Chemical syntheses and in vitro antibacterial activity of two desferrioxamine B-ciprofloxacin conjugates with potential esterase and phosphatase triggered drug release linkers

Article abstract of DOI:10.1016/j.bmc.2012.04.034

Two desferrioxamine B-ciprofloxacin conjugates with 'trimethyl-lock' based linkers that are designed to release the antibiotic after esterase or phosphatase-mediated hydrolysis were synthesized. The potential esterase-sensitive conjugate 13 displayed mode

Full text of DOI:10.1016/j.bmc.2012.04.034

Bioorganic & Medicinal Chemistry 20 (2012) 3828–3836
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemical syntheses and in vitro antibacterial activity of two desferrioxamine
B-ciprofloxacin conjugates with potential esterase and phosphatase
triggered drug release linkers
⇑
Cheng Ji, Marvin J. Miller
Department of Chemistry and Biochemistry, 251 Nieuwland Science Hall, University of Notre Dame, Notre Dame, Indiana, 46556, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Two desferrioxamine B-ciprofloxacin conjugates with ‘trimethyl-lock’ based linkers that are designed to
release the antibiotic after esterase or phosphatase-mediated hydrolysis were synthesized. The potential
esterase-sensitive conjugate 13 displayed moderate to good antibacterial activities against selected fer-
rioxamine-utilizing bacteria, although the activities were lower than the parent drug ciprofloxacin. How-
ever, the potential phophatase-sensitive conjugate 23 was inactive against the same panel of organisms
tested. These properties appeared to be related to the activating efficiency of the linker by the enzyme
and to the outer membrane protein recognition of the chemically modified siderophore used in the
conjugate.
Received 28 February 2012
Revised 10 April 2012
Accepted 17 April 2012
Available online 2 May 2012
Keywords:
Iron transport
Antibiotics
Siderophore conjugates
Drug delivery
Drug release
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
recognized as a suitable target for developing new antibiotic strat-
egies. For example, using siderophore-drug conjugates as a ‘Trojan
The emergence of pathogenic bacteria that are resistant to
existing antibiotics has increased at an alarming rate.1,2 Due to
the misuse and overuse of antibiotics, bacteria have developed var-
ious mechanisms for resistance, including (1) changing membrane
permeability via porin modification; (2) decreasing intracellular
drug concentration by efflux pump systems and (3) enzymatically
deactivating the antibiotic.3 (Fig. 1) Among these modes of resis-
tance, the lack of cell wall permeability resulting from a limitation
of passive diffusion through size restricted porins is especially sig-
nificant.4 Therefore, the development of effective and efficient drug
delivery processes for combating porin-mediated antibiotic resis-
tance is of particular interest.
One attractive strategy for antibiotic delivery is to exploit the
microbial iron acquisition system which is essential to all patho-
genic bacteria. Iron is a crucial element for almost all forms of life.
At physiological pH, however, the bioavailability of iron is
restricted by its low solubility. To assimilate iron for survival,
microorganisms synthesize and secrete low molecular weight iron
chelators called siderophores which bind iron(III) effectively by
forming high affinity complexes. The resulting siderophore-iro-
n(III) complexes can be recognized by specific outer membrane
proteins and then be actively transported into the cell via certain
protein channels.5 The indispensable iron uptake system has been
Horse’ for the delivery of antibiotics can bypass the membrane-
associated resistance mechanism as the conjugates are actively
transported by the iron uptake system in the target microbe. This
‘Trojan Horse’ approach has been adopted by certain bacteria dur-
ing evolution to compete with other microorganisms for necessary
nutrients. Naturally occurring siderophore-drug conjugates (sider-
omycins) such as albomycins6–8 and salmycins9,10 are found to be
effective antibacterial agents and to enter bacterial cells via active
iron uptake pathways. (Scheme 1)
Structurally, a typical siderophore-drug conjugate consists of
three parts: siderophore, linker and drug. (Scheme 1) For a long
time, the linker was considered as a subunit of the drug. However,
it actually is a key component of the conjugate. In some cases in
which the drug must be cleaved from the siderophore moiety for
optimum potency, incorporation of a linker with a drug release
function in the conjugate becomes crucial. In the aforementioned
albomycins, after being internalized by non-producing strains of
bacteria, the drug-moiety, namely the thionucleoside, is enzymat-
ically cleaved by the action of a serine peptidase N. The drug-re-
lease process is essential for antibiotic activity of albomycins as
microorganisms lacking serine peptidase N to cleave the drug are
not albomycin-sensitive.6–8 Intracellular drug release has also been
shown to be necessary for maximum activity of synthetic sidero-
phore-drug conjugates. For example, fluoroquinolones have been
conjugated to pseudomonal siderophores pyoverdin and pyochelin
to target Pseudomonas aeruginosa.11–13 These conjugates displayed
⇑
Corresponding author.
E-mail address: mmiller1@nd.edu (M.J. Miller).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.034

C. Ji, M. J. Miller / Bioorg. Med. Chem. 20 (2012) 3828–3836
3829
Figure 1. Antibiotic resistance in bacteria and iron-transport mediated drug delivery (the ‘Trojan Horse’ strategy).
Scheme 1. General siderophore drug conjugates and two naturally occurring examples.
greatest activity with a hydrolysable linkage that releases the anti-
biotic, however, in a random manner. Therefore, novel linkers with
drug release function controlled by a biochemical process need to
be further explored.
We reasoned that a ‘trimethyl lock’ induced lactonization trig-
gered by the action of enzymes such as esterases/phosphatases
could be used for the drug release process. The chemical structure
of the ‘trimethyl lock’ 1 is an o-hydroxycinnamic acid derivative in
which unfavorable steric interactions between the three methyl
groups encourage rapid lactonization to form a hydrocouma-
rin.14–16 The rate enhancements resulting from the ‘trimethyl lock’
are on the order of 105 relative to an unsubstituted dihydrocoum-
arin acid.14,17,18 The ‘trimethyl lock’ structure has been applied to
several prodrug strategies, including those sensitive to esterases
Scheme 2. ‘Trimethyl lock’ derived siderophore-antibiotic conjugates.

3830
C. Ji, M. J. Miller / Bioorg. Med. Chem. 20 (2012) 3828–3836
esterase triggered conjugate started with TBDMS-protected diol 6
which was prepared from 3,5-dimethylphenol according to the
methods of Borchardt.21 DCC-mediated coupling of 6 with monob-
enzyl succinate provided the TBDMS ether 7 in 77% yield. Treat-
ment of 7 with a mixture of acetic acid, THF and water removed
the TBDMS group to give the primary alcohol 8. A two-step oxida-
tion with PCC and sodium chlorite proceeded in excellent yield to
convert alcohol 8 to acid 10 which was ready to couple with cipro-
floxacin. To increase the coupling efficiency and simplify the puri-
fication, ciprofloxacin was converted to its benzyl ester. Coupling
of acid 10 with O-benzyl ciprofloxacin using EDC and HOBt affor-
ded ciprofloxacin derivative 11 in 70% yield. Both benzyl protecting
groups in 11 were removed by hydrogenolysis to give acid 12,
which was coupled with desferrioxamine to yield potential ester-
ase triggered desferrioxamine B-ciprofloxacin conjugate 13
(Scheme 4).
The synthesis of the intended phosphatase triggered conjugate
started from lactone 14, which was prepared using chemistry
developed by Carpino.32 After protection of the free phenolic hy-
droxyl group in 14 with a TBDMS group, reductive ring opening
of the protected lactone 15 afforded alcohol 16 in moderate yield.
Using NaH as the base, selective phosphorylation of the phenol in
16 was achieved with tetrabenzyl pyrophosphate33 to give alcohol
17. Similar to the process used to make the potential esterase trig-
gered conjugate, treatment of 17 with PCC followed by sodium
chlorite provided acid 19. EDC-mediated coupling of 19 with O-
benzyl ciprofloxacin generated the ciprofloxacin derivative 20 in
59% yield. The silyl protecting group was removed with KF to ex-
pose the phenolic hydroxyl group in 21, which served as the cou-
pling site with O-benzyl desferrioxamine succinic acid to yield
the protected conjugate 22. Global deprotection of 22 by hydrog-
enolysis produced the potential phosphatase triggered desferriox-
amine B-ciprofloxacin conjugate 23. (Scheme 5) As a control
compound, conjugate 26 with a succinyl linkage which is stable
under physiological conditions was also synthesized and included
in the biological studies (Scheme 6).
Scheme 3. Structures of desferrioxamine B (DFO) and Ciprofloxacin (Cip).
and phosphatases.19–27 In bacteria and fungi, esterases and phos-
phatases are known to be not only widely distributed but also of-
ten substrate non-specific, which led to the hypothesis that they
have evolved to enable access to carbon or phosphorus sources
for uptake and use by the microbes.28,29 Therefore, linkers with a
‘trimethyl lock’ structure triggered by potential esterases and
phosphatases could be incorporated in a ‘Trojan Horse’ approach
where a siderophore is attached to an acyl or phosphoryl phenolic
derivative of the ‘trimethyl lock’ structure 1, and a drug is con-
densed with the carboxyl group as an ester or amide to give sider-
ophore-antibiotic conjugate 2. Enzymatic hydrolysis of conjugate 2
with potential esterases or phosphatases could produce the tran-
sient ‘trimethyl lock’ structure 3 which is anticipated to undergo
rapid lactonization with concomitant release of the drug.
(Scheme 2)
Our choice for drug attachment was ciprofloxacin (Scheme 3), a
quinolone antibiotic used to treat a wide variety of both Gram-neg-
ative and Gram-positive bacteria infections.30 It targets bacterial
DNA gyrase and DNA topoisomerase IV, both of which are required
for cell growth and division. Previous studies showed that some
siderophore-fluoroquinolone conjugates are up to 50 times less ac-
tive than the parent drug in terms of gyrase inhibition activity,
most likely due to decreased target recognition from steric hin-
drance caused by the presence of the siderophore in the conju-
gate.12 Therefore, an intracellular drug release process to ensure
efficacy of the conjugates is highly preferred. Desferrioxamine B
(Scheme 3) was selected as the siderohore in the conjugates. It is
an important and well-characterized trihydroxamate siderophore
produced by several species of Nocardia, Streptomyces, Micromonos-
pora, Arthrobacter, Chromobacterium, and Pseudomonas, and is uti-
lized by a variety of other bacteria and fungi.31 Herein we
describe the syntheses of two desferrioxamine B-ciprofloxacin con-
jugates with potential esterase and phosphatase triggered ‘tri-
methyl lock’ linkers, respectively, with the aim of exploring their
antibacterial activities and possible drug release mechanisms.
2.2. Biological studies
The desferrioxamine B-ciprofloxacin conjugates 13 and 23 were
evaluated for their ability to inhibit the growth of bacteria using
the agar well diffusion test. Representative Gram-positive strains
included Bacillus subtilis, Staphylococcus aureus and Micrococcus lu-
teus. The Gram-negative strains studied included a wild type
(K799/wt) and a penetration mutant strain (K799/61) of Pseudomo-
nas aeruginosa and an Escherichia coli test strain. Previous studies
showed that desferrioxamine B is a growth promoter of all the
strains used in the assay except E. coli. The parent drug ciprofloxa-
cin and conjugate 26 with a stable succinyl linker were also in-
cluded in the assay as controls.
2. Results and discussion
The results of assays for antibacterial activity (Table 1) showed
that all three conjugates displayed moderate activities against the
E. coli strain, indicating that they can reach their cellular target
regardless of the drug being released or not. Conjugate 26 with a
stable succinyl linker has moderate activity against both B. subtilis
and S. aureus but showed virtually no activity against M. luteus,
against which ciprofloxacin itself is only weakly active. A similar
activity profile was observed in the structurally related desferrida-
noxamine B-ciprofloxacin conjugate.34 Conjugate 13 with the po-
tential esterase triggered linker was almost equipotent relative to
conjugate 26 against Gram-positive strains and the P. aeruginosa
penetration mutant strain. However, in the case of wild type P.
aeruginosa, conjugate 13 displayed higher activity (larger inhibi-
tion zone) than conjugate 26 which is completely inactive, suggest-
ing that the drug was partially released, although the possibility of
hydrolysis caused by some extracellular esterase cannot be ruled
2.1. Synthesis of conjugates
In the design of the potential esterase triggered conjugate, the
phenolic oxygen in the linker was masked with a succinyl group
with the desferrioxamine attached. In the potential phosphatase
triggered conjugate, the phenolic oxygen was masked with a phos-
phate group while the desferrioxamine was attached on an addi-
tional phenolic oxygen at the para position. We anticipated that
both conjugates could restore the ‘trimethyl lock’ structure after
the masking acyl or phosphoryl groups were enzymatically cleaved
to release the ciprofloxacin.
Due to the facile lactonization of the ‘trimethyl lock’ system, the
incorporation of the linker into the conjugates required derivatiza-
tion of the phenolic hydroxyl group. Therefore, synthesis of the

C. Ji, M. J. Miller / Bioorg. Med. Chem. 20 (2012) 3828–3836
3831
Scheme 4. Synthesis of the potential esterase triggered desferrioxamine B-ciprofloxacin conjugate 13. Reagents and conditions: (a) Monobenzyl succinate, DCC, DMAP,
CH2Cl2, rt (ca. 23 °C), 77%; (b) AcOH/THF/H2O = 3:3:1, rt, 97%; (c) PCC, CH2Cl2, rt, 82%; (d) NaClO2, H2O2, NaH2PO4, CH3CN/H2O, 0 °C-rt, 97%; (e) EDC, HOBt, O-benzyl
ciprofloxacin, Et3N, DMAP, CH2Cl2, rt, 70%; (f) Pd/C, H2 balloon, rt, 86%; (g) EDC, NHS, DMF then DFO mesylate, Et3N, 47%.
Scheme 5. Synthesis of the potential phosphatase triggered desferrioxamine B-ciprofloxacin conjugate 23. Reagents and conditions: (a) TBDMSCl, DBU, CH2Cl2, 90%; (b)
LiAlH4, THF, rt-reflux, 51%; (c) NaH, tetrabenzyl pyrophosphate, THF–DMF, 0 °C-rt, 53%; (d) PCC, CH2Cl2, rt, 78%; (e) NaClO2, H2O2, NaH2PO4, CH3CN/H2O, 0 °C rt-99%; (f) EDC,
HOBt, O-benzyl ciprofloxacin, Et3N, DMAP, CH2Cl2, rt, 59%; (g) KF, DMF–H2O, rt, 74%; (h) EDC, O-benzyl desferrioxamine succinic acid 24, DMF, rt, 45%; (i) Pd/C, H2 balloon,
THF–H2O, 35%.
out.35 In all the strains tested, conjugate 13 was less active than the
parent drug ciprofloxacin, implying that 13 may be a poor sub-
strate for microbial esterases. Conjugate 23 with the phosphatase
triggered linker was not active against all the strains in the panel
tested except the E. coli penetration mutant, suggesting that this
conjugate was no longer recognized by siderophore outer
membrane receptors thus could not enter bacterial cells via the
iron uptake pathway.

3832
C. Ji, M. J. Miller / Bioorg. Med. Chem. 20 (2012) 3828–3836
Scheme 6. Synthesis of the desferrioxamine B-ciprofloxacin conjugate 26 with a stable linker. Reagents and conditions: (a) EDC, HOBt, O-benzyl ciprofloxacin, Et3N, DMAP,
CH2Cl2, rt, 75%; (b) Pd/C, H2 balloon, MeOH, 82%.
Table 1
Diameter of growth inhibition zones (mm) in the agar diffusion antibacterial susceptibility assay
Compds
Gram-positive bacteria
Staphylococcus aureus
SG 511
Gram-negative bacteria
Pseudomonas aeruginosa
Bacillus subtilis
Micrococcus luteus
Escherichia coli
ATCC 6633
ATCC 10240
K799/wt
K799/61
X580
Ciprofloxacin
28b
21
26
0
28a
18
16
0
12Pa
21b
0
26
0
24/32pb
26P
27/37p
0
31c
30/33p
29
26
13
23
0
0
0
21
p, partially clear inhibition zone/colonies in the inhibition zone; P, unclear inhibition zone/many colonies in the inhibition zone.
Exactly 50 L of a 0.2 mM solution of each compound dissolved in 1:9 DMSO/MeOH was filled in 9 mm wells in agar media (Standard I Nutrient Agar, Serva or Mueller Hinton
II Agar, Becton, Dickinson and Company). Inhibition zones read after incubation at 37 °C for 24 h.
l
a,b,c
Ciprofloxacin was tested at (a) 5 lg/mL, (b) 1.66 lg/mL, (c) 0.33 lg/mL in H2O.
Table 2
MIC90 values of conjugate 13 and ciprofloxacin against ESKAPEE panel of bacteria
Compds
MIC90(l
M) Against ESKAPEE Test Organismsa,b
E. faecium
S. aureus
K. pneunonia
A. baumanii
P. aeruginosa
E. aerogenes
E. coli
NCTC 7171
ATCC 29213
ATCC 700603
ATCC 17961
ATCC 27853
ATCC 35029
ATCC 25922
13
>128
8
32
NT
16
0.25
8
0.25
2
1
1
Ciprofloxacin
0.125
<0.015
<0.015
NT, not tested.
a
MIC90 values (lM) were determined using the broth microdilution method in Mueller-Hinton broth No.2 (MHII) with visual end point analysis according to the CLSI
guidelines.36
b
Each compound was tested in triplicate.
Conjugate 13 was also tested for antibacterial activity against a
panel of ESKAPEE bacteria2 by determining their minimum inhibi-
tory concentrations (MIC90) using the broth microdilution assay
(Table 2). Conjugate 13 displayed moderate to good antibacterial
rial strains, but the activity was weaker than that of the parent
drug ciprofloxacin, most likely because 13 is a poor substrate for
the esterase. The potential phosphatase triggered conjugate 23
was inactive in the same panel of bacteria tested, probably due
to the failure of using iron uptake pathway to enter bacterial cells.
To further develop siderophore-drug conjugates with microbial
triggered linkers, an appropriate choice of siderophore to ensure
active transport after chemical modification is essential. Mean-
while, linkers that are truly inert to the extracellular environment
of bacterial growth cultures but only selectively activated inside
microbial cells are required. Taking both factors into consideration,
new conjugates based on the ‘trimethyl lock’ concept are being
synthesized and their biological studies will be reported in due
course.
activity (1–32
lM) against all the strains except E. faecium
(>128 M). In all the strains tested, conjugate 13 showed reduced
l
activities compared to the free drug ciprofloxacin, which is consis-
tent with the trend observed in the agar diffusion assay, further
suggesting that conjugate 13 might be a poor substrate for the
esterases needed to release the drug.
3. Conclusion
Two desferrioxamine B-ciprofloxacin conjugates with potential
esterase and phosphatase triggered linkers were synthesized. The
linkers, with a ‘trimethyl-lock’ structure, were designed to release
the antibiotic by a fast and spontaneous lactonization process after
enzymatic hydrolysis of the conjugates. Thus, the conjugates had
the potential to use active iron transport processes to deliver the
antibiotic to bacteria cells and release the drug intracellularly to
negate potential steric hindrance due to the presence of the sider-
ophore-linker moiety. The potential esterase triggered conjugate
13 had moderate to good antibiotic activity against several bacte-
4. Experimental section
4.1. General
All reactions were carried out under argon by using standard
techniques. All solvents and reagents were obtained from commer-
cial sources and used without further purification unless otherwise
stated. NMR spectra were recorded on a Varian Unityplus 300 MHz

C. Ji, M. J. Miller / Bioorg. Med. Chem. 20 (2012) 3828–3836
3833
spectrometer, Varian Inova 500 MHz spectrometer, or Varian
600 MHz spectrometer at ambient temperature. Silica gel column
chromatography was performed using Sorbent Technologies silica
138.0, 137.1, 135.8, 133.0, 132.8, 128.8, 128.5, 128.4, 123.4, 66.9,
56.8, 38.3, 31,8, 30.1, 29.1, 26.1, 25.6, 20.4; HRMS (ESI) calcd for
C
24H28NaO5 (M+Na)+: 419.1815, found 419.1829.
gel 60 (32–63 lM). Reverse Phase C18 Silica Gel was a generous
gift from Eli Lilly and Co.
4.1.4. 3-(2-(4-(Benzyloxy)-4-oxobutanoyloxy)-4,6-
The following compounds were prepared according to pub-
lished procedures: TBDMS-protected diol 6,21 lactone 14,32 tetrab-
enzyl pyrophosphate,33 O-benzyl ciprofloxacin,37 O-benzyl
desferrioxamine succinic acid 24.38
dimethylphenyl)-3-methylbutanoic acid (10)
To a solution of aldehyde 9 (4.76 g, 12.0 mmol) in 12 mL of
CH3CN and 6 mL of H2O, were added sodium dihydrogen phos-
phate (450 mg, 3.26 mmol) and 30% H2O2 (1 mL). The mixture
was cooled to 0 °C and a solution of sodium chlorite (2.00 g,
22.2 mmol) in 18 mL of H2O was added slowly. The reaction was
kept at 0 °C for 1 h and warmed to room temperature. Then
30 mL of sat. Na2S2O3 was added slowly to destroy excess H2O2
and sodium chlorite. The solution was acidified with 3 N HCl to
pH = 1–2 and extracted with EtOAc (200 mL ꢁ 3). The combined
organic layers were washed sequentially with H2O, brine, dried
over Na2SO4 and evaporated to give acid 10 (4.82 g, 97%) as a light
4.1.1. Benzyl 2-(4-(tert-butyldimethylsilyloxy)-2-methylbutan-
2-yl)-3,5-dimethylphenyl succinate (7)
Monobenzyl succinate (4.35 g, 20.9 mmol) was dissolved in
200 mL of anhydrous dichloromethane. The solution was cooled
to 0 °C and DCC (4.70 g, 23.0 mmol) was added in three portions.
The solution was kept at 0 °C for 10 min before
6 (8.09 g,
25.1 mmol) and DMAP (25 mg, 0.21 mmol) were added. The reac-
tion mixture was warmed to room temperature (ca. 23 °C). After
stirring at room temperature for 16 h, the solution was filtered to
remove the precipitated DCU. The filtrate was washed sequentially
with sat. NaHCO3, 5% HCl, brine, dried over Na2SO4 and filtered.
The solvent was removed under vacuum to give the crude product
which was purified by chromatography on a silica gel column (10%
ethyl acetate in hexanes) to give 7 (8.68 g, 77%) as a colorless oil: IR
(thin film) 2953, 2927, 2855, 1740, 1472 cmꢀ1; 1H NMR (300 MHz,
CDCl3) d 7.36–7.47 (m, 5H), 6.81 (s, 1H), 6.54 (s, 1H), 5.17 (s, 2H),
3.48 (t, J = 7.5 Hz, 2H), 2.78–2.91 (m, 4H), 2.53 (s, 3H), 2.22 (s,
3H), 2.04 (t, J = 7.5 Hz, 2H), 1.58 (s, 6H), 0.85 (s, 9H), ꢀ0.02 (s,
6H); 13C NMR (75 MHz, CDCl3) d 172.2, 171.6, 149.9, 138.6,
136.2, 135.9, 134.2, 132.6, 128.8, 128.5, 128.4, 123.2, 66.9, 61.0,
46.2, 39.2, 32.0, 30.1, 29.3, 26.2, 25.5, 20.4, 18.4, ꢀ5.1; HRMS
(ESI) calcd for C30H45O5Si (M+H)+: 513.3031, found 513.3015.
yellow oil: IR (thin film) 2926, 2850, 1772, 1739, 1647, 1473 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 7.33–7.38 (m, 5H), 6.82 (s, 1H), 6.58 (s,
1H), 5.18 (s, 2H), 2.89–2.94 (m, 2H), 2.84 (s, 2H), 2.78–2.83 (m, 2H),
2.54 (s, 3H), 2.22 (s, 3H), 1.58 (s, 6H); 13C NMR (75 MHz, CDCl3) d
177.4, 172.2, 171.6, 149.5, 138.2, 136.5, 135.9, 133.5, 132.7,
128.8, 128.5, 128.4, 123.1, 66.9, 47.7, 38.8, 31.4, 30.0, 29.2, 25.4,
20.4; HRMS (ESI) calcd for C24H28NaO6 (M+Na)+: 435.1778, found
435.1760.
4.1.5. O-Bn ciprofloxacin conjugate (11)
To a solution of acid 10 (500 mg, 1.21 mmol) in 20 mL of anhy-
drous CH2Cl2 cooled to 0 °C were added EDC (470 mg, 2.45 mmol)
and HOBt (331 mg, 2.45 mmol). The mixture was stirred at that
temperature for 5 min before O-benzyl ciprofloxacin (530 mg,
1.26 mmol), Et3N (0.67 mL, 4.80 mmol) and DMAP (5 mg) were
added. The reaction mixture was warmed to room temperature
and stirred for 18 h. The mixture was diluted with 30 mL of CH2Cl2
and the organic layer was washed sequentially with water, sat.
NaHCO3, brine, dried over Na2SO4 and filtered. The solvent was re-
moved under vacuum and the residue was purified by chromatog-
raphy on a silica gel column (5% methanol in CH2Cl2) to give 11
(690 mg, 70%) as a light yellow oil: 1H NMR (600 MHz, CDCl3) d
8.52 (s, 1H), 8.03 (d, J = 13.20 Hz, 1H), 7.29–7.54 (m, 10H), 7.19
(d, J = 7.04 Hz, 1H), 6.82 (d, J = 2.05 Hz, 1H), 6.57 (d, J = 2.05 Hz,
1H), 5.38 (s, 2H), 5.15 (s, 2H), 3.76 (br. s., 2H), 3.49 (br. s., 2H),
3.38 (m, 1H), 3.12 (br. s., 2H), 3.04 (br. s., 2H), 2.85–2.93 (m, 4H),
2.78–2.84 (m, 2H), 2.57 (s, 3H), 2.20 (s, 3H), 1.62 (s, 6H), 1.28 (m,
2H), 1.09 (m, 2H); 13C NMR (150 MHz, CDCl3) d 173.1, 172.0,
171.3, 170.0, 165.4, 154.1, 152.4, 149.5, 148.4, 144.1, 138.2,
137.9, 136.4, 136.2, 135.5, 134.2, 132.5, 128.6, 128.5, 128.3,
128.1, 128.0, 127.9, 123.3, 122.9, 113.4, 110.1, 104.9, 66.7, 66.4,
50.3, 49.4, 45.8, 45.0, 41.2, 39.5, 34.5, 32.0, 30.0, 28.9, 25.6, 22.6,
20.2, 8.1; HRMS (ESI) calcd for C48H51FN3O8 (M+H)+: 816.3655,
found 816.3627.
4.1.2. Benzyl 2-(4-hydroxy-2-methylbutan-2-yl)-3,5-
dimethylphenyl succinate (8)
Silyl ether 7 (8.50 g, 16.6 mmol) was dissolved in a mixture of
50 mL of THF, 50 mL of H2O and 150 mL of AcOH. The solution
was vigorously stirred at room temperature for 3 h. After removal
of the solvents under vacuum, the residue was dissolved in 100 mL
of EtOAc and washed sequentially with water, sat. NaHCO3, brine,
dried over Na2SO4 and filtered. The combined organic layers were
evaporated to give alcohol 8 (6.40 g, 97%) as a colorless oil: IR (thin
film) 2925, 1736, 1615, 1473 cmꢀ1 1H NMR (300 MHz, CD3OD) d
;
7.29–7.37 (m, 5H), 6.84 (s, 1H), 6.52 (s, 1H), 5.16 (s, 2H), 3.37 (t,
J = 6.9 Hz, 2H), 2.86–2.90 (m, 2H), 2.75–2.79 (m, 2H), 2.52 (s, 3H),
2.19 (s, 3H), 2.05 (t, J = 6.9 Hz, 2H), 1.46 (s, 6H); 13C NMR
(75 MHz, CD3OD) d 173.9, 173.7, 151.4, 139.6, 137.6, 137.5,
135.1, 133.4, 129.7, 129.3, 124.4, 67.7, 60.7, 40.1, 32.6, 31.0, 30.0,
25.6, 20.4; HRMS (ESI) calcd for C24H30NaO5 (M+Na)+: 421.1985,
found 421.1981.
4.1.3. Benzyl 3,5-dimethyl-2-(2-methyl-4-oxobutan-2-yl)phenyl
succinate (9)
4.1.6. Ciprofloxacin conjugate (12)
Alcohol 8 (6.33 g, 15.9 mmol) was dissolved in 200 mL of
dichloromethane at room temperature under argon. PCC (6.87 g,
31.8 mmol) was added in three portions. The dark slurry was stir-
red at room temperature for 3 h. After removal of the solvent, the
residue was suspended in 30 mL of dichloromethane and filtered
through a short silica gel column. The column was further washed
with 20% ethyl acetate in hexanes. The solvents were removed un-
der reduced pressure to give aldehyde 9 (5.17 g, 82%) as a yellow
Conjugate 11 (122 mg, 0.17 mmol) was dissolved in 5 mL of 10%
H2O in THF under argon. 10% Pd/C (10 mg) was added and the flask
was flushed with H2 gas and left to stir under a hydrogen atmo-
sphere (balloon) at room temperature for 3 h. After purging with
argon, the reaction mixture was filtered over Celite and the solvent
was evaporated to give acid 12 (92 mg, 86%) as a light yellow solid:
mp = 128–129 °C (dec.); IR (thin film) 2922, 2853, 1729, 2626,
1466 cmꢀ1 1H NMR (300 MHz, CD3OD) d 8.78 (s, 1H), 7.89 (d,
;
oil: IR (thin film) 2922, 2851, 1736, 1352 cmꢀ1
;
1H NMR
J = 13.2 Hz), 7.53 (bs, 1H), 6.84 (s, 1H), 6.63 (s, 1H), 3.66–3.79 (m,
3H), 3.58 (s, 2H), 3.26 (s, 2H), 3.13 (s, 2H), 2.96 (s, 2H), 2.86–2.90
(m, 2H), 2.70–2.74 (m, 2H), 2.57 (s, 3H), 2.18 (s, 3H), 1.63 (s, 6H),
1.42 (s, 2H), 1.21 (s, 2H)); 13C NMR (150 MHz, DMSO-d6) d 176.4,
173.9, 169.7, 168.0, 165.9, 153.8, 152.1, 151.1, 148.1, 145.0,
(300 MHz, CDCl3) d 9.54 (t, J = 2.4 Hz, 1H), 7.32–7.37 (m, 5H),
6.86 (s, 1H), 6.60 (s, 1H), 5.17 (s, 2H), 2.87–2.92 (m, 2H), 2.83 (d,
J = 2.4 Hz, 2H), 2.78–2.83 (m, 2H), 2.54 (s, 3H), 2.23 (s, 3H), 1.57
(s, 6H); 13C NMR (75 MHz, CDCl3) d 203.1, 172.1, 171.6, 149.5,

3834
C. Ji, M. J. Miller / Bioorg. Med. Chem. 20 (2012) 3828–3836
144.9, 139.2, 136.9, 136.0, 129.0, 126.8, 118.8, 115.4, 111.1, 110.9,
106.8, 106.7, 49.6, 49.2, 44.6, 44.3, 40.8, 35.9, 34.5, 28.9, 28.7, 28.5,
27.4, 27.2, 22.6, 20.1, 7.6; HRMS (ESI) calcd for C34H39FN3O8
(M+H)+: 636.2716, found 636.2723.
(t, J = 6.7 Hz, 2H), 2.30 (s, 3H), 2.19 (t, J = 6.7 Hz, 2H), 2.11 (s, 3H),
1.56 (s, 6H), 1.03 (s, 9H), 0.14 (s, 6H); 13C NMR (150 MHz, CDCl3)
d 149.3, 146.8, 130.6, 129.6, 127.1, 117.7, 61.8, 45.1, 40.0, 32.5,
26.3, 18.9, 18.6, 17.6, ꢀ3.0; HRMS (ESI) calcd for C19H35O3Si
(M+H)+: 339.2350, found 339.2326.
4.1.7. Ciprofloxacin conjugate (13)
To a solution of acid 12 (80 mg, 0.12 mmol) in anhydrous
CH2Cl2 (5 mL) were added EDC (46 mg, 0.24 mmol) and N-
hydroxysuccinimide (28 mg, 0.24 mmol). The reaction mixture
was stirred at room temperature for 18 h and diluted with 20 mL
of CH2Cl2. The solution was sequentially washed with sat. NaHCO3,
water, brine, dried over Na2SO4 and filtered. Removal of the solvent
in vacuo to give the crude NHS ester as a yellow foam which was
4.1.10. Dibenzyl-(4-((tert-butyldimethylsilyl)oxy)-2-(4-hydroxy-
2-methylbutan-2-yl)-3,5-dimethylphenyl) phosphate (17)
To a solution of 16 (1.63 g, 4.81 mmol) in anhydrous THF/
DMF = 4:1 (20 mL) at 0 °C was added NaH (60% dispersion in min-
eral oil, 0.21 g, 5.25 mmol). The mixture was kept at that temper-
ature for 30 min before tetrabenzyl pyrophosphate (3.88 g,
7.21 mmol) was added in one portion. The mixture was warmed
to room temperature and stirred for additional 8 h. The reaction
was quenched by adding sat. NH4Cl (100 mL). The aqueous layer
was extracted with EtOAc (50 mL ꢁ 3). The combined organic lay-
ers were washed with brine, dried over Na2SO4, filtered and the
solvent was removed under reduced pressure. The crude oil was
purified by chromatography on a silica gel column (hexanes/
EtOAc = 2:1) to yield 17 as a clear oil (1.54 g, 53%): 1H NMR
(600 MHz, CDCl3) d 7.32–7.37 (m, 10H), 7.00 (s, 1H), 5.07–5.14
(m, 4H), 3.52 (t, J = 7.6 Hz, 2H), 2.31 (s, 3H), 2.12 (t, J = 7.6 Hz,
2H), 2.07 (s, 3H), 1.50 (s, 6H), 1.02 (s, 9H), 0.15 (s, 6H); 13C NMR
(150 MHz, CDCl3) d 149.9, 144.0, 135.6, 134.6, 130.1, 128.6,
128.0, 127.0, 120.2, 69.8, 60.6, 46.1, 39.8, 32.2, 26.1, 18.7, 18.5,
17.5, ꢀ3.2; HRMS (ESI) calcd for C33H47O6PSi (M+H)+: 599.2952,
found 599.2942.
used directly in the next step. Desferrioxamine
B mesylate
(40 mg, 0.06 mmol) and triethylamine (34 L, 0.24 mmol) were
l
dissolved in 10 mL anhydrous DMF (gentle heating was required
to generate a homogeneous solution), and the NHS ester of 12
was added in one portion. The solution was stirred at room tem-
perature for 18 h and the solvent was removed under reduced
pressure. The residue was purified by chromatography on a re-
verse-phase silica gel column (C18, CH3CN/H2O = 1:1) to give conju-
gate 13 (33 mg, 47%) as a white solid: mp = 173–175 °C; IR (thin
film) 2927, 2852, 1754, 1615, 1472, 1265 cmꢀ1
;
1H NMR
(600 MHz, CD3OD) d 8.76 (s, 1H), 7.87 (d, J = 13.2 Hz, 1H), 7.51 (d,
J = 6.75 Hz, 1H), 6.84 (s, 1H), 6.64 (s, 1H), 4.59 (s, 4H), 3.75 (s,
1H), 3.70 (s, 2H), 3.53–3.60 (m, 6H), 3.27 (s, 2H), 3.15 (m, 6H),
2.96 (s, 2H), 2.89 (t, J = 6.75 Hz, 2H), 2.72–2.77 (m, 4H), 2.62 (t,
J = 6.75 Hz, 2H), 2.57 (s, 3H), 2.42–2.45 (m, 4H), 2.19 (s, 3H), 2.09
(s, 3H), 1.56–1.65 (m, 6H), 1.61 (s, 6H), 1.48–1.54 (m, 6H), 1.39–
1.43 (m, 2H), 1.29–1.35 (m, 6H), 1.20–1.23 (m, 2H); 13C NMR
(150 MHz, CD3OD) d 178.4, 175.0, 174.6, 174.0, 173.8, 173.7,
172.6, 169.8, 155.9, 154.3, 151.4, 149.5, 146.9, 140.9, 139.8,
137.5, 135.7, 133.3, 124.3, 112.8, 112.6, 107.4, 51.1, 50.6, 47.1,
46.4, 42.6, 40.8, 40.5, 40.4, 37.1, 33.0, 31.7, 31.5, 31.3, 30.1, 29.1,
29.0, 27.5, 26.1, 25.0, 20.4, 8.7; HRMS (ESI) calcd for C59H85FN9O15
(M+H)+: 1178.6144, found 1178.6152.
4.1.11. Dibenzyl (4-((tert-butyldimethylsilyl)oxy)-3,5-dimethyl-
2-(2-methyl-4-oxobutan-2-yl) phenyl) phosphate (18)
Alcohol 17 (492 mg, 0.82 mmol) was dissolved in 20 mL of
dichloromethane at room temperature under argon. PCC (264 mg,
1.23 mmol) was added in three portions. The dark slurry was stir-
red at room temperature for 3 h. After filtered through a Celite pad
to remove the insoluble matters, the solvent was removed and the
crude oil was purified by chromatography on a silica gel column
(hexanes/EtOAc = 3:1) to yield aldehyde 18 as a clear oil (370 mg,
78%): 1H NMR (600 MHz, CDCl3) d 9.46 (t, J = 2.3 Hz, 1H), 7.29–
7.34 (m, 10H), 7.05 (s, 1H), 5.06–5.12 (m, 4H), 2.87 (d, J = 2.3 Hz,
2H), 2.31 (s, 3H), 2.09 (s, 3H), 1.56 (s, 6H), 1.02 (s, 9H), 0.15 (s,
6H); 13C NMR (150 MHz, CDCl3) d 203.4, 150.3, 143.7, 135.7,
133.4, 129.7, 128.8, 128.7, 128.2, 127.9, 120.5, 70.1, 57.2, 38.8,
31.7, 26.3, 18.9, 18.8, 17.8, ꢀ2.9; HRMS (ESI) calcd for C33H46O6PSi
(M+H)+: 597.2796, found 597.2774.
4.1.8. 6-((tert-Butyldimethylsilyl)oxy)-4,4,5,7-
tetramethylchroman-2-one (15)
Lactone 14 (3.0 g, 13.6 mmol) and TBDMSCl (3.5 g, 22.8 mmol)
were dissolved in 100 mL of CH2Cl2. To this solution was added
DBU (3.4 mL, 22.8 mmol). After 1 h, the reaction was quenched
with 100 mL of sat. NH4Cl solution. The organic phase was sepa-
rated and washed with brine, dried over Na2SO4, filtered and the
solvent was removed under reduced pressure. The residue was
purified by chromatography on a silica gel column (hexanes/
EtOAc = 6:1) to give 15 as a waxy solid (4.1 g, 90%): 1H NMR
(600 MHz, CDCl3) d 6.72 (s, 1H), 2.57 (s, 2H), 2.31 (s, 3H), 2.18 (s,
3H), 1.43 (s, 6H), 1.04 (s, 9H), 0.18 (s, 6H); 13C NMR (150 MHz,
CDCl3) d 168.7, 149.0, 145.4, 128.3, 128.2, 127.1, 117.1 46.0, 35.4,
27.5, 26.0, 18.7, 17.6, 16.6, ꢀ3.1; HRMS (ESI) calcd for C19H31O3Si
(M+H)+: 335.2037, found 335.2065.
4.1.12. 3-(6-((bis(Benzyloxy)phosphoryl)oxy)-3-((tert-
butyldimethylsilyl)oxy)-2,4-dimethylphenyl)-3-
methylbutanoic acid (19)
To a solution of 18 (341 mg, 0.57 mmol) in 6 mL of CH3CN and
3 mL of H2O were added sodium dihydrogen phosphate (230 mg,
1.65 mmol) and 30% H2O2 (0.2 mL). The mixture was cooled to
0 °C and a solution of sodium chlorite (92 mg, 1.03 mmol) in
1 mL of H2O was added slowly. The reaction was kept at 0 °C for
1 h and warmed to room temperature. Sat. Na2S2O3 (2 mL) was
added slowly to destroy excess H2O2 and NaClO2. The solution
was acidified with 3 N HCl to pH = 1–2 and the mixture was ex-
tracted with EtOAc (20 mL ꢁ 3). The combined organic layers were
washed sequentially with H2O, brine, dried over Na2SO4, filtered
and the solvent was evaporated to give acid 19 (345 mg, 99%) as
a colorless oil: 1H NMR (600 MHz, CDCl3) d 7.30–7.35 (m, 10H),
6.94 (s, 1H), 5.07–5.13 (m, 4H), 2.86 (s, 2H), 2.32 (s, 3H), 2.05 (s,
3H), 1.60 (s, 6H), 1.01 (s, 9H), 0.13 (s, 6H); 13C NMR (150 MHz,
CDCl3) d 150.0, 143.7, 135.4, 134.0, 130.2, 128.7, 128.6, 128.0,
127.3, 120.1, 70.0, 47.4, 39.6, 31.7, 26.1, 18.7, 18.4, 17.5, ꢀ3.2;
HRMS (ESI) calcd for C33H45NaO7PSi (M+Na)+: 635.2564, found
635.2577.
4.1.9. 4-((tert-Butyldimethylsilyl)oxy)-2-(4-hydroxy-2-
methylbutan-2-yl)-3,5-dimethylphenol (16)
Lactone 15 (3.5 g, 10.4 mmol) was dissolved in 20 mL of anhy-
drous THF. The solution was added dropwise to a suspension of
LiAlH4 (1.0 g, 26.3 mmol) in 100 mL of anhydrous THF cooled to
0 °C. The mixture was warmed to room temperature and heated
to reflux for 16 h. The excess LiAlH4 was quenched with 1 mL of
cold water. After some MgSO4 was added, the mixture was allowed
to stir for an additional 30 min and then filtered to remove the salt.
The solvent was removed under reduced pressure to give the crude
product which was purified by chromatography on a silica gel col-
umn (hexanes/EtOAc = 2:1) to give 16 (1.8 g, 51%) as a light yellow
solid: 1H NMR (600 MHz, CDCl3) d 6.34 (s, 1H), 5.46 (bs, 1H), 3.64

C. Ji, M. J. Miller / Bioorg. Med. Chem. 20 (2012) 3828–3836
3835
4.1.13. O-Bn-O0-TBS ciprofloxacin conjugate (20)
173.9, 173.3, 172.4, 171.2, 171.2, 170.3, 165.8, 154.3, 152.7,
148.5, 147.1, 146.3, 144.4, 138.2, 136.6, 135.7, 135.5, 135.4,
134.6, 131.8, 129.4, 129.4, 129.4, 129.2, 129.1, 129.0, 128.9,
128.9, 128.9, 128.7, 128.3, 128.2, 128.1, 123.5, 120.0, 110.4,
105.2, 76.9, 76.5, 76.5, 70.3, 70.2, 66.6, 49.6, 45.7, 45.5, 45.1,
41.2, 40.1, 39.6, 35.0, 34.7, 32.2, 32.1, 31.1, 30.8, 30.8, 29.9, 29.9,
29.8, 29.8, 29.7, 29.6, 29.5, 29.4, 29.4, 29.2, 29.0, 28.8, 28.6, 28.3,
28.2, 28.0, 27.5, 27.2, 26.7, 26.6, 26.1, 24.1, 24.0, 23.9, 23.8, 22.9,
To a solution of acid 19 (143 mg, 0.23 mmol) in 5 mL of anhy-
drous CH2Cl2 cooled to 0 °C were added EDC (88 mg, 0.46 mmol)
and HOBt (62 mg, 0.46 mmol). The mixture was stirred at that tem-
perature for 5 min before O-benzyl ciprofloxacin (106 mg,
0.25 mmol), Et3N (130 lL, 0.92 mmol), DMAP (2 mg) were added.
The reaction mixture was warmed to room temperature and stirred
for 18 h. The mixture was diluted with 30 mL of CH2Cl2 and the or-
ganic layer was washed sequentially with water, sat. NaHCO3, brine,
dried over Na2SO4 and filtered. The solvent was removed in vacuo
and the crude product was purified by chromatography on a silica
gel column (5% methanol in CH2Cl2) to yield 20 (138 mg, 59%) as a
light yellow oil: 1H NMR (500 MHz, CDCl3) d 8.54 (s, 1H), 8.08 (d,
J = 13.2 Hz, 1H), 7.52–7.54 (m, 2H), 7.37–7.40 (m, 2H), 7.26–7.33
(m, 11H), 7.15 (d, J = 7.0 Hz, 1H), 6.98 (s, 1H), 5.40 (s, 2H), 5.04–
5.13 (m, 4H), 3.68–3.70 (m, 2H), 3.43–3.45 (m, 2H), 3.31–3.36 (m,
1H), 3.05–3.07 (m, 2H), 2.94–2.96 (m, 2H), 2.95 (s, 2H), 2.38 (s,
3H), 2.06 (s, 3H), 1.63 (s, 6H), 1.25–1.29 (m, 2H), 1.06–1.09 (m,
2H), 1.02 (s, 9H), 0.15 (s, 6H); 13C NMR (125 MHz, CDCl3) d 170.5,
165.9, 150.1, 148.6, 144.0, 136.7, 135.7, 130.4, 129.0, 128.9, 128.8,
128.3, 128.2, 128.1, 127.2, 119.8, 113.8, 113.6, 110.5, 105.2, 70.1,
66.7, 45.8, 45.5, 41.2, 40.1, 34.7, 32.3, 26.3, 19.0, 18.9, 17.8, 8.4, -
2.9; HRMS (ESI) calcd for C57H67FN3NaO9PSi (M+Na)+: 1038.4260,
found 1038.4257.
20.7, 16.9, 16.5, 14.3, 8.3; HRMS (ESI) calcd for C101H121FN9NaO19
P
(M+Na)+: 1836.8393, found 1836.8407.
4.1.16. Desferrioxamine B-ciprofloxacin conjugate (23)
Compound 22 (17.5 mg, 9.6 lMol) was dissolved in 4 mL of
THF/H2O = 10:1 solution under argon. To this solution was added
10% Pd/C (5 mg) and the flask was flushed with H2 gas and left to
stir under a hydrogen atmosphere (balloon) at room temperature
for 6 h. After removal of the catalyst by filtration, the solvent was
evaporated under reduced pressure. The resulting yellow oil was
dissolved in 0.5 mL of CH3CN/H2O = 1:1 solution and further puri-
fied by chromatography on a reverse-phase silica gel column (C18
,
100% H2O to CH3CN/H2O = 1:1) to afford 23 (4.3 mg, 35%) as a yel-
low foam: 1H NMR (600 MHz, CD3OD) d 8.82 (s, 1H), 7.92 (d,
J = 13.2 Hz, 2H), 7.52 (d, J = 7.34 Hz, 1H), 7.40 (s, 1H), 3.72–3.79
(m, 2H), 3.57–3.62 (m, 6H), 3.52–3.54 (m, 2H), 3.13–3.22 (m,
8H), 3.06–3.13 (m, 4H), 2.87 (m, 2H), 2.71–2.80 (m, 4H), 2.52 (m,
2H), 2.41–2.50 (m, 4H), 2.23 (s, 3H), 2.09 (s, 3H), 2.02 (s, 3H),
1.74 (s, 6H), 1.20–1.64 (m, 22H); 13C NMR (150 MHz, CD3OD) d
177.1, 173.5, 173.0, 173.0, 172.4, 172.2, 172.1, 171.1, 168.3,
154.5, 152.9, 150.1, 147.9, 145.6, 145.6, 144.0, 139.4, 134.0,
133.9, 130.2, 128.2, 119.3, 119.2, 119.2, 111.0, 110.8, 106.6,
106.3, 105.0, 49.2, 49.0, 47.2, 45.8, 43.6, 41.2, 40.5, 38.9, 38.8,
35.6, 30.1, 30.0, 29.8, 28.6, 28.5, 28.5, 27.5, 27.5, 25.9, 23.5, 23.5,
18.8, 15.6, 15.3, 7.2; HRMS (ESI) calcd for C59H86FN9O19P (M+H)+:
1274.5756, found 1274.5799.
4.1.14. O-Bn ciprofloxacin conjugate (21)
Compound 20 (101 mg, 97 lMol) was dissolved in a DMF/
H2O = 5:1 solution (2 mL). Argon was bubbled through the solution
for 5 min before KF (12 mg, 0.21 mmol) was added and the mixture
was vigorously stirred for 30 min. The reaction was quenched by
adding 10 mL of sat. NH4Cl and extracted with chloroform
(5 mL ꢁ 3). The organic layers were combined and washed sequen-
tially with brine, dried over Na2SO4 and filtered. After removal of
the solvent under vacuum, the residue was applied to a preparative
silica gel TLC plate, eluted with 5% MeOH in CHCl3 to afford 21
(65 mg, 74%) as a clear oil: 1H NMR (600 MHz, CD3OD) d 8.60 (s,
1H), 7.82 (d, J = 13.2 Hz, 1H), 7.47–7.48 (m, 2H), 7.36–7.38 (m,
2H), 7.28–7.33 (m, 12H), 6.84 (s, 1H), 5.31 (s, 2H), 5.06–5.18 (m,
4H), 3.61–3.62 (m, 2H), 3.50–3.54 (m, 1H), 3.46–3.47 (m, 2H),
3.11–3.12 (m, 2H), 2.98–2.99 (m, 2H), 2.98 (s, 2H), 2.41 (s, 3H),
2.06 (s, 3H), 1.60 (s, 6H), 1.23–1.28 (m, 2H), 1.06–1.09 (m, 2H);
13C NMR (150 MHz, CD3OD) d 172.4, 155.5, 153.9, 152.4, 152.3,
150.1, 145.8, 145.8, 143.9, 143.9, 139.7, 137.9, 136.9, 136.8,
136.2, 136.1, 129.9, 129.7, 129.7, 129.6, 129.6, 129.4, 129.3,
129.2, 129.2, 128.5, 128.4, 127.8, 127.7, 124.5, 120.6, 120.6,
113.2, 113.0, 107.2, 71.4, 71.4, 71.2, 71.2, 67.2, 51.0, 50.5, 47.0,
46.8, 42.3, 40.7, 36.3, 32.5, 31.9, 17.3, 17.2, 16.6, 16.5, 8.5; HRMS
(ESI) calcd for C51H54FN3O9P (M+H)+: 902.3576, found 902.3599.
4.1.17. Succinyl O-Bn desferrioxamine B-ciprofloxacin
conjugate (25)
To a solution of O-benzyl desferrioxamine succinic acid 24
(84 mg, 0.09 mmol) in 3 mL of anhydrous CH2Cl2 cooled to 0 °C
were added EDC (35 mg, 0.18 mmol) and HOBt (24 mg,
0.18 mmol). The mixture was stirred at that temperature for
5 min before O-benzyl ciprofloxacin (50 mg, 0.12 mmol), Et3N
(50 lL, 0.36 mmol) and DMAP (2 mg) were added. The reaction
mixture was warmed to room temperature and stirred for 18 h.
The mixture was diluted with 20 mL of CH2Cl2 and the organic
layer was washed sequentially with water, sat. NaHCO3, brine,
dried over Na2SO4 and filtered. The solvent was removed in vacuo
and the crude oil was purified by chromatography on a silica gel
column (5% methanol in CH2Cl2) to give 25 (90 mg, 75%) as a light
yellow oil: 1H NMR (600 MHz, CDCl3) d 8.55 (s, 1H), 8.10 (d,
J = 12.91 Hz, 1H), 7.29–7.53 (m, 20H), 7.26 (d, J = 7.5 Hz, 1H),
6.51–6.62 (m, 2H), 6.37 (br. s., 1H), 5.40 (s, 2H), 4.85 (s, 4H), 4.81
(s, 2H), 3.82 (m, 2H), 3.73 (m, 2H), 3.64 (br. s., 6H), 3.41 (m, 1H),
3.27 (m, 2H), 3.20 (m, 8H), 2.82 (m, 4H), 2.73 (t, J = 6.90 Hz, 2H),
2.58 (t, J = 7.04 Hz, 2H), 2.49 (m, 4H), 2.09 (s, 3H), 1.60–1.66 (m,
6H), 1.49–1.53 (m, 6H), 1.26–1.31 (m, 8H), 1.08–1.15 (m, 2H);
13C NMR (150 MHz, CDCl3) d 173.2, 172.3, 170.9, 165.8, 154.4,
152.7, 148.6, 144.3, 144.2, 138.2, 136.6, 129.4, 129.4, 129.2,
128.9, 128.7, 128.2, 128.1, 123.9, 113.9, 113.8, 110.5, 105.3, 76.6,
76.5, 66.6, 50.4, 50.0, 45.5, 41.7, 39.6, 34.7, 31.4, 30.8, 30.7, 29.3,
28.7, 26.8, 26.6, 24.2, 23.9, 23.8, 8.4; HRMS (ESI) calcd for
4.1.15. O-Bn desferrioxamine B-ciprofloxacin conjugate (22)
To a solution of 21 (33 mg, 37
amine succinic acid (70 mg, 75
was added EDC (30 mg, 156
lMol) and O-benzyl desferriox-
l
Mol) in 1 mL anhydrous DMF,
l
Mol) in one portion. After stirring
at room temperature for 24 h, the reaction was diluted with
30 mL of CHCl3. The solution was washed sequentially with sat.
NaHCO3, brine, dried over Na2SO4 and filtered. After removal of
the solvent under vacuum, the residue was applied to a preparative
silica gel TLC plate and eluted with 10% MeOH in CHCl3 to afford 22
(30 mg, 45%) as a white foam: 1H NMR (600 MHz, CDCl3) d 8.53 (s,
1H), 8.08 (d, J = 13.2 Hz, 1H), 7.24–7.52 (m, 30H), 7.16 (d, J = 7.0 Hz,
1 H), 7.06 (s, 1H), 6.40 (br. s., 3H), 5.40 (s, 2H), 5.07 (m, 4H), 4.77–
4.89 (m, 6H), 3.56 - 3.71 (m, 8H), 3.40 (m, 2H), 3.35 (m, 1H), 3.12–
3.24 (m, 6H), 3.05 (m, 2H), 2.94 (m, 4H), 2.80 (m, 4H), 2.57–2.67
(m, 4H), 2.42–2.53 (m, 4H), 2.30 (s, 3H), 2.06–2.12 (m, 3H), 1.98
(s, 3H), 1.55–1.68 (m, 12H), 1.42–1.54 (m, 6H), 1.21–1.37 (m,
8H), 1.05–1.08 (m, 2H); 13C NMR (150 MHz, CDCl3) d 174.3,
C
74H93FN9O13 (M+H)+: 1334.6871, found 1334.6874.
4.1.18. Succinyl desferrioxamine B-ciprofloxacin conjugate (26)
Compound 25 (40 mg, 0.03 mmol) was dissolved in 5 mL of
MeOH under argon. 10% Pd/C (4 mg) was added and the flask

3836
C. Ji, M. J. Miller / Bioorg. Med. Chem. 20 (2012) 3828–3836
8. Hartmann, A.; Fiedler, H. P.; Braun, V. Eur. J. Biochem. 1979, 99, 517.
9. Möllmann, U.; Dong, L.; Vertesy, L.; Miller, M. J. The 2nd international Biometals
symposium; Garmisch-Partenkirchen, 2004.
10. Vertesy, L.; Aretz, W.; Fehlhaber, H. W.; Kogler, H. Helv. Chim. Acta 1995, 78, 46.
11. Rivault, F.; Liebert, C.; Burger, A.; Hoegy, F.; Abdallah, M. A.; Schalk, I. J.; Mislin,
G. L. A. Bioorg. Med. Chem. Lett. 2007, 17, 640.
12. Hennard, C.; Truong, Q. C.; Desnottes, J. F.; Paris, J. M.; Moreau, N. J.; Abdallah,
M. A. J. Med. Chem. 2001, 44, 2139.
13. Noel, S.; Gasser, V.; Pesset, B.; Hoegy, F.; Rognan, D.; Schalk, I. J.; Mislin, G. L. A.
Org. Biomol. Chem. 2011, 9, 8288.
14. Milstien, S.; Cohen, L. A. J. Am. Chem. Soc. 1972, 94, 9158.
15. Borchard, R. T.; Cohen, L. A. J. Am. Chem. Soc. 1972, 94, 9166.
16. Milstien, S.; Cohen, L. A. Proc. Natl. Acad. Sci. U.S.A. 1970, 67, 1143.
17. King, M. M.; Cohen, L. A. J. Am. Chem. Soc. 1983, 105, 2752.
18. Caswell, M.; Schmir, G. L. J. Am. Chem. Soc. 1980, 102, 4815.
19. Houghton, T. J.; Tanaka, K. S. E.; Kang, T.; Dietrich, E.; Lafontaine, Y.; Delorme,
D.; Ferreira, S. S.; Viens, F.; Arhin, F. F.; Sarmiento, I.; Lehoux, D.; Fadhil, I.;
Laquerre, K.; Liu, J.; Ostiguy, V.; Poirier, H.; Moeck, G.; Parr, T. R.; Far, A. R. J.
Med. Chem. 2008, 51, 6955.
was flushed with H2 gas and left to stir under a hydrogen atmo-
sphere (balloon) for 6 h. After purging with argon, the mixture
was filtered and concentrated in vacuo to give compound 26
(24 mg, 82%) as a light yellow foam: 1H NMR (600 MHz, DMSO-
d6) d 9.67 (br. s., 1H), 9.62 (br. s., 2H), 8.67 (s, 1H), 7.94 (d,
J = 13.2 Hz, 1H), 7.74–7.84 (m, 3H), 7.58 (d, J = 7.6 Hz, 1H), 3.82
(m, 2H), 3.68 (m, 4H), 3.45 (m, 6H), 3.25–3.31 (m, 2H), 2.94–3.05
(m, 6H), 2.53–2.64 (m, 6H), 2.34 (t, J = 6.9 Hz, 2H), 2.26 (t,
J = 7.0 Hz, 4H), 1.96 (s, 3H), 1.49 (br. s., 6H), 1.28–1.43 (m, 8H),
1.13–1.27 (m, 8H); 13C NMR (151 MHz, DMSO-d6) d 176.4, 172.0,
171.2, 170.1, 166.0, 148.1, 139.2, 106.8, 47.1, 46.8, 38.5, 35.9,
30.4, 29.9, 28.8, 27.8, 27.6, 26.0, 23.5, 20.4, 7.6; HRMS (ESI) calcd
for C46H68FN9NaO13 (M+Na)+: 996.4813, found 996.4848.
Acknowledgments
20. Greenwald, R. B.; Choe, Y. H.; Conover, C. D.; Shum, K.; Wu, D. C.; Royzen, M. J.
Med. Chem. 2000, 43, 475.
21. Wang, B. H.; Gangwar, S.; Pauletti, G. M.; Siahaan, T. J.; Borchardt, R. T. J. Org.
Chem. 1997, 62, 1363.
22. Pauletti, G. M.; Gangwar, S.; Wang, B. H.; Borchardt, R. T. Pharm. Res. 1997, 14,
11.
23. Nicolaou, M. G.; Yuan, C. S.; Borchardt, R. T. J. Org. Chem. 1996, 61, 8636.
24. Wang, B. H.; Zhang, H. J.; Wang, W. Bioorg. Med. Chem. Lett. 1996, 6, 945.
25. Wang, B. H.; Wang, W.; Zhang, H. J.; Shan, D. X.; Smith, T. D. Bioorg. Med. Chem.
Lett. 1996, 6, 2823.
26. Amsberry, K. L.; Gerstenberger, A. E.; Borchardt, R. T. Pharm. Res. 1991, 8, 455.
27. Amsberry, K. L.; Borchardt, R. T. Pharm. Res. 1991, 8, 323.
28. Bornscheuer, U. T. FEMS Microbiol. Rev. 2002, 26, 73.
29. Beacham, I. R. Int. J. Biochem. 1979, 10, 877.
This research was supported by Grant AI054193 from the NIH.
We gratefully acknowledge the use of the NMR facilities provided
by the Lizzadro Magnetic Resonance Research Center at the Uni-
versity of Notre Dame (UND) under the direction of Dr. Jaroslav
Zajicek 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 under CHE-0741793). We gratefully thank Mrs. Patricia
A. Miller (UND) for antibacterial susceptibility testing and the
group of Prof. Shahriar Mobashery for the ESKAPEE assays.
30. Mitscher, L. A. Chem. Rev. 2005, 105, 559.
31. Muller, G.; Raymond, K. N. J. Bacteriol. 1984, 160, 304.
32. Carpino, L. A.; Triolo, S. A.; Berglund, R. A. J. Org. Chem. 1989, 54, 3303.
33. Nelson, T. D.; Rosen, J. D.; Bhupathy, M.; McNamara, J.; Sowa, M. J.; Rush, C.;
Crocker, L. S. Org. Synth. 2003, 80, 219.
34. Wencewicz, T. A.; Möllmann, U.; Long, T. E.; Miller, M. J. Biometals 2009, 22,
633.
35. Wilhelm, S.; Tommassen, J.; Jaeger, K. E. J. Bacteriol. 1999, 181, 6977.
36. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow
Aerobically, 8th ed.; Clinical and Laboratory Standards Institute (CLSI):
Villanova, PA, USA, 2009, approved standard document M07–A7.
37. Wencewicz, T. A. Dissertation; University of Notre Dame: Notre Dame, IN, 2011.
38. Roosenberg, J. M., II Dissertation; University of Notre Dame: Notre Dame, IN,
2000.
References and notes
1. Spellberg, B.; Guidos, R.; Gilbert, D.; Bradley, J.; Boucher, H. W.; Scheld, W. M.;
Bartlett, J. G.; Edwards, J. Clin. Infect. Dis. 2008, 46, 155.
2. Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.;
Scheld, M.; Spellberg, B.; Bartlett, J. Clin. Infect. Dis. 2009, 48, 1.
3. Walsh, C. Antibiotics: Actions, Origins, Resistance; American Society for
Microbiology (ASM) Press: Washington, DC, 2003.
4. Pages, J. M.; James, C. E.; Winterhalter, M. Nat. Rev. Microbiol. 2008, 6, 893.
5. Roosenberg, J. M.; Lin, Y. M.; Lu, Y.; Miller, M. J. Curr. Med. Chem. 2000, 7, 159.
6. Braun, V.; Pramanik, A.; Gwinner, T.; Koberle, M.; Bohn, E. Biometals 2009, 22, 3.
7. Braun, V.; Gunthner, K.; Hantke, K.; Zimmermann, L. J. Bacteriol. 1983, 156, 308.