AHL-dependent quorum sensing inhibition: Synthesis and biological evaluation of α-(N-alkyl-carboxamide)-γ-butyrolactones and α-(N-alkyl-sulfonamide)-γ-butyrolactones

Article abstract of DOI:10.1016/j.bmcl.2011.09.010

New N-acylhomoserine lactone (AHL) analogues in which the amide function is replaced by a reverse-amide one have been studied as AHL QS modulators. The series of compounds consists of α-(N-alkyl-carboxamide)-γ- butyrolactones, α-(N-alkyl-sulfonamide)-γ-bu

Full text of DOI:10.1016/j.bmcl.2011.09.010

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6876–6879
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
AHL-dependent quorum sensing inhibition: Synthesis and biological
evaluation of
a-(N-alkyl-carboxamide)-
c-butyrolactones
and -(N-alkyl-sulfonamide)-
a
c-butyrolactones
a,b,c
, Mohamad Sabbah a,b, Laurent Soulère a,b, , Mohamed L. El Efrit c,
⇑
Mohamed Boukraa
Yves Queneau
a
a,b,
⇑
a,b
, Alain Doutheau
INSA Lyon, ICBMS, Bât J. Verne, 20 av A. Einstein, 69621 Villeurbanne Cedex, France
CNRS, UMR 5246, ICBMS, Université Lyon 1, INSA-Lyon, CPE-Lyon, Bât CPE, 43 bd du 11 Novembre 1918, 69622 Villeurbanne Cedex, France
Laboratoire de Synthèse Organique et Hétérocyclique, Faculté des Sciences de Tunis, Campus Universitaire, 2092 Manar II, Tunis, Tunisia
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2011
Revised 1 September 2011
Accepted 2 September 2011
Available online 10 September 2011
New N-acylhomoserine lactone (AHL) analogues in which the amide function is replaced by a reverse-
amide one have been studied as AHL QS modulators. The series of compounds consists of
boxamide)- -butyrolactones, -(N-alkyl-sulfonamide)- -butyrolactones, and 2-(N-alkyl-carboxamide)-
cyclopentanones and cyclopentanols. Most active compounds exhibited antagonist activities against LuxR
reaching the 30 M range.
a-(N-alkyl-car-
c
a
c
l
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Quorum sensing
Inhibition
AHL
Reverse-amide
Reverse-sulfonamides
The communication system referred to as quorum sensing (QS)
is used by bacteria for adapting themselves to their environment.
QS is based on the regulation of the expression of genes encoding
for phenotype expression via small molecules called autoinducers
function, and (3) the lactone moiety (Fig. 1). In the context of
AHL-dependent QS modulation, the strategies used in the design
of active compounds have included the synthesis of close struc-
tural analogues of AHLs in which the three areas have been modi-
1–6
8,9,17,18
which interact with their cognate receptor proteins.
Important
fied.
With respect to structural variations of the central
phenotypes such as bioluminescence, virulence expression, biofilm
formation are regulated by QS. Therefore modulation of QS can
have significant impact on human health and environment. QS
modulation can be achieved by several approaches, one of them
being the design of compounds able to act as agonist or antagonists
amide zone, our group has reported the synthesis and the evalua-
tion of several families of active analogues, including sulfona-
1
9
20
21
mides, ureas, and sulfonylureas,
considered as amide bioisosteres.
all this functions being
In keeping with our work focusing on the amide function of
AHLs, we were interested in testing new analogues having a ‘re-
verse-amide’ linkage. This approach is a common one for altering
amide containing compounds, notably for the design of peptide
7
–13
of autoinducers.
New QS modulators can bring insights into
bacterial signaling from the fundamental and applied points of
view, including the possible development of new antibacterial
strategies.
2
2,23
analogues.
Surprisingly, despite the known antagonist activity
N-Acyl-homoserine lactones (AHLs) are an important family of
1
4–
QS autoinducers, and the main one in Gram negative bacteria.
1
7
AHLs are rather simple molecules for which one can delimit
O
O
three areas ideal for chemical modification: (1) the acyl chain
including the characteristic carbonyl or hydroxyl substitution or
lack thereof at the C-3 position, (2) the central amide connective
O
H
N
O
⇑
Corresponding authors. Tel.: +33 4 72 43 61 69; fax: +33 4 72 43 88 96 (Y.Q.);
A
B
C
tel.: +33 4 72 43 83 42; fax: +33 4 72 43 88 96 (L.S.).
E-mail addresses: laurent.soulere@insa-lyon.fr (L. Soulère), yves.queneau@
insa-lyon.fr (Y. Queneau).
Figure 1. The three areas of AHLs: the lactone moiety (A), the central amide
function (B), and the side acyl chain (C).
0
960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.010

M. Boukraa et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6876–6879
6877
1
2
3
3
a : R = C H , R = H ; 85 %
4
9
O
O
O
1
2
b : R = C H , R = H ; 82 %
5
11
a
b
R1
COOH
COOH
1
2
COOH
O
3c : R = C
6
H
13, R = H ; 41 %
O
N
R2
1
2
3
d : R = C H , R = H ; 32 %
8
17
1
2
1
2
3e : R = C10H21, R = H ; 36 %
3
a-g
1
2
3
3
f : R = C H , R = Me; 54 %
4 9
1
2
g : R = C H , R = Me; 57 %
6
13
1
2
Scheme 1. Preparation of reverse amides 3a–g. Reagents and conditions: (a) NBu
4
Br, CH
3
2 2
CN, 75 °C, 16 h; (b) R R NH, DCC, DMAP, CH Cl , 0 °C, rt, 14 h.
O
O
N2
O
O
O
a
O
O
b
O
C
R
OH
N
H
O
c
C H
5 11
N
7
7
a : R = C H ; 76 %
b : R = C H ; 68 %
5 11
17
H
4
5
6
8
8 ; 75 %
Scheme 2. Preparation of cyclopentyl reverse amides 7 and 8. Reagents and conditions: (a) TsN
h.
3
, Et
3
N, CH
2
Cl
2
, 0 °C, 1 h, 77%; (b) RNH
2
, xylene, reflux 1 h; (c) NaBH
4
, MeOH, rt,
2
Table 1
O
O
O
O
O
O
Inhibition of bioluminescence obtained with compounds 3, 7, 8,
and 11
O
11a : R = C ; 42 %
11b : R
= C H11; 49 %
5
4
H
9
a
b
S
O
O
S
O
Cl
R
N
H
IC50a,b
(lM)
Compounds
9
10
11
3a
96 (±4)
34 (±5)
54 (±3)
52 (±3)
75 (±1)
40 (±2)
77 (±1)
200 (±8)
>200
70 (±5)
3b
Scheme 3. Preparation of reverse-sulfonamides 11. Reagents and conditions: (a)
SOCl , ClSO H, 120 °C, 2 h; (b) RNH , CH Cl , À10 °C, rt, 1.5 h.
3c
3d
2
3
2
2
2
3
7
7
8
e
a
b
of some homoserine lactone amides,13 and sulfonamides,19 to our
knowledge, only the reverse-amides analogues of oroidin, a marine
11a
1b
1
alkaloid possessing anti-biofilm properties, have been designed
and tested as QS inhibitors.2
4,25
We report hereafter the synthesis
a
Concentration (
l
M) required to reduce to 50% intensity
-HSL.
and the biological evaluation of some various AHL analogues
having a reverse-amide or a reverse-sulfonamide functions, as well
compounds in which the lactone ring is also modified as a cyclo-
pentanone or cyclopentanol one.
(IC50) the bioluminescence induced by 200 nM of 3-oxo-C
6
b
Values are the means of three experiments; standard devi-
ation is given in brackets.
Reverse-amide AHLs analogues, namely
a-(N-alkyl-carboxam-
ide)- -butyrolactones 3, were obtained in two steps (Scheme 1).
Reaction of butyrolactone 2 prepared from commercially avail-
c
promoter region of Vibrio fischeri to the luxCDABE operon of Photor-
habdus luminescens. None of them proved to display any agonistic
26
29
able cyclopropane dicarboxylic acid (1) with primary alkylamines
(C , C , C , C , C10) led to the desired amides 3a–e in 36–85% iso-
4 5 6 8
lated yields. Reaction with secondary amines N-methyl-N-butyl
and N-methyl-N-hexyl led to the corresponding N-methylated
amides 3f–g.
activity at any concentration (0.1–200
evaluated by measuring the decrease of the bioluminescence in-
duced by 3-oxo-C -HSL, the main autoinducer in Vibrio fischeri (Ta-
ble 1). Most compounds displayed antagonist activity in the whole
concentration range (1–200 M). The reverse amides 3a–e were
first tested, all of them exhibiting antagonist activity, with IC50
ranging from 34 to 96 M, the most active one being the N-pentyl
derivative. Surprisingly, a slightly shorter N-butyl chain resulted in
a significantly lower activity (96 M), whereas compounds having
lM). QS inhibition was then
6
l
Cyclopentanones analogues 7 were prepared in three steps from
commercially available cyclohexane-1,3-dione (4) (Scheme 2).
l
2
7
Reaction of 4 with tosylazide in the presence of triethylamine
led to the 2-diazo 5 which underwent a Wolff rearrangement to
give the intermediate ketene 6, which reacted with pentylamine
or octylamine to give 2-(N-alkyl-carboxamide)-cyclopentanones
l
chain length elongated to six, eight or 10 carbon atoms proved to
be only moderately less efficient antagonists. Since native AHLs
with various chain lengths have been reported to be either agonists
or antagonists with activities reaching the micromolar range,8
7
a and 7b in good yields. Reduction of compound 7a with sodium
,9,17
borohydride in methanol led to alcool 8a obtained in 75% yield, as a
mixture of two diasteroiomers, from which one of them could be
isolated as a pure sample.
this suggests that the
a-amidolactone motif in AHLs is an impor-
tant structural signature for efficient binding to the transcriptional
factors LuxR type proteins. N-methylamides 3f–g were inactive,
showing the importance of the NH system for binding within the
receptor active site. The cyclopentanones 7a and 7b also exhibited
antagonistic activity, in the same range of activity measured for the
corresponding lactones 3a and 3d, respectively. Alteration of the
Reverse-sulfonamides were prepared in two steps (Scheme 3)
from
c-butyrolactone (9) via its a-chlorosulfonyl 10 derivative ob-
tained by reaction with thionyl chloride in presence of chlorosul-
2
8
fonic acid.
pentylamine led to the corresponding
butyrolactones 11a–b in moderate yields.
Subsequent reaction of 10 with butylamine or
a-(N-alkyl-sulfonamide)-c-
lactone ring would be of limited effect once the
a-amidolactone
Compounds 3a–g, 7a,b, 8, and 11a,b were first tested for their
ability to induce luminescence in an Escherichia coli biosensor
strain containing a plasmid that couples the luxR and luxICDABE
linkage is replaced by a 1,3-dicarboxy system. By contrast, the
presence of an hydroxyl group instead of the keto one in cyclopent-
anols 8 having the pentyl chain resulted in loss of activity either for

6
878
M. Boukraa et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6876–6879
A molecular modeling study was investigated with the LuxR
3
0
model and compounds 3b, 11b as well as with the previously
1
9
31
6
reported C5 sulfonamide and 3-oxo-C -HSL. All compounds
showed similar binding modes interacting with conserved residues
Trp66, Tyr62, Asp79 (Figs. 2A and B). Distances between key atoms
involved in hydrogen bonds were not significantly different for
C@O or S@O groups and basically identical for the NH group (Table
2
3
). Modeling of compound 3b compared to the natural ligand
-oxo-C -HSL, (Fig. 2A) showed a different orientation of the lac-
6
3
1
tone and the alkyl chain. For compound 11b (Fig. 2B) versus C5 sul-
fonamide, a different orientation of the sulfonamide function is
observed with two hydrogen bonds with Tyr62 for 11b and only
one for C5 sulfonamide. In both cases, it is seen that the NH func-
tion locations are nearly superimposable. Antagonist activity of
reverse analogues can be explained by the necessary adaptation
of the location of the molecule when forced to match as much as
possible the H-bond network imposed by the binding site.
In conclusion, new AHL analogues, designed by replacing the
amide function by a reverse-amide or a reverse-sulfonamide one,
have been prepared and their activity as QS modulators has been
evaluated. Some reverse-amides
butyrolactones] and reverse-sulfonamides
amide)- -butyrolactones] having short alkyl side chains exhibited
significant QS antagonist activity, the most active having a 30
[a
-(N-alkyl-carboxamide)-
c-
[
a-(N-alkyl-sulfon-
c
lM
IC50
.
Altering the lactone to a cyclopentanone in the reverse carbox-
amides did not result in significant change in the activity. Finally,
N-methylated reveres-amides proved to be inactive. Besides the
interest of enlarging the structural scope of AHL antagonists, these
results also point out the key role of the NH group in the position-
ing of the AHL analogue in the binding site.
Acknowledgments
6
Figure 2. (A) Proposed binding modes of reverse amide 3b (in blue) and 3-oxo-C -
HSL (in green). (B) Proposed binding modes of reverse-sulfonamide 10b (in blue)
and C5 sulfonamide (in pink).
Financial support from MESR, CNRS, and the ‘Cluster de recher-
che chimie/infectiologie de la Région Rhône-Alpes’ is gratefully
acknowledged. M.S. is the recipient of a scholarship from the clus-
ter. We warmly thank Dr. Sylvie Reverchon for helpful discussions
and assistance during biological evaluation.
Table 2
Hydrogen bonds network with distances in Å between residues Trp66, Tyr62 and
Asp79 and different functions of 3-oxo-C
6
-HSL, reverse-amide 3b, C5-sulfonamide19
and reverse-sulfonamide 11b
Supplementary data
Trp66
Tyr62
Asp79
Supplementary data (experimental section for synthesis, bio-
logical evaluation and modeling) associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.2011.09.010.
3
-oxo-C
Reverse amide 3b
C5) Sulfonamide
6
-HSL
2.3 (C@O
lactone)
2.3 (C@O
lactone)
2.6 (C@O
lactone)
2.3 (C@O
lactone)
3.0 (C@O amide)
3.0
(NH)
3.0
(NH)
3.0
(NH)
2.9
2.8 (C@O amide)
3.1 (S@O)
(
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