Click-based synthesis of bromotyrosine alkaloid analogs as potential anti-biofilm leads for SAR studies

Article abstract of DOI:10.1016/j.bmcl.2015.10.073

A library of triazole-based analogs of bromotyramine alkaloids such as verongamines, hemibastadins, pseudoceramine D and clavatidine E was designed in order to identify promising leads that may help in the control of bacterial biofilms. Twenty-three compounds were screened for their biofilm inhibitory activity against three strains of Gram-negative bacteria. SAR studies revealed that hemibastadins analogs were the most active compounds which act as inhibitors of biofilm development (EC₅₀ 8.8-29 μM) without effect on bacterial growth even at high concentrations (100 μM).

Full text of DOI:10.1016/j.bmcl.2015.10.073

Bioorganic & Medicinal Chemistry Letters 25 (2015) 5762–5766
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Click-based synthesis of bromotyrosine alkaloid analogs as potential
anti-biofilm leads for SAR studies
⇑
S. Andjouh, Y. Blache
Université de Toulon, MAPIEM, EA 4323, 83957 La Garde, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of triazole-based analogs of bromotyramine alkaloids such as verongamines, hemibastadins,
pseudoceramine D and clavatidine E was designed in order to identify promising leads that may help
in the control of bacterial biofilms. Twenty-three compounds were screened for their biofilm inhibitory
activity against three strains of Gram-negative bacteria. SAR studies revealed that hemibastadins analogs
Received 4 August 2015
Revised 20 October 2015
Accepted 23 October 2015
Available online 24 October 2015
were the most active compounds which act as inhibitors of biofilm development (EC50 8.8–29
out effect on bacterial growth even at high concentrations (100 M).
lM) with-
l
Keywords:
Click chemistry
Biofilm
Ó 2015 Elsevier Ltd. All rights reserved.
1,2,3-Triazole
Bromotyrosine alkaloïds
Biofilms, are structured communities of bacterial cells protected
by a self-produced polymeric matrix and adherent to an inert or a
living surface.1 Among the benefits that biofilms can provide to
bacteria, protection against antibiotics or disinfectants is one of
the major problems particularly in the medical sector, where they
colonize implants such as artificial joints and catheters.2 Similarly,
in marine environment, formation of biofilms on immersed sub-
strata increase fuel consumption and cause material damages.3–5
Although the control of the development of planktonic bacteria
communities is well known, the control of bacteria within a biofilm
is still a challenge, as they are much more resistant to antibiotic
and to biocide treatment (up to 1000-fold increased resistance).
Therefore, targeting the formation of biofilms is of utmost interest
in order to parsimonious use of antibiotics and/or biocides. In this
context, development of original biofilm inhibitors may have the
potential to be used in a preventive treatment of a wide diversity
of industrial and medical surfaces. Some of the anti-biofilm strate-
gies that are tested) explored today consist in studying secondary
metabolites from marine organisms such as sponges or soft corals
in order to find molecules that inhibit and/or disperse bacterial
biofilm specifically through non-microbicidal mechanisms. These
compounds will likely serve as potential adjuvants for conven-
tional antibiotic therapies or friendly environmentally biocides
that can mitigate the increasing microbial resistance derived from
biofilms.6 Among this large variety of organisms, marine sponges
of the order Verongida are well-known to produce a diverse array
of bromotyrosine alkaloids that have been linked to antimicrobial
and/or cytotoxic activities.7–9 Bromotyrosine derivatives repre-
sented in Figure 1 possess common structural features: a western
region constituted of a bromoaromatic cycle, a central core includ-
ing an oxime function and an amide link, finally eastern regions
which allows an interesting chemical diversity for structure–activ-
ity relationships studies. Verongamine (1), a histamine derivative,
was originally isolated from Veronlula gigantea.10 Its 5-bromod-
erivative (2) was reported to possess anti-biofouling properties
through inhibition of barnacle larvae settlement without toxic
effects,11 and antibacterial activity toward diverse clinical
strains.12 Clavatadine E (3), a guanidine derivative extracted from
Suberea clavata showed antithrombotic activity.13 The general class
of Hemibastadins, possessing a tyramime eastern moiety, are rep-
resented by hemibastadin-1 (4), and 10-methoxyhemibastadin-1
(5). These two last bromotyrosine alkaloids were purified from
the pacific elephant ear sponge (Ianthella basta) and have been
shown to inhibit blue mussel phenoloxydase in vitro.14 Pseudoce-
ramine D (6), extracted from a marine sponge Pseudoceratina sp.
was identified through a bioassay-guided screening for its capacity
to inhibit the type III secretion (T3S) system of the Gram-negative
bacterium Yersinia pseudotuberculosis as well as for its antibacterial
activity targeting bacterial growth.15,16
In addition, instead of searching for additional bioactive com-
pounds, some efforts have been directed toward the optimization
of the structure of such simple bioactive alkaloids based on
structure–activity relationships studies (SAR). (As) For examples,
Niemann et al. have recently reported the preparation of hemibas-
tadins analogs,14 while Richards et al. have described series of
⇑
Corresponding author.
E-mail address: blache@univ-tln.fr (Y. Blache).
http://dx.doi.org/10.1016/j.bmcl.2015.10.073
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

S. Andjouh, Y. Blache / Bioorg. Med. Chem. Lett. 25 (2015) 5762–5766
5763
Central core Eastern region
Western region
R
OH
OH
O
MeO
HO
Br
N
N
NH
NH2
H
N
H
N
Br
N
H
N
HN
O
1 : R= H, Verongamine
3 : Clavatidine E
2 : R= Br, 5-bromoverongamine
R2
Br
OH
OH
R1O
Br
MeO
Br
N
H
N
H
H
N
N
R3
OH
N
O
O
Br
4 : R1 = H, R2 = Br, R3 = H, Hemibastadin-1
5 : R1 = CH3, R2= H, R3=Br, 1'-methoxyhemibastadin-1
6 : Pseudoceramine D
Figure 1. Examples of bromotyrosine alkaloids of biological interest.
Amide bond formation
peptide coupling
Cu-catalyzed
cycloaddition
Central core
N
Br
Br
C CO2H
( )
( )
N
Br
( )
HC
R2 NH2
N
N
3
n
N
n
N
n
N
H
N
O
O
R1
O
R2
CO2H
R1
R1
X
X
X
O
Western region
Eastern region
Figure 2. Structure of the targeted library and retrosynthetic approach.
n
n
Cl
n
N
X
N
X
X
( )
( )
N3
( )
(ii)
(i)
N
R1
O
R1
R1
O
O
CO2H
7a : n=1, R1=OMe, X= Br
7b : n=2, R1=OMe, X= Br
7c : n=2, R1=H, X=Br
7d : n=2, R1=H, X=H
8a : 99%
9a : 90%
9b : 90%
9c : 81%
9d : 78%
8b : 94%
8c : 96%
8d : 92%
(i) : NaN3, DMF, 5h, 90°C. (ii) : CuSO4, Na ascorbate, EtOH/H2O, 12h, RT
Scheme 1. General synthesis of 1,2,3-triazoles carboxylic acids.
oroidin analogs as potent anti-biofilm molecules.17 For our part
and in the same lines, we showed that ‘click chemistry’ methodolo-
gies could be retained as interesting high through processes to
design naamine and isonaamine alkaloids analogs, highlighting
the 1,2,3-triazole ring as a non-classical bioisostere of the imida-
zole ring.18 In parallel, we demonstrated that when associated with
linear terpenoid skeletons, 1,2,3-triazole ring afforded interesting
non-toxic anti-biofilm derivatives.19,20 Taking into account these
results, and since the bioisosteric replacement of an oxime by a
1,2,3-triazole was recently described in the diversity-oriented syn-
thesis of pyrrolidinyl triazoles as bioisosteres of some pyrrolidinyl
oxime (an mPTP blocker as a viable therapeutic target for the treat-
ment of Alzheimer’s disease),21 we felt that the bioisosteric
replacement of the oxime group of bromotyrosine alkaloids by a
triazole heterocycle could afford some interesting elements of
SAR in the field of anti-biofilm compounds. In this study, we will
describe our investigations to design a new library of bromoty-
rosine alkaloids possessing a triazolic core in which three point
of SAR can be considered: the eastern region which is accessible
by the nature amine reagent for the second step, the western
region and length of linker which are both accessible by the nature
of starting azido compound (Fig. 2).
The first step of this work was the preparation of intermediary
carboxylic acids (9a–d). These compounds were obtained in excel-
lent yield in two steps. Treatment of benzyl chlorides (7a–d) by
sodium azide in dimethylformamide afforded the corresponding
azides (8a–e). Then, Synthesis of the targeted carboxylic acids
(9a–d) was achieved by performing the copper(I)-catalyzed
1,3-dipolar cycloaddition of the organic azides with propargylic
acid resulting in the formation of 1,2,3-triazoles. In general, these
reactions usually proceed to completion in 6–36 h at ambient
temperature in water with a variety of organic co-solvents, such
as tert-butanol, ethanol, DMF, DMSO, THF, or CH3CN, and this
reaction is useful for large classes of azides and alkynes.22,23
Ethanol was chosen rather than DMF to allow an easier workup,
and a better purity of products as reported in our previous work.18
In practice, propargylic acid was added to a solution of appropriate
azide (8a–d), CuSO4/sodium ascorbate in water/ethanol mixture
(50/50) and the reaction time was optimized to 12 h at room
temperature (see Scheme 1).

5764
S. Andjouh, Y. Blache / Bioorg. Med. Chem. Lett. 25 (2015) 5762–5766
n
N
n
N
N
N
X
X
( )
( )
N
N
H
N
H
N
Y
R1
R1
O
O
N
HN
O
O
OH
Y
10a-d
11a-d : Y=H
11e : Y=Br obtained from 11a by (iii)
11f : Y=Br obtained from 11b by (iii)
11g : Y=Br obtained from 11d by (iii)
OH
N
(i),
(i),
H2N
H2N
N
H
n
N
N
X
( )
N
R1
O
CO2H
9a-d
NHBoc
m
(i),
(ii)
H2N
( )
S
NHBoc
(iv),
n
n
N
NHBoc
N
X
N
N
X
( )
N
N
NH3
H
H
N
m
m
N
R1
( )
R1
O
( )
O
NH3,CF3CO2
N
NH,CF3CO2
H
O
O
13a-f
12a-f
(i) DCC, HOBt, DIPEA, DMF/CH2Cl2, 48h, RT
(ii) TFA,CH2Cl2
(iii) NBS,THF,RT
(iv) a: DMF/CH2CL2/TEA/HgCl2 followed by b: TFA,CH2Cl2
Scheme 2. Synthesis of targeted bromotyrosine analogs from carboxylic acid 9a–d.
Table 1 (continued)
Table 1
Yielda (%)
Selected 1,4-disubstituted 1,2,3-triazoles
Compound
R1
X
n
m
R2
13c
13d
13e
13f
CH3
CH3
CH3
CH3
Br
Br
Br
Br
1
2
1
2
2
2
3
3
83
80
59
67
H
N
n
N
NH2
N
X
( )
( )
m
N
H
N
NH3, CF3CO2
R1
O
R2
a
O
Yield of amide formation step for 10a–d, 11a–d, 12a–f, yield of bromination
step for 11e–g, yield of guanilation/deprotection step for 13a–f.
Compound
R1
X
n
m
R2
Yielda (%)
Verongamine analogs
10a
10b
10c
10d
CH3
CH3
H
H
Br
Br
Br
H
1
2
2
2
—
—
—
—
89
88
51
74
N
Access to the different classes of bromotyramine analogs 10a–d,
11a–d, 12a–f, was allowed by a peptide coupling step using
DCC/HOBt methodology (followed by a deprotection step for
12a–f).24 All amides were obtained in good yields. Further access
to 11e–g was allowed from 11a–c, respectively, by means of a
bromination step using N-bromosuccinimide.25 Preparation of
clavatidine E analogs 13a–g necessitated a guanilation step of com-
pounds 12a–f. This step was performed with N,N0-di(tert-butoxy-
carbonyl)-thiourea26 to afford the corresponding protected
guanidines which were deprotected by trifluoroacetic acid in
dichloromethane to give 13a–f (Scheme 2 and Table 1).27
N
H
Hemibastadin analogs
OH
11a
11b
11c
11d
CH3
CH3
H
H
Br
Br
Br
H
1
2
2
2
—
—
—
—
84
80
75
75
11e
11f
11g
CH3
CH3
H
Br
Br
3,5-diBr
1
2
2
—
—
—
Br
81
77
80
OH
Br
In order to assess the anti-biofilm activity of these compounds
against representative Gram-negative bacterial biofilms, three
strains were chosen for their capacity to form biofilms: Pseudoal-
teromonas lipolytica (TC8), Pseudoalteromonas ulvae (TC14) and a
Paracoccus sp. strain (4M6).28 All compounds were tested for their
ability to modulate biofilm formation at an initial concentration of
Pseudoceramine D analogs
12a
12b
CH3
CH3
Br
Br
1
2
1
2
1
2
1
1
2
2
3
3
m
89
63
84
81
95
81
( )
NH3,CF3CO2
12c
12d
12e
12f
CH3
CH3
CH3
CH3
Br
Br
Br
Br
200 lM by using our previous method adapted from Leroy et al.
Clavatidine E analogs
using the specific fluorophore SytoÒ61.29–31 Results of this screen
are outlined in Table 2.
13a
13b
CH3
CH3
Br
Br
1
2
1
1
87
78
Partial information about structure–activity relationship (SAR)
can be highlighted at this stage. Results of this initial screening

S. Andjouh, Y. Blache / Bioorg. Med. Chem. Lett. 25 (2015) 5762–5766
5765
Table 2
Biological screening against bacteria biofilms
Compound
TC14
TC8
4M6
% Of adhesiona
4.9 1.8
EC50
% Of adhesiona
8.9 2.5
EC50
% Of adhesiona
33.9 6.0
EC50
Ampicillin
9.3 1.0
17.9 1.0
144.1 0.6
Hemibastadin analogs
11a
11b
11c
11d
11e
11f
20.7 5.9
18.7 2.5
31.4 3.8
16 9.4
23.3 1.5
5.9 1.5
29.3 5.2
40 6.2
38.3 3.4
35 4.9
134.2 14.7
104.4 22.8
113.2 11.1
170.5 1.5
123.6 24.2
67.8 8.3
31.2 10.33
97.3 16.7
72.9 4.8
155.5 21.2
195.6 22
84 23.2
32.6 10.2
69.3 19.4
72.9 11.5
40.2 1.6
8.8 2.3
28.2
43.4
3
1
35
43
1
1
44.5 2.4
37.9 7.4
40 4.3
29.7 0.5
40.32 3.7
28.8
2
40.7 10.6
83.2 8.9
11g
26.6 7.8
96 10.7
38.7 4.2
Verongamine analogs (10a–d)
Pseudoceramine D analogs (12a–f)
Clavatidine E analogs (13a–f)
Inactives compounds (% of adhesion at 200 lM > 75%)
a
Percentage of adhesion at 200 lM compared to untreated samples.
11a
11b
11c
11d
11e
11f
11g
Ampicillin
11a
11b
11c
11d
11e
11f
11g
Ampicillin
0.3
0.25
0.2
0.4
0.35
0.3
0.25
0.2
0.15
0.1
untreated sample
0.15
0.1
untreated sample
0.05
0
0.05
0
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
Time (hours)
Time (hours)
Figure 3a. Effect on TC14 growth of compounds 11a–g at concentrations of
100 M.
Figure 3b. Effect on TC8 growth of compounds 11a–g at concentrations of 100 lM.
l
showed clearly that the eastern region of this class of natural com-
pounds derived triazoles is of great importance for assessing a
good anti-biofilm activity. Verongamine, pseudoceramine D and
clavatidine E analogs were inactive and only the hemibastadin
analogs 11a–g, possessing a phenol substituent, were able to mod-
ulate biofilm formation of the three strains when compared to
ampicillin. More precisely, these compounds were active on all
11a
11b
11c
11d
11e
11f
11g
0.3
0.25
0.2
0.15
0.1
Ampicillin
untreated sample
the three strains with a better efficiency at 200 lM on TC 14
(8–29% of adhesion) than on TC8 (29–43% of adhesion) and 4M6
(28–49% of adhesion). In order to precise structure–activity rela-
tionships, effective concentrations to inhibit 50% of the bacterial
adhesion (expressed as EC50) were determined for 11a–g. In this
way, first observation was to note that the TC14 strain was more
sensitive to this class of triazoles than TC8 and 4M6 strains. Fur-
thermore, and in respect with the natural frameworks, influence
of substituents on the aromatic ring of the western region was
examined. It can be seen that compounds possessing a methoxy
group (11a, 11b, 11f) vs an hydroxyl group (11c, 11d) at the 4-posi-
tion of the aromatic ring are more potent anti-biofilm candidates.
We can also remark that deletion of the bromine on the 3-position
(compound 11d) resulted in reduction of effectiveness especially
with the TC8 and 4M6 strains. In the same way, high degrees of
bromination on both aromatic rings (11e, 11f, 11g) gave rise to
the best anti-biofilm activities. Finally, when looking to the
influence of length of spacer (n = 1 for 11a and 11e or n = 2 for
11b and 11f), we can conclude that best activities were obtained
with n = 2, leading to the most potent compound 11f with
0.05
0
0
1
2
3
4
5
6
7
Time (hours)
Figure 3c. Effect on 4M6 growth of compounds 11a–g at concentrations of 100
l
M.
The final goal of the study was to investigate if these com-
pounds exhibited a specific anti-biofilm activity or if this observa-
tion was simply related to a general toxic effect on the bacteria. For
this purpose, a growth inhibition31 and viability assay was per-
formed.32 Active compounds 11a–g were tested for their capacity
to inhibit the growth of the three strains TC14, TC8 and 4M6.
Experiments were performed at high concentrations of 100
(Figs. 3a–3c).
The results showed that when compared to untreated samples
and in contrast with ampicillin, these compounds exhibit no effect
on the bacterial growth of TC8 and 4M6 strains, while the most
active derivative 11f exhibited a slight effect on the growth of
lM
an EC50 = 8.8
on 4M6).
lM on TC14 strain (29.7 lM on TC8 and 28.8 lM

5766
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15. Yin, S.; Davis, R. A.; Shelper, T.; Sykes, M. L.; Avery, V. M.; Elofsson, M.; Sundin,
TC8
TC14
4M6
140
120
100
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C.; Quinn, R. J. Org. Biomol. Chem. 2011, 9, 6755.
16. Kauppi, A. M.; Nordfelth, R.; Uvell, H.; Wolf-Watz, H.; Elofsson, M. Chem. Biol.
2003, 241.
17. Richards, J. J.; Ballard, T. E.; Melander, C. Org. Biomol. Chem. 2008, 1356.
18. Linares, D.; Bottzeck, O.; Pereira, O.; Praud-Tabariès, A.; Blache, Y. Bioorg. Med.
Chem. Lett. 2011, 21, 6751.
19. Praud-Tabariès, A.; Dombrowsky, L.; Bottzek, O.; Briand, J. F.; Blache, Y.
Tetrahedron Lett. 2009, 50, 1645.
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20. Sall, C.; Dombrowsky, L.; Bottzeck, O.; Praud-Tabaries, A.; Blache, Y. Bioorg.
Med. Chem. Lett. 2011, 21, 1493.
21. Jung, S. H.; Choi, K.; Ae Kyun Lee, N. P. J.; Choo, H.; Keum, G.; Choa, Y. S.; Min, S.
J. Org. Biomol. Chem. 2014, 12, 9674.
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22. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
20
23. Huisgen,R.1,3-DipolarCycloadditionChemistry;Wiley:NewYork,1984. pp. 1–176.
24. Shearman, J. W.; Myers, M. R.; Beale, T. M.; Brenton, J. D.; Steven, V.; Ley, S. V.
Tetrahedron Lett. 2010, 51, 4812.
0
25. Andersen, J. R.; Faulkner, J. D. J. Org. Chem. 2001, 66, 3111.
26. Rodriguez, F.; Rozas, I.; Ortega, J. E.; Erdozain, A. M.; Meana, J. J.; Callado, L. F. J.
Med. Chem. 2009, 52, 601.
Compound
27. Spectral data for selected compounds: N-(2-(1H-Imidazol-5-yl)ethyl)-1-(3-
bromo-4-methoxybenzyl)-1H-1,2,3-triazole-4-carboxamide (10a): 1H NMR
(400 MHz, D6-DMSO-) d 8.62 (s, 1H), 8.59 (t, J = 5.8 Hz, 1H, NHCO), 7.66 (d,
J = 2.2 Hz, 1H), 7.56 (s, 1H), 7.38 (dd, J = 8.5, 2.2 Hz, 1H), 7.11 (d, J = 8.5 Hz, 1H),
6.82 (s, 1H), 5.56 (s, 2H), 3.83 (s, 3H), 3.56–3.41 (m, 2H), 2.73 (t, J = 7.3 Hz, 2H).
13C NMR (100 MHz, D6-DMSO) d 159.3, 155.2, 142.9, 134.5, 134.3, 132.7, 129.0
(2C), 126.1, 116.2, 112.7, 110.4, 56.1, 51.7, 38.4, 26.7. 1-(3-Bromo-4-
methoxybenzyl)-N-(4-hydroxyphenethyl)-1H-1,2,3-triazole-4-carboxamide
(11a): 1H NMR (400 MHz, D6-DMSO) d 9.16 (s, 1H, OH), 8.60 (s, 1H), 8.48 (t,
J = 5.8 Hz, 1H, NHCO), 7.65 (d, J = 2.2 Hz, 1H), 7.38 (dd, J = 8.5, 2.2 Hz, 1H), 7.12
(d, J = 8.5 Hz, 1H), 7.02–6.97 (m, 2H), 6.68–6.63 (m, 2H), 5.56 (s, 2H), 3.83 (s,
3H), 3.44–3.35 (m, 2H), 2.68 (t, J = 7.2 Hz, 2H). 13C NMR (100 MHz, D6-DMSO) d
159.4, 155.6, 155.4, 143.1, 132.9, 129.5, 129.4, 129.2 (2C), 126.3, 115.1, 112.9,
110.6, 56.3, 51.9, 40.3, 34.4. 1-(3-Bromo-4-methoxybenzyl)-N-(2-aminoethyl)-
1H-1,2,3-triazole-4-carboxamide (12a): 1H NMR (400 MHz, CD3OD) d 8.39 (s,
1H), 7.56 (d, J = 2.1 Hz, 1H), 7.33 (dd, J = 8.5, 2.1 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H),
5.56 (s, 2H), 3.86 (s, 3H), 3.68 (t, J = 6.4 Hz, 2H), 3.18 (t, J = 6.4 Hz, 2H). 13C NMR
(100 MHz, CD3OD) d 163.5, 157.7, 143.8, 134.2, 130.1, 129.8, 127.2, 113.5,
112.7, 56.8, 53.9, 40.9, 38.0. 1-(3-Bromo-4-methoxybenzyl)-N-(2-
amidinoethyl)-1H-1,2,3-triazole-4-carboxamide (13a): 1H NMR (400 MHz,
CD3OD) d 8.38 (s, 1H), 7.56 (d, J = 2.2 Hz, 1H), 7.33 (dd, J = 8.5, 2.2 Hz, 1H),
7.01 (d, J = 8.5 Hz, 1H), 5.55 (s, 2H), 3.85 (s, 3H), 3.56 (t, J = 6.1 Hz, 2H), 3.41 (t,
J = 6.1 Hz, 2H). 13C NMR (101 MHz, CD3OD) d 163.15, 158.93, 157.68, 143.88,
134.23, 130.07, 129.80, 127.13, 113.45, 112.76, 56.79, 53.90, 41.93, 39.20.
28. Brian-Jaisson, F.; Ortalo-Magne, A.; Guentas-Dombrowsky, L.; Armaougon, F.;
Blache, Y.; Molmeret, M. Microb. Ecol. 2014, 68, 94.
Figure 3d. Effect on TC14, TC8, and 4M6 viability of compounds 11a–g at
concentrations of 100 M.
l
the TC14 strain. For viability, the same methodology used for anti-
adhesion assay with SytoÒ61 was applied using resazurin test
(Fig. 3d). These results clarify that the compounds 11a–g were
not lethal at 100 lM to the bacteria. This suggests that the anti-
biofilm activities of such compounds were not connected to
antibacterial effect, while those observed for ampicillin are directly
connected to a toxic effect. Furthermore, we can note that some of
them (11d, 11f, 11g) may act as non-toxic bacteriostatic agents
against TC14 strain since growth of bacterium was slowed.
In conclusion, we have used click chemistry to generate tria-
zole-substituted marine alkaloids analogs. A biological screening
allowed us to design potent inhibitors of biofilm formation of gram
negative bacteria. Finally the low toxicity of the more potent anti-
biofilm leads allows us to focus our interest in the development of
these molecules as non-toxic anti-biofilm compounds for potential
use as non-toxic co-biocides or co-antibiotic in view of rational
eradication of persistent biofilms. Further structure–activity
relationships studies are actually in progress in order to optimize
the activity as well as to define the mechanism of action of this
original class of compounds.
29. Camps, M.; Briand, J. F.; Guentas-Dombrowsky, L.; Culioli, G.; Bazire, A.; Blache,
Y. Mar. Pollut. Bull. 2011, 1032, 62.
30. Leroy, C.; Delbarre-Ladrat, C.; Ghillebaert, F.; Rochet, M. J.; Compère, C.;
Combes, D. Lett. Appl. Microbiol. 2007, 44, 372.
31. Bacterial adhesion assays (adapted from Leroy et al. 28). Pseudoalteromonas
lipolytica (TC8) was grown on VNSS (Vaatanen Nine Salt solution) at 20 °C and
sampled at the stationary phase. After centrifugation, cells were suspended in
sterile artificial sea water (ASW) until an optical density of 0.2–0.4 at 600 nm
Acknowledgment
was achieved. 200
96-well microtiter plates (sterile black polystyrene NUNC), and 100
bacterial suspension on other wells using an eight-channel pipette. 100
diluted standard biocide (Seanine) and purified molecules were added in the
latter wells. All the concentrations were tested in triplicates. 100 L of ASW
l
L of ASW was inoculated on the border-row wells of the
L of the
L of
l
The Paracoccus sp. strain 4M6 was provided by the LBCM
(Université de Bretagne Sud).
l
l
was added in six wells to constitute the bacterial adhesion control. After 15 h
at 20 °C, the non-adhered bacteria were eliminated by three successive washes
(36 g/L of sterile NaCl solution). The adhered bacteria were stained 10 min at
References and notes
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room temperature by adding 200
The excess stain was removed by two washes (36 g/L NaCl solution). The SytoÒ
61 was then solubilized in 200 L of 36 g/L NaCl solution and fluorescence was
lL of 40 l
mol/L SytoÒ 61 (Invitrogen, USA).
l
measured (kexc = 615 nm, kem = 670 nm) using an Infinit 200 micro-plate
fluorescence reader (TECAN, Lyon, France). The dose-response curves fitting
and the determination of the EC50 for each molecule were achieved using
GraphPad Prism software.
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shaking conditions (120 rpm) and collected during the exponential phase. After
centrifugation, cells were suspended in sterile VNSS (OD600 nm = 0.1). 180 lL
at different concentrations for each tested compounds (standard biocides,
natural or natural-derived products) were added in four wells of the microtiter
plates (sterile transparent PS; Nunc, Fisher Scientific). All the concentrations
were tested in triplicate and the fourth well was filled for control. The
maximum percentage of solvent used for the dilution of biocides was also
tested in triplicate as additional control. For the growth inhibition control,
180
was inoculated on all the wells except the border-row wells and all the wells
were filled out to 200 L with VNSS. Turbidity (OD600 nm) was measured
every hour during 6 h. Then, resazurin (50 M) was added in all the wells, and
lL of VNSS was added in six wells. Then 20 lL of the bacterial suspension
l
l
fluorescence was measured after 2 h to quantify the percent of bacterial
viability. The same methodology used with SYTO 61 was applied to calculate a
percent of viability after resazurin staining.