Synthesis and biological evaluation of new acinetoferrin homologues for use as iron transport probes in mycobacteria

Article abstract of DOI:10.1021/jm049805y

Four new acinetoferrin homologues were synthesized using a modular synthetic approach. Two linear and two cyclic imide derivatives were generated and evaluated for growth stimulating behavior in Mycobacterium avium subsp paratuberculosis. The yield for the tandem coupling of a functionalized aminohydroxamic acid motif (2 equiv) to a tert-butyl citrate derivative was significantly improved using DCC and N-hydroxysuccinimide. ¹H NMR spectroscopy (CD₃OD) provided a convenient method for monitoring the final imidization step in TFA using the doublet patterns between 2.5 and 3.06 ppm. New protocols demonstrated that only a 20% growth enhancement was observed with M. avium subsp. paratuberculosis using the imide of acinetoferrin. Last, a siderophore from Streptomyces pilosus, deferrioxamine B, was shown to cross-feed M. avium subsp. paratuberculosis with the same efficiency as the more costly, native chelator, mycobactin J.

Full text of DOI:10.1021/jm049805y

J . Med. Chem. 2004, 47, 4933-4940
4933
Syn th esis a n d Biologica l Eva lu a tion of New Acin etofer r in Hom ologu es for Use
a s Ir on Tr a n sp or t P r obes in Mycoba cter ia
Richard A. Gardner,† George Ghobrial,‡ Saleh A. Naser,‡ and Otto Phanstiel, IV*,†
Department of Chemistry and Department of Molecular Biology and Microbiology, University of Central Florida,
Orlando, Florida 32816-2366
Received March 8, 2004
Four new acinetoferrin homologues were synthesized using a modular synthetic approach. Two
linear and two cyclic imide derivatives were generated and evaluated for growth stimulating
behavior in Mycobacterium avium subsp paratuberculosis. The yield for the tandem coupling
of a functionalized aminohydroxamic acid motif (2 equiv) to a tert-butyl citrate derivative was
significantly improved using DCC and N-hydroxysuccinimide. 1H NMR spectroscopy (CD3OD)
provided a convenient method for monitoring the final imidization step in TFA using the doublet
patterns between 2.5 and 3.06 ppm. New protocols demonstrated that only a 20% growth
enhancement was observed with M. avium subsp. paratuberculosis using the imide of
acinetoferrin. Last, a siderophore from Streptomyces pilosus, deferrioxamine B, was shown to
cross-feed M. avium subsp. paratuberculosis with the same efficiency as the more costly, native
chelator, mycobactin J .
In tr od u ction
Iron transport and storage in mammals are carried
out by high molecular weight proteins such as trans-
ferrin (transport) and ferritin (storage).1,2 In contrast,
iron acquisition in microorganisms is the responsibility
of siderophores, which are low molecular weight iron-
specific ligands.1,2 In the environment, iron is mainly
found in its insoluble ferric (Fe3+) form as ferric hy-
droxide polymers. Siderophores allow bacteria to solu-
bilize and sequester iron(III) from these insoluble
extracellular sources. Once bound to the siderophore,
the iron (Fe3+) is made available to the cell by intra-
cellular modification, reduction to Fe2+, or siderophore
decomposition.2
Most bacteria produce distinct siderophore constructs.
As shown in Figure 1, many of these have been isolated
including mycobactin J 1 and carboxymycobactin 2 from
Mycobacterium tuberculosis,2 deferrioxamine B 3 from
Streptomyces pilosus,3 nannochelin A 4 from Nannocys-
tis exedens,4 acinetoferrin 5 from Acinetobacter haemo-
lyticus,5 schizokinen 6 from Bacillus megaterium,6 and
aerobactin 7 from Aerobacter strains.7 Although the
siderophores differ in their overall structure, several
iron-binding motifs have been conserved. These ligands
primarily include the hydroxamic acids, catechols, and/
or R-hydroxycarboxylic acids.1 Several siderophores
(4-7) also share a common feature of a central citric
acid component.
One antibiotic strategy could rely on the molecular
recognition events involved in the internalization of the
siderophore-iron complex for targeted drug delivery
into specific microbes.2 Siderophores could act as anti-
biotic agents through several mechanisms. First, they
could act as deferration agents, which complex all
* To whom correspondence should be addressed. Phone: (407) 823-
5410. Fax: (407) 823-2252. E-mail: ophansti@mail.ucf.edu.
† Department of Chemistry.
‡ Department of Molecular Biology and Microbiology.
F igu r e 1. Structures of iron chelators.
10.1021/jm049805y CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/26/2004

4934 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Gardner et al.
available iron and make it unavailable for microbial use.
This action would in effect sequester this essential
micronutrient and “iron-starve” the bacteria, thereby
limiting its growth.2 Second, bioconjugates between
siderophores and antimicrobial agents may provide
targeted delivery into microbes that have transporters
for the import of siderophore-iron complexes. Alterna-
tively these agents could inhibit microbial iron uptake
by blocking cell-surface binding sites with a rogue
siderophore-iron complex.8,9 Therefore, understanding
the structure-activity relationships involved in sidero-
phore-mediated transport has real value in terms of
antibiotic agent development.
late their growth. Once a suitable surrogate siderophore
was identified, then bioconjugates could be made to
target this bacterial class with appended antibiotic
agents. To study mycobacterial iron transport, Myco-
bacterium avium subsp. paratuberculosis was chosen as
an alternative pathogenic model for M. tuberculosis.
This less-pathogenic strain is responsible for J ohne’s
disease in cattle and Crohn’s disease in humans.16
A
key feature in selecting this model was that M. avium
subsp. paratuberculosis requires the addition of exog-
enous siderophore (mycobactin J , 1) in order to grow.
In this regard, it is an excellent screen for determining
how non-native siderophores influence mycobacterial
growth.
The virulence of many microbes depends on their
ability to compete for and obtain essential elements such
as iron(III). Perhaps most pernicious is the bacterium
(Mycobacterium tuberculosis) responsible for tubercu-
losis (TB) in humans, which continues to infect millions
of people each year.10 Although approximately 40% of
the world’s population is infected with M. tuberculosis,
it grows slowly and the immune system can indefinitely
suppress it. Still, according to the World Health Orga-
nization (WHO), the disease killed more people in 1995,
when effective treatment was available, than in 1900
when no drugs were available to combat the disease.10
The first antibiotic made available to fight tuberculosis
was streptomycin, which was discovered in 1944. While
other antituberculosis drugs have also been discovered,
the current increase in mortality is due to the emer-
gence of multiple drug resistant (MDR) strains of M.
tuberculosis.10,11 The WHO reports that the death rate
of people with MDR-TB in the U.S. in 1995 was 70%
and TB continues to be a public health threat to the
extent that the WHO has declared the disease as a
global public health emergency.10
Prior work involved cross-feeding M. avium subsp.
paratuberculosis with several siderophores to evaluate
whether non-native iron chelators could serve as sur-
rogate siderophores and promote growth.17 During the
biological evaluation of several citrate-containing sid-
erophores,17 several trends became apparent. First, the
number of carbons separating the bidentate ligand sites
within each siderophore was vital. Indeed, longer teth-
ers were more efficacious in cross-feeding M. avium
subsp. paratuberculosis. Second, citrate derivatives that
contained 2-octenoyl “tails” were better growth stimu-
lants than those containing cinnamoyl tails. Third,
derivatives that contained cyclic imides were initially
shown to be 5 times more efficient in cross-feeding M.
avium subsp. paratuberculosis. Collectively, these trends
warranted the further investigation of these structural
components. Using our earlier findings as a guide, we
now report the synthesis of four new acinetoferrin
analogues (5b, 5c, 8b, and 8c) that incorporate these
trends. These systems represent important new probes
to study iron transport processes in M. avium subsp.
paratuberculosis.
Like most mycobacteria, the cell walls of M. tubercu-
losis are rich in lipids and consist mainly of mycolic
acids containing long hydrocarbon chains. The high
degree of saturation and length of these hydrocarbon
chains contribute to a cell wall that is very tightly
packed with extremely low fluidity.12 The cell wall,
therefore, has a low permeability to antibiotics and other
chemotherapeutic agents.12,13 A second feature of M.
tuberculosis is the production of two kinds of sidero-
phore: an intracellular mycobactin 1 that is associated
with the cell wall and an extracellular exochelin 2. The
absorption of iron through the cell wall of M. tubercu-
losis is thought to involve an “iron handoff” between the
two types of siderophores. In low-iron environments a
hydrophilic exochelin is synthesized and excreted to
bind exogenous ferric iron. This bound iron can then be
transferred to the lipophilic mycobactin in the cell wall
so that the iron can be stored or released into the cell
by mycobactin reductases.2
Resu lts a n d Discu ssion
The synthetic strategy was predicated upon the
modular approach developed for the synthesis of acineto-
ferrin 5a and acinetoferrin imide 8a . Four new acineto-
ferrin homologues were generated (5b, 5c, 8b, and 8c).
The procedure allowed for installation of a longer tether,
which significantly increased the number of carbons
between the bidentate ligands (e.g., from 5a (n ) 1) to
5b (n ) 3) in Figure 2). The 2-E-octenoyl tail was
retained because of its observed enhanced cross-feeding
capabilities. The approach to the cyclic imides 8b and
8c was via their respective precursors: the acyclic
R-hydroxycarboxylic acids 5b and 5c. Therefore, any
improvements in the methodology to access 5 directly
impacted the overall yield of 8. Moreover, an improved
cyclization procedure was also developed for the direct
synthesis of 8b and 8c from their respective tert-butyl
esters, 20 and 21.
Since iron is necessary for the growth and survival
of M. tuberculosis, several approaches have been put
forward for the development of antibiotics predicated
upon siderophores and their analogues.8,9 However,
targeting the iron transport system of M. tuberculosis
is very challenging because of the complex architecture
of the mycobactins.14 This structural complexity also
hampers the synthesis of bioconjugates.15
The discussion will first focus on the construction of
the linear analogues 5b and 5c. The modular synthetic
approach involved the synthesis of a protected N-
(alkylamino)-N-hydroxyl-trans-octenoyl precursor that
could be deprotected and coupled to the terminal car-
boxylic acids of an activated citric acid derivative.17
series of protection and deprotection steps were needed
for the synthesis of the N-(alkylamino)-N-hydroxyl-
trans-octenoyl precursor. As shown in Scheme 1, the
A
Our initial goal was to identify non-native sidero-
phores, which deliver iron to mycobacteria and stimu-

Acinetoferrin Homologues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4935
and 12) in 72% and 73% yields, respectively. The
tandem oxidation-acylation of a primary amine group
to introduce the O-benzoyl-protected hydroxamate has
been shown by Milewska et al.18
This approach led to our improved amine-oxidation
method involving biphasic conditions, which was used
successfully in this and many other reports.17,19-21 The
monoprotected diamines 11 and 12 were each dissolved
in a biphasic mixture containing a pH 10.5 carbonate
buffer and benzoyl peroxide (BPO) dissolved in CH2-
Cl2.19,20 After the oxidation step, each mixture was
acylated in situ with trans-octenoyl chloride to the give
the desired compounds 13 (61%) and 14 (65%), respec-
tively.
The synthesis of the tert-butylacinetoferrin homo-
logues (20 and 21) required the coupling of the func-
tionalized pentylamine and hexylamine fragments to
the derivatized citric acid framework. Previous experi-
ence revealed that higher yields were obtained if the
benzoyl group was removed prior to the amide forma-
tion.4,5,22 Therefore, compounds 13 and 14 were treated
with a solution of 10% concentrated NH4OH in MeOH
solution at -23 °C to give the “free” hydroxamic acids
15 (84%) and 16 (86%), respectively. Although the
hydroxamic acids were purified for analytical purposes,
they were typically used in the following steps without
purification. Removal of the tert-butyl carbamate groups
of 15 and 16 using trifluoroacetic acid (TFA) at 0 °C
gave the respective TFA salts 17 and 18. These were
used without further purification in the next step, which
F igu r e 2. Acinetoferrin analogues and bis-NHS ester 19.
Sch em e 1a
4-6,17
involved coupling to the citric acid derivative, 19.
Prior methods for the selective coupling of amines to
the carboxylic acid groups of the citric acid portion have
involved using anhydromethylene citric acid or 2-sub-
stituted-1,3-bis-activated esters of citric acid.6,7 How-
ever, the reported imide formation associated with the
use of the anhydromethylene unit and the low overall
isolated yield achieved in the synthesis of the 1,3-bis-
N-hydroxysuccinimide esters of citric acid (20%) fol-
lowed by the coupling step (80%, 16% overall) prompted
the investigation for an improved method.17
A number of different reagent combinations were
tried for the coupling step including HOBt/EDC, NHS/
EDC, and NHS/DCC. In these reactions the activated
1,3-bis-N-hydroxy containing ester was not isolated but
used directly in the coupling step. Ironically, the most
efficient reagent was the NHS and DCC combination.
Indeed, significantly higher yields were obtained when
the bis-N-hydroxysuccinimide ester 19 (Figure 2) was
not isolated but used directly in the subsequent coupling
step. Therefore, the condensation of a solution of the
respective TFA salt (17 or 18) and triethylamine with
a solution of crude 1917 gave the corresponding tert-
butylacinetoferrin homologue 20 or 21 in yields of 60%
and 70%, respectively. Finally treatment of 20 and 21
with TFA gave the final homologues 5b and 5c in yields
of 77% and 67%, respectively.
Initially, the imides 8b and 8c were generated in
refluxing TFA for 3 h from 5b and 5c, respectively. The
reactions were monitored by 1H NMR. In particular the
two doublets at δ 2.68 and δ 2.58, integrating for two
protons each, were monitored. These are the citrate
methylene groups, which flank the prochiral center.
Upon treatment of 5b in refluxing TFA, these two
a
Reagents: (a) di-tert-butyl dicarbonate, 10% TEA/MeOH; (b)
benzoyl peroxide (1.1 equiv)/CH2Cl2, pH 10.5 aqueous sodium
carbonate buffer, R; (c) trans-octenoyl chloride, CH2Cl2; (d) 10%
NH4OH/MeOH; (e) TFA, 0 °C; (f) 19, TEA, room temp; (g) TFA,
reflux.
reaction of the diamines 9 and 10 with di-tert-butyl
dicarbonate afforded the monoprotected diamines (11

4936 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Ta ble 1. BACTEC Dataa
Gardner et al.
ies had suggested that systems containing longer teth-
ers gave higher GI values.17 We speculated that the
longer tether allowed for a more conformationally flex-
ible ligand to properly coordinate to iron and provides
an incremental increase in hydrophobicity. Indeed, such
tether effects have been observed with other siderophore
systems such as DFO 3. Molecular modeling studies
with DFO suggest that tethers, which are too short (<5
C), may compromise the optimum binding geometry
around iron by perturbing the octahedral complex.1b
av BACTEC reading
growth index (GI)
compd
( standard deviation relative to mycobactin J
mycobactin J , 1
DFO, 3
511 ( 38
555 ( 53
489 ( 23
483 ( 24
619 ( 58
600 ( 11
515 ( 98
1.00
1.09
0.96
0.95
1.21
1.17
1.01
5C linear, 5b
6C linear, 5c
3C imide, 8a
5C imide, 8b
6C imide, 8c
a
Siderophores were dosed at 2.4 µM and run in triplicate. All
siderophores were dosed in the same media with the same cell
type and uniformly cultured under the same environmental
conditions (see Experimental Section). In the absence of added
chelator, the bacterial growth was extremely slow and gave a
relative BACTEC average reading near 100.
Caveat. Earlier studies showed a nearly 5-fold growth
increase of M. avium subsp. paratuberculosis in the
presence of acinetoferrin imide 8a (GI ) 4.6)17 vs the
control platform (mycobactin J ). Using a revised proto-
col, we observed an increase in growth index for media
supplemented with acinetoferrin imide compared to
those with mycobactin J . However, the increase in
growth index was lower than reported in an earlier trial
(Table 1, 8a : 1.2). We believe that this earlier result
was correct in identifying the growth enhancement
trend but incorrect in its magnitude. The earlier experi-
ments were also run in triplicate as reported here and
had both mycobactin J and DFO as separate controls
run in parallel. The different observations with the same
siderophore 8a warranted further investigation.
doublets become four distinct doublets at δ 2.99, δ 2.89,
δ 2.72, and δ 2.59 (integrating for one proton each),
respectively. Isolation and characterization of the prod-
1
uct by H NMR confirmed these doublets were derived
from the cyclic imide 8b. Although the resonances near
δ 2.7 and δ 2.6 appear to overlap, the doublets at δ 2.99
and δ 2.89 are clearly resolved. Therefore, one can
routinely monitor the conversion by integrating these
respective doublets. Using this approach, we observed
70% conversion of the tert-butylacinetoferrins 5b and
5c to the imides 8b and 8c, respectively. At this point
the reactions were stopped because evidence for the
formation of an elimination product (an alkene) could
Careful scrutiny of the bacteria-dosing protocol found
that the mycobacteria tend to clump upon standing. The
original bacteria-injection protocol inoculated each vial
in a successive manner using a stock suspension of
mycobacteria. Therefore, it was possible that more
bacterial count (e.g., a rogue clump) could be introduced
into one vial over another. This clumping phenomenon
could then skew the results because the BACTEC
instrument would detect more bacteria over time. To
remove the possibility of this event, we altered our
protocol to include a resuspension of the bacterial
culture just before each inoculation. In this manner the
bacterial clumps were broken up and resuspended just
prior to injection into the respective BACTEC vial.
Additionally, the new protocol was shown to be very
reproducible, where repeated trials gave the same
relative growth indices.
1
be seen in the H NMR at δ 5.8. Purification by flash
column chromatography on silica gel gave the cyclic
imides 8b and 8c in isolated yields of 43% and 41%,
respectively. An alternative imidization method using
4 N HCl in glacial acetic acid (21 days at 25 °C)
overcame alkene formation.
In summary, improvements to previous methods for
the synthesis of acinetoferrin were developed during the
synthesis of four new homologues of acinetoferrin (5b,
5c, 8b, and 8c). The synthesis of these homologues
successfully incorporated the key features of the previ-
ous research: namely, the longer tether length, the 2-E-
octenoyl tails, and the cyclic imide unit. Because of the
previous findings with 5a and 8a , the four ligands
reported herein were expected to be superior sidero-
phore surrogates for mycobacteria.
Biologica l Eva lu a tion . All derivatives tested were
capable of forming hexadentate-binding architectures
to iron(III). The respective siderophores (5b, 5c, 8b, and
8c) were evaluated for their ability to act as growth
factors for M. avium subsp. paratuberculosis at 2.4 µM
(a standard concentration used routinely with 1 in M.
avium subsp. paratuberculosis cultures).14,23 Several
controls (DFO 3 and 8a ) were run in tandem. Mycobac-
tin J (the native chelator, 1) provided the benchmark
control. In this manner, systems, which provided sig-
nificantly higher growth index (GI) values than the
native chelator 1 (GI ) 1), were identified as superior
growth stimulants and more efficacious iron delivery
agents (vide infra). In addition, DFO 3 probed the effect
of simply providing solubilized ferric ion as the deter-
mining factor in stimulating growth. The results are
listed in Table 1.
Tr en d s. As shown in Table 1, the BACTEC readings
were normalized to the growth observed with native
mycobactin J , 1. By use of the revised protocol, 8a was
only a modest growth stimulant (Table 1, 8a GI ) 1.21)
for M. avium subsp. paratuberculosis. The related C5
analogue 8b also had enhanced growth activity (GI )
1.17). However, the C6 homologue 8c was equivalent
to 1 (GI ) 1.01). [Note: since the error associated with
the BACTEC reading of 8c (515 ( 98) was large, the
experiment was repeated and gave the same relative
activities.] Therefore, the longer pentylene tether length
of 8b did not radically improve the GI found with 8a .
However, this modest enhancement was lost upon
further extension of the tether (e.g., hexylene 8c, GI )
1.01). It is possible that this reduction in growth-
stimulating activity may be due to increasing entropic
considerations associated with the highly flexible ligand
8c.
Comparison of acyclic 5b (C5 tether, GI ) 0.96) and
imide 8b (C5 tether, GI ) 1.17) reaffirmed the signifi-
cance of the cyclic imide architecture. However, this
trend was only significant when the proper tether was
As shown in Table 1, most derivatives were virtually
equivalent or superior to mycobactin J in stimulating
M. avium subsp. paratuberculosis growth. Earlier stud-

Acinetoferrin Homologues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4937
31.69, 30.02, 29.38, 27.49, 25.04, 23.71, 14.56; HRMS (FAB)
calcd for C32H57N4O9 (M + 1), 641.4126; found (M + 1),
641.4112.
in place [e.g., acyclic 5c (C6 tether, GI ) 0.95) and imide
8c (C6 tether, GI ) 1.01)]. The fact that both the native
chelator (1) and 8 each contain a cyclic bidentate ligand
within their architectures may be more than a curious
coincidence and deserves further study.
Acin etofer r in C6 Hom ologu e (5c). TFA (15 mL) was
added dropwise to 21 (0.266 g, 0.37 mmol) at 0 °C. After the
addition was complete, the stirred solution was allowed to
warm to room temperature. TLC (10% MeOH/CHCl3) showed
that no starting material remained after 3 h. The volatiles
were removed in vacuo to give a light-brown oil that was
purified by column chromatography using LH-20 Sephadex (30
g, 8% EtOH/toluene) to give 5c as a white solid (0.165 g, 67%).
Last, the fact that all ligands tested had growth
activity near the native chelator 1 is noteworthy. While
the bacteria’s structural preferences for the iron complex
seem less important than our original estimates, the
tolerances accommodated by the M. avium subsp.
paratuberculosis iron transport system provide new
opportunities for drug delivery by this pathway. In
addition, there may be significant cost savings associ-
ated with harvesting these slow-growing organisms by
using alternative chelators. Since one is able to replace
ferric mycobactin J (Allied Monitor, Inc.: $6500/g) with
a less expensive chelator like DFO 3 (mesylate salt,
Sigma-Aldrich, Inc.: $70/g), these studies have also
provided an economic alternative to the mycobacteria
community.
1
5c: Rf ) 0.35 (10% MeOH/CHCl3); mp 118-119 °C; H NMR
(CD3OD) δ 6.83 (dt, 2H, olefinic), 6.61 (d, 2H, olefinic), 3.65 (t,
4H, CH2NO), 3.16 (t, 4H, CH2NHCO), 2.74 (d, 2H, CH2CdO),
2.62 (d, 2H, CH2CdO), 2.25 (ddd, 2H, CH2CdC), 1.65 (quin,
4H, CH2), 1.48 (m, 8H, 4 × CH2), 1.42-1.21 (m, 16H, 8 × CH2),
0.92 (t, 6H, CH3); 13C NMR (CD3OD) δ 176.88, 171.96, 168.40,
148.03, 120.51, 75.24, 44.98, 40.38, 33.50, 32.70, 30.35, 29.40,
27.71, 27.47, 23.72, 14.57; HRMS (FAB) calcd for C34H61N4O9
(M + 1), 669.4439; found (M +1), 669.4417.
Im id e of Acin etofer r in C5 Hom ologu e (8b). TFA (15
mL) was added dropwise to 20 (0.12 g, 0.17 mmol) at 0 °C.
After the addition was complete, the stirred solution was
allowed to warm to room temperature. TLC (10% MeOH/
CHCl3) showed that no starting material remained after 3 h.
The mixture was then refluxed for a further 3 h. TLC (10%
MeOH/CHCl3) showed a new spot with a higher Rf. The
volatiles were removed in vacuo to give a light-brown oil that
was purified by flash chromatography (5% MeOH/CHCl3) using
“pretreated” silica gel to give the product 8b as a white solid
(0.046 g, 43%). 8b: Rf ) 0.28 (5% MeOH/CHCl3), Rf ) 0.57
(10% MeOH/CHCl3); mp 142-144 °C; 1H NMR (CD3OD) δ 6.83
(dt, 2H, olefinic), 6.61 (d, 2H, olefinic), 3.64 (t, 4H, CH2NO),
3.49 (t, 2H, CH2NHCO), 3.11 (t, 2H, CH2NHCO), 2.99 (d, 1H,
CH2CdO), 2.89 (d, 1H, CH2CdO), 2.72 (d, 1H, CH2CdO), 2.59
(d, 1H, CH2CdO), 2.24 (q, 2H, CH2CdC), 1.63 (m, 6H, CH2),
1.48 (m, 6H, CH2), 1.38-1.27 (m, 12H, 6 × CH2), 0.91 (t, 6H,
CH3); 13C NMR (CD3OD) δ 180.47, 176.83, 171.17, 168.31,
148.05, 120.53, 73.77, 40.26, 39.66, 33.71, 32.72, 30.07, 29.42,
28.35, 27.55, 25.09, 23.74, 14.58; HRMS (FAB) calcd for
Con clu sion s
In summary, several acinetoferrin homologues were
synthesized and showed good activity in cross-feeding
M. avium subsp. paratuberculosis with similar efficiency
as the native chelator, mycobactin J , 1. In this regard,
significant cost savings can be obtained by using alter-
native chelators to grow mycobacteria.
Exp er im en ta l Section
Gen er a l. Trans-2-octenoic acid (97.1% pure) was purchased
from Lancaster Synthesis, Inc. All solutions are expressed in
vol %. Ammonia-containing solutions were prepared by mea-
suring out concentrated aqueous NH4OH in the listed vol %.
The pH 10.5 carbonate buffer solution was prepared by
combining 222 mL of 0.75 N aqueous NaHCO3 and 78 mL of
1.5 N aqueous NaOH. “Iron-free” glassware was obtained by
soaking the glassware in 6 N HCl bath overnight, then
washing with deionized water. Deionized water was obtained
from a “B-pure” filtration system by collecting the water when
the “in-line” electrical resistance was 17.0 MΩ‚cm. “Iron-free”
silica gel was made by washing with methanol/acetone/10 M
HCl (45:45:10), followed by 10% (w/w) Na2CO3 solution, then
rinsing with deionized water until pH 7 was attained, and air-
drying. Every effort was made to prepare the final acinetof-
errin analogues as metal-free ligands. While it is possible that
trace iron contaminants could be present, all tested materials
passed elemental analysis as metal-free ligands (see Support-
ing Information). The respective metal complexes are formed
when these ligands are dosed into a culture broth containing
ferric ammonium citrate (0.04 g/L media) during the bioassay.
Acin etofer r in C5 Hom ologu e (5b). TFA (15 mL) was
added dropwise to 20 (0.287 g, 0.41 mmol) at 0 °C. After the
addition was complete, the stirred solution was allowed to
warm to room temperature. TLC (10% MeOH/CHCl3) showed
that no starting material remained after 3 h. The volatiles
were removed in vacuo to give a light-brown oil that was
purified by column chromatography using LH-20 Sephadex (30
g, 8% EtOH/toluene) to give the acinetoferrin analogue 5b as
a white solid (0.182 g, 77%). 5b: Rf ) 0.3 (10% MeOH/CHCl3);
mp 122-124 °C; 1H NMR (CDCl3) δ 7.15 (br s, 2H, NH), 6.85
(m, 2H, olefinic), 6.65 and 6.05 (m, 2H, olefinic), 3.67 (m, 4H,
CH2NO), 3.20 (q, 4H, CH2NHCO), 2.67 (d, 2H, CH2CdO), 2.52
(d, 2H, CH2CdO), 2.21 (q, 2H, CH2CdC), 1.68 (m, 4H, CH2),
1.58-1.11 (m, 29H, 10 × CH2, C(CH3)3), 0.83 (t, 6H, CH3); 1H
NMR (CD3OD) δ 6.83 (dt, 2H, olefinic), 6.61 (d, 2H, olefinic),
3.65 (t, 4H, CH2NO), 3.17 (t, 4H, CH2NHCO), 2.73 (d, 2H,
CH2CdO), 2.63 (d, 2H, CH2CdO), 2.25 (ddd, 2H, CH2CdC),
1.67 (quin, 4H, CH2), 1.52 (m, 8H, 4 × CH2), 1.41-1.27 (m,
12H, 6 × CH2), 0.92 (t, 6H, CH3); 13C NMR (CD3OD) δ 176.87,
171.96, 168.26, 148.07, 120.46, 75.20, 44.99, 40.27, 33.58,
C
32H55N4O8 (M + 1), 622.4020; found (M + 1), 622.4046.
Im id e of Acin etofer r in C6 Hom ologu e (8c). TFA (15
mL) was added dropwise to 21 (0.095 g, 0.13 mmol) at 0 °C.
After the addition was complete, the stirred solution was
allowed to warm to room temperature. TLC (10% MeOH/
CHCl3) showed that no starting material remained after 3 h.
The mixture was then refluxed for a further 3 h. TLC (10%
MeOH/CHCl3) showed a new spot with a higher Rf. The
volatiles were removed in vacuo to give a light-brown oil that
was purified by flash chromatography (5% MeOH/CHCl3) using
“pretreated” silica gel to give the product 8c as a white solid
(0.035 g, 41%). 8c: Rf ) 0.33 (5% MeOH/CHCl3), Rf ) 0.62
(10% MeOH/CHCl3); mp 154-155 °C; 1H NMR (CD3OD) δ 6.83
(dt, 2H, olefinic), 6.61 (d, 2H, olefinic), 3.63 (t, 4H, CH2NO),
3.48 (t, 2H, CH2NHCO), 3.09 (t, 2H, CH2NHCO), 3.00 (d, 1H,
CH2CdO), 2.89 (d, 1H, CH2CdO), 2.72 (d, 1H, CH2CdO), 2.59
(d, 1H, CH2CdO), 2.23 (q, 2H, CH2CdC), 1.62 (m, 6H, CH2),
1.47 (m, 6H, CH2), 1.38-1.29 (m, 16H, 8 × CH2), 0.91 (t, 6H,
CH3); 13C NMR (CD3OD) δ 180.39, 176.79, 171.12, 147.88,
120.63, 73.95, 43.34, 40.33, 39.83, 33.53, 32.65, 30.39, 29.37,
28.56, 27.70, 27.59, 27.47, 27.41, 23.64, 14.45; HRMS (FAB)
calcd for
651.4354.
C34H59N4O8 (M + 1), 651.4333; found (M +1),
5-(ter t-Bu toxyca r bon yla m in o)p en tyla m in e (11). 1,5-
Diaminopentane 9 (10.2 g, 0.1 mol) was dissolved in a solution
of triethylamine and methanol (10% TEA in MeOH, 220 mL).
A solution of di-tert-butyl dicarbonate (7.27 g, 0.033 mol) in
methanol (20 mL) was added to this mixture with vigorous
stirring. The mixture was refluxed for 2 h and left to stir at
room temperature overnight. The tert-butoxy-carbonylation
was complete as evidenced by TLC (4% NH4OH/MeOH). The
excess 9, methanol, and TEA were removed in vacuo to yield
an oily residue that was dissolved in CH2Cl2 and washed with
a solution of 10% aqueous Na2CO3. The organic layer was

4938 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Gardner et al.
separated, dried over anhydrous Na2SO4, and filtered, and the
solvent was removed in vacuo. The oily residue was purified
by flash column chromatography to give the product 11 as a
yellow oil (4.88 g, 73%). 11: Rf ) 0.3 (1:10:89 NH4OH/MeOH/
dried over anhydrous Na2SO4, filtered, and concentrated in
vacuo to give the crude product. The crude product was
purified by flash column chromatography (20% EtOAc/hexane)
to yield the product 14 as an oil (3.69 g, 65%). 14: Rf ) 0.35
(25% EtOAc/hexane); 1H NMR (CDCl3) δ 8.11 (d, 2H, phenyl),
7.67 (t, 1H, phenyl), 7.52 (t, 2H, phenyl), 7.01 (dt, 1H, olefinic),
6.04 (dt, 1H, olefinic), 4.61 (br s, 1H, NH), 3.85 (t, 2H, CH2-
NO), 3.08 (t, 2H, CH2NBOC), 2.14 (ddd, 2H, CH2CdC), 1.67
(quin, 2H, CH2), 1.58-1.05 (m, 21H, 6 × CH2, C(CH3)3), 0.83
(t, 3H, CH3); 13C NMR (CDCl3) δ 164.65, 156.14, 149.40, 134.60,
130.20, 129.10, 127.03, 118.29, 79.40, 48.70, 40.77, 32.80,
31.58, 30.26 28.78, 28.13, 27.43, 26.74, 26.67, 22.75, 14.31;
HRMS (FAB) calcd for C26H41N2O5 (M + 1), 461; found (M +
1), 461.
1
CHCl3); H NMR (CDCl3) δ 4.59 (br s, 1H, NH), 3.10 (q, 2H,
CH2NBOC), 2.68 (t, 2H, CH2N), 1.55-1.27 (m, 15H, 3 × CH2,
C(CH3)3); 13C NMR (CDCl3) δ 156.12, 79.02, 42.12, 40.63, 33.38,
30.11, 28.64, 24.27; HRMS (FAB) calcd for C10H23N2O2 (M +
1), 203.1760; found (M + 1), 203.1757.
6-(ter t-Bu toxyca r bon yla m in o)h exyla m in e (12). 1,6-Di-
aminohexane 10 (11.6 g, 0.1 mol) was dissolved in a solution
of triethylamine and methanol (10% TEA in MeOH, 220 mL).
A solution of di-tert-butyl dicarbonate (7.27 g, 0.033 mol) in
methanol (20 mL) was added to this mixture with vigorous
stirring. The mixture was refluxed for 2 h and left to stir at
room temperature overnight. The tert-butoxy-carbonylation
was complete as evidenced by TLC (4% NH4OH/MeOH). The
excess 10, methanol, and TEA were removed in vacuo to yield
an oily residue that was dissolved in CH2Cl2 and washed with
a solution of 10% aqueous Na2CO3. The organic layer was
separated, dried over anhydrous Na2SO4, and filtered, and the
solvent was removed in vacuo. The oily residue was purified
by flash column chromatography (1:10:89 NH4OH/MeOH/
CHCl3) to give the product 12 as a yellow oil (5.17 g, 72%).
12: Rf ) 0.35 (1:10:89 NH4OH/MeOH/CHCl3); 1H NMR
(CDCl3) δ 4.90 (br s, 1H, NH), 3.11 (q, 2H, CH2NBOC), 2.67
(t, 2H, CH2N), 1.55-1.27 (m, 17H, 4xCH2, C(CH3)3); 13C NMR
(CDCl3) δ 156.13, 79.07, 42.29, 40.73, 33.86, 30.33, 28.72, 26.92,
26.82; HRMS (FAB) calcd for C11H25N2O2 (M + 1), 217.1916;
found (M + 1), 217.1717.
N -(5-(t er t -Bu t oxyc a r b on yla m in o)p e n t yl)-N -(b e n z-
oyloxy)-2(E)-octen a m id e (13). A solution of benzoyl peroxide
(BPO, 3.29 g, 0.014 mol) in CH2Cl2 (50 mL) was added
dropwise to a vigorously stirred mixture of 11 (2.5 g, 0.0124
mol) in a carbonate buffer solution (pH 10.5, 50 mL) at room
temperature. The buffer solution was prepared by combining
0.75 N aqueous NaHCO3 (37 mL) and 1.5 N aqueous NaOH
(13 mL). The starting material was consumed overnight as
shown by TLC (4% NH4OH/MeOH). Acylation of the oxidized
intermediate was carried out in situ by the dropwise addition
of a solution of trans-2-octenoyl chloride (1.99 g, 0.0124 mol)
in CH2Cl2 (20 mL). Note: the acid chloride was generated from
trans-2-octenoic acid using oxalyl chloride24 and was freshly
made prior to use. The disappearance of the N-benzoy-
loxyamine was monitored by TLC (40% EtOAc/hexane). After
the acylation was complete, the organic layer was separated
off and the remaining water layer was extracted with CH2-
Cl2. The organic layers were combined, dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo to give the crude
product. The crude product was purified by flash column
chromatography (20% EtOAc/hexane) to yield the product 13
as a brown oil (3.36 g, 61%). 13: Rf ) 0.3 (25% EtOAc/hexane);
1H NMR (CDCl3) δ 8.10 (d, 2H, phenyl), 7.67 (t, 1H, phenyl),
7.52 (t, 2H, phenyl), 7.01 (dt, 1H, olefinic), 6.05 (d, 1H, olefinic),
4.64 (br s, 1H, NH), 3.86 (t, 2H, CH2NO), 3.10 (q, 2H, CH2-
NBOC), 2.14 (q, 2H, CH2CdC), 1.69 (quin, 2H, CH2), 1.59-
1.07 (m, 19H, 5 × CH2, C(CH3)3), 0.83 (t, 3H, CH3); 13C NMR
(CDCl3) δ 164.74, 156.24, 149.55, 134.70, 130.28, 129.19,
127.05, 118.32, 79.39, 48.72, 40.83, 32.88, 31.66, 30.06 28.84,
28.20, 27.25, 24.29, 22.82, 14.39; HRMS (FAB) calcd for
N-(5-(ter t-Bu toxyca r bon yla m in o)p en tyl)-N-(h yd r oxy)-
2-(E)-octen a m id e (15). A 10% NH4OH/MeOH solution (50
mL) was added dropwise to 13 (3.12 g, 7.0 mmol) under
nitrogen at -20 °C using a dry ice/MeOH bath. The reaction
was monitored by TLC (40% EtOAc/hexane). After 3 h the
reaction was complete. The mixture was concentrated in vacuo
to give a light-brown oil that was coevaporated with benzene
and CHCl3. The crude product was purified by flash column
chromatography (20% EtOAc/hexane) using “pretreated” silica
gel to give the product 15 as a pink solid (2.02 g, 84%). 15: Rf
1
) 0.25 (40% EtOAc/hexane); mp 56-58 °C; H NMR (CDCl3)
δ 6.90 (m, 1H, olefinic), 6.64 and 6.03 (m, 1H, olefinic), 4.67
(m, 1H, NH), 3.70 (t, 2H, CH2NO), 3.11 (m, 2H, CH2NBOC),
2.20 (m, 2H, CH2CdC), 1.70 (m, 2H, CH2), 1.58-1.12 (m, 19H,
5 × CH2, C(CH3)3), 0.83 (t, 3H, CH3); 13C NMR (CDCl3) δ
167.52, 156.22, 147.96, 147.13, 119.61, 116.32, 79.70, 49.26,
47.85, 40.12, 39.88, 32.87, 31.74, 30.07, 28.78, 28.34, 25.80,
23.97, 22.81, 14.36; HRMS (FAB) calcd for C18H35N2O4 (M +
1), 343.2591; found (M + 1), 343.2619.
N-(6-(ter t-Bu toxyca r bon yla m in o)h exyl)-N-(h yd r oxy)-
2(E)-octen a m id e (16). A 10% NH4OH/MeOH solution (50
mL) was added dropwise to 14 (3.42 g, 7.4 mmol) under
nitrogen at -20 °C using a dry ice/MeOH bath. The reaction
was monitored by TLC (40% EtOAc/hexane). After 3 h the
reaction was complete. The mixture was concentrated in vacuo
to give a light-brown oil that was coevaporated with benzene
and CHCl3. The crude product was purified by flash column
chromatography (30% EtOAc/hexane) using “pretreated” silica
gel to give the product 16 as a pink solid (2.26 g, 86%). 16: Rf
) 0.3 (40% EtOAc/hexane); mp 61-63 °C; 1H NMR (CDCl3) δ
6.95 (dt, 1H, olefinic), 6.02 (d, 1H, olefinic), 4.50 (br s, 1H, NH),
3.68 (t, 2H, CH2NO), 3.09 (t, 2H, CH2NBOC), 2.22 (q, 2H,
CH2CdC), 1.70 (m, 2H, CH2), 1.58-1.06 (m, 21H, 6 × CH2,
C(CH3)3), 0.83 (t, 3H, CH3); 13C NMR (CDCl3) δ 167.43, 156.39,
147.68, 146.98, 119.58, 116.58, 79.45, 49.04, 48.24, 41.98,
40.60, 32.86, 31.71, 30.08, 28.74, 28.47, 28.32, 26.62, 26.39,
22.79, 14.35; HRMS (FAB) calcd for C19H37N2O4 (M + 1),
357.2748; found (M + 1), 357.2765.
N-(5-Am in op en tyl)-N-(h yd r oxy)-2 (E)-octen a m id e, Tr i-
flu or oa cetic Acid Sa lt (17). TFA (50 mL) was added drop-
wise over 5 min to 15 (1.95 g, 5.7 mmol) at 0 °C under a
nitrogen atmosphere. The ice bath was removed, and the
solution was stirred at room temperature for 15 min. The
volatiles were removed in vacuo, and the residue was coevapo-
rated with benzene and CHCl3 to give the product 17 as the
TFA salt (2.02 g). The oil was consumed immediately in the
synthesis of the tert-butylacinetoferrin homologue, 20.
C
25H39N2O5 (M + 1), 447.2859; found (M +1), 447.2846.
N-(6-Am in oh exyl)-N-(h yd r oxy)-2(E)-octen a m id e, Tr i-
flu or oa cetic Acid Sa lt (18). TFA (50 mL) was added drop-
wise over 5 min to 16 (2.2 g, 6.2 mmol) at 0 °C under a nitrogen
atmosphere. The ice bath was removed, and the solution was
stirred at room temperature for 15 min. The volatiles were
removed in vacuo and the residue was coevaporated with
benzene and CHCl3 to give the product 18 as the TFA salt
(2.28 g). The oil was consumed immediately in the synthesis
of the tert-butyl acinetoferrin homologue, 21.
Acin etofer r in C5 Hom ologu e ter t-Bu tyl Ester (20). In
an “iron-free” round-bottom flask 3-tert-butyl citrate5,17 (0.21
g, 0.85 mmol) was dissolved in dry THF (20 mL). N-Hydroxy-
succinimide (NHS, 0.214 g, 1.86 mmol) and dicyclohexylcar-
N-(6-(ter t-Bu toxycar bon ylam in o)h exyl)-N-(ben zoyloxy)-
2(E)-octen a m id e (14). A solution of BPO (3.29 g, 0.014 mol)
in CH2Cl2 (50 mL) was added dropwise at room temperature
to a vigorously stirred mixture of 12 (2.67 g, 0.0124 mol) in a
carbonate buffer solution (pH 10.5, 50 mL). The starting
material was consumed overnight as shown by TLC (4% NH4-
OH/MeOH). Acylation of the oxidized intermediate was carried
out in situ by the dropwise addition of a solution of trans-2-
octenoyl chloride (1.99 g, 0.0124 mol) in CH2Cl2 (20 mL). The
disappearance of the N-benzoyloxyamine was monitored by
TLC (40% EtOAc/hexane). After the acylation was complete
the organic layer was separated and the remaining water layer
was extracted with CH2Cl2. The organic layers were combined,

Acinetoferrin Homologues
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20 4939
bodiimide (DCC) (0.385 g, 1.86 mmol) were added to this
solution, and the reaction mixture was stirred at room tem-
perature for 3 h producing the activated 1, 3-bis-N-hydroxy-
succinimide ester 19 and a white precipitate. The solution was
concentrated in vacuo, giving a white solid residue that was
used directly for the next coupling step. The crude white solid
was dissolved in dry dioxane (20 mL). A solution of 17 (0.659
g, 0.186 mmol, 2.2 equiv) in CH2Cl2 (10 mL) was first
neutralized with 1.4 mL of TEA at 0 °C and then added to the
dioxane solution. The mixture was stirred at room temperature
overnight. The dicyclohexylurea was removed by filtration, and
the solvents were removed in vacuo. The oily residue was
dissolved in CHCl3 and washed with 0.5 N HCl solution. The
organic layer was separated, and the aqueous layer was
extracted with CHCl3. The organic layers were combined, dried
over anhydrous Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by flash chromatography (gradient
from 2% MeOH/CHCl3 to 6% MeOH/CHCl3) using “pretreated”
silica gel to give the product 20 as a pale-yellow oil (0.36 g,
60%). 20: Rf ) 0.6 (10% MeOH/CHCl3); 1H NMR (CDCl3) δ
7.15 (br s, 2H, NH), 6.85 (m, 2H, olefinic), 6.65 and 6.05 (m,
2H, olefinic), 3.67 (m, 4H, CH2NO), 3.20 (q, 4H, CH2NHCO),
2.67 (d, 2H, CH2CdO), 2.52 (d, 2H, CH2CdO), 2.21 (q, 2H,
CH2CdC), 1.68 (m, 4H, CH2), 1.58-1.11 (m, 29H, 10 × CH2,
bottle. The bottles therefore contain 7H9 and other ingredients
such as ammonium sulfate, glutamic acid, sodium citrate,
pyridoxine, biotin, disodium phosphate, ferric ammonium
citrate (0.04 g/L media), magnesium sulfate, calcium chloride,
zinc sulfate, and copper sulfate. The 7H9 broth was also
supplemented with 100 mL/L OADC (oleic acid, bovine albu-
min, dextrose, catalase enrichment from the Becton Dickinson
Company) and 0.05% Tween 80. Tween 80 is a detergent added
to reduce clumping (Sigma-Aldrich Chemical Co.). Ferric
mycobactin J (Allied Monitor, Fayette, MO) at a final concen-
tration of 2.4 µM was added to the M. avium subsp. paratu-
berculosis strain ATCC 43015 culture (MAP) as a positive
control. When the bacterium was challenged with other
siderophores, ferric mycobactin J was not added to the culture
media. Siderophores were added to the Bactec 7H12 B+
bottled liquid media using Becton Dickinson B-D “1 cm3” sterile
syringes to a final concentration of 2.4 µM. To each of these
Bactec bottles, 100 µL of freshly suspended Bactec 7H12 B+
cultured MAP (GI reading of 100-300 with an average of 1 ×
105 cfu/mL) was inoculated using Becton Dickinson B-D “1
cm3” sterile syringes. Microbial growth activity in this culture
medium is indicated by the release of 14CO2 into the atmo-
sphere of the sealed vial following the metabolism of 14C-
labeled palmitic acid by the microorganism.
1
C(CH3)3), 0.83 (t, 6H, CH3); H NMR (CD3OD) δ 6.83 (dt, 2H,
The atmosphere of the sealed BACTEC 12B vial is aspirated
from the vial via a sterile 1.0 cm3 needle attached to a robotic
arm inside the BACTEC 460 TB analyzer system. The
BACTEC analyzer operates by initially drawing room air
through a dust filter, a flush valve, and an ion chamber
transferring all 14CO2 into a CO2 trap, where it is retained.
This process cleans the electrometer and leaves it ready to
start the next cycle. During the next cycle, a pair of 18G
needles are heated. A pump produces a partial vacuum in the
ion chamber used to lower the testing needles through the
rubber septum of the vial being tested. A vacuum draws
culture gas from the vial to the ion chamber. The electrometer
measures the very small current that the radioactive 14CO2
produces in the ion chamber. Following removal of the
radioactive culture gas, fresh 5% CO2 is introduced into the
medium headspace every time a vial is tested, enhancing the
growth of mycobacteria. The current measured by the elec-
trometer is amplified and displayed as a numeric reading. The
reading is measured on a scale of 0-999 and is an indication
of microbial growth activity in the bottle. Usually, a reading
of 10 or higher is an indication of definite microbial growth.
The growth indices (GI values) listed in Table 1 were calcu-
lated by dividing the instrument reading obtained for the
chelator by that obtained for the mycobactin J control. As is
often the circumstance when looking at relative microbiological
growth rates over time, the error in making these measure-
ments can be appreciable (up to (98 reading units). Slow-
growing cultures may bias the results because their early
growth is more time-sensitive, whereas faster growing cultures
may give disproportionately higher values at early sample
times. In this regard, one looks for significant differences in
growth rates and overall trends. In the absence of any added
chelator, the bacterial growth is extremely slow and well below
the listed values. A typical negative control BACTEC reading
is near 100 compared to the 450-620 BACTEC reading values
obtained when the respective chelators are added (Table 1).
Note: the acinetoferrin-iron complex was recently reported
by Groves et al.25
olefinic), 6.61 (d, 2H, olefinic), 3.65 (t, 4H, CH2NO), 3.16 (t,
4H, CH2NHCO), 2.68 (d, 2H, CH2CdO), 2.58 (d, 2H, CH2Cd
O), 2.25 (q, 2H, CH2CdC), 1.67 (m, 4H, CH2), 1.58-1.11 (m,
29H, 10 × CH2, C(CH3)3), 0.83 (t, 6H, CH3); 13C NMR (CDCl3)
δ 173.33, 170.35, 167.42, 147.26, 119.77, 118.32, 83.10, 76.92,
48.11, 44.20, 38.93, 32.90, 31.80, 29.08, 28.42, 28.14, 25.98,
23.87, 22.84, 14.41; HRMS (FAB) calcd for C36H65N4O9 (M +
1), 697.4752; found (M + 1), 697.4770.
Acin etofer r in C6 Hom ologu e ter t-Bu tyl Ester (21). In
an “iron-free” round-bottom flask 3-tert-butyl citrate5,17 (0.23
g, 0.93 mmol) was dissolved in dry THF (20 mL). N-Hydroxy-
succinimide (0.235 g, 2.0 mmol) and dicyclohexylcarbodiimide
(DCC) (0.42 g, 2.0 mmol) were added to this solution, and the
reaction mixture was stirred at room temperature for 3 h
producing the activated 1, 3-bis-N-hydroxysuccinimide ester
19 and a white precipitate. The solution was concentrated in
vacuo giving a white solid residue that was used directly for
the next coupling step. The crude white solid was dissolved in
dry dioxane (20 mL). A solution of 18 (0.75 g, 2.0 mmol, 2.2
equiv) in CH2Cl2 (10 mL) was first neutralized with 2.0 mL of
TEA at 0 °C and then added to the dioxane solution. The
mixture was stirred at room temperature overnight. The
dicyclohexylurea (DCU) was removed by filtration, and the
solvents were removed in vacuo. The oily residue was dissolved
in CHCl3 and washed with a 0.5 N HCl solution. The organic
layer was separated, and the aqueous layer was extracted with
CHCl3. The organic layers were combined, dried over anhy-
drous Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (gradient from 4%
MeOH/CHCl3 to 6% MeOH/CHCl3) using “pretreated” silica
gel to give the product 21 as a pale-yellow oil (0.47 g, 70%).
21: Rf ) 0.65 (10% MeOH/CHCl3); 1H NMR (CDCl3) δ 7.10
(br s, 2H, NH), 6.81 (m, 2H, olefinic), 6.62 and 6.03 (m, 2H,
olefinic), 3.64 (m, 4H, CH2NO), 3.16 (m, 4H, CH2NHCO), 2.69
(d, 2H, CH2CdO), 2.55 (d, 2H, CH2CdO), 2.19 (m, 2H, CH2-
CdC), 1.62 (m, 4H, CH2), 1.58-1.05 (m, 33H, 12 × CH2,
C(CH3)3), 0.83 (t, 6H, CH3); 13C NMR (CDCl3) δ 173.44, 170.17,
167.11, 147.79, 147.17, 119.62, 118.32, 83.06, 74.32, 49.23,
48.17, 44.04, 39.44, 32.90, 31.75, 29.44, 29.17, 28.39, 28.26,
28.14, 26.74, 26.39, 22.82, 14.40; HRMS (FAB) calcd for
Su p p or tin g In for m a tion Ava ila ble: Elemental analyses
for compounds 5b , 5c, 8b , 8c, 11-16, 20, and 21. This
material is available free of charge via the Internet at
http://pubs.acs.org.
C
38H69N4O9 (M + 1), 725.5065; found (M + 1), 725.5060.
Bioa ssa y. BACTEC 12B mycobacteria, also known as
Middlebrook 7H12 medium, was purchased from Becton
Dickinson (Pittsburgh, PA).14,23 It contains 7H9 broth base,
casein hydrolysate, bovine serum albumin, catalase, and
palmitic acid labeled with 14C. It is specific for growing
mycobacteria and is used in conjunction with the BACTEC
brand 460 TB analyzer. The Middlebrook 7H9 broth base
media consists of 4 mL of broth mixture included in a sealed
Refer en ces
(1) (a) Bergeron, R. J .; Brittenham, G. M. The Development of Iron
Chelators for Clinical Use; CRC Press: Boca Raton, FL, 1994;
pp 279-284. (b) Bergeron, R. J .; Brittenham, G. M. The
Development of Iron Chelators for Clinical Use; CRC Press: Boca
Raton, FL, 1994; p 258.

4940 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 20
Gardner et al.
(2) Roosenberg, J . M., II; Lin, Y.-M.; Lu, Y.; Miller, M. J . Studies
and Syntheses of Siderophores, Microbial Iron Chelators, and
Analogs as Potential Drug Delivery Agents. Curr. Med. Chem.
2000, 7, 159-197 and references therein.
(3) Bergeron, R. J .; McManis, J . S.; Phanstiel, O., IV; Vinson, J . R.
T. Total Synthesis of DFO. J . Org. Chem. 1995, 60, 109-114.
(4) Bergeron, R. J .; Phanstiel, O, IV. The Total Synthesis of
Nannochelin: A Novel Cinnamoyl Hydroxamate Containing
Siderophore. J . Org. Chem. 1992, 57, 7140-7143.
(5) Phanstiel, O., IV; Wang, Q. Total Synthesis of Acinetoferrin. J .
Org. Chem. 1998, 63, 1491-1495.
(15) (a) Maurer, P. J .; Miller, M. J . Total Synthesis of a Mycobactin:
Mycobactin S2. J . Am. Chem. Soc. 1983, 105, 240-245. (b) Hu,
J .; Miller, M. J . Total Synthesis of a Mycobactin S, a Siderophore
and Growth Promoter of Mycobacterium Smegmatis, and De-
termination of Its Growth Inhibitory Activity against Myco-
bacterium tuberculosis. J . Am. Chem. Soc. 1997, 119, 3462-
3468. (c) Hu, J . Total Synthesis of Mycobactins, Siderophore-
Drug Conjugates and Biological Studies. Ph.D. Dissertation,
Department of Chemistry and Biochemistry, University of Notre
Dame, 1996.
(16) (a) Barclay, R.; Ratledge, C. Iron binding compounds of M.
avium, M. intracellularae, M. scrofulaceum, and mycobactin-
dependent M. paratuberculosis and M. avium. J . Bacteriol. 1983,
153, 1138-1146. (b) Chamberlin, W.; Graham, D. Y.; Hulten,
K.; El-Zimaity, H. M. T.; Schwartz, M. R.; Naser, S.; Shafran,
I.; El-Zaatari, F. A. K. Mycobacterium avium subsp. Paratuber-
culosis as one cause of Crohn’s disease. Aliment Pharmacol.
Ther. 2001, 15, 337-346. (c) Chiodini, R. J .; Van Kruiningen,
H. J .; Merkal, R. S. Ruminant paratuberculosis (J ohne’s Dis-
ease): The current status and future prospects. Cornell Vet.
1984, 74, 218-262.
(6) Milewska, M. J .; Chimiak, A. I.; Glowacki, Z. Synthesis of
Schizokinen, Homoschizokinen, Its Imide and the Detection of
Imide with 13C-NMR Spectroscopy. J . Prakt. Chem. 1987, 329
(3), 447-456.
(7) Maurer, P. J .; Miller, M. J . Microbial iron chelators: total
synthesis of aerobactin and its constituent amino acid, N6-acetyl-
N6-hydroxylysine. J . Am. Chem. Soc. 1982, 104, 3096-3101.
(8) Malouin, F.; Miller, M. J . Microbial Iron Chelators as Drug
Delivery Agents: The Rational Design and Synthesis of Sidero-
phore-Drug Conjugates. Acc. Chem. Res. 1993, 26, 241-249.
(9) Lin, Y.; Helquist, P.; Miller, M. J . Synthesis and Biology
Evaluation of a Siderophore-Virginiamycin Conjugate. Synthe-
sis 1999, 1510-1514.
(17) Guo, H.; Naser, S. A.; Ghobrial, G.; Phanstiel, O., IV. Synthesis
and Biological Evaluation of New Citrate-Based Siderophores
as Potential Probes for the Mechanism of Iron Uptake in
Mycobacteria. J . Med. Chem. 2002, 45, 2056-2063.
(10) (a) World Health Organization. Global Tuberculosis Control;
WHO Report WHO/CDS/TB/2001.287; WHO:
Geneva,
Switzerland, 2001. (b) World Health Organization. Tuber-
culosis Fact Sheet, No. 104, 2000 (see http://www.
who.int/inf-fs/en/fact104.html).
(18) Milewska, M. J .; Chimiak, A. An Alternative Synthesis of N5-
Acetyl-N5-hydroxy-L-ornithine from L-Ornithine. Synthesis 1990,
233-234.
(11) (a) Miller, M. J . Synthesis and Therapeutic Potential of Hydrox-
amic Acid Based Siderophores and Analogues. Chem. Rev. 1989,
89, 1563-1579; (b) Haeym, B.; Honore´, N.; Truffot-Pernot, C.;
Banerjee, A.; Schurra, C.; J acobs, W. R., J r.; van Embden, J . D.
A.; Grosset, J . H.; Cole, S. T. Implications of Multidrug Resis-
tance for the Future of Short Course Chemotherapy of Tuber-
culosis: A Molecular Study. Lancet 1994, 344, 293-298.
(12) (a) Rouhi, A. M. Tuberculosis: A Tough Adversary. Chem. Eng.
News 1999, May 17, 52-69. (b) Xu, Y.; Miller, M. J . Total
Syntheses of Mycobactin Analogues as Potent Antimycobacterial
Agents Using a Minimal Protecting Group Strategy. J . Org.
Chem. 1998, 63, 4314-4322.
(19) Wang, Q. X,; King, J .; Phanstiel, O., IV. An Improved Synthesis
of O-Benzoyl Protected Hydroxamates. J . Org. Chem. 1997, 62,
8104-8108.
(20) Phanstiel, O., IV; Wang, Q. X.; Powell, D. H.; Ospina, M. P.;
Leeson, B. A. Synthesis of Secondary Amines via N-(Benzoyloxy)-
amines and Organoboranes. J . Org. Chem. 1999, 64, 803-806.
(21) Badescu, V. Synthesis and Thermal Study of
a Series of
N-Benzoyloxyamines and the Scope and Limitations of an Amine
Oxidation Process. Masters Thesis, Department of Chemistry,
University of Central Florida, 2000.
(13) (a) Connell, N. D.; Nikaido, H. Membrane Permeability and
Transport in Mycobacterium Tuberculosis. In Tuberculosis:
Pathogenesis, Protection, and Control; Bloom, B. R., Ed.; ASM
Press: Washington, DC, 1994; p 333. (b) Besra, G. S.; Chatterjee,
D. Lipid and Carbohydrates of Mycobacterium Tuberculosis. In
Tuberculosis: Pathogenesis, Protection, and Control; Bloom, B.
R., Ed.; ASM Press: Washington, DC, 1994; p 285.
(14) (a) McCullough, W. G.; Merkal, R. S. Structure of Mycobactin
J . Curr. Microbiol. 1982, 7, 337-341. (b) Mathews, P. R.;
McDiarimid, J . A.; Collins, P.; Brown, A. The dependence of some
strains of M. avium on mycobactin for initial and subsequent
growth. J . Med. Microbiol. 1979, 11, 53-57. (c) Lambrecht, R.
S.; Collins, M. T. Mycobacterium paratuberculosis. Factors that
influence mycobactin dependence. Diagn. Microbiol. Infect. Dis.
1992, 15 (3), 239-246.
(22) Wang, L.; Phanstiel, O., IV Synthesis of N-(Hydroxy)amide and
N-(Hydroxy)thioamide-Containing Peptides. J . Org. Chem. 2000,
65, 1442-1447.
(23) Naser, S. A.; Gillespie, R.; Naser, N. A.; El-Zaatari, F. A. K. Effect
of IS900 gene of Mycobacterium paratuberculosis on Mycobac-
terium smegmatis. Curr. Microbiol. 1998, 37, 373-379.
(24) Rastetter, W. H.; Erickson, T. J .; Venuti, M. C. Synthesis of iron
chelators. Enterobactin, enantioenterobactin, and a chiral ana-
log. J . Org. Chem. 1981, 46, 3579-3590.
(25) Fadeev, E.; Luo, M.; Groves, J . T. Synthesis and characterization
of acinetoferrin, schizokinen and their metal complexes. Abstr.
Pap.sAm. Chem. Soc. 2002, 224 (August 18), 099-INOR.
J M049805Y