Disubstituted 4-chloro-3-nitrophenylthiourea derivatives: antimicrobial and cytotoxic studies

Article abstract of DOI:10.3390/molecules23102428

4-Chloro-3-nitrophenylthioureas 1-30 were synthesized and tested for their antimicrobial and cytotoxic activities. Compounds exhibited high to moderate antistaphylococcal activity against both standard and clinical strains (MIC values 2-64 g/mL). Among th

Full text of DOI:10.3390/molecules23102428

molecules
Article
Disubstituted 4-Chloro-3-nitrophenylthiourea
Derivatives: Antimicrobial and Cytotoxic Studies
Anna Bielenica 1,
*
, Giuseppina Sanna 2, Silvia Madeddu 2, Gabriele Giliberti 2,
Joanna Stefan´ska 3, Anna E. Kozioł 4, Oleksandra Savchenko 5, Paulina Strzyga-Łach 1
,
Alicja Chrzanowska , Grazyna Kubiak-Tomaszewska and Marta Struga 1,7
1
6
˙
1
Department of Biochemistry, Medical University, 02-097 Warszawa, Poland;
paulina.strzyga@gmail.com (P.S.-Ł.); chrzanow.a@gmail.com (A.C.); mstruga@wum.edu.pl (M.S.)
Department of Biomedical Sciences, Section of Microbiology and Virology, University of Cagliary,
Cittadella Universitaria, 09042 Monserrato, Italy; g.sanna@unica.it (G.S.); silvia.madeddu@unica.it (S.M.);
ggiliberti@unica.it (G.G.)
Department of Pharmaceutical Microbiology, Medical University, 02-007 Warszawa, Poland;
jstefanska@wum.edu.pl
Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland;
anna.koziol@poczta.umcs.lublin.pl
Ecotech Complex, Maria Curie-Sklodowska University, 20-031 Lublin, Poland;
oleksandra.savchenko@poczta.umcs.lublin.pl
2
3
4
5
6
Department of Biochemistry and Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw,
02-097 Warszawa, Poland; grazynakubiak@poczta.onet.pl
Laboratory of Centre for Preclinical Research, Medical University of Warsaw, 02-097 Warszawa, Poland
Correspondence: abielenica@wum.edu.pl; Tel.: +48-22-572-06-93
7
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 5 September 2018; Accepted: 19 September 2018; Published: 21 September 2018
Abstract: 4-Chloro-3-nitrophenylthioureas
cytotoxic activities. Compounds exhibited high to moderate antistaphylococcal activity against both
standard and clinical strains (MIC values 2–64 g/mL). Among them derivatives with electron-donating
alkyl substituents at the phenyl ring were the most promising. Moreover, compounds and 19
were cytotoxic against MT-4 cells and various other cell lines derived from human hematological tumors
(CC50 10 M). The influence of derivatives 11 13 and 25 on viability, mortality and the growth rate of
1–30 were synthesized and tested for their antimicrobial and
µ
1–6
8–
≤
µ
,
immortalized human keratinocytes (HaCaT) was observed.
Keywords: antimicrobial activity; biofilm; cytotoxicity; thiourea derivatives; X-ray crystallography
1. Introduction
In the field of medical chemistry, thiourea derivatives are of great interest due to their synthetic
utility and biological importance. The thiourea moiety is a structural part of many bioactive compounds,
that display antimicrobial [
1
–
5], antifungal [6], antitubercular [2,7,8], antiviral [9–12], and central
nervous system stimulating [13
–
16] properties. Derivatives of this group are also viewed as one of
the most promising classes of anticancer agents. 1,2,4-Triazole-linked (thio)urea conjugates have been
described as cytotoxic and apoptosis inducing substances [17]. The same properties, supported by
DNA topoisomerase II inhibition, were proved for podophylltoxin-thiourea congeners [18]. Diaryl
thiourea derivatives bearing 1H-indazole-3-amine scaffold displayed anti-angiogenic potential as
selective multi-target receptor tyrosine kinase (RTK) inhibitors [19]. Particularly, they have blocked
activities of VEGFR-2, Tie-2 and EphB4 kinases, thus as a result can prevent tumorigenesis. On the other
hand, bis-thiazoles derived from disubstituted thioureas are supposed to be involved in the regulation
of the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), overexpressed in
Molecules 2018, 23, 2428; doi:10.3390/molecules23102428
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2428
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pathological conditions; e.g., cancer [20]. Another group, thioureido benzenesulfonamide derivatives,
was found to inhibit carbonic anhydrase CA IX, which triggers the migration of tumor cells and
results in a raise of the aggressive/invasive phenotype of cancer [6]. Pyrimidinyl acyl thioureas have
shown the ability to block Heat Shock Protein 90 (Hsp90) ATP binding site [21]. The role of this
protein is stabilization and regulation of oncogenic client proteins, therefore designing its inhibitors
is a novel cancer therapy method. What is more, in different types of cancer, overexpression and
mutation of the epidermal growth factor receptor (EGFR) have been observed; thus, finding inhibitors
of EGFR kinases among (thio)ureido-quinazoline derivatives is also a promising approach to tumor
treatment [22,23]. It was proven that the anticancer potency of symmetrical 1,3-phenyl bis-thiourea
compounds were combined with their tubulin-binding properties [24]. By halting mitosis of tumor
cells in prometaphase, this lead to their death via apoptosis. A series of pyrimidine-thiourea conjugates
were found to exhibit an inhibitory effect against histone lysine demethylase 1 (LSD1), overexpressed
in gastric, prostate, lung, and breast cancer cells [25,26]. Moreover, miscellaneous thiourea derivatives
carrying sulfonamide moieties were proven to possess antiproliferative properties by docking to
the active site of the mitogen kinase enzyme (MK-2), controlling the signal transduction pathway
in cell proliferation [5,27,28]. The cytotoxicity of acridinylthioureas originates from their ability to
intercalate with DNA and as a result inhibit telomerase or topoisomerase action [29]. Thiourea-derived
compounds were also employed in the field of coordination chemistry as versatile chelating agents.
The Ru(II), Pd(II) and Pt(II)-based complexes of N,N-disubstituted thioureas have exerted cytotoxic
properties via DNA binding ability [30–32].
A noticeable cytotoxic potential of mentioned thiourea-derived compounds was observed against
a wide range of solid tumors, such as breast [
prostate [ 18 31 33], cervical [ 29 30], brain [20 24
human leukemia [29]. Simultaneously some of them, when tested on normal human cells, were found
to possess a low cytotoxicity profile [ 17 20]. The main structural element of anticancer active thioureas
were the presence of “the butterfly-like” tricyclic ring of acridine [29] or other heterocycles, such as
pyrimidine [ 21], quinoline [27], quinazoline [22 23], furane [32], benzimidazole [ ], chrysene [33],
benzodioxole [18], triazole [17] or thiazole [20]. On the other hand, the most promising functionalities of a
terminal aromatic ring were mainly electron-withdrawing substituents, such as nitro [27 33 35], cyano [17],
trifluoromethyl/methoxy [17 19], halogeno [ 20 22 23], but also electron-withdrawing—methoxy [18 21 25
and methyl [24] groups. Among them, compounds containing the nitro group attached to the aromatic
moiety (e.g., nitrofurans, bisnaphthalimides) are typically used as antibiotics and antimicrobials [36 37].
4,
6
,
17
,
21
,
23
,
32
,
33], lung [
4
,
20
,
22
,
23
,
27
,
29
,
31
,
34], liver [
5],
4,
,
–
4,
,
,
,
29], gastric [25], or colon [27
,
29
,35] carcinoma, as well as
1, ,
5,6,
,
4
,
,
,
5,
,
,
, , ]
,
The introduction of an additional deactivating substituent, such as a halogen atom, might increase the
bioactivity of a disubtituted derivative, because of a stronger electronegativity effect achieved [1,2].
Prompted by the above facts and as a continuation of our interest in biologically active
thioureas [1,2,38], we report the synthesis of some new 4-chloro-3-nitrophenylthiourea derivatives and
complete the cytotoxic profile of compounds of this group [2].
2. Results and Discussion
2.1. Chemistry
In this paper new 1,3-disubstituted thioureas 20–24 and 27–30 were synthesized from 4-chloro-3-
nitroaniline and variety of isothiocyanates (Scheme 1, Table 1). Reactions were conducted in anhydrous
acetonitrile at room temperature. The structures of new compounds were established by spectroscopic
methods (1H NMR, 13C NMR, MS, and IR). The derivatives
1–19 and 25–26 were obtained previously [2].

Molecules 2018, 23, 2428
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Scheme 1. Synthesis of 4-chloro-3-nitrophenylthiourea derivatives 20–24, 27–30.
Table 1. Cytotoxicity and anti-HIV activity of 4-chloro-3-nitrophenylthiourea derivatives 1–30.
MT-4 *
CC50 [µM]
HIV-1IIIB
EC50 [µM]
*
Comp.
R
1
2
1
2
2-chlorophenyl
3-chlorophenyl
8.8
7.0
>8.8
>7.0
3
4
4-chlorophenyl
2-bromophenyl
7.0
9.6
>7.0
>9.7
5
6
3-bromophenyl
4-bromophenyl
8.0
6.6
>8.0
>6.6
7
8
2-fluorophenyl
3-fluorophenyl
13.0
9.0
>13.0
>9.0
9
4-fluorophenyl
7.0
7.3
7.9
8.0
8.0
8.2
8.0
7.0
>7.0
>7.3
>7.9
>8.0
>8.0
>8.2
>8.0
>7.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3-chloro-4-fluorophenyl
3,4-dichlorophenyl
2,4-dichlorophenyl
3-chloro-4-methylphenyl
5-chloro-2-methylphenyl
3-(trifluoromethyl)phenyl
4-(trifluoromethyl)phenyl
4-iodophenyl
7.8
7.0
8.2
>7.8
>7.0
>8.2
4-nitrophenyl
4-cyanophenyl
4-methylphenyl
14.6
35.0
18.7
23.0
16.0
36.7
16.7
15.5
43.0
33.7
25.6
>14.6
>35.0
>18.7
>23.0
>16.0
>36.7
>16.7
>15.5
43.0
4-methoxyphenyl
4-butyl-2-methylphenyl
phenyl
bicyclo[2.2.1]hept-2-yl
benzyl
1-phenylethyl
(2-methyl)prop-2-en-1-yl
prop-2-en-1-yl
3-(methylsulfanyl)propyl
furan-2-ylmethyl
>33.7
>25.6
1
Compound concentration (
µ
M) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined
2
by the MTT method; Compound concentration (
µM) was required to achieve 50% protection of MT-4 cells
from the HIV-1 induced cytopathogenicy, as determined by the MTT method; * Reference compound—Efavirenz
(CC50 = 46 µM, EC50 = 0.002 µM).
2.2. Antimicrobial Studies
Newly obtained 4-chloro-3-nitrophenylthioureas 20
antimicrobial activity against a set of bacterial and fungal strains. The initial testing by the disc
diffusion method [39 40] chose derivatives with meaningful antimicrobial properties. Considering the
fact that all compounds exhibited activity against Gram-positive strains (GIZ 13 mm), in the next
step the minimal inhibitory concentrations (MIC) were assessed by the twofold serial microdilution
method [41 42]. The results showed that all investigated compounds (except 21) exhibited the potential
–24 and 27–30 were evaluated for their in vitro
,
≥
,
to moderate antibacterial potency, mainly against standard staphylococcal and M. luteus strains
(Table 2).

Molecules 2018, 23, 2428
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A close examination of the structures of the active compounds revealed that the nature of the
substituent at the thiourea moiety governs their antimicrobial potential. In general, the most prominent
activity against standard strains was confined to derivatives comprising the electron-donating alkyl
groups at the C2/C4 position of the aromatic ring (20
from 2 to 4 g/mL. The moderate activity (MIC range 8–16
for bicyclo[2.2.1]hept-2-yl- (24), phenyl- (23), furan-2-ylmethyl- (30) and (thio)alkylthioureas (27
What is more, compounds possessing an alkyl chain at the thiourea nitrogen (27 29) exerted some
activity against P. vulgaris, representing Gram-negative strains (MIC 32–128 g/mL). Only one analog
,
22). For these derivatives the MIC values ranged
g/mL) towards staphylococci was denoted
29).
µ
µ
,
–
µ
of the series, 4-methoxyphenylthiourea (21), was devoid of activity against tested microorganisms.
The potency of thioureas 22 and 24 towards all hospital methicillin-resistant strains of S. epidermidis
was 2–16 stronger than the reference Ciprofloxacin (Table 3). The antistaphylococcal activity of other
tested compounds was equal or 2–4 times higher than observed for the standard antibiotic. Selected
strains (MRSE 405/11, 406/11, 409/11, 411/11, 422/11) were remarkably less susceptible for the presence
of synthesized thiourea than for the reference drug.

Molecules 2018, 23, 2428
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Table 2. Activity of compounds against standard bacterial strains, expressed as minimal inhibitory concentrations (MIC, µg/mL).
S. aureus
NCTC 4163
S. aureus
ATCC 25923
S. aureus
ATCC 6538
S. aureus
ATCC 29213
S. epidermidis
ATCC 35984
S. epidermidis
ATCC 12228
B. subtilis
ATCC 6633
B. cereus
ATCC 11778
E. faecalis
ATCC 29212
M. luteus
ATCC 9341
M. luteus
ATCC 10240
Comp.
20
22
23
24
27
28
29
30
* Ref.
2
4
16
8
16
64
16
16
0.25
2
2
16
8
16
32
16
8
4
2
16
8
16
32
16
16
0.25
2
2
16
8
16
64
16
16
0.5
4
4
4
4
4
4
8
4
4
8
8
8
32
8
16
0.25
-
-
16
-
64
64
64
32
1
4
4
4
32
16
32
16
16
1
4
2
8
16
32
32
32
16
2
16
16
16
32
16
32
0.25
16
16
16
32
16
32
0.25
8
16
32
16
16
<0.12
0.5
* Ref.—Ciprofloxacin.
Table 3. Activity of compounds against hospital methicillin-resistant strains of Staphylococcus epidermidis (MRSE)—minimal inhibitory concentrations (MIC,
µg/mL).
S. epidermidis
MRSE 403/11
S. epidermidis
MRSE 404/11
S. epidermidis
MRSE 405/11
S. epidermidis
MRSE 406/11
S. epidermidis
MRSE 407/11
S. epidermidis
MRSE 409/11
S. epidermidis
MRSE 411/11
S. epidermidis
MRSE 412/11
S. epidermidis
MRSE 413/11
S. epidermidis
MRSE 422/11
Comp.
20
22
23
16
4
16
4
16
8
16
4
16
4
16
4
16
8
16
8
16
4
16
4
16
4
16
4
16
4
16
4
32
8
32
4
16
8
16
4
32
8
16
4
24
27
28
29
30
32
64
32
32
16
32
64
32
32
64
32
64
32
32
4
32
64
32
64
0.5
32
64
32
32
64
32
64
32
32
2
32
64
32
32
4
64
128
32
32
64
32
64
32
32
32
64
128
32
32
4
* Ref.
* Ref.—Ciprofloxacin.

Molecules 2018, 23, 2428
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It is worth noting that the factor influencing the biological activity could be a molecular flexibility.
It is evident from the determined crystal structures of 20 and 24 that molecules of this group of
derivatives are able to adopt different conformations (Figure 1). Variable orientations of the thiourea
C=S bond versus either 4-chloro-3-nitrophenyl or the second substituent could be described as cis-trans
and trans-cis, respectively. Further, the patterns of intermolecular hydrogen bonding are specific for
given conformers.
20 conformer cis-trans
24 conformer trans-cis
Figure 1. Perspective view of molecules 20 and 24. Conformers are defined by relative orientation of
4-chloro-3-nitrophenyl and aryl/alkyl groups to thiourea C=S bond.
2.3. Cytotoxicity and Anti-HIV Activity
Due to the previously reported anti-HIV activities of thiourea derivatives [11
,43], both formerly
(1
–
19 25 26) and newly synthesized compounds (20 24 27 30) were tested in cell-based assay
,
,
–
,
–
against the human immunodeficiency virus type-1 (HIV-1), using Efavirenz as the reference inhibitor.
The cytotoxicity against MT-4 cells was evaluated in parallel with the antiviral activity (Table 1).
None of tested compounds showed selective antiviral activity against HIV-1. However, all compounds
turned out interestingly cytotoxic for exponentially growing MT-4 cells, with many of them cytotoxic in
the low micromolar range (CC50 < 10
line derived from a hematological human tumor prompted us to evaluate the antiproliferative activity of a
group of cytotoxic compounds ( 10 11 13 14 17 19), also for a panel of other human and solid
µM). The antiproliferative activity against the CD4+ human T cell
1,
4,
6,
9,
,
,
,
,
,
tumors, as well as for cell lines derived from normal human tissues (Table 4).
Interestingly, the tested compounds showed CC50 values comparable to that obtained with MT-4
cells, but they did not result highly selective, showing cytotoxicity also against the “normal” CRL7065
cell lines, even if with lower values. The highest selectivity versus CCRF-CEM and SK-MEL-28 cells
was observed for 4-bromophenyl (
6) and 4-iodophenylthiourea (17) derivatives. None of the tested
compounds showed CC50 values comparable with Camptotecin, used as the reference compound.

Molecules 2018, 23, 2428
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Table 4. Antiproliferative activity of selected compounds against human leukaemia/lymphoma, solid
tumor and normal cell lines.
MT-4
CCRF-CEM
WIL-2NS
CCRF-SB
SK-MEL-28
DU145
CRL7065
Comp.
1
CC50
1
4
6
8.8
9.6
6.6
7.0
7.3
7.9
8.0
8.2
7.8
13
22
14
30
7.0
11
7.0
9.9
7.6
10
7.0
10
14
22
8.7
15
6.0
20
6.6
8.5
6.0
9.0
6.0
9.0
0.07
7.6
8.0
6.0
8.4
6.5
6.0
7.0
7.8
11
18
20
30
22
16
16
16
18
30
20
0.3
6.0
9.1
5.0
8.0
4.0
9.4
6.0
9.1
0.003
8.0
9.4
8.8
8.6
6.7
9.8
8.3
9.2
0.004
9
10
11
13
14
17
19
* Ref
8.2
0.004
8.0
0.08
0.005
Data represent mean values for three independent determinations; 1 Compound concentration (
µM) required to
reduce cell proliferation by 50% under conditions allowing untreated controls to undergo at least three consecutive
rounds of multiplication, as determined by the MTT method; * Ref.—Camptotecin.
2.4. Cytotoxic Activity in HaCaT Cells
The most promising antistaphylococcal (11, 13) and antimycobacterial (25) agents among
derivatives presented in our previous study [ ] were now screened with respect to their toxicity
2
against HaCaT cell line (health cell line from adult human skin). The HaCaT cell line was chosen,
because it exhibits normal differentiation capacity, and its DNA fingerprint pattern is unaffected by
long-term cultivations, transformations, and multiple chromosomal alterations [44].
Human keratinocyte cell lines were exposed to different concentrations of synthesized compounds
for 24 h. Derivatives were tested in concentrations equal to their cytotoxic concentrations against
MT-4 cells (i.e., 8
µM for 11 and 13, and 37
µM for 25), as well as in concentrations 2 and 3 times
higher. At the concentration 8
µM, the thiourea compound 11 caused approximately 20% decrease in
cell viability, in comparison with controlled and untreated cells (Figure 2a), that indicated its weak
cytotoxic influence against normal cells. On the other hand, cytotoxicity of antibacterial agents 13 and
25 observed at the level of 50%, was found to be moderate according to ISO 10993-5 standards [45].
Results of the LDH (lactate dehydrogenase release) assay indicated that cell mortality in the
presence of compounds 11 and 13 increased to 20% and 30%, respectively (Figure 2b). The LD10
value (concentration at which 10% of cells were dead) was determined at the lowest investigated
concentration of the compound 11 (8 µM). Substance 13 at concentrations 8 and 16 µM was found to
be lowly cytotoxic, resulting in an approximate 10% release of LDH. Compound 25 had the strongest
impact on the mortality of tested cells, however, its concentration was the highest among all tested
compounds (37 µM). All applied concentrations of that derivative resulted in 7-fold higher cytotoxicity
as compared to the LD10 value.
Significant concentration-dependent changes in shape, size, and density of HaCaT cells were
observed under a light microscope after 24 h treatment with lowest concentrations of all investigated
compounds (Figure 3). At given concentrations, derivatives inhibited viable cell proliferation by about
30–40% (compounds 13 and 25) or 65% (11) (Figure 4). This suggests a direct effect on the HaCaT
viability, which was confirmed by the MTT assay. Derivatives 13 and 25 have demonstrated an
increased proliferation rate.
The tested substances modified cell viability as well as mortality and growth rate of HaCaT
cultures. The presented results demonstrate that derivative 11, one of the strongest antistaphylococcal
and antibiofilm agents, at its lowest concentration, possessed the lowest toxic effect on HaCaT cells.
Our studies suggest that all tested substances in their lowest concentrations were less toxic in normal
immortalized cell lines in comparison to cancer cell lines, however, further studies are needed to
describe the underlining mechanism for future application.

Molecules 2018, 23, 2428
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(a)
120
100
80
60
40
20
0
**
*
**
*
**
*
*
*
*
control solvent
11 - 11 - 11 -
13 - 13 - 13 - 25 -
25 -
25-
24 μM 16 μM 8 μM 24 μM 16 μM 8 μM 111 74 μM 37 μM
μM
Tested compounds
(b)
90
80
70
60
50
40
30
20
10
0
*
*
*
*
**
*
*
control solvent
11 -
11 -
11 -
13 -
13 -
13 -
25 - 25 -
25-
24 μM 16 μM 8 μM 24 μM 16 μM 8 μM 111 μM 74 μM 37 μM
Tested compounds
Figure 2. Cell viability assessed by MTT mitochondrial reduction (
of cell death in HaCaT cells treated for 24 h with compounds 11 13 and 25. Data are expressed as means
SD from three independent experiments performed in triplicate. Statistical significance: * p < 0.01 and
** p < 0.001 refer to the control (untreated) cells.
a), and LDH release (b) as a marker
,
±
(a)
(b)
Figure 3. Cont.

Molecules 2018, 23, 2428
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(c)
Figure 3. Morphological changes of HaCaT cells after 24 h treatment with compounds 11
observed under the light microscope (800 ): Untreated cells ( ). Cells treated for 24 h with compounds
(d)
,
13 and 25,
×
a
11, 13 and 25 in concentrations 8 µM, 8 µM and 37 µM, respectively (b–d).
120
viable cells
dead cells
100
80
60
40
20
0
*
*
**
*
**
*
Comp. 11
Comp. 13
Comp. 25
Figure 4. Proliferation rate assessment in HaCaT cells treated for 48 h with compounds 11
,
13 and 25
,
applied at concentrations 8 M, 8 M and 37 M, respectively. Data are expressed as means
from three independent experiments performed in triplicate. Statistical significance: * p < 0.01 and
** p < 0.001 refer to the control (untreated) cells.
µ
µ
µ
±
SD
3. Materials and Methods
3.1. Chemistry
All reagents and solvents were purchased from Alfa Aesar, Sigma Aldrich or POCh (Polskie
Odczynniki Chemiczne, Gliwice, Poland). The infrared (IR) spectra were obtained on Perkin Elmer
Spectrum 1000 spectrometer. The nuclear magnetic resonance (NMR) spectra were recorded on
Varian VNMRS 300 Oxford NMR spectrometer, using tetramethylsilane (TMS) as the internal
reference. Mass spectral electrospray ionization (ESI) measurements were carried out on Waters
ZQ Micro-mass instruments with quadruple mass analyzer, at a declustering potential of 40–60 V.
Flash chromatography was performed on Merck silica gel 60 (200–400 mesh) using chloroform eluent.
General procedure for the synthesis of (4-chloro-3-nitrophenyl)thiourea derivatives (20–24, 27–30)
To a solution of 4-chloro-3-nitroaniline (0.0029 mol; 0.5g) in dried acetonitrile (10 mL),
a corresponding isothiocyanate (0.0029 mol) was added. The mixture was stirred at room temperature

Molecules 2018, 23, 2428
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for 12 h. Next the solvent was evaporated and the solid residue was either crystallized from acetonitrile
(chloroform) or purified by column chromatography (chloroform).
The synthesis of derivatives 1–19, 25, 26 was published formerly [2].
1-(4-chloro-3-nitrophenyl)-3-(4-methylphenyl)thiourea (20)
◦
Yield 30%, yellow crystals, m.p. 156–158 C. FT-IR (KBr, cm−1): 3326.2, 3196.1 (
ν
N-H); 3113.6, 3024.4
ar(1,4)C-C); 1358.6
C=S); 864.0 ( C-Cl); 703.7
(
ν
ar C-H); 2977.6 (
ν
alC-H); 1589.6 (
δ
N-H); 1533.8 (
alC-C); 1159.7, 1145.4 (
ν
N-O); 1482.0 (
ν
arC-C); 1407.0 (
δ
(ν
N-O); 1299.9 (
(ν C=S). H NMR (300 MHz, DMSO)
δ
arC-H); 1236.4 (
ν
δ
arC-H); 1043.3 (
ν
δ
1
δ
: 10.07–10.05 (m, 2H, NH), 8.35 (d, 1H, J = 2.7 Hz, H-2), 7.80 (dd,
1H, J1 = 2.4 Hz, J2 = 2.7 Hz, H-6), 7.69 (d, 1H, J = 8.7 Hz, H-5), 7.34–7.29 (m, 2H, H30, H-50), 7.18–7.16 (d,
2H, J = 8.1 Hz, H-20, H-60), 2.29 (s, 3H, H-40a). 13C NMR (75.4 MHz, DMSO)
δ: 179.63 (C=S), 146.78 (C-3),
139.86 (C-1), 136.13 (C-10), 134.40 (C-40), 131.20 (C-4), 129.13 (C-30, C-50), 128.34 (C-5), 124.11 (C-20, C-60),
119.69 (C-2), 119.28 (C-6), 20.52 (C-40a). ESI MS: m/z = 320.0 [M − H]− (100%). Rf for TLC (CHCl3:MeOH
(1%)): 0.12.
Crystal data: crystal system triclinic, space group P-1, unit cell dimensions at 293 K: a = 7.442(1) Å
b = 8.012(2) Å, c = 12.944(3) Å,
= 98.74(3)◦, = 94.81(3)◦, = 100.17(3)◦, V = 746.0(3) Å3,
Z = 2, Dcalc = 1.433 g/cm3, F(000) = 332, = 3.647 mm−1 range = 3.48 to 75.15◦, reflections
,
α
β
γ
µ
, θ
collected/independent/observed 3147/3042/957, max. and min. transmission 0.7118 and 0.4283;
Goodness-of-fit on F3 0.929, final R indices [I > 2σσI)], R1 = 0.0479, wR2 = 0.1266, R indices (all
data), R1 = 0.2455, wR2 = 0.1857, residual electron density max./min. 0.38 and
No. 1849698.
−
0.36 e Å−3. CCDC
1-(4-chloro-3-nitrophenyl)-3-(4-methoxyphenyl)thiourea (21)
Yield 28%, yellow powder, m.p. 149–150.5 ◦C. FT-IR (KBr, cm−1): 3241.8, 3201.5 (
ν
N-H); 3056.2
N-O); 1241.5
(
(
ν arC-H); 1588.4 (
δ
N-H); 1548.9 (
ν
N-O); 1484.8 (
ν
arC-C); 1405.6 (
δ
ar(1,4)C-C); 1345.5 (
ν
ν
C-O-C); 1175.9, 1125.5 (
δ
arC-H); 1038.3 (
ν
C=S); 880.2 (
δ
C-Cl); 697.2 (ν
C=S). 1H NMR (300 MHz,
DMSO)
δ: 9.98 (s, 2H, NH), 8.34 (d, 1H, J = 2.4 Hz, H-2), 7.80 (dd, 1H, J1 = 2.4 Hz, J2 = 2.7 Hz, H-6),
7.68 (d, 1H, J = 8.7 Hz, H-5), 7.34–7.29 (m, 2H, H-30, H-50), 6.96–6.91 (m, 2H, H-20, H-60), 3.76 (s, 3H,
H-40a). 13C NMR (75.4 MHz, DMSO) : 179.54 (C=S), 156.65 (C-40), 146.46 (C-3), 139.63 (C-1), 131.15
δ
(C-10), 130.88 (C-4), 128.06 (C-5), 125.88 (C-20, C-60), 119.41 (C-2), 118.93 (C-6), 113.61 (C-30, C-50), 54.97
(C-40a). ESI MS: m/z = 336.0 [M − H]− (100%). Rf for TLC (CHCl3:MeOH (1%)): 0.17.
1-(4-butyl-2-methylphenyl)-3-(4-chloro-3-nitrophenyl)thiourea (22)
◦
Yield 40%, dark yellow solid, m.p. 67–69 C. FT-IR (KBr, cm−1): 3341.6, 3187.5 (
ν N-H);
3098.1 (ν arC-H); 2954.5, 2859.9 (
(δ ar(1,4)C-C); 1342.5 ( N-O); 1126.3 (
(300 MHz, DMSO)
ν
alC-H); 1584.5 (
δ
N-H); 1533.7 (
ν
N-O); 1481.5 ( arC-C); 1407.5
ν
ν
δ
arC-H); 1045.6 (
ν
C=S); 872.3 (
δ
C-Cl); 715.2 (ν
C=S). 1H NMR
δ
: 9.95 (s, 1H, NH), 9.66 (s, 1H, NH), 8.36 (d, 1H, J = 2.1 Hz, H-2), 7.82 (dd, 1H,
J1 = J2 = 2.7 Hz, H-6), 7.68 (d, 1H, J = 9.0 Hz, H-5), 7.17–6.98 (m, 3H, H-30, H-50, H-60), 2.57–2.55 (m,
2H, H-4a0), 2.20 (s, 3H, H-20a), 1.58–1.50 (m, 2H, H-40b), 1.35–1.27 (m, 2H, H-40c), 0.93–0.87 (m, 3H,
δ
H-40d). 13C NMR (75.4 MHz, DMSO) : 180.42 (C=S), 146.69 (C-3), 141.03 (C-20), 140.57 (C-40), 139.92
(C-1), 134.53 (C-10), 131.14 (C-4), 130.356 (C-30), 128.40 (C-5), 127.65 (C-50), 126.16 (C-60), 119.74 (C-2),
119.26 (C-6), 34.35 (C-40a), 33.04 (C-40b), 21.77 (C-40c), 17.75 (C-20a), 13.76 (C-40d). ESI MS: m/z = 376.1
[M − H]− (100%). Rf for TLC (CHCl3:MeOH (1%)): 0.08.
1-(4-chloro-3-nitrophenyl)-3-phenylthiourea (23)
◦
Yield 56%, pale yellow powder, m.p. 163–165 C. FT-IR (KBr, cm−1): 3323.5, 3161.3 (
ν N-H); 3089.6,
2982.8 (
1298.8, 1187.6, 1143.4 (
DMSO)
(d, 1H, J = 9.0 Hz, H-5), 7.47–7.44 (m, 2H, H-20, H-60), 7.37 (t, 2H, J = 7.8 Hz, H-30, H-50), 7.18 (t, 1H,
J = 7.35 Hz, H-40). 13C NMR (75.4 MHz, DMSO) : 179.95 (C=S), 147.09 (C-3), 140.05 (C-10), 139.08
ν
arC-H); 1592.0 (
δ
N-H); 1527.6 (
ν
N-O); 1482.0 (
ν
arC-C); 1407.5 (
δ
ar(1,4)C-C); 1362.7 (ν N-O);
C=S). H NMR (300 MHz,
1
δ
arC-H); 1047.1 (
ν
C=S); 863.7 (
δ
C-Cl); 695.2 (
ν
δ: 10.15 (s, 2H, NH), 8.35 (d, 1H, J = 2.4 Hz, H-2), 7.81 (dd, 1H, J1 = J2 = 2.4 Hz, H-6), 7.70
δ

Molecules 2018, 23, 2428
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0
0
0
0
0
(C-1), 131.53 (C-4), 128.92 (C-3 , C-5 ), 128.62 (C-5), 125.31 (C-4 ), 124.20 (C-2 , C-6 ), 119.98 (C-2), 119.65
(C-6). ESI MS: m/z = 330.0 [M + Na]+ (100%). Rf for TLC (CHCl3:MeOH (1%)): 0.15.
1-bicyclo[2.2.1]hept-2-yl-3-(4-chloro-3-nitrophenyl)thiourea (24)
◦
Yield 30%, dark yellow solid, m.p. 94–96 C. FT-IR (KBr, cm−1): 3304.1, 3196.0 (
3033.5 (ν arC-H); 2953.0, 2869.5 ( alC-H); 1576.1 ( N-H); 1540.6 ( N-O); 1478.9 ( arC-C); 1408.6
N-O); 1258.5, 1133.8, 1109.6 ( arC-H); 1047.2 ( C=S); 883.3 ( C-Cl); 714.7
ν N-H);
ν
δ
ν
ν
(
δ ar(1,4)C-C); 1343.4 (
ν
δ
ν
δ
1
(ν C=S). H NMR (300 MHz, DMSO)
δ
: 9.65 (s, 1H, NH), 8.47 (s, 1H, NH), 8.06 (m, 1H, H-2), 7.74
(dd, 1H, J1 = J2 = 2.4 Hz, H-6), 7.65 (d, 1H, J = 9.0 Hz, H-5), 3.98 (br. s, 1H, H-10), 2.29–2.26 (m,
2H, H-70), 1.77–1.70 (m, 1H, H-20), 1.54–1.30 (m, 4H, H-30, H-60), 1.22–1.08 (m, 3H, H-40, H-50). 13C
NMR (75.4 MHz, DMSO) δ: 178.76 (C=S), 146.20 (C-3), 139.47 (C-1), 130.68 (C-4), 126.10 (C-5), 117.27
(C-2, C-6), 56.34 (C-70), 40.97 (C-20, C-60), 34.70 (C-50), 34.67 (C-10), 27.29 (C-40), 25.48 (C-30). ESI MS:
m/z = 324.1 [M − H]− (100%). Rf for TLC (CHCl3:MeOH (1%)): 0.09.
Crystal data: crystal system triclinic, space group P-1, unit cell dimensions at 120 K: a = 7.273(2) Å
,
b = 9.652(2) Å, c = 11.862(3) Å,
Z = 2, Dcalc = 1.431 g/cm3, = 3.600 mm−1, F(000) = 340,
collected/independent/observed 10755/2994/2701; max. and min. transmission 1 and 0.75929;
Goodness-of-fit on F2 1.084, final R indices [I > 2
(I)] R1 = 0.0395, wR2 = 0.0943; R indices (all data)
R1 = 0.0456, wR2 = 0.0975, residual electron density max/min 0.45 and
0.54 e Å−3. The cycloalkyl
α
= 105.57(2)◦,
β
= 105.66(3)◦,
γ
= 96.76(2)◦, V = 756.0(3) Å3,
µ
θ
range 4.08 to 73.77◦, reflections
σ
−
group, viz. 1-bicyclo[2.2.1]hept-2-yl, is disordered over two positions with the site occupancy factors
being 0.64:0.36. CCDC No. 1849699.
1-(4-chloro-3-nitrophenyl)-3-(2-methylprop-2-en-1-yl)thiourea (27)
Yield 29%, light brown powder, m.p. 96–98 ◦C. FT-IR (KBr, cm−1): 3336.7, 3198.9 (
3022.8 ( arC-H); 1652.3 ( C=C); 1577.4 ( N-H); 1535.1 ( N-O); 1470.4 ( arC-C,
N-O); 1312.2, 1194.6, 1140.4 ( arC-H); 1040.5 ( C=S); 882.0 (
: 9.99 (br. s, 1H, NH), 8.42 (br. s, 1H, H-2), 8.23 (br. s, 1H, NH),
ν
N-H); 3075.6,
alC-H); 1407.5
C-Cl); 683.2
ν
ν
δ
ν
ν
δ
(δ ar(1,4)C-C); 1347.5 (
ν
δ
ν
δ
1
(ν C=S). H NMR (300 MHz, DMSO)
δ
0
0
7.76 (dd, 1H, J1 = J2 = 2.4 Hz, H-6), 7.68 (d, 1H, J = 9.0 Hz, H-5), 4.84 (s, 2H, H-3 ), 4.09 (br. s, 2H, H-1 ),
1.72 (s, 3H, H-20a). 13C NMR (75.4 MHz, DMSO)
δ: 181.06 (C=S), 147.07 (C-3), 141.78 (C-1), 140.09
(C-5), 131.60 (C-4), 127.49 (C-30), 118.79 (C-20), 110.78 (C-2, C-6), 49.19 (C-10), 20.65 (C-20a). ESI MS:
m/z = 284.0 [M − H]− (100%). Rf for TLC (CHCl3:MeOH (1%)): 0.10.
1-(4-chloro-3-nitrophenyl)-3-prop-2-en-1-ylthiourea (28)
◦
Yield 31%, pale yellow crystals, m.p. 112–114 C. FT-IR (KBr, cm−1): 3370.1, 3173.8 (
3002.6 ( arC-H, =C-H); 1641.6 ( C=C); 1600.7 ( N-H); 1534.5 ( N-O); 1471.6 (
(δ =C-H); 1409.4 ( ar(1,4)C-C); 1349.5 ( alC-H, =C-H); 1311.5 ( N-O); 1195.9, 1127.9 (
1041.4 ( C=S); 839.4 ( C-Cl); 711.5 ( : 9.97 (s, 1H, NH), 8.40 (d,
C=S). 1H NMR (300 MHz, DMSO)
1H, J = 2.1 Hz, H-2), 8.24 (s, 1H, NH), 7.76 (dd, 1H, J1 = 2.4 Hz, J2 = 2.7 Hz, H-6), 7.68 (d, 1H, J = 9.0 Hz,
ν
N-H); 3072.7,
arC-C); 1431.1
arC-H);
ν
ν
ν
δ
ν
ν
δ
δ
δ
ν
δ
ν
δ
ν
δ
H-5), 5.96–5.83 (m, 1H, H-2 ), 5.25–5.11 (m, 2H, H-3 ), 4.15 (m, 2H, H-1 ). 13C NMR (75.4 MHz, DMSO)
0
0
0
δ
: 180.52 (C=S), 146.82 (C-3), 139.78 (C-1), 134.19 (C-5), 131.32 (C-4), 127.31 (C-30), 118.64 (C-20), 116.08
(C-2, C6), 46.02 (C-10). ESI MS: m/z = 270.0 [M − H]− (100%). Rf for TLC (CHCl3:MeOH (1%)): 0.11.
1-(4-chloro-3-nitrophenyl)-3-[3-(methylsulfanyl)propyl]thiourea (29)
Yield 35%, light brown powder, m.p. 102–103.5 ◦C. FT-IR (KBr, cm−1): 3330.7, 3171.5 (
3070.3, 2999.3 ( arC-H); 2941.3, 2915.6 ( alC-H); 1572.8 ( N-H); 1531.5 ( N-O); 1475.5 (
1455.1 ( alC-H); 1410.5 ( ar(1,4)C-C); 1387.7 ( alC-H); 1351.8 ( N-O); 1298.9, 1154.7, 1134.1 (
1043.6 ( C=S); 887.3 (
C=S). 1H NMR (300 MHz, DMSO)
ν N-H);
arC-C);
δ arC-H);
: 9.88 (br. s, 1H, NH), 8.37
ν
ν
δ
ν
ν
δ
δ
δ
ν
ν
δ
C-Cl); 701.7 (
ν
δ
(d, 1H, J = 2.1 Hz, H-2), 8.17 (br. s, 1H, NH), 7.73 (dd, 1H, J1 = J2 = 1.8 Hz, H-6), 7.67 (d, 1H, J = 8.7 Hz,
H-5), 3.56–3.54 (m, 2H, H-10), 2.51–2.49 (m, 2H, H-30), 2.06–2.05 (m, 3H, H-40), 1.87–1.78 (m, 2H, H-20).
13C NMR (75.4 MHz, DMSO)
δ: 179.85 (C=S), 146.25 (C-3), 139.24 (C-1), 130.78 (C-4), 126.57 (C-5),

Molecules 2018, 23, 2428
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117.86 (C-2, C-6), 42.28 (C-10), 30.04 (C-20), 27.03 (C-30), 14.04 (C-40). ESI MS: m/z = 318.0 [M
(100%). Rf for TLC (CHCl3:MeOH (1%)): 0.09.
−
H]−
1-(4-chloro-3-nitrophenyl)-3-(furan-2-ylmethyl)thiourea (30)
Yield 58%, brown powder, m.p. 144–146 ◦C. FT-IR (KBr, cm−1): 3299.1, 3180.4 (
(ν arC-H); 1578.2 ( N-H); 1537.3 ( N-O); 1478.9 ( arC-C); 1448.8 ( alC-H); 1407.9 (
1364.3 ( alC-H); 1339.2 ( N-O); 1297.7, 1190.4, 1139.0 ( arC-H); 1018.2 ( C=S); 884.6 (
C=S). 1H NMR (300 MHz, DMSO)
δ: 9.98 (s, 1H, NH), 8.49 (s, 1H, NH), 8.41 (d, 1H, J = 2.1 Hz, H-2),
ν N-H); 3009.7
δ
ν
ν
δ
δ
ar(1,4)C-C);
δ
ν
δ
ν
δ C-Cl); 714.8
(
ν
7.76 (dd, 1H, J1 = J2 = 2.4 Hz, H-6), 7.68 (d, 1H, J = 8.7 Hz, H-5), 7.62–7.61 (m, 1H, H-50), 6.42 (dd, 1H,
J1 = 2.1 Hz, J2 = 1.8 Hz, H-40), 6.35 (d, 1H, J1 = 3.3 Hz, H-30), 4.73 (d, 2H, J = 5.1 Hz, H-10). 13C NMR
(75.4 MHz, DMSO) δ
: 180.27 (C=S), 150.75 (C-20), 146.55 (C-3), 142.09 (C-50), 139.42 (C-1), 131.07 (C-4),
127.09 (C-5), 118.62 (C-2), 118.37 (C-6), 110.25 (C-40), 107.44 (C-30). ESI MS: m/z = 310.0 [M
(100%). Rf for TLC (CHCl3:MeOH (1%)):0.15.
−
H]−
The NMR spectra of compounds 20–24, 27–30 can be found in Supplementary Materials.
3.2. X-ray Crystalography
The X-ray diffraction intensities were measured for 20 and 24 on a SuperNova diffractometer with
monochromatized CuK radiation ( = 1.54184 Å). Data sets were collected using the scan technique.
α
λ
ω
The programs CrysAlisPro [46] were used for data collection, cell refinement and data reduction;
empirical absorption correction was applied using spherical harmonics implemented in SCALE3
ABSPACK scaling algorithm. The structure was solved by direct methods using SHELXS-97 and
refined by the full-matrix least-squares on F2 using the SHELXL-97 [47]. Non-hydrogen atoms were
refined with anisotropic displacement parameters. Hydrogen atoms were positioned geometrically
and allowed to ride on their parent atoms, with Uiso(H) = 1.2 Ueq(C).
3.3. In Vitro Evaluation of Antimicrobial Activity
The antimicrobial activity of the compounds was tested on Gram-positive bacteria (Staphylococcus
aureus NCTC 4163, Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococcus
aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Staphylococcus epidermidis ATCC 35984,
Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 11778, Micrococcus
luteus ATCC 9341, Micrococcus luteus ATCC 10240); Gram-negative rods (Escherichia coli ATCC 10538,
Escherichia coli ATCC 25922, Escherichia coli NCTC 8196, Proteus vulgaris NCTC 4635, Pseudomonas
aeruginosa ATCC 15442, Pseudomonas aeruginosa NCTC 6749, Pseudomonas aeruginosa ATCC 27863,
Bordetella bronchiseptica ATCC 4617) and yeasts (Candida albicans ATCC 10231, Candida albicans ATCC
90028, Candida parapsilosis ATCC 22019). Other microorganisms used were obtained from the collection
of the Department of Pharmaceutical Microbiology, Medical University of Warsaw, Poland.
Antibacterial activity was examined by the disc-diffusion method under standard conditions
using Mueller-Hinton II agar medium (Becton Dickinson) according to CLSI (previously NCCLS)
guidelines [39]. Antifungal activities were assessed using Mueller-Hinton agar + 2% glucose and
0.5
µg/mL Methylene Blue Dye Medium [40]. Sterile filter paper discs (9 mm diameter, Whatman No
3 chromatography paper) were dripped with tested compound solutions (in DMSO) to load 400
µg
of a given compound per disc. Dry discs were placed on the surface of appropriate agar medium.
◦
The results (diameter of the growth inhibition zone) were read after 18 h of incubation at 35 C. Minimal
Inhibitory Concentration (MIC) was tested by the twofold serial microdilution method (in 96-well
microtiter plates) using Mueller-Hinton Broth medium (Beckton Dickinson) for bacteria or RPMI-1640
medium for Candida species according to CLSI guidelines [41
was prepared in DMSO and diluted in sterile water. Concentrations of tested agents ranged from
0.125 to 512
g/mL. The final inoculum of all studied microorganisms was 105 CFU/ mL−1 (colony
forming units per mL). Minimal inhibitory concentrations (the lowest concentration of a tested agent
,42]. The stock solution of tested agent
µ

Molecules 2018, 23, 2428
13 of 18
that prevents visible growth of a microorganism) were read after 18 h (bacteria) or 24 h (yeasts) of
incubation at 35 ◦C.
3.4. Cytotoxicity and Anti-HIV Assays
Cell line supporting the multiplication of Human Immunodeficiency Virus type-1 (HIV-1) was
the CD4+ human T-cells, derived from human hematological tumors, containing an integrated HTLV-1
genome (MT-4). Cells were purchased from American Type Culture Collection (ATCC). Human
Immunodeficiency Virus type-1 (HIV-1) IIIB laboratory strain was obtained from the supernatant of
the persistently infected H9/IIIB cells (NIH 1983).
Exponentially growing MT-4 cells were seeded at an initial density of 1
96-well plates in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 units/mL
penicillin G and 100
g/mL streptomycin. Cell cultures were then incubated at 37 ◦C in a humidified,
×
105 cells/mL in
µ
5% CO2 atmosphere, in the absence or presence of serial dilutions of tested compounds. Cell viability
◦
was determined after 96 h at 37 C by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) method.
The activity of compounds against HIV-1 was based on inhibition of virus-induced cytopathogenicity
in MT-4 cell acutely infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly, 50
µ
L of RPMI containing
1
×
104 MT-4 cells were added to each well of flat-bottom microtiter trays, containing 50
µL of RPMI
without or with serial dilutions of test compounds. Then, 20 µL of a HIV-1 suspension containing 100
CCID50 were added. After a 4-day incubation at 37 ◦C, cell viability was determined by the MTT method.
3.5. Antiproliferative Assays
Cell lines derived from human hematological tumors were: CD4+ human T-cells containing
an integrated HTLV-1 genome (MT-4); CD4+ human acute T-lymphoblastic leukemia (CCRF-CEM);
human splenic B-lymphoblastoid cells (WIL-2NS); human acute B-lymphoblastic leukemia (CCRF-SB).
Cells were seeded at an initial density of 1
supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin G and 100
×
105 cells/mL in 96 well plates in RPMI-1640 medium
g/mL streptomycin.
µ
Cell lines derived from human solid tumors were: Skin melanoma (SK-MEL-28); prostate
carcinoma (DU-145). Normal tissues foreskin fibroblasts (CRL-7065) were also used. Cells were
seeded at 1
×
105 cells/mL in 96 well plates in specific media supplemented with 10% FCS and
antibiotics, as above. Cell cultures were then incubated at 37 ◦C in a humidified, 5% CO2 atmosphere
in the absence or presence of serial dilutions of test compounds. Cell viability was determined after
96 h at 37 ◦C by the MTT method.
3.6. Cytotoxic Activity in HaCaT Cells
3.6.1. Cell Culture: Conditions and Treatments
Human immortal keratinocyte cell line from adult human skin (HaCaT) was purchased from
American Type Culture Collection (Rockville, MD, USA), and cultured in Dulbecco’s Modified Eagle’s
Medium (DMEM), supplemented with antibiotics (penicillin and streptomycin), 10% heat-inactivated
◦
FBS-fetal bovine serum (Gibco Life Technologies, Carlsbad, CA, USA) at 37 C and 5% CO2 atmosphere.
Cells were passaged using trypsin-EDTA (Gibco Life Technologies, Carlsbad, CA, USA) and cultured
in 24-well plates (2.5 × 104 cells per well). Experiments were conducted in DMEM with 2% FBS.
3.6.2. Cell Viability Assessment (Mitochondrial Function Assessment)
The cell viability assay was assessed by the determination of MTT salt (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich Chemie, Buchs, Germany) conversion by
mitochondrial dehydrogenase. The cells were incubated for 24 h in 24-well plates with tested
compounds (11
, 13 and 25) at given concentrations, and subsequently for another 2 h with 0.5 mg/mL
of MTT solution, which was converted in living cells under the effect of mitochondrial dehydrogenase

Molecules 2018, 23, 2428
14 of 18
into insoluble formazan. Then the converted dye was solubilized with the use of 0.04 M HCl in
absolute isopropanol. Absorbance of solubilized formazan was measured spectrophotometrically at
570 nm (using Epoch microplate reader, BioTek Inc., Winooski, VT, USA, equipped with Gen5 software
(BioTech Instruments, Inc., Biokom, Janki, Polska).
Cell viability was presented as a percent of MTT in the treated cells versus the control (cells
incubated in serum-free DMEM without extracts). The relative MTT level (%) was calculated as [A]/[B]
×
100, where [A] is the absorbance of the test sample and [B] is the absorbance of the control sample
containing the untreated cells. The decreased relative MTT level indicates decreased cell viability.
3.6.3. Lactate Dehydrogenase Release Assay (Cellular Membrane Integrity Assessment)
Release of lactate dehydrogenase (LDH) from the cytosol to culture medium is a marker of cell
death. The assay was performed after 24 h incubation of HaCaT cells in 24-well plates with investigated
concentrations of each compound. The activity of LDH released from the cytosol of damaged cells to
the supernatant was measured using the protocol of the cytotoxicity detection kit LDH test described
by the manufacturer (Roche Diagnostics, Berlin, Germany). Absorbance was measured at 490 nm
using a microplate reader (using Epoch microplate reader, BioTek Inc., Winooski, VT, USA) equipped
with Gen5 software (BioTech Instruments, Inc., Biokom, Janki, Polska)
Compounds mediated cytotoxicity expressed as the LDH release (%) was determined by the
following equation: [(A test sample—A low control)/(A high control—A low control)]
×
100%
(A-absorbance); where “low control” were cells in DMEM with 2% FBS without tested compounds,
and “high control” were cells incubated in DMEM with 2% FBS with 10% Triton X-100 (100% LDH
release).
3.6.4. Proliferation Rate in Cultures of HaCaT Cells
After 48 h incubation with the lowest concentration of the tested compound the increased number
of viable cells were assessed by direct counting of cells using trypan blue with a Countess Automated
Cell Counter (Invitrogen, Carlsbad, CA, USA). The results are presented as a % of viable and dead
cells versus control.
4. Conclusions
To sum up, this work has revealed the synthesis of new thiourea derivatives and their in vitro
antibacterial activity against clinically relevant pathogens. The title compounds showed potent
to moderate antimicrobial activity. Representative aryl-thioureas 20, 22, 23 and bicyclic-thiourea
derivative 24 were even more active towards selected clinical staphylococci than the reference drug,
Ciprofloxacin. To complete the biological profile of the presented thiourea series, their cytotoxic
activities in MT-4 cells, as well as human hematological and solid tumors cell lines were assessed.
Several compounds (1, 4, 6, 9–11, 13, 14, 17, 19) turned out cytotoxic in the low micromolar
range (CC50 < 10 µM). Performed studies proved that synthesized derivatives weakly to moderately
influenced the viability and growth of the human immortal keratinocyte cell line (HaCaT).
Supplementary Materials: Supplementary materials can be found online. The experimental details and final atomic
parameters for 20 and 24 have been deposited with the Cambridge Crystallographic Data Centre as supplementary
material (CCDC ID: 1849698 and 1849698, respectively). Copies of the data can be obtained free of charge on request via
www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
Author Contributions: Conceptualization, A.B. and G.S.; Methodology, J.S.; Software, A.E.K.; Validation, A.C.
and P.S.-Ł.; Formal Analysis, S.M.; Investigation, G.S.; S.M.; G.G.; Resources, J.S.; Data Curation, A.E.K. and
O.S.; Writing—Original Draft Preparation, A.B. and G.G.; Writing—Review & Editing, A.B.; Visualization, P.S.-Ł.;
Supervision, M.S.; Project Administration, M.S.; Funding Acquisition, G.K.-T.

Molecules 2018, 23, 2428
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Funding: This work was supported by the Medical University of Warsaw and carried out with the use of
CePT infrastructure financed by the European Union—the European Regional Development Fund within the
Operational Programme Innovative Economy for 2007–2013. The research was carried out with the facilities
purchased thanks to the financial support of the European Regional Development Fund in the framework of the
Operational Program Development of Eastern Poland 2007–2013 (Contract No. POPW.01.03.00-06-009/11-00,
Equipping the laboratories of the Faculties of Biology and Biotechnology, Mathematics, Physics and Informatics,
and Chemistry for studies of biologically active substances and environmental samples.
Acknowledgments: Authors would like to thank Sylwester Krukowski from the Department of Inorganic and
Analytical Chemistry, Faculty of Pharmacy, Medical University of Warsaw, for recording the IR spectra.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to
publish the results.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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