Antagonism of quorum sensing phenotypes by analogs of the marine bacterial secondary metabolite 3-methyl-N-(20-phenylethyl)-butyramide

Article abstract of DOI:10.3390/md17070389

Quorum sensing (QS) antagonists have been proposed as novel therapeutic agents to combat bacterial infections. We previously reported that the secondary metabolite 3-methyl-N-(2⁰-phenylethyl)-butyramide, produced by a marine bacterium identifie

Full text of DOI:10.3390/md17070389

marine drugs
Communication
Antagonism of Quorum Sensing Phenotypes
by Analogs of the Marine Bacterial Secondary
Metabolite 3-Methyl-N-(20-Phenylethyl)-Butyramide
Susan M. Meschwitz 1,*, Margaret E. Teasdale 2, Ann Mozzer 1, Nicole Martin 1, Jiayuan Liu 2,
Stephanie Forschner-Dancause 2 and David C. Rowley 2,
*
1
Department of Chemistry, Salve Regina University, Newport, RI 02840, USA
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy,
University of Rhode Island, Kingston, RI 02881, USA
2
*
Correspondence: susan.meschwitz@salve.edu (S.M.M.); drowley@uri.edu (D.C.R.);
Tel.: +1-401-341-3125 (S.M.M.); +1-401-874-9228 (D.C.R.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 7 June 2019; Accepted: 27 June 2019; Published: 1 July 2019
Abstract: Quorum sensing (QS) antagonists have been proposed as novel therapeutic agents
to combat bacterial infections. We previously reported that the secondary metabolite
3-methyl-N-(20-phenylethyl)-butyramide, produced by a marine bacterium identified as Halobacillus
salinus, inhibits QS controlled phenotypes in multiple Gram-negative reporter strains. Here we
report that N-phenethyl hexanamide, a structurally-related compound produced by the marine
bacterium Vibrio neptunius, similarly demonstrates QS inhibitory properties. To more fully explore
structure–activity relationships within this new class of QS inhibitors, a panel of twenty analogs
was synthesized and biologically evaluated. Several compounds were identified with increased
attenuation of QS-regulated phenotypes, most notably N-(4-fluorophenyl)-3-phenylpropanamide
against the marine pathogen Vibrio harveyi (IC50 = 1.1 µM). These findings support the opportunity to
further develop substituted phenethylamides as QS inhibitors.
Keywords: quorum sensing; Vibrio; phenethylamide
1. Introduction
Bacterial populations synchronize gene expression via the release, detection, and biochemical
response to small signaling molecules called autoinducers [
this chemical communication process, also referred to as quorum sensing (QS), coordinates phenotypes
such as production of virulence factors, biofilms, and swarming motility [ ]. Hence, interference
with QS pathways provides an opportunity to attenuate pathogenicity, thereby representing a novel
mechanism for battling bacterial infections [ 11]. Furthermore, several studies have demonstrated
1,2]. In the case of pathogenic bacteria,
3–7
8
–
increased susceptibility of pathogenic bacteria to antibiotics when used in combination with QS
inhibitors [12,13].
Natural product investigations have yielded structurally distinct quorum sensing inhibitors
(QSIs), such as the brominated furanones isolated from the marine red alga Delisea pulchra [14
,15]
and the -lactone, plakofuranolactone, isolated from the marine sponge Plakortis cf. lita [16]. Marine
γ
organisms, including plants, animals, and microorganisms, have proven to be a particularly rich
source of QSIs with diverse structures [17]. Moreover, synthetic modifications to many of these
naturally-occurring scaffolds have led to higher affinity antagonists against bacteria pathogens [18].
For example, the marine honaucins, isolated from the bloom-forming cyanobacterium Leptolyngbya
crossbyana, were found to inhibit QS signaling-dependent phenotypes in V. harveyi and an engineered
Mar. Drugs 2019, 17, 389; doi:10.3390/md17070389
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Escherichia coli strain, and synthetic modifications resulted in improved QS inhibition as compared to
the natural products [19].
Gram-negative bacteria commonly use N-acyl homoserine lactones (AHL) as signals, which bind
their cognate receptor proteins to activate gene expression. These autoinducers share a conserved
L-homoserine lactone moiety, while the length and sites of oxidation on the acyl chain dictate the
species-specificity [20]. Antagonist activity can arise from simple structural variations to the native
autoinducer [21
–
23]. For example, replacement of the lactone with a thiolactone in the native
25]. Modifying the
autoinducer can provide both improved potency and hydrolytic stability [24
,
length of the acyl chain can also impart antagonist activity, as demonstrated in the case of the plant
pathogen Agrobacterium tumefaciens [26]. Previous studies also demonstrate that incorporation of aryl
functionality with electron withdrawing groups onto the acyl side chain renders many AHL mimics
as potent QSIs [27–29]. For example, termination of the acyl chain of the Pseudomonas aeruginosa
autoinducer butanoyl-homoserine lactone with 4-bromophenyl interrupts AHL-mediated biofilm
formation [30]. Hence, synthetic modifications to the natural substrates have proven to be useful in
creating QSIs.
We previously reported that phenethylamide secondary metabolites (1 and 2, Figure 1),
produced by marine Halobacillus salinus strain C42 obtained from the surface of a seagrass
sample, inhibit QS regulated phenotypes in three Gram-negative reporter strains. Specifically,
3-methyl-N-(20-phenylethyl)-butyramide (
1) inhibits bioluminescence by the marine pathogen
V. harveyi, violacein production by Chromobacterium violaceum, and green fluorescent protein
(GFP) production by the QS sensor strain Escherichia coli JB525 [31]. The close congener
2-methyl-N-(20-phenethyl)-propionamide (
2) demonstrated reduced potencies against these three
reporter strains. Here we report the identification of N-phenethyl hexanamide ( ), produced by a Vibrio
3
neptunius strain, as a closely related QSI. The variable potencies of these QSIs encouraged the synthesis
of twenty analogs to help define structure–activity relationships (SAR), resulting in the identification
of more potent compounds against these reporter strains.
Figure 1. Chemical structures of phenethylamide natural products.
2. Results
2.1. N-Phenethyl Hexanamide from Vibrio neptunius RIP07-147
Using our previously described cell–cell co-cultivation assay to identify marine bacteria with
QSI potential against V. harveyi BB120 [17], we found that strain RIP07-147 (GenBank accession
number MK821060), identified as a V. neptunius by 16S rRNA sequence comparison, demonstrated
both antibiotic and bioluminescence inhibition activities. We were unaware of any previous
natural product investigations of this species, and therefore undertook further study of this strain.
RIP07-147 was cultivated on marine agar trays at 24 ◦C for 48 h. Following extraction of the whole
cultures with ethyl acetate, bioassay-guided fractionation was pursued on the resulting extract using
repeated reversed-phase chromatography, and bioactivity was followed by monitoring QS-controlled
bioluminescence in the sensor strain V. harveyi BB120 as previously described [31]. These studies
revealed that the hybrid PKS-NRPS secondary metabolite andrimid [32] was responsible for antibiotic
activity, while QSI activity was due to N-phenethyl hexanamide (3) [33]. The structures were confirmed
by comparison of 1H NMR and MS data with literature values, and later by synthesis in the case of
(see below).
3

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2.2. Bioassay Testing
Compounds were tested in triplicate against three established QSI sensor strains [31].
V. harveyi causes disease in a variety of marine animals, especially shrimp [34], and has
been previously used in the discovery of QSIs [35–37]. V. harveyi BB120 responds to the
autoinducers 3-hydroxybutanoyl-L-homoserine lactone (HBHL) AI-1, the furanosyl borate diester AI-2,
and (S)-3-hydroxytridecan-4-one (CAI-1) to regulate a variety of bacteria behaviors [38]. C. violaceum is
a Gram-negative bacterium that produces violacein, an antibiotic purple pigment, under QS control
using the autoinducer N-hexanoyl-L-homoserine lactone (HHL) [39]. Finally, the panel was screened
for QSI activity using E. coli JB525, a mutant E. coli harboring the gfp plasmid pJBA132 linked to
the LuxI/R quorum sensing system of Vibrio fischeri. This sensor strain produces an unstable green
fluorescent protein (GFP) in response to exogenous C6-C8 AHL autoinducers [40]. Broth dilution
assays with E. coli JB525 were conducted in the presence of 32 nM HHL, as we found this autoinducer
provided the most consistent results and was used as a positive control in a similar E. coli reporter
system [41].
Phenethylamide
3 inhibited V. harveyi bioluminescence (IC50 = 99 µM) and violacein production
by C. violaceum (ZOI = 14 mm), but lacked activity against E. coli JB525, demonstrating that modest
changes in the alkyl chain impacts the anti-QS activity (Table 1).
Table 1. Activity of natural products and their analogs against three reporter strains.
V. harveyi
BB120
E. coli
JB525
C. violaceum
ATCC 12472
Compound
IC50 (µM) Std. Dev.
IC50 (µg/mL)
Std. Dev.
Zone of Inhibition a (mm)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
110
NA
99
89
NA
17
94
29
6.2
15
48
3.3
5.6
86
3.5
1.1
3.0
6.0
12
12
11
NA
NA
NA
NA
NA
NA
NA
5.2
NA
1.1
>200
32
3.5
20
12
14
NA
21
NA
NA
NA
11
5.9
13
2.9
7.0
3.0
0.40
1.0
0.36
12
NA
22
12
7.6
1.9
3.6
2.7
1.6
0.60
0.37
2.0
9
>200
3.8
25
NA
NA
9
1.0
13
14
69
NA
NA
NA
NA
NA
NA
>150
NA
NA
56
8.1
NA
>200
19
13 b
11
1.2
17
2.2
4.0
82
13
a
IC50 values greater than 500 were considered inactive and are designated as NA (no activity). Zones of inhibition
determined for 500
b
µ
g/disc. Includes 8 mm zone of growth inhibition followed by zone of no violacein production.
2.3. Analog Design and Biological Results
The structural simplicity of the phenethylamide QSIs, along with their variable activities
against three different sensor strains, encouraged the preparation of synthetic analogs to explore
structure–activity relationships. Specifically, a panel of analogs was designed to explore the effects of

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substitutions on the phenyl rings (X and Y), distance of the amide bond from the phenyl group (n),
and chain length (m) and modifications (R) to the acyl group (Figure 2).
Figure 2. Chemical structures of synthetic analogs. Panel
A compounds represent modifications to the
acyl chain (R). Panel compounds represent modifications to the chain length on either side of the of
B
the amide bond. Panel C compounds represent modifications to the phenyl rings.
The initial analogs were designed to investigate modifications to the acyl chain (R) and included
compounds
demonstrated that extending the length of the aliphatic chain (>C10) resulted in the creation of
antagonists [42]. In compound , extending R by four carbons (decanoyl) relative to abolished activity
against C. violaceum but was equipotent against V. harveyi. In compound , installation of a shortened
butanoyl chain had minimal effect on activity versus C. violaceum (ZOI = 21 mm) but abolished activity
against V. harveyi. Interestingly, all modifications to the 3-methylbutanoyl chain of had detrimental
4–6 (Figure 2A). Prior SAR studies of HHL, the native autoinducer of C. violaceum,
4
3
5
1
effects on the activity against E. coli JB525 (Table 1). These results demonstrate modifications to the acyl
chain length can be used to tune the QSI to a particular QS system.
Previous studies aimed at designing QSIs demonstrate the benefit of installing a terminal phenyl
ring on the AHL acyl side chain or as a replacement for the AHL lactone ring. For example,
4-phenylbutanoyl-homoserine lactone and 3-oxo-C12-2-aminophenol [23] were previously reported
as potent Lux-R type antagonists [29]. With this in mind, compound
6
was synthesized and found
. However,
to increase potency by nearly 6-fold against V. harveyi (IC50 = 17 M) in comparison to
µ
1
compound 6 lacked activity against either C. violaceum or E. coli JB525.
The diphenyl motif was further expanded (Figure 2B, compounds 7–11) by investigating
modifications to the chain length on either side of the amide bond (n and m). Increasing the m
linker by one carbon (m = 3) proved detrimental to the potency against V. harveyi ( , IC50 = 94 M),
while increasing n to three ( , IC50 = 29 M) had a modest negative impact. Conversely, anilines
n = 0) resulted in much improved potency against V. harveyi. Compound was 16-fold more active in
V. harveyi versus the natural product , 2-fold more active against E. coli JB52, and retained activity
against C. violaceum (ZOI = 11 mm). Interestingly, compound 11, which replaces the phenylethyl group
of with the pentyl chain of natural product , demonstrated a 5-fold improved activity against E. coli
JB525 (IC50 = 1.1 µg/mL).
We next turned our attention to installing substituents on the phenyl rings of
We noted that several previous reports demonstrated the benefit of electron withdrawing groups in
a para position for improved QSI activity [21 22 28]. Initially, we investigated fluorine or a methoxy
group in the para positions to compare the effects of an electron withdrawing and electron donating
substituents (compounds 12 19, Figure 2C). Our previous work on aryl beta-keto esters as QSIs of V.
7
µ
8
µ
(
9
1
9
3
9
(Figure 2C).
,
,
–
harveyi BB120 showed the most active derivatives to be 4-fluoro and 4-methoxy phenyl substituted
analogs [35]. Here, the 4-fluoro derivative 16 resulted in nearly 6-fold improved inhibition against

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V. harveyi as compared to the non-substituted
9, while a methoxy substituent (12) improved potency
by less than 2-fold. Installing a fluorine (15) or methoxy (18) substituent in the para-position of
the opposite phenyl ring resulted in only a 2-fold improvement versus V. harveyi as compared to
Installing para-substituents on both phenyl rings had variable effects on potency (14 17 19), with the
9.
,
–
addition of a methoxy group to both phenyl rings (14) having a detrimental effect in all three assays,
suggesting either an electronic or steric limitation.
Replacement of the fluorine on the amide phenyl ring with an iodine atom (20), or inclusion of
a para-bromine atom on the acyl phenyl ring (21) had drastic effects, abrogating activity against all
three sensor strains, suggesting either a steric or electronic effect [43]. Replacing the methoxy with
a hydroxyl group on either phenyl ring (22 and 23) was also detrimental to the activity against V. harveyi
but improved activity against E. coli, suggesting a variable hydrogen bonding role for the hydroxyl
group in binding to certain Lux-R type receptors, such as the V. fischeri homolog in JB525.
Compound 11 was the most potent inhibitor of the LuxR construct E. coli JB525 (IC50 = 1.1 µg/mL).
To explore if 11 is a competitive antagonist of AHLs at the Lux R receptor, it was tested in serial
dilutions against rising concentrations of N-(3-oxohexanoyl)-L-homoserine lactone (OHHL, 16–512 nM).
We previously used this strategy to determine that
1 was a competitive antagonist of AHL mediated
QS in E. coli JB525 [31]. Increased OHHL surmounted the inhibitory effects of 11 (Figure 3), consistent
with an agonist-antagonist relationship, and suggesting a similar mechanism as
1 for inhibition of the
LuxR controlled GFP expressed in E. coli JB525 [31].
100.00
80.00
60.00
16 nM OHHL
32 nM OHHL
64 nM OHHL
128 nM OHHL
40.00
20.00
0.00
3.2
6.41
12.81
25.63
51.25
102.5
Concentration (μg/mL)
Figure 3. Bar graph showing green fluorescent protein (GFP) production (fluorescence) at various
concentrations of antagonist (11) and agonist (OHHL). The inhibitory activity of 11 is surmounted
by higher concentrations of OHHL agonist, consistent with an antagonist-agonist relationship.
The concentration of 11 is in
µg/mL and OHHL in nM. There were significant effects of compound 11
concentrations, OHHL concentrations and interactions (two-way ANOVA F23,71 = 342.3098, p < 0.0001
,
Supplementary Material Table S1). Standard deviation error bars are included in a 2D version of the
graph (Supplementary Material Figure S5).
Growth curves were conducted on all of the diphenyl analogs of compound
9 (compounds
12
–
19, Figure 2C) and N-phenylhexanamide (11) at 100 M, which is well above the IC50 value of
µ
the compounds, to ensure that the observed inhibition of luminescence was not due to inhibition or
delay in growth of V. harveyi by the analogs. None of the compounds demonstrated a delay in growth
(Supplementary Material Figures S1–S3). Additionally, a luminescence curve for compound 16—the

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most active compound—demonstrated that the luminescence inhibition persists for the duration of
luminescence production by the untreated bacteria (Supplementary Material Figure S4).
3. Discussion
N-phenethyl hexanamide (3) was isolated from a marine V. neptunius strain and identified as a QSI
against the pathogen V. harveyi. To date, this is the first secondary metabolite reported for this bacteria
species. N-phenethyl hexanamide adds to a small group of previously described phenethylamide QSIs
produced by marine bacteria strains belonging to the genera Halobacillus [31,33] and Oceanobacillus [44],
and bears structural resemblance to cyclic dipeptide QSIs comprising phenylalanine [45]. These natural
products provided the motivation for synthesizing a panel of derivatives to more fully investigate
this QSI class. The simple structures facilitated rapid assembly, frequently in one synthetic step with
crystallization to prepare the desired pure product, further encouraging exploration of structure–activity
relationships for these cell–cell signaling antagonists.
Many analogs were more active than the natural product
compounds having IC50 values in the low micromolar range. In particular, diphenyl analogs appear
to be the most promising QSIs against V. harveyi, and four of these ( 13 16, and 23) demonstrated
1 in the V. harveyi assay, with several
9
,
,
activity against all three reporter strains. While a general QSI might be desirable for treating infections
caused by pathogens that use AHL-mediated signaling, it appears that the phenethylamide scaffold is
more easily modified to optimize activity against specific QS systems.
Substitution of phenethylamine with aniline yielded N-phenylhexanamide (11), which was the
only analog demonstrating equal or more potent activity than
1 against all three reporter strains.
Other groups have identified non-natural modulators of AHL-based QS in which the native homoserine
lactone moiety has been replaced with a phenyl ring, which suggests that the lactone moiety is not
necessary for receptor binding. For example, Smith et al. demonstrated that 2-(3-oxo-C12-amino)phenol
inhibits GFP production in a Pseudomonas aeruginosa strain constructed to express GFP in its LasR QS
circuit [23].
Taken together, our results suggest that further exploration of the diphenyl analogs of the
marine phenylethyl amide secondary metabolites (1–3) may yield more potent QSIs and highlights
the need for increased investigation of marine microbes for the discovery and development of new
anti-QS compounds.
4. Materials and Methods
4.1. Media
Bioassay media consisted of the following: (1) Marine broth (MB) containing 1 g yeast extract and
5 g peptone (Alpha Biosciences, Baltimore, MD, USA) per L synthetic seawater (Instant Ocean; 36 g per
L); (2) Luria–Bertani broth containing 4 g sodium chloride (LB4); and (3) nutrient broth (NB) containing
5 g yeast extract and 10 g tryptone per L DI H2O. For agar media, 15 g agar per L of H2O was used.
4.2. Reporter Strains
◦
Vibrio harveyi BB120 [
3
] a wild-type, bioluminescent strain, was cultivated at 30 C in MB.
◦
C. violaceum ATCC 12472 was cultured at 29 C with shaking in NB. E. coli JB525 is E. coli MT102
harboring the gfp plasmid pJBA132 and produces an unstable green GFP in response to C6-C8 AHL
autoinducers [40]. E. coli JB525 was cultured in LB4 at 30 ◦C.
4.3. Isolation and Sequencing RIP07-147
The bacterial strain RIP07-147 (GenBank accession number MK821060) was isolated from
a suspended marine particle collected in August 2007 in the East Passage of Narragansett Bay,
Rhode Island, USA. The isolate was grown overnight at 24 ◦C in YP media and DNA was extracted
using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) per the manufacturer’s protocol.

Mar. Drugs 2019, 17, 389
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PCR amplification of the bacterial 16S rRNA gene was accomplished using universal bacterial primers
27F and 1392R [46]. The isolate was identified based on 97.01% homology to V. neptunius strain LMG
20536 (22 February 2019) [47,48].
4.4. Co-Cultivation with V. harveyi BB120
An overnight culture of V. harveyi BB120 was diluted into 5 mL of molten MB soft agar at 40 ◦C and
poured atop a MB agar plate. Two µL of an overnight culture of the test isolate, RIP07-147, was spotted
onto the V. harveyi lawn. The plate was incubated at 24 ◦C overnight and imaged with a Typhoon 9410
variable mode imager (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) in chemiluminescence mode.
Zone of no light bioluminescence was measured to the nearest mm.
4.5. Isolation of N-Phenethyl Hexanamide
RIP07-147 was inoculated on yeas◦t and peptone (YP) agar media in three 16
×
30 cm aluminum
pans. The pans were incubated at 24 C for 48 h. The agar was then extracted with ethyl acetate,
filtered, and the liquid portion concentrated in vacuo. The extract was adsorbed onto C18 resin
and fractionated by vacuum liquid chromatography with step-wise gradients of 100% water to 100%
methanol (20% methanol increments) with a final acetone wash. Using the V. harveyi BB120 assay
(see below), the active constituent was determined to be in the 60% and 80% methanol fractions.
These fractions were combined and further purified by HPLC (Waters Xterra RP18 19
80% methanol over 30 min at 5 mL/min) to yield 3 as the single active compound. Compound 3 was
identified as N-phenethyl hexanamide by comparison of 1H NMR spectroscopy and mass spectrometry
×
100 mm, 20 to
data in comparison with literature data [33].
4.6. Bioassays
4.6.1. V. harveyi BB120 Broth Dilution Assay
An overnight culture of V. harveyi BB120 in MB was diluted (OD600 = 0.1), and 200
µL of the
diluted culture was added to 10 mL of MB. One L of test compounds dissolved in DMSO at 50 mg/mL
µ
was added to a 96-well clear bottom, white microtiter plate (Corning, 0.5% DMSO final concentration).
The diluted cell culture was added to the wells of the opaque microtiter plate and incubated at 30 ◦C
with shaking for 5 h. The plates were read on a SpectraMax Multimode Microplate Reader (Molecular
Devices, Sunnyvale, CA, USA). Relative luminescence units (RLU) were normalized by the OD600
values. Percent luminescence was calculated by defining the untreated cells (no compound) as 100%.
4.6.2. C. violaceum Disc Diffusion Assay
Disc diffusion assays were performed with pure compounds at 500
µg/disc. One hundred
microliters of overnight bacterial culture were added to 10 mL of NB, vortexed, and then 100
µL of the
diluted culture was spread atop an NB agar plate. Impregnated, sterile discs (6 mm) were laid onto
the test plates and incubated overnight. Zones of inhibition (ZOI), as indicated by lack of pigment
production, were measured to the nearest mm.
4.6.3. E. coli JB525 Bioassay
Inhibition of fluorescence was determined using a method modified from Teasdale et al. [31].
Briefly, an overnight culture of E. coli JB525 in LB4 broth was diluted (OD450 0.25) with fresh media.
Cultures were treated with 32 nM HHL and test compound ranging from 2 to 250
final DMSO concentration in 200 L) in a 96-well clear bottom, black microtiter plate. To determine
antagonist–agonist relationships, each serial dilution of test compound (8–1000 M) was challenged
µg/mL (0.5%
µ
µ
with each increasing OHHL concentration (16–512 nM) in three biologically separate replicates (0.7%
DMSO final concentration). Plates were incubated with shaking at 30 ◦C for 3 h. Fluorescence was
detected with an excitation at 475 nm and emission at 515 nm on the SpectraMax i3 multi-mode

Mar. Drugs 2019, 17, 389
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microplate reader (Molecular Devices, Sunnyvale, CA, USA). Growth was evaluated after 3 h by optical
density at 450 nm. Fluorescence values were normalized by optical density. For IC50 determination
at 32 nM HHL, percent fluorescence was determined by defining control wells with 32 nM HHL as
100% fluorescence.
4.6.4. Statistical Analysis
Assays were performed in biological and technical triplicate. Data were analyzed using GraphPad
Prism 7. IC50 values were calculated using non-linear regression analysis and the values of each trial
were averaged for the final reported value.
4.7. Chemical Syntheses
1H NMR spectra were recorded on a Bruker Avance (300 MHz) or a Bruker Biospin (400 MHz)
spectrometer and mass spectra were recorded on a SCIEX QTOF 4600 using flow injection in 75%
aqueous CH3OH containing 0.1% HCOOH. All reagents and compounds were purchased from
Sigma-Aldrich or Acros Chemicals. Purification of the desired products was accomplished by either
recrystallization (ethyl acetate and hexane), automated column chromatography on silica (CombiFlash,
Teledyne Isco, Lincoln, NE, USA) using a linear gradient of hexanes in ethyl acetate (0%–100%), or by
reverse-phase HPLC (Waters X-Terra Prep RP18 column, 19
formic acid, 5 mL/min). HPLC was performed on a Waters 600 with a 2487 dual wavelength detector
set to λ 220 nm and λ 254 nm. Compounds were synthesized as follows.
×
100 mm, gradient of MeOH in H2O (0.1%
4.7.1. General Procedure for Coupling Reactions
The appropriate carboxylic acid in 50 mL acetonitrile was treated with HBTU
(N,N,N0,N0-Tetramethyl-O-(1H-benzotriazol-1-yl)uranium
hexafluorophosphate)
(1.2
eq),
diisopropylethyamine (1.5 eq), and the requisite amine. The reaction was stirred overnight
at ambient temperature, concentrated in vacuo, and then partitioned between ethyl acetate and
0.1 M HCl. The organic phase was separated, sequentially washed with saturated sodium bicarbonate
and water, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The resulting
products were purified by either crystallization or chromatography as described. Percent yields ranged
from 30%–89%.
4.7.2. Synthesis and Characterization of Compounds 1–23
3-Methyl-N-(20-phenylethyl)-butyramide (
purified by crystallization (white crystals). ESI-MS [M + H]+ = 206.15; 1H NMR (400 MHz, CDCl3):
0.92 (d, J = 8.0 Hz, 6H), 1.98 (d, J = 8.0 Hz, 2H), 2.07 (m, 1H), 2.82 (t, J = 6.8 Hz, 2H), 3.53 (m, 2H),
5.46 (s, 1H) 7.15–7.35 (m, 5H).
1). Iso-valeric acid and phenethylamine. Desired product was
δ
N-Phenethylhexanamide (3). Hexanoic acid (4 mmol, 1 eq) and phenethylamine (4 mmol, 1 eq). Desired
product was purified by HPLC (50%–75% MeOH in H2O over 10 min, white solid). ESI-MS [M + H]+
1
= 220.17; H NMR (400 MHz, CDCl3):
δ 0.88 (t, J = 7.0 Hz, 3H), 1.29 (m, 4H), 1.59 (m, 2H), 2.09 (t,
J = 7.5 Hz, 2H), 2.82 (t, J = 7.0 HZ, 2H), 3.52 (m, 2H), 5.44 (s, 1H), 7.19 (m, 5H).
N-Phenethyldecanamide ( ). Decanoic acid (5 mmol, 1 eq) and phenethylamine (5 mmol, 1 eq). Desired
product was purified by crystallization (white crystals). ESI-MS [M + H]+ = 276.12; 1H NMR (400 MHz,
CDCl3): 0.88 (t, J = 8 Hz, 3H), 1.26 (s, 12H), 1.58 (t, J = 8 Hz, 2H), 2.11 (t, J = 8 Hz, 2H), 2.81 (t, J = 8 Hz
2H), 3.51 (dt, J = 8 Hz, 2H), 5.61 (s, 1H), 7.18–7.32 (m, 5H).
N-Phenethylbutryamide ( ). Butyric acid (4 mmol, 1 eq) and phenethylamine (4 mmol, 1 eq). Desired
product was purified by HPLC (50% MeOH in H2O to 75% MeOH over 10 min, white solid). ESI-MS
[M + H]+ = 192.06; 1H NMR (400 MHz, CDCl3):
0.91 (t, J = 7.5 Hz, 3H), 1.61 (m, 2H), 2.10 (t, J = 7.5 Hz
2H), 2.81 (t, J = 7.5 Hz, 2H), 3.51 (m, 2H), 5.54 (s, 1H), 7.18–7.33 (m, 5H).
4
δ
,
5
δ
,

Mar. Drugs 2019, 17, 389
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N-Phenethyl-3-phenylpropanamide (
6
). Hydrocinnamic acid (4 mmol, 1 eq) and phenethylamine (4 mmol,
1 eq). Desired product was purified by crystallization (white crystals). ESI-MS [M + H]+ = 254.17;
1H NMR (400 MHz, CDCl3):
3.48 (m, 2H), 5.42 (s, 1H), 7.04–7.31 (m, 10H).
δ 2.43 (t, J = 6.8 Hz, 2H), 2.74 (t, J = 6.8 Hz, 2H), 2.95 (t, J = 6.8 Hz 2H),
N-Phenethyl-4-phenylbutanamide ( ). 4-phenyl-butyric acid (5 mmol, 1 eq) and phenethylamine (5 mmol,
1 eq). Desired product was purified by crystallization (white crystals). ESI-MS [M + H]+ = 268.18;
1H NMR (400 MHz, CDCl3):
1.95 (m, 2H), 2.13 (t, J = 7.0 Hz, 2H), 2.63 (t, J = 8.0 Hz, 2H), 2.82 (t,
J = 8.0 Hz, 2H), 5.46 (s, 1H), 7.14–7.33 (m, 10H).
3-Phenyl-N-(3-phenylpropyl) propanamide ( ).
7
δ
8
Hydrocinnamic acid (4 mmol, 1 eq) and
3-phenyl-1-propylamine (4 mmol, 1 eq). Desired product was purified by HPLC (50% MeOH
1
in H2O to 75% MeOH over 10 min, white solid). ESI-MS [M+Na]+ = 290.03; H NMR (400 MHz,
CDCl3):
δ 1.77 (m, 2H), 2.43 (t, J = 8Hz, 2H), 2.57 (t, J = 8Hz, 2H), 2.95 (t, J = 8Hz, 2H), 3.25 (t, J = 8Hz,
2H), 5.32 (s, 1H), 7.13–7.30 (m, 10H).
N,3-Diphenylpropanamide (9). Hydrocinnamic acid (4 mmol, 1 eq) and aniline (4 mmol, 1 eq). Desired
product was purified by crystallization (white crystals). ESI-MS [M + H]+ = 226.04; 1H NMR (400 MHz,
CDCl3): δ 2.66 (t, J = 8 Hz, 2H), 3.05 (t, J = 8 Hz, 2H), 7.10 (t, J = 8 Hz, 1H), 7.22–7.45 (m, 10H).
N,4-Diphenylbutanamide (10). 4-phenyl-butyric acid (4 mmol, 1 eq) and aniline (4 mmol, 1 eq). Desired
product was purified by crystallization (white crystals). ESI-MS [M + H]+ = 240.04; 1H NMR (400 MHz,
CDCl3):
δ 2.08 (m, 2H), 2.35 (t, J = 8 Hz, 2H), 2.72 (t, J = 8 Hz, 2H), 7.11 (t, J = 8Hz, 1H), 7.21–7.34 (m,
8H), 7.51 (d, J = 8 Hz, 2H).
N-Phenylhexanamide (11). Hexanoic acid (4 mmol, 1 eq) and aniline (4 mmol, 1 eq). Desired product
was purified by crystallization (white crystals, 89%). ESI-MS [M + H]+ = 192.14; 1H NMR (400 MHz,
CDCl3):
δ 0.91 (t, J = 6.5 Hz, 3H), 1.35 (m, 4H), 1.73 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H), 7.26 (s, 1H), 7.10 (t,
J = 7.3 Hz, 1H), 7.31 (t, J = 8.0 Hz, 2H), 7.52 (d, J = 7.8 Hz, 2H).
N-4-Methoxyphenyl-3-phenylpropanamide (12). Hydrocinnamic acid and p-anisidine. Desired product
1
was purified by crystallization (pale purple crystals, 30%). ESI-MS [M + H]+ = 256.14; H NMR
(300 MHz, CDCl3):
δ 2.61 (t, J = 7.6 Hz, 2H), 3.75 (s, 3H), 3.02 (t, J = 7.7 Hz, 2H), 6.8 (d, J = 9Hz, 2H),
7.19–7.32 (m, 7H), 7.33 (bs, 1H).
N-Phenyl-3-(4-methoxyphenyl)-propanamide (13). 3-(4-methoxyphenyl) propanoic acid and aniline.
Desired product was purified by crystallization (white crystals, 31%). ESI-MS [M+Na]+= 278.12;
1H NMR (300 MHz, CDCl3):
δ 2.59 (t, J = 7.6 Hz, 2H), 2.95 (t, J = 7.6 Hz, 2H), 3.76 (s, 3H), 6.80 (d,
J = 8.6 Hz, 2H), 7.13 (m, 3H), 7.26 (t, J = 7.0 Hz, 2H), 7.44 (d, J = 7.7 Hz, 3H).
N-4-Methoxyphenyl-3-(4-methoxyphenyl)-propanamide (14). 3-(4-methoxyphenyl) propanoic acid and
p-anisidine. Desired product was purified by crystallization (white crystals, 78%). ESI-MS [M+Na]+
= 308.13; 1H NMR (300 MHz, CDCl3):
δ 2.58 (t, J = 7.7 Hz, 2H), 2.95 (t, J = 7.6 Hz, 2H), 3.77 (s, 6H),
6.82 (d, J = 8.6Hz, 4H), 7.14 (d, J = 8.4 Hz, 2H), 7.34 (bs, 1H), 7.37 (d, J = 8.8 Hz, 2H).
N-Phenyl-3-(4-fluorophenyl) propanamide (15). 3-(4-fluorophenyl) propanoic acid and Aniline. Desired
product was purified by crystallization (white crystals, 51%). ESI-MS [M+Na]+i = 266.09; 1H NMR
(300 MHz, CDCl3):
δ 2.61 (t, J = 7.5 Hz, 2H), 3.00 (t, J = 7.5 Hz, 2H), 6.96 (t, J = 8.7 Hz, 2H), 7.13 (m, 2H),
7.28 (t, J = 7.9 Hz, 2H), 7.27 (d, J = 8.01 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H).
N-(4-Fluorophenyl)-3-phenylpropanamide (16). 4-fluoroaniline and hydrocinnamic acid. Desired products
were purified by crystallization (white crystals, 42%). ESI-MS [M+Na]+ = 266.09; 1H NMR (300 MHz,
CDCl3):
δ 2.62 (t, J = 7.6 Hz, 2H), 3.01 (t, J = 7.6 Hz, 2H), 6.94 (t, J = 8.7 Hz, 2H), 7.17–7.37 (m, 7H),
7.43 (bs, 1H).

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N-4-Fluorophenyl-3-(4-fluorophenyl) propanamide (17).
3-(4-fluorophenyl) propanoic acid and
4-fluoroaniline. Desired product was purified by crystallization (white crystals, 28%). ESI-MS
1
[M+Na]+ = 284.09; H NMR (300 MHz, CD3OD)
δ 2.63 (t, J = 8.5 Hz, 2H), 2.95 (t, J = 7.6 Hz, 2H),
6.99 (m, 5H), 7.24 (dd, J = 8.6 Hz, 5.5 Hz, 2H), 7.49 (dd, J = 9.2 Hz, 4.9 Hz, 2H).
N-(4-Fluorophenyl)-3-(4-methoxyphenyl) propanamide (18). 3-(4-methoxyphenyl)propanoic acid and
4-fluoroaniline. Desired product was purified by crystallization (white crystals, 39%). ESI-MS [M+Na]+
= 296.11; 1H NMR (300 MHz, CD3OD):
δ 2.63 (t, J = 7.6 Hz, 2H), 2.92 (t, J = 7.7 Hz, 2H), 3.74 (s, 3H),
6.82 (d, J = 8.7 Hz, 1H), 7.04 (t, J = 8.9Hz, 2H), 7.17] (d, J = 8.9 Hz, 1H), 7.65 (d, J = 9.2 Hz, 2H), 7.65 (d,
J = 9.2 Hz, 2H), 7.69 (bs, 1H).
N-(4-Methoxyphenyl)-3-(4-fluorophenyl) propanamide (19). 3-(4-fluorophenyl) propanoic acid and
p-anisidine. Desired product was purified by crystallization (pale purple crystals, 62%). ESI-MS
[M+Na]+ = 296.11; 1H NMR (300 MHz, CDCl3):
δ 2.63 (t, J = 7.6 Hz, 2H), 2.97 (t, J = 7.6 Hz, 2H), 3.75 (s,
3H), 6.84 (d, J = 9.0 Hz, 2H), 7.02 (t, J = 8.8 Hz, 2H), 7.28 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H),
7.53 (d, J = 9 Hz, 2H), 8.98 (bs, 1H).
N-(4-Iodophenyl)-3-phenylpropanamide (20). 4-Iodoaniline and hydrocinnamic acid. Desired product was
purified by crystallization (pale purple crystals, 40%). ESI-MS [M+Na]+ = 374.00; 1H NMR (300 MHz,
CDCl3):
2H).
δ 2.65 (t, J = 7.5 Hz, 2H), 3.05 (t, J = 7.5 Hz, 2H),6.92 (bs, 1H), 7.26 (m, 7H), 7.59 (d, J = 8.8 Hz,
3-(4-Bromophenyl)-N-phenylpropanamide (21). 3-(4-bromophenyl) propionic acid (4 mmol, 1 eq) and
aniline (4 mmol, 1 eq). Desired product purified by crystallization. ESI-MS [M+Na]+ = 325.88; 1H NMR
(400 MHz, CDCl3):
δ 2.63 (t, J = 7.5 Hz, 2H), 3.03 (t, J = 7.5 Hz, 2H), 7.10 (m, 4H), 7.26 (d, J = 8 Hz, 1H),
7.32 (t, J = 7 Hz, 2H), 7.44 (m, 3H).
3-(4-Hydroxyphenyl)-N-phenylpropanamide (22). Compound 12 (1 mmol) was dissolved in 2 mL DMF,
treated with iodocyclohexane (10 mmol), and refluxed under nitrogen for 14 h. The reaction was
then cooled, poured into water (20 mL), and extracted with ethyl acetate (3
×
20 mL). The organic
layer was washed sequentially with saturated aq. NaHSO3 and brine, dried over Na2SO4, filtered,
and concentrated. The crude product was purified by column chromatography (white solid, 54%).
ESI-MS [M+Na]+ = 264.10; 1H NMR (300 MHz, CD3OD):
δ 2.61 (t, J = 7.0 Hz, 2H), 2.97 (t, J = 7.0 Hz,
2H), 8.9 (t, J = 7.7 Hz, 2H), 6.70 (d, J = 8.9 Hz, 2H), 7.25 (m, 5H), 7.25 (d, J = 9.0 Hz, 2H).
N-Phenyl-3-hydroxyphenylpropanamide (23). Prepared from 13 using identical method as for compound
22 and purified by column chromatography (white solid, 53%). ESI-MS [M + H]+ = 242.12; 1H NMR
(300 MHz, CDCl3): d 2.62 (t, J = 7.6 Hz, 2H), 2.89 (t, J = 7.7 Hz, 2H), 6.75 (t, J = 8.6 Hz, 2H), 7.06 (m,
1H), 7.08 (d, J = 8.6 Hz, 2H), 7.27 (t, J = 7.9 Hz, 2 H), 7.64 (d, J = 7.6 Hz, 2H), 9.12 (bs, 1H).
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/7/389/s1,
Figures S1–S3: 24-h growth curves of V. harveyi in the presence of phenethylamide analogues, Figure S4:
24-h luminescence curves by V. harveyi BB120 in the presence of phenethylamide analogues 14 (100 µM) and 16
(10 µM) at concentrations that are above their IC50 values. Figure S5: 2D version of Figure 3 with error bars
added. Bar graph showing GFP production (fluorescence) at various concentrations of antagonist (11) and agonist
(OHHL). Error bars reflect at least three experiments each done in triplicates. Table S1: One-way ANOVA for
effect of Compound 11 concentration.
Author Contributions: D.C.R., S.M.M., and M.E.T. conceived and designed the experiments. M.E.T, A.M., N.M.,
J.L., and S.F.D. performed the syntheses, bioassays, and chemical purifications. S.M.M., S.F.D., and D.C.R. edited
and approved the final version of the manuscript.
Funding: This research was funded in part by an Institutional Development Award (IDeA) Network for
Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes
of Health under grant number P20GM103430, NSF Grant MCB 04538743, and by NOAA Grant NA040AR4600193.
Some NMR measurements were acquired on instrumentation provided by National Science Foundation EPSCoR
Cooperative Agreement #EPS-1004057.

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Acknowledgments: We gratefully acknowledge Michael Givskov for his donation of bacterial strain E. coli JB525
and Jameson F. Chace for his assistance with data analysis and ANOVA calculations. Abstract image credit:
The National Wildlife Federation.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
References
1.
Fuqua, C.; Parsek, M.R.; Greenberg, E.P. Regulation of gene expression by cell-to-cell communication:
Acyl-homoserine lactone quorum sensing. Annu. Rev. Genet. 2001, 35, 439–468. [CrossRef] [PubMed]
Kleerebezem, M.; Quadri, L.E.; Kuipers, O.P.; de Vos, W.M. Quorum sensing by peptide pheromones and
two-component signal-transduction systems in Gram-positive bacteria. Mol. Microbiol. 1997, 24, 895–904.
[CrossRef] [PubMed]
2.
3.
4.
Bassler, B.L. Small talk. Cell-to-cell communication in bacteria. Cell 2002, 109, 421–424. [CrossRef]
Bjarnsholt, T.; Jensen, P.Ø.; Rasmussen, T.B.; Christophersen, L.; Calum, H.; Hentzer, M.; Hougen, H.-P.;
Rygaard, J.; Moser, C.; Eberl, L.; et al. Garlic blocks quorum sensing and promotes rapid clearing of
pulmonary Pseudomonas aeruginosa infections. Microbiology 2005, 151, 3873–3880. [CrossRef] [PubMed]
Camilli, A.; Bassler, B.L. Bacterial Small-Molecule Signaling Pathways. Science 2006, 311, 1113–1116.
[CrossRef]
Eberl, L.; Winson, M.K.; Sternberg, C.; Stewart, G.S.; Christiansen, G.; Chhabra, S.R.; Bycroft, B.; Williams, P.;
Molin, S.; Givskov, M. Involvement of N-acyl-L-hormoserine lactone autoinducers in controlling the
multicellular behaviour of Serratia liquefaciens. Mol. Microbiol. 1996, 20, 127–136. [CrossRef] [PubMed]
Hentzer, M.; Givskov, M. Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial
infections. J. Clin. Investig. 2003, 112, 1300–1307. [CrossRef]
5.
6.
7.
8.
9.
Bjarnsholt, T.; Givskov, M. The role of quorum sensing in the pathogenicity of the cunning aggressor
Pseudomonas aeruginosa. Anal. Bioanal. Chem. 2007, 387, 409–414. [CrossRef]
Clatworthy, A.E.; Pierson, E.; Hung, D.T. Targeting virulence: A new paradigm for antimicrobial therapy.
Nat. Chem. Biol. 2007, 3, 541–548. [CrossRef]
10. Martin, C.A.; Hoven, A.D.; Cook, A.M. Therapeutic frontiers: Preventing and treating infectious diseases by
inhibiting bacterial quorum sensing. Eur. J. Clin. Microbiol. Infect. Dis. 2008, 27, 635–642. [CrossRef]
11. Defoirdt, T. Quorum-sensing systems as targets for antivirulence therapy. Trends Microbiol. 2018, 26, 313–328.
[CrossRef] [PubMed]
12. Busetti, A.; Shaw, G.; Megaw, J.; Gorman, S.P.; Maggs, C.A.; Gilmore, B.F. Marine-derived quorum-sensing
inhibitory activities enhance the antibacterial efficacy of tobramycin against Pseudomonas aeruginosa. Mar.
Drugs 2014, 13, 1–28. [CrossRef] [PubMed]
13. Brackman, G.; Cos, P.; Maes, L.; Nelis, H.J.; Coenye, T. Quorum sensing inhibitors increase the susceptibility
of bacterial biofilms to antibiotics in vitro and in vivo. Antimicrob. Agents Chemother. 2011, 55, 2655–2661.
[CrossRef] [PubMed]
14. Givskov, M.; de Nys, R.; Manefield, M.; Gram, L.; Maximilien, R.; Eberl, L.; Molin, S.; Steinberg, P.D.;
Kjelleberg, S. Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling. J. Bacteriol.
1996, 178, 6618–6622. [CrossRef] [PubMed]
15. Gram, L.; de Nys, R.; Maximilien, R.; Givskov, M.; Steinberg, P.; Kjelleberg, S. Inhibitory Effects of Secondary
metabolites from the red alga Delisea pulchra on swarming motility of Proteus mirabilis. Appl. Environ.
Microbiol. 1996, 62, 4284–4287. [PubMed]
16. Costantino, V.; Della Sala, G.; Saurav, K.; Teta, R.; Bar-Shalom, R.; Mangoni, A.; Steindler, L. Plakofuranolactone
as a quorum quenching agent from the Indonesian sponge Plakortis cf. lita. Mar. Drugs 2017, 15, 59. [CrossRef]
17. Teasdale, M.E.; Donovan, K.A.; Forschner-Dancause, S.R.; Rowley, D.C. Gram-positive marine bacteria as
a potential resource for the discovery of quorum sensing inhibitors. Mar. Biotechnol. 2011, 13, 722–732.
[CrossRef]
18. Hentzer, M.; Riedel, K.; Rasmussen, T.B.; Heydorn, A.; Andersen, J.B.; Parsek, M.R.; Rice, S.A.; Eberl, L.;
Molin, S.; Høiby, N.; et al. Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by
a halogenated furanone compound. Microbiology 2002, 148, 87–102. [CrossRef]

Mar. Drugs 2019, 17, 389
12 of 13
19. Choi, H.; Mascuch, S.J.; Villa, F.A.; Byrum, T.; Teasdale, M.E.; Smith, J.E.; Preskitt, L.B.; Rowley, D.C.;
Gerwick, L.; Gerwick, W.H. Honaucins A-C, potent inhibitors of inflammation and bacterial quorum sensing:
Synthetic derivatives and structure-activity relationships. Chem. Biol. 2012, 19, 589–598. [CrossRef]
20. Parsek, M.R.; Greenberg, E.P. Acyl-homoserine lactone quorum sensing in gram-negative bacteria: A signaling
mechanism involved in associations with higher organisms. Proc. Natl. Acad. Sci. USA 2000, 97, 8789–8793.
[CrossRef]
21. Geske, G.D.; O’Neill, J.C.; Blackwell, H.E. N-Phenylacetanoyl-l-homoserine lactones can strongly antagonize
or superagonize quorum sensing in Vibrio fischeri. ACS Chem. Biol. 2007, 2, 315–319. [CrossRef] [PubMed]
22. Geske, G.D.; O’Neill, J.C.; Miller, D.M.; Mattmann, M.E.; Blackwell, H.E. Modulation of bacterial quorum
sensing with synthetic ligands: Systematic evaluation of N-Acylated homoserine lactones in multiple species
and new insights into their mechanisms of action. J. Am. Chem. Soc. 2007, 129, 13613–13625. [CrossRef]
[PubMed]
23. Smith, K.M.; Bu, Y.; Suga, H. Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic
autoinducer analogs. Chem. Biol. 2003, 10, 81–89. [CrossRef]
24. Eberhard, A.; Widrig, C.A.; McBath, P.; Schineller, J.B. Analogs of the autoinducer of bioluminescence in
Vibrio fischeri. Arch. Microbiol. 1986, 146, 35–40. [CrossRef] [PubMed]
25. Boursier, M.E.; Combs, J.B.; Blackwell, H.E. N-Acyl l-homocysteine thiolactones are potent and stable
synthetic modulators of the RhlR quorum sensing receptor in Pseudomonas aeruginosa. ACS Chem. Biol. 2019
14, 186–191. [CrossRef] [PubMed]
,
26. Zhu, J.; Beaber, J.W.; Moré, M.I.; Fuqua, C.; Eberhard, A.; Winans, S.C. Analogs of the autoinducer
3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J.
Bacteriol. 1998, 180, 5398–5405. [PubMed]
27. Geske, G.D.; O’Neill, J.C.; Miller, D.M.; Wezeman, R.J.; Mattmann, M.E.; Lin, Q.; Blackwell, H.E. Comparative
analyses of N-acylated homoserine lactones reveal unique structural features that dictate their ability to
activate or inhibit quorum sensing. Chembiochem 2008, 9, 389–400. [CrossRef] [PubMed]
28. Geske, G.D.; Mattmann, M.E.; Blackwell, H.E. Evaluation of a focused library of N-aryl L-homoserine lactones
reveals a new set of potent quorum sensing modulators. Bioorg. Med. Chem. Lett. 2008, 18, 5978–5981.
[CrossRef]
29. Reverchon, S.; Chantegrel, B.; Deshayes, C.; Doutheau, A.; Cotte-Pattat, N. New synthetic analogues of
N-acyl homoserine lactones as agonists or antagonists of transcriptional regulators involved in bacterial
quorum sensing. Bioorg. Med. Chem. Lett. 2002, 12, 1153–1157. [CrossRef]
30. Geske, G.D.; Wezeman, R.J.; Siegel, A.P.; Blackwell, H.E. Small molecule inhibitors of bacterial quorum
sensing and biofilm formation. J. Am. Chem. Soc. 2005, 127, 12762–12763. [CrossRef]
31. Teasdale, M.E.; Liu, J.; Wallace, J.; Akhlaghi, F.; Rowley, D.C. Secondary metabolites produced by the marine
bacterium Halobacillus salinus that inhibit quorum sensing-controlled phenotypes in gram-negative bacteria.
Appl. Environ. Microbiol. 2009, 75, 567–572. [CrossRef] [PubMed]
32. Fredenhagen, A.; Tamura, S.Y.; Kenny, P.T.M.; Komura, H.; Naya, Y.; Nakanishi, K.; Nishiyama, K.; Sugiura, M.;
Kita, H. Andrimid, a new peptide antibiotic produced by an intracellular bacterial symbiont isolated from
a brown planthopper. J. Am. Chem. Soc. 1987, 109, 4409–4411. [CrossRef]
33. Maskey, R.P.; Asolkar, R.N.; Kapaun, E.; Wagner-Döbler, I.; Laatsch, H. Phytotoxic arylethylamides from
limnic bacteria using a screening with microalgae. J. Antibiot. 2002, 55, 643–649. [CrossRef] [PubMed]
34. Austin, B.; Zhang, X.-H. Vibrio harveyi: A significant pathogen of marine vertebrates and invertebrates. Lett.
Appl. Microbiol. 2006, 43, 119–124. [CrossRef] [PubMed]
35. Forschner-Dancause, S.; Poulin, E.; Meschwitz, S. Quorum sensing inhibition and structure-activity
relationships of β-Keto Esters. Molecules 2016, 21, 971. [CrossRef] [PubMed]
36. Manefield, M.; Harris, L.; Rice, S.A.; de Nys, R.; Kjelleberg, S. Inhibition of luminescence and virulence in
the black tiger prawn (Penaeus monodon) pathogen Vibrio harveyi by intercellular signal antagonists. Appl.
Environ. Microbiol. 2000, 66, 2079–2084. [CrossRef] [PubMed]
37. Defoirdt, T.; Boon, N.; Sorgeloos, P.; Verstraete, W.; Bossier, P. Quorum sensing and quorum quenching in
Vibrio harveyi: Lessons learned from in vivo work. ISME J. 2008, 2, 19–26. [CrossRef]
38. Henke, J.M.; Bassler, B.L. Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi. J.
Bacteriol. 2004, 186, 6902–6914. [CrossRef]

Mar. Drugs 2019, 17, 389
13 of 13
39. McClean, K.H.; Winson, M.K.; Fish, L.; Taylor, A.; Chhabra, S.R.; Camara, M.; Daykin, M.; Lamb, J.H.;
Swift, S.; Bycroft, B.W.; et al. Quorum sensing and Chromobacterium violaceum: Exploitation of violacein
production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 1997, 143 Pt 12,
3703–3711. [CrossRef]
40. Andersen, J.B.; Heydorn, A.; Hentzer, M.; Eberl, L.; Geisenberger, O.; Christensen, B.B.; Molin, S.; Givskov, M.
gfp-Based N-Acyl Homoserine-lactone sensor systems for detection of bacterial communication. Appl.
Environ. Microbiol. 2001, 67, 575–585. [CrossRef]
41. Olsen, J.A.; Severinsen, R.; Rasmussen, T.B.; Hentzer, M.; Givskov, M.; Nielsen, J. Synthesis of new 3- and
4-substituted analogues of acyl homoserine lactone quorum sensing autoinducers. Bioorg. Med. Chem. Lett.
2002, 12, 325–328. [CrossRef]
42. McLean, R.J.C.; Pierson, L.S.; Fuqua, C. A simple screening protocol for the identification of quorum signal
antagonists. J. Microbiol. Methods 2004, 58, 351–360. [CrossRef] [PubMed]
43. Champagne, P.; Desroches, J.; Paquin, J.-F. Organic fluorine as a hydrogen-bond acceptor: Recent examples
and applications. Synthesis 2015, 47, 306–322.
44. Chen, X.; Chen, J.; Yan, Y.; Chen, S.; Xu, X.; Zhang, H.; Wang, H. Quorum sensing inhibitors from marine
bacteria Oceanobacillus sp. XC22919. Nat. Prod. Res. 2018, 33, 1–5. [CrossRef] [PubMed]
45. Abed, R.M.M.; Dobretsov, S.; Al-Fori, M.; Gunasekera, S.P.; Sudesh, K.; Paul, V.J. Quorum-sensing inhibitory
compounds from extremophilic microorganisms isolated from a hypersaline cyanobacterial mat. J. Ind.
Microbiol. Biotechnol. 2013, 40, 759–772. [CrossRef]
46. Lane, D.J. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics; Stackebrandt, E.,
Goodfellow, M., Eds.; John Wiley and Sons: New York, NY, USA, 1991; pp. 115–175.
47. Thompson, F.L.; Hoste, B.; Vandemeulebroecke, K.; Swings, J. Genomic diversity amongst Vibrio isolates
from different sources determined by fluorescent amplified fragment length polymorphism. Syst. Appl.
Microbiol. 2001, 24, 520–538. [CrossRef]
48. Thompson, F.L.; Li, Y.; Gomez-Gil, B.; Thompson, C.C.; Hoste, B.; Vandemeulebroecke, K.; Rupp, G.S.;
Pereira, A.; De Bem, M.M.; Sorgeloos, P.; et al. Vibrio neptunius sp. nov., Vibrio brasiliensis sp. nov. and Vibrio
xuii sp. nov., isolated from the marine aquaculture environment (bivalves, fish, rotifers and shrimps). Int. J.
Syst. Evol. Microbiol. 2003, 53, 245–252. [CrossRef]
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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