Synthetic homoserine lactone-derived sulfonylureas as inhibitors of Vibrio fischeri quorum sensing regulator.
ABSTRACT A series of 9 homoserine lactone-derived sulfonylureas substituted by an alkyl chain, some of them bearing a phenyl group at the extremity, have been prepared. All compounds were found to inhibit the action of 3-oxo-hexanoyl-L-homoserine lactone, the natural inducer of bioluminescence in the bacterium Vibrio fischeri, the aliphatic compounds being more active than their phenyl-substituted counterparts. Molecular modelling studies performed on the most active compound in each series suggest that the antagonist activity could be related to the perturbation of the hydrogen-bond network in the ligand-protein complexes.
- SourceAvailable from: europepmc.org[Show abstract] [Hide abstract]
ABSTRACT: Bacteria use a cell-to-cell communication activity termed "quorum sensing" to coordinate group behaviors in a cell density dependent manner. Quorum sensing influences the expression profile of diverse genes, including antibiotic tolerance and virulence determinants, via specific chemical compounds called "autoinducers". During quorum sensing, Gram-negative bacteria typically use an acylated homoserine lactone (AHL) called autoinducer 1. Since the first discovery of quorum sensing in a marine bacterium, it has been recognized that more than 100 species possess this mechanism of cell-to-cell communication. In addition to being of interest from a biological standpoint, quorum sensing is a potential target for antimicrobial chemotherapy. This unique concept of antimicrobial control relies on reducing the burden of virulence rather than killing the bacteria. It is believed that this approach will not only suppress the development of antibiotic resistance, but will also improve the treatment of refractory infections triggered by multi-drug resistant pathogens. In this paper, we review and track recent progress in studies on AHL inhibitors/modulators from a biological standpoint. It has been discovered that both natural and synthetic compounds can disrupt quorum sensing by a variety of means, such as jamming signal transduction, inhibition of signal production and break-down and trapping of signal compounds. We also focus on the regulatory elements that attenuate quorum sensing activities and discuss their unique properties. Understanding the biological roles of regulatory elements might be useful in developing inhibitor applications and understanding how quorum sensing is controlled.Frontiers in Microbiology 01/2013; 4:114. · 3.90 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Many bacterial species are capable of assessing their local population densities through a cell-cell signaling mechanism termed quorum sensing (QS). This intercellular communication process is mediated by small molecule or peptide ligands and their cognate protein receptors. Numerous pathogens use QS to initiate virulence once they achieve a threshold cell number on a host. Consequently, approaches to intercept QS have attracted considerable attention as potential anti-infective therapies. Our interest in the development of small molecule tools to modulate QS pathways motivated us to evaluate triazole-containing analogs of natural N-acyl l-homoserine lactone (AHL) signals as non-native QS agonists and antagonists in Gram-negative bacteria. We synthesized 72 triazole derivatives of five broad structure types in high yields and purities using efficient Cu(i)-catalyzed azide-alkyne couplings. These compounds were evaluated for their ability to activate or inhibit two QS receptors from two prevalent pathogens - LasR from Pseudomonas aeruginosa and AbaR from Acinetobacter baumannii - using bacterial reporter strains. Several triazole derivatives were identified that were capable of strongly modulating the activity of LasR and AbaR. These compounds represent a new and synthetically accessible class of AHL analogs, and could find utility as chemical tools to study QS and its role in bacterial virulence.Organic & Biomolecular Chemistry 12/2012; · 3.57 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Bacteria belonging to the Pectobacterium genus are the causative agents of the blackleg and soft-rot diseases that affect potato plants and tubers worldwide. In Pectobacterium, the expression of the virulence genes is controlled by quorum-sensing (QS) and N-acylhomoserine lactones (AHLs). In this work, we screened a chemical library of QS-inhibitors (QSIs) and AHL-analogs to find novel QSIs targeting the virulence of Pectobacterium. Four N,N'-bisalkylated imidazolium salts were identified as QSIs; they were active at the µM range. In potato tuber assays, two of them were able to decrease the severity of the symptoms provoked by P. atrosepticum. This work extends the range of the QSIs acting on the Pectobacterium-induced soft-rot disease.International Journal of Molecular Sciences 01/2013; 14(10):19976-19986. · 2.46 Impact Factor
Synthetic homoserine lactone-derived sulfonylureas as inhibitors
of Vibrio fischeri quorum sensing regulator
Marine Frezza,a,bLaurent Soule `re,a,bSylvie Reverchon,cNicolas Guiliani,dCarlos Jerez,d
Yves Queneaua,band Alain Doutheaua,b,*
aINSA Lyon, Institut de Chimie et Biochimie Mole ´culaires et Supramole ´culaires, Laboratoire de Chimie Organique,
Ba ˆt J. Verne, 20 av A. Einstein, 69621 Villeurbanne Cedex, France
bCNRS, UMR 5246 ICBMS, Universite ´ Lyon 1, INSA-Lyon, CPE-Lyon, Ba ˆt CPE,
43 bd du 11 novembre 1918, 69622 Villeurbanne Cedex, France
cUnite ´ Microbiologie, Adaptation et Pathoge ´nie, UMR CNRS-UCBL-INSA-BAYER CROPSCIENCE 5240,
Universite ´ Lyon 1, INSA-Lyon, Domaine Scientifique de la Doua, Villeurbanne, France
dLaboratorio de Microbiologı ´a Molecular y Biotecnologı ´a, Unidad de Comunicacio ´n Bacterian,
Departamento de Biologı ´a—Facultad de Ciencias, Universidad de Chile, Las Palmeras, 3425 N˜un ˜oa, Santiago, Chile
Abstract—A series of 9 homoserine lactone-derived sulfonylureas substituted by an alkyl chain, some of them bearing a phenyl
group at the extremity, have been prepared. All compounds were found to inhibit the action of 3-oxo-hexanoyl-L L-homoserine lac-
tone, the natural inducer of bioluminescence in the bacterium Vibrio fischeri, the aliphatic compounds being more active than their
phenyl-substituted counterparts. Molecular modelling studies performed on the most active compound in each series suggest that
the antagonist activity could be related to the perturbation of the hydrogen-bond network in the ligand–protein complexes.
Quorum sensing (QS) is a cell-to-cell communication
system allowing bacteria to coordinate the expression
of specific genes according to their population density.
This process is based on the synthesis, diffusion and
detection of small signal molecules, called auto-inducers
(AIs). When these molecules reach a critical threshold
concentration, they interact with transcriptional regula-
tor proteins.1A great variety of bacteria use QS to coor-
dinate the expression of many diverse genes. In various
pathogenic bacteria, in particular, either the expression
of virulence factors or biofilm development is regulated
by QS. Consequently, QS disruption has been suggested
as a promising new strategy to control microbial infec-
tion or biofilm formation.2Among several conceivable
approaches to interfering with QS, the most widely ex-
plored to date has been the synthesis of AI analogues
displaying antagonist activity. Many Gram-negative
bacteria use acyl-homoserine lactones (AHLs) as AIs
with LuxR type proteins as transcriptional regulators.3
This QS mechanism has been the most studied, by far,
and different types of AHL analogues have been synthe-
sized and evaluated as potential inhibitors of LuxR.
Most of the synthetic analogues were prepared either
by varying the nature of the acyl chain or by altering
the lactone ring.4We recently reported two new different
classes of AHL analogues bearing either a sulfonamide
(compounds 1)5aor a urea (compounds 2)5bfunction in-
stead of the amide function, both of which showing a
pronounced inhibitory activity in the Vibrio fischeri QS
system. The inhibitory activity of compounds 1 was ex-
plained by the tetrahedral geometry around the sulfone
allowing the formation of an additional hydrogen bond
with a tyrosine residue of the ligand pocket,5a,6while the
activity of ureas 2 was interpreted by the presence of the
external NH of the urea function that enforces hydro-
gen-bonding with an aspartic acid residue.5b,6In keeping
with the study of the influence of modification of the
amide linkage in AHLs on their biological properties,
Keywords: QS; AHLs; Analogues; Antagonist; V. fischeri; Molecular
*Corresponding author. Fax: +33 472 43 88 96; e-mail: alain.
we report here the synthesis and the biological evalua-
tion of AHL-derived sulfonylureas of type 3 whose
structure contains both the two NH groups of the urea
function and maintains the tetrahedral geometry of the
sulfonamide one. In compounds 3 the R2group is an al-
kyl chain, some of them bearing a phenyl group at the
extremity (Scheme 1).
2. Results and discussion
2.1. Synthesis of sulfonylureas 3
Sulfonylureas 3 were prepared by submitting the cyclic
catechol sulfate 47to the action of diverse amines in ba-
sic conditions to give the intermediates 5, which further
react with homoserine lactone.8Sulfonylureas 3a–i were
thus obtained in overall yields ranging from 12% to 74%
2.2. Biological activity
Compounds 3a,i were tested for their ability to inhibit
the induction of luminescence by N-3-oxo-hexanoyl-L L-
homoserine lactone (3-oxo-C6-HSL) in an Escherichia
coli biosensor strain containing a plasmid that couples
the luxR and luxICDABE promoter region of V. fischeri
to the luxCDABE operon of Photorhabdus luminescens.
The assays were performed following the protocol previ-
ously described.9Both the sulfonylurea and 3-oxo-C6-
HSL were introduced in the culture medium, the latter
compound at the final concentration of 200 nM as re-
quired for 1/2 maximal induction of luminescence under
As shown by the results depicted in Figures 1 and 2, all
compounds proved to be active. For aliphatic com-
pounds, with a chain ranging from 4 to 10 carbon
atoms, the inhibitory activity is slightly dependent on
the chain length, the n-pentyl-sulfonylurea 3b being the
most active with an IC50value of 2 lM (Fig. 1).
Sulfonylureas bearing a phenyl substituent at the
extremity of the alkyl chain are less active than their ali-
phatic counterparts, the most active compound being 3f
which has the shortest distance between the urea func-
tion and the aromatic ring (Fig. 2).
2.3. Molecular modelling
Compounds 3b and 3f showing the best antagonist activ-
binding site of TraR, used as a model for the putative li-
to be appropriate since the docking of the natural ligand
of either LuxR (3-oxo-C6-HSL) or TraR (3-oxo-C8-
HSL) within the ligand-binding site of TraR led to very
similar binding modes (Fig. 3A and B). Preferential con-
formations of 3b and 3f were first calculated by varying
the key torsion angles and then compared to the confor-
ers were then docked in the active site of TraR.
2.4. Structure–activity relationship
In view of the structural similarity of the sulfonylureas 3
to the native AI, we hypothesized that these compounds
target the LuxR ligand-binding site, in the same way as
the preceding synthetic AHL analogues prepared in our
laboratory.5This hypothesis is reinforced by the molec-
ular modelling study which shows that the ligand-bind-
ing site of this protein can readily accommodate
The analysis of the docking results suggests that the
binding of 3b (Fig. 3C–D) looks quite different from that
of 3f (Fig. 3E–F). In the case of 3b, in contrast with the
NH of the amide function in the natural AI (Fig. 3B),
R1= -(CH2)n-CH3, -CH2-CO-(CH2)n-CH3
R2= -(CH2)n-CH3, -(CH2)n-Ph
Scheme 1. Structure of AHLs and of synthetic analogues 1–3.
a R = -C4H9
b R = -C5H11
c R = -C6H13
d R = -C8H17
e R = -C10H21
f R = -CH2-Ph
g R = -(CH2)2-Ph
h R = -(CH2)3-Ph
i R = -(CH2)4-Ph
Scheme 2. Synthetic route to sulfonylureas 3a–i.
the internal NH is more than 3.5 A˚away from Asp70 (as
well as from Tyr61 and Trp57) and, thus, is probably
not involved in a hydrogen bond with these residues.
On the other hand, the external NH and one atom of
oxygen of the sulfone group are both capable of forming
a hydrogen bond with the Tyr53 residue. A limited
number of conformations for the alkyl chain fits well
in the hydrophobic pocket. For the sulfonylurea 3f, a
higher number of conformations of the alkyl chain are
tolerated within the hydrophobic pocket. The internal
NH is now involved in a hydrogen bond with the
Asp70 residue, as was the NH of the amide function
of the natural AI, and the oxygen atoms of the sulfone
group could both form hydrogen bonds, one with the
Tyr53 and one with the Tyr61 residue.
The activity of 3b, the most active of the sulfonylureas,
was compared to that of the closest analogues in the sul-
fonylamide and urea series, namely the pentyl-sulfonyl-
amide and the butyl-urea.5The values of IC50, calculated
from the data obtained when these compounds were
activity is within the same micro-molar range (Fig. 4).
Thus, no synergistic effect on antagonist activity of the
tetrahedral arrangement of the SO2group and the urea
function was observed. Molecular modelling suggests
that this could result from the incapacity of the sulfonyl-
urea to establish more than two (for aliphatic ureas) or
three (for phenyl-substituted ureas) hydrogen bonds, as
donor or acceptor, within the ligand-binding site.
In this report, we have described the preparation of
various sulfonylureas substituted by an alkyl chain,
some of them bearing a phenyl group at their extrem-
ity, and the evaluation of their ability to inhibit the QS
regulator in V. fischeri bacteria. All compounds display
antagonist activities, aliphatic analogues being, how-
ever, more active than their phenyl-substituted coun-
terparts. Though this new series of AHL analogues
does not display improved antagonist activity when
compared to the synthetic analogues of AHLs previ-
ously prepared by us, the reported results provide
new structure–activity data giving a better understand-
ing of the ligand–protein interaction of this important
class of bacterial QS regulators. In particular, molecu-
lar modelling confirms that only a relatively slight per-
turbation ofthe hydrogen-bond
proximity of the amide-lactone moiety in the ligand–
protein complex is enough to induce a significant
All chemicals were purchased from Sigma–Aldrich
(France). Organic solutions were dried over anhydrous
sodium sulfate. The reactions were performed under a
constant flow of nitrogen and were monitored by t.l.c.
0 102030 40
Residual luminescence (%)
IC50 =3.6 µM
IC50 =2.0 µM
IC50 =5.0 µM
IC50 =7.2 µM
IC50 =7.8 µM
Figure 1. Inhibitory activity of aliphatic sulfonylureas 3a–e.
Residual luminescence (%)
IC50 = 6.4 µM
IC50 = 13 µM
IC50 = 9.4 µM
IC50 = 13 µM
Figure 2. Inhibitory activity of phenyl-substituted sulfonylureas 3f–i.
Figure 3. Docking results in the active site of TraR obtained for natural ligands (A), 3b (C) and 3f (E). Schematic overview of the ligand-binding site
complex for natural ligands (B), 3b (D) and 3f (F) displaying the hydrogen bond network.
on Silica Gel F254(Merck); detection was carried out by
charring a 5% phosphomolybdic acid solution in ethanol
containing 10% of H2SO4. Silica gel (Kieselgel 60, 70–
230 mesh ASTM, Merck) was used for flash-column
chromatographies. Melting points were determined on
a Kofler block apparatus. The
300 MHz) and13C NMR (50 MHz or 75 MHz) spectra
DRX300 spectrometer. The signal of the residual pro-
tonated solvent was taken as reference. Chemical shifts
(d) and coupling constants (J) are reported in ppm
and Hz, respectively. Elemental analyses were per-
formed by ‘Service Central d’Analyse du CNRS’ 69390
1H (200 MHz or
4.1.1. General procedure for the synthesis of sulfamates
(5a–i).8To a solution of the amine (1 mmol) in CH2Cl2
(2 mL) were added, at 0 ?C, triethylamine (160 lL,
1.1 mmol) and a solution of 47(192 mg, 1.1 mmol) in
CH2Cl2(1 mL). After 2 h (5a, 5g–i), 3h (5f) or 5 h (5b–
e) of stirring at 0 ?C, the reaction mixture was hydro-
lyzed (HClaq1%, 10 mL) and the organic layer was dec-
anted. The aqueous layer was extracted (Et2O, 2·
30 mL) and the combined organic phases were dried, fil-
tered and concentrated in vacuo. The residue thus ob-
tained was purified by column chromatography to give
the pure compounds 5.
Chromatography: CHCl3/MeOH (95:5). Colourless oil
(m, 1H), 7.31 (dd, J = 1.6 Hz and 8.1 Hz, 1H), 7.01
1H), 3.28 (t, J = 7.1 Hz, 2H), 1.66–1.52 (m, 2H), 1.48–
1.30 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H).13C NMR (ace-
ton-d6, 50 MHz): d 150.9, 139.8, 128.9, 124.9, 121.3,
118.9, 45.2, 32.9, 20.9, 14.5.
1H NMR (aceton-d6, 200 MHz): d 7.19–7.10
4J = 1.6 Hz and
3J = 8.1 Hz, 1H), 6.91–6.82 (m,
184.108.40.206. 2-Hydroxy-phenyl N-pentylsulfamate (5b).
Chromatography: CHCl3/MeOH (96:4). Colourless oil
2H), 7.06 (m, 1H), 6.90 (m, 1H), 3.26 (t, J = 7.2 Hz, 2H),
1.60–1.55 (m, 2H), 1.33–1.29 (m, 4H), 0.90 (t, J = 7.2 Hz,
3H).13C NMR (CDCl3, 75 MHz): d 148.4, 137.7, 128.3,
123.1, 121.0, 118.2, 44.8, 29.2, 28.5, 22.2, 13.9.
1H NMR (CDCl3, 300 MHz): d 7.23–7.16 (m,
2H), 7.05 (m, 1H), 6.92 (m, 1H), 6.25 (br s, 1H), 4.73
(br s, 1H), 3.28 (t, J = 6.8 Hz, 2H), 1.60–1.52 (m, 2H),
1.35–1.23 (m, 6H), 0.88 (t, J = 6.8 Hz, 3H).13C NMR
(CDCl3, 75 MHz): d 148.4, 137.7, 128.4, 123.3, 121.1,
118.3, 44.9, 31.3, 29.5, 26.1, 22.5, 14.0.
1H NMR (CDCl3, 300 MHz): d 7.22–7.16 (m,
Chromatography: CHCl3/MeOH (95:5). Colourless oil
(57%).1H NMR (CDCl3, 300 MHz): d 7.23–7.173 (m,
2H), 7.05 (m, 1H), 6.92 (m, 1H), 6.21 (br s, 1H), 4.82
(br s, 1H), 3.27 (t, J = 7.1 Hz, 2H), 1.62–1.54 (m, 2H),
1.27 (m, 10H), 0.88 (t, J = 6.6 Hz, 3H).
(CDCl3, 75 MHz): d 148.4, 137.7, 128.4, 123.1, 121.1,
118.6, 44.8, 31.7, 29.5, 29.1, 29.0, 26.3, 22.8, 14.0.
Chromatography: CHCl3/MeOH (99:1). Colourless oil
7.15 (m, 1H), 7.02 (m, 1H), 6.89 (m, 1H), 6.54 (br s,
1H), 5.23 (br s, 1H), 3.20 (m, 2H), 1.53–1.49 (m, 2H),
1.24(m,14H),0.88(t,J = 6.8 Hz,3H).13CNMR(CDCl3,
75 MHz): d 148.4, 137.7, 128.4, 123.1, 121.1, 118.3, 44.9,
32.0, 29.7, 29.6, 29.5, 29.4, 29.1, 26.4, 22.8, 14.2.
1H NMR (CDCl3, 300 MHz): d 7.23 (m, 1H),
220.127.116.11. 2-Hydroxy-phenyl N-benzylsulfamate (5f).
Chromatography: P/EtOAc (75:25). White solid (85%).
1H NMR (aceton-d6, 300 MHz): d 7.41–7.28 (m, 6H),
7.15 (ddd, J = 1.5, 7.2 and 8.1 Hz, 1H), 7.03 (d, J = 1.8
and 8.1 Hz, 1H), 6.87 (ddd, J = 1.8, 6.3 and 8.1 Hz,
1H), 4.47 (s, 2H).
150.3, 139.0, 138.2, 129.2, 128.7, 128.4, 128.3, 124.3,
120.6, 118.3, 48.3.
13C NMR (aceton-d6, 75 MHz): d
mate (5g). Chromatography: P/EtOAc (70:30). White
7.18 (m, 6H), 7.10 (m, 1H), 7.01 (m, 1H), 6.84 (m,
1H), 3.52 (t, J = 7.8 Hz, 2H), 2.91 (t, J = 7.8 Hz, 2H).
13C NMR (aceton-d6, 75 MHz): d 150.2, 139.3, 138.9,
129.5, 129.2, 128.3, 127.1, 124.2, 120.5, 118.2, 46.1, 36.4.
1H NMR (aceton-d6, 300 MHz): d 7.31–
18.104.22.168. 2-Hydroxy-phenyl N-(3-phenyl)propyl-sulfa-
mate (5h). Chromatography: P/EtOAc (70:30). White
solid (89%).1H NMR (CDCl3, 300 MHz): d 7.26–7.04
(m, 7H), 6.96 (m, 1H), 6.79 (m, 1H), 3.18 (t,
J = 7.2 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 1.79 (qui,
J = 7.2 Hz, 2H).13C NMR (CDCl3, 75 MHz): d 149.5,
140.9, 138.2, 128.4, 128.3, 128.0, 126.0, 123.2, 120.0,
118.4, 44.1, 32.5, 31.1.
mate (5i). Chromatography: P/EtOAc (70:30). White solid
(97%).1H NMR (aceton-d6, 300 MHz): d 7.30–7.10 (m,
7H), 6.99 (m, 1H), 6.85 (m, 1H), 3.31 (t, J = 7.2 Hz, 2H),
2.63(t,J = 7.2 Hz,2H),1.73–1.60(m,4H).13CNMR(ace-
ton-d6, 75 MHz): d 150.1, 142.9, 138.9, 129.0, 128.9, 128.2,
126.4, 124.1, 120.5, 118.2, 44.5, 35.8, 29.7, 29.0.
4.1.2. General procedure for the synthesis of sulfonylureas
(3a–i). To a suspension of D D,L L-a-amino-c-butyrolactone
IC50= 3 μ μM
IC50= 5 μ μM
IC50= 6 μ μM
Figure 4. Antagonist activity of sulfonylurea 3b and of its sulfonylamide and urea counterparts.
hydrobromide (200 mg, 1.1 mmol) in dioxane (1 mL),
were added triethylamine (160 lL, 1.1 mmol) and then
a solution of 5 (1 mmol) in dioxane (1 mL). The reaction
mixture was refluxed for 3 h and then cooled to rt and
filtered. The solvent was evaporated in vacuo and the
residue was hydrolysed (water, 20 mL) The aqueous
solution was extracted (CHCl3, 30 mL) and the organic
layer was dried, filtered and concentrated in vacuo. The
residue thus obtained was purified by column chroma-
tography to give the compounds 3a–i as white solids.
The biological tests were made on recrystallised samples.
nylurea (3a). Chromatography: CHCl3/MeOH (95:5).
Yield: 35%. Recrystallisation: P/toluene. Mp: 75–
76 ?C.1H NMR (CDCl3, 300 MHz): d 5.39 (br s, 1H),
4.92 (t, J = 5.4 Hz, 1H), 4.43 (t, J = 8.7 Hz, 1H), 4.30–
4.20 (m, 2H), 3.06 (m, 2H), 2.76–2.68 (m, 1H), 2.35–
2.21 (m, 1H), 1.57–1.48 (m, 2H), 1.41–1.29 (m, 2H),
0.90 (t, 3H, J = 7.2 Hz).13C NMR (CDCl3, 75 MHz):
d 175.4, 66.2, 52.2, 43.2, 31.5, 30.4, 19.9, 13.7. Anal.
Calcd for C8H16N2O4S: C, 40.66; H, 6.83; N, 11.86.
Found: C, 40.78; H, 6.92; N, 11.76.
fonylurea (3b). Chromatography: P/EtOAc (60:40).
Yield: 12%. Recrystallisation: P/toluene. Mp: 65–
66 ?C.1H NMR (CDCl3, 300 MHz): d 5.25 (br s, 1H),
4.76 (br s, 1H), 4.45 (t, J = 9.0 Hz, 1H), 4.30–4.22 (m,
2H), 3.08 (m, 2H), 2.79–2.70 (m, 1H), 2.36–2.21 (m,
1H), 1.60–1.51 (m, 2H), 1.33–1.28 (m, 4H), 0.88 (t,
3H, J = 6.8 Hz).13C NMR (CDCl3, 75 MHz): d 175.0,
66.0, 52.2, 43.4, 30.5, 29.1, 28.7, 22.2, 13.9. Anal. Calcd
for C9H18N2O4S: C, 43.18; H, 7.25; N, 11.19. Found: C,
43.34; H, 7.28; N, 10.76.
fonylurea (3c). Chromatography: CHCl3/MeOH (95:5).
Yield: 38%. Recrystallisation: P/toluene. Mp: 73–74 ?C.
1H NMR (CDCl3, 300 MHz): d 4.82 (br s, 1H), 4.48 (t,
J = 9.0 Hz, 1H), 4.38 (t, J = 6.8 Hz, 1H), 4.32–4.17 (m,
2H), 3.11 (q, J = 6.8 Hz, 2H), 2.83–2.74 (m, 1H), 2.37–
2.22 (m, 1H), 1.60–1.53 (m, 2H), 1.37–1.26 (m, 6H),
0.88 (t, 3H, J = 6.8 Hz).13C NMR (CDCl3, 75 MHz): d
175.4, 66.2, 52.3, 43.5, 31.4, 30.4, 29.5, 26.4, 22.6, 14.1.
Anal. Calcd for C10H20N2O4S: C, 45.44; H, 7.63; N,
10.60. Found: C, 45.22; H, 7.57; N, 10.55.
nylurea (3d). Chromatography: P/EtOAc (60:40).Yield:
39%. Recrystallisation: P/toluene. Mp: 73–74 ?C.
NMR (CDCl3, 300 MHz): d 5.39 (br s, 1H), 4.88 (t,
J = 6.0 Hz, 1H), 4.43 (t, J = 9.0 Hz, 1H), 4.29–4.20 (m,
2H), 3.06 (q, J = 6.4 Hz, 2H), 2.76–2.68 (m, 1H), 2.35–
2.21 (m, 1H), 1.56–1.51 (m, 2H), 1.35–1.24 (m, 10H),
0.85 (t, 3H, J = 6.8 Hz).13C NMR (CDCl3, 75 MHz): d
175.3, 66.2, 52.3, 43.5, 31.8, 30.5, 29.6, 29.3, 29.2, 26.8,
22.7, 14.1. Anal. Calcd for C12H24N2O4S: C, 49.29; H,
8.27; N, 9.58. Found: C, 48.65; H, 8.16; N, 9.31.
nylurea (3e). Chromatography: P/EtOAc (60:40).Yield:
62%. Recrystallisation: P/toluene. Mp: 87–88 ?C.
NMR (CDCl3, 300 MHz): d 5.47 (br s, 1H), 5.00 (t,
J = 5.7 Hz, 1H), 4.39 (t, J = 8.7 Hz, 1H), 4.27–4.19 (m,
2H), 3.03 (m, 2H), 2.74–2.65 (m, 1H), 2.33–2.19 (m,
1H), 1.52–1.48 (m, 2H), 1.22 (m, 14H), 0.84 (t, 3H,
J = 6.8 Hz).13C NMR (CDCl3, 75 MHz): d 175.5, 66.1,
52.2, 43.4, 31.9, 30.2, 29.7, 29.6, 29.5, 29.3, 29.2, 26.7,
22.6, 14.1. Anal. Calcd for C14H28N2O4S: C, 52.47; H,
8.81; N, 8.74. Found: C, 52.43; H, 8.85; N, 8.61.
fonylurea (3f). Chromatography: P/EtOAc (50:50).Yield:
74%. Recrystallisation: P/CHCl3. Mp: 101–102 ?C.1H
NMR (aceton-d6, 300 MHz): d 7.43–7.26 (m, 5H), 6.55
(br s, 1H), 6.35 (br s, 1H), 4.43–4.21 (m, 5H), 2.77–2.66
(m, 1H), 2.37–2.22 (m, 1H).
75 MHz): d 175.7, 138.7, 129.1, 128.9, 128.1, 66.2, 52.7,
47.6, 30.6. Anal. Calcd for C11H14N2O4S: C, 48.88; H,
5.22; N, 10.36. Found: C, 48.78; H, 5.13; N, 10.42.
13C NMR (aceton-d6,
sulfonylurea (3g). Chromatography: P/EtOAc (50:50).
Yield: 74%. Recrystallisation: P/EtOAc. Mp: 118–
119 ?C.1H NMR (CDCl3, 300 MHz): d 7.34–7.21 (m,
5H), 5.08 (d, J = 5.1 Hz, 1H), 4.61 (t, J = 6.4 Hz, 1H),
4.39 (t, J = 8.7 Hz, 1H), 4.20–4.08 (m, 1H), 3.99–3.91
(m, 1H), 3.38 (q, J = 6.8 Hz, 2H), 2.88 (t, J = 6.9 Hz,
2H), 2.64–2.55 (m, 1H), 2.20–2.11 (m, 1H).13C NMR
(CDCl3, 75 MHz): d 174.9, 138.1, 129.1, 128.9, 127.6,
66.1,52.1, 44.5, 35.7,
C12H16N2O4S: C, 50.69; H, 5.67; N, 9.85. Found: C,
50.81; H, 5.65; N, 9.78.
30.6. Anal.Calcd for
an-3-yl)-sulfonylurea (3h). Chromatography: P/EtOAc
(60:40). Yield: 52%. Recrystallisation: P/CHCl3. Mp:
123–125 ?C.1H NMR (CDCl3, 300 MHz): d 7.30–7.16
(m, 5H), 5.31 (br s, 1H), 4.92 (t, J = 6.0 Hz, 1H), 4.39
(t, J = 8.7 Hz, 1H), 4.26–4.15 (m, 2H), 3.12 (q,
J = 6.4 Hz, 2H), 2.71–2.63 (m, 3H), 2.30–2.16 (m, 1H),
1.89 (quint., J = 7.5 Hz, 2H).
75 MHz): d 175.3, 141.1, 128.6, 128.5, 126.2, 66.2,
C13H18N2O4S: C, 52.33; H, 6.08; N, 9.39. Found: C,
52.53; H, 6.10; N, 9.38.
13C NMR (CDCl3,
(50:50). Yield: 71%. Recrystallisation: P/EtOAc. Mp:
7.16 (m, 5H), 6.37 (d, J = 7.2 Hz, 1H), 5.92 (t,
J = 6.4 Hz, 1H), 4.41–4.22 (m, 3H), 3.14–3.06 (m, 2H),
2.73–2.66 (m, 1H), 2.62 (t, J = 7.5 Hz, 2H), 2.34–2.20
(m, 1H), 1.68–1.55 (m, 4H).
75 MHz): d 175.5, 143.0, 129.0, 128.9, 126.3, 66.1,
52.5, 43.4, 35.9, 31.0, 30.2, 29.9. Anal. Calcd for
C14H20N2O4S: C, 53.83; H, 6.45; N, 8.97. Found: C,
53.95; H, 6.43; N, 8.94.
1H NMR (aceton-d6, 300 MHz): d 7.29–
13C NMR (aceton-d6,
4.2. Biological tests
Measurement of inhibitory activity was performed
according to our previously reported protocol.9Pre-
sented data are from one culture but are representative
of three independently performed experiments where
standard variation was less than 15% in each case. The
IC50values were obtained from the graphs of Figures
1 and 2.
4.3. Molecular modelling
All calculations were performed on a Dell OPTIPLEX
GW 620 PC equipped with a double processor and with
the Sybyl 7.2 package for Linux,10ArgusLab11and YA-
SARA12as software. Conformation analyses were car-
ried out using the grid search module of Sybyl.
Docking experiments were performed with both the
docking module of Sybyl and of ArgusLab. The docking
box was generated by selecting residues within a dis-
tance of 3.5 A˚from the ligand. Conformation analyses
and minimization calculations were performed using
the TRIPOS force field with the conjugate Gradient
method and Gasteiger-Hu ¨ckel charges. The systematic
search was carried out on each compound by varying
key torsion angles. Resulting conformers were then clas-
sified by increasing order of energy to analyse conforma-
tions of lowenergy. Representative
conformers (20–30 fixed conformers) obtained were then
docked as rigid ligands in the docking box. Docking re-
sults were analysed for the conformers with the best li-
gand pose to describe interactions between the ligand
and the active site of TraR, hydrogen bonds were as-
signed within a distance of 3 A˚.
Financial support from MENESR and CNRS is grate-
fully acknowledged. M.F. thanks the MENESR for
her scholarship. This work was supported, in part, by
a ECOS (France-Chili; C05B04) grant. 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, 25, 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; (e) Gonzalez, J. E.; Kesh-
avan, N. D. Microbiol. Mol. Biol. Rev. 2006, 70, 859–875;
(f) Rasmussen, T. B.; Givskov, M. Microbiology 2006,
152, 895–904; (g) Reading, N. C.; Sperandio, V. FEMS
Microbiol. Lett. 2006, 254, 1–11.
2. (a) Suga, H.; Smith, K. M. Curr. Opin. Chem. Biol. 2003,
7, 586–591; (b) Hentzer, M.; Givskov, M. J. Clin. Invest.
2003, 112, 1300–1307; (c) Hentzer, M.; Eberl, L.; Nielsen,
J.; Givskov, M. BioDrugs 2003, 17, 241–250; (d) Persson,
T.; Givskov, M. M.; Nielsen, J. Curr. Med. Chem. 2005,
12, 3103–3115; (e) 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; (f) Rice, S. A.; McDougald, D.; Kumar, N.;
Kjelleberg, S. Curr. Opin. Investig. Drugs 2005, 6, 168–184;
(g) Rasmussen, T. B.; Givskov, M. Int. J. Med. Microbiol.
2006, 296, 149–161; (h) Abraham, Wolf-Rainer A. Curr.
Med. Chem. 2006, 13, 1509–1524; (i) Sperandio, V. Expert
Rev. Anti Infect. Ther. 2007, 5, 271–276.
3. (a) Eberhard, A.; Burlingame, A. L.; Eberhard, C.;
Kenyon, G. L.; Nealson, K. H.; Oppenheimer, N. J.
Biochemistry 1981, 20, 2444–2449; (b) Smith, D.; Wang,
J.-H.; Swatton, J. E.; Davenport, P.; Price, B.; Mikkelsen,
H.; Stickland, H.; Nishikawa, K.; Gardiol, N.; Spring, D.
R.; Welch, M. Sci. Prog. 2006, 89, 167–211; (c) Nasser,
W.; Reverchon, S. Anal. Bioanal. Chem. 2007, 387, 381–
4. (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) Zhu, J.; Beaber, J. W.; More, M. I.; Fuqua, C.;
Eberhard, A.; Winans, S. C. J. Bacteriol. 1998, 180, 5398–
5405; (f) Kline, T.; Bowman, J.; Iglewski, B. H.; de Kievit,
T.; Kakai, Y.; Passador, L. Biorg. Med. Chem. Lett. 1999,
9, 3447–3452; (g) Smith, K. M.; Bu, Y.; Suga, H. Chem.
Biol. 2003, 10, 81–89; (h) Smith, K. M.; Bu, Y.; Suga, H. .
Chem. Biol. 2003, 10, 563–571; (i) 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; (j) Persson, T.; Hansen, T. H.; Rasmussen, T. B.;
Skinderso, M. E.; Givskov, M.; Nielsen, J. Org. Biomol.
Chem. 2005, 3, 253–262; (k) Geske, G. D.; Wezeman, R.
J.; Siegel, A. P.; Blackwell, H. E. J. Am. Chem. Soc. 2005,
127, 12762–12763; (l) Geske, G. D.; O’Neill, J. C.;
Blackwell, H. E. ACS Chem. Biol. 2007, 2, 315–319; (m)
Ishida, T.; Ikeda, T.; Takiguchi, N.; Kuroda, A.; Ohtake,
H.; Kato, J. Appl. Environ. Microbiol. 2007, 73, 3183–
3188; (n) Janssens, J. C. A.; Metzger, K.; Daniels, R.;
Ptacek, D.; Verhoeven, T.; Habel, L. W.; Vanderleyden,
J.; De Vos, D. E.; De Keersmaecker, S. C. Appl. Environ.
Microbiol. 2007, 73, 535–544.
5. (a) 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; (b) Frezza, M.; Castang,
S.; Estephane, J.; Soulere, L.; Deshayes, C.; Chantegrel,
B.; Nasser, W.; Queneau, Y.; Reverchon, S.; Doutheau, A.
Bioorg. Med. Chem. 2006, 14, 4781–4791.
6. Soule `re, L.; Frezza, M.; Queneau, Y.; Doutheau, A.
J. Mol. Graphics Modell. 2007, 26, 581–590.
7. Tickner, A. M.; Liu, C.; Hild, E.; Mendelson, W. Synth.
Commun. 1994, 24, 1631–1637.
8. DuBois, G. E.; Stephenson, R. A. J. Org. Chem. 1980, 45,
9. Reverchon, S.; Chantegrel, B.; Deshayes, C.; Doutheau,
A.; Cotte-Pattat, N. Bioorg. Med. Chem. Lett. 2002, 12,
10. SYBYL, Molecular Modeling System, Tripos Associates
Inc., S. Hanley Road, St. Louis, MO 63144-62913, USA,
11. Thompson, M. A. ArgusLaB 4.01, Seattle, WA, Planetaria
Software LLC, 2004.
12. Krieger, E.; Koraimann, G.; Vriend, G. Proteins 2002, 47,