2-substituted aniline as a simple scaffold for LuxR-regulated QS modulation

Article abstract of DOI:10.3390/molecules22122090

The ability of the 2-substituted aniline motif to serve as a scaffold for designing potential LuxR-regulated quorum sensing (QS) modulators has been investigated, using docking experiments and biological evaluation of a series of 15 specially synthesized compounds. Aniline, 2-acetyl-aniline and 2-nitroaniline were considered, as well as their N-acylated derivatives. Docking experiments showed that the 2-substituted aniline motif fits within the LuxR binding site at the place of the lactone moiety of AHL, and the biological evaluation revealed QS antagonisitic activity for several compounds, validating the hypothesis that this scaffold acts on QS. Structure activity relationships are discussed regarding interactions with the key residues of the LuxR binding site, showing significant variations in the H-bonding pattern.

Full text of DOI:10.3390/molecules22122090

molecules
Article
2-Substituted Aniline as a Simple Scaffold for
LuxR-Regulated QS Modulation
Sizhe Li 1, Julien Wawrzyniak 2, Yves Queneau 1,* and Laurent Soulère 1,
*
1
Univ Lyon, INSA LYON, Université Lyon 1, CPE Lyon, UMR 5246, CNRS, ICBMS, Bât. J. Verne,
20 Avenue A. Einstein, F-69621 Villeurbanne, France; sizhe.li@insa-lyon.fr
Univ Lyon, INSA Lyon, Université Lyon 1, UMR 5240, CNRS, MAP, 10 rue Raphaël Dubois,
F-69622 Villeurbanne, France; julien.wzk@gmail.com
2
*
Correspondence: yves.queneau@insa-lyon.fr (Y.Q.); laurent.soulere@insa-lyon.fr (L.S.);
Tel.: +33-472-436-169 (Y.Q.); +33-472-438-342 (L.S.)
Received: 29 September 2017; Accepted: 27 November 2017; Published: 29 November 2017
Abstract: The ability of the 2-substituted aniline motif to serve as a scaffold for designing potential
LuxR-regulated quorum sensing (QS) modulators has been investigated, using docking experiments
and biological evaluation of a series of 15 specially synthesized compounds. Aniline, 2-acetyl-aniline
and 2-nitroaniline were considered, as well as their N-acylated derivatives. Docking experiments
showed that the 2-substituted aniline motif fits within the LuxR binding site at the place of the
lactone moiety of AHL, and the biological evaluation revealed QS antagonisitic activity for several
compounds, validating the hypothesis that this scaffold acts on QS. Structure activity relationships are
discussed regarding interactions with the key residues of the LuxR binding site, showing significant
variations in the H-bonding pattern.
Keywords: quorum sensing; antagonists; 2-nitroaniline scaffold; molecular docking
1. Introduction
Bacterial quorum sensing (QS) is a communication system based on the biosynthesis of small
diffusible molecules, called autoinducers, able to interact with proteins, in particular transcriptional
factors [
to fight bacterial virulence, has been extensively studied [
systems have been identified [ ]. Two of them are regulated by acyl homoserine lactones (AHLs),
1]. This communication system, and, more specifically, its quenching using small molecules
2–7
]. In Pseudomonas, to date, three QS
8
namely N-3-oxo-dodecanoyl-homoserine lactone and N-butanoyl-homoserine lactone, and they
interact, respectively, with LasR and RhlR. Another system, called PQS (Pseudomonas quinolone
signal), is regulated via quinolone derivatives. These compounds are biosynthesized from anthranilic
acid, yielding HHQ [9]. Recently, a secondary metabolite from this biosynthetic pathway, namely
2-aminoacetophenone (2-AA), has been shown, by Kviatkovski and co-workers [10], to activate LuxR.
In their work, they also used molecular modeling to demonstrate that 2-AA replaces the lactone, within
the ligand binding site of a LuxR model, with hydrogen bonds between the C=O and Trp66 and between
the amine and Asp79. The 2-nitrobenzamide derivative TP-1 [11] (Figure 1) and other 2-nitrobenzamide
derivatives have also been shown to be LasR modulators [12]. The X-ray-analyzed structure of the
TP-1/LasR complex [11] shows that the 2-nitrobenzamide moiety, like 2-AA, is positioned at the same
place as the lactone of AHL, with hydrogen bonding between the nitro group and Trp60 and also
between the NH of the amide function and Asp73. These observations led us to investigate whether
other 2-substituted anilines, in particular 2-nitroaniline (2-NA), act as new simple scaffolds for QS
modulation. We report here the results of our study, using docking and biological screening, on a series
of 2-acetyl-aniline and 2-nitroaniline compounds, as well as their N-acylated derivatives, as potential
QS modulators.
Molecules 2017, 22, 2090; doi:10.3390/molecules22122090
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Figure 1. Structure-based design of 2-nitroaniline (2-NA) and derivatives (2-NA).
2. Results and Discussion
Flexible docking investigations [13
,14] of N-3-oxo-hexanoyl-homoserine (OHHL) [15] and
2-nitroaniline using a LuxR model described in 2007 by Soulère and co-workers [16] showed that
2-nitroaniline is mimicking the lactone ring, notably forming hydrogen bonds between the nitro group
(replacing the ester function of OHHL) and Trp66 (Figure 2).
Figure 2. Proposed binding mode obtained as a result of docking experiments of 2-NA (magenta)
within the LuxR model binding site. OHHL is also represented, in cyan. Docking experiments were
performed using ArgusLab as software with a genetic algorithm as the docking engine (GADock)
(see experimental section).
In keeping with the same structural analogy, in which the amine function of 2-NA or 2-AA
(see supplementary data) and the NH group of the amide function of OHHL appear in similar positions
within the binding site, we included in this study their N-acylated derivatives, thus mimicking the
amide side chain of AHLs. For comparison, we also investigated acyl anilines to examine the effect of
this substituent in position 2 (Figure 3).

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Figure 3. Structure of substituted anilines and their acylated derivatives.
All compounds were easily prepared from 2-NA
corresponding acyl chloride (Figure 4). For compounds
[
17], 2-AA or aniline by direct acylation with the
10, and 15, the acyl chloride was synthesized
5,
using oxalyl chloride from 4-phenylbutyric acid with catalytic dimethylformamide.
Figure 4. Synthesis and structure of acyl aniline derivatives as LuxR modulators.
For the biological evaluation [18], all compounds were evaluated as agonists for their ability
to induce bioluminescence in the V. fischeri QS system with a recombinant Escherichia coli biosensor.
This biosensor strain produces luminescence with exogenously-provided AHL. None of them were
found to be agonists, i.e., they did not induce bioluminescence. Compounds were also tested, at various
concentrations, as antagonists in competition with 200 nM of 3-oxo-C6-HSL (Figure 5).
Interestingly, these assays revealed that 2-NA, N-(2-nitrophenyl)butanamide (
N-(2-nitrophenyl)hexanamide ( ) all displayed antagonist activity whereas other acyl derivatives,
were totally inactive. Based on the LuxR-QS system, compounds and exhibited IC50 values of
58 M and 94 M, respectively, comparable with many other compounds described in the literature,
thus validating our rational approach [ ]. For acyl nitroaniline , with longer acyl chains (C8 and
C10) or with a terminal phenyl group, their inactivity can be explained by an inappropriate size of the
substituent R2, like what was observed for other structurally close AHL analogues with long chains [ ].
As shown in Figure 6, docking experiments, with compounds and (nitro-C4 and nitro-C6) within
1) and
2
3–
10
,
1
2
µ
µ
5
3–5
5
1
2
the LuxR model binding site, revealed a satisfactory fit of these compounds, with a binding mode
of hydrogen bonds between Trp66 and the nitro function and between Asp79 and the NH group.

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The aromatic part of the compound fits well and replaces the lactone moiety. The difference in activity
for compounds 1 and 2 may be due to the change in the orientation of the alkyl chain.
Figure 5. Antagonistic activity of 2-NA, and acyl 2-nitroaniline derivatives 1–2 and acyl aniline
12. Other derivatives were inactive (not shown). Bioluminescence was induced with 200 nM of
3-oxo-C6-HSL. The experiments were carried out in triplicate shown in different colors.
Figure 6. Proposed binding mode obtained as a result of the docking experiment with compound
1
(magenta) and
binding site.
2 (cyan) showing that the phenyl group and the alkyl chain fit within the LuxR model
A comparison (biological evaluation and docking experiments) of 2-unsubstituted anilines was
then performed. As already reported with LuxR for compound 14 19], compounds 13 and 15
were found to be inactive. Interestingly, the hexanamide 12 showed significant antagonist activity,
with an IC50 value of 79 M, in the same range as N-(2-nitrophenyl)hexanamide ( ). However, to our
[
µ
2
surprise, further shortening of the chain to C4 (compound 11) led to a total loss of activity, whereas
the corresponding nitrated counterpart was found to be the most active compound in this study.
This shows that the two families of compounds behave differently, with a crucial nitro-substituent
which anchors the molecules within the binding site, via a hydrogen bond with Trp66, and a less

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important chain length effect rendering both the C4 and the C6 derivatives active. On the other hand,
for the unsubstituted aniline series, only one very specific derivative, the C6 amide, displays sufficient
favorable interaction and sterical balance. This hypothesis is supported by docking experiments
showing only one favorable binding mode for hexanoylaniline 12 within the LuxR binding site
(Figure 7) whereas several binding modes, all unfavorable, were found for compound 11 within the
LuxR binding site.
Figure 7. The binding mode (molecular docking) of compound 12 (cyan) shows a good fit, through
hydrogen bonding, with Asp79 (NH).
We next examined the H-bond interactions involving the important and conserved residues of the
binding site (Trp66, Asp79 and Tyr62) for 2-AA, 2-NA, and compounds 1–2, 6–7 and 11–12, with the
appropriate R2 substituents (Table 1).
Table 1. Occurrence and distances for hydrogen bonds between Trp66, Asp79, and Tyr 62 and the main
chemical functions of studied compounds with distances (Å) a,b
.
Compounds
Trp66
Asp79
Tyr62
OHHL (natural ligand) +2.34 (C=O lactone) a
+3.01 (NH amide)
+3.03 (NH2)
+3.01 (C=O amide)
No function
2-AA (agonist)
2-NA (antagonist)
1 (antagonist)
2 (antagonist)
6 (No activity)
7 (No activity)
11 (No activity)
12 (antagonist)
a
+2.20 (C=O)
+2.96 and 2.37 (NO2)
+2.47 (NO2)
-NH2 too far
No function
+3.00 (NH amide)
+3.01 (NH amide)
+3.10 (NH amide)
+2.98 (NH amide)
-
-C=O too far
-C=O too far
+2.97 (C=O amide)
+2.94 (C=O amide)
-
+2.20 (NO2)
+1.99 (C=OCH3)
+2.01 (C=OCH3)
-
No function
+3.04 (NH amide)
-C=O too far
+ indicates a possible H-bond; b the function implicated is indicated in brackets.
The autoinducer OHHL displays three hydrogen bonds with Trp66, Asp79, and Tyr62. The agonist
2-AA displays two hydrogen bonds with the same residues (none with Tyr62 due to the absence of the
carboxamide function). In contrast, 2-NA, which shows antagonist activity, interacts only with Trp66,
but in this case through two hydrogen bonds between Trp66 and the nitro group, and it does not interact
with Asp79. The binding modes for 2-AA (agonist) and 2-NA (antagonist) are, therefore, significantly

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different with respect to the H-bond interactions. The acylated nitroanilines
1
and
2
display two
hydrogen bonds, one with Trp66 and one with Asp79, whereas the simpler hexanoylaniline 12 shows
only one hydrogen bond with Asp79. Thus, antagonists and 12 all display hydrogen bonds with
1, 2
Asp79 and Trp66 (if possible) but not with Tyr62. The combination of satisfactory overall binding and
the absence of any interaction with Tyr62 appear to be a common feature among compounds showing
antagonistic activity. For the inactive compound 11, docking experiments showed several binding
modes due to the size of this compound and the absence of the nitro group. For compounds 6 and 7,
they adopt a binding mode with the same hydrogen bond network as OHHL, including the interaction
with Tyr62.
3. Materials and Methods
3.1. Binding Mode Studies
Docking experiments were performed with the docking module of the ArgusLab software
4.0.1 (Planetaria Software LLC.: Seattle, WA, USA) [14]. The protein model of LuxR [16] was built
using SWISS-MODEL [20] with ClustalW [21]. The binding mode of OHHL was obtained using the
method described by Estephane and co-workers [15]. Docking experiments were performed with
the following parameters: Docking box: X = Y = Z = 15 Å, ligand option: flexible; calculation type:
Dock; Docking engine: GADock (Genetic Algorithm) [13]; Genetic algorithm dock settings: default
advanced parameters; hydrogen bonds were assigned within a distance of 3 Å. Figures were generated
using PyMOL. The binding mode with hydrogen bonds of 2-AA (magenta) is shown in the following
figure (Figure 8) and it is in keeping with that obtained by Kviatkovski and co-workers [10].
Figure 8. The binding mode (molecular docking) of 2-AA (magenta) within the LuxR model binding
site. OHHL is also represented, in cyan.
3.2. Biological Evaluation
Compounds were evaluated for their ability to induce bioluminescence (agonistic activity) in
the V. fischeri QS system. For this purpose, we used the recombinant Escherichia coli biosensor strain
NM522 containing the plasmid pSB401. In this plasmid, the luxR and the luxI promoters from V.
fischeri are associated with the lux structural operon (luxCDABE) from Photorhabdus luminescens [18,22].
This biosensor strain produces luminescence with exogenously provided AHL. The activity was
measured using a microtiter plate format (Fisher Scientific, Waltham, MA, USA). A competition assay

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(antagonistic activity) was also performed in the presence of 200 nM of 3-oxo-C6-HSL and with
simultaneously added analogs. The experiments were carried out in triplicate with a maximum of 2%
DMSO. A negative 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 performed in the presence of
3-oxo-C6-HSL only.
3.3. Synthesis
Chemical reagents were purchased from Sigma Aldrich (St. Louis, MO, USA). Flash chromatography
was performed using 60 M silica gel. All reactions were monitored with thin-layer chromatography (TLC)
carried out on Merck aluminum silica gel 60-F254 (Darmstadt, Germany) using UV light and a KMnO4
solution as the stain. 1H nuclear magnetic resonance (NMR) spectra were obtained on Bruker ALS300 and
DRX300 spectrometers (Billerica, MA, USA).
3.3.1. General Procedure for the Synthesis of N-(2-Nitrophenyl) Amide
To a solution of 2-nitroaniline (1.0 eq), in 10 mL anhydrous dichloromethane, was added
triethylamine (2.5 eq) and acyl chloride (2.5 eq: butyryl chloride, hexanoyl chloride, octanoyl chloride,
decanoyl chloride, 4-phenylbutanoyl chloride was prepared from 4-phenylbutanoic acid and oxalyl
◦
chloride with catalytic DMF) at 0 C. The reaction mixture was left to warm to room temperature,
with stirring, for 16 h. The mixture was diluted in 30 mL dichloromethane and washed with 40 mL
2 M HCl, 40 mL 1 M NaOH and 40 mL of brine. The organic layer was dried over anhydrous Na2SO4,
evaporated and then the residue was purified by flash chromatography column to give the required
products (29% to 85% yield).
3.3.2. General Procedure for the Synthesis of N-phenyl Amide Compounds and N-(2-acetylphenyl)
Amide Compounds
To a solution of aniline or 2-Aminoacetophenone (1.0 eq), in 10 mL anhydrous dichloromethane,
was added triethylamine (1.1 eq) and acyl chloride (1.1 eq: butyryl chloride, hexanoyl chloride, octanoyl
chloride, decanoyl chloride, 4-phenylbutanoyl chloride was prepared from 4-phenylbutanoic acid and
◦
oxalyl chloride with catalytic DMF) at 0 C. The reaction mixture was left to warm to room temperature,
with stirring, for 16 h. The mixture was diluted in 30 mL dichloromethane and washed with 40 mL
1 M HCl, 40 mL sat. NaHCO3 and 40 mL brine. The organic layer was dried over anhydrous Na2SO4,
evaporated and then the residue was purified by flash chromatography column to give the required
products (36% to 94% yields).
N-(2-Nitrophenyl)butyramide (1) see reference [17].
N-(2-Nitrophenyl)hexanamide (2) see reference [17].
N-(2-Nitrophenyl)octanamide (3) see reference [17].
N-(2-Nitrophenyl)decanamide (
4
). Purification: pentane/diethyl ether = 10/1; yellow solid (43%);
1H-NMR (300 MHz, Chloroform-d)
δ
10.30 (s, 1H, NH), 8.74 (dd, J = 8.6, 1.4 Hz, 1H, Ph-H),
8.15 (dd, J = 8.5, 1.6 Hz, 1H, Ph-H), 7.58 (ddd, J = 8.8, 7.2, 1.6 Hz, 1H, Ph-H), 7.10 (ddd, J = 8.5,
7.2, 1.4 Hz, 1H, Ph-H), 2.42 (t, J = 7.5 Hz, 2H, COCH2), 1.69 (p, J = 7.5 Hz, 2H, CH2), 1.35–1.11
(m, 12H, 6CH2), 0.81 (t, J = 7.4 Hz, 3H, CH3). 13C-NMR (75 MHz, Chloroform-d)
δ 172.4, 136.3, 136.1,
135.2, 125.9, 123.2, 122.3, 38.9, 32.0, 29.5, 29.43, 29.37, 29.26, 25.5, 22.8, 14.2. HR-MS (ESI positive mode)
calculated for C16H25N2O3+: 293.1860. Found: 293.1846.
N-(2-Nitrophenyl)-4-phenylbutanamide (
5). Purification: pentane/AcOEt =15/1; yellow liquid (29%);
1H-NMR (300 MHz, Chloroform-d)
δ
10.38 (d, J = 5.5 Hz, 1H, NH), 8.83 (d, J = 8.3 Hz, 1H, Ph-H),
8.46–8.15 (m, 1H, Ph-H), 7.85–7.49 (m, 1H, Ph-H), 7.44–7.02 (m, 6H, Ph-H), 2.77 (t, J = 7.3 Hz,
2H, PhCH2), 2.55 (t, J = 7.3 Hz, 2H, COCH2), 2.16 (q, J = 7.4 Hz, 2H, CH2). 13C-NMR (75 MHz,

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Chloroform-d)
δ 171.0, 140.2, 135.4, 135.1, 134.0, 127.6, 125.3, 124.9, 122.3, 121.3, 36.9, 34.2, 25.8. HR-MS
(ESI positive mode) calculated for C16H17N2O3+: 285.1234. Found: 285.1231.
1
N-(2-Acetylphenyl)butyramide ( ). Purification: pentane/AcOEt =15/1; white solid (36%); H-NMR
6
(300 MHz, Chloroform-d) 11.71 (s, 1H, NH), 8.95–8.61 (m, 1H, Ph-H), 7.97–7.79 (m, 1H, Ph-H),
δ
7.55 (dddd, J = 8.5, 7.3, 1.6, 0.5 Hz, 1H, Ph-H), 7.10 (ddd, J = 8.0, 7.3, 1.2 Hz, 1H, Ph-H),
2.67 (s, 3H, COCH3), 2.41 (t, J = 7.5 Hz, 2H, COCH2), 1.92–1.66 (m, 2H, CH2), 1.01 (t, J = 7.4 Hz,
3H, CH3). 13C-NMR (75 MHz, Chloroform-d)
δ 202.9, 172.7, 141.2, 135.3, 131.7, 122.3, 121.7, 120.8, 40.8,
28.7, 19.1, 13.9. HR-MS (ESI positive mode) calculated for C12H15NNaO2+: 228.0995. Found: 228.0985.
N-(2-Acetylphenyl)hexanamide (7) see reference [23].
N-(2-Acetylphenyl)octanamide (
(300 MHz, Chloroform-d) 11.70 (s, 1H, NH), 8.77 (dd, J = 8.5, 1.2 Hz, 1H, Ph-H), 7.89 (dd, J = 8.0,
8
). Purification: pentane/AcOEt =15/1; colorless oil (85%); 1H-NMR
δ
1.6 Hz, 1H, Ph-H), 7.54 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H, Ph-H), 7.10 (ddd, J = 8.0, 7.3, 1.2 Hz, 1H, Ph-H),
2.66 (s, 3H, COCH3), 2.43 (t, J = 7.5 Hz, 2H, COCH2), 1.83–1.62 (m, 2H, CH2), 1.44–1.16 (m, 8H, 4CH2),
0.96–0.72 (t, J = 7.4 Hz, 3H, CH3). 13C-NMR (75 MHz, Chloroform-d)
δ 202.9, 172.9, 141.3, 135.3, 131.7,
122.3, 121.8, 120.9, 38.9, 31.8, 29.3, 29.1, 28.7, 25.7, 22.7, 14.2. HR-MS (ESI positive mode) calculated for
C16H24NO2+: 262.1802. Found: 262.1793.
N-(2-Acetylphenyl)decanamide (9) see reference [24].
N-(2-Acetylphenyl)-4-phenylbutanamide (10). Purification: pentane/AcOEt =13/1; colorless oil (44%);
1H-NMR (300 MHz, Chloroform-d)
δ 11.75 (s, 1H, NH), 8.80 (dd, J = 8.5, 1.2 Hz, 1H, Ph-H), 7.92 (dd, J = 8.0,
1.6 Hz, 1H, Ph-H), 7.70–7.50 (m, 1H, Ph-H), 7.41–7.06 (m, 6H, Ph-H), 2.75 (t, J = 7.4 Hz, 2H, PhCH2),
2.69 (s, 3H, COCH3), 2.50 (t, J = 7.5 Hz, 2H, COCH2), 2.20–2.05 (m, 2H, CH2). 13C-NMR (75 MHz,
Chloroform-d) δ 202.9, 172.4, 141.6, 141.2, 135.3, 131.7, 128.6, 128.5, 126.1, 122.4, 121.8, 120.9, 38.1, 35.3, 28.7,
27.1. HR-MS (ESI positive mode) calculated for C18H20NO2+: 282.1489. Found: 282.1483.
N-Phenylbutyramide (11) see reference [25].
N-Phenylhexanamide (12) see reference [26].
N-Phenyloctanamide (13) see reference [27].
N-Phenyldecanamide (14) see reference [28].
N,4-Diphenylbutanamide (15) see reference [29].
4. Conclusions
In our search for a novel scaffold that is active on LuxR-regulated QS and based on a benzenic
backbone, we designed an original combination of substituents, notably the nitro and acylated
amine groups. Docking experiments demonstrated that this scaffold replaces exactly the lactone
moiety of AHL within the binding site of LuxR. However, variations were observed in the hydrogen
network between the ligands and some key amino acids of the binding site, with some significant
results revealed by the biological evaluation. In particular, the absence of the hydrogen bond
with Tyr62 appears to be essential for antagonistic activity in this new family of aniline and
nitroaniline QS inhibitors. The design and the docking experiments were validated by biological assays
showing significant inhibitory activity, notably for N-(2-nitrophenyl)butyramide (1). Thus, 2-nitro
aniline, a very simple and versatile scaffold, has proved to be a valuable basis for designing novel
QS-active candidates.
1
Supplementary Materials: Supplementary materials are available online. H-NMR spectra.
Acknowledgments: Financial support from MESR and CNRS is gratefully acknowledged. Sizhe Li would like to
thank the Chinese Scholarship Council for his grant. The authors thank Valerie James for improving the English
of the manuscript.

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Author Contributions: Y.Q. and L.S. conceived and designed the experiments; S.L. performed the experiments;
Y.Q. and L.S. analyzed the data; J.W. contributed reagents/materials/analysis tools; Y.Q. and L.S. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–15 are available from the authors.
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