Benzaldehyde thiosemicarbazone derivatives against replicating and nonreplicating Mycobacterium tuberculosis

Article abstract of DOI:10.1038/s41429-019-0140-9

In this article, we report a series of benzaldehyde thiosemicarbazone derivatives possessing high activity toward actively replicating Mycobacterium tuberculosis strain with minimum inhibitory concentration (MIC) values in the range from 0.14 to 2.2 μM. Among them, two compounds—2-(4-phenethoxybenzylidene)hydrazine-1-carbothioamide (13) and 2-(3-isopropoxybenzylidene)hydrazine-1-carbothioamide (20) also demonstrate submicromolar antimycobacterial activity against M. tuberculosis under hypoxia with MIC values of 0.68 and 0.74 μM, respectively. The activity of compounds 13 and 20 toward five investigated isoniazid-, rifampicin-, and fluoroquinolone-resistant M. tuberculosis isolates is similar to commercially available antituberculosis drugs. The compounds 13 and 20 possess good ADME properties and have low cytotoxicity toward human liver cells (HepG2). Therefore, 2-(4-phenethoxybenzylidene)hydrazine-1-carbothioamide (13) and 2-(3-isopropoxybenzylidene)hydrazine-1-carbothioamide (20) are valuable candidates for further preclinical studies.

Full text of DOI:10.1038/s41429-019-0140-9

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0140-9
ARTICLE
Benzaldehyde thiosemicarbazone derivatives against replicating
and nonreplicating Mycobacterium tuberculosis
Galyna P. Volynets1 Michail A. Tukalo1 Volodymyr G. Bdzhola1 Nataliia M. Derkach2 Mykola I. Gumeniuk2
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Sergiy S. Tarnavskiy1 Sergiy A. Starosyla1 Sergiy M. Yarmoluk1
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Received: 2 October 2018 / Revised: 27 December 2018 / Accepted: 7 January 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
In this article, we report a series of benzaldehyde thiosemicarbazone derivatives possessing high activity toward
actively replicating Mycobacterium tuberculosis strain with minimum inhibitory concentration (MIC) values in the range
from 0.14 to 2.2 μM. Among them, two compounds—2-(4-phenethoxybenzylidene)hydrazine-1-carbothioamide (13) and
2-(3-isopropoxybenzylidene)hydrazine-1-carbothioamide (20) also demonstrate submicromolar antimycobacterial activity
against M. tuberculosis under hypoxia with MIC values of 0.68 and 0.74 μM, respectively. The activity of compounds 13
and 20 toward five investigated isoniazid-, rifampicin-, and fluoroquinolone-resistant M. tuberculosis isolates is similar to
commercially available antituberculosis drugs. The compounds 13 and 20 possess good ADME properties and have low
cytotoxicity toward human liver cells (HepG2). Therefore, 2-(4-phenethoxybenzylidene)hydrazine-1-carbothioamide (13)
and 2-(3-isopropoxybenzylidene)hydrazine-1-carbothioamide (20) are valuable candidates for further preclinical studies.
Introduction
Food and Drug Administration (FDA) for MDR tubercu-
losis treatment. This antibiotic was the first new medicine to
fight tuberculosis in more than 40 years. Recently, the cases
of bedaquiline resistance also were reported [1–5]. This fact
highlights the need to keep pursuing the research on novel
antituberculosis drugs able to kill resistant isolates of
Mycobacterium tuberculosis.
Concerning antituberculosis drug discovery, the pheno-
typic high-throughput screening has demonstrated to be a
more efficient method to produce lead candidates in com-
parison to the target-based approaches. For example,
bedaquiline was discovered using whole cell phenotypic
assay.
Tuberculosis (TB) is one of the most serious human dis-
eases and public problems throughout the world. The
treatment of TB is impaired by the long duration of therapy,
which leads to a high risk of drug resistance development.
Resistance to the first-line antituberculosis antibiotics such
as rifampicin and isoniazid is called multidrug resistant
(MDR) tuberculosis. The resistance to fluoroquinolones,
which are key second-line anti-tuberculosis drugs, increases
the chances of getting extremely drug resistant (XDR) and
totally drug resistant (TDR) tuberculosis. Therefore, there is
an urgent need for new drugs with improved anti-
tuberculosis efficiency. Unfortunately, the development of
novel antituberculosis agents has been very slow. In 2012,
the novel antibiotic bedaquiline was approved by the U.S.
Using phenotypic screening assay, we have identified
benzaldehyde thiosemicarbazone derivatives with anti-
tuberculosis activity. Thiosemicarbazone derivatives have
been already reported in the literature as antibacterial,
antifungal, and anti-tubercular agents [6–9].
The molecular mechanism of action for thiosemicarba-
zone derivatives is still not clearly understood. There
are some indications concerning the mechanism of
action for antituberculosis drug from this chemical class—
thiacetazone. It has been shown that thiacetazone is a
prodrug that requires activation by the mycobacterial
monooxygenase EthA. The activated thiacetazone affects
mycolic acid synthesis, probably by inhibiting certain
* Galyna P. Volynets
galina.volinetc@gmail.com
1
Institute of Molecular Biology and Genetics, NAS of Ukraine, 150
Zabolotnogo Street, 03143 Kyiv, Ukraine
2
National Institute of Phthisiology and Pulmonology named after F.
G. Yanovsky, NAMS of Ukraine, 10, M. Amosova Street, 03038
Kyiv, Ukraine

G. P. Volynets et al.
cyclopropane mycolic acid synthases [10, 11]. Moreover, it
is known that thiosemicarbazone group chelates metal
cations strongly and the antitubercular action of thiaceta-
zone is enhanced by copper [12]. Therefore, anti-
tuberculosis action of thiosemicarbazone derivatives may be
due to complex action.
Taking into account the biological potential of thiose-
micarbazone derivatives, we have investigated a series of
benzaldehyde thiosemicarbazones for their activity toward
pathogenic M. tuberculosis strain H37Rv and isoniazid,
rifampicin, and fluoroquinolone resistant isolates.
(100 µM rifampicin (Sigma-Aldrich)), and maximum
growth (DMSO only), as well as a rifampicin dose response
curve. Plates were inoculated with M. tuberculosis and
incubated for 5 days: growth was measured by OD590 and
fluorescence (Ex 560/Em 590) using a BioTek™ Synergy 4
plate reader. Growth was calculated separately for OD590
and RFU. To calculate the MIC, the 10-point dose response
curve was plotted as % growth and fitted to the Gompertz
model using GraphPad Prism 5. The MIC was defined as
the minimum concentration at which growth was com-
pletely inhibited and was calculated from the inflection
point of the fitted curve to the lower asymptote (zero
growth).
Materials and methods
Antibacterial activity under hypoxic conditions
Chemical synthesis
The antimicrobial activity of compounds under hypoxic
conditions was assessed using the low oxygen recovery
assay (LORA) [18]. Test compounds were prepared as
20-point two-fold serial dilutions in DMSO and diluted
into DTA medium (6.5 g L−1 Dubos broth base (Fisher),
10% (v/v) Dubos medium albumin, 0.05% w/v Tween 80)
in 96-well plates with a final DMSO concentration of 2%.
Control compounds were prepared as 20-point two-fold
serial dilutions in DMSO and diluted into DTA medium
in 96-well plates with a final DMSO concentration
of 2%. The highest concentration of compound was
200 µM.
M. tuberculosis constitutively expressing the luxABCDE
operon was inoculated into DTA medium in gas-impermeable
glass tubes and incubated for 18 days to generate hypoxic
conditions (Wayne model of hypoxia). At this point, bacteria
are in a non-replicating state (NRP stage 2) induced by
oxygen depletion.
Thiosemicarbazide (1 mmol) was dissolved in 20 ml of
ethanol under heating and appropriate aldehyde (1 mmol)
was added. The resulting mixture was heated to solid for-
mation. Solid products were filtered, washed with ethanol
(2 × 5 ml), and dried. The compounds have been already
patented by us [13].
Antibacterial activity under aerobic conditions
The antimicrobial effect of compounds against M. tuber-
culosis H37Rv grown under aerobic conditions was asses-
sed by determining the minimum inhibitory concentration
(MIC). The assay is based on the measurement of growth in
liquid medium of a fluorescent reporter strain H37Rv
expressing dsRED under the control of constitutively
expressed rpsA promoter, where the readout is either optical
density (OD) or fluorescence [14–17]. The use of two
readouts minimizes the problems caused by compound
precipitation or autofluorescence. A linear relationship
between OD and fluorescence readout has been established
justifying the use of fluorescence as a measure of bacterial
growth. MICs generated from the OD are reported in the
“Results and discussion” section.
Oxygen-deprived bacteria were inoculated into com-
pound assay plates and incubated under anaerobic condi-
tions for 10 days followed by incubation under aerobic
conditions (outgrowth) for 28 h. Growth was measured by
luminescence.
The antimicrobial activity of compounds against five
resistant isolates of M. tuberculosis was assessed only by
OD readout.
Intracellular activity assay
The intracellular activity of compounds was measured using
a macrophage cell line infected with M. tuberculosis [19].
The activity of compounds against intracellular bacteria was
determined by measuring viability in infected THP-1 human
monocytic cell line (ATCC) after 3 days in the presence of
test compounds. Compounds were prepared as 10-point
three-fold serial dilutions in DMSO (Fisher). The highest
concentration of compound tested was 50 µM where com-
pounds were soluble in DMSO at 10 mM.
The MIC of compound was determined by measuring
bacterial growth after 5 days in the presence of test com-
pounds. Compounds were prepared as 10-point two-fold
serial dilutions in DMSO (Fisher) and diluted into 7H9-Tw-
OADC medium (4.7 g L−1 7H9 base broth (VWR), 0.05%
w/v Tween 80 (Fisher), 10% v/v OADC Supplement
(VWR)) in 96-well plates with a final DMSO concentration
of 2%. The highest concentration of compound was
200 µM. Each plate included assay controls for background
(medium/DMSO only, no bacterial cells), zero growth
THP-1 cells were cultured in complete RPMI (RPMI-1640
(Fisher), 10% v/v FBS (Fisher), 2 mM GlutaMAX (Fisher),

Benzaldehyde thiosemicarbazone derivatives against replicating and nonreplicating Mycobacterium. . .
1 mM sodium pyruvate (Fisher)) and differentiated into
macrophage-like cells using 80 nM PMA (Sigma-Aldrich)
overnight at 37 °C, 5% CO2. THP-1 cells were infected with a
luminescent strain of H37Rv (which constitutively expresses
luxABCDE) at a multiplicity of infection of 1 and incubated
overnight at 37 °C, 5% CO2. Infected cells were recovered
using Accutase/EDTA solution (Accutase, 5 mM EDTA),
washed twice with PBS (Fisher) to remove extracellular
bacteria and seeded into assay plates. Compound dilutions
were added to a final DMSO concentration of 0.5%.
Assay plates were incubated for 72 h at 37 °C, 5% CO2.
Each run included isoniazid (Sigma-Aldrich) as a control.
Relative luminescent units (RLU) were measured using a
Biotek Synergy 2 plate reader. The dose response curve
was fitted using the Levenberg–Marquardt algorithm.
The IC50 and IC90 were defined as the compound con-
centrations that produced 50% and 90% inhibition of growth,
respectively.
Quality control
The A-side contained 100 μM Lucifer yellow (Sigma) in
transport buffer (1.98 g L−1 glucose (Teknova) in 10 mM
HEPES (Sigma), 1× Hank’s Balanced Salt Solution (Lonza)
pH 6.5), and the B-side contained transport buffer (1.98 g L−1
glucose (Teknova) in 10 mM HEPES (Sigma), 1× Hank’s
Balanced Salt Solution (Lonza) pH 7.4). Caco-2 cells were
incubated with these buffers for either 1 or 2 h, and the
receiver side buffer was removed for analysis by LC-MS/MS.
Aliquots of the cell buffers were analyzed by fluorescence to
determine the transport of the impermeable dye Lucifer yel-
low to verify the Caco-2 cell monolayers were properly
formed. Any deviations from control values were reported.
Data are expressed as permeability, Papp = (dQ/dt)/(C0/A), in
which dQ/dt is the rate of permeation, C0 is the initial con-
centration of compound, and A is the area of monolayer.
HepG2 cytotoxicity
Plasma protein binding
The cytotoxicity of compounds towards eukaryotic cells was
determined using the human liver cells (HepG2) [26–29].
The cytotoxicity of compounds was determined by mea-
suring HepG2 (ATCC) cell viability growth after 3 days in
the presence of test compounds. Compounds were prepared
as 10-point three-fold serial dilutions in DMSO. The highest
concentration of compound tested was 100 µM where
compounds were soluble in DMSO at 10 mM. HepG2
cells were cultured in complete DMEM (DMEM medium
(Invitrogen), 1× penicillin–streptomycin solution (Fisher),
2 mM Corning Glutogro supplement (Fisher), 1 mM sodium
pyruvate (Fisher), 10% v/v Fetal Bovine Serum (Fisher)),
inoculated into 384-well assay plates containing compounds
and incubated for 24 h at 37 °C, 5% CO2. Compounds were
added and cells were cultured for a further 72 h. The
final DMSO concentration was 1%. Cell viability was
determined using the CellTiter-Glo® Luminescent Cell
Viability Assay (Promega) and measuring RLU. The dose
response curve was fitted using the Levenberg–Marquardt
algorithm. The IC50 was defined as the compound con-
centration that produced 50% decrease in viable cells. Each
run included staurosporine (Santa Cruz Biotechnology) as a
control.
Plasma protein binding for compounds 13 and 20 was
determined by equilibrium dialysis [20]. Compounds were
added to human plasma (Bioreclamation Inc.) at a fixed
concentration of 5 µM. The mixture was dialyzed in a RED
device (Rapid Equilibrium Dialysis, Pierce) against PBS
and incubated on an orbital shaker for 4 h at 37 °C. Aliquots
from plasma and PBS sides were collected; an equal volume
of PBS was added to the plasma sample, and an equal
volume of plasma was added to the PBS sample. Three
volumes of methanol (containing the internal binding
standard propranolol (Sigma)) was added to precipitate the
proteins and release the compound. Samples were cen-
trifuged, the supernatant was recovered and analyzed by
LC-MS/MS. Each experiment included warfarin (Sigma) as
a high-binding control.
Caco-2 cell permeability
The permeability of test compounds was assessed using a
Caco-2 cell monolayer [21–25]. Caco-2 cells (ATCC) were
trypsinized, resuspended in medium, and dispensed into a
Millipore 96-well Caco-2 plate. The cells were allowed to
grow and differentiate for 3 weeks, with feeding at 2-day
intervals. For apical to basolateral (A→B) permeability, the
compound was added to the apical (A) side and amount of
permeation was determined on the basolateral (B) side; for
basolateral to apical (B→A) permeability, the compound
was added to the B side and the amount of permeation was
determined on the A side. Each experiment included the
control compounds atenolol (MP Biomedicals) (low per-
meability), propranolol (Sigma) (high permeability), and
talinolol (TRC) (P-gp efflux control).
Results and discussion
In order to find novel antituberculosis compounds, we have
performed phenotypic screening of the library containing
about 1000 compounds from different chemical classes
against M. tuberculosis H37Rv strain. As a result, a series of
benzaldehyde thiosemicarbazone derivatives possessing
high activity toward actively replicating M. tuberculosis

G. P. Volynets et al.
Table 1 Structures and antimycobacterial activity of benzaldehyde
thiosemicarbazone derivatives toward H37Rv strain under aerobic
conditions
strains has been identified. As shown in Table 1, a number
of investigated benzaldehyde thiosemicarbazones demon-
strate moderate to excellent anti-tubercular activities under
aerobic conditions.
We have analyzed the impact of substituents on the
antimycobacterial activity of benzaldehyde thiosemi-
carbazone derivatives. It was revealed that the substitution
of hydrogen atom (compound 1) in the position R1
with fluorine atom (compound 25) or pentoxy group
(compound 27) leads to the decrease of antimycobacterial
activity (corresponding MIC values are 3.2, 6.3,
and 17 μM).
It was identified that the antimycobacterial activity of
benzaldehyde thiosemicarbazone derivatives depends on the
structure of R3 substituents. The presence of pentoxy group
as R3 substituent (compound 2, MIC = 0.14 μM) is more
favored than propoxy (compound 3, MIC = 0.22 μM) or
ethoxy group (compound 4, MIC = 0.54 μM). Therefore, the
increase in the inhibition of M. tuberculosis growth is
attributed to the increase in alkyl chain length. The sub-
stitution of alkyl chain with benzyloxy group (compound 5),
4-chloro-benzyloxy (compound 6), or 4-bromo-benzyloxy
(compound 7) leads to decrease in antimycobacterial activity
(corresponding MIC values are 0.78, 2.2, and 2.9 μM). From
the obtained results, the order of potency for the substituent
R3 in a series of compounds with R2 = methoxy group,
could be proposed as following: pentoxy group > propoxy
group > ethoxy group > benzyloxy > 4-chloro-benzyloxy >
4-bromo-benzyloxy. It has been observed that for a series of
compounds 13–18 with R2 = hydrogen atom the order of
potency for R3 substituent is following: 2-phenylethoxy >
dimethylamine > methyl > > bromine > hydroxyl
group >
nitro group (corresponding MIC values are 0.18, 0.32, 0.48,
0.86, 1.5, and 3.2 μM).
The insertion of additional substituent R2 (methoxy
group) in the structure of R3-substituted benzaldehyde
thiosemicarbazone derivatives leads to the decrease in
antimycobacterial activity. To compare this effect, one can
refer to compound pairs such as 15, 6; 18, 8 (corresponding
MIC values are 0.43, 2.2; 1.5, 2.8 μM). The substitution
of methoxy group (compound 9) with ethoxy group
(compound 10) does not have a significant influence on the
potency of investigated compounds (MIC = 19 μM).
As shown in Table 1, the substitution of hydrogen atom
(compound 1) at the position R4 with isopropoxy group
(compound 20) results in appreciable rise of anti-
mycobacterial activity (MIC equal to 3.2 and 0.71 μM,
respectively). The introduction of bromine atom (compound
9) instead of hydrogen (compound 8) at the position R4
leads to significant decrease in antimycobacterial activity
(corresponding MIC values are 19 and 2.8 μM).

Benzaldehyde thiosemicarbazone derivatives against replicating and nonreplicating Mycobacterium. . .
Table 3 Intracellular activity of compounds 13 and 20 toward THP-1
human monocytic cell line infected with M. tuberculosis
It has been found that R5 also impacts compound's
antimycobacterial activity. The substitution of hydrogen
atom (compound 25) with chlorine atom (compound 26)
results in the increase of antimycobacterial activity (corre-
sponding MIC values are 6.3 and 9 μM).
Therefore, structure–activity relationships (SAR) of ben-
zaldehyde thiosemicarbazone derivatives with respect to their
antitubercular activity revealed that the antimycobacterial
activity of the studied compounds depends on the structure of
all substituents. The results of SAR studies can be used for
further chemical optimization of benzaldehyde thiosemi-
carbazone derivatives.
Compound
ІС50, μM
ІС90, μM
MIC, μM
13
<0.098
<0.098
0.315
<0.098
<0.098
0.38
<0.098
<0.098
1.46
20
Isoniazid
Table 4 Minimum inhibitory concentration (MIC, μM) of compounds
13 and 20 toward resistant isolates of M. tuberculosis
Compound
INH-R1a INH-R2b RIF-R1c RIF-R2d FQ-R1e
The most active 16 compounds identified in the primary
screening were also evaluated against M. tuberculosis
H37Rv under hypoxic conditions (Table 2). This method is
used for screening of antimicrobial agents against non-
replicating persistence M. tuberculosis. The compounds 13
and 20 which possess submicromolar activity have been
taken for further biological investigations.
The intracellular activity of compounds 13 and 20 was
measured using a macrophage cell line infected with M.
tuberculosis. This is important because M. tuberculosis can
survive inside macrophages, which contributes to the
treatment failure and disease relapse [30–32]. Accordingly
to the results presented in Table 3, MIC, ІС50, and ІС90
values of compounds 13 and 20 are lower than for isoniazid.
Also, it should be noted that intracellular activities of these
compounds are significantly lower than the activities in
axenic media.
13
0.21
0.11
0.18
0.011
>200
2.3
0.079
0.39
3.0
0.25
0.61
>50
0.71
1.6
0.25
0.64
0.015
0.80
26
20
0.38
Rifampicin
Isoniazid
0.020
>200
0.36
1.3
Levofloxacin 1.4
aINH-R1—isoniazid resistant strain derived from H37Rv and is a katG
mutant (Y155* = truncation)
bINH-R2—isoniazid resistant strain ATCC35822 which has
complete deletion of the katG gene
cRIF-R1—rifampicin resistant strain derived from H37Rv and is an
rpoB mutant (S522L)
dRIF-R2—rifampicin resistant strain ATCC35828 with a single G
nucleotide absent at position 288 in the pncA gene
eFQ-R1—fluoroquinolone-resistant strain derived from H37Rv which
a
is a gyrB mutant (D94N)
The compounds 13 and 20 were tested for antibacterial
activity against five resistant isolates of M. tuberculosis
under aerobic conditions. Strains tested were two isoniazid
resistant strains (INH-R1 and INH-R2), two rifampicin
resistant strains (RIF-R1 and RIF-R2), and a fluor-
oquinolone resistant strain (FQ-R1). The assay was based
on the measurement of growth in liquid medium of each
strain where the readout is OD. INH-R1 was derived from
H37Rv and is a katG mutant (Y155* = truncation). INH-R2
is strain ATCC35822 which has a complete deletion of the
katG gene. RIF-R1 was derived from H37Rv and is an rpoB
mutant (S522L). RIF-R2 is strain ATCC35828 with a single
G nucleotide absent at position 288 in the pncA gene.
FQ-R1 is a fluoroquinolone-resistant strain derived from
H37Rv which is a gyrB mutant (D94N). Accordingly to the
results of investigation, compounds 13 and 20 are highly
potent toward all tested resistant isolates of M. tuberculosis
(Table 4).
Plasma protein binding for compounds 13 and 20 was
determined by equilibrium dialysis. As it can be seen from
Table 5, compounds 13 and 20 are highly bound to plasma
protein.
The permeability of test compounds was assessed using a
Caco-2 cell monolayer. Compound permeability was mea-
sured in both directions. The experiment included the
control compounds atenolol (low permeability), propranolol
Table 2 Antituberculosis activity for benzaldehyde thiosemicarbazone
derivatives toward H37Rv strain under hypoxic conditions
Compound
MIC, μM
2
1.2
3
20
4
15
5
71
6
>200
92
7
8
>200
0.68
>200
8.9
13
14
15
16
17
18
19
20
21
58
>200
>200
>200
0.74
82

G. P. Volynets et al.
Table 5 Plasma protein binding for compounds 13 and 20, %
National Institute of Allergy and Infectious Diseases (Contract No.
HHSN272201100009I).
Compound Mean plasma
Mean plasma
Recovery, %
fraction unbound, % fraction bound, %
Compliance with ethical standards
13
20
0.8
99.2
86.8
75.6
99.4
87.6
96
13.2
Conflict of interest The authors declare that they have no conflict of
interest.
Propranolol 24.4
Warfarin 0.62
98.4
106
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Table 6 Caco-2 permeability for compounds 13 and 20
References
Compound
Assay
duration (h) Papp
Mean A→B Mean B→A Efflux
Papp
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(10−6 cm/s)
(10−6 cm/s)
13
1
1
2
2
1
2
1
2
1
2
6.8
9.7
1.4
20
37.5
8.2
24.7
3.5
0.66
0.43
0.51
8.7
13
20
28.2
0.036
0.31
17.3
25.4
0.017
0.072
14.3
0.31
0.42
43.3
21.1
5.04
6.3
Atenolol
Atenolol
Propranolol
Propranolol
Talinolol
Talinolol
1.4
2.5
0.83
298
87.6
aPapp is the apparent permeability rate coefficient = (dQ/dt)/(C0A)
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active transporters
(high permeability), and talinolol (P-gp efflux control).
As it can be seen from Table 6, compounds 13 and 20 have
good permeability across Caco-2 cell monolayer in both
directions.
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cells was determined using the human liver cells (HepG2).
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In conclusion, we have identified two compounds—2-(4-
phenethoxybenzylidene)hydrazine-1-carbothioamide (13)
and 2-(3-isopropoxybenzylidene)hydrazine-1-carbothioa-
mide (20) possessing high antibacterial activity toward M.
tuberculosis H37Rv under aerobic and hypoxic conditions.
The compounds are active against isoniazid-, rifampicin-,
and fluoroquinolone-resistant strains and also have good
ADME properties and low cytotoxicity toward human liver
cells (HepG2). These obtained data indicate that the com-
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Acknowledgements This work was supported by the grants from the
National Academy of Sciences of Ukraine (0117U003914) and by the

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