Evaluation of 4,5-Disubstituted-2-Aminoimidazole-Triazole Conjugates for Antibiofilm/Antibiotic Resensitization Activity Against MRSA and Acinetobacter baumannii

Article abstract of DOI:10.1002/cmdc.201100316

A library of 4,5-disubstituted-2-aminoimidazole-triazole conjugates (2-AITs) was synthesized, and the antibiofilm activity was investigated. This class of small molecules was found to inhibit biofilm formation by methicillin-resistant Staphylococcus aureus (MRSA) at low-micromolar concentrations; 4,5-disubstituted-2-AITs were also able to inhibit and disperse Acinetobacter baumannii biofilms. The activities of the lead compounds were compared against the naturally occurring biofilm dispersant cis-2-decenoic acid and were revealed to be more potent. The ability of selected compounds to resensitize MRSA to traditional antibiotics (resensitization activity) was also determined. Lead compounds were observed to resensitize MRSA to oxacillin by 2-4-fold. Fight against the film: 4,5-Disubstituted-2-aminoimidazole-triazole conjugates (2-AITs) were developed and investigated for antibiofilm activity and antibiotic resensitization. This class of small molecules was found to inhibit biofilm formation by methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii at micromolar concentrations. Conventional antibiotic resensitization was also observed for lead compounds.

Full text of DOI:10.1002/cmdc.201100316

DOI: 10.1002/cmdc.201100316
Evaluation of 4,5-Disubstituted-2-Aminoimidazole–Triazole
Conjugates for Antibiofilm/Antibiotic Resensitization
Activity Against MRSA and Acinetobacter baumannii
Zhaoming Su, Lingling Peng, Roberta J. Worthington, and Christian Melander*[a]
A library of 4,5-disubstituted-2-aminoimidazole–triazole conju-
gates (2-AITs) was synthesized, and the antibiofilm activity was
investigated. This class of small molecules was found to inhibit
biofilm formation by methicillin-resistant Staphylococcus aureus
(MRSA) at low-micromolar concentrations; 4,5-disubstituted-2-
AITs were also able to inhibit and disperse Acinetobacter bau-
mannii biofilms. The activities of the lead compounds were
compared against the naturally occurring biofilm dispersant
cis-2-decenoic acid and were revealed to be more potent. The
ability of selected compounds to resensitize MRSA to tradition-
al antibiotics (resensitization activity) was also determined.
Lead compounds were observed to resensitize MRSA to oxacil-
lin by 2–4-fold.
Introduction
Antibiotic-resistant pathogens have become an emerging crisis
during the last decade.[1] Bacteria have demonstrated the abili-
ty to develop resistance to virtually every antibiotic introduced
by the medical community.[2] Among the many defense mech-
anisms that bacteria have developed, biofilm formation plays
an important role in establishing protection against antibacte-
rial agents.[3] Biofilms are defined as sessile communities of mi-
croorganisms that exist as differentiated entities embedded in
an extracellular matrix of biomolecules (typically exopolysac-
charides).[4] Bacteria within a biofilm display up to 1000-fold in-
creased resistance against traditional antibiotics relative to
their planktonic counterparts.[5] The US National Institutes of
Health (NIH) have estimated that three of every four cases of
bacterial infection are mediated by biofilms. Biofilms are the
preferred living mode for bacteria, as ~80% of the world’s mi-
crobial mass exists as a biofilm.[6]
nisms (Figure 2).[8–16] Among these molecules, dihydrosventrin
4 (DHS)[8] and reverse amide 5 (RA-11)[9] were found to possess
outstanding antibiofilm activity against Pseudomonas aerugino-
sa (PA), while the 2-aminoimidazole–triazole conjugate (2-AIT)
6
[10] was identified as the first small molecule to inhibit and dis-
There are a number of naturally occurring molecular scaf-
folds that are capable of inhibiting biofilm formation[7] includ-
ing brominated furanone 1, cis-2-decenoic acid 2, and oroidin
3 (Figure 1). Our research group has focused on studying the
ability of oroidin derivatives to modulate biofilm formation
and, through a focused synthesis and screening effort, we
have developed a number of oroidin analogues that inhibit
and disperse bacterial biofilms via non-microbicidal mecha-
Figure 2. Small molecules inspired by oroidin that have antibiofilm activity.
[a] Z. Su, Dr. L. Peng, Dr. R. J. Worthington, Prof. Dr. C. Melander
Department of Chemistry, North Carolina State University
Raleigh, NC 27695-8204 (USA)
Fax: (+1)919-515-5079
E-mail: Christian_melander@ncsu.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100316.
Figure 1. Antibiofilm natural products.
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C. Melander et al.
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perse bacterial biofilms across order, class, and phylum. 2-AIT 6
was also reported to work synergistically with conventional an-
tibiotics.[17] In an effort to augment the activity of lead 2-AIT 6,
we recently reported the synthesis of a second-generation 2-
AIT library.[11] Subsequent screening of the library demonstrat-
ed that 2-AIT 7 is better able to inhibit methicillin-resistant
Staphylococcus aureus (MRSA) biofilm formation and to dis-
perse preformed Acinetobacter baumannii biofilms than com-
pound 6.
In a parallel structure–activity relationship (SAR) study, 4,5-
disubstituted-2-AI analogues with simple substituent patterns
were synthesized by a nitroenolate approach, and we identi-
fied a number of broad-spectrum
kylation of the protected glycine ethyl ester 8 with iodoalkyne
9 delivered the protected a-amino ester 10 in 71% yield. A
click reaction was then performed between 10 and vinyl bro-
mide 11 to generate triazole 12. Suzuki–Miyaura cross-coupling
was then used to install both phenyl and naphthyl moieties
(13a and 13b).[11] Weinreb amides (14a and 14b) were then
accessed from 13 by treatment with N,O-dimethylhydroxya-
mine hydrochloride and isopropylmagnesium chloride.
With Weinreb amides 14 in hand, diversity was installed
through reaction of each respective amide with a variety of
commercially available Grignard reagents (Scheme 2). Each of
the resulting ketones was then deprotected (2n hydrochloric
antimicrobial reagents against a
wide range of terrestrial and
marine bacteria.[18] Given the suc-
cess of our 2-AIT 6 toward both
modulating biofilm formation and
resensitizing multidrug-resistant
bacteria to conventional antibiot-
ics (resensitization activity), we
elected to study the biological
impact of imparting a 4,5-disub-
stitution pattern on 2-AITs 6 and
7 in the context of antibiofilm
and antibiotic resensitization ac-
Scheme 1. Synthetic route to Weinreb amides. Reagents and conditions: a) tBuOK, THF, À788C, 69 h (71%);
b) sodium ascorbate, CuSO4, tBuOH/H2O/CH2Cl2, RT, 12 h (82%); c) RB(OH)2, PdCl2(PPh3)2, Na2CO3 (aq.), THF, 698C,
20 h (13a 96%, 13b 98%); d) O,N-dimethylhydroxyamine·HCl, iPrMgCl, THF, À208C, 2 h (14a 69%, 14b 56%)
tivity. Specifically, we were inter-
ested in investigating the out-
come that augmenting the anti-
biofilm properties of 2-AITs 6 and
7
through analogue synthesis
would have upon resensitization
activity. Our original nitroenolate
approach, however, was incom-
patible with accessing 2-AIT ana-
logues based on 6 and 7. To over-
come this limitation, we report
herein a general approach to 4,5-
disubstituted-2-AIT
derivatives
based on the use of Weinreb
amides. Furthermore, we show
that imparting the 4,5-disubstitu-
tion pattern augments antibiofilm
properties without compromising
antibiotic resensitization activity.
The activity of our lead com-
pounds were also compared with
the naturally occurring biofilm
dispersant cis-2-decenoic acid 2.
Results and Discussion
The synthesis of our Weinreb in-
termediate and subsequent gen-
eration of a 4,5-disubstituted-2-
aminoimidazole–triazole pilot li-
Scheme 2. Pilot libraries of 4,5-disubstituted-2-AITs. Reagents and conditions: a) R’MgBr, THF; b) NH4Cl (aq.); c) 2n
brary is outlined in Scheme 1. Al- HCl, EtOH; d) NH2CN, EtOH, pH 4.3. [a] Yield over three steps.
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2-AITs: Antibiofilm/Antibiotic Resensitizers
acid/ethanol) and cyclized with cyanamide to deliver the
target 4,5-disubstituted-2-AIT. All derivatives, after purification,
were prepared as their respective hydrochloride salts for bio-
logical screening.
The pilot library was first assessed for its ability to inhibit
MRSA biofilm formation at 100 mm using the crystal violet re-
porter assay.[19] To our delight, all members in the library were
capable of inhibiting biofilm formation by >95% at this con-
centration. A dose–response study was subsequently undertak-
en to determine the IC50 value of each compound for biofilm
inhibition (Table 1). All compounds were subjected to growth-
curve analysis (A600) at their respective IC50 concentrations, and
their antibiofilm activity was established to be non-microbici-
dal except for compounds 16bb, 16bc, 16be, and 16bf. We
performed follow-up colony-count analysis with 16ae and veri-
fied that biofilm modulation occurs via a non-microbicidal
mechanism.
Figure 3. Second-generation library.
Table 2. Biofilm inhibition activity of second-generation compounds
against MRSA.
Compd
IC50 [mm][a]
Compd
IC50 [mm][a]
Table 1. Biofilm inhibition activity of pilot libraries against MRSA.
16ah
16ai
16aj
16ak
1.50Æ0.21
1.45Æ0.05
1.89Æ0.11
1.39Æ0.08
16al
1.52Æ0.30
1.34Æ0.10
1.96Æ0.06
1.95Æ0.05
IC50 [mm][a]
Compd
IC50 [mm][a]
16am
16an
16ao
Compd
16aa
16ab
16ac
16ad
16ae
16af
16ag
25.73Æ0.28
2.25Æ0.04
1.91Æ0.02
2.01Æ0.01
1.42Æ0.18
2.10Æ0.13
1.89Æ0.05
16ba
2.95Æ0.37
2.19Æ0.03
10.08Æ0.13
2.23Æ0.07
2.15Æ0.23
8.20Æ0.10
1.97Æ0.03
16bb[b]
16bc[b]
16bd
[a] Values represent the mean ÆSD as determined by at least three inde-
pendent parallel experiments.
16be[b]
16bf[b]
16bg
Table 2. To our delight, two compounds displayed improved
activity, with 16am being the most active (1.34 mm). Growth-
curve and colony-count analyses demonstrated that none of
the second-generation compounds show toxicity against bac-
teria at their IC50 concentrations.
[a] Values represent the mean ÆSD as determined by at least three inde-
pendent parallel experiments. [b] Growth curve defects were noted at
these concentrations.
According to the data outlined in Table 1, it was generally
noted that compounds with the phenyl substituent (16a
series) present an overall trend toward increased antibiofilm
activity over the 16b series, which contains the naphthyl subu-
nit. Additionally, several of the compounds with the naphthyl
moiety were found to be microbicidal during growth-curve
analysis, whereas all compounds with the phenyl moiety were
determined to inhibit biofilm formation via a non-microbicidal
mechanism. We also noted, as previously observed, that activi-
ty is modulated by introduction of the 4,5-disubstitution pat-
tern. Overall, compound 16ae is the most potent compound
of the pilot library, registering an IC50 value of 1.42 mm.
One of the most striking observations is that a propyl group
on the newly introduced phenyl ring correlates with increased
activity. Based on this observation, a second-generation library
of eight compounds was synthe-
Once the effect of these 4,5-disubstituted-2-AITs had been
evaluated on a representative Gram-positive strain (MRSA), we
assessed the antibiofilm activity of these compounds against
A. baumannii, a representative Gram-negative bacterial strain.
All compounds were initially screened for the ability to inhibit
biofilm formation against A. baumannii at 100 mm. Each com-
pound that showed >75% antibiofilm activity was subjected
to a dose–response study to determine the IC50 value for bio-
film inhibition. Several compounds displayed a precipitous de-
crease in their biofilm inhibition properties (16ad, 16ag, 16ah,
16ai, 16aj, 16ak, 16an, 16ba, 16bf, and 16bg) over a narrow
concentration range, which typically indicates that biofilm in-
hibition occurs through a traditional microbicidal mechanism
(IC50 not determined). Other compounds with applicable IC50
values are summarized in Table 3. Growth-curve analysis was
sized (Figure 3), in which we fur-
Table 3. Biofilm inhibition and dispersion activity of representative compounds against A. baumannii.
ther varied functionalities on the
phenyl ring, including: methoxy,
isopropyl, fluoro/difluoro, pyrroli-
dinyl, phenyl, and 1,3-benzodiox-
olyl. Antibiofilm activities of
second-generation compounds
were evaluated, and the IC50
values of biofilm inhibition
against MRSA are outlined in
Compd
IC50 [mm][a]
EC50 [mm][a]
Compd
IC50 [mm][a]
EC50 [mm][a]
16ab[b]
16ac
16ae
16af[b]
16al
15.65Æ0.27
15.57Æ0.31
11.28Æ0.68
61.74Æ2.54
60.67Æ3.18
–
16am
16ao
16bb[b]
16bd[b]
16be[b]
32.03Æ2.88
20.35Æ0.07
19.73Æ0.64
30.46Æ0.57
22.92Æ2.14
64.55Æ17.34
50.03Æ8.54
60.80Æ9.52
44.61Æ1.96
–
–
–
–
–
[a] Values represent the mean ÆSD as determined by at least three independent parallel experiments.
[b] Growth curve defects were noted at these concentrations.
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C. Melander et al.
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conducted on each of the com-
pounds outlined in Table 3 at
their respective IC50 concentra-
tions. 2-AIT conjugates 16ac,
16ae, 16al, 16am, and 16ao
were found to inhibit biofilm for-
mation through a non-microbici-
dal mechanism, as no defect in
the growth curve was noted
over 24 h. Colony-count analysis
on lead compounds 16ae and
16ao verified these results.
Table 4. Resensitization of MRSA to oxacillin.
Compd
MICOxa [mgmLÀ1
]
Compd
MICOxa [mgmLÀ1
]
Compd
MICOxa [mgmLÀ1
]
16aa (20.0 mm)
16ab (2.1 mm)
16ac (3.6 mm)
16ad (8.6 mm)
16ae (1.0 mm)
16af (18.8 mm)
16ag (2.1 mm)
2 (50.0 mm)
32
32
16
64
64
32
64
64
16ah (4.1 mm)
16ai (2.0 mm)
16aj (2.0 mm)
16ak (2.1 mm)
16al (14.7 mm)
16am (1.8 mm)
16an (4.0 mm)
16ao (2.0 mm)
64
32
64
64
32
32
64
32
16ba (8.9 mm)
16bb (1.0 mm)
16bc (6.6 mm)
16bd (2.0 mm)
16be (3.6 mm)
16bf (4.2 mm)
16bg (1.0 mm)
Control[a]
32
32
32
32
16
16
32
64
[a] No compound was combined with oxacillin.
The ability of lead compounds
to disperse preformed A. baumannii biofilms was also evaluat-
ed. EC50 values were determined and are summarized in
Table 3. Here, we define EC50 as the concentration that elicits
50% dispersion of a preformed biofilm. All lead compounds
were able to disperse preformed A. baumannii biofilms at low-
micromolar concentrations, with 16ae being the most active
compound.
Compounds 16ac, 16be, and 16bf resensitized MRSA to the
effects of oxacillin by fourfold (roughly twofold over parent
molecule 6). Next, the lead compounds were subjected to the
checkerboard assay,[22] in which the synergistic activity of the
combined agents was evaluated. The SFIC values of 16ac,
16be, and 16bf were determined to be 0.25, 0.19, and 0.5 re-
spectively, indicating that these three 2-AIT derivatives work
synergistically with oxacillin (SFIC=FICCompd +FICOxa, for which
FICCompd =[MICCompd in combination]/[MICCompd alone], and
FICOxa =[MICOxa in combination]/[MICOxa alone]).
cis-2-Decenoic acid 2 was recently reported as a signaling
molecule that possesses the ability to inhibit and disperse bio-
films from both Gram-positive and Gram-negative bacterial
strains as well as a representative fungal strain.[20] Given this ac-
tivity, we were eager to evaluate how this naturally occurring
molecule compares with our 2-AIT derivatives under identical
conditions. Compound 2 was synthesized as described by
Rappe[21] and evaluated for its antibiofilm activity by initially
screening at 400 mm for biofilm inhibition/dispersion activity
against A. baumannii, MRSA, and methicillin-sensitive S. aureus
(MSSA). The ability of 2 to disperse preformed biofilms against
all three bacterial strains and to inhibit biofilm formation
against A. baumannii at 400 mm was <10%. Nonetheless, 2 dis-
played moderate biofilm inhibition activity against MRSA and
MSSA. IC50 values were determined to be 220Æ14 mm against
MRSA and 121Æ35 mm against MSSA. A stability study of 2
was performed by 1H NMR spectroscopy and is described in
detail in the Supporting Information.
In the resensitization and checkerboard assays we also ob-
served that in between the thoroughly clear and turbid wells,
there were wells that contained a minute amount of bacteria
that were barely visible by the naked eye. This is in contrast to
MIC assays with conventional antibiotics in which there is an
abrupt transition between turbid and clear wells. Although all
our MIC values were recorded from completely clear wells, we
were interested in determining the bacterial density of these
wells that contained the barely visible bacterial precipitate. We
performed colony-count analysis on selected wells from the
checkerboard assay of 16be, and these results are summarized
in Table 5. Comparing the activity of 16be plus 16 mgmLÀ1 ox-
acillin against 16 mgmLÀ1 oxacillin alone reveals that the com-
bination is 99.9% more effective. However, we note from
growth-curve analysis that 16be does alter early (0–8 h) MRSA
growth characteristics, although bacterial CFUs are identical
after 16 h.
Next, we studied the ability of our 2-AIT library to resensitize
MRSA to oxacillin. This antibiotic was specifically chosen be-
cause the parent compound 6 had minimal effect on the abili-
ty to resensitize MRSA to oxacillin (about twofold). Therefore, it
represented an ideal canvas to test whether our 4,5-disubsti-
tuted-2-AIT library possesses enhanced activity. We were also
interested in determining whether, like 2-AI-derived antibiofilm
agents, cis-2-decenoic acid modulates planktonic response to
antibiotics. We first determined the minimum inhibitory con-
centration (MIC) values of each compound against MRSA. A
fourth of each respective MIC value was subsequently used as
the concentration to test antibiotic resensitization. This con-
centration was chosen because in previous studies, no microbi-
cidal activity is typically observed at 25% of the MIC values of
our 2-AI antibiofilm agents.
Conclusions
In summary, we have successfully synthesized a variety of 4,5-
disubstituted-2-aminoimidazole–triazole conjugates through a
Weinreb amide approach. The antibiofilm activity of these
compounds was evaluated, and compounds 16ae, 16ak, and
16am were identified as the most active. Growth-curve and
colony-count analyses indicated that lead compounds inhibit
biofilm formation via a non-microbicidal mechanism. Further
comparison of lead compounds against the naturally occurring
dispersant cis-2-decenoic acid revealed that appropriately de-
signed 2-AI derivatives are two orders of magnitude more
potent. 2-AI-based antibiofilm agents can also be simultane-
ously augmented for both their antibiofilm properties and
their antibiotic resensitization activity. The fact that not all lead
compounds have the ability to resensitize MRSA to the effects
Bacteria were pretreated with each compound for 30 min,
and the MIC of oxacillin was subsequently determined. All MIC
values of oxacillin combined with each of the 4,5-disubstitut-
ed-2-AITs and cis-2-decenoic acid 2 are summarized in Table 4.
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2-AITs: Antibiofilm/Antibiotic Resensitizers
MRSA (ATCC #BAA-44), MSSA (ATCC #29213) and A. baumannii
(ATCC #19606) were obtained from the American Type Culture Col-
lection. Mechanically defibrinated sheep blood (DSB100) was ob-
tained from Hemostat Labs. Oxacillin sodium salt was purchased
from Fluka (cat. #28221). All other reagents were purchased from
commercially available sources.
Table 5. Colony-count comparison between wells in checkerboard assay
against MRSA.
Synthesis of N-methoxy-N-methylamide–triazole precursors
(14a, 14b): Ethyl 2-(diphenylmethyleneamino)non-8-ynoate (10):
N-(Diphenylmethylene)glycine ethyl ester 8 (4.010 g, 15 mmol) and
anhydrous THF (110 mL) were added to a 250 mL round-bottomed
flask. The solution was stirred under N2 at À788C for 15 min.
tBuOK (2.020 g, 18 mmol) was added, and the resulting solution
was allowed to warm to 08C and stirred for 1 h. 7-Iodohept-1-yne
9 (4.329 g, 19.5 mmol) was prepared as described by Knapp
et al.[23] and added to the mixture. The mixture was then warmed
and stirred at room temperature for 69 h. The reaction was
quenched by 100 mL H2O and extracted with EtOAc (2ꢂ100 mL).
The combined organic extracts were washed with 100 mL brine,
dried over Na2SO4, and the solvent was evaporated under reduced
pressure. Purification of the residue was carried out on a silica gel
column and eluted with CH2Cl2/MeOH (9:1) to give the desired
product 10 (3.854 g, 71%) as a colorless oil: 1H NMR (400 MHz,
CDCl3): d=7.65 (m, 1H), 7.64 (m, 1H), 7.44 (m, 2H), 7.39 (m, 1H),
7.38 (m, 1H), 7.35 (m, 1H), 7.33 (m, 1H), 7.17 (m, 2H), 4.17 (m, 2H),
4.04 (t, J=6.8 Hz, 1H), 2.13 (td, J=2.4, 7.2 Hz, 2H), 1.92 (m, 2H),
1.91 (t, J=2.8 Hz, 1H), 1.49 (m, 2H), 1.32 (m, 4H), 1.26 (t, J=7.6 Hz,
3H) ppm; 13C NMR (100 MHz, CDCl3): d=172.7, 170.6, 139.8, 136.8,
130.5, 129.0, 128.8, 128.7, 128.3, 128.1, 84.8, 68.4, 65.6, 61.1, 33.7,
28.7, 28.5, 25.7, 18.5, 14.5 ppm; IR nmax =3280, 3136, 3064, 2923,
2855, 1955, 1735, 1660, 1599, 1447, 1317, 1278, 1177, 1073, 1030,
941, 920, 763, 700 cmÀ1; lmax =252 nm; HRMS (FAB) calcd for
C24H27NO2 [M+H]+: 362.2115, found: 362.2117.
Count [CFUmLÀ1
]
MICOxa [mgmLÀ1
]
Oxacillin+16be[a]
Oxacillin alone
64
32
16
8
4
2
1
0.5
0.25
Control
–
–
7.60Æ0.57ꢂ105
1.11Æ0.06ꢂ108
1.96Æ0.20ꢂ105
2.04Æ0.20ꢂ107
4.70Æ0.57ꢂ107
9.55Æ1.34ꢂ107
1.50Æ0.26ꢂ108
1.38Æ0.11ꢂ108
2.03Æ0.42ꢂ108
8.35Æ0.64ꢂ108
1.75Æ0.16ꢂ108
–
–
–
–
–
–
8.00Æ1.27ꢂ108
[a] [16be] in assay: 3.6 mm; values represent the mean ÆSD as deter-
mined by at least three independent parallel experiments.
(E)-Ethyl-7-[1-(3-bromo-2-methylallyl)-1H-1,2,3-triazol-4-yl]-2-(di-
phenylmethyleneamino)heptanoate (12): Compound 10 (3.354 g,
9.29 mmol) was dissolved in a solvent mixture of 18 mL CH2Cl2,
36 mL tBuOH, and 36 mL H2O in a 150 mL round-bottomed flask.
(E)-3-Azido-1-bromo-2-methylprop-1-ene 11 (1.950 g, 11.14 mmol)
was prepared as described by Handa et al.[24] and added to the so-
lution at room temperature. With vigorous stirring, CuSO4 (0.222 g,
1.39 mmol) and sodium ascorbate (0.736 g, 3.71 mmol) were
added, and the resulting solution was stirred at room temperature
for 12 h. The mixture was extracted with CH2Cl2 (3ꢂ20 mL). The
combined organic extracts were washed with 20 mL brine, dried
over Na2SO4, and the solvent was removed in vacuo. The resulting
residue was purified by column chromatography (hexane/EtOAc,
1:1) to afford the target compound 12 (4.105 g, 82%) as a yellow
of oxacillin indicates that antibiofilm properties and resensitiza-
tion potentially act through distinct mechanisms. However, we
note that a single molecular scaffold based on the 2-AI frame-
work can be designed to target both properties simultaneous-
ly.
Experimental Section
General experimental
All reagents used for chemical synthesis were purchased from
commercially available sources were and used without further pu-
rification. Chromatography was performed with 60 ꢁ mesh stan-
dard-grade silica gel from Sorbtech (Atlanta, GA, USA). Infrared
spectra were collected on an FT/IR-4100 spectrophotometer (nmax
in cmÀ1). UV absorbance was recorded on a Genesys 10 scanning
UV/Vis spectrophotometer (lmax in nm). NMR solvents were ob-
tained from Cambridge Isotope Labs and used as is. 1H NMR
(400 MHz) and 13C NMR (100 MHz) spectra were recorded at 258C
on Varian Mercury spectrometers. Chemical shifts (d) are given in
ppm relative to tetramethylsilane or the respective NMR solvent;
coupling constants (J) are in Hertz (Hz). Abbreviations used are: s=
singlet, bs=broad singlet, d=doublet, dd=doublet of doublets,
t=triplet, dt=doublet of triplets, td=triplet of doublets, tt=trip-
let of triplets, bt=broad triplet, q=quartet, m=multiplet, bm=
broad multiplet. Mass spectra were obtained at the NCSU Depart-
ment of Chemistry Mass Spectrometry Facility. Purity of tested
compounds was confirmed to be >95% by LC–MS analysis unless
otherwise stated.
1
oil: H NMR (400 MHz, CDCl3): d=7.50 (d, J=8.0 Hz, 2H), 7.29 (m,
3H), 7.18 (m, 4H), 7.03 (d, J=6.8 Hz, 2H), 6.14 (s, 1H), 4.74 (s, 2H),
4.01 (m, 2H), 3.93 (m, 1H), 2.51 (t, J=7.2 Hz, 2H), 1.80 (m, 2H),
1.56 (s, 3H), 1.50 (m, 2H), 1.14 (m, 4H), 1.10 ppm (t, J=7.2 Hz, 3H);
13C NMR (100 MHz, CDCl3): d=172.4, 170.4, 148.7, 139.6, 136.5,
136.5, 130.5, 128.9, 128.8, 128.7, 128.2, 127.9, 121.0, 108.0, 65.5,
60.9, 55.6, 33.7, 29.3, 29.1, 25.8, 25.7, 17.2, 14.4 ppm; IR nmax =3136,
3061, 3024, 2929, 2856, 2110, 1959, 1896, 1732, 1623, 1445, 1369,
1289, 1181, 1030, 957, 781, 699 cmÀ1; lmax =250 nm; HRMS (FAB)
calcd for C28H33BrN4O2 [M+H]+: 537.1860, found: 537.1867.
(E)-Ethyl-2-(diphenylmethyleneamino)-7-[1-(2-methyl-3-phenyl-
allyl)-1H-1,2,3-triazol-4-yl]heptanoate (13a): Compound 12
(0.802 g, 1.50 mmol) and THF (8 mL) were added to a vial (23ꢂ
85 mm). PhB(OH)2 (0.367 g, 3.00 mmol) was then added and subse-
quently treated with dichlorobis(triphenylphosphine)palladium(II)
(0.056 g, 0.08 mmol) and an aqueous solution of Na2CO3 (2m,
ChemMedChem 2011, 6, 2243 – 2251
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C. Melander et al.
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2.38 mL) under vigorous stirring. The resulting mixture was initially
sonicated for 15 s and then stirred at 698C for 20 h. The reaction
was extracted with EtOAc (3ꢂ3 mL). The combined organic ex-
tracts were washed with brine, dried over Na2SO4, and the solvent
was evaporated under reduced pressure. The residue was dissolved
in CH2Cl2 and purified by column chromatography (hexane/EtOAc,
1:1) to give the desired compound 13a (0.771 g, 96%) as a yellow
(E)-2-(Diphenylmethyleneamino)-N-methoxy-N-methyl-7-{1-[2-
methyl-3-(naphthalen-1-yl)allyl]-1H-1,2,3-triazol-4-yl}heptana-
mide (14b): Compound 13b (0.664 g, 1.14 mmol) was treated with
N,O-dimethylhydroxyamine·HCl (0.444 g, 4.55 mmol) and iPrMgCl
(2m, 4.55 mL) according to the procedure above. Purification by
column chromatography gave the desired compound 14b
1
(0.378 g, 56%) as a yellow oil: H NMR (400 MHz, CDCl3): d=7.83
1
oil: H NMR (400 MHz, CDCl3): d=7.65 (d, J=7.2 Hz, 2H), 7.42 (m,
(d, J=8.0 Hz, 1H), 7.76 (d, J=7.2 Hz, 1H), 7.69 (d, J=8.4 Hz, 1H),
7.62 (d, J=8.4 Hz, 2H), 7.38 (m, 7H), 7.24 (m, 4H), 7.11 (m, 2H),
6.79 (s, 1H), 5.03 (s, 2H), 4.30 (t, J=5.6 Hz, 1H), 3.16 (s, 3H), 3.07 (s,
3H), 2.67 (t, J=7.2 Hz, 2H), 2.02 (m, 2H), 1.64 (m, 2H), 1.60 (s, 3H),
1.29 ppm (m, 4H); 13C NMR (100 MHz, CDCl3): d=173.3, 169.7,
148.8, 139.7, 137.1, 134.7, 133.8, 133.7, 131.9, 130.4, 129.0, 128.7,
128.7, 128.5, 128.2, 128.0, 128.0, 127.6, 126.9, 126.4, 126.2, 125.5,
124.9, 121.1, 63.0, 61.1, 57.9, 33.8, 32.4, 29.5, 29.2, 26.2, 25.9,
16.0 ppm; IR nmax =3396, 3135, 3058, 2935, 2857, 1682, 1623, 1594,
1577, 1506, 1445, 1286, 1045, 955, 784, 700 cmÀ1; lmax =259,
287 nm; HRMS (FAB) calcd for C38H41N5O2 [M+H]+: 600.3333,
found: 600.3329.
3H), 7.31 (m, 9H), 7.17 (m, 2H), 6.46 (s, 1H), 4.95 (s, 2H), 4.15 (m,
2H), 4.05 (t, J=7.2 Hz, 1H), 2.68 (t, J=7.6 Hz, 2H), 1.94 (m, 2H),
1.77 (s, 3H), 1.65 (m, 2H), 1.31 (m, 4H), 1.23 ppm (t, J=7.2 Hz, 3H);
13C NMR (100 MHz, CDCl3): d=172.7, 170.6, 148.8, 139.8, 136.7,
132.5, 130.6, 130.3, 130.0, 129.2, 129.1, 128.9, 128.8, 128.5, 128.3,
128.1, 127.4, 120.8, 65.7, 61.1, 58.7, 33.8, 29.5, 29.2, 26.0, 25.9, 15.9,
14.5 ppm; IR nmax =3277, 3136, 3097, 2931, 2858, 2092, 1948, 1713,
1593, 1563, 1452, 1437, 1369, 1298, 1248, 1024, 986, 742, 700 cmÀ1
;
lmax =229 nm; HRMS (FAB) calcd for C34H38N4O2 [M+H]+: 535.3068,
found: 535.3056.
(E)-Ethyl-2-(diphenylmethyleneamino)-7-[1-(2-methyl-3-(naph-
thalen-1-yl)allyl]-1H-1,2,3-triazol-4-yl)heptanoate (13b): Com-
pound 12 (0.856 g, 1.60 mmol) and 1-naphthylboronic acid
(0.549 g, 3.19 mmol) was treated with dichlorobis(triphenylphos-
phine)palladium(II) (0.056 g, 0.08 mmol) and an aqueous solution
of Na2CO3 (2m, 2.40 mL) according to the procedure above. Purifi-
cation by column chromatography gave the desired compound
Procedure for one-pot synthesis of 4,5-disubstituted-2-aminoi-
midazole–triazole conjugates from the Weinreb amide (16aa
and 16ba): (E)-5-Methyl-4-{5-[1-(2-methyl-3-phenylallyl)-1H-
1,2,3-triazol-4-yl]pentyl}-1H-imidazol-2-amine (16aa): Compound
14a (0.046 g, 0.084 mmol) was dissolved in 1.5 mL anhydrous THF
in a vial (23ꢂ85 mm). The solution was stirred under N2 at À208C,
and MeMgBr (3m, 0.09 mL) was added dropwise. The resulting
mixture was allowed to warm to room temperature and stirred for
4 h. The reaction was cooled to 08C and quenched with NH4Cl
(20%, 2 mL), then extracted with EtOAc (3ꢂ2 mL). The combined
organic extracts were washed with brine, dried over Na2SO4, and
the solvent was removed under reduced pressure. The residue was
redissolved in 1.5 mL EtOH, and HCl (2m, 0.75 mL) was added and
stirred at room temperature for 12 h. The solution was then adjust-
ed to pH 4.3 with NaOH (0.1m). Cyanamide (0.016 g, 0.38 mmol)
was then added, and the resulting mixture was heated at 958C for
3 h. The EtOH was removed in vacuo and the residue was purified
by column chromatography (CH2Cl2/MeOH satd. NH3 4:1) to afford
the target product 16aa (0.025 g, 82%) in its free-base form. Addi-
tion of concentrated HCl to a MeOH solution (2 mL) of the free
base followed by solvent evaporation under reduced pressure de-
1
13b (0.919 g, 98%) as a yellow oil: H NMR (400 MHz, CDCl3): d=
7.87 (m, 2H), 7.78 (d, J=8.4 Hz, 1H), 7.63 (d, J=7.2 Hz, 2H), 7.42
(m, 8H), 7.32 (m, 3H), 7.15 (m, 2H), 6.87 (s, 1H), 5.12 (s, 2H), 4.16
(m, 2H), 4.04 (t, J=7.2 Hz, 1H), 2.71 (t, J=7.6 Hz, 2H), 1.93 (m, 2H),
1.67 (m, 5H), 1.34 (m, 4H), 1.25 ppm (t, J=7.2 Hz, 3H); 13C NMR
(100 MHz, CDCl3): d=172.7, 170.6, 149.0, 139.8, 136.7, 134.6, 133.8,
133.7, 131.9, 130.5, 129.1, 128.9, 128.8, 128.3, 128.1, 128.0, 128.0,
127.8, 127.0, 126.4, 126.2, 125.5, 124.8, 120.8, 65.7, 61.1, 58.1, 33.8,
29.6, 29.3, 26.0, 25.9, 16.1, 14.5 ppm; IR nmax =3137, 3058, 2926,
2855, 1958, 1732, 1621, 1445, 1369, 1286, 1180, 1044, 1029, 783,
698 cmÀ1; lmax =234, 287 nm; HRMS (FAB) calcd for C38H40N4O2
[M+H]+: 585.3224, found: 585.3226.
(E)-2-(Diphenylmethyleneamino)-N-methoxy-N-methyl-7-[1-(2-
1
livered the corresponding 16aa as its HCl salt: H NMR (400 MHz,
methyl-3-phenylallyl)-1H-1,2,3-triazol-4-yl]heptanamide
(14a):
CD3OD): d=8.56 (s, 1H), 7.33 (m, 4H), 7.26 (m, 1H), 6.78 (s, 1H),
5.33 (s, 2H), 2.92 (t, J=7.6 Hz, 2H), 2.47 (t, J=7.2 Hz, 2H), 2.06 (s,
3H), 1.85 (s, 3H), 1.81 (m, 2H), 1.63 (m, 2H), 1.43 ppm (m, 2H);
13C NMR (100 MHz, CD3OD): d=146.2, 145.3, 136.4, 132.4, 130.2,
128.9, 128.2, 127.4, 126.4, 121.7, 117.6, 60.9, 28.3, 28.0, 27.9, 23.2,
22.8, 14.7, 7.6 ppm; IR nmax =3399, 2920, 2850, 2110, 1682, 1457,
1403, 1180, 1052, 797, 699 cmÀ1; lmax =202, 237 nm; HRMS (FAB)
calcd for C21H28N6 [M+H]+: 365.2448, found: 365.2438.
Compound 13a (0.441 g, 0.83 mmol) and anhydrous THF (2 mL)
were added to a vial (23ꢂ85 mm). The solution was stirred under
N2 at À208C until 13a was dissolved. The solution was treated
with N,O-dimethylhydroxyamine·HCl (0.242 g, 2.48 mmol), and
iPrMgCl (2m, 2.48 mL) was then added dropwise in order to keep
the reaction temperature below À58C. The resulting solution was
stirred at 08C for 2 h. The reaction was quenched with NH4Cl
(20%, 2 mL) and then extracted with EtOAc (3ꢂ2 mL). The com-
bined organic extracts were washed with brine, dried over Na2SO4,
and the solvent was removed in vacuo. Purification of the residue
took place on a silica gel column and was eluted with EtOAc to
give the desired product 14a (0.311 g, 69%) as a colorless oil:
1H NMR (400 MHz, CDCl3): d=7.64 (d, J=7.6 Hz, 2H), 7.42 (m, 3H),
7.30 (m, 9H), 7.16 (m, 2H), 6.48 (s, 1H), 4.98 (s, 2H), 4.31 (t, J=
6.4 Hz, 1H), 3.22 (s, 3H), 3.13 (s, 3H), 2.67 (t, J=7.2 Hz, 2H), 2.02
(m, 2H), 1.79 (s, 3H), 1.64 (m, 2H), 1.29 ppm (m, 4H); 13C NMR
(100 MHz, CDCl3): d=173.6, 169.7, 149.0, 139.8, 137.2, 136.7, 132.5,
130.4, 130.0, 129.2, 129.0, 128.7, 128.7, 128.5, 128.2, 128.1, 127.4,
120.7, 63.1, 61.1, 58.7, 33.8, 32.5, 29.4, 29.2, 26.2, 25.9, 15.9 ppm; IR
nmax =3057, 3024, 2933, 2856, 1666, 1621, 1597, 1576, 1445, 1386,
1285, 1178, 1046, 1021, 990, 782, 699 cmÀ1; lmax =246 nm; HRMS
(FAB) calcd for C34H39N5O2 [M+H]+: 550.3177, found: 550.3177.
(E)-5-Methyl-4-{5-[1-(2-methyl-3-{naphthalen-1-yl}allyl)-1H-1,2,3-
triazol-4-yl]pentyl}-1H-imidazol-2-amine (16ba): According to the
procedure above, 14b (0.082 g, 0.14 mmol) was treated with
MeMgBr (3m, 0.09 mL) followed by deprotection with HCl (2m,
1.2 mL) and cyclization with cyanamide (0.027 g, 0.65 mmol). Purifi-
cation by column chromatography gave the desired product 16ba
(0.050 g, 88%) in its free-base form. Addition of concentrated HCl
to a MeOH solution (2 mL) of the free base followed by solvent
evaporation under reduced pressure delivered the corresponding
1
16ba as its HCl salt: H NMR (400 MHz, CD3OD): d=7.95 (s, 1H),
7.88 (m, 1H), 7.84 (m, 1H), 7.79 (d, J=8.4 Hz, 1H),7.48 (m, 3H),
7.31 (d, J=7.6 Hz, 1H), 6.86 (s, 1H), 5.22 (s, 2H), 2.73 (t, J=7.2 Hz,
2H), 2.37 (t, J=7.2 Hz, 2H), 1.97 (s, 3H), 1.71 (m, 2H),1.62 (s, 3H),
2248
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ChemMedChem 2011, 6, 2243 – 2251

2-AITs: Antibiofilm/Antibiotic Resensitizers
1
1.56 (m, 2H), 1.34 ppm (m, 2H); 13C NMR (100 MHz, CD3OD): d=
148.2, 146.2, 134.8, 133.9, 133.9, 131.9, 128.3, 127.6, 127.2, 126.5,
126.0, 125.8, 125.1, 124.6, 122.7, 121.7, 117.5, 57.3, 29.0, 28.4, 28.1,
24.9, 22.8, 14.8, 7.5 ppm; IR nmax =3410, 2925, 2853, 2104, 1958,
1680, 1402, 1261, 1180, 1021, 954, 805, 698 cmÀ1; lmax =207,
286 nm; HRMS (FAB) calcd for C25H30N6 [M+H]+: 415.2605, found:
415.2600.
15am (0.059 g, 77%) as a colorless oil: H NMR (400 MHz, CDCl3):
d=7.98 (d, J=8.4 Hz, 2H), 7.68 (m, 2H), 7.59 (m, 4H), 7.42 (m, 5H),
7.27 (m, 10H), 7.09 (m, 2H), 6.45 (s, 1H), 4.94 (s, 2H), 4.79 (t, J=
7.2 Hz, 1H), 2.67 (t, J=8.0 Hz, 2H), 2.03 (m, 2H), 1.77 (s, 3H), 1.64
(m, 2H), 1.32 ppm (m, 4H); 13C NMR (100 MHz, CDCl3): d=199.8,
169.6, 148.6, 145.4, 139.8, 139.4, 136.6, 136.4, 134.7, 132.2, 130.3,
129.7, 129.6, 128.9, 128.9, 128.7, 128.7, 128.6, 128.2, 128.1, 128.0,
127.7, 127.2, 127.1, 127.0, 120.4, 70.1, 58.4, 35.0, 29.2, 28.9, 26.2,
25.6, 15.6 ppm; IR nmax =3062, 2928, 2854, 1948, 1683, 1600, 1447,
1277, 1120, 1044, 856, 748, 697 cmÀ1; lmax =229, 252 nm; HRMS
(FAB) calcd for C44H42N4O [M+H]+: 643.3431, found: 643.3422.
Synthesis of selected 4,5-disubstituted-2-aminoimidazole–tria-
zole conjugates (16ae, 16ak, and 16am): General procedure for
the preparation of a-diphenylmethyleneamino ketones: Com-
pound 14a (or 14b) was dissolved in 1.5 mL anhydrous THF in a
vial (23ꢂ85 mm). The solution was stirred under N2 at À208C, and
the appropriate Grignard reagent was added dropwise. The result-
ing mixture was allowed to warm to room temperature and stirred
for 2–4 h until completion of the reaction was confirmed by TLC
analysis. The reaction was cooled to 08C and quenched with NH4Cl
(20%, 2 mL), then extracted with EtOAc (3ꢂ2 mL). The combined
organic extracts were washed with brine, dried over Na2SO4, and
the solvent was removed under reduced pressure. The residue was
dissolved in CH2Cl2 and purified by column chromatography
(hexane/EtOAc, 1:1) to give the desired a-diphenylmethyleneamino
ketones. All a-diphenylmethyleneamino ketones were then sub-
jected to deprotection/cyclization (detailed below).
Deprotection/cyclization procedure to access 2-aminoimidazoles
from diphenylmethyleneamino ketones: The appropriate a-di-
phenylmethyleneamino ketone was dissolved in 1.5 mL EtOH. HCl
(2m) was added to the solution, which was stirred at room temper-
ature for 12 h. The resulting solution was adjusted to pH 4.3 with
NaOH (0.1m). Then the mixture was treated with cyanamide and
heated at 958C for 3 h. The EtOH was removed under reduced
pressure, and the resulting solid was washed with Et2O (1ꢂ3 mL).
Purification by column chromatography (CH2Cl2/MeOH satd. NH3
4:1) afforded the desired compound in its free-base form. Addition
of concentrated HCl to a MeOH solution (2 mL) of the free base fol-
lowed by solvent evaporation under reduced pressure delivered
the corresponding 2-aminoimidazole as its HCl salt.
(E)-2-(Diphenylmethyleneamino)-7-[1-(2-methyl-3-phenylallyl)-
1H-1,2,3-triazol-4-yl]-1-(4-propylphenyl)heptan-1-one
(15ae):
(E)-4-{5-[1-(2-Methyl-3-phenylallyl)-1H-1,2,3-triazol-4-yl]pentyl}-
Compound 14a (0.050 g, 0.091 mmol) was treated with (4-propyl-
phenyl)MgBr (0.5m, 1.82 mL) according to the general procedure.
Purification by column chromatography gave the desired product
5-(4-propylphenyl)-1H-imidazol-2-amine
(16ae):
Compound
15ae (0.053 g, 0.087 mmol) was treated with HCl (2m, 0.44 mL),
then reacted with cyanamide (0.110 g, 2.61 mmol) according to the
general procedure. Purification by column chromatography afford-
ed the target compound 16ae (0.030 g, 74%) over two steps as a
yellow oil: 1H NMR (400 MHz, CD3OD): d=7.94 (s, 1H), 7.33 (m,
4H), 7.28 (m, 5H), 6.57 (s, 1H), 5.11 (s, 2H), 2.72 (t, J=7.6 Hz, 2H),
2.64 (t, J=8.0 Hz, 2H), 2.59 (t, J=8.0 Hz, 2H), 1.77 (d, J=1.2 Hz,
3H), 1.70 (m, 4H), 1.64 (m, 2H), 1.39 (m, 2H), 0.92 ppm (t, J=
7.6 Hz, 3H); 13C NMR (100 MHz, CD3OD): d=148.8, 148.1, 144.6,
138.0, 133.3, 131.5, 130.4, 130.1, 129.5, 128.4, 128.3, 127.1, 124.5,
124.0, 123.7, 60.0, 38.8, 29.9, 29.8, 29.5, 25.8, 25.7, 25.0, 15.9,
14.2 ppm; IR nmax =3360, 2919, 2849, 1955, 1682, 1457, 1128, 1081,
702 cmÀ1; lmax =204, 250 nm; HRMS (FAB) calcd for C29H36N6 [M+
H]+: 469.3074, found: 469.3086.
1
15ae (0.049 g, 88%) as a colorless oil: H NMR (400 MHz, CDCl3):
d=7.80 (d, J=8.4 Hz, 2H), 7.66 (m, 2H), 7.28 (m, 12H), 7.19 (d, J=
8.0 Hz, 2H), 7.07 (m, 2H), 6.47 (s, 1H), 4.96 (s, 2H), 4.76 (dd, J=5.6,
7.6 Hz, 1H), 2.66 (t, J=7.6 Hz, 2H), 2.60 (t, J=7.6 Hz, 2H), 1.98 (m,
2H), 1.78 (s, 3H), 1.63 (m, 4H), 1.30 (m, 4H), 0.93 ppm (t, J=7.6 Hz,
3H); 13C NMR (100 MHz, CDCl3): d=200.0, 169.6, 148.8, 148.4,
139.6, 136.8, 136.6, 133.9, 132.4, 130.3, 129.8, 129.2, 129.0, 128.9,
128.7, 128.7, 128.6, 128.4, 128.1, 127.8, 127.3, 120.6, 69.9, 58.6, 38.1,
35.2, 29.3, 29.0, 26.4, 25.7, 24.3, 15.7, 13.9 ppm; IR nmax =3058,
2926, 2855, 1958, 1661, 1604, 1447, 1277, 1047, 941, 741, 700 cmÀ1
;
lmax =248 nm; HRMS (FAB) calcd for C41H44N4O [M+H]+: 609.3588,
found: 609.3571.
(E)-2-(Diphenylmethyleneamino)-1-(4-fluorophenyl)-7-[1-(2-
methyl-3-phenylallyl)-1H-1,2,3-triazol-4-yl]heptan-1-one (15ak):
Compound 14a (0.063 g, 0.11 mmol) was treated with (4-fluoro-
phenyl)MgBr (1m, 1.15 mL) according to the general procedure.
Purification by column chromatography gave the desired product
(E)-5-(4-Fluorophenyl)-4-{5-[1-(2-methyl-3-phenylallyl)-1H-1,2,3-
triazol-4-yl]pentyl}-1H-imidazol-2-amine (16ak): Compound 15ak
(0.053 g, 0.091 mmol) was treated with HCl (2m, 0.91 mL), then re-
acted with cyanamide (0.153 g, 3.63 mmol) according to the gener-
al procedure. Purification by column chromatography afforded the
target compound 16ak (0.038 g, 95%) over two steps as a color-
1
15ak (0.053 g, 79%) as a colorless oil: H NMR (400 MHz, CDCl3):
1
d=8.01 (m, 2H), 7.65 (m, 2H), 7.41 (m, 3H), 7.32 (m, 5H), 7.28 (m,
4H), 7.05 (m, 4H), 6.47 (s, 1H), 4.97 (s, 2H), 4.69 (dd, J=6.0, 8.4 Hz,
1H), 2.67 (t, J=8.0 Hz, 2H), 1.98 (m, 2H), 1.78 (d, J=1.2 Hz, 3H),
1.63 (m, 2H), 1.29 ppm (m, 4H); 13C NMR (100 MHz, CDCl3): d=
198.9, 169.9, 166.9, 164.4, 148.7, 139.4, 136.6, 136.6, (132.4, 132.4,
132.4), (132.1, 132.0), 130.5, 129.9, 129.0, 128.9, 128.8, 128.4, 128.2,
127.8, 127.3, 120.6, 115.7, 115.5, 70.6, 58.6, 35.2, 29.3, 29.0, 26.3,
25.7, 15.8 ppm; IR nmax =3077, 2918, 2849, 1961, 1682, 1597, 1446,
1230, 1156, 1049, 781, 698 cmÀ1; lmax =248 nm; HRMS (FAB) calcd
for C38H37FN4O [M+H]+: 585.3024, found: 585.3026.
less oil: H NMR (400 MHz, CD3OD): d=8.50 (s, 1H), 7.48 (m, 2H),
7.33 (m, 4H), 7.22 (m, 3H), 6.76 (s, 1H), 5.31 (s, 2H), 2.87 (t, J=
7.6 Hz, 2H), 2.66 (t, J=7.2 Hz, 2H), 1.85 (d, J=1.2 Hz, 3H), 1.76 (m,
4H), 1.44 ppm (m, 2H); 13C NMR (100 MHz, CD3OD): d=165.4,
162.9, 148.2, 146.6, 137.7, 133.7, 131.5, 130.8, 130.7, 130.2, 129.5,
128.7, 127.5, 126.0, 124.4, 122.8, (117.3, 117.1), 62.1, 29.6, 29.5, 29.0,
24.8, 24.5, 16.0 ppm; IR nmax =3349, 2923, 2855, 1683, 1515, 1456,
1226, 1159, 1066, 838, 701 cmÀ1; lmax =204, 242 nm; HRMS (FAB)
calcd for C26H29FN6 [M+H]+: 445.2510, found: 445.2508.
(E)-5-(Biphenyl-4-yl)-4-{5-[1-(2-methyl-3-phenylallyl)-1H-1,2,3-tri-
azol-4-yl]pentyl}-1H-imidazol-2-amine (16am): Compound 15am
(0.059 g, 0.092 mmol) was treated with HCl (2m, 0.92 mL), then re-
acted with cyanamide (0.154 g, 3.67 mmol) according to the gener-
al procedure. Purification by column chromatography afforded the
target compound 16am (0.026 g, 57%) over two steps as a color-
(E)-1-(Biphenyl-4-yl)-2-(diphenylmethyleneamino)-7-[1-(2-
methyl-3-phenylallyl)-1H-1,2,3-triazol-4-yl]heptan-1-one (15am):
Compound 14a (0.066 g, 0.12 mmol) was treated with biphenyl-4-
yl-MgBr (0.5m, 2.4 mL) according to the general procedure. Purifi-
cation by column chromatography gave the desired product
ChemMedChem 2011, 6, 2243 – 2251
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C. Melander et al.
MED
less oil: 1H NMR (400 MHz, CD3OD): d=8.41 (s, 1H), 7.72 (d, J=
8.4 Hz, 2H), 7.64 (m, 2H), 7.54 (d, J=8.0 Hz, 2H), 7.46 (t, J=8.0 Hz,
2H), 7.30 (m, 6H), 6.70 (s, 1H), 5.23 (s, 2H), 2.84 (t, J=8.0 Hz, 2H),
2.74 (t, J=7.6 Hz, 2H), 1.80 (d, J=1.2 Hz, 3H), 1.74 (m, 4H),
1.45 ppm (m, 2H); 13C NMR (100 MHz, CD3OD): d=148.3, 146.8,
142.5, 141.3, 137.7, 133.4, 131.6, 130.2, 130.2, 129.5, 129.0, 128.8,
128.7, 128.7, 128.6, 128.0, 127.2, 124.6, 123.4, 61.8, 29.6, 29.4, 29.1,
25.0, 24.6, 15.9 ppm; IR nmax =3394, 2918, 2847, 1957, 1683, 1456,
1121, 1024, 702 cmÀ1; lmax =204, 240, 289 nm; HRMS (FAB) calcd
for C32H34N6 [M+H]+: 503.2918, found: 503.2917.
filled (100 mL per well) from the remaining 3 mL bacterial subcul-
tures, allowing the concentration of compound to be kept uniform
throughout the antibiotic dilution procedure. After standing for
10 min, aliquots (200 mL) of the samples containing antibiotic were
distributed to the corresponding first-row wells of the microtiter
plate. Row 1 wells were mixed six to eight times, and then 100 mL
were transferred to row 2. Row 2 wells were mixed six to eight
times, followed by a 100 mL transfer from row 2 to row 3. This pro-
cedure was repeated to serially dilute the rest of the rows of the
microtiter plate, with the exception of the final row, to which no
antibiotic was added (to check for growth of bacteria in the pres-
ence of compound alone). The plate was then covered and incu-
bated under stationary conditions at 378C. After 16 h, MIC values
were recorded as the lowest concentration of antibiotic at which
no visible growth of bacteria was observed.
Biological screening
Inhibition assays of selected compounds against MRSA and
A. baumannii: Inhibition assays were performed by taking over-
night cultures of MRSA or A. baumannii and subculturing at an
OD600 of 0.01 into tryptic soy broth with a 0.5% glucose supple-
ment (TSBG) for MRSA or Luria–Bertani (LB) media for A. baumannii.
Stock solutions of predetermined concentrations of the test com-
pound were prepared in the resulting bacterial suspension, and ali-
quots of 100 mL from stock solutions were distributed to the wells
of a 96-well plate. Plates were covered, sealed (GLAD Press’n Seal
film) and incubated under stationary conditions at 378C for 24 h.
The media was then discarded, and the plates were washed thor-
oughly with H2O. Each well was stained with crystal violet (CV;
110 mL, 0.1% solution) at room temperature for 30 min. After thor-
ough washing with H2O again, the remaining stain was dissolved
in 95% EtOH (200 mL) and 125 mL was transferred to corresponding
wells of a polystyrene microtiter dish. Biofilm inhibition was quanti-
fied by measuring the OD540 value of each well. Blank wells were
employed as background controls.
Checkerboard assay: MHB was inoculated with MRSA (5ꢂ
105 CFUmLÀ1) and 100 mL aliquots were distributed to all wells of a
96-well plate except well 1A. Inoculated MHB (200 mL) containing
compound (at 2ꢂ the highest concentration being tested) was
added to well 1A, and 100 mL of the same sample was placed in
each of wells 2A–12A. Column A wells were mixed six to eight
times, and then 100 mL were withdrawn and transferred to col-
umn B. Column B wells were mixed six to eight times, followed by
a 100 mL transfer to column C. This procedure was repeated to seri-
ally dilute the rest of the columns of the plate up to column G (col-
umn H was not mixed to allow determination of the MIC of antibi-
otic alone). Inoculated media (100 mL) containing antibiotic at 2ꢂ
the highest concentration being tested was placed in wells A1–H1
and serially diluted in the same manner to row 11 (row 12 was not
mixed to allow determination of the MIC of compound alone). The
plates were incubated for 16 h at 378C. The MIC values (lowest
concentration at which there was no visible growth of bacteria) of
both compound and antibiotic in the combination were recorded,
as well as the MIC values of compound alone (from row 12) and
antibiotic alone (from column H). The SFIC values were calculated
as follows: SFIC=FICCompd +FICAntibiotic, for which FICCompd =[MICCompd
in combination]/[MICCompd alone], and FICAntibiotic =[MICAntibiotic in
combination]/[MICAntibiotic alone]. The combination is considered
synergistic if SFICꢀ0.5, indifferent if 0.5<SFIC<2, and antagonis-
tic if SFICꢁ2.
Dispersion assays of selected compounds against A. baumannii:
Dispersion assays were performed by taking overnight cultures of
A. baumannii and subculturing at an OD600 of 0.01 into LB media.
Aliquots (100 mL) of the resulting bacterial suspension were distrib-
uted to the wells of a 96-well plate. Plates were then wrapped
(GLAD Press’n Seal film) followed by incubation under stationary
conditions at 378C for 24 h. The media was then discarded from
the wells, and the plates were washed thoroughly with H2O. Stock
solutions of predetermined concentrations of the test compound
were prepared in LB, and 100 mL was transferred from stock solu-
tions into the 96-well plate with preformed biofilms. Media alone
was added as a control. Sample plates were incubated at 378C for
24 h. The media was discarded from the wells after incubation, and
the plates were washed thoroughly with H2O. Each well was
stained with CV (110 mL, 0.1% solution) at room temperature for
30 min and then washed with H2O again. The remaining stain in
each well was dissolved in 95% EtOH (200 mL) and 125 mL was
transferred to corresponding wells of a polystyrene microtiter dish.
The biofilm dispersal effect was quantified by measuring the OD540
value of each well. Blank wells were employed as background con-
trols.
Acknowledgements
The authors thank the UNCGA competitive research fund and the
US DoD Defense Medical Research and Development Program
(DMRDP, # W81XWH-11-2-0115) for support of this work. The
DMRDP is administered by the Department of the Army; the US
Army Medical Research Acquisition Activity [820 Chandler Street,
Fort Detrick, MD 21702-5014 (USA)] is the awarding and adminis-
tering acquisition office. The content of this manuscript does not
necessarily reflect the position or the policy of the US Govern-
ment, and no official endorsement should be inferred.
Broth microdilution method for antibiotic resensitization: Muel-
ler–Hinton broth (MHB) was inoculated (5ꢂ105 CFUmLÀ1) with
MRSA. Aliquots (4 mL) of the resulting bacterial suspension were
distributed to culture tubes and compound, from 100 mm DMSO
stock, was added to give the final testing concentration. Bacteria
not treated with the tested 2-AI derivative served as the control.
After sitting for 30 min at room temperature, 1 mL of each sample
was transferred to a new culture tube and oxacillin sodium salt
was added from 128 mgmLÀ1 H2O stock to give a final concentra-
tion of 128 mgmLÀ1. Rows 2–12 of a 96-well microtiter plate were
Keywords: aminoimidazoles
biofilms · MRSA · synergism
·
antibiotic resensitization
·
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