N-Acyl-3-amino-5H-furanone derivatives as new inhibitors of LuxR-dependent quorum sensing: Synthesis, biological evaluation and binding mode study

Article abstract of DOI:10.1016/j.bmcl.2008.06.090

New N-acyl homoserine lactone analogues, N-acyl-3-amino-5H-furanone derivatives and some 4-halogeno counterparts, were synthesised and tested for their ability to modulate LuxR-dependent bacterial quorum sensing. Both types of analogues proved to be inhib

Full text of DOI:10.1016/j.bmcl.2008.06.090

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4321–4324
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Acyl-3-amino-5H-furanone derivatives as new inhibitors
of LuxR-dependent quorum sensing: Synthesis, biological evaluation
and binding mode study
c
Jane Estephane a,b, Julien Dauvergne a,b, Laurent Soulère a,b, , Sylvie Reverchon ,
*
Yves Queneau a,b, Alain Doutheau a,b,
*
a INSA Lyon, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Laboratoire de Chimie Organique, Bât J. Verne, 20 av A. Einstein, 69621 Villeurbanne Cedex, France
b CNRS, UMR 5246 ICBMS, Université Lyon 1, INSA-Lyon, CPE-Lyon, Bât CPE, 43 bd du 11 novembre 1918, 69622 Villeurbanne Cedex, France
c Unité Microbiologie, Adaptation et Pathogénie, UMR CNRS-UCBL-INSA-BAYER CROPSCIENCE 5240, Université Lyon 1, INSA-Lyon, Domaine Scientifique de la Doua,
Villeurbanne, France
a r t i c l e i n f o
a b s t r a c t
Article history:
New N-acyl homoserine lactone analogues, N-acyl-3-amino-5H-furanone derivatives and some 4-halo-
geno counterparts, were synthesised and tested for their ability to modulate LuxR-dependent bacterial
quorum sensing. Both types of analogues proved to be inhibitors, the halogenated compounds being sig-
nificantly more active. Molecular modelling suggested that the conjugated enamide group induces two
preferential conformations leading to specific binding modes. In addition, the presence of the halogen
atom could enhance the fitting of the lactone ring through specific interactions with strictly conserved
or conservatively replaceable residues in the LuxR protein family, namely Asp79, Trp94 and Ile81.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 14 May 2008
Revised 25 June 2008
Accepted 25 June 2008
Available online 28 June 2008
Keywords:
Quorum sensing
AHLs
LuxR
Antagonist
V. fischeri
Molecular modelling
Docking
Bacteria are able to communicate through small diffusible mol-
ecules called autoinducers. The concentration of these chemical
messengers in a specific environment reflects the bacterial density
and, when a critical concentration is reached, bacteria change their
biological activities. This cell to cell communication system, named
‘quorum sensing’, allows bacteria to coordinate their behaviour for
a specific activity, such as biofilm formation or virulence,1–4 ren-
dering it an attractive target for the conception of new antibacte-
rial agents.5–12 In Gram-negative bacteria, the quorum sensing is
logues, namely 3-amino-5H-furanone derivatives 5–7 and some 4-
halogenated counterparts 8–10 (Scheme 1). Compounds 5–7 were
designed to evaluate the modulation of the antagonist activity in-
duced by conformational and H-bonding modifications resulting
from the replacement of the amide by an enamide function. Be-
cause of their analogy with natural brominated furanones, known
to be quorum sensing inhibitors which display antimicrobial activ-
ities,19–21 the halogenated analogues 8–10 were also prepared.
The 3-amino-5H-furanone derivatives 5–10 were obtained from
known22 3-amino-5H-furan-2-one (2) prepared by reacting so-
orchestrated by N-acyl-L-homoserine lactones (AHLs) acting as
autoinducers, AHL synthase, an AHL-dependent transcriptional
regulator and specific genes encoding different phenotypes.13 Pre-
viously we have reported the antagonist activity and molecular
modelling studies of AHL analogues with structural modifications
on the acyl moiety, either with a terminal aromatic group14 or
resulting from the replacement of the amide function by sulfon-
amide or urea functions.15–18 We present here the synthesis, the
biological evaluation and the binding mode study of new AHL ana-
dium azide with a-bromo-c-butyrolactone (1) in dimethylform-
amide (Scheme 1).23 This compound 2 was further halogenated
with N-bromo or N-chloro-succinimide to afford known24 3-ami-
no-4-bromo-5H-furan-2-one (3) and 3-amino-4-chloro-5H-furan-
2-one (4), respectively. The furanones 2–4 were then acylated with
acyl chlorides under standard conditions to furnish the compounds
5 and 7–10. Compound 6 was prepared by acylation of Meldrum’s
acid with butyryl chloride and then by further reaction with 2. Pure
6 was obtained in low yield because crude product was a mixture
of 6 and of N-butanoyl-3-amino-5H-furan-2-one, arising from
reaction between 2 and butyryl chloride, two compounds that
proved to be difficult to separate by column chromatography.
* Corresponding authors. Fax: +33 472 43 88 96 (A.D.).
E-mail addresses: laurent.soulere@insa-lyon.fr (L. Soulère), alain.doutheau@insa-
lyon.fr (A. Doutheau).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.06.090

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J. Estephane et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4321–4324
O
O
O
i
O
O
ii
O
NH2
NH2
Br
3 X = Br
4 X = Cl
1
2
X
iii
iii
O
O
O
O
R
R
O
O
NH
NH
X
8 X = Br, R = -(CH2)4-CH3
9 X = Br, R = -(CH2)3-Ph
10 X = Cl, R = -(CH2)4-CH3
5 R = -(CH2)4-CH3
6 R = -CH2-CO-(CH2)2-CH3
7 R = -(CH2)3-Ph
Scheme 1. Synthesis of 3-amino-5H-furanone derivatives 5–10. Reagents and conditions: (i) NaN3, Et3N, DMF, 80 °C, 5 h, 60%; (ii) N-bromosuccinimide or N-
chlorosuccinimide, CH2Cl2, 50 °C, 2 h, 82% (3), 66% (4); (iii) RCOCl, diisopropylethylamine or triethylamine, THF, 0 °C then rt, 5–18 h, 95% (5), 95% (7), 85% (8), 70% (9), 69%
(10); Meldrum’s acid, butyryl chloride, pyridine, CH2Cl2, 0 °C then rt, 2 h, 2, dichloroethane, 80 °C, 24 h, 12% (6).
Compounds 5–10 were evaluated for their ability to inhibit the
induction of luminescence obtained with 3-oxo-hexanoylhomoser-
ine lactone (OHHL), the natural ligand of LuxR in the V. fischeri QS
system (Table 1).16 The structurally closest analogues 5 and 6 of
OHHL displayed an IC50 of 85 and 55 lM, respectively, whereas
compound 7, bearing a phenyl group on the terminal alkyl chain,
was found to be inactive, in contrast with previous results obtained
with phenyl-substituted OHHL analogues.6,14,25 Interestingly, the
brominated analogues in position 4, 8 and 9, exerted a stronger
inhibitory activity compared with their hydrogenated counter-
parts, which are either inactive (7) or about 10 times less active
(5). In order to confirm the effect of the halogen atom on antago-
nist activity, we prepared the chlorinated enamide 10. This com-
pound proved to be as active as 8 with about the same IC50 value
of 6.9 lM.
A molecular modelling study was performed to understand the
structure–activity relationship observed. Amino acid sequence
alignments revealed a high homology between the two proteins,
LuxR and TraR, especially for the strictly conserved residues lo-
cated in the ligand binding site (Tyr62, Trp66, Tyr70, Asp72,
Trp94, Ala113 and Gly121 for LuxR). A LuxR homology model
was developed17 based on a TraR structure (PDB code: 1L3L).26
To perform docking experiments with 3-amino-5H-furanone deriv-
atives, we defined the ligand binding site as a docking box with the
natural ligand 3-oxohexanoylhomoserine lactone (OHHL). The
docking result (Fig. 1A) showed a very similar binding mode of
OHHL as compared to the binding mode of OOHL co-crystallised
in the active site of TraR, with the same hydrogen bond network
and only slight differences in terms of inter-atomic distances
(Fig. 1B).26
Table 1
Inhibition of bioluminescence obtained with compounds 5–10
a,b
Compound
R
X
IC50
(lM)
5
6
7
–(CH2)4-CH3
–CH2-CO-(CH2)2-CH3
–(CH2)3-Ph
H
H
H
85 ( 2)
55 ( 2)
nac
Figure 1. (A) Docking result of OHHL in the ligand binding site of LuxR; (B)
schematic overview displaying the hydrogen bond network with distances; strictly
conserved residues and conservatively replaceable residues are coloured, respec-
tively, in red and in blue.
8
9
10
–(CH2)4-CH3
–(CH2)3-Ph
–(CH2)4-CH3
Br
Br
Cl
6.5 ( 0.1)
84 ( 1)
6.9 ( 0.4)
a
Concentration (lM) required to reduce to 50% intensity (IC50) the biolumines-
cence induced by 200 nM of 3-oxo-hexanoylhomoserine lactone.
Docking experiments were then conducted on compounds 5
and 8. The binding modes I and II were obtained as a result of
the flexible docking experiments with these compounds (Fig. 2).
b
Values are the means of three experiments; standard deviation is given in
brackets.
c
Not active at concentrations up to 100 lM.

J. Estephane et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4321–4324
4323
Figure 2. (A and B) Binding modes obtained as a result of flexible docking experiments with compounds 5 and 8 in the LuxR binding site; (C and D) magnified view displaying
hydrogen bonds with distances for binding mode I; (E and F) magnified view displaying hydrogen bonds with distances for binding mode II.
The binding mode I for compounds 5 and 8, coloured, respectively,
in blue and in magenta, involved the same hydrogen bond network
as the natural ligand with residues Asp79, Tyr62 and Trp66 (Fig. 2C
and D and see Fig. 1). The binding mode II for compounds 5 and 8,
coloured, respectively, in green and in cyan, is different since the
amide carbonyl group is involved in a hydrogen bond with
Trp66, with distances of 3.0 Å (5) and 2.8 Å (8) (Fig. 2E and F). In-
deed, the enamide function induces a conformational change as
compared to the saturated lactone involving two different binding
modes I and II which could be a plausible explanation for the
antagonist activity observed.
The comparison of binding modes between compounds 5 and 8
showed a different orientation of the lactone moiety for compound
8 due to specific hydrophobic and electrostatic interactions27 of the
bromine atom with residues strictly conserved or conservatively
replaceable in the LuxR protein family (Fig. 1), Ile81 and Trp94
(binding mode I) or with Asp79 and Ile81 (binding mode II). These
specific interactions provide a plausible explanation for the in-
creased antagonist activity of halogenated N-acyl-3-amino-5H-
furanones derivatives 8–10.
furanone have been identified. Halogenated compounds were
found to be more active than their hydrogenated counterparts,
most likely due to particular interactions between the halogen
atom and specific residues of LuxR. These results open up possibil-
ities for the design of potential QS inhibitors derived from haloge-
nated heterocyclic derivatives.
Acknowledgments
Financial support from MENESR and CNRS is gratefully
acknowledged. The authors thank V. James for improving the Eng-
lish of the manuscript.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.06.090.
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