Identification of aryl 2-aminoimidazoles as biofilm inhibitors in Gram-negative bacteria

Article abstract of DOI:10.1016/j.bmcl.2010.04.042

The synthesis and biofilm inhibitory activity of a 30-member aryl amide 2-aminoimidazole library against the three biofilm forming Gram-negative bacteria Escherichia coli, Psuedomonas aeruginosa, and Acinetobacter baumannii is presented. The most active compound identified inhibits the formation of E. coli biofilms with an IC₅₀ of 5.2 μM and was observed to be non-toxic to planktonic growth, demonstrating that analogues based on an aryl framework are viable options as biofilm inhibitors within the 2-aminoimidazole family.

Full text of DOI:10.1016/j.bmcl.2010.04.042

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3797–3800
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of aryl 2-aminoimidazoles as biofilm inhibitors
in Gram-negative bacteria
*
Cynthia A. Bunders, Justin J. Richards, Christian Melander
Department of Chemistry, North Carolina State University, Raleigh, NC 27695, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biofilm inhibitory activity of a 30-member aryl amide 2-aminoimidazole library
against the three biofilm forming Gram-negative bacteria Escherichia coli, Psuedomonas aeruginosa, and
Acinetobacter baumannii is presented. The most active compound identified inhibits the formation of
E. coli biofilms with an IC50 of 5.2 lM and was observed to be non-toxic to planktonic growth, demon-
strating that analogues based on an aryl framework are viable options as biofilm inhibitors within the
Received 11 March 2010
Revised 8 April 2010
Accepted 12 April 2010
Available online 18 April 2010
2-aminoimidazole family.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
2-Aminoimidazoles
Bacterial biofilms
Bacterial biofilms, which are defined as a surface-attached com-
munity of bacteria protected by an extracellular matrix of biomol-
ecules,1 are increasingly being recognized as an important
pathogenic target.2 Within a biofilm state, bacteria are upwards
of 1000-fold more resistant to antibiotics and are generally resis-
tant to the host immune response.3 This presents a tremendous
hurdle for the treatment of bacterial infections as the NIH esti-
mates 75% of all bacterial infections are biofilm-based. Given this
situation, there has been a significant effort to identify molecules
that are capable of inhibiting and dispersing biofilms.2
Our research group has been studying the ability of simple ana-
logues of the marine natural products bromoageliferin and oroidin
(Fig. 1) to control biofilm development.4–14 Bromoageliferin is a
sponge-derived alkaloid that was reported to inhibit the formation
of bacterial biofilms from marine sources, presumably as a defense
mechanism against biofouling.15 We reasoned that core structures
derived from this complex molecule could be used as structural
inspiration for the development of potent and synthetically acces-
sible anti-biofilm agents. One of these scaffolds developed was
based upon an aryl framework.16 From a medicinal chemistry
standpoint, this scaffold was deemed a promising platform to de-
velop further functionalized 2-aminoimidazole (2-AI) derivatives
as diversity could rapidly be introduced through manipulation of
the benzene ring. Herein we describe the synthesis and anti-bio-
film activity of these aryl 2-AI derivatives against three commonly
studied Gram-negative bacteria.
ages around a core phenyl ring obtained through an acylation reac-
tion with an aniline intermediate. It was hoped that this might lend
insight into what effect, if any, the position of the amide side group
would have on biofilm inhibitory activity. Furthermore, this study
would allow us to gauge whether an aryl ring could serve as a suit-
able surrogate for an aliphatic linker in the family of 2-aminoimi-
dazole anti-biofilm agents. Previous studies examining the role of
aliphatic linkers between the 2-AI head and tail of these com-
pounds has indicated a correlation in activity.6,12
The first scaffold prepared bore the aniline functionality para to
the 2-AI head (Scheme 1). This was achieved by employing similar
methodology described in accessing aryl halide 2-AI substrates for
palladium catalyzed Sonogoshira cross-couplings.16 Synthesis
commenced through activation of commercially available p-azido-
benzoic acid with oxalyl chloride and subsequent reaction with
diazomethane followed by quenching with concentrated HBr to
deliver the
a-bromoketone. Installation of the Boc-protected 2-
aminoimidazole motif was affected through condensation of the
a-bromoketone with Boc-guanidine to deliver azido intermediate
We envisioned a straightforward synthesis to access aryl 2-AIs
with variation in the location of structurally diverse amide append-
* Corresponding author. Tel.: +1 919 513 2960; fax: +1 919 515 5079.
E-mail address: Christian_Melander@NCSU.edu (C. Melander).
Figure 1. 2-Aminoimidazole-based anti-biofilm molecules.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.042

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C. A. Bunders et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3797–3800
Scheme 1. Synthesis of para substituted acylated 2-AI. Reactions and conditions:
(a) (i) (COCl)2, DMF, CH2Cl2, rt; (ii) CH2N2 Et2O, 0 °C; (iii) concd HBr; (b) Boc-
guanidine, DMF, rt; (c) Boc2O, Et3N, THF, rt; (d) H2 (1 atm), 10% Pd/C, THF, rt; (e)
RCOCl, Et3N, CH2Cl2, THF, À78 °C to rt; (f) (i) TFA, CH2Cl2, 0 °C; (ii) 2 M HCl in Et2O.
Scheme 2. Synthesis of ortho and meta acylated 2-AI. Reactions and conditions:
(a) CbzCl, NaHCO3, H2O, acetone, 0 °C to rt; (b) Br2, AlCl3 (cat.), 0 °C; (c) Boc-
guanidine, DMF, rt; (d) H2 (35 psi), 10% Pd/C, EtOH, rt; (e) RCOCl, Et3N, CH2Cl2, THF,
À78 °C to rt; (f) (i) TFA, CH2Cl2, 0 °C; (ii) 2 M HCl in Et2O.
1. Exhaustive Boc protection of the exocyclic amino functionality
and reduction of the azide under mild hydrogenation conditions
followed by in situ acylation of the aniline with various acid chlo-
rides delivered the Boc-protected targets. However, analogous to
our report utilizing a similar reductive acylation strategy,11 the di-
rect use of intermediate 1 in the acylation sequence afforded good
yields of mono-acylated product thereby allowing for circumven-
tion of the extra synthetic step. Acidic removal of the Boc group
with TFA followed by counterion exchange (chloride for trifluoro-
acetate) gave the target para oriented 2-AI analogues. These com-
pounds were synthesized by employing the corresponding acid
chlorides of the groups depicted in Figure 2 (Supplementary data
for complete list of synthesized compounds).
Due to the commercial unavailability of the corresponding ortho
and meta oriented azido benzoic acids, each remaining 2-AI aniline
scaffold was accessed through its readily available aminoacetophe-
none building block (Scheme 2). These starting materials were first
protected as their benzyl carbamates.17 This would allow for their
selective removal under neutral conditions later on in the synthesis
once the Boc-protected 2-AI residues had been installed into the
scaffolds. The methyl ketones were mono-brominated under the
action of Br2 and catalytic AlCl3 before again undergoing cycliza-
tion with Boc-guanidine. Removal of the Cbz groups required
elevated pressure (35 psi), yet in each case delivered the desired
2-AI aniline products. Unlike the in situ acylation reaction applied
to the synthesis of the para derivatives, scaffolds 4 and 5 could not
be directly carried onto the acylation step after reduction as each
intermediate required purification and isolation. Regardless, acyla-
tion and deprotection proceeded smoothly to yield the requisite
derivatives through implementing the various acid chlorides avail-
able listed in Figure 2 (Supplementary data for complete list of syn-
thesized compounds).
One aspect worth noting is in reference to the compatibility of
the acylation reaction with acylating reagents other than acid
chlorides. Unlike the previous reductive acylation approach we dis-
closed involving an aliphatic-based amine intermediate6 (compat-
ible with acid chlorides, anhydrides, trichloromethylketones, and
succinate esters), each aniline scaffold was unable to react with
anything other than acid chlorides even when trying to modify
the reaction conditions through the addition of acyl transfer cata-
lysts (such as DMAP) or the application of heat.
Escherichia coli bacterial infections account for significant med-
ical cost and morbidity worldwide.18 Surgical site infection, Pneu-
monia, Meningitis, and Urinary tract infections are just a few
health problems caused by E. Coli bacteria. For anti-biofilm assess-
ment, each compound in the library was initially screened at
100 lM for its ability to inhibit the formation of E. coli biofilms
using a crystal violet reporter assay.19 Compounds that demon-
strated >95% inhibition were then subjected to dose response stud-
ies to determine IC50 values. Under this criteria, compounds 6–11
Table 1
Hits identified from the E. coli biofilm inhibition assays
Compound
R@
E. coli biofilm IC50 (lM)
6
5.2 1.1
7
8
16.3 3.7
39.4 1.5
9
47.0 1.0
43.4 1.3
10
11
16.5 1.0
Figure 2. Side chains employed in the acylation reaction of the aryl 2-AI aniline
scaffolds.

C. A. Bunders et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3797–3800
3799
Table 2
showed the highest activities and, after dose response studies,
were determined to exhibit IC50’s of 5.2 M, 16.3 M, 39.4 M,
47.0 M, 43.4 M, and 16.5 M, respectively (Table 1). Follow-up
growth curve analysis of each analogue at its respective IC50
concentration indicated that only compound 6, the most active
Summary of biofilm inhibition results for compounds 6–14 against three Gram-
negative strains of bacteria
l
l
l
l
l
l
Compound
E. coli
P. aeruginosab
A. baumanniic
Biofilm inhibition
IC50 valuesa
Biofilm inhibition
IC50 valuesa
compound, was eliciting activity through
a non-microbicidal
mechanism. All other compounds showed varying degrees of
E. coli growth inhibition (Supplementary data).
6
7
8
5.2 1.1
16.3 3.7
39.4 1.5
47.0 1.0
43.4 1.3
16.5 1.0
nd
27.6 2.0b
nd
nd
nd
nd
Once we had demonstrated that the aryl 2-AI scaffold provided
a basis for the discovery of active anti-biofilm compounds, we as-
sayed the derivatives for activity in preventing the formation of
Pseudomonas aeruginosa PA14 and Acinetobacter baumannii bio-
films. Both P. aeruginosa and A. baumannii are opportunistic
Gram-negative bacteria that place a severe burden on the global
health care system. P. aeruginosa is known for driving morbidity
and mortality of cystic fibrosis patients20 while A. baumannii is
known for causing closures of intensive care wards and infecting
wounded soldiers in the Middle East.21 As with the E. coli experi-
9
10
11
12
13
14
nd
16.8 1.7b
39.4 1.4b
43.0 1.9c
nd
nd
All values are reported at
nd = not determined.
l
M concentrations.
a
Values are means of three or more experiments.
P. aeruginosa.
A. baumannii.
b
c
ments, each compound was initially screened at 100 lM and IC50
values were determined for compounds that demonstrated >95%
inhibition (Fig. 3, all data summarized in Table 2). Against P. aerugin-
osa, the three compounds 6, 12, and 13 revealed IC50’s of 27.6
16.8 M, and 39.4 M, respectively. Against A. baumannii, only com-
pound 14 displayed noteworthy activity (IC50 = 43.0 M). Follow-up
lM,
l
l
l
growth curve analysis at each compounds respective IC50 concentra-
tion, however, indicated that these compounds elicited reduction in
bacterial growth in comparison to untreated controls.
Analysis of the structure–activity relationship (SAR) profile of
the aryl 2-AI compound set indicated a few interesting features
with respect to biofilm inhibition. First, a number of these deriva-
tives demonstrated a toxic effect to the growth of planktonic E. coli,
P. aeruginosa, and A. baumannii. The exception was compound 6,
which was observed to be non-toxic to E. coli. Moreover, the posi-
tion of the difluorobenzyl appendage on the aryl 2-AI scaffold was
critical for eliciting maximum activity (Fig. 4). When this group
was para to the 2-AI head (6), the analogue exhibited an E. coli
Figure 4. Observed activity of difluorobenzyl analogues on biofilm formation in
E. coli.
used to treat established biofilm infections. Therefore, molecules
that disperse established biofilms presumably have more thera-
peutic potential than molecules that simply inhibit their formation.
Our active hits were assayed for the ability to disperse existing bio-
films from all three strains of bacteria employed in the current
study. Biofilms were first allowed to form in the absence of any
compound. Media and planktonic bacteria were then washed away
and either media alone or media containing compound was added
to the residual biofilm. After 24 h, media and planktonic bacteria
were again washed away and the remaining biofilm was stained
with crystal violet. Only compounds 6 and 7 were able to disperse
IC50 of 5.2
ortho (16) positions on the ring severely compromised E. coli activ-
ity. A modest 64% inhibition at 100 M was observed in the case of
15 while 16 essentially demonstrated no activity within the con-
centration range analyzed (<5% biofilm inhibition at 100 M). This
trend was also observed against P. aeruginosa. Compound 6 exhib-
ited an IC50 of 27.6 M, while compounds 15 and 16 both had <5%
biofilm inhibition at 100 M.
lM. Relocation of this group to either the meta (15), or
l
l
l
l
established E. coli biofilms (51% and 67%, respectively at 50
while compound 14 was able to disperse 40% of A. baumannii bio-
films at 100 M. None of the compounds were observed to possess
lM),
From a medical standpoint, molecules that inhibit the formation
of a bacterial biofilm could be used as a prophylactic measure;
however, molecules that disperse pre-formed biofilms could be
l
the ability to disperse pre-formed P. aeruginosa biofilms.
Finally, given the success of previous ventures into studying the
anti-biofilm activities of 2-aminoimidazole/triazole conju-
gates,10,12 we elected to functionalize intermediate 1 with a tria-
zole isostere to determine if potency could be improved within
the aryl 2-AI framework (Scheme 3). Facile preparation of 17
through treatment of 1 with LiHMDS and one equivalent of Boc
anhydride22 followed by subjection to standard conditions for the
copper mediated [3+2] cycloaddition reaction employing 1-ethy-
nyl-3,5-difluorobenzene as the alkyne quickly afforded target 18
after deprotection. Unfortunately, preliminary inhibition screens
Scheme 3. Synthesis of triazole isostere 18. Reactions and conditions; (a) LiHMDS,
Boc2O, THF, 0 °C; (b) CuSO4Á5H2O, sodium ascorbate, 1-ethynyl-3,5-difluoroben-
zene, CH2Cl2, EtOH, H2O, rt; (c) (i) TFA, CH2Cl2, 0 °C; (ii) 2 M HCl in Et2O.
Figure 3. Aryl 2-aminoimidazoles found to inhibit biofilm formation in either P.
aeruginosa (6, 12, and 13) or A. baumannii (14).

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C. A. Bunders et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3797–3800
at 100
l
M against all strains revealed very weak activity against all
growth curves) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2010.04.042.
strains (35% inhibition against E. coli, <5% biofilm inhibition against
P. aeruginosa, 21% inhibition against A. baumannii). Therefore, it ap-
pears that within the aryl 2-AI scaffold, substitution of a triazole is
detrimental to anti-biofilm activity when 18 is compared to the
References and notes
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most potent analogue 6 (E. coli IC50 = 5.2 lM).
In conclusion, we have developed a synthetic route to access
ortho, meta, and para-substituted aryl amide 2-aminoimidazoles.
Using this approach, a set of 30 unique small molecules with vary-
ing substitution patterns were screened as potential inhibitors and
dispersal agents against biofilms formed by the medically relevant
Gram-negative bacteria E. coli, P. aeruginosa, and A. baumannii
(Table 2). Compound 6 was identified as a low micromolar E. coli
biofilm inhibitor and was also found to be non-toxic to the bacte-
rium despite other analogues in the compound set demonstrating
poorer activity and greater toxicity towards bacterial growth in all
strains. Overall, the data suggests the feasibility of aryl derived 2-
aminoimidazoles for development as effective leads for the discov-
ery and further optimization of unique chemical probes for deter-
mining the importance of E. coli biofilm formation in a variety of
E. coli driven infections.
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Acknowledgments
Financial support for this work is gratefully acknowledged from
Jimmy V. Cancer Therapeutics Training Program (predoctoral fel-
lowship to J.J.R.), Agile Sciences, and the University of North Caro-
lina Competiveness Research Fund.
16. Richards, J. J.; Melander, C. J. Org. Chem. 2008, 73, 5191.
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Supplementary data
Supplementary data (experimental procedures, characteriza-
tion data, inhibition and dispersion assay protocols, and bacterial