Investigation of the biological properties of (hetero)aromatic thiosemicarbazones

Article abstract of DOI:10.3390/molecules171113483

Two series of thiosemicarbazone-based iron chelators (twenty-seven compounds) were designed and synthesized using a microwave-assisted approach. Quinoline and halogenated phenyl were selected as parent scaffolds on the basis of a similarity search. The li

Full text of DOI:10.3390/molecules171113483

Molecules 2012, 17, 13483-13502; doi:10.3390/molecules171113483
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Investigation of the Biological Properties of (Hetero)Aromatic
Thiosemicarbazones
Maciej Serda 1, Anna Mrozek-Wilczkiewicz 1,2, Josef Jampilek 3,4, Matus Pesko 5,
Katarina Kralova 6, Marcela Vejsova 7, Robert Musiol 1,*, Alicja Ratuszna 2 and
Jaroslaw Polanski 1
1
Department of Organic Chemistry, Institute of Chemistry, University of Silesia, PL-40006
Katowice, Poland
2
Institute of Physics, University of Silesia, PL-40007 Katowice, Poland
3
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical
Sciences, Palackeho 1/3, CZ-61242 Brno, Czech Republic
4
Research Institute for Pharmacy and Biochemistry, Lidicka 1879/48, CZ-60200 Brno,
Czech Republic
5
Department of Ecosozology and Physiotactics, Faculty of Natural Sciences, Comenius University,
Mlynska dolina Ch-2, SK-84215 Bratislava, Slovakia
6
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina Ch-2,
SK-84215 Bratislava, Slovakia
7
Department of Clinical Microbiology, Charles University Medical School and Teaching Hospital,
Sokolska 581, CZ-50005 Hradec Kralove, Czech Republic
* Author to whom correspondence should be addressed; E-Mail: robert.musiol@us.edu.pl;
Tel.: +48-032-359-1514.
Received: 8 October 2012; in revised form: 7 November 2012 / Accepted: 8 November 2012 /
Published: 14 November 2012
Abstract: Two series of thiosemicarbazone-based iron chelators (twenty-seven
compounds) were designed and synthesized using a microwave-assisted approach.
Quinoline and halogenated phenyl were selected as parent scaffolds on the basis of a
similarity search. The lipophilicity of the synthesized compounds was measured using
HPLC and then calculated. Primary in vitro screening of the synthesized compounds was
performed against eight pathogenic fungal strains. Only a few compounds showed
moderate activity against fungi, and (E)-2-(quinolin-2-ylvinyl)-N,N-dimethylhydrazine-
carbothioamide appeared to be more effective than fluconazole against most of the fungal
strains tested. Antiproliferative activity was measured using a human colon cancer cell line

Molecules 2012, 17
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(HCT-116). Several of the tested compounds showed submicromolar antiproliferative
activity. Compounds were also tested for their activity related to the inhibition of
photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. The
structure-activity relationships are discussed for all of the compounds.
Keywords: thiosemicarbazones; lipophilicity; photosynthetic electron transport inhibition;
spinach chloroplasts; iron chelators; in vitro antifungal activity; in vitro anticancer activity
1. Introduction
Iron is an element of great importance for virtually all living organisms. It is present in various
biological processes such as metabolic pathways, reproduction or photosynthesis. On the basis of its
importance, the iron-sulphur theory of the origin of life has been proposed [1,2]. Despite being so
pervasive, iron rarely exists alone or in the form of simple cations. It is commonly chelated by larger
organic molecules such as porphyrin. This form is present in hemoglobin or in most of the metabolic
enzymes of cytochrome P450 [3]. Clearly, all living organisms have their ways of acquiring and
managing such an important element. Any alteration of or damage to the iron metabolism system may
lead to serious diseases. In humans, this may cause hemochromatosis or iron deficiency anemia [4]. As
a target for therapy, iron is used mainly in the treatment of iron overload using iron chelators such as
deferoxamine (DFO, also known as desferrioxamine B) [5]. More recently, iron chelators have been
proposed for cancer treatment [5–10] because rapidly proliferating cancer cells have a higher demand
for this element compared to the normal cells [5,11]. Although the exact mechanism of the anticancer
activity of iron chelators remains unknown, several possible modes of action can be implemented,
e.g., the inhibition of iron uptake [12] and the inhibition of ribonucleotide reductase (RR) [13] or the
formation of reactive oxygen species (ROS) [14].
Other potential therapeutic or agricultural application of iron chelators may be derived from
processes or pathways that are dependent on iron in pathological organisms. Fungi and bacteria
require an appropriate amount of iron for correct growth, and apparently to develop their resistance to
drugs [15,16]. Pathogenic microbes have evolved their unique way of acquiring this important metal
from a host organism through the overexpression of Fe receptors or the utilization of siderophores [17]. If
the iron level is reduced, fungi lose some of their virulence and have a decreased potency to invade the
epithelium. Hence, lactoferrin and deferoxamine have been tested for their antifungal potency. Early
attempts have consisted in the co-administration of lactoferrin with conventional antifungals such as
amphotericin B [18,19]. DFO was tested as an ancillary drug with amphotericin B in antifungal
therapy in a clinical trial [20,21]. The study was terminated prematurely due to an increased mortality
in patients who had taken DFO when compared to those who had taken a placebo. However, this
seems to be an effect of the more complex prerequisites connected with the unknown site of
administration of DFO rather than with synergic toxicity. Nevertheless, this is a trigger to search for
structurally new iron chelators with an antifungal activity. Several reports describing the synthesis and
antifungal activity of thiosemicarbazones have been published [22–26]. Li et al. described a series of
benzyl thiosemicarbazones whose substitution with a chlorine atom triggered the activity of mushroom

Molecules 2012, 17
13485
tyrosinase [27]. Moreover, chlorine derivatives of benzaldehyde 4-phenyl-3-thiosemicarbazone and
their cyclization products were tested against Toxoplasma gondii and several bacterial strains [28].
Some of these derivatives were more active than chloramphenicol and rifampicin.
In this paper, some disubstituted benzaldehyde thiosemicarbazones are described along with their
anticancer and antifungal activity. These structures were tested on the basis of a former study on
antifungal compounds [29–33] and quinolone-based thiosemicarbazones with anticancer activity [34].
Figure 1 illustrates the fragment-based similarity of the compounds investigated with known and
effective iron chelators [5,35–37]. The design of compounds with polypharmacological activity was
the aim of our investigation. Such compounds may be of great value in anticancer therapy where
fungal infections are very common. Furthermore, these compounds, due to their simple and cheap
synthesis, are suitable for agricultural use. However, antifungal agents such as azoles used for crop
protection raise some concerns, e.g., triggering resistance [38] and have a negative impact on the
environment. In fact, iron is also important in plants where it plays a crucial role in photosystems I and
II as a part of ferredoxin [39] and cytochrome b559 [40–43]. Taking this aspect into account,
thiosemicarbazone iron chelators may exert some unwanted effects on the green parts of plants. Thus,
the compounds prepared for the inhibition of photosynthetic electron transport were also tested.
Figure 1. (A) Structures of known iron chelators 3-AP [5], 311 [35], Dp44mT [36,37].
(B) investigated benzylidenethiosemicarbazones 3a–o, quinolinylvinylthiosemicarbazones
4a–l.
A
N
N
NH2
N
CH3
N
H
H
H
N
N
NH2
N
N
N
N
CH3
O
S
N
S
OH
3-aminopyridine-2-carboxaldehyde
2-hydroxy-1-naphthaldehyde
di-2-pyridylketone 4,4-dimethyl-
thiosemicarbazone (triapine, 3-AP)
isonicotinoyl hydrazone (311)
3-thiosemicarbazone (Dp44mT)
B
Cl
R´
R´
H
H
N
Cl
N
N
N
N
N
N
R´´
N
R´´
S
S
S
3a-e
4a-d
R´
N
OH
R´
N
H
N
H
N
Cl
Cl
N
R´´
N
R´´
S
3f-j
4e-h
R´
R´
N
N
H
H
N
N
N
R´´
N
R´´
N
S
S
OH
Br
3k-o
4i-l

Molecules 2012, 17
13486
2. Results and Discussion
2.1. Chemistry
Compounds 3a–o were obtained as shown in Scheme 1. Microwave irradiation was found to be
helpful for enhancing the yield of the syntheses and the purity of the final products (the purity of all of
the compounds was assessed using HPLC and was higher than 98%, while the overall average yield
exceeded 80%). All of the synthesized compounds were in the E-configuration, which was confirmed
using 1H-NMR spectroscopy (the signal of the NH group was in the 10–11 ppm range, in comparison
to Z-isomer, which possesses a characteristic NH signal in the 14–15 ppm range), the COSY and
NOESY experiments and by crystal structure as was reported recently [44]. Exemplary spectra are
available in Supplementary Material for this article.
Scheme 1. Synthesis of the discussed benzylidenethiosemicarbazones 3a–o and
quinolinylvinylthiosemicarbazones 4a–l.
S
Z
Y
Z
Y
H2N
R´
S
N
N
H
H
O
O
N
R´
N
N
EtOH, AcOH, MW
H
H
X
X
S
S
H2N
R´
N
N
N
R´
H
H
N
N
H
H
N
N
EtOH, AcOH, MW
R´´
R´´
2.2. Lipophilicity
Lipophilicity is a property that has a major effect on absorption, distribution, metabolism, excretion
and toxicity properties as well as on pharmacological activity because drugs cross biological
membranes through passive transport, which is strongly dependent on their lipophilicity. Lipophilicity
has been studied and applied as an important drug property for decades. The lipophilicity of the
compounds studied was determined using RP-HPLC as a capacity factor logarithm (log k) and
calculated as log P/Clog P using two commercially available software programs (ChemOffice and
ACD/LogP) [45,46]. The results for benzylidenethiosemicarbazones 3a–o are shown in Table 1, they
are summarized for quinolinylvinylthiosemicarbazones 4a–l in Table 2 and are illustrated for both
series in Figure 2.
Reversed phase high performance liquid chromatography (RP-HPLC) methods have become
popular and are widely used for lipophilicity measurements. The general procedure is the measurement
of the directly accessible retention time under isocratic conditions using varying amounts of methanol
as an organic modifier in the mobile phase using RP columns and calculating the logarithm of the
capacity factors (log k). Log k is the logarithm of the capacity factors in chromatographic approaches
that is related to the partitioning of a compound between a mobile and a (pseudo-)stationary phase.
Log k is used as the lipophilicity index converted to log P scale.

Molecules 2012, 17
13487
Table 1. Structure of aryl compounds 3a–o and comparison of the calculated lipophilicities
(log P/Clog P) with determined log k values.
2
R
H
N
N
3
N
R
1
R
S
log P/Clog P
log P
Comp.
R1
R2
R3
log k
ChemOffice ACD/LogP
3a
3b
3c
3d
3e
3f
2,3-Cl
2,3-Cl
H
H
H
0.6892
0.7357
0.7214
0.7958
0.9230
0.6974
0.7563
0.7555
0.8415
0.9774
0.6580
0.6993
0.6784
0.7568
0.8772
2.81/3.473
3.33/3.177
3.71/3.533
3.67/3.706
5.00/5.266
2.81/3.473
3.33/3.177
3.71/3.533
3.67/3.706
5.00/5.266
2.53/2.952
3.05/2.734
3.42/3.090
3.39/3.263
4.71/4.823
3.22 ± 0.38
3.14 ± 0.59
3.11 ± 0.60
3.68 ± 0.59
4.90 ± 0.59
3.28 ± 0.38
3.21 ± 0.59
3.17 ± 0.60
3.74 ± 0.59
4.96 ± 0.59
2.79 ± 0.39
2.72 ± 0.61
2.68 ± 0.62
3.25 ± 0.61
4.47 ± 0.61
CH3
CH3
C2H5
C6H5
H
2,3-Cl CH3
2,3-Cl
2,3-Cl
3,4-Cl
3,4-Cl
H
H
H
H
3g
3h
3i
CH3
CH3
C2H5
C6H5
H
3,4-Cl CH3
3,4-Cl
3,4-Cl
4-Br
H
H
3j
3k
3l
H
4-Br
H
CH3
CH3
C2H5
C6H5
3m
3n
3o
4-Br
CH3
H
4-Br
4-Br
H
Commercially available chemical software does not resolve the various lipophilicity values of
individual positional isomers, etc. The software calculates lipophilicity contributions according to
different internal databases/libraries and uses different approaches. It can be concluded that for small,
highly functionalized molecules with many heteroatoms, a number of intermolecular forces and
intramolecular interactions are typical. The more of these interactions that can be expected, the less the
predictability of common software can be, thus experimental lipophilicity determination is of great
importance. Therefore, we decided to perform our measurements using the water-methanol system as
the mobile phase. Log k derived from RP-HPLC retention factors and computational log P values are
given in the manuscript and biological data are related to log k data because log k data specify
lipophilicity within the series of compounds more precisely than the available chemical software.
The results obtained with all the compounds show that the experimentally-determined
lipophilicities (log k) of the compounds discussed are relatively in accordance with the calculated
values of compounds 3a–o and 4a–l as is shown in Figure 2. The minimum match of experimental (log k)
versus calculated (log P) values was found for ChemOffice. Generally, benzylidenethiosemicarbazones
3a–o showed a higher match (see Figure 2A) than quinolinylvinylthiosemicarbazones 4a–l (see
Figure 2B), which indicates inter- and intramolecular interactions in the last mentioned series.

Molecules 2012, 17
13488
Table 2. Structure of heteroaryl compounds 4a–l and comparison of calculated
lipophilicities (log P/Clog P) with determined log k values.
2
R
H
1
R
N
N
3
N
R
S
log P/Clog P
log P
Comp.
R1
R2
R3
log k
ChemOffice ACD/LogP
4a
4b
4c
4d
H
CH3
H,
CH3
CH3
0.6329
0.6184
0.6892
0.7553
2.73/1.968
3.10/2.114
3.06/2.497
4.39/4.057
2.06 ± 0.59
2.02 ± 0.60
2.59 ± 0.59
3.81 ± 0.59
N
N
N
N
N
C2H5
C6H5
H
4e
4f
H
CH3
H
CH3
C6H5
C2H5
C6H5
CH3
0.6236
0.5927
0.6810
0.7548
0.6193
2.34/2.064
2.71/2.163
2.67/2.593
4.00/4.153
1.91/2.131
1.85 ± 0.84
1.81 ± 0.85
2.38 ± 0.84
3.60 ± 0.84
1.62 ± 1.09
OH
OH
OH
N
N
N
4g
4h
4i
H
OH
N
OH
OH
OH
OH
H
N
N
N
4j
4k
4l
CH3
H
CH3
C2H5
C6H5
0.5831
0.6539
0.6748
2.29/2.163
2.25/2.660
3.58/4.220
1.58 ± 1.10
2.15 ± 1.09
3.37 ± 1.09
H
Lipophilicity calculations of these small and highly functionalized molecules do not cover inter-
and intramolecular interactions that can be found by experimental lipophilicity determination. As was
expected, quinolinylvinylthiosemicarbazones 4a–l showed a lower lipophilicity (log k) than
benzylidene-thiosemicarbazones 3a–o. Within the series of benzylidenethiosemicarbazones, the
determined log k values increased as follows: 4-Br (3k–o) < 2,3-Cl (3a–e) < 3,4-Cl (3f–j), while
within the series of quinolinylvinylthiosemicarbazones, the determined log k values increased in the
following order: 7-hydroxyquinolin-8-yl (4i–l) < 8-hydroxyquinolin-2-yl (4e–h) < quinoline-2-yl (4a–d).
The influence of R2 and R3 substituents on lipophilicity is as follows: 2H < 2CH3 < H, CH3 < H, C2H5 < H,
C6H5. Based on the above-mentioned data, it can be assumed that compounds 4e–l are possible
tautomeric isomers. This seems to be in agreement with our former findings [47]. Therefore, it can be
concluded that log k values specify lipophilicity within individual series of the compounds studied
more precisely than the calculated log P/Clog P data.

Molecules 2012, 17
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Figure 2. Comparison of log P/Clog P data calculated using two programs with log k
values found experimentally. (A) match of calculated data with log k values of 3a–o found
experimentally; (B) match of calculated data with log k values of 4a–l found experimentally.
A
1.0
0.9
0.8
R2 = 0.8603
R
2 = 0.8399
R2 = 0.9195
0.7
0.6
2.4
2.9
3.4
3.9
4.4
4.9
5.4
log P /Clog P
log P [ChemOffice] Clog P [ChemOffice] log P [ACD/LogP]
B
0.8
0.7
0.6
0.5
R2 = 0.6544
R2 = 0.6774
R
2 = 0.8553
1.5
2.0
2.5
3.0
3.5
4.0
4.5
log P /Clog P
log P [ChemOffice] Clog P [ChemOffice] log P [ACD/LogP]
2.3. Biological Activities
The compounds under investigation can be divided into two groups based on their chemical
structure: Group 1 included benzylidenethiosemicarbazones 3a–o and Group 2 contained
quinolinylvinylthiosemicarbazones 4a–l. The compounds showed a wide range of biological activities
and some interesting structure-activity relationships were observed. All of the results are summarized
in Table 3.
2.3.1. Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
The activity of all of the evaluated derivatives related to the inhibition of photosynthetic electron
transport (PET) in spinach (Spinacia oleracea L.) chloroplasts was moderate or rather low relative to
the standard (see Table 3). Diuron® (DCMU), a marketed herbicide with its activity mechanism
directed to photosystem II, was used as the standard. The compounds within Group 2 could not be
evaluated for their structure-activity relationships because the majority of these compounds showed no
PET-inhibiting activity. (E)-2-(3,4-Dichlorobenzylidene)-N-methylhydrazinecarbothioamide (3g)
expressed the highest PET-inhibiting activity (IC50 = 135.6 µmol/L) within Group 1.

Molecules 2012, 17
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Table 3. IC50 [μmol/L] values related to PET inhibition in spinach chloroplasts of the
substituted thiosemicarbazones in comparison with 3-(3,4-dichlorophenyl)-1,1-dimethylurea
(DCMU) as the standard; in vitro antifungal activity (IC80/IC50 [μmol/L]) of compounds
compared to fluconazole (FLU) as the standard; in vitro antiproliferative activity IC50
[μmol/L] of compounds compared to doxorubicin as the standard. ND = not determined
due to precipitation during the experiment or interaction with 2,6-dichlorophenol-
indophenol (DCPIP).
1,2 MIC (a IC80 / b IC50) [µmol/L]
PET
IC50
CA a
24 h
CT a CK a CG a TB a AF b AC b TM b
HCT-116
IC50
Comp.
24 h 24 h 24 h 24 h
48 h 48 h 48 h 48 h
24 h
48 h
24 h
72 h
48 h
48 h 120 h
3a
3b
3c
3d
3e
3f
ND
170.1
ND
125
500
125
500
125
500
125
15.62
31.25
500
125
500
125 125 125 125 125 125 125
500 500 500 500 500 500 500
125 125 125 125 125 125 125
500 500 500 500 500 500 500
125 125 125 125 125 125 125
500 500 500 500 500 500 500
125 125 125 125 125 125 125
41.5 ± 1.7 3
>60
-
ND
58.5 ± 0.2 3
>60
499.3
283.3
135.6
46.0 ± 0.9
55.5 ± 6.2
3g
500
500
500
500
500
500
125 >500 >500 31.25
500 >500 >500 62.50
3h
329.3
47.0 ± 1.3
3i
3j
280.2
425.9
500 500 500 500 500 500 500
125 125 125 125 125 125 125
500 500 500 500 500 500 500
500 500 500 500 500 500 500
500 500 500 500 500 500 500
500 500 500 15.62 500 500 62.50
500 500 500 62.50 500 500 62.50
500 500 500 500 500 500 500
500 500 500 500 500 500 500
500 500 500 500 500 500 500
48.5 ± 2.8
>60
3k
3l
586.4
ND
>60
>60
500
500
62.50
250
3m
594.6
47.5 ± 2.4
3n
3o
4a
ND
ND
ND
500
500
500
1.95
>60 3
>60
>25
1.95 3.90
7.81 3.90
1.95
1.95
1.95
1.95
3.90
3.90
3.90
3.90
3.90
3.90
4b
1368
4.86 ± 1.48 3
1.95
4e
4f
302
ND
125
500
125
125
500
500
125
125 125 125 125 125 125 125
500 500 500 500 500 500 500
125 125 125 125 125 125 125
125 125 125 125 125 125 125
500 500 500 500 500 500 500
500 500 500 500 500 500 500
1.71 ± 0.34 3
-
4h
4i
ND
ND
24.97 ± 4.29 3
>25 3
250 250 125
250 250 125 250 250 250
125 250 250
125
125
4j
4k
4l
520.4
ND
20.75 ± 5.34 3
>25 3
250
500
500
31.25
62.50
500 500 62.50 500 500 500 62.50
500 500 500 500 500 500 62.50
125 31.25 15.62 15.62 125 15.62 15.62
ND
16.28 ± 1.69 3
125
125 31.25 125
125 62.50 15.62

Molecules 2012, 17
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Table 3. Cont.
1,2 MIC (a IC80 / b IC50) [µmol/L]
PET
Comp.
CA a
24 h
48 h
-
CT a CK a CG a TB a AF b AC b TM b
24 h 24 h 24 h 24 h 24 h 24 h 72 h
48 h 48 h 48 h 48 h 48 h 48 h 120 h
HCT-116
IC50
IC50
DCMU
FLU
1.9
-
-
-
-
-
-
-
0.06
0.12
-
0.12 3.91 0.98 0.24 125 125 1.95
125 15.62 3.91 0.48 125 125 3.91
-
-
-
DXR
-
-
-
-
-
-
-
10 ± 1.1
1
a
The MIC determination was performed according to the CLSI reference protocol: M27-A2 for
b
yeasts (IC80 value) and M38-A for moulds (IC50 value); CA = Candida albicans, CT = Candida
tropicalis, CK = Candida krusei, CG = Candida glabrata, TB = Trichosporon beigelii,
AF = Aspergillus fumigatus, AC = Absidia corymbifera, and TM = Trichophyton mentagrophytes.
2
All compounds were tested for short and long term activity. When inactive only one value is
3
presented. Synthesis and anticancer activity described elsewhere [34,44]. HCT-116 human colon
cancer cells, DXR- doxorubicin as the standard.
Figure 3 illustrates the dependence of the PET-inhibiting activity, expressed by the negative
logarithm of the log (1/IC50 [mol/L]) value on the lipophilicity, expressed as log k, of
benzylidenethiosemicarbazones 3a–o. Although the dependence seems to be clear, it is not possible to
draw a simple relationship. The regression with parabolic function did not provide a good correlation.
This suggests that lipophilicity is only one of the important factors. Nevertheless, despite the relatively
low inhibitory activity of the compounds studied, the correlations between log (1/IC50) and the
lipophilicity showed a bilinear course: in the range of log k = 0.6580 (3k) to log k = 0.7563 (3g)
PET-inhibiting activity linearly increased with increasing compound lipophilicity; however, with a further
increase in lipophilicity, the inhibitory activity declined. Generally, 4-Br-benzylidenethiosemi-
carbazones 3k–o showed the worst PET inhibition. Disubstitution by 3,4-Cl of the benzylidene ring
seems to be more advantageous than disubstitution by 2,3-Cl of the benzylidene moiety. It can be
stated that within the 3,4-Cl-benzylidenethiosemicarbazones 3f–j N,N-disubstitution caused a decrease
in PET inhibition (compare 3g/3h). Based on these facts, it can be concluded that PET-inhibiting
activity would probably be negatively influenced by bulky or long-chain alkyls or by the substitution
of the second hydrogen of the terminal amino moiety.
2.3.2. In Vitro Antifungal Susceptibility Testing
The antifungal activity of the compounds studied was tested against several pathogenic fungi and
the results of the screening are shown in Table 3. The values are correlated to the standard drug
fluconazole. The majority of the compounds studied appeared to be inactive against the fungal strains
tested. Nevertheless, some of them, 3h, 3m, and 4l, showed moderate activity, especially against
Candida albicans, Trichosporon beigelii, and Trichophyton mentagrophytes. (E)-2-(Quinolin-2-
ylvinyl)-N,N-dimethylhydrazinecarbothioamide (4b) expressed the strongest activity among all of the
compounds tested. Compound 4b was more active than fluconazole against Aspergillus fumigatus and
Absidia corymbifera. Furthermore, this compound showed an interesting pattern of long terminal

Molecules 2012, 17
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activity and was active at a comparable level for 24 h and 48 h protocols. The (Candida tropicalis and
Candida krusei) activity of fluconazole decreased significantly for some fungi.
Figure 3. Relationships between PET inhibition log (1/IC50) [mol/L] in spinach
chloroplasts and lipophilicity of studied benzylidenethiosemicarbazones 3a–o.
4.0
3g
3.8
3b
3.6
3f
3i
3h
3.4
3j
3e
3m
3k
3.2
3.0
0.6
0.7
0.8
0.9
1.0
log k
As most compounds showed only a moderate or no activity, no thorough structure-activity
relationships could be established. According to the results presented in Table 3, it can be concluded
that compounds with a quinoline nucleus, i.e., quinolinylvinylthiosemicarbazones (Group 2) seem to be
more effective antifungals than Group 1, i.e., benzylidenethiosemicarbazones. Based on recent results [32],
it was expected that 8-hydroxyquinolinylvinylthiosemicarbazones would possess a higher antifungal
activity than antifungal tests showed. It can be stated, contrary to PET-inhibiting activity, that
N,N-disubstitution of the terminal amino moiety by two methyl groups is crucial for a high antifungal
effect. This fact corresponds to results obtained by Opletalova et al. [26].
2.3.3. In Vitro Antiproliferative Activity
The antiproliferative activity of all of the compounds was examined against the human colon cancer
(HCT-116) cell line using an MTS assay. The HCT-116 p53 wild type cell line was chosen on the
basis of our former experience and some results on the activity of quinoline-based thiosemicarbazones
that was published recently. Namely, compounds 4a–l have been tested previously for their wide-
ranging anticancer activity [34]. The antiproliferative activity of the compounds evaluated was
assessed using Doxorubicin as a reference compound. In general, quinoline-based thiosemicarbazones
4 seem to have a much better anticancer activity than halogenophenyl compounds 3. Thus, the
quinoline nucleus can be defined as being crucial for antiproliferative activity. A more detailed
analysis of the anticancer activity of quinoline-based thiosemicarbazone is available [34].
Benzylidenethiosemicarbazones were much less active against the cells tested. Compound 3a appeared
to be the most active in this assay. Nevertheless, some more general conclusions can be drawn from
the gathered data. Dichlorosubstituted compounds are in general more active than monobromo
equivalents. The free amino group or substitution pattern with small substituents such as methyl or
ethyl are favorable for activity. According to our former results the chelating ability of the compounds
may be one of the prerequisites of the antiproliferative activity. In this study we have measured the
UV-VIS spectra of the compounds in presence of various concentration of Fe3+ cations to observe its

Molecules 2012, 17
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chelating potency (supplementary material). Isosbestic points are observable in case of more active
compounds like 4a–l series while in 3a–o only minor or no interaction between ligand and metal can
be noticed. Compound 4b one of the most active against HCT-116 cells and in antifungal assays gave
clear isosbestic points at 375 nm. However as 4j give also two points at 330 and 385 nm, iron
chelating ability should be regarded only as one of the factor influencing the biological activity.
3. Experimental
3.1. General
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Princeton Chemicals
Ltd. (Luton, Bedfordshire, UK). Kieselgel 60, 0.040–0.063 mm (Merck, Darmstadt, Germany) was
used for the column chromatography. Syntheses were performed in a CEM DISCOVERY microwave
reactor (Matthews, NC, USA) with temperature and pressure control. TLC experiments were
performed on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt, Germany). The plates were
illuminated under UV (254 nm) and evaluated in an iodine vapor. The melting points were determined
on an Optimelt MPA100 instrument (SRS, USA) and are uncorrected. Infrared (IR) spectra were
recorded on a Smart MIRacle™ ATR ZnSe for Nicolet™ Impact 410 FT-IR spectrometer (Thermo
Fisher Scientific, Waltham, MA, USA). The spectra were obtained by the accumulation of 256 scans with
1
13
2 cm−1 resolution in the region of 4000–600 cm−1. All H and C-NMR spectra were recorded on a
1
Bruker Avance III 400 MHz FT-NMR spectrometer (400 MHz for H and 100 MHz for 13C,
BrukerBioSpin Comp., Karlsruhe, Germany). Chemicals shifts are reported in ppm () using internal
Si(CH3)4 as the reference with diffuse, easily exchangeable signals being omitted. HR-MS (EI)
analysis was performed on a Finnigan MAT95 spectrometer (ThermoFinnigan, San Jose, CA, USA)
for all new compounds.
3.2. Synthesis
3.2.1. General Procedure for Synthesis of Thiosemicarbazones 3a–o
Equimolar quantities of an appropriate thiosemicarbazide and benzaldehyde derivative were mixed
and 2 drops of glacial acetic acid were added. The resulting mixture was heated in a microwave reactor
at 85 °C for 12 min (max microwave power 75 W). After cooling, the precipitated solids were filtered
off, washed with ether and crystallized from methanol. The compounds 3a–o studied are presented
in Table 1.
1
(E)-2-(2,3-Dichlorobenzylidene)hydrazinecarbothioamide (3a). Yield: 78%, H-NMR (d6-DMSO,
400 MHz, ppm): 11.69 (bs, 1H, NH), 8.49 (s, 1H, CH), 8.36 (s, 1H, NH), 8.30 (d, J = 8.0 Hz, 1H,
13
ArH), 8.18 (bs, 1H, NH), 7.66 (d, J = 7.9 Hz, 1H, ArH), 7.38 (t, J = 8.0 Hz, 1H, ArH). C-NMR
(d6-DMSO, 100 MHz, ppm): 178.8, 138.2, 134.2; 132.6, 131.6, 131.2, 128.5, 126.5. MP: 226–227 °C
HR-MS (EI): 246.9745 (calc. for C8H7Cl2N3S: 246.9738).
(E)-2-(2,3-Dichlorobenzylidene)-N-methylhydrazinecarbothioamide (3b). Yield: 74%, 1H-NMR
(d6-DMSO, 400 MHz, ppm): 11.76 (s, 1H, NH), 8.68 (d, J = 4.4 Hz, 1H, NH), 8.49 (s, 1H, CH), 8.29

Molecules 2012, 17
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(d, J = 8.0 Hz, 1H, ArH), 7.67 (d, J = 7.9 Hz, 1H, ArH), 7.41 (t, J = 7.9 Hz, 1H, ArH), 3.02 (d, J = 4.5 Hz,
13
CH3). C-NMR (d6-DMSO, 100 MHz, ppm): 178.5, 137.7, 134.5, 132.5, 131.4, 131.3, 128.4, 126.2,
31.4. MP: 223–224 °C, HR-MS (EI): 260.9891 (calc. for C9H9Cl2N3S: 260.9894).
1
(E)-2-(2,3-Dichlorobenzylidene)-N,N-dimethylhydrazinecarbothioamide (3c). Yield: 69%, H-NMR
(d6-DMSO, 400 MHz, ppm): 11.27 (s, 1H, NH), 8.64 (s, 1H, CH), 7.92 (dd, J1 = 7.9 Hz, J2 = 1.4 Hz,
1H, ArH), 7.66 (dd, J1 = 7.9 Hz, J2 = 1.5 Hz, 1H, ArH), 7,42 (t, J = 8.0 Hz, 1H, ArH), 3.31 (s, 6H,
13
CH3). C-NMR (d6-DMSO, 100 MHz, ppm): 180.8, 140.0, 134.7, 132.7, 131.5, 131.0, 128.8, 125.7,
42.4. MP: 145–146 °C, HR-MS (EI): 275.0047 (calc. for C10H11Cl2N3S: 275.0051).
(E)-2-(2,3-Dichlorobenzylidene)-N-ethylhydrazinecarbothioamide (3b). Synthesis and more detailed
analysis of the structure is available [44]. Yield: 67%, 1H-NMR (d6-DMSO, 400 MHz, ppm): 11.70 (s,
1H, NH), 8.70 (m, 1H, NH), 8.49 (s, 1H, CH), 8.31 (d, J = 7.9 Hz, 1H, ArH), 7.67(d, J = 7.9 Hz, 1H,
ArH), 7.41 (t, J = 7.9 Hz, 1H, ArH), 3.70–3.52 (m, 2H, CH2), 1.16 (t, J = 7.0 Hz, 3H, CH3). 13C-NMR
(d6-DMSO, 100 MHz, ppm): 177.4, 137.6, 134.4, 132.5, 131.5, 131.3, 128.5, 126.02, 38.9, 14.7. MP:
213–214 °C HR-MS (EI): 275.0054 (calc. for C10H11Cl2N3S: 275.0051), IR: 3349 (NH), 3142 (PhH),
2983 (CH), 1584 (C=N), 1542 (C-N), 1520 (CH), 1452 (CH/NH), 1416 (CH3), 1311 (CNH), 1264
(C/NH), 1160(N-N), 1108 (), 1042 (NH), 929 , 906 (CS), 782 (CS), 737, 704 (ring), 626 (C-N).
UV-VIS (methanol; log): 323.0 (3.96), 237.0 (3.67), 204.0 (3.91).
(E)-2-(2,3-dichlorobenzylidene)-N-phenylhydrazinecarbothioamide (3b). Yield: 79%, 1H-NMR
(d6-DMSO, 400 MHz, ppm): 12.08 (bs, 1H, NH), 10.26 (s, 1H, NH), 8.62 (s, 1H, CH), 8.47 (d,
J = 9.3 Hz, 1H, ArH), 7.68 (t, J = 7.1 Hz, 1H, ArH), 7.55 (d, J = 7.7 Hz, 2H, ArH), 7.43–7.37 (m, 3H,
ArH), 7.23 (t, J = 7.6 Hz, 1H, ArH). 13C-NMR (d6-DMSO, 100 MHz, ppm): 176.9, 139.5, 138.9,
134.3, 132.6, 131.8, 131.5, 128.6, 126.8, 126.6, 126.0. MP: 203–204 °C, HR-MS (EI): 323.0066
(calc. for C14H11Cl2N3S: 323.0051).
(E)-2-(3,4-Dichlorobenzylidene)hydrazinecarbothioamide (3f). Synthesis and more detailed analysis
1
of the structure is available [44]. Yield: 85%, H-NMR (d6-DMSO, 400 MHz, ppm): 11.56 (s, 1H,
NH), 8.28 (s, 1H, NH), 8.24 (d, J = 1.7 Hz, 1H, ArH), 8.00 (s, 1H, CH), 7.72 (dd, J = 8.4, 1.8 Hz, 1H,
ArH), 7.64 (d, J = 8.3 Hz, 1H, ArH). 13C-NMR (d6-DMSO, 100 MHz, ppm): 178.7, 139.9, 135.6,
132.3, 132.3, 131.2, 128.7, 128.2. MP: 205–206 °C [34]. IR: 3395, 3256 (NH), 3154 (PhH), 2992
(CH), 1596 (NH2), 1538 (C=N), 1471 (CH/NH), 1291 NH), 1129 (N-N), 1100 (), 1065 (NH),
1031(ring), 938(CH), 870, 823 (CS), 625 C-N). UV-VIS (MeOH; log : 320.0 (4.01), 239.0 (3.63),
203.0 (3.92).
(E)-2-(3,4-Dichlorobenzylidene)-N-methylhydrazinecarbothioamide (3f). Yield: 89% 1H-NMR
(d6-DMSO, 400 MHz, ppm): 11.63 (s, 1H, NH), 8.68 (d, J = 3.8 Hz, 1H, NH), 8.20 (s, 1H, CH), 8.00
(s, 1H, ArH), 7.69 (dd, J1 = 23.9, J2 = 8.3 Hz, 2H), 3.03 (d, J = 4.3 Hz, 3H). 13C-NMR (d6-DMSO, 100
MHz, ppm): 178.4, 139.4, 135.7, 132.2, 132.2, 131.2, 128.5, 128.1, 31.62. MP: 202–203 °C.
HR-MS (EI): 260.9902 (calc. for C9H9Cl2N3S: 260.9894).

Molecules 2012, 17
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1
(E)-2-(3,4-Dichlorobenzylidene)-N,N-dimethylhydrazinecarbothioamide (3h). Yield: 79% H-NMR
(d6-DMSO, 400 MHz, ppm): 11.12 (s, 1H, NH), 8.17 (s, 1H, CH), 7.86 (d, J = 1.7 Hz, 1H, ArH),
7.70–7.63 (m, 1H, ArH), 3.30 (s, 6H, CH3). 13C-NMR (d6-DMSO, 100 MHz, ppm): 181.0, 141.5,
136.0, 132.1, 132.0, 131.5, 128.4, 127.0, 42.5. MP: 138–139 °C HR-MS (EI): 275.0048 (calc. for
C10H11Cl2N3S: 275.0051).
(E)-2-(3,4-Dichlorobenzylidene)-N-ethylhydrazinecarbothioamide (3i). Yield: 83%, 1H-NMR
(d6-DMSO, 400 MHz, ppm): 11.56 (s, 1H, NH), 8.73 (t, J = 5.7 Hz, 1H, NH), 8.19 (d, J = 1.7 Hz, 1H,
ArH), 8.00 (s, 1H, CH), 7.78–7.70 (m, 1H, ArH), 7.67 (d, J = 8.3 Hz, 1H), 3.61 (m, 2H, CH2), 1.16
(t, J = 7.1 Hz, 3H, CH3). 13C-NMR (d6-DMSO, 100 MHz, ppm): 177.3, 139.5, 135.6, 132.2, 132.2, 131.3,
128.6, 128.1, 38.8, 15.0. MP: 177–178 °C, HR-MS (EI): 275.0058 (calc. for C10H11Cl2N3S: 275.0051).
(E)-2-(3,4-Dichlorobenzylidene)-N-phenylhydrazinecarbothioamide (3j). Yield: 86%, 1H-NMR
(d6-DMSO, 400 MHz, ppm): 11.96 (s, 1H, NH), 10.27 (s, 1H, NH), 8.35 (s, 1H, NH), 8.12 (s, 1H, Ar),
7.82 (dd, J = 8.4, 1.7 Hz, 1H, ArH), 7.68 (d, J = 8.3 Hz, 1H, ArH), 7.53 (d, J = 7.7 Hz, 2H, Ph), 7.39
13
(t, J = 7.8 Hz, 2H, Ph), 7.24 (t, J = 7.4 Hz, 1H, Ph). C-NMR (d6-DMSO, 100 MHz, ppm): 176.9,
140.6, 139.5, 135.4, 132.5, 132.3, 131.2, 128.9, 128.6, 126.9, 126.1. MP: 200–201 °C [28].
(E)-2-(4-Bromobenzylidene)hydrazinecarbothioamide (3k). Yield: 69% 1H-NMR (d6-DMSO,
400 MHz, ppm): 11.48 (s, 1H, NH), 8.23 (s, 1H, NH), 8.07 (s, 1H, NH), 8.02 (s, 1H, CH), 7.77 (d,
13
J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH). C-NMR (d6-DMSO, 100 MHz, ppm): 178.6,
141.4, 134.0, 132.1, 129.6, 123.5. MP: 222 °C [48].
(E)-2-(4-bromobenzylidene)-N-methylhydrazinecarbothioamide (3l). Yield: 72%; 1H-NMR (d6-
DMSO, 400 MHz, ppm): 11.54 (s, 1H, NH), 8.57 (d, J = 4.0 Hz, 1H), 8.01 (s, 1H, CH), 7.77 (d,
J = 8.3 Hz, 2H, ArH), 7.61 (d, J = 8.4 Hz, 2H, ArH), 3.02 (d, J = 4.4 Hz, 3H, CH3). 13C-NMR
(d6-DMSO, 100 MHz, ppm): 178.3, 140.8, 134.1, 132.0, 129.4, 123.4, 31.2. MP: 204–205 °C [49].
(E)-2-(4-bromobenzylidene)-N,N-dimethylhydrazinecarbothioamide (3m). Yield: 61%, 1H-NMR
(d6-DMSO, 400 MHz, ppm): 11.00 (s, 1H, NH), 8.17 (s, 1H, CH), 7.71–7.45 (m, 4H, ArH), 3.29
13
(s, 6H, CH3). C-NMR (d6-DMSO, 100 MHz, ppm): 181.0, 143.0, 134.4, 132.2, 129.0, 123.1, 42.6.
MP: 150–151 °C [50].
(E)-2-(4-Bromobenzylidene)-N-ethylhydrazinecarbothioamide (3n). Synthesis and more detailed
1
analysis of the structure is available [44]. Yield: 78%, H-NMR (d6-DMSO, 400 MHz, ppm): 11.47
(s, 1H, NH), 8.61 (t, J = 5.8 Hz, 1H, NH), 8.02 (s, 1H, CH), 7.77 (d, J = 8.5 Hz, 2H, ArH), 7.61
(d, J = 8.5 Hz, 2H, ArH), 3.63–3.56 (m, J = 7.0 Hz, 2H, CH2), 1.15 (t, J = 7.1 Hz, 3H, ArH).
13C-NMR (d6-DMSO, 100 MHz, ppm): 177.2, 140.9, 134.1, 132.0, 129.6, 123.4, 38.8, 15.0. MP:
199–200 °C, HR-MS(EI): 284.9944 (calc. for C10H12BrN3S Exact Mass: 284,9935), IR: 3363 NH),
3139 PhH), 2980 CH), 1589 C=N), 1538 C-N), 1522 CH), 1485 CH/NH), 1397 CNH), 1293
(CNH), 1104 N-N), 1081  1066 NH), 1006 ring), 921 CH), 816 CS), 623 C-N).
UV-VIS (MeOH, log : 330 (sh), 319.5 (3.98), 233.0 (3.65), 218 (sh), 202.5 (3.91).

Molecules 2012, 17
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(E)-2-(4-bromobenzylidene)-N-phenylhydrazinecarbothioamide (3o). Yield: 68%, 1H-NMR
(d6-DMSO, 400 MHz, ppm): 11.88 (s, 1H, NH), 10.17 (s, 1H, NH), 8.13 (s, 1H, CH), 7.89 (d,
J = 8.5 Hz, 2H, ArH), 7.63 (d, J = 8.5 Hz, 2H, ArH), 7.56 (d, J = 7.6 Hz, 2H, Ph), 7.38 (t, J = 7.8 Hz,
2H, Ph), 7.22 (t, J = 7.4 Hz, 1H, Ph). 13C-NMR (d6-DMSO, 100 MHz, ppm):176.6, 142.0, 139.5,
133.9, 132.1, 130.0, 128.5, 126.5, 125.9, 123.7. MP: 197–198 °C [49].
3.2.2. General Procedure for the Synthesis of Thiosemicarbazones 4a–l
All quinoline-based thiosemicarbazones were prepared according to a recently published procedure
[34]. Equimolar quantities of an appropriate thiosemicarbazide and benzaldehyde derivative were
mixed and 2 drops of glacial acetic acid were added. The resulting mixture was heated in a microwave
reactor at 85 °C for 12 min (max microwave power 75 W). After cooling, the precipitated soli were
filtered and washed with ether and crystallized from methanol. The compounds 4a–l studied are presented
in Table 2.
3.3. Lipophilicity Determination Using HPLC (Capacity Factor k/Calculated log k)
A Waters Alliance 2695 XE HPLC separation module and a Waters Photodiode Array Detector
2996 (Waters Corp., Milford, MA, USA) were used. A Symmetry® C18 5 μm, 4.6  250 mm, Part No.
WAT054275 (Waters Corp.) chromatographic column was used. The HPLC separation process was
monitored using Empower™ 2 Chromatography Data Software, Waters 2009 (Waters Corp.). A
mixture of MeOH p.a. (55%) and H2O-HPLC-Mili-Q Grade (45%) was used as a mobile phase. The
total flow of the column was 0.9 mL/min, injection volume 30 μL, column temperature 30 °C and
sample temperature 10 °C. A detection wavelength of 210 nm was chosen. A KI methanolic solution
was used for the determination of dead time (tD). Retention times (tR) were measured in minutes. The
capacity factors k were calculated using the Empower™ 2 Chromatography Data Software according
to the formula k = (tR − tD)/tD, where tR is the retention time of the solute, whereas tD denotes the dead
time obtained using an unretained analyte. Log k, calculated from the capacity factor k, is used as the
lipophilicity index converted to log P scale. The log k values of the individual compounds are shown
in Tables 1 and 2.
3.4. Lipophilicity Calculations
Log P, i.e., the logarithm of the partition coefficient for n-octanol/water, was calculated using
the programs ChemOffice [45] and ACD/LogP [46]. Clog P values (the logarithm of n-octanol/water
partition coefficient based on established chemical interactions) were generated using ChemOffice
software [45]. The results are shown in Tables 1 and 2.
3.5. Study of the Inhibition Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and
Kralova [51]. The inhibition of the photosynthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Fisher Scientific, Waltham, MA, USA) using
an artificial electron acceptor 2,6-dichlorophenol-indophenol (DCIPP) according to Kralova et al. [52]

Molecules 2012, 17
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and the rate of photosynthetic electron transport was monitored as a photoreduction of DCPIP. The
measurements were carried out in a phosphate buffer (0.02 mol/L, pH 7.2) containing sucrose
(0.4 mol/L), MgCl2 (0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll content was 30 mg/L in these
experiments and the samples were irradiated (~100 W/m2 with 10 cm distance) with a halogen lamp
(250 W) using a 4 cm water filter to prevent warming of the samples (suspension temperature 22 °C).
The compounds studied were dissolved in DMSO due to their limited water solubility. The applied
DMSO concentration (up to 4%) did not affect the photochemical activity in the spinach chloroplasts.
The inhibitory efficiency of the compounds studied was expressed by IC50 values, i.e., by the molar
concentration of the compounds that caused a 50% decrease in the oxygen evolution rate relative to the
untreated control. The comparable IC50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1-
dimethylurea, DCMU (Diurone®) was about 1.9 μmol/L. The results are summarized in Table 3.
3.6. In Vitro Antifungal Susceptibility Testing
The broth microdilution test [53–55] was used for the assessment of the in vitro antifungal activity
of the synthesized compounds against Candida albicans ATCC 44859 (CA), Candida tropicalis
156 (CT), Candida krusei ATCC 6258 (CK), Candida glabrata 20/I (CG), Trichosporon beigelii
1188 (TB), Aspergillus fumigatus 231 (AF), Absidia corymbifera 272 (AC), and Trichophyton
mentagrophytes 445 (TM). Fluconazole (FLU) was used as the standard since it is a clinically used
antimycotic drug. The procedure was performed using a twofold dilution of the compounds in RPMI
1640 (Sevapharma a.s., Prague, Czech Republic) buffered to pH 7.0 with 0.165 mol of 3-morpholino-
propane-1-sulfonic acid (MOPS, Sigma, Prague, Czech Republic). The final concentrations of the
compounds ranged from 500 to 0.975 μmol/L. Drug-free controls were included. The determination of
minimum inhibitory concentration (MIC) was performed according to the Clinical and Laboratory
Standards Institute (CLSI, formerly NCCLS) reference protocol M27-A2 for yeasts (IC80 value) and
M38-A for moulds (IC50 value). IC80 and IC50 were defined as an 80% resp. 50% or greater reduction
in growth in comparison with the control. The values of MICs were determined after 24 and 48 h of
static incubation at 35 °C. The final MICs for T. mentagrophytes, were determined after 72 and 120 h
of incubation. The results are summarized in Table 3.
3.7. Cell Culture and in Vitro Antiproliferative Activity
The human colon cancer cell line HCT116 with the wild type p53 gene (p53+/+) were obtained from
Maria Sklodowska-Curie Memorial Cancer Center in Gliwice, Poland. Briefly, the cells were seeded
in 96-well plates (3 × 103 cells/well) in cellular proliferation assays 24 h before the addition of the
chelators. The assay was performed following a 72 h incubation with varying concentrations of the
agents. Each compound was tested in triplicate in a single experiment with each experiment being
repeated 3 times. After 72 h incubation with the compounds being investigated, 20 µL of MTS
solution was added to each well (100 µL) and incubated for 1 h at 37 °C. The optical densities of the
samples were analyzed at 490 nm. Results were expressed as a percentage of the control. The
inhibitory concentration (IC50) was defined as the compound concentration necessary to reduce the
absorbance to 50% of the untreated control. The results are summarized in Table 3.

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4. Conclusions
A series of fifteen substituted benzylidenethiosemicarbazones and twelve substituted
quinolinylvinylthiosemicarbazones were designed, prepared and characterized. The synthesis of these
compounds was carried out in microwave conditions, which improves the yield and purity of products
isolated. The prepared compounds were tested for their ability to inhibit photosynthetic electron
transport (PET) in spinach chloroplasts (Spinacia oleracea L.), for their antifungal activity and for
their anticancer activity against human colon cancer. All of the compounds were primarily designed as
potential thiosemicarbazone-based iron chelators. All of the compounds discussed showed moderate
PET inhibition as well as an antifungal effect. (E)-2-(quinolin-2-ylvinyl)-N,N-dimethylhydrazine-
carbothioamide (4b) expressed the strongest activity among all of the compounds tested; it was
comparable to or more active than fluconazole against Aspergillus fumigatus, Absidia corymbifera,
Candida tropicalis and Candida krusei. Compounds 4b and 4e expressed micromolar antiproliferative
activity better than standard doxorubicin. According to the results, the quinoline nucleus and the
N,N-dimethylhydrazinecarbothioamide chain can be considered as basic structural fragments for high
antiproliferative and antifungal activity. Iron chelation was assumed in UV-VIS spectra analysis.
According to the results we can concluded that iron chelating ability of the compounds is one of
factors necessary for the activity.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/17/11/13483/s1.
Acknowledgements
This study was supported by the grant from the Polish Ministry of Science R 0504303, by the
Slovak Grant Agency VEGA Grant No. 1/0612/11 and by Sanofi-Aventis Pharma Slovakia. M.S. was
supported by a TWING fellowship and NCN grant DEC-2011/01/N/NZ4/01166. A.M.-W. appreciates
the support of the UPGOW fellowship, NCN grant N405/068440 and Dokto-RIS studentship.
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distributed under the terms and conditions of the Creative Commons Attribution license
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