Substituent effects on desferrithiocin and desferrithiocin analogue iron-clearing and toxicity profiles

Article abstract of DOI:10.1021/jm300509y

Desferrithiocin (DFT, 1) is a very efficient iron chelator when given orally. However, it is severely nephrotoxic. Structure-activity studies with 1 demonstrated that removal of the aromatic nitrogen to provide desazadesferrithiocin (DADFT, 2) and introdu

Full text of DOI:10.1021/jm300509y

Article
pubs.acs.org/jmc
Substituent Effects on Desferrithiocin and Desferrithiocin Analogue
Iron-Clearing and Toxicity Profiles
Raymond J. Bergeron,* Jan Wiegand, Neelam Bharti, and James S. McManis
Department of Medicinal Chemistry, University of Florida, Box 100485 JHMHC, Gainesville, Florida, 32610-0485
S
* Supporting Information
ABSTRACT: Desferrithiocin (DFT, 1) is a very efficient iron chelator when given orally. However, it is severely nephrotoxic.
Structure−activity studies with 1 demonstrated that removal of the aromatic nitrogen to provide desazadesferrithiocin (DADFT,
2) and introduction of either a hydroxyl group or a polyether fragment onto the aromatic ring resulted in orally active iron
chelators that were much less toxic than 1. The purpose of the current study was to determine if a comparable reduction in renal
toxicity could be achieved by performing the same structural manipulations on 1 itself. Accordingly, three DFT analogues were
synthesized. The iron-clearing efficiency and ferrokinetics were evaluated in rats and primates; toxicity assessments were carried
out in rodents. The resulting DFT ligands demonstrated a reduction in toxicity that was equivalent to that of the DADFT
analogues and presented with excellent iron-clearing properties.
example, in the presence of Fe(II), endogenous H2O2 is
reduced to the hydroxyl radical (HO•), a very reactive species,
and HO−, the Fenton reaction. 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 and produce carcinogens.13,15
The liberated Fe(III) is reduced back to Fe(II) via a variety of
biological reductants (e.g., ascorbate, glutathione), a problem-
atic cycle.
Iron-mediated damage can be focal, as in reperfusion
damage,16 Parkinson's,17 Friedreich's ataxia,18 macular degener-
ation,19 and hemorrhagic stroke,20 or global, as in transfusional
iron overload, for example, thalassemia,21 sickle cell disease,21,22
and myelodysplasia,23 with multiple organ involvement. The
INTRODUCTION
■
Nearly all life forms require iron as a micronutrient. However, the
low solubility of Fe(III) hydroxide (Ksp = 1 × 10−39),1 the
predominant form of the metal in the biosphere, required the
development of sophisticated iron storage and transport systems
in nature. Microorganisms utilize low molecular weight, ferric
iron-specific ligands, siderophores;2 eukaryotes tend to employ
proteins to transport and store iron.3−5 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 metal.6 Whether derived from trans-
fused red blood cells7−9 or from increased absorption of dietary
iron,10,11 without effective treatment, body iron progressively
increases with deposition in the liver, heart, pancreas, and
elsewhere (iron overload disease).
In patients with iron overload disease, the toxicity derives
Received: April 10, 2012
Published: August 13, 2012
from iron's interaction with reactive oxygen species.12−14 For
© 2012 American Chemical Society
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solution in both scenarios is the same: chelate and promote the
excretion of excess unmanaged iron.
of (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thiazolecar-
boxylic acid (2, Chart 1) to yield (S)-2-(2,4-dihydroxyphenyl)-
4,5-dihydro-4-methyl-4-thiazolecarboxylic acid (deferitrin, 3,41
Chart 1), or the introduction of polyether fragments42,43 to 2, for
example, (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecy-
loxy)]-4-methyl-4-thiazolecarboxylic acid (4, Chart 1), led to
the discovery of orally active iron chelators that were much less
toxic than 1. This begs the question of what the impact of the
hydroxylation of DFT itself, or fixing a polyether fragment to
DFT, would have on the nephrotoxicity and iron clearance
properties of the new analogues.
Treatment with a chelating agent capable of sequestering iron
and permitting its excretion from the body is the only therapeutic
approach available.24,25 Some of the iron-chelating agents that
are now in use or that have been clinically evaluated include
desferrioxamine B mesylate (DFO),26 1,2-dimethyl-3-hydroxy-
4-pyridinone (deferiprone, L1),27−30 and 4-[3,5-bis(2-hydroxy-
phenyl)-1,2,4-triazol-1-yl]benzoic acid (desferasirox,
ICL670A).31−34 Each of these ligands presents with short-
comings. DFO must be given subcutaneously (sc) for protracted
periods of time, for example, 12 h a day, 5 days a week, a serious
patient compliance issue.7,35,36 Deferiprone, while orally active,
simply does not remove enough iron to maintain patients in a
negative iron balance.27−30 Desferasirox did not show non-
inferiority to DFO and is associated with numerous side effects,
including some serious renal toxicity issues.31−34
RESULTS AND DISCUSSION
■
Design Concept. DFT (1, Table 1) is a natural product iron
chelator isolated from Streptomyces antibioticus.44 It forms a 2:1
complex with Fe(III) with a cumulative formation constant of
4 × 1029 M−1.45,46 Although the compound was shown to be an
excellent deferration agent when administered orally (po) to
rats47 and primates,48,49 it caused severe nephrotoxicity in rats.37
However, the compound's oral activity spurred SAR studies
focused on the DFT platform aimed at identifying an orally active
and safe DFT analogue.38−40
In addition to the above chelators, two desferrithiocin [(S)-
4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecar-
boxylic acid (DFT, 1, Chart 1) analogues have made it to clinical
Chart 1. Extensive Structural Alterations of the Tridentate
Chelator 1 Were Carried Out, Including Simple
Hydroxylation of the Aromatic Ring of 2 To Yield 3, or the
Introduction of Polyether Fragments to 2, For Example, 4 and
(S)-4,5-Dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)]-4-
methyl-4-thiazolecarboxylic Acid (5)
Our approach first entailed simplifying the platform. Removal
of the pyridine nitrogen of 1 provided 2 (Chart 1 and Table 1),
the parent ligand of the desazadesferrithiocin (DADFT) series.40
Interestingly, although 2 was not overtly nephrotoxic, it elicited
serious gastrointestinal (GI) problems.37,38,40 In spite of its GI
toxicity, the ligand's excellent iron-clearing efficiency (ICE) and
the absence of nephrotoxicity prompted further SAR studies
predicated on this pharmacophore. This led to the discovery that
the lipophilicity (partition between octanol and water, expressed
as the log of the fraction in the octanol layer, log Papp)
50 of the
DADFT analogues could have a profound effect on the ligand's
ICE, organ distribution, and toxicity profile.38,51,52
Ultimately, it was determined that hydroxylation of DADFT
and a number of different analogues in the 3′-, 4′-, or 5′-position
allowed for ligands that were very efficient, orally active iron
chelators with profoundly less toxicity than 1 or 2.38,51 Clearly,
hydroxylation had a significant effect on toxicity reduction. One
of these ligands, 3, was taken into human clinical trials by
Genzyme and was moving forward until the once daily dosing
was changed to twice daily.41 Patients presented with increases in
blood urea nitrogen (BUN) and serum creatinine (SCr) levels,
and the trial was terminated.41
The molecule (3) was reengineered, introducing a 3,6,9-
trioxadecyloxy group in the 4′- position of 2 (4, Chart 1).42 This
provided a remarkably efficient orally active iron chelator, which,
given to rats po once or twice daily, was virtually nephrotoxicity-
free.42 This turned out also to be true when a variety of polyether
backbones were fixed at the 3′-, 4′-, or 5′-position of the DADFT
pharmacophore.43,53−55 In fact, (S)-4,5-dihydro-2-[2-hydroxy-3-
(3,6,9-trioxadecyloxy)]-4-methyl-4-thiazolecarboxylic acid (5,
Chart 1) has now been moved forward to clinical trials. Thus,
it appeared as though fixing a polyether fragment to the DADFT
framework was also a uniformly effective tool in further reducing
3-induced nephrotoxicity. These observations drive the current
study.
A series of questions based on the results of the SAR studies of
DADFT analogues are addressed as follows: (1) How does
hydroxylation of 1 itself affect its iron-clearing properties? (2)
How does hydroxylation of 1 affect its renal toxicity? (3) What is
the effect of fixing polyether fragments to 1 on its iron clearance?
(4) What is the effect of fixing polyether fragments to 1 on its
trials. As will be described below, although 1 itself is severely
nephrotoxic,37 extensive structure−activity relationship (SAR)
studies,38−40 including simple hydroxylation of the aromatic ring
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Table 1. Iron-Clearing Efficiency of DFT Analogues Administered to Rodents and Primates with the Respective Log Papp Values
a
In the rodents [n = 3 (7), 4 (2 and 10), 5 (1, 6, 8, and 9), or 8 (3)], the drugs were given po at a dose of 150 (1 and 2) or 300 μmol/kg (3 and 6−
10). The drugs were administered in capsules (7), solubilized in 40% Cremophor RH-40/water (1 and 2), or given as their monosodium salts,
prepared by the addition of 1 equiv of NaOH to a suspension of the free acid in distilled water (3, 6, and 8−10). The efficiency of each compound
was calculated by subtracting the 24 or 48 h iron excretion of control animals from the iron excretion of the treated animals. The 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
b
brackets. The ICE data for 1 is from ref 47, 2 is from ref 38, 3 is from ref 51, 7 is from ref 54, and 9 is from ref 55. ICE is based on a 48 h sample
c
collection period. In the primates [n = 4 (1, 2, 6, and 7 in capsules and 8−10), or 6 (3), or 7 (7 as the monosodium salt)], the chelators were given
po at a dose of 75 (2 and 6−10) or 150 μmol/kg (1 and 3). Ligand 10 was also given to the primates sc at a dose of 75 μmol/kg. The drugs were
administered in capsules (7), solubilized in 40% Cremophor RH-40/water (1 and 2), or given as their monosodium salts, prepared by the
addition of 1 equiv of NaOH to a suspension of the free acid in distilled water (1−3 and 6−10). The efficiency 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 ICE data for 1−3 are from ref 38, 7 is from ref 54, and 9 is from
d
ref 55. The relative percentages of the iron excreted in the feces and urine are in brackets. The performance ratio (PR) is defined as the mean
ICEprimates/ICErodents
.
renal toxicity? To answer these questions, three ligands were
assembled (Table 1), (S)-4,5-dihydro-2-(3,5-dihydroxy-2-pyr-
idinyl)-4-methyl-4-thiazolecarboxylic acid (6), (S)-4,5-dihydro-
2-[3-hydroxy-5-(3,6-dioxaheptyloxy)-2-pyridinyl]-4-methyl-4-
thiazolecarboxylic acid (8), and (S)-4,5-dihydro-2-[3-hydroxy-4-
(3,6-dioxaheptyloxy)-2-pyridinyl]-4-methyl-4-thiazolecarboxylic
acid (10).
Synthesis. The preparation of 5′-hydroxydesferrithiocin (6)
and its 5′-nor polyether (8) (Table 1) began with 2-cyano-3,5-
difluoropyridine (11), which was converted to 2-cyano-3,5-
dihydroxypyridine (13) in two steps (Scheme 1). Heating 11
with 4-methoxybenzyl alcohol in the presence of NaH (2.5 equiv
each) in DMF56,57 at 95 °C for 18 h gave protected diol 12 in
73% yield. Removal of the 4-methoxybenzyl groups of 12 using
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a
Scheme 1. Synthesis of 6 and 8
a
Reagents and conditions: (a) 4-Methoxybenzyl alcohol, 60% NaH (2.5 equiv each), DMF, 95−100 °C, 18 h, 73%. (b) TFA, pentamethylbenzene,
22 h, quantitative. (c) CH3OH, 0.1 M pH 6 buffer, NaHCO3, 73−76 °C, 45 h. (d) EtI, DIEA (1.5 equiv each), DMF, 47 h, 70%. (e) K2CO3
(1.6 equiv), acetone, reflux, 1 d, 65%. (f) 50% NaOH(aq), CH3OH, then HCl, 96% (6), 97% (8).
excess trifluoroacetic acid (TFA)58 and pentamethylbenzene59 at
room temperature for 22 h provided nitrile 13 in quantitative
yield. Cyclocondensation of 13 with (S)-2-methyl cysteine (14)
in aqueous CH3OH buffered at pH 6 at 75 °C for 45 h followed
by esterification of crude acid 6 with iodoethane and N,N-
diisopropylethylamine (DIEA) (1.5 equiv each) in DMF
produced ethyl (S)-4,5-dihydro-2-(3,5-dihydroxy-2-pyridinyl)-
4-methyl-4-thiazolecarboxylate (15) in 70% yield. Hydrolysis of
15 with aqueous NaOH in CH3OH at room temperature
generated 6 as a solid in 96% yield. Also, ester 15 was alkylated at
the less hindered phenol54 in the presence of the pyridine
nitrogen with tosylate 16 and K2CO3 in refluxing acetone,
affording ligand precursor 17 in 65% yield. The carboxylate
was unmasked under alkaline conditions to give 8 in 97% yield as
an oil.
providing aldehyde 21. Specifically, 19 was treated with 3-
chloroperbenzoic acid (m-CPBA) in CH2Cl2, and the resulting
N-oxide was heated at reflux in acetic anhydride. Cleavage of the
acetate ester with base gave the 2-pyridinemethanol 20 in 87%
overall yield. Primary alcohol 20 was further oxidized to aldehyde
21 in 83% yield with sulfur trioxide−pyridine complex and NEt3
in DMSO and CHCl3. The oxime 22, generated in 90% yield
under standard conditions,42 was heated at reflux with acetic
anhydride, furnishing the corresponding nitrile 23 in 94% yield.
Removal of the benzyl-protecting group from 23 by hydro-
genolysis (1 atm, 10% Pd−C, CH3OH) in the presence of the
cyano group and pyridine ring produced 4-(3,6-dioxaheptyloxy)-
3-hydroxy-2-pyridinecarbonitrile (24) in 85% yield. Heating 24
with amino acid 14 in aqueous CH3OH buffered at pH 6
generated 10 in 95% yield.
Synthesis of the 4′-nor polyether DFT analogue 10, an
isomer of iron chelator 8 (Table 1), started with 2-methyl-3-
(benzyloxy)-4-pyridone (18), available in two steps from
maltol60 (Scheme 2). O-Alkylation of 18 with tosylate 16 and
K2CO3 in refluxing acetonitrile61 afforded 2-methyl-3-(benzyl-
oxy)-4-(3,6-dioxaheptyloxy)pyridine (19) in 68% yield. The
methyl group of 19 was oxidized by known methodology,60
Stoichiometry of the Ligand−Fe(III) Complexes. Earlier
studies with 1 by Anderegg and Raber showed the chelator to
̈
form a 2:1 complex with Fe(III).46 The cumulative formation
constant for this complex was determined to be 4 × 1029 M−1.
Hahn et al. were ultimately able to isolate both the Δ and λ 1−
Cr(III) complexes, with chromium serving as a surrogate for
Fe(III).45 As expected, the crystal structures of the complexes
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a
Scheme 2. Synthesis of 10
a
Reagents and conditions: (a) K2CO3 (2 equiv), CH3CN, 68%. (b) m-CPBA, CH2Cl2. (c) Ac2O, reflux. (d) NaOH(aq), EtOH, reflux, 4 h, 87%.
(e) SO3·pyridine, NEt3, DMSO, CHCl3, 16 h, 83%. (f) H2NOH·HCl, NaOAc, CH3OH, reflux, 2 h, 90%. (g) Ac2O, reflux, 94%. (h) H2, 10% Pd−C,
CH3OH, 85%. (i) CH3OH, 0.1 M pH 6 buffer, NaHCO3, 75 °C, 48 h, 95%.
unequivocally demonstrated a 2:1 ligand to metal ratio. In later
studies in our laboratories, Job's plots with 338 (Table 1) and
the corresponding desmethyl analogue40 also showed that
these ligands formed 2:1 complexes with Fe(III). This is of
course in keeping with the fact that the donor groups of
the chelators, the aromatic hydroxyl, the thiazoline nitrogen,
and the carboxylate are the same as in 1 itself. Furthermore,
the cumulative distance (9.77 Å) between the donor groups in
our x-ray crystal structure of free ligand 362 is identical to the
corresponding value in the Cr(III) complex of 1.45 In the
current study, Job's plots were run on 6, 8, and 10 (Figure 1).
In each instance, the ligands formed 2:1 complexes with Fe(III).
Partition Properties. The partition values between octanol
and water (at pH 7.4, Tris buffer) were determined using a “shake
flask” direct method of measuring log Papp values.50 The fraction
of drug in the octanol is then expressed as log Papp. While the
values vary widely (Table 1), one observation stands out: DFT
and its analogues are always more hydrophilic than their DADFT
counterparts, that is, 1 vs 2, 6 vs 3, 8 vs 7, and 10 vs 9. This, of
course, is likely due to the presence of the aromatic nitrogen, a
moderately good hydrogen bond acceptor, on the DFT
analogues. Relative to the differences in lipophilicity between
DFT and DADFT, fixing a polyether backbone to either the DFT
or the DADFT pharmacophore had a much more moderate
effect (Table 1).
Chelator-Induced Iron Clearance in Noniron-Over-
loaded Bile Duct-Cannulated Rodents. A measure of the
amount of iron excretion induced by a chelator is best described
by its ICE. The ICE, expressed as a percent, is calculated as
(ligand-induced iron excretion/theoretical iron excretion) ×
100. To illustrate, the theoretical iron excretion after
administration of 1 mmol of DFO, a hexadentate chelator that
forms a 1:1 complex with Fe(III), is 1 milli-g-atom of iron. Two
millimoles of DFT (1, Table 1), a tridentate chelator that forms a
2:1 complex with Fe(III), is required for the theoretical
expression of 1 milli-g-atom of iron.
The ICE values for compounds 1−3, 7, and 9 (Table 1) are
historical and included for comparative purposes.38,42,47,54,55
The biliary ferrokinetics profiles of ligands 3 and 6−10 are
presented in Figure 2. Each of the rats in these studies was given
a single po dose of the chelator at 300 μmol/kg. Note that the
biliary ferrokinetics data for compounds 1 and 2 are not
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Figure 1. Job's plots of the Fe(III) complexes of ligands 6, 8, and 10. Solutions containing different ligand/Fe(III) ratios were prepared such that
[ligand] + [Fe(III)] = 1.0 mM in Tris-HCl buffer at pH 7.4. The theoretical mole fraction maximum for a 2:1 ligand:Fe complex is 0.667 (arrow). The
observed maxima for 6, 8, and 10 are 0.669, 0.676, and 0.677, respectively. The optical density (y-axis) was determined at 498, 484, and 485 nm for 6, 8,
and 10, respectively.
Figure 2. Biliary ferrokinetics of DFT analogues (6, 8, and 10) and DADFT analogues (3, 7, and 9) in bile duct-cannulated rats. The compounds were
given po at 300 μmol/kg. The iron excretion (y-axis) is reported as μg of iron per kg body weight.
included, simply because these animals were dosed at 150 μmol/kg
and the curves are not strictly comparable with the 300 μmol/kg
data. Although these results are published elsewhere,38,47 we will
comment on this briefly.
DFT (1) given to the rats po at a dose of 150 μmol/kg had an
ICE of 5.5 3.2%.47 Maximum iron clearance (MIC) occurred at
3 h, but deferration had returned to baseline levels by 12 h. The
desaza analogue of DFT, 2, at 150 μmol/kg had an ICE of 2.7
0.5%.38 The ligand reached MIC at 6 h and had returned to
baseline iron excretion by 12 h postdrug.
Compound 3 was the least effective ligand, with an ICE of
1.1 0.8%.42 It presented with an MIC at 3 h; deferration was
virtually over at 9 h (Figure 2). The DFT analogue of 3, ligand 6,
had an ICE that was significantly better than 3, 9.0 3.8% (p <
0.005). MIC occurred at 6 h, and its iron decorporation slowly
dropped to near baseline levels by 24 h. The most efficient
chelator, 7, had an ICE of 26.7 4.7%.54 The ligand also had a
very protracted iron clearance; even though its MIC occurred
at around 12 h, it was still active at 48 h. Note that although
the biliary ferrokinetics curve of 7 may appear to be biphasic
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(Figure 2), the reason for this unusual line shape is that several
animals had temporarily obstructed bile flow. While the con-
centration of iron in the bile remained the same, the bile volume,
and thus overall iron excretion, decreased. Once the obstruction
was resolved, bile volume and overall iron excretion normalized.
The DFT analogue 8 had an ICE that was significantly less
than that of 7 (11.7 1.2 vs 26.7 4.7% for 8 and 7, respectively,
p < 0.02). Ligand 8 also achieved MIC earlier than 7, 6 vs 12 h
(Figure 2), and the iron clearance induced by 8 was basically over
by 21 h. In addition, DADFT analogue 955 and its corresponding
DFT analogue 10 presented with similar ICEs (∼15%) but with
very different biliary ferrokinetics (Figure 2). Both ligands
achieved MIC at 6 h. However, while ligand 9-induced iron
clearance had returned to baseline by 24 h, 10 was still quite
active.
achieved by the corresponding DADFT ligand 7. Likewise, the
ICE of 10 given po was also less than that of DADFT analogue 9
(Table 1). In fact, the DADFT analogues were consistently
better deferrating agents in the primates than the corresponding
DFT ligands (Table 1).
Several generalizations can be derived from Table 1. The
performance ratios, PR values, ICEprimate/ICErodent (Table 1),
show that the ligands are either as effective or better at iron
clearance in primates than in rodents. The exception to this is
ligand 10. The ICE of this drug given po to the primates is 6.1
1.8%, while in the rats, it is 14.2 2.4%. Its PR ratio was 0.4,
showing it to be far less efficient in primates than rodents. The
poor iron clearance in primates relative to rodents was surprising.
Two scenarios were evaluated in search of an explanation:
ligand−plasma binding and a potential GI absorption problem.
A ligand−plasma binding experiment was performed in which
rodent and primate plasma were incubated separately with
chelator 10 at 37 °C for 4 h. Each sample was then passed
through a Millipore Amicon Ultra regenerated cellulose filter
(3000 MWCO). The filtrate was assayed for 10. The results
indicated there was little, if any, binding of the ligand to either the
rodent or the primate plasma. This suggests that ligand−plasma
binding does not explain the difference in the rodent vs primate
ICE values. However, when primates were given ligand 10 sc, the
ICE rose to 16.9 7.3%, which is similar to what was seen in
rodents given 10 orally (Table 1). This observation is consistent
with the idea that the primates do not absorb 10 well when the
drug is administered orally.
Finally, there is an excellent correlation between ICE and
log Papp in rodents among the DFT analogues 6, 8, and 10
(Figure 3). The most lipophilic ligands are also the most
Effect of Hydroxylation or Introduction of a Polyether
Fragment on DFT Toxicity. In a previous study, 1 was given
to rats with normal iron stores po once daily at a dose of
384 μmol/kg/day (100 mg/kg/day). All of the rats were dead by
day 5 of a planned 10 day experiment. The chelator was found to be
severely nephrotoxic.37 The pathologist noted vacuolar changes
of the proximal tubules that were diffuse and severe, with
multifocal vaculolar degeneration and necrosis. Nevertheless, the
drug's remarkable oral activity initiated a series of SAR studies
aimed at the development of orally active, nontoxic DFT
analogues. This led to the development of 3 (Table 1), which
made it to clinical trials.41 This chelator, when given once daily,
cleared iron from the patients and was proceeding forward.
Unfortunately, it was discovered that the drug induced proximal
tubule nephrotoxicity when it was administered twice daily, and
the trial was halted.41 The chelator was reengineered, and it was
determined that by fixing polyether fragments to the 3′- or
4′-position of 2, for example, 5 and 4 (Chart 1), 7 (Table 1),
the renal toxicity virtually disappeared.42,53−55 This outcome
suggested that the introduction of either a hydroxyl group or
a polyether fragment directly into DFT itself might reduce
the drug's nephrotoxicity. Accordingly, ligands 6, 8, and 10
were synthesized and evaluated for their toxicity in rodents
relative to 1.
Assessment of chelator-induced impaired renal function
has traditionally relied on the detection of a rise in BUN
and/or SCr. However, because of the functional reserve of
the kidney, these parameters are often unreliable indica-
tors of acute kidney injury; the ultimate answer requires
histopathology. The Critical Path Institute's Preventive
Safety Testing Consortium (PSTC) has identified kidney
injury molecule-1 (Kim-1, rat) or (KIM-1, human) as an early
diagnostic biomarker for monitoring acute kidney tubular
toxicity.63 Kim-1 is a type 1 transmembrane protein located
in the epithelial cells of proximal tubules.64,65 After injury, for
Figure 3. ICE, expressed as a percentage (y-axis), vs log Papp for DFT
analogues 6, 8, and 10 in bile duct-cannulated rats given po at a dose of
300 μmol/kg.
active. We have observed this ICE vs log Papp pattern time and
again.38,51,52
Chelator-Induced Iron Clearance in Iron-Overloaded
Primates. The primate iron clearance data are provided in Table 1.
The ICE values for compounds 1, 2, 3, 7, and 9 are again
historical and included for comparative purposes.38,54,55 The
chelators were given to the primates po at a dose of 75 (2 and 6−
10) or 150 μmol/kg (1 and 3); 10 was also given to the primates
sc at a dose of 75 μmol/kg. Ligand 1 was found to have an ICE of
16.1
8.5%.38 Removal of the pyridine nitrogen to yield 2
increased the ICE to 21.5 12%.38 However, the increase in ICE
was not significant (p > 0.05). The introduction of a hydroxyl
group at the 4′-position of 2 to provide analogue 3 resulted in a
chelator with an ICE of 16.8 7.2%,38 which is within error of
the ICE found for 1 and 2 (p > 0.05). The reintroduction of the
pyridine nitrogen into DADFT ligand 3 to provide DFT
analogue 6 (Table 1) decreased the ICE to 10.0
2.9%,
significantly less than its DADFT counterpart, 3 (p < 0.05).
When a polyether fragment was attached to the 5′-position of 6
to yield 8, the ICE increased to 18.0 5.2%, again less than that
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Figure 4. Urinary Kim-1 excretion (y-axis) is expressed as Kim-1 (ng/kg/24 h) of rats treated with DFT (1), DFT analogues 6, 8, and 10 or DADFT
analogues 3 and 7. The rodents were given the drugs po twice daily (b.i.d.) at a dose of 237 μmol/kg/dose (474 μmol/kg/day) for up to 7 days. Note that
none of the rats survived the planned 7 day exposure to 1. N = 5 for 1, 6−8, and 10; N = 3 for ligand 3.
example, exposure to a nephrotoxic agent or ischemia, the
ectodomain of Kim-1 is shed from the proximal tubular
kidney epithelial cells into the urine.66−68 BioAssay Works
has recently developed RenaStick, a direct lateral flow
immunochromatographic assay, which allows for the rapid
detection (less than 30 min) and quantitation of urinary
Kim-1 (rat) or KIM-1 (human) excretion.69 In the current
study, rats were treated with 1, 6, 8, and 10 given po twice
daily at a dose of 237 μmol/kg/dose (474 μmol/kg/day) for
up to 7 days. Urinary Kim-1 levels were assessed at 24 h
intervals (Figure 4). The studies were performed on rats
with normal iron stores; each animal served as its own
control. The data for ligands 3 and 7 are historical and are
included for comparative purposes.55
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of all of the 8- or 10-treated rats were well within the normal
range. Thus, as with the DADFT pharmacophore, fixing a
polyether fragment to the DFT framework was an effective tool
in further reducing nephrotoxicity.
None of the 1-treated rats (n = 5) survived the planned 7 day
exposure to the drug. Two rats became moribund and were
sacrificed after being given the drug for 4 days. The three
remaining animals were found dead the morning of day 6; they
had received the chelator for 5 days. None of the rodents
produced any urine on day 5. The 1-treated rats' baseline (day 0)
urinary Kim-1 value was <20 ng/kg/24 h (Figure 4). After
1 day of 1, the Kim-1 had increased nearly 10-fold, to 192
316 ng/kg/24 h. After 3 days of 1, the Kim-1 had further increased
CONCLUSION
■
We previously demonstrated that the severe nephrotoxicity
associated with 1 could be ameliorated by the removal of the
pyridine nitrogen of 1 to provide 2 and simple hydroxylation of
the aromatic ring of 2 to yield 3 (Chart 1).38,51 Further reduction
in 3-induced nephrotoxicity, observed when the chelator was
given po at 237 μmol/kg twice daily, was accomplished by the
addition of polyether fragments, for example, 4, 5, and 7 (Chart 1
and Table 1).42,53−55 The purpose of the current study was to
determine how these same structural modifications to DFT itself
would impact the new ligands' ICE and nephrotoxicity.
Accordingly, three DFT analogues, 6, 8, and 10, were synthesized
and assessed for their lipophilicity, ICE properties in rats and
primates, and their toxicity in rats.
to 1528
539 ng/kg/24 h. Blood was taken from the two
moribund animals immediately prior to sacrifice; the serum
was assessed for its BUN and SCr content. The rats' BUN was
139 8 mg/dL (normal 9−30 mg/dL),70 while their SCr was
5.1 0.3 mg/dL (normal 0.4−1 mg/dL).70 In addition, as no
blood was obtained from the three animals that were found dead,
these values likely underestimate the actual impact of 1 on these
parameters.
In contrast, in a previous study assessing the impact of 3 po on
urinary Kim-1 excretion,55 all of the treated rats (n = 3) survived
the 237 μmol/kg twice daily (474 μmol/kg/day) × 7 day dosing
period. The rats' baseline (day 0) urinary Kim-1 content was
<20 ng/kg/24 h (Figure 4). After 3 days of exposure to 3, the
urinary Kim-1 had increased to 69 47 ng/kg/24 h. At the end
of the 7 day dosing period, the urinary Kim-1 had further
increased to 189 187 ng/kg/24 h (Figure 4). The rats were
euthanized on day 8; their BUN at that time was 32 13 mg/dL,
while their SCr was 1.3 1.0 mg/dL.
In the current study, all of the animals treated po with the
corresponding hydroxylated DFT analogue, 6, at 237 μmol/kg
twice daily (474 μmol/kg/day) also survived the full 7 days of
treatment. The rats' baseline (day 0) urinary Kim-1 content was
<20 ng/kg/24 h and remained <50 ng/kg/24 h until day 5
(Figure 4). On day 6, the Kim-1 increased to 125 48 ng/kg/24 h
and further increased to 435 269 on day 7 (Figure 4). Although
the increase in Kim-1 is greater with the 6-treated rats than
with the 3-treated animals, the increase is not statistically
significant (p > 0.05). The animals were euthanized on day 8;
their BUN at that time was 13 2 mg/dL, while their SCr was
0.5 0.1 mg/dL. Thus, simple hydroxylation of the aromatic ring
of both DADFT and DFT, for example, 3 and 6, respectively,
resulted in ligands that were much less toxic than DFT itself.
We previously demonstrated that introducing a polyether
fragment in the 3′- or 4′-position of the DADFT pharmacophore
provided remarkably efficient orally active iron chelators that
were much less toxic than 3.42,43,53−55 For example, the impact of
ligand 7 on urinary Kim-1 excretion was determined when the
drug was given po: (1) once daily during the course of 28 day
toxicity trials, (2) once daily at a dose of 384 μmol/kg/day ×
10 days, and (3) twice daily at a dose of 237 μmol/kg/dose
(474 μmol/kg/day) × 7 days.55 All of the rats survived the dosing
period. The rats' baseline (day 0) urinary Kim-1 content was
<20 ng/kg/24 h and stayed within error of this value for the
duration of the drug exposure. The data from the 237 μmol/kg
twice daily (474 μmol/kg/day) × 7 days regimen55 are depicted
in Figure 4. In addition, the BUN and SCr of all of the 7-treated
rats were well within the normal range.
DFT (1, Table 1) and its analogues were all significantly more
water-soluble (lower log Papp) than the corresponding DADFT
analogues, for example, 1 vs 2, 6 vs 3, 8 vs 7, and 10 vs 9. There
was an excellent correlation between ICE and log Papp in rodents
among the DFT analogues 6, 8, and 10 (Figure 3), with the more
lipophilic ligands having a greater ICE. This trend is in keeping
with previous observations that more lipophilic ligands have
better ICE properties. The biliary ferrokinetics of the DFT and
DADFT ligands in the bile duct-cannulated rats (Figure 2) have
similar temporal properties, except for ligand 7. This drug has the
highest ICE and a very protracted iron clearance time.
In the primates, the DADFT analogues were consistently
better deferration agents than the corresponding DFT ligands
(Table 1). The most unusual finding was with ligand 10, a DFT
analogue with a 4′-(3,6-dioxaheptyloxy) ether functionality fixed
to the 4′-position of 1. When the drug was given po to the
primates, its ICE was only 6.1 1.8 vs 14.2 2.4% in the rats and
a PR value of 0.4 (Table 1). However, when the monkeys were
given the chelator sc, its ICE increased to 16.9 7.3%, with a PR
value now at 1.2. This is consistent with the idea that ligand 10
simply was not absorbed well orally in primates.
The effects of structural modification of DFT on its renal
toxicity were assessed in rats using a urinary Kim-1 (kidney injury
molecule) assay,69 as well as monitoring BUN and SCr. The most
notable finding was that fixing a hydroxyl group or a polyether
fragment to the DFT aromatic ring resulted in a nearly identical
reduction in renal toxicity as seen after the same modification to
DADFT (Figure 4). Although some nephrotoxicity was noted
with both hydroxylated DADFT and DFT analogues, 3 and 6,
respectively, the introduction of polyether groups into either
pharmacophore resulted in ligands with little to no impact on
renal function, for example, 7, 8, and 10 (Figure 4).
In summary, rather simple manipulation of the DFT aromatic
ring, for example, hydroxylation, or the introduction of a
polyether functionality, can have a marked effect on the ligand's
ICE and renal toxicity (Table 1 and Figure 4). Although the
resulting DFT chelators were generally as effective in the rodents
as their DADFT counterparts, they were less active in the
primates. However, the tissue distribution of 6, 8, and 10 in
rodents remains to be elucidated. Higher levels of these
analogues in the critical target organs, that is, the liver, heart,
and pancreas, could easily compensate for their somewhat lower
ICE values. Nevertheless, at least one of the DFT polyethers (8)was
sufficiently effective at iron clearance in rodents (ICE 11.7 1.2%)
In the current study, we evaluated the impact that affixing a
polyether fragment to DFT itself would have on nephrotoxicity.
Accordingly, groups of rats (n = 5) were given 8 or 10 po twice
daily at 237 μmol/kg/dose (474 μmol/kg/day) × 7 days. All of
the animals survived the drug-dosing regimen. The animals'
urinary Kim-1 excretion remained within error of that of the
baseline (day 0) levels (Figure 4). In addition, the BUN and SCr
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1
furnished 1.49 g of 8 (97%) as a yellow oil: [α] +25.3° (c 0.88). H
and primates (ICE 18.0 5.2%) and had an acceptable toxicity
NMR: δ 1.73 (s, 3 H), 3.22 (d, 1 H, J = 12.0), 3.41 (s, 3 H), 3.59−3.61
(m, 2 H), 3.72−3.74 (m, 2 H), 3.83 (d, 1 H, J = 11.6), 3.88 (t, 2 H, J =
4.8), 4.19 (t, 2 H, J = 4.4), 6.84 (d, 1 H, J = 2.4), 7.94 (d, 1 H, J = 2.4). 13C
NMR: δ 24.62, 39.13, 58.98, 67.99, 69.28, 70.63, 71.81, 82.83, 107.67,
126.98, 131.64, 158.15, 158.44, 174.59, 175.94. HRMS m/z calcd for
C15H21N2O6S, 357.1115 (M + H); found, 357.1125. Anal.
(C15H20N2O6S) C, H, N.
profile to merit further studies.
EXPERIMENTAL SECTION
■
Materials. Reagents were purchased from Aldrich Chemical Co.
(Milwaukee, WI). Compound 11 was obtained from Matrix Scientific
(Columbia, SC). Fisher Optima grade solvents were routinely used.
Reactions were run under a nitrogen atmosphere, and organic extracts
were dried with sodium sulfate. Silica gel 40−63 from SiliCycle, Inc.
(Quebec City, Quebec, Canada) was used for column chromatography.
Melting points are uncorrected. Glassware that was presoaked in 3 N
HCl for 15 min, washed with distilled water and distilled EtOH, and
oven-dried, was used during the isolation of 6, 8, and 10. Optical
rotations were run at 589 nm (sodium D line) and 20 °C on a Perkin-
Elmer 341 polarimeter, with c being concentration in grams of
(S)-4,5-Dihydro-2-[3-hydroxy-4-(3,6-dioxaheptyloxy)-2-pyridin-
yl]-4-methyl-4-thiazolecarboxylic Acid (10). Compound 14 (0.78 g,
4.58 mmol), pH 6 phosphate buffer (30 mL), and NaHCO3 (0.44 g,
5.23 mmol) were successively added to a solution of 24 (0.78 g, 3.27 mmol)
in degassed CH3OH (30 mL). The reaction mixture was heated at 75 °C
for 48 h with stirring, cooled, and concentrated by rotary evaporation.
The residue was dissolved in distilled H2O (25 mL), and the aqueous
layer was acidified with cold 2 N HCl to pH <2 followed by extraction
with EtOAc (5 × 50 mL). Concentration in vacuo resulted in 1.15 g
of 10 (95%) as a light yellow oil: [α] +52.8° (c 0.40). 1H NMR: δ 1.73 (s,
3 H), 3.24 (d, 1 H, J = 11.6), 3.39 (s, 3 H), 3.56−3.58 (m, 2 H), 3.73−
3.75 (m, 2 H), 3.85 (d, 1 H, J = 11.6), 3.94 (t, 2 H, J = 4.8), 4.27 (t, 2 H,
J = 4.8), 6.88 (d, 1 H, J = 5.2), 8.08 (d, 1 H, J = 4.8). 13C NMR: δ 24.65,
39.52, 59.08, 68.57, 69.31, 70.87, 71.94, 83.67, 109.81, 133.20, 141.56,
147.16, 154.39, 175.11, 176.26. HRMS m/z calcd for C15H21N2O6S,
357.1115 (M + H); found, 357.1115. Anal. (C15H20N2O6S) C, H, N.
3,5-Bis(4-methoxybenzyloxy)pyridine-2-carbonitrile (12). Sodium
hydride (60%, 3.66 g, 91.5 mmol) was added to 4-methoxybenzyl
alcohol (11.5 mL, 92.6 mmol) in DMF (89 mL). The reaction mixture
was stirred for 50 min and was cooled in an ice water bath, followed by
addition of 11 (5.13 g, 36.6 mmol). After it was stirred at rt for 30 min
and heated at 95−100 °C for 18 h, the reaction was quenched at 0 °C
with EtOH and was concentrated by rotary evaporation under high
vacuum. The residue was treated with H2O (250 mL) and extracted with
warm EtOAc (400 mL, 2 × 100 mL). The organic extracts were washed
with saturated NaCl (150 mL). Purification by flash column
chromatography using 2% acetone/CH2Cl2 gave 10.11 g of 12 (73%)
as a white solid; mp 122−122.5 °C. 1H NMR: δ 3.82 (s, 3 H), 3.83 (s, 3
H), 5.04 (s, 2 H), 5.11 (s, 2 H), 6.85 (d, 1 H, J = 2.0), 6.92 (dd, 4 H, J =
8.6, 6.6), 7.31 (t, 4 H, J = 8.6), 8.01 (d, 1 H, J = 2.0). 13C NMR: δ 55.45,
55.47, 70.98, 71.00, 106.86, 114.41, 115.63, 116.26, 126.83, 126.94,
129.04, 129.56, 131.98, 158.41, 159.10, 160.00, 160.14. HRMS m/z
calcd for C22H21N2O4, 377.1496 (M + H); found, 377.1500. Anal.
(C22H20N2O4) C, H, N.
1
compound per 100 mL of solvent (CHCl3, not indicated). H NMR
spectra were run in CDCl3 (not indicated) at 400 MHz, and chemical
shifts (δ) are given in parts per million downfield from tetramethylsilane.
13C NMR spectra were measured at 100 MHz, and chemical shifts (δ)
are referenced to the residual solvent resonance of δ 77.16 for CDCl3
(not indicated) or δ 39.52 for DMSO-d6. Coupling constants (J) are in
hertz. ESI-FTICR mass spectra are reported. The iron content of the
Fe(III)−NTA solution was verified using a Perkin-Elmer 5100 PC
Atomic Absorption Spectrophotometer (AAS). Data for the Job's plots
were recorded on a UV-2550 UV−vis spectrophotometer.
Elemental analyses were performed by Atlantic Microlabs (Norcross,
GA) and were within 0.4% of the calculated values. Purity of the
compounds is supported by high-pressure liquid chromatography
(HPLC) (≥95% for 6, 8, and 10) and by elemental analyses.
Male Sprague−Dawley rats were procured from Harlan Sprague−
Dawley (Indianapolis, IN). Male Cebus apella monkeys (3.5−4 kg) were
obtained from World Wide Primates (Miami, FL). Ultrapure salts were
obtained from Johnson Matthey Electronics (Royston, United
Kingdom). All hematological and biochemical studies were performed
by Antech Diagnostics (Tampa, FL). Atomic absorption (AA) measure-
ments were made on a Perkin-Elmer model 5100 PC (Norwalk, CT). A
R-Rena-strip Lateral-flow Kit for the detection of Kim-1 in rat urine was
obtained from BioAssay Works (Ijamsville, MD). A Chromatoreader
ReaScan (Otsuka Electronics Co., Japan) was utilized to read the test
strips and to allow for the quantitation of Kim-1 in rat urine.
Synthetic Methods. (S)-4,5-Dihydro-2-(3,5-dihydroxy-2-pyridin-
yl)-4-methyl-4-thiazolecarboxylic Acid (6). A solution of 50% (w/w)
NaOH (13.7 g, 0.171 mol) in CH3OH (135 mL) was added to 15
(4.85 g, 17.2 mmol) in CH3OH (125 mL) over 13 min at 0 °C. The reaction
mixture was warmed to rt over 19 h, and the bulk of the solvent was
removed by rotary evaporation. The concentrate was treated with 3 M
aqueous NaCl (150 mL) and was extracted with Et2O (2 × 100 mL).
The aqueous layer was cooled in ice, acidified with cold 6 N HCl
(30 mL), and extracted with EtOAc (250 mL, 2 × 100 mL). The EtOAc
extracts were washed with saturated NaCl (80 mL). Solvent was
removed in vacuo, providing 4.18 g of 6 (96%) as an off white solid; mp
226−227 °C (dec): [α] +46.0° (c 0.82, DMF). 1H NMR (DMSO-d6): δ
1.58 (s, 3 H), 3.27 (d, 1 H, J = 11.3), 3.69 (d, 1 H, J = 11.7), 6.72 (d, 1 H,
J = 2.4), 7.80 (d, 1 H, J = 2.0), 10.82 (s, 1 H), 12.32 (s, 1 H), 13.20 (s, 1
H). 13C NMR (DMSO-d6): δ 24.32, 38.48, 82.98, 108.59, 125.88,
131.13, 156.68, 157.58, 173.30, 173.74. HRMS m/z calcd for
C10H11N2O4S, 255.0434 (M + H), 277.0253 (M + Na), 299.0073
(M − H + 2Na), 320.9892 (M − 2H + 3Na); found, 255.0439,
277.0255, 299.0077, 320.9899. Anal. (C10H10N2O4S) C, H, N.
(S)-4,5-Dihydro-2-[3-hydroxy-5-(3,6-dioxaheptyloxy)-2-pyridin-
yl]-4-methyl-4-thiazolecarboxylic Acid (8). A solution of 50% (w/w)
NaOH (1.46 mL, 47.0 mmol) in CH3OH (40 mL) was added dropwise
to a solution of 17 (1.66 g, 4.32 mmol) in CH3OH (20 mL) at 0 °C. The
reaction mixture was stirred at rt for 6 h, and the bulk of the solvent was
removed under reduced pressure. The residue was treated with 3 M
aqueous NaCl (50 mL) and was extracted with Et2O (2 × 30 mL). The
aqueous layer was cooled in ice, acidified with 2 N HCl to pH 2, and
extracted with EtOAc (5 × 40 mL). Combined EtOAc layers were
washed with saturated NaCl (60 mL). Solvent removal in vacuo
3,5-Dihydroxy-2-pyridinecarbonitrile (13). TFA (477 g) was added
over 26 min to 12 (9.63 g, 25.6 mmol) and pentamethylbenzene (38.2 g,
0.258 mol) with ice bath cooling. The reaction mixture was stirred at rt
for 22 h, and volatiles were removed by rotary evaporation. The residue
was partitioned between cold 2 N NaOH (180 mL) and Et2O (350 mL)
and separated. The Et2O layer was back extracted with 0.5 N NaOH
(80 mL). The combined aqueous phase was extracted with Et2O (100 mL),
cooled in an ice water bath, and combined with cold 2 M HCl (220 mL)
and saturated NaCl (100 mL). The aqueous layer was extracted with
EtOAc (250 mL, 2 × 120 mL). The latter organic extracts were washed
with saturated NaCl (150 mL) and concentrated in vacuo, giving 3.70 g
of 13 (quantitative) as a light tan solid. 1H NMR (DMSO-d6): δ 6.80 (d,
1 H, J = 2.4), 7.74 (d, 1 H, J = 2.0), 10.98 (s, 1 H), 11.39 (s, 1 H). 13C
NMR (DMSO-d6): δ 108.69, 111.17, 116.74, 132.54, 158.10, 159.21.
HRMS m/z calcd for C6H3N2O2, 135.0200 (M − H); found, 135.0196.
An analytical sample was recrystallized from aqueous EtOH. At > 300 °C,
the sample was dark but not melted. Anal. (C6H4N2O2) C, H, N.
Ethyl (S)-4,5-Dihydro-2-(3,5-dihydroxy-2-pyridinyl)-4-methyl-4-
thiazolecarboxylate (15). A degassed solution of 0.1 M phosphate
buffer (pH 6, 310 mL) and CH3OH (300 mL) was added to 13 (4.04 g,
29.7 mmol) and 14 (6.95 g, 40.5 mmol). The pH of the reaction solution
was adjusted to 6.0 with NaHCO3 (4.92 g, 58.6 mmol). The reaction
mixture was heated at 73−76 °C for 45 h with stirring, cooled to 0 °C,
and reduced in volume by rotary evaporation. The residue was acidified
to pH ∼ 1 with cold 2 N HCl (61 mL) followed by extraction with
EtOAc (300 mL, 2 × 100 mL). The organic layer was washed with
saturated NaCl (100 mL), concentrated in vacuo, and dried with
toluene, resulting in 6.30 g of 6. Iodoethane (3.0 mL, 37.5 mmol) and
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DIEA (6.5 mL, 37.3 mmol) were successively added to 6 in DMF
(130 mL), and the solution was stirred at rt for 47 h. After solvent removal
under high vacuum, the residue was treated with 12:5 0.5 M HCl/
saturated NaCl (170 mL) followed by extraction with EtOAc (150 mL,
4 × 70 mL). The EtOAc layers were washed with 100 mL portions of 1%
NaHSO3 and saturated NaCl, and the solvent was evaporated.
Purification by column chromatography using (5% acetone/CH2Cl2)
gave 5.88 g of 15 (70%) as a pale yellow solid; mp 85−87.5 °C, [α]
+35.6° (c 0.74). 1H NMR: δ 1.32 (t, 3 H, J = 7.2), 1.69 (s, 3 H), 3.20 (d, 1
H, J = 11.7), 3.79 (d, 1 H, J = 11.7), 4.27 (q, 2 H, J = 7.2), 6.77 (d, 1 H, J =
2.4), 7.82 (d, 1 H, J = 2.3). 13C NMR: δ 14.23, 24.78, 39.60, 62.33, 83.67,
110.11, 127.71, 130.80, 156.17, 157.79, 173.21, 174.02. HRMS m/z
calcd for C12H15N2O4S, 283.0747 (M + H), 305.0567 (M + Na); found,
283.0751, 305.0573. Anal. (C12H14N2O4S) C, H, N.
Ethyl (S)-4,5-Dihydro-2-[3-hydroxy-5-(3,6-dioxaheptyloxy)-2-pyr-
idinyl]-4-methyl-4-thiazolecarboxylate (17). Flame-activated K2CO3
(0.72 g, 5.21 mmol) was added to a mixture of 16 (0.96 g, 3.2 mmol) and
15 (0.90 g, 3.19 mmol) in dry acetone (25 mL). The reaction mixture
was heated at reflux for 24 h. After it was cooled to rt, the solvent was
removed by rotary evaporation. The residue was treated with 1:9 0.2 N
HCl/saturated NaCl (50 mL) and was extracted with EtOAc (4 ×
30 mL). The organic extracts were washed with saturated NaCl
(50 mL), and solvent was removed in vacuo. Column chromatography
using 1:2:7 CH3OH/hexane/CH2Cl2 furnished 0.80 g of 17 (65%) as a
viscous oil; [α] +30.9° (c 1.12). 1H NMR: δ 1.30 (t, 3 H, J = 7.0), 1.67 (s,
3 H), 3.19 (d, 1 H, J = 11.3), 3.40 (s, 3 H), 3.56−3.62 (m, 2 H), 3.70−
3.75 (m, 2 H), 3.80 (d, 1 H, J = 11.7), 3.86−3.93 (m, 2 H), 4.19 (t, 2 H,
J = 4.7), 4.25 (q, 2 H, J = 7.0), 6.80 (d, 1 H, J = 2.3), 7.95 (d, 1 H, J = 2.3),
12.37 (s, 1 H). 13C NMR: δ 14.23, 24.77, 39.45, 59.25, 62.04, 68.11,
69.46, 70.99, 72.01, 83.84, 107.63, 127.72, 131.49, 157.39, 158.22,
172.87, 173.96. HRMS m/z calcd for C17H25N2O6S, 385.1428 (M + H),
407.1247 (M + Na); found, 385.1432, 407.1266. Anal. (C17H24N2O6S)
C, H, N.
20 as a light brown oil. 1H NMR: δ 3.34 (s, 3 H), 3.52−3.54 (m, 2 H),
3.69−3.71 (m, 2 H), 3.92 (t, 2 H, J = 5.2), 4.28 (t, 2 H, J = 4.4), 4.65 (s, 2
H), 5.09 (s, 2 H), 6.83 (d, 1 H, J = 5.2) 7.32−7.39 (m, 3 H), 7.40−7.44
(m, 2 H), 8.19 (d, 1 H, J = 5.6). 13C NMR: δ 59.20, 60.23, 68.13, 69.44,
70.96, 72.04, 74.79, 107.83, 128.49, 128.54, 128.63, 137.15, 140.53,
144.66, 152.98, 157.52. HRMS m/z calcd for C18H24NO5, 334.1649
(M + H), 356.1468 (M + Na); found, 334.1648, 356.1455. Anal.
(C18H23NO5) C, H, N.
4-(3,6-Dioxaheptyloxy)-3-(benzyloxy)pyridine-2-carboxaldehyde
(21). Triethylamine (70 mL, 0.29 mol) followed by DMSO (70 mL) was
added to 20 (16.5 g, 49.0 mmol) in CHCl3 (100 mL). Sulfur trioxide−
pyridine complex (35 g, 0.22 mol) was slowly added over 35 min to the
reaction mixture with ice bath cooling. After it was warmed to rt, the
reaction mixture was stirred overnight and was diluted with CHCl3
(200 mL). The organic phase was washed with H2O (3 × 200 mL) and
saturated NaCl (100 mL). After the solvent was removed in vacuo,
column chromatography using 5:5:1 EtOAc/CHCl3/CH3OH furnished
13.61 g of 21 (83%) as a viscous colorless oil. 1H NMR: δ 3.34 (s, 3 H),
3.52−3.54 (m, 2 H), 3.70−3.72 (m, 2 H), 3.95 (t, 2 H, J = 4.4), 4.30 (t, 2
H, J = 4.4), 5.24 (s, 2 H), 7.02 (d, 1 H, J = 5.2), 7.32−7.39 (m, 3 H),
7.41−7.46 (m, 2 H), 8.39 (d, 1 H, J = 5.6), 10.25 (s, 1 H). 13C NMR: δ
59.17, 68.54, 69.23, 70.94, 71.97, 76.24, 111.69, 128.68, 128.75, 128.87,
136.16, 145.87, 146.90, 148.14, 159.49, 189.87. HRMS m/z calcd for
C18H21NNaO5, 354.1312 (M + Na); found, 354.1326. Anal.
(C18H21NO5) C, H, N.
4-(3,6-Dioxaheptyloxy)-3-(benzyloxy)pyridine-2-carboxaldehyde
Oxime (22). Hydroxylamine hydrochloride (4.2 g, 60.0 mmol) and
NaOAc (5.2 g, 60.0 mmol) were added to a solution of 21 (13.5 g,
40.7 mmol) in CH3OH (50 mL), and the reaction mixture was heated at
reflux for 2 h. The reaction mixture was concentrated by rotary
evaporation, and the residue was treated with saturated NaCl (100 mL)
and 0.1 M aqueous citric acid (100 mL) and then was extracted with
EtOAc (2 × 100 mL). The organic layers were washed with H2O
(100 mL) and saturated NaCl (100 mL). The solvent was removed in vacuo,
providing 12.7 g (90%) of 22 as a pale solid; mp 72−73 °C. 1H NMR: δ
3.34 (s, 3 H), 3.46−3.51 (m, 2 H), 3.64−3.71 (m, 2 H), 3.92 (t, 2 H, J =
4.4), 4.27 (t, 2 H, J = 4.4), 5.10 (s, 2 H), 6.85 (d, 1 H, J = 5.6), 7.29−7.46
(m, 5 H), 8.28 (d, 1 H, J = 5.2), 8.46 (s, 1 H). 13C NMR: δ 59.14, 68.12,
69.28, 70.86, 71.94, 75.67, 108.62, 128.42, 128.55, 128.59, 136.70,
143.43, 144.81, 145.23, 146.71, 158.66. HRMS m/z calcd for
C18H21N2O5, 345.1450 (M − H); found, 345.1426. Anal.
(C18H22NO5) C, H, N.
2-Methyl-3-(benzyloxy)-4-(3,6-dioxaheptyloxy)pyridine (19).
Flame-activated K2CO3 (27.6 g, 0.20 mol) and 16 (27.4 g, 0.10 mol)
were added to 18 (21.5 g, 0.10 mol) in dry CH3CN (500 mL). The
reaction mixture was heated at reflux for 24 h. After it was cooled to rt,
the solvent was evaporated by rotary evaporation. The residue was
treated with 1.7 M aqueous NaCl (200 mL) and was extracted with
CH2Cl2 (4 × 150 mL). The organic extracts were washed with saturated
NaCl (300 mL). After the solvent was removed in vacuo, column
chromatography using 4:4:2 EtOAc/petroleum ether/acetone fur-
1
nished 21.5 g of 19 (68%) as a colorless viscous oil. H NMR: δ 2.42
4-(3,6-Dioxaheptyloxy)-3-(benzyloxy)pyridine-2-carbonitrile (23).
Compound 22 (12.15 g, 36.71 mmol) was dissolved in Ac2O (40 mL)
and heated at reflux for 8 h under a Drierite tube. The reaction mixture
was concentrated by rotary evaporation and was dissolved in 8%
aqueous NaHCO3 (100 mL) and extracted with CHCl3 (100 mL, 2 ×
50 mL). Combined organic fractions were washed with 4% NaHCO3
(50 mL) and saturated NaCl (100 mL) followed by solvent removal in
vacuo. Purification by flash chromatography eluting with 10% CH3OH/
(s, 3 H), 3.34 (s, 3 H), 3.51−3.53 (m, 2 H), 3.69−3.71 (m, 2 H), 3.91 (t,
2 H, J = 4.8), 4.24 (t, 2 H, J = 4.4), 5.02 (s, 2 H), 6.72 (d, 1 H, J = 5.6),
7.31−7.40 (m, 3 H), 7.44−7.49 (m, 2 H), 8.12 (d, 1 H, J = 5.6). 13C
NMR: δ 19.34, 59.16, 67.86, 69.45, 70.92, 71.99, 74.57, 106.68, 128.21,
128.45, 128.49, 137.53, 142.32, 145.41, 153.40, 157.64. HRMS m/z
calcd for C18H24NO4, 318.1700 (M + H); found, 318.1714. Anal.
(C18H23NO4·0.2H2O) C, H, N.
4-(3,6-Dioxaheptyloxy)-3-(benzyloxy)-2-pyridinemethanol (20).
An ice-cooled solution of m-CPBA (3.67 g, 36.0 mmol) in CH2Cl2
(75 mL) was added slowly to 19 (10.4 g, 32.8 mmol) in CH2Cl2 (50 mL)
over 15 min at 0 °C. The reaction mixture was warmed to rt, stirred for
6 h, and diluted with CH2Cl2 (150 mL). The reaction mixture was washed
with 5% Na2CO3 (3 × 100 mL) and saturated NaCl (100 mL) and was
concentrated under reduced pressure to give a colorless oil. Acetic
anhydride (80 mL, 0.85 mol) was added, and the reaction mixture was
heated at 130 °C for 2 h. The solvent was removed under reduced
pressure, and the residue was dissolved in H2O (100 mL). The pH of the
aqueous solution was adjusted to 8 with 2 N NaOH, and the aqueous
solution was extracted with CH2Cl2 (3 × 100 mL). The organic fractions
were combined and washed with saturated NaCl (100 mL) and
concentrated in vacuo. The residue was dissolved in CH3OH, treated
with decolorizing charcoal, filtered, and concentrated to yield a brown
oil, which was dissolved in EtOH (40 mL). Aqueous 1 M NaOH
(80 mL) was added, and the reaction mixture was refluxed for 4 h and
cooled. Extraction with CH2Cl2 (4 × 100 mL), washing with saturated
NaCl (100 mL), concentration under reduced pressure, and column
chromatography using 10% CH3OH/CHCl3 provided 9.52 g (87%) of
1
CH2Cl2 gave 11.31 g (94%) of 23 as a pale solid; mp 34−35 °C. H
NMR: δ 3.34 (s, 3 H), 3.53−3.55 (m, 2 H), 3.70−3.72 (m, 2 H), 3.93 (t,
2 H, J = 4.8), 4.27 (t, 2 H, J = 4.4), 5.31 (s, 2 H), 6.98 (d, 1 H, J = 5.6),
7.31−7.38 (m, 3 H), 7.49−7.52 (m, 2 H), 8.21 (d, 1 H, J = 5.2). 13C
NMR: δ 59.14, 68.59, 69.09, 70.92, 71.94, 75.84, 111.27, 115.43, 128.60,
128.66, 128.73, 128.84, 135.78, 147.21, 148.28, 158.42. HRMS m/z
calcd for C18H20N2NaO4, 351.1315 (M + Na); found, 351.1325. Anal.
(C18H20N2O4) C, H, N.
4-(3,6-Dioxaheptyloxy)-3-hydroxy-2-pyridinecarbonitrile (24).
Palladium on carbon (10%, 0.065 g) was added to a solution of 23
(1.30 g, 3.95 mmol) in CH3OH (15 mL), and the mixture was stirred
under H2 at 1 atm for 2 h. The reaction mixture was filtered through
Celite, and the solids were washed with CH3OH (3 × 5 mL). The filtrate
was concentrated under reduced pressure, and the residue was subjected
to column chromatography eluting with 10% CH3OH/EtOAc,
furnishing 0.80 g (85%) of 24 as a colorless oil. 1H NMR: δ 3.42 (s, 3
H), 3.61−3.63 (m, 2 H), 3.75−3.77 (m, 2 H), 3.92 (t, 2 H, J = 4.8), 4.24
(t, 2 H, J = 4.4), 6.91 (d, 1 H, J = 4.8), 8.10 (d, 1 H, J = 5.6). 13C NMR: δ
58.92, 68.66, 69.05, 70.52, 71.78, 110.68, 115.28, 120.29, 143.49, 148.84,
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Article
153.72. HRMS m/z calcd for C11H14N2NaO4, 261.0846 (M + Na);
found, 261.0849. Anal. (C11H14N2O4) C, H, N.
Collection of Urine for Kim-1 Studies. The rats were housed in
individual metabolic cages. Urine samples were collected from the
metabolic cages at 24 h intervals. A baseline (day 0) urine sample was
collected and assessed for its Kim-1 content; each animal served as its
own control. The urine was collected chilled as previously described.55
Performance of Urinary Kim-1 Studies. The chilled urine was
collected, vortexed, and warmed to room temperature; any sediment in
the samples was allowed to settle. The Kim-1 content was assessed using
a Rat Kim-1 Rapid Test Kit according to the manufacturer's instructions.
The result was read using a ReaScan Test Reader. The quantity of Kim-1
excreted in the urine per day was calculated by multiplying the
concentration of Kim-1 (ng/mL urine) × 24 h urine volume, divided by
the weight of the animal. The result is expressed as urinary Kim-1
(ng/kg/24 h). 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.
Job's Plots for 6, 8, and 10. The stoichiometries of the ligand−
Fe(III) complexes of 6, 8, and 10 were determined spectrophotometri-
cally using Job's plots. Solutions were monitored at the visible λmax of the
Fe(III) complexes (498 nm for 6, 484 nm for 8, and 485 nm for 10). A
100 mM Tris HCl buffer was used to maintain the pH at 7.4. Solutions
containing different ligand/Fe(III) ratios were prepared by mixing
appropriate volumes of 1.0 mM ligand solution and 1.0 mM Fe(III)−
nitriloacetate (NTA) in Tris-HCl buffer. The 1.0 mM Fe(III)−NTA
solution was prepared immediately prior to use by dilution of a 41.6 mM
Fe(III)−NTA stock solution with the Tris HCl buffer, whereas the
ligand's stock solution was prepared by dissolving the ligand as its
monosodium salt in Tris HCl buffer at pH 7.4. The Fe(III)−NTA stock
solution was prepared by mixing equal volumes of 90 mM of FeCl3 and
180 mM trisodium NTA. The iron content of the Fe(III)−NTA
solution was verified by AAS.
Biological Methods. All animal experimental treatment protocols
were reviewed and approved by the University of Florida's Institutional
Animal Care and Use Committee.
Cannulation of Bile Duct in Noniron-Overloaded Rats. The
cannulation has been described previously.37,48 Bile samples were
collected from male Sprague−Dawley rats (400−450 g) at 3 h intervals
for up to 48 h. The urine sample(s) was taken at 24 h intervals. Sample
collection and handling are as previously described.37,48
ASSOCIATED CONTENT
■
S
* Supporting Information
Elemental analytical data for synthesized compounds. This
material is available free of charge via the Internet at http://pubs.
acs.org.
Iron Loading of C. apella Monkeys. The monkeys were iron
overloaded with intravenous iron dextran as specified in earlier
publications48,71 to provide about 500 mg of iron per kg of body
weight; the serum transferrin iron saturation rose to between 70 and
80%. At least 20 half-lives, 60 days,72 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.37,48,73
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 concentrations were determined by flame absorption spectroscopy
as presented in other publications.48,74
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 352-273-7725. Fax: 352-392-8406. E-mail: rayb@ufl.edu.
Notes
The authors declare the following competing financial interest(s):
Patents filed.
ACKNOWLEDGMENTS
■
The project described was supported by grant number
R37DK049108 from the National Institutes of Health. The
content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institute of
Diabetes and Digestive and Kidney Diseases or the National
Institutes of Health. We thank Elizabeth M. Nelson and Katie
Ratliff-Thompson for their technical assistance and Miranda E.
Coger for her editorial and organizational support. We
acknowledge the spectroscopy services in the Chemistry
Department, University of Florida, for the mass spectrometry
analyses.
Drug Preparation and Administration: Iron Clearance. In the
iron-clearing experiments, the rats were given 6, 8, and 10 po at a dose of
300 μmol/kg. The primates were given 6, 8, and 10 po at a dose of
75 μmol/kg; ligand 10 was also given sc at a dose of 75 μmol/kg. The drugs
were administered to the rats and primates as their monosodium salts
(prepared by the addition of 1 equiv of NaOH to a suspension of the free
acid in distilled water). Drug preparation for the rodent urinary Kim-1
excretion studies involving 1, 6, 8, and 10 are described below.
Calculation of Iron Chelator Efficiency. In the text below, the
term “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 administration of 1 mmol of DFO, a
hexadentate chelator that forms a 1:1 complex with Fe(III), is 1 milli-g-
atom of iron. Two millimoles of DFT (1, Table 1), a tridentate iron
chelator that forms a 2:1 complex with Fe(III), is required for the
theoretical excretion of 1 milli-g-atom of iron. The theoretical iron
outputs of the chelators were generated on the basis of a 2:1 ligand:iron
complex. The efficiencies in the rats and monkeys were calculated as set
forth elsewhere.38,73 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.
Drug Preparation and Administration: Rodent Toxicity/
Urinary Kim-1 Excretion Studies. The impact of ligands 1, 6, 8,
and 10 on urinary Kim-1 excretion was evaluated in rodents. The
chelators were administered to the rats po as their monosodium salts,
prepared as described above, twice daily at a dose of 237 μmol/kg/dose
(474 μmol/kg/day) for up to 7 days. The studies were performed on rats
with normal iron stores. The rats were fasted overnight and were given
the first dose of the chelator first thing in the morning. The rats were fed
∼3 h postdrug and had access to food for ∼5 h before being fasted
overnight.
ABBREVIATIONS USED
■
DFO, desferrioxamine B mesylate; DFT, desferrithiocin, (S)-4,5-
dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarbox-
ylic acid; ICE, iron-clearing efficiency; DADFT, desazadesferri-
thiocin; SAR, structure−activity relationship; BUN, blood urea
nitrogen; SCr, serum creatinine; PR, performance ratio; Kim-1,
kidney injury molecule-1; MIC, maximum iron clearance; b.i.d.,
twice daily
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