Synthetic transition from thiourea-based compounds to tetrazole derivatives: Structure and biological evaluation of synthesized new n-(furan-2-ylmethyl)-1h-tetrazol-5-amine derivatives

Article abstract of DOI:10.3390/molecules26020323

Twelve novel derivatives of N-(furan-2-ylmethyl)-1H-tetrazol-5-amine were synthesized. For obtained compound 8, its corresponding substrate single crystals were isolated and X-ray diffraction experiments were completed. In the initial stage of research, in silico structure-based pharmacological prediction was conducted. All compounds were screened for their antibacterial and antimycobacterial activities using standard and clinical strains. The cytotoxic activity was evaluated against a panel of human cancer cell lines, in contrast to normal (HaCaT) cell lines, by using the MTT method. All examined derivatives were found to be noncytotoxic against normal cell lines. Within the studied group, compound 6 showed the most promising results in antimicrobial studies. It inhibited four hospital S. epidermidis rods’ growth, when applied at the amount of 4 μg/mL. However, the most susceptible to the presence of compound 6 was S. epidermidis T 5501 851/19 clinical strain, for which the MIC value was only 2 μg/mL. Finally, a pharmacophore model was established based on lead compounds from this and our previous work.

Full text of DOI:10.3390/molecules26020323

molecules
Article
Synthetic Transition from Thiourea-Based Compounds to Tetrazole
Derivatives: Structure and Biological Evaluation of Synthesized
New N-(Furan-2-ylmethyl)-1H-tetrazol-5-amine Derivatives
Daniel Szulczyk 1,
* ,
, Anna Bielenica 1, Piotr Roszkowski 2 , Michał A. Dobrowolski 2 , Wioletta Olejarz 3
Sebastian Kmiecik 4, Małgorzata Podsiad 1 and Marta Struga 1
1
Chair and Department of Biochemistry, Medical University of Warsaw, 02-097 Warszawa, Poland;
anna.bielenica@wum.edu.pl (A.B.); malgorzata.podsiad@wum.edu.pl (M.P.); mstruga@wum.edu.pl (M.S.)
Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warszawa, Poland;
roszkowski@chem.uw.edu.pl (P.R.); miked@chem.uw.edu.pl (M.A.D.)
Department of Biochemistry and Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw,
02-097 Warszawa, Poland; wioletta.olejarz@wum.edu.pl
2
3
4
Biological and Chemical Research Centre, Faculty of Chemistry, University of Warsaw,
02-089 Warsaw, Poland; sekmi@chem.uw.edu.pl
*
Correspondence: daniel.szulczyk@wum.edu.pl
Abstract: Twelve novel derivatives of N-(furan-2-ylmethyl)-1H-tetrazol-5-amine were synthesized.
For obtained compound , its corresponding substrate single crystals were isolated and X-ray
8
diffraction experiments were completed. In the initial stage of research, in silico structure-based
pharmacological prediction was conducted. All compounds were screened for their antibacterial and
antimycobacterial activities using standard and clinical strains. The cytotoxic activity was evaluated
against a panel of human cancer cell lines, in contrast to normal (HaCaT) cell lines, by using the MTT
method. All examined derivatives were found to be noncytotoxic against normal cell lines. Within the
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Szulczyk, D.; Bielenica, A.;
Roszkowski, P.; Dobrowolski, M.A.;
Olejarz, W.; Kmiecik, S.; Podsiad, M.;
Struga, M. Synthetic Transition from
Thiourea-Based Compounds to
studied group, compound
four hospital S. epidermidis rods’ growth, when applied at the amount of 4
most susceptible to the presence of compound was S. epidermidis T 5501 851/19 clinical strain, for
which the MIC value was only 2 g/mL. Finally, a pharmacophore model was established based on
6
showed the most promising results in antimicrobial studies. It inhibited
µg/mL. However, the
Tetrazole Derivatives: Structure and
Biological Evaluation of Synthesized
New N-(Furan-2-ylmethyl)-1H-
tetrazol-5-amine Derivatives.
6
µ
lead compounds from this and our previous work.
Molecules 2021, 26, 323. https://
doi.org/10.3390/molecules26020323
Keywords: antimicrobial; antibacterial; tetrazole; cytotoxicity; crystal structure
Academic Editor: Helen Osborn
Received: 7 December 2020
Accepted: 7 January 2021
1. Introduction
Recently, there is a need for research and development of new antimicrobials. Past
years showed an increasing number of patients suffering from antibiotic-resistant bacterial
strains. Antimicrobial resistance is the ability of microorganisms such as bacteria, fungi,
or protozoans to grow despite exposure to antimicrobial substances designed to inhibit
their growth.
A number of studies were engaged to help solve this issue. Scientists worldwide are
trying to identify new natural antimicrobials isolated from plants or design new small
molecules that might serve as future antimicrobial medicines. In some of the published
papers it can be found that thiourea derivatives are recognized as promising antimicrobial
Published: 10 January 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
agents [1–8]. Thiourea derivatives are relatively easy to synthesize and can be used for
further structural modifications. One of the interesting scaffolds that can be obtained from
them is the tetrazole ring. The 1,5-disubstituted tetrazoles can be generated by oxidative
desulfurization of 1,3-disubstituted thioureas. This method acquires external nucleophile—
sodium azide and leads to corresponding 1,5-disubstituted tetrazoles. Mercury salts serve
as desulfurization agents.
Molecules 2021, 26, 323. https://doi.org/10.3390/molecules26020323
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 323
2 of 15
In past years a few interesting studies of tetrazole-based compounds possessing antimi-
crobial activity were published [9–11]. Moreover, clinicians have already used tetrazole-
derived antimicrobial drugs such as Cefamandole, Ceftezole, both second-generation
broad-spectrum cephalosporin antibiotics and an oxazolidinone-class antibiotic Tadalizolid
(Figure 1).
Figure 1. Structures of tetrazole-based antibiotics—Cefamandole, Ceftezole, and Tadalizolid, respectively.
We can also find very promising studies concerning tetrazoles as antibacterial agents.
Dis¸li et al. reported the successful synthesis and antimicrobial activity of new
6-dimethoxypyrimidine compounds incorporating a 1H-tetrazol-5-ylthio moiety. The
synthesized compounds were evaluated for in vitro antibacterial activity against Gram-
positive (Staphylococcus aureus ATCC 25923, Streptococcus agalactiae from Institute of Pasteur,
55118) and Gram-negative (Klebsiella pneumoniae ATCC 700603) bacteria strains. The results
showed that some of these compounds exhibited as good antibacterial activity as that
of standard antibiotics Penicillin, Ampicillin, and Erythromycin [9]. In another paper,
tetrazole-1,2,3-triazole hybrids displayed promising in vitro antibacterial and antifungal
activities against a panel of clinically important bacteria such as S. aureus, Bacillus subtilis
or Escherichia coli, and six of them were comparable to Gatifloxacin [10]. Other research
showed that a series of tetrazole-norfloxacin conjugates and their derivatives were found
active against various strains of S. aureus including drug-resistant strains MRSA and
Gentamycin-resistant S. aureus (GRSA), E. coli and K. pneumoniae. The majority of them
showed great potency against tested Gram-positive and Gram-negative pathogens, with
MIC values ranging from 0.39 to 50 mg/mL, and they were comparable or more effective
than Ciprofloxacin and Gentamycin against staphylococci [11]. Above-mentioned exam-
ples and many more are well disclosed in a review of tetrazole-based compounds with
antimicrobial activity [12].
During previous years, our team also participated in research for new antimicrobial
agents built on a tetrazole scaffold [13
working with other heterocyclic skeleton-based compounds [16
design and synthesize a series of heterocyclic scaffold tetrazole hybrids [20]. The best
–
15]. On the other hand, using our experience in
–
19] we have decided to

Molecules 2021, 26, 323
3 of 15
results were found for N-(furan-2-ylmethyl)-1H-tetrazol-5-amine derivatives, therefore
it was obvious to prepare new compounds possessing that moiety and evaluate them as
antimicrobial agents.
2. Results and Discussion
2.1. In Silico Structure-based Pharmacological Prediction
2.1.1. Antibacterial Activity
Using AntiBac-Pred [21] web services of the Way2Drug platform, activity against Gram-
positive and Gram-negative bacteria was predicted for twelve synthesized compounds. It
was found that the whole group might be active against Mycobacterium tuberculosis (con-
fidence values were ranging from 0.1861 to 0.0123) and Sarcina (confidence values were
ranging from 0.2401 to 0.2197). It should be mentioned that interesting probability scores
were found for compounds 7 (confidence value 0.4039), 10 (confidence value 0.2411), and
12 (confidence value 0.1718) for activity against Staphylococcus aureus subsp. aureus MW2
resistant strain.
2.1.2. Toxicological Parameters
The pkCSM [22] software was used for the evaluation of toxicological parameters
in silico. Evaluation revealed that none of synthesized compounds were positive in the
AMES test, therefore recognizing it as non-carcinogenic. Furthermore, all derivatives were
found not to be hERG I/II (the human Ether-à-go-go-Related Gene) inhibitors, which are
the principal causes of acquire long QT syndrome. Flathead minnow larvae test revealed
the low environmental hazard in the case of all derivatives, since all results were higher
than 0.5 nM over two times. Results are consistent in terms of the skin sensitization test,
as all compounds should not develop this effect. The maximum recommended tolerated
dose (MRTD) should be considered as low. In most of the test results (Table 1), it could be
stated that they are consistent along synthesized compounds.
Table 1. The toxicological parameters of N-(furan-2-ylmethyl)-1H-tetrazol-5-amine derivatives estimated using pkCSM software.
Toxicological Test
Unit
1
2
3
4
5
6
7
8
9
10
11
12
AMES toxicity
(Yes/No)
No
No
No
No
No
No
No
No
No
No
No
No
Max. tolerated dose
(human)
(log mg/kg/day)
0.056
0.14
0.016
0.038
0.074
0.045
0.031
0.039
0.055
0.032
0.028
0.021
hERG I inhibitor
hERG II inhibitor
(Yes/No)
(Yes/No)
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Oral Rat Acute
Toxicity (LD50)
(mol/kg)
2.069
0.99
2.211
1.001
2.148
0.966
2.145
0.947
2.254
0.898
2.052
0.977
2.154
0.947
2.151
0.953
2.064
0.983
2.16
2.133
0.941
2.141
0.941
Oral Rat Chronic
Toxicity (LOAEL)
(log
0.954
mg/kg_bw/day)
Hepatotoxicity
Skin Sensitisation
T.Pyriformis toxicity
Minnow toxicity
(Yes/No)
(Yes/No)
(log ug/L)
(log mM)
Yes
No
Yes
No
No
No
Yes
No
No
No
Yes
No
Yes
No
Yes
No
Yes
No
No
No
Yes
No
Yes
No
0.34
1.698
0.341
1.426
0.342
1.079
0.34
1.435
0.346
1.177
0.337
1.749
0.341
1.289
0.342
1.365
0.338
1.768
0.343
1.219
0.34
1.416
0.34
1.27
2.2. Chemistry
Thiourea substrates were obtained in our past project. Synthesis, physicochemical
and biological evaluation were extensively described in the published paper [23]. The
main goal in the current design was to replace the thiourea arrangement with a tetrazole
scaffold. Twelve new N-(furan-2-ylmethyl)-1H-tetrazol-5-amine derivatives were synthe-
sized according to the well-known procedure [15]. Reaction conditions are depicted below
(Scheme 1). All compounds were obtained in good yields, ranging from 79% to 92%. Yield
differences are linked to the amount of unreacted substrates in each reaction.

Molecules 2021, 26, 323
4 of 15
Scheme 1. Transition from thiourea-based substrates to tetrazole derivatives.
It should be stated that two additional compounds possessing a furan-2yl-methyl moi-
ety were synthesized on an earlier stage of the research and investigated for antimicrobial
activity (Figure 2) [20].
Both (Ref 1 and Ref 2) were synthesized in the same manner as the new twelve
derivatives. These compounds were recognized as leading structures, possessing the
highest antimicrobial activity, thus encouraging our team to design different furan-2yl-
methyl tetrazole hybrids for further evaluation. In subsequent parts of the paper, these
compounds will be used as the reference for newly synthesized derivatives.
Figure 2. The structure of 1-(3-chloro-4-methylphenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine
(Ref 1) and 1-(3-chloro-4-fluorophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (Ref 2).
2.3. X-ray Studies
The structure of 1-(4-chlorophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine 8 and 1-
(4-chlorophenyl)-3-(furan-2-ylmethyl)thiourea 8a were determined by X-ray diffraction on a
single crystal. The thiourea-based substrate was characterized in previously published pa-
pers [23,24], but no X-ray studies were presented. Both (8a) and (8) crystallize in a monoclinic
system, with one molecule in an asymmetric part of the unit cell—the former in centrosym-
metric space group P21/c, the latter in non-centrosymmetric P21. Crystal data and structure
refinement were recorded (Table S1, X-ray studies file, Supplementary Materials).
Molecular structures of thiourea-based substrates 8a and 8 are presented in Figure 3.
Going from substrate to product twists the furan moiety with respect to chlorophenylth-
iourea/tetrazole skeleton (Figure 4). Compound 8a is more bent, the angle between best
planes calculated for furan and phenyl rings equals 65.7◦, while for 8 it is 75.2◦.
The crystal structure of 8a forms a chain along the y axis stabilized by a set of strong
hydrogen bonds N1-H1 S1 (2.658Å, Figure 5), whereas
(2.183Å, Figure 6). Additionally, there are intermolecular interactions C11-H11 N4 (2.689Å,
see Figure S1, X-ray studies file, Supplementary Materials) for and C-H S in the range of
8 does so along x axis N1-H1 N4
8

Molecules 2021, 26, 323
5 of 15
2.783-2.853Å, C-H O in the range of 2.614-2.738Å for 8a (see Figure S2, X-ray studies file,
Supplementary Materials).
To show structural diversities three compounds with similar phenyltetrazole substi-
tuted moieties have been found in Cambridge Structural Database [25]. The torsion angles
between the tetr◦azole and phenyl ring are significantly different and equal for 8-140.62◦;
◦
◦
JODHOA-127.63 ; YICNAA-89.10 and JIKRAZ-161.44 (Figure 7). This comparison shows
how labile this fragment is and a small structural feature such as an additional ring or
substituent may completely change the crystal structure. It may be important for the
starting geometry in molecular docking studies, when real solid-state parameters are used
instead of in silico data.
In a subsequent article, the comparisons between the two groups will be made in order
to be able to draw unambiguous conclusions as to the change of the thiourea fragment to
the tetrazole with a description of the physicochemical changes in the solid state. Firstly, a
greater group of suitable thiourea-tetrazole derivative pairs must be collected.
Figure 3. The molecular structure of (8a, top) and (8, bottom) showing displacement ellipsoids at the
50% probability level.

Molecules 2021, 26, 323
6 of 15
Figure 4. Superposition of 8a and 8.
Figure 5. Intermolecular interactions in the crystal lattice along [010] direction for 8a.

Molecules 2021, 26, 323
7 of 15
Figure 6. Intermolecular interactions in the crystal lattice along [100] direction for 8.
Figure 7. The comparison of four molecules taken from crystal structures—1-(4-chlorophenyl)-
N-(furan-2-ylmethyl)-1H-tetrazol-5-amine ; JODHOA-1-(4-Nitrophenyl)-1H-tetrazol-5-amine [26];
8
YICNAA-1-(1-(4-Nitrophenyl)-1H-tetrazol-5-yl)-1H-1,2,3-benzotriazole [27]; JIKRAZ-(1-(4-nitrophenyl)-
N-(4H-1,2,4-triazol-4-yl)-1H-tetrazol-5-aminido)-hexakis(aqua)-calcium 1-(4-nitrophenyl)-N-(4H-1,2,4-
triazol-4-yl)-1H-tetrazol-5-aminide dehydrate [20].

Molecules 2021, 26, 323
8 of 15
2.4. Biological Studies
2.4.1. In Vitro Antibacterial Activity Studies
Both disubstituted N-(furan-2-ylmethyl)-1H-tetrazol-5-amines studied by us recently [20
exerted excellent antimicrobial profiles. The 3-chloro-4-fluorophenyl derivative Ref 2 was
active against various standard bacterial strains at the range of 0.25-4 g/mL, being also a
strong inhibitor of the growth of clinical cocci (MICs of 2-16 g/mL). Very similar results were
]
µ
µ
also noted for its 3-chloro-4-methylphenyl Ref 1 analogue. Moreover, these tetrazol-5-amines
were severalfold more active than the referential Ciprofloxacin.
Prompted by this, all obtained N-(furan-2-ylmethyl)-1H-tetrazol-5-amine derivatives
1–12 were tested in vitro against a number of bacteria, including representative standard
Gram-positive and Gram-negative rods. Compounds were screened for their minimal
inhibitory concentrations (MICs) [28]. The results revealed that half of investigated tetra-
zole derivatives exhibited moderate-to-weak antimicrobial properties, with MIC values
ranging from 8 to 256
table. The active compounds came mainly from the group of para-substituted
disubstituted tetrazoles, and the 2-fluorophenyl analogue was the most promising.
µg/mL (Table 2). Inactive compounds were not included in the
1, 3
or
3
6
Its growth-inhibitory properties towards standard Staphylococci were in the range of 8–32
µg/mL, whereas it prevented the rise of some Gram-negative strains at the concentration
of 16
completed the antimicrobial profile of derivative
S. epidermidis rods growth, when applied at the amount of 4
the rest of studied Staphylococci were at the level of 8 g/mL, except of S. aureus 155/6
isolate (MIC = 16 g/mL). However, the most susceptible to the presence of tested 2-
µ
g/mL. Next, the tests performed against the hospital staphylococcal isolates panel
(Table 3). It inhibited four hospital
g/mL. The MICs towards
6
µ
µ
µ
fluorophenyltetrazole
MIC value was only 2
6
µ
was the S. epidermidis T 5501 851/19 clinical strain, for which the
g/mL. It is worth noting that this derivative’s growth inhibitory
properties towards two clinical S. epidermidis strains was 8–16-fold times stronger than
the referential chemotherapeutic. In addition, it exerted half of the Ciprofloxacin strength
against other the staphylococcal KR 4047 825/19 isolate.
Results presented for Ref 1 and Ref 2 in the table above are not complete since the
other strain collection was used in the previous project [20]. For the same reason we cannot
compare the results for clinical strains. Collected clinical strains for current and previous
antimicrobial evaluation are different. However, we want to point out that compound Ref 1
and Ref 2 possessed the highest antimicrobial potential against standard and clinical strains.
We want to emphasize that derivative
6 can also be recognized as a leading compound
because of a comparable level of antimicrobial activity.
Table 2. Activity of compounds against standard bacteria strains, expressed by minimal inhibitory concentrations (µg/mL).
Ciprofloxacin
Compound
[µg/mL]
Strain
1
64
2
64
64
256
64
128
>
3
4
6
8
8
128
128
>
Ref 1
Ref 2
S. aureus NCTC 4163
S. aureus ATCC 29213
S. epidermidis ATCC 12228
E. hirae ATCC 10541
64
256
256
512
512
>
4
-
2
-
0.125
0.5
64
64
8
256
128/64
128
>
128
128
128
>
32
16
16
>
2
8
-
2
4
-
0,25
2
>
E. coli ATCC 25922
>
0.015
0.25
0.015
0.125
P. aeruginosa ATCC 27853
E. coli NCTC 8196
>
>
4
-
2
-
128
>
128
>
128
512
512
>
512
>
>
P. aeruginosa ATCC 15442
>
32
64

Molecules 2021, 26, 323
9 of 15
Table 3. Activity of derivative 6 against clinical isolates, expressed by minimal inhibitory concentrations (µg/mL).
Strain
Compound 6 [µg/mL]
Ciprofloxacin [µg/mL]
S. epidermidis KR 4047 825/19
S. epidermidis KR 4243 829/19
S. epidermidis KR 4268 830/19
S. epidermidis KR 4313 834/19
S. epidermidis KR 4358/2 840/19
S. epidermidis T 5253 845/19
S. epidermidis T 5399 848/19
S. epidermidis T 5501 851/19
S. aureus T5384/48
8
4
8
4
4
8
4
2
8
8
8
8
8
16
8
8
4
0.015
0.03
64
0.25
64
0.015
0.03
0.2
S. aureus 203/19
0.1
S. aureus T5403/51
0.2
S. aureus 176/13
0.5
S. aureus T5241/46
0.1
S. aureus 155/6
0.1
S. aureus T5591/68
0.1
S. aureus T5595/69
0.1
2.4.2. In Vitro Antimycobacterial Activity Studies
Inspired by biological properties observed for another series of 1H-tetrazol-5-amine
derivatives [14], all new tetrazoles 12 were screened for their in vitro antitubercular prop-
1–
erties against M. tuberculosis H37Rv strain and two “wild-type” mycobacteria isolated from
tuberculosis patients—multidrug-resistant Spec. 210 (with resistance to p-aminosalicylic
acid (PAS), isoniazid (INH), ethambutol (EMB) and rifampicin (RMP)) and Spec. 192,
fully susceptible to established tuberculostatics (Table S2, Supplementary Materials). The
compounds did not exert expected antimycobacterial activity (MIC values from 256 to
512 µg/mL).
2.4.3. Cytotoxic Studies
To get information about cytotoxicity of new compounds 1–12 in mammalian cells,
in vitro assays on three cancer cell lines were performed—HTB-140 (human melanoma), A549
(human epithelial lung carcinoma), Caco-2 (Caucasian colon adenocarcinoma), as well as on
the normal HaCaT cell line were performed. The results (IC50 >100 µM) showed that studied
tetrazoles did not influence the viability of these cells (Table S3, Supplementary Materials).
2.4.4. Structure-activity Relationship Studies (SAR)
The molecule conformations were obtained using the ATB topology builder tool
3.0 quantum mechanical calculations combined with a knowledge-based approach [29].
Panel (d) shows aligned structures of (a), (b), and (c) together with pharmacophore units
presented as spheres (Figure 8). The pharmacophore was detected via multiple alignments
of the lead molecules using the PharmaGist tool [30]. We identified eight pharmacophore
units—three aromatic, three acceptors in the tetrazole ring and two donors, oxygen in
the furan ring, and the amine group’s nitrogen. Halogen substituents of the phenyl
ring (marked by green circles) were not identified as pharmacophore units because of
their different spatial positions, either as substituents in the ring and the ring’s different
orientations that obviously may change in vivo. Thus, the role of fluorine or chlorine

Molecules 2021, 26, 323
10 of 15
substituents in the antimicrobial activity is not evident in the pharmacophore structure
analysis but may be important as indicated in our studies.
Figure 8. The leading compounds and their pharmacophore. Panels (
a–
c) show the conformations of the three leading
molecules—compound from this work and compound Ref 2 and Ref 1 from our previous work [20]. Panel (
6
d) shows
aligned structures of (a–c) together with pharmacophore units presented as spheres.
3. Materials and Methods
3.1. Apparatus, Materials, and Analysis
The reagents were supplied from Alfa Aesar (Haverhill, MA, USA) or Sigma Aldrich
(Saint Louis, MO, USA). Organic solvents (acetonitrile, DMF, chloroform, and methanol)
were supplied from POCh (Polskie Odczynniki Chemiczne, Gliwice, Poland). All chemicals
were of analytical grade. Before use, dried DMF and acetonitrile were kept in crown cap
bottles over anhydrous phosphorus pentoxide (Carl Roth, Karlsruhe, Germany).
The NMR spectra were recorded on a Bruker AVANCE DMX400 (Bruker, Billerica,
MA, USA) spectrometer, operating at 500 MHz (1H NMR) and 125 MHz (13C NMR). The
chemical shift values are expressed in ppm relative to TMS as an internal standard. Mass
spectral ESI measurements were carried out on Waters ZQ Micro-mass instruments (Waters,
Milford, MA, USA) with a quadrupol mass analyzer. The spectra were performed in the
positive ion mode at a de-clustering potential of 40–60 V. The sample was previously
separated on a UPLC column (C18) using the UPLC ACQUITYTM system by Waters
connected with a DPA detector. Flash chromatography was performed on Merck silica
gel 60 (200–400 mesh) using pure chloroform and chloroform/methanol (max. 19:1 vol)
mixture as eluent. Analytical TLC was carried out on silica gel F254 (Merck) plates (0.25 mm
thickness).
X-ray diffraction experiments were performed at 100(2) K for 8a and
on a Bruker D8 Venture Photon100 diffractometer (Bruker, Billerica, MA, USA) with a
TRIUMPH monochromator and MoK fine-focus sealed tube ( = 0.71073 Å). The crystals
were positioned 40 and 46 mm from the CCD camera for 8a and , respectively. A total
8, respectively,
α
λ
8

Molecules 2021, 26, 323
11 of 15
of 901 and 689 frames were measured with exposure times of 30 min and 2,4 h for 8a
and 8. Data were collected using the APEX2 program [31], integrated with the Bruker
SAINT software package [32] and corrected for absorption effects using the multi-scan
method (SADABS) [33]. The structures were solved and refined using the SHELX Software
Package [34] with the atomic scattering factors taken from the International Tables [35].
The figures were generated with Mercury (ver. 2020.2.0) [36].
CCDC 2047281, 2047282 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html.
3.2. Derivatives of N-(furan-2-ylmethyl)-1H-tetrazol-5-amine
Triethylamine (503 µL, 3.75 mmol, 1–3 drops) was added to a suspension of a corre-
sponding thiourea substrate (1.25 mmol), sodium azide (244 mg, 3.75 mmol), and mercuric
chloride (373 mg, 1.38 mmol) in 20 mL of dry DMF. The resulting suspension was stirred for
6 h at room temperature or until TLC indicated complete consumption of starting material.
The suspension was filtered through a pad of celite, washing with CH2Cl2. The filtrate
was diluted with water, and extracted with 3
×
15 mL of CH2Cl2. The combined organics
were dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting
residue was purified by silica gel chromatography using pure chloroform initially as eluent,
then increasingly adding methanol (max. 19:1 vol).
1-(4-fluorophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (
CD3OD): 4.53 (s, 2H, CH2), 6.31–6.33 (m, 2H, Fur), 7.31–7.36 (m, 2H), 7.39–7.40 (m, 1H,
Fur), 7.53–7.57 (m, 2H). 13C NMR (125 MHz, CD3OD):
41.6, 108.6 (Fur), 111.3 (Fur), 118.0
1
), Yield 83%. 1H NMR (500 MHz,
δ
δ
(d, 2J = 14.1 Hz, 2C), 128.4 (d, 3J = 5.5 Hz, 2C), 130.6 (d, 4J = 2.0 Hz), 143.4 (Fur), 152.9 (Fur),
156.5 (Tet), 164.5 (d, 1J = 148.4 Hz). HRMS (ESI) calc. for C12H10FN5O [M
found: 258,2441.
−
H]−: 258,2444
1
1-(5-chloro-2-methylphenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (
(500 MHz, CD3OD):
1H, Fur), 7.40–7.42 (m, 2H), 7.44–7.46 (m, 1H, Fur), 7.52–7.54 (m, 2H). 13C NMR (125 MHz,
CD3OD): 16.8, 41.4, 108.6 (Fur), 111.3 (Fur), 128.7, 132.2, 133.6, 133.9, 134.0, 136.3, 143.4
2), Yield 92%. H NMR
δ
2.02 (s, 3H), 4.51 (s, 2H, CH2), 6.29–6.30 (m, 1H, Fur), 6.33–6.34 (m,
δ
(Fur), 152.9 (Fur), 157.0 (Tet). HRMS (ESI) calc. for C13H12ClN5O [M
found: 288,7227.
−
H]−: 288,7230
1
N-(furan-2-ylmethyl)-1-(4-iodophenyl)-1H-tetrazol-5-amine (
CD3OD):
Fur), 7.95–7.98 (m, 2H). 13C NMR (125 MHz, CD3OD):
3
), Yield 89%. H NMR (500 MHz,
δ
4.53 (s, 2H, CH2), 6.32-6.34 (m, 2H, Fur), 7.31–7.33 (m, 2H), 7.40–7.41 (m, 1H,
41.6, 97.0, 108.7 (Fur), 111.4 (Fur),
δ
127.4 (2C), 134.3, 140.4 (2C), 143.4 (Fur), 152.9 (Fur), 156.3 (Tet). HRMS (ESI) calc. for
C12H10IN5O [M − H]−: 366,1505 found: 366,1503.
1-(3-chlorophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (4
), Yield 79%. 1H NMR (500 MHz,
CD3OD):
δ
4.54 (s, 2H, CH2), 6.33 (s, 2H, Fur), 7.41 (s, 1H), 7.48–7.50 (m, 1H, Fur), 7.57–7.60
(m, 3H). 13C NMR (125 MHz, CD3OD):
δ
41.6, 108.7 (Fur), 111.4 (Fur), 124.1, 125.8, 131.1,
132.4, 135.7, 136.5, 143.3 (Fur), 152.8 (Fur), 156.3 (Tet). HRMS (ESI) calc. for C12H10ClN5O
[M − H]−: 274,6960 found: 274,6859.
1-(3,4-dichlorophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (5
), Yield 88%. 1H NMR
(500 MHz, CD3OD):
δ 4.54 (s, 2H, CH2), 6.33 (d, J = 0.9 Hz, 2H, Fur), 7.41 (t, J = 0.9 Hz, 1H),
7.50 (dd, J1 = 1.5 Hz, J2 = 5.4 Hz, 1H, Fur), 7.75 (d, J = 5.1 Hz, 1H), 7.78 (d, J = 1.5 Hz, 1H).
13C NMR (125 MHz, CD3OD):
δ 41.6, 108.8 (Fur), 111.4 (Fur), 125.5, 127.8, 132.7, 134.1, 134.7,
135.0, 143.5 (Fur), 152.7 (Fur), 156.3 (Tet). HRMS (ESI) calc. for C12H9Cl2N5O [M − H]−
:
309,1380 found: 309,1378.
1-(2-fluorophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (
CD3OD): 4.53 (s, 2H, CH2), 6.29-6.33 (m, 2H, Fur), 7.39–7.44 (m, 3H), 7.52–7.56 (m, 1H,
Fur), 7.62–7.67 (m, 1H). 13C NMR (125 MHz, CD3OD):
41.5, 108.5 (Fur), 111.3 (Fur), 118.3
(d, 2J = 11.3 Hz), 121.9 (d, 3J = 7.6 Hz), 126.6 (d, 4J = 2.3 Hz), 130.0, 134.0 (d, 3J = 4.7 Hz), 143.4
6
), Yield 90%. 1H NMR (500 MHz,
δ
δ

Molecules 2021, 26, 323
12 of 15
(Fur), 152.9 (Fur), 157.2 (Tet), 158.4 (d, 1J = 151.0 Hz). HRMS (ESI) calc. for C12H10FN5O
[M − H]−: 258,2444 found: 258,2441.
1-(3-bromophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (
CD3OD):
(m, 2H). 13C NMR (125 MHz, CD3OD):
7
), Yield 85%. 1H NMR (500 MHz,
δ
4.54 (s, 2H, CH2), 6.33 (s, 2H, Fur), 7.41 (s, 1H), 7.7.49
−
7.52 (m, 2H), 7.71-7.74
δ
41.6, 108.7 (Fur), 111.4 (Fur), 124.1, 124.5, 128.7,
132.6, 134.1, 135.8, 143.4 (Fur), 152.8 (Fur), 156.3 (Tet). HRMS (ESI) calc. for C12H10BrN5O
[M − H]−: 319,1500 found: 319,1498.
1-(4-chlorophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (8
), Yield 81%. 1H NMR (500 MHz,
CD3OD):
δ
4.53 (s, 2H, CH2), 6.32–6.34 (m, 2H, Fur), 7.40–7.41 (m, 1H, Fur), 7.51–7.54 (m,
2H), 7.60–7.63 (m, 2H). 13C NMR (125 MHz, CD3OD):
δ
41.6, 108.7 (Fur), 111.4 (Fur), 127.3
(2C), 131.2 (2C), 133.2, 136.8, 143.4 (Fur), 152.9 (Fur), 156.4 (Tet). HRMS (ESI) calc. for
C12H10ClN5O [M − H]−: 274,6960 found: 274,6859.
1-(3-fluorophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (9
), Yield 89%. 1H NMR (500 MHz,
CD3OD): δ 4.55 (s, 2H, CH2), 6.33−6.34 (m, 2H, Fur), 7.30–7.34 (m, 1H), 7.36–7.39 (m, 2H),
7.41–7.42 (m, 1H, Fur), 7.60–7.65 (m, 1H). 13C NMR (125 MHz, CD3OD):
δ
4
41.6, 108.7
2
2
(Fur), 111.4 (Fur), 113.2 (d, J = 15.1 Hz), 117.8 (d, J = 12.7 Hz), 121.5 (d, J = 2.0 Hz),
3
132.8 (d, 3J = 5.4 Hz), 135.9 (d, J = 6.2 Hz), 143.4 (Fur), 152.9 (Fur), 156.3 (Tet), 164.3 (d,
1J = 147.9 Hz). HRMS (ESI) calc. for C12H10FN5O [M − H]−: 258,2444 found: 258,2441.
1-(4-bromophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (10), Yield 83%. 1H NMR (500 MHz,
CD3OD):
δ
4.54 (s, 2H, CH2), 6.32–6.34 (m, 2H, Fur), 7.40–7.41 (m, 1H, Fur), 7.45–7.48 (m,
41.6, 108.7 (Fur), 111.4 (Fur), 124.7,
2H), 7.75–7.78 (m, 2H). 13C NMR (125 MHz, CD3OD):
δ
127.35 (2C), 133.7, 134.3 (2C), 143.4 (Fur), 152.9 (Fur), 156.3 (Tet). HRMS (ESI) calc. for
C12H10BrN5O [M − H]−: 319,1500 found: 319,1498.
1
1-(2-chlorophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (11), Yield 80%. H NMR (500 MHz,
CD3OD):
δ
4.51 (s, 2H, CH2), 6.29–6.33 (m, 2H, Fur), 7.39–7.40 (m, 1H, Fur), 7.53–7.55 (m,
2H), 7.60–7.64 (m, 1H), 7.68–7.70 (m, 1H). 13C NMR (125 MHz, CD3OD):
δ
41.5, 108.5 (Fur),
111.3 (Fur), 129.7, 130.9, 131.7, 132.0, 133.4, 133.7, 143.4 (Fur), 152.9 (Fur), 157.1 (Tet). HRMS
(ESI) calc. for C12H10ClN5O [M − H]−: 274,6960 found: 274,6859.
1-(2-bromophenyl)-N-(furan-2-ylmethyl)-1H-tetrazol-5-amine (12), Yield 91%. 1H NMR (500 MHz,
CD3OD):
δ
4.51 (s, 2H, CH2), 6.30–6.33 (s, 2H, Fur), 7.39–7.40 (m, 1H, Fur), 7.51–7.56 (m,
2H), 7.58–7.61 (m, 1H), 7.85–7.87 (m, 1H). 13C NMR (125 MHz, CD3OD):
δ
41.4, 108.5 (Fur),
111.3 (Fur), 123.1, 130.3, 131.1, 133.3, 133.9, 135.2, 143.3 (Fur), 152.9 (Fur), 157.0 (Tet). HRMS
(ESI) calc. for C12H10BrN5O [M − H]−: 319,1500 found: 319,1498.
Copies of NMR spectra were added to supplementary part (NMR file).
3.3. Biological Assays
3.3.1. Cytotoxic Studies (MTT Assay)
The cell viability was assessed by using an enzymatic conversion of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) to insoluble formazan
crystals by mitochondrial dehydrogenases, occurring in living cells. Cells were cultured
in 96-well plates at a density of l
×
104 cells per well, and cultured to adhere for 24 h at
37 ◦C in a CO2 incubator. After 24 h of incubation, culture medium was replaced with a
fresh medium, then cells were treated with various concentrations of the compounds and
incubated for 72 h at 37 ◦C in a CO2 incubator. Untreated cells were used as the control.
Subsequently, MTT solution (0,5 mg/mL in free-serum medium) was added, and samples
◦
were incubated for 4 h at 37 C in a CO2 incubator. Then, the medium was aspirated,
and formed formazan crystals were solubilized by adding an isopropanol-DMSO mixture
(1:1 vol). The intensity of the dissolved crystals was measured using a UVM 340 reader
(ASYS Hitech GmbH, Austria), at a wavelength of 570 nm. Cell viability was presented as
a percent of MTT reduced in treated cells versus control cells (incubated without tested
compounds). 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 IC50 value was estimated using CalcuSyn version 1.0.

Molecules 2021, 26, 323
13 of 15
3.3.2. Antimicrobial Activity
The antimicrobial assays were conducted using reference strains of bacteria derived
from international microbe collections—American Type Culture Collection (ATCC) and
National Collection of Type Culture (NCTC). Among the reference strains there were
4 Gram-positive isolates (S. aureus: NCTC 4163, ATCC 29213, S. epidermidis ATCC 12228
and E. hirae ATCC 10541) and 4 Gram-negative rods (P. aeruginosa ATCC 15442 and ATCC
27853, E. coli NCTC 8196 and ATCC 25922). The antimicrobial activity of compounds
was expressed by minimum inhibitory concentration values (MICs), according to CLCI
reference procedures with some modifications. MIC was tested by the two-fold serial
microdilution method (in 96-well microtiter plates) on MH II liquid medium. The final
inoculum of all studies bacteria was 106 CFU/mL (colony forming unit per ml). The stock
solution of tested compounds was prepared in DMSO, and diluted in sterile medium (to a
maximum of 1% of solvent content). The MIC value was the lowest concentration of the
compound at which bacterial growth was no longer observed after 18 h of incubation at
35 ◦C. Clinical microorganisms used in this study were obtained from the collection of the
Department of Pharmaceutical Microbiology, Medical University of Warsaw, Poland.
A detailed description related to all conducted biological studies including cell culture,
suitable conditions, and methodology was presented in our previous papers [13–20,37].
4. Conclusions
Designed transition from thiourea substrates to appropriate N-(furan-2-ylmethyl)-
1H-tetrazol-5-amines was successfully performed in the course of a single reaction. The
synthesis was based on results from past studies and in silico conducted predictions. Within
the obtained twelve derivatives, the 2-fluorophenyltetrazole
against a panel of standard and clinical bacterial strains. In some cases of evaluation against
clinical isolates, results were even better than for the reference Ciprofloxacin. For and
6 showed interesting activity
8
its corresponding substrate 8a, single crystals were isolated and data gained related to
physicochemical properties of the synthesized compounds. In the near future, for two
groups, X-ray evaluation will be made to be able to draw unambiguous conclusions as to
the change of the thiourea fragment with regards to the tetrazole one.
Biological evaluation results confirmed that none of tested derivatives were cyto-
toxic against normal HaCaT cell lines. None of synthesized compounds was recognized
as a candidate for additional tests for antitubercular activity. In terms of seeking new
antimicrobials, compounds
four hospital S. epidermidis rods’ growth, when applied at the amount of 4
ever, the most susceptible to the presence of tested 2-fluorophenyltetrazole
S. epidermidis T 5501 851/19 clinical strain, for which the MIC value was only 2
6
was found as an interesting leading structure. It inhibited
g/mL. How-
was the
g/mL. In
µ
6
µ
general, towards two clinical S. epidermidis strains it was 8–16-fold times stronger than the
reference Ciprofloxacin.
Summarizing, N-(furan-2-ylmethyl)-1H-tetrazol-5-amines derivatives should be con-
sidered as promising in the field of antimicrobial action. The pharmacophore model was
established. We identified eight pharmacophore units—three aromatic, three acceptors in
the tetrazole ring and two donors, oxygen in the furan ring, and the amine group’s nitrogen.
Phenyl substituents were not recognized as pharmacophore units. Further research will be
performed on a broader group of compounds, especially those with fluorine-containing
moieties attached to the phenyl ring.
1
Supplementary Materials: The following are available online. NMR file: H and 13C NMR, Table S2
:
In vitro evaluation of antitubercular properties, Table S3: Antiproliferative activity of tested com-
pounds, X-ray studies file: Crystal data.

Molecules 2021, 26, 323
14 of 15
Author Contributions: D.S. performed the syntheses of compounds and wrote the manuscript. A.B.
collected biological data and wrote part of the paper. P.R. completed the spectral data analyses.
M.A.D. collected and analysed X-ray data. W.O. performed the cytotoxicity assay. S.K. performed
SAR studies. M.P. conducted antimicrobial studies, M.S. was the principal investigator of the project
and provided the research funding. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: Authors are grateful to Medical University of Warsaw CePT infrastructure for
support during initial stage of research. The Medical University of Warsaw supported this work and
it was 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.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
References
1.
Bielenica, A.; Stefan´ska, J.; Ste˛pien´, K.; Napiórkowska, A.; Augustynowicz-Kopec´, E.; Sanna, G.; Madeddu, S.; Boi, S.;
Giliberti, G.; Wrzosek, M.; et al. Synthesis, cytotoxicity and antimicrobial activity of thiourea derivatives incorporating 3-
(trifluoromethyl)phenyl moiety. Eur. J. Med. Chem. 2015, 101, 111–125. [CrossRef] [PubMed]
2.
Abbas, S.Y.; El-Sharief, M.A.M.S.; Basyouni, W.M.; Fakhr, I.M.I.; El-Gammal, E.W. Thiourea derivatives incorporating a hip-
puric acid moiety: Synthesis and evaluation of antibacterial and antifungal activities. Eur. J. Med. Chem. 2013, 64, 111–120.
[CrossRef] [PubMed]
3.
4.
Suresha, G.P.; Suhas, R.; Kapfo, W.; Channe Gowda, D. Urea/thiourea derivatives of quinazolinone–lysine conjugates: Synthesis
and structure–activity relationships of a new series of antimicrobials. Eur. J. Med. Chem. 2011, 46, 2530–2540. [CrossRef] [PubMed]
Saeed, S.; Rashid, N.; Jones, P.G.; Ali, M.; Hussain, R. Synthesis, characterization and biological evaluation of some thiourea
derivatives bearing benzothiazole moiety as potential antimicrobial and anticancer agents. Eur. J. Med. Chem. 2010, 45, 1323–1331.
[CrossRef] [PubMed]
5.
6.
7.
8.
9.
Vega-Pérez, J.M.; Periñán, I.; Argandoña, M.; Vega-Holm, M.; Palo-Nieto, C.; Burgos-Morón, E.; López-Lázaro, M.; Vargas, C.;
Nieto, J.J.; Iglesias-Guerra, F. Isoprenyl-thiourea and urea derivatives as new farnesyl diphosphate analogues: Synthesis and
in vitro antimicrobial and cytotoxic activities. Eur. J. Med. Chem. 2012, 58, 591–612. [CrossRef]
Stefanska, J.; Szulczyk, D.; Koziol, A.E.; Miroslaw, B.; Kedzierska, E.; Fidecka, S.; Busonera, B.; Sanna, G.; Giliberti, G.; La Colla, P.;
et al. Disubstituted thiourea derivatives and their activity on CNS: Synthesis and biological evaluation. Eur. J. Med. Chem. 2012
55, 205–213. [CrossRef]
,
Dobrikov, G.M.; Valcheva, V.; Nikolova, Y.; Ugrinova, I.; Pasheva, E.; Dimitrov, V. Efficient synthesis of new (R)-2-amino-1-butanol
derived ureas, thioureas and acylthioureas and in vitro evaluation of their antimycobacterial activity. Eur. J. Med. Chem. 2013, 63,
468–473. [CrossRef]
Joshi, S.D.; Dixit, S.R.; Kirankumar, M.N.; Aminabhavi, T.M.; Raju, K.V.S.N.; Narayan, R.; Lherbet, C.; Yang, K.S. Synthesis,
antimycobacterial screening and ligand-based molecular docking studies on novel pyrrole derivatives bearing pyrazoline,
isoxazole and phenyl thiourea moieties. Eur. J. Med. Chem. 2016, 107, 133–152. [CrossRef]
Dis¸li, A.; Mercan, S.; Yavuz, S. Synthesis and Antimicrobial Activity of New Pyrimidine Derivatives Incorporating 1H-Tetrazol-5-
ylthio Moiety. J. Het. Chem. 2013, 50, 1446–1450. [CrossRef]
10. Naresh, E.; Bhaskar, K.; Linga goud, G. Synthesis of Some New 5-(1-Phenyl-1H-1,2,3-Triazol-4-Yl)-1H-Tetrazoles and Evaluation
of Their Antimicrobial Activity. Russ. J. Gen. Chem. 2016, 86, 2862–2864. [CrossRef]
11. Chauhan, K.; Singh, P.; Kumar, V.; Shukla, P.K.; Siddiqi, M.I.; Chauhan, P.M.S. Investigation of Ugi-4CC Derived 1H-Tetrazol-5-Yl-
(Aryl) Methyl Piperazinyl-6-Fluoro-4-Oxo-1,4-Dihydroquinoline-3-Carboxylic Acid: Synthesis, Biology and 3D-QSAR Analysis.
Eur. J. Med. Chem. 2014, 78, 442–454. [CrossRef] [PubMed]
12. Gao, F.; Xiao, J.; Huang, G. Current Scenario of Tetrazole Hybrids for Antibacterial Activity. Eur. J. Med. Chem. 2019, 184, 111744.
[CrossRef] [PubMed]
13. Bielenica, A.; Szulczyk, D.; Olejarz, W.; Madeddu, S.; Giliberti, G.; Materek, I.B.; Koziol, A.E.; Struga, M. 1H-Tetrazol-5-Amine and
1,3-Thiazolidin-4-One Derivatives Containing 3-(Trifluoromethyl)Phenyl Scaffold: Synthesis, Cytotoxic and Anti-HIV Studies.
Biomed. Pharmacother. 2017, 94, 804–812. [CrossRef] [PubMed]
14. Szulczyk, D.; Bielenica, A.; Głogowska, A.; Augustynowicz-Kopec´, E.; Dobrowolski, M.; Roszkowski, P.; Ste˛pien´, K.; Chrzanowska,
A.; Struga, M. Development of (4-Methoxyphenyl)-1H-Tetrazol-5-Amine Regioisomers as a New Class of Selective Antitubercular
Agents. Eur. J. Med. Chem. 2020, 186, 111882. [CrossRef] [PubMed]

Molecules 2021, 26, 323
15 of 15
15. Szulczyk, D.; Bielenica, A.; Roszkowski, P.; Dobrowolski, M.A.; Olejarz, W.; Napiórkowska, M.; Struga, M. Cytotoxicity Evaluation
of Novel Bis(2-aminoethyl)amine Derivatives. Molecules 2020, 25, 2861. [CrossRef]
16. Stefanska, J.; Nowicka, G.; Struga, M.; Szulczyk, D.; Koziol, A.E.; Augustynowicz-Kopec, E.; Napiorkowska, A.; Bielenica,
A.; Filipowski, W.; Filipowska, A.; et al. Antimicrobial and Anti-Biofilm Activity of Thiourea Derivatives Incorporating a
2-Aminothiazole Scaffold. Chem. Pharm. Bull. 2015, 63, 225–236. [CrossRef]
17. Stefanska, J.; Stepien, K.; Bielenica, A.; Szulczyk, D.; Miroslaw, B.; Koziol, A.E.; Sanna, G.; Iuliano, F.; Madeddu, S.; Jozwiak, M.;
et al. Antimicrobial and Anti-Biofilm Activity of Thiourea Derivatives Bearing 3-Amino-1H-1,2,4-Triazole Scaffold. Med. Chem.
2016, 12, 478–488. [CrossRef]
18. Dobrowolski, M.A.; Roszkowski, P.; Struga, M.; Szulczyk, D. The Unexpected Product of Diels-Alder Reaction between “In-
danocyclon” and Maleimide. J. Mol. Struct. 2017, 1130, 573–578. [CrossRef]
19. Szulczyk, D.; Tomaszewski, P.; Józ´wiak, M.; Kozioł, A.E.; Lis, T.; Collu, D.; Iuliano, F.; Struga, M. Synthesis and Biological
Activities of Ethyl 2-(2-Pyridylacetate) Derivatives Containing Thiourea, 1,2,4-Triazole, Thiadiazole and Oxadiazole Moieties.
Molecules 2017, 22, 409. [CrossRef]
20. Szulczyk, D.; Dobrowolski, M.A.; Roszkowski, P.; Bielenica, A.; Stefan´ska, J.; Kolin´ski, M.; Kmiecik, S.; Józ´wiak, M.; Wrzosek, M.;
Olejarz, W.; et al. Design and Synthesis of Novel 1H-Tetrazol-5-Amine Based Potent Antimicrobial Agents: DNA Topoisomerase
IV and Gyrase Affinity Evaluation Supported by Molecular Docking Studies. Eur. J. Med. Chem. 2018, 156, 631–640. [CrossRef]
21. Pogodin, P.V.; Lagunin, A.A.; Rudik, A.V.; Druzhilovskiy, D.S.; Filimonov, D.A.; Poroikov, V.V. AntiBac-Pred: A Web Application
for Predicting Antibacterial Activity of Chemical Compounds. J. Chem. Info. Mod. 2019, 59, 4513–4518. [CrossRef] [PubMed]
22. Pires, D.E.V.; Blundell, T.L.; Ascher, D.B. PkCSM: Predicting Small-Molecule Pharmacokinetic and Toxicity Properties Using
Graph-Based Signatures. J. Med. Chem. 2015, 58, 4066–4072. [CrossRef] [PubMed]
23. Bielenica, A.; Stepien, K.; Sawczenko, A.; Lis, T.; Koziol, A.E.; Madeddu, S.; Collu, D.; Iuliano, F.; Kosmider, A.; Struga, M.
Synthesis, Structural Studies and Biological Evaluation of Halogen Derivative of 1,3-Disubstituted Thiourea. Lett. Drug Des.
Discov. 2017, 14, 636–646. [CrossRef]
24. Herschhorn, A.; Hizi, A. Virtual screening, identification, and biochemical characterization of novel inhibitors of the reverse
transcriptase of human immunodeficiency virus type-1. J. Med. Chem. 2008, 51, 5702–5713. [CrossRef]
25. Groom, C.R.; Bruno, I.J.; Lightfoot, M.P.; Ward, S.C. The Cambridge Structural Database. Acta Cryst. 2016, B72, 171–179. [CrossRef]
26. Lyakhov, A.S.; Vorobiov, A.N.; Ivashkevich, L.S.; Gaponik, P.N. Two derivatives of 1,5-disubstituted tetrazoles: 1-(4-nitrophenyl)-
1H-tetrazol-5-amine and {(E)-[1-(4-ethoxyphenyl)-1H-tetrazol-5-yl]iminomethyl}dimethylamine. Acta Cryst. 2008, 64, o414–o416.
27. Lyakhov, A.S.; Egorova, N.G.; Artamonova, T.V.; Gaponik, P.N.; Koldobskii, G.I. 1-[1-(4-Nitrophenyl)-1H-tetrazol-5-yl]-1H-1,2,3-
benzotriazole. Acta Cryst. 2007, 63, o2486–o2487. [CrossRef]
28. Clinical and Laboratory Standards Institute Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;
Approved Standard M7-A7; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2006.
29. Stroet, M.; Caron, B.; Visscher, K.M.; Geerke, D.P.; Malde, A.K.; Mark, A.E. Automated Topology Builder Version 3.0: Prediction
of Solvation Free Enthalpies in Water and Hexane. J. Chem. Theory. Comput. 2018, 14, 5834–5845. [CrossRef]
30. Schneidman-Duhovny, D.; Dror, O.; Inbar, Y.; Nussinov, R.; Wolfson, H.J. PharmaGist: A webserver for ligand-based pharma-
cophore detection. Nucleic Acids Res. 2008, 36, W223–W228. [CrossRef]
31. APEX2 (Version 2009.1); Bruker AXS Inc.: Madison, WI, USA.
32. SAINT (Version 4.050); Bruker AXS Inc.: Madison, WI, USA.
33. SADABS (Version 2004/1); Bruker AXS Inc.: Madison, WI, USA.
34. Sheldrick, G.M. Crystal structure refinement with SHELXL. Acta Crystallogr. C 2015, 71, 3–8. [CrossRef]
35. Wilson, A.J.C.; Geist, V. International Tables for Crystallography; BKluwer Academic Publishers: Dordrecht, The Netherlands, 1993;
Volume C, p. 883.
36. Macrae, C.F.; Edgington, P.R.; McCabe, P.; Pidcock, E.; Shields, G.P.; Taylor, R.; Towler, M.; van de Streek, J. Mercury Visualization
and Analysis of Crystal Structures. J. Appl. Crystallogr. 2006, 39, 453–457. [CrossRef]
37. Chrzanowska, A.; Roszkowski, P.; Bielenica, A.; Olejarz, W.; Ste˛pien´, K.; Struga, M. Anticancer and Antimicrobial Effects of Novel
Ciprofloxacin Fatty Acids Conjugates. Eur. J. Med. Chem. 2020, 185, 111810. [CrossRef] [PubMed]