Structure-activity relationship studies of microbiologically active thiosemicarbazides derived from hydroxybenzoic acid hydrazides

Article abstract of DOI:10.1111/cbdd.12392

Forty-five derivatives of thiosemicarbazide were synthesized, and their antibacterial activity against Gram-positive and Gram-negative bacteria was evaluated. Some of the described compounds exhibited interesting activity against reference strains of Gram

Full text of DOI:10.1111/cbdd.12392

Chem Biol Drug Des 2014
Research Article
Structure–activity Relationship Studies of
Microbiologically Active Thiosemicarbazides Derived
from Hydroxybenzoic Acid Hydrazides
Tomasz Plech1,*, Agata Paneth1, Barbara
proteins characterized by a low affinity to antibiotics (1). For
2
2
ꢀ1
many years, the methicillin-resistant strain of Staphylococ-
cus aureus (MRSA) posed the most serious epidemiological
risk. It was first identified in 1961 in the United Kingdom (2).
Since that time, the infection rate exhibited by the MRSA
strains has been rising constantly. This increase is particu-
larly pronounced in developed countries; in the USA, for
example, approximately 60% of staphylococcal infections
are caused by methicillin-resistant strains (3). Mortality rate
arising from such infections has already exceeded that
related to AIDS (4). Currently, the world’s attention has
turned to the occurrence and fast spreading of the so-called
super-resistance gene NDM–1 (5,6). The gene is responsi-
ble for generating resistance to virtually all b-lactam antibiot-
ics, including carbapenems, to date considered as
particularly insensitive to b–lactamases. Due to these facts,
there is a high demand for the efforts aimed at finding novel
chemical compounds endowed with antibacterial activity.
One of the groups, in which novel antibacterial substances
are actively sought, is thiosemicarbazide derivatives (7–10).
For many years, designing novel members of this group
was based only on the available data on the structure–activ-
ity relationships. However, in the recent years, researchers
have focused on determining the molecular mechanism of
action of thiosemicarbazides (11–13). The results of both
biological tests and in silico analyses have indicated the inhi-
bition of bacterial topoisomerase IV as one of those mecha-
nisms (12,13). Topoisomerases are essential in the process
of replication of genetic material and constitute molecular
targets for antibacterial drugs belonging to other chemical
classes (14). This work is a continuation of our previous
studies on potential thiosemicarbazide-based inhibitors of
type IIa topoisomerases.
Kapron , Urszula Kosikowska , Anna Malm ,
Aleksandra Strzelczyk3 and Paweł Staczek3
z
1Department of Organic Chemistry, Faculty of Pharmacy,
Medical University, Chodzki 4A, Lublin 20-093, Poland
2Department of Pharmaceutical Microbiology, Faculty of
Pharmacy, Medical University, Chodzki 1, Lublin 20-093,
Poland
3Department of Genetics of Microorganisms, University of
Lodz, Banacha 12/16, Lodz 90-237, Poland
*Corresponding author: Tomasz Plech,
tomasz.plech@umlub.pl
Forty-five derivatives of thiosemicarbazide were
synthesized, and their antibacterial activity against
Gram-positive and Gram-negative bacteria was
evaluated. Some of the described compounds exhib-
ited interesting activity against reference strains of
Gram-positive bacteria, whereas only two derivatives
had the ability to inhibit the growth of Gram-negative
species (Escherichia coli ATCC 25922, Klebsiella pneu-
moniae ATCC 13883, Proteus mirabilis ATCC 12453).
The most potent antimicrobial activity was observed in
the cases of salicylic acid hydrazide derivatives. The
differences in activity inspired us to conduct conforma-
tional analysis using molecular mechanics level. The
obtained results suggest that the molecule geometry,
especially at the N4–terminus of thiosemicarbazide
skeleton, determines the antibacterial activity. Unfortu-
nately, in opposition to what we expected, only one of
the tested compounds inhibited the activity of the
topoIV enzyme, and none of them was active against
DNA gyrase.
Key words: drug design, enzymatic mechanism, structure–
activity relationships, thiosemicarbazide derivatives
Literature data reveals that promising antibacterial activity
is demonstrated by 1,4-disubstituted thiosemicarbazides
obtained from benzoic or 3-chlorobenzoic acid hydrazides
(15,16), for example, 1,4-dibenzoylthiosemicarbazide
inhibited the growth of Gram-positive bacteria at the
concentrations ranged between 3.1 and 6.3 lg/mL (15),
while minimal inhibitory concentration (MIC) value for
4-(4-bromophenyl)-1-(3-chlorobenzoyl)thiosemicarbazide
was 15.63 lg/mL (16). We presumed that an exchange of
chlorine substituent for a hydroxyl group may be beneficial
for the compound’s interaction with its potential molecular
target. The -OH group, as a potent electron-donating
Received 12 March 2014, revised 11 May 2014 and accepted
for publication 2 July 2014
Uncontrolled use of antibiotics has caused the incidence of
selection of antibiotic-resistant strains. Bacterial resistance
to antibiotics is mostly a result of the activation of the follow-
ing defensive mechanisms: (i) reduction in bacterial cell wall
permeability; (ii) active (energy-dependent) efflux of the drug
from the interior of the cell; (iii) production of enzymes that
inactivate antibiotics; and (iv) production of enzymatic
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12392
1

Plech et al.
substituent, may significantly change the electrostatic
properties of a molecule and enable formation of additional
hydrogen bonds.
BioSpin GmbH, Rheinstetten, Germany) using TMS as an
internal standard. The IR spectra (KBr) were obtained on a
Perkin-Elmer 1725X FTIR spectrophotometer. Elemental
analyses were performed on an AMZ 851 CHX analyser
ꢀ
With the above information in mind, we designed a group
(PG, Gdansk, Poland), and the results were within Æ0.4%
of derivatives characterized by the presence of
a
of the theoretical value.
thiosemicarbazide skeleton and a hydroxyphenyl moiety.
Applying rational principles of designing novel compounds
involves a stage of in silico tests, in which various compu-
tational algorithms are used to check the affinity of the
newly designed molecules to the crystal structure of an
enzymatic protein. The topoisomerase IV (topoIV) inhibitors
we described (Figure 1) were used to validate the proce-
dure of docking ligands to enzymatic proteins, whose
structures were derived from the RCSB PDB database. In
the calculations, forms of the topoIV enzymes originating
from Gram-positive (PDB id: 4LPB) and Gram-negative
(PDB id: 3FV5) bacteria were used. The results of in vitro
enzymatic tests performed with compounds A–D (Figure 1)
ideally corresponded to their docking scores obtained for
the active site of 4LPB enzyme model (data not shown).
The obtained results are also in accordance with the
selective activity of the aforementioned compounds A–D
against Gram-positive bacteria observed in the microbio-
logical assays. Therefore, we considered that the model of
the active site of topoIV—4LPB—would be appropriate for
testing the inhibitory potential of the designed compounds
(1–45). The free binding energies (DGb) of the ligands ran-
ged between -24.64 and -31.15 kJ/mol (versus
À30.31 kJ/mol for native ligand; detailed data not shown),
which allowed us to hypothesize that we might identify fur-
ther bacterial topoisomerase inhibitors in this group, with a
potent antibacterial activity.
Synthesis of 1,4-disubstituted derivatives of
thiosemicarbazide (1–45)
A solution of 0.01 mol of hydroxybenzoic acid hydrazide
and equimolar amount of appropriate isothiocyanate in
25 mL of anhydrous ethyl alcohol were heated under
reflux for 15 min. After cooling, the solid formed was fil-
tered off, washed with diethyl ether, dried, and recrystal-
lized from ethyl alcohol. Information on all compounds,
except 14, 30, 32, and 34, may be retrieved in the Chemi-
cal Abstract Service database.
4-Benzyl-1-(2-hydroxybenzoyl)thiosemicarbazide (1)
Yield: 82%, CAS: 94494-02-7
4-(4-Fluorobenzyl)-1-(2-hydroxybenzoyl)thiosemicarbazide
(2)
Yield: 75%, CAS: 891101-93-2
4-(4-Chlorobenzyl)-1-(2-hydroxybenzoyl)thiosemicarbazide
(3)
Yield: 85%, CAS: 891376-50-4
1-(2-Hydroxybenzoyl)-4-(4-methoxybenzyl)thiosemicarbaz-
ide (4)
Experimental
Yield: 74%, CAS: 1013521-31-7
General comments
All reagents and solvents were purchased from Alfa Aesar
(Ward Hill, MA, USA) and Merck Co. (Darmstadt, Ger-
many). Melting points were determined using Fisher-Johns
apparatus (Fisher Scientific, Schwerte, Germany) and are
uncorrected. The 1H-NMR and 13C-NMR spectra were
1-(2-Hydroxybenzoyl)-4-phenylthiosemicarbazide (5)
Yield: 86%, CAS: 37536-32-6
4-(4-Fluorophenyl)-1-(2-hydroxybenzoyl)thiosemicarbazide
recorded on
a
Bruker Avance spectrometer (Bruker
(6)
A
B
C
D
Figure 1: Structures of thiosemicarbazide-
based inhibitors of bacterial topoisomerase
IV (12,13).
2
Chem Biol Drug Des 2014

Thiosemicarbazides as Antibacterials
4-Benzyl-1-(3-hydroxybenzoyl)thiosemicarbazide (17)
Yield: 88%, CAS: 891093-06-4
Yield: 79%, CAS: 343-70-4
4-(4-Chlorophenyl)-1-(2-hydroxybenzoyl)thiosemicarbazide
(7)
4-(4-Fluorobenzyl)-1-(3-hydroxybenzoyl)thiosemicarbazide
Yield: 85%, CAS: 26131-23-7
(18)
1-(2-Hydroxybenzoyl)-4-(4-methoxyphenyl)thiosemicarbaz-
Yield: 78%, CAS: 891102-88-8
ide (8)
4-(4-Chlorobenzyl)-1-(3-hydroxybenzoyl)thiosemicarbazide
Yield: 80%, CAS: 94565-92-1
(19)
4-(3,4-Dichlorophenyl)-1-(2-hydroxybenzoyl)thiosemicar-
Yield: 74%, CAS: 891375-94-3
bazide (9)
1-(3-Hydroxybenzoyl)-4-(4-methoxybenzyl)thiosemicarbaz-
Yield: 82%, CAS: 891577-67-6
ide (20)
4-(2,4-Dichlorophenyl)-1-(2-hydroxybenzoyl)thiosemicar-
Yield: 78%, CAS: 878946-30-6
bazide (10)
1-(3-Hydroxybenzoyl)-4-phenylthiosemicarbazide (21)
Yield: 81%, CAS: 26036-20-4
Yield: 74%, CAS: 891577-24-5
4-(3,5-Dichlorophenyl)-1-(2-hydroxybenzoyl)thiosemicar-
bazide (11)
4-(4-Fluorophenyl)-1-(3-hydroxybenzoyl)thiosemicarbazide
(22)
Yield: 78%, CAS: 891577-60-9
Yield: 71%, CAS: 896901-32-9
4-(4-Bromo-2-chlorophenyl)-1-(2-hydroxybenzoyl)thiose-
micarbazide (12)
4-(4-Chlorophenyl)-1-(3-hydroxybenzoyl)thiosemicarbazide
(23)
Yield: 72%, CAS: 893343-72-1
Yield: 72%, CAS: 891109-57-2
4-(3-Chloro-4-methylphenyl)-1-(2-hydroxybenzoyl)thiose-
micarbazide (13)
1-(3-Hydroxybenzoyl)-4-(4-methoxyphenyl)thiosemicarbaz-
ide (24)
Yield: 86%, CAS: 891376-43-5
Yield: 81%, CAS: 891084-72-3
4-[4-chloro-3-(trifluoromethyl)phenyl]-1-(2-hydroxybenzoyl)
thiosemicarbazide (14)
4-(3,4-Dichlorophenyl)-1-(3-hydroxybenzoyl)thiosemicar-
bazide (25)
Yield: 84%, m.p. 180–182 °C. 1H-NMR (300 MHz): 6.96–
7.02 (m, 1H, Ar-H), 7.48 (t, 1H, Ar-H, J = 7.1 Hz), 7.69–
8.32 (m, 5H, Ar-H), 10.17, 10.77, 11.79 (3s, 3H, 3NH),
11.45 (s, 1H, OH). 13C-NMR (90 MHz): 14.35, 117.76,
119.44, 121.27, 124.90, 125.92, 126.62, 130.87, 131.77,
132.51, 134.74, 139.27, 159.86, 169.24, 188.18. IR (KBr):
3264 (OH), 3179 (NH), 3007 (Ar-H), 1664 (C=O), 1315
(C=S). Anal. calc. for C15H11ClF3N3O2S: C 46.22, H 2.84,
N 10.78. Found: C 46.40, H 2.95, N 10.73.
Yield: 85%, CAS: 891578-39-5
4-(2,4-Dichlorophenyl)-1-(3-hydroxybenzoyl)thiosemicar-
bazide (26)
Yield: 74%, CAS: 891578-47-5
4-(3,5-Dichlorophenyl)-1-(3-hydroxybenzoyl)thiosemicar-
bazide (27)
4-Cyclohexyl-1-(2-hydroxybenzoyl)thiosemicarbazide (15)
Yield: 75%, CAS: 26036-12-4
Yield: 77%, CAS: 891577-39-2
4-(4-Bromo-2-chlorophenyl)-1-(3-hydroxybenzoyl)thiose-
4-Benzoyl-1-(2-hydroxybenzoyl)thiosemicarbazide (16)
Yield: 80%, CAS: 130136-99-1
micarbazide (28)
Yield: 78%, CAS: 893341-77-0
Chem Biol Drug Des 2014
3

Plech et al.
4-(3-Chloro-4-methylphenyl)-1-(3-hydroxybenzoyl)thiose-
Yield: 78%, CAS: 314767-23-2
micarbazide (29)
4-(4-Chlorophenyl)-1-(4-hydroxybenzoyl)thiosemicarbazide
Yield: 80%, CAS: 891375-66-9
(37)
4-[4-chloro-3-(trifluoromethyl)phenyl]-1-(3-hydroxybenzoyl)
Yield: 74%, CAS: 136414-15-8
thiosemicarbazide (30)
1-(4-Hydroxybenzoyl)-4-(4-methoxyphenyl)thiosemicarbaz-
ide (38)
Yield: 78%, m.p. 161–163 °C. 1H-NMR (300 MHz): 6.98–
7.96 (m, 7H, Ar-H), 9.77, 10.07, 10.54 (3s, 3H, 3NH),
11.45 (s, 1H, OH). 13C-NMR (90 MHz): 14.33, 115.38,
117.65, 119.38, 121.26, 124.88, 125.95, 126.89, 127.27,
128.50, 129.82, 132.49, 157.82, 168.21, 188.14. IR (KBr):
3387 (OH), 3268 (NH), 3098 (Ar-H), 1677 (C=O), 1321
(C=S). Anal. calc. for C15H11ClF3N3O2S: C 46.22, H 2.84,
N 10.78. Found: C 46.31, H 2.74, N 10.74.
Yield: 81%, CAS: 136414-17-0
4-(3,4-Dichlorophenyl)-1-(4-hydroxybenzoyl)thiosemicar-
bazide (39)
Yield: 80%, CAS: 891577-74-5
4-Cyclohexyl-1-(3-hydroxybenzoyl)thiosemicarbazide (31)
Yield: 73%, CAS: 26036-19-1
4-(2,4-Dichlorophenyl)-1-(4-hydroxybenzoyl)thiosemicar-
bazide (40)
Yield: 82%, CAS: 891578-16-8
4-Benzoyl-1-(3-hydroxybenzoyl)thiosemicarbazide (32)
4-(3,5-Dichlorophenyl)-1-(4-hydroxybenzoyl)thiosemicar-
bazide (41)
Yield: 79%, m.p. 208–209 °C. 1H-NMR (300 MHz): 7.04
(d, 1H, Ar-H, J = 7.6 Hz), 7.34-7.43 (m, 3H, Ar-H), 7.57 (t,
2H, Ar-H, J = 7.7 Hz), 7.70 (m, 1H, Ar-H), 8.02 (d, 2H,
Ar-H, J = 7.3 Hz), 9.85, 11.08, 11.82 (3s, 3H, 3NH),
12.40 (br. s, 1H, OH). 13C-NMR (90 MHz): 115.05,
118.72, 119.57, 128.98, 129.26, 130.12, 132.40, 133.69,
133.94, 157.91, 165.08, 168.37, 180.93. IR (KBr): 3374
(OH), 3211 (NH), 3057 (Ar-H), 1670, 1653 (C=O), 1323
(C=S). Anal. calc. for C15H13N3O3S: C 57.13, H 4.16, N
13.33. Found: C 57.25, H 4.02, N 13.38.
Yield: 77%, CAS: 891577-88-1
4-(4-Bromo-2-chlorophenyl)-1-(4-hydroxybenzoyl)thiose-
micarbazide (42)
Yield: 84%, CAS: 894230-65-0
4-(3-Chloro-4-methylphenyl)-1-(4-hydroxybenzoyl)thiose-
micarbazide (43)
4-Benzyl-1-(4-hydroxybenzoyl)thiosemicarbazide (33)
Yield: 82%, CAS: 891376-15-1
Yield: 76%, CAS: 708285-32-9
4-Cyclohexyl-1-(4-hydroxybenzoyl)thiosemicarbazide (44)
Yield: 79%, CAS: 26036-24-8
1-(4-Hydroxybenzoyl)-4-(4-methoxybenzyl)thiosemicarbaz-
ide (34)
Yield: 80%, m.p. 194–196 °C. 1H-NMR (300 MHz): 3.75
(s, 3H, OCH3), 4.68 (d, 2H, -CH2-, J = 5.7 Hz), 6.84–6.90
(m, 4H, Ar-H), 7.28 (d, 2H, Ar-H, J = 8.6 Hz), 7.83 (d, 2H,
Ar-H, J = 8.6 Hz), 8.56 (br. s, 1H, OH), 9.34, 10.11, 10.17
(3s, 3H, 3NH). 13C-NMR (90 MHz): 46.67, 55.54, 113.91,
115.25, 123.60, 129.00, 130.40, 131.92, 158.61, 161.20,
166.20, 184.16. IR (KBr): 3387 (OH), 3268 (NH), 3025
(Ar-H), 2879 (C-Haliph.) 1677 (C=O), 1321 (C=S).Anal. calc.
for C16H17N3O3S: C 57.99, H 5.17, N 12.68. Found: C
58.11, H 5.03, N 12.84.
4-Benzoyl-1-(4-hydroxybenzoyl)thiosemicarbazide (45)
Yield: 72%, CAS: 332393-58-5
Antimicrobial Activity Evaluation
The antimicrobial activity of the compounds was tested on
the Gram-positive strains (Staphylococcus aureus ATCC
25923, Staphylococcus aureus ATCC 6538, Staphylococ-
cus aureus Microbank 14001, Staphylococcus epidermidis
ATCC 12228, Bacillus subtilis ATCC 6633, Bacillus cereus
ATCC 10876, Micrococcus luteus ATCC 10240) and on
the Gram-negative ones (Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Proteus mirabilis
ATCC 12453, Pseudomonas aeruginosa ATCC 9027).
Ampicillin, cefuroxime, and vancomycin were used as con-
trol antibacterial agents. Microbial suspensions with an
1-(4-Hydroxybenzoyl)-4-phenylthiosemicarbazide (35)
Yield: 80%, CAS: 26036-25-9
4-(4-Fluorophenyl)-1-(4-hydroxybenzoyl)thiosemicarbazide
(36)
4
Chem Biol Drug Des 2014

Thiosemicarbazides as Antibacterials
optical
density
of
0.5
McFarland
standard
plasmid DNA (0.5 lg) was incubated with 1 U gyrase, in the
dedicated supercoiling assay buffer supplied by the manu-
facturer, in the presence of varying concentrations of the
test compounds. Reactions were carried out at 37 °C for
1 h and then terminated by the addition of equal volume of
29 STOP Buffer (40% sucrose, 100 mM Tris–Cl pH 7.5,
1 mM EDTA, 0.5 mg/mL bromophenol blue) and chloro-
form/isoamyl 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.
150 9 106 CFU/mL (CFU—colony forming units) were
prepared in sterile 0.85% NaCl. All stock solutions of the
tested compounds were dissolved in dimethyl sulfoxide
(DMSO). The medium with DMSO in the final concentration
and without the tested compounds served as a control—
no microbial growth inhibition was observed. Preliminary
antibacterial in vitro potency of the tested compounds was
screened using an agar dilution method on the basis of
the growth inhibition on a Mueller–Hinton agar to which
the tested compounds in concentration of 1000 lg/mL
were added. The plates were incubated at 37ᵒC for 18 h.
In the next assay, in vitro antibacterial activity of the com-
pounds with inhibitory effect was determined by a broth
microdilution method. The 96-well microplates were used;
198 lL of Mueller–Hinton broth with a series of twofold dilu-
tions of the tested compound in the range of the final con-
centrations from 0.24 to 1000 lg/mL was inoculated with
2 lL of microbial suspension. After incubation (at 37 °C for
18 h), spectrophotometric measurements of optical density
(OD600) of the bacterial cultures with the tested compounds
were performed to determine MIC. OD600 of bacterial cul-
tures in the medium without the tested compounds was
used as a control. The activity was expressed as the mini-
mal concentration of the compound that inhibits the visible
growth of the bacteria (MIC). The minimal bactericidal con-
centration (MBC), defined as the lowest concentration of
each compound that resulted in >99.9% reduction in CFU
of the initial inoculum, was also assessed. Both MIC and
MBC values were given in lg/mL.
The decatenation assay was performed using S. aureus
and E. coli topoisomerase IV decatenation kits (Inspiralis).
Interlinked kDNA substrate (0.5 lg) was incubated with
1 U topoisomerase IV (Inspiralis), in the dedicated decate-
nation assay buffer supplied by the manufacturer, in the
presence of varying concentrations of the test compounds.
Reactions were carried out at 37 °C for 1 h and then ter-
minated by the addition of equal volume of 29 STOP Buf-
fer (40% sucrose, 100 mM Tris–Cl pH 7.5, 1 mM EDTA,
0.5 mg/mL bromophenol blue) and chloroform/isoamyl
alcohol. Samples were vortexed, centrifuged, and run
through a 15 cm 1% agarose gel in TAE buffer for 1.5 h
at 80 V. Gels were stained with ethidium bromide and
visualized under UV light. Concentrations of inhibitor that
prevented 50% of the kinetoplast DNA from being con-
verted into decatenated minicircles (IC50 values) were
determined by plotting the results obtained from the densi-
tometric analyses of the gel images using QUANTITY ONE soft-
ware (Bio-Rad, Hercules, CA, USA).
Computational Part
Conformational search and electrostatic potential surfaces
were calculated using HYPERCHEM 8.0.3a Conformational
searches were conducted using the molecular mechanics
level with Amber force field. The most stable structures
obtained were next optimized to the closest local minimum
at the semiempirical level using AM1 parametrization. Con-
vergence criteria for Amber and AM1 calculations were set
Results and Discussion
Chemistry
1-(ortho/meta/para)-Hydroxybenzoyl-4-substituted thiose-
micarbazide derivatives (1–45) were prepared following the
procedure known from the literature (16). Synthetic route
is depicted in Scheme 1. In short, hydroxybenzoic acid
hydrazides and different isothiocyanates were allowed to
react in boiling ethyl alcohol for 15 min. Information on all
compounds, except 14, 30, 32, and 34 may be retrieved
in the Chemical Abstract Service database (CAS numbers
are given in the experimental section).
ꢁ
to 0.1 and 0.01 kcal/mol*A, respectively.
Docking studies were performed using FLEXX program (17)
as implemented in LEADIT software packageb using models
of topoIV (PDB codes: 4LPB, 3FV5) complexed with refer-
ence ligands. The active site was defined to include all
ꢁ
atoms within 6.5 A radius of the native ligand. To validate
the docking protocol, reference ligands were initially
docked into the crystal structure of the appropriate
enzymes.
Antimicrobial Activity
Antimicrobial activity evaluation was conducted using
Gram-positive and Gram-negative bacterial strains. In the
first stage, the activity was determined using agar-well dif-
fusion method. The compounds which inhibited bacterial
growth were then subjected to assays determining their
MIC and MBC values. The comparison of MBC and MIC
values of the same compounds allows the determination
whether the compound has bactericidal (MBC/MIC ≤ 4) or
bacteriostatic (MBC/MIC > 4) effect. It was observed that
Evaluation of Inhibition of Bacterial Type IIa
Topoisomerases
The inhibitory activity of DNA gyrase from E. coli and
S. aureus was evaluated using gyrase supercoling assay
kits (Inspiralis, Norwich, UK). Briefly, supercoiled pBR322
Chem Biol Drug Des 2014
5

Plech et al.
Scheme 1: Synthetic route to compounds
1–45 (R1 and R2 substituents are presented
in Table 1).
Table 1: Antibacterial activity of compounds 1–45
S. aureus S. aureus S. aureus S. epidermidis B. subtilis
ATCC 12228
B. cereus
M. luteus
R1
R2
MSSA-1
MSSA-2
MRSA
ATCC 6633 ATCC 10876 ATCC 10240
1
2
3
4
5
6
7
8
9
2-OH Benzyl
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2-OH 4-F-benzyl
2-OH 4-Cl-benzyl
2-OH 4-OCH3-benzyl
2-OH Phenyl
2-OH 4-F-phenyl
2-OH 4-Cl-phenyl
2-OH 4-OCH3-phenyl
2-OH 3,4-diCl-phenyl
250
7.82
31.25
–
250
31.25
62.5
–
500
7.82
31.25
–
62.5
7.82
7.82
7.82
7.82
1.95
7.82
15.63
500
7.82
15.63
–
250
250
31.25
31.25
–
31.25
62.5
–
1000
125
–
31.25
3.91
3.91
3.91
7.82
1.95
7.82
15.63
62.5
–
125
7.82
7.82
7.82
3.91
7.82
7.82
15.63
125
–
31.25
1.95
3.91
1.95
3.91
1.95
7.82
7.82
15.63
–
62.5
7.82
3.91
15.63
3.91
15.63
15.63
31.25
3.91
–
500
31.25
1.95
3.91
1.95
3.91
3.91
3.91
31.25
1.95
–
7.82
15.63
15.63
10 2-OH 2,4-diCl-phenyl
11 2-OH 3,5-diCl-phenyl
12 2-OH 2-Cl-4-Br-phenyl
13 2-OH 3-Cl-4-CH3-phenyl
14 2-OH 3-CF3-4-Cl-phenyl
15 2-OH Cyclohexyl
16 2-OH Benzoyl
500
31.25
7.82
1000
125
7.82
17 3-OH Benzyl
–
–
–
–
–
–
–
–
18 3-OH 4-F-benzyl
19 3-OH 4-Cl-benzyl
20 3-OH 4-OCH3-benzyl
21 3-OH Phenyl
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
250
1000
–
500
–
1000
–
3.91
125
–
125
–
62.5
–
31.25
–
500
250
–
1000
–
1.95
125
–
125
–
62.5
–
15.63
–
125
1000
1000
500
1000
125
500
7.82
–
1.95
22 3-OH 4-F-phenyl
23 3-OH 4-Cl-phenyl
24 3-OH 4-OCH3-phenyl
25 3-OH 3,4-diCl-phenyl
26 3-OH 2,4-diCl-phenyl
27 3-OH 3,5-diCl-phenyl
28 3-OH 2-Cl-4-Br-phenyl
29 3-OH 3-Cl-4-CH3-phenyl
30 3-OH 3-CF3-4-Cl-phenyl
31 3-OH Cyclohexyl
32 3-OH Benzoyl
–
1000
–
–
–
–
3.91
125
–
3.91
125
–
125
–
125
–
31.25
–
15.63
250
15.63
125
62.5
–
125
–
–
–
–
–
62.5
125
125
125
–
125
–
31.25
–
62.5
–
15.63
500
15.63
–
7.82
–
–
15.63
33 4-OH Benzyl
34 4-OH 4-OCH3-benzyl
35 4-OH Phenyl
–
–
125
500
500
1000
3.91
62.5
1.95
31.25
–
–
–
–
–
–
–
–
250
250
31.25
500
7.82
31.25
3.91
31.25
–
250
500
1000
1000
15.63
62.5
15.63
62.5
–
125
500
500
500
3.91
31.25
1.95
31.25
–
125
125
15.63
500
62.5
62.5
15.63
62.5
–
250
62.5
36 4-OH 4-F-phenyl
37 4-OH 4-Cl-phenyl
38 4-OH 4-OCH3-phenyl
39 4-OH 3,4-diCl-phenyl
40 4-OH 2,4-diCl-phenyl
41 4-OH 3,5-diCl-phenyl
42 4-OH 2-Cl-4-Br-phenyl
43 4-OH 3-Cl-4-CH3-phenyl
44 4-OH Cyclohexyl
45 4-OH Benzoyl
500
1000
1000
125
15.63
125
1.95
31.25
1.95
31.25
–
15.63
62.5
7.82
62.5
–
–
–
–
–
–
–
–
31.25
nd
0.49
0.98
31.25
nd
0.98
0.49
31.25
nd
nd
31.25
nd
0.24
0.98
15.63
nd
15.63
nd
15.63
62.5
31.25
nd
7.82
nd
0.98
nd
Ampicillin
Cefuroxime
Vancomycin
0.98
MSSA-1, Staphylococcus aureus ATCC 25923; MSSA-2, Staphylococcus aureus ATCC 6538; MRSA, Staphylococcus aureus Mikrobank
14001; ‘–’, MIC value higher than 1000 lg/mL; nd, not determined; MBC, minimal bactericidal concentration; MIC, minimal inhibitory con-
centration; compounds with bactericidal effect (MBC/MIC ≤ 4) are indicated in bold.
6
Chem Biol Drug Des 2014

Thiosemicarbazides as Antibacterials
only two derivatives had the ability to inhibit the growth of
Gram-negative bacteria (Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Proteus mirabilis
ATCC 12453). However, in both cases, the MIC values
were as high as 250–1000 lg/mL (data not shown in
Table 1). A considerably more pronounced antibacterial
activity was observed toward Gram-positive bacteria
(Table 1).
(S. aureus Microbank 14001), demonstrated the highest
susceptibility to 4–(disubstituted-phenyl)thiosemicarbazide
derivatives. 4-(3-Chloro-4-methylphenyl)-1-(2-hydroxybenz
oyl)thiosemicarbazide (13) and 4-(3,5-dichlorophenyl)-1-(4-
hydroxybenzoyl)thiosemicarbazide (41) were characterized
by an activity almost comparable to that of vancomycin,
an antibiotic used in the treatment of severe infections
caused by methicillin-resistant strains of S. aureus. Even
higher susceptibility to the tested compounds was demon-
strated by Staphylococcus epidermidis. These bacteria
constitute a normal component of the human bacterial
Both MSSA strains (S. aureus ATCC 25923, S. aureus
ATCC 6538), as well as a particularly virulent MRSA strain
Figure 2: Charge distribution (calculated
using AM1 parametrization) on the surface
of respective 4-cyclohexyl derivatives.
Chem Biol Drug Des 2014
7

Plech et al.
flora and do not pose threat to people with a correctly
functioning immune system. At the same time, epidemio-
logical data demonstrate that S. epidermidis is one of the
most common causes of hospital-acquired infections (18).
In the cases of compounds 9, 11, 13, 25, and 41, MICs
against S. epidermidis ATCC 12228 were as low as
1.95 lg/mL. Finally, the most promising results were
obtained in the cases of Bacillus subtilis ATCC 6633 and
Bacillus cereus ATCC 10876. As for the first strain, four
derivatives of salicylic acid hydrazide (9, 10, 12, 16)
revealed higher activity than that of the reference antibiotic,
and three of those compounds (10, 12, 16) exhibited a
fourfold more potent activity than that of cefuroxime. Even
higher susceptibility to the studied derivatives was demon-
strated by B. cereus ATCC 10876, an opportunistic Gram-
positive species which (especially in neonates) may cause
sepsis, severe infections of the central nervous system,
pneumonia, and myocarditis (19–21). As was shown by
the MIC values of ampicillin (MIC = 62.5 lg/mL) and
cefuroxime (MIC = 31.25 lg/mL), the efficacy of classical
antibiotics in combating infections caused by B. cereus
may be limited. Therefore, it is essential that some of the
investigated derivatives exhibit better antibacterial activity
than both aforementioned antibiotics, for example, N4-di-
chlorophenyl (9, 41), N4-[(4-chloro-3-trifluoromethyl)phenyl]
(14), and N4-benzoyl (16) derivatives exert an eightfold
stronger effect than ampicillin and a fourfold stronger
effect than cefuroxime. By establishing the MBC values, it
was possible to determine whether among the synthesized
derivatives, there were substances endowed with bacterio-
static or bactericidal activity (substances that have bacteri-
cidal effect are indicated in Table 1 in bold).
tional monocarbon group (-CH2- or C=O) between the
nitrogen atom and the phenyl ring. However, while all ben-
zyl derivatives (1, 17, 33) lacked antibacterial activity, the
MIC values of benzoyl derivatives (16, 32, 45) reached
even 1.95 lg/mL. The observed differences indicate that it
is not the molecule’s ‘length’ but the particular properties
of the carbonyl group (e.g., the potential of forming hydro-
gen bonds) that is the source of such large differences in
bioactivity. It also seemed interesting that the 4-phenyl (5)
and 4-cyclohexyl (15) derivatives demonstrated virtually no
difference in activity, even though the electron properties
of both substituents differ significantly. This may indicate
that the electron properties of the substituent at position 4
may be of less importance. However, a completely differ-
ent correlation in the case of other cyclohexyl (31, 44) and
phenyl (21, 35) derivatives inspired us to conduct confor-
mational analyses of compounds 15, 31, and 44. Molecule
geometry optimization was performed using Amber force
field which yields good results for geometries and the
energetics of thiosemicarbazide derivatives (22). The most
stable structures obtained were optimized to the closest
local minimum using AM1 parametrization (implemented in
HyperChem)a. Charge distribution (Figure 2) on the surface
A
Structure–activity Relationship Studies
B
Thanks to a large amount of microbiological data (Table 1);
it was possible to determine many important correlations
between the structure of thiosemicarbazide derivatives
(1–45) and their antibacterial activity. The obtained
information may enable designing potential novel drugs
efficient in treating infectious diseases.
The most potent antimicrobial activity was observed with
2-hydroxybenzoic (salicylic) acid derivatives. By comparing
the activity of respective benzyl (1–4) and phenyl
derivatives (5–8), it may be seen that the introduction of an
additional torsion angle results in a complete or significant
loss of activity. This suggests that the molecule geometry,
especially at the N4–terminus of thiosemicarbazide
skeleton, determines the antibacterial activity. The same
correlation was confirmed in the case of compounds syn-
thesized from 3-hydroxybenzoic and 4-hydroxybenzoic
acid hydrazides. Our previous studies enabled us to
hypothesize that the molecule’s ‘length’ may be one of the
factors determining the inhibiting properties of 1-aroyl-4-
substituted thiosemicarbazide derivatives (12). In the case
of three compound pairs, 1/16, 17/32, and 33/45, mole-
cule elongation was a result of the introduction of an addi-
C
Figure 3: Overlay of structures of the active (15) and inactive (31,
44) derivatives.
8
Chem Biol Drug Des 2014

Thiosemicarbazides as Antibacterials
of the molecules was so similar in all three compounds
that the hypothesis of electrostatic interactions determining
their activity could be excluded. But interestingly, overlay-
ing optimized structures of active (15) and inactive (31, 44)
derivatives revealed that the geometry of the active com-
pound was completely different from the geometries of the
other two inactive compounds (Figure 3).
Therefore, it indicates that the described 1-aroyl-4-aryl
derivatives of thiosemicarbazide endowed with a high bio-
activity play the role of the key as in the classical lock-
and-key theory (postulated by Emil Fischer in the 19th
century).
Enzymatic Studies
Subsequently, it was investigated whether the substitution
pattern of N4-aryl substituent had any influence on the
antibacterial activity. As can be seen from the results in
Table 1, the presence of substituent in the para position
was not well tolerated, for example, 4-(4-chlorophenyl)-1-
(2-hydroxybenzoyl)thiosemicarbazide (7) was inactive, as
opposed to the compound containing an unsubstituted
phenyl ring (5). However, the introduction of another
substituent in ortho or meta position yielded compounds
endowed with a high bioactivity. This demonstrates that
substitutions of ortho and meta positions are well tolerated
(MICs reached 1.95 lg/mL). The analysis of geometry and
electrostatic properties of N4–disubstituted aryl (Figure S1
and S2) derivatives revealed no simple correlations
between these factors and biological activity (i.e.,
compounds with a similar geometry and a similar distribu-
tion of the electron cloud did not present similar activities).
In such case, the most probable explanation is that the
positions of substituents in both phenyl rings rather than
their physicochemical properties (electron-donating/elec-
tron-withdrawing properties; bulkiness) are of great impor-
tance. Thus, as expected, compounds 10 and 12 (2,4–-
disubstituted derivatives), as well as 9, 13, and 14 (3,4-
disubstituted derivatives), had quite similar antibacterial
activity. An identical correlation also occured in the cases
of the following pairs of compounds: 26/28 and 40/42.
Among the synthesized thiosemicarbazide derivatives
(1–45), a group of nine compounds characterized by vari-
ous levels of antibacterial activity was selected for enzy-
matic tests. The group consisted of derivatives endowed
with potent (9, 13, 41) and moderate (15, 16, 45) antibac-
terial activity, as well as derivatives with no such activity (7,
31, 43). In a preliminary assay (Figure 4), the ability of the
above compounds to inhibit the type IIa topoisomerases
(DNA gyrase and topoIV) derived from S. aureus and
E. coli strains was tested. As opposed to what we
expected, only one of the tested compounds inhibited the
activity of the topoIV enzyme, and none of them was
active against DNA gyrase. The subsequent tests allowed
us to determine the IC50 values for 41, which were 105 lM
(for the topoIV obtained from S. aureus) and 155 lM (for
the E. coli enzyme; Figure 5). The lack of antibacterial
activity of compound 41 against E. coli strain, despite the
observed affinity to topoIV, may be explained by the lim-
ited diffusion of the compound across the Gram-negative
bacteria cell wall, whose structure is significantly different
from that of the surrounding Gram-positive bacteria cells.
Another cause of the observed phenomenon may be an
efficient efflux mechanism removing the drug molecules
from the cell. The lack of inhibitory activity of the other
active compounds confirms the earlier hypothesis that
1,4-disubstituted thiosemicarbazides may demonstrate
Figure 4: Assay results of topoisomerase IV
from Escherichia coli (left) and Staphylococcus
aureus (right) with the selected
thiosemicarbazides.
Figure 5: Assay results of topoisomerase IV
from Escherichia coli (left) and Staphylococcus
aureus (right) with compound 41.
Chem Biol Drug Des 2014
9

Plech et al.
antibacterial activity by activating various mechanisms,
among which the inhibition of bacterial topoisomerases is
only one of the options.
7. El-Sabbagh O.I., El-Sadek M.E., Lashine S.M., Yassin
S.H., El-Nabtity S.M. (2009) Synthesis of new 2(1H)-
quinoxalinone derivatives for antimicrobial and antiin-
flammatory evaluation. Med Chem Res;18:782–797.
8. Keshk E.M., El-Desoky S.I., Hammouda M.A.A.,
Abdel-Rahman A.H., Hegazi A.G. (2008) Synthesis and
reactions of some new quinoline thiosemicarbazide
derivatives of potential biological activity. Phosphorus,
Sulfur Silicon Relat Elem;183:1323–1343.
9. Abdel-Rahman H.M., Hussein M.A. (2006) Synthesis of
b-hydroxypropanoic acid derivatives as potential anti-
inflammatory, analgesic and antimicrobial agents. Arch
Pharm (Weinheim);339:378–387.
10. Demirbas A., Sahin D., Demirbas N., Karaoglu S.A.
(2009) Synthesis of some new 1,3,4-thiadiazol-2-ylm-
ethyl-1,2,4-triazole derivatives and investigation of their
antimicrobial activities. Eur J Med Chem;44:2896–2903.
11. Farag N.A.H., El-Tayeb W. (2010) Design, synthesis and
docking studies of new furobenzopyranones and pyra-
nobenzopyranones as photoreagent towards DNA and
as antimicrobial agents. Eur J Med Chem;45:317–325.
Conclusions
To sum up, the analysis of structure and activity of
45 thiosemicarbazide derivatives allowed us to draw the
following conclusions: (i) the antibacterial activity of 4-cy-
clohexyl derivatives depends on the molecule’s
geometry; (ii) not the molecule’s ‘length’ but the particu-
lar properties of the carbonyl group has more effect on
the biological activity; and (iii) the activity of 4–aryl
derivatives is determined to a greater extent by the posi-
tion (substitution pattern) than the type of substituent.
Noteworthy, among the described derivatives, we
managed to identify compounds endowed with potent
antibacterial activity.
ꢀ
Acknowledgments
12. Siwek A., Staczek P., Wujec M., Stefanska J.,
z
Kosikowska U., Malm A., Jankowski S., Paneth P.
(2011) Biological and docking studies of topoisomer-
The project was funded by the National Science Centre
(decision number: 2012/05/D/NZ7/02278).
ase IV inhibition by thiosemicarbazides.
J
Mol
Model;17:2297–2303.
ꢀ
13. Siwek A., Staczek P., Stefanska J. (2011) Synthesis
z
Conflict of interest statement
and structure-activity relationship studies of 4-arylthi-
osemicarbazides as topoisomerase IV inhibitors with
Gram-positive antibacterial activity. Search for molecu-
lar basis of antibacterial activity of thiosemicarbazides.
Eur J Med Chem;46:5717–5726.
The authors declare that there are no conflicts of interest.
References
14. Tse-Dinh Y.C. (2007) Exploring DNA topoisomerases as
targets of novel therapeutic agents in the treatment of
infectious diseases. Infect Disord Drug Targets;7:3–9.
15. Cunha S., Macedo F.C. Jr, Costa G.A.N., Rodrigues
M.T. Jr, Verde R.B.V., de Souza Neta L.C., Vencato I.,
1. Tenover F.D. (2006) Mechanisms of antimicrobial resis-
tance in bacteria. Am J Med;119:3–10.
2. Barber M. (1961) Methicillin-resistant staphylococci. J
Clin Pathol;14:385–393.
ꢀ
Lariucci C., Sa F.P. (2007) Antimicrobial activity and
3. Klevens R.M., Edwards J.R., Tenover F.C., McDonald
L.C., Horan T., Gaynes R. (2006) Changes in the epi-
demiology of methicillin-resistant Staphylococcus aur-
eus in intensive care units in U.S. hospitals 1992–
2003. Clin Infect Dis;42:389–391.
structural study of disubstituted thiourea derivatives.
Monatsh Chem;138:511–516.
16. Plech T., Wujec M., Siwek A., Kosikowska U., Malm
A. (2011) Synthesis and antimicrobial activity of thiose-
micarbazides, s-triazoles and their Mannich bases
4. Klevens R.M., Morrison M.A., Nadle J., Petit S., Gersh-
man K., Ray S., Harrison L.H. et al. (2007) Invasive
methicillin-resistant Staphylococcus aureus infections in
the United States. JAMA;298:1763–1771.
5. Yong D., Toleman M.A., Giske C.G., Cho H.S., Sund-
man K., Lee K., Walsh T. (2009) Characterization of a
new metallo-b-lactamase gene, blaNDM-1, and a novel
erythromycin esterase gene carried on a unique genetic
structure in Klebsiella pneumoniae sequence type 14
from India. Antimicrob Agents Chemother;53:5046–
5054.
bearing 3-chlorophenyl moiety. Eur
Chem;46:241–248.
17. Kramer B., Rarey M., Lengauer T. (1999) Evaluation of
the FlexX incremental construction algorithm for pro-
tein-ligand docking. Proteins;37:228–241.
J
Med
€
18. Ziebuhr W., Hennig S., Eckart M., Kranzler H., Batzilla
C., Kozitskaya S. (2006) Nosocomial infections by
Staphylococcus epidermidis: how a commensal bacte-
rium turns into
a
pathogen. Int
J
Antimicrob
Agents;28:14–20.
19. Miller J.M., Hair J.G., Hebert M., Hebert L., Roberts
F.J. Jr (1997) Fulminating bacteremia and pneumo-
nia due to Bacillus cereus. J Clin Microbiol;35:504–
507.
6. Rolain J.M., Parola P., Cornaglia G. (2010) New Delhi
metallo-beta-lactamase (NDM-1): towards a new pan-
demia? Clin Microbiol Infect;16:1699–1701.
10
Chem Biol Drug Des 2014

Thiosemicarbazides as Antibacterials
20. Kotiranta A., Lounatmaa K., Haapasalo M. (2000) Epi-
demiology and pathogenesis of Bacillus cereus infec-
tions. Microbes Infect;2:189–198.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
21. Bottone E.J. (2010) Bacillus cereus, a volatile human
pathogen. Clin Microbiol Rev;23:382–398.
ꢀ
22. Siwek A., Swiderek K., Jankowski S. (2012) Prob-
Figure S1. Charge distribution on the surface of the com-
lems with molecular mechanics implementations on
the example of 4-benzoyl-1-(4-methyl-imidazol-5-yl)-
pounds.
carbonylthiosemicarbazide.
849.
J
Mol Model;18:843–
Figure S2. Overlay of structures of some active and inac-
tive compounds.
Notes
aHyperChem 8.0.3. HyperCube Inc., Gainsville, FL, USA,
2007.
bLeadIT 2.1.0, BioSolveIT GmbH, St. Augustin, Germany,
2012.
Chem Biol Drug Des 2014
11