Design, synthesis, and evaluation of alkyl-quinoxalin-2(1h)-one derivatives as anti-quorum sensing molecules, inhibiting biofilm formation in aeromonas caviae Sch3

Article abstract of DOI:10.3390/molecules23123075

With the increasing antibiotic resistance of bacterial strains, alternative methods for infection control are in high demand. Quorum sensing (QS) is the bacterial communication system based on small molecules. QS is enables bacterial biofilm formation and pathogenic development. The interruption of QS has become a target for drug discovery, but remains in the early experimental phase. In this study, we synthesized a set of six compounds based on a scaffold (alkyl-quinoxalin-2(1H)-one), new in the anti-QS of Gram-negative bacteria Aeromonas caviae Sch3. By quantifying biofilm formation, we were able to monitor the effect of these compounds from concentrations of 1 to 100 μM. Significant reduction in biofilm formation was achieved by 3-hexylylquinoxalin-2(1H)-one (11), 3-hexylylquinoxalin-2(1H)-one-6-carboxylic acid (12), and 3-heptylylquinoxalin-2(1H)-one-6-carboxylic acid (14), ranging from 11% to 59% inhibition of the biofilm. This pilot study contributes to the development of anti-QS compounds to overcome the clinical challenge of resistant bacteria strains.

Full text of DOI:10.3390/molecules23123075

molecules
Communication
Design, Synthesis, and Evaluation of
Alkyl-Quinoxalin-2(1H)-One Derivatives as
Anti-Quorum Sensing Molecules, Inhibiting
Biofilm Formation in Aeromonas caviae Sch3
René Blöcher 1, Ariel Rodarte Ramírez 2, Graciela Castro-Escarpulli 2, Everardo Curiel-Quesada 3
and Alicia Reyes-Arellano 1,
*
1
Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional (ENCB-IPN),
Departamento de Química Orgánica, Ciudad de México 11340, México; rene.bloecher@googlemail.com
Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional (ENCB-IPN),
Laboratorio de Investigación Clínica y Ambiental, Departamento de Microbiología,
Ciudad de México 11340, México; rodar_t@hotmail.com (A.R.R.); chelacastro@hotmail.com (G.C.-E.)
Escuela Nacional de Ciencias Biológicas del Instituto Politécnico Nacional (ENCB-IPN),
Departamento de Bioquímica, Ciudad de México 11340, México; ecqmixcoacdf@gmail.com
Correspondence: areyesarellano@yahoo.com.mx; Tel.: +52-57-29-6000 (ext. 62526)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 18 October 2018; Accepted: 20 November 2018; Published: 24 November 2018
Abstract: With the increasing antibiotic resistance of bacterial strains, alternative methods for
infection control are in high demand. Quorum sensing (QS) is the bacterial communication
system based on small molecules. QS is enables bacterial biofilm formation and pathogenic
development. The interruption of QS has become a target for drug discovery, but remains
in the early experimental phase. In this study, we synthesized a set of six compounds
based on a scaffold (alkyl-quinoxalin-2(1H)-one), new in the anti-QS of Gram-negative bacteria
Aeromonas caviae Sch3. By quantifying biofilm formation, we were able to monitor the effect of these
compounds from concentrations of 1 to 100
achieved by 3-hexylylquinoxalin-2(1H)-one (11), 3-hexylylquinoxalin-2(1H)-one-6-carboxylic acid
12), and 3-heptylylquinoxalin-2(1H)-one-6-carboxylic acid (14), ranging from 11% to 59% inhibition
µM. Significant reduction in biofilm formation was
(
of the biofilm. This pilot study contributes to the development of anti-QS compounds to overcome
the clinical challenge of resistant bacteria strains.
Keywords: anti-quorum sensing; biofilm; quinoxalin-2(1H)-one; Aeromonas caviae; bioisosteres;
Grignard reaction
1. Introduction
The significant improvement of human health by the discovery of penicillin is undeniable.
With the increase in bacterial strains resistant to virtually every clinically approved antibiotic,
drug discovery research is facing the challenge of sustaining infection therapy [1,2]. The development
of new antibiotics is limited and the result is often short lasting. Therefore, the need for alternative
therapies has emerged and anti-quorum sensing represents a promising approach [1–3].
Quorum sensing describes the bacterial cell–cell communication based on small molecules called
autoinducers [
density, which they sense by means of extracellular signal molecules such as N-acylated homoserine
lactones (AHLs) in the QS system [ ]. In Gram-negative bacteria, QS is also a key function for the
formation of biofilm. Biofilm is a packed community of bacterial species embedded in a polymeric
4]. Gram-negative bacteria regulate their virulence factors as a function of population cell
5
Molecules 2018, 23, 3075; doi:10.3390/molecules23123075
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3075
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extracellular matrix providing channels for water and nutrients circulation [
6]. The biofilm maintains
bacterial viability, shields bacteria from antibiotic influences, and provides an environment for
pathogen development [ ]. Aeromonas caviae Sch3 is a Gram-negative bacterium and an environmental
opportunistic pathogen of animals and humans [ ]. Aeromonas cause gastroenteritis, wound infections,
bacteremia, and, less frequently, respiratory infections, hepatobiliary infection, peritonitis, urinary tract
infections, and ocular infections [ ]. Aeromonas have a remarkable ability to colonize a broad variety of
environments, relying on their biofilm production and cell–cell communication [ ]. The disruption of
7
7
7
7
biofilm formation is therefore a valuable target for novel anti-infective treatments. QS autoinducers
are categorized into different types. For our investigation, only the type 1 autoinducer (AI-1) system,
which is widespread among Gram-negative bacteria, is relevant [
N-acyl homoserine lactones (AHLs), which are derived from common components of the bacterial
metabolism [ ]. The major autoinducers produced by Aeromonas are N-butanoyl homoserine lactone
(C4-AHL) and N-hexanoyl homoserine lactone (C6-AHL) [ ], shown in Scheme 1. To inhibit the
QS mediated by these autoinducers, analogues containing increased N-acyl carbon chains (C10- to
C14-AHL) have been used successfully [ ]. There has been a focus on alterations of the N-acyl carbon
chain within the QS inhibition of C4- and C6-AHL [10 11]. To develop innovative anti-QS compounds,
7]. The AI-1 system small molecules
8
9
9
,
we changed the lactone moiety of AHL by introducing a pharmacophores-oriented bioisoster [12,13],
and to integrate the N-acyl carbon chain into a hetero cycle. Our structural solution resulted in the
alkyl-quinoxalin-2(1H)-one shown in Scheme 1. We demonstrated its ability to significantly reduce
biofilm formation in Aeromonas caviae Sch3 at the lowest compound concentration of 1 µM.
2. Results and Discussion
2.1. Design of Quorum Sensing Inhibitors and Structural Development
The majority of N-acyl homoserine-lactone analogues provide their inhibition activity through an
extended N-acyl carbon chain [14–16], while preserving the lactone moiety. As such, we decided to
introduce a bioisosteric replacement of the lactones carbonyl proton acceptor with a tertiary amine as
part of a closed ring heterocyclic structural solution. In Scheme 1, the pharmacophore and bioisosteres
are marked with a green dashed-lined circle. By focusing on the pharmacophore proton-binding
character of the autoinducer, changing the lactone fragment and introducing a bioisostere, we produced
a valuable anti-quorum sensing scaffold. In our design, we adapted the N-acyl fragment of the N-acyl
homoserine-lactone by integrating it into the 3-alkyl quinoxalin-2(1H)-one hetero cycle. In Scheme 1,
the adapted structural motives are highlighted in red. This adaption enabled us to introduce different
carbon chain lengths within our integrated N-acyl motive, inspired by the previous-stated importance
of this structural element in the context of inhibitory strength [14–16].
Scheme 1. Design of anti-quorum sensing compounds based on the autoinducer structural characteristics,
AHL, and bioisosteric replacement. The general core structure of the anti-quorum sensing compounds
presented here is shown on the left. In the middle and on the right are illustrations of two N-acyl
homoserine-lactone autoinducers (C4-AHL, C6-AHL) responsible for bacterial cell communication via
quorum sensing. The N-acyl carbon chain is a consistent structural fragment in all three molecules and
is marked in red. The bioisosteric proton acceptor substitution of the carbonyl moiety with the tertiary
amine is highlighted with a green dashed line rectangle, visualizing the proton-binding area.

Molecules 2018, 23, 3075
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During the structural development of these inhibitors, we realized a water solubility limitation
under assay conditions with the hexyl carbon chain (11, 3-hexylylquinoxalin-2(1H)-one), so we were
therefore not able to evaluate the heptyl carbon chain derivative (13, 3-hepylquinoxalin-2(1H)-one).
To increase the water solubility and still evaluate the heptyl carbon chain derivative, we introduced a
carboxylic acid functional group at position 6 (14, 3-heptylquinoxalin-2(1H)-one-6-carboxylic acid).
The acid function was placed in a position where it should not interfere with the theoretical structure
described above. To enable a direct comparison of acid and non-acid substitution in position 6,
a derivative of the 3-hexylylquinoxalin-2(1H)-one (11), containing the acid substitution in position 6
(12, 3-hexylylquinoxalin-2(1H)-one-6-carboxylic acid), was synthesized and evaluated.
2.2. Synthesis
Scheme 2 shows the pathway for the synthesis of the anti-QS compounds (
9–14). The synthetic
route starts with the synthesis of required Grignard reagents ( ) (Scheme 2a). Ethyl-, butyl-, hexyl-,
1–4
and heptyl-bromide were reacted with magnesium turnings in tetrahydrofuran (THF) and a catalytic
◦
amount of iodine [17]. The activated Grignard reagents had to be used immediately at
with diethyl oxalate in dry THF (Scheme 2b). The resulting -keto esters (
further purification in a condensation/cyclization reaction with o-diamino phenyl or 3-carboxyl
−
78 C
β
5–8) were used without
o-diamino phenyl (Scheme 2c) [18]. The final 3-alkyl quinoxalin-2(1H)-one (9–14) were purified by
column chromatography.
Scheme 2. Synthesis pathway for 3-alkyl quinoxalin-2(1H)-one. (a) Dry THF, room temperature (rt;
◦
◦
◦
25 C) −40 C, 45 min; (b) dry THF, –78 C, 2 h; and (c) ethanol (EtOH), 12 h.
2.3. Inhibition of Biofilm Formation by Anti-Quorum Sensing Compounds
The determination of the inhibition of biofilm formation in A. caviae Sch3 at a starting concentration
of 6
570 nm, 0.50 absorbance (OD570 of 0.50) produced by five synthesized compounds (
at 1, 10, and 100 M) demonstrated a significant effect by compounds 11 and 12 at all but the
×
108 colony forming units (CFU)/ml bacteria, measured as absorbance of the culture at
9–12 and 14,
µ
lowest concentration and by 14 at all concentrations tested (Figure 1 and Table 1). The biofilm
quantification was determined through measuring the absorbance at 570 nm of the dye staining the
biofilm. Figure 1 shows the biofilm formation influenced by the five synthesized compounds. Table 1
shows the reduction in biofilm formation caused by the addition of the five synthesized compounds.
The most significant reduction in biofilm formation was achieved by 3-heptylquinoxalin-
2(1H)-one-6-carboxylic acid (14), with a biofilm inhibition of 59% at 100
M (Table 1). 3-Hexylquinoxalin-2(1H)-one-6-carboxylic acid (12) and 3-hexylquinoxalin-2(1H)-one
11) showed similar and significant reduction in biofilm formation with 21% (12) and 25% (11) at
a 100 M compound concentration, as well as 12% (12) and 11% (11) inhibition at 10 M. At 1 M,
both compounds (11 12) were not able to significantly decrease the biofilm formation, nor were
3-butylquinoxalin-2(1H)-one ( ) or 3-ethylquinoxalin-2(1H)-one ( ) able to significantly inhibit the
biofilm formation at any used compound concentration.
µM, 24% at 10 µM, and 19% at
1
µ
(
µ
µ
µ
,
9
8

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Figure 1. Biofilm formation influenced by compounds 9, 10, 11, 12, and 14 in Aeromonas caviae Sch3 at
6 × 108 colony forming units (CFU)/mL bacteria. The data are presented as percentages of the biofilm
formation of the control, which received no inhibitor treatment and was set as 100%. The compounds
were evaluated at 100, 10, and 1 µM concentrations. Dimethyl sulfoxide (DMSO) had no significant
effect on biofilm formation; the control experiment data can be found in the Supplementary Materials.
Significance was confirmed by the Student’s t-test with an accuracy of * p < 0.005 and # p < 0.05 at the
marked bars. Experiments were performed with n = 6. Compound number (Cpd No.)
Table 1. Inhibition of biofilm formation by anti-quorum sensing compounds. * p < 0.005, # p < 0.05.
% Reduction of Biofilm Formation
ID
R1
R2
100 µM
10 µM
1 µM
9
Ethyl
Butyl
Hexyl
Hexyl
Heptyl
Heptyl
-
-
-
6
3
25 *
21 *
-
4
4
5
3
6
1
-
10
11
12
13
14
11 #
12 #
-
COOH
-
COOH
59 *
24 *
19 *
From the analysis of the structure activity-relationship of our compound set, we concluded that
the correlation of the N-acyl carbon chain length with biofilm inhibition reported in literature [14 16
and mentioned earlier in this work, is reflected in our results. The short length of our integrated N-acyl
carbon chain, such as ethyl and butyl in compounds and 10, had no significant effect on biofilm
formation, whereas compounds 11 12, and 14 with extended hexyl and heptyl carbon chains showed
–
]
9
,
significant inhibition. We additionally recognized that the introduction of a carboxylic acid functional
group in position 6 increased water solubility, but did not affect biofilm inhibition in Aeromonas caviae.
This was shown by the similar biofilm reduction of compounds 11 and 12, whose structure only differs
by the carboxylic acid substitution in position 6. This study provides anti-quorum sensing compounds
for Aeromonas caviae Sch3 based on a scaffold benefiting future studies.

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2.4. Evaluation of Quorum Sensing Inhibition Through Reduction of Violacein Production in Chromobacterium
Violaceum CV026
The five synthesized compounds (9–12 and 14, at 1, 10, and 100 µM) were added to bacteria in
L-broth, optical density at 600 nm, 0.1 absorbance (OD600 of 0.1) supplemented with C6-AHL and
cells were incubated for 40 h. After quantifying violacein production from Chromobacterium violaceum
CV026, we confirmed the QS inhibition observed before via the biofilm reduction in Aeromonas caviae.
Figure 2 shows the specific production of violacein influenced by the added compounds.
Figure 2. Production of violacein by Chromobacterium violaceum CV026 in the presence of compounds
(9, 10, 11, 12, and 14 at 1, 10, and 100 µM). The specific production of violacein was determined by the
ratio of detectable pigment (OD577) per amount bacteria (OD720). The data were normalized on the
untreated violacein production of the culture. Experiments were performed as duplicates. Compound
number (Cpd No.).
The QS inhibition evaluated in Chromobacterium violaceum CV026 (Figure 2) describes a similar
trend as the QS inhibition in Aeromonas caviae (Figure 1), which was demonstrated via biofilm reduction.
The violacein production in Chromobacterium violaceum CV026 is a more sensitive assay for QS inhibition
than the quantification of biofilm formation. Therefore, stronger QS inhibition is reported in Figure 2.
Compound 14 shows the most significant effect throughout all tested concentrations. The major effects
of all five compounds were observed at the highest concentration of 100
dose-dependent response.
µM, with a clearly recognizable
3. Materials and Methods
3.1. Chemistry
All reagents and solvents were purchased from Sigma Aldrich, Toluca, Mexico and used directly
without further purification. All reactions were carried out at room temperature, if not stated otherwise.
Reactions were monitored by thin-layer chromatography (TLC) on Merck F253 silica gel aluminum
sheets, and spots were revealed with ultraviolet (UV) light (254 nm) (Darmstadt, Mercury apparatus
at 300 MHz and 75 MHz, respectively, or on a Varian at 500 and 125 MHz, respectively (Palo Alto,
) are referenced to internal (CH3)4Si with δ 1H = 0 13C = 0, and given
CA, USA). The chemical shifts (δ , δ
in ppm. High Resolution Mass Spectra (HRMS) were recorded on a Bruker micrOTOF Q and processed
with the Software Bruker Compass DataAnalysis 4.1 (Karlsruhe, Germany).

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3.1.1. General Method for the Synthesis of Compounds 9–14, Described at the Example of
3-Ethylquinoxalin-2(1H)-one (8)
Magnesium turnings (Mg◦), 0.2 g (8 mmol), were washed with 100 mL hexane by repeated
decanting. The Mg◦ was dried in vacuum under heat support. The Mg◦ was allowed to cool down
while still under vacuum. The reaction vessel, a 25-mL 2-neck round bottom flask, was supplied with a
dropping-funnel and sealed with septa. The apparatus was flushed with nitrogen gas while heated with
a heat-gun. The apparatus was allowed to cool down while still flushed in a nitrogen stream. The dried
Mg◦ was transferred into the dry apparatus. A catalytic amount of iodine was added to the metal as
a reaction indicator [17] and catalyst [19]. In the dropping-funnel above, we placed 0.6 mL (8 mmol)
1-bromoethan in 8 mL THF (1 molar concentration). Under stirring in the nitrogen atmosphere, 1–2 mL
of the halide solution were added to the magnesium-iodine, just enough to cover it. To start the
reaction, the magnesium was cracked with a spatula or glass rod to break the magnesium-oxide layer
while covered with THF. Silver shining magnesium became visible. Under strong stirring, the reaction
started within 15 min (long chain alkyl bromides needed up to a maximum of 45 min before reaction
onset). Activation of the reagent was observed by a color change of the iodine. From an initial orange,
the mixture turned into yellow and the color finally disappeared, leaving a grey solution. At this
point, the remaining alkyl-halide solution was added slowly dropwise to the round-bottom flask [17].
◦
After completing addition, the mixture was heated to 40 C and stirred for another 30 min to guarantee
total activation. The magnesium turnings disappeared and a cloudy grey solution remained with few
black particles. For the next step, a second apparatus was prepared. One 100-mL round-bottom flask
with a dropping-funnel on top was placed in a Dewar and dried as previously described in a nitrogen
stream. In the round-bottom flask, we placed 0.98 mL (7.2 mmol) diethyl oxalate in 3 mL dry THF and
cooled down to
−
78 ◦C in a dry-ice/acetone bath. After the Grignard reagent was activated, it was
transferred into the second dropping-funnel. Here, it was important to work quickly and precisely
as the reagent becomes inactivated by air and moisture. Likewise, side products can be produced,
such as salts that can block the second dropping funnel, if addition is postponed too long. After the
active Grignard reagent was placed in the second dropping-funnel, a slow/drop-wise addition into
the
−
78 ◦C cold round-bottom flask followed. A fast addition of the Grignard reagent resulted in the
occurrence of side products. After the addition was completed, the mixture was stirred at
−
78 ◦C for
◦
2 h, followed by a fast quenching of the reaction at
solution. The reaction was allowed to warm up to room temperature, and was subsequently extracted
with 50 mL of dichloromethane. The organic layer was washed with water, dried over sodium sulfate,
−
78 C with 20 mL of 3 N hydrochloric acid aqueous
and removed under reduced pressure, leaving a clear liquid—the ketovaline ethyl ester (
was used without further purification. Then, 0.78 g (7.2 mmol) O-phenylendiamine were dissolved in
6 mL ethanol under nitrogen atmosphere [18]. The synthesized ketovaline ethyl ester ( ) was dissolved
in 2 mL ethanol and added slowly to the o-phenylendiamine ethanolic solution. The reaction was
stirred for 12 h. The 3-ethylquinoxalin-2(1H)-one ( ) precipitated in the reaction solution. The crude
1)—which
1
9
product was collected through filtration and washed with 3–5 mL cold ethanol. Final purification was
performed by column chromatography with silica gel and a solvent mixture of ethyl acetate/hexane
1/2. In the purification process of the acidic derivatives, 3-hexylquinoxalin-2(1H)-one-6-carboxylic
acid (12), and 3-heptylquinoxalin-2(1H)-one-6-carboxylic acid (14), 1% acetic acid was added to the
chromatography eluent mixture. The pure compounds all remained as white solid powder.

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3.1.2. Analytics
3-Ethylquinoxalin-2(1H)-one (9)
Total yield: 29%. 1H NMR (500 MHz, dmso)
δ
12.31 (s, 1H;
1
), 7.71 (dd, J = 7.9, 0.7 Hz, 1H;
8
), 7.49–7.43
(m, 1H;
5), 7.29–7.23 (m, 2H;
6, 7
), 2.79 (q, J = 7.4 Hz, 2H; 10), 1.23–1.19 (m, 3H; 11). 13C NMR (126 MHz,
DMSO)
δ
167.76 ( ), 159.77 (3), 139.98 (9), 136.86 (4), 134.63 (5), 128.23 (6), 122.58 (7), 119.82 (8),
2
27.9 (10), 9.5 (11); IR: KBr v: 3436 (N–H) cm−1, 2946 (C–H) cm−1, 1663 (C=O) cm−1, 1567 (N=C) cm−1
;
HRMS: measured 174.0873 (calculated for C10H10N2O: 174.0866 ).
3-Butylquinoxalin-2(1H)-one (10)
1
Total yield: 55%. H NMR (500 MHz, DMSO)
δ
12.34 (s, 1H;
), 2.83–2.75 (m, 2H; 10), 1.74–1.64 (m, 2H; 11),
161.88 ( ), 154.58 ( ), 131.74 ( ), 129.36 ( ),
6), 28.26 (10), 22.10 (11), 18.59 (12), 13.90 (13); IR: KBr v:
1), 7.73 (d, J = 7.8 Hz, 1H; 8),
1
7.48 (d, J = 7.7 Hz, H;
5), 7.28 (t, J = 8.1 Hz, 2H;
6
,
7
1.47–1.34 (m, 2H; 12), 0.98–0.89 (m, 3H; 13). 13C NMR
128.06 ( ), 123.06 ( ), 112.91 ( ), 115.21 (
δ
2
3
9
4
8
7
5
3436 (N–H) cm−1, 2946 (C–H) cm−1, 1663 (C=O) cm−1, 1562 (N=C) cm−1; HRMS: measured 203.1118
(calculated for C12H14N2O: 203.1179).
3-Hexylquinoxalin-2(1H)-one (11)
Total yield: 42%. 1H NMR (500 MHz, DMSO)
7.31–7.22 (m, 2H; ), 2.82–2.69 (m, 2H; 10), 1.65 (qd, J = 14.9, 7.5 Hz, 2H; 11), 1.44–1.13 (m, 6H; 12
14), 0.91–0.79 (m, 3H; 15). 13C NMR (126 MHz, DMSO)
167.26 ( ), 160.01 ( ), 136.90 ( ), 136.82 (9
δ
12.30 (s, 1H;
1
), 7.74–7.66 (m, 1H;
8
), 7.52–7.41 (m, 1H; 5),
13
),
6,
7
,
,
δ
2
3
4
134.47 (5), 133.37 (6), 128.23 (7), 120.41 (8), 33.66 (12), 33.76 (13), 31.21 (10), 27.22 (11), 22.9 (14), 19.15 (15);
IR: KBr v: 2951 (C–H) cm−1, 1667 (C=O) cm−1, 1562 (N=C) cm−1; HRMS: measured 231.1496 (calculated
for C14H18N2O: 231.1492 ).
3-Hexylquinoxalin-2(1H)-one-6-carboxylic acid (12)
1
Total yield: 21%. H NMR (500 MHz, DMSO)
δ
13.24 (s, 1H; 10), 12.52 (s, 1H;
1), ), 7.97 (dd, J = 8.5,
1.9 Hz, 1H;
5), 7.87–7.72 (m, 1H;
7
), 7.35–7.27 (m, 1H; ), 2.81–2.69 (m, 2H; 11), 1.71–1.60 (m, 2H; 12),
8

Molecules 2018, 23, 3075
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1.38–1.15 (m, 6H; 13
,
14,
15), 0.89–0.77 (m, 3H; 16). 13C NMR (126 MHz, DMSO)
δ
167.07, 163.36,
155.12, 135.56, 131.32, 130.23, 129.88, 125.83, 117.09, 115.83, 31.61, 29.17, 28.94, 22.46, 14.35; IR: KBr v:
3430 (N–H) cm−1, 1658 (C=O) cm−1, HRMS: measured 274.3169 (calculated for C15H18N2O3: 274.3150).
3-Heptylquinoxalin-2(1H)-one (13)
1
Total yield: 27%. H NMR (500 MHz, DMSO)
δ
12.30 (s, 1H;
), 2.80–2.69 (m, 2H; 10), 1.68 (dt, J = 15.2, 7.5 Hz,
15), 0.83 (dt, J = 14.8, 8.8 Hz, 3H; 16). 13C NMR (126 MHz,
), 136.90 ( ), 136.82 ( ), 134.46 ( ), 133.23 ( ), 128.28 ( ), 120.40 ( ),
1), 7.70 (d, J = 7.5 Hz, 1H; 8), 7.49–7.43
(m, 1H;
2H; 11), 1.44–1.14 (m, 8H; 12
DMSO) 167.06 ( ), 159.77 (
5
), 7.25 (dd, J = 12.0, 4.5 Hz, 2H;
6, 7
,
13, 14
,
δ
2
3
9
4
6
5
7
8
36.43 (14), 34.03 (13), 33.67 (12), 31.25 (10), 27.28 (11), 23.8 (15), 19.11 (16); IR: KBr v: 3436 (N–H) cm−1
,
2915 (C–H) cm−1, 1663 (C=O) cm
C15H20N2O: 245.1648).
−
1, 1565 (N=C) cm−1; HRMS: measured 245.1650 (calculated for
3-Heptylquinoxalin-2(1H)-one-6-carboxylic acid (14)
1
Total yield: 23%. H NMR (500 MHz, DMSO)
δ
13.09 (s, 1H; 10), 12.63 (s, 1H;
1
), 8.12–8.04 (m, 1H;
5),
7.99–7.74 (m, 1H;
13 14 15
16), 0.98–0.88 (m, 3H; 17); 13C NMR (126 MHz, DMSO)
137.91 ( ), 135.92 ( ), 134.25 ( ), 131.23 ( ), 129.28 ( ), 123.40 ( ), 35.43 (14), 33.23 (13), 31.67 (15),
30.23 (11), 26.18 (12), 21.5 (16), 17.09 (17); IR: KBr v: 3414 (N–H) cm−1, 2915 (C–H) cm−1, 1661 (C=O)
7
), 7.51–7.32 (m, 1H;
8
), 2.90–2.82 (m, 2H; 11), 1.82–1.73 (m, 2H; 12), 1.48–1.27 (m, 8H;
,
,
,
δ
166.06 (10), 163.9 ( ), 157.67 ( ),
2
3
9
4
6
5
7
8
cm
1, 1568 (N=C) cm−1; HRMS: measured 289.1549 (calculated for C16H20N2O3: 289.1547). Selected
−
Spectra: IR, 1H, 13C, HSQC, HMBC and HRMS can be found in the Supplementary Materials.
3.2. Bacteriology
For the evaluation of biofilm reduction in Aeromonas caviae Sch3, previous published protocols
were used [20]. A brief description of the process is provided here as well as individual changes.
3.2.1. Bacterial Strain Aeromonas caviae Sch3
We used the Aeromonas caviae Sch3 strain isolated from diarrheal feces of a 5-year-old child patient,
kindly provided by Dr. Jonathan Shaw from the University of Sheffield Medical School (Sheffied,
United Kingdom). Genetic and biochemical characterization of A. caviae Sch3 was performed by
Dr. Castro-Escarpulli from the “Escuela Nacional de Ciencias Biólogicas del Instituto Politécnico
Nacional” (Mexico City, Mexico).
3.2.2. Culture Conditions Aeromonas caviae Sch3
Aeromonas caviae Sch3 strain was grown on 1.5% trypticase soy agar medium (TSA) (Bioxon,
◦
Mexico City, Mexico) at 37 C for 16 h. Short-term storage of isolates was carried out in minimal
maintenance medium: 1% v/v polypeptone, 0.3% v/v yeast extract, 0.5% v/v bacteriological agar,

Molecules 2018, 23, 3075
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and 0.85% v/v NaCl) at room temperature (RT). Long-term storage was carried out in Todd Hewitt
broth (Oxoid, Mexico City, Mexico) containing 30% (v/v) glycerol at –70 ◦C.
3.2.3. Evaluation of Biofilm Reduction
In transparent flat bottom 24-well plates, Aeromonas caviae-Sch3 was used at a concentration of
6 × 108 CFU/mL. Bacterial concentration was adjusted to an OD570 of 0.50. Compounds
9, 10, 11, 12,
and 14 were evaluated at 100, 10, and 1 µM, and compared to a control of the exact same mixture
without compound within the same plate. Each well contained a volume of 2000
evaluation, the composition consisted of 1880 L broth (with 1% TWEEN80), 20
stock of 10, 1, and 0.1 mM concentration (resulting in assay concentrations of 100, 10, and 1
µ
L. For the compound
L compound DMSO
M with 1%
108 CFU/mL bacteria in broth. Plates were incubated for 16 h
µ
µ
µ
DMSO, respectively), and 100
µL of 6
×
◦
at 37 C and processed as previously described [20]. Briefly, all the liquid was disposed. The remaining
biofilm attached to the surface was washed once with distilled water and fixed with absolute methanol.
The dried biofilm was stained with 2.5 mL per well of 0.4% (w/v) crystal violet solution for 15 min at
room temperature. After eliminating excess staining solution from the wells, the attached dye was
dissolved from the biofilm with 2.5 mL 33% glacial acetic acid solution. The absorbance of the obtained
solution, equivalent to the biofilm quantity, was detected in an Epoch BioTek (Winooski, VT, USA)
reader at 570 nm and visualized with the Gen5 software (Bio Tek, Winooski, VT, USA). Experiments
were performed with n = 6, and statistical significance was evaluated with the Student’s t-test in
Microsoft Office (Redmond, WA, USA).
3.2.4. Culture Conditions Chromobacterium violaceum CV026
Luria Bertani (LB) broth was prepared in 1 L of distilled water by adding 10 g peptone, 5 g yeast
extract, and 5 g NaCl, then sterilized in an autoclave at 15 psi and 121 ◦C for 15 min. For the LB solid
medium, 15 g of bacteriological agar were added to 1 L of distilled water. C. violaceum CV026 was
always grown in the presence of 30 µg/mL kanamycin. From the cryovials containing C. violaceum
CV026, a roast was taken◦and crosswise streaked in a box with LB agar and 30
µg/mL kanamycin,
which was incubated at 29 C for 24 h. A roast was then taken from an isolated colony and inoculated in
◦
5 mL LB medium with 30
µg/mL kanamycin for CV026, which was incubated at 29 C and 200 rpm for
15 h. Finally, the boxes were stored in refrigeration. For each experiment, a cryo-stock was unthawed
and cultured in 60 mL LB medium with 30
to 600 nm.
µg/mL kanamycin until reaching an optical density of 0.1
3.2.5. Evaluation of Compounds as Quorum Sensing Inhibitors in Chromobacterium violaceum CV026
C. violaceum CV026 was cultured in 60 mL LB medium with 30 g/mL kanamycin until reaching
an optical density of 0.1 to 600 nm. Subsequently, in 2 mL tubes, 980 L of this culture, 80
of C6-AHL (800 nM final concentration), and 10 L of the dilutions of the test compounds were
added until reaching the final concentrations of 1 mM, 100 M, 10 M, 1 M, 100 nM, 10 nM, 1 nM,
µ
µ
µM
µ
µ
µ
µ
and 100 pM. Then, the tubes were incubated at 29 ◦C and 700 rpm for 24 h. Upon completion of the
incubation, cell density was determined by absorbance at 720 nm by using LB medium as the blank.
Finally, the absorbance of violacein was measured. Next, 500
µL of the bacterial culture were placed in
a 2 mL tube and 500 L of acetone were added. The tubes were vortexed and centrifuged at 15,000 rpm
µ
for 4 min, followed by determination of the absorbance of violacein in the supernatants at 577 nm.
The readings at 577 nm and 720 nm were employed to calculate the specific production of violacein
(dividing the value at 577 nm by that at 720 nm). Experiments were performed in duplicates.
3.2.6. Statistical Analysis
The data of biofilm formation influenced by compounds 9, 10, 11, 12, and 14 in Aeromonas caviae
Sch3 are presented as percentages of the biofilm formation of the control, which received no inhibitor
treatment and was considered as 100%. Significance was confirmed by the Student’s t-test in Microsoft

Molecules 2018, 23, 3075
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Office with an accuracy of * p < 0.005 and # p < 0.05 at the marked bars. Experiments were performed
with n = 6. Figure 1.
The data of production of violacein by Chromobacterium violaceum CV026 under the influence
of the added compounds 9, 10, 11, 12, and 14 were normalized on the violacein production
without compound addition and this was considered as 100%. Experiments were performed as
duplicates (Figure 2).
Experiments were performed with n = 6, and statistical significance was evaluated with the
Student’s t-test in Microsoft Office.
4. Conclusions
Given the urgent need for new therapies to overcome the current bacterial antibiotic resistance
problem, our study contributes to the research effort by providing a compound set and a valuable
scaffold new for anti-QS in Aeromonas caviae. The introduction of alkyl-quinoxalin-2(1H)-one was
shown to be successful in the reduction of biofilm formation. Within the hetero cyclic-integrated
N-acyl structural solution, increased N-acyl chain length showed inhibition according to the
current literature [7,9,21]. The substitution of the lactone moiety due to an aromatic feature
has the potential for better metabolic stability due to the resistance against lactamases. The QS
inhibition of these compounds was also confirmed in Chromobacterium violaceum by reduced violacein
production. Based on our compound set, quinoxalin-2(1H)-one as anti-QS compounds could be further
developed in the search for novel anti-infection treatments, whose importance is rapidly increasing,
as anti-bacterial approaches keep losing effectiveness due to growing bacterial resistance. Future steps
in the drug developing process would include the synthesis of more quinoxalin-2(1H)-one analogs
to intensify the structure activity relationship study in Aeromonas caviae. The goal of this future work
would be to improve the compound properties in terms of activity, water solubility, toxicity, selectivity,
and bioavailability.
Supplementary Materials: 1H NMR-, 13C NMR-, HSQC-, IR-, HRMS-spectra; control measurement for DMSO
effect on biofilm formation.
Author Contributions: Conceptualization, R.B., A.R.-A.; Methodology, R.B., A.R.-A.; Software, R.B.; Validation,
R.B., A.R.-A., G.C.-E., E.C.-Q, A.R.R; Formal Analysis, R.B. A.R.-A; Investigation, R.B, A.R.-A. E.C-Q;
Resources, A.R.-A., G.C.-E. E.C.-Q.; Data Curation, R.B., A.R.-A.; Writing-Original Draft Preparation, R.B.;
Writing-Review & Editing, A.R.-A., G.C.-E., A.R.R.; Visualization, R.B., A.R.-A.; Supervision, R.B., A.R.-A; Project
Administration, A.R.-A.; Funding Acquisition, A.R.-A.
Funding: This research was funded by CONACYT grant number 240808 and by IPN grant SIP20181765 (A.R.-A.)
and grant SIP20180060 (G.C.-E.).
Acknowledgments: R.B. thanks Conacyt for postdoctoral position granted. We are grateful to CNMN (IPN) for
the HRMS and to C.E. of the ENCB (IPN) for the IR. We thank the collective group support by the scientist in the
Reyes-Arellano laboratory as well as the Castro-Escarpulli laboratory. The constructive atmosphere should be
acknowledged here. We thank Dr. Rogelio Jiménez Juárez for giving us 3,4-diamino benzoic acid. We thank Sofia
Mulia for kindly correcting the style of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 9, 10, 11, 12, 13 and 14 are available from the corresponding
author Dr. Alicia Reyes-Arellano.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).