Benzimidazole-based antibacterial agents against Francisella tularensis

Article abstract of DOI:10.1016/j.bmc.2013.02.059

Francisella tularensis is a highly virulent pathogenic bacterium. In order to identify novel potential antibacterial agents against F. tularensis, libraries of trisubstituted benzimidazoles were screened against F. tularensis LVS strain. In a preliminary

Full text of DOI:10.1016/j.bmc.2013.02.059

Accepted Manuscript
Benzimidazole-Based Antibacterial Agents Against F. tularensis
Kunal Kumar, Divya Awasthi, Seung-Yub Lee, Jason E. Cummings, Susan E.
Knudson, Richard A. Slayden, Iwao Ojima
PII:
S0968-0896(13)00260-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.02.059
BMC 10695
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 December 2012
11 February 2013
19 February 2013
Please cite this article as: Kumar, K., Awasthi, D., Lee, S-Y., Cummings, J.E., Knudson, S.E., Slayden, R.A., Ojima,
I., Benzimidazole-Based Antibacterial Agents Against F. tularensis, Bioorganic & Medicinal Chemistry (2013),
doi: http://dx.doi.org/10.1016/j.bmc.2013.02.059
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Benzimidazole-Based Antibacterial Agents
Against F. tularensis
Leave this area blank for abstract info.
Kunal Kumar, Divya Awasthi, Seung-Yub Lee,
Jason E. Cummings, Susan E. Knudson,
Richard A. Slayden, Iwao Ojima
Preliminary Screening:
23 compounds with 90 %
growth inhibition at 1
µg/mL
O
R1
N
R3
NH
H
N
H
N
R1'
R2
R2
N
N
HN
R3
2,5,6- and 2,5,7-Trisubstituted Benzimidazoles
HN
F. tularensis
O
EtO
O
21 compounds
MIC90 ~ 0.35-48.6 µg/mL

Bioorganic & Medicinal Chemistry
journal hom epage: www.elsevier.com
Benzimidazole-Based Antibacterial Agents Against F. tularensis
Kunal Kumara , Divya Awasthia, Seung-Yub Leea, Jason E. Cummingsb, Susan E. Knudsonb, Richard A.
Slaydenb, Iwao Ojimaa, ∗
aDepartment of Chemistry and Institute of Chemical Biology & Drug Discovery, Stony Brook University, Stony Brook, NY-11794-3400, United States
bDepartment of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado 80523-0922, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Francisella tularensis is a highly virulent pathogenic bacterium. In order to identify novel
potential antibacterial agents against F. tularensis, libraries of trisubstituted benzimidazoles
were screened against F. tularensis LVS strain. In a preliminary screening assay, remarkably, 23
of 2,5,6- and 2,5,7-trisubstituted benzimidazoles showed excellent activity exhibiting greater
than 90 % growth inhibition at 1 µg/mL. Among those hits, 21 compounds showed MIC90 values
in the range of 0.35-48.6 µg/mL after accurate MIC determination. In ex-vivo efficacy assays,
four of these compounds exhibited 2-3 Log reduction in colony forming units (CFU) per mL at
concentrations of 10 and 50 µg/mL.
Revised
Accepted
Available online
Keywords:
Antibacterial
2009 Elsevier Ltd. All rights reserved.
Benzimidazole
Francisella tularensis
———
∗ Corresponding author. Tel.: 631-632-1339; fax: 631-632-7942; e-mail: iwao.ojima@stonybrook.edu

strains. Also, the target of these benzimidazoles was validated by
Mtb FtsZ polymerization inhibition by light scattering and TEM
as well as GTPase assay.24 For the Mtb project, we created a
library of ca. 1,100 novel trisubstituted benzimdazoles.
Accordingly, it was very logical for us to screen this compound
library against F. tularensis since it had been shown that FtsZ
was highly conserved and ubiquitous cell division protein among
a wide range of bacteria,25-28 and the validity of FtsZ as a novel
antibacterial drug target has been confirmed by the work of
various groups including ours.19-22, 24, 29-37
1. Introduction
Bacterial infections due to pathogens such as methicillin-
resistant Staphylococcus aureus (MRSA),1 vancomycin-resistant
enterococci (VRE),2 F. tularensis,3 Y. pestis4 and others have re-
emerged as major health concerns throughout the world. F.
tularensis is one such pathogen which has attracted widespread
attention as a potential bioterrorism weapon.5 It is a highly
contagious gram-negative bacteria which is the causative agent of
tularemia or “Rabbit fever”, a zoonotic disease.6 Symptoms of
tularemia generally depend on the mode of infection.
Predominantly the symptoms are fever, ulcers, dyspnea, and
others.5, 7 Normally the bacteria reside in rabbit or rodents but it
can infect humans through vectors such as mosquitos and fleas.5
Bacteria primarily infect macrophages of the host organism
where it multiplies and later invades the lungs, spleen, liver, and
kidneys.5, 8 Among the four known subspecies of F. tularensis,
Type A is the most virulent and is associated with most lethal
pulmonary infections.9 It is highly virulent as it can cause a fatal
infection on exposure to doses as low as 10 colony forming
units.10,11 Due to its high infectivity that enables airborne
transmission, difficult diagnosis and a very low infection dose, F.
tularensis has been classified as category A list of select agents
Thus, we have screened the in-house libraries of 2,5,6- and
2,5,7-trusubstituted benzimidazoles in the hope of discovering
novel antibacterial agents against F. tularensis, and in fact found
a dozen promising hit compounds. We describe here the
screening, identification of hit compounds, resynthesis, MIC90
determination, ex-vivo efficacy evaluation of selected hit
compounds against RAW 264.7 macrophages infected by F.
tularensis LVS.
2. Chemistry
Screening of in- house libraries against F. tularensis LVS
presented 23 hits at MIC <1 µg/mL. A general procedure for the
synthesis of these libraries of 2,5,6- and 2,5,7-trisubstituted
benzimidazoles have been reported in the previous work.219
Chemically pure 2,5,6-trisubstituted benzimidazole hit
compounds were synthesized following the synthetic protocol as
outlined in scheme 1. The aromatic nucleophilic substitution of
commercially available 2,4-dinitro-5-fluoroaniline (1) with
various amines afforded 5-amino-2,4-dinitroanilines 2a-2e in 94-
98 % yield as yellow solids. The acylation of intermediates 2a-2e
with different acyl chlorides afforded the corresponding N-
acylanilines 3a-3i in 75-95% yield. One-pot reduction and
subsequent cyclization in the presence of stannous chloride
dihydrate and 4 M hydrochloric acid gave 5-aminobenzimida-
zoles 4a-4i in 65-79% yield. The derivatization of the 5-
aminobenzimidazoles 4a-4i using various acyl chloride, acids,
sulfonylchlorides, chloroformates or corresponding N-
hydroxysuccinimide ester generated the desired analytically pure
hit compounds 5a-5k in 42-62% yields.
8
by NIAID.5, Airborne infection by F. tularensis could be
potentially used as a biological weapon.3, 5, 8 Once people inhale
the bacteria, they can develop life threatening respiratory illness,
including fatal systemic infection.12 It has been also estimated
that if the infection is left untreated, the mortality rate can be as
high as 30 to 60% of the cases.11, 13 According to studies done in
1970, the WHO predicted that the release of 50 kg of F.
tularensis in air over a city with a population of 5 million would
result in 250,000 cases of disease.14
Treatment against this pathogen still relies on a rather
limited number of aging antibacterial drugs which target cell wall
biosynthesis, nucleic acid synthesis, protein synthesis, etc.15 The
infection is largely treated by antibiotics like aminoglycosides,
streptomycin, tetracycline, chloramphenicol, and quinolones.12, 16
However, these antibiotics have either shown high toxicity or a
high relapse rate.5, 17 As an alternative treatment option, use of
vaccines has acquired considerable interest and is in a very early
phase of development. Live vaccine strain (LVS) was successful
enough in decreasing the incidence rates of laboratory-acquired
tularemia.18 However, due to the unknown role of the vaccine,
FDA rejected applications to license the LVS.5 Additionally, the
widespread bacterial resistance to existing antibiotics due to poor
drug compliance has been another crucial obstacle in finding
suitable and more efficient treatments.17
Chemically pure 2,5,7-trisubstituted benzimidazole hit
compounds were synthesized as outlined in scheme 2.
Commercially available 5-amino-2,4-dinitrobenzamide (6) was
hydrolyzed to give 4-amino-3,5-dinitrobenzoic acid, which was
converted to acyl chloride 7. The reaction of 7 with sodium azide
afforded the corresponding acyl azide, which was subjected to
the Curtius rearrangement to give the corresponding isocyanate.
The isocyanate was treated with corresponding alcohol to form
intermediate 8a-8b as bright yellow solid in 85 % yield. The
reduction of 8a-8b followed by cyclocondensation with the
bisulfite salts of different aldehydes afforded 7-
aminobenzimidazoles 9a-9e. The derivatization of the 7-
aminobenzimidazole using various acyl chloride, chloroformates
or the corresponding N-hydroxysuccinimide esters generated the
desired hit compounds 10a-10i in analytically pure form in 41-
53% yields.
About 10 years ago, we launched our research program on
the discovery and development of new antibacterial agents for M.
tuberculosis (Mtb), which would overcome bacterial drug
resistance by exploiting a drug target that had not been
extensively studied. Then, we chose FtsZ, an essential protein for
bacterial cell division, as the target since the disruption of cell
division should lead to inhibition and arrest of bacterial growth.
Since FtsZ is a homolog of tubulin/microtubules in eukaryotes,
known tubulin inhibitors were screened against Mtb, and several
potent pyridopyrazine- and pteridine-based FtsZ inhibitors with
anti-TB activity were identified.19-21 We found that novel C-seco-
taxoids exhibited significant antibacterial activity against Mtb by
blocking the FtsZ depolymerization.22 Also, thiabendazole and
albendazole, known tubulin inhibitors, were found to interfer and
delay the Mtb cell division processes.23 Based on the structural
similarity of the pyridopyrazine moiety, pteridine moeity,
albendazole and thiabendazole,19-21 we selected the
benzimidazole scaffold for the development of novel FtsZ
inhibitors. Then, we investigated novel trisubstituted-
benzimidazoles for antibacterial activity against Mtb, and found a
series of highly active compounds against drug resistant Mtb

O
R3
NH
R1
N
R1
N
F
NH2
NO2
NH2
NO2
c
b
R2
a
R2
75-90%
95-100%
40-60%
O2N
O2N
O2N
: R R N = Et2N,
NO2
3
1
2
2a: R1R2N = piperidin-1-yl
1
: R R N = Et2N
R3 = 4-FC6H4
R3 = 2-MeOC6H4
R3 = 4-MeC6H4
R3 = Ph
1
2
3a
3b
2
1 2
2b
: R R N = Et2N,
3c: R1R2N = Et2N,
3d: R1R2N = Et2N,
1
2
2c
: R R N = Me2NCH2CH2(Et)N
2d: R1R2N = Me2NCH2CH2(Me)N
2e: R1R2N = pyrrolidin-1-yl
1
: R R N = piperidin-1-yl,
2
R3 = 2-thienyl
R3 = 2-furyl
R3 = c-C6H11
R3 = c-C6H11
3e
3f
1
2
: R R N = piperidin-1-yl,
1
2
3g
: R R N = pyrrolidin-1-yl,
3h: R1R2N = Me2NCH2CH2(Et)N,
3i: R1R2N = Me2NCH2CH2(Me)N, R3 = c-C6H11
R1
R1
N
H
N
H
N
N
d
R2
R2
R3
R3
N
N
H2N
HN
4
R4
O
5
5a: R1R2N = Et2N,
R3 = 4-FC6H4,
R4 = c-Bu
4a: R1R2N = Et2N,
4b: R1R2N = Et2N,
R3 = 4-FC6H4
R3 = 2-MeOC6H4
R3 = 4-MeC6H4
R3 = Ph
R3 = 2-thienyl
R3 = 2-furyl
1
2
: R R N = Et2N,
1
R3 = 2-MeOC6H4, R4 = c-Bu
R3 = 4-MeC6H4, R4 = c-Bu
R3 = 2-MeOC6H4, R4 = 2-MeOC6H4
5b
5c
2
1
2
: R R N = Et2N,
4c
4d
: R R N = Et2N,
1
2
1
2
5d
: R R N = Et2N,
: R R N = Et2N,
5e: R1R2N = piperidin-1-yl,
5f: R1R2N = piperidin-1-yl,
2
: R R N = piperidin-1-yl,
R3 = 2-thienyl,
R3 = 2-thienyl,
R3 = 2-furyl,
R3 = 2-furyl,
R4 = (C6H4)CH2O
R4 = 2-MeOC6H4
R4 = 2-MeOC6H4
R4 = 1-naphthyl
R4CO = PhSO2
R4CO = PhSO2
R4 =c-Pr
4e: R1R2N = piperidin-1-yl,
4f: R1R2N = piperidin-1-yl,
4g: R1R2N = pyrrolidin-1-yl,
1
R3 = c-C6H11
R3 = c-C6H11
5g
5h
5i
1
2
1
2
: R R N = piperidin-1-yl,
4h
4i
: R R N = Me2NCH2CH2(Et)N,
1
1
2
: R R N = Me2NCH2CH2(Et)N,
5j: R1R2N = Me2NCH2CH2(Et)N,
5k: R1R2N = pyrrolidin-1-yl,
R3 = c-C6H11
,
: R R N = Me2NCH2CH2(Me)N, R3 = c-C6H11
2
R3 = c-C6H11
R3 = c-C6H11
R3 = Ph,
,
,
1
2
R4 = 4-MeC6H4
5l
: R R N = Et2N,
Scheme 1. Synthesis of 2,5,6-trisubstituted benzimidazoles. Reagents and conditions: (a) R1R2NH, DIPEA, THF, 2 h, room temperature (RT);
(b) R3COCl, pyridine, reflux, overnight; (c) SnCl2·2H2O, 4 N HCl, EtOH, reflux,4 h; (d) R4COCl/R4COOCl/R4COOH/R4SO2Cl, CH2Cl2,
overnight, RT.
NO2
NO2
NO2
NH2
NO2
NH2
NO2
NH2
NO2
f,g
c,d,e
a,b
O
H2N
Cl
55%
(5 steps)
R2O
N
H
60%
O
6
O
7
8
: R2 = Et
: R2 = t-butyl
8a
8b
O
R4
NH
NH2
H
N
H
N
: R2 = Et,
R3 = 4-BrC6H4,
R3 = 4-BrC6H4,
R3 = 4-BrC6H4,
R4 = 2-MeOC6H4
R4 = c-Pr
R4 = C6H5
O
O
h
10a
R3
R3
10b: R2 = Et,
10c: R2 = Et,
10d
R2O
N
H
R2O
N
N
N
H
10
9
: R2 = t-butyl, R3 = Ph,
: R2 = Et,
R4 = C6H4CH2O
: R2 = Et,
R3 = 4-BrC6H4
R3 = 3,5-Me2C6H4
R3 = 2-furyl
R3 = 4-MeOCOC6H4, R4 = c-C6H11
R3 = 4-MeOCOC6H4, R4 = 4-MeOC6H4
R3 = 4-MeOCOC6H4, R4 = Ph
R3 = 2-furyl,
R3 = 2-furyl,
9a
10e
10f
: R2 = Et,
: R2 = Et,
10g: R2 = Et,
10h: R2 = Et,
9b
2
9c
: R =Et,
9d: R2=Et,
9e: R2=t-butyl, R3 = Ph
R3 = 4-MeOCOC6H4
R4 = 4-FC6H4
R4 = Ph
: R2 = Et,
10i
Scheme 2. Synthesis of 2,5,7-trisubstituted benzimidazoles. Reagents and conditions: (a) 6 M HCl, reflux, 36 h; (b) SOCl2, reflux, 12 h; (c)
NaN3, acetone, 0 oC, 30 min; (d) toluene, reflux, 4 h; (e) R2OH, RT, 24 h; (f) 10% wt Pd/C, HCO2NH4, EtOH, 1 h, RT; (g) R3CH(OH)SO3Na,
EtOH:H2O (1:1), 14 h; (h) R4COCl/R4OCOCl/ R4COOH , DMF, 12 h, RT.

In order to confirm their activity, these compounds were
synthesized in analytically pure form following the synthesis
protocol outlined in scheme 1 and scheme 2. These compounds
were then re-tested for their activity against F. tularensis LVS to
determine MIC values. Among the hits, 21 compounds showed
MIC90 values in the range of 0.35-48.6 µg/mL with no
appreciable cytotoxicity (IC50 >200 µg/mL) against Vero cells,
except 10h (IC50 <50 µg/mL) (Table 1). It should be noted that
two compounds 10f and 10g did not show any activity, even
though they were found to be active in preliminary screening. As
Table 1 shows, the most active compounds (MIC90 <1.0 µg/mL)
are 5e. 10b, 10c and 10d (most potent: MIC90 0.34 µg/mL).
3. Results and discussions:
3.1 In-vitro preliminary screening of libraries of 2,5,6- and
2,5,7-trisubstituted benzimidazoles against F. tularensis
The libraries of 2,5,6- and 2,5,7-trisubstituted
benzimidazoles (~1100 compounds) were screened for their
activity against F. tularensis LVS strain using the “Microplate
Alamar Blue assay (MABA)” in a 96-well format (single point
assay in triplicates). Remarkably, 23 compounds showed
significant activity exhibiting greater than 90 % growth inhibition
at 1 µg/mL (Fig. 1). Of these hit compounds, 7 compounds
exhibited 40-50 % growth inhibition at 0.2 µg/mL (Fig. 1). It is
interesting to note that none of the hit compounds against Mtb,
showed appreciable activity against F. tularensis. The absence of
apparent cross activity between Mtb and F. tularensis is
discussed later.
(A)
R1
MeO
H
N
H
N
H
N
H
N
H
N
N
N
N
N
N
R2
X
R3
N
N
N
N
N
HN
MeO
HN
HN
HN
HN
O
S
O
O
R4
O
O
O
5a: R3 = 4-FC6H4
5d
5e
5f
: R4 = C6H4CH2O, X = S
5k
5i: R1R2N = Me2NCH2CH2(Et)N
5j: R1R2N = Me2NCH2CH2(Me)N
: R3 = 2-MeOC6H4
: R3 = 4-MeC6H4
: R4 = 2-MeOC6H4, X = S
5g: R4 = 2-MeOC6H4, X = O
5h: R4 = 1-naphthyl, X = O
5b
5c
R4
NH2
(B)
(C)
R4
N
O
O
NH
R3
10e: R4
=
c-C6H11
NH
O
N
H
N
10f
EtO
N
H
: R4 = 4-MeOC6H4
: R4 = Ph
O
CO2Me
10g
N
N
O
N
H
: R3 = 4-BrMeOC6H4
9b: R3 = 3,5-Me2C6H3
EtO
N
H
Br
9a
R4
EtO
N
H
H
H
N
N
O
O
NH
R3
N
N
O
: R4 = 2-MeOC6H4
: R4 = c-pentyl
: R4 = Ph
O
NH
HN
10a
10b
10c
O
H
N
N
H
O
R4
: R3 = 4-MeC6H4, R4 = c-Bu
: R3 = Ph, R4 = 4-MeC6H4
O
EtO
N
H
5c
5l
N
: R4 = 4-FC6H4
O
N
H
10h
10i: R4 = Ph
10d
Figure 1. Hit trisubstituted benzimidazoles against F. tularensis.
(A) Structures of 2,5,6-trisubstituted benzimidazoles against F. tularensis LVS at 1 µg/mL with >90 % growth inhibition. (B) Structures of
2,5,7-trisubstituted benzimidazoles against F. tularensis LVS at 1 µg/mL with >90 % growth inhibition. (C) Structures of hit benzimidazoles
against F. tularensis LVS with 40-50 % growth inhibition at 0.2 µg/mL
H
N
N
S
N
thiabendazole
H
N
N
HN
S
N
O
O
5e
Figure 2. Effect of benzimidazoles on F. tularensis LVS infection in RAW 264.7 macrophages ex vivo.

RAW macrophages infected with F. tularensis LVS were treated with various benzimidazoles at different concentrations. After the treatment
the cells were lysed and lysate transferred to CHAB plates for CFU enumeration.
Table 1
MIC90 of hit compounds against F. tularensis
O
R4
R2
N
R2
N
NH
NH2
H
N
H
N
H
N
H
N
R1
R1
R3
R3
R3
R3
N
N
N
N
HN
R4
HN
R4
HN
HN
R2O
5i-5j
10a-10j
5a-5h
5k-5l
9a-9b
R2O
O
O
O
µ
( g/mL) Cytotoxicity
Compound R1R2N
MIC90
F. tularensis
MIC90 (µg/mL) Cytotoxicity
F. tular ensis
R3
R4
R4
R2
R3
Compound
µ
Vero cells
(
g/mL)
(µg/mL)
Vero cells
LVS
LVS
∗
∗
∗
∗
5a
4.84
> 200
10a
1.4
F
Br
>200
∗
∗
∗
N
∗
OMe
5b
5c
7.59
1.16
> 200
> 200
>200
>200
∗
∗
10b
10c
∗
∗
0.75
0.48
Br
Br
∗
∗
N
N
∗
∗
MeO
∗
∗
OMe
∗
∗
∗
5d
5e
4.85
0.98
> 200
>200
9a
9b
2.99
Br
>200
∗
∗
N
N
∗
∗
MeO
S
∗
∗
∗
∗
>200
>200
O
4.33
0.35
OMe
S
5f
1.6
>200
10d
∗
∗
∗
∗
O
N
∗
∗
OMe
O
5g
5h
5i
2.1
2.4
>200
>200
>200
∗
∗
>200
>200
10e
10f
∗
∗
1.5
∗
∗
CO2Me
CO2Me
∗
∗
N
N
∗
∗
O
>256
∗
∗
O
∗
10g
35.8
N
S
∗
∗
∗
∗
>256
2.8
CO2Me
_>200
O
∗
∗
∗
∗
N
N
O
∗
∗
O
O
< 50
_
N
F
∗
5j
48.6
> 200
10h
10i
S
O
O
∗
∗
5k
5l
2.1
>200
>200
∗
3.8
∗
>200
N
N
∗
∗
∗
12.9
∗
3.2 Activity of lead benzimidazoles against F. tularensis LVS in the
ex vivo model of efficacy.
well as maintain that activity ex vivo, and low to no associated
toxicity to mammalian cells make these lead compounds
attractive for further evaluation in mouse models for their
efficacy in vivo.
To assess the efficacy potential of active compounds identified
with potency against F. tularensis LVS, selected lead compounds
were further evaluated for activity in the ex vivo efficacy model.
The lead compounds 5e, 5f, 5g and 5 h were subjected to ex vivo
efficacy assay against F. tularensis LVS infection of RAW
macrophages. With the exception of 5f, the lead compounds were
highly effective in reducing bacterial numbers in a dose-
dependent manner over the concentration range of 10 and 50
µg/mL (Fig. 2). There was a 2-3 Log reduction in the colony
forming units (CFU) per mL which is considered to be
significant. Among the four lead compounds examined, 5e was
found to be the most potent, which is consistent with its smallest
MIC90 value as compared to other three. Doxycycline and
thiabendazole, which are known to be effective against F.
tularensis, were used as reference compounds for this study. The
assay indicates that these compounds are very effective in killing
intercellular bacteria in macrophages. Given the high activity of
the trisubstituted benzimidazole compound class against the
bacteria cells, ability to permeate mammalian cell membranes as
As mentioned above, none of the hit compounds for Mtb
exhibited appreciable activity against F. tularensis. This apparent
lack of cross activity implies a couple of possibilities: (i) the
target of the hit compounds for F. tularensis is not FtsZ of this
bacteria although two groups of trisubstituted benzimidazoles are
structurally very similar to each other; (ii) the hit compounds for
F. tularensis target FtsZ, but not at the highly conserved GTP
binding catalytic domain, i.e., bind an allosteric site of FtsZ. In
fact, the exact binding site of the lead benzimidazoles active
against Mtb in FtsZ has not been identified yet, although it has
been verified that Mtb FtsZ is the target of these lead compounds.
Accordingly, we expressed and purified F. tularensis FtsZ for
target verification. However, unfortunately, the F. tularensis
FtsZ, thus obtained, has not shown appreciable polymerization in
the presence of GTP, to date, under the conditions similar to
those for Mtb FtsZ. Thus, at present, we do not have evidence to
claim that these hit/lead benzimidazoles target F. tularensis FtsZ.

Nevertheless, this work has clearly demonstrated that at least
several novel trisubstituted benzimidazoles exhibit highly
promising activity against F. tularensis cells as well as
macrophages infected by F. tularensis in ex-vivo evaluation,
which warrants further active investigation.
2 H, J = 1.2 Hz, 2.4 Hz), 7.30 (d, 1 H, J = 3.6 Hz), 7.63 (m, 1 H),
8.68 (s, 1 H), 8.85 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 23.60,
25.34, 52.01, 108.4, 112.9, 116.9, 125.8, 127.4, 132.7, 138.7,
145.7, 146.9, 150.5, 156.7; MS (ESI) m/z 361.3 (M+1)+.
5-Amino-6-piperidinyl-2-thiophenyl-1H-benzo[d]imidazole
(4e)
4. Conclusion
To a solution of 3e (800 mg, 2.12 mmol) in dioxane (50 mL)
and ethanol (50 mL) was added ammonium formate (3.5 g) and
10 wt. % Pd-C (340 mg) with stirring. The reaction mixture
turned from yellow to red and then to colorless in 60 min at room
temperature. The catalyst and excess ammonium formate were
filtered off to give the product solution. Concentrated
hydrochloric acid was added to this solution and adjusted to 4 M
HCl concentration. Then, the mixture was refluxed for 6 h. The
reaction mixture was basified by addition of ammonium
hydroxide, extracted with ethyl acetate (40 mL x 3) and dried
over anhydrous magnesium sulfate. The crude product was
purified by flash chromatography on silica gel using ethyl
acetate/hexanes (gradient: AcOEt/hexanes = 20/80–40/60) as
eluent to give pure 4e as a pale yellow solid; 50 % yield; mp 145-
148oC; 1H NMR (400 MHz, CDCl3) δ 1.64 (m, 6 H), 2.73 (broad
s, 4 H), 6.80 (s, 1 H), 6.94 (t, 1 H, J = 4.8 Hz), 7.19 (s, 1 H), 7.24
(d, 1 H,J = 5.2 Hz), 7.64 (d, 1 H,J = 3.6 Hz); 13C NMR (100
MHz, CDCl3) δ 24.17, 26.87, 53.44, 98.05, 107.4, 125.9, 127.0,
127.9, 133.4, 134.5, 139.0, 139.2, 145.6; HRMS (ESI) m/z calcd
for C16H18N4SH+: 299.1330, Found: 299.1330 (∆ = 0.0 ppm).
In summary, in the present investigation, libraries of
trisubstituted benzimidazoles were screened using high
throughput screening method against F. tularensis LVS strain to
identify novel anti-bacterial compounds. In
a preliminary
screening assay, 23 of 2,5,6- and 2,5,7-trisubstituted
benzimidazoles were identified exhibiting greater than 90 %
growth inhibitory activity at 1 µg/mL. Upon resynthesis of hit
compounds, 21 compounds showed MIC90 values in the range of
0.35-48.6 µg/mL. Subsequently, in ex-vivo efficacy assays,
selected lead compounds exhibited 2-3 Log reduction in colony
forming units (CFU) per mL at concentrations of 10 and 50
µg/mL. Further investigation on the mechanism of action as well
as the proof of concept study against animal models in vivo are
actively underway in these laboratories.
Experimental
The chemicals were purchased from Sigma Aldrich Co.,
Synquest Inc., Alfa Aesar and purified before use by standard
methods. Tetrahydrofuran was freshly distilled from sodium
metal and benzophenone. Dichloromethane was also distilled
immediately prior to use under nitrogen from calcium hydride. 1H
and 13C NMR spectra were measured on a Varian and Brucker
300, 400 or 500 MHz NMR spectrometer. Melting points were
measured on a Thomas Hoover Capillary melting point apparatus
and are uncorrected. TLC was performed on Merck DCalufolien
with Kieselgel 60F-254 and column chromatography was carried
out on silica gel 60 (Merck; 230-400 mesh ASTM). High-
resolution mass spectra were obtained from Mass Spectrometry
Laboratory, University of Illinois at Urbana-Champaign, Urbana,
IL. Purity was determined by Agilent 1100 series HPLC
assembly.
A similar procedure was used for the synthesis of 4f.
5-Amino-2-furyl-6-piperidinyl-1H-benzo[d]imidazole (4f)
Dark brown solid; 55 % yield; mp 124-126 C; H NMR
o
1
(400 MHz, CDCl3) δ 1.68 (m, 6 H), 2.78 (broad s, 4 H), 6.45 (m,
1 H), 6.85 (s, 1 H), 7.09 (m, 1 H), 7.24 (s, 1 H), 7.40 (s, 1 H); 13
C
NMR (100 MHz, CDCl3) δ 24.19, 26.88, 53.48, 98.04, 107.5,
109.4, 112.1, 133.3, 133.9, 139.2, 139.3, 142.1, 143.0, 145.6;
HRMS (ESI) m/z calcd for C16H18N4OH+: 283.1559, Found:
283.1561 (∆ = 0.7 ppm).
4-Amino-3,5-dinitro-1-(tert-butoxycarbonylamino)benzene
(8b)
Compounds, 2a-2e, 3a-3d, 3g-3i, 4a-4d, 4g-4i, 8 and 9a-9d
were prepared by using the procedures reported earlier by these
laboratories.28
A suspension of 4-amino-3,5-dinitrobenzamide (6) (543 mg,
2.4 mmol) in 4 N HCl (20 mL) was refluxed overnight. The
reaction mixture was cooled and the precipitate was filtered off to
give 4-amino-3,5-dinitrobenzoic acid as a yellow solid, which
was dissolved in thionyl chloride (4 mL) and refluxed overnight.
The reaction mixture was cooled down to room temperature and
excess thionyl chloride was distilled off to give compound 7. The
crude product 7 was immediately dissolved in acetone (2.4 mL)
in an ice-bath. To this solution was added dropwise NaN3 (448
mg, 5.94 mmol) in ice-water (0.88 mL). The mixture was stirred
2,4-Dinitro-1-(2-thiophenyl)carboxamido-5-piperidinyl-
benzene (3e)
A solution of 1 (800 mg, 3 mmol) and 2-thiophenecarbonyl
chloride (529 mg, 3.61 mmol) in pyridine (8 mL) was refluxed
overnight. The reaction mixture was diluted with
dichloromethane, washed with water (30 mL x 3) and dried over
anhydrous magnesium sulfate. The solvent was removed to give
yellow oil which was dried under vacuum to afford yellow solid.
The solid was washed with methanol to give 3e as a yellow solid
o
for 20 min at 0 C until a solid precipitated out. After dilution
with ice-water (12 mL), the reaction mixture was extracted with
dichloromethane (6 mL x 2), dried over anhydrous magnesium
o
1
(959 mg, 85 % yield): mp 178-180 C; H NMR (400 MHz,
CDCl3) δ 1.72 (m, 6 H), 3.30 (t, 4 H, J = 4.8 Hz), 7.19 (dd, 1 H, J
= 0.8 Hz, 4 Hz), 7.67 (d, 1 H, J = 4.8 Hz), 7.75 (d, 1 H, J = 4 Hz),
8.66 (s, 1 H), 8.86 (s, 1 H), 11.7 (s, NH); 13C NMR (100 MHz,
CDCl3) δ 23.60, 25.36, 52.03, 108.1, 125.5, 127.5, 127.6, 128.3,
129.5, 132.7, 133.0, 138.6, 139.0, 150.6, 160.6; MS (ESI) m/z
377.3 (M+1)+.
o
sulfate at 0 C for 1 h, and filtered. The filtrate was concentrated
on a rotary evaporator (below room temperature), and the residue
dissolved in toluene (15 mL). After refluxing for 2 h, the reaction
mixture was cooled down to room temperature, and tert-butanol
(10 mL) was added. After stirring overnight at room temperature,
the reaction mixture was concentrated in vacuo and purified by
flash chromatography on silica gel (hexane/ethylacetate = 1/1) to
afford 4-amino-3,5-dinitro-1-(tert-butoxycarbonylamino)benzene
A similar procedure was used for the synthesis of 3f.
o
1
(8b) (84% yield) as a bright red solid: mp 178-180 C; H NMR
(400 MHz, DMSO-d6) δ 1.48 (s, 9 H), 8.19(s, 2 H), 8.65 (s, 2 H),
9.69 (s, 1 H); 13C NMR (100 MHz, DMSO-d6) δ28.02, 79.97,
123.3, 126.6, 134.3, 136.9, 152.8; MS (ESI) m/z 299.2 (M+1)+.
2,4-Dinitro-1-(2-furyl)carboxamido-5-piperidinylbenzene
(3f)
Yellow solid; 81 % yield; mp 196-198oC;1H NMR (400
MHz, CDCl3) δ 1.72 (m, 6 H), 3.30 (t, 4 H, J = 4.8 Hz), 6.61 (dd,

White solid; 42 % yield; mp 110-112 °C; 1H NMR (400
MHz, CDCl3) δ 0.95 (t, 6 H, J = 7.2 Hz), 1.93 (m, 1 H), 2.01 (m,
1 H), 2.28 (m, 2 H), 2.35 (m, 2 H), 2.96 (m, 4 H, J = 7 Hz), 3.23
(m, 1 H), 4.07 (s, 3 H), 7.05 (d, 1 H, J = 8 Hz), 7.10 (t, 1 H, J = 8
Hz),), 7.39 (m, 1 H), 7.59 (s, 1 H), 8.51 (d, 1 H, J = 7.6 Hz), 8.69
(s, 1 H), 9.26 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 12.90,
18.01, 25.53, 41.40, 50.35, 55.94, 100.4, 111.4, 113.1, 117.9,
121.6, 129.6, 130.8, 132.6, 135.5, 149.8, 156.6, 172.8; HRMS
(ESI) m/z calcd for C23H28N4O2H+: 393.2289, Found: 393.2291
(∆ = -0.5 ppm).
7-Amino-2-phenyl-5-(tert-butoxycarbonylamino)-1H-benzo-
[d]imidazole (9e)
To a suspension of 8b (324 mg, 1.08 mmol) in ethanol (24
mL), was added ammonium formate (1.8 g) and 10 wt. % Pd-C
(170 mg) under nitrogen. The mixture was stirred at room
temperature overnight. The catalyst and excess ammonium
formate were filtered off on a Celite pad. The filtrate was treated
with the sodium bisulfite adduct of benzaldehyde (228 mg, 1.08
mmol) at 0 °C. After the solution was stirred for 12-16 h at room
temperature under nitrogen, a trace of insoluble material was
removed by filtration and the filtrate was concentrated on a rotary
evaporator until approximately 60-70% of the solvent was
removed. To the residue was added an equal volume of ethyl
acetate and the mixture was transferred to a separatory funnel.
The organic layer was separated, and the water layer was
extracted with ethyl acetate (40 mL). The combined organic
layers were washed with brine, dried over anhydrous sodium
sulfate. The crude product was purified by flash chromatography
on silica gel (hexane/ethyl acetate = 1/1) to afford 9e (169 mg,
6-N,N-Diethylamino-5-(2-methoxybenzamido)-2-(2-methoxy-
phenyl)-1H-benzo[d]imidazole (5d)
White solid; 54 % yield; mp >200 °C; 1H NMR (400 MHz,
CDCl3) δ 0.96 (t, 6 H, J = 7.2 Hz), 3.10 (q, 4 H, J = 7 Hz), 4.04
(s, 3 H), 4.06 (s, 3 H), 7.04 (t, 2 H, J = 7.6 Hz), 7.09 (q, 2 H, J =
7.2 Hz), 7.39 (t, 1 H, J = 3.2 Hz), 7.45 (t, 1 H, J = 3.2 Hz), 7.65
(s, 1 H), 8.35 (d, 1 H, J = 8 Hz), 8.54 (d, 1 H, J = 8 Hz), 9.00 (s, 1
H), 11.50 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 12.32, 50.54,
55.62, 55.89, 101.8, 111.2, 11.5, 113.3, 117.9, 121.2, 121.6,
122.6, 129.6, 130.8, 132.4, 132.7, 133.9, 135.9, 149.8, 155.6,
157.2, 162.8; HRMS (ESI) m/z calcd for C26H28N4O3H+:
445.2240, Found: 445.2240 (∆ = -0.0 ppm).
1
48% yield) as brown solid: mp >200 °C; H NMR (400MHz,
CD3OD) δ 1.51 (s, 9 H), 6.49 (s, 1 H), 7.15 (s, 1 H), 7.48 (m, 3
H), 8.00 (d, 2 H, J = 8.4 Hz); 13C NMR (100 MHz, CD3OD)
δ28.94, 80.71, 93.64, 101.0, 127.4, 130.1, 130.8, 131.4, 137.4,
139.1, 151.2, 155.7; HRMS (ESI) m/z calcd for C18H20N4O2H+:
325.1665, Found: 325.1669 (∆ = 1.2 ppm).
5-Benzyloxycarbonylamino-6-piperidin-1-yl-2-thiophen-1H-
benzo[d]imidazole (5e)
To a solution of 4e (200 mg, 0.67 mmol) in dichloromethane
(10 mL) was added a solution of N-benzyloxycarbonyloxy-
succinimide (167 mg, 0.67 mmol) in dichloromethane (5 mL)
dropwise at room temperature. After the addition, the mixture
was stirred at room temperature overnight. After the completion
of the reaction, the reaction mixture was concentrated under
reduced pressure. The crude product was purified via flash
chromatography on silica gel (gradient: AcOEt/hexanes = 20/80 –
40/60) to give 5e as white solid (206 mg, 48 % yield): mp 105-
107 °C; 1H NMR (400 MHz, CDCl3) δ 1.69 (m, 6 H), 2.72 (broad
s, 4 H), 5.22 (s, 2 H), 6.93 (t, 1 H, J = 3.9 Hz), 7.27 (d, 1 H, J =
4.8 Hz), 7.37 (m, 6 H), 8.21 (s, 1 H), 8.27 (s, 1 H); 13C NMR
(100 MHz, CDCl3) δ 24.12, 25.80, 27.03, 54.60, 66.97, 101.2,
109.9, 126.5, 127.8, 128.1, 128.2, 128.4, 128.7, 130.1, 133.0,
136.5, 139.8, 147.0, 153.9, 171.3, 173.6; HRMS (ESI) m/z calcd
for C24H24N4O2SH+: 433.1698, Found: 433.1698 (∆ = -0.0 ppm).
5-(Cyclobutanamido)-6-N,N-diethylamino-2-tolyl-1H-benzo-
[d]imidazole (5c)
To
dichloromethane (5
a
solution of 4c28 (115 mg, 0.39 mmol) in
mL) was added
a
solution of
cyclobutanecarbonyl chloride (0.45 ml, 0.39 mmol) in
dichloromethane (4 mL) dropwise at room temperature. After the
addition, the mixture was stirred at room temperature overnight.
After the completion of the reaction, the reaction mixture was
washed with a saturated solution of sodium bicarbonate. The
organic layer was separated and dried over anhydrous magnesium
sulfate, filtered and concentrated under reduced pressure. The
crude product was purified by flash chromatography on silica gel
(gradient: AcOEt/hexanes = 20/80–40/60) to give 5c as a white
solid (85 mg, 58 %): mp >200 °C; 1H NMR (400 MHz, CDCl3) δ
0.92 (t, 6 H, J = 7.2 Hz), 1.83 (m, 1 H), 1.95 (m, 1 H), 2.22-2.37
(m, 7 H), 2.92 (q, 4 H, J = 7.2 Hz), 3.29 (m, 1 H), 7.17 (d, 2 H, J
= 8 Hz), 7.52 (s, 1 H), 7.99 (d, 2 H, J = 8 Hz), 8.79 (s, 1 H), 9.40
(s, 1 H); 13C NMR (100 MHz, CDCl3) δ 12.90, 17.93, 21.27,
25.69, 41.44, 50.30, 101.6, 112.7, 126.6, 127.4, 129.3, 132.0,
133.1, 135.5, 139.6, 152.6, 173.1; HRMS (ESI) m/z calcd for
C23H28N4OH+: 377.2341, Found: 377.2341 (∆ = 0.5 ppm).
5-(2-Methoxybenzamido)-6-piperidinyl-2-thiophen-1H-benzo-
[d]imidazole (5f).
White solid; 52 % yield; mp 165-167 °C; 1H NMR (400
MHz, CDCl3) δ 1.65 (m, 6 H), 2.74 ( broad s, 4 H), 4.01 (s, 3 H),
6.71 (t, 1 H, J = 3.6 Hz), 7.01 (m, 2 H), 7.17 (d, 1 H, J = 4.8 Hz),
7.37 (d, 1 H, J = 3.2 Hz), 7.43 (m, 2 H), 8.89 (s, 1 H), 10.70 (s, 1
H); 13C NMR (100 MHz, CDCl3) δ 23.87, 26.65, 54.39, 56.16,
104.0, 109.6, 111.5, 121.3, 122.6, 126.1, 127.1, 127.5, 129.5,
132.1, 132.9, 133.6, 139.6, 140.8, 147.6, 157.3, 164.0; HRMS
(ESI) m/z calcd for C24H24N4O2SH+: 433.1698, Found: 433.1699
(∆ = 0.2 ppm).
A similar procedure was used for the synthesis of 5a, 5b, 5d,
5f, 5g, 5h, 5k and 5l. For the synthesis of 5i and 5j,
benzenesulfonyl chloride was used.
5-Cyclobutanamido-6-N,N-diethylamino-2-(4-fluorophenyl)-
1H-benzo[d]imidazole (5a)
White solid; 48 % yield; mp 170-172 °C; 1H NMR (400
MHz, CDCl3) δ 0.93 (t, 6 H, J = 7.2 Hz), 1.81 (m, 1 H), 2.02 (m,
1 H), 1.35-2.19 (m, 4 H), 2.94 (q, 4 H, J = 7 Hz), 3.30 ( m, 1 H),
7.06 (t, 2 H, J = 8.8 Hz), 7.55 (s, 1 H), 8.08 (m, 2 H), 8.78 (s, 1
H), 9.41 (s, 1 H); 13C NMR (100 MHz, CDCl3) δ 12.91, 17.96,
25.71, 41.44, 50.32, 101.7, 112.9, 115.6, 115.9, 126.4, 128.7,
132.2, 133.2, 135.9, 139.3, 162.6, 164.8, 173.3; HRMS (ESI) m/z
calcd for C22H25N4FOH+: 381.2091, Found: 381.2090 (∆ = -0.3
ppm).
2-Furyl-5-(2-Methoxybenzamido)-6-piperidinyl-1H-benzo[d]-
imidazole (5g)
White solid; 56 % yield; mp 155-157 °C; 1H NMR (400
MHz, CDCl3) δ 1.66 (m, 6 H), 2.74 (broad s, 4 H), 4.06 (s, 3 H),
6.32 (d, 1 H, J = 3.6 Hz), 6.97 (d, 2 H, J = 3.6 Hz), 7.06 (m, 2 H),
7.42 (s, 1 H), 7.51 (t, 1 H, J = 8.4 Hz), 9.01 (s, 1 H), 10.81 (s, 1
H); 13C NMR (100 MHz, CDCl3) δ 23.86, 26.62, 54.33, 56.20,
104.1, 109.6, 111.5, 111.8, 121.3, 122.6, 126.1, 129.8, 131.4,
132.5, 132.9, 139.2, 140.9, 142.9, 144.1, 145.7, 157.3, 163.9;
HRMS (ESI) m/z calcd for C24H24N4O3H+: 417.1927, Found:
417.1924 (∆ = -0.7 ppm).
5-Cyclobutanamido-6-N,N-diethylamino-2-(2-methoxyphenyl)-
1H-benzo[d]imidazole (5b)

7.90 (m, 1 H), 8.19 (d, 1 H, J = 8.28 Hz), 8.21 (m, 1 H), 8.35 (s, 1
H), 8.73 (s, 1 H); 13C NMR (100 MHz, acetone-d6) δ 14.11,
55.88, 56.15, 60.16, 95.77, 103.6, 112.2, 120.8, 121.1, 121.5,
121.6, 123.0, 127.5, 129.7, 131.2, 131.9, 132.0, 133.4, 134.1,
157.8; HRMS (ESI) m/z calcd for C24H21BrN4O4H+: 509.0818,
Found: 509.0814 (∆ = 0.8 ppm).
2-Furyl-5-(1-naphthylamido)-6-piperidinyl-1H-benzo[d]-
imidazole (5h)
White solid; 62 % yield; mp 202-204 °C; 1H NMR (400
MHz, CDCl3) δ 1.58 (broad s, 6 H), 2.86 (broad s, 4 H), 6.10 (m,
1 H), 6.55 (m, 2 H7.49 (m, 2 H), 7.59 (m, 2 H), 7.86 (dd, 1 H, J =
0.8 Hz, 6.96 Hz ), 7.92 (m, 1 H), 8.04 (d, 1 H, J = 8.28 Hz), 8.51
(d, 1 H, J = 9.4 Hz), 9.32 (s, 1 H), 9.82 (s, 1 H); 13C NMR (100
MHz, CDCl3) δ 23.78, 26.90, 54.63, 102.3, 109.4, 111.4, 124.7,
124.9, 125.2, 126.7, 127.3, 128.2, 129.9, 130.2, 130.7, 133.8,
135.3, 139.7, 143.0, 144.4, 145.2, 167.9; HRMS (ESI) m/z calcd
for C27H24N4O2H+: 437.1971, Found: 437.1964 (∆ = 1.6 ppm).
2-(4-Bromophenyl)-5-ethoxycarbonylamino-7-cyclopentyl-
amido-1H-benzo[d]midazole (10b)
o
1
Pale brown solid; 41 % yield; mp >200 C; H NMR (400
MHz, acetone-d6) δ 1.28 (t, 3 H, J = 7.08 Hz), 1.64 (m, 2 H), 1.77
(m, 2 H), 1.96 (m, 4 H), 3.08 (m, 1 H), 4.18 (m, 2 H), 7.70 (d, 1
H, J = 8.8 Hz), 7.73 (s, 1 H), 8.10 (d, 1 H, J = 8.8 Hz), 8.69 (s, 1
H); 13C NMR (100 MHz, acetone-d6) δ 15.06, 26.80, 31.30,
47.00, 61.12, 96.69, 105.0, 124.1, 128.9, 130.5, 132.9, 136.7,
149.6, 154.7, 175.6; HRMS (ESI) m/z calcd for C22H23BrN4O3H+:
471.1024, Found: 471.1021 (∆ = 0.6 ppm).
5-Benzenesulfonamido-2-cyclohexyl-6-(N’,N’-dimethyl-N-
ethylethylenediamino)-1H-benzo[d]imidazole (5i)
White solid; 19 % yield; mp 187-188 °C;
1H NMR (400
MHz, METHANOL-d4) δ 0.60 (t, 3 H, J = 7.2 Hz), 1.21 - 1.51
(m, 7 H), 1.56 - 1.70 (m, 2 H), 1.73 - 1.82 (m, 2 H), 1.82 - 1.93
(m, 2 H), 2.00 - 2.10 (m, 2 H), 2.32 - 2.43 (m, 8 H), 2.74 (q, 2 H,
J = 7.2 Hz), 2.80 - 2.94 (m, 3 H), 7.29 (s, 1 H), 7.37 - 7.45 (m, 2
H), 7.46 - 7.52 (m, 1 H), 7.71 (s, 1 H), 7.73 - 7.78 (m, 2 H);13C
NMR (100 MHz, METHANOL-d4) δ 11.9, 25.6, 25.8, 31.4, 38.4,
43.6, 49.3, 53.7, 56.3, 100.0, 126.7, 128.5, 131.5, 132.1, 138.1,
140.9, 160.2; HRMS (ESI) m/z calcd for C25H35N5O2SH+:
470.2590, Found: 470.2590 (∆ = 0.0 ppm).
2-(4-Bromophenyl)-5-ethoxycarbonylamino-7-benzamido-1H-
benzo[d]midazole (10c)
o
1
Off-white solid; 50 % yield; mp >200 C; H NMR (400 MHz,
acetone-d6) δ 1.28 (t, 3 H, J = 7.1 Hz), 4.18 (m, 2 H), 7.63-7.72
(m, 4 H), 7.86 (s, 1 H), 8.12 (d, 1 H, J = 8.8 Hz), 8.80 (s, 1 H),
9.62 (s, 1 H); 13C NMR (100 MHz, acetone-d6) δ 15.04, 61.18,
124.2, 128.3, 129.0, 129.2, 129.6, 130.3, 132.7, 132.8, 132.9,
135.8, 154.87; HRMS (ESI) m/z calcd for C23H19BrN4O3H+:
479.0723, Found: 479.0719 (∆ = 0.8 ppm).
5-Benzenesulfonamido-2-cyclohexyl-6-(N’,N’,N-trimethyl-
ethylenediamino)-1H-benzo[d]imidazole (5j)
1
White solid; 47 % yield; mp >200 °C; H NMR (400 MHz,
7-(Benzyoxycarbonylamino)-2-phenyl-5-(tert-butoxycarbonyl-
amino)-1H-benzo[d]imidazole (10d)
CD3OD) δ 1.19 - 1.53 (m, 7 H), 1.57 - 1.69 (m, 2 H), 1.77 (m, 1
H), 1.86 (m, 2 H), 2.04 (m, 2 H 2.54 (s, 3 H), 2.90 (m, 7 H), 7.40
(d, 2 H, J = 12.00 Hz), 7.52 (t, 2 H, J = 16.00 Hz), 7.61 (t, 1 H, J
= 16.00 Hz), 7.86 (d, 2 H, J = 4.00 Hz);13C NMR (100 MHz,
METHANOL-d4) δ 25.4, 25.6, 31.2, 38.1, 42.6, 43.3, 50.6, 55.0,
107.5, 107.8, 126.9, 128.9, 132.9, 139.9, 141.1, 160.4; HRMS
(ESI) m/z calcd for C24H33N5O2SH+: 456.2430, Found: 456.2433
(∆ = -0.7 ppm).
o
1
White solid, 55 % yield; mp >200 C H NMR (400 MHz,
acetone-d6) δ 1.50 (s, 9 H), 5.26 (s, 2 H), 7.34-7.40 (m, 3 H),
7.49-7.51 (m, 5 H), 7.72 (s, 1 H), 8.15 (d, 2 H, J = 7.0 Hz), 8.48
(s, 1 H); 13C NMR (100 MHz, acetone-d6) δ 28.68, 67.33, 79.78,
127.4, 128.9, 129.0, 129.4, 129.8, 130.4, 131.3, 137.9, 154.0;
HRMS (ESI) m/z calcd for C26H26N4O4H+: 459.2027, Found:
459.2032 (∆ = - 1.1 ppm).
7-(Cyclohexylamido)-5-ethoxycarbonylamino-2-(4-methoxy-
carbonylphenyl)-1H-benzo[d]midazole (10e)
2-Cyclohexyl-5-cyclopropylamido-6-pyrrolidinyl-1H-benzo-
[d]imidazole (5k)
o
1
White solid; 53 % yield; mp >200 C; H NMR (400 MHz,
acetone-d6) δ 1.24-1.28 (m, 6 H), 1.55-1.66 (m, 3 H), 1.76 (m, 2
H), 1.99 (m, 2 H), 2.58 (m, 1 H), 3.90 (s, 3 H), 4.16 (q, 2 H, J =
7.2 Hz), 7.81 (s, 1 H), 8.10 (m, 3 H), 8.25 (m, 2 H), 8.70 (s, 1 H),
9.17 (s, 1 H); 13C NMR (100 MHz, acetone-d6) δ 15.03, 26.61,
46.53, 52.56, 61.15, 97.71, 105.4, 127.0, 131.6, 135.1, 136.9,
149.5, 154.8, 166.8, 175.4; HRMS (ESI) m/z calcd for
C25H28N4O5H+: 465.2129, Found: 465.2138 (∆ = - 1.9 ppm).
1
Light brown solid; 44 % yield; mp 165-166 °C; H NMR
(400 MHz, CDCl3) δ 0.77 - 0.93 (m, 4 H), 1.00 - 1.40 (m, 10 H),
1.46 - 1.73 (m, 5 H), 1.76 - 1.79 (m, 1 H), 1.87 - 2.07 (m, 6 H),
2.68 - 2.84 (m, 1 H), 2.99 (br. s., 4 H), 7.46 (s, 1 H), 8.44 (s, 1
H), 8.93 (s, 1 H);13C NMR (100 MHz, CDCl3) δ 8.0, 14.3, 16.4,
22.8, 24.6, 26.0, 26.3, 29.9, 31.9, 38.6, 53.8, 103.0, 109.5, 129.5,
136.0, 159.6, 172.0; HRMS (ESI) m/z calcd for C21H28N4OH+:
353.2336, Found: 353,2341 (∆ = -1.4 ppm).
5-Ethoxycarbonylamino-7-(4-methoxybenzamido)-2-(4-
methoxycarbonylphenyl)-1H-benzo[d]midazole (10f)
Off-white solid; 52 % yield; m.p. >200 oC; 1H NMR
(400MHz, d6-acetone) δ 1.28 (t, 3 H, J = 9.6 Hz), 3.92 (s, 6 H),
4.22 (q, 2 H, J = 9.3 Hz), 7.14 (d, 2 H, J = 11.8), 7.88 (s, 1 H),
7.68 (s, 1 H), 8.07-8.33 (m, 6 H), 8.54 (s, 1 H), 8.82 (s, 1 H), 9.33
(s, 1 H), 12.16 (s, 1 H);13C NMR (100 MHz, d6-acetone) δ 14.09,
51.61, 55.06, 60.21, 96.00, 103.9, 113.9, 126.2, 129.1, 129.9,
134.3, 135.2, 136.3, 148.8, 154.0, 162.8, 165.8 ; HRMS (ESI)
m/z calcd for C26H24N4O6H+: 489.1774, Found: 489.1774 (∆ = -
0.0 ppm).
6-N,N-Diethylamino-2-phenyl-5-*4-methylbenzamido)-1H-
benzo[d]imidazole (5l)
White solid; 44 % yield; mp >200 °C; 1H NMR (400 MHz,
CDCl3) δ 0.96 (t, 6 H, J = 7.2 Hz), 2.44 (s, 3 H), 3.02 (q, 4 H, J =
7 Hz), 7.09 (t, 2 H, J = 7.6 Hz), 7.24 (m, 3 H), 7.64 (s, 1 H), 7.84
(d, 2 H, J = 8.4 Hz), 7.94 (d, 2 H, J = 7.6 Hz), 9.14 (s, 1 H), 10.36
(s, 1 H); 13C NMR (100 MHz, CDCl3) δ 12.98, 21.48, 50.53,
101.6, 113.3, 126.5, 127.0, 128.5, 129.3, 129.6, 129.9, 132.3,
132.5, 133.1, 135.9, 142.1, 152.5, 165.6; HRMS (ESI) m/z calcd
for C25H26N4OH+: 399.2185, Found: 399.2184 (∆ = - 0.3 ppm).
2-(4-Bromophenyl)-5-ethoxycarbonylamino-7-(2-methoxy-
benzamido)-1H-benzo[d]midazole (10a)
Off-white solid; 52 % yield; mp >200 oC; 1H NMR
(400MHz, acetone-d6) δ 1.28 (t, 3 H, J = 7.08 Hz), 4.01 (s, 3 H),
4.18 (m, 2 H), 7.08 (m, 1 H), 7.16-7.22 (m, 2 H), 7.31 (d, 1 H, J =
8.32 Hz), 7.55 (m, 1 H), 7.71 (d, 1 H, J = 8.32 Hz ), 7.85 (s, 1 H),
7-Benzamido-5-ethoxycarbonylamino-2-(4-methoxycarbonyl-
phenyl)-1H-benzo[d]midazole (10g)
Brown solid; 51 % yield; m.p. > 200 C; H NMR (400
MHz, acetone-d6) δ 1.28 (t, 3 H, J = 7.2 Hz) 3.91 (s, 3 H), 4.20
(q, 2 H, J = 7.2 Hz), 7.61 (m, 3 H), 7.87 (s, 1 H), 8.10 (m, 4 H),
o
1

8.28 (d, 2 H, J = 6.3 Hz), 7.79 (s, 1 H); 13C NMR (100 MHz,
acetone-d6) δ 15.09, 52.55, 61.20, 97.20, 105.0, 127.2, 128.1,
129.7, 130.7, 131.7, 132.8, 135.2, 136.0, 154.7, 166.8; HRMS
(ESI) m/z calcd for C25H22N4O5H+: 459.1660, Found: 459.1668
(∆ = - 1.7 ppm).
Manassas, VA) were used to assess intracellular efficacy of our
compounds. RAW cells were cultured in complete growth
medium (ATCC) supplemented with 10% fetal bovine serum
(Atlas Biologicals, Fort Collins, CO. Bacteria were added to
2.5x105 RAW cells per well in a 24-well tissue culture plate at a
multiplicity of infection of 50 CFU per cell in a 0.5 mL media.
The plates were then centrifuged at 2,400 x g for 2 min and
5-Ethoxycarbonylamino-7-(4-fluorobenzamido)-2-(1-furyl)-
1H-benzo[d]imidazole (10h)
o
placed at 37 C/5% CO2 to incubate for 1 h. Supernatant was
then removed; plate washed once with 2 ml PBS and 1 mL of
0.05 mg/mL gentamicin (Sigma-Aldrich) in complete media
added. Plates were then incubated for 1 h and washed 2 times
with 2 mL PBS.
Selected compounds were then added to each well at 50, 10,
and 2 µg/mL in complete media in triplicate. Untreated, 1 µg/mL
doxycycline (Sigma-Aldrich), and 50 µg/mL thiabendazole
Light brown solid; 51 % yield; mp turned black at 140 °C;
1H NMR (400 MHz, CDCl3) δ 1.25 (t, 3 H, J = 7.2 Hz) 4.17 (q, 2
H, J = 7.2 Hz) 6.42 (br. s., 1 H) 7.01 (br. s., 1 H) 7.08 (t, 2 H, J =
8.3 Hz) 7.30 - 7.51 (m, 2 H) 7.69 (br. s., 3 H) 7.96 (m, 3 H) 9.12
(br. s., 1 H); 13C NMR (100 MHz, CDCl3) δ 14.68, 61.51, 110.84,
112.35, 115.74, 115.95, 130.03, 130.12, 130.64, 134.42, 143.19,
144.05, 144.97, 154.81, 163.93, 165.41, 166.44; HRMS (ESI)
m/z calcd for C21H17FN4O4H+: 409.1316, Found: 409.1312 (∆ =
1.0 ppm).
(Sigma-Aldrich) were included as controls.
Plates were
incubated for 18 h and cells observed for signs of infection. Cells
were washed 3 times with 2 mL PBS and 1 mL sterile ddH2O
added to each well. Each well was thoroughly mixed/scraped to
insure complete cell lysis. Lysates were then serially diluted 1:10
and inoculum plated from the 10-1, 10-2, 10-3 and 10-4 dilutions
onto CHAB agar plates. Plates were incubated for 48 h at 37 oC.
Colonies from each plate was counted and CFU/ml lysate was
calculated. MIC90 was calculated using linear regression analysis
of compound dilution series to determine specific compound
concentration that exhibits 90% growth reduction of bacteria.
7-Benzamido-5-ethoxycarbonylamino-2-(1-furyl)-1H-benzo-
[d]imidazole (10i)
Light brown solid; 52 % yield; mp turned black at 140 °C;
1H NMR (400 MHz, CDCl3) δ 1.26 (t, 3 H, J = 7.2 Hz), 4.18 (q, 2
H, J = 7.2 Hz), 6.43 (br. s, 1 H), 7.04 (br. s, 1 H), 7.31 - 7.68 (m,
5 H), 7.97 (m, 3 H), 9.14 (br. s., 1 H); 13C NMR (100 MHz,
CDCl3) δ 14.7, 61.5, 112.4, 127.7, 128.8, 132.2, 134.5, 143.1,
144.1, 154.8, 166.5; HRMS (ESI) m/z calcd for C21H18N4O4H+:
391.1401, Found: 391.1406 (∆ = -1.3 ppm).
Cytotoxicity Assay. The cytotoxicity of the compounds was
tested against Vero cells. Epithelial cells from the kidneys of the
African Green Monkey were used to start the Vero cell line. Vero
cells were grown in L15 media without CO2. Serial 2-fold
dilutions of the drugs were prepared (200 µg/mL to 50 µg/mL) in
triplicate using 96-well microtiter plates. The cells were added to
the plates to a final concentration of 1.25 × 105/well in media
containing Resazurin for a final concentration 25 µg/mL. The
plates were incubated for 3 days at 37 oC. The LD was the lowest
drug concentration that inhibited cell growth and the resazurin
was not reduced.
Antibacterial activity. F. tularensis LVS provided by Dr. J.
Petersen (Centers for Disease Control, Fort Collins, CO) was
grown to an OD600 of ~0.6, frozen at -80oC in 10 % glycerol and
used as a standard bacterial stock for these studies. For each
evaluation bacteria were prepared fresh by growth from the
standard stocks on Cystine Heart Agar supplemented with 2%
o
hemoglobin (BD, Franklin Lakes, NJ) grown at 37 C for 48-72
h. Bacteria recovered from the Cystine Heart Agar-Hemoglobin
(CHAB) plates were used to inoculate 50 mL Mueller-Hinton
broth (BD) supplemented with 0.025% ferric pyrophosphate
(Sigma-Aldrich), 2% IsoVitaleX (BD), 0.1% glucose (Sigma-
Aldrich). Broth cultures were then incubated for 18 h at 37oC
passed 1:20 and incubated for an additional 8 h at 37 oC.
Bacteria were then diluted to a concentration of 1x107 colony
forming units (CFU)/mL in modified Mueller-Hinton (MMH)
broth and 50 µL added to each well for each test plate.
Trisubstituted benzimidazole compound library was screened in a
percent inhibition high-throughput fashion against F. tularensis
LVS in a 96-well plate format. All compounds were diluted in
MMH broth to concentrations of 10, 2 and 0.4 µg/mL in a 50µL
volume/well. For MIC determination, compounds were added to
the 96-well plate starting at 512 µg/mL in the first column and
serially diluted 1:2 to column 12 for a final concentration of 0.25
in MMH broth. MIC plates were incubated at 37oC for 18 h at
which time 10 µL of Alamar Blue (Invitrogen, Carlsbad,
California) was added to each well and plates were incubated for
an additional 4 h. Reduction of Alamar Blue was determined by
absorbance readings at wavelengths of 570 and 600 nm using a
microplate reader (Biotek, Winooski, VT). Percent growth
reduction was calculated and MIC90 values were determined by
5. Acknowledgements
This research was supported by grants from the
National Institutes of Health (Grant AI078251 to
I.O.) and (U01082164 to R.A. S.), as well as the NYSTAR (to
I.O.).
6. References
1. Gould, I. M.; David, M. Z.; Esposito, S.; Garau, J.; Lina, G.;
Mazzei, T.; Peters, G. Int. J. Antimicrob. Agents 2012, 39, 96.
2. Wang, J.-L.; Hsueh, P.-R. Expert Opin. Pharmacother. 2009,
10, 785.
3. McLendon, M. K.; Apicella, M. A.; Allen, L.-A. H. Annu. Rev.
Microbiol. 2006, 60, 167.
4. Feodorova, V. A.; Corbel, M. J. Expert Rev. Vaccines 2009, 8,
1721.
plotting
the
percentage
inhibition
calculated
from
5. Oyston, P. C. F.; Sjoestedt, A.; Titball, R. W. Nature Reviews
Microbiology 2004, 2, 967.
spectrophotometric readings over the drug concentration series.
Bacterial growth and MIC was confirmed by optical density.
Non-linear regression analysis was performed on % growth
inhibition curves to determine MIC90 values.
6. Ryan, K. J.; Ray, C. G. Sherris Medical Microbiology: An
Introduction to Infectious Diseases: McGraw Hill, 2004.
7. Evans, M. E.; Gregory, D. W.; Schaffner, W.; McGee, Z. A.:
United States FIELD Citation:, 1985, 251.
Francisella ex vivo model of efficacy. RAW 264.7 mouse
macrophages (American Type Tissue Collection (ATCC),

8. Dennis, D. T.; Inglesby, T. V.; Henderson, D. A.; Bartlett, J. G.;
Ascher, M. S.; Eitzen, E.; Fine, A. D.; Friedlander, A. M.;
Hauer, J.; Layton, M.; Lillibridge, S. R.; McDade, J. E.;
Osterholm, M. T.; O'Toole, T.; Parker, G.; Perl, T. M.; Russell,
P. K.; Tonat, K. Jama 2001, 285, 2763.
33. Jaiswal, R.; Beuria, T. K.; Mohan, R.; Mahajan, S. K.; Panda,
D. Biochemistry 2007, 46, 4211.
34. Stokes, N. R.; Sievers, J.; Barker, S.; Bennett, J. M.; Brown, D.
R.; Collins, I.; Errington, V. M.; Foulger, D.; Hall, M.; Halsey,
R.; Johnson, H.; Rose, V.; Thomaides, H. B.; Haydon, D. J.;
Czaplewski, L. G.; Errington, J. J. Biol. Chem. 2005, 280,
39709.
9. Larsson, P.; Oyston, P. C. F.; Chain, P.; Chu, M. C.; Duffield,
M.; Fuxelius, H.-H.; Garcia, E.; Haelltorp, G.; Johansson, D.;
Isherwood, K. E.; Karp, P. D.; Larsson, E.; Liu, Y.; Michell, S.;
Prior, J.; Prior, R.; Malfatti, S.; Sjoestedt, A.; Svensson, K.;
Thompson, N.; Vergez, L.; Wagg, J. K.; Wren, B. W.; Lindler,
L. E.; Andersson, S. G. E.; Forsman, M.; Titball, R. W. Nat.
Genet. 2004, 37, 153.
35. Ito, H.; Ura, A.; Oyamada, Y.; Tanitame, A.; Yoshida, H.;
Yamada, S.; Wachi, M.; Yamagishi, J.-i. Microbiol. Immunol.
2006, 50, 759.
36. Paradis-Bleau, C.; Beaumont, M.; Sanschagrin, F.; Voyer, N.;
Levesque, R. C. Bioorg. Med. Chem. 2007, 15, 1330.
37. Kumar, K.; Awasthi, D.; Berger, W. T.; Tonge, P. J.; Slayden,
R. A.; Ojima, I. Future Med. Chem. 2010, 2, 1305.
10. Saslaw, S.; Eigelsbach, H. T.; Wilson, H. E.; Prior, J. A.;
Carhart, S. Arch Intern Med 1961, 107, 689.
11. Saslaw, S.; Eigelsbach, H. T.; Prior, J. A.; Wilson, H. E.;
Carhart, S. Arch Intern Med 1961, 107, 702.
12. CDC. "Tularemia: FAQ About Tularemia". Available at
http://www.cdc.gov/tularemia/Tul_Fact.html 2010.
13. Dienst, F. T., Jr. J La State Med Soc 1963, 115, 114.
14. "Health Aspects of Chemical and Biological Weapons" World
Health Organization 1970.
15. Miller, J. R.; Waldrop, G. L. Expert Opin. Drug Discovery
20105, 145.
16. Enderlin, G.; Morales, L.; Jacobs, R. F.; Cross, J. T. Clinical
infectious diseases an official publication of the Infectious
Diseases Society of America 1994, 19, 42.
17. Ikaheimo, I.; Syrjala, H.; Karhukorpi, J.; Schildt, R.; Koskela,
M. J. Antimicrob. Chemother. 2000, 46, 287.
18. Burke, D. S. J Infect Dis 1977, 135, 55.
19. White, E. L.; Ross, L. J.; Reynolds, R. C.; Seitz, L. E.; Moore,
G. D.; Borhani, D. W. J. Bacteriol. 2000, 182, 4028
20. White, E. L.; Suling, W. J.; Ross, L. J.; Seitz, L. E.; Reynolds,
R. C. J. Antimicrob. Chemother. 2002, 50, 111.
21. Reynolds, R. C.; Srivastava, S.; Ross, L. J.; Suling, W. J.;
White, E. L. Bioorg. Med. Chem. Lett. 2004, 14, 3161.
22. Huang, Q.; Kirikae, F.; Kirikae, T.; Pepe, A.; Amin, A.;
Respicio, L.; Slayden, R. A.; Tonge, P. J.; Ojima, I. J. Med.
Chem. 2006, 49, 463.
23. Slayden, R. A.; Knudson, D. L.; Belisle, J. T. Microbiology
2006, 152, 1789.
24. Kumar, K.; Awasthi, D.; Lee, S.-Y.; Zanardi, I.; Ruzsicska, B.;
Knudson, S.; Tonge Peter, J.; Slayden Richard, A.; Ojima, I. J
Med Chem 2011, 54, 374.
25. Margolin, W. FEMS Microbiology Reviews 2000, 24, 531.
26. Rothfield, L.; Justice, S.; Garcia-Lara, J. Annual Review of
Genetics 1999, 33, 423.
27. Lock, R. L.; Harry, E. J. Nature Reviews Drug Discovery 2008,
7, 324.
28. Awasthi, D.; Kumar, K.; Ojima, I. Expert Opin Ther Pat 2011,
21, 657.
29. Margalit, D. N.; Romberg, L.; Mets, R. B.; Hebert, A. M.;
Mitchison, T. J.; Kirschner, M. W.; RayChaudhuri, D. Proc.
Natl. Acad. Sci. U. S. A. 2004, 101, 13969.
30. Urgaonkar, S.; La Pierre, H. S.; Meir, I.; Lund, H.; Ray
Chaudhuri, D.; Shaw, J. T. Org. Lett. 2005, 7, 5609.
31. Rai, D.; Singh, J. K.; Roy, N.; Panda, D. Biochem. J. 2008, 410,
147.
32. Domadia, P.; Swarup, S.; Bhunia, A.; Sivaraman, J.; Dasgupta,
D. Biochem. Pharmacol. 2007, 74, 831.