A facile synthesis of 1,5-disubstituted-2-aminoimidazoles: Antibiotic activity of a first generation library

Article abstract of DOI:10.1016/j.bmcl.2011.05.123

An efficient synthetic route to 1,5-disubstituted 2-aminoimidazoles from readily available amino acids and aldehydes has been developed. A library of simple analogues was synthesized and several compounds were shown to exhibit notable antibiotic activity

Full text of DOI:10.1016/j.bmcl.2011.05.123

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4516–4519
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A facile synthesis of 1,5-disubstituted-2-aminoimidazoles: Antibiotic activity
of a first generation library
⇑
Tyler L. Harris, Roberta J. Worthington, Christian Melander
Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthetic route to 1,5-disubstituted 2-aminoimidazoles from readily available amino acids
and aldehydes has been developed. A library of simple analogues was synthesized and several com-
pounds were shown to exhibit notable antibiotic activity against a variety of bacterial strains including
multi-drug resistant isolates.
Received 25 April 2011
Revised 28 May 2011
Accepted 31 May 2011
Available online 15 June 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
2-Aminoimidazole
Amino aldehyde
Biofilm
Antibiotic
The number of antibiotic-resistant infections encountered glob-
ally continues to rise and the current supply of anti-bacterial drugs
is becoming increasingly ineffective. This has lead to a resurgence
in research into anti-infective strategies.1 Bacterial strains resistant
to many, or even all, currently available antibiotics are increasingly
common, while at the same time fewer new antibiotic classes are
being developed. Resistance is mediated by many different mech-
anisms including inactivation by enzymes, bypass of antibiotic tar-
gets and efflux of antibiotics from the bacterial cell.2 In addition to
the search for novel antibiotics, other approaches to the control of
bacterial infections will also prove valuable. One such approach is
the control and maintenance of bacteria within the biofilm state.3
Biofilms are microbially derived sessile communities, character-
ized by cells that are irreversibly attached to a substratum or inter-
face or to each other, and are embedded in a matrix of extracellular
polymeric substances that they have produced. They exhibit an al-
tered phenotype with respect to growth rate and gene transcrip-
tion compared to their planktonic counterparts.4 Biofilms are
increasingly recognized as being significant in human disease,
accounting for over 80% of microbial infections in the body.5 Dis-
eases associated with bacterial biofilms include, lung infections
of cystic fibrosis (CF), colitis, urethritis, conjunctivitis, otitis, endo-
carditis and periodontitis.4,5 Bacteria within biofilms are inherently
insensitive to antiseptics and host immune responses, and residing
within the biofilm state confers resistance to conventional antibi-
otics upwards of 1000 times that of planktonic bacteria.3 Biofilm
infections are therefore rarely resolved by host defense mecha-
nisms and while antibiotic therapy generally reverses the symp-
toms caused by planktonic cells released from the biofilm, it fails
to kill bacteria residing within the biofilm. For this reason biofilm
infections typically show recurring symptoms, even after cycles
of antibiotic therapy.6 One mechanism of biofilm resistance to anti-
biotics is the inability of the antibiotic to penetrate the full depth of
the biofilm, as the polymeric matrix of a biofilm is known to retard
the diffusion of antibiotics. A second hypothesis is the fact that
some cells within a biofilm experience nutrient limitation and
therefore exist in a slow-growing or starved state making them
less susceptible to many antimicrobial agents. This heterogeneity
of biofilms is an important survival strategy as at least some of
the cells are almost certain to survive any metabolically directed
attack.6
We have developed several diverse libraries of compounds with
anti-biofilm activity against a variety of bacterial strains, both
Gram-positive and Gram-negative, in addition to compounds with
fungal anti-biofilm activity.7–25 These compounds are based on the
structures of natural products with demonstrated anti-biofilm
activity. The majority are based on oroidin 1 and bromoageliferin
2 (Fig. 1), secondary metabolites of the marine sponge family Agel-
asidae, which were initially reported to possess anti-biofouling
activity against the Gram-negative marine
a-proteobacterium
Rhodospirillum salexigens,26 and contain a key 2-aminoimidazole
moiety. Many of these anti-biofilm compounds inhibit the forma-
tion of a bacterial biofilm without exhibiting microbicidal activity
towards planktonic bacteria. There is significant potential for mol-
ecules that possess the ability to inhibit and/or disperse bacterial
biofilms via a non-toxic mechanism in infectious disease therapy,
⇑
Corresponding author. Tel.: +1 919 515 2960; fax: +1 919 515 5079.
E-mail address: Christian_melander@ncsu.edu (C. Melander).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.123

T. L. Harris et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4516–4519
4517
To this end, we envisioned that the desired 1,5-disubstituted-2-
aminoimidazoles derivatives could be readily prepared from the
condensation of N-substituted
In turn, these N-substituted
a
-aminoaldehydes with cyanamide.
a
-aminoaldehydes could be prepared
from a reductive amination reaction between commercially avail-
able aldehydes and readily available amino acids, followed by
reduction of the carboxylic acid to the aldehyde (Fig. 2).
The first step of our route to 1,5-substituted 2-aminoimidazoles
involved the formation of N-substituted
a-amino acids. We tested
several conditions for the reductive amination of
L
-phenylalanine
with benzaldehyde and determined the best method to be a two-
step procedure using sodium borohydride as the reducing agent,
which, after Boc-protection, afforded the desired N-substituted
phenylalanine 5a in 70% yield. We then turned our attention to
the conversion of the
a-amino acid to its corresponding a-amino
aldehyde. Several methods were investigated including thiol ester
formation followed by reduction with triethylsilane33 and sodium
amalgam (Akabori) reduction15 of the methyl ester of compound
5a. It was decided that the most efficient route, and that which al-
lowed for the greatest introduction of diversity, was via the forma-
tion of the N-methoxy-N-methylamide (Weinreb amide) and
subsequent reduction.33 Conversion of the Boc protected amino
acid 5a to the Weinreb amide 6a was achieved using (ben-
zotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluoro-
phosphate (BOP) as the coupling reagent. Reduction of the
Weinreb amide 10a to the aldehyde was carried out using diisobu-
tylaluminum hydride (DIBAL-H) followed by in situ Boc-deprotec-
tion with HCl, and cyclization with cyanamide to afford the 1,5-
disubstituted-2-aminoimidazole 15a (Scheme 1). It is worth noting
Figure 1. Natural products possessing anti-biofilm activity, bromoageliferin 1 and
oroidin 2, lead 4,5-disubstituted-2-aminoimidazole 3 and the general structure of
1,5-disubstituted-2-aminoimidazoles 4.
as if a molecule does not directly kill bacteria there is a reduced
likelihood of the bacteria acquiring resistance to the molecule.
Additionally we have shown that these anti-biofilm compounds
can be used in combination with traditional antibiotics to re-sensi-
tize bacteria to the antibiotic.25
We recently reported the synthesis of a pilot library of 4,5-
disubstituted-2-aminoimidazoles via a nitroenolate approach.24
Many of these compounds displayed promising biological activity,
with the lead compound, 3, shown in Figure 1. Methods to access
2-aminoimidazoles with other pre-defined substitution patterns
will allow examination of the effect of specific substitution pat-
terns on the antibiotic and antibacterial activity of 2-aminoimidazole
derivatives. We have therefore developed a facile route to 1,5-
disubstituted-2-aminoimidazoles 4, in which a substituent is
placed at one of the endocyclic nitrogen atoms (Fig. 1).
that the use of
allowing for the future preparation of 1,4,5-substituted 2-amino-
imidazoles via -amino ketone formation from Grignard addition
to these -amino Weinreb amides.
a-amino Weinreb amides also has the advantage of
a
a
Following these test reactions, an initial 20-member library was
generated, possessing a variety of substituents including, straight
chain and branched alkyl groups, phenyl and alkyl phenyl groups
and a more polar carbamate group (Scheme 1). Briefly, imine for-
mation between equimolar amounts of the commercially available
amino acids and aldehydes in methanol in the presence of lithium
hydroxide was followed by reduction with 2 equiv of sodium boro-
hydride. The crude secondary amines were Boc protected, using
Boc anhydride in acetonitrile with tetramethylammonium hydrox-
ide as base, to furnish the N-substituted Boc amino acids 5–9a–d.
Amide coupling of these protected amino acids with N,O-dim-
ethylhydroxylamine, using BOP as the coupling reagent in CH2Cl2
in the presence of triethylamine, afforded the desired Weinreb
amides 10–14a–d. The Weinreb amides were then reduced with
DIBAL-H in THF and after quenching of the reaction, the crude
aldehydes were extracted with diethyl ether then treated with
diethyl ether/aqueous HCl or TFA/CH2Cl2 to remove the Boc group.
Following solvent removal the crude amino aldehydes were dis-
solved in a 1:1 mixture of ethanol/water, the pH adjusted to 4.3,
and allowed to react with cyanamide at 95 °C. Following purifica-
tion the desired 1,5-disubstituted-2-aminoimidazoles 15–19a–d
were converted to their HCl salts for biological testing.
Reported methods for the preparation of 1,5-disubstituted-
2-aminoimidazoles do not allow for the introduction of a diverse
array of C-5 substituents. Guanylation of 1-amidino-3-trityl-thio-
ureas followed by reaction with
a-bromo ketones allows the
preparation of 2-aminoimidazoles with an acyl group at the
5-position.27 The preparation of 5-aryl 1,5-disubstituted-2-amino-
imidazoles has been reported via the formation of imidazo
(1,2-a)pyrimidinium salts from substituted 2-aminopyrimidines
and
a-bromocarbonyl compounds, followed by opening of the
pyrimidine ring with hydrazine.28 A library of N-1-substituted
4(5)-phenyl-2-aminoimidazoles was recently reported to exhibit
biofilm inhibitory activity against Salmonella typhimurium and
Pseudomonas aeruginosa.29 In order to rapidly assemble a library
for antibiotic and anti-biofilm screening we desired a method that
utilized readily available building blocks and allowed for the intro-
duction of diversity at both positions. Such a method would be of
use in many areas of medicinal chemistry as, in addition to pos-
sessing anti-bacterial activity, substituted 2-aminoimidazoles have
been identified in many pharmacologically active substances
including compounds with anti-cancer activity,30,31 anti-fungal
compounds,31 and nitric oxide synthase inhibitors.32
Following the discovery that our 4,5-dialkylated 2-aminoimid-
azoles24 exhibited significant microbicidal activity, we opted to ini-
tially investigate the antibiotic activity of this 1,5-disubstituted-2-
Figure 2. Retrosynthetic analysis of 1,5-disubstituted-2-aminoimidazoles.

4518
T. L. Harris et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4516–4519
A
B
Bacterial strain
18e
1
18f
2
E. coli
A. baumannii
MDRAB
8
64
8
>128
>128
0.5
1
K. pneumoniae
S. epidermidis
MRSE
32
0.25
1
MSSA
1
2
MRSA
1
2
Figure 3. (A) Structures of compounds 18e and 18f. (B) MIC values (
lg/mL) for
compounds 18e and 18f.
Scheme 1. Synthesis and structures of 1,5-disubstituted 2-aminoimidazoles 15–
19a–d. Reagents and conditions: (a) (i) LiOHÁH2O, CH3OH, rt, 18 h, (ii) NaBH4, rt, 1 h;
(b) Boc2O, TMAH, CH3CN, rt, 18 h; (c) HN(OCH3)CH3, Et3N, BOP, DCM, rt 18 h; (d)
DIBAL-H/hexanes, THF, À78 °C to rt, 1 h; (e) 4 M HCl(aq)/Et2O or (1:10) rt, 2 h; (f)
EtOH/H2O, pH 4.3, H2NCN, 95 °C 2.5 h.
MDRAB, K. pneumoniae, S. epidermidis, MRSE, MSSA and MRSA,
respectively. This compound demonstrated greater antibiotic
activity against the Gram-positive strains tested than against the
Gram-negative strains, and has particularly notable activity against
the opportunistic bacterium S. epidermidis. For compounds 18c and
18d, it can be seen that increasing the chain length of the N-1 sub-
stituent leads to an increase in activity from 8- or 16-fold for the
Gram-negative strains up to 64-fold for S. epidermidis. We there-
fore decided to synthesize two more compounds with the same
C-5 substituent and increasing alkyl chain lengths of the N-1 sub-
stituent to see whether this trend would be continued. These com-
pounds (18e and 18f, Fig. 3) were synthesized in the same manner
as the initial library and were tested for antibiotic activity against
the same eight bacterial strains (Fig. 3). Unfortunately, a significant
increase in antibiotic activity from compound 18d was not ob-
served, though these compounds still possess considerable antibi-
otic activity against S. epidermidis, MRSE, MSSA, MRSA and E. coli.
We then screened the 1,5-disubstituted 2-aminoimidazole li-
brary for the ability to inhibit bacterial biofilm formation. For this
investigation five biofilm forming bacterial strains were selected,
aminoimidazole library. This was carried out by measurement of
the minimum inhibitory concentration (MIC) of each derivative
against a variety of representative pathogenic bacterial strains,
both Gram-negative and Gram-positive, using a standard broth
microdilution protocol.34 We tested activity against: Escherichia
coli, Acinetobacter baumannii, multi-drug resistant A. baumannii
(MDRAB), Staphylococcus epidermidis, methicillin resistant S. epide-
rmidis (MRSE), methicillin susceptible Staphylococcus aureus
(MSSA), methicillin resistant S. aureus (MRSA) and a carbapenem
resistant strain of Klebsiella pneumoniae which produces the re-
cently reported New Delhi metallo-b-lactamase (NDM-1).35 The re-
sults of this screen are outlined in Table 1. It can be seen that
compound 18d is the most active antibiotic, with MIC values of
2, 8, 8, 32, 0.125, 2, 2 and 2 lg/mL against E. coli, A. baumannii,
Table 1
MIC values (lg/mL) for compounds 15–19a–d against several bacterial strains
E. coli
A. baumannii
MDRAB
K. pneumoniae
S. epidermidis
MRSE
MSSA
MRSA
15a
15b
15c
15d
16a
16b
16c
16d
17a
17b
17c
17d
18a
18b
18c
18d
19a
19b
19c
19d
128
32
64
128
8
32
128
64
128
16
64
2
32
8
32
2
64
64
32
8
>128
32
64
128
128
32
64
16
128
32
64
16
16
16
64
8
64
32
128
64
>128
32
64
128
16
32
64
64
128
32
64
16
16
32
64
8
>128
128
128
>128
128
128
>128
128
128
128
128
64
64
64
64
32
128
128
>128
128
128
16
32
128
2
128
16
64
128
8
32
128
64
128
32
128
8
16
16
32
2
32
16
64
16
>128
32
64
128
8
32
64
64
128
64
>128
16
16
8
32
2
32
16
32
16
>128
32
128
128
4
32
64
64
128
32
128
8
32
8
32
2
32
16
128
16
8
16
16
16
4
32
2
16
4
8
0.125
32
16
16
2
64
32
128
64

T. L. Harris et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4516–4519
4519
Table 2
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94 0.1
97 0.5
96 0.3
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92
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1
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1
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1
96 0.3
98 0.6
Not active
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97 0.1
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73
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2
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2
83 10
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via a microbicidal mechanism. IC50 values for biofilm inhibition
activity for this library of compounds were therefore not
determined.
In conclusion, we have developed a facile route to 1,5-disubsti-
tuted-2-aminoimidazoles that allows rapid assembly of a diverse
array of 2-aminoimidazole derivatives from readily available start-
ing materials. The lead compounds identified in this study show
significant antibiotic activity against a wide variety of bacterial
strains. Several of the simple analogues developed in this study
demonstrated the ability to inhibit biofilm formation, albeit
through a microbicidal mechanism. This chemistry can now be
used to access 1,5-substituted derivatives of our lead anti-biofilm
compounds, in which the 5-position substituent has been finely
tuned through numerous cycles of analogue synthesis and testing.
These compounds are currently being developed in our lab and will
be tested to determine whether this substitution pattern leads to
enhanced anti-biofilm activity.
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The authors would like to thank the North Carolina Biotechnol-
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Supplementary data
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Supplementary data (experimental protocols for the synthesis
and biological assays, and characterization of new compounds)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2011.05.123.
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