A nitroenolate approach to the synthesis of 4,5-disubstituted-2- aminoimidazoles. Pilot library assembly and screening for antibiotic and antibiofilm activity

Article abstract of DOI:10.1039/c001479f

A library of 4,5-disubstituted-2-aminoimidazoles was synthesized using a nitroenolate route and then screened for antibiofilm and antimicrobial activity. These compounds displayed notable biofilm dispersal and planktonic microbicidal activity against various Gram-positive and Gram-negative bacteria.

Full text of DOI:10.1039/c001479f

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A nitroenolate approach to the synthesis of 4,5-disubstituted-2-
aminoimidazoles. Pilot library assembly and screening for antibiotic and
antibiofilm activity†
Zhaoming Su, Steven A. Rogers, W. Steve McCall, Alicia C. Smith, Sindhu Ravishankar, Trey Mullikin and
Christian Melander*
Received 22nd January 2010, Accepted 30th March 2010
First published as an Advance Article on the web 29th April 2010
DOI: 10.1039/c001479f
A library of 4,5-disubstituted-2-aminoimidazoles was synthesized using a nitroenolate route and then
screened for antibiofilm and antimicrobial activity. These compounds displayed notable biofilm
dispersal and planktonic microbicidal activity against various Gram-positive and Gram-negative
bacteria.
As a therapeutic strategy, molecules that inhibit/disperse
biofilms through non-microbicidal mechanisms will most likely
Introduction
The global rise of antibiotic resistant pathogens1 has driven a
revitalization of the field of anti-infective research. This research
emphasis has been multi-faceted, including the obvious search for
novel antibiotics. However, as bacteria have shown the ability to
evolve resistance to any antibiotics placed in the human healthcare
system,2 additional approaches to controlling bacterial infections
are being explored. These approaches include small molecules
that target: (1) quorum sensing (QS),3 (2) plasmids that harbor
antibiotic resistance genes,4 and (3) the formation/maintenance
of bacteria within the biofilm state.5
In an effort to address these needs in biomedical science, our
research group has spent a number of years developing molecules
that inhibit/disperse bacterial biofilms.6 Bacterial biofilms, defined
as surface-attached communities of microorganisms encased in a
protective extracellular matrix of biomolecules,7 are specfically
targeted because within a biofilm state, bacteria are upwards
of 1000-fold more resistant to conventional antibiotics and are
inherently insensitive to the host immune response in comparison
to their free-floating (planktonic) counterparts.8 The biofilm
mode of existence is the preferred lifestyle for bacteria as ap-
proximately 80% of the world’s microbial mass is in a biofilm
at any given time and the NIH has estimated that ca. 75%
of bacterial infections are driven by a biofilm.9 To this end,
we have developed a number of 2-aminoimidazole derivatives
based upon the natural products bromoageliferin and oroidin
that inhibit and disperse biofilms from both Gram-positive and
Gram-negative bacteria.10–18 We have also developed a class of
2-aminobenzimidazoles that inhibit and disperse gram-positive
biofilms via a Zn(II)-dependent mechanism.19 Both classes of
molecules control biofilm development and maintenance via a
non-microbicidal mechanism.
serve as adjuvants to conventional antibiotics.20 The anti-biofilm
molecules will keep the bacteria within their more sensitive plank-
tonic state, while a combination of conventional antibiotics and the
host immune response will serve to eliminate the bacterial threat.
In this regard, we have developed new antibiotics based upon a 2-
aminobenzimidazole/amide-conjugate framework that are active
against MRSA and multi-drug resistant Acinetobacter baumannii
and are not affected by current mechanisms of antibiotic resistance
(multidrug efflux pumps, etc.).21
As the 2-aminoimidazole heterocycle is a key pharmacophore
(isolated or fused to an aromatic ring) in all the aforemen-
tioned molecules developed by our group, methods to access
2-aminoimidazoles with pre-defined substitution patterns are nec-
essary to systematically evaluate the impact these substitution pat-
terns have upon both anti-biofilm and antibiotic activity. Recently,
Looper and co-workers have developed an elegant solution to the
synthesis of substituted 2-aminoimidazoles via an intramolecular
La(III)-catalyzed addition/hydroamination sequence.22 Parallel
to this approach, we have developed a nitroenolate approach
to access 4,5-disubstituted-2-aminoimidazoles. Herein, we detail
the use of the nitroenolate reaction to access a-nitroketones,
that following reduction and condensation with cyanamide
yield 4,5-disubstituted-2-aminoimidazoles. Using this methodol-
ogy, we have assembled a pilot library of 4,5-disubstituted-2-
aminoimidazoles that have been subsequently screened for both
anti-biofilm and antibiotic activity against E. coli, multi-drug
resistant Acinetobacter baumannii (MDRAB), methicillin resistant
Staphylococcus aureus (MRSA), methicillin sensitive Staphylo-
coccus aureus (MSSA), Staphylococcus epidermidis, vancomycin
resistant Enterococcus facium (VRE), Vibrio cholerae, Vibrio
vulnificus, Listonella anguillarum and Rhodospirillum salexigens.
Results and discussion
Department of Chemistry, North Carolina State University, Raleigh, USA.
E-mail: Christian_Melander@ncsu.edu; Tel: 1-919-513-2960
Synthesis and pilot library assembly
† Electronic supplementary information (ESI) available: 1H NMR and 13
C
NMR spectra for all new compounds, synthetic protocols and charac-
terization for all a-nitro ketones and 4,5-disubstituted-2-aminoimidazoles
not presented in the experimental section, and biofilm dispersion and
hemolysis graphs. See DOI: 10.1039/c001479f
Fundamentally, our approach to pilot library synthesis relies
upon the use of readily available building blocks to rapidly
access 2-aminoimidazoles with pre-defined substitution patterns
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Table 2 Effects of reaction time and temperature on product yield
for biological evaluation. Therefore, we were interested in devel-
oping the synthesis of 4,5-disubstituted-2-aminoimidazoles via a
nitroenolate approach (Scheme 1) because the starting building
blocks, activated carboxylic acids and alkyl nitro derivatives, are
either commercially available or available in one step from either
carboxylic acids or alkyl halides, respectively. Once assembled, the
target a-nitro ketones could simply be reduced and then condensed
with cyanamide to yield 4,5-disubstituted-2-aminoimidazoles.
Entry
T/◦C
Time/h
Yield [%]
1
2
3
4
5
6
7
8
9
RT
40
45
50
55
66
55
55
55
12
12
12
12
12
12
6
20
27
47
46
50
20
43
24
—
24
48
Scheme 1 4,5-Disubstituted-2-aminoimidazole synthetic route.
Once we had the a-nitro ketone, we verified that the re-
duction/condensation sequence would deliver the target 2-
aminoimidazole. We reduced the nitro group to the corresponding
amine using H2, 5 eq. HCl and 5% Pd/C in ethanol. The resulting
crude reaction was then simply filtered over Celite, and the ethanol
and excess HCl removed in vacuo to deliver the crude a-amino
ketone as its HCl salt. The salt was then redissolved in ethanol,
5 eq. cyanamide was then added, the pH adjusted to 4.5 and
the resulting solution was heated to 95 ◦C for two hours. After
purification, the target 4,5-disubstituted-2-aminoimidazole was
isolated in 55% yield.
This synthetic approach23 was first optimized by using
4-phenylbutyric acid and nitromethane as the building blocks.
We sought to determine the optimal combination of enolization
base, carboxylate activation and solvent that would deliver the
highest yield of the targeted a-nitro ketone (Table 1). We screened
THF and CH2Cl2 as potential solvents, DBU and t-BuO-K+ as
enolization bases and CDI and ethyl chloroformate as carboxylate
activators. From this initial screen, we determined that 2.5
equivalents of DBU as the enolization base, THF as the solvent
and CDI as the activating agent generated the target a-nitro ketone
in the highest yield (76%) when the reaction was run at 40 ◦C
overnight.
Once we had determined the optimized conditions of the
nitroenolate approach, we sought to apply these conditions to the
synthesis of 4,5-disubstituted-2-aminoimidazoles. We first applied
the aforementioned reaction conditions to the coupling of 4-
phenylbutyric acid and 1-nitropropane. Under these conditions,
we were able access the target a-nitro ketone in 27% yield. Given
this low yield, we sought to further optimize the reaction by study-
ing the optimal reaction temperature and reaction time (Table 2).
The◦temperature was first varied from room temperature to reflux
(66 C, ◦entries 1–6) and we determined the optimal temperature
was 55 C. Heating the reaction to higher temperatures led to a
decreased reaction yield, most likely from decomposition. We then
sought to determine if reaction time would affect reaction yield
by running the reaction for either 6, 12, 24, or 48 h. As predicted
by our temperature studies, the longer the reaction is heated, the
lower the yield of the target a-nitro ketone. At 6 h, the reaction
yield was slightly lower. Under these optimized conditions (2.5 eq.
DBU, 2 eq. CDI, THF, 55 ◦C, 12 h), we were able to achieve a 50%
yield of the target a-nitro ketone.
Using this approach, we assembled a 15-member pilot library
where we varied both the length of the alkyl chain and the number
of methylene units between the 2-aminoimidazole and phenyl
group (Fig. 1).
Fig. 1 Composition of pilot library.
Biological screening
We first assessed the ability of each member of the pilot library
to inhibit either E. coli or MDRAB biofilm formation at 100 mM
using a crystal violet reporter assay.24 The results of this initial
screen are summarized in Table 3. From this screen, 12 compounds
inhibited E. coli biofilm formation >95%, while eight compounds
inhibited MDRAB biofilm formation >95%.
Table 1 Effects of reaction solvent and base on product yield
When we subjected each compound that showed >80% activity
to a dose–response study to determine the IC50 value for biofilm
inhibition, we noted that biofilm inhibition dropped precipitously
over a narrow concentration range. This is typically indicative
of biofilm inhibition via a traditional microbicidal mechanism
instead of a mechanism that modulates biofilm formation through
non-microbicidal mechanisms. The microbicidal activity of
Solvent
Enolization base
Activating base
Yield [%]
THF
THF
THF
CH2Cl2
CH2Cl2
DBU
Ethyl chloroformate
—
76
—
—
46
DBU
CDI
CDI
CDI
CDI
t-BuO-K+
t-BuO-K+
DBU
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Table 3 Test compound inhibition of biofilm formation at 100 mM
MDRAB, MRSA, VRE, S. Epidermidis and MSSA. In general,
we noted that activity correlated with the length of the alkyl chain,
with 5 carbons being most active. We also observed that the linker
between the 2-AI and phenyl ring also modulated activity, as the
5-carbon spacer was most active.
Once we noted that increasing chain length of both the
alkyl chain and the linker correlated with increased activity, we
synthesized five second generation compounds (Fig. 2) where
we further varied these two parameters to maximize activity.
The MIC values of the second generation compounds against
each of the aforementioned bacterial strains are outlined in
Table 5. Unfortunately, none of the second generation compounds
displayed augmented activity in comparison to lead compound 15;
however, compound 19 had similar activity.
Compound
E. coli
MDRAB
1
<5
>99
>99
>99
>99
>99
86.4 7.9
97.9 0.6
98.2 0.03
96.6 2.9
96.3 0.08
>99
95.8 1.5
96.9 1.1
83.0 0.8
<5
>99
>99
>99
>99
>99
72.9 0.4
96.5 2.6
>99
94.3 2.2
>99
2
3
4
5
6
7
8
9
10
11
12
13
14
15
94.5 0.2
87.8 0.3
85.4 0.7
80.3 8.1
representative compound 15 was verified by conducting a growth
curve analysis (A600) against E. coli at its IC50 concentration
(13 mM). At this concentration, we observed a 97% reduction
in bacterial growth.
Once we determined that this specific 4,5-disubstitution pattern
imparted microbicidal activity onto the 2-AI framework, we
quantified this activity by measuring the MIC of each derivative
against a variety of representative pathogenic bacterial strains
using the microdilution protocol.25 We chose E. coli, MDRAB,
MRSA, MSSA, S. epidermidis and VRE for initial evaluation.
The results of this study are outlined in Table 4. From this screen,
compound 15 was determined to be our lead compound and had
MIC values (mg mL-1) of 2, 2, 1, 0.5, 0.25, and 1 against E. coli,
Fig. 2 Composition of second generation compounds.
Once we had determined the effect that these 4,5-disubstituted-
2-aminoimidazoles had on representative terrestrial pathogens, we
determined their activity against a representative set of marine
Table 4 Microdilution MIC of pilot library of compounds for representative pathogenic bacterial strains
Compound
E. colia
MDRABa
MRSAa
MSSAa
S. epidermidisa
VREa
1
128
32
16
32
8
32
32
8
32
8
8
16
8
4
128
32
32
64
8
16
32
16
32
8
8
32
8
4
2
64
16
8
16
4
8
32
8
8
2
4
8
32
8
4
16
4
2
16
4
8
2
2
8
2
4
4
1
0.25
4
2
2
0.5
1
2
0.5
0.25
0.25
256
16
8
16
2
16
32
4
4
1
8
8
2
3
4
5
6
7
8
9
10
11
12
13
14
15
4
4
2
1
4
2
1
4
2
0.5
2
a MIC values are in mg mL-1.
Table 5 Microdilution MIC values of second generation compounds
Compound
E. colia
MDRABa
MRSAa
MSSAa
S. epidermidisa
VREa
16
17
18
19
20
8
32
8
2
8
8
1
1
1
1
1
2
—
2
1
2
0.5
—
0.5
0.5
0.5
1
1
1
0.5
1
16
16
4
8
a MIC values are in mg mL-1.
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Table 6 Marine bacteria microdilution MIC data for both pilot and
Table 7 Marine bacteria biofilm dispersion data for lead compounds
second generation libraries
Compound
15a
19a
Compound V. choleraea V. vulnificusa L. anguillaruma R. salexigensa
E. coli
MDRAB
MRSA
MSSA
S. epidermidis
VRE
V. cholerae
V. vulnificus
L. anguillarum
R. salexigens
12.0 1.6
7.1 1.3
42.5 6.1
11.1 0.1
74.9 9.8
26.1 0.7
17.7 0.3
14.1 0.2
14.7 1.8
3.20 1.3
13.8 1.5
17.6 3.2
1
>50
53.2
27.9
58.8
15.4
26.6
55.9
29.4
30.8
16.1
27.9
29.4
15.4
8.05
4.20
8.40
69.9
18.2
2.19
4.20
50.4
26.6
27.9
58.8
15.4
26.6
13.9
14.7
30.8
8.05
13.9
29.4
3.85
4.02
2.10
8.40
69.9
18.2
2.19
4.20
50.4
26.6
13.9
58.8
7.70
13.9
13.9
14.7
30.8
8.05
13.9
14.7
7.70
4.02
2.10
4.20
69.9
18.2
2.19
4.20
50.4
13.3
13.9
29.4
3.85
13.3
13.9
7.35
7.70
2.01
7.00
14.7
3.85
8.05
2.10
2.10
69.9
2.27
1.09
2.10
2
33.9 3.9
19.9 3.3
24.7 5.8
17.3 2.3
7.12 0.3
18.1 2.3
14.7 1.7
6.60 1.1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
a EC50 values are in mM.
a MIC values are in mg mL-1.
Scheme 2 Structure of 21 and the synthesis of 22 and 23.
bacteria. This was driven by a number of factors, including our
interest in the development of antibiofoulants26 and the prevalence
of vibrio infections in human populations.27 Vibrio cholerae, Vibrio
vulnificus, Listonella anguillarum and Rhodospirillum salexigens
were chosen for evaluation. Given the microbicidal activity that
was observed against terrestrial bacterial strains, we determined
the MIC values of each compound against all the aforementioned
strains. This data is summarized in Table 6. Paralleling our
previous results, compounds 15 and 19 were the lead compounds.
Compound 15 had MIC values of 4.2, 2.1, 2.1 and 2.1 mg mL-1
against V. cholerae, V. vulnificus, L. anguillarum and R. salexigens,
respectively, while compound 19 had MIC values of 2.2 mg mL-1
against V. cholerae, V. vulnificus and L. anguillarum, and 1.1 mg
mL-1 against R. salexigens.
We also determined the ability of 15 and 19 to disperse pre-
formed biofilms against all the aforementioned bacterial strains
(both marine and terrestrial). To quantify this effect, we deter-
mined the EC50 value of each compound against each bacterial
strain, where EC50 is defined as the concentration of compound
that elicits 50% dispersion of a pre-formed biofilm. This data is
summarized in Table 7. Both compounds are able to disperse pre-
formed biofilms at low micromolar concentrations, albeit these
concentrations are microbicidal to planktonic bacteria.
To examine the necessity of the 4,5-disubstitution pattern for an-
timicrobial activity of the 2-aminoimidazole scaffold, two mono-
substituted 2-aminoimidazole analogues of compound 19 were
synthesized and screened alongside the parent 2-aminoimidazole
for antibiotic activity (Scheme 2). Heptanoic acid and phenyl-
hexanoic acid were treated with oxalyl chloride with catalytic
dimethyl formamide in dichloromethane to produce the respective
acid chlorides which were then reacted with diazomethane and
quenched with hydrobromic acid to make the respective a-
bromo ketones. These a-bromo ketones were separately cyclized
with boc-guanidine, providing a boc-protected 2-aminoimidazole
scaffold that was subsequently deprotected upon treatment with
trifluoroacetic acid in dichloromethane to yield 22 and 23.
2-Aminoimidazole 21 was found to have no antibiotic activity
at the highest concentration tested (36 mg mL-1) against every
bacterial strain. Compound 22 demonstrated MIC values of
greater than 41 mg mL-1 against all of the test strains except for S.
epidermidis in which a remarkable MIC value of 0.04 mg mL-1 was
found; making 22 the most potent antibiotic in this study. Lastly,
compound 23 was also found to contain no notable antibiotic
activity against any of the test strains except for S. epidermidis, for
which an MIC value of 0.05 mg mL-1 was found. This demonstrates
the necessity for substitution on the 2-AI scaffold to elicit the
antibiotic response with broad spectrum activity being attributed
to 4, 5-disubstitution.
Once we had evaluated both the anti-biofilm and antibacterial
properties of each 2-AI derivative, we assessed the hemolytic
potential of compounds 15, 19 and 1.28 Compounds 15 and 19
are the leads from our first generation and second generation
compounds, respectively, while compound 1 was chosen for
comparison purposes to evaluate the activity of a bacterial inactive
2-AI. Hemolysis was measured using difibrinated sheep’s blood.
Hemolytic potential was quantified by determining the HD50
of compounds 15, 19 and 1, where HD50 is defined as the
concentration that elicits 50% hemolysis. From this assay, we
determined that the HD50 of compounds 15, 19 and 1 were 66,
61 and >400 mM. This corresponds to 22 and 21 mg mL-1 for
compounds 15 and 19.
Finally, given that active compounds are characterized by
a polar head group and a lipophilic tail, we probed whether
compounds 15 and 19 were eliciting their activity via a pore-
forming mechanism. As a control, we also included the inac-
tive compound 1. This was probed via a dye dispersion assay
from synthetic vesicles.29 Two types of vesicles were prepared,
one containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
(POPC) and the other containing 1-palmitoyl-2-oleoyl-sn-glycero-
3-phosphoglycerol (POPG). POPC mimics typical cell membranes
encountered for mammalian red blood cells, while POPG mimics
the negatively charged lipids encountered in bacterial membranes.
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In 5 min, at 25 mM, compounds 15 and 19 elicited 20% dye
leakage from the POPG vesicles, while they elicited 10 and 15% dye
leakage from the POPC vesicles, respectively. However, in 5 min
at 500 mM, compounds 15 and 19 elicited 78 and 85% dye leakage
from the POPG vesicles, respectively. In 5 min at 25 mM, inactive
compound 1 elicited 9 and 18% dye leakage from the POPC and
POPG vesicles, respectively, while in 5 min at 500 mM 1 elicited
15 and 50% leakage from the POPC and POPG vesicles. Given
this differential response, it’s probable that compounds 15 and 19
are inducing microbicidal activity through a simple pore-forming
mechanism; however, further experiments are necessary to validate
this.
and 1,8-diazabicyclo[5.4.0]undec-7-ene, dissolved in THF (2 mL)
that was pre-stirred for 20 min, was then added dropwise to the
resulting solution. The resulting mixture was stirred for 12 h at
55 ◦C. The mixture was cooled down to 0 ◦C and 1 N HCl (2 mL)
was added, then extracted with ethyl acetate (3 ¥ 2 mL). The com-
bined organic extracts were washed with water (1 ¥ 3 mL), brine
(1 ¥ 3 mL), dried over Na2SO4 and the solvent evaporated under
reduced pressure. Purification of the residue took place on a silica-
gel column eluted with dichloromethane to give the target a-nitro
ketones. Resulting a-nitro ketones judged to be >85% pure by 1H
NMR were then subjected to hydrogenation/cyclization (detailed
below). Representative a-nitro ketones and 4,5-disubstituted-2-
aminoimidazoles are detailed below. All other characterization is
supplied in the supplementary information.
Conclusions
2-Nitro-8-phenyloctan-3-one. 6-Phenylhexanoic acid (0.154 g,
0.80 mmol) and CDI (0.261 g, 1.60 mmol) were added together
and then reacted with nitroethane (0.090 g, 1.21 mmol) and
DBU (0.306 g, 2.00 mmol) according to the general procedure.
Purification by column chromatography gave 0.060 g (30%) as a
yellow oil: 1H NMR (400 MHz, CDCl3) d 7.31 (m, 2H), 7.21(m,
3H), 5.24 (q, J = 7.2 Hz, 1H), 2.64 (t, J = 7.6 Hz, 2H), 2.58 (td,
J = 3.2, 6.8 Hz, 2H), 1.69 (d, J = 7.2 Hz, 3H), 1.68 (m, 4H),
1.37 (m, 2H) ppm; 13C NMR (100 MHz, CDCl3) d 200.1, 142.6,
128.7, 128.6, 126.0, 89.1, 39.3, 35.9, 31.4, 28.7, 23.3, 15.2 ppm;
IR nmax/cm-1 3026, 2933, 2858, 1732, 1559, 1453, 1362, 1030, 749,
701; HRMS (FAB) calcd for C14H19NO3 (MNa+) 272.1257, found
272.1255.
In summary, we have developed a route to 4,5-disubstituted-2-
aminoimidazoles that allows rapid assembly of 2-AI derivatives
from readily available building blocks. Using this approach, we
have identified two lead compounds, 15 and 19, that are both
microbicidal and have the ability to disperse pre-formed biofilms.
Given this activity and the simple substituent patterns employed
in this study, it is highly probable that more functionalized
4,5-disubstituted-2-aminoimidazoles, as well as 2-aminoimidzoles
with alternative substitution patterns, will yield 2-AI derivatives
with either enhanced antibiotic or anti-biofilm activity. These
studies are ongoing in our lab and will be reported in due course.
Experimental
3-Nitro-9-phenylnonan-4-one. 6-Phenylhexanoic acid (0.100 g,
0.52 mmol) and CDI (0.169 g, 1.04 mmol) were added together
and then reacted with nitropropane (0.070 g, 0.78 mmol) and
DBU (0.198 g, 1.30 mmol) according to the general procedure.
Purification by column chromatography gave 0.059 g (43%) as a
yellow oil: 1H NMR (400 MHz, CDCl3) d 7.28 (m, 2H), 7.17 (m,
3H), 5.04 (dd, J = 4.4, 9.6 Hz, 1H), 2.60 (m, 4H), 1.62 (m, 6H),
1.31 (m, 2H) 1.02 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (100 MHz,
CDCl3) d 199.3, 142.5, 128.6, 128.5, 126.0, 95.8, 39.5, 35.9, 31.3,
28.7, 23.5, 23.3, 10.6 ppm; IR nmax/cm-1 2922, 2855, 1730, 1559,
1454, 1363, 1030, 747, 699; HRMS (FAB) calcd for C15H21NO3
(MNa+) 286.1414, found 286.1410.
General experimental
All reagents used for chemical synthesis were purchased from com-
mercially available sources and used without further purification.
Chromatography was performed using 60 A mesh standard grade
˚
silica gel from Sorbtech (Atlanta, GA, USA). NMR solvents were
obtained from Cambridge Isotope Labs and used as is. 1H NMR
(300 MHz or 400 MHz) and 13C NMR (75 MHz or 100 MHz)
spectra were recorded at 25 ◦C on Varian Mercury spectrometers.
Chemical shifts (d) are given in ppm relative to tetramethylsilane
or the respective NMR solvent; coupling constants (J) are in Hertz
(Hz). Abbreviations used are s = singlet, bs = broad singlet, d =
doublet, dd = doublet of doublets, t = triplet, dt = doublet of
triplets, td = triplet of doublets, bt = broad triplet, q = quartet,
m = multiplet, bm = broad multiplet and br = broad. Mass
spectra were obtained at the NCSU Department of Chemistry
Mass Spectrometry Facility.
S. aureus (ATCC # 29213), S. epidermidis (ATCC # 29886),
MRSA (ATCC # BAA-44), MDRAB (ATCC # BAA-1605),
vancomycin resistant Enterococcus faecium (VRE) (ATCC #
51559) and E. coli (ATCC # 35695) were obtained from the
ATCC. Mechanically difibrinated sheep blood (DSB100) was
obtained from Hemostat Labs. Mueller-Hinton medium was
purchased from Fluka (# 70192).
4-Nitro-10-phenyldecan-5-one. 6-Phenylhexanoic
acid
(0.100 g, 0.52 mmol) and CDI (0.169 g, 1.04 mmol) were
added together and then reacted with nitrobutane (0.080 g,
0.78 mmol) and DBU (0.198 g, 1.30 mmol) according to the
general procedure. Purification by column chromatography gave
1
0.084 g (59%) as a yellow oil: H NMR (400 MHz, CDCl3) d
7.30 (m, 2H), 7.20 (m, 3H), 5.17 (dd, J = 4.4, 10.0 Hz, 1H), 2.63
(m, 4H), 1.65 (m, 6H), 1.36 (m, 4H) 1.00 (t, J = 7.2 Hz, 3H)
ppm; 13C NMR (100 MHz, CDCl3) d 199.5, 142.6, 128.7, 128.6,
126.0, 94.3, 39.5, 35.9, 31.8, 31.4, 28.7, 23.3, 19.5, 13.6 ppm; IR
n
max/cm-1 3027, 2935, 2858, 1730, 1560, 1454, 1373, 1031, 749,
700; HRMS (FAB) calcd for C16H23NO3 (MNa+) 300.1570, found
300.1575.
Synthesis of 4,5-disubstituted 2-aminoimidazoles
7-Nitro-1-phenylundecan-6-one. 6-Phenylhexanoic
acid
General procedure for the preparation of a-nitro ketones. To a
vial (23 ¥ 85 mm) was added appropriate phenyl carboxylic acids
and 1,1¢-carbonyldiimidazole in THF (3 mL) and stirred at room
temperature for 20 min. A mixture of appropriate nitroalkanes
(0.100 g, 0.52 mmol) and CDI (0.169 g, 1.04 mmol) were added
together and then reacted with nitropentane (0.091 g, 0.78 mmol)
and DBU (0.198 g, 1.30 mmol) according to the general procedure.
Purification by column chromatography gave 0.049 g (32%) as a
2818 | Org. Biomol. Chem., 2010, 8, 2814–2822
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yellow oil: 1H NMR (400 MHz, CDCl3) d 7.28 (m, 2H), 7.17 (m,
3H), 5.10 (dd, J = 4.4, 10.0 Hz, 1H), 2.61 (m, 4H), 1.63 (m, 6H),
1.35 (m, 6H) 0.92 (t, J = 6.8 Hz, 3H) ppm; 13C NMR (100 MHz,
CDCl3) d 199.5, 142.5, 128.6, 128.5, 126.0, 94.5, 39.5, 35.9, 31.3,
29.6, 28.7, 28.1, 23.3, 22.2, 13.9 ppm; IR nmax/cm-1 3027, 2931,
2859, 1731, 1559, 1454, 1363, 1030, 748, 700; HRMS (FAB) calcd
for C17H25NO3 (MNa+) 314.1727, found 314.1722.
128.1, 125.5, 121.9, 117.5, 35.5, 31.1, 28.6, 28.1, 22.9, 7.5 ppm; IR
n
max/cm-1 3169, 2929, 2857, 1680, 1453, 1020, 749, 700; HRMS
(FAB) calcd for C15H21N3 (MH+) 244.1808, found 244.1814.
5-Ethyl-4-(5-phenylpentyl)-1H-imidazol-2-amine
(12). 3-
Nitro-9-phenylnonan-4-one (0.050 g, 0.19 mmol) reacted with
concentrated HCl (0.95 mmol) and palladium, 5 wt.% on
activated carbon (0.081 g, 0.038 mmol) under H2, then reacted
with cyanamide (0.040 g, 0.95 mmol) according to the general
procedure. Purification by column chromatography gave 0.014 g
(29%) over two steps as a yellow oil: 1H NMR (300 MHz,
CD3OD) d 7.25 (m, 2H), 7.14 (m, 3H), 2.59 (t, J = 7.5 Hz, 2H),
2.42 (m, 4H), 1.63 (m, 4H), 1.28 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H)
ppm; 13C NMR (75 MHz, CD3OD) d 146.2, 142.4, 128.2, 128.1,
125.5, 123.6, 121.3, 35.5, 31.1, 28.7, 28.1, 22.9, 16.5, 13.1 ppm; IR
7-Nitro-1-phenyldodecan-6-one. 6-Phenylhexanoic
acid
(0.150 g, 0.78 mmol) and CDI (0.253 g, 1.56 mmol) were added
together and then reacted with nitrohexane (0.153 g, 1.17 mmol)
and DBU (0.297 g, 1.95 mmol) according to the general procedure.
Purification by column chromatography gave 0.090 g (38%) as a
yellow oil: 1H NMR (400 MHz, CDCl3) d 7.28 (m, 2H), 7.17 (m,
3H), 5.11 (dd, J = 4.8, 10.4 Hz, 1H), 2.61 (m, 4H), 1.63 (m, 6H),
1.33 (m, 8H) 0.90 (t, J = 6.8 Hz, 3H) ppm; 13C NMR (100 MHz,
CDCl3) d 199.5, 142.5, 128.6, 128.5, 126.0, 94.6, 39.5, 35.9, 31.3,
31.2, 29.9, 28.7, 25.7, 23.3, 22.4, 14.1 ppm; IR nmax/cm-1 3027,
2930, 2858, 1731, 1559, 1454, 1363, 1030, 748, 699; HRMS (FAB)
calcd for C18H27NO3 (MNa+) 328.1883, found 328.1881.
n
max/cm-1 3327, 2934, 1680, 1453, 1016, 749; HRMS (FAB) calcd
for C16H23N3 (MH+) 258.1965, found 258.1973.
4-(5-Phenylpentyl)-5-propyl-1H-imidazol-2-amine
(13). 4-
Nitro-10-phenyldecan-5-one (0.084 g, 0.30 mmol) reacted with
concentrated HCl (1.50 mmol) and palladium, 5 wt.% on
activated carbon (0.129 g, 0.060 mmol) under H2, then reacted
with cyanamide (0.064 g, 1.50 mmol) according to the general
procedure. Purification by column chromatography gave 0.025 g
(30%) over two steps as a yellow oil: 1H NMR (300 MHz,
CD3OD) d 7.20 (m, 2H), 7.15 (m, 3H), 2.60 (t, J = 7.2 Hz,
2H), 2.40 (m, 4H), 1.56 (m, 6H), 1.30 (m, 2H), 0.92 (t, J =
7.2 Hz, 3H) ppm; 13C NMR (100 MHz, CD3OD) d 146.4, 142.5,
128.3, 128.1, 125.6, 122.1, 121.9, 35.5, 31.1, 28.8, 28.2, 25.0, 23.0,
22.3, 12.6 ppm; IR nmax/cm-1 3165, 2931, 1680, 1453, 1031, 747,
699; HRMS (FAB) calcd for C17H25N3 (MH+) 272.2121, found
272.2127.
7-Nitro-1-phenyltridecan-6-one. 6-Phenylhexanoic
acid
(0.150 g, 0.78 mmol) and CDI (0.253 g, 1.56 mmol) were added
together and then reacted with nitroheptane (0.170 g, 1.17 mmol)
and DBU (0.297 g, 1.95 mmol) according to the general procedure.
Purification by column chromatography gave 0.104 g (42%) as a
yellow oil: 1H NMR (400 MHz, CDCl3) d 7.28 (m, 2H), 7.17 (m,
3H), 5.11 (dd, J = 4.8, 10.0 Hz, 1H), 2.61 (m, 4H), 1.63 (m, 6H),
1.33 (m, 10H) 0.89 (t, J = 7.2 Hz, 3H) ppm; 13C NMR (100 MHz,
CDCl3) d 199.5, 142.5, 128.6, 128.5, 126.0, 94.6, 39.4, 35.9, 31.5,
31.3, 29.9, 28.8, 28.7, 26.0, 23.3, 22.7, 14.2 ppm; IR nmax/cm-1
3027, 2931, 2859, 1731, 1559, 1454, 1363, 1031, 748, 700; HRMS
(FAB) calcd for C19H29NO3 (MNa+) 342.2040, found 342.2038.
5-Butyl-4-(5-phenylpentyl)-1H-imidazol-2-amine
(14). 7-
General procedure for 2-aminoimidazole synthesis
Nitro-1-phenylundecan-6-one (0.049 g, 0.17 mmol) reacted
with concentrated HCl (0.85 mmol) and palladium, 5 wt.% on
activated carbon (0.072 g, 0.034 mmol) under H2, then reacted
with cyanamide (0.035 g, 0.84 mmol) according to the general
procedure. Purification by column chromatography gave 0.025 g
(52%) over two steps as a yellow oil: 1H NMR (400 MHz,
CD3OD) d 7.22 (m, 2H), 7.15 (m, 3H), 2.60 (t, J = 7.2 Hz, 2H),
2.43 (m, 4H), 1.64 (m, 6H), 1.34 (m, 4H), 0.94 (t, J = 7.6 Hz, 3H)
ppm; 13C NMR (75 MHz, CD3OD) d 146.4, 142.4, 128.2, 128.1,
125.6, 122.1, 121.9, 35.5, 31.2, 31.1, 28.7, 28.2, 23.0, 22.8, 21.9,
12.9 ppm; IR nmax/cm-1 3166, 2930, 2857, 1680, 1453, 1030, 748,
699; HRMS (FAB) calcd for C18H27N3 (MH+) 286.2278, found
286.2288.
The appropriate a-nitro ketone (>85% pure, judged by 1H NMR)
was dissolved in ethanol (3 mL), concentrated HCl and 5%
palladium on carbon (0.2 equivalents) were added and the reaction
was stirred under H2 for 24 h. The mixture was filtered through
Celite and the solvent was evaporated under reduced pressure. The
residue was dissolved in ethanol (3 mL) and the pH was adjusted
to 4.5 with 0.1 N NaOH. To the solution was added cyanamide
and heated at 95 ◦C for 2 h. The ethanol was then evaporated
under reduced pressure and the resulting residue was purified
by column chromatography (CH2Cl2–MeOH sat. NH3 80 : 20) to
afford the desired compound in its free base form. Addition of
concentrated HCl to a methanol solution (2 mL) of the free base
followed by solvent evaporation under reduced pressure delivered
the corresponding 2-aminoimidazole as its HCl salt.
5-Pentyl-4-(5-phenylpentyl)-1H-imidazol-2-amine
(15). 7-
Nitro-1-phenyldodecan-6-one (0.090 g, 0.29 mmol) reacted
with concentrated HCl (1.45 mmol) and palladium, 5 wt.% on
activated carbon (0.125 g, 0.060 mmol) under H2, then reacted
with cyanamide (0.062 g, 1.47 mmol) according to the general
procedure. Purification by column chromatography gave 0.071 g
(81%) over two steps as a yellow oil: 1H NMR (300 MHz,
CD3OD) d 7.20 (m, 2H), 7.14 (m, 3H), 2.59 (t, J = 7.5 Hz, 2H),
2.42 (m, 4H), 1.56 (m, 6H), 1.32 (m, 6H), 0.90 (t, J = 7.2 Hz,
3H) ppm; 13C NMR (75 MHz, CD3OD) d 146.3, 142.5, 128.3,
128.1, 125.6, 122.1, 121.8, 35.5, 31.1, 31.1, 28.7, 28.7, 28.2, 23.1,
23.0, 22.3, 13.3 ppm; IR nmax/cm-1 3169, 2928, 2857, 1680, 1453,
5-Methyl-4-(5-phenylpentyl)-1H-imidazol-2-amine
(11). 2-
Nitro-8-phenyloctan-3-one (0.025 g, 0.10 mmol) reacted with
concentrated HCl (0.50 mmol) and palladium, 5 wt.% on
activated carbon (0.043 g, 0.020 mmol) under H2, then reacted
with cyanamide (0.021 g, 0.50 mmol) according to the general
procedure. Purification by column chromatography gave 0.010 g
(41%) over two steps as a yellow oil: 1H NMR (300 MHz,
CD3OD) d 7.22 (m, 2H), 7.14 (m, 3H), 2.59 (t, J = 7.5 Hz, 2H),
2.42 (t, J = 6.9 Hz, 2H), 2.01 (s, 3H) 1.60 (m, 4H), 1.31 (m,
2H) ppm; 13C NMR (75 MHz, CD3OD) d 146.2, 142.5, 128.3,
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1202, 1030, 746, 699; HRMS (FAB) calcd for C19H29N3 (MH+)
300.2434, found 300.2441.
1-Bromo-7-phenylhexan-2-one. To a 100 mL round bottomed
flask, phenyl hexanoic acid (2.08 g, 10.81 mmol) was added and
then dissolved in dichloromethane and a stir bar was added and
allowed to stir. Then, three drops of dimethylformamide was added
and then the reaction mixture was cooled to 0 ◦C. Oxalyl chloride
(4.12 g, 32.43 mmol) was added dropwise and left to continue
stirring for one hour. Separately in a diazomethane kit, a stir
bar, KOH (10.00 g, 178.2 mmol), 24 mL of ethanol, 17 mL of
water was added to the top of the diazomethane apparatus and
was heated to 65 ◦C and stirred. To a diazomethane kit liquid
addition funnel diazald (10.00 g, 46.67 mmol) and 100 mL of
diethyl ether was added. The diethyl ether/diazald mixture was
allowed to add dropwise to the KOH–water–ethanol mixture so
that the diazomethane was generate◦d and distilled over to the
collection flask that was cooled to 0 C. Once the diazomethane
had been completely collected, and the heptanoic acid had reacted
with the oxalyl chloride for one hour, the heptanoic acid reaction
mixture was concentrated in vacuo without heating in excess of
25 ◦C, dissolved in 3 mL of dichloromethane and was added
slowly to the flask containing the diazomethane while still being
cooled to 0 ◦C. The reaction mixture was allowed to stir for
5-Hexyl-4-(5-phenylpentyl)-1H-imidazol-2-amine
(19). 7-
Nitro-1-phenyltridecan-6-one (0.104 g, 0.33 mmol) reacted
with concentrated HCl (1.65 mmol) and palladium, 5 wt.% on
activated carbon (0.139 g, 0.065 mmol) under H2, then reacted
with cyanamide (0.068 g, 1.63 mmol) according to the general
procedure. Purification by column chromatography gave 0.049 g
(48%) over two steps as a yellow oil: 1H NMR (400 MHz,
CD3OD) d 7.23 (m, 2H), 7.15 (m, 3H), 2.60 (t, J = 7.6 Hz, 2H),
2.42 (m, 4H), 1.63 (m, 6H), 1.30 (m, 8H), 0.89 (t, J = 6.8 Hz, 3H)
ppm; 13C NMR (100 MHz, CD3OD) d 146.4, 142.4, 128.2, 128.1,
125.6, 122.1, 121.8, 35.5, 31.5, 31.1, 29.0, 28.7, 28.6, 28.2, 23.1,
23.0, 22.5, 13.3 ppm; IR nmax/cm-1 3165, 2929, 2857, 1680, 1453,
1202, 1029, 747, 699; HRMS (FAB) calcd for C20H31N3 (MH+)
314.2591, found 314.2596.
Control 2-aminoimidazole synthesis
22 and 23 were synthesized from their corresponding car-
boxylic acids as outlined below, using established literature pro-
tocols.12
◦
one hour at 0 C. Then 4 mL of concentrated hydrobromic acid
was added slowly to the reaction mixture and allowed to stir for
20 min. Then, 100 mL of a saturated sodium bicarbonate solution
was added slowly to the reaction mixture and allowed to stir for
30 min. The resulting mixture was then extracted with ethyl acetate,
washed twice with a brine solution, concentrated in vacuo and then
purified via column chromatography with a 10% ethyl acetate–
hexanes solution providing 1-bromo-7-phenylheptan-2-one as a
light yellow oil (2.53 g, 87% yield). 1H NMR (300 MHz, CDCl3)
d 7.32 (t, J = 7.5 Hz, 2H), d 7.20 (d, J = 7.5 Hz, 3H), d 3.86
(s, 2H), d2.63 (t, J = 7.2 Hz, 4H), d 1.65 (q, J = 7.8, 7.2 Hz,
4H), d 1.34 (m, 2H) ppm; 13C NMR (75 MHz, CDCl3) d 202.3,
142.7, 128.7, 128.6, 126.0, 39.9, 35.9, 34.9, 31.5, 28.9, 23.8 ppm;
IR nmax/cm-1 3434, 2930, 2848, 2113, 1641, 1393; HRMS (ESI)
calcd for C13H1779BrO (M+) 268.0462, found 268.0467.
1-Bromooctan-2-one. To a 100 mL round bottomed flask,
heptanoic acid (1.41 g, 10.81 mmol) was added and then dissolved
in dichloromethane and a stir bar was added and allowed to
stir. Then, three drops of dimethylformamide was added and
then the reaction mixture was cooled to 0 ◦C. Oxalyl chloride
(4.12 g, 32.43 mmol) was added dropwise and left to continue
stirring for one hour. Separately in a diazomethane kit, a stir
bar, KOH (10.00 g, 178.2 mmol), 24 mL of ethanol, 17 mL of
water was added to the top of the diazomethane apparatus and
was heated to 65 ◦C and stirred. To a diazomethane kit liquid
addition funnel diazald (10.00 g, 46.67 mmol) and 100 mL of
diethyl ether was added. The diethyl ether/diazald mixture was
allowed to add dropwise to the KOH–water–ethanol mixture so
that the diazomethane was generate◦d and distilled over to the
collection flask that was cooled to 0 C. Once the diazomethane
had been completely collected, and the heptanoic acid had reacted
with the oxalyl chloride for one hour, the heptanoic acid reaction
mixture was concentrated in vacuo without heating in excess of
25 ◦C, dissolved in 3 mL of dichloromethane and was added
slowly to the flask containing the diazomethane while still being
cooled to 0 ◦C. The reaction mixture was allowed to stir for
one hour at 0 ◦C. Then, 4 mL of concentrated hydrobromic acid
was added slowly to the reaction mixture and allowed to stir for
20 min. Then, 100 mL of a saturated sodium bicarbonate solution
was added slowly to the reaction mixture and allowed to stir for
30 min. The resulting mixture was then extracted with ethyl acetate,
washed twice with a brine solution, concentrated in vacuo and then
purified via column chromatography with a 10% ethyl acetate–
hexanes solution providing 1-bromooctan-2-one as a light yellow
oil (2.19 g, 97% yield) 1H NMR (300 MHz, CDCl3) d 3.79 (s, 2H),
d 2.49 (t, J = 7.2 Hz, 2H), d 1.44 (m, 2H), d 1.13 (bs, 6H), d 0.72
(t, J = 4.5 Hz, 3H) ppm; 13C NMR (75 MHz, CDCl3) d 202.15,
39.9, 34.9, 31.6, 28.8, 23.9, 22.6, 14.1 ppm; IR nmax/cm-1 3423,
306, 3025, 2933, 2856, 1716, 1495, 1453, 1030, 748; HRMS (ESI)
calcd for C8H1579BrO (M+) 206.0306, found 206.0301.
4-Hexyl-1H-imidazol-2-amine hydrochloride. 1-Bromooctan-
2-one (1.00 g, 4.83 mmol) was placed in a 50 mL round bottomed
flask with a stir bar and dissolved in 10 mL of DMF. Boc guanidine
(2.31 g, 14.89 mmol) was then added to the reaction mixture
and it was allowed to stir for 48 h. Water was then added to
the reaction mixture and it was placed in a separating funnel.
The mixture was then extracted twice with ethyl acetate, washed
twice with water, washed twice with brine, concentrated in vacuo
and then purified via column chromatography (5% methanol–
95% dichloromethane) to provide tert-butyl 2-amino-4-hexyl-1H-
imidazole-1-carboxylate as a hygroscopic light yellow solid (0.74 g,
1
57% yield). H NMR (300 MHz, CDCl3) d 6.69 (s, 2H), d 6.34
(s, 1H), d 2.20 (t, J = 7.5 Hz, 2H), d 1.44 (bs, 11H), 1.17 (s, 6H),
d 0.76 (s, 3H) ppm; 13C NMR (75 MHz, CDCl3) d 151.1, 149.6,
139.3, 105.7, 84.1, 31.8, 29.2, 28.4, 28.3, 28.0, 22.7, 14.2 ppm;
IR nmax/cm-1 3421, 2109, 1644, 1442, 1204, 1142; HRMS (ESI)
calcd for C14H25N3O2 (M+) 267.1946, found 267.1940. Then, tert-
butyl 2-amino-4-hexyl-1H-imidazole-1-carboxylate was dissolved
in a 1 : 4 mixture of trifluoroacetic acid–dichloromethane and
allowed to stir for five hours and then concentrated in vacuo. The
sample was then dissolved with a dilute HCl–methanol (5 drops
HCl/50 mL of methanol) and then concentrated in vacuo to
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provide 4-(5phenylpentyl)-1H)-imidazol-2-amine hydrochloride
as a hygroscopic light yellow solid (0.57 g, 57% overall yield).
1H NMR (300 MHz, CD3OD) d 6.34 (bs, 3H), d 2.41 (t, J =
7.5 Hz, 2H), d1.56 (m, 2H), d 1.28 (bs, 6H), d 0.82 (t, J =
6.6 Hz, 3H) ppm; 13C NMR (75 MHz, CDCl3) d 151.4, 131.9,
111.9, 35.3, 32.4, 31.9, 28.3, 26.3, 17.1 ppm; IR nmax/cm-1 3411,
2099, 1638; HRMS (ESI) calcd for C9H17N3 (M+) 167.1422, found
167.1416.
rows of the microtier plate. The microtiter plate sample was then
covered with a microtiter plate lid and then placed in a covered
plastic container. The chamber was incubated under stationary
conditions at 37◦ C. After 16 h, the lid was removed and MIC
values were recorded.
Red blood cell hemolysis assay. Hemolysis assays were per-
formed on mechanically difibrinated sheep blood (Hemostat Labs:
DSB100). 1.5 mL of blood was placed into a microcentrifuge
tube and centrifuged at 10000 rpm for ten minutes. The super-
natant was removed and then the cells were resuspended with
1 mL of phosphate-buffered saline (PBS). The suspension was
centrifuged, the supernatant was removed and cells resuspended
two more times. The final cell suspension was then diluted tenfold.
Test compound solutions were made in PBS and then added to
aliquots of the tenfold suspension dilution. PBS alone was used as
a negative control and as a zero hemolysis marker, whereas a 1%
Triton X sample was used as a positive control and the 100% lysis
marker. Samples were then placed in an incubator at 37 ◦C while
being shaken at 200 rpm for one hour. After one hour, the samples
were transferred to microcentrifuge tubes and then centrifuged
at 10000 rpm for ten minutes. The resulting supernatant was
diluted by a factor of 40 in distilled water. The absorbance of the
supernatant was measured with a UV spectrometer at a 540 nm
wavelength.
4-(5-Phenylpentyl)-1H)-imidazol-2-amine hydrochloride. 1-
Bromo-7-phenylheptan-2-one (1.00 g, 3.71 mmol) was placed in
a 50 mL round bottomed flask with a stir bar and dissolved in
10 mL of DMF. Boc guanidine (1.77 g, 11.13 mmol) was then
added to the reaction mixture and it was allowed to stir for
48 h. Water was then added to the reaction mixture and it was
placed in a separating funnel. The mixture was then extracted
twice with ethyl acetate, washed twice with water, washed
twice with brine, concentrated in vacuo and then purified via
column chromatography (5% methanol–95% dichloromethane)
to provide tert-butyl 2-amino-4-(5-phenylpentyl)-1H-imidazole-
1-carboxylate as a hygroscopic light yellow solid (0.77 g, 63%
1
yield). H NMR (300 MHz, CDCl3) d 7.31 (m, 2H), d 7.26 (m,
3H), d 6.66 (s, 2H), d 6.51 (s, 1H), d 2.63 (t, J = 7.5 Hz, 2H), d
2.39 (t, J = 7.5 Hz, 2H), d 1.67 (m, 4H), d 1.53 (s, 9H), d 1.40
(m, 2H) ppm; 13C NMR (75 MHz, CDCl3) d 151.1, 149.8, 143.0,
130.3, 128.7, 128.5, 125.8, 106.2, 84.5, 36.1, 31.6, 29.2, 28.5, 28.4,
28.2 ppm; IR nmax/cm-1 3433, 2098, 1639, 1454, 1204; HRMS
(ESI) calcd for C19H27N3O2 (M+) 329.2103, found 329.2109.
Then, tert-butyl 2-amino-4-(5-phenylpentyl)-1H-imidazole-1-
carboxylate was dissolved in a 1 : 4 mixture of trifluoroacetic
acid–dichloromethane and allowed to stir for five hours and then
concentrated in vacuo. The sample was then dissolved with a
dilute HCl–methanol (5 drops HCl/50 mL of methanol) and then
concentrated in vacuo to provide 4-(5phenylpentyl)-1H)-imidazol-
2-amine hydrochloride as a hygroscopic light yellow solid (0.62 g,
Procedure to determine the dispersal effect of test compounds
on E. faecium (VRE), MRSA, S. aureus, S. epidermidis, E.
coli, R. salexigens, V. cholerae, V. vulnificus and L. anguillarum
preformed biofilms. Dispersion assays were performed by taking
an overnight culture of bacterial strain and subculturing it at an
OD600 of 0.01 into the necessary medium (brain heart infusion
for E. faecium, tryptic soy broth with a 0.5% glucose supplement
(TSBG) for MRSA, S. aureus and S. epidermidis, Luria-Bertani
(LB) medium for MDRAB and E. coli, and tryptic soy broth with
a 0.5% glucose supplement and a 3.0% NaCl supplement (TGN)
for R. salexigens, V. cholerae, V. vulnificus and L. anguillarum).
The resulting bacterial suspension was aliquoted (100 mL) into
the wells of a 96-well PVC microtiter plate. Plates were then
1
63% overall yield). H NMR (300 MHz, CD3OD) d 7.20 (t, J =
7.2 Hz, 2H), d 7.12 (d, J = 7.2 Hz, 3H), d 6.37 (s, 1H), d 2.56 (t,
J = 7.5 Hz, 2H), d 2.41 (t, J = 7.5 Hz, 2H), d 1.57 (m, 4H), d 1.34
(m, 2H) ppm; 13C NMR (75 MHz, CDCl3) d 147.3, 142.5, 128.3,
128.1, 127.7, 125.6, 108.2, 35.5, 31.1, 28.3, 27.0, 24.2 ppm; IR
R
wrapped in GLAD Press n’ Sealꢀ followed by an incubation
n
max/cm-1 3385, 2933, 2857, 1679, 1536, 1495, 1453, 1335, 1172,
under stationary conditions at 37 ◦C to establish the biofilms.
After 24 h, the medium was discarded from the wells and the
plates were washed thoroughly with water. Stock solutions of
predetermined concentrations of the test compound were then
made in the necessary medium. These stock solutions were
aliquoted (100 mL) into the wells of the 96-well PVC microtiter
plate with the established biofilms. Medium alone was added to a
subset of the wells to serve as a control. Sample plates were then
incubated for 24 h at 37 ◦C. After incubation, the medium was
discarded from the wells and the plates were washed thoroughly
with water. Plates were then stained with 100 mL of 0.1% solution
of crystal violet (CV) and then incubated at ambient temperature
for 30 min. Plates were washed with water again and the remaining
stain was solubilized with 200 mL of 95% ethanol. A sample of
125 mL of solubilized CV stain from each well was transferred to
the corresponding wells of a polystyrene microtiter dish. Biofilm
dispersion was quantitated by measuring the OD540 of each well
in which a negative control lane wherein no biofilm was formed
served as a background and was subtracted out.
748, 700; HRMS (ESI) calcd for C14H19N3 (M+) 229.1578, found
229.1571.
Biological screening experimental
Broth microdilution method for MIC determination. Overnight
cultures of bacterial strain were subcultured to 5 ¥ 105 CFU/mL in
Mueller-Hinton medium (Fluka # 70192). The resulting bacterial
suspension was aliquoted (1.0 mL) into culture tubes. Samples
were prepared from these culture tubes containing either 256 mg
mL-1 of specified antibiotic or no test compound as a control.
Samples were then aliquoted (200 mL) into the first row of wells
of a 96-well microtiter plate in which subsequent wells were
prefilled with 100 mL of Mueller-Hinton medium based 5 ¥ 105
CFU/mL bacterial subculture. Using the multichannel pipettor
set at 100 mL, row one wells were mixed 8–10 times. Then, 100 mL
were withdrawn and transferred to row two. Row two wells were
mixed 8–10 times followed by a 100 mL transfer from row two to
row three. This procedure was used to serial dilute the rest of the
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13 J. J. Richards, T. E. Ballard, R. W. Huigens III and C. Melander,
ChemBioChem, 2008, 9, 1267–1279.
Acknowledgements
14 R. W. Huigens III, L. Ma, C. Gambino, A. Basso, P. D. R. Moeller,
J. Cavanagh, D. J. Wozniak and C. Melander, Mol. BioSyst., 2008, 4,
614–621.
The authors would like to thank the North Carolina Biotechnol-
ogy Center (grant# 2008-MRG-1114) and the V foundation for
funding (fellowships to SAR and SR), as well as Anne Young
and Professor Neville Kallenbach (NYU) for performing the dye
dispersion assay.
15 T. E. Ballard, J. J. Richards, A. L. Wolfe and C. Melander, Chem.–
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