(S)-4,5-dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid polyethers: A solution to nephrotoxicity

Article abstract of DOI:10.1021/jm0508944

Previous studies revealed that within a family of ligands the more lipophilic chelators have better iron-clearing efficiency. The larger the log P_app value of the compound, the better the iron-clearing efficiency. What is also clear from the da

Full text of DOI:10.1021/jm0508944

2772
J. Med. Chem. 2006, 49, 2772-2783
(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylic Acid Polyethers:
A Solution to Nephrotoxicity
Raymond J. Bergeron,* Jan Wiegand, James S. McManis, John R. T. Vinson, Hua Yao, Neelam Bharti, and James R. Rocca†
Department of Medicinal Chemistry and the AdVanced Magnetic Resonance Imaging and Spectroscopy Facility, UniVersity of Florida,
GainesVille, Florida 32610-0485
ReceiVed September 9, 2005
Previous studies revealed that within a family of ligands the more lipophilic chelators have better iron-
clearing efficiency. The larger the log Papp value of the compound, the better the iron-clearing efficiency.
What is also clear from the data is that although the relative effects of log Papp changes are essentially the
same through different families, there are differences in absolute value between families. However, there
also exists a second, albeit somewhat more disturbing, relationship. In all sets of ligands, the most lipophilic
chelator is always the most toxic. The current study focuses on designing ligands that balance the lipophilicity/
toxicity problem while iron-clearing efficiency is maintained. Earlier studies with (S)-4,5-dihydro-2-(2-
hydroxy-4-methoxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-4′-(CH3O)-DADFT, 6] indicated that
this methyl ether was a ligand with excellent iron-clearing efficiency in both rodents and primates; however,
it was too toxic. On the basis of this finding, a less lipophilic, more water-soluble ligand than 6 was assembled,
(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid [(S)-4′-
(HO)-DADFT-PE, 11], a polyether analogue, along with its ethyl and isopropyl esters. The parent polyether
and its isopropyl and ethyl esters were all shown to be highly efficient iron chelators in both rodents and
primates. A comparison of 11 in rodents with the desferrithiocin analogue (S)-4,5-dihydro-2-(2,4-
dihydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-4′-(HO)-DADFT, 1] revealed the polyether to be
more tolerable, achieving higher concentrations in the liver and significantly lower concentrations in the
kidney. The lower renal drug levels are in keeping with the profound difference in the architectural changes
seen in the kidney of rodents given 1 versus those treated with 11.
Introduction
reaction of Fe(II) with endogenous hydrogen peroxide to
produce the hydroxyl radical, a very reactive species, and the
hydroxide anion, is repudiated to be a major source of the tissue
damage associated with excess iron. The hydroxyl radical reacts
very quickly with a variety of cellular constituents and can
initiate free radicals and radical-mediated chain processes that
damage DNA and membranes, as well as produce carcinogens.
The Fe(III) liberated can be reduced back to Fe(II) via a variety
of biological reductants, a problematic cycle. Some iron
chelators inhibit the Fenton reaction [e.g., desferrioxamine B
(DFO18)]; paradoxically, others stimulate it [e.g., 1,2-dimethyl-
3-hydroxypyridin-4-one (deferiprone, L1)].20 Although the
precise nature of iron-mediated damage is complex18 and
remains somewhat elusive, the solution is clear: identify
chelators that will sequester this transition metal and render it
excretable without promoting Fenton chemistry.
In the majority of patients with thalassemia major or other
transfusion-dependent refractory anemias, treatment with a
chelating agent capable of sequestering iron and permitting its
excretion from the body is the only therapeutic approach
available. The iron-chelating agents now in use or under clinical
evaluation are DFO, L1, the Novartis triazole 4-[3,5-bis(2-
hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid (ICL670A), and
our own desferrithiocin [(S)-4,5-dihydro-2-(3-hydroxy-2-py-
ridinyl)-4-methyl-4-thiazolecarboxylic acid, DFT] analogue (S)-
4,5-dihydro-2-(2,4-dihydroxyphenyl)-4-methyl-4-thiazolecar-
boxylic acid [(S)-4′-(HO)-DADFT, 1] (Chart 1).
Humans have evolved a highly efficient iron management
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.1 Two systemic “iron processing” proteins are involved,
one for transport, transferrin, and the other for storage, ferritin.
Introduction of excess iron into this closed system, whether
derived from transfused red blood cells2-4 or from increased
absorption of dietary iron,5,6 leads to a build up of the metal in
the liver, heart, pancreas, and elsewhere. Such iron accumulation
eventually produces (i) liver disease that may progress to
cirrhosis,7-9 (ii) diabetes related both to iron-induced decreases
in pancreatic â-cell secretion10,11 and increases in hepatic insulin
resistance, and (iii) heart disease, still the leading cause of death
in thalassemia major12-14 and related forms of transfusional iron
overload. The precise pathways of iron deposition and its
regulation are incompletely understood, but the 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. Recent
evidence suggests that L-type Ca2+ channels constitute a major
pathway for iron entry into cardiomyocytes from plasma
nontransferrin bound iron (NTBI).15 Similar L-type Ca2+
channels are present in pancreatic â-cells and cells of the anterior
pituitary but are not found in hepatocytes or Kupffer cells.
There are a number of disease states in which the body iron
stores and plasma NTBI levels are sufficiently high to cause
significant oxidative tissue damage. The Fenton reaction,16-19
DFO, a naturally occurring hexadentate trihydroxamic acid,
although used clinically, is poorly absorbed from the gas-
trointestinal (GI) tract and rapidly eliminated from the circula-
tion, necessitating prolonged (8-12 h daily) parenteral [sub-
cutaneous (sc) or intravenous (iv)] infusion.21-24 Not surprisingly,
* Corresponding Author. Phone (352) 846-1956. Fax (352) 392-8406.
E-mail: rayb@ufl.edu.
† Advanced Magnetic Resonance Imaging and Spectroscopy Facility.
10.1021/jm0508944 CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/04/2006

Thiazolecarboxylic Acid Polyethers as Fe Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2773
Chart 1. Initial Structural Alterations in the Tridentate Chelator
vulnerable to iron-induced toxicity. Ligands that are more
effective would be able to more rapidly reduce dangerous body
iron burdens to safer levels and forestall the development 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 progression of injury. Especially with
respect to heart disease, still the cause of death in 70% or more
of patients with thalassemia major, the availability of such agents
could be life-saving.
Desferrithiocin (DFT)a
However, the underlying problem of nephrotoxicity is the
subset of the current work, in particular as this issue relates to
DFT analogues. This study focuses on the design, synthesis,
and testing of DFT analogues that are very efficient iron
chelators with minimal nephrotoxicity.
Results and Discussion
Design Concept. DFT is a tridentate siderophore33 that forms
a stable 2:1 complex with Fe(III); the cumulative formation
constant is 4 × 1029 M-1 34,35
.
It performed well when given
orally (po) in both the bile duct-cannulated rodent model (ICE,
5.5%)36 and in the iron-overloaded Cebus apella primate (ICE,
16%).37,38 Unfortunately, DFT is severely nephrotoxic.38 Nev-
ertheless, 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.
a Abbreviations used throughout the text were derived from the names
of 2 and 3, as shown by the boldface letters. The ligating sites are also in
bold text.
patients have difficulty with this demanding regimen, and
considerable effort has been devoted to finding suitable alterna-
tives.
Our approach first entailed simplifying the platform. Removal
of the pyridine nitrogen of DFT gave (S)-4,5-dihydro-2-(2-
hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-DAD-
FT, 2], the parent ligand of the desaza (DA) series. Substitution
of the 4-methyl with hydrogen, as well, led to (S)-4,5-dihydro-
2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid [(S)-DADMDFT,
3], the very simple desazadesmethyl (DADM) system (Chart
1). Although chelators 2 and 3 were still very effective iron
chelators, they were plagued with GI toxicity rather than
nephrotoxicity.39 Further structure-activity studies were aimed
at ameliorating these toxicity problems. By altering the lipo-
philicity (i.e., partition properties, 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.
The orally active bidentate chelator L1 is licensed in Europe
and some other countries as second-line therapy to DFO.
Unfortunately, although it is orally active, it is less efficient
than DFO at removing iron.25-28 Whereas the orally active
tridentate chelator ICL670A has now been approved by the
FDA, it did not demonstrate noninferiority to DFO. Furthermore,
it apparently has a somewhat narrow therapeutic window, owing
to potential nephrotoxicity.29-31
Ligand 1 is an orally active tridentate DFT analogue now in
phase I/II trials in patients. Although the preclinical toxicity
profile of 1 was relatively benign, i.e., no geno- or reproductive
toxicity and only mild nephrotoxicity at high doses, the clinical
results remain to be elucidated.
It was ultimately determined that hydroxylation, as in the
systems (S)-4′-(HO)-DADFT (1) or (S)-2-(2,4-dihydroxyphe-
nyl)-4,5-dihydro-4-thiazolecarboxylic acid [(S)-4′-(HO)-DAD-
MDFT, 4] (Chart 1), was compatible with iron clearance in the
primate model and also profoundly reduced toxicity in
rodents.39-41 Previous studies indicate that the dose-limiting
toxicity of 1 in rodents was nephrotoxicity.40 Although the origin
of the nephrotoxicity is unclear, the target seems to be centered
largely in the proximal tubules. The current study focuses on
structural alterations of 1 that, which while not diminishing the
iron-clearing efficiency of the parent drug, reduce the level of
chelator achieved in the kidney. These alterations are shown to
have a profound effect both on how the drug distributes to the
kidneys, as well as how it impacts the proximal tubules.
All of these agents predominantly chelate iron derived 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. Almost daily administration of near
maximal tolerated doses of the first three agents is required to
keep pace with rates of transfusion iron loading in patients with
thalassemia major and other refractory anemias. As a conse-
quence of the major systemic source of chelated iron, each of
these agents is maximally effective when given to prevent the
accumulation of a toxic iron burden and to maintain a low level
of body iron stores. In patients who accumulate substantial body
iron burdens, the metal builds up in vulnerable organs.
Prolonged, intensive therapy is then needed to arrest progression
of the complications of iron overload or, in the case of heart
disease, to reverse cardiac dysfunction. For example, in patients
with thalassemia major who present with cardiac failure, daily
24-h infusions of DFO for periods of years are required to
reverse the iron-induced heart disease.32
Previous studies revealed that within a family of ligands the
more lipophilic compounds have better iron-clearing efficiency.
For example, in an earlier study42 we evaluated the impact of
altering the lipophilicity, log Papp, of both (S)-4′-(HO)-DADFTs
(compounds 1, 4-6, Table 1) and (S)-2-(2,3-dihydroxyphenyl)-
4,5-dihydro-4-thiazolecarboxylic acids [(S)-3′-(HO)-DADFTs],
(compounds 7-10, Table 1). We found that the larger the log
We believe that there is a pressing need for the continued
development of new iron-chelating agents that are more effective
and/or that can selectively remove iron from organs especially

2774 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Bergeron et al.
Table 1. Iron-Clearing Activity of Desferrithiocin Analogues When Administered Orally to Iron-Loaded C. apella Primates and the Partition
Coefficients of the Compoundsa
a Adapted from ref 42. b In the monkeys, n ) 4 (1, 4, 5, 8), 7 (6), 5 (10), 6 (9), or 8 (7). The primates were given a single 150 µmol/kg dose of the
chelators. 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. c Data are expressed as the log of the fraction in the octanol layer (log
P
app); measurements were done in Tris buffer, pH 7.4, using a “shake flask” direct method. The values obtained for compounds 1 and 4-6 are from ref 41.
d Data are from ref 39. e Data are from ref 40. f Data are from ref 41. g Data are from ref 42.
Papp value of the compound, the greater the iron-clearing
efficiency. What is also clear from the data is that although the
relative effects of log Papp changes are essentially the same for
(S)-4′-(HO)-DADFTs and (S)-3′-(HO)-DADFTs, there are dif-
ferences in absolute value between families.
suggests a reduction in iron clearance. In fact, the data was
consistent with improved iron-clearing efficiency. However, this
alkylation, methylation of 1 to 6, was found to elicit pronounced
nephrotoxicity when 6 was given sc to rats at a dose of 300
µmol/kg/d for 5 d (unpublished data). Rodents treated with 1
under an identical dosing regimen did not exhibit any renal
toxicity. Nevertheless, the fact that 6 was a significantly better
iron chelator in rodents than the parent 1 encouraged the pursuit
of a less lipophilic, more water-soluble ligand than 6. It was
felt that such a ligand was likely to be cleared from the kidneys
more quickly, thus lowering the steady-state tissue concentration
and therefore toxicity. The current study focuses on designing
ligands that balance the lipophilicity/toxicity interaction while
iron-clearing efficiency is maintained. The ligand of choice was
the polyether (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxade-
cyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid [(S)-4′-(HO)-
DADFT-PE, 11].
However, there also exists a second, albeit somewhat more
disturbing, relationship. The greater the lipophilicity of a
chelator, the more toxic it is. The ligands in Table 2 can be
separated into three families: the DADMDFTs (3 and 4), the
DADFTs (1, 2, 6 and 11), and the Me2-DADMDFTs (12 and
13). In each family, as the lipophilicity decreases, i.e., the log
Papp becomes more negative, the toxicity also decreases.
Reduction in the lipophilicity of (S)-DADMDFT (3) was
accomplished by simple 4′-hydroxylation of the aromatic ring
to generate 4. In the DADFT series, again, hydroxylation of
the 4′ aromatic position moving from 2 to 1 or, as will be seen
below, introduction of the polyether into 2 to produce (S)-4′-
(HO)-DADFT-PE (11) produced similar results: profound
reductions in toxicity. Finally, hydroxylation of (S)-5,5-Me2-
DADMDFT (12) to (S)-5,5-Me2-4′-(HO)-DADMDFT (13) is
also consistent with the idea that 4′-hydroxylation and/or
reduction in lipophilicity can have a significant effect on ligand
toxicity within a family of chelators. The data suggested it would
be possible to further operate on a ligand to optimize the
lipophilicity/iron clearance/toxicity profile.
Synthetic Methods. The synthesis of (S)-4′-(HO)-DADFT-
PE (11) began with the conversion of (S)-4′-(HO)-DADFT (1)
to its isopropyl ester 14 in 99% yield with 2-iodopropane (1.9
equiv) and N,N-diisopropylethylamine (1.9 equiv, DIEA) in
DMF (Scheme 1). Ester 14 was then alkylated at the 4′-hydroxyl
using an equimolar amount of tri(ethylene glycol) monomethyl
ether under Mitsunobu conditions [diisopropyl azodicarboxylate
(1.2 equiv, DIPAD) and triphenylphosphine (1.2 equiv) in THF],
providing (S)-4′-(HO)-DADFT-PE-iPrE (15) in 76% yield.
Hydrolysis of isopropyl ester 15 with 50% NaOH (13 equiv)
in methanol followed by acidification with dilute HCl furnished
(S)-4′-(HO)-DADFT-PE (11) in 95% yield. Esterification of
Earlier studies with (S)-4,5-dihydro-2-(2-hydroxy-4-methoxy-
phenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-4′-(CH3O)-
DADFT (6)] indicated it to be a ligand with excellent iron-
clearing efficiency in both rodents and primates.42 Thus, there
is nothing implicit in the alkylation of the 4′-(HO) of 1 that

Thiazolecarboxylic Acid Polyethers as Fe Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2775
Table 2. Partition Coefficients and Tolerability of DFT Analoguesa
Figure 1. 1H resonances and pertinent homonuclear NOE correlations
for (S)-4′-(HO)-DADFT-PE-iPrE (15); the percent NOE is indicated
next to the dotted lines.
group (b), enhanced the upfield thiazoline methine doublet at
δ 3.18 (c); thus, these ring substituents are syn.
Chelator-Induced Iron Clearance in Non-Iron-Overloaded
Rodents. When describing the effects of structural alterations
on the DFT skeleton, the iron-clearing efficiencies of the
compounds in rats are shown (Table 3), along with the relative
fractions of the iron excreted in the bile and in the urine. A
single 300 µmol/kg dose of drug was administered to male
Sprague-Dawley rats (400-450 g) po and/or sc. We will begin
with a brief discussion of (S)-4′-(HO)-DADFT (1) and (S)-4′-
(CH3O)-DADFT (6). Ligand 1 performed poorly in rodents
when given either po or sc. The iron-clearing efficiencies, 1.1
( 0.8% and 1.1 ( 0.6%, respectively, were minimal at best.
When 1 was 4′-(HO) methylated to provide 6, the iron-clearing
efficiency after po administration increased substantially to 6.6
( 2.8%; 98% of the iron was in the bile and 2% was in the
urine. Thus, 6 performed better than the parent drug 1 (p <
0.02). The polyether of 1, (S)-4′-(HO)-DADFT-PE (11), in
which a 3,6,9-trioxadecyl group was fixed to the 4′-(HO) of 1,
also performed well, with an iron-clearing efficiency of 5.5 (
1.9% and a 90/10 distribution of iron in the bile/urine when
administered po. Given sc, the efficiency increased to 8.7 (
2.6%, slightly more than po (p < 0.05). We also investigated
the iron-clearing efficiencies of the corresponding isopropyl
a All compounds were given to the rats orally by gavage at a dose of
384 µmol/kg/d for a maximum of 10 days.
polyether acid 11 with iodoethane (1.6 equiv) by the method
of 14 afforded (S)-4′-(HO)-DADFT-PE-EE (16) in 83%
yield.
That substitution of resorcinol derivative 14 occurred at the
4′-hydroxyl and not the more sterically hindered 2′-hydroxyl,
giving monoadduct 15, was verified by NOE difference
spectroscopy. Irradiation at δ 4.14, assigned to methylene (h)
of the triether chain, significantly enhanced two aromatic
signalssδ 6.46 (j) and 6.49 (k)sas well as the adjacent
methylene of the linear chain at δ 3.86 (g) (Figure 1). Moreover,
irradiation at δ 1.64, the signal corresponding to the 4-methyl
Scheme 1. Synthesis of (S)-4′-(HO)-DADFT-PE (11) and Its Isopropyl (15) and Ethyl Ester (16)a
a Reagents: (a) i-PrI (1.9 equiv), DIEA (1.9 equiv), DMF, rt, 14 d, 99%; (b) CH3[O(CH2)2]3OH (1.0 equiv), DIPAD (1.2 equiv), PPh3 (1.2 equiv), THF,
5 °C, 1 d, 76%; (c) 50% NaOH (13 equiv), CH3OH, then 1 N HCl, rt, 1 d, 95%; (d) EtI (1.6 equiv), DIEA (1.6 equiv), DMF, rt, 50 h, 83%.

2776 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Bergeron et al.
Table 3. Iron-Clearing Efficiency of DFT Analogues in Non-Iron-Overloaded Rodentsa
a The rodents were given a single 300 µmol/kg dose of the chelators. The efficiency of each compound was calculated by subtracting the iron excretion
of control animals from the iron excretion of the treated animals. This number was then divided by the theoretical output; the result is expressed as a percent.
The relative percentages of the iron excreted in the bile and urine are in brackets. b The compounds were solubilized by the addition of 1 equiv of NaOH
to a suspension of the free acid in distilled H2O.
ester, (S)-4′-(HO)-DADFT-PE-iPrE (15). Although the isopropyl
ester (15) is only a bidentate ligand, nonspecific esterase
cleavage would liberate the tridentate chelator 11. Such required
cleavage of 15 to the active compound (11) could also increase
its half-life, possibly increasing iron-clearing efficiency. When
administered po, the efficiency of 15 was significantly higher
than that of the polyether parent 11, 13.9 ( 3.3% versus 5.5 (
1.9%, (p < 0.002) with 95% of the iron in the bile. When
administered sc, the isopropyl ester (15) was also significantly
better than 11, 22.1 ( 7.6% versus 8.7 ( 2.6% (p < 0.003)
with 95% of the iron in the bile. It is important to point out
that because of the poor water solubility of this compound, it
was administered in 50% ethanol-water. The profound differ-
ence in iron-clearing efficiencies between 15 and any other
compound given sc compelled us to be sure that 15 was not
being transesterfied in the aqueous ethanol. The ethyl ester, (S)-
4′-(HO)-DADFT-PE-EE (16), would likely be cleaved by
nonspecific esterases more efficiently than the isopropyl ester
(15) to the parent ligand (11). When we gave rodents the ethyl
ester 16 in 50% aqueous ethanol sc, the iron-clearing efficiency
was 11.4 ( 0.8%, significantly lower than that of the isopropyl
ester 15 (p < 0.01). The distribution of iron was similar, 96%
of the iron being excreted in the bile.
µmol/kg. The efficiency of the parent drug (1) in the primates
was 16.8 ( 7.2% when given po and 15.9 ( 2.7% when given
sc. The iron distribution was largely biliary, 88/12. A similar
scenario unfolded with the 4′-(CH3O) analogue (6). The
efficiency of the chelator given po was 24.4 ( 10.8%, with a
similar iron excretion ratio of 91/9. The polyether (11) given
po had an efficiency of 25.4 ( 7.4%. When given sc the
clearance was also very impressive, 30.4 ( 7.2%. Both methods
of dosing presented with largely fecal iron excretion, 96 and
99%, respectively. With the isopropyl ester (15) the efficiency
was 19.5 ( 6.8% after po dosing, with a stool/urine iron
excretion ratio of 77/23. With the polyether ethyl ester (16),
the iron-clearing efficiency was 17.9 ( 1.0% when given sc
with a feces/urine distribution of 81/19.
In addition, both 1 and 11 demonstrate excellent oral
bioavailability. The po vs sc iron clearing efficiencies are nearly
identical: 16.8 ( 7.2% vs 15.9 ( 2.7% for ligand 1 and 25.4
( 7.4% vs 30.4 ( 7.2% for 11. The efficiency of 15 po and 16
sc are within the error of the efficiency of 1 given po or sc (p
> 0.05). Finally, although the efficiency of 11 given po is greater
than that of 1 po, the increase is just shy of being significant (p
) 0.06). However, 11 given sc is significantly more effective
than 1 sc (p < 0.02). Nevertheless, the po and sc iron-clearing
efficiencies of 11 are 500% greater than that of sc-administered
DFO.38
Iron Clearance in Iron-Loaded Primates. Not unexpect-
edly, all of the ligands performed better in the iron-loaded
primates than in the non-iron-overloaded rodents (Table 4). The
drugs were given po and/or sc to the monkeys at a dose of 150
Toxicity Assessment of (S)-4′-(CH3O)-DADFT (6) and (S)-
4′-(HO)-DADFT-PE (11). Male Sprague-Dawley rats (n ) 6

Thiazolecarboxylic Acid Polyethers as Fe Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2777
Table 4. Iron-Clearing Efficiency of DFT Analogues in Iron-Loaded Primatesa
a The primates were given a single 150 µmol/kg dose of the analogues. The efficiency of each compound was calculated by averaging the iron output for
4 days before 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 The compounds were
solubilized by the addition of 1 equiv of NaOH to a suspension of the free acid in distilled H2O. c Data are from ref 42.
per group) were given 6 or 11 po once daily for up to 10 d at
a dose of 384 µmol/kg/d (equivalent to 100 mg/kg/d of the DFT
sodium salt). In a second experiment, rats were given 11 po at
a dose of 63.3 µmol/kg/d for 30 days. The latter dose was
selected on the basis of primate iron clearance data, i.e., this
dose represents enough ligand to clear 450 µg of Fe/kg of body
weight from the primates. Dosing with 6 had to be stopped after
five doses; severe nephrotoxicity, reminiscent of what was found
when the ligand was given sc at 300 µmol/kg/d for 5 days
(unpublished data), resulted. However, histopathological results
of extensive tissues including the heart, liver, pancreas, kidney,
stomach, and intestine from animals treated with 11 for 10 or
30 days were normal in both sets of animals.
Tissue Distribution of (S)-4′-(HO)-DADFT-PE (11). One
of the toxicity issues surrounding iron chelation therapy is the
problem of renal damage. Many iron chelators have been shown
to have untoward effects at the proximal tubule level. This is
certainly one of the dose-limiting toxicities of several of the
DFT analogues and the Novartis triazole ICL670A. Although
a structure-activity study made it possible to largely ameliorate
the nephrotoxicity of DFT, as realized in (S)-4′-(HO)-DADFT
(1), it is not entirely absent at high doses. This observation
compelled us to further search for a DFT analogue that would
maintain an iron-clearing efficiency similar to 1 but would
accumulate to a lesser extent in the kidney. In the current study,
we compare the tissue distribution of the polyether 11 and its
metabolite 1 with those for 1 itself and (S)-4′-(CH3O)-DADFT
(6) and its metabolite 1 (Figure 2). The organs chosen in addition
to the kidneys, e.g., the liver, pancreas, and heart, are the tissues
most negatively affected by iron overload.
Each rat was given a single sc 300 µmol/kg dose of the drug,
and the animals were sacrificed 2, 4, 6, and 8 h after dosing. In
the plasma, although neither 1 nor 11 ever achieved levels above
10 µM, very small amounts of the polyether metabolite 1 were
observed. In contrast, 6 rose to levels in the plasma of 470 µM
at 2 h, dropping to 75 µM at 8 h. Very little of 1, the metabolite
of 6, was observed (Figure 2A).
All of the drugs achieved fairly high levels in the liver (Figure
2B). The concentration of the parent drug 1 was 48 ( 20 nmol/g
wet weight at 2 h, dropping to 12 ( 0.8 nmol/g wet weight at
8 h. The 4′-(CH3O) analogue (6) and its metabolite (1) reached
155 ( 13 nmol/g wet weight 2 h postdrug. The metabolite
represents 28% of the total of the components. At 8 h the total
drug dropped to 106 ( 19 nmol/g wet weight, 23% of which is
the metabolite. The polyether (11) and its metabolite (1) reached
134 ( 32 nmol/g wet weight at 2 h, 2% as the metabolite. At
8 h the polyether dropped to 25 ( 5 nmol/g wet weight with
no measurable metabolite. The liver levels relative to the plasma
of ligands 1 and 11 suggest first-pass clearance.
The ligands do not concentrate to a great extent in the
pancreas (Figure 2C). At 2 h postdosing 1 has achieved 6 ( 1
nmol/g wet weight and is not detectable at future time points.
The polyether (11) reaches a concentration of 30 ( 25 nmol/g
wet weight at 2 h, dropping to 3 ( 3 nmol/g wet weight at 8 h.
The 4′-(CH3O) ligand (6) also rises to 38 ( 5 nmol/g wet weight

2778 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Bergeron et al.
Figure 2. Distribution in plasma (A), liver (B), pancreas (C), heart (D), and kidney (E) of (S)-4′-(HO)-DADFT (1), (S)-4′-(CH3O)-DADFT (6),
and (S)-4′-(HO)-DADFT-PE (11) after a single sc administration. For all time points, n ) 3. The asterisks indicate a level below detectable limits
(ca. 5 nmol/g wet wt). Data for 1 and 6 are from ref 42.

Thiazolecarboxylic Acid Polyethers as Fe Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2779
at 2 h, dropping to around 8 nmol/g wet weight at 8 h. A small
amount of 1, the metabolite of 6, was observed at 2 h.
The drug levels in the heart are very interesting (Figure 2D).
The parent drug (1) is found only at 2 h postdrug and even
then only at 5 ( 3 nmol/g wet weight. Ligand 11 also achieves
very low concentrations. At 2 h it is just 9 ( 3 nmol/g wet
weight, dropping to 5 ( 1 nmol/g wet weight at 4 h, not
detectable at 6 h, and only 2 ( 2 nmol/g wet weight at 8 h. In
contrast, 6 and its metabolite (1) are present at 73 ( 3 nmol/g
wet weight at 2 h; 9% is in the form of the metabolite (1). This
drops to 33 ( 4 nmol/g wet at 4 h and remains fairly steady
through the 8 h time point.
The kidney drug levels are probably most cogent to the
current study (Figure 2E). Both the parent ligand (1) and its
methoxy analogue (6) achieve fairly high levels in the kidney.
Ligand 1 is nearly 100 nmol/g wet weight at 2 h, dropping to
9 ( 3 nmol/g wet weight at 8 h. Ligand 6 and its metabolite
(1) reach nearly 150 nmol/g wet weight at 2 h; 34% is in the
form of the metabolite. The concentration then remains >80
nmol/g wet weight through 8 h postdrug. However, the renal
concentration of polyether (11) is only 41 ( 3 nmol/g wet
weight at 2 h and decreases to less than 10 nmol/g wet weight
at 8 h. These tissue concentrations (6 > 1 > 11) seem to be in
keeping with the compounds’ nephrotoxicity profiles.
A Comparison of the Impact of (S)-4′-(HO)-DADFT-PE
(11) and (S)-4′-(HO)-DADFT (1) on Rodent Kidneys. In a
preliminary dose-range finding study, male Sprague-Dawley
rats were given 1 twice daily for 7 days at doses of 237, 355,
or 474 µmol/kg/dose (474, 711, or 947 µmol/kg/d). The drug
was found to cause moderate to severe vacuolization in the renal
proximal tubules at all doses. It was decided that the 237 µmol/
kg/dose (474 µmol/kg/d) would be used to compare the renal
toxicity of 1 with that of the polyether (11).
A total of 12 rats were used for this study: four controls,
four treated with 11, and four treated with 1. The two drugs 11
and 1 were given po by gavage to the rodents twice daily for 7
days at equimolar doses of 237 µmol/kg/dose (474 µmol/kg/d).
One day postdrug the kidneys were perfusion-fixed and one
kidney from each rat was dissected. Tissue samples of 1 mm3
were cut from the kidney cortexes. Under light microscopy the
proximal and distal tubules of kidneys from the control animals
show normal tubular architecture (Figure 3A). The proximal
tubules of kidneys from the polyether (11) treated rodents
(Figure 3B) are indistinguishable from those of the control
animals (Figure 3A); the distal tubules present with occasional
vacuolization but are otherwise normal. However, animals
treated with 1 (Figure 3C) show regional, moderate to severe
vacuolization in the proximal tubules, a loss of the brush border,
and tubular extrusions toward the lumen; the distal tubules show
moderate to severe vacuolization.
Under electron microscopy the kidneys from the control
animals show normal proximal and distal tubular architecture
(photographs not shown). Kidneys from animals treated with
the polyether (11) present with occasional vacuolization and
apoptotic nuclei and have some abnormal giant lysosomes at
the basolateral side of the proximal tubule but are otherwise
normal. The same is true of the distal tubules. Finally, animals
treated with 1 show regional, moderate to severe vacuolization
of the proximal tubules (mainly S2 segments), loss of the brush
border, golgi dilations, tubular extrusions toward the lumen, and
apoptotic nuclei. The distal tubules demonstrate moderate to
severe vacuolization. While the distal tubules of both the
polyether 11 and the parent 1 treated animals do demonstrate
some vacuolization, the changes to the kidneys of the animals
Figure 3. The impact of (S)-4′-(HO)-DADFT-PE (11) and (S)-4′-(HO)-
DADFT (1) on rat kidney proximal tubules as compared with untreated
rats (control). The drugs were given po twice daily for 7 days at a
dose of 237 µmol/kg/dose (474 µmol/kg/d). The proximal tubules from
rats treated with 11 (B) are almost indistinguishable from those of the
control animals (A). However, the proximal tubules from rats treated
with 1 (C) display moderate to severe damage (tubules marked with
an asterisk). Magnification ) 400×.
treated with 1 are much more pronounced. These findings are
in keeping with the level of 11 achieved in the kidney relative
to that of 1.
Conclusion
Our previous studies revealed that within a family of ligands
the more lipophilic chelators have better iron-clearing efficiency,
i.e., the larger the log Papp value of the compound, the better
the iron-clearing efficiency. What is also clear from the data is
that although the relative effects of log Papp changes are
essentially the same through different families, there are real

2780 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Bergeron et al.
differences in absolute value. For example, increasing the
lipophilicity of (S)-4′-(HO)-DADMDFT (4) and (S)-3′-(HO)-
DADMDFT (7) increased the iron-clearing efficiencies about
as effectively (Table 1). However, the (S)-3′-(HO)-DADFT
ligands (7-10) were consistently more efficient.42
compounds’ nephrotoxicity profiles. The lower renal concentra-
tions of 11 relative to 1 are in keeping with the profound
difference in architectural changes seen in the kidneys of rodents
given each drug.
Under light microscopy, the proximal and distal tubules of
kidneys from the control animals show normal tubular archi-
tecture. The proximal tubules of kidneys from the polyether (11)
treated rodents are indistinguishable from those of the control
animals; the distal tubules present with occasional vacuolization
but are otherwise normal. However, animals treated with 1 show
regional, moderate to severe vacuolization in the proximal
tubules, a loss of the brush border, and tubular extrusions toward
the lumen; the distal tubules show moderate to severe vacu-
olization.
Under electron microscopy the kidneys from the control
animals show normal proximal and distal tubular architecture.
Kidneys from animals treated with the polyether (11) present
with occasional vacuolization and apoptotic nuclei and have
some abnormal giant lysosomes at the basolateral side of the
proximal tubule but are otherwise normal. The same is true of
the distal tubules. Finally, animals treated with 1 show regional,
moderate to severe vacuolization of the proximal tubules (mainly
S2 segments), loss of the brush border, golgi dilations, tubular
extrusions toward the lumen, and apoptotic nuclei. The distal
tubules demonstrate moderate to severe vacuolization. While
the distal tubules of both the polyether 11 and the parent 1
treated animals do demonstrate some vacuolization, the changes
to the kidneys of the animals treated with 1 are much more
pronounced. These findings are in keeping with the level of 11
achieved in the kidney relative to that of 1.
There also exists a second, albeit somewhat more disturbing,
relationship. In all sets of ligands, the more lipophilic chelator
is always the more toxic. The current study focuses on designing
ligands that balance the lipophilicity/toxicity interaction while
iron-clearing efficiency is maintained. Earlier studies with (S)-
4′-(CH3O)-DADFT (6) indicated that this methyl ether had
excellent iron-clearing efficiency in both rodents and primates;
however, it was too toxic. This established that the 4′-(HO)
functionality of (S)-4′-(HO)-DADFT (1) was not requisite for
iron-clearing function. On the basis of this finding, a less
lipophilic, more water-soluble ligand than 6 was assembled, the
polyether (S)-4′-(HO)-DADFT-PE (11), along with its ethyl (16)
and isopropyl (15) esters. The key step in the synthesis of this
ligand was the coupling of tri(ethylene glycol) monomethyl ether
with isopropyl 2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-
4-thiazolecarboxylate (14) under Mitsunobu conditions in the
presence of diisopropyl azodicarboxylate and triphenylphos-
phine.
When 1 was 4′-(HO) methylated to provide 6, the iron-
clearing efficiency after po administration increased substantially
to 6.6 ( 2.8% in a rodent model. The polyether (11), in which
a 3,6,9-trioxadecyl group was fixed to the 4′-(HO) of 1, also
performed well, with an iron-clearing efficiency of 5.5 ( 1.9%
when administered po. Given sc, the efficiency increased to 8.7
( 2.6%, slightly more than po (p < 0.05). The iron-clearing
efficiency of the corresponding isopropyl ester, (S)-4′-(HO)-
DADFT-PE-iPrE (15), was significantly higher than the poly-
ether parent 11 when administered po, 13.9 ( 3.3% versus 5.5
( 1.9% (p < 0.002). When administered sc, the isopropyl ester
(15) was also significantly better than 11, 22.1 ( 7.6% versus
8.7 ( 2.6% (p < 0.003). Although the isopropyl ester (15) is
only a bidentate ligand, nonspecific esterase cleavage would
liberate the tridentate chelator, 11.
The efficiency of the 4′-(CH3O) analogue (6) given po to
primates was 24.4 ( 10.8%. The corresponding polyether (11)
given po performed very well in primates, with an efficiency
of 25.4 ( 7.4%. When given sc the efficiency was 30.4 ( 7.2%,
not significantly better than when given po.
In addition, both the parent ligand (1) and the polyether
analogue (11) demonstrate excellent oral bioavailability in the
iron-loaded primates. The po vs sc iron clearing efficiencies
are nearly identical: 16.8 ( 7.2% vs 15.9 ( 2.7% for ligand 1
and 25.4 ( 7.4% vs 30.4 ( 7.2% for 11. The po and sc iron
clearing efficiencies of 11 are 500% greater than that of sc
administered DFO.38
A comparison of 11 in rodents with 1 revealed the polyether
to be more tolerable, achieving higher concentrations in the liver
and significantly lower concentrations in the kidney. The kidney
drug levels are probably most cogent to the current study. Both
the parent ligand (S)-4′-(HO)-DADFT (1) and its methoxy
analogue 6 achieve fairly high levels in the kidney. Ligand 1 is
nearly 100 nmol/g wet weight at 2 h, dropping to 9 ( 3 nmol/g
wet weight at 8 h. Ligand 6 and its metabolite 1 reach nearly
150 nmol/g wet weight at 2 h; 34% is in the form of the
metabolite. The concentration then remains >80 nmol/g wet
weight through 8 h postdrug. However, the polyether (11) renal
concentration is only 41 ( 3 nmol/g wet weight at 2 h and
decreases to less than 10 nmol/g wet weight at 8 h. These tissue
concentrations (6 > 1 > 11) seem to be in keeping with the
These data are consistent with the idea that further preclinical
studies of 11 are warranted, particularly longer-term toxicity
studies and pharmacokinetics.
Experimental Section
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 hematologi-
cal and biochemical studies38 were performed by Antech Diagnos-
tics (Tampa, FL). Atomic absorption (AA) measurements were
made on a Perkin-Elmer model 5100 PC (Norwalk, CT). Histo-
pathological analysis was carried out by Florida Vet Path (Bushnell,
FL).
Cannulation of Bile Duct in Non-Iron-Overloaded Rats. The
cannulation has been described previously.37,38 Bile samples were
collected from male Sprague-Dawley rats (400-450 g) at 3-h
intervals for 24 h. The urine sample was taken at 24 h. Sample
collection and handling are as previously described.37,38
Iron Loading of C. apella Monkeys. The monkeys (3.5-4 kg)
were iron overloaded with iv iron dextran as specified in earlier
publications to provide about 500 mg of iron per kg of body
weight;43 the serum transferrin iron saturation rose to between 70
and 80%. At least 20 half-lives, 60 d,44 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.37,38,45 Briefly, the collections began 4 d prior to the
administration of the test drug and continued for an additional 5 d
after the drug was given. Iron concentrations were determined by
flame atomic absorption spectroscopy as presented in other publica-
tions.37,46
Drug Preparation and Administration. In the iron-clearing
experiments the rats were given a single 300 µmol/kg dose of the
drugs po and/or sc. The compounds were administered as (1) a
solution in water (11); (2) solubilized in 40% Cremophor RH-40/

Thiazolecarboxylic Acid Polyethers as Fe Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2781
water (15, po); (3) the monosodium salt of the compound of interest
(prepared by addition of the free acid to 1 equivalent of NaOH)
(1, 6); or (4) in 50% ethanol-water (15 sc and 16). Cremophor
was utilized as the vehicle for the po (but not sc) dosing of 15
because of the potential for 50% aqueous ethanol to cause gastric
ulceration that could lead to increased drug absorption and falsely
increased iron clearance.
The drugs were given to the monkeys po and/or sc at a dose of
150 µmol/kg. The drugs were prepared as for the rats, except that
6 was solubilized in 40% Cremophor RH-40/water.
removed, and immersed in a glass vial containing fresh fixative at
room temperature. Four hours later, the kidneys were again rinsed
with Tyrode’s buffer. The tissue samples were placed in sodium
cacodylate. After two rinses with cacodylate buffer, the samples
were dehydrated with graded concentrations of ethyl alcohol and
infiltrated with propylene oxide overnight. Finally, they were
embedded in TAAB with DMP-30. The TAAB was polymerized
at 60 °C for 48 h.
Thick sections (500 nm) were cut from TAAB embedded tissue,
mounted onto glass slides and stained with Toluidine Blue. The
tissue slices were photographed with a Zeiss Axioskop II at 400×
magnification.
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 rats and monkeys were calculated
as set forth elsewhere.40 Data are presented as the mean ( the
standard error of the mean; P-values were generated via a one-
tailed Student’s t-test, in which the inequality of variances was
assumed; and a P-value of <0.05 was considered significant.
Toxicity Evaluation of (S)-4′-CH3O-DADFT (6) and the
Polyether Acid (11) in Rodents. Male Sprague-Dawley rats
(250-300 g) were fasted overnight and were given 6 or 11 po by
gavage once daily for up to 10 d at a dose of 384 µmol/kg/d. This
dose is equivalent to 100 mg/kg/d of the DFT sodium salt. The
animals were fed ∼3 h postdrug and had access to food for 5 h
before being fasted overnight. The rats given 11 in the 30-day
experiment were given the drug po at a dose of 63.3 µmol/kg/d, a
dose that, in the primates, results in the excretion of 450 µg of
Fe/kg. Due to the protracted nature of the latter experiment, the
animals were not fasted. To minimize food content in the stomach,
the drug was not given until the afternoon. Additional animals
served as age-matched controls.
Collection of Tissue Samples from Rodents. Male Sprague-
Dawley rats (250-350 g) were given a single sc injection of 1, 6,
and 11 at a dose of 300 µmol/kg prepared as described above. At
times 2, 4, 6, and 8 h after dosing (n ) 3 rats per time point) the
animals were euthanized by exposure to CO2 gas. Blood was
obtained via cardiac puncture into vacutainers containing sodium
citrate. The blood was centrifuged and the plasma separated for
analysis. The liver, heart, kidneys, and pancreas were then removed
from the animals.
For further demonstration of architectural differences, thin slices
(50-90 nm) were mounted onto copper grids, stained with uranyl
acetate, counterstained with lead nitrate, and evaluated via a Zeiss
transmission electron microscope.
Synthetic Methods. Compound 1 was synthesized using the
method published by this laboratory.40 Reagents were purchased
from Aldrich Chemical Co. (Milwaukee, WI), and Fisher Optima-
grade solvents were routinely used. DMF and THF were distilled,
the latter from sodium and benzophenone. Reactions were run under
a nitrogen atmosphere, and organic extracts were dried with sodium
sulfate. Silica gel 40-63 from SiliCycle, Inc. (Quebec City, QC,
Canada) was used for flash column chromatography. Optical
rotations were run at 589 nm (sodium D line) utilizing a Perkin-
Elmer 341 polarimeter, with c being concentration in grams of
compound per 100 mL of CHCl3 solution. Chemical shifts (δ) for
1H NMR spectra are given in parts per million downfield from
tetramethylsilane for CDCl3 (not indicated) or sodium 3-(trimeth-
ylsilyl)propionate-2,2,3,3-d4 for D2O. NOE difference spectra at
500 and 750 MHz were obtained as follows: specified resonances
of non-degassed samples in CDCl3 were irradiated for 2.5-3.0 s
prior to acquisition of the spectra with saturation, and from these
were subtracted control spectra with irradiation off resonance.
Chemical shifts (δ) for 13C NMR spectra are given in parts per
million referenced to 1,4-dioxane (δ 67.19) in D2O or to the residual
solvent resonance in CDCl3 (δ 77.16). Coupling constants (J) are
in hertz. Elemental analyses were performed by Atlantic Microlabs
(Norcross, GA).
Tissue Analytical Methods. The tissue samples were prepared
for HPLC analysis by homogenizing them 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. The 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 Discovery RP Amide
C16 HPLC system with UV detection at 310 nm as described
previously.47,48 Mobile phase and chromatographic conditions were
as follows: solvent A, 5% CH3CN:95% buffer; solvent B, 60%
CH3CN:40% buffer. The gradients for compounds 1, 6, and 11 were
a linear ramp from 100% A to 50% A (9 min), followed by a hold
of 10 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.
A Comparison of the Impact of (S)-4′-(HO)-DADFT-PE (11)
and (S)-4′-(HO)-DADFT (1) on Rodent Kidneys. Male Sprague-
Dawley rats (200-250 g) were housed two to a cage and were
fasted overnight. The rats were given 11 or 1 po twice daily at a
dose of 237 µmol/kg/dose (474 µmol/kg/d) for 7 days. The rats
were fed approximately 3 h postdrug and had access to food for 5
h before being fasted again.
Isopropyl 2-(2,4-Dihydroxyphenyl)-4,5-dihydro-4-methyl-4-
thiazolecarboxylate (14). 2-Iodopropane (4.0 mL, 40 mmol) and
DIEA (7.0 mL, 40 mmol) were successively added to 1 (5.32 g,
21.0 mmol) in DMF (65 mL), and the solution was stirred at room
temperature for 14 d. After solvent removal under high vacuum,
the residue was treated with 1:1 0.5 M citric acid/saturated NaCl
(200 mL) and was extracted with EtOAc (200 mL, 2 × 50 mL).
The combined extracts were washed with 100-mL portions of 0.25
M citric acid, 1% NaHSO3, H2O, and saturated NaCl, and solvent
was evaporated. Purification by flash column chromatography using
20% EtOAc/toluene furnished 6.1 g (99%) of 14 as a yellow oil:
[R]26 +64.3° (c 1.00); 300 MHz 1H NMR δ 1.27 and 1.29 (2 d, 6
H, J ) 5.5), 1.64 (s, 3 H), 3.18 (d, 1 H, J ) 11.3), 3.82 (d, 1 H,
J ) 11.2), 5.08 (septet, 1 H, J ) 6.3), 6.37 (dd, 1 H, J ) 8.5, 2.5),
6.43 (d, 1 H, J ) 2.4), 7.27 (d, 1 H, J ) 8.5) 12.8 (br s, 1 H);
125.8 MHz 13C NMR δ 21.70, 24.46, 39.86, 69.89, 83.18, 103.20,
107.58, 109.97, 132.24, 160.79, 161.28, 171.03, 172.75. Anal.
(C14H17NO4S) C, H, N.
Isopropyl (S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecy-
loxy)phenyl]-4-methyl-4-thiazolecarboxylate (15). Tri(ethylene
glycol) monomethyl ether (1.6 mL, 10 mmol) and diisopropyl
azodicarboxylate (2.3 mL, 12 mmol) were successively added to a
solution of 14 (2.89 g, 9.78 mmol) and triphenylphosphine (3.07
g, 11.7 mmol) in THF (60 mL) with ice bath cooling. The solution
was stirred at room temperature for 3 h and was maintained at 5
°C for 16 h. Solvent was removed by rotary evaporation, and 40%
EtOAc/petroleum ether (40 mL) was added. The solution was kept
at 5 °C for 12 h; solid formed and was filtered and washed with
solvent (90 mL). The filtrate was concentrated in vacuo and was
purified by flash chromatography (48% EtOAc/petroleum ether)
to give 3.29 g of 15 (76%) as a yellow oil: [R]26 +40.6° (c 1.16);
750 MHz 1H NMR δ, see Figure 1; 188.6 MHz 13C NMR δ 21.76,
One day postdrug, the rats were anesthetized with sodium
pentobarbital. The abdominal aorta was isolated and cleansed of
extraneous tissue and cannulated. The kidneys were perfusion-fixed
with glutaraldehyde in Tyrode’s buffer containing 3% PVP,

2782 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9
Bergeron et al.
(8) Bonkovsky, H. L.; Lambrecht, R. W. Iron-Induced Liver Injury. Clin.
LiVer Dis. 2000, 4, 409-429, vi-vii.
(9) Pietrangelo, A. Mechanism of Iron Toxicity. AdV. Exp. Med. Biol.
2002, 509, 19-43.
24.51, 39.89, 59.19, 67.71, 69.61, 69.66, 70.75, 70.82, 71.04, 72.10,
83.36, 101.60, 107.41, 110.09, 131.78, 161.35, 163.12, 170.75,
172.39. Anal. (C21H31NO7S) C, H, N.
(S)-4,5-Dihydro-2-[2-hydroxy-4-[2-[(3,6,9-trioxadecyloxy)phe-
nyl]-4-methyl-4-thiazolecarboxylic Acid (11). A solution of 50%
(w/w) NaOH (4.9 mL, 94 mmol) in CH3OH (50 mL) was added to
15 (3.24 g, 7.34 mmol) in CH3OH (100 mL) with ice bath cooling.
The reaction mixture was stirred at room temperature for 17 h,
and the bulk of the solvent was removed by rotary evaporation.
The residue was treated with dilute NaCl (150 mL) and was
extracted with ether (3 × 50 mL). The basic aqueous phase was
cooled in ice, acidified with 1 N HCl (120 mL), and extracted with
EtOAc (200 mL, 2 × 100 mL). After the EtOAc layers were washed
with saturated NaCl (100 mL), glassware that was presoaked in 3
N HCl for 15 min was employed henceforth. After solvent removal
by rotary evaporation, the residue was dissolved in distilled H2O
(70 mL) and lyophilized to furnish 2.78 g of 11 (95%) as an orange
(10) Cario, H.; Holl, R. W.; Debatin, K. M.; Kohne, E. Insulin Sensitivity
and â-Cell Secretion in Thalassaemia Major with Secondary Haemo-
chromatosis: Assessment by Oral Glucose Tolerance Test. Eur. J.
Pediatr. 2003, 162, 139-146.
(11) Wojcik, J. P.; Speechley, M. R.; Kertesz, A. E.; Chakrabarti, S.;
Adams, P. C. Natural History of C282Y Homozygotes for Hemo-
chromatosis. Can. J. Gastroenterol. 2002, 16, 297-302.
(12) Brittenham, G. M.; Griffith, P. M.; Nienhuis, A. W.; McLaren, C.
E.; Young, N. S.; Tucker, E. E.; Allen, C. J.; Farrell, D. E.; Harris,
J. W. Efficacy of Deferoxamine in Preventing Complications of Iron
Overload in Patients with Thalassemia Major. N. Engl. J. Med. 1994,
331, 567-573.
(13) Brittenham, G. M. Disorders of Iron Metabolism: Iron Deficiency
and Overload. In Hematology: Basic Principles and Practice, 3rd
ed.; Hoffman, R., Benz, E. J., Shattil, S. J., Furie, B., Cohen, H. J.,
et al., Eds.; Churchill Livingstone: New York, 2000; pp 397-428.
(14) Zurlo, M. G.; De Stefano, P.; Borgna-Pignatti, C.; Di Palma, A.;
Piga, A.; Melevendi, C.; Di Gregorio, F.; Burattini, M. G.; Terzoli,
S. Survival and Causes of Death in Thalassaemia Major. Lancet 1989,
2, 27-30.
(15) Oudit, G. Y.; Sun, H.; Trivieri, M. G.; Koch, S. E.; Dawood, F.;
Ackerley, C.; Yazdanpanah, M.; Wilson, G. J.; Schwartz, A.; Liu,
P. P.; Backx, P. H. L-type Ca2+ Channels Provide a Major Pathway
for Iron Entry into Cardiomyocytes in Iron-Overload Cardiomyopa-
thy. Nat. Med. 2003, 9, 1187-1194.
1
oil: [R]25 +53.1° (c 0.98); 400 MHz H NMR (D2O) δ 1.76 (s, 3
H), 3.35 (s, 3 H), 3.54-3.61 (m, 3 H), 3.64-3.72 (m, 4 H), 3.74-
3.78 (m, 2 H), 3.90-3.94 (m, 2 H), 3.96 (d, 1 H, J ) 12.0), 4.25-
4.29 (m, 2 H), 6.53 (d, 1 H, J ) 2.4), 6.64 (dd, 1 H, J ) 9.0, 2.2),
7.61 (d, 1 H, J ) 9.2); 100 MHz 13C NMR (D2O) δ 23.65, 39.56,
58.65, 68.34, 69.33, 70.07, 70.18, 70.44, 71.62, 77.58, 102.11,
106.72, 109.66, 134.67, 161.27, 167.07, 176.86, 180.70. Anal.
(C18H25NO7S) C, H, N.
(16) Graf, E.; Mahoney, J. R.; Bryant, R. G.; Eaton, J. W. Iron-Catalyzed
Hydroxyl Radical Formation. Stringent Requirement for Free Iron
Coordination Site. J. Biol. Chem. 1984, 259, 3620-3624.
(17) Halliwell, B. Free Radicals and Antioxidants: A Personal View. Nutr.
ReV. 1994, 52, 253-265.
(18) Halliwell, B. Iron, Oxidative Damage, and Chelating Agents. In The
DeVelopment of Iron Chelators for Clinical Use; Bergeron, R. J.,
Brittenham, G. M., Eds.; CRC: Boca Raton, FL, 1994; pp 33-56.
(19) Koppenol, W. Kinetics and Mechanism of the Fenton Reaction:
Implications for Iron Toxicity. In Iron Chelators: New DeVelopment
Strategies; Badman, D. G., Bergeron, R. J., Brittenham, G. M., Eds.;
Saratoga: Ponte Vedra Beach, FL, 2000; pp 3-10.
(20) Dean, R. T.; Nicholson, P. The Action of Nine Chelators on Iron-
Dependent Radical Damage. Free Radic. Res. 1994, 20, 83-101.
(21) Porter, J. B. Deferoxamine Pharmacokinetics. Semin. Hematol. 2001,
38, 63-68.
(22) Lee, P.; Mohammed, N.; Marshall, L.; Abeysinghe, R. D.; Hider, R.
C.; Porter, J. B.; Singh, S. Intravenous Infusion Pharmacokinetics
of Desferrioxamine in Thalassaemic Patients. Drug Metab. Dispos.
1993, 21, 640-644.
(23) Pippard, M. J.; Callender, S. T.; Finch, C. A. Ferrioxamine Excretion
in Iron-Loaded Man. Blood 1982, 60, 288-294.
(24) Pippard, M. J. Desferrioxamine-Induced Iron Excretion in Humans.
Bailliere’s Clin. Haematol. 1989, 2, 323-343.
(25) Hoffbrand, A. V.; Al-Refaie, F.; Davis, B.; Siritanakatkul, N.;
Jackson, B. F. A.; Cochrane, J.; Prescott, E.; Wonke, B. Long-term
Trial of Deferiprone in 51 Transfusion-Dependent Iron Overloaded
Patients. Blood 1998, 91, 295-300.
(26) Olivieri, N. F. Long-term Therapy with Deferiprone. Acta Haematol.
1996, 95, 37-48.
(27) Olivieri, N. F.; Brittenham, G. M.; McLaren, C. E.; Templeton, D.
M.; Cameron, R. G.; McClelland, R. A.; Burt, A. D.; Fleming, K.
A. Long-Term Safety and Effectiveness of Iron-Chelation Therapy
with Deferiprone for Thalassemia Major. N. Engl. J. Med. 1998, 339,
417-423.
(28) Richardson, D. R. The Controversial Role of Deferiprone in the
Treatment of Thalassemia. J. Lab. Clin. Med. 2001, 137, 324-329.
(29) Nisbet-Brown, E.; Olivieri, N. F.; Giardina, P. J.; Grady, R. W.;
Neufeld, E. J.; Sechaud, R.; Krebs-Brown, A. J.; Anderson, J. R.;
Alberti, D.; Sizer, K. C.; Nathan, D. G. Effectiveness and Safety of
ICL670 in Iron-Loaded Patients with Thalassaemia: A Randomised,
Double-Blind, Placebo-Controlled, Dose-Escalation Trial. Lancet
2003, 361, 1597-1602.
(30) Galanello, R.; Piga, A.; Alberti, D.; Rouan, M.-C.; Bigler, H.;
Se´chaud, R. Safety, Tolerability, and Pharmacokinetics of ICL670,
a New Orally Active Iron-Chelating Agent in Patients with Transfu-
sion-Dependent Iron Overload Due to â-Thalassemia. J. Clin.
Pharmacol. 2003, 43, 565-572.
(31) Cappellini, M. D. Iron-Chelating Therapy with the New Oral Agent
ICL670 (Exjade). Best Pract. Res. Clin. Haematol. 2005, 18, 289-
298.
Ethyl (S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)-
phenyl]-4-methyl-4-thiazolecarboxylate (16). Iodoethane (0.25
mL, 3.1 mmol) and DIEA (0.55 mL, 3.2 mmol) were successively
added to 11 (0.781 g, 1.95 mmol) in DMF (31 mL), and the solution
was stirred at room temperature for 50 h. After solvent removal
under high vacuum, the residue was treated with 1:1 0.5 M citric
acid/saturated NaCl (85 mL) and was extracted with EtOAc (4 ×
60 mL). The combined extracts were washed with 50-mL portions
of 1% NaHSO3, H2O, and saturated NaCl, and solvent was
evaporated. Purification by flash column chromatography using 1.5:
2.5:6 EtOAc/petroleum ether/CH2Cl2 furnished 0.690 g (83%) of
1
16 as an oil: [R]23 +40.2 (c 1.09); 400 MHz H NMR δ 1.30 (t,
3 H, J ) 7.2) 1.66 (s, 3 H), 3.19 (d, 1 H, J ) 11.2), 3.38 (s, 3 H),
3.54-3.57 (m, 2 H), 3.64-3.70 (m, 4 H), 3.72-3.76 (m, 2 H),
3.81-3.88 (m, 3 H), 4.12-4.17 (m, 2 H), 4.20-4.28 (m, 2 H),
6.46 (dd, 1 H, J ) 8.8, 2.4), 6.49 (d, 1 H, J ) 2.4), 7.28 (d, 1 H,
J ) 8.4), 12.69 (s, 1 H). Anal. (C20H29NO7S) C, H, N.
Acknowledgment. Funding was provided by the National
Institutes of Health Grant No. RO1-DK49108. We thank
Elizabeth M. Nelson and Katie Ratliff-Thompson for their
technical assistance and Carrie A. Blaustein for her editorial
and organizational support.
Supporting Information Available: Elemental analytical data
for synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(1) Ponka, P. Physiology and Pathophysiology of Iron Metabolism:
Implications for Iron Chelation Therapy in Iron Overload. In The
DeVelopment of Iron Chelators for Clinical Use; Bergeron, R. J.,
Brittenham, G. M., Eds.; CRC: Boca Raton, FL, 1994; pp 1-29.
(2) Olivieri, N. F.; Brittenham, G. M. Iron-chelating Therapy and the
Treatment of Thalassemia. Blood 1997, 89, 739-761.
(3) Vichinsky, E. P. Current Issues with Blood Transfusions in Sickle
Cell Disease. Semin. Hematol. 2001, 38, 14-22.
(4) Kersten, M. J.; Lange, R.; Smeets, M. E.; Vreugdenhil, G.;
Roozendaal, K. J.; Lameijer, W.; Goudsmit, R. Long-Term Treatment
of Transfusional Iron Overload with the Oral Iron Chelator Defer-
iprone (L1): A Dutch Multicenter Trial. Ann. Hematol. 1996, 73,
247-252.
(5) Conrad, M. E.; Umbreit, J. N.; Moore, E. G. Iron Absorption and
Transport. Am. J. Med. Sci. 1999, 318, 213-229.
(6) Lieu, P. T.; Heiskala, M.; Peterson, P. A.; Yang, Y. The Roles of
Iron in Health and Disease. Mol. Aspects Med. 2001, 22, 1-87.
(7) Angelucci, E.; Brittenham, G. M.; McLaren, C. E.; Ripalti, M.;
Baronciani, D.; Giardini, C.; Galimberti, M.; Polchi, P.; Lucarelli,
G. Hepatic Iron Concentration and Total Body Iron Stores in
Thalassemia Major. N. Engl. J. Med. 2000, 343, 327-331.
(32) Davis, B. A.; Porter, J. B. Long-Term Outcome of Continuous 24-
Hour Deferoxamine Infusion via Indwelling Intravenous Catheters
in High-Risk â-Thalassemia. Blood 2000, 95, 1229-1236.

Thiazolecarboxylic Acid Polyethers as Fe Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 9 2783
(33) Naegeli, H.-U.; Za¨hner, H. Metabolites of Microorganisms. Part 193.
Ferrithiocin. HelV. Chim. Acta 1980, 63, 1400-1406.
(34) Hahn, F. E.; McMurry, T. J.; Hugi, A.; Raymond, K. N. Coordination
Chemistry of Microbial Iron Transport. 42. Structural and Spectro-
scopic Characterization of Diastereomeric Cr(III) and Co(III) Com-
plexes of Desferriferrithiocin. J. Am. Chem. Soc. 1990, 112, 1854-
1860.
(35) Anderegg, G.; Ra¨ber, M. Metal Complex Formation of a New
Siderophore Desferrithiocin and of Three Related Ligands. J. Chem.
Soc. Chem. Commun. 1990, 1194-1196.
(36) Bergeron, R. J.; Wiegand, J.; Dionis, J. B.; Egli-Karmakka, M.; Frei,
J.; Huxley-Tencer, A.; Peter, H. H. Evaluation of Desferrithiocin and
Its Synthetic Analogues as Orally Effective Iron Chelators. J. Med.
Chem. 1991, 34, 2072-2078.
(37) Bergeron, R. J.; Streiff, R. R.; Wiegand, J.; Vinson, J. R. T.; Luchetta,
G.; Evans, K. M.; Peter, H.; Jenny, H.-B. A Comparative Evaluation
of Iron Clearance Models. Ann. N. Y. Acad. Sci. 1990, 612, 378-
393.
(38) Bergeron, R. J.; Streiff, R. R.; Creary, E. A.; Daniels, R. D., Jr.;
King, W.; Luchetta, G.; Wiegand, J.; Moerker, T.; Peter, H. H. A
Comparative Study of the Iron-Clearing Properties of Desferrithiocin
Analogues with Desferrioxamine B in a Cebus Monkey Model. Blood
1993, 81, 2166-2173.
(39) Bergeron, R. J.; Wiegand, J.; Weimar, W. R.; Vinson, J. R. T.;
Bussenius, J.; Yao, G. W.; McManis, J. S. Desazadesmethyldesfer-
rithiocin Analogues as Orally Effective Iron Chelators. J. Med. Chem.
1999, 42, 95-108.
(40) Bergeron, R. J.; Wiegand, J.; McManis, J. S.; McCosar, B. H.;
Weimar, W. R.; Brittenham, G. M.; Smith, R. E. Effects of C-4
Stereochemistry and C-4′ Hydroxylation on the Iron Clearing
Efficiency and Toxicity of Desferrithiocin Analogues. J. Med. Chem.
1999, 42, 2432-2440.
(41) Bergeron, R. J.; Wiegand, J.; McManis, J. S.; Bussenius, J.; Smith,
R. E.; Weimar, W. R. Methoxylation of Desazadesferrithiocin
Analogues: Enhanced Iron Clearing Efficiency. J. Med. Chem. 2003,
46, 1470-1477.
(42) Bergeron, R. J.; Wiegand, J.; McManis, J. S.; Weimar, W. R.; Park,
J.-H.; Eiler-McManis, E.; Bergeron, J.; Brittenham, G. M. Partition-
Variant Desferrithiocin Analogues: Organ Targeting and Increased
Iron Clearance. J. Med. Chem. 2005, 48, 821-831.
(43) Bergeron, R. J.; Streiff, R. R.; Wiegand, J.; Luchetta, G.; Creary, E.
A.; Peter, H. H. A Comparison of the Iron-Clearing Properties of
1,2-Dimethyl-3-hydroxypyrid-4-one, 1,2-Diethyl-3-hydroxypyrid-4-
one, and Deferoxamine. Blood 1992, 79, 1882-1890.
(44) Wood, J. K.; Milner, P. F.; Pathak, U. N. The Metabolism of Iron-
dextran Given as a Total-dose Infusion to Iron Deficient Jamaican
Subjects. Br. J. Haematol. 1968, 14, 119-129.
(45) Bergeron, R. J.; Wiegand, J.; Brittenham, G. M. HBED: A Potential
Alternative to Deferoxamine for Iron-Chelating Therapy. Blood 1998,
91, 1446-1452.
(46) Bergeron, R. J.; Wiegand, J.; Wollenweber, M.; McManis, J. S.;
Algee, S. E.; Ratliff-Thompson, K. Synthesis and Biological Evalu-
ation of Naphthyldesferrithiocin Iron Chelators. J. Med. Chem. 1996,
39, 1575-1581.
(47) Bergeron, R. J.; Wiegand, J.; Weimar, W. R.; McManis, J. S.; Smith,
R. E.; Abboud, K. A. Iron Chelation Promoted by Desazadesfer-
rithiocin Analogues: An Enantioselective Barrier. Chirality 2003,
15, 593-599.
(48) Bergeron, R. J.; Wiegand, J.; Ratliff-Thompson, K.; Weimar, W. R.
The Origin of the Differences in (R)- and (S)-Desmethyldesferrithio-
cin: Iron-Clearing Properties. Ann. N.Y. Acad. Sci. 1998, 850,
202-216.
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