Design and synthesis of a novel protected mixed ligand siderophore

Article abstract of DOI:10.1016/j.tetlet.2008.02.007

A novel siderophore analog (4) has been designed to facilitate iron transport-mediated drug delivery and drug release. This mixed ligand siderophore analog includes three bidentate ligands intended to octahedrally coordinate iron (III). The ligands includ

Full text of DOI:10.1016/j.tetlet.2008.02.007

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Tetrahedron Letters 49 (2008) 2306–2310
Design and synthesis of a novel protected mixed ligand siderophore
Pingyu Ding, Clara E. Schous, Marvin J. Miller *
Walther Cancer Research Center and Department of Chemistry and Biochemistry, University of Notre Dame, 251 Nieuwland Science Hall,
Notre Dame, IN 46556, United States
Received 3 October 2007; revised 31 January 2008; accepted 1 February 2008
Available online 7 February 2008
Abstract
A novel siderophore analog (4) has been designed to facilitate iron transport-mediated drug delivery and drug release. This mixed
ligand siderophore analog includes three bidentate ligands intended to octahedrally coordinate iron (III). The ligands include a 2,3-di-
hydroxy benzoic acid moiety, N5-acetyl-N5-hydroxy-L-ornithine, and a b-N-hydroxy-a,b-diaminopropionic acid derivative. The total
synthesis of 15, a form of 4 that is suitably protected, yet contains a free carboxylic acid for subsequent drug conjugation, is described.
Ó 2008 Elsevier Ltd. All rights reserved.
The evolution of antibiotic resistance is inevitable as
bacteria compete for limited resources for survival. Bacte-
ria exhibit antibiotic resistance by several mechanisms.
Examples include the (1) production of enzymes, such as
b-lactamases, to destroy antibiotics, (2) adaptation of an
efflux mechanism to shuttle drugs out of the cell, (3) chem-
ical alteration of the target of the antibiotic, and (4) altered
cell wall permeability to prevent diffusion of the drugs into
the cells.1 The growing antibacterial resistance observed in
the world today translates into staggering consequences for
the medical community and pharmaceutical industry.2 In
addition to searching for new antibiotics, attention must
also be paid to make wiser use of the effective medicines
available today. The utilization of specific drug delivery
agents coupled with known and new antibiotics is under
development in our group, among others, as a potentially
new avenue for antimicrobial drug therapy.3
Due to the low solubility of iron at physiological pH,
bacteria biosynthesize low molecular weight iron chelators
called siderophores to sequester and deliver the Fe3+ essen-
tial to their survival.4 The most effective siderophores con-
sist of three bidentate ligands, most commonly catechol,
hydroxamic acid, or a-hydroxycarboxylic acid iron-binding
moieties. These structures reflect the high affinity of oxygen
for ferric ions and allow for complete octahedral coordina-
tion of Fe3+. By the conjugation of a drug to a siderophore,
the iron transport process can be exploited to smuggle
antibiotics into microorganisms. The concept of utilizing
a siderophore as a transport agent parallels the mode of
action of several naturally occurring antibiotics, such as
the albomycins, ferrimycin, and salmycins (Fig. 1).5 These
antibiotics consist of a siderophore component and an anti-
microbial component, and are meant to provide the parent
organism with a selective growth advantage. When other
microbes try to utilize the iron from these siderophores,
they also assimilate the antibiotic and are killed.
Several biological studies have proven the utility of
siderophore drug conjugates for use as antibacterial, anti-
tumor, and antiviral agents. This method of disguising a
drug has been called the ‘Trojan Horse’ microbe selective
drug delivery concept.3 The conjugates usually consist of
a synthetic siderophore for binding the iron, a linker for
attaching the drug to the siderophore, and a drug for
conveying the antibacterial activity (Fig. 2). Once the
microorganism absorbs the siderophore–drug conjugate,
the complex may kill the cell in a variety of ways: by releas-
ing the drug, by acting as an intact antibacterial agent, or
by blocking further iron assimilation. The linker plays a
vital role in the conjugate because it can dictate if the drug
is released (chemically or enzymatically) within the cell and
could potentially allow controlled release of drugs.3
*
Corresponding author. Tel.: +1 574 631 7571; fax: +1 574 631 6652.
E-mail address: mmiller1@nd.edu (M. J. Miller).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.02.007

P. Ding et al. / Tetrahedron Letters 49 (2008) 2306–2310
2307
O
O
CH3
N
N
2
O
N
R
H
HO
HO
O
HN
N
O
N
O
N
O
CO2H
OH
OH
OH
S
H
N
O
OH
N
H
H N
H
N
OH
3
5
O
MeO
N
OH
OH
NH
NH2
O
1, Albomycins (R=O, NH, or NHCONH2)
(shown without Fe)
O
2, Ferrimycin
(shown without Fe)
OH
H2N
OH
CH3
O
O
NH
N
NH
N
O
O
O
OH
O
O
HN
HN
Fe
O
O
Intracellular
Ferric reductase
O
OH
O
N
N
O N
(CH2)n-OH
HO N
(CH2)n-OH
O
Fe(II)Ln
O
O
O
O
O
O
HO
HO
HO
CH3
CH3
HO
HO
HN
HO
HN
R
R
OH
O
OH
O
O
O
OH
CH2OH
CH2OH
OH
3a, Salmycin A R = NOH, n = 5
3b, Salmycin B R = O or (OH)2, n = 4
3c, Salmycin C R = O or (OH)2, n = 5
3d, Salmycin D R = NOH, n = 4
HO
O
HO
HO
CH3
HN
HO
Antibiotic
effect ??
R
OH
O
O
OH
CH2OH
Fig. 1. Naturally occurring siderophore–antibiotic conjugates and a potential ‘drug release’ process.
siderophores (containing more than one type of iron-bind-
ing moiety) can gain access to the cell by more than one
transport pathway, or type of receptor, possibly lending
them an advantage as drug delivery agents.6 One additional
benefit of siderophore–drug conjugates is that because the
membrane no longer serves as a barrier to the drug, active
transport of the complexes can increase the rate of entry
into the cell beyond the passive diffusion rate. This
phenomenon is reflected in lower MIC values by more than
two orders of magnitude.7
Thus far, many natural and artificial siderophores have
been synthesized and shown to exhibit good microbial
growth promoting activity. Several recent papers review
many of the chemical syntheses of these siderophores, high-
lighting much of the previous work that has been done in
our laboratory and others.3,4a The original report on the
isolation3 of the salmycins and our subsequent total syn-
theses of salmycins and analogs8 indicated that they were
less stable in the deferri (iron free) form. We hypothesize
that this might be due to an intramolecular nucleophilic
reaction of a released hydroxamate to induce cleavage of
the linker to the aminoglycoside antibiotic component of
the salmycins (Fig. 1). If so, the result would be another
Siderophore
Fe (III)
Linker
Drug
Fig. 2. Schematic representation of a siderophore–drug conjugate.
The most appealing aspect of the ‘Trojan Horse’ princi-
ple is the wide array of opportunities for specific drug
delivery. Because bacteria have specific siderophore-bind-
ing receptor proteins, harmful bacteria can potentially be
selectively targeted with the most effective and appropriate
drugs. The arena is also open for the introduction of more
toxic drugs, and the reinstitution of drugs that currently
meet with resistance. If toxicity can be diminished by con-
jugation to a siderophore, perhaps some of the most deadly
compounds could be added to the arsenal in the fight
against virulent bacteria. Additionally, this strategy may
be particularly useful for combating common resistance
mechanisms, especially those associated with efflux of the
drug or blocked diffusion.3b Several biological studies have
shown that some siderophore–drug conjugates have been
able to overcome previous permeability problems for
certain drugs.3 Studies have also shown that mixed ligand

2308
P. Ding et al. / Tetrahedron Letters 49 (2008) 2306–2310
O
O
O
O
OH
OH
NH OH
O
N
O
NH OH
O
N
O
OH
OH
N
O
N
O
OH
OH
O
OH
O
H
N
H
N
N
N
N
N
H
H
H
H
O
O
Drug
4
4-drug Conjugate
Fig. 3. Targeted mixed ligand siderophore.
clever extension of natural siderophore antibiotics that are
not only recognized and actively transported by microbial
iron assimilation systems, but also contain an iron reduc-
tase that would trigger intracellular release of the anti-
biotic. While studies are in progress to demonstrate this
concept with derivatives of danoxamine, the siderophore
component of the salmycins,8 we initiated studies to design
artificial siderophores capable of similar anchimerically
assisted drug release that is triggered by the reductive
removal of the metal inside the cell. Herein we report the
synthesis of a suitably protected form of a novel mixed
ligand siderophore analog, 4, for the subsequent prepara-
tion of drug conjugates to further test this hypothesis. As
described previously,3,4a the use of mixed ligands is
intended to facilitate broader microbial recognition and
assimilation through multiple siderophore receptors while
the existence of the short side chain on the C-terminal
amino acid may induce intracellular release of the drug
by an intramolecularly assisted cleavage as suggested for
the salmycins (Fig. 3).
As shown in Scheme 1, the synthesis of the catechol
ligand 7 began with the selective benzylation of 2,3-di-
hydroxybenzoic acid which was accomplished with excess
benzyl bromide and KOH in DMSO to yield 5 in 89%
yield.9 Benzoic acid 5 was then coupled to methyl 6-amino-
caproate hydrochloride, which was prepared in quantita-
tive yield from 6-aminocaproic acid in HCl (g) MeOH
solution. Several different coupling additives were experi-
mented with and HOAt gave the highest and the most
reproducible yield, thus the two fragments were coupled
in the presence of HOAt, EDC, and DIPEA in CH3CN
to give 6 in 97% yield. The methyl ester of the coupled
product 6 was then saponified in 98% yield with 2 equiv
of LiOH in a 1:1 solution of THF and water. This com-
pleted the synthesis of the protected catechol-containing
ligand 7.
As shown in Scheme 2, the protected ornithine-derived
hydroxamate 810,11 was subjected to deprotection with
HBr–HOAc. The resulting free amine HBr salt was cou-
pled to acid 7 using HOAt and EDC as coupling reagents
in the presence of DIPEA to afford dipeptide methyl ester 9
in 91% yield. Hydrolysis of 9 then provided dipeptide acid
10 in 92% yield. Dipeptide acid 10 was next coupled to
glycine methyl ester or glycine tert-butyl ester in the pres-
ence of HOAt and EDC to give the corresponding tripep-
tide methyl ester 11a or tert-butyl ester 11b, both in 95%
yield. Methyl ester 11a was hydrolyzed by LiOH to provide
tripeptide acid 12 in 97% yield; while the removal of the
tert-butyl protecting group by the treatment of 11b with
TFA in CH2Cl2 also gave tripeptide acid 12 in 92% yield.
The final peptide-bond-forming reaction required to
synthesize protected tetrapeptide acid 15 is shown in
Scheme 3. Tripeptide acid 12 was first coupled to
methyl L-3-(N-acetyl-N-benzyloxyamino)aminoalaninate
13a using HOAt and EDC in CH3CN to provide tetrapep-
tide methyl ester 14a in 88% yield. Unfortunately, however,
the saponification of 14a was not always reproducible
within an optimized reaction time. On several occasions,
the saponification reaction took up to three days to com-
plete. The lengthy reaction time led to racemization of
the terminal a-center and the formation of other by-prod-
ucts, so the isolated yield was extremely low. In further
studies, the problem was solved by using an allyl ester as
an alternative to the methyl ester. Allyl ester 14b was
OBn
OH
HCl.NH2(CH2)5CO2Me
BnO
CO2H
HO
CO2H BnBr, KOH, DMSO
89%
HOAt, EDCI, CH3CN
97%
5
OBn O
OBn O
LiOH, THF-H2O
98%
BnO
BnO
N
CO2H
N
CO2Me
H
H
7
6
Scheme 1.

P. Ding et al. / Tetrahedron Letters 49 (2008) 2306–2310
2309
O
O
O
O
O
OBn
OBn
N
NH OBn
O
N
NH OBn
O
N
1. 33% HBr-HOAc, CH2Cl2
LiOH, THF-H2O
92%
OBn
OBn
OBn
2, 7, HOAt, EDC, DIPEA
91%
OMe
OH
CbzHN
CO2Me
N
H
N
H
O
O
8
10
9
O
O
R = tBu:
TFA, CH2Cl2
O
O
OBn
OBn
NH OBn
O
N
NH OBn
O
N
OBn
OBn
HCl.NH2CH2CO2R
HOAt, EDCI, DIPEA
92%
R = Me:
LiOH, THF-H2O
97%
O
O
H
N
H
N
N
H
OR
N
H
OH
O
O
11a: R = Me, 95%
11b: R = tBu, 95%
12
Scheme 2.
O
O
O
O
O
R' = Me:
N
OBn
OBn
OBn
OR'
LiOH, THF-H2O
trace of product
NH OBn
O
N
O
NH OBn
O
N
O
OBn
O
OBn
O
H2N
12
N
N
O
R' = Allyl:
Et3N-HCO2H, Pd(PPh3)4, THF
94%
O
OBn
OR'
OBn
OH
H
N
H
N
13a: R = Me
13b: R = Allyl
N
H
N
N
H
N
H
H
O
O
O
HOAt, EDCI, CH3CN
14a: R = Me, 88%
14b: R = Allyl, 91%
15
Scheme 3.
readily prepared by coupling tripeptide acid 12 to allyl L-3-
(N-acetyl-N-benzyloxyl)aminoalaninate 13b in good yield.
An allyl ester 14b was initially deprotected with catalytic
palladium(0) using O-benzylhydroxylamine as the allyl cat-
ion scavenger.12 However, this reaction was only com-
pleted after 36 h and required the addition of up to 20%
mmol palladium(0) and 3 equiv of O-benzylhydroxyl-
amine. The long reaction time also resulted in some racemi-
zation. Alternatively, the allyl protecting group was readily
cleaved to furnish the desired tetrapeptide acid 15 by the
treatment of 14b with triethylammonium formate (5 equiv)
in the presence of a catalytic amount of Pd(PPh3)4.13
In conclusion, the total synthesis of a novel suitably pro-
tected mixed ligand siderophore 15 has been accomplished
using fully protected hydroxamate 8 as the starting mate-
rial and using appropriate protecting groups. Syntheses
of antibacterial and antifungal siderophore drug conju-
gates are underway in our laboratory. Subsequent bio-
logical test results and the drug release assays of these
siderophore drug conjugates along with their iron (III)
complexes will provide us useful information to further
understand the drug transportation and release mecha-
nism. Those results will be reported in due course.
Acknowledgments
We gratefully acknowledge the Department of Defense,
Walther Cancer Research Center at the University of
Notre Dame, NIH, and Eli Lilly & Company for support
of this research and the Lizzadro Magnetic Resonance
Research Center at Notre Dame for NMR facilities, as well
as Dr. B. Boggess and N. Sevova for mass spectrometry
facilities.
Supplementary data
Experimental procedures, NMR spectra and analytic
data for all new compounds are provided. Supplementary
data associated with this article can be found, in the online
version, at doi:10.1016/j.tetlet.2008.02.007.
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