Synthesis and structure-activity relationship studies of 4-arylthiosemicarbazides as topoisomerase IV inhibitors with Gram-positive antibacterial activity. Search for molecular basis of antibacterial activity of thiosemicarbazides

Article abstract of DOI:10.1016/j.ejmech.2011.09.034

1-(indol-2-carbonyl)-4-(4-nitrophenyl)-thiosemicarbazide was synthesized and antibacterial and type IIA topoisomerases (DNA gyrase and topoisomerase IV) activity was evaluated. It was found that it shows activity against Gram-positive bacteria with MICs o

Full text of DOI:10.1016/j.ejmech.2011.09.034

European Journal of Medicinal Chemistry 46 (2011) 5717e5726
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Laboratory note
Synthesis and structureeactivity relationship studies of 4-arylthiosemicarbazides
as topoisomerase IV inhibitors with Gram-positive antibacterial activity. Search
for molecular basis of antibacterial activity of thiosemicarbazides
,
b
,
c
d
Agata Siwek a , Pawe1 Sta˛czek , Joanna Stefanska
*
ꢀ
a
ꢀ
Department of Organic Chemistry, Faculty of Pharmacy, Medical University, Chodzki 4a, 20-093 Lublin, Poland
b Institute of Applied Radiation Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland
c Department of Genetics of Microorganisms, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland
d Department of Pharmaceutical Microbiology, Medical University, Oczki 3, 02-007 Warszawa, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2011
Received in revised form
16 September 2011
Accepted 20 September 2011
Available online 28 September 2011
1-(indol-2-carbonyl)-4-(4-nitrophenyl)-thiosemicarbazide was synthesized and antibacterial and type
IIA topoisomerases (DNA gyrase and topoisomerase IV) activity was evaluated. It was found that it shows
activity against Gram-positive bacteria with MICs of 50
mg/mL and inhibitory action against topoisom-
erase IV with an IC50 of 14 M. Although modification of its structure resulted in molecules with a lower
m
biological profile, our observations strongly implicate that thiosemicarbazide derivatives participate in at
least two different mechanisms of antibacterial activity; one is connected with the inhibition of topo-
isomerase IV, while the nature of the other cannot be elucidated from the limited data collected thus far.
The differences in bioactivity further investigated by the molecular modeling approach and docking
studies suggest that inhibitory activity of 4-arylthiosemicarbazides is connected with electronic structure
rather than the geometry of the molecule.
Keywords:
Thiosemicarbazide derivatives
Antibacterial activity
Bacterial topoisomerase IV
SAR
Ó 2011 Elsevier Masson SAS. All rights reserved.
Molecular docking
1. Introduction
dependent strand-passing reaction plays an essential role in
modulating DNA supercoiling, knotting, and catenation. DNA
Increasing antibiotic resistance of bacterial pathogens has been
observed for many years, both in Gram-negative and Gram-positive
bacteria, concerning the isolates from hospitalized and ambulatory
patients. [1e5] Therefore, significant efforts have been made by
many research groups to improve the potency and antibacterial
spectrum of existing drugs. [5e9] A major goal, however, is that of
finding new chemical entries rather than continuing the search for
new members of already defined chemical classes. [10e13]
Attractive targets in the search for new antibiotics are well-
studied bacterial type IIA topoisomerases DNA gyrase and topo-
isomerase IV, two highly homologous enzymes that play essential
roles in bacterial DNA replication, chromosome segregation, and
DNA compaction. [14e21] These enzymes act by transporting one
segment of the DNA duplex through transient protein-linked
double-strand breaks in another DNA segment. This ATP-
gyrase and topoisomerase IV are structurally and mechanistically
related but have acquired distinct characteristics during evolution.
DNA gyrase facilitates DNA unwinding at replication forks, and
topoisomerase IV has a specialized function in mediating the dec-
atenation of interlocked daughter chromosomes. Both DNA gyrase
and topoisomerase IV are heterotetramers consisting of two A and
two B subunits (A2B2). The A subunit contains the active-site
tyrosine involved in the DNA breakageereunion activity while the
B subunit catalyzes ATP hydrolysis. [19,22]
Recently, [23] in the search for novel bacterial topoisomerase
inhibitors, we have reported that two of our compounds, 4-benzoyl-
1-(indol-2-yl)-carbonylthiosemicarbazide (compound 1p, Fig.1), and
4-benzoyl-1-(4-methyl-imidazol-5-yl)-carbonylthiosemicarbazide
(compound 5p, Fig. 1) represent a new class of topoisomerase IV
inhibitors. Based on the DFTand docking studies it was proposed that
the inhibitory activity of 4-benzoylthiosemicarbazides is strongly
connected with the geometry of the molecule. In order to expand our
initial disclosure with further details on the structural features
required for bioactivity of thiosemicarbazide derivatives, new
analogs with4-arylthiosemicarbazideskeletonwere synthesized and
tested for their antibacterial and topoisomerases (DNA gyrase and
* Corresponding author. Department of Organic Chemistry, Faculty of Pharmacy,
ꢀ
Medical University, Chodzki 4a, 20-093 Lublin, Poland. Tel.: þ48 081 532 05 19;
fax: þ48 081 532 45 46.
E-mail address: agata.siwek@am.lublin.pl (A. Siwek).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.034

5718
A. Siwek et al. / European Journal of Medicinal Chemistry 46 (2011) 5717e5726
compared to 1o. Moreover, it had not shown inhibitory action
against both DNA gyrase and topoisomease IV. Based on these
results it was proposed that either 4-nitrophenylthiosemicarbazide
scaffold is not the key functionality required for antibacterial and
topoisomerase IV inhibitory activity or physicochemical properties
of molecule have very pronounced influence on its bioactivity. In
hope to provide deeper insight into molecular basis of antibacterial
activity of thiosemicarbazide derivatives, two series with indo-
5p
1p
Fig. 1. Structures of bacterial topoisomerase IV inhibitors with 4-benzoylthiosemi-
carbazide skeleton.
leethiosemicarbazide
1 and thiadiazoleethiosemicarbazide 2
topoisomerase IV) inhibitory activities. Thus, the present study
reports the synthesis, the biological profile, and the structuree
activity relationship (SAR) evaluation of three series with
4-nitrophenyl-thiosemicarbazide (series o), indoleethiosemicarba-
zide (series 1), and thiadiazoleethiosemicarbazide (series 2) core. In
SAR studies, biological properties of these molecules were compared
with several theoretical parameters frequently used in SAR consid-
erations. Since the geometry of the molecule may be important for
a topoisomerase IV inhibitory action, conformational studies were
carried out. In addition, the distribution of the frontierorbitals HOMO
and LUMO as well as the electrostatic potentials for geometries that
resultedfromdockingstudies wereanalyzedasthesefeaturesmay be
related to the interaction of the molecule with the target receptor.
skeleton were synthesized and their biological potency was eval-
uated. As can be seen from the results collected in Table 2, for series
of 4-aryl-1-(indol-2-yl)-carbonylthiosemicarbazides 1 substitution
of the para-position of phenyl ring with an electron withdrawing
fluoro group, exemplified by 1h, resulted in 2-fold increase in
activity against M. luteus ATCC 10240 as compared to the lead 1o. Its
antibacterial activity was also observed against the other Gram-
positive strains tested, however with 2-fold higher MICs than
that of 1o. Addition of a second fluoro group in the ortho-position,
exemplified by 1i, decreased slightly the activity in comparison to
1h. Substitution of ortho-position of the phenyl ring with fluoro 1f
or bromo 1k groups was not well tolerated; the molecules were
found to be 2e4-fold less active than that of 1o. Subsequently, the
antibacterial potency of 4-aryl-1-(4-methyl-thiadiazol-5-yl)-car-
bonylthiosemicarbazides 2 was tested. Compounds with an elec-
tron donating para-methoxy 2d and ortho-methyl 2b groups were
inactive. Among molecules with an electron withdrawing substit-
uents, para-chloro 2m showed the highest antibacterial potency,
2. Results and discussion
2.1. Chemistry
The title 4-arylthiosemicarbazides were synthesized based on
known procedure [24e27] in the reaction of related hetero-
carboxylic acid hydrazide with phenyl isothiocyanate that con-
tained electron-rich or electron-poor substituent (Scheme 1). In
this way, twenty compounds with the general formula Rie(C¼O)e
NHeNH-(C¼S)eNHeRj were obtained. Substituents Ri (i ¼ 1, 2, 3
.) at the carbon atom and Rj (j ¼ a, b, c .) at the nitrogen atom that
were used in this study are listed in Table 1. A combination of the
number “i” and the letter “j” allows for the unique identification of
a compound, e.g., 1o corresponds to 1-(indol-2-carbonyl)-4-(4-
nitrophenyl)-thiosemicarbazide, and is used in the following
sections.
MIC range 50e100 mg/mL. Biological activity diminishes regardless
of whether chlorine atom in 2m was replaced by less hydrophobic
fluorine 2h or more hydrophobic bromine 2l. Addition of a second
chloro group in ortho-position, exemplified by 2n, led to w2-fold
decrease in activity. The same trend was observed when antibac-
terial activities of 2h and 2i were analyzed; para-fluoro 2h had
antibacterial potency with MIC range 100e400 mg/mL while 2,4-
difluoro 2i was inactive. These observations together seem to
indicate that ortho-substitution is not well tolerated in this chem-
ical series. This hypothesis is supported by the antibacterial results
for 2j and 2k.
Presented above results of antibacterial assay suggest that the
position and the identity of individual substitutions are important
parameter influencing the activity of studied 4-arylthiosemi-
carbazides. Para-substitution seemed to be more important to
increase the antibacterial activity than substitution at the ortho-
position of the phenyl ring. Substitution of the phenyl ring by an
electron withdrawing groups seemed to be better than that by an
electron donating substituents. In order to clarify the effect of steric
and electronic properties of substituents on antibacterial potency of
tested thiosemicarbazides, some parameters such as the bulk
parameter based on molar refraction MR and electronic parameters F
and R were taken from the literature [28] and correlated with bio-
logical activity, see Table 3. Experiments have shown [29,30] that
a parameter representing the volume of the substituents on each
compound, relativetoothermembers of thesameseries, mayoftenbe
linearly correlated with the biological response. However, lack of
relationship between antibacterial activity of series 1 and 2 and the
bulk parameter MR or positionally weighted parameters F and R were
observed. Thus, physicochemical properties of substituents are
only secondary parameters for antibacterial activity of title
4-arylthiosemicarbazides.
2.2. Biological evaluation
As starting compound for our study, 1-(indol-2-carbonyl)-4-(4-
nitrophenyl)-thiosemicarbazide 1o, new analog of previously
described 1p, [23] was synthesized and antibacterial and type IIA
topoisomerases (DNA gyrase and topoisomerase IV) activity eval-
uated. It was found that it shows activity against Gram-positive
bacteria with MICs of 50
mg/mL (see Table 2) and inhibitory
activity against topoisomerase IV with an IC50 of 14
mM. To confirm
that 4-nitrophenylthiosemicarbazide scaffold can be developed
into an efficient Gram-positive antibacterials targeting topoisom-
erase IV, three new analogs 2oe4o with five-membered hetero-
cyclic ring were synthesized. Based on our previous experiments it
was expected that replacement of the indole with a smaller sized
heterocyclic ring would result in improved antibacterial activity.
Surprisingly, among tested compounds only 2o was found to be
active against Gram-positive stains. However, except for Micro-
coccus luteus ATCC 10240, it was w2-fold weaker in activity as
RjNCS
Considering the results of the in vitro antibacterial assay, we
have conducted the DNA gyrase and topoisomerase IV inhibition
tests for 1i which showed high therapeutic potential against M.
R
i
Rj
i
luteus ATCC 10240 with MIC of 25
mg/mL. Among series 2 we have
selected the most active 2m, two less active 2h and 2l, and, in order
to exclude permeability barrier, efflux, in vivo affinity, [31] two
Ri = 1-5; Rj = a-o (for symbols used to identify studied compounds see Table 1)
Scheme 1.

A. Siwek et al. / European Journal of Medicinal Chemistry 46 (2011) 5717e5726
5719
Table 1
Symbols used to identify studied compounds using substituents at thiosemicarbazide moiety.
CH3
1
a
b
N
H
2
3
c
e
d
CH3
CF3
f
4
5
g
h
F
i
j
k
l
m
n
o
p
inactive 2i and 2k. Unfortunately, among tested compounds only 1i
and 2l were found as weak topoisomerase IV inhibitor with IC50 of
2.3. Computational part
295
m
M and 403
m
M, respectively. Based on these findings we can
Initially we have calculated several parameters that are
frequently used in SAR studies to identify their role in modulating
the biological profile of title 4-arylthiosemicarbazides. As known,
lipophilicity of molecules has an important effect on their biological
activity. For the compounds, however, no close correlation between
lipophilicity, expressed as clogP, and biological activity was found.
support our previously conclusion that thiosemicarbazides partic-
ipate in at least two different mechanisms of antibacterial activity;
one is connected with inhibition of topoisomerase IV, while the
nature of the other cannot be elucidated from the limited data
collected thus far.

5720
A. Siwek et al. / European Journal of Medicinal Chemistry 46 (2011) 5717e5726
Table 2
inhibitory effect of 4-benzoylthiosemicarbazides on topoisomerase
In vitro antibacterial activity (MIC [mg/mL]) of thiosemicarbazides.
IV activity is the geometry of the molecule. It was observed that
the geometries of biologically active inhibitors 1p and 5p were
almost similar and considerably different from the structure of
inactive ap. Thus, in the search for the steric motif that may be
responsible for the inhibitory action of 4-arylthiosemicarbazides,
which differ from 4-benzoylthiosemicarbazides in the “length” of
the molecule, conformational analysis of thiosemicarbazides from
series o, 1, and 2 has been performed. The 4-arylthiosemi-
carbazides skeleton has 6 rotatable single bonds thus huge
conformational space that cannot be searched systematically at
either ab initio or DFT levels should be sampled in order to find the
most stable conformations. We have therefore carried out exten-
sive conformational searches at the molecular mechanics level
with OPLS force field implemented in HyperChem which gives
very good results for geometries and the energetics of this class of
compounds. [36] The most stable structures obtained were
subsequently optimized to the closest local minimum at the
semiempirical level using RM1 parametrization, which has been
shown to perform very well for s-triazoles, cyclic derivatives of
thiosemicarbazides. [37] As illustrated in Fig. 2, the most stable
conformations of inhibitors 1o, 1i, 2l are similar. Confusingly, the
superposition of the conformers of active 1o and inactive 2o also
revealed only minor deviations while structures of active 2l and
inactive 2k are almost superimposable. This may imply that the
binding of 4-arylthiosemicarbazides by the receptor depends on
their electronic rather than structural properties. It was reasonable
to suppose that the electrostatic interaction of thiosemicarbazides
with the active site of topoisomerase IV could be explained in
terms of the frontier molecular orbitals maps. [38] As expected,
the molecular modeling analysis of frontier molecular orbitals
revealed that the most active 1o differs from the others in low
distribution of both HOMO and LUMO around the sulfur atom, see
Fig. 3, suggesting the importance of these orbitals for
thiosemicarbazide-topoisomerase IV complex formation.
The thiosemicarbazideereceptor complexes were further
analyzed by means of AutoDock Vina docking program using the
same as the previous [23] model of the ATP binding site of Thermus
thermophilus gyrase B (PDB 1KIJ) complexed with novobiocin as
template. Default docking parameters and flexible space of
24 ꢁ 24 ꢁ 24 Å [3] were validated by re-docking native ligand
which docked exactly in the position present in the crystal struc-
ture with affinity of ꢀ11.0 kcal/mol. Subsequently, the binding
affinity of title thiosemicarbazides was evaluated. The overall good
correlation between the bioactivity of studied compounds and the
binding affinities predicted by AutoDock Vina was found as indi-
cated in Table 4. Although all molecules, with “length” very similar
to that of novobiocin, were found to be well fitted in the binding
pocket of the topoisomerase (see Fig. 4) our bioassay protocol
indicated that only 1o has a significant inhibitory action most
probably due to its ability to form strong hydrogen bond with
Compound
S. aureus
S. epidermidis M. luteus B. subtilis B. cereus
1o
2o
50a,b,c,d
100a,b,c
200d
50e
50f,g
50h
50i
100e
100f
100h
100i
50g
1f
200a,c,d
100b
200e
100e
100e
200e
400e
200e
100f,g
100h
100h
100h
200h
400h
200h
200i
100i
100i
200i
400i
400i
1h
1i
100a,b,c,d
50f
25g
100a,b,c
200d
100f
25g
1k
2a
2e
200a,c
100f,g
100b,d
400a,b,c,d
100f
200g
200f,g
100a
50b
400c
200d
2g
2h
2j
200a,b
400c,d
400a,c,d
200b
400e
200e
400e
200e
50e
200f
400h
200h
200h
100h
50h
400i
200i
400i
100i
50i
100g
100f,g
400a,c,d
200b
100f
200g
50f
2l
200a,c,d
100b
100g
100f
50g
2m
100a,c
50b,d
2n
100a,b,c,d
0.5a,b,c,d
100e
100f,g
4f
100h
100i
1i
Ciprofloxacin
0.5e
<0.125h
2g
Strains:
a
b
c
d
e
f
NCTC 4163.
ATCC 25923.
ATCC 6538.
ATCC 29213.
ATCC 12228.
ATCC 9341.
ATCC 10240.
ATCC 6633.
ATCC 11778.
g
h
i
As can be seen from results collected in Table 3, 2j, 2k, and 2m have
similar level of lipophilicity but considerably different level of
antibacterial activity. In the series of 4-nitrophenylthiosemi-
carbazides o two antibacterials 1o and 2o have clogP 3.10 and
0.44, respectively while 3o with clogP 0.92, 4o with clogP 0.40, and
5o with clogP ꢀ3.51 were inactive. Moreover, 1o exhibited a strong
inhibitory action against topoisomerase IV while 1i, 2m, 2k, and 2l,
with comparable level of lipophilicity to 1o, were slightly active or
inactive. No correlation was also observed between the positionally
weighted distributive parameter
parameter of choice for correlating both binding to biological
macromolecules and transport through biological system,
p, which is considered the
a
[32e35] and biological activity of studied thiosemicarbazides. On
the other hand, in analyzing the physicochemical parameters pre-
sented in Table 3 there are some SAR trends that were evident. As
can be seen, the most active 1o presents the highest values of
surface area (SA), volume (V), coreecore interaction (ICeC), and heat
of formation (HF). Moreover, it possesses higher values of refrac-
Glu49 as well as
a
large electrostatic contribution to
ligandereceptor binding. As can be seen from Fig. 5, all studied
compounds are characterized by a charge distribution substantially
different than that of novobiocin. The electrostatics of 1o shows
negative charge around carbonyl oxygen larger than in other
compounds and resembling high charge density of the amid part of
novobiocin as well as a large positive charge on the indole NeH
proton resembling that of hydroxyl of novobiocin. Finally, geome-
tries of the molecules in the active site of topoisomerase were
compared with those optimized without the presence of the
enzyme. No correlation between the strain energy defined as the
difference of heats of formation of the structure fully optimized and
the structure in the active site and the bioactivity defined by IC50
was observed.
tivity (Rf) and polarizabilitiy (a) and lower values of total energy
(ET), isolated atomic energy (EIA), and electronic energy (EE) than the
other compounds. In this way it can be proposed that both struc-
tural and electronic properties of 4-arylthiosemicarbazides are
important for their biological potency while lipophilicity is only
a secondary parameter.
As it was mentioned in the introduction, in our previous paper
[23] we have proposed that a very important factor influencing the

A. Siwek et al. / European Journal of Medicinal Chemistry 46 (2011) 5717e5726
5721
Table 3
SAR parametersa of studied thiosemicarbazides.
Comp
MR
R
p
V
a
m
HOMO
LUMO
HLG
c
EB
EE
HF
F
clogP
SA
Rf
h
ET
EIA
ICeC
1o
2o
3o
4o
5o
1f
6.0
1.11
6.0
1.11
6.0
1.11
6.0
1.11
6.0
1.11
ꢀ0.4
0.88
ꢀ0.4
0.71
0.16
3.10
0.16
0.44
0.16
0.92
0.16
0.40
0.16
ꢀ3.51
ꢀ0.29
3.29
ꢀ0.34
3.29
e
0.22
434.71
0.22
388.93
0.22
395.83
0.22
376.11
0.22
397.32
0.00
389.26
0.15
390.51
e
918.58
97.43
817.84
88.49
804.79
84.80
788.94
80.60
820.94
85.72
876.73
90.32
867.61
90.32
874.24
90.54
915.69
97.73
751.36
81.67
793.16
86.71
836.30
88.13
818.91
87.64
767.27
81.88
770.62
81.88
765.86
82.10
38.26
3.38
33.66
3.05
33.24
4.77
30.99
4.70
31.99
5.43
36.33
6.32
36.33
5.02
36.24
5.34
39.04
6.43
31.82
3.88
33.65
4.04
34.29
5.30
33.38
4.39
31.73
4.47
31.73
2.83
31.63
2.27
ꢀ9.06
ꢀ1.24
ꢀ9.33
ꢀ1.67
ꢀ9.14
ꢀ1.21
ꢀ9.20
ꢀ1.30
ꢀ9.34
ꢀ1.42
ꢀ8.92
ꢀ0.65
ꢀ8.92
ꢀ0.63
ꢀ8.97
ꢀ0.71
ꢀ8.87
ꢀ0.64
ꢀ8.93
ꢀ1.44
ꢀ8.91
ꢀ1.43
ꢀ8.95
ꢀ1.31
ꢀ9.15
ꢀ1.62
ꢀ9.08
ꢀ1.49
ꢀ9.15
ꢀ1.40
ꢀ9.11
ꢀ1.56
ꢀ9.12
ꢀ1.59
ꢀ9.01
ꢀ1.54
ꢀ9.09
ꢀ1.52
ꢀ9.07
ꢀ1.50
ꢀ9.15
ꢀ1.56
ꢀ7.82
3.91
5.15
ꢀ3240.93
ꢀ723,604.30
622,739.80
ꢀ629,588.74
535,027.38
ꢀ571,316.25
483,505.90
ꢀ580,415.93
491,793.28
ꢀ633,407.46
539,624.32
ꢀ634,705.81
541,932.98
ꢀ633,619.74
540,846.94
ꢀ695,294.52
591,598.28
ꢀ626,521.90
536,743.71
ꢀ493,417.47
417,873.45
ꢀ541,813.27
462,708.59
ꢀ589,644.89
503,249.20
ꢀ728,866.77
616,974.21
ꢀ550,565.85
464,095.96
ꢀ546,963.43
460,492.09
ꢀ606,441.76
509,049.79
ꢀ608,541.73
511,148.52
ꢀ540,850.80
457,375.35
ꢀ539,278.50
455,802.92
ꢀ540,372.12
456,302.85
ꢀ592,273.66
499,679.61
980.71
822.67
969.25
764.65
699.80
734.37
734.41
637.07
739.47
675.90
702.08
760.21
392.49
576.14
574.68
480.14
478.91
581.01
580.89
447.99
220.55
ꢀ100,864.51
ꢀ97623.58
ꢀ2567.62
ꢀ91,993.74
ꢀ2365.93
ꢀ85,444.42
ꢀ2669.23
ꢀ85,953.42
ꢀ2899.19
ꢀ90,883.95
ꢀ3274.04
ꢀ89,498.80
ꢀ3274.00
ꢀ89,498.80
3338.13
ꢀ7.66
5.50
3.83
ꢀ94,561.36
5.18
ꢀ7.93
3.97
ꢀ87,810.35
ꢀ7.90
5.25
3.95
ꢀ88,622.65
5.38
ꢀ7.92
3.96
ꢀ93,783.14
ꢀ8.27
4.79
4.14
ꢀ92,772.84
4.78
92,772.80
4.84
1h
1i
ꢀ8.29
4.15
ꢀ0.8
ꢀ8.26
e
3.43
ꢀ0.15
3.94
e
398.37
0.84
413.37
e
4.13
ꢀ103,696.24
ꢀ100,358.11
ꢀ3276.79
ꢀ86,501.40
ꢀ2534.37
ꢀ73,009.65
ꢀ2783.28
ꢀ76,321.39
ꢀ2784.72
ꢀ83,610.98
ꢀ2993.24
ꢀ108,899.32
ꢀ2600.92
ꢀ83,868.96
ꢀ2602.38
ꢀ83,868.96
ꢀ2663.70
ꢀ94,728.27
ꢀ2664.94
ꢀ94,728.27
ꢀ2603.90
ꢀ80,871.56
ꢀ2604.02
ꢀ80,871.56
ꢀ2739.16
ꢀ81,330.11
ꢀ2943.49
ꢀ89,650.56
1k
2a
2b
2d
2e
2g
2h
2i
7.6
0.91
ꢀ8.23
4.12
4.76
ꢀ89,778.19
5.19
e
e
ꢀ7.49
2.42
ꢀ0.12
2.89
ꢀ0.50
2.17
0.16
3.30
ꢀ0.12
2.56
ꢀ0.34
2.56
e
328.39
0.49
350.50
ꢀ0.12
391.57
1.04
358.28
0.22
337.81
0.15
3.75
ꢀ75,544.02
4.7
ꢀ7.48
3.74
5.17
ꢀ0.07
ꢀ79,104.68
5.13
6.5
ꢀ7.64
0.41
4.00
0.79
ꢀ0.40
0.69
ꢀ0.40
0.71
ꢀ0.80
e
3.82
ꢀ86,395.69
ꢀ7.53
3.77
5.39
ꢀ111,892.56
5.29
ꢀ7.59
3.80
ꢀ86,469.88
ꢀ7.75
3.88
5.23
345.56
e
ꢀ86,471.34
5.34
ꢀ7.55
2.70
e
350.93
e
3.78
ꢀ97,391.97
2j
ꢀ0.80
e
774.89
82.10
811.43
89.29
816.94
89.29
791.75
86.47
31.63
3.83
34.44
4.09
34.44
3.11
33.75
2.72
ꢀ7.53
3.77
5.36
2.70
ꢀ0.15
3.21
ꢀ0.18
3.21
ꢀ0.16
2.94
e
347.64
0.84
367.37
1.19
374.23
0.73
368.45
e
ꢀ97,393.21
5.28
2k
2l
7.60
0.91
7.60
0.73
4.80
0.69
9.60
e
ꢀ7.47
3.74
ꢀ83,475.46
ꢀ7.57
3.79
5.31
ꢀ83,475.58
5.29
2m
2n
ꢀ7.57
3.79
ꢀ84,069.27
831.13
91.28
35.67
2.00
ꢀ7.59
3.80
5.36
3.46
397.76
ꢀ92,594.05
a
Surfaces and refractivity in Å2, volumes and polarizability in Å3, moment dipoles in D, energies in kcal/mol.
3. Conclusions
electronic structure rather than geometry of the molecule, (iv) the
H-bond interactions seem to be the prevalent factors modulating
inhibitory activity of both 4-benzoyl- and 4-arylthiosemicarbazides.
The above results allow for rational design of new thio-
semicarbazide derivatives that should show higher affinities to ATP
binding pocket of topoisomerase and thus with increased inhibi-
tory activity.
In this contribution we have synthesized twenty 4-arylthio-
semicarbazides with six rotatable bonds to evaluate their antibac-
terial and inhibitory activity against type IIA topoisomerases DNA
gyrase and topoisomerase IV and have correlated biological results
with some molecular properties. The study revealed new lead 1-
(indol-2-carbonyl)-(4-nitrophenyl)-thiosemicarbazide with signif-
icant action against topoisomerase IV. Although modification of its
structure resulted in molecules with a lower biological profile, some
indications for a molecular basis of antibacterial activity of thio-
semicarbazides have been interred from SAR studies and can be
summarized as follows: (i) the type and the position of substitu-
tion at phenyl ring are of great importance for antibacterial activity
of 4-arylthiosemicarbazides; the most appropriate is substitution
on the para-position by the electron withdrawing group, (ii)
both 4-benzoylthiosemicarbazides and 4-arylthiosemicarbazides
participate in at least two different mechanisms of antibacterial
activity; one is connected with inhibition of topoisomerase IV, while
the nature of the other cannot be elucidated from the limited data
collected thus far, (iii) in contrast to 4-benzoylthiosemicarbazides,
inhibitory activity of 4-arylthiosemicarbazides is connected with
4. Experimental section
4.1. Chemistry
All commercial reactants and solvents were purchased from
either Sigma-Aldrich or Lancaster with the highest purity and used
without further purification. Melting points were determined on
a FischereJohns block and are uncorrected. Elemental analyses
were determined by a AMZ-CHX elemental analyzer (are within
ꢂ0.4% of the theoretical values). IR spectra were recorded in KBr
using a Specord IR-75 spectrophotometer. 1H-NMR spectra were
recorded on a Bruker Avance (300 MHz). Analytical thin layer
chromatography (TLC) was performed with Merc 60F254 silica gel
plates and visualized by UV irradiation (254 nm).

5722
A. Siwek et al. / European Journal of Medicinal Chemistry 46 (2011) 5717e5726
Fig. 2. Overlay of structures of the most active topoisomerase IV inhibitor 1o, slightly active 1i and 2l, and inactive 2k and 2o.
4.1.1. General procedure for synthesis of 1-substituted-4-aryl-
thiosemicarbazides
4.1.1.4. 4-(2-Fluorophenyl)-1-(indol-2-yl)-carbon-
ylthiosemicarbazide 1f. Yield: (2.96 g, 90%). Mp: 210e12 ꢃC. IR (
n,
A reaction mixture of appropriate heterocarboxylic hydrazide
(0.01 mol) and related isothiocyanate (0.01 mol) was heated in an
oil bath at 80 ꢃC and progress of reaction was monitored by thin
layer chromatography. After 12 h, the reaction was completed and
the crude reaction mixture was washed with diethyl ether and
crystallized from ethanol.
cmꢀ1) 3299, 3215 (NH), 3122, 1621, 1595, 1503, 1474, 744 (AreH),
1664 (C¼O) 1314 (C¼S). 1H-NMR (DMSO-d6) dH 7.02e7.07 (m, 1H,
CH), 7.15e7.32 (m, 6H, 6 ꢁ CH), 7.43e7.46 (d, 1H, CH), 7.63e7.66 (d,
1H, CH), 9.69, 9.92, 10.62, 11.74 (4s, 4H, 4 ꢁ NH). Anal. C16H13FN4OS
(C, H, N).
Physicochemical characterization of 2a and 2b was presented
previously. [39] Compound 3o is commercially available.
4.1.1.5. 4-(4-Fluorophenyl)-1-(indol-2-yl)-carbon-
ylthiosemicarbazide 1h. Yield: (2.89 g, 88%). Mp: 198e200 ꢃC. IR (
n,
cmꢀ1) 3310, 3212 (NH), 1623, 1580, 1491, 1455 (AreH), 1664 (C¼O),
1316 (C¼S). 1H-NMR (DMSO-d6) dH 7.02e7.08 (m, 1H, CH), 7.13e7.23
(m, 4H, 4 ꢁ CH), 7.43e7.47 (m, 3H, 3 ꢁ CH), 7.64e7.66 (d, 1H, CH),
9.79, 9.88, 10.55, 11.73 (4s, 4H, 4 ꢁ NH). Anal. C16H13FN4OS (C, H, N).
4.1.1.1. 1-(Indol-2-carbonyl)-4-(4-nitrophenyl)-thiosemicarbazide
1o. Yield: (3.23 g, 91%). Mp: 230e2 ꢃC. IR ( , cmꢀ1) 3100 (NH), 1661
n
(C¼O), 1625, 1577, 1510, 1491 (AreH), 1320 (C¼S). 1H-NMR (DMSO-
d6) dH 7.00e7.09 (m, 1H, CH), 7.14e7.26 (m, 2H, 2 ꢁ CH), 7.41e7.47
(m, 1H, CH), 7.65e7.68 (d, 1H, CH), 7.92e7.94 (d, 2H, 2 ꢁ CH),
8.20e8.23 (d, 2H, 2 ꢁ CH), 10.19, 10.23, 10.65, 11.60 (4s, 4H, 4 ꢁ NH).
Anal. C16H13N5O3S (C, H, N).
4.1.1.6. 4-(2,4-Difluorophenyl)-1-(indol-2-yl)-carbon-
ylthiosemicarbazide 1i. Yield: (2.94 g, 85%). Mp: 178e80 ꢃC. IR (
n,
cmꢀ1) 3319 (NH), 1630, 1589, 1499 (AreH), 1660 (C¼O) 1322 (C¼S).
1H-NMR (DMSO-d6) dH 7.03e7.09 (m, 2H, 2 ꢁ CH), 7.18e7.31 (m, 4H,
4 ꢁ CH), 7.44e7.47 (m, 1H, CH), 7.64e7.66 (d, 1H, CH), 9.66, 9.96,
10.63, 11.75 (4s, 4H, 4 ꢁ NH). Anal. C16H12F2N4OS (C, H, N).
4.1.1.2. 1-(4-Methyl-thiadiazol-5-carbonyl)-4-(4-nitrophenyl)-thio-
semicarbazide 2o. Yield: (3.01 g, 89%). Mp: 191e3 ꢃC. IR ( , cmꢀ1
n )
3225 (NH), 2925, 2850, 1455, 1358 (Aliph.), 1665 (C¼O), 1610, 1580,
1512, 1479 (AreH), 1319 (C¼S). 1H-NMR (DMSO-d6) dH 2.86 (s, 3H,
CH3), 7.85e7.90 (m, 2H, 2 ꢁ CH), 8.20e8.26 (m, 2H, 2 ꢁ CH), 10.29,
10.51, 10.97 (3s, 3H, 3 ꢁ NH). Anal. C11H10N6O3S2 (C, H, N).
4.1.1.7. 4-(2-Bromophenyl)-1-(indol-2-yl)-carbon-
ylthiosemicarbazide 1k. Yield: (3.43 g, 88%). Mp: 191e3 ꢃC. IR (
n,
cmꢀ1) 3305, 3212 (NH), 1618, 1579, 1501, 1470, (AreH), 1658 (C¼O),
1327 (C¼S). 1H-NMR (DMSO-d6) dH 7.02e7.08 (m, 2H, 2 ꢁ CH),
7.14e7.26 (m, 4H, 4 ꢁ CH), 7.34e7.46 (m, 2H, 2 ꢁ CH), 7.62 (d, 1H,
CH), 9.64, 9.88, 10.60, 11.72 (4s, 4H, 4 ꢁ NH). Anal. C16H13BrN4OS (C,
H, N).
4.1.1.3. 4-(4-nitrophenyl)-1-(pyrrol-2-yl)-carbonylthiosemicarbazide
4o. Yield: (2.9 g, 95%). Mp: 230e32 ꢃC. IR ( , cmꢀ1) 3250 (NH), 1656
n
(C¼O), 1620, 1579, 1521, 1487 (AreH), 1320 (C¼S). 1H-NMR (DMSO-
d6) dH 6.15 (s, H, CH), 6.96 (s, 2H, 2 ꢁ CH), 7.92 (d, 2H, 2 ꢁ CH),
8.18e8.21 (d, 2H, 2 ꢁ CH), 10.07, 10.17, 10.54, 11.69 (4s, 4H, 4 ꢁ NH).
Anal. C12H11N5O3S (C, H, N).
4.1.1.8. 4-(4-Methoxyphenyl)-1-(4-methyl-thiadiazol-5-yl)-carbon-
ylthiosemicarbazide 2d. Yield: (2.91 g, 90%). Mp: 118e20 ꢃC. IR (
n,

A. Siwek et al. / European Journal of Medicinal Chemistry 46 (2011) 5717e5726
5723
Fig. 3. HOMO (left) and LUMO (right) maps of the most active topoisomerase IV inhibitor 1o, slightly active 1i and 2l, and inactive 2h, 2i, 2k, 2m, and 2o.
cmꢀ1) 3300 (NH), 3100, 1590, 1509, 1463 (AreH), 2865, 1446
(Aliph.), 1675 (C¼O), 1190 (C¼S). 1H-NMR (DMSO-d6) dH 2.83 (s, 3H,
CH3), 3.75 (s, 3H, OCH3), 6.88e6.94 (m, 2H, 2 ꢁ CH), 7.23e7.32 (m,
2H, 2 ꢁ CH), 9.76, 9.83, 10.80 (3s, 3H, 3 ꢁ NH). Anal. C12H13N5O2S2
(C, H, N).
(Aliph.), 1668 (C¼O), 1223 (C¼S). 1H-NMR (DMSO-d6) dH 2.83 (s, 3H,
CH3), 7.47e7.60 (m, 2H, 2 ꢁ CH), 7.65e7.75 (m, 2H, 2 ꢁ CH), 9.78,
10.01, 10.97 (3s, 3H, 3 ꢁ NH). Anal. C12H10F3N5OS2 (C, H, N).
4.1.1.10. 4-(3-Fluorophenyl)-1-(4-methyl-thiadiazol-5-yl)-carbon-
ylthiosemicarbazide 2g. Yield: (2.68 g, 86%). Mp: 154e6 ꢃC. IR (
n,
4.1.1.9. 4-(2-Trifluoromethylphenyl)-1-(4-methyl-thiadiazol-5-yl)-
carbonylthiosemicarbazide 2e. Yield: (3.29 g, 91%). Mp: 184e6 ꢃC. IR
cmꢀ1) 3322 (NH), 3110, 1591, 1522, 1466 (AreH), 3033, 1629
(Aliph.), 1667 (C¼O), 1235 (C¼S). 1H-NMR (DMSO-d6) dH 2.85 (s, 3H,
(n
, cmꢀ1) 3318 (NH), 3112,1587,1519,1468 (AreH), 3030,1622,1487
Table 4
Binding free energy, H-bond contacts, and (RM1) heats of formation of the structures
fully optimized and the structures of the best docking poses.
Compd.
D
Gb
Hydrogen bonds
RM1opt
RM1bind
DRM1
(kcal/mol) between atoms of
compounds and
(kcal/mol) (kcal/mol) (kcal/mol)
amino acids
Atom of
Amino
compound acid
1i
ꢀ9.4
ꢀ8.6
ꢀ7.8
ꢀ7.8
ꢀ7.8
ꢀ7.7
ꢀ7.6
ꢀ7.5
e
e
637.0706
693.6096 56.5
36.9
1o
2h
2i
2k
2m
2o
2l
NHNHC(]S) Glu49 980.7148 1017.589
e
e
e
e
e
e
e
e
e
e
e
e
574.6769
480.1422
581.0132
447.9944
822.6685
580.8924
600.9854 26.3
507.1369 27.0
617.0674 36.1
458.6842 10.7
843.5225 20.9
606.3458 25.5
Fig. 4. Superimposition of the native ligand, novobiocin (rendered as tubes) and the
best conformations of 1o, 1i, 2l, 2h, 2i, 2k, 2m, and 2o docked to the ATP binding
pocket of gyrase B (1KIJ).

5724
A. Siwek et al. / European Journal of Medicinal Chemistry 46 (2011) 5717e5726
Fig. 5. Comparison of the electrostatic potential surfaces of novobiocin (top) and geometries of thiosemicarbazides that resulted from docking studies.
CH3), 7.46e7.66 (m, 2H, 2 ꢁ CH), 7.69e7.72 (m, 2H, 2 ꢁ CH), 9.78,
10.06, 10.99 (3s, 3H, 3 ꢁ NH). Anal. C11H10FN5OS2 (C, H, N).
4.1.1.16. 4-(4-Chlorophenyl)-1-(4-methyl-thiadiazol-5-yl)-carbon-
ylthiosemicarbazide 2m. Yield: (2.79 g, 85%). Mp: 185e7 ꢃC. IR (
n,
cmꢀ1) 3270 (NH), 3089, 1589, 1498, 1463 (AreH), 2964, 2888, 1366
(Aliph.), 1659 (C¼O), 1241 (C¼S). 1H-NMR (DMSO-d6) dH 2.84 (s, 3H,
CH3), 7.38e7.49 (m, 4H, 4 ꢁ CH), 9.98, 10.41, 10.87 (3s, 3H, 3 ꢁ NH).
Anal. C11H10ClN5OS2 (C, H, N).
4.1.1.11. 4-(4-Fluorophenyl)-1-(4-methyl-thiadiazol-5-yl)-carbon-
ylthiosemicarbazide 2h. Yield: (2.74 g, 88%). Mp: 155e7 ꢃC. IR (
n,
cmꢀ1) 3335 (NH), 3088, 1589, 1509, 1466 (AreH), 3055, 1632,
1479 (Aliph.), 1670 (C¼O), 1239 (C¼S). 1H-NMR (DMSO-d6) dH
2.83 (s, 3H, CH3), 7.14e7.23 (m, 2H, 2 ꢁ CH), 7.39e7.43 (m, 2H,
2 ꢁ CH), 9.91, 10.35, 10.85 (3s, 3H, 3 ꢁ NH). Anal. C11H10FN5OS2
(C, H, N).
4.1.1.17. 4-(2,4-Dichlorophenyl)-1-(4-methyl-thiadiazol-5-yl)-car-
bonylthiosemicarbazide 2n. Yield: (3.26 g, 90%). Mp: 167e9 ꢃC. IR (
n,
cmꢀ1) 3290 (NH), 3079, 1577, 1500, 1468 (AreH), 2968, 2890, 1368
(Aliph.), 1667 (C¼O), 1243 (C¼S). 1H-NMR (DMSO-d6) dH 2.83 (s, 3H,
CH3), 7.40e7.48 (m, 4H, 4 ꢁ CH), 9.83, 10.26, 10.93 (3s, 3H, 3 ꢁ NH).
Anal. C11H9Cl2N5OS2 (C, H, N).
4.1.1.12. 4-(2,4-Difluorophenyl)-1-(4-methyl-thiadiazol-5-yl)-car-
bonylthiosemicarbazide 2i. Yield: (2.83 g, 86%). Mp: 148e50 ꢃC. IR
(n
, cmꢀ1) 3323 (NH), 3080, 1592, 1511, 1455 (AreH), 3015, 1630
(Aliph.), 1666 (C¼O), 1243 (C¼S). 1H-NMR (DMSO-d6) dH 2.84 (s, 3H,
CH3), 7.07e7.13 (m, 1H, CH), 7.29e7.36 (m, 2H, 2 ꢁ CH), 9.74, 10.09,
10.93 (3s, 3H, 3 ꢁ NH). Anal. C11H9F2N5OS2 (C, H, N).
4.2. Antibacterial assay
The following microorganisms were used in this study:
Staphylococcus aureus (ATCC 25923, ATCC 6538, ATCC 29213, NCTC
4163), Staphylococcus epidermidis ATCC 12228, Bacillus subtilis
ATCC 6633, Bacillus cereus ATCC 11778, M. luteus (ATCC 9341, ATCC
10240), Escherichia coli (ATCC 10538, ATCC 25922, NCTC 8196),
Pemphigus vulgaris NCTC 4635, Pseudomonas aeruginosa (ATCC
15442, ATCC 27853, NCTC 6749), and Bordetella bronchiseptica
ATCC 4617.
4.1.1.13. 4-(2,5-Difluorophenyl)-1-(4-methyl-thiadiazol-5-yl)-car-
bonylthiosemicarbazide 2j. Yield: (2.90 g, 88%). Mp: 156e8 ꢃC. IR (
n,
cmꢀ1) 3300 (NH), 3079, 1591, 1518, 1460 (AreH), 3010, 1633
(Aliph.), 1669 (C¼O), 1250 (C¼S). 1H-NMR (DMSO-d6) dH 2.68 (s, 3H,
CH3), 7.08e7.29 (m, 3H, 3 ꢁ CH), 9.75, 10.10, 10.88 (3s, 3H, 3 ꢁ NH).
Anal. C11H9F2N5OS2 (C, H, N).
Initially, antibacterial activity of thiosemicarbazides was
screened on the basis of growth inhibition zone (giz) utilizing the
disc diffusion method. For compounds showing the inhibitory
effect on the growth of tested bacteria, monitored as an appearance
of giz, the minimal inhibitory concentrations (MICs) were deter-
mined as the lowest concentration of the compound preventing
growth of the tested microorganism using agar dilution method.
[40,41]
4.1.1.14. 4-(2-Bromophenyl)-1-(4-methyl-thiadiazol-5-yl)-carbon-
ylthiosemicarbazide 2k. Yield: (3.54 g, 91%). Mp: 107e9 ꢃC. IR (
n,
cmꢀ1) 3357 (NH), 3056, 1622, 1580, 1493 (AreH), 2966, 1460, 1391
(Aliph.), 1669 (C¼O), 1242 (C¼S). 1H-NMR (DMSO-d6) dH 2.83 (s, 3H,
CH3), 7.20e7.26 (m, 2H, 2 ꢁ CH), 7.36e7.43 (m, 2H, 2 ꢁ CH), 9.81,
9.98, 10.91 (3s, 3H, 3 ꢁ NH). Anal. C11H10BrN5OS2 (C, H, N).
4.1.1.15. 4-(4-Bromophenyl)-1-(4-methyl-thiadiazol-5-yl)-carbon-
ylthiosemicarbazide 2l. Yield: (3.05 g, 82%). Mp: 181e3 ꢃC. IR (
n
,
4.3. Inhibition of bacterial type IIA topoisomerases
cmꢀ1) 3269 (NH), 3078, 1585, 1491, 1455 (AreH), 2962, 2870,
1466, 1365 (Aliph.), 1666 (C¼O), 1240 (C¼S). 1H-NMR (DMSO-d6)
dH 2.84 (s, 3H, CH3), 7.42e7.43 (m, 2H, 2 ꢁ CH), 7.51e7.56 (m, 2H,
2 ꢁ CH), 9.98, 10.33, 10.87 (3s, 3H, 3 ꢁ NH). Anal. C11H10BrN5OS2
(C, H, N).
4.3.1. Supercoiling assay
The assays were performed using S. aureus Gyrase Supercoiling
Assay Kits (Inspiralis). Briefly, supercoiled pBR322 plasmid DNA
(0.5
mg) was incubated with 1 unit of gyrase, in the dedicated

A. Siwek et al. / European Journal of Medicinal Chemistry 46 (2011) 5717e5726
5725
supercoiling assay buffer supplied by the manufacturer, in the
References
presence of varying concentrations of the test compounds. Reac-
tions were carried out at 37 ꢃC for 1 h and then terminated by the
addition of equal volume of 2x 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.
[1] S.B. Levy, Multidrug resistance e a sign of the times, N. Eng. J. Med. 338 (1998)
1376e1378.
[2] W. Witte, Antibiotic resistance in gram-positive bacteria: epidemiological
aspects, J. Antimicrob. Chem. 44 (1999) 1e9.
[3] R.F. Service, Antibiotics that resist resistance, Science 270 (1995) 724e727.
[4] J.A. Heinemann, R.G. Ankenbauer, C.F. Amábile-Cuevas, Do antibiotics main-
tain antibiotic resistance? Drug Discov. Today 5 (2000) 195e204.
[5] T. Komine, A. Kojima, Y. Asahina, T. Saito, H. Takano, T. Shibue, Y. Fukusa,
Synthesis and structureeactivity relationship studies of highly potent novel
oxazolidinone antibacterials, J. Med. Chem. 51 (2008) 6558e6562.
[6] A.R. Renslo, G.W. Luehr, M.F. Gordeev, Recent developments in the identifi-
cation of novel oxazolidinone antibacterial agents, Bioorg. Med. Chem. 14
(2006) 4227e4240.
4.3.2. Decatenation assay
The assay was performed using S. aureus topoisomerase IV
decatenation kits (Inspiralis). Interlinked kDNA substrate (0.5 mg)
[7] D.J. Bast, J.C.S. de Azavedo, Quinolone resistance: older concepts and newer
developments, Curr. Infect. Dis. Rep. 3 (2001) 20e28.
was incubated with 1 unit of topoisomerase IV (Inspiralis), 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 ꢃC for 1 h and then terminated by
the addition of equal volume of 2x STOP Buffer (40% sucrose,
100 mM Tris-Cl pH 7.5, 1 mM EDTA, 0.5 mg/ml bromophenol blue)
and chloroform/iso-amyl alcohol. Samples were vortexed, centri-
fuged 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. The concentrations of the inhibitor that prevented
50% of the kinetoplast DNA from being converted into decatenated
minicircles (IC50 values) were determined by plotting the results
obtained from the densytometric analyses of the gel images using
Quantity One software (BioRad).
[8] R.J. Kerns, M.J. Rybak, G.W. Kaatz, F. Vaka, R. Cha, R.G. Grucz, V.U. Diwadkar,
Structural features of piperazinyl-linked ciprofloxacin dimmers required for
activity against drug-resistant strains of Staphylococcus aureus, Bioorg. Med.
Chem. Lett. 13 (2003) 2109e2112.
ꢁ
ꢁ ꢀ
ꢀ
ꢁ ꢀ
ꢀ
ꢀ
[9] M. Bukvic Krajacic, P. Novak, M. Dumic, M. Cindric, H. Cipcic Paljetak,
ꢁ ꢀ
N. Kujundzic, Novel ureas and thioureas of 15-membered azalides with
antibacterial activity against key respiratory pathogens, Eur. J. Med. Chem. 44
(2009) 3459e3470.
[10] L. Xu, A.K. Farthing, J.F. Dropinski, P.T. Meinke, Ch. McCallum, P.S. Leavitt,
E.J. Hickey, L. Colwell, J. Barrett, K. Liu, Nocathiacin analogs: synthesis and
antibacterial activity of novel water-soluble amides, Bioorg. Med. Chem. Lett.
19 (2009) 3531e3535.
[11] M.S. Kabir, K. Engelbrecht, R. Polanowski, S.M. Krueger, R. Ignasiak, M. Rott,
W.R. Schwan, M.E. Stemper, K.D. Reed, D. Sherman, J.M. Cook, A. Monte, New
classes of Gram-positive selective antibacterials: inhibitors of MRSA and
surrogates of the causative agents of anthrax and tuberculosis, Bioorg. Med.
Chem. Lett. 18 (2008) 5745e5749.
[12] Y. Hirokawa, H. Kinoshita, T. Tanaka, T. Nakamura, K. Fujimoto, S. Kashimoto,
T. Kojima, S. Kato, Pleuromutilin derivatives having a purine ring. Part 1: new
compounds with promising antibacterial activity against resistant Gram-
positive pathogens, Bioorg. Med. Chem. Lett. 18 (2008) 3556e3561.
[13] G. Aridoss, S. Amirthaganesan, N.A. Kumar, J.T. Kim, K.T. Lim, S. Kabilan,
Y.T. Jeong, A facile synthesis, antibacterial, and antitubercular studies of some
piperidin-4-one and tetrahydropyridine derivatives, Bioorg. Med. Chem. Lett.
18 (2008) 6542e6548.
4.4. Computational details
Conformational search, physicochemical parameters, and
HOMO/LUMO maps were calculated using HyperChem8.0.1 [42]
Extensive conformational searches were carried out using the
molecular mechanics level with OPLS force field. The most stable
structures obtained were subsequently optimized to the closest
local minimum at the semiempirical level using RM1 parametri-
zation. Convergence criteria were set to 0.1 and 0.01 kcal molꢀ1 Åꢀ1
for OPLS and RM1 calculations, respectively. Electrostatic potentials
were calculated for the geometries that resulted from docking
using Gaussian 03 and GaussView 5 at the HF/6-31G level.
[14] B.J. Bradbury, M.J. Pucci, Recent advances in bacterial topoisomerase inhibi-
tors, Curr. Opin. Pharmacol. 8 (2008) 574e581 and references therein.
[15] D.A. Ostrov, J.A. Hernández Prada, P.E. Corsino, K.A. Finton, N. Le, T.C. Rowe,
Discovery of novel DNA gyrase inhibitors by high-throughput virtual
screening, Antimicrob. Agents Chemother. 51 (2007) 3688e3698.
ꢁ
ꢁ
[16] M. Oblak, S.G. Grdadolnik, M. Kotnik, R. Jerala, M. Filipic, T. Solmajer, In silico
fragment-based discovery of indolin-2-one analogues as potent DNA gyrase
inhibitors, Bioorg. Med. Chem. Lett. 15 (2005) 5207e5210.
ꢁ
[17] M. Oblak, S. Golic Grdadolnik, M. Kotnik, A. Poterszman, R.A. Atkinson,
H. Nierengarten, D. Desplancq, D. Moras, T. Solmajer, Biophysical character-
ization of an indolinone inhibitor in the ATP-binding site of DNA gyrase,
Biochem. Biophys. Res. Commun. 349 (2006) 1206e1213.
Abbreviations of physicochemical parameters collected in
Table 3 are as follows: surface area (SA), volume (V), refractivity (Rf),
[18] C. Kus¸ , G. Ayhan-Kılcıgil, S. Özbey, F. Betül Kaynak, M. Kaya, T. Çoban, B. Can-
Eke, DNA topoisomerases and their poisoning by anticancer and antibacterial
drugs, Chem. Biol. 17 (2010) 421e433.
[19] J.J. Champoux, DNA topoisomerases: structure, function, and mechanism,
Annu. Rev. Biochem. 70 (2001) 369e413.
polarizabilitiy
(a), moment dipole (m), the highest occupied
molecular orbital energy (EHOMO), the lowest unoccupied molecular
orbital energy (ELUMO), the difference between HOMO and LUMO
energy levels (HLG), hardness (
h
) obtained from the equation
) obtained
[20] J.C. Wang, Cellular roles of DNA topoisomerases: a molecular perspective, Nat.
Rev. Mol. Cell. Biol. 3 (2002) 430e440.
h
¼ (ELUMO ꢀ EHOMO)/2, Mulliken electronegativity (
c
[21] A.M. Wilstermann, N. Osheroff, Stabilization of eukaryotic topoisomerase II-
DNA cleavage complexes, Curr. Top. Med. Chem. 3 (2003) 321e338.
[22] C. Levine, H. Hiasa, K.J. Marians, DNA gyrase and topoisomerase IV:
biochemical activities, physiological roles during chromosome replication,
and drug sensitivities, Biochem. Biophys. Acta 1400 (1998) 29e43.
from the equation
c
¼ ꢀ(EHOMO þ ELUMO)/2, total energy (ET),
binding energy (EB), isolated atomic energy (EIA), electronic energy
(EE), coreecore interaction (ICeC), heat of formation (HF) and
HOMO/LUMO maps were calculated using HyperChem8.0.1
program at RM1 level of theory.
ꢀ
[23] A. Siwek, P. Sta˛czek, M. Wujec, J. Stefanska, U. Kosikowska, A. Malm,
S. Jankowski, P. Paneth, Biological and docking studies of topoisomerase IV
inhibition by thiosemicarbazides, J. Mol. Model. 17 (2011) 2297e2303.
ꢀ
[24] I.R. Ezabadi, C. Camoutsis, P. Zoumpoulakis, A. Geronikaki, M. Sokovic,
ꢀ
ꢀ
4.5. Automated docking setup
J. Glamocilija, A. Ciri&cacute, Sulfonamide-1,2,4-triazole derivatives as anti-
fungal and antibacterial agents: synthesis biological evaluation, lipophilicity,
and conformational studies, Bioorg. Med. Chem. 16 (2008) 1150e1161.
[25] C. Kus¸ , G. Ayhan-Kılcıgil, S. Özbey, F.B. Kaynak, M. Kaya, T. Çoban, B. Can-Eke,
Synthesis and antioxidant properties of novel N-methyl-1,3,4-thiadiazol-2-
amine and 4-methyl-2H-1,2,4-triazole-3(4H)-thione derivatives of benz-
imidazole class, Bioorg. Med. Chem. 16 (2008) 4294e4303.
Flexible ligandereceptor docking was performed using Auto-
dock Vina program [43] using the default settings. Model of the ATP
binding site based on the structure deposited in the Protein Data
Bank [44] under the PDB ID 1KIJ [45] was employed. Default
docking parameters and flexible space of 24 ꢁ 24 ꢁ 24 Å [3] were
validated by re-docking novobiocin which docked exactly in the
position present in the crystal structure with affinity of ꢀ11.0 kcal/
mol. Subsequently, eight small molecule compounds were docked
using same docking parameters.
ꢂ
[26] T. Önkol, D.S. Dogruer, L. Uzun, S. Adak, S. Özkan, M.F.J. S¸ ahin, J. Enzym. Inhib.
Med. Chem. 23 (2008) 277e284.
[27] H.Z. Shams, R.M. Mohareb, M.H. Helal, A.E. Mahmoud, Phosphorus Sulfur 182
(2007) 237e263.
[28] F.E. Norrington, R.M. Hyde, S.G. Williams, R. Wootton, Physiochemical-activity
relations in practice. 1. A rational and self-consistent data bank, J. Med. Chem.
18 (1975) 604e607.

5726
A. Siwek et al. / European Journal of Medicinal Chemistry 46 (2011) 5717e5726
[29] A. Cammarata, Quantitated structureeactivity relationships, Annu. Rep. Med.
Chem. 6 (1971) 245e253.
[38] ÉC. Moreno Nascimento, J.B.L. Martins, Electronic structure and PCA analysis
of covalent and non-covalent acetylcholinesterase inhibitors, J. Mol. Model. 17
(2011) 1371e1379.
[39] A. Siwek, M. Wujec, M. Dobosz, I. Wawrzycka-Gorczyca, Study of direction of
cyclization of 1-azolil-4-aryl/alkyl-thiosemicarbazides, Heteroatom. Chem. 21
(2010) 521e532.
[40] Clinical and Laboratory Standards Institute, Performance Standards for Anti-
microbial Disc Susceptibility Tests; Approved Standard M2eA9. Clinical and
Laboratory Standards Institute, Wayne, PA, USA, 2006.
[30] E. Kutter, C. Hansch, Steric parameters in drug design. Monoamine oxidase
inhibitors and antihistamines, J. Med. Chem. 12 (1969) 647e652.
[31] C. Oefner, A. Douangamath, A. D’Arcy, S. Häfeli, D. Mareque, A. Mac Sweeney,
J. Padilla, S. Pierau, H. Schulz, M. Thormann, S. Wadman, G.E. Dale, The 1.15A
crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and
complexes with triazole based inhibitors, J. Mol. Biol. 332 (2003) 13e21.
[32] T. Fujita, J. Iwasa, C. Hansch, A new substituent constant,
p, derived from
partition coefficients, J. Am. Chem. Soc. 86 (1964) 5175e5180.
[33] C. Hansch, W.J. Dunn, Linear relationship between lipophilic character and
biological activity of drugs, J. Pharm. Sci. 61 (1972) 1e19.
[34] C. Hansch, J.M. Clayton, Lipophilic character and biological activity of drugs II:
the parabolic case, J. Pharm. Sci. 62 (1973) 1e21.
[41] Clinical and Laboratory Standards Institute, Methods for Dilution Antimicro-
bial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Stan-
dard M7eA7. Clinical and Laboratory Standards Institute, Wayne, PA, USA,
2006.
[42] Hyperchem 8.0.3. HyperCube Inc., Gainsville, FL, USA, 2007.
[43] O. Trott, A.J. Olson, AutoDockVina: improving the speed and accuracy of
[35] A.E. Bird, A.C. Marshall, Correlation of serum binding of penicillins with
partition coefficients, Biochem. Pharmacol. 16 (1967) 2275e2290.
docking with
a new scoring function, efficient optimization and multi-
ꢀ
[36] A. Siwek, K. Swiderek, S. Jankowski, Problems with molecular mechanics
threading, J. Comput. Chem. 31 (2010) 455e461.
implementations on the example of 4-benzoyl-1-(4-methyl-imidazol-5-yl)-
carbonylthiosemicarbazide, J. Mol. Model., online first doi: 10.1007/s00894-
011-1117-1.
[44] www.pdb.org and the following citation: H.M. Berman, J. Westbrook, Z. Feng,
G. Gilliland, T.N. Bhat, H. Weissig, I.N. Shindyalov, P.E. Bourne, The protein
data bank, Nucleic Acids Res. 28 (2000) 235e242.
[45] V. Lamour, L. Hoermann, J.M. Jeltsch, P. Oudet, D. Moras, An open confor-
mation of the Thermus thermophilus gyrase B ATP-binding domain, J. Biol.
Chem. 277 (2002) 18947e18953.
ꢀ
[37] A. Siwek, M. Wujec, J. Stefanska, P. Paneth, Antimicrobial properties of 4-aryl-
3-(2-methyl-furan-3-yl)-D2-1,2,4-triazoline-5-thiones, Phosphorus Sulfur
184 (2009) 3149e3159.