Conjugates of desferrioxamine B (DFOB) with derivatives of adamantane or with orally available chelators as potential agents for treating iron overload

Article abstract of DOI:10.1021/jm9016703

Desferrioxamine B (DFOB) conjugates with adamantane-1-carboxylic acid, 3-hydroxyadamantane-1-carboxylic acid, 3,5-dimethyladamantane-1-carboxylic acid, adamantane-1-acetic acid, 4-methylphenoxyacetic acid, 3-hydroxy-2-methyl-4-oxo- 1-pyridineacetic acid (

Full text of DOI:10.1021/jm9016703

1370 J. Med. Chem. 2010, 53, 1370–1382
DOI: 10.1021/jm9016703
Conjugates of Desferrioxamine B (DFOB) with Derivatives of Adamantane or with Orally Available
Chelators as Potential Agents for Treating Iron Overload
Joe Liu,† Daniel Obando,† Liam G. Schipanski,† Ludwig K. Groebler,‡ Paul K. Witting,‡
Danuta S. Kalinowski,‡ Des R. Richardson,‡ and Rachel Codd*,†
†School of Medical Sciences (Pharmacology) and Bosch Institute, University of Sydney, New South Wales 2006, Australia and ‡School of Medical
Sciences (Pathology) and Bosch Institute, University of Sydney, New South Wales 2006, Australia
Received November 12, 2009
Desferrioxamine B (DFOB) conjugates with adamantane-1-carboxylic acid, 3-hydroxyadamantane-
1-carboxylic acid, 3,5-dimethyladamantane-1-carboxylic acid, adamantane-1-acetic acid, 4-methylphe-
noxyacetic acid, 3-hydroxy-2-methyl-4-oxo-1-pyridineacetic acid (N-acetic acid derivative of deferi-
prone), or 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid (deferasirox) were prepared and
the integrity of Fe(III) binding of the compounds was established from electrospray ionization mass
spectrometry and RP-HPLC measurements. The extent of intracellular 59Fe mobilized by the DFOB-
3,5-dimethyladamantane-1-carboxylic acid adduct was 3-fold greater than DFOB alone, and the IC50
value of this adduct was 6- or 15-fold greater than DFOB in two different cell types. The relationship
between logP and 59Fe mobilization for the DFOB conjugates showed that maximal mobilization of
intracellular 59Fe occurred at a logP value ∼2.3. This parameter, rather than the affinity for Fe(III),
appears to influence the extent of intracellular 59Fe mobilization. The low toxicity-high Fe mobilization
efficacy of selected adamantane-based DFOB conjugates underscores the potential of these compounds
to treat iron overload disease in patients with transfusional-dependent disorders such as β-thalassemia.
Introduction
agents that coordinate the excess iron and form complexes
that are excreted via the urinary and/or fecal route.6,7 Before
the advent of chelation therapy in 1962, β-thalassemia pa-
tients maintained on prophylactic blood transfusions would
die in early adulthood from complications arising from iron
overload.3
Inheritable disorders of hemoglobin arising from mono-
genic defects are the most common diseases in the world, with
about 7% of people estimated as carriers.1,2 Each year,
300000-500000 children are born with severe hemoglobin
disorders, which include sickle cell anemia and the thalasse-
mias.1 Without treatment for their anemia, these infants die
in the first few years of life.1 Patients with severe forms of
β-thalassemia require lifelong blood transfusions at 2-4
weekly intervals.3 These regular blood transfusions increase
macrophage-induced heme catabolism, which releases iron
into the serum. This saturates the iron transport protein,
transferrin, resulting in an increased pool of nontransferrin-
bound-iron (NTBIa).4 Humans do not have an active iron
excretion mechanism, and levels of NTBI in excess of the
normal, tightly regulated Fe(III) concentrations (about 10-24
M) can generate reactive oxygen species (ROS) that can cause
dysfunction of the heart, liver, anterior pituitary, and pan-
creas.5 Therefore, β-thalassemia patients must undergo, in
addition to their blood transfusions, treatment with chelating
The first-linetreatment for iron overload isthe mesylatesalt
of the trihydroxamic acid-based siderophore, desferrioxamine
B (DFOB; 1), produced bythe bacterium Streptomyces pilosus
(Figure 1).8 Siderophores are low-molecular-weight organic
compounds produced by nonpathogenic and pathogenic
bacteria in response to Fe deprivation.9-12 With poor gastro-
intestinal absorption and a short plasma half-life (t1/2 ∼ 12
min),13 DFOB mesylate is not orally active, which requires
3
that patients are treated via subcutaneous or intravenous
infusion for about 60 h per week.3 This arduous treatment
regimen has a negative impact upon the quality of life of
patients. Poor compliance with chelation therapy can lead to
siderotic cardiac disease, which accounts for 71% of the
mortality from thalassemia.5,14 Significant drawbacks in the
efficacy of the monocationic, hydrophilic 1 (water solubility ∼
0.4 M) as a chelation agent, include both its rapid clearance
(reflected in the treatment regimen) and its inability to readily
cross cell membranes to access intracellular iron pools.15 The
distinguishing attribute of 1, which confers value upon its
clinical use, is the very high affinity toward Fe(III), forming a
stable 1:1 Fe(III):1 hexadentate complex via the three hydro-
xamic acid functional groups (logβ110 = 30.5).16-18 The
drawbacks of 1 have prompted research efforts to find orally
available iron chelating agents.5,6,19 Two of these candidates,
deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one, L1 (2)) and
*To whom correspondence should be addressed. Phone: þ61-2-9351-
6738. Fax: þ61-2-9351-4717. E-mail: rcodd@med.usyd.edu.au.
aAbbreviations: AdAc, adamantane-1-acetic acid; AdA, adaman-
tane-1-carboxylic acid; LDX, 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-
1-yl]benzoic acid (deferasirox); DFOB, desferrioxamine B; AdAdMe
,
3,5-dimethyladamantane-1-carboxylic acid; ESI-MS, electrospray ioni-
zation mass spectrometry; 59Fe-Tf, 59Fe-labeled transferrin; AdAOH, 3-
hydroxyadamantane-1-carboxylic acid; Dp44mT, di-2-pyridyl ketone
4,4-dimethyl-3-thiosemicarbazone; MPOAc, 4-methylphenoxyacetic
acid; L1D, 3-hydroxy-2-methyl-4-oxo-1-pyridineacetic acid; NTBI, non-
transferrin-bound-iron; ROS, reactive oxygen species; RP-HPLC,
reversed-phase high pressure liquid chromatography.
r
pubs.acs.org/jmc
Published on Web 12/30/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1371
siderophores have a rich research profile as potential anti-
bacterial and anticancer agents.44,53,54
Here, we describe the synthesis, characterization, and
structure-activity relationships of seven DFOB conjugates
(4-10, Figure 2). These studies include examination of the
integrity of Fe(III)-binding using electrosprayionization mass
spectrometry (ESI-MS) and reversed-phase high pressure
liquid chromatography (RP-HPLC). We report the determi-
nation of the logP values of 4-10 in the absence and presence
of Fe(III) and the iron chelation efficacy of 4-10 with regard
to their ability to mobilize intracellular 59Fe from SK-N-MC
neuroepithelioma cells and to prevent 59Fe uptake from 59Fe-
transferrin (59Fe-Tf). Furthermore, we have examined the
antiproliferative activity of 4-10 in SK-N-MC cells and in a
renal epithelial cell type. Together, the results indicate that
selected conjugates of 1 should be further evaluated as poten-
tial new agents for the treatment of iron overload disease.
Figure 1. Schematic of the clinically used iron chelators used in
this study: desferrioxamine B, DFOB (1); deferiprone (2); defera-
sirox (3).
deferasirox (4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-
benzoic acid, ICL670 (3)) (Figure 1), both of which are orally
active, have varied profiles in the clinic.19-21
Deferiprone (2) is an orally active, three-times-daily,
R-ketohydroxypyridine-based bidentate iron chelator, which
can remove iron from noncardiac parenchyma, macrophages,
transferrin, ferritin, and hemosiderin.19 Deferiprone is not as
effective as 1 at removing Fe(III) and has toxicity issues,
including agranulocytosis.5,22,23 Its use is largely limited to the
European Union as a second-line treatment for patients
intolerant to 1. Currently, 2 is not approved for use in the
USA. Deferasirox (3) is a tridentate, one dose-per-day, orally
administered iron chelator in use since 2005 in the USA,
Switzerland, and Europe.19,20 Deferasirox crosses hepatocyte
and cardiac myocyte cell membranes and has shown good
tolerance and safety with side effects that include mild gastro-
intestinal symptoms and mild aminotransferase elevation.24,25
The first-in-class thiazolecarboxylic acid-based iron chelator,
desferrithiocin, showed good iron clearance in monkeys but
was severely nephrotoxic.26,27 The less toxic derivative, defer-
itrin, showed favorable pharmacokinetics in rats, dogs, and
monkeys28 and is currently in phase II clinical trials.19 Renal
toxicity has also been observed in some patients treated
with 3.29,30 The development of acceptable iron chelating
drugs for iron overload, therefore, requires consideration of
efficacy of Fe-binding, renal toxicity, and other potential side
effects. Other drugs in development as iron chelators include
the deferiprone derivative L1NA11,19 hydroxypyridinone-
based compounds,31 isonicotinoyl hydrazones,32-35 and
thiosemicarbazones;32,36-40 the latter two classes of com-
pounds being developed principally as anticancer agents.
An alternative approach toward the design of new iron
chelating compounds for the treatment of iron overload
involves 1-based semisynthesis, where ancillary compounds
are appended to 1 via the free primary amine group, which
itself is not involved in the Fe(III)-1 coordination sphere.17 In
this approach, the integrity of the 1-derived Fe(III)-binding
hydroxamic acid groups of the conjugate are retained, yet the
properties of the compound may be tuned as a function of the
ancillary fragments. Conjugates of 1 havebeen previously pre-
pared with a variety of groups appended at the amine termi-
nus, including fluorophores,41-44 ferrocene,45 hydroxypyridi-
none-, or catecholate-based ligands46-48 and others.49-52
Most recently, the octanol-water partition coefficients of a
series of alkylated 1 compounds were determined to be
200-3900 times that of free 1 at 25 °C.51 This may have
implications for improving the ability of free 1 to traverse cell
membranes to access intracellular iron stores. Starch poly-
mers of 1 have also been explored as a mechanism to improve
the plasma half-life and toxicity.52 In a more general context,
conjugates of hydroxamic acid-based and catechol-based
Results and Discussion
Rationale for Chelator Design. Semisynthesis was used to
prepare conjugates between 1 and lipophilic compounds
with structures similar to those of selected orally available
compounds in clinical use. Our rationale was that the favor-
able properties of the ancillary fragments (oral availability,
lipophilicity, low toxicity) may be conferred upon the con-
jugates of 1. We selected adamantane-1-carboxylic acid-
based ancillary fragments for conjugation to 1, with several
compounds of this class in use clinically to treat influenza A
(amantadine, rimantadine),55 Parkinson’s disease (amanta-
dine),56 Alzheimer’s disease (memantine),57 and pulmonary
tuberculosis (N-adamantan-2-yl-N0-((2E)-3,7-dimethyl-2,
6-octadien-1-yl)-1,2-ethanediamine (SQ109)).58 Amanta-
dine, rimantadine, and memantine are orally active and are
generally well tolerated by patients. Among our target com-
pounds, we included conjugates between 1 and (i) 4-methyl-
phenoxyacetic acid, which mimics the internal fragment
of the orally available compounds rosiglitazone and pro-
pranolol, (ii) 3-hydroxy-2-methyl-4-oxo-1-pyridineacetic
acid, which mimics deferiprone (2), and (iii) deferasirox (3)
itself.
Chemistry.
3-Hydroxy-2-methyl-4-oxo-1-pyridineacetic
acid (L1D), which is the N-acetic acid derivative of 2, was
prepared according to literature methods,59,60 and the purity
of the compound was confirmed by 1H and 13C NMR spectro-
scopy. Seven carboxylic acid derivatives, adamantane-1-
carboxylic acid (AdA), 3-hydroxyadamantane-1-carboxylic acid
(AdAOH), 3,5-dimethyladamantane-1-carboxylic acid (AdAdMe),
adamantane-1-acetic acid (AdAc), 4-methylphenoxyacetic acid
(MPOAc), L1D, or 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]-
benzoic acid (LDX, 3), were conjugated to 1 to yield 1-AdA (4),
1-AdAOH (5), 1-AdAdMe (6), 1-AdAc (7), 1-MPOAc (8), 1-L1D
(9), or 1-LDX (10), respectively (Figure 2). Initially, 4 was
prepared via the conjugation of 1 with NHS-activated
adamantane-1-carboxylic acid.61 A more streamlined
synthesis of 4-10 used HOBt-based, EDC-activated con-
jugation62 (Scheme 1) and the compounds were purified to
>95% using preparative RP-HPLC. Conjugation in each
1
of 4-10 was evident from the absence in the H NMR
spectra of the amine group signal (δ = 2.7 ppm) of 1 and
of the CO2H signal in each of AdA, AdAOH, AdAdMe
,
AdAc, MPOAc, L1D, or 3 (δ ∼11.9 ppm). Additionally, the
appearance of the signal due to the amide peak (δ ∼ 7.3
ppm) and of the signals assigned to the ancillary ligands
confirmed conjugation.

1372 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Liu et al.
Figure 2. Schematic of the new DFOB conjugates prepared in this investigation: DFOB-AdA (4), DFOB-AdAOH (5), DFOB-AdAdMe (6),
DFOB-AdAc (7), DFOB-MPOAc (8), DFOB-L1D (9), and DFOB-LDX (10).
Scheme 1. Synthesis of 4-10
Charge of 4-10 in the Absence and Presence of Fe(III) and
Fe(III)/(II) Redox Potentials. Upon the basis of the range of
the pKa values determined for the three hydroxamic acid-
based protons of DFOB (pKa 8.32-9.71)63 and that com-
pounds 4-8 have ancillary fragments with protons that will
not ionize under biologically relevant conditions (as a re-
ference point for 5, 1-adamantanol pKa = 1864), 4-8 will be
neutral at physiological pH values. While compounds 9 and
10 have ancillary fragments with ionizable protons, the pKa
values of the hydroxyl group of 2 (pKa = 9.75)65,66 and of the
phenolate groups of 3 (pKa = 10.13 and 12.09)67 prescribe
that 9 and 10 will be neutral at physiological pH values.
Therefore, 4-10 would be expected to be able to readily cross
cell membranes as neutral species, which is likely to be
favorable for accessing intracellular iron pools. The 1:1
complex formed between Fe(III) and the triple deprotonated
DFOB fragment of 4-8 will also be neutral. Therefore,
Fe(III)-loaded 4-8 are expected to exit the cell as neutral
complexes. Depending upon metal:ligand stoichiometry, the
charge of Fe(III)-loaded complexes of 9 and 10 may deviate
from neutral. Upon the basis of the established coordination
chemistry of Fe(III) and 268 or 3,67,69 it is likely that there will
be more than one type of Fe(III)-loaded complex formed
with 9 and 10.
The negative Fe(III)/(II) redox potentials reported for
[Fe(1(3-))]þ/0 (E1/2 -0.48 V vs NHE at pH 7.5),70,71
[Fe(2(1-))3]0/1- (E1/2 -0.62 to -0.54 V vs NHE)72 and
[Fe(3(3-))2]3-/4- (E1/2 -0.6 V vs NHE),69 indicate that the
Fe-loaded complexes of 4-10 will exist as redox stable
Fe(III) complexes. Thus, it is unlikely that these complexes
would engage in one-electron reduction reactions to yield the
corresponding Fe(II) complexes under physiological condi-
tions. Therefore, the possibility of the generation of ROS
from Fe(II)-based Fenton reactions is unlikely for 4-10.
This enhances the potential for these compounds as Fe(III)
chelators for iron overload rather than as iron chelators for
cancer treatment.32 The latter class of compounds, which
includes di-2-pyridyl ketone 4,4-dimethyl-3-thiosemicarba-
zone (Dp44mT), are thought to depend upon Fe(III)/(II)
redox cycling mechanisms for cytotoxicity.32,33,36,37

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1373
Table 1. ESI-MS Data (Positive Ion Mode) from 4-10 in the Absence and Presence of Fe(III)
Fe(III) free
Fe(III) loaded
compd
[M]
exp
calcd
species
exp
calcd
species
4
722.9
723.1
745.3
723.9
745.9
[M þ Hþ]þ
776.5
798.5
776.8
798.8
[M - 3Hþ þ Fe3þ þ Hþ]þ
[M þ Naþ]þ
[2 M þ Naþ]þ
[M þ Hþ]þ
[M - 3Hþ þ Fe3þ þ Naþ]þ
1467.5
737.2
1468.8
739.9
5
6
7
738.9
751.0
736.9
792.5
814.5
804.5
826.6
790.6
812.6
792.8
814.8
804.8
826.8
790.8
812.8
[M - 3Hþ þ Fe3þ þ Hþ]þ
[M - 3Hþ þ Fe3þ þ Naþ]þ
[M - 3Hþ þ Fe3þ þ Hþ]þ
[M - 3Hþ þ Fe3þ þ Naþ]þ
[M - 3Hþ þ Fe3þ þ Hþ]þ
[M - 3Hþ þ Fe3þ þ Naþ]þ
751.1
752.0
[M þ Hþ]þ
737.4
759.6
782.3
710
737.9
759.9
781.9
709.8
731.8
1440.6
726.8
748.8
917.0
939.0
[M þ Hþ]þ
[M þ Naþ]þ
[M - Hþ þ 2Naþ]þ
[M þ Hþ]þ
8
708.8
762.3
784.3
887.2
868.1
762.7
784.7
888.6
868.9
[M - 3Hþ þ Fe3þ þ Hþ]þ
732
1439.2
727
[M þ Naþ]þ
[2 M þ Naþ]þ
[M þ Hþ]þ
[M - 3Hþ þ Fe3þ þ Naþ]þ
[M - 3Hþ þ Fe3þ þ Hþ]þ 2NaCl 0.5H2O
3
3
9
725.8
916.0
[M - 4Hþ þ 2Fe3þ þ Cl-]þ
748.5
916.5
938.7
[M þ Naþ]þ
[M þ Hþ]þ
10
1022.2
1022.7
[M - 5Hþ þ 2Fe3þ þ
]
[M þ Naþ]þ
Fe(III) Coordination. The Fe(III) coordination of 4-10
was examined using ESI-MS (Table 1), electronic absorption
spectroscopy, and RP-HPLC measurements. For Fe(III)-
loaded solutions of 4-7, the dominant species in the positive
ion ESI-MS formulated as the protonated ([M - 3Hþ þ Fe3þ þ
Hþ]þ) or sodiated ([M - 3Hþ þ Fe3þ þ Naþ]þ) form of the
intrinsically uncharged species, [M - 3Hþ þ Fe3þ ], in which
Fe(III) was bound to the triple deprotonated 1 motif. For 8,
RP-HPLC of Compounds in the Absence and Presence of
Fe(III). All compounds were purified by preparative scale
RP-HPLC because the modest solubility of 4-10 in water or
methanol prevented purification by recrystallization or flash
chromatography. A single major peak was observed in the
analytical RP-HPLC of 4-10, which demonstrated that the
purity of the compounds was >95% (Figure 5). In the
presence of Fe(III), the values of the retention time (tr) of
the peaks attributable to Fe(III)-loaded 4-9 decrease (range
of 0.5 min (6) to 2.1 min (9)), which indicates that the Fe(III)-
loaded complexes are more water-soluble that the free
ligand. This is consistent with the function of bacterial
siderophores in nature to increase the aqueous solubility of
Fe(III) under aerobic conditions10 and with the decrease in
logD7.4 values for Fe(III)-loaded complexes of 1-3, relative
to the values of the respective free ligands.74
Two peaks eluted in the RP-HPLC trace of a solution of
Fe(III)-loaded 9 (tr 12.2 and 10.9 min). Analysis from RP-
HPLC-MS (positive ion mode) showed a distribution of
Fe(III)-loaded 9 species. The HPLC-MS trace from the
fraction eluting at tr = 12.2 min yielded signals at m/z:
390.32 (60), m/z 416.79 (1), 779.22 (100), and 1557.90 (5),
corresponding to [M - 3Hþ þ Fe3þ þ 2Hþ]2þ (m/zcalc
390.34), [M - 3Hþ þ 2Fe3þ - Hþ]2þ (m/zcalc 416.75), [M -
3Hþ þ Fe3þ þ Hþ]þ (m/zcalc 779.67), and [M - 4Hþ þ Fe3þ þ
the [M - 3Hþ þ Fe3þ þ Hþ]þ and [M - 3Hþ þ Fe3þ
þ
Naþ]þ ions were present in low abundance (both ∼12%),
with the major signal (100%) ascribed to [M - 3Hþ þ Fe3þ þ
Hþ]þ 2NaCl 0.5H2O. The Fe(III):ligand ratio of 1:1 for
3
3
4-8 determined from ESI-MS measurements was also
established from Job’s plots analyses. The isotope pattern
of the signal at m/z = 868.1 for Fe(III)-loaded 9 simulated as
[M - 4Hþ þ 2Fe3þ þ Cl-]þ ([C33H51N7O11ClFe2]þ requires
868.9). For Fe(III)-loaded 10, the observed isotope pattern
(m/z = 1022.2) was consistent with [M - 5Hþ þ 2Fe3þ þ
]
([C46H56N9O11Fe2]þ requires 1022.7) (Figure 3). For 9 and
10, which feature pendant groups with the capacity to bind
iron, it is possible that species exist where the metal:ligand
ratio is >1. This is evident from the ESI-MS analysis,
with Fe(III):9 or 10 = 2:1, indicating that 9 and 10 could
potentially carry a greater than stoichiometric load of Fe.
Under iron saturation, Fe(III)-loaded 9 or 10 could yield
[Fe4(9(4-))3] or [Fe3(10(5-))2]-, respectively (Figure 4). The
argument against Fe(III)-saturated complexes of 9 and 10 as
efficacious Fe mobilizing agents relates to the high molecular
weights of each of these complexes, [Fe4(9(4-))3] (Mr =
2388.8 g mol-1) and [Fe3(10(5-))2]- (Mr = 1989.6 g mol-1),
which may impede cellular efflux. Models of Fe(III)-loaded
complexes of 1-10 were built in HyperChem 7.5 using data
from X-ray crystal structures of [Fe(1(3-))]þ,17 2,5-dioxo-
pyrrolidin-1-yl adamantane-1-carboxylate,61 [Cr(2(1-))3],73
M - Hþ þ Fe3þ þ
]
or [2(M - 3Hþ þ Fe3þ) þ Hþ]þ (m/zcalc
1558.34), respectively. The major Fe(III)-9 species present in
the fraction eluting at tr = 12.2 min occurred as 1:1 or 2:2
species. The HPLC-MS trace from the fraction eluting at tr
= 10.9 min yielded signals at m/z 390.32 (60), m/z 416.79
(100), 779.22 (75), 868.09 (25), and 946.04 (40). The latter
two signals were unique to the peak eluting at tr = 10.9 min
and corresponded with [M - 3Hþ þ 2Fe3þ - Hþ þ Cl-]þ
2-
(m/zcalc 868.96) and [M - 3Hþ þ 2Fe3þ þ SO4 0.5CH3-
and [Fe{(3,5-bis(2-hydroxyphenyl)-1-phenyl-1,2,4-triazole)-
OH]þ (m/zcalc 946.61). The three major Fe(III)-9 species
present in the peak eluting at tr = 10.9 min occurred as 2:1
species. Therefore, the HPLC-MS analysis indicated that
there was a distribution of Fe(III)-9 species present with
different Fe(III):ligand ratios with charges that may con-
tribute a distribution coefficient (logD) based effect upon the
tr values in the RP-HPLC for the two groups of species. In
support of this idea is the Job’s plot analysis for 9, which
showed diffuse isosbestic points at 260, 310, and 370 nm,
(2-)}2].69 The volumes of [Fe4(9(4-))3] (5335 A ) and
3
˚
3
[Fe3(10(5-))2]- (4436 A ) were significantly greater than
˚
3
˚
the volume of [Fe(6(3-))] (1984 A ). The size and shape of
the Fe(III) complex may be an important structure-activity
relationship with regard to the ability of 9 and 10 to mobilize
cellular Fe, although the variable stoichiometry of Fe(III):9
or 10 complexes makes it difficult to establish such a relation-
ship with certainty.

1374 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Liu et al.
Figure 3. ESI-MS (positive ion) from Fe(III)-loaded solutions of 6, 9, or 10 as experiment (A) and (B) simulated. For 6, m/z 826.6 (obs) [M -
3Hþ þ Fe3þ þ Naþ]þ requires 826.8 (calcd) [C38H63N6O9FeNa]þ. For 9, m/z 868.1 (obs) [M - 4Hþ þ 2Fe3þ þ Cl-]þ requires 868.9 (calcd)
[C33H51N7O11ClFe2]þ. For 10, m/z 1022.2 (obsd) [M - 5Hþ þ 2Fe3þ þ
]
requires 1022.7 (calcd) [C46H56N9O11Fe2]þ.
indicative of the presence of more than two species in
solution. A distribution of species formed between Fe(III)
and 2 has been detected within the limits of ESI-MS,68 which
further supports the presence of more than one species in
solutions of Fe(III) and 9.
m/z = 969.29, which corresponded to [M - 3Hþ þ Fe3þ þ
Hþ]þ. The sloping front of the peak gave a signal at m/z =
1022.13, which corresponded to [M - 5Hþ þ 2Fe3þ]þ, as
observed in the ESI-MS experiments (Table 1). Therefore,
similarly to Fe(III)-loaded 9, there appeared to be a distribu-
tion of species of Fe(III)-loaded 10, which accords with
the complex pH-dependent species distribution previously
observed for 3.69
Determination of LogP Values of 4-10. The tr values for
4-10 were greater than the tr value of 1, which is congruous
with the predicted increase in the logP values of 4-10,
compared to 1. The water solubility of 1 is attributable, in
part, to the charged amine group at physiological pH values.
The charge neutrality of 4-10, in addition to the lipophilicity
inherent to the ancillary fragments (Figure 2), would expect
to yield compounds that are more lipophilic than the parent
1. Previous work has shown this to be the case for alkylated
adducts of 1.51
For 10, there was not a clear shift in the retention time of
the peak ascribed to Fe(III)-loaded 10 compared to the free
ligand. However, Fe(III) binding was evident from the
significant change in the absorbance value of the peak of
Fe(III)-loaded 10. The absorbance value at 220 nm of an
electronic absorption spectrum from a 1:1 Fe(III):10 solu-
tion (0.1 mM) and 10 (0.1 mM) from the Job’s plot analysis
was 1.8 and 0.15, respectively. A similar fold difference in
absorbance was observed in the RP-HPLC traces of 10 in the
absence and presence of Fe(III). The phenomenon of a
change in electronic absorption spectrum but not in RP-
HPLC retention time for hydrophobic siderophores has been
reported for those derived from bacteria.75 The apex of the
major peak from a solution of Fe(III)-loaded 10 analyzed
by RP-HPLC-MS (positive-ion mode) gave a signal at
The logP values of 4-10 in the absence and presence of
Fe(III) were estimated using RP-HPLC (Table 2). This

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1375
Figure 4. Models built using HyperChem 7.5 of [Fe(6(3-))], [Fe4(9(4-))3] and [Fe3(10(5-))2]- based on data from X-ray crystal structures of
related fragments.17,61,69,73 Hydrogen atoms have been omitted for clarity.
logP values are not able to be calculated reliably using RP-
HPLC, alongside values for 1-3 and for Fe(III)-loaded 1-3.
The tr values for 4, 6-8, and 10 fell within or close to the tr
values of the four standard compounds used to generate the
regression analysis.76 For 5 and 9, the tr values fell outside the
range of the standards, which prompted parallel determina-
tions of logP values for 4-10 using the shake-flask method.
The experimentally determined logP values for 4-10 using
both RP-HPLC and shake-flask methods compared reason-
ably well (Table 2) and were also in broad agreement with
logP values calculated using Advanced Chemistry Develop-
ment Software V8.14 or for models of 4-10 that were built
using HyperChem 7.5 (Table 2).
Cellular 59Fe Mobilization. The ability of 1-10 and
Dp44mT to mobilize intracellular 59Fe from human SK-N-
MC neuroepithelioma cells prelabeled with 59Fe-Tf was
examined (Figure 6A). The Fe metabolism of this cell type
and the effect of a variety of chelators on this cell type is well
characterized,77,78 which underscores its choice for measur-
ing Fe mobilization. The ability of 1 (logP(av) = -2.1) to
induce the mobilization of intracellular 59Fe is rather modest
(12 ( 1%), relative to control medium alone (5 ( 1%;
Figure 6A). This is consistent with our previous findings
using this assay where DFOB mesylate was unable to readily
3
access intracellular iron stores over short incubation
times.77,78
Of the four adamantane-1-carboxylic acid-based conju-
gates of 1, three compounds (4, 6, and 7) were effective in
increasing the mobilization of intracellular 59Fe in compar-
ison to free 1 by factors of 2.2, 3, and 2.8, respectively.
Compound 6 increased 59Fe release to an extent that was
comparable (p > 0.05) to the positive controls, 3, and
Dp44mT (Figure 6A). Compound 7 showed 59Fe mobilizing
efficacy comparable (p > 0.05) to that of 3. In contrast, 5
Figure 5. RP-HPLC traces of 1, 4, 5, 6, 9, and 10 in the absence
showed activity that was similar to control medium and was
(black) or presence (gray) of Fe(III).
significantly (p < 0.001) less efficient as an 59Fe mobilizing
agent than 1.
method is valid in this case because, under the conditions
used for RP-HPLC measurements, 4-10 will be neutral and
there will be no logD contribution to the logP values. For
neutral, Fe(III)-loaded complexes of 4-8, the logP values
will also be valid, as determinable via RP-HPLC. However,
due to the non-neutral charge on Fe(III)-loaded 9 and 10,
Because no major steric or electronic perturbations were
made to the 1 motif of the monofunctional 1-adamantyl
adducts 4-7, the affinities of 4-7 toward Fe(III) will
be similar to the affinity between 1 and Fe(III) (logβ110
=
30.5).16,17 These values, or the pFe(III) values (pFe(III) =

1376 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Liu et al.
Table 2. LogP Values of 1-10 in the Absence and Presence of Fe(III) as Determined from RP-HPLC, the Shake-Flask Method, and from Calculation
Fe(III) free
Fe(III) loaded
tr (min)b
LogPexp
LogPexp
LogPcalc
LogPcalc
LogPav
tr (min)b
LogPexp
c
d
e
f
c
3
V (A )
˚
compd
1
12.04
NDh
NCg
ND
ND
-1.02i
-2.74
-0.22
6.43
ND
-1.45
-0.81
5.18
-2.10 ( 0.91
-0.68 ( 0.41
5.14 ( 1.32
1.58 ( 0.46
0.10 ( 0.20
2.36 ( 0.50
1.65 ( 0.49
1.14 ( 0.48
-1.04 ( 0.33
10.40
ND
NC
1470
1155
1766
1903
1922
1984
2002
1918
5335k
2
3
4
5
6
7
8
9
ND
NC
25.43
19.31
15.39
21.54
19.92
18.21
13.04
NC
3.8j
25.39
18.56
14.51
21.08
19.38
17.48
10.93
12.23
25.8
1.29
-0.11
1.95
1.47
0.93
-1.16
2.11
0.29
2.92
2.21
1.68
-1.29
1.35
0.11
1.04
-0.48
1.82
1.31
0.68
NC
ND
ND
2.22
1.28
ND
ND
0.80
-0.66
ND
NC
NC
10
25.8
3.02
ND
ND
ND
ND
2.17
ND
1.64
2.70
3.45
4.12
4.17
1.51
2.19
3.05
3.11
3.12 ( 1.00
1.59 ( 0.07
2.48 ( 0.26
3.27 ( 0.20
3.76 ( 0.56
4436l
ND
ND
ND
ND
BDMEa
BDEEa
NAPHa
DBFNa
20.02
24.09
26.99
28.88
1.61m
2.54m
3.32
ND
ND
ND
ND
ND
ND
ND
ND
4.04
a BDME = 1,2-benzenedicarboxylic acid 1,2-dimethyl ester; BDEE = 1,2-benzenedicarboxylic acid 1,2-diethyl ester; NAPH = naphthalene;
DBFN = dibenzofuran. b to = 1.81 min. c Determined from RP-HPLC (multiple runs showed reproducibility; given data is from a single series of
experiments). d Determined from shake-flask. e As reported as calculated using Advanced Chemistry Development Software V8.14.on SciFinder Scholar
Database. f Calculated from models built using HyperChem 7.5. g NC = not calculable. h ND = not determined. i From ref 91. j From ref 92. k Modeled
for Fe(III) saturated 9:Fe(III) = 3:4 complex (Figure 4). l Modeled for Fe(III) saturated 10:Fe(III) = 2:3 complex (Figure 4). m From ref 93.
-log[Fe(III)] when [Fe(III)]total=10-6 M and [ligand]total
=
Inhibition of Cellular 59Fe Uptake from 59Fe-Transferrin.
The ability of 1-10 or Dp44mT to prevent the internaliza-
tion of 59Fe from 59Fe-Tf was analyzed in the human SK-N-
MC neuroepithelioma cell line (Figure 6B). Generally, these
results reflected those of the intracellular 59Fe mobilization
study (Figure 6A), demonstrating that compounds with high
59Fe mobilization efficacy were also efficient at preventing
the uptake of 59Fe from 59Fe-Tf. The ability of 1 to prevent
59Fe uptake from 59Fe-Tf was poor, inhibiting 59Fe uptake
to 87% of the control (Figure 6B), as shown in previous
studies.77,78 Of the compounds in this work, 4, 6-8, and 10
were significantly (p < 0.01) more active than 1. Compound
6 was the most efficient compound, inhibiting 59Fe uptake
from 59Fe-Tf to 18 ( 2% of the untreated control. This
efficiency was significantly (p < 0.001) greater than the
positive control 3, which reduced 59Fe uptake to 39 ( 2%
of the control and was slightly less effective than the highly
potent antitumor chelator, Dp44mT, which inhibited
59Fe uptake to 12 ( 1% of the control.
10-5 M at pH 7.4; for 1, pFe(III) = 26),79 are not expected to
differ significantly among this subgroup of compounds.
Therefore, the decreased release of 59Fe mediated by 5,
compared to the 59Fe mobilization mediated by 4, 6, and 7,
suggests the efficacy of a chelator is not solely determined by
the affinity toward Fe(III). In fact, multiple factors are
involved in terms of optimal Fe chelation efficacy, as found
for other types of ligands.80
Of the remaining 1 conjugates (8, 9, and 10), 8 and 10
showed significantly (p < 0.001) greater 59Fe cellular efflux
activity than 1. The conjugate between L1D and 1 (9)
demonstrated little activity, not being significantly (p >
0.05) more active that control medium at mobilizing 59Fe
from cells. Compared to the control, the clinically used orally
active chelator, L1 (2), also showed little ability to mobilize
59Fe (Figure 6A) and was less effective than the other positive
controls (1, 3 and Dp44mT). Previous studies using a similar
protocol have demonstrated that relatively high concentra-
tions of L1 (i.e., 0.5 mM) are necessary to induce only
moderate 59Fe mobilization from cells.81 Hence, in terms
of structure-activity relationships, conjugates between 1
and 2 do not appear optimal. The conjugate between 1 and
3 (10) showed 59Fe mobilization efficacy that was signifi-
cantly (p < 0.001) less effective than 3 alone.
An ideal iron chelating molecule for iron overload treat-
ment should have properties that allow the compound to:
(i) cross the cell membrane to access intracellular iron stores,
(ii) selectively form a redox-inactive Fe(III) complex, and
(iii) exit the cell as a stable, Fe(III)-loaded complex for
excretion.7,32 Criteria (i) and (iii) are described in part by the
charge and the partition coefficient of the free compound and
ofthe Fe(III)-loadedcomplex, respectively, and criterion (ii) is
described by the thermodynamics and kinetics of Fe(III)
coordination. In the case of some of the 1 conjugates
(namely, 4, 6-8), these properties have been favorably altered
in comparison to 1. In contrast, for other conjugates such as 5
and 9, structure-activity relationships involving properties
such as relatively low logP values and/or the high molecular
weight of Fe(III)-loaded complexes may explain the hindered
membrane permeability and, thus, Fe chelation efficacy.
In summary, examining both the Fe efflux and Fe uptake
studies (Figure 6A,B), the most effective 1 conjugates in
terms of Fe chelation efficacy were 4, 6, 7, and 10, with
activities that are at least twice that of 1.
Structure-Activity Relationship between LogP Values and
59Fe Mobilization. A parabolic relationship is evident bet-
ween the logP values of 4-10 and the cellular efflux of 59Fe
(Figure 7A), giving an optimal logP value of 2.3 for maximal
Fe efflux. The data from 1, 2, and Dp44mT36 also fit well
onto this parabola, with data for 3 an outlier. Because the
descending parabola is described by only one data point with
a broad error margin (10), there is some uncertainty as to
whether the relationship between the logP values and 59Fe
efflux is truly parabolic. However, the ascending data is
populated by sufficient data points to claim at least a
sigmoidal relationship between 59Fe efflux and logP values
<2.3. This optimal logP value for Fe mobilization is also
supported by the parabolic, or at a minimum, sigmoidal
relationship, between logP and 59Fe uptake from 59Fe-Tf
(Figure 7B). Thus, the lipophilicity of the chelator appears to
be an important factor in determining the ability of the
ligand to mobilize cellular Fe.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1377
Figure 7. LogP values (average of values determined by RP-
HPLC, shake-flask, and calculation) of 1-10 and Dp44mT as a
function of (A) cellular 59Fe released (%) from human SK-N-MC
neuroepithelioma cells prelabeled with 59Fe-transferrin (59Fe-Tf) or
(B) 59Fe uptake (% of control) from 59Fe-transferrin (59Fe-Tf) by
SK-N-MC neuroepithelioma cells.
Table 3. IC50 Values (μM) of 1-10 and Dp44mT in Madin-Darby
Canine Kidney Type II (MDCK II) or in SK-N-MC Neuroepithelioma
cell types
IC50 (μM)
MDCK II
canine kidney
SK-N-MC neuro-
epithelioma
compd
DFOB
deferiprone
1
2
9.49 ( 1.24
NDa
NDa
16.04 ( 0.47
165.88 ( 1.90
20.54 ( 0.56
174.04 ( 1.61
>200
3
deferasirox
4
DFOB-AdA
DFOB-AdAOH
DFOB-AdAdMe
DFOB-AdAc
DFOB-MPOAc
DFOB-L1D
118.86 ( 1.16
>100
5
6
163.37 ( 1.52
225.56 ( 1.28
>150
92.36 ( 1.66
167.14 ( 2.82
>200
7
8
Figure 6. The effect of the new DFOB conjugates (4-10) in
comparison with the clinically used chelators DFOB (1), deferi-
prone (2), deferasirox (3), or Dp44mT on: (A) cellular 59Fe released
(%) from human SK-N-MC neuroepithelioma cells prelabeled with
59Fe-transferrin (59Fe-Tf), or (B) 59Fe uptake (% of control) from
59Fe-transferrin (59Fe-Tf) by SK-N-MC neuroepithelioma cells.
Results are mean ( SD of three experiments with three determina-
tions in each experiment.
9
10
>300
23.41 ( 1.53
NDa
>200
20.62 ( 1.79
0.01 ( 0.01
DFOB-LDX
Dp44mT
a ND = not determined.
trends as observed in the MDCK II cell line. Importantly, in
both cell types, all of the 1 conjugates (4-10) showed less
cytotoxicity than 1. Moreover, the most active 1 conjugates
at mobilizing 59Fe from cells and reducing cellular 59Fe
uptake from 59Fe-Tf, namely 4, 6, 7, and 10, were signifi-
cantly (p < 0.005) less cytotoxic than 1, demonstrating that
these compounds are highly tolerated and do not remove
cellular Fe pools vital for cellular proliferation. This low
antiproliferative activity is vital in the design of an iron
chelator for the treatment of iron overload. The antiproli-
ferative activity of all of the 1 conjugates are 1600 to >20000
times less effective than the known cytotoxic iron chelator,
Dp44mT (IC50 0.01 μM; Table 3).40 Hence, this analysis
Cell Viability. The ability of selected compounds from
1-10 and Dp44mT to inhibit cellular proliferation of
Madin-Darby canine kidney type II (MDCK II) cells and
human SK-N-MC neuroepithelioma cells was assessed using
the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide] (MTT) assay82 (Table 3). Compounds that are well
tolerated by cells will not affect regular cellular proliferation
or growth and will have high IC50 (or LD50) values relative to
more cytotoxic compounds. The cell viability data using the
human SK-N-MC neuroepithelioma cell type reflects similar

1378 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Liu et al.
suggests that the 1 conjugates are more appropriate for
the treatment of iron overload disease rather than cancer.
Finally, a recent study showed a positive correlation between
the logP value and toxicity of desferrithiocin analogues.27
For 4-10, no structure-activity relationships were evident
between logP and toxicity.
(ESI-MS) were recorded using positive ionization on a Finnigan
LCQ or a Finnigan MAT 900 XL ion trap mass spectrometer
(San Jose, CA) with the following parameters. Mobile phase,
methanol; flow rate, 0.30 mL min-1; injection volume, 25 μL,
spray voltage, 4.50 kV; capillary voltage, 35 V; capillary tem-
perature, 210 °C; tube lens-offset, 10 V. Analytical thin layer
chromatography (TLC) was performed using precoated silica
gel plates (Sigma-Aldrich), which were eluted with MeOH:
CHCl3 = 1:3 and visualized using solid iodine or by dipping
the plate into an ethanolic solution of FeCl3 (60 mM). Reversed-
phase high pressure liquid chromatography (RP-HPLC) used a
Waters system (Milford, MA) consisting of a GBC 1460 degas-
ser, a Rheodyne 7725i injector (analytical 20 μL loop, prepara-
tive 1700 μL loop) (Apple Valley, MN), a Waters 486 tunable
absorbance detector, and a Waters Empower 2 software with
Waters Sunfire C18 columns (particle size 5 μm, column dimen-
sion 4.6 mm ꢀ 150 mm i.d. (analytical), or particle size 5 μm,
column dimension 19 mm ꢀ 150 mm i.d. (preparative)) and a
flow rate of 0.2 mL/min (analytical) or 7.0 mL/min (prepara-
tive), with a mobile phase of water (0.1% TFA, solvent A) and
acetonitrile (0.1% TFA, solvent B). HPLC-MS was conducted
on a Thermo Separation system with a ThermoQuest Finnigan
LCQ Deca mass spectrometer (San Jose, CA, USA).
General Procedure. 3-Hydroxy-2-methyl-4-oxo-1-pyridine-
acetic acid (L1D) was synthesized according to the literature59,60
from an aqueous solution (75 mL) of glycine (30 g, 400 mmol),
maltol (12.7 g, 100 mmol), and NaOH (11 g, 275 mmol), which was
reacted at 35 °C for 5 d. The solution was acidifed with 5 M HCl (4
mL) and was refrigerated for 2 h to yield a cream colored residue,
which was filtered and washed with cold water. The solid product
was redissolved in basic aqueous solution (pH 10.8) using 2 M
NaOH (20 mL) and was acidified with 5 M HCl (4 mL) to give L1D
as a pale-white solid (3.28 g, 18%). 1H NMR (300 MHz, DMSO-
d6) δH: 7.3 (1H, s, OH), 2.1 (3H, s, CH), 1.9 (6H, s, CH2), 1.7
(6H, s, CH2).
Compounds 4-10 were prepared based upon a literature
bioconjugation method62 from a solution of DMF (10 mL)
containing AdA, AdAOH, AdAdMe, AdAc, MPOAc, L1D, or 3 (1
mmol), EDC (1.5 mmol), 1 (1 mmol), and HOBt (0.19 g, 1.5
mmol), which was heated to 45 °C. After the reagents had
dissolved, DIPEA (2 mmol) was added to the solution and the
mixture was stirred overnight at room temperature under
nitrogen. The solution was evaporated to dryness in vacuo
and the solid residue was washed with diethylether (3 mL) and
distilled water (3 mL) before redissolving the solid in methanol
and removing the solvent under reduced pressure. The progress
of all syntheses was monitored using TLC. Compounds 4-10
were not sufficiently soluble in water or methanol to enable
purification using either recrystallization or flash chromatogra-
phy and were, therefore, purified to >95% purity, using pre-
parative RP-HPLC. This level of purity was confirmed by CHN
microanalysis.
DFOB-AdA (4). The residue was triturated with diethylether
(5 ꢀ 5 mL), recrystallized from methanol and purified by
preparative RP-HPLC (90:10 (A:B) to 25:75 (A:B) over 30 min)
to give 4 as a off-white solid (0.56 g, 70%). Solubility in ethanol
(25 °C): 28 mg mL-1 (∼38mM). 1H NMR(300 MHz, DMSO-d6)
δH: 9.6 (2H, m, NH), 7.7 (3H, s, OH), 7.3 (1H, s, NH), 3.4 (6H, t,
J = 9 Hz, CH2), 3.0 (4H, q, J = 6 Hz, CH2), 2.6 (4H, t, J = 6 Hz,
CH2), 2.3 (4H, t, J = 6 Hz, CH2), 1.9 (3H, s, CH3), 1.7 (3H, s,
CH), 1.6 (12H, m, CH2), 1.1-1.5 (18H, m, CH2). 13C NMR (300
MHz, DMSO-d6) δC: 177.1, 172.3, 171.6, 47.5, 47.2, 36.5, 30.3,
29.2, 28.0, 27.9, 26.4, 23.9, 23.8, 20.7. MS: m/z ESI (positive ion).
Found [M þ Hþ]þ 723.07 (98), [M þ Naþ]þ 745.27 (100),
[C36H62N6O9Na]þ requires 745.91. MeOH (320 nm), ε = 18.27
M-1 cm-1. Anal. Calcd for C36H62N6O9: C, 59.81% H; 8.64%;
N, 11.63%. Found: C, 57.44% H; 7.50%; N, 11.15%.
Conclusions
The high solubility of 1 in water, which impacts negatively
on its biological activity, can be significantly reduced by
conjugating an ancillary fragment to the amine group of 1
without adversely affecting the Fe(III) coordinating ability of
the conjugate. This is a crucial structure-activity relationship
that may be useful to exploit in the future. We conjugated to 1:
(i) polycyclic-cage based compounds (adamantane-based
derivatives), which have analogues in the clinic (amantadine,
rimantadine, and memantine) that are orally active and that
are generally well-tolerated by patients, (ii) 4-methylphenoxy-
acetic acid, and (iii) a deferiprone mimic (3-hydroxy-2-
methyl-4-oxo-1-pyridineacetic acid), and deferasirox itself.
Given that the affinity toward Fe(III) will not vary signifi-
cantly among 4-8, the relatively poor Fe mobilization ability
of 5 illustrates that Fe(III) affinity is not the sole determinant
ofFechelatingefficacy. FromFeeffluxand Feuptake studies,
4, 6, 7, and 10 were at least twice as active as 1 with regard to
Fe chelation efficacy and were significantly less cytotoxic than
1, as determined using two different cell types. Therefore,
these compounds may have promise as compounds for the
treatment of iron overload disease rather than as anticancer
agents. Neurological conditions that have been associated
with transition metal ion dys-homeostasis, such as Parkin-
son’s disease, Alzheimer’s disease, and Huntington’s dis-
ease,83 or other conditions that have shown benefits from
treatment via Fe chelation, such as malaria,41,84 could also be
potentially targeted by this class of compounds.
Experimental Section
Chemical Studies. Chemicals. Desferrioxamine B mesylate
3
(DFOB, 95%), 3-hydroxy-2-methyl-4-pyrone (maltol, 99%),
glycine (>99%) adamantane-1-carboxylic acid (AdA, >99%),
3-hydroxyadamantane-1-carboxylic acid (AdAOH, 97%), 3,5-
dimethyladamantane-1-carboxylic acid (AdAdMe, 97%), ada-
mantane-1-acetic acid (AdAc, 98%), 4-methylphenoxyacetic acid
(MPOAc, 98%), N-(3-dimethylaminopropyl)-N0-ethylcarbodii-
mide HCl (EDC, protein sequence grade), Fe(ClO4)3 H2O,
3
3
dimethylformamide (DMF, biotech grade), and acetonitrile
(CH3CN, biotech grade) were obtained from Sigma-Aldrich
(St. Louis, MO). 1-Octanol (99.5% GC grade) was from Fluka
(Buchs, Switzerland). N-Hydroxybenzotriazole (HOBt) was ob-
tained from Auspep (Parkville, VIC, Australia), and 4-[3,5-bis-
(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid (LDX, defera-
sirox) was obtained from AmplaChem (Carmel, IN). N,N-Diiso-
propylethylamine (DIPEA) (99%) was purchased from Lan-
caster Synthesis, Inc. (Pelham, NH), and methanol (99%) was
obtained from Mallinckrodt Chemicals (Phillipsburg, NJ). All
chemicals and solvents were used as received.
General Instrumentation. 1H Nuclear magnetic resonance
spectra were recorded using a Bruker Avance DPX 200
(Rheinstetten, Germany) at a frequency of 200.13 MHz or a
Bruker Avance DPX 300 at a frequency of 300.10 MHz.
Chemical shifts are reported as parts per million (ppm) with
DMSO-d6 (δH = 2.50) or CD3OD (δH 3.31) used as an internal
reference. 13C Nuclear magnetic resonance spectra were re-
corded using a Bruker Avance DPX 200 spectrometer at a
frequency of 50.3 MHz or a Bruker Avance DPX 300 at a
frequency of 75.5 MHz. Electron spray ionization mass spectra
DFOB-AdAOH (5). The residue was triturated with diethyl-
ether (5 ꢀ 5 mL), recrystallized from methanol and purified by
RP-HPLC (90:10 (A:B) to 25:75 (A:B) over 30 min) to give 5 as

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1379
(320 nm), Anal. Calcd for
99.6 M-1 cm-1
C33H55N7O11 2H2O: C, 52.02% H; 7.81%; N, 12.87%. Found:
an off-white solid (0.45 g, 61%). Solubility in ethanol (25 °C):
15.2 mg mL-1 (∼20 mM). Solubility in water (25 °C): 5 mg mL-1
ε
=
.
3
1
(∼6.5 mM). H NMR (300 MHz, DMSO-d6) δH: 9.6 (2H, m,
C, 49.69% H; 6.42%; N, 11.93%.
NH), 7.7 (3H, s, OH), 7.3 (1H, s, NH), 3.4 (6H, t, J = 9 Hz,
CH2), 3.2 (1H, s, OH), 3.0 (4H, q, J = 6 Hz, CH2), 2.6 (4H, t, J =
6 Hz, CH2), 2.3 (4H, t, J = 6 Hz, CH2), 1.9 (3H, s, CH3), 1.7 (2H,
s, CH), 1.5-1.6 (12H, m, CH2), 1.2-1.5 (18H, m, CH2). 13C
NMR (300 MHz, DMSO-d6) δC: 176.3, 171.7, 67.0, 56.4, 47.2,
44.7, 38.2, 31.0, 30.3, 29.2, 27.9, 26.4, 23.9, 23.8, 20.7. MS: m/z
DFOB-LDX (10). The residue was triturated with diethylether
(5 ꢀ 5 mL), recrystallized from methanol, and purified by RP-
HPLC (95:5 (A:B) to 45:55 (A:B) over 30 min) to give 10 as an
off-white solid (0.14 g, 65%). 1H NMR (200 MHz, CD3OD) δH:
8.2 (2H, d, J = 6.9 Hz, CH), 7.9 (2H, d, J = 6.5 Hz, CH), 7.6
(2H, d, J = 6.8 Hz, CH), 7.4 (2H, m, CH), 6.9-7.0 (4H, m, CH),
3.6 (6H, t, J = 6.8 Hz, CH2), 3.2 (4H, m, CH2), 2.8 (4H, m, CH2),
2.4 (4H, m, CH2), 2.1 (3H, s, CH3), 1.6-1.7 (8H, m, CH2), 1.5
(4H, m, CH2), 1.3 (6H, m, CH2), (OH not observed). 13C NMR
(200 MHz, CD3OD) δC: 19.2, 23.7, 23.9, 26.3, 27.9, 29.0, 30.5,
39.2, 39.9, 44.7, 116.3, 119.6, 119.8, 123.8, 127.2, 128.3, 130.9,
131.4, 133.5, 141.5, 157.2, 160.1, 173.2, 173.5. MS: m/z ESI
(positive ion). Found [M þ Hþ]þ 916.5 (85), [M þ Naþ]þ 938.7
(100), [C46H61N9O11Na]þ requires 939.03. MeOH (320 nm), ε =
3.127 ꢀ 103 M-1 cm-1. Anal. Calcd for C46H61N9O11: C,
60.31% H; 6.91%; N, 13.76%. Found: C, 59.40% H; 5.63%;
N, 13.38%.
Extinction Coefficients and Job’s Plot Analysis. A plot of
absorbance/path length (cm-1) at 320 nm vs concentration (M)
in methanol of 4-10 was measured using a SpectraMax
M5/M5e UV-vis (Molecular Devices, Sunnyvale, CA) and the
slope of the line determined as ε (M-1 cm-1). Job’s Plots
analyses were carried out for 4-10, as detailed in the literature,85
with compounds (0.1 mM) dissolved in a solution of 20% (v/v)
ESI (positive ion). Found [M
þ
Hþ]þ 737.20 (100),
[C36H63N6O10]þ requires 739.92. MeOH (320 nm), ε = 49.74
M-1 cm-1. Anal. Calcd for C36H62N6O10: C, 58.51% H; 8.46%;
N, 11.38%. Found: C, 56.88% H; 8.21%; N, 10.92%.
DFOB-AdAdMe (6). The residue was triturated with diethyl-
ether (5 ꢀ 5 mL), recrystallized from methanol, and purified by
RP-HPLC (90:10 (A:B) to 40:60 (A:B) over 30 min) to give 6 as
an off-white solid (0.57 g, 80%). Solubility in ethanol (25 °C): 15
mg mL-1 (∼20 mM). 1H NMR (300 MHz, DMSO-d6) δH: 9.6
(2H, m, NH), 7.7 (3H, s, OH), 7.3 (1H, s, NH), 3.4 (6H, t, J = 15
Hz, CH2), 3.0 (4H, q, J = 6 Hz, CH2), 2.6 (4H, t, J = 9 Hz,
CH2), 2.3 (4H, t, J = 6 Hz, CH2), 1.9 (3H, s, CH3), 1.6-1.1
(31H, m, CH2, CH), 0.8 (6H, s, CH3). 13C NMR (300 MHz, d6-
DMSO) δC: 176.8, 172.3, 171.7, 50.7, 45.4, 42.8, 37.8, 31.1, 28.2,
27.9, 26.4, 23.9, 23.8, 20.7. MS: m/z ESI (positive ion). Found
[M þ Hþ]þ 751.13 (100), [C38H67N6O9]þ requires 751.98.
MeOH (320 nm), ε = 32.17 M-1 cm-1. Anal. Calcd for C38H66-
N6O9: C, 60.77% H; 8.86%; N, 11.19%. Found: C, 58.85% H;
8.04%; N, 10.71%.
DFOB-AdAc (7). The residue was triturated with diethylether
(5 ꢀ 5 mL), recrystallized from methanol, and purified by RP-
HPLC (90:10 (A:B) to 25:75 (A:B) over 30 min) to give 7 as an
off-white solid (0.55 g, 80%). Solubility in ethanol (25 °C): 15 mg
mL-1 (∼20 mM). 1H NMR (300 MHz, DMSO-d6) δH: 9.6 (2H,
m, NH), 7.7 (3H, s, OH), 7.3 (1H, s, NH), 3.4 (6H, t, J = 6 Hz,
CH2), 3.0 (4H, q, J = 6 Hz, CH2), 2.6 (4H, t, J = 6 Hz, CH2), 2.3
(4H, t, J = 6 Hz, CH2), 1.9 (3H, s, CH3), 1.8 (2H, s, CH2), 1.7
(3H, s, CH), 1.5-1.6 (12H, m, CH2), 1.0-1.5 (18H, m, CH2).
13C NMR (300 MHz, d6-DMSO) δC: 171.6, 170.1, 50.4, 42.5,
36.8, 30.2, 29.2, 28.4, 27.9, 26.4, 23.9, 20.7. MS: m/z ESI
(positive ion). Found [M þ Hþ]þ 737.44 (100), [C37H65N6O9]þ
requires 737.95. MeOH (320 nm), ε = 16.53 M-1 cm-1. Anal.
Calcd for C37H64N6O9: C, 60.30% H; 8.75%; N, 11.41%.
Found: C, 58.37% H; 8.53%; N, 11.72%.
MeOH in Tris HCl (100 mM, pH 7.4) and the pH value of the
3
solution adjusted to pH 7.4 prior to making the solution to
volume. A stock solution of Fe(III) (0.1 mM) was prepared from
FeCl3 6H2O in a similar fashion to the compounds (20% v/v
3
MeOH in 100 mM Tris, pH 7.4). Molar ratios of Fe:compound
were 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1, with 0
containing no iron and 1 containing no ligand. All samples had a
final concentration ([Fe(III)] þ [ligand]) of 0.1 mM and electro-
nic absorption spectra were acquired from 200 to 800 nm, with
the absorbance value of 450 nm absorbance used to construct
the Job’s plot.
Fe(III)-Loading of 4-10 for ESI-MS and RP-HPLC. An
aliquot of
a freshly prepared methanolic solution of
FeCl3 6H2O (100 μL of 10 mM) was added to an aliquot
3
(100 μL of 10 mM) of 4-8 in methanol. For 9 and 10, the
volume ratio of FeCl3 6H2O:compound was 150:50 μL.
3
Partition Coefficients. Analytical RP-HPLC was used to
estimate the log of the partition coefficients (logP) of 4-10.
Samples (5 mM) of 4-10 in MeOH (HPLC grade) were filtered
(0.22 μm) and analyzed by analytical RP-HPLC, together with a
set of compounds with known logP values (1,2-benzenedicarbo-
xylic acid dimethylester (logP = 1.59), 1,2-benzenedicarboxylic
acid diethylester (logP = 2.48), naphthalene (logP = 3.27), and
dibenzofuran (logP = 3.76)). The capacity factor (k) was deter-
mined for the samples using eq 1, where tr was the retention time of
the compound and to was the deadtime (to = 1.81 min). The log of
the partition coefficient (logP) was calculated according to eq 2.76
DFOB-MPOAc (8). The residue was triturated with diethyl-
ether (5 ꢀ 5 mL), recrystallized from methanol, and purified by
RP-HPLC (90:10 (A:B) to 40:60 (A:B) over 30 min) to give 8 as
an off-white solid (0.23 g, 73%). 1H NMR (200 MHz, DMSO-
d6) δH: 7.1 (2H, d, J = 7.1 Hz, CH), 6.8 (2H, d, J = 7.0 Hz, CH),
4.4 (2H, s, CH2), 3.5 (6H, t, J = 6.8 Hz, CH2), 3.0 (4H, m, CH2),
2.5 (4H, m, CH2), 2.3 (4H, m, CH2), 2.2 (3H, s, CH3), 1.9 (3H, s,
CH3), 1.3-1.5 (12H, m, CH2), 1.2 (6H, m, CH2). 13C NMR (200
MHz, DMSO-d6) δC: 21.1, 21.4, 24.5, 27.1, 28.5, 29.8, 31.0, 47.8,
48.2, 68.2, 115.6, 130.8, 156.7, 168.6, 172.3, 173.0. MS: m/z ESI
(positive ion). Found [M þ Naþ]þ 731.7 (100), [C34H56N6-
O10Na]þ requires 731.40. MeOH (320 nm), ε = 101.63 M-1
cm-1. Anal. Calcd for C34H56N6O10: C, 57.61% H; 7.96%; N,
11.86%. Found: C, 56.60% H; 7.31%; N, 11.73%.
DFOB-L1D (9). The residue was triturated with diethylether
(5 ꢀ 5 mL), recrystallized from methanol, and purified by RP-
HPLC (95:5 (A:B) to 40:60 (A:B) over 30 min) to give 9 as a very
pale-pink solid (0.11 g, 70%). 1H NMR (300 MHz, CD3OD) δH:
8.2 (1H, d, J = 7.0 Hz, CH), 7.2 (1H, d, J = 7.0 Hz, CH), 5.2
(2H, s, CH2), 3.6 (6H, t, J = 5.6 Hz, CH2), 3.2 (4H, t, J = 6.9 Hz,
CH2), 2.8 (4H, t, J = 6.8 Hz, CH2), 2.5 (4H, t, J = 7.0 Hz, CH2),
2.1 (3H, s, CH3), 1.5-1.7 (12H, m, CH2), 1.3-1.4 (6H, m, CH2),
(OH not observed). 13C NMR (300 MHz, CD3OD) δC: 19.2,
23.9, 26.2, 26.3, 27.9, 28.8, 28.9, 30.4, 39.3, 39.7, 48.9, 58.0,
113.1, 139.8, 142.8, 143.9, 159.7, 165.6, 172.5, 173.5, 173.9. MS:
m/z ESI (positive ion). Found [M þ Hþ]þ 726.6 (65), [M þ
Naþ]þ 748.7 (100), [C46H61N9O11Na]þ requires 748.8. MeOH
k ¼ ðtr -toÞ=to
(1Þ
log k ¼ a þ blog P
(2Þ
Partition coefficients were also determined using the shake-
flask method with presaturated 1-octanol and water solutions
as follows. An aliquot (1 mL) of water was added to an aliquot of
1-octanol containing dissolved 4-10 (1 or 2 mg) in a glass vial,
and the mixture was shaken overnight at RT. Aliquots (20 μL) of
each phase were analyzed by analytical RP-HPLC using the
conditions described above.
Molecular Modeling. Models were built in HyperChem 7.5
using data from X-ray crystal structures of [Fe(1(3-))]þ,17 2,5-
dioxopyrrolidin-1-yl adamantane-1-carboxylate,61 [Cr(2(1-))3],73
and [Fe{(3,5-bis(2-hydroxyphenyl)-1-phenyl-1,2,4-triazole)(2-)}2].69
The structure of the organic ancillary fragment was minimized

1380 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Liu et al.
using the Polak-Ribiere algorithm and the MMþ force field
and the structure of the Fe(III)-loaded ligand was frozen. No
conformational searching procedure was used.
J. G., Measham, A. R., Alleyne, G., Claeson, M., Evans, D. B., Jha, P.,
Mills, A., Musgrove, P., Eds.; Oxford University Press: New York,
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Biological Studies. Cell Culture. The human SK-N-MC neu-
roepithelioma and Madin-Darby canine kidney type II
(MDCK II) cell lines were obtained from the American Type
Culture Collection (ATCC; Manassas, VA). The SK-N-MC cell
type was grown as described previously,77 while MDCK II cells
were cultured by standard procedures.86
(3) Nick, H. Iron Chelation, Quo Vadis? Curr. Opin. Chem. Biol. 2007,
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59Fe-Transferrin Labeling. Human transferrin (Tf) was la-
beled with 59Fe (Dupont NEN, MA) to produced 59Fe2-Tf
(59Fe-Tf), as previously described.87,88
59Fe Efflux from SK-N-MC Neuroepithelioma Cells. Efflux of
59Fe from SK-N-MC neuroepithelioma cells were measured for
1-10 and for Dp44mT at concentrations of 50 μM using
established techniques.38,77,89 Briefly, following prelabeling of
cells with 59Fe-Tf (0.75 μM) for 3 h at 37 °C, the cell cultures
were washed four times with ice-cold PBS and then subsequently
incubated with each chelator (50 μM) for 3 h at 37 °C. The
overlying media containing released 59Fe was then separated
from the cells using a Pasteur pipet. Radioactivity was measured
in both the cell pellet and supernatant using a γ-scintillation
counter (Wallac Wizard 3, Turku, Finland). In these studies, the
new ligands were compared to the previously characterized
chelators, 1, 2, 3, and Dp44mT.
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Effect of 1-10 at Preventing 59Fe Uptake from Transferrin by
SK-N-MC Neuroepithelioma Cells. The uptake of 59Fe from
59Fe-labeled transferrin was measured for 1-10 and for
Dp44mT at concentrations of 50 μM in SK-N-MC neuroepithe-
lioma cells using standard techniques.38,77,89 Briefly, cells were
incubated with 59Fe-Tf (0.75 μM) for 3 h at 37 °C in the presence
of each of the chelators (50 μM). The cells were then washed four
times with ice-cold PBS and internalized 59Fe was determined by
standard techniques by incubating the cell monolayer for 30 min
at 4 °C with the general protease Pronase (1 mg/mL; Sigma).87,88
The cells were removed from the monolayer using a plastic
spatula and centrifuged for 1 min at 14000 rpm. The supernatant
represents membrane-bound, Pronase-sensitive 59Fe that was
released by the protease, while the Pronase-insensitive fraction
represents internalized 59Fe. The new ligands were compared to
the previously characterized chelators, 1, 2, 3, and Dp44mT.
Effect of 1-10 on Cell Viability. This was examined in
Madin-Darby canine kidney type II (MDCK II) cells and in
human SK-N-MC neuroepithelioma cells using the MTT assay
by standard methods.77,82,90 MTT color formation was directly
proportional to the number of viable cells measured by Trypan
blue staining (SK-N-MC)77 or total cell protein (MDCK II).86
Statistical Analysis. Experimental data were compared using
Student’s t-test. Results were expressed as mean or mean ( SD
(number of experiments) and considered to be statistically
significant when p < 0.05.
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Acknowledgment. This work was supported by the Na-
tional Health and Medical Research Council of Australia
(grant to RC; Senior Principal Research Fellowship and
Project grants to DRR), the Australian Research Council
(Discovery grants to DRR and PKW), and The University of
Sydney (Faculty of Medicine Strategic Research grant to
RC, Sydnovate Fund grant to RC; Faculty of Medicine
cofunded postgraduate scholarship to JL). DSK was sup-
ported by a Cancer Institute New South Wales Early Career
Development Fellowship. Microanalysis was performed at
the Chemical Analysis Facility at Macquarie University,
Sydney, Australia.
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