Synthesis and biological evaluation of homoserine lactone derived ureas as antagonists of bacterial quorum sensing

Article abstract of DOI:10.1016/j.bmc.2006.03.017

A series of 15 racemic alkyl- and aryl-N-substituted ureas, derived from homoserine lactone, were synthesized and tested for their ability to competitively inhibit the action of 3-oxohexanoyl-l-homoserine lactone, the natural inducer of bioluminescence in

Full text of DOI:10.1016/j.bmc.2006.03.017

Bioorganic & Medicinal Chemistry 14 (2006) 4781–4791
Synthesis and biological evaluation of homoserine lactone
derived ureas as antagonists of bacterial quorum sensing
a
b
a
a
`
Marine Frezza, Sandra Castang, Jane Estephane, Laurent Soulere,
Christian Deshayes,a Bernard Chantegrel,a William Nasser,b Yves Queneau,a
Sylvie Reverchonb and Alain Doutheaua,*
aLaboratoire de Chimie Organique, UMR CNRS-UCBL-INSA 5181, Institut National des Sciences Applique´es,
20 avenue A. Einstein, 69621 Villeurbanne, France
b
ˆ
´
Unite´ de Microbiologie et Ge´ne´tique, UMR CNRS-INSA-UCBL 5122, Bat. A. Lwoff, Universite Lyon 1,
69622 Villeurbanne, France
Received 19 January 2006; revised 1 March 2006; accepted 14 March 2006
Available online 29 March 2006
Abstract—A series of 15 racemic alkyl- and aryl-N-substituted ureas, derived from homoserine lactone, were synthesized and tested
for their ability to competitively inhibit the action of 3-oxohexanoyl-L-homoserine lactone, the natural inducer of bioluminescence
in the bacterium Vibrio fischeri. N-alkyl ureas with an alkyl chain of at least 4 carbon atoms, as well as certain ureas bearing a phenyl
group at the extremity of the alkyl chain, were found to be significant antagonists. In the case of N-butyl urea, it has been shown that
the antagonist activity was related to the inhibition of the dimerisation of the N-terminal domain of ExpR, a protein of the receptor
LuxR family. Molecular modelling suggested that this would result from the formation of an additional hydrogen bond in the
protein acylhomoserine lactone binding cavity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
disrupting agents were also shown to be active against
biofilm formation, whereas traditional antibiotics were
ineffective for killing bacteria which adopt a biofilm
life-style.3
In many bacteria, the expression of some sets of genes is
regulated in response to population density. This pro-
cess is based on the production, by bacteria, of small sig-
nalling molecules, called autoinducers (AI), which are
released in the extracellular medium and sensed by other
individual bacteria through specific receptors.1 This cell-
to-cell communication system is termed quorum sensing
(QS) and has been detected in a growing number of
pathogenic bacteria. In these bacteria, QS prevents pre-
mature virulence factor production, and the subsequent
activation of host defence responses, and allows them to
invade an infected host once their numbers are sufficient
enough to carry out a full-scale attack. Thus, QS has
emerged as a possible target pathway for the design of
a novel antimicrobial therapy since QS antagonists, that
is, molecules that can bind to quorum sensing receptors
but block them in an inactive conformation, would con-
stitute a new class of potential antibacterial agents.2 QS
More than 20 years ago Eberhard et al.4 demonstrat-
ed that the marine bacteria Vibrio fischeri use
acylhomoserine lactones (AHLs), particularly N-hexa-
noyl-L-homoserine lactone (C6-HSL) 1 and N-3-oxo-
hexanoyl-L-homoserine lactone (3-oxo-C6-HSL)
2
(Scheme 1), to regulate bioluminescence in response to
cell density. The LuxI protein is responsible for AHL
biosynthesis, while the LuxR quorum sensing receptor
is a transcriptional regulator that activates biolumines-
cence genes. The current model for quorum sensing in
V. fischeri maintains that the association of 3-oxo-C6-
HSL with LuxR is a key step enabling the activator to
bind to lux box DNA at the luxICDABEG operon, there-
by facilitating the binding and activation of RNA poly-
merase at the luxI promoter.5 Since these initial
observations, more than 40 Gram negative bacterial spe-
cies have been shown to possess QS systems using AHLs
as autoinducers and containing proteins of the LuxI–
LuxR family.6
Keywords: Quorum sensing; Vibrio fischeri; AHLs; Ureas; Antagonists.
*
Corresponding author. Tel.: +33 0 472 43 82 21; fax: +33 0 472 43 88
96; e-mail: alain.doutheau@insa-lyon.fr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.03.017

4782
M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
2. Chemistry
O
O
N
R
In order to examine the effect of the substitution on the
activity, a series of 15 racemic10 alkyl- and aryl-N-
substituted ureas derived from homoserine lactone, as
well as 3 related compounds, were synthesized and test-
ed. Ureas 8–21 were easily prepared following three
routes (Scheme 2). Ureas 8–15 were obtained (route a)
by reacting homoserine lactone with isocyanates 7,
either commercially available (synthesis of 8–10, 13
and 14), or obtained in situ by the Curtius rearrange-
ment of the corresponding acyl azide. The latter com-
pounds were obtained either (11,15) by reacting
sodium azide with an acid chloride or (12) from the reac-
tion of diphenylphosphoryl azide with 6-phenylhexanoic
acid. The reaction of homoserine lactone with isocya-
nates was achieved in the presence of dimethylamino-
pyridine (DMAP) except for urea 8, for which these
experimental conditions led only to hydantoin 22 result-
ing from the opening of the lactone ring by the urea moi-
ety.11 This particular behaviour was probably due to the
acidity of the urea group in 8 which gave rise, in the ba-
sic reaction mixture, to a nucleophilic amidure type spe-
cies. By replacing DMAP with the less basic Et3N, we
obtained a mixture of urea 8 and hydantoin 22 from
which pure 8 was obtained by two consecutive separa-
tions, using silica gel column chromatography.
H
O
R = CH3-(CH2)4- (1)
R = CH3-(CH2)2-CO-CH2- (2)
O
O
N
R
H
O
R = Ph-(CH2)3- (3)
R = Ph-CH2-CO-CH2- (4)
O O
O
S
N
R
H
O
R = C5H11- (5)
R = Ph-(CH2)n- (6)
Scheme 1. Structure of compounds 1–6.
Different types of AHL analogs have already been syn-
thesized and evaluated as potential inhibitors of QS by
several research groups.7,8 As part of an ongoing pro-
gram aimed at obtaining such compounds, we have pre-
viously identified two families of antagonists of QS in
V. fischeri bacteria: AHL analogs bearing an aromatic
ring at the extremity of the alkyl side chain8a and sulfo-
nylamides,8b the most active compounds being, respec-
tively, 3 and 4 in the first series and 5 and 6a (n = 2)
in the second.
Ureas 16–18 resulted (route b) from the reaction be-
tween amines 25 with a-isocyanato-c-butyrolactone 24
prepared in situ by adding diphenylphosphoryl azide
to known12a-carboxy-c-butyrolactone 23. Ureas 19–21
were prepared (route c) by reacting 4-nitro-phenylchlo-
roformate with benzylamines 26 to generate the corre-
sponding intermediate carbamate 27, followed by the
addition of homoserine lactone.
In keeping with our research into new antagonists of QS
based on the replacement of the amide function of
AHLs by other linkages,8b we considered it to be of
interest to evaluate urea analogs. Indeed, acyclic and
cyclic ureas are often biologically active, nontoxic com-
pounds, probably due to the fact that the ureido (–NH-
CO-NH–) unit is a pseudodipeptide motif.9 We report
here the synthesis and the results of the biological assays
of a series of these new AHL analogs.
Ureas 30 and 32 were prepared according to the synthet-
ic sequences depicted in Scheme 3. Enamino-urea 30 was
obtained by first reacting the known13 enamino lactone
28 with 4-nitro-phenylchloroformate to produce the
intermediate carbamate 29, which was then submitted
R = Ph- (8)
R = Ph-CH2- (9)
R = Ph-CH2-CH2- (10)
R = Ph-(CH2)2-CH2- (11)
R = Ph-(CH2)4-CH2- (12)
R = C4H9- (13)
route a
O
R
C
N
7
O
H2N
O
route b
R = C5H11- (14)
R = C6H11-CH2-CH2- (15)
R = Ph-(CH2)3-CH2- (16)
R = C3H7- (17)
R = C6H13- (18)
R = p-CH3-Ph-CH2- (19)
R = p-CF3-Ph-CH2- (20)
R = p-Cl-Ph-CH2- (21)
RNH2
O
O
O
25
O
C
O
R
HOOC
23
N
N
N
O
H
H
O
O
24
route c
O
H2N
O
NO2
O
HO
O
N-Ph
O
RNH2
RNH
O
HN
26
27
22
Scheme 2. Synthesis of ureas 8–21. Structure of hydantoin 22.

M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
4783
O2N
O
O
O
O
C4H9
O
O
H2N
N
N
O
N
H
H
H
O
O
O
O
28
29
30
O
O
O
O
H2N
N
H
C3H7
N
N
H
H
O
31
32
Scheme 3. Synthesis of ureas 30 and 32.
to the action of butylamine. Acylurea 32 was prepared
by reacting known14 urea 31 with butanoyl chloride.
3. Biological results and molecular modelling
3.1. Inhibitory activity
The N-methyl urea 33 (Scheme 4) was obtained accord-
ing to route b, using N-methylbutylamine. The thiourea
34 was prepared according to route a: the action of
homoserine lactone on commercially available butyl iso-
thiocyanate gave rise to a mixture of expected 34 and
thiohydantoin 35.15
The above ureas 8–21, 30 and 32–34 were evaluated for
their ability to interfere with the induction of lumines-
cence by N-3-oxohexanoyl-L-homoserine lactone in the
V. fischeri bacteria QS system (see Section 6.1).
3.1.1. Aryl ureas 8–12, and 16. Since AHL analogs, bear-
ing a phenyl group at the extremity of the alkyl chain,
were often shown to display antagonist activities,7d,i,j,8
we first prepared, and evaluated, the biological activities
of phenyl substituted ureas 8-12 and 16 with 4–9
r-bonds between the aromatic and the lactone rings.
With the exception of compound 8, all these ureas
proved to be antagonists (Fig. 1). Compounds 9, 11,
12 and 16, in which the phenyl group is distant from
the lactone ring of 5, 7, 8 and 9 bonds, respectively, dis-
played inhibitory activity of about the same magnitude
(IC50 ꢀ 4–7 lM). Urea 10, in which this distance is of
6 bonds, is significantly less active. It is interesting to
note that a similar effect was observed in the case of
phenylalkylsulfonyl HSLs 6 when the alkyl spacer
length was varied from n = 1 to n = 3, corresponding,
respectively, to 4- to 6-bond distances between the phen-
yl and lactone rings.8b The strongest inhibitory activity
was for a 5-bond spacing (compound 6a, n = 2), whereas
6 (6b, n = 3) or 4-bond (6c, n = 1) spacing led to a lower,
or nearly absent, activity, respectively.
O
O
C4H9
N
N
H
O
33
S
O
C4H9
HO
N
N
H
H
O
34
O
N-C4H9
HN
S
35
Scheme 4. Structures of compounds 33, 34 and 35.
O
120
100
80
60
40
20
0
O
N
H
N
H
8
O
O
O
O
O
O
O
(IC50 = 18 µM)
(IC50 = 6.5 µM)
N
H
N
H
10
9
N
H
N
H
O
O
O
O
O
(IC50 = 5.5 µM)
(IC50 = 4.9 µM)
16
12
11
N
H
N
H
O
O
O
N
H
N
H
O
(IC50 = 4.4 µM)
N
H
N
H
0
5
10
15
20
25
30
35
40
Ureas [µM]
Figure 1. Inhibitory activity of ureas 8–12 and 16. Effect of the distance between the lactone and the phenyl groups on antagonist activity.

4784
M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
3.1.2. 4-Substituted phenyl ureas 19–21. The effect of the
aromatic ring substitution on the antagonist activity was
then briefly investigated with the 4-substituted analogs
19–21 of 9, easily prepared from commercially available
amines. These new analogs displayed inhibitory activity
of about the same significant magnitude as the parent
compound 9 (Fig. 2). However, a slight enhancement
of the inhibitory activity was observed when the phenyl
group was substituted with a methyl group or a chlorine
atom as electrodonating substituents (19 and 21
IC50 ꢀ 3 lM), whereas substitution by the strongly elec-
tron-withdrawing trifluoromethyl group was without
any significant effect (20 and 9, IC50 ꢀ 7 lM).
a fairly similar inhibitory effect as n-hexyl urea 18.
Alkylation of the urea nitrogen with a methyl group
(urea 33) resulted in a drastic decrease in antagonist
activity. Finally, we observed that compounds 30, 32
and 34 were deprived of any inhibitory activity (data
not shown).
3.2. Dimerisation of the N-terminal domain of a protein of
the LuxR family
To analyse conformational transition induced by 3-oxo-
C6-HSL in a protein of the LuxR family, we tried to puri-
fy the N-terminal domain of ExpR, an Erwinia chrysant-
hemi quorum sensing regulator which is responsive to 3-
oxo-C6-HSL as LuxR. Attempts to overproduce LuxR
in the absence of ligand were unsuccessful, suggesting
that LuxR was unstable under such conditions.5 By con-
trast, we obtained large amounts of the ExpR N-terminal
domain(His)6, henceforth designated as ExpR-N. Addi-
tion of 3-oxo-C6-HSL to this purified domain modified
its electrophoretic mobility in a concentration dependent
manner (Fig. 4A). This finding suggested that 3-oxo-C6-
HSL induced a conformational transition in ExpR-N.
In size-exclusion chromatography, purified ExpR-N
was eluted with a single peak corresponding to a
3.1.3. Alkyl ureas and related compounds. We then tested
the alkyl ureas 13–15, 17, 18 and the bis-alkyl urea 33, as
well as the enamino-urea 30, the acylurea 32 and the
thiourea 34. Among these compounds, the n-alkyl ureas
13–15 and 18 displayed good antagonist activity (Fig. 3).
If the propyl urea 17 is essentially inactive, the inhibito-
ry activity of 13, 14, and 18 increased with alkyl chain
length. However, beyond 5 carbon atoms the activity
reached a maximum and 14 (pentyl) and 18 (hexyl) both
displayed about the same activity (IC50 ꢀ 1 lM). Inter-
estingly 15, bearing a cyclohexyl substituent, exhibited
120
100
80
60
40
20
0
O
O
(IC50 = 7.0 µM)
N
H
N
H
20
9
O
O
F3C
O
O
O
O
(IC50 = 6.5 µM)
(IC50 = 2.9 µM)
N
H
N
H
N
H
N
H
21
19
O
Cl
O
O
(IC50 = 2.7 µM)
N
H
N
H
O
H3C
0
5
10
15
20
25
30
35
40
Ureas [µM]
Figure 2. Inhibitory activity of ureas 9 and 19–21. Effect of the phenyl group substitution on antagonist activity.
O
O
(IC50 = 56 µM)
(IC50 = 36 µM)
(IC50 = 4.8 µM)
(IC50 = 1.8 µM)
(IC50 = 1.1 µM)
(IC50 = 1.0 µM)
33
17
13
15
14
18
120
100
80
60
40
20
0
N
N
H
O
O
O
O
O
O
O
O
N
H
N
H
O
O
N
H
N
H
O
O
O
O
N
H
N
H
O
O
N
H
N
H
N
H
N
H
0
5
10
15
20
25
30
35
40
Ureas [µM]
Figure 3. Inhibitory activity of ureas 13–15, 17, 18 and 33. Effect of the alkyl chain on antagonist activity.

M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
4785
Figure 4. Electrophoretic mobility of the ExpR-N terminal domain(His)6. (A) Influence of 3-oxo-C6-HSL (OHHL) on electrophoretic mobility of the
ExpR-N terminal domain(His)6. (B) Electrophoretic mobility of the ExpR-N terminal domain(His)6 in the presence of 20 lM OHHL and an
increasing concentration of urea 13.
molecular mass of 25.5 kDa, a value analogous with the
calculated size of 20 kDa for one monomer of this pro-
tein. On the other hand, ExpR-N complexed with 3-
oxo-C6-HSL eluted with a single peak corresponding to
a molecular mass of 40.2 kDa, a value in agreement with
the calculated size of one homodimer of this protein (data
not shown). These results demonstrated that 3-oxo-C6-
HSL induces the dimerisation of ExpR-N.
4. Discussion
Molecular modelling suggests that the geometry of the
urea function fits well in the AHL-binding cavity of
LuxR. Moreover, this functional group is capable of
forming two hydrogen bonds with the residue Asp79
˚
(3.0 A). This residue was shown to be essential for
AHL-binding in different proteins of the LuxR family
since alteration of the corresponding residues of TraR
(Asp70)17, LasR (Asp73)18 and of LuxR (Asp79)19 com-
pletely abolishes the AHL-binding capacity of these pro-
teins, which failed to dimerise and lacked any detectable
transcriptional activity. Why the enforced hydrogen
bonding might be a determining factor in conferring
an antagonist activity to the AHL derived ureas is not
clear, but the importance of the hydrogen atom of the
external NH of the urea function was confirmed by
the inactivity of the N-methylated urea 33.
3.3. Urea-HSL analogs inhibit dimerisation of the ExpR
N-terminal domain
To gain insights into the molecular effects of urea-HSL
analogs on quorum sensing inhibition, we analysed the
effect of urea 13 on the electrophoretic mobility of
ExpR-N complexed with 3-oxo-C6-HSL (Fig. 4B). In
the presence of 20 lM of 3-oxo-C6-HSL, approximately
50% of the ExpR-N was in a dimer state (Fig. 4A). In-
creased amounts (0–20 mM) of 13 were added to
ExpR-N in the presence of 20 lM 3-oxo-C6-HSL
(Fig. 4B). At the highest concentration of urea 13 the
dimer was no longer present. This observation suggested
that 13 competes with 3-oxo-C6-HSL to occupy the
ligand-binding pocket of ExpR and inhibits dimerisa-
tion of the N-terminal domain.
With the aryl substituted ureas 8–12 and 16, it seems that
the antagonist activity depends on the distance between
the phenyl group and the lactone ring. This could result
from the relative positions of the phenyl group and the
aromatic residues, Tyr62 and Tyr70, located in the hydro-
phobic pocket of the active site (Fig. 5A). Depending on
the distance between the lactone and the aromatic rings,
urea 9 (5 bond spacing) and 11 (7 bond spacing) would
develop an attractive interaction with either Tyr62 or
Tyr 70. On the other hand, such a favourable interaction
would not be permitted for ureas equipped with an inap-
propriate spacer, either too short in 8 (4-bond spacing) or
intermediate in 10 (6-bond spacing). The strong activity
of 12 and 16, similar to that of 11, is probably due to
the increased hydrophobic character of a longer alkyl
chain. The slight enhancement of the activity of ureas
19 and 21, compared to 9, appears to be consistent with
the effect of electrodonating substituents. The biological
results obtained with the alkyl substituted derived ureas
13–15, 17 and 18 showed that the antagonist activity
increases with the hydrophobic character of the ligand
to reach a maximum value for the hexyl substituent
(IC50 = 1 lM), probably correlated with the enhanced
affinity for the hydrophobic protein pocket. Moreover,
alkyl substituted ureas seem to show stronger activity
compared to more sterically hindered aryl substituted
ureas. In enaminourea 30 the flattening of the lactone
ring, resulting from the introduction of a carbon–carbon
3.4. Molecular modelling
To investigate the structure–activity relationships of
AHL derived ureas, correlated with their possible inter-
actions with the transcriptional regulator LuxR, the
compound 13 was modelled in the active site of
the LuxR model,8b whose construction was based on
the folding of TraR.16 Preferential conformations of
13 were first calculated by varying the key torsion angles
and they were compared to the natural ligand of TraR.16
The resulting conformation was then docked as a ligand
in the active site of LuxR. The analysis of the docking
result (Fig. 5A) suggests that (i) the urea function per-
mits the formation of an additional hydrogen bond with
˚
the residue Asp79 (3.0 A) in addition to the one already
present with the amide function of the AHLs (see
Fig. 5B for the hydrogen bond network); (ii) the planar
urea functional group allows a favourable orientation of
the alkyl chain in the hydrophobic pocket (Fig. 5C); and
(iii) the urea moiety fits well in the hydrophilic domain
of the active site (Fig. 5D).

4786
M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
A
B
C
D
Figure 5. Docking result of urea 13 in the active site of the LuxR model. (A) Visualisation of the simplified active site with urea 13. (B) Schematic
overview of the active site displaying the hydrogen bond network with urea 13. (C) The interactions between the alkyl chain and the hydrophobic
pocket and (D) shows the interactions of the urea functional group with polar residues (residues are represented with van der Waals surfaces).
double bond, would be the cause of its inactivity. As in the
case of sulfonylamides8b, we observed that the presence
of an additional carbonyl function in urea 32 resulted in
a nearly total loss of activity. The data obtained from
molecular modelling (not shown) indicated that the intro-
duction of a sp2 carbon resulted in a conformation of the
acyl chain which is not tolerated in the binding site. The
loss of activity when changing the carbonyl function of
the urea to a thiocarbonyl one (compound 34) could re-
sult either from the bigger size of the sulfur atom or from
the weaker capacity of sulfur, compared with oxygen, to
act as a hydrogen bonding acceptor.
activity was related to the dimerisation of the N-termi-
nal domain of ExpR, a protein of the LuxR family.
Molecular modelling suggested that this would result
from the formation of an additional hydrogen bond in
the protein AHL-binding cavity.
6. Experimental
6.1. Antagonist activity measurement
Compounds 8–21, 30 and 32–34 were evaluated for their
ability to interfere with the induction of luminescence by
N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-
HSL, 2) in the V. fischeri QS system For that purpose,
we used the recombinant Escherichia coli biosensor
strain NM522 containing the plasmid pSB401.20 In this
plasmid, the luxR and the luxI promoter from V. fischeri
have been coupled to the entire lux structural operon
(luxCDABE) from Photorhabdus luminescens. This bio-
sensor strain produces luminescence in response to exog-
enously provided AHL. AHL activity was measured in a
microtitre plate format, with bioluminescence quantified
using a luminescence reader (Luminoskan Ascent, Lab-
systems). Competition assays were performed in LB
5. Conclusion
In this report, we have prepared 15 homoserine lactone
derived ureas and three related compounds, and evalu-
ated their ability to inhibit the QS system in V. fischeri
bacteria. We observed that two classes of these new
AHL analogs displayed significant antagonist activity:
(1) certain ureas bearing a phenyl group at the extremity
of the alkyl chain, (2) N-alkyl substituted ureas with an
alkyl chain of at least 4 carbon atoms long. We ob-
served, in the case of N-butyl urea, that the antagonist

M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
4787
growth medium in the presence of 200 nM of 3-oxo-
C6-HSL and with simultaneously added analogs, in
concentrations ranging from 20 nM to 40 lM. After
inoculation by the biosensor strain, the plate was incu-
bated at 30 ꢁC. The amount of light produced by the
bacteria was detected after 4–5 h and was expressed in
relative light units (RLU). The experiments were carried
out in triplicate and the standard deviation (data not
shown) did not exceed 10% of the mean value. A nega-
tive control was performed in the absence of 3-oxo-C6-
HSL and this gave the basal level of bioluminescence in
the absence of an inducer. A positive control was per-
formed in the presence of 3-oxo-C6-HSL only. We also
checked that the analogs did not inhibit bacterial growth
by measuring the turbidity of the bacterial culture at OD
600 nm for the highest analog concentration used. Sim-
ilarly, to be sure that the analogs inhibited induction of
luxCDABE genes and did not inhibit Lux enzyme activ-
ity, an additional control was performed in which the
highest analog concentration was added to glowing bac-
teria after the 4–5 h growth period and we verified that
bioluminescence was not suppressed.
fate. After dilution with one volume of extraction buffer
without KCl and filtration on a 0.45 lm filter, the protein
solution was applied to a PAK DEAE 15HR column
(50 mm · 100 mm; Waters). The column was washed
with extraction buffer and eluted using a linear KCl gra-
dient (0.075–0.6 M) at a flow rate of 1.4 ml.minꢁ1. After
SDS–PAGE analysis, the ExpR N-terminal protein(His)6
was then observed in the fraction corresponding to the
injection peak. This fraction was dialysed against phos-
phate buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl,
10 mM imidazole, 2 mM b-mercaptoethanol, 10% glycer-
ol, 1 mM PMSF) and then purified on Ni–NTA resin
(QIAGEN) with empty columns (QIAGEN), according
to the manufacturer’s instructions. Fractions were ana-
lysed by SDS–PAGE and those containing the ExpR
N-terminal domain(His)6 were pooled and dialyzed
against 3· 1.5 L of buffer (10 mM Tris–HCl, pH 7.9,
200 mM NaCl, 2 mM b-mercaptoethanol, 1 mM EDTA
and 1 mM PMSF). The resulting ExpR N-terminal
domain(His)6 preparation was >95% pure, as determined
by SDS–PAGE, and was stored at 4 ꢁC. Protein concen-
tration was measured using the Bradford assay.
6.2. Inhibition of dimerisation
6.2.2. 3-oxo-C6-HSL effect on electrophoretic mobility of
the ExpR N-terminal domain. Purified ExpR N-terminal
domain(His)6 (20 lM) was incubated with increasing
amounts of 3-oxo-C6-HSL (from 0 to 200 lM) at
30 ꢁC for 30 min. After the addition of an adequate vol-
ume of Laemmli sample buffer, without SDS and
b-mercaptoethanol, the reaction mixtures were electro-
phoresed on native 12% Tris–glycine polyacrylamide
gels (1:30 bis-acrylamide to acrylamide ratio) at
10 V/cm and at room temperature. Gels were then
stained with Coomassie blue and dried.
6.2.1. Overproduction and purification of the N-terminal
domain of ExpR. The ExpR N-terminal domain-coding
region (amino acids 1–163) was amplified from Erwinia
chrysanthemi strain 3937 chromosome by PCR, using
the two primers ExpR deb (50-GGAATCCATATGTC
TATATCATTCTCTAACG-30) and ExpR-Nterm fin
(50-CCGCTCGAGTCGGTACAGAGAGGTGAG-30).
Artificial NdeI and XhoI restriction sites were incorporat-
ed in the primers (restriction sites are underlined). The
resulting PCR product was digested with NdeI and XhoI,
and then cloned into pET20(b+) (Novagen) to form
pSC2833. In the resulting pSC2833 plasmid, the ExpR
N-terminal domain is fused with a His-tag at the C-termi-
nus. The integrity of the N- terminal expR coding region
was confirmed by sequencing. For ExpR N-terminal
domain(His)6 overproduction, colonies of E. coli
BL21(DE3) pLysS (Novagen) carrying pSC2833 were
grown overnight on LB-ampicillin–chloramphenicol
plates and were used to inoculate 200 ml of pre-warmed
LB containing 100 lg/ml ampicillin and 20 lg/ ml chlor-
amphenicol. The culture was incubated at 25 ꢁC until
reaching an optical density of 0.6 at 600 nm. Bacterial
cells were then induced with isopropyl-b-D-thiogalacto-
side (0.2 mM final concentration), and incubated at
25 ꢁC for an additional 2.5 h. Cells were harvested by cen-
trifugation at 5000g for 10 min. All subsequent steps were
carried out at 4 ꢁC. The cell pellets from 200 ml culture
were washed and resuspended in 25 ml of extraction
buffer (10 mM Tris–HCl, pH 7.9, 300 mM KCl, 2 mM
b-mercaptoethanol, 10% glycerol and 1 mM phenylmeth-
ylsulfonyl fluoride (PMSF)). Crude protein extract was
obtained by disrupting bacteria at 138,000 kPa in a
French pressure cell (AMINCO). Insoluble material
was removed by 2 steps of centrifugation (20,000g, for
10 min). The cellular extract obtained was then submitted
to a 0–20% ammonium sulfate precipitation. After centri-
fugation, the supernatant was dialysed against 2· 2 L of
extraction buffer to eliminate the residual ammonium sul-
6.2.3. Analysis of the oligomeric state of the ExpR N-
terminal domain. For gel filtration analysis, 0.2 ml of the
ExpR N-terminal domain(His)6 (25 lM) was chromato-
graphed on a Superdex 75 HR 10/30 column (Amersham),
at 15 ꢁC, using a Bio-Rad Biologic fast performance
liquid chromatography apparatus. This experiment was
performed either in the absence or presence of 250 lM
3-oxo-C6-HSL. Samples were incubated at 30 ꢁC for
30 min before filtration and injection. The column was
equilibrated and eluted with gel filtration buffer (50 mM
Tris–HCl, pH 7.9, 150 mM NaCl and 1 mM EDTA) at
a flow rate of 0.5 ml/min and fractions were collected at
0.25 ml/min. 3-Oxo-C6-HSL was not incorporated in
the gel filtration buffer. However, we observed that when
dimerisation is achieved, the dimer is quite stable and is
not dissociated during elution. Similar results were
obtained with two other quorum sensing regulators of
the LuxR family, namely TraR and LasR, which tightly
bind their cognate AHL and did not dissociate even after
prolonged dialysis.21,22 The column was calibrated with
protein standards from a molecular weight gel filtration
kit (SERVA). Fractions were then analysed by dot blot
using anti-ExpR polyclonal antibodies.
6.3. Molecular modelling
A conformational study was carried out on the butyl-
urea using the Tripos Force-field method with Sybyl

4788
M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
7.0 for Linux. Stable conformations were superimposed
on the natural ligand and the closest was then manually
docked into the active site of the LuxR model8b to ana-
lyse intermolecular interactions. Hydrogen bonds were
assigned based on the distance measured between the
donor and the acceptor. Figure 5 was created using
YASARA.23
(t, J = 4.7 Hz, 1H), 7.33–7.52 (m, 5H), 8.50 (s, 1H). In
agreement with reported data.10 13C NMR (DMSO-d6,
50 MHz): d 34.5, 53.8, 56.7, 126.7, 127.6, 128.6, 132.4,
155.8, 173.8.
6.4.2.2. 1-Benzyl-3-(2-oxo-tetrahydro-furan-3-yl)-urea
(9). Reaction time: 36 h. Chromatography: EtOAc/pen-
tane, 9/1. Recrystallisation: EtOAc/pentane. White solid
(22%). Mp: 143–144 ꢁC. 1H NMR (DMSO-d6,
200 MHz): d 2.05–2.27 (m, 1H), 2.34–2.44 (m, 1H),
4.13–4.54 (m, 3H), 4.24 (d, J = 6 Hz, 2H), 6.51
(d, J = 7 Hz, 1H), 6.69 (t, J = 5.9 Hz, 1H), 7.20–7.37
(m, 5H). 13C NMR (DMSO-d6, 50 MHz): d 29.1, 42.9,
48.9, 65.1, 126.6, 127.0, 128.2, 140.6, 157.4, 176.3. Anal.
Calcd for C12H14N2O3: C, 61.53; H, 6.02; N, 11.96.
Found: C, 60.54; H, 6.13; N, 11.77.
6.4. Chemistry
6.4.1. General remarks. All chemicals were purchased
from Aldrich. Organic solutions were dried over anhy-
drous sodium sulfate. The reactions were performed un-
der a constant flow of nitrogen and were monitored by
thin-layer chromatography on Silica Gel 60 F254
(Merck); detection was carried out by charring with a
5% phosphomolybdic acid solution in ethanol contain-
ing 10% of H2SO4. Silica gel (Kieselgel 60, 70–230 mesh
ASTM, Merck) was used for flash chromatography.
Melting points were determined on a Kofler block
apparatus. The 1H (200 MHz or 300 MHz) and
13C NMR (50 MHz or 75 MHz) spectra were recorded
with a Bruker AC200, ALS300 or DRX300 spectrome-
ter. The signal of the residual protonated solvent was
taken as reference. Chemical shift (d) and coupling
constants (J) are reported in ppm and Hz, respectively.
Elemental analyses were performed by ‘Service Central
de Microanalyses du CNRS’ 69360 Solaize (France).
6.4.2.3. 1-(2-Oxo-tetrahydro-furan-3-yl)-3-phenethyl-
urea (10). Reaction time: 24 h. Chromatography:
EtOAc. Recrystallisation: EtOAc/pentane. White solid
1
(84%). Mp: 113–114 ꢁC. H NMR (CDCl3, 200 MHz):
d
2.05–2.27 (m, 1H), 2.56–2.69 (m, 1H), 2.77
(t, J = 7.2 Hz, 2H), 3.33–3.43 (q, J = 6.7 Hz, 2H),
4.16–4.29 (m, 1H), 4.34–4.49 (m, 2H), 5.47
(t, J = 5.6 Hz, 1H), 5.79 (d, J = 6.6 Hz, 1H), 7.15–7.32
(m, 5H).13C NMR (CDCl3, 50 MHz): d 30.5, 36.6,
41.7, 49.9, 66.2, 126.4, 128.6, 128.8, 139.2, 158.3,
177.3. Anal. Calcd for C13H16N2O3: C, 62.89; H, 6.50;
N, 11.28. Found: C, 62.92; H, 6.68; N, 11.35.
6.4.2. General procedure for the synthesis of ureas 8–
10, 13–14 and 34. To a solution of D,L-a-amino-c-
butyrolactone hydrobromide (0.50 g, 2.75 mmol) and
triethylamine (0.28 g, 2.75 mmol) (8 and 34) or
4-dimethylaminopyridine (0.33 g, 2.75 mmol) (9, 10,
13 and 14) in dry THF (30 mL) was added at 0 ꢁC
the appropriate commercially available isocyanate or
isothiocyanate (2.75 mmol). The reaction mixture was
allowed to warm at rt and stirred for 8 to 48 h. The
solution was filtered and the solvent was evaporated.
The residue was purified by flash chromatography to
give a solid (except for 34 and 35), which was
recrystallised.
6.4.2.4. 1-Butyl-3-(2-oxo-tetrahydro-furan-3-yl)-urea
(13). Reaction time: 48 h. Chromatography: EtOAc.
Recrystallisation: EtOAc/pentane. White solid (83%).
Mp: 100–101 ꢁC. 1H NMR (acetone-d6, 200 MHz):
d 0.90 (t, J = 7.3 Hz, 3H), 1.24–1.51 (m, 4H), 2.11–2.33
(m, 1H), 2.50–2.64 (m, 1H), 3.09–3.18 (q, J = 6.5 Hz,
2H), 4.19–4.57 (m, 3H), 5.78 (s, 1H), 5.90 (s, 1H). 13C
NMR (CDCl3, 50 MHz): d 13.8, 20.1, 30.4, 32.3, 40.1,
49.9, 66.2, 158.4, 177.4. Anal. Calcd for C9H16N2O3:
C, 53.98; H, 8.05; N, 13.99. Found: C, 54.18; H, 8.42;
N, 14.12.
6.4.2.5. 1-(2-Oxo-tetrahydro-furan-3-yl)-3-pentyl-urea
(14). Reaction time: 24 h. Chromatography: EtOAc/
pentane, 9/1. Recrystallisation: EtOAc/pentane. White
solid (75%). Mp: 106–107 ꢁC. 1H NMR (CDCl3,
200 MHz): d 0.85 (t, J = 6.6 Hz, 3H), 1.22–1.33 (m,
4H), 1.35–1.47 (m, 2H), 2.17–2.33 (m, 1H), 2.53–2.66
(m, 1H), 3.05–3.14 (q, J = 6.5 Hz, 2H), 4.17–4.55 (m,
3H), 5.76 (t, J = 5.3 Hz, 1H), 6.06 (d, J = 7.0 Hz, 1H).
13C NMR (CDCl3, 50 MHz): d 14.0, 22.4, 29.1, 29.9,
30.1, 40.4, 49.8, 66.1, 158.4, 177.4. Anal. Calcd for
C10H18N2O3: C, 56.06; H, 8.47; N, 13.07. Found: C,
56.40; H, 8.39; N, 13.03.
6.4.2.1. 1-(2-Oxo-tetrahydro-furan-3-yl)-3-phenyl-urea
(8) and 5-(2-hydroxy-ethyl)-3-phenyl-imidazolidine-2,4-
dione (22). Reaction time: 8 h. Chromatography:
EtOAc/pentane, 8:2. Compound 8 was first eluted
(Rf: 0.48) and then 22 (Rf: 0.18). The urea 8 was further
purified by a second chromatography (EtOAc/pentane,
9:1). Compound 8. Recrystallisation: EtOAc/pentane.
White solid (14%). Mp: 161–162 ꢁC. 1H NMR (ace-
tone-d6, 200 MHz): d 2.21–2.42 (m, 1H), 2.58–2.73 (m,
1H), 4.24–4.47 (m, 2H), 4.53–4.67 (m, 1H), 6.24
(d, J = 6 Hz, 1H), 6.90–6.99 (t, J = 7.3 and J = 1.2 Hz,
1H), 7.20–7.28 (t, J = 7.5 Hz, 2H), 7.47–7.52 (dd,
J = 7.5 and J = 1.2 Hz, 2H), 8.19 (s, 1H). 13C NMR
(DMSO-d6, 50 MHz): d 28.8, 48.8, 65.2, 117.9, 121.4,
128.7, 140.0, 154.7, 176.0. Anal. Calcd for
C11H12N2O3: C, 59.99; H, 5.49; N, 12.72. Found: C,
60.24; H, 5.66; N, 12.70. Compound 22. Recrystallisa-
6.4.2.6. 1-Butyl-3-(2-oxo-tetrahydro-furan-3-yl)-thio-
urea (34) and 3-butyl-5-(2-hydroxy-ethyl)-2-thioxo-imi-
dazolidine-4-one
Chromatography: EtOAc/pentane, 55:45. Compound
(35).
Reaction
time:
24 h.
34 was first eluted (Rf: 0.38) and then 35 (Rf: 0.22). Com-
pound 34: colourless oil (21%). H NMR (acetone-d6,
1
1
tion: EtOAc/pentane. White solid. Mp: 119–120 ꢁC. H
NMR (200 MHz, DMSO-d6): d 1.80–2.04 (m, 2H),
3.53–3.69 (m, 2H), 4.29 (t, J = 5.7 Hz, 1H), 4.70
200 MHz): d 0.92 (t, J = 7.2 Hz, 3H), 1.28–1.46 (m,
2H), 1.51–1.66 (m, 2H), 2.11–2.28 (m, 1H), 2.75–2.88

M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
4789
(m, 1H), 3.50–3.53 (m, 2H), 4.28–4.46 (m, 2H), 5.32–
5.46 (m, 1H), 7.04 (s, 1H), 7.27 (s, 1H). 13C NMR (ace-
tone-d6, 50 MHz): d 14.7, 21.3, 31.6, 32.5, 45.5, 54.5,
66.9, 176.8, 185.3. HR-MS (EI): M+. Calcd for
C9H16N2O2S: 216.0932. Found: 216.0934. Compound
35: colourless oil. 1H NMR (CDCl3, 200 MHZ):
d 0.94 (t, J = 7.2 Hz, 3H), 1.24–1.44 (m, 2H), 1.57–1.72
(m, 2H), 1.82–2.05 (m, 1H), 2.12–2.26 (m, 1H), 2.65 (s,
1H), 3.79 (t, J = 7.5 Hz, 2H), 3.88–3.93 (m, 2H), 4.23
(dd, J = 3.9 Hz and 9.0 Hz, 1H). 13C NMR (CDCl3,
50 MHz): d 13.7, 20.0, 29.7, 33.6, 41.2, 58.1, 59.6,
175.1, 183.8.
at rt, the solvent was evaporated. The residue was puri-
fied by flash chromatography (EtOAc/pentane, 9:1) to
give a solid, which was recrystallised (EtOAc/pentane).
White solid (54%). Mp: 78–79 ꢁC.1H NMR (acetone-
d6, 300 MHz): d 1.32–1.42 (m, 2H), 1.47–1.57 (m, 2H),
1.59–1.69 (m, 2H), 2.16–2.30 (m, 1H), 2.52–2.60
(m, 1H), 2.61 (t, J = 7.8 Hz, 2H), 3.15 (q, J = 6.6 Hz,
2H), 4.21–4.30 (m, 1H), 4.37 (dt, J = 1.8 and J = 9 Hz,
1H), 4.48–4.57 (m, 1H), 5.86 (t, J = 4.8 Hz, 1H), 5.99
(d, J = 7.2 Hz, 1H), 7.14–7.30 (m, 5H). 13C NMR (ace-
tone-d6, 75 MHz): d 27.2, 30.8, 31.0, 32.1, 36.4, 40.6,
50.1, 66.0, 126.4, 129.0, 129.2, 143.5, 158.5, 176.8. Anal.
Calcd for C16H22N2O3: C, 66.18; H, 7.64; N, 9.65.
Found: C, 66.34; H, 7.89; N, 9.64.
6.4.3. General procedure for the synthesis of ureas 11 and
15. To a solution of sodium azide (1.04 g, 16.0 mmol) in
water (4 mL) was added dropwise a solution of acyl
chloride (5.3 mmol) in acetone (4 mL). After 1 h of stir-
ring at rt, the solution was extracted with dichorome-
thane (2· 20 mL). The organic phase was washed with
water and dried. The solvent was evaporated to give
the corresponding carboxylic azide as an oil, which
was refluxed in dry THF (50 mL) for 1 h. The reaction
mixture was then cooled to rt and D,L-a-amino-c-
butyrolactone hydrobromide (0.92 g, 5.0 mmol) fol-
lowed by 4-dimethylaminopyridine (0.68 g, 5.6 mmol)
were added. After 24 h, the reaction mixture was filtered
and the solvent was evaporated. The residue was puri-
fied by flash chromatography to give a solid, which
was recrystallised.
6.4.5. General procedure for the synthesis of ureas 16–18
and 33. To a solution of a-carboxy-c-butyrolactone11
(0.26 g, 2.0 mmol) in dry THF (30 mL) were added
diphenylphosphorylazide (0.55 g, 2.0 mmol) and trieth-
ylamine (0.20 g, 2.0 mmol). The solution was refluxed
for 3.5 h and then cooled to rt. The appropriate amine
(2.0 mmol) was then added. After 18 h of stirring at rt,
the solvent was evaporated. The residue was purified
by flash chromatography to give a solid (except 33),
which was recrystallised.
6.4.5.1. 1-(2-Oxo-tetrahydro-furan-3-yl)-3-(4-phenyl-
butyl)-urea (16). Chromatography: EtOAc/pentane,
85:15. Recrystallisation (EtOAc/pentane). White solid
1
(21%). Mp: 81–82 ꢁC. H NMR (CDCl3, 300 MHz): d
6.4.3.1. 1-(2-Oxo-tetrahydro-furan-3-yl)-3-(3-phenyl-
propyl)-urea (11). Chromatography: EtOAc. Recrystalli-
sation: EtOAc/cyclohexane. White solid (82%). Mp:
80–81 ꢁC. 1H NMR (CDCl3, 200 MHz): d 1.71–1.86
(m, 2H), 2.09–2.31 (m, 1H), 2.58–2.71 (m, 3H), 3.13–
3.23 (q, J = 6.6 Hz, 2H), 4.16–4.29 (m, 1H), 4.34–4.56
(m, 2H), 5.63 (t, J = 5.6 Hz, 1H), 5.90 (d, J = 6.6 Hz,
1H), 7.13–7.30 (m, 5H). 13C NMR (CDCl3, 50 MHz):
d 30.6, 31.8, 33.1, 40.0, 50.0, 66.3, 126.0, 128.4, 128.5,
141.6, 158.2, 177.5. Anal. Calcd for C14H18N2O3: C,
64.10; H, 6.92; N, 10.68. Found: C, 64.44; H, 7.17; N,
10.64.
1.43–1.67 (m, 4H), 2.05–2.28 (m, 1H), 2.57–2.71
(m, 3H), 3.11–3.21 (q, J = 6.4 Hz, 2H), 4.15–4.55 (m,
3H), 5.49 (s, 1H), 5.79 (s, 1H), 7.13–7.31 (m, 5H). 13C
NMR (CDCl3, 75 MHz): d 28.7, 29.8, 30.6, 35.6, 40.2,
50.0, 66.2, 125.8, 128.3, 128.4, 142.3, 158.2, 177.4. Anal.
Calcd for C15H20N2O3: C, 65.20; H, 7.30; N, 10.14.
Found: C, 65.27; H, 7.40; N, 9.92.
6.4.5.2. 1-(2-Oxo-tetrahydro-furan-3-yl)-3-propyl-urea
(17). Chromatography: EtOAc. Recrystallisation:
EtOAc/pentane. White solid (24%). Mp: 109–110 ꢁC.
1H NMR (CDCl3, 300 MHz): d 0.88 (t, J = 7.2 Hz,
3H), 1.42–1.52 (m, 2H), 2.16–2.28 (m, 1H), 2.64–2.73
(m, 1H), 3.10 (q, J = 7.0 Hz, 2H), 4.25–4.31 (m, 1H),
4.42 (dt, J = 1.5 and J = 9 Hz, 1H), 4.47–4.56 (m, 1H),
5.49 (t, J = 5.3 Hz, 1H), 5.80 (d, J = 6.8 Hz, 1H). 13C
NMR (acetone-d6, 75 MHz): d 12.6, 25.2, 31.6, 43.4,
51.1, 67.0, 159.6, 177.9. Anal. Calcd for C8H14N2O3:
C, 51.60; H, 7.58; N, 15.04. Found: C, 51.33; H, 7.72;
N, 14.90.
6.4.3.2. 1-(2-Cyclohexyl-ethyl)-3-(2-oxo-tetrahydro-
furan-3-yl)-urea (15). Chromatography: EtOAc. Recrys-
tallisation: EtOAc/cyclohexane. White solid (61%). Mp:
1
117–118 ꢁC. H NMR (CDCl3, 200 MHz): d 0.87–0.97
(m, 2H), 1.15–1.44 (m, 6H), 1.67–1.72 (m, 5H), 2.05-
2.31 (m, 1H), 2.69–2.83 (m, 1H), 3.19 (t, J = 7.2 Hz,
2H), 4.22–4.35 (m, 1H), 4.39–4.57 (m, 2H), 5.26
(s, 2H). 13C NMR (CDCl3, 50 MHz): d 26.3, 26.6,
30.6, 33.2, 35.8, 37.6, 38.3, 49.9, 66.2, 158.3, 177.4. Anal.
Calcd for C13H22N2O3: C, 61.39; H, 8.72; N, 11.01.
Found: C, 61.57; H, 8.62; N, 11.18.
6.4.5.3. 1-Hexyl-3-(2-oxo-tetrahydro-furan-3-yl)-urea
(18). Chromatography: EtOAc/pentane, 9:1. Recrystalli-
sation: EtOAc/pentane. White solid (16%). Mp: 96–
1
97 ꢁC. H NMR (acetone-d6, 300 MHz): d 0.88 (t, J =
6.4.4. 1-(2-Oxo-tetrahydro-furan-3-yl)-3-(5-phenyl-pen-
tyl)-urea (12). To a solution of 6-phenyl-hexanoic acid
(0.38 g, 2.0 mmol) and triethylamine (0.20 g, 2.0 mmol)
in dry THF (10 mL) was added diphenylphosphorylaz-
ide (0.55 g, 2.0 mmol). The solution was refluxed for
2 h and then cooled to rt D,L-a-amino-c-butyrolactone
hydrobromide (0.37 g, 2.0 mmol) and triethylamine
(0.20 g, 2 mmol) were then added. After 16 h of stirring
6.6 Hz, 3H), 1.23–1.35 (m, 6H), 1.42–1.48 (m, 2H),
2.15–2.29 (m, 1H), 2.51–2.61 (m, 1H), 3.12 (q,
J = 6.6 Hz, 2H), 4.21–4.29 (m, 1H), 4.33–4.39 (dt,
J = 1.5 Hz and J = 8.8 Hz, 1H), 4.46–4.55 (m, 1H), 5.85
(s, 1H), 5.98 (d, J = 6.6 Hz, 1H).13C NMR (acetone-d6,
75 MHz): d 15.1, 24.0, 28.1, 31.6, 31.8, 33.1, 41.5, 50.9,
66.7, 159.3, 177.6. Anal. Calcd for C11H20N2O3: C,
57.87;H,8.83;N,12.27.Found:C, 57.33;H,8.87;N,12.12.

4790
M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
6.4.5.4. 1-Butyl-1-methyl-3-(2-oxo-tetrahydro-furan-3-
yl)-urea (33). Chromatography: EtOAc. Yellow oil
(13%). 1H NMR (CDCl3, 300 MHz):
0.89
(t, J = 7.5 Hz, 3H), 1.22–1.34 (m, 2H), 1.43–1.53 (m,
2H), 2.09–2.23 (m, 1H), 2.68–2.77 (m, 1H), 2.86 (s,
3H), 3.13–3.30 (m, 2H), 4.19–4.28 (m, 1H), 4.38–4.50
(m, 2H), 5.19 (d, J = 5.1 Hz, 1H). 13C NMR (CDCl3,
75 MHz): d 14.1, 20.2, 30.3, 31.1, 34.5, 48.9, 50.7, 66.4,
157.7, 177.2. HR-MS (EI): M+. Calcd for
C10H18N2O3: 214.1317. Found: 214.1318.
6.4.7. 1-Butyl-3-(2-oxo-2,5-dihydro-furan-3-yl)-urea (30).
To a solution of 4-nitrophenyl chloroformate (0.48 g,
2.4 mmol) and triethylamine (0.24 g, 2.4 mmol) in dry
THF (15 mL) was added dropwise a solution of 3-ami-
no-5H-furan-2-one 2812 (0.20 g, 2.0 mmol) in THF
(5 mL). After 4 h of stirring at rt, butylamine (0.15 g,
2.0 mmol) was added. After 48 h, the solution was fil-
tered and the solvent was evaporated. The residue was
purified by flash chromatography (EtOAc/pentane:
7:3). White solid (26%). Mp: 101–103 ꢁC. 1H NMR
(CDCl3, 300 MHz): d 0.93 (t, J = 7.2 Hz, 3H), 1.30–
1.44 (m, 2H), 1.48–1.57 (m, 2H), 3.26 (t, J = 6.8 Hz,
2H), 4.90 (d, J = 1.8 Hz, 2H), 5.92 (s, 1H), 7.27 (t,
J = 1.8 Hz, 1H), 7.83 (s, 1H). 13C NMR (acetone-d6,
75 MHz): d 14.8, 21.4, 33.7, 40.9, 71.8, 122.1, 128.3,
156.1, 172.0. HR-MS (EI): M+. Calcd for C9H14N2O3:
198.1004. Found: 198.1005.
d
6.4.6. General procedure for the synthesis of ureas 19–
21. To a solution of 4-nitrophenyl chloroformate
(0.55 g, 2.7 mmol) and diisopropylethylamine (0.36 g,
2.7 mmol) in dry THF (10 mL) was added dropwise
a solution of the appropriate amine (2.7 mmol) in
THF (5 mL). After 4 h of stirring at rt, D,L-a-amino-
c-butyrolactone hydrobromide (0.5 g, 2.7 mmol) fol-
lowed by diisopropylethylamine (0.355 g, 2.7 mmol)
were added. After 18 h, the solution was filtered and
the solvent was evaporated. The residue was purified
by flash chromatography to give a solid which was
recrystallised.
6.4.8.
1-Butyryl-3-(2-oxo-tetrahydro-furan-3-yl)-urea
(32). To a solution of (2-oxo-tetrahydro-furan-3-yl)-urea
(31)13 (0.45 g, 3.1 mmol) in dry pyridine was added
dropwise at 0 ꢁC butyryl chloride (0.33 g, 3.1 mmol).
After 18 h of stirring at room temperature, the solvent
was evaporated. The residue was purified by flash chro-
matography (EtOAc/pentane: 5:5) to give 32 (0.44 g,
66%) as a white solid. Mp: 168–169 ꢁC. 1H NMR
(CDCl3, 200 MHz): d 0.99 (t, J = 7.3 Hz, 3H), 1.61–
1.79 (m, 2H), 2.32 (t, J = 7.3 Hz, 2H), 2.29–2.45 (m,
1H), 2.64–2.74 (m, 1H), 4.23–4.36 (m, 1H), 4.45–4.60
(m, 2H), 8.98 (s, 2H). 13C NMR (CDCl3, 50 MHz): d
13.6, 18.3, 29.3, 38.8, 49.3, 65.7, 154.5, 174.3, 175.2.
Anal. Calcd for C9H14N2O4: C, 50.46; H, 6.59; N,
13.08. Found: C, 50.83; H, 6.87; N, 13.04.
6.4.6.1. 1-(4-Methyl-benzyl)-3-(2-oxo-tetrahydro-fu-
ran-3-yl)-urea (19). Chromatography: EtOAc/pentane,
9:1. Recrystallisation: EtOAc/pentane. White solid
(70%). Mp: 161–162 ꢁC. 1H NMR (DMSO-d6,
300 MHz): d 2.08–2.22 (m, 1H), 2.27 (s, 3H), 2.35–2.44
(m, 1H), 4.14–4.28 (m, 1H), 4.15 (d, J = 5.7 Hz, 2H),
4.31 (dt, J = 1.9 and 9 Hz, 1H), 4.40–4.49 (m, 1H),
6.46 (d, J = 7.8 Hz, 1H), 6.60 (t, J = 6.0 Hz, 1H), 7.10–
7.16 (m, 4H). 13C NMR (DMSO-d6, 75 MHz): d 20.7,
29.2, 42.7, 48.9, 65.1, 127.1, 128.8, 135.7, 137.4, 157.4,
176.4. Anal. Calcd for C13H16N2O3: C, 62.89; H, 6.50;
N, 11.28. Found: C, 62.61; H, 6.63; N, 11.20.
Acknowledgments
´
ˆ
Financial support from MENESR, the Region Rhone-
Alpes (Programme Emergence 2002) and CNRS is
gratefully acknowledged. M.F. thanks the MENESR
for a scholarship. S.C. was supported by a Region
6.4.6.2. 1-(2-Oxo-tetrahydro-furan-3-yl)-3-(4-trifluo-
romethyl-benzyl)-urea (20). Chromatography: EtOAc.
Recrystallisation: EtOAc/pentane. White solid (60%).
Mp: 132–133 ꢁC. 1H NMR (DMSO-d6, 300 MHz): d
2.09–2.24 (m, 1H), 2.35–2.45 (m, 1H), 4.14–4.23
(m, 1H), 4.28–4.34 (m, 3H), 4.41–4.51 (m, 1H), 6.61
(d, J = 7.5 Hz, 1H), 6.81 (t, J = 6.0 Hz, 1H), 7.46
(d, J = 8.1 Hz, 2H), 7.67 (d, J = 8.1 Hz, 2H). 13C
NMR (DMSO-d6, 75 MHz): d 29.1, 42.6, 48.9, 65.1,
124.4 (q, J = 270 Hz), 125.0 (q, J = 3.5 Hz), 127.3 (q,
J = 31.5 Hz), 127.6, 145.7, 157.4, 176.3. Anal. Calcd
for C13H13F3N2O3: C, 51.66; H, 4.34; N, 9.27. Found:
C, 51.52; H, 4.48; N, 9.25.
ˆ
Rhone-Alpes Emergence fellowship. We thank V. James
for improving the English of the manuscript.
References and notes
1. For recent reviews, see: (a) Miller, M. B.; Bassler, B. L.
Annu. Rev. Microbiol. 2001, 55, 165–199; (b) Whitead, N.
A.; Barnard, A. M. L.; Slater, H.; Simpson, N. J. L.;
Salmond, G. P. C. FEMS Microbiol. Rev. 2001, 5, 365–
404; (c) Lyon, G. J.; Muir, T. W. Chem. Biol. 2003, 10,
1007; (d) Waters, C. M.; Bassler, B. L. Annu. Rev. Cell.
Dev. Biol. 2005, 21, 319–346.
2. (a) Suga, H.; Smith, K. M. Curr. Opin. Chem. Biol. 2003,
7, 586–591; (b) Persson, T.; Givskov, M.; Nielsen, J. Curr.
Med. Chem. 2005, 12, 3103–3115; (c) Raffa, R. B.;
Iannuzzo, J. R.; Levine, D. R.; Saeid, K. K.; Schwartz,
R. C.; Sucic, N. T.; Terleckyj, O. D.; Young, J. M.
J. Pharmacol. Exp. Ther. 2005, 312, 417–423.
6.4.6.3. 1-(4-Chloro-benzyl)-3-(2-oxo-tetrahydro-fu-
ran-3-yl)-urea (21). Chromatography: EtOAc/pentane,
9:1. Recrystallisation: EtOAc/pentane. White solid
(46%). Mp: 149–150 ꢁC. 1H NMR (DMSO-d6,
300 MHz): d 2.07–2.21 (m, 1H), 2.34–2.43 (m, 1H),
4.13–4.20 (m, 3H), 4.30 (t, J = 7.8 Hz, 1H), 4.39–4.48
(m, 1H), 6.52 (d, J = 7.8 Hz, 1H), 6.70 (t, J = 6.0 Hz,
1H), 7.26 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H).
13C NMR (DMSO-d6, 75 MHz): d 29.1, 42.3, 48.9,
65.1, 128.2, 128.9, 131.1, 139.8, 157.4, 176.4. Anal.
Calcd for C12H13ClN2O3: C, 53.64; H, 4.88; N, 10.43.
Found: C, 53.39; H, 4.85; N, 10.13.
3. (a) Kjelleberg, S.; Molin, S. Curr. Opin. Microbiol. 2002, 5,
254–258; (b) Hentzer, M.; Eberl, L.; Nielsen, J.; Givskov,
M. BioDrugs 2003, 17, 241–250; (c) Rice, S. A.; McDoug-
ald, D.; Kumar, N.; Kjelleberg, S. Curr. Opin. Investig.
Drugs 2005, 6, 168–184.

M. Frezza et al. / Bioorg. Med. Chem. 14 (2006) 4781–4791
4791
4. Eberhard, A.; Burlingame, A. L.; Eberhard, C.; Kenyon,
G. L.; Nealson, K. H.; Oppenheimer, N. J. Biochemistry
1981, 20, 2444–2449.
5. Urbanowski, M. L.; Lostroh, C. P.; Greenberg, E. P.
J. Bacteriol. 2004, 186, 631–637.
6. (a) Lerat, E.; Moran, N. A. Mol. Biol. Evol. 2004, 21, 903–
913; (b) Parsek, M. R.; Greenberg, E. P. Proc. Natl. Acad.
Sci. USA 2000, 97, 8789–8793.
10. When both L- and D-isomers of natural AHLs were tested
on P. aeruginosa, bacteria with an AHL-dependent QS
system, only the L-isomers displayed autoinducing activ-
ity, whereas the D-isomers were inactive. Moreover, the
activity of the L-isomers was not inhibited by the presence
of the D-isomer: Ikeda, T.; Kajiyama, K.; Kita, T.;
Takiguchi, N.; Kuroda, A.; Kato, J.; Ohtake, H. Chem.
Lett. 2001, 314–315.
7. (a) Eberhard, A.; Widrig, C. A.; McBath, P.; Schineller, J.
B. Arch. Microbiol. 1986, 146, 35–40; (b) Chaabra, S. R.;
Stead, P.; Bainton, N. J.; Salmond, G. P. C.; Stewart, G.
S. A. B.; Williams, P.; Bycroft, B. W. J. Antibiot. 1993, 46,
441–454; (c) Passador, L.; Tucker, K. D.; Guertin, K. R.;
Journet, M. P.; Kende, A. S.; Iglewski, B. H. J. Bacteriol.
1996, 5995–6000; (d) Schaefer, A. L.; Hanzelka, B. L.;
Eberhard, A.; Greenberg, E. P. J. Bacteriol. 1996, 2897–
2901; (e) Kline, T.; Bowman, J.; Iglewski, B. H.; de Kievit,
T.; Kakai, Y.; Passador, L. Biorg. Med. Chem. Lett. 1999,
9, 3447–3452; (f) Smith, K. M.; Bu, Y.; Suga, H. Chem.
Biol 2003, 10, 81–89; (g) Smith, K. M.; Bu, Y.; Suga, H.
Chem. Biol. 2003, 10, 563–571; (h) Welch, M.; Dutton, J.
M.; Glansdorp, F. G.; Thomas, G. L.; Smith, D. S.;
Coulthurst, S. J.; Barnard, A. M. L.; Salmond, G. P. C.;
Spring, D. R. Biorg. Med. Chem. Lett. 2005, 15, 4235–
4238; (i) Persson, T.; Hansen, T. H.; Rasmussen, T. B.;
Skinderso, M. E.; Givskov, M.; Nielsen, J. Org. Biomol.
Chem. 2005, 3, 253–262; (j) Geske, G. D.; Wezeman, R. J.;
Siegel, A. P.; Blackwell, H. E. J. Am. Chem. Soc. 2005,
127, 12762–12763, and references cited therein.
8. (a) Reverchon, S.; Chantegrel, B.; Deshayes, C.; Dou-
theau, A.; Cotte-Pattat, N. Bioorg. Med. Chem. Lett. 2002,
12, 1153–1157; (b) Castang, S.; Chantegrel, B.; Deshayes,
C.; Dolmazon, R.; Gouet, P.; Haser, R.; Reverchon, S.;
Nasser, W.; Hugouvieux-Cotte-Pattat, N.; Doutheau, A.
Bioorg. Med. Chem. Lett. 2004, 14, 5145–5149.
9. For recent reviews, see: (a) Chrusciel, R. A.; Strohbach, J.
W. Curr. Top. Med. Chem. 2004, 4, 1097–1114; (b) Dumas,
J. Curr. Opin. Drug Discov. Dev. 2002, 5, 718–727.
11. Kraatz, U.; Wamhoff, H.; Korte, F. Liebigs Ann. Chem.
1971, 744, 33–41.
12. Rao, V. S. Synth. Commun. 1989, 19, 1389–1393.
13. Kraatz, U.; Hasenbrink, W.; Wamhoff, H.; Korte, F.
Chem. Ber. 1971, 104, 2458–2466.
14. Knobler, Y.; Bonni, E.; Frankel, M. Tetrahedron 1967, 23,
1557–1563.
15. Silica gel chromatography of the reaction mixture
provided 34 still contaminated by
(ꢀ10%) of 35.
a small amount
16. Zhang, R. G.; Pappas, T.; Brace, J. L.; Miller, P. C.;
Oulmassov, T.; Molyneaux, J. M.; Anderson, J. C.;
Bashkin, J. K.; Winans, S. C.; Joachimiak, A. Nature
2002, 417, 971–974.
17. Luo, Z. Q.; Smyth, A. J.; Gao, P.; Qin, Y.; Farrand, S. K.
J. Biol. Chem. 2003, 278, 13173–13182.
18. Kiratisin, P.; Tucker, K. D.; Passador, L. J. Bacteriol.
2002, 184, 4912–4919.
19. Shadel, G. S.; Young, R.; Baldwin, T. O. J. Bacteriol.
1990, 172, 3980–3987.
20. Winson, M. K.; Swift, S.; Fish, L.; Throup, J. P.;
Jorgensen, F.; Chhabra, S. R.; Bycroft, B. W.; Williams,
P.; Stewardt, M. K. FEMS Microbiol. Lett. 1998, 163,
185–192.
21. Schuster, M.; Urbanowski, M. L.; Greenberg, E. P. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 15833–15839.
22. Zhu, J.; Winans, S. C. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 4832–4837.
23. Krieger, E.; Koraimann, G.; Vriend, G. Proteins 2002, 47,
393–402.