Discovery of novel 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2, 4-dione inhibitors of MurD ligase

Article abstract of DOI:10.1021/jm100285g

We have designed, synthesized, and evaluated 5-benzylidenerhodanine-and 5-benzylidenethiazolidine-2,4-dione-based compounds as inhibitors of bacterial enzyme MurD with E. coli IC₅₀ in the range 45-206 μM. The high-resolution crystal structure o

Full text of DOI:10.1021/jm100285g

6584 J. Med. Chem. 2010, 53, 6584–6594
DOI: 10.1021/jm100285g
Discovery of Novel 5-Benzylidenerhodanine and 5-Benzylidenethiazolidine-2,4-dione
Inhibitors of MurD Ligase†
‡
‡
‡
‡
ꢀ ꢁ ‡
Delphine Patin, Didier Blanot, Carlos Contreras Martel, Andrea Dessen, Manica Muller Premru,
ꢀ ‡
ꢀ
Nace Zidar, Tihomir Tomasic, Roman Sink, Veronika Rupnik, Andreja Kovac, Samo Turk,
§
§
^
ꢁ
€
Anamarija Zega, Stanislav Gobec, Lucija Peterlin Masic,*,‡ and Danijel Kikelj*,‡
‡
‡
ꢀ ꢀ
‡
§
Faculty of Pharmacy, University of Ljubljana, 1000 Ljubljana, Slovenia, Enveloppes Bacteriennes et Antibiotiques, IBBMC, UMR 8619 CNRS,
ꢁ
Universite Paris-Sud 11, 91405 Orsay, France, Institut de Biologie Structurale, J. P. Ebel, CEA, CNRS, UJF, UMR5075, F-38027 Grenoble,
ꢁ
France, and ^Medical Faculty, Institute of Microbiology and Immunology, University of Ljubljana, 1105 Ljubljana, Slovenia
Received March 4, 2010
We have designed, synthesized, and evaluated 5-benzylidenerhodanine- and 5-benzylidenethiazolidine-
2,4-dione-based compounds as inhibitors of bacterial enzyme MurD with E. coli IC50 in the range 45-
206 μM. The high-resolution crystal structure of MurD in complex with (R,Z)-2-(3-[{4-([2,4-dioxothia-
zolidin-5-ylidene]methyl)phenylamino}methyl)benzamido)pentanedioic acid [(R)-32] revealed details
of the binding mode of the inhibitor within the active site and provides a good foundation for structure-
based design of a novel generation of MurD inhibitors.
1. Introduction
from UDP-N-acetylmuramic acid (UDP-MurNAc) to yield
UDP-N-acetylmuramoylpentapeptide (UDP-MurNAc-penta-
peptide) by sequentially adding L-Ala (MurC), D-Glu (MurD),
meso-diaminopimelic acid or L-Lys (MurE), and the D-Ala-
D-Ala dipeptide (MurF).6 The kinetic properties and the cata-
lytic mechanism of Mur ligases, which catalyze the formation
of a peptide bond with concomitant cleavage of ATP into
ADP and inorganic phosphate, are well-known. The enzymes
operate by essentially similar chemical mechanisms. This entails
activation of the C-terminal carboxyl group of the nucleotide
substrate to an acyl phosphate intermediate, followed by
nucleophilic attack by the amino group of the condensing amino
acid or dipeptide, elimination of phosphate, and subsequent
peptide bond formation.6 All Mur ligases have the same three-
domain topology. The N-terminal and central domains bind
UDP precursor and ATP, respectively, while the incoming
amino acid or dipeptide binds to the C-terminal part.7,8 Seq-
uence alignments of Mur ligase orthologues and paralogues
show relatively low homology, with the exception of residues
comprising the active sites.9,10 Therefore, the key features to
be considered in the design of Mur ligases inhibitors are the
conserved binding motifs and the common chemical mecha-
nism of MurC-F. Despite extensive research on the discovery
of new inhibitors of Mur ligases,5,6,11,12 to date, none has
found therapeutic application, probably because of problems
associated with bacterial cell entry.3 Most known MurD
inhibitors containD-glutamicacid, suggestedtobeanessential
structural element of a potent inhibitor.13-17 It has been
demonstrated that MurD displays a high substrate stereo-
specificity toward D-Glu18,19 and that the binding site for
D-Glu is conserved across different bacterial species.10 Rhoda-
nineandthiazolidine-2,4-dionerings arewell-known scaffolds
in medicinal chemistry and have been incorporated in com-
pounds displaying a variety of pharmacological effects, inclu-
ding antibacterial, antifungal, and antidiabetic activity.20 Rhoda-
nine derivatives have also been reported as inhibitors of Mur
Infectious diseases remain the leading cause of death in low-
incomecountriesand the second leading causeof death world-
wide.1 The escalating rate of bacterial resistance to currently
available antibiotics is a well-known problem, and the need for
novel antibacterial drugs is increasingly important clinically.2
The cell wall of bacteria remains a viable source of targets for
novel antibacterials.3 Peptidoglycan is an essential bacterial
cell-wall polymer, unique to prokaryotic cells and therefore
a prime target for the selective targeting of microbial vital path-
ways. Its main function is to preserve cell integrity by with-
standing the internal osmotic pressure and maintaining a
defined cell shape.4 Biosynthesis of peptidoglycan is a multi-
stage process, which begins in the cytoplasm, where the
monomeric peptidoglycan building block is formed. This is
then translocated through the cellular membrane to its outer
side and incorporated in the peptidoglycan macromolecule.
A large number of antibiotics in clinical use, e.g., β-lactams
and glycopeptides, act by inhibiting the later, extracellular
steps of peptidoglycan biosynthesis. Enzymes involved in the
earlier steps of the biosynthesis of cytoplasmic peptidogly-
can precursor are more difficult drug targets that have been
subjected to many HTS efforts to no avail.5 Mur ligases
(MurC, MurD, MurE and MurF)a constitutea groupof cyto-
plasmic enzymes that catalyze a series of reactions, starting
†PDB code: 2x5o.
*To whom correspondence should be addressed. For L.P.M.: phone,
+386-1-4769635; fax, +386-1-4258031; e-mail, Lucija.PeterlinMasic@
ffa.uni-lj.si. For D.K.: phone, þ386-1-4769561; fax, þ386-1-4258031;
e-mail, danijel.kikelj@ffa.uni-lj.si.
a Abbreviations: UDP, uridine 50-diphosphate; UMA, UDP-N-acetylmura-
moyl-L-alanine; UMAG, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate;
MurC, UDP-N-acetylmuramate:L-alanine ligase; MurD, UDP-N-acetylmur-
amoyl-L-alanine:D-glutamate ligase; MurE, UDP-N-acetylmuramoyl-L-alanyl-
D-glutamate:meso-diaminopimelate ligase; MurF, UDP-N-acetylmuramoyl-L-
alanyl-γ-D-glutamyl-meso-diaminopimelate:D-alanyl-D-alanine ligase.
r
pubs.acs.org/jmc
Published on Web 08/30/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6585
ligases.13,21,22 In the search for novel peptidoglycan biosynthesis
inhibitors we published a preliminary report on MurD inhibi-
tory activity of novel compounds that combine glutamic acid
and 5-benzylidenerhodanine or 5-benzylidenethiazolidine-2,4-
dione structural elements (Figure 1). These were designed based
on the 2,4-diaminoquinazoline-containing highest ranked com-
pound from virtual screening.13 (R)-21, which was the most
potent in the series and inhibited MurD with an IC50 of 174 μM,
stimulated us to undertake an intensive structure modification
program with the aim of improving MurD inhibition.
We report here a full account of the synthesis and biochemi-
cal evaluation of a series of new 5-benzylidenerhodanine- and
5-benzylidenethiazolidine-2,4-dione-based analogues as inhi-
bitors of MurD. We examined the effects of (i) different
substitution patterns in rings A and B, (ii) the replacement
of glutamic acid, (iii) the distance between the glutamic acid
and thiazolidine moieties on MurD inhibition, and (iv) the
difference between thiazolidine-2,4-dione- and rhodanine-
based derivatives. This endeavor resulted in (R)-32 and (R)-
33 with improved inhibitory potency (IC50 = 85 and 45 μM,
respectively). Compound (R)-32 was cocrystallized with
MurD, and the crystal structure of the complex was deter-
˚
mined to 1.5 A resolution. The details of the binding mode of
(R)-32 in the MurD active site will help us to understand the
structure-activity relationships and provide a good founda-
tion for structure-based design of a novel generation of MurD
inhibitors.
2. Results and Discussion
2.1. Design. Encouraged by the promising MurD inhibi-
tory activity of (R)-21, whose docking into the MurD active
site suggested binding of D-glutamic acid residue in the D-Glu
pocket of the enzyme and the positioning of the rhodanine
moiety in the region normally occupied by the diphosphate
group of UDP,13 we started a systematic structure modifica-
tion program to increase the potency by optimizing the
interactions of the inhibitor with the enzyme active site. To
this end, four structural types of compound, comprising
meta- or para-substitution patterns in rings A and B, were
Figure 1. Design of improved MurD inhibitors by optimization of
the initial hit compound (R)-21.
Scheme 1. Synthesis of 5-Benzylidenerhodanine and 5-Benzylidenethiazolidine-2,4-dione Derivatives (R)-20, (S)-20, (R)-21, (S)-21,
(R)-24, (S)-24, (R)-25, (S)-25, (R)-28, (R)-29, (R)-32, and (R)-33a
a Reagents and conditions: (a) EDC/HOBt, DMF, room temp, 24 h; (b) piperidine, AcOH, EtOH, microwave, 150 ꢀC, 20 min. (c) For the preparation
of 14 and 16: H2/Pd-C, MeOH/THF, room temp, 5 h. For the preparation of 15 and 17: SnCl2, EtOH, reflux, 2 h. (d) (R)-4, (R)-5, or (S)-5, NaCNBH3,
DMF, room temp, 24 h; (e) 2.2 M LiOH, MeOH/H2O, room temp, 15 h.

6586 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Zidar et al.
Scheme 2. Synthesis of 5-Benzylidenerhodanine and 5-Benzylidenethiazolidine-2,4-dione Derivatives with an Alkanediyl Linkera
a Reagents and conditions: (a) Et3N, CH2Cl2, 0 ꢀC to room temp, 3 h; (b) K2CO3, KI, CH3CN, 85 ꢀC, 3 h; (c) 8 or 9, piperidine, AcOH, EtOH,
microwave, 150 ꢀC, 20 min; (d) 2.2 M LiOH, MeOH/water, room temp, 15 h.
conceived. R- and S-enantiomeric series were first envisaged
and later limited to (R)-glutamic acid derivatives because of
the small difference in observed inhibitory potency of enantio-
mers. Rhodanine and thiazolidine-2,4-dione compounds were
devised to assess the role of an exocyclic sulfur substituent
for binding to the enzyme. Since rhodanine or thiazolidine-
2,4-dione moieties and glutamic acid were considered crucial
for interaction with the MurD active site and since the mode
of binding of this class of MurD inhibitors was not known at
the beginning of our studies, (R)-53 and (R)-54 with a more
flexible linker, together with (R)-45, (R)-46, (R)-51, and (R)-
52 with a shorter linker between glutamic acid and thiazoli-
dine moieties, were also considered. Compared to (R)-32 and
(R)-33, the number of bonds between the amide group and
benzylidene moiety was retained in (R)-53 and (R)-54, while
the number of rotatable bonds was increased by 2. This
modification gave (R)-53 and (R)-54 more conformational
freedom and allowed shorter distances between glutamic
acid and thiazolidine moieties. To further assess the role of
conformational freedom on the interactions between the
inhibitor and MurD, (R)-62, (R)-66 and (R)-68 possessing
a reduced exocylic carbon-carbon double bond were also
taken into consideration. These compounds can be regarded
as more flexible analogues of (R)-33, (R)-21, and (R)-24,
respectively, with a loss of conjugation and planarity in the
eastern part of the molecules. Finally, analogues of (R)-21 in
which D-glutamic acid was replaced with L-lysine were desig-
ned to explore possible ionic interactions with active site amino
acids possessing anionic side chains.
benzylidene)rhodanines 11 and 13 were obtained in good
yields via Knoevenagel condensation of nitrobenzaldehyde 6
or 7 with thiazolidine-2,4-dione (8) or rhodanine (9) in a
microwave reactor. The presence of only one signal for the
methyne proton at 7.7-8.0 ppm in 1H NMR spectra of
10-13 suggested that a single isomer was present, which was
assigned Z-configuration with the aid of an 1H-coupled 13
C
NMR spectrum,23 as described previously.24 The exclusive
formation of the thermodynamically stable Z-isomers of 10-13
is in agreement with literature reports for similar com-
pounds.24-26 The reduction of the nitro group in 10 and 12
was achieved through catalytic hydrogenation, using Pd-C
as a catalyst. Reduction of rhodanine derivatives 11 and 13,
which could not be transformed into amines 15 and 17 by
catalytic hydrogenation, was achieved with tin(II) chloride.
In the next step, R- and S-isomers of 18, 19, 22, and 23 and
R-isomers of 26, 27, 30 and 31 were obtained by sodium
cyanoborohydride effected reductive amination of amines
14-17 with benzaldehydes (R)-4, (R)-5, and (S)-5. The latter
compounds were prepared via EDC/HOBt promoted coup-
ling of L- and D-glutamic acid methyl esters 1 with carboxy-
benzaldehydes 2 and 3. Finally, hydrolysis of methyl esters
with aqueous lithium hydroxide solution afforded targets
(R)-20, (S)-20, (R)-21, (S)-21, (R)-24, (S)-24, (R)-25, (S)-25,
(R)-28, (R)-29, (R)-32, and (R)-33.
The synthesis of (R)-45, (R)-46, and (R)-51-(R)-54 with a
more flexible alkanediyl linker between glutamic acid and
thiazolidine moieties, which would allow shorter distances
between the two residues, is outlined in Scheme 2. The reac-
tion of D-glutamic acid dimethyl ester [(R)-1] with acyl halides
34 and 35 gave (R)-36 and (R)-37, respectively, which upon
heating with hydroxylbenzaldehyde 38 or 39 in the presence
of potassium iodide and potassium carbonate yielded (R)-40-
(R)-42. Knoevenagel condensation of (R)-40-(R)-42 with
thiazolidine-2,4-dione (8) or rhodanine (9) in a microwave
reactor afforded (R)-43, (R)-44, and (R)-47-(R)-50, which,
after hydrolysis with aqueous lithium hydroxide solution,
2.2. Chemistry. The syntheses of both enantiomers of com-
pounds 20 and 21 with para-substitution in ring A and meta-
substitution in ring B, R- and S-isomers of 24 and 25 possessing
para-substitution in both phenyl rings, (R)-28 and (R)-29
with meta-substitution in both aromatic rings, and (R)-32 and
(R)-33 with meta-substitution in ring A and para-substitution
in ring B are outlined in Scheme 1. In the first step, 5-(nitro-
benzylidene)thiazolidine-2,4-diones 10 and 12 and 5-(nitro-

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6587
Scheme 3. Synthesis of Reduced 5-Benzylrhodanine and 5-Benzylthiazolidine-2,4-dione Derivatives (R)-62, (R)-66, and (R)-68a
a Reagents and conditions: (a) diethyl 2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate, silica gel 60, toluene, 100 ꢀC, 24 h. (b) For the preparation
of 59: H2/Pd-C, MeOH/THF, room temp, 5 h. For the preparation of 58 and 60: SnCl2, EtOH, reflux, 2 h. (c) (R)-4, NaCNBH3, MeOH, room temp,
24 h; (d) 3, NaCNBH3, MeOH, room temp, 24 h; (e) (R)-1, EDC/HOBt, DMF, room temp, 24 h; (f) 2,2 M LiOH, MeOH/water, room temp, 15 h.
Scheme 4. Synthesis of Derivatives with a Lysine Residuea
a Reagents and conditions: (a) for the preparation of 69, 70 and 72: NaCNBH3, MeOH, room temp, 24 h. For the preparation of 71: NaCNBH3,
DMF, room temp, 24 h. (b) N-Boc- L-Lys-OMe, EDC/HOBt, DMF, room temp, 24 h; (c) 2.2 M LiOH, MeOH/H2O, room temp, 15 h; (d) HCl(g), AcOH,
room temp, 0.5 h.
gave target compounds (R)-45, (R)-46, and (R)-51-(R)-54.
For the synthesis of 5-benzylthiazolidin-4-one derivatives
(R)-62, (R)-66, and (R)-68 with reduced exocyclic double
bond (reduced analogues of (R)-33, (R)-21, and (R)-24) the
crucial step was the reduction of 11-13 using diethyl 2,6-
dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate (Hantsch
ester)27 and activated silica gel28 to yield products 55-57,
which were further converted into final compounds (R)-62,
(R)-66, and (R)-68 using the synthetic strategy described in
Scheme 3.
in Scheme 4. Compounds 69-72 were obtained by reductive
amination of amines 14-17 with carboxybenzaldehydes 2
and 3, using sodium cyanoborohydride as a reducing agent.
EDC/HOBt-promoted coupling of 69-72 with N-Boc-L-
lysine methyl ester afforded the S-isomers of 73, 76, 77,
and 82, which were converted into products (S)-75, (S)-80,
(S)-81, and (S)-84 upon hydrolysis with aqueous lithium
hydroxide solution and subsequent cleavage of the Boc pro-
tecting group with gaseous hydrochloric acid in glacial acetic
acid.
The synthesis of (S)-75, (S)-80, (S)-81, and (S)-84, in
which D-glutamic acid was replaced with L-lysine, is shown
2.3. Biological Activity. Both isomers of targets 20, 21, 24,
and 25, R-isomers of 28, 29, 32, 33, 45, 46, 51-54, 62, 66, and

6588 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Zidar et al.
Table 1. MurD Inhibitory Potencies of 5-Benzylidenerhodanine- and
5-Benzylidenethiazolidine-2,4-dione-Based Target Compounds (Scheme 1)
(R)-20, (S)-20, and (R)-29 vs (R)-28], in the most active sub-
set of compounds with a meta, para substitution pattern the
inhibitory potencies of rhodanine (R)-33 and thiazolidine-
2,4-dione (R)-32 were comparable (45μM comparedto 85μM).
These results were confirmed by additionally testing (R)-32
and (R)-33 for MurD inhibition in a radioactivity assay30
which gave IC50 of 35 and 49 μM, respectively.
The reduction of the exocyclic double bond in (R)-21,
(R)-24, and (R)-33 to give (R)-66, (R)-68, and (R)-62 reduced
MurD inhibitory activity (Supporting Information, Table 2).
Compounds with flexible alkyl linkers replacing the benzyl-
amine moiety were devoid of inhibitory activity with the
exception of (R)-46, for which a weak inhibition was ob-
served. The absence of inhibitory activity in both classes of
compounds is probably due to an increased internal entropy
term because of additional rotational freedom in the more
flexible molecules, whereas in (R)-62, (R)-66, and (R)-68,
the loss of conjugation may also play a role. (S)-75, (S)-80,
(S)-81, and (S)-84, in which glutamic acid was replaced with
L-Lys, were inactive as MurD inhibitors, providing further
evidence for the importance of the glutamic acid γ-carboxy-
late for interaction with the enzyme active site (Supporting
Information Table 2).
Antimicrobial activity of (R)-21 and (R)-33 against two Gram-
positive (S. aureus ATCC 29213, E. faecalis ATCC 29212)
and two Gram-negative (E. coli ATCC 25922, P. aeruginosa
ATCC 27853) bacteria was evaluated. For both compounds
the minimal inhibitory concentrations against all four bac-
teria tested were found to be greater than 128 μg/mL. Inacti-
vity of (R)-21 and (R)-33 against tested bacteria is probably
due to problems associated with bacterial cell entry.
substitution
MurD
compd
X
ring A
ring B
RAa (%)
(R)-20
(S)-20
(R)-21
(S)-21
(R)-24
(S)-24
(R)-25
(S)-25
(R)-28
(R)-29
(R)-32
(R)-33
O
O
S
1,4
1,4
1,4
1,4
1,4
1,4
1,4
1,4
1,3
1,3
1,3
1,3
1,3
1,3
1,3
1,3
1,4
1,4
1,4
1,4
1,3
1,3
1,4
1,4
85
87
IC50 = 174 ( 2 μM
IC50 = 206 ( 3 μM
70
S
O
O
S
88
74
S
68
87
O
S
39
IC50 = 85 ( 3(35 ( 1)b μM
O
S
IC50 = 45 ( 1(49 ( 1)b μM
a Residual activity of the enzyme. Concentration of inhibitor was
250 μM. Results represent the mean of two independent experiments.
Standard deviations were within (10% of the mean. For IC50 determi-
nation assays were run in triplicate. b Radioactivity assay.
68, and S-isomers of 75, 80, 81, and 84 were tested for their
inhibitory activities on E. coli MurD using the Malachite
green assay29 for detecting orthophosphate generated during
the enzymatic reaction (Table 1 and Supporting Information
Table 2). The results are presented as residual activities (RAs)
of the enzyme in the presence of 250 or 500 μM of each com-
pound and as IC50 values for the most active compounds.
Some of the dimethyl ester precursors of target compounds,
i.e., (R)-18, (S)-18, (R)-19, (S)-19, (R)-22, (S)-22, (R)-23, and
(S)-23, have also been tested against MurD but were found to
be essentially noninhibitory, with residual activities in the
range 86-98% (data not shown). These results were expected,
considering the high affinity of the enzyme for D-Glu, and
demonstrate the importance of the free carboxylate groups
which, in the case of the MurD product UMAG, form seve-
ral hydrogen bonds and electrostatic interactions with amino
acid residues in the MurD active site.7
The results in Table 1 show no significant changes in inhi-
bitory potency upon replacing D-Glu by L-Glu. This is in
accordance with steady-state kinetics and X-ray diffraction
studies, which demonstrated that the L-Glu moiety of N-sub-
stituted Glu derivatives can bind into the D-Glu-binding site
of MurD in a manner similar to that of D-Glu in UMAG.15
The results (Table 1) demonstrate that a change in the
substitution pattern on both phenyl rings has an important
effect on MurD inhibitory potency. Whereas extended struc-
tures with para-substitution on both phenyl rings [(R)-24,
(S)-24, (S)-25, (R)-25] displayed only weak inhibition, com-
pounds with meta-substitution in ring A [(R)-32, (R)-33],
meta-substitution in ring B [(R)-21, (S)-21], or meta-substi-
tution in both rings [(R)-29] were found to be promising
inhibitors of MurD. The most potent MurD inhibitors of the
series were (R)-32 and (R)-33 with the Malachite green assay
IC50 of 85 and 45 μM, respectively. Although it appears that
rhodanine derivatives are generally better inhibitors than
thiazolidine-2,4-dione-type compounds [cf. (R)-21, (S)-21 vs
2.4. X-ray Crystallography. The crystal structure of MurD
˚
in complex with compound (R)-32 was solved to 1.5 A reso-
lution (PDB code 2x5o) to determine the binding mode of the
inhibitor within the MurD active site (Figure 2). In the
MurD-(R)-32 complex the D-Glu moiety occupies the same
site as the D-Glu residue of the product UMAG of the
enzymatic reaction catalyzed by MurD.7 The R-carboxyl
group of the D-Glu moiety of the inhibitor forms a charge-
based interaction with Nξ of Lys348 and is additionally
hydrogen-bonded with a conserved water molecule W2526
which is further hydrogen-bonded to Oγ of Thr321 and the
carboxyl group of Asp182. The carboxyl group of the D-Glu
side chain is held in place by hydrogen bonds with Ser415 and
Phe422. One of the D-Glu side chain carboxylic oxygen
atoms forms hydrogen bonds with both the backbone nitro-
gen and the Oγ of Ser415, whereas the other oxygen atom
interacts with the backbone nitrogen of Phe422 and also with
water molecule W2528. The latter is further hydrogen-
bonded to water molecule W2530, which is in direct contact
with the amide nitrogen of the D-Glu moiety of the inhibitor,
forming an extended ring-type system. The carbonyl oxygen
in the amide bond is hydrogen-bonded with W2532 and with
guanidine nitrogen of Arg186. Additional interactions be-
tween the amino group in the para position of the 5-benzy-
lidenethiazolidine-2,4-dione ring and W2527 also contribute
to recognition within the active site. The water molecule
W2527 is hydrogen-bonded to W2138 which itself interacts
with the Oγ of Ser71 and W2033 that is hydrogen-bonded to
the backbone carbonyl group of Leu13. The crystal struc-
tures of MurD in complex with small-molecule inhibitors
have been solved before,14,15 enabling the comparison of
their binding modes with that of (R)-32. The increase of
affinity observed for (R)-32 can be explained by additional

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6589
Figure 2. (a) Binding mode of compound (R)-32 in the active site of E. coli MurD. The Fo - Fc residual map is contoured at 3σ (green).
(b) Superposition of compound (R)-32 (pink) and UMAG (gray) in the MurD active site.
interactions, especially in the uracyl-binding pocket, which is
occupied by the thiazolidine-2,4-dione ring of the inhibitor.
Superposition of (R)-32 and UMAG in the MurD active site
is represented in Figure 2b. The ring nitrogen forms a hydro-
gen bond with the Oγ of Thr36, and the carbonyl oxygen at
position 2 interacts with the Thr36 backbone NH group. The
other carbonyl oxygen of the thiazolidine-2,4-dione ring
interacts, via water molecule W2529, with the Oγ of Thr36.
In addition, the thiazolidine-2,4-dione ring participates in
interplane stacking with a salt bridge formed between Asp35
and Arg37. The crystal structure of (R)-32 in complex with
MurD also reveals that the exocyclic double bond exists in the
Z-configuration, which has been reported as the thermo-
dynamically stable configuration of similar compounds.24-26
4.2. Radioactivity Inhibition Assay. Selected compounds were
tested for inhibition of the addition of the radiolabelled amino-
acid substrate D-[14C]Glu to UDP-MurNAc-L-Ala catalyzed by
MurD, using a mixture (final volume, 50 µL) containing 0.1 M
Tris-HCl, pH 8.6, 5 mM MgCl2, 5 mM ATP, 25 µM UDP-
MurNAc-L-Ala, 25 µM D-[14C]Glu (50,000 cpm), 5% (v/v)
DMSO, 30 µM Tween 20, purified MurD (diluted in 20 mM
potassium phosphate, pH 7.0, 1 mM dithiothreitol, 1 mg/mL
BSA), and 250 µM of each test compound. The mixture was
incubated at 37 ꢀC for 30 min, and the reaction was stopped by
adding 10 µL of glacial acetic acid. The resulting mixture was
lyophilized and taken up in water (∼10 µL). The radioactive
substrate and product were separated by TLC on LK6D silica
gel plates (Whatman), which were developed in 1-propanol/
ammonium hydroxide/water 6:3:1 (v/v) and quantified with a
radioactivity scanner (model Multi-Tracemaster LB285, Berthold-
France, Thoiry, France). IC50 values were determined by
measuring the residual activities at seven different compound
concentrations.
3. Conclusion
We have prepared a series of novel 5-benzylidenerhodanine
and 5-benzylidenethiazolidine-2,4-dione derivatives as promis-
ing inhibitors of MurD ligase, with IC50 in the range 45-206 μM
for MurD from E. coli. The crystal structure of the complex of
MurD with (R)-32, one of the most potent MurD inhibitors
in the series, reveals its binding mode in the enzyme active
site and makes (R)-32 an excellent candidate for further
structure-based optimization toward an effective novel anti
bacterial drug.
4.3. Determination of Antibacterial Activity. With the macro-
dilution method, the susceptibility of four standard strains
(E. coli ATCC 25922, P. aeruginosa 27853, S. aureus ATCC
29213, and E. faecalis ATCC 29212) to compounds (R)-21 and
(R)-33 was tested.
Compounds (R)-21 and (R)-33 (10 mg) were dissolved in 5 mL
of dimethyl sulfoxide (DMSO) to get a stock solution of the
concentration 2 mg/mL. Working solutions were made by
serially diluting stock solution in cation-adjusted Mueller–
Hinton broth (CAMHB) as described by Amsterdam and
Barry.32,33 Briefly, 34 mL of CAMHB was added to 5 mL of
stock to obtain a 256 μg/mL concentration and filter sterilized.
The compound was further serially diluted to obtain 14 dilutions
down to the lowest concentration of 0.031 μg/mL33 and stored
frozen for a maximum of 2 weeks. Just prior to bacterial
inoculation, 0.5 mL dilutions in range of the compounds were
pipetted into 13 ꢀ 100 mm screw cap tubes. The inoculum was
prepared in such a way that four colonies of a fresh overnight
culture on a nonselective agar plate were inoculated into saline.
The turbidity was adjusted to match that of 0.5 McFarland
standard (approximately 108 CFU/mL). A portion of a stan-
dardized suspension was diluted to approximately 1:1000 (105
CFU/mL). Afterwards, 0.5 mL of this dilution was added within
30 min to each tube containing 0.5 mL of the tested compound
diluted in CAMHB and incubated at 35 ꢀC for 18–24 h. After
inoculum was added, dilutions of 0.016–128 μg/mL of the
compound were achieved. The broth not containing any com-
pound was inoculated as a growth control. If the compound
inhibited bacterial growth, it was considered a potential anti-
microbial agent. The lowest concentration of antimicrobial agent
that resulted in complete inhibition of visible growth represented
4. Experimental Section
4.1. Colorimetric Inhibition Assay. The compounds were
tested for their ability to inhibit the addition of D-Glu to
UDP-MurNAc-L-Ala catalyzed by MurD from E. coli. Detection
of the orthophosphate generated during the reaction was based
on the colorimetric Malachite green method, as described,29
with slight modifications. A mixture with a final volume of 50 μL
contained 50 mM Hepes, pH 8.0, 3.25 mM MgCl2, 6.5 mM
(NH4)2SO4, 0.005% Triton X-114, 80 μM UDP-MurNAc-L-
Ala, 100 μM D-Glu, 400 μM ATP, purified MurD from E. coli31
(diluted with 20 mM Hepes, pH 7.2, 5 mM dithiothreitol), and
500 or 250 μM of the tested compound dissolved in DMSO. The
final concentration of DMSO was 5% (v/v). The reaction
mixture was incubated at 37 ꢀC for 15 min, then quenched with
100 μL of Biomol reagent. Absorbance at 650 nm was measured
after 5 min. Residual activities were calculated with respect to
control assays without the compounds and with DMSO. IC50
values, the concentrations of the compounds at which the resi-
dual activities were 50%, were determined by measuring the
residual activities at seven different compound concentrations.

6590 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Zidar et al.
the minimal inhibitory concentration (MIC). Quality control of
the methods performance was done by testing S. aureus ATCC
29213 and gentamicin. Dilutions of antibiotic were made in
the same way as for tested compound, and the MICs obtained
were in the range proposed by Clinical Laboratory Standards
Institute.34
ethyl acetate/petroleum ether (2:1) as eluent. Yield, 25% (0.181 g);
white solid; mp 66-69 ꢀC; [R]2D3 þ1.3 (c 0.235, DMF); IR (KBr)
ν=3412, 3312, 2954, 2849, 1734, 1702, 1636, 1524, 1337, 1415,
1374, 1304, 1265, 1200, 1166, 1104, 1080, 980, 945, 902, 882,
829, 814, 780, 767, 749, 678, 668, 650 cm-1; 1H NMR (DMSO-
d6) δ 1.97-2.21 (m, 2H, CHCH2CH2), 2.48 (t, 2H, J=7.5 Hz,
CHCH2CH2), 3.59 (s, 3H, CH3), 3.66 (s, 3H, CH3), 4.48-4.55
(m, 1H, CH), 7.73 (t, 1H, J = 7.7 Hz, H-5), 8.10 (dt, 1H, 3J =
7.7 Hz, 4J=1.4 Hz, Ar-H-4/6), 8.19 (dt, 1H, 3J=7.7 Hz, 4J=
1.4 Hz, Ar-H-4/6), 8.42 (t, 1H, J=1.4 Hz, Ar-H-2), 9.01 (d, 1H,
J=7.5 Hz, NH), 10.09 (s, 1H, CHO); MS (ESI) m/z (%)=308
(MHþ). Anal. (C15H17NO6) C, H, N.
(Z)-5-(4-Nitrobenzylidene)thiazolidine-2,4-dione (12). To a sus-
pension of thiazolidine-2,4-dione (8, 0.616 g, 5.26 mmol) and
4-nitrobenzaldehyde (7, 0.870 g, 5.78 mmol) in absolute
ethanol (5 mL) in a 10 mL process vial, glacial acetic acid
(25 μL, 0.26 mmol) and piperidine (26 μL, 0.26 mmol) were
added. The vial was sealed, placed in a microwave reactor,
and heated at 150 ꢀC for 20 min (max power 30 W). The
mixture was cooled and the precipitate filtered off and dried.
Yield, 85% (1.12 g); yellow crystals; mp 274-278 ꢀC (lit.36 280 ꢀC);
IR (ATR) ν=3187, 3113, 3047, 2731, 1747, 1704, 1669, 1606,
1591, 1525, 1342, 1311, 1143, 1007, 902, 844 cm-1; 1H NMR
(DMSO-d6) δ 7.86 (d, 2H, J=8.7 Hz, Ar-H-2,6), 7.90 (s, 1H,
CH), 8.34 (d, 2H, J=8.7 Hz, Ar-H-3,5), 12.8 (s, 1H, NH); MS
(EI) m/z = 250 (Mþ).
4.4. Crystallization, Preparation of the Inhibitor Complexes,
and Data Collection. Crystals of the conformationally closed
MurD ligase were obtained by cocrystallizing the 6 ꢀ His-tagged
enzyme at 293 K with UMA and a nonhydrolyzable analogue of
ATP (AMP-PNP)7,15,35 with the vapor-diffusion method and
hanging-drop system in Linbro plates. An amount of 2 μL of
protein solution drops (8.9 mg/mL purified enzyme, 20 mM
HEPES, pH 7.4, 200 mM NaCl, 5 mM dithiothreitol, 1 mM
UMA, and 5 mM AMP-PNP) was mixed with 2 μL of reservoir
solution (0.1 M HEPES, pH 7.5, 1.8–1.9 M ammonium sulfate,
7–8% (w/v), PEG 400, and 100 mM NaCl). Crystals were grown
in 4–6 days. They were subsequently incubated with 1 mM
concentration of compound (R)-32, with 5% (v/v) DMSO. After
6 h of soaking, crystals were rapidly frozen in liquid nitrogen
using Paratone oil as a cryoprotectant. The data set was collected
at the European Synchrotron Radiation Facility (ESRF, Grenoble,
France) ID13-EH2 beamline. The atomic coordinates and the
related experimental data were deposited at Protein Data Bank
under PDB ID code 2x5o.
4.5. Chemistry. Chemicals were obtained from Acros, Aldrich
Chemical Co., and Fluka and used without further purification.
THF was kept over sodium and distilled immediately prior to
use. Analytical TLC was performed on silica gel Merck 60
F254 plates (0.25 mm), using visualization with UV light and
ninhydrin. Column chromatography was carried out on silica gel
60 (particle size 240–400 mesh). HPLC analyses were performed
on an Agilent Technologies 1100 instrument with a G1365B
UV–vis detector, a G1316A thermostat, and a G1313A auto-
sampler, using a Phenomenex Luna 5 μm C18 column (4.6 mm ꢀ
150 mm). The eluent consisted of trifluoroacetic acid (0.1% in
water) and methanol or acetonitrile. Microwave-assisted reac-
tions were performed using a CEM Discover microwave reactor
(CEM Corp.). Melting points were determined on a Reichert hot
stage microscope and are uncorrected. 1H NMR and 13C NMR
spectra were recorded at 300 and 75 MHz on a Bruker
AVANCE DPX300 spectrometer in CDCl3 or DMSO-d6 solu-
tion, with TMS as the internal standard. Spectra were assigned
using gradient COSY, HSQC, and 1H-coupled 13C NMR
experiments. IR spectra were recorded on a Perkin-Elmer
1600 FT-IR spectrometer. Mass spectra were obtained using a
VGAnalytical Autospec Q mass spectrometer. Optical rotations
were measured on a Perkin-Elmer 1241 MC polarimeter. The
reported values for specific rotation are average values of 10
successive measurements using an integration time of 5 s. Since
chiral 5-benzylidenerhodanines and 5-benzylidenethiazolidine-
2,4-diones showed ellipticity and circular dichroism, their spe-
cific rotation values are not reported. Microanalyses were
performed on a Perkin-Elmer C, H, N analyzer 240 C. Analyses
indicated by the symbols of the elements were within 0.4% of the
theoretical values. The purity of the tested compounds was
established to be g95%.
(Z)-5-(4-Aminobenzylidene)thiazolidine-2,4-dione (16). (Z)-5-(4-
Nitrobenzylidene)thiazolidine-2,4-dione (12, 0.500 g, 2.00 mmol)
was dissolved in a mixture of tetrahydrofuran (15 mL) and metha-
nol (15 mL), 10% Pd-C (150 mg) was added, and the mixture was
stirred under hydrogen atmosphere for 5 h. The catalyst was filte-
red off and the solvent removed under reduced pressure. Yield,
95% (0.418 g); yellow crystals; mp 225-228 ꢀC (lit.36 231 ꢀC); 1H
NMR (DMSO-d6) δ 6.08 (br s, 2H, NH2), 6.66 (d, 2H, J=8.7 Hz,
Ar-H-3,5), 7.28 (d, 2H, J = 8.7 Hz, Ar-H-2,6), 7.60 (s, 1H, CH),
12.20 (br s, 1H, NH).
(R,Z)-Dimethyl 2-(3-((4-((2,4-Dioxothiazolidin-5-ylidene)methyl)-
phenylamino)methyl)benzamido)pentanedioate [(R)-30]. To a stirred
solution of amine 16 (0.750 g, 3.41 mmol) in anhydrous DMF
(40 mL), benzaldehyde (R)-4 (1.048 g, 3.41 mmol) and NaCNBH3
(0.258 g, 4.09 mmol) were added. The mixture was stirred overnight
at room temperature under an argon atmosphere. The solvent was
evaporated under reduced pressure and the crude product recrys-
tallized from ethyl acetate. Yield, 32% (0.558 g); yellow crystals; mp
148-153 ꢀC; IR (KBr) ν = 3583, 3488, 3434, 2850, 2040, 1718,
1639, 1561, 1541, 1510, 1456, 1341, 1292, 1185, 1147, 812, 626 cm-1
;
1H NMR (DMSO-d6) δ 1.95-2.19 (m, 2H, CHCH2CH2), 2.45 (t,
2H, J=7.5 Hz, CHCH2CH2), 3.58 (s, 3H, CH3), 3.64 (s, 3H, CH3),
4.41-4.50 (m, 3H, CH2NH, CHCH2CH2), 6.72 (d, 2H, J=8.7 Hz,
Ar-H-2,6), 7.28 (t, 1H, J=6.0HzCH2NH), 7.33 (d, 2H, J=8.7Hz,
Ar-H-3,5), 7.44 (t, 1H, J=7.5 Hz, Ar-H-50), 7.52 (d, 1H, J=7.5
Hz, Ar-H-40), 7.60 (s, 1H, CHdC), 7.77 (d, 1H, J=7.5 Hz, Ar-H-
60), 7.88 (s, 1H, Ar-H-20), 8.74 (d, 1H, J= 7.5 Hz, CONH), 12.28
(br s, 1H, CONHCO); MS (ESI-) m/z (%)=510([M - H]-, 100),
478 (5), 439 (20), 407 (20), 281 (30), 255 (30). Anal. (C25H25N3O7S)
C, H, N.
(R,Z)-2-(3-((4-((2,4-Dioxothiazolidin-5-ylidene)methyl)phenyl-
amino)methyl)benzamido)pentanedioic Acid [(R)-32]. To a stirred
solution of the methyl ester (R)-30 (0.115 g, 0.226 mmol) in MeOH/
water (1:1) (10 mL), 2.2 M LiOH (0.41 mL, 0.902 mmol) was
added. The mixture was stirred overnight at room temperature.
The solution was neutralized with 1 M HCl and concentrated under
reduced pressure. The residual aqueous solution was acidified with
1 M HCl to pH 2 and the product extracted with ethyl acetate
(3ꢀ15 mL). The combined organic phases were washed with brine
(2ꢀ15mL), driedoverNa2SO4, filtered and the solvent evaporated
under reduced pressure. Yield, 73% (79.8 mg); yellow crystals; mp
125-128 ꢀC; IR (KBr) ν = 3412, 3237, 2776, 2044, 1719, 1685,
1639, 1618, 1574, 1561, 1538, 1524, 1421, 1329, 1291, 1185, 1148,
4.5.1. Synthesis of (R)-32. (R)-Dimethyl 2-(3-Formylbenzamido)-
pentanedioate [(R)-4]. A solution of H-Glu(OMe)-OMe HCl
3
(0.500 g, 2.36 mmol), 3-carboxybenzaldehyde (2, 0.426 g, 2.83
mmol), and HOBt (0.488 g, 3.07 mmol) in DMF (25 mL) was
prepared and the pH adjusted to 8 with N-methylmorpholine.
EDC (0.634 g, 3.31 mmol) was added and the reaction mixture
stirred overnight at room temperature. The solvent was evapo-
rated in vacuo and the oily residue dissolved in ethyl acetate
(60 mL) and washed successively with 10% citric acid (3ꢀ20 mL),
saturated aqueous NaHCO3 solution (3 ꢀ 20 mL), and brine
(20 mL). The organic phase was dried over Na2SO4, filtered
and the solvent evaporated under reduced pressure. The crude
product was purified with flash column chromatography using
1
1027, 815, 690, 610 cm-1; H NMR (DMSO-d6) δ 1.89-2.16

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6591
(m, 2H, CHCH2CH2), 2.36 (t, 2H, J = 7.5 Hz, CHCH2CH2),
4.37-4.43 (m, 3H, CH2NH, CHCH2CH2), 6.72 (d, 2H, J=8.7Hz,
Ar-H-2,6), 7.27 (t, 1H, J = 5.7 Hz, CH2NH), 7.33 (d, 2H,
J= 8.7 Hz, Ar-H-3,5), 7.44 (t, 1H, J= 7.7 Hz, Ar-H-50), 7.51 (d,
1H, J=7.7 Hz, Ar-H-40), 7.60 (s, 1H, CHdC), 7.78 (d, 1H, J=7.7
Hz, Ar-H-60), 7.89 (s, 1H, Ar-H-20), 8.58 (d, 1H, J = 7.5 Hz,
CONH), 12.32 (br s, 3H, CONHCO, 2 ꢀ COOH); MS (ESI-)
m/z (%)=482 ([M - H]-, 100), 464 (3), 353 (22). Anal. (C23H21-
N3O7S) C, H, N.
3200, 2925, 2850, 1716, 1606, 1574, 1549, 1491, 1422, 1275, 1207,
1063, 678 cm–1. 1H NMR (DMSO-d6) δ 1.80–2.10 (m, 2H,
CHCH2CH2), 2.27 (t, 2H, J = 7.5 Hz, CHCH2CH2), 4.27–
4.34 (m, 1H, CHCH2CH2), 4.63 (s, 2H, CH2O), 7.11 (dd, 1H,
3J = 8.1 Hz, 4J = 2.3 Hz, Ar-H-6), 7.17–7.22 (m, 2H, Ar-H-2,4),
7.47 (t, 1H, J = 8.1 Hz, Ar-H-5), 7.61 (s, 1H, CHC), 8.38 (d, 1H,
J = 8.1 Hz, CONH), 12.44 (br s, 2H, 2 ꢀ COOH), 13.84 (br s,
1H, CSNHCO); 13C NMR (DMSO-d6, 75 MHz) δ 26.95 (CH2),
30.95 (CH2), 51.90 (CHNH), 67.60 (CH2O), 117.20 (Ar-C/
CHC), 118.26 (Ar-C/CHC), 123.97 (Ar-C/CHC), 127.01 (Ar-
C/CHC), 131.38 (Ar-C/CHC), 132.20 (Ar-C/CHC), 135.19 (Ar-
C/CHC), 159.11 (Ar-C/CHC), 168.46 (CO), 170.26 (CO), 173.78
(CO), 174.61 (CO), 196.59 (CS); MS (ESI) m/z (%) = 423 ([M –
H]–, 100), 369 (25), 336 (80), 302 (35). HRMS for C17H15N2-
O7S2: calculated 423.0321; found 423.0332. Anal. (C17H16N2O7S2)
C, H, N.
4.5.2. Synthesis of (R)-46. (R)-Dimethyl 2-(2-Chloroacet-
amido)pentanedioate [(R)-36]. To a solution of H-Glu(OMe)-OMe
3
HCl [(R)-1)] (1.00 g, 4.72 mmol) and triethylamine (1.97 mL,
14.18 mmol) in dichloromethane (30 mL), cooled to 0 ꢀC on an ice
bath, a solution of chloroacetyl chloride (414 μL, 5.20 mmol) in
dichloromethane (5 mL) was added dropwise. The mixture was
allowed to reach room temperature and stirred for 3 h, after which it
was washed with water (2 ꢀ 15 mL), 10% citric acid (2 ꢀ 15 mL),
and brine (2 ꢀ 10 mL), dried over Na2SO4, and concentrated under
4.5.3. Synthesis of (R)-66. 5-(3-Nitrobenzyl)-2-thioxothiazo-
lidin-4-one (55). To a stirred suspension of 11 (1.98 g, 7.45 mmol)
in toluene (100 mL) was added diethyl 2,6-dimethyl-1,4-dihy-
dro-3,5-pyridinedicarboxylate (2.45 g, 9.69 mmol) and silica gel
60 (7.45 g, 1 g/mmol), previously activated by heating at 120 ꢀC
for 5 h. The mixture was heated to 100 ꢀC for 24 h under an
argon atmosphere in the dark, cooled, and filtered. The filter
cake was rinsed with ethyl acetate. The combined filtrate and
rinse were evaporated to dryness. The residue was redissolved in
ethyl acetate (100 mL), washed with 1 M HCl (3ꢀ50 mL), and
brine (50 mL), dried over Na2SO4, filtered and the solvent
evaporated under reduced pressure. The crude product was
purified by flash column chromatography using dichloro-
methane/methanol (40:1) as an eluent. Yield, 84% (1.68 g);
yellow crystals; mp 165–168 ꢀC; IR (KBr) ν = 3068, 2874, 1718,
1581, 1529, 1454, 1349, 1307, 1269, 1234, 1208, 1103, 1083, 939,
908, 833, 814, 736, 722, 680, 662, 585, 509 cm–1; 1H NMR
23
reduced pressure. Yield, 90% (1.07 g); colorless oil; [R]546 þ37.0
(c 0.319, DMF); IR (neat) ν = 3319, 3008, 2957, 2851, 2090, 1738,
1732, 1682, 1539, 1436, 1372, 1338, 1219, 1176, 1123, 1087, 1015, 985,
825, 778 cm–1 (lit.37,38 3317, 2956, 1733, 1667, 1528, 1206, 1170 cm–1);
1H NMR (DMSO-d6) δ 1.81–2.09 (m, 2H, CHCH2CH2), 2.39
(t, 2H, J = 7.5 Hz, CHCH2CH2), 3.59 (s, 3H, CH3), 3.64 (s, 3H,
CH3), 4.11 (s, 2H, CH2Cl), 4.28–4.36 (m, 1H, CHCH2CH2), 8.63 (d,
1H, J = 7.5 Hz, NH).
(R)-Dimethyl 2-(2-(3-Formylphenoxy)acetamido)pentane-
dioate [(R)-40]. (R)-36 (252 mg, 1.00 mmol), 3-hydroxybenzal-
dehyde (38, 122 mg, 1.00 mmol), potassium carbonate (276 mg,
2.00 mmol), and potassium iodide (199 mg, 1.20 mmol) were
suspended in acetonitrile (10 mL) and heated at 85 ꢀC for 3 h.
The solvent was evaporated, the residue dissolved in ethyl
acetate (20 mL), washed with water (2ꢀ10 mL), and brine
(2 ꢀ 10 mL), dried over Na2SO4, and concentrated under
2
3
(DMSO-d6) δ 3.40 (dd, 1H, AB system, J = 14.1 Hz, J =
23
2
reduced pressure. Yield, 89% (300 mg); colorless oil; [R]546
7.9 Hz, HA from Ar-CH2CH), 3.52 (dd, 1H, AB system, J =
14.1 Hz, 3J =5.4 Hz, HB from Ar-CH2CH), 5.12 (dd, 1H, J1 =
7.9 Hz, J2 = 5.4 Hz, SCHCO), 7.63 (dt, 1H, 3J = 7.7 Hz,
4J = 0.8 Hz, Ar-H-5), 7.73 (d, 1H, J = 7.7 Hz, Ar-H-6), 8.12–
8.16 (m, 2H, Ar-H-2,4), 13.17 (s, 1H, CSNHCO); MS (ESI) m/z
(%) = 267 ([M – H]–, 100). HRMS (ESI) for C10H7N2O3S2:
calculated 266.9898; found 266.9888.
þ22.1 (c 0.348, DMF); IR (neat) ν = 3355, 3067, 3002, 2955,
2850, 2736, 2091, 1717, 1616, 1594, 1539, 1488, 1456, 1436, 1386,
1374, 1260, 1173, 1082, 1062, 1016, 996, 870, 792, 744, 683 cm–1;
1H NMR (DMSO-d6) δ 1.84–2.12 (m, 2H, CHCH2CH2), 2.35 (t,
2H, J = 7.5 Hz, CHCH2CH2), 3.58 (s, 3H, CH3), 3.63 (s, 3H,
CH3), 4.34–4.42 (m, 1H, CHCH2CH2), 4.63 (d, 1H, AB system,
2J = 15.0 Hz, HA from CH2O), 4.69 (d, 1H, AB system, 2J =
15.0 Hz, HB from CH2O), 7.29–7.33 (m, 1H, Ar-H), 7.44 (d, 1H,
J = 2.7 Hz, Ar-H), 7.52–7.59 (m, 2H, 2 ꢀ Ar-H), 8.55 (d, 1H,
J = 7.5 Hz, NH), 9.98 (s, 1H, CHO); MS (ESI) m/z (%) = 360
(MNaþ, 100), 338 (MHþ, 63), 278 (50), 246 (48), 218 (45).
HRMS for C16H19NO7Na: calculated 360.1059; found 360.1068.
Anal. (C16H19NO7) C, H, N.
5-(3-Aminobenzyl)-2-thioxothiazolidin-4-one (58). To a stirred
solution of 55 (1.71 g, 6.38 mmol) in ethanol (100 mL), tin(II)
chloride (6.05 g, 31.9 mmol) was added, and the mixture was
heated under reflux for 2 h. The solvent was evaporated under
reduced pressure. The residue was dissolved in saturated aque-
ous NaHCO3 solution (100 mL) and the product extracted with
ethyl acetate (5 ꢀ 50 mL). The combined organic extracts were
washed with brine (2 ꢀ 50 mL), dried over Na2SO4 and the
solvent removed under reduced pressure. Yield, 96% (1.46 g);
yellow crystals; mp 71–74 ꢀC; IR (KBr) ν = 3414, 2368, 2345,
1618, 1431, 1296, 1254, 1220, 1182, 1066, 863, 778, 723, 618, 475
(R,Z)-Dimethyl 2-(2-(3-((2-Oxo-4-thioxothiazolidin-5-ylidene)-
methyl)phenoxy)acetamido)pentanedioate [(R)-44]. (R)-44 was
prepared from (R)-40 (500 mg, 1.48 mmol) and rhodanine (9,
179 mg, 1.35 mmol) according to the procedure described in
the synthesis of 12. Yield, 57% (348 mg); yellow crystals; mp
121–124 ꢀC; IR (KBr) ν = 3417, 2920, 2849, 1721, 1641, 1436,
1296, 1254, 1213, 1176, 1059, 776 cm–1; 1H NMR (DMSO-d6) δ
1.85–2.13 (m, 2H, CHCH2CH2), 2.35 (t, 2H, J = 7.5 Hz,
CHCH2CH2), 3.58 (s, 3H, CH3), 3.63 (s, 3H, CH3), 4.34–4.42
(m, 1H, CHCH2CH2), 4.61 (d, 1H, AB system, 2J = 15.6 Hz, HA
1
2
cm–1; H NMR (DMSO-d6) δ 2.96 (dd, 1H, AB system, J =
14.0 Hz, 3J =9.7 Hz, HA from Ar-CH2CH), 3.23 (dd, 1H, AB
system, 2J = 14.0 Hz, 3J =4.3 Hz, HB from Ar-CH2CH), 4.93
(dd, 1H, J1 = 9.7 Hz, J2 = 4.3 Hz, SCHCO), 6.36–6.46 (m, 3H,
Ar-H-2,4,6), 6.95 (t, 1H, J = 7.6 Hz, Ar-H-5), signals for Ar-
NH2 and CSNHCO not seen; MS (ESI) m/z (%) = 239 (MHþ,
33), 108 (85), 77 (100). HRMS (ESI) for C10H11N2OS2: calcu-
lated 239.0313; found 239.0311.
2
from CH2O), 4.66 (d, 1H, AB system, J = 15.6 Hz, HB from
CH2O), 7.10 (dd, 1H, 3J = 8.0 Hz, 4J = 2.3 Hz, Ar-H-6), 7.15 (s,
1H, Ar-H-2), 7.22 (d, 1H, J = 7.8 Hz, Ar-H-4), 7.48 (dd, 1H,
J1 = 8.0 Hz, J2 = 7.8 Hz, Ar-H-5), 7.61 (s, 1H, CHC), 8.55 (d,
1H, J = 7.8 Hz, CONH), 13.84 (br s, 1H, CSNHCO); MS (ESI)
m/z (%) = 475 (MNaþ, 100), 453 (MHþ, 90), 421 (43), 393 (50),
184 (50), 77 (70). Anal. (C19H20N2O7S2) C, H, N.
4-((3-((4-Oxo-2-thioxothiazolidin-5-yl)methyl)phenylamino)-
methyl)benzoic Acid (63). A suspension of amine 58 (0.751 g,
3.15 mmol) and 4-carboxybenzaldehyde (3, 0.543 g, 3.62 mmol)
in methanol (60 mL) was heated at 65 ꢀC for 2 h and then cooled
to room temperature. NaCNBH3 (257 mg, 4.10 mmol) was
added and the mixture stirred overnight under an argon atmo-
sphere. The solvent was evaporated under reduced pressure. The
residue was dissolved in ethyl acetate (40 mL) and washed with
water (2ꢀ20 mL) and brine (20 mL). The organic phase was
dried over Na2SO4, filtered and the solvent removed under
(R,Z)-2-(2-(3-((2-Oxo-4-thioxothiazolidin-5-ylidene)methyl)-
phenoxy)acetamido)-pentanedioic acid [(R)-46]. (R)-46 was pre-
pared by hydrolysis of (R)-44 (200 mg, 0.442 mmol) according to
the procedure described in the synthesis of (R)-32. Yield, 75%
(141 mg); yellow crystals; mp 195–198 ꢀC; IR (KBr) ν = 3371,

6592 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Zidar et al.
reduced pressure. The crude product was purified with flash
column chromatography using dichloromethane/methanol (20:1)
as an eluent. Yield, 45% (0.528 g); yellow crystals; mp 79–
81 ꢀC; IR (KBr) ν = 2852, 1686, 1605, 1426, 1222, 1176, 1069,
844, 763, 694, 670, 498 cm–1. 1H NMR (DMSO-d6) δ 2.97 (dd,
1H, AB system, 2J = 14.0 Hz, 3J =9.8 Hz, HA from Ar-
CH2CH), 3.23 (dd, 1H, AB system, 2J = 14.0 Hz, 3J = 4.2 Hz,
HB from Ar-CH2CH), 4.32 (s, 2H, CH2NH), 4.93 (dd, 1H, J1 =
9.8 Hz, J2 = 4.2 Hz, SCHCO), 6.36–6.46 (m, 4H, Ar-H-2,4,6,
CH2NH), 6.97 (t, 1H, 3J = 7.7 Hz, Ar-H-5), 7.45 (d, 2H, J =
8.2 Hz, Ar-H-30,50), 7.89 (d, 2H, J = 8.2 Hz, Ar-H-20,60), 12.79
(br s, 1H, COOH), 13.13 (br s, 1H, CSNHCO); MS (ESI)
m/z (%) = 373 (MHþ, 94), 355 (100). Anal. (C18H16N2O3S2) C,
H, N.
1573, 1521, 1493, 1454, 1421, 1336, 1292, 1218, 1182, 1160, 1080,
1020, 975, 911, 820, 796, 755, 694 cm–1; 1H NMR (DMSO-d6) δ
4.44 (d, 2H, J = 6.0 Hz, CH2NH), 6.71 (d, 2H, J = 8.7 Hz, Ar-
H-20,60), 7.27–7.34 (m, 3H, CH2NH, Ar-H-30,50), 7.47 (t, 1H,
J = 7.7 Hz, Ar-H-5), 7.59–7.61 (m, 2H, CHC, Ar-H-4), 7.82 (d,
1H, J = 7.7 Hz, Ar-H-6), 7.94 (s, 1H, Ar-H-2), 12.29 (br s, 1H,
CONHCO/COOH), 12.92 (br s, 1H, CONHCO/COOH); MS
(ESI) m/z (%) = 353 ([M – H]–, 72), 325 (10), 311 (10), 282 (100),
255 (15), 218 (30). HRMS for C18H13N2O4S: calculated
353.0596; found 353.0603. Anal. (C18H14N2O4S 0.2H2O) C,
3
H, N.
(S,Z)-Methyl 6-(tert-Butoxycarbonylamino)-2-(3-((4-((2,4-di-
oxothiazolidin-5-ylidene)methyl)phenylamino)methyl)benzamido)-
hexanoate [(S)-73]. A solution of H-Lys(Boc)-OMe HCl (352
3
mg, 1.19 mmol), carboxylic acid 69 (400 mg, 1.13 mmol), and
HOBt (215 mg, 1.35 mmol) in DMF (25 mL) was prepared and
the pH adjusted to 8 with N-methylmorpholine. EDC (281 mg,
1.47 mmol) was added and the mixture stirred overnight at room
temperature. The solvent was evaporated under reduced pres-
sure. The residue was dissolved in ethyl acetate (50 mL) and
washed successively with 10% citric acid (3 ꢀ 15 mL) and brine
(2 ꢀ 10 mL). The organic phase was dried over Na2SO4, filtered
and the solvent evaporated under reduced pressure. The crude
product was crystallized from ethyl acetate. Yield, 40% (270
mg); yellow crystals; mp 125–130 ꢀC; IR (KBr) ν = 3551, 3475,
3413, 2940, 2771, 2066, 1734, 1718, 1684, 1636, 1617, 1560, 1577,
1540, 1522, 1337, 1288, 1225, 1184, 1149, 1018, 820, 756, 632,
608 cm–1; 1H NMR (DMSO-d6) δ 1.29–1.35 (m, 13H, t-Bu,
CH2CH2CH2CH2CH), 1.78–1.82 (m, 2H, CH2CH2CH2CH2-
CH), 2.89–2.91 (m, 2H, CH2CH2CH2CH2CH), 3.64 (s, 3H,
CH3), 4.36–4.43 (m, 3H, CH2NHAr, CH2CH2CH2CH2CH),
6.70–6.75 (m, 3H, BocNH/CH2NHAr, Ar-H-20,60), 7.26–7.34
(m, 3H, BocNH/CH2NHAr, Ar-H-30,50), 7.44 (t, 1H, J = 7.5
Hz, Ar-H-5), 7.52 (d, 1H, J = 7.5 Hz, Ar-H-4), 7.61 (s, 1H,
CHC), 7.78 (d, 1H, J = 7.5 Hz, Ar-H-6), 7.88 (s, 1H, Ar-H-2),
8.67 (d, 1H, J = 7.2 Hz, CONH), 12.28 (br s, 1H, CONHCO);
MS (ESI) m/z (%) = 595 ([M – H]–, 100), 524 (2), 495 (3), 397
(3), 265 (20). HRMS for C30H35N4O7S: calculated 595.2226;
(2R)-Dimethyl 2-(4-((3-((4-Oxo-2-thioxothiazolidin-5-yl)-
methyl)phenylamino)methyl)benzamido)pentanedioate [(R)-65].
A solution of H-Glu(OMe)-OMe HCl [(R)-1] (212 mg, 1.00
3
mmol), 63 (372 mg, 1.00 mmol), and HOBt (191 mg, 1.20 mmol)
in DMF (25 mL) was prepared and pH adjusted to 8 with
N-methylmorpholine. EDC (249 mg, 1.47 mmol) was added and
the mixture stirred overnight at room temperature. The solvent
was evaporated under reduced pressure. The residue was dis-
solved in ethyl acetate (50 mL) and washed successively with
10% citric acid (3 ꢀ 15 mL) and brine (2 ꢀ 10 mL). The organic
phase was dried over Na2SO4, filtered and the solvent evapo-
rated under reduced pressure. The crude product was purified by
flash column chromatography using dichloromethane/methanol
(20:1) as an eluent. Yield, 22% (117 mg); yellow crystals; mp
114–116 ꢀC; IR (KBr) ν = 3419, 3340, 3251, 2948, 1742, 1637,
1607, 1526, 1499, 1437, 1337, 1281, 1217, 1181, 1108, 1070, 1018,
986, 837, 768, 699, 500 cm–1; 1H NMR (DMSO-d6) δ 1.94–2.18
(m, 2H, CHCH2CH2), 2.45 (t, 2H, J = 7.5 Hz, CHCH2CH2),
2.97 (dd, 1H, AB system, 2J = 14.1 Hz, 3J = 9.8 Hz, HA3from
2
Ar-CH2CH), 3.24 (dd, 1H, AB system, J = 14.1 Hz, J =
4.3 Hz, HB from Ar-CH2CH), 3.58 (s, 3H, CH3), 3.64 (s, 3H,
CH3), 4.32 (d, 2H, J = 5.0 Hz, CH2NH), 4.42–4.49 (m, 1H,
CHCH2CH2), 4.93 (dd, 1H, J1 = 9.8 Hz, J2 = 4.3 Hz, SCHCO),
6.33 (t, 1H, J = 6.2 Hz, CH2NH), 6.38–6.44 (m, 2H, Ar-H-4,6),
6.48 (s, 1H, Ar-H-2), 6.97 (t, 1H, J = 7.8 Hz, Ar-H-5), 7.44 (d,
2H, J = 8.3 Hz, Ar-H-30,50), 7.82 (d, 2H, J = 8.3 Hz, Ar-H-
20,60), 8.66 (d, 2H, J=7.5 Hz, CONH), 13.14 (br s, 1H, CSNHCO);
MS (ESI) m/z (%) = 530 (MHþ, 100). Anal. (C25H27N3O6S2) C,
H, N.
found 595.2211. Anal. (C30H36N4O7S 0.5H2O) C, H, N.
3
(S,Z)-6-(tert-Butoxycarbonylamino)-2-(3-((4-((2,4-dioxothia-
zolidin-5-ylidene)methyl)phenylamino)methyl)benzamido)hexanoic
Acid [(S)-74]. (S)-74 was prepared by hydrolysis of (S)-73 (200
mg, 0.335 mmol) according to the procedure described in the
synthesis of (R)-32 (instead of 4.0 equiv of LiOH, only an
amount of 2.5 equiv was used). Yield, 96% (187 mg); yellow
crystals; mp 110–115 ꢀC; IR (KBr) ν = 3545, 3414, 3236, 2976,
2931, 2757, 2042, 1637, 1617, 1590, 1540, 1364, 1330, 1283, 1184,
(2R)-2-(4-((3-((4-Oxo-2-thioxothiazolidin-5-yl)methyl)phenyl-
amino)methyl)benzamido)pentanedioic Acid [(R)-66]. (R)-66 was
prepared from (R)-65 (100 mg, 0.189 mmol) according to the
procedure described in the synthesis of (R)-32. Yield, 96% (91
mg); yellow crystals; mp 89–91 ꢀC; IR (KBr) ν = 2926, 2060,
1735, 1607, 1542, 1438, 1184, 1071, 696 cm–1. 1H NMR (DMSO-
d6) δ 1.90–2.13 (m, 2H, CHCH2CH2), 2.35 (t, 2H, J = 7.4 Hz,
CHCH2CH2), 2.97 (dd, 1H, AB system, 2J = 14.2 Hz, 3J = 9.7
Hz, HA from Ar-CH2CH), 3.24 (dd, 1H, AB system, 2J = 14.2
Hz, 3J = 4.2 Hz, HB from Ar-CH2CH), 4.31 (s, 2H, CH2NH),
4.36–4.43 (m, 1H, CHCH2CH2), 4.94 (dd, 1H, J1 = 9.7 Hz, J2 =
4.2 Hz, SCHCO), 6.37–6.44 (m, 2H, Ar-H-4,6), 6.49 (s, 1H, Ar-
H-2), 6.97 (t, 1H, J = 7.8 Hz, Ar-H-5), 7.44 (d, 2H, J = 8.2 Hz,
Ar-H-30,50), 7.83 (d, 2H, J = 8.2 Hz, Ar-H-20,60), 8.52 (d, 2H,
J = 7.7 Hz, CONH), 12.46 (br s, 2H, COOH), 13.15 (s, 1H,
CSNHCO), signal for CH2NH not seen; MS (ESI) m/z (%) =
1
1147, 1020, 820, 745, 607 cm–1; H NMR (DMSO-d6) δ 1.30–
1.39 (m, 13H, t-Bu, CH2CH2CH2CH2CH), 1.74–1.80 (m, 2H,
CH2CH2CH2CH2CH), 2.87–2.92 (m, 2H, CH2CH2CH2CH2-
CH), 4.30–4.43 (m, 3H, CH2NHAr, CH2CH2CH2CH2CH),
6.70–6.77 (m, 3H, BocNH/CH2NHAr, Ar-H-20,60), 7.26–7.34
(m, 3H, BocNH/CH2NHAr, Ar-H-30,50), 7.43 (t, 1H, J =7.5
Hz, Ar-H-5), 7.51 (d, 1H, J = 7.5 Hz, Ar-H-4), 7.60 (s, 1H,
CHC), 7.78 (d, 1H, J = 7.5 Hz, Ar-H-6), 7.88 (s, 1H, Ar-H-2),
8.52 (d, 1H, J = 7.5 Hz, CONH), 12.28 (br s, 1H, CONHCO/
COOH), 12.56 (br s, 1H, CONHCO/COOH); MS (ESI) m/z
(%) = 581 ([M – H]–, 100), 555 (1), 538 (1), 507 (10), 265 (20).
HRMS for C29H33N4O7S: calculated 581.2070; found 581.2075.
Anal. (C29H34N4O7S) C, H, N.
500 ([M – H]–, 100). Anal. (C23H23N3O6S2 0.25EtOAc) C, H, N.
3
4.5.4. Synthesis of (S)-75. (Z)-3-((4-((2,4-Dioxothiazolidin-
5-ylidene)methyl)phenylamino)methyl)benzoic Acid (69). The
suspension of amine 16 (220 mg, 2.11 mmol) and 3-carboxy-
benzaldehyde (2, 365 mg, 2.43 mmol) in methanol (60 mL) was
heated at 65 ꢀC for 2 h, then cooled to room temperature.
NaCNBH3 (172 mg, 2.74 mmol) was added and the mixture
stirred overnight under an argon atmosphere. The solid was
filtered off, washed with methanol, and dried in air. Yield, 70%
(0.523 mg); yellow crystals; mp 270–275 ꢀC; IR (KBr) ν = 3551,
3415, 3333, 3237, 3133, 3037, 2788, 2040, 1721, 1688, 1639, 1616,
(S,Z)-5-Carboxy-5-(3-((4-((2,4-dioxothiazolidin-5-ylidene)-
methyl)phenylamino)methyl)benzamido)pentan-1-aminium Chloride
[(S)-75]. A solution of (S)-74 (146 mg, 0.250 mmol) in glacial
acetic acid (10 mL) was saturated with gaseous HCl and stirred
at room temperature for 1 h. The solvent was removed under
reduced pressure, and the solid was filtered off and washed
with diethyl ether. Yield, 67% (87 mg); yellow crystals; mp
165–170 ꢀC; IR (KBr) ν = 3552, 3475, 3414, 3235, 2934, 2737,
1734, 1700, 1636, 1617, 1323, 1289, 1186, 1151, 1014, 822, 750,
687, 610 cm–1. 1H NMR (DMSO-d6) δ 1.34–1.64 (m, 4H,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6593
CH2CH2CH2CH2CH), 1.78–1.86 (m, 2H, CH2CH2CH2CH2-
CH), 2.74–2.80 (m, 2H, CH2CH2CH2CH2CH), 4.34–4.43 (m,
3H, CH2NHAr, CH2CH2CH2CH2CH), 6.73 (d, 2H, J = 8.7 Hz,
Ar-H-20,60), 7.33 (d, 2H, J = 8.7 Hz, Ar-H-30,50), 7.43 (t, 1H,
J = 7.5 Hz, Ar-H-5), 7.52 (d, 1H, J = 7.5 Hz, Ar-H-4), 7.60 (s,
1H, CHC), 7.79–7.92 (m, 5H, NH3þ, Ar-H-2,6), 8.52 (d, 1H,
J = 7.5 Hz, CONH), 12.29 (br s, 1H, CONHCO), broad signals
for COOH and CH2NHAr not seen; MS (ESI) m/z (%) = 481
([M – HCl – H]–, 100), 470 (1), 438 (3), 410 (3), 286 (12), 255 (15),
212 (22). HRMS for C24H25N4O5S: calculated 481.1546; found
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(18) Michaud, C.; Blanot, D.; Flouret, B.; van Heijenoort, J. Partial
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481.1542. Anal. (C24H27ClN4O5S 2.2H2O) C, H, N.
3
Acknowledgment. This work was supported by the EU FP6
Integrated Project EUR-INTAFAR (Project LSHM-CT-2004-
512138) and by the Slovenian Research Agency (Grant P1-0208).
ꢀ ꢁ
ꢀ ꢀ
(20) Tomasic, T.; Peterlin Masic, L. Rhodanine as a privileged scaffold
in drug discovery. Curr. Pharm. Des. 2009, 16, 1596–1629.
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Supporting Information Available: Experimental procedures
and characterization of intermediate and target compounds; IR
and 13C spectra of representative compounds; results from
elemental analysis and HRMS. This material is available free
of charge via the Internet at http://pubs.acs.org.
ꢀ
ꢀ
ꢀ
(22) Kotnik, M.; Oblak, M.; Stefanic Anderluh, P.; Prezelj, A.; Huml-
ꢀ
jan, J.; Plantan, I.; Urleb, U.; Solmajer, T. Preparation of Benzo-
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A1, 2008.
(23) Splitting pattern and coupling constant of the signal of rhodanine
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due to a long-range coupling with methyne proton. On the basis of
13C and X-ray data of similar compounds, coupling constants of 6
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