Targeting quorum sensing by designing azoline derivatives to inhibit the N-hexanoyl homoserine lactone-receptor CviR: Synthesis as well as biological and theoretical evaluations

Article abstract of DOI:10.1016/j.bmc.2015.10.046

To counteract bacterial resistance, we investigated the interruption of quorum sensing mediated by non-classical bioisosteres of the N-hexanoyl homoserine lactone with an azoline core. For this purpose, a set of selected 2-substituted azolines was synthesized, establishing the basis for a new protocol to synthesize 2-amino imidazolines. The synthesized compounds were evaluated as inhibitors of violacein production in Chromobacterium violaceum. Theoretical studies on bioisostere-protein interactions were performed using CviR. The results show that some azolines decreased violacein production, suggesting an antiquorum sensing profile against Gram-negative bacteria. Docking and molecular dynamic simulations together with binding free energy calculations revealed the exact binding and inhibitory profiles. These theoretical results show relationship with the in vitro activity of the azoline series.

Full text of DOI:10.1016/j.bmc.2015.10.046

Bioorganic & Medicinal Chemistry 23 (2015) 7565–7577
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Targeting quorum sensing by designing azoline derivatives to inhibit
the N-hexanoyl homoserine lactone-receptor CviR: Synthesis as well
as biological and theoretical evaluations
a
a,
⇑
b
b
Alejandro Bucio-Cano , Alicia Reyes-Arellano , José Correa-Basurto , Martiniano Bello ,
a
a
a
a
Jenifer Torres-Jaramillo , Héctor Salgado-Zamora , Everardo Curiel-Quesada , Javier Peralta-Cruz ,
a
Alcives Avila-Sorrosa
a
Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Química Orgánica, Campus Casco de Santo Tomás, Carpio y Plan de Ayala S/N, Colonia Santo
Tomás, 11340 México, D.F., Mexico
b
Instituto Politécnico Nacional, Escuela Superior de Medicina, Laboratorio de Modelado Molecular y Diseño de Fármacos, Campus Casco de Santo Tomás, 11340 México, D.F., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
To counteract bacterial resistance, we investigated the interruption of quorum sensing mediated by non-
classical bioisosteres of the N-hexanoyl homoserine lactone with an azoline core. For this purpose, a set of
selected 2-substituted azolines was synthesized, establishing the basis for a new protocol to synthesize
Received 1 September 2015
Revised 23 October 2015
Accepted 31 October 2015
Available online 2 November 2015
2-amino imidazolines. The synthesized compounds were evaluated as inhibitors of violacein production
in Chromobacterium violaceum. Theoretical studies on bioisostere–protein interactions were performed
using CviR. The results show that some azolines decreased violacein production, suggesting an antiquo-
rum sensing profile against Gram-negative bacteria. Docking and molecular dynamic simulations together
with binding free energy calculations revealed the exact binding and inhibitory profiles. These theoretical
results show relationship with the in vitro activity of the azoline series.
Keywords:
Azolines
Quorum sensing
Non classical bioisosteres
Chromobacterium violaceum
Molecular dynamic simulation
Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1
. Introduction
The AHL chemical structure contains a c-lactone ring and an
alkyl amide side chain with an asymmetric center (S). The side
chain depends on the type of bacteria. Today it is known that the
chain has up to 18C, and that the C-b to C@O can contain another
Quorum sensing (QS) is a mechanism of regulation of gene-
expression mediated by detection of chemical messengers called
1
5
autoinducers. This chemical biological phenomenon allows bacte-
C@O or a hydroxyl group.
ria (as well as fungi and yeast) to communicate among themselves
and determine the appropriate moment to carry out group and
synchronic actions, thus functioning as an excellent cooperative
Many bacterial processes are controlled through QS, including
two that can protect bacteria from antibiotics: biofilm production
5
,6
and virulence factors. Through these mechanisms, Gram-negative
bacteria are capable of becoming highly resistant to antimicrobial
2
,3
society. This ability is activated when the bacteria reach an opti-
2,3
6–8
mized population density, in order to effectively deploy their
energy resources.
treatment. The fact that these bacteria produce many nosocomial
infections has created the need to produce ever more novel
In Gram-negative bacteria the chemical signal (semiochemical4)
is based on the synthesis and release of autoinducers, which are
small diffusible signaling molecules, such as N-acyl-homoserine
lactones (AHLs), that bind to their cognate transcriptional regula-
tory proteins. This binding triggers a wide range of intracellular
antibiotics.
8
This situation requires totally new strategies to face the prob-
lem of drug resistance, and an excellent alternative is the disrup-
tion or blocking of bacterial communication. Since QS is not
necessary for growth, its disruption is unlikely to affect primary
metabolic pathways essential for survival, consequently there is
no associated selection pressure for the bacteria to develop resis-
1
,2
reaction cascades to carry out the required biological processes.
8
–11
tance against such treatments.
One means of interrupting bacterial communication is through
the synthesis of novel bioisosteres from AHL. Most bioisosteres
⇑
Corresponding author.
E-mail address: areyesarellano@yahoo.com.mx (A. Reyes-Arellano).
12
http://dx.doi.org/10.1016/j.bmc.2015.10.046
968-0896/Ó 2015 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
0

7
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A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
(
LBD) connected to a DNA binding domain (DBD) through a highly
flexible coil. The LBD is the larger domain, composed of
and b-sheets, while the DBD is composed of a few a-helices.
a-helices
2
3
Pursuing our ongoing efforts to identify new QS inhibitors, we
herein designed and synthesized non-classical bioisosteres
of C6-HSL (N-hexanoyl-homoserine lactone) by replacing the
lactone ring and performing chemical modifications in the amide
group and the aliphatic chain. These structural analogs were then
biologically tested as anti-QS molecules. In order to understand
the structure–activity relationship experimentally observed, we
performed docking, MD simulations and binding free energy calcu-
lations between the receptor protein (CviR) of Chromobacterium
violaceum and the synthesized bioisosteres.
Figure 1. Type of AHL bioisosteres.
2. Results and discussion
2
.1. Drug design
ofAHL used, are AHL analogs because of the ease ofpunctualreplace-
1
3
14
ment (Fig. 1, 1 ). This subject has been covered in several
In order to build totally new compounds with QS inhibition, we
1
5,16
reviews.
that have no classical partial replacements.
moiety has been replaced with another cycle, for example,
However, some bioisosteres have been synthesized
considered AHL as the lead compound comprised of three moieties
(Scheme 1).
16,17
Thus the lactone
1
8
19
thiolactone (Fig. 1, 2, 4) and oxazolidinone, or the chain has
sometimes been drastically modified (Fig. 1, 3 ). There are few
20
non-classical bioisosteres with the replacement of both moieties
Lactone
2
1
Chain
Amide
(Fig. 1, 4 ). Very few works have been deal with the moiety amide,
however, Song et al. have synthesized and evaluated some
N-sulfonyl homoserine lactone derivatives as quorum sensing
inhibitors.
2
2
Scheme 1.
On the other hand, molecular dynamics (MD) simulation studies
have yielded important information about the inherent flexibility of
LuxR proteins (including CviR) in water solution. This flexibility
In a previous report we showed that some imidazolines (Fig. 2)
exhibit anti-QS activity against Chromobacterium violaceum and
2
4
Serratia marcescens.
Therefore, we designed the new bioisosteres of AHL following
Scheme 2.
This approach allowed the design of several types of
2
3
enables different proteins to carry out their functions properly.
The structural topology of CviR reveals a homo-dimer composed of
two identical and overlapping chains of about 250 amino acids each,
where each monomeric chain consists of a ligand binding domain
1
3
bioisosteres, summarized as follows:
Type of bioisoster
Functional group
exchange/chain
Amide/bioisosteres
Connector
Lactone bioisosteres
Compounds
I
II
III
IV
V
Aryl group
Aryl group
Chain
Chain
Chain
Amide bioisoster
Amide
Amide
Ether
Ether
Methylene phenoxy
—
—
Phenyl
Phenyl
Imidazoline
Imidazoline
Thiazoline
Oxazoline
11–13
14
15–19
20
Tetrahydropyrimidine
21
Figure 2. Imidazolines evaluated in a previous report.
Scheme 2.

A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
7567
We decided to design these types of bioisosteres because, dur-
ing the evaluation of inhibitory activity by some imidazolines on
Chromobacterium violaceum and Serratia marcescens, we observed
the following: the imidazoline ring was indispensable for the inhi-
bitory activity, the 4-phenoxy group played an important role in
this activity (5, 6), and the imidazoline 10 containing the amide
group was active even at nanomolar concentrations.24 For these
reasons, we combined these two groups in the same molecule,
2
2
.2. Chemistry
.2.1. Acetamide synthesis
0
Two approaches were followed to synthesize N-[4 -chlorophe-
0
0
000
nyl-2-(4 -imidazolin-2 -yl)-phenoxy]-acetamide. In the first one
Path A, Scheme 3), the key intermediate was 8-hydroxy phenyl
-imidazoline (23). In the second strategy (Path B, Scheme 4), the
key intermediate was aldehyde (27).
(
2
00
000
resulting in the series of N-[aryl-2-(4 -imidazolin-2 -yl)-phe-
noxy]-acetamides, 11–13.
2
.2.1.1. Path A.
8-Hydroxy phenyl 2-imidazoline (23) was
obtained in 69% yield, whereas the alkylation of the imidazoline
to give product 11 yielded only 30% (Fig. 3).
1
1
1
1
2
3
R= 4-Cl-C
6
H -
4
R= 4-MeO-C
6
H
4
R= Phenyl
2
.2.1.2. Path B.
In a second strategy, the alkylation of
-haloacetic acid.
p-hydroxybenzaldehyde was attempted using an
a
The best results were obtained using ethyl-2-bromoacetate.
Thereafter, the ester was hydrolyzed and the acid activated with
either DCC or through the mixed anhydride prepared from pivaloyl
chloride (t-BuCOCl). Condensation with the latter gave better
results (60–70% yield) as compared to DCC activation (60–62%
yield).
On the other hand, we wanted to know the effect when the
amide group is directly bonded to the imidazoline ring, leading
us to synthesize benzamide 14.
The synthesis of imidazolines 11–13 by the alkylation of
p-hydroxybenzaldehyde, followed by the synthesis of the amide
and its cyclization (Path B; Fig. 4), gave better yields than synthetic
path A.
2
.2.2. Benzamide synthesis
The thiazolylamide series (15–19) was synthesized to evaluate
the effect of a divalent bioisosteric replacement (i.e., –S– by
nitrogen).
2
5
Butler reported the synthesis of tetrahydropyrimidines from
-imidazolines. In another report, the synthesis of 2-amino benz-
2
imidazole from guanidinium hydrochloride and 1,2-phenylenedi-
2
6
amine was described. Based on these reports, the synthesis of
the benzamide 14 was carried out from 2-aminoimidazoline
hydrochloride 30, which was acylated with the corresponding
carboxylic acid. The latter compound was synthesized from
guanidinium hydrochloride 29 under solvent-free conditions in
quantitative yield (Table 1). To the best of our knowledge, this
methodology for the preparation of 2-aminoimidazoline has not
previously been reported. This compound has been synthesized
1
5 R= n-Pentyl
6 R= n-Octyl
1
1
1
1
7 R= n-Undecyl
8 R= Phenyl
9 R= Benzyl
Two additional bioisosteric replacements were carried out on
the imidazoline ring: nitrogen was exchanged for oxygen and the
ring was expanded to obtain oxazoline 20 and tetrahydropyrim-
idine 21, respectively.
27
by addition–elimination reactions of 2-thiomethyl-2-imidazoline
28,29
and other methodologies.
To obtain the benzamide 14, the carboxylic acid was
activated with thionyl chloride followed by the addition of
2
-aminoimidazoline hydrochloride under various conditions
(Fig. 5). The best condition for achieving good yields was by
using an ether/water mixture, KOH and 0 °C (80%). The synthe-
sis was also performed with the mixed anhydride from pivaloyl
chloride (t-BuCOCl), achieving similar yields. Compound 14 was
Scheme 3. Path A.

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A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
Scheme 4. Path B.
Figure 3. Synthesis of imidazoline 11 from 8-hydroxy phenyl 2-imidazoline.
O
O
1)
Br
O
O
H
H
DBU, CH
3
CN, 120 °C, 60 %
HO
2
3
) LiBr, Et
3
N, CH
3
CN, H O, rt
2
HO
O
) HCl 10 %
22
28 quantitative
O
1
3
) t-BuCOCl, Et N, THF, -20 °C
2
) RNH , LiCl/THF, RT
2
O
N
H
N
H
NH
2
H
N
H
2
H N
N
R
O
R
O
K CO , I , t-BuOH
2
3
2
O
O
70 °C, 4h
R = 4-ClC H
11 93 %
6
4
R = 4-ClC H
27a 60 %
27b 65 %
6
4
R = 4-CH OC H 12 95 %
3
6 4
R = 4-CH OC H
3
6 4
R = C H
13 quantitative
6
5
R = C H
27c
60 %
6
5
Figure 4. Synthesis of imidazolines 11–13 by path B.
previously prepared by Augustin30 as the tautomer, N-benzoyl
imino imidazolidine.
hydrochloride and the corresponding carboxylic acid in acetoni-
trile. Thiazolines were obtained in good yields (Fig. 6). The spec-
troscopic data are given in the Supplementary section.
Compounds 15–19 were obtained in similar yields by adding
DCC and triethylamine to methylene chloride and stirring for
12 h at rt. Thiazoline activity as QS inhibitors has not been pre-
viously explored.
2
.2.3. Synthesis of N-thiazoline amides
Thiazolines 15–19 are available commercially, although their
spectroscopic data were not found in literature. Therefore, we
synthesized them at home starting from 2-aminothiazoline

A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
7569
Table 1
-Amine-imidazoline synthesis from guanidinium hydrochloride
2
Solvent
Catalyst
—
Ethylenediamine (equiv)
t (h)
Isolated yield (%)
3
0
31
1
2
3
4
5
6
7
—
—
EtOH
EtOH
4
4
1
1
1
1
1.2
8
12
12
12
12
16
16
0
0
5
65
80
42
90
0
0
0
—
*
Cl
Cu
—
—
—
—
2
CH CN
3
—
Quantitative
78
0
5
—
ꢁ
3
All reactions were carried out to reflux with one eq. of compound 29. In reaction 3, 15 mL of solvent were utilized for every 4.2 ꢀ 10 mol of 29. In reactions 4 and 5, 1 mL of
ꢁ
3
solvent was utilized for every 4.2 ꢀ 10 mol of 29.
*
Trace.
observe the inhibitory effect. In our previous work,24 it was deter-
mined that 500 nM is a good concentration of C6-HSL for observing
the violacein inhibition process.
Every compound was tested at the following concentrations: 0
(
blank), 0.001, 0.01, 0.1, 1, 10, 100 and 1000
tometric readings were performed for every compound, one at
20 nm corresponding to the optical density (OD) of the culture
lM. Two spectropho-
7
and the other at 577 nm corresponding to the violacein dye after
acetone extraction of the culture. The specific production of viola-
cein was calculated by dividing the OD found at 577 nm by that
obtained at 720 nm (577/720). The production percentage was cal-
culated considering the average specific production of the control
as 100%. Results were used to build Graph 1. It is important to
add that, all compounds were tested on C. violaceum in the absence
of AHL. None of them showed an agonistic activity.
Figure 5. Synthesis of imidazolyl benzamide, 14.
Oxazoline 20 was synthesized according to the procedure24
Graph 1 shows a multiphasic curve for every compound. Marti-
nelli and coworkers have also observed a non-linear effect in the
evaluation of several compounds as inhibitors of violacein produc-
tion, concluding that the same compound may be an activator or
inhibitor. It can be appreciated that 11 did not display inhibitory
activity, while 12 and 13 were active at concentrations ranging
reported for imidazoline 5 and 6 in 75% yield. Tetrahydropyrim-
idine 21 was synthesized in 85% yield by following the procedure
used for imidazolines 5 and 6, but with the use of 1,3-diamine
propane in the cyclization step.
3
1
from 10
for 12 and 66.08
activity was 19, showing approximately 80% inhibition.
However, the IC50 value was found to be 670.27 M only at a high
concentration of 1000 M. Tetrahydropyrimidine 21 presented
activity similar to that observed with 6, since the inhibitory effect
observed in the graph at a concentration of 1000 M is due to the
toxic effect of the compound. Oxazoline 20 showed an inhibitory
effect at 100 M, which is similar to that observed for 5. The IC50
for 20 was 97.4 M. Compound 33 was evaluated in C. violaceum
l
M to 1000
lM. The IC50 was found to be 340.73 lM
2
.3. Evaluation of the synthesized compounds in
lM for 13. The only thiazoline with inhibitory
Chromobacterium violaceum CV026
l
Chromobacterium violaceum CV026 was chosen for evaluating
the synthesized products (Fig. 7) as anti-QS agents since this strain
does not synthesize its own C6-HSL. This fact allows the control of
bacterial violacein production by varying the concentration of C6-
HSL that is added to medium. This control is very important, given
that a high concentration of violacein can make it difficult to
l
l
l
l
with the aim of proving that the presence of the oxyphenyl group
is not enough to give activity. In fact, 33 has no anti-QS effect. Since
compounds 14–18 and 33 showed no activity, they were not
included in the graph.
O
O
N
1
2 2 3 3
) SOCl /K CO /CH CN
2
)
R
OH
N
R
N
H
S
The most active compounds known currently are decanoyl
Cl
H N
22,32
0
S
homoserine lactone, (C10-HSL),
2(4 chlorophenoxy)-N-buta-
3
33
noyl homoserine lactone (ClPhBu-HSL) and N-[4-Phenyl-(imi-
dazo-2-yl)]-nonamide, 10. The last compound is considered by
1
5 R = n-Pentyl
87 %
83 %
24
1
6 R = n-Octyl
us among the most active because it started its activity at 1 nM.
Recently Song et al. found, using a spot test method, that fluo-
rophenyl sulfamoyl lactone and chlorophenyl sulfamoyl lactone
showed activity at very low concentrations (IC50 = 1.66 and
1
7 R = n-Undecyl 80 %
1
8 R = Phenyl
88 %
86 %
1
9 R = n-Benzyl
2
2
Figure 6. Synthesis of N-thiazolin-2-yl amides.
1.64 lM respectively).

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A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
Figure 7. Molecules evaluated in Chromobacterium violaceum CVO26.
Graph 1. Evaluation of the violacein production in C. violaceum CV026 with the azolines: 11 (black), 12 (red), 13 (green), 19 (yellow), 20 (blue) and 21 (pink). Data were
calculated by using a two way ANOVA test with Duncan correction and are displayed as the mean ± SE (n = 6).
Graph 1 allows observe that the most active compounds in this
work are 12 and 13. The activity was measured by spectrophotom-
etry using C6-HSL as control. Compound 13 showed activity in
results (see Experimental Section 4.3). Then, with the aim of
including conformational dynamics and solvation properties in
the complexes, the conformers obtained through docking calcula-
tions with the lowest binding free energy values were used as ini-
tial conformers for 50-ns long MD simulations in explicit solvent
conditions. Overall, the binding modes exhibited by the eleven
compounds through docking calculations demonstrate that they
have affinity toward the LBD from CviR, as observed by the super-
position between C6-HSL and three compounds (9, 11 and 13),
which through docking and MD simulations showed the best bind-
ing free energy values (Fig. 8).
range 10–1000
l with IC50 of 66.08 lM, in contrast C10-HSL,
the most active compound of a reported series of acyl homoserine
lactones, showing a 51% inhibition of violacein at 100 nM, the IC50
32
in the presence of constant (500 nM) C6-HSL are 208 nM (Song
2
2
et al. reported IC50 of 0.32 ± 0.07
gave a total inhibition of violacein at 10 M. Finally compound
0 showed 14% activity at 1 nM and 49% at 100 M. Considering
lM). In addition ClPhBu-HSL
ꢁ
4
1
l
this data, compound 13 can be considered with similar activity
than 10; but not as active as C10-HSL, ClPhBu-HSL, fluorophenyl
sulfamoyl lactone and chlorophenyl sulfamoyl lactone.
After evaluating the interactions between ligand and receptor,
the conformational stability of the complexes was assessed
through a root–mean squared deviation (RMSD) analysis. The
2
.4. Docking and molecular dynamics simulations
RMSD plots are shown for the Ca atoms of each protein during
the production simulation period (Fig. 9, see Supplementary mate-
rial). As can be observed in the plot, some complexes exhibited a
higher stability than others (1–5, 3–20, 4–21, 5–9, 6–10, 34–33,
and 38-C6-HSL vs 2–6, 10–11, 11–13, 12–12, 35–7 and 3qp6-a-
19). This analysis showed that although some complexes reached
equilibrium in the first 10 ns, all the complexes reached
convergence within the first 25 ns of simulation, with average
RMSD values ranging from 5.66 to 12.20 (Table 2, Supplementary
The proposed binding mode of the eleven derived compounds
Table 2, Supplementary section) was obtained through docking
(
calculations between the derived compounds and the tridimen-
sional structure of CviR (a LuxR protein) co-crystallized with the
C6-HSL molecule (PDB entry, 3QP6). To validate our docking
results, the co-crystallized ligand (C6-HSL) was re-docked into
the LBD, obtaining binding poses consistent with the experimental

A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
7571
Figure 8. Structural superposition between 38-C6-HSL and the three compounds (5–9, 10–11 and 11–13) that showed the best binding free energy values through docking
and MD simulations (Table 4).
material). Therefore, all subsequent analyses excluded the initial
5 ns period. To examine the origin of this different stability of
the complexes, a more detailed analysis on a per-residue basis
was conducted (see Supplementary material).
ing free energy (DGbind) with the contribution of entropy was
2
found to be energetically favorable for all complexes except for
the 3qp6-a-19 complex, for which the marginally favorable bind-
ing free energy turns unfavorable when the entropic contribution
is considered. The main factor favoring binding was the existence
2
.4.1. Total and decomposed MM-GBSA binding energies
Binding free energy values for all the protein–ligand complexes
of non-polar interactions (
interactions ( vdw) contributed significantly. Contrarily, the polar
interactions ( polar) exhibited an unfavorable behavior that
DEnon-polar), to which the van der Waals
DE
DE
were calculated according to the MM-GBSA approach imple-
mented in Amber 12 package.34 For this purpose, the last 25 ns
of MD simulations were used (Fig. 9 and Table 2 Supplementary
material), a time period in which the RMSD values converged for
all systems. As can be appreciated in Table 3, the calculated bind-
diminished the binding, suggesting that this molecular recognition
process is hydrophobic in nature (Table 3).
ꢁ
1
The highest
D
G
bind found was ꢁ24.04 kcal mol for 11, fol-
ꢁ1
lowed by ꢁ22.95, ꢁ21.55 and ꢁ19.69 kcal mol for 9, C6-HSL,
Table 3
Binding free energy components of protein–ligand complexes (in units of kcal/mol)
System/compound
D
E
vdw
D
E
ele
D
G
ele,sol
D
G
npol,sol
D
E
polar
D
E
non-polar
D
G
mmgbsa
ꢁTD
S
DG
b
1
2
3
4
5
6
1
1
1
3
3
3
3
–5
ꢁ41.09
0.52)
ꢁ8.83
25.30
(0.67)
ꢁ5.33
16.47
11.94
14.30
7.7
ꢁ46.42
ꢁ47.71
ꢁ43.63
ꢁ31.94
ꢁ46.51
ꢁ49.86
ꢁ52.46
ꢁ49.25
ꢁ50.81
ꢁ36.26
ꢁ44.13
ꢁ20.05
ꢁ39.17
ꢁ29.95
ꢁ17.04
ꢁ12.91
ꢁ14.17
ꢁ5.09
(
(0.64)
(0.02)
(0.40)
(2.21)
–6
ꢁ41.36
0.53)
ꢁ17.3
(0.81)
29.24
(0.66)
ꢁ6.35
ꢁ35.77
ꢁ21.6
(
(0.06)
(0.45)
(2.46)
–20
ꢁ38.3
ꢁ6.13
20.43
(0.40)
ꢁ5.33
ꢁ29.33
ꢁ24.24
(
0.45)
(0.34)
(0.02)
(0.33)
(6.05)
–21
ꢁ27.4
ꢁ202.30
210
(2.7)
ꢁ4.54
ꢁ24.24
ꢁ11.1
ꢁ13.14
ꢁ22.95
ꢁ14.89
ꢁ24.04
ꢁ19.69
ꢁ15.92
ꢁ10.81
ꢁ5.18
(
0.66)
(3.0)
(0.07)
(1.0)
(2.2)
–9
ꢁ41.17
ꢁ28.7
41.1
(0.45)
ꢁ5.34
12.4
ꢁ34.11
ꢁ11.16
(
0.53)
(0.7)
(0.02)
(0.51)
(3.7)
–10
ꢁ44
ꢁ29.4
39.17
(0.64)
ꢁ5.86
9.77
ꢁ40.09
ꢁ25.2
(
0.50)
(0.73)
(0.03)
(0.53)
(4.34)
0–11
1–13
2–12
4–33
5–7
ꢁ46.16
ꢁ28.77
41.28
(0.60)
ꢁ6.30
12.51
13.65
12.12
12.53
10.85
15.92
1.92
ꢁ39.95
ꢁ15.91
(
0.51)
(0.92)
(0.02)
(0.44)
(4.14)
ꢁ43.37
0.47)
ꢁ39.1
52.75
(0.57)
ꢁ5.88
ꢁ35.6
ꢁ15.91
(
(0.74)
(0.02)
(0.46)
(4.14)
ꢁ44.41
0.45)
ꢁ41.95
54.07
(0.47)
ꢁ6.4
ꢁ38.69
ꢁ22.77
(
(0.75)
(0.02)
(0.41)
(3.3)
ꢁ31.56
0.42)
0.29
(0.38)
12.24
(0.37)
ꢁ4.70
ꢁ23.73
ꢁ12.92
(
(0.02)
(0.39)
(2.63)
ꢁ38.62
0.36)
ꢁ17.33
28.18
(0.92)
ꢁ5.51
ꢁ33.28
ꢁ28.1
(
(1.5)
(0.02)
(0.79)
(4.24)
qp6-a-19
8-C6-HSL
ꢁ17.35
0.60)
ꢁ37.18
53.10
(1.8)
ꢁ2.7
ꢁ4.13
ꢁ12.0
7.87
(
(1.1)
(0.22)
(0.40)
(2.5)
ꢁ34.81
0.33)
ꢁ39.0
40.92
(0.57)
ꢁ4.36
ꢁ37.25
ꢁ15.70
ꢁ21.55
(
(0.69)
(0.02)
(0.37)
(3.42)

7
572
A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
and 13, respectively. Hence, two of the twelve complexes
showing the highest
major contribution to the
W84, L85, Y88, I99, M100 and W111 via strong
D
G
bind values (Table 3). In these cases, the
total is from I57, V59, M72, V75,
stacking with
showed a higher
lized complex.
D
Gbind value than that found for the co-crystal-
DG
p–p
Decomposition of the binding energy on a per-residue basis is
very important for understanding the binding mode and assess-
ing the role of each residue in the binding process. Figure 10
displays the key residues for binding and their total free energy
W84, Y88. For the remaining complexes, the key residues for bind-
ing (see Figs. 11A and B, available as Supplemental material) indi-
cate that the major contribution to the
DGtotal is from hydrophobic
interactions, consistent with the observations from the docking
study (data not shown).
contributions (DGtotal) for the complexes (11, 9, 13 and C6-HSL)
Figure 10. MM-GBSA approaches for getting the free energy values and the principal no-covalent interactions depicted during the MD simulations. (A and B) Receptor protein
with 5–9; (C and D) protein with 10–11; (E and F) protein with 11–9, and (G and H) protein with C6-HSL.

A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
total were W84, Y88,
I99, M100, W111 and M135, whereas for 9, 11 and 13 the main
contributors were I57, V59, M72, V75, L85, Y88, I99, M100 and
W111. Overall, these results show that the new non-classical bioi-
sosteres proposed in this contribution are stabilized by a higher
number of residues than the classical bioisostere (C6-HSL),
7573
For C6-HSL, the main contributors to the
D
G
aliphatic chain improves inhibition but causes a less optimized
map of interactions. Compound 12 showed inhibition similar to
that of compound 13, which is also interesting considering that
the former has an almost identical atomic structure as the latter.
The only dissimilarity is the methoxy substituent on the aromatic
ring of 12, supporting our hypothesis that the chloride substituent
could be responsible for the lack of inhibition (Table 4).
explaining the higher
DGbind observed for 9 and 11 than C6-HSL
(Table 3). Furthermore, this analysis is in line with previous studies
In general, these results point out that although compounds 9,
reporting the importance of the phenyl ring in the azoline struc-
ture for anti-QS activity23 in CviR, which is due to the fact that
the phenyl ring of compounds 9, 11, and 13 interact with the aro-
10, 12 and 13 show good DGbind values and inhibitory properties,
the best combination is found with 9 and 13. Hence, these two
compounds can be proposed as a new non-classical bioisosteres,
and the phenyl ring in their azoline structure can be used as phar-
macophore models to design new non-classical bioisosteres. It is
clear that imidazoline and phenyl rings are important but not suf-
ficient for QS inhibitory activity. It appears that the bioisostere
needs flexibility as well. Finally, it is important to note that com-
pound 13 was constructed based on the replacement of all AHL
moieties and the introduction of a connector. Since this replace-
ment can be classified as non-classical, compound 13 is a non-clas-
sical bioisostere in every sense of the word.
matic amino acids from CviR through
W111 and W84 (Fig. 10).
p–p interactions with Y88,
ele vwd npol,sol
The polar (DE + DGele,sol) and non-polar (DE + DG )
contributions. All energies are averaged over 250 snapshots and
are in kcal/mol (±standard error of the mean).
2
.5. Structure–activity relationship
By comparingthe
that of C6-HSL (Table 3), the activity of the compounds can be pre-
dicted. According to this relationship, the compounds with a
from ꢁ24.04 to ꢁ19.69 are strongly active (9, 11 and 13), those with
bind from ꢁ15.92 to ꢁ12.91 (5, 6, 21, 10 and 12) are moderately
active, and those with a
DGbind values obtained for the compounds with
DG
bind
3
. Conclusions
a
DG
In this contribution a series of AHL analogs were designed, syn-
D
G < ꢁ12.9 (20, 33, 7 and 3qp6-a-19) are
thesized, and tested theoretically and experimentally to evaluate
their possible interruption of anti-QS activity in CviR. We chose
AHL analogs by considering previous reports that identified the
best combinations of chemical groups for inhibitory activity on
CviR. Among these AHL analogs, eight compounds combined the
imidazoline ring and the phenoxy group in the same molecule (5,
inactive. Nevertheless, to make a proper activity comparison, it is
necessary to correlate theoretical and experimental data.
This data shows that among the compounds classified as
strongly active (9, 11 and 13), only 9 and 13 showed inhibition
(
at 0.1 lM, in the range of 10–1000 lM, respectively) whereas 11
did not show inhibition (Table 4). Interestingly, compounds 11
and 13 have almost identical atomic structure, the only difference
being the chloride substituent on the aromatic ring of 11 (Fig. 8).
This Cl-atom allowed 11 to reach more optimized map of interac-
tions into the LBD from CviR than 13 (Fig. 10), indicating that the
lack of inhibition by 11 could be caused by external factors rather
than molecular recognition. Such external factors could be protein
metabolism or the incapacity of the drug to pass through biological
barriers.
6
, 9–13, and 21). For the remaining complexes, some modifications
were introduced in the imidazoline ring (20 and 21) or between
the imidazoline ring and the phenoxy group (19).
The combination of experimental and theoretical approaches
allowed for the correlation of inhibitory activity with
for all the compounds. Of the compounds classified as strongly
active based on bind values (9, 11 and 13), only 9 and 13 exhibited
inhibitory activity (at 0.1 M and in the range of 10–1000 M,
respectively). Although 11 had a similar atomic structure, the chlo-
ride substituent on the aromatic ring might be responsible for the
lack of inhibitory activity. This could be caused by external factors,
such as protein metabolism or the incapacity of the drug to pass
through biological barriers. In contrast, this chloride substituent
enables 11 to reach the best optimized map of interactions into
the LBD of CviR.
DGbind values
D
G
l
l
Among the compounds classified as moderately active, 10 and
1
2 stand out. Despite not having the best thermodynamic proper-
ties, compound 10 shows inhibition at as low as 1 nM and 12 in the
range of 10–1000 M (Table 4). Compound 10 has a chemical
l
structure similar to that of compound 9, although in the former
the aliphatic chain is longer (3 carbon atoms). Hence, a longer
Although two compounds classified as moderately active (10
and 12) did not show the best thermodynamic properties, they
exhibited good inhibitory activity. While 10 showed the maximum
inhibitory activity (at 100 nM), 12 had inhibitory activity similar to
13. Fascinatingly, 10 has a chemical structure similar to 9, differing
only in the longer aliphatic chain for the former (3 carbon atoms).
This suggests that a longer aliphatic chain improves the inhibitory
properties, despite the fact that it affects the map of interactions
Table 4
Summary of the experimental activity and theoretical data from the synthesized
compounds
Compound Experimental activity
IC50
M)
Docking
(kcal/mol) dynamics
kcal/mol)
Molecular
(l
(
5
6
Active 100
Bacteriostatic 100
and bactericidal 1 mM
Active 0.1
Active from 1 nM
Inactive
l
M
90.9
—
ꢁ7.56
ꢁ9.59
ꢁ12.91
ꢁ14.17
l
M
and therefore the
DGbind value. On the other hand, 12 has a struc-
ture similar to 13, differing only in the methoxy substituent on the
second aromatic ring. Although with this difference the map of
interactions of 12 was affected (compared to 13), the inhibitory
activity was not.
9
1
1
1
1
1
1
2
2
3
lM
ND
ND
—
ꢁ10.03
ꢁ8.31
ꢁ9.21
ꢁ22.95
ꢁ14.89
ꢁ24.04
ꢁ15.92
ꢁ19.69
—
0
1
2
3
4
9
0
1
3
Active 10–1000
Active 10–1000
Inactive
l
l
M
M
340.73 ꢁ11.70
66.08
—
ꢁ12.34
Overall, four compounds showed good
DGbind values (9, 10, 12
—
and 13), two constituted by the imidazoline plus two aromatic
rings (12 and 13) and two by the imidazoline plus phenyl ring
and aliphatic chain (9 and 10). Of these four compounds, only
two exhibited good inhibitory properties (12 and 13). The one hav-
Active 1000
Active 100
l
M
670.27
97.4
—
—
—
ꢁ8.36
ꢁ9.31
ꢁ9.98
ꢁ6.94
ꢁ7.26
7.87
l
M
ꢁ5.06
ꢁ13.14
ꢁ10.81
ꢁ21.55
Bactericidal 1000 lM
Inactive
—
C6-HSL
ing the best combination of
DGbind values and inhibitory properties
ND = not determined.
was 13. Therefore, this compound can be proposed as a new non-

7
574
A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
classical bioisostere, and the imidazoline and two aromatic rings
shared by 9 and 13 can be used as pharmacophore models to
design new non-classical bioisosteres.
4.1.5. 2-Amine imidazoline (30)
2
To a 4.2 mmol of guanidinium hydrochloride under N atmo-
sphere, 4.2 mmol ethylenediamine were added and the mixture
was heated to reflux for 16 h. Then the mixture was kept in Kugel-
rohr equipment at 80 °C (0.001 Torr) for 8 h. The remaining residue
was a very dense oil, which was analyzed spectroscopically and
4
4
. Experimental section
used for the follow reaction.
.1. Experimental chemistry
1
) d: 7.50 (s, NH) 3.56 (s, 4H). 13C NMR
H NMR (DMSO-d
) d: 160.15 (NAC@N), 42.54 (CH
lated for C (ESI, M+1):86.0713; Found 86.0745.
6
(
DMSO-d
6
2 2
ACH ). HRMS EI: Calcu-
Melting points were determined on an Electrothermal appara-
3 7 3
H N
tus and are uncorrected. Infrared spectra were recorded on a Perkin
Elmer 599-B spectrophotometer. 1H and C NMR were recorded
with a Varian Mercury apparatus at 300 MHz and 75 MHz, respec-
tively, or on a Varian at 500 and 125 MHz, respectively. The chem-
13
4
.1.6. N-Imidazolin-2-yl benzamide (14)
To 4.2 mmol of 2-amine imidazoline hydrochloride was added
1
13
KOH (7.14 mmol). Afterward, 1 mL deionized water was added
and the mixture was stirred for 5 min, then immediately cooled
to 0 °C, at which time 7 mL of ether were added, and stirring was
continued for 30 min. Finally, benzoyl chloride (0.0042 mol) was
added and the reaction mixture was stirred for 12 h, allowing the
temperature to gradually reach room temperature. The solid resi-
due was recrystallized with ethanol/hexane (8:2).
3 4
ical shifts (d) are referenced to internal (CH ) Si, (d H = 0, d C = 0)
and are given in ppm. HRMS were determined using a JEOL-JSM-GC
mate II and HRMS ESI(+) were recorded using a BRUKER MicrOTOF
QII .
00
000
4
.1.1. N-[Aryl-2-(4 -imidazolin-2 -yl)-phenoxy]-acetamides
synthesis. General procedure
t-Butanol (15 mL) was added to the corresponding 2-oxo(4 -formyl
phenyl)-N-arylacetamide (0.0008 mol), then 15 mL of ethylene-
Yield 80%. mp = 183–185 °C, EtOH/Hexane (8:2); [tautomer,
00
27
ꢁ1
1
1
(
1
91–192 °C , EtOH]. IR KBr
m
: 3062 (NAH) cm , 2917 (CAH),
1
758 (C@O), 1673 (NAC@N). H NMR (DMSO-d
m, 5H, Ph), 3.90 (s, 4H, H-4 , H-5 ). C NMR (DMSO-d
69.47 (C@O), 150.70 (C-2 ), 134.20 (C-2), 131.29 (C-5), 128.53
6
) d: 7.53–7.28
diamine under N
for 1 h. Afterward, iodine (0.0008 mol) and K
2
were added and the mixture was stirred at rt
CO (0.0024 mol)
0
0
13
6
) d:
2
3
0
were added and the mixture was heated to 70 °C. The reaction
was monitored with TLC. Upon termination, the reaction was
cooled to rt and the solid was washed with acetone and
solvent evaporated. The residue was purified by recrystallization
0
0
(
C-3), 127.46 (C-4), 40.16 (C-4 , C-5 ). HRMS: Calculated for
10 11 3
C H N O (ESI, M+H): 190.0975; Found 190.0980.
4
.1.7. N-Thiazoline amides synthesis, general procedure
A mixture of K CO (15.0 mmol), 10 mL acetonitrile, and the
(
EtOH/hexane 8:2).
2
3
corresponding carboxylic acid (5.0 mmol) was stirred for 5 min,
then cooled to 0 °C and stirred for 15 min longer. Afterward, thio-
nyl chloride (6.0 mmol) was added and the mixture was stirred for
30 min before removing the ice bath. Then the reaction mixture
was stirred for 30 min again. Finally, 2-amine thiazoline
(5.5 mmol) was added to the reaction mixture and stirred for
12 h, after which time acetonitrile was evaporated and the product
purified by chromatographic column with gradient hexane/AcOEt.
0
00
000
4
.1.2. N-[4 -Chlorophenyl-2-(4 -imidazolin-2 -yl)-phenoxy]-
acetamide (11)
Mp 194–196 °C. IR KBr
C@O) 1608 (C@N). H NMR (DMSO-d
AA BB , 4H, H-2 , H-3 ), 7.48 (AA BB , 4H, H-2 , H-3 ) 4.74 (s, 2H,
H-2), 3.58 (s, 4H, H-4 y H-5 ). 13C NMR (DMSO-d
), 163.79 (C-2 ), 159.96 (C-1 ), 137.59 (C-4 ), 129.29 (C-3 ),
29.02 (C-3 ), 127.84 (C-1 ), 123.26 (C-4 ), 121.73 (C-2 ), 114.76
ꢁ
m
: 3131 (NAH) cm 1, 2923 (CAH), 1677
1
(
(
6
) d: 8.28 (s, 1H, NAH), 7.37
0
0
00
00
0
0
0
0
000
000
6
) d: 166.83 (C-
000
00
0
00
1
1
(
0
0
00
0
0
0
000
000
0
C-2 ), 67.55 (C-2), 49.42 (C-4 y C-5 ). HRMS EI: Calculated for
4.1.8. N-2 -Thiazoline hexanamide (15)
C
17
H
16
N
3
O
2
Cl: 329.0931; Found 329.0922.
Yield 80%. Mp = 108–110 °C recrystallized from hexane/AcOEt.
ꢁ1
IR KBr
C@N). H NMR (CDCl
.25 (t, 2H, 5 ), 2.34 (t, 2H, H2), 1.68 (m, 2H, H3), 1.33 (m, 4H,
m
: 3135 (NAH) cm , 2926 (CAH), 1707 (C@O) 1630
1
0
(
3
3
) d: 9.43 (s, 1H, NHC@O), 3.95 (t, 2H, 4 ),
0
00
000
4
.1.3. N-[4 -Methoxyphenyl-2-(4 -imidazolin-2 -yl)-phenoxy]-
0
acetamide (12)
13
H4, H5), 0.89 (t, 3H, CH
3
). C NMR (CDCl
3
) d: 173.3 (C@O), 162.0
: 3390 (NAH) cm 1, 2924, 1674
ꢁ
Mp = 185–187 °C. IR KBr
m
0
0
0
(
2
+
C-2 ), 55.5 (C-4 ), 36.6 (C-3), 32.2 (C-5 ), 31.4 (C-2), 24.5 (C-4),
1
(
C@O), 1238, 825. H NMR (DMSO-d
6
0
) d: 10.05 (s, 1H, NH), 7.50
9 2
2.4 (C-5), 13.9 (C-6). HRMS: Calculated for C H16SN O (ESI, M
H): 201.0985; Found 201.0759.
0
0
00
00
0
0
0
(
AA BB , 4H, H-2 , H-3 ), 7.22 (AA BB , 4H, H-2 , H3 ), 4.79 (s, 2H,
000
000
13
H-2), 3.78 (s, 4H, H-4 , H5 ), 3.72 (s, 3H, CH
3
O).
C NMR
000
00
(
DMSO-d
6
) d: 166.18 (C@O), 164.38 (C-2 ), 161.64 (C-1 ), 156.19
0
4
.1.9. N-2 -Thiazolin nonamide (16)
0
0
00
0
00
(
C-4 ), 132.05 (C-1 ), 130.26 (C-3 ), 121.93 (C-2 ), 115.50 (C-4 ),
Yield 81%; mp = 102–103 °C (Hexane/AcOEt). IR KBr
m: 3138
00
0
000
000
1
14.51 (C-2 , C-3 ), 67.70 (C-2), 55.84 (OCH
3
), 47.54 (4 , 5 ). HRMS
ꢁ1
1
(
(
NAH) cm , 2919 (CAH), 17109 (C@O) 1631 (C@N). H NMR
EI: Calculated for C18
H
19
N
3
3
O : 325.1426; Found 325.1418.
0
0
CDCl
3
). d: 3.95 (t, 2H, H-4 ), 3.25 (t, 2H, H-5 ), 2.33 (t, 2H, H-2),
1
.66 (q, 2H, H-3), 1.26 (m, 10H, H-4 H-5, H-6, H-7, H-8,), 0.87 (t,
0
0
000
13
´
4
(
.1.4. N-[Phenyl-2-(4 -imidazolin-2 -yl)-phenoxy]-acetamide
3H, H-9). C NMR (CDCl
(C-4 ), 36.6 (C-3), 32.3 (C-2), 31.8 (C-5), 29.3 (C-4), 29.2 (C-5),
29.0 (C-6), 24.9 (C-7), 22.6 (C-8), 13.9 (C-9). HRMS: Calculated
3
) d: 173.1 (C@O), 161.8 (C-2), 55.8
0
´
13)
Mp = 203–205 °C. IR KBr
C@O) 1612 (C@N). H NMR (DMSO-d
d, 2H, H-2 ), 7.40 (AA BB , 4H, H-2 , H-3 ), 7.32 (t, 2H, H-3 )
.08 (t, 2H, H-4 ) 4.76 (s, 2H, H-2), 3.58 (s, 4H, H-4 , H-5 ). NMR
) d: 166.94 (C-1), 163.80 (C-2 ), 160.03 (C-1 ),
39.02 (C-4 ), 129.43 (C-3 ), 129.34 (C-3 ), 124.39 (C-1 ), 124.15
C-4 ), 120.33 (C-2 ), 114.90 (C-2 ), 67.71 (C-2), 50.06 (C-4 , C-
). HRMS EI: Calculated for
95.1278.
ꢁ
m
: 3185 (NH) cm 1, 2923 (CH), 1685
1
(
(
6
00
) d: 10.14 (s, 1H, NH), 7.63
for C12
2
H22SN O (ESI, M+H): 243.1455; Found 243.1540.
0
0
0
00
0
0
000
000
0
7
4.1.10. N-2 -Thiazolin dodecanamide (17)
Yield 76%; mp 98–100 °C (Hex/AcOEt). IR KBr
cm , 2917 (CAH), 1700 (C@O) 1635 (C@N). H NMR (CDCl ) d:
3.91 (t, 2H, H-4 ), 3.39 (t, 2H, H-5 ), 2.20 (t, 2H, H-2), 1.57 (q, 2H,
H-3), 1.21 (m, 14H, H-4, H-5, H-6, H-7, H-8, H-9, H-10, H-11),
3
0.85 (t, 3H, H-12). C NMR (CDCl ) d: 181.6 (C-1), 169.9 (C-2 ),
1
3
000
00
C (DMSO-d
6
m
: 3128 (NAH)
0
00
0
0
ꢁ1
1
1
3
0
0
0
00
000
0
0
(
5
2
0
00
17 17 3 2
C H N O : 295.1321; Found
1
3
0

A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
7575
0
0
5
2
1
2
2.6 (C-4 ), 37.2 (C-3), 33.0 (C-2), 31.9 (C-5 ), 29.7 (C-4), 29.7 (C-5),
14.07 (C-19). HRMS: Calculated for
303.2431; Found 303.2400.
C
19
H
30
N
2
O
(ESI, M+1):
9.6 (C-6), 29.5 (C-7), 29.4 (C-8), 29.3 (C-9), 26.0 (C-10), 23.0 (C-
1), 14.2 (C-12). HRMS: Calculated for C15
85.1995; Found 285.2016.
2
H28SN O (ESI, M+H):
4
4
.2. Biological evaluation
4
.1.11. N-Thiazoline benzamide (18)
Yield 70%; mp 133–135 °C (Hexane/AcOEt). IR KBr
cm ), 3195, 1664 (C@O), 1599, 757. H NMR (CDCl ) d: 8.13–7.39
m, 5H, Ph), 3.55 (t, 2H, H-4 ), 3.22 (t, 2H, H-5 ). C NMR (CDCl
.2.1. Medium and growth conditions for C. violaceum
ꢁ
1
m
: cm 3405
Thioglycolate medium BBL (Becton–Dickinson. Sparks, MD) was
prepared at one half of the concentration indicated by the provider
and autoclaved. Afterward, filter-sterilized supplements were
ꢁ1
1
(
(
3
0
0
13
3
:
0
DMSO-d
1
6
) d: 167.4 (C@O), 163.0 (C-2 ), 129.5 (C-2), 127.3 (C-5),
2
added to reach the following final concentrations: 17 mM MgCl ,
0
0
27.0 (C-4), 126.1 (C-3), 53.9 (C-4 ), 31.6 (C-5 ). HRMS: Calculated
2
5
5
6 mM
K
2
HPO
4
,
2.8 mM
K
2
SO
4
,
3 mg/mL
L-methionine and
for C10 O (ESI, M+H): 207.0587; Found 207.0595.
H
10SN
2
00 ng/mL vitamin B12. LB agar was prepared with 10 g peptone,
g yeast extract, 5 g NaCl and 15 g agar per liter and sterilized
4
.1.12. N-Thiazoline phenylacetamide (19)
Yield 83%; mp 113–116 °C. IR KBr
927, 1702 (C@O), 1629. H NMR (CDCl
Ph), 3.98 (t, 2H, H-4 ), 3.89 (s, 2H, H-2), 3.68 (t, 2H, H-5 ).
for 15 min at 121 °C. When it was required, filter-sterilized antibi-
otics were added.
ꢁ1
ꢁ1
m
: cm 3491 (cm ), 3082,
1
2
3
) d: 7.35–7.25 (m, 5H,
0
0
13
C
0
4.2.2. Evaluation of synthesized compounds on CV026
To evaluate violacein synthesis an overnight C. violaceum CV026
culture was added to a flask containing 30 mL thioglycolate med-
NMR (CDCl
3
) d: 172.5 (C@O), 161.59 (C-2 ), 134.25 (C-3), 128.7
0
0
(
C-6), 127.8 (C-5), 126.2 (C-4), 53.4 (C-4 ), 43.2 (C-2), 31.4 (C-5 ).
HRMS: Calculated for C11 O (ESI, M+H): 221.0743; Found
21.0756.
H12SN
2
ium plus 30
OD600 = 0.14 and 980
l
g/mL kanamycin. Cultures were adjusted to an
L aliquots were dispensed into 2 mL sterile
2
l
filter microfuge tubes. Ten microliters of the test compound dis-
solved in DMSO were added to 0, 0.001, 0.01 0.1, 1.0, 10, 100 and
4
.1.13. 8-n-hexyloxyphenyl-2-oxazoline (20)
To 1 equiv of 4-hexyloxybenzaldehyde dissolved in t-BuOH was
1
000
l
M final concentrations, followed by the addition of 10
lL
added 1.1 equiv of ethanolamine at rt under N
with constant stirring. The reaction mixture was held under these
conditions for 1 h, then 1.25 equiv of I and 3 equiv of K CO were
added. The resulting mixture was stirred at 70 °C for 30 h. Then the
mixture was filtered and the residue was poured in water and
extracted with EtOAc 5 times. The organic phase was washed with
a saturated solution of Na
solvent was evaporated in a rotatory evaporator and the remnant
residue was purified by chromatography on neutral Al
ane/EtOAc gradient) to furnish the pure compound.
2
atmosphere and
5
0
l
M C
6
-AHL. Tubes were capped, incubated at 29 °C in a Ther-
momix R mixer with shaking at 900 rpm for 15 h. At the end of this
period cell growth was measured at 720 nm, using a thioglycolate
medium as blank. To estimate violacein production, 500 lL ali-
quots of cultures were mixed with an equal volume of acetone
and centrifuged for 4 min at 15,000 rpm. Supernatants were recov-
ered and 577 nm absorbances registered. Each experiment was
repeated 6 times.
2
2
3
2 3 2 4
SO and dried over Na SO anhyd. The
2 3
O (hex-
Mp 47–48 °C. IR KBr m
: 3425 (NAH) cmꢁ1, 2930, 1649 (C@N),
4.2.3. Viable count
1
0
0
To determine the viable count, 100 lL aliquots of cultures
1
254, 846. H NMR (CDCl
3
) d: 7.37 (AA BB , 4H, Ph), 4.39 (t, 2H,
ꢁ6
ꢁ7
ꢁ8
diluted 10 , 10 and 10 were spread onto LB plates and incu-
H-5), 4.02 (t, 2H, H-10), 3.97 (t, 2H, H-4), 1.78 (m, 2H, H-11),
bated overnight at 37 °C.
1
.45 (m, 2H, H-12), 1.33 (m, 4H, H-13, H-14), 0.90 (t, 3H, H-15).
1
3
3
C NMR (CDCl ) d: 164.40 (C-2), 161.70 (C-9), 129.91 (C-7),
1
3
1
2
20.00 (C-6), 114.14 (C-8), 68.07 (C-10), 67.45 (C-5), 54.72 (C-4),
1.52 (C-11), 29.09 (C-12), 25.64 (C-13), 22.54 (C-14), 13.99 (C-
4
4
.3. Theoretical calculations
5). HRMS EI: Calculated for
47.1572.
15 2
C H21NO : 247.1572; Found
.3.1. Docking calculations
For docking studies, we utilized CviR (Chromobacterium vio-
laceum 12472 located at the Protein Data Bank with the PDB code:
3QP6) as a protein target. First, the initial geometric optimization
of ligands was performed with HYPERCHEM at the MM+ level.36
Then, the compounds were optimized at the AM1 and DFT
(B3LYP/6-31G(d,p)) levels using the Gaussian 09 program.3
The AutoDock (4.2) program was selected for docking studies
because this algorithm maintains a rigid macromolecule while
4
.1.14. 8-n-Nonyloxyphenyl-2-tetrahydropyrimidine (21)
To 4.8 mmol of 4-nonyloxybenzaldehyde dissolved in t-BuOH
was added 5.0 mmol of 1,3-propylenediamine, at rt under N
atmosphere and with constant stirring. The reaction mixture was
held under these conditions for 1 h, then 4.8 mmol of I and
4.4 mmol of K CO were added. The resulting mixture was stirred
2
7,38
2
1
2
3
3
9
under reflux for 4 h. Then the mixture was filtered and the residue
allowing ligand flexibility. This program has been widely used
because it displays good free energy correlation values between
was poured in water and extracted with EtOAc (5 ꢀ 20 ml). The
4
0
organic phase was washed with a saturated solution of Na
2
SO
3
docking simulations and experimental data. A GRID-based proce-
dure was utilized to prepare the structural inputs and to define all
and dried over Na
reduced pressure and the remanent residue was purified by chro-
matography on neutral Al
the pure compound.
2 4
SO anhyd. The solvent was evaporated under
3
9
of the binding sites. A rectangular lattice (126 ꢀ 126 ꢀ 126 Å)
with points separated by 0.375 Å was superimposed on the entire
protein structure to achieve a blind docking procedure.
2
O
3
(hexane/EtOAc gradient) to furnish
ꢁ
Mp 78–79 °C. IR KBr
m
: 3211 (NAH) cm 1, 2925, 2852, 1610
All docking simulations were conducted using the hybrid
Lamarckian genetic algorithm with an initial population of 100
1
0
0
(
C@N), 1249, 842. H NMR (CDCl
3
) d: 7.27 (AA BB , 4H, Ph), 5.87
7
(
NH), 3.96 (t, 2H, H-11), 3.42 (t, 4H, H-4, H-6), 1.88–1.75 (m, 4H,
randomly placed individuals and a maximum of 1.0 ꢀ 10 energy
H-12, H-13), 1.45 (m, 2H, H-14), 1.30 (m, 8H, H-15, H-16, H-17,
H-18), 0.90 (t, 3H, H-19).
evaluations. All other parameters were maintained at their default
settings. The resulting docked orientations were clustered
together, within a RMSD of 0.5 Å. The lowest energy cluster for
each ligand was subjected to further free energy and binding
13
3
C NMR (CDCl ) d: 161.60 (C-2),
1
1
56.77 (C-10), 128.36 (C-8), 125.06 (C-7), 114–40 (C-9), 68.22 (C-
1), 40.69 (C-4, C6), 31.81 (C-5), 29.46 (C-12), 29.33 (C-13), 29.19
4
1
(
C-14), 29.09 (C-15), 25.94 (C-16), 22.60 (C-17), 19.66 (C-18),
geometry analyses, as previously reported.

7
576
A. Bucio-Cano et al. / Bioorg. Med. Chem. 23 (2015) 7565–7577
4
.3.2. Molecular dynamics simulations
reported for the MMGBSA calculations can be termed absolute
The nine complexes generated through docking procedures
binding free energies.
together with the crystallized complex (PDB entry 3QP6) were sub-
mitted to MD simulations to corroborate the map of interactions
obtained through docking calculations.
The entropy contributions were calculated using the MMPBSA.
3
5
py module present in AMBER analysis tools. Contributions were
evaluated for only 40 snapshots taken at time intervals of
1000 ps from the last 40 ns production runs. In order to detect
the hot spot residues, the absolute binding free energies were
decomposed into the contribution of individual residues using
A series of MD simulations were carried out for the complexes
with the highest energies. The MD simulations were performed
with the Amber 12 package35 using the ff99SB force field.
Topologies for the protein–ligand complexes were constructed
by the Leap module, minimized through the sander module,
and the MD simulations were run with the ‘pmemd’ module.
Ligand topologies were obtained through the antechamber mod-
42
5
2
the theory of free energy per residue decomposition.
Acknowledgments
43
ule, based on the generalized Amber force field (GAFF). AM1-
J.C. gives thanks to CONACYT – México (132352), SIP-COFFA/
IPN – México (SIP: 20140252) and CYTED (214RT0482). A.R. is
grateful to IPN and SECITI – México for financial support through
Grants from SIP IPN, México (20150549), CONACYT – México
(240808) and ICyTDF – México (325/2011). A.B.C. and J.T.J. thank
CONACYT – México and the IPN – México for scholarships. We
are grateful to CNMN (IPN) for the HRMS and to CE of the ENCB
(IPN) for the IR.
4
4
BCC atomic charges
were calculated with the antechamber
module.4 Cl ions were placed at different places by Leap to neu-
tralize the positive charges around the complex models at pH 7. A
rectangular-shaped box of water was constructed using the TIP3P
water model. The water model extended 12.0 Å between the
complex and the edge of the box. Then, the systems were equili-
brated by carrying out minimization through 1000 steps of steep-
est descent minimization followed by 1000 steps of conjugate
gradient minimization. 200 picoseconds (ps) of heating and
5
ꢁ
4
6
Supplementary data
2
00 ps of density equilibration with weak restraints on the com-
plex were followed by 600 ps of constant pressure equilibration
at 300 K, using the SHAKE algorithm47 on hydrogen atoms, a time
step of 2 fs and langevin dynamics for temperature control. The
equilibration run was followed by a 50 ns MD run without posi-
tion restraints under periodic boundary conditions (PBC). The
unbound list was generated by using an atom-based cutoff of
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.10.046.
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