Synergistic effects of thiosemicarbazides with clinical drugs against S. Aureus

Article abstract of DOI:10.3390/molecules25102302

Antimicrobial resistance spurred by the overuse and misuse of antibiotics is a major global health concern, and of the Gram positive bacteria, S. aureus is a leading cause of mortality and morbidity. Alternative strategies to treat S. aureus infections, s

Full text of DOI:10.3390/molecules25102302

molecules
Article
Synergistic Effects of Thiosemicarbazides with
Clinical Drugs against S. aureus
Beata Chudzik-Rza˛d 1, Anna Malm 1 , Nazar Trotsko 2 , Monika Wujec 2 , Tomasz Plech 3
and Agata Paneth 2,
*
1
Department of Pharmaceutical Microbiology with Laboratory for Microbiological Diagnostics,
Faculty of Pharmacy, Medical University of Lublin, Chodz´ki 1, 20-093 Lublin, Poland;
BeChudz@poczta.fm (B.C.-R.); anna.malm@umlub.pl (A.M.)
Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Lublin, Chodzki 4a,
20-093 Lublin, Poland; nazar.trotsko@umlub.pl (N.T.); monika.wujec@umlub.pl (M.W.)
Department of Pharmacology, Faculty of Health Sciences, Medical University of Lublin, Chodz´ki 4a,
20-093 Lublin, Poland; tomasz.plech@umlub.pl
2
3
*
Correspondence: agata.paneth@umlub.pl
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 January 2020; Accepted: 9 May 2020; Published: 14 May 2020
Abstract: Antimicrobial resistance spurred by the overuse and misuse of antibiotics is a major
global health concern, and of the Gram positive bacteria, S. aureus is a leading cause of mortality
and morbidity. Alternative strategies to treat S. aureus infections, such as combination therapy,
are urgently needed. In this study, a checkerboard method was used to evaluate synergistic
interactions between nine thiosemicarbazides (4-benzoyl-1-(2,3-dichloro-benzoyl)thiosemicarbazides
1–5 and 4-aryl-1-(2-fluorobenzoyl)thiosemicarbazides 6–9) and conventional antibiotics against
S. aureus ATCC 25923, which were determined as the fractional inhibitory concentration indices
(FICIs). For these experiments, amoxicillin, gentamicin, levofloxacin, linezolid, and vancomycin
were selected to represent the five antimicrobial classes most commonly used in clinical practice.
With one exception of
combinations tested had an antagonistic effect, showing promising results with respect to a
combination therapy. The synergic effect was observed for the -linezolid, -levofloxacin,
-linezolid, -gentamicin, -linezolid, and -levofloxacin combinations. No interactions were
seen in combination of the thiosemicarbazide with gentamicin or vancomycin, whereas all
combinations with linezolid acted in additive or synergism, except for -gentamicin and -linezolid.
The 4-(4-chlorophenyl)-1-(2-fluorobenzoyl)thiosemicarbazide showed a clear preference for the
7-vancomycin combination, none of the forty-five thiosemicarbazide-antibiotic
2
4
5
6
6
7
6
7
6
potency; it affected synergistically in combinations with gentamicin or linezolid and additively in
combinations with amoxicillin, levofloxacin, or vancomycin. In further studies, the inhibitory potency
of the thiosemicarbazides against S. aureus DNA gyrase and topoisomerase IV was examined to
clarify the molecular mechanism involved in their synergistic effect in combination with levofloxacin.
The most potent synergist
6 at concentration of 100 µM was able to inhibit ~50% activity of S. aureus
DNA gyrase, thereby suggesting that its anti-gyrase activity, although weak, may be a possible factor
contributing to its synergism effect in combination with linezolid or gentamycin.
Keywords: thiosemicarbazides; antibacterial activity; synergistic effect; bacterial topoisomerases
1. Introduction
Antimicrobial resistance spurred by the overuse and misuse of antibiotics is a major global health
concern, and of the Gram positive bacteria, S. aureus is a leading cause of mortality and morbidity [
1–7].
The important clinical S. aureus infections are bacteraemia, infective endocarditis, as well as skin and soft
Molecules 2020, 25, 2302; doi:10.3390/molecules25102302
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2302
2 of 13
tissue, osteoarticular, pleuropulmonary, and device-related infections. Other clinical manifestations
of S. aureus infections include epidural abscess, meningitis, toxic shock syndrome, and urinary
tract infections [8,9]. Originally, S. aureus was predominately a nosocomial pathogen. Over time,
epidemiologically distinct clones emerged in the community setting to cause a global public health
concern. In fact, S. aureus deploys a specific combinations of virulence factors, such as adhesins, toxins,
and immunomodulatory molecules, that facilitate infection of host tissues and evading from host
immune response [10
difficult to treat. Indeed, over decades, S. aureus isolates have developed resistance to several classes of
antibiotics, like -lactams, fluoroquinolones, macrolides, glycopeptides, and oxazolidinones [12 13].
Especially, emergence and spread of methicillin-resistant S. aureus (MRSA) resulted in high morbidity
and mortality which demands prompted clinical attention [14 15]. Vancomycin remained as one of
the last resort therapies to tackle these strains for years, but its slow bactericidal activity, low tissue
penetration, and the emergence of resistance restricted its clinical utility [16 19]. Although daptomycin
remains as one of the main treatment options for MRSA, sporadic resistance cases reported in patients
treated with daptomycin are a growing concern [20 21]. This situation has resulted in an urgent need
,11]. The ability to resist the activity of multiple antibiotics classes makes S. aureus
β
,
,
–
,
to identify and develop novel antibacterial candidates to treat infections caused by S. aureus.
To explore and discover novel chemical scaffold with potent antibacterial activity, a library of
about 1000 thiosemicarbazides constructed in our lab was tested in the broth microdilution assay
against the panel of reference Gram positive and Gram negative bacteria. The most potent of
them with 4-benzoylthiosemicarbazide scaffold inhibited the growth of S. aureus clinical isolates
at concentrations equipotent or even lower compared to ampicillin, vancomycin, streptomycin,
and nitrofurantoin [22]. In addition, they gave successful inhibition effect against growth and biofilm
formation by clinical isolates of MSSA (methicillin-sensitive S. aureus), MRSA, and MDR-MRSA
(multidrug-resistant MRSA) [23]. Thus, the unique chemical scaffold, low cytotoxicity and effectiveness
of thiosemicarbazides against S. aureus spurred us to further explore their antibacterial potential and
safety profile. Several studies published in recent years have shown that the combination therapy is
one of the important strategies to improve efficacy and bioavailability, as well as treat mixed diseases in
the clinic. One example is the study by Saisubramanian and co-workers [24] describing the potentiated
effect of ciprofloxacin in combination with benzochromene derivatives against drug resistant strains
of S. aureus. The rationale for the combination therapy includes synergistic effects in vitro, overcome
multi-drug resistance, prevention of dissemination by more rapid bactericidal activity, optimized
biofilm activity, and intracellular penetration. Taking this into account, we performed the microdilution
broth checkerboard assay to evaluate synergism effects between five 4-benzoylthiosemicarbazides
with previously confirmed antibacterial potential [23] and conventional antibiotics against S. aureus
ATCC 25923. For these experiments four 4-arylthiosemicarbazides were also included for comparison,
whereas amoxicillin, gentamicin, levofloxacin, linezolid, and vancomycin were selected to represent the
five antimicrobial classes most commonly used in clinical practice. Our results indicate that is possible
to establish a combination therapy between these substances, since none of the combinations tested
had an antagonistic effect, except for one. Although most combinations had indifferent or additive
effects, six of them showed synergistic interactions. Since combinations of antibacterial agents with
similar mechanisms of action or those that influence the same target exert synergistic effects more
readily than others, in further studies, the inhibitory potency of the thiosemicarbazides against bacterial
topoisomerases was examined to clarify the molecular mechanism involved in their synergistic effect
in combination with levofloxacin. The results from these experiments are presented in this paper.

Molecules 2020, 25, 2302
3 of 13
2. Results and Discussion
2.1. Rationale and Synthesis
So far, the most promising antibacterial agents that emerged from our studies on antibacterial
potency of thiosemicarbazides [20 21 23 25 26] are those with 4-benzoyl-1-(2,3-dichlorobenzoyl)
thiosemicarbazide scaffold. The most potent of them, , have been shown to possess potent,
broad spectrum of antibacterial activities against Gram positive strains with minimal inhibitory
concentrations (MICs) between 0.49 and 15.63 g/mL, thereby indicating, in most cases, equipotent
or even more effective action than cefuroxime [23]. In further studies, the inhibitory efficacy of
para-nitro compound on planktonic cells and biofilms formation in clinical isolates of S. aureus was
,
, , ,
1–5
µ
5
also tested, and it was found that the compound was able to eradicate MSSA and MRSA biofilms
at low concentrations, with minimal biofilm inhibitory concentrations (MBICs) between 7.82 and
15.63
their synergistic effects in combination with clinical drugs were tested, and the results of these studies
are presented in this paper. For these studies, four 4-arylthiosemicarbazides with much weaker
antibacterial potency against reference strains of Gram-positive bacteria as compared to were
also included for comparison (Table 1). All compounds were prepared using an established
23], in one-step reaction of appropriate hydrazide with aryl or benzoyl
µg/mL [23]. Thus, to further explore antibacterial potential of potent thiosemicarbazides 1–5,
6–9
1–5
1–9
route described elsewhere [22
isothiocyanate (Scheme 1).
,
Scheme 1. Synthetic route for 4-benzoylthiosemicarbazides (
R1 = 2,3-diCl ( ); R2 = 2-MeC(=O)Ph (
), 2-F ( ), 3-MeC(=O)Ph (
), 4-NO2C(=O)Ph ( ), 4-ClPh ( ), 3-ClPh ( ), 3-CF3Ph ( ), and 3-BrPh (
see Table 2.
1
–
5
) and 4-arylthiosemicarbazides (
), 2-ClC(=O)Ph ( ), 4-ClC(=O)Ph
). For full structures of 1–9,
6–9).
1
–5
6–
9
1
2
3
(
4
5
6
7
8
9

Molecules 2020, 25, 2302
4 of 13
Table 1. Antibacterial data (MIC, µg/mL) for 4-benzoylthiosemicarbazides 1–5, 4-arylthiosemicarbazides 6–9, and antibiotics.
1
2
3
4
5
6
7
8
9
AMX GN
LEV LZD
VA
S. a. ATCC 6538
S. a. ATCC 25923
S. e. ATCC 12228
B. s. ATCC 6633
B. c. ATCC 10876
M. l. ATCC 10240
15.63
15.63
7.81
3.91
3.91
1.95
7.81
7.81
3.91
0.49
1.95
0.98
15.63 15.63
15.63 31.25
1.95
1.95
1.95
0.98
0.98
0.49
250
500
250
500
31.25 31.25
7.82 7.82
31.25 15.63 62.50
62.50 15.63 62.50
0.12
0.12
7.81
0.12
0.98
1.95
0.12
0.24
0.24
0.24
0.12
0.12
0.12
0.98
0.98
1.95
0.49
0.98
0.98
0.98
0.49
0.98
0.98
0.24
0.98
0.24
7.81
3.91
3.91
0.98
7.81
0.98
3.91
1.95
31.25
15.63
7.82
7.82
7.82
7.82
31.25
15.63
15.63 >250 15.63
7.82 0.24 0.98
Note: S. a.—S. aureus, S. e.—S. epidermidis, B. s.—B. subtilis, B. c.—B. cereus, M. l.—M. luteus. AMX—amoxicillin, GN—gentamicin, LEV—levofloxacin, LZD—linezolid, and VA—vancomycin.
MICs for 4-benzoylthiosemicarbazides against reference strains of Gram-positive bacteria are taken from Reference [23]. MICs for and antibiotics against clinical isolates of S. aureus
are presented in Table S1; all the thiosemicarbazides were inactive against these clinical isolates (MICs 62.5 g/mL). All the thiosemicarbazides were inactive against Gram
negative strains (E. coli ATCC 25922, K. pneumoniae ATCC 13883, P. mirabilis ATCC 12453, and P. aeruginosa ATCC 9027), as well.
1–
5
1–9
1–
9
≥
µ
1–9

Molecules 2020, 25, 2302
5 of 13
2.2. Cytotoxicity Assay
A critical factor in research and development of new antibacterial agents is their abilities to
selectively target bacterial cells without toxicity to mammalian cells. Thus, in order to test if
antibacterial activity of title thiosemicarbazides is not due to their general toxicity, their effect on
mammalian cell viability was assessed using human foreskin fibroblast Hs27 cells (CRL-1634) via
MTT assay. In Table 2, the data are presented as the relative viability at the concentration of 25
µg/mL
compared to control vehicle-treated samples (100% viability) because except of , the compounds
2
reached their solubility limit before their IC50 values could be determined. The results confirmed that
all tested thiosemicarbazides produced very limited effects on the cellular viabilities of Hs27 cells at
the concentration of 25
µg/mL after 24 h of treatment. Important to note, all potent antibacterial agents
with 4-benzoylthiosemicarbazide core structure (1–5) reached their effective inhibitory effect against S.
aureus ATCC 25923 strain much before the concentration at which their cytotoxic affect against Hs27
cell line could be observed. The second important observation from the cytotoxic assay is that the
trend in antibacterial potency against S. aureus ATCC 25923;
(MIC 15.63 g/mL) > (MIC 31.25 g/mL) >
5
9
(MIC 1.95
(MIC 62.5
µ
g/mL) >
g/mL) >
2
6
(MIC 7.81 g/mL)
(MIC 500 µg/mL)
µ
>
1
=
3
=
8
µ
4
µ
7
=
µ
is fortunately not linked directly with cytotoxic effect. Indeed, the weakest antibacterial agents
and differ substantially in their cytotoxicity; the compound was the most cytotoxic, whereas
7 were at the same least cytotoxic against Hs27 cells among all thiosemicarbazides tested.
For the compound with the best solubility ( ), its cytotoxicity against L929 and HeLa cells was
6, 7,
9
9
6 and
2
presented previously [23]. The cytotoxicity was expressed as rigorous IC30 values, defined as the
highest dilutions of test samples that cause 30% or greater destruction of cells. According to these
results, the compound
2 gave the IC30 value of 30.78 µg/mL against the L929 cell line and the IC30
value of 30.97 g/mL against the HeLa cell line. Thus, as evidenced by these data, the compound
µ
2
display antibacterial effect against S. aureus ATCC 25923 at non-toxic concentrations in mammalian
cells, with the estimated selectivity indexes SI (SI = IC30/MIC) against L929 and HeLa cell lines 3.94
and 3.97, respectively.
Table 2. Viability of human foreskin fibroblast Hs27 cells (CRL-1634) determined by MTT assay after
24 h of treatment with 4-benzoylthiosemicarbazides (1–5) and 4-arylthiosemicarbazides (6–9).
1
2
3
4
5
6
7
8
9
LEV
IC50
(µg/Ml)
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
Viability at 25 µg/mL
(%) *
78.48 76.68 80.97 80.72 70.91 96.84 93.21 65.24 63.11
100.51
Note: LEV—levofloxacin; * Mean from 3 independent measurements done in triplicate (SD values were in the range
of ±5% of the reported values).
2.3. Synergy Assay
The microdilution checkerboard method was used to evaluate the synergistic effects between the
thiosemicarbazides and antibiotics, which were determined as the fractional inhibitory concentration
1–9
indices (FICIs). For these experiments, amoxicillin, gentamicin, levofloxacin, linezolid, and vancomycin
were selected to represent the five antimicrobial classes most commonly used in clinical practice.
In this way, a total of 45 combinations of thiosemicarbazide-amoxicillin, thiosemicarbazide-gentamicin,
thiosemicarbazide-levofloxacin, thiosemicarbazide-linezolid, and thiosemicarbazide-vancomycin
against S. aureus ATCC 25923 were tested to identify in vitro synergy effects.
As presented in Table 3, when the 4-benzoylthiosemicarbazide
ortho-methyl substitution was combined with tested antibiotics, an additive effect with amoxicillin
or linezolid was observed, whereas the other three -antibiotic combinations showed no interactions.
Moving the ortho-methyl group of to the meta-position to give results in synergistic effect of the
-linezolid combination and additive effect of the -amoxicillin combination. As for compound
1 with electron-donating
1
1
2
2
2

Molecules 2020, 25, 2302
6 of 13
1
, the other three
2-antibiotic combinations showed no interactions. In turn, replacing the
electron-donating ortho-methyl group of
the compound that affected additively by
combinations, while no interaction were seen when
1
with the electron-withdrawing ortho-chloro group results in
-amoxicillin, -gentamicin, -levofloxacin, and -linezolid
and vancomycin was combined. Moving the
results in synergistic effect of the -levofloxacin
-linezolid combinations. As for other compounds
3
3
3
3
3
3
ortho-chloro group of
combination and additive effect of
tested, the combination of
replacing the para-chloro group in
3
to the para-position to give
4
4
4
4
-amoxicillin and
4
with vancomycin did not display any synergism nor additivity. Finally,
with the electron-withdrawing para-nitro results in analogue that
-linezolid combination, whereas the other four -antibiotic combinations
4
5
affected synergistically by
5
5
showed no interactions.
Further investigations were performed for set of 4-arylthiosemicarbazides
the para-chloro compound with synergistic interactions in combinations with gentamicin or linezolid
and additive interactions in combinations with amoxicillin, levofloxacin, or vancomycin showed a clear
preference for the potency among this series. Its meta isomer affected synergistically in combination
6–9. As shown in Table 3,
6
7
with levofloxacin and additively in combination with amoxicillin, whereas no interactions or even
antagonistic were detected for its combinations with gentamicin, linezolid, or vancomycin, respectively.
In turn, the other two 4-arylthiosemicarbazides with meta electron-withdrawing trifluoromethyl
bromo substitutions showed the same trend in potency; they effected additively with levofloxacin or
linezolid and indifferently in the combinations with amoxicillin, gentamicin, or vancomycin.
Overall, except for -vancomycin combination, none of the forty-five thiosemicarbazide-antibiotic
combinations tested had an antagonistic effect, showing promising results with respect to a
combination therapy. Among them, the synergic effect was observed for the -linezolid, -levofloxacin,
-linezolid, -gentamicin, -linezolid, and -levofloxacin combinations. Except for , and
no interactions were seen when gentamicin or vancomycin with were combined, whereas
all thiosemicarbazide-linezolid combinations acted in additive or synergism, except of -linezolid
combination. The 4-arylthiosemicarbazide with para-chloro substitution showed a clear preference
8 or
9
7
2
4
5
6
6
7
3,
6
7,
1–9
7
6
for the potency among all the thiosemicarbazides tested; it affected synergistically in combinations with
gentamicin or linezolid and additively in combinations with amoxicillin, levofloxacin, or vancomycin.
It is important to note, however, although the checkerboard broth dilution method is suitable for
in vitro experiments aiming to mimic in vivo conditions [27], in vitro FIC index is still unlikely to
predict in vivo therapeutic efficacy [28]. The most important explanation of the lack in vitro-in vivo
correlation of combination therapy is that there are marked differences between in vitro and in vivo
conditions due to serum protein binding that affects drug’s efficiency [29]. Consequently, an in vitro
synergistic or antagonistic combination may be additive or indifferent in vivo if the administered dose
levels result in drug’s concentrations lower or higher that those at which synergism or antagonism was
observed in vitro
[
28]. Finally, the conclusion of the present in vitro studies is that the combinations of
-linezolid, -gentamicin, -linezolid, and -levofloxacin are applicable to
2
-linezolid, -levofloxacin,
4
5
6
6
7
in vivo synergy studies, genotoxicity/mutagenicity tests, and pharmacokinetic/pharmacodynamics
analyses to provide more information on their mechanism of action, safety profile, and potential
clinical relevance. Such experiments are now in progress in our laboratory, and their results will soon
be presented.

Molecules 2020, 25, 2302
7 of 13
Table 3. In vitro evaluation of antibacterial combinations.
+ AMX
+ GN
+ LEV
+ LZD
+ VA
Compound
FIC Effect
FIC Effect
FIC Effect
FIC Effect
FIC Effect
0.750
additive
1.125
indifferent
1.490
Indifferent
0.750
additive
1.500
indifferent
0.562
additive
1.062
indifferent
1.031
indifferent
0.281
synergistic
1.063
indifferent
0.750
additive
0.750
additive
1.000
additive
0. 563
additive
1.250
indifferent
0.508
additive
1.500
indifferent
0.498
synergistic
0.563
additive
1.500
indifferent
1.250
indifferent
2.125
indifferent
1.500
indifferent
0.375
synergistic
3.000
indifferent
0.563
additive
0.375
synergistic
0. 625
additive
0.500
synergistic
0.625
additive
0.750
additive
1.125
indifferent
0.373
synergistic
1.031
indifferent
31.254
antagonistic
1.500
indifferent
1.266
indifferent
1.000
additive
0.750
additive
1.500
indifferent
1.250
indifferent
1.125
indifferent
0.748
additive
0.750
additive
1.500
indifferent
Note: FIC is interpreted as synergistic (FIC
or antagonistic (FIC > 4.0). FIC—fractional inhibitory concentration indices, AMX—amoxicillin, GN—gentamicin,
≤
0.5), additive (0.5 > FIC
≤
1.0), indifferent (1 < FIC
≤
4),
LEV—levofloxacin, LZD—linezolid, and VA—vancomycin.
2.4. Enzymatic Assay
Several studies have shown that closely related analogues and structurally similar scaffolds
produce qualitatively the same biologic response and share a common function or mechanism of
action [30]. Therefore, it is quite strange that our structurally and electronically closely related
thiosemicarbazides tested in the present study exert synergistic effects with antibiotics that have
totally different mechanisms. In essence, two 4-benzoylthiosemicarbazides with electron-donating
meta-methyl substitution
2 and electron-withdrawing para-nitro substitution 5 share very similar
molecular geometries, which only slightly differ in the orientation of the N4 aryl ring (Figure 1, left),
and exerted synergistic effect when combined with linezolid that molecular mechanism involves
inhibition of the initiation process of protein production [31]. In contrast, compound with para-chloro
substitution 4, which geometry is superimposable with 2 (Figure 1, middle), showed no synergy when
combined with linezolid but acted synergistically with levofloxacin that involves inhibition of the
enzymatic activity of DNA gyrase and topoisomerase IV; the enzymes essential for DNA replication,
transcription, repair, and recombination. Likewise, within subclass of 4-arylthiosemicarbazides
the chloro isomers and share very similar stretched geometries, only slightly different in the
orientation of the N4 benzoyl ring (Figure 1, right), whereas produce quite different antibacterial
response; isomer para exerts synergistic effect in combinations with linezolid or gentamicin, while its
isomer meta 7 acts synergistically in combination with levofloxacin.
6–9,
6
7
6

Molecules 2020, 25, 2302
8 of 13
Figure 1. Overlay of structures of the 4-benzoylthiosemicarbazides
and . Note: The geometry of is superimposable with , whereas the geometry of
with 2 and 4. The geometries of 8 and 9 are superimposable with 6.
2
,
4
,
5
, and 4-arylthiosemicarbazides
6
7
1
5
3
is superimposable
Thus, based on SAR (structure activity relationship) analysis one can conclude that tested
thiosemicarbazides have a complex molecular-level antibacterial mechanism, which requires
1–9
further investigation. When combined with our previous studies on molecular basis of antibacterial
activity of thiosemicarbazides, these data provide further evidence that at least two molecular
mechanisms for this class of compounds should be really expected. Thus far, the first one proposed
by us assumed that antibacterial activity of thiosemicarbazides might be related to inhibition of
bacterial topoisomerases [22,25,32,33]. Taking this fact into account, we have submitted 4 and 7
for enzyme inhibition assay against S. aureus bacterial topoisomerases (Figures S1–S3) to clarify
the molecular mechanism involved in their synergistic effects observed in the combination with
levofloxacin. No inhibitory actions, however, were recorded for both the compounds against DNA
gyrase and topoisomerase IV. Finally, the inhibitory effects of 2, 5, and 6 that showed synergism in
combination with linezolid or gentamycin against S. aureus DNA gyrase and topoisomerase IV were
also assayed for comparison (Figures S1–S3). Surprisingly, the most potent synergist in our studies
6
at concentration of 100 µM was able to inhibit ~50% activity of S. aureus DNA gyrase (Table 4),
suggesting that its anti-gyrase activity, although weak, may be a possible factor contributing to its
synergism effect in combination with linezolid or gentamycin. For remaining thiosemicarbazides
1, 3, 8, and 9 no inhibitory effects against S. aureus topoisomerase IV was observed (Figures S1–S3).
Evidently, more research is needed to understand the molecular mechanism behind the identified
synergy. Such studies are in progress in our laboratory.
Table 4. Inhibitory activity of 6 and 7 against S. aureus DNA gyrase.
S. aureus Gyrase DNA Inhibition (%) ± SD
Compound
50 µM
30.76 ± 3.19
23.84 ± 5.02
n.d.
100 µM
6
7
49.22 ± 3.18
33.14 ± 1.04
92.38 ± 3.74
LEV
Note: S. aureus gyrase DNA inhibition was quantified densitometrically from two independent experiments and
expressed as mean ± SD. n.d.—not determined.
3. Materials and Methods
3.1. Chemistry
All commercial reactants and solvents were purchased from either Sigma-Aldrich (St. Louis,
MO, USA) or Alfa Aesar (Karlsruge, Germany) with the highest purity and used without further
purification. The melting points were determined on a Fischer-Johns block (Fischer Scientific, Schwerte,
Germany) and are uncorrected. Elemental analyses were determined by a AMZ-CHX elemental
1
analyzer (PG, Gdan´sk, Poland) and are within
±
0.4% of the theoretical values. H-NMR spectra were
recorded on a Bruker Avance (300 MHz) spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany).
Analytical thin layer chromatography was performed with Merck 60F254 silica gel plates (Merck,
Darmstadt, Germany) and visualized by UV irradiation (254 nm). Physicochemical characterizations of

Molecules 2020, 25, 2302
9 of 13
the compounds
1
–6
were reported elsewhere [23
,34]. The structures of compounds
7
and
8
are known
(CAS numbers 443666-45-3 and 905231-62-1, respectively); however, there is no references reporting
their preparation and physicochemical characterization; therefore, these data have been included into
the manuscript.
3.2. General Procedure for Synthesis of the Thiosemicarbazides 1–9
A solution of 2,3-dichlorobenzoic hydrazide or 2-fluorobenzoic hydrazide (0.01 mol) and an
equimolar amount of aryl or benzoyl isothiocyanate (0.01 mole) in anhydrous ethanol (25 mL) was
heated under reflux for 10–30 min. After cooling, the solid formed was filtered off, dried, and crystallized
from ethanol.
◦
1
4-(3-chlorophenyl)-1-(2-fluorobenzoyl)thiosemicarbazide (
(DMSO-d6) (ppm): 7.26–7.93 (m, 8H, 8 CHar); 9.91, 10.06, 10.41 (3s, 3H, 3
C14H11ClFN3OS (%): C 51.93, H 3.42, N 12.98. Found: C 51.79, H 3.71, N 12.89.
7
). Yield: 84%. M.p. 158–160 C. H-NMR
δ
×
×
NH). Anal. calc. for
1-(2-fluorobenzoyl)-4-(3-trifluoromethylphenyl)thiosemicarbazide (
). Yield: 81%. M.p. 176–178 ◦C. 1H-NMR
(DMSO-d6) (ppm): 7.37–7.91 (m, 8H, 8 CHar); 10.02, 10.03, 10.14 (3s, 3H, 3 NH). Anal. calc. for
C15H11F4N3OS (%): C 50.42, H 3.10, N 11.76. Found: C 50.35, H 3.32, N 11.88. Anal. (C, H, N).
8
δ
×
×
◦
1
4-(3-bromophenyl)-1-(2-fluorobenzoyl)thiosemicarbazide (
9
). Yield: 86%. M.p. 166–168 C. H-NMR
(DMSO-d6) (ppm): 7.30–7.91 (m, 8H, 8 CHar); 9.89, 10.04, 10.41 (3s, 3H, 3
C14H11BrFN3OS (%): C 45.67, H 3.01, N 11.41. Found: C 45.82, H 3.22, N 11.17. Anal. (C, H, N).
δ
×
×
NH). Anal. calc. for
3.3. In Vitro Evaluation of Antibacterial Activity
Minimal inhibitory concentration (MIC) of each antibacterial agents was determined by standardized
broth microdilution method with inoculum of 5
to the recommendation of CLSI (Clinical Laboratory Standards Institute) [35]. The antibacterial
activity of the thiosemicarbazides and antibiotics (amoxicillin, gentamicin, levofloxacin, linezolid,
×
105 CFU/mL and incubated for 24 h at 35 ◦C according
1–9
and vancomycin) was tested on the Gram-positive strains (Microbiologics, St. Cloud, MN, USA)
(S. aureus ATCC 25923, S. aureus ATCC 6538, S. epidermidis ATCC 12228, B. subtilis ATCC 6633, B. cereus
ATCC 10876, M. luteus ATCC 10240), the Gram-negative strains (Microbiologics, St. Cloud, MN, USA)
(E. coli ATCC 25922, K. pneumoniae ATCC 13883, P. mirabilis ATCC 12453, P. aeruginosa ATCC 9027), and
clinical isolates of S. aureus (Medical University of Lublin, Lublin, Poland) (MSSA-1, MSSA-2, MSSA-3,
MSSA-4, MRSA-1). Similarly to previous experiments with the thiosemicarbazides 1–5, cefuroxime
was used as positive control antibiotic [23], whereas sterile distilled water served as negative controls.
3.4. Fractional Inhibitory Concentration Indices (FICIs)
Interaction of the thiosemicarbazides 1–9 in combination with amoxicillin, gentamicin, levofloxacin,
linezolid or vancomycin against S. aureus ATCC 25923 were analyzed using the checkerboard broth
dilution method to determine the fractional inhibitory concentration indices (FICIs), calculated as:
FIC = MIC of drug A in combination/MIC of drug A alone + MIC of drug B in combination/MIC of
drug B alone. The calculated FICIs were interpreted as synergistic (FIC
≤
0.5), additive (0.5 < FIC < 1),
indifferent (1 FIC < 4.0), or antagonistic (FIC 4.0) [36 37]. The checkerboard assay included
≤
≥
,
ciprofloxacin-carvedilol combination as positive control [38], whereas cefuroxime in combination with
4-(4-bromophenyl)-1-(3-fluorobezoyl)thiosemicarbazide served as negative control [39]. The Z-factor
value for the bioassay was 0.683.
3.5. Cytotoxicity Assay
Cytotoxic properties of the compounds were evaluated using human foreskin fibroblast cells Hs27
(ATCC CRL-1634) (ATCC, Manassas, VA, USA) that were cultured in DMEM-high glucose (Sigma
Aldrich, St. Louis, MO, USA). Medium was supplemented with 10% fetal bovine serum (FBS, Sigma

Molecules 2020, 25, 2302
10 of 13
Aldrich), 100 U/mL of penicillin, and 100 mg/mL of streptomycin (PenStrep, Sigma Aldrich, St. Louis,
MO, USA). Cells were incubated at 37 ◦C in a humidified atmosphere of 5% CO2. Stock solutions
of the tested compounds were prepared dissolving in a sterile dimethyl sulfoxide (DMSO) to the
concentration of 50 mg/mL. The cells were seeded into 96-well sterile plates (Nunc) at a cell density of
1 × 105 cells/mL. After 24 h of incubation, the medium was removed from each well, and then cells
were incubated for the next 24 h with tested compounds (in a fixed dose of 25 µg/mL) in DMEM-high
glucose containing 2% FBS. Control cells were cultured only with medium containing 2% addition of
FBS. Cytotoxicity of the compounds was evaluated using MTT assay, which is based on the conversion
of 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) into dark-blue formazan
crystals. Briefly, after 24-h incubation of cells with varying concentrations of the tested compounds,
culture medium was removed from the plates. Cells were washed with PBS, and then 100
medium containing 10% MTT solution (5 mg/mL) was added to each well. Cells were incubated for
the next 3 h at 37 ◦C in an atmosphere of 5% CO2. Next, 100
L (per well) of 10% sodium dodecyl
µL of
µ
sulfate (SDS) buffer solution was added to dissolve formazan crystals and after an overnight incubation
the absorbance was measured at 570 nm using a microplate reader (Epoch, BioTek Instruments, Inc.,
Winooski, VT, USA). Z-factor value for the bioassay was 0.996.
3.6. Inhibition of Bacterial type IIA Topoisomerases
Supercoiling assay: The assays were performed using S. aureus Gyrase Supercoiling Assay Kits
(Inspiralis, Norwich, UK). Briefly, supercoiled pBR322 plasmid DNA (0.5 µg) was incubated with 1 unit
of gyrase, in the dedicated supercoiling assay buffer supplied by the manufacturer, in the presence of
varying concentrations of the test compounds. Reactions were carried out at 37 ◦C for 1 h and then
terminated by the addition of equal volume of 2
×
STOP Buffer (40% sucrose, 100 mM Tris-Cl pH
7.5, 1 mM EDTA, and 0.5 mg/mL bromophenol blue) and chloroform/iso-amyl alcohol. Samples were
vortexed, centrifuged, and run through a 15 cm 1% agarose gel in TAE buffer (40 mM Tris-acetate,
2 mM EDTA) for 3 h at 50 V. Gels were stained with ethidium bromide and visualized under UV light.
Inhibition of decatenation activity of S. aureus topoisomerase IV: The assay was performed using
S. aureus topoisomerase IV decatenation kits (Inspiralis, Norwich, UK). Interlinked kDNA substrate
(0.5 µg) was incubated with 1 unit of topoisomerase IV (Inspiralis, Norwich, UK), in the dedicated
decatenation assay buffer supplied by the manufacturer, in the presence of varying concentrations of
the test compounds. Reactions were carried out at 37 ◦C for 1 h and then terminated by the addition of
equal volume of 2
×
STOP Buffer and chloroform/iso-amyl alcohol. Samples were vortexed, centrifuged,
and run through a 15 cm 1% agarose gel in TAE buffer for 1.5 h at 80 V. Gels were stained with ethidium
bromide and visualized under UV light.
3.7. Computational Details
Conformational search was carried out using the molecular mechanics level with Amber99 force
field [40], as implemented in HyperChem 8.0.3 (Hypercube, Gainesville, FL, USA) [41].
4. Conclusions
The checkerboard method was used to evaluate synergistic interactions between nine
thiosemicarbazides 1–9 and five conventional antibiotics against S. aureus ATCC 25923. For these
experiments, amoxicillin, gentamicin, levofloxacin, linezolid, and vancomycin were selected to
represent the five antimicrobial classes most commonly used in clinical practice. With one
exception of
tested had an antagonistic effect, showing promising results with respect to a combination therapy.
The synergic effect was observed for the -linezolid, -levofloxacin, -linezolid, -gentamicin, -linezolid,
and -levofloxacin combinations. No interactions were seen in combination of the thiosemicarbazide
with gentamicin or vancomycin, whereas all combinations with linezolid acted in additive or synergism,
except for -gentamicin and -linezolid. The thiosemicarbazide showed a clear preference for the
7-vancomycin combination, none of the thiosemicarbazide-antibiotic combinations
2
4
5
6
6
7
6
7
6

Molecules 2020, 25, 2302
11 of 13
potency; it affected synergistically in combinations with gentamicin or linezolid and additively in
combinations with amoxicillin, levofloxacin, or vancomycin. In further studies, the results of enzymatic
assay confirmed that anti-gyrase activity of 6 may be a possible factor contributing to its synergism
effect in combination with linezolid or gentamycin.
Supplementary Materials: The following are available online, Figure S1: Inhibition of decatenation activity of
S. aureus topoisomerase IV by compounds 1–5 and levofloxacin. Figure S2: Inhibition of decatenation activity of
S. aureus topoisomerase IV by compounds 6–9 and levofloxacin. Figure S3: Inhibition of decatenation activity of
S. aureus topoisomerase IV by compounds
chromatograms for 4-benzoylthiosemicarbazides (
data (MIC, g/mL) for 4-benzoylthiosemicarbazides
clinical isolates of S. aureus.
1
–
9
and ciprofloxacin. Figure S4: The TLC (thin layer chromatography)
) and 4-arylthiosemicarbazides ( ). Table S1: Antibacterial
, 4-arylthiosemicarbazides , and antibiotics against
1–
5
6–9
6–9
µ
1
–
5
Author Contributions: Conceptualization, A.P.; methodology, A.P., A.M., B.C.-R., T.P.; software, A.P.; validation,
M.W., N.T., T.P.; formal analysis, A.P.; investigation, B.C.-R., M.W., N.T., T.P; resources, A.P.; data curation, A.P.;
writing—Original draft preparation, A.P.; writing—Review and editing, A.P.; visualization, A.P., T.P. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by Medical University of Lublin, Poland, grant number DS 15.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
Brown, E.D.; Wright, G.D. Antibacterial drug discovery in the resistance era. Nature 2016, 529, 336–34310.
[CrossRef]
Antibiotic/Antimicrobial Resistance (AR/AMR). Centers for Disease Control and Prevention, Atlanta, GA. 2019.
Available online: https://www.cdc.gov/drugresistance/biggest_threats.html (accessed on 2 January 2020).
Vicetti Miguel, C.P.; Mejias, A.; Leber, A.; Sanchez, P.J. A decade of antimicrobial resistance in Staphylococcus
aureus: A single center experience. PLoS ONE 2019, 14, e0212029. [CrossRef] [PubMed]
Craft, K.M.; Nguyen, J.M.; Berg, L.J.; Townsend, S.D. Methicillin-resistant Staphylococcus aureus (MRSA):
Antibiotic-resistance and the biofilm phenotype. Med. Chem. Commun. 2019, 10, 1231–1241. [CrossRef]
Turner, N.A.; Sharma-Kuinkel, B.K.; Maskarinec, S.A.; Eichenberger, E.M.; Shah, P.P.; Carugati, M.;
Holland, T.L.; Fowler Jr, V.G. Methicillin-resistant Staphylococcus aureus: An overview of basic and clinical
research. Nat. Rev. Microbiol. 2019, 17, 203–218. [CrossRef]
6.
7.
O’brien, W.J.; Gupta, K.; Itani, K.M.F.; Longitudinal, A. Study of S. aureus infection in a national cohort of
surgical patients. Open Forum Infect. Dis. 2019, 6, ofz350. [CrossRef] [PubMed]
Kavanagh, K.T. Control of MSSA and MRSA in the United States: Protocols, policies, risk adjustment and
excuses. Antimicrob. Resist. Infect. Control 2019, 8, 103. [CrossRef] [PubMed]
8.
9.
Archer, G.L. Staphylococcus aureus: A well-armed pathogen. Clin. Infect. Dis. 1998, 26, 1179–1181. [CrossRef]
Tong, S.Y.C.; Davis, J.S.; Eichenberger, E.; Holland, T.L.; Fowler Jr, V.G. Staphylococcus aureus infections:
Epidemiology, pathophysiology, clinical manifestations, and management. Clin. Microbiol. Rev. 2015, 28,
603–661. [CrossRef]
10. Sinha, B.; Fraunholz, M. Staphylococcus aureus host cell invasion and post-invasion events. Int. J. Med. Microbiol.
2010, 300, 170–175. [CrossRef]
11. Foster, T.J. Colonization and infection of the human host by staphylococci: Adhesion, survival and immune
evasion. Vet. Dermatol. 2009, 20, 456–470. [CrossRef]
12. Liu, C.; Bayer, A.; Cosgrove, S.E.; Daum, R.S.; Fridkin, S.K.; Gorwitz, R.J.; Kaplan, S.L.; Karchmer, A.W.;
Levine, D.P.; Murray, B.E.; et al. Clinical practice guidelines by the Infectious Diseases Society of America
for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children: Executive
summary. Clin. Infect. Dis. 2011, 52, 285–292. [CrossRef] [PubMed]
13. Drebes, J.; Künz, M.; Pereira, C.A.; Betzel, C.; Wrenger, C. MRSA infections: From classical treatment to
suicide drugs. Curr. Med. Chem. 2014, 21, 1809–1819. [CrossRef] [PubMed]
14. Kalita, S.; Kandimalla, R.; Bhowal, A.C.; Kotoky, J.; Kundu, S. Functionalization of B-lactam antibiotic on
lysozyme capped gold nanoclusters retrogress MRSA and its persisters following awakening. Sci. Rep. UK
2018, 8, 5778. [CrossRef]

Molecules 2020, 25, 2302
12 of 13
15. Gandhi, T.N.; Malani, P.N. Combination therapy for methicillin-resistant Staphylococcus aureus bacteremia:
Not ready for prime time. JAMA 2020, 323, 515–516. [CrossRef] [PubMed]
16. Mohammad, H.; Mayhoub, A.S.; Ghafoor, A.; Soofi, M.; Alajlouni, R.A.; Cushman, M.; Seleem, M.N. Discovery
and characterization of potent thiazoles versus methicillin-and vancomycin-resistant Staphylococcus aureus.
J. Med. Chem. 2014, 57, 1609–1615. [CrossRef]
17. Rybak, M.; Lomaestro, B.; Rotschafer, J.C.; Moellering, R.; Craig, W., Jr.; Billeter, M.; Dalovisio, J.R.; Levine, D.P.
Therapeutic monitoring of vancomycin in adult patients: A consensus review of the American Society of
Health-System Pharmacists, the Infectious Diseases Society of America, and the Society of Infectious Diseases
Pharmacists. Am. J. Health Syst. Pharm. 2009, 66, 82–98. [CrossRef]
18. Han, J.H.; Edelstein, P.H.; Lautenbach, E. Reduced vancomycin susceptibility and staphylococcal cassette
chromosome mec (SCCmec) type distribution in methicillin-resistant Staphylococcus aureus bacteraemia.
J. Antimicrob. Chemother. 2012, 67, 2346–2349. [CrossRef]
19. Cong, Y.; Yang, S.; Rao, X. Vancomycin resistant Staphylococcus aureus infections: A review of case updating
and clinical features. J. Adv. Res. 2020, 21, 169–176. [CrossRef]
20. Kullar, R.; Casapao, A.M.; Davis, S.L.; Levine, D.P.; Zhao, J.J.; Crank, C.W.; Segreti, J.; Sakoulas, G.;
Cosgrove, S.E.; Rybak, M.J. A multicentre evaluation of the effectiveness and safety of high-dose daptomycin
for the treatment of infective endocarditis. J. Antimicrob. Chemother. 2013, 68, 2921–2926. [CrossRef] [PubMed]
21. Sharma, M.; Riederer, K.; Chase, P.; Khatib, R. High rate of decreasing daptomycin susceptibility during the
treatment of persistent Staphylococcus aureus bacteremia. Eur. J. Clin. Microbiol. Infect. Dis. 2008, 27, 433–437.
[CrossRef]
22. Paneth, A.; Sta˛czek, P.; Plech, T.; Strzelczyk, A.; Janowska, D.; Stefan´ska, J.; Dzitko, K.; Wujec, M.;
Kosiek, S.; Paneth, P. Synthesis and antibacterial activity of 1,4-dibenzoylthiosemicarbazide derivatives.
Biomed. Pharmacother. 2017, 88, 1235–1242. [CrossRef]
23. Paneth, A.; Plech, T.; Kapron´, B.; Hagel, D.; Kosikowska, U.; Kus´mierz, E.; Dzitko, K.; Paneth, P.
Design, synthesis and biological evaluation of 4-benzoyl-1-dichlorobenzoylthiosemicarbazides as potent
Gram-positive antibacterial agents. J. Enzym. Inhib. Med. Chem. 2016, 31, 434–440. [CrossRef] [PubMed]
24. Ganesan, A.; Christena, R.C.; Subbarao, H.M.V.; Venkatasubramanian, U.; Thiagarajan, R.;
Sivaramakrishnan, V.; Kasilingam, K.; Saisubramanian, N.; Ganesan, S.S. Identification of benzochromene
derivatives as a highly specific NorA efflux pump inhibitor to mitigate the drug resistant strains of S. aureus.
RSC Adv. 2016, 6, 30258–30267. [CrossRef]
25. Paneth, A.; Sta˛czek, P.; Plech, T.; Strzelczyk, A.; Dzitko, K.; Wujec, M.; Kus´mierz, E.; Kosikowska, U.;
Grzegorczyk, A.; Paneth, P. Biological evaluation and molecular modelling study of thiosemicarbazide
derivatives as bacterial type IIA topoisomerases inhibitors. J. Enzym. Inhib. Med. Chem. 2016, 31, 14–22.
[CrossRef]
26. Janowska, D. Synthesis and Antibacterial Screening of New Thiosemicarbazide Derivatives. Ph.D. Thesis,
Medical University of Lublin, Lublin, Poland, 2018.
27. Nageeb, W.; Metwally, L.; Kamel, M.; Zakaria, S. In vitro antimicrobial synergy studies of
carbapenem-resistant Acinetobacter baumannii isolated from intensive care units of a tertiary care hospital
in Egypt. J. Infect. Public. Heal. 2015, 8, 593–602. [CrossRef]
28. Meletiadis, J.; Pournaras, S.; Roilides, E.; Walsh, T.J. Defining Fractional Inhibitory Concentration Index cutoffs
for additive interactions based on self-drug additive combinations, Monte Carlo simulation analysis, and
in vitro-in vivo correlation data for antifungal drug combinations against Aspergillus fumigatus. Antimicrob.
Agents Chemother. 2010, 54, 602–609.
29. Brochmann, R.P.; Helmfrid, A.; Jana, B.; Magnowska, Z.; Guardabassi, L. Antimicrobial synergy between
carprofen and doxycycline against methicillin-resistant Staphylococcus pseudintermedius ST71. BMC Vet.
Res. 2016, 12, 126. [CrossRef]
30. Wu, X.; Li, Z.; Li, X.; Tian, Y.; Fan, Y.; Yu, C.; Zhou, B.; Liu, Y.; Xiang, R.; Yang, L. Synergistic effects of
antimicrobial peptide DP7 combined with antibiotics against multidrug-resistant bacteria. Drug Des. Dev.
Ther. 2017, 11, 939–946. [CrossRef]
31. Coleman, J.P.; Smith, C.J. Microbial nucleic acid and protein synthesis. In xPharm: The Comprehensive
Pharmacology Reference; Elsevier: Amsterdam, The Netherlands, 2007.

Molecules 2020, 25, 2302
13 of 13
32. Plech, T.; Paneth, A.; Kapron´, B.; Kosikowska, U.; Malm, A.; Strzelczyk, U.; Sta˛czek, P. Structure-activity
relationship studies of microbiologically active thiosemicarbazides derived from hydroxybenzoic acid
hydrazides. Chem. Biol. Drug Des. 2015, 85, 315–325. [CrossRef] [PubMed]
33. Siwek, A.; Sta˛czek, P.; Wujec, M.; Bielawski, K.; Bielawska, A.; Paneth, P. Cytotoxic effect and molecular
docking of 4-ethoxycarbonylmethyl-1-(piperidin-4-ylcarbonyl)-thiosemicarbazide-a novel topoisomerase II
inhibitor. J. Mol. Model. 2013, 19, 1319–1324. [CrossRef]
34. Joshi, K.C.; Mehta, D.S. Synthesis of some 3-(fluorinated aryl)-4-alkyl-/aryl-5-mercapto-1,2,4-triazoles and
related compounds as possible CNS [central nervous system] depressants. J. Indian Chem. Soc. 1974, 51,
613–615.
35. CLSI. Performance Standards for Antimicrobial Susceptibility Testing, 27th ed.; Clinical and Laboratory Standards
Institute: Wayne, PA, USA, 2017.
36. Zuo, G.Y.; Zhang, X.J.; Han, J.; Li, Y.Q.; Wang, G.C. In vitro synergism of magnolol and honokiol in
combination with antibacterial agents against clinical isolates of methicillin-resistant Staphylococcus aureus
(MRSA). BMC Complement. Altern. Med. 2015, 15, 425. [CrossRef]
37. Hauser, C.; Hirzberger, L.; Unemo, M.; Furrer, H.; Endimiani, A. In vitro activity of fosfomycin alone and in
combination with ceftriaxone or azithromycin against clinical Neisseria gonorrhoeae isolates. Antimicrob.
Agents Chemother. 2015, 59, 1605–1611. [CrossRef]
38. Zawadzka, K.; Marta Nowak, M.; Piwon´ski, I.; Lisowska, K. The synergy of ciprofloxacin and carvedilol
against Staphylococcus aureus—Prospects of a new treatment strategy? Molecules 2019, 24, 4104. [CrossRef]
[PubMed]
39. Paneth, A.; Stefan´ska, J.; Ste˛pien´, K.; Plech, T.; Janowska, D.; Trotsko, N.; Wujec, M. The synergy
of thiosemicarbazide derivatives with antibiotics in the perspective of their potential applications in
bacterial deseases. Presented at the Third Symposium “School of Medicinal Chemistry”, Wroclaw, Poland,
6–8 September 2017.
´
40. Siwek, A.; Swiderek, K.; Jankowski, S. Problems with molecular mechanics implementations on the example
of 4-benzoyl-1-(4-methyl-imidazol-5-yl)-carbonylthiosemicarbazide. J. Mol. Model. 2012, 18, 843–849.
[CrossRef] [PubMed]
41. HyperCube Inc. Hyperchem 8.0.3; HyperCube Inc.: Gainsville, FL, USA, 2007.
Sample Availability: Samples of the compounds 1–9 are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).