Structure-activity relationship of brominated 3-alkyl-5-methylene-2(5H)- furanones and alkylmaleic anhydrides as inhibitors of Salmonella biofilm formation and quorum sensing regulated bioluminescence in Vibrio harveyi

Article abstract of DOI:10.1016/j.bmc.2010.05.055

A library of 25 1′-unsubstituted and 1′-bromo or 1′-acetoxy 3-alkyl-5-methylene-2(5H)-furanones and two 3-alkylmaleic anhydrides was synthesized using existing and new methods. This library was tested for the antagonistic effect against the biofilm format

Full text of DOI:10.1016/j.bmc.2010.05.055

Bioorganic & Medicinal Chemistry 18 (2010) 5224–5233
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship of brominated 3-alkyl-5-methylene-
2(5H)-furanones and alkylmaleic anhydrides as inhibitors
of Salmonella biofilm formation and quorum sensing regulated
bioluminescence in Vibrio harveyi
Hans P. Steenackers a,b, , Jeremy Levin b, , Joost C. Janssens a,b, Ami De Weerdt a, Jan Balzarini c,
Jos Vanderleyden a, Dirk E. De Vos b, Sigrid C. De Keersmaecker a,
*
a Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium
b Centre for Surface Chemistry and Catalysis, Katholieke Universiteit Leuven, Kasteelpark Arenberg 23, B-3001 Leuven, Belgium
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
A library of 25 10-unsubstituted and 10-bromo or 10-acetoxy 3-alkyl-5-methylene-2(5H)-furanones and
two 3-alkylmaleic anhydrides was synthesized using existing and new methods. This library was tested
for the antagonistic effect against the biofilm formation by Salmonella Typhimurium and the quorum
sensing regulated bioluminescence of Vibrio harveyi. The length of the 3-alkyl chain and the bromination
pattern of the ring structure were found to have a major effect on the biological activity of the 10-unsub-
stituted furanones. Remarkably, the introduction of a bromine atom on the 10 position of the 3-alkyl chain
did drastically enhance the activity of the furanones in both biological test systems. The introduction of
an acetoxy function in this position did in general not improve the activity. Finally, the potential of the
(bromo)alkylmaleic anhydrides as a new and chemically easily accessible class of biofilm and quorum
sensing inhibitors was demonstrated.
Article history:
Received 8 March 2010
Revised 7 May 2010
Accepted 19 May 2010
Available online 25 May 2010
Keywords:
Halogenated furanones
Biofilm formation
Quorum sensing
Salmonella Typhimurium
Vibrio harveyi
Ó 2010 Elsevier Ltd. All rights reserved.
Pd catalysis
1. Introduction
genated furanones isolated from the macro-algae Delisea pulchra
and their synthetic analogues are providing to be promising candi-
In the last decade, we faced a rapid increase in pathogenic bac-
teria that were resistant to antibiotics. This resistance was a result
of two factors. First, by blocking the growth, antibiotics placed the
bacteria under harsh selective pressure to develop resistance. Sec-
ond, bacteria that grew in specialized surface-attached communi-
ties, called biofilms,1 had an innate tolerance to antibiotics.
Bacteria in biofilms were shown to be up to 1000-fold more resis-
tant to antibiotics than their planktonic counterparts.2 This is of
great concern since, according to the National Institutes of Health,
approximately 80% of persistent bacterial infections in the US are
associated with biofilms.3
dates in the search for anti-pathogenic drugs as they have been
shown to act as inhibitors of biofilm formation for several bacterial
species.5–11 Interference with the N-acyl-
L-homoserinelactone
(AHL) and autoinducer 2 (AI-2) mediated quorum sensing systems
(cell–cell-communication systems) has been proven as a mecha-
nism for this reduced biofilm formation, at least in a subset of
the bacterial species tested.8,12–14 As in some species virulence is
also mediated by quorum sensing, halogenated furanones have
also been shown to block virulence in these species.15–21 Despite
the fact that considerable effort has been made to study the biolog-
ical effects of a small group of halogenated furanones (espe-
cially (Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone
In an attempt to tackle this problem, strategies have been pur-
sued to find alternative drugs, referred to as ‘anti-pathogenic’
drugs,4 that selectively block virulence and/or biofilm formation,
without affecting the planktonic growth of the bacteria. The halo-
and (Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone), only
a
few studies have focused on determining elaborate structure–
activity relationships.9,10,22–25 Thus, for this study we focused on
determining elaborate structure–activity relationships by synthe-
sizing a broad range of 25 10-unsubstituted and 10-subsituted
3-alkyl-5-methylene-2(5H)-furanones. All of these furanones have
been previously described, except for the mono-brominated
furanones 3-hexyl-5-bromomethylene-2(5H)-furanone 11c and
* Corresponding author. Tel.: +32 16321631; fax: +32 16321966.
E-mail address: Sigrid.DeKeersmaecker@biw.kuleuven.be (S.C. De Keersmaeck-
er).
Equal contribution.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.055

H. P. Steenackers et al. / Bioorg. Med. Chem. 18 (2010) 5224–5233
5225
cases the infection may spread to the blood stream and can cause
death unless the patient is treated with antibiotics. While non-ty-
phoid Salmonella infections are commonly treated with fluoroquin-
olones such as ciprofloxacin and third-generation cephalosporins
(such as ceftriaxone), there are alarming reports concerning the
development of resistance against these antibiotics.26 In addition,
Salmonella has been able to form biofilms on different surfaces,
ranging from abiotic surfaces (e.g., concrete, plastics, glass, poly-
styrene,. . .),27 to biotic surfaces (gallstones,28 plant surfaces,29
and epithelial cell layers30). These biofilms are an important sur-
vival strategy in all stages of infection, from transmission to
chronic infection. Alternative anti-Salmonella strategies are ur-
gently needed. Previously, we reported that several furanones were
able to inhibit Salmonella biofilm formation at non-growth-inhibit-
ing concentrations and that pre-treatment with furanones ren-
dered the biofilms more susceptible to antibiotics.10 Others also
recently reported that the effect of disinfectants on Salmonella in
biofilms is significantly enhanced in the presence of furanones.31
Although halogenated furanones are generally considered to inter-
fere with the quorum sensing systems of gram-negative bacteria,
no evidence was found that furanones act on the currently known
quorum sensing systems of Salmonella.10 Since (Z)-4-bromo-5-bro-
momethylene-3-alkyl-2(5H)-furanones with chain lengths of two
to six carbon atoms were found to be the most interesting com-
pounds in our previous study, these furanones served as structural
templates for the development of more potent analogues in this
study.
Scheme 1. Reaction scheme for the synthesis of furanones 1–3, adapted from
Kumar and Read (2002).
3-hexyl-4-bromo-5-methylene-2(5H)-furanone 13c (Scheme 2).
These mono-brominated compounds could not be synthesized by
using existing procedures. Furthermore the known synthesis
pathways towards halogenated furanones have some important
drawbacks regarding environmental friendliness and selectivity.
These shortcomings prompted us to develop a new, more versatile,
synthesis procedure. We evaluated the biological activity of the
halogenated furanones and some 3-alkylmaleic anhydrides, which
are intermediates in the new synthesis pathway, in two model sys-
tems: biofilm formation by Salmonella enterica Typhimurium and
quorum sensing by Vibro harveyi. In an iterative process of synthe-
sis and biological testing, we tried to identify the features of the
molecules mediating their biological response.
We also wanted to test the influence of our library of haloge-
nated furanones on a test system, which has been shown to be reg-
ulated by quorum sensing. Therefore, as a second model system we
made use of the quorum sensing regulated bioluminescence of V.
harveyi. This marine bacterium and closely related species, such
As a first biological model system, we used the biofilm forma-
tion by Salmonella Typhimurium. Salmonella enterica is worldwide
one of the most prevalent food borne pathogens. Most patients in-
fected develop a self-limiting gastroenteritis, however, in severe
Scheme 2. Reaction schemes for the synthesis of 3-alkyl-5-methylene-2(5H)-furanones 4a–f, 5a–c, 6a–b, 11c and 13c and 3-alkyl-4-bromo-maleic anhydride 20c. Pathway
A: procedure developed by Manny et al. (1997). Pathway B: new procedure for the synthesis of (Z)-3-alkyl-4-bromo-5-bromomethylene-2(5H)-furanones and (Z)-3-alkyl-5-
bromomethylene-2(5H)-furanones. Pathway C: pathway for the synthesis of 3-alkyl-4-bromo-5-methylene-2(5H)-furanones. Pathway D: pathway for the synthesis of 4-
bromo-3-alkylmaleic anhydrides.

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H. P. Steenackers et al. / Bioorg. Med. Chem. 18 (2010) 5224–5233
as Vibrio campbellii, are important pathogens in the intensive rear-
ing of marine fish and invertebrates like penaeid shrimp. Due to
large-scale (mis)use of antibiotics in aquaculture, multi-resistant
Vibrio strains have emerged and antibiotics are no longer effective
in the treatment of luminescent vibriosis.32 Therefore, alternative
methods will need to be developed to prevent the outbreak of vib-
riosis in aquaculture.21 One possible strategy could be interference
with quorum sensing. V. harveyi makes use of three parallel quo-
rum sensing systems (resp. HAI-1, CAI-1 and AI-2 system) that con-
verge to control the production of the master regulator LuxR,33
which regulates the expression of phenotypes such as biolumines-
cence, siderophore production, biofilm formation, and virulence. A
natural furanone as well as several synthetic analogues have been
shown to inhibit luminescence and virulence of Vibrio spe-
cies.5,8,19,20,24,34–37 Recently, the natural furanone was shown to de-
crease the DNA-binding capacity of the master regulator LuxR.36
However, in vivo experiments revealed toxic effects of this natural
furanone and the synthetic furanone C-30 at low concentrations
against the rotifer Brachionus plicatilis, the brine shrimp Artemia
franciscana, and the rainbow trout.19,20,35 Therefore, we screened
our library of furanones for their ability to inhibit luminescence
production of V. harveyi, in an attempt to find analogues with a
higher anti-quorum sensing activity and possibly a higher thera-
peutic index.
sion yield of 95% with a selectivity of 90% of the desired isomers
9c–d, according to the literature.42 The crude product was directly
used in the deprotection step, which employs trifluoroacetic acid
in dry chloroform. The final step consists of a one-pot Hunsdiecker
bromination/debromocarboxylation and a bromination/dehydro-
bromination promoted by microwave, adapted from a simple bro-
mination/dehydrobromination procedure.43 The brominating
agent in the reaction is N-bromosuccinimide (NBS) and the base
used for dehydrobromination is lithium acetate (LiOAc). When
we used an excess of NBS of 6 equiv and an excess of lithium ace-
tate of 2 equiv, this reaction permitted us to obtain up to 30% yield
of the dibrominated compounds 4c–d. Because of the difference in
reactivity of the two double bonds, we could easily perform the
selective bromination of the methylene position. In the microwave
promoted reaction, we simply decreased the proportion of NBS to
2 equiv and LiOAc to 0.5 equiv, leading to 45% yield of product 11c.
With this synthesis pathway, we can produce compounds 4 and 11
with any 3-alkyl side chain, except for a methyl group because of
possible bromination of the methyl group.9 Conclusively, these
four steps gave stable products, with good yields and predictable
results. Moreover, the last step is relatively selective and permits
some adaptations. Full details and insights in this new route will
be published in an upcoming paper.
In order to synthesize mono-brominated 3-alkyl-2(5H)-fura-
nones that are selectively brominated at the 4-position of the ring,
we followed an alteration of an existing synthesis pathway,44 also
starting from the anhydride. As depicted in Scheme 2 (pathway C),
the first reaction is a classical Grignard reaction implying an anhy-
dride with methylmagnesium iodide as Grignard agent. Yields of
compound 12c evolved around 50%, as expected from the litera-
ture.44 The second reaction is the bromination of the double bound
using an excess of Br2. After the reaction, we surprisingly found
that a dehydrobromination and a dehydration of the molecule oc-
curred in the same reaction mixture which had been treated with
Br2. This unexpected behaviour gave us directly the desired com-
pound 13c, with 24% yield. This allowed us to avoid the dehydro-
bromination and the dehydration steps.
2. 3-Alkyl-5-methylene-2(5H)-furanones
2.1. Chemistry
The brominated furanones 1–3 without a 3-alkyl chain were
synthesized by using an adaptation of the protocol of Kumar and
Read (2002), as shown in Scheme 1. The brominated 3-alkyl-5-
methylene-2(5H)-furanones 4a–f, 5a–c and 6a–b, shown in
Scheme 2, were initially synthesized by the method developed
by Beechan and Sims38 and reinvestigated by Manny et al.39
Although this pathway, depicted in Scheme 2 (pathway A), is the
most versatile procedure available, it has some major drawbacks.
First, the bromination step (step 3) and the acid-catalyzed oxida-
tive cyclodehydration step (step 4) make use of harsh, environ-
mentally unfriendly conditions. Second, the cyclodehydration
step is not selective, as mixtures of different di- and tribrominated
compounds are formed, resulting in ratios that are difficult to con-
trol. This means that extensive purification and separation of com-
pounds are required. Finally, mono-brominated compounds that
are specifically brominated at the 4-position of the ring or the
methylene position are not (or only in trace amounts) observed
in the reaction mixture. The latter is especially problematic in
the context of this study, since our previous results indicate a dras-
tic influence of the bromination pattern of the ring structure on the
biological activity of the compounds.10 To study the influence of
the bromination pattern more in detail, a selective synthesis of
these mono-brominated furanones was needed. Taking these
shortcomings of the Manny synthesis into account, it was decided
to develop a new synthesis procedure.
This new synthesis pathway, shown in Scheme 2 (pathway B), is
built around the Wittig reaction on maleic anhydride derivatives.
The first step consisted of the synthesis of an alkylmaleic anhy-
dride 8b–d starting from the corresponding commercially avail-
able alkyne 7b–d. The reaction was performed in an atmosphere
composed by CO/CO2/air with the following proportions: 3/8/1.40
The couple PdI2/KI was used as catalytic system. Maleic anhydrides
with alkyl chain lengths of four (8b), six (8c) and eight (8d) carbon
atoms have been synthesized, with yields around 60–70%. The sec-
ond step, the Wittig reaction,24,41 required the use of a protected
ylide.42 The coupling reaction with anhydrides 8c–d gave a conver-
Finally, it was attempted to simplify the structure of the active
molecules. Considering all efforts required to prepare furanones
starting from anhydrides, it was decided to test the unbrominated
alkylmaleic anhydride 8c and the brominated alkylmaleic anhy-
dride 20c, which has, to the best of our knowledge, never been de-
scribed before. The synthesis pathway used for 20c is based on two
reactions already described, as shown in Scheme 2 (pathway D).
2.2. Biological results
Firstly, the activity of the furanones 4a–c, 5a–c, 6a, 11c and 13c
on biofilm formation of S. Typhimurium ATCC14028 was tested,
using a 96-well microtiter plate assay with polystyrene pegs and
crystal violet staining. In our previous study, which was focused
on the effect of the length of the 3-alkyl chain, we found that com-
pounds with ethyl, butyl, and hexyl side chains have the most
interesting activity as they show a broad concentration range in
which they inhibit biofilm formation without affecting the plank-
tonic growth. Compounds without a 3-alkyl chain were found to
be toxic to the planktonic cells, while compounds with a longer
side chain were shown not to reduce biofilm formation.10
In this study, focus is on the effect of the bromination pattern of
the ring structure on the activity of the furanones with the most
interesting side chain lengths (ethyl, butyl and hexyl). Table 1 lists
the concentrations of furanones needed to inhibit Salmonella bio-
film formation by 50% (IC50s). Within this range from ethyl to
hexyl, the length of the alkyl chain does not have a major effect
on the antagonistic activity. However, the bromination pattern of
the ring has a large impact on the biofilm inhibition. Dibrominated

H. P. Steenackers et al. / Bioorg. Med. Chem. 18 (2010) 5224–5233
5227
Table 1
Antagonistic effect of compounds 4–6, 11c, 13c, 8c and 20c against S. Typhimurium biofilm formation and planktonic growth
a
Compound
IC50
(
lM)
95% Confidence interval for IC50
Effect on growth at 250
l
M
Effect on growth at 100
lM
Effect on growth at 10 lM
b
c
d
4a
5a
6a
4b
5b
4c
5c
11c
13c
8c
17.91
199.9
57.46
23.12
148
10.74
160.1
>1000
19.42
>1000
65.89
12.57–25.54
126.6–315.6
36.17–91.29
14.52–36.80
64.76–338.3
8.056–14.33
103.4–247.8
+
+ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
nde
+
nd
+ꢀ
nd
nd
+
nd
ꢀ
nd
nd
+
nd
ꢀ
nd
nd
ꢀ
12.97–29.09
42.90–101.2
nd
ꢀ
nd
nd
20c
a
b
c
IC50: Concentration of inhibitor needed to inhibit biofilm formation by 50%.
+: The planktonic growth is completely or almost completely inhibited when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor.
+ꢀ: The planktonic growth is slightly slowed down when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor.
ꢀ: The planktonic growth is not affected when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor.
nd: Not determined.
d
e
compounds 4a–c with one bromine atom on the 4-position of the
ring and one bromine atom on the methylene group (di1-configu-
ration) are much more active than, respectively compounds 5a–c
with a dibrominated methylene group (di2-configuration). More-
over, the mono-brominated compound 13c with its bromine atom
on the 4-position had a similar activity as compound 4c, while the
mono-brominated compound 11c with its bromine atom on the
methylene group was not active at the highest concentration
tested. This indicates that the bromine at position 4 is crucial for
the biofilm inhibitory activity. To validate that the compounds
are true inhibitors of biofilm formation and not acting as bacterici-
dal agents, growth curves were determined in the presence of dif-
ferent concentrations of the most active furanones by measuring
the OD600 in function of time using an automated system (Bio-
screen, Oy Growth Curves Ab Ltd). All the furanones tested inhib-
ited biofilm formation by 50% at a concentration that did not
affect planktonic growth (Table 1).
very interesting result as 20c is chemically much more easily avail-
able. Moreover, this compound did not affect the planktonic
growth of Salmonella at 250 lM, which was the highest concentra-
tion tested.
The ability of furanones 1–3, 4a–f, 5a–c, 6a–b and 13c to inhibit
the quorum sensing regulated bioluminescence of V. harveyi BB120
was also investigated. Both the IC50 values (concentrations inhibit-
ing the luminescence expression by 50%) and the GIC50 values
(concentrations reducing the growth (OD600) of the V. harveyi cul-
tures by 50%) are listed in Table 2. The compounds 1–3 without
side chain and the compounds 4a and 6a with an ethyl side chain
inhibited the bioluminescence expression at low concentrations,
but also inhibited the growth at low concentrations. Also the tri-
brominated compound 6b with a butyl side chain was highly toxic
for the bacteria. For these compounds, inhibition of the lumines-
cence could at least be partly due to a reduction of the cell number.
However, all mono- and dibrominated compounds with alkyl
chains of 4 to 10 carbon atoms showed a clear concentration range
with only inhibition of bioluminescence and no effect on the
growth. Only compound 4f, with a dodecyl side chain, did not af-
fect the bioluminescence, which could be explained by a low solu-
bility in the growth medium and/or difficult access to the cell. In
accordance with the Salmonella biofilm inhibitory activity, the ef-
fect on the bioluminescence was clearly influenced by the bromin-
The unbrominated maleic anhydride 8c, which is an intermedi-
ate in the new synthesis pathway towards halogenated furanones,
did not inhibit biofilm formation at the highest concentration
tested. However, the C4-brominated maleic anhydride 20c, inhib-
ited Salmonella biofilm formation with an IC50 of 65.89 lM (Ta-
ble 1), which is about six times higher than the IC50 of the
corresponding 3-hexyl-2(5H)furanone 4c. Nevertheless, this is a
Table 2
Antagonistic effect of compounds 1–6, 8c, 13c and 20c against V. harveyi bioluminescence
a
b
Compound
IC50
95% Confidence interval
GIC50
95% Confidence interval
1
2
3
4.071
2.708
2.878
9.268
104.6
1.152
1.362
18.9
3.207–5.168
1.687–4.348
1.640–5.051
8.686–9.889
81.88–133.6
1.000–1.328
1.167–1.589
17.18–20.80
13.38–15.41
10.35–13.69
47.98–147.2
2.522–4.623
19.81–35.78
—
15.7
13.22–18.65
2.854–3.489
15.99–40.68
10.46–11.12
232.8–453.6
1.991–2.574
56.44–75.48
124.2–159.8
19.31–21.77
28.66–40.44
164.3–237.1
86.85–820.6
3.155
25.51
10.79
325
2.264
65.27
140.9
20.5
34.04
197.4
267
>300
—
4a
5a
6a
4b
5b
6b
4c
5c
4d
4e
4f
14.36
11.91
84.05
3.414
26.62
c
—
—
13c
8c
20c
1.357
97.77
10.56
1.046–1.761
89.94–106.3
8.257–13.51
36.30
639.5
208.3
30.49–43.22
538.9–759.0
185.9–233.5
a
b
c
IC50: Concentration of inhibitor needed to inhibit luminescence by 50%.
GIC50: Concentration of inhibitor needed to inhibit bacterial growth by 50%.
ꢀ: No effect observed at the highest concentration tested (1000
lM).

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H. P. Steenackers et al. / Bioorg. Med. Chem. 18 (2010) 5224–5233
ation pattern of the ring structure. Dibrominated molecules 4a–c
with a di1-configuration showed a higher activity than, respec-
tively compounds 5a–c with a di2-configuration. Interestingly,
the chemically easily accessible 3-bromo-4-hexylmaleic anhydride
20c also inhibited the bioluminescence of Vibrio at low concentra-
tions (IC50 = 10.56 lM).
Compound 4b has previously been shown to have a similar im-
pact on the bioluminescence of wildtype V. harveyi BB120 and the
constitutively luminescent mutant strains JAF553,45 JAF483,45 and
BNL258,46 which are affected in the genes encoding, respectively
LuxU, LuxO and Hfq.36 LuxU is the most upstream protein in the
common part of the signal transduction pathway that is shared
by the 3 quorum sensing systems, while LuxO is located immedi-
ately downstream of LuxU. Hfq is a chaperone, which plays a role
at the end of the pathway, immediately upstream of the response
regulator LuxR. The fact that compound 4b still blocked biolumi-
nescence in the
Dhfq mutant was given as evidence by Defoirdt
et al. that this compound acts downstream of Hfq, that is, at the le-
vel of the luxR mRNA and/or the LuxR protein.36 In order to get a
tentative idea whether the mono-brominated furanone 13c and
the brominated anhydride 20c act on the same target as the dibro-
minated furanone 4b, we compared the influence of these 3 com-
pounds on the bioluminescence of wildtype V. harveyi BB120 and
the mutant strains JAF553, JAF483 and BNL258. As depicted in Fig-
ure 1, compounds 13c and 20c had a similar effect on the biolumi-
nescence of the different strains as observed for compound 4b.
From this we may conclude that compounds 13c and 20c like com-
pound 4b act downstream of Hfq.
The potential application of the compounds as anti-pathogenic
drugs depends on the therapeutic window between the IC50 for
biofilm or quorum sensing inhibition and the toxicity in eukaryotic
cells. To compare the cytotoxicity of the new compounds 13c and
20c to the previously described compound 4a, we determined the
influence of the three compounds on the proliferation of five
eukaryotic cell lines. As shown in Table 3, the new compounds
do not seem to have an improved therapeutic index as compared
to compound 4a.
3. 10-Substituted 3-alkyl-5-methylene-2(5H)-furanones
3.1. Chemistry
To study the influence of functionalization of the alkyl chain,
compounds 14a–c, 15a–b, 16a and 17–19 with a bromine atom or
acetoxy group on the 10 position of the side chain were synthesized
using methods developed by Kumar and Read,22 as shown in
Scheme 3. Since the furanones with an ethyl, butyl and hexyl side
chain showed the most interesting activity, the functionalization
was only performed on furanones with these particular side chain
lengths. Briefly, bromination of the side chain was performed via a
radical reaction with NBS in CCl4 in the presence of small amounts
of the initiator benzoyl peroxide. The reaction mixture was irradi-
ated with light and refluxed during an 18 h period. After purification,
some of the 3-(10-bromoalkyl)-5-methylene-2(5H)-furanones were
converted into 3-(10-acetoxyalkyl)-5-methylene-2(5H)-furanones
via a nucleophilic substitution with sodium acetate in acetic acid.
Figure 1. Influence of a dilution series of compounds 4b, 13c and 20c on the
bioluminescence of wildtype V. harveyi BB120 and the mutant strains BNL258
(hfq::Tn5lacZ), JAF483 (luxO D47A linked to KanR) and JAF553 (luxU H58A linked to
KanR). The bioluminescence of each strain in the presence of a compound is
expressed as a percentage compared to an untreated control for that strain.
3.2. Biological results
chain overrules the necessity of a bromine atom on the 4-position
of the ring. The increased activity could be explained by the hypoth-
esis that the target receptor binds covalently to the halogenated
furanones via a nucleophilic substitution of a bromine atom. The
bromine atom on the side chain of the furanone is much more prone
to nucleophilic substitution than the other bromine atoms. Indirect
support for this hypothesis is provided by Hjelmgaard et al., who
tested an array of brominated and non-brominated furanones for
As shown in Table 4 the molecules with a bromine atom on the
side chain were remarkably more active in the Salmonella biofilm
inhibition assay than the molecules without this substitution. Sur-
prisingly, there was no clear difference in activity between the mol-
ecules with a di1- or di2-bromination pattern of the ring structure,
which indicates that the presence of a bromine atom on the alkyl

H. P. Steenackers et al. / Bioorg. Med. Chem. 18 (2010) 5224–5233
5229
Table 3
showed that the furanones do not affect the expression of AI-2 reg-
ulated genes in Salmonella.10 Bioscreen analysis revealed that the
molecules 14a and 15a–b retarded the planktonic growth of the
bacteria slightly at concentrations near the IC50 value for biofilm
inhibition, which means that it cannot be excluded that biofilm for-
mation is (at least partly) inhibited by reducing the number of
planktonic cells before the biofilms are established. However, mol-
ecules 14b–c are clearly selective biofilm inhibitors as they showed
a wide concentration window with only biofilm inhibition and no
effect on the planktonic growth. Molecules 17b and 18a with an
acetoxy function at the first carbon atom of the side chain did not
show an improved activity as compared to their unsubstituted
counterparts. Compound 19a did have a slightly improved activity.
Similar structure–activity relationships were found in the case
of the inhibition of the quorum sensing regulated bioluminescence
of V. harveyi (Table 5). In general introduction of a bromine atom
on the side chain improved the anti-bioluminescence activity. This
effect was most pronounced for the compounds 15a–b with a di2-
configuration. However, all compounds with a bromine on the side
chain also inhibited the growth of the bacteria, starting from con-
centrations that are similar or only slightly higher than the concen-
trations at which inhibition of bioluminescence occurred (the
GIC50 is only slightly higher than the IC50). This means that the bio-
luminescence inhibition is partly due to a reduction of the cell
number. Nevertheless, we continue to observe a dose-dependent
inhibition when the output is expressed as relative light units
(RLU) that is, light intensity divided by OD600 (data not shown).
This could suggest that these 10-brominated furanones have a spe-
cific, non-growth related effect on the bioluminescence. As in the
case of Salmonella biofilm inhibition, substitution with an acetoxy
group did not improve the activity.
Inhibitory effect of compounds 4a, 13c and 20c on the proliferation of murine
leukemia cells (L1210/0), murine mammary carcinoma cells (FM3A) and human T-
lymphocyte cells (CEM/0), human cervix carcinoma cells (HeLa) and human
osteosarcoma cells (OST TKꢀ
)
a
Compound
L1210/0
IC50
(lM)
FM3A/0
>500
CEM/0
Hela
39
48 20
156 31
OST TKꢀ
4a
13c
20c
284
8.5 0.4
208 13
9
208 191
7.5 2.3
90 49
0
6.3 3.2
2.0 0.9
16
2
243 17
43
7
a
IC50: Concentration of compound needed to inhibit the proliferation by 50%.
In conclusion, we synthesized a library of 25 10-unsubstituted
and 10-subsituted 3-alkyl-5-methylene-2(5H)-furanones and two
3-alkylmaleic anhydrides and tested their antagonistic effect
against the biofilm formation by S. Typhimurium and the quorum
sensing regulated bioluminescence of V. harveyi. Firstly, we found
that the length of the 3-alkyl chain plays an important role in
the antagonistic activity.10 The molecules without a 3-alkyl chain
were shown to be highly toxic for both Salmonella and Vibrio, while
the 10-unsubstituted furanones with a long 3-alkyl chain did not
reduce biofilm formation (octyl chain and longer) nor biolumines-
cence (dodecyl chain). However, the 10-unsubstituted furanones
with ethyl, butyl or hexyl side chains inhibited biofilm formation
at low concentrations, without affecting the planktonic growth at
these concentrations. Similarly, the 10-unsubstituted furanones
with a butyl to decyl side chain inhibited bioluminescence without
affecting the planktonic growth of the bacteria at the same concen-
trations. Secondly, the bromination pattern of the ring structure
was shown to have a large influence on the antagonistic activity
Scheme 3. Synthesis of 10-bromo- and 10-acetoxy-3-alkyl-2(5H)-furanones with
different bromination patterns of the ring structure.
quorum sensing antagonism and found that the brominated com-
pounds were much more efficient.23 This hypothesis is further sup-
ported by the finding of Zang et al. that furanone 4b is able to
covalently modify purified recombinant Bacillus subtilis LuxS en-
zymes (which is the synthase of AI-2).14 However, in Salmonella
the target is probably not LuxS, as our previous experiments
Table 4
Antagonistic effect of compounds 14–19 against S. Typhimurium biofilm formation and planktonic growth
a
Compound IC50
95% Confidence interval for
IC50
Effect on growth at
Effect on growth at
Effect on growth at
Effect on growth at
(
l
M)
250
l
M
100
lM
10
lM
1
lM
b
14a
15a
16a
14b
15b
14c
17b
18a
19a
4.505
1.166
4.094
5.714
1.31
3.235
131.6
161.6
7.295
3.170–6.401
0.8035–1.693
2.679–6.256
4.503–7.251
1.115–1.541
2.389–4.379
96.03–180.5
124.4–209.8
3.375–15.77
+
+
+
+ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
c
+ꢀ
+ꢀ
+ꢀ
+
+
ꢀ
+
+
+ꢀ
ꢀ
+ꢀ
ꢀ
ꢀ
ꢀ
+ꢀ
d
ꢀ
ꢀ
+ꢀ
ꢀ
+
ꢀ
a
b
c
IC50: Concentration of inhibitor needed to inhibit biofilm formation by 50%.
+: The planktonic growth is completely or almost completely prevented when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor.
+ꢀ: The planktonic growth is slightly slowed down when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor.
ꢀ: The planktonic growth is not affected when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor.
d

5230
H. P. Steenackers et al. / Bioorg. Med. Chem. 18 (2010) 5224–5233
Table 5
Antagonistic effect of compounds 14–19 against V. harveyi bioluminescence
a
b
Compound
IC50
95% Confidence interval
GIC50
95% Confidence interval
14a
15a
16a
14b
15b
14c
17b
19a
6.339
0.7742
2.761
1.180
0.1999
5.207
50.05
6.311
5.700–7.050
0.6119–0.9795
2.379–3.205
0.9382–1.484
0.1459–0.2740
4.273–6.345
36.05–69.48
5.640–7.062
16.14
3.073
9.738
4.042
1.074
7.271
147.2
25.38
14.49–17.97
2.639–3.579
8.571–11.07
3.451–4.735
0.9306–1.240
6.401–8.260
136.5–158.7
22.16–29.06
a
IC50: Concentration of inhibitor needed to inhibit luminescence by 50%.
GIC50: Concentration of inhibitor needed to inhibit bacterial growth by 50%.
b
of the 10-unsubstituted furanones: the dibrominated compounds
with one bromine atom on the 4-position of the ring and one bro-
mine atom on the methylene group (di1-configuration) were much
more active than the compounds with a dibrominated methylene
group (di2-configuration), both in the Salmonella and Vibrio test
system. Furthermore, we found that the introduction of a bromine
atom on the first carbon atom of the alkyl side chain drastically im-
proved the activity of the furanones in both test systems. This ef-
and filled, respectively with 5 atm of air, 40 atm of CO2 and
16 atm of CO. The reactor was heated at 80 °C for 40 h. After degas-
sing, the solution was filtered. The filtrate was concentrated under
vacuum. The resulting mixture was filtered on silica with DCM as
eluent.
4.1.4. Generalprocedurefor3-alkyl-4-bromo-5-(bromomethylene)-
2(5H)-furanones and 3-alkyl-5-bromomethylene-2(5H)-furanones
starting from alkylmaleic anhydrides (Scheme 2 (pathway B))
4.1.4.1. Wittig coupling procedure. A solution of (tert-butoxy-
carbonylmethyl)-triphenylphosphonium bromide (4.5 mmol) in
DCM (10 ml) was vigorously stirred with 10 ml of a solution of
Na2CO3 (10% w/w in water) for 30 min. The phases were separated
and the aqueous phase was washed twice with DCM. The com-
bined organic phases were concentrated under vacuum. The ob-
tained solid was poured in a solution of alkylmaleic anhydride
(4.5 mmol in 30 ml benzene). This solution was flushed with argon
and then stirred during 2.5 h at reflux. After the reaction was com-
pleted, the solvent was removed under vacuum. The residue was
triturated by hexane and filtrated. The filtrate was concentrated
under vacuum and purified by chromatography.
fect was most pronounced for the molecules with
a di2-
configuration. On the other hand, in general the placement of an
acetoxy function at this position did not improve the activity of
the molecules. Finally, we demonstrated the potential of the bro-
moalkylmaleic anhydrides as a new and easily accessible class of
biofilm and quorum sensing inhibitors.
4. Experimental section
4.1. Chemistry
All reagents used for synthesis were purchased from commer-
cial sources and used without further purification. Chromatogra-
phy was performed using a Biotage flash Purification system SP1
and adapted cartridges KP-Sil. NMR solvents were obtained from
Acros. 1H NMR and 13C NMR spectra were recorded at room tem-
perature on a 300 MHz Bruker spectrometer. Chromatograms and
mass spectra were obtained from an Agilent 6890N GC–MS. Micro-
wave enhanced reactions were performed on a CEM Single-Mode
Microwave oven.
4.1.4.2. Deprotection procedure. The protected furanone (5 mmol)
was stirred at room temperature for 30 min in a mixture of 15 ml
DCM and 7.5 ml of CF3COOH. The solution was concentrated under
strong vacuum to remove the excess of trifluoroacetic acid.
4.1.4.3. Bromination/debromocarboxylation and bromination/
dehydrobromination procedure. In an adapted vial we placed
the (E/Z)-2-(4-alkyl-5-oxofuran-2(5H)-ylidene)acetic acid com-
pound (1 mmol) with NBS and LiOAcꢁ2H2O. The proportions were
depending on the desired product: for the synthesis of 3-alkyl-4-
bromo-5-(bromomethylene)-2(5H)-furanones we used 6 equiv
NBS and 2 equiv LiOAc; for the synthesis of 3-alkyl-4-bromo-5-
methylene-2(5H)-furanones we used 2 equiv NBS and 0.5 equiv
LiOAc. We added 2 ml of acetonitrile just before the reaction. The
microwave oven was limited to 100 W of power, the cut-off pres-
sure inside the vial was 280 psi, and the temperature was 100 °C.
The program started with 2 min of gradual heating, followed by
90 s at maximum temperature. After reaction, the tube was cooled
down. The solvent was removed under vacuum and the residue
was purified by chromatography.
4.1.1. General procedure for 5-methylene-2(5H)-furanones
without 3-alkyl side (Scheme 1)
The molecules were prepared following a method adapted from a
procedure described in the literature.47 4-Oxopentanoic acid was bro-
minated with an equimolar amount of Br2 to yield mono-brominated
4-oxopentanoic acid. Subsequently this crude product was cyclized
using 100% H2SO4 to yield a mixture of compounds 1–3. Analytical
data of the compounds are provided as Supplementary data.
4.1.2. General procedure for 3-alkyl-5-methylene-2(5H)-
furanones via protocol of Manny et al. (1999) (Scheme 2
(pathway A))
The molecules were prepared following a procedure described
in the literature.39 Analytical data of the compounds have been
provided as Supplementary data.
4.1.4.4. (E/Z)-2-(4-Hexyl-5-oxofuran-2(5H)-ylidene)acetic acid
(10c). White solid-1H NMR (300 MHz, CDCl3): d 8.02 (1H, s,
–OH); 5.84 (1H, s, –CHCO); 2,41 (2H, t, J 7.5 Hz, (H10)2), 1.63 (2H,
q, J 7.4 Hz, (H20)2), 1.30 (6H, m, (H30)2 to (H50)2), 0.88 (3H, t, J
6.3 Hz). 13C NMR (CDCl3): d 14.2; 22.7; 25.8; 27.6; 29; 31.6; 90.8;
135.5; 136.4; 161.5; 168.6; 170.4. Mass spectrum: m/z 224 (M,
2.2%), 206 (5.8),180 (9.6),179 (79.7), 178 (5.6), 155 (10.6), 154
(100), 141 (13.1), 136 (52.1), 126 (20.3), 108 (16.7), 69 (32.8), 67
(12.3), 55 (16.2), 53 (13.7), 43 (46.6), 42 (10.2), 41 (35.7), 39 (21.7).
4.1.3. General procedure for alkylmaleic anhydrides (8b–d)
(Scheme 2 (pathway B))
4.1.3.1. Oxidative carbonylation procedure. 50 ml of dioxane
was poured into a 300 ml stainless steel Parr reactor. Alkyne
(8 mmol), H2O (20 mmol), KI (0.4 mmol) and PdI2 (0.04 mmol)
were added to the solvent. The Parr reactor was carefully closed

H. P. Steenackers et al. / Bioorg. Med. Chem. 18 (2010) 5224–5233
5231
4.1.4.5. (E/Z)-2-(4-Octyl-5-oxo-furan-2(5H)-ylidene)acetic acid
(10d). White solid- 1H NMR (300 MHz, CDCl3): d 7.95 (1H, s,
–OH); 5.83 (1H, s, –CHCO); 2,44 (2H, td, J 7.3 Hz, (H10)2), 1.62
(2H, q, J 7.4 Hz, (H20)2), 1.31 (10H, m, (H30)2 to (H70)2), 0.90 (3H,
t, J 6.4 Hz). 13C NMR (CDCl3): d 14; 22.4; 25.8; 27.3; 28.9; 31.4;
99.7; 128.5; 134.7; 141.1; 161.5; 168.5; 170.4. Mass spectrum:
m/z 252 (M, 2.4%), 234 (3.8), 208 (11.7), 207 (81.2), 164 (10), 155
(14.7), 154 (100), 141 (15.8), 137 (13.9), 136 (49), 126 (17.7), 108
(15.9), 69 (28.5), 67 (11.6), 57 (20.4), 55 (18.4), 43 (24.4), 41
(33.6), 39 (13.4).
(M(79Br), 9.7), 216.9 (1.4), 215 (0.9), 203 (3.8), 200.9 (4.3), 190.9
(10.5), 190 (44.4), 189 (19.4), 188 (45.8), 187 (9.4), 179 (79.0),
162 (12.1), 159.9 (12.6), 151 (55.5), 137 (83.6), 135 (18,0), 133
(16.2), 123 (19.0), 121.9 (20.9), 119.9 (18.4), 111 (15.7), 110
(28.1), 109 (100.0), 107 (15.8), 95 (23.5), 93 (14.2), 91 (14.2), 81
(46.4), 79 (32.1), 69 (30.6), 67 (58.3), 65 (29.6), 55 (37.7), 53
(51.2), 51 (32.7), 43 (99.5), 41 (92.7), 39 (83.9).
4.1.6. General procedure for bromoalkylmaleic anhydrides
starting from alkylmaleic anhydrides (Scheme 2 (pathway D))
The bromination of the alkylmaleic anhydrides was performed
via the bromo/dehydrobromination procedure described above
for the bromination of 3-alkyl-5-hydroxy-5-methyl-2(5H)-
furanones.
4.1.4.6. 3-Hexyl-5-bromomethylene-2(5H)-furanone (11c). Light
yellow solid - 1H NMR (300 MHz, CDCl3): d 7.03 (1H, s, H4); 5.95
(1H, s, 5-CHBr); 2.34 (2H, t, J 7.6 Hz, (H10)2), 1.58 (2H, q, J
7.46 Hz, (H20)2), 1.30 (6H, m, (H30)2 to (H50)2), 0.88 (3H, t, J
6.3 Hz). 13C NMR (CDCl3): d 14.6; 22.9; 25.8; 27.7; 29.2; 31.83;
89.8; 135.3; 136.5; 151.8; 169.5. Mass spectrum: m/z 261 (0.4),
260 (M(81Br), 2.7%), 259 (0.5), 258 (M(79Br), 2.7%), 217 (0.4), 215
(0.4), 203 (2.6), 201 (2.6), 190 (18.6), 189 (4.7), 188 (20), 179
(100), 162 (5.5), 160 (5.9), 133 (4.2), 122 (7.8), 120 (7.4), 109
(12.5), 95 (3.5), 81 (5.8), 79 (3.8), 65 (5.2), 53 (5.8), 43 (11.6), 41
(10.5).
4.1.6.1. Bromohexylmaleic anhydride (20c). White solid - 1H
NMR (300 MHz, CDCl3): d 2.55 (2H, t, J 7.4 Hz, (H10)2); 1.64 (2H,
q, J 7.3 Hz, (H20)2); 1.27 (6H, m, (H30)2 to (H50)2); 0.88 (3H, t, J
6.5 Hz, (H60)3). 13C NMR (CDCl3): d 14.5; 23; 26.4; 27.2; 29.4;
32.1; 125.4; 126.8; 149.5; 160.6. Mass spectrum: m/z 262
(M(81Br), 0.1), 260 (M(79Br),0.1), 243 (0.2), 240.9 (0.3), 233.9
(0.7), 232 (0.8), 217 (1.5), 215 (1.5), 205 (3.2), 203 (2.8), 192
(63.1), 190 (66.0), 181 (5.4), 164 (17.9), 161,9 (17.6), 161 (4.9),
159 (5.5), 153 (7.8), 147 (5.2), 145 (3.7), 135 (12.6), 125 (8.4),
118.9 (8.6), 117 (8.9), 111 (11.8), 107 (15.3), 81 (6.6), 79 (17.6),
77 (9.2), 71 (13.5), 70 (15.4), 69 (10.7), 67 (15.0), 66 (4.2), 55
(26.7), 53 (13.4), 51 (17.4), 43 (100.0), 41 (58.8), 39 (314).
4.1.5. General procedure for 3-alkyl-4-bromo-5-methylene-
2(5H)-furanones starting from alkylmaleic anhydrides
(Scheme 2 (pathway C))
4.1.5.1. Grignard methylation procedure. To
a solution of
8.9 mmol of alkylmaleic anhydride in 15 ml of anhydrous diethyl-
ether, under argon, at ꢀ20 °C, a solution of methylmagnesium io-
dide was added dropwise (1.1 equiv). After addition, the reaction
was stirred for 2 h at the same temperature. Afterwards, the reac-
tion was quenched with a saturated NH4Cl solution. The biphasic
mixture was extracted with ethyl acetate. The organic phase was
washed with brine, dried with magnesium sulfate, and concen-
trated under vacuum. The residue was purified by chromatography.
4.1.7. General procedure for 3-(10-bromoalkyl)-5-methylene-
2(5H)-furanones (Scheme 3)
The molecules were prepared from the 10-unsubstituted 3-al-
kyl-5-methylene-2(5H)-furanones by following a procedure de-
scribed in the literature.22 Analytical data are provided as
Supplementary data.
4.1.8. General procedure for 3-(10-acetoxalkyl)-5-methylene-
2(5H)-furanones (Scheme 3)
4.1.5.2. Bromo/dehydrobromination procedure. 3-Alkyl-5-hy-
droxy-5-methyl-2(5H)-furanone (1 mmol) and Br2 (1 mmol) were
placed in a vial. The vial was sealed and the mixture was stirred
for 3–4 days. After the reaction was completed, the vial was de-
gassed. Then 4 ml of CHCl3 solvent was added. A solution of pyri-
dine (1 mmol) in 1 ml of CHCl3 was added dropwise over 20 min.
After this addition, the solution was stirred for 1.5 h. The mixture
was concentrated under vacuum and the residue was filtered
through silica with DCM as eluent.
The molecules were prepared from the 3-(10-bromoalkyl)-5-
methylene-2(5H)-furanones by following a procedure described
in the literature.22 Analytical data are provided as Supplementary
data.
4.2. Biological assays
4.2.1. Static peg assay for prevention of S. Typhimurium biofilm
formation10
4.1.5.3. 3-Hexyl-5-hydroxy-5-methyl-2(5H)-furanone(12c). Pale
yellow oil - 1H NMR (300 MHz, CDCl3): d 5.64 (1H, s, –CHCO–); 5.13
(1H, broad s, –OH); 2.27 (2H, m, (H10)2), 1.57 (3H, s, –CH3), 1.55
(2H, q, J 7.8 Hz, (H20)2), 1.26 (6H, m, (H30)2 to (H50)2), 0.83 (3H, t, j
6.5 Hz). 13C NMR (CDCl3): d 14.4; 22.9; 24.1; 26.8; 26.9; 29.3; 31.8;
107.8; 115.6; 172.1; 173.8. Mass spectrum: m/z 198 (0.3%), 184
(9.4), 183 (84), 180 (2.7), 156 (5.5), 155 (54.6), 153 (2.3), 152 (6.3),
138 (3.4), 137 (18.6), 124 (5.2), 123 (8.2), 113 (19.4), 111 (30.1),
110 (46.5), 109 (33.7), 97 (13.9), 95 (31.8), 94 (16.9), 82 (21.8), 81
(55.6), 69 (27.1), 68 (31.3), 67 (41), 53 (19.5), 43 (100), 41 (37.8), 39
(27.8).
The device used for biofilm formation is a platform carrying 96
polystyrene pegs (Nunc no. 445497) that fits as a microtiter plate
lid with a peg hanging into each microtiter plate well (Nunc no.
269789).48
Twofold serial dilutions of the compounds in 100 ll liquid TSB
1/20 broth per well were prepared in the microtiter plate. Subse-
quently, an overnight culture of S. Typhimurium ATCC14028
(grown in Luria-Bertani medium49) was diluted 1:100 into Tryptic
Soy Broth diluted 1/20 (TSB 1/20; BD Biosciences) and 100 ll (ca.
1 ꢂ 106 cells) was added to each well of the microtiter plate, result-
ing in a total amount of 200 ll medium per well. The pegged lid
was placed on the microtiter plate and the plate was incubated
for 48 h at 16 °C without shaking. During this incubation period,
biofilms were formed on the surface of the pegs. After 24 h, the
lid was transferred into a new plate containing medium and the
specific molecules used for testing. For quantification of biofilm
4.1.5.4. 3-Hexyl-4-bromo-5-methylene-2(5H)-furanone (13c).
Brown oil - 1H NMR (300 MHz, CDCl3): d 6.09 (1H, t, J 1.8 Hz, –
CO@CH2); 5.90 (1H, broad s, –CO@CH2); 2.64 (1H, qd,
J
8.2 Hz,(H10)1); 2.35 (1H, qd, J 8.5 Hz, (H10)1); 1.73 (2H, qd, J
7.5 Hz, (H20)2); 1.45 (6H, m, (H30)2 to (H50)2); 0.92 (3H, t, J 6.9 Hz,
(H60)3). 13C NMR (CDCl3): d 14.1; 22.5; 26.4; 27.3; 28.7; 31.4;
43.5; 94.6; 116.7; 167.3; 171.6. Mass spectrum: m/z 260,9
(M+1(81Br), 0.6%), 260 (M(81Br), 8.3), 258.9 (M+1(79Br), 0.8), 258
formation, the pegs were washed once in 200
fered saline (PBS). The remaining attached bacteria were stained
for 30 min with 200 l 0.1% (w/v) crystal violet in an isopropa-
nol/methanol/PBS solution (v/v 1:1:18). Excess stain was rinsed
ll phosphate buf-
l

5232
H. P. Steenackers et al. / Bioorg. Med. Chem. 18 (2010) 5224–5233
off by placing the pegs in a 96-well plate filled with 200
water per well. After the pegs were air dried (30 min), the dye
bound to the adherent cells was extracted with 30% glacial acetic
l
l distilled
(CoE EF/05/007 SymBioSys), and by the Institute for the Promo-
tion of Innovation through Science and Technology in Flanders
(IWT-Vlaanderen) through scholarships to H.S. and J.J. J.V. and
D.D.V. are grateful for support in the frame of the IAP program
Functional Supramolecular Systems. We thank Mrs. Lizette van
Berckelaer for excellent technical assistance. We gratefully
acknowledge B. Bassler and T. Defoirdt for kindly providing the
V. harveyi strains.
acid (200 ll). The OD570 of each well was measured using a VERSA-
max microtiter plate reader (Molecular Devices). The IC50 value for
each compound was determined from concentration gradients in
two or three independent experiments (with two or three repeats
per experiment), by using the GraphPad software of Prism.
4.2.2. Bioscreen assay for measuring S. Typhimurium growth
inhibition
Supplementary data
The Bioscreen device (Oy Growth Curves Ab Ltd) was used for
measuring the influence of the chemical compounds on the plank-
tonic growth of S. Typhimurium. The Bioscreen is a computer con-
trolled incubator/reader/shaker that uses 10 ꢂ 10 well microtiter
plates and measures light absorbance of each well at a specified
wave length in function of time. An overnight culture of S.
Typhimurium ATCC14028 (grown up in LB medium) was diluted
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.05.055.
References and notes
1. Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Science 1999, 284,
1318.
2. Olson, M. E.; Ceri, H.; Morck, D. W.; Buret, A. G.; Read, R. R. Can. J. Vet. Res. 2002,
66, 86.
3. National Institutes of Health. Minutes of the National Advisory dental and
Craniofacial Research Council-153rd Meeting, Bethesda, MD, 1997.
4. Rasmussen, T. B.; Givskov, M. Int. J. Med. Microbiol. 2006, 296, 149.
5. Ren, D.; Sims, J. J.; Wood, T. K. Environ. Microbiol. 2001, 3, 731.
6. Baveja, J. K.; Willcox, M. D.; Hume, E. B.; Kumar, N.; Odell, R.; Poole-Warren, L.
A. Biomaterials 2004, 25, 5003.
7. De Nys, R.; Givskov, M.; Kumar, N.; Kjelleberg, S.; Steinberg, P. D. Prog. Mol.
Subcell. Biol. 2006, 42.
8. Lonn-Stensrud, J.; Petersen, F. C.; Benneche, T.; Scheie, A. A. Oral Microbiol.
Immunol. 2007, 22, 340.
9. Han, Y.; Hou, S.; Simon, K. A.; Ren, D.; Luk, Y.-Y. Bioorg. Med. Chem. Lett. 2008,
18, 1006.
10. Janssens, J. C.; Steenackers, H.; Robijns, S.; Gellens, E.; Levin, J.; Zhao, H.;
Hermans, K.; De Coster, D.; Verhoeven, T. L.; Marchal, K.; Vanderleyden, J.; De
Vos, D. E.; De Keersmaecker, S. C. Appl. Environ. Microbiol. 2008, 74, 6639.
11. Lonn-Stensrud, J.; Landin, M. A.; Benneche, T.; Petersen, F. C.; Scheie, A. A. J.
Antimicrob. Chemother. 2009, 63, 309.
12. Manefield, M.; de Nys, R.; Kumar, N.; Read, R.; Givskov, M.; Steinberg, P.;
Kjelleberg, S. Microbiology 1999, 145, 283.
13. Manefield, M.; Rasmussen, T. B.; Henzter, M.; Andersen, J. B.; Steinberg, P.;
Kjelleberg, S.; Givskov, M. Microbiology 2002, 148, 1119.
14. Zang, T.; Lee, B. W.; Cannon, L. M.; Ritter, K. A.; Dai, S.; Ren, D.; Wood, T. K.;
Zhou, Z. S. Bioorg. Med. Chem. Lett. 2009, 19, 6200.
15. Manefield, M.; Welch, M.; Givskov, M.; Salmond, G. P.; Kjelleberg, S. FEMS
Microbiol. Lett. 2001, 205, 131.
16. Hentzer, M.; Riedel, K.; Rasmussen, T. B.; Heydorn, A.; Andersen, J. B.; Parsek,
M. R.; Rice, S. A.; Eberl, L.; Molin, S.; Hoiby, N.; Kjelleberg, S.; Givskov, M.
Microbiology 2002, 148, 87.
17. Hentzer, M.; Wu, H.; Andersen, J. B.; Riedel, K.; Rasmussen, T. B.; Bagge, N.;
Kumar, N.; Schembri, M. A.; Song, Z.; Kristoffersen, P.; Manefield, M.; Costerton,
J. W.; Molin, S.; Eberl, L.; Steinberg, P.; Kjelleberg, S.; Hoiby, N.; Givskov, M.
EMBO J. 2003, 22, 3803.
18. Wu, H.; Song, Z.; Hentzer, M.; Andersen, J. B.; Molin, S.; Givskov, M.; Hoiby, N. J.
Antimicrob. Chemother. 2004, 53, 1054.
1:200 TSB 1/20 broth. We added 300 ll of the diluted overnight
culture to each well of the 10 ꢂ 10 well microtiter plate. Subse-
quently, serial dilutions of the chemical compounds were prepared
in EtOH. We added 3
(containing the 300
l of the appropriate solvent was also added to the plate in three-
ll of each diluted stock solution to the wells
ll bacterial culture) in threefold. As a control,
3
l
or fourfold. The microtiter plate was incubated in the Bioscreen de-
vice at 16 °C for at least 36 h, with continuous medium shaking.
The absorbance of each well was measured at 600 nm each
15 min. Excel was used to generate the growth curves for the trea-
ted wells and the untreated control wells.
4.2.3. Luminescence assay for inhibition of V. harveyi quorum
sensing
Twofold serial dilutions of the compounds in 100 ll liquid LM
broth per well were prepared in white microtiter plates (Cliniplate,
Thermo Life sciences). Subsequently, an overnight culture (grown
in AB medium50) of V. harveyi strain BB120 (wildtype), BNL258
(hfq::Tn5lacZ),45 JAF483 (luxO D47A linked to KanR),46 or JAF553
(luxU H58A linked to KanR)46 was diluted 1:100 into Luria-Marine
broth46 (after adjusting the OD600 to 0.4) and 100
l
l was added to
each well of the microtiter plates, resulting in a total amount of
200 l medium per well. The microtiter plates were covered with
l
Breathable Sealing Membranes (Greiner Bio-One N.V.) and incu-
bated with aeration for 5 h at 30 °C. After incubation, the lumines-
cence was measured using a CCD camera (PerkinElmer Life
Science) and the OD600 of 135 ll of each well was measured with
a VERSAmax microtiter plate reader. The IC50 and the GIC50 value
for each compound was determined from concentration gradients
in two or three independent experiments (with two or three re-
peats per experiment), by using the GraphPad software of Prism.
19. Defoirdt, T.; Crab, R.; Wood, T. K.; Sorgeloos, P.; Verstraete, W.; Bossier, P. Appl.
Environ. Microbiol. 2006, 72, 6419.
20. Tinh, N. T.; Linh, N. D.; Wood, T. K.; Dierckens, K.; Sorgeloos, P.; Bossier, P. J.
Appl. Microbiol. 2007, 103, 194.
21. Defoirdt, T.; Boon, N.; Sorgeloos, P.; Verstraete, W.; Bossier, P. Isme J. 2008, 2,
19.
22. Read, R. W.; Kumar, N. PCT WO99/54323, 1999.
23. Hjelmgaard, T.; Perssona, T.; Rasmussen, T. B.; Givskov, M.; Nielsen, J. Bioorg.
Med. Chem. 2003, 11, 3261.
24. Benneche, T.; Hussain, Z.; Scheie, A. A.; Lönn-Stensrud, J. New J. Chem. 2008, 32,
1567.
25. Wright, A. D.; de Nys, R.; Angerhofer, C. K.; Pezzuto, J. M.; Gurrath, M. J. Nat.
Prod. 2006, 69, 1180.
26. Parry, C. M.; Threlfall, E. J. Curr. Opin. Infect. Dis. 2008, 21, 531.
27. Latasa, C.; Roux, A.; Toledo-Arana, A.; Ghigo, J. M.; Gamazo, C.; Penades, J. R.;
Lasa, I. Mol. Microbiol. 2005, 58, 1322.
28. Prouty, A. M.; Schwesinger, W. H.; Gunn, J. S. Infect. Immun. 2002, 70, 2640.
29. Barak, J. D.; Whitehand, L. C.; Charkowski, A. O. Appl. Environ. Microbiol. 2002,
68, 4758.
4.2.4. Determination of cytostatic activity
Murine leukemia L1210, murine mammary carcinoma FM3A,
human T-lymphocyte CEM, human cervix carcinoma (HeLa) and
human osteosarcoma (OST) cells were suspended at 300,000–
500,000 cells/mL of culture medium, and 100
sion was added to 100 L of an appropriate dilution of the test
compounds in 200 L-wells of 96-well microtiter plates. After
lL of a cell suspen-
l
l
incubation at 37 °C for two (L1210, FM3A), three (CEM) or four
(HeLa, OST) days, the cell number was determined using a Coulter
counter. The IC50 was defined as the compound concentration re-
quired to inhibit cell proliferation by 50%.
30. Boddicker, J. D.; Ledeboer, N. A.; Jagnow, J.; Jones, B. D.; Clegg, S. Mol. Microbiol.
2002, 45, 1255.
31. Vestby, L. K.; Lonn-Stensrud, J.; Moretro, T.; Langsrud, S.; Aamdal-Scheie, A.;
Benneche, T.; Nesse, L. L. J. Appl. Microbiol. 2009.
32. Cabello, F. C. Environ. Microbiol. 2006, 8, 1137.
Acknowledgements
33. Ng, W. L.; Bassler, B. L. Annu. Rev. Genet. 2009, 43, 197.
34. Manefield, M.; Harris, L.; Rice, S. A.; de Nys, R.; Kjelleberg, S. Appl. Environ.
Microbiol. 2000, 66, 2079.
This work was supported by the Industrial Research Fund of
K.U. Leuven (KP/06/014), the Research Council of K.U. Leuven

H. P. Steenackers et al. / Bioorg. Med. Chem. 18 (2010) 5224–5233
5233
35. Rasch, M.; Buch, C.; Austin, B.; Slierendrecht, W. J.; Ekmann, K. S.; Larsen, J. L.;
43. Bazin, M. A. et al Tetrahedron Lett. 2007, 48, 4347.
44. Haval, K. P.; Argade, N. P. Synthesis 2007, 14, 2198.
45. Lenz, D. H.; Mok, K. C.; Lilley, B. N.; Kulkarni, R. V.; Wingreen, N. S.; Bassler, B. L.
Cell 2004, 118, 69.
46. Freeman, J. A.; Bassler, B. L. Mol. Microbiol. 1999, 31, 665.
47. Kumar, N.; Read, R. W. WO2002000639, 2002.
48. De Keersmaecker, S. C.; Varszegi, C.; van Boxel, N.; Habel, L. W.; Metzger, K.;
Daniels, R.; Marchal, K.; De Vos, D.; Vanderleyden, J. J. Biol. Chem. 2005, 280,
19563.
Johansen, C.; Riedel, K.; Eberl, L.; Givskov, M.; Gram, L. Syst. Appl. Microbiol.
2004, 27, 350.
36. Defoirdt, T.; Miyamoto, C. M.; Wood, T. K.; Meighen, E. A.; Sorgeloos, P.;
Verstraete, W.; Bossier, P. Environ. Microbiol. 2007, 9, 2486.
37. Lowery, C. A.; Abe, T.; Park, J.; Eubanks, L. M.; Sawada, D.; Kaufmann, G. F.;
Janda, K. D. J. Am. Chem. Soc. 2009, 131, 15584.
38. Beechan, C. M.; Sims, J. J. Tetrahedron Lett. 1979, 19, 1649.
39. Manny, A. J.; Kjelleberg, S. K. N.; de Nys, R.; Read, R. W.; Steinberg, P.
Tetrahedron 1997, 53, 15813.
49. Sambrook, J.; Fritsch, E. F.; Maniatis, T. Cold spring Harbor Laboratory Press,
Cold spring Harbor, NY, 1989.
40. Gabriele, B. et al Eur. J. Org. Chem. 2003, 1722.
41. Benneche, T.; Lönn, J.; Scheie, A. A. Synth. Commun. 2006, 36, 1401.
42. Kayser, M. M.; Breau, L. Tetrahedron Lett. 1988, 29, 6203.
50. Greenberg, E. P.; Hastings, J. W.; Ulitzer, S. Arch. Microbiol. 1979, 120, 87.