1H-Tetrazol-5-amine and 1,3-thiazolidin-4-one derivatives containing 3-(trifluoromethyl)phenyl scaffold: Synthesis, cytotoxic and anti-HIV studies

Article abstract of DOI:10.1016/j.biopha.2017.07.152

On the basis of recently reported biologically active 3-(trifluoromethyl)phenylthioureas, a series of diaryl derivatives incorporating 1H-tetrazol-5-yl (1a–11a, 1a’–11a’) and 1,3-thiazolidin-4-one (1b–11b) scaffolds were synthesized. The synthesis pathway

Full text of DOI:10.1016/j.biopha.2017.07.152

Biomedicine & Pharmacotherapy 94 (2017) 804–812
Available online at
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Original article
1H-Tetrazol-5-amine and 1,3-thiazolidin-4-one derivatives containing
3-(trifluoromethyl)phenyl scaffold: Synthesis, cytotoxic and anti-HIV
studies
b
Anna Bielenicaa, , Daniel Szulczyk , Wioletta Olejarzb,c, Silvia Madeddud,
*
Gabriele Gilibertid, Ilona B. Matereke, Anna E. Koziole, Marta Strugaa,c
Chair and Department of Biochemistry, Medical University of Warsaw, 02-097 Warszawa, Poland
Department of Biochemistry and Pharmacogenomics, Medical University of Warsaw, 02-097 Warszawa, Poland
Laboratory of Centre for Preclinical Research, Medical University of Warsaw, 02-097 Warszawa, Poland
a
b
c
d
Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria, 09042 Monserrato, Cagliari, Italy
Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland
e
A R T I C L E I N F O
A B S T R A C T
Article history:
On the basis of recently reported biologically active 3-(trifluoromethyl)phenylthioureas, a series of diaryl
derivatives incorporating 1H-tetrazol-5-yl (1a–11a, 1a’–11a’) and 1,3-thiazolidin-4-one (1b–11b)
scaffolds were synthesized. The synthesis pathway was confirmed by an X-ray crystallographic studies
of 3a’, 6a, 8a, 6b and 8b. The cytotoxicity against MT-4 cells and anti-HIV properties of new derivatives
were evaluated. As compared to initial thiourea connections, the cyclisation reduced the cytotoxicity of
compounds by 2–15 times. The most promising N-(4-nitrophenyl)-1H-tetrazol-5-amine 7a was found to
be more active than the origin thiourea. Its cytotoxicity was evaluated on A549, HTB-140 and HaCaT cell
lines using MTT assay. The compound shows significant influence on cancer, but not on normal cells.
Obtained results can provide some constructive data for further designing of novel family of potentially
bioactive analogs.
Received 9 June 2017
Received in revised form 17 July 2017
Accepted 30 July 2017
Keywords:
1,3-Thiazolidin-4-one
1H-Tetrazol-5-amine
Cytotoxicity
X-ray crystallography
© 2017 Published by Elsevier Masson SAS.
1. Introduction
microorganisms [9–14]. As a result, a 1H-tetrazolyl-based com-
pound, a novel antifungal drug candidate, is currently in phase II
Among various heterocycles reported to date, thiazolidine,
tetrazole and their derivatives have received much attention due to
their various bioactivities. Numerous 1H-tetrazol-5-amine con-
nections were discovered as potent, low-toxic angiotensin II
receptor type (AT1) blockers [1–3], thus acted as efficient
antihypertonic agents. Their analogs, bearing pyrazolyl ring,
exerted cardiotonic activity [4,5], acting via inhibition of phos-
phodiesterase (PDE3). 1,4-Diaryltetrazoles were found to be potent
P2X7 antagonists, demonstrating a role in reduction of symptoms
of chronic inflammatory diseases, such as osteoarthritis [6,7].
Investigations on hypoglycemic properties of 1,4-diphenyltetra-
zoles with urea moiety were also conducted [8]. Some literature
data has revealed that tetrazole-based molecules, e.g. cephalospo-
rin antibiotics, are inhibitors of the growth of various
clinical trials [15]. The 5H-tetrazolo[1,5-a]azepine known as
Cardiazol, and its analog Gabapentin, were used in clinical practice
as anticonvulsants and the central nervous system stimulants
[16,17]. The substitution of a tetrazol-5-yl ring allowed to create
new neurotransmitters [18] and pain relievers, effective also in the
treatment of atherosclerosis [16].
What is more, tetrazolylhydrazides [19], tetrazol-5-ones and
tetrazol-5-thiones [20] exhibited anticancer properties, reducing
the expression of the Ki-67 marker of cell proliferation in
mammary tumor cells and the rate of DNA synthesis in leukemia
cells. 1,5-Disubstituted tetrazoles were found as particularly active
against liver carcinoma (HepG2), prostate (DU145) and lung
adrenocarcinoma (A549) cancer cell lines [21]. Among 1,2-
disubstituted derivatives, these with methoxyphenyl [22] and
nitrophenyl [22,23] groups have been revealed as the strongest
inhibitors of breast cancer cells growth. The synthesis of tetrazolyl
compounds [24,25] with established antiviral activity was also
documented. Within this group, analogs of HIV-1 reverse
* Corresponding author.
E-mail address: abielenica@wum.edu.pl (A. Bielenica).
http://dx.doi.org/10.1016/j.biopha.2017.07.152
0753-3322/© 2017 Published by Elsevier Masson SAS.

A. Bielenica et al. / Biomedicine & Pharmacotherapy 94 (2017) 804–812
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Fig. 1. Synthesis of compounds 1a–11a, 1a’–11a’ and 1b–11b.
Fig. 2. Molecular structure of compounds 6a and 6b.

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A. Bielenica et al. / Biomedicine & Pharmacotherapy 94 (2017) 804–812
transcriptase (HIV-1 RT) [26,27], protease [28,29], as well as
integrase [30,31] inhibitors were found.
bonded H-atoms were found in the difference-Fourier map and
refined with isotropic displacement parameters. The crystal data
for 3a’, 6a, 8a, 6b and 8b are collected in Tables 1S and 2S
(Supplementary material), and have been deposited with the
Cambridge Crystallographic Data Centre as Supplementary mate-
rial (CCDC Nos. 1553352–1553357). Copies of the data can be
obtained free of charge on application: e-mail: deposit@ccdc.cam.
ac.uk or www: http://www.ccdc.cam.ac.uk).
Molecules containing another five-membered ring, thiazolidi-
none, are associated mainly with antibacterial [32–35], antituber-
cular [36], antidiabetic [37] and anticancer [38–40] activities.
According to Chandrappa et al. [41] estimations, a substitution
with electron donating groups (CH3, OCH3) at aromatic ring
resulted in an increase in anticancer potency by inducing the cell
death, while cyclic compounds containing electron withdrawing
functionalities (CN, F, CF3) exhibited decreased activity. The
mechanism of action of 2,3-diaryl-1,3-thiazolidin-4-ones as
antiretroviral agents was attributed to the inhibition of HIV-1
RT [42–44] or integrase [45], with simultaneously low toxicity
towards MT-4 cells. It was estimated that thiazolidin-4-ones could
behave as first-generation of non-nucleoside reverse transcriptase
inhibitors (NNRTIs) like nevirapine, and bind at the allosteric site of
RT in a “butterfly-like conformation” [46–50]. Rawal et al. [51]
underlined the importance of overall lipophilicity of the potential
agents, as well as steric and electronic parameters of meta/para-
substituted 3-aryl terminal fragment. Since the binding site of RT
consists of hydrophobic amino acids, the thiazolidinones-RT
complex is stabilized by extensive hydrophobic and some
hydrogen-bonding interactions [50]. Additionally, the presence
of halogen atoms on the phenyl ring restricts its rotation and
preserves the favorable conformation of a ligand.
The synthesis of 3-[3-(trifluoromethyl)phenyl]thiourea deriv-
atives (1, 3–11) [52] and the preparation of starting thiourea 2 [53]
were published previously.
2.1.1.1. General procedure for the synthesis of N-[3-(trifluoromethyl)
phenyl]-1H-tetrazol-5-amine (1a–11a). A solution of the thiourea
derivative 1–11 (1.25 mmol) in dried DMF was treated with sodium
azide (3.75 mmol) in the presence of mercuric chloride
(1.38 mmol) and triethylamine (0.5 mL) for 3 h. After that the
inorganic residue was filtrated, rinsed with chloroform, and the
filtrate was evaporated. The product was purified by column
chromatography (chloroform).
2.1.1.2. General procedure for the synthesis of 3-[3-(trifluoromethyl)
phenyl]-1,3-thiazolidin-4-ones (1b–11b). A solution of derivatives
1–11 (0.001 mol) was heated with chloroacetamide (0.003 mol) in
dried acetonitrile (20 mL) for 24 h. After the solvent was
evaporated, the solid residue was purified by column
chromatography (chloroform).
Considering above, we have modified the structure of bioactive
derivatives bearing thiourea branch [52,53], by introducing the 1H-
tetrazol-5-yl or 1,3-thiazolidin-4-one core instead, and tested
them for cytotoxic and anti-HIV-1 activities. It allowed to evaluate
the impact of cyclisation on compounds’ biological potency, as
compared to parent linear molecules.
The details of the synthesis of each compound (1a–11a,1b–11b)
are provided in the Supplementary material.
2.1.2. Cytotoxicity studies
2. Material and methods
2.1.2.1. Cell cultures and viruses. Human Immunodeficiency Virus
type-1 (HIV-1) IIIB laboratory strain was obtained from the
supernatant of the persistently infected H9/IIIB cells (NIH 1983).
Cell lines supporting the multiplication of HIV-1, the CD4+ human
T-cells containing an integrated HTLV-1 genome (MT-4), Human
immortal keratinocyte cell line from adult human skin (HaCaT),
Human epithelial lung carcinoma cell line (A549) and Human
melanoma cell line (HTB-140) were bought from American Type
Culture Collection (Rockville, USA), and cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with 1%
antibiotics (penicillin and streptomycin), and 10% heat-
inactivated FBS-fetal bovine serum (Gibco Life Technologies,
USA), at 37 ꢀC and 5% CO2 atmosphere. Cells were passaged
using trypsin-EDTA (Gibco Life Technologies, USA) and cultured in
96-well plates (1 Â104 cells per well). Experiments were
conducted in DMEM with 2% FBS.
2.1. Chemistry
2.1.1. General procedure
The reagents were supplied from Alfa Aesar or Sigma Aldrich.
Organic solvents (N,N-dimethylformamide, acetonitrile, DMF,
chloroform and methanol) were supplied from POCh (Polskie
Odczynniki Chemiczne). All chemicals were of analytical grade.
Before use, dried DMF and acetonitrile were kept in crown cap
bottles over anhydrous phosphorus pentoxide (Carl Roth). The
NMR spectra were recorded on a Varian VNMRS 300 Oxford NMR
spectrometer, operating at 300 MHz (1H NMR) and 75.4 MHz (13C
NMR). Chemical shifts (d) were expressed in parts per million
relative to tetramethylsilane used as the internal reference. Mass
spectral ESI measurements were carried out on Waters ZQ Micro-
mass instruments with quadruple mass analyzer. The spectra were
performed in the negative or positive ion mode at a declustering
potential of 40–60 V. The sample was previously separated on a
UPLC column (C18) using an UPLC ACQUITYTM system by Waters
connected with DPA detector. Flash chromatography was per-
formed on Merck silica gel 60 (230–400 mesh) using chloroform
eluent. Analytical TLC was carried out on silica gel F254 (Merck)
plates (0.25 mm thickness). The X-ray diffraction data were
collected at 120(2) K on a SuperNova diffractometer (Oxford
2.1.2.2. Cytotoxicity and antiviral assays. Exponentially growing
MT-4 cells were seeded at an initial density of 4 Â105 cells/ml in
96-well plates in RPMI-1640 medium, supplemented with 10%
fetal bovine serum (FBS), 100 units/mL penicillin G and 100 mg/mL
streptomycin. Cell cultures were then incubated at 37 ꢀC in a
humidified, 5% CO2 atmosphere, in the absence or presence of
serial dilutions of tested compounds. Cell viability was determined
after 48–96 h at 37 ꢀC by MTT method. Compound’s activity against
HIV-1 was based on inhibition of virus-induced cytopathogenicity
in exponentially growing MT-4 cell, acutely infected with a
Diffraction) equipped with the microfucus X-ray source (Cu K
a,
l
= 1.54184 Å) and CCD detector. The data were corrected for
Lorentz and polarization effects. A multi-scan absorption correc-
tion was applied. The structure was solved using direct methods
(SHELXS-97 program), and refined by the full-matrix least-squares
on F2 with the SHELXL-97 program [54]. All non-H atoms were
refined with anisotropic displacement parameters. The H-atoms
attached to carbon were positioned geometrically and refined
using the riding model with Uiso(H) = 1.2Ueq(C). The nitrogen
multiplicity of infection (m.o.i.) of 0.01. Briefly, 50 mL of RPMI
containing 1 Â104 MT-4 cells were added to each well of flat-
bottom microtitre trays, containing 50 mL of RPMI without or with
serial dilutions of tested samples. Then, 20 mL 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

A. Bielenica et al. / Biomedicine & Pharmacotherapy 94 (2017) 804–812
807
Table 1
[B] Â 100, where [A] is the absorbance of the tested sample and [B]
means the absorbance of the control sample containing untreated
cells. Decreasing of the relative MTT level indicates decreased cell
viability.
Cytotoxicity, anti-HIV activity and ClogP values of thiourea derivatives 1a–11a and
1b–11b.
Compd
R1
MT-4
CC50
2 HIV-1IIIB
EC50
ClogPc
a
b
1a
2a
3a
4a
5a
6a
7a
8a
3-Cl, 4-F-Ph
3-F-Ph
4-OCH3-Ph
3-Cl, 4-Cl-Ph
4-Cl-Ph
4-CN-Ph
4-NO2-Ph
CH2-Ph
3-Br-Ph
2-F-Ph
3-Cl-Ph
3-Cl, 4-F-Ph
3-F-Ph
4-OCH3-Ph
3-Cl, 4-Cl-Ph
4-Cl-Ph
3-CF3-Ph
4-NO2-Ph
CH2-Ph
3-Br-Ph
2-F-Ph
3-Cl-Ph
–
41
48
72
45
48
18
3.2
>100
20
>41
>48
>72
>45
>48
>18
>3.2
>100
>20
5.07
4.36
4.21
5.52
4.93
3.66
3.97
3.66
5.08
4.36
4.93
4.01
3.30
3.08
4.46
3.87
4.04
2.90
4.56
4.02
3.30
3.87
–
2.1.2.4. LDH assay. Release of lactate dehydrogenase (LDH) from
the cytosol to culture medium (cellular membrane integrity
assessment) is a marker of a cell death. The assay was performed
after 72 h incubation of cells in 96-well plates with the investigated
compound, according to previously used methodology [52]. The
activity of LDH released from cytosol of damaged cells to the
supernatant was measured using the protocol of the cytotoxicity
detection kit (LDH; Roche Diagnostics, Germany). The absorbance
was measured at 490 nm using a microplate reader (by Epoch
microplate reader, BioTek Inc., USA), equipped with Gen5 software
(BioTech Instruments, Inc., Biokom). Compounds mediated
cytotoxicity expressed as the LDH release (%) was determined
by the following equation: [(A test sample–A low control)/(A high
controlÀA low control)] Â 100% (A-absorbance); where “low
control” means cells in DMEM or FGM with 2% FBS without
tested compound, and “high control” are cells incubated in DMEM
or FGM with 2% FBS and 10% Triton X-100 (100% LDH release). The
cytotoxicity was expressed as percentage of LDH release, as
compared to the maximum release of LDH from Triton-X100-
treated cells.
9a
10a
11a
1b
2b
3b
4b
5b
6b
7b
8b
9b
10b
11b
EFV
50
29
>50
>29
>100
>100
44.0
>100
>100
>100
48
43.0
54.0
>100
>100
46.0
>100
>100
>44.0
>100
>100
>100
>48
>43
>54.0
>100
>100
0.003
Data represent mean values for three independent determinations, made with
duplicate samples. The variations among them were less than 15%.
a
Compound concentration (mM) required to reduce the viability of cells by 50%,
3. Results
as determined by the MTT method.
b
Compound concentration (mM) required to achieve 50% protection of MT-4
3.1. Chemistry
cells from the HIV-1 induced cytopathogeneticy, as determined by the MTT method.
c
Calculated by ChemBioDraw Ultra 14.
The scheme for the modification of the structures of previously
obtained thiourea derivatives 1-11 [52,53] is shown in Fig. 1. The
aim of the first route was to synthesize 5-aminotetrazoles using
the mercury(II)-promoted attack of azide anion on a thiourea. The
main products of cyclisation were N-substituted-1-[3-(trifluoro-
methyl)phenyl]-1H-tetrazol-5-amines 1a–11a (40–79% yield),
however, small amounts of isomeric 1-substituted-N-[3-(trifluor-
omethyl)phenyl]-1H-tetrazol-5-amines 1a’–11a’ (3–11%) were
also obtained. The dual synthesis route was confirmed by an X-
ray crystallographic studies of isomeric 5-amino-tetrazoles: 3a’, 6a
and 8a (Fig. 2 and Supplementary material Figs. 1S–3S).
method. Concentrations resulting in 50% inhibition (CC50 or EC50
were determined by linear regression analysis.
)
2.1.2.3. MTT assay. The cell viability was assessed by
determination of MTT salt 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide, converted by mitochondrial
dehydrogenase. The cells were incubated for 72 h in 96-well
plates with given concentration of tested compound, and
subsequently for another 4 h with 0.5 mg/mL of MTT solution,
which in live cells
– under the effect of mitochondrial
On the other hand, the same starting thioureas underwent
cyclisation in the presence of chloroacetamide to give correspond-
ing 1,3-thiazolidin-4-ones 1b–11b (30–40%). The structures of
newly synthesized compounds were verified by spectral analyses
and finally determined by the X-ray structural analysis of 6b and 8b
(Fig. 2 and Supplementary material Figs. 4S and 5S).
The derivatives containing both 1H-tetrazol-5-amine and 1,3-
thiazolidin-4-one cores show conformational flexibility, which
may influence a manner of intermolecular contacts formation. In
the solid state for 6a, 6b and 8b, three, two and four conformers are
observed, respectively (Supplementary material Figs. 6S –9S).
dehydrogenase – converts to insoluble formazan. The converted
dye was then solubilized with the use of 0.04 M HCl in absolute
isopropanol. The absorbance of solubilized formazan was
measured spectrophotometrically at 570 nm (using Epoch
microplate reader, BioTek Inc., USA), equipped with Gen5
software (BioTech Instruments, Inc., Biokom). Cell viability was
presented as a percent of MTT reduction in the treated cells versus
the control (cells incubated in serum-free DMEM without studied
compounds). The relative MTT level (%) was calculated as [A]/
Table 2
Antiproliferative activity of compound 7a.
3.2. Biological activity studies
Compound
Cancer cells
A549c
Normal cells
HaCaTe
HTBÀ140d
3.2.1. Cytotoxicity
IC50
SIb
IC50
SI
IC50
a
Derivatives 1a-11a and 1b-11b were tested in cell-based assay
against the human immunodeficiency virus type-1 (HIV-1), using
Efavirenz as the reference drug. Simultaneously, the cytotoxicity
against MT4 cells was evaluated. The results, expressed as CC50 and
EC50 were summarized in Table 1. None of the studied compounds
showed anti-HIV-1 activity. Most of the new cyclic molecules
showed a moderate cytotoxicity similar to the reference drug. The
higher cytotoxic potential was observed for 1H-tetrazol-5-yl
derivatives.
7a
5.70 Æ 0.68
1.95 Æ 0.83
0.63 Æ 0.21
2.22
1.46
1.73
4.10 Æ 1.25
1.13 Æ 0.19
0.47 Æ 0.18
3.09
2.51
2.32
12.70 Æ 2.94
2.84 Æ 1.06
1.09 Æ 0.23
Cisplatin
Doxorubicin
a
Concentration of
a
compound
(mM) that corresponds to a 50% growth
inhibition.
b
Value calculated using formula: SI = IC50 for normal cell line/IC50 cancer cell line.
Data are expressed as mean Æ SD.
c
Human epithelial lung carcinoma cell line (A549).
Human melanoma cell line (HTB-140).
Human immortal keratinocyte cell line from adult human skin (HaCaT).
d
e

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A. Bielenica et al. / Biomedicine & Pharmacotherapy 94 (2017) 804–812
Fig. 3. LDH release as a marker of cell death in the HaCaT, A549 and HTB-140 cells, treated for 72 h with compound 7a at concentration 40 mM, 20 mM, 10 mM, 5 mM, 1 mM,
respectively. Data values are expressed as the mean Æ SD from three independent experiments performed in triplicate. ***p ꢁ 0,001 and **p ꢁ 0,01 as compared to control
cells.
As the most promising (CC50 = 3.2
m
M), the compound 7a was
keratinocyte cell line from adult human skin). Cell lines were
exposed to different concentrations of the thiourea derivative for
72 h. The tested tetrazole demonstrated significant cytotoxic effect
screened for its cytotoxic activity against two tumor cell lines:
human epithelial lung carcinoma (A549) and human melanoma
cell line (HTB-140), as well as normal HaCaTcells (human immortal
on cancer cells (IC50 = 4.10–5.70 mM), being less toxic to normal
Fig. 4. Cell viability assessed by MTT mitochondrial conversion in cells treated for 6 and 24 h with IC50 concentration ANF and next by 48 h with A. Doxorubicin, B. Cisplatin.
The relative MTT level (%) was calculated as [A]/[B] Â 100, where [A] is the absorbance of the test sample and [B] is the absorbance of control sample containing the untreated
cells. Decreased relative MTT level indicates decreased cell viability. Data are expressed as means Æ SD. ***p ꢁ 0,001 and **p ꢁ 0,01 as compared to Doxorubicin or Cisplatin.

A. Bielenica et al. / Biomedicine & Pharmacotherapy 94 (2017) 804–812
809
Fig. 5. Microscopic examination of cells migration. A. migration of HaCaTcells, B. migration of A549 cells, C. migration of HTB-140 cells. The digital images of viable cells were
visualized using the phase-contrast inverted microscopy Eclipse TS 100F (Nikon, USA) equipped with the Nikon Digital Sight DS-U2 camera using NIS-Elements Nikon BR 2.30
software.
cells than the reference drugs – Cisplatin and Doxorubicin (Table 2).
Evaluation of LDH release by dead cells (Fig. 3) showed that the
studied thiourea after 72 h incubation, at the concentration 10–
tested derivative for 6 and 24 h, and then incubated for 72 h with a
chemotherapeutic agent (Cisplatin and Doxorubicin). The cell
viability was determined by MTT method (Fig. 4).
40
m
M, induced HTB-140 and A549 cells death. However, it was not
Tumor cells (A549 and HTB-140) showed higher sensitivity to
compound 7a as compared to normal cells (HaCaT). The viability of
the HaCaT cells after 6 h pre-incubation with the derivative 7a has
not been changed. A significant inhibition of cells proliferation was
observed after 24 h. Among cancer cells the strongest influence
was observed in HTB-140 cells. Their viability after 24 h treatment
was inhibited by 30.7% for Cisplatin and 33.7% for Doxorubicin. It
cytotoxic towards HaCaT cells. At lower doses (1–5 mM) no toxic
effect to normal and cancer cells was observed.
3.2.2. In vitro drug sensitivity of HaCaT, A549 and HTB-140 cells
In order to evaluate the sensitivity of cancer and normal cells to
the presence of compound 7a, they were pre-incubated with the

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A. Bielenica et al. / Biomedicine & Pharmacotherapy 94 (2017) 804–812
was noted that 7a inhibited a viability of A549 cells after 72 h from
51.8 to 41.6% for Cisplatin, and 51.8% to 47.9% for Doxorubicin.
The cytotoxic potency of the tetrazole core combined with the
nitrophenyl ring has been previously noticed by Arshad et al. [23].
As expected, the biological profile of the tetrazole-derived 7a is
found to be very beneficial. The compound at the applied
concentration caused cancer cells death, however had no toxic
influence on normal HaCaT cells. The results of this study clearly
indicate that human lung cells (A549) and melanoma cells (HTB-
140) were highly sensitive on the derivative 7a exposure,
compared to human normal keratinocyte cells (HaCaT). It was
also noted that the selectivity index (SI) for 7a was higher than
observed for the reference chemotherapeutics. The HTB-140 cells
were more susceptible for the presence of the mentioned tetrazole,
suggesting its preferential effect against human melanoma cells
then lung cancer cells.
3.2.3. Cell migration assay
Cells were seeded in 96-well plates. When 70% confluence was
reached, a cutting line in the middle of the plate was made, and
compound 7a was added at the concentration of 6 and 12
mM (for
HaCaT cells), 3 and 6 M (for A549 cells), 2 and 4 M (for HTB-140
m
m
cells). Cells were incubated for 72 h, and then the pictures were
obtained through Nikon Eclipse TS 100 microscope.
As shown in Fig. 5, derivative 7a significantly inhibited cell
migration. It is noteworthy that at 6 and 12 mM it exerted stronger
effect on A549 and HTB-140 cells, than on HaCaT cells.
3.2.4. Statistical analysis
Both the cytotoxicity and the migration tests show a
The results are expressed as the mean Æ SD from the indicated
number of experiments. Comparisons were made using Student’s
t-test. Differences between experimental groups were considered
to be statistically significant at p < 0.05.
concentration-dependent inhibition of cell growth, although with
a different degree of efficacy as expressed by the different IC50
values. The combination of potent anticancer activity of the
studied tetrazole with its low toxicity makes it the promising lead
compound for cancer chemotherapy.
4. Discussion
On the other hand, the lack of anti-HIV potency of new
derivatives could be associated with the deficiency in their
structural flexibility. Previous studies by other groups proved that
2,3-diaryl-1,3-thiazolidin-4-ones [46,47] maintained the “butterfly
conformation”, therefore entered the RT binding hydrophobic
pocket effectively. Particularly, new cyclic benzyl (8a, 8b) or
dihalogenophenyl (1a, 1b, 4a, 4b) derivatives, due to their
predicted molecular flexibility, were expected to exert an
antiretroviral potency. However, none of newly synthesized
compounds has fulfilled required conformational elasticity.
The cyclisation of thiourea branch lead to stiffening of the
whole molecule, as well as shifting of its lone electron pairs. It can
result in higher bioactivity of obtained derivatives, in comparison
with their linear equivalents. Because of the difficulty of the
cyclocondensation, heterocyclic analogs of thiourea are rarely
tested for any biological activity, in consequence À are not
frequently applied in pharmacology. In this study, the use of polar
aprotic solvents at various temperatures gave different types of
products. In the presence of DMF at room temperature, two
isomeric tetrazolyl forms were obtained: N-substituted-1-[3-
(trifluoromethyl)phenyl]-1H-tetrazol-5-amines 1a–11a (main
products) and 1-substituted-N-[3-(trifluoromethyl)phenyl]-1H-
tetrazol-5-amines 1a’–11a’ (side products). The cyclisation to
thiazolidinones carried out by heating in acetonitrile yielded only
3-substituted-2-{[3-(trifluoromethyl)phenyl]imino}-1,3-thiazoli-
din-4-ones (1b–11b).
Physicochemical parameters of trifluoromethyl group are
unique among all electron-accepting functionalities. As it is highly
lipophilic, it contributes in a faster and more even distribution of a
drug, that favors a stronger biological potency. The presence of this
group prolongs biological half-life of a compound and enhances
reactivities of other functional groups within a molecule. Finally, it
produces a strong inductive effect, which improves whole acidity.
As a result solubility, permeability and binding affinity of a
potential drug considerably increase [55,56].
Acknowledgements
This paper was supported by Medical University of Warsaw,
1WK/N/2016.
Cytotoxicity studies were carried out with the use of CePT
infrastructure financed by the European Union À the European
Regional Development Fund within the Operational Programme
“Innovative economy for 2007–2013”.
The X-ray crystallographic studies were 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).
As compared to starting thiourea-based analogs [52,53], the
transformation of the thioureidic branch into the tetrazole ring
reduced the cytotoxicity of compounds by 2–7 times. Similarly, the
formation of the thiazolidin-4-one moiety resulted in 2–15 lower
cytotoxicity. It is worth to note that the N-(4-nitrophenyl)tetrazo-
1-yl derivative 7a turned out to be cytotoxic for exponentially
growing MT4 cells in the low micromolar range, being 3 times
more potent than its parent thiourea. The changes in cytotoxicity
could be explained by differences in lipophilicity (expressed as
ClogP), caused by closing of a thiourea branch (Table 1). To
compare, 1H-tetrazol-5-yl derivatives (1a–11a) have slightly
higher ClogP values than initial thioureas [52,53]. Considering
1,3-thiazolidin-4-ones (1b–11b), the cyclisation process has
resulted in significant decreasing of compounds’ lipophilicity.
The opposite trend was observed only for both benzyl derivatives
(8a, 8b). Changes in hydrophobicity could affect the permeability
by biological membranes, therefore the pharmacological activity of
new derivatives has reduced (except from highly cytotoxic 7a).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.
biopha.2017.07.152.
References
[1] X.F. Han, X. He, M. Wang, D. Xu, L.P. Hao, A.H. Liang, J. Zhang, Z.M. Zhou,
Discovery of novel, potent and low-toxicity angiotensin II receptor type 1 (AT1)
blockers: design, synthesis and biological evaluation of 6-substituted
aminocarbonyl benzimidazoles with a chiral center, Eur. J. Med. Chem. 103
(2015) 473–487.
[2] H. Yanagisawa, Y. Amemiya, T. Kanazaki, Y. Shimoji, K. Fujimoto, Y. Kitahara, T.
Sada, M. Mizuno, M. Ikeda, S. Miyamoto, Y. Furukawa, H. Koike, Nonpeptide
angiotensin II receptor antagonists: synthesis, biological activities, and
structure-activity relationships of imidazole-5-carboxylic acids bearing alkyl,
alkenyl, and hydroxyalkyl substituents at the 4-position and their related
compounds, J. Med. Chem. 39 (1996) 323–338.
[3] Y. Shibouta, Y. Inada, M. Ojima, T. Wada, M. Noda, T. Sanada, K. Kubo, Y. Kohara,
T. Naka, K. Nishikawa, Pharmacological profile of a highly potent and long-

A. Bielenica et al. / Biomedicine & Pharmacotherapy 94 (2017) 804–812
811
acting angiotensin II receptor antagonist 2-ethoxy-1-[[2’-(1H-tetrazol-5-yl)
biphenyl-4-yl]methyl]-1H-benz-imida-zole-7-carboxylic acid (CV-11974),
and its prodrug, (+/À)-1-(cyclohexylocarbony-loxy)-ethyl-2-ethoxy-1-[[2’-
(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylate
(TCV-116), J. Pharmacol. Exp. Ther. 266 (1993) 114–120.
[25] R.B. Perni, J. Pitlik, S.D. Britt, J.J. Court, L.F. Courtney, D.D. Deininger, L.J. Farmer,
C.A. Gates, S.L. Harbeson, R.B. Levin, C. Lin, K. Lin, Y.C. Moon, Y.P. Luong, E.T.
O’Malley, B.G. Rao, J.A. Thomson, R.D. Tung, J.H. Van Drie, Y. Wei, Inhibitors of
hepatitis C virus NS3.4A protease 2. warhead SAR and optimization, Bioorg.
Med. Chem. Lett. 14 (2004) 1441–1446.
[4] L.M. Duan, H.Y. Yu, Y.L. Li, C.J. Jia, Design and discovery of 2-(4-(1H-tetrazol-5-
yl)-1H-pyrazol-1-yl)-4-(4-phenyl)thiazole derivatives as cardiotonic agents
via inhibition of PDE3, Bioorg. Med. Chem. 23 (2015) 6111–6117.
[5] Z.W. Mao, Q.L. Li, P. Wang, Y.L. Sun, Y. Liu, Design and development of novel 4-
(4-(1H-tetrazol-5-yl)-1H-pyrazol-1-yl)-6-morpholino-N-(4-nitrophenyl)-
1,3,5-triazin-2-amine as cardiotonic agent via inhibition of PDE3, Arch. Pharm.
(Weinheim). 349 (2016) 268–276.
[6] P.G. Ruminski, M. Massa, J. Strohbach, C.E. Hanau, M. Schmidt, J.A. Scholten, T.R.
Fletcher, B.C. Hamper, J.N. Carroll, H.S. Shieh, N. Caspers, B. Collins, M.
Grapperhaus, K.E. Palmquist, J. Collins, J.E. Baldus, J. Hitchcock, H.P. Kleine, M.
D. Rogers, J. McDonald, G.E. Munie, D.M. Messing, S. Portolan, L.O. Whiteley, T.
Sunyer, M.E. Schnute, Discovery of N-(4-Fluoro-3-methoxybenzyl)-6-(2-
(((2S,5R)-5-(hydroxymethyl)-1,4-dioxan-2-yl)methyl) À2H-tetrazol-5-yl)-2-
methylpyrimidine-4-carboxamide. A highly selective and orally bio-available
matrix metalloproteinase-13 inhibitor for the potential treatment of
osteoarthritis, J. Med. Chem. 59 (2016) 313–327.
[26] T. Jiang, K.L. Kuhen, K. Wolff, H. Yin, K. Bieza, J. Caldwell, B. Bursulaya, T.
Tuntland, K. Zhang, D. Karanewsky, Y. He, Design synthesis, and biological
evaluations of novel oxindoles as HIV-1 non-nucleoside reverse transcriptase
inhibitors. Part 2, Bioorg. Med. Chem. Lett. 16 (2006) 2109–2112.
[27] E. Muraglia, O.D. Kinzel, R. Laufer, M.D. Miller, G. Moyer, V. Munshi, F. Orvieto,
M.C. Palumbi, G. Pescatore, M. Rowley, P.D. Williams, V. Summa, Tetrazole
thioacetanilides: potent non-nucleoside inhibitors of WT HIV reverse
transcriptase and its K103N mutant, Bioorg. Med. Chem. Lett. 16 (2006) 2748–
2752.
[28] B.C.H. May, A.D. Abell,
a-Methylene tetrazole-based peptidomimetics:
synthesis and inhibition of HIV protease, J. Chem. Soc. Perkin Trans. 1
(2002) 172–178.
[29] A.D. Abell, G.J. Foulds, Synthesis of a cis-conformationally restricted peptide
bond isostere and its application to the inhibition of the HIV-1 protease, J.
Chem. Soc. Perkin Trans. 1 (1997) 2475–2482.
[30] D.C. Crosby, X. Lei, C.G. Gibbs, B.R. McDougall, W.E. Robinson, M.G. Reinecke,
Design synthesis, and biological evaluation of novel hybrid dicaffeoyltartaric/
diketo acid and tetrazole-substituted L-chicoric acid analogue inhibitors of
human immunodeficiency virus type 1 integrase, J. Med. Chem. 53 (2010)
8161–8175.
[7] A. Perez-Medrano, D.L. Donnelly-Roberts, A.S. Florjancic, D.W. Nelson, T. Li, M.
T. Namovic, S. Peddi, C.R. Faltynek, M.F. Jarvis, W.A. Carroll, Synthesis and in
vitro activity of N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine P2X(7)
antagonists, Bioorg. Med. Chem. Lett. 21 (2011) 3297–3300.
[8] O.M. Antypenko, S.I. Kovalenko, G.O. Zhernova, Search for compounds with
hypoglycemic activity in the series of 1-[2-(1H-tetrazol-5-yl)-R(1)-phenyl]-3-
R(2)-phenyl (ethyl)ureas and R(1)-tetrazolo[1,5-c]quinazolin-5(6H)-ones, Sci.
Pharm. 84 (2016) 233–254.
[9] L.L. Dai, H.Z. Zhang, S. Nagarajan, S. Rasheeda, C.H. Zhou, Synthesis of tetrazole
compounds as a novel type of potential antimicrobial agents and their
synergistic effects with clinical drugs and interactions with calf thymus DNA,
Med. Chem. Commun. 6 (2015) 147–154.
[31] M.A. Walker, T. Johnson, Z. Ma, J. Banville, R. Remillard, O. Kim, Y. Zhang, A.
Staab, H. Wong, A. Torri, H. Samanta, Z. Lin, C. Deminie, B. Terry, M. Krystal, N.
Meanwell, Triketoacid inhibitors of HIV-integrase: a new chemotype useful for
probing the integrase pharmacophore, Bioorg. Med. Chem. Lett. 16 (2006)
2920–2924.
[32] C.V. Kavitha, S.N. Basappa, K. Swamy, S. Mantelingu, M.A. Doreswamy, J.
Sridhar, Shashidhara Prasad, K.S. Rangappa, Synthesis of new bioactive
venlafaxine analogs: novel thiazolidin-4-ones as antimicrobials, Bioorg. Med.
Chem. 14 (2006) 2290–2299.
[10] K. Waisser, J. Adamec, J. Kuneš, J. Kaustová, Antimycobacterial 1-Aryl-5-
benzyl-sulfanyltetrazoles, Chem. Pap. 58 (2004) 214–219.
[33] C.G. Bonde, N.J. Gaikwad, Synthesis and preliminary evaluation of some
pyrazine containing thiazolines and thiazolidinones as antimicrobial agents,
Bioorg. Med. Chem. 12 (2004) 2151–2161.
[34] S. Bondock, W. Khalifa, A.A. Fadda, Synthesis and antimicrobial evaluation of
some new thiazole thiazolidinone and thiazoline derivatives starting from 1-
chloro-3,4-dihydronaphthalene-2-carboxaldehyde, Eur. J. Med. Chem. 42
(2007) 948–954.
[11] K. Waisser, J. Kunes, A. Hrabalek, M. Machacek, Z. Odlerova, New groups of
potential antituberculotics À 5-alkylthio-1-aryltetrazoles, Coll. Czech. Chem.
Commun. 61 (1996) 791–798.
[12] Z.H. Chohan, C.T. Supuran, A. Scozzafava, Metalloantibiotics: synthesis and
anti-bacterial activity of cobalt(II), copper(II), nickel(II) and zinc(II) complexes
of kefzol, J. Enzyme Inhib. Med. Chem. 19 (2004) 79–84.
[13] L.W. Tremblay, H. Xu, J.S. Blanchard, Structures of the Michaelis complex (1.2 Å)
[35] D. Maclean, F. Holden, A.M. Davis, R.A. Scheuerman, S. Yanofsky, C.P. Holmes,
W.L. Fitch, K. Tsutsui, R.W. Barrett, M.A. Gallop, Agonists of the follicle
stimulating hormone receptor from an encoded thiazolidinone library, J.
Comb. Chem. 6 (2004) 196–206.
and the covalent acyl intermediate (2.0 Å) of cefamandole bound in the active
sites of the Mycobacterium tuberculosis
b-lactamase K73A and E166A
mutants, Biochemistry 49 (2010) 9685–9687.
[14] S. Vilain, P. Cosette, G.A. Junter, T. Jouenne, Phosphate deprivation is associated
with high resistance to latamoxef of gel-entrapped, sessile-like Escherichia
coli cells, J. Antimicrob. Chemother. 49 (2002) 315–320.
[15] W.J. Hoekstra, T.Y. Hargrove, Z. Wawrzak, D. da Gama Jaen Batista, C.F. da Silva,
A.S. Nefertiti, G. Rachakonda, R.J. Schotzinger, F. Villalta, N. Soeiro Mde, G.I.
Lepesheva, Clinical candidate VT-1161’s antiparasitic affect in vitro, activity in
a murine model of chagas disease, and structural characterization in complex
with the target enzyme CYP51 from Trypanosoma cruzi, Antimicrob, Agents
Chemother. 60 (2015) 1058–1066.
[36] A. Verma, S.K. Saraf, 4-Thiazolidinone – a biologically active scaffold, Eur. J.
Med. Chem. 43 (2008) 897–905.
[37] R. Ottanà, R. Maccari, M. Giglio, A. Del Corso, M. Cappiello, U. Mura, S.
Cosconati, L. Marinelli, E. Novellino, S. Sartini, C. La Motta, F. Da Settimo,
Identification of 5-arylidene-4-thiazolidinone derivatives endowed with dual
activity as aldose reductase inhibitors and antioxidant agents for the
treatment of diabetic complications, Eur. J. Med. Chem. 46 (2011) 2797–2806.
[38] R. Ottanà, S. Carotti, R. Maccari, I. Landini, G. Chiricosta, B. Caciagli, M.G.
Vigorita, E. Mini, In vitro antiproliferative activity against human colon cancer
cell lines of representative 4-thiazolidinones. Part I, Bioorg. Med. Chem. Lett.
15 (2005) 3930–3933.
[16] V.A. Ostrovskii, G.I. Koldobskii, R.E. Trifonov, 6.07–Tetrazoles, Comp.
Heterocycl. Chem. III 6 (2008) 257–423.
[17] C.E. Burgos-Lepley, L.R. Thompson, C.O. Kneen, S.A. Osborne, J.S. Bryans, T.
Capiris, N. Suman-Chauhan, D.J. Dooley, C.M. Donovan, M.J. Field, M.G.
Vartanian, J.J. Kinsora, S.M. Lotarski, A. El-Kattan, K. Walters, M. Cherukury, C.P.
Taylor, D.J. Wustrow, J.B. Schwarz, Carboxylate bioisosteres of gabapentin,
Bioorg. Med. Chem. Lett. 16 (2006) 2333–2336.
[18] H. Bräuner-Osborne, J. Egebjerg, E.O. Nielsen, U. Madsen, P. Krogsgaard-Larsen,
Ligands for glutamate receptors: design and therapeutic prospects, J. Med.
Chem. 43 (2000) 2609–2645.
[39] X. Li, S.R. Srinivasan, J. Connarn, A. Ahmad, Z.T. Young, A.M. Kabza, E.R.P.
Zuiderweg, D. Sun, J.E. Gestwicki, Analogs of the allosteric heat shock protein
70 (Hsp70) inhibitor MKT-077, as anti-cancer agents, ACS Med. Chem. Lett. 4
(2013) 1042–1047.
[40] K. Appalanaidu, R. Kotcherlakota, T.L. Dadmal, V.S. Bollu, R.M. Kumbhare, C.R.
Patra, Synthesis and biological evaluation of novel 2-imino-4-thiazolidinone
derivatives as potent anti-cancer agents, Bioorg. Med. Chem. Lett. 26 (2016)
5361–5368.
[19] N. Rüger, M. Roatsch, T. Emmrich, H. Franz, R. Schüle, M. Jung, A. Link,
Tetrazolyl-hydrazides as selective fragment-like inhibitors of the Jumonji C-
domain-containing histone demethylase KDM4A, ChemMedChem 10 (2015)
1875–1883.
[20] A.S. Gundugola, K.L. Chandra, E.M. Perchellet, A.M. Waters, J.P. Perchellet, S.
Rayat, Synthesis and antiproliferative evaluation of 5-oxo and 5-thio
derivatives of 1,4-diaryl tetrazoles, Bioorg. Med. Chem. Lett. 20 (2010) 3920–
3924.
[21] C.N.S.S.P. Kumar, D.K. Parida, A. Santhoshi, A.K. Kota, B. Sridhar, V.J. Rao,
Synthesis and biological evaluation of tetrazole containing compounds as
possible anticancer agents, Med. Chem. Commun. 2 (2011) 486–492.
[22] R. Romagnoli, P.G. Baraldi, M.K. Salvador, D. Preti, M.A. Tabrizi, A. Brancale, X.H.
Fu, J. Li, S.Z. Zhang, E. Hamel, R. Bortolozzi, G. Basso, G. Viola, Synthesis and
evaluation of 1,5-disubstituted tetrazoles as rigid analogues of combretastatin
A-4 with potent antiproliferative and antitumor activity, J. Med. Chem. 55
(2012) 475–488.
[41] S. Chandrappa, C.V. Kavitha, M.S. Shahabuddin, K. Vinaya, C.S. Ananda Kumar,
S.R. Ranganatha, S.C. Raghavan, K.S. Rangappa, Synthesis of 2-(5-((5-(4-
chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic
acid derivatives and evaluation of their cytotoxicity and induction of apoptosis
in human leukemia cells, Bioorg. Med. Chem. 17 (2009) 2576–2584.
[42] Y.S. Prabhakar, R.K. Rawal, M.K. Gupta, V.R. Solomon, S.B. Katti, Topological
descriptors in modeling the HIV inhibitory activity of 2-aryl-3-pyridyl-
thiazolidin-4-ones, Comb. Chem. High Throughput Screen. 8 (2005) 431–437.
[43] R.K. Rawal, Y.S. Prabhakar, S.B. Katti, E. De Clercq, 2-(Aryl)-3-furan-2-ylmethyl-
thiazolidin-4-ones as selective HIV-RT inhibitors, Bioorg. Med. Chem. 13
(2005) 6771–6776.
[44] H. Chen, Z. Guo, Q. Yin, X. Duan, Y. Gu, X. Li, Design, synthesis and HIV-RT
inhibitory activity of novel thiazolidin-4-one derivatives, Front. Chem. Sci. Eng.
5 (2011) 231–237.
[45] K. Ramkumar, V.N. Yarovenko, A.S. Nikitina, I.V. Zavarzin, M.M. Krayushkin, L.
V. Kovalenko, A. Esqueda, S. Odde, N. Neamati, Design, synthesis and structure-
activity studies of rhodanine derivatives as HIV-1 integrase inhibitors,
Molecules 15 (2010) 3958–3992.
[23] M. Arshad, A.R. Bhat, S. Pokharel, J.E. Kim, E.J. Lee, F. Athar, I. Choi, Synthesis,
characterization and anticancer screening of some novel piperonyl-tetrazole
derivatives, Eur. J. Med. Chem. 71 (2014) 229–236.
[24] V.V. Zarubaev, E.L. Golod, P.M. Anfimov, A.A. Shtro, V.V. Saraev, A.S. Gavrilov, A.
V. Logvinov, O.I. Kiselev, Synthesis and anti-viral activity of azolo-
adamantanes against influenza A virus, Bioorg. Med. Chem.18 (2010) 839–848.
[46] Y. Tian, P. Zhan, D. Rai, J. Zhang, E. De Clercq, X. Liu, Recent advances in the
research of 2,3-diaryl-1,3-thiazolidin-4-one derivatives as potent HIV-1 non-
nucleoside reverse transcriptase inhibitors, Curr. Med. Chem. 19 (2012) 2026–
2037.

812
A. Bielenica et al. / Biomedicine & Pharmacotherapy 94 (2017) 804–812
ꢀ
ꢀ
[47] M.L. Barreca, A. Chimirri, L.D. Luca, A.M. Monforte, P. Monforte, A. Rao, M.
Zappalà, J. Balzarini, E. De Clercq, C. Pannecouque, Discovery of 2,3-diaryl-1,3-
thiazolidin-4-ones as potent anti-HIV-1 agents, Bioorg, Med. Chem. Lett. 11
(2001) 1793–1796.
[48] M.L. Barreca, J. Balzarini, A. Chimirri, E. De Clercq, L. De Luca, H.D. Höltje, M.
Höltje, A.M. Monforte, P. Monforte, C. Pannecouque, Design synthesis,
structure-activity relationships, and molecular modeling studies of 2,3-diaryl-
1,3-thiazolidin-4-ones as potent anti-HIV agents, J. Med. Chem. 45 (2002)
5410–5413.
[49] A. Rao, J. Balzarini, A. Carbone, A. Chimirri, E. De Clercq, A.M. Monforte, P.
Monforte, C. Pannecouque, M. Zappalà, 2-(2,6-Dihalophenyl)-3-(pyrimidin-2-
yl)-1,3-thiazolidin-4-ones as non-nucleoside HIV-1 reverse transcriptase
inhibitors, Antivir. Ees. 63 (2004) 79–84.
[50] R.K. Rawal, A. Kumar, M.I. Siddiqi, S.B. Katti, Molecular docking studies on 4-
thiazolidinones as HIV-1 RT inhibitors, J. Mol. Model. 13 (2007) 155–161.
[51] R.K. Rawal, V.R. Solomon, Y.S. Prabhakar, S.B. Katti, E. De Clercq, Synthesis and
QSAR studies on thiazolidinones as anti-HIV agents, Comb. Chem. High
Throughput Screening 8 (2005) 439–443.
[52] A. Bielenica, J. Stefanska, K. Ste˛pien, A. Napiórkowska, E. Augustynowicz-
ꢀ
Kopec, G. Sanna, S. Madeddu, S. Boi, G. Giliberti, M. Wrzosek, M. Struga,
Synthesis, cytotoxicity and antimicrobial activity of thiourea derivatives
incorporating 3-(trifluoromethyl)phenyl moiety, Eur. J. Med. Chem. 101 (2015)
111–125.
ꢀ
ꢀ
[53] A. Bielenica, E. Ke˛dzierska, M. Kolinski, S. Kmiecik, A. Kolinski, F. Fiorino, B.
Severino, E. Magli, A. Corvino, I. Rossi, P. Massarelli, A.E. Kozioł, A. Sawczenko,
M. Struga, 5-HT2 receptor affinity, docking studies and pharmacological
evaluation of a series of 1, 3-disubstituted thiourea derivatives, Eur. J. Med.
Chem. 116 (2016) 173–186.
[54] G.M. Sheldrick, A short history of SHELX, Acta Cryst. A 64 (2008) 112–122.
[55] H.J. Böhm, D. Banner, S. Bendels, M. Kansy, B. Kuhn, K. Müller, U. Obst-Sander,
M. Stahl, Fluorine in medicinal chemistry, Chembiochem 5 (2004) 637–643.
[56] W.K. Hagmann, The many roles for fluorine in medicinal chemistry, J. Med.
Chem. 51 (2008) 4359–4369.