,
1
H-imidazol-4-one 48 and 7.6 mg (45 μmol) of copper (II)
chloride dihydrate. Yield: 12 mg (52%). HRMS calculated
+
+
400.0545 (C19
Elemental analysis: calculated (%) for C19
H 4.06, N 8.90, found C 48.30, H 3.77, N 8.87.
H19CuN
3
OS) , found 400.0536 (C19 OS) .
H
19CuN
3
H
19CuCl N OS C 48.36,
2 3
a
Preparation of the coordination compound 5-(Z)-3-(4-
methylthiophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 49 with copper (II)
+
+
834 54 Cu
(C36
2
15%. HRMS (pos.) calculated
771.1121
) , UV-vis
+
+
chloride dihydrate (49k). 49k was obtained as
a
black
H
32
O
6
N
6
CuS
2
) , found 771.1121 (C36
H
32
O
6
N
6
CuS
2
-
1
-1
crystalline precipitate from 15 mg (44 μmol) of 5-(Z)-3-(4-
methylthiophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 49 and 7.4 mg (44 μmol) of
(λ, nm/ε, l mol cm ): 395/4233. Elemental analysis: calculated
H32Cu Cl N O S C 44.22, H 3.30, N 8.60, found C
2 4 6 6 2
44.19, H 3.26, N 8.59.
Preparation of the coordination compound (4Z,4'Z)-2,2'-
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(4-t-butylphenyl)-2,3-dihydro-4H-
imidazol-5-one) 55 with copper (II) chloride dihydrate
(55k). 55k was obtained as a black crystalline precipitate from
(%) for C36
copper (II) chloride dihydrate. Yield: 9 mg (43%). HRMS
+
calculated 403.9953 (C17
H15CuN
3
OS
2
) , found 403.9946
+
(
C
17
H
15CuN
15CuCl
N 8.86.
3
OS
2
) . Elemental analysis: calculated (%) for
N OS C 42.90, H 3.18, N 8.83, found C 42.93, H 3.20,
2 3 2
C
17
H
Preparation of the coordination compound 5-(Z)-3-(2,5-
difluorophenyl)-2-(methylthio)-5-(pyridin-2-
ylmethylidene)-1H-imidazol-4-one 50 with copper (II)
15
mg
(22
μmol)
of
(4Z,
4'Z)-2,2'-(ethane-1,2-
diyldisulfandiyl)bis(4-(2-pyridylmethylidene)-1-(4-
tretbutylphenyl)-2,3-dihydro-4H-imidazole-5-one) 55 as a
result of reaction with 7.5 mg (44 μmol) of copper (II) chloride
dihydrate. Yield: 6.1 mg (33%). MALDI: m/z = 763 55Cu 100%,
chloride dihydrate (50k). 50k was obtained as
a
black
+
crystalline precipitate from 15 mg (45 μmol) of 5-(Z)-3-(2,5-
difluorophenyl)-2-(methylthio)-5-(pyridin-2-ylmethylidene)-
+
825 55Cu
(C40
2
20%. HRMS (pos.) calculated
763.1950
+
+
1
H-imidazol-4-one 50 and 7.6 mg (45 μmol) of copper (II)
chloride dihydrate. Yield: mg (24%). HRMS calculated
93.9882 found 393.9892
OS)+,
H
40
O
2
N
6
CuS
2
) , found 763.1948 (C40
H
40
O
2
N
6
CuS
2
) , UV-vis
1
-1
5
(λ, nm/ε, l
mol- cm ): 375/46311. Elemental analysis:
3
2 3
(C16H11CuF N
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Page 29 of 34
Journal of Medicinal Chemistry
1
2
calculated (%) for C40
H40Cu
2
Cl
4
N
6
O
2
S
2
C 49.54, H 4.16, N 8.67,
Preparation of the coordination compound (4Z,4'Z)-2,2'-
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
found C 49.59, H 4.20, N 8.72.
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
Preparation of the coordination compound (4Z,4'Z)-2,2'-
ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(3-bromophenyl)-2,3-dihydro-4H-
imidazol-5-one) 61 with copper (II) chloride dihydrate
(61k). 61k was obtained as a black crystalline precipitate from
15 mg (21 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-diyl
disulfandiyl)bis(4-(2-pyridylmethylidene)-1-(3-bromophenyl)-
2,3-dihydro-4H-imidazole-5-one) 61 as a result of reaction with
7.0 mg (42 μmol) of copper (II) chloride dihydrate. Yield: 5.7 mg
(
pyridylmethylidene)-1-(2,4,6-trimethylphenyl)-2,3-
dihydro-4H-imidazol-5-one) 56 with copper (II) chloride
dihydrate (56k). 56k was obtained as a black crystalline
precipitate from 15 mg (22 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-
diyldisulfandiyl)bis(4-(2-pyridylmethylidene)-1-(2,4,6-
+
+
trimethylphenyl)-2,3-dihydro-4H-imidazole-5-one) 56 as
result of reaction with 7.5 mg (44 μmol) of copper (II) chloride
a
(30%). MALDI: m/z = 806 61Cu 100%, 869 61Cu
2
5%. HRMS
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
(pos.) calculated 858.9433 (C32
H
22
O
2
N
6
CuBr
2
S
2
+ CH
3
OH + H
O) , UV-vis (λ,
2
O) ,
+
dihydrate. Yield: 10 mg (49%) HRMS (pos.) calculated 718.9919
found 858.9431 (C32 CuBr
H
22
O
2
N
6
2
S
2
+ CH OH + H
3
2
+
+
-1
-1
(
C
32
H
22Cl
2
CuN
Elemental analysis: calculated (%) for C32
48.46, H 3.85, N 8.92, found C 48.52, H 4.10, N 8.69.
6
O
2
S
2
) , found 718.9883 (C32
H
22Cu
22Cl
2
2
CuN
Cl
6
O
2
S
2
2
nm/ε, l mol cm ): 395/28600. Elemental analysis: calculated
(%) for C32 Cl Br C 39.22, H 2.26, N 8.58, found C
39.25, H 2.26, N 8.60.
H
4
N
6
O
2
S
C
H22Cu
2
3
2 6 2 2
N O S
Preparation of the coordination compound (4Z,4'Z)-2,2'-
ethane-1,2-diyldisulfanediyl)bis(4-(2-
Preparation of the coordination compound (4Z,4'Z)-2,2'-
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
(
pyridylmethylidene)-1-(2-t-butylphenyl)-2,3-dihydro-4H-
imidazol-5-one) 57 with copper (II) chloride dihydrate
pyridylmethylidene)-1-(2-bromophenyl)-2,3-dihydro-4H-
imidazol-5-one) 62 with copper (II) chloride dihydrate
(62k). 62k was obtained as a black crystalline precipitate from
15 mg (21 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-diyl
disulfandiyl)bis(4-(2-pyridylmethylidene)-1-(2-bromophenyl)-
2,3-dihydro-4H-imidazole-5-one) 62 as a result of reaction with
7.0 mg (42 μmol) of copper (II) chloride dihydrate. Yield: 6.1 mg
(
57k). 57k was obtained as a black crystalline precipitate from
15 mg (22 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-
diyldisulfandiyl)bis(4-(2-pyridylmethylidene)-1-(4-
tretbutylphenyl)-2,3-dihydro-4H-imidazole-5-one) 57 as
result of reaction with 7.5 mg (44 μmol) of copper (II) chloride
dihydrate. Yield: 7 mg (35%) HRMS (pos.) calculated 763.2023
a
+
+
(33%). MALDI: m/z = 806 62Cu 100%, 870 62Cu
2
5%. HRMS
+
+
+
(
C
40
H
41CuN
6
O
l
2
S
2
) , found 763.1978 (C40
H
41CuN
6
O
2
S
2
) . UV-vis
mol- cm ): 367/43300. Elemental analysis:
calculated (%) for C42 Cl (57k + 2CH OH) C 48.79,
H 4.68, N 8.13, found C 48.57, H 4.67, N 8.24.
(pos.) calculated 858.9433 (C32
H
22
O
2
N
6
CuBr O) ,
3
2
S
2
+ CH
3
OH + H
2
1
-1
+
(λ, nm/ε,
found 858.9435 (C32 CuBr
H
22
O
2
N
6
2
S
2
+ CH OH + H O) , UV-vis (λ,
2
-
1
-1
H48Cu
2
4
N
6
O
4
S
2
3
nm/ε, l mol cm ): 395/36083. Elemental analysis: calculated
(%) for C32 Cl Br C 39.22, H 2.26, N 8.58, found C
39.24, H 2.25, N 8.61.
H22Cu
2
3
2 6 2 2
N O S
Preparation of the coordination compound (4Z,4'Z)-2,2'-
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
Preparation of the coordination compound (4Z,4'Z)-2,2'-
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(4-fluorophenyl)-2,3-dihydro-4H-
imidazol-5-one) 58 with copper (II) chloride dihydrate
pyridylmethylidene)-1-(2-chlorophenyl)-2,3-dihydro-4H-
imidazol-5-one) 63 with copper (II) chloride dihydrate
(63k). 63k was obtained as a black crystalline precipitate from
15 mg (23.5 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-diyl
disulfandiyl)bis(4-(2-pyridylmethylidene)-1-(2-chlorophenyl)-
2,3-dihydro-4H-imidazole-5-one) 63 as a result of reaction with
8.0 mg (47 μmol) of copper (II) chloride dihydrate. Yield: 5.7 mg
(
1
58k). 58k was obtained as a black crystalline precipitate from
mg (25 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-diyl
disulfandiyl)bis(4-(2-pyridylmethylidene)-1-(4-fluorophenyl)-
,3-dihydro-4H-imidazole-5-one) 58 as a result of reaction with
8.5 mg (50 μmol) of copper (II) chloride dihydrate. Yield: 5.8 mg
5
2
+
+
(30%). MALDI: m/z = 687 58Cu 100%, 750 58Cu
pos.) calculated 739.1034 (C32 CuF + CH
found 739.1253 (C32 CuF + CH OH + H
2
10%. HRMS
+
+
+
2
(
H
22
O
2
N
6
2
S
2
3
OH + H
+
2
O) ,
(32%). MALDI: m/z = 717 63Cu 100%, 781 63Cu
(pos.) calculated 771.0443 (C32 CuCl + CH
found 771.0440 (C32 CuCl + CH OH + H
15%. HRMS
+
H
22
O
2
N
6
2
S
2
3
2
O) , UV-vis (λ,
H
22
O
2
N
6
2
S
2
3
OH + H
O) , UV-vis (λ,
2
O) ,
nm/ε, l mol- cm ): 470/37109. Elemental analysis: calculated
%) for C32 Cl C 44.79, H 2.58, N 9.79, found C
44.85, H 2.60, N 9.82.
1
-1
H
22
O
N
S
2
+
2
6
2
3
2
-1
-1
(
H22Cu
2
3
F
2
S
2
N
6
O
2
S
2
nm/ε, l mol cm ): 370/10874. Elemental analysis: calculated
(%) for C32 Cl C 43.13, H 2.49, N 9.43, found C
43.17, H 2.51, N 9.41.
H22Cu
2
5 6 2 2
N O S
Preparation of the coordination compound (4Z,4'Z)-2,2'-
ethane-1,2-diyldisulfanediyl)bis(4-(2-
(
Preparation of the coordination compound (4Z,4'Z)-2,2'-
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
pyridylmethylidene)-1-(2-methoxy-4-chloro-5-
methylphenyl)-2,3-dihydro-4H-imidazol-5-one) 64 with
copper (II) chloride dihydrate (64k). 64k was obtained as a
black crystalline precipitate from 15 mg (23.5 μmol) of (4Z, 4'Z)-
pyridylmethylidene)-1-(3-fluorophenyl)-2,3-dihydro-4H-
imidazol-5-one) 59 with copper (II) chloride dihydrate
(
59k). 59k was obtained as a black crystalline precipitate from
15 mg (25 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-diyl
disulfandiyl)bis(4-(2-pyridylmethylidene)-1-(3-fluorophenyl)-
2
8
,3-dihydro-4H-imidazole-5-one) 59 as a result of reaction with
.5 mg (50 μmol) of copper (II) chloride dihydrate. Yield: 5.9 mg
2,2'-(ethane-1,2-diyl
disulfandiyl)bis(4-(2-
pyridylmethylidene)-1-(2-methoxy-4-chloro-5-methylphenyl)-
2,3-dihydro-4H-imidazole-5-one) 64 as a result of reaction with
8.0 mg (47 μmol) of copper (II) chloride dihydrate. Yield: (after
+
+
(
(
32%). MALDI: m/z = 686 59Cu 100%, 749 59Cu
pos.) calculated 739.1034 (C32 CuF + CH
CuF + CH OH + H
2
13%. HRMS
+
H
22
O
2
N
6
2
S
2
3
OH + H
+
2
O) ,
found 739.1233 (C32
H
22
O
2
N
6
2
S
2
3
2
O) , UV-vis (λ,
purification by column chromatography CH
2
Cl
calculated
CuN
2
:MeOH 40:1) 6.1
nm/ε, l mol- cm ): 395/24800. Elemental analysis: calculated
%) for C32 22CuCl C 44.79, H 2.58, N 9.79, found C
4.81, H 2.63, N 9.81.
Preparation of the coordination compound (4Z,4'Z)-2,2'-
ethane-1,2-diyldisulfanediyl)bis(4-(2-
1
-1
mg
(C36
(31%).
HRMS
(pos.)
808.0516
+
+
(
4
H
3
F
2
N
6
O
2
S
2
H
31Cl
2
CuN
6
O
4
S) , found 808.0518 (C36
H
31Cl
2
6
O
4
S) . UV-
-
1
-1
vis (λ, nm/ε, l mol cm ): 367/45000. Elemental analysis:
calculated (%) for C43 Cl (64k + 2Et O + CH OH +
CH CN): C 44.45, H 4.08, N 8.44, found C 44.14, H 4.02, N 8.33.
H47Cu
2
6
N
7
O
6
S
2
2
3
(
3
pyridylmethylidene)-1-(2-fluorophenyl)-2,3-dihydro-4H-
imidazol-5-one) 60 with copper (II) chloride dihydrate
Preparation of the coordination compound (4Z,4'Z)-2,2'-
(ethane-1,2-diyldisulfanediyl)bis(4-(2-
(
1
60k). 60k was obtained as a black crystalline precipitate from
mg (25 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-diyl
disulfandiyl)bis(4-(2-pyridylmethylidene)-1-(2-fluorophenyl)-
,3-dihydro-4H-imidazole-5-one) 60 as a result of reaction with
8.5 mg (50 μmol) of copper (II) chloride dihydrate. Yield: 6.1 mg
pyridylmethylidene)-1-(2-bromo-4-fluorophenyl)-2,3-
dihydro-4H-imidazol-5-one) 65 with copper (II) chloride
dihydrate (65k). 65k was obtained as a black crystalline
precipitate from 15 mg (21 μmol) of (4Z, 4'Z)-2,2'-(ethane-1,2-
diyl disulfandiyl)bis(4-(2-pyridylmethylidene)-1-(2-bromo-4-
fluorophenyl)-2,3-dihydro-4H-imidazole-5-one) 65 as a result
of reaction with 7.0 mg (42 μmol) of copper (II) chloride
5
2
+
+
(
33%). MALDI: m/z = 687 60Cu 100%, 750 60Cu
pos.) calculated 739.1034 (C32 CuF + CH
CuF + CH OH + H
2
16%. HRMS
+
(
H
22
O
2
N
6
2
S
2
3
OH + H
2
O) ,
+
found 739.1053 (C32
H
22
O
2
N
6
2
S
2
3
2
O) , UV-vis (λ,
dihydrate. Yield: 8.2 mg (32%) HRMS (pos.) calculated 845.8817
nm/ε, l mol- cm ): 364/36150. Elemental analysis: calculated
%) for C32 Cl C 44.79, H 2.58, N 9.79, found C
4.85, H 2.67, N 9.83.
1
-1
(C32
(C32
H
H
21Br
21Br
CuF
CuF
N
N
O
O
S
S
) ,
+
found
845.8860
2
2
6
2
2
+
mol-1 cm ):
-1
(
4
H22Cu
2
3
F
2
N
6
O
2
S
2
2
2
6
2
2
) . UV-vis (λ, nm/ε,
l
367/44700. Elemental analysis: calculated (%) for
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