Antimicrobial activity of rhodanine-3-acetic acid derivatives

Article abstract of DOI:10.1016/j.bmc.2017.01.045

Twenty-four 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (rhodanine-3-acetic acid)-based amides, esters and 5-arylalkylidene derivatives were synthesized, characterized and evaluated as potential antimicrobial agents against a panel of bacteria, mycobact

Full text of DOI:10.1016/j.bmc.2017.01.045

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
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Bioorganic & Medicinal Chemistry
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Antimicrobial activity of rhodanine-3-acetic acid derivatives
a,
a
b
⇑
´
ˇ
ˇ
Martin Krátky , Jarmila Vinšová , Jirina Stolaríková
a Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 50005 Hradec Králové, Czech Republic
b Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in Ostrava, Partyzánské námĕstí 7, 702 00 Ostrava, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty-four 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (rhodanine-3-acetic acid)-based amides,
esters and 5-arylalkylidene derivatives were synthesized, characterized and evaluated as potential
antimicrobial agents against a panel of bacteria, mycobacteria and fungi. All of the derivatives were active
against mycobacteria. N-(4-Chlorophenyl)-2-[5-(2-hydroxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl]
acetamide demonstrated the highest activity against Mycobacterium tuberculosis with minimum inhibi-
Received 4 October 2016
Revised 27 January 2017
Accepted 28 January 2017
Available online xxxx
tory concentrations (MIC) of 8–16
to 2-[5-(2-hydroxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl]acetic acids (MIC values P32
highest antibacterial activity against Gram-positive bacteria including methicillin-resistant
Staphylococcus aureus exhibited 4-(trifluoromethyl)phenyl 2-(4-oxo-2-thioxothiazolidin-3-yl)acetate
l
M. Non-tuberculous mycobacteria were the most susceptible
M). The
Keywords:
Amides
Antibacterial activity
Antifungal activity
Antimycobacterial activity
Condensation
l
(MIC P 15.62
lM). Several structure-activity relationships were identified. The activity against Gram-
negative and fungal pathogens was marginal.
Esters
Ó 2017 Elsevier Ltd. All rights reserved.
In vitro activity
2-(4-Oxo-2-thioxothiazolidin-3-yl)acetic
acid
Rhodanine
1. Introduction
aldose reductase,6 dolicholphosphate mannose synthase,7
deoxyxylulose 5-phosphate reductoisomerase,8 gyrase B,9 Mur
ligases,10 as well as growth of pathogenic protozoa,7 fungi,11 bacte-
ria,8,10,12,13 and Mycobacterium tuberculosis14 in whole-cell screen-
ing assays. It is beyond doubt that rhodanines including those
with C-5 substitution are a well-established scaffold for both
microbial enzymes inhibition and in vitro antimicrobial activity.
Based on here mentioned facts and as a part of our research
effort focused on synthesis and identification of novel antimicro-
bial agents,15–19 we evaluated previously reported N-phenylamides
and phenyl esters of rhodanine-3-acetic acid,4 novel salicylalde-
hyde-based C-5 arylmethylidene derivatives of the RAA and their
mutual conjugates against mycobacteria, Gram-positive and
Gram-negative bacteria and also eight fungal species. The selected
panel of twenty microbes covers a wide range of important human
pathogens including strains with an acquired resistance. It is a use-
ful screening tool for an initial identification of potential antimi-
crobial activity of new compounds.
Currently, novel antimicrobial agents active especially against
drug-resistant bacteria and fungi are required to overcome limita-
tions of the treatment opportunities. Microorganisms that are
resistant to most of the clinically used antibiotics are emerging
at a rapid rate. The spread of methicillin-resistant Staphylococcus
aureus, biofilm-producing bacteria, nosocomial infections caused
by Enterococci and Streptococci, a wide spectrum of Gram-negative
pathogens or fungal species, increasing incidence of tuberculosis,
onset of HIV/AIDS epidemic may serve as examples of serious
threats for public health. One additional reason for developing
new antibiotics is related to their own toxicity and side effects.
Unfortunately, the number of innovative classes of the antimicro-
bial agents being developed has decreased dramatically recently
and it is lagging behind clinical requirements.1–3
Rhodanine (2-thioxothiazolidin-4-one) as a privileged scaffold
in medicinal chemistry offers various possibilities of chemical
modification. Principally, N-3 and/or ‘‘active methylene” C-5 sub-
stitution has brought a wide range of potentially bioactive com-
pounds. Illustratively, derivatives of rhodanine-3-acetic acid
(RAA; 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid) are able to
inhibit various enzymes, e.g., cholinesterases,4,5 15-lipoxygenase,5
2. Results and discussion
2.1. Chemistry
The synthesis of amides 1 and esters 2 from RAA using predom-
inantly N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride (EDC)-based coupling was described previously.4
⇑
Corresponding author.
E-mail address: martin.kratky@faf.cuni.cz (M. Krátky´).
http://dx.doi.org/10.1016/j.bmc.2017.01.045
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Krátky´ M., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.045

´
M. Krátky et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
2
O
mers was also confirmed by the comparison with previously
reported NMR data of known or structurally close similar
compounds.6,10,21
COOH
CH3COO-Na+
CH3COOH
O
N
N
R
S
O
R
+
OH
S
X
X
S
O
S
3
2.2. Pharmacology
Scheme 1. Synthesis of 5-arylidenerhodanine-3-acetic acids 3 (X = CH, N; R = H, 2-
OH, 3-OH, 4-OH, 2-OH-5-Br, 2-OH-5-Cl).
2.2.1. Antimycobacterial activity
Initially, we evaluated previously reported amides 1 and esters
2 of rhodanine-3-acetic acid,4 parent RAA and newly synthesized
5-arylmethylidenerhodanine-3-acetic acids 3 as potential antimy-
cobacterial agents. A drug-susceptible strain of Mycobacterium
tuberculosis (Mtb.) 331/88 (H37Rv) and three strains of atypical
(non-tuberculous) mycobacteria (M. avium, M. kansasii including
one clinical isolate – 6509/96 strain) were involved (Table 1). With
one exception (5b), there were no solubility problems in the test-
ing medium up to the concentration of 1 mM.
Among the RAA N-phenylamides 1 and phenyl esters 2, only 4-
chloroderivative 1b exhibited a moderate activity against Mtb.
with MIC values of 32–62.5 lM and 125–250 lM for M. kansasii
6509/96. The removal or the replacement of chlorine by other sub-
stituent decreases the antimycobacterial activity of 1b especially
against Mtb. The switch of the N-(4-chlorophenyl)-2-(4-oxo-2-
thioxothiazolidin-3-yl)acetamide 1b to the corresponding ester
2b modulates the action in the same way. In general, the esters 2
are not superior to the amides 1. M. avium displayed the lowest
C-5 substituted rhodanine-3-acetic acids were synthesized by
heating of parent RAA together with 1.1 equivalents of appropriate
hydroxybenzaldehydes (Scheme 1, X = CH) and pyridine-2-car-
baldehyde (X = N, R is missing) in glacial acetic acid in the presence
of sodium acetate. This procedure takes 5 h proving satisfactory
yields of 78–97%. We selected salicylaldehyde as an initial
compound (derivative 3a), followed by their positional isomers
3-/4-hydroxybenzaldehyde (3b and 3c) and 5-halogenated salicy-
laldehydes (chlorine and bromine derivatives 3d and 3e, respec-
tively). This halogenation of salicylic ring has been reported to be
beneficial for enhancing of antimicrobial activity.15,16 Supported
by a previous report of Dolezel et al.,11 we also performed conden-
sation of rhodanine-3-acetic acid with pyridine-2-carbaldehyde
leading to conjugate 3f.
To discover the possible importance of 2-hydroxybenzylidene
free phenolic group or to reveal potential advantage of its esterifi-
cation,20 we treated 3a with acetic anhydride (yield of 78%;
Scheme 2).
susceptibility to all the derivatives 1 and 2 (MIC P 1000
Unsubstituted RAA is virtually inactive with a uniform MIC value
of 1000 M.
Salicylaldehyde-based derivatives
in vitro growth inhibition of mycobacteria (MIC within the range
of 32–250 M). Obviously, the employment of isomeric 3-hydrox-
lM).
Based on results of antimycobacterial activity evaluation, we
decided to combine both active fragments, N-(4-chlorophenyl)-2-
(4-oxo-2-thioxothiazolidin-3-yl)acetamide 1a and 2-[5-(2-hydrox-
ybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl]acetic acids 3a, 3d
and 3e, in one molecular entity. A conjugate with the pyridine
derivative 3f was synthesized too. From two possible two-step
synthetic pathways, we opted for the reaction of amide 1d with
a mild excess of aromatic aldehydes under identical conditions
(acetic acid, sodium acetate, reflux; Scheme 3) used for the synthe-
sis of the compounds 3. Yields of this step were within the range of
77–85%. The reaction in a reverse order, i.e., synthesis of 3 followed
by conversion of free carboxyl to N-(4-chlorophenyl)amide, could
be complicated by a side reaction of free hydroxyl group.
Compounds were characterized by melting points, IR and NMR
spectra; their purity was checked by thin-layer chromatography,
melting point and elemental analysis.
l
3
provide more efficient
l
ybenzaldehyde (condensate 3b) and 4-hydroxybenzaldehyde (3c)
or pyridine-2-carbaldehyde (3f) led to the negligible active mole-
cules, thus indicating the importance of phenolic group at the posi-
tion 2. The acetylation of 3a (compound 4) and introduction of
chlorine (3d) or bromine (3e) into the salicylic moiety did not
influence the antimycobacterial action sharply; their activities
are mostly comparable each other. This means that none of these
modifications is preferred over unsubstituted salicylaldehyde (2-
hydroxybenzaldehyde). The susceptibility rate of all mycobacterial
strains were similar, only 5-bromosalicylidene-RAA is somewhat
less active against clinically isolated M. kansasii 6509/96.
5-Salicylidene or pyridin-2-ylmethylene derivatives 3–5 can
form two geometric isomers (E and Z) due to the presence of exo-
cyclic double bond. In NMR spectra of all of the derivatives 3–5,
only single isomer was observed uniformly. According to the liter-
ature11 and references therein, this type of synthesis leads to the
thermodynamically more stable Z-isomers. The identity of Z-iso-
Keeping in mind activity data of the amide 1b and salicylalde-
hyde condensates (3a, 3d, 3e), we decided to combine both these
antimycobacterial pharmacophores in one molecule to determine
if this ‘‘amide-condensates” conjugation will result also in a more
potent suppression of mycobacteria. This initial assumption was
confirmed in the case of Mtb. Conjugate 5a derived from unsubsti-
tuted salicylaldehyde exhibited the highest potency in this study
with MIC values of 8–16
lM, followed by 5-chloro derivative 5b
O
O
(16–32 M). The activity against atypical mycobacteria was
l
retained in the case of the salicylaldehyde as a starting material
(3a vs. 5a), but slightly diminished for 5-halogeno-salicylaldehydes
(3d vs. 5b and 3e vs. 5c). MIC values of pyridine-2-carboxaldehyde
H2SO4 (catal.)
(CH3CO)2O
N
N
OH
OH
S
S
O
OH
O
O
S
S
O
CH3
4
conjugate 5d were higher than 250 lM. The evaluation at higher
3a
concentrations was prevented due to solubility problems. These
data are a proof of the importance of the salicylidene structural
Scheme 2. Esterification of 3a.
O
H
O
N
R
O
CH3COO-Na+
CH3COOH
N
O
N
HN
S
1a
Cl
+
O
S
X
Cl
R
S
Y
X
Y
S
5
Scheme 3. Synthesis of 2-(5-arylmethylidene-4-oxo-2-thioxothiazolidin-3-yl)-N-(4-chlorophenyl)acetamides 5 (X = N, C; if X = N then Y is missing, if X = C then Y = OH;
R = H, Cl, Br).
Please cite this article in press as: Krátky´ M., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.045

´
3
M. Krátky et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
Overview and antimycobacterial activity of rhodanine derivatives 1–5.
Code
X
R
MIC [lM]
Mtb. 331/88
14 d
M. avium 330/88
M. kansasii 235/80
M. kansasii 6509/96
21 d
14 d
21 d
7 d
14 d
1000
21 d
7 d
14 d
21 d
RAA
- (free acid)
1000
1000
1000
1000
1000
1000
1000
1000
1000
O
O
N
X
R
S
S
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
NH
H
Cl
1000
32
1000
62.5
1000
500
1000
500
1000
500
1000
1000
1000
500
1000
1000
>1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
>1000
1000
>1000
>1000
>1000
1000
1000
1000
1000
1000
1000
1000
1000
250
1000
250
500
500
500
250
1000
1000
1000
500
1000
500
1000
500
500
500
1000
500
1000
1000
1000
500
1000
500
1000
500
1000
1000
1000
1000
1000
1000
1000
1000
1000
125
1000
500
500
500
500
250
1000
1000
1000
500
1000
250
1000
500
500
500
1000
500
1000
1000
1000
500
1000
250
1000
500
1000
1000
1000
500
1000
1000
1000
500
NH
NH
NH
NH
NH
O
O
O
O
O
CF3
CH3
OCH3
NO2
H
500
500
500
500
1000
250
500
1000
1000
500
Cl
CF3
CH3
OCH3
NO2
O
O
N
S
R
OH
X
S
O
3a
3b
3c
3d
3e
3f
4
C
2-OH
3-OH
4-OH
2-OH-5-Cl
2-OH-5-Br
–
32
32
125
125
32
62.5
1000
1000
62.5
62.5
1000
62.5
62.5
1000
1000
62.5
62.5
1000
62.5
32
62.5
1000
1000
62.5
125
62.5
1000
1000
125
250
1000
62.5
CH
CH
C
C
N
1000
1000
32
32
250
32
1000
1000
62.5
62.5
500
32
>1000
1000
62.5
62.5
1000
62.5
>1000
1000
62.5
62.5
1000
62.5
1000
1000
32
62.5
500
62.5
500
1000
32
62.5
500
62.5
500
62.5
C
2-AcO
O
S
H
N
N
O
Cl
R
S
X
5a
5b
5c
5d
INH
C-OH
C-OH
C-OH
N
H
8
16
16
32
62.5
250a
1
62.5
250
125
250
32
62.5
125
>500
250a
>250
125
250
>500
250a
>250
62.5
125
125
250a
4
125
250
250
250a
8
125
250
500
250a
16
Cl
Br
H
125
500
250a
>250
32
500
500
250a
0.5
250a
>250
250a
>250
Mtb: Mycobacterium tuberculosis. INH: isoniazid.
The best values for each strain and each group of the derivatives are provided in bold.
a
At presented concentration the growth of strain was observed, at duplex concentration, there was present precipitate and/or turbidity, therefore it was not possible to
determine exact MIC value.
fragment for the antimycobacterial activity, preferably without any
halogen substitution at the position 5.
Generally, the Gram-positive strains were more susceptible
than the Gram-negative species (see Table 2). None of the rhodani-
nes 1–5 was able to block the growth of P. aeruginosa and both
strains of K. pneumoniae, while E. coli was inhibited by only three
When compared to antimycobacterial agent isoniazid (INH),
none of the rhodanine derivatives 1–5 exceeded its in vitro potency
against Mtb. H37Rv and M. kansasii 6509/96. The situation is differ-
ent in the case of INH-resistant non-tuberculous strains (M. avium
330/88, M. kansasii 235/80). Six derivatives (3a, 3d, 3e, 4, 5a, and
salicylidene derivatives (3a, 3d, 3e) with MIC of 500
ues for Staphylococci and Enterococcus were similar each other
within the range of 15.62–500 M. There was not any difference
lM. MIC val-
l
5b) showed lower MIC values (P32
The growth inhibition of the atypical mycobacteria was indepen-
dent of the presence of INH-resistance or susceptibility.
l
M) than this first-line drug.
in the activity against a methicillin-susceptible S. aureus and a
MRSA strain, thus indicating no cross-resistance with beta-lac-
tams. However, several rhodanine-based molecules were inactive
(parent RAA, amides 1c-1e, esters 2b and 2d, condensate 3f and
conjugate 5a). The highest antibacterial activity exhibited 4-(triflu-
oromethyl)phenyl ester 2c (MIC = 15.62 and 62.5 lM after 24 and
48 h of incubation, respectively), followed by 5-salicylidene deriva-
tive 3a and its acetylated analogue 4.
4-Nitrophenyl amide 1f showed slightly improved activity
when compared to other amides 1. The reaction of RAA with
hydroxybenzaldehydes improves antibacterial activity uniformly.
The presence of a hydroxyl group at the position 2 of the benzyli-
dene moiety is an optimal choice (3a and 4); its positional isomers
(3-hydroxybenzaldehyde and 4-hydroxybenzaldehyde derivatives
2.2.2. In vitro antibacterial activity
Antibacterial activity was evaluated by the microdilution broth
method against eight strains: Staphylococcus aureus (SA), methi-
cillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epi-
dermidis (SE) and Enterococcus sp. (EF) were representatives of
Gram-positive bacteria, while Gram-negative strains involved in
our study were Escherichia coli (EC), Klebsiella pneumoniae, ESBL-
positive Klebsiella pneumoniae and Pseudomonas aeruginosa. An
established drug bacitracin (BAC) used for treatment of topical bac-
terial infections was used as a reference drug for the comparison.
Please cite this article in press as: Krátky´ M., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.045

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M. Krátky et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4
Table 2
Antibacterial activity of rhodanines 1–5.
Code
MIC [
SA
lM]
MRSA
24 h
SE
EF
EC
24 h
48 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
1a
1b
1f
2a
2c
2e
2f
3a
3b
3c
3d
3e
4
5b
5c
5d
BAC
500
>500
500
500
>500
500
>500
>500
500
>125
15.62
>500
500
62.5
500
500
125
125
62.5
125
500
>500
>500
500
>125
62.5
>500
500
62.5
>500
>500
125
500
500
250
>125
15.62
500
500
125
>500
250
62.5
62.5
31.25
250
500
>125
15.62
500
>500
250
>125
62.5
>500
500
125
>500
500
62.5
62.5
31.25
>250
500
500
500
250
125
15.62
500
500
125
500
>500
125
500
62.5
>250
500
500
>500
250
>125
62.5
>500
500
>500
>500
>500
>125
>500
>500
>500
500
>500
>500
>500
>125
>500
>500
>500
>500
>500
>500
>500
500
>500
>250
>500
>125
>500
>125
>125
62.5
>500
500
62.5
>500
>500
250
15.62
>500
250
62.5
>500
500
250
250
62.5
250
500
125
>500
>500
500
500
125
>250
500
>125
31.25
>500
>500
500
250
125
500
62.5
>250
500
>125
31.25
62.5
>250
500
>125
31.25
>500
>250
>500
>125
>500
>125
15.62
>125
15.62
>125
31.25
125
31.25
SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-resistant Staphylococcus aureus H 5996/08; SE: Staphylococcus epidermidis H 6966/08; EF: Enterococcus sp. J 14365/
08. Escherichia coli CCM 4517. BAC: bacitracin.
The best values for each strain are provided in bold.
3b and 3c, respectively) share a lower activity. The acetylation of
3a to form 4 decreased MIC values for S. epidermidis (125 M vs.
31.25 M), otherwise the activities are comparable. On the other
2.2.3. In vitro antifungal activity
l
The antifungal action of the rhodanines 1–5 were screened
in vitro against eight species: Candida albicans, C. tropicalis, C. krusei,
C. glabrata, Trichosporon asahii, Aspergillus fumigatus, Absidia
corymbifera and Trichophyton mentagrophytes (Table 3). Flucona-
zole was used as a reference drug.
l
hand, halogenation of 3a to more lipophilic chlorine and bromine
derivatives 3d and 3e led to an unexpected reduction of the activ-
ity towards methicillin-susceptible S. aureus and Enterococcus sp.;
MIC values for MRSA and S. epidermidis are analogous. The conju-
gates 5a-5d joining RAA, N-(4-chlorophenyl) and C-5 arylalkyli-
dene structural motifs showed only a limited (5b-5d) or no
antibacterial properties (5a). This modification mostly retained or
slightly improved the activity of amide 1b, but it abolished the
activity of 3a completely.
Two fungal strains, namely Candida krusei E28 and Absidia
corymbifera showed a total resistance to all rhodanines involved.
Unfortunately, parent RAA, five amides (1b-f), three esters
(2a, 2d, 2e), four C5-substituted derivatives (3b, 3d-f) and all of
the conjugates 5a-d avoided any antifungal activity. Surprisingly,
our results did not confirm previously reported an excellent anti-
fungal activity of the compound 3f against yeasts (MIC values
Based on MIC values, the ester 2c was comparable ( one dilu-
tion) to bacitracin against all Gram-positive strains and 2-[5-(2-
P0.98
and Aspergillus fumigatus was only sporadic at the concentration
of 500 M. Only Trichophyton mentagrophytes showed a compara-
l
M).11 The activity against Candidae, Trichosporon asahii
acetoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl]acetic
acid
4against S. epidermidis.
l
Interestingly, resembling catechol-rhodanine-3-acetic acid con-
jugates were reported as inhibitors of deoxyxylulose 5-phosphate
tively higher susceptibility. The growth of this mould strain was
inhibited by five rhodanines (2b-2f, 3a, 4) with MIC values of
reductoisomerase, an enzyme involved in 2-C-methyl-
D
-erythritol
125–500 lM. In general, the esters 2 and derivatives of salicylalde-
4-phosphate pathway. However, this enzyme inhibition with low
micromolar IC50 values did not result in the growth inhibition of
E. coli for five derivatives from total six.8
hyde were superior to others. However, the rhodanines 1–5 failed
in the searching for potential antimycotic drugs active at low
micromolar concentrations.
Table 3
Antifungal activity of selected rhodanine derivatives.
MIC/IC80/IC50 [lM]
CA
CT
CG
TA
AF
TM
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
72 h
120 h
1a
2b
2c
2f
3a
3c
4
>500
>500
>500
>500
500
500
>500
0.24
>500
>500
>500
>500
500
500
>500
0.24
500
500
>500
>500
500
>500
>500
>500
>500
62.5
>500
>500
>500
>500
>500
>500
>500
500
>500
>500
500
>500
>500
>500
>500
250
>500
>500
>500
>500
>500
>500
>500
500
>500
>500
>500
500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
500
125
125
250
>500
250
7.81
>500
500
250
250
250
>500
500
125
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
FLU
CA: Candida albicans ATCC 44859; CT: Candida tropicalis; CG: Candida glabrata 20/I; TA: Trichosporon asahii 1188; AF: Aspergillus fumigatus 231; TM: Trichophyton menta-
grophytes 445. FLU: fluconazole.
The positive hits are provided in bold.
Please cite this article in press as: Krátky´ M., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.045

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5
M. Krátky et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
234 °C decomp. IR (ATR): 3270, 3050, 2982, 2943, 1729, 1703,
1687, 1585, 1484, 1416, 1401, 1392, 1366, 1329, 1292, 1239,
1193, 1134, 1100, 1060, 960, 918, 893, 817, 746, 720, 690,
3. Material and methods
3.1. Chemistry
673 cmꢀ1 1H NMR (500 MHz, DMSO-d6): d 13.16 (1H, s, COOH),
.
11.51 (1H, s, OH), 7.88 (1H, s, -CH@), 7.52–7.46 (2H, m, H4, H6),
6.94 (1H, d, J = 8.7 Hz, H3), 4.69 (2H, s, CH2). 13C NMR (126 MHz,
DMSO-d6): d 193.63, 167.44, 166.61, 156.86, 135.54, 132.09,
128.56, 122.33, 122.18, 118.68, 110.82, 45.47. Anal. Calcd. for
3.1.1. General
All of the reagents and solvents were purchased from Sigma-
Aldrich (Darmstadt, Germany) or Penta Chemicals (Prague, Czech
Republic), and they were used as received. The progress of the
reactions and the purity of the products were monitored by thin-
layer chromatography using a mixture of toluene with ethyl acet-
ate (4:1, v/v); plates were coated with 0.2 mm Merck 60 F254 silica
gel and were visualised by UV irradiation (254 nm). Melting points
were determined on a Büchi Melting Point machine B-540 appara-
tus using open capillaries, and the reported values are uncorrected.
Elemental analysis (C, H, N) was performed on an automatic
microanalyser CHNS-O CE instrument (FISONS EA 1110, Milano,
Italy). Infrared spectra (ATR) were recorded on FT-IR spectrometer
Nicolet 6700 FT-IR in the range of 400–4000 cmꢀ1. The NMR spec-
tra were measured in DMSO-d6 at ambient temperature on a Var-
ian V NMR S500 instrument (500 MHz for 1H and 125 MHz for 13C;
Varian Comp. Palo Alto, CA, USA). The chemical shifts, d, are given
in ppm, with respect to tetramethylsilane as an internal standard.
The coupling constants (J) are reported in Hz.
C12H8BrNO4S2 (374.23): C, 38.51; H, 2.15; N, 3.74. Found: C,
38.69; H, 2.11; N, 3.93.
3.1.2.2.6.
2-[4-Oxo-5-(pyridin-2-ylmethylene)-2-thioxothiazo-
lidin-3-yl]acetic acid11 3f. Yellow solid; yield 97%; mp 257–
259.5 °C (lit. 260–261 °C11). Anal. Calcd. for C11H8N2O3S2
(280.32): C, 47.13; H, 2.88; N, 9.99. Found: C, 46.98; H, 3.00; N,
9.84.
3.1.2.3. Synthesis of 2-[5-(2-acetoxybenzylidene)-4-oxo-2-thioxothia-
zolidin-3-yl]acetic acid 4. Compound 3a (0.4 mmol) was suspended
in 4 mL of Ac2O and then 2 drops of concentrated sulphuric acid
was added. The mixture was refluxed for 2 h, and then poured in
cold water. The resulting precipitate was filtered off and washed
with a small volume of cold water to provide pure crystals of 4.
3.1.2.3.1. 2-[5-(2-Acetoxybenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl]acetic acid 4. Yellow solid; yield 78%; mp 137.5–138.5 °C. IR
(ATR): 2917, 1746, 1720, 1432, 1399, 1364, 1318, 1240, 1220,
3.1.2. Synthesis
1208, 1125, 1103, 1038, 980, 880, 857, 778, 761, 721 cmꢀ1 1H
.
3.1.2.1. Synthesis of amides and esters of rhodanine-3-acetic acid. The
synthesis and characterization (m.p., NMR and IR spectra,
elemental analyses) of substituted 2-(4-oxo-2-thioxothiazolidin-
3-yl)-N-phenylacetamides 1a-f and phenyl 2-(4-oxo-2-thioxothia-
zolidin-3-yl)acetates 2a-f were published recently.4
NMR (500 MHz, DMSO-d6): d 13.46 (1H, s, COOH), 7.72 (1H, s,
ACH@), 7.62–7.57 (2H, m, H4, H6), 7.47 (1H, td, J = 7.6 Hz,
J = 1.2 Hz, H5), 7.35 (1H, dd, J = 7.6 Hz, J = 1.4 Hz, H3), 4.73 (2H, s,
CH2), 2.36 (3H, s, CH3). 13C NMR (126 MHz, DMSO-d6): d 193.34,
169.13, 167.39, 166.24, 150.03, 132.63, 129.19, 127.20, 126.76,
125.79, 124.66, 124.00, 45.24, 20.85. Anal. Calcd. for C14H11ClNO5-
S2 (337.37): C, 49.84; H, 3.29; N, 4.15. Found: C, 49.72; H, 3.05; N,
4.10.
3.1.2.2. Synthesis of 2-(5-arylmethylidene-4-oxo-2-thioxothiazolidin-
3-yl)acetic acids 3. An equivalent of 2-(4-oxo-2-thioxothiazolidin-
3-yl)acetic acid (rhodanine-3-acetic acid) (1 mmol) together with
1 equivalent of anhydrous sodium acetate were dissolved in glacial
acetic acid (8 mL) followed by addition of 1.1 equivalents of substi-
tuted benzaldehyde/pyridine-2-carbaldehyde. The reaction mix-
ture was refluxed for 5 h. After cooling down of the mixture,
resulting crystals were filtered off, washed with a small volume
of cold water and dried to give products 3. If necessary, they were
recrystallized from acetic acid.
The identity of known compounds (i.e., 3a, 3b, 3c, 3d, and 3f)
was confirmed by NMR and IR spectroscopy. All spectroscopic
characteristics were in accordance with previously reported data.
The purity was checked additionally by melting points measure-
ment and elemental analysis.
3.1.2.4. Synthesis of 2-(5-arylmethylidene-4-oxo-2-thioxothiazolidin-
3-yl)-N-(4-chlorophenyl)acetamides
5. N-(4-Chlorophenyl)-2-(4-
oxo-2-thioxothiazolidin-3-yl)acetamide 1b (0.5 mmol) together
with 1 equivalent of anhydrous sodium acetate were dissolved in
glacial acetic acid (5 mL) followed by addition of 1.1 equivalents
of substituted benzaldehyde/pyridine-2-carbaldehyde. The reac-
tion mixture was refluxed for 5 h. After cooling down of the mix-
ture, resulting crystals were filtered off, washed with a small
volume of cold water and dried to give products 5a-d. If necessary,
they were recrystallized from ethyl acetate/n-hexane.
3.1.2.4.1. N-(4-Chlorophenyl)-2-[5-(2-hydroxybenzylidene)-4-oxo-
2-thioxothiazolidin-3-yl]acetamide 5a. Buff solid; yield 83%; mp
195–198 °C decomp. IR (ATR): 3295, 3040, 1725, 1678, 1593,
1530, 1489, 1454, 1403, 1375, 1356, 1311, 1264, 1242, 1201,
1187, 1175, 1123, 1092, 1036, 1011, 991, 941, 825, 808, 764,
3.1.2.2.1. 2-[5-(2-Hydroxybenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl]acetic acid5,7 3a. Yellow solid; yield 81%; mp 227–230 °C
decomp. Anal. Calcd. for C12H9NO4S2 (295.33): C, 48.80; H, 3.07;
N, 4.74. Found: C, 48.70; H, 3.19; N, 4.87.
749, 739, 694, 669 cmꢀ1 1H NMR (500 MHz, DMSO-d6): d 10.86
.
3.1.2.2.2. 2-[5-(3-Hydroxybenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl]acetic acid7 3b. Yellow solid; yield 82%; mp 292–295 °C
decomp. Anal. Calcd. for C12H9NO4S2 (295.33): C, 48.80; H, 3.07;
N, 4.74. Found: C, 48.98; H, 3.25; N, 4.94.
3.1.2.2.3. 2-[5-(4-Hydroxybenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl]acetic acid7,21 3c. Yellow solid; yield 82%; mp > 300 °C (lit.
>300 °C21). Anal. Calcd. for C12H9NO4S2 (295.33): C, 48.80; H,
3.07; N, 4.74. Found: C, 48.89; H, 2.94; N, 4.66.
(1H, s, OH), 10.56 (1H, s, NH), 8.04 (1H, s, ACH@), 7.59–7.55 (2H,
m, H2, H6), 7.41–7.35 (4H, m, H3, H5, H40, H60), 7.00–6.95 (2H,
m, H30, H50), 4.88 (2H, s, CH2). 13C NMR (126 MHz, DMSO-d6): d
194.20, 166.98, 163.62, 157.90, 137.53, 133.50, 129.99, 129.70,
128.96, 127.43, 120.90, 120.70, 120.20, 120.01, 116.55, 46.87. Anal.
Calcd. for C18H13ClN2O3S2 (404.88): C, 53.40; H, 3.24; N, 6.92.
Found: C, 53.68; H, 3.36; N, 7.13.
3.1.2.4.2.
2-[5-(5-Chloro-2-hydroxybenzylidene)-4-oxo-2-
3.1.2.2.4. 2-[5-(5-Chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-
thiazolidin-3-yl]acetic acid6,9 3d. Yellow solid; yield 78%; mp
248.5–251 °C decomp. (lit. 243–247 °C6). Anal. Calcd. for C12H8-
ClNO4S2 (329.78): C, 43.70; H, 2.45; N, 4.25. Found: C, 43.79; H,
2.19; N, 4.44.
thioxothiazolidin-3-yl]-N-(4-chlorophenyl)acetamide 5b. Yellow-
orange solid; yield 85%; mp 221–224 °C. IR (ATR): 3296, 3194,
3118, 3076, D2975, 2930, 1739, 1687, 1661, 1619, 1600, 1539,
1492, 1403, 1359, 1325, 1256, 1219, 1198, 1176, 1108, 1094,
1045, 1013, 974, 958, 881, 833, 782, 730, 702 cmꢀ1 1H NMR
.
3.1.2.2.5. 2-[5-(5-Bromo-2-hydroxybenzylidene)-4-oxo-2-thioxo-
thiazolidin-3-yl]acetic acid 3e. Yellow solid; yield 81%; mp 231–
(500 MHz, MSO-d6): d 11.26 (1H, s, OH), 10.56 (1H, s, NH), 7.91
(1H, s, ACH@), 7.59–7.54 (2H, m, H2, H6), 7.42–7.35 (4H, m, H3,
Please cite this article in press as: Krátky´ M., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.045

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M. Krátky et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
6
H5, H40, H60), 6.98 (1H, d, J = 8.7 Hz, H30), 4.87 (2H, s, CH2). 13C NMR
(126 MHz, DMSO-d6): d 193.93, 166.83, 163.55, 156.44, 137.51,
132.75, 129.20, 128.96, 128.56, 127.44, 123.44, 122.58, 121.60,
120.89, 118.26, 46.90. Anal. Calcd. for C18H12Cl2N2O3S2 (439.33):
C, 49.21; H, 2.75; N, 6.38. Found: C, 48.95; H, 3.00; N, 6.32.
3.1.2.4.3. 2-[5-(5-Bromo-2-hydroxybenzylidene)-4-oxo-2-thioxo-
thiazolidin-3-yl]-N-(4-chlorophenyl)acetamide 5c. Yellow-orange
solid; yield 77%; mp 215.5–217 °C decomp. IR (ATR): 3261, 3058,
1686, 1670, 1583, 1546, 1491, 1416, 1403, 1369, 1358, 1335,
1292, 1254, 1187, 1136, 1093, 1062, 1015, 984, 957, 847, 830,
were analysed visually. The MIC values were determined after 24
and 48 h of incubation in the dark at 35 °C ( 0.1) in a humid
atmosphere.22
3.2.3. Antifungal activity
The antifungal properties were evaluated against four Candida
strains (Candida albicans ATCC 44859, Candida tropicalis 156, Can-
dida krusei E28, and Candida glabrata 20/I), Trichosporon asahii
1188 and three strains of filamentous fungi (Aspergillus fumigatus
231, Absidia corymbifera 272, and Trichophyton mentagrophytes
445).
817, 750, 712, 693, 669 cmꢀ1 1H NMR (500 MHz, DMSO-d6): d
.
11.47 (1H, s, OH), 10.56 (1H, s, NH), 7.88 (1H, s, ACH@), 7.59–
7.54 (2H, m, H2, H6), 7.50–7.47 (2H, m, H40, H60), 7.39–7.35 (2H,
m, H3, H5), 6.90 (1H, d, J = 9.4 Hz, H30), 4.87 (2H, s, CH2). 13C
NMR (126 MHz, DMSO-d6): d 194.01, 172.16, 166.87, 163.58,
137.52, 135.61, 132.31, 128.95, 127.42, 122.24, 122.04, 120.89,
118.97, 110.27, 46.88. Anal. Calcd. for C18H12BrClN2O3S2 (483.78):
C, 44.69; H, 2.50; N, 5.79. Found: C, 44.60; H, 2.75; N, 6.01.
The microdilution broth method was used according to the CLSI
M27-A3 and M38-A2 guidelines in RPMI 1640 with glutamine
(KlinLab, Prague, Czech Republic) buffered to pH 7.0 with
0.165 mol of 3-morpholino-propane-1-sulphonic acid (Sigma-
Aldrich, Darmstadt, Germany). DMSO served as a diluent for all
the compounds. In yeast, the final size of the inoculum was
5 ꢁ 103 0.2 CFU/mL, and in the case of the moulds, the final size
of the inoculum was 0.5–5 ꢁ 104 CFU/mL. Fluconazole (FLU) was
involved as a comparative drug. The MIC values for yeasts and fil-
amentous fungi were assayed as a reduction of growth of at least
80% (IC80) or of at least 50% (IC50) compared with the control,
respectively. The results were analysed visually and/or spectropho-
tometrically at 540 nm. The MIC values were determined after 24
and 48 h of incubation in the dark at 35 °C ( 0.1) in a humid atmo-
sphere, but for T. mentagrophytes, the final MIC values were deter-
mined after 72 and 120 h of incubation.23
3.1.2.4.4.
N-(4-Chlorophenyl)-2-[4-oxo-5-(pyridin-2-ylmethy-
lene)-2-thioxothiazolidin-3-yl]acetamide 5d. Yellow-green solid;
yield 84%; mp 293–296 °C decomp. IR (ATR): 3244, 3044, 2934,
1709, 1672, 1661, 1611, 1594, 1544, 1492, 1433, 1402, 1377,
1326, 1301, 1251, 1227, 1211, 1198, 1125, 1096, 1053, 1014,
979, 951, 910, 827, 783, 743, 728, 705, 692 cmꢀ1 1H NMR
.
(500 MHz, DMSO-d6): d 10.67 (1H, s, NH), 8.83 (1H, d, J = 4.9 Hz,
H60), 8.00–7.89 (3H, m, ACH@, H30, H40), 7.60–7.56 (2H, m, H2,
H6), 7.50–7.45 (1H, m, H50), 7.39–7.34 (2H, m, H3, H5), 4.89 (2H,
s, CH2). 13C NMR (126 MHz, DMSO-d6): d 200.08, 166.87, 163.67,
151.19, 149.73, 137.92, 137.58, 129.33, 128.94, 128.59, 127.39,
126.68, 124.50, 120.90, 46.46. Anal. Calcd. for C17H12ClN3O2S2
(389.87): C, 52.37; H, 3.10; N, 10.78. Found: C, 52.70; H, 3.12; N,
10.92.
4. Conclusions
Twenty-four derivatives of rhodanine-3-acetic acid were evalu-
ated as potential antimicrobial agents. Amides and esters were
taken over one of our previous work,4 condensates of RAA with
aromatic aldehydes and ‘‘mutual conjugates” of N-(4-chlorophe-
nyl)-2-(4-oxo-2-thioxothiazolidin-3-yl)acetamide and aromatic
aldehydes were synthesized originally. All of these derivatives
underwent in vitro assays against mycobacteria, eight bacterial
and fungal strains.
3.2. Pharmacology
3.2.1. Antimycobacterial activity
The antimycobacterial activity against Mycobacterium tubercu-
losis (Mtb.) 331/88 (H37Rv; dilution of this strain was 10ꢀ3),
Mycobacterium avium 330/88 (dilution of 10ꢀ5), and two strains
of Mycobacterium kansasii, namely 235/80 (dilution of 10ꢀ4) and
the clinically isolated strain 6509/96 (dilution of 10ꢀ5) was evalu-
ated using a previously described method.22 The following concen-
trations were used: 1000, 500, 250, 125, 62.5, 32, 16, 8, 4, 2, and
All of the derivatives were active against mycobacteria. Among
the amides and esters, only the N-(4-chlorophenyl)amide exhibited
a potent antimycobacterial activity (MIC against Mtb. of 32–
1
l
M. MIC is the lowest concentration at which complete inhibi-
62.5
inhibition of Gram-positive bacteria comparable to bacitracin at
low concentrations starting from 15.62 M. Salicylaldehyde-based
rhodanine C-5 condensates produced a more efficient and consis-
tent inhibition of mycobacteria. An additional substitution of sali-
cylaldehyde does not improve antibacterial and antimycobacterial
properties significantly with an exception of S. epidermidis. Iso-
meric 3- and 4-hydroxybenzaldehydes and pyridine-2-carbalde-
hyde-based derivatives lack the activity. Based on these results,
the conjugates combining both these pharmacophores were
lM) and only 4-(trifluoromethyl)phenyl ester showed an
tion of mycobacterial growth was observed. INH was chosen as a
reference compound. The experiments were prepared in quadru-
plicates and the determination was repeated twice.
l
3.2.2. Antibacterial activity
The antibacterial activities were assayed against a panel of eight
Gram-positive and Gram-negative strains: Staphylococcus aureus
CCM 4516/08, methicillin-resistant Staphylococcus aureus
H
5996/08 (MRSA), Staphylococcus epidermidis H 6966/08, Enterococ-
cus sp. J 14365/08; Escherichia coli CCM 4517, Klebsiella pneumoniae
D 11750/08, extended-spectrum b-lactamases (ESBL) positive Kleb-
siella pneumoniae J 14368/08, and Pseudomonas aeruginosa CCM
1961.
The microdilution broth method modified according to stan-
dard M07-A07 in Mueller-Hinton broth (HiMedia Laboratories,
Mumbai, India) adjusted to pH 7.4 ( 0.2) was used. The
tested compounds were dissolved in DMSO to final concentrations
designed.
oxo-2-thioxothiazolidin-3-yl]acetamide was identified as the most
active agent against Mtb. with MIC of 8–16 M. These derivatives
N-(4-Chlorophenyl)-2-[5-(2-hydroxybenzylidene)-4-
l
were also able to affect the growth of INH-resistant atypical
mycobacteria, thus indicating no cross-resistance. Gram-negative
bacteria and most of the fungi tolerate the majority of the investi-
gated rhodanines. In comparison, only fungus Trichophyton menta-
grophytes was inhibited more considerably, although at higher
concentrations.
ranging from 500 to 0.49 lM. Bacitracin (BAC) was used as a refer-
ence drug. A bacterial inoculum in sterile water was prepared to
reach 0.5 on the McFarland scale (1.5 ꢁ 108 CFU/mL). The mini-
mum inhibitory concentrations were assayed as a reduction in
growth of at least 95% (IC95) compared with the control. The results
Importantly, all of the reported rhodanines 1–5 meet the
requirements of Lipinski’s rule of five (MW < 500, no more than
five hydrogen bond donors and ten hydrogen bond acceptors, log
P not > 5).
Please cite this article in press as: Krátky´ M., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.045

´
7
M. Krátky et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3. Kong C, Eng S-A, Lim M-P, Nathan S. Front Microbiol. 2016;7 Article Number
Funding
This work was supported by the Czech Science Foundation pro-
1956.
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Declaration of interest
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The authors declare no conflict of interest.
Acknowledgements
We want to thank to Ida Dufková for the excellent performance
of susceptibility tests for bacteria and fungi, the staff of the Depart-
ment of Inorganic and Organic Chemistry, Faculty of Pharmacy, for
the technical assistance and Jaroslava Urbanová, M.A., for the lan-
guage help provided.
15. Krátky´ M, Vinšová J. Bioorg Med Chem. 2016;24:1322.
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Chem. 2014;22:4073.
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18. Krátky M, Vinšová J, Volková M, Buchta V, Trejtnar F, Stolaríková J. Eur J Med
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Please cite this article in press as: Krátky´ M., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.01.045