Partition-variant desferrithiocin analogues: Organ targeting and increased iron clearance

Article abstract of DOI:10.1021/jm049306x

Altering the lipophilicity (log P_app) of desferrithiocin analogues can change the organ distribution of the chelators and lead to enhanced iron clearance. For example, alkylation of (S)-2-(2,4-dihydroxyphenyl)- 4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-4′-(HO)-DADFT] and its analogues to more lipophilic compounds, such as (S)-4,5-dihydro-2-(2-hydroxy-4- methoxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-4′-(CH ₃O)-DADFT], provides ligands that achieved between a 3- and 8-fold increase in chelator concentrations in the heart, liver, and pancreas (the organs most at risk in iron-overload disease) of treated rodents. The 4′-O-methylated compounds are demethylated to their hydroxylated counterparts in rodents; furthermore, this O-demethylation takes place in both rodent and human liver microsomes. The relationship between chelator lipophilicity and iron-clearing efficacy in the iron-overloaded Cebus apella primate is further underscored by a comparison of the iron-clearing efficiency of (S)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-3′-(HO)-DADFT] and its 3′-(CH₃O) counterpart. Finally, these DFT analogues are shown to be both inhibitors of the iron-mediated oxidation of ascorbate as well as effective radical scavengers.

Full text of DOI:10.1021/jm049306x

J. Med. Chem. 2005, 48, 821-831
821
Partition-Variant Desferrithiocin Analogues: Organ Targeting and Increased
Iron Clearance1
Raymond J. Bergeron,* Jan Wiegand, James S. McManis, William R. Weimar,† Jeong-Hyun Park,
Eileen Eiler-McManis, Jennifer Bergeron,‡ and Gary M. Brittenham§
Departments of Medicinal Chemistry and Educational Psychology, University of Florida, Gainesville, Florida 32610-0485, and
Departments of Pediatrics and Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032
Received August 23, 2004
Altering the lipophilicity (log Papp) of desferrithiocin analogues can change the organ distribution
of the chelators and lead to enhanced iron clearance. For example, alkylation of (S)-2-(2,4-
dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-4′-(HO)-DADFT] and its
analogues to more lipophilic compounds, such as (S)-4,5-dihydro-2-(2-hydroxy-4-methoxyphenyl)-
4-methyl-4-thiazolecarboxylic acid [(S)-4′-(CH3O)-DADFT], provides ligands that achieved
between a 3- and 8-fold increase in chelator concentrations in the heart, liver, and pancreas
(the organs most at risk in iron-overload disease) of treated rodents. The 4′-O-methylated
compounds are demethylated to their hydroxylated counterparts in rodents; furthermore, this
O-demethylation takes place in both rodent and human liver microsomes. The relationship
between chelator lipophilicity and iron-clearing efficacy in the iron-overloaded Cebus apella
primate is further underscored by a comparison of the iron-clearing efficiency of (S)-2-(2,3-
dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-3′-(HO)-DADFT] and its
3′-(CH3O) counterpart. Finally, these DFT analogues are shown to be both inhibitors of the
iron-mediated oxidation of ascorbate as well as effective radical scavengers.
Introduction
available evidence suggests that these differ in the liver,
physiologically a major systemic iron depot, and in the
pancreas and heart, which normally do not serve as iron
storage sites. Nontransferrin-bound plasma iron, a
heterogeneous pool of iron in the circulation that is not
bound to the physiological iron transporter, transferrin,
seems to be a principal source of the abnormal tissue
distribution of iron that develops with chronic iron
overload. Nontransferrin-bound plasma iron is rapidly
taken up by hepatocytes in the liver, the major iron
storage organ, perhaps through the divalent metal
transporter 120 and other pathways that remain poorly
characterized. Nontransferrin-bound plasma iron also
seems to gain entry into cardiomyocytes, pancreatic
â-cells, and anterior pituitary cells specifically via L-type
voltage-dependent Ca2+ channels not found in hepato-
cytes or Kupffer cells.21 These differences in modes of
iron uptake also may underlie clinical observations of
differences in the course and pace of iron deposition and
damage to the liver, pancreas, and heart in patients
with all forms of iron overload.7
The toxicity associated with excess iron, whether a
systemic or a focal problem, derives from its interaction
with reactive oxygen species, for instance, endogenous
hydrogen peroxide (H2O2).22-25 In the presence of Fe(II),
H2O2 is reduced to the hydroxyl radical (HO•), a very
reactive species, and HO-, a process known as the
Fenton reaction. The Fe(III) liberated can be reduced
back to Fe(II) via a variety of biological reductants (e.g.,
ascorbate), a problematic cycle. The hydroxyl radical
reacts very quickly with a variety of cellular constitu-
ents and can initiate free radicals and radical-mediated
chain processes that damage DNA and membranes, as
well as produce carcinogens.23,26,27 Radical traps can
help to attenuate the already ongoing free-radical-
Essentially all prokaryotes and eukaryotes have a
strict requirement for iron. Although this element
comprises 5% of the earth’s crust, living systems have
great difficulty in accessing and managing this vital
micronutrient. The low solubility of Fe(III) hydroxide
(Ksp ) 1 × 10-39),2 the predominant form of the metal
in the biosphere, has led to the development of sophis-
ticated iron storage and transport systems in nature.
Microorganisms utilize low molecular weight, virtually
ferric-iron-specific, ligands, siderophores;3 higher eu-
karyotes tend to employ proteins to transport and store
iron (e.g., transferrin and ferritin, respectively).4-6
Humans have evolved a highly efficient iron manage-
ment system in which we absorb and excrete only about
1 mg of the metal daily; there is no mechanism for the
excretion of excess iron.7 Whether derived from trans-
fused red blood cells8-10 or from increased absorption
of dietary iron,11,12 without effective treatment, body
iron progressively increases with deposition in the liver,
heart, pancreas, and elsewhere. Iron accumulation
eventually produces (i) liver disease that may progress
to cirrhosis,13-15 (ii) diabetes related both to iron-
induced decreases in pancreatic â-cell secretion and
increases in hepatic insulin resistance,16,17 and (iii) heart
disease, still the leading cause of death in thalassemia
major and related forms of transfusional iron over-
load.7,18,19 The precise pathways of iron deposition and
its regulation are incompletely understood, but the
* Corresponding author. Phone: (352) 846-1956. Fax: (352) 392-
8406. E-mail: bergeron@mc.cop.ufl.edu.
‡ Department of Educational Psychology, University of Florida.
§ Columbia University College of Physicians and Surgeons.
† Present address: Department of Pharmaceutical Sciences, South
University School of Pharmacy, 709 Mall Blvd., Savannah, GA 31406.
10.1021/jm049306x CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/14/2005

822 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Bergeron et al.
Chart 1. Structures of Desferrithiocin (DFT, Top Left)
and Historical Analogues (S)-Desmethyldesferrithiocin
[(S)-DMDFT, Top Right], (S)-Desazadesferrithiocin
[(S)-DADFT, Bottom Left], and Desazadesmethyldes-
ferrithiocin [(S)-DADMDFT, Bottom Right]
mediated damage; for example, N,N′-bis(2-hydroxyben-
zyl)ethylenediamine-N,N′-diacetic acid (HBED)28 and
desferrioxamine B (DFO)29,30 are excellent radical traps.
A major concern in the design of iron chelators for
therapeutic use is related to their potential for facilitat-
ing this reduction of Fe(III) to Fe(II), thus promoting
toxic Fenton chemistry.22,31 Some agents inhibit the
Fenton reaction (e.g., DFO24); paradoxically, certain
ligands, such as ethylenediaminetetraacetic acid
(EDTA),31 nitrilotriacetic acid (NTA),22 5-aminosalicylic
acid (5-ASA),32,33 and 1,2-dimethyl-3-hydroxypyridin-4-
one (deferiprone, L1),31,34 actually enhance the reduction
of Fe(III) to Fe(II). Although the precise nature of iron-
mediated damage is complex24 and remains somewhat
elusive, the solution is clear: identify chelators that will
sequester this transition metal and lead to its excretion
without promoting Fenton chemistry.
In the majority of patients with thalassemia major
or other transfusion-dependent refractory anemias, the
severity of the anemia precludes phlebotomy therapy
as a means of removing toxic accumulations of iron.
Treatment with a chelating agent capable of sequester-
ing iron and permitting its excretion from the body is
then the only therapeutic approach available. The iron-
chelating agents now in use or under clinical evalua-
tion35 include Desferal (DFO, mesylate salt), defer-
iprone, ICL670A (4-[3,5-bis(2-hydroxyphenyl)-1,2,4-
triazol-1-yl]benzoic acid), and our desferrithiocin (DFT)
analogue, (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-
methyl-4-thiazolecarboxylic acid [(S)-4′-(HO)-DADFT].
Each of these agents chelate iron derived predomi-
nantly from systemic storage sites, either from iron
released by macrophages after catabolism of senescent
red blood cells, from iron in hepatic pools, or from both.35
Small but detectable amounts of intracellular desferri-
oxamine and ferrioxamine are seen within a single hour
of incubation with these compounds in vitro.36 In
patients, treatment with DFO can sometimes improve
cardiomyopathy within days, before the total iron
burden could have changed appreciably.37 Nonetheless,
the amounts removed from the heart are small; daily
24-h infusions of DFO are then required for periods of
years to reverse iron-induced heart disease completely.37
The efficacy of deferiprone in patients with heart disease
remains uncertain, and ICL670A likely does not enter
the heart in appreciable amounts. In any event, almost
daily administration of near maximal tolerated doses
of the first three agents is required to keep pace with
rates of transfusional iron loading in patients with
thalassemia major and other refractory anemias38 and
thereby minimize the amounts of circulating nontrans-
ferrin-bound iron. With increasing body iron, deposits
develop in the liver, heart, pancreas, and other endo-
crine organs. Prolonged, intensive chelation therapy is
then needed to arrest progression of the complications
of iron overload or, in the case of established heart
disease, to reverse cardiac dysfunction.
or progression of complications. Iron-chelating agents
that could selectively enter cardiac, pancreatic, or
hepatic cells could help immediately reduce toxic iron
pools and provide prompt protection against the pro-
gression of injury. Especially with respect to heart
disease, still the cause of death in 70% or more of
patients with thalassemia major,8,18,19 the development
of such agents could be life-saving.
Iron-clearing efficiency (ICE) is used as a measure of
the amount of iron excretion induced by a chelator. The
ICE, expressed as a percent, is calculated as (ligand-
induced iron excretion/theoretical iron excretion) × 100.
To illustrate, the theoretical iron excretion after admin-
istration of 1 mmol of DFO, a hexadentate chelator
which forms a 1:1 complex with Fe(III), is 1 mg-atom
of iron. In reality, DFO in clinical use is only about
5-7% efficient.39
Desferrithiocin. (S)-4,5-Dihydro-2-(3-hydroxy-2-
pyridinyl)-4-methyl-4-thiazolecarboxylic acid (DFT, Chart
1) is a tridentate siderophore40 that forms a stable 2:1
complex with Fe(III); the cumulative formation constant
is 4 × 1029 M-1 41,42
.
The donor groups include a phenolic
oxygen, a thiazoline nitrogen, and a carboxyl. Desferri-
thiocin was one of the first iron chelators that was
shown to be orally active.43 It performed well in both
the bile duct-cannulated rodent model (ICE, 5.5%)44 and
in the iron-overloaded Cebus apella primate (ICE,
16%).45,46 Unfortunately, DFT was severely nephro-
toxic.46 Nevertheless, the outstanding oral activity
spurred a structure-activity study to identify an orally
active and safe DFT analogue. Our initial goal was to
define the minimal structural platform compatible with
iron clearance upon po administration.
Our approach entailed simplifying the platform. The
thiazoline methyl of DFT was deleted to produce
(S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-thiazolecar-
boxylic acid [(S)-DMDFT, Chart 1], reducing the ICE
(4.8%) by two-thirds in the primate model.46 Removal
of DFT’s aromatic nitrogen left (S)-4,5-dihydro-2-(2-
hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-
DADFT, Chart 1], modestly diminishing the com-
pound’s ICE to 13% in C. apella.47 Abstraction of the
thiazoline methyl from (S)-DADFT, leaving (S)-4,5-
dihydro-2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid
[(S)-DADMDFT, Chart 1], had little effect on efficacy,
12% vs 13% ICE.46,48 These observations suggested that
more apparently lipophilic chelators are more active, for
example, DFT, (S)-DADFT, or (S)-DADMDFT vs (S)-
DMDFT.
Consequently, there is a pressing need for the con-
tinued development of new iron-chelating agents that
are more effective that can selectively remove iron from
organs and tissues especially vulnerable to iron-induced
toxicity, or both. Ligands that are more effective would
be able to more rapidly reduce dangerous body iron
burdens to safer levels and forestall the development

Partition-Variant Desferrithiocin Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 823
Table 1. Desferrithiocin Analogues’ Iron-Clearing Activity When Administered Orally to C. apella Primates and the Partition
Coefficients of the Compounds
a In the monkeys [n ) 4 (1, 2, 3, 6), 7 (4), 5 (8), 6 (7), or 8 (5)], the dose was 150 µmol/kg. The efficiency of each compound was calculated
by averaging the iron output for 4 days before the administration of the drug, subtracting these numbers from the 2-day iron clearance
after the administration of the drug, and then dividing by the theoretical output; the result is expressed as a percent. The relative
percentages of the iron excreted in the stool and urine are in brackets. b Data are expressed as the log of the fraction in the octanol layer
(log Papp); measurements were done in TRIS buffer, pH 7.4, using a “shake flask” direct method. The values obtained for compounds 1-4
are from ref 53. c Data are from ref 48. d Data are from ref 47. e Data are from ref 53.
Few further structural changes could be made to the
(S)-DADMDFT framework without an adverse effect on
ICE. Alterations of the distances between the donor
centers results in loss of activity.48 Thiazoline ring
modifications, that is, expansion (dihydrothiazine), oxi-
dation (thiazole), or reduction (thiazolidine), abrogated
iron-clearing activity.48,49 Likewise, replacement of the
sulfur with oxygen (oxazolines), with nitrogen (dihydro-
imidazole), or with a methylene (dihydropyrrole) re-
sulted in significant loss of efficacy.48,49 Changes in
configuration at C-4 also had a profound effect on ICE
in the C. apella model,47,48,50,51 demonstrating a poten-
tial stereoselective barrier in iron clearance; an (S)-
configuration at the C-4 carbon is optimal.51,52 Benzo-
fusions, designed to improve the ligands’ tissue residence
time and possibly ICE, were ineffective; both the naph-
thyl analogues and the quinoline systems performed
poorly.48,50
Finally, the (S)-DADMDFT framework was subjected
to a structure-activity study aimed at ameliorating its
toxicity. Both DADFT analogues, (S)-DADFT and (S)-
DADMDFT, were quite toxic: severe GI toxicity was
prominent, rather than nephrotoxicity, as with DFT.46-48
This structure-activity analysis was based on the idea
that by altering the lipophilicity (i.e., partition proper-
ties, log Papp) and/or the redox potential of the aromatic
ring, the drug’s toxicity profile, organ distribution
properties, and potential metabolic disposition could
change.
thiazoline methyl. It was determined that hydroxyl-
ation, as in the systems (S)-2-(2,4-dihydroxyphenyl)-
4,5-dihydro-4-thiazolecarboxylic acid [(S)-4′-(HO)-
DADMDFT, 1] or (S)-4′-(HO)-DADFT (2) (Table 1), was
compatible with iron clearance in the primate model and
also profoundly reduced toxicity in rodents.47,48,53 For
example, rats that were treated with (S)-DADFT or (S)-
DADMDFT were dead by day 5 of a planned 10-day
dosing regimen;46 those administered 1 and 2 at the
same dose did not display any frank toxicity.47 In fact,
(S)-4′-(HO)-DADFT (2) is now our lead compound in
clinical trials. These initial results suggested an inverse
correlation between toxicity and lipophilicity. In the C.
apella model, the hydroxylated compounds were gener-
ally less active than their corresponding parent drugs,
e.g., a 5.3% ICE for (S)-4′-(HO)-DADMDFT47 [vs 12.4%
for (S)-DADMDFT when administered po at a dose of
300 µmol/kg]. This result further underscores the im-
portance of lipophilicity in the chelator’s iron-clearing
efficiency.
The challenge was to increase lipophilicity and ICE
without increasing toxicity. In the current study, con-
sistent with our previous results,53 we demonstrate that
this goal can be achieved by methoxylation of (S)-
DADMDFT or (S)-DADFT. Either 3′- or 4′-methoxyl-
ation increases lipophilicity, altering the octanol-water
partition coefficients (log Papp), and improving ICE in
the iron-overloaded C. apella primate. Further, we show
that the more lipophilic compounds, as exemplified by
(S)-4′-(CH3O)-DADFT (4), have better access to the
organs most affected by iron overload, that is, the liver,
heart, and pancreas. Our studies also measured the
ability of representative systems to prevent ascorbate-
The redox potential of the analogues was modified by
addition of several different substituents to the aromatic
ring;48 log Papp was also changed by addition of these
same substituents and/or the presence or absence of the

824 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Bergeron et al.
Scheme 1. Synthesis of Desferrithiocin Analogues 6
and 8a
mediated reduction of Fe(III) to Fe(II) and to act as
radical scavengers. Finally, we verified that the methoxy
group, while increasing the ligand’s lipophilicity and
ICE, did not appreciably alter the compound’s toxicity
profile.
Results and Discussion
Design Concept. Initial considerations48 suggested
that the correlation between chelator lipophilicity, that
is, octanol-water partition coefficients (log Papp), and
ICEs exists principally within “families” of ligands. For
example, although the octanol-water partition coef-
ficients of DFT and (S)-DMDFT are very similar (-1.77
and -1.87, respectively), their ICEs in primates were
quite different, 16.1 ( 8.5% and 4.8 ( 2.7%, respectively.
However, considerations within the same structural
group afforded very different results. For example, a
comparison of the partition properties of DADMDFT
and (S)-4′-(HO)-DADMDFT (1) (-0.93 and -1.2, respec-
tively) and their corresponding ICEs (12.4 ( 7.6% vs
5.3 ( 1.7%) does imply a relationship between lipophi-
licity and iron-clearing efficiency. Accordingly, we have
assembled a group of structurally similar desferrrithio-
cins that have graded variations in their lipophilic
properties. On the basis of the substitutions in the
aromatic ring, two different core systems were consid-
ered, 2,4-dihydroxyphenyl (1-4) from earlier studies53
and 2,3-dihydroxyphenyl (5-8) compounds in the present
work (Table 1). The 4′-substituted systems included (S)-
4′-(HO)-DADMDFT (1), (S)-4′-(HO)-DADFT (2), (S)-4,5-
dihydro-2-(2-hydroxy-4-methoxyphenyl)-4-thiazolecar-
boxylic acid [(S)-4′-(CH3O)-DADMDFT, 3], and (S)-4,5-
dihydro-2-(2-hydroxy-4-methoxyphenyl)-4-methyl-4-
thiazolecarboxylic acid [(S)-4′-(CH3O)-DADFT, 4]. The
3′-substituted systems encompassed (S)-2-(2,3-dihydroxy-
phenyl)-4,5-dihydro-4-thiazolecarboxylic acid [(S)-3′-
(HO)-DADMDFT, 5], (S)-2-(2,3-dihydroxyphenyl)-4,5-
dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-3′-(HO)-
DADFT, 6], (S)-4,5-dihydro-2-(2-hydroxy-3-methoxy-
a Reagents: (a) BBr3, CH2Cl2, 84%; (b) NaHCO3, pH ) 6.0
phosphate buffer, CH3OH, 72-75 °C, 18 h, 88% (6); 39% (8).
amino acid 12 gave (S)-3′-(CH3O)-DADFT (8) in 39%
yield.
Iron Clearance. The animal model selected for this
series of experiments was the iron-overloaded C. apella
monkey. In this model, we are able to measure both total
iron excretion, expressed as ICE, and the modes of iron
clearance, that is, biliary (stool) and renal. The results
for compounds 1-4 are historical47,48,53 and are included
in Table 1 for comparison with the present findings.
Data are presented as the mean ( standard error of the
mean. For comparison of the means between two
compounds, a two-sample t-test (without the assumption
of equality of variances) was performed. All tests were
two-tailed, and a significance level of P < 0.05 was used.
We had observed that increasing the lipophilicity of
4′-hydroxylated DADFT analogues increased their ICE;53
the situation was similar with the 2′,3′-dihydroxy
derivatives. When a methyl group was introduced into
the 4-position of the thiazoline ring of (S)-3′-(HO)-
DADMDFT (5) to afford (S)-3′-(HO)-DADFT (6), the
iron-clearing efficiency changed. The efficiency of com-
pound 5 was 5.8 ( 3.4%; the stool:urine ratio was 91:9.
Upon methylation of the thiazoline ring to produce (6),
the efficiency increased to 23.1 ( 5.9% (P < 0.01 vs 5).
Eighty-three percent of the iron was in the stool, and
17% was in the urine. Although an increase in iron-
clearing efficiency was also observed upon addition of
a 4-methyl to the 2′-hydroxy-3′-methoxy system (ana-
logues 7 and 8, Table 1), the difference between these
two ligands was not statistically significant, 15.5 ( 7.3%
for derivative 7 vs 22.5 ( 7.1% for 4-methyl compound
8 (P > 0.05). 4-Methylation had no significant effect on
the proportion of the iron in the stool, 91% for 8 versus
87% for 7. Although methylation of the 3′-hydroxyl also
increased the iron-clearing efficiency (7 vs 5, P ) 0.02),
phenyl)-4-thiazolecarboxylic
acid
[(S)-3′-(CH3O)-
DADMDFT, 7], and (S)-4,5-dihydro-2-(2-hydroxy-3-
methoxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-
3′-(CH3O)-DADFT, 8].
Synthetic Methods. (S)-4′-(HO)-DADMDFT (1),48
(S)-4′-(CH3O)-DADMDFT (3),53 and (S)-3′-(CH3O)-
DADMDFT (7)48 were synthesized in these laboratories
by cyclization of D-cysteine with the requisite nitrile.
The reaction of the unusual amino acid D-R-methyl-
cysteine40 under these conditions provided (S)-4′-(HO)-
DADFT (2)47 and (S)-4′-(CH3O)-DADFT (4).53
(S)-2-(2,3-Dihydroxyphenyl)-4,5-dihydro-4-thiazole-
carboxylic acid (5), the desmethyl analogue of (S)-3′-
(HO)-DADFT (6), had been prepared by heating D-
cysteine with 2,3-dihydroxybenzimidate54 at a pH of 6.48
However, when D-R-methylcysteine was treated with
this imidate, none of the chelator 6 resulted. Thus, 2,3-
dihydroxybenzonitrile (10) was generated by unmasking
the chemically sensitive catechol group of 2,3-dimethoxy-
benzonitrile (9) with boron tribromide in CH2Cl255,56 in
84% yield (Scheme 1). Cyclocondensation of D-R-methyl-
cysteine (12) onto the cyano group of 10 in heated
phosphate-buffered methanol at pH 6 afforded (S)-3′-
(HO)-DADFT (6) in 88% yield. Analogously, condensa-
tion of 3-methoxy-2-hydroxybenzonitrile (11)57,58 with

Partition-Variant Desferrithiocin Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 825
Figure 2. Effect of various chelators on the iron-mediated
oxidation of ascorbate (Fenton chemistry) (percent of control,
y-axis): nitrilotriacetic acid (NTA), 1,2-dimethyl-3-hydroxy-
pyridin-4-one (L1), desferrioxamine (DFO), and DFT analogues
1-8 at several ligand:metal ratios (x-axis). Typically, each
assay contained three controls with only ascorbate and FeCl3;
these varied less than 5% ((SD). Each assay also usually
included a “negative control” containing ascorbate, FeCl3, and
L1 at a ligand:iron ratio of 2:1; this value was 174 ( 27%.
Figure 1. Iron-clearing efficiency (percent) of 4′-substituted
ligands 1-4 (open circles) and 3′-substituted analogues 5-8
(filled squares) plotted versus the respective partition coef-
ficients (log Papp) of the compounds. The plot of ligands 1-4 is
from ref 53 and is included for comparison.
methylation of the thiazoline ring exerted a more
profound effect (6 vs 5, P < 0.01).
Relationship between Partition Coefficients and
Iron-Clearing Efficacy. The partition coefficients of
the analogues were evaluated in buffered octanol-
water. The log Papp values (Table 1) ranged from -0.70
for 4 to -1.67 for 5. Attaching alkyl groups to the
ligands increases a compound’s lipophilicity (solubility
in octanol); furthermore, when the iron-clearing ef-
ficiency is plotted versus partition coefficient (Figure 1),
it is obvious that increasing the lipophilicity also aug-
ments the ICE within a particular family. Weighted
regression analyses of the log Papp/ICE data for the 2′,4′-
disubstituted analogues demonstrated that there was
sufficient evidence to conclude a significant relationship
between efficiency and log Papp. Furthermore, methyl-
ation of the 4′-hydroxyl of the parent 1, as in 3, seemed
to have a more dramatic effect on lipophilicity and iron-
clearing efficiency than did appending a methyl to the
4-position of the thiazoline ring (2).53
In the case of the 2′,3′-dihydroxy compounds (5-8),
there was also an apparent relationship between log Papp
and iron-clearing efficiency. However, what is most
interesting is the difference in the impact that changing
log Papp has on the efficiency of the 2′,3′-substituted
derivatives vs the effect on that of the 2′,4′-substituted
analogues. Linear regression analyses were conducted
to find whether the relationship between log Papp and
iron-clearing efficiency was the same for both series of
compounds. The data consisted of the log Papp for all of
the drug groups within both families. A preliminary
inspection of the data indicated that ordinary least
squares estimation could be applied to test for the
relationship between iron-clearing efficiency and log
Papp. Although the data do not appear to be perfectly
linear (Figure 1), any curvature was negligible so that
ordinary least squares could still be applied. A group ×
log Papp interaction was tested to determine whether the
slopes of the lines for both families were equal (i.e., Ho:
group × log Papp ) 0), that is, that the relationship
between iron-clearing efficiency and log Papp was the
same for both groups. The results indicated that the
interaction was not significant, suggesting that the
slopes for the two groups were not significantly different
from each other (P ) 0.815). The interaction term (group
× log Papp) was then dropped from the model. The effect
for the group was tested to indicate for which group iron-
clearing efficacy was the greater (i.e., Ho: groups ) 0).
These results clearly show a significant group effect with
the 2′,3′-disubstituted set of analogues having a greater
overall efficiency (Figure 1).
Antioxidant and Free-Radical-Scavenging Ac-
tivity. The principal reason for removing excess iron
from tissue is to prevent iron-mediated oxidative dam-
age. Thus, one of the issues of major concern is related
to the effect of any potential therapeutic ligand on the
reduction of Fe(III) to Fe(II). In the +2 oxidation state,
iron drives Fenton chemistry. Thus, a situation in which
the reduction of Fe(III) to Fe(II) is promoted by a
chelator would worsen any Fenton chemistry-mediated
cell damage. Because some iron chelators are known to
catalyze this reduction,22,31 a determination of whether
a ligand promotes, prevents, or has no effect on Fe(III)
reduction is critical in designing therapeutic chelators
for clinical use. Of the many physiological reductants
that can reduce Fe(III) to Fe(II), ascorbate is a fre-
quently used model. Ascorbate oxidation is easy to follow
qualitatively by its disappearance spectrophotometri-
cally.31,34 The data in Figure 2 are plotted as the change
in ascorbate concentration versus the ligand/metal ratio.
Diminished Fe(III)-mediated ascorbate oxidation is
indicated by a value less than 100% of control. Because
the equilibria unfolding during the reaction are quite
complicated, these results should not be overinterpreted.

826 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Bergeron et al.
Table 2. ABTS Radical Cation Quenching Activity of
indicates a more effective radical scavenger. As antici-
pated, the 3′-methoxylated compounds were less effec-
tive radical scavengers than were the corresponding 3′-
hydroxylated molecules; nevertheless, 7 and 8 were as
effective as Trolox and their corresponding positional
analogues (3 and 4, respectively) at trapping free
radicals and much better than the parent DFT. Com-
pounds 5 and 6 scavenged the model radical cation at a
level considerably less than those of the corresponding
4′-hydroxy analogues 1 and 2.
Disposition of Desferrithiocin Analogues. From
a drug design perspective, the relationship between ICE
and lipophilicity (log Papp) described above was very
useful. Nonetheless, the pharmacology behind this
phenomenon needed clarification. Why, for example,
should (S)-4′-(CH3O)-DADMDFT (3) be nearly 4 times
more efficient than (S)-4′-(HO)-DADMDFT (1), or (S)-
4′-(CH3O)-DADFT (4) be nearly twice as efficient as (S)-
4′-(HO)-DADFT (2), beyond the fact that both meth-
oxylated ligands partition into octanol to a greater
degree than do their hydroxylated counterparts?
To explore the possible relationship between tissue
disposition and ICE, we studied the behavior of the 4′-
methoxylated compounds (3 and 4) as representatives.
To avoid potential bioavailability problems associated
with GI absorption, we first examined tissues from
rodents given these compounds sc. The liver, heart, and
pancreas were removed from the animals 0.5, 1, 2, 4, 6,
and 8 h after single-dose administration of 4 or 2 (300
µmol/kg); compounds 4 and 2 were detected in tissue
homogenates by HPLC (Figure 3). Liver tissue taken
from animals given 4 sc revealed two peaks in the
chromatogram, one corresponding to the drug admin-
istered, 4, and the second corresponding to the 4′-
demethylated metabolite 2.
The differences in total chelator (either methoxylated
derivative 4 plus putative metabolite 2 or 2 when
administered alone) tissue concentrations seen were
profound (Figure 3). In the liver after sc administration
(panel A), the difference at 0.5 h was greater than
2-folds275 ( 85 nmol/g wet wt of tissue for [4] plus [2]
vs 123 ( 17 nmol/g for [2] (P < 0.05). The contrast
became more prominent, roughly 9-fold, at 8 h after
dosing, 106 ( 19 vs 12 ( 0.8 nmol/g, respectively (P <
0.007). A different, and more interesting, scenario is
unfolding in the heart (panel C). Although the total
chelator concentration ([4] + [2]) in the hearts of
animals treated with 4 is about one-half of that mea-
sured in the liver of the same animals, for example, 119
( 20 nmol/g at 0.5 h, this total is almost 5-fold that of
2 (27 ( 7 nmol/g) in animals treated with 2 (P < 0.005,
panel C). By 4 h, whereas the concentration of 4 in the
heart has dropped nearly 5-fold and 2 as the metabolite
of 4 is near detectable limits, in 2-treated animals, the
compound is below detectable limits (<4.8 nmol/g).
Similar proportions of 4 to 2 are found in the pancreas
(panel E), although the levels of compound achieved are
lower than those in the liver and heart. Excellent oral
bioavailability of the methoxy compound is suggested
by the comparable concentrations of methoxylated
compound and hydroxylated metabolite attained when
po administration is employed and the same time course
is examined (Figure 3, panels B, D, and F).
Desferrithiocin Derivatives
compd
slope × 103 OD units/µMa
-0.9b
DFT
7
-35 (-33c,d
-37b
)
Trolox
8
6
5
-39 (-36d)
-58 (-106b)
-62 (-102b)
a The slope was derived from A734 versus concentration data
after a 6-min reaction period between the chelator of interest and
the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical
cation (ABTS•+), which was formed from the reaction between
ABTS and persulfate. A negative slope represents a decrease in
the amount of highly colored radical cation over the time interval
from an initial A734 of about 0.900. Trolox, an analogue of vitamin
E, served as a positive control. b Data are from ref 30. c Numbers
in parentheses are the slopes obtained with the corresponding 4′-
substituted compound, e.g., the results for compound 3 are shown
in parentheses next to the slope for analogue 7. d Data are from
ref 53.
The question addressed is, simply, in the presence of a
test ligand, is the rate of ascorbate oxidation/iron
reduction increased, decreased, or the same relative to
controls containing iron(III) and ascorbate without the
compound? Desferrioxamine functions as a positive
control, as it decreases ascorbate loss;31 L1, which
increases ascorbate disappearance at ratios less than
3:1, serves as a negative control. Recall that the des-
ferrithiocin analogues form 2:1 complexes with Fe(III);
accordingly, the ligand-to-metal ratios from 0.5 to 2 are
measured for ligands 1-8 (Figure 2). The 4′-substituted
(1-4),53 3′-hydroxy (5 and 6), and 3′-methoxy (7 and 8)
analogues slowed Fe(III) reduction considerably, even
at a 0.5:1 ratio, as did the parent compound DFT (data
not shown).30,59 The results with the parent ligand are
consistent with those reported in this same assay31,34
and in a cultured cell system.34 These results indicate
that an enhancement of Fenton chemistry is unlikely
to account for the toxicity of desferrithiocin and some
of its derivatives.
Radical traps can help to attenuate Fenton chemistry-
induced, free-radical-mediated damage; thus, the
radical-scavenging properties of a particular ligand are
of importance. Two hexacoordinate iron chelators,
HBED28 and DFO,29,30 are excellent radical traps. The
issue of Fenton chemistry and chelator design has a
dimension beyond the prevention of the reduction of
Fe(III). The liberated HO• molecules are very short-
lived, reacting with most surrounding molecules at a
diffusion-controlled rate. Ultimately, less active, more
selective radicals, which can initiate a radical chain
process, are produced and can cause significant cell
damage.60
We have evaluated the 3′-hydroxylated compounds (5
and 6) and their respective O-methylated derivatives
(7 and 8) in a free-radical-scavenging assay; the previ-
ously reported results obtained for the 4′-substituted
compounds (1-4)53 are included in Table 2 for compari-
son. The capacity of each of these ligands to function
as a one-electron donor to the preformed radical mono-
cation of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS•+) was compared with that of Trolox, an
analogue of vitamin E.61 Table 2 shows the calculated
slopes of the decrease in A734 versus ligand concentra-
tion line for each compound; a more negative slope

Partition-Variant Desferrithiocin Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 827
Figure 3. Tissue distribution in liver (panels A and B), heart (panels C and D), and pancreas (panels E and F) of (S)-4′-(CH3O)-
DADFT (4) vs that of (S)-4′-(HO)-DADFT (2) upon sc administration (panels A, C, and E) and those of 4 upon po administration
(panels B, D, and F). The concentrations (y-axis) are reported as nmol compound per g wet wt of tissue. For all time points, n )
3. The asterisks indicate a level below detectable limits (approx. 5 nmol/g wet wt).
A similar measurement was made comparing (S)-4′-
(CH3O)-DADMDFT (3) vs (S)-4′-(HO)-DADMDFT (1) at
times from 2 to 8 h postdosing. The rats were given
either compound sc at 300 µmol/kg. Only the 2- and 4-h
time points were remarkable; the levels of both ligands
in all tissues were below detectable limits by 6 h
postdosing. In like fashion to 4, 3 was demethylated to
1. Further, the total chelator concentration ([3] + [1])
in the liver of animals treated with 3 was 6-fold (2 h
postdosing: 203 ( 62 vs 35 ( 29 nmol/g, P < 0.02) and
almost 10-fold (4 h postdosing: 45 ( 8 vs < 4.8 nmol/g)
higher than that of 1 when given alone. In the heart
and pancreas, the only measurable concentrations of
ligands were found at 2 h. In the heart, the total
chelator concentration ([3] + [1]) was approximately 10-
fold higher than that found when 1 was given alone, 62
( 31 vs < 4.8 nmol/g, respectively (P < 0.05).
most adversely affected by iron overload, that is, liver,
heart, and pancreas.
Metabolism of (S)-4′-(CH3O)-DADFT by Micro-
somes in Vitro. Although the details of the demethyl-
ation shown in Figure 3 remained uncharacterized, such
a demethylation is not without precedent. Demethyl-
ation of codeine, catalyzed by an isoform of cytochrome
P450, is responsible for its conversion to morphine in
the liver.62-64 It seemed likely that a similar process was
occurring with the 4′-methoxylated compounds (3 and
4) to their hydroxylated counterparts (1 and 2, respec-
tively) in the rodents. It is critical to note that when a
putative rodent-deactivated metabolite of DADFT, (S)-
5′-(HO)-DADFT, was given to primates, it was quite
active (unpublished observations), underscoring our
view that hydroxylated DFT analogues can be handled
differently by primates vs rodents.
Apparently, more total chelator gets into the tissues
of animals treated with 4 than of those treated with 3.
Clearly, much more total ligand is present in the tissues
of animals treated with the methoxy compounds than
those treated with their hydroxylated counterparts. The
metabolites found in the heart and pancreas are prob-
ably derived from metabolism of the methoxylated
compounds by the liver, but we have not yet examined
this pathway. Moreover, the total chelator tissue resi-
dence time is longer for (S)-4′-(CH3O)-DADFT (4) than
for (S)-4′-(CH3O)-DADMDFT (3). Taken collectively, this
is in keeping with the increased ICE (i.e., 3 vs 1; 4 vs
2). Overall, we emphasize that the methoxylated ligands
achieve these concentrations in the very organs that are
Unwilling to sacrifice a primate at this stage, we
compared the metabolism of 4 in rat and human liver
microsomes.65 In brief, pooled human or rat liver mi-
crosomes were used as the source of cytochrome P450.
The viability of the microsomes was evaluated using
resorufin benzyl ether, a substrate of cytochrome P450
3A4 (CYP3A4).65 The reaction mixture contained mi-
crosomes and an NADPH-generating system in buffer.
The reaction was initiated by the addition of compound
4 and was incubated for up to 2 h. The results (Figure
4) are presented as concentration of either the starting
drug (4) or metabolite (2) (as quantified by HPLC of the
trichloroacetic acid supernatant) in the mixture, nor-
malized to microsomal protein concentration. This sug-

828 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Bergeron et al.
difference in ICE of the (S)-3′-(HO)-DADMDFTs (5-8)
over the (S)-4′-(HO)-DADMDFTs (1-4).
Furthermore, although methylation of the 3′- or 4′-
hydroxyl did not alter the ability of the ligand to prevent
ascorbate reduction of Fe(III), it did decrease the
ligands’ free-radical-scavenging capacity relative to their
respective parent nonmethylated systems. Nevertheless,
these compounds were still at least as active as Trolox.30
Experimental Section
D-R-Methylcysteine as its hydrochloride salt (12) was ob-
tained from DSM Fine Chemicals, Linz, Austria. Reagents
were purchased from Aldrich Chemical Co. (Milwaukee, WI),
and Fisher Optima-grade solvents were routinely used. Dis-
tilled solvents and glassware that had been presoaked in 3 N
HCl for 15 min were employed in reactions involving chelators.
Phosphate buffer was made up to 0.1 M at a pH of 5.9566 and
was degassed. Organic extracts were dried with sodium
sulfate. Silica gel 32-63 from Selecto Scientific, Inc. (Suwanee,
GA) was used for flash column chromatography, and Sephadex
LH-20 was obtained from Amersham Biosciences (Piscataway,
NJ). Melting points are uncorrected. NMR spectra were
obtained at 300 MHz (1H) on a Varian Unity 300, with
chemical shifts (δ) given in parts per million referenced to
tetramethylsilane. Coupling constants (J) are in hertz.
Elemental analyses were performed by Atlantic Microlabs
(Norcross, GA).
Desferrioxamine B in the form of the methanesulfonate salt,
Desferal (Novartis Pharma AG, Basel, Switzerland), was
obtained from a hospital pharmacy. 1,2-Dimethyl-3-hydroxy-
pyridin-4-one (L1) was a generous gift from Dr. H. H. Peter
(Ciba-Geigy, Basel, Switzerland). Analogues 1-5 and 7 were
accessed as described in our earlier publications.47,48,53
Spectrophotometric readings (Aλ) for the ascorbate and
radical cation assays were taken on a Perkin-Elmer Lambda
3B spectrophotometer (Norwalk, CT).
Figure 4. Metabolism of (S)-4′-(CH3O)-DADFT (4) by rat and
human liver microsomes. The reaction mixture contained
microsomes and an NADPH-generating system in buffer. The
reaction was initiated by the addition of compound 4 and was
incubated up to 2 h (times shown on x-axis). The results are
shown as concentration of compound, normalized to protein
concentration present (y-axis, mean ( SD), as quantified by
HPLC of triplicate reaction mixtures after the incubation
period indicated.
gests that, in a human liver, roughly 50% of ligand 4 is
demethylated to 2 in 2 h. On the basis of these findings,
further in vivo measurement, i.e., following the metabo-
lism of selected compounds in primates via liver biop-
sies, is warranted. This will allow us to correlate the in
vivo rodent data with the in vivo primate data as they
relate to the respective microsomal numbers.
In Vivo Toxicity Assessment. At this point, it was
clear that we had to verify that the methoxylated
compounds, although more lipophilic, did not elicit any
unexpected toxic effects. Accordingly, 12 male Sprague-
Dawley rats, six with normal iron stores and six iron-
loaded to a level of 350 mg/kg, were treated with (S)-
4′-(CH3O)-DADMDFT (3) at a daily po dose of 100 µmol
(25 mg)/kg for 30 days. This dose was selected on the
basis of primate iron clearance data, that is, this dose
represents enough ligand to clear 450 µg Fe/kg of body
weight per day from the primates. Histopathological
results from extensive tissues, including the heart, liver,
kidney, pancreas, stomach, and intestine, were normal
in both sets of animals; this was similar to our earlier
findings with hydroxylated counterpart 1.48 Thus, at
least in this series of compounds, lipophilicity and iron
clearance can be increased without a corresponding
increase in toxicity.
C. apella monkeys were obtained from World Wide Primates
(Miami, FL). Male Sprague-Dawley rats were procured from
Harlan Sprague-Dawley (Indianapolis, IN). Cremophor RH-
40 was obtained from BASF (Parsippany, NJ). Ultrapure salts
were obtained from Johnson Matthey Electronics (Royston,
UK). All hematological and biochemical studies46 were per-
formed by Antech Diagnostics (Tampa, FL). Atomic absorption
(AA) measurements were made on a Perkin-Elmer model 5100
PC (Norwalk, CT).
(S)-2-(2,3-Dihydroxyphenyl)-4,5-dihydro-4-methyl-4-
thiazolecarboxylic Acid (6). Compound 12 (2.19 g, 12.8
mmol) was added to a solution of 10 (1.15 g, 8.51 mmol) in
degassed CH3OH (100 mL). Phosphate buffer (50 mL) and
NaHCO3 (2.15 g, 25.6 mmol) were added, and the reactants
were heated at 70 °C for 18 h with stirring under nitrogen.
The reaction mixture was cooled to room temperature, and the
solvents were removed by rotary evaporation. The residue was
partitioned between 8% NaHCO3 (175 mL) and EtOAc (85 mL).
After further extraction with 8% NaHCO3 (35 mL), the
combined aqueous portion was shaken with EtOAc (85 mL)
and was acidified with cold 1 N HCl (255 mL). Extraction with
EtOAc (3 × 175 mL), washing with saturated NaCl (120 mL),
Conclusion
and solvent removal in vacuo gave 1.90 g of 6 (88%) as a green
These observations have led to two hypotheses. (1)
Increasing the lipophilicity of selected series of DFT
analogues should enhance iron removal from the liver,
heart and pancreas, the organs at the greatest risk of
iron-induced injury. Extrapolating from the extent to
which ligands 3 and 4 enter the liver, heart, and
pancreas, one would expect excellent iron clearance from
these organs. (2) Increasing the lipophilicity of selected
series of DFT analogues can substantially augment
their systemic iron-clearing efficiency without increas-
ing their toxicity. Implicit in these hypotheses is the
question of whether we have the best platform; for
example, as shown in Figure 1, there is a significant
1
foam: [R]24 +67.0 ° (c 0.10, DMF); H NMR (CD3OD) δ 1.67
D
(s, 3 H), 3.33 (d, 1 H, J ) 11.7), 3.87 (d, 1 H, J ) 11.4), 6.74 (t,
1 H, J ) 7.8), 6.93 (dd, 1 H, J ) 3.3, 1.8), 6.96 (dd, 1 H, J )
2.7, 1.5). Anal. (C11H11NO4S) C, H, N.
(S)-4,5-Dihydro-2-(2-hydroxy-3-methoxyphenyl)-4-meth-
yl-4-thiazolecarboxylic Acid (8). Compound 12 (0.68 g, 4.0
mmol) was added to a solution of 1157,58 (0.5 g, 3.4 mmol) in
degassed CH3OH (10 mL). Phosphate buffer (5 mL) was added,
and the pH was adjusted to 6 with NaHCO3 (0.42 g, 5.0 mmol).
The reactants were heated at reflux for 12 h with stirring
under nitrogen. The reaction mixture was cooled to room
temperature and was concentrated by rotary evaporation. The
residue was dissolved in NaHCO3 (20 mL), and the pH was
adjusted to 2-3 with 1 N HCl. Extraction with ethyl acetate

Partition-Variant Desferrithiocin Analogues
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3 829
(2 × 50 mL) was carried out, and the extracts were con-
centrated in vacuo. LH-20 column chromatography (6%
CH3CH2OH/toluene) generated 0.350 g of 8 (39%) as a yellow
solid: mp 158-160 °C; [R]24D +61.6 ° (c 1.02, DMF); 1H NMR
(DMSO-d6) δ 1.60 (s, 3 H), 3.40 (d, 1 H, J ) 11.4), 3.80 (s, 3
H), 3.82 (d, 1 H, J ) 11.1), 6.89 (t, 1 H, J ) 7.8), 7.03 (dd, 1 H,
J ) 8.1, 1.4), 7.15 (dd, 1 H, J ) 8.0, 1.4). 12.6 (br s, 1 H), 13.3
(br s, 1 H). Anal. (C12H13NO4S) C, H, N.
metabolism studies, the rodents were given the monosodium
salt of the compound of interest (prepared by addition of the
free acid to 1 equiv of NaOH) either po or sc at a single dose
of 300 µmol/kg. In the toxicity trial, the monosodium salt of 3
was administered po by gavage at a daily dose of 100 µmol
(25 mg)/kg.
Calculation of Iron Chelator Efficiency. The theoretical
iron outputs of the chelators were generated on the basis of a
2:1 complex. The efficiencies in the monkeys were calculated
2,3-Dihydroxybenzonitrile (10). Boron tribromide (1 M
in CH2Cl2, 300 mL, 0.3 mol) was added dropwise to 9 (12.24
g, 75.00 mmol) in CH2Cl2 (30 mL) with dry ice/acetone cooling.
The reaction solution was allowed to warm to room temper-
ature and was stirred for 18 h. After quenching with H2O (50
mL) with ice-bath cooling, solids were filtered, and the layers
of the filtrate were separated. The aqueous phase was diluted
with H2O (200 mL), and the pH was adjusted to 2 with 1 N
HCl. Extraction with ethyl acetate (3 × 150 mL) was per-
formed, and the extracts were concentrated in vacuo to produce
as set forth elsewhere.47
Data are presented as the mean (
the standard error of the mean; P-values were generated via
a two-tailed Student’s t-test, in which the inequality of
variances was assumed, and a P-value of <0.05 was considered
significant.
Collection of Tissue Samples from Rodents. Male
Sprague-Dawley rats (250-350 g) were given the compounds
prepared as described above either po following an overnight
fast or sc. At times 0.5, 1, 2, 4, 6, and 8 h after dosing with
compounds 2 and 4 or at times 2, 4, 6, and 8 h after dosing
with compounds 1 and 3, the liver, heart, and pancreas were
removed from three animals.
1
8.5 g of 10 (84%) as a pale yellow solid: mp 153-157 °C; H
NMR (CD3OD) δ 6.74 (t, 1 H, J ) 7.9), 6.96 (dd, 1 H, J ) 11.7,
1.5), 6.98 (dd, 1 H, J ) 11.7, 1.5). Anal. (C7H5NO2) C, H, N.
Prevention of Iron-Mediated Oxidation of Ascorbate.
The iron chelators (NTA, L1, DFO, 1-8) were assessed for
their ability to diminish the iron-mediated oxidation of ascor-
bate by a literature method.31 Briefly, a solution of freshly
prepared ascorbate (100 µM) in sodium phosphate buffer (5
mM, pH 7.4) was incubated in the presence of FeCl3 (30 µM)
and chelator (ligand/Fe ratios varied from 0 to 2) for 40 min.
The A265 was read at 10 and 40 min; the ∆A265 in the presence
of ligand was compared to that in its absence.
Tissue Analytical Methods. The tissue samples were
prepared for HPLC analysis by homogenizing in water at a
ratio of 1:2 (w/v). Then, to precipitate proteins, three times
the volume of CH3OH was added, and the mixture was stored
at -20 °C for 20 min. This homogenate was centrifuged; the
supernatant was filtered with a 0.2 µm membrane. This
filtrate was injected directly onto the column or diluted with
mobile phase A [95% buffer (25 mM KH2PO4, pH 3.0):5%
CH3CN), vortexed, and filtered as above prior to injection.
Analytical separation was performed on a C18 reversed-
phase HPLC system with UV detection at 310 nm as described
previously.51,52 Mobile phase and chromatographic conditions
were as follows: solvent A, 5% CH3CN:95% buffer; solvent B,
60% CH3CN:40% buffer. The gradients for each compound
were as follows: for 2 and 4, linear ramp from 100% A to 50%
A (9 min), followed by a hold of 10 min; for 3, linear ramp
from 100% A to 20% B (20 min), followed by a hold of 5 min,
then a linear ramp to 100% B (15 min), followed by a hold of
20 min; for 1, linear ramp from 100% A to 20% B (20 min),
followed by a hold of 5 min, then a linear ramp to 100% B (8
min), followed by a hold of 5 min.
The concentrations were calculated from the peak area fitted
to calibration curves by nonweighted least squares linear
regression with Rainin Dynamax HPLC Method Manager
software (Rainin Instrument Co.). The method had a detection
limit of 0.5 µM and was reproducible and linear over a range
of 1-1000 µM.
Toxicity Evaluations in Rodents. Male Sprague-Dawley
rats (225-250 g) (n ) 12) were iron overloaded by ip admin-
istration of iron dextran (Sigma, 100 mg of Fe/mL) to a level
of 350 mg/kg. The rats were given four doses of the iron (105
mg/kg/dose) over a two-week period on a Monday, Friday,
Monday, Friday schedule. After a two-week equilibration
period, the animals were weighed again, and their total iron
burden was determined (total mg of iron/final body weight).
The rats were then assigned to groups so that the body weight
and iron burden between groups were within error of each
other.
Quenching of the ABTS Radical Cation. The iron
chelators were tested for their ability to quench the radical
cation formed from 2,2′-azinobis(3-ethylbenzothiazoline-6-sul-
fonic acid) (ABTS) by a published method.61 Briefly, a stock
solution of ABTS radical cation was generated by mixing ABTS
(10 mM, 2.10 mL) with K2S2O8 (8.17 mM, 0.90 mL) in H2O
and allowing the solution to sit in the dark at room temper-
ature for 18 h. This stock solution of deep blue-green ABTS
radical cation was diluted in sufficient sodium phosphate (10
mM, pH 7.4) to give an A734 of about 0.900. Test compounds
were added to a final concentration ranging from 1.25 to 15
µM, and the decrease in A734 was read after 1, 2, 4, and 6 min.
Assays were performed in triplicate at each concentration. The
reaction was largely complete by 1 min, but the data presented
are based on a 6-min reaction time.
Determination of Partition Coefficients. The octanol-
H2O partition data are expressed as distribution coefficients
uncorrected for partial ionization of the acids and were all
measured at pH 7.4 (50 mM TRIS buffer) using UV spectrom-
etry. The measurements were done using a “shake flask” direct
measurement.67 Quadruplicate samples were vigorously agi-
tated with 1-octanol (HPLC grade 1, Sigma-Aldrich, St. Louis,
MO) overnight in a Parr shaker, and the layers were allowed
to settle for 1-2 h prior to separation. The experiments were
conducted at room temperature (22-24 °C) using a Shimadzu
UV-265 spectrophotometer (Columbia, MD).
Iron Loading of C. apella Monkeys. The monkeys were
iron overloaded with iv iron dextran as specified in earlier
publications to provide about 500 mg of iron per kg of body
weight;68 the serum transferrin iron saturation rose to between
70 and 80%. At least 20 half-lives, 60 days,69 elapsed before
any of the animals were used in experiments evaluating iron-
chelating agents.
Primate Fecal and Urine Samples. Fecal and urine
samples were collected at 24-h intervals and processed as
described previously.45,46,70 Briefly, the collections began 4 days
prior to the administration of the test drug and continued for
an additional 5 days after the drug was given. Iron concentra-
tions were determined by flame atomic absorption spectroscopy
as presented in other publications.45,50
Additional animals (n ) 12) were left unloaded to serve as
age-matched counterparts. Both sets of animals were then
divided into control (H2O) and treated (3) groups (n ) 6 each).
The animals were given 3 orally by gavage as described above
or an equivalent volume of H2O for 30 days; the trial was
otherwise conducted as described in an earlier publication.47
Microsomal Assays.65 Pooled human liver microsomes or
rat liver microsomes (BD Biosciences, Bedford, MA) were used
as the source of cytochrome P450. Resorufin benzyl ether
(Molecular Probes, Eugene OR), a substrate of CYP3A4, was
used to assess the viability of the microsomes.65 One milliliter
of reaction buffer contained microsomes (0.8 mg) and an
NADPH-generating system (NADP+, 1.3 mM; glucose-6-
phosphate, 3.3 mM; glucose-6-phosphate dehydrogenase, 0.4
U/mL; MgCl2, 3.3 mM) in potassium phosphate buffer (100
Drug Preparation and Administration. For iron clear-
ance experiments, the compounds were administered as a
solution or a suspension in 40% Cremophor RH-40/water (v/
v) given po to the monkeys at a dose of 150 µmol/kg. In the

830 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 3
Bergeron et al.
(20) Chua, A. C. G.; Olynyk, J. K.; Leedman, P. J.; Trinder, D.
Nontransferrin-Bound Iron Uptake by Hepatocytes Is Increased
in the Hfe Knockout Mouse Model of Hereditary Hemochroma-
tosis. Blood 2004, 104, 1519-1525.
(21) Oudit, G. Y.; Sun, H.; Trivieri, M. G.; Koch, S. E.; Dawood, F.;
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mM, pH 7.4). The reaction was initiated by the addition of
compound 4 (100 µM) and was incubated for up to 2 h at 37
°C in a shaking water bath. Ice-cold CH3OH (3 volumes) was
added to stop the reaction after either 30, 60, 90, or 120 min;
the mixture was kept on ice for 20 min prior to centrifugation
at 12 000g for 5 min. The collected supernatant was then
injected onto a C18 column at a 1:10 dilution for quantitation
of standard, chelator, and metabolite by HPLC (for resorufin
standard, λex 550 nm, λem 585 nm). The results are normalized
to protein content and are expressed as µM/mg of protein.
Acknowledgment. Funding was provided by the
National Institutes of Health Grant No. R01-DK49108
and by Genzyme Drug Discovery and Development,
Waltham, MA. We thank Elizabeth M. Nelson, Harold
Snellen, J. R. Timothy Vinson, Samuel E. Algee, and
Katie Ratliff-Thompson for their technical assistance.
Supporting Information Available: Elemental analyti-
cal data for synthesized compounds 6, 8, and 10. This ma-
terial is available free of charge via the Internet at http://
pubs.acs.org.
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