Acinetobacter baumannii OxPhos inhibitors as selective anti-infective agents

Article abstract of DOI:10.1016/j.bmcl.2014.11.020

The Gram-negative bacterium Acinetobacter baumannii is an opportunistic pathogen in humans and infections are poorly treated by current therapy. Recent emergence of multi-drug resistant strains and the lack of new antibiotics demand an immediate action for development of new anti-Acinetobacter agents. To this end, oxidative phosphorylation (OxPhos) was identified as a novel target for drug discovery research. Consequently, a library of ～10,000 compounds was screened using a membrane-based ATP synthesis assay. One hit identified was the 2-iminobenzimidazole 1 that inhibited the OxPhos of A. baumannii with a modestly high selectivity against mitochondrial OxPhos, and displayed an MIC of 25 μM (17 μg/mL) against the pathogen. The 2-iminobenzimidazole 1 was found to inhibit the type 1 NADH-quinone oxidoreductase (NDH-1) of A. baumannii OxPhos by a biochemical approach. Among various derivatives that were synthesized to date, des-hydroxy analog 5 is among the most active with a relatively tight SAR requirement for the N′-aminoalkyl side chain. Analog 5 also showed less cytotoxicity against NIH3T3 and HepG2 mammalian cell lines, demonstrating the potential for this series of compounds as anti-Acinetobacter agents. Additional SAR development and target validation is underway.

Full text of DOI:10.1016/j.bmcl.2014.11.020

Bioorganic & Medicinal Chemistry Letters 25 (2015) 378–383
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Acinetobacter baumannii OxPhos inhibitors as selective anti-infective
agents
a
a
b
b
Harvey Rubin a, , Trevor Selwood , Takahiro Yano , Damian G. Weaver , H. Marie Loughran ,
⇑
Michael J. Costanzo b, Richard W. Scott b, Jay E. Wrobel b, Katie B. Freeman b, Allen B. Reitz b,
⇑
a University of Pennsylvania, 522 Johnson Pavilion, Philadelphia, PA 19104, United States
b Fox Chase Chemical Diversity Center, Inc., 3805 Old Easton Rd., Doylestown, PA 18902, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The Gram-negative bacterium Acinetobacter baumannii is an opportunistic pathogen in humans and infec-
tions are poorly treated by current therapy. Recent emergence of multi-drug resistant strains and the lack
of new antibiotics demand an immediate action for development of new anti-Acinetobacter agents. To
this end, oxidative phosphorylation (OxPhos) was identified as a novel target for drug discovery research.
Consequently, a library of ꢀ10,000 compounds was screened using a membrane-based ATP synthesis
assay. One hit identified was the 2-iminobenzimidazole 1 that inhibited the OxPhos of A. baumannii with
Received 5 September 2014
Revised 4 November 2014
Accepted 6 November 2014
Available online 22 November 2014
Keywords:
a modestly high selectivity against mitochondrial OxPhos, and displayed an MIC of 25 lM (17 lg/mL)
Acinetobacter baumannii
Oxidative phosphorylation
Anti-infective
against the pathogen. The 2-iminobenzimidazole 1 was found to inhibit the type 1 NADH–quinone oxi-
doreductase (NDH-1) of A. baumannii OxPhos by a biochemical approach. Among various derivatives that
were synthesized to date, des-hydroxy analog 5 is among the most active with a relatively tight SAR
requirement for the N0-aminoalkyl side chain. Analog 5 also showed less cytotoxicity against NIH3T3
and HepG2 mammalian cell lines, demonstrating the potential for this series of compounds as anti-Aci-
netobacter agents. Additional SAR development and target validation is underway.
2-Iminobenzimidazole
Ó 2014 Elsevier Ltd. All rights reserved.
Acinetobacter baumannii (Ab) is a bacterial pathogen that is a
major source of Gram-negative bacterial infections within the
hospital setting.1–3 Multidrug resistant (MDR) forms of A. bau-
mannii, defined as resistance to three or more antibiotic drugs,
account for 63% of A. baumannii varieties and are a primary cause
of pneumonia or bloodstream infections among critically ill
patients (CDC webpage). The risk of mortality is high, especially
among ventilator-associated pneumonia (VAP) patients.4 A 2004
study indicated A. baumannii infection in burn wound patients
increased the cost of treatment by almost $100,000 per patient.5
A. baumannii is the second most commonly isolated non-ferment-
ing bacterium in humans, and infection can result in pneumonia,
skin and wound infections, bacteremia and meningitis. In addi-
tion, A. baumannii biofilms have been implicated in diseases such
as cystic fibrosis, periodontitis and urinary tract infections, partly
because of the bacteria’s ability to colonize indwelling medical
devices. The continual appearance of strains resistant to b-lac-
tams, cephalosporins, aminoglycosides, quinolones and, lately,
carbapenems has compromised treatment options. Therefore,
the development of new therapeutic agents to treat A. baumannii
NH
Cl
N
N
Et2N
O
HO
Cl
Figure 1. Compound 1 (bisTFA salt).
infection is of great need. We have focused on developing novel
antibiotics that selectively target the bacterial oxidative phos-
phorylation (OxPhos) system, which plays an essential role in
energy production in the form of ATP. Inhibition of this pathway
is known to significantly impair viability of pathogenic bacteria.
As an obligate aerobe, A. baumannii represents an ideal
target organism. High throughput screening for inhibition of
A. baumannii OxPhos with a library of compounds uncovered
several promising scaffolds. Derivatives of one of these scaffolds,
1H-benzo[d]imidazol-2(3H)-imine (BDIs), namely
1 (Fig. 1),
showed a good spectrum of antibacterial activity against A. bau-
mannii. Additional structure–activity relationship studies based
upon the synthesis of >100 analogs led to 5 (described below)
with improved potency and good activity against a battery of
multi-drug resistant clinical isolates of A. baumannii.
⇑
Corresponding authors. Tel.: +1 215 746 4234 (H.R.), +1 215 589 6435 (A.B.R.).
E-mail addresses: rubinh@upenn.edu (H. Rubin), areitz@fc-cdci.com (A.B. Reitz).
http://dx.doi.org/10.1016/j.bmcl.2014.11.020
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

H. Rubin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 378–383
379
The OxPhos system is composed of the electron transport chain
(ETC) and ATP synthase, and plays a central role in ATP synthesis in
various Gram-positive and Gram-negative bacterial pathogens.
Recent biochemical6–10 and genetic11,12 studies on the OxPhos
pathway in bacteria indicate that these coupled processes of the
oxidation of NADH, the transfer of electrons along the ETC and
the establishment, and maintenance and utilization of the electro-
chemical proton gradient to drive ATP synthesis are essential for
1.9 lM IC50 for the inhibition of ATP synthesis, an MIC of 25 lM
against A. baumannii, and the best selectivity for A. baumannii
OxPhos over mammalian (SMP) OxPhos. A correlation between
inhibition of ATP synthesis and MIC was established as more com-
pounds were studied. Interestingly, no correlation was found
between inhibition of SMP ATP synthesis and cytotoxicity, limiting
the utility of this counterscreen in determining the selection of
compounds to progress into medicinal chemistry and animal
studies.
bacterial survival under
a wide range of conditions. While
sequence overlap exists between some bacterial and mammalian
mitochondrial OxPhos components, there is excellent evidence
that marked differences exist between the components of the bac-
terial ETC/ATP synthase and those of mammalian mitochondria.
For example, cytochrome bd oxidases and type II NADH:quinone
oxidoreductase (NDH-2) have no counterpart in human mitochon-
dria, but appear to be nearly ubiquitous in aerobic bacteria. In
addition, bioinformatic analyses indicate structural divergence
between the enzymes of the OxPhos pathway of bacteria and mito-
chondria. For instance, the a and c subunits of ATP synthases have
been suggested to be good targets for specific antibacterial
agents.13 This approach has been validated by the use of atovaqu-
one, which targets complex III of the ETC of malarial-causing Plas-
Replacing the imino NH of 1 with O to afford urea 2 led to a six-
fold increase in ATP synthesis IC50 (Ab IC50), and lower antibacterial
activity (Ab MIC, Fig. 2 and Table 1). Removing the hydrophobic
side chain (3) led to a precipitous drop in potency, while removing
the 2-(diethylamino)ethyl side chain (4) retained activity but lost
selectivity (SMP IC50). Compound 5 in which the OH moiety was
removed from the alkyl dichlorophenoxy side chain showed 3-fold
improved activity for ATP synthesis, and greater Ab MIC potency.
Time-kill assays at 8ꢁ the MIC demonstrated 5 to be bactericidal
against Ab. Selectivity was similar to lead 1. Shorter hydrophobic
side chains (6 and 7) were 10-fold less potent than 5.
The isolated membranes used for screening contain a fully func-
tional OxPhos system and are amenable to biochemical investiga-
tion of the mechanism of action of compounds. Consequently,
the mechanism of action of 5 has been investigated in these mem-
branes. Using this system NDH-1 was identified as a target of 1.
Figure 3a shows the result of monitoring O2 consumption by A.
baumannii membranes that were exposed to 5. The blue trace
shows initial O2 consumption with NADH (1 mM) as the substrate
which is abruptly terminated upon addition of 5 to give a concen-
modium, and by the discovery of
a potent inhibitor of
Mycobacterium tuberculosis ATP synthase, the diarylquinoline
bedaquiline,13,14 which was approved in late 2012, although a
Phase II study showed this compound to have a higher death rate
than comparators.15 In addition, we have reported that inhibitors
of ETC component type 2 NADH–quinone oxidoreductase (NDH-
2) are effective anti-M. tuberculosis agents in culture and in animal
models.8,9
tration of 50 lM. Addition of ascorbate/TMPD (10 mM/0.1 mM in
Isolated A. baumannii membranes were screened against a
ꢀ10,000 member small-molecule library that was designed and
collated based on previous antibiotic screening. Submitochondrial
particles (SMP), prepared from bovine heart, were used to estimate
selectivity relative to mammalian OxPhos as a secondary assay.
The primary screen identified several drug-like scaffolds such as
1 that inhibited A. baumannii ATP synthesis. We have investigated
the 1H-benzo[d]imidazol-2(3H)-imines (viz. 1) to the greatest
extent, as 1 was the most potent among the original hits with a
the assay) returned O2 consumption. These results can be rational-
ized by 5 inhibiting NDH-1 (preventing transfer of electrons to the
quinone (Q) pool) and ascorbate/TMPD directly supplying
electrons to the terminal oxidases, cytochrome bo3 oxidase and
cytochrome bd oxidase, thereby circumventing the inhibited
NDH-1. This is depicted in Figure 3b which shows the sequence
of proteins that shuttle electrons through the OxPhos system of
A. baumannii to create a membrane potential to facilitate ATP syn-
thase function. Similarly, the green trace in Figure 3a shows initial
O
NH2
N
NH2
N
Cl
Cl
N
N
Et2N
N
N
Et2N
O
O
HO
Cl
Cl
3
4
2
Cl
NH
·HBr
NH
A
Cl
Cl
N
N
Et2N
N
N
Et2N
O
6 ·2TFA (A = O)
7 ·2TFA (A = CH2)
Cl
5
O
N
NEt2
n
OH
O
HN
NH
NH
Cl
Cl
Cl
N
N
HN
O
·2TFA
N
R
N
·2TFA
Cl
Cl
N
O
HO
9 ·2TFA (n = 1, R = OH)
10 ·2TFA (n = 2, R = OH)
8
Cl
12
11 ·2TFA (n = 2, R = H)
Figure 2. Subset of analogs of 1 that were prepared and investigated.

380
H. Rubin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 378–383
O2 consumption with succinate (50 mM) as the substrate and no
inhibition of O2 consumption upon addition of 5 to a 50 M con-
centration or upon a second addition to give a 100 M concentra-
of these findings, the IC50 values obtained by monitoring ATP syn-
thesis or by direct monitoring of NADH consumption at 340 nm
match closely for both 1 and 5.
l
l
tion. As can be seen from Figure 3b the succinate pathway, via
succinate dehydrogenase (SDH), would not be affected by inhibi-
tion of NDH-1. These characteristics mirror those of 1. In support
In continuing SAR studies of 5, moving the position of the alkyl
dichlorophenoxy side chain from the ring benzimidazole nitrogen
to 2-CH2NH (8) led to poor activity. Compounds 9–12 were pre-
pared that move the point of attachment of the 2-aminoethyl side
chain and structurally related side chains from the internal
benzimidazole nitrogens of 1 to the external nitrogen. These
analogs generally showed good potency but poor selectivity, with
Table 1
A. baumannii activity for compounds in Figure 2
little or no MIC activity at 100
permeability.
lM, possibly due to poor membrane
Compd
Ab IC50
(
lM)
SMP IC50
(l
M)
Ratio SMP
Ab MIC3
(lM)
1
2
For the purpose of the additional SAR analysis presented here,
the dichlorophenoxy moiety was kept constant; however, o-chlo-
rophenyl and biphenyl substitution here has also looked promising
so far (data not shown). The SAR aspects of 5 were explored fur-
ther. Diverse side chain replacements were made to the 2-(diethyl-
amino)ethyl moiety with the reasoning that this side chain was
likely to contribute to binding with the OxPhos protein NDH-1
involved in activity. A dialkylamino group is often subject to phase
I oxidative metabolism via cytochrome P450 mediated N-dealkyla-
tion, which was observed for 5 (see below). An assortment of ana-
logs varying the 2-aminoethyl side chain is compared in Table 2
keeping the rest of the molecule constant, including ethyl 13, butyl
14 and phenylpropyl 15, di(alkyl)aminoethyl derivatives 16 and
17, heterocycles 18–27, amide 28, and diether 29. Of this group,
most of the compounds showed low micromolar activity in the A.
baumannii ATP synthesis screen, and a few had antibacterial
IC50/Ab IC50
1
2
3
4
5
6
7
8
9
1.9
12
40
0.9
0.6
6.9
5
41
5
10
8
15
21
7.9
1.8
2.5
0.9
5.2
2.7
1.9
2.6
9.4
1.3
2.5
1.2
25
>100
>100
25
13
100
25
>100
>100
>100
50
100
0.8
3.2
18.3
9.4
106
47
13
20
9.6
10
11
12
7.8
>100
1
A. baumannii membrane ATP synthesis, n = 2, standard deviations between the
duplicates were on average 9% of the mean.
2
Sub-mitochondrial particle (SMP, bovine) ATP synthesis, n = 2, standard devi-
ations between the duplicates were on average 9% of the mean.
A. baumannii minimum inhibitory concentration, MICs were performed in
duplicate and all reproduced to within one well. In cases where the MICs did not
duplicate the lower MIC value is reported.
3
activity at or below 50
lM. With this in mind we focused on more
conservative changes to 2-(diethylamino)ethyl moiety of 5.
Figure 3. Panel a, the effect of compound 5 on O2 consumption. Panel b, the Ab OxPhos.

H. Rubin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 378–383
381
Table 2
Further SAR studies on 5 involving the (diethylamino)ethyl side chain
NH
Cl
R
N
N
O
Cl
Ab
Ab
SMP
IC50
NIH3T3 HepG2
NIH3T3 HepG2
IC50 IC50
M)4 M)4
Ab
Ab
Ratio
SMP IC50
/ Ab IC50
Ratio SMP
IC50 / Ab
IC50
IC50
IC50
µ
IC50
µ
IC50
SMP IC50
M)2
MIC
MIC
FW (µM)1
(
µ
M)2
(
µ
M)3
(
M)4
(
M)4
FW
(
µ
M)1
(
µ
(
µ
M)3
(
µ
(
µ
R (Salt form)
R (Salt form)
(CH2)2-2-pyridyl
(2TFA)
13
Et (TFA)
478
6.4
8.8
1.4
50
24
96
21
22
669
3.2
6.6
2.1
100
36
60
Boc
N
·TFA
14
15
nBu (TFA)
(CH2)3Ph (TFA)
506
568
3.8
2.1
1.8
2.2
0.5
1.1
50
25
28
36
72
65
662
430
1.7
18.3
1.0
0.6
2.7
50
25
61
42
137
23 (CH2)2-N-imidazoyl
49.7
3.1
>100
>100
O
N
·TFA
516
20
83
561
99
63
5
(CH2)2NEt2 (HBr)
0.61-1.2 2.9 - 3.2 3.0-5.2
13
24
3.6
0.9
Ph
N
··2TFA
N
767
723
400
22
1305
35
16 (CH2)2N(nPr)2 (HBr) 544
2.8
1.7
2.9
2.5
1.0
1.5
25
13
28
19
49
39
25
1.0
0.9
0.8
0.9
0.8
1.0
>100
50
O
CF3
N
·2TFA
17 (CH2)2N(iPr)2 (2TFA) 692
26
(CH2)2-N-pyrrolidinyl
N
·TFA
O
18
19
(2TFA)
(CH2)3-N-piperidinyl
(2TFA)
661
690
2.1
3.6
4.9
2.3
6.2
13
50
12
37
67
83
27
559
563
1.9
6.0
6.1
3.2
1.9
100
94
68
168
143
22.4
28 CH2C(O)NEt2 (TFA)
11.4
>100
(CH2)2-N-
20 morpholinyl (2TFA) 677
(CH2)2O(CH2)2OEt
9.2
25.5
2.8
>100
76
215
29
(TFA)
566
5.6
6.0
1.1
>100
62
151
Highlights compound 5, which is the compound investigated to the greatest extent.
1 A. baumannii membrane ATP synthesis, n = 2, standard deviations between the duplicates were on average 9% of the mean.
2 Sub-mitochondrial particle (SMP, bovine) ATP synthesis, n = 2, standard deviations between the duplicates were on average 9% of the mean.
3 A. baumannii minimum inhibitory concentration, MICs were performed in duplicate and all reproduced to within one well. In cases where the MICs did not duplicate the
lower MIC value is reported.
4 Cells are exposed to compounds for 1 h in the absence of serum. Compounds are removed and metabolically active cells are measured using MTS reduction, N = 3.
R'
N
RX (1.1 equiv)
Cs2CO3
R'X (1.2 to 2.0 equiv)
K2CO3
N
N
N
NH2
NH2
NH
NH
N
R
DMF
DMF
R
50 oC
100-120 oC
30
32
34
31
3-7, 13-29
R"
N
35 or R"X (1.1 equiv)
amine
N
N
Cl
NRR'
NRR'
DIPEA
K2CO3
NH
NH
N
9-12
Neat or NMP
DMF
100 - 145 oC
80 oC
33
Cl
O
O
N
35
Cl
8
NH
NH2
Cs2CO3
DMF
50 oC
H2C
CH3
O
Br
K2CO3, DMF, 50 oC
1)
2) 4M HCl, 65 oC
3)
N
N(Et)2
2
NH
35, K2CO3, DMF,100-120 oC
36
Figure 4. Synthetic schemes for the preparation of 2–29.
Congeners 17 and 18 (Table 2, highlighted in green) were as active
as 5, although somewhat less selective. Cytotoxicity was evaluated
in NIH3T3 and HepG2 cells, showing selectivity for the potentially-
therapeutic effect. The separation of cytotoxicity and MIC activity
was not large and is a primary focus of optimization in future
analogs.
Benzimidazoles 2–29 were prepared using the synthetic
schemes shown in Figure 4. Compounds 3–7 and 13–29 were pre-
pared by sequential alkylation of 2-aminobenzimidazole (30) under
relatively mild conditions to give 31 followed by a second alkylation
under more rigorous conditions. 2-Aminobenzimidazoles 9–12
were prepared by sequential reaction of 2-chlorobenzimidazole

382
H. Rubin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 378–383
Table 3
two species (E. coli and K. pneumonia) were tested in the presence
MIC distribution for 5 and comparator agents
and absence of 40% serum. In both cases the MIC rose in the pres-
ence of serum suggesting that free drug was reduced upon serum
addition.
Test Agent
MIC (µ g/mL)
0.25
0.5
1
2
4
8
16
32
64
>64
Number of A. baumannii isolates that test compd has demonstrated this MIC
Compound 5 showed low metabolic stability in mouse and
human liver microsomes (13 and 9.2 min t1/2, respectively), which
translates to the potential for high hepatic clearance rates in vivo.
It was determined that the major metabolite generated in mouse
liver microsomes was N-dealkylation of the diethyl amine, and
minor amounts of hydroxylated products were also observed.
Because of this finding, other compounds not possessing the dial-
kylamino side chain were evaluated in mouse liver microsome sta-
bility assays including 15, 25 and 26 (indicated in blue in Table 2).
Each of these three compounds showed considerably greater sta-
bility than 5 (48.2, 25.2 and >90 min, respectively). Although these
compounds were not in themselves potent, this result suggests
that we are able to improve metabolic stability through structural
modifications, and that decreasing the dealkylation metabolic
pathway did not result in some other pathway becoming overly
prevalent.
Herein, a new mechanistic approach to target A. baumannii by
inhibition of bacterial OxPhos, specifically NDH-1, has been dem-
onstrated. Selectively targeting OxPhos in bacteria, without effect
upon host OxPhos, represents a new approach to attack the grow-
ing problem of resistant Gram-negative bacteria, such as A. bau-
mannii. It is possible that this novel mechanism of action may
afford stand alone agents or agents that are useful in combination
with other agents to minimize potential drug resistance over time.
We have developed a validated and robust assay for identifying
inhibitors of the OxPhos pathway. Further biochemical and genetic
characterization of the interactions of test compounds with target
membranes are being done to substantiate the role of NDH-1 in the
antibacterial activity and identify other potential OxPhos targets.
The application of the ‘drug-like’ 2-iminobenzimidazole scaffold
for Gram-negative bacteria is a relatively novel approach. The
demonstrated activity for 4 and 5 in a battery of >20 multi-drug
resistant A. baumannii clinical isolates is a promising indicator.
With this in mind further SAR and target validation studies are
underway.
0
0
1
0
0
0
0
0
0
0
0
7
0
1
1
1
0
2
1
20
2
0
2
2
3
0
0
2
5
0
0
1
7
0
0
15
3
Compd 5
Ceftazidime
Imipenem
Colistin
15
0
0
32 to give 2-aminobenzimidazoles 33 followed by alkylation on
an internal nitrogen. Amino-alcohol 8 was prepared by reaction
of 2-aminomethyl benzimidazole 34 with epoxide 35 derived
upon reaction of epichlorohydrin and 2,4-dichlorophenol. The bis
substituted imidazole 2 was prepared from commercially available
36 albeit in poor overall yield (ꢀ5%). The method of preparation of 5
is given in the reference section.16
Compound 5 (highlighted in grey in Table 2) was evaluated in a
panel of 23 clinical isolates of A. baumannii including multi-drug
resistant (MDR) strains from various regions around the US, Europe
and Latin America. The compound showed impressive activity for
this early stage (Table 3). The MIC90 for 5 was 8
and the range was tight (4–16 g/mL). The compound performed
better against the drug-resistant strains than commercial antibiot-
ics ceftazidime and imipenem, and was generally only a few fold
less potent than standard of care colistin (MIC90 = 2 lg/mL), which
displays dose-limiting toxicities. Similar results were obtained
with 4 in a second screen of the same panel of clinical isolates;
MIC90 = 8 lg/mL and MIC range = 8–16 lg/mL (data not shown).
lg/mL (15.5 lM)
l
Importantly, susceptibility to either compound was comparable
between drug-sensitive and drug-resistant strains, indicating that
the resistance phenotypes were not resistant to the OxPhos
inhibitors.
Compounds 4 and 5 were also screened against drug-suscepti-
ble (ATCC 17978) and multi-drug resistant (BAA-1606) strains of
A. baumannii17,18 and there were no significant differences in sus-
ceptibility (MICs = 6.25 and 12.5 lg/ml, respectively). These results
coupled with those from the clinical isolate screens argue that cel-
lular responses, such as overexpression of efflux pumps, that con-
fer resistance against multiple classes of antibiotics, including
cephalosporins, carbapenems, fluoroquinolones and tetracyclines
are not effective against the OxPhos inhibitors.
The spectrum of activity of 5 was investigated by determining
its MIC against bacterial species other than A. baumannii. The
MIC values obtained with Staphylococcus aureus, methicillin-resis-
tant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneu-
moniae and Pseudomonas aeruginosa are listed in Table 4.
Comparable MIC values to that obtained for A. baumannii were
obtained with MRSA and one strain of K. pneumonia. In addition,
Acknowledgments
Financial support was provided by the National Institutes of
Health (R41 AI 108196-01). We also acknowledge the encourage-
ment and support of RMH Sciences, Ben Franklin Partners, BioAd-
vance, George Hobbs and Glenn Mattes.
References and notes
1. Hidron, A. I.; Edwards, J. R.; Patel, J.; Horan, T. C.; Sievert, D. M.; Pollock, D. A.;
Fridkin, S. K. Infect. Control Hosp. Epidemiol. 2008, 29, 996.
2. Perez, F.; Hujer, A. M.; Hujer, K. M.; Decker, B. K.; Rather, P. N.; Bonomo, R. A.
Antimicrob. Agents Chemother. 2007, 52, 3471.
3. Magill, S. S.; Edwards, J. R.; Bamberg, W.; Beldavs, Z. G.; Dumyati, G.; Kainer, M.
A., et al. New Eng. J. Med. 2014, 370, 1198.
Table 4
MIC determinations for 5 against a panel of bacterial species1
Species
MIC (lg/mL)
4. Dijkshoorn, L.; Nemec, A.; Seifert, H. Nat. Rev. Microbiol. 2007, 5, 939.
5. Wilson, S. J.; Knipe, C. J.; Zieger, M. J.; Gabehart, K. M.; Goodman, J. E.; Volk, H.
M.; Sood, R. J. Infect. Control 2004, 32, 342.
6. de Graef, M. R.; Alexeeva, S.; Snoep, J. L.; Teixeira de Mattos, M. J. J. Bacteriol.
1999, 181, 2351.
7. Gyan, S.; Shiohira, Y.; Sato, I.; Takeuchi, M.; Sato, T. J. Bacteriol. 2006, 188, 7062.
8. Yano, T.; Li, L. S.; Weinstein, E.; Teh, J. S.; Rubin, H. J. Biol. Chem. 2006, 281,
11456.
9. Weinstein, E. A.; Yano, T.; Li, L. S.; Avarbock, D.; Avarbock, A.; Helm, D.;
McColm, A. A.; Duncan, K.; Londsdale, J. T.; Rubin, H. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 4548.
10. Noda, S.; Takezawa, Y.; Mizutani, T.; Asakura, T.; Nishiumi, E.; Onoe, K.; Wada,
M.; Tomita, F.; Matsushita, K.; Yokota, A. J. Bacteriol. 2006, 188, 6869.
11. Green, J.; Paget, M. S. Nat. Rev. Microbiol. 2004, 2, 954.
12. Partridge, J. D.; Scott, C.; Tang, Y.; Poole, R. K.; Green, J. J. Biol. Chem. 2006, 281,
27806.
Staphylococcus aureus, ATCC 25923
MRSA2, USA 300
Escherichia coli, ATCC 25922
11
5
11
>22
5
22
Escherichia coli, ATCC 25922 (Serum)3
Klebsiella pneumoniae, ATCC 4352
Klebsiella pneumoniae, ATCC 13883
Klebsiella pneumoniae, ATCC 13883 (Serum)
Pseudomonas aeruginosa, ATCC 10145
>22
>22
1
MICs were determined by serial dilution of 5 in Mueller–Hinton broth at a
starting concentration of bacteria of 105–106 CFU/mL. Values are reported after 18 h
incubation at 37 °C.
2
Methicillin-resistant Staphylococcus aureus.
The medium contained 40% serum.
3

H. Rubin et al. / Bioorg. Med. Chem. Lett. 25 (2015) 378–383
383
13. Maeda, M. J. Bioenerg. Biomembr. 2008, 40, 117.
product, which was purified by silica chromatography using a gradient of
0–100% (90:9:1 DCM/methanol/ammonium hydroxide) to DCM to obtain N,N-
diethyl-2-(2-imino-2,3-dihydro-1H-benzo[d]imidazol-yl)ethanamine (240 mg,
1.03 mmol, 39% yield). A mixture of N,N-diethyl-2-(2-imino-2,3-dihydro-1H-
14. Andries, K.; Verhasseit, P.; Guillemont, J.; Gohlmann, H. W.; Neefs, J. M.;
Winkler, H.; Van Gestel, J.; Timmerman, P.; Zhu, M.; Lee, E.; Williams, P.; de
Chaffoy, D.; Hultric, E.; Hoffner, S.; Cambau, E.; Truffot-Pernot, C.; Lounis, N.;
Jarlier, V. Science 2005, 307, 223.
benzo[d]imidazol-yl)ethanamine
(50 mg,
0.215 mmol)
and
1-(3-
15. Cox, E.; Laessig, K. New Eng. J. Med. 2014, 371, 689.
bromopropoxy)-2,4-dichlorobenzene (73 mg, 0.258 mmol) in toluene (1 mL)
was heated at 110 °C for 15 h. Upon cooling, the precipitate was isolated to
afford the title compound as the HBr salt (73 mg, 0.142 mmol, 66%). HRMS
(ESI+): calculated for C22H29Cl2N4O m/z [M+H]+: 435.1713, observed 435.1624;
1H NMR (300 MHz, CD3OD) d = 7.50 (td, J = 1.7, 7.6 Hz, 2H), 7.41–7.20 (m, 4H),
6.95 (d, J = 8.8 Hz, 1H), 4.42 (t, J = 6.6 Hz, 2H), 4.27–4.19 (m, 2H), 4.13 (t,
J = 5.3 Hz, 2H), 2.90–2.82 (m, 2H), 2.60 (q, J = 7.3 Hz, 4H), 2.36 (td, J = 6.1,
11.6 Hz, 2H), 1.01–0.91 (m, 6H).
16. 2-(3-(3-(2,4-Dichlorophenoxy)propyl)-2-imino-2,3-dihydro-1H-benzo[d]imidazol-
1-yl)-N,N-diethylethanamine (5). A mixture of 2-aminobenzimidazole (350 mg,
2.63 mmol), 2-bromo-N,N-diethylethanamine (520 mg, 2.89 mmol), and
cesium carbonate (1880 mg, 5.77 mmol) in DMF (19 ml) was heated at 50 °C
for 3 h. Additional 2-bromo-N,N-diethylethanamine (260 mg, 1.45 mmol) and
cesium carbonate (940 mg, 2.88 mmol) was added and the reaction was heated
for an additional 14 h. The reaction was concentrated in vacuo and partitioned
between 3 N NaOH and ethyl acetate. The aqueous phase was extracted with 2
more portions of ethyl acetate. The combined ethyl acetate extracts were dried
over sodium sulfate, filtered, and concentrated in vacuo to afford the crude
17. Chopra, S.; Torres-Ortiz, M.; Hokama, L.; Madrid, P.; Tanga, M.; Mortelmans, K.,
et al. J. Antimicrobial. Chemother. 2010, 65, 2598.
18. Chopra, S.; Ramkissoon, K.; Anderson, D. C. J. Proteom. 2013, 84, 17.