Chemical synthesis and biological screening of 2-aminoimidazole-based bacterial and fungal antibiofilm agents

Article abstract of DOI:10.1002/cbic.200900617

A collection of 2-aminoimidazole/triazole amides has been synthesized and screened for antibiofilm activity. This class of small molecules was found to modulate the biofilm activity of Pseudomonas aeruginosa, a multidrug-resistant strain of Acinetobacter

Full text of DOI:10.1002/cbic.200900617

DOI: 10.1002/cbic.200900617
Chemical Synthesis and Biological Screening of
2-Aminoimidazole-Based Bacterial and Fungal
Antibiofilm Agents
Steven A. Rogers, Joseph D. Bero, and Christian Melander*[a]
A collection of 2-aminoimidazole/triazole amides has been syn-
thesized and screened for antibiofilm activity. This class of
small molecules was found to modulate the biofilm activity of
Pseudomonas aeruginosa, a multidrug-resistant strain of Acine-
tobacter baumannii (MDRAB), a methicillin-resistant Staphylo-
coccus aureus strain (MRSA), Escherichia coli, Rhodospirillum
salexigens, Staphylococcus epidermidis, Vibrio vulnificus, and
vancomycin-resistant Enterococcus faecium as well as the yeast
Candida albicans and Cryptococcus neoformans. Furthermore,
lead compounds were found to not lyse red blood cells at
active concentrations.
Introduction
A biofilm is defined as a surface-accreted microcolony that is
surrounded by a self-produced extracellular polymeric matrix
(EPS).[1] This EPS is a biosynthetic polymer composed of nucleic
acids, polysaccharides, phospholipids, and proteins that acts
dually as a cement and protective barrier against antibiotics
and other generic microbicidal agents. It has been estimated
that bacteria within a biofilm can display up to 1000-fold in-
creased resistance to antibiotic treatment in comparison to
their planktonic counterparts.[2] Since biofilms drive 80% of all
bacterial infections,[3] this increased tolerance to antimicrobial
therapy is detrimental to human health; causing many biofilm-
related infections to become chronic.[4] Prolonged disease
states can increase mortality rates as well as place significant fi-
nancial burden on our health-care system.
Due to the ubiquitous nature of biofilm-driven disease,
much attention has been focused on the development of
small-molecule modulators of biofilm development.[5] We have
previously shown that 2-aminoimidazole analogues (Scheme 1)
are highly successful in inhibiting and dispersing bacterial bio-
films through nonmicrobicidal mechanisms.[6–18] Within this
class of molecules, 2-aminoimidazole triazole (2-AIT) conju-
gates[13] were the first small molecules reported to inhibit and
disperse biofilms across bacterial order, class, and phylum. Fur-
ther analogue development has yielded the dibromopyrrole
TAGE-triazole conjugate 3[11] as a potent inhibitor of Staphylo-
coccus aureus biofilm formation and RA-11[17] as a highly
potent inhibitor of Pseudomonas aeruginosa biofilm formation.
Recent advances in our understanding of pathogen behavior
suggest that fungal and bacterial species can coordinate their
behavior using small-molecule signals.[19] Therefore, molecules
that control bacterial behavior might be able to control fungal
behavior. Furthermore, given that fungal strains also form bio-
films and represent an important pathogen target for biomedi-
cal science,[20] we became interested in determining whether
we could identify 2-AI derivatives that were capable of inhibit-
ing and dispersing fungal biofilms.
Scheme 1. Previously studied 2-AI antibiofilm agents.
Results and Discussion
Our first attempt focused on evaluating the ability of 2-AIT 1
to inhibit the formation of Candida albicans biofilms. Triazole 1
was deemed the most likely 2-AI derivative to inhibit the for-
mation of fungal biofilms because of its broad-spectrum
activity.[13]
[a] S. A. Rogers, J. D. Bero, Dr. C. Melander
Chemistry, North Carolina State University
2620 Yarborough Drive, Raleigh, NC, 27695-8204 (USA)
Fax: (+1)919-513-2960
E-mail: christian_melander@ncsu.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.200900617.
396
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Aminoimidazole-Based Antibiofilm Agents
Compound 1 was screened at 100 mm for its ability to inhibit
to the formation of C. albicans biofilms by using a crystal violet
(CV) reporter assay.[21] Briefly, biofilms were allowed to form for
24 h in a 96-well microtiter plate in the absence or presence of
100 mm of 1. The wells were subsequently washed thoroughly
with water to remove free-floating and loosely adherent
fungus and then treated with crystal violet. Crystal violet stains
the remaining surface-attached fungus (i.e., the biofilm), which
following solubilization, can be quantified by spectrophotome-
try (A540).
With this initial success, we asked the question whether ana-
logue synthesis could deliver alternative 2-AIT derivatives with
enhanced anti-fungal-biofilm activity. Previous work in our lab-
oratory had demonstrated that 2-AI-based inhibitors of bio-
films can be subdivided into three separate sections:[18] 1) the
2-AI head, 2) the linker region, and 3) the tail region
(Scheme 2). Structure–activity relationship (SAR) data indicate
that selectivity and activity can be tuned/enhanced by modifi-
cation of the tail region.[6–18] Based upon these data, we elect-
ed to synthesize a new pilot collection of 2-AI derivatives for
antibiofilm testing in which diversity could be rapidly achieved
by substituents on the triazole ring through commercially
available carboxylic acids.
Compound 1 was able to inhibit C. albicans formation by
12% at 100 mm. Follow-up growth curves at 100 mm demon-
strated that this antibiofilm activity was nonfungicidal.
The synthetic approach to this 2-AIT derivatization is out-
lined in Scheme 3. In our previous synthesis of 2-AIT conju-
gates,[13] we employed the alkyne-derived 2-AI as a precursor
for the CuI-mediated[3+2] alkyne/azide cycloaddition (click reac-
tion).[22] Although this reaction worked well, purification of the
resulting product was cumbersome, due to the need for copi-
ous amounts of ammonia saturated methanol for column chro-
matography. Therefore, we decided to revise the route by em-
ploying a Boc-protected 2-AI alkyne that would allow more tra-
ditional means of purification (i.e., methanol/dichloromethane
Scheme 2. 2-AI structure analysis.
Scheme 3. Synthesis of triazole-substituted 2-AI derivatives. a) i: (COCl)2, CH2Cl2, DMF (cat.), ii: CH2N2, Et2O/CH2Cl2, iii: HBr (82%); b) Boc-guanidine, DMF, RT
(59%); c) azide, CuSO4, Na ascorbate, EtOH, CH2Cl2, H2O; d) trifluoroacetic acid, CH2Cl2 (58–94% over two steps).
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C. Melander et al.
columns). The Boc-protected scaffold was synthesized from
oct-7-ynoic acid by treatment with oxayl chloride followed by
diazomethane and quenching of the resulting a-diazo ketone
with HBr to generate the intermediate a-bromo ketone. Cycli-
zation with Boc-guanidine then delivered the target 2-AI
alkyne 5.[23]
Once 5 had been synthesized, we assembled a diverse array
of azido amides to employ in the click reaction to create our
pilot collection of 2-AIT conjugates. Briefly, 2-bromo-ethylamine
was treated with sodium azide to deliver 2-azido-ethylamine,
which following acylation (via the respective acid chloride)
generated the azido amides for elaboration into the 2-AIT col-
lection. Each azido amide was then subjected to the click reac-
tion with 5. Boc-deprotection (TFA/CH2Cl2) followed by coun-
terion exchange (trifluoroacetate for chloride) delivered the
target 2-AIT compounds for antibiofilm screening.
tion, molecules that are effective against a preformed biofilms
are more clinically significant.
Once we had established that these next-generation 2-AIT
conjugates had the ability to inhibit and disperse C. albicans
biofilms, we addressed whether these compounds would also
inhibit and disperse biofilms from Cryptococcus neoformans, an
opportunistic fungal strain known to infect immunosuppressed
patients, especially those with HIV infections.[24] Initial screen-
ing of our 2-AIT derivatives showed that 7e and 7m had
potent antibiofilm activity against C. neoformans. Follow up
dose–response studies revealed IC50 values of 1.3 mm and
8.0 mm (Table 1). Comparison of fungal growth in the presence
or absence of either compound indicated that they were not
fungicidal. Unfortunately, neither compound was able to dis-
perse preformed C. neoformans biofilms. We are currently
trying to identify analogues of these two compounds that
would.
Each 2-AIT conjugate was assayed at 100 mm for its ability to
inhibit the formation of C. albicans biofilms by using the CV re-
porter assay. From this assay, derivatives 7 f and 7m were de-
termined to be the most potent. Subsequent dose–response
studies revealed that 7 f had an IC50 of 2.9 mm while 7m had
an IC50 of 3.3 mm (Table 1). Growth-curve and colony-count
analysis of 7 f and 7m at respective IC50 values demonstrated
their antibiofilm activity to be nonfungicidal.
After demonstrating that the 2-AIT conjugates had activity
against opportunistic fungal strains, we wanted to investigate
this library’s activity against Gram-negative and -positive bacte-
rial biofilms. (It has been shown that a select number of small
molecules are employed in cross-kingdom communication.[19]
)
As representative Gram-negative pathogens, we chose multi-
drug-resistant Acinetobacter baumannii (MDRAB), E. coli, and
Pseudomonas aeruginosa (strains PAO1 and PA14). Two repre-
sentative marine-based bacteria were chosen: Vibrio vulnificus
and Rhodospirillum salexigens. As representative Gram-positive
pathogens, MRSA, vancomycin-resistant enterococci (VRE) and
Staphylococcus epidermidis were chosen.
Table 1. Fungal biofilm data.
Compound IC50 [mm] EC50 [mm] Compound IC50 [mm]
EC50 [mm]
Each 2AIT was screened at 100 mm against each bacterial
strain for the ability to inhibit biofilm formation as assessed by
the CV reporter assay.[25] As with the fungal studies delineated
above, the strongest compounds with the highest percentage
inhibition were screened at various concentrations so that IC50
values could be calculated from the dose–response curves.
Active compounds were then screened for their ability to dis-
perse preformed biofilms. The dispersal capability of each
active compound was quantified by determining their EC50
values. Finally, toxicity studies were performed for all com-
pounds reported, and they were found to be nonmicrobicidal
at their IC50 values.
C. albicans
7 f
7m
C. neoformans
7e
S. cerevisiae
2.9ꢀ0.7 37.2ꢀ5.7 7g
2.7ꢀ0.1
207.4ꢀ7.3
353.7ꢀ7.3
3.3ꢀ1.6 24.7ꢀ4.5 7h
50.4ꢀ2.2
7j
130.6ꢀ16.9 >400
1.3ꢀ0.3
8.0ꢀ3.4
–
–
7m
Next, we addressed whether 7 f and 7m could disperse pre-
formed C. albicans biofilms. C. albicans was allowed to establish
biofilms in a 96-well microtiter plate for 24 h. The plate was
then washed to remove any free floating or loosely adherent
fungus. 2-AIT 7 f or 7m was then added to each well at 75 mm,
and the plate was incubated at 378C for 24 h. The wells were
then washed with water and stained with CV to quantify any
remaining biofilms. In comparison to biofilms treated with
medium only, compound 7 f dispersed 56%, while 7m dis-
persed 62% of the biofilm. Once we had established that both
compounds could disperse preformed biofilms, the effect was
quantified by determining 7 f and 7m’s EC50 values against
preformed C. albicans biofilms. Here, EC50 is defined as the con-
centration at which the compound will disperse 50% of a pre-
formed biofilm. Dose–response studies revealed EC50 values of
37.2 mm and 24.7 mm for 7 f and 7m, respectively (Table 1).
From a medical perspective, molecules that simply inhibit the
formation of a biofilm could be used in a prophylactic sense;
however, given that a majority of patients already have an es-
tablished biofilm infection when they seek medical interven-
Against the Gram-negative bacterial strains, 2-AIT conjugates
with para-alkyl phenyl substituents displayed the strongest an-
tibiofilm activity, which could be tuned by methylene chain
length (Table 2). For example, compound 7u, which has seven
methylene units para to the amide, displayed the strongest
biofilm-development modulation against MDRAB and E. coli,
giving IC50/EC50 values of 1.9 mm/7.9 mm and 11.2 mm/23.4 mm,
respectively. Compound 7w, with nine methylene units, dem-
onstrated nanomolar IC50 values against PA14 and PAO1 giving
IC50/EC50 values of 0.8 mm/2.5 mm against PA14 and 0.5 mm/
15.4 mm against PAO1. Compound 7w shows an increase of
PAO1-biofilm inhibition in comparison to 1, which gave a
5.6 mm IC50, as well as an increase of PA14 biofilm dispersal ac-
tivity, in which 1 has a reported 22 mm EC50. Finally, 7t, with six
methylene units, gave an IC50/EC50 of 21.4 mm/36.2 mm against
V. vulnificus and an IC50 of 8.9 mm against R. salexigens. Interest-
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Aminoimidazole-Based Antibiofilm Agents
previously shown that these strains develop equally well in
TSB in both the planktonic and biofilm forms.[27] Compounds
7 f and 7m gave IC50 values of 40 mm and 62 mm, respectively,
this indicates that 2-AI-based antibiofilm agents are active
against multispecies biofilms.
Table 2. Gram-negative bacterial biofilm data.
Compound IC50 [mm] EC50 [mm] Compound IC50 [mm] EC50 [mm]
MDRAB
7s
7t
7u
7v
7w
PA14
7s
7t
7u
7v
7w
V. vulnificus
7t
7u
E. coli
15.1ꢀ2.4 17.2ꢀ0.4 7e
43.8ꢀ4.2 114.1ꢀ18.79
40.1ꢀ0.3 93.1ꢀ3.6
11.2ꢀ0.4 23.4ꢀ7.9
4.5ꢀ0.2 5.4ꢀ0.7 7s
1.9ꢀ0.6 7.9ꢀ1.4 7u
Once we had established the cross-kingdom activity of 2-
AIT-based antibiofilm agents, we wanted to examine if the 2-AI
scaffold was necessary for the antibiofilm activity. Analogues of
compounds 7g and 7w were synthesized that did not have
the 2-aminoimidazole scaffold (Scheme 4). Both compounds 8
and 9 were tested and found not to have antibiofilm activity
at 100 mm against any of the bacterial and yeast strains.
5.5ꢀ2.4
–
–
7v
7w
PAO1
53.1ꢀ3.0
13.5ꢀ0.7
–
–
11.2ꢀ3.6
34.4ꢀ0.8 39.3ꢀ4.8 7t
38.2ꢀ0.7 42.7ꢀ2.3 7u
2.2ꢀ0.4 4.3ꢀ1.4 7v
5.8ꢀ1.9 13.8ꢀ3.2 7w
0.8ꢀ0.1 2.5ꢀ1.6 7bb
1.1ꢀ0.5
7.2ꢀ1.8
1.2ꢀ0.3 45.9ꢀ1.2
62.5ꢀ2.2 67.9ꢀ2.4
0.5ꢀ0.1 15.4ꢀ5.3
64.5ꢀ2.3 119.7ꢀ9.9
R. salexigens
21.4ꢀ1.8 36.2ꢀ5.5 7t
56.9ꢀ3.7 94.7ꢀ5.2 7u
8.9ꢀ0.4
–
–
27.1ꢀ3.1
ingly, the marine-sponge-derived alkaloids bromoageliferin and
oroidin, which served as structural inspiration for the 2-AI anti-
biofilm agents, have reported IC50 values for R. salexigens of
1.2 mm (1.7 mgmLꢁ1) and 24.5 mm (63 mgmLꢁ1), respectively.
Compound 7u shows better inhibitory activity than oroidin
and similar activity to bromoageliferin.
Scheme 4. Analogues of 4g and 4w without 2-aminoimidazole scaffolds.
As we have seen with other 2-AIT-based antibiofilm agents,
these 2-AIT derivatives also displayed substantial antibiofilm
activity against Gram-positive bacteria. This information is sum-
marized in Table 3. Compound 7u displayed the highest bio-
Finally, red-blood-cell hemolysis analysis of 7u and 7 f was
performed with defibrinated sheep’s blood.[29] These com-
pounds were chosen because 7u was the lead compound
against most bacterial strains and 7 f was a leading fungal anti-
biofilm agent. The HD50 (hemolytic dose that lyses 50% of the
red blood cells) was found to be 194 mm for 7u and 210 mm
for 7 f. Even more promising is that neither compound showed
more than 5% lysis until after 50 mm, a concentration well
above their respective IC50 values.
Table 3. Gram-positive bacterial biofilm data.
Compound IC50 [mm]
EC50 [mm] Compound IC50 [mm] EC50 [mm]
VRE
MRSA
7s
7t
7u
14.4ꢀ2.8 15.9ꢀ0.9 7s
4.3ꢀ1.9 80.6ꢀ9.5
19.5ꢀ5.3 21.2ꢀ1.5 7t
0.7ꢀ0.4
1.9ꢀ0.1
4.7ꢀ0.1
0.9ꢀ0.3
5.5ꢀ0.5
–
0.5ꢀ0.1
0.7ꢀ0.3 7u
Conclusions
S. epidermidis
7 f
7m
7u
7w
7v
5.2ꢀ0.4
2.8ꢀ0.9
–
–
We have shown that 2-aminoimidazole/triazole conjugates can
be designed to have cross-kingdom activity in that they can
regulate prokaryotic and eukaryotic biofilm development in a
nonmicrobicidal manner. Furthermore, we have also successful-
ly explored and executed a more efficient synthesis to these 2-
aminoimidazoles that avoids undesirable purification tech-
niques. Finally, we have demonstrated that these compounds
do not lyse red blood cells until concentrations well past their
IC50 and EC50 values. This information, coupled with previous
results from our group that established that 2-AI-based anti-
biofilm agents are nontoxic to both mammalian cell culture[6]
and model organisms,[9] makes these molecules candidates for
exploring in vivo remediation efforts for biofilm infections.
0.6ꢀ0.3 28.1ꢀ7.5
0.9ꢀ0.2
–
film regulatory activity of any reported 2-AI against VRE, giving
an IC50/EC50 of 0.5 mm/0.7 mm. Compound 7t gave a nanomolar
IC50/EC50 of 0.7 mm/0.9 mm against MRSA, and 7u an IC50/EC50 of
0.6 mm/28.1 mm against S. epidermidis.
We then studied the ability of lead compounds to inhibit
the formation of mixed-species biofilms. S. epidermidis and
C. albicans are common sources of medical-implant-based in-
fections, and studies show that these species coexist well in
the biofilm form.[26,27] These mixed-species biofilm infections
represent a major hurdle for health care, as treatment with an
antibiotic or antifungal alone would be less likely to be suc-
cessful. Given the success of compounds 7 f and 7m against
both S. epidermidis and C. albicans, we investigated the ability
of these compounds as mixed-species antibiofilm agents. We
used tryptic soy broth (TSB) as a growth medium since it was
Experimental Section
General: MRSA (ATCC BAA-44), S. aureus (ATCC 29213), E. coli
(ATCC 35695), vancomycin resistant Enterococcus faecium (VRE;
ATCC 51559), MDRAB (ATCC BAA-1605), R. salexigens (ATCC 35888),
S. cerevisiae (ATCC 204508), C. albicans (ATCC 76615), C. neoformans
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399

C. Melander et al.
(ATCC MYA-422), and Staphylococcus epidermidis (ATCC 29886)
were obtained from the ATCC. P. aeruginosa strain PAO1 was pro-
vided by Dr. Wozniak (Wake Forest School of Medicine). All other
reagents were purchased from commercially available sources.
1.8 Hz, 1H), 3.28 (t, J=5.7 Hz, 2H), 3.03 (q, J=6.3, 5.7 Hz, 2H),
2.85 ppm (s, 6H); 13C NMR (75 MHz, CDCl3): d=152.3, 134.8, 130.9,
130.1, 129.7, 128.9, 123.4, 118.9, 115.6, 51.0, 45.6, 42.6 ppm; HRMS
(ESI) calcd for C14H17N5O2S: 319.1103 [M]+, found 319.1104.
Synthesis: All reagents used for chemical synthesis were pur-
chased from commercial sources and used without further purifica-
tion. Chromatography was performed by using 60 ꢁ mesh stan-
dard-grade silica gel from Sorbtech (Atlanta, MA, USA). NMR sol-
vents were obtained from Cambridge Isotope Labs and used as
N-(2-Azidoethyl)-2,3,4,5,6-pentamethylbenzenesulfonamide: As
in the synthesis of N-(2-azidoethyl)tetradecanamide, 2,3,4,5,6-pen-
tamethylbenzene-1-sulfonyl chloride (0.304 g, 1.23 mmol) was
treated with 2-azidoethanamine (0.106 g, 1.23 mmol) and triethyl-
amine (0.249 g, 2.46 mmol) in dichloromethane (5 mL) to give N-(2-
1
supplied. H NMR (300 or 400 MHz) and 13C NMR (75 or 100 MHz)
azidoethyl)-2,3,4,5,6-pentamethylbenzenesulfonamide
(0.296 g,
81% yield). 1H NMR (300 MHz, CDCl3): d=5.12 (t, J=3.9 Hz, 1H),
3.35 (t, J=5.4 Hz, 2H), 3.05 (q, J=6.0, 5.1 Hz, 2H), 2.59 (s, 6H), 2.28
(s, 3H), 2.24 ppm (s, 6H); 13C NMR (75 MHz, CDCl3): d=139.9, 136.1,
135.2, 134.2, 51.1, 42.3, 19.2, 17.9, 17.3 ppm; HRMS (ESI) calcd for
C13H20N4O2S: 296.1307 [M]+, found 296.1304.
spectra were recorded at 258C on Varian Mercury spectrometers.
Chemical shifts (d) are given in ppm relative to tetramethylsilane
or the respective NMR solvent. Mass spectra were obtained at the
NCSU Department of Chemistry Mass Spectrometry Facility.
N-(2-Azidoethyl)tetradecanamide: 2-Azidoethanamine (0.102 g,
1.18 mmol), DCM (5 mL), and then triethylamine (0.239 g,
N-(2-Azidoethyl)benzenesulfonamide: As in the synthesis of N-(2-
azidoethyl)tetradecanamide, benzenesulfonyl chloride (0.201 g,
1.16 mmol) was treated with 2-azidoethanamine (0.100 g,
1.16 mmol) and triethylamine (0.235 g, 2.32 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)benzenesulfonamide (0.289 g,
84% yield). 1H NMR (300 MHz, CDCl3): d=7.90 (d, J=1.8 Hz, 2H),
7.55 (m, 3H), 5.47 (s, 1H), 3.39 (t, J=5.1 Hz, 2H), 3.12 ppm (t, J=
5.4 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=139.8, 133.2, 129.5, 127.2,
50.9, 42.6 ppm; HRMS (ESI) calcd for C6H8N4O2S: 226.0524 [M]+,
found 226.0523.
2.37 mmol) were added to
a 25 mL round-bottomed flask
equipped with a magnetic stirrer. To this reaction mixture, tetrade-
canoyl chloride (0.292 g, 1.18 mmol) was added dropwise, and the
mixture was allowed to stir at room temperature for 24 h. The re-
action mixture was concentrated in vacuo and then purified by
silica gel column chromatography (dichloromethane!methanol/
dichloromethane 1:40) to give N-(2-azidoethyl)tetradecanamide
(0.245 g, 79% yield). 1H NMR (300 MHz, CDCl3): d=5.86 (s, 1H),
3.42 (s, 4H), 2.18 (t, J=7.8 Hz, 2H), 1.62 (m, 2H), 1.24 (m, 20H),
0.87 ppm (t, J=3.9 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=173.7,
51.2, 39.1, 36.9, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5, 25.9, 22.9,
14.4 ppm; HRMS (ESI) calcd for C16H32N4O: 296.2576 [M]+, found
296.2566.
(E)-N-(2-Azidoethyl)-4-phenylbut-3-enamide: As in the synthesis
of N-(2-azidoethyl)tetradecanamide, (E)-4-phenylbut-3-enoyl chlo-
ride (0.198 g, 1.19 mmol) was treated with 2-azidoethanamine
(0.114 g, 1.19 mmol) and triethylamine (0.239 g, 2.37 mmol) in di-
chloromethane (5 mL) to give (E)-N-(2-azidoethyl)-4-phenylbut-3-
enamide (0.168 g, 55% yield). 1H NMR (300 MHz, CDCl3): d=7.68
(d, J=15.6 Hz, 1H), 7.45 (d, J=3.3 Hz, 2H), 7.29 (m, 3H), 7.04 (t, J=
4.1 Hz, 1H), 6.59 (d, J=15.9 Hz, 1H), 3.55 (q, J=6.0, 5.1 Hz, 2H),
3.47 ppm (t, J=1.5 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=166.9,
141.6, 134.9, 130.1, 129.1, 128.1, 120.8, 51.1, 39.4 ppm; HRMS (ESI)
calcd for C11H12N4O: 216.1011 [M]+, found 216.1005.
N-(2-Azidoethyl)octadec-9-enamide: As in the synthesis of N-(2-
azidoethyl)tetradecanamide, octadec-9-enoyl chloride (0.374 g,
1.24 mmol) was treated with 2-azidoethanamine (0.107 g,
1.24 mmol) and triethylamine (0.252 g, 2.49 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)octadec-9-enamide (0.309 g,
1
71% yield). H NMR (300 MHz, CDCl3): d=6.30 (s, 1H), 5.29 (m, 2H),
3.39 (d, J=2.1 Hz, 2H), 3.38 (d, J=2.4 Hz, 2H), 2.19 (t, J=7.2 Hz,
2H), 1.97 (m, 4H), 1.59 (t, J=7.5 Hz, 2H), 1.26 (m, 20H), 0.84 ppm
(t, J=6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=173.9, 130.2, 129.9,
51.1, 39.1, 36.8, 32.1, 30.0, 29.9, 29.7, 29.5, 29.4, 29.3, 27.4, 27.3,
25.9, 22.9, 14.3 ppm; HRMS (ESI) calcd for C20H38N4O: 350.3046
[M]+, found 350.3039.
N-(2-Azidoethyl)-2-(phenylthio)acetamide: As in the synthesis of
N-(2-azidoethyl)tetradecanamide, 2-(phenylthio)acetyl chloride
(0.222 g, 1.19 mmol) was treated with 2-azidoethanamine (0.102 g,
1.19 mmol) and triethylamine (0.240 g, 2.37 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-2-(phenylthio)acetamide
(0.179 g, 64% yield). 1H NMR (300 MHz, CDCl3): d=7.29 (m, 4H),
7.18 (m, 2H), 3.60 (s, 2H), 3.34 ppm (m, 4H); 13C NMR (75 MHz,
CDCl3): d=168.6, 134.8, 129.5, 128.6, 127.1, 50.8, 39.3, 37.7 ppm;
HRMS (ESI) calcd for C10H12N4OS: 236.0732 [M]+, found 236.0729.
N-(2-Azidoethyl)thiophene-2-sulfonamide: As in the synthesis of
N-(2-azidoethyl)tetradecanamide, thiophene-2-sulfonyl chloride
(0.218 g, 1.19 mmol) was treated with 2-azidoethanamine (0.103 g,
1.19 mmol) and triethylamine (0.241 g, 2.39 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)thiophene-2-sulfonamide
(0.221 g, 80% yield). 1H NMR (300 MHz, CDCl3): d=7.62 (d, J=
1.2 Hz, 1H), 7.59 (d, J=1.2 Hz, 1H), 7.08 (t, J=3.9 Hz, 1H), 5.52 (s,
1H), 3.41 (t, J=5.4 Hz, 2H), 3.17 ppm (q, J=5.4, 3.8 Hz, 2H);
13C NMR (75 MHz, CDCl3): d=140.6, 132.7, 132.6, 127.9, 50.9,
42.9 ppm; HRMS (ESI) calcd for C6H8N4O2S2: 232.0089 [M]+, found
232.0084.
N-(2-Azidoethyl)palmitamide: As in the synthesis of N-(2-azido-
ethyl)tetradecanamide, palmitoyl chloride (0.338 g, 1.23 mmol) was
treated with 2-azidoethanamine (0.106 g, 1.23 mmol) and triethyl-
amine (0.249 g, 2.46 mmol) in dichloromethane (5 mL) to give N-(2-
azidoethyl)palmitamide (0.336 g, 84% yield). 1H NMR (300 MHz,
CDCl3): d=6.11 (s, 1H), 3.41 (s, 4H), 2.17 (t, J=7.5 Hz, 2H), 1.60 (m,
2H), 1.23 (m, 24H), 0.85 ppm (t, 6.3 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d=173.9, 51.1, 39.1, 36.9, 32.1, 29.9, 29.8, 29.8, 29.7, 29.6,
29.5, 25.9, 22.9, 14.3 ppm; HRMS (ESI) calcd for C18H36N4O:
324.2889 [M]+, found 324.2879.
N-(2-Azidoethyl)-5-(dimethylamino)naphthalene-1-sulfonamide:
As in the synthesis of N-(2-azidoethyl)tetradecanamide, 5-(dimeth-
ylamino)naphthalene-1-sulfonyl chloride (0.321 g, 1.19 mmol) was
treated with 2-azidoethanamine (0.103 g, 1.19 mmol) and triethyl-
amine (0.241 g, 2.38 mmol) in dichloromethane (5 mL) to give N-(2-
azidoethyl)-5-(dimethylamino)naphthalene-1-sulfonamide (0.328 g,
86% yield). 1H NMR (300 MHz, CDCl3): d=8.52 (d, J=8.4 Hz, 1H),
8.28 (d, J=8.4 Hz, 1H), 8.22 (d, J=0.9 Hz, 1H), 7.53 (t, J=8.1 Hz,
1H), 7.50 (t, J=7.5 Hz, 1H), 7.17 (d, J=7.8 Hz, 1H), 5.58 (t, J=
N-(2-Azidoethyl)decanamide: As in the synthesis of N-(2-azido-
ethyl)tetradecanamide, decanoyl chloride (0.251 g, 1.32 mmol) was
treated with 2-azidoethanamine (0.114 g, 1.32 mmol) and triethyl-
amine (0.267 g, 2.64 mmol) in dichloromethane (5 mL) to give N-(2-
azidoethyl)decanamide (0.262 g, 83% yield). 1H NMR (300 MHz,
400
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ChemBioChem 2010, 11, 396 – 410

Aminoimidazole-Based Antibiofilm Agents
CDCl3): d=6.49 (s, 1H), 3.36 (s, 2H), 3.35 (s, 2H), 2.14 (t, J=7.2 Hz,
2H), 1.46 (m, 2H), 1.21 (m, 12H), 0.81 ppm (t, J=6.0 Hz, 3H);
13C NMR (75 MHz, CDCl3): d=174.1, 50.9, 39.1, 36.8, 32.0, 29.7, 29.6,
29.5, 29.4, 25.9, 22.8, 14.3 ppm; HRMS (ESI) calcd for C12H24N4O:
240.1950 [M]+, found 240.1947.
thane (5 mL) to give N-(2-azidoethyl)-4-heptylbenzamide (0.260 g,
75% yield). 1H NMR (300 MHz, CDCl3): d=7.73 (d, J=7.8 Hz, 2H),
7.19 (d, J=7.8 Hz, 3H), 3.57 (q, J=5.7, 5.4 Hz, 2H), 3.47 (t, J=
5.7 Hz, 2H), 2.61 (t, J=7.5 Hz, 2H), 1.61 (m, 2H), 1.26 (m, 8H),
0.87 ppm (t, J=6.0 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=168.3,
147.3, 131.7, 128.8, 127.4, 50.9, 39.7, 36.1, 32.0, 31.4, 29.4, 29.3,
22.9, 14.3 ppm; HRMS (ESI) calcd for C16H24N4O: 288.1950 [M]+,
found 288.1943.
N-(2-Azidoethyl)-2-iodobenzamide: As in the synthesis of N-(2-azi-
doethyl)tetradecanamide,
2-iodobenzoyl
chloride
(0.312 g,
1.17 mmol) was treated with 2-azidoethanamine (0.101 g,
1.17 mmol) and triethylamine (0.237 g, 2.34 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-2-iodobenzamide (0.272 g,
74% yield). 1H NMR (300 MHz, CDCl3): d=7.84 (d, J=7.2 Hz, 1H),
7.33 (m, 2H), 7.08 (t, J=3.3 Hz, 1H), 6.51 (s, 1H), 3.54 (s, 2H),
3.53 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=169.9, 141.9, 140.0,
131.5, 128.4, 128.4, 92.7, 50.8, 39.6 ppm; HRMS (ESI) calcd for
C9H9IN4O: 315.9821 [M]+, found 315.9820.
N-(2-Azidoethyl)-4-butylbenzamide: As in the synthesis of N-(2-
azidoethyl)tetradecanamide, 4-butylbenzoyl chloride (0.242 g,
1.22 mmol) was treated with 2-azidoethanamine (0.105 g,
1.22 mmol) and triethylamine (0.246 g, 2.43 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-4-butylbenzamide (0.224 g,
75% yield). 1H NMR (300 MHz, CDCl3): d=7.73 (d, J=8.4 Hz, 2H),
7.39 (t, J=1.8 Hz, 1H), 7.17 (d, J=8.1 Hz, 2H), 3.55 (q, J=8.7,
5.7 Hz, 2H), 3.45 (t, J=5.7 Hz, 2H), 2.60 (t, J=7.8 Hz, 2H), 1.56 (m,
2H), 1.30 (m, 2H), 0.90 ppm (t, J=4.2 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d=168.5, 147.2, 131.7, 128.8, 127.4, 50.9, 39.7, 35.7, 33.5,
22.5, 14.1 ppm; HRMS (ESI) calcd for C13H18N4O: 246.1481 [M]+,
found 246.1476.
N-(2-Azidoethyl)-4-tert-butylbenzamide: As in the synthesis of N-
(2-azidoethyl)tetradecanamide,
4-tert-butylbenzoyl
chloride
(0.244 g, 1.23 mmol) was treated with 2-azidoethanamine (0.106 g,
1.23 mmol) and triethylamine (0.249 g, 2.47 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-4-tert-butylbenzamide
(0.221 g, 73% yield). 1H NMR (300 MHz, CDCl3): d=7.73 (d, J=
6.6 Hz, 2H), 7.43 (d, J=9.0 Hz, 2H), 6.95 (t, J=3.9 Hz, 1H), 3.59 (q,
J=5.4, 5.7 Hz, 2H), 3.50 (t, J=5.1 Hz, 2H), 1.31 ppm (s, 9H);
13C NMR (75 MHz, CDCl3): d=168.2, 155.4, 131.4, 127.2, 125.8, 51.1,
39.7, 35.2, 31.4 ppm; HRMS (ESI) calcd for C13H18N4O: 246.1480
[M]+, found 246.1479.
N-(2-Azidoethyl)-4-hexylbenzamide: As in the synthesis of N-(2-
azidoethyl)tetradecanamide, 4-hexylbenzoyl chloride (0.293 g,
1.30 mmol) was treated with 2-azidoethanamine (0.112 g,
1.30 mmol) and triethylamine (0.264 g, 2.61 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-4-hexylbenzamide (0.299 g,
84% yield). 1H NMR (300 MHz, CDCl3): d=7.75 (d, J=8.1 Hz, 2H),
7.43 (t, J=3.9 Hz, 1H), 7.17 (d, J=7.8 Hz, 2H), 3.55 (q, J=6.3,
5.7 Hz, 2H), 3.46 (t, J=5.7 Hz, 2H), 2.59 (t, J=7.5 Hz, 2H), 1.58 (m,
2H), 1.29 (m, 4H), 0.87 ppm (t, J=6.9 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d=168.6, 147.3, 131.7, 130.3, 128.8, 127.5, 50.9, 39.7, 35.9,
31.6, 31.1, 22.7, 14.2 ppm; HRMS (ESI) calcd for C15H22N4O:
274.1794 [M]+, found 274.1789.
N-(2-Azidoethyl)-3,5-difluorobenzamide: As in the synthesis of N-
(2-azidoethyl)tetradecanamide,
3,5-difluorobenzoyl
chloride
(0.219 g, 1.24 mmol) was treated with 2-azidoethanamine (0.107 g,
1.24 mmol) and triethylamine (0.251 g, 2.48 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-3,5-difluorobenzamide
(0.280 g, 59% yield). 1H NMR (300 MHz, CDCl3): d=7.32 (s, 2H),
7.29 (s, 1H), 6.91 (t, J=2.4 Hz, 1H), 3.58 (q, J=5.7, 5.1 Hz, 2H),
3.51 ppm (t, J=4.8 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=165.9,
164.7, 161.5, 161.4, 137.6, 110.7, 106.9, 50.7, 39.9 ppm; HRMS (ESI)
calcd for C9H8F2N4O2S: 226.0666 [M]+, found 226.0662.
N-(2-Azidoethyl)-4-pentylbenzamide: As in the synthesis of N-(2-
azidoethyl)tetradecanamide, 4-pentylbenzoyl chloride (0.283 g,
1.35 mmol) was treated with 2-azidoethanamine (0.116 g,
1.35 mmol) and triethylamine (0.273 g, 2.69 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-4-pentylbenzamide (0.267 g,
76% yield). 1H NMR (300 MHz, CDCl3): d=7.74 (d, J=8.1 Hz, 2H),
7.61 (t, J=5.1 Hz, 1H), 7.15 (d, J=8.4 Hz, 2H), 3.54 (q, J=5.7,
5.1 Hz, 2H), 3.44 (t, J=5.7 Hz, 2H), 2.58 (t, J=7.5 Hz, 2H), 1.57 (m,
2H), 1.26 (m, 4H), 0.86 ppm (t, J=6.6 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d=168.6, 147.2, 131.7, 128.7, 127.5, 50.8, 39.7, 35.9, 31.6,
31.1, 22.7, 14.2 ppm; HRMS (ESI) calcd for C14H20N4O: 260.1637
[M]+, found 260.1632.
N-(2-Azidoethyl)-2,4,6-trichlorobenzamide: As in the synthesis of
N-(2-azidoethyl)tetradecanamide, 2,4,6-trichlorobenzoyl chloride
(0.301 g, 1.23 mmol) was treated with 2-azidoethanamine (0.106 g,
1.23 mmol) and triethylamine (0.249 g, 2.47 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-2,4,6-trichlorobenzamide
(0.317 g, 88% yield). 1H NMR (300 MHz, CDCl3): d=7.27 (2H, s),
6.88 (s, 1H), 3.54 (s, 2H), 3.53 ppm (s, 2H); 13C NMR (75 MHz,
CDCl3): d=164.4, 135.9, 134.3, 132.9, 128.2, 50.7, 39.4 ppm; HRMS
(ESI) calcd for C9H7N4O: 291.9685 [M]+, found 291.9681.
N-(2-Azidoethyl)biphenyl-4-carboxamide: As in the synthesis of
N-(2-azidoethyl)tetradecanamide, biphenyl-4-carboxylic acid chlo-
ride (0.283 g, 1.31 mmol) was treated with 2-azidoethanamine
(0.113 g, 1.31 mmol) and triethylamine (0.264 g, 2.61 mmol) in di-
chloromethane (5 mL) to give N-(2-azidoethyl)biphenyl-4-carbox-
N-(2-Azidoethyl)-2-naphthamide: As in the synthesis of N-(2-azi-
doethyl)tetradecanamide,
2-naphthoyl
chloride
(0.237 g,
1.24 mmol) was treated with 2-azidoethanamine (0.107 g,
1.24 mmol) and triethylamine (0.252 g, 2.49 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-2-naphthamide (0.234 g, 77%
1
amide (0.297 g, 85% yield). H NMR (300 MHz, CDCl3): d=7.88 (d,
2H), 7.67 (m, 4H), 7.44 (m, 3H), 6.63 (s, 1H), 3.67 (q, J=6.0, 1.8 Hz,
2H), 3.58 ppm (t, J=6.0 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=
167.8, 144.8, 140.1, 132.9, 129.2, 128.3, 127.8, 127.5, 127.4, 51.2,
39.7 ppm; HRMS (ESI) calcd for C15H14N4O: 266.1168 [M]+, found
266.1162.
1
yield). H NMR (300 MHz, CDCl3): d=8.69 (s, 1H), 7.84 (m, 4H), 7.50
(m, 2H), 6.97 (t, J=4.2 Hz, 1H), 3.68 (q, J=4.8, 1.2 Hz, 2H),
3.56 ppm (t, J=3.0 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=168.3,
135.1, 132.8, 131.5, 129.2, 128.8, 128.0, 127.9, 127.1, 123.8, 51.2,
39.8 ppm; HRMS (ESI) calcd for C13H12N4O: 240.1011 [M]+, found
240.1007.
N-(2-Azidoethyl)-4,5-dibromo-1-methyl-1H-pyrrole-2-carbox-
amide: As in the synthesis of N-(2-azidoethyl)tetradecanamide, 4,5-
dibromo-1-methyl-1H-pyrrole-2-carboxylic acid chloride (0.529 g,
1.38 mmol) was treated with 2-azidoethanamine (0.119 g,
1.38 mmol) and triethylamine (0.279 g, 2.75 mmol) in dichlorome-
thane (5 mL) to give N-(2-azidoethyl)-4,5-dibromo-1-methyl-1H-pyr-
N-(2-Azidoethyl)-4-heptylbenzamide: As in the synthesis of N-(2-
azidoethyl)tetradecanamide, 4-heptylbenzoyl chloride (0.289 g,
1.21 mmol) was treated with 2-azidoethanamine (0.104 g,
1.21 mmol) and triethylamine (0.245 g, 2.42 mmol) in dichlorome-
ChemBioChem 2010, 11, 396 – 410
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401

C. Melander et al.
role-2-carboxamide (0.331 g, 69% yield). 1H NMR (300 MHz,
CD3OD): d=6.84 (s, 1H), 3.91 (s, 3H), 3.50 (t, J=3.6 Hz, 2H),
3.25 ppm (t, J=1.5 Hz, 2H); 13C NMR (75 MHz, CD3OD): d=161.7,
127.7, 114.7, 111.4, 97.8, 50.4, 38.9, 35.1 ppm; HRMS (ESI) calcd for
C8H9N5O: 348.9174 [M]+, found 348.9183.
with a magnetic stir bar. Oxalyl chloride (0.400 g, 3.15 mmol) was
added dropwise, and the reaction mixture was allowed to stir for
1 h, then concentrated in vacuo. Dichloromethane (10 mL), 2-azi-
doethanamine (0.299 g, 3.47 mmol), and then triethylamine
(0.351 g, 3.47 mmol) was added to the crude mixture, and it was
allowed to stir for 2 h. The reaction mixture was then concentrated
in vacuo and purified by silica gel column chromatography (di-
chloromethane!methanol/dichloromethane 1:40 ) to give (E)-N-
(2-azidoethyl)-2-methyl-3-phenylacrylamide (0.698 g, 96% yield).
1H NMR (300 MHz, CDCl3): d=7.39 (m, 6H), 7.01 (s, 1H), 3.51 (t, J=
4.5 Hz, 2H), 3.45 (t, J=4.8 Hz, 2H), 2.13 ppm (s, 3H); 13C NMR
(75 MHz, CDCl3): d=170.5, 136.2, 134.5, 132.0, 130.3, 129.6, 128.9,
128.6, 128.3, 50.9, 39.8, 14.5 ppm; HRMS (ESI) calcd for C12H14N4O:
230.1168 [M]+, found 230.1165.
N-(2-Azidoethyl)-1H-pyrrole-2-carboxamide: As in the synthesis
of N-(2-azidoethyl)tetradecanamide, 1H-pyrrole-2-carboxylic acid
chloride (0.249 g, 1.17 mmol) was treated with 2-azidoethanamine
(0.101 g, 1.17 mmol) and triethylamine (0.237 g, 2.34 mmol) in di-
chloromethane (5 mL) to give N-(2-azidoethyl)-1H-pyrrole-2-carbox-
1
amide (0.137 g, 66% yield). H NMR (300 MHz, CD3OD): d=6.88 (d,
J=1.5 Hz, 1H), 6.77 (d, J=2.4 Hz, 1H), 6.14 (t, J=2.4 Hz, 1H), 3.46
(t, J=5.7 Hz, 2H), 3.37 ppm (q, J=5.7, 14.7 Hz, 2H); 13C NMR
(75 MHz, CD3OD): d=162.9, 125.6, 122.2, 121.9, 110.9, 109.2, 50.6,
38.9 ppm; HRMS (ESI) calcd for C7H9N5O: 179.0807 [M]+, found
179.0803.
N-(2-Azidoethyl)-2’,4’-difluoro-3-hydroxybiphenyl-4-carbox-
amide: 2’,4’-Difluoro-3-hydroxybiphenyl-4-carboxylic acid (0.301,
1.20 mmol), N,N-dimethylformamide (5 mL), N,N’-dicyclohexylcarbo-
diimide (0.248 g, 1.20 mmol), and n-methylmorpholine (0.25 mL)
were added to a 25 mL round-bottomed flask equipped with a
magnetic stirrer. The reaction mixture was cooled to 08C and al-
lowed to stir. Then, 2-azidoethanamine (0.1037 g, 1.20 mmol) was
added dropwise, and the mixture was allowed to warm slowly to
room temperature with stirring over 24 h. The reaction mixture
was diluted with water, extracted with dichloromethane, washed
with 1n HCl, saturated sodium bicarbonate, and brine, and then
concentrated in vacuo. The resulting residue was then purified by
silica gel column chromatography (methanol/dichloromethane
1:40!1:10) to give N-(2-azidoethyl)-2’,4’-difluoro-3-hydroxybiphen-
yl-4-carboxamide (0.232 g, 61% yield). 1H NMR (300 MHz, CDCl3):
d=7.60 (s, 1H), 7.47 (d, J=6.0 Hz, 1H), 7.29 (m, 2H), 7.02 (d, J=
6.6 Hz, 1H), 6.88 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d=170.3,
160.8, 135.1, 131.3, 131.2, 131.3, 131.2, 126.8, 118.8, 114.6, 112.0,
111.8, 104.8, 104.6, 104.3, 50.7, 39.3 ppm; HRMS (ESI) calcd for
C15H12F2N4O3: 318.0928 [M]+, found 318.0933.
N-(2-Azidoethyl)-4-bromo-1H-pyrrole-2-carboxamide: As in the
synthesis of N-(2-azidoethyl)tetradecanamide, 4-bromo-1H-pyrrole-
2-carboxylic acid chloride (0.408 g, 1.34 mmol) was treated with 2-
azidoethanamine (0.115 g, 1.34 mmol) and triethylamine (0.270 g,
2.67 mmol) in dichloromethane (5 mL) to give N-(2-azidoethyl)-4-
bromo-1H-pyrrole-2-carboxamide (0.299 g, 82% yield). 1H NMR
(300 MHz, CD3OD): d=6.93 (s, 1H), 6.80 (s, 1H), 3.49 (t, J=0.6 Hz,
2H), 3.41 ppm (t, J=0.9 Hz, 2H); 13C NMR (75 MHz, CD3OD): d=
161.7, 126.1, 122.0, 112.4, 96.4, 50.5, 38.9 ppm; HRMS (ESI) calcd for
C7H8BrN5O: 256.9912 [M]+, found 256.9908.
N-(2-Azidoethyl)-4-nonylbenzamide: As in the synthesis of N-(2-
azidoethyl)tetradecanamide, the acid chloride (0.354 g, 1.40 mmol)
was treated with 2-azidoethanamine (0.121 g, 1.40 mmol) and tri-
ethylamine (0.284 g, 2.80 mmol) in dichloromethane (5 mL) to give
N-(2-azidoethyl)-4-nonylbenzamide (0.339 g, 80% yield). 1H NMR
(300 MHz, CDCl3): d=6.21 (s, 1H), 3.40 (s, 4H), 2.19 (t, J=7.2 Hz,
2H), 1.62 (t, J=6.0 Hz, 2H), 1.22 (brs, 14H), 0.842 ppm (t, J=
6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=173.8, 51.1, 30.1, 36.8,
32.1, 29.8, 29.7, 29.6, 29.5, 29.2, 25.9, 22.9, 14.3 ppm; HRMS (ESI)
calcd for C14H28N4O: 268.2263 [M]+, found 268.2259.
General procedure for click reactions and subsequent Boc de-
protection: The terminal alkyne (1.0 equiv) was dissolved in etha-
nol/water/dichloromethane (1:1:1; ca. 9 mL per 0.300 g of terminal
alkyne). The appropriate azide (1.0 equiv) was added to this solu-
tion at room temperature, with vigorous stirring. Copper(II) sulfate
(15 mol%) and sodium ascorbate (45 mol%) were then added to
the solution sequentially. Mixtures were allowed to stir until com-
pletion of the reaction by TLC analysis (12–24 h). The solvents
were then removed in vacuo, and the resulting residue was dis-
solved in dichloromethane and purified by silica gel column chro-
matography (methanol/dichloromethane 1:40!1:10). To remove
the Boc protecting group, the resulting product was dissolved in
trifluoroacetic acid/dichloromethane (1:4), and the solution was al-
lowed to stir for 5 h. Upon completion, the reaction mixture was
concentrated in vacuo and then left under a high vacuum, over-
night. Then, methanol supplemented with HCl was added to the
product to form the HCl salt of the deprotected product, and this
was then concentrated in vacuo. The resulting residue was washed
N-(2-Azidoethyl)-4-nonylbenzamide: As in the synthesis of N-(2-
azidoethyl)tetradecanamide, the acid chloride (0.383 g, 1.43 mmol)
was treated with 2-azidoethanamine (0.124 g, 1.43 mmol) and tri-
ethylamine (0.290 g, 2.87 mmol) in dichloromethane (5 mL) to give
N-(2-azidoethyl)-4-nonylbenzamide (0.441 g, 97% yield). 1H NMR
(300 MHz, CDCl3): d=7.72 (d, 2H), 7.26 (t, 1H), 7.18 (d, 2H), 3.57 (q,
2H), 3.45 (t, 2H), 2.60 (t, 2H), 1.58 (m, 2H), 1.27 (m, 12H), 0.86 ppm
(t, 3H); 13C NMR (75 MHz, CDCl3): d=168.5, 147.3, 131.7, 128.8,
127.4, 50.9, 39.7, 36.1, 32.1, 31.5, 29.8, 29.7, 29.6, 29.5, 22.9,
14.4 ppm; HRMS (ESI) calcd for C18H28N4O: 316.2263 [M]+, found
316.2268.
N-(2-Azidoethyl)-4-octylbenzamide: As in the synthesis of N-(2-
azidoethyl)tetradecanamide, the acid chloride (0.262 g, 1.19 mmol)
was treated with 2-azidoethanamine (0.103 g, 1.19 mmol) and tri-
ethylamine (0.242 g, 2.39 mmol) in dichloromethane (5 mL) to give
N-(2-azidoethyl)-4-octylbenzamide (0.220 g, 69% yield). 1H NMR
(300 MHz, CDCl3): d=7.73 (d, 2H), 7.37 (t, 1H), 7.17 (d, 2H), 3.56 (q,
2H), 3.44 (t, 2H), 2.59 (t, 2H), 1.58 (m, 2H), 1.27 (m, 10H), 0.86 ppm
(t, 3H); 13C NMR (75 MHz, CDCl3): d=168.4, 147.3, 131.7, 128.8,
127.4, 50.9, 39.7, 36.1, 32.1, 31.5, 29.7, 29.5, 29.5, 22.9, 14.3 ppm;
HRMS (ESI) calcd for C17H28N4O: 302.2107 [M]+, found 302.2104.
with diethyl ether and then placed under
overnight.
a high vacuum
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-2-(phenylthio)acetamide hydrochloride (7q): tert-Butyl
2-amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.112 g,
0.405 mmol) was treated with N-(2-azidoethyl)-2-(phenylthio)acet-
amide (0.096 g, 0.405 mmol) according to the general click proce-
dure to give tert-butyl 2-amino-4-(5-(1-(2-(2-(phenylthio)acetami-
do)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR (300 MHz, CDCl3): d=7.45 (s, 1H), 7.19 (m, 5H), 7.13 (s,
(E)-N-(2-Azidoethyl)-2-methyl-3-phenylacrylamide: (E)-2-Methyl-3-
phenylacrylic acid (0.0.512 g, 3.15 mmol) and dichloromethane
(10 mL) were added to a 25 mL round-bottomed flask equipped
402
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 396 – 410

Aminoimidazole-Based Antibiofilm Agents
1H), 6.43 (s, 1H), 6.13 (brs, 2H), 4.29 (s, 2H), 3.68 (s, 2H), 3.57 (s,
2H), 2.56 (s, 2H), 2.35 (s, 2H), 1.52 ppm (m, 15H); 13C NMR
(75 MHz, CDCl3): d=187.8, 172.0, 169.1, 162.4, 156.9, 135.0, 129.5,
128.3, 126.8, 121.8, 85.00, 49.4, 39.9, 37.4, 29.4, 28.9, 28.4, 28.2,
27.6, 25.6 ppm; HRMS (ESI) calcd for C25H35N7O3S: 513.2522 [M]+,
found 513.2522. The carboxylate was subsequently deprotected to
give 7q (0.133 g, 73% yield). 1H NMR (300 MHz, CD3OD): d=8.47
(s, 1H), 7.30 (s, 4H), 7.19 (s, 1H), 6.58 (s, 1H), 4.66 (s, 2H), 3.76 (s,
2H), 3.64 (s, 2H), 2.84 (s, 2H), 2.34 (s, 2H), 1.73 (s, 2H), 1.64 (s, 2H),
1.26 ppm (s, 2H); 13C NMR (75 MHz, CD3OD): d=170.9, 159.3, 158.9,
147.2, 135.6, 128.1, 128.9, 127.6, 126.6, 108.7, 108.6, 52.0, 39.1, 37.5,
37.2, 36.6, 36.1, 30.8, 28.1, 27.9, 27.7, 24.2, 23.7, 23.6 ppm; HRMS
(ESI) calcd for C20H27N7OS: 413.1998 [M]+, found 413.1991.
cedure to give tert-butyl 2-amino-4-(5-(1-(2-(phenylsulfon-
amido)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxyl-
ate. H NMR (300 MHz, CDCl3): d=7.81 (d, J=6.9 Hz, 2H), 7.44 (m,
1
5H), 6.44 (s, 1H), 6.07 (s, 2H), 4.42 (t, J=5.7 Hz, 2H), 3.36 (t, J=
5.4 Hz, 2H), 2.57 (t, J=7.2 Hz, 2H), 2.20 (s, 2H), 1.47 (m, 13H),
1.29 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=202.9, 149.5, 148.2,
140.2, 132.8, 129.4, 127.1, 122.5, 106.5, 85.0, 53.7, 50.3, 42.9, 32.7,
31.2, 29.9, 29.2, 28.9, 28.2, 26.3, 25.5 ppm; HRMS (ESI) calcd for
C23H33N7O4S: 503.2315 [M]+, found 503.2310. The carboxylate was
1
subsequently deprotected to give 7n (0.142 g, 64% yield). H NMR
(300 MHz, CD3OD): d=7.81 (d, J=6.6 Hz, 2H), 7.56 (m, 4H), 6.47 (s,
1H), 4.62 (s, 2H), 3.41 (s, 2H), 2.78 (s, 2H), 2.48 (s, 2H), 1.72 (s, 2H),
1.63 (s, 2H), 1.42 ppm (s, 2H); 13C NMR (75 MHz, CD3OD): d=176.2,
165.6, 159.9, 147.3, 140.2, 132.8, 129.3, 127.7, 126.8, 108.5, 108.4,
515.8, 42.3, 28.1, 27.9, 27.6, 24.1 ppm; HRMS (ESI) calcd for
C18H25N7O2S: 403.1790 [M]+, found 403.1781.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)thiophene-2-sulfonamide hydrochloride (7i): tert-Butyl 2-
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.105 g,
0.379 mmol) was treated with N-(2-azidoethyl)thiophene-2-sulfon-
amide (0.099 g, 0.379 mmol) according to the general click proce-
dure to give tert-butyl 2-amino-4-(5-(1-(2-(thiophene-2-sulfon-
amido)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxyl-
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-2,3,4,5,6-pentamethylbenzenesulfonamide hydrochlo-
ride (7a): tert-Butyl 2-amino-4-(hept-6-ynyl)-1H-imidazole-1-carbox-
ylate (0.120 g, 0.434 mmol) was treated with N-(2-azidoethyl)-
2,3,4,5,6-pentamethylbenzenesulfonamide (0.141 g, 0.434 mmol)
according to the general click procedure to give tert-butyl 2-
amino-4-(5-(1-(2-(2,3,4,5,6-pentamethylphenylsulfonamido)ethyl)-
1
ate. H NMR (300 MHz, CDCl3): d=7.49 (s, 3H), 7.39 (s, 1H), 6.99 (s,
1H), 6.41 (brm, 3H), 4.43 (s, 2H), 3.42 (s, 2H), 2.54 (s, 2H), 2.09 (s,
2H), 1.51 (m, 13H), 1.19 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=
150.8, 149.4, 148.1, 141.1, 138.3, 132.2, 132.1, 127.7, 122.6, 85.2,
53.8, 50.2, 43.2, 31.2, 29.9, 29.2, 28.8, 28.1, 25.5 ppm; HRMS (ESI)
calcd for C21H31N7O4S2: 509.1879 [M]+, found 509.1879. The carbox-
ylate was subsequently deprotected to give 7i (0.098 g, 58%
1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR
(300 MHz, CDCl3): d=7.30 (s, 1H), 7.01 (s, 1H), 6.44 (s, 1H), 6.17 (s,
2H), 4.35 (s, 2H), 3.34 (s, 2H), 2.47 (s, 2H), 2.48 (s, 6H), 2.20 (m,
11H), 1.43 ppm (m, 15H); 13C NMR (75 MHz, CDCl3): d=149.6,
148.1, 139.7, 136.2, 134.9, 134.3, 122.4, 84.9, 53.7, 50.2, 42.5, 31.1,
29.3, 28.9, 28.4, 28.2, 25.6, 19.1, 17.9, 17.2 ppm; HRMS (ESI) calcd
for C28H43N7O4S: 573.3097 [M]+, found 573.3086. The carboxylate
was subsequently deprotected to give 7a (0.143 g, 76% yield).
1H NMR (400 MHz, CD3OD): d=8.11 (s, 1H), 6.42 (s, 1H), 4.48 (s,
2H), 3.34 (s, 2H), 2.65 (s, 2H), 2.40 (s, 8H), 2.12 (s, 9H), 1.58 (s, 4H),
1.19 ppm (s, 2H); 13C NMR (100 MHz, CD3OD): d=159.9, 147.3,
139.6, 136.1, 134.8, 134.0, 127.8, 127.6, 108.6, 108.4, 54.1, 51.5, 41.8,
36.5, 28.2, 28.1, 27.7, 24.1, 23.7, 18.2, 16.8, 16.1 ppm; HRMS (ESI)
calcd for C23H35N7O2S: 473.2573 [M]+, found 473.2565.
1
yield). H NMR (400 MHz, CD3OD): d=8.37 (s, 1H), 7.76 (s, 1H), 7.57
(s, 1H), 7.11 (s, 1H), 6.46 (s, 1H), 4.79 (s, 2H), 3.55 (s, 2H), 2.50 (s,
2H), 1.66–1.18 ppm (brm, 8H); 13C NMR (100 MHz, CD3OD): d=
175.6, 155.6, 147.2, 140.9, 132.7, 132.3, 127.8, 127.7, 108.8, 53.1,
42.2, 36.8, 28.1, 27.8, 27.5, 24.9, 24.3, 23.8 ppm; HRMS (ESI) calcd
for C16H23N7O2S2: 409.1355 [M]+, found 409.1354.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-5-(dimethylamino)naphthalene-1-sulfonamide hydro-
chloride (7x): tert-Butyl 2-amino-4-(hept-6-ynyl)-1H-imidazole-1-
carboxylate (0.112 g, 0.403 mmol) was treated with N-(2-azidoeth-
yl)-5-(dimethylamino)naphthalene-1-sulfonamide
(0.140 g,
(E)-N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-
1-yl)ethyl)-2-methyl-3-phenylacrylamide hydrochloride (7l): tert-
Butyl 2-amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate (0.1103 g,
0.397 mmol) was treated with (E)-N-(2-azidoethyl)-2-methyl-3-phe-
nylacrylamide (0.092 g, 0.397 mmol) according to the general click
procedure to give (E)-tert-butyl 2-amino-4-(5-(1-(2-(2-methyl-3-phe-
nylacrylamido)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-car-
0.403 mmol) according to the general click procedure to give tert-
butyl 2-amino-4-(5-(1-(2-(5-(dimethylamino)naphthalene-1-sulfon-
amido)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxyl-
1
ate. H NMR (300 MHz, CDCl3): d=8.48 (d, J=8.4 Hz, 1H), 8.24 (d,
J=8.4 Hz, 1H), 8.17 (d, J=7.2 Hz, 1H), 7.44 (m, 3H), 7.09 (s, 1H),
7.07 (d, J=7.5 Hz, 1H), 6.44 (s, 1H), 6.09 (brs, 2H), 4.32 (s, 2H),
3.36 (s, 2H), 2.52 (s, 2H), 2.22 (s, 2H), 1.43 (m, 13H), 1.22 ppm (s,
2H); 13C NMR (75 MHz, CDCl3): d=176.9, 152.1, 149.6, 148.0, 138.6,
135.1, 130.7, 130.1, 129.7, 129.4, 128.5, 123.3, 122.4, 119.1, 115.5,
84.9, 67.5, 50.3, 50.2, 45.6, 42.9, 37.9, 29.1, 28.9, 28.2, 28.0,
25.5 ppm; HRMS (ESI) calcd for C29H40N8O4S: 596.2893 [M]+, found
596.2881. The carboxylate was subsequently deprotected to give
1
boxylate. H NMR (300 MHz, CDCl3): d=7.28 (m, 7H), 6.94 (s, 2H),
6.06 (s, 2H), 4.46 (s, 2H), 3.79 (s, 2H), 2.61 (s, 2H), 2.27 (s, 2H), 1.99
(s, 3H), 1.49 (brs, 11H), 1.18 ppm (s, 4H); 13C NMR (75 MHz, CDCl3):
d=192.4, 191.3, 170.4, 150.9, 140.2, 136.2, 134.8, 131.6, 129.6,
128.6, 128.1, 122.1, 95.8, 85.2, 74.3, 53.7, 52.6, 50.7, 49.4, 40.1, 29.9,
29.3, 28.9, 28.2, 25.6, 14.4 ppm; HRMS (ESI) calcd for C27H37N7O3:
507.2958 [M]+, found 507.2942. The carboxylate was subsequently
deprotected to give 7l (0.148 g, 84% yield). 1H NMR (400 MHz,
CD3OD): d=8.78 (s, 1H), 7.33 (s, 4H), 7.26 (s, 1H), 7.19 (s, 1H), 6.42
(s, 1H), 4.78 (s, 2H), 3.84 (s, 2H), 2.87 (s, 2H), 2.44 (s, 2H), 1.99 (s,
3H), 1.76 (s, 2H), 1.59 (s, 2H), 1.42 ppm (s, 2H); 13C NMR (100 MHz,
CD3OD): d=174.7, 136.0, 134.4, 131.5, 129.3, 128.7, 128.3, 128.0,
127.6, 108.6, 94.6, 53.1, 39.2, 27.6, 27.9, 27.7, 27.6, 24.1, 23.8, 23.1,
14.3, 13.4 ppm; HRMS (ESI) calcd for C22H29N7O: 407.2434 [M]+,
found 407.2429.
1
7x (0.139 g, 65% yield). H NMR (400 MHz, CD3OD): d=8.70 (t, J=
4.8 Hz, 2H), 8.31 (s, 1H), 8.08 (s, 2H), 7.82 (s, 2H), 6.43 (s, 1H), 4.55
(s, 2H), 3.32 (s, 8H), 2.63 (s, 2H), 2.48 (s, 2H), 1.66 (s, 4H), 1.40 ppm
(s, 2H); 13C NMR (100 MHz, CD3OD): d=159.9, 147.3, 140.7, 136.8,
129.9, 129.3, 127.8, 127.7, 126.5, 119.3, 108.6, 108.4, 76.7, 67.3, 51.5,
42.3, 37.4, 36.5, 28.3, 27.9, 27.6, 24.1, 24.0, 23.7 ppm; HRMS (ESI)
calcd for C24H32N8O2S: 496.2369 [M]+, found 496.2359.
tert-Butyl 2-amino-4-(5-(1-(2-(phenylsulfonamido)ethyl)-1H-1,2,3-
triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate
hydrochloride
(7n): tert-Butyl 2-amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.114 g, 0.410 mmol) was treated with N-(2-azidoethyl)benzenesul-
fonamide (0.104 g, 0.410 mmol) according to the general click pro-
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)cinnamamide hydrochloride (7d): tert-Butyl 2-amino-4-
(hept-6-ynyl)-1H-imidazole-1-carboxylate (0.106 g, 0.384 mmol) was
ChemBioChem 2010, 11, 396 – 410
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
403

C. Melander et al.
treated with N-(2-azidoethyl)cinnamamide (0.083 g, 0.384 mmol)
according to the general click procedure to give (E)-tert-butyl 2-
amino-4-(5-(1-(2-cinnamamidoethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-
(75 MHz, CD3OD): d=175.7, 147.3, 128.5, 127.6, 125.1, 120.8, 108.6,
108.4, 105.6, 63.1, 59.3, 52.5, 48.6, 48.3, 48.1, 47.9, 47.7, 47.5, 57.3,
38.7, 336.6, 35.8, 36.6, 35.8, 31.8, 30.6, 29.4, 29.3, 29.3, 29.2, 28.0,
27.8, 27.7, 25.8, 24.1, 23.9, 23.3, 22.6 ppm; HRMS (ESI) calcd for
C22H39N7O: 417.3216 [M]+, found 417.3209.
1
imidazole-1-carboxylate. H NMR (300 MHz, CDCl3): d=7.67 (s, 1H),
7.51 (d, J=11.1 Hz, 1H), 7.28 (m, 6H), 6.45 (t, J=14.1 Hz, 2H), 6.08
(s, 2H), 4.47 (s, 2H), 3.79 (s, 2H), 2.57 (s, 2H), 2.23 (s, 2H), 1.50 (brs,
13H), 1.28 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=166.9, 150.4,
149.5, 148.3, 141.2, 138.9, 134.9, 129.9, 128.9, 128.0, 122.1, 120.8,
106.5, 84.8, 53.7, 49.5, 39.9, 31.1, 29.3, 28.9, 28.3, 28.2, 25.6 ppm;
HRMS (ESI) calcd for C26H35N7O3: 493.2801 [M]+, found 493.2800.
The carboxylate was subsequently deprotected to give 7d
(0.097 g, 59% yield). 1H NMR (400 MHz, CD3OD): d=8.10 (s, 1H),
7.49 (s, 3H), 7.32 (s, 3H), 6.30 (d, J=10.8 Hz, 1H), 6.39 (s, 1H), 4.66
(s, 2H), 3.83 (s, 2H), 2.73 (s, 2H), 2.40 (s, 2H), 1.57 (s, 2H), 1.55 (s,
2H), 1.36 ppm (s, 2H); 13C NMR (100 MHz, CD3OD): d=167.8, 160.5,
150.2, 147.3, 141.1, 134.8, 130.7, 129.9, 128.8, 127.8, 127.6, 124.9,
123.9, 120.2, 108.5, 108.3, 53.1, 39.3, 36.5, 30.6, 28.4, 28.1, 27.6,
26.5, 24.2, 24.1, 23.7 ppm; HRMS (ESI) calcd for C21H27N7O:
393.2277 [M]+, found 393.2273.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)dodecanamide hydrochloride (7 f): tert-Butyl 2-amino-4-
(hept-6-ynyl)-1H-imidazole-1-carboxylate (0.107 g, 0.387 mmol) was
treated with N-(2-azidoethyl)dodecanamide (0.104 g, 0.387 mmol)
according to the general click procedure to give tert-butyl 2-
amino-4-(5-(1-(2-dodecanamidoethyl)-1H-1,2,3-triazol-4-yl)pentyl)-
1H-imidazole-1-carboxylate. 1H NMR (300 MHz, CDCl3): d=7.36 (s,
1H), 6.94 (s, 1H), 6.49 (s, 1H), 6.23 (s, 2H), 4.46 (t, J=5.1 Hz, 2H),
3.74 (q, J=5.1, 5.7 Hz, 2H), 2.68 (t, J=7.5 Hz, 2H), 2.34 (t, J=
6.9 Hz, 2H), 2.16 (t, J=7.5 Hz, 2H), 1.54 (m, 13H), 1.47 (m, 2H), 1.24
(m, 18H), 0.87 ppm (t, J=6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=
174.3, 150.4, 149.5, 148.3, 138.5, 121.9, 106.5, 84.9, 49.5, 39.5, 36.6,
34.1, 32.1, 29.8, 29.8, 29.7, 29.6, 29.5, 29.3, 28.9, 28.2, 28.1, 29.0,
26.9. 25.6, 22.9, 14.3 ppm; HRMS (ESI) calcd for C29H51N7O3:
545.4053 [M]+, found 545.4053. The carboxylate was subsequently
deprotected to give 7 f (0.155 g, 83% yield). 1H NMR (400 MHz,
CD3OD): d=8.31 (s, 1H), 6.49 (s, 1H), 4.63 (s, 2H), 3.97 (s, 2H), 2.81
(s, 2H), 2.51 (t, J=7.2 Hz, 2H), 2.14 (t, J=7.2 Hz, 2H), 1.76 (s, 2H),
1.67 (s, 2H), 1.51 (s, 2H), 1.44 (s, 2H), 1.26 (brs, 16H), 0.71 ppm (s,
3H); 13C NMR (100 MHz, CD3OD): d=175.6, 159.4, 147.3, 127.6,
108.5, 51.3, 48.6, 38.8, 35.8, 31.9, 29.6, 29.5, 29.4, 29.3, 29.2, 28.2,
28.1, 27.7, 24.8, 24.2, 23.9, 22.6, 22.5, 13.4, 13.3 ppm; HRMS (ESI)
calcd for C24H43N7O: 445.3429 [M]+, found 445.3524.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)heptadec-8-enamide hydrochloride (7m): tert-Butyl 2-
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.098 g,
0.353 mmol) was treated with N-(2-azidoethyl)octadec-9-heptadec-
8-enamide (0.124 g, 0.353 mmol) according to the general click
procedure to give tert-butyl 2-amino-4-(5-(1-(2-heptadec-8-enami-
doethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR (300 MHz, CDCl3): d=7.26 (s, 1H), 6.81 (s, 1H), 6.42 (s, 2H),
5.88 (s, 1H), 5.27 (s, 2H), 4.38 (s, 2H), 3.65 (s, 2H), 2.60 (s, 2H), 2.09
(s, 2H), 1.53–1.01 (m, 35H), 0.79 ppm (t, J=6.3 Hz, 3H); 13C NMR
(75 MHz, CDCl3): d=174.2, 173.8, 148.3, 130.3, 130.1, 129.9, 129.8,
121.9, 85.1, 67.1, 49.5, 39.5, 39.1, 38.0, 37.7, 36.7, 36.6, 32.8, 32.3,
32.1, 31.7, 29.9, 29.8, 29.7, 29.5, 29.3, 29.2, 28.9, 28.7, 28.1, 28.0,
27.9, 27.4, 27.3, 27.3, 26.9, 25.9, 25.6, 25.3, 22.9, 22.8, 22.6, 14.3,
14.2 ppm; HRMS (ESI) calcd for C35H61N7O3: 627.4836 [M]+, found
627.4823. The carboxylate was subsequently deprotected to give
7m (0.128 g, 66% yield). 1H NMR (400 MHz, CD3OD): d=8.36 (s,
1H), 6.96 (s, 1H), 6.45 (s, 1H), 6.09 (s, 1H), 4.58 (s, 2H), 3.58 (s, 2H),
2.75 (s, 2H), 2.10 (s, 2H), 1.96 (s, 4H), 1.69 (s, 2H), 1.41 (m, 4H),
1.23 (s, 24H), 0.84 ppm (s, 3H); 13C NMR (100 MHz, CD3OD): d=
175.5, 158.8, 155.7, 147.3, 129.7, 129.6, 127.6, 108.5, 94.6, 76.7, 51.3,
50.4, 48.6, 48.4, 48.2, 47.9, 47.8, 47.5, 47.3, 38.9, 37.5, 36.6, 45.9,
45.8, 33.9, 32.6, 31.9, 31.5, 29.8, 29.5, 29.4, 29.3, 29.2, 29.1, 28.2,
27.8, 27.0, 25.8, 24.9, 24.2, 23.8, 22.5, 13.5, 13.3 ppm; HRMS (ESI)
calcd for C30H53N7O: 527.4312 [M]+, found 527.4298.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)tetradecanamide hydrochloride (7g): tert-Butyl 2-amino-
4-(hept-6-ynyl)-1H-imidazole-1-carboxylate (0.111 g, 0.401 mmol)
was treated with N-(2-azidoethyl)tetradecanamide (0.130 g,
0.401 mmol) according to the general click procedure to give tert-
butyl 2-amino-4-(5-(1-(2-tetradecanamidoethyl)-1H-1,2,3-triazol-4-
yl)pentyl)-1H-imidazole-1-carboxylate. 1H NMR (300 MHz, CDCl3):
d=7.26 (s, 1H), 6.85 (s, 1H), 6.42 (s, 1H), 6.05 (s, 1H), 4.38 (s, 2H),
3.66 (s, 2H), 2.60 (s, 2H), 2.11 (s, 2H), 2.08 (t, J=3.6 Hz, 2H), 1.41
(m, 13H), 1.32 (s, 2H), 1.17 (s, 18H), 0.77 ppm (t, J=6.6 Hz, 3H);
13C NMR (75 MHz, CDCl3): d=202.9, 174.3, 149.6, 148.3, 121.9,
100.1, 84.8, 49.5, 42.3, 39.5, 36.7, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5,
29.5, 29.3, 28.9, 28.1, 25.9, 25.6, 22.9, 14.3 ppm; HRMS (ESI) calcd
for C31H55N7O3: 573.4366 [M]+, found 573.4365. The carboxylate
was subsequently deprotected to give 7g (0.118 g, 87% yield).
1H NMR (400 MHz, CD3OD): d=8.51 (s, 1H), 6.53 (s, 1H), 4.71 (s,
2H), 3.75 (s, 2H), 2.89 (s, 2H), 2.54 (t, J=6.8 Hz, 2H), 2.17 (t, J=
6.8 Hz, 2H), 1.80 (s, 2H), 1.69 (s, 2H), 1.54 (s, 2H), 1.48 (s, 2H), 1.28
(s, 20H), 0.88 ppm (t, J=6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD):
d=175.6, 161.8, 147.2, 127.6, 127.4, 108.5, 52.3, 38.8, 36.6, 35.8,
31.9, 30.6, 29.7, 29.6, 29.5, 29.4, 29.2, 28.0, 27.9, 27.7, 25.8, 24.1,
23.6, 23.4, 22.6, 13.4 ppm; HRMS (ESI) calcd for C26H47N7O:
473.3842 [M]+, found 473.3834.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)decanamide hydrochloride (7e): tert-Butyl 2-amino-4-
(hept-6-ynyl)-1H-imidazole-1-carboxylate (0.114 g, 0.412 mmol) was
treated with N-(2-azidoethyl)decanamide (0.099 g, 0.412 mmol) ac-
cording to the general click procedure to give tert-butyl 2-amino-4-
(5-(1-(2-decanamidoethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-
1-carboxylate. 1H NMR (400 MHz, CDCl3): d=7.23 (s, 1H), 6.57 (s,
1H), 6.43 (s, 1H), 6.09 (s, 2H), 4.38 (s, 2H), 3.67 (s, 2H), 2.61 (t, J=
5.4 Hz, 2H), 2.08 (s, 2H), 2.08 (t, J=5.7 Hz, 2H), 1.42 (m, 15H), 1.17
(m, 14H), 0.79 ppm (t, J=4.5 Hz, 3H); 13C NMR (100 MHz, CDCl3):
d=174.2, 149.7, 148.4, 138.8, 121.9, 106.6, 84.9, 49.5, 39.5, 36.7,
32.1, 31.3, 30.6, 29.9, 29.7, 29.6, 29.5, 29.5, 29.3, 29.0, 28.3, 28.2,
25.9, 25.6, 25.5, 22.9, 14.3 ppm; HRMS (ESI) calcd for C27H47N7O3:
517.3740 [M]+, found 517.3736. The carboxylate was subsequently
deprotected to give 7e (0.137 g, 73% yield). 1H NMR (300 MHz,
CD3OD): d=8.55 (s, 1H), 6.53 (s, 1H), 4.72 (s, 2H), 3.75 (s, 2H), 2.89
(s, 2H), 2.54 (t, J=6.8 Hz, 2H), 2.16 (t, J=7.2 Hz, 2H), 1.81 (s, 2H),
1.69 (s, 2H), 1.49 (m, 4H), 1.28 (s, 12H), 0.89 ppm (t, 3H); 13C NMR
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)palmitamide hydrochloride (7h): tert-Butyl 2-amino-4-
(hept-6-ynyl)-1H-imidazole-1-carboxylate (0.087 g, 0.313 mmol) was
treated with N-(2-azidoethyl)palmitamide (0.102 g, 0.313 mmol) ac-
cording to the general click procedure to give tert-butyl 2-amino-4-
(5-(1-(2-palmitamidoethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imid-
1
azole-1-carboxylate. H NMR (300 MHz, CDCl3): d=7.29 (s, 1H), 6.78
(t, J=4.5 Hz, 1H), 6.41 (s, 1H), 6.19 (s, 2H), 4.39 (t, J=5.4 Hz, 2H),
3.67 (q, J=5.5, 4.8 Hz, 2H), 2.61 (t, J=6.9 Hz, 2H), 2.12 (t, J=
6.5 Hz, 2H), 2.07 (t, J=7.5 Hz, 2H), 1.42 (m, 13H), 1.42 (m, 2H), 1.32
(m, 22H), 0.81 ppm (t, J=5.4 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=
404
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 396 – 410

Aminoimidazole-Based Antibiofilm Agents
176.8, 174.3, 173.9, 149.5, 148.3, 121.9, 106.4, 85.0, 67.2, 51.1, 49.5,
39.5, 39.1, 38.1, 36.8, 36.7, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5, 29.3,
28.9, 28.3, 28.2, 27.9, 25.9, 25.6, 22.9, 14.3 ppm; HRMS (ESI) calcd
for C33H59N7O3: 601.4679 [M]+, found 601.4671. The carboxylate
was subsequently deprotected to give 7h (0.155 g, 92% yield).
1H NMR (300 MHz, CD3OD): d=8.59 (s, 1H), 6.53 (s, 1H), 4.72 (s,
2H), 3.75 (s, 2H), 2.90 (s, 2H), 2.54 (s, 2H), 2.16 (t, J=7.2 Hz, 2H),
1.80 (s, 2H), 1.69 (s, 2H), 1.51 (s, 4H), 1.28 (brs, 24H), 0.71 ppm (t,
J=6.3 Hz, 3H); 13C NMR (75 MHz, CD3OD): d=175.7, 147.2, 145.6,
128.4, 127.6, 108.6, 67.2, 52.7, 37.4, 36.5, 35.8, 31.9, 29.7, 29.5, 29.4,
29.2, 28.0, 27.9, 27.8, 27.7, 25.8, 24.1, 23.6, 23.2, 22.6 ppm; HRMS
(ESI) calcd for C28H51N7O: 501.4155 [M]+, found 501.4143.
1H NMR (300 MHz, CDCl3): d=8.18 (s, 1H), 7.36 (d, J=5.7 Hz, 2H),
7.24 (s, 1H), 6.84 (t, J=8.1 Hz, 1H), 6.43 (s, 1H), 6.04 (s, 2H), 4.49 (s,
2H), 3.89 (s, 2H), 2.53 (s, 2H), 2.09 (s, 2H), 1.44 (m, 13H), 1.24 ppm
(s, 2H); 13C NMR (75 MHz, CDCl3): d=165.9, 164.8, 164.6, 161.5,
137.5, 122.3, 111.0, 110.7, 107.5, 107.1, 106.8, 85.1, 49.4, 40.5, 29.3,
28.9, 28.2, 25.5 ppm; HRMS (ESI) calcd for C22H32N8O3: 456.2597
[M]+, found 456.2590. The carboxylate was subsequently depro-
1
tected to give 7c (0.129 g, 94% yield). H NMR (400 MHz, CD3OD):
d=8.65 (s, 1H), 7.41 (s, 2H), 7.17 (s, 1H), 6.50 (s, 1H), 4.74 (s, 2H),
3.98 (s, 2H), 2.88 (s, 2H), 2.49 (s, 2H), 1.78 (m, 4H), 1.44 ppm (s,
2H); 13C NMR (100 MHz, CD3OD): d=166.3, 164.5, 162.0, 161.9,
137.4, 110.6, 110.3, 110.2, 108.5, 107.1, 106.8, 59.3, 52.4, 39.5, 36.5,
27.9, 27.8, 27.6, 24.1, 23.5, 23.3 ppm; HRMS (ESI) calcd for
C17H24N8O: 356.2073 [M]+, found 356.2080.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-4,5-dibromo-1-methyl-1H-pyrrole-2-carboxamide hydro-
chloride (7k): tert-Butyl 2-amino-4-(hept-6-ynyl)-1H-imidazole-1-
carboxylate (0.116 g, 0.417 mmol) was treated with N-(2-azidoeth-
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-4-tert-butylbenzamide hydrochloride (7p): tert-Butyl 2-
yl)-4,5-dibromo-1-methyl-1H-pyrrole-2-carboxamide
0.417 mmol) according to the general click procedure to give tert-
butyl 2-amino-4-(5-(1-(2-(4,5-dibromo-1-methyl-1H-pyrrole-2-car-
(0.145 g,
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.070 g,
0.253 mmol) was treated with N-(2-azidoethyl)-4-tert-butylbenz-
amide (0.062 g, 0.253 mmol) according to the general click proce-
dure to give tert-butyl 2-amino-4-(5-(1-(2-(4-tert-butylbenzami-
do)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR (300 MHz, CDCl3): d=7.71 (d, J=7.5 Hz, 2H), 7.64 (t, J=
5.7 Hz, 1H), 7.36 (d, J=7.5 Hz, 2H), 7.27 (s, 1H), 6.42 (s, 1H), 5.99
(s, 2H), 4.51 (t, J=5.7 Hz, 2H), 3.88 (q, J=5.1, 5.3 Hz, 2H), 2.58 (t,
J=7.5 Hz, 2H), 2.22 (t, J=7.5 Hz, 2H), 1.52 (m, 13H), 1.21 ppm (m,
11H); 13C NMR (75 MHz, CDCl3): d=168.2, 155.3, 150.4, 149.6, 148.3,
138.9, 131.2, 127.3, 125.6, 122.2, 106.5, 84.8, 49.4, 40.2, 35.1, 31.3,
31.1, 29.3, 28.9, 28.3, 28.2, 28.1, 25.6 ppm; HRMS (ESI) calcd for
C28H41N7O3: 523.3271 [M]+, found 523.3270. The carboxylate was
boxamido)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carbox-
1
ylate. H NMR (300 MHz, CDCl3): d=7.76 (s, 1H), 7.32 (s, 1H), 6.81
(s, 1H), 6.49 (s, 1H), 6.17 (s, 2H), 4.52 (s, 2H), 3.94 (s, 3H), 3.84 (s,
2H), 2.65 (t, J=7.2 Hz, 2H), 2.30 (m, 2H), 1.58 (m, 13H), 1.35 (m,
2H); 13C NMR (75 MHz, CDCl3): d=161.2, 149.6, 148.2, 138.8, 127.4,
122.2, 114.9, 111.9, 106.5, 98.2, 84.5, 49.5, 39.7, 35.9, 29.3, 28.9, 28.2,
28.0, 27.7, 25.6 ppm; HRMS (ESI) calcd for C23H32N8O3: 626.0964
[M]+, found 626.0960. The carboxylate was subsequently depro-
1
tected to give 7k (0.151 g, 64% yield). H NMR (400 MHz, CD3OD):
d=8.24 (s, 1H), 6.86 (s, 1H), 6.56 (s, 1H), 4.71 (s, 2H), 3.81 (s, 5H),
2.78 (s, 2H), 2.44 (t, J=7.2 Hz, 2H), 1.69 (s, 2H), 1.59 (s, 2H),
1.37 ppm (s, 2H); 13C NMR (100 MHz, CD3OD): d=161.4, 150.2,
159.8, 159.4, 147.3, 127.6, 127.3, 114.9, 111.6, 108.4, 97.9, 51.3, 39.0,
35.2, 28.3, 28.0, 27.6, 24.2, 23.8 ppm; HRMS (ESI) calcd for
C18H24Br2N8O: 526.0439 [M]+, found 526.0425.
1
subsequently deprotected to give 7p (0.105 g, 90% yield). H NMR
(400 MHz, CD3OD): d=8.48 (s, 1H), 7.76 (d, J=6.3 Hz, 2H), 7.48 (d,
J=6.3 Hz, 2H), 6.50 (s, 1H), 4.84 (s, 2H), 3.95 (s, 2H), 2.85 (s, 2H),
2.46 (t, J=5.4 Hz, 2H), 1.73 (s, 2H), 1.57 (m, 2H), 1.39 (s, 2H),
1.31 ppm (s, 9H); 13C NMR (100 MHz, CD3OD): d=169.2, 155.6,
147.3, 130.8, 129.9, 127.6, 127.2, 127.1, 126.7, 125.4, 125.3, 119.5,
108.6, 108.5, 52.3, 39.4, 34.6, 30.4, 27.9, 27.8, 27.6, 24.1, 23.2,
22.2 ppm; HRMS (ESI) calcd for C23H33N7O: 423.2747 [M]+, found
423.2741.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-4-bromo-1H-pyrrole-2-carboxamide hydrochloride (7z):
tert-Butyl
2-amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.104 g, 0.375 mmol) was treated with N-(2-azidoethyl)-4-bromo-
1H-pyrrole-2-carboxamide (0.131 g, 0.375 mmol) according to the
general click procedure to give tert-butyl 2-amino-4-(5-(1-(2-(4-
bromo-1H-pyrrole-2-carboxamido)ethyl)-1H-1,2,3-triazol-4-yl)pen-
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-2-naphthamide hydrochloride (7b): tert-Butyl 2-amino-4-
(hept-6-ynyl)-1H-imidazole-1-carboxylate (0.095 g, 0.344 mmol) was
treated with N-(2-azidoethyl)-2-naphthamide (0.083 g, 0.344 mmol)
according to the general click procedure to give tert-butyl 4-(5-(1-
(2-(2-naphthamido)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-2-amino-1H-
1
tyl)-1H-imidazole-1-carboxylate. H NMR (300 MHz, CDCl3): d=11.44
(s, 1H), 7.84 (s, 1H), 7.28 (s, 1H), 6.84 (s, 1H), 6.74 (s, 1H), 6.46 (s,
1H), 4.89 (s, 2H), 3.82 (s, 2H), 2.57 (s, 2H), 2.28 (s, 2H), 1.55 (m,
13H), 1.29 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d=186.8, 161.4,
150.7, 149.3, 148.2, 137.6, 126.3, 122.5, 122.1, 113.1, 100.4, 96.9,
85.4, 49.6, 29.1, 28.7, 28.2, 27.7, 25.5 ppm; HRMS (ESI) calcd for
C22H31BrN8O3: 534.1703 [M]+, found 534.1705. The carboxylate was
1
imidazole-1-carboxylate. H NMR (300 MHz, CDCl3): d=8.24 (s, 1H),
8.01 (s, 1H), 7.72 (m, 4H), 7.39 (m, 2H), 7.26 (s, 1H), 6.33 (s, 1H),
6.04 (s, 2H), 4.51 (s, 2H), 3.88 (s, 2H), 2.48 (t, 2H), 2.12 (s, 2H), 1.47
(m, 13H), 1.17 ppm (s, 2H); 13C NMR (75 MHz, CDCl3): d=168.5,
150.4, 149.5, 148.3, 138.6, 134.9, 132.7, 131.4, 129.2, 128.5, 128.1,
127.9, 126.8, 124.0, 122.3, 106.5, 84.9, 49.4, 40.4, 29.9, 29.2, 28.9,
28.6, 28.2, 27.9, 28.6, 27.9, 27.8, 25.6 ppm; HRMS (ESI) calcd for
C28H35N7O3: 517.2801 [M]+, found 517.2792. The carboxylate was
1
subsequently deprotected to give 7z (0.129 g, 73% yield). H NMR
(400 MHz, CD3OD): d=8.42 (s, 1H), 6.93 (s, 1H), 6.82 (s, 1H), 6.47 (s,
1H), 4.76 (s, 2H), 3.87 (s, 2H), 2.79 (s, 2H), 2.45 (s, 2H), 1.69 (s, 2H),
1.60 (s, 2H), 1.37 ppm (s, 2H); 13C NMR (100 MHz, CD3OD): d=
161.4, 159.5, 159.1, 147.3, 147.2, 127.6, 125.8, 122.3, 122.1, 112.8,
108.6, 108.5, 96.4, 51.9, 38.9, 28.0, 27.9, 27.6, 24.1, 23.6 ppm; HRMS
(ESI) calcd for C17H23BrN8O: 434.1178 [M]+, found 434.1171.
1
subsequently deprotected to give 7b (0.140 g, 90% yield). H NMR
(400 MHz, CD3OD): d=8.38 (s, 1H), 7.96 (m, 3H), 7.83 (t, J=7.2 Hz,
2H), 7.58 (t, J=6.4 Hz, 2H), 7.33 (s, 1H), 6.22 (s, 1H), 5.89 (s, 2H),
4.54 (t, J=5.6 Hz, 2H), 3.72 (q, J=5.2, 3.3 Hz, 2H), 2.56 (q, J=7.6,
7.2 Hz, 2H), 2.24 (t, J=7.6 Hz, 2H), 1.54 (t, J=6.8 Hz, 2H), 1.47 (t,
J=7.2 Hz, 2H), 1.23 (m, 4H) ppm; 13C NMR (100 MHz, CD3OD): d=
167.8, 161.1, 148.7, 147.4, 134.8, 132.8, 132.2, 129.5, 128.9, 128.6,
128.3, 128.2, 127.5, 126.4, 124.5, 122.9, 110.4, 65.8, 49.1, 32.3, 29.4,
28.7, 26.3, 25.7 ppm; HRMS (ESI) calcd for C23H27N7O: 417.2277
[M]+, found 417.2274.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-1H-pyrrole-2-carboxamide hydrochloride (7c): tert-Butyl
2-amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.097 g,
0.349 mmol) was treated with N-(2-azidoethyl)-1H-pyrrole-2-carbox-
amide (0.063 g, .349 mmol) according to the general click proce-
dure to give tert-butyl 4-(5-(1-(2-(1H-pyrrole-2-carboxamido)ethyl)-
1H-1,2,3-triazol-4-yl)pentyl)-2-amino-1H-imidazole-1-carboxylate.
ChemBioChem 2010, 11, 396 – 410
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
405

C. Melander et al.
1
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-2-iodobenzamide hydrochloride (7y): tert-Butyl 2-amino-
4-(hept-6-ynyl)-1H-imidazole-1-carboxylate (0.083 g, 0.299 mmol)
was treated with N-(2-azidoethyl)-2-iodobenzamide (0.095 g,
0.299 mmol) according to the general click procedure to give tert-
butyl 2-amino-4-(5-(1-(2-(2-iodobenzamido)ethyl)-1H-1,2,3-triazol-4-
yl)pentyl)-1H-imidazole-1-carboxylate. 1H NMR (300 MHz, CDCl3):
d=7.74 (d, J=7.5 Hz, 2H), 7.57 (t, J=4.5 Hz, 1H), 7.36 (s, 1H), 7.22
(s, 2H), 6.97 (m, 1H), 6.39 (s, 1H), 5.97 (s, 2H), 4.48 (t, J=5.1 Hz,
2H), 3.85 (m, 2H), 2.44 (t, J=7.5 Hz, 2H), 2.18 (t, J=6.6 Hz, 2H),
1.45 (m, 13H), 1.18 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d=
170.3, 150.4, 149.5, 148.2, 141.8, 139.9, 138.7, 131.3, 128.4, 128.3,
122.3, 106.5, 92.8, 84.9, 49.2, 40.5, 29.2, 28.9, 28.3, 28.2, 28.0,
25.5 ppm; HRMS (ESI) calcd for C24H32IN7O3: 593.1611 [M]+, found
593.1603. The carboxylate was subsequently deprotected to give
7y (0.125 g, 79% yield). 1H NMR (400 MHz, CD3OD): d=8.35 (s,
1H), 7.88 (d, J=7.8 Hz, 1H), 7.45 (t, J=6.9 Hz, 1H), 7.34 (d, J=
6.9 Hz, 1H), 7.15 (t, J=7.5 Hz, 1H), 6.47 (s, 1H), 4.78 (s, 2H), 3.99 (s,
2H), 2.83 (s, 2H), 2.44 (t, J=6.9 Hz, 2H), 1.76 (s, 2H), 1.64 (s, 2H),
1.29 ppm (s, 2H); 13C NMR (75 MHz, CD3OD): d=171.6, 159.2, 147.3,
142.1, 139.8, 131.2, 128.2, 127.7, 124.4, 92.1, 52.2, 40.8, 39.4, 31.2,
28.3, 28.1, 27.7, 24.1, 20.9, 15.9, 10.7 ppm; HRMS (ESI) calcd for
C19H24IN7O: 493.1087 [M]+, found 493.1085.
give 7t (0.164 g, 96% yield). H NMR (400 MHz, CD3OD): d=8.28 (s,
1H), 7.67 (d, J=7.6 Hz, 2H), 7.21 (d, J=7.6 Hz, 2H), 6.43 (s, 1H),
4.72 (s, 2H), 3.87 (s, 2H), 2.74 (s, 2H), 2.58 (t, J=7.6 Hz, 2H), 2.31 (t,
J=6.8 Hz, 2H), 1.66 (s, 2H), 1.55 (s, 4H), 1.35 (s, 2H), 1.25 (s, 6H),
0.66 ppm (t, J=8.8 Hz, 3H); 13C NMR (100 MHz, CD3OD): d=169.2,
159.5, 147.4, 147.3, 131.2, 108.4, 51.2, 48.5, 48.3, 48.1, 47.9, 47.7,
47.5, 47.3, 39.5, 36.5, 35.6, 31.6, 31.7, 31.2, 28.8, 28.2, 27.9, 27.7,
24.1, 23.9, 22.6, 13.3, 9.9 ppm; HRMS (ESI) calcd for C25H37N7O:
451.3059 [M]+, found 451.3058.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-4-butylbenzamide hydrochloride (7r): tert-Butyl 2-
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
0.443 mmol) was treated with N-(2-azidoethyl)-4-butylbenzamide
(0.109 g, 0.443 mmol) according to the general click procedure to
give tert-butyl 2-amino-4-(5-(1-(2-(4-butylbenzamido)ethyl)-1H-
(0.123 g,
1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR
(300 MHz, CDCl3): d=7.67 (s, 1H), 7.67 (d, J=7.5 Hz, 2H), 7.29 (s,
1H), 7.11 (d, J=7.5 Hz, 2H), 6.42 (s, 1H), 6.25 (s, 2H), 4.48 (s, 2H),
3.83 (s, 2H), 2.56 (t, J=7.2 Hz, 4H), 2.21 (s, 2H), 1.47 (m, 13H), 1.21
(m, 4H), 0.83 ppm (t, J=7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=
168.3, 140.6, 149.5, 148.2, 147.1, 138.4, 131.5, 128.7, 127.4, 122.2,
106.4, 84.9, 49.4, 40.2, 35.7, 33.5, 29.2, 28.9, 28.3, 28.1, 27.9, 25.5,
22.4, 14.1 ppm; HRMS (ESI) calcd for C28H41N7O3: 523.3271 [M]+,
found 523.3261. The carboxylate was subsequently deprotected to
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-4-heptylbenzamide hydrochloride (7u): tert-Butyl 2-
1
give 7r (0.191 g, 94% yield). H NMR (300 MHz, CD3OD): d=8.43 (s,
1H), 7.69 (s, 2H), 7.22 (s, 2H), 6.44 (s, 1H), 4.73 (s, 2H), 3.89 (s, 2H),
3.34 (s, 2H), 2.59 (s, 4H), 2.41 (s, 2H), 1.54 (s, 4H), 1.29 (s, 4H),
0.88 ppm (t, J=6.9 Hz, 3H); 13C NMR (75 MHz, CD3OD): d=169.2,
165.1, 160.3, 158.1, 147.4, 141.4, 131.2, 128.5, 127.7, 127.3, 126.9,
92.9, 39.6, 36.3, 33.4, 30.9, 28.0, 27.7, 24.1, 22.2, 13.2, 12.1, 10.9, 8.9,
6.2, 2.5 ppm; HRMS (ESI) calcd for C23H33N7O: 423.2747 [M]+, found
423.2738.
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.125 g,
0.449 mmol) was treated with N-(2-azidoethyl)-4-heptylbenzamide
(0.129 g, 0.449 mmol) according to the general click procedure to
give tert-butyl 2-amino-4-(5-(1-(2-(4-heptylbenzamido)ethyl)-1H-
1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR
(400 MHz, CDCl3): d=7.78 (s, 1H), 7.65 (d, 2H), 7.27 (s, 1H), 7.09 (d,
2H), 6.39 (s, 1H), 6.22 (s, 2H), 4.47 (s, 2H), 3.81 (s, 2H), 3.96 (s, 4H),
2.20 (s, 2H), 2.05 (s, 2H), 1.48 (m, 12H), 1.17 (m, 8H), 0.77 (t, J=
6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=168.3, 149.6, 148.2,
147.1, 138.6, 131.5, 128.6, 127.4, 122.1, 106.4, 84.8, 67.1, 49.4, 40.2,
37.9, 36.9, 31.9, 31.3, 31.1, 29.8, 28.4, 29.3, 28.9, 28.3, 28.1, 28.0,
25.5, 22.8, 14.3 ppm; HRMS (ESI) calcd for C31H47N7O3: 565.3704
[M]+, found 656.3737. The carboxylate was subsequently depro-
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-4-pentylbenzamide hydrochloride (7s): tert-Butyl 2-
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
0.408 mmol) was treated with N-(2-(4-(5-(2-amino-1H-imidazol-4-yl)-
pentyl)-1H-1,2,3-triazol-1-yl)ethyl)biphenyl-4-carboxamide hydro-
(0.113 g,
chloride (0.106 g, 0.408 mmol) according to the general click proce-
dure to give tert-butyl 2-amino-4-(5-(1-(2-(4-pentylbenzamido)eth-
yl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate. 1H NMR
(300 MHz, CDCl3): d=7.75 (s, 1H), 7.67 (d, J=7.5 Hz, 2H), 7.27 (s,
1H), 7.12 (d, J=7.8 Hz, 2H), 4.48 (s, 2H), 3.84 (s, 2H), 2.55 (m, 4H),
2.22 (s, 2H), 1.50 (s, 13H), 1.23 (s, 8H), 0.80 ppm (t, J=5.7 Hz, 3H);
13C NMR (75 MHz, CDCl3): d=168.3, 150.5, 149.5, 148.2, 147.2,
138.6, 131.5, 128.7, 127.4, 106.4, 84.8, 49.4, 40.2, 35.9, 31.6, 31.0,
29.3, 28.9, 28.1, 27.9, 25.6, 22.6, 14.2 ppm; HRMS (ESI) calcd for
C29H43N7O3: 537.3427 [M]+, found 537.3420. The carboxylate was
1
tected to give 7u (0.194 g, 86% yield). H NMR (300 MHz, CD3OD):
d=8.25 (s, 1H), 7.71 (d, J=5.4 Hz, 2H), 7.24 (d, J=5.7 Hz, 2H), 6.45
(s, 1H), 4.74 (s, 2H), 3.90 (s, 2H), 2.63 (s, 2H), 2.61 (t, 2H), 2.41 (s,
2H), 1.58 (m, 6H), 1.26 (m, 10H), 0.85 ppm (t, J=4.8 Hz, 3H);
13C NMR (100 MHz, CD3OD): d=169.2, 150.2, 147.4, 131.2, 128.5,
127.6, 127.3, 108.4, 51.4, 39.6, 36.5, 35.6, 31.8, 331.3, 29.1, 29.1,
28.3, 28.0, 27.7, 24.1, 22.6, 13.4, 13.3 ppm; HRMS (ESI) calcd for
C26H39N7O: 465.3216 [M]+, found 465.3207.
1
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-4-hexylbenzamide hydrochloride (7t): tert-Butyl 2-
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
0.349 mmol) was treated with N-(2-azidoethyl)-4-hexylbenzamide
(0.096 g, 0.349 mmol) according to the general click procedure to
give tert-butyl 2-amino-4-(5-(1-(2-(4-hexylbenzamido)ethyl)-1H-
subsequently deprotected to give 7s (0.178 g, 92% yield). H NMR
(300 MHz, CD3OD): d=8.41 (s, 1H), 7.69 (d, J=7.5 Hz, 2H), 7.21 (d,
J=7.2 Hz, 2H), 6.46 (s, 1H), 4.84 (s, 2H), 3.90 (s, 2H), 2.79 (s, 2H),
2.58 (t, J=7.8 Hz, 2H), 2.39 (t, J=7.2 Hz, 2H), 1.68 (s, 2H), 1.55 (s,
4H), 1.26 (s, 6H), 0.84 ppm (t, J=6.6 Hz, 3H); 13C NMR (75 MHz,
CD3OD): d=169.2, 159.5, 158.9, 147.5, 147.3, 131.1, 128.5, 127.6,
127.4, 108.5, 54.2, 51.9, 39.5, 35.6, 31.4, 30.9, 30.6, 28.4, 27.9, 27.6,
26.9, 24.1, 23.5, 22.9, 22.4, 13.3 ppm; HRMS (ESI) calcd for
C24H35N7O: 437.2903 [M]+, found 437.2892.
(0.097 g,
1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR
(300 MHz, CDCl3): d=7.65 (d, J=8.1 Hz, 2H), 7.55 (t, J=6.7 Hz, 1H),
7.26 (s, 1H), 7.09 (d, J=7.8 Hz, 2H), 6.39 (s, 1H), 6.04 (s, 2H), 4.48
(t, J=4.8 Hz, 2H), 3.83 (q, J=5.6, 4.8 Hz, 2H), 2.56 (q, J=7.5,
8.1 Hz, 4H), 2.24 (t, J=6.9 Hz, 2H), 1.50 (m, 13H), 1.21 (m, 1H),
0.77 ppm (t, J=5.7 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=168.3,
148.3, 147.3, 138.7, 131.4, 128.7, 127.4, 122.2, 106.4, 84.9, 73.9, 49.5,
40.2, 36.0, 31.8, 31.3, 29.9, 29.3, 29.1, 28.9, 28.2, 28.1, 28.0, 25.6,
22.8, 14.3 ppm; HRMS (ESI) calcd for C30H45N7O3: 551.3584 [M]+,
found 551.3581. The carboxylate was subsequently deprotected to
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-2,4,6-trichlorobenzamide hydrochloride (7o): tert-Butyl
2-amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.088 g,
0.318 mmol) was treated with N-(2-azidoethyl)-2,4,6-trichlorobenz-
amide (0.093 g, 0.318 mmol) according to the general click proce-
dure to give tert-butyl 2-amino-4-(5-(1-(2-(2,4,6-trichlorobenzami-
406
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 396 – 410

Aminoimidazole-Based Antibiofilm Agents
do)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR (300 MHz, CDCl3): d=8.22 (s, 1H), 7.34 (s, 1H), 7.26 (s, 2H),
6.42 (s, 1H), 5.97 (s, 2H), 4.51 (s, 2H), 3.94 (s, 2H), 2.45 (t, 2H), 2.20
(s, 2H), 1.47 (m 13H), 1.21 ppm (m, 2H); 13C NMR (75 MHz, CDCl3):
d=164.7, 148.0, 135.7, 134.6, 132.9, 128.7, 128.1, 127.3, 122.3, 84.9,
53.7, 49.3, 39.9, 31.8, 29.2, 28.9, 28.2, 25.5, 22.8, 14.3 ppm; HRMS
(ESI) calcd for C24H30Cl3N7O3: 569.1476 [M]+, found 569.1477. The
carboxylate was subsequently deprotected to give 7o (0.116 g,
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-4-octylbenzamide hydrochloride (7v): tert-Butyl 2-
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.078 g,
0.282 mmol) was treated with N-(2-azidoethyl)-4-octylbenzamide
(0.085 g, 0.282 mmol) according to the general click procedure to
give tert-butyl 2-amino-4-(5-(1-(2-(4-octylbenzamido)ethyl)-1H-1,2,3-
triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate. 1H NMR (300 MHz,
CDCl3): d=7.64 (s, 2H), 7.47 (s, 1H), 7.21 (s, 1H), 7.09 (s, 2H), 6.47
(brs, 3H), 4.48 (s, 2H), 3.84 (s, 2H), 2.53 (s, 4H), 2.28 (t, J=15.9 Hz,
2H), 1.51 (brs, 6H), 1.18 (brs, 10H), 0.77 ppm (t, J=6.6 Hz, 3H);
13C NMR (75 MHz, CDCl3): d=180.7, 180.2, 168.3, 147.4, 142.4,
139.2, 131.4, 128.8, 127.4, 107.4, 49.6, 40.2, 36.1, 32.1, 31.4, 29.9,
29.6, 29.5, 28.8, 28.2, 28.1, 25.4, 22.9, 14.3 ppm; HRMS (ESI) calcd
for C32H49N7O3: 579.3896 [M]+, found 579.3890. The carboxylate
was subsequently deprotected to give 7v (0.097 g, 67% yield).
1H NMR (400 MHz, CD3OD): d=7.65 (s, 2H), 7.23 (s, 3H), 6.45 (s,
1H), 3.97 (s, 4H), 2.60 (s, 4H), 2.47 (s, 2H), 1.57 (m, 6H), 1.27 (brm,
12H), 0.83 ppm (t, J=6.4 Hz, 3H); 13C NMR (100 MHz, CD3OD): d=
183.5, 172.0, 169.3, 161.8, 153.9, 147.4, 131.2, 128.5, 127.7, 127.3,
119.1, 108.8, 104.7, 94.6, 63.4, 38.7, 35.6, 31.8, 31.3, 29.3, 29.2, 29.1,
28.1, 27.7, 27.1, 24.2, 22.5, 13.3, 12.6 ppm; HRMS (ESI) calcd for
C27H41N7O: 479.3372 [M]+, found 479.3378.
1
72% yield). H NMR (300 MHz, CD3OD): d=8.36 (s, 1H), 7.45 (s, 2H),
6.46 (s, 1H), 4.77 (s, 2H), 3.97 (s, 2H), 2.80 (s, 2H), 2.48 (t, J=6.9 Hz,
2H), 1.73 (s, 2H), 1.62 (s, 2H), 1.41 ppm (s, 2H); 13C NMR (75 MHz,
CD3OD): d=165.5, 147.3, 136.9, 134.5, 132.7, 128.2, 128.0, 127.6,
108.6, 51.2, 39.0, 28.1, 27.7, 24.1, 23.8 ppm; HRMS (ESI) calcd for
C19H22Cl3N7O: 469.0951 [M]+, found 469.0941.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-3,5-difluorobenzamide hydrochloride (7aa): tert-Butyl 2-
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.123 g,
0.442 mmol) was treated with N-(2-azidoethyl)-3,5-difluorobenz-
amide (0.100 g, 0.442 mmol) according to the general click proce-
dure to give tert-butyl 2-amino-4-(5-(1-(2-(3,5-difluorobenzami-
do)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR (300 MHz, CDCl3): d=8.25 (s, 1H), 7.39 (m, 3H), 6.88 (t, J=
4.8 Hz, 1H), 6.43 (s, 1H), 6.38 (brs, 2H), 4.55 (s, 2H), 3.93 (s, 2H),
2.56 (t, J=5.1 Hz, 2H), 2.24 (s, 2H), 1.55 (m, 14H), 1.23 ppm (s, 2H);
13C NMR (75 MHz, CDCl3): d=165.9, 164.3, 164.2, 161.9, 150.3,
149.3, 148.2, 137.6, 122.4, 111.0, 110.8, 107.3, 107.1, 106.8, 106.6,
85.5, 49.3, 40.4, 29.9, 29.5, 29.1, 28.7, 28.2, 27.9, 27.5, 25.4 ppm;
HRMS (ESI) calcd for C24H31F2N7O3: 503.2456 [M]+, found 503.2458.
The carboxylate was subsequently deprotected to give 7aa
(0.115 g, 59% yield). 1H NMR (400 MHz, CD3OD): d=8.55 (s, 1H),
7.42 (d, J=2.0 Hz, 2H), 7.29 (t, J=2.40 Hz, 1H), 6.51 (s, 1H), 4.84 (t,
J=4.8 Hz, 2H), 3.96 (q, J=13.2, 4.8 Hz, 2H), 2.89 (t, J=7.2 Hz, 2H),
2.49 (t, J=7.2 Hz, 2H), 1.79 (m, 2H), 1.75 (m, 2H), 1.44 ppm (m,
2H); 13C NMR (100 MHz, CD3OD): d=164.5, 164.4, 127.6, 126.9,
110.6, 110.6, 110.3, 108.5, 107.1, 106.8, 106.6, 52.5, 39.5, 30.4, 27.9,
27.8, 27.6, 24.0, 23.8, 23.1 ppm; HRMS (ESI) calcd for C19H23F2N7O:
403.1932 [M]+, found 403.1926.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-4-nonylbenzamide hydrochloride (7w): tert-Butyl 2-
amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.636 g,
2.29 mmol) was treated with N-(2-azidoethyl)-4-nonylbenzamide
(0.726 g, 2.29 mmol) according to the general click procedure to
give tert-butyl 2-amino-4-(5-(1-(2-(4-nonylbenzamido)ethyl)-1H-
1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR
(300 MHz, CDCl3): d=7.70 (d, J=7.2 Hz, 2H), 7.29 (s, 1H), 7.27 (t,
J=8.7 Hz, 1H), 7.16 (d, J=7.2 Hz, 2H), 4.53 (s, 2H), 3.91 (s, 2H),
2.61 (m, 4H), 2.41 (m, 1H), 2.14 (s, 1H), 1.56 (brs, 15H), 1.23 (brs,
14H), 0.83 ppm (t, J=6.6 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=
188.1, 169.7, 168.3, 164.4, 148.0, 147.4, 131.4, 128.8, 127.4, 122.2,
51.6, 49.5, 40.2, 36.0, 32.1, 31.4, 29.7, 29.6, 29.5, 29.4, 29.3, 28.2,
25.6, 22.9, 14.3 ppm; HRMS (ESI) calcd for C33H51N7O3: 593.4053
[M]+, found 593.4049. The carboxylate was subsequently depro-
1
tected to give 7w (0.183 g, 74% yield). H NMR (400 MHz, CD3OD):
d=8.66 (s, 1H), 7.69 (s, 2H), 7.25 (s, 2H), 6.54 (s, 1H), 4.81 (s, 2H),
3.62 (s, 2H), 2.85 (s, 2H), 2.63 (s, 2H), 2.46 (s, 2H), 1.75 (s, 2H), 1.39
(s, 4H), 1.29 (m, 14H), 0.88 ppm (s, 3H); 13C NMR (100 MHz,
CD3OD): d=205.0, 169.2, 147.5, 147.3, 131.2, 128.5, 127.3, 108.9,
108.6, 96.0, 52.4, 51.6, 39.3, 38.9, 35.6, 31.9, 31.3, 30.6, 29.5, 29.4,
29.3, 29.2, 28.1, 27.9, 27.7, 24.1, 23.6, 22.6, 13.4 ppm; HRMS (ESI)
calcd for C28H43N7O: 493.3529 [M]+, found 493.3522.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)biphenyl-4-carboxamide hydrochloride (7bb): tert-Butyl
2-amino-4-(hept-6-ynyl)-1H-imidazole-1-carboxylate
(0.093 g,
0.334 mmol) was treated with N-(2-azidoethyl)biphenyl-4-carbox-
amide (0.089 g, 0.334 mmol) according to the general click proce-
dure to give tert-butyl 2-amino-4-(5-(1-(2-biphenyl-4-carboxami-
doethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carboxylate.
1H NMR (300 MHz, CDCl3): d=7.91 (d, J=5.1 Hz, 3H), 7.57 (t, J=
8.4 Hz, 4H), 7.44 (t, J=6.9 Hz, 3H), 7.34 (1H), 4.60 (t, J=5.1 Hz,
2H), 3.96 (q, J=5.1, 5.4 Hz, 2H), 2.63 (t, J=7.2 Hz, 2H), 2.27 (t, J=
7.5 Hz, 2H), 1.62 (m, 13H), 1.33 ppm (m, 2H); 13C NMR (75 MHz,
CDCl3): d=168.0, 149.6, 148.3, 144.6, 140.1, 138.8, 132.8, 129.1,
128.2, 128.0, 127.4, 127.3, 122.2, 84.9, 49.4, 40.3, 29.3, 28.9, 28.3,
28.2, 28.1, 25.6 ppm; HRMS (ESI) calcd for C30H37N7O3 [M]+
543.2958, found 543.2949. The carboxylate was subsequently de-
protected to give 7bb (0.093 g, 58% yield). 1H NMR (300 MHz,
CD3OD): d=8.35 (s, 1H), 7.88 (d, J=8.1 Hz, 2H), 7.65 (d, J=8.4 Hz,
2H), 7.57 (d, J=7.5 Hz, 2H), 7.39 (t, J=7.5 Hz, 2H), 7.34 (t, J=
7.2 Hz, 1H), 6.39 (s, 1H), 4.81 (s, 2H), 3.96 (s, 2H), 2.78 (s, 2H), 2.37
(t, J=7.5 Hz, 2H), 1.67 (s, 2H), 1.56 (t, 2H), 1.33 ppm (s, 2H);
13C NMR (75 MHz, CD3OD): d=203.6, 158.9, 160.1, 159.5, 159.1,
159.0, 158.5, 149.8, 147.2, 144.6, 132.4, 128.9, 127.9, 127.6, 126.9,
126.4, 108.4, 51.7, 39.5, 27.9, 27.6, 24.0, 23.5, 7.2 ppm; HRMS (ESI)
calcd for C25H29N7O: 443.2434 [M]+, found 443.2423.
N-(2-(4-(5-(2-Amino-1H-imidazol-4-yl)pentyl)-1H-1,2,3-triazol-1-
yl)ethyl)-2’,4’-difluoro-3-hydroxybiphenyl-4-carboxamide hydro-
chloride (7j): tert-Butyl 2-amino-4-(hept-6-ynyl)-1H-imidazole-1-car-
boxylate (0.085 g, 0.306 mmol) was treated with N-(2-azidoethyl)-
2’,4’-difluoro-3-hydroxybiphenyl-4-carboxamide
0.306 mmol) according to the general click procedure to give tert-
butyl 2-amino-4-(5-(1-(2-(2’,4’-difluoro-3-hydroxybiphenyl-4-ylcar-
(0.097 g,
boxamido)ethyl)-1H-1,2,3-triazol-4-yl)pentyl)-1H-imidazole-1-carbox-
1
ylate. H NMR (300 MHz, CDCl3): d=8.54 (s, 1H), 7.83 (s, 1H), 7.37
(d, J=7.2 Hz, 1H), 7.28 (s, 1H), 7.18 (s, 1H), 6.89 (d, J=7.8 Hz, 1H),
6.72 (s, 2H), 6.39 (s, 1H), 4.57 (s, 2H), 3.81 (s, 2H), 3.34 (s, 1H), 2.47
(s, 2H), 2.07 (s, 2H), 1.49 (m, 13H), 1.17 ppm (s, 2H); 13C NMR
(75 MHz, CDCl3): d=169.5, 163.7, 161.3, 160.5, 159.9, 150.5, 149.2,
148.2, 137.1, 136.3, 134.5, 131.4, 12.8, 126.8, 124.4, 122.3, 117.9,
116.1, 111.9, 111.6, 106.7, 104.8, 104.4, 104.1, 85.8, 50.5, 49.4, 46.6,
50.0, 37.9, 29.9, 28.9, 28.5, 28.3, 28.1, 27.4, 25.3, 25.0 ppm; HRMS
(ESI) calcd for C30H35F2N4O4: 595.2729 [M]+, found 595.2717. The
ChemBioChem 2010, 11, 396 – 410
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
407

C. Melander et al.
carboxylate was subsequently deprotected to give 7j (0.137 g,
84% yield). H NMR (400 MHz, CD3OD): d=8.60 (s, 1H), 7.89 (s, 1H),
cans biofilm formation: Inhibition assays were performed by
taking an overnight culture of yeast or yeast/bacteria strain and
subculturing it at an OD600 of 0.05 into YPD (yeast extract, peptone
and dextrose (BD 242820)) medium for the yeast alone or tryptic
soy broth for the S. epidermidis/C. albicans. Stock solutions of pre-
determined 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. Sample
plates were then wrapped in GLAD Press n’ Seal followed by an in-
cubation under stationary conditions for 24 h at 378C. After incu-
bation, the medium was discarded from the wells, and the plates
were washed thoroughly with water. Plates were then stained with
a 0.1% solution of CV (100 mL) and incubated at ambient tempera-
ture for 30 min. Plates were washed with water again, and the re-
maining stain was solubilized with 95% ethanol (200 mL). A sample
of solubilized CV stain from each well (125 mL) was transferred to
the corresponding wells of a polystyrene microtiter dish. Biofilm in-
hibition was quantitated by measuring the OD540 of each well. A
negative control wherein no biofilm was formed served as a back-
ground and was subtracted out.
1
7.52 (s, 2H), 6.97 (t, J=6.3 Hz, 3H), 6.45 (s, 1H), 4.81 (s, 2H), 2.78 (s,
2H), 2.42 (s, 2H), 1.72 (s, 2H), 1.61 (d, J=7.8 Hz, 2H), 1.38 ppm (s,
2H); 13C NMR (100 MHz, CD3OD): d=172.9, 169.8, 161.6, 159.5,
147.2, 134.4, 131.7, 128.4, 127.6, 126.0, 117.5, 115.6, 111.7, 111.5,
108.5, 104.2, 104.0, 103.7, 94.6, 51.9, 39.0, 27.9, 27.6, 26.8, 24.1,
23.8 ppm; HRMS (ESI) calcd for C25H27F2N7O2: 459.2194 [M]+, found
459.2190.
Procedure to determine the inhibitory effect of test compounds
on E. faecium, MRSA, S. epidermidis, PAO1, PA14, E. coli, and
MDRAB biofilm formation: Inhibition 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, LB medium for MDRAB, LB medium without NaCl
(LBNS) for PAO1 and PA14, LB medium for E. coli and tryptic soy
broth with a 0.5% glucose supplement and a 3.0% NaCl supple-
ment (TGN) for S. epidermidis). Stock solutions of predetermined
concentrations of the test compound were then made in the nec-
essary medium. These stock solutions were aliquoted (100 mL) into
the wells of the 96-well PVC microtiter plate. Sample plates were
then wrapped in GLAD Press n’ Seal and incubated under station-
ary conditions for 24 h at 378C. After incubation, the medium was
discarded from the wells, and the plates were washed thoroughly
with water. Plates were then stained with a 0.1% solution of CV
(100 mL) and incubated at ambient temperature for 30 min. The
plates were washed with water again, and the remaining stain was
solubilized with 95% ethanol (200 mL). A sample of solubilized CV
stain from each well (125 mL) was transferred to the corresponding
wells of a polystyrene microtiter dish. Biofilm inhibition was quanti-
tated by measuring the OD540 of each well. A negative control
wherein no biofilm was formed served as a background and was
subtracted out.
MRSA, S. epidermidis, PAO1, PA14, E. coli, and MDRAB 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 (as above for dispersal effects). The re-
sulting bacterial suspension was aliquoted (100 mL) into the wells
of a 96-well PVC microtiter plate. Plates were then wrapped in
GLAD Press n’ Seal and incubated under stationary conditions at
ambient temperature 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 nec-
essary 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
378C. After incubation, the medium was discarded from the wells,
and the plates were washed thoroughly with water. The plates
were then stained with a 0.1% solution of CV (100 mL) and incubat-
ed at ambient temperature for 30 min. The plates were washed
with water again, and the remaining stain was solubilized with
95% ethanol (200 mL). A sample (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. A negative control lane wherein
no biofilm was formed served as a background and was subtracted
out.
Procedure to determine the dispersal effect of test compounds
on E. faecium, MRSA, S. epidermidis, PAO1, PA14, E. coli, and
MDRAB 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 (as above). The re-
sulting bacterial suspension was aliquoted (100 mL) into the wells
of a 96-well PVC microtiter plate. Plates were then wrapped in
GLAD Press n’ Seal and incubated under stationary conditions at
ambient temperature 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 nec-
essary 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
378C. After incubation, the medium was discarded from the wells,
and the plates were washed thoroughly with water. Plates were
then stained with a 0.1% solution of CV (100 mL) and incubated at
ambient temperature for 30 min. The plates were washed with
water again, and the remaining stain was solubilized with 95% eth-
anol (200 mL). A sample of solubilized CV stain (125 mL) 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. A negative-control wherein no biofilm was
formed served as a background and was subtracted out.
Procedure to determine the dispersal effect of test compounds
on C. albicans and C. neoformans preformed biofilms: Dispersion
assays were performed by taking an overnight culture of bacterial
strain and subculturing it at an OD600 of 0.01 into yeast extract,
peptone, and dextrose (BD 242820) medium. The resulting bacteri-
al suspension was aliquoted (100 mL) into the wells of a 96-well
PVC microtiter plate. The plates were then wrapped in GLAD Press
n’ Seal and incubated under stationary conditions at ambient tem-
perature to establish the biofilms. After 24 h, the medium was dis-
carded 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 378C. After incuba-
Procedure to determine the inhibitory effect of test compounds
on C. albicans, C. neoformans, and a mixed S. epidermidis/C. albi-
408
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ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 396 – 410

Aminoimidazole-Based Antibiofilm Agents
tion, the medium was discarded from the wells, and the plates
were washed thoroughly with water. The plates were then stained
with a 0.1% solution of CV (100 mL) and then incubated at ambient
temperature for 30 min. The plates were washed with water again,
and the remaining stain was solubilized with 95% ethanol (200 mL).
A sample (125 mL) of solubilized CV stain from each well was trans-
ferred to the corresponding wells of a polystyrene microtiter dish.
Biofilm dispersion was quantitated by measuring the OD540 of each
well. A negative control lane wherein no biofilm was formed
served as a background and was subtracted out.
200 rpm until the OD600 of the control samples reached approxi-
mately 1.2. At this point, 100 mL was taken from each culture tube
and then diluted serially into LB medium. Then, 10 mL was re-
moved from each serial dilution and plated out on a square grid-
ded Petri dish. The dishes were incubated for 16 h at 378C to grow
viable colonies, which were quantified by using the track-dilution
method.
Red blood cell hemolysis assay: Hemolysis assays were performed
on mechanically defibrinated sheep’s blood (DSB100, Hemostat
Labs, Dixon, USA). A sample (1.5 mL) of blood was placed into a
microcentrifuge tube and separated at 10000 rpm for 10 min. The
supernatant was removed, and the cells were resuspended in
phosphate-buffered saline (PBS; 1 mL). The suspension was centri-
fuged, the supernatant was removed, and the cells were resus-
pended two more times. The final cell suspension was then diluted
tenfold. Test compound solutions were made in PBS in small cul-
ture tubes and then added to aliquots of the tenfold-diluted sus-
pension. PBS alone was used as a negative control and as a zero
hemolysis marker, while a 1% Triton X sample was used as a posi-
tive control and the 100% lysis marker. Samples were then placed
in an incubator at 378C while being shaken at 200 rpm. After 1 h,
the samples were transferred to microcentrifuge tubes and then
separated at 10000 rpm for 10 min. The resulting supernatant was
diluted by a factor of 40 in distilled water. The absorbance of the
supernatant was measured on a UV spectrometer at a 540 nm
wavelength.
Procedure to determine the effect of leading test compounds
on E. faecium, MRSA, S. epidermidis, PAO1, PA14, E. coli, and
MDRAB planktonic viability by growth curve analysis: Growth
curves were generated by taking an overnight culture of bacterial
strain and subculturing it at an OD600 of 0.01 into the necessary
medium (as above for dispersal effects, save nutrient broth for
S. epidermidis). The resulting bacterial suspension was then aliquot-
ed (3.0 mL) into culture tubes. The test compound was then added
at a predetermined concentration to the medium of the test sam-
ples. Controls were employed in which no test compound was
added to the bacterial suspension. Samples were then placed in an
incubator at 378C and shaken at 200 rpm. The OD600 of the sam-
ples was measured at time intervals starting at 2 h and ending at
24 h.
Procedure to determine the effect of leading test compounds
on C. albicans and C. neoformans planktonic viability by growth
curve analysis: Growth curves were generated by taking an over-
night culture of yeast strain and subculturing it at an OD600 of 0.01
into YPD medium. The resulting bacterial suspension was then ali-
quoted (3.0 mL) into culture tubes. The test compound was added
at a predetermined concentration to the medium of the test sam-
ples. Controls were employed in which no test compound was
added to the bacterial suspension. Samples were then incubated
at 378C and shaken at 200 rpm. The OD600 of the samples was
measured at time intervals starting at 2 h and ending at 24 h.
Acknowledgements
The authors would like to thank the UNC General Administration
Competitiveness Grant and the V foundation for funding (S.A.R.
as a predoctoral Jimmy V Scholar). Further funding was obtained
from the North Carolina Biotechnology Center and the NCSU De-
partment of Chemistry.
Colony-count procedure to determine the effect of leading test
compounds on E. faecium, MRSA, S. epidermidis, PAO1, PA14,
E. coli, and MDRAB planktonic viability: Colonies were counted
by taking an overnight culture of bacterial strain and subculturing
it at an OD600 of 0.01 into the necessary medium (as above for
effect of leading test compounds). The resulting bacterial suspen-
sion was then aliquoted (3.0 mL) into culture tubes. A test com-
pound was added to the medium of the test samples at a prede-
termined concentration. Controls were employed in which no test
compound was added to the bacterial suspension. Samples were
then incubated at 378C and shaken at 200 rpm until the OD600 of
the control samples reached approximately 1.2. At this point,
100 mL was taken from each culture tube and then diluted serially
into LB medium. Then, 10 mL were removed from each serial dilu-
tion and plated out on a square gridded Petri dish. The dishes
were incubated for 16 h at 378C to grow viable colonies, which
were quantified by using the track-dilution method.
Keywords: aminoimidazoles · biofilms · MDRAB · MRSA ·
multidrug-resistant bacteria
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ChemBioChem 2010, 11, 396 – 410