Structure-activity relationship of 4(5)-aryl-2-amino-1 H -imidazoles, N 1-substituted 2-aminoimidazoles and imidazo[1,2- a ]pyrimidinium salts as inhibitors of biofilm formation by salmonella typhimurium and pseudomonas aeruginosa

Article abstract of DOI:10.1021/jm1011148

A library of 112 4(5)-aryl-2-amino-1H-imidazoles, 4,5-diphenyl-2-amino-1H- imidazoles, and N1-substituted 4(5)-phenyl-2-aminoimidazoles was synthesized and tested for the antagonistic effect against biofilm formation by Salmonella Typhimurium and Pseudomo

Full text of DOI:10.1021/jm1011148

472 J. Med. Chem. 2011, 54, 472–484
DOI: 10.1021/jm1011148
Structure-Activity Relationship of 4(5)-Aryl-2-amino-1H-imidazoles, N1-Substituted
2-Aminoimidazoles and Imidazo[1,2-a]pyrimidinium Salts as Inhibitors of Biofilm Formation by
Salmonella Typhimurium and Pseudomonas aeruginosa
Hans P. L. Steenackers,†,§ Denis S. Ermolat’ev,‡ Bharat Savaliya,‡, Ami De Weerdt,† David De Coster,† Anamik Shah,
Erik V. Van der Eycken,‡ Dirk E. De Vos,§ Jozef Vanderleyden,† and Sigrid C. J. De Keersmaecker*,†
†Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium,
‡Laboratory for Organic & Microwave-Assisted Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium,
§Centre for Surface Chemistry and Catalysis, Katholieke Universiteit Leuven, Kasteelpark Arenberg 23, B-3001 Leuven, Belgium, and
Department of Chemistry, Saurashtra University, Rajkot-360 005, Gujarat, India
Received May 19, 2010
A library of 112 4(5)-aryl-2-amino-1H-imidazoles, 4,5-diphenyl-2-amino-1H-imidazoles, and N1-substituted
4(5)-phenyl-2-aminoimidazoles was synthesized and tested for the antagonistic effect against biofilm
formation by Salmonella Typhimurium and Pseudomonas aeruginosa. The substitution pattern of the 4(5)-
phenyl group and the nature of the N1-substituent were found to have a major effect on the biofilm inhibitory
activity. The most active compounds of this series were shown to inhibit the biofilm formation at low
micromolar concentrations. Furthermore, the influence of 6 imidazo[1,2-a]pyrimidines and 18 imidazo[1,2-
a]pyrimidinium salts on the biofilm formation was tested. These compounds are the chemical precursors of the
2-aminoimidazoles in our synthesis pathway. 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 are possibly cleaved by cellular nucleophiles to form the active
2-aminoimidazoles. However, the imidazo[1,2-a]pyrimidines did not show any biofilm inhibitory activity,
indicating that these molecules are not susceptible to in situ degradation to 2-aminoimidazoles. Finally, we
demonstrated the lack of biofilm inhibitory activity of an array of 37 2N-substituted 2-aminopyrimidines,
which are the chemical precursors of the imidazo[1,2-a]pyrimidinium salts in our synthesis pathway.
Introduction
to inhibit biofilm formation.9 Among the few molecular scaf-
folds that have been identified, the most intensively studied
examples are (1) the halogenated furanones, which were origin-
ally isolated from the seaweed Delisea pulchra,10,11,12 (2) analogues
of the homoserine lactone signaling molecules,13 and (3) anal-
ogues of the sponge-derived marine natural alkaloids oroidin
and bromoageliferin.14-21 As bacteria develop resistance against
compounds with a microbiocidal activity, the development of
compounds that specifically target the biofilm formation in a
nontoxic manner is a valuable approach. These nontoxic biofilm
inhibitors have the potential to be used in a preventive treatment
of a wide diversity of industrial and medical surfaces. Further-
more, the potential to codose biofilm inhibitors and classical
antibiotics for the treatment of biofilm infections is also an
attractive option.11
Salmonella enterica serovar Typhimurium and Pseudomonas
aeruginosa are two well-studied organisms in terms of biofilm
formation. Salmonella enterica is worldwide one of the most
important foodborne pathogens. Salmonella is able to form
biofilms on different surfaces, ranging from abiotic surfaces
(e.g., concrete, plastics, glass, polystyrene,...),22,23 to biotic
surfaces (gallstones,24 plant surfaces,25,26 and epithelial cell
layers27). These biofilms are an important survival strategy in
all stages of infection, from transmission to chronic infection.
While severe nontyphoid Salmonella infections are commonly
treated with fluoroquinolones and third-generation cepha-
losporins, there are alarming reports concerning the deve-
lopment of resistance against these antibiotics.28 Alternative
Biofilms are defined as structured communities of bacterial
cells enclosed in a self-produced polymeric matrix and adherent
to an inert or living surface.1 Biofilms as compared with
planktonic cells provide the bacterial cells with an array of
advantages including the ability to resist challenges from pre-
dators, antibiotics, disinfectants, and host immune systems.2-5
This causes serious problems and high associated costs in health
care and industrial settings. In medicine, biofilms play a role in
infectious diseases, both for specific conditions such as cystic
fibrosis and periodontitis and in bloodstream and urinary tract
infections as a result of indwelling medical devices.3 According
to the National Institutes of Health, more than 80% of micro-
bial infections are related to biofilms.6 This is particularly
problematic given the fact that bacteria in biofilms can be up
to 1000-fold more resistant to antibiotics.7 Industry related
biofilms can contribute, e.g., to contamination of installations
in food industry, decreasing passage through pipelines by
colonization of the interior of the pipes, mild steel corrosion,
and enhanced resistance of vessels by initiating biofouling on the
vessel hulls. The yearly economic loss caused by “biofouling” in
the marine industry is estimated at $ 6.5 billion.8
Given the extent of problems caused by biofilms, there has
been a significant effort to develop small molecules that are able
*To whom correspondence should be addressed. Phone: 32-
16321631. Fax: 32-16321966. E-mail: Sigrid.DeKeersmaecker@biw.
kuleuven.be.
r
pubs.acs.org/jmc
Published on Web 12/20/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 473
Scheme 1. Overview and Synthesis of the Different Classes of Compounds of Which the Antibiofilm Activity was Investigated in the
Present Studya
a (A) Synthesis of 4(5)-mono and 4,5-disubstituted 2-amino-1H-imidazoles with aryl substituents from imidazo[1,2-a]pyrimidines.32 (B) Synthesis of
N1-substitued 2-aminoimidazoles from 2-aminopyrimidines via N1-substituted imidazo[1,2]pyrimidinium salts.37,38
anti-Salmonella strategies are thus urgently needed. Given the
importance of biofilms in the spread of Salmonella, the
development of Salmonella biofilm inhibitors seems a promis-
ing approach. P. aeruginosa is an opportunistic pathogen
implicated in a myriad of infections. Patients with compromised
host defenses, such as those infected with human immunodefi-
ciency virus, burn patients, or those with cystic fibrosis (80%
colonization rate of P. aeruginosa29) are particularly susceptible
to P. aeruginosa infections.30 There is evidence that biofilms are
involved in chronic P. aeruginosa infections such as recurrent
ear infections, chronical bacterial prostatitis, and lung infec-
tions in CF patients, the latter being extremely harmful as
P. aeruginosa colonization and chronic lung infection is the major
causative agent of morbidity and mortality in CF patients.31
Moreover, P. aeruginosa 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.
Recently, we have published a method for the microwave-
assisted synthesis of substituted 2-amino-1H-imidazoles from
imidazo[1,2-a]pyrimidines (Scheme 1, route A),32 focusing on
4(5)-mono- or 4,5-disubstituted 2-amino-1H-imidazoles with
aryl substituents. Some representatives of this class of imida-
zoles have been documented to inhibit plaque formation by
Streptococcus mutans and biofilm formation by Acinetobacter
baumanii.33,34 Moreover, some representatives of the class of
imidazo[1,2-a]pyrimidines, which are the chemical precursors
of the 2-amino-1H-imidazoles in our synthesis pathway, have
been shown to possess antibacterial and antifungal activity.35,36
Therefore, we decided to initiate an in depth study on the
structure-antibiofilm activity of both classes of compounds.
Additionally, we previously reported on a procedure for the
synthesis of N1-substituted 2-aminoimidazoles from 2-amino-
pyrimidines via N1-substituted imidazo[1,2-a]pyrimidin-1-ium
salts (Scheme 1, route B).37,38 The availability of these N1-
substituted 2-aminoimidazoles allowed us to study the effect of
different kinds of N1-substituents on the antibiofilm activity of
the 2-aminoimidazoles. We hypothesized that the imidazo-
[1,2-a]pyrimidinium salts could also in situ (i.e., in the biofilm
or in the cell) be degraded to 2-aminoimidazoles by attack of
cellular nucleophiles. Therefore, we also studied the potential of
the imidazo[1,2-a]pyrimidinium salts as biofilm inhibitors. Some
of the 2-aminopyrimidines were previously shown to possess
antibacterial and antifungal activity.39,40 Therefore, we decided
to investigate the influence of a broad array of 2N-substituted
2-aminopyrimidines on biofilm formation. Initially, the com-
pounds were screened for antibiofilm activity against S. Typhi-
murium. In a second stage, the most active compounds were
evaluated against P. aeruginosa biofilm formation.
Results and Discussion
4(5)-Monosubstituted 2-amino-1H-imidazoles. The effect
of the para-substituted 4(5)-phenyl-2-aminoimidazoles
1-10 (Table 1) on the biofilm formation of S. Typhimurium
ATTC14028 was studied. The compounds were initially
screened at 25 °C, mimicking conditions outside the host where
biofilms are supporting survival and transmission of Salmonella.
The basic compound 1 with a nonsubstituted phenyl group at
the C4(5) position of the imidazole ring shows a moderate
biofilm inhibitory activity (IC50 = 130 μM). Interestingly, sub-
stitution at the para-position of the phenyl ring with a chlorine,
nitro, or phenyl group enhances the biofilm inhibitory activity
about 10 times (IC50s ∼ 15 μM). Substitution with a bromine,
fluorine, methyl, or methoxy group does also improve biofilm
inhibition moderately (IC50s ∼ 45-120 μM), while substitution
with a methanesulfonyl or nitrile function reduces biofilm inhibi-
tory activity drastically. To validate that the compounds are true
inhibitors of biofilm formation and not acting as bactericidal
agents, growth curves of planktonic cells were measured at 25 °C
in the presence of different concentrations of the most active
compounds. Compounds 1-5 do not or only slightly slow down
the planktonic growth at the IC50 for biofilm inhibition, indicating
that the reduced biofilm formation is not a consequence of kill-
ing the bacteria (Table 1). Compound 9, however, retards the
bacterial growth substantially at the IC50 for biofilm inhibition,
suggesting that the biofilm inhibition is at least partly due to killing
the planktonic bacteria before biofilms could be established.
Salmonella can also form biofilms as a strategy to induce
chronic infections24,41,42 and even colonize host organisms.
To mimic the conditions inside the host, we also determined
the influence of the most active compounds 1-5 and 9 on
S. Typhimurium biofilm formation at 37 °C. As depicted in
Table 1, all the tested compounds inhibit biofilm formation

474 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Steenackers et al.
Table 1. Influence of para-Substituted 4(5)-Phenyl-2-amino-1H-imidazoles 1-10 on the Biofilm Formation and the Planktonic Growth of S.
Typhimurium ATCC14028 at 25 and 37 °C
25 °C
37 °C
effect on growth atb
95% confidence 400 200 150 40 25 20 10
interval for IC50 μM μM μM μM μM μM μM μM (μM)
effect on growth atb
a
a
IC50
(μM)
4
IC50
95% confidence 400 250 150 100 50 25
interval for IC50 μM μM μM μM μM μM
compd
R
1
2
3
4
5
6
7
8
9
H
F
130.2
84.4
16.0
47.9
17.6
112.6-150.7
69.7-102.3
14.3-17.9
36.6-62.8
15.0-20.5
þ
-
-
þ
-
-
o
-
-
-
-
146.4
238.4
30.1
54.3
395.7
ndc
67.7-316.5
169.2-335.8
20.5-44.0
38.5-76.6
305.1-513.2
-
-
-
-
-
-
-
Cl
Br
o
o
o
-
-
NO2
-
-
-
o
SO2Me >800
OMe
Me
Ph
119.7
77.7
17.3
106.9-134.1
70.4-85.8
15.9-18.8
nd
nd
o
þ
348.9
nd
277.9-437.9
o
10 CN
316.4
224.4-446.1
a IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%. b o: the planktonic growth is completely or almost completely inhibited
when the bacteria are grown in the presence of theindicated concentration of biofilm inhibitor; þ: the planktonicgrowth 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.
Table 2. Influence of para-Substituted 4(5)-Phenyl-2-amino-1H-imidazoles 1-9 on the Biofilm Formation and the Planktonic Growth of P. aeruginosa
at 25 and 37 °C
25 °C
37 °C
effect on growth atb
effect on growth atb
a
a
IC50
95% confidence
IC50
(μM)
95% confidence
interval for IC50 800 μM 400 μM 100 μM 40 μM 20 μM
compd (μM) interval for IC50 100 μM 50 μM 40 μM 10 μM 5 μM
1
2
3
4
5
6
7
8
9
72.6
15.0
3.5
38.7-136.2
8.6-26.2
2.3-5.2
2.3-4.5
20.4-58.5
-
22.7
∼47.6
29.3
8.5-61.1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
11.4-75.2
39.1-67.3
10.6-829.5
3.2
þ
51.3
94.0
ndc
þ
-
-
34.5
>800
186.3
45.8
8.6
o
-
-
136.2-254.8
32.1-65.3
4.8-15.6
nd
nd
-
-
∼364.6
o
þ
a IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%. b o: the planktonic growth is completely or almost completely inhibited
when the bacteria are grown in the presence of theindicated concentration of biofilm inhibitor; þ: the planktonicgrowth 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.
at 37 °C. Although the IC50 values at 37 °C are remarkably
higher than those at 25 °C, this is still an interesting result, as
to the best of our knowledge no Salmonella biofilm inhibitors
acting both at room temperature and body temperature have
been reported before. The halogenated furanones for in-
stance are able to inhibit Salmonella biofilm formation at
16 °C and to a lesser extent at 25 °C, but they do not show
activity at 37 °C.11 Growth curve analysis at 37 °C revealed
that compounds 1 and 4 have a broad concentration range
with only biofilm inhibition and almost no effect on the
planktonic growth. Compounds 5 and 9, on the other hand,
completely block the planktonic growth at the IC50 for
biofilm inhibition, indicating that the biofilm inhibition in
these cases is due to a bactericidal effect.
Next, the influence of compounds 1-9 on P. aeruginosa
strain PA14 was investigated, both at 25 and 37 °C. As presented
in Table 2, the biofilm formation at 25 °C is very sensitive to
low (IC50s<15 μM), nongrowth inhibiting concentrations of the
compounds 2-4 and 9, while compounds 1, 5, 7, and 8 inhibit
the biofilm formation at moderate concentrations (IC50s ∼
50-200 μM). Also, at 37 °C, the biofilm formation is clearly
affected at concentrations lower than 100 μM for all compounds,
except for compound 9, which has an IC50 higher than 300 μM
(Table 2). No substantial effect on the planktonic growth of
Pseudomonas was observed for compounds 1-5 at the IC50 for
biofilm inhibition, indicating that the biofilm inhibition is not
due to antibacterial properties of the compounds. Compound 9,
however, does reduce the planktonic growth at the biofilm-
inhibiting concentrations.
4,5-Disubstituted 2-amino-1H-imidazoles. To determine
whether the introduction of a second aryl group at the
C-C double bond of the 2-aminoimidazole ring could
enhance activity, we also synthesized a series of 4,5-disub-
stituted 2-amino-1H-imidazoles via our previously estab-
lished procedure.32 We screened these compounds against
inhibition of S. Typhimurium ATCC14028 biofilm forma-
tion at 25 °C. Compounds 12, 14, 15, and 17 were found to
inhibit the biofilm formation at low concentrations (IC50s
∼20 μM) (Table 3), comparable with the active concentra-
tions of the most promising para-substituted 4(5)-phenyl-2-
amino-imidazoles. The other compounds are active at mod-
erate concentrations (IC50 values of 50-200 μM). In general,
each disubstituted compound was found to be more active
than the least active of the two monosubstituted compounds

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 475
Table 3. Influence of 4,5-Disubstituted 2-Amino-1H-imidazoles 11-18 on the Biofilm Formation and the Planktonic Growth of S. Typhimurium
ATCC14028 at 25 °C
effect on growth atb
95% confidence
interval for IC50
a
compd
Rl
R2
IC50
400 μM
80 μM
40 μM
20 μM
10 μM
4 μM
11
12
13
14
15
16
17
18
H
H
H
Cl
Cl
Cl
F
4-OMe
4-CF3
4-C1
77.1
29.2
46.9
12.9
10.8
71.8
18.2
182.1
60.6-98.0
22.7-37.6
35.2-62.5
10.7-15.6
6.5-17.8
50.2-102.5
17.0-19.5
116.6-284.4
o
-
-
4-Me
4-CF3
3,5-diF
4-F
o
o
o
o
o
o
þ
þ
-
-
o
o
þ
-
-
-
þ
-
a IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%. b o: the planktonic growth is completely or almost completely inhibited
when the bacteria are grown in the presence of theindicated concentration of biofilm inhibitor; þ: the planktonicgrowth 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.
Scheme 2. Structures of the 2,3-Disubstituted Imidazo[1,2-a]pyrimidines 19-24
obtained by theoretically removing one substituent. To
answer the question as to whether the tested compounds
specifically interfere with biofilm formation or are bacter-
icidal in nature, we determined the influence of the most
active compounds on the planktonic growth of Salmonella.
Compound 12 does only very slightly influence planktonic
growth at the IC50 for biofilm inhibition, suggesting that
biofilm inhibition is mediated by a biofilm specific mode
of action. However, compound 17 slows down planktonic
growth at the IC50 for biofilm inhibition, while compounds
14 and 15 block the growth almost completely.
2,3-Disubstituted Imidazo[1,2-a]pyrimidines. The effect of
the 2,3-disubstituted imidazo[1,2-a]pyrimidines 19-24, which
are the chemical precursors of the 4,5-disubstituted 2-aminoi-
midazoles in our synthesis pathway (Scheme 2), against Salmo-
nella biofilm formation, was assayed at 16, 25, and 37 °C. None
of the compounds are able to reduce the biofilm formation at
400 μM, irrespective of the incubation temperature used (results
not shown). In our chemical synthesis procedure, the 2-aminoi-
midazoles (containing active biofilm inhibitors, Tables 1, 2, and
3) are formed by cleavage of the imidazo[1,2-a]pyrimidines with
a nucleophile such as hydrazine under conventional heating
or microwave irradiation (Scheme 1).32 Our rationale to test the
antibiofilm effect of the 2,3-disubstituted imidazo[1,2-a]pyri-
midines was based on the reported antibacterial and antifungal
activity of some members of this class of compounds35,36 but
also on the hypothesis that the imidazo[1,2-a]pyrimidines could
in situ, i.e. in the biofilm assay, be cleaved by cellular nucleo-
philes to form the active 2-aminoimidazoles. However, the lack
of antibiofilm activity of the 2,3-disubstituted imidazo[1,2-a]pyri-
midines means that this hypothesis cannot be substantiated for
the tested 2,3-disubstituted imidazo[1,2-a]pyrimidines.
N1-Substituted 2-Aminoimidazoles. In an attempt to im-
prove their biofilm inhibitory activity, we substituted the N1-
position of some of the para-substituted 4(5)-phenyl-2-ami-
noimidazoles and some 4,5-disubstituted 2-aminoimidazoles
with n-alkyl, cyclo-alkyl, and aromatic groups by using our
previously established chemistry (Scheme 1).37,38
n-Alkyl Substituents. In the first instance, a broad variety
of alkyl groups, with lengths ranging from one carbon atom
to 14 carbon atoms, were introduced at the N1-position of
the 4(5)-monosubstituted 2-aminoimidazoles 1, 3, and 9,
resulting in compounds 25-38, 39-51, and 52-63, respec-
tively (Table 4). Each compound was assayed for the ability
to inhibit S. Typhimurium ATCC14028 biofilm formation at
25 °C. As depicted in Figure 1, a clear correlation was found
between the length of the alkyl substituent and the biofilm
inhibitory activity. In general, compounds with a short alkyl
chain substitution (C1, C2, C3) at the N1-position have a
lower activity as compared to their respective unsubstituted
counterparts. This effect is most pronounced for the methyl
substituent, which causes a reduction in the activity of at

476 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Steenackers et al.
Table 4. Influence of N1-Alkylated 2-Aminoimidazoles 25-63 on the Biofilm Formation and the Planktonic Growth of S. Typhimurium ATCC14028
and P. aeruginosa PA14 at 25 °C
S. Typhimurium
effect on growth atb
P. aeruginosa
effect on growth atb
a
IC50 95% confidence
a
IC50 95% confidence
compd
R
R1
Me
(μM) interval for IC50 80 μM 40 μM 20 μM 10 μM 5 μM (μM) interval for IC50 80 μM 40 μM 20 μM 10 μM 5 μM
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
51
52
53
54
55
56
57
58
59
60
61
62
63
H
H
H
H
H
H
H
H
H
H
H
H
H
H
431.5
206.0
175.5
51.9
48.4
39.7
12.2
11.9
∼6.0
∼6.1
7.2
261.6-711.7
168.7-251.5
139.7-220.6
45.1-59.8
37.5-62.4
30.0-52.5
9.9-15.1
95.2
20.7
4.5
67.2-134.7
9.7-44.2
3.2-6.3
1.1-4.3
1.2-3.7
Et
i-Pr
-
-
-
-
-
-
-
n-Bu
2.1
2.1
n-Pen
n-Hex
n-Hept
n-Oct
n-Non
n-Dec
n-Und
n-Dod
N-Trd
N-Tet
o
-
-
-
-
-
-
-
-
-
19.1
6.8
15.3-23.9
4.0-11.5
8.4-40.7
6.0-11.7
9.3-24.2
10.5-25.2
8.0-20.6
20.5-36.3
6.6-14.6
87.0-143.6
7.2-17.7
11.1-26.0
4.8-7.5
4.1-7. 1
2.2-3.2
3.0-5.2
6.0-10.7
16.3-30.6
29.6-65.4
30.1-82.0
15.0-76.4
11.8-31.0
-
-
-
þ
-
-
-
-
10.2-13.8
þ
o
-
þ
o
18.5
8.4
-
-
-
-
-
-
-
-
-
o
15.0
16.3
12.8
27.3
9.8
5.8-8.8
5.8-8.7
5.4-6.6
7.1-10.7
75.3-155.2
35.0-80.8
34.7-96.5
5.8-11.1
3.5-6.9
o
þ
þ
o
7.1
6.0
o
-
-
-
-
-
o
-
8.7
-
-
-
-
þ
-
-
-
Cl Me
Cl Et
108.1
53.2
57.8
8.0
111.8
11.3
16.7
6.0
þ
þ
-
-
-
-
-
Cl n-Bu
Cl n-Pen
Cl n-Hex
Cl n-Hept
Cl n-Oct
Cl n-Non
Cl n-Dec
Cl n-Und
Cl n-Dod
Cl n-Trd
Cl n-Tet
Ph Et
-
o
-
o
-
þ
-
þ
-
-
-
-
-
-
þ
þ
-
-
-
-
-
4.9
9.5
5.4
2.6
5.4-16.8
o
∼5.9
4.0
o
o
o
4.0
8.0
o
o
3.1-5.5
2.4-7.0
2.9-5.6
þ
o
4.1
4.1
22.3
44.0
49.7
33.9
19.1
∼95.1
41.6
73.9
32.1
25.2
8.9
-
-
-
o
þ
-
6.2
4.5-8.5
-
-
19.5
453.3
14.0
∼21.1
9.2
11.1-34.5
319.6-643.1
13.0-15.1
-
-
-
-
o
o
o
-
o
-
-
Ph n-Bu
Ph n-Pen
16.1-107.5
57.5-94.9
23.0-44.7
15.6-40.9
3.5-22.5
7.1-11.8
Ph n-Hex ∼12.1
o
o
Ph n-Hept
Ph n-Oct
Ph n-Non
Ph n-Dec
Ph n-Und
9.3
3.3
6.9-12.4
1.2-9.0
8.0-33.6
þ
þ
-
-
-
þ
þ
o
-
-
-
-
16.4
5.7
þ
þ
-
>400
>400
>400
>400
>400
>400
3.2-10.3
-
-
60.2
17.7-204.7
100.7-275.5
Ph n-Dod 166.6
Ph N-Trd >800
Ph N-Tet >800
-
-
-
-
a IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%. b o: the planktonic growth is completely or almost completely inhibited
when the bacteria are grown in the presence of theindicated concentration of biofilm inhibitor; þ: the planktonicgrowth 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.
least 3 times. To a lesser extent, ethyl and isopropyl substit-
uents also cause a reduction in activity. An exception to this
rule is compound 52 (with an ethyl substituent), which is
slightly more active than the unsubstituted molecule 9.
Substitution with a butyl group does not have an unequi-
vocal effect. In the case of compound 28, the activity is
enhanced 3-fold, while in the other cases the activity is
reduced. Remarkably, all the compounds substituted with
an alkyl group with an intermediate length between 5 and
10 carbon atoms are very active biofilm inhibitors, with IC50
values in general below 12 μM. Compound 57, substituted
with an octyl group, is the most potent inhibitor, with an IC50
of 3.25 μM. The effect of substitution with a longer alkyl
chain drastically depends on the nature of the substituent on
the C5-position of the ring (phenyl, p-chlorophenyl, or
biphenyl). Indeed, replacement of the N1-hydrogen of com-
pound 1 (phenyl on C5 of the ring) by a long alkyl chain
(C11-C14) raises the activity drastically, from an IC50 of
130 μM for compound 1 to values below 9 μM for the sub-
stituted compounds (35-38). In the case of compound 3 (p-
chlorophenyl on C5 of the ring), substitution with an undecyl
(48) and dodecyl (49) group results in very active compounds
(IC50 values below 6.5 μM), while introduction of a tridecyl
group (50) does not have a clear effect on the biofilm inhibi-
tory activity and introduction of a tetradecyl (51) group
drastically reduces the activity (IC50 = 453 μM). In the case

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 477
N1-alkyl chain but to a lesser extent than in the case of
compound 9 (compounds 47-51). Interestingly, growth
curve analysis revealed that these compounds are much less
toxic to P. aeruginosa than to S. Typhimurium. Most of the
compounds tested show a broad concentration range with
only biofilm inhibition and no effect on the planktonic
growth (Table 4).
To consolidate the structure-activity relationship de-
scribed above, we first synthesized some additional compounds
with short alkyl substituents at the N1-position and compared
their biofilm inhibitory activity with their unsubstituted counter-
parts (Table 5 and Figure 2). As expected, introduction of a
methyl, ethyl, and iso-propyl group does in general markedly
decrease the biofilm inhibitory activity, while introduction of a
n-butyl group does not have an unequivocal effect. Because the
introduction of a n-octyl side chain at the N1-position of com-
pounds 1, 3, and 9 does result in very active inhibitors of both
the Salmonella and Pseudomonas biofilm formation, we also
decided to introduce a n-octyl chain at the N1-position of
some other 4(5)-aryl-2-amino-1H-imidazoles and to evaluate
the biofilm inhibitory activity of the substituted compounds
(Table 6). All the compounds (75-84) are active inhibitors of
both the S. Typhimurium and P. aeruginosa biofilm formation
at 25 °C, except for compounds 82 and 83. These compounds
have very bulky C5-substituents, which could diminish the
solubility of the compounds in the growth medium, reduce the
uptake into the cell, or cause steric hindrance in the putative
receptor binding pocket. Comparison of the activities of com-
pounds 75-78 with their unsubstituted counterparts 2, 5, 6, and
7 (Table 1 and 2) confirms that introduction of a n-octyl chain at
the N1-position of the 4(5)-phenyl-2-amino-1H-imidazoles in
most cases improves the biofilm inhibitory activity of the
compounds. Growth curve analysis confirmed the previously
described finding that the N1-alkylated 2-aminoimidazoles in
general show a broad concentration range in which they spe-
cifically inhibit the biofilm formation of P. aeruginosa, without
reducing the planktonic growth of the bacteria, while this
concentration range is smaller in the case of S. Typhimurium.
Compounds 81 and 84 are exceptions to this rule, as we found
them to slow down the planktonic growth of P. aeruginosa at the
IC50 for biofilm inhibition.
Cyclo-alkyl Substituents. To determine whether the intro-
duction of an intermediate or long cyclo-alkyl chain could
also enhance the activity of the compounds, a broad variety of
cyclo-alkyl groups, with lengths ranging from 4 to 12 carbon
atoms, were introduced at the N1-position of the 4(5)-mono-
substituted 2-aminoimidazoles 1, 3, and 9 (Table 7). The com-
pounds were tested against biofilm formation of S. Typhimurium
and P. aeruginosa at 25 °C. As depicted in Figure 3, introduc-
tion of an intermediate cyclo-alkyl chain does improve the
biofilm inhibitory activity of compound 1 against S. Typhimurium
and even more drastically against P. aeruginosa. In the case
of Salmonella, introduction of a cyclo-heptyl chain yields the
most active compound (compound 89), with an IC50 of 27 μM,
as compared to 130 μM for compound 1. In the case of
Pseudomonas, cyclo-hexyl and cyclo-octyl are the best substitu-
ents as introduction of these groups improves the activity
drastically from an IC50 of 73 μM for compound 1 to 5 μM
for the substituted compounds. All compounds derived from 3
and 9 by introduction of an intermediate length side chain are
very strong inhibitors of the Salmonella and Pseudomonas
biofilm formation (Table 7), although their activity is not
markedly better than the activity of the unsubstituted com-
pounds. Introduction of a dodecyl side chain abolishes the
Figure 1. Effect of introduction of alkyl chains with different lengths
(1-14 C-atoms; 0 means no alkyl substituent) at the N1-position of
compound 1 (black bars), 3 (dark-gray bars), and 9 (light-gray bars) on
the IC50 (μM) for inhibition of the biofilm formation of S. Typhimur-
ium ATCC14028 (A) and P. aeruginosa PA14 (B) at 25 °C in TSB 1/20.
All the substituents are n-alkyl groups, except for the alkyl group with 3
carbon atoms, which is an iso-propyl group.
of compound 9 (biphenyl on C5 of the ring), however,
substitution with all the long alkyl chains (C11-C14)
(60-63) drastically reduces the activity, with the strongest
reduction for the longest alkyl chains. Subsequently, we
investigated the influence of some of the most active repre-
sentatives of this class of compounds on the planktonic
growth curve of S. Typhimurium at 25 °C. Most compounds
only show a very small concentration range with a specific
effect on biofilm formation and no effect on the planktonic
growth (Table 4). Therefore, we cannot exclude the possibi-
lity that the biofilm inhibitory effect of these compounds (at
the higher concentrations) is partially due to a reduction of
the planktonic growth of the bacteria in the growth medium
used in the setup to monitor biofilm formation. Interestingly,
compounds 38, 49, and 59, with long 3-alkyl chains, do not
follow this general trend, as no effect on the growth was
observed at 40 μM while the biofilm formation is inhibited at
concentrations lower than 10 μM.
As depicted in Table 4 and Figure 1, a similar structure
-activity relationship was found for the inhibition of
P. aeruginosa biofilm formation at 25 °C. Again, introduction
of a short alkyl chain at the N1-position generally results in a
decreased activity (except for compound 26), while all the
compounds with an intermediate alkyl chain length show
very strong activities. The effect of substitution with a long
alkyl chain is in this case also dependent on the nature of the
C5-subsitutent. In the case of compound 1, substitution with
a long alkyl chain results in very active compounds (34-38),
while these substitutions yield almost completely inactive
compounds (58-63) in the case of compound 9. The activity
of compound 3 is also decreased after introduction of a long

478 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Steenackers et al.
Table 5. Influence of 2-Aminoimidazoles 64-74 with Short Alkyl Substituents at the N1-Position on the Biofilm Formation and the Planktonic Growth
of S. Typhimurium ATCC14028 at 25 °C
S. Typhimurium
P. aeruginosa
compd
R
Rl
Et
IC50a (μM)
95% confidence interval for IC50
IC50a(u.M)
95% confidence interval for IC50
64
65
66
67
68
69
70
71
72
73
74
F
290.4
56.2
221.1-381.5
45.20-69.89
67.65-235.1
12.16-21.45
104.5-250.6
227.2-358.8
311.9-414.9
92.12-141.9
48.56-79.85
173.4-238.4
67.8
55.7
39.32-116.9
47.57-65.14
53.71-68.21
10.06-34.91
54.43-89.43
95.69-165.3
F
Br
n-Bu
Et
126.1
16.2
60.5
18.7
Br
n-Bu
Me
Et
NO2
NO2
NO2
NO2
Me
Me
CN
161.8
285.5
359.7
114.3
62.3
69.8
125.8
>800
372.6
31.7
i-Pr
n-Bu
Me
i-Pr
Me
218.2-636.3
25.89-38.79
191.2-302.1
250.4-438.8
203.3
>400
240.3
331.4
a IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.
Salmonella biofilm inhibition depends on the nature of the C5-
group. Indeed, compound 91, derived from 1 by introduction of
a cyclo-dodecyl group, is a very strong biofilm inhibitor, while
compound 97, derived from 3, is a moderate biofilm inhibitor,
and compound 103, derived from 9, does not have any activity.
As in the case of the n-alkyl substituents, growth curve analysis
revealed that most compounds with cyclo-alkyl substituents
only possess a very small concentration range with a selective
effect on the Salmonella biofilm formation and no effect on the
planktonic growth. An exception is compound 91, with a cyclo-
dodecyl side chain, which does not affect the planktonic growth
of Salmonella at 80 μM (the highest concentration tested),
while the IC50 for biofilm inhibition is 15 μM. Interestingly,
the concentration range between biofilm inhibition and
growth inhibition is much broader in the case of P. aeruginosa
(Table 7). We also tested the antibiofilm effect of N1-cyclohexyl
4,5-diphenyl-2-aminoimidazole 104. This 1,4,5-trisubstituted
compound shows a good effect against S. Typhimurium
(IC50=24 μM) and P. aeruginosa (IC50=5 μM) biofilm forma-
tion, while only a slight Salmonella growth inhibition was
observed at 80 μM.
Aromatic Substituents. Finally, we also tested the influence
of substituting the N1-hydrogen with some aromatic groups
on inhibition of the biofilm formation by S. Typhimurium
and P. aeruginosa. As shown in Table 8, introduction of a
phenyl group at the N1-position of the 4,5-disubstituted-2-
amino-imidazole 14 (resulting in compound 105) abolishes
its biofilm inhibitory activity completely. Substitution of the
N1-position of compound 1 with a benzyl group (resulting in
106) enhances the activity against both Salmonella and
Pseudomonas 2- to 3-fold. The same substitution also en-
hances the activity of compound 2 (compound 107) against
Salmonella, while it lowers the activity against Pseudomonas.
Substitution of the N1-position of compounds 3 and 4 with a
benzyl group (resulting in compounds 108 and 109, re-
spectively) on the other hand lowers the activity against both
species. Substitution of the N1-hydrogen with a 3,4-di-
methoxybenzyl group (110) decreases the activity of com-
pound 1 against Pseudomonas, while no clear effect on the
activity against Salmonella could be observed. Also substitu-
tion of the N1-hydrogen of compound 1 with a veratryl
group (111) or a piperonyl group (112) does not have a
substantial effect on the biofilm inhibitory activity.
Figure 2. Effect of introduction of short alkyl chains (Me, Et, i-Pr, or
n-Bu) at the N1-position of compounds 2, 4, 5, 8, and 10 on the IC50
(μM) for inhibition of the biofilm formation of S. Typhimurium
ATCC14028 (A) and P. aeruginosa PA14 (B) at 25 °C in TSB 1/20.
activity of all three compounds against P. aeruginosa biofilm
formation, while the effect of a cyclo-dodecyl substituent on the

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 479
Table 6. Influence of N1-Octyl-2-aminoimidazoles 75-84 on the Biofilm Formation and the Planktonic Growth of S. Typhimurium ATCC14028 and
P. aeruginosa PA14 at 25 °C
S. Typhimurium
effect on growth atb
P. aeruginosa
effect on growth atb
a
IC50 95% confidence
a
IC50 95% confidence
compd
R
R1
n-Oct ∼10.8
n-Oct 35.4
(μM) interval for IC50 80 μM 40 μM 20 μM 10 μM 5 μM (μM) interval for IC50 40 μM 20 μM 10 μM 5 μM
75
76
77
78
79
80
81
82
83
84
4-F
o
-
4.9
3.8-6.2
o
þ
þ
-
-
4-NO2
4-SO2Me
4-OMe
4-SMe
3,4-diCI
3-Br
25.2-49.7
þ
þ
þ
n-Oct ∼29.1
-
þ
4.9
1.2
2.8
2.6-9.3
0.9-1.7
1.9-4.0
-
-
o
n-Oct
n-Oct
n-Oct
n-Oct
8.5
6.7
7.8-9.3
6.0-7.5
-
þ
þ
-
-
-
3.7
11.2
3.4-4.1
10.3-12.1
54.6-248.5
þ
40.6
>800
>800
17.6
29.5-56.1
þ
-
4-(40-NO2Ph) n-Oct 116.5
4-(40-biphenyl) n-Oct 850.2 677.0-1068.0
6.6
6.2-7.0
-
-
11.1-28.1
þ
-
a IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%. b o: the planktonic growth is completely or almost completely inhibited
when the bacteria are grown in the presence of theindicated concentration of biofilm inhibitor; þ: the planktonicgrowth 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 7. Influence of N1-Cyclo-alkyl-2-aminoimidazoles 85-104 on the Biofilm Formation and the Planktonic Growth of S. Typhimurium and
P. aeruginosa PA14 at 25 °C
S. Typhimurium
effect on growth atb
P. aeruginosa
effect on growth atb
95% confidence 80
95% confidence 80
40 20 10
5
compd
R
R1
IC50a(μM) interval for IC50 μM 40 μM 20 μM 10 μM 5 μM IC50a(μM) interval for IC50 μM μM μM μM μM
85
86
H
H
H
H
H
H
H
c-Pr
c-Bu
89.6
116.3
70.7
76.0-105.7
91.5-147.9
56.0-89.2
54.1-134.3
16.5-43.7
25.9-63.2
11.1-19.1
47.0-64.0
8.5-15.8
25.8-42.8
13.0-18.9
11.2-14.8
70.3-205.4
21.8-31.6
5.5-10.3
35.0
19.9
12.6
4.6
21.8-56.3
14.4-27.6
7.2-22.1
2.8-7.68
9.7-20.9
3.1-6.6
-
-
-
-
87
88
c-Pen
c-Hex
c-Hep
c-Oct
c-Dod
85.2
26.9
-
-
-
-
-
-
-
-
89
90
14.2
4.5
-
-
40.5
14.6
o
-
-
-
91
92
-
-
>800
13.0
6.0
Cl c-Pr
54.8
11.6
7.0-24.2
4.8-7.5
8.6-14.7
2.78-5.8
15.6-151.6
-
93
94
Cl c-Bu
Cl c-Hex
Cl c-Hep
Cl c-Oct
Cl c-Dod
Ph c-Bu
Ph c-Pen
Ph c-Hex
Ph c-Hep
Ph c-Oct
Ph c-Dod
o
þ
-
-
þ
-
-
-
-
-
33.2
15.7
11.2
4.0
-
-
95
96
12.9
o
o
48.7
>800
∼24.5
353.1
69.7
6.6
97
98
120.2
26.3
7.5
99
o
o
o
o
o
o
o
o
-
-
þ
-
151.2-824.6
36.7-132.3
2.9-14.7
100
101
102
103
104
∼12.6
∼12.5
-22.9
>800
23.9
3.2
>800
5.0
1.5-6.7
-
-
-
-
-
-
17.49-32.60
-
-
-
-
1.5-16.5
a IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%. b o: the planktonic growth is completely or almost completely inhibited
when the bacteria are grown in the presence of theindicated concentration of biofilm inhibitor; þ: the planktonicgrowth 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.
N1-Substituted Imidazo[1,2-a]pyrimidinium Perchlorate Salts.
In our chemical synthesis procedure, the N1-substituted 2-ami-
noimidazoles are formed by cleavage of the N1-substituted
imidazo[1,2-a]pyrimidinium perchlorate salts with a nucleo-
phile such as hydrazine, under conventional heating or micro-
wave irradiation.37,38 After cleavage, the N1-substituent of

480 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Steenackers et al.
2-aminoimidazole will be the same as the N1-substituent of
the salt (Scheme 1). A possible mechanism for the pyrimidine
ring cleavage of the salts upon reaction with amines involves
a nucleophilic attack by a first molecule of amine at the C5-
position, resulting in the formation of azabutadienes. 1,4-Addi-
tion of a second molecule of amine results in the formation of the
2-aminoimidazole.38 As previously reported,38 we found that the
imidazo[1,2-a]pyrimidinium salts can also be cleaved to 2-ami-
noimidazoles at room temperature upon stirring with excess
of secondary amine. Therefore, we hypothesized that these
imidazo[1,2-a]pyrimidinium salts could be more susceptible to
cleavage by cellular nucleophiles (e.g., proteins, metalloproteins,
polyamines, amino acids,...) than the 4,5-disubstituted imidazo-
[1,2-a]pyrimidines, leading to the in situ formation of the active
2-aminoimidazoles. To strengthen this hypothesis, we deter-
mined whether 1-ethyl-2-(40-nitrophenyl)-imidazo[1,2-a]pyrim-
idin-1-ium perchlorate (compound 114 (Table 9)) could be
degraded to the corresponding 2-aminoimidazole 69 under
influence of 2 equiv of the amino acid analogue L-alanine ethyl
ester hydrochloride at room temperature. We found the salt to
be almost completely degraded to the imidazole after 12 h of
reaction, indicating that the imidazo[1,2-a]pyrimidinium salts
could indeed be cleaved by cellular nucleophiles within the time
frame of our biofilm assay. To further substantiate our hypothe-
sis, we synthesized a series of imidazo[1,2-a]pyrimidinium salts
112-130 with N1-alkyl substituents of different lengths and
determined whether the structure-activity relationship found
for these salts is in agreement with the previously described
structure-activity relationship for the corresponding 2-aminoi-
midazoles. As depicted in Table 9, salts 113-117 (correspond-
ing imidazoles 69-71) withshortN1-alkyl chains (C2-C4) have
IC50 values higher than 400 μM both for the Salmonella and
Pseudomonas biofilm inhibition at 25 °C, while compound 118
(corresponding imidazole 76) with a n-octyl side chain is a
good biofilm inhibitor, with IC50 values of 19 and 68 μM for
P. aeruginosa and S. Typhimurium biofilm inhibition, respec-
tively. Hence, we may conclude that the structure-activity rela-
tionship delineated for the N1-substituted 2-aminoimidazoles does
also apply to the corresponding N1-substituted salts, which con-
tributes to the plausibility of our hypothesis. As introduction of a
n-octyl substituent at the N1-position of the 2-aminoimidazoles
in general improves their biofilm inhibitory activity, we decided
to synthesize a series of 2-aryl-imidazo[1,2-a]pyrimidinium
perchlorate salts with n-octyl substituents at the N1-position. In
agreement with the structure-activity relationship of the 2-ami-
noimidazoles, most of the N1-octyl substituted imidazo[1,2-
a]pyrimidinium salts were found to be strong inhibitors of both
the S. Typhimurium and P. aeruginosa biofilm formation, with
IC50 values in general below 35 μM. However, in contrast to the
almost inactive 2-aminoimidazoles 82 and 83, the corresponding
salts 127 and 130 are very active, with IC50 values below 10 μM.
This could possibly be explained by a reduction of the planktonic
growth of the bacteria in the growth medium used in the setup to
Figure 3. Effect of introduction of cyclo-alkyl chains with different
lengths (1-12 C-atoms; 0 means no alkyl substituent) at the N1-position
of compound 1 (black bars), 3 (dark-gray bars) and 9 (light-gray bars)
on the IC50 (μM) for inhibition of the biofilm formation of S.
Typhimurium ATCC14028 (A) and P. aeruginosa PA14 (B) at 25 °C.
Table 8. Influence of 2-Aminoimidazoles 105-112 with Aromatic Substituents at the N1-Position on the Biofilm Formation of S. Typhimurium
ATCC14028 at 25 °C
S. Typhimurium
P. aeruginosa
compd
R1
R4
R5
IC50a(μM)
95% confidence interval for IC50
IC50a(μM)
95% confidence interval for IC50
105
106
107
108
109
110
111
112
Ph
Bn
Bn
Bn
Bn
p-MePh
p-CIPh
Ph
>400
55.1
37.3
>800
26.1
32.0
H
H
H
H
H
H
H
45.3-67.0
32.3-43.2
22.0-36.9
48.6-113.1
109.8-221.1
147.6-231.9
72.97-129.7
17.0-39.9
27.1-37.9
11.9-18.5
36.2-92.4
115.0-220.6
43.0-62.6
78.6-138.8
p-FPh
p-CIPh
p-BrPh
H
28.5
74.2
14.8
57.8
3,4-diMeOBn
veratryl
piperonyl
155.8
185.0
97.3
159.3
51.9
104.4
H
H
a IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 481
Table 9. Influence of N1-Substituted Imidazo[1,2-a]pyrimidinium Salts 113-130 on the Biofilm Formation and the Planktonic Growth of S.
Typhimurium ATCC14028 and P. aeruginosa PA14 at 25°C
S. Typhimurium
effect on growth ata
P. aeruginosa
effect on growth atb
95% confidence
95% confidence
compd
R1
Me
R
IC50 μM interval for IC50 40 μM 20 μM 10 μM 5 μM IC50 μM interval for IC50 40 μM 20 μM 10 μM 5 μM
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
3-NO2
427.3
>800
>800
>400
405.2
63.6
561.0
9.8
233.0-783.6
>800
>800
>800
>800
>800
18.7
249.5
15.1
>800
8.7
Et
4-NO2
4-NO2
4-NO2
4-NO2
i-Pr
n-Bu
i-Bu
300.5-546.3
54.1-74.7
277.9-1133.0
6.4-15.1
59.4-487.3
26.3-45.9
27.9-45.7
3.2-4.7
n-Oct 4-NO2
c-Hex 4-NO2
c-Dod 4-NO2
12.6-27.8
140.7-442.3
11.2-20.3
þ
-
-
-
-
þ
-
-
o
þ
Bn
4-NO2
170.1
34.8
35.7
3.8
n-Oct 4-OMe
n-Oct 4-F
o
þ
3.5-21.8
14.1-26.9
10.7-15.1
4.5-10.9
2.8-16.9
0.4-3.0
-
þ
o
-
-
þ
-
-
-
19.5
12.7
7.0
n-Oct 3.4-diF
n-Oct 3-Br
þ
-
-
7.3
6.0-8.9
þ
þ
o
n-Oct naphtyl
n-Oct 4-(40N02Ph)
n-Oct 4-SO2Me
n-Oct 4-SMe
n-Oct 4-(40-biphenyl)
∼6.0
1.5
þ
o
-
þ
6.8
1.0
þ
o
þ
-
o
1.2-1.8
56.4-119.1
17.4-24.5
4.1-6.2
o
o
82.0
20.7
5.0
49.6
9.3
ndc
23.8-103.6
7.0-12.3
o
þ
þ
-
þ
-
a IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%. b 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
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.
Scheme 3. Structures of the N2-Substituted 2-Aminopyrimidines 131-136
monitor biofilm formation, as growth curve analysis revealed a
toxic effect of salts 127 and 130. Most of the other compounds
tested show a small concentration range with only biofilm inhibition
and no effect on the planktonic growth. To exclude the possibility
that part of the toxic or biofilm inhibitory effect of the salts is due to
the perchlorate anion, we tested the influence of different concen-
trations of sodium perchlorate on both the planktonic growth and
biofilm formation of Salmonella and Pseudomonas. No effect was
found at the highest concentration tested (500 μM).
2N-Substituted 2-Aminopyrimidines. Some members of
the 2-aminopyrimidine class of compounds have previously
been shown to possess antibacterial and antifungal activity.39,40
These molecules are the precursors of the N1-substituted imidazo-
[1,2-a]pyrimidinium salts in our synthesis pathway (Scheme 1).
The availability of a broad array of 2-aminopyrimidines,
substituted with n-alkyl, cyclo-alkyl, and aromatic groups at
the N2-position, prompted us to investigate the potential of this
class of compounds as biofilm inhibitors. The influence of
compounds 131-136 (Scheme 3) on the biofilm formation
of S. Typhimurium was tested at 25 °C. Remarkably, none of
the compounds do have an effect on the biofilm formation at
400 μM, which was the highest concentration tested.
Conclusion
Recently, we published a method for the microwave-
assisted synthesis of substituted 2-amino-1H-imidazoles from
imidazo[1,2-a]pyrimidines, focusing on 4(5)-mono and 4,5-
disubstituted 2-amino-1H-imidazoles with aryl substituents.
Furthermore, we reported on a procedure for the synthesis of
N1-substituted 2-aminoimidazoles from 2-aminopyrimidines
via N1-substituted imidazo[1,2-a]pyrimidinium salts. In the pre-
sent study, we investigated the effect of the different reaction

482 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2
Steenackers et al.
products (2-aminoimidazoles), intermediates (imidazopyrimi-
dines, imidazo[1,2-a]pyrimidinium salts), and reagents (2-amino-
pyrimidines) on the biofilm formation of S. Typhimurium and
P. aeruginosa. We found that our simplest 2-aminoimidazole
structure, 4(5)-phenyl-2-amino-1H-imidazole 1, showed a mod-
erate biofilm inhibitory activity both against S. Typhimurium
and P. aeruginosa. Substitution of the para-position of the 4(5)-
phenyl ring with a chlorine, nitro, or phenyl group enhanced the
activity against Salmonella biofilms (25 °C) about 10 times, while
substitution with a bromine, fluorine, methyl, or methoxy group
did improve the biofilm inhibition more moderately and sub-
stitution with a methanesulfonyl or nitrile function reduced the
biofilm inhibitory activity drastically. In case of Pseudomonas,
para-substitution with a chlorine, bromine or phenyl group
increased the biofilm inhibitory (25 °C) activity about 20 times.
The 4,5-disubstituted 2-amino-1H-imidazoles showed a high
or moderate activity against S. Typhimurium biofilm formation,
but they were in general highly toxic to Salmonella. We were
able to delineate a relationship between the length of the
N1-alkyl or N1-cyclo-alkyl chain of the N1-substituted 2-amino-
imidazoles and their biofilm inhibitory activity. In general,
introduction of a short alkyl chain at the N1-position resulted
in a decreased activity against S. Typhimurium and P. aeruginosa
biofilm formation, while all compounds with an intermediate
alkyl or cyclo-alkyl chain length showed very strong activities in
both test systems. The introduction of a long alkyl or cyclo-alkyl
chain could either drastically enhance the activity or totally
abolish the activity, depending on the nature of the C5-sub-
situtent and the bacterial species tested. Growth curve analysis
revealed that the N1-(cyclo-)alkylated 2-aminoimidazoles show
a broad concentration range in which they specifically inhibit the
biofilm formation of P. aeruginosa, without retarding the plank-
tonic growth of the bacteria. However, these compounds were
much more toxic to S. Typhimurium. Therefore, we cannot
exclude the possibility that part of the inhibitory effect against
Salmonella biofilms (at the higher concentrations) is due to a
reduction of the planktonic growth of the bacteria in the growth
medium surrounding the surface on which the biofilms are
formed. To test the hypothesis that the 2,3-disubstituted
imidazo[1,2-a]pyrimidines and the imidazo[1,2-a]pyrimidinium
salts, which are the chemical precursors of the 2-aminoidazoles,
could also in situ be cleaved by cellular nucleophiles to form
the active 2-aminoimidazoles, we tested their influence on the bio-
film formation. We found a good correlation between the activity
of the imidazo[1,2-a]pyrimidinium salts and their corresponding
2-aminoimidazoles, supporting our hypothesis. The disubstituted
imidazo[1,2-a]pyrimidines, however, did not show any biofilm
inhibitory activity, indicating that these molecules are less suscep-
tible to degradation by cellular nucleophiles. Finally, we tested the
influence of a broad array of 2N-substituted 2-aminopyrimidines,
which are the chemical precursors of the imidazo[1,2-a]pyrimi-
dinium salts, and demonstrated their lack of activity against the
biofilm formation of S. Typhimurium and P. aeruginosa, although
some members of the class of 2-aminopyrimidines were previously
reported to have antibacterial activity. In conclusion, the 2-amino-
imidazoles and the 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.
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%.
Microwave Irradiation Experiments. A multimode Milestone
MicroSYNTH microwave reactor was used in the standard config-
uration as delivered, including proprietary software. All experiments
were carried out in sealed microwave process vials (15, 30, and
50 mL). After completion of the reaction, the vial was cooled down
to 25 °C via air jet cooling before opening. Reaction temperatures
were monitored by an IR sensor on the outside wall of the reaction
vial and a fiber-optic sensor inside the reaction vial.
4(5)-Monosubstituted 2-Amino-1H-imidazoles and 4,5-Disub-
stituted 2-Amino-1H-imidazoles. The 4(5)-Monosubstituted
2-amino-1H-imidazoles and 4,5-disubstituted 2-amino-1H-imi-
dazoles were synthesized according to our previously estab-
lished protocols.32
2,3-Disubstituted Imidazo[1,2-a]pyrimidines. The 2,3-disubsti-
tuted imidazo[1,2-a]pyrimidines were synthesized by using our
previously described protocols.43
N1-Substituted 2-Aminoimidazoles and Imidazo[1,2-a]pyrimidi-
nium Salts. The N1-substituted 2-aminoimidazoles and imidazo-
[1,2-a]pyrimidinium salts were synthesized following our previously
published methods.37,38 A general procedure is described below:
(1) To a solution of substituted 2-aminopyrimidine (6 mmol) and
R-bromoketone (7.2 mmol, 1.2 equiv) in acetonitrile (12 mL)
was added 4-dimethylaminopyridine (6 mg, 0.05 mmol). After
stirring at 85 °C for 5 h, the reaction mixture was diluted with
acetone (20 mL) and the precipitate was filtered and washed
with acetone (2 ꢀ 20 mL) and ether (2 ꢀ 20 mL) and dried
over P2O5 to give a white solid (2,3-dihydro-2-hydroxyi-
midazo[1,2-a]pyrimidin-1-ium bromide).
(2) A mixture of 84% polyphosphoric acid (3 g) and 2,3-
dihydro-2-hydroxyimidazo[1,2-a]pyrimidin-1-ium bromide
(3 mmol) was heated in 50 mL beaker upon intensive stirring
at 150 °C for 15 min. After cooling to the room temperature,
the resulting viscous mass was dissolved in 30 mL of water
and 1 mL (10 mmol) of 70% HClO4 was added dropwise
upon mild stirring. The white precipitate was washed with
distilled water (3 ꢀ 10 mL) and ether (2 ꢀ 10 mL) until a
neutral reaction of the pH paper and then dried over P2O5 to
give salt (imidazo[1,2-a]pyrimidin-1-ium perchlorate) as fine
white crystals.
(3) Perchlorate salt (1 mmol) was dissolved in acetonitrile (5 mL)
and hydrazine hydrate (0.15 mL, 3 mmol of a 64% aqueous
solution, 3 equiv) was added, and the mixture was irradiated
in the sealed reaction tube for 10 min at a ceiling temperature
of 100 °C at 150 W maximum power. After cooling down,
hydrazine hydrate was evaporated with toluene (3 ꢀ 20 mL).
The resulting residue was purified by column chromatogra-
phy (silica gel; MeOH-DCM 1:9 v/v with 5% of 6 M NH3 in
MeOH) to afford 2-aminoimidazole as bright-yellow needles.
The analytical data of the new compounds are listed as
Supporting Information.
Substituted 2-Aminopyrimidines. General procedure for the
preparation of substituted 2-aminopyrimidines: in a 50 mL micro-
wave vial were successively dissolved in EtOH (20 mL) 2-chloro-
pyrimidine (3.43 g, 30 mmol), primary amine (39 mmol, 1.3 equiv),
and triethylamine (6.2 mL, 45 mmol, 1.5 equiv). The reaction tube
was sealed and irradiated in the cavity of a microwave reactor at a
ceiling temperature of 120 °C at 80 W maximum power for 5 min.
After the reaction mixture was cooled with an air flow for 15 min,
it was diluted with water (100 mL), extracted with CH2Cl2 (2 ꢀ
150 mL), and dried over Na2SO4. The solvent was removed under
reduced pressure, and the residue was subjected to silica gel flash
Experimental Section
Chemistry. General Experimental Methods. Chemicals were
purchased from commercial sources and used without further
1
treatment. Melting points are uncorrected. H NMR spectra

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 2 483
chromatography (from 0% to 5% MeOH-CH2Cl2) to afford
substituted 2-aminopyrimidine.
The analytical data of the new compounds are listed as
Supporting Information.
of the diluted overnight culture was added to each well of the 10 ꢀ
10 well microtiter 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 the 300μLofbacterial culture) in3-fold. Asa control,
3 μL of the appropriate solvent was also added to the plate in 3- or
4-fold. The microtiter plate was incubated in the Bioscreen device
at 25 or 37 °C for at least 24 h, with continuous medium shaking.
The absorbance ofeachwellwas measuredat600 nmeach15min.
Excel was used to generate the growth curves for the treated wells
and the untreated control wells.
Reaction of Imidazo[1,2-a]pyrimidin-1-ium Salt with Amino
Acid Derivative. 1-Ethyl-2-(40-nitrophenyl)-imidazo[1,2-a]pyrimi-
din-1-ium perchlorate (185 mg, 0.5 mmol, 1 equiv) was dissolved
in methanol (3 mL), and triethylamine was added (210 μL, 1.5
mmol, 3 equiv). Then L-alanine ethyl ester hydrochloride (155 mg,
1 mmol, 2 equiv) was added and the reaction mixture was allowed to
stir for 12 h. After reaction completion (as judged by TLC), satd
NH4Cl solution (30 mL) was added and the mixture was extracted
by DCM (2 ꢀ 50 mL). The combined organic extracts were washed
with water (100 mL) and brine (50 mL). After drying over anhy-
drous MgSO4 and evaporating the solvent, the resulting residue was
subjected to flash chromatography using MeOH-DCM mixture
(1:9) as an eluent to afford 1-ethyl-5-(40-nitrophenyl)-2-aminoimi-
dazole as red crystals in 83% yield (97 mg).
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,
indicated by the symbol “-”.
(2) The planktonic growth is retarded, indicated by the
symbol “þ”.
(3) The planktonic growth is completely or almost complete-
ly inhibited, indicated by the symbol “o”.
Biological Assays. Static Peg Assay for Prevention of Salmonella
Typhimurium and Pseudomonas aeruginosa Biofilm Formation.11
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).44 Two-fold serial dilutions of the compounds in 100
μL of liquid broth per well were prepared in the microtiter plate
(two or three repeats per compound). For the S. Typhimurium
biofilm experiments at 25 °C, Tryptic Soy Broth diluted 1/20 (TSB
1/20; BD Biosciences) was used, while Colonization factor antigen
(CFA)45 broth was used for the experiments at 37 °C. For the P.
aeruginosa biofilm experiments at 25 °C, TSB 1/20 was used, while
Luria-Bertani medium46 without salt (LBNS) was used for the
experiments at 37 °C. Subsequently, an overnight culture of S.
Typhimurium ATCC14028 (grown in Luria-Bertani medium46)
or P. aeruginosa (grown in TSB) was diluted 1:100 into the
respective liquid broth and 100 μL (∼106 cells) was added to each
well of the microtiter plate, resulting in a total amount of 200 μL of
medium per well. The pegged lid was placed on the microtiter plate,
and the plate was incubated for 24 h at 25 or 37 °C without shaking.
During this incubation 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 indication of the effect of the compounds on the
planktonic growth. For quantification of biofilm formation, 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 extracted with
30% glacial acetic acid (200 μL). The OD570 of each well
was measured using a VERSAmax microtiter plate reader
(Molecular Devices). The IC50 value for each compound
was determined from the concentration gradient by using
the GraphPad software of Prism.
The following criterium was used to decide between the first
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 4 consecu-
tive hours, then the effect on the planktonic growth is classified
in category 2.
Acknowledgment. This work was supported by the Indus-
trial Research Fund of KU Leuven (KP/06/014), the Research
Council of KU Leuven (CoE EF/05/007 SymBioSys), the FWO
(Fund for Scientific Research;Flanders (Belgium)) and by the
Institute for the Promotion of Innovation through Science and
Technology in Flanders (IWT;Vlaanderen) through scholar-
ships to H.S. J.V. and D.D.V. are grateful for support in the
frame of the IAP program Functional Supramolecular Systems.
E.D.S. is grateful to the KU Leuven for obtaining a postdoctoral
fellowship and S.B. to Erasmus Mundus External Cooperation
Window Lot 15 India (EMECW15) for obtaining a scholarship.
Supporting Information Available: Analytical data of N1-
substituted 2-aminoimidazoles and imidazo[1,2-a]pyrimidinium
salts and substituted 2-aminopyrimidines. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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