Indole-triazole conjugates are selective inhibitors and inducers of bacterial biofilms

Article abstract of DOI:10.1039/c3md00064h

Herein is described a method of accessing indole-triazole and benzothiophene-triazole analogues that selectively promote or inhibit biofilm formation by Gram-positive and Gram-negative bacteria. Structure/function studies revealed that the addition of a b

Full text of DOI:10.1039/c3md00064h

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Indole–triazole conjugates are selective inhibitors and
inducers of bacterial biofilms†
Cite this: Med. Chem. Commun., 2013,
, 916
4
Marine J. Minvielle, Cynthia A. Bunders and Christian Melander*
Herein is described a method of accessing indole–triazole and benzothiophene–triazole analogues that
selectively promote or inhibit biofilm formation by Gram-positive and Gram-negative bacteria.
Structure/function studies revealed that the addition of a bromine atom at the 2-position of the indole–
triazole scaffold altered activity against both Gram-negative and Gram-positive bacteria and could
transform a biofilm inhibitor into a biofilm inducer. Isosteric replacement of the indole core by a
benzothiophene significantly impaired anti-biofilm activity. A competition assay exposing Escherichia coli
to the most potent biofilm inducer and an inhibitor of E. coli biofilm formation was performed.
The inducer exhibited the ability to mute the effect of the anti-biofilm compound for this targeted
bacterial population.
Received 26th February 2013
Accepted 5th April 2013
DOI: 10.1039/c3md00064h
www.rsc.org/medchemcomm
Bacterial biolms are structured communities of bacteria biolm communities of commensal bacteria unaffected or
attached to each other and/or to a surface and encased in a actually promoting their development.
1
self-produced matrix of extracellular polymeric substance. It
In this regard, we have been exploring indole derivatives as
is known that biolms offer protective advantages to the potential agents for the selective modulation of biolm devel-
embedded population as they provide shelter, nutrient and opment. Indole is a ubiquitous small molecule signal that
2
metabolic diversity. Bacteria within a biolm have been controls a variety of phenotypes in both Gram-positive and
shown to be upwards of 1000-fold more resistant to antibiotics Gram-negative bacteria. Over 85 species of bacteria have been
as compared to their planktonic counterparts, rendering their documented to produce indole, while both indole-producing
3
eradication difficult. It has been estimated that bacterial and non-producing species will modify their behavior in
9–11
biolms cause 80% of microbial infections in the human response to exogenous indole. Indole has been shown to play
body, including lung infections of cystic brosis patients, a role in acid tolerance, antibiotic resistance and biolm
4,5
gingivitis and infections of indwelling medical devices.
formation. Indole decreases the biolm formation of E. coli in a
Although strategies to develop small molecules to modulate non-toxic manner by repressing motility, chemotaxis and cell
12
biolm development typically focus on pathogenic bacteria, adherence. In contrast with E. coli, indole increases biolm
biolm formation by a number of commensal bacteria is formation in Vibrio cholerae and the non-indole producing
6
13
benecial for their host. Extensive studies of the human Pseudomonas aeruginosa. Many bacteria produce oxygenases
microbiome have established the symbiotic relationship that oxidize indole to generate indole derivatives that may also
14
between human health and changes in the microbiota. For affect biolm formation.
example, bacterial species of the commensal ora present in Our approach to harnessing the potential of synthetic indole
the gastrointestinal tract such as Escherichia coli provide derivatives to control bacterial behavior is underpinned by
crucial health benets, strengthening the mucosal barrier, studying the activity of marine natural product derivatives
promoting polysaccharide digestion and protecting the host containing a deep-seated indole core. To this end, the indole
7
against colonization by pathogenic bacteria. Antimicrobial derived ustramine family of natural compounds has been
agents oen eradicate commensal bacteria as well as patho- previously investigated in our lab and we have demonstrated
genic bacterial populations, which can lead to opportunistic that simple ustramine derivatives have the ability to inuence
8
15,16
infections and a temporarily weakened immune system. In the formation of bacterial biolms.
We established a pyr-
this regard, it is potentially important to generate selective roloindoline–triazole-amide scaffold inspired by ustramine C
therapies that disrupt pathogenic biolms while leaving (Fig. 1) and identied several compounds in this class that
inhibit E. coli, Acinetobacter baumannii, Staphylococcus aureus
15
and methicillin-resistant S. aureus (MRSA) biolm formation.
Department of Chemistry, North Carolina State University, Raleigh, NC, 27695-8204,
USA. E-mail: ccmeland@ncsu.edu; Fax: +1 919-515-5079; Tel: +1 919-513-2960
The lead compound of the pyrroloindoline–triazole library,
compound 1, exhibits non-toxic anti-biolm activity against
E. coli and MRSA. Based upon this initial study, a number of
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3md00064h
1
9
16 | Med. Chem. Commun., 2013, 4, 916–919
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15
are amphipathic, various hydrophobic amides were intro-
duced at the triazole tail. All amide azides were prepared using
a two-step procedure from 2-bromoethylamine hydrobromide
or chloroacetyl chloride via nucleophilic displacement and
21
acylation.
Since many antibiolm agents also present a bromine atom
16,22
in their structure,
we elected to study the impact of bromi-
Fig. 1 Generated scaffold incorporating flustramine C inspiration.
nation upon activity in the context of our simplied ustramine
derivatives by introducing a bromine atom at the 2-position of
the indole ring. To this end, the treatment of 3-propargylindole
structure/function questions arose. Specically, we were inter-
ested in probing: (1) the necessity of the tricyclic pyrroloindo-
line scaffold; (2) the impact of halogenation on activity; (3) the
effect of an isostere replacement of the indole core, and (4)
whether small molecules based upon this scaffold could be
employed as selective biolm agents (Fig. 1).
The synthetic approaches to probe these structure/function
questions are outlined in Schemes 1 and 2. Indole–triazole
analogues could be accessed rapidly in only two steps. The rst
step involved the installation of a terminal alkyne at the 3-
2
with NBS in a mixture of AcOH : HCO H (3 : 1) afforded
2
selective bromination and delivered intermediate 4 in 35%
yield. Minor amounts of isomer brominated in the 4-position
contributed to the observed moderate yield. Finally, the alkyne/
azide cycloaddition was performed to afford the nal products
5c–g in 18–61% yield (Scheme 1).
The synthetic route to the benzothiophene–triazole-amide
conjugates allowed the generation of target compounds in three
steps (Scheme 2). Commercially available 3-bromobenzothio-
phene 6 was converted into the corresponding Grignard reagent
17
position of indole via a zinc-mediated Barbier reaction, which
entailed treatment of indole with propargyl bromide and zinc
dust in THF at ambient temperature to deliver 3-propargylindole
by reaction with Mg and I
2
, then subsequently reacted with
3-(trimethylsilyl)propargyl bromide to afford the TMS-protected
alkyne 7 in 80% yield. Upon treatment with aqueous silver
nitrate and triuoroacetic acid, intermediate 8 was obtained in
quantitative yield. Finally, the alkyne was elaborated through
cycloaddition chemistry to generate the benzothiophene–tri-
azole conjugates 9a–h in 11–73% yield (Scheme 2). To assess the
effect of the benzothiophene core on the biological activity in
comparison to an indole core, the same hydrophobic amides
were employed as previously introduced in the indole–triazole
scaffold.
2
in 30% yield. Other reported methods to synthesize 2 were
18,19
explored;
however, each method failed to deliver 2 or deliv-
ered it in inferior yields to the Barbier reaction. Formation of the
triazole was achieved through Huisgen Cu(I) – catalyzed alkyne/
azide cycloadditions (click chemistry) under standard condi-
tions to generate a small pilot library of indole–triazole
analogues 3a–h in 23–82% yield. Since most biolm modulators
20
Compounds were screened initially at 150 mM to investi-
gate their effects on biolm development using E. coli,
A. baumannii, S. aureus and MRSA as our model bacteria.
Biolm development was monitored under static conditions
23
using a crystal violet reporter assay. For compounds showing
the ability to either inhibit biolm formation, dose–response
studies were subsequently performed to quantify activity. Here
we dene the compound concentration to inhibit 50% of
biolm growth relative to an untreated control as the IC50
value. Analogues inducing biolm formation less than 50% at
5
0 mM were qualied as “inducers” and analogues inducing
biolm formation more than 50% at 50 mM were qualied as
strong inducers”. For the strong inducers of E. coli biolms,
“
we generated dose–response curves and dened the concen-
tration required to promote 50% of biolm formation as the
PC50 value. PC25 and PC75 values were also determined and
dene the concentration of a compound that promotes bio-
Scheme 1 Synthesis of indole–triazole-amide analogues 3a–h and 5c–g. (a) Zn
ꢀ
dust, THF, 0 C to rt; (b) azide, CuSO
4
, sodium ascorbate, H
2
O : tBuOH : DCM
(
2 : 2 : 1); and (c) NBS, HOAc : HCO
2
H (3 : 1), rt.

lm growth by 25% and 75% respectively. Growth curve and
colony counts analyses were performed for each active
compound to determine their toxicity towards planktonic
bacteria at the IC , PC , PC and PC75 values. These anal-
5
0
25
50
yses reveal if a compound inhibits or promotes biolm
formation via a biocidal or non-microbicidal mechanism.
Molecules that modulate biolm formation through non-
microbicidal mechanisms would potentially be highly desir-
able, as they would have a reduced likelihood of exerting
Scheme 2 Synthesis of benzothiophene–triazole analogues 9a–h. (a) Mg, I
pellets, THF, reflux; (b) 3-bromo-1-(trimethylsilyl)-1-propyne; (c)AgNO aq,
CF CO H, acetone, dark, rt; and (d) azide, CuSO sodium ascorbate,
O : tBuOH : DCM (2 : 2 : 1).
2
3
3
2
4
,
H
2
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evolutionary pressure on the bacteria to adapt and become bacterial strain when brominated at the 2-position (5d). This
resistant. result is also observed for Gram-negative bacteria, as moderate
The results of these studies are summarized in Tables 1 and A. baumannii anti-biolm analogues 3d and 3e become inducers
. Each compound affects biolm formation in a unique way for this bacterial strain when brominated (5d and 5e).
2
against the four strains of bacteria. Although most analogues
Interestingly, benzothiophene–triazole-amide 9d was shown
exhibited moderate inhibitory activity, some of them displayed to be selectively active against the formation of S. aureus bio-
remarkable selectivity towards individual bacterial populations. lms in a non-toxic manner while promoting biolm formation
In general, compounds 3b–d strongly promote biolm of the three other bacterial strains. However, this activity
formation in E. coli while selectively inhibiting the formation of appeared to be unique as most of the benzothiophene deriva-
S. aureus biolms with IC50 values of 44.4 mM, 173.6 mM and tives displayed marginal ability to modulate biolm develop-
174.8 mM respectively. The four analogues 3a–d structurally ment. Therefore, it appears that there is no bioisosteric
differ from each other in the para-alkylphenyl chain of the tri- relationship between indole and benzothiophene since the
azole amide substituent. Compounds 3b–d that present a long analogues of these two libraries do not have similar biological
para-substituted alkyl chain strongly promote biolm forma- activity. Growth curve analysis was performed for each inducer
tion of E. coli while compound 3a, which does not present an at a concentration of 50 mM. It was determined that promotion
alkyl appendage at the 4-position, moderately inhibits biolm of biolm was occurring in a non-toxic manner.
formation of this strain. Analogue 5f promotes biolm forma-
Since it is desirable to tune the activity of small molecules
tion of S. aureus and inhibits biolm formation of E. coli, that can selectively inhibit biolm formation in specic bacte-
A. baumannii and MRSA while analogue 5d induces biolm rial strains and promote biolm formation in a targeted
growth for all four bacterial strains. Interestingly, each analogue bacterial population we decided to further investigate the
except 5f demonstrated an opposite effect to that of indole by inducing effect upon biolm formation by E. coli, a bacterial
inducing biolm formation of E. coli. For each compound pre- species present in the gut ora. Of all the synthesized
senting an IC50 value, growth curve analysis and colony counts analogues, compound 3c exhibits the most potent anti-biolm
were performed to evaluate their toxicity. None of these
analogues affected planktonic bacterial growth. In order to
determinate if promoters were inducing biolm formation via a
toxic mechanism, growth curve and colony counts analyses were
Table 2 Efficiency of compounds 3b–d, 9b and 9d in promoting the formation
of E. coli biofilms
completed at the PC , PC and PC75 values. Inducers were
2
5
50
shown to promote biolm formation in a non-toxic manner.
When comparing brominated and non-brominated
analogues, it was established that incorporating a bromine
atom at the 2-position of the indole core attenuated anti-biolm
Compound
PC25
PC50
PC75
3
3
3
b
c
d
48.3 Æ 1.17 mM
47.8 Æ 1.20 mM
50.5 Æ 1.82 mM
26.7 Æ 0.06 mM
22.2 Æ 0.62 mM
74.8 Æ 0.08 mM
82.6 Æ 2.80 mM
80.7 Æ 2.76 mM
99.8 Æ 5.03 mM
56.3 Æ 5.57 mM
111.6 Æ 0.17 mM
160.7 Æ 6.13 mM
119.7 Æ 2.19 mM
151.9 Æ 4.85 mM
93.8 Æ 1.36 mM
activity. Compound 3d, which exhibited an IC50 value of 9b
74.8 mM against S. aureus, becomes a strong inducer of this 9d
1
Table 1 Biological activity of the indole–triazole analogues and the benzothiophene–triazole analogues
Compound
E. coli
A. baumannii
S. aureus
MRSA
3
3
3
3
3
3
3
3
5
5
5
5
5
9
9
9
9
9
9
9
9
a
b
c
d
e
f
g
h
c
d
e
f
g
a
b
c
d
e
f
<5%
<5%
<5%
23% at 150 mM
44.4 Æ 3.57 mM
173.6 Æ 3.16 mM
174.8 Æ 2.12 mM
Inducer
<5%
<5%
<5%
40% at 150 mM
Inducer
Inducer
Strong inducer
<5%
<5%
<5%
<5%
<5%
Strong inducer
Strong inducer
Strong inducer
<5%
Inducer
Inducer
<5%
Inducer
Inducer
Inducer
12% at 150 mM
<5%
14% at 150 mM
Strong inducer
<5%
Strong inducer
<5%
Inducer
25% at 150 mM
30% at 150 mM
15% at 150 mM
<5%
25% at 150 mM
20% at 150 mM
22% at 150 mM
Inducer
312.6 Æ 1.89 mM
<5%
35% at 150 mM
<5%
<5%
<5%
25% at 150 mM
Strong inducer
20% at 150 mM
151.4 Æ 0.91 mM
Inducer
<5%
<5%
<5%
<5%
<5%
Inducer
325.5 Æ 4.63 mM
17% at 150 mM
<5%
<5%
<5%
Inducer
<5%
Inducer
20% at 150 mM
20% at 150 mM
<5%
83.0 Æ 4.45 mM
Strong inducer
214.9 Æ 1.87 mM
<5%
20% at 150 mM
<5%
<5%
g
h
<5%
<5%
<5%
9
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Acknowledgements
The authors would like to thank the National Institutes of
Health (GM055769) for their support.
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