Synthesis and antibacterial activity of 5-ylidenethiazolidin-4-ones and 5-benzylidene-4,6-pyrimidinediones

Article abstract of DOI:10.1016/j.ejmech.2009.12.030

5-Benzylidenethiazolidin-4-ones and 5-benzylidenepyrimidine-4,6-diones (compounds 1-9), carrying 2,3,4-trifluoro or 3,4,5-trimethoxy groups on the benzylidene moiety, and rhodanine derivatives 10 and 11 were synthesized and assayed in vitro for their anti

Full text of DOI:10.1016/j.ejmech.2009.12.030

European Journal of Medicinal Chemistry 45 (2010) 1667–1672
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and antibacterial activity of 5-ylidenethiazolidin-4-ones and
5-benzylidene-4,6-pyrimidinediones
a
b
a,
,
ˇ ˇ a
*
ˇ ´ a
*
Tihomir Tomasic , Nace Zidar , Manica Mueller-Premru , Danijel Kikelj , Lucija Peterlin Masic
a
ˇ
ˇ
University of Ljubljana, Faculty of Pharmacy, Askerceva 7, 1000 Ljubljana, Slovenia
b
ˇ
University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, Zaloska 4, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Benzylidenethiazolidin-4-ones and 5-benzylidenepyrimidine-4,6-diones (compounds 1–9), carrying
2,3,4-trifluoro or 3,4,5-trimethoxy groups on the benzylidene moiety, and rhodanine derivatives 10 and
11 were synthesized and assayed in vitro for their antimicrobial activity against four standard bacterial
strains (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922
and Pseudomonas aeruginosa ATCC 27853). Compounds 1–3 and 9 that were active against S. aureus, were
also tested against methicillin-resistant S. aureus (MRSA) ATCC 43300, Streptococcus pneumoniae ATCC
49619 and Streptococcus pyogenes ATCC 19615. (Z)-5-(2,3,4-Trifluorobenzylidene)rhodanine (1) inhibited
Received 27 October 2009
Received in revised form
6 December 2009
Accepted 14 December 2009
Available online 22 December 2009
Keywords:
the growth of S. aureus at 0.5 mg/mL and MRSA at 32 mg/mL. Stronger antimicrobial activity against S.
Antibacterial agents
Knoevenagel condensation
Rhodanine
aureus was observed for compounds bearing the rhodanine ring than those containing other heterocyclic
moieties. Neither of the compounds 1–11 inhibited the growth of Gram-negative bacteria E. coli or
P. aeruginosa.
Thiazolidin-4-one
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
known for more than 50 years, there have been several attempts
to design antibacterial agents based on this heterocycle [4].
The treatment of bacterial infections still remains an important
and challenging therapeutic problem because of factors that
include emerging infectious diseases and the increasing number of
multi-drug resistant microbial pathogens. In spite of the large
number of antibiotics and chemotherapeutics available for medical
use, the emergence of old and new antibiotic resistant bacterial
strains in the last decades constitutes a substantial need for new
classes of antibacterial agents [1].
The diverse biological activities of rhodanines (2-thioxothia-
zolidin-4-ones) and their analogues have been known from the
beginning of the 20th century. Rhodanines and thiazolidine-2,4-
diones have become a pharmacologically important class of
heterocyclic compounds since the introduction of various glita-
zones and epalrestat into clinical use for the treatment of type II
diabetes and diabetic complications, respectively [2,3]. Chemical
modifications of these heterocycles have consistently resulted in
compounds with a broad spectrum of other pharmacological
activities. Since the antimicrobial activity of rhodanines has been
5-Arylmethylidenerhodanines have a number of structural
features responsible for their antimicrobial activity, such as an aryl-
methylidene group at position 5 of the rhodanine, and the non-
substituted rhodanine ring nitrogen at position 3 [5–7]. The incor-
poration of a halide-substituted benzylidene group at position 5 of 4-
thioxothiazolidin-2-one, in which, in comparison to the rhodanine
ring, only 4-thioxo and 2-oxo groups are exchanged, resulted in
antimicrobial activity against Gram-positive bacteria, including
multi-drug-resistant isolates [8]. Although the structure–activity
relationships of thiazolidin-4-one derivatives have been studied
extensively, very little is known about the contributions to antibac-
terial activity of the trifluoro- and trimethoxybenzylidene moieties
attached to position 5 of rhodanine, 2-(4-oxo-2-thioxothiazolidin-3-
yl)acetic acid (rhodanine-N-acetic acid), thiazolidine-2,4-dione,
pyrimidine-2,4,6(1H,3H,5H)-trione (barbituric acid) and 2-thio-
xodihydropyrimidine-4,6(1H,5H)-dione (thiobarbituric acid).
In view of these facts, and as part of our ongoing studies
in developing new antimicrobial agents [9–12], we synthesized
5-benzylidenerhodanines 1, 2, 5 and 6, 5-benzylidenethiazolidine-
2,4-diones 3 and 4, 5-benzylidenethiobarbituric acid 7, 5-benzyli-
denebarbituric acids 8 and 9, and rhodanine derivatives 10 and 11
(Fig. 1). They were evaluated as antimicrobial agents on four stan-
dard bacterial strains (Staphylococcus. aureus ATCC 29213, Entero-
coccus faecalis ATCC 29212, Escherichia coli ATCC 25922 and
* Corresponding authors. Tel.: þ386 1 476 95 00; fax: þ386 1 425 80 31.
E-mail addresses: danijel.kikelj@ffa.uni-lj.si (D. Kikelj), lucija.peterlin@ffa.uni-lj.si
ˇ ˇ
(L.P. Masic).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.030

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T. Tomasi´c et al. / European Journal of Medicinal Chemistry 45 (2010) 1667–1672
Fig. 1. Thiazolidin-4-ones and pyrimidine-4,6-diones 1–11.
Pseudomonas aeruginosa ATCC 27853). The most potent compounds
were also tested on MRSA ATCC 43300, Streptococcus pneumoniae
ATCC 49619 and Streptococcus pyogenes ATCC 19615.
3. Results and discussion
3.1. Antimicrobial activity
All the compounds 1–11 were tested for their in vitro antibac-
terial activity against S. aureus ATCC 29213, E. faecalis ATCC 29212, E.
coli ATCC 25922 and P. aeruginosa ATCC 27853. The results are given
in Table 1. The most active compounds 1–3 and 9 against S. aureus
2. Chemistry
5-Benzylidene-substituted compounds 1–9 were prepared in
good yields via a Knoevenagel condensation between the corre-
sponding heterocyclic cores of rhodanine, rhodanine-N-acetic acid,
thiazolidine-2,4-dione, barbituric acid and thiobarbituric acid and
a range of substituted benzaldehydes (Schemes 1 and 2). Thiazolidin-
4-one-based compounds were synthesized using microwave-
assisted synthesis, with piperidine and glacial acetic acid as catalysts.
The reaction conditions were optimized variously, depending on the
heterocyclic nucleus. For example, rhodanine-N-acetic acid deriva-
tives 5 and 6 were synthesized at lower temperature and prolonged
reaction times than for their rhodanine and thiazolidine-2,4-dione
counterparts 1–4. Conventional heating of barbituric or thio-
barbituric acid with the corresponding benzaldehyde derivative at
reflux temperatures had to be employed for the synthesis of
compounds 7–9, since applicationof microwave irradiationproved to
beunsuccessful. The synthesis ofalkylidenerhodaninecompounds10
and 11 has been described elsewhere [13]. In theory, E and Z
geometrical isomers around the exocyclic double bond (CH]C) are
possible for 5-benzylidene derivatives 1–6 and 5-alkylidene
compounds 10 and 11. 1H NMR spectra of compounds 1–6 show only
one signal for the methyne proton in the range 7.46–7.82 ppm, at
lower field values than those expected for the E-isomers, which
strongly indicates that the compounds have the Z-configuration.
The latter has been reported as thermodynamically more stable
than the E-configuration [14,15]. The Z-configuration of compounds
10 and 11 was confirmed from the 1H-coupled 13C NMR spectrum
of compound 11 followed by examination of the splitting pattern
and coupling constant of the signal of the C]O group in the rho-
danine system [13].
(minimal inhibitory concentration (MIC) < 32
mg/mL) were also
tested against methicillin-resistant S. aureus (MRSA isolate ATCC
43300), S. pneumoniae ATCC 49619 and S. pyogenes ATCC 19615
(Table 2). Their antimicrobial activities were compared, using
gentamicin as the reference antibacterial agent. Significant inhibi-
tory activity against Gram-positive S. aureus is exhibited by
compounds 1–3 and 9–11, with MIC values in the 0.5–32
range. Compounds 1–3 and 9 also demonstrated moderate inhibi-
tion of MRSA growth (MICs 32–128 g/mL). None of the compounds
1–11 inhibited the growth of Gram-negative E. coli or P. aeruginosa
(MIC > 128 g/mL).
mg/mL
m
m
Thiazolidine-2,4-diones and 4-thioxo-thiazolidine-2-ones have
been reported to be active against S. aureus but inactive against
Gram-negative bacteria [7,8]. The significant activity of compounds
1–3 and the inactivity of compound I (Fig. 2), previously identified
as a multitarget inhibitor of enzymes MurD-F [12a] that catalyze
the intracellular steps of bacterial peptidoglycan biosynthesis [16],
against S. aureus can be attributed to the hydrophilic character of
the 2,3,4-trihydroxybenzylidene group that hinders the penetra-
tion of the molecule into the bacterial cell. Indeed, the calculated
log P value, c Log P, for compound I is ꢀ0.13 [17]. Replacement of the
2,3,4-trihydroxybenzylidene group of I with the more lipophilic
2,3,4-trifluorobenzylidene group increased c Log P to 2.1, resulting
in the most potent antibacterial compound in the series (1), which
inhibited the growth of S. aureus with an MIC value of 0.5
Compound 1 was also evaluated for antibacterial activity against
MRSA (MIC ¼ 32 g/mL), S. pneumoniae (minimal bactericidal
concentration (MBC) ¼ 64 g/mL) and S. pyogenes (MBC > 128 g/
mg/mL.
m
m
m
Scheme 1. Reagents and conditions: (a) piperidine, AcOH, EtOH, 30 W, 18 bar, 140 ꢁC, 30 min for 1–4 or 110 ꢁC, 40 min for 5 and 6.

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T. Tomasi´c et al. / European Journal of Medicinal Chemistry 45 (2010) 1667–1672
1669
Scheme 2. Reagents and conditions: (a) water, reflux, 10 h.
mL). Attachment of the 3,4,5-trimethoxybenzylidene moiety to the
rhodanine heterocycle at position 5, instead of 2,3,4-tri-
fluorobenzylidene, resulted in compound 2 (c Log P ¼ 1.1) which
the importance of the rhodanine moiety for potent antibacterial
activity, since replacement of the rhodanine ring in compound 2 by
the thiobarbituric or barbituric acid rings (compounds 7 and 8)
was active against S. aureus (MIC
¼
8
m
g/mL) and MRSA
resulted in inactivity against S. aureus (MIC ¼ 8
mg/mL compared to
MIC >128 g/mL). Although the incorporation of the thiocarbonyl
(MIC ¼ 128
m
g/mL). In contrast, replacement of the rhodanine
m
nucleus by thiazolidine-2,4-dione (compounds 3–4) led to signifi-
cantly weaker antibacterial activity. Thiazolidine-2,4-dione-based
compound 3, possessing the 2,3,4-trifluorobenzylidene moiety,
group (compound 7) instead of the carbonyl group (compound 8)
increased the lipophilicity of the compound (c Log P ¼ 0.96 versus
0.09, respectively), it did not improve antibacterial activity of
compound 7. On the other hand, replacement of the 3,4,5-trime-
thoxybenzylidene moiety (compound 8 with c Log P ¼ 0.09) by
a more lipophilic 2,3,4-trifluorobenzylidene moiety (compound 9
with c Log P ¼ 1.1) had an important influence on the antibacterial
activity, since 2,3,4-trifluorobenzylidenebarbituric acid (9)
was 16-times less active against S. aureus (MIC ¼ 32
mg/mL) and 2-
times less active against MRSA (MIC ¼ 64 g/mL) than its rhodanine
m
counterpart 1, while the (Z)-5-(3,4,5-trimethoxybenzylid-
ene)thiazolidine-2,4-dione (4) was inactive against S. aureus and
other bacterial strains, although the c Log P values for compounds 3
(c Log P ¼ 2.0) and 4 (c Log P ¼ 1.0) are comparable to those of
compounds 1 and 2, respectively. Considering the comparable lip-
ophilicity of the thiocarbonyl- and carbonyl-based compounds
(compound 1 and 2 versus compounds 3 and 4, respectively), the
thiocarbonyl group seems to play a very important role for the
potent antibacterial activity of these compounds. The difference
between the activities of the rhodanines and thiazolidine-2,4-dio-
nes tested confirms reports that various rhodanines possess anti-
bacterial activity and inhibit bacterial enzymes, while their
thiazolidine-2,4-dione counterparts usually display weaker activity
or are inactive [4]. However, the inability of 1 to inhibit MurC-F
ligases [12b] indicates that other targets may be involved in the
antibacterial action of compounds 1–3 against S. aureus. Moreover,
in our series of compounds, the antibacterial activity was also
dependent on the substitution of the rhodanine nitrogen at posi-
tion 3. Both rhodanine-based compounds 5 and 6 that incorporate
an acetic acid group at this position 3 were inactive against all
possessed activity against S. aureus (MIC ¼ 32
mg/mL) and MRSA
(MIC ¼ 128 g/mL).
m
In the course of our studies towards the discovery of novel Mur
ligase inhibitors, recently described 2H-1,4-benzoxazin-3(4H)-one-
based inhibitors of bacterial histidine protein kinase [18,19]
attracted our attention due to similarities in the structure of Mur
enzymes and various kinases. Thus, compounds 10 and 11, in which
the rhodanine moiety is connected to the 2H-1,4-benzoxazin-
3(4H)-one scaffold via an ethylidene linker, were prepared as rho-
danine analogues of histidine protein kinase inhibitors II and III
(Fig. 2), for which docking experiments had predicted promising
interaction also with bacterial enzymes MurC and MurD (unpub-
lished results). While N4-(4-chlorobenzyl)-substituted compound
II showed 8-times more potent antibacterial activity against S.
aureus (MIC ¼ 8
m
g/mL) than N4-(4-cianobenzyl)-substituted
g/mL) [11], the effect of N4-substituent of
compound III (MIC ¼ 64
m
the rhodanine-based analogues 10 and 11 on the antibacterial
tested bacterial strains, with MIC values greater than 128
mg/mL.
activity was less pronounced, since both compounds inhibited the
Replacing the rhodanine ring in compounds 1 and 2 with thio-
barbituric or barbituric acid rings gave compounds 7–9. The results
of the antibacterial activity of compounds 7 and 8 further support
growth of S. aureus at 32
ability to inhibit the growth of E. faecalis (MIC > 128
compared to its analogue II (MIC ¼ 16 g/mL), while compound 11
g/mL) displayed 2-times lower MIC values against E.
m
g/mL. Moreover, compound 10 lost its
mg/mL) as
m
(MIC ¼ 32
m
faecalis than its counterpart III (MIC ¼ 64
mg/mL).
Table 1
Antibacterial activity of compounds 1–11 and I–III against S. aureus and E. faecalis
(MIC, mg/mL) and their c Log P values [17].
4. Conclusion
Compound
S. aureus ATCC 29213
E. faecalis ATCC 29212
c Log P
The synthesis and antibacterial activity are reported for a series
of rhodanine-, rhodanine-N-acetic acid-, thiazolidine-2,4-dione-,
1
2
3
4
5
6
7
8
0.5
8
32
>128
>128
>128
>128
>128
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
2.1
1.1
2.0
1.0
Table 2
1.5
Antibacterial activity of compounds 1–3 against MRSA, S. pneumoniae and S.
0.47
0.96
0.09
1.1
3.3
2.0
pyogenes (MIC,
m
g/mL).
Compound
MRSA ATCC
S. pneumoniaea ATCC
S. pyogenesa ATCC
9
10
11
43300
49619
19615
32
32
1
2
3
9
32
128
64
64a
>128a
>128a
ND
>128a
ND
I
II
III
>128
8
64
>128
16
64
ꢀ0.13
4.7
3.4
128
ND
ND
a
MBC, mg/mL. ND – not determined.

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T. Tomasi´c et al. / European Journal of Medicinal Chemistry 45 (2010) 1667–1672
Fig. 2. (Z)-5-(2,3,4-Trihydroxybenzylidene)rhodanine (I) and 2H-1,4-benzoxazin-3(4H)-one-based inhibitors of histidine protein kinase II and III.
barbituric- and thiobarbituric acid-based compounds bearing an
0.1 equiv) were added. The reaction mixture was heated with
microwave irradiation (max. power of 30 W) to 140 ꢁC and the
heating continued for 30 min to maintain the temperature. The
pressure limit was set at 18 bar. The reaction vessel was cooled in an
ice-bath and the precipitate was filtered off, washed with ice-
cooled ethanol and dried under vacuum.
ylidene substituent at position 5. The results of antibacterial
activity of the synthesized compounds against selected Gram-
positive and Gram-negative bacteria show that the 2,3,4-
trifluorobenzylidene moiety and the thiocarbonyl group on the
thiazolidin-4-one ring are important for potent inhibition of S.
aureus and MRSA growth. The most potent compound of the series,
(Z)-5-(2,3,4-trifluorobenzylidene)rhodanine (1), inhibited the
5.1.1.1. (Z)-2-Thioxo-5-(2,3,4-trifluorobenzylidene)thiazolidin-4-one
(1). Orange crystalline solid. Yield: 87.8%. Melting range 198–200 ꢁC.
IR (KBr): nmax ¼ 3552, 3474, 3413, 3231, 1718, 1637, 1615, 1590, 1515,
1466, 1438, 1302, 1216, 1052, 1010, 976, 798, 671, 638, 601 cmꢀ1. 1H
growth of S. aureus at 0.5 mg/mL and MRSA at 32 mg/mL. When
2,3,4-trifluorobenzylidene moiety was replaced by a 3,4,5-trime-
thoxybenzylidene moiety or when thiazolidine-2,4-dione, rhoda-
nine-N-acetic acid, barbituric or thiobarbituric acid ring was used
instead of the rhodanine ring, thus obtained compounds 2–9
always possessed weaker antibacterial activity than compound 1.
2H-1,4-Benzoxazin-3(4H)-one-based compound 11 displayed
NMR (DMSO-d6):
d
¼ 7.35–7.43 (m, 1H, Ar-H5), 7.46–7.55 (m, 2H, CH,
Ar-H6), 13.93 (br s, 1H, CONHCS) ppm. MS (ESIþ): m/z (%) ¼ 276
([M þ H]þ, 85), 327 (100). Anal. calcd for C10H4F3NOS2: C, 43.63%; H,
1.46%; N, 5.09%; found C, 43.88%; H, 1.49%; N, 5.12%.
activity also against E. faecalis (MIC ¼ 32
mg/mL), but all of the
synthesized compounds were inactive against Gram-negative
bacteria. Potent antibacterial activity makes compound 1 promising
starting point for further optimization. However, additional studies
regarding the mechanism of action are necessary for a complete
understanding of the antimicrobial activity of these compounds.
5.1.1.2. (Z)-2-Thioxo-5-(3,4,5-trimethoxybenzylidene)thiazolidin-4-
one (2). Orange crystalline solid. Yield: 71.3%. Melting range 196–
198 ꢁC (202–203 ꢁC [20]). IR (KBr): nmax ¼ 3546, 3003, 1686, 1596,
1572, 1499, 1442, 1332, 1300, 1250, 1216, 1154, 1128, 1068, 991, 824,
712, 668, 614, 550 cmꢀ1
.
1H NMR (DMSO-d6):
d
¼ 3.74 (s, 3H, 4-
OCH3), 3.85 (s, 6H, 3-OCH3, 5-OCH3), 6.92 (s, 2H, Ar-H2, H6), 7.61 (s,
1H, CH), 13.82 (s, 1H, CONHCS) ppm. MS (ESIþ): m/z (%) ¼ 312
([M þ H]þ, 60), 334 (100). Anal. calcd for C13H13NO4S2: C, 50.11%; H,
4.12%; N, 4.45%; found C, 50.14%; H, 4.21%; N, 4.50%.
5. Experimental protocols
Chemicals were obtained from Acros, Aldrich Chemical Co. and
Fluka and used without further purification. Yields refer to purified
products and were not optimized. Analytical thin-layer chromatog-
raphy (TLC) was performed using silica gel 60F254 pre-coated plates
(0.25 mm thick) with a fluorescent indicator from Merck (Germany).
Flash column chromatography was carried out on silica gel 60
(particle size 0.040–0.063 mm; Merck, Germany). The components
of chromatographic eluents are given as volume-to-volume ratios
(v/v). Melting points were determined on a Reichert hot stage
microscope and are uncorrected. Structures of compounds were
confirmed by routine spectrometric analysis. 1H NMR spectra were
recorded at 300 MHz on a Bruker AVANCE DPX300 spectrometer in
DMSO-d6 solution with TMS as the internal standard. IR spectra were
recorded on a Perkin–Elmer 1600 FT-IR spectrometer. Elemental
analyses were performed on a Perkin–Elmer C, H, N analyzer 240C
and were within ꢂ0.4% of the theoretical values. Mass spectra were
obtained using a VG-Analytical Autospec Q mass spectrometer.
Microwave-assisted reactions were performed using a focused
microwave reactor (DiscoverÔ, CEM Corporation, Matthews, NC).
Reactionswerecarriedoutinseptum-sealedglassvials(10mL)which
enable high-pressure reaction conditions (max 20 bar). The temper-
ature of the reaction mixture was monitored using a calibrated
infrared temperature controller mounted under the reaction vessel.
5.1.2. General procedure for microwave-assisted synthesis of (Z)-5-
benzylidenethiazolidine-2,4-diones 3 and 4
To a suspension of thiazolidine-2,4-dione (0.200 g, 1.71 mmol,
1.0 equiv) in dry ethanol (5 mL), the corresponding aldehyde
(1.71 mmol, 1.0 equiv),
a catalytic amounts of piperidine
(0.171 mmol, 0.1 equiv) and glacial acetic acid (0.171 mmol,
0.1 equiv) were added. The reaction mixture was heated with
microwave irradiation (max. power of 30 W) to 140 ꢁC and the
heating continued for 30 min to maintain the temperature. The
pressure limit was set at 18 bar. The reaction vessel was cooled in an
ice-bath and the precipitate was filtered off, washed with ice-
cooled ethanol and dried under vacuum.
5.1.2.1. (Z)-5-(2,3,4-Trifluorobenzylidene)thiazolidine-2,4-dione
(3). Yellow crystalline solid. Yield: 85.8%. Melting range 120–
122 ꢁC. IR (KBr): nmax ¼ 3414, 3034, 2769, 1745, 1690, 1616, 1509,
1466, 1343, 1304, 1291, 1176, 1153, 1047, 1013, 974, 896, 808, 728,
676, 634, 602, 536, 503, 474 cmꢀ1. 1H NMR (DMSO-d6, 300 MHz):
d
¼ 7.36–7.55 (m, 2H, Ar-H5, H6), 7.70 (s, 1H, CH), 12.79 (br s, 1H,
CONHCO) ppm. MS (ESIþ): m/z (%) ¼ 260 ([M þ H]þ, 60), 164 (100).
Anal. calcd for C10H4F3NO2S: C, 46.34%; H, 1.56%; N, 5.40%; found C,
46.43%; H, 1.70%; N, 5.65%.
5.1. Chemistry
5.1.2.2. (Z)-5-(3,4,5-Trimethoxybenzylidene)thiazolidine-2,4-dione
(4). The precipitate was recrystallized from ethyl acetate to obtain
compound 4 as a pale yellow crystalline solid. Yield: 39.0%. Melting
range 170–172 ꢁC (184–186 ꢁC [21]). IR (KBr): nmax ¼ 3165, 2371,
1742,1708,1609,1508,1313,1252,1132, 999, 621, 556 cmꢀ1. 1H NMR
5.1.1. General procedure for microwave-assisted synthesis of (Z)-2-
thioxo-5-benzylidenethiazolidin-4-ones 1 and 2
To
a suspension of 2-thioxo-thiazolidin-4-one (0.200 g,
1.50 mmol, 1.0 equiv) in dry ethanol (5 mL), the corresponding
aldehyde (1.50 mmol, 1.0 equiv), a catalytic amounts of piperidine
(0.150 mmol, 0.1 equiv) and glacial acetic acid (0.150 mmol,
(DMSO-d6):
d
¼ 3.73 (s, 3H, 4-OCH3), 3.83 (s, 6H, 3-OCH3, 5-OCH3),
6.92 (s, 2H, Ar-H2, H6), 7.75 (s, 1H, CH), 12.58 (s, 1H, CONHCO) ppm.

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T. Tomasi´c et al. / European Journal of Medicinal Chemistry 45 (2010) 1667–1672
1671
MS (ESIꢀ): m/z (%) ¼ 294 ([M ꢀ H]ꢀ, 100). HRMS (ESIꢀ): calcd for
100 ꢁC and the mixture stirred at 100 ꢁC for 1 h. After cooling to
room temperature, the product was filtered off and washed with
petroleum ether. Compound 9 was obtained as a white crystalline
solid. Yield: 68.0%. Melting range 320–325 ꢁC. IR (KBr): nmax ¼ 3524,
3256, 3124, 3092, 2819, 1770, 1673, 1583, 1516, 1439, 1348, 1300,
C13H14NO5S: 294.0436, found: 294.0440.
5.1.3. General procedure for microwave-assisted synthesis of (Z)-2-
(4-oxo-2-thioxo-5-benzylidenethiazolidin-3-yl)acetic acids 5 and 6
To a suspension of rhodanine-3-acetic acid (0.200 g, 1.05 mmol,
1.0 equiv) in dry ethanol (5 mL), aldehyde (1.05 mmol, 1.0 equiv),
a catalytic amounts of piperidine (0.105 mmol, 0.1 equiv) and
glacial acetic acid (0.105 mmol, 0.1 equiv) were added. The reaction
mixture was heated with microwave irradiation (max. power of
30 W) to 110 ꢁC and the heating continued for 40 min to maintain
the temperature. The pressure limit was set at 18 bar. The reaction
vessel was cooled in an ice-bath, the precipitate was filtered off,
washed with ice-cooled ethanol and dried under vacuum.
1040 cmꢀ1. 1H NMR (DMSO-d6):
d
¼ 7.36–7.45 (m, 1H, Ar-H), 7.71–
7.80 (m, 1H, Ar-H), 8.17 (s, 1H, CH),11.33 (s,1H, NH),11.50 (s,1H, NH)
ppm. MS (ESIꢀ): m/z (%) ¼ 269 ([M ꢀ H]ꢀ, 83), 249 (100). Anal.
calcd for C11H5F3N2O3: C, 48.90%; H, 1.87%; N, 10.37%; found C,
48.89%; H, 2.09%; N, 10.32%.
5.2. Determination of antibacterial activity
The susceptibilities of four standard strains (E. coli ATCC 25922,
P. aeruginosa ATCC 27853, S. aureus ATCC 29213 and E. faecalis ATCC
29212) to compounds 1–11 were determined by the macrodilution
method. Compounds (10 mg) were dissolved in 5 mL of DMSO to
give a stock solution of 2 mg/mL concentration. Working solutions
were made by serially diluting stock solution in cation-adjusted
Mueller–Hinton broth (CAMHB), as described by Amsterdam and
Barry [22,23]. 34 mL of CAMHB was added to 5 mL stock to give
5.1.3.1. (Z)-2-(4-Oxo-2-thioxo-5-(2,3,4-trifluorobenzylidene)thiazoli-
din-3-yl)acetic acid (5). Yellow crystalline solid. Yield: 40.5%.
Melting range 169–171 ꢁC. IR (KBr): nmax ¼ 3431, 3033, 2982, 1721,
1634,1614, 1591, 1512, 1468, 1436, 1411, 1374, 1329, 1308, 1249, 1215,
1118, 1060, 1042, 985, 945, 904, 796, 749, 724, 664, 619, 600, 564,
550, 531, 501, 476 cmꢀ1. 1H NMR (DMSO-d6):
d
¼ 4.72 (s, 2H, CH2-
COOH), 7.45–7.58 (m, 2H, Ar-H5, H6), 7.76 (s, 1H, CH) ppm, broad
signal for COOH not seen. MS (EI): m/z (%) ¼ 333 (M, 41), 188 (100).
HRMS (EI): calcd for C12H6F3NO3S2: 332.9741, found: 332.9745.
a 256
was further serially diluted to provide 14 dilutions down to the
lowest concentration of 0.031 g/mL [22] that were stored frozen
mg/mL concentration, then filter sterilized. The compound
m
for a maximum of two weeks. 0.5 mL of these dilutions were
pipetted into 13 ꢃ 100 mm screw cap tubes just prior to bacterial
inoculation.
Four colonies of a fresh overnight culture on a non-selective agar
plate were inoculated into saline. The turbidity was adjusted to
match that of 0.5 McFarland standard (approx. 108 CFU/mL). A
portion of the standardized suspension was diluted approx. 1:1000
(to 105 CFU/mL). 0.5 mL of this dilution was then added within
30 min to each tube containing 0.5 mL of test compound diluted in
CAMHB and incubated at 35 ꢁC for 18–24 h. Dilutions from 0.016 to
5.1.3.2. (Z)-2-(4-Oxo-2-thioxo-5-(3,4,5-trimethoxybenzylidene)thi-
azolidin-3-yl)acetic acid (6). Dark yellow crystalline solid. Yield:
35.4%. Melting range 217–219 ꢁC. IR (KBr): nmax ¼ 3446, 1706, 1603,
1577, 1503, 1465, 1417, 1322, 1250, 1149, 1128, 1053, 1003, 830, 764,
739, 629, 575 cmꢀ1. 1H NMR (DMSO-d6):
d
¼ 3.76 (s, 3H, 4-OCH3),
3.86 (s, 6H, 3-OCH3, 5-OCH3), 4.66 (s, 2H, CH2-COOH), 6.99 (s, 2H,
Ar-H2, H6), 7.82 (s,1H, CH) ppm, broad signal for COOH not seen. MS
(ESIꢀ): m/z (%) ¼ 368 ([M ꢀ H]ꢀ, 20), 324 (100). HRMS (ESIꢀ): calcd
for C15H14NO6S2: 368.0263, found: 368.0270.
128
mg/mL of the compound were achieved on addition of the
5.1.4. General procedure for the synthesis of compounds 7 and 8
inoculum. Broth not containing any compound was inoculated as
a growth control. The lowest concentration of antimicrobial agent
that resulted in complete inhibition of visible growth was the
minimal inhibitory concentration (MIC). In the case of agar dilution,
A
suspension of 3,4,5-trimethoxybenzaldehyde (200 mg,
1.02 mmol) and barbituric or 2-thiobarbituric acid (1.02 mmol) in
water (20 mL) was refluxed for 10 h, after which the reaction
mixture was cooled to room temperature, the product filtered off
and washed with diethyl ether.
an inoculum of 106 CFU/ml was prepared. 20 l, containing 104 CFU/
m
spot was inoculated on sheep blood agar plates, each containing
different concentrations of test compounds, and incubated at 35 ꢁC
for 18–24 h in CO2 atmosphere. Quality control of the methods was
performed by testing S. aureus ATCC 29213 and gentamicin. Dilu-
tions of antibiotic were made in the same way as for tested
compound and the MICs obtained were in the range proposed by
the Clinical Laboratory Standards Institute [24].
5.1.4.1. 2-Thioxo-5-(3,4,5-trimethoxybenzylidene)dihydropyrimidine-
4,6(1H,5H)-dione (7). Orange crystalline solid. Yield: 84.1%. Melting
range 238–250 ꢁC. IR (KBr): nmax ¼ 3524, 3104, 3062, 2994, 2907,
1646, 1573, 1556, 1539, 1495, 1423, 1302, 1123, 998 cmꢀ1. 1H NMR
(DMSO-d6):
d
¼ 3.81 (s, 3H, 4-OCH3), 3.84 (s, 6H, 3-OCH3, 5-OCH3),
7.89 (s, 2H, Ar-H2, H6), 8.28 (s,1H, CH),12.31 (s,1H, NH),12.43 (s,1H,
NH) ppm. MS (ESIþ): m/z (%) ¼ 345 ([M þ Na]þ, 2), 323 ([M þ H]þ,
29), 77 (100). HRMS (ESIþ): calcd for C14H15N2O5S: 323.0702, found
323.0697.
Acknowledgements
This work was supported by the Slovenian Research Agency
(P1-0208, P1-0230, J1-6693). The authors thank Professor Dr. Roger
Pain for critical reading of the manuscript.
5.1.4.2. 5-(3,4,5-Trimethoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-
trione (8). Yellow crystalline solid. Yield: 78.3%. Melting range 274–
276 ꢁC. IR (KBr): nmax ¼ 3506, 3241, 3139, 3016, 2839, 1753, 1734,
1667, 1654, 1550, 1507, 1418, 1301, 1125, 996 cmꢀ1. 1H NMR (DMSO-
References
d6):
d
¼ 3.80 (s, 3H, 4-OCH3), 3.83(s, 6H, 3-OCH3, 5-OCH3), 7.84 (s, 2H,
Ar-H2, H6), 8.26 (s, 1H, CH), 11.22 (s, 1H, NH), 11.53 (s, 1H, NH) ppm.
MS (ESIþ): m/z (%) ¼ 329 ([M þ Na]þ, 5), 307 ([M þ H]þ, 38), 77 (100).
HRMS (ESIþ): calcd. for C14H15N2O6: 307.0930, found 307.0919.
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