5-benzylidenethiazolidin-4-ones as multitarget inhibitors of bacterial Mur ligases

Article abstract of DOI:10.1002/cmdc.200900449

Mur ligases participate in the intracellular path of bacterial peptidoglycan biosynthesis and constitute attractive, although so far underexploited, targets for antibacterial drug discovery. A series of hydroxy-substituted 5-benzylidenethiazolidin-4-ones

Full text of DOI:10.1002/cmdc.200900449

MED
DOI: 10.1002/cmdc.200900449
5-Benzylidenethiazolidin-4-ones as Multitarget Inhibitors
of Bacterial Mur Ligases
[a]
[a]
ˇ ´ [a]
ˇ [a]
ˇ ˇ [b]
Tihomir Tomasic, Nace Zidar, Andreja Kovac, Samo Turk, Mihael Simcic,
[c]
[d]
[b]
ˇ [e]
ˇ
Didier Blanot, Manica Mꢀller-Premru, Metka Filipic, Simona Golic Grdadolnik,
Anamarija Zega,[a] Marko Anderluh,[a] Stanislav Gobec,[a] Danijel Kikelj,*[a] and Lucija Peterlin
[a]
ˇ ˇ
Masic*
Mur ligases participate in the intracellular path of bacterial
peptidoglycan biosynthesis and constitute attractive, although
so far underexploited, targets for antibacterial drug discovery.
A series of hydroxy-substituted 5-benzylidenethiazolidin-4-ones
were synthesized and tested as inhibitors of Mur ligases. The
most potent compound 5a was active against MurD–F with
IC50 values between 2 and 6 mm, making it a promising multi-
target inhibitor of Mur ligases. Antibacterial activity against dif-
ferent strains, inhibitory activity against protein kinases, muta-
genicity and genotoxicity of 5a were also investigated, and ki-
netic and NMR studies were conducted.
Introduction
The search for new antibacterial agents with novel mecha-
nisms of action is one of the key strategies emerging for com-
bating drug-resistant bacteria.[1] The biochemical machinery in-
volved in peptidoglycan biosynthesis remains a viable source
of novel, previously unexploited targets for antibacterial
drugs.[2] A large number of important antibiotic classes act by
inhibiting extracellular steps of peptidoglycan biosynthesis.
Peptidoglycan is an essential cell-wall polymer unique to pro-
karyotic cells that provides the rigidity, flexibility and strength
required for bacterial cells to grow and divide, while with-
standing high internal osmotic pressure.[3] Recently, there has
been an increased interest in exploiting the enzymes involved
in the early intracellular steps of cytoplasmic peptidoglycan
precursor biosynthesis for antibacterial drug discovery. Among
them are the ATP-dependent Mur ligases (MurC–F) that cata-
lyze a series of reactions leading to UDP-MurNAc-pentapeptide
(Park’s nucleotide) by sequentially adding l-Ala (MurC), d-Glu
(MurD), l-Lys or meso-diaminopimelic acid (MurE) and d-Ala–
d-Ala dipeptide (MurF) to the starting MurC substrate UDP-
MurNAc. The fact that Mur enzymes are vital for the survival of
bacteria makes them promising targets for antibacterial drug
discovery.[4]
Figure 1. Catalytic mechanism of Mur ligases.
UDP substrate and finally the condensing amino acid or dipep-
tide.[7] All the Mur ligases share the same three-domain topolo-
gy, with the N-terminal and central domains binding UDP pre-
cursor and ATP respectively, while the C-terminal domain binds
the condensing amino acid or dipeptide residue.[3,8]
ˇ ´
ˇ
[a] T. Tomasic, N. Zidar, Dr. A. Kovac, S. Turk, Prof. Dr. A. Zega,
Prof. Dr. M. Anderluh, Prof. Dr. S. Gobec, Prof. Dr. D. Kikelj,
ˇ ˇ
Prof. Dr. L. Peterlin Masic
University of Ljubljana, Faculty of Pharmacy
ˇ
ˇ
Askerceva 7, 1000 Ljubljana (Slovenia)
Fax: (+386)1-425-8031
E-mail: lucija.peterlin@ffa.uni-lj.si
danijel.kikelj@ffa.uni-lj.si
ˇ ˇ
[b] M. Simcic, Prof. Dr. S. G. Grdadolnik
Mur ligases catalyze the formation of an amide or peptide
bond between the UDP substrate and the condensing amino
acid. Initially, the terminal carboxyl group of the UDP substrate
is activated by ATP phosphorylation, resulting in the formation
of an acylphosphate intermediate that is subsequently at-
tacked by the amino group of the incoming amino acid or di-
peptide. The tetrahedral high-energy intermediate formed col-
lapses with elimination of inorganic phosphate and concomi-
tant formation of the amide or peptide bond (Figure 1).[3,5,6]
Moreover, based on biochemical studies of MurC and MurF,
Mur ligases exhibit an ordered kinetic mechanism in which ATP
binds first to the free enzyme, followed by the corresponding
Laboratory of Biomolecular Structure, National Institute of Chemistry
Hajdrihova 19, 1001 Ljubljana (Slovenia)
[c] Dr. D. Blanot
Enveloppes Bactꢁriennes et Antibiotiques, IBBMC, UMR 8619, CNRS
Universitꢁ Paris-Sud, 91405 Orsay (France)
[d] Prof. Dr. M. Mꢀller-Premru
Institute of Microbiology and Immunology, Medical Faculty
University of Ljubljana, Zaloska 4, 1105 Ljubljana (Slovenia)
ˇ
ˇ
[e] Prof. Dr. M. Filipic
National Institute of Biology, Department of Genetic Toxicology and
Cancer Biology, Vecna pot 111, 1000 Ljubljana (Slovenia)
ˇ
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900449.
286
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ChemMedChem 2010, 5, 286 – 295

Multitarget Inhibitors of Bacterial Mur Ligases
There have been several attempts to design inhibitors of the
Mur ligases by mimicking substrates, products or tetrahedral
intermediates.[4,9] The crystal structures of apoenzymes and en-
zymes in complex with their substrates, products or inhibi-
tors,[5,10] have enabled structure-based design of inhibitors and
recently led to the discovery of diverse Mur ligase inhibitors by
virtual screening of compound libraries.[11]
ligases. Indeed, some N-acylhydrazones incorporating the
2,3,4-trihydroxyphenyl group have already shown inhibition of
MurC and MurD.[17] For these reasons, we synthesized a series
of hydroxy-substituted 5-benzylidenethiazolidin-4-one deriva-
tives and assayed them as inhibitors of the MurC–F enzymes.
It is now widely recognized that compounds designed to
bind to more than one target (designed multitarget ligands)
can be more therapeutically beneficial than highly target-spe-
cific ligands. Designing multitarget ligands is usually a de-
manding challenge, with the need to appropriately balance af-
finities for different targets while preserving their drug-like
properties. Nevertheless, designed multitarget ligands have
rapidly become a paradigm in drug discovery in different ther-
apeutic areas, such as cancer, hypertension, allergic, psychiatric
and metabolic diseases.[12a] Using network models of antimicro-
bial drugs, Csermely et al.[12b] showed that multitarget attacks
perturb complex systems more effectively than focused at-
tacks, even if the number of targeted interactions is the same.
Consequently, we believe that inhibition of multiple Mur ligas-
es would result in potent antibacterial activity. Moreover, it
should be beneficial in combating the proliferation of bacterial
resistance caused by mutation,[2] a major advantage for multi-
target inhibition in bacteria. Since Mur ligases share the same
catalytic mechanism and possess several conserved residues in
their active sites, particularly in the ATP-binding site,[13] it
should be possible to inhibit all Mur ligases (MurC–F) with a
single molecular entity.[4b] Indeed, Mansour et al. recently re-
ported naphthyl tetronic acids as multitarget inhibitors of bac-
terial peptidoglycan biosynthesis that inhibit MurB–F from
Staphylococcus aureus and Escherichia coli, and MurA from
E. coli.[12c] There are at least three possible ways by which inhib-
ition of multiple Mur ligases could be achieved: 1) by ATP-com-
petitive inhibition, 2) by an inhibitor mimicking the UDP-
MurNAc moiety of the UDP substrate and 3) by binding at an
allosteric site common to all four Mur ligases.
Results and Discussion
Chemistry
The thiazolidine-4-one-based compounds 5a–d, 6a–c and 7b–
d (Table 1) were synthesized via a Knoevenagel condensation
between the 2-thioxothiazolidin-4-one (rhodanine, 1), thiazoli-
Table 1. Structures of compounds 5a–d, 6a–c and 7b–d.
Compd
X
R1
R2
R3
R4
R5
5a
5b
5c
5d
6a
6b
6c
7b
7c
7d
S
S
S
S
O
O
O
S
S
S
OH
OH
H
OH
OH
OH
H
OH
H
OH
OH
H
OH
H
OH
H
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
CH2COOH
CH2COOH
CH2COOH
OH
H
OH
dine-2,4-dione (2) or 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic
acid (rhodanine-N-acetic acid, 3) and the corresponding ben-
zaldehyde 4a–d (Scheme 1). The target compounds were pre-
As part of our efforts to discover new small-molecule inhibi-
tors of the intracellular steps of peptidoglycan biosynthesis,
we recently reported glutamic-acid-based selective MurD in-
hibitors containing a rhodanine moiety that act as MurD prod-
uct mimics.[14] Since compounds bearing a rhodanine ring
often exhibit antibacterial activity due to inhibition of bacterial
enzymes,[15] we have further explored rhodanine-based inhibi-
tors of Mur ligases. The rhodanine ring has already been em-
ployed as a diphosphate surrogate or phosphate mimetic,[16]
which makes it a convenient scaffold for the design of Mur
ligase inhibitors. Since the ATP-binding and UDP-binding pock-
ets of MurC–F active sites are the most highly conserved, and
since visual inspection of the crystal structures suggests several
possible hydrogen-bond interactions with the substituted rho-
danine scaffold, compounds based on the 5-benzylidenerhoda-
nine scaffold could bind to either the ATP- or UDP-binding
pocket of the Mur enzymes, which is expected to result in mul-
tiple Mur ligase inhibition. Attachment of hydroxy groups to
the benzylidene moiety would offer potential hydrogen-bond
formation and should improve inhibitory activity against Mur
Scheme 1. Reagents and conditions: a) piperidine, AcOH, EtOH, 30 W, 18 bar,
1408C, 30 min for 5a–d and 6a–c or 1108C, 40 min for 7b–d.
pared under a variety of reaction conditions under microwave
irradiation, using piperidine and glacial acetic acid as catalysts.
Compounds 5a–d and 6a–c were obtained in good yields by
heating the reaction mixtures at 1408C for 30 min. In contrast,
the use of the same conditions for the synthesis of 7b–d did
not give the target compounds. Nevertheless, by varying the
temperature and reaction time, 7b–d could be synthesized by
heating the reactants at 1108C for 40 min, but pure reaction
products could only be isolated in low yields. In theory, there
are two possible geometrical isomers (E/Z) for 5-benzylidene-
1
thiazolidin-4-ones; H NMR spectra of compounds 5a–d, 6a–c
and 7b–d show only one signal for the methyne proton in the
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287

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L. P. Masic, D. Kikelj et al.
MED
range 7.47 to 8.01 ppm, at lower field values than those ex-
pected for the E isomers, which strongly indicates that the
compounds have the Z configuration. The latter has been re-
ported as thermodynamically more stable than the E configura-
tion.[18a,b] The Z configuration of compounds 5a–d, 6a–c and
7b–d can also be inferred from the X-ray crystal structures[18c]
and from the splitting pattern and coupling constants of the
use the Hantzsch ester method, or 2) prevent phenolate gener-
ation and use lithium borohydride reduction. The treatment of
5a with acetic anhydride in the presence of potassium carbon-
ate in diethyl ether gave compound 10, which was successfully
reduced to compound 11 in moderate yield using the
Hantzsch ester method. Finally, hydrazinolysis[22] of the ester
groups of 11 gave the target compound 12.
1
proton signal in H-coupled 13C NMR spectrum of similar com-
pounds arising due to interaction with the C=O group of the
rhodanine system.[18d] 2-Thioxodihydropyrimidine-4,6(1H,5H)-
dione (9), a thiobarbituric acid derivative, was prepared by re-
fluxing thiobarbituric acid (8) and 2,3,4-trihydroxybenzalde-
hyde (4a) in water overnight (Scheme 2), since application of
the procedure described above proved unsuccessful.
Biology
Following our idea to combine rhodanine with a hydroxyphen-
yl moiety, we first synthesized compound 5a and tested its
ability to inhibit Mur ligases (MurC, MurD and MurF from E. coli
and MurE from S. aureus) using the malachite green assay for
detecting orthophosphate generated during the enzymatic re-
action.[23] To exclude possible nonspecific (promiscuous) inhibi-
tion, the compounds were tested in the presence of detergent
(Triton X-114, 0.005%).[24] Compound 5a inhibited MurD and
MurF with an IC50 value of 2 mm, and MurE with IC50 of 6 mm. It
thus possessed well-balanced inhibitory activity against en-
zymes MurD–F. Inspired by this promising result, we synthe-
sized a series of analogues by varying the number and position
of hydroxy groups on the benzylidene ring (compounds 5b–d)
in order to obtain some insight into the structure–activity rela-
tionships (SAR). The similarity of thiazolidine-2,4-dione (2) to
rhodanine (1) prompted us to synthesize analogues 6a–c. The
SAR were further exploited by the synthesis of compounds
bearing the N-substituted rhodanine ring (7b–d) or thiobarbi-
turic acid moiety (compound 9).
Scheme 2. Reagents and conditions: a) H2O, reflux, o/n.
Only a few methods have been reported for reducing the
exocyclic double bond in 5-benzylidenethiazolidin-4-ones. Lith-
ium borohydride can be used as a reducing agent, however,
long reaction times and poor conversion have been reported
for phenol-containing compounds, because the phenolates
generated under the reaction conditions were insoluble in the
reaction media.[19] We therefore considered reduction using di-
Compounds 5b–d, 6a–c and 7b–d were first tested for in-
hibition of MurE ligase; those compounds inhibiting MurE
were further tested on the other Mur ligases. The results are
presented in Table 2 as percent inhibition of the enzymes in
the presence of 100 or 500 mm of the tested compound, or IC50
values for the most active compounds.
ethyl
2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate
(Hantzsch ester)[20] and activated silica gel.[21] Our first attempts
to reduce the exocyclic double bond of 5a were unsuccessful
due to the insolubility of 5a in toluene, even at elevated tem-
peratures. To increase its solubility, different solvents (THF, DMF
and EtOAc) were used instead of toluene but these also failed
to yield the desired product. Therefore, we decided to acety-
late the hydroxy groups of 5a (compound 10, Scheme 3) in
order to 1) increase the solubility of 5a in toluene and then
Some SAR can be deduced from structural analysis of the
tested compounds and their inhibitory activities: 1) The most
potent multitarget MurD–F inhibitors 5a and 5d bear a trihy-
Table 2. Inhibitory activities of compounds against Mur ligases.
Compd
Inhibition[a] [%] or IC50 [mm]
MurC
MurD
MurE
MurF
5a
5b
5c
5d
6a
6b
6c
7b
7c
7d
9
52%
nt
nt
41%
56%
nt
nt
32%
nt
42%
nt
nt
2 mm
nt
nt
8 mm
59%
nt
nt
21%
nt
46%
12%
10%
40%
6 mm
14%
11%
9 mm
3 mm
15%
14%
39 mm
0%
19 mm
nt
24%[b]
33%[b]
2 mm
nt
nt
4 mm
3 mm
nt
nt
14 mm
nt
6 mm
20%
11 %
38%
11
12
nt
Scheme 3. Reagents and conditions: a) Ac2O, K2CO3, Et2O, 08C!RT, 15 h;
b) diethyl 2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate, silica gel 60,
toluene, 1008C, 24 h; c) NH2NH2·H2O, CH3CN, RT, 20 min.
[a] Enzyme inhibition (%) in the presence of 100 mm concentration of test
compound. nt, not tested. [b] Compound tested at 500 mm concentration.
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Multitarget Inhibitors of Bacterial Mur Ligases
droxyphenyl-substituted rhodanine moiety. The replacement of
the rhodanine ring by the thiazolidine-2,4-dione (6a) or rhoda-
nine-N-acetic acid (7d) moiety led to decreased inhibition of
MurD, while MurE and MurF inhibition remained in the low mi-
cromolar range (IC50 =3–19 mm). 2) In the case of MurE, com-
pounds bearing the dihydroxyphenyl-substituted rhodanine
(5b, 5c) or thiazolidine-2,4-dione moiety (6b, 6c) were devoid
of inhibitory activity, while 2,4-dihydroxybenzylidenerhoda-
nine-N-acetic acid (7b) inhibited MurE with an IC50 value of
39 mm.
Compounds 5a, 6a and 7d were tested for their antibacteri-
al activity against two Gram-negative (E. coli ATCC 25922 and
Pseudomonas aeruginosa ATCC 27853) and two Gram-positive
(S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212)
bacterial strains using the macrodilution method (Table 3) and
several protein kinases, raised questions regarding the mecha-
nism-of-action of these two compounds.
A literature survey suggested that 5-benzylidenerhodanines
could act as substrates for reversible Michael-type 1,4-conjuga-
tive addition of nucleophilic cysteine residues of proteins.[15,26]
However, such a mechanism is contradicted by the fact that di-
hydroxybenzylidene-substituted compounds 5b and 5c, analo-
gous to trihydroxybenzylidene compound 5a, were found to
be inactive. Moreover, thiobarbituric acid derivative 9, which
could also act as Michael acceptor, was only very weakly active
against MurD and MurF, with 12% and 20% inhibition at
100 mm. If these compounds act via the above mechanism, the
inhibition of E. coli MurD, which was shown to possess a reac-
tive cysteine in its active site,[27] would be stronger. In order to
obtain further insight into the role of the exocyclic double
bond, 5a was reduced to give the racemic compound 12
(Scheme 3) that displayed some inhibition of MurD and MurF
(inhibition=38–40% at 100 mm), while inhibition of MurE was
practically lost, even at higher concentrations of compound 12
(inhibition=33% at 500 mm). We believe that the observed de-
crease in inhibitory potency of 12 as compared to that of 5a is
exclusively due to the flexibility of the molecule and to the
loss of conjugation between the rhodanine and the phenyl
ring.
Table 3. MIC and MBC values of selected compounds against selected
bacterial strains.[a]
Compd
MIC [mgmLꢁ1
]
E. coli
P. aeruginosa
E. faecalis
S. aureus
5a
6a
7d
>128
>128
>128
128[b]
>128
>128
>128
>128
128
>128
128
128
2,3,4-Trihydroxyphenyl and 2,4,5-trihydroxyphenyl moieties
have recently been recognized as functional groups that are
not correlated with false-positive activity in high-throughput
screening of chemical libraries against b-lactamase.[28] To check
whether the presence of these functionalities might be the
sole reason for the inhibition of Mur enzymes, we tested the
susceptibility of MurD to pyrogallol (benzene-1,2,3-triol) and
found that it was inactive at 100 mm concentration. The ab-
sence of potent inhibition of Mur enzymes by compounds 9
and 12, which also possess the trihydroxyphenyl motif, further
excludes inhibition due to the presence of the trihydroxyphen-
yl moiety alone. However, a weaker inhibition by 11 as com-
pared to 12 lends support to the role of the phenol groups.
Furthermore, the inactivity of (Z)-5-(ethoxymethylidene)rhoda-
nine in the MurD inhibition assay (inhibition=1% at 100 mm)
also indicates that the rhodanine moiety alone cannot be re-
sponsible for inhibiting Mur enzymes.
[a] E. coli ATCC 25922; P. aeruginosa ATCC 27853; E. faecalis ATCC 29212;
S. aureus ATCC 29213. [b] Minimal bactericidal concentration (MBC) for
compound 5a against P. aeruginosa ATCC 27853 was 128 mgmLꢁ1
.
were found to be weak inhibitors of bacterial growth in vitro.
According to the bacterial reverse mutation assay with Salmo-
nella typhimurium (TA98 and TA100), compound 5a can be
considered as a bacterial nonmutagen (see the Experimental
Section and table S1 in the Supporting Information for details).
Compound 5a was also not genotoxic in human hepatoma
HepG2 cells at noncytotoxic concentrations (see the Experi-
mental Section and table S2 in the Supporting Information for
details).
The inhibition of multiple Mur ligases by thiazolidin-4-ones
5a, 5d, 6a, 7b and 7d, together with their, albeit weak, anti-
bacterial activity, stimulated us to investigate the mode-of-
action and selectivity of these Mur inhibitors. Following our hy-
pothesis of putative ATP-competitive binding of these Mur
ligase inhibitors, we evaluated the selectivity of compounds
5a and 6a against a diverse panel of 76 protein kinases using
the gold standard radioactive (33P-ATP) filter-binding assay.[25]
This revealed that both compounds are also inhibitors of some
protein kinases (see table S3 in the Supporting Information for
details).
Next we investigated the mode-of-action of compound 5a
with a steady-state kinetic study on MurD ligase. Kinetic analy-
sis revealed that compound 5a acts as a noncompetitive MurD
inhibitor with respect to all three substrates, namely ATP, d-Glu
and UDP-MurNAc-l-Ala (UMA), with Ki values of 2.2ꢀ0.12 mm,
2.2ꢀ0.10 mm and 1.8ꢀ0.12 mm, respectively (Figure 2). Due to
known promiscuity of the rhodanine scaffold,[30] to support the
specific action of the inhibitors by structural data, an NMR
study of the interactions of 5a with MurD was performed. This
study showed that 5a mainly interacts with the residues flank-
ing the UMA-binding site, while binding to the ATP-binding
site or other parts of the protein was not observed. These con-
clusions are based on monitoring the 1H/13C chemical shift
changes of MurD selectively labeled with 13C at the methyl
groups of Ile, Val, and Leu[31] upon binding of 5a, UMA, adenyl-
yl 5’-(b,g-methylene)diphosphonate (AMPPCP), and 6-butoxy-
Compounds 5a and 6a were also tested against d-ala-
nine:d-alanine ligase (Ddl from E. coli), another ATP-dependent
enzyme responsible for supplying MurF ligase with the sub-
strate d-alanyl-d-alanine.[4] They were found to be inactive at
100 mm (inhibition=19 and 13%, respectively). Different activi-
ties against various ATP-dependent enzymes, inactivity against
Ddl and some protein kinases, and inhibition of MurD–F and
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289

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L. P. Masic, D. Kikelj et al.
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Figure 2. Kinetic analysis of MurD (E. coli) inhibition by compound 5a. The reciprocal of the initial velocity was plotted according to Dixon vs the inhibitor
*
*
concentration (compound 5a) at different concentrations of one substrate and fixed concentrations of the other two: a) differing ATP (25 ( ), 100 ( ), 400
!
!
!
*
* *
), 800 mm ( ) ATP; 80 mm UMA; 100 mm d-Glu); b) differing d-Glu (25 ( ), 100 ( ), 400 mm ( ) d-Glu; 80 mm UMA, 400 mm ATP); c) differing UMA (10 ( ), 20
(
(
!
!
*
), 40 ( ), 80 mm ( ) UMA; 100 mm d-Glu, 400 mm ATP). Concentrations of 5a were 0, 0.5, 0.9, 1.5, 2.5 and 4.0 mm. Data were fitted to competitive, noncom-
petitive and uncompetitive inhibition models using SigmaPlot 11.0 software[29] and Ki values for the best fitted model were calculated.
naphthalene-N-sulfonyl-d-glutamic acid. The binding mode of
several naphthalene-N-sulfonyl-d-Glu derivatives as novel in-
hibitors of MurD has recently been determined by X-ray and
NMR.[10f–g,32] The binding interactions of ADP and UMA with
MurD are known from the X-ray co-crystal structures.[5] Several
pronounced effects on the MurD methyl chemical shifts are
observed in 1H/13C HSQC spectra upon binding of these li-
gands (see figure S1 in the Supporting Information). The UMA
binding has the most pronounced influence on MurD resonan-
ces. The effect of 5a is more local and limited to the signals,
which are also influenced by UMA binding. Apparently 5a in-
teracts with part of the UMA-binding site in a way that does
not prevent the binding of UMA. Several resonances with pro-
nounced chemical-shift perturbations induced by binding of
5a are strongly influenced also by UMA binding, while the
effect of 6-butoxy-naphtalene-N-sulfonyl-d-Glu is strikingly
lower. This observation indicates that 5a extends towards the
uracil-binding pocket, which is occupied by the C6 substituents
of naphthalene-N-sulfonyl-d-Glu derivatives.[10f,g,32] Due to
highly conserved active sites of MurD–F, and similar structures
of inhibitors 5a, 5d, 6a, 7b and 7d, we hypothesize that
these compounds bind to the same regions of MurD–F.
Conclusions
To conclude, the 5-(trihydroxybenzylidene)rhodanines 5a and
5d were shown to be inhibitors of multiple MurD–F ligases
with well-balanced IC50 values in the low micromolar range.
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Multitarget Inhibitors of Bacterial Mur Ligases
1257, 1196, 1070, 1045, 1014, 967, 776, 722, 691, 636, 562, 541,
477 cmꢁ1; MS (ESI+): m/z (%): 270 (70) [M+H]+, 214 (100); Anal.
calcd for C10H7NO4S2: C, 44.60; H, 2.62; N, 5.20; found: C, 44.70; H,
2.85; N, 5.10.
The most potent compound 5a inhibited MurD and MurF with
IC50 values of 2 mm, and MurE with an IC50 value of 6 mm. In
contrast, the N-substituted (Z)-5-(di-/tri-hydroxybenzylidene)r-
hodanines 7b and 7d strongly inhibited only MurE and MurF
ligases. The molecular interactions of (Z)-5-(hydroxybenzylide-
ne)rhodanines and (Z)-5-(hydroxybenzylidene)thiazolidine-2,4-
diones with the MurD–F enzymes are currently the subject of
further investigation using NMR and X-ray crystallography to
obtain further insight into their inhibition mechanism. The ob-
served inhibition of different Mur enzymes, involved in the bio-
synthesis of bacterial peptidoglycan, makes these compounds
interesting leads in the search for multitarget antibacterial
agents.
(Z)-5-(2,4-Dihydroxybenzylidene)-2-thioxothiazolidin-4-one (5b):
Purification by column chromatography (CH2Cl2/CH3OH; 20:1) gave
compound 5b as a brown crystalline solid (0.209 g, 55%): Rf =0.38
(CH2Cl2/MeOH/CH3COOH; 9:1:0.1); mp: >3008C (literature value,[34]
1
162–1638C); H NMR (300 MHz, [D6]DMSO): d=13.52 (br s, 1H, NH),
10.58 (s, 1H, OH), 10.27 (s, 1H, OH), 7.79 (s, 1H, CH), 7.15 (d, J=
9.3 Hz, 1H, ArH(6)), 6.43–6.40 ppm (m, 2H, ArH(3,5)); 13C NMR
(75 MHz, [D6]DMSO): d=195.8, 170.0, 162.3, 159.8, 131.0, 127.8,
119.0, 111.9, 108.8, 102.5 ppm; IR (KBr): n˜ =3413, 3067, 2845, 2364,
1690, 1637, 1617, 1560, 1516, 1466, 1335, 1272, 1187, 1110, 974,
837, 791, 754, 693, 623, 563, 513, 478 cmꢁ1; MS (EI): m/z (%): 253
(65) [M]+, 166 (100); HRMS (ESIꢁ): m/z [MꢁH]ꢁ calcd for
C10H6NO3S2: 251.9789, found: 251.9792.
Experimental Section
(Z)-5-(3,4-Dihydroxybenzylidene)-2-thioxothiazolidin-4-one (5c):
Recrystallization from MeOH gave 5c as a brown crystalline solid
(0.229 g, 60%): Rf =0.45 (CH2Cl2/MeOH/CH3COOH; 9:1:0.1); mp:
>3008C (literature value,[35] 270–2808C); 1H NMR (300 MHz,
[D6]DMSO): d=13.66 (s, 1H, NH), 9.93 (s, 1H, OH), 9.51 (s, 1H, OH),
7.47 (s, 1H, CH), 7.02–6.98 (m, 2H, ArH(2,6)), 6.88 ppm (d, 1H, ArH(5));
13C NMR (75 MHz, [D6]DMSO): d=195.4, 169.4, 149.1, 146.0, 132.8,
124.8, 124.3, 120.6, 116.6, 116.4 ppm; IR (KBr): n˜ =3445, 2054, 1677,
1640, 1611, 1584, 1527, 1435, 1288, 1259, 1166, 1117, 968, 906, 844,
758, 615, 556, 510, 484 cmꢁ1; MS (ESI+): m/z (%): 254 (100)
[M+H]+; HRMS (ESIꢁ): m/z [MꢁH]ꢁ calcd for C10H6NO3S2: 251.9789,
found: 251.9796.
Chemistry
General Methods: Chemicals were obtained from Acros, Sigma–Al-
drich and Fluka and used without further purification. Analytical
thin-layer chromatography (TLC) was performed using silica gel
60 F254 precoated plates (0.25 mm) from Merck (Germany). Flash
column chromatography was carried out on silica gel 60 (particle
size 0.040–0.063 mm; Merck, Germany). Melting points were deter-
mined on a Reichert hot-stage microscope and are uncorrected.
1H NMR and 13C NMR spectra were recorded on a Bruker AVANCE
DPX300 spectrometer at 300 MHz and 75 MHz, respectively. Samples
were prepared in CDCl3 or [D6]DMSO solution with TMS as the in-
ternal standard. IR spectra were recorded on a Perkin–Elmer
1600 FTIR spectrometer. Elemental analyses were performed on a
Perkin–Elmer C, H, N analyzer 240 C and were within ꢀ0.4% of the
theoretical values. Mass spectra were obtained using a VG-Analyti-
cal Autospec Q mass spectrometer. (Z)-5-(Ethoxymethylidene)rho-
danine was synthesized as described previously.[33]
(Z)-5-(2,4,5-Trihydroxybenzylidene)-2-thioxothiazolidin-4-one
(5d): Recrystallization from MeOH gave 5d as a red crystalline
solid (0.080 g, 20%): Rf =0.23 (CH2Cl2/CH3OH; 9:1); mp: 270–2728C;
1H NMR (300 MHz, [D6]DMSO): d=13.45 (s, 1H, NH), 10.01 (d, 2H,
J=5.1 Hz, OH), 8.93 (s, 1H, OH), 7.80 (s, 1H, CH), 6.72 (s, 1H, ArH(6)),
6.43 ppm (s, 1H, ArH(3)); 13C NMR (75 MHz, [D6]DMSO): d=194.8,
169.0, 152.8, 151.0, 138.9, 127.3, 117.1, 113.2, 109.9, 102.8 ppm; IR
(KBr): n˜ =3451, 3206, 1684, 1578, 1533, 1465, 1441, 1400, 1360,
1326, 1294, 1242, 1169, 1132, 1065, 930, 902, 853, 747, 684, 630,
557, 511 cmꢁ1; MS (ESIꢁ): m/z (%): 268 (83) [MꢁH]ꢁ, 209 (100);
HRMS (ESIꢁ): m/z [MꢁH]ꢁ calcd for C10H6NO4S2: 267.9738, found:
267.9737.
Microwave-assisted reactions were performed using a focused mi-
crowave reactor (DiscoverTM, CEM Corporation, Matthews, USA). Re-
actions were performed in septum-sealed glass vials (10 mL) which
enable high-pressure reaction conditions (20 bar). The temperature
of the reaction mixture was monitored using a calibrated infrared
temperature controller mounted under the reaction vessel. The
maximum power used was 30 W and the pressure limit was set at
18 bar, unless otherwise stated.
General procedure for microwave-assisted synthesis of 5-benzyl-
idenethiazolidine-2,4-diones (6a–c): A suspension of thiazolidine-
2,4-dione (0.200 g, 1.71 mmol, 1.0 equiv) in dry EtOH (5 mL) was
treated with aldehyde (1.71 mmol, 1.0 equiv), piperidine
(0.171 mmol, 0.1 equiv) and glacial AcOH (0.171 mmol, 0.1 equiv).
The reaction mixture was heated by microwave irradiation to
1408C and the temperature maintained for 30 min. The reaction
vessel was cooled in an ice bath; the precipitate was filtered off,
washed with ice-cold EtOH and dried in vacuo.
General procedure for microwave-assisted synthesis of 5-benzyl-
idenerhodanines (5a–d): A suspension of rhodanine (0.200 g,
1.50 mmol, 1.0 equiv) in dry EtOH (5 mL) was treated with alde-
hyde (1.50 mmol, 1.0 equiv), piperidine (0.150 mmol, 0.1 equiv) and
glacial AcOH (0.150 mmol, 0.1 equiv). The reaction mixture was
heated by microwave irradiation to 1408C and the temperature
maintained for 30 min. The reaction vessel was cooled in an ice
bath; the precipitate was filtered off, washed with ice-cold EtOH
and dried in vacuo.
(Z)-5-(2,3,4-Trihydroxybenzylidene)thiazolidine-2,4-dione
(6a):
Recrystallization from MeOH gave 6a as a brown crystalline solid
(0.344 g, 74%): Rf =0.36 (CH2Cl2/MeOH/CH3COOH; 9:1:0.1); mp:>
(Z)-5-(2,3,4-Trihydroxybenzylidene)-2-thioxothiazolidin-4-one
(5a): Recrystallization from MeOH gave 5a as a brown crystalline
solid (0.285 g, 71%): Rf =0.31 (CH2Cl2/MeOH/CH3COOH; 9:1:0.1);
1
3008C; H NMR (300 MHz, [D6]DMSO): d=12.31 (s, 1H, NH), 10.03
(s, 1H, OH), 8.74 (s, 1H, OH), 8.40 (s, 1H, -OH), 8.01 (s, 1H, CH), 6.73
(d, J=8.7 Hz, 1H, ArH(6)), 6.48 ppm (d, J=8.7 Hz,1H, ArH(5));
13C NMR (75 MHz, [D6]DMSO): d=168.3, 167.7, 149.1, 147.3, 132.8,
127.5, 119.0, 117.3, 112.6, 107.8 ppm; IR (KBr): n˜ =3436, 3050, 2789,
2043, 1775, 1729, 1673, 1640, 1624, 1590, 1511, 1481, 1384, 1347,
1311, 1256, 1159, 1052, 1023, 965, 910, 790, 692, 637, 615, 549,
498, 481 cmꢁ1; MS (ESI+): m/z (%): 254 (100) [M+H]+; HRMS
1
mp: >3008C; H NMR (300 MHz, [D6]DMSO): d=13.54 (s, 1H, NH),
10.16 (s, 1H, OH), 9.57 (s, 1H, OH), 8.77 (s, 1H, OH), 7.84 (s, 1H,
CH), 6.71 (d, J=8.7 Hz, 1H, ArH(6)), 6.51 ppm (d, J=8.7 Hz, 1H,
ArH(5)); 13C NMR (75 MHz, [D6]DMSO): d=195.6, 169.4, 150.0, 147.8,
132.9, 128.4, 120.4, 119.1, 112.7, 108.3 ppm; IR (KBr): n˜ =3552,
3409, 3088, 2841, 1695, 1621, 1570, 1509, 1445, 1391, 1326, 1301,
ChemMedChem 2010, 5, 286 – 295
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
291

ˇ ˇ
L. P. Masic, D. Kikelj et al.
MED
(ESIꢁ): m/z [MꢁH]ꢁ calcd for C10H6NO5S: 251.9967, found:
(Z)-2-(5-(2,4,5-Trihydroxybenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl)acetic acid (7d): Orange crystalline solid (0.028 g, 8%): Rf =
0.08 (CH2Cl2/MeOH/CH3COOH; 9:1:0.1); mp: 260–2628C; 1H NMR
(300 MHz, [D6]DMSO): d=13.34 (s, 1H, COOH), 10.16 (s, 1H, OH),
8.99 (s, 1H, OH), 7.99 (s, 1H, CH), 6.79 (s, 1H, ArH(6)), 6.46 (s, 1H,
ArH(3)), 4.72 ppm (s, 2H, CH2)—OH signal was not seen; 13C NMR
(75 MHz, [D6]DMSO): d=192.4, 166.8, 165.9, 153.3, 151.7, 139.1,
129.4, 113.3, 113.1, 109.9, 102.8, 44.3 ppm; IR (KBr): n˜ =3247, 1719,
1657, 1608, 1570, 1531, 1459, 1396, 1342, 1307, 1244, 1190, 1106,
1090, 1065, 989, 890, 850, 743, 599, 515 cmꢁ1; MS (ESIꢁ): m/z (%):
326 (38) [MꢁH]ꢁ, 209 (100); HRMS (ESI+): m/z [M+H]+ calcd for
C12H10NO6S2: 327.9950, found: 327.9950.
251.9964.
(Z)-5-(2,4-Dihydroxybenzylidene)thiazolidine-2,4-dione (6b): Pu-
rification by column chromatography (CH2Cl2/CH3OH; 20:1) gave
compound 6b as a brown crystalline solid (0.280 g, 69%): Rf =0.50
(CH2Cl2/MeOH/CH3COOH; 7:1:0.1); mp: 276–2788C; 1H NMR
(300 MHz, [D6]DMSO): d=12.30 (s, 1H, NH), 10.42 (s, 1H, OH), 10.14
(s, 1H, OH), 7.98 (s, 1H, CH), 7.17 (d, J=8.1 Hz, 1H, ArH(6)),
6.40 ppm (m, 2H, ArH(3,5)); 13C NMR (75 MHz, [D6]DMSO): d=167.9,
167.2, 161.2, 158.9, 129.4, 127.0, 116.4, 111.2, 107.8, 102.1 ppm; IR
(KBr): n˜ =3418, 2060, 1638, 1465, 1343, 1323, 1272, 1159, 1099,
1029, 844, 805, 697, 627, 482 cmꢁ1; MS (EI): m/z (%): 237 (70) [M]+,
166 (100); HRMS (ESIꢁ): m/z [MꢁH]ꢁ calcd for C10H6NO4S:
236.0018, found: 236.0020.
2-Thioxo-5-(2,3,4-trihydroxybenzylidene)dihydropyrimidine-
4,6(1H,5H)-dione (9): A solution of 2-thiobarbituric acid (0.100 g,
0.649 mmol)
and
2,3,4-trihydroxybenzaldehyde
(0.094 g,
(Z)-5-(3,4-Dihydroxybenzylidene)thiazolidine-2,4-dione (6c): Re-
crystallization from MeOH gave 6c as a brown crystalline solid
(0.265 g, 66%): Rf =0.57 (CH2Cl2/MeOH/CH3COOH; 7:1:0.1); mp:
>3308C (literature value,[36] 266–2688C); 1H NMR (300 MHz,
[D6]DMSO): d=12.41 (s, 1H, NH), 9.81 (s, 1H, OH), 9.43 (s, 1H, OH),
7.61 (s, 1H, CH), 7.00–6.95 (m, 2H, ArH(2,6)), 6.87 ppm (d, J=8.1 Hz,
1H, ArH(5)); 13C (75 MHz, [D6]DMSO): d=167.8, 167.2, 148.3, 145.5,
132.4, 124.0, 123.6, 118.4, 116.1, 116.0 ppm; IR (KBr): n˜ =3491, 3257,
3048, 2783, 1734, 1664, 1589, 1515, 1451, 1379, 1332, 1314, 1276,
1178, 1153, 1111, 1029, 963, 919, 859, 799, 778, 738, 695, 630, 611,
510 cmꢁ1; MS (ESI+): m/z (%): 238 (100) [M+H]+; HRMS (ESIꢁ): m/z
[MꢁH]ꢁ calcd for C10H6NO4S: 236.0018, found: 236.0016.
General procedure for microwave-assisted synthesis of 5-benzyl-
idenerhodanine-3-acetic acids (7b–d): A suspension of rhoda-
nine-3-acetic acid (0.200 g, 1.05 mmol, 1.0 equiv) in dry EtOH
(5 mL) was treated with aldehyde (1.05 mmol, 1.0 equiv), piperidine
(0.105 mmol, 0.1 equiv) and glacial AcOH (0.105 mmol, 0.1 equiv).
The reaction mixture was heated by microwave irradiation to
110 8C and the temperature maintained for 40 min. The solvent
was evaporated in vacuo and the residue purified by column chro-
matography (CH2Cl2/CH3OH; 9:1).
0.649 mmol) in H2O (15 mL) was heated at reflux overnight. The
precipitate was filtered, washed with H2O and Et2O and dried
(Na2SO4) to give 9 as a red-brown solid (0.110 g, 61%): Rf =0.10
1
(CH2Cl2/MeOH/CH3COOH; 9:1:0.1); mp: >3008C; H NMR (300 MHz,
[D6]DMSO): d=12.18–12.09 (m, 2H, NH), 10.77 (s, 1H, OH), 9.99 (s,
1H, OH), 8.84 (s, 1H, CH), 8.79 (s, 1H, OH), 8.42 (d, J=9.2 Hz, 1H,
ArH(6)), 6.43 ppm (d, J=9.2 Hz, 1H, ArH(5)); 13C NMR (75 MHz,
[D6]DMSO): d=208.7, 204.1, 189.1, 174.6, 173.2, 164.9, 138.1, 132.6,
116.1, 112.1, 100.4 ppm;; IR (KBr): n˜ =3359, 1654, 1506, 1398, 1308,
1257, 1191, 1145, 1050, 967, 804, 788, 754, 718, 568, 517, 496,
471 cmꢁ1; MS (ESIꢁ): m/z (%): 279 (83) [MꢁH]ꢁ, 309 (100) [M+K]+;
Anal. calcd for C11H8N2O5S·H2O: C, 44.29; H, 3.38; N, 9.39; found: C,
44.48; H, 3.36; N, 9.39.
(Z)-4-((4-Oxo-2-thioxothiazolidin-5-ylidene)methyl)benzene-
1,2,3-triyl triacetate (10): A stirred suspension of compound 5a
(1.60 g, 5.96 mmol) and K2CO3 (3.29 g, 23.8 mmol) in Et2O (60 mL)
was cooled to 08C and treated dropwise with Ac2O (8.45 mL,
89.3 mmol). After stirring at RT for 15 h, EtOAc (50 mL) and H2O
(50 mL) were added to the reaction mixture. The combined organic
extracts were washed successively by H2O (2ꢃ40 mL), saturated aq
NaHCO3 (2ꢃ20 mL) and brine (2ꢃ20 mL), then dried (Na2SO4), fil-
tered and concentrated in vacuo to give compound 10 as a yellow
solid (2.11 g, 89.6%): Rf =0.39 (CH2Cl2/CH3OH; 20:1); mp: >3008C;
1H NMR (300 MHz, [D6]DMSO): d=13.92 (br s, 1H, NH), 7.50 (d, J=
8.7 Hz, 1H, ArH(6)), 7.44 (d, 1H, J=8.7 Hz, ArH(5)), 7.39 (s, 1H,
CH),2.41 (s, 3H, CH3), 2.34 (s, 3H, CH3), 2.31 ppm (s, 3H, CH3);
13C NMR (75 MHz, CDCl3): d=192.2, 167.9, 167.4, 167.3, 166.5,
145.3, 143.6, 135.8, 128.0, 125.6, 125.3, 125.1, 121.5, 20.7, 20.3,
20.1 ppm; IR (KBr): n˜ =3418, 3170, 3078, 2840, 2056, 1789, 1768,
1704, 1637, 1592, 1490, 1446, 1432, 1369, 1335, 1274, 1223, 1190,
1100, 1063, 1022, 897, 850, 798, 776, 678, 664, 644, 598, 585, 550,
521, 506, 458 cmꢁ1; MS (ESIꢁ): m/z (%): 394 (100) [MꢁH]ꢁ; HRMS
(ESIꢁ): m/z [MꢁH]ꢁ calcd for C16H12NO7S2: 394.0055, found:
394.0045.
(Z)-2-(5-(2,4-Dihydroxybenzylidene)-4-oxo-2-thioxothiazolidin-3-
yl)acetic acid (7b): Brown crystalline solid (0.027 g, 8%): Rf =0.10
(CH2Cl2/MeOH/CH3COOH; 7:1:0.1); mp: 215–2178C; 1H NMR
(300 MHz, [D6]DMSO): d=13.29 (s, 1H, COOH), 10.80 (s, 1H, OH),
10.46 (s, 1H, OH), 8.00 (s, 1H, CH), 7.24 (d, J=8.6 Hz, 1H, ArH(6)),
6.45 (d, J=7.8 Hz, 2H, ArH(3,5)), 4.71 ppm (s, 2H, CH2); 13C NMR
(75 MHz, [D6]DMSO): d=193.1, 167.0, 166.3, 162.6, 159.7, 131.4,
129.9, 114.6, 111.5, 108.7, 102.1, 44.6 ppm; IR (KBr): n˜ =3220, 1724,
1679, 1614, 1579, 1514, 1467, 1438, 1398, 1322, 1286, 1252,
1191.95, 1136, 1094, 1055, 959, 853, 785, 748, 682, 636, 614, 550,
495 cmꢁ1; MS (ESIꢁ): m/z (%): 310 (100) [MꢁH]ꢁ; HRMS (ESIꢁ): m/z
[MꢁH]ꢁ calcd for C12H8NO5S2: 309.9844, found: 309.9852.
(Z)-2-(5-(3,4-Dihydroxybenzylidene)-4-oxo-2-thioxothiazolidin-3-
yl)acetic acid (7c): Pale yellow crystalline solid (0.037 g, 12%): Rf =
0.26 (CH2Cl2/MeOH/CH3COOH; 9:1:0.1); mp: 217–2198C (literature
4-((4-Oxo-2-thioxothiazolidin-5-yl)methyl)benzene-1,2,3-triyl tri-
acetate (11): A stirred suspension of 10 (0.513 g, 1.30 mmol) in tol-
uene (50 mL) was treated with diethyl 2,6-dimethyl-1,4-dihydro-3,5-
pyridinedicarboxylate (0.427 g, 1.69 mmol) and silica gel 60 (1.3 g,
1 gmmꢁ1ol), previously activated by heating at 1208C for 5 h. The
mixture was heated to 1008C for 24 h in the dark under Ar. The re-
action mixture was cooled and filtered. The filter cake was rinsed
with EtOAc. The combined filtrate and rinse were concentrated to
dryness. The residue was redissolved in EtOAc (30 mL) and washed
with aq HCl (1m, 3ꢃ30 mL) and brine (30 mL). The organic phase
was dried (Na2SO4), filtered and concentrated in vacuo. Purification
by column chromatography (CH2Cl2/CH3OH; 20:1) gave compound
11 as a yellow solid (0.286 g, 57.0%): Rf =0.41 (CH2Cl2/CH3OH;
1
value,[37] 328–3298C); H NMR (300 MHz, [D6]DMSO): d=7.64 (s, 1H,
CH), 7.05–7.00 (m, 2H, ArH(2,6)), 6.89 (d, 1H, J=8.0 Hz, ArH(5)),
4.41 ppm (s, 2H, CH2)—OH and COOH signals were not seen;
13C NMR (75 MHz, [D6]DMSO): d=192.7, 166.9, 166.5, 149.4, 145.9,
133.5, 123.9, 117.1, 116.5, 116,1, 43.1, 21.9 ppm; IR (KBr): n˜ =3430,
3200, 2509, 1696, 1563, 1452, 1407, 1365, 1304, 1283, 1203, 1102,
1051, 922, 862, 798, 671, 632, 609, 526 cmꢁ1; MS (ESIꢁ): m/z (%):
310 (45) [MꢁH]ꢁ, 165 (100); HRMS (ESIꢁ): m/z [MꢁH]ꢁ calcd for
C12H8NO5S2: 309.9844, found: 309.9850.
292
www.chemmedchem.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 286 – 295

Multitarget Inhibitors of Bacterial Mur Ligases
1
20:1); mp: 145–1468C; H NMR (300 MHz, [D6]DMSO): d=13.25 (br
ATCC 29212) to compounds 5a, 6a, 6c, 7d and 9 were tested,
using the macrodilution method. For the compounds that showed
activity against S. aureus, susceptibility of a standard strain ATCC
43300 of methicillin-resistant S. aureus (MRSA) was additionally
tested using the same method. The test compound (10 mg) was
s, 1H, NH), 7.28 (d, J=8.6 Hz, 1H, ArH(6)), 7.21 (d, J=8.6 Hz, 1H,
ArH(5)), 4.96 (dd, J=5.0 Hz, J=9.2 Hz, 1H, SCHCO), 3.27–3.13 (m,
2H, CH2CH), 2.34 (s, 3H, CH3), 2.29 (s, 3H, CH3), 2.26 ppm (s, 3H,
CH3); 13C NMR (75 MHz, CDCl3): d=199.0, 175.6, 167.4, 167.1, 166.2,
142.5, 141.6, 134.8, 127.1, 126.6, 120.6, 54.2, 33.0, 20.1, 19.8,
19.6 ppm; IR (KBr): n˜ =3430, 2935, 2867, 2081, 1779, 1759, 1637,
1496, 1449, 1372, 1284, 1194, 1106, 1080, 1039, 1015, 968, 923,
864, 815, 769, 724, 675, 659, 579, 535, 524 cmꢁ1; MS (ESI+): m/z
(%): 398 (72) [M+H]+, 420 (100) [M+Na]+; Anal. calcd for
C16H15NO7S2: C, 48.35; H, 3.80; N, 3.52; found: C, 48.20; H, 3.93; N,
3.53.
dissolved in 5 mL of DMSO to give a stock solution of 2 mgmLꢁ1
.
Working solutions were made by serially diluting stock solution in
cation-adjusted Mueller–Hinton broth (CAMHB) as described by
Amsterdam and Barry.[39,40] CAMHB (34 mL) was added to a 5 mL
stock solution to give 256 mgmLꢁ1 concentration and then passed
through a sterilized filter. The compound was further serially dilut-
ed to give 14 dilutions down to the lowest concentration of
0.031 mgmLꢁ1 and stored frozen for a maximum of two weeks.[39]
Just prior to bacterial inoculation, 0.5 mL dilutions in the range of
the compounds were pipetted into 13ꢃ100 mm screw-cap tubes.
2-Thioxo-5-(2,3,4-trihydroxybenzyl)thiazolidin-4-one (12): A solu-
tion of 11 (0.118 g, 0.297 mmol) and N2H4·H2O (0.028 g,
0.892 mmol) in CH3CN (5 mL) was stirred at RT for 20 min. The re-
action mixture was neutralized with glacial AcOH and the solvent
evaporated in vacuo. The residue was dissolved in EtOAc and
washed successively with H2O (2ꢃ10 mL) and brine (10 mL). The
organic phase was dried (Na2SO4), filtered and concentrated in va-
cuo. Purification by flash column chromatography (CH2Cl2/CH3OH/
AcOH; 90:10:1) gave compound 12 as a yellow solid (0.081 g,
73.2%): Rf =0.33 (CH2Cl2/MeOH/CH3COOH; 9:1:0.1); mp: 208–
The inoculum was prepared in such a way that four colonies of a
fresh overnight culture on a nonselective agar plate were inoculat-
ed into saline. The turbidity was adjusted to match that of 0.5
McFarland standard (~108 CFUmLꢁ1). A portion of a standardized
suspension was diluted (~1:1000; 105 CFU mLꢁ1), and a 0.5 mL ali-
quot of this dilution was then added to each tube containing
0.5 mL of the tested compound diluted in CAMHB within 30 min
and incubated at 358C for 18–24 h. After inoculum was added, di-
lutions 0.016 to 128 mgmLꢁ1 of the compound were achieved.
Broth not containing any compound was inoculated as a growth
control. If the compound inhibited bacterial growth, it was consid-
ered a potential antimicrobial agent. The lowest concentration of
antimicrobial agent that resulted in complete inhibition of visible
growth was the minimal inhibitory concentration (MIC). A sample
from each tube that displayed no visible growth portion was
plated to blood agar to determine the minimal bactericidal con-
centration (MBC). Quality control of the methods was done by test-
ing S. aureus ATCC 29213 and gentamycin. Dilutions of antibiotic
were made in the same way as for tested compounds and the MIC
values obtained were in the range proposed by the Clinical Labora-
tory Standards Institute.[41]
1
2108C; H NMR (300 MHz, [D6]DMSO): d=13.08 (br s, 1H, NH), 9.01
(s, 1H, OH), 8.46 (s, 1H, OH), 8.29 (s, 1H, OH), 6.36 (d, J=8.2 Hz,
1H, ArH(6)), 6.22 (d, J=8.2 Hz, 1H, ArH(5)), 4.89 (dd, J=4.5 Hz, J=
10.3 Hz,1H, SCHCO), 3.41–3.33 ppm (m, 2H, CH2CH, signal over-
lapped with residual H2O); 13C NMR (75 MHz, [D6]DMSO): d=203.5,
177.9, 145.1, 144.2, 132.6, 119.3, 114.6, 106.0, 54.9, 31.9 ppm; IR
(KBr): n˜ =3460, 3103, 2903, 2849, 2094, 1719, 1636, 1540, 1514,
1449, 1388, 1310, 1293, 1227, 1189, 1153, 1107, 1080, 1035, 960,
905, 761, 728, 688, 621, 582, 503, 481, 458 cmꢁ1; MS (ESI+): m/z
(%): 272 (60) [M+H]+, 294 (65) [M+Na]+, 252 (100); Anal. calcd for
C10H9NO4S2: C, 44.27; H, 3.34; N, 5.16; found: C, 44.43; H, 3.59; N,
5.41.
Biology
Enzyme assays
Mutagenicity and genotoxicity of compound 5a
The inhibition of Mur ligases and Ddl was determined using the
malachite green assay, as previously reported.[11a,38] For compound
5a, which showed inhibitory activity against MurD–F ligases, Ki
values were determined against MurD from E. coli. Ki determina-
tions were performed under similar conditions as described for the
Mur ligases and Ddl inhibition assay: different concentrations of
one substrate and fixed concentrations of the other two. First, the
concentration of ATP (25, 100, 400 and 800 mm) was varied at fixed
concentrations of UMA (80 mm) and d-Glu (100 mm), then the con-
centration of d-Glu (25, 100 and 400 mm) was modified at fixed
concentrations of ATP (400 mm) and UMA (80 mm), and finally, the
concentration of UMA (10, 20, 40 and 80 mm) was changed at fixed
concentrations of ATP (400 mm) and d Glu (100 mm). The concentra-
tions of 5a used were 0, 0.5, 0.9, 1.5, 2.5 and 4.0 mm. After incuba-
tion for 15 min at 378C, the enzyme reaction was terminated by
adding Biomol greenTM reagent and the absorbance was read at
650 nm. Initial velocity data were fitted to competitive, noncompe-
titive and uncompetitive inhibition models using SigmaPlot 11.0
software[29] and Ki values were calculated for the best fitted model.
Bacterial strains and human hepatoma HepG2 cells: Salmonella ty-
phimurium strain TA98, which detects frame-shift mutations, and
TA100, which detects base-pair substitution mutations, were ob-
tained from Professor B. N. Ames (University of California, Berkeley,
USA). The strains were kept at ꢁ808C and were checked for their
histidine/biotin dependence, rfa marker (crystal violet), uvrB dele-
tion (UV sensitivity), and the presence of the plasmid pKM101 (am-
picillin resistance). The HepG2 cell line was obtained from the
ECACC (European Collection of Cell Culture, UK). The cells were
grown in monolayer culture in EMEM medium supplemented with
4 mm l-glutamine, 1% nonessential amino acids and 15% FBS in a
humidified atmosphere of 5% CO2 at 378C.
Ames test
Mutagenicity of 5a was tested using the S. typhimurium reverse
mutation assay (Ames test) with strains TA98 and TA100, with and
without metabolic activation as described by Maron and Ames.[42]
0.1 mL of diluted 5a solution (in DMSO, vehicle or positive control),
0.1 mL of overnight grown bacterial culture and 0.5 mL of S9 mix
(containing 4% S9—Arachlor-induced rat liver microsomal fraction;
Moltox, USA) or phosphate-buffer solution (for treatment without
activation) were added to 2 mL of molten top agar containing a
limited amount of histidine/biotin at 458C, gently mixed and
Antibacterial activity
The susceptibilities of four standard strains (E. coli ATCC 25922,
P. aeruginosa ATCC 27853, S.aureus ATCC 29213 and E. faecalis
ChemMedChem 2010, 5, 286 – 295
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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ˇ ˇ
L. P. Masic, D. Kikelj et al.
MED
poured onto Vogel–Bonner minimal glucose-agar plates. His+ re-
vertants were counted after 48 h incubation at 378C. The back-
ground lawn was inspected for signs of toxicity or compound pre-
cipitation. All experiments were carried out in triplicate using three
concentrations (0.019, 0.095 and 0.476 mm per plate) of 5a. The
highest tested concentration was selected based on previously de-
termined MIC values against P. aeruginosa and S. aureus. Benzo[a]-
pyrene (B[a]P, 10 mg per plate) and 4-nitroquinoline oxide (4-
NQNO, 0.5 mg per plate) were used as positive controls in tests
with and without metabolic activation, respectively.
mixture (9:1, v/v) containing HEPES buffer (20 mm, pH 7.2),
(NH4)2SO4 (7 mm), MgCl2 (3.5 mm), DTT (0.3 mm) and ATP (0.4 mm).
The concentration of MurD selectively labeled with 13C at the
methyl groups of Ile (d1 only), Val, and Leu was 0.07 mm. The pro-
tein was titrated by the ligands in MurD:ligand molar ratios of 0.5,
1, 2, 5, and 10. Spectra were acquired with 1024 data points in t2,
32 scans, 64 complex points in t1, and a relaxation delay of 1 s. The
1H and 13C sweep widths were 9470 and 3340 Hz, respectively.
Spectra were processed and analyzed with Sparky software.[48]
Spectra were zero-filled twice and apodized with a squared sine
bell function shifted by p/2 in both dimensions, using linear pre-
diction of the data in the incremented dimension. The combined
chemical-shift perturbations Dd were calculated from 1H and 13C
Cytotoxicity assay
chemical-shift changes using the equation: Dd=(D1H + (0.252ꢃ
The cytotoxicity of compound 5a was measured using the MTT re-
duction assay.[43] The HepG2 cells were seeded onto 96-well micro-
plates at a density of 104 cellswellꢁ1 and incubated for 24 h at
378C to attach. The medium was then replaced with fresh com-
plete medium containing graded concentrations of (0.000128–
0.4 mm) 5a and incubated for 24 h. MTT (final concentration=
0.5 mgmLꢁ1) was then added and the plates were incubated for an
additional 3 h. At the end of the incubation with MTT, the medium
was removed and the formazan crystals dissolved in DMSO. Optical
density (OD) was measured at 570 nm (reference filter 690 nm)
using a microplate reading spectrofluorometer (GENiosꢄ, Tecan,
Mꢅnnedorf, Switzerland). Viability was determined by comparing
the OD of the wells containing the cells treated with 5a with the
vehicle-treated cells (DMSO, 0.1% v/v). The Student’s t test was
used to evaluate the statistical significance between exposed and
control cells; P<0.05 was considered significant.
1
2
13
2
[49]
D C) ) .
Acknowledgements
This work was supported financially by the European Union FP6
Integrated Project EUR-INTAFAR (Project No. LSHM-CT-2004–
512138) under the thematic priority of Life Sciences, Genomics,
and Biotechnology for Health, and the Slovenian Research
Agency (P1–0208, P1–0230, J1–6693). The authors thank Kristina
ˇ
ˇ
Vrtacnik, Ana Poje and Natasa Lesar for technical assistance, and
Professor Roger Pain for critical reading of the manuscript.
Keywords: antibacterial agents · inhibitors · Mur ligases ·
protein NMR · thiazolidine-4-ones
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Received: October 29, 2009
Published online on December 18, 2009
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295