Effect of New Analogs of Hexyloxy Phenyl Imidazoline on Quorum Sensing in Chromobacterium violaceum and in Silico Analysis of Ligand-Receptor Interactions

Article abstract of DOI:10.1155/2020/8735190

The increasing common occurrence of antibiotic-resistant bacteria has become an urgent public health issue. There are currently some infections without any effective treatment, which require new therapeutic strategies. An attractive alternative is the design of compounds capable of disrupting bacterial communication known as quorum sensing (QS). In Gram-negative bacteria, such communication is regulated by acyl-homoserine lactones (AHLs). Triggering of QS after bacteria have reached a high cell density allows them to proliferate before expressing virulence factors. Our group previously reported that hexyloxy phenylimidazoline (9) demonstrated 71% inhibitory activity of QS at 100 μM (IC₅₀ = 90.9 μM) in Chromobacterium violaceum, a Gram-negative bacterium. The aim of the present study was to take 9 as a lead compound to design and synthesize three 2-imidazolines (13-15) and three 2-oxazolines (16-18), to be evaluated as quorum-sensing inhibitors on C. violaceum CV026. We were looking for compounds with a higher affinity towards the Cvi receptor of this bacterium and the ability to inhibit QS. The binding mode of the test compounds on the Cvi receptor was explored with docking studies and molecular dynamics. It was found that 8-pentyloxyphenyl-2-imidazoline (13) reduced the production of violacein (IC₅₀ = 56.38 μM) without affecting bacterial growth, suggesting inhibition of quorum sensing. Indeed, compound 13 is apparently one of the best QS inhibitors known to date. Molecular docking revealed the affinity of compound 13 for the orthosteric site of N-hexanoyl homoserine lactone (C₆-AHL) on the CviR protein. Ten amino acid residues in the active binding site of C₆-AHL in the Cvi receptor interacted with 13, and 7 of these are the same as those interacting with AHL. Contrarily, 8-octyloxyphenyl-2-imidazoline (14), 8-decyloxyphenyl-2-imidazoline (15), and 9-decyloxyphenyl-2-oxazoline (18) bound only to an allosteric site and thus did not compete with C₆-AHL for the orthosteric site.

Full text of DOI:10.1155/2020/8735190

Hindawi
Journal of Chemistry
Volume 2020, Article ID 8735190, 18 pages
https://doi.org/10.1155/2020/8735190
Research Article
EffectofNewAnalogsofHexyloxyPhenylImidazolineon Quorum
Sensing in Chromobacterium violaceum and In Silico Analysis of
Ligand-Receptor Interactions
1
2
3
´
´
Jose Luis Herrera-Arizmendi, Everardo Curiel-Quesada, Jose Correa-Basurto ,
Martiniano Bello,3 and Alicia Reyes-Arellano
1
1Departamento de Qu´ımica Orga´nica, Escuela Nacional de Ciencias Biolo´gicas, Instituto Polite´cnico Nacional,
´
´
Carpio y Plan de Ayala S/N, Colonia Santo Tomas, 11340 Ciudad de Mexico, Mexico
2
´
´
´
Departamento de Bioquımica, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional,
´
´
Carpio y Plan de Ayala S/N, Colonia Santo Tomas, 11340 Ciudad de Mexico, Mexico
3
´
´
´
Escuela Superior de Medicina, Laboratorio de Diseño y Desarrollo de Nuevos Farmacos e Innovacion Biotecnologica,
Instituto Polite´cnico Nacional, 11340 Ciudad de Me´xico, Mexico
Correspondence should be addressed to Alicia Reyes-Arellano; areyesarellano@yahoo.com.mx
Received 16 October 2019; Accepted 30 December 2019; Published 25 February 2020
Academic Editor: Oliver Sutcliffe
Copyright © 2020 Jose´ Luis Herrera-Arizmendi et al. .is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
.e increasing common occurrence of antibiotic-resistant bacteria has become an urgent public health issue. .ere are currently
some infections without any effective treatment, which require new therapeutic strategies. An attractive alternative is the design of
compounds capable of disrupting bacterial communication known as quorum sensing (QS). In Gram-negative bacteria, such
communication is regulated by acyl-homoserine lactones (AHLs). Triggering of QS after bacteria have reached a high cell density
allows them to proliferate before expressing virulence factors. Our group previously reported that hexyloxy phenylimidazoline (9)
demonstrated 71% inhibitory activity of QS at 100 μM (IC50 = 90.9 μM) in Chromobacterium violaceum, a Gram-negative
bacterium. .e aim of the present study was to take 9 as a lead compound to design and synthesize three 2-imidazolines (13–15)
and three 2-oxazolines (16–18), to be evaluated as quorum-sensing inhibitors on C. violaceum CV026. We were looking for
compounds with a higher affinity towards the Cvi receptor of this bacterium and the ability to inhibit QS. .e binding mode of the
test compounds on the Cvi receptor was explored with docking studies and molecular dynamics. It was found that 8-penty-
loxyphenyl-2-imidazoline (13) reduced the production of violacein (IC50 = 56.38 μM) without affecting bacterial growth, sug-
gesting inhibition of quorum sensing. Indeed, compound 13 is apparently one of the best QS inhibitors known to date. Molecular
docking revealed the affinity of compound 13 for the orthosteric site of N-hexanoyl homoserine lactone (C6-AHL) on the CviR
protein. Ten amino acid residues in the active binding site of C6-AHL in the Cvi receptor interacted with 13, and 7 of these are the
same as those interacting with AHL. Contrarily, 8-octyloxyphenyl-2-imidazoline (14), 8-decyloxyphenyl-2-imidazoline (15), and
9-decyloxyphenyl-2-oxazoline (18) bound only to an allosteric site and thus did not compete with C6-AHL for the orthosteric site.
mechanism is based on the detection of small signaling
molecules called autoinducers or semiochemicals, which are
synthesized and released when bacteria reach a certain
population density [4].
Interrupting QS is an attractive strategy in the fight
against bacteria, especially against pharmacoresistant bac-
teria because the QS break does not directly affect the
1. Introduction
.e quorum sensing (QS) is a mechanism of bacterial
communication involved in regulating the expression of
genes linked to the production of virulence factors, among
other functions. Such virulence factors include lytic en-
zymes, proteases, siderophores, and adhesins [1–3]. .is

2
Journal of Chemistry
survival of these microorganisms [5]. Given the increasingly
common antibiotic resistance displayed by bacteria, par-
ticularly in the case of hospital infections, there is an intense
search for novel antibacterial agents. Hence, the develop-
ment of quorum-sensing inhibitors (QSIs), also known as
antiquorum-sensing (antiQS) molecules, could possibly be
an important element in the development of new antimi-
crobial agents.
Compounds that function as QSIs can be obtained from
plants (2 and 3) [6], bacteria [7], marine algae (4 and 5) [8],
fungi (6) [9], and synthetic procedures (7, 8, and 9) [10–12].
In Gram-negative bacteria, the QS system is regulated by acyl-
homoserine lactones (AHLs). Bacterial enzymes of some
Gram-positive bacteria like the Bacillus species interfere with
QS via an AHL-lactonase [13], Figure 1. Various analogs and
bioisosteres of AHLs have been synthesized as inhibitors of
Gram-negative bacteria [12], and some of them designed to
interfere with QS in Chromobacterium violaceum [14].
Chromobacterium violaceum is a Gram-negative bacte-
rium whose autoinducer is hexanoyl homoserine lactone
(C6-AHL, 1). C. violaceum was herein used as a biosensor
[15].
Our group previously observed [12] the potential of
imidazoline 9 as a lead compound, evidenced by its 71%
inhibitory activity of QS in C. violaceum at 100 μM
(IC50 = 90.9 μM). In the present study, we sought analogs of
compound 9 with improved inhibitory activity. In addition
to carrying out inhibition assays of the test compounds on C.
violaceum, docking and molecular dynamics studies were
performed with the same compounds to gain insights into
the interactions responsible for the desired activity.
2.2. Biological Activity
2.2.1. Evaluation of Azolines 13–18 as Quorum-Sensing In-
hibitors in Chromobacterium violaceum CV026. After syn-
thesizing the azolines 13–18, all were examined as possible
QSIs in C. violaceum CV026. Since this Gram-negative
bacterial strain does not synthesize its own C6-AHL, the
production of violacein depends on the concentration of
exogenous C6-AHL added to the medium.
Compounds 13–18 were evaluated as QSIs at 0 (blank),
0.1, 1, 10, 100, and 1000 μM (Figure 2). Two readings were
taken on a spectrophotometer to determine optical density,
one at 720 nm for the culture and the other at 577 nm for the
extraction of violacein. Bacterial density is usually measured
at 600 or 660 nm. Since violacein absorbs at these wave-
lengths, we had to use 720 nm. .ese readings were used to
calculate the relative violacein production, dividing the
absorbance value at 577 nm by that found at 720 nm. For
calculating the percentage of relative violacein production
after the application of each test compound, the average of
the values of the blanks was taken as 100%. .e resulting
percentage of violacein production was plotted as a function
of the concentration of the compound. Our group previously
reported [12] that a concentration of 500 nM of C6-AHL
makes it easier to detect the production of violacein and its
inhibition by a compound.
In the range of 0.1 to 1 μm, compounds 13–18 did not
cause a decrease in the percentage of violacein production.
However, when the bacterium was exposed to a concen-
tration ≥10 μm of compounds 14, 15, 16, and 18, there was
a significantly higher percentage of violacein production
compared to the control (Figure 2). In contrast, com-
pounds 13 and 17 at the same concentration did not
stimulate such an increase in production. In fact, violacein
production significantly declined with 13 between 100 μM
and 1000 μM due to a toxic effect on bacteria at these
concentrations.
Although three oxazolines elevated the production of
violacein at 100 μM, only two of them, oxazolines 17 and 18,
did so at 1000 μM. .e latter two compounds did not have
any effect on the growth of C. violaceum CV026. On the
contrary, imidazoline 15 at a concentration of 100 and
1000 μM did not trigger violacein production, but instead
had an inhibitory effect on the growth of bacteria, which was
verified by the viable count. .e same effect was observed
with 1000 μM of oxazoline 16. Since the 10 and 100 μM
concentrations of imidazoline 13 prompted an abrupt de-
crease in violacein production, an experiment was con-
ducted with concentrations of 0–100 μM (Figure 3).
Imidazoline 13 showed a dose-dependent behavior
(Figure 3). A viable count was made between 30 and 100 μM.
Colony growth was observed between 30 and 90 μM. At
100 μM, colony growth was not observed; therefore, the
reduction in violacein production can be attributed to QSI
activity between 30 and 90 μM, Figure 3. According to the
experimental data, the IC50 was calculated to be 56.38 μM.
.is value can be compared to those reported by Bucio-Cano
et al. for 9, 19–23 [12, 14], 24, and the other synthesized
inhibitors [19] (Figure 4).
2. Results and Discussion
2.1. Chemistry. Six azolines were synthesized, including
three 8-alkyloxyphenyl-2-imidazolines and three 9-alky-
loxyphenyl-2-oxazolines, with moderate and good yields,
respectively (Table 1).
Compound 13 has been synthesized and mediated by
pentyloxybenzene, 4-aminobutanoic acid, phosporic acid,
and P2O5, with yield 48% [16]. It was here synthesized with a
better yield as shown in Table 1. Compound 14 has already
been synthesized by 4-octyloxybenzonitrile and ethyl-
enediamine, with yield 50% [17]. Here, it was obtained with a
yield of 79%. Compound 15 was reported by Gossens et al.
[18] as a precursor of an ionic liquid, but melting point, yield
and, spectroscopic data are missing. .ey obtained com-
pound 15 mediated by ethylenediamine and NBS. Attempts
to obtain the 16, 17, and 18 compounds by conventional
heat gave only traces of the compounds after a long time.
Contrariwise, microwave energy gave good yields in short
times. Compounds 16–18 are new. Compounds 13–18 have
never been employed as a QSI.
All compounds were characterized by spectroscopic
methods (IR, NMR, and HRMS), identifying 8-pentylox-
yphenyl-2-imidazoline (13), 8-octyloxyphenyl-2-imidazo-
line (14), 8-decyloxyphenyl-2-imidazoline (15), 9-
pentyloxyphenyl-2-oxazoline (16), 9-octyloxyphenyl-2-
oxazoline (17), and 9-decyloxyphenyl-2-oxazoline (18).

Journal of Chemistry
3
HO
HO
OH
O
OH
O
O
O
HO
OH
OH
O
O
O
O
HO
OH
N
H
O
O
O
O
OH
HO
HO
OH
‧xH2O
O
HO
OH
1
2
3
R1
O
O
8
Br
R2
F
O
NO2
OH
N
O
N
6
O
O
O
O
7
4 = R1, R2 = Br
N
5 = R1 = H R2 = Br
N
H
O
9
Figure 1: C6-AHL (1) and some known quorum-sensing inhibitors (2–9).
Table 1: Synthesis of azolines.
O
N
X
X
H
H N
2
I , t-BuOH, K CO
(a) 70°C, 7h
(b) 78°C, MW, 1h 40 min
2
2
3
RO
RO
10–12
13–18
R
X
Reaction conditions
Compound number
Isolated yield (%)
n-C5H11
n-C8H17
n-C10H21
n-C5H11
n-C8H17
n-C10H21
NH
NH
NH
O
(a)
(a)
(a)
(b)
(b)
(b)
13
14
15
16
17
18
80
79
69
89
80
68
O
O
180
∗
∗
∗
∗
160
140
120
100
80
∗
60
40
20
0
0
0.1
1
10
100
1000
µM
9-Decyloxyphenyl-2-oxazoline, 18
8-Pentyloxyphenyl-2-imidazoline, 13
9-Octyloxyphenyl-2-oxazoline, 17
8-Decyloxypheyl-2-imidazoline, 15
9-Pentyloxyphenyl-2-oxazoline, 16
8-Octyloxyphenyl-2-imidazoline, 14
Figure 2: Production of violacein upon exposure to azolines 13–18. Asterisks denote statistically significant differences.

4
Journal of Chemistry
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
140
120
100
80
∗
∗
∗
60
∗
∗
∗
∗
40
20
0
0
20
30
40
50
60
70
80
90
µM
OD 720
Percentage specific production of violacein
Figure 3: Bacterial growth (OD720 nm) and percentage of violacein production (mean SE; n � 6) in Chromobacterium violaceum CV026
when exposed to different concentrations (0–100 μM) of 13. Asterisks indicate statistically significant activity (p < 0.05, calculated with
ANOVA).
N
N
N
O
N
N
H
N
H
N
H
N
H
S
H
N
H
N
O
O
O
MeO
O
21
O
IC50 = 670.27μM
20
19
9
IC50 = 340.73μM
IC50 = 66.08μM
IC50 = 90.9μM
(a)
(b)
(c)
(d)
N
N
O
CI
O
O
N
H
N
H
N
H
O
O
N
N
H
H
24
22
23
Active from 1nM
Active 0.1μM
(e)
(f)
(g)
Figure 4: IC50 of some azolines [12, 14] and chlorophenoxy-N-butanoyl homoserine lactone 24 [19] evaluated as QSIs in Chromobacterium
violaceum CV026. ND � not determined.
Imidazoline 13(IC50 = 56.38 μM) is more active than 9
(90.9 μM) [12], 19 (66.08 μM), 20 (340.73 μM), and 21
(670.27 μM) [14] and is comparable to 2-(4′-chlor-
ophenoxy)-N-butanoyl homoserine lactone 24 [19]. .e
latter is proposed as the most active analog of AHL known so
far, giving 100% inhibition of violacein at 10− 4 M.
violaceum CV026 was incubated in the presence of each of
the compounds at concentrations of 0.1, 1, 10, 100, and
1000 μM, at 29°C and 700 rpm for 24 h, without exogenous
C6-AHL. A positive control with C6-AHL and a negative
control without C6-AHL were included. No violacein pro-
duction was observed in any of the tubes containing the
compounds.
Other cases of overproduction of violacein have been
documented [12, 15, 20]. In 2004, Martinelli et al. [20]
investigated several compounds in relation to their capacity
to affect bacteria growth and/or activate QS in C. violaceum
CV026 to improve the production of violacein. .e most
outstanding of their findings for the purposes of the current
contribution is that some compounds are capable of stim-
ulating violacein production, but only in the presence of C6-
AHL. .ey proposed that these compounds may either bind
synergistically with C6-AHL at the protein receptor site or
interact with a second autoinductor system like the one
detected in V. harveyi. On the contrary, Kothari and co-
workers [15] suggested that enhanced violacein production
is due to overexpression of the genes participating in glucose
metabolism, which after some steps would affect tryptophan
Bucio et al. [12] did not give the IC50 value for N-[4-
phenyl-(imidazo-2-yl)]-hexylamide, 23, or N-[4-phenyl-
(imidazo-2-yl)]-nonamide, 22. However, 23 was described as
QSI active from 0.1 μM. Compound 22 showed an inhibitory
effect on violacein production starting at a concentration of
1 nM, but the highest inhibition was 49% at 100 μM. Imida-
zoline 13 was herein found to be active as of 30 μM. Since it
was essential to examine the possibility that 13 works as an
antagonist of C6-AHL in C. violaceum CV026, a docking study
was carried out for this ligand on the CviR protein.
2.2.2. Evaluation of Imidazolines and Oxazolines as Agonists
of C6-AHL in Chromobacterium violaceum CV026. It was
also crucial to determine whether the oxazolines 16–18 work
as agonists of C6-AHL in C. violaceum. .erefore, C.

Journal of Chemistry
5
biosynthesis and finally violacein biosynthesis. According to
the current results, the compounds capable of elevating
violacein production only did so in the presence of C6-AHL,
clearly showing that the test compounds did not perform the
same function as AHL.
Compound 16 forms two hydrogen bonds, one between
the oxygen of the ether and Ser155 and the other between the
N of the oxazoline ring and the hydrogen of Trp84. .ere are
also hydrophobic interactions with amino acids Ile57, Tyr80,
Leu85, Tyr88, Asp97, Ile99, Trp111, Phe115, Met135, and
Ile153, Figure 10.
Oxazoline 17 exhibits only hydrophobic interactions
with amino acids Ile57, Val59, Met72, Val75, Tyr80, Trp84,
Leu85, Tyr88, Ile99, Met100, Trp111, Met135, Ile153, and
Ser155, Figure 11.
2.3. Docking Simulations. Ligand-protein exploration was
made by means of molecular docking as to whether each
compound can decrease or increase violacein production by
binding to the target site on the protein. Docking was carried
out with the AutoDock 4.2 Program [21]. .e prior vali-
dation of docking studies involved the natural ligand C6-
AHL and the CviR protein, yielding a free energy value (ΔG)
of − 7.11 kcal/mol. .e C6-AHL protein made four hydrogen
bond interactions with amino acid residues of the CviR
active site region: (1) the carbonyl of the lactone ring with
Tyr80, (2) the alpha oxygen of the lactone ring with Trp84,
(3) the hydrogen of the amide with Asp97, and (4) the
oxygen of the amide with Ser155. .e respective distances of
Regarding the interactions between the ligands and the
allosteric site, imidazoline 14 displays hydrophobic inter-
actions with the amino acids Ile28, Ala31, Gly32, His177,
Gln180, Ala181, Val183, Arg184, Leu188, and Pro190. A
hydrogen bond exists between the hydrogen of the imida-
˚
zoline ring and Pro189, at a distance of 3.08 A, Figure 12.
Ligand 15 makes a hydrogen bond between the N-H of
˚
imidazoline and Pro189, at a distance of 2.87 A. In addition,
there are hydrophobic interactions with the amino acid
residues Ile28, Ala31, Gly32, His177, Gln180, Ala181,
Val183, Arg184, Leu188, and Pro190, the same interactions
that were found for imidazoline 14, Figure 13.
˚
these four bonds were 2.56, 2.95, 2.94, and 3.28 A, Figure 5.
.e present data from the docking study of C6-AHL on
CviR coincide with the results obtained by Bucio-Cano et al.
[14], who found a ΔG of − 7.26 kcal/mol and the following
amino acid residues in the binding region: Tyr80, Trp84,
Tyr88, Asp97, Ile99, Trp111, Phe115, and Ser155, Figure 6.
Once the docking method was validated by a simulation
using a natural ligand, theoretical calculations were made for
the other ligands, finding the ΔG values for the interactions
and identifying the amino acid residues of CviR involved in
protein-ligand binding with each compound, Table 2.
.e map of residues coordinating the binding of the test
ligands, Table 2, suggests that there are two binding sites for
the CviR protein. One is the orthosteric site of C6-AHL, the
binding location of 8-pentyloxyphenyl-2-imidazoline, 9-
pentyloxyphenyl-2-oxazoline, and 9-octyloxyphenyl-2-oxa-
zoline. .e amino acid residues most frequently involved in
interactions with these compounds are Tyr80, Trp84, Tyr88,
Asp97, Ile99, Trp111, Phe115, Phe126, Met135, Ile153, and
Ser155. .e other binding site for the CviR protein is al-
losteric, the binding location of 16, 17, and 20, most fre-
quently involving the following residues: Ile28, Ala31, Gly32,
His177, His179, Gln180, Ala181, Val183, Arg184, Leu188,
and Pro190. Based on the aforementioned data, a 3D image
was constructed to illustrate the overlap of the ligands in
their respective binding sites, Figure 7.
Finally, compound 18 does not establish hydrogen
bonds, but has many hydrophobic interactions with Ala31,
Gly32, His177, His179, Gln180, Ala181, Val183, Arg184,
Leu188 Pro189, Ile28, and Pro190, Figures 14 and 15.
Molecular coupling assays indicate that imidazoline 13
can attach to the orthostatic site of C
6-AHL, opening the
possibility of an antagonistic effect. .is compound proved
to decrease violacein production experimentally, which is in
agreement with the findings reported by Bucio for hexyloxy
phenylimidazoline [13].
Since ligands 14, 15, and 18 only interact with the amino
acid residues of the allosteric site, they do not compete with
C6-AHL for the orthostatic site. Such an allosteric binding
mode may be due to the number of carbons in the aliphatic
chain of each compound.
One hypothesis is that these compounds function as
positive allosteric modulators. By binding to the allosteric
site, they might trigger a modification in the orthostatic site
that allows for better binding by C6-AHL. Consequently, the
protein-C6-AHL complex would form and bind to the
promoter site (vioBox), thus activating and causing an
enhanced transcription of the genes encoding for the en-
zymes involved in the synthesis of violacein.
Regarding oxazoline 16 and oxazoline 17, the docking
studies show that they bind to the orthosteric site of C6-
AHL. Although these compounds elicited a high production
of violacein experimentally, they were unable to produce
violacein in the absence of C6-AHL. Hence, they are not
agonists.
.e noncovalent interactions of each ligand at the
orthosteric site of the CviR protein are presently described.
Two hydrogen bonds are formed by the interaction of 8-
pentyloxyphenyl-2-imidazoline,13, with the CviR protein,
one between the oxygen of the ether and the Ser155 residue
˚
at a distance of 2.76 A, and the other between the hydrogen
˚
of the imidazoline ring and Asp97 at a distance of 3.16 A.
.ere are also hydrophobic interactions (Van der Waals),
which occur with the amino acid residues Val59, Met72,
Tyr80, Trp84, Leu85, Tyr88, Ile99, Met100, Trp111, Phe115,
Ph126, Met135, and I153, Figure 8. Additionally, π-π in-
teractions are observed between the phenyl ring and Trp111,
Figure 9.
2.4. Molecular Dynamics, Binding Free Energy Calculations,
and Per-Residue Decomposition. During docking simula-
tions, all atoms of the receptor remained fixed. It was
necessary to incorporate flexibility and solvation for all
atoms of the protein-ligand complex during the docking
calculations of the predicted complexes to achieve a more

6
Journal of Chemistry
S155
I99
Y80
W111
D97
Y88
F115
W84
(a)
(b)
Figure 5: Schematic 2D diagram showing the interactions between N-hexanoyl homoserine lactone and CviR. .e hydrophobic residues of
the protein receptor that make contact with the ligand are represented by semicircles and radiant lines.
S155
I99
Y80
W111
D97
Y88
F115
W84
(a)
(b)
Figure 6: .e interaction of N-hexanoyl homoserine lactone with CviR portrayed in 3D.
reliable result. .erefore, molecular dynamics simulations
.e relative binding free energy (ΔGmmgbsa), established
with the MM/GBSA approach for examining CviR-ligand
interactions show that all these complexes are thermody-
namically favorable for binding. .e main energetic contri-
were conducted to examine the preservation of the CviR-
ligand complexes for compounds 13, 14, and 16.
To assess the stability of the complexes, first an RMSD
analysis was made to determine the appropriate time period
for a structural and energetic evaluation once reaching the
equilibration stages. .e CviR-13, CviR-14, and CviR-16
systems reached equilibrated RMSD fluctuations during the
bution to ΔGmmgbsa was guided by the nonpolar contributions
(ΔEnon-polar), while the polar contributions (ΔEpolar) were
found to be unfavorable for the protein-ligand association
(Table 3). .e comparison of the different protein-ligand
systems of the test compounds revealed that 13 and 16, which
both target the orthosteric site, bind with similar affinity.
Interestingly, they exhibit a less favorable ΔGmmgbsa value
than 14, even though the latter targets the allosteric site.
˚
first 10 ns, with average values ranging from 5.65 to 8.7 A.
Accordingly, the first 10 ns were excluded from the 50-ns-
long MD simulations for the subsequent clustering analysis
and binding free energy calculations.

Journal of Chemistry
7
Table 2: .e ΔG values for the ligand-protein interactions and the amino acid residues of the protein involved in binding.
ΔG
Ligand
Amino acid residues of the interaction site
(kcal/mol)
O
O
O
▲
▲
− 7.11
Tyr80, Trp84 , Tyr88, Asp97 , Ile99, Trp111, Phe115, Ser155
N
H
C6-AHL, 1
N
▲
■
▲
− 7.56
Tyr80, Trp84, Tyr88, Asp97 , Ile99, Trp111 , Phe126, Met135, Ile153, Ser155
N
H
O
13
N
▲
N
H
− 5.95
− 6.02
− 7.62
Ile28, Ala31, Gly32, His177, GlnQ180, Val183, Arg184, Pro189
O
14
N
▲
N
H
Ile28, Ala31, Gly32, His177, Gln180, Val183, Arg184, Pro189
O
15
N
▲
Ile57, Tyr80, Trp84 , Leu85, Tyr88, Asp97, Ile99, Trp111, Phe115, Met135,Ile153,
▲
O
Ser155
O
16
N
Ile57, Val59, Met72, Val75, Tyr80, Trp84, Leu85, Tyr88, Ile99, Met100, Trp111,
Met135, Ile153, Ser155
− 7.28
− 6.14
O
O
17
N
O
Ala31, Gly32,His177, His179, Gln180, Ala181, Val183, Arg184, Leu188, Pro190
O
18
▲
■
Hydrogen bonds ( ); π-π interactions ( ).
.ese
equations
show
the
nonpolar
polar
that the CviR-13, Figures 16(a) and 16(b) and CviR-16,
(ΔEpolar = ΔEele + ΔGele,sol
)
and
(ΔEnon-
Figures 16(c) and 16(d) , complexes are stabilized by many
of the same residues. Nonetheless, the chemical differences
between them leads distinct residues to provide greater
contributions to the ΔGmmgbsa value: Ile57, Ile99, Met100,
and Trp111 for CviR-13, Figures 16(a) and 16(b); Ile57,
Met72, Tyr88, and Met100 for CviR-16, Figures 16(c) and
16(d). Ile57 and Met100 are present in the stabilization of
both 13 and 16, suggesting a crucial role for these residues
polar = ΔEvwd + ΔGnpol,sol) contributions of ligand-protein
interactions. Energy is expressed in kcal/mol ( standard
error of the mean) and averaged over 400 snapshots at time
intervals of 100 ps taken during 40 ns (after ignoring the first
10 ns of MD simulations).
To dissect the contribution of each residue to binding,
analysis was made of the per-residue decomposition of each
of the residues contributing to ΔGmmgbsa. .e key residues
involved in binding and the per-residue free energy (ΔEper-
residue) were examined for the CviR-13, Figures 16(a) and
16(b); CviR-16, Figures 16(C) and 16(d); and CviR-14,
Figures 16(e) and 16(f) complexes, Figure 16. It turns out
in molecular recognition at the orthosteric site. For 14,
Figures 16(e) and 16(f), on the other hand, the nonpolar
protein environment implicated in stabilization is lesser for
the allosteric than the orthosteric site. Pro6, Gln180,
Arg184, and Pro190 contribute most to the ΔGmmgbsa value.

8
Journal of Chemistry
Figure 7: 3D illustration of the interaction of the ligands at two binding sites of the CviR protein, consisting of the orthosteric site and an
allosteric site of C6-AHL. (1.A) Approach of the overlap of C6-AHL, 13, 16, and 17 with the amino acid residues of the active site of C6-AHL.
(1.B) .e same approach with a 45° rotation. (2.A) Approach of the overlap of 14, 15, and 18 with the amino acid residues of the allosteric
site. (2.B) .e same approach with a 45° rotation.
I153
S155
I99
Y88
Y80
W111
D97
M135
W84
F126
(a)
(b)
Figure 8: .e noncovalent interaction of 13 with CviR. .e ligand-protein interaction is illustrated (a) in 3D and (b) in a 2D schematic
˚
diagram. Hydrogen bonds are depicted with a dotted green line, indicating the bond distance in A (
). .e hydrophobic residues of
3.28
Phe115
the protein in contact with the ligand are portrayed by red semicircles and radiant lines:
.
.ey may be crucial residues for stabilization at the allo-
steric site.
this case with metallic sodium, and refluxed under nitrogen
atmosphere for 1 h. Ethylenediamine was stored at 5°C under
nitrogen atmosphere and protected from light.
3. Materials and Methods
3.2. Characterization of the Synthesized Compounds. .e
3.1. Drying and Purification of Solvents. Ethyl acetate,
methylene chloride, and hexane were purified by fractional
distillation with calcium oxide (CaO) as the drying agent. .e
solvents were refluxed for 5 h before carrying out distillation.
Ethylenediamine was also purified by fractional distillation, in
organic compounds were characterized by NMR spectroscopy,
1
mass spectrometry (MS), and infrared (IR) spectroscopy. H
and 13C NMR spectra were recorded on a Varian Mercury
spectrometer at 300 and 75 MHz and on a Varian 500 Mercury

Journal of Chemistry
9
I153
S155
Y88
I99
Y80
W111
D87
W84
M135
F126
(a)
(b)
Figure 9: 3D portrayal of the π-π interactions between the phenyl ring of 13 and W111.
I153
I57
S155
I99
W111
Y80
D97
Y88
L85
F115
M135
W84
(a)
(b)
Figure 10: .e interactions between oxazoline 16 and CviR (a) in a 2D schematic diagram and (b) in 3D.
spectrometer at 500 and 125 MHz, respectively. Infrared (IR)
spectra were obtained on a Perkin Elmer FT-IR spectrum 2000
spectrometer from the ENCB-IPN spectroscopy instrumenta-
tion center. HRMS was performed with a JEOL-JSM-GC mate
II and LRMS ESI(+), and spectra were recorded using a
BRUKER MicrOTOF QII. Melting points were determined on
an electrothermal apparatus and are uncorrected.
Subsequently, 10 mL of distilled acetone were injected into
the flask and the mixture was heated to reflux with constant
stirring for 90 min. .en, the corresponding alkyl halide
(5.05 ×10− 3 moles, 1.5 eq.) was added and the reaction
mixture was maintained at reflux with constant stirring
while being monitored by TLC. After the reaction ended,
the mixture was cooled to room temperature (rt), filtered,
and extracted with methylene chloride (CH2Cl2) and the
solvent was evaporated under reduced pressure in a rotary
evaporator. .e product was purified by silica gel column
chromatography by using a polarity gradient of hexane-
ethyl acetate. .e fractions containing pure product were
evaporated under reduced pressure and finally dried under
3.3. Synthesis of Alkylated Aldehydes [13]. In a 25 mL two-
neck flask, adapted with a refrigerant and magnetic stirrer and
kept under nitrogen atmosphere (N2), 4-hydroxybenzaldehyde
(4.09 ×10− 3 mmol) and potassium carbonate (K2CO3)
(8.19 ×10− 3 mmol, 2 eq.) were added with a funnel for solids.

10
Journal of Chemistry
M72 I153
V59
V75
S155
M100
I57
I99
Y80
Y88
W111
L85
M135
W84
(a)
(b)
Figure 11: A 2D schematic diagram of the interaction between 17 and CviR.
P189
G32
R184
I28
A31
Q180
H177
V183
(a)
(b)
Figure 12: A 2D schematic diagram of the interaction between 14 and CviR.
P189
G32
I28
Q180
R184
A31
H177
V183
(a)
Figure 13: A 2D schematic diagram depicting the interactions between 15 and CviR.
(b)

Journal of Chemistry
11
P190
L188
G32
A31
R184
Q180
V183
H177
A181
H179
(a)
(b)
Figure 14: A 2D schematic diagram of the interaction of 18 with CviR.
P190
L188
G32
A31
R184
V183
H177
A181
H179
(a)
(b)
Figure 15: .e interaction of 18 with CviR shown in 3D.
a high vacuum. .e resulting substances were characterized
by NMR, IR, and HRMS.
] � 3074 (C-H), 2931, 2870 (C-H), 1688 (C�O), 1262,
1027 (�C-O-C), and 830.
1H NMR (CDCl3)
3.3.1. para-Pentyloxy Benzaldehyde (10)
δ � 9.82 (s, 1H, CHO), 7.35 (AA′ BB′, 4H, Ar), 3.98 (t,
2H, OCH2), 1.76 (q, 2H, H-7), 1.38 (m, 4H, H-8y
H-9), and 0.90 (t, 3H, CH3).
O
3
2
4
H
1
10
13C NMR (CDCl3)
8
6
5
O
9
7
δ � 190.47 (CHO), 164.06 (C-5), 131.73 (C-3), 129.56
(C-2), 114.54 (C-4), 68.19 (C-6), 28.55 (C-7), 27.91 (C-
8), 22.21 (C-9), and 13.78 (C-10).
Oil at rt
IR (KBr)

12
Journal of Chemistry
Table 3: Binding free energy components of protein-ligand complexes (in units of kcal/mol).
System
ΔEvdw
ΔEele
ΔGele,sol
ΔGnpol,sol
ΔEnon-polar
ΔEpolar
ΔGmmgbsa
13
15
16
− 37.86 (0.22)
− 45.77 (0.27)
− 33.95 (0.30)
− 6.07 (0.21)
− 11.32 (0.30)
− 14.21 (0.26)
21.38 (0.18)
24.02 (0.26)
24.69 (0.20)
− 5.35 (0.02)
− 5.96 (0.02)
− 5.08 (0.03)
− 43.21
− 51.73
− 39.03
15.31
12.7
− 27.90 (0.23)
− 39.03 (0.35)
− 28.55 (0.27)
10.48
Ile153
Met135
Phe115
Trp111
Met100
Ile99
Tyr88
Leu85
Trp84
Tyr80
Val75
Met72
Val59
Ile57
–2.0
–1.5
–1.0
–0.5
0.0
ΔEper-residue (kcal)
(a)
(b)
(d)
(f)
Ala191
Pro190
Arg184
Val183
Ala181
Gln180
Hie177
Gly32
Ala31
Hie29
Ile28
Arg10
Ala9
Asn8
Ile7
Pro6
Lys5
–2.0 –1.8 –1.6 –1.4 –1.2 –1.0 –0.8 –0.6 –0.4 –0.2 0.0
ΔEper-residue (kcal)
(c)
Ser155
Ile153
ꢀr140
Trp111
Met100
Ile99
Ala94
Ser89
Tyr88
Leu85
Tyr80
Val75
Met72
Val59
Ile57
–1.6 –1.4 –1.2 –1.0 –0.8 –0.6 –0.4 –0.2 0.0
ΔEper-residue (kcal)
(e)
Figure 16: Per-residue free energy (ΔEper-residue) and map of interactions for the most populated conformation of the CviR-13 (a and b),
CviR-16 (c and d), and CviR-14 (e and f) complexes. .e map of interactions was constructed with Maestro Schro¨dinger version 10.5 [22].

Journal of Chemistry
13
3.3.2. para-Octyloxy Benzaldehyde (11)
solution, and subsequently dried with anhydrous sodium
sulfate. .e organic phases were combined and the solvent
was evaporated. .e remaining residue was recrystallized
from ethyl acetate.
O
3
2
4
5
H
1
12
10
8
6
O
13
Oil at rt
11
9
7
3.4.1. 8-Pentyloxyphenyl-2-imidazoline (13)
IR (KBr)
3
4
N
] � 3074 (C-H), 2925, 2855 (C-H), 1693 (C�O), 1255,
1019 (�C-O-C), and 830.
6
4′
2
5
7
8
N
H
1
11
9
13
6′
1H NMR (CDCl3)
O
10
12
7′
δ � 9.71 (s, 1H, CHO), 7.24 (AA′ BB′, 4H, Ar), 3.86 (t,
2H, OCH2), 1.65 (q, 2H, H-7), 1.31 (m, 2H, H-8), 1.18
(m, 8H, H-9 al H-12), and 0.76 (t, 3H, CH3).
m.p.105–107°C from AcOEt. 55°C from pet−ro1leum ether;
b.p.125°/0.01Torr [16]. IR (KBr), υ � 3203 cm (NH) and
1
1618 (C�N). H NMR (DMSO-D6) δ � 7.34 (AA′ BB′, 4H,
13C NMR (CDCl3)
Ph), 3.98 (t, 2H, H-9), 3.56 (s, 4H, H-4, H-4′), 1.71 (qi, 2H,
H-10), 1.36 (m, 4H, H-11, H-12), and 0.89 (t, 3H, CH3). 13
C
δ � 189.91 (CHO), 163.77 (C-5), 131.39 (C-3), 129.40
(C-2), 114.27 (C-4), 67.92 (C-6), 31.39 (C-7), 28.92 (C-
8), 28.82 (C-9), 28.66 (C-10), 25.56 (C-11), 22.24 (C-
12), and 13.65 (C-13).
NMR (DMSO-D6) δ � 163.03 (C-2), 160.00 (C-8), 128.45 (C-
6), 122.78 (C-5), 113.70 (C-7), 67.34 (C-9), 49.17 (C-4, C-4′),
28.14 (C-10), 27.51 (C-11), 21.17 (C-12), and 13.71 (C-13).
HRMS, m/z � calculated for C14H21N2O (M+): 233.1648;
found 233.1625.
3.3.3. para-Decyloxy Benzaldehyde (12)
O
3.4.2. 8-Octyloxyphenyl-2-imidazoline (14)
3
2
4
5
H
1
3
4
14
12
10
8
6
N
O
6
4′
2
5
15
Oil at rt
13
11
9
7
7
8
N
1
H
15
9
13
11
6′
O
IR (KBr)
16
14
10
12
7′
] � 3074 (C-H), 2923, 2854 (C-H), 1696 (C�O), 1258,
1016 (�C-O-C), and 831.
m.p. 115–117°C from AcOEt. m.p.109°C from benzene :
hexane [17]. IR (KBr) υ � 3209 cm− 1 (NH) and 1618 (C�N).
1H NMR (CDCl3)
1H NMR (DMSO-D6)δ � 6.95 (AA′ BB′, 4H, Ph), 3.99 (t, 2H,
H-9), 3.60 (s, 4H, H-4, H-4′), 1.72 (qi, 2H, H-10), 1.39 (m,
2H, H-11), 1.27 (m, 8H, H-12, H-13, H-14, H-15), and 0.86
(t, 3H, CH3). 13C NMR (DMSO-D6) δ � 163.28 (C-2), 160.43
(C-8), 128.77 (C-6), 122.23 (C-5),113.96 (C-7), 67.57 (C-9),
49.10 (C-4,C-4′), 31.12 (C-10), 28.72 (C-11), 28.65 (C-12),
28.60 (C-13), 25.48 (C-14), 22.07 (C-15), and 13.93 (C-16).
HRMS, m/z � calculated for C17H26N2O (M+): 275.2118;
found 275.2099.
δ � 9.81 (s, 1H, CHO), 7.33 (AA′ BB′, 4H, Ar), 3.96 (t,
2H, OCH2), 1.75 (q, 2H, H-7), 1.41 (m, 2H, H-8), 1.27
(m, 12H, H-9 al H-14), and 0.84 (t, 3H, CH3).
13C NMR (CDCl3)
δ � 190.37 (CHO), 164.06 (C-5), 131.70 (C-3), 129.58
(C-2), 114.53 (C-4), 68.20 (C-6), 31.71 (C-7), 29.38 (C-
8), 29.17 (C-9), 29.14 (C-10), 28.88 (C-11), 25.78 (C-
12), 22.49 (C-13), 13.95 (C-14), and 13.87 (C-15).
3.4.3. 8-Decyloxyphenyl-2-imidazoline (15)
3.4. Synthesis of Imidazolines. In a two-neck 25 mL balloon
flask adapted with a refrigerant and magnetic stirrer and
kept under nitrogen atmosphere and at rt, 1 eq. of the
corresponding aldehyde was added followed by the injection
of 8 mL of tert-butanol and 1.1 eq. of ethylenediamine.
Ninety min later, 3 eq. of K2CO3 and 1.25 eq. of molecular I2
were added and the temperature was raised to 70°C. .e
reaction of the mixture lasted 5 h and was monitored by
TLC. .e system was allowed to reach rt before adding water
to the reaction flask and carrying out extractions with ethyl
acetate. .e organic phase was washed with a saturated
solution of sodium sulfite and then with a 20% NaCl
3
4
N
6
2
4′
1
N
H
7
8
17
9
15
13 11
12
6′
O
18 16
14
7′
10
m.p. 151–153°C IR (KBr), υ � 3132 cm− 1 (NH) and
1614 (C�N). 1H NMR (DMSO-D6) d � 7.56 (AA′ BB′, 4H,
Ph), 4.09 (t, 2H, H-9), 3.98 (s, 4H, H-4, H-4′), 1.73 (qi,
2H, H-10), 1.41 (m, 2H, H-11), 1.29 (m, 12H, H-12, H-13,
H-14, H-15, H-16, H-17), and 0.84 (t, 3H, CH3). 13C

14
Journal of Chemistry
3.5.2. 9-Octyloxy phenyl-2-oxazoline (17)
NMR (DMSO-D6) δ � 164.43 (C-2), 163.67 (C-8), 130.79
(C-6), 115.32 (C-7), 113.93 (C-5), 68.38 (C-9), 44.35 (C-
4,C-4′), 31.42 (C-10), 29.12 (C-11), 29.07.18 (C-12),
28.84 (C-13), 28.82 (C-14), 28.56 (C-15), 25.53 (C-16),
22.23 (C-17), and 14.08 (C-18). HRMS, m/z � calculated
for C19H30N2O (M+): 303.2431; found 303.2467.
3
4
N
5′
7
2
6
8
9
O
1
10
16
12
14
7′
O
17
15
13
11
8′
m.p. 41–43°C. IR (KBr), υ � 1644 cm− 1 (C�N). 1H NMR
3.5. Synthesis of Oxazolines. To a 200 mL MW reactor flask,
adapted with a refrigerant and a magnetic stirrer, 1 eq. of
the corresponding aldehyde (depending on the oxazoline
to be synthesized) followed by the injection of 5 mL of
t-BuOH and 1.1 eq. of ethanolamine were added. .e flask
was placed in a chemical microwave oven (model MIC-1,
Prendo), and the conditions for the reaction were pro-
grammed (50°C, 3 min 14 s, 1290 rpm, and 60% power at
762 watts). After this cycle, 3 eq. of K2CO3 and then of 1.5
eq. of I2 were added and the reaction conditions were
changed (78°C, 20 min 14 s, 1290 rpm, and 60% power at
762 Watts). .e reaction, monitored by TLC, ended upon
completion of 5 cycles. Once the system reached rt, the
organic phase was separated and water was added to the
reaction flask. Potassium carbonate was solubilized
(aqueous phase), and extractions were carried out by
using ethyl acetate. .e organic phase was washed with a
saturated solution of sodium sulfite (Na2SO3) and then
with a 20% NaCl solution, and subsequently dried with
anhydrous sodium sulfate. .e ethyl acetate was evapo-
rated under reduced pressure. .e mixture of the product
and remaining raw material were separated by silica gel
column chromatography by using a polarity gradient of
hexane-ethyl acetate. .e solvent was evaporated from the
pure fractions and the purified product dried under a high
vacuum.
(CDCl3) δ � 6.88 (AA′ BB′, 4H, Ph), 4.39 (t, 2H, H-5), 4.03 (t,
2H, H-10),3.98 (t, 2H, H-4), 1.78 (m, 2H, H-11), 1.45 (m, 2H,
H-12), 1.32 (m, 8H, H-13, H-14, H-15, H-16), and 0.88 (t,
3H, CH3). 13C NMR (CDCl3) δ � 165.16 (C-2), 161.69 (C-9),
127.01 (C-7),117.05 (C-6), 111.37 (C-8), 65.33 (C-10), 64.68
(C-5), 52.08 (C-4), 29.04 (C-11), 26.55 (C-12), 26.43 (C-13),
26.38 (C-14), 23.23 (C-15), 19.88 (C-16), and 11.31 (C-17).
HRMS, m/z � calculated for C17H25NO2 (M+): 276.1958;
found 276.2000.
3.5.3. 9-Decyloxy phenyl-2-oxazoline (18)
3
4
N
7
5′
2
6
8
9
O
1
18
10
16
14
12
7′
O
19
17
11
15
8′
13
m.p. 48–50°C. IR (KBr), υ � 1650 cm− 1 (C�N). 1H NMR
(DMSO-D6) δ � 6.97(AA′ BB′, 4H, Ph), 4.35 (t, 2H, H-5),
4.00 (t, 2H, H-10), 3.91 (t, 4H, H-4, H-4′), 1.71 (m, 2H,
H-11), 1.40 (m, 2H, H-12), 1.28 (m, 12H, H-13, H-14, H-15,
H-16, H-17, H18), and 0.85 (t, 3H, CH3). 13C NMR (DMSO-
D6) δ � 162.65 (C-2), 161.00 (C-9), 129.41 (C-7),119.75 (C-
6),114.31 (C-8), 67.63 (C-10), 67.13 (C-5), 54.31 (C-4), 31.28
(C-11), 28.98 (C-12), 28.93 (C-13), 28.73 (C-14), 28.68 (C-
15), 28.55 (C-16), 25.44 (C-17), 22.08 (C-18), and 13.92 (C-
19). HRMS, m/z � calculated for C19H29N02 (M+):304.246;
found 304.2316.
3.5.1. 9-Pentyloxy phenyl-2-oxazoline (16)
3
4
N
7
5′
2
6
8
9
3.6. Preparation of Culture Media, Test Compounds, and
Inoculum. .e Luria Bertani (LB) broth was prepared in one
liter of distilled water by adding 10 g peptone, 5 g yeast
extract, and 5 g NaCl and then sterilized in an autoclave at
15 psi and 121°C for 15 min. For the LB solid medium, 15 g of
bacteriological agar was added to a liter of distilled water. C.
violaceum CV026 was always grown in the presence of 30 μg/
mL of kanamycin.
.e amount of each compound required for a con-
centration of 100 mM was weighed. With the resulting so-
lution, serial dilutions 1 :10 were made to obtain the
concentrations of 1000 μM, 100 μM, 10 μM, 1 μM, and
0.1 μM.
O
1
12
10
14
7′
O
11
8′
13
m.p. 36–38°C. IR (KBr), υ � 1649 cm− 1 (C�N). 1H NMR
(DMSO-D6) δ � 6.96 (AA′ BB′,4H, Ph), 4.35 (t, 2H, H-5),
3.99 (t, 2H H-10), 3.90 (t, 2H, H-4), 1.71 (m, H-11), 1.35 (m,
H-12, H-13), and 0.88 (t, 3H, CH3). 13C NMR (DMSO-D6)
δ � 162.70 (C-2), 161.06 (C-9), 129.46 (C-7), 119.81 (C-6),
114.35 (C-8), 67.68 (C-10), 67.18 (C-5), 54.37 (C-4), 28.31
(C-11), 27.67 (C-12), 21.91 (C-13), and 13.97 (C-14). HRMS,
m/z � calculated for C14H29NO2 (M+): 234.1489; found
234.1529.

Journal of Chemistry
15
From the cryovials containing C. violaceum CV026, a
roast was taken and crosswise streaked in a box containing
LB agar and 30 μg/mL kanamycin, followed by incubation at
29°C for 24 h. A roast was also taken from an isolated colony
and inoculated in 5 mL LB medium with 30 μg/mL kana-
mycin for CV026, followed by incubation at 29°C and
200 rpm for 15 h. Finally, the boxes were stored in a
refrigerator.
3.11. Determination of MIC in Chromobacterium violaceum
CV026. .e procedure described in section 3.7 was fol-
lowed, except that the final concentration of the compounds
was from 10 to 1000 μM. .e MIC was assigned to the lowest
concentration of the compound yielding no bacterial
development.
3.12. Modeling and Optimization of Ligands. Docking
studies were carried out on the ligands tested experi-
mentally in C. violaceum CB026 and on the natural ligand
of the CviR protein. .e ligands were built with the ACD/
ChemSketch program, creating a geometric preoptimiza-
tion in 3D (respecting the stereochemistry and spatial
configuration). .e structures were saved in .mol format,
and these files served as an input to create the Z matrix for
each molecule on the GaussView 5.0 graphical visualizer.
.e files were saved in .gjp input format for their use in
Gaussian 09. .e structures obtained (and their respective
matrices Z) were submitted to a geometric and energetic
optimization at the AM1 semiempirical level with the
chemical-quantum package of Gaussian 09. .e output files
in .out format were transformed into 3D format.pdb with
the GaussView 5.0 program. .e latter files were utilized for
simulations by molecular coupling.
3.7. Evaluation of the Compounds as Quorum-Sensing In-
hibitors in Chromobacterium violaceum CV026. C. viola-
ceum CV026 was cultured in 60 mL of the LB medium with
30 μg/mL kanamycin until reaching an optical density of 0.1
to 600 nm. Subsequently, in 2 mL tubes, 980 μL of this
culture, 80 μM C6-AHL (800 nM final concentration), and
10 μL of the dilutions of the test compounds were added
until reaching the final concentration of 1000 μM, 100 μM,
10 μM, 1 μM, and 0.1 μM. .en, the tubes were incubated at
29°C and 700 rpm for 24 h. Upon completion of the incu-
bation time, cell density was determined by absorbance at
720 nm by using the LB medium as the blank. Finally, the
absorbance of violacein was measured.
3.8. Evaluation of Violacein. 500 μL of the bacterial culture
weas placed in a 2 mL tube and 500 μL of acetone were
added. .e tubes were vortexed and centrifuged at
15000 rpm for 4 min to prepare for the determination of the
absorbance of violacein in the supernatants at 577 nm. .e
specific production of violacein was calculated by dividing
the value of the reading at 577 nm by that at 720 nm. Each
experiment was performed 6 times/compound, and the
results were graphed. Statistical significance was analyzed by
ANOVA.
3.13. Molecular Studies. With docking studies, an initial
examination was made of each ligand-binding site on the
CviR protein as well as the ligand-receptor interactions. C.
violaceum 12472 (located in the Protein Data Bank under the
PDB code: 3QP6) was chosen as the target protein for this
analysis on the AutoDock 4.2 program, which maintains the
macromolecule rigid while allowing flexibility in the ligand
[23]. .e AutoDock 4.2 program has shown good correla-
tion between the free energy values of the binding simu-
lations and the experimental data [24]. Blind docking was
3
˚
carried out with a grid box of 126 ×126 ×126 A
and a
3.9. Viable Count. From the 24 h cultures of the test com-
pounds, 10 μL were taken to make decimal dilutions and
5 μL of each dilution was dripped onto plates containing the
LB agar. After incubation at 29°C for 24 h, the colonies on
each spot were counted. .ese assays were performed in
triplicate, and viable counts were confirmed by standard
bacterial plating.
3
˚
0.375 A space between grid points and by using the Hybrid
Genetic Algorithm of Lamarckian with an initial population
of 100 randomized individuals and a maximum number of
energy evaluations of 1 × 107. .e results of the simulations
were examined by means of the PyMol visualizer, observing
the amino acid residues of the protein involved in the in-
teractions with the ligands. 2D protein-ligand interaction
diagrams were generated with the LIGPLOT program, re-
vealing additional amino acid residues that interact with the
ligands.
3.10. Assay of the Compounds as Agonists. A flask containing
60 mL of the LB medium with kanamycin was adjusted to an
optical density of 0.1 to 600 nm with C. violaceum CV026.
Subsequently, 990 μL of this solution were placed in 2 mL
tubes and 10 μL of the dilutions of the compounds were
added until reaching a final concentration of 1000 μM,
100 μM, 10 μM, 1 μM, and 0.1 μM. Moreover, a positive
control (990 μL culture + 10 μL of an 80 μM solution of C6-
AHL) and a negative control (990 μL culture + 10 μL of the
LB medium) were included. .e tubes were incubated at
29°C while subjected to shaking at 700 rpm for 24 h. .e
presence or absence of pigment was then observed to make
an evaluation of violacein.
3.14. Molecular Dynamics Simulations and Binding Free
Energy Calculations. MD simulations of the CviR-ligand
systems were conducted with the PMEMD module AM-
BER12 package [25], the ff99SB force field [26], and the
˚
generalized Amber force field (GAFF) [27]. A 12 A rect-
angular-shaped box of TIP3P water molecules [28] was
constructed to solvate the CviR-ligand complexes, and
counterions were placed at different locations to neutralize
the charges of the complexes at pH 7. Systems were mini-
mized and equilibrated by carrying out a protocol that began

16
Journal of Chemistry
with 1000 steps of steepest descent minimization and
continued with 1000 steps of conjugate gradient minimi-
zation. Equilibrations began by heating the systems from 0 to
310 K during 200 picoseconds (ps) of MD simulations, with
position restraints set at a constant volume. Successive MD
simulations were conducted under periodic boundary
conditions (PBCs) using an isothermal isobaric (NpT) en-
semble of 200 ps to adjust the solvent density, followed by
800 ps of constant pressure equilibration at 310 K (with the
SHAKE algorithm) [29] on hydrogen atoms using a time
step of 2 femtoseconds (fs) and Langevin dynamics for
temperature control. Subsequent to equilibrations, 50-ns-
long MD simulations were conducted in the absence of
position restraints, under PBCs and with an NpT ensemble
antiquorum sensing effect. .is was the most active com-
pound in the homologous series. Of the imidazolines cur-
rently under study, those with anti-QS activity had an
aliphatic chain similar in size to that of C6-AHL. An in-
hibitory effect on the growth of C. violaceum CV026 was
elicited by 100 and 1000 μM of imidazoline 17 (15), 100 μM
of imidazoline 16 (14), and 1000 μM of oxazoline 18 (16).
Quorum-sensing agonist activity was not found for any of
the test compounds. .e experimental inhibitory effect on
violacein production promoted by 15 (13) was in agreement
with the docking study. Ten amino acid residues in the active
site of the receptor protein were involved in the interactions
with 8-pentyloxyphenyl-2-imidazoline. Seven of these 10
also interacted with AHL. Additionally, one of them is
implicated in π-π interactions.
˚
at 310 K. A 10 A cutoff was applied for the van der Waals
interactions. .e electrostatic term was described via the
particle mesh Ewald method [30], and bond lengths were
constrained at their equilibrium values with the SHAKE
algorithm [29]. Temperature and pressure were maintained
by utilizing the weak-coupling algorithm [31] with coupling
constants τT and τP of 1.0 and 0.2 ps, respectively (310 K,
1 atm). .e time dependence of the MD simulation runs was
analyzed by employing AmberTools from Amber12. On the
other hand, structural representations were created with
Data Availability
.e data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
.e authors declare that there are no conflicts of interest
regarding the publication of this paper.
¨
PyMOL v0.99 [32] and Maestro Schrodinger version 10.5
[22].
Authors’ Contributions
A.R.-A. was involved in conceptualization; A.R.-A., E.C.-Q.,
and J.C.-B. were involved in methodology; J.C.-B., M.B., and
J.L.H.-A were responsible for software; A.R.-A., E.C.-Q., and
J.C.-B. validated the study; A.R.-A., E.C.-Q., J.C.-B., J.L.H-
A., and M.B. performed formal analysis; J.L.H-A. and M.B.
performed investigation; A.R.-A., E.C.-Q., and J.C.-B. were
responsible for resources; A.R.-A. curated the data ; A.R.-A.,
E.C.-Q., and J.C.-B. wrote and prepared the original draft;
A.R.-A. wrote, reviewed, and edited the manuscript; A.R.-A.
was involved in visualization; A.R.-A. supervised the study;
A.R.-A. was involved in project administration; A.R.-A was
involved in funding acquisition.
3.15. Calculation of Relative Binding Free Energies and Per-
Residue Contributions. Relative binding free energies were
calculated according to the MMGBSA [33–36] provided in
Amber12 [25]. For this purpose, 400 snapshots at time
intervals of 100 ps were extracted during 40 ns, ignoring the
first 10 ns of the 50-ns-long MD simulations, using a salt
concentration of 0.1 M and the Born implicit solvent model
[36] after removing water molecules and counterions. .e
analyses were performed with the MMPBSA Perl script [35].
.e binding free energy of each complex can be calculated as
follows:
ΔGmmgbsa � Gcomplex − Greceptor − Gligand
,
(1)
Acknowledgments
ΔGbind � ΔEMM + ΔGsolvation − TΔS.
AR is grateful to IPN for the financial support through grant
SIP20181666 and to CONACyTfor grants 240808 and 255420.
JC is grateful to CONACyT for the financial support through
grant 254600. JLHA is appreciative of scholarships provided by
CONACYTand the IPN. .e authors are beholden to CNMN,
IPN, for HRMS and to CE, ENCB, for IR spectra.
4. Conclusions
Six azolines were synthesized, including three imidazolines
obtained in moderate yields by conventional heating and
three oxazolines afforded in good yields by MW. .e per-
formance of the azolines was dependent on the size of the
chains of the alkoxy benzaldehydes. A significant increase in
violacein production was induced (in the presence of hex-
anoyl homoserine lactone) by 8-decyloxyphenyl-2-imida-
zoline 17 (15) (10 μM), 9-decyloxyphenyl-2-oxazoline 20
(18) (10, 100, and 1000 μM), 9-pentyloxyphenyl-2-oxazoline
18 (16) (10 and 100 μM), and 9-octyloxyphenyl-2-oxazoline
19 (17) (100 and 1000 μM). An inhibitory effect on violacein
production was shown by 8-pentyloxyphenyl-2-imidazoline
15 (13), with an IC50 of 56.38 μM, strongly suggesting an
Supplementary Materials
Selected 1H NMR-, 13C NMR-, HSQC- HMBC- IR-, and
HRMS-spectra. (Supplementary Materials)
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