Highly efficient microwave synthesis of rhodanine and 2-thiohydantoin derivatives and determination of relationships between their chemical structures and antibacterial activity

Article abstract of DOI:10.1039/c9ra08690k

Here we report studies on the synthesis of 12 new heterocyclic derivatives that differ in three structural motifs and the simultaneous evaluation of the impact of these three variables on the biological properties. The examined compounds are based on rhodanine and 2-thiohydantoin cores equipped with hydrogen or carboxymethyl substituents at the N-3 position and linked to a triphenylamine moiety through 1,4-phenylene, 1,4-naphthalenylene and 1,9-anthracenylene spacers at the C-5 position of the heterocycles. All the compounds were synthesized very quickly, selectively and in high yields according to the developed microwave-assisted Knoevenagel condensation protocol, and they were characterized thoroughly with NMR, FT-IR and ESI-HRMS techniques. The derivatives were tested for their activity against selected strains of Gram-positive and Gram-negative bacteria and yeast. Two compounds showed good activity against Gram-positive bacteria, and all of them showed low cytotoxicity against three cell lines of the human immune system. Based on membrane permeability assays it was demonstrated that the active compounds do not penetrate the cell membrane, and thus they must act on the bacterial cell surface. Finally, we proved that the evaluated structure modifications had a synergistic effect and the simultaneous presence of a 1,4-phenylene spacer and carboxymethyl group at N-3 caused the highest boost in antimicrobial activity.

Full text of DOI:10.1039/c9ra08690k

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PAPER
Highly efficient microwave synthesis of rhodanine
and 2-thiohydantoin derivatives and determination
of relationships between their chemical structures
and antibacterial activity†
Cite this: RSC Adv., 2019, 9, 39367
a
Bartosz Orwat, *bc Izabela Korona-Głowniak,
d
Waldemar Tejchman,
a
bc
e
Anna Barbasz,
Ireneusz Kownacki,
Gniewomir Latacz,
e
a
d
Jadwiga Handzlik,
Ewa Z˙ esławska
and Anna Malm
Here we report studies on the synthesis of 12 new heterocyclic derivatives that differ in three structural
motifs and the simultaneous evaluation of the impact of these three variables on the biological
properties. The examined compounds are based on rhodanine and 2-thiohydantoin cores equipped with
hydrogen or carboxymethyl substituents at the N-3 position and linked to a triphenylamine moiety
through 1,4-phenylene, 1,4-naphthalenylene and 1,9-anthracenylene spacers at the C-5 position of the
heterocycles. All the compounds were synthesized very quickly, selectively and in high yields according
to the developed microwave-assisted Knoevenagel condensation protocol, and they were characterized
thoroughly with NMR, FT-IR and ESI-HRMS techniques. The derivatives were tested for their activity
against selected strains of Gram-positive and Gram-negative bacteria and yeast. Two compounds
showed good activity against Gram-positive bacteria, and all of them showed low cytotoxicity against
three cell lines of the human immune system. Based on membrane permeability assays it was
demonstrated that the active compounds do not penetrate the cell membrane, and thus they must act
on the bacterial cell surface. Finally, we proved that the evaluated structure modifications had
a synergistic effect and the simultaneous presence of a 1,4-phenylene spacer and carboxymethyl group
at N-3 caused the highest boost in antimicrobial activity.
Received 23rd October 2019
Accepted 15th November 2019
DOI: 10.1039/c9ra08690k
rsc.li/rsc-advances
pathogens, many dangerous ones belong to the kingdom of
bacteria. These organisms can be classied into two groups
Introduction
Throughout evolution, many pathogenic microorganisms have based on the Gram staining result that indicates differences in
gained resistance to antibiotics, and this constitutes a threat to their structure, physiology and drug resistance. Gram-negative
1
public health. This ongoing process is a serious challenge for bacteria have an outer membrane of their cell walls that
2–4
modern medicinal chemistry, but it also stimulates develop- makes it difficult for antibiotics to penetrate into the cell. In
ment in the eld. For this reason, many research groups seek addition, in the periplasmic space located between the outer
new substances that can efficiently act against pathogenic and the inner membrane, there are enzymes that deactivate
5
microorganisms without posing a risk to patients. Among the antibiotics. Gram-positive bacteria do not have an outer
membrane, but the structure of their peptidoglycan is more
6
complicated than in Gram-negative bacteria. The lack of an
a
Department of Chemistry, Institute of Biology, Pedagogical University of Cracow,
outer membrane facilitates the penetration of antibiotics into
the cells and increases the number of molecular targets on the
cell wall surface that can be attacked by substances with anti-
Podchor a˛ ˙z ych 2, 30-084 Krak ´o w, Poland
b
Faculty of Chemistry, Adam Mickiewicz University, Uniwersytetu Pozna n´ skiego 8, 61-
6
14 Pozna n´ , Poland. E-mail: b.orwat@amu.edu.pl
c
Centre for Advanced Technology, Uniwersytetu Pozna n´ skiego 10, 61-614 Pozna n´ , bacterial activity.
Poland
There are well-known and particularly dangerous lethal
d
Department of Pharmaceutical Microbiology, Medical University of Lublin, Chod ´z ki 1,
diseases such as tuberculosis, leprosy and gas gangrene which
are caused by Gram-positive bacteria; cholera, brucellosis, pla-
gue, syphilis and salmonellosis that are caused by Gram-
negative bacteria. In the search for a cure against them, many
2
0-093 Lublin, Poland
e
Department of Technology and Biotechnology of Drugs, Jagiellonian University
Medical College, Medyczna 9, 30-688 Krak ´o w, Poland
†
Electronic supplementary information (ESI) available: Synthetic procedures,
NMR, IR, LR-MS and ESI-HRMS spectra, MIC and MBC values for selected scientists have synthesized various heterocyclic compounds
Gram-positive bacteria, LD50 values for human immune cell lines, and PAMPA
7,8
and studied their bactericidal properties. Among others,
experiments HPLC chromatograms. See DOI: 10.1039/c9ra08690k
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36
derivatives of ve-membered heterocyclic systems based on the the series of various chlorinated 5-arylidene rhodanines. It is
core of 1,3-thiazolidine or imidazolidine have been studied due noteworthy that compounds containing lipophilic, condensed
9,10
to their broad biological activity.
Of particular interest are aromatic moieties like naphthalene might exhibit better anti-
their derivatives containing exocyclic sulfur and oxygen atoms, bacterial activity than those containing unfused aromatics in
11
0
such as rhodanine (2-sulfanylidene-1,3-thiazolidin-4-one) or 2- the C-5 position (e.g. 1,1 -biphenyl or p-methox-
12
37,38
thiohydantoin (2-sulfanylidene-1,3-diazolidin-4-one).
The yphenylazosalicylidene moiety).
The incorporation of such
presence of these double-bonded atoms with other functional moieties might also affect other biological properties. Sing et al.
groups results in particularly high density of binding sites for proved that the incorporation of larger and more hydrophobic
polar interactions and hydrogen bonds, which are responsible substituents in the C-5 position of rhodanine 3-acetic acid can
13
for their interesting biological properties. A spectrum of their increase HCV NS3 protease inhibition selectivity up to 25 times
1
4,15
16–18
39
biological activity includes antifungal,
anticancer,
anti- compared to a derivative bearing biphenyl moiety. It is
anticonvul- therefore prerequisite that the size and lipophilicity of C-5
19,20
21
22,23
diabetic,
anti-inammatory,
antiviral,
2
4–26
27,28
sant
and enzyme-inhibiting properties.
Another substituents can modulate biological activity of 5-substituted
noteworthy feature of such cyclic systems is their strong polar rhodanine and its analogues. We mentioned before that the C-5
and electron-withdrawing character, making them popular substituent effect might not be the only parameter modulating
anchoring and electron-accepting groups in construction of activity. As Trotsko et al. showed, antibacterial activity of 2-thi-
merocyanine dyes for organic dye-sensitized solar cells ohydantoin derivatives was slightly higher than the corre-
29–31
40
(
DSSC).
building blocks is supported by the feasibility of their multi- that the heterocycle is another binding point for interaction
functionalization in various positions (Scheme 1). with enzymes or receptors, and that its core structure might
Additionally, the popularity of these heterocycles as sponding rhodanine derivatives. Consequently, we believe
In our previous research we proved that antibacterial activity alter the nal compound's activity. Finally, the substituent in
of the examined rhodanine-3-carboxylic acid series were the N-3 position is also very important. As Sim et al. observed,
strengthened with the increase in hydrophobic character of the rhodanine derivatives containing alkyl or aryl groups in this
32
substituent in position 5. Moreover, the lowest minimal position do not show antibacterial activity. The highest anti-
inhibitory concentration (MIC) and minimal bactericidal bacterial activity is exhibited in compounds equipped with
34
concentration (MBC) were observed for derivatives equipped hydrogen or carboxylic acid in the N-3 position. To sum up, we
with a triphenylamine moiety. This result indicated that are convinced that in order to design a substance with high
a coexistence of aromatic rings and multi-interacting hetero- antibacterial activity, the total effect of three variable points,
cycles within the same molecule might be a key requirement for namely the C-5 substituent, the heterocycle core structure and
enabling its activity. This conclusion prompted us to explore the the N-3 substituent, should be taken into account. These vari-
topic for similar reports in the literature. In the search, we ables should be examined together within the same molecule
encountered some works describing activity of familiar deriva- since they might synergistically inuence binding to active
tives. Toma ˇs i ´c et al. also reported that the increase of hydro- centers, but we did not nd such an approach for rhodanine
phobicity of a benzylidene substituent in the rhodanine C-5 and 2-thiohydantoin derivatives in the literature.
33
position led to an increase in antimicrobial activity. Among
Due to the lack of thorough evaluation of aromatic linker size
all examined compounds, 5-(2,3,4-triuorobenzylidene)- effect evaluation on the biological properties of rhodanine and
rhodanine exhibited the highest activity, inhibiting Staphylo- 2-thiohydantoin derivatives, we decided to ll in this gap.
coccus aureus and MRSA growth at concentrations of 0.5 and Therefore, we designed and synthesized a series of merocyanine
ꢀ1
3
2 mg L , respectively. A series of substituted rhodanines dyes consisting of the mentioned heterocycles with a triphenyl-
bearing benzylidene-derived fragments of various steric amine moiety bonded through a hydrocarbon aromatic linker
34
hindrance in position 5 showed also activity towards MRSA.
in position C-5. For this purpose, 1,4-phenylene, 1,4-naph-
Derivatives of hydantoin, an oxygen analogue of 2-thio- thalenylene and 1,10-anthracenylene were selected as the
hydantoin bearing a bulky 4-methyl-2-phenyl-furo[(3,2-d)pyr- simplest homologue linkers. Guided by the literature, we have
azole(3,2-d)imidazole] moiety in C-5, also showed antimicrobial
35
activity against Escherichia coli and Staphylococcus aureus.
Furthermore, it was proved that the steric and electronic
properties of the C-5 substituent affect bactericidal activity in
Scheme 1 General structure of rhodanine (left) and 2-thiohydantoin Scheme 2 Illustration of the concept and the differences between the
(
right). R groups mark positions of their potential functionalization.
compounds investigated in this work.
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undertaken to nd a cumulative effect of linker size, heterocycle
Aer successful preparation of intermediates 1a–c, we
core component and ancillary N-3 substituent (Scheme 2) on focused on the Knoevenagel condensation of the aldehydes with
biological properties. Our goal was to determine how the vari- selected heterocycles. Such transformation is a well-known
ation of the three interacting points inuence antibacterial method for C]C bond formation; however, the results of this
activity and cytotoxicity of the tested compounds, and to shed reaction are not always satisfactory. Additionally, in the most
some light on their mechanisms of activity.
reported protocols, prolonged heating of the reaction mixture in
reux conditions followed by recrystallization of the product
47–49
were required, resulting in medium yields.
During our
Results and discussion
Synthesis
thorough literature review, we encountered the promising
concept of microwave-accelerated Knoevenagel condensation.
50
Because this unconventional heat source has recently been
The proposed synthetic protocol for the target molecules was
based on Knoevenagel condensation of 4 -(diphenylamino)-
0
proved to increase the efficiency of various catalytic and stoi-
51
0
chiometric reactions in our group, we decided to examine its
effectiveness in our current work. Preliminary tests in which
anthracene-9-carbaldehyde and rhodanine were involved as
a model reagent system conrmed the superior efficiency of
microwave irradiation compared to a classical oil bath. Thus, we
decided to apply the former as a heat source in our synthetic
protocol. The rst reported reactions were conducted with
rhodanines 2a and 2b according to Scheme 4.
[
1,1 -biphenyl]-4-carbaldehyde
(1a),
4-(4-(diphenylamino)
(1b) and 10-(4-
diphenylamino)phenyl)anthracene-9-carbaldehyde (1c) with
phenyl)-1-naphthalenecarbaldehyde
(
rhodanines (2a, 2b) and thiohydantoins (3a, 3b). The former
aldehydes were prepared in the course of a Suzuki–Miyaura
cross-coupling reaction, the most commonly used of the
palladium-mediated reactions in the synthesis of active phar-
4
1,42
maceutical ingredients (APIs).
synthetic work, 4-(diphenylamino)phenylboronic acid, 4-
bromo-1-naphthalenecarbaldehyde and 10-bromo-9-
anthracenecarbaldehyde were prepared with slightly modied
Therefore, in the rst step of
During the synthesis of the desired compounds, we observed
that the reactions started almost immediately at elevated
temperature, evidenced by the change of reaction mixture color
mostly from yellow to intense red or orange. At this point, we
noticed that the progress of the condensation correlated with
the size of the aromatic linker. The reason for this might be the
increase in steric hindrance in the surrounding of the formyl
group from 1a to 1c, resulting in a decrease in yield from 4a to
43–45
literature methods.
Then we started the synthesis of the
intermediate aldehydes 1a–c. At the beginning we decided to
examine the reliability of the reported synthetic protocol for
46
1
a. To our surprise, we obtained the product in 70% yield,
which differs signicantly from the reported value of 98%.
Moreover, this method proved to be completely ineffective in
the coupling of the two other bromoarenecarbaldehydes. We
suppose that this was due to their low solubility in the 2-
propanol/water reaction medium used. Even aer raising the
4
c and 5a to 5c. Because of this, reaction time was extended to 6
and 7 minutes for aldehydes bearing 1,4-naphthalenylene and
,10-anthracenylene linkers in their structures, respectively.
9
Nevertheless, a slight trend in decreasing yield was still
observed.
ꢁ
reaction temperature to 100 C, there were formed sticky
The same reaction conditions were applied in the conden-
sation processes involving 2-thiohydantoins 3a and 3b. It is
worth emphasizing that in the condensations involving 3a, the
substrate was equipped with an acetyl group in position N-1 due
to superior feasibility of its synthesis compared to related
unprotected 2-thiohydantoin. However, the acetyl group was
cleaved in situ in an acidic environment during the Knoevenagel
reaction, yielding N-1 deprotected derivatives of 2-thio-
hydantoin (Scheme 5).
clumps which probably hampered further progress of the
reaction. In the face of this obstacle, we decided to use
a different solvent system to facilitate the solubility of the
reactants. Fortunately, changing the reaction medium to
2 3
a mixture of toluene, ethanol and 2 M aqueous K CO solution
52
enabled us to efficiently perform all cross-coupling reactions
with very good yields (Scheme 3). As a result, aldehydes 1a–c
were isolated by the means of ash chromatography purica-
tion in the form of yellow or orange solids.
Scheme 3 Synthesis of the aldehyde intermediates through Suzuki–Miyaura cross-coupling.
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Scheme 4 Knoevenagel condensation of rhodanines (2a, 2b) with aldehydes (1a, 1b, 1c).
Table 1 summarizes the yields of all isolated target Antibacterial activity
compounds. It is evident that 2-thiohydantoin derivatives are
In order to examine biological activity of the synthesized
compounds, MIC and MBC values were determined to quantify
their inuence on selected strains of Gram-positive and Gram-
negative bacteria, as well as yeast. Escherichia coli ATCC25922,
Salmonella typhimurium ATCC14028, Klebsiella pneumoniae
ATCC13883, Pseudomonas aeruginosa ATCC9027 and Proteus
mirabilis ATCC12453 were selected as model strain of Gram-
negative bacteria. Staphylococcus aureus ATCC25923, Staphylo-
coccus aureus ATCC6538, Staphylococcus aureus ATCC43300,
Staphylococcus epidermidis ATCC12228, Micrococcus luteus
ATCC10240, Bacillus subtilis ATCC6633, Bacillus cereus
ATCC10876, Streptococcus pyogenes ATCC19615, Streptococcus
pneumoniae ATCC49619 and Streptococcus mutans ATCC25175
were selected as Gram-positive bacteria. Finally, the yeast
Candida albicans ATCC10231, Candida albicans ATCC2091,
Candida parapsilosis ATCC22019, Candida glabrata ATCC 90030
and Candida crusei ATCC14243 were selected. Ciprooxacin and
vancomycin were used as reference positive controls (Table
characterized by lower yields than rhodanines. This difference
is most likely due to the replacement of the less electronegative
sulfur atom in position 1 for an –NH– moiety. Such a trend is
more pronounced for the compounds bearing a 1,4-phenylene
and 9,10-anthracenylene linker in their structures than those
comprising
a 1,4-naphthalenylene moiety. It should be
emphasized that all the compounds were obtained with good to
excellent yields, and most were isolated by simple ltration and
rinsing. In the case of compounds 6c and 7c, conversion of the
starting aldehyde was insufficiently low, causing contamination
of the crude product with an observable amount of unreacted 1c
that could not be separated by simple rinsing of the obtained
solid. This was probably caused by p–p stacking of condensed
aromatic linker that is more favored with the increase in
aromatic moiety size. It may also explain the lack of contami-
nation problems in the case of products bearing smaller
aromatic linkers in their structures. Nonetheless, this problem
was solved by simple recrystallization. In this way we have
3
S†). None of the tested compounds demonstrated activity

nally developed a successful protocol for the synthesis of the
ꢀ1
against Gram-negative bacteria and yeast (MIC > 1000 mg L
,
desired compounds, providing ourselves materials for further
studies of their biological activity. Additionally, aldehydes 1a–c
and heterocyclic derivatives 4a–7c were fully characterized by
nuclear magnetic resonance spectroscopy (NMR), high resolu-
tion mass spectrometry (HRMS) and Fourier-transform infrared
spectroscopy (FTIR), providing high quality identication of the
new compounds. All spectra were included in the ESI.†
data not shown). Tables 1S and 2S,† containing a complete set
of measured data against Gram-positive bacteria, are included
in the ESI.†
Comparison of the data compiled in Tables 1S and 2S†
prompted us to conclude that rhodanine derivatives are more
biologically active than 2-thiohydantoin derivatives in general.
Among all tested strains, M. luteus ATCC 10240 appeared to be
Scheme 5 Knoevenagel condensation of 2-thiohydantoins (3a, 3b) with aldehydes (1a, 1b, 1c).
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Table 1 Comparison of the yields of the target compounds
outstanding values of the determined parameters. Activity of
these compounds is discussed in detail below.
Fig. 1 presents the activity of compound 5a. MIC values for 5a
ꢀ
1
range from 0.98 mg L for S. epidermidis ATCC12228 and M.
ꢀ
1
luteus ATCC10240, to a level of 1000 mg L against M. mutans
ATCC25175 (not presented in the chart to maintain clarity).
MBC values were also determined in order to establish whether
the examined substance has bacteriostatic or bactericidal effect.
For most strains, MBC was signicantly higher than MIC,
allowing us to assume less bactericidal activity. For this quan-
tication the MBC/MIC ratio was calculated, since if it is #4, the
examined compound is recognized as bactericidal. 5a showed
the satisfactory MBC/MIC ratios against B. subtilis ATCC 6633, S.
aureus ATCC 6538, S. aureus ATCC 43300 and B. cereus ATCC
Molecule's fragment
9
9
6% (4a)
4% (5a)
94% (4b)
90% (5b)
90% (4c)
89% (5c)
53
1
0876, while for the other strains the determined ratios were in
7
8
2% (6a)
4% (7a)
86% (6b)
85% (7b)
74% (6c)
74% (7c)
the range of 8–32.
Fig. 2 presents activity of 7a with exclusion of data for S.
ꢀ1
mutans ATCC 25175 (MBC > 1000 mg L ). Measured MIC values
range from 1.95 mg L against S. aureus ATCC6538, S. aureus
ATCC43300, S. epidermidis ATCC12228 and M. luteus
ATCC10240 to 125 mg L for B. cereus ATCC10876. The corre-
sponding MBC values range from 3.9 mg L to 250 mg L
which indicates a less varied response of 7a compared to 5a. For
ꢀ1
ꢀ1
ꢀ
1
ꢀ1
,
the most sensitive to the examined compounds, while S. mutans the nine presented strains, MIC/MBC ratios were not satisfac-
ATCC 25175 was the most resistant (sensitive only to 7a tory only in the case of M. luteus ATCC 10240, which had a value
ꢀ1
however, in the high concentration of 62.5 mg L ). As one can of 16.
see, compounds 5a and 7a are characterized by signicantly
Comparison of the MBC/MIC ratios, determined for the two
most active compounds, leads to the conclusion that 7a has
Fig. 1 Activity of 5a against selected strains of Gram-positive bacteria.
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Fig. 2 Activity of 7a against selected strains of Gram-positive bacteria.
better bactericidal characteristics than 5a. In the case of 7a, the enzymes/receptors. In consequence, their antibacterial
values of MBC/MIC ratio usually range from 1 to 4, while for 5a activity is much lower than the derivatives that can form
the typical range is from 1 to 32. However, compound 5a is hydrogen bonds.
0
generally characterized by lower MIC values, proving its higher
A
previously
examined
5-(4 -N,N-
inhibitory activity. It should be emphasized that the mentioned diphenylaminobenzylidene)-rhodanine-3-acetic acid demon-
ꢀ
1
differences are caused only by the replacement of the sulfur strated biological activity (MIC 1.95–500 mg L , MBC/MIC 2–4)
32
atom with the –NH– moiety in position 1 of the heterocycle. similar to the activity of 5a. The chemical structures of both
Another noted relationship is the inuence of a carboxymethyl compounds differed by the presence of an additional 1,4-phe-
substituent on MIC and MBC values. If we compare 4a vs. 5a nylene linker between the triphenylamine and rhodanine
and 6a vs. 7a, it is seen that the presence of the mentioned moieties. Introducing the phenylene linker to the structure of 5-
0
substituent in position 3 of the heterocycle has an enormous (4 -N,N-diphenylaminobenzylidene)-rhodanine-3-acetic
acid
boosting effect on biological activity. This very signicant resulted in a slight increase in the biological activity, especially
observation might serve as conrmation of the role of the against S. pyogenes ATCC19615 and S. pneumoniae ATCC49619.
carboxylic group in the activity of the examined compounds. However, bactericidal properties of the obtained derivative
Our ndings might be supported by the results of Talele et al., became lower at the same time. Further increase in the
who investigated the antiviral activity of 5-(3-phenox- aromatic linker size (1,4-naphthalenylene or 9,10-anthraceny-
ybenzylidene) rhodanine-3-acetic acid derivatives. Their target lene) resulted in a decrease or even total block of biological
54
was HC5 hepatitis C virus (HCV) polymerase. They observed activity. Unfortunately, we could not perform a similar
0
that a carboxylic group in the N-3 position of the rhodanine ring comparison
for
7a
since
analogous
5-(4 -N,N-
formed a hydrogen bond with the guanidine group of arginine. diphenylaminobenzylidene)-2-thiohydantoin-3-acetic acid has
The exocyclic oxygen atom in the C-4 position of the rhodanine not been reported in literature.
ring also took part in the formation of hydrogen bonds. Based
Based on these results, we concluded that the presence of
on the former, we suppose that rhodanine and 2-thiohydantoin both a carboxymethyl group in the N-3 position and a triphe-
derivatives without a carboxyl group in the N-3 position have nylamine moiety connected through a 1,4-phenylene linker in
fewer binding centers and thus cannot produce sufficiently the C-5 position are crucial for the desirable biological activity
strong hydrogen bonds with amino acid side chains present in of rhodanine and 2-thiohydantoin derivatives. Moreover, the
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activity can be modulated to some extent with introduction of bearing 1,4-phenylene (compounds a) and 9,10-anthracenylene
an aromatic linker.
(compounds c) linkers. It is also worth noting that among
compounds 4–7c, derivatives containing a carboxymethyl group
in the N-3 position always exhibited higher cytotoxicity toward
all examined cell lines, compared to derivatives with a hydrogen
Cytotoxicity toward human immune cell lines
Aer evaluation of the antibacterial properties of 4a–7c, we substituent in the same position.
focused our research on their inuence on three human
To sum up this section, we conclude that all examined
immune system cell lines. The immune cells present in the compounds showed very low cytotoxicity towards the examined
body are responsible for protection against infections caused by U-937, HUT-78 and COLO-720L cell lines. For all the
microorganisms or xenobiotics; thus, potential antimicrobial compounds, signicantly higher LD50 values than MIC values
compounds should be characterized by low cytotoxicity towards were measured for compounds 5a and 7a, thus conrming their
immune cells in order to support its efficacy. The group of three selective toxicity against bacteria and non-toxicity towards
chosen cell lines consisted of U-937 monocytic cells, a non- selected human immune system cells. Based on the results
adherent cells differentiable to monocytes and macro- obtained so far, we decided to briey explore their probable
5
5,56
phages;
features of a mature T-cell line with inducer/helper phenotype
and the B lymphoblastoid COLO-720L cell line, growing in the
T-cell lymphoma HUT-78 line, exhibiting the mechanisms of action.
57
58
Membrane permeability
in the graphical form of charts, while the raw data are grouped Our attention was drawn to the differences in general activity of
in Table 4S.† 4a–7c against various microorganisms. The fact that the
form of characteristic clumps. The results are presented below
Fig. 3 (top) presents the LD50 of rhodanine derivatives compounds affected only Gram-positive bacteria, and not
against the examined cell lines. As can be clearly seen, the Gram-negative bacteria or yeast, allowed us to speculate about
cytotoxicity for series 4a–c is inversely proportional to the the mechanism of interaction. According to the literature,
increase of the aromatic linker size, for all cell lines. In contrast, potential antibiotics can interfere with various molecular
59
introduction of an acidic moiety in position N-3 results in targets, causing malfunction of pathogens' systems. These
higher cytotoxicity in general. In the series 5a–c, LD50 is less interactions can be classied into ve main categories: (1)
diversied with only one anomalously high value of 5a for the inhibition of specic enzymes (especially gene expression
HUT-78 line. However, the origin of this deviation remains enzymes e.g., topoisomerases); (2) inhibition of RNA synthesis;
unknown. In the case of rhodanine derivatives, the highest (3) inhibition of protein synthesis; (4) inhibition of cytoplasmic
cytotoxicity was observed for compounds bearing a 1,4-phenyl- membrane precursor synthesis; and (5) blocking the functions
ene linker, for all cell lines (except for the mentioned example of of the cytoplasmic membrane. The rst four actions can occur
5a against the HUT-78 cell line).
only if the antibiotic is successfully transferred through the cell
A similar set of data for the derivatives of 2-thiohydantoin is membrane and into the cytoplasm. In contrast, the last process
presented in the Fig. 3 (bottom). The demonstrated compounds requires antibiotic molecules to bind to receptors or enzymes
show more varied cytotoxicity with respect to the rhodanine present on the cell surface, as penicillin and other b-lactam
analogs. Compounds 6a–c are characterized by the same rela- antibiotics do. In the search for potential targets for rhodanine
tive values of LD50 against U-937 and COLO-720L lines; however, and 2-thiohydantoin derivatives, we referred to the literature.
their activity towards HUT-78 line does not follow these trends.
In almost all bacteria, the main component of the cell wall is
Interestingly, compounds 7a–c exhibited similar behavior. peptidoglycan. This polymer protects the cell from changes in
Moreover, the highest LD50 values from all the compounds were osmotic pressure as well as physical, chemical and mechanical
recorded for 6c and 7b against the HUT-78 line, representing agents. Therefore, disturbance of its synthesis or rendering it
60,61
the highest anomalies in the 6a–c and 7a–c series. Leaving dysfunctional might induce the cell's death.
Mendgen et al.
behind these exceptions, 2-thiohydantoin derivatives 6b, 6c, 7b examined the inhibitory effect of 5-arylideno rhodanines and 2-
and 7c showed almost unchanged cytotoxicity against the three thiohydantoins towards MurA, one of the enzymes responsible
62
examined cell lines.
for peptidoglycan synthesis. They discovered that their activity
The measured LD for line U-937 was between 17.81 and might be caused by the special electronic properties conferred
5
0
ꢀ1
3
6
4.44 mg L ; for cell line HUT-78 between 15.93 and by the exocyclic double-bonded sulfur atom, empowering
ꢀ1
7.63 mg L ; and for cell line COLO-720L it was between 21.34 molecule-to-enzyme binding. Another important enzyme, MurB
ꢀ
1
and 44.58 mg L . As demonstrated, the examined compounds reductase, was also examined regarding its interaction with 5-
were shown to be the most cytotoxic against U-937 line and the substituted rhodanines. In the reported theoretical studies, it
least toxic against HUT-78 line. Moreover, the latter cell line was shown that molecular docking might be particularly
showed signicantly variable response to treatment with 4a–7c, preferred in the case of naphthyl-derived rhodanines due to
thus proving its sensitivity towards the chemical structure of the their interaction with the lipophilic area of the enzyme's active
examined compounds. However, no regularity was observed. In site. On the other hand, the disturbance of peptidoglycan
contrast to that, cytotoxicity towards U-937 and COLO-720L was synthesis might also occur on the cell's surface as a result of
much less variable. Notwithstanding, we observed very similar interaction with penicillin-binding proteins (PBPs). These
trends of activity towards these two cell lines for compounds proteins catalyze the nal steps of peptidoglycan synthesis
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Fig. 3 LD50 of rhodanine (4a–c, top left), rhodanine-3-acetic acid (5a–c, top right), 2-thiohydantoin (6a–c, bottom left), and 2-thiohydantoin-3-
acetic acid (7a–c, bottom right) derivatives, determined for three human immune system cell lines.
63
(
maturing), thus affecting its proper construction. Distur- penetrate the cell membrane. During PAMPA tests we observed
bance of such a process was examined for 5-arylalkylidenerho- that the examined compounds underwent isomerization
danines and 5-arylalkylidene-2-iminothiazolidin-4-ones, and it process which might be attributed to the sunlight irradia-
6
5,66
proved to inhibit cell wall growth in Gram-positive bacteria in tion
or the interaction with the membrane. Due to the
64
the case of some tested compounds. As one can see, it is structural similarities of all studied compounds, we suppose
possible that the compounds examined in our work can hamper that none of 4a–7c will be able to reach the interior of the cells.
the peptidoglycan synthetic pathway due to structural Despite the inability to penetrate the cell wall, compounds 5a
similarity.
and 7a showed high antibacterial activity. Based on the PAMPA
In order to test whether our compounds inhibit the activity test results, their Gram-positive antibacterial activity must be
of enzymes operating in the cytoplasm, or rather act only on the due to outer-cell interaction. We suspect that the compounds
cell's surface, we performed a parallel articial membrane exhibiting antibacterial activity may bind to the active centers of
permeability assay (PAMPA) test for several of them. We chose the enzymes responsible for the cross-linking of the peptido-
the most active compound 5a along with 5b and 5c in order to glycan structure, causing them to inactivate and consequently
verify any possible relationship between linker size and impair the protective function of the cell wall. This may
membrane permeability. The results of the studies clearly particularly occur for derivatives containing a carboxymethyl
showed that none of the tested compounds had the ability to group in the N-3 position, a group capable of forming hydrogen
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bonds with nucleophilic sites of the enzymes. However, butyllithium (1.6 M solution in hexanes), trimethyl borate, tri-
a necessary condition also appears to be the presence of a 1,4- phenylphosphine, naphthalene, bromine, potassium
phenylene linker in combination with a triphenylamine moiety, carbonate, potassium hydroxide, sodium hydroxide and 9-
which results in a good t of the entire C-5 substituent to the anthracenecarbaldehyde were obtained from Sigma-Aldrich; 2-
hydrophobic pocket of the enzyme. If both conditions are met, propanol (IPA), hexanes, ethyl acetate, 4-bromobenzaldehyde
the change of –S– to –NH– group in the C-1 position of the and palladium(II) chloride from POCH, while deuterated
heterocycle shows a little effect on MIC reduction and a signif- solvents were purchased from Deutero GmbH and Sigma-
67
68
icant improvement in bactericidal properties by reducing the Aldrich. Rhodanine, rhodanine-3-acetic acid, 1-acetyl-2-thi-
5
0,69
70
MIC/MBC ratio. The signicantly lower antibacterial activity of ohydantoin,
2-thiohydantoin-3-acetic acid and dichlorobis
71
the other derivatives, or even its lack, conrms the key role of (triphenylophosphine)palladium(II) were prepared following
the abovementioned structural motifs. In addition, signicantly the cited papers. 4-(diphenylamino)phenylboronic acid, 1,4-
lower activity of compounds subjected to a single structure dibromonaphthalene,
modication in reference to 5a and 7a indicates a synergistic and 10-bromo-9-anthracenecarbaldehyde,
4-bromo-1-naphthalenecarbaldehyde
72,73
were synthesized
effect of the three structural variables. Due to inactivity of 4a–7c according to the published procedures with slight
towards Gram-negative bacteria, we believe that these modications.
compounds are neutralized within the periplasmic space.
Lithiation reactions and Suzuki–Miyaura couplings were
performed under an inert atmosphere, using standard vacuum-
gas manifold (99.999% argon). For these transformations, all
reagents and solvents were deoxygenated and moreover, DMF
Conclusions
In summary, we obtained two series of new rhodanine and 2- and THF were dehydrated. DMF was dried over calcium hydride,
thiohydantoin derivatives containing a triphenylamine moiety, while THF over sodium/benzophenone, and both distilled prior
differing in the heterocyclic core as well as the number of to use. Other reactions were conducted under air atmosphere
condensed aromatic rings in the linker and the N-3 substituent without special precautions, using reagents and solvents as
of the heterocycles. The target compounds were obtained in received from the suppliers. Reactions accelerated with micro-
a two-step process involving Suzuki–Miyaura cross-coupling wave irradiation were conducted in CEM microwave 10 mL vials
and highly efficient microwave-assisted Knoevenagel conden- with CEM Discover, a pressurized microwave reactor.
sation. In the course of further research, we determined anti-
The reaction progress was monitored by means of a gas
bacterial properties, cytotoxicity against human immune chromatograph (Bruker 450-GC) coupled to a mass detector
system cells and permeability through cell membranes. The (Bruker MS-320) and thin layer chromatography (TLC). Chem-
most active turned out to be derivatives containing a 1,4-phe- ical structures of the obtained compounds were conrmed by
nylene moiety, and as the size of the aromatic linker increased, nuclear magnetic resonance (NMR) spectroscopy using Bruker
their activity decreased noticeably. In addition, the antibacterial Ultrashield 300 MHz and Bruker Ascend 600 MHz spectrome-
properties of the compounds bearing a 1,4-phenylene linker ters. CDCl
3 2 2 6
, CD Cl and DMSO-d were used for NMR analysis
were signicantly enhanced by introducing the carboxymethyl with residual solvent peak as the internal standard (chloroform
1
13
1
substituent in the N-3 position, proving the synergy of these
structural modications. Among the tested compounds, 5a and 5.32 ppm, C d ¼ 54.00 ppm; DMSO H d ¼ 2.50 ppm, C d ¼
a showed the highest activity, while rhodanine derivative 5a 39.52 ppm). Infrared spectra were recorded using Thermo
H d ¼ 7.26 ppm, C d ¼ 77.16 ppm; dichloromethane H d ¼
13 1 13
7
was characterized by a higher activity in the inhibition of the Fisher Nicolet iS50 FT-IR spectrometer with attenuated total
bacterial growth, whereas 2-thiohydantoin derivative 7a was reectance (ATR) unit. Low- and high-resolution mass spec-
more bactericidal. In addition, the cytotoxicity of the examined troscopy (LR- and HR-MS) analysis was conducted with mass
compounds against three cell lines of the human immune spectrometer equipped with deep exposure probe (Bruker MS-
system: U-937, HUT-78 and COLO-720L was found to be very 320, GCMS or DEPMS mode) and electrospray ionization (ESI)
low. PAMPA tests excluded the ability of the compounds to time of ight (TOF) mass detector (ABSciex QTOF 5600),
penetrate the cells which suggested that the activity of 4a–7c is respectively. ESI-HRMS spectra were recorded in negative or
associated only with their interaction with molecular targets positive ionization modes and the analytes were dissolved in
located on the surface of the cell membrane. A more detailed HPLC grade ACN or 1 : 1 DCM : methanol mixture. Elemental
explanation of the antimicrobial mechanism of activity of analysis was performed on a Thermo Scientic Flash 2000
compounds 5a and 7a will constitute the next stage of our work. analyzer. Chromatographic purications were carried out with
preparative ash chromatogram Isolera One from Biotage using
˚
self-packed columns with silica gel 60 A 230–400 mesh (Merck).
Experimental section
General information
Celite 545 0.02–0.1 mm (Merck) was used as a Samplet® ller.
All target compounds were screened for antibacterial and
Acetic acid (AA), acetic anhydride, acetonitrile (ACN), amino- antifungal activities by micro-dilution broth method using
ethanoic acid, ammonium thiocyanate, carbon disulde, Mueller–Hinton broth and Mueller–Hinton broth with 5% lysed
chloroacetic acid, ethanol, dichloromethane (DCM), N,N- sheep blood for growth of non-fastidious and fastidious
dimethylformamide (DMF), methanol, pentane, tetrahydro- bacteria or Mueller–Hinton broth with 2% glucose for growth of
furan (THF), toluene, 4-bromo-N,N-diphenylaniline, n- fungi. Minimal inhibitory concentration (MIC) and minimal
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bactericidal concentration (MBC) of the tested compounds were (Waters, Milford, USA). The analyte/internal standard (IS) peak
evaluated for the panel of the reference microorganism from height ratios were used to determine the concentrations of
American Type Culture Collection (ATCC), including Escherichia caffeine (CFN) in donor and acceptor wells. The permeability
ꢀ1
coli ATCC25922, Salmonella typhimurium ATCC14028, Klebsiella coefficient (P , cm s ) was calculated using the following
e
pneumoniae ATCC13883, Pseudomonas aeruginosa ATCC9027 formula: P
and Proteus mirabilis ATCC12453 were selected as exemplary ¼ lter area (0.3 cm ), V
Gram-negative bacteria; Staphylococcus aureus ATCC25923, acceptor well volume (0.2 mL), t ¼ incubation time (s), C
e
¼ {ꢀln[1 ꢀ C
A
/Ceq]}/[A ꢂ t ꢂ (1/V
D A
+ 1/V )] where: A
2
D
¼ donor well volume (0.3 mL), V
A
¼
A
¼
Staphylococcus aureus ATCC6538, Staphylococcus aureus compound concentration in the acceptor well aer incubation,
ATCC43300, Staphylococcus epidermidis ATCC12228, Micro-
C
D
¼ compound concentration in the donor well aer incuba-
coccus luteus ATCC10240, Bacillus subtilis ATCC6633, Bacillus tion, C ¼ [C ꢂ V + C ꢂ V ]/(V + V ).
eq
D
D
A
A
D
A
cereus ATCC10876, Streptococcus pyogenes ATCC19615, Strepto-
coccus pneumoniae ATCC49619 and Streptococcus mutans General procedure for the synthesis of 1a, 1b and 1c
ATCC25175 as Gram-positive bacteria; and Candida albicans
ATCC10231, Candida albicans ATCC2091, Candida parapsilosis
ATCC22019, Candida glabrata ATCC 90030 and Candida crusei
ATCC14243 as yeast. A detailed procedure for the determination
A 100 mL Schlenk ask was loaded with 4-(diphenylamino)
phenylboronic acid, aryl bromoaldehyde and dichlorobis(-
triphenylophosphine)palladium(II). Subsequently, the ask was
evacuated and backlled with argon three times. Next, toluene,
of the MIC and MBC value has been presented in the earlier
ethanol and deoxygenated aqueous potassium carbonate solu-
32
publication.
tion was added. The reaction mixture was vigorously stirred at
Human histiocytic lymphoma cell line U-937 (obtained from
American Type Culture Collection), human T-cell lymphoma
HUT-78 and human normal B lymphocytes COLO-720L (ob-
tained from European Collection of Authenticated Cell
Cultures) were cultured in suspension in RPMI 1640 containing
ꢁ
9
0 C for 6 h. Aer complete consumption of bromoaldehyde
conrmed by GC-MS, the volume of the organic phase was
almost completely reduced under reduced pressure. Then,
20 mL of DCM was added and water phase was removed. The
organic phase was washed three times with 50 mL of water and
evaporated to dryness under reduced pressure. The residue was
adsorbed on Celite and loaded into a Biotage Samplet®. The
product was puried by column chromatography using gradient
elution from 100% hexanes to 60% DCM : 40% hexanes. The
fractions containing desired product were collected and
concentrated under reduced pressure. The residue was recrys-
tallized by slow evaporation from DCM : hexanes and subse-
quently washed twice with pentane.
5% FBS and 0.01% penicillin–streptomycin. Stock solution of
compounds 1–12 were diluted with DMSO to the required
concentration.
The MTT tetrazolium salt colorimetric assay was used to
detect cytotoxicity of compounds 1–12. Cells were cultured in
6
9
6-well plates in amount of 0.2 ꢂ 10 U-937 cells per well in
a volume of 0.2 mL. Aer incubation 50 mL MTT solution (5 mg
mL ) in sterile water was applied to each well and le for 2 h
incubation at 37 C. Then 0.4 mL of dimethylsulfoxide (DMSO)
ꢀ1
ꢁ
was added to each Eppendorf tube and le for 5 min. Aer
centrifugation the optical density of the supernatant at 570 nm
was read. Data were expressed as mean ꢃ SD of at least 5
independent experiments.
0
0
-(Diphenylamino)-[1,1 -biphenyl]-4-carbaldehyde (1a)
4
The product was synthesized according to the general proce-
dure, using the following substances: 4-(diphenylamino)phe-
nylboronic acid (1.272 g, 4.40 mmol), 4-bromobenzaldehyde
0.74 g, 4.0 mmol), dichlorobis(triphenylophosphine)palladiu-
m(II) (28.0 mg, 0.04 mmol), 2 M potassium carbonate aqueous
solution (11.99 mL, 23.96 mmol), 23 mL of toluene 7.6 mL of
Based on the results of the cell survival presented in this
paper, the values of the median lethal dose (LD50 median lethal
(
74
dose) for cells were calculated using Behrens' method with the
following assumptions: the number of cells per measurement
point is constant, and the absorbance determined in the MTT
method is correlated with the number of live cells. Besides, if
the cell is experiencing a higher dose, it has survived all lower
doses; if the cell dies at the lower dose, it would die at all higher
doses. Then, the percentage of mortality was calculated for each
dose of the compound and LD50 values were obtained from
appropriate graphs.
ethanol. The product was obtained as yellow needles (1.36 g,
1
3
1
8
.90 mmol). Yield: 90%. H NMR (300 MHz, CD Cl ) d 10.02 (s,
2
2
H), 7.92 (d, J ¼ 8.4 Hz, 2H), 7.75 (d, J ¼ 8.4 Hz, 2H), 7.55 (d, J ¼
1
3
.8 Hz, 2H), 7.36–7.23 (m, 4H), 7.18–7.02 (m, 8H). C NMR (75
Cl ) d 192.20, 149.00, 147.93, 146.97, 135.40, 133.30,
30.72, 129.95, 128.54, 127.39, 125.45, 124.05, 123.57. IR (ATR,
MHz, CD
1
2
2
ꢀ1
n
max, cm ): 1690 (C]O). MS (DEP-LRMS): 350.3 (28.0%), 349.2
100.0%). MS (ESI-HRMS): calculated for [C25H20NO] 350.1539,
measured 350.1565 (error 7.4 ppm).
+
(
Pre-coated PAMPA Plate System Gentest™ was purchased
from Corning, Tewksbury, MA, USA. The stock solutions of 5a,
5
b, 5c and caffeine (10 mM) were prepared in DMSO. Before the
assay the examined compounds were diluted in PBS buffer (pH
.4) to 200 mM and added to the donor wells (300 mL per well).
4
-(4-(Diphenylamino)phenyl)-1-naphthalenecarbaldehyde
(1b)
7
The PBS buffer was added to the acceptor wells (200 mL per well). The product was synthesized according to the general proce-
Each compound was tested in triplicate. The plate was incu- dure, using the following substances: 4-(diphenylamino)phe-
bated next at room temperature without shaking for ve hours. nylboronic acid (1.178 g, 4.08 mmol), 4-bromo-1-
The solutions from donor and acceptor wells were analyzed by naphthalenecarbaldehyde (0.871 g, 3.71 mmol), dichlorobis(-
LC/MS Waters ACQUITY™ TQD system with the TQ Detector triphenylophosphine)palladium(II) (26.0 mg, 0.037 mmol), 2 M
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potassium carbonate aqueous solution (11.12 mL, 23.96 mmol), Aer cooling down, a precipitate was formed, which was next
2 mL of toluene 7 mL of ethanol. The product was obtained as ltered and recrystallized from water. The product was obtained
2
1
1
yellow powder (1.05 g, 2.63 mmol). Yield: 71%. H NMR (300 as a white crystalline solid (21.49 g, 0.275 mol), yield: 55%. H
1
3
MHz, CD Cl ) d 10.40 (s, 1H), 9.34 (d, J ¼ 7.6 Hz, 1H), 8.13 (d, J ¼ NMR (300 MHz, DMSO-d ) d 4.55 (s, 2H), 4.40 (s, 2H). C NMR
2
2
6
8
7
7
1
1
.4 Hz, 1H), 8.03 (d, J ¼ 7.4 Hz, 1H), 7.71 (t, J ¼ 6.9 Hz, 1H), 7.65– (75 MHz, DMSO-d
6
) d 202.96, 173.88, 167.47, 44.90, 36.08. MS
ꢀ
.53 (m, 2H), 7.42–7.26 (m, 6H), 7.24–7.14 (m, 6H), 7.09 (t, J ¼ (ESI-HRMS): calculated for [C
5
H
4
NO
3
S
2
]
189.9638, measured
1
3
2 2
.3 Hz, 2H). C NMR (75 MHz, CD Cl ) d 193.74, 148.51, 148.11, 189.9625 (error 6.8 ppm).
47.70, 136.88, 133.82, 132.56, 131.73, 131.30, 130.85, 129.95,
29.25, 127.40, 127.36, 126.60, 125.48, 125.37, 123.92, 123.28. IR 1-Acetyl-2-thiohydantoin (3a)
ꢀ1
(ATR, nmax, cm ): 1680 (C]O). MS (DEP-LRMS): 400.3 (31.4%),
A ask containing a mixture of aminoethanoic acid (37.5 g, 0.5
mol), ammonium thiocyanate (38.06 g, 0.5 mol), 150 mL of
acetic anhydride and 15 mL of acetic acid was heated in a water
bath at 110 C for 30 minutes under reux. Next, the ask was
cooled to 60 C and heated again at 100 C for 15 minutes. Then,
+
3
4
99.3 (100%). MS (ESI-HRMS): calculated for [C H NO]
29 22
00.1696, measured 400.1728 (error 8.0 ppm).
0-(4-(Diphenylamino)phenyl)anthracene-9-carbaldehyde (1c)
ꢁ
ꢁ
1
ꢁ
The product was synthesized according to the general proce- the reaction mixture was poured into a beaker containing
dure, using the following substances: 4-(diphenylamino)phe- 1000 mL of cold water. The resulting precipitate was ltered and
nylboronic acid (0.829 g, 2.87 mmol), 10-bromo-9- recrystallized from acetic acid. The product was obtained as
1
anthracenecarbaldehyde (0.743 g, 2.61 mmol), dichlorobis(- a brown crystalline solid (55.3 g, 0.35 mol), yield: 70%. H NMR
6
triphenylophosphine)palladium(II) (18.0 mg, 0.026 mmol), 2 M (300 MHz, DMSO-d ) d 12.58 (s, 1H), 4.39 (s, 2H), 2.67 (s, 3H).
1
3
potassium carbonate aqueous solution(7.82 mL, 15.63 mmol),
C NMR (75 MHz, DMSO-d
6
) d 182.57, 170.46, 169.44, 52.26,
ꢀ
1
5 mL of toluene 5 mL of ethanol. The product was obtained as 26.70, 26.67. MS (ESI-HRMS): calculated for [C H N O S]
orange powder (1.06 g, 2.36 mmol). Yield: 90%. H NMR (300 157.0077, measured 157.0052 (error 15.9 ppm).
5 5 2 2
1
MHz, CD Cl ) d 11.55 (s, 1H), 9.00 (d, J ¼ 9.0 Hz, 2H), 7.90 (d, J ¼
2
2
8
.8 Hz, 2H), 7.73–7.62 (m, 2H), 7.53–7.42 (m, 2H), 7.41–7.31 (m, 2-Thiohydantoin-3-acetic acid (3b)
1
3
4
H), 7.30–7.22 (m, 8H), 7.10 (t, J ¼ 7.2 Hz, 2H). C NMR (75
Cl ) d 193.79, 148.32, 148.20, 146.06, 132.20, 132.09,
32.01, 130.68, 129.98, 129.08, 128.67, 125.98, 125.53, 125.36,
A solution of sodium hydroxide (12.0 g, 0.3 mol) was added to
a well stirred suspension of aminoethanoic acid (18.75 g, 0.25
mol) in 20 mL of water. To the resulting solution carbon
disulde (11.42 g, 0.15 mol) was added under nitrogen atmo-
sphere and the reaction mixture was reuxed for 8 h. Aer the
heating was nished, the excess of carbon disulde was evap-
orated on a rotary evaporator. Next, a mixture of 100 mL of
concentrated hydrochloric acid and 200 mL of water was added.
The resulting mixture was reuxed for 2 h. Aer cooling,
MHz, CD
2
2
1
1
ꢀ1
24.00, 123.90, 123.29. IR (ATR, nmax, cm ): 1664 (C]O). MS
(DEP-LRMS): 450.3 (36.2%), 449.3 (100.0%). MS (ESI-HRMS):
+
calculated for [C H NO] 450.1852, measured 400.1876
3
3
24
(error 5.3 ppm).
Rhodanine (2a)
A solution of chloroacetic acid (23.62 g, 0.25 mol) and ammo- a precipitate was formed, which was ltered and subsequently
nium thiocyanate (38.06 g, 0.50 mol) in 150 mL of water was recrystallized from water. The product was obtained as a white
1
heated for 20 minutes under reux. Aer cooling down, crystalline solid (39.15 g, 0.225 mol), yield: 75%. H NMR (300
a precipitate was formed. The crude product was ltered and MHz, DMSO-d
6
) d 10.34 (s, 1H), 4.34 (s, 2H), 4.23 (d, J ¼ 1.3 Hz,
1
3
recrystallized from water. The product was obtained as 2H). C NMR (75 MHz, DMSO-d
6
) d 182.87, 172.27, 168.44,
1
ꢀ
5 5 2 3
a yellowish powder (14.96 g, 0.113 mol), yield: 45%. H NMR 48.64, 41.47. MS (ESI-HRMS): calculated for [C H N O S]
1
3
(
6
300 MHz, DMSO-d ) d 13.14 (s, 1H), 4.26 (s, 2H). C NMR (75 173.0026, measured 173.0008 (error 10.4 ppm).
MHz, DMSO-d ) d 205.43, 176.81, 39.42. MS (ESI-HRMS):
6
ꢀ
calculated for [C H NOS ] 131.9583, measured 131.9572 General procedure for the synthesis of 4a–7c
3
2
2
(error 8.3 ppm).
1a (0.1 g, 0.286 mmol), 1b (0.114 g, 0.286 mmol) or 1c (0.129 g,
0
.286 mmol), respective rhodanine (0.046 g, 0.343 mmol),
Rhodanine-3-acetic acid (2b)
rhodanine-3-acetic acid (0.066 g, 0.343 mmol), 1-acetyl-2-
A solution of potassium hydroxide (28.00 g, 0.5 mol) in 100 mL thiohydantoin (0.054 g, 0.343 mmol) or 2-thiohydantoin-3-
of water was added to the suspension of aminoethanoic acid acetic acid (0.06 g, 0.343 mmol) and ammonium acetate
ꢁ
(
18.75 g, 0.25 mol). The resulting solution was cooled to 5 C (44 mg, 0.57 mmol) were added to a CEM microwave vial.
and carbon disulde (19.03 g, 0.25 mol) was added. The content Subsequently, 2 mL of AA was added and the vessel was closed
ꢁ
of the ask was mixed at 5 C for 6 h. The cooling bath was with a cap. The mixture was heated with maximum power 200 W
ꢁ
removed and mixing was continued at room temperature for at 180 C for 5–7 min (5 min with 1a, 6 min with 1b and 7 min
2
1
8
0 h. Then, a solution of chloroacetic acid (23.62 g, 0.25 mol) in with 1c). During heating the color of the mixtures turned red
00 mL of water was added. The reaction mixture was stirred for very quickly and became homogenous. Aer cooling back to
h at the temperature below 15 C. Next, a mixture of 150 mL room temperature the precipitate was formed (some products
ꢁ
concentrated hydrochloric acid and 200 mL of water was added tended to form supercooled solutions, in that case precipitation
ꢁ
slowly. The resulting mixture was heated at 90 C for 25 min. was initiated by ultrasonication). The solid was centrifuged
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and the supernatant was sucked off with a syringe. The solid (Z)-2-(5-((4-(4-(Diphenylamino)phenyl)naphthalen-1-yl)
was washed with 1 mL of AA and 4 times with 4 mL portions of methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5b)
water in the same manner. The obtained product was dried in
1
Orange crystalline solid (148 mg, 0.258 mmol). Yield: 90%. H
ꢁ
an oven at 110 C overnight. In some cases the product was
puried by subsequent recrystallization from 2-propanol.
NMR (300 MHz, DMSO-d
6
) d 8.55 (s, 1H), 8.26 (d, J ¼ 8.3 Hz, 1H),
.02 (d, J ¼ 8.3 Hz, 1H), 7.79 (d, J ¼ 7.6 Hz, 1H), 7.75–7.61 (m,
H), 7.59 (d, J ¼ 7.6 Hz, 1H), 7.45–7.31 (m, 6H), 7.17–7.04 (m,
8
2
8
1
1
1
1
3
6
H), 4.78 (s, 2H). C NMR (151 MHz, DMSO-d ) d 193.84,
0
0
(
Z)-5-((4 -(Diphenylamino)-(1,1 -biphenyl)-4-yl)methylene)-2-
thioxothiazolidin-4-one (4a)
Brick-red crystalline solid (128 mg, 0.276 mmol). Yield: 96%. H
NMR (300 MHz, DMSO-d
67.51, 166.03, 147.34, 147.06, 142.95, 132.76, 131.79, 131.32,
30.97, 130.73, 129.89, 129.21, 127.82, 127.41, 127.18, 126.79,
26.73, 124.95, 124.73, 124.09, 123.78, 122.29, 45.22. IR (ATR,
1
ꢀ
1
6
) d 7.79 (d, J ¼ 8.2 Hz, 2H), 7.71–7.55
13
nmax, cm ): 1725 (C]O), 1708 (C]O), 1062 (C]S). MS (ESI-
ꢀ
(
m, 5H), 7.33 (t, J ¼ 7.8 Hz, 4H), 7.14–6.95 (m, 8H). C NMR (75
HRMS): calculated for [C34
71.1137 (error 3.3 ppm).
H
23
N
2
O
3
S
2
]
571.1156, measured
6
MHz, DMSO-d ) d 195.64, 169.62, 147.75, 146.89, 141.66, 132.02,
5
1
1
31.50, 131.42, 129.84, 127.94, 126.90, 124.86, 124.72, 123.82,
ꢀ
1
22.64. IR (ATR, nmax, cm ): 1689 (C]O), 1068 (C]S). MS (ESI-
(
Z)-2-(5-((10-(4-(Diphenylamino)phenyl)anthracen-9-yl)
ꢀ
HRMS): calculated for [C28
63.0947 (error 0.6 ppm).
H
19
N
2
OS
2
]
463.0944, measured
methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (5c)
4
1
Red crystalline solid (159 mg, 0.255 mmol). Yield: 89%. H NMR
(
(
300 MHz, DMSO-d
d, J ¼ 8.7 Hz, 2H), 7.69–7.58 (m, 2H), 7.59–7.48 (m, 2H), 7.38 (t,
6
) d 8.83 (s, 1H), 8.06 (d, J ¼ 8.7 Hz, 2H), 7.76
(
Z)-5-((4-(4-(Diphenylamino)phenyl)naphthalen-1-yl)
J ¼ 7.8 Hz, 4H), 7.28 (d, J ¼ 8.5 Hz, 2H), 7.23–7.07 (m, 8H), 4.79
methylene)-2-thioxothiazolidin-4-one (4b)
1
3
(s, 2H). C NMR (151 MHz, DMSO-d ) d 193.63, 167.57, 165.05,
6
Dark-brown crystalline solid (139 mg, 0.270 mmol). Yield: 94%.
1
1
1
1
6
47.18, 139.73, 132.57, 131.94, 131.10, 130.85, 129.92, 129.53,
1
H NMR (300 MHz, DMSO-d ) d 8.31 (s, 1H), 8.23 (d, J ¼ 8.4 Hz,
6
27.89, 127.51, 127.27, 126.99, 126.31, 125.30, 124.76, 123.75,
1
6
1
1
1
1
H), 8.01 (d, J ¼ 8.1 Hz, 1H), 7.75–7.53 (m, 4H), 7.45–7.30 (m,
ꢀ1
22.25, 45.29. IR (ATR, nmax, cm ): 1727 (C]O), 1713 (C]O),
1
3
H), 7.18–7.04 (m, 8H). C NMR (151 MHz, DMSO-d ) d 196.17,
ꢀ
6
25 2 3 2
056 (C]S). MS (ESI-HRMS): calculated for [C38H N O S ]
68.97, 147.16, 146.97, 142.33, 132.77, 131.66, 131.19, 130.85,
29.75, 129.34, 128.60, 128.17, 127.52, 127.17, 126.68, 126.63,
21.1312, measured 621.1289 (error 3.7 ppm).
ꢀ
1
26.57, 124.58, 123.93, 123.60, 122.26. IR (ATR, nmax, cm ):
0
0
(
Z)-5-((4 -(Diphenylamino)-(1,1 -biphenyl)-4-yl)methylene)-2-
thioxoimidazolidin-4-one (6a)
Orange crystalline solid (92 mg, 0.206 mmol). Yield: 72%. H
NMR (300 MHz, DMSO-d ) d 12.38 (s, 1H), 12.20 (s, 1H), 7.80 (d, J
8.2 Hz, 2H), 7.67 (t, J ¼ 8.2 Hz, 4H), 7.33 (t, J ¼ 7.8 Hz, 4H),
683 (C]O), 1073 (C]S). MS (ESI-HRMS): calculated for
ꢀ
[C
32
H
21
N
2
OS
2
]
513.1101, measured 513.1103 (error 0.4 ppm).
1
6
¼
(
Z)-5-((10-(4-(Diphenylamino)phenyl)anthracen-9-yl)
1
3
7
.13–6.97 (m, 8H), 6.51 (s, 1H). C NMR (151 MHz, DMSO-d
6
)
methylene)-2-thioxothiazolidin-4-one (4c)
Orange powder (145 mg, 0.257 mmol). Yield: 90%. H NMR (300
MHz, DMSO-d
) d 8.52 (s, 1H), 8.10 (d, J ¼ 8.6 Hz, 2H), 7.75 (d, J
8.7 Hz, 2H), 7.68–7.58 (m, 2H), 7.58–7.47 (m, 2H), 7.39 (t, J ¼
d 179.17, 166.05, 147.45, 147.05, 140.32, 132.81, 131.06, 129.88,
1
127.83, 127.61, 126.46, 124.59, 123.73, 123.05, 111.63. IR (ATR,
6
ꢀ1
nmax, cm ): 1720 (C]O), 1080 (C]S). MS (ESI-HRMS): calcu-
¼
ꢀ
lated for [C28
ppm).
H
20
N
3
OS] 446.1333, measured 446.1321 (error 2.7
1
3
7
.9 Hz, 4H), 7.30 (d, J ¼ 8.5 Hz, 2H), 7.24–7.08 (m, 8H). C NMR
(151 MHz, DMSO-d ) d 147.24, 132.02, 129.97, 129.56, 127.88,
6
1
27.42, 127.04, 126.27, 125.58, 124.76, 124.70, 123.78, 122.36. IR
(
Z)-5-((4-(4-(Diphenylamino)phenyl)naphthalen-1-yl)
methylene)-2-thioxoimidazolidin-4-one (6b)
Yellow crystalline solid (122 mg, 0.245 mmol). Yield: 86%. H
NMR (300 MHz, DMSO-d
) d 12.34 (s, 2H), 8.15 (d, J ¼ 8.2 Hz,
H), 7.98 (d, J ¼ 7.9 Hz, 1H), 7.85 (d, J ¼ 7.5 Hz, 1H), 7.70–7.53
(m, 2H), 7.49 (d, J ¼ 7.5 Hz, 1H), 7.45–7.31 (m, 6H), 7.17–7.05
ꢀ1
(ATR, nmax, cm ): 1734 (C]O), 1068 (C]S). MS (ESI-HRMS):
ꢀ
calculated for [C36
H
23
N
2
OS
2
]
563.1257, measured 563.1259
1
(error 0.4 pm).
6
1
0
0
(
Z)-2-(5-((4 -(Diphenylamino)-(1,1 -biphenyl)-4-yl)methylene)-
1
3
4
-oxo-2-thioxothiazolidin-3-yl)acetic acid (5a)
Red powder (140 mg, 0.268 mmol). Yield: 94%. H NMR (300
MHz, DMSO-d
) d 7.90 (s, 1H), 7.84 (d, J ¼ 8.3 Hz, 2H), 7.70 (dd, J
8.5, 6.3 Hz, 4H), 7.34 (t, J ¼ 7.8 Hz, 4H), 7.15–7.04 (m, 6H),
6
(m, 9H). C NMR (151 MHz, DMSO-d ) d 130.99, 129.93, 124.64,
123.72. IR (ATR, nmax, cm ): 1714 (C]O), 1087 (C]S). MS (ESI-
HRMS): calculated for [C H N OS] 496.1489, measured
4
ꢀ
1
1
ꢀ
3
2
22 3
6
96.1465 (error 4.8 ppm).
¼
1
3
7
.02 (d, J ¼ 8.6 Hz, 2H), 4.74 (s, 2H). C NMR (151 MHz, DMSO)
(
Z)-5-((10-(4-(Diphenylamino)phenyl)anthracen-9-yl)
d 193.21, 167.49, 166.56, 147.89, 146.88, 142.17, 133.79, 131.89,
methylene)-2-thioxoimidazolidin-4-one (6c)
1
1
1
5
31.76, 131.28, 129.87, 128.01, 127.01, 124.80, 123.90, 122.56,
ꢀ
1
21.12, 45.19. IR (ATR, nmax, cm ): 1722 (C]O), 1701 (C]O), The pure product was obtained aer recrystallization from 2-
ꢀ
056 (C]S). MS (ESI-HRMS): calculated for [C30
H
21
N
2
O
3
S
2
]
propanol. Red crystalline solid (116 mg, 0.212 mmol). Yield:
74%. H NMR (300 MHz, DMSO-d ) d 12.37 (s, 1H), 11.67 (s, 1H),
6
1
21.0999, measured 521.0994 (error 1.0 ppm).
39378 | RSC Adv., 2019, 9, 39367–39380
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Paper
RSC Advances
8
4
6
.06 (d, J ¼ 8.4 Hz, 2H), 7.74 (d, J ¼ 8.6 Hz, 2H), 7.62–7.46 (m,
H), 7.46–7.35 (m, 4H), 7.34–7.25 (m, 3H), 7.22 (d, J ¼ 7.1 Hz,
Acknowledgements
1
3
H), 7.12 (t, J ¼ 7.3 Hz, 2H). C NMR (151 MHz, DMSO-d ) The research was partially nanced from the Polish Statutory
6
d 179.13, 164.64, 147.15, 146.88, 137.76, 133.63, 131.86, 131.51, Research Projects K/ZDS/007886 and BS-465/G/2018.
1
1
29.79, 129.64, 129.08, 127.09, 126.77, 126.18, 125.85, 125.68,
ꢀ1
24.57, 123.54, 122.38, 108.17. IR (ATR, nmax, cm ): 1742 (C]
Notes and references
ꢀ
O), 1099 (C]S). MS (ESI-HRMS): calculated for [C36
46.1646, measured 546.1629 (error 3.1 ppm).
24 3
H N OS]
5
1 S. N. Farouk, J. Adv. Microbiol., 2017, 7, 1.
2
M. Exner, S. Bhattacharya, B. Christiansen, J. Gebel,
P. Goroncy-Bermes, P. Hartemann, P. Heeg, C. Ilschner,
A. Kramer, E. Larson, W. Merkens, M. Mielke,
P. Oltmanns, B. Ross, M. Rotter, R. M. Schmithausen,
H. G. Sonntag and M. Trautmann, GMS Hyg. Infect. Control,
2017, 12, Doc05, DOI: 10.3205/dgkh000290.
0
0
(
4
Z)-2-(5-((4 -(Diphenylamino)-(1,1 -biphenyl)-4-yl)methylene)-
-oxo-2-thioxoimidazolidin-3-yl)acetic acid (7a)
1
Orange crystalline solid (122 mg, 0.241 mmol). Yield: 84%. H
NMR (300 MHz, DMSO-d ) d 12.58 (s, 1H), 7.85 (d, J ¼ 8.2 Hz,
6
2
7
H), 7.72 (d, J ¼ 8.2 Hz, 2H), 7.68 (d, J ¼ 8.4 Hz, 2H), 7.34 (t, J ¼
3
K. C. Huanga, R. Mukhopadhyay, B. Wena, Z. Gitaia and
N. S. Wingreena, Proc. Natl. Acad. Sci. U. S. A., 2008, 105,
13
.7 Hz, 4H), 7.15–6.97 (m, 8H), 6.72 (s, 1H), 4.51 (s, 2H).
) d 178.26, 168.37, 163.78, 147.49,
47.00, 140.67, 132.65, 131.27, 130.76, 129.84, 127.84, 126.47,
25.72, 124.60, 124.58, 123.73, 122.93, 113.61, 41.97. IR (ATR,
C
NMR (151 MHz, DMSO-d
6
1
9282.
N. Kashef, Y. Y. Huang and M. R. Hamblin, Nanophotonics,
017, 6, 853.
S. I. Miller and N. R. Salama, PLoS Biol., 2018, 16, e2004935.
1
1
4
5
2
ꢀ
1
n
max, cm ): 1735 (C]O), 1647 (C]O), 1076 (C]S). MS (ESI-
ꢀ
HRMS): calculated for [C H N O S] 504.1387, measured
3
0
22
3
3
6 K. Li, X. X. Yuan, H. M. Sun, L. S. Zhao, R. Tang, Z. H. Chen,
504.1383 (error 0.8 ppm).
Q. L. Qin, X. L. Chen, Y. Z. Zhang and H. N. Su, Microbiology,
2018, 9, 620, DOI: 10.3389/fmicb.2018.00620.
7
8
9
A. Al-Mulla, Der Pharma Chem., 2017, 9, 141.
(
Z)-2-(5-((4-(4-(Diphenylamino)phenyl)naphthalen-1-yl)
R. Kumar and S. Patil, Hygeia J. Drugs Med., 2017, 9, 80.
S. K. Manjal, R. Kaur, R. Bhatia, K. Kumar, V. Singh,
R. Shankar, R. Kaur and R. K. Rawal, Bioorg. Chem., 2017,
methylene)-4-oxo-2-thioxoimidazolidin-3-yl)acetic acid (7b)
1
Yellow powder (135 mg, 0.243 mmol). Yield: 85%. H NMR (300
MHz, DMSO-d
1
6
) d 8.17 (d, J ¼ 8.2 Hz, 1H), 7.99 (d, J ¼ 8.3 Hz,
75, 406.
H), 7.88 (d, J ¼ 7.5 Hz, 1H), 7.70–7.55 (m, 2H), 7.52 (d, J ¼
1
1
1
1
1
1
1
1
0 S. Vengurlekar, R. Sharma and T. Trivedi, Lett. Drug Des.
7
.5 Hz, 1H), 7.44–7.29 (m, 7H), 7.17–7.05 (m, 8H), 4.54 (s, 2H).
C NMR (151 MHz, DMSO) d 178.48, 168.44, 163.38, 147.15,
47.09, 141.08, 133.36, 131.75, 131.19, 130.94, 129.88, 128.57,
28.37, 128.14, 127.25, 126.86, 126.73, 126.48, 124.63, 124.58,
24.38, 123.69, 123.64, 122.59, 122.53, 110.33, 42.00. IR (ATR,
Discov., 2012, 9, 549.
1
3
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5, 743.
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1
1
1
2
ꢀ
1
n
max, cm ): 1707 (C]O), 1649 (C]O), 1073 (C]S). MS (ESI-
ꢀ
HRMS): calculated for [C34
54.1527 (error 3.1 ppm).
H
24
N
3
O
3
S] 554.1544, measured
5
5
2
(
Z)-2-(5-((10-(4-(Diphenylamino)phenyl)anthracen-9-yl)
methylene)-4-oxo-2-thioxoimidazolidin-3-yl)acetic acid (7c)
The pure product was obtained aer recrystallization from 2-
propanol. Orange powder (128 mg, 0.211 mmol). Yield: 74%. H
1
NMR (300 MHz, DMSO-d ) d 12.09 (s, 1H), 8.05 (d, J ¼ 8.6 Hz,
6
2
7
7
1
1
1
H), 7.76 (d, J ¼ 8.6 Hz, 2H), 7.64–7.48 (m, 5H), 7.40 (t, J ¼
61, 149.
.7 Hz, 4H), 7.33–7.26 (m, 2H), 7.25–7.17 (m, 6H), 7.12 (t, J ¼
1
1
2
2
2
2
8 A. Y. Hassan, M. M. Said, M. T. Sarg, H. S. Al-Zahabi and
1
3
6
.3 Hz, 2H), 4.55 (s, 2H). C NMR (151 MHz, DMSO-d ) d 178.29,
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72.10, 168.35, 162.43, 147.16, 146.93, 138.11, 131.85, 131.81,
31.42, 129.81, 129.66, 129.09, 127.15, 126.36, 125.76, 125.69,
9 S. Prachand, A. K. Gupta and R. Gilhotra, Int. J. Pharm. Sci.
Nanotechnol., 2018, 11, 3977.
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1 S. Rekha, U. Shantharam and C. Vineet, Int. Res. J. Pharm.,
ꢀ
1
24.61, 123.58, 122.36, 110.38, 41.85. IR (ATR, nmax, cm ): 1718
(C]O), 1643 (C]O), 1075 (C]S). MS (ESI-HRMS): calculated
ꢀ
for [C H N O S] 604.1700, measured 604.1692 (error 1.3
ppm).
3
8
26 3 3
2011, 2, 81.
2 Y. Huang, Z. Guo, H. Song, Y. Liu, L. Wang and Q. Wang, J.
Agric. Food Chem., 2018, 66, 8253.
3 C. Tintori, G. Iovenitti, E. R. Ceresola, R. Ferrarese,
C. Zamperini, A. Brai, G. Poli, E. Dreassi, V. Cagno,
Conflicts of interest
There are no conicts of interest to declare.
This journal is © The Royal Society of Chemistry 2019
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