Synthesis and evaluation of thiazolidinedione and dioxazaborocane analogues as inhibitors of AI-2 quorum sensing in Vibrio harveyi

Article abstract of DOI:10.1016/j.bmc.2012.11.055

Two focused libraries based on two types of compounds, that is, thiazolidinediones and dioxazaborocanes were designed. Structural resemblances can be found between thiazolidinediones and well-known furanone type quorum sensing (QS) inhibitors such as N-ac

Full text of DOI:10.1016/j.bmc.2012.11.055

Bioorganic & Medicinal Chemistry 21 (2013) 660–667
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of thiazolidinedione and dioxazaborocane
analogues as inhibitors of AI-2 quorum sensing in Vibrio harveyi
Gilles Brackman a, , Abed Al Aziz Al Quntar b,c, Claes D. Enk d, Izet Karalic e, Hans J. Nelis a,
⇑
Serge Van Calenbergh e, Morris Srebnik b, Tom Coenye a
a Laboratory of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
b Institute of Drug Research, Hebrew University, Jerusalem, Israel
c Department of Material Engineering, Al Quds University, Jerusalem, Israel
d Department of Dermatology, Hadassah-Hebrew University Medical School, Jerusalem, Israel
e Laboratory of Medicinal Chemistry, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2012
Revised 26 November 2012
Accepted 29 November 2012
Available online 11 December 2012
Two focused libraries based on two types of compounds, that is, thiazolidinediones and dioxazaborocanes
were designed. Structural resemblances can be found between thiazolidinediones and well-known fura-
none type quorum sensing (QS) inhibitors such as N-acylaminofuranones, and/or acyl-homoserine lac-
tone signaling molecules, while dioxazaborocanes structurally resemble previously reported
oxazaborolidine derivatives which antagonized autoinducer 2 (AI-2) binding to its receptor. Because of
this, we hypothesized that these compounds could affect AI-2 QS in Vibrio harveyi. Although all com-
pounds blocked QS, the thiazolidinediones were the most active AI-2 QS inhibitors, with EC50 values in
the low micromolar range. Their mechanism of inhibition was elucidated by measuring the effect on bio-
luminescence in a series of V. harveyi QS mutants and by DNA-binding assays with purified LuxR protein.
The active compounds neither affected bioluminescence as such nor the production of AI-2. Instead, our
results indicate that the thiazolidinediones blocked AI-2 QS in V. harveyi by decreasing the DNA-binding
ability of LuxR. In addition, several dioxazaborocanes were found to block AI-2 QS by targeting LuxPQ.
Ó 2012 Elsevier Ltd. All rights reserved.
We dedicate this article to the memory of
our friend and colleague Professor M.
Srebnik who passed away in December 2011
Keywords:
Quorum sensing
AI-2
Vibrio harveyi
Thiazolidinedione
Dioxazaborocane
1. Introduction
LuxS, a key enzyme in the production of AI-2, catalyzes the conver-
sion of S-ribosyl-L-homocysteine to 4,5-dihydroxy-2,3-pentanedi-
Quorum sensing (QS) is a mechanism by which bacteria regulate
gene expression in response to population density. This form of sig-
nal-dependent communicationis present in both Gram-positive and
Gram-negative bacteria. At low population density only basal
amounts of signal are produced, not provoking an effect. At a certain
threshold, signal concentrations will be high enough to result in
binding of the signal molecule to the receptor, ultimately leading
to altered gene expression.1 Many bacteria use QS to regulate the
production of virulence factors. Hence, QS systems have been pro-
posed as attractive targets for the development of anti-pathogenic
compounds.2,3 Typically, acyl-homoserine lactone (AHL) signal mol-
ecules are used by Gram-negative bacteria, while peptides are used
as QS molecules in Gram-positive bacteria.4,5 A mixture of signaling
molecules, collectively referred to as autoinducer-2 (AI-2) is thought
to function as a universal signal for interspecies communication.1
one (DPD).6,7 DPD then undergoes several spontaneous cyclization
steps to form the AI-2 mixture. The structure of one of these
molecules was identified as (2S,4S)-2-methyl-2,3,3,4-tetra-
hydroxytetrahydrofuran-borate.8 In Vibrio harveyi, QS is regulated
by three synergistically acting signaling molecules, that is, 3-
hydroxy-butanoyl-homoserine lactone (3-OH-C4-HSL), cholera-
autoinducer-1 (CAI-1) and AI-2, which are sensed by the LuxN, CqsS
and LuxPQ receptors, respectively.9,10 At low population density, the
receptors act as kinases, resulting in the phosphorylation of the
downstream response regulator LuxO, through a cascade involving
LuxU.11 The phosphorylation of LuxU results in the production of
small RNA’s which, together with the chaperone protein Hfq, desta-
bilize mRNA encoding the response regulator LuxR.12,13 At high cell
density, binding of the signals to their cognate receptor leads to a
switch from kinase to phophatase activity. LuxO will be dephospho-
rylated, no small RNA’s will be formed, mRNA of LuxR remains stable
and will be transcribed.13 Although AI-2 controls gene expression in
differentbacteria, QS-regulated bioluminescence in V. harveyi serves
⇑
Corresponding author. Tel.: +32 (0) 9 264 80 93; fax: +32 (0) 9 264 81 95.
E-mail address: Gilles.brackman@UGent.be (G. Brackman).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.055

G. Brackman et al. / Bioorg. Med. Chem. 21 (2013) 660–667
661
concentrations below the minimal inhibitory concentration. To
rule out direct interference with bioluminescence, the direct effect
of all compounds on bioluminescence itself was investigated using
the constitutively luminescent Escherichia coli DH5apBluelux
strain. None of the compounds tested had an effect on biolumines-
cence (data not shown).
2.3. Effect on AI-2 QS
The effect of all compounds on AI-2 regulated bioluminescence
of V. harveyi BB170 and MM32 was determined. All thiazolidined-
iones (1–6) and dioxazaborocanes (7–15) interfered with AI-2 QS
(Table 1). Compounds 1 and 2 reduced the AI-2 QS signal by more
than 99% (V. harveyi BB170) or 90% (V. harveyi MM32) at 100 lM
(Table 1). A decrease in activity was observed when the alkylidene
chain length of the thiazolidinedione derivatives was increased
from 10 to 12 carbons (compounds 2, 3 and 4, respectively) or de-
creased from 10 to 8 carbons (compounds 2 and 1, respectively)
(Table 1). The pyrrolidinedione compound 6 was less active than
its thiazolidinedione counterpart 2. A loss in activity was also ob-
served when the double bond of the alkylidene chain was satu-
Figure 1. Basic structure of (A) a thiazolidinedione, (B) a synthetic furanone, (C) an
dioxazaborocane and (D) AI-2.
as a model to study AI-2 QS inhibition.14 Several QS inhibitors (QSI)
have been described targeting AI-2 QS.15–18 Structural resemblances
can be found between thiazolidinediones (Fig. 1A) and well-known
furanone type QSI (Fig. 1B) such as N-acylaminofuranones and/or
AHL signaling molecules, while dioxazaborocanes (Fig. 1C) structur-
ally resemble previously reported oxazaborolidine derivatives
which antagonized AI-2 (Fig. 1D) binding to its receptor.18–21 For this
reason, we hypothesized that thiazolidinedione and dioxazoboro-
cane derivatives could affect QS in V. harveyi.
rated (EC50 of 21.9 and 37.9 lM for compound 5 in V. harveyi
BB170 and MM32, respectively). In general, these results indicate
that a thiazolidinedione with a 10 carbon-alkylidene chain without
N-substitution yields the highest QS-inhibitory activity. Although
the other compounds were still active, alterations in the alkylidene
chain, saturation of the double bond and replacement of the thia-
zolidine ring by a pyrrolidine ring led to a decrease in QS-inhibitory
activity.
2. Results and discussion
2.1. Library design and synthesis
An increase in activity was observed when the phenyl moiety of
the 2-phenyl-1,3,6,2-dioxazaborocanes was substituted with a hy-
In the present study two libraries of 5-substituted thiazolidin-
edione and 2-phenyl-1,3,6,2-dioxazaborocane analogues were
investigated. The compounds in the first library (Fig. 2) were de-
signed to investigate the influence of varying the length of the alki-
lydene chain, saturation of this chain and replacement of the
thiazolidine ring by a pyrrolidine ring on AI-2 QS. The dioxazaboro-
cane library (Fig. 3) was designed to evaluate the effect of different
substitution patterns on the 2-phenyl-1,3,6,2-dioxazaborocane
scaffold. 1,3,6,2-Dioxazaborocanes were previously reported to af-
fect AI-2 QS and 2-phenyl substitution was shown to confer higher
activity than alkyl substitution.15
The 5-alkylidene substituted thiazolidine-2,4-diones 1, 2, 3 and
4 were prepared by treatment of 2,4-thiazolidinedione with the
appropriate aliphatic aldehyde in ethanol in the presence of a cat-
alytic amount of piperidine. The desired products were precipi-
tated from the reaction mixture upon addition of 1 M HCl and
water and were obtained in analytically pure form after several
washing steps. 5-Decylthiazolidine-2,4-dione (5) was obtained by
catalytic hydrogenation of 2. The pyrrole-2,5-dione derivative 6
was synthesized via a Wittig reaction between decanal and 3-(tri-
phenylphosphoranylidene)-2,5-pyrrolidinedione, obtained from
the addition of triphenylphosphine to maleimide.
droxy, amino or a carboxyl group (EC50 of 12.2
lM, 29.2 and
46.9 M for compounds 13, 8 and 7 respectively in V. harveyi
l
BB170). Compound 11 resulted in a decrease of 65.4 8.4% in bio-
luminescence signal (Table 1). In addition, a decrease in activity
was observed when the carboxyl function was moved from para
to meta position (EC50 38.8 lM and 46.9 lM for compounds 9
and 7, respectively in V. harveyi BB170) and when the amino group
was substituted with hydroxyalkyl moieties (EC50 of 35.7 and
41.3 lM for compounds 14 and 15, respectively in V. harveyi
BB170) (Table 1). Strong AI-2 QS inhibitory activity was observed
for compound 10 (decrease of 95.8 0.2% and 84.2 7.3% in biolu-
minescence signal of V. harveyi BB170 and MM32, respectively,
compared to the control).
2.4. Identification of the molecular target in the V. harveyi QS
system
To identify the molecular target of the different QSI we mea-
sured the effect of selected compounds on bioluminescence pro-
duction in different V. harveyi QS-mutants (Table 2). The signal
synthase mutants V. harveyi BB152 (does not produce AHL but pro-
duces AI-2 and CAI-1) and MM30 and MM32 (does not produce AI-
2 but responds to exogenously added AI-2 from E. coli K12 or to
synthetic DPD) and the signal receptor mutants V. harveyi BB170
(does not respond to 3-OH-C4-HSL), BB886 (does not respond to
AI-2) and JMH597 (does not respond to 3-OH-C4-HSL and CAI-1)
were used. Compound 13 did not block bioluminescence in a mu-
tant which does not produce AI-2 (V. harveyi MM30) or lacks the
AI-2 receptor LuxPQ (V. harveyi BB886) (Fig. 4; Supplementary data
Fig. S16). Although the supernatants of E. coli K12 treated with the
compounds revealed no difference in AI-2 activity compared with
the control (data not shown), differences in inhibitory activity
were observed for the V. harveyi MM30 mutant (Fig. 4). Com-
pounds 1 and 2 blocked bioluminescence in V. harveyi MM30,
while no inhibitory effects were observed with compound 13 in
The 6-alkyl-2-phenyl-1,3,6,2-dioxazaborocanes 7–15 were syn-
thesized by esterification of the appropriate phenyl boronic acid
with the suitable 2,20-(alkylimino)bisethanol, in the presence of
molecular sieves, followed by precipitation and recrystallization.
2.2. Compounds do not affect bacterial growth or
bioluminescence
The antimicrobial activity of all compounds was evaluated
against a number of bacterial strains. Even when used in concen-
trations up to 1000 lM, the different thiazolidinediones and diox-
azaborocane derivatives did not affect growth (data not shown)
and in all subsequent experiments, compounds were used in

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G. Brackman et al. / Bioorg. Med. Chem. 21 (2013) 660–667
Figure 2. Thiazolidinediones and a pyrroledinedione.
Figure 3. Dioxazaborocanes.
the absence of exogenous AI-2 (Fig. 4). However, compound 13 re-
duced bioluminescence when supernatants containing AI-2 or syn-
thetic DPD was added to V. harveyi MM30, indicating that these
compounds exert their activity at the level of the AI-2 receptor
(data not shown). Although the boronated-ring in AI-2 does not
contain an aromatic substituent or nitrogen, a structural relation-

G. Brackman et al. / Bioorg. Med. Chem. 21 (2013) 660–667
663
Table 1
Inhibition of QS-regulated bioluminescence in V. harveyi BB170 or V. harveyi MM32 (activity is expressed as the % inhibition of the bioluminescence signal of the untreated
control standard deviation; n P30)
V. harveyi BB170
V. harveyi MM32
Compound code
Reduction in bioluminescence (% SD)
EC50
Reduction in bioluminescence (% SD)
EC50
50
lM
100
l
M
lM
50
l
M
100
lM
lM
Thiazolidinediones
1
2
3
4
5
6
96.1 3.6
98.3 1.0
91.1 7.5
75.0 18.8
65.5 5.2
58.9 15.2
99.6 0.3
99.4 0.5
97.6 1.7
88.3 10.9
84.6 5.3
79.2 10.6
8.2
2.1
17.0
29.4
21.9
24.5
79.1 1.9
92.3 0.3
72.6 8.3
62.6 7.6
56.7 13.9
52.1 8.8
93.5 0.5
97.5 9.8
96.9 0.9
77.5 11.2
75.6 11.4
67.6 3.4
16.8
9.8
23.7
34.6
37.9
39.8
Dioxazaborocanes
7
8
9
10
11
12
13
14
15
52.3 3.0
64.6 13.7
51.6 7.0
95.8 0.2
65.4 8.4
51.8 18.3
98.1 2.6
51.7 20.3
50.1 16.6
73.7 7.3
75.4 12.3
74.7 10.7
99.2 0.9
92.5 1.7
71.9 14.9
99.8 0.1
71.9 17.5
67.8 16.6
46.9
29.2
38.8
10.7
35.4
39.0
12.2
35.7
41.3
39.5 5.4
51.8 13.5
53.4 15.9
84.2 7.3
67.5 5.4
41.1 9.3
87.3 8.1
51.2 9.8
47.3 7.9
70.1 16.1
61.8 4.1
68.4 9.5
95.5 1.3
93.5 0.6
67.7 6.3
96.9 0.9
71.2 2.7
68.2 10.6
54.1
47.1
49.1
15.9
29.2
61.2
12.6
49.3
57.4
Table 2
Strains used in this study
Strain
Characteristics
References
V. harveyi BB120
V. harveyi BB152
V. harveyi MM30
V. harveyi BB170
V. harveyi BB886
V. harveyi MM32
V. harveyi JAF375
V. harveyi JMH597
V. harveyi JMH612
V. harveyi JAF553
V. harveyi JAF483
V. harveyi BNL258
Wild-type
luxM::Tn5
luxS::Tn5
luxN::Tn5
27
12
28
29
12
28
11
30
30
11
13
31
17
26
32
luxPQ::Tn5
luxN::CmR luxS::Tn5
luxN::CmR luxQ::KanR
luxN::Tn5 cqsS::CmR
luxPQ::Tn5 cqsS::CmR
luxU H58A linked to KanR
luxO D47A linked to KanR
hfq::Tn5lacZ
E. coli DH5
apBluelux
Strain (not producing AI-2) containing pBluelux polylinker and luxCDABE genes
Strain containing the gst-luxR overexpression construct
AI-2 producing strain
E. coli BL21 pGET-1
E. coli K12
ship of compound 13 with known AI-2 QSI is evident.15 The diox-
azaborocane compounds contain a negatively charged boron in a
hydrated heterocyclic ring-structure. In addition, a phenyl substi-
tuted dioxazaborocane was previously shown to induce AI-2 regu-
lated bioluminescence in V. harveyi,15 while phenyl substituted
dioxazaborocanes in the present study were capable of blocking
AI-2 QS. This strongly suggests that the phenyl substituent can
interact with the AI-2 binding site of LuxPQ, leading to either acti-
vation or inhibition of AI-2 signal transduction. Only few specific
inhibitors of the V. harveyi AI-2 receptor have been described. Sev-
eral para-substituted phenylboronic acids (EC50 in low micromolar
contain a point mutation in the luxU and luxO genes, respectively,
thereby abolishing their phosphorelay capacity.11,13 V. harveyi
BNL258 has a Tn5 insertion in the hfq gene, resulting in a non-
functional Hfq protein.31 V. harveyi strains JAF553, JAF483 and
BNL258 are all constitutively luminescent, hence a decrease in bio-
luminescence signal would indicate that the compounds act down-
stream of the mutation. Compounds 1 and 2 blocked QS-controlled
bioluminescence in all mutants, including V. harveyi JAF553,
JAF483 and BNL258, which suggests that they possibly act down-
stream of the AI-2 QS signal transduction pathway at the level of
LuxR. To further investigate their effect on LuxR, a fluorescently la-
beled fragment of a V. harveyi consensus LuxR binding sequence
was incubated together with purified LuxR protein in the presence
and absence of compounds 1 and 2. Incubation of LuxR with this
DNA fragment in the absence of compounds resulted in a signifi-
cant increase in anisotropy (Fig. 5). When LuxR was incubated with
this DNA fragment in the presence of compounds 1 or 2, binding to
DNA was strongly inhibited (Fig. 5), indicating that these com-
pounds inhibit AI-2 mediated QS by decreasing the DNA-binding
ability of LuxR. However, in order to gather additional support
for this proposed mechanism and in order to identify the LuxR
site(s) that interact with compounds 1 and 2, the protein–inhibitor
complex should be investigated in more detail. In the signal trans-
duction mutants no effects on bioluminescence (Fig. 5) and on the
LuxR DNA-binding ability (data not shown) were observed for
range), pyrogallol derivatives (EC50 below 10 lM) and sulfonyl
compounds (EC50 below 40 lM) were reported to block AI-2 QS
in a way similar to the compounds reported in this study.22–24 In
addition, LMC-21, an adenosine derivative with a p-methoxyphe-
nylpropionamide moiety at C-30 blocked AI-2 QS (EC50 of 20
lM).
Although the available evidence suggests that this compound
interferes with the LuxPQ receptor, the molecular interaction of
LMC-21 with this receptor remains to be determined.18
In contrast, compounds 1 and 2 blocked QS-controlled biolumi-
nescence in all mutants, indicating that they block all three chan-
nels of the QS system and act downstream at the level of signal
transduction. To reveal their target, the effect of the compounds
on bioluminescence production was evaluated in three signal
transduction mutants. The V. harveyi JAF553 and JAF483 mutants

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G. Brackman et al. / Bioorg. Med. Chem. 21 (2013) 660–667
Figure 4. Effect of compounds on the bioluminescence of the different Vibrio harveyi QS mutants. The percentage of bioluminescence of the Vibrio harveyi with 50 lM of
compound 1, 2 and 13 are presented. Measurements were performed 6 h after the addition of the compounds. Bioluminescence of the control (without addition of compound)
was set at 100% and the response for the other samples was normalized accordingly. The error bars represent the standard deviation.
DNA-binding ability of LuxR. In addition, several dioxazaborocanes
were found to block AI-2 QS by targeting LuxPQ.
4. Materials and methods
4.1. Synthesis of the thiazolidinediones
4.1.1. Synthesis of thiazolidinediones—general procedure
2,4-Thiazolidinedione (1 equiv) is added to a solution of a long
chain aldehyde in ethanol in the presence of a catalytic amount of
piperidine at room temperature. The reaction is kept at 80–90 °C
for 24 h and then cooled to 0 °C in an ice bath. Thereafter 1 M HCl
and water are added and the precipitate is filtered over a glass sin-
tered filter, washed with water and petroleum ether and dried in
vacuum to obtain an analytically pure thiazolidinedione derivative.
Figure 5. DNA binding of LuxR in the absence and presence of inhibitors (10
l
M).
The fractional change in anisotropy,
LuxR (nM).
DF/Fo, is plotted against the concentration of
4.1.2. (Z)-5-octylidenethiazolidine-2,4-dione (1)
compound 13. Cinnamaldehyde and cinnamaldehyde derivatives
were previously reported to act in a similar manner by decreasing
the DNA-binding ability of LuxR.17,25 However, compounds 1 and 2
are much more active inhibitors of AI-2 QS than cinnamaldehyde,
(Z)-5-octylidenethiazolidine-2,4-dione (1) was prepared follow-
ing the general procedure, using 2,4-thiazolidinedione and octanal
as reactants (55%). 1H NMR (500 MHz): d 0.90 (t, 3H, JHH 6.0 Hz),
1.29–1.34 (m, 8H), 1.53–1.57 (m, 2H), 2.21–2.25 (q, 2H,
JHH = 7.5 Hz), 7.06 (t, 1H, JHH = 8 Hz) ppm; 13C NMR (75.9 MHz,
CDCl3): d 14.33, 22.87, 28.04, 29.43, 29.46, 29.50, 29.64, 32.08,
126.48, 140.03, 166.03, 167.78 ppm. m/z: 227.0980 (100%),
228.1014 (11.9%), 229.0938 (4.5%). elemental analysis: C,58.12;
H, 7.54; N 6.16; O, 14.08; S, 14.11. (M+H+NH4), calcd: 226.0902;
found: 226.0907 (Supplementary data file 1).
with concentrations of 8.2 and 2.1
l
M exhibiting similar QS-inhib-
itory activity as 100
l
M of cinnamaldehyde.17,25
3. Conclusion
Thiazolidinedione and dioxazaborocane compounds were syn-
thesized and their effect on AI-2 QS was evaluated. Although all
compounds blocked QS, the thiazolidinediones were the most ac-
tive AI-2 QS inhibitors, with EC50 values in the low micromolar
range, making them among the most active AI-2 QS inhibitors
reported to date. Our results indicate that the thiazolidinedione
compounds blocked AI-2 QS in V. harveyi by decreasing the
4.1.3. (Z)-5-decylidenethiazolidine-2,4-dione (2)
(Z)-5-decylidenethiazolidine-2,4-dione (2) was prepared fol-
lowing the general procedure, using 2,4-thiazolidinedione and dec-
enal as reactants (57%). 1H NMR f d 0.85 (t, JHH = 6.6 Hz, 3H), 1.25 (s,
12H), 1.56 (m, 2H), 2.20 (q, JHH = 7.2 HZ, 2H), 7.05 (t, JHH = 7.5 HZ,

G. Brackman et al. / Bioorg. Med. Chem. 21 (2013) 660–667
665
1H) ppm; 13C NMR (75.9 MHz, CDCl3): d 14.33, 22.87, 28.04, 29.43,
4.3. Synthesis of the dioxazaborocanes
29.46, 29.50, 29.64, 32.08, 126.48, 140.03, 166.03, 167.78 ppm. m/
z: 255.1293 (100%), 256.1327 (14.1%), 257.1251 (4.5%). Elemental
4.3.1. Synthesis of dioxazaborocanes—general procedure
analysis: C, 61.14; H, 8.29;
(M+H+NH4), calcd: 254.1220; found: 254.1224 (Supplementary
data file 1).
N
5.48; O, 12.53; S, 12.56.
To a solution of phenyl boronic acid (7.14 mmol) in dry toluene
(30 ml) stuffed with 5 g of molecular sieves, was added the appro-
priate 2,20-azanediyldiethanol (7.14 mmol) at room temperature.
After stirring for 5 h at room temperature, the solvent was evapo-
rated and the solid was recrystallized from ether/petroleum ether.
4.1.4. (Z)-5-undecylidenethiazolidine-2,4-dione (3)
(Z)-5-undecylidenethiazolidine-2,4-dione (3) was prepared fol-
lowing the general procedure, using 2,4-thiazolidinedione and
undecanal as reactants (60%). 1H NMR (300 MHz): d 0.88 (t, 3H,
JHH 6.9 Hz), 1.26 (s, 14H), 1.45–1.55 (m, 2H), 2.19–2.25 (q, 2H,
JHH = 7.5 Hz), 7.04 (t, 1H, JHH = 7.8 Hz) ppm; 13C NMR (75.9 MHz,
CDCl3): d 14.44, 22.92, 23.11, 28.12, 29.46, 29.55, 29.56, 29.59,
32.11, 126.54, 139.95, 165.97, 167.82 ppm. m/z: 269.1449 (100%),
270.1483 (15.1%), 271.1407 (4.5%), 271.1517 (1.1%). Elemental
4.3.2. 3-(6-Methyl-1,3,6,2-dioxazaborocan-2-yl)benzoic acid (7)
Identical to the procedure used in the general procedure for the
synthesis of oxazoborolines using 3-boromobenzoic acid and N-
methyl diethanol amine (58%). 1H NMR (300 MHz, DMSO-d) d:
2.23(s, 3 H), 3.05 (m, 2H), 3.22 (m, 2H), 4.19 (m, 4H), 7.20–8.10
(overlap, 4H); 13C NMR (75.9 MHz, DMSO-d) d: 47.0, 60.5, 62.2,
128.5, 131.2, 133.5, 135.3, 140.1, 168.8; 11B NMR (125 MHz,
DMSO-d) d: 12.0.m/z: 249.1172 (100%), 248.1209 (24.8%),
250.1206 (13.0%), 249.1242 (3.2%). Elemental analysis: C, 57.87;
H, 6.47; B, 4.34; N, 5.62; O, 25.69. (M+H+NH4), calcd: 267.1511;
found: 267.1414 (Supplementary data file 1).
analysis: C, 62.42; H, 8.61;
N 5.20; O, 11.88; S, 11.90.
(M+H+NH4), calcd: 268.1377; found: 268.1381 (Supplementary
data file 1).
4.1.5. (Z)-5-dodecylidenethiazolidine-2,4-dione (4)
(Z)-5-dodecylidenethiazolidine-2,4-dione (4) was prepared fol-
lowing the general procedure, using 2,4-thiazolidinedione and
dodecanal as reactants (52%). 1H NMR (300 MHz): d 0.88 (t, 3H,
JHH 6.6 Hz), 1.26 (s, 16H), 1.49–1.56 (m, 2H), 2.17–2.25 (q, 2H,
JHH = 7.0 Hz), 7.04 (t, 1H, JHH = 7.8 Hz) ppm; 13C NMR (75.9 MHz,
CDCl3): d 14.36, 22.84, 22,92, 23.15, 28.00, 29.48, 29.47, 29.60,
29.57, 32.00, 126.40, 140.11, 166.13, 167.68 ppm. m/z: 283.1606
(100%), 284.1640 (16.2%), 285.1564 (4.5%), 285.1673 (1.2%). Ele-
mental analysis: C, 63.56; H, 8.89; N, 4.94; O, 11.29; S, 11.31.
(M+H+NH4), calcd: 282.1533; found: 282.1543 (Supplementary
data file 1).
4.3.3. 3-(6-Methyl-1,3,6,2-dioxazaborocan-2-yl)aniline (8)
Identical to the procedure used in the general procedure for the
synthesis of oxazoborolines using 3-aminophenylboronic acid and
N-methyl diethanol amine (52%). 1H NMR (300 MHz, DMSO-d) d:
2.25 (s, 3 H), 3.01 (m, 2H), 3.15 (m, 2H), 4.15 (m, 4H), 6.82–7.25
(overlap, 4H); 13C NMR (75.9 MHz, DMSO-d) d: 47.8, 60.0, 62.0,
115.4, 123.2, 129.2, 131.9, 146.4; 11B NMR (125 MHz, DMSO-d) d:
11.4.m/z: 220.1383 (100%), 219.1419 (24.8%), 221.1417 (11.9%),
220.1453 (3.0%). Elemental analysis: C, 60.03; H, 7.79; B, 4.91; N,
12.73; O, 14.54. (M+H+NH4), calcd: 238.1721; found: 238.1747
(Supplementary data file 1).
4.1.6. 5-Decylthiazolidine-2,4-dione (5)
4.3.4. 4-(6-Methyl-1,3,6,2-dioxazaborocan-2-yl)benzoic acid (9)
Identical to the procedure used in the general procedure for the
synthesis of oxazoborolines using 4-boronobenzoic acid and N-
methyl diethanol amine (45%). 1H NMR (300 MHz, DMSO-d) d:
2.22(s, 3 H), 3.01 (m, 2H), 3.15 (m, 2H), 4.18 (m, 4H), 6.92 (dt,
2H), 7.63 (dt, 2H), 10.5; 13C NMR (75.9 MHz, DMSO-d) d: 47.2,
60.2, 62.0, 112.9, 113.8, 134.2, 134.5, 168.2; 11B NMR (125 MHz,
DMSO-d) d: 10.7. m/z: 249.1172 (100%), 248.1209 (24.8%),
250.1206 (13.0%), 249.1242 (3.2%). Elemental analysis: C, 57.87;
H, 6.47; B, 4.34; N, 5.62; O, 25.69. (M+H+NH4), calcd: 267.1516;
found: 267.1551 (Supplementary data file 1).
5-Decylthiazolidine-2,4-dione (5) was prepared following gen-
eral procedure, using 2,4-tiazoldinedione and decenal as reactants.
Then, the product was hydrogenated in methanol under a suspen-
sion of Pd/C at 29 psi (48%). 1H NMR (300 MHz, CDCl3): d 0.85 (t,
JHH = 6.6 Hz, 3H), 1.25 (overlap, 14H), 1.56 (m, 2H), 2.20 (q,
JHH = 7.2 Hz, 2H), 3.72 (m, 1H); 13C NMR (75.9 MHz, CDCl3): d
14.31, 22.88, 24.80, 28.01, 29.48, 29.43, 29.54, 29.69, 32.11,
55.00, 166.00, 167.82 ppm. m/z: 257.1449 (100%), 258.1483
(14.1%), 259.1407 (4.5%). Elemental analysis: C, 60.66; H, 9.01; N,
5.44; O, 12.43; S, 12.46. (M+H+NH4), calcd: 256.1377; found:
256.1364 (Supplementary data file 1).
4.3.5. 4-(6-Methyl-1,3,6,2-dioxazaborocan-2-yl)benzaldehyde
(10)
4.2. Synthesis of (E)-3-decylidenepyrrolidine-2,5-dione (6)
Identical to the procedure used in the general procedure for the
synthesis of oxazoborolines using 4-formylphenylboronic acid and
N-methyl diethanol amine (43%). 1H NMR (300 MHz, DMSO-d) d:
2.23(s, 3 H), 3.00 (m, 2H), 3.15 (m, 2H), 4.20 (m, 4H), 7.02 (dt,
2H), 7.80 (dt, 2H), 8.50 (s, 1H); 13C NMR (75.9 MHz, DMSO-d) d:
47.8, 59.9, 61.6, 114.2, 114.9, 135.4, 135.2, 189.3; 11B NMR
(125 MHz, DMSO-d) d: 11.4. m/z: 233.1223 (100%), 232.1260
(24.8%), 234.1257 (13.0%), 233.1293 (3.2%). Elemental analysis: C,
61.84; H, 6.92; B, 4.64; N, 6.01; O, 20.59. (M+H+NH4), calcd:
251.1567; found: 251.1452 (Supplementary data file 1).
Compound 6 was prepared by mixing maleimide (0.1994 g,
2 mmol) and triphenyl phosphine (0.5232 g, 1.994 mmol), fol-
lowed by reflux for 1 h in acetone (10 ml). A precipitate was ob-
tained which was washed with several portions of acetone and
then dried under reduced pressure to yield 100%. This product
(2 mmol) and decanal (0.376 ml, 2 mmol) are mixed in methanol
(10 ml) and refluxed for 12 h. Thereafter the reaction mixture
was cooled and HCl (1 M, 0.03 ml and water 10 ml) was added.
The precipitate (45%) was filtered and washed with petroleum
ether and purified by flash chromatography (silica, ethyl acetate:
petroleum ether, 30:70) to yield pure 6. 1H NMR (300 MHz, CDCl3):
d 0.88 (t, 3H, JHH = 6 Hz), 1.29 (s, 10H), 1.46–1.51 (o, 2H), 2.13–2.25
4.3.6. 2-(4-Bromophenyl)-6-methyl-1,3,6,2-dioxazaborocane
(11)
(o, 2H), 3.25 (s, 2H), 6.80 (t, 1H, JHH = 7.8 Hz), 8.00 (s, 1H) ppm; 13
C
Identical to the procedure used in the general procedure for the
synthesis of oxazoborolines using 4-bromophenylboronic acid and
N-methyl diethanol amine (44%). 1H NMR (300 MHz, DMSO-d) d:
2.25(s, 3 H), 3.00 (m, 2H), 3.20 (m, 2H), 4.18 (m, 4H), 6.90 (dt,
2H), 7.60 (dt, 2H). 13C NMR (75.9 MHz, DMSO-d) d: 47.4, 60.2,
62.0, 113.8, 114.0, 134.6, 134.7; 11B NMR (125 MHz, DMSO-d) d:
NMR (75.0 MHz, CDCl3): d 13.60, 22.21, 23.33, 25.58, 27.69, 28.63,
28.82, 29.48, 31.30, 32.51, 126.43, 138.89, 170.71, 175.32 ppm. m/
z: 237.1729 (100%), 238.1762 (15.1%), 239.1796 (1.1%). Elemental
analysis: C, 70.85; H, 9.77; N, 5.90; O, 13.48. (M+H+NH4), calcd:
236.1656; found: 236.1646 (Supplementary data file 1).

666
G. Brackman et al. / Bioorg. Med. Chem. 21 (2013) 660–667
10.7. m/z: 283.0379 (100%), 285.0359 (97.3%), 282.0416 (24.8%),
284.0395 (24.2%), 284.0413 (11.9%), 286.0392 (11.6%), 283.0449
(3.0%), 285.0429 (2.9%). Elemental analysis: C, 46.53; H, 5.32; B,
3.81; Br, 28.14; N, 4.93; O, 11.27. (M+H+NH4), calcd: 301.0717;
found: 301.0732 (Supplementary data file 1).
in the presence of ampicillin (100 lg/ml). E. coli K12 was routinely
cultured in TSB at 37 °C.
4.5. Determination of the MIC
MICs of the compounds were determined in triplicate according
to the EUCAST broth microdilution protocol, using flat-bottom 96-
well microtiter plates (TPP, Trasadingen, Switzerland). The inocu-
lum was standardized to approximately 5 ꢀ 105 CFU/ml. The plates
were incubated at 37 °C for 20 h, and the optical density at 590 nm
was determined by using a multilabel microtiter plate reader (Envi-
sion; Perkin–Elmer LAS, Waltham, MA). The lowest concentration
of compound for which the optical density was not different from
that in the uninoculated control wells was recorded as the MIC.
4.3.7. 4-(6-Methyl-1,3,6,2-dioxazaborocan-2-
yl)phenyl)methanol (12)
Identical to the procedure used in the general procedure for the
synthesis of oxazoborolines using 4-(hydroxymethyl)phenylbo-
ronic acid and N-methyl diethanol amine (45%). 1H NMR
(300 MHz, DMSO-d) d: 2.26 (s, 3H), 3.09 (m, 2H), 3.27 (m, 2H),
4.21 (m, 4H), 4.50 (s, 2H), 6.91 (dt, 2H), 7.62 (dt, 2H). 13C NMR
(75.9 MHz, DMSO-d) d: 47.2, 60.2, 62.5, 65.0, 113.2, 114.3, 134.7,
134.3; 11B NMR (125 MHz, DMSO-d) d: 11.0. m/z: 235.1380
(100%), 234.1416 (24.8%), 236.1413 (13.0%), 235.1450 (3.2%). Ele-
mental analysis: C, 61.31; H, 7.72; B, 4.60; N, 5.96; O, 20.42.
(M+H+NH4), calcd: 253.1723; found: 253.1713 (Supplementary
data file 1).
4.6. Determination of the QS inhibitory effect
The bioluminescence assay with E. coli DH5apBlueLux and the
AI-2 QS inhibition assay using V. harveyi BB170 and E. coli K12
supernatans were conducted as described previously.18 The AI-2
QS inhibition assay using V. harveyi MM32 was conducted in a sim-
ilar way using synthetic DPD (5 lM; OMM Scientific, US). Each
compound was tested at least six times in triplicate (n P18).
4.3.8. 4-(6-Methyl-1,3,6,2-dioxazaborocan-2-yl)phenol (13)
Identical to the procedure used in the general procedure for the
synthesis of oxazoborolines using 4-hydroxyphenylboronic acid
and N-methyl diethanol amine (55%). 1H NMR (300 MHz, DMSO-
d) d: 2.26 (s, 3H), 3.05 (m, 2H), 3.28 (m, 2H), 4.19 (m, 4H), 6.91
(dt, 2H), 7.62 (dt, 2H); 13C NMR (75.9 MHz, DMSO-d) d: 47.0,
60.3, 62.5, 113.7, 114.4, 134.5, 134.5; 11B NMR (125 MHz, DMSO-
d) d: 11.2. m/z: 221.1223 (100%), 220.1260 (24.8%), 222.1257
(11.9%), 221.1293 (3.0%). Elemental analysis: C, 59.77; H, 7.30; B,
4.89; N, 6.34; O, 21.71. (M+H+NH4), calcd: 239.1567; found:
239.1454 (Supplementary data file 1).
4.7. Identification of the molecular target of the QS inhibitors
The bioassay for LuxS inhibition (using V. harveyi MM30 and
MM32) and assays to determine the molecular target of the com-
pounds tested (using V. harveyi BB120, BB152, BB170, BB886,
JAF375, JMH597, JMH612, JAF553, JAF483 and BNL258) were con-
ducted as described previously.17,18 Each compound was tested
at least ten times in triplicate (n P30).
4.3.9. 2-(2-Phenyl-1,3,6,2-dioxazaborocan-6-yl)ethanol (14)
Identical to the procedure used in the general procedure for the
synthesis of oxazoborolines using phenylboronic acid and trietha-
nol amine (60%). 1H NMR (300 MHz, DMSO-d) d: 2.23 (broad, 2H),
2.80–3.15 (overlap, 2H), 3.50 (broad, 2H), 3.90 (broad, 4H), 7.15–
7.60 (overlap, 5H); 13C NMR (75.9 MHz, DMSO-d) d: 57.2, 57.7,
60.7, 60.3, 127.3, 127.7, 133.3; 11B NMR (125 MHz, DMSO-d) d:
13.2. m/z: 235.1380 (100%), 234.1416 (24.8%), 236.1413 (13.0%),
235.1450 (3.2%). Elemental analysis: C, 61.31; H, 7.72; B, 4.60; N,
5.96; O, 20.42. (M+H+NH4), calcd: 253.1216; found: 253.1493
(Supplementary data file 1).
4.8. LuxR-DNA binding assay
Induction of GST-LuxR overexpression and protein purification
were conducted as previously described.26 Fluorescence polariza-
tion measurements in the presence and absence of QS inhibitors
(10 l
M) were conducted as described previously.26
4.9. Statistics
The normal distribution of the data was checked using the
Shapiro–Wilk test. Normally distributed data were analyzed using
an independent sample T-test. Statistical analyses were carried out
using SPSS software, version 19.0 (SPSS, Chicago, IL, USA).
4.3.10. 1-(4,8-Dimethyl-2-phenyl-1,3,6,2-dioxazaborocan-6-
yl)propan-2-ol (15)
Identical to the procedure used in the general procedure for the
synthesis of oxazoborolines using phenylboronic acid and triiso-
propyl amine (60%). 1H NMR (300 MHz, DMSO-d) d: 1.05 (d, 3H),
1.09 (d, 3H), 2.86 (m, 2H), 3.10 (m, 2H), 3.60 (m, 1H), 4.15 (overlap,
2H), 6.95–7.70 (overlap, 5H); 13C NMR (75.9 MHz, DMSO-d) d: 20.5,
20.90, 62.2, 62.4, 64.3, 64.5, 65.1, 66.00, 113.6, 113.8, 134.2, 134.5;
11B NMR (125 MHz, DMSO-d) d: 10.7. m/z: 277.1849 (100%),
276.1886 (24.8%), 278.1863 (16.2%), 277.1919 (4.0%), 279.1916
(1.2%). Elemental analysis: C, 65.00; H, 8.73; B, 3.90; N, 5.05; O,
17.32. (M+H+NH4), calcd: 295.2193; found: 295.2079 (Supplemen-
tary data file 1).
Acknowledgements
We would like to thank the ‘Fund for scientific research Flan-
ders’ (FWO-Vlaanderen) and the ‘Special Research fund of Ghent
University’ (BOF09/GOA/011) for financial support. G.B. is a post-
doctoral fellow of FWO-Vlaanderen.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.055.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
4.4. Strains and culture conditions
All bacterial strains used in this study are listed in Table 2. V.
harveyi strains were cultured in Marine-Broth (MB) (BD) in the
presence of antibiotics at 30 °C with shaking. E. coli BL21 pGET-1
(containing the gst-luxR overexpression construct) and E. coli
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