Synthesis, antibacterial and antiproliferative potential of some new 1-pyridinecarbonyl-4-substituted thiosemicarbazide derivatives

Article abstract of DOI:10.1007/s00044-016-1599-6

In this study, the antibacterial, cytotoxic and antiproliferative activities of novel thiosemicarbazide derivatives were assessed. Our results demonstrated that some of the novel compounds possess good antibacterial properties against Staphylococcus epidermidis, Streptococcus mutans and Streptococcussanguinis and are only slightly cytotoxic; thus, they exhibit an excellent therapeutic index, which is higher than that of ethacridine lactate. Moreover, our data showed that compounds 2 and 4 have an antiproliferative activity against human breast adenocarcinoma and human hepatocellular carcinoma cell lines. We expect that the novel thiosemicarbazide derivatives can be used as agents for treatment of dental caries and also for chemotherapy support.

Full text of DOI:10.1007/s00044-016-1599-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1599-6
ORIGINAL RESEARCH
Synthesis, antibacterial and antiproliferative potential of some
new 1-pyridinecarbonyl-4-substituted thiosemicarbazide
derivatives
1
Monika Pitucha1 Maciej Wos Malgorzata Miazga-Karska2 Katarzyna Klimek2
•
•
•
•
´
Barbara Mirosław3 Anna Pachuta-Stec1 Agata Gładysz4 Grazyna Ginalska2
•
•
•
Received: 17 January 2016 / Accepted: 17 May 2016
Ó The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract In this study, the antibacterial, cytotoxic and
antiproliferative activities of novel thiosemicarbazide
derivatives were assessed. Our results demonstrated that
some of the novel compounds possess good antibacterial
properties against Staphylococcus epidermidis, Strepto-
coccus mutans and Streptococcus sanguinis and are only
slightly cytotoxic; thus, they exhibit an excellent thera-
peutic index, which is higher than that of ethacridine lac-
tate. Moreover, our data showed that compounds 2 and 4
have an antiproliferative activity against human breast
adenocarcinoma and human hepatocellular carcinoma cell
lines. We expect that the novel thiosemicarbazide deriva-
tives can be used as agents for treatment of dental caries
and also for chemotherapy support.
Introduction
For many years, new drugs of an interesting structure,
unknown molecular target, low toxicity and a high thera-
peutic index have been looked for. This is due to the
impossibility of treating many serious diseases, such as
bacterial infections or cancer. For a few years, the attention
of researchers has been focused on thiosemicarbazide
derivatives, which were investigated as a pharmacophore
¨
for antimicrobial and anticancer activity (Salgın-Goks¸en
et al., 2007). In vitro screening of some thiosemicarbazides
demonstrated activities against Escherichia coli, Klebsiella
pneumoniae (recultured), methicillin-resistant Staphylo-
coccus aureus, methicillin-sensitive Staphylococcus aureus
and Mycobacterium tuberculosis (Sheikly et al., 2012;
Umadevi et al., 2012; Patel et al., 2014; Tan et al., 2012).
Many of the compounds showed a good antibacterial
activity against K. pneumoniae (Alagarsamy et al., 2010)
and S. aureus in comparison with the standard drug—
ciprofloxacin (Rane et al., 2014). Additionally, thiosemi-
carbazides are one of the most promising biologically
active compounds which can be used in cancer treatment
(Arora et al., 2014; Mohsen et al., 1981). These derivatives
have been effectively used against a number of carcinoma
Keywords Thiosemicarbazide Á Antibacterial activity Á
Cytotoxicity Á Cell proliferation
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1599-6) contains supplementary
material, which is available to authorized users.
& Monika Pitucha
monika.pitucha@umlub.pl
´
cell lines (Perkovic et al., 2012; Bhata et al., 2008; Malki
et al., 2014; Zhang et al., 2011). It has been found that
thiosemicarbazide derivatives demonstrated cytotoxic and
antiproliferative activity against HeLa, HepG2, MDA-MB-
231 and HT-29 cell lines (Mavrova et al., 2014).
1
Department of Organic Chemistry, Medical University,
Chodzki 4A, 20-093 Lublin, Poland
2
Chair and Department of Biochemistry and Biotechnology,
Medical University, Chodzki 1, 20-093 Lublin, Poland
In this study, we synthesized new thiosemicarbazide
derivatives and investigated their antibacterial, cytotoxic
and antiproliferative properties. We expected the presence
of the pyridine ring to significantly affect the biological
activity of the tested derivatives. Additionally, because the
literature lacks information about the effectiveness of
3
Department of Crystallography, Faculty of Chemistry, Maria
Curie-Sklodowska University, pl. M. Curie-Skłodowskiej 3,
20-031 Lublin, Poland
4
Department of Medicinal Chemistry, Medical University of
Lublin, Jaczewskiego 4, 20-090 Lublin, Poland
123

Med Chem Res
thiosemicarbazide derivatives against oral bacteria, we
decided to perform in vitro tests against Streptococcus
mutans and Streptococcus sanguinis. It is extremely
important because bacterial infections co-occurring with
dental caries may be the cause of chronic diseases such as
endocarditis, myocardial infarction (Cognasse et al., 2014;
Kerrigan et al., 2002) and cancer, for example, pancreatic
and gastrointestinal cancer (Meurman, 2010). It is worth
highlighting that cancer patients undergoing chemotherapy
often suffer from oral complications.
Results and discussion
Chemistry
The 1-pyridinecarbonyl-4-substituted thiosemicarbazide
derivatives (1–10) were prepared by the reactions of 2-, 3- or
4-pyridine carboxylic acid hydrazide with isothiocyanates.
The reaction was carried out in methanol as solvent and was
refluxed for 30 min. The synthesis of all compounds was
accomplished using the reaction illustrated in Scheme 1.
O
C
O
C
O
C
H
N
H
N
NH2
i
N
H
ii
OCH2CH3
N
H
R
S
N
N
N
1-10
R
F
2-pyridine
2-pyridine
1
Cl
2
2-pyridine
2-pyridine
3
4
N
O
Cl
Cl
2-pyridine
3-pyridine
3-pyridine
5
6
7
SCH3
SCH3
Cl
Cl
4-pyridine
4-pyridine
8
9
SCH3
F
Cl
4-pyridine
10
Cl
Scheme 1 Synthesis 1-pyridinecarbonyl-4-substituted thiosemicarbazide derivatives. Reagents and conditions: (i) NH2NH2ÁH2O, C2H5OH,
reflux; (ii) RNCS, CH3OH, reflux
123

Med Chem Res
Compounds 9 and 10 were obtained earlier (Byung
et al., 2004; Goldfarb, 2009). According to the Chemical
Abstracts Service (SciFinder), some of the compounds
(1–4 and 7) have the CAS number, but there is no method
synthesis and references.
The structures of obtained compounds were confirmed
by spectral analysis (1H, 13C NMR, IR and MS). For
compound 2 was performed X-ray diffraction analysis.
Figure 1 shows that this compound crystallizes in the
triclinic P-1 space group. The molecule of compound 2 has
an extended but not planar conformation with the dihedral
angle of 47° between the mean planes of aryl rings.
Fig. 1 Molecular structure with atom numbering scheme for com-
pound 2
In this compound, molecules interact through two N–H…O
˚
hydrogen bonds (dN1…O1 = 2.860(3) A, \N1–H1…O1
=
˚
153(4)° and dN2…O1 = 2.800(4) A, \N2–H2…O1 = 141(4)°)
forming dimers. Between the chlorophenyl rings, there is a
p…p interaction with a distance between the ring centroids
˚
of 3.728(4) A. The molecules stack in columns along the
a axis (Fig. 2).
Antibacterial activity evaluation
All synthesized compounds were initially screened for their
potential in vitro antibacterial activity using the agar
dilution technique. It was found that seven out of ten
thiosemicarbazide derivatives (1, 2, 4–7, 10) effectively
inhibited some of the tested strains (Table 1).
Two thiosemicarbazides (1, 4) showed potential activity
against all tested aerobic Gram-positive, aerobic Gram-
negative and microaerobic Gram-positive bacterial strains.
Additionally, zones of bacterial growth inhibition of some
Fig. 2 Crystal packing in compound 2
Table 1 Zones of bacterial growth inhibition (mm) produced by 100 lg for the more active compounds and standards
Compound
Zone of bacterial growth inhibition (mm)
S.a
S.e
E.c
P.a
S.m
S.s
L.a
L.spp.
1
18
17
0
26
25
7
22
20
0
12
4
10
24
0
10
0
12
16
0
24
24
0
2
3
0
0
4
22
10
10
0
27
10
9
20
11
9
14
0
35
0
25
15
19
10
0
20
19
0
24
11
0
5
6
0
11
24
0
7
10
0
9
0
5
7
8
0
0
0
0
0
9
0
0
0
0
0
0
0
0
10
16
38
23
10
13
36
23
8
7
0
28
32
26
24
20
29
29
24
0
0
Cefepime
35
15
12
29
16
4
31
24
15
29
24
10
CLX
Ethacridine lactate
S.a—Staphylococcus aureus ATCC 25923, S.e—Staphylococcus epidermidis ATCC 12228, E.c—Escherichia coli ATCC 25922, P.a—Pseu-
domonas aeruginosa ATCC 9027, S.m—Streptococcus mutans PCM 2502, S.s—Streptococcus sanguinis PCM 2335, L.a—Lactobacillus aci-
dophilus PCM 210, L.spp.—Lactobacillus spp.
123

Med Chem Res
Table 2 Minimum inhibitory concentration (MIC [lg/mL]) of the tested compounds against bacterial strains
Compound
S. aureus
S. epidermidis
E. coli
P. aeruginosa
S. mutans
S. sanguinis
L. acidophilus
1
31.25
31.25
NA
15.6
31.25
NA
62.5
62.5
NA
500
250
500
125
2
1000
NA
250
250
62.5
NA
3
NA
NA
4
31.25
250
31.25
125
62.5
125
500
31.25
31.25
250
7.81
31.25
15.63
1000
NA
31.25
500
5
[1000
[1000
[1000
NA
6
250
500
62.5
[1000
NA
[1000
[1000
NA
7
[1000
NA
500
500
8
NA
NA
9
NA
NA
NA
NA
NA
NA
NA
10
125
250
1000
0.015
0.488
15.63
1000
0.488
15.6
250
1000
0.122
7.81
31.25
1000
3.9
Cefepime
0.976
0.488
31.25
0.976
0.488
31.25
0.122
0.488
62.5
CLX
0.976
31.25
Ethacridine lactate
125
NA means inactive
The thiosemicarbazide derivatives (1, 2, 4, 5) showed
significant activity (MIC = 7.81–500 lg/mL) against the
tested pathogenic microaerobic bacteria (causing dental
caries). The strongest antibacterial properties were exhib-
ited by compound 4, whose MIC was 7.81 lg/mL against
S. sanguinis and 31.25 lg/mL against S. mutans, Lacto-
bacillus acidophilus and Lactobacillus spp. Substance 6
also showed significant activity against the pathogenic oral
bacteria S. sanguinis, S. mutans but simultaneously did not
limit the growth of the probiotic dental flora: L. aci-
dophilus, Lactobacillus spp. These data suggest the possi-
bility of using compounds especially 6 and 4 as well as 1
and 2 in the treatment of caries (Table 2). It is worth noting
that among the tested pyridine derivatives, compounds 4
and 5 showed greater or equal activity against S. mutans
and S. sanguinis, and compound 6 against S. sanguinis,
than the commonly used antiseptic ethacridine lactate.
Additionally, the activity of compound 4 against S. san-
guinis was equal to that of chlorhexidine (CLX)
(MIC = 7.81 lg/mL). CLX is an antiseptic drug used in
the prophylaxis and treatment of dental caries (Autio-Gold
2008). However, the in vitro activity of the newly syn-
thesized compounds against other tested bacterial strains
was found to be lower compared to the controls (Cefepime,
CLX and ethacridine lactate).
Fig. 3 In vitro cytotoxic activity (CC50) of the compounds against
normal human skin fibroblasts. The data were expressed as mean
values SD from three independent experiments. CLX Chlorhexi-
dine, EL Ethacridine lactate
compounds were higher compared with CLX and ethacri-
dine lactate. Only cefepime was characterized by large
zones of inhibition (29–38 mm) in comparison with
thiosemicarbazide derivatives. The detailed in vitro
antibacterial activity of the potentially active compounds
was later determined using the broth microdilution method
on the basis of minimal inhibitory concentration (MIC). Six
of the compounds (especially 1, 2, 4, but also 5, 6, 10) had
a potential activity against aerobic Gram-positive bacteria
(MIC = 15.6–500 lg/mL). The antimicrobial activity of
derivatives 1, 2 and 4 against these bacteria was greater or
similar to the activity of the control ethacridine lactate. The
same compounds (1, 2, 4, 5 and 6) were also found to
effectively inhibit the growth of Gram-negative E. coli at a
concentration between 62 and 125 lg/mL. The growth of
Pseudomonas aeruginosa was moderately inhibited only
by compounds 1 and 4 (MIC = 500 lg/mL for both)
(Table 2).
Cytotoxic activity evaluation
The synthesized compounds and the reference antibacterial
agents were evaluated for the in vitro cytotoxic activity
against the BJ cell line (normal human skin fibroblasts)
using the MTT assay. The calculated response parameter
was CC50, which corresponds to the concentration required
123

Med Chem Res
Table 3 In vitro therapeutic potential of the compounds
Compound
TI
Bacterial species
S.a.
S.e.
E.c.
S.m.
S.s.
L.a.
L.spp.
P.a.
1
6.82
0.72
ND
13.70
0.72
ND
3.41
0.36
ND
0.85
0.09
ND
0.43
0.09
ND
1.70
0.36
ND
6.83
0.72
ND
0.62
0.09
ND
ND
ND
ND
ND
34.6
ND
0.43
ND
ND
0.43
ND
ND
ND
ND
ND
ND
0.54
ND
2
3
4
0.62
0.19
3.67
ND
0.62
0.38
1.83
0.18
ND
0.31
0.38
14.68
0.08
ND
0.62
1.53
3.67
0.18
ND
2.50
1.53
58.70
0.08
ND
0.62
0.10
ND
5
6
7
ND
8
ND
ND
9
ND
ND
ND
ND
ND
ND
10
2.25
17.33
0.22
1.12
17.33
0.44
ND
1.12
17.33
0.11
0.56
1.08
0.44
0.28
8.66
0.22
Chlorhexidine (CLX)
Ethacridine lactate
17.33
0.44
TI (therapeutic index): the ratio between CC50 and MIC values, ND means not determined, due to lack of CC50 or MIC values, S.a—
Staphylococcus aureus ATCC 25923, S.e—Staphylococcus epidermidis ATCC 12228, E.c—Escherichia coli ATCC 25922, P.a—Pseudomonas
aeruginosa ATCC 9027, S.m—Streptococcus mutans PCM 2502, S.s—Streptococcus sanguinis PCM 2335, L.a—Lactobacillus acidophilus
PCM 210, L.spp.—Lactobacillus spp.
for a 50 % reduction of cell viability. The in vitro cytotoxic
activity of the synthesized compounds and the reference
antibacterial agents is summarized in Fig. 3.
claim that the profile of in vitro cytotoxicity of antibacterial
agents may be characterized by the CC50/MIC ratio
(Kashyap et al., 2012; Panchal et al., 2009; Zoraghi et al.,
2011). For this reason, in the present study, we attempted to
evaluate the in vitro therapeutic potential of novel
thiosemicarbazide derivatives and compared them to the
reference antibacterial agents (Table 3).
In the cytotoxic study, the novel thiosemicarbazide
derivatives showed significant differences in cytotoxicity.
The CC50 values of the synthesized compounds ranged
from 19.5 to 917.4 lg/mL. Among all tested compounds,
4-(2,4-dichlorophenyl)-1-(pyridin-2-yl)carbonylthiosemi-
carbazide (4) exhibited the highest cytotoxic activity with a
CC50 value of 19.5 lg/mL. Nevertheless, compound 4
showed lower cytotoxicity than the two reference
antibacterial agents (CLX and ethacridine lactate), whose
CC50 values were 8.46 lg/mL and 6.88 lg/mL, respec-
tively. It is worth noting that 4-(4-methylthiophenyl)-1-
(pyridin-3-yl)carbonylthiosemicarbazide (6) only slightly
decreased the BJ cell viability with a CC50 value of
917.4 lg/mL. This result indicated that compound 6
showed the lowest cytotoxic activity in comparison with
both the tested thiosemicarbazide derivatives and the ref-
erence antibacterial agents. Most interestingly, 4-(2,4-
dichlorophenyl)-1-(pyridin-3-yl)carbonylthiosemicar-
bazide (7 with a CC50 of 88.3 lg/mL) significantly
decreased cell viability compared to compound 6. It was
not possible to calculate the CC50 value for cefepime as it
did not reduce cell viability by 50 % at the highest tested
concentration of 1500 lg/mL (data not shown). It is widely
known that all drugs applicable in the treatment of bacterial
infections should exhibit high antibacterial efficiency and
low toxicity toward human cells. Thus, many researchers
The TI values below 1 obtained by the tested substances
correspond to the lack of therapeutic safety. Among the
synthesized compounds, derivatives 1, 6 and 10 showed the
highest values of therapeutic index. Compound 6 exhibited
the in vitro therapeutic potential against S. aureus, S. epi-
dermidis, E. coli and, which is important, against S. mutans
and S. sanguinis, with the TI values of 3.67, 1.83, 14.68,
3.67 and 58.7, respectively. The essential observation is
that the in vitro therapeutic indices of compound 6 were
approximately 4–133 times higher than the in vitro TI
values of ethacridine lactate and 58 times higher than the
TI value obtained by CLX against S. mutans. Compound 1
also showed high in vitro TI values (6.82, 13.7, 3.41, 6.83
against S. aureus, S. epidermidis, E. coli and L. species,
respectively). Additionally, the TI values of compound 10
against S. aureus, S. epidermidis and S. mutans were
greater than those of compound 1. Nevertheless, among all
the tested agents, CLX exhibited the highest in vitro TI
values. It should be noted that antibacterial agents which
possess a value of therapeutic index higher than 10 can be
administered to perform in vivo evaluation (Kashyap et al.,
2012; Ghareb et al., 2012). Our two newly synthesized
123

Med Chem Res
bFig. 4 Antiproliferative activity of the synthesized compounds
against normal human skin fibroblasts (a), human hepatocellular
carcinoma (b) and human breast adenocarcinoma (c). The results
were expressed as mean values SEM from three independent
experiments. The IC50 values were presented as mean values SD.*
Statistical significance obtained at p \ 0.01 compared to the control
A
BJ
120
2
4
*
*
*
100
80
60
40
20
0
*
*
*
compounds (1 and 6) had antibacterial activity and exhib-
ited excellent TI values higher than 10 against some bac-
terial strains.
*
*
*
*
*
IC50=3.06 0.46
IC50=1.86 0.44
*
*
*
*
*
*
Antiproliferative activity evaluation
0
1
2
3
4
5
The synthesized compounds were also evaluated for
in vitro antiproliferative activity against various cell lines,
i.e., BJ (normal human skin fibroblasts), HepG2 (human
hepatocellular carcinoma) and MCF-7 (human breast ade-
nocarcinoma). In order to evaluate cell proliferation, the
cells were treated with compounds at concentrations of
0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100 and 200 lg/mL for 96 h.
Among the investigated compounds, only two (2 and 4)
exhibited antiproliferative activity. Both compounds
strongly decreased the BJ, HepG2 and MCF-7 cell prolif-
eration in a concentration-dependent manner (Fig. 4).
The results were expressed as mean values SEM from
three independent experiments. The IC50 values were pre-
sented as mean values SD. Statistical significance was
obtained at p \ 0.01 compared to the control. In the case of
normal human cell line (BJ), compound 4 decreased cell
proliferation more potently than compound 2. The statis-
tical significance for compound 4 against BJ cells was even
obtained at 0.1 lg/mL (Fig. 4a).
Log [Concentration] x 10-2, [µg/mL]
HepG2
B
120
100
80
60
40
20
0
2
4
*
*
*
*
*
*
*
*
*
*
IC50=2.71 0.23
IC50=2.09 0.11
*
*
0
1
2
3
4
5
Log [Concentration] x 10-2, [µg/mL]
Nevertheless, it should be noted that compound 4 sup-
pressed the HepG2 and MCF-7 cell proliferation more
effectively than compound 2, with IC50 of 2.09 lg/mL
(6.12 lM) and 8.63 lg/mL (25.3 lM), respectively
(Fig. 4b, c).
MCF-7
C
120
2
4
Therefore, the IC50 value of compound 4 against the
MCF-7 cell line was approximately two times lower than
the IC50 value of compound 2. On the other hand, com-
pound 2 suppressed cell division of hepatocellular carci-
noma slightly more potently than that of normal skin
fibroblasts. The most pronounced effect was observed with
10 lg/mL of compound 2, which reduced cell proliferation
to 32 % (BJ) and to 18.9 % (HepG2) compared to the
control (Fig. 4a, b). Thus, these data showed that com-
pound 2 at 10 lg/mL is more effective against tumor than
normal cells. According to the available literature data, our
compounds exhibited a very high antiproliferative poten-
tial. The diarylthiosemicarbazide derivatives containing
*
100
80
60
40
20
0
*
*
*
*
*
*
*
*
*
IC50=14.80 1.03
IC50=8.63 1.75
*
*
*
*
0
1
2
3
4
5
Log [Concentration] x 10-2, [µg/mL]
123

Med Chem Res
Table 4 Molecular properties of the thiosemicarbazides (1–10)
No
Ar
R
M.W. [amu]
logP
miLogP
n
ON
n
OHNH
PSA
[A ]
TPSA
2
˚
1
2
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-2-yl
Pyridin-3-yl
Pyridin-3-yl
Pyridin-4-yl
Pyridin-4-yl
Pyridin-4-yl
2-FC6H4
290.32
306.77
385.48
341.22
317.41
317.41
341.22
317.41
290.32
341.22
1.46
1.087
1.601
-0.213
2.255
1.405
1.339
2.189
1.289
0.968
2.137
5
5
7
5
5
5
5
5
5
5
3
3
3
3
3
3
3
3
3
3
93.5
92.5
66.044
66.044
78.516
66.044
66.044
66.044
66.044
66.044
66.044
66.044
2-ClC6H4
1.89
-1.43
2.52
2.04
1.56
2.05
1.56
0.98
2.04
3
CH2CH2morph.
2,4-Cl2C6H3
4-CH3SC6H4
4-CH3SC6H4
2,4-Cl2C6H3
4-CH3SC6H4
2-FC6H4
111.0
94.1
4
5
117.7
117.0
93.8
6
7
8
117.1
93.1
9
10
2,4-Cl2C6H3
93.5
urea group and pyridine group at the para position, which
also occur in the structure of our compounds, exhibited
various antiproliferative activities against alveolar epithe-
lial, lung and colorectal cell lines. After a 72-h incubation,
the IC50 values of these compounds ranged from 1.8 to
82.4 lmol (Xin et al., 2013). Moreover, Ghareb and col-
leagues reported that the thiosemicarbazide and semicar-
bazide derivatives of benzimidazole hydrazides with
hydrazine hydrate afforded N3-substituted-5-((2-phenyl-
1H-benzo[d]imidazol-1-yl)methyl)-4H-1,2,4-triazole-3,4-
diamines that have antiproliferative potential against the
MCF-7 cell line. Two of them after a 48-h exposure
inhibited cell proliferation with an IC50 of 13.7 and
16.2 lg/mL (Ghareb et al., 2012). Hence, our results
indicated that the synthesized compounds 2 and 4 have a
good antiproliferative potential against some tumor cells
and may be promising candidates for further anticancer
study.
bond donors (-NH and -OH) and fewer than ten hydrogen
bond acceptors (N, O). This is very important information
because a decreased number of donors are known to reduce
the affinity of P-glycoprotein, and the more the acceptors,
the more water molecules are connected. In addition, the
amount of donors and acceptors of hydrogen bonds affect
the magnitude of the compound’s polar surface area (PSA),
which is defined as the sum of surfaces of polar atoms
(usually of oxygen, nitrogen and attached hydrogen atoms)
in a molecule. This is a useful parameter for the prediction
of molecular transport properties, particularly in intestinal
absorption and blood–brain barrier penetration (Fernandes
and Gattass, 2009). Referring to our findings (Table 4), the
PSA values of the tested thiosemicarbazide derivatives
2
2
˚
ranged from 92[A ] to 118[A ]. Compounds 2 and 4 have
˚
demonstrated the highest antibacterial and antiproliferative
2
activities, and their PSA values were 92.5[A ] and
˚
2
94.1[A ], respectively.
˚
The obtained values of topological polar surface area
(TPSA) confirmed this relationship (Table 4). It seems that
this may be an important parameter for searching for a
relation between structure and activity for this group of
compounds.
Structure–activity analysis
An important feature of a potential drug is its bioavail-
ability which determines how an investigated compound
can penetrate a biological membrane. Thus, the physio-
chemical analysis of a molecule known as Lipinski’s rule
of five is used (Lipinski et al., 1997). For this purpose, all
thiosemicarbazides were analyzed in silico estimating their
bioavailability via calculating such parameters as molecu-
lar weight (MW), partition coefficient (logP), the number
of donors and acceptors of hydrogen bonds and the polar
surface area (PSA). The obtained data showed that all
compounds meet the criteria of Lipinski’s rule (Lipinski
et al., 1997). The molecular weight of the tested derivatives
ranged from 290 to 385 Da (\500 Da), and the log p val-
ues ranged from -1.43 to 2.52 (\5), respectively. All the
researched compounds have no more than five hydrogen
Conclusions
In this study, we reported the synthesis and antibacterial
activity of new compounds with pyridinecarbonyl group
connected to the thiosemicarbazide system. It should be
noted that two thiosemicarbazide derivatives, i.e., 2 and 4,
exhibited good or moderate inhibition of all the most
common caries-associated Gram-positive and Gram-nega-
tive bacterial strains. Moreover, these compounds strongly
suppressed human hepatocellular carcinoma and human
breast adenocarcinoma cell proliferation. The structure–
123

Med Chem Res
(DMSO-d6, 300 MHz) d: 7.16–7.66 (m, 4H, CHphenyl),
8.01–8.69 (m, 4H, CHpyridine), 9.52 (s, 1H, –NH
exchangeable with D2O), 9.88 (s, 1H, NH exchangeable
activity relationship of the compounds showed that sub-
stitution at the position 2 of the pyridine ring enhances
biological activity. The prominent antibacterial and
antiproliferative effect of compounds 2 and 4 may be due
to changing the number of chlorine atoms in the phenyl
ring. Thus, it is worth underlying that 4-(2-chloro/2,4-
dichlorophenyl)-1-(pyridine-2yl)carbonylthiosemicarbazide
derivatives will be auspicious as potential agents for caries
treatment and caries-associated cancer diseases. The
physicochemical analysis indicates that the polar surface
area is an important parameter for biological activity of the
investigated compounds. Our results will have an impact
on further investigation in this field in search of
thiosemicarbazide compounds as antibacterial and
antiproliferative agents.
with D2O), 10.81 (s, 1H, NH exchangeable with D2O). 13
C
NMR (DMSO-d6, 75 MHz) d: 116.01, 116.14, 122.98,
124.30, 127.41, 128.34, 130.87, 138.12, 148.95, 149.92,
164.25, 182.48. FT-IR m: 3312, 1658, 1351 cm-1. MS (Cl)
m/z = 291 [M?]. Anal.: Calcd. for C13H11N4OSF (290.31):
C (53.78), H (3.82), N (19.29). Found: C (53.81), H (3.87),
N (19.19) (CAS: 891086-61-6).
4-(2-Chlorophenyl)-1-(pyridin-2-yl)carbonylthiosemicar-
bazide (2) Yield 83 %, m.p. 172–174 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 7.25–7.66 (m, 4H, CHphenyl),
8.02–8.71 (m, 4H, CHpyridine), 9.54 (s, 1H, –NH
exchangeable with D2O); 9.88 (s, 1H, NH exchangeable
with D2O); 10.82 (s, 1H, NH exchangeable with D2O). 13
C
NMR (DMSO-d6, 75 MHz) d: 122.99, 127.47, 128.18,
129.74, 131.00, 138.17, 149.01, 149.87, 164.27, 182.17.
FT-IR m: 3246, 1655, 1354 cm-1. MS (CI) m/z: 307 (M?).
Anal.: Calcd. for C13H11N4OSCl (306.77): C (50.89), H
(3.61), N (18.29). Found: C (50.91), H (3.64), N (18.33)
(CAS: 894234-77-6).
Experimental
Chemicals and instruments
The chemicals used for synthesis and analysis were purchased
from Merck Co. or Alfa Aesar and used without further
purification. Melting points were determined on a Fisher-
4-(2-Morpholinoethyl)-1-(pyridin-2-yl)carbonylthiosemi-
1
carbazide (3) Yield 90 %, m.p. 196–198 °C. H NMR
1
Johns block and presented without any corrections. The H
and 13C NMR spectra were recorded on a Bruker Avance
300 MHz spectrometer in solution noted and with TMS as an
internal standard. The IR spectra were recorded on a Thermo
(DMSO-d6, 300 MHz) d: 2.35–2.59 (m, 4H, 2xCH2 mor-
pholine), 3.45–3.52 (4H, 2xCH2 morpholine), 3.57–3.58
(m, 2H, –NH–CH2–CH2–), 3.74–3.76 (m, 2H, –NH–CH2–
CH2–), 7.64–8.69 (m, 4H, CHpyridine), 8.69 (s, 1H, NH
exchangeable with D2O), 9.43 (s, 1H, NH exchangeable
Nicolet 6700 ATR device in the range of 500–3500 cm-1
.
The elementary analysis was performed with the application
of Perkin-Elmer analyzer (940 Winter St., Waltham, MA,
USA). The obtained results were within 0.4 % of the the-
oretical value. Follow-up of the reactions and the purity of the
newly obtained compounds were checked using TLC on
aluminum oxide 60 F254 plates (Merck) in a CHCl3/C2H5OH
(10:1 and 10:2) solvent system with UV visualization. The
carboxylic acid hydrazides were synthesized via the reaction
of the appropriate carboxylic acid ester with 98 % hydrazine
hydrate in the solution of anhydrous ethanol using the method
described earlier (Idhayadhulla et al., 2013; Priebe et al.,
2008; Zamani, et al., 2002).
with D2O), 10.63 (s, 1H, NH exchangeable with D2O). 13
C
NMR (DMSO-d6, 75 MHz) d: 53.22, 53.59, 53.82, 56.50,
56.89, 57.27, 66.44, 66.55, 66.68, 122.94, 127.50, 138.23,
149.03, 149.66, 181.70. FT-IR m: 3292, 1627, 1338 cm-1
.
MS (CI) m/z: 308 (M?). Anal.: Calcd. for C13H19N5O2S
(309.38): C (50.46), H (6.18), N (22.63). Found: C (50.51),
H (6.22), N (22.57) (CAS: 455314-30-4).
4-(2,4-Dichlorophenyl)-1-(pyridin-2-yl)carbonylthiosemi-
carbazide (4) Yield 91 %, m.p. 158–160 °C. 1H (DMSO-
d6, 300 MHz) d: 7.42–7.66 (m, 3H, CHphenyl), 8.02–8.69
(m, 4H, CHpyridine), 9.57 (s, 1H, NH exchangeable with
D2O), 9.96 (s, 1H, NH exchangeable with D2O), 10.83 (s,
1H, NH exchangeable with D2O). 13C NMR (DMSO-d6,
75 MHz) d: 123.06, 127.46, 127.68, 129.20, 131.72,
132.52, 136.70, 138.15, 148.97, 164.27, 182.35. FT-IR m:
3242, 3108, 1652, 1346 cm-1. MS (CI) m/z: 342 (M?).
Anal.: Calcd. for C13H10N4OSCl2 (341.21): C (45.75), H
(2.95), N (16.41). Found: C (45.80), H (2.97), N (16.37)
(CAS: 891538-65-4).
General procedure for the synthesis of 1-
pyridinecarbonyl-4-substituted thiosemicarbazide
derivatives (1–10)
A mixture of 2-, 3- or 4-pyridinecarboxylic acid hydrazide
(0.01 mol), isothiocyanate (0.01 mol) and methanol
(15 mL) was heated in a water bath reflux temperature for
0.5 h. The product was filtered, dried and crystallized from
mixture methanol–acetonitrile (1:1).
4-(4-Methylthiophenyl)-1-(pyridin-2-yl)carbonylthiosemi-
1
carbazide (5) Yield 88 %, m.p. 184–186 °C. H NMR
(DMSO-d6, 300 MHz) d: 2.28 (s, 3H, CH3), 7.01–7.25 (m,
4-(2-Fluorophenyl)-1-(pyridin-2-yl)carbonylthiosemicar-
bazide (1) Yield 87 %, m.p. 182–184 °C. 1H NMR
123

Med Chem Res
4H, CHphenyl), 7.51–8.25 (m, 4H, CHpyridine), 8.66 (1 s, 1H,
NH exchangeable with D2O), 10.53 (s, 1H, NH
exchangeable with D2O), 12.01 (s, 1H, NH exchangeable
with D2O). 13C NMR (DMSO-d6, 75 MHz) d: 14.79,
124.50, 125.47, 125.89, 129.26, 132.24, 137.81, 139.69,
145.62, 149.68, 150.00, 169.69. FT-IR m: 3236, 3111,
1654, 1324 cm-1. MS (CI) m/z: 318 (M?). Anal.: Calcd.
for C14H14N4OS2 (318.41): C (52.80), H (4.43), N (17.59).
Found: C (52.71), H (4.38), N (17.51).
165.02, 182.69. FT-IR m: 3265, 3113, 1677, 1368 cm-1
.
MS (CI) m/z: 291 (M?). Anal.: Calcd. for C13H11N4OSF
(290.31): C (53.78), H (3.82), N (19.29). Found: C (53.65),
H (3.74), N (19.42) (Byung et al., 2004).
4-(2,4-Dichlorophenyl)-1-(pyridin-4-yl)carbonylthiosemi-
1
carbazide (10) Yield 84 %, m.p. 164–166 °C. H NMR
(DMSO-d6, 300 MHz) d: 7.37–7.45 (m, 3H, CHphenyl),
7.68–8.78 (m, 4H, CHpyridine), 9.76 (s, 1H, NH exchange-
able with D2O), 10.05 (s, 1H, NH exchangeable with D2O),
10.95 (s, 1H, NH exchangeable with D2O). 13C NMR
(DMSO-d6, 75 MHz) d: 122.24, 127.81, 129.30, 132.12,
132.92, 133.27, 136.55, 150.65, 165.09, 182.53. FT-IR m:
3309, 3117, 1677, 1380 cm-1. MS (CI) m/z (%): 341 (M?).
Anal.: Calcd. for C13H10N4OSCl2 (341.21): C (45.75), H
(2.95), N (16.41). Found: C (45.69), H (2.90), N (17.01)
(Goldfarb, 2009).
4-(4-Methylthiophenyl)-1-(pyridin-3-yl)carbonylthiosemi-
1
carbazide (6) Yield 89 %, m.p. 176–177 °C. H NMR
(DMSO-d6, 300 MHz) d: 2.47 (s, 3H, CH3), 7.23–7.57 (m,
4H, CHphenyl), 8.27–8.76 (m, 4H, CHpyridine), 9.11 (s, 1H,
NH exchangeable with D2O), 9.81 (s, 1H, NH exchange-
able with D2O), 10.76 (s, 1H, NH exchangeable with D2O).
13C NMR (DMSO-d6, 75 MHz) d: 15.58, 123.90, 126.24,
127.14, 128.72, 134.96, 136.05, 136.83, 149.40, 152.84,
165.14, 181.53. FT-IR m: 3284, 1632, 1341 cm-1. MS (CI)
m/z (%): 319 (M?). Anal.: Calcd. for C14H14N4OS2
(318.41): C (52.80), H (4.43), N (17.59). Found: C (52.72),
H (4.49), N (17.64).
X-ray analysis
The X-ray diffraction intensities were collected at 100 K
on an Oxford Diffraction Xcalibur CCD diffractometer
with graphite-monochromatized MoKa radiation (k =
4-(2,4-Dichlorophenyl)-1-(pyridin-3-yl)carbonylthiosemi-
carbazide (7) Yield 92 %, m.p. 196–197 °C. 1H (DMSO-
d6, 300 MHz) d: 7.37–7.68 (m, 3H, CHphenyl), 8.27–9.11
(m, 4H, CHpyridine), 9.75 (s, 1H, NH exchangeable with
D2O), 10.02 (s, 1H, NH exchangeable with D2O), 10.86 (s,
1H, NH exchangeable with D2O). 13C NMR (DMSO-d6,
75 MHz) d: 123.96, 129.36, 135.14, 148.55, 152.23,
164.80. FT-IR m: 3331, 3150, 1700, 1359 cm-1. MS (CI)
m/z: 342 (M?). Anal.: Calcd. for C13H10N4OSCl2 (341.21):
C (45.75), H (2.95), N (16.41). Found: C (45.98), H (2.91),
N (16.52) (CAS: 475180-05-3).
˚
0.71073 A) using the x scan technique, with an angular
scan width of 1.0°. The programs CrysAlis CCD and
CrysAlis Red (Oxford Diffraction, Xcalibur CCD System,
CRYSALIS Software System, Version 1.171, Oxford
Diffraction Ltd. 2009) were used for data collection, cell
refinement and data reduction. Absorption corrections were
applied using the multi-scan method by Blessing (Bless-
ing, 1995). The structures were solved via direct methods
using SHELXS-97 and refined by the full-matrix least-
squares on F2 using the SHELXL-97 (Sheldrick, 2008).
Non-hydrogen atoms were refined with anisotropic dis-
placement parameters. The N-bonded H atoms were found
in the difference Fourier maps and then remained fixed
during the least-squares refinements. All the remaining H
atoms were positioned geometrically and allowed to ride
on their parent atoms, with Uiso(H) = 1.2 Ueq(C). The
molecular plots were drawn with Olex2 (Dolomanov
et al., 2009).
4-(4-Methylthiophenyl)-1-(pyridin-4-yl)carbonylthiosemi-
1
carbazide (8) Yield 86 %, m.p. 197–198 °C. H NMR
(DMSO-d6, 300 MHz) d: 2.47 (s, 3H, CH3), 7.23–7.86 (m,
4H, CHphenyl), 8.77–8.78 (m, 4H, CHpyridine), 9.83 (s, 2H,
NH exchangeable with D2O), 10.86 (s, 1H, NH
exchangeable with D2O). 13C NMR (DMSO-d6, 75 MHz)
d: 15.57, 122.15, 126.26, 127.04, 134.97, 136.79, 140.07,
150.67, 164.93, 181.45. FT-IR m: 3097, 2936, 1667,
1378 cm-1. MS (CI) m/z (%): 319 (M?). Anal.: Calcd. for
C14H14N4OS2 (318.41): C (52.80), H (4.43), N (17.59).
Found: C (52.96), H (4.51), N (52.68).
Antibacterial activity
Panel reference strains of bacteria from the American Type
Culture Collection or Polish Collection of Microorganisms,
including aerobic Gram-positive bacteria: Staphylococcus
aureus ATCC 25923 and Staphylococcus epidermidis
ATCC 12228, and aerobic Gram-negative bacteria:
Escherichia coli ATCC 25922 and Pseudomonas aerugi-
nosa ATCC 9027, as well as microaerobic Gram-positive
bacteria: Lactobacillus spp., Lactobacillus acidophilus
PCM 2105, Streptococcus mutans PCM 2502 and
4-(2-Fluorophenyl)-1-(pyridin-4-yl)carbonylthiosemicar-
bazide (9) Yield 78 %, m.p. 202–204 °C. 1H NMR
(DMSO-d6, 300 MHz) d: 7.18–7.31 (m, 4H, CHphenyl),
7.86–8.78 (m, 4H, CHpyridine), 9.70 (s, 1H, NH exchange-
able with D2O), 9.99 (s, 1H, exchangeable with D2O),
10.94 (s, 1H, NH exchangeable with D2O). 13C NMR
(DMSO-d6, 75 MHz) d: 116.14, 116.27, 122.21, 124.44,
127.51, 128.74, 131.17, 140.00, 150.65, 157.08, 158.70,
123

Med Chem Res
Streptococcus sanguinis PCM 2335, were used. Microbial
suspensions with an optical density of 0.5 McFarland
standard at 1.5 9 108 CFU/mL (CFU: colony forming
unit) were prepared in sterile 0.9 % NaCl. Mueller–Hinton
(M–H) broth and M–H agar (Oxoid Ltd., England) for
aerobic strains, and MRS Broth Lactobacillus, MRS Agar
Lactobacillus (BioMaxima S.A., Poland), BHI Broth and
BHI agar (BioMaxima S.A., Poland) for microaerobic
strains were used in the microbial tests. All stock solutions
of the newly synthesized compounds were prepared in
DMSO (the final DMSO concentration used in bacterial
tests did not inhibit microbial growth and was less than
1.5 %). The antibacterial activity of the newly synthesized
compounds was compared with the controls: cefepime
dihydrochloride (Maxipime, Bristol-Myers Squibb Latina),
chlorhexidine digluconate ((CLX) Amara Poland) and
ethacridine lactate (Rivanolum, PharmaSwiss, Czech
Republic).
incubation, the panel was digitally analyzed at 600 nm
using the microplate reader Bio Tech Synergy (USA) with
a dedicated software system. The growth intensity in each
well was compared with the negative and positive controls.
Cell lines
Normal human skin fibroblasts (BJ), human hepatocellular
carcinoma (HepG2) and human breast adenocarcinoma
(MCF-7) were obtained from American Type Culture
Collection (ATCC, England, UK). The cells were cultured
in Eagle’s minimum essential medium (EMEM, ATCC)
supplemented with 10 % fetal bovine serum (FBS, PAA
Laboratories), 100 U/mL penicillin and 100 lg/mL strep-
tomycin (Sigma-Aldrich). In the case of the MCF-7 cell
line, the culture medium was additionally supplemented
with 0.01 lg/mL of human recombinant insulin (Sigma-
Aldrich). The cells were grown in 75-cm2 flasks and
maintained at 37 °C in a humidified atmosphere of 5 %
CO2 and 95 % air.
Disk diffusion method
The preliminary antibacterial activity of the carbazide
derivatives against human pathogenic Gram-positive,
Gram-negative aerobic and microaerobic bacteria was
evaluated by measuring the zones of inhibition in the disk
diffusion method (Murray et al., 1995). Each compound
(100 lg) was placed on Petri plates with agar medium
(previously inoculated with 0.5 McFarland standards with
the tested bacterial strains). After 18 h of incubation at
37 °C (for aerobic strains) or 40 h at 35 °C (for
microaerobic strains), zones of microbial growth produced
around the tested substances were measured and recorded
as the diameters of inhibition.
Cytotoxicity assay
In order to determine the cytotoxicity, BJ cells were seeded
in flat-bottom 96-well plates in 100 lL of a complete
growth medium at a concentration of 1.7 9 104 cells/well
and incubated for 24 h at 37 °C in a humidified atmosphere
of 5 % CO2. Immediately before drug treatment, the syn-
thesized compounds (1, 2, 4, 5, 6, 7, 10) were dissolved in
dimethyl sulfoxide (DMSO, Sigma-Aldrich) and then
diluted in cell culture medium supplemented with 2 %
FBS. Moreover, cefepime dihydrochloride (Maxipime,
Bristol-Myers Squibb Latina), chlorhexidine digluconate
((CLX) Amara Poland) and ethacridine lactate (Rivanolum,
PharmaSwiss, Czech Republic) were used as reference
antibacterial agents. After incubation, the growth medium
was replaced with 100 lL of the appropriate serial dilu-
tions of the investigated compounds. Untreated cells were
used as negative controls, and different concentrations of
DMSO were used as the solvent control. The cell cultures
were incubated at 37 °C for 24 h. The cytotoxicity was
estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay as described by
Mosmann with some modifications (Mosmann, 1983).
Briefly, the cells were incubated for 3 h with 25 lL of
MTT solution (5 lg/mL in PBS buffer) per well. The MTT
assay is a rapid colorimetric method based on the conver-
sion of mitochondrial succinate dehydrogenase from yel-
low, soluble tetrazolium salt to blue formazan crystals to
determine the number of viable cells. In order to dissolve
formazan crystals in live cells, 100 lL of SDS-HCl solu-
tion (10 % SDS in 0.01 N HCl) was added per well. After
overnight incubation, absorbance was measured at 570 nm
Broth microdilution method
A broth microdilution method was used to evaluate the
minimum inhibition concentration (MIC) according to the
CLSI document (CLSI performance standards for antimi-
crobial susceptibility testing, 2008, Eighteenth Interna-
tional Supplement, CLSI document M7-MIC, Clinical
Laboratory Standards Institute, Wayne) with some modi-
fications. The lowest concentration of the tested compound
(expressed in lg/mL) which did not allow any visible
growth of bacteria was considered as MIC. A serial dou-
bling dilution of the compounds was prepared in 96-well
plates (200 lL per well). A suitable medium (M-H Broth,
MRS Broth Lactobacillus, BHI Broth) was used as a
diluent. The final concentrations of derivatives were
1000–0.015 lg/mL. Finally, 2 lL of inoculum of the tested
bacterial strain (1.5 9 108 CFU/mL) was added to each
well. The tests were performed either at 36 °C for 18 h
(aerobic strains) or at 40 h (microaerobic strains). After
123

Med Chem Res
using microplate reader (BioTek ELx50). The MTT assay
was repeated in three independent experiments performed
in octuplicates. The half-maximal cytotoxic concentration
(CC50) was defined as the compound concentration (lg/
mL) required to reduce cell viability to 50 %. Therapeutic
index (TI) is a widely accepted parameter to represent the
specificity of antibacterial agents for human (Begg et al.,
1999). In this study, the in vitro TI values were calculated
as the ratio of CC50 (cytotoxic activity) and MIC (an-
tibacterial activity); thus, greater values of in vitro thera-
peutic index indicate safer specificity for eukaryotic cells.
The geometry and energy of the tested compounds were
optimized by AM1 semiempirical method (Dewar et al.,
1985). The TPSA, miLogP and hydrogen bond donors and
acceptors were calculated by Molinspiration program
(http://www.molinspiration.com/cgi-bin/properties-accessed
1 February, 2015).
Acknowledgments This study was partially supported by a DS2
grant (Medical University in Lublin, Poland). The paper was devel-
oped using the equipment purchased within agreement No.
PORPW.01.03.00-06-010/09-00 Operational Program Development
of Eastern Poland 2007-2013, Priority Axis I, Modern Economy,
Operations 1.3. Innovations Promotion.
Cell proliferation assay
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
In order to evaluate cell proliferation, the cells were seeded
in flat-bottom 96-well plates in 100 lL of a complete
growth medium at a concentration of 2 9 103 cells/well
(BJ), 1.5 9 104 cells/well (HepG2) and 2.5 9 104 cells/
well (MCF-7) and incubated for 24 h at 37 °C in a
humidified atmosphere of 5 % CO2. Before drug exposure,
the synthesized compounds (1–10) were dissolved in
DMSO and then diluted in a complete culture medium
supplemented with 10 % FBS. Subsequently, the growth
medium was gently removed and the cells were exposed to
100 lL of serial dilutions of the investigated compounds at
concentrations ranging from 0.05 to 200 lg/mL. Untreated
cells were used as negative controls, and different con-
centrations of DMSO were used as the solvent control.
After 96-h incubation at 37 °C in a humidified atmosphere
of 5 % CO2, cell proliferation was assessed using the MTT
test as described previously (Cytotoxicity assay). The MTT
assay was repeated in three independent experiments in
quadruplicates. The half-maximal inhibitory concentration
(IC50) was defined as the compound concentration (lg/mL)
required to inhibit cell proliferation to 50 %.
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