Antibacterial activity of fluorobenzoylthiosemicarbazides and their cyclic analogues with 1,2,4-triazole scaffold

Article abstract of DOI:10.3390/MOLECULES26010170

The development of drug-resistant bacteria is currently one of the major challenges in medicine. Therefore, the discovery of novel lead structures for the design of antibacterial drugs is urgently needed. In this structure–activity relationship study, a l

Full text of DOI:10.3390/MOLECULES26010170

molecules
Article
Antibacterial Activity of Fluorobenzoylthiosemicarbazides and
Their Cyclic Analogues with 1,2,4-Triazole Scaffold
Urszula Kosikowska 1, Monika Wujec 2 , Nazar Trotsko 2 , Wojciech Płonka 3 , Piotr Paneth 4
and Agata Paneth 2,
*
1
2
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Lublin, Chodz´ki 1,
20-093 Lublin, Poland; urszula.kosikowska@umlub.pl
Department of Organic Chemistry, Faculty of Pharmacy, Medical University of Lublin, Chodz´ki 4a,
20-093 Lublin, Poland; monika.wujec@umlub.pl (M.W.); nazar.trotsko@umlub.pl (N.T.)
FQS-Fujitsu Poland, Parkowa 11, 33-332 Kraków, Poland; w.plonka@fqs.pl
3
4
Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology,
˙
Zeromskiego 116, 90-924 Lodz, Poland; piotr.paneth@p.lodz.pl
*
Correspondence: agata.paneth@umlub.pl; Tel.: +48-81-448-7243
Abstract: The development of drug-resistant bacteria is currently one of the major challenges in
medicine. Therefore, the discovery of novel lead structures for the design of antibacterial drugs is
urgently needed. In this structure–activity relationship study, a library of ortho-, meta-, and para-
fluorobenzoylthiosemicarbazides, and their cyclic analogues with 1,2,4-triazole scaffold, was created
and tested for antibacterial activity against Gram-positive bacteria strains. While all tested 1,2,4-
triazoles were devoid of potent activity, the antibacterial response of the thiosemicarbazides was
highly dependent on substitution pattern at the N4 aryl position. The optimum activity for these
compounds was found for trifluoromethyl derivatives such as 15a, 15b, and 16b, which were active
against both the reference strains panel, and pathogenic methicillin-sensitive and methicillin-resistant
Staphylococcus aureus clinical isolates at minimal inhibitory concentrations (MICs) ranging from 7.82
to 31.25 µg/mL. Based on the binding affinities obtained from docking, the conclusion can be reached
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Kosikowska, U.; Wujec, M.;
Trotsko, N.; Płonka, W.; Paneth, P.;
Paneth, A. Antibacterial Activity of
Fluorobenzoylthiosemicarbazides
and Their Cyclic Analogues with
1,2,4-Triazole Scaffold. Molecules 2021,
26, 170. https://doi.org/10.3390/
molecules26010170
that fluorobenzoylthiosemicarbazides can be considered as potential allosteric D-alanyl-D-alanine
ligase inhibitors.
Keywords: thiosemicarbazides; 1,2,4-triazoles; antibacterial activity; S. aureus clinical isolates;
SAR/QSAR analysis
1. Introduction
Received: 2 November 2020
Accepted: 28 December 2020
Published: 31 December 2020
A huge number of drugs and clinical candidates for development contain halogen
atoms. For a long time, predominantly only the steric and hydrophobic factors of halogens
were considered when trying to exploit their role in ligand recognition and target–ligand
binding complex stabilization. Today, the ability of halogens to form favorable intermolec-
ular interactions, such as halogen bonds, hydrogen bonds, and multipolar interactions that
significantly contribute to the stability of target–ligand complexes has been well recognized,
and widely utilized in the rational design of new therapeutics against clinically significant
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
targets [1–4]. According to an analysis by Hernandes and co-workers [5], the majority of
halogenated drugs are fluorine drugs (57%), followed by chlorine ones (38%), while those
with bromine are rare (4%), and only a few iodinated drugs are known (1%) due to their
relatively instability, caused by the high polarizability of the C–I bond.
Regarding the fluorinated drugs and drug candidates, fluorine substitutions have been
extensively investigated in drug research and there are some important reasons that justify
their predominance. For instance, several studies have documented that selective introduc-
tion of a trifluoromethyl group or even a single fluorine atom into a in a key position of a
therapeutic molecule significantly enhances its pharmacokinetic and physicochemical prop-
Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
erties, such as improved metabolic stability and membrane permeability [6]. The increased
Molecules 2021, 26, 170. https://doi.org/10.3390/molecules26010170
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 170
2 of 18
binding affinity of fluorine-containing drug candidates to molecular targets has also been
proved in several cases [7–19]. Currently, several fluorinated pharmaceuticals are widely
used in clinical practice, and nearly one-third of the 100 top-selling drugs are organoflu-
orine compounds. The most common among these are fluoroquinolone antibiotics, anti-
malarials, antidepressants, antivirals, anesthetics, anticancers, cholesterol-lowering drugs,
anti-inflammatory drugs, and asthma drugs, and several reviews on potential therapeutic
applications of fluorinated molecules have been published in recent years [4,20–22]. For
these reasons, we explored the antibacterial potential within the structure–activity rela-
tionship of a library of 1-fluorobenzoyl-4-aryl(alkyl)thiosemicarbazides. Previously, we
had searched for the N1 substituents which would improve the antibacterial potency of
thiosemicarbazide-based compounds and had found that the fluorobenzoyl group might
constitute a particularly useful series [23]. Other research groups also confirmed the
thiosemicarbazide structure as a novel scaffold for potential antibacterial agents [24–33],
although a systematic survey of the effects of the N1 and N4 terminal substituents on
antibacterial potency has not been presented thus far. Thus, in an attempt to more closely
define the factors controlling the potency of these compounds, we prepared three series,
with ortho-, meta-, and para-fluorobenzoyl groups, and determined their antibacterial ac-
tivity in vitro with the structure–activity relationship. In carrying out this study of the
structure–activity relationship, our strategy was four-stage. First, we sought to investigate
the effect of the aromatic fluorine substitution (ortho, meta, and para) and the N4 alkyl and
aryl groups. Second, we sought to extend the antibacterial assay on methicillin-sensitive
and methicillin-resistant Staphylococcus aureus clinical isolates. Third, we set out to alter
the linear structure of the thiosemicarbazide by the synthesis of cyclic analogues with a
1,2,4-triazole scaffold, and to compare these with the corresponding thiosemicarbazide
counterparts. Finally, our results of docking our analogues to the allosteric site of D-alanyl-
D-alanine ligase concurred with the pioneering reports made by Ameryckx et al. [24
the molecular basis of the antibacterial activity of benzoylthiosemicarbazides.
,25] on
2. Results and Discussion
2.1. Synthetic Protocols
The synthesis of ortho-, meta-, and para-fluorobenzoylthiosemicarbazides (sets
and their cyclic analogues with a 1,2,4-triazole-5-thione scaffold (sets IV VI) was per-
formed according to a well-established synthetic route, described elsewhere [23 34
I–III)
–
,
]
(Scheme 1). The reaction of the corresponding fluorobenzoylhydrazide with related alkyl/
aryl isothiocyanate in boiling ethanol gave 1-fluorobenzoyl-4-aryl/(alkyl)thiosemicarbazides
(sets I–III) in 40–90% yields. Resulting compounds were cyclized in boiling aqueous
sodium hydroxide to give corresponding 1,2,4-triazole-3-thiones (sets IV–VI), which were
obtained in 48–90% yields.
R1
R1
R1
+ R2NCS
2% NaOH
R2
R2
sets I-III
Scheme 1. Synthetic route for 1-fluorobenzoyl-4-aryl(alkyl)thiosemicarbazides (sets
IV a, set IV at), m-F (set II set V bt), p-F (set III , set VI
VI). R1 = o-F (set ct). R2 = Pr (
sets IV-VI
I–
III) and 1,2,4-triazole-3-thiones (sets
), Bu ( ), Ph ( ), 1-naph ( ),
–
I
-
-
-b,
-
-
c
-
1
2
3
4
m-tol (5), p-tol (6), m-F (7), p-F (8), m-Cl (9), p-Cl (10), m-Br (11), p-Br (12), m-I (13), p-I (14), m-CF3 (15), p-CF3 (16).
2.2. Antibacterial Screening
The thiosemicarbazides (sets
I–III) were evaluated for their antibacterial activity
against a panel of the reference Gram-positive bacteria using the standard two-fold serial

Molecules 2021, 26, 170
3 of 18
microdilution assay described by the Clinical and Laboratory Standards Institute [35].
The results for the minimum inhibitory concentrations (MICs) are reported in Table 1.
Commercially available antibiotic cefuroxime was used as the reference control.
Table 1. In vitro antibacterial activities of the fluorobenzoylthiosemicarbazides of the ortho set
I
(1a–
16a), meta set II
(1b–
16b),
and para set III (1c–16c) expressed as minimum inhibitory concentrations (MICs) (µg/mL, [mM]).
R1
R2
cLogP
S. a.*
S. a.**
S. e.
B. s.
B. c.
M. l.
500
[1.73]
500
[1.73]
500
[1.73]
250
[0.87]
250
[0.87]
250
[0.87]
3a
4a
2F
Ph
2.26
250
[0.74]
125
[0.37]
250
[0.74]
125
[0.37]
125
[0.37]
125
[0.37]
2F
2F
2F
2F
2F
2F
2F
3F
3F
3F
3F
3F
3F
3F
3F
3F
3F
3F
3F
3F
1-naph
3.42
2.42
2.91
2.94
3.04
3.13
3.15
2.28
3.44
2.71
2.73
2.42
2.44
2.94
2.96
3.07
3.09
3.34
3.15
3.18
250
[0.81]
250
[0.81]
250
[0.81]
125
[0.41]
125
[0.41]
62.5
[0.20]
8a
para-FPh
meta-ClPh
para-ClPh
meta-BrPh
31.25
[0.10]
62.5
[0.19]
31.25
[0.10]
15.63
[0.05]
31.25
[0.10]
7.82
[0.02]
9a
250
[0.77]
500
[1.54]
250
[0.77]
500
[1.54]
31.25
[0.10]
7.82
[0.02]
10a
11a
15a
16a
3b
62.5
[0.17]
62.5
[0.17]
31.25
[0.09]
15.63
[0.04]
15.63
[0.04]
7.82
[0.02]
meta-
CF3Ph
15.63
[0.04]
15.63
[0.04]
7.82
[0.02]
7.82
[0.02]
7.82
[0.02]
7.82
[0.02]
para-
CF3Ph
500
[1.40]
500
[1.40]
125
[0.35]
7.82
[0.02]
15.63
[0.04]
7.82
[0.02]
250
[0.87]
250
[0.87]
1000
[3.46]
250
[0.87]
250
[0.87]
125
[0.44]
Ph
250
[0.74]
125
[0.37]
250
[0.74]
125
[0.37]
125
[0.37]
125
[0.37]
4b
1-naph
125
[0.41]
250
[0.83]
5b
meta-tol
n.a.
n.a.
n.a.
n.a.
1000
[3.30]
1000
[3.30]
1000
[3.30]
1000
[3.30]
1000
[3.30]
1000
[3.30]
6b
para-tol
62.5
[0.20]
62.5
[0.20]
125
[0.41]
62.5
[0.20]
62.5
[0.20]
31.25
[0.10]
7b
meta-FPh
para-FPh
meta-ClPh
para-ClPh
meta-BrPh
para-BrPh
meta-IPh
250
[0.81]
125
[0.41]
500
[1.62]
125
[0.41]
250
[0.81]
250
[0.81]
8b
15.63
[0.05]
15.63
[0.05]
31.25
[0.10]
31.25
[0.10]
15.63
[0.05]
7.82
[0.02]
9b
15.63
[0.05]
7.82
[0.02]
7.82
[0.02]
15.63
[0.05]
62.5
[0.19]
7.82
[0.02]
10b
11b
12b
13b
15b
16b
31.25
[0.09]
15.63
[0.04]
62.5
[0.17]
15.63
[0.04]
15.63
[0.04]
15.63
[0.04]
62.5
[0.17]
31.25
[0.09]
1000
[2.72]
125
[0.34]
62.5
[0.17]
15.63
[0.04]
31.25
[0.08]
15.63
[0.04]
62.5
[0.15]
15.63
[0.04]
15.63
[0.04]
15.63
[0.04]
meta-
CF3Ph
15.63
[0.04]
31.25
[0.09]
31.25
[0.09]
31.25
[0.09]
31.25
[0.09]
15.63
[0.04]
para-
CF3Ph
15.63
[0.04]
15.63
[0.04]
31.25
[0.09]
31.25
[0.09]
15.63
[0.04]
15.63
[0.04]

Molecules 2021, 26, 170
4 of 18
Table 1. Cont.
R1
R2
cLogP
S. a.*
S. a.**
S. e.
B. s.
B. c.
M. l.
250
[0.81]
62.5
[0.20]
250
[0.81]
125
[0.41]
125
[0.41]
62.5
[0.20]
8c
9c
4F
4F
4F
4F
4F
4F
4F
4F
4F
para-FPh
2.47
250
[0.77]
62.5
[0.19]
62.5
[0.19]
15.63
[0.05]
meta-ClPh
para-ClPh
meta-BrPh
para-BrPh
meta-IPh
para-IPh
2.96
2.98
3.09
3.12
3.36
3.39
3.18
3.20
n.a.
n.a.
62.5
[0.19]
31.25
[0.10]
125
[0.39]
62.5
[0.19]
31.25
[0.10]
15.63
[0.05]
10c
11c
12c
13c
14c
15c
16c
Cef.
62.5
[0.17]
125
[0.34]
125
[0.34]
31.25
[0.09]
31.25
[0.09]
15.63
[0.04]
62.5
[0.17]
31.25
[0.09]
62.5
[0.17]
31.25
[0.09]
31.25
[0.09]
15.63
[0.04]
31.25
[0.08]
15.63
[0.04]
125
[0.30]
62.5
[0.15]
31.25
[0.08]
15.63
[0.04]
125
[0.30]
62.5
[0.15]
125
[0.30]
62.5
[0.15]
62.5
[0.15]
15.63
[0.04]
meta-
CF3Ph
125
[0.35]
125
[0.35]
125
[0.35]
125
[0.35]
125
[0.35]
62.5
[0.18]
para-
CF3Ph
125
[0.35]
62.5
[0.18]
125
[0.35]
62.5
[0.18]
62.5
[0.18]
31.25
[0.09]
0.98
[0.002]
0.49
[0.001]
0.24
[0.0006]
15.63
[0.04]
31.25
[0.07]
0.98
[0.002]
Note: S. a.*—S. aureus ATCC 6538, S. a.**—S. aureus ATCC 25923, S. e.—S. epidermidis ATCC 12228, B. s.—B. subtilis ATCC 6633, B. c.—B. cereus
ATCC 10876, M. l.—M. luteus ATCC 10240. Cef.—cefuroxime. n.a.—no active. The remaining thiosemicarbazides (1a 2a 5a 7a 12a 14a
1b, 2b, 14b, 1c–7c) showed no antibacterial activity. The cLogP values were obtained through the online data server, Molinspiration [36].
,
,
–
,
–
,
A few general conclusions arose from the antibacterial assay, which can be summarized
as follows. The most important is that within set of ortho-fluorobenzoylthiosemicarbazides
1a 16a, the compounds that bear substituents at the N4 aryl position that are electron
acceptors were more active than those that are either unsubstituted or bear electron donors.
Electron-withdrawing substituents in the meta position (7a 9a 11a 13a 15a) generally
increased the antibacterial potency, and within meta electron-withdrawing compounds the
activity follows the trend: meta-CF3 15a (MIC 7.82–15.63 g/mL; 0.02–0.04 mM) > meta-Cl 9a
(MIC 7.82–62.5 g/mL; 0.02–0.19 mM) ~ meta-Br 11a (MIC 7.82–62.5 g/mL; 0.02–0.17 mM
> meta-F 7a = meta-I 13a (n.a.; MICs at 2000 g/mL; 6.51 and 4.82 mM, respectively). The
assessment of the results of the meta electron-withdrawing compounds was made in three
terms: (i) an electronic one, expressed by the Hammett substituent constant 37 39
and inductive substituent constant σI; (ii) a steric, expressed by the molar refractivity MR,
the Taft’s constant Es [39 42], and the Charton steric parameter σν 39 43 46]; and (iii) a
hydrophobic, expressed by cLogP and , the Hansch substituent constant [39 47], and led to
the conclusion that the single most important physicochemical property that could explain
the variance in the MICs of these compounds is the Hammett substituent constant
Indeed, as presented in Figure 1, a good correlation exists between the MICs of the meta-
substituted compounds 7a 9a 11a 13a 15a, and the constants, whereas no correlation
could be found between their MICs and steric or hydrophobic parameters. Comparative
I
–
,
,
,
,
µ
µ
µ
)
µ
σ
[ – ]
m
–
[ , –
π
,
σ
.
m
,
,
,
,
σ
m
σ
,
m
σI
activity of these compounds is generally correlated with the Hammett substituent constant
(F < I < Cl < Br < CF3) suggests that the electronic properties of the meta electron
, σν, MR, Es, π, and cLogP values are listed in Table S1. The fact that the improvement in
σ
m
withdrawing substituents control the antibacterial activity. While it remains unclear what
role, if any, the electronegativity of the meta substituents plays in antibacterial activity,
possible interpretations of the correlation between the antibacterial activity of 7a
13a 15a, and are therefore that: (i) antibacterial activity increases with decreasing
electron density on the thiosemicarbazide scaffold because enhancement in binding of
, 9a, 11a,
,
σ
m

Molecules 2021, 26, 170
5 of 18
the thiosemicarbazide to the molecular target is created through the increased positive
charge character of the N4 aryl ring or whole thiosemicarbazide scaffold; or (ii) antibacterial
activity increases with decreasing pKa of the thiosemicarbazide core (NH-NH-C(=S)-NH),
because the compound must be deprotonated to be active (deprotonation hypothesis) [48].
A combination of both effects is possible. A second important observation is that although
the steric substituent constants, such as the Taft Es constant or Charton σν parameter, do
not directly explain the trend in antibacterial activity of meta compounds 7a, 9a, 11a, 13a,
and 15a, the most potent antibacterial meta-CF3 15a proved to possess the lowest Taft Es
constant and the highest Charton σν parameter, thus suggesting that the bulkiness effect
of the CF3 substituent induced an improvement in antibacterial activity. The last but not
least observation that arose from the antibacterial assay of the thiosemicarbazides of set I,
is that substitutions in the para position of the N4 phenyl ring abolished activity (Table 1,
compounds 6a
10a, which inhibited the growth of Bacillus cereus at a concentration similar to cefuroxime,
and Micrococcus luteus at the MIC of 7.82 g/mL (0.02 mM). The fact that 10a and 16a still
, 8a, 10a, 12a, and 14a). The only exceptions were para-CF3 16a and para-Cl
µ
possess antibacterial activity suggest that although the protein binding pocket provides
sufficient space for additional groups at the para position, the combination of both steric
and specific, i.e., hydrophobic and electrostatic or intermolecular interactions might be the
most prominent factor regulating the activity of the para substituted thiosemicarbazides
of set I.
Staphylococcus aureus ATCC 6538
Staphylococcus aureus ATCC 25923
Staphylococcus epidermidis ATCC 12228
Bacillus subtilis ATCC 6633
Bacillus cereus ATCC 10876
Micrococcus luteus ATCC 10240
Figure 1. Plot of values of log(1/MIC) for bacterial strains determined experimentally for the compounds of set
I
with
meta electron-withdrawing substitution (7a, 9a, 11a, 13a, 15a), versus the appropriate values of the σ Hammett electronic
m
substituent constant.
For the meta-fluorobenzoylthiosemicarbazides of set II (1b
antibacterial activity was observed compared to ortho set I. Clearly, within set II, para-Cl
10b with MICs in the range of 7.82–15.63 g/mL (0.02–0.19 mM) against Staphylococcus
–16b), a reverse trend of
µ
spp., B. subtilis, and M. luteus showed a clear preference for the potency whereas the
antibacterial activity of para-CF3 16b was comparable to that of meta-CF3 15b. Furthermore,
meta-I 13b, in contrast to inactive meta-I 13a, was able to inhibit the growth of S. aureus,
B. subtilis, B. cereus, and M. luteus at the concentration of 15.63
µg/mL (0.04 mM), whereas
the antibacterial activity of meta-Cl 9b and meta-Br 11b against S. aureus strains was two to

Molecules 2021, 26, 170
6 of 18
four times higher compared to their isomers, 9a and 11a, respectively. Clearly, within set
II, meta-Cl and para-Cl compounds (9b, 10b), and meta-CF3 and para-CF3 compounds (15b,
16b), proved to be the most effective. No important contribution(s), however, of electronic,
steric, and/or hydrophobic factors on antibacterial trend within set II was found.
Finally, the antibacterial activity of the para-fluorobenzoylthiosemicarbazides of set III
(
1c
ortho (set
10c), bromo (11c
cefuroxime. In addition, these compounds with MICs at 15.63
10c, and 0.04 mM for 11c 12c 13c 14c) showed good inhibitory action against M. luteus.
–
16c) was tested. Although these compounds were generally less effective than their
) and meta (set II) isomers, the efficiency of the compounds with chloro (9c
12c), and iodo (13c 14c) substitutions against B. cereus was still close to
g/mL (0.05 mM for 9c
I
,
,
,
µ
,
,
,
,
The presence of other substituents, both an electron withdrawing (CF3, F) and an electron
donating (CH3), reduced potency, whereas the presence of a propyl, butyl, phenyl, or
naphthyl group abolished activity; similarly to the antibacterial trends observed for sets I
and II.
To confirm antibacterial potential of the most potent thiosemicarbazides, 15a
, 16a,
9b
,
10b 11b 15b 16b, these compounds were subsequently assayed against a panel of
,
,
,
pathogenic methicillin-sensitive and methicillin-resistant Staphylococcus aureus clinical
isolates. As indicated from the data collected in Table 2, the best antibacterial response,
with MICs in the range of 3.91–15.63
µg/mL, was noted for trifluoromethyl derivatives;
meta 15a (0.02–0.04 mM) and para 16b (0.04–0.09 mM). Meta trifluoromethyl derivative 15b
showed an inhibitory action at a slightly higher concentration (MICs 7.82–31.25
0.04–0.08 mM), while with the remaining compounds activity was lost.
µg/mL;
Table 2. In vitro antibacterial activities of 15a
, 15b, and 16b against clinical isolates of methicillin-
sensitive and methicillin-resistant Staphylococcus aureus, expressed as MICs (µg/mL, [mM]).
Strain
15a
15b
16b
Vancomycin
MSSA-1
MSSA-2
MSSA-3
MSSA-4
MSSA-5
MSSA-6
MSSA-7
MSSA-8
MRSA-11
MRSA-12
MRSA-13
MRSA-14
MRSA-15
MRSA-16
MRSA-17
MRSA-18
15.63 [0.04]
15.63 [0.04]
7.82 [0.02]
15.63 [0.04]
7.82 [0.02]
7.82 [0.02]
7.82 [0.02]
7.82 [0.02]
15.63 [0.04]
7.82 [0.02]
7.82 [0.02]
15.63 [0.04]
15.63 [0.04]
7.82 [0.02]
15.63 [0.04]
15.63 [0.04]
31.25 [0.09]
31.25 [0.09]
15.63 [0.04]
31.25 [0.09]
15.63 [0.04]
15.63 [0.04]
15.63 [0.04]
15.63 [0.04]
31.25 [0.09]
31.25 [0.09]
31.25 [0.09]
15.63 [0.04]
31.25 [0.09]
15.63 [0.04]
15.63 [0.04]
15.63 [0.04]
7.82 [0.02]
15.63 [0.04]
7.82 [0.02]
15.63 [0.04]
15.63 [0.04]
15.63 [0.04]
7.82 [0.02]
15.63 [0.04]
7.82 [0.02]
3.92 [0.01]
7.82 [0.02]
7.82 [0.02]
7.82 [0.02]
7.82 [0.02]
3.92 [0.01]
7.82 [0.02]
0.39 [0.0003]
0.78 [0.0005]
0.39 [0.0003]
0.78 [0.0005]
0.78 [0.0005]
0.78 [0.0005]
0.78 [0.0005]
1.56 [0.001]
0.78 [0.0005]
0.39 [0.0003]
0.78 [0.0005]
0.78 [0.0005]
0.78 [0.0005]
0.78 [0.0005]
0.78 [0.0005]
0.78 [0.0005]
Note: multidrug-sensitive clinical isolates of S. aureus (MSSA1–MSSA8) and multidrug-resistant clini-
cal isolates of S. aureus (MRSA11–MRSA18) from hospital sources were obtained from wound swabs.
Since SAR analysis of the fluorobenzoylthiosemicarbazides (sets
more complicated than we expected, as mentioned above, we also prepared their cyclic
analogues with a 1,2,4-triazole-3-thione scaffold (sets IV VI), and submitted them to
I–III) seemed to be
–
antimicrobial assay to determine the role, if any, of the thiosemicarbazide core NH-NH-
C(=S)-NH on antibacterial activity. As shown in Table 3, all tested triazoles showed much
lower antibacterial potency compared to their acyclic precursors (e.g., 15a vs. 15at) or were
even inactive (e.g., 13b vs. 13bt). It is important to note, that none of them showed potent
antibacterial activity. Thus, these results confirmed that, although the linear NH-NH-C(=S)-
NH core is a key structural element for antibacterial activity, the electronic effect of the N1
and N4 substituents on the whole thiosemicarbazide skeleton is equally important. It seems
equally possible, however, that the N1 and N4 substituents could lead to structural benefits,

Molecules 2021, 26, 170
7 of 18
either by providing favorable conformation of the thiosemicarbazide in the molecular target
binding site, or by providing favorable intermolecular contacts. Of course, a combination of
both electronic and steric effects is also possible. Another important observation is that the
hydrophobic factor cLogP, alone, had a negligible influence on the antibacterial response of
the tested thiosemicarbazides and s-triazoles.
Table 3. In vitro antibacterial activities of the s-triazoles of ortho set IV
(1ct–16ct) expressed as MICs (µg/mL, [mM]).
(1at–
16at), meta set V (1bt–16bt), and para set VI
R1
R2
cLogP
S. a.*
S. a.**
S. e.
B. s.
B. c.
M. l.
1000
[4.21]
125
[0.53]
1000
[4.21]
1000
[4.21]
500
[2.11]
500
[2.11]
1at
8at
2F
Pr
2.56
500
[1.73]
250
[0.86]
250
[0.86]
500
[1.73]
125
[0.43]
2F
2F
2F
2F
2F
2F
2F
3F
3F
3F
3F
4F
4F
4F
4F
4F
4F
para-FPh
meta-BrPh
para-BrPh
meta-IPh
3.12
3.95
3.76
4.22
4.04
4.03
3.85
3.84
3.65
3.97
3.87
3.16
3.68
3.81
4.27
4.08
3.90
n.a.
500
[1.43]
125
[0.36]
125
[0.36]
125
[0.36]
500
[1.43]
500
[1.43]
11at
12at
13at
14at
15at
16at
9bt
1000
[2.86]
500
[1.43]
1000
[2.86]
1000
[2.86]
1000
[2.86]
1000
[2.86]
125
[0.31]
62.5
[0.16]
125
[0.31]
15.63
[0.04]
125
[0.31]
125
[0.31]
62.5
[0.16]
125
[0.31]
62.5
[0.16]
62.5
[0.16]
250
[0.63]
62.5
[0.16]
para-IPh
62.5
[0.18]
62.5
[0.18]
125
[0.37]
62.5
[0.18]
31.25
[0.09]
62.5
[0.18]
meta-CF3Ph
para-CF3Ph
meta-ClPh
para-ClPh
meta-BrPh
para-CF3Ph
para-FPh
250
[0.74]
62.5
[0.18]
250
[0.74]
250
[0.74]
125
[0.37]
62.5
[0.18]
125
[0.41]
62.5
[0.20]
250
[0.82]
250
[0.82]
125
[0.41]
62.5
[0.20]
250
[0.82]
31.25
[0.10]
500
[1.64]
125
[0.41]
250
[0.82]
31.25
[0.10]
10bt
11bt
16bt
8ct
62.5
[0.18]
62.5
[0.18]
125
[0.36]
125
[0.36]
62.5
[0.18]
125
[0.36]
250
[0.74]
125
[0.37]
500
[1.47]
250
[0.74]
250
[0.74]
125
[0.37]
500
[1.73]
500
[1.73]
1000
[3.46]
500
[1.73]
500
[1.73]
500
[1.73]
125
[0.41]
31.25
[0.10]
125
[0.41]
125
[0.41]
125
[0.41]
125
[0.41]
10ct
12ct
13ct
15ct
16ct
Cef.
para-ClPh
para-BrPh
meta-IPh
125
[0.36]
62.5
[0.18]
125
[0.36]
62.5
[0.18]
125
[0.36]
62.5
[0.18]
125
[0.31]
62.5
[0.16]
125
[0.31]
62.5
[0.16]
62.5
[0.16]
62.5
[0.16]
125
[0.37]
250
[0.74]
125
[0.37]
125
[0.37]
62.5
[0.18]
meta-CF3Ph
para-CF3Ph
250
n.a.
125
[0.37]
62.5
[0.18]
n.a.
n.a.
n.a.
0.98
[0.002]
0.49
[0.001]
0.24
[0.0006]
15.63
[0.04]
31.25
[0.07]
0.98
[0.002]
Note: S. a.*—S. aureus ATCC 6538, S. a.**—S. aureus ATCC 25923, S. e.—S. epidermidis ATCC 12228, B. s.—B. subtilis ATCC 6633, B. c.—B. cereus
ATCC 10876, M. l.—M. luteus ATCC 10240. Cef.—cefuroxime. n.a.—no active. Remaining triazoles (2at 7at 9at 10at 1bt 8bt 12bt 15bt
1ct 7ct 9ct 11ct 14ct) showed no antibacterial activity. The cLogP values were obtained through the online data server, Molinspiration [36].
–
,
,
,
–
,
-
,
–
,
,
,

Molecules 2021, 26, 170
8 of 18
2.3. Assessing Bactericidal/Bacteriostatic Characteristics
Although the molecular mechanism of the antibacterial activity of thiosemicarbazides
is still unknown, their inhibitory action towards bacterial topoisomerases has been pro-
posed as one possible explanation [49–51]. Experiments aimed at identifying possible
additional targets of these compounds are currently ongoing, but so far the only conclu-
sive results have been yielded by Ameryckx et al., for ortho- hydroxybenzoylthiosemi-
carbazides [24,25]. According to these results, D-alanyl-D-alanine ligase (Ddl) might be
considered as a potential bacterial target for the thiosemicarbazides, with the N1 ortho-
hydroxybenzoyl substitution and subsequent time kill assay for representative analogue,
with 2,3-dichlorophenyl group at the N4 position (PR), proving that it acts as a bactericidal
agent. Generally, when considering the molecular mechanism by which antibacterial agents
control microorganisms, the mode of their action can also be classified according to whether
they lead to the death of the microbe (bactericidal action), or inhibit its growth (bacterio-
static action). Thus, to distinguish whether our thiosemicarbazides were bactericidal or
bacteriostatic in nature, we next submitted them to a minimal bactericidal concentration
(MBC) assay. Upon analysis, all compounds were found to be bacteriostatic (MBC/MIC
>4) toward all bacteria strains tested (data not shown). Subsequent results from the con-
centration dependent time-kill assay, for the most potent antibacterial agents 15a, 15b, 16b
against S. aureus ATCC 25923 over a period of 24 h, confirmed this observation (Figure 2).
A concentration-dependent and time-dependent bacteriostatic activity was found for all
the compounds tested, even at a high concentration of 4
activity, defined as a reduction of at least 99.9% of the total count of CFU/mL in the original
inoculum within a period of 24 h, was not observed.
×
MIC or 8
×
MIC. Bactericidal
15a
15b
16b
Figure 2. Time-kill kinetics of 15a
,
15b, and 16b against S. aureus ATCC 25923 over a period of 24 h. Control—S. aureus
ATCC 25923 growth without tested compounds.
As a general rule, antibacterial agents that disrupt the cell wall or cell membrane, or
interfere with essential enzymes are often bactericidal, whereas those that inhibit protein
synthesis, e.g., bacterial topoisomerases, or interfere with metabolic processes tend to be
bacteriostatic [52
–54]. Taking this fact into account, the results of the MBC assay are not
surprising. In fact, in our previous studies [23], on the thiosemicarbazides 9a and 10a, we

Molecules 2021, 26, 170
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showed that their inhibitory action against S. aureus DNA gyrase was very weak; compound
10a was able to inhibit ~50% of S. aureus DNA gyrase activity at as high a concentration
as 100
(~33%). Collectively, the results of the MBC assay further confirm the assumption that, for
our bacteriostatic thiosemicarbazides of sets III, bacterial topoisomerases are not primary
µg/mL, whereas the inhibitory activity of 9a at this concentration was even lower
I
–
factors contributing to their antibacterial activity.
2.4. Thiol−ThioneTautomerismintheThiosemicarbazides
In an attempt to extract a general background of the relationship between the antibacte-
rial activity and physicochemical properties of the tested thiosemicarbazides and s-triazoles,
we also performed a QSAR analysis. Before experiments, however, the relative stabilities
of the thiosemicarbazide tautomeric forms were studied, based on computational analysis.
As is known, the proton transfers influence conformational and electronical changes
in a molecule, and thus induce changes in its flexibility, polarity, lipophilicity, and acceptor–
donor capacity. All these factors have a significant impact on biological activity, including
cell membrane permeability, metal complexation, and inter- and intra-molecular interac-
tions. Since the thiosemicarbazides studied here formally can exist in tautomeric thione
(C=S) and thiole (C–S) forms [24
ative Gibbs free energies. For this purpose, geometries of the thione and thiole forms of
the thiosemicarbazides were optimized at the DFT level of theory, using the B97X-D
,25,55,56] (Figure 3), we evaluated theoretically their rel-
ω
functional [57] expressed in the def2-TZVP basis set [58], as implemented in the Gaussian16
program [59]. Vibrational analysis was used to ensure that the optimized geometry corre-
sponded to a stationary point representing a minimum on the potential energy surface (3n
−
6 real vibrations). A SMD continuum solvent model [60] was used to model the aqueous
solution. Our results indicated that the thiosemicarbazides in the thiole tautomeric form
were less stable by about 21.8 kcal/mol, and thus their presence in the aqueous solution can
be disregarded. The relative stability in the tautomers of s-triazole was studied previously,
and it was established that thione tautomer is also the energetically most stable form [61].
R
F
R
F
Figure 3. Thione-thiole tautomeric forms of the thiosemicarbazides.
2.5. QSAR Analysis
Having determined the relative stabilities of the thiosemicarbazides and s-triazoles,
QSAR analysis was performed using SCIGRESS software (SCIGRESS v 3.4.4 www.scigress.
com), to find correlations between the structure and the minimal inhibitory concentration
for each microbial species separately. For these studies, only the thiosemicarbazides and
s-triazoles that were active in the antibacterial assay were included. For the purpose of
the analysis the values of the MICs were converted to Log10(1/MIC). The 466 descriptors
used included both topological and quantum descriptors, calculated by the PM6/COSMO
method (see Supplementary Materials). A fully automated procedure of descriptor selection
by the enhanced replacement method (ERM) was applied to select five descriptors for
best fitting of the linear regression equation (Table 4). The obtained values of R2 are in
the range of 0.64–0.72, so, while some correlation was observed, it is not high enough for
predicting unknown compounds. It is worth noticing, however, that the presence of a
chlorine atom in the molecule appears to be beneficial for activity, while a bromine atom
apparently decreases activity against S. aureus ATCC 25923. The resulting equations and
fit (R2 and leave-one-out cross validation R2) are shown in Table 4. The graphs of linear
regressions and cross validations are shown in Figure 4.

Molecules 2021, 26, 170
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Table 4. Parameters of the five descriptor multiple linear regression QSAR models of antibacterial activity.
Species
R2
CV r2
Equation
Log10(1/MIC) = 0.4570 × Chlorine count + 5851.7193 × highest electrophilic
susceptibility on O − 275.6544 × ring count all aromatic/MW − 1600.7357 ×
highest nucleophilic susceptibility/MW − 0.7411 × ln(highest nucleophilic
susceptibility on H) − 3.6088
S. a.*
0.7229
0.6346
Log10(1/MIC) = −0.8910 × Bromine count + 68.6203 × highest electrophilic
susceptibility on C − 10.8611 × highest radical susceptibility on C + 3544.5345 ×
highest electrophilic susceptibility on O + 0.0205 × hydrophobicity weighted
positive area − 6.2195
S. a.**
0.6376
0.5042
Log10(1/MIC) = 0.6767
×
Chlorine count + 23.1042
×
highest radical susceptibility
on C + 0.0282 × atomic charge weighted positive area-atomic charge weighted
negative area − 5541.1048 × highest nucleophilic susceptibility on C/MW −
1.7821 × ln(Csp2 bonded to 2 C) + 4.8737
S. e.
B. s.
B. c.
0.6589
0.6477
0.6493
0.5465
0.5163
0.5250
Log10(1/MIC) = 0.3005 × Chlorine count − 40.9730 × all count/MW − 5.3291 ×
highest nucleophilic susceptibility2 + 1.3923 × ln(rotatable bond count) +
2.26699e-03 × 1.0/highest nucleophilic susceptibility on H − 0.0468
Log10(1/MIC) = 8728.8101
×
highest electrophilic susceptibility on O
−
16.0298
×
highest radical susceptibility on C2 − 16.8597 × 1.0/log P + 1.64514e-03 ×
1.0/highest nucleophilic susceptibility on H − 2.7091 × sqrt(ring count all
aromatic) + 4.5796
Log10(1/MIC) = 0.7892 × Chlorine count + 5623.4960 × highest electrophilic
susceptibility on O − 344.9316 × ring count all aromatic/MW − 1613.2362 ×
highest nucleophilic susceptibility/MW + 1.84660e-03 × 1.0/highest radical
susceptibility on H + 0.3114
M. l.
0.6858
0.5990
Note: S. a.*—S. aureus ATCC 6538, S. a.**—S. aureus ATCC 25923, S. e.—S. epidermidis ATCC 12228, B. s.—B. subtilis ATCC 6633, B. c.—B. cereus
ATCC 10876, M. l.—M. luteus ATCC 10240.
2.6. Docking Studies
As mentioned above, recently Ameryckx et al. [24,25] reported the discovery of a
series of 4-aryl-1-(2-hydroxybenzoyl)thiosemicarbazides as promising allosteric inhibitors
of D-alanyl-D-alanine ligase (Ddl); an essential enzyme in bacterial cell wall biosynthesis,
and an important target for developing new antibacterial agents. This enzyme catalyzes
the formation of D-alanile:D-alanine dipeptide, the crucial precursor of bacterial cell wall
peptidoglycan, by two half-reactions. In the first half-reaction, Ddl uses one D-alanine
and one ATP as substrates to produce a phosphorylated D-alanine intermediate. In the
second half-reaction, Ddl uses a second D-alanine substrate to complete the reaction to
the normal D-alanine:D-alanine dipeptide product [62]. According to previously reported
results, the 4-(3,4-dichlorophenyl)-1-(2-hydroxybenzoyl)thiosemicarbazide (PR, Figure 5)
was identified as one of the most potent Ddl inhibitors, with a bactericidal activity against
Gram-positive bacteria, including multidrug resistant strains and, at the same time, very
low cytotoxicity on a THP-1 human monocytic cell line. Although the authors concluded
their extensive experiments with the judgment that the ortho hydroxy group is critical
for the inhibitory action of benzoylthiosemicarbazides against Ddl, none of the tested
compounds that were inactive on ligase exhibited an antibacterial activity at the same time.
This fact supports the hypothesis that the inhibition of Ddl activity could explain, at least
in part, the antibacterial effect observed for the benzoylthiosemicarbazides.
Given the above, we decided to dock the best of our benzoylthiosemicarbazides, 9a
,
11a
,
15a 16a 9b 10b 11b 13b 15b, and 16b, to the allosteric site of S. aureus Ddl (PDB
,
,
,
,
,
,
code: 2I80). The best docking scores for the thiosemicarbazides, and previously reported
inhibitor PR, are listed in Table 5. In Figure 5 the best binding pose for representative model
compound 9a is presented. The best binding poses of the remaining thiosemicarbazides at
the allosteric site of S. aureus Ddl are available in the Supplementary Materials.

Molecules 2021, 26, 170
11 of 18
Figure 4. Cont.

Molecules 2021, 26, 170
12 of 18
Figure 4. Predicted vs. experimental log10(1/MIC) values for the respective species for all active compounds (red squares)
and leave-one-out model validation results (blue circles). Note: S. a.*—S. aureus ATCC 6538, S. a.**—S. aureus ATCC 25923,
S. e.—S. epidermidis ATCC 12228, B. s.—B. subtilis ATCC 6633, B. c.—B. cereus ATCC 10876, M. l.—M. luteus ATCC 10240.

Molecules 2021, 26, 170
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PR
9a
NI
Figure 5. Interactions of previously reported Ddl inhibitor PR, our representative model thiosemicarbazide (9a), and native
ligand NI in the allosteric binding site of Ddl.
Table 5. FleXx docking scores of the thiosemicarbazides 9a
,
11a
,
15a
,
16a
,
9b
,
10b
,
11b
,
13b
,
15b, and
16b in relation to S. aureus d-alanyl-d-alanine ligase (Ddl).
9a
25.9
11a
23.4
15a
28.4
16a
25.4
9b
27.9
10b
28.3
11b
28.5
13b
26.0
15b
27.9
16b
28.2
PR
NI
−
−
−
−
−
−
−
−
−
−
−
27.7
−
14.3
Note: PR—4-(3,4-dichlorophenyl)-1-(2-hydroxybenzoyl)thiosemicarbazide; previously reported Ddl
inhibitor [24]; NI—native inhibitor.
According to the docking results presented in Table 5, all the thiosemicarbazides are
identified as binding at an allosteric site of Ddl, with much higher affinity than the native
inhibitor. Their predicted binding sites overlap well with the binding site of the native
inhibitor NI, and in all cases at least one hydrogen bond is observed between the NH group
of the thiosemicarbazide skeleton and Pro311, Met 310, or Pro93 (see Supporting Material).
The binding of the thiosemicarbazides 9a, 11a, 15a, 16a, 9b, 10b, 11b, 13b, 15b, and 16b is
further stabilized by numerous hydrophobic interactions with surrounding residues, and
most of these interactions are identical to those in the crystal structure of native amide NI
in complex with S. aureus Ddl [62]. Notably, the binding mode of our thiosemicarbazides in
the allosteric site of Ddl is stabilized by similar intermolecular interactions as predicted for
previously discovered inhibitor PR (see Figure 5). Thus, the docking results indicate that
our fluorobenzoylthiosemicarbazides can be at least theoretically considered as potential D-

Molecules 2021, 26, 170
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alanyl-D-alanine allosteric ligase inhibitors. Enzymatic experiments, however, are needed
to confirm this assumption.
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 (Karlsruhe, 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 were uncorrected. Elemental analyses
were determined by a AMZ-CHX elemental analyzer (PG, Gdan´sk, Poland) and were
1
within
±
0.4% of the theoretical values. H-NMR spectra were recorded on a Bruker Avance
(300 MHz) spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). Physicochemical
characterizations of the compounds 3a
3c 6c 8c 9c 10c 12c 3at 6at 8at 10at
9ct 10ct 12ct, were reported elsewhere [51
,
6a
,
8a
2bt
63
,
9a
,
10a
,
12a
,
15a
,
2b
,
,
3b
1ct
,
8b
,
10b
,
,
12b
6ct
,
1c
,
,
,
,
,
,
,
,
,
,
,
,
12at
,
,
3bt
,
8bt
,
10bt
,
12bt
,
3ct
,
4ct
,
8ct
,
,
,
–
70]. These compounds were prepared in
our lab for the purpose of this work, and their characteristics (melting points, 1H-NMR
spectra) compared well with the characteristics already reported. The structures of the
compounds 1a, 2a, 5a, 7a, 1b, 4b, 5b, 6b, 7b, 9b, 15b, 2c, 4c, 5c, 7c, 15c, 1at, 2at, 4at, 5at,
9at 1bt 2ct, and 5ct are known (CAS numbers); however, there are no references reporting
,
,
their preparation and physicochemical characterization; therefore, these data have been
included into the manuscript, and are presented in the Supplementary Materials file.
3.1.1. General Procedure for Synthesis of the Thiosemicarbazides
A solution of appropriate fluorobenzoic hydrazide (0.01 mol) and an equimolar
amount of aryl or alkyl isothiocyanate (0.01 mol) in anhydrous ethanol (25 mL) was
heated under reflux for 10–40 min. After cooling, the solid formed was filtered off, dried,
and crystallized from ethanol.
3.1.2. General Procedure for Synthesis of the 1,2,4-Triazole-3-thiones
A solution of appropriate thiosemicarbazide (0.01 mol) in 2% NaOH (20 mL) was
heated under reflux for 2 h. After cooling, the solution was acidified with 3M HCl where-
upon a solid separated out. The solid formed was filtered off, dried, and crystallized
from ethanol.
3.2. Antibacterial Screening
The antimicrobial activity of the title compounds was tested on six reference Gram-
positive strains: S. aureus ATCC 25923, S. aureus ATCC 6538, Staphylococcus epidermidis
ATCC 12228, Bacillus subtilis ATCC 6633, B. cereus ATCC 10876, Micrococcus luteus ATCC
10240, eight MSSA clinical isolates (MSSA 1–MSSA 8), and eight MRSA clinical isolates
(MRSA 11–MRSA 18). For this purpose, microbial suspensions were prepared in sterile
saline (0.85% NaCl) with an optical density of 0.5 McFarland standard; 150
(CFU, colony-forming units). All the stock solutions of the tested compounds were dis-
solved in DMSO. Mueller-Hinton broth medium was used with a series of 2-fold dilutions
×
106 CFU/mL
of the tested substances in the range of final concentrations from 3.91 to 1000
µg/mL. For
SAR analysis, compounds 7a and 13a were also tested at the concentration of 2000
µg/mL.
Cefuroxime was used as positive control.
The in vitro antibacterial activity of the tested compounds was screened on the basis
of MIC (minimal inhibitory concentration), defined as the lowest concentration of the
compound at which there was no visible growth of the tested microorganisms. Deter-
mination of the MIC values was achieved by a broth microdilution method, according
to CLSI recommendation [71]. MBC (minimal bactericidal concentration), defined as the
lowest concentration of compound that resulted in a > 99.9% reduction in CFU of the initial
inoculum, was determined by plating out the contents of wells (20 µL), that showed no

Molecules 2021, 26, 170
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visible growth of bacteria, onto Mueller-Hinton agar plates, and incubating at 35 ◦C for
18 h. Both MIC and MBC values are given in
µg/mL, according to the CLSI reference [35].
Time-Kill Assay
The time-kill curve analyses were performed by culturing Staphylococcus aureus
ATCC 25923 in Mueller-Hinton broth medium (bioMerieux, France), in the presence of
seven antimicrobial-concentrations, in doubling dilutions ranging from 3.9 to 250
µ
g/mL
1
(
× MIC to 8
×
MIC) of compounds 15a
108 CFU/mL, colony-forming units per milliliter) was prepared in MHB
L of inoculum
, 15b, and 16b. Subsequently, 0.5 McFarland
S2tandard (1.5
×
medium from culture grown in MHB medium overnight at 35 ◦C. Next, 1
µ
was refreshed into 10 mL MHB medium in tubes with antimicrobial-free (control) and
1
with various concentrations of compounds 15a
,
15b, and 16b (multiples MICs—
×
MIC;
2
◦
1 × MIC, 2
×
MIC, 4
×
MIC and 8
×
MIC), and incubated at 35 C and shaking at 150 rpm.
L) of the cultures were removed at 0, 2, 4, 6, 8, 16, 20, and 24 h, and serial
Aliquots (1
µ
10-fold dilution were prepared in PBS buffer before planting on a Mueller-Hinton agar
(bioMerieux, France, Grenoble) plate. For samples obtained from each time-point, the
number of viable colonies was determined on a compound-free MHA plate following
incubation at 24 h at 35 ◦C (number of colonies as reciprocal of the dilution factor). The
lower limit for the colony counts was 5 to 300 CFU on the plate with proper dilution.
Antimicrobial compounds were considered to the bactericidal at the lowest concentration
that reduced the original inoculum by 99.9% for each of the indicated time-points [72].
3.3. Docking Studies
The docking simulations were performed using the FlexX docking module of the
LeadIT environment as implemented in the LeadIT 2.1.9 program (BioSolveIT GmbH,
Augustin, Germany) using the crystal structure of Staphylococcus aureus D-alanine:D-alanine
ligase in complex with 3-chloro-2,2-dimethyl-N-[4-(trifluoromethyl)phenyl]propanamide
(PDB code 2I80). The active sites were defined to include all of the atoms within 6.5 Å
radius of the native ligand. To validate the docking protocol, ligands co-crystallized with
the proteins were initially docked into the crystal structure of Ddl; the best conformations
obtained were practically identical with the experimental ones. Subsequently, the studied
thiosemicarbazides were docked using the same docking parameters. The first 100 top
ranked docking poses were saved for each docking run.
4. Conclusions
We have shown that for a range of fluorobenzoylthiosemicarbazides, alteration of
the electronic or structural nature of the thiosemicarbazide, by incorporation of the N4
alkyl or electron-donating aryl system or by the preparation of cyclic derivatives with the
1,2,4-triazole scaffold, offers no advantages over the N4 electron-withdrawing aryl system.
The activity of the N4 electron-withdrawing aryl thiosemicarbazides is highly dependent
on the steric effects of the substituents. The optimum activity lies with trifluoromethyl
derivatives such as 15a
by Ameryckx et al. [24
,
15b, and 16b. The docking results support the idea presented
,25] that these compounds are able to act as allosteric inhibitors
of D-alanyl-D-alanine ligase. Enzymatic experiments, however, are needed to provide
evidence for this assumption.
Supplementary Materials: The following are available online: steric and electronic parameters for
F, Cl, Br, I, and CF3 substituents, physicochemical characterization of the thiosemicarbazides and
s-triazoles, list of descriptors used in QSAR modeling, docking binding poses obtained for all studied
compounds.
Author Contributions: Conceptualization, A.P., M.W; methodology, U.K., M.W., N.T., W.P., P.P.;
validation, U.K.; formal analysis, A.P.; investigation, U.K., A.P; resources, U.K., M.W.; data curation,
M.W.; writing—original draft preparation, A.P.; writing—review and editing, A.P.; visualization,
A.P., N.T., W.P.; supervision, A.P.; project administration, M.W., A.P; funding acquisition, U.K.,

Molecules 2021, 26, 170
16 of 18
M.W.; QSAR calculations, W.P.; computational studies, P.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.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Edyta Stefaniszyn and Dr Szymon Kosiek for the synthesis of
title compounds.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of title compounds are available from the authors.
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