The chemical synthesis and antibiotic activity of a diverse library of 2-aminobenzimidazole small molecules against MRSA and multidrug-resistant A. baumannii

Article abstract of DOI:10.1016/j.bmc.2009.12.003

Multidrug-resistant bacterial infections continue to be a rising global health concern. Herein is described the development of a class of novel 2-aminobenzimidazoles with antibiotic activity. These active 2-aminobenzimidazoles retain their antibiotic activity against several strains of multidrug-resistant Staphylococcus aureus and Acinetobacter baumannii when compared to susceptible strains.

Full text of DOI:10.1016/j.bmc.2009.12.003

Bioorganic & Medicinal Chemistry 18 (2010) 663–674
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The chemical synthesis and antibiotic activity of a diverse library
of 2-aminobenzimidazole small molecules against MRSA and
multidrug-resistant A. baumannii
Robert W. Huigens III, Samuel Reyes, Catherine S. Reed, Cynthia Bunders, Steven A. Rogers,
*
Andrew T. Steinhauer, Christian Melander
North Carolina State University Chemistry Department, 2620 Yarborough Drive, Raleigh, NC 27695-8204, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Multidrug-resistant bacterial infections continue to be a rising global health concern. Herein is described
the development of a class of novel 2-aminobenzimidazoles with antibiotic activity. These active
2-aminobenzimidazoles retain their antibiotic activity against several strains of multidrug-resistant
Staphylococcus aureus and Acinetobacter baumannii when compared to susceptible strains.
Ó 2009 Published by Elsevier Ltd.
Received 12 August 2009
Revised 1 December 2009
Accepted 2 December 2009
Available online 6 December 2009
Keywords:
2-Aminobenzimidazole
Chemical library synthesis
Antibiotic
Multidrug-resistant bacteria
Staphylococcus aureus
Acinetobacter baumannii
1. Introduction
possess both antibiotic and biofilm inhibition activities
(Fig. 1).25,26 Although this appears to be contradictory, our group
There is a growing need for new and effective antibiotic agents
due to the emergence of life threatening, multidrug-resistant
(MDR) bacterial infections1–6 such as methicillin-resistant Staphy-
lococcus aureus (MRSA).7–9 MRSA infections are the leading cause
of nosocomial infections and cause more deaths than complica-
tions due to HIV infection in the US every year.10,11 These infec-
tions are not restricted to hospital settings, as community-
acquired MRSA infections are on the rise.12 Vancomycin-resistant
strains of S. aureus have also emerged, depriving the medical com-
munity of their last line of defense for treating MRSA infections.10
Nosocomial infections from Acinetobacter baumannii have also re-
ceived considerable attention as the incidence of multidrug-resis-
tant infections from this Gram-negative pathogen has steadily
been on the rise and has also resulted in several cases of severe
infections to American soldiers that were deployed to Iraq and
Kuwait.13–17
has synthesized and evaluated several synthetic analogues of these
natural products and determined that these analogues can be
either toxic or non-toxic to planktonic bacteria depending on the
compound concentration, bacterial species, and analogue architec-
ture.18–20 In continuing to develop small molecules inspired by
bromoageliferin 1, we have synthesized and screened a library fo-
cused on the 2-aminobenzimidazole (2ABI) ring system for anti-
bacterial activity (Fig. 2).
Over the past few years, our group has been interested in the
design and synthesis of biologically active small molecules derived
from 2-aminoimidazole marine alkaloids.18–24 Bromoageliferin 1
and oroidin 2 are two such alkaloids that have been reported to
* Corresponding author. Tel.: +1 919 513 2960; fax: +1 919 515 5079.
E-mail address: christian_melander@ncsu.edu (C. Melander).
Figure 1. Bromoageliferin 1 and oroidin 2 have reported to have antibiotic and
antibiofilm activities against various bacteria.
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.12.003

664
R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
2ABI sulfonamides (Scheme 3). These sulfonamides were synthe-
sized in an analogous approach as the 2ABI amides; however, the
overall yields were lower over the two steps (20–84% yield). As
with the synthesis of the 2ABI amides, the sulfonylation reaction
was quenched with dilute acid upon completion, extracted, passed
through a small plug of silica gel and subsequently subjected to
Boc-group deprotection by treatment with TFA in methylene chlo-
ride. The resulting TFA salt was then dissolved in HCl/methanol,
concentrated then washed with ether to give pure 2ABI sulfon-
amide as an HCl salt (Scheme 3).
We were also interested in evaluating triazole molecules since
our group has had success with triazole conjugates as biofilm mod-
ulators.20,21 As an extension of the amide library, we synthesized
alkyne 47 from amine 11 to be used in click reactions with various
azides to make 2ABI triazoles (Scheme 4). Azide diversity was ac-
cessed through a two-step procedure to generate azide amides
from chloroacetyl chloride 49. This was accomplished by adding
chloroacetyl chloride 49 dropwise to an amine at 0 °C to give the
Figure 2. Structural simplification of the core ring system of bromoageliferin 1 to
give the general structure of 2ABI 3.
Benzimidazole derivatives have received attention in recent
years due to their biological activities.27–30 These heterocyclic com-
pounds have demonstrated antimicrobial activity against many
bacterial and fungal strains and prove to be promising starting
points for studies toward simplified structures of bromoageliferin’s
tetrahydrobenzimidazole core.31–38 Furthermore, we have shown
that 2ABI small molecules can be appropriately designed to inhibit
and disperse biofilms of Gram-positive bacteria.39
corresponding
purification, and treated with sodium azide in DMF overnight at
elevated temperatures to give the -azido amide 51 in 78–96%
a-chloroamide 50. This was then used, without
a
yield over two steps. These azide amides were triturated or recrys-
tallized in hexanes and did not require chromatography for
purification.
2. Results and discussion
2.1. Chemical synthesis of 2-aminobenzimidazole (2ABI) library
2.2. Screening the 2ABIs library for antibiotic activity against
MRSA (ATCC BAA-44)
The chemical synthesis of simple 2ABIs was accomplished via
the straightforward condensation between commercially available
1,2-phenyldiamines and cyanogen bromide (Scheme 1). The yields
were generally good for this condensation, required no chromatog-
raphy for purification, were scalable, and after Boc-protection al-
lowed for analogue synthesis using 11 as the key building block
of this synthesis. Amine 11 is a mixture of Boc-group constitutional
isomers and was not separated, but used as a mixture to elaborate
amide and sulfonamide libraries. Once these constitutional iso-
mers were acylated, sulfonylated, or used to make various tria-
zoles, deprotection generated one regiomeric product, thus we
didn’t feel it necessary to separate the constitutional isomers of
11 at any point.
All 2ABIs were evaluated for antibiotic activity by determining
MIC values using standard microdilution protocols.41 With this in
mind, we used a 96-well microtiter plate microdilution suscepti-
bility testing starting at 400
ing 12, twofold serial dilutions (entire range of this screen:
400 M–391 nM) in PVC wells. MIC values were determined as
lM of each compound and then mak-
l
the lowest concentration at which no growth was observed upon
visual inspection after incubating for 16 h at 37 °C. Pellets formed
on the bottom of wells were considered bacterial growth even if
the wells were clear of turbidity.
Initially, 44 2ABIs were screened against MRSA (ATCC BAA-44).
From this initial assay, 37 compounds were completely inactive
The synthesis of the 2-aminobenzimidazole amide library was
adapted from a previously reported route to 11 starting from 10
(Scheme 2).40 It was envisioned to use amine 11 to systematically
construct several small classes of 2ABI small molecules for screen-
ing. Amine 11 was acylated with various acid chlorides or anhy-
drides followed by Boc-group removal with TFA in methylene
chloride, followed by anion exchange to give 2ABI amides as HCl
salts in yields between 57% and 94%. Amine 11 was also treated
with various sulfonyl chlorides to generate a diverse set of 10
against BAA-44 with MIC values of >400
showed marginal antibiotic activity (MIC = 400
fonamide derivatives (38 and 45) demonstrated moderate antibi-
otic activity against BAA-44 (MIC = 100 M). Three amide
derivatives (20, 21, and 33), however, showed good antibiotic
activity against BAA-44 with MIC values of 12.5–25 M (Fig. 3).
Two members from the 2ABI amide collection, 21 and 33, dem-
onstrated the most potent antibiotic activity against BAA-44 with
MIC values of 12.5 lM while 20 had an MIC of 25 lM. Interesting,
l
M. Two compounds
lM) and two sul-
l
l
Scheme 1. Synthesis of the simple 2ABIs.

R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
665
Scheme 2. 2ABI Amides provide structural diversity through amide formation.
Scheme 3. Synthesis of 2ABI sulfonamides.
these three compounds can occupy nearly the same chemical space
of the carbon atoms of the benzene ring of 33 (Fig. 3). There was no
when the alkyl chains of 20 and 21 are orientated to lay over four
antibiotic activity against BAA-44 at the highest concentration

666
R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
Scheme 4. Synthesis of 2ABI triazoles using click chemistry.
Figure 3. Lead 2ABIs amides against BAA-44.
tested (400
l
M) from the shorter aliphatic chain of amide 19. Lack
Boc protected synthetic precursor of 33 (69, Fig. 5). Although 69
is a mixture of two regioisomers, testing of this mixture in the mic-
rodilution susceptibility assays enables us to identify if an unpro-
tected 2-aminoimidazole moiety is required for antibiotic activity
against BAA-44. Two other control compounds were also synthe-
sized that incorporated the active 4-pentylphenyl tail of 33 while
eliminating the 2-aminobenzimidazole moiety. Complete removal
of the 2-aminoimidazole moiety of 33 was accomplished by syn-
thesizing 70. The aniline amide moiety of 33 was completely elim-
inated in the design of control structure 71, which is aimed at
determining the role of the 2-aminoimidazole head fused to the
4-pentylphenyl tail in the antibiotic activity of BAA-44 (Fig. 5).
The synthesis of 70 was accomplished by simply adding 4-pent-
ylbenzoyl chloride 72 to aniline (Scheme 5). Control analogue 71
was synthesized by adding 72 to diazomethane in ether and allow-
ing the resulting reaction mixture to stir for one hour. This was fol-
lowed by treatment with concentrated HBr (aq), and the resulting
of activity was also observed for the longer aliphatic chain in amide
22 at 400 M.
l
2.3. Second-generation library developed from 33 and
simplified analogues to determine the necessary structural
elements for antibiotic activity against BAA-44
Due to its structural rigidity, 33 was chosen as the lead struc-
ture for further SAR studies. A second-generation library of eight
2ABIs was synthesized in an attempt to enhance antibiotic activity,
where the focus of this library was derivatizing the 4-position of
the phenyl amide moiety in 33. Both extension and retraction of
hydrocarbon chain length, chain branching, substitution with an-
other aromatic ring, and azide substitution was investigated
(Fig. 4).
In addition to these modifications, we synthesized and assayed
a number of control compounds to evaluate the necessity of the
2ABI subunit. We focused on the 2-aminoimidazole ring contained
within the 2-aminobenzimidazole moiety of 33 by testing the tri-
a-bromoketone was taken on without purification and treated
with Boc-guanidine in DMF for three days at room temperature.
Following flash chromatography, 71 was formed after Boc-removal

R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
667
Figure 4. A focused library was synthesized based on the 4-position of the phenyl amide moiety in 33.
with TFA and final ion exchange to the HCl salt. 2-Aminoimidazole
71 was formed in 41% yield over these three steps.
This second-generation library was then screened in microdilu-
tion susceptibility assays to give a structure-activity relationship
for these 2ABI amides against BAA-44. The two 4-substituted 2ABI
triazoles 67 and 68 demonstrated no activity against BAA-44 as
did the 4-phenyl derivative 2ABI amide 65 (MIC >400
stitution of a 4-azido group in 2ABI amide analogue 66 demon-
strated weak antibiotic activity against BAA-44 (MIC = 400 M).
lM). The sub-
l
There was an interesting structural trend in activity in the ali-
phatic substitution at the 4-position of the phenyl amide moiety of
the lead 33. The 4-tert-butyl group substitution of 64 showed a de-
crease in antibiotic activity from 33, but still retained some antibi-
otic activity (MIC = 50
one methylene unit shorter than 33, also demonstrated decreased
potency against BAA-44 (MIC = 25 M for 61 vs MIC = 12.5 M for
lM). The 4-butyl side chain of 61, which is
l
l
33). When 2ABI amide 62, which has an additional methylene unit
to its straight chain than 33, was screened against BAA-44, it demon-
strated the same potency as 33 (MIC = 12.5 lM). When this chain
Figure 5. Determining the essential structural elements of 33 for antibiotic activity
towards BAA-44 through the screening of various structural analogues related to
33.
Scheme 5. Synthesis of 70 and 71.

668
R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
Table 1
length was extended out one methylene unit further the activity
against BAA-44 began to decrease (MIC = 25 M). It is apparent that
MIC values of antibiotics and 2ABIs against S. aureus strains
l
chain length from the 4-position of the phenyl amide moiety is crit-
ical for optimal antibiotic activity against BAA-44 with n-pentyl and
n-hexyl chains being optimal against this multidrug-resistant
bacterium.
Compounds 69, 70, and 71 were also screened for anti-MRSA
activity. As predicted, the tri-Boc-protected 69 had no activity
Compound
MRSA BAA-44 ATCC 29213
391 nM 391 nM
>400 3.13
ATCC 29740
ATCC 25923
Vancomycin
Methicillin
Ciprofloxacin
Gentamycin
Streptomycin >200
2ABIs:
195 nM
n.a.
391 nM
6195 nM
391 nM
391 nM
n.a.
n.a.
n.a.
n.a.
l
M
lM
12.5
200
l
M
391 nM
391 nM
lM
l
M
1.56
l
M
against BAA-44 at 400
also unable to demonstrate antibiotic activity against BAA-44 at
400 M. However, 2-aminoimidazole control compound 71 dem-
onstrated the same anti-MRSA activity as 33 with a MIC value of
12.5 M. From these studies, we conclude that the 2-aminoimidaz-
lM. As in the case with 69, control 70 was
20
21
33
38
45
61
62
63
64
65
66
67
68
69
70
71
25
lM
12.5
l
M
12.5
12.5
6.25
100
l
l
l
M
M
M
25
50
l
l
M
M
12.5
12.5
100
100
l
l
M
M
25 lM
12.5
100
100
l
M
12.5
n.a.
n.a.
l
M
l
lM
l
M
M
lM
lM
l
n.a.
l
25
lM
25
l
M
12.5
6.25
12.5
lM
lM
lM
25
l
M
ole heterocycle is required in its unprotected form, either as its
own heterocycle or as part of a larger 2-aminobenzimidazole,
along with the 4-pentylphenyl or 4-hexylphenyl tail to maximized
anti-MRSA activity in microdilution susceptibility testing (Fig. 6).
12.5
l
M
M
M
12.5
l
M
M
M
12.5
l
M
25
50
l
l
25
50
l
l
50
l
M
50
lM
100 lM
>400
400 lM
>400
>400
>400
>400
l
M
>400
n.a.
n.a.
n.a.
n.a.
>400
n.a.
l
M
M
>400
lM
>400
lM
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
l
l
l
l
M
2.4. Activity of 2-aminobenzimidazoles screened against other
S. aureus strains
M
M
M
l
>400
n.a.
lM
>400
n.a.
lM
12.5 lM
The 2ABIs were further evaluated in the microdilution suscepti-
bility assays against three other S. aureus strains (ATCC 29213,
ATCC 25923, and ATCC 29740; Table 1) in comparison to five clin-
ically used antibiotics. Results from the microdilution susceptibil-
ity testing demonstrated antibiotic-resistance of BAA-44 to every
clinical antibiotic tested here (Table 1), except for vancomycin,
when compared to the other S. aureus strains. As outlined by the
ATCC, BAA-44 demonstrates resistant towards methicillin, cipro-
floxacin, gentamycin and streptomycin while the other S. aureus
strains (ATCC 29213, ATCC 25923, ATCC 29740) were found to be
susceptible. Using the microdilution susceptibility testing, BAA-
44 demonstrated >125-fold increase in resistance towards methi-
cillin, 500-fold increase in resistance towards gentamycin, 400-fold
increase in resistance to ciprofloxacin and >125-fold increase in
Note: n.a.—not applicable/not determined.
resistance to streptomycin when compared to S. aureus strain ATCC
29213.
BAA-44 did not show drug-resistance to these 2ABIs as the rel-
ative potencies were consistent against all of the S. aureus strains.
The lead 2ABIs were found to be more effective against BAA-44 in
the microdilution susceptibility screens than methicillin, gentamy-
cin, and streptomycin (Table 1). The active 2ABI leads were as ac-
tive as ciprofloxacin in the microdilution susceptibility assays (all
having MIC = 12.5
lM).
Figure 6. A detailed SAR for these novel 2ABIs against BAA-44.

R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
669
2.5. Determining microbicidal activity and vancomycin
enhancement with 33
screen selected 2ABIs against five A. baumannii strains. Four of
these A. baumannii strains were MDR. Five clinically used antibiot-
ics were tested in parallel to 12 2ABIs for direct comparison of anti-
biotic activity and to evaluate the drug-resistance of the clinical
isolates.
Further evaluation of 33 against BAA-44 allowed us to determine
its Minimum Bactericidal Concentration (MBC) to be 25 lM, thus
demonstrating that 33 has bactericidal activity and not bacterio-
static activity against BAA-44. The MBC is the lowest concentration
at which there is P99.9% reduction in CFU mLÀ1 when compared to
an untreated control.42 Bacteriostatic activity is generally defined as
an agent that inhibits bacterial growth, whereas bactericidal activity
is defined as an agent which actually kills bacteria.42 This activity is
determined by MBC/MIC ratios for antibacterial agents. Antibacte-
rial activity is considered to be bactericidal when a compound’s
MBC is 64 times the concentration of the MIC, while a ratio is >4
the activity is said to be bacteriostatic. Since the MBC/MIC ratio for
First, we generated a preliminary structure–activity relation-
ship of these 2ABIs against a non-MDR strain of A. baumannii (ATCC
19606). The 2ABI amides 20, 21, and 33 were found to demonstrate
the most potent antibiotic activity against S. aureus strains and
were also found to be the leads against A. baumannii with MIC val-
ues of 25
with an MIC value of 50
activity (MIC = 100 M). Compounds 19 (with a shorter chain)
and 22 (with a longer chain) demonstrated no antibiotic activity
at 400 M. Although the potency of these 2ABIs was slightly re-
lM. Analogues 61, 62, and 63 were slightly less active
lM, while 64 showed reduced antibiotic
l
l
33 was 2 (MBC = 25
l
M/MIC = 12.5
l
M against BAA-44) we con-
duced, the structure–activity relationship was the same as it was
clude the activity of 33 is bactericidal.
against S. aureus strains.
The use of bactericidal agents, such as 33, is preferred from a
clinical standpoint compared to bacteriostatic agents since bacte-
rial infections are better cured with greater reduction in viable bac-
An MDR strain of A. baumannii was purchased from ATCC (BAA-
1605) and screened to determine the 2ABI analogue’s activity. As
with BAA-44, all but one clinically used antibiotic showed in-
creased resistance when subjected to microdilution susceptibility
testing. Tobramycin showed increased activity against this strain,
but this MDR A. baumannii strain demonstrated >125-fold increase
in resistance towards imipenem, >4-fold increase in resistance to-
wards gentamycin and >125-fold increase in resistance towards
tetracycline (Table 2).
As previously discovered when the 2ABIs were screened against
BAA-44, the active 2ABIs against A. baumannii strain ATCC 19606
were at least as active against BAA-1605. Analogues 21, 33, 61,
and 62 all demonstrated a twofold increase in antibiotic activity.
The most active 2ABI analogues against this MDR A. baumannii
strain were found to be 21, 33, and 62 which had MIC values of
teria or bacterial load.42,43 At 12.5
l
M (the MIC value against BAA-
44), 33 demonstrated a 99.15 0.75% reduction in BAA-44 CFU mL
À1. The MBC90 of 33 was determined to be 11.15 0.12
M and the
MBC50 is 5.88 0.50 M.
l
l
There is also clinical value in combination therapy options
when treating MRSA infections.44 We were interested to see if 33
could synergistically enhance vancomycin activity against BAA-
44 at concentrations well below the MIC value of 33. BAA-44
was treated with 2, 4, and 6 lM of 33 and allowed to incubate with
twofold dilutions of vancomycin against MRSA. After 16 h, all con-
centrations of 33 tested increased vancomycin activity by twofold.
Without 33, vancomycin’s MIC is 391 nM, but with sub-MIC con-
centrations of two ABI 33, vancomycin recorded an MIC value of
12.5
imipenem (MIC = 50
for tobramycin (MIC = 6.25
l
M and demonstrated more potent antibacterial activity than
M) and the other antibiotics tested except
M).
195 nM against BAA-44. It was determined that at 2
lM of 33 there
l
was a 21% reduction in the CFU mLÀ1 of BAA-44, demonstrating a
synergistic twofold enhancement in antibiotic activity of vancomy-
cin when paired with 33.
l
Once we had established increased antibiotic activity against
the MDR strain of A. baumannii we evaluated these 2ABIs against
three additional clinical isolates (AB0043, UH8407, and 3340).
The most well characterized clinical specimen in this study was
AB0043, an isolate from a patient suffering an A. baumannii infec-
tion at Walter Reed Army Medical Center. This bacterial strain
was isolated in 2004 from the blood of a soldier that was deployed
to Iraq/Kuwait, and was found to be resistant to 7 of the 9
2.6. 2-Aminobenzimidazole microdilution susceptibility testing
against A. baumannii
Next, we assessed the 2ABI’s activity against a Gram-negative
pathogen and used the microdilution susceptibility approach to
Table 2
MIC value of antibiotics and 2ABIs against A. baumannii
Compound
A. baumannii ATCC 19606
MDRAB BAA-1605
50
>400
UH8407
3.125
>400
3340
AB0043
50
>400
Imipenem
Ampicillin
Tobramycin
Gentamycin
Tetracycline
2ABIs:
19
20
21
22
33
61
62
63
64
65
51
391 nM
l
M
l
l
M
200
l
M
l
M
l
>400
25
l
M
M
M
lM
>400
>400
>400
l
l
l
M
M
M
M
l
6.25
>400
>400
l
l
l
M
M
M
200
lM
200
200
6.25
l
l
l
M
M
M
100
lM
>400
lM
3.13
lM
>400
l
M
M
25
l
M
>400
l
M
M
>400
lM
>400
l
>400
25
12.5
>400
25
100
lM
400
lM
25
25
l
M
25
12.5
>400
12.5
25
l
M
25
25
l
M
l
M
25
12.5
>400
12.5
25
l
M
lM
lM
lM
lM
lM
lM
l
M
>400
l
l
M
>400
lM
lM
25
50
50
50
l
l
l
l
M
lM
25
50
25
l
l
l
M
M
M
l
M
M
M
M
lM
l
M
lM
12.5
n.a.
100
n.a.
l
M
50
50
l
l
M
M
12.5
12.5
l
l
M
M
n.a.
100
n.a.
100
lM
lM
lM
100
l
M
50 lM
>400
>400
>400
>400
l
l
l
l
M
M
M
M
>400
n.a.
n.a.
lM
>400
>400
>400
n.a.
lM
lM
lM
>400
>400
n.a.
l
l
M
M
>400
>400
n.a.
l
l
M
M
59
70
n.a.
Note: n.a.—not applicable/not determined.

670
R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
antibiotics used in the study to determine its MDR phenotype. Ge-
netic analysis showed that this isolate demonstrated enhanced ef-
flux pump activity via adeR expression, which typically makes
isolates resistant to aminoglycosides, quinolones, tetracycline
and trimethoprim. Several aminoglycosides-modifying enzymes
(AMEs) were expressed in this clinical isolate as well. Isolate
AB0043 was found to express the AME gene aadB which was often
associated with tobramycin resistance of A. baumannii clinical
isolates.45
The isolates UH8407, 3340, and AB0043 were found to be resis-
tant against tobramycin and gentamycin by use of the microdilu-
tion susceptibility assay. There was increased resistance towards
imipenem with isolate 3340 showing >500-fold more resistance
when compared to the wild-type ATCC 19606 strain. Tetracycline
demonstrated antibiotic activity against two of the four MDRAB
strains. None of the clinical antibiotics maintained their antibiotic
activity against all clinical isolates and MDR A. baumannii strains
compared to the wild-type A. baumannii ATCC 19606 strain.
When the 2ABI analogues were screened against the clinical
specimens we observed the same level of antibiotic activity against
the wild-type (ATCC 19606) and MDRAB strains. In terms of com-
pound potency, 2ABI analogues 20, 21, 33, and 62 consistently
demonstrate more potent antibiotic activity towards the A. bau-
mannii clinical isolates than any of the clinically used antibiotics
during this study. These analogues do not appear to be affected
by the developed mechanisms of drug-resistance, including in-
creased drug efflux, and maintain their relative antibiotic activity
across the five A. baumannii strains (Table 2).
From these studies, we have identified 2ABI 33 which dem-
onstrates bactericidal activity against the MRSA strain BAA-44.
In addition, 2ABI 33 is active against several MDR A. baumannii
strains, including clinical isolates. This same compound was
also found to synergistically increase vancomycin activity two-
fold at 2
12.5 M).
lM, a concentration well under its MIC value (MIC =
l
4. Experimental
4.1. Chemistry
All chemicals and solvents used were purchased from commer-
cially available sources and used without further purification. Silica
gel was used for column chromatography and was performed with
60 Å mesh standard grade silica gel from Sorbtech. 1H and 13C NMR
spectra were performed using Varian 300 MHz and 400 MHz spec-
trometers. Chemical shifts are reported in parts per million relative
to CDCl3 (d 7.26), CD3OD (d 3.31) and DMSO-d6 (d 2.50) for 1H NMR
and relative to CDCl3 (d 77.23), CD3OD (d 49.51) and DMSO-d6
13
(d 39.51) for C NMR with TMS as an internal standard. Abbrevi-
ations used for 1H NMR splitting are as follows: s = singlet, br
s = broad singlet, d = doublet, dd = doublet of doublets, t = triplet,
q = quartet, m = multiplet, br m = broad multiplet. High-resolution
mass spectra were obtained at the North Carolina State Mass Spec-
trometry Laboratory for Biotechnology. Fluorescence measure-
ments were recorded using a Hitachi Fluorescence Spectrometer
(F-2500). All compounds that were tested for antibacterial activity
were synthesized using standard techniques and are described in
complete detail in the Supplementary data. Selected 2ABIs are
characterized here.
2.7. Potential of lead compound 33 to lyse red blood cells and
synthetic vesicles
Given that active 2ABI analogues contain a lipophilic tail cou-
pled to a polar head group, we investigated whether these com-
pounds were potentially eliciting their activity through a pore-
forming mechanism. Two experiments were run to probe this
mode of action, a red blood hemolysis assay46 and a dye dispersion
assay47 from synthetic vesicles. Compound 33 was subjected to red
blood cells (RBCs) in hemolysis assays along with inactive com-
pound 29 which served as our control. Each compound was as-
4.1.1. General procedure for the formation of simple 2ABIs
from 1,2-phenylenediamine starting materials. 5-Nitro-1H-
benzo[d]imidazol-2-amine (8)
Cyanogen bromide (3.55 g, 33.5 mmol) was added to a solution
of 4-nitro-1,2-phenylenediamine (5.06 g, 33.1 mmol) in water/ace-
tonitrile (80:10 mL). This reaction was refluxed for 4 h while turn-
ing a deep red color. After concentrating nearly half of the water/
acetonitrile reaction solvent, concentrated ammonia hydroxide
(aq) was added to the solution until a yellow precipitate began
to form. The mixture was then filtered, and the precipitate was
dried under reduced pressure to give 5.97 g of 8.
sayed at 25 lM, the highest MIC observed for 33. At this
concentration, both compounds showed <1% hemolysis.
Next, we evaluated the potential of 33 to induce dye leakage from
synthetic vesicles. Two types of vesicles were prepared, one contain-
ing 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) and
the other containing 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phospho-
glycerol (POPG). POPC mimics typical cell membranes encountered
for mammalian red blood cells, while POPG mimics the negatively
charged lipids encountered in bacterial membranes. At 25 lM 33 in-
duced only 16% dye leakage in the POPG lipids while it induced 9%
dye leakage in POPC lipids. Furthermore, >50% dye leakage in the
POPG vesicles was only observed at >500 lM 33, concentrations that
4.1.2. General procedure for the formation of 2ABI amides (14)
To a vial of 11 (100 mg, 0.22 mmol) and triethylamine (70 lL,
0.50 mmol) in methylene chloride (5 mL) was added the respective
acid chloride or anhydride (0.21 mmol) either dropwise or as a so-
lid. The reaction was then allowed to stir overnight (16–20 h) at
ambient temperatures. Upon the completion of the reaction, it
was quenched with 5% HCl (aq) and transferred to a separatory
funnel. The crude amide was formed as two regioisomers (no at-
tempts at separating these isomers was attempted) and extracted
two times with methylene chloride and the combined organic lay-
ers were dried with sodium sulfate and concentrated. This mixture
was quickly flashed through a plug of silica gel and concentrated in
a vial. The mixture of regioisomer amide products were taken on
without characterization, however, a select few were character-
ized. The resulting tri-Boc–protected 2ABI amide was then dis-
solved in 2 mL of methylene chloride and 2 mL of triflouroacetic
acid was added slowly to give a 1:1 mixture (CH2Cl2/TFA). This
was allowed to stir for 2 h at room temperature after which the
solvent was removed under reduced pressure to give the 2ABI
amide as a TFA salt. To this was added 2 mL of saturated HCl/meth-
anol to give the HCl salt of the 2ABI amide. Then methanol and
are significantly above its MIC. Taken together, this data suggests
that 33 is not eliciting its antibacterial activity through a pore-form-
ing mechanism.
3. Conclusion
In conclusion, a collection of 55 small molecules has been syn-
thesized and screened for antibiotic activity against several mul-
tidrug-resistant bacterial strains. Active 2ABIs from this library
demonstrate broad-spectrum antibiotic activity. In all, nine
strains of S. aureus and A. baumannii were used to determine
the effectiveness of this library using microdilution susceptibility
testing.

R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
671
ether was added to the vial to triturate the resulting product 14.
This two step procedure gave yields between 57% and 94%.
4.1.9. N-(2-Amino-1H-benzo[d]imidazol-5-yl)-6-bromohexana-
mideÁHCl (23)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 23 which was formed as a light yellow so-
lid in 71% yield. Light yellow solid: mp 222–225 °C; 1H NMR
(400 MHz, CD3OD) d 7.93 (m, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.27
(dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 3.46 (t, J = 6.4 Hz, 2H), 2.41 (t,
J = 7.2 Hz, 2H), 1.90 (quintet, J = 7.2 Hz, 2H), 1.74 (quintet,
J = 7.6 Hz, 2H), 1.54 (quintet, J = 7.2, 2H); 13C NMR (100 MHz,
CD3OD) d 174.6, 152.6, 136.6, 131.3, 127.3, 117.3, 112.5, 104.9,
37.8, 34.3, 33.8, 28.9, 26.1; HRMS (ESI) calcd for C13H17BrN4O
(MH+) 324.0586, found 324.0590.
4.1.3. N-(2-Amino-1H-benzo[d]imidazol-5-yl)acetamideÁHCl (15)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 15 which was formed as a white solid
in 89% yield. White solid: mp 313–318 °C; 1H NMR (400 MHz,
DMSO-d6) d 12.39 (br s, 2H), 10.13 (s, 1H), 8.43 (s, 2H), 7.91 (s,
1H), 7.25 (s, 2H), 2.05 (s, 3H); 13C NMR (100 MHz, DMSO-d6) d
168.3, 150.6, 135.3, 129.7, 125.1, 114.5, 111.2, 102.4, 24.0; HRMS
(ESI) calcd for C9H10N4O (MH+) 190.0855, found 190.0857.
4.1.4. N-(2-Amino-1H-benzo[d]imidazol-5-yl)-2,2,2-trifluoroace-
tamideÁHCl (16)
4.1.10. N-(2-Amino-1H-benzo[d]imidazol-5-yl)cyclobutanecarb-
oxamideÁHCl (25)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 16 which was formed as a white solid
in 62% yield. White solid: mp 234–238 °C; 1H NMR (400 MHz,
CD3OD) d 7.93 (s, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 8.8 Hz,
1H); 13C NMR (100 MHz, CD3OD) d 157.1, 156.7, 152.9, 134.1,
131.3, 128.7, 118.3, 112.7, 105.9; HRMS (ESI) calcd for C9H7F3N4O
(MH+) 244.0572, found 244.0575.
The general procedure for the formation for 2ABI amides was
used for the synthesis of 25 which was formed as a white solid
in 59% yield. White solid: mp 199–203 °C; 1H NMR (400 MHz,
CD3OD) d 7.93 (s, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.24 (dd, J = 1.6 Hz,
J = 1.2 Hz, 1H), 2.34 (quintet, J = 1.6 Hz, 2H), 2.23 (m, 2H), 2.18–
2.01 (sextet, J = 8.8 Hz, 2H), 1.92 (m, 1H); 13C NMR (100 MHz,
CD3OD) d 176.3, 152.5, 136.6, 131.1, 127.1, 117.4, 112.4, 104.9,
41.7, 26.2, 19.1; HRMS (ESI) calcd for C12H14N4O (MH+) 230.1168,
found 230.1166.
4.1.5. N-(2-Amino-1H-benzo[d]imdazol-5-yl)propionamideÁHCl
(18)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 18 which was formed as a white solid
in 84% yield. 1H NMR (400 MHz, CD3OD) d 7.93 (m, 1H), 7.26 (m,
2H), 2.40 (q, J = 14.8 Hz, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H);
13C NMR (100 MHz, CD3OD) d 175.6, 152.5, 136.6, 131.2, 127.2,
117.3, 117.3, 112.4, 104.8, 31.1, 10.4; HRMS (ESI) calcd for
C10H12N4O (MH+) 204.1011, found 204.1009.
4.1.11. N-(2-Amino-1H-benzo[d]imidazol-5-yl)cyclopentanecarb-
oxamideÁHCl (26)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 26 which was formed as a white solid
in 91% yield. 1H NMR (400 MHz, CD3OD) d 7.86 (d, J = 1.6 Hz, 1H),
7.30 (dd, J = 8.6 Hz, J = 1.8 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 2.86
(quintet, J = 8.0 Hz, 1H), 1.97–1.90 (br m, 2H), 1.84–1.70 (br m,
4H), 1.66–1.57 (br m, 2H); 13C NMR (100 MHz, CD3OD) d 177.9,
152.4, 136.7, 131.1, 127.1, 117.4, 112.4, 104.9, 47.3, 31.7, 27.2;
HRMS (ESI) calcd for C13H16N4O (MH+) 244.1324, found 244.1321.
4.1.6. N-(2-Amino-1H-benzo[d]imidazole-5-yl)heptanamideÁHCl
(19)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 19 which was formed as a white solid
in 58% yield. 1H NMR (400 MHz, DMSO-d6) d 12.51 (br s, 2H),
10.14 (s, 1H), 8.47 (s, 2H), 7.91 (s, 1H), 7.31 (dd, J = 8.8 Hz,
1.8 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 2.31 (t, J = 7.6 Hz, 2H), 1.56
(m, 2H), 1.27 (m, 6H), 0.84 (m, 3H); 13C NMR (100 MHz, DMSO-
d6) d 171.2, 150.5, 135.3, 129.7, 125.1, 114.4, 111.2, 102.4, 36.4,
31.0, 28.3, 25.1, 22.0, 13.9; HRMS (ESI) calcd for C14H20N4O
(MH+) 260.1637, found 260.1642.
4.1.12. N-(2-Amino-1H-benzo[d]imidazol-5-yl)cyclohexanecarb-
oxamideÁHCl (27)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 27 which was formed as a white solid
in 86% yield. 1H NMR (400 MHz, CD3OD) d 7.88 (s, 1H), 7.29 (d,
J = 8.4 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 2.41 (t, J = 11.6 Hz, 1H),
1.87 (d, J = 12.8 Hz, 2H), 1.80 (d, J = 12.8 Hz, 2H), 1.70 (d,
J = 12.0 Hz, 1H), 1.51 (q, J = 25.0 Hz, J = 12.4 Hz, 2H), 1.40–1.22
(m, 3H); 13C NMR (100 MHz, CD3OD) d 177.9, 152.5, 136.7, 131.1,
127.2, 117.5, 112.4, 104.9, 47.2, 30.8, 27.0, 26.9; HRMS (ESI) calcd
for C14H18N4O (MH+) 258.1481, found 258.1480.
4.1.7. N-(2-Amino-1H-benzo[d]imidazol-5-yl)decanamideÁHCl (20)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 20 which was formed as a white solid
in 94% yield. 1H NMR (400 MHz, CD3OD) d 7.93 (m, 1H), 7.28 (dd,
J = 8.8 Hz, J = 0.8 Hz, 1H), 7.26 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 2.39
(t, J = 3.6 Hz, 2H), 1.70 (m, 2H), 1.36–1.28 (m, 12 H), 0.89 (t,
J = 6.4 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 174.9, 152.5, 136.6,
131.1, 127.2, 117.3, 112.4, 104.8, 38.1, 33.1, 30.7, 30.6, 30.5, 30.5,
27.1, 23.8, 14.6; HRMS (ESI) calcd for C17H26N4O (MH+) 302.2107,
found 302.2109.
4.1.13. N-(2-Amino-1H-benzo[d]imidazole-5-yl)-3-cyclopentyl-
propanamideÁHCl (28)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 28 which was formed as a light yellow so-
lid in 73% yield. 1H NMR (400 MHz, CD3OD) d 9.35 (s, 2H), 8.75 (s,
1H), 8.19 (d, J = 1.2 Hz, 1H), 8.17 (d, J = 1.6 Hz, 1H), 8.07 (d,
J = 8.4 Hz, 1H), 3.13 (t, J = 7.6 Hz, 2H), 2.48 (br m, 3H), 2.37 (m,
2H), 2.30 (m, 2H), 2.20 (m, 2H), 1.83 (br m, 2H); 13C NMR
(100 MHz, CD3OD) d 181.1, 160.2, 145.1, 139.3, 134.7, 124.2,
120.8, 112.1, 49.0; HRMS (ESI) calcd for C15H20N4O (MH+)
272.1637, found 272.1630.
4.1.8. N-(2-Amino-1H-benzo[d]imidazol-5-yl)dodecanamideÁHCl
(21)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 21 which was formed as a light orange so-
lid in 79% yield. 1H NMR (400 MHz, CD3OD) d 7.93 (s, 1H), 7.29 (d,
J = 8.4 Hz, 1H), 7.26 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 2.39 (t, J = 7.6 Hz,
2H), 1.70 (quintet, J = 7.2 Hz, 2H), 1.36–1.26 (m, 16H), 0.88 (t,
J = 6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 165.3, 143.0, 127.0,
121.6, 117.7, 107.7, 102.9, 95.3, 28.5, 23.5, 21.2 (2), 21.1, 20.9 (2),
20.8, 17.4, 14.2, 4.9; HRMS (ESI) calcd for C19H30N4O (MH+)
330.2420, found 330.2422.
4.1.14. N-(2-Amino-1H-benzo[d]imidazol-5-yl)benzamideÁHCl
(29)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 29 which was formed as a white solid
in 83% yield. White solid: mp 245–250 °C; 1H NMR (400 MHz,
DMSO-d6) d 12.53 (br s, 2H), 10.41 (s, 1H), 8.49 (s, 2H), 8.03 (d,

672
R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
J = 2.0 Hz, 1H), 7.98 (s, 1H), 7.96 (s, 1H), 7.59 (m, 1H), 7.53 (m, 3H),
7.33 (d, J = 8.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) d 165.5,
150.7, 134.9, 131.6, 129.7, 128.4, 127.7, 125.8, 115.9, 111.1,
103.7; HRMS (ESI) calcd for C14H12N4O (MH+) 252.1011, found
252.1017.
32.9, 32.4, 30.1, 23.8, 14.5; HRMS (ESI) calcd for C20H24N4O
(MH+) 336.1950, found 336.1945.
4.1.20. N-(2-Amino-1H-benzo[d]imidazol-5-yl)-4-heptylbenza-
mideÁHCl (63)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 63 which was formed as a white solid
in 82% yield. White solid: mp 279–283 °C; 1H NMR (400 MHz,
CD3OD) d 7.96 (d, J = 2 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H), 7.46 (dd,
J = 8.8 Hz, J = 2.0 Hz, 1H), 7.29 (dd, J = 8.4 Hz, J = 5.2 Hz, 3H), 2.65
(t, J = 8.0 Hz, 2H), 1.62 (quintet, J = 7.2 Hz, 2H), 1.29 (br m, 8H),
0.87 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 169.0,
152.6, 148.8, 136.6, 133.4, 131.2, 129.8, 128.9, 127.6, 118.5,
112.4, 105.9, 36.9, 33.1, 32.5, 30.4 (2), 23.8, 14.6; HRMS (ESI) calcd
for C21H26N4O (MH+) 350.2107, found 350.2101.
4.1.15. N-(2-Amino-1H-benzo[d]imidazol-5-yl)-4-pentylbenza-
mideÁHCl (33)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 33 which was formed as a white solid
in 80% yield. White solid: mp 263–267 °C; IR (KBr) 3303, 3142,
2952, 1686, 1639, 1562, 1356 cmÀ1 1H NMR (400 MHz, CD3OD)
;
d 8.01 (s, 1H), 7.88 (d, J = 2.8 Hz, 1H), 7.85 (d, J = 2.8 Hz, 1H), 7.45
(dd, J = 8.4 Hz, J = 1.6 Hz, 1H), 7.34 (m, 2H), 7.32 (m, 1H), 2.69 (t,
J = 6.8 Hz, 2H), 1.66 (quintet, J = 7.2 Hz, 2H), 1.35 (m, 4H), 0.91 (t,
J = 6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 169.0, 152.6, 148.8,
136.6, 133.5, 131.2, 129.8, 128.9, 127.6, 118.5, 112.4, 105.9, 36.9,
32.7, 32.2, 23.7, 14.5; HRMS (ESI) calcd for C19H22N4O (MH+)
322.1794, found 322.1788.
4.1.21. N-(2-Amino-1H-benzo[d]imidazol-5-yl)-4-tert-butylben-
zamideÁHCl (64)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 64 which was formed as a white solid in
88% yield. White solid: mp 337–341 °C; 1H NMR (400 MHz, CD3OD)
d 7.99 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 6.8 Hz, 2H), 7.47 (dd,
J = 8.4 Hz, J = 1.2 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 1.35 (s, 9H); 13C
NMR (100 MHz, CD3OD) d 167.0, 156.9, 136.6, 133.2, 131.2, 128.7,
127.7, 126.7, 126.5, 118.5, 112.5, 106.0, 36.0, 31.7; HRMS (ESI) calcd
for C18H20N4O (MH+) 308.1637, found 308.1633.
4.1.16. N-(2-Amino-1H-benzo[d]imidazol-5-yl)furan-2-carbox-
amideÁHCl (34)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 34 which was formed as a white solid
in 64% yield. White solid: mp 225–229 °C; 1H NMR (400 MHz,
DMSO-d6) d 10.36 (s, 1H), 8.43 (s, 2H), 7.97 (d, J = 2.0 Hz, 1H),
7.94 (m, 1H), 7.52 (dd, J = 8.8 Hz, J = 1.6 Hz, 1H), 7.40 (d,
J = 3.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 6.70 (m, 1H); 13C NMR
(100 MHz, DMSO-d6) d 156.3, 150.9, 147.4, 145.8, 134.2, 129.8,
126.0, 115.9, 114.9, 112.1, 111.2, 103.9; HRMS (ESI) calcd for
C12H10N4O2 (MH+) 242.0804, found 242.0810.
4.1.22. General procedure for the formation of 2ABI sulfon-
amides (36)
To a vial containing 11 (100 mg, 0.22 mmol) and triethylamine
(70 lL, 0.50 mmol) stirring in methylene chloride (5 mL) was
added sulfonyl chloride (0.23 mmol) either dropwise or as a solid
to the reaction mixture which was allowed to stir overnight
(ꢀ16 h) at ambient temperatures. The reaction was then quenched
with 5% HCl (aq) and transferred to a separatory funnel. The result-
ing sulfonamide was extracted two times with dichloromethane
and the combined organic layers were dried with sodium sulfate
and concentrated. This mixture was then flashed through a plug
of silica gel and concentrated in a vial. This gave an inseparable
mixture of regioisomer sulfonamide products. The resulting sul-
fonamide 2ABI was then dissolved in 2 mL of methylene chloride
and 2 mL of triflouroacetic acid was added slowly to give a 1:1
mixture (CH2Cl2/TFA). This was allowed to stir for 2 h at room tem-
perature after which the solvent was removed under reduced pres-
sure to give the TFA salt of the desired 2ABI sulfonamide. To this
was added 2 mL of saturated HCl/methanol and the TFA salt was
exchanged to give the HCl salt of the 2ABI sulfonamide. Then ether
was added to the vial to triturate the resulting product and the
ether was then decanted giving pure 2ABI sulfonamide (36) as an
HCl salt between 20% and 84% yield.
4.1.17. N-(2-Amino-1H-benzo[d]imidazol-5-yl)thiophene-2-car-
boxamideÁHCl (35)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 35 which was formed as a white solid
in 79% yield. White solid: mp 99–104 °C; 1H NMR (400 MHz,
DMSO-d6) d 10.42 (s, 1H), 8.45 (s, 2H), 8.09 (d, J = 3.6 Hz, 1H),
7.95 (s, 1H), 7.86 (d, J = 5.2 Hz, 1H), 7.51 (dd, J = 8.8 Hz, J = 2.0 Hz,
1H), 7.32 (d, J = 8.8 Hz, 1H), 7.23 (t, J = 4.4 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) d 160.0, 150.8, 140.1, 134.5, 131.8, 129.8,
129.5, 128.2, 125.9, 116.0, 111.2, 103.9; HRMS (ESI) calcd for
C12H10N4OS (MH+) 258.0575, found 258.0578.
4.1.18. N-(2-Amino-1H-benzo[d]imidazol-5-yl)-4-butylbenza-
mideÁHCl (61)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 61 which was formed as a white solid
in 83% yield. 1H NMR (400 MHz, CD3OD) d 7.95 (s, 1H), 7.85 (d,
J = 8.4 Hz, 2H), 7.46 (dd, J = 8.6 Hz, J = 1.8 Hz, 1H), 7.29 (m, 3H),
2.65 (t, J = 7.8 Hz, 2H), 1.59 (quintet, J = 7.6 Hz, 2H), 1.35 (sextet,
J = 7.6 Hz, 2H), 0.95 (t, J = 12.8 Hz, 3H); 13C NMR (100 MHz, CD3OD)
d 167.0, 148.7, 136.5, 133.4, 131.1, 130.9, 129.8, 128.9, 127.6,
118.5, 112.4, 106.0, 37.0, 34.7, 23.4, 14.4; HRMS (ESI) calcd for
C18H20N4O (MH+) 308.1637, found 308.1636.
4.1.23. N-(2-Amino-1H-benzo[d]imidazol-5-yl)hexane-1-sulfon-
amideÁHCl (38)
The general procedure for the formation for 2ABI sulfonamides
was used for the synthesis of 38 which was formed as a light yel-
low solid in 68% yield. Light yellow solid: mp 214–219 °C; 1H NMR
(400 MHz, CD3OD) d 7.44–7.34 (m, 3H), 3.62 (t, J = 7.2 Hz, 2H), 1.85
(m, 2H), 1.47 (m, 2H), 1.35 (m, 4H), 0.90 (m, 3H); 13C NMR
(100 MHz, CD3OD) d 153.4, 132.3, 131.4, 131.0, 128.5, 116.0,
112.8, 56.5, 32.4, 28.9, 24.4, 23.5, 14.4; HRMS (ESI) calcd for
C13H20N4O2S (MH+) 296.1307, found 296.1316.
4.1.19. N-(2-Amino-1H-benzo[d]imidazol-5-yl)-4-hexylbenza-
mideÁHCl (62)
The general procedure for the formation for 2ABI amides was
used for the synthesis of 62 which was formed as a white solid
in 91% yield. 1H NMR (400 MHz, CD3OD) d 7.95 (s, 1H), 7.85 (d,
J = 8.4 Hz, 2H), 7.46 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 7.28 (m, 3H),
2.63 (t, J = 7.6 Hz, 2H), 1.60 (br m, 2H), 1.29 (m, 6H), 0.87 (t,
J = 3.4 Hz, 3H); 13C NMR (100 MHz, CD3OD) d 168.9, 148.7, 136.5,
133.4, 131.1, 130.9, 129.7, 128.9, 127.5, 118.5, 112.4, 105.9, 36.9,
4.1.24. N-(2-Amino-1H-benzo[d]imidazol-5-yl)-4-methylbenze-
nesulfonamideÁHCl (39)
The general procedure for the formation for 2ABI sulfonamides
was used for the synthesis of 39 which was formed as a red residue

R. W. Huigens et al. / Bioorg. Med. Chem. 18 (2010) 663–674
673
in 84% yield. 1H NMR (400 MHz, CD3OD) d 7.61 (s, 1H), 7.59 (m,
1H), 7.27 (s, 1H), 7.25 (s, 1H), 7.20 (d, J = 1.2 Hz, 1H), 7.18 (d,
J = 8.4 Hz, 1H), 6.90 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H), 2.35 (s, 3H);
13C NMR (100 MHz, CD3OD) d 171.7, 143.9, 136.6, 134.1, 130.2,
129.4, 128.5, 127.1, 118.1, 111.4, 105.4, 20.2; HRMS (ESI) calcd
for C14H14N4O2S (MH+) 302.0838, found 302.0829.
rodilution susceptibility testing. Serial dilutions were made with
the samples that were taken from the wells and they were then
plated out on TSA plates and incubated overnight to grow BAA-
44 colonies. From these BAA-44 colonies, CFU mLÀ1 were deter-
mined. The MBC is the lowest concentration that gave P99.9%
reduction in CFU mLÀ1. The dose–response was plotted with con-
centrations of 33 that gave less than the P99.9% reduction in
CFU mLÀ1. This was then used to give MBC50 and MBC90 values
4.1.25. N-(2-Amino-1H-benzo[d]imidazole-5-yl)naphthalene-2-
sulfonamideÁHCl (45)
for 33. The MBC value for 33 was found to be 25
was found to be 5.88 0.50 M and the MBC90 was found to be
11.15 0.12 M.
lM. The MBC50
The general procedure for the formation for 2ABI sulfonamides
was used for the synthesis of 45 which was formed as a white solid
in 72% yield. White solid: mp 265–269 °C; 1H NMR (400 MHz,
CD3OD) d 8.36 (s, 1H), 8.08 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 8.03 (d,
J = 8.4 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.87 (m, 1H), 7.76 (m, 1H),
7.66 (m, 1H), 7.29 (dd, J = 8.4 Hz, J = 2.4 Hz, 1H), 6.99 (m, 1H),
6.93 (m, 1H); 13C NMR (100 MHz, CD3OD) d 153.4, 137.3, 137.1,
133.4, 132.6, 131.9, 131.4, 131.2, 131.1, 130.8, 130.8, 129.3,
129.3, 128.9, 124.2, 116.2, 112.8; HRMS (ESI) calcd for C17H14N4O2S
(MH+) 338.0838, found 338.0834.
l
l
4.2.5. Vancomycin enhancement assay with 33
Serial dilutions were made with Vancomycin according to the
methods previously described to determine MBC values. Inocu-
lated media was prepared and added to the appropriate row of
wells in the microtiter plates with 2, 4, and 6
lM 33 before vanco-
mycin was added to a well at 400 M. Serial dilutions were then
l
made and incubated for 16–20 h. The plates were then removed
after incubation and inspected for bacterial growth. Vancomycin
4.2. Biology
was enhanced with each treatment of 33 (2, 4, and 6 lM) twofold.
Vancomycin’s MBC without 33 is 391 nM against BAA-44, but after
4.2.1. Bacterial strains
adding 33 to the vancomycin treated wells the MBC is 195 nM.
The S. aureus strains were purchased from ATCC (ATCC 29213,
ATCC 25923, and ATCC 29740), including MRSA strain BAA-44.
Two A. baumannii strains were also purchased from ATCC (ATCC
19606 and BAA-1605). Three A. baumannii clinical isolates
(AB0043, UH8407, and 3340) were obtained from Dr. Robert
Bonomo.
4.3. Red blood hemolysis
Hemolysis assays were performed on mechanically difibrinated
sheep blood (Hemostat Labs: DSB100). Difibrinated blood (1.5 mL)
was placed into a microcentrifuge tube and centrifuged for 10 min
at 10,000 rpm. The supernatant was then removed and then the
cells were resuspended with 1 mL of phosphate-buffered saline
(PBS). The suspension was centrifuged, the supernatant was re-
moved and cells were resuspended two additional times. The final
cell suspension was then diluted 10-fold. Test compound solutions
were made in PBS in small culture tubes and then added to aliquots
of the 10-fold suspension dilution of blood. PBS was used as a neg-
ative control and a zero hemolysis marker. Triton X (a 1% sample)
was used as a positive control serving as the 100% lysis marker.
Samples were then placed in an incubator at 37 °C while being sha-
ken at 200 rpm for one hour. After one hour, the samples were
transferred to microcentrifuge tubes and centrifuged for 10 min
at 10,000 rpm. The resulting supernatant was diluted by a factor
of 40 in distilled water. The absorbance of the supernatant was
then measured with a UV spectrometer at a 540 nm wavelength.
4.2.2. Reagents
All compounds that were tested were stored as 100 mM stock
solutions in dimethyl sulfoxide (DMSO) and frozen at À80 °C until
used for screening.
4.2.3. Microdilution susceptibility testing
All antibiotic susceptibility testing was completed with a start-
ing inoculum of 5 Â 105 CFU mLÀ1 according to NCCLS41 standards
and incubated for 16–20 h at 37 °C. After this time bacterial growth
was visually inspected and the lowest concentration at which no
observable bacterial growth or turbidity was observed was consid-
ered to be the MIC value. Pellets that formed at the bottom of 96
well microtiter plates were considered growth and although there
was essentially no turbidity, the pellet was considered bacterial
growth in these studies. Microdilutions were made in the 96 well
microtiter plates from a starting concentration of 400
the addition of 200 L to a predetermined well. From this, twofold
serial dilutions were made by transferring 100 L of the initially
treated well (of 400 M) into the next well and mixing once using
a multichannel pipet, this next well then contained 200 L of bac-
terial, media and test 2ABI at a concentration of 200 M. This
transfer of 100 L was done in succession for a total of 12 twofold
serial dilutions giving a range of 400 M–391 nM for tested antibi-
otic in 96 well microtiter plates. The final dilution then had 200
(of the 391 nM wells) and then 100 L was removed and thrown
l
M with
4.4. Dye leakage from synthetic vesicles
l
l
The leakage of vesicle contents was monitored by the release of
calcein encapsulated in large unilamellar vesicles. Vesicles were
prepared by reverse-phase evaporation. After evaporating the or-
ganic solvent, residue was hydrated with the calcein solution
(100 mM) in buffer (10 mM Tris–HCl pH 7.4). The free calcein
was removed by elution through a Sephadex G-50 size-exclusion
column in the same buffer. The leakage process was monitored
by following the increase of calcein fluorescence intensity at
l
l
l
l
l
lL
l
away. A plastic lid was then used to cover the wells and the plates
with their lids were wrapped in suran wrap and placed in a humid-
ified chamber and incubated for 16–20 h at 37 °C. In one plate, 8
antibiotics could be run in parallel with 12 twofold dilutions. After
the incubation time, the wells were observed for bacterial growth
and MICs were determined accordingly (described above).
515 nm (excitation at 490 nm) after 20
ent concentrations were added to 10 L of vesicle solution mixed
in 2 mL of TBS buffer. Complete leakage was achieved by addition
of 100 L of 20% Triton X-100 to the 2 mL solution, and the corre-
lL of compound at differ-
l
l
sponding fluorescence intensity was used as 100% leakage for the
calculation of leakage fraction.
4.2.4. Determining MBC values for 33
Acknowledgements
The MBC was determined by taking 50 lL from individual
microtiter wells from untreated BAA-44 and treated BAA-44 wells
with 33 at several concentrations after a 16 h incubation from mic-
The authors would like to thank the UNC General Admission
Competitiveness Grant and the V Foundation for funding (R.W.H.

674
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