Synthesis and biological testing of (5Z)-2-aryl-5-arylmethylidene-3,5-dihydro-4H-imidazol-4-ones as antimitotic agents

Article abstract of DOI:10.1007/s00044-016-1566-2

Compounds interacting with cell protein tubulin and microtubules represent an important type of antimitotic agents. A series of tubulin-targeted 2-aryl-4-benzoyl-imidazoles were reported to possess high cytotoxicity, and so, we prepared a series of structural isomers of these to be evaluated as antimitotic agents. The synthesis of the novel (Z)-2-aryl-5-arylmethylidene-3,5-dihydro-4H-imidazol-4-ones involved coupling of substituted hippuric acids with aromatic aldehydes. Subsequent conversion of the resulting oxazolones to the corresponding imidazolones was carried out under microwave irradiation in the presence of urea and ammonium acetate. The cytotoxicity of the majority of the compounds to human epithelial carcinoma cancer cell line A549 was in the sub-micromolar range and was found to be more sensitive to the substituents on the 5-arylmethylidene fragment than on the 2-aryl ring in general. The cytotoxicities of the synthesized compounds were lower than those of the previously reported isomeric 2-aryl-4-benzoyl-imidazoles, and the basic structure–activity relationships in the isomeric pairs were different. Synthesized (5Z)-5-[(4-bromophenyl)methylidene]-2-(4-methylphenyl)-3,5-dihydro-4H-imidazol-4-one, which had the highest cytotoxicity (IC₅₀?~?440?nM) in the series of novel compounds, had a definite cytostatic effect on the A549 cells, but its antiproliferative properties were not linked to action on the microtubules. This would be an interesting lead compound for additional investigation into the mechanism of cytostatic action, and further structural optimization.

Full text of DOI:10.1007/s00044-016-1566-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1566-2
ORIGINAL RESEARCH
Synthesis and biological testing of (5Z)-2-aryl-5-arylmethylidene-
3,5-dihydro-4H-imidazol-4-ones as antimitotic agents
Anastasia A. Beloglazkina1 Birgit Wobith2 Elena S. Barskaia1 Nikolay A. Zefirov1 Alexander G. Majouga1
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•
•
•
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Elena K. Beloglazkina1 Nikolay V. Zyk1 Sergei A. Kuznetsov2 Olga N. Zefirova1,3
•
•
•
Received: 7 January 2016 / Accepted: 29 March 2016
Ó Springer Science+Business Media New York 2016
Abstract Compounds interacting with cell protein tubu-
lin and microtubules represent an important type of
antimitotic agents. A series of tubulin-targeted 2-aryl-4-
benzoyl-imidazoles were reported to possess high cyto-
toxicity, and so, we prepared a series of structural isomers
of these to be evaluated as antimitotic agents. The synthesis
of the novel (Z)-2-aryl-5-arylmethylidene-3,5-dihydro-4H-
imidazol-4-ones involved coupling of substituted hippuric
acids with aromatic aldehydes. Subsequent conversion of
the resulting oxazolones to the corresponding imidazolones
was carried out under microwave irradiation in the pres-
ence of urea and ammonium acetate. The cytotoxicity of
the majority of the compounds to human epithelial carci-
noma cancer cell line A549 was in the sub-micromolar
range and was found to be more sensitive to the sub-
stituents on the 5-arylmethylidene fragment than on the
2-aryl ring in general. The cytotoxicities of the synthesized
compounds were lower than those of the previously
reported isomeric 2-aryl-4-benzoyl-imidazoles, and the
basic structure–activity relationships in the isomeric pairs
were different. Synthesized (5Z)-5-[(4-bromophenyl)
methylidene]-2-(4-methylphenyl)-3,5-dihydro-4H-imidazol-
4-one, which had the highest cytotoxicity (IC50 * 440 nM)
in the series of novel compounds, had a definite cytostatic
effect on the A549 cells, but its antiproliferative properties
were not linked to action on the microtubules. This would be
an interesting lead compound for additional investigation
into the mechanism of cytostatic action, and further struc-
tural optimization.
Keywords Dihydroimidazolones Á Cytotoxicity Á
Microtubule network Á Human lung carcinoma A549
Introduction
The antimitotic activity of the natural compound colchicine
is induced by binding to the cell protein tubulin, causing
cessation of protein polymerization to microtubules, which
play a key role in cell division (Pellegrini and Budman,
2005; Stanton et al., 2011). There are a lot of structurally
diverse ligands known which bind in the colchicine domain
in tubulin (Chen et al., 2009, 2010a, b; Lu et al., 2012),
including the natural product combretastatin A (CA-4,
Fig. 1). Because of its simple structure, high cytotoxicity
and tumor vasculature-disrupting ability, CA-4 became a
lead compound in the design of numerous series of ana-
logues as putative anticancer agents (Gill et al., 2014;
Hsieh et al., 2005; Ji et al., 2015; Nam, 2003; Singh and
Kaur, 2009).
& Elena K. Beloglazkina
bel@org.chem.msu.ru
Olga N. Zefirova
olgaz@med.chem.msu.ru
The vast majority of CA-4 analogues were obtained by
linker modifications with different heterocycles (or other
fragments), along with variations of the substituents on the
phenyl rings (see Beale et al., 2012; O’Boyle et al., 2013;
Gill et al., 2014; Hsieh et al., 2005; Ji et al., 2015; Lee
et al., 2011; Liu et al., 2012; Maya et al., 2005; Nam, 2003;
Romagnoli et al., 2012, 2013; Singh and Kaur, 2009;
Tsyganov et al., 2013; Zheng et al., 2014 for recent
1
M.V. Lomonosov Moscow State University, Moscow,
Russian Federation 119992
2
Institute of Biological Sciences, University of Rostock,
18059 Rostock, Germany
3
Institute of Physiologically Active Compounds,
Chernogolovka, Russian Federation 142432
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Med Chem Res
OCH3
d obtained from aromatic acyl halides and glycine) were
coupled with aromatic aldehydes using potassium acetate
as a base. Addition of a small amount of acetic anhydride
helped to increase the yield of the target Z-isomer. The
vinyl proton in the product is observed in the 7.1–7.4 ppm
OCH3
CH3O
OCH3
CH3O
OCH3
CH3O
CH3O
HO
O
1
range in the H NMR (Chuang et al., 2011).
b
CH3CONH
a
Oxazolones 2a–n were converted to the corresponding
imidazolones 3a–n under microwave irradiation in the
presence of urea and ammonium acetate in ethylene glycol.
The large variation in the reaction yields (6–92 %; see
experimental section) may be due to the differences in
solubility of the target compounds. The higher yields of the
products isolated in oily form in comparison with the ones
for the solid imidazolones might be explained by a sig-
nificant loss of material during recrystallization. The imi-
dazolone proton appears at *10 ppm in the 1H NMR
spectrum of most of the obtained imidazolones, and cor-
responds to the amide proton [NH–C(O)]. However, in the
case of compounds 3b, 3c, 3f and 3g, the enol-imine tau-
tomer prevails, and the corresponding proton [N=COH)] is
observed at *12 ppm.
Fig. 1 Structures of colchicine (a) and combretastatin A (b)
examples). The activity of the resulting compounds was
found to be highly sensitive to the distance between the
rings and their relative spatial orientation. Recently, Chen
et al. (2010a, b) synthesized a series of 2-aryl-4-benzoyl-
imidazoles with high cytotoxicity to different types of
cancer cells (Fig. 2). A radio-ligand method was used to
assess the ability of these compounds to compete with
colchicine for binding with tubulin.
In the present work, we design and prepare a series of
structural isomers of 2-aryl-4-benzoyl-imidazoles, namely
2-aryl-5-arylmethylidene-3,5-dihydro-4H-imidazol-4-ones
(Fig. 2). The rationale for the design was based on the
comparison of the most stable conformations of the iso-
meric molecules, calculated by molecular dynamic meth-
ods using an open-source molecular builder and
visualization tool Avogadro (Hanwell et al., 2012, Fig. 3).
As shown in Fig. 3, the basic templates of the 2-aryl-4-
benzoyl-imidazoles show good overlap of the aromatic
rings in the (Z)-configuration of the designed compounds.
Thus, we synthesized a series of (5Z)-2-aryl-5-aryl
methylidene-3,5-dihydro-4H-imidazol-4-ones to assess if
these isomers maintain a high level of cytotoxicity.
Cytotoxicity
(5Z)-2-Aryl-5-arylmethylidene-3,5-dihydro-4H-imidazol-
4-ones 3a–n were tested to determine their cytotoxicity
against the growth of A549 human lung carcinoma cancer
cells in a standard colorimetric MTT assay (Mosmann,
1983). The results are presented in Table 1.
As shown in the table, most of the synthesized imida-
zolones display noticeable cytotoxicity (in the sub-micro-
molar range). Comparison of the cytotoxicity values of
isomeric pairs 2-aryl-4-benzoyl-imidazoles I–V (Fig. 2,
Chen et al., 2010a, b) and 2-aryl-5-arylmethylidene-3,5-
dihydro-4H-imidazol-4-ones 3a, 3d, 3e, 3j and 3n indicates
that the latter were less active. Activity was lower by about
one order of magnitude for pairs I/3j and V/3n, but dra-
matically lower for pairs II/3d, III/3e and IV/3a. Although
introducing p-methoxy, p-methyl or p-fluorine groups to
the A ring distinctly increased the cytotoxicity in the
Results and discussion
Chemistry
The target imidazolones, with different substituents on the
A and B rings, were synthesized in three steps according to
Scheme 1. Substituted hippuric acids (N-acylglycines 1a–
R'
R'
B
B
R
R'
IC50 (nM)
I
4-F
3,4,5-(OCH3)3 73-320
A
R
A
R
II 4-CH3 3,4,5-(OCH3)3 9-46
III 4-CH3 4-F
IV 4-OCH3 3,4,5-(OCH3)3 31-108
V H
N
N
O
45-222
N
H
N
H
4-F
570-2100
O
a
b
Fig. 2 Structures of 2-aryl-4-benzoyl-imidazoles obtained (Chen et al., 2010a, b) (a; some cytotoxicity data are marked in blue) and (5Z)-2-aryl-
5-arylmethylidene-3,5-dihydro-4H-imidazol-4-ones synthesized in this work (b)
123

Med Chem Res
Fig. 3 Most
stable conformations of the
basic structural templates of
2-aryl-4-benzoyl-imidazoles
(a) and (5Z)-2-aryl-5-
arylmethylidene-3,5-dihydro-
4H-imidazol-4-ones (b)
Scheme 1 Synthesis of target
(5Z)-2-aryl-5-arylmethylidene-
3,5-dihydro-4H-imidazol-4-
ones. Reagents and conditions.
(i) 1. NaOH, H2O, 1–2 h, rt. 2.
HCl, 2 h, rt. (ii) Aromatic
aldehyde, Ac2O, CH3COOK,
Ar, 100 °C, 2–4 h; (iii)
O
O
N
H
COOH
Cl
i
ii
+
H2N
COOH
R
R
1a R=OCH3 1c R=F
1b R=CH3
1d R=H
H2NC(O)NH2, CH3COONH4,
160–165 °C, then MW
(300–350 W), 10 min
R'
R'
N
N
iii
R
R
N
H
O
O
O
3a R=OCH3, R'=3,4,5-(OCH3)3
3b R=OCH3, R'=4-OCH3
3c R=OCH3, R'=4-OH
3d R=CH3, R'=3,4,5-(OCH3)3
3e R=CH3, R'=4-F
2a R=OCH3, R'=3,4,5-(OCH3)3
2b R=OCH3, R'=4-OCH3
2c R=OCH3, R'=4-OH
2d R=CH3, R'=3,4,5-(OCH3)3
2e R=CH3, R'=4-F
3f R=CH3, R'=4-Cl
2f R=CH3, R'=4-Cl
3g R=CH3, R'=4-Br
3h R=CH3, R'=4-OH
3i R=CH3, R'=H
3j R=F, R'=3,4,5-(OCH3)3
3k R=F, R'=4-F
3l R=F, R'=2,3,4-(OCH3)3
3m R=F, R'=2,4,5-(OCH3)3
3n R=H, R'=4-F
2g R=CH3, R'=4-Br
2h R=CH3, R'=4-OH
2i R=CH3, R'=H
2j R=F, R'=3,4,5-(OCH3)3
2k R=F, R'=4-F
2l R=F, R'=2,3,4-(OCH3)3
2m R=F, R'=2,4,5-(OCH3)3
2n R=H, R'=4-F
2-aryl-4-benzoyl-imidazole series (Fig. 2), there was no
such effect in this new imidazolone series (Table 1).
Moreover, replacement of 3,4,5-trimethoxy-substitution
with para-fluoro substitution in the B ring of imidazolones
3d and 3f led to loss of activity, so it was not bioisosteric as
had been observed in the isomeric pair II and III. This data
indicates that in spite of the structural similarity of these
isomers, their action in the tumor cells is quite different.
Among the obtained 2-aryl-5-arylmethylidene-3,5-
dihydro-4H-imidazol-4-ones, a curious structure–activity
relationship was observed in series 3d–3i with a para-
methyl substituted A ring. The introduction of fluoro and
hydroxy groups to the para-position of the B ring abolished
cytotoxicity (compare 3i and 3e, 3h), while the introduc-
tion of a bromine substituent (3g) increased cytotoxicity by
approximately one order of magnitude. The bulkiness of
bromine, in combination with its ability to form both
hydrogen and halogen bonds, may play an important role in
binding to the molecular target. As compound 3g was the
most cytotoxic of the synthesized imidazolones
(IC50 * 440 nM in A459 cells), we tested it in a series of
additional experiments.
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Med Chem Res
Table 1 Cytotoxicity of 2,5-disubstituted imidazolone derivatives against human cell lung carcinoma A549
a
The average of three to six experiments
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Med Chem Res
Cell growth and microtubule dynamics
their isomeric 2-aryl-4-benzoyl-imidazoles, but para-
bromo compound b40 was the most cytotoxic compound
(IC50 * 440 nM) in the new series. It had a cytostatic
effect on A549 cells, but that its antiproliferative properties
were not connected with action on the microtubules.
The reported data may be useful for further refinement
of the pharmacophore model of ligands binding to the
colchicine domain in tubulin, and (5Z)-5-[(4-bro-
mophenyl)methylidene]-2-(4-methylphenyl)-3,5-dihydro-
4H-imidazol-4-one (3g) represents an interesting lead
compound for additional investigation into the mecha-
nism of its cytostatic action and further structural
optimization.
First, we confirmed the MTT data by studying the effect of
3g on cell growth using direct cell counting by microscopy
over 48 h of culturing. As expected, compound 3g strongly
inhibited the cell growth (Fig. 4a).
The ability of 3g to alter the microtubule dynamics in
cells was studied using immunofluorescence microscopy as
described by Zefirova et al. (2011, 2013). However,
treating cells with 3g at concentration as high as 10 lM
over 48 h had no effect on microtubule arrays (Fig. 4b).
Moreover, after 36 h of incubation, several cells were
observed at different mitosis stages. This correlates with
the cell growth data, showing some growth recovery after
24 h of incubation (Fig. 4a). Additional microscopy anal-
ysis also revealed that cells treated with 3g possessed
normal adhesion and morphology. The absence of nucleus
fragmentation and chromatin condensation indicates that
cells had not undergone apoptosis after treatment with 3g
for 48 h. These results demonstrate that 3g has a real
cytostatic effect on the A549 cells, but its antiproliferative
properties are not linked to action on the microtubules.
Thus, a series of 2-aryl-5-arylmethylidene-3,5-dihydro-
4H-imidazol-4-ones, which are structural isomers of highly
cytotoxic tubulin-targeting 2-aryl-4-benzoyl-imidazoles
previously described in the literature, was synthesized via a
three-step synthetic procedure, and were evaluated for
cytotoxicity to human epithelial carcinoma cancer cell line
A549. The cytotoxicity of the majority of synthesized
compounds was in the sub-micromolar range, and was
found to be more sensitive to the substituents on the B ring
than on the A ring in general.
Experimental
Chemistry
General information
Solvents for extraction and chromatography were technical
grade and distilled under standard drying agents. Reaction
control was carried out by thin-layer chromatography on
1
‘‘Silufol-UV254’’ plates. H NMR and 13C NMR spectra
were recorded on Bruker Avance spectrometer (at
400 MHz and 100 MHz correspondingly) and are refer-
enced to residual signal of the solvent. Chemical shifts are
given in ppm (d); multiplicities are indicated by s (singlet),
d (doublet), dd (double doublet), t (triplet), m (multiplet).
Infrared spectra (IR) were registered on UR-20 appara-
tus (in Nujol) or on Thermo Nicolet IR200 spectrometer (in
KBr plates) and reported in cm-1. Elemental analysis of
the synthesized compounds was performed on CNH
The cytotoxicities of the (Z)-2-aryl-5-arylmethylidene-
3,5-dihydro-4H-imidazol-4-ones were lower than those of
Fig. 4 Cell growth analysis in the absence (Control) and in the presence of 10 lM 3g (a). B. Microtubule networks in A549 cells treated for
48 h with 10 lM 3g; bar 10 lm (b)
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1
analyzer Vario Micro Cube (Elementar). Melting points
were measured in block with open capillaries and are
uncorrected. The syntheses of oxazolones 2b, 2f, 2h, 2k,
2n were described previously.
NMR H (CDCl3): 8.21 (d, J = 7.8 Hz, 2H, Ar), 8.11
(d, J = 8.1 Hz, 2H, Ar), 7.19 (d, J = 8.1 Hz, 2H, Ar), 7.13
(s, 1H, HC=), 7.01 (d, J = 7.8 Hz, 2H, Ar), 3.89 (s, 3H,
CH3), 3.71 (s, 3H, CH3).
High resolution mass spectra were obtained on the
Bruker microTOF II apparatus by electrospray ionization
(ESI). The operations were made in positive ion mode
(capillary voltage—4500 V). Scanned mass range—m/z
50–3000 D, internal or external standard (Electrospray
Calibration Solution, Fluka), nitrogen gas vaporizer (4 l/
min), interface temperature 180 °C. Samples were injected
as the solutions in acetonitrile, the flow rate—3 mkl/min.
(4Z)-2-(4-Methoxyphenyl)-4-[(4-Hydroxyphenyl)methyli-
dene]-1,3-oxazol-5(4H)-one (2a) was synthesized from
0.23 g (1.9 mmol) of 4-hydroxybenzaldehyde, 0.40 g
(1.9 mmol) of 4-methoxy-hippuric acid, 0.19 g (1.9 mmol)
of potassium acetate. Yield 56 % (0.315 g), yellow crys-
tals, mp 191–192 °C.
1
NMR H (CDCl3): 8.17 (d, J = 7.8 Hz, 2H, Ar), 8.11
(d, J = 7.8 Hz, 2H, Ar), 7.14 (s, 1H, HC=), 6.97–7.01 (m,
4H, Ar), 3.89 (s, 3H, CH3).
Synthetic procedures
C17H13NO4 requires (%): C, 69.15; H, 4.46; N 4.75.
Found: C, 69.31; H, 4.50; N, 4.68.
4-Methoxy-hippuric acid (1a) and 4-methyl-hippuric acid
(1b) were synthesized from 4-methoxybenzoic acid as
described in Wang et al. (2011).
(4Z)-2-(4-Methylphenyl)-4-[(3,4,5-trimethoxyphenyl)
methylidene]-1,3-oxazol-5(4H)-one (2d) was synthe-
sized from 0.3 ml (1.5 mmol) of 3,4,5-trimethoxyben-
zaldehyde, 0.45 g (1.5 mmol) of 4-methyl-hippuric acid,
0.15 g (1.5 mmol) of potassium acetate. Yield 44 %
(0.235 g), yellow crystals, mp 171–173 °C.
NMR 1H (CDCl3): 8.04 (d, J = 7.7 Hz, 2H, Ar), 7.57 (s,
2H), 7.35 (d, J = 7.7 Hz, 2H, Ar), 7.17 (s, 1H, HC=), 4.00
(s, 6H, CH3), 3.97 (s, 3H, CH3), 2.49 (s, 1H, CH3).
C20H19NO5 requires (%): C, 67.99; H, 5.43; N 3.97.
Found: C, 68.10; H, 5.63; N, 4.00.
4-Fluoro-hippuric acid (1c) was synthesized from the
4-fluorobenzoyl chloride as described in El-Araby et al.
(2012).
Hippuric acid (1d) was synthesized from benzoyl chlo-
ride as described in Mokale et al. (2012).
General protocol for preparation of (4Z)-1,3-oxazol-
5(4H)-ones Aromatic aldehyde (1 equiv.) was added to
the solution of hippuric acid (1 equiv.) and potassium
acetate (1 equiv.) in acetic anhydride under stirring. The
mixture was refluxed under argon for 2–4 h at 100 °C and
then cooled for the product precipitation. The precipitate
was filtered, washed by distilled water and ether and dried
on air.
(4Z)-2-(4-Methylphenyl)-4-[(4-fluorophenyl)methylidene]-
1,3-oxazol-5(4H)-one (2e) was synthesized from 0.35 ml
(2.4 mmol) of 4-fluorobenzaldehyde, 0.45 g (2.4 mmol) of
4-methyl-hippuric acid, 0.24 g (2.4 mmol) of potassium
acetate. Yield 72 % (0.48 g), yellow crystals, mp 240–245 °C.
1
NMR H (CDCl3): 8.25 (dd, J1 = 7.2 Hz, J2 = 8.1 Hz,
(4Z)-2-(4-Methoxyphenyl)-4-[(3,4,5-trimethoxyphenyl)
methylidene]-1,3-oxazol-5(4H)-one (2a) was synthe-
sized from 0.37 g (1.9 mmol) of 3,4,5-trimethoxyben-
zaldehyde, 0.40 g (1.9 mmol) of 4-methoxy-hippuric acid,
0.19 g (1.9 mmol) of potassium acetate. Yield 52 %
(0.365 g), yellow crystals, mp 166–167 °C.
2H, Ar), 8.10 (d, J = 7.8 Hz, 2H, Ar), 7.37 (d, J = 7.8 Hz,
2H, Ar), 7.21 (s, 1H, HC=), 7.19 (dd, J1 = 7.9 Hz,
J2 = 8.1 Hz, 2H, Ar), 2.49 (s, 3H, CH3).
C17H12NO2F requires (%): C, 72.59; H, 4.27; N 4.98.
Found: C, 72.61; H, 4.45; N, 4.81.
NMR 1H (CDCl3): 8.10 (d, J = 8.0 Hz, 2H, Ar), 7.56 (s,
2H, Ar), 7.13 (s, 1H, HC=), 7.05 (d, J = 8.0 Hz, 2H, Ar),
4.00 (s, 6H, CH3), 3.97 (s, 3H, CH3), 3.93 (s, 3H, CH3).
C20H19NO6 requires (%): C, 65.04; H, 5.15; N 3.79.
Found: C, 64.75; H, 5.05; N, 3.77.
(4Z)-2-(4-Methylphenyl)-4-[(4-chlorophenyl)methylidene]-
1,3-oxazol-5(4H)-one (2f) was synthesized from 0.5 g
(2.4 mmol) of 4-chlorobenzaldehyde, 0.45 g (2.4 mmol)
of 4-methyl-hippuric acid, 0.24 g (2.4 mmol) of potas-
sium acetate. Yield 79 % (0.55 g), yellow crystals, spec-
tral data correspond to the described (Hoshina et al.,
2000).
(4Z)-2-(4-Methoxyphenyl)-4-[(4-methoxyphenyl)methyli-
dene]-1,3-oxazol-5(4H)-one (2b) was synthesized from
0.23 ml (1.9 mmol) of 4-methoxybenzaldehyde, 0.40 g
(1.9 mmol) of 4-methoxy-hippuric acid, 0.19 g (1.9 mmol)
of potassium acetate. Yield 55 % (0.32 g), yellow crystals,
spectral data correspond to the described (Mariappan et al.,
2011).
1
NMR H (CDCl3): 8.13 (d, J = 8.4 Hz, 2H, Ar), 8.05
(d, J = 8.2 Hz, 2H, Ar), 7.43 (d, J = 8.4 Hz, 2H, Ar), 7.32
(d, J = 8.0 Hz, 2H, Ar), 7.14 (s, 1H, HC=), 2.45 (s, 3H,
CH3).
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Med Chem Res
(4Z)-2-(4-Methylphenyl)-4-[(4-bromophenyl)methylidene]-
1,3-oxazol-5(4H)-one (2g) was synthesized from 0.44 g
(2.4 mmol) of 4-bromobenzaldehyde, 0.45 g (2.4 mmol) of
4-methyl-hippuric acid, 0.24 g (2.4 mmol) of potassium
acetate. Yield 99 % (0.81 g), yellow crystals, mp
190–195 °C.
(1.5 mmol) of 4-fluorobenzaldehyde, 0.3 g (1.5 mmol) of
4-fluoro-hippuric acid, 0.15 g (1.5 mmol) of potassium
acetate. Yield 68 % (0.29 g), yellow crystals, spectral data
correspond to the described (Blanco-Lomas et al., 2013).
1
NMR H (CDCl3): 8.21 (dd, J1 = 4.1 Hz, J2 = 8.1 Hz,
2H, Ar), 8.06 (d, J = 8.1 Hz, 2H, Ar), 7.33 (d, J = 8.1 Hz,
1
NMR H (CDCl3): 8.25 (d, J = 7.5 Hz, 2H, Ar), 8.02
2H, Ar), 7.13–7.18 (m, 3H, Ar and HC=).
(d, J = 7.5 Hz, 2H, Ar), 7.55 (d, J = 7.5 Hz, 2H, Ar), 7.45
(d, J = 7.5 Hz, 2H, Ar), 7.00 (s, 1H, HC=), 2.49 (s, 3H,
CH3).
NMR 13C (DMSO-d6): 167.3, 144.8, 135.1, 134.2,
133.8, 132.3, 131.9, 130.8, 130.5, 126.3, 124.7, 21.8.
C17H12NO2Br requires (%): C, 59.65; H, 3.51; N 4.09.
Found: C, 60.02; H, 3.74; N, 4.13.
(4Z)-2-(4-Fluorophenyl)-4-[(2,3,4-trimethoxyphenyl)
methylidene]-1,3-oxazol-5(4H)-one (2l) was synthe-
sized from 0.295 g (1.5 mmol) of 2,3,4-trimethoxyben-
zaldehyde, 0.3 g (1.5 mmol) of 4-fluoro-hippuric acid,
0.15 g (1.5 mmol) of potassium acetate. Yield 12 % (0.065
u), yellow crystals, mp 255–257 °C.
1
NMR H (CDCl3): 8.64 (dd, J1 = 3.6 Hz, J2 = 7.9 Hz,
(4Z)-2-(4-Methylphenyl)-4-[(4-hydroxyphenyl)methylidene]-
1,3-oxazol-5(4H)-one (2h) was synthesized from 0.29 g
(2.4 mmol) of 4-hydroxybenzaldehyde, 0.45 g (2.4 mmol)
of 4-methyl-hippuric acid, 0.24 g (2.4 mmol) of potassium
acetate. Yield 19 % (0.125 g), yellow crystals, mp
205–207 °C.
2H, Ar), 8.15 (dd, J = 7.7 Hz, J2 = 7.9 Hz, 2H, Ar), 8.01
(d, J = 7.7 Hz, 1H, Ar), 7.30 (d, J = 7.7 Hz, 1H, Ar), 7.24
(s, 1H, HC=), 3.98 (s, 3H, CH3), 3.94 (s, 3H, CH3), 3.87 (s,
3H, CH3).
HRMS (ESI, m/z) [M ? H]? calcd. for C19H16FNO5
358.1079, found 358.1085.
1
NMR H (CDCl3): 8.27 (d, J = 7.9 Hz, 2H, Ar), 8.09
(4Z)-2-(4-Fluorophenyl)-4-[(2,4,5-trimethoxyphenyl)
methylidene]-1,3-oxazol-5(4H)-one (2m) was syn-
thesized from 0.55 g (2.5 mmol) of 2,4,5-trimethoxyben-
zaldehyde, 0.5 g (2.5 mmol) of 4-fluoro-hippuric acid,
0.25 g (2.5 mmol) of potassium acetate. Yield 35 %
(0.3 g), yellow crystals, mp 259–260 °C.
(d, J = 7.5 Hz, 2H, Ar), 7.37 (d, J = 7.8 Hz, 2H, Ar), 7.28
(s, 1H, HC=), 7.37 (d, J = 7.5 Hz, 2H, Ar), 2.49 (s, 3H,
CH3).
C17H13NO3 requires (%): C, 73.12; H, 4.66; N 5.02.
Found: C, 72.79; H, 4.92; N, 4.87.
(4Z)-2-(4-Methylphenyl)-4-(phenylmethylidene)-1,3-oxazol-
5(4H)-one (2i) was synthesized from 0.3 ml (3 mmol) of
benzaldehyde, 0.75 g (3 mmol) of 4-methyl-hippuric acid,
0.3 g (3 mmol) of potassium acetate. Yield 94 % (0.75 g),
yellow crystals, spectral data correspond to the described
(Blanco-Lomas et al., 2013).
1
NMR H (CDCl3): 8.61 (s, 1H, Ar), 8.12–8.15 (m, 3H,
Ar), 7.85 (s, 1H, Ar), 7.21–7.28 (m, 3H, Ar and HC=), 6.52
(s, 1H, Ar), 4.04 (s, 3H, CH3), 4.01 (s, 3H, CH3), 3.95 (s,
3H, CH3).
IR (m, cm-1): 3407 (H2O), 1763 (C=O), 1641 (C=N).
C19H16NO5F 9 H2O requires (%): C, 60.80; H, 4.83; N
3.92. Found: C, 60.87; H, 4.44; N, 3.89.
1
NMR H (CDCl3): 8.19 (dd, J1 = 2 Hz, J2 = 6.1 Hz,
2H, Ar), 8.07 (d, J = 8.4 Hz, 2H, Ar), 7.79–7.43 (m, 3H,
Ar), 7.32 (d, J = 8.4 Hz, 2H, Ar), 7.21 (s, 1H, HC=), 2.45
(s, 3H, CH3).
(4Z)-2-Phenyl-4-[(4-fluorophenyl)methylidene]-1,3-oxazol-
5(4H)-one (2n) was synthesized from 0.37 ml
(2.5 mmol) of 4-fluorobenzaldehyde, 0.5 g (2.5 mmol) of
hippuric acid, 0.25 g (2.5 mmol) of potassium acetate.
Yield 30 % (0.2 g), yellow crystals, spectral data corre-
spond to the described in Jat et al. (2012).
(4Z)-2-(4-Fluorophenyl)-4-[(3,4,5-trimethoxyphenyl)
methylidene]-1,3-oxazol-5(4H)-one (2j) was synthe-
sized from 0.3 g (1.5 mmol) of 3,4,5-trimethoxybenzalde-
hyde, 0.3 g (1.5 mmol) of 4-fluoro-hippuric acid, 0.15 g
(1.5 mmol) of potassium acetate. Yield 44 % (0.22 g),
yellow crystals, mp 185–190 °C.
1
NMR H (CDCl3): 8.26 (dd, J1 = 6.1 Hz, J2 = 7.6 Hz,
2H, Ar), 8.21 (d, J = 8.9 Hz, 2H, Ar), 7.64 (t, J = 8.8 Hz,
1H, Ar), 7.57 (t, J = 8.8 Hz, 2H, Ar), 7.28 (s, 1H, HC=),
7.18–7.22 (m, 2H, Ar).
1
NMR H (CDCl3): 8.15–8.18 (m, 2H, Ar), 7.55 (s, 2H,
Ar), 7.24 (dd, J1 = 7.9 Hz, J2 = 8.1 Hz, 2H, Ar), 7.20 (s,
1H, HC=), 4.00 (s, 6H, CH3), 3.97 (s, 3H, CH3).
C19H16NO5F requires (%): C, 63.86; H, 4.51; N 3.92.
Found: C, 63.95; H, 4.68; N, 3.91.
General protocol for preparation of 2-aryl-5-arylmethyli-
dene-3,5-dihydro-4H-imidazol-4-ones A solution of (4Z)-
1,3-oxazol-5(4H)-ones (1 equiv.), urea (*1.5 equiv.),
ammonium acetate (*1.5 equiv.) in ethylene glycol
(1–5 ml) was heated at 160–165 °C under stirring till
(4Z)-2-(4-Fluorophenyl)-4-[(4-fluorophenyl)methylidene]-
1,3-oxazol-5(4H)-one (2k) was synthesized from 0.2 ml
123

Med Chem Res
(5Z)-2-(4-Methylphenyl)-5-[(3,4,5-trimethoxyphenyl)
homogenization of the mixture and then exposed to
microwave irradiation (300–350 W) for 10 min. The mix-
ture was cooled, diluted with water and the precipitated
solid or oily product was isolated, washed with water, then
with ethanol and crystallized from chloroform (in the case
of solid product) or dried in vacuo (in the case of oily
product).
methylidene]-3,5-dihydro-4H-imidazol-4-one
(3d) was
synthesized from 0.22 g (0.62 mmol) of 2d, 0.056 g
(1 mmol) of urea, 0.073 g (1 mmol) of ammonium acetate.
Yield 70 % (0.154 g), yellow oil.
NMR 1H (DMSO-d6): 9.98 (s, 1H, NH), 7.94 (d,
J = 8.0 Hz, 2H, Ar), 7.46 (s, 1H, HC=), 7.32 (d,
J = 8.0 Hz, 2H, Ar), 7.06 (s, 2H, Ar), 3.40 (s, 6H, CH3),
3.33 (s, 3H, CH3), 2.38 (s, 3H, CH3).
(5Z)-2-(4-Methoxyphenyl)-5-[(3,4,5-trimethoxyphenyl)
methylidene]-3,5-dihydro-4H-imidazol-4-one (3a) was
synthesized from 0.34 g (0.92 mmol) of 2a, 0.083 g
(1.4 mmol) of urea, 0.11 g (1.4 mmol) of ammonium
acetate. Yield 10 % (0.031 g), brown solid, mp
275–278 °C.
NMR 13C (DMSO-d6): 166.5, 165.7, 159.4, 132.5,
129.5, 128.2, 124.9, 116.0, 66.9, 53.3, 21.5.
HRMS (ESI, m/z) [M ? H]? calcd. for C20H21N2O4
353.1501, found 353.1487.
1
NMR H (DMSO-d6): 8.30 (s, 2H, NH), 7.75–7.80 (m,
(5Z)-2-(4-Methylphenyl)-5-[(4-fluorophenyl)methylidene]-
3,5-dihydro-4H-imidazol-4-one (3e) was synthesized
from 0.238 g (0.85 mmol) of 2e, 0.077 g (1.26 mmol) of
urea, 0.01 g (1.26 mmol) of ammonium acetate. Yield
39 % (0.093 g), brown oily liquid.
4H, Ar), 7.15 (d, J = 7.7 Hz, 2H, Ar), 6.91 (s, 1H, HC=),
3.87 (s, 6H, CH3), 3.74 (s, 3H, CH3).
NMR 13C (DMSO-d6): 173.2, 162.0, 161.5, 153.1,
142.6, 140.3, 137.4, 129.7, 128.0, 120.7, 113.41, 109.3,
61.0, 56.2, 55.3.
NMR 1H (DMSO-d6): 9.93 (s, 1H, NH), 7.85 (d,
J = 7.8 Hz, 2H, Ar), 7.69 (dd, J1 = 7.8 Hz, J2 = 7.7 Hz,
2H, Ar), 7.42 (s, 1H, HC=), 7.29 (d, J = 7.8 Hz, 2H, Ar),
7.16 (m, 2H, Ar), 2.46 (s, 3H, CH3).
HRMS (ESI, m/z) [M ? H]? calcd. for C20H21N2O5
369.1445, found 369.1443.
(5Z)-2-(4-Methoxyphenyl)-5-[(4-methoxyphenyl)methylidene]-
3,5-dihydro-4H-imidazol-4-one (3b) was synthesized from
0.309 g (1 mmol) of 2b, 0.09 g (1.5 mmol) of urea, 0,
1155 g (1.5 mmol) of ammonium acetate. Yield 6 %
(0.02 g), brown waxy solid.
NMR 13C (DMSO-d6): 171.6, 161.4, 161.0, 143.3,
136.4, 132.0, 131.1, 129.4, 128.5, 127.4, 125.6, 114.3,
21.0.
HRMS (ESI, m/z) [M ? H]? calcd. for C17H14N2OF
281.1090, found 281.1135.
NMR 1H (DMSO-d6): 11.92 (s, 1H, NH), 8.29 (d,
J = 9.2 Hz, 2H, Ar), 8.12 (d, J = 8.5 Hz, 2H, Ar), 7.14 (d,
J = 8 Hz, 2H, Ar), 7.05 (d, J = 11 Hz, 2H, Ar), 6.93 (s,
1H, HC=), 3.87 (s, 3H, OCH3), 3.84 (s, 3H, OCH3).
NMR 13C (DMSO-d6): 160.7, 153.3, 142.6, 135.8,
134.1, 129.5, 128.1, 63.2, 58.2.
(5Z)-2-(4-Methylphenyl)-5-[(4-chlorophenyl)methylidene]-
3,5-dihydro-4H-imidazol-4-one (3f) was synthesized
from 0.086 g (0.3 mmol) of 2f, 0.027 g (0.45 mmol) of
urea, 0.035 g (0.45 mmol) of ammonium acetate. Yield
6 % (0.005 g), brown solid, mp 214–215 °C.
HRMS (ESI, m/z) [M ? H]? calcd. for C18H17N2O3
1
NMR H (DMSO-d6): 12.02 (br. s, 1H, NH), 8.39 (d,
309.1234, found 309.1235.
J = 7.5 Hz, 2H, Ar), 8.02 (d, J = 7.5 Hz, 2H, Ar), 7.34 (d,
J = 7.5 Hz, 2H, Ar), 7.51 (d, J = 7.5 Hz, 2H, Ar), 7.05 (s,
1H).
NMR 13C (DMSO-d6): 171.9, 161.2, 143.3, 141.1,
133.3, 133.1, 131.4, 130.4, 128.6, 128.4, 127.3, 125.7,
21.0.
IR (m, cm-1): 3100 (br., OH), 1787, 1764 (C=O), 1653
(C=N).
(5Z)-2-(4-Methoxyphenyl)-5-[(4-hydroxyphenyl)methylidene]-
3,5-dihydro-4H-imidazol-4-one (3c) was synthesized from
0.295 g (1 mmol) of 2c, 0.09 g (1.5 mmol) of urea,
0.116 g (1.5 mmol) of ammonium acetate. Yield 14 %
(0.039 g), brown solid, mp 273–274 °C.
1
NMR H (DMSO-d6): 10.00 (s, 1H, OH), 9.55 (s, 1H,
NH), 7.97 (d, J = 7.6 Hz, 2H, Ar), 7.58 (d, J = 7.4 Hz,
2H, Ar), 7.38 (s, 1H, HC=), 7.18 (d, J = 7.4 Hz, 2H, Ar),
6.62 (d, J = 7.6 Hz, 2H, Ar), 3.85 (s, 3H, CH3).
NMR 13C (DMSO-d6): 183.4, 165.5, 157.5, 145.5,
140.6, 133.3, 130.4, 127.7, 125.0, 118.7, 114.9, 100.3,
55.9.
C17H13N2OCl requires (%): C, 68.80; H, 4.42; N 9.44.
Found: C, 68.58; H, 4.12; N, 9.74.
(5Z)-2-(4-Methylphenyl)-5-[(4-bromophenyl)methylidene]-
3,5-dihydro-4H-imidazol-4-one (3g) was synthesized
from 0.342 g (1 mmol) of 2g, 0.09 g (1.5 mmol) of urea,
0.117 g (1.5 mmol) of ammonium acetate. Yield 19 %
(0.065 g), brown solid, mp 281–283 °C.
C17H14N2O3 requires (%): C, 69.39; H, 4.79; N 9.52.
Found: C, 69.55; H, 4.61 N, 9.66.
123

Med Chem Res
NMR 1H (DMSO-d6): 12.01 (s, 1H, NH), 8.25 (d,
J = 7.6 Hz, 2H, Ar), 8.07 (d, J = 7.7 Hz, 2H, Ar), 7.68 (d,
J = 7.7 Hz, 2H, Ar), 7.46 (d, J = 7.6 Hz, 2H, Ar), 6.98 (s,
1H, HC=), 2.42 (s, 3H, CH3).
NMR 13C (DMSO-d6): 172.0, 160.9, 143.2, 139.9,
133.7, 131.7, 130.5, 129.0, 128.5, 127.4, 125.6, 123.1,
21.1.
IR (m, cm-1): 3150 (br., OH), 1703 (C=O), 1644 (C=N).
C17H13N2OBr 9 0.5H2O requires (%): C, 58.30; H,
4.03; N 8.00. Found: C, 58.32; H, 3.99; N, 7.56.
HRMS (ESI, m/z) [M ? H]? calcd. for C19H18FN2O4
357.1239, found 357.1243.
(5Z)-2-(4-Fluorophenyl)-5-[(4-fluorophenyl)methylidene]-
3,5-dihydro-4H-imidazol-4-one (3k) was synthesized
from 0.066 g (0.32 mmol) of 2k, 0.021 g (0.35 mmol) of
urea, 0.027 g (0.35 mmol) of ammonium acetate. Yield
7 % (0.004 g), brown solid, mp 207–210 °C.
NMR 1H (DMSO-d6): 9.98 (s, 1H, NH), 7.82 (d,
J = 7.3 Hz, 2H, Ar), 7.73 (dd, J1 = 5.0 Hz, J2 = 7.7 Hz,
2H, Ar), 7.48 (s, 1H, HC=), 7.30 (d, J = 7.3 Hz, 2H, Ar),
7.24 (dd, J1 = 7.4 Hz, J2 = 7.7 Hz, 2H, Ar).
NMR 13C (DMSO-d6): 170.9, 163.3, 160.7, 160.6,
136.3, 132.0, 131.1, 130.3, 129.3, 123.5, 115.8, 115.4.
HRMS (ESI, m/z) [M ? H]? calcd. for C16H11N2OF2
285.1085, found 285.1089.
(5Z)-2-(4-Methylphenyl)-5-[(4-hydroxyphenyl)methylidene]-
3,5-dihydro-4H-imidazol-4-one (3h) was synthesized
from 0.119 g (0.45 mmol) of 2h, 0.038 g (0.67 mmol) of
urea, 0.05 g (0.67 mmol) of ammonium acetate. Yield
44 % (0.052 g), yellow oil.
1
NMR H (DMSO-d6): 9.95 (s, 1H, OH), 9.82 (s, 1H,
(5Z)-2-(4-Fluorophenyl)-5-[(2,3,4-trimethoxyphenyl)methyli-
dene]-3,5-dihydro-4H-imidazol-4-one (3l) was synthe-
sized from 0.062 g (0.2 mmol) of 2l, 0.018 g (0.3 mmol)
of urea, 0.023 g (0.3 mmol) of ammonium acetate. Yield
20 % (0.0125 g), yellow solid, mp 217–219 °C.
NH), 7.89 (d, J = 7.8 Hz, 2H, Ar), 7.54 (d, J = 8.0 Hz,
2H, Ar), 7.41 (s, 1H, HC=), 7.32 (d, J = 8.0 Hz, 2H, Ar),
6.75 (d, J = 7.8 Hz, 2H, Ar), 2.51 (s, 3H, CH3).
NMR 13C (DMSO-d6): 172.0, 161.2, 158.9, 143.2,
139.9, 134.3, 132.6, 128.5, 127.7, 127.4, 125.7, 116.8,
21.0.
1
NMR H (DMSO-d6): 9.96 (s, 1H, NH), 8.74 (m, 1H,
HRMS (ESI, m/z) [M ? H]? calcd. for C17H15N2O2
Ar), 8.03 (m, 2H, Ar), 7.32–7.40 (m, 2H), 7.05 (s, 1H,
HC=), 3.89 (s, 3H, CH3), 3.79 (s, 3H, CH3), 3.76 (s, 3H,
CH3).
279.1455, found 279.1492.
(5Z)-2-(4-Methylphenyl)-5-(phenylmethylidene)-3,5-dihydro-
4H-imidazol-4-one (3i) was synthesized from 0.249 g
(1 mmol) of 2i, 0.09 g (1.5 mmol) of urea, 0.116 g
(1.5 mmol) of ammonium acetate. Yield 25 % (0.061 g),
brown solid, mp 304–305 °C.
NMR 1H (DMSO-d6): 10.04 (s, 1H, NH), 7.83 (d,
J = 7.9 Hz, 2H, Ar), 7.62 (d, J = 7.9 Hz, 2H, Ar), 7.40 (d,
J = 7.9 Hz, 2H, Ar), 7.25 (s, 1H, HC=), 7.22 (d,
J = 7.9 Hz, 2H, Ar), 2.39 (s, 3H, CH3).
NMR 13C (DMSO-d6): 173.9, 161.2, 143.3, 139.6, 135.1,
134.1, 132.7, 132.4, 128.8, 128.6, 127.3, 125.6, 21.0.
IR (m, cm-1): 3411 (NH), 1708 (C=O), 1697 (C=N).
HRMS (ESI, m/z) [M ? H]? calcd. for C17H15N2O)
263.1184, found 263.1176.
NMR 13C (DMSO-d6): 170.7, 163.8, 161.1, 154.2,
154.0, 141.5, 141.2, 130.6, 125.9, 124.4, 123.6, 116.2,
115.5, 106.7, 60.7, 60.4, 55.8.
HRMS (ESI, m/z) [M ? H]? calcd. for C19H18N2O4F
357.1243, found 357.1239.
(5Z)-2-(4-Fluorophenyl)-5-[(2,4,5-trimethoxyphenyl)
methylidene]-3,5-dihydro-4H-imidazol-4-one
(3m)
was synthesized from 0.071 g (0.2 mmol) of 2m, 0.018 g
(0.3 mmol) of urea, 0.023 g (0.3 mmol) of ammonium
acetate. Yield 7 % (0.005 g), brown solid, mp 263–265 °C.
1
NMR H (DMSO-d6): 9.87 (s, 1H, NH), 8.81 (s, 1H,
Ar), 8.00–8.03 (m, 2H, Ar), 7.24–7.28 (m, 3H, Ar b HC=),
6.53 (s, 1H, Ar), 4.01 (s, 3H, CH3), 3.97 (s, 3H, CH3), 3.77
(s, 3H, CH3).
(5Z)-2-(4-Fluorophenyl)-5-[(3,4,5-trimethoxyphenyl)methyli-
dene]-3,5-dihydro-4H-imidazol-4-one (3j) was synthe-
sized from 0.2 g (0.56 mmol) of 2j, 0.05 g (0.84 mmol) of
urea, 0.066 g (0.84 mmol) of ammonium acetate. Yield
55 % (0.11 g), yellow oil.
NMR 13C (DMSO-d6): 171.3, 163.7, 161.0, 153.9,
148.2, 144.1, 141.5, 130.3, 125.9, 123.6, 116.2, 115.2,
114.3, 98.8, 56.1, 56.8, 55.6.
HRMS (ESI, m/z) [M ? H]? calcd. for C19H18N2O4F,
357.1243, found 357.1237.
NMR 1H (DMSO-d6): 10.1 (s, 1H, NH), 7.96 (d,
J = 8.8 Hz, 2H, Ar), 7.5 (s, 1H, CH =), 7.36 (d,
J = 8.5 Hz, 2H, Ar), 7.02 (s, 2H, Ar), 3.40 (s, 6H, OCH3),
3.33 (s, 3H, OCH3).
NMR 13C (DMSO-d6): 171.2, 163.2, 160.6, 153.1,
142.6, 140.4, 137.3, 131.0, 129.4, 123.7, 115.8, 109.4,
60.9, 56.1.
(5Z)-2-Phenyl-5-[(4-fluorophenyl)methylidene]-3,5-dihy-
dro-4H-imidazol-4-one (3n) was synthesized from 0.2 g
(0.75 mmol) of 2n, 0.07 g (1.13 mmol) of urea, 0.088 g
(1.13 mmol) of ammonium acetate. Yield 91 % (0.182 g),
brown solid, mp 236–238 °C.
123

Med Chem Res
NMR 1H (DMSO-d6): 10.04 (s, 1H, NH), 7.95 (d, labeled with mouse monoclonal antibody against a-tubulin
J = 7.8 Hz, 2H, Ar), 7.72 (dd, J1 = 6.6 Hz, J2 = 7.9 Hz, at a dilution of 1:400 (Sigma, St. Louis. USA), followed by
2H, Ar), 7.60 (t, J = 7.8 Hz, 1H, Ar), 7.53 (d, J = 7.6 Hz, incubation of Alexa Fluor488 labeled goat anti-mouse IgG
2H, Ar), 7.46 (s, 1H, HC=), 7.25 (dd, J1 = 7.6 Hz, at a dilution of 1:200 (Invitrogen, Germany). Images of all
J2 = 7.9 Hz, 2H, Ar).
samples were acquired with a Nikon Diaphot 300 inverted
NMR 13C (DMSO-d6): 166.6, 153.0, 147.6, 139.2, microscope (Nikon GmbH, Du¨sseldorf, Germany) equip-
135.0, 136.8, 132.6, 132.5, 116.2, 115.9, 115.2, 92.6.
ped with a cooled charge-couple device camera system
HRMS (ESI, m/z) [M ? H]? calcd. for C16H12N2OF (SenSys; Photometrics, Munich, Germany).
267.0928, found 267.0930.
Acknowledgments This work was done in the frame of a Scientific
Cooperation Agreement between Moscow and Rostock Universities
and supported by Russian Foundation for Basic Research (No. 15-03-
04894) and Russian Academy of Sciences.
Biology
MTT cytotoxicity assay
A549 human lung epithelial carcinoma cells (CCL-185TM
´
were cultured with Dulbeccos Modified Eagle medium
)
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a
humidified 5 % CO2 atmosphere. Cells were seeded in
96-well plates at a density of 3 9 103 cells per well. Stock
solutions of test compounds were prepared in dimethyl-
sulfoxide (DMSO). Cells were treated for 24 h with
selected compounds at 1–52000 nM or with colchicine at
5–100 nM as positive controls (8 wells for each concen-
tration). DMSO (0.4 %) served as a negative control. The
number of surviving cells was determined by the colori-
metric MTT assay (Mosmann, 1983). MTT [3-(4,5-
dimethylthiazolyl-2)-2,5-diphenyl-2H-tetrazoliumbromid,
Roth GmbH, Karlsruhe, Germany] was prepared at 5 mg/
ml in phosphate-buffered saline (PBS) and filtered through
a 0.22-lm filter. Twenty microliter of sterile MTT solution
(final concentration 0.5 mg/ml) was added to each well 2 h
before the end of compound exposure. Then, the super-
natant was removed, and 100 ll of DMSO containing
10 % SDS and 0.6 % acetic acid was added to each well.
Resulting formazan crystals were solubilized by thorough
mixing on a plate shaker. Optical density was measured at
590 nm with 690 nm reference filter using a EL808 Ultra
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