Trihydroxamate siderophore-fluoroquinolone conjugates are selective sideromycin antibiotics that target staphylococcus aureus

Article abstract of DOI:10.1021/bc300610f

Siderophores are multidentate iron(III) chelators used by bacteria for iron assimilation. Sideromycins, also called siderophore-antibiotic conjugates, are a unique subset of siderophores that enter bacterial cells via siderophore uptake pathways and deliv

Full text of DOI:10.1021/bc300610f

Article
pubs.acs.org/bc
Trihydroxamate Siderophore−Fluoroquinolone Conjugates Are
Selective Sideromycin Antibiotics that Target Staphylococcus aureus
Timothy A. Wencewicz,† Timothy E. Long,†,# Ute Mollmann,‡ and Marvin J. Miller*,†
̈
†Department of Chemistry and Biochemistry, 251 Nieuwland Science Hall, University of Notre Dame, Notre Dame, Indiana 46556,
United States
‡Leibniz Institute for Natural Product Research and Infection Biology, Hans Knoll Institute, Beutenbergstrasse 11a, 07745 Jena,
̈
Germany
S
* Supporting Information
ABSTRACT: Siderophores are multidentate iron(III) chela-
tors used by bacteria for iron assimilation. Sideromycins, also
called siderophore−antibiotic conjugates, are a unique subset
of siderophores that enter bacterial cells via siderophore
uptake pathways and deliver the toxic antibiotic in a “Trojan
horse” fashion. Sideromycins represent a novel antibiotic
delivery technology with untapped potential for developing
sophisticated microbe-selective antibacterial agents that limit
the emergence of bacterial resistance. The chemical synthesis of a series of mono-, bis-, and trihydroxamate sideromycins are
described here along with their biological evaluation in antibacterial susceptibility assays. The linear hydroxamate siderophores
used for the sideromycins in this study were derived from the ferrioxamine family and inspired by the naturally occurring
salmycin sideromycins. The antibacterial agents used were a β-lactam carbacepholosporin, Lorabid, and a fluoroquinolone,
ciprofloxacin, chosen for the different locations of their biological targets, the periplasm (extracellular) and the cytoplasm
(intracellular). The linear hydroxamate-based sideromycins were selectively toxic toward Gram-positive bacteria, especially
Staphylococcus aureus SG511 (MIC = 1.0 μM for the trihydroxamate−fluoroquinolone sideromycin). Siderophore−sideromycin
competition assays demonstrated that only the fluoroquinolone sideromycins required membrane transport to reach their
cytoplasmic biological target and that a trihydroxamate siderophore backbone was required for protein-mediated active transport
of the sideromycins into S. aureus cells via siderophore uptake pathways. This work represents a comprehensive study of linear
hydroxamate sideromycins and teaches how to build effective hydroxamate-based sideromycins as Gram-positive selective
antibiotic agents.
associated with bringing a new drug to market and the limited
market lifetime of antibiotics, investing in drug delivery
technology is a prudent choice for antibiotic discovery
programs.8
INTRODUCTION
■
Bacterial resistance to antibiotics is developing at an alarming
rate.1 As the current pipeline of useful antibacterial agents
diminishes, the crisis worsens and there is now a desperate need
for new methods of limiting the emergence of resistance to
antibiotics. One promising approach involves the development
of more sophisticated, pathogen-targeted antibiotic delivery
technologies that bypass or limit exposure of the antibacterial
agent to known resistance mechanisms. This approach is
attractive because it does not require the discovery of new
antibacterial scaffolds or validation of new biological targets,
which has proven to be an extremely difficult and time-
consuming task.2,3 New antibiotic delivery methodologies
present the opportunity to recycle old antibiotics rendered
useless by resistance that have already been structurally
optimized to interact with their biological target, revisit potent
antibiotics abandoned due to toxicity associated with the
existence of human orthologs to the biological target,4 bring
new life to antibiotic scaffolds that failed due to membrane
permeability problems,5,6 and extend the useful clinical lifetime
of new antibiotics coming to market by better managing
resistance.7 Considering the serious financial investment
One of the biggest challenges for targeted antibiotic delivery
is finding useful biological pathways in bacteria to exploit for
membrane transport.9 The ideal membrane transport pathway
for antibiotic delivery should be specific to bacterial cells (to
eliminate toxicity toward eukaryotic cells), essential for
virulence (to eliminate resistance development via deletion of
uptake pathway), and general enough to accept unnatural
substrates (to ensure successful uptake of the delivery vector
derivatized to carry the antibiotic). Bacterial iron-acquisition
pathways have been identified as suitable pathways for
developing such antibiotic delivery systems.10−12
The most common pathway for bacterial iron acquisition
involves the biosynthesis and excretion of low-molecular-weight
multidentate iron(III)-chelators, known as siderophores, used
Received: November 17, 2012
Revised: January 10, 2013
Published: January 28, 2013
© 2013 American Chemical Society
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Figure 1. Generic schematic of siderophore-mediated iron uptake and genetic regulation in Gram-negative and Gram-positive bacteria. Iron
metabolism in bacteria is under genetic control by the ferric uptake regulator (Fur) transcription factor protein. During times of iron abundance, the
Fur protein complexes Fe(II) and adopts a conformation that binds to a region of DNA known as a “Fur box” which represses the expression of
siderophore-related proteins, including biosynthetic enzymes. During times of iron deficiency, the apo-Fur protein dissociates from the “Fur box”
allowing expression of siderophore-related genes. The siderophore system is now fully functional and begins with siderophore biosynthesis and efflux
to the extracellular environment. For Gram-negative bacteria, after the siderophore chelates Fe(III) to form a soluble complex it is transported into
the periplasm by a high-affinity outer membrane transport protein (OMT; formerly called OMR). This transport is driven by the proton motive
force with energy transfer mediated by the membrane-spanning TonB-ExbB/D protein complex. The Fe(III)-loaded siderophore is then bound by a
siderophore periplasmic binding protein (SPBP) and trafficked to an ATP-binding cassette transporter (ABC) that moves the siderophore-Fe(III)
complex into the cytoplasm. The iron nutrient is then removed from the siderophore, typically via reduction of Fe(III) to Fe(II) by an iron reductase
enzyme (Red), and is distributed to parts of the cell in need or stored in bacterioferritin. In most cases, the siderophore is then recycled for further
use. Gram-positive bacteria lack the outer membrane found in Gram-negative bacteria and therefore also lack the OMTs and TonB complex. Instead,
extracellular siderophore-Fe(III) complexes are recognized and transported by membrane-anchored SPBPs. The remaining steps of the siderophore
pathway in Gram-positive bacteria are analogous to those described for Gram-negative bacteria. This figure was inspired by a variety of siderophore
transport diagrams reported in the literature, which also describe these pathways in great detail.13−15
for extracellular solubilization of otherwise insoluble iron(III)
minerals followed by protein-mediated active transport of the
metal complexes into bacterial cells (Figure 1).13−15 To ensure
a competitive growth advantage, bacteria commonly possess the
ability to obtain iron(III) using siderophores produced by
competing organisms.16 Along this evolutionary path, some
bacteria have learned to exploit this “iron thievery” for
antibiotic delivery by covalently attaching a toxic antibacterial
agent to the siderophore. This unique subset of siderophores,
known as sideromycins (Figure 2a), enter competing bacterial
cells via siderophore uptake pathways (Figure 2b) and deliver
the toxic agent in a “Trojan horse” fashion.17
bacteria with extremely potent minimum inhibitory concen-
trations (MIC) as low as 5 ng/μL.17 This impressive potency is
attributed to active transport into bacteria via the ferrichrome
membrane transport proteins FhuA20 and FhuD.21 Once
internalized, the seryl-t-RNA synthetase inhibitor is released
by a serine protease, peptidase N, which cleaves the amide
bond linkage to the siderophore.22 The albomycins are highly
effective in mouse infection models,17 and recently, the
biosynthetic gene cluster was discovered that makes possible
the production of novel analogues and engineering of
superproducers.23
Far less is known about the salmycins that were isolated from
The albomycins and salmycins (Figure 2a) are two naturally
occurring classes of sideromycins. The albomycins are the best-
studied of all sideromycins.17 They were originally reported in
1947 from a variety of Streptomyces strains18 and the structure
was correctly elucidated in 1982.19 The albomycins consist of a
ferrichrome-like trihydroxamate siderophore joined through an
amide bond to a thioribosyl pyrimidine inhibitor of seryl-t-RNA
synthetase and display in vitro and in vivo broad-spectrum
antibacterial activity against Gram-positive and Gram-negative
́
Streptomyces violaceus DSM 8286 in 1995 by Vertesy and co-
workers.24 A total synthesis of the salmycins was completed by
Miller and co-workers in 2002 which corrected the structure
assignment.25 The salmycins consist of a linear trihydroxamate
siderophore from the ferrioxamine family, known as danox-
amine, and an aminoglycoside antibiotic joined through a
succinoyl linker (Figure 3). They show potent and selective in
vitro antibacterial activity against Gram-positive bacteria,
including highly antibiotic resistant strains, with reported
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Figure 2. (A) Structures of generic and natural sideromycins (albomycins and salmycins). (B) Generic schematic of sideromycins as “Trojan horse”
antibiotic delivery agents. Sideromycins (siderophore−antibiotic conjugates) act as normal siderophores for sideromycin producing microbes, which
can self-protect from the antibiotic, by scavenging extracellular Fe(III) via the pathways described in Figure 1. For competing microbes attempting to
steal the sideromycin-bound Fe(III) nutrient, active transport of the sideromycin via siderophore uptake pathways exposes the competing microbe to
the antibiotic, for which it lacks the inherent resistance found in the producer, and ultimately causes cell death.
Figure 3. Structures of desferrisalmycin B, desferridanoxamine (3a), desferrioxamine B (DFO-B), and synthetic desferridanoxamine−antibiotic
conjugates from this study.
MIC values as low as 10 ng/μL.17 Similar to the albomycins,
the incredible potency is attributed to active transport of the
salmycins through hydroxamate siderophore membrane trans-
port proteins.26 Unfortunately, the salmycins only show weak in
vivo activity in mouse infection models probably due to
extracellular hydrolysis of the labile ester linkage.17 We
hypothesized that intracellular hydrolysis of the aminoglycoside
antibiotic was required for activity and that the ferrioxamine
siderophore was responsible for the narrow spectrum of
antibacterial activity against Gram-positive bacteria.27
In this report, we set out to better understand the selective
antibiotic activity of the salmycins using a series of synthetic
mono-, bis-, and trihydroxamate sideromycins (Figure 3) and
exploit this knowledge for building more effective synthetic
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Figure 4. Structures of synthetic hydroxamate siderophores (1a−3a) and sideromycins (1b−3b and 1c−3c) used in this work.
a
Scheme 1. Syntheses of Iron-Free (1a−3a) and Iron-Bound (3a-Fe) Hydroxamate Siderophores
a
Reagents and conditions: (a) 10% Pd−C, H2 (1 atm), MeOH; 6 h, 88% for 1a; 4.5 h, 90% for 2a; 11 h, 86% for 3a. (b) Fe(acac)3, MeOH; 4 h, 99%
for 3a-Fe.
sideromycins. Using broad-spectrum antibiotics with different
biological targets, β-lactams and fluoroquinolones, we demon-
strate that the full trihydroxamate siderophore backbone is
required for active membrane transport and that this type of
delivery vector is selective for Gram-positive bacteria that
possess hydroxamate siderophore transport proteins, as
reported for the salmycins and observed here against S. aureus.
We also establish that sideromycins with intracellular antibiotic
targets, which require membrane transport to be reached,
should be used to target Gram-positive pathogens.
corresponding deprotected, iron-free siderophores (1a, 2a, and
3a) were prepared and isolated in high yield and purity using
Pd-catalyzed hydrogenolysis followed by precipitation from
MeOH/Et2O. The iron(III)-complexed siderophore, danox-
amine (3a-Fe), was isolated in quantitative yield as an orange
solid after treatment of desferridanoxamine (3a) with Fe(acac)3
and precipitation from MeOH/Et2O (Scheme 1).30
Antibiotic derivatives 8 and 10 were synthesized for use as
coupling partners with the O-benzyl-protected siderophores 4,
5, and 6. N-Boc-O-PNB-Lorabid (7; a gift from Eli Lilly and
Co.) was treated with anhydrous TFA to give TFA salt 8.
Ciprofloxacin (9) was converted to O-benzyl-ciprofloxacin HCl
salt (10) by sequential N-Boc protection, carboxylate
benzylation, and N-Boc deprotection according to a literature
protocol (Scheme 2).31 The free amines of the protected β-
lactam and fluoroquinolone antibiotics, 8 and 10, were poised
for conjugation to the succinoyl group of benzyl-protected
siderophore precursors 4−6 through amide bond formations.
Importantly, β-lactam32 and fluoroquinolone33 antibiotics are
known to tolerate bulky substituents at these points of
derivatization and representative siderophore-conjugates of
these classes of antibiotics have been shown to retain affinity
for their distinct biological targets.34,35
RESULTS AND DISCUSSION
■
Syntheses of Siderophores and Sideromycins. The
synthetic sideromycins used in this study were rationally
designed to mimic the structure of the naturally occurring
salmycins (Figure 3). The linear hydroxamate siderophores
(1a−3a) were all derived from the natural siderophore portion
of the salmycins containing a succinoyl linker. Sideromycins
1b−3b featured the broad-spectrum β-lactam carbacephalo-
sporin antibiotic Lorabid which has a periplasmic biological
target. Sideromycins 1c−3c featured the broad-spectrum
fluoroquinolone antibiotic ciprofloxacin, which has a cytoplas-
mic biological target (Figure 4).28
The fully protected sideromycins (11−16) were assembled
using EDC-mediated amide couplings between the free
succinoyl carboxylate of the protected siderophores (4−6)
and the antibiotic amine salts (8 and 10) under basic
conditions (Scheme 3). The protected Lorabid sideromycins
The hydroxamate siderophores and sideromycins were
synthesized using a benzyl-protecting group strategy. The bis-
O-benzyl (4), tris-O-benzyl (5), and tetra-O-benzyl (6)
protected siderophore precursors were synthesized using a
literature protocol developed by Roosenberg and Miller.29 The
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after recrystallization from MeOH/Et2O with no need for
chromatographic purification.
Scheme 2. Synthesis of Protected Antibiotics (8 and 10)
Suitable for Siderophore Conjugation
a
Antibacterial Activity of Siderophores and Side-
romycins Against ESKAPEE Pathogens. The siderophores,
sideromycins, and control antibiotics (Lorabid and ciproflox-
acin) were tested for antibacterial activity against a panel of
ESKAPEE bacteria (E. faecium, S. aureus, K. pneumonia, A.
baumannii, P. aeruginosa, E. aerogenes, E. coli)1 by determining
the minimum inhibitory concentrations (MIC) using the broth
microdilution assay (Table 1).40 As expected, the siderophores
(1a−3a and 3a-Fe) had no antibacterial activity (MIC values
>128 μM) since these molecules should be growth-promoting
factors. The β-lactam−hydroxamate conjugates (1b−3b) had
moderate activity against S. aureus (MIC values from 32 to 64
μM), but at a reduced potency relative to the parent antibiotic,
Lorabid (MIC value of 1 μM). Conjugates 1b−3b also had
moderate activity against A. baumannii (MIC values from 32 to
128 μM), while Lorabid had no measurable activity (MIC value
of >128 μM). Overall, the β-lactam−hydroxamate conjugates
(1b−3b) had a narrowed spectrum of activity and reduced
potency (higher MIC values) against the ESKAPEE bacteria
relative to Lorabid alone.
a
Reagents and conditions: (a) N-Boc-O-PNB-Lorabid (7), TFA,
CH2Cl2, 30 min, 99%. (b) ciprofloxacin (9), Boc2O, NaHCO3, DMF,
2.5 h; then, BnBr, 90 °C, 25.5 h, 98%. (c) conc. HCl, CH2Cl2, 40 min,
92%.
(11, 13, and 15) were universally deprotected using hydro-
genolysis conditions optimized to preserve the vinyl chloride
functionality of the β-lactam bicycle.36−39 Purification by
preparative HPLC and recrystallization from MeOH/Et2O
provided analytically pure samples of the β-lactam sideromycins
(1b, 2b, and 3b). The O-benzyl protected ciprofloxacin
sideromycins (12, 14, and 16) were universally deprotected
by Pd-catalyzed hydrogenolysis, and the fluoroquinolone
sideromycins (1c, 2c, and 3c) were obtained in pure form
The fluoroquinolone−hydroxamate conjugates (1c−3c) also
showed a reduced spectrum of activity relative to the broadly
active parent antibiotic, ciprofloxacin (Table 1). The mono-
hydroxamate conjugate 1c had activity against S. aureus (32
μM), K. pneumonia (16 μM), A. baumannii (16 μM), P.
aeruginosa (128 μM), E. aerogenes (4 μM), and E. coli (2 μM),
a
Scheme 3. Syntheses of Hydroxamate Sideromycins (1b−3b and 1c−3c)
a
Reagents and conditions: (a) 8, iPr2EtN, DMAP, CH2Cl2; 18 h, 73% for 11; 16 h, 66% for 13; 20 h, 87% for 15. (b) 10% Pd−C, H2 (1 atm), HCl
(3 equiv), DMF:H2O (95:5); 41 h, 38% for 1b; 24 h, 42% for 2b; 24 h, 42% for 3b. (c) 10, iPr2EtN, DMAP, CH2Cl2; 5 h, 92% for 12; 23 h, 66% for
14; 4.5 h, 84% for 16. (d) 10% Pd−C, H2 (1 atm), MeOH; 12 h, 70% for 1c; 16 h, 86% for 2c; 16 h, 97% for 3c.
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a,b
Table 1. MIC Values of Compounds 1b−3b and 1c−3c against ESKAPEE Panel of Bacteria
test organisms
E. faecium
S. aureus SG K. pneumonia ATCC
511 700603
A. baumannii
P. aeruginosa
ATCC 27853
E. aerogenes ATCC E. coli ATCC
entry
compound
1a
NCTC 7171
ATCC 17961
35029
25922
1
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
4
>128
2
2a
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
>128
>128
16
>128
>128
>128
≥128
32
>128
>128
>128
>128
≥128
128
3
3a
4
3a-Fe
5
1b
>128
>128
>128
128
6
2b
64
7
3b
64
64
8
1c
32
16
2
9
2c
64
128
64
64
4
2
10
11
12
3c
1
>128
128
≥128
>128
0.25
>128
>128
0.125
64
>128
2
Lorabid
ciprofloxacin
32
1
>128
8
0.5
0.25
<0.015
<0.015
a
MIC values were determined using the broth microdilution method in Mueller-Hinton broth No. 2 (MHII) using visual end point analysis
according to the CLSI guidelines.40 Each compound was tested in triplicate.
b
but at significantly reduced potencies relative to ciprofloxacin
(MIC values of 0.5, 0.25, 0.25, 0.125, <0.015, and <0.015 μM,
respectively). The bishydroxamate conjugate 2c had similar
activity to 1c against S. aureus (64 μM), K. pneumonia (128
μM), A. baumannii (64 μM), P. aeruginosa (64 μM), E.
aerogenes (4 μM), and E. coli (2 μM), but again at significantly
reduced potencies compared to ciprofloxacin. Trihydroxamate
conjugate 3c showed selective antibacterial activity toward S.
aureus (MIC value of 1 μM) at approximately equivalent
potency as ciprofloxacin (MIC value of 0.5 μM). Mono- and
bishydroxamate conjugates, 1c and 2c, had spectra of
antibacterial activity similar to that of ciprofloxacin (broad
spectrum with increased potency toward Gram-negative
bacteria) at a reduced potency, while the trihydroxamate
conjugate 3c had a narrow spectrum of activity primarily against
Gram-positive bacteria, especially S. aureus.
Effect of Fe(III) Concentration on Sideromycin
Antibacterial Activity. Since the hydroxamate−antibiotic
conjugates (1b−3b and 1c−3c) all had activity toward S.
aureus SG511, this organism was chosen for further experi-
ments to determine if the synthetic sideromycins were actively
transported into the bacterial cells via siderophore uptake
pathways. Siderophore transport pathways are known to be
upregulated during times of Fe(III)-restriction,41 and con-
sequently, it has been discovered that the potency of
antibacterial activity of a sideromycin increases (lower MIC
value) under these circumstances.42 As shown in Table 2, MIC
values against S. aureus were determined for all the conjugates
using Fe(III)-supplemented (MHII + Fe) and Fe(III)-
restricted (MHII − Fe) testing media.43 None of the MIC
values observed for the β-lactam−hydroxamate conjugates
(1b−3b) under varying Fe(III) concentrations changed
significantly (a significant change for the broth microdilution
assay is >2-fold). Only trihydroxamate conjugate 3c produced a
significant (4-fold) decrease in MIC value when going from
Fe(III)-supplemented media (4 μM) to Fe(III)-restricted
media (1 μM). The MIC values for the control antibiotics,
Lorabid and ciprofloxacin, showed no dependence on the
Fe(III) concentration. These data suggest that the fluoroqui-
nolone−trihydroxamate conjugate 3c might be utilizing side-
rophore-transport pathways.
Table 2. MIC Values (μM) of Compounds 1b−3b and 1c−3c
against S. aureus SG511 under Varying Concentrations of
Fe(III)
a,b
test organism Staphylococcus aureus
SG511
c
d
entry
compound
1b
MHII + Fe
MHII − Fe
1
2
3
4
5
6
8
16
128
128
64
64
4
16
64
64
32
64
1
2b
3b
1c
2c
3c
Lorabid
ciprofloxacin
1
1
9
0.5
0.5
a
MIC values were determined using the broth microdilution method
using visual end point analysis according to the CLSI guidelines.40
b
c
Each compound was tested in triplicate. MHII + Fe: Mueller-Hinton
d
Broth No. 2 + 100 μM FeCl3. MHII − Fe: Mueller-Hinton Broth No.
2 + 100 μM 2,2′-bipyridine.
known to be antagonized in the presence of siderophores
competing for uptake via the same membrane transport
systems.44 To screen for this type of antagonism, MIC values
against S. aureus were determined under Fe(III)-restricted
conditions using 1:1 molar mixtures of the hydroxamate−
antibiotic conjugates (1b−3b and 1c−3c) with the correspond-
ing parent hydroxamate compounds (1a−3a and 3a-Fe) and
the commercially available trihydroxamate siderophore desfer-
rioxamine B (DFO-B) or its Fe(III) complex ferrioxamine B
(FO-B), as shown in Table 3. The siderophore DFO-B and
siderophore-Fe(III) complex FO-B were chosen, since they are
known to be utilized by S. aureus for siderophore-mediated
Fe(III)-acquisition and are structurally similar to danoxamine,
the siderophore portion of the salmycins.45 The antibacterial
activity of the β-lactam−monohydroxamate conjugate 1b was
not antagonized by the monohydroxamate 1a (Table 3, Entry
1) or DFO-B (Table 3, Entry 2). The activity of the β-lactam−
bishydroxamate conjugate 2b was not antagonized, but was
enhanced by the presence of bishydroxamate 2a (Table 3,
Entry 3) or DFO-B (Table 3, Entry 4). Similarly, the activity of
the β-lactam−trihydroxamate conjugate 3b was not antago-
nized by the presence of trihydroxamate-Fe(III) complex 3a-Fe
(Table 3, Entry 6) or FO-B (Table 3, Entry 8) and was slightly
Effect of Competing Siderophores on Sideromycin
Antibacterial Activity. Sideromycin antibacterial activity is
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Table 3. MIC Values (μM) of Compounds 1b−3b and 1c−3c
against S. aureus SG511 in the Presence of Competing
a
Siderophores
test organism Staphylococcus aureus SG511
b
c
entry
compound
MHII − Fe
1
1b + 1a
16
16
32
16
32
64
32
64
32
16
64
64
1
2
1b + DFO-B
2b + 2a
3
4
2b + DFO-B
3b + 3a
5
6
3b + 3a-Fe
3b + DFO-B
3b + FO-B
1c + 1a
7
8
9
10
11
12
13
14
15
16
1c + DFO-B
2c + 2a
2c + DFO-B
3c + 3a
Figure 5. Agar diffusion competition assay for conjugates 3b and 3c
and siderophores 3a, 3a-Fe, DFO-B, and FO-B tested against S. aureus
SG511. Siderophores 3a, 3a-Fe, DFO-B, and FO-B clearly antagonize
the anti-S. aureus activity of the fluoroquinolone conjugate 3c (c,d),
but not the β-lactam conjugate 3b (a,b). Images are not to actual scale.
3c + 3a-Fe
3c + DFO-B
3c + FO-B
1
128
>128
a
MIC values were determined using the broth microdilution method
using visual end point analysis according to the CLSI guidelines.40
Each compound was tested in triplicate. Data presented here should be
compared to MIC values in Table 1 for compounds 1b−3b and 1c−
3c. Compounds were tested as 1:1 molar mixtures. MHII − Fe:
Mueller-Hinton Broth No. 2 + 100 μM 2,2′-bipyridine.
Fe, DFO-B, and FO-B), while ciprofloxacin was unaffected. In
agreement with the MIC data (Table 3), DFO-B and FO-B
antagonized the activity of 3c more than siderophores 3a and
3a-Fe (Figure 5c,d).
b
c
enhanced by the trihydroxamate 3a (Table 3, Entry 5) and
DFO-B (Table 3, Entry 7).
Further Sideromycin Antibacterial Susceptibility Test-
ing in Agar Diffusion Assays. A more quantitative agar
diffusion assay was used to further verify the observed affects of
variable Fe(III) concentrations and competing siderophores on
the anti-S. aureus activity of the hydroxamate−antibiotic
conjugates (Figures 6 and 7). The quantitative agar diffusion
assay used was a modified46 Kirby-Bauer assay.47 Compound
solutions were added to 9 mm wells of an agar Petri dish
inoculated with S. aureus SG511 according to the conditions
indicated in the captions and color-coded legends of Figures 6
and 7. After incubation, the diameters of growth inhibition
zones were measured and are reported on the vertical axes as
the average of three independent trials. As shown in Figure 6,
the antibiotic activities of the β-lactam−hydroxamate con-
jugates 1b−3b and Lorabid against S. aureus were not
significantly affected by changes in the concentration of Fe(III)
in the testing media or by the presence of competing
siderophores. These observations are consistent with the
MIC values discussed previously (Tables 2 and 3).
As shown in Figure 7, all three of the fluoroquinolone−
hydroxamate conjugates (1c−3c) showed decreased potency
(smaller diameter zone of growth inhibition) against S. aureus
in Fe(III)-supplemented media (MHII + Fe) relative to
Fe(III)-restricted media (MHII − Fe). Furthermore, the
potency of conjugates 1c−3c all decreased in the presence of
an equimolar amount of the competing parent siderophores
(1a−3a, respectively) or DFO-B. Again, in agreement with
MIC data from Table 3, antagonism of the fluoroquinolone−
trihydroxamate conjugate 3c by DFO-B and FO-B was more
pronounced than by the parent siderophore 3a and its Fe(III)-
complex 3a-Fe. These data again strongly support that the
trihydroxamate conjugate 3c is utilizing the same siderophore-
transport pathway as DFO-B to deliver the fluoroquinolone
antibiotic to its cytoplasmic target (DNA gyrase) in S. aureus.
Neither the mono- or bishydroxamate−fluoroquinolone
conjugates 1c and 2c, respectively, were antagonized by the
presence of the parent hydroxamate compounds (1a and 2a,
respectively) or DFO-B (Table 3, Entries 9−12). The
fluoroquinolone−trihydroxamate conjugate 1c was not antag-
onized by the parent trihydroxamate siderophore 3a (Table 3,
Entry 13) or the Fe(III)-complex 3a-Fe (Table 3, Entry 14).
However, conjugate 3c was strongly antagonized by the
trihydroxamate siderophore DFO-B (Table 3, Entry 15) and
the Fe(III)-complex FO-B up to >128-fold. Antagonism of the
antibacterial activity of 3c by DFO-B and FO-B suggests that
these molecules are competing for entrance into S. aureus cells
via siderophore-uptake pathways.
The influence of competing siderophores 3a, 3a-Fe, DFO-B,
and FO-B on the antibacterial activity of the β-lactam−
trihydroxamate conjugate 3b (Figure 5a,b) and the fluoroqui-
nolone−trihydroxamate conjugate 3c (Figure 5c,d) against S.
aureus was also studied using a qualitative agar diffusion
competition experiment (Figure 5). Sterile paper strips soaked
in solutions of the test compounds were layed in a “criss-cross”
pattern on Fe(III)-deficient MHII agar inoculated with S.
aureus. After incubation and growth of the organism, analysis of
the intersecting and nonintersecting points of the paper strips
revealed the effects of the compound mixtures relative to the
single compounds alone. Data from these studies were in
agreement with the data from the MIC studies (Table 3). The
antibacterial activity of the β-lactam−trihydroxamate conjugate
3b was not antagonized by the presence of 3a-Fe and FO-B
and was slightly enhanced by the presence of 3a and DFO-B,
while the activity of Lorabid was unaffected (Figure 5a,b). The
antibacterial activity of the fluoroquinolone−trihydroxamate
conjugate 3c was antagonized by all the siderophores (3a, 3a-
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Figure 6. Effect of varying Fe(III) concentrations and the presence of competing siderophores (1a, 2a, 3a, 3a-Fe, DFO-B) on the antibacterial
activity of β-lactam sideromycins (1b, 2b, and 3b) against S. aureus SG511 in the agar diffusion assay. The vertical axis indicates the diameter of the
growth inhibition zone in millimeters and the horizontal axis represents the test compound. The assay was performed in triplicate and standard
deviations are indicated by error bars. MHII-Fe: Mueller-Hinton agar No. 2 + 100 μM 2,2′-bipyridine. MHII+Fe: Mueller-Hinton agar No. 2 + 100
μM FeCl3. Parent siderophore refers to the iron-free siderophore (1a, 2a, and 3a) with the same compound numeral as the corresponding
sideromycin (1b, 2b, and 3b, respectively). Data for all mixtures corresponds to a 1:1 molar ratio of test compounds. 50 μL of sample at the
concentration indicated in parentheses under the compound numbers was added to 9 mm wells as described in the Experimental Section. The
tabulated numerical data for this chart are provided in the Supporting Information (Tables S2 and S3).
Figure 7. Effect of varying Fe(III) concentrations and presence of competing siderophores (1a, 2a, 3a, 3a-Fe, DFO-B, and FO-B) on the
antibacterial activity of fluoroquinolone sideromycins (1c, 2c, and 3c) against S. aureus SG511 in the agar diffusion assay. The vertical axis indicates
the diameter of the growth inhibition zone in millimeters and the horizontal axis indicates the test compound. The assay was performed in triplicate.
Ciprofloxacin was used as a control. MHII-Fe: Mueller-Hinton agar No. 2 + 100 μM 2,2′-bipyridine. MHII+Fe: Mueller-Hinton agar No. 2 + 100
μM FeCl3. Parent siderophore refers to the iron-free siderophore (1a, 2a, and 3a) with the same compound numeral as the corresponding
sideromycin (1c, 2c, and 3c, respectively). Data for all mixtures correspond to a 1:1 molar ratio of test compounds. 50 μL of sample at the
concentration indicated in parentheses under the compound numbers was added to 9 mm wells as described in the Experimental Section. The
tabulated numerical data for this chart are provided in the Supporting Information (Tables S2 and S3).
broad-spectrum aminoglycoside antibiotic. The precise reason
for the selective antibacterial activity against Gram-positive
bacteria is still up for debate, but one possibility is that the
more promiscuous membrane-anchored hydroxamate side-
rophore binding proteins in S. aureus, known as FhuD1 and
FhuD2, are capable of binding to the salmycins and the
trihydroxamate−ciprofloxacin conjugate with high affinity,
while the more discriminative hydroxamate siderophore
outer-membrane proteins found in Gram-negative bacteria
have reduced affinity for the derivatized siderophores.45,48,49
Antibacterial susceptibility testing in both the broth micro-
dilution and agar diffusion assays revealed that the antibacterial
activity of the hydroxamate−β-lactam conjugates (1b−3b)
against S. aureus showed no dependence on Fe(III)
concentration in the media and was unaffected by the presence
of competing siderophores. These observations are consistent
with the fact that the β-lactam biological targets, the penicillin
binding proteins (PBPs), are in the periplasmic peptidoglycan
FURTHER DISCUSSION
■
The hydroxamate−β-lactam (1b−3b) and hydroxamate−
fluoroquinolone conjugates (1c−3c) from this study all showed
a narrowed spectrum of activity compared to the parent
antibiotics. The hydroxamate−antibiotic conjugates showed
high selectivity toward the Gram-positive pathogen Staph-
ylococcus aureus SG511. The trihydroxamate−fluoroquinolone
conjugate 3c showed the most selective activity and retained
equivalent potency as the parent antibiotic, ciprofloxacin,
against S. aureus (MIC = 1 μM). Sideromycin 3c represents
one of the few synthetic siderophore−antibiotic conjugates
using an antibiotic with an intracellular target that maintains the
potency of the parent drug.27,35 The trihydroxamate side-
rophore, desferridanoxamine (3a), clearly was responsible for
the narrowed spectrum of activity of the trihydroxamate−
ciprofloxacin conjugate (3c). Analogously, desferridanoxamine
is also the likely source of the exquisite Gram-positive
selectivity observed for the salmycins which contain a normally
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which is outside of the cell membrane for S. aureus. Therefore,
active transport into the S. aureus cells actually denied the β-
lactam sideromycins access to S. aureus PBPs. The hydrox-
amate−fluoroquinolone conjugates required active transport
across the cell membrane to reach their biological target (DNA
gyrase) in the cytoplasm. This clearly demonstrated that
siderophore−antibiotic conjugates targeting Gram-positive
bacteria should only use antibiotics with cytoplasmic targets.
Furthermore, antibacterial activity of the trihydroxamate−
ciprofloxacin conjugate (3c) showed a significant dependence
on Fe(III) concentration in the testing media and was
antagonized by competing siderophores, while the antibacterial
activities of the mono- and bishydroxamate−ciprofloxacin
conjugates (1c and 2c, respectively) were less influenced by
Fe(III) and competing siderophores in the testing media. This
suggested that the entire trihydroxamate backbone of the
siderophore is necessary to facilitate efficient active transport of
the sideromycin by S. aureus. The likely reason for needing the
full trihydroxamate siderophore is for competitive chelation of
environmental Fe(III) and recognition of the siderophore-
Fe(III) complex by the siderophore-binding receptor proteins,
which preferentially bind to octahedral, trihydroxamate-Fe(III)
complexes.14
The ability of the ferrioxamine siderophore DFO-B and its
Fe(III) complex FO-B to antagonize the anti-S. aureus activity
of sideromycin 3c to a much greater extent than the structurally
similar trihydroxamate parent siderophore desferridanoxamine,
3a, and its Fe(III)-complex danoxamine, 3a-Fe, is attributed to
the drastic difference in Fe(III)-affinity observed for these
molecules. Desferrioxamine B has an Fe(III)-affinity (∼1030)
which is ∼7 orders of magnitude greater than the Fe(III)-
affinity of trihydroxamate siderophore 3a (∼1023) (unpublished
data from our laboratory). In work unpublished so far, we have
shown that a competing siderophore must have an Fe(III)-
affinity equal to or greater than that of the sideromycin in order
to antagonize the antibacterial activity of the sideromycin. This
observation is attributed to the fact that the sideromycin can
only be transported into bacterial cells, as the Fe(III)-complex
and the molecule with the higher Fe(III)-affinity will “win” the
competition for entry into the bacterial cell by “winning” the
competition for binding the scarce Fe(III) nutrient.
bacteria to the active ingredient. Sideromycin 3c has the
potential for decreasing mammalian toxicity of fluoroquino-
lones since it selectively enters S. aureus cells via membrane
transport systems that are absent in eukaryotic cells.51
Trihydroxamate ferrioxamine siderophores, including FDA-
approved DFO-B, have been proven safe for human
consumption at extremely large doses (4−12 g/day) when
given to patients for chelation treatment of iron-overload
diseases.52 It has been documented that the use of DFO-B to
treat iron-overload during the course of an S. aureus infection
can worsen the infection since S. aureus can use the Fe(III)-
complex FO-B for iron assimilation.45 Using sideromycin 3c to
treat iron-overload diseases might be an effective method for
simultaneously treating or preventing S. aureus infections. In
fact, the salmycins have been patented for such an intended
use.53,54 Additionally, ferrioxamine siderophores are not bound
by the mammalian siderophore-binding protein siderocalin, so
the bioavailability is predicted to be high and consequently the
required dosing to treat bacterial infections might be low.55
One foreseeable limitation of sideromycin antibiotics, and
most pathogen-targeted antibiotic agents, is that they might not
be effective at treating intracellular infections caused by
pathogenic microbes that survive inside mammalian cells
since the delivery agent is not engineered to penetrate higher
eukaryotic cells.56 Fortunately, alternative methods for treating
these types of infections with engineered delivery systems are in
development.57 Another potential limitation to sideromycin
antibiotic chemotherapy born from their exquisite microbe
selectivity is the need for sensitive pathogen detection and rapid
diagnostics. Unlike broad-spectrum antibiotics, microbe-selec-
tive antibiotics such as sideromycins will require early pathogen
identification to direct accurate antibacterial drug prescription.2
Fortunately, major advancements in bacterial pathogen
detection using siderophore-based sensors are in development
by several groups and will certainly play an important role in
the future for sideromycin antibiotics in the clinic.58,59
SUMMARY AND CONCLUSIONS
■
Similar to the salmycins, the synthetic linear hydroxamate
sideromycins studied here showed selective antibacterial activity
against Gram-positive bacteria, especially Staphylococcus aureus
SG511 (MIC = 1 μM for the trihydroxamate−fluoroquinolone
sideromycin 3c). Siderophore−sideromycin competition assays
and bacterial growth inhibition assays using variable Fe(III)
concentrations showed that only the fluoroquinolone side-
romycins (1c−3c) required membrane passage to reach their
cytoplasmic biological target and that the full trihydroxamate
siderophore delivery vector was required for siderophore-
associated protein-mediated active transport into S. aureus cells.
This work supports the hypothesis that linear trihydroxamate
siderophores are selective antibiotic delivery vectors for Gram-
positive pathogens, as observed for the naturally occurring
salmycins and the synthetic desferridanoxamine−ciprofloxacin
sideromycin (3c), and teaches how to engineer more effective
hydroxamate-based sideromycins.
Since a stable amide bond linkage was formed between the
antibiotic ciprofloxacin and the siderophore desferridanox-
amine, it was suspected that the entire desferridanoxamine−
ciprofloxacin was the active antibacterial agent inhibiting the
action of DNA gyrase. This hypothesis is supported in the
literature by examples of siderophore−ciprofloxacin conjugates
linked through an amide bond on the distal nitrogen of the
piperazine unit that still show high in vitro binding to DNA
gyrase.35 The question of whether or not the aminoglycoside
antibiotic of the salmycins is released inside bacterial cells is still
up for debate, but it is proposed that the ideal synthetic
sideromycin should feature a microbe-triggered linker that
releases the antibiotic inside the cell to give optimal affinity for
the biological target.27 In fact, the development of new linker
systems that make such intracellular release possible have been
the focus of much work in our group.50
EXPERIMENTAL SECTION
The trihydroxamate−ciprofloxacin sideromycin 3c developed
in this work has many potential applications. The exquisite
selectivity against S. aureus makes sideromycin 3c an ideal
candidate for a targeted microbe-selective antibacterial chemo-
therapy that could limit the spread of fluoroquinolone
multidrug resistance by limiting exposure of nontargeted
■
Materials and Instrumentation. All reactions were
performed under a dry argon atmosphere, unless otherwise
stated. All solvents and reagents were obtained from
commercial sources and used without further purification
unless otherwise stated. Dichloromethane (CH2Cl2) was
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distilled from calcium hydride. Dimethylformamide (DMF)
and diisopropylethylamine (iPr2EtN) were used from Acros
Seal anhydrous bottles. N-Boc-O-PNB-Lorabid (7) was a gift
from Eli Lilly and Company. Ciprofloxacin (9) was purchased
from Sigma-Aldrich (St. Louis, MO). Benzyl-protected
hydroxamate siderophores 4−6 were prepared according to a
previously described method.29 Silica gel column chromatog-
raphy was performed using Sorbent Technologies silica gel 60
(32−63 μm).
All microbiological media and liquids were sterilized by
autoclaving (121 °C, 15 min) before use unless otherwise
stated. All aqueous solutions and media were prepared using
distilled, deionized, and filtered water (Millipore Milli-Q
Advantage A10 Water Purification System). Luria broth (LB)
was purchased from VWR. Mueller-Hinton No. 2 broth (MHII
broth; cation adjusted) was purchased from Sigma-Aldrich (St.
Louis, MO). Iron-deficient MHII broth (MHII − Fe) was
prepared by adding 0.8 mL of a 1 mg/mL filter-sterilized
aqueous solution of 2,2′-bipyridine to 49.2 mL of sterile MHII
broth. Iron-supplemented MHII broth (MHII + Fe) was
prepared by adding 0.8 mL of a freshly prepared 1 mg/mL
filter-sterilized aqueous solution of FeCl3 to 49.2 mL of sterile
MHII broth. Mueller-Hinton No. 2 agar (MHII agar; HiMedia
Laboratories) was purchased from VWR. Iron-deficient MHII
agar (MHII − Fe) was prepared by adding 0.5 mL of a 1 mg/
mL filter-sterilized aqueous solution of 2,2′-biypridine to 34 mL
of melted MHII agar with gentle mixing. Iron-supplemented
MHII agar (MHII + Fe) was prepared by adding 0.5 mL of a
freshly prepared 1 mg/mL filter-sterilized aqueous solution of
FeCl3 to 34 mL of melted MHII agar with gentle mixing.
McFarland BaSO4 turbidity standards were purchased from
under argon. The flask was charged with 10% Pd−C (50.0 mg)
and exposed to a balloon of hydrogen gas (∼1 atm). Reaction
progress was monitored by RP-C18 TLC (1.5:1 CH3CN:H2O;
FeCl3 stain), and after 11 h, there was no remaining starting
material (6). The flask was flushed with argon, and the mixture
was diluted with warm MeOH, vacuum filtered through a pad
of Celite using a fine glass frit, and concentrated under reduced
pressure. The resulting solid was dissolved in a minimal amount
of MeOH and precipitated by addition of cold Et2O. After
trituration with Et2O, the siderophore desferridanoxamine (3a)
was obtained in 97% yield as a white solid (208.0 mg, 0.34
1
mmol). Mp 134−140 °C; H NMR (600 MHz, DMSO-d6) δ
11.87 (br s, COOH, 1 H), 9.62 (br s, N−OH, 3 H), 7.78 (t, J =
5.3 Hz, 2 H), 4.35 (br s, 1 H), 3.47−3.42 (m, 6 H), 3.39−3.36
(m, 2 H), 3.02−2.97 (m, 4 H), 2.57 (t, J = 6.6 Hz, 6 H), 2.39 (t,
J = 6.6 Hz, 2 H), 2.26 (t, J = 6.9 Hz, 4 H), 1.49 (dt, J = 14.2, 7.1
Hz, 6 H), 1.43−1.34 (m, 6 H), 1.27−1.17 (m, 6 H); 13C NMR
(150 MHz, DMSO-d6) δ 174.1, 172.0, 171.9, 171.6, 171.3, 60.6,
47.2, 47.1, 38.4, 32.2, 29.9, 28.8, 28.7, 27.6, 27.5, 27.1, 26.2,
26.0, 23.5, 23.4, 22.7; HRMS-ESI (m/z): [M+H]+ calcd. for
C27H50N5O11: 620.3501, found 620.3506.
Danoxamine (3a-Fe). Prepared as described previously.29,30
Briefly, siderophore 3a (154 mg, 0.25 mmol) was dissolved in
10 mL of MeOH at 40 °C (oil bath temperature). Fe(acac)3
(97.0 mg, 0.28 mmol) was added, and the clear, orange solution
was stirred for 2 h. The solution was cooled to rt and the
MeOH was evaporated under reduced pressure. The resulting
orange residue was dissolved in a minimal amount of MeOH
and precipitated by addition of cold Et2O. After trituration with
Et2O, the Fe(III)-complexed siderophore danoxamine (3a-Fe)
was isolated in 99% yield as an orange powder (165.2 mg, 0.25
mmol). Mp 151−154 °C (dec.); HRMS-ESI (m/z): [M+H]+
calcd. for C27H47FeN5O11: 673.2616, found 673.2647.
́
bioMerieux, Inc. Sterile plastic Petri dishes (145 mm × 20 mm;
Greiner Bio-One) and sterile polystyrene 96-well plates (BD
Falcon) used for antibacterial susceptibility testing were
purchased from VWR. Ferric chloride (anhydrous, reagent
grade) and 2,2′-bipyridine (reagent plus grade) were purchased
from Sigma-Aldrich. All test organisms used in this work were
from sources given in the Supporting Information (Table S1).
1H NMR and 13C NMR spectra were obtained on a 300, 500,
or 600 MHz Varian DirectDrive spectrometer and FIDs were
processed using ACD/ChemSketch version 10.04. Chemical
shifts (δ) are given in parts per million (ppm) and are
referenced to residual solvent. Coupling constants (J) are
reported in hertz (Hz). High-resolution, accurate mass
measurements were obtained with a Bruker micrOTOF II
electrospray ionization time-of-flight mass spectrometer in
positive ion mode. Sample was introduced via flow injection at
a rate of 4 μL/min, and mass spectra were accumulated from 50
to 3000 m/z for 2 min. Preparative HPLC purifications were
performed on a Waters preparative binary pump system at a
flow rate of 15 mL/min with UV detection at 254 nm using a
YMC-Pack Pro C18 column (150 × 20 mm; 5 μm particle size)
fit with a guard column. Thin layer chromatography (TLC) was
performed with Al-backed Merck 60-F254 or Al-backed Merck
RP-C18 F256 silica gel plates using a 254 nm lamp and aqueous
FeCl3 for visualization. Melting points were determined in
capillary tubes using a Thomas-Hoover melting point apparatus
and are uncorrected.
O-PNB-Lorabid TFA Salt (8). N-Boc-O-PNB-Lorabid (7;
208.0 mg, 0.36 mmol) was dissolved in 7 mL of anhydrous
TFA:CH2Cl2 (1:1). After 30 min, TLC (2:1 hexanes:EtOAc)
showed complete consumption of the starting material. The
TFA/CH2Cl2 were removed by evaporation under reduced
pressure giving TFA salt 8 as a light yellow viscous oil (208.0
mg, 0.36 mmol) that was used immediately in the next reaction
without purification or characterization.
O-Benzyl-ciprofloxacin HCl Salt (10). Prepared by a
modified literature procedure.31 Ciprofloxacin (9; 1.01 g, 3.05
mmol), Boc anhydride (820.0 mg, 3.76 mmol), and NaHCO3
(1.28 g, 15.24 mmol) were suspended in 25 mL of anhydrous
DMF and stirred at rt for 2 h. Benzyl bromide (1.62 mL, 13.64
mL) was added and the mixture was heated to 90 °C (oil bath
temperature). After 1 h of heating, the reaction mixture became
homogeneous. After 25.5 h of heating, the mixture was cooled
to rt and the DMF was removed using high-vacuum rotary
evaporation (∼1 mmHg). The resulting solid was dissolved in
75 mL of CHCl3 and 50 mL of H2O. The layers were separated
and the CHCl3 was washed with H2O (25 mL) and brine (25
mL), dried over anhydrous MgSO4, filtered, and concentrated
to give an orange solid. The crude solid was suspended in Et2O,
filtered, and washed several times with Et2O. This provided N-
Boc-O-benzyl-ciprofloxacin in 98% yield as a pale yellow solid
1
(1.56 g, 2.99 mmol). Mp 191−193 °C; H NMR (600 MHz,
Representative Synthetic Procedures for the Syn-
thesis of Siderophores and Sideromycins. Desferridanox-
amine (3a). Prepared as described previously.29,30 Briefly, tetra-
O-benzyldanoxamine 6 (340.0 mg, 0.35 mmol) was dissolved in
35 mL MeOH in an HCl-washed round-bottom flask sealed
CDCl3) δ 8.52 (s, 1 H), 8.05 (d, J = 13.5 Hz, 1 H), 7.52 (d, J =
7.0 Hz, 2 H), 7.38 (t, J = 7.5 Hz, 2 H), 7.33−7.29 (m, 1 H),
7.26 (d, J = 7.0 Hz, 1 H), 5.39 (s, 2 H), 3.69−3.62 (m, 4 H),
3.44−3.37 (m, 1 H), 3.24−3.19 (m, 4 H), 1.50 (s, 9 H), 1.32−
1.27 (m, 2 H), 1.15−1.09 (m, 2 H); 13C NMR (150 MHz,
482
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CDCl3) δ 173.0, 165.5, 154.6, 153.3 (d, JC−F = 248.5 Hz),
148.3, 144.4 (d, JC−F = 10.1 Hz), 137.9, 136.4, 133.3, 129.3,
mmol) and concentrated HCl (33.0 μL, 24.0 mmol) were
dissolved in 1.1 mL of DMF:H2O (95:5) in an HCl-washed, 10
mL round-bottom flask sealed under argon. The flask was
charged with 10% Pd−C (23.0 mg) and exposed to a balloon of
hydrogen gas (∼1 atm). Reaction progress was monitored by
RP-C18 TLC (2.5:1 H2O:CH3CN; FeCl3 stain) and after 24 h
there was no remaining starting material (15). The flask was
flushed with argon and the mixture was diluted with MeOH,
vacuum filtered through Celite, and concentrated using high-
vacuum rotary evaporation (∼1 mmHg). The crude product
was sequentially purified by size exclusion chromatography (0.5
× 6 in Sephadex G50; 1:1 MeOH:EtOAc eluent), preparative
HPLC using a 150 × 20 mm YMC-Pack Proc C18 column fit
with a guard column, 0.1% TFA in H2O (A) and 0.1% TFA in
CH3CN (B) as mobile phases, and a flow rate of 15 mL/min. A
gradient was formed from 20−50% of B over 4 min where the
desired compound (3b) eluted at 4.1 min. Pure fractions were
lyophilized and the obtained solid was recrystallized from
MeOH/Et2O to give the desired product (3b) in 42% yield as
an off-white solid (30.0 mg, 0.03 mmol). Mp 103−105 °C
128.5, 127.9, 127.8, 123.3 (d, JC−F = 6.2 Hz), 113.4 (d, JC−F
=
24.1 Hz), 110.2, 105.0, 80.2, 66.3, 49.9, 34.5, 28.4, 8.1; HRMS-
ESI (m/z): [M+H]+ calcd. for C29H33FN3O5: 522.2399, found
522.2423. The solid (1.50 g, 2.88 mmol) was dissolved in 25
mL of CH2Cl2 and concentrated HCl (5 mL) was added slowly
giving a dark orange solution. After 40 min, the CH2Cl2/HCl
were evaporated giving a yellow solid. The solid was dissolved
in hot MeOH (25 mL), activated charcoal was added, and the
mixture was vacuum filtered through Celite. The MeOH was
evaporated and the solid was recrystallized from CH2Cl2/Et2O
at −20 °C, which gave the desired O-benzyl-ciprofloxacin
monohydrochloride salt (10) in 92% as a yellow solid (1.20 g,
1
2.62 mmol). Mp 250−255 °C (dec.); H NMR (600 MHz,
DMSO-d6) δ 9.71 (br s, 1 H), 9.62 (br s, 1 H), 8.48 (d, J = 3.5
Hz, 1 H), 7.84−7.77 (m, 1 H), 7.51−7.44 (m, 3 H), 7.42−7.36
(m, 2 H), 7.32 (t, J = 7.0 Hz, 1 H), 5.27 (s, 2 H), 3.65−3.53
(m, 1 H), 3.53−3.46 (m, 4 H), 3.34−3.24 (m, 4 H), 1.28−1.23
(m, 2 H), 1.12−1.07 (m, 2 H); 13C NMR (150 MHz, DMSO-
d6) δ 171.6, 164.5, 152.4 (d, JC−F = 246.8 Hz), 148.6, 142.8 (d,
JC−F = 10.7 Hz), 138.0, 136.6, 128.4, 127.7, 127.6, 122.5, 111.8
(d, JC−F = 24.1 Hz), 108.9, 106.7, 65.2, 46.4, 42.5, 34.9, 7.6;
HRMS-ESI (m/z): [M+H]+ calcd. for C24H25FN3O3: 422.1874,
found 422.1871.
1
(color change), 170−175 °C (dec.); H NMR (600 MHz,
CD3OD) δ 7.45−7.32 (m, 5 H), 5.43−5.42 (m, 1 H), 5.37 (d, J
= 5.0 Hz, 1 H), 3.88 (td, J = 7.9, 3.8 Hz, 1 H), 3.83 (ddd, J =
14.0, 7.3, 7.1 Hz, 1 H), 3.62−3.53 (m, 4 H), 3.30−3.26 (m, 1
H), 3.18−3.13 (m, 4 H), 2.84 (t, J = 6.3 Hz, 2 H), 2.76 (td, J =
7.1, 3.1 Hz, 3 H), 2.70−2.54 (m, 5 H), 2.53 (d, J = 5.6 Hz, 1
H), 2.50−2.43 (m, 4 H), 2.40 (dt, J = 15.2, 5.5 Hz, 1 H), 1.78−
1.72 (m, 2 H), 1.68−1.57 (m, 6 H), 1.57−1.54 (m, 2 H), 1.54−
1.47 (m, 4 H), 1.39−1.35 (m, 2 H), 1.34−1.27 (m, 4 H); 13C
NMR (150 MHz, CD3OD) δ 175.6, 175.1, 174.6, 174.6, 174.6,
174.5, 173.9, 166.3, 163.9, 138.3, 130.0, 129.7, 129.4, 129.1,
126.0, 62.9, 59.9, 59.6, 54.1, 40.5, 40.4, 33.4, 32.5, 31.7, 31.6,
31.3, 30.5, 30.2, 30.1, 30.0, 29.9, 29.1, 29.0, 28.9, 27.6, 27.4,
27.2, 25.0, 24.8, 24.1, 23.1; HRMS-ESI (m/z): [M+H]+ calcd.
for C43H64ClN8O14: 951.4225, found 951.4234.
tetra-O-Benzyldanoxamine-O-PNB-Lorabid Conjugate
(15). O-PNB-Lorabid TFA salt (8; 85.7 mg, 0.15 mmol) was
dissolved in 10 mL of anhydrous CH2Cl2 and iPr2EtN (0.13
mL, 0.75 mmol) was added slowly until the solution was basic
(pH paper). tetra-O-Benzyl-trihydroxamate 6 (155.5 mg, 0.16
mmol), DMAP (8.0 mg, 0.06 mmol), and EDC-HCl (95.0 mg,
0.50 mmol) were then added, respectively. After 20 h at rt,
TLC (3% MeOH in EtOAc; FeCl3 stain) showed no remaining
starting material (8). The CH2Cl2 was removed under reduced
pressure and the resulting oil was dissolved in EtOAc (20 mL),
washed with 1 N HClaq (15 mL), H2O (10 mL), 10% aqueous
NaHCO3 (10 mL), and brine (10 mL), dried over anhydrous
MgSO4, filtered, and concentrated. The crude product was
purified by silica gel column chromatography (1 × 6 in silica
gel; 3%−5% MeOH in EtOAc) to give the desired product
(15) in 87% yield as a clear, colorless oil (183.0 mg, 0.100.13
tetra-O-Benzyldanoxamine-O-benzylciprofloxacin Conju-
gate (16). O-Benzyl-ciprofloxacin hydrochloride salt (10) was
free-based using Amberlite IR400(OH−) resin in CHCl3 for 4
h. The resulting O-benzyl-ciprofloxacin amine (46.0 mg, 0.11
mmol), tetra-O-Benzyl-trihydroxamate 6 (103.0 mg, 0.10
mmol), iPr2EtN (0.04 mL, 0.23 mmol), DMAP (3.2 mg, 0.03
mmol), and EDC-HCl (30.2 mg, 0.16 mmol) were dissolved in
10 mL of anhydrous CH2Cl2, respectively. After 4.5 h at rt,
TLC (9% MeOH in CH2Cl2; FeCl3 stain) showed no
remaining starting material 6. The mixture was diluted with
CH2Cl2 (20 mL), washed with 10% aqueous citric acid (15
mL), H2O (15 mL), 10% aqueous NaHCO3 (15 mL), and
brine (15 mL), dried over anhydrous MgSO4, filtered, and
concentrated. The crude product was purified by silica gel
column chromatography (1 × 4 in silica gel; 3% MeOH in
CH2Cl2) to give the desired product (16) in 84% yield as a
1
mmol). H NMR (500 MHz, CDCl3) δ 8.61 (d, J = 7.4 Hz, 1
H), 8.21 (d, J = 8.6 Hz, 2 H), 7.60 (d, J = 8.8 Hz, 2 H), 7.45−
7.24 (m, 25 H), 6.75 (br s, 1 H), 6.45 (br s, 1 H), 5.65 (d, J =
7.0 Hz, 1 H), 5.46−5.42 (m, 1 H), 5.41 (d, J = 14.4 Hz, 1 H),
5.33−5.28 (m, 1 H), 4.86−4.77 (m, 5 H), 4.75 (d, J = 10.2 Hz,
1 H), 4.47 (s, 2 H), 4.03−3.93 (m, 1 H), 3.91−3.85 (m, 1 H),
3.66−3.56 (m, 2 H), 3.52−3.46 (m, 1 H), 3.46−3.37 (m, 3 H),
3.34−3.10 (m, 4 H), 3.00−2.92 (m, 1 H), 2.87−2.71 (m, 6 H),
2.65−2.50 (m, 5 H), 2.51−2.41 (m, 4 H), 1.68−1.55 (m, 6 H),
1.55−1.46 (m, 2 H), 1.46−1.39 (m, 3 H), 1.39−1.30 (m, 4 H),
1.24−1.16 (m, 4 H), 1.15−1.06 (m, 1 H); 13C NMR (125
MHz, CDCl3) δ 174.3, 174.2, 173.8, 172.4, 172.2, 171.9, 171.4,
165.2, 160.0, 147.8, 142.1, 138.5, 136.9, 134.4, 134.2, 134.1,
130.8, 129.2, 129.1, 129.1, 129.1, 129.1, 129.0, 128.9, 128.8,
128.8, 128.7, 128.7, 128.7, 128.6, 128.6, 128.5, 128.3, 128.2,
127.7, 127.5, 127.4, 123.9, 123.7, 123.6, 123.1, 77.2, 76.3, 76.1,
72.8, 70.1, 66.2, 58.8, 57.6, 52.8, 45.5, 44.8, 43.7, 39.3, 39.2,
31.9, 30.6, 30.3, 30.1, 29.6, 29.3, 28.8, 28.1, 27.8, 27.7, 27.5,
26.7, 26.2, 25.7, 23.7, 23.4, 23.3, 21.3; HRMS-ESI (m/z): [M
+H]+ calcd. for C78H93ClN9O16: 1446.6423, found 1446.6416.
Desferridanoxamine−Lorabid Conjugate (3b). tetra-O-
Benzyl-trihydroxamate-Lorabid conjugate 15 (110.2 mg, 0.08
1
clear, yellow oil (121.5 mg, 0.09 mmol). H NMR (600 MHz,
CDCl3) δ 8.53 (s, 1 H), 8.07 (d, J = 12.9 Hz, 1 H), 7.52 (d, J =
7.3 Hz, 2 H), 7.43−7.24 (m, 24 H), 6.46 (br s, 1 H), 6.33 (br s,
1 H), 5.39 (s, 2 H), 4.91 (s, 2 H), 4.84 (s, 4 H), 4.47 (s, 2 H),
3.86−3.82 (m, 2 H), 3.76−3.73 (m, 2 H), 3.69−3.58 (m, 6 H),
3.44 (t, J = 6.6 Hz, 2 H), 3.42−3.37 (m, 1 H), 3.31−3.27 (m, 2
H), 3.24−3.16 (m, 6 H), 2.86−2.76 (m, 6 H), 2.70 (t, J = 6.0
Hz, 2 H), 2.47 (t, J = 6.2 Hz, 4 H), 1.68−1.58 (m, 8 H), 1.52−
1.45 (m, 4 H), 1.42−1.24 (m, 8 H), 1.12−1.08 (m, 2 H); 13C
NMR (150 MHz, CDCl3) δ 174.0, 173.8, 173.7, 173.0, 172.9,
172.1, 172.0, 170.4, 165.5, 153.3 (d, JC−F = 249.1 Hz), 148.3,
144.1 (d, JC−F = 10.7 Hz), 138.5, 137.9, 136.4, 134.5, 134.3,
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Article
129.1, 129.0, 128.9, 128.8, 128.8, 128.6, 128.5, 128.3, 127.9,
homogeneous solution. The DMSO solution was then diluted
with sterile broth media to give a solution at 512 μM for both
compounds. Finally, 50 μL of the mixed compound broth
solution was used in the assay exactly as described previously
for single compound experiments.
127.8, 127.5, 127.4, 123.5 (d, JC−F = 6.7 Hz), 113.5 (d, JC−F
=
23.6 Hz), 110.2, 105.1 (d, JC−F = 2.24 Hz), 76.4, 76.3, 76.2,
72.8, 70.1, 66.3, 50.3, 50.2, 49.6, 49.5, 45.7, 45.5, 45.2, 45.1,
44.8, 41.5, 39.3, 34.5, 30.7, 30.5, 29.3, 29.0, 28.5, 28.1, 27.9,
27.3, 27.2, 26.6, 26.5, 26.4, 23.9, 23.6, 23.4, 8.1; HRMS-ESI (m/
z): [M+H]+ calcd. for C79H96FN8O13: 1383.7075, found
1383.7064.
General Procedure for the Agar Diffusion Paper Strip
Siderophore−Sideromycin Competition Assay. A culture
of S. aureus SG511 was grown in LB broth for 18−24 h and a
standard cell suspension of 1.5 × 106 CFU/mL was prepared in
saline solution (0.9% NaCl) according to a 0.5 BaSO4
McFarland Standard.60 The standardized cell suspension (0.1
mL) was added to 34 mL of sterile, melted MHII − Fe iron-
deficient agar tempered to 47−50 °C. After gentle mixing, the
inoculated agar media was poured into a sterile plastic Petri
dish (145 mm × 20 mm) and allowed to solidify near a flame
with the lid cracked for ∼30 min. Solutions of the test
compounds dissolved in a 1:10 mixture of DMSO:MeOH (3a,
3b, and 3c) or sterile, distilled, and deionized H2O (3a-Fe,
DFO-B, FO-B, Lorabid, and ciprofloxacin) were prepared at
the desired concentration (2.0 mM for 3a, 3a-Fe, 3b, DFO-B,
and FO-B; 0.5 mM for 3c; 0.1 mM for Lorabid; 5 μg/mL for
ciprofloxacin). Sterile filter paper strips (Whatman No. 1
Standard grade; cut to ∼1 × 8 cm) were soaked in the test
compound and laid on the surface of the inoculated agar media
as shown in Figure 5. The Petri dishes were incubated at 37 °C
for 18−24 h and photographed after 48 h.
General Procedure for the Quantitative Agar Diffu-
sion Antibiotic Susceptibility Assay. Antibacterial activity
of the compounds was determined by a modified Kirby-Bauer
agar diffusion assay.46,47 Overnight cultures of test organisms
were grown in LB broth for 18−24 h and standard cell
suspensions of 1.5 × 106 CFU/mL were prepared in saline
solution (0.9% NaCl) according to a 0.5 BaSO4 McFarland
Standard.60 Each standardized cell suspension (0.1 mL) was
added to 34 mL of sterile, melted agar (MHII, MHII − Fe, or
MHII + Fe) tempered to 47−50 °C. After gentle mixing, the
inoculated agar media was poured into a sterile plastic Petri
dish (145 mm × 20 mm) and allowed to solidify near a flame
with the lid cracked for ∼30 min. Wells of 9.0 mm diameter
were cut from the Petri dish agar and filled with exactly 50 μL
of the test sample solution. For studies using mixtures of
compounds, both test compounds were diluted to equal
concentrations as one homogeneous solution and 50 μL of the
mixed compound solution was added to the sample well. The
Petri dish was incubated at 37 °C for 18−24 h and the
inhibition zone diameters were measured (mm) with an
electronic caliper after 24−48 h.
Desferridanoxamine−Ciprofloxacin Conjugate (3c). tetra-
O-Benzyl-trihydroxamate-O-benzylciprofloxacin conjugate (16;
55.0 mg, 0.04 mmol) was dissolved in 6 mL of MeOH in an
HCl-washed, 10 mL rb flask sealed under argon. The flask was
charged with 10% Pd−C (15.0 mg) and exposed to a balloon of
hydrogen gas (∼1 atm). Reaction progress was monitored by
RP-C18 TLC (1.5:1 CH3CN:H2O; FeCl3 stain) and after 16 h
there was no remaining starting material (16). The flask was
flushed with argon and the mixture was diluted with MeOH,
vacuum filtered through Celite, and concentrated under
reduced pressure. The resulting solid was dissolved in a
minimal amount of MeOH and precipitated by addition of cold
Et2O. After trituration with Et2O, the desired product (3c) was
obtained in 97% yield as a light yellow solid (36.1 mg, 0.039
mmol). Mp 94−96 °C (color change), 153−160 °C (dec.); 1H
NMR (600 MHz, DMSO-d6) δ 9.66 (br s, N−OH, 1 H), 9.62
(br s, N−OH, 2 H), 8.67 (s, 1 H), 7.93 (d, J = 13.2 Hz, 1 H),
7.78 (t, J = 4.4 Hz, 1 H), 7.58 (d, J = 7.3 Hz, 1 H), 4.35 (t, J =
5.0 Hz, 1 H), 4.04 (s, 1 H), 3.86−3.79 (m, 1 H), 3.74−3.66 (m,
4 H), 3.49−3.41 (m, 6 H), 3.39−3.34 (m, 4 H), 3.31−3.27 (m,
2 H), 3.03−2.95 (m, 4 H), 2.65 (t, J = 6.5 Hz, 2 H), 2.61−2.53
(m, 6 H), 2.30−2.21 (m, 4 H), 1.54−1.45 (m, 6 H), 1.43−1.34
(m, 6 H), 1.34−1.30 (m, 2 H), 1.27−1.16 (m, 8 H); 13C NMR
(150 MHz, DMSO-d6) δ 176.3, 172.0, 171.9, 171.9, 171.3,
171.2, 170.2, 165.9, 152.9 (d, JC−F = 249.1 Hz), 148.1, 144.9 (d,
JC−F = 10.7 Hz), 139.1, 111.0 (d, JC−F = 23.0 Hz), 106.6, 60.6,
60.5, 49.6, 49.6, 49.2, 47.2, 47.1, 47.1, 44.4, 40.8, 38.4, 38.4,
38.3, 38.3, 35.9, 32.2, 32.1, 29.9, 29.8, 28.8, 27.6, 27.2, 26.9,
26.2, 26.1, 26.0, 23.5, 22.7, 7.6; HRMS-ESI (m/z): [M+H]+
calcd. for C44H66N8O14: 933.4728, found 933.4744.
Determination of MIC Values by the Broth Micro-
dilution Method. Antibacterial activity of the compounds was
determined by measuring their minimum inhibitory concen-
trations (MICs) using the broth microdilution method
according to the Clinical and Laboratory Standards Institute
(CLSI, formerly the NCCLS) guidelines.40 Each well of a 96-
well microtiter plate was filled with 50 μL of sterile broth media
(MHII, MHII − Fe, or MHII + Fe). Each test compound was
dissolved in DMSO or H2O making a 20 mM solution, then
diluted with sterile MHII broth to 512 μM. Exactly 50 μL of the
compound solution was added to the first well of the microtiter
plate and 2-fold serial dilutions were made down each row of
the plate. Exactly 50 μL of bacterial inoculum (5 × 105 CFU/
mL) was then added to each well giving a total volume of 100
μL/well and a final compound concentration gradient of 128
μM−0.0625 μM. The plate was incubated at 37 °C for 18 h and
then each well was examined for bacterial growth. The MIC
was recorded as the lowest compound concentration (μM)
inhibiting visible bacterial growth as judged by turbidity of the
culture media relative to a row of wells filled with a DMSO
standard. Ciprofloxacin was included in a control row at a
concentration gradient of 32−0.0156 μM as well as a solvent
control row.
ASSOCIATED CONTENT
■
S
* Supporting Information
List of strains, markers, and origins for microorganisms used in
this work (Table S1). Tabulated biological data collected
during this study (Tables S2−S3). Experimental procedures for
the syntheses of compounds 1a−c, 2a−c, and 11−14. Copies
1
of H NMR and 13C NMR spectra for compounds 1a-c, 2a-c,
3a-c, and 11−16. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
For MIC studies using mixtures of compounds, both test
compounds were diluted to 20 mM in DMSO as one
*Phone (574) 631-7571; fax (574) 631-6652; e-mail
mmiller1@nd.edu.
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Article
mitochondrial sequestration in human cells. J. Am. Chem. Soc. 133,
3260−3263.
Present Address
#Department of Pharmaceutical and Biomedical Sciences,
University of Georgia, Athens, GA 30602, United States.
(5) Silver, L. L. (2008) Are natural products still the best source for
antibacterial discovery? The bacterial entry factor. Expert Opin. Drug
Discovery 3, 487−500.
Notes
The authors declare no competing financial interest.
(6) O’Shea, R., and Moser, H. E. (2008) Physiochemical properties
of antibacterial compounds: Implications in drug discovery. J. Med.
Chem. 51, 2871−2878.
ACKNOWLEDGMENTS
■
(7) Ranny, D. F. (2000) Biomimetic transport and rational drug
delivery. Biochem. Pharmacol. 59, 105−114.
We gratefully acknowledge the use of NMR facilities provided
by the Lizzadro Magnetic Resonance Research Center at The
University of Notre Dame (UND) and the mass spectrometry
services provided by The UND Mass Spectrometry &
Proteomics Facility (Mrs. N. Sevova, Dr. W. Boggess, and Dr.
M. V. Joyce; supported by the National Science Foundation
(8) Christofferson, R. E. (2006) Antibiotics: An investment worth
making? Nat. Biotechnol. 24, 1512−1514.
(9) Braun, V., and Endriβ, F. (2007) Energy-coupled outer
membrane transport proteins and regulatory proteins. Biometals 20,
219−231.
́ ́
(10) Ji, C., Juarez-Hernandez, R. E., and Miller, M. J. (2012)
̌ ́
under CHE-0741793). We thank Dr. Viktor Krchnak (UND)
Exploiting bacterial iron acquisition: siderophore conjugates. Future
Med. Chem. 4, 297−313.
for assistance with HPLC. We are grateful to Prof. Shahriar
Mobashery (UND) and Dr. Sergei Vakulenko (UND) for
providing the ESKAPEE panel of bacteria and Dr. Nuno Tiago
GiaoAntunes (UND) for determination of some preliminary
MIC values against the panel. TAW gratefully acknowledges
The UND Chemistry-Biochemistry-Biology (CBBI) Interface
Program funded by the NIH (T32GM075762) for three years
of fellowship support and funding an internship at the Hans
(11) Roosenberg, J. M., Lin, Y.-M., Lu, Y., and Miller, M. J. (2000)
Studies and syntheses of siderophores, microbial iron chelators, and
analogs as potential drug delivery agents. Curr. Med. Chem. 7, 159−
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Knoll Institute (HKI) in Jena, Germany to learn micro-
̈
biological assays. T.A.W. sincerely thanks the HKI for hosting
such an internship. T.A.W. also thanks the UND Department
of Chemistry and Biochemistry (Grace Fellowship), the UND
Center for Environmental Science and Technology (CEST),
and Bayer for additional fellowship support. We acknowledge
The University of Notre Dame and the NIH for financial
support of this work.
(16) Challis, G. L., and Hopwood, D. A. (2003) Synergy and
contingency as driving forces for the evolution of multiple secondary
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ABBREVIATIONS
■
(17) Braun, V., Pramanik, A., Gwinner, T., Koberle, M., and Bohn, E.
̈
ABC, ATP-binding cassette transporter; acac, acetylacetonate;
ATCC, American Type Culture Collection; Bn, benzyl; Boc,
tert-butoxycarbonyl; CFU, colony forming unit; DFO-B,
desferrioxamine B; DMAP, 4-dimethylaminopyridine; DMF,
dimethylformamide; DMSO, dimethylsulfoxide; ESI, electro-
spray ionization; ExbB/D, inner membrane bound protein of
TonB complex; FO-B, ferrioxamine B; Fur, ferric uptake
regulator protein; HPLC, high-performance liquid chromatog-
raphy; HRMS, high-resolution mass spectrometry; MHII,
Mueller−Hinton media no. II; MIC, minimum inhibitory
concentration; NMR, nuclear magnetic resonance; OMR, outer
membrane receptor protein; OMT, TonB-dependent outer
membrane siderophore transport protein; PNB, para-nitro-
benzyl; Red, iron reductase enzyme; SPBP, siderophore
periplasmic binding protein; TFA, trifluoroacetic acid; TLC,
thin layer chromatography; TonB, membrane-spanning protein
complex involved in siderophore transport
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