Targeting Mobilization of Ferrous Iron in Pseudomonas aeruginosa Infection with an Iron(II)-Caged LpxC Inhibitor

Article abstract of DOI:10.1021/acsinfecdis.9b00057

Iron is essential to all life, and competition for this vital nutrient is central to host-pathogen interactions during infection. The opportunistic Gram-negative pathogen Pseudomonas aeruginosa utilizes a diverse array of iron-acquisition strategies, including those enabling import of extracellular ferrous iron. We hypothesize that soluble and redox-active ferrous iron can be employed to activate caged antibiotics at sites of infection in vivo. Here we describe new chemistry that expands the application of our laboratory's Fe²⁺-activated-prodrug chemistry to cage hydroxamic acids, a class of drugs that present manifold development challenges. We synthesize the caged form of a known LpxC inhibitor and show that it is efficacious in an acute P. aeruginosa mouse-lung infection model, despite showing little activity in cell-culture experiments. Overall, our results are consistent with the Fe²⁺-promoted uncaging of an antibacterial payload at sites of infection in an animal and lend support to recent reports indicating that extracellular pools of ferrous iron can be utilized by bacterial pathogens like P. aeruginosa during infection.

Full text of DOI:10.1021/acsinfecdis.9b00057

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Article
Targeting Mobilization of Ferrous Iron in Pseudomonas
aeruginosa Infection with an Iron(II)-Caged LpxC Inhibitor
Brian Blank, Poulami Talukder, Ryan Muir, Erin Green, Eric P Skaar, and Adam R. Renslo
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00057 • Publication Date (Web): 29 May 2019
Downloaded from http://pubs.acs.org on May 30, 2019
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Targeting Mobilization of Ferrous Iron in Pseudomonas aeruginosa
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Infection with an Iron(II)-Caged LpxC Inhibitor
†,‡
†,‡
†
§
§
Brian R. Blank, Poulami Talukder, Ryan K. Muir, Erin R. Green, Eric P. Skaar, and Adam R.
Renslo
†,*
th
†Department of Pharmaceutical Chemistry, University of California, San Francisco, 600 16 Street, San Francisco, CA
94158.
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Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, 1161 21st Avenue
South, Medical Center North, Nashville, TN 37232
E-mail: adam.renslo@ucsf.edu
acquisition strategies and pathways. The Gram-negative
bacterium Pseudomonas aeruginosa, for example, is able
to express transporters for the iron siderophores of
competing bacterial species in addition to those for its
Iron is essential to all life and competition for this vital
nutrient is central to host-pathogen interaction during
infection. The opportunistic Gram-negative pathogen
Pseudomonas aeruginosa utilizes a diverse array of iron
acquisition strategies, including those enabling import of
extracellular ferrous iron. We hypothesize that soluble
and redox-active ferrous iron can be employed to activate
caged antibiotics at site(s) of infection in vivo. Here we
describe new chemistry that expands the application of
3
cognate siderophores pyoveridine and pyochelin. The
pathogen also utilizes non-siderophore based iron uptake
via dedicated transporters of heme(II)iron and inorganic
2+
Fe present in the extracellular milieu, or pried from host
4
heme/iron proteins (Figure 1). Iron acquisition and
2
+
withholding similarly lies at the heart of host-pathogen
our laboratory’s Fe -activated prodrug chemistry to cage
hydroxamic acids, a class of drugs that present manifold
development challenges. We synthesize the caged form
of a known LpxC inhibitor and show that it is efficacious
in an acute P. aeruginosa mouse lung infection model,
despite showing little activity in cell culture experiments.
4-7
8-9
interactions,
nutritional immunity,
and the
mammalian response to bacterial infection. This response
includes the production of lactoferrin to sequester Fe
and the release of hepcidin, a negative regulator or iron
export, which leads to host withholding of iron (as Fe )
in macrophages and the liver.
3
+
3
+
2
+
Overall, our results are consistent with the Fe -promoted
uncaging of an antibacterial payload at sites of infection
in an animal, and lend support to recent reports indicating
that extracellular pools of ferrous iron can be utilized by
bacterial pathogens like P. aeruginosa during infection.
Keywords: host-pathogen interaction, iron acquisition,
nutrient acquisition, targeted prodrugs, caged antibiotics,
tissue targeting
Iron is the fourth most abundant element in the earth’s
crust and may have played a key role in the emergence of
1
life in the reducing atmosphere of the pre-biotic earth .
The ubiquity of iron-mediated chemistry in contemporary
biology is thus unsurprising. Iron cofactors redox cycle
Figure 1. Diverse iron uptake strategies of P. aeruginosa are
united by the mobilization of ferrous iron (yellow spheres)
in various cellular and extracellular compartments. In the
approach described herein, an antibacterial prodrug (in box,
green) is uncaged and activated by ferrous iron.
2
+
3+
between the ferrous (Fe ) and ferric (Fe ) states,
performing a wide array of useful redox chemistry and
thereby enabling cellular processes. Under aerobic
conditions at physiological pH, iron exists in the insoluble
ferric state, and so the transport and storage of iron in
biology requires various chaperones (e.g. transferrin,
lactoferrin, ferritin, siderophores, etc.) that bind,
1
0
In a recent report, Nolan and co-workers showed that
sequestration of the reduced Fe ion by the human
calprotectin protein induces an iron starvation response in
P. aeruginosa. This finding implies that the Fe ion is a
bioavailable source of iron during infection by certain
bacterial pathogens. Notably, antibacterial strategies that
2
+
3
+
solubilize, and render redox-inert the Fe ion. The much
2
+
2+
more soluble Fe ion redox cycles, promoting Fenton
2
chemistry that is toxic to cells.
Competition between bacterial populations for iron
resources has seen remarkable adaptions in iron
2
+
specifically target pools of the reduced Fe ion have been
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essentially unknown to date. Instead, prior efforts have
focused on the ferric ion (Fe ) and the various
siderophores that are required to solubilize and transport
iron in this oxidation state. Thus, a wide array of
siderophore-antibiotic conjugates or ‘sideromycins’ have
enhanced tolerability and efficacy in xenograft models.25
The same chemistry has likewise been applied to produce
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3+
26
27
TRX-PURO and ICL-1, new cellular and in vivo
2+
probes of Fe that exhibit remarkable metal ion and
oxidation-state specificity, and that are stable toward
other cellular reductants such as glutathione and cysteine.
In the context of a bacterial infection, TRX-antibiotic
conjugates were envisioned to exploit extracellular pools
been described, many with impressive activity and an
11-15
expanded antimicrobial spectrum.
not without challenges
This approach is
Preclinical
however.
2+
investigations of the siderophore-monobactam agents
MB-1 and SMC-3176, for example, have revealed an
adaptive resistance mechanism in which certain P.
of Fe and possibly also periplasmic and cytoplasmic
2+
Fe liberated during the unloading of iron from
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siderophores (Figure 1). The in vitro and in vivo activities
of such antibiotic conjugates might also provide insights
into reduced forms of iron in the microenvironment of
aeruginosa strains overexpress pyoveridine siderophore
16-17
as a means to prevent sideromycin uptake.
As well,
27
selection of SMC-3176-resistant strains identified various
resistance mutations to bacterial iron uptake genes.
infection. Indeed, a recent study of the TRX-based
2+
probe ICL-1 revealed a mobilization of Fe in mice
Nevertheless, the siderophore-cephalosporin cefiderocol
infected with the Gm-negative pathogen A. baumannii.
Many existing antibiotics possess the requisite amine
functionality required for conjugation the TRX moiety.
However, to expand the scope of this approach, we
describe here new chemistry that enables conjugation of
hydroxamate drug payloads (Figure 2). We use this
chemistry to prepare the iron(II)-caged form of a known
LpxC inhibitor and study its antimicrobial effects across
a large panel of Gm-negative pathogens and in a P.
aeruginosa mouse acute lung infection model. Notably,
we find the iron(II)-caged agent to be efficacious in vivo,
despite having little activity in the context of an in vitro
MIC experiment. Taken together, our findings support the
idea that ferrous iron is a bioavailable iron source during
infection, and suggest this redox-active species can be
exploited to activate iron(II)-caged antibiotics at site(s) of
infection.
18
(
S-649266) is currently progressing through human
clinical trials and is apparently less impacted by these
resistance mechanisms than the earlier siderophore-
monobactams.
Whereas exploiting uptake of Fe3+ suggests a siderophore-
2
+
based approach, we reasoned that targeting pools of Fe
could instead leverage a reactivity-based approachinvolving
Fenton-type chemistry of organic peroxides. In fact, safe and
well-tolerated antimalarials including the artemisinins and
19
20
synthetic 1,2,4-trioxolanes arterolane and artefenomel
provide clinical precedent for iron(II)-dependent
pharmacology based on Fenton chemistry. In recent years,
2
1-22
our lab has described arterolane-inspired scaffolds
(denoted TRX) that re-imagine the 1,2,4-trioxolane ring as a
2
+
caging moiety and molecular sensor of the Fe ion, and that
incorporate a traceless linker to drug payloads (Figure 2).
Previous work (refs. 21-27)
O
O
RESULTS and DISCUSSION
O
O
HN DRUG
The
emergence
of
carbapenem-resistant
O
Fe2+
Enterobacteriaceae (CRE) and multidrug resistant P.
aeruginosa highlights the need for new drug targets and
H N DRUG
2
2
8-30
•
•
amine or aniline payloads
mouse malaria and cancer models
technologies to address these pathogens.
Among
several promising drug targets being explored is UDP-3-
O-(R-3-hydroxymyristoyl)-N-acetylglucosamine
This work
deacetylase (LpxC, a metallo-deacetylase that performs
the first committed step in Lipid A biosynthesis. Lipid A
in turn serves as the membrane-anchoring component of
lipopolysaccharide (LPS), an essential component of the
O NH
O
O
DRUG
Fe2+
O
O
HO NH
O
DRUG
3
1
outer membrane of Gm-negative bacteria. Several in
vivo-active hydroxamate-based LpxC inhibitors (LpxCi)
•
•
hydroxamate payloads
mouse P. aeruginosa infection model
3
2
have been described in the past decade, including PF-
3
3
Figure 2. Representative structure of iron(II)-sensing
trioxolane prodrugs described by our group previously
5081090 and ACHN-975, which entered human clinical
3
2
safety evaluation (Figure 3). Frequencies of resistance
to LpxC inhibitors are generally very low, with a value of
(top) and the new caged hydroxamates described herein.
-
10
<
5.0 x 10
reported for PF-5081090 against P.
However, the clinical challenges of
3
3
Previous in vivo studies of TRX conjugates have
aeruginosa.
demonstrated enhanced efficacy and tolerability in
malaria infection models for iron(II)-caged forms of a
advancing hydroxamate-based agents, including matrix
3
4
35
metalloproteinase and histone deacetylases inhibitors,
are well documented. These include inhibition of off-
target metalloenzymes, metabolic and proteolytic
instability of the hydroxamate function, and mutagenic
2
3
preclinical parasite cysteine protease inhibitor and the
2
4
antimalarial mefloquine. Similarly, TRX-caged forms
of a duocarmycin-class chemotherapeutic showed
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2
BCl •SMe proceeded in 89% yield to afford alcohol 3.
potential that likely results from Lossen rearrangement of
3
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O-activated hydroxamates to form DNA-reactive
isocyanates.
Unsurprisingly, oxidation of 3 to afford 4 was complicated
by the propensity of 4 to undergo beta-elimination.
Ultimately, we found that this conversion could be
accomplished in good to high yields with the Dess-Martin
reagent, or alternatively by Swern oxidation under carefully
controlled conditions as detailed in the experimental section.
Griesbaum co-ozonolysis reaction of 4 with adamantan-2-
one O-methyl oxime afforded 1,2,4-trioxolane 5 in 92%
35
OH
F
OMe
O
O
H
HO
N
HO
N
N
N
H
H
O
SO2Me
O
H N
2
36
ACHN-975
PF-5081090
yield based on 4. We have reported that this process
Figure 3. Clinical and pre-clinical stage LpxC inhibitors
ACHN-975 and PF-5081090, respectively, based on
hydroxamate metal-binding moieties (shown in red).
generally proceeds with high diastereoselectivity favoring
the trans isomer, and in the present case furnished 5 in an
excellent 13:1 diastereomeric ratio (d.r.). Deprotection of 5
with hydrazine proceeded smoothly to afford the desired
aminooxy TRX intermediate (±)-6. To further enable study
of TRX conjugates derived from (±)-6, we also developed a
route to the corresponding aminomethyl intermediate (±)-7
0
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Caging a hydroxamate drug at the oxygen atom of the
hydroxamate with the TRX moiety offers a possible
means to address these liabilities. Blocking this crucial
site would serve several purposes, namely 1) temporarily
(see Supporting Information). This material can be
2
+
blocking on-target activity prior to activation by Fe , 2)
sterically occluding interaction with host metalloenzyme
off-targets while in the caged form, and 3) preventing or
mitigating Lossen rearrangement, which proceeds via
oxygen atom activation (e.g., acylation). Here we aimed
to prepare a TRX conjugate of the known LpxC inhibitor
PF-5081090. A retro-synthetic analysis of putative TRX-
LpxCi conjugates suggested a convergent disconnection
at C–N of the hydroxamate (Figure 4). Following this
disconnection required access to the LpxCi as a
carboxylate and an aminooxy-substituted TRX
intermediate. The former is typically the penultimate
intermediate in the synthesis of hydroxamate-based
agents, while the latter could, in principle, be prepared by
Griesbaum co-ozonolysis reaction of a phthalimide
protected 3-(aminooxy)cyclohexan-1-one (Figure 4).
employed to prepare peroxidic control conjugates in which
the drug payload is inactivated (i.e., amide rather than
hydroxamate form).
Next, intermediates (±)-6 and (±)-7 were coupled to the
3
7
known carboxylic acid (±)-8 (Scheme 2). This provided
the final drug conjugate 9 and the amide-linked control
1
0 as diastereomeric mixtures that were evaluated as such
in all subsequent bioassays (Scheme 2). We confirmed
that 9 reacts cleanly with ferrous ammonium sulfate in
PBS pH 7.4 and DMSO to liberate the ketone
intermediate 9* and then (±)-11 as desired (Scheme 2 and
Supporting Information). Control 10 possesses the same
peroxide bond as 9 but cannot liberate an active
hydroxamate, thus controlling for any peroxide-based
contribution to the antimicrobial activity of 9. In fact, 10
exhibited no measurable MIC in any of the strains tested,
confirming that the TRX moiety in 9 serves as iron(II)-
sensor and caging group, without significant antibacterial
action of its own.
O
NH
O
O
O
DRUG
O
C-N bond formation
5000
4500
4000
11
9
O
NH2
+
O
HO
O
O
DRUG
3
3
2
500
000
500
O
Griesbaum
O
co-ozonolysis
2000
1500
1000
O
N
N
OMe
+
O
O
500
Figure 4. Retrosynthetic analysis of trioxolane-hydroxamate
conjugates in which the hydroxamate oxygen atom serves as
the site of conjugation to the TRX moiety.
0
0
100
200
300
400
500
Time (min)
Figure 5. Plasma exposure profile of prodrug 9 (red) and
systemically released 11 (blue) following a single IP dose of
After considerable experimentation, an efficient synthetic
approach amenable to multi-gram scales was identified
9
(16 mg/kg) to healthy female NSG mice (n = 9).
(Scheme 1). Thus, mono-benzylation of cyclohexane-1,3-
diol afforded the desired ether 1, which was subjected to
Mitsunobu reaction with N-hydroxyphthalimide to afford 2.
Selective removal of the benzyl protecting group with
To evaluate its in vivo stability and pharmacokinetic
profile, 9 was administered by IP injection to female NSG
mice and the resulting plasma samples analyzed for both
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intact 9 and free LpxCi payload 11 that may have been
released from 9 systemically by Fe dependent or
independent processes. Compound 9 exhibited a
Gram-negative pathogens and control strains. These
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9
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2+
included strains expressing common resistance factors
(mainly -lactamases) and variable efflux pump expression
phenotypes (Table 1). MICs were determined according to
relatively rapid elimination profile (T ~ 0.7 hr) similar
1
/2
3
8-39
37
CLSI standards
in both in cation adjusted Mueller
to that reported for PF-5081090 (Figure 5). We also
observed some systemic release of payload 11 from 9,
amounting to ca. 19% of the dose by AUC, or roughly 2-
Hinton II broth (CAMHB) and also in ID-CAMHB media
that had been depleted of iron by treatment with Chelex.
Iron-depleted media is typically used to study siderophore-
antibiotic conjugates in MIC experiments, as these
conditions are expected to activate the bacterial iron uptake
pathways that are exploited by such agents. Here, we
employed iron-depleted media both to evaluate the possible
3
times the extent of release observed for TRX conjugates
25
based on carbamate linker chemistry . Although such
systemic activation is undesirable, compound
9
0
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0
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0
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8
9
0
nevertheless remained ~80% intact in the experiment, and
exhibited sufficient in vivo exposure for evaluation in
animal infection models.
First however, we performed in vitro microbiological
evaluation of compounds 9, 10 and 11 across a panel of
3
+
effect of activating Fe uptake pathways on prodrug
activation, and also to control for spurious extracellular
activation of 9 by iron in normal media.
Scheme 1. Stereocontrolled synthesis of key trioxolane intermediate 6 bearing a 3’-aminooxy function.
O
HO
N
BnO
OH
BnO
O
HO
O
HO
OH
BnBr, NaH
DMF
NPhth
BCl3•SMe2
NPhth
O
CH2Cl2
rt, 64 h
87%
PPh , DIAD
3
0˚C-rt, 64 h
THF, 0˚C-rt, 20 h
1
1.6 g
1
2
3
49%
62%
100 mmol
N
OMe
O
NH2
O
NPhth
O
O
O
O
2.5 equiv.
O3
H2NNH2
Dess-Martin
CH2Cl2
O
O
NPhth
O
O
CHCl /EtOH
rt, 20 h
3
0
˚C, 5 h
2%
CCl4/CH2Cl2 (2:1)
0 ˚C, 2 h
6
4
(±)-5
3:1 d.r
78%
(±)-6
92%
1
Scheme 2 Synthesis of hydroxamate- and amide-linked TRX conjugates 9 and 10 from known LpxC inhibitor 8 (top
left). Compound 10 is chemically unable to release an LpxCi and serves as a control for any peroxide-derived
antibacterial activity in 9. Reaction of 9 with ferrous ammonium sulfate in PBS/DMSO (1:1) cleanly produces the
intermediate 9* and then free 11 as revealed by LC/MS (scheme below left and UV absorbance spectra at right).
Me
Me
O
O
X
NH2
+
O
O
S
O
HATU, DIEA
rt, 30 min
H
S
O
O
O
X
N
O
HO
N
O
O
N
O
24-47%
O
O
F
O
F
O
9 (X = O)
(±)-6 (X = O) or
(±)-8 (ref 37)
10 (X = CH2)
(±)-7 (X = CH2)
Me
Me
O
O
O
O
S
O
S
O
H
H
O
N
O
N
N
HO
N
Fe2+
9
O
O
F
O
O
F
O
(
±)-11
9*
2
+
Accordingly, compounds 9-11 and antibiotic controls
were tested under both conditions against twenty strains
representing five different pathogens. We observed no
measurable MIC for control 10 under any of the conditions
the compounds and access of 9 to intracellular Fe , and 2)
the efficiency of iron(II)-promoted uncaging of 9 assuming
2
+
it can access intracellular Fe . In fact, we observed the
MICs of 9 to be ~8-fold higher than for 11 in Escherichia
coli strains and ~16-fold higher in Klebsiella pneumoniae,
Enterobacter cloacae, and P. aeruginosa strains. Both
compounds were subject to efflux based on comparison of
MICs for pump knockout and parental strains (Table 1).
The MIC data thus suggest that 9 has limited access to
(
MICs > 64 mg/mL), indicating that the TRX moiety
present in 10 and 9 lacks antimicrobial activity of its own
Table 1). The MICs obtained for 9 can therefore be
(
inferred to derive primarily or solely from uncaging of the
active LpxC inhibitor 11. Differences in the MICs of 9 and
2
+
1
1 are thus interpreted as arising primarily from a
intracellular Fe in an MIC experiment, presumably due
to poor membrane permeability and/or drug efflux.
combination of two factors, 1) the relative permeability of
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We found that the MICs for both 9 and 11 were
Infection of the cystic fibrosis (CF) lung by bacterial
essentially unchanged whether performed in standard
CAMHB media or in ID-CAMHB media (see
Supporting Information for full data set). The lone
exception was a single E. coli strain (MMX 119) in
which the MICs of 9 and 11 were 16-fold and 4-fold
higher, respectively, in ID-CAMHB media. Since this
MIC shift was observed for both iron(II)-sensitive (9)
and insensitive (11) compounds, the shift in the MMX
pathogens, including P. aeruginosa, is associated with
dramatic changes to iron homeostasis. Ferrous iron
levels in the sputum of CF patients can be correlated
with severity of disease, and reach aberrant (mid-M)
concentrations in advanced disease, likely due to an
acidic and hypoxic microenvironment in the infected
lung. The likely role of extracellular Fe in P.
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9
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3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
40
41
2+
aeruginosa infection is further supported by the
2+
1
19 strain appears to be unrelated to iron uptake or
existence in the pathogen of transporters for the Fe ion
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mobilization. The lack of MIC shift for 9 in ID media
also implies that the compound is stable in the standard
media and does not undergo iron(II)-promoted
activation outside the cell in MIC experiments. This is
consistent with the good stability of TRX conjugates in
(FeoA/B system) and by utilization of the host protein
2+
calprotectin to sequester the Fe ion in response to
1
0
infection. Accordingly, the CF lung appears to possess
a substantial reservoir of extracellular ferrous iron that
promotes bacterial colonization and virulence, but
might also be exploited for the tissue-selective delivery
of antibiotics. Since a tractable and robust mouse model
of CF is unavailable, we compared the efficacy of
conjugate 9 to its parent LpxCi payload (11) in an acute
lung infection model using the PA14 strain of P.
26
mammalian cell culture media and the expectation that
extracellular iron in an aerobic MIC experiment will
exist in the TRX-inert ferric state. We next sought to test
the efficacy of 9 in an in vivo infection model where
host-pathogen competition for iron resources was
2+
27
42
predicted to mobilize Fe in infected tissues.
aeruginosa.
Table 1. MICs of compounds 9-11 and antibiotic controls against diverse Gm-negative pathogens in CAMBH media.a
MIC (g/mL) of Test Compounds and Controls
Test Organisms
MMX./ATCC No.
684/13352
Phenotype
Ceftazidime/
Avibactam
0.5/4
1
1
10
9
Imipenem
5
TEM-10
1
>64
8
0.25
6
839/BAA-2326
5980
CTX-M-15
NDM-1
0.5
>64
>64
4
2
0.12
8
0.12/4
>64/4
E. coli
0.25
1
1
19
21
Parent (TolC
Knockout)
TolC Knockout
0.25
>64
2
0.12
0.12/4
0.12/4
<0.06
0.5
>64
>64
0.12
16
0.25
0.12
102/25922
37/700603
QC
1/4
(0.06-0.25)
(1/4-4/4)
5
SHV-1
4
>64
>64
0.25
1/4
K. pneumoniae
4
4
5
6
7
5
683
692
979
308
941
981
KPC-2
KPC-3
NDM-1
MEM-R
AmpC
4
2
8
4
0.5
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
16
>64
8
>64
64
16
0.25
>64
0.5/4
2/4
>64/4
4/4
1/4
>64/4
E. cloacae
NDM-1
Parent (mexAB/oprM
KO)
3
3
476
477
1
>64
>64
16
1
1
2/4
P. aeruginosa
mexAB/oprM
Knockout
VIM-2
<0.06
0.5
0.5/4
4
4
698
654
2
2
1.5
>64
>64
–
64
>64
30
>64
>64
–
32/4
>64/4
–
IMP-7
PA14
1
1-4)
2/4
(0.5/4-4/4)
1
03/27853
QC
2
>64
>64
(
A. baumannii
1630/19606
651
Wild type
OXA-27
>64
>64
>64
>64
>64
>64
0.25
64
64/4
64/4
4
aCLSI QC ranges shown below MIC where applicable; see Supporting Information for MICs in CAMHB-ID media. All
MIC determinations (except PA14) were performed at Micromyx, LLC (Kalamazoo, MI).
ACS Paragon Plus Environment

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Page 6 of 12
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Bacterial colony forming units (CFU) in lung, heart and spleen in mice infected with PA14 strain P. aeruginosa 24
hours after being treated with dosing vehicle (Veh), a single IP dose of 11 (10 mg/kg), an equimolar IP dose of 9 (16 mg/kg),
or two- or four-fold higher doses of 9. * p < 0.05, ** p < 0.01. Error bars represent standard error of the mean.
First, we confirmed that the LpxCi payload 11
delivered from 9 was active against PA14 P.
aeruginosa. Indeed, compound 11 exhibited activity
(PA14 MIC = 1.5 g/mL) at concentrations that seemed
likely to be achieved in vivo, assuming effective
uncaging of 9 (to liberate 11) at the infection site. The
MIC of 9 against this strain was weaker, as expected
The superior efficacy of 9 compared to 11 in this model
is consistent with the selective uncaging of 9 at sites of
2
+
infection, most likely by extracellular Fe in the
microenvironment. This inference is based on the
observation that intact 9 exhibited little antimicrobial
effect in the MIC experiment where only intracellular or
2
+
periplasmic Fe is available for activation. The possible
role of the endoperoxide function in the superior in vivo
efficacy of 9 is ruled out by the complete lack of activity
observed for control compound 10. Although we cannot
strictly rule out the contributions of alternate
mechanisms of in vivo activation, extensive
characterization of diverse TRX conjugates by our
(MIC = 30 g/mL), and consistent with the MIC shifts
observed across the larger panel of isolates (Table 1).
Next, wildtype female BALB/c mice were inoculated
5
intranasally with 1x10 CFU/mL of PA14 strain P.
42
aeruginosa as described previously. At two and six
hours
post-inoculation,
mice
were
injected
2
6
27
laboratory
and others
suggests that, among
intraperitoneally with vehicle, active LpxCi 11 (10
mg/kg), or the iron(II)-caged LpxCi 9 at an equimolar
dose (16 mg/kg), or at a two-fold (32 mg/kg), or four-
fold (64 mg/kg) higher dose. Bacterial colony forming
units (CFU) were assessed at 24 hrs post-inoculation in
lung, liver, and spleen (Figure 6). Bacterial load was
highest in the lung and so this tissue provided the data
most useful for comparison of treatment arms.
Surprisingly, administration of 11 directly had only a
modest effect on bacterial load that was not statistically
different from vehicle treated controls. By contrast, and
despite its much weaker MICs, the iron(II)-caged form
2
+
biologically relevant reducing agents, Fe is by far the
most likely to be involved in their activation, and
particularly in the mode of activation that leads to
release of free drug payload.
Notably, access of 9 to intracellular Fe in the MIC
experiment was quite limited, leading to inferior MICs
compared to 11 (Table 1). This suggests that host-
pathogen interaction in the live animal mobilizes Fe in
infected tissues to an extent that is not mimicked in MIC
experiments. In part this difference may derive from
liberation of reduced iron from host proteins (carriers
and enzymes) during active infection, producing
reservoirs of extracellular Fe that can uncage TRX
prodrugs and concentrate antibiotic payload in the
infection microenvironment. This proposed mechanism
of action for 9 would be consistent with the in vivo
2
+
2
+
9
significantly reduced bacterial load over vehicle
2
+
control at both the equimolar dose (16 mg/kg) and at the
two higher dosing levels. Compound 9 was well
tolerated even at the highest, 64 mg/kg dose. Bacterial
CFU counts were significantly lower in liver, below the
limit of detection in some animals. Nevertheless,
compound 9 at the 64 mg/kg dose exhibited a significant
reduction in CFU over vehicle controls in liver.
Bacterial loads in spleen were below the limit of
detection in the majority of the animals and thus
provided no interpretable data.
27
2+
behavior of ICL-1 , wherein Fe -mediated release of
aminoluciferin payload was correlated with A. baumanii
infection in several tissues.
Here we have presented a prototypical iron(II)-caged
LpxC inhibitor and in vivo data suggesting its activation
at sites of infection in an animal. The use of such agents
could enable concentration of antibiotic at sites of
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diastereomer), 34.1 (minor diastereomer), 34.1, 30.3,
infection while mitigating inhibition of metalloenzyme
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
off-targets, though the latter feature remains to be tested
30.1 (minor diastereomer), 19.0, 18.3 (minor
diastereomer); MS (ESI) calculated for C H O [M +
H] m/z 207.14, found 206.92.
explicitly. The in vivo results presented here are
1
3
19
2
10, 27, 41
+
consistent with emerging evidence
that
2+
3+
competition for Fe , like that for Fe , is an important
aspect of host-pathogen interaction during infection.
We propose here that this reactive iron pool can be
exploited for tissue-selective uncaging of antibacterial
agents in vivo, and that this approach may enhance
efficacy and tolerability. Further studies, including
optimization of the TRX moiety for antibacterial
applications and use of this new approach to deliver
other antibacterial payloads are ongoing in our
laboratories and will be reported in due course.
3-(benzyloxy)cyclohexyl phthalimidyl ether (2). A
round bottom flask equipped with an argon inlet
adapter, stirbar, and rubber septum was charged with a
solution of alcohol 1 (6.188 g, 30 mmol, 1.0 equiv) in
THF (300 mL). To the solution was added N-
hydroxyphthalimide (5.550 g, 33 mmol, 1.10 equiv) and
triphenylphosphine (8.743 g, 33 mmol, 1.10 equiv).
After cooling the solution to 0˚C, diisopropyl
azodicarboxylate (6.86 mL, 34.5 mmol, 1.15 equiv) was
added dropwise and the mixture maintained at 0˚C for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9
0 min, after which it was allowed to warm to room
METHODS
temperature and stirred for 19 hours. The reaction
mixture was then concentrated under reduced pressure
and diluted with 200 mL of EtOAc and 100 mL of satd.
37
Synthesis. Intermediate 8 was prepared as described.
General synthetic methods and specific procedures for
the synthesis of intermediate 7 and control 10 are
provided as Supporting Information.
aq. NaHCO . The layers were separated and the organic
3
phase was washed with four 100 mL portions of satd.
3
-(benzyloxy)cyclohexan-1-ol (1). To a round bottom
aq. NaHCO . The aqueous layer was then extracted with
3
flask equipped with an argon inlet adapter, stirbar, and
rubber septum was charged with a solution of 1,3-
cyclohexanediol (11.62 g, 100 mmol, 1.0 equiv.) in
DMF (170 mL). The solution was cooled at 0 ˚C to
which was slowly added sodium hydride (60%
dispersion in mineral oil, 4.2 g, 105 mmol, 1.05 equiv).
The suspension was allowed to warm to room
temperature and stirred for 1 hour, and then cooled to 0
a 100 mL portion of EtOAc and the combined organic
layers washed with 100 mL satd. aq. NaCl, dried over
Na SO , filtered, and concentrated to yield a yellow oil.
2
4
The crude residue was purified by flash column
chromatography (0–30% EtOAc/Hexanes gradient
elution) to afford 2 (6.618 g, 62%) as a clear colorless
1
oil that solidified upon standing at 4˚C. H NMR (400
MHz, CDCl )  7.85–7.78 (m, 2H), 7.76–7.70 (m, 2H),
3
˚C. Benzyl bromide (12.74 mL, 105 mmol, 1.05 equiv)
7.36–7.17 (m, 5H), 4.61–4.44 (m, 3H), 4.02–3.88 (m,
1
3
was then added and the reaction mixture stirred at 0 ˚C
for 50 minutes, after which the cooling bath was
removed and the mixture stirred for 63 hours at ambient
temperature. The reaction mixture was then diluted with
1H), 2.08–1.94 (m, 2H), 1.90–1.56 (m, 6H); C NMR
(
100 MHz, CDCl )  164.2, 138.9, 138.6 (minor
3
diastereomer), 134.4 (minor diastereomer), 134.3,
28.9, 128.9 (minor diastereomer), 128.3 (minor
diastereomer), 128.2, 127.5 (minor diastereomer),
27.5, 127.3, 123.5 (minor diastereomer), 123.4, 84.2
minor diastereomer), 83.6, 75.5 (minor diastereomer),
3.7, 70.2, 70.1 (minor diastereomer), 37.1 (minor
diastereomer), 35.3, 31.3 (minor diastereomer), 30.4
minor diastereomer), 30.3, 29.7, 20.2 (minor
1
1
50 mL of satd. aq. NaCl, 200 mL of 10% HCl and 100
mL of EtOAc. The aqueous layer was separated and
extracted with two 100 mL portions of EtOAc. Solid
NaCl was then added to the aqueous layer and further
extracted with four 100 mL portions of EtOAc. The
combined organic layers were washed with 100 mL of
1
(
7
(
satd. aq. NaCl, dried over Na SO , filtered, and
2
4
diastereomer), 18.9; MS (ESI) calculated for
+
concentrated under reduced pressure to afford a yellow
oil. The crude product was purified by column
chromatography (0–50% EtOAc/hexanes gradient
elution) to yield 1 (10.133 g, 49%) as a yellow oil. Based
C H NO [M + H] m/z 352.15, found 352.07.
2
1
22
4
3
-(phthalimidyloxy)cyclohexan-1-ol (3). A round
bottom flask equipped with an argon inlet adapter,
stirbar, and rubber septum was charged with a solution
of benzyl ether 2 (2.108 g, 6 mmol, 1.0 equiv) in CH Cl
1
on H NMR analysis, the product exists as a 6:1 mixture
2
2
1
of diastereomers. H NMR (400 MHz, CDCl )  7.35–
(100 mL). To the solution was added boron trichloride
dimethyl sulfide solution (2.0 M in CH Cl , 32 mL, 64
3
7
4
1
.27 (m, 4H), 7.27–7.22 (m, 1H), 4.58–4.46 (m, 2H),
.09–4.00 (m, 1H, minor diastereomer), 3.81–3.74 (m,
H, minor diastereomer), 3.73–3.64 (m, 1H), 3.55–3.45
2
2
mmol, 10.7 equiv) dropwise via syringe. The mixture
was stirred at room temperature for 63 hours, at which
point the reaction mixture was added in 10 mL portions
to an Erlenmeyer flask containing a stirring mixture of
CH Cl (100 mL) and satd. aq. NaHCO (100 mL). The
(m, 1H), 2.14–2.04 (m, 1H), 1.88–1.68 (m, 3H), 1.68–
1
.54 (m, 1H), 1.53–1.33 (m, 2H), 1.31–1.18 (m, 1H);
1
3
C NMR (100 MHz, CDCl )  138.9 (minor
3
2
2
3
diastereomer),
138.5,
128.3,
128.2
(minor
organic phase was collected and washed with two 50
diastereomer), 127.4, 127.4 (minor diastereomer),
27.3, 127.2 (minor diastereomer), 75.3, 73.9 (minor
diastereomer), 70.1, 69.8 (minor diastereomer), 68.2,
6.8 (minor diastereomer), 39.4, 39.1 (minor
mL portions of satd. aq. NaHCO . The combined
3
1
aqueous layers were extracted thrice with 100 mL
portions of CH Cl and the combined organic phases
2
2
6
washed with 100 mL satd. aq. NaCl, dried over Na SO ,
2
4
7
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Page 8 of 12
filtered, and concentrated under reduced pressure to layers were then washed with saturated aqueous
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
afford a brown oil. The crude residue was purified by
flash column chromatography (0–75% EtOAc/Hexanes
NaHCO (50 mL x 3), and brine (50 mL), dried over
3
anhydrous Na SO , filtered, and concentrated under
2
4
gradient elution) to yield 3 (1.361 g, 87%) as a light
reduced pressure to afford 4 (1.23 g, 94% crude yield)
as a yellow-orange solid.
1
orange solid. H NMR (400 MHz, CDCl )  7.87–7.80
3
(m, 2H), 7.79–7.72 (m, 2H), 4.64–4.55 (m, 1H), 4.35–
3''-phthalimidyloxydispiro[adamantane-2,3'-
[1,2,4]trioxolane-5',1''-cyclohexane] (5). A round
bottom flask equipped with a stirbar was charged with a
solution of ketone 4 (0.438 g, 1.689 mmol, 1.0 equiv) in
carbon tetrachloride (20 mL) and dichloromethane (10
mL). To this solution was added adamantan-2-one O-
methyl oxime (0.606 g, 3.379 mmol, 2.0 equiv) and the
4
1
.26 (m, 1H), 2.15–2.05 (m, 1H), 1.94–1.76 (m, 5H),
.76–1.54 (m, 2H), 1.53–1.42 (m, 1H); C NMR (100
13
MHz, CDCl )  164.2, 134.4, 128.9, 123.5, 83.5, 66.6,
3
3
7.9, 33.6, 29.4, 18.8; MS (ESI) calculated for
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
C H NO [M + H] m/z 262.11, found 261.98.
1
4
16
4
3
-(phthalimidyloxy)cyclohexan-1-one (4). A round
solution cooled to 0 ˚C and sparged with O for 10
bottom flask equipped with an argon inlet adapter,
stirbar, and rubber septum was charged a solution of
alcohol 3 (0.638 g, 2.4 mmol, 1.0 equiv.) in CH Cl (25
2
minutes. The reaction mixture was maintained at 0˚C
while ozone was bubbled (2 L/min, 40% power) through
the solution. After stirring for 50 mins, additional oxime
2
2
mL). The solution was cooled at 0˚C and then Dess-
Martin periodinane (1.363 g, 3.1 mmol, 1.2 equiv.) was
added in one portion. The solution was allowed to come
to room temperature and stirred for 5 hours. The
reaction mixture was then treated with 50 mL of CH Cl
(
(
0.151 g, 0.842 mmol, 0.5 equiv), carbon tetrachloride
10 mL) and dichloromethane (10 mL) were added in a
single portion to the reaction. Ozone was bubbled
through the reaction for another 50 mins, at which point
the reaction was judged complete by LCMS and TLC
2
2
and 50 mL of satd. aq. NaHCO . The organic layer was
3
analysis. The solution was sparged with O for 10
collected and washed with two 50 mL portions of satd.
2
minutes to remove dissolved ozone, and then sparged
with argon gas for 10 minutes to remove any dissolved
oxygen. The solution was concentrated under reduced
pressure to provide a viscous oil. The crude residue was
purified by flash column chromatography (0–20%
EtOAc/hexanes gradient elution) to afford 5 (0.665 g,
aq. NaHCO , one 50 mL portion of satd. aq. NaCl, dried
3
over anhydrous Na SO , filtered, and concentrated
2
4
under reduced pressure to deliver a colorless oil. The
crude residue was purified by column chromatography
(
0-40% EtOAc/hexanes) to afford the product 4 (0.390
1
g, 62%) as a colorless oil H NMR (400 MHz, DMSO)
9
2%) as a white foamy solid. NMR analysis suggested

4
7.86 (s, 4H), 4.70–4.58 (m, 1H), 2.80 (dd, J = 14.9,
.6 Hz, 1H), 2.62 (dd, J = 14.8, 6.8 Hz, 1 H), 2.38–2.25
1
a 13:1 d.r. in favor of the trans diastereomer. H NMR
(
400 MHz, CDCl )  7.87–7.80 (m, 2H), 7.79–7.72 (m,
(
1
1
m, 2 H), 2.19–2.00 (m, 2H), 2.00–1.90 (m, 1H), 1.73–
3
1
3
2H), 4.49–4.40 (m, 1 H, minor diastereomer), 4.31–4.18
m, 1H), 2.50–2.41 (m, 1H), 2.41–2.35 (m, 1H, minor
diastereomer), 2.24–2.15 (m, 1H), 2.03–1.92 (m, 5H),
.60 (m, 1H); C NMR (100 MHz, DMSO)  207.5,
(
63.7, 134.7, 128.6, 123.2, 84.6, 45.5, 40.1, 27.7, 19.5;
+
MS (ESI) calculated for C H NO [M + H] m/z
1
4
14
4
1
2
1
3
.92–1.72 (m, 7H), 1.72–1.61 (m, 6H), 1.52–1.36 (m,
2
60.09, found 259.90.
Alternate Conditions (Swern): To a solution of DMSO
1.1 mL, 15.5 mmol, 3.07 equiv) in CH Cl (25 mL),
1
3
H); C NMR (100 MHz, CDCl )  164.1, 134.5,
3
28.9, 123.5, 111.7, 108.8, 83.9, 39.5, 36.7, 36.2, 36.2,
(
2
2
4.8, 34.7, 34.7, 33.6, 29.6, 26.8, 26.4, 19.6; MS (ESI)
cooled to –78 ˚C, was added oxalyl chloride (0.641 mL,
.58 mmol, 1.5 equiv) dropwise via syringe. This
+
calculated for C H NO Na [M + Na] m/z 448.17,
7
24 27
6
found 448.04.
O-(dispiro[adamantane-2,3'-[1,2,4]trioxolane-5',1''-
cyclohexan]-3''-yl)hydroxylamine (6). round
solution was allowed to stir at –78 ˚C for 30 minutes,
after which a solution of alcohol 3 (1.32 g, 5.05 mmol,
A
1
.0 equiv) pre-dissolved in CH Cl (15 mL) was added
2 2
bottom flask equipped with an argon inlet adapter,
stirbar, and rubber septum was charged with a solution
of trioxolane 5 (0.640 g, 1.504 mmol, 1.0 equiv) in
chloroform (18 mL) and ethanol (7 mL). To the solution
was added hydrazine monohydrate (0.285 mL, 3.761
mmol, 2.5 equiv) and the mixture allowed to stir at room
temperature for 20 hours. At this point, the solution was
filtered through a glass fritted funnel to remove
precipitated material and the filter cake washed well
dropwise to the mixture over a period of 5 minutes,
rinsing the vessel with two additional 5 mL portions of
CH Cl . Triethylamine (5 mL, 35.9 mmol, 7.1 equiv;
2
2
use of excess important) was then added dropwise to the
solution over 2 minutes, and the resulting mixture was
allowed to stir at –78 ˚C for 90 min. At this time,
saturated aqueous citric acid (40 mL) was added directly
to the reaction mixture while at –78 ˚C (important). To
the frozen mixture was added additional CH Cl (40
2
2
with CH Cl . The resulting filtrate was dried over
mL) and 20 mL of saturated aqueous citric acid, after
which the frozen mixture was allowed to warm to ~4 ˚C
over ca. 20 minutes. Once the mixture had thawed, the
layers were separated and the organic layer was washed
with additional saturated aqueous citric acid (20 mL).
The combined aqueous layers were then extracted thrice
2
2
anhydrous Na SO , filtered, and concentrated under
2
4
reduced pressure to afford a white solid. The crude
residue was then purified by flash column
chromatography (0–20% EtOAc/hexanes gradient
elution) to yield 6 (0.346 g, 78%) as a slightly orange
1
solid. H NMR analysis revealed a 13:1 d.r. in favor of
with 40 mL portions of CH Cl . The combined organic
2
2
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1
the trans diastereomer. H NMR (400 MHz, CDCl ) 
American Type Culture Collection (ATCC) were
included as directed by CLSI. The MIC assay method
followed the procedure described by CLSI and
employed automated liquid handling to conduct serial
dilutions and liquid transfers.
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
3
1
.27 (br s, 2H), 3.85–3.76 (m, 1H, minor diastereomer),
.70–3.56 (m, 1H), 2.35–2.24 (m, 1H), 2.20–2.13 (m,
H, minor diastereomer), 2.08–1.86 (m, 8H), 1.86–1.63
m, 10H), 1.63–1.50 (m, 1H), 1.49–1.35 (m, 1H), 1.21–
.07 (m, 1H); C NMR (100 MHz, CDCl )  111.5
minor diastereomer), 111.3, 109.2, 108.9 (minor
diastereomer), 79.7, 79.5 (minor diastereomer), 39.3,
8.8 (minor diastereomer), 36.7, 36.3, 36.3, 34.9 (minor
diastereomer), 34.8, 34.8, 34.7, 34.7, 34.1, 29.7, 29.4
minor diastereomer), 26.8, 26.4, 19.8, 19.5 (minor
diastereomer); MS (ESI) calculated for C H NO [M
(
1
1
3
A standardized inoculum of each organism was
prepared per CLSI methods. Colonies were picked from
the primary plate and a suspension was prepared to
equal a 0.5 McFarland turbidity standard. Suspensions
were diluted 1:20 in CAMBH (or ID-CAMBH) medium
and then transferred to compartments of sterile
reservoirs divided by length. A liquid handler was used
dispense 10 L of standardized inoculum into each well
of the appropriate daughter plate for a final
3
(
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
1
6
26
4
+
+
H] m/z 296.19, found 295.96.
N-(((trans-dispiro[adamantane-2,3'-
1,2,4]trioxolane-5',1''-cyclohexan]-3''-yl)oxy)-4-(4-
5
[
concentration of approximately 5 x 10 CFU per well.
(
2-fluoro-4-methoxyphenyl)-2-oxopyridin-1(2H)-
The wells of the daughter plates ultimately contained
185 L of media, 5 L of drug solution, and 10 L of
inoculum. Plates were stacked 3 high, covered with a lid
on the top plate, placed into plastic bags, and incubated
at 35ºC for 18-20 hr. Plates were viewed from the
bottom using a plate viewer. An un-inoculated solubility
control plate was observed for evidence of drug
precipitation. MICs were read where visible growth of
the organism was inhibited.
MIC testing of P. aeruginosa PA14 were performed in
the Skaar lab as follows. Overnight cultures were sub-
cultured 1:50 into fresh LB broth and grown for 1 hour.
Bacteria were diluted 1:100 into medium containing
increasing concentrations of either 9 (25-50 µg/mL) or
11 (0.25-1.5 µg/mL). Growth assays were carried out in
96 well plates in a 100 µL volume, and growth was
measured by optical density absorbance at 600nm at 30
minute intervals. The MIC was defined as the minimum
concentration of compound that inhibited growth over a
period of 18 hours.
Pharmacokinetics Studies. Female NSG mice (n = 9 in
three groups) were treated with a single intraperitoneal
injection of 9 formulated in 50:40:10 PEG 400/20% 2-
hydroxypropylcylcodextrine in water/DMSO. Blood
samples were collected 5 min, 15 min, 30 min, 1 h, 2 h,
4 h, 8 h, 12 h, and 24 h after dosing (3 mice were
sampled at each time point, with each group of three
sampled thrice over 24 hrs). Plasma samples were
analyzed for concentrations of 9 and 11 using MS/MS
analysis at Integrated Analytical Solutions, Inc.
(Berkeley, CA). The resulting data were analyzed with
WinNonlin software to calculate standard PK
parameters.
yl)-2-methyl-2-(methylsulfonyl)butanamide (9). To a
vial charged with a stirred solution of acid 8 (13.0 mg,
0
.034 mmol, 1 equiv) in DMF (0.5 mL) was added
HATU (22.0 mg, 0.057 mmol, 1.5 equiv) and N,N-
diisopropylethylamine (10 L, 0.057 mmol, 1.5 equiv)
at room temperature. The orange solution was allowed
to stir for 15 mins, at which point a solution of
intermediate 6 (12.0 mg, 0.042 mmol, 1.1 equiv)
dissolved in DMF (0.5 mL) was added. The reaction
mixture was stirred for an additional 15 mins, at which
point the reaction was judged complete by TLC and
LCMS analysis. The reaction mixutre was then diluted
with with brine (20 mL) and extracted with two 10 mL
portions of EtOAc. The combined organic layers were
then dried over anhydrous Na SO , filtered, and
concentrated under reduced pressure to afford an orange
oil. The crude residue was purified by flash column
chromatography (25 g silica gel cartridge, 0–100%
EtOAc/hexanes, gradient elution) to afford the product
contaminated with starting material. Further
2
4
purification by rpHPLC provided pure 9 (6.0 mg, 24%)
as a white solid. H NMR (400 MHz, CDCl ) δ 12.24 -
1
3
1
6
4
3
2
1
2.32 (m, 1H), 7.33 - 7.46 (m, 2H), 6.79 - 6.85 (m, 2H),
.71 - 6.77 (m, 1H), 6.58 (br d, J = 6.82 Hz, 1H), 4.21 -
.30 (m, 1H), 4.10 (br d, J = 4.87 Hz, 2H), 3.87 (s, 3H),
.22 (d, J = 3.90 Hz, 3H), 2.46 - 2.61 (m, 3H), 2.35 -
.45 (m, 1H), 2.20 - 2.27 (m, 1H), 1.83 - 2.08 (m, 10H),
1
3
.66 - 1.80 (m, 10H), 1.43 (br d, J = 6.82 Hz, 2H); C
NMR (100 MHz, CDCl ) δ 165.2, 165.1, 163.3, 163.3,
3
1
1
1
4
3
2
62.3, 162.2, 162.1, 159.7, 148.8, 137.0, 130.5, 130.5,
18.7, 118.7, 117.4, 117.3, 111.7, 111.0, 111.0, 109.2,
09.0, 109.0, 102.7, 102.4, 81.8, 81.6, 76.8, 69.0, 55.9,
8.4, 39.4, 39.3, 38.6, 38.5, 36.9, 36.4, 36.4, 36.3, 35.0,
4.9, 34.8, 34.5, 29.7, 29.6, 28.7, 27.0, 27.0, 26.6, 21.6,
Infection Model. Wildtype BALB/c mice were
purchased from Jackson Laboratories. All animal
experiments were approved by the Vanderbilt
University Medical Center Institutional Animal Care
and Use Committee. Animal studies were not blinded.
The murine model of P. aeruginosa pneumonia was
1.5, 19.91, 19.88; MS (ESI) calculated for
+
C H FN O S [M + H] m/z 675.28, found 675.11.
34 44
2
8
Microbiology. MICs for the panel of strains presented
in Table 1 were performed under contract by Micromyx
LLC (Kalamazoo, MI). The test isolates consisted of
Gram-negative pathogens from the Micromyx
repository; relevant quality control organisms from the
4
2
performed as previously described . Briefly, overnight
cultures of P. aeruginosa PA14 were sub-cultured
1:1000 into fresh LB broth and grown for 3.5 hours to
mid-exponential phase. Cultures were washed twice
9
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with PBS, and resuspended in PBS at a density of
Page 10 of 12
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7
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9
1
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2
.5×10 CFU/mL. Five to six-week old female mice
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AUTHOR INFORMATION
Corresponding Author
Vergne, A.; Roosenberg, J. M.; Moraski, G.; Minnick, A. A.; McKee-
Dolence, J.; Hu, J.; Fennell, K.; Kurt Dolence, E.; Dong, L.;
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Phone: 415-514-9698; Fax: 415-514-4507;
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Adam R. Renslo: 0000-0002-1240-2846
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Mislin, G. L.; Schalk, I. J., Siderophore-dependent iron
Author Contributions
uptake systems as gates for antibiotic Trojan horse strategies against
Pseudomonas aeruginosa. Metallomics 2014, 6 (3), 408-420 DOI
10.1039/c3mt00359k.
‡
These authors contributed equally. B.R.B., P.T., and
A.R.R. conceived of experiments. B.R.B, P.T., and
R.K.M. synthesized compounds. E.P.S and E.R.G.
designed the mouse infection model, which E.R.G.
carried out. A.R.R. and R.K.M. drafted the manuscript
and all authors reviewed and edited the manuscript.
1
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Ghosh, M.; Miller, P. A.; Mollmann, U.; Claypool, W. D.;
Schroeder, V. A.; Wolter, W. R.; Suckow, M.; Yu, H.; Li, S.; Huang,
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against Multidrug Resistant Acinetobacter baumannii Both in Vitro
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The authors declare the following competing financial
interest(s): A.R.R. holds equity in Tatara Therapeutics,
Inc. which seeks to develop iron sensing therapies for
cancer and infectious disease.
1
Banevicius, M. A.; Finegan, S. M.; Irvine, R. L.; Brown, M. F.;
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DOI 10.1128/aac.00629-13.
ACKNOWLEDGMENT
A. R. Renslo acknowledges funding from the US National
Institutes of Health, R01 Grant AI105106. R. K. Muir
acknowledges funding from the NIH Research Training
Grant in Chemistry and Chemical Biology, T32
GM064337. Work in the E.P. Skaar laboratory was
supported by R01 AI101171-08 and E. R. Green was
supported by 5T32HL094296.
1
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