Inhibition of biofilm formation by conformationally constrained indole-based analogues of the marine alkaloid oroidin

Article abstract of DOI:10.1016/j.bmcl.2014.03.094

Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC₅₀ = 20 μM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.

Full text of DOI:10.1016/j.bmcl.2014.03.094

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2530–2534
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of biofilm formation by conformationally constrained
indole-based analogues of the marine alkaloid oroidin
a
b
a
a
a
b
ˇ
ˇ
ˇ
´
´
Ziga Hodnik , Joanna M. Łos , Aleš Zula , Nace Zidar , Ziga Jakopin , Marcin Łos ,
Marija Sollner Dolenc a, Janez Ilaš a, Grzegorz We˛grzyn b, , Lucija Peterlin Mašic , Danijel Kikelj a
ˇ a,
⇑
⇑
a
ˇ
University of Ljubljana, Faculty of Pharmacy, Aškerceva 7, 1000 Ljubljana, Slovenia
b University of Gdan´sk, Department of Molecular Biology, Wita Stwosza 59, 80-308 Gdan´sk, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhib-
itors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge,
the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This
study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus
and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-
Received 3 March 2014
Revised 28 March 2014
Accepted 29 March 2014
Available online 8 April 2014
based analogue 4b (MBIC50 = 20
itors of S. aureus and S. mutans biofilms.
lM), emerged as a promising hit for further optimisation of novel inhib-
Keywords:
Biofilm
Indole
Ó 2014 Elsevier Ltd. All rights reserved.
Inhibition
Marine
Oroidin
The growth of bacteria is faster on surfaces that present a pro-
tective and nutrient-rich environment compared to growth in
water and in liquids with low nutrient contents.1 The adhesion of
bacterial cells to inert or living surfaces results in the formation
of biofilms, that is, structured communities that are encased in a
self-produced extracellular matrix of biomolecules.1,2 The extracel-
lular matrix, which consists of polysaccharides, proteins and
nucleic acids, is the primary reason for profound protection from
the host’s immune system and for a nearly 1000-fold increase in
biofilm resistance to conventional antibiotics when compared to
planktonic bacteria.3,4
The robust resistance of biofilms to the hazards of their environ-
ment, for example, predators, antibiotics and host immune sys-
tems,2,5,6 results not only in biofouling in the food and marine
industries but also in various nosocomial and chronic infections
in patients, which result in perpetual inflammation and tissue
damage.3,7 The formation of biofilms on inert surfaces includes
indwelling medical devices, catheters and contact lenses, and
infections of living surfaces most often include lung infections in
cystic fibrosis patients, periodontitis, endocarditis and urinary tract
infections.4,8 Furthermore, an estimate by the National Institutes of
Health states that 80% of microbial infections are biofilm-based.9
Facultative anaerobic cocci Staphylococcus aureus and Strepto-
coccus mutans are among common Gram-positive bacteria that
form infectious biofilms.1,10 Nosocomial infections are frequently
caused by omnipresent S. aureus bacteria, which are commonly
found in the anterior nares in humans and can enter the circulatory
system through an epithelial breach.11,12 Osteomyelitis, indwelling
medical device infections, chronic wound infections, endocarditis
and periodontitis exemplify the most prevalent biofilm-based
infections caused by S. aureus. While periodontitis is sometimes
caused by S. mutans, its most common biofilm-based infection
remains dental caries. S. mutans is considered to be one of the ear-
liest colonisers of human teeth after their eruption, and its fermen-
tation of dietary carbohydrates and consequential production of
acids poses one of the most important factors for the formation
of dental caries.10
Ubiquitous biofouling presents a considerable threat to immo-
bile marine organisms such as sponges and seaweeds, which do
not possess an immune system that can protect them against
microbes. Hence, they have evolved to biosynthesise various
secondary metabolites that function as a defence system against
bacterial infections.1,13 Among the most investigated anti-biofilm
agents from marine sponges are the halogenated furanones from
the seaweed Delisea pulchra,14,15 terpenoids16,17 and especially 2-
aminoimidazole-based alkaloids.18–20 The 2-aminoimidazole-based
⇑
Corresponding authors. Tel.: +48 58 523 6024; fax: +48 58 523 5501 (G.W.);
tel.: +386 1 4769 635; fax: +386 1 4258 031 (L.P.M.).
E-mail addresses: grzegorz.wegrzyn@biol.ug.edu.pl (G. We˛grzyn), lucija.
ˇ
peterlin@ffa.uni-lj.si (L. Peterlin Mašic).
http://dx.doi.org/10.1016/j.bmcl.2014.03.094
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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Ziga Hodnik et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2530–2534
2531
marine alkaloid oroidin (Fig. 1) and various series of its analogues
were extensively studied as biofilm inhibitors, primarily by Meland-
er and coworkers.18,20–27
indole-based series of compounds by synthesising conformational-
ly constrained analogues (Fig. 1) in which the central alkene region
of oroidin was replaced with synthetically more favourable 1,3-
phenylene (compounds 3a–14) and 1,4-phenylene (compounds
15a–17b) moieties. To study the structure–activity relationship
of our small library of conformationally constrained indole-based
analogues of oroidin we (i) evaluated the effect of the rigidification
of analogues 1 and 2 on anti-biofilm activity, (ii) compared the 1,3-
substituted series to the 1,4-substituted series of analogues and
(iii) studied the influence of various substituents on indole and
2-aminoimidazole moieties on anti-biofilm activity (Fig. 1).
Syntheses of oroidin and compounds 1–5, 7, 8, 9 and 11–15b
were performed as previously described.31,32 Compounds 10a
(cf. Supplementary material), 16a and 17a were synthesised using
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluorobo
rate (TBTU)-promoted coupling of tert-butyl 2-amino-4-(3-amino-
phenyl)-1H-imidazole-1-carboxylate31 or tert-butyl 2-amino-4-(4-
aminophenyl)-1H-imidazole-1-carboxylate31 with the corresponding
indole-2-carboxylic acids.33 Subsequent treatment of compounds
10a, 16a and 17a with gaseous hydrogen chloride gave compounds
10, 16b and 17b. Compound 6a was synthesised from 10a, using
Although numerous analogues of oroidin have been observed to
control biofilm development,18,20–27 to our knowledge, indole-
based analogues of oroidin have not yet been studied. In contrast,
various studies outlined the ability of indole to regulate the growth
of various bacterial biofilms and therefore represents an important
quorum sensing molecule.28–30 In the present work, we first
replaced the dibromopyrrole moiety of oroidin with the isosteric
indole and 5-fluoroindole moieties, and evaluated the anti-biofilm
activity of synthesised analogues 131 and 231 (Table 1) against two
Gram-positive biofilm-forming strains of methicillin-resistant
Staphylococcus aureus (MRSA) and Streptococcus mutans and
against a Gram-negative biofilm-forming strain of Pseudomonas
aeruginosa (cf. Supplementary material). While indole-based ana-
logue 1 did not exhibit any anti-biofilm activity against the tested
strains, its 5-fluoroindole analogue 2 attenuated the formation of
MRSA and S. mutans biofilms to 8.1% and 18.7%, respectively, at
100 lM (Table 1). Based on these encouraging anti-biofilm activi-
ties we further explored the structure–activity relationship of
Br
HN
HN
H
H2N
Br
N
N
O
oroidin
R1
R1 = H or F
HN
HN
H
N
H2N
N
O
1-2
rigidification
O
H
N
N
HN
R1
1,4-substitution
H2N
R2
N
HN
O
N
H
H
N
HN
R3
H
N
NH
O
H2N
N
N
N
1,3-substitution
R1
3a-14
R2
R1
15a 17b
-
R1 = H, F, Cl,...
R2 = H or Boc
R3 = H or CH3
R1 = H, F or Cl
2 = H or Boc
R
Figure 1. Design of conformationally constrained indole-based analogues of oroidin with potential anti-biofilm activity.
Table 1
Anti-biofilm activities of oroidin and its indole-based analogues
HN
R1
HN
H
N
H2N
N
O
1-2
Compounds
R1
Staphylococcus aureus
% Of biofilm formationa
Streptococcus mutans
% Of biofilm formationa
b
b
MBIC50
(l
M)
MBIC50 (lM)
Oroidin
1
2
—
H
F
n.a.
n.a.
8.1 0.7
—
—
60
18.0 8.6
n.a.
18.7 9.7
—
—
60
a
Percentage of biofilm formation compared to the negative control. Compounds were tested at 100
n.a. = not active.
lM. Each value is the mean of three independent experiments.
b
The concentration of compound that inhibits biofilm formation by at least 50%. Each MBIC50 value is the mean of three independent experiments.

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Ziga Hodnik et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2530–2534
2532
standard catalytic hydrogenation procedure. Following treatment
of 6a with gaseous hydrogen chloride gave compound 6b (cf.
Supplementary material). The synthesised compounds were
screened for their anti-biofilm activity in strains of MRSA,
Streptococcus mutans and Pseudomonas aeruginosa using a modified
biofilm formation assay (cf. Supplementary material), described by
Christensen et al.34 The MBIC50 values of analogues 2, 4b, 12, 14,
15b, 16a and 16b were determined at multiple concentrations
using a modified biofilm formation assay.35 The anti-biofilm
activities against MRSA and Streptococcus mutans are presented
in Tables 1–3. Oroidin and its indole-based analogues (1–17b)
did not display any anti-biofilm activity against Pseudomonas
aeruginosa (results not shown).
while most of the 1,3-substituted indole-based analogues attenu-
ated the formation of S. mutans biofilms (Table 2). The substitution
at position 5 of the indole ring was also found to be important for
anti-biofilm activity of 1,3-substituted analogues. In contrast to
unsubstituted analogue 3b (MRSA BF: 13.9%, S. mutans BF: 14.2%),
the hydroxy-(5b) and nitro-substituted (10) analogues displayed
weaker activities against S. mutans biofilm (BF: 53.4% and 33.3%,
respectively) and were inactive against MRSA biofilm formation.
The amino-substituted analogues 6a and 6b did not display any
anti-biofilm activities in both tested strains. The weak inhibitions
of hydroxy-(5a and 5b) and amino-substituted analogues (6a and
6b) further suggest that polar 5-indole substituents negatively
affect the activities against MRSA and S. mutans biofilms. The influ-
ence of the indole moiety substitution pattern on the anti-biofilm
activity is further illustrated by 3-carboxamidoindole-based
1,3-Substituted indole-based analogues 3b, 4b, 8, 11, 12 and 14
exhibited moderate to strong inhibition of MRSA biofilm formation,
Table 2
Anti-biofilm activities of conformationally restricted indole-based analogues of oroidin
O
O
H+Cl-
N
H
N
R3
HN
H
N
O
H+Cl-
N
O
H
N
H+Cl-
N
N
N
H
N
H
H2N
O
N
H
H2N
N
H
H2N
HN
N
HN
3b - 6b
12
HN
NH
S
-
R1
3a - 6a
13
14
7
O
R1
R3
Compounds
R1
Staphylococcus aureus
Streptococcus mutans
b
b
% Of biofilm formationa
MBIC50
(
lM)
% Of biofilm formationa
MBIC50 (lM)
3a
3b
4a
4b
5a
5b
6a
6b
7
H
H
—
H
—
H
—
H
—
H
H
H
H
H
H
n.a.
13.9 2.3
n.a.
34.1 6.1
n.a.
n.a.
n.a.
n.a.
n.a.
44.0 8.3
n.a.
—
—
—
20
—
—
—
—
—
—
—
—
—
70
—
90
30.6 5.2
14.2 2.0
n.a.
17.0 3.9
57.6 11.5
53.4 7.0
n.a.
—
—
—
20
—
—
—
—
—
—
—
—
—
80
—
60
OCF3
OCF3
OH
OH
NH2
NH+3Clꢀ
F
Cl
OCH3
NO2
OBn
H
n.a.
8.7 3.5
6.8 3.3
10.5 1.8
33.3 4.9
8.2 2.4
8.8 1.9
23.9 2.9
10.1 1.3
8
9
10
11
12
13
14
n.a.
31.4 12.2
19.1 6.4
n.a.
CH3
—
—
—
—
23.0 10.6
a
Percentage of biofilm formation compared to the negative control. Compounds were tested at 100
n.a. = not active.
lM. Each value is the mean of three independent experiments.
b
The concentration of compound that inhibits biofilm formation by at least 50%. Each MBIC50 value is the mean of three independent experiments.
Table 3
Anti-biofilm activities of conformationally restricted indole-based analogues of oroidin
H2N
N
H
N
NH
O
N
R2
R1
15a 17b
-
Compounds
R1
R2
Staphylococcus aureus
% Of biofilm formationa
Streptococcus mutans
% Of biofilm formationa
b
b
MBIC50
(l
M)
MBIC50 (lM)
15a
H
H
F
F
Cl
Cl
Boc
H
Boc
H
Boc
H
n.a.
—
22.6 7.2
8.1 3.5
56.0 20.6
3.9 3.0
n.a.
—
15bc
16a
11.1 3.0
13.1 4.7
10.9 4.7
78.0 20.3
n.a.
90
80
60
—
60
120
50
—
16bc
17a
17bc
—
38.2 13.3
—
a
Percentage of biofilm formation compared to the negative control. Compounds were tested at 100
n.a. = not active.
lM. Each value is the mean of three independent experiments.
b
The concentration of compound that inhibits biofilm formation by at least 50%. Each MBIC50 value is the mean of three independent experiments.
HCl salt.
c

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Ziga Hodnik et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2530–2534
2533
analogue 13 (MRSA BF: not active, S. mutans BF: 23.9%), which in
contrast to 2-carboxamidoindole-based analogue 3b, exhibited
weaker activity against both tested strains. However, thieno
[3,2-b]pyrrole-based analogue 14 (MRSA BF: 23.0%, S. mutans
BF: 10.1%) displayed comparable potencies to those of ana-
logue 3b.
To conclude, several novel synthetic indole-based analogues of
the marine alkaloid oroidin exhibited antibiofilm activities in the
low micromolar range against the Gram-positive biofilm-forming
strains MRSA and S. mutans and therefore represent a novel class
of 2-aminoimidazole-based inhibitors of S. aureus biofilm forma-
tion and, to the best of our knowledge, the first reported 2-amino-
imidazole-based inhibitors of S. mutans biofilm formation. The
anti-biofilm activities of the majority of the synthesized indole-
based oroidin analogues illustrate the applicability of the indole
moiety as a dibromopyrrole surrogate for generating inhibitors of
MRSA and S. mutans biofilm formation. The latter suggests that
indole-based oroidin analogues could act as inhibitors of bacterial
biofilms by dispersing their structure as 2-aminoimidazol-based
analogues18,23 and inhibit the quorum sensing mechanisms due
to possession of the indole moiety.28–30 The SAR study revealed
promising substituents at position 5 of the indole moiety for
anti-biofilm activity against the tested bacterial strains. Finally,
this study resulted in a potent, 5-(trifluoromethoxy)indole-based
analogue 4b as a promising hit for further optimisation to generate
novel inhibitors of S. aureus and S. mutans biofilms formation.
The influence on anti-biofilm activity was also evident in the
case of substitution at the position 1 of the 2-aminoimidazole ring.
Hence, the tert-butyloxycarbonyl (Boc)-substituted analogues 3a,
4a and 5a exhibited weaker anti-biofilm activities against S.
mutans biofilm (BF: 30.6%, not active and 57.6%, respectively) com-
pared to their unsubstituted analogues 3b (BF: 14.2%), 4b (BF:
17.0%) and 5b (BF: 53.4%) (Table 2). Similar effect was also evident
in the case of MRSA biofilm, where in contrast to the unsubstituted
analogues 3b (BF: 13.9%) and 4b (BF: 34.1%), analogues 3a and 4a
did not display any activity. This suggests that introduction of lar-
ger substituents at position 1 of the 2-aminoimidazole moiety
weakens the inhibition of biofilm formation. Comparison of the
biofilm formation assay results for compounds 3b and 12 (MRSA
BF: 19.1%, S. mutans BF: 8.8%) suggests that the substitution of 2-
amino group of the imidazole moiety does not contribute signifi-
cantly to the inhibition of biofilm formation. In general, compared
to the biofilm of MRSA strain, 1,3-substituted analogues displayed
stronger inhibitions of S. mutans biofilm formation.
Similar to 1,3-substituted analogues, 1,4-substituted indole-
based analogues 15a–17b also inhibited the formation of MRSA
or S. mutans biofilms (Table 3). Comparison of the potencies of
indole (15b; MRSA BF: 11.1%, S. mutans BF: 8.1%) and 5-fluoroin-
dole (16b; MRSA BF: 10.9%, S. mutans BF: 3.9%) analogues with
the 5-chloroindole analogue 17b (MRSA BF: not active, S. mutans
BF: 38.2%) suggests that in the case of 1,4-substituted analogues,
introduction of larger substituents in position 5 of the indole moi-
ety leads to the loss of anti-biofilm activity. As in the case of 1,3-
substituted analogues, 1,4-substituted analogues 15a–17b also
indicated the relevance of substitution at position 1 of the 2-
aminoimidazole moiety for anti-biofilm activity. The Boc-substi-
tuted analogues 15a, 16a and 17a exhibited weaker inhibitions of
MRSA and S. mutans biofilms, compared to unsubstituted ana-
logues 15b, 16b and 17b (Table 3).
Acknowledgments
This work was supported by the European Union FP7 Integrated
Project MAREX: Exploring Marine Resources for Bioactive Com-
pounds: From Discovery to Sustainable Production and Industrial
Applications (Project No. FP7-KBBE-2009-3-245137), by the Slove-
nian Research Agency (Grant No. P1-0208 and Grant No. Z1-5458)
and by the Ministry of Science and Higher Education, Poland (Grant
No. 1544/7.PR UE/2010/7).
M.L. acknowledges a support by the European Union, within the
European Regional Development Fund, through the Grant Innova-
tive Economy (POIG.01.01.02-00-008/08).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.03.
094.
With an aim to supplement the results of the biofilm formation
assay, seven structurally diverse analogues (2, 4b, 12, 14, 15b, 16a,
16b) were selected for minimal biofilm inhibitory concentration
(MBIC50) determination. The resulting MBIC50 values in the lower
micromolar range were consistent with the screening results from
the biofilm formation assay (Tables 1–3). Model compound 5-flu-
oroindole analogue 2 displayed an MBIC50 value of 60 lM against
MRSA and S. mutans biofilms, while the most potent, 5-(trifluoro-
methoxy)indole-based analogue 4b exhibited an MBIC50 value of
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(MBIC50 = 60
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and the mixture stirred at 35 °C for 24 h. The solvent was evaporated in vacuo,
the residue dissolved in ethyl acetate (30 mL), and washed successively with
water (2 ꢁ 10 mL), saturated aqueous NaHCO3 solution (2 ꢁ 10 mL), and brine
(1 ꢁ 10 mL). The organic phase was dried over Na2SO4, filtered and the solvent
evaporated under reduced pressure. The crude product was purified by flash
column chromatography using ethyl acetate/petroleum ether or
dichloromethane/methanol as an eluent, to afford 16a (178 mg, 45% yield) as
a white solid; mp 191–193 °C; IR (KBr)
m
= 3291, 3179, 1679, 1664, 1590, 1514,
1488, 1448, 1409, 1335, 1315, 1257, 1237, 1204, 1143, 1114, 833, 755 cmꢀ1
.
1H
NMR (DMSO-d6) d 1.60 (s, 9H, t-Bu), 6.61 (s, 2H, NH2), 7.10 (dt, 1H, 3J = 8.8 Hz,
4J = 2.4 Hz, Ar-H), 7.31 (s, 1H, Ar-H), 7.43–7.50 (m, 3H, 3ꢁAr-H), 7.74 (d, 2H,
3J = 8.4 Hz, 2ꢁAr-H), 7.80 (d, 2H, 3J = 8.4 Hz, 2ꢁAr-H), 10.29 (s, 1H, NH), 11.86
(s, 1H, NH); 13C NMR (DMSO-d6) d 27.85 (CCH3), 84.37 (CCH3), 104.09 (d, 1C, JC–
F = 6 Hz), 105.90 (d, 1C, JC–F = 21 Hz), 108.84, 113.61 (d, 1C, JC–F = 10 Hz),
120.25, 122.96, 124.73, 126.42, 127.01 (d, 1C, JC–F = 11 Hz), 132.95, 133.59,
135.75, 136.33, 138.61, 147.48, 156.23 (d, 1C, JC–F = 203 Hz), 159.35; MS (ESI)
m/z (%) = 436.2 (MH+, 100). HRMS for C23H23N5O3F: calculated 436.1785; found
436.1780. Anal. Calcd for C23H22N5O3F ꢁ 0.35 H2O (%): C, 62.53; H, 5.18; N,
15.85. Found: C, 62.83; H, 5.21; N, 15.44.
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35. MBIC50 determination: The MBIC50 (minimal biofilm inhibitory concentration)
value was determined by measurement of effects of the tested compound at
various concentrations. Several different concentrations between 0 and 120
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ˇ
ˇ
31. Zidar, N.; Montalvão, S.; Hodnik, Z.; Nawrot, D. A.; Zula, A.; Ilaš, J.; Kikelj, D.;
ˇ
Tammela, P.; Peterlin Mašic, L. Mar. Drugs 2014, 12, 940.
ˇ
32. Zidar, N.; Jakopin, Z.; Madge, D. J.; Chan, F.; Tytgat, J.; Peigneur, S.; Dolenc, M.
l
M were used and the minimal concentration giving at least 50% inhibition
´
ˇ
S.; Tomašic, T.; Ilaš, J.; Peterlin Mašic, L.; Kikelj, D. Eur. J. Med. Chem. 2014, 74,
was determined. A series of dilutions changing by 10 M was employed rather
l
23.
than the doubling dilution technique, as in our preliminary experiments we
found that effects of particular oroidin derivatives were not linearly
proportional to their concentrations. Instead, a more or less sharp threshold
concentration was noted, below which relatively small changes were
determined with a concentration increase, and above which the inhibition
was significantly more pronounced with a relatively small increase in the
tested compound level.
33. General procedure for the syntheses of compounds 16a and 17a (with 16a as an
example): To
a suspension of 5-fluoroindole-2-carboxylic acid (122 mg,
1.09 mmol) and TBTU (380 mg, 1.18 mmol) in dichloromethane (5 mL) N-
methylmorpholine (0.501 mL, 4.56 mmol) was added, and the mixture stirred
at rt for 0.5 h upon which a clear solution formed. tert-Butyl 2-amino-4-(4-
aminophenyl)-1H-imidazole-1-carboxylate (250 mg, 0.91 mmol) was added