Novel acyl thiourea derivatives: Synthesis, antifungal activity, gene toxicity, drug-like and molecular docking screening

Article abstract of DOI:10.1002/ardp.201800275

Nine novel acyl thioureas were synthesized. Their identities and purities were confirmed by LC-MS spectra; each structure was elucidated by elemental analysis, IR, ¹Н and ¹³C NMR spectra. Applying an in vitro screening of their antifungal potential, three substances (3, 5, and 6) could be selected as showing high activity against 11 fungi and 3 Phytophthora strains of phytopathogenic significance. Analysis of gene toxicity with the Salmonella reverse mutagenicity test, as an assessment of drug likeness, lipophilicity, and calculations of frontier molecular orbitals assign a low toxicity profile to these compounds. Molecular docking studies point to 14α-demethylase (CYP51) and N-myristoyltransferase (NMT) as possible fungal targets for growth inhibition. The findings are discussed with respect to structure–activity relationship (SAR).

Full text of DOI:10.1002/ardp.201800275

Received: 16 September 2018
DOI: 10.1002/ardp.201800275
Revised: 3 November 2018
Accepted: 11 November 2018
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FULL PAPER
Novel acyl thiourea derivatives: Synthesis, antifungal activity,
gene toxicity, drug-like and molecular docking screening
Lyudmyla Antypenko1
Fatuma Meyer1
Olena Kholodniak2
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Zhanar Sadykova1
Tereza Jirásková1
Anastasiia Troianova1
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Vladlena Buhaiova1
Surui Cao1
Sergiy Kovalenko2
Leif-Alexander Garbe1
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Karl G. Steffens1
1 Faculty of Agriculture and Food Science,
Abstract
Neubrandenburg University, Neubrandenburg,
Germany
Nine novel acyl thioureas were synthesized. Their identities and purities were
confirmed by LC-MS spectra; each structure was elucidated by elemental analysis, IR,
1Н and 13C NMR spectra. Applying an in vitro screening of their antifungal potential,
three substances (3, 5, and6) could beselected asshowing high activityagainst 11 fungi
and 3 Phytophthora strains of phytopathogenic significance. Analysis of gene toxicity
with the Salmonella reverse mutagenicity test, as an assessment of drug likeness,
lipophilicity, and calculations of frontier molecular orbitals assign a low toxicity profile
to these compounds. Molecular docking studies point to 14α-demethylase (CYP51)
and N-myristoyltransferase (NMT) as possible fungal targets for growth inhibition. The
findings are discussed with respect to structure–activity relationship (SAR).
2 Department of Organic and Bioorganic
Chemistry, Zaporizhzhya State Medical
University, Zaporizhzhya, Ukraine
Correspondence
Lyudmyla Antypenko, Department of Food
and Bioproduct Technology, Neubrandenburg
University, Brodaer Str. 2, 17033
Neubrandenburg, Germany.
Email: antypenkol@gmail.com
Funding information
Enamine Ltd.; German Federal Ministry of
Education and Research, Grant number: FKZ
03FH025IX4
K E Y W O R D S
anti-phytopathogens, drug likeness, gene toxicity, molecular docking, N-(2-
carbamothioylhydrazine-1-carbonothioyl)cyclopropanecarboxamide, N-substituted N-
(hydrazinecarbonothioyl)-cyclopropanecarbox(benz)amides
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1
INTRODUCTION
based on thiourea core warrant special scrutiny, since some of them
are already described having antifungal activity.[2] Thiourea was found
Substances with antifungal activity are of eminent importance in
human health care, veterinary medicine, and agriculture. Low toxicity
and environmental friendliness are further mandatory properties
making such chemicals ready for commercialization. Unfortunately,
the occurrence of fungal strains resistant toward standard antifungals
has developed to an extent threatening established treatment
regimens against fungal infections and food security for a growing
world population.[1] Therefore, the search for novel, potent biologically
active compounds is an ongoing need to cope with challenges caused
by the emergence of new resistant fungi. In this context derivatives
to occur naturally in laburnum shrubs, and as a metabolite of
Verticillium albo-atrum and Bortrylio cinerea.[3] A literature survey[4]
revealed findings of antifungal activity of some thiourea structural
analogues (A and B, Figure 1). Namely, it was reported, that
thiosemicarbazone A inhibited growth of different Aspergillus strains:
A. nomius, A. ochraceus, and A. parasiticus with a minimum inhibition
concentration (MIC) of 125 µg/mL and of A. flavus and Fusarium
verticillioides with MICs of 250 and 500 µg/mL, respectively.
A comparison of the results of the thiosemicarbazone A and other
studied semicarbazone antifungals revealed that sulfur instead of
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Arch Pharm Chem Life Sci. 2018;e1800275.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800275

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FIGURE 1 Structural analogues (A–D) with antifungal activity and substances to be synthesized
oxygen in the core structure conferred a higher activity. Pyrimidine
substituted B showed antifungal activity against A. flavus, A. parasiticus,
and F. verticillioides, but only at an elevated concentration of 500 µg/
mL. Among the tested series of compound C a complete lack of
bioactivity was noted for a derivatives with naphthalene or isoquino-
line ring, whereas replacement by smaller indole (C1) resulted in a
significant antifungal activity (MICs of 200 µg/mL against C. albicans
and 100 µg/mL against C. parapsilosis).[5] An antifungal response was
also noted at a higher concentration (MICs > 400 µg/mL toward all
screened Candida species for pyrazine derivative C2, whereas the
remaining compounds with five-membered heterocyclic ring were
inactive. Siwek et al.[5] also observed that the replacement of an aryl
ring in 4-arylthiosemicarbazides with a flexible chain dramatically
optimized the synthesis method of compound 8 from accessible
reagents (Figure 2).
A one-pot synthesis was easily carried out in acetonitrile with the
consecutive addition of equimolecular amounts of ammonium
isothiocyanate (80°C for 30 min) and isonicotinic acid hydrazide
(80°C for 90 min) to benzoyl chloride with constant stirring.
Subsequently, compounds 1–7 and
9 were synthesized with
satisfactory yields from the acyl chloride of the cyclopropanecarbox-
ylic acid (a), ammonium isothiocyanate (b) and the corresponding acyl
hydrazides ((thio)carbazides) using the given method (Figure 2). This
method is selective and characterized by good yields and high purity of
the final products.
The purities of compounds were confirmed by LC-MS spectra,
structure elucidated by elemental analysis, IR, 1Н and 13C NMR spectra.
When characterizing the LC-MS spectra, increasing molecular
mass at acyl hydrazidic (1–7) or thiosemicarbazidic (9) fragments and
registration of quasimolecular positive ions [M+1] definitely proved
the structure of novel compounds.
In 1H NMR-spectra of substances 1–9 the characteristic signals of
protons were amide (-С(О)NH-), thioamide (-(C(S)NH-) and acyl
hydrazide (-NHNHC(O)-R1) fragments, which were registered at the
low field at the 12.69–12.27 ppm, 11.80–11.34 ppm, and 11.34–
8.88 ppm, appropriately. The electronic effects of the substituents
significantly effected the chemical shift of acyl hydrazide (-NHNHC-
(O)-R1) proton's signal.

reduced antifungal response, and that the NH-NH-C( S)-NH core
structure seemed to be important for antifungal activity of thiosemi-
carbazides. 2-(2-((4-Methyl-2-oxo-2H-chromen-7-yl)oxy)acetyl)-N-
(2,4,6-trichloro-phenyl)hydrazine-1-carbothioamide (D) also exhibited
moderate antifungal activity (46% of growth inhibition) at 10 µg/mL.
Generally, among studied series of coumarinyl thiosemicarbazides,
compounds with aromatic substituents showed a better antifungal
activity than those with methyl or ethyl rest groups.[6]
Here we describe the synthesis, structure analysis, and antifungal
activity of nine novel acyl thioureas (Figure 1, target substances); their
potential gene toxicity is analyzed using the Salmonella reverse
mutagenicity assay (Ames test). Physicochemical drug-likeness de-
scriptors and frontier molecular orbitals energies calculations are
applied to estimate potential toxicity. Finally, in silico methods are used
to evaluate the structure–activity relationship (SAR), and to predict the
potential affinity to the most common antifungal enzymatic targets by
molecular docking.
The protons peaks of cyclopropylic fragment (1–7) in position 1
were found in the high field as multiplets at the 2.24–1.79 ppm. And
signals protons in the second and third positions appeared as
broadened multiplets at the 1.18–0.51 ppm. An exception was
spectrum of the substance 9, which had Н-1 signal as doublet of
triplets with corresponding coupling constants (12.5 and 6.3 Hz).
Besides, the spectra of compounds 1 and 9 were characterized by
signals of the NH2 group of carbazide residues at the 6.21 and
7.68 ppm, and compounds 5 and 6 – by signals of the -CH2 group at the
4.65 and 3.76 ppm, correspondingly. In the 1H NMR spectra of
compounds 3–8, also signals of protons from aromatic substituents
were recorded, which, depending on the proton environment, had a
corresponding multiplicity.
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
Preparation of acyl thioureas may be conducted as with isolation of
intermediate acyl isothiocyanates,[7] so using insufficient studied one-
pot-synthesis method.[8] So, at the first stage of the study, we

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FIGURE 2 Synthetic route of novel N-substituted N-(hydrazinecarbonothioyl)-cyclopropane-carboxamides 1–7, N-(2-
isonicotinoylhydrazine-1-carbonothioyl)benzamide (8) and N-(2-carbamothioylhydrazine-1-carbonothioyl)cyclopropanecarboxamide (9)
Uniquely and additionally, the structures of compounds 1, 5, and 9
were proven by 13С NMR spectra. Hence, the signals of sp2-carbon
atom were strongly shifted to low field and were registered at the
183.44–178.29 ppm, namely, -С(О)NHС(S)-) at the 175.74–
175.23 ppm; (-С(О)NHС(S)-) and (-NHNHC(O)-R1) at the 169.59–
156.41 ppm. Besides, the mentioned compounds were characterized
by specific signals of sp3-carbon of cyclopropyl fragment at 14.37–
14.27 ppm (С-1) and at 9.87–9.57 ppm (С-2, 3).
Besides, compound 9 was highly active against Colletotrichum
higginsianum, Gibberella zeae, and Lecanicillium lecanii; in contrast to
substances 3, 5, and 6 its activity against other strains was poor. It may
be speculated that substance 9 due to different functional groups
(Figure 1) targets other physiologically important functions than 3, 5,
and 6. So, the average antifungal dosage of majority of substances was
lower (10–50 μg/mL) than of reported compounds A–D (200–500 μg/
mL, Figure 1).[4–6]
Since compounds 3, 5, and 6 were the most promising, a mixture of
50 μg/mL or 16.6 μg/mL of them was tested against more resistant
strains (Figure 3).
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2.2 Antifungal activity
The mycelial growth rate assay[9] was used to determine antifungal
activity in different concentrations from 50 to 1 µg/mL. The standard
antifungal hymexazol was used as reference. Antifungal activities of
nine acyl thioureas and hymexazol against all tested fungi are listed in
Table 1.
With A. niger and F. equiseti a significant increase of the inhibitory
effects was observed when compared with the most active single
compound. Interestingly, against F. equiseti the mixture was more
effective at its lower concentration. With respect to the other tested
strains the most active single substance (mostly 3) was in the same
range or even more active as within a corresponding cocktail. A deeper
understanding of the molecular mode of action may offer further
guidelines how to apply acyl thioureas as mixtures in the most efficient
way.
Notably, Penicillium digitatum, Mucor indicus, and Fusarium
equiseti were quite resistant toward acyl thioureas. Nevertheless,
with the exception of F. equiseti, also hymexazol was inefficient
against these strains. Comparing the activity of all acyl thioureas
substances 3, 5, and 6 were the most effective, even exceeding the
activity of hymexazol. They inhibited 9 of 14 studied strains to
more than 90% at 50 μg/mL. Also more resistant fungi Aspergillus
niger (3: 67.3%), Botrytis cinerea (3: 76.4%), and P. digitatum (3:
55.9%) were susceptible. Remarkably, substance 3 was active
against all tested strains with the lowest efficiency against
M. indicus (28.3%).
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2.3 SAR
Summing up the obtained data, SAR can be derived with the following
traits (Figure 4).
A phenyl ring (3), methylenoxyphenyl (5) or methylenethio- (6)
substituent increases the rate and diversity of antifungal activity.
Comparing substances 7 and 8, the replacement of a cyclopropyl ring
by a phenyl ring confers an increase of antifungal activity.
Derivatives of acyl thioureas that had pyridine (7), 2-amino group
in phenyl (4) or just an amino group (1) exhibited greatly reduced
activity. Interestingly, the exchange of a carbonyl oxygen to a sulfur
(1 vs. 9) has a considerable effect on activity. At the same time, the
replacement of oxygen in ether group to sulfur to obtain thioether
The interesting observation also is that in some cases substances
with the lowest shown activity (1, 4, 7) even enhanced the fungi
growth (negative values, Table 1). This result should be taken into
account for further investigations as growth stimulators.
At a lower concentration range from 25 to 1 μg/mL also significant
antifungal activities against at least nine fungi were detected. Again,
compounds 3, 5, and 6 were among the most effective substances.

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Future chemical modification to enhance the antifungal potential
should include phenyl ring systems impact, e.g., methylenoxyphenyl or
methylenethiophenyl rest groups, or aryls with electron withdrawing
substituents from hydrazide site of acyl thioureas with thiosemicar-
bazide fragment instead of semicarbazide one (Figure 4).
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2.4 Salmonella reverse mutagenicity test
The detected antifungal activity renders acyl thioureas to an attractive
starting point to develop themas useful agrochemicals, whichmay come
into widespread contact to farm operators and the environment. An
evaluation of their safety must therefore be an integral part of this
development. Here the potential gene-toxicity, which often associates
with mutagenicity, was analyzed applying the Salmonella reverse
mutagenicity assay (“Ames test”).[10] Frame shift (TA 98) and base
substitution (TA 100) his- mutated Salmonella test strains were
challenged with acyl thioureas at doses of 50 or 500 μg/plate.
Substances were also analyzed in the presence of rat liver extract
(“S9-mix”) in order to simulate potential metabolic activation. Grown
colonies of revertants (reverse mutations) were counted after 48 h
incubation on a minimal agar medium. As an indication of gene toxicity a
mutagenic index (Mi; ratio of colony number of revertants in the
presence of test compound vs. revertants of negative control (i.e.,
spontaneous reversion rate)) exceeding 2 was set.[10,11]
FIGURE 3 Antifungal activity of acyl thiourea mixtures. Acyl
thiourea derivatives 3, 5, and 6 were applied as a mix of 50 μg/mL
(each) or 16.6 μg/mL (each) and compared to the single compound
(inlet number) which gave the highest activity (Table 1). Designation
of strains is as in Table 1. Data represent means and standard
deviations (error bars) from experiments carried out in triplicate. All
other experimental procedures were as described in Experimental
part
(5 vs. 6) does not alter the activity. In these structures, the electron
density and delocalization of the phenyl ring probably prevented
interaction with enzymatic targets of the mentioned atoms.
FIGURE 4 Structure–antifungal activity relationship of novel acyl thioureas

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In all tests the mutagenicity index was below 2, i.e., the number of
revertants did not increase; also in the presence of S9-mix a mutagenic
effect was not observed (Table 2).
activity and drug likeness of novel substances.[14,15] If two or more of
“drug-like” requirements are not met, there is a high probability of poor
bioavailability for the drug candidate[16–20] (Table 3). So, if the
molecular weight (MW) is less than 500 g/mol, it may penetrate
through biological membranes more easily and rapidly, may have a
minimum number of undesirable targeting adhesions, and therefore
has less toxic side effects. Hydrogen bond donor (HBD) and acceptor
(HBA) numbers indicate the compound's tendency to form hydrogen
bonds and to dissolve in water. When the value is high, the adhesion to
biological targets could become too strong and cause pathological
changes for organism. If the logarithm of the substance distribution
ratio (log D) between n-octanol and water is below −0.5, the substance
does not dissolve in the lipid phase and penetrate through the cell
membrane, i.e., it will lose the ability to be absorbed into the blood
stream from the gastrointestinal tract. As far as studied acyl thioureas
are ionizable substances, the log D was calculated assuming different
levels of pH (3.0, 7.4, and 9.0).[21] Special attention was given to
pH = 7.4 considering it is the H+ concentration of human plasma. The
topological polar surface area (TPSA) of a molecule is defined as the
surface sum over all polar atoms, showing a low permeability through
cell membranes, when it is larger than 140 angstroms2 (Å2).[22] In order
to penetrate through the blood–brain barrier, a TPSA must be less than
90 Å2.[23] But when lower than 75 Å2 it associates with an increased
A 10-fold increase in the substance amount to 500 µg per plate
revealed a biocidal effect against Salmonella for all substances except
for 1, 7, and 9. With these compounds there was no dose-dependent
correlation in the number of revertants. At a dose of 500 μg/plate the
inclusion of S9-mix lowered the amount of revertants for TA 100. The
significance of this finding is difficult to evaluate since at this high dose
bactericidal effects may interfere with mutagenic effects. Generally, it
may be assumed that oxidation of amino group mediated by
cytochrome P450 of S9-mix could lead to formation of highly
mutagenic N-hydroxylamines, nitroso compounds or nitrenium
ions.[12,13] Nevertheless, we did not find any evidence that S9-mix
was active in this sense. Therefore, according to our test results we
classify novel thiourea derivatives as “non-mutagenic” so far.
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2.5 Physicochemical characteristics
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2.5.1 Drug likeness
Substance promiscuity and frontier orbitals energies are physico-
chemical parameters to get an in-depth understanding of biological
TABLE 2 Mutagenicity indexes (Mi) calculated from numbers of revertants
TA 98
TA 100
Substances
Dosage, μg/plate
+S9-mix
+S9-mix
2-Nitrofluorene
Methyl methansulfonate
2-Aminofluorene
1
10
58.40
1
8.34
1.01
1.82
1.17
1.36
ng
10
1.56
0.60
0.73
0.84
ng
47.35
0.55
0.88
1.12
ng
4.58
0.76
0.69
0.90
ng
50
500
50
2
3
4
5
6
7
8
9
500
50
0.66
ng
0.52
ng
1.39
ng
0.67
ng
500
50
0.66
ng
0.75
0.13
0.95
0.05
0.66
ng
1.37
ng
0.94
ng
500
50
0.60
0.02
0.57
ng
1.81
ng
0.82
ng
500
50
1.58
ng
0.84
ng
500
50
0.73
0.55
0.76
0.06
0.60
0.60
1.12
0.52
1.07
ng
1.64
0.63
1.58
ng
0.96
0.42
0.78
ng
500
50
500
50
0.81
0.68
0.95
1.84
0.74
1.00
500
Substances for positive controls and acyl thioureas (1–9) were solved in DMSO; all assays, including negative controls (spontaneous reversion rate), were
carried out with 100 μL DMSO in top agar. For Mi calculations mean numbers were taken from experiments carried out in triplicate. ng, no growth.

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TABLE 3 Calculated parameters of lead-like and structure optimization
Log D
Log D
Log D
#
1
2
3
4
5
SMILES
MW
pH = 3
−0.63
0.48
pH = 7.4
pH = 10
TPSA
96.25
70.22
70.22
96.25
79.46
HBA
HBD
nrotb
NC(=O)NNC(=S)NC(=O)C1CC1
O=C(NNC(=S)NC(=O)C1CC1)C2CC2
O=C(NC(=S)NNC(=O)C1=CC=CC=C1)C2CC2
NC1=CC=CC=C1C(=O)NNC(=S)NC(=O)C2CC2
202.24
227.29
263.32
278.34
293.35
−0.63
0.48
1.56
1.38
1.22
−1.18
−0.05
0.91
6
5
5
6
6
5
3
3
5
3
4
5
5
5
7
1.56
1.23
0.79
O=C(COC1=CC=CC=C1)NNC(=S)NC(=O)
C2CC2
1.22
0.67
6
O=C(CSC1=CC=CC=C1)NNC(=S)NC(=O)
C2CC2
309.42
1.70
1.70
1.16
70.22
5
3
7
7
8
O=C(NC(=S)NNC(=O)C1=CC=NC=C1)C2CC2
264.31
300.34
0
0.34
1.41
−0.43
83.11
83.11
6
6
3
3
5
5
O=C(NNC(=S)NC(=O)C1=CC=CC=C1)
C2=CC=NC=C2
1.07
0.78
9
NC(=S)NNC(=S)NC(=O)C1CC1
218.31
0.26
0.26
−0.27
79.17
5
5
5
Drug lead-like criteria
≤500
≤5
≤140
≤10
≤5
≤10
SMILES, simplified molecular input line entry system; MW, molecular weight; Log D, n-octanol/water distribution coefficient; TPSA, molecular polar surface
area; HBA, hydrogen bonds acceptors; HBD, hydrogen bonds donors; nrotb, number of rotatable bonds.
risk of adverse effects due to non-specific toxicity, particularly when
combined with a high lipophilicity (log D > 4). An increase in the
number of rotatable bonds (nrotb) increases free rotation axes of the
molecule and thereby provides an enhanced flexibility.
any approved drug (true means it was found in the base; and false − not
found). A high pScore value combined with a “true” inDrug finding are
strong predictors to an enhanced promiscuity and potential toxicity.
Such an analysis revealed that pyridine scaffolds of 7 and 8 have a
pScore higher than 300 (Table 4). Taking under consideration the
presence of pyridine in their structure, a mutagenic potential can be
assumed. At least this was not confirmed by bacterial mutagenicity
analysis (Table 2). Since compounds 7 and 9 showed low antifungal
activity, they will not be taken under consideration for further
antifungal studies. The substances 5 and 8 had pScore/inDrug profile
determined as low/false. The pScores of 1–7 and 9 are calculated as
moderate (185) reflecting the presence of the cyclopropyl ring.
Calculating the promiscuity of 2–4, 6, and 7 no data were generated,
which meant a neutral result with respect to toxicity prediction. Still
lacking heterocyclic rings the substances are assumed to have a low
toxicity profile.
As calculations revealed (Table 3), all substances do not violate
drug-like criteria and fulfill the requirements of the bioavailability
rules.[18–20]
Still there are some aspects worth to be discussed: the molecular
TPSAs for all acyl thioureas were not more than 140 Å2, but lower than
75 Å2 for substances 2, 3, and 6; these could possibly penetrate the
blood brain barrier. Compounds 1, 4, and 9 have the limit amount of
HBD (5) and could remain in the cell due to strong hydrogen bonding.
At pH = 7.4 substance 1 is quite hydrophilic with a log D of −0.63. At
the same pH compounds 2, 7, and 9 have the lowest lipophilicity (log
D = 0.48, 0.34, and 0.26, respectively). In contrast, 3, 5, and 6 appeared
as the most penetrable ones, due to highest lipophilicity and lowest
TPSA. It is notable that these above-mentioned profiles correlate with
their distinctive antifungal activity.
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2.5.3 Frontier molecular orbitals energy
Next, molecular mechanics, namely, calculations of the frontier
molecular orbitals energies to predict substances toxicity by HOMO
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2.5.2 Promiscuity
The next analysis was to determine how far acyl thioureas may
categorize as “Pan Assay Interference Compounds” (PAINS).[24] PAINS
are compounds that turn up as frequent hitters in many biochemical
high-throughput drug discovery screens and so look as promising
starting point for further drug development. Unfortunately, often
these substances are “promiscuous,” i.e., reacting simultaneously with
many targets in a non-specific or even indirect mode.[25] Correspond-
ingly, substances were screened by means of bioactivity data using an
associative promiscuity pattern learning engine[26,27] (Table 4). The
pScore column points to promiscuity, whereas “inDrug” column
indicates whether the corresponding molecular scaffold exists within
TABLE
4
Calculated promiscuity scores by bioactivity data
associative promiscuity pattern learning engine
Substance
Scaffold
pScore
inDrug
True
1–7, 9
Cyclopropane
Whole molecule
Whole molecule
Pyridine
185 (moderate)
30 (low)
5
False
False
True
8
2 (low)
7, 8
328 (high)
False, not found in reported drug; true, found.

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(highest occupied molecular orbital) and LUMO (lowest unoccupied
molecular orbital) (Table 5) were carried out.[14,28] Also energy gap and
molecular descriptors computed at the level of semiempirical
molecular mechanics (MM+ and MNDO (modified neglect of diatomic
overlap)) by the means of HyperChem Professional 8.0[29] are shown
as bioactivity indicators.
hydrogen bond between sulfur and MET A:508 (3.72 Å), π-alkyl bonds
of cyclopropyl fragment with TYR A:132 and A:118 (4.97 and 3.58 Å)
and two unfavorable positive–positive repulsion of the phenyl ring and
HEM-nitrogen A:601 (3.98 and 4.78 Å). Additionally, substance 3 also
interacts with GLY A:307 and LEU A:376 of target due to van der
Waals attraction.
The binding ability of the molecule gets stronger with increasing
HOMO and decreasing LUMO energy values. If LUMO energy is
negative, the substance is considered electrophilic. When the HOMO–
LUMO energy gap (ΔE) increases, the molecule becomes harder (η),
more stable and less reactive. Vice versa, the higher the electronega-
tivity (χ) and the electronic chemical potential (μ), the less stable or
more reactive the molecule will be. Electrophilicity index (ω) is also an
indicator of the stabilization in energy after a system accepts additional
amount of electronic charge from the environment. A highly bioactive
substance typically has an elevated electrophilicity index and low
hardness. As it is shown in Table 5, the studied substances are not
highly reactive hard electrophiles. According to the index of
electrophilicity, substances 7–9 are predicted as the most bioreactive
among tested series. In contrast they are low with antifungal activity,
and therefore are not in the focus of future antifungal studies.
The binding of substance 5 is similar to substance 3 with some
additional aspects: a further hydrogen bond is predicted between
protonated nitrogen and GLY A:307 (2.70 Å). Besides
a third
hydrophobic π-alkyl bond is found between cyclopropyl and LEU
A:121 (5.48 Å). Also the same type of bond is demonstrated for phenyl
ring with ILE A:131 (5.01 Å) in parallel to π-sigma one with HEM A:601
(3.80 Å). Also van der Waals interactions with VAL A:509, PHE A:228,
and LEU A:376 are shown.
Considering N-myristoyltransferase enzyme, two times more
bonds are predicted (Figure 6) with substances 3 or 5.
Compound 3, it forms a hydrogen bond with sulfur by TYR B:354
(3.78 Å), π-sulfur bond with PHE B:117 (5.06 Å). LEU B:451 binds to
three positively charged nitrogens (2.20, 4.07, and 4.19 Å) due to
electrostatic attractive charge interaction. Also cyclopropyl interacts
with two TYR B:119 (5.47 Å) and B:107 (4.43 Å), LEU B:337 (3.85 Å) by
alkyl bonding. π–π T-shaped bond is shown between TYR B:225
(4.97 Å) and phenyl ring, with additionally π-σ type to LEU B:394
(3.64 Å). Additionally, compound 3 binds by three van der Waals
interactions comprising LEU B:415; B:450, and TYR B:335.
Substance5fits intotheenzymeactivesite duetotwo conventional
hydrogen bonds between sulfur and THR B:211 (4.46 Å), oxygen and
TYRB:335(2.93 Å). Furtherinteractionsarepredicted:carbon hydrogen
bond of OCH2 fragment with LEU B:450 (3.75 Å), attractive charge type
of nitrogen with LEU B:451 (3.15 Å), three alkyl bonds to cyclopropyl by
TYR B:107 (4.68 Å), PHE B:117 (3.84 Å) and LEU B:337 (5.18 Å), π-σ
bond by LEU B:394 (3.64 Å), two π–π T-shaped bonds by TYR B:354
(5.36 Å) and B:225 (5.13 Å) to the phenyl ring. But, surprisingly, no van
der Waals attractions were present.
|
2.6 Molecular docking studies
Analysis of the in silico molecular docking analysis[30] was used as a tool
to predict affinity scores of hymexazol and acyl thioureas 1–9 to
common antifungal targets[5] (Table 6).
The 14α-demethylase (CYP51) and N-myristoyltransferase (NMT)
are found to be possible target enzymes for lead-compounds 3 and 5
according to their best affinity scores (−7.8 to −8.2) (Table 6). This
finding is in agreement with earlier studies of antifungal activity of
thiosemicarbazides.[5]
The visualization obtained by molecular docking results indicates
(Figure 5) that substance 3 binds to CYP51 due to the presence of a
TABLE 5 Frontier molecular orbitals
Electrochem.
HOMO/
LUMO/
|electron
affinity|
potential, μ,
|electronegat.,
HOMO
|ionization
potential|
LUMO
+1 MO
Energy
Hardness,
Softness,
Electrophilicity
#
−1 MO
gap, ΔE
χ|
η
σ
index, ω
1
−9.6533
−9.5638
−9.6172
−9.2885
−9.5897
−9.5851
−10.0023
−9.5560
−9.2455
−10.4157
−9.4418
−9.2448
−9.3720
−9.2428
−9.1785
−9.5536
−9.5875
−9.8320
−9.0340
−10.1161
−0.7390
−0.5587
−0.6992
−0.6146
−0.8196
−0.7934
−0.8866
−0.7814
−0.9731
−0.1170
0.5070
8.7028
8.6861
8.6727
8.6282
8.3589
8.7602
8.7009
9.0506
8.0609
9.9992
5.09
4.90
5.04
4.93
5.00
5.17
5.24
5.31
5.00
5.12
4.35
4.34
4.34
4.31
4.18
4.38
4.35
4.53
4.03
5.00
0.23
0.23
0.23
0.23
0.24
0.23
0.23
0.22
0.25
0.20
2.98
2.77
2.92
2.82
2.99
3.06
3.15
3.11
3.11
2.62
2
0.2751
3
−0.4345
−0.4159
0.0918
4
5
6
−0.2975
−0.5951
−0.4916
−0.1738
−0.9532
7
8
9
Hymexazol
Energies of frontier molecular orbitals, the energy gap and molecular descriptors computed at the level of semi-empirical MM+ and MNDO Hamiltonians, eV.

ANTYPENKO ET AL.
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TABLE 6 Affinity to binding sites
Affinity, kcal/mol
Candida albicans
Escherichia coli
Sacchromyces cerevisiae
Sub.
5TZ1
−4.3
−6.1
−6.9
−8.2
−8.2
−8.1
−8.0
−7.7
−8.8
−5.6
−7.5
1IYL
1EAG
−3.9
−5.7
−6.0
−7.1
−7.0
−7.3
−7.1
−6.3
−7.4
−5.3
−6.6
1UAG
−4.4
−6.4
−5.9
−6.5
−6.8
−6.6
−6.8
−6.6
−7.3
−5.7
−6.5
1XFF
−5.1
−6.3
−6.0
−5.6
−5.6
−6.3
−6.2
−5.5
−6.0
−5.9
−5.9
1Q1D
−4.6
−6.3
−6.5
−6.4
−6.4
−6.2
−5.8
−5.8
−7.1
−6.2
−6.3
Mean
−4.6
−6.2
−6.3
−6.9
−6.9
−7.1
−6.9
−6.5
−7.6
−5.7
–
Hymexazol
−5.0
−6.1
−6.6
−7.8
−7.6
−7.8
−7.7
−7.1
−9.1
−5.7
−7.3
1
2
3
4
5
6
7
8
9
Mean for 1–9
The calculated affinity of substances to binding sites of sterol 14α-demethylase (CYP51) 5TZ1, N-myristoyltransferase (NMT) 1IYL, secreted aspartic
proteinase (SAP2) 1EAG, UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) 1UAG, L-glutamine:D-fructose-6-phosphate amidotransferase (GlcN-
6-P) 1XFF and topoisomerase II (Topo II) 1Q1D.
A 3-D fit of substances within above-mentioned enzymes active
sites is shown on the right of Figures 5 and 6.
descriptors in order to identify those parameters having the highest
impact on the activity presence for tested series of compounds
(Table 7).
The highest correlation coefficient was found between average
antifungal activity of each substance and their TPSA (0.6672), whereas
MW influenced much less (R2 = 0.2093). Also protonation at pH = 3
caused the moderate result of correlation − 0.5339, and deprotonation
|
2.7 Correlation activity – structural descriptors
Correlation coefficients were determined between average antifungal
properties of each acyl thiourea derivative and its calculated
FIGURE 5 Visual representation (2D and 3D) of the lead compounds 3 and 5 showing bonds formation and position in the active site of
14α-demethylase (5TZ1). Red − unfavorable positive–positive interaction, pale green − van der Waals interaction, green − classical
conventional hydrogen bond, violet − hydrophobic π-σ bond, pink − hydrophobic alkyl and π-alkyl bonds

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ANTYPENKO ET AL.
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FIGURE 6 Visual representation (2D and 3D) of the lead-compounds 3 and 5 showing bonds formation and position in the active site of N-
myristoyltransferase (1IYL). Pale green − van der Waals interaction, green − classical conventional hydrogen bond, light blue − non-classical
carbon hydrogen bond, dark orange − electrostatic attractive charge interaction, light orange − miscellaneous π-sulfur bond, violet −
hydrophobic π-σ bond, magenta − hydrophobic π–π T-shaped bond, pink − hydrophobic alkyl and π-alkyl bonds
at pH = 10 lead to decrease of R2 with 0.4575. And at pH = 7.4, R2 was
the lowest among them (0.4250). Electrophilicity was not linearly
related to detected antifungal activity. Among enzymatic targets
affinity to SAP2 had the highest correlation to found antifungal
properties (0.2551).
frame-shift mutated TA98 strain a weak correlation (0.2191) with the
LUMO was detected. This result confirms our finding of the absence of
a mutagenic potential of this series of acyl thioureas.
|
3
CONCLUSION
Lipophilic compounds with low-energy LUMOs are considered
likely to be mutagenic, hence possibly carcinogenic.[31–33] Therefore,
this parameter was also calculated against found results (mean of Mi-
values of all acyl thioureas of both tester strains with/without S9-mix,
respectively; Table 2) from Salmonella mutagenicity assay. Only to the
Among nine novel acyl thioureas potent non-mutagenic antifungal
substances with a low toxicity profile were identified, which warrant
follow-up studies: further investigations of antifungal activity of active
TABLE 7 Correlation coefficients between average antifungal activity of 1–9 to their calculated physico-chemical descriptors, and LUMO to
mutagenicity or lipophilicity results
Average antifungal activity against
Log D at pH
Descriptor
R2
MW
TPSA
3
7.4
10
Electrophilicity index
0.2093
0.6672
0.5339
0.4250
0.4575
0.0181
Affinity
Enzyme
CYP51
NMT
SAP2
0.2551
Topo II
MurD
GlcN-6-P
R2
0.1368
0.1684
0.0030
0.0034
0.0597
LUMO energy against
TA 98, 50 µg/plate
TA 100, 50 µg/plate
+ S9
Log D at pH
Salmonella strain/descriptor
+ S9
0.0048
3
7.4
0.0315
10
R2
0.2191
0.0114
0.0524
0.0441
0.0390
MW, molecular weight; TPSA, molecular topological polar surface area; Log D, distribution coefficient; Salmonella strain TA 98 and TA 100 without and with
addition of S9-mix; affinity to 14α-demethylase (CYP51), N-myristoyltransferase (NMT), secreted aspartic proteinase (SAP2), topoisomerase II (Topo II),
UDP-N-acetyl-muramoyl-L-alanine:D-glutamate ligase (MurD), L-glutamine:D-fructose-6-phosphate aminotransferase (GlcN-6-P), LUMO, energy of lowest
unoccupied molecular orbital.

ANTYPENKO ET AL.
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substance mixtures to broaden spectrum of their properties; ecotoxicity
tests for lead substances 3, 5, and 6 to be used in agriculture; antifungal
spectrum widening to human pathogens for active compounds;
chemical modification of the main core according to SAR results;
studies of in vitro enzymatic affinity to validate the predicted
interactions with target structures as identified by docking studies.
N-(2-(Cyclopropanecarbonyl)hydrazine-1-carbonothioyl)-
cyclopropanecarboxamide (2)
Yield: 52.6%; mp 192–192°C; IR (cm−1): 3193, 1683, 1651, 1391,
1138, 1102, 940, 882, 866, 818, 709, 676, 642; 1H NMR, δ, ppm (J, Hz):
12.58 (s, 1H, -C(O)NHC(S)-), 11.58 (s, 1H, -C(S)NHNHC(O)-), 10.77 (s,
1H, -C(S)NHNHC(O)-), 2.13–1.98 (m, 1H, cyclopropanecarboxamide
H-1), 1.93–1.79 (m, 1H, cyclopropanecarbonylhydrazine, H-1), 1.15–
0.51 (m, 8H, cyclopropanecarboxamide H-2,2′,3,3′, cyclopropanecar-
bonylhydrazine H-2,2′,3,3′); LC-MS: m/z = 228 [M+1]. Anal. calcd. for
C9H13N3O2S: C, 47.56; H, 5.77; N, 18.49; S, 14.11. Found: C, 47.67; H,
5.65; N, 18.54; S, 14.18.
|
4
EXPERIMENTAL
|
4.1 Chemistry
|
4.1.1 General
N-(2-Benzoylhydrazine-1-carbonothioyl)cyclopropanecarboxamide
(3)
Melting points were determined in open capillary tubes and were
uncorrected. The elemental analyses (C, H, N, S) were performed using
a vario EL Cube analyzer (Elementar Americas, NJ, USA). Analyses
were indicated by the symbols of the elements or functions within
0.3% of the theoretical values. The 1H NMR spectra (400 MHz) were
recorded on a Varian-Mercury 400 (Varian Inc., Palo Alto, CA, USA)
spectrometer with TMS as an internal standard in DMSO-d6 solution.
LC-MS were recorded using the chromatography/mass spectrometric
system consisting of an “Agilent 1100 Series” high performance liquid
chromatograph (Agilent, Palo Alto, CA, USA) equipped with an “Agilent
LC/MSD SL” diode-matrix.
Yield: 75.4%; mp 204–206°C; IR (cm−1): 3230, 1627, 1519, 1455,
1290, 1217, 1155, 950, 912, 707, 670, 619; 1H NMR, δ, ppm (J, Hz):
12.40 (s, 1H, −C(O)NHC(S)-), 11.76 (s, 1H, -C(S)NHNHC(O)-), 10.81 (s,
1H, -C(S)NHNHC(O)-), 7.91 (d, J = 7.2, 2H, Ph H-2,6), 7.54 (t, J = 7.2,
1H, Ph H-4), 7.46 (t, J = 7.1, 2H, Ph H-3,5), 2.22–1.93 (m, 1H, Cpr H-1),
1.18–0.75 (m, 4H, Cpr H-2,2′,3,3′); LC-MS: m/z = 264 [M+1], 265
[M+2]. Anal. calcd. for C12H13N3O2S: C, 54.74; H, 4.98; N, 15.96; S,
12.18. Found: C, 54.79; H, 5.04; N, 16.02; S, 12.22.
N-(2-(2-Aminobenzoyl)hydrazine-1-carbonothioyl)-
Starting materials and solvents were obtained from commercially
available sources and used without additional purification.
IR, LC-MS and 1H, 13C NMR spectra of the novel substances 1–9
are provided as Supporting Information. The InChI codes of the
investigated compounds together with some biological activity data
are also provided as Supporting Information.
cyclopropanecarboxamide (4)
Yield: 81.6%; mp 187–189°C; IR (cm−1): 3155, 1652, 1515, 1390,
1158, 931, 668; 1H NMR (DMSO-d6), δ, ppm (J, Hz): 12.52 (s, 1H, -C(O)
NHC(S)-), 11.74 (s, 1H, -C(S)NHNHC(O)-), 7.95–7.62 (m, 2H, NH2),
7.53 (d, J = 7.7, 1H, Ar H-6), 7.15 (t, J = 7.6, 1H, Ar H-4), 6.72 (d, J = 8.2,
1H, Ar H-3), 6.53 (t, J = 7.4, 1H, Ar H-5), 2.24–1.95 (m, 1H, Cpr H-1),
1.12–0.79 (m, 4H, Cpr H-2,2′,3,3′); LC-MS: m/z = 279 [M+1], 280
[M+2]. Anal. calcd. for C12H14N4O2S: C, 51.78; H, 5.07; N, 20.13; S,
11.52. Found: C, 51.82; H, 5.11; N, 20.18; S, 11.56.
|
4.1.2 General procedure for the synthesis of
substances 1–9
To a solution of proper chloroanhydrides (0.01 mol) in 20 mL of
acetonitrile 0.76 g of ammonium isothiocyanate (0.01 mol) was added
and stirred at 80°C for 30 min. The mixture was cooled down to r.t. and
0.01 mol of proper acylhydrazide, or hydrazinecarboxamide, or hydrazi-
necarbothioamide was added and stirred at 80°C for further 90 min. The
solution was cooled down, poured into the water and the formed
precipitate was filtrated, dried, and recrystallized from methanol.
N-(2-(2-Phenoxyacetyl)hydrazine-1-carbonothioyl)-
cyclopropanecarboxamide (5)
Yield: 70.8%; mp 194–196°C; IR (cm−1): 1651, 1549, 1472, 1441,
1394, 1224, 1158, 1036, 936, 885, 836, 749, 668, 628; 1H NMR, δ,
ppm (J, Hz): 12.62 (s, 1H, -C(O)NHC(S)-), 11.77 (s, 1H, -C(S)NHNHC-
(O)-), 10.73 (s, 1H, -C(S)NHNHC(O)-), 7.27 (t, J = 7.8, 2H, Ph H-3,5),
7.01–6.85 (m, 3H, Ph H-2,4,6), 4.65 (s, 2H, -CH2OPh), 2.16–1.98 (m,
1H, Cpr H-1), 1.04–0.66 (m, 4H, Cpr H-2,2′,3,3′); 13C NMR, δ, ppm:
178.29 (-C(O)NHC(S)-), 175.74 (C(O)NHC(S)-), 165.59 (-C(S)NHNHC
(O)-), 158.11 (Ph C-1), 129.96 (Ph C-3,5), 121.77 (Ph C-4), 115.18 (Ph
C-2,4), 66.02 (-CH2OPh), 14.37 (Cpr. С-1), 9.87 (Cpr. С-2,3). LC-MS:
m/z = 294 [M+1], 295 [M+2]. Anal. calcd. for C13H15N3O3S: C, 53.23;
H, 5.15; N, 14.32; S, 10.93. Found: C, 53.29; H, 5.22; N, 14.38; S, 10.97.
2-((Cyclopropanecarbonyl)carbamothioyl)hydrazine-1-
carboxamide (1)
Yield: 75.2%; mp 199–201°C; IR (cm−1): 3212, 1692, 1504, 1395, 1233,
1186, 1153, 1074, 1031, 945, 882, 763, 710, 668; 1H NMR, δ, ppm
(J, Hz): 12.56 (s, 1H, C(O)NHC(S)-), 11.45 (s, 1H, -C(S)NHNHC(O)-),
8.88 (s, 1H, -C(S)NHNHC(O)-), 6.21 (s, 2H, NH2), 2.14–1.93 (m, 1H, Cpr
H-1), 1.02–0.77 (m, 4H, H-2,2′,3,3′); 13C NMR, δ, ppm: 183.44 (-C(O)
NHC(S)-), 175.57 (-C(O)NHC(S)-), 156.41 (NHC(O)NH2), 14.27 (Cpr С-
1), 9.57 (Cpr С 2,3); LC-MS: m/z = 203 [M+1], 204 [M+2]. Anal. calcd. for
C6H10N4O2S: C, 35.64; H, 4.98; N, 27.70; S, 15.85. Found: C, 35.72; H,
5.05; N, 27.76; S, 15.89.
N-(2-(2-(Phenylthio)acetyl)hydrazine-1-carbonothioyl)-
cyclopropanecarboxamide (6)
Yield: 76.4%; mp 187–189°C; IR (cm−1): 1680, 1643, 1436, 1386,
1213, 1156, 873, 738, 670; 1H NMR, δ, ppm (J, Hz): 12.69 (d, J = 5.0,
1H, -C(O)NHC(S)-), 11.68 (s, 1H, -C(S)NHNHC(O)-), 10.99 (d, J = 4.9,

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1H, -C(S)NHNHC(O)-), 7.40 (d, J = 7.6, 2H, Ph-H 2,6), 7.28 (t, J = 7.6,
2H, Ph H-3,5), 7.17 (t, J = 7.2, 1H, Ph H-4), 3.76 (s, 2H, -CH2SPh), 2.18–
1.96 (m, 1H, Cpr. H-1), 1.20–0.68 (m, 4H, Cpr H-2,2′,3,3′); LC-MS:
m/z = 310 [M+1], 312 [M+3]. Anal. calcd. for C13H15N3O2S2: C, 50.47;
H, 4.89; N, 13.58; S, 20.72. Found: C, 50.53; H, 4.93; N, 13.64; S, 20.79.
dextrose agar (PDA) was purchased from C. Roth (Karlsruhe,
Germany). Hymexazol (98%) was obtained from Prosperity World
Store (Hebei, China). Strains were cultivated on PDA for 6 d at 25°C.
Spores from each strain were gently harvested with a sterile glass rod
from plate surfaces with deionized water. Spore concentration
numbers in suspension were determined microscopically and adjusted
to 7.5 × 106 spores/mL. Clear stock solutions of 5 mg/mL were made
of 0.050 g of reference substance hymexazol or acyl thiourea in 10 mL
of sterile dimethyl sulfoxide (DMSO). A total of 1 mL of each stock
solution was mixed in situ into 99 mL of PDA prior to solidification to
obtain a final concentration of 50 μg/mL. In the same way, series of
PDA with tested compounds were prepared with final concentrations
of 25, 10, 5, and 1 μg/mL. A total of 9 mL of each mixture were poured
into 6 cm diameter petri dishes. After solidification central hole
(diameter: 2.5 mm) was cut out and inoculated with 6.5 μL spore
suspension. Plates were incubated at 25°C ( 1°C) for 6 d. Control
plates containing only PDA and water were prepared in the same way.
Inhibitory effects (I %) were determined by analyzing growth zone
diameters and calculated as described by Tang et al.[9]: I % = [(C − T)/(C
− 2.5 mm)]) × 100, where C (mm) represents the growth zone of control
PDA, and T (mm) is the average growth zone in the presence of
reference or test substances. All growth experiments were carried out
in triplicate. Means and standard deviations were calculated with
software “Excel 2016” (Microsoft, USA).
N-(2-Isonicotinoylhydrazine-1-carbonothioyl)-
cyclopropanecarboxamide (7)
Yield: 72.3%; mp 183–186°C; IR (cm−1): 2846, 1655, 1472, 1217,
1162, 1113, 944, 910, 870, 735, 674; 1H NMR, δ, ppm (J, Hz): 12.27 (s,
1H, -C(O)NHC(S)-), 11.80 (s, 1H, -C(S)NHNHC(O)-), 11.20 (s, 1H, -C(S)
NHNHC(O)-), 8.70 (d, J = 5.2, 2H, Py H-2,6), 7.80 (d, J = 5.3, 2H, Py H-
3,5), 2.20–1.96 (m, 1H, Cpr H-1), 1.17–0.69 (m, 4H, Cpr H-2,2′,3,3′);
LC-MS: m/z = 265 [M+1], 266 [M+2]. Anal. calcd. for C11H12N4O2S: C,
49.99; H, 4.58; N, 21.20; S, 12.13. Found: C, 50.06; H, 4.63; N, 21.26; S,
12.16.
N-(2-Isonicotinoylhydrazine-1-carbonothioyl)benzamide (8)
Yield: 89.3%; mp 265–267 °C; IR (cm−1): 1669, 1486, 1274, 1179, 756,
714, 668; 1H NMR, δ, ppm (J, Hz): 12.59 (s, 1H, -C(O)NHC(S)-), 11.66 (s,
1H, -C(S)NHNHC(O)-), 11.34 (s, 1H, -C(S)NHNHC(O)-), 8.72 (d, J = 4.9,
2H, Py H-2,6), 8.05 (d, J = 7.5, 2H, Ph H-2,6), 7.84 (d, J = 4.9, 2H, Py H-
3,5), 7.61 (t, J = 7.1, 1H, Ph H-4), 7.49 (t, J = 7.5, 2H, Ph H-3,5); LC-MS:
m/z = 301 [M+1], 302 [M+2]. Anal. calcd. for C14H12N4O2S: C, 55.99;
H, 4.03; N, 18.66; S, 10.67. Found: C, 56.05; H, 4.06; N, 18.72; S, 10.71.
|
4.3 Salmonella reverse mutagenicity test
N-(2-Carbamothioylhydrazine-1-carbonothioyl)-
cyclopropanecarboxamide (9)
The mutagenicity test was applied as a standard plate incorporation
assay with Salmonella typhimurium strains TA 98 and TA 100 as
described by Maron and Ames.[10] Tested Salmonella strains were
obtained from culture collection, University of Göteborg (Göteborg,
Sweden). 2-Nitrofluorene (2-NF), dimethylsulfoxide (DMSO), 2-
aminofluorene (2-AF), methyl methanesulfonate (MMS), β-nicotin-
amide adenine dinucleotide phosphate hydrate (β-NADP), and
glucose-6-phosphate were purchased from Sigma–Aldrich (Steinheim,
Germany), whereas D(+)-biotin, D(+)-glucose anhydrous, L-histidine and
NaNH4HPO4 were sourced from Carl Roth GmbH & Co. KG (Karlsruhe,
Germany). Citric acid monohydrate, NaCl, NaH2PO4, K2HPO4 anhy-
drous, MgCl2*6H2O, KCl were purchased from Applichem GmbH
(Darmstadt, Germany). NaOH solution was obtained from Riedel
deHaen/Seelze (Hannover, Germany), MgSO4 anhydrous was ob-
tained from Merck (Darmstadt, Germany). Stock solutions of controls
and acyl thioureas were solved in DMSO. Their final doses in top agar
were adjusted to 50 and 500 μg/plate. The positive controls were 2-
NF (10 mg/mL in DMSO; 10 µL/plate) for TA98 and methyl-methane-
sulfonate (MMS; 10 % (v/v) in DMSO; 1 µL/plate) for TA100; buffer
with 100 µL of DMSO for both strains was used as negative control
(i.e., determination of spontaneous reversion rate). In parallel experi-
ments with metabolic activation were carried out by adding activated
rat liver extract (S9-mix, Trinova Biochem, Giessen/Germany) instead
of sodium buffer. Activity of S9-mix was confirmed with Salmonella TA
98 and 2-aminofluorene (2-AF, 10 mg/mL in DMSO, 10 μL/plate). All
further experimental procedures were as described.[10]
Yield: 60.3%; mp 252–254°C; IR (cm−1): 3123, 1686, 1615, 1524,
1465, 1221, 1170, 1145, 943, 828, 734, 680, 639; 1H NMR, δ, ppm (J,
Hz): 13.37 (s, 1H, -C(O)NHC(S)-), 11.53 (s, 1H, -C(S)NHNHC(O)-),
10.49 (s, 1H, -C(S)NHNHC(O)-), 7.68 (s, 2H, NH2), 2.05 (dt, J = 12.5,
6.3 Hz, 1H, Cpr H-1), 1.02–0.67 (m, 4H, Cpr H-2,2′,3,3′); 13C NMR, δ,
ppm: 182.05 (-C(O)NHC(S)-), 175.23 (-C(O)NHC(S)-), 169.59 (NHC(S)
NH2), 14.31 (Cpr С-1), 9.62 (Cpr С-2,3); LC-MS: m/z = 219 [M+1]. Anal.
calcd. for C6H10N4OS2: C, 33.01; H, 4.62; N, 25.67; S, 29.37. Found: C,
33.17; H, 4.69; N, 25.72; S, 29.43.
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4.2 Antifungal activity
The mycelial growth rate assay was used for antifungal studies.[9]
Strains of filamentous fungi were obtained from the following sources:
Asperillus niger (AN) DSM 246, Altenaria alternata (AA) DSM 1102,
F. equiseti (FE) DSM 21725, F. graminearum (FG) DSM 1095 and
F. fujikuroi (FF) DSM 893, Verticillium lecanii (VL), M. indicus (MI) DSM
2185, P. digitatum (PD) DSM 2731 from DSMZ (Braunschweig,
Germany); Fusarium oxysporum (FO) 39/1201 St. 9336 and Botrytis
cinerea from the Technische Universität Berlin (Germany); C.
higginsianum (CH) MAFF 305635, originally isolated in Japan, via the
Department of Biology, Friedrich-Alexander-Universität (Erlangen,
Germany). Oomycete strains Phytophthora infestans (PI GL-1) GL-1 01/
14 wild strain, p-3 (PI p-3) (4/91; R+) and p-4 (PI p-4) (4/91; R−) were
kindly donated by Julius Kühn-Institut (Quedlinburg, Germany). Potato

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4.4 Frontier molecular orbitals calculations
03FH025IX4) for financial support of this work; Federal Research
Centre for Cultivated Plants (Quedlinburg, Germany) for presenting
the strain Phytophthora infestans; Department of Biology, Friedrich
Alexander University, Erlangen, Nürnberg, Germany for the strain
Colletotricum higginsianum, and Dr. Oleksii Antypenko (Zaporizhzhya
State Medical University, Ukraine) for conducting molecular docking.
All calculations based on semi-empirical molecular orbital theory have
been carried out using ChemDraw 15.0[34] and HyperChem Profes-
sional 8.0[29] molecular modeling softwares. The structures of the
investigated molecules in ground state were optimized using MM+
(molecular mechanics) and MNDO (modified neglect of the diatomic
overlap) semi-empirical methods with RHF (restricted Hartree–Fock)
basis. All calculations referred to the isolated molecule (gas phase). In
this respect, the conjugate gradients algorithm (Polak-Ribier) was
employed for the geometry optimization using a convergence set to
the value of 0.01 kcal/(Å mol). The geometry optimization was done by
minimization of the binding energy of the molecule. Based on
theoretical MOs energy spectra the following molecular descriptors
have been calculated: ionization potential (IP), electron affinity (EA),
electronegativity (χ), chemical hardness (η) and softness (σ), and
electrophilicity index (ω) according to the reported equations.[14,28]
CONFLICT OF INTEREST
The authors declare no competing financial interest.
ORCID
Lyudmyla Antypenko
Sergiy Kovalenko
http://orcid.org/0000-0003-0057-1551
http://orcid.org/0000-0001-8017-9108
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How to cite this article: Antypenko L, Meyer F, Kholodniak
O, et al. Novel acyl thiourea derivatives: Synthesis, antifungal
activity, gene toxicity, drug-like and molecular docking
screening. Arch Pharm Chem Life Sci. 2018;1–14.
https://doi.org/10.1002/ardp.201800275
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