Structure-activity relationship of 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1, 2-a]pyrimidinium salts and 2N-substituted 4(5)-aryl-2-amino-1H-imidazoles as inhibitors of biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.bmc.2011.04.026

A library of 80 1-substituted 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-a] pyrimidinium salts and 54 2N-substituted 4(5)-aryl-2-amino-1H-imidazoles was synthesized and tested for the antagonistic effect against biofilm formation by Salmonella Typhimurium a

Full text of DOI:10.1016/j.bmc.2011.04.026

Bioorganic & Medicinal Chemistry 19 (2011) 3462–3473
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship of 2-hydroxy-2-aryl-2,3-dihydro-
imidazo[1,2-a]pyrimidinium salts and 2N-substituted 4(5)-aryl-2-
amino-1H-imidazoles as inhibitors of biofilm formation
by Salmonella Typhimurium and Pseudomonas aeruginosa
Hans P. L. Steenackers a,c, Denis S. Ermolat’ev b, Bharat Savaliya b,d, Ami De Weerdt a,
David De Coster a, Anamik Shah d, Erik V. Van der Eycken b, Dirk E. De Vos c,
Jozef Vanderleyden a, Sigrid C. J. De Keersmaecker a,
⇑
a Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium
b Laboratory for Organic & Microwave-Assisted Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium
c Centre for Surface Chemistry and Catalysis, Katholieke Universiteit Leuven, Kasteelpark Arenberg 23, B-3001 Leuven, Belgium
d Department of Chemistry, Saurashtra University, 360 005 Rajkot, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 December 2010
Revised 8 April 2011
Accepted 13 April 2011
Available online 19 April 2011
A library of 80 1-substituted 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts and 54 2N-
substituted 4(5)-aryl-2-amino-1H-imidazoles was synthesized and tested for the antagonistic effect
against biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa. The nature of the
substituent at the 1-position of the salts was found to have a major effect on their biofilm inhibitory
activity. Salts with an intermediate length n-alkyl or cyclo-alkyl chain (C7–C10) substituted at the 1-posi-
tion in general prevented the biofilm formation of both species at low micromolar concentrations, while
salts with a shorter n-alkyl or cyclo-alkyl chain (C1–C5) or longer n-alkyl chain (C11–C14) were much less
potent. Salts with a long cyclo-alkyl chain however were found to be strong biofilm inhibitors. Further-
more, we demonstrated the biofilm inhibitory potential of salts with certain aromatic substituents at the
1-position, such as piperonyl or 3-methoxyphenetyl. The activity of the 2-aminomidazoles was found to
be dependent on the nature of the 2N-substituent. Compounds with a n-butyl, iso-butyl, n-pentyl, cyclo-
pentyl or n-hexyl chain at the 2N-position have an improved activity as compared to their unsubstituted
counterparts, whereas compounds with shorter 2N-alkyl chains do have a reduced activity and com-
pounds with longer 2N-alkyl chains do have an effect that is dependent on the nature of the substitution
pattern of the 4(5)-phenyl ring. Finally, we demonstrated that introduction of a 3-methoxyphenethyl or
piperonyl group at the 2N-position of the imidazoles could also result in an enhanced biofilm inhibition.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Biofilm formation
Biofilm prevention
Salmonella Typhimurium
Pseudomonas aeruginosa
2-Aminoimidazoles
2-Hydroxy-2-aryl-2,3-dihydro-
imidazo[1,2-a]pyrimidinium salts
1. Introduction
a high risk for the development of biofilm related infections.2 This
high prevalence of biofilm related infections is particularly prob-
Bacteria are able to switch between a planktonic life style and a
biofilm mode of growth, in which they live as structured commu-
nities of bacterial cells enclosed in a self-produced polymeric ma-
trix and adherent to an inert or living surface.1–3 As bacteria in
biofilms are more resistant to challenges from predators, antibiot-
ics, disinfectants and host immune systems,2,4–8 serious problems
and high costs have been associated with biofilms, both in medi-
cine and industrial settings. According to the National Institutes
of Health, more than 80% of microbial infections are related to bio-
films.9 Especially the use of indwelling medical devices comprises
lematic given the fact that bacteria in biofilms can be up to
1000-fold more resistant to antibiotics.10,11 In industry, biofilms
have been implicated, for example, in the contamination of instal-
lations in food industry, mild steel corrosion, decreased passage
through pipelines by colonization of the interior of the pipes, and
enhanced resistance of vessels by initiation of ‘biofouling’ on the
vessel hulls. The yearly economic loss caused by ‘biofouling’ in
the marine industry is estimated at $ 6.5 billion.12
One way to deal with the problem of biofilms is the develop-
ment of small molecules that are able to prevent or destroy biofilm
formation.13–15 Only a few molecular scaffolds have been identified
to date, among which the best-studied examples are (1) the
halogenated furanones, which were originally isolated from the
seaweed Delisea pulchra,16–18 (2) analogues of the homoserine
⇑
Corresponding author. Tel.: +32 16321631; fax: +32 16321966.
E-mail address: Sigrid.DeKeersmaecker@biw.kuleuven.be (S.C.J. De Keersmaecker).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.026

H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
3463
lactone signalling molecules19 and (3) analogues of the sponge-de-
rived marine natural alkaloids oroidin and bromoageliferin.20–27
oles (pathway A), while the hydrazinolysis of 2-hydroxy-2,3-dihy-
dro-imidazopyrimidinium salts results in formation of 2N-
substituted 2-amino-1H-imidazoles (pathway B). Recently, we
reported on the biofilm inhibitory activity of N1-substituted 5-phe-
nyl-2-aminoimidazoles and their precursor imidazo[1,2-a]pyri-
midinium salts,42 which were synthesized via pathway A. A good
correlation was found between the activity of the imidazo[1,2-
a]pyrimidinium salts and their corresponding 2-aminoimidazoles,
supporting the hypothesis that the imidazo[1,2-a]pyrimidinium
salts could also in situ be cleaved by cellular nucleophiles to form
the active 1-substituted 2-aminoimidazoles. In the same report we
described the biofilm inhibitory activity of para-substituted
4(5)-phenyl-2-amino-1H-imidazoles and 4,5-distubstituted 2-
amino-1H-imidazoles, which were synthesized via a different pro-
cedure.42,43 In the present study, we focus on the biofilm inhibitory
activity of compounds synthesized via pathway B of Scheme 1. We
tested the influence of a series of 2-hydroxy-2-phenyl-2,3-dihy-
dro-imidazopyrimidinium salts with alkyl, cyclo-alkyl and
aromatic substituents at the 1-position and their corresponding
2N-substituted 2-aminoimidazoles on the biofilm formation of
Salmonella enterica serovar Typhimurium and P. aeruginosa and
we delineated a structure–activity relationship.
A
particularly valuable approach is the development of small mole-
cules that specifically target the biofilm formation in a non-toxic
manner, as it is expected that resistance against these compounds
will emerge much slower than against classical microbiocidal com-
pounds. As a consequence, non-toxic biofilm inhibitors have the
potential to be used in a preventive treatment of a wide diversity
of industrial and medical surfaces. Furthermore, the potential to
co-dose biofilm inhibitors and antibiotics for the treatment of bio-
film infections is also an attractive option.17
Salmonella enterica serovar Typhimurium (S. Typhimurium) and
Pseudomonas aeruginosa are two well studied organisms in terms of
biofilm formation. Salmonella enterica is worldwide one of the most
important causes of foodborne infections. Salmonella is able to form
biofilms (as reviewed by Steenackers et al.28) on a variety of both
biotic surfaces (such as gallstones,29 plant surfaces,30 and epithelial
cell layers31) and abiotic surfaces (such as concrete, plastics, glass,
polystyrene, . . .).32,33 These biofilms are an important survival
strategy in all stages of infection, from transmission to chronic
infection. Severe non-typhoid Salmonella infections are commonly
treated with fluoroquinolones and third-generation cephalospo-
rins. Unfortunately, there are alarming reports concerning the
development of resistance against these antibiotics,34 underlining
the urgent need of alternative anti-Salmonella strategies. Given
the importance of biofilms in the spread of Salmonella, the develop-
ment of Salmonella biofilm inhibitors seems a promising approach.
P. aeruginosa is an ubiquitous Gram-negative microorganism found
in many environments, such as soil and water. P. aeruginosa is also
an opportunistic pathogen implicated in a myriad of infections. Pa-
tients with compromised host defenses, such as burn patients, HIV
patients and cystic fibrosis patients (80% colonization rate of P.
aeruginosa35) are particularly susceptible to P. aeruginosa infec-
tions.36 P. aeruginosa biofilms have been related to recurrent ear
infections, chronical bacterial prostatitis and lung infections in CF
patients, the latter being extremely harmful as P. aeruginosa coloni-
zation and chronic lung infection is the major causative agent of
morbidity and mortality in CF patients.37–39 Moreover, P. aerugin-
osa can colonize as biofilms a variety of medical devices such as
intravascular catheters and urinary catheters. Obviously there is
an urgent need of agents that can prevent or eradicate P. aeruginosa
biofilms on infected tissues and on medical devices.
2. Results and discussion
2.1. 2-Hydroxy-2,3-dihydro-imidazopyrimidinium salts
A broad array of 1-substituted 2-hydroxy-2-aryl-2,3-dihydro-
imidazopyrimidinium salts were synthesized via pathway B of
Scheme 1 and their ability to prevent the biofilm formation of
S. Typhimurium and P. aeruginosa was tested. These molecules
differ in the substitution pattern of the 2-phenyl ring and in the
nature of the substituent at the 1-position, that is, n-alkyl, iso-alkyl,
cyclo-alkyl or aromatic.
2.1.1. n-Alkyl or iso-alkyl substituents at 1-position
In first instance, a series of 2-hydroxy-2-aryl-2,3-dihydro-
imidazopyrimidinium salts with a broad variety of n-alkyl or iso-
alkyl substituents at the 1-position, with lengths ranging
from one carbon atom to 14 carbon atoms, was synthesized. The
2-phenyl group of these salts is either unsubstituted (compounds
1–14), para-substituted with a chlorine atom (compounds 15–
27), a nitro group (compounds 28–36), a fluorine atom (com-
pounds 37–40), a bromine atom (compounds 41–43) or a methoxy
group (compounds 45–47), meta-substituted with a nitro group
(Compound 44) or 3,4-disubstituted with chlorine atoms
(compounds 48–50) (Table 1). Each compound was assayed for
Previously, we published a divergent synthesis of substituted 2-
aminoimidazoles from 2-aminopyrimidines and a-bromocarbonyl
compounds.40,41 As described in Scheme 1, the cleavage of 1,2,3-
trisubstituted imidazo[1,2-a]pyrimidinium salts with hydrazine
or secondary amine leads to 1,4,5-trisubstituted 2-aminoimidaz-
Scheme 1. Divergent synthesis of substituted 2-aminoimidazoles from 2-aminopyrimidines and
a
-bromocarbonyl compounds.41 (A) Synthesis of N1-substituted 2-
aminoimidazoles from 2-aminopyrimidines via 1-substituted imidazo[1,2-a]pyrimidinium salts. (B) Synthesis of 2N-substituted 2-aminoimidazoles from 2-aminopyrim-
idines via 1-substituted 2-hydroxy-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts.

3464
H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
the ability to inhibit S. Typhimurium ATCC14028 biofilm formation
at 25 °C. As depicted in Figure 1A, a clear correlation was found be-
tween the length of the alkyl substituent and the biofilm inhibitory
activity. In general the activity of the molecules with a short alkyl
chain (C1, C2 and C3) was found to be very low (IC50 >400 lM).
Within a series of molecules with the same substitution of the 2-
phenyl ring, in general a gradual increase in biofilm inhibitory
activity was observed by raising the length of the alkyl chain from
Table 1
Influence of 1-alkylated 2-hydroxy-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts 1–50 on the biofilm formation of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at 25 °C
Compound
R1
R
S. Typhimurium
P. aeruginosa
Effect on growth atb
IC50
(l
M)
95% confidence
Effect on growth atb
a
a
IC50
(lM)
95% confidence
interval for IC50
interval for IC50
a
a
IC50
40
lM
IC50
40 lM
1
2
3
4
5
6
7
8
9
Me
i-Pr
n-Bu
i-Bu
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-F
>800
>400
>400
>400
540.5
409.5
131.2
278.2
102.0
23.4
9.8
420.4–695.0
348.4–481.3
64.0–268.9
197.4–392.2
77.9–133.7
19.6–27.8
8.3–11.5
289.4
565.4
405.8
25.5
14.4
26.8
29.9
>800d
>800d
>800d
>800d
169.6
24.9
83.4
163.9
79.8
36.9
15.7
20.2
83.3
>800d
>800
>800
>800
>400
>800
191.9
145.2
191.7
41.5
12.4
12.1
22.3
ndc
148.3–564.6
269.6–1186.0
183.5–897.4
16.5–39.4
10.3–20.3
16.0–44.9
n-Pen
n-Hex
n-Hep
n-Oct
n-Non
n-Dec
n-Und
n-Dod
n-Trd
n-Tet
Me
o
o
o
o
o
o
+
+
o
o
8.3
7.2–9.5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
15.0
24.6
27.5
81.6
179.6
225.0
29.7
31.4
32.6
13.7
6.7
13.4–16.8
20.6–29.4
21.5–35.2
70.5–94.6
142.3–226.8
176.1–287.5
23.6–37.3
21.1–46.6
27.0–39.3
11.5–16.3
5.7–7.7
20.1–44.4
o
o
34.5–834.0
15.9–39.1
60.2–115.6
105.7–254.2
29.9–213.0
24.2–56.4
10.6–23.3
13.5–30.2
50.1–138.3
n-Bu
i-Bu
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
n-Pen
n-Hex
n-Hep
n-Oct
n-Non
n-Dec
n-Und
n-Dod
n-Trd
n-Tet
Me
Et
i-Pr
n-Bu
i-Bu
t-Bu
n-Hex
n-Oct
n-Dec
Me
n-Bu
n-Oct
n-Dec
n-Bu
i-Bu
n-Pen
Me
n-Bu
n-Oct
n-Dec
i-Bu
o
7.0
6.1–8.0
o
o
o
o
o
o
18.3
51.3
61.9
122.1
316.1
>800
698.3
578.8
466.5
292.8
322.5
211.1
54.7
6.6
15.3–22.0
42.4–62.2
50.7–75.4
101.7–146.5
240.9–414.7
446.1–1093.0
432.9–774.0
375.4–579.8
220.7–388.3
252.1–412.5
162.2–274.7
41.1–72.8
5.5–7.968
8.056–12.3
526.1–6328.0
136.8–194.2
6.334–9.058
14.9–27.0
76.2–106.7
3.2–8.6
1.1–2.5
470.3–675.9
226.5–436.3
8.2–13.6
14.5–23.8
6.4–10.0
91.1–404.2
54.2–388.6
102.8–357.5
29.1–59.2
9.6–16.0
+
+
o
+
+
o
8.3–17.6
17.4–28.5
9.9
1825.0
163.0
7.6
20.0
90.1
5.2
4-F
4-F
4-F
nd
nd
nd
nd
6.3
4.2
nd
nd
nd
nd
nd
nd
4-Br
4-Br
4-Br
3-NO2
4-OMe
4-OMe
4-OMe
3,4-diCl
3,4-diCl
3,4-diCl
ꢀ
ꢀ
+
ꢀ
3.7–10.6
2.4–7.5
ꢀ
ꢀ
ꢀ
ꢀ
1.7
563.8
314.4
10.5
18.6
8.0
o
ꢀ
o
n-Oct
n-Dec
7.1
32.4
6.2–8.2
21.0–50.1
nd
a
IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.
o: the planktonic growth is completely or almost completely inhibited when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; +:
b
the planktonic growth is retarded when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; ꢀ: the planktonic growth is not or only
slightly affected when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; No symbol indicated: effect not determined.
c
nd: not determined.
d
Although IC50 >800
l
M, the compound inhibits biofilm formation to a certain extent, but the dose response curve reaches a steady state level of 25–45% biofilm inhibition
starting from concentrations between 25 and 50
lM.

H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
3465
1 to 8 carbon atoms. The compounds with an octyl substituent
show a maximal activity with IC50 values in the range of 6–
11 lM. By further raising the alkyl chain length from 8 to 14 car-
dro-imidazopyrimidinium salts 51–56 with more variation in the
substitution pattern of the 2-phenyl ring. As depicted in Table 2
these compounds generally inhibit biofilm formation of S.
Typhimurium at low concentrations. However, no improved activ-
ity was observed in comparison with the previously described
compounds (Table 1). The effect of the n-octyl substituted com-
pounds 51–56 (Table 2) on the biofilm formation of P. aeruginosa
strongly depends on the substitution pattern of the 2-phenyl ring,
as some of the compounds (51, 52 and 56) have low IC50 values
bon atoms, a gradual decrease in biofilm inhibitory activity was ob-
served. For the molecules with a heptyl, octyl, nonyl and decyl side
chain (which are the most active molecules), the nature of the sub-
stituent of the 2-phenyl group does not have a substantial effect on
the biofilm inhibitory activity.
Next, we assayed the influence of a subset of the 2-hydroxy-2-
aryl-2,3-dihydro-imidazopyrimidinium salts (compounds 1–36)
for their ability to prevent the biofilm formation of P. aeruginosa.
As represented in Table 1 and Figure 1B, a similar structure–
activity relationship was found as in the case of Salmonella biofilm
inhibition. Compounds with short alkyl substituents (C1–C6) in
general only have a slight effect on the biofilm formation (IC50’s
(10–40
lM), while the other compounds only have a moderate
activity (IC50 = 100–400
l
M).
Finally, we determined the influence of some of the most active
compounds on the planktonic growth of S. Typhimurium and P.
aeruginosa in function of time. As depicted in Table 1, the tested
compounds with a butyl or pentyl chain at the 1-position do not
reduce the planktonic growth at concentrations equal to the IC50
for biofilm inhibition (except for compound 18). This demonstrates
the potential of the class of the 2-hydroxy-2,3-dihydro-imi-
dazo[1,2-a]pyrimidinium salts as specific inhibitors of biofilm for-
mation. However, all the tested compounds with alkyl side chains
between 6 and 12 carbon atoms in length were found to strongly
reduce or completely prevent the planktonic growth at concentra-
tions equal to the IC50 for biofilm inhibition. This indicates that
these compounds are more toxic and are likely to prevent biofilm
formation by inhibiting the planktonic growth of the bacteria in
the growth medium around the surface on which the biofilms is
formed, that is, being toxic for planktonic cells.
>80
(C7–C10) have IC50 values between 20 and 40
with long side chains (C11–C14) have IC50 values higher than
800 M. However, it should be mentioned that some of the com-
l
M), while compounds with medium length side chains
lM and compounds
l
pounds with long side chain (11–14 and 24) do reduce the biofilm
formation to a certain extent, but their dose response curves reach
a steady state level of 25–45% biofilm inhibition starting from con-
centrations between 25 and 50 lM.
Since the compounds with a n-octyl chain substituted at the 1-
position have the highest activity against Salmonella biofilms and
also have a high activity against Pseudomonas biofilms, we decided
to synthesize the additional 1-octyl-2-hydroxy-2-aryl-2,3-dihy-
Figure 1. Effect of the length of the n-alkyl or iso-alkyl chain at the 1-position of 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts, substituted with Cl, H or
nitro at the para-position of the 2-phenyl ring, on the IC50 M) for inhibition of biofilm formation of S. Typhimurium ATCC14028 (Graph A) and P. aeruginosa (Graph B).
(l

3466
H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
2.1.2. Cyclo-alkyl substituents at 1-position
to the structure–activity relationship delineated for the n-alkyl
substituted salts, a gradual increase in the inhibitory activity
against Salmonella biofilms was observed by raising the length of
the cyclo-alkyl chain from 3 to 8 carbon atoms. However, in
contrast to the salts with a n-dodecyl chain at the 1-position, the
salts with a cyclo-dodecyl chain do have a very strong effect
We synthesized and tested a series of 2-hydroxy-2-aryl-2,3-
dihydro-imidazopyrimidinium salts with a broad variety of cyclo-
alkyl substituents at the 1-position, with lengths ranging from 3
to 12 carbon atoms (Table 3). We also tested one compound with
an adamantyl group substituted at the 1-position (compound
70). The 2-phenyl group of these salts was either unsubstituted
(compounds 57–60), or para-substituted with a chlorine atom
(compounds 61–67) or a nitro group (compounds 68–70). Similarly
against Salmonella biofilms (IC50 values <6.25
adamantyl substituted compound 70 has a strong anti-Salmonella
biofilm activity (IC50 = 9 M). By analogy with the effect on
lM). Also the
l
Table 2
Influence of N1-octyl 2-hydroxy-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts 51–56 on the biofilm formation of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at 25 °C
Compound
R
S. Typhimurium
P. aeruginosa
a
a
IC50
(lM)
95% confidence interval for IC50
IC50
(lM)
95% confidence interval for IC50
51
52
53
54
55
56
4-SMe
ꢁ13.2b
14.7
31
60.3
ꢁ52.8
7.1
36.5
15.1
215.2
99.9
336.1
29.0
30.1–44.3
10.2–22.2
82.1–564.0
66.8–149.4
217.8–518.6
20.6–40.8
13.4–16.2
27.1–35.5
42.9–84.7
4-(40-NO2Ph)
4-SO2Me
4-(40-Biphenyl)
3-Br
6.2–8.2
a
IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.
ꢁ: the IC50 value could not be determined accurately due to the steepness of the dose response curve.
b
Table 3
Influence of 1-cyclo-alkyl-2-hydroxy-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts 57–70 on the biofilm formation of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at
25 °C
Compound
R1
R
S. Typhimurium
95% confidence
P. aeruginosa
95% confidence
a
a
IC50
(l
M)
Effect on growth atb
IC50
(
lM)
Effect on growth atb
interval for IC50
interval for IC50
a
a
IC50
80
lM
IC50
80 lM
57
58
59
60
61
62
63
64
65
66
67
68
69
70
c-Bu
H
H
H
H
493.0
139.0
84.2
58.4
82.7
120.8
41.8
33.0
27.9
12.6
5.6
352.7–689.1
100.6–192.0
72.8–97.3
>400
22.4
191.3
48.6
179.2
371.5
41.9
c-Hex
c-Hep
c-Oct
c-Pr
+
10.5–47.9
83.9–436.5
22.4–105.5
90.3–355.8
273.4–504.6
23.0–76.2
9.2–29.43
37.9–153.0
ꢀ
ꢀ
o
+
o
ꢀ
42.9–79.5
o
o
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-NO2
4-NO2
4-NO2
65.0–105.2
94.1–155.2
30.2–57.8
26.9–40.48
22.7–34.25
10.9–14.68
4.8–6.715
c-Bu
c-Pen
c-Hex
c-Hep
c-Oct
c-Dod
c-Pr
+
+
o
o
o
+
o
+
ꢀ
o
+
o
+
o
o
16.4
76.2
ꢁ22.7c
6.8
5.6–8.2
10.6–39.0
3.9–7.4
125.1
<6.3
8.8
88.5–177.0
20.4
5.4
14.4
c-Dod
Adamantyl
o
o
6.8–11.4
10.4–20.1
a
IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.
o: the planktonic growth is completely or almost completely inhibited when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; +:
b
the planktonic growth is retarded when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; ꢀ: the planktonic growth is not or only
slightly affected when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; No symbol indicated: effect not determined.
c
ꢁ: the IC50 value could not be determined accurately due to the steepness of the dose response curve.

H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
3467
Salmonella, we observed that all the cyclo-alkyl substituted salts
with a short side chain do only have a slight effect on the biofilm
formation of P. aeruginosa (IC50’s >150 lM), while the compounds
with a medium length side chain have a stronger biofilm inhibitory
Growth curve analysis revealed that compounds 73, 79 and 80 do
not affect the plantkonic growth of Salmonella at concentrations
higher than the IC50 of biofilm inhibition. However, the most active
compounds 77 and 78 have a toxic effect on Salmonella at concen-
trations equal to the IC50 for biofilm inhibition, implicating that we
cannot exclude the possibility that part of the inhibitory effect
against Salmonella biofilms is due to a reduction of the planktonic
growth in the growth medium surrounding the surface on which
the biofilms are formed.
activity. The salts with a cyclo-dodecyl chain do drastically reduce
the Pseudomonas biofilm formation (IC50’s ꢁ7
lM), in sharp con-
trast to the compounds with a n-dodecyl side chain. Growth curve
analysis revealed that some of the compounds have a specific effect
on biofilm formation as they do not affect the planktonic growth at
the IC50 for biofilm formation, that is, compound 59 in case of Sal-
monella biofilm inhibition and compounds 58, 59 and 64 in case of
Pseudomonas biofilm inhibition (Table 3). The other compounds
tested do strongly decrease the planktonic growth of the bacteria
at the IC50 for biofilm inhibition, which indicates that we cannot
exclude the possibility that (part) of the biofilm inhibitory activity
of the compounds is a consequence of the reduction of the amount
of cells in the growth medium around the biofilm substrate.
2.2. 2N-substituted 2-aminoimidazoles
In a previous publication, the biofilm inhibitory activity of the
class of the 2N-unsubstituted 4(5)-aryl-2-amino-1H-imidazoles
was described.42 We also reported that introduction of a medium
length n-alkyl or cyclo-alkyl chain at the N1-position of these
imidazoles could drastically enhance the biofilm inhibitory
activity. Also introduction of a benzyl or piperonyl group at the
N1-position was described to enhance the biofilm inhibitory
activity, although this effect is strongly dependent on the nature
of the substitution pattern of the 4(5)-phenyl ring. These N1-
substituted 2-aminoimidazoles were synthesized via synthesis
pathway A of Scheme 1. In the present study, we aimed to inves-
tigate whether introduction of a n-alkyl, iso-alkyl, cyclo-alkyl or
aromatic group at the 2N-position of the 4(5)-aryl-2-amino-1H-
imidazoles could also enhance their biofilm inhibitory activity.
Therefore a broad range of 2N-substituted 4(5)-aryl-2-amino-
1H-imidazoles were synthesized by using pathway B (Scheme
1) and their activity was compared with the activity of the 2N-
unsubstituted 4(5)-aryl-2-amino-1H-imidazoles.
2.1.3. Aromatic substituents at the 1-position
Finally, we synthesized and tested an array of 2-hydroxy-2-
aryl-2,3-dihydro-imidazopyrimidinium salts, which are substi-
tuted at the 1-position with a benzyl (compounds 71–73), para-
methoxybenzyl (compound 74), 3-methoxyphenethyl (compounds
75–78) or piperonyl group (compounds 79 and 80) (Table 4). The
compounds with a benzyl and para-methoxybenzyl substituent
only inhibit the biofilm formation of Salmonella moderately (IC50
values between 36 and 212 lM), while the activity of the com-
pounds with a 3-methoxyphenetyl group is dependent on the nat-
ure of the substitution pattern of the 2-phenyl ring. Indeed,
compounds 75 and 76, bearing respectively a para-nitro and
para-fluoro substituted 2-phenyl ring, inhibit Salmonella biofilm
formation only at moderate concentrations, while compounds 77
and 78, bearing respectively a 3-dichlorobenzyl group and a
[1,10:40,100-terphenyl]-4-yl group at the 2-position have a strong
2.2.1. n-Alkyl or iso-alkyl substituents at 2N-position
We synthesized and tested an array of 4(5)-aryl-2-aminoimid-
azoles 2N-substituted with either a short n- or iso-alkyl chain
(C1–C5) or a n-octyl or n-nonyl chain. As depicted in Table 5, a
broad diversity of 4(5)-aryl groups were included such as phenyl
(compounds 81–84), para-chlorophenyl (compounds 85–89),
activity (IC50’s 7–12
lM). Compounds 79 and 80, substituted with
a piperonyl ring at the 2-position inhibit Salmonella biofilm forma-
tion at low to moderate concentrations (IC50’s resp. 17 and 53 lM).
Table 4
Influence of 2-hydroxy-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts 71–80 with aromatic substituents at the N1-position on the biofilm formation of S. Typhimurium
ATCC14028 at 25 °C
Effect on growth atb
a
Compound
R
IC50
(lM)
95% confidence interval for IC50
a
IC50
80
l
M
40 lM
71
72
73
74
75
76
77
78
79
80
H
4-NO2
4-I
4-Br
4-NO2
4-F
3,4-diCl
4-(40-Biphenyl)
4-FPh
211.9
174.3
36.7
109.8
123.5
105.2
7.2
12.4
17.2
53.7
171.0–262.6
204.0–446.6
28.5–47.21
85.8–140.5
99.3–153.7
70.9–155.9
5.6–9.3
9.3–16.7
12.1–24.5
29.6–97.6
ꢀ
ꢀ
+
o
o
ꢀ
ꢀ
3,4-MethylenedioxyPh
ꢀ
ꢀ
a
IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.
o: the planktonic growth is completely or almost completely inhibited when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; +:
b
the planktonic growth is retarded when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; ꢀ: the planktonic growth is not or only
slightly affected when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; No symbol indicated: effect not determined.

Table 5
Influence of 2N-alkylated 2-aminoimidazoles 81–117 on the biofilm formation and the planktonic growth of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at 25 °C
H
N
H
N
R1 N
R1 N
N
H
N
H
R
81-113
and
114
115-117
Compound R1
R
S. Typhimurium
Effect on growth atb
P. aeruginosa
Effect on growth at:b
IC50
(l
M)a 95% confidence
interval for IC50
IC50
(l
M)a 95% confidence
interval for IC50
a
a
IC50
200
lM
150
l
M
80
l
M
40
l
M
20
l
M
IC50
100
l
M
80
lM
40
lM
20
l
M
10
lM
5
lM
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
H
H
H
H
H
Cl
Cl
Cl
Cl
130.2
>800
25.3
4.9
16.0
2.0
112.6–150.7
ꢀ
ꢀ
72.6
38.7–136.2
156.9–434.9
21.3–47.3
0.6–2.5
2.3–5.2
0.5–1.8
ꢀ
ꢀ
i-Pr
n-Bu
i-Bu
H
i-Bu
n-Pen
n-Oct
n-Non Cl
H
Me
Et
261.2
31.8
1.2
23.3–27.6
3.5–6.7
14.3–17.9
1.6–2.5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3.5
0.9
ꢀ
>400
>400
>800
17.6
170.4
171.3
701.5
3.8
10.9
84.4
101.7
15.5
>400
>400
47.9
7.1
ꢁ6.3d,e
ꢁ1.6e
ndc
NO2
15.0–20.5
113.3–256.2
123.9–236.9
460.8–1068.0
3.0–4.8
ꢀ
o
34.5
56.1
103.9
>800
22.9
ꢁ25.0e
15.0
25.2
10.8
ꢁ12.5e
ꢁ3.1e
3.2
20.4–58.5
31.4–100.3
40.7–265.0
ꢀ
ꢀ
+
ꢀ
NO2
NO2
NO2
NO2
NO2
F
F
F
F
F
ꢀ
n-Bu
i-Bu
n-Oct
H
ꢀ
ꢀ
ꢀ
ꢀ
5.8–91.2
ꢀ
7.6–15.4
ꢀ
ꢀ
69.7–102.3
85.7–120.6
12.6–19.2
ꢀ
8.6–26.2
21.1–30.2
7.8–14.9
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Me
98
99
n-Bu
n-Hex
n-Oct
H
n-Bu
i-Bu
n-Pen
n-Oct
H
n-Bu
n-Oct
Pr
n-Pen
n-Oct
Me
ꢀ
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
Br
Br
Br
Br
36.6–63.0
3.7–13.9
2.6–3.4
2.0–5.0
1.1–3.4
2.3–4.5
6.8–14.1
0.6–2.3
2.9–35.9
ꢀ
ꢀ
ꢀ
ꢀ
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
9.8
1.2
ꢀ
ꢀ
2.9
3.1
1.9
+
+
ꢀ
ꢀ
10.2
>800
186.3
46.3
>800
27.7
0.7
>800
nd
nd
Br
4-OMe
4-OMe
4-OMe
3,4-diCl
3,4-diCl
3,4-diCl
3,4-diF
4-OH
119.7
52.6
ꢁ400e
10.9
2.2
106.9–134.1
16.3–169.0
136.2–254.8
28.5–75.1
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
9.6–12.4
1.7–2.8
71.7–195.2
288.1–1257.0
115.8–221.7
132.1–430.3
ꢀ
11.7–65.4
0.4–1.1
118.3e
601.9
160.3
238.4e
Me
n-Oct
H
n-Oct
n-Oct
>800
>800
ꢁ3.1e
>800
4-SO2Me >800
4-SO2Me 41.8
3-Br
30.6–57.1
22.9–87.0
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
44.64
a
IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.
o: the planktonic growth is completely or almost completely inhibited when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; +: the planktonic growth is retarded when the bacteria are
b
grown in the presence of the indicated concentration of biofilm inhibitor; ꢀ: the planktonic growth is not or only slightly affected when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; No
symbol indicated: effect not determined.
c
nd: not determined.
d
ꢁ: the IC50 value could not be determined accurately due to the steepness of the dose response curve.
e
The compound is not able to completely prevent biofilm formation, as the dose response curve either reaches a steady state level between 50% and 70% biofilm inhibition or shows a maximum between 50% and 70% biofilm
inhibition and decreases again with higher compound concentrations.

H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
3469
para-nitrophenyl (compounds 90–95), para-fluorophenyl (com-
higher IC50 values (100–400
l
M). Moreover, these compounds are
pounds 96–100), para-bromophenyl (compounds 101–105), para-
methoxyphenyl (compounds 106–108), 3,4-dichlorophenyl (com-
pounds 109–111), 3,4-difluorophenyl (compound 112), para-
hydroxyphenyl (compound 113), naphthyl (compound 114),
para-methylsulphonylphenyl (115–116) and meta-bromophenyl
(117). To be able to easily compare the activities of the 2N-substit-
ed and 2N-unsubstituted imidazoles, also the (previously de-
scribed) activities of the 2N-unsubstituted imidazoles were
included in Table 5 (compounds 81, 85, 90, 96, 101, 106 and
115). The 2-aminoimidazoles substituted at the 2N-position with
a methyl, ethyl or (iso-)propyl were found to have lower activities
as compared to their 2N-unsubstituted counterparts against both
Salmonella and Pseudomonas biofilm formation, although they still
not able to completely prevent biofilm formation at higher concen-
trations, as their dose response curves either reach a steady state
level between 50% and 70% biofilm inhibition or show a maximum
between 50% and 70% biofilm inhibition and decrease again with
higher compound concentrations. Finally, the compounds with a
para-chlorophenyl or para-fluorophenyl at the 5-position and hex-
yl, octyl or nonyl at the 2N-position had a very low or no biofilm
inhibitory activity (IC50 >800 lM). In case of P. aeruginosa, intro-
duction of an octyl chain at the 2N-position of imidazoles bearing
3,4-dichlorophenyl, naphtyl, para-methoxyphenyl or bromophenyl
at the 5-position results in compounds that do not have an effect
on the biofilm formation at the highest concentration tested
(800 lM). On the other hand, the imidazoles with a chlorine atom,
show a moderate activity with IC50 values in the range of 10
l
M
fluorine atom, nitro group or sulphonylmethyl group at the para-
position of the 5-phenyl ring and a pentyl, hexyl or octyl chain at
the 2N-position decrease the biofilm formation at low concentra-
(compound 107) to 170 M, except for compounds 82 and 112
l
which are almost completely inactive. On the other hand, the
compounds with a n-butyl, iso-butyl or n-pentyl substitution at
the 2N-position were found to be in general more active than their
2N-unsubstituted counterparts, with respect to both the Salmonella
and Pseudomonas biofilm formation. The best activity was found
for compound 110, (bearing a 3,4-dichlorophenyl group at the
4(5)-position and a n-pentyl group at the 2N-position), which
inhibits the biofilm formation of both Salmonella and Pseudomonas
tions (IC50’s 1.5–25 lM), however, they are not able to completely
prevent biofilm formation as their dose response curves either
reach a steady state level around 50% biofilm inhibition or show
a maximum around 50% biofilm inhibition and decrease again with
higher compound concentrations. Finally, we tested the effect of
some of the most active imidazoles on the planktonic growth by
growth curve analysis. Interestingly, all the imidazoles tested show
a broad concentration range with only an effect on the biofilm for-
mation and no effect on the planktonic growth, indicating that
these compounds have potential to be used as selective biofilm
inhibitors.
by 50% at concentrations below 2.5
87, which inhibits Salmonella biofilm formation only at high con-
centrations (IC50 >400 M). Furthermore this compound has a
low IC50 value for Pseudomonas biofilm inhibition (IC50 ꢁ6 M),
lM. An exception is compound
l
l
but is not able to prevent Pseudomonas biofilm formation by more
than 50%. The effect of introduction of longer alkyl side chains (C6–
C9) at the 2N-position was found to be strongly dependent on the
substitution pattern of the 5-phenyl ring and the specific model
organism studied. In case of S. Typhimurium, introduction of an
octyl group at the 2N-position of compounds bearing a para-
bromophenyl, meta-bromophenyl, para-nitrophenyl or para-meth-
ylsulphonyl group at the 5-position of the imidazole ring results in
compounds with a high biofilm inhibitory activity (IC50 = 2–
2.2.2. Cyclo-alkyl substituents at 2N-position
As described in the previous section, we found that the 2-ami-
noimidazoles substituted at the 2N-position with an intermediate
length n-alkyl or iso-alkyl chain were very potent inhibitors of
the biofilm formation of both Salmonella and Pseudomonas. In an
attempt to even improve this biofilm inhibitory activity, we
decided to synthesize an array of 2-aminoimidazoles substituted
at the 2N-position with cyclo-alkyl groups with intermediate
length, that is, cyclo-pentyl (compounds 118–122) and cyclo-hexyl
(compounds 123–126). As depicted in Table 6, the compounds
with a cyclo-pentyl chain at the 2N-positon have an improved
45 lM). On the other hand, introduction of an octyl group at
compounds bearing a para-methoxyphenyl, naphtyl or 3,4-dichlo-
rophenyl group at the 5-position results in compounds with much
Table 6
Influence of 2N-cyclo-alkyl-2-aminoimidazoles 118–127 on the biofilm formation and the planktonic growth of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at 25 °C
Compound R1
R
S. Typhimurium
M) 95% confidence
Effect on growth atb
P. aeruginosa
IC50 (lM) 95% confidence
Effect on growth atb
a
a
IC50
(l
interval for IC50
interval for IC50
IC50 80
l
M
40
lM
20
lM
IC50 80
l
M
40
lM
20 lM
118
119
120
121
122
123
124
125
126
127
c-Pen
H
52.9
4.4
11.8
12.1
42.4–66.1
4.0–4.8
7.9–17.7
6.5–22.5
3.8–8.5
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
33.8
13.5
20.2
7.2
23.9–47.7
9.0–20.5
12.3–33.1
4.4–12.0
4.2–14.8
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
c-Pen
c-Pen
c-Pen
c-Pen
4-Cl
4-NO2
4-Br
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3,4-diCl 5.7
7.9
ꢀ
ꢀ
c-Hex 4-Cl
>800
36.7
629.5
ꢁ25c,d
ꢁ25d
4.4
c-Hex 4-NO2
c-Hex 3-NO2
c-Hex 4-OMe
c-Hex 4-SMe
29.7–45.3
274.6–1443.0
ꢀ
ꢀ
2.6–7.5
21.9–136.9
9.5–26.1
ꢀ
ꢁ377.6
54.8
15.8
ꢀ
ꢀ
ꢀ
191.3
129.1–283.6
ꢀ
a
IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.
o: the planktonic growth is completely or almost completely inhibited when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; +:
b
the planktonic growth is retarded when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; ꢀ: the planktonic growth is not or only
slightly affected when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; No symbol indicated: effect not determined.
c
ꢁ: the IC50 value could not be determined accurately due to the steepness of the dose response curve.
d
The compound is not able to completely prevent biofilm formation, as the dose response curve either reaches a steady state level at about 50% biofilm inhibition.

3470
H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
activity against Salmonella biofilms as compared to the 2N-unsub-
stituted compounds. However, no further improvement of the Sal-
monella biofilm inhibitory activity of the n-alkyl and iso-alkyl
substituted imidazoles could be achieved. The compounds with a
cyclo-pentyl chain also inhibit Pseudomonas biofilm formation at
2.3. 2N-substituted 2-aminoimidazoles versus 1-substituted 2-
hydroxy-2-aryl-2,3-dihydro-imidazopyrimidinium salts
In the divergent chemical synthesis procedure, 2N-substituted
4(5)-aryl-2-aminoimidazoles are formed by cleavage of the 1-
substituted 2-hydroxy-2-aryl-2,3-dihydro-imidazopyrimidinium
salts with a nucleophile such as hydrazine, under conventional
heating or microwave irradiation (Scheme 1, pathway B). After
cleavage, the 4(5)-aryl substituent and the 2N-substituent of the
2-aminoimidazole will be the same as respectively the 2-aryl
substituent and the 1-substituent of the salt. Analogously, N1-
substituted 2-aminoimidazoles are formed by cleavage of the
1-substituted imidazo[1,2-a]pyrimidinium perchlorate salts
(Scheme 1, pathway A). In a previous publication, we described a
correlation between the activity of the 1-substituted imidazo[1,2-
a]pyrimidinium perchlorate salts and their corresponding
N1-substituted 2-aminoimidazoles, supporting the hypothesis that
these imidazo[1,2-a]pyrimidinium perchlorate salts could also
in situ be degraded to the corresponding N1-substituted 2-amino-
imidazoles, for example, by cellular nucleophiles.42 To determine
whether a similar correlation could be found between the biofilm
inhibitory activity of the 1-substituted 2-hydroxy-2-aryl-2,3-
dihydro-imidazopyrimidinium salts and their corresponding 2N-
substituted 2-aminoimidazoles, in this section we compare the
structure–activity relationship of both classes of compounds.
The 2-aminoimidazoles substituted at the 2N-position with a
methyl, ethyl or (iso-)propyl were found to have a low to moderate
activity against the biofilm formation. This is essentially in agree-
ment with the structure-activity relationship found for the 2-hy-
droxy-2-aryl-2,3-dihydro-imidazopyrimidinium salts, although
comparison of the IC50 values reveals that the activity of the imida-
zoles is in general a bit higher than the activity of the correspond-
ing salts. Also the activity of the 2-aminoimidazoles substituted
with an n-butyl, iso-butyl, n-pentyl and cyclo-pentyl group was
in general found to be higher than the activity of the corresponding
salts. The higher activity of the 2-aminoimidazoles compared to
their precursor imidazopyrimidinium salts could possibly be
explained by an incomplete in situ degradation of the salts to
imidazoles. Therefore this difference in activity between salts
low to moderate concentrations (IC50’s 7–35 lM), although their
activities were not in all cases better than these of their unsubsti-
tuted counterparts. Interestingly, analogously to the 2N-n-alkyl
substituted 2-aminoimidazoles, growth curve analysis revealed
that all the 2N-cyclopentyl 2-aminozoles tested possess a concen-
tration range with only biofilm inhibition and no effect on the
planktonic growth, both in the case of Salmonella and Pseudomonas.
The compounds 2N-substituted with a cyclo-hexyl, on the other
hand, in general only show a moderate to low activity against Sal-
monella biofilm formation, with IC50 values between 50 and
>800
lM, while they inhibit Pseudomonas biofilms at lower con-
centrations (IC50 4–50
l
M). Interestingly, all the 2-aminoimidaz-
oles tested have a concentration range in which they specifically
inhibit Salmonella and Pseudomonas biofilm formation, without
affecting the planktonic growth (Table 6).
2.2.3. Aromatic substituents at 2N-position
Finally we synthesized and tested an array of 4(5)-phenyl-2-
aminoimidazoles, which are substituted at the 2N-position with a
benzyl (compound 128), para-methoxybenzyl (compound 129),
3-methoxyphenethyl (compounds 130–132) or piperonyl group
(compounds 133–134) (Table 7). Compound 128, bearing a benzyl
group, has a good activity against both Salmonella and Pseudomo-
nas biofilm formation (IC50’s ꢁ30
lM), although these activities
are lower than those of the 2N-unsubstituted counterpart. Com-
pound 129, bearing a para-methoxybenzyl group, has no activity.
The activity of the compounds 2N-substituted with a 3-methoxy-
phenethyl group is clearly dependent on the substitution of the
4(5)-phenyl group, as compound 130, with para-nitrophenyl group
at the 4-position is more active than its unsubstituted counterpart,
while compounds 131 and 132, bearing respectively a 4,5-dichlo-
robenzyl group and a [1,10:40,100-terphenyl]-4-yl group at the
4(5)-position, are inactive at the highest concentration tested
(400 lM). Finally, the compounds with a 2N-piperonyl group
(133–134) show a moderate activity both against Salmonella and
and imidazoles does not have to lead to
a rejection of
Pseudomonas biofilm formation.
the hypothesis that the biofilm inhibitory activity of the
Table 7
Influence of 2-aminoimidazoles 127–133 with aromatic substituents at the 2N-position on the biofilm formation of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at 25 °C
Compound
R
S. Typhimurium
95% confidence
P. aeruginosa
95% confidence
Effect on growth atb
IC50
(l
M)
Effect on growth atb
a
a
IC50
(l
M)
interval for IC50
24.55–38.47
11.85–30.67
interval for IC50
13.61–50.71
1.913–7.172
IC50
40
lM
IC50
40 lM
128
129
130
131
132
133
134
4-Cl
4-NO2
4-NO2
3,4-diCl
4-(40-Biphenyl)
4-F
30.73
>800
19.07
>800
>800
66.59
261
ꢀ
ꢀ
26.27
>800
3.704
>800
>800
455.5
10.52
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
53.29–83.21
170.5–399.7
ꢀ
296.8–699.1
6.158–17.99
a
IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.
o: the planktonic growth is completely or almost completely inhibited when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; +:
b
the planktonic growth is retarded when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; ꢀ: the planktonic growth is not or only
slightly affected when the bacteria are grown in the presence of the indicated concentration of biofilm inhibitor; No symbol indicated: effect not determined.

H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
3471
imidazopyrimidinium salts is mediated by the 2-aminoimidazoles,
formed by in situ cleavage of the salts. However, remarkably, some
of the 2N-octyl 2-aminoimidazoles tested were found to have
much higher IC50 values than their corresponding 1-octyl-2-hydro-
xy-2-aryl-2,3-dihydro-imidazopyrimidinium salts. Furthermore,
the 2N-cylcohexyl 2-aminoimidazole 123 is much less active
against Salmonella biofilms than its corresponding salt 64. This dis-
crepancy could be explained by the toxic effect of the salts at con-
centrations lower than the biofilm inhibitory concentrations of the
corresponding imidazoles, which causes a non-specific inhibition
of the biofilm formation by reducing the planktonic growth of
the bacteria around the surface on which the biofilms are formed.
Therefore, these data do not exclude the possibility that the biofilm
inhibitory activity of the imidazopyrimidinium salts is mediated by
the 2-aminoimidazoles, formed by in situ cleavage of the salts.
2,3-dihydro-imidazopyrimidinium salts and 2N-substituted 2-
amino-1H-imidazoles as inhibitors of the biofilm formation by S.
Typhimurium and P. aeruginosa. The results of the previous study
and the present study are summarized in Scheme 2. We found that
2-hydroxy-2,3-dihydro-imidazopyrimidinium salts with interme-
diate length n-alkyl chains (C7–C10) substituted at the 1-position
in general prevent the biofilm formation of both species at low
micromolar concentrations (IC50’s 5–50 lM). Salts with a shorter
(C1–C5) or longer (C11–C14) n-alkyl chain at the 1-position were
found to be much less potent. Consistent with this, we observed
that salts with an intermediate length cyclo-alkyl chain are much
more active against biofilm formation of both species as compared
to the salts with a short cyclo-alkyl chain. However, remarkably,
salts with a long cyclo-dodecyl side chain were found to have even
better activities than salts with an intermediate length cyclo-alkyl
side chain. Furthermore, we demonstrated the potential of 2-hy-
droxy-2,3-dihydro-imidazopyrimidinium salts with certain aro-
matic substituents at the 1-position, such as piperonyl or 3-
methoxyphenetyl as inhibitors of Salmonella and Pseudomonas bio-
film formation. In the framework of the 2-aminoimidazoles, we
delineated that introduction of a butyl or pentyl side chain at
the 2N-position of the 2-aminoimdazoles results in an enhanced
biofilm inhibitory activity against both species, while introduction
of a shorter n-alkyl chain reduces the biofilm inhibitory activity.
The effect of introduction of longer n-alkyl chains, however, seems
to be strongly dependent on the substitution pattern of the 5-phe-
nyl ring and the bacterial species studied. Also introduction of a
3. Conclusion
Previously, we reported the biofilm inhibitory activity of
N1-substituted 5-aryl-2-aminoimidazoles and their precursor imi-
dazo[1,2-a]pyrimidinium salts.42 These compounds were synthe-
sized by utilizing the first path of our divergent synthesis
pathway towards 2-aminoimidazoles40,41 The second path of this
divergent pathway describes the synthesis of 2N-substituted 2-
amino-1H-imidazoles and their precursor 1-substituted 2-hydro-
xy-2,3-dihydro-imidazopyrimidinium salts. In the present study,
we investigated the potential of these 1-substituted 2-hydroxy-
imidazo[1,2-a]pyrimidinium salts
(previous study)
2-hydroxy-2-aryl-2,3-dihydro-
imidazo[1,2-a]pyrimidinium salts
(present study)
ClO4
N R1
N
N
N
N
N R1
OH
Br
R
R
Strong anti-biofilm activity for:
Strong anti-biofilm activity for:
R1=intermediate length n-alkyl chain (C7-C10)
R1=intermediate length cyclo-alkyl chain (C6-C8)
R1=long cyclo-alkyl chain (C12)
R1=intermediate length n-alkyl chain (C8)
R1=intermediate length cyclo-alkyl chain (C6)
R1=long cyclo-alkyl chain(C12)
(for all R groups tested)
R1=3-methoxyphenethyl or piperonyl
(for all R groups tested)
substituted 4(5)-aryl-2-amino-1H-imidazoles
(present and previous study)
Strong anti-biofilm activity for (present study):
H
N
R1 = n-Bu,i-Bu,n-Pen
R1 N
R1 = cyclo-Pen
Strong anti-biofilm activity for
(previous study42):
N
(for R2 = H and all R groups tested)
R1 = n-Oct
R2
R
R = Cl, Ph, NO2, Br, Me, F
R1 = benzyl, 3-methoxyphenethyl or piperonyl
(for R2 = H and a subset of the R groups tested)
(for R1 and R2 = H)
Strong anti-biofilm activity for (previousstudy42):
R2 = intermediate length n-alkyl chain (C5-C10)
R2 = intermediate length cyclo-alkyl chain (C5-C8)
(for R2 = H and all R groups tested)
R2 = long n-alkyl or cyclo-alkyl chain (C11-C14)a
R2 = benzyl
(for R2 = H and a subset of the R groups tested)
Scheme 2. Overview of the substituents important for anti-biofilm activity of 2-hydroxy2-aryl-2,3-dihydroimidazo[1,2-a]pyrimidinium salts, imidazo[1,2-a]pyrimidinium
salts and 4(5)-aryl-2-amino-1H-imidazoles.

3472
H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
cyclo-pentyl side chain in general results in an improved activity.
Finally, we showed that introduction of a 3-methoxyphenethyl or
piperonyl group at the 2N-position can also result in an enhanced
biofilm inhibition, although this effect is also dependent on the
nature of the substitution pattern of the 5-phenyl ring. Interest-
ingly, growth curve analysis revealed that the 2-aminoimidazoles
in general have a broad concentration range in which they specif-
ically inhibit biofilm formation without affecting the planktonic
growth, indicating that these compounds have potential to be used
as selective biofilm inhibitors. As described in our previous publi-
cation,42 we found a good correlation between the activity of the
N1-substituted 4(5)-aryl-2-aminoimidazoles and their precursor
imidazo[1,2-a]pyrimidinium salts, leading to the hypothesis that
the salts could also in situ be cleaved by cellular nucleophiles to
form the active 2-aminoimidazoles. However, in the present study
no good correlation was found between the activity of some of the
active 1-octyl-2-hydroxy-2-aryl-2,3-dihydro-imidazopyrimidini-
um salts (and 1-cyclohexyl-2-hydroxy-2-aryl-2,3-dihydro-imi-
dazopyrimidinium salts) and their corresponding less efficient 2-
aminoimidazoles. However, this could be explained by the toxic
effect of the salts at concentrations lower than the biofilm inhibi-
tory concentrations of the corresponding imidazoles, which causes
a non-specific inhibition of the biofilm formation. Therefore this
difference in activity between salts and imidazoles does not neces-
sarily falsify the hypothesis that the biofilm inhibitory activity of
the imidazopyrimidinium salts is mediated by the 2-aminoimidaz-
oles, formed by in situ cleavage of the salts. In conclusion, the 2N-
substituted 2-aminoimidazoles and 2-hydroxy-2-aryl-2,3-dihy-
dro-imidazopyrimidinium salts of the present study are valuable
candidates in the development of therapeutics and sanitizers for
the combat of biofilm formation by S. Typhimurium, P. aeruginosa
and possibly other pathogenic bacteria.
In a 30 mL microwave vial were successively brought acetoni-
trile (15 mL), N-(1,3-benzodioxol-5-ylmethyl)pyrimidin-2-amine
(1.15 g, 5 mmol), 4-fluorophenacylbromide (1.3 g, 6 mmol,
1.2 equiv), and a catalytic amount of4-dimethylaminopyridine
(6 mg, 0.05 mmol). The reaction tube was sealed and irradiated
in a microwave reactor at a ceiling temperature of 80 °C at
150 W maximum power for 30 min. After the reaction mixture
was cooled with an air flow for 15 min, the precipitate was washed
with acetone (25 mL), ether (20 mL) and dried in vacuum to afford
79 (1.98 g, 89% yield) as a white powder.
The analytical data of the new compounds are listed as Supple-
mentary data.
4.1.4. 2N-Substituted 4(5)-aryl-2-amino-1H-imidazoles
All 2N-substituted 4(5)-aryl-2-amino-1H-imidazoles were
synthesized by using previously described protocols.41 A general
procedure is described below (with compound 133 as an exam-
ple): To a suspension of salt 79 (2 mmol) in acetonitrile (5 mL)
was added hydrazine hydrate (0.7 mL, 14 mmol of a 64% solu-
tion, 7 equiv), and the mixture was irradiated in the sealed reac-
tion tube for 10 min at a ceiling temperature of 100 °C at 150 W
maximum power. After the mixture was cooled, hydrazine hy-
drate was evaporated with toluene (3 ꢂ 20 mL). The resulting
residue was purified by column chromatography (silica gel;
MeOH-DCM 1:4 v/v with 5% of 6 M NH3 in MeOH) to afford
2-amino-1H-imidazole 133 (473 mg, 76% yield) as an amorphous
solid.
The analytical data of the new compounds are listed as Supple-
mentary data.
4.2. Biological assays
4.2.1. Static peg assay for prevention of Salmonella
Typhimurium and Pseudomonas aeruginosa biofilm
formation17
4. Experimental section
4.1. Chemistry
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.
4.1.1. General experimental methods
269789).44 Twofold serial dilutions of the compounds in 100
ll li-
Chemicals were purchased from commercial sources and used
without further treatment. Melting points are uncorrected. 1H
NMR spectra were recorded at 300 MHz, 13C NMR spectra at 75 with
tetramethylsilane or solvent (CDCl3, CD3OD, DMSO-d6) as internal
standard (d ppm). The ion source temperature was 150–250 °C, as
required. High-resolution EI-mass spectra were performed with a
resolution of 10 000. The low-resolution spectra were obtained with
a HP5989A MS instrument. For column chromatography 70–230
mesh silica gel was used. The purity of the compounds was checked
by HPLC. All compounds were obtained with a purity >95%.
quid broth (Tryptic Soy Broth diluted 1/20 (TSB 1/20)) per well
were prepared in the microtiter plate (2 or 3 repeats per com-
pound). Subsequently, an overnight culture of S. Typhimurium
ATCC14028 (grown in Luria-Bertani medium45) or P. aeruginosa
PA14 (grown in TSB) was diluted 1:100 into the respective liquid
broth and 100
l
l (ꢁ106 cells) was added to each well of the micro-
titer plate, resulting in a total amount of 200 ll medium per well.
The pegged lid was placed on the microtiter plate and the plate
was incubated for 24 h at 25 °C without shaking. During this incu-
bation period biofilms were formed on the surface of the pegs.
After 24 h, the optical density at 600 nm (OD600) was measured
for the planktonic cells in the microtiter plate using a VERSAmax
microtiter plate reader (Molecular Devices). This gives a first indi-
cation of the effect of the compounds on the planktonic growth. For
quantification of biofilm formation, the pegs were washed once in
4.1.2. Microwave irradiation experiments
A multimode Milestone MicroSYNTH microwave reactor was
used in the standard configuration as delivered, including proprie-
tary software. All experiments were carried out in sealed micro-
wave process vials (15, 30 and 50 mL). After completion of the
reaction, the vial was cooled down to 25 °C via air jet cooling be-
fore opening. Reaction temperatures were monitored by an IR sen-
sor on the outside wall of the reaction vial and a fibre-optic sensor
inside the reaction vial.
200
ll phosphate buffered saline (PBS). The remaining attached
bacteria were stained for 30 min with 200
ll 0.1% (w/v) crystal vio-
let in an isopropanol/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
(30 min), the dye bound to the adherent cells was extracted with
30% glacial acetic acid (200 l). The OD570 of each well was mea-
ll distilled water per well. After the pegs were air dried
4.1.3. 1-Substituted 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-
a]pyrimidinium salts
l
sured using a VERSAmax microtiter plate reader (Molecular De-
vices). The IC50 value for each compound was determined from
the concentration gradient by using the GraphPad software of
Prism.
All 1-substituted 2-hydroxy-2-aryl-2,3-dihydro-imidazo[1,2-
a]pyrimidinium salts were synthesized by using previously de-
scribed protocols.41 A general procedure is described below (with
compound 79 as an example):

H. P. L. Steenackers et al. / Bioorg. Med. Chem. 19 (2011) 3462–3473
3473
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4.2.2. Bioscreen assay for measuring Salmonella Typhimurium
and P. aeruginosa growth inhibition
The Bioscreen device (Oy Growth Curves Ab Ltd) was used for
measuring the influence of the chemical compounds on the plank-
tonic growth of Salmonella Typhimurium and P. aeruginosa PA14.
The Bioscreen is a computer controlled incubator/reader/shaker
that uses 10 ꢂ 10 well microtiter plates and measures light absor-
bance of each well at a specified wave length in function of time.
An overnight culture of S. Typhimurium ATCC14028 (grown up in
LB medium) or P. aeruginosa (grown up in TSB) was diluted
1:200 in liquid broth (TSB 1/20). 300 ll of the diluted overnight
culture was added to each well of the 10 ꢂ 10 well microtiter plate.
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prepared in DMSO or EtOH. 3
was added to the wells (containing the 300
in threefold. As a control 3 l of the appropriate solvent was also
l
l of each diluted stock solution
ll bacterial culture)
l
added to the plate in three- or four-fold. The microtiter plate was
incubated in the Bioscreen device at 25 °C for at least 24 h, with
continuous medium shaking. The absorbance of each well was
measured at 600 nm each 15 min. Excel was used to generate the
growth curves for the 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:
(1) The planktonic growth is not or only slightly affected, indi-
cated by the symbol ‘ꢀ’.
(2) The planktonic growth is retarded, indicated by the symbol
‘+’.
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and the second category:
If the absorbance (measured at 600 nm) of the bacterial culture
treated with the compound is at least 0.5 (for Salmonella) or 0.8 (for
Pseudomonas) units lower than the absorbance of the untreated
culture during four consecutive hours, then the effect on the plank-
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Acknowledgments
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This work was supported by the Industrial Research Fund of K.
U. Leuven (KP/06/014), the Research Council of K. U. Leuven (CoE
EF/05/007 SymBioSys), the F.W.O. (Fund for Scientific Research –
Flanders (Belgium) and by the Institute for the Promotion of
Innovation through Science and Technology in Flanders (IWT-Vla-
anderen) through a scholarship to H.S. J.V. and D.D.V. are grateful
for support in the frame of the IAP program Functional Supramo-
lecular Systems. E.D.S. is grateful to the K. U. Leuven for obtaining
a postdoctoral fellowship and S.B. to Erasmus Mundus External
Cooperation Window Lot 15 India (EMECW15) for obtaining a
scholarship.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.04.026.
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