Microwave-assisted one-pot synthesis and anti-biofilm activity of 2-amino-1H-imidazole/triazole conjugates

Article abstract of DOI:10.1039/c3ob42282h

A microwave-assisted protocol was developed for the construction of 2-amino-1H-imidazole/triazole conjugates starting from the previously described 2-hydroxy-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium salts. The process involves a one-pot hydrazinolysis/Dimroth-rearrangement of these salts followed by a ligand-free copper nanoparticle-catalyzed azide-alkyne Huisgen cycloaddition. The 2-amino-1H-imidazole/triazole conjugates showed moderate to high preventive activity against biofilms of S. Typhimurium, E. coli, P. aeruginosa and S. aureus. The most active compounds had BIC₅₀ values between 1.3 and 8 μM. A remarkable finding was that introduction of the triazole moiety into the side chain of 2-aminoimidazoles with a long (C ₈-C₁₃) 2N-alkyl chain did drastically improve their activity. Conclusively, the 2-amino-1H-imidazole/triazole scaffold provides a lead structure for further design and development of novel biofilm inhibitors. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3ob42282h

Organic &
Biomolecular Chemistry
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Microwave-assisted one-pot synthesis and
anti-biofilm activity of 2-amino-1H-imidazole/
triazole conjugates†
Cite this: Org. Biomol. Chem., 2014,
12, 3671
Hans Steenackers,‡a Denis Ermolat’ev,‡b Tran Thi Thu Trang,b Bharat Savalia,b,c
Upendra K. Sharma,b Ami De Weerdt,a Anamik Shah,c Jozef Vanderleyden*a and
Erik V. Van der Eycken*b
A microwave-assisted protocol was developed for the construction of 2-amino-1H-imidazole/triazole
conjugates starting from the previously described 2-hydroxy-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-
ium salts. The process involves a one-pot hydrazinolysis/Dimroth-rearrangement of these salts followed
by a ligand-free copper nanoparticle-catalyzed azide–alkyne Huisgen cycloaddition. The 2-amino-1H-
imidazole/triazole conjugates showed moderate to high preventive activity against biofilms of S. Typhi-
murium, E. coli, P. aeruginosa and S. aureus. The most active compounds had BIC50 values between 1.3
and 8 µM. A remarkable finding was that introduction of the triazole moiety into the side chain of 2-
aminoimidazoles with a long (C8–C13) 2N-alkyl chain did drastically improve their activity. Conclusively,
the 2-amino-1H-imidazole/triazole scaffold provides a lead structure for further design and development
of novel biofilm inhibitors.
Received 15th November 2013,
Accepted 18th March 2014
DOI: 10.1039/c3ob42282h
www.rsc.org/obc
anti-biofilm activity of diverse 5-aryl-substituted 2-AI
(Fig. 1a).2c–f Moreover, it has recently been reported that
Introduction
The 2-amino-1H-imidazole (2-AI) structural motif is of particu- 2-amino-1H-imidazole/triazole (2-AIT) conjugates, in which a
lar interest within the realm of medicinal and bio-organic triazole moiety is coupled to the 4(5)-position of the 2-AI-ring
chemistry. Many compounds possessing this framework occur via an alkyl linker, inhibit and disperse both Gram-positive
in nature and display a broad range of biological properties.1 and Gram-negative bacterial biofilms through a non-microbio-
For example, among them are the potent modulators of the cidal mechanism (Fig. 1b).5 The effect of coupling a triazole
formation and dispersion of bacterial biofilms,2 human moiety to the exocyclic 2N-position of the 2AI-ring via an alkyl
β-secretase (BACE-1) inhibitors3 and tubulin-binding agents.4 linkage has however not been explored (Fig. 1c).
Biofilms in particular account for more than 80% of all bac-
Despite the number of existing approaches to 2-AI, most of
terial infections and are responsible for the mortality and mor- them involve long experimental procedures and the use of
bidity of almost all cystic fibrosis patients.1c Moreover biofilms unstable precursors. There are only a few approaches that
cause major problems in industrial and household settings.2f describe the direct synthesis of 2-AI. The earliest method
Given the biomedical and industrial prominence of biofilms, involves condensation of α-aminocarbonyl compounds with
there have been significant efforts to discover potent com- cyanamide or their synthetic equivalents.6,7 Other general
pounds that modulate or/and inhibit the biofilm growth. applicable strategies are cyclocondensation of α-bromoketones
In this vein, we have recently described the synthesis and with N-acetyl- or N-Boc-protected guanidine,8 iminophosphor-
ane-mediated cyclization of α-azido esters,9 ammonolysis of
2-amino-1,3-oxazol-3-ium salts,10 and the sequential
aCentre of Microbial and Plant Genetics (CMPG), Department of Microbial and
functionalization of the 1,2-diprotected imidazole ring with
Molecular Systems, KU Leuven, Kasteelpark Arenberg 20, Box 2460, B-3001 Leuven,
different electrophiles.11
Belgium. E-mail: Jozef.Vanderleyden@biw.kuleuven.be
bLaboratory for Organic & Microwave-Assisted Chemistry (LOMAC), Department of
Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium.
E-mail: Erik.VanderEycken@chem.kuleuven.be
cDepartment of Chemistry, Saurashtra University, 361 005 Rajkot, India
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ob42282h
“Click chemistry”, of which the CuAAC is the most impor-
tant representative, has emerged as a fast and efficient
approach to the synthesis of novel compounds with desired
functionality employing selected “near perfect” reactions.12,13
In the last few years, a large number of protocols have been
developed for this most studied and reliable “click” reaction
‡Equal contribution.
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Fig. 1 (a) Classes of 5-aryl-substituted 2-AI with anti-biofilm activity previously reported by our lab; (b) 2-amino-1H-imidazole/triazole (2-AIT)
conjugates with anti-biofilm activity, reported by Melander et al.; (c) 2-amino-1H-imidazole/triazole conjugates investigated in the present study.
employing copper(I) complexes, copper on charcoal, solid
phase-supported copper(I) catalysts and copper nano-
particles.14
Herein, we report a rapid and highly efficient microwave-
assisted one-pot synthesis of the 2-AIT framework, in which
the triazole moiety is coupled to the 2N-position of the 2-AI
ring via an alkyl linkage (Fig. 1c). The protocol starts from our
previously described 2-hydroxy-2,3-dihydro-1H-imidazo[1,2-a]-
pyrimidin-4-ium salts, combining a Cu(I)-catalyzed azide–
alkyne Huisgen cycloaddition (CuAAC) and
a Dimroth-
rearrangement.15 The applicability of the protocol is demon-
strated by the synthesis of a library of 36 compounds, of which
we show the preventive anti-biofilm activity against Gram-nega-
tive and Gram-positive bacterial species, delineating a struc-
ture–activity relationship.
Scheme 1 Sequential synthesis of the 2-AIT framework.
surmised that the required copper(0) nanocatalyst could be
generated14 in situ upon reduction of a Cu(II) salt with hydra-
zine. The procedure was evaluated employing the hydroxy salt
3a (R1 = Ph, R2 = H) and phenylacetylene as a model system
(Table 1). To our satisfaction the reaction progressed well
when 2 equiv. of hydrazine hydrate in combination with
10 mol% Cu(OAc)2 were employed upon microwave irradiation
at a ceiling temperature of 100 °C for 20 min (Table 1, entry 3).
When only 1 equiv. of hydrazine hydrate was used, the product
was obtained in a moderate yield of 51% (Table 1, entry 1), but
increasing the amount of hydrazine hydrate did not influence
the yield (Table 1, entry 2). The reaction time could be
decreased to a mere 2 min while a further shortening resulted
in an incomplete Dimroth rearrangement reaction (Table 1,
entries 3–7). Also lowering the reaction temperature to 90 °C
resulted in a decreased yield (Table 1, entry 9). Replacement of
Cu(OAc)2 with CuSO4 gave the product in 67% yield (Table 1,
entry 11), while with Cu-powder (200 mesh) only trace
amounts of the desired compound were observed (Table 1,
entry 13). As expected, no product was formed in the absence
Results and discussion
Synthesis of 2-amino-1H-imidazole/triazole conjugates
In order to develop a new strategy to the targeted 2-AIT 5, we
carefully examined the reaction conditions (Scheme 1). A
mixture of N-(3-azidopropyl)pyrimidin-2-amine (1a) and phen-
acyl bromide (2a, R1 = Ph, R2 = H) in MeCN was heated at
75 °C for 3 h resulting in the formation of the 2-hydroxy-2,3-
dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium salt 3a. This salt
smoothly underwent the Dimroth-type rearrangement15 upon
treatment with 7 equiv. of hydrazine hydrate yielding N-(3-azido-
propyl)-1H-imidazol-2-amine 4a. Subsequent CuAAC was per-
formed upon treatment of 2-AI 4a with phenylacetylene
(1.5 equiv.) and CuI (10 mol%) as the catalyst under microwave
irradiation at a ceiling temperature of 100 °C and a maximum
power of 40 W for 10 min, delivering the desired 2-AIT 5a
(R1, R3 = Ph; R2 = H; n = 2) in 70% yield (Scheme 1).
Now we were keen to know whether the Dimroth rearrange-
ment and the CuAAC could be run in a one-pot fashion, as we
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Table 1 Optimization of the one-pot synthesis of the 2-AITa
in the case of the morpholylamide substituent, lower yields
were observed (Table 2, entries 22 and 23), probably due to
hydrazinolysis of the starting hydroxy salts 3h and 3i. Remark-
ably, 4,5-disubstituted hydroxy salts 3l and 3m were success-
fully reacted, giving the desired products in 84 and 56% yield,
respectively. Generally, the cycloaddition reaction proceeded
efficiently with aliphatic and aromatic terminal acetylenes pro-
viding the corresponding 2-AIT 5 in good yields. Thus, a
variety of substituted 2-AIT bearing an aromatic (Table 2,
entries 1, 2, 9, 10, 11, 16, 17, and 24), aliphatic (Table 2,
entries 3–5, 12, 13, 22, 23, 25, and 27), cyclic (Table 2, entries
7, 8, 14, 15, 20, 26, and 28) or heterocyclic (Table 2, entry 19) sub-
stituent at the C-4 position of the triazole ring were obtained.
Regarding the mechanism of the transformation of the
2-hydroxy-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium salts
3 into 5, we presume that the reaction proceeds via an unusual
Dimroth-type rearrangement15 (Scheme 2).
In the first step at lower temperatures the 2-hydroxy-2,3-
dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium salt 3 undergoes
mild cleavage of the pyrimidinium ring via intermediate A,
resulting in the generation of the pyrazole and 2-amino-
5-hydroxyimidazolidine. Copper nanoparticles14 generated by
reduction of Cu(OAc)2 with hydrazine hydrate at elevated temp-
eratures catalyse the CuAAC and generate intermediate B. The
latter is in equilibrium with the open form C, which cyclizes to
the more stable rearranged 2-AIT 5 at high temperatures.
Entry
Time (min)
Temp. (°C)
Cu source, mol%
Yieldb (%)
1c
2d
3
4
5
6
7
8
9
10
11
12
13
14
20
20
20
10
5
2
1
2
2
2
2
100 °C
100 °C
100 °C
100 °C
100 °C
100 °C
100 °C
100 °C
90 °C
100 °C
100 °C
100 °C
100 °C
rt
Cu(OAc)2, 10
Cu(OAc)2, 10
Cu(OAc)2, 10
Cu(OAc)2, 10
Cu(OAc)2, 10
Cu(OAc)2, 10
Cu(OAc)2, 10
Cu(OAc)2, 5
Cu(OAc)2, 5
Cu(OAc)2, 2
CuSO4·5H2O, 5
—
51
84
86
80
90
90
53
90
73
57
67
—
Traces
24
2
2
24 h
Cu-powder, 10
Cu(OAc)2, 5
a All reactions were conducted on a 0.25 mmol scale of 3a, applying
hydrazine hydrate (2 equiv.), and phenylacetylene (1.5 equiv.) in EtOH–
H2O (4 : 1, 1 mL) under microwave irradiation; the mixture was
irradiated in a sealed tube at the indicated ceiling temperature and
35 W maximum power for the stipulated time. b Isolated yields.
c 1.0 equiv. of hydrazine hydrate was used. d 5.0 equiv. of hydrazine
hydrate were used.
Anti-biofilm activity of 2-amino-1H-imidazole/triazole
conjugates and intermediates of their synthesis
of the catalyst (Table 1, entry 12). The optimal reaction con-
ditions were achieved when a mixture of the hydroxy salt 3a
(0.25 mmol), hydrazine hydrate (2 equiv.), phenyl acetylene
(1.5 equiv.), and Cu(OAc)2 (5 mol%) in EtOH–H2O (4 : 1; 1 mL)
was irradiated for 2 min at a ceiling temperature of 100 °C
applying a maximum power of 35 W. The desired compound
5a was isolated in 90% yield (Table 1, entry 8). When the reac-
tion was performed at rt for 24 h, the product 5a was obtained
in very low yield (Table 1, entry 14). The novelty of this one-pot
procedure is in the fact that the hydrazine is doing two totally
different tasks in the same pot, namely the decomposition of
the pyrimidinium salt on the one hand, and the in situ gene-
ration of copper nanoparticles on the other hand. Moreover, to
the best of our knowledge, the in situ generation of the
required copper catalyst to catalyze the CuAAC-reaction (“click
reaction”) is unprecedented, and is a valuable addition to the
arsenal of catalytic conditions for the CuAAC-reaction.
Encouraged by these findings, we explored the scope of this
protocol. First, an array of hydroxy salts was prepared using
our optimized conditions (Table 2). Special attention was given
to the use of α-bromoketones bearing halogenated phenyl sub-
stituents that were previously found to be crucial for the anti-
biofilm activity.2c,d In most cases, the reactions were run for
3 h in MeCN at 75 °C affording the hydroxy salts 3b–l in good
to excellent yields as white precipitates.
Compounds 5a–ab were assayed for their ability to prevent the
biofilm formation of Salmonella Typhimurium ATCC14028 and
Pseudomonas aeruginosa PA14 in TSB 1/20 at 25 °C, mimicking
conditions outside the host. As indicated in Tables 2 and S1,†
many of the compounds were active against both Salmonella
and Pseudomonas with BIC50 values (i.e. IC50 for biofilm inhi-
bition) in the range of 10–40 µM. Compounds 5g, 5h, 5s, 5z
and 5ab inhibited Salmonella biofilm formation even stronger,
with BIC50 values between 2 and 8 µM. These activities are
similar to those of the most potent 2N-substituted 5-aryl-2-
aminoimidazoles previously reported.2d To validate that the
compounds are specific inhibitors of biofilm formation and
do not act as bactericidal agents, also their effect on the plank-
tonic growth was measured. IC50 values for planktonic growth
inhibition are provided in Table 2, while effects on the growth
curve are provided in Table S1.† Most of the compounds are
specific biofilm inhibitors with a broad difference between
BIC50 and IC50 values. However, for some of the most active
compounds, i.e. 5g and 5s, it cannot be excluded that the
biofilm inhibition is at least partly due to killing the plank-
tonic bacteria before biofilms could be established.
More detailed analysis of the data allows us to draw a
number of conclusions with regard to the structure–activity
relationship. (i) Comparison of compounds 5a, 5c, 5l, and 5v
with respectively 5b, 5d, 5m, and 5w indicates that the length
(n = 2 or 3) of the alkyl linker between triazole and 2-aminoimid-
azole does not markedly affect the activity. (ii) However, the
The thus prepared hydroxy salts 3b–m (n = 1, 2) were
reacted with different (hetero)aromatic and alkyl acetylenes
(Table 2). All reactions were completed within 2 min delivering
the desired compounds in good to excellent yields. However,
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Table 2 Scope of the microwave-assisted one-pot synthesis of the 2-AIT framework and anti-biofilm activity against S. Typhimurium and
P. aeruginosaa
Anti-biofilm activity
Salmonella Typhimurium
ATCC14028
Pseudomonas aeruginosa PA14
Compounds 3
Compounds 5 Compounds 3 Compounds 5
b
c
Yield of Yield of BIC50
IC50
BIC50 IC50
BIC50
(µM)
IC50
(µM)
BIC50
(µM)
IC50
(µM)
Entry Compound R1
R2
n
R3
3 (%)
5 (%)
(µM)
(µM)
(µM)
(µM)
1
2
3
4
5
6
7
8
3a, 5a
3b, 5b
3c, 5c
3d, 5d
3c, 5e
3d, 5f
3c, 5g
3c, 5h
3c, 5i
3d, 5j
3c, 5k
3e, 5l
3f, 5m
3e, 5n
3f, 5o
3f, 5p
3e, 5q
3f, 5r
Ph
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2
1
1
2
1
2
1
1
1
2
1
1
2
1
2
2
1
2
2
1
1
1
Ph
Ph
Pr
Pr
Hept
C(CH3)2(NH2) 83
c-Pr
c-Hex
4-MeC6H4
4-PentylC6H4
4-MeOC6H4
Pr
Pr
c-Hex
83
89
90
83
90
91
84
94
75
73
75
80
71
73
80
66
85
91
89
75
85
84
81
91
80
68
39
71.7
142.6
45.1
19.5
>400
>400
162.6 25.3
40.0
36.5
>400
>400
∼94.5 43.6
∼96.3 63.3
23.5
29.3
365.5
>400
>400
133.1
19.0
>400
48.7
27.2
>400
21.5
71.6
12.5d
>400
>400
42.7
27.4
24.4
∼25d
>400
>400
>400
>400
>400
>400
20.1
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
4-BrC6H4
3,4-DiClC6H3
3,4-DiClC6H3
3,4-DiClC6H3
3,4-DiClC6H3
3,4-DiClC6H3
3,4-DiClC6H3
3,4-DiClC6H3
3,4-DiClC6H3
4-FC6H4
>400
35.3
188.8 >400
30.9
2.0
8.4
>400
>400
91.2
35.6
31.8
55.2
26.8
17.8
>800
10.8
2.0
∼91.4
2.4
90
90
90
83
90
75
77
75
77
77
75
77
>400
26.2
18.5
>400
>400
>400
>400
60.9
>400
>400
>400
>400
67.5
5.4
9
>400
>400
>400
>400
>400
>400
>400
10
11
12
13
14
15
16
17
18
19
20
21
22
15.3
12.5
>400
>400
46.6
50.5
196.0
120.3
c-Hex
4-tertBuC6H4
4-HeptylC6H4
CH2NHMe
Thiophen-3-yl 77
c-Pr
∼12.5d >400
>400
8.1
>400
6.7
>400
>400
>400
>400
3f, 5s
3g, 5t
3g, 5u
3h, 5v
3.8e
69
69
76
172.8
>400
>400
>400
>400
>400
93.3
128.3 >400
>400
>400
>400
19.0
>400
>400
>400
∼50d
32.9
>400
4-FC6H4
c-Pr-CH2
Pr
Morpholino-
methanone
Morpholino-
methanone
Naphtha-2-yl
CHPh2
CHPh2
Ph
4-ClC6H4
>400
>400
>400
∼91.0 23.1
>400
>400
>400
332.8
349.2
42.6
23
3i, 5w
H
2
Pr
65
45
>400
>400
24
25
26
27
28
3j, 5x
H
H
H
Ph
4-Me
C6H4
1
1
1
2
1
4-BuC6H4
tertBu
c-Pen
Hept
c-Pen
69
72
81
75
72
64
67
68
84
56
22.4
34.1
66.3
>400
>400
30.9
8.4
10.8
6.5
189.1
210.6
>400
10.9
>400
>400
>400
137.7
>400
>400
>400
3k, 5y
3k, 5z
3l, 5aa
3m, 5ab
169.4
49.1
>400
>400
∼200d >400
115.2
∼384.6 >400
a All reactions were conducted on a 0.25 mmol scale of 3b–m, applying hydrazine hydrate (2 equiv.), acetylene (1.5 equiv.), and Cu(OAc)2 (5 mol%) in EtOH–
H2O (4 : 1) (1 mL); the mixture was irradiated in a sealed tube at a ceiling temperature of 100 °C and 35 W maximum power for 2 min; isolated yields are
given. b BIC50: the compound concentration at which the biofilm formation is inhibited with 50%; 95% confidence intervals are provided in Table S1. c IC50
:
the compound concentration at which the planktonic growth is inhibited with 50%; 95% confidence intervals are provided in Table S1. The effect of the
compounds on the planktonic growth curves is also provided in Table S2. d The compound is not able to completely prevent biofilm formation, as the dose
response curve reaches a steady state level at about 50% biofilm inhibition. e With increasing concentrations, the dose response curve reaches a maximum of
90% biofilm inhibition at a concentration of ∼25 µM. At higher concentrations the % inhibition decreases again.
activity is strongly dependent on the nature of the substituents differences in activity were observed within the series of com-
at the 4(5)-position of the imidazole ring (R1 and R2). Indeed, pounds with the same R1 and R2 groups, i.e. compounds
compounds 5v and 5w, in which R1 is a morpholinometha- 5c–5k, 5l–5s, 5t–5u and 5y–5z. (iv) Remarkably, compound 5l,
none group, are not active at the highest concentration tested in which R1 is 3,4-dichorophenyl and R3 is propyl, has a good
(400 µM), while compounds 5c, 5d, 5l and 5m, which have a activity against both Salmonella and Pseudomonas biofilms.
halogenated phenyl group as R1 (but are further identical to 5v Meanwhile, compound A (Fig. 2), which has a side chain of the
and 5w) have BIC values around 50 µM. (iii) Also the substitu- same length at the 2N-position of the imidazole, but lacks the
ent at the triazole ring (R3) is of major importance as strong triazole moiety, was previously shown to be inactive.2d Also, com-
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Scheme 2 Proposed mechanism for the one-pot formation of the 2-AIT framework.
could be explained by (a partial) in situ cleavage of these salts
by cellular nucleophiles to form the active 2-aminoimidazoles.
Consistently, compound B (Fig. 2), which is formed after clea-
vage of compound 3m, inhibits the biofilm formation of Sal-
monella and Pseudomonas at BIC50 values which are slightly
lower than those of compound 3m.
Synthesis and anti-biofilm activity of 2-amino-1H-imidazole/
triazole conjugates with alkyl chain substitutions at the
triazole ring
Fig. 2 A. 2-Octyl-4(5)-(m,p-dichlorophenyl)-2-aminoimidazole, pre-
viously reported to be inactive against Salmonella and Pseudomonas
biofilms.2d
B.
2N-(2-Azidoethyl)-4-(p-chlorophenyl)-5-(p-methyl-
We previously reported that 5-phenyl-2-aminoimidazoles
bearing a long alkyl chain (C6–C9) at the 2N-position in many
cases have a low activity against biofilms, depending on the
substitution pattern of the 5-phenyl ring and the model organ-
ism studied.2d The results above suggest that incorporation of
a triazole moiety into this long alkyl chain can strongly
enhance the activity. To further consolidate this finding, we
synthesized an array of 2-AIT 5ac–aj (n = 1 or 2) in which R3
ranges from butyl to heptyl, and compared their anti-biofilm
activities with those of 5-aryl-2-aminoimidazoles 6a–i, bearing
2N-alkyl chains with the same total length. Next to S. Typhi-
murium and P. aeruginosa, also Escherichia coli and Staphylo-
coccus aureus were included in these tests. As indicated in
Tables 3 and S2,† the introduction of the triazole moiety
makes the compounds in general (except for 5ae and 5aj)
much more active (up to >100 times) against S. Typhimurium
and P. aeruginosa biofilms. Most of the 2-AIT have a broad con-
centration range between BIC50 and IC50, indicating that they
specifically affect Salmonella and Pseudomonas biofilm for-
mation. Incorporation of the triazole moiety makes the com-
pounds also much more potent against E. coli (except for 5ae).
However, the concentration range between BIC50 and IC50 is in
general much narrower. Finally, in the case of S. aureus, the
effect of introducing a triazole moiety is much less pro-
nounced. Both imidazoles and 2-AIT inhibit biofilm formation
in an aspecific way, as S. aureus biofilm formation and plank-
tonic growth are affected at similar concentrations.
phenyl)-2-aminoimidazole, formed after cleavage of compound 3m.
pound 5t in which R1 is 4-fluorophenyl and R3 is c-propyl has
a moderate activity against biofilms, while 4(5)-(4-fluoro-
phenyl)-2-aminoimidazoles with a long alkyl chain (>C5) at the
2N-position were previously shown to be inactive against
Salmonella biofilms.2d This suggests that introduction of the
triazole moiety into the long alkyl chains at the 2N-position of
the 2-aminoimidazoles can increase their activity.
As indicated in Tables 2 and S1,† also most of the
2-hydroxy-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium salts
3 (except for 3h and 3i) showed activity against both Salmonella
and Pseudomonas biofilms, with BIC50 values between 10 and
150 µM (Table 2). None of the compounds (except for 3j) had
an effect on the planktonic growth at the BIC50, indicating that
they have a specific anti-biofilm activity. These compounds
thus have a similar activity range and profile to the previously
described 1-pentyl- and 1-hexyl-2-hydroxy-2,3-dihydro-1H-
imidazo[1,2-a]pyrimidin-4-ium salts, which have a side chain
of the same length at the 1-position, but lack the azide func-
tion. As indicated in Scheme 1, 2-hydroxy-2,3-dihydro-1H-
imidazo[1,2-a]pyrimidin-4-ium salts can be cleaved in vitro
with a nucleophile such as hydrazine to yield 2-aminoimid-
azoles. We previously hypothesized that the activity of the
2-hydroxy-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium salts
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Table 3 Effect of incorporation of a triazole moiety into the long 2N-alkyl chain of 5-aryl-2-aminoimidazoles on the anti-biofilm activity against S.
Typhimurium, E. coli, P. aeruginosa and S. aureus
S. Typhimurium
ATCC14028
E. coli TG1
P. aeruginosa PA14
S. aureus SH100
Compound R1
R2
n
2
2
2
1
2
1
1
1
R3
R4
BIC50 a (µM) IC50 b (µM) BIC50 (µM) IC50 (µM) BIC50 (µM) IC50 (µM) BIC50 (µM) IC50 (µM)
5ac
6a
5ad
6b
4-ClC6H4
4-ClC6H4
4-FC6H4
4-FC6H4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Bu
23.9
>400
186.9
>400
114.5
49.70
28.1
38.2
41.5
46.5
137.2
>400
9.7
56.9
12.2
>400
∼100
∼199.5
71
47.3
6.7
19
>400
340
5.0c
>400
>400
>400
>400
159.7
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
∼94.7
∼200.8
304.3
∼75
∼150
∼55.7
∼50.1
∼70.9
45.7
∼91.4
>400
∼201.9
36.7
∼141.1
134.2
236.0
390.8
∼88.8
130.9
32.9
349.3
43.4
125.4
43.9
>400
440.8
109.5
141.4
∼51.0
42.1
Dec
Dec
Dec
Non
Dec
Non
Dec
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
>400
Bu
3.7c
∼400
∼75
>400
∼25
>400
∼25
∼50
>400
>400
∼25
>400
∼100
>400
19.0d
6.9c
5ae
6c
5af
6d
5ag
6e
5ah
6f
5ai
6g
5aj
6h
4-OMeC6H4
4-OMeC6H4
3,4-DiClC6H3
3,4-DiClC6H3
3,4-DiClC6H3
3,4-DiClC6H3
Naphth-2-yl
Naphth-2-yl
Naphth-2-yl
Naphth-2-yl
CHPh2
Bu
>400
3.1
Bu
9.6
14.9d
Bu
10.3
44.8
52.7
>400
13.2
187.6
40.2
∼200
6.6c
>400
3.6c
Bu
>400
Pen
Hept
1.3c
>400
31.9
>400
37.4
24.61c
51.9
∼55.0
CHPh2
Dodec >400
a BIC50: the compound concentration at which the biofilm formation is inhibited with 50%; 95% confidence intervals are provided in Table S2.
b IC50: the compound concentration at which the planktonic growth is inhibited with 50%; 95% confidence intervals are provided in Table S2.
c With increasing concentrations, the dose response curve reaches a maximum of 70 to 90% biofilm inhibition at a concentration between 6.25
and 50 µM. At higher concentrations the % inhibition decreases again. d With increasing concentrations, the dose response curve reaches a
maximum of 50 to 60% biofilm inhibition at a concentration between 12.5 and 25 µM. At higher concentrations the % inhibition decreases
again.
S. aureus undiluted TSB was used. Subsequently, overnight cul-
Experimental
General procedure for the preparation of 2-aminoimidazole-
tures (grown in Luria–Bertani medium)17 of S. Typhimurium
ATCC14028, P. aeruginosa PA14, E. coli TG1 and S. aureus TH1
were diluted 1 : 100 into the respective liquid broth and 100 μL
triazoles (2-AIT)
To a cooled (0 °C) solution of hydrazine hydrate (2 equiv.) in (∼106 cells) was added to each well of the microtiter plate,
EtOH (0.8 mL) was added a solution of Cu(OAc)2 (5 mol%) in resulting in a total amount of 200 μL of medium per well. The
water (0.2 mL) and the mixture was stirred for 2 min at 0 °C. pegged lid was placed on the microtiter plate, and the plate
Then acetylene (1.5 equiv., 0.38 mmol) and hydroxy salt 3 was incubated for 24 h at 25 °C (S. Typhimurium, P. aerugi-
(0.25 mmol) were added, the vial was sealed and the mixture nosa, E. coli) or 48 h at 37 °C (S. aureus) without shaking.
was irradiated (microwaves) at a ceiling temperature of 100 °C During this incubation period, biofilms were formed on the
applying a maximum power of 35 W for 2 min. After com- surface of the pegs. After incubation, the optical density at
pletion of the reaction, the solvent was removed under 600 nm (OD600) was measured for the planktonic cells in the
reduced pressure. The crude product was purified by column microtiter plate using a microtiter plate reader (Multimode
chromatography over silica gel using DCM–MeOH–NH3 (7 N Synergy MX, Biotek). The IC50 value for planktonic growth
soln in MeOH) (96 : 3 : 1) as the eluent.
inhibition was determined for each compound from the con-
centration gradient using the GraphPad software of Prism.
This gives the first indication of the effect of the compounds
on the planktonic growth. For quantification of biofilm for-
mation, the pegs were washed once in 200 μL of phosphate
buffered saline (PBS). The remaining attached bacteria were
stained for 30 min with 200 μL of 0.1% (w/v) crystal violet in
an 2-propanol–methanol–PBS solution (v/v 1 : 1 : 18). Excess
stain was rinsed off by placing the pegs in a 96-well plate filled
with 200 μL of distilled water per well. After the pegs were air-
dried (30 min), the dye bound to the adherent cells was
Static peg assay for prevention of biofilm formation
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).16 Two-fold serial dilutions of the com-
pounds in 100 μL of liquid broth per well were prepared in the
microtiter plate (two or three repeats per compound). For
S. Typhimurium, E. coli, and P. aeruginosa Tryptic Soy Broth
diluted 1/20 (TSB 1/20; BD Biosciences) was used, while for
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extracted with 30% glacial acetic acid (200 μL). The OD570 of grateful to Erasmus Mundus External Cooperation Window Lot
each well was measured using the Multimode Synergy MX, 15 India (EMECW15) for obtaining a scholarship. U.K.S. is
Biotek. The BIC50 value (i.e. IC50 for biofilm inhibition) for grateful to the Research Fund of KU Leuven for obtaining an
each compound was determined from the concentration gradi- F+ postdoctoral fellowship. T.T.T.T. is grateful to the 322-
ent using the GraphPad software of Prism.
program of Vietnamese Government for obtaining a PhD-grant.
The effect of the chemical compounds on the growth curve
of S. Typhimurium and P. aeruginosa was assayed using the
Bioscreen device (Oy Growth Curves AB Ltd). An overnight
culture of S. Typhimurium ATCC14028 or P. aeruginosa was
diluted 1 : 100 in liquid TSB 1/20. 300 μL of the diluted over-
night culture was added to each well of the 10 × 10 well micro-
titer plate. Subsequently, serial dilutions of the chemical
compounds were prepared in DMSO or EtOH. Three μL of each
diluted stock solution was added to the wells (containing
300 μL of bacterial culture) in 3-fold. As a control, 3 μL of the
appropriate solvent was also added to the plate in 3-fold. The
microtiter plate was incubated in the Bioscreen device at 25 °C
for at least 24 h, with continuous medium shaking. The absor-
bance of each well was measured at 600 nm at an interval of
15 min. The Excel was used to generate the growth curves for
the treated wells and the untreated control wells. The effect of
each compound concentration on the planktonic growth was
classified into one of the following categories:
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indicated by the symbol “−”.
(2) The planktonic growth is reduced, indicated by the
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Acknowledgements
Support was provided by the Research Fund of the KU Leuven
under grant BOF-IDO/11/008, by the Institute for the Pro-
motion of Innovation through Science and Technology in Flan-
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Scientific Research (FWO) – Flanders (Belgium). H.S. and D.E.
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