The design, synthesis, and evaluation of organ-specific iron chelators

Article abstract of DOI:10.1021/jm0608816

A series of iron chelators, three (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4- methyl-4-thiazolecarboxylic acid (DADFT) and three (S)-4,5-dihydro-2-(2- hydroxyphenyl)-4-thiazolecarboxylic acid (DADMDFT) analogues are synthesized and assessed for their lipophilicity (log P_app), iron-clearing efficiency (ICE) in rodents and iron-loaded primates (Cebus apella), toxicity in rodents, and organ distribution in rodents. The results lead to a number of generalizations useful in chelator design strategies. In rodents, while log P_app is a good predictor of a chelator's ICE, chelator liver concentration is a better tool. In primates, log P_app is a good predictor of ICE, but only when comparing structurally very similar chelators. There is a profound difference in toxicity between the DADMDFT and DADFT series: DADMDFTs are less toxic. Within the DADFT family of ligands, the more lipophilic ligands are generally more toxic. Lipophilicity can have a profound effect on ligand organ distribution, and ligands can thus be targeted to organs compromised in iron overload disease, for example, the heart.

Full text of DOI:10.1021/jm0608816

7032
J. Med. Chem. 2006, 49, 7032-7043
The Design, Synthesis, and Evaluation of Organ-Specific Iron Chelators
Raymond J. Bergeron,* Jan Wiegand, James S. McManis, and Neelam Bharti
Department of Medicinal Chemistry, UniVersity of Florida, GainesVille, Florida 32610-0485
ReceiVed July 26, 2006
A series of iron chelators, three (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid
(DADFT) and three (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid (DADMDFT) analogues
are synthesized and assessed for their lipophilicity (log Papp), iron-clearing efficiency (ICE) in rodents and
iron-loaded primates (Cebus apella), toxicity in rodents, and organ distribution in rodents. The results lead
to a number of generalizations useful in chelator design strategies. In rodents, while log Papp is a good
predictor of a chelator’s ICE, chelator liver concentration is a better tool. In primates, log Papp is a good
predictor of ICE, but only when comparing structurally very similar chelators. There is a profound difference
in toxicity between the DADMDFT and DADFT series: DADMDFTs are less toxic. Within the DADFT
family of ligands, the more lipophilic ligands are generally more toxic. Lipophilicity can have a profound
effect on ligand organ distribution, and ligands can thus be targeted to organs compromised in iron overload
disease, for example, the heart.
Introduction
that these differ in the liver, physiologically a major systemic
iron depot, and in the pancreas and heart, which normally do
not serve as iron storage sites. Nontransferrin-bound plasma iron
(NTBI), a heterogeneous pool of iron in the circulation that is
not bound to the physiological iron transporter, transferrin, seems
to be a principal source of the abnormal tissue distribution of
iron that develops with chronic iron overload. NTBI is rapidly
taken up by hepatocytes in the liver, the major iron storage
organ, perhaps through the divalent metal transporter 125 and
other pathways that remain poorly characterized. NTBI also
seems to gain entry into cardiomyocytes, pancreatic â-cells, and
anterior pituitary cells specifically via L-type voltage-dependent
Ca2+ channels not found in hepatocytes or Kupffer cells.26 These
differences in modes of iron uptake also may underlie clinical
observations of differences in the course and pace of iron
deposition and damage to the liver, pancreas, and heart in
patients with all forms of iron overload.6
Iron is essential to redox processes in all prokaryotes and
eukaryotes. Although this element comprises 5% of the earth’s
crust, living systems have great difficulty in accessing and
managing this vital micronutrient. The low solubility of Fe(III)
hydroxide (Ksp ) 1 × 10-39),1 the predominant form of the
metal in the biosphere, has led to the development of sophis-
ticated iron storage and transport systems in nature. Microorgan-
isms utilize low molecular weight, virtually ferric iron-specific
ligands, siderophores;2 higher eukaryotes tend to employ
proteins to transport and store iron (e.g., transferrin and ferritin,
respectively).3-5
Higher life forms, for example, humans, have developed 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.6 Whether a systemic
or a focal problem, the toxicity associated with excess iron
derives from its interaction with reactive oxygen species, for
instance, endogenous hydrogen peroxide (H2O2).7-10 In the
presence of Fe(II), H2O2 is reduced to the hydroxyl radical (HO•)
and HO-, a process known as 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, as well as produce
carcinogens.8,11,12 The Fe(III) liberated in the peroxide reduction
can be converted back to Fe(II) via a variety of biological
reductants (e.g., ascorbate), a problematic cycle.
Whether derived from transfused red blood cells13-15 or from
increased absorption of dietary iron,16,17 without effective treat-
ment, body iron progressively increases with deposition in the
liver, heart, pancreas, and elsewhere. Iron accumulation eventu-
ally produces liver disease that may progress to cirrhosis,18-20
diabetes related both to iron-induced decreases in pancreatic
â-cell secretion and to increases in hepatic insulin resistance,21,22
and heart disease, still the leading cause of death in thalassemia
major and related forms of transfusional iron overload.6,23,24 The
precise pathways of iron deposition and its regulation are
incompletely understood, but the available evidence suggests
In the majority of patients with thalassemia major or other
transfusion-dependent refractory anemias, the severity of the
anemia precludes phlebotomy therapy as a means of removing
toxic accumulations of iron. Treatment with a chelating agent
capable of sequestering iron and permitting its excretion from
a Abbreviations: DADFT, desazadesferrithiocin [(S)-4,5-dihydro-2-(2-
hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid]; DADMDFT, desaza-
desmethyldesferrithiocin [(S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazole-
carboxylic acid]; ICE, iron-clearing efficiency; log Papp, octanol-water
partition coefficient (lipophilicity); DFO, desferrioxamine B mesylate; DFT,
desferrithiocin [(S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thia-
zolecarboxylic acid]; NTBI, nontransferrin-bound iron; (S)-4′-(HO)-DADFT,
1, (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic
acid; (S)-4′-(CH3O)-DADFT, 2, (S)-4,5-dihydro-2-(2-hydroxy-4-methoxy-
phenyl)-4-methyl-4-thiazolecarboxylic acid; (S)-5′-(HO)-DADFT, 3, (S)-
2-(2,5-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid;
(S)-5′-(CH3O)-DADFT, 4, (S)-4,5-dihydro-2-(2-hydroxy-5-methoxyphenyl)-
4-methyl-4-thiazolecarboxylic acid; (S)-3′,4′-(CH3O)2-DADFT, 5, (S)-4,5-
dihydro-2-(3,4-dimethoxy-2-hydroxyphenyl)-4-methyl-4-thiazolecarboxyl-
ic acid; (S)-4′-(HO)-DADFT-PE, 6, (S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-
trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylic acid; (S)-4′-(HO)-
DADMDFT, 7, (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-thiazolecarboxylic
acid; (S)-4′-(CH3O)-DADMDFT, 8, (S)-4,5-dihydro-2-(2-hydroxy-4-meth-
oxyphenyl)-4-thiazolecarboxylic acid; (S)-5′-(HO)-DADMDFT, 9, (S)-2-
(2,5-dihydroxyphenyl)-4,5-dihydro-4-thiazolecarboxylic acid; (S)-5′-(CH3O)-
DADMDFT, 10, (S)-4,5-dihydro-2-(2-hydroxy-5-methoxyphenyl)-4-thiazole-
carboxylic acid; (S)-3′,4′-(CH3O)2-DADMDFT, 11, (S)-4,5-dihydro-2-(3,4-
dimethoxy-2-hydroxyphenyl)-4-thiazolecarboxylic acid.
* To whom correspondence should be addressed. Phone: (352) 846-
1956. Fax: (352) 392-8406. E-mail: rayb@ufl.edu.
10.1021/jm0608816 CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/07/2006

Organ-Specific Iron Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7033
Table 1. Iron-Clearing Activity of Desferrithiocin Analogues When Administered Orally to Rodents and the Partition Coefficients of the Compounds
a In the rodents [n ) 3 (9), 4 (2, 3, 4, 7, 8, 11), 5 (5, 6, 10), or 8 (1)], the dose was 150 µmol/kg for 9 and 300 µmol/kg for 1-8, 10, and 11. The
compounds were solubilized in either distilled water (6) or 40% Cremophor (7 and 9) or were given as their monosodium salts, prepared by the addition of
1 equiv of NaOH to a suspension of the free acid in distilled water (1-5, 8, 10, and 11). 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 Data are expressed as the log of the fraction in
the octanol layer (log Papp); measurements were done in TRIS buffer, pH 7.4, using a “shake flask” direct method. The values obtained for compounds 1,
2, 7, and 8 are from ref 38; the value for 6 is from ref 39. c Data are from ref 39. d Data are from ref 52. e ICE is based on a 48 h sample collection.
the body is then the only therapeutic approach available. The
iron-chelating agents now in use or under clinical evaluation are
desferrioxamine B mesylate (DFOa), 1,2-dimethyl-3-hydroxy-
pyridin-4-one (deferiprone, L1), 4-[3,5-bis(2-hydroxyphenyl)-
1,2,4-triazol-1-yl]benzoic acid (deferasirox, ICL670A), and our
own desferrithiocin (DFT) analogue, (S)-2-(2,4-dihydroxyphe-
nyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [deferitrin,
(S)-4′-(HO)-DADFT, 1, Table 1]. Each of these agents chelates
iron derived predominantly from systemic storage sites, from
iron released by macrophages after catabolism of senescent red
blood cells, from iron in hepatic pools, or from both.27
DFO are then required for periods of years to reverse iron-
induced heart disease completely.28 The efficacy of deferiprone
in patients with heart disease remains uncertain, and deferasirox
likely does not enter the heart in appreciable amounts. In any
event, almost daily administration of near maximal tolerated
doses of the first three agents is required to keep pace with
rates of transfusional iron loading in patients with thalassemia
major and other refractory anemias29 and thereby minimize the
amounts of circulating NTBI. Consequently, there is a pressing
need for the continued development of new iron-chelating agents
that are more efficient and that can selectively remove iron from
organs and tissues especially vulnerable to iron-induced toxicity.
Ligands that are more efficient would be able to more rapidly
reduce dangerous body iron burdens to safer levels and forestall
the development or progression of complications. Iron-chelating
In patients, treatment with DFO can sometimes improve
cardiomyopathy within days, before the total iron burden could
have changed appreciably.28 Nonetheless, the amounts of iron
removed from the heart are small; daily 24-hour infusions of

7034 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Bergeron et al.
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. This is
especially true with respect to heart disease, still the cause of
death in 70% or more of patients with thalassemia major;13,23,24
the development of such agents could be life-saving.
profile. Again, iron-mediated cardiac damage is the main cause
of mortality in iron overload disease.
Two ligand families are assessed, the (S)-4,5-dihydro-2-(2-
hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acids (DADFTs,
1-6) and the (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazole-
carboxylic acids (DADMDFTs, 7-11; Table 1). The new
ligands in the DADFT series include (S)-2-(2,5-dihydroxyphe-
nyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-5′-
(HO)-DADFT, 3], (S)-4,5-dihydro-2-(2-hydroxy-5-methoxyphe-
nyl)-4-methyl-4-thiazolecarboxylic acid [(S)-5′-(CH3O)-DADFT,
4], and (S)-4,5-dihydro-2-(3,4-dimethoxy-2-hydroxyphenyl)-4-
methyl-4-thiazolecarboxylic acid [(S)-3′,4′-(CH3O)2-DADFT, 5].
The new ligands in the DADMDFT series include (S)-2-(2,5-
dihydroxyphenyl)-4,5-dihydro-4-thiazolecarboxylic acid [(S)-
5′-(HO)-DADMDFT, 9], (S)-4,5-dihydro-2-(2-hydroxy-5-meth-
oxyphenyl)-4-thiazolecarboxylic acid [(S)-5′-(CH3O)-DADMDFT,
10], and (S)-4-5-dihydro-2-(3,4-dimethoxy-2-hydroxyphenyl)-
4-thiazolecarboxylic acid [(S)-3′,4′-(CH3O)2-DADMDFT, 11].
The DADMDFT chelators previously studied in this laboratory
consist of (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-thiazole-
carboxylic acid [(S)-4′-(HO)-DADMDFT, 7]36 and (S)-4,5-
dihydro-2-(2-hydroxy-4-methoxyphenyl)-4-thiazolecarboxylic acid
[(S)-4′-(CH3O)-DADMDFT, 8].38
Results and Discussion
Design Concept. DFT is a tridentate siderophore30 that forms
a stable 2:1 complex with Fe(III)31,32 and clears the metal well
when the ligand is given orally (po) in both the bile duct-
cannulated rodent model (iron-clearing efficiency, ICE, 5.5%)33
and in the iron-overloaded Cebus apella primate (ICE, 16%).34,35
However, DFT is severely nephrotoxic.35 Nevertheless, its oral
activity spurred a structure-activity study focused on identifying
an orally active and safe DFT analogue.
The structure-activity study first entailed simplifying the
platform. For example, removal of the pyridine nitrogen of DFT
led to (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thiazole-
carboxylic acid [(S)-DADFT], still a very effective iron chelator,
although the ligand now presented with gastrointestinal toxicity
rather than nephrotoxicity.36 It was next demonstrated that
hydroxylation of this and related desaza ligands was compatible
with iron clearance in the primate model and provided much
less toxic chelators.36-38 As previously mentioned, one of these
ligands, (S)-4′-(HO)-DADFT (1; Table 1), is now in clinical
trials for treatment of iron overload. However, animal studies
indicate that the dose-limiting toxicity of this chelator will still
likely be nephrotoxicity.37 Although the origin of the nephro-
toxicity is unclear, the target seems to be centered largely in
the proximal tubules. Recently, we were able to show that
conversion of 1 to the corresponding polyether (S)-4,5-dihydro-
2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thia-
zolecarboxylic acid [(S)-4′-(HO)-DADFT-PE, 6, Table 1]
ameliorated much of the problem simply by reducing the level
of drug in the kidney. The polyether had a substantially better
ICE in both the rat and the primate than the parent drug (1)
and greatly reduced nephrotoxicity.39
In the course of our structure-activity studies focused on
developing an acceptable toxicity profile, the significance of
lipophilicity, log Papp, in chelator design became apparent. The
ligands’ log Papp often correlated with iron-clearing efficiency
(Tables 1 and 2), toxicity (Table 3), and organ distribution
(Figures 1 and 2). Within a series, or family, of ligands, the
more lipophilic compounds generally have better iron-clearing
efficiency, that is, the larger the log Papp value of the compound,
the greater the iron-clearing efficiency. As will be seen below,
the impact of lipophilicity on chelator toxicity can be very
significant, although it is very much dependent on the family
of ligands. Finally, data consistently shows lipophilicity to have
a profound effect on organ distribution of the chelator (Figures
1 and 2); the more lipophilic ligands typically achieve higher
concentrations of drug and/or remain longer in organs. For
example, (S)-4,5-dihydro-2-(2-hydroxy-4-methoxyphenyl)-4-
methyl-4-thiazolecarboxylic acid [(S)-4′-(CH3O)-DADFT, 2]
achieves between two and three times the concentration of
chelator in the heart and pancreas than does administration of
the parent (S)-4′-(HO)-DADFT (1; Figure 1). If it were not for
the poor toxicity profile of 2,39 it might be of great value for
removing cardiac iron.
Synthetic Methods. Cyclocondensation of the appropriately
substituted 2-hydroxybenzonitrile (12-14) with (S)-R-methyl
cysteine (15) in aqueous CH3OH buffered at pH 6 furnished
DADFTs 3-5; use of (S)-cysteine (16) instead gave the
corresponding DADMDFTs 9-11 (Scheme 1). Treatment of
2,5-dimethoxybenzonitrile (17) with boron tribromide in CH2-
Cl2 produced 2,5-dihydroxybenzonitrile (12) in 80% yield
(Scheme 2). Upon heating nitrile 12 with amino acid 15 or 16
near neutrality, (S)-5′-(HO)-DADFT (3; 97% yield) or (S)-5′-
(HO)-DADMDFT (9; 75% yield), respectively, was obtained.
2-Hydroxy-5-methoxybenzonitrile (13),40 when heated with
amino acid 15, led to (S)-5′-(CH3O)-DADFT (4) in 99% yield.
Condensation of nitrile 13 with D-cysteine (16) gave (S)-5′-
(CH3O)-DADMDFT (10) in 77% yield (Scheme 1). 3,4-
Dimethoxy-2-hydroxybenzaldehyde (18)41,42 was converted to
its oxime 19 in 84% yield using hydroxylamine hydrochloride
and NaOAc in CH3OH. Dehydration of 19 with refluxing acetic
anhydride and saponification of the aryl acetate intermediate
gave 3,4-dimethoxy-2-hydroxybenzonitrile (14) in 82% yield
(Scheme 3). Cyclization of amino acid 15 or 16 with aryl
cyanide 14 at pH 6 produced (S)-3′,4′-(CH3O)2-DADFT (5; 60%
yield) or (S)-3′,4′-(CH3O)2-DADMDFT (11; 76% yield), respec-
tively (Scheme 1).
Chelator-Induced Iron Clearance in Non-Iron-Overloaded
Rodents. Iron-clearing efficiencies of the compounds in rats
are shown (Table 1), along with the relative fractions of the
iron excreted in the bile and in the urine. A single 150 µmol/kg
(9) or 300 µmol/kg (1-8, 10, 11) dose of drug was administered
orally by gavage to male Sprague-Dawley rats. The data will
be discussed within the sets of ligands, that is, DADFT chelators
[1, 2], [3, 4], [5], [6] and DADMDFT ligands [7, 8], [9, 10],
[11]. Recall that iron clearing efficiency (ICE) is defined as
(net iron excretion)/(total iron-binding capacity of the chelator),
expressed as a percent. The previous studies with ligands [1,
2] and [7, 8] clearly indicated that the more lipophilic chelators
had greater ICE values.43 In the current study with [3, 4] and
[9, 10], again the more lipophilic ligand was the more efficient
(4 > 3, p < 0.001 and 10 > 9, p < 0.003). While the polyether
(6) follows the expected trend, the dimethoxy systems 5 and
11 were not well behaved within the DADFT and DADMDFT
families; their ICEs were higher than anticipated. However, this
The current study further defines the role of ligand lipo-
philicity in iron-clearing efficiency, toxicity, and organ distribu-
tion with the intent of identifying ligands that achieve high and
prolonged cardiac levels and present with an acceptable toxicity

Organ-Specific Iron Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7035
Table 2. Iron-Clearing Activity of Desferrithiocin Analogues When Administered Orally to Cebus apella Primates and the Partition Coefficients of the
Compounds
a In the monkeys [n ) 4 (3, 4, 6-9), 3 (10), 6 (1, 11), or 7 (2)], the dose was 150 µmol/kg. The compounds were solubilized in either distilled water (6)
or 40% Cremophor (2, 7, 8) or were 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, 4, 9-11). The efficiency of each compound was calculated by averaging the iron output for 4 days before the administration of the drug,
subtracting these numbers from the 2-day iron clearance after the administration of the drug, and then dividing by the theoretical output; the result is
expressed as a percent. The relative percentages of the iron excreted in the stool and urine are in brackets. b Data are expressed as the log of the fraction in
the octanol layer (log Papp); measurements were done in TRIS buffer, pH 7.4, using a “shake flask” direct method. The values obtained for compounds 1,
2, 7, and 8 are from ref 38; the value for 6 is from ref 39. c Data are from ref 37. d Data are from ref 36. e Data are from ref 38. f Not tested in the primates
due to acute toxicity in mice (data not shown). g Data are from ref 39.
apparent discrepancy may be explained by liver chelator concen-
trations (Figures 1 and 2). This is consistent with the idea that
for the lipophilicity/ICE relationship to hold, the extent of
aromatic ring functionalization must remain similar. Addition-
ally, there was no clear trend between log Papp and modes of
iron excretion, for example, urinary versus biliary. Finally, it is
noteworthy that moving 4′-hydroxyl of 7 to the 5′-position
providing ligand 9 had a significant effect on both decreasing
lipophilicity and reducing the ICE, 3% to <0.05%, p < 0.05.
This was not the case with ligands 1 and 3 of the DADFT series
(Table 1). Although the log Papp of these ligands changed by
a similar magnitude as 7 to 9, there was little effect on ICE
(p > 0.05).
Relationship between Rodent ICE and Liver Chelator
Concentration. In rodents, while the ICE of the monosubstituted
DADFT and DADMDFT analogues correlated well with the
lipophilicity of the ligands, this was not the case with the
disubstituted chelators 5 and 11; their ICE was much higher
than expected. However, an examination of the liver concentra-
tion of the DADFT ligands 0.5 h postdrug versus the ICE of
the drugs (Figure 3) reveals that as the liver tissue concentration
of the chelators increases, the ICE also increases (r2 ) 0.965).
This scenario was also seen with the DADMDFT analogues
(r2 ) 0.953, data not shown). Note that although the chelators
were given subcutaneously (sc) in the tissue distribution studies
and po in the ICE experiments, we have previously demon-

7036 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Bergeron et al.
Table 3. Partition Coefficients and Rodent Tolerability of DADFT and DADMDFT Analogues
a The compounds were given to the rats orally by gavage at a dose of 384 µmol/kg/d for a maximum of 10 days. b Toxicity data are from ref 37.
c Toxicity data are from ref 39. d Toxicity data are from ref 43.
strated, for example, (S)-4′-(CH3O)-DADFT (2), that the ligands
have a very high oral bioavailability and that there was little
difference in the tissue distribution of po versus sc administered
chelators.43 Thus, liver chelator concentration may be a more
efficiency (Table 2). This polyether behaves more like its
(S)-4′-(CH3O)-DADFT analogue (2) than would be predicted
on the basis of lipophilicity. While we were unwilling to evaluate
the iron clearance properties of dimethoxy DADFT analogue 5
in primates because of its unexpected acute toxicity in mice
(not described), the dimethoxy DADMDFT analogue 11 was
more efficient at iron clearance than would be predicted from
its log Papp value.
Again, there was no clear relationship between modes of
excretion, biliary versus urinary, and log Papp. Once again, it is
noteworthy that a simple shift of the 4′-(HO) to the 5′ position
decreased the ICE from 4 to 2% for the [7, 9] set (p < 0.02)
and from 17 to 13% for the [1, 3] set, although the decrease
was not significant (Table 2).
Relationship between Toxicity and Lipophilicity. The
toxicity trials were carried out in male Sprague-Dawley rats.
The animals were given the chelators orally by gavage at a dose
of 384 µmol/kg/d for a maximum of 10 days. The dose, which
is equivalent to 100 mg/kg of the DFT monosodium salt, was
chosen to compare toxicity with previous data.37,39,43 There are
predictive tool of ICE in rodents than log Papp
.
Chelator-Induced Iron Clearance in Iron-Overloaded
Primates. The drugs were given po to the monkeys at a dose
of 150 µmol/kg (Table 2). In every instance, the ligands’ ICE
values were higher in iron-overloaded primates than in the non-
iron-loaded rodents (Table 1). The difference in ICE values
between species is not surprising in view of the larger, accessible
iron pool in the iron-overloaded primates. Again, looking at
the sets of DADFT chelators [1, 2], [3, 4], [6] and DADMDFT
ligands [7, 8], [9, 10], [11] (Table 2), the trend is similar to the
rodents regarding lipophilicity, with some leveling effect
probably due to more available iron for chelation. With the sets
[1, 2] and [7, 8], the more lipophilic molecules had the better
ICE values.43 Again, the more lipophilic compounds are more
active (3 vs 4, p < 0.01) and (9 vs 10, p ) 0.05). However,
with the polyether (6) there is a rather surprising jump in

Organ-Specific Iron Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7037
Figure 1. Tissue distribution in plasma, kidney, liver, heart, and pancreas of rats treated with DADFT analogues 1-5 given sc at a dose of 300
µmol/kg. The concentrations (y-axis) are reported as µM (plasma) or as nmol compound per g wet weight of tissue. For all time points, n ) 3. The
demethylation in the liver of 2 f 1 is also shown.
two obvious relationships bearing on the toxicity profiles of
the DFT analogues evaluated (Table 3): (1) the DADMDFT
ligands (7-11) are generally less toxic than the corresponding
DADFT series (1-5) and (2) while the DADMDFT ligand
toxicity profiles are independent of partition properties, this is
not the case with the DADFT analogues. Within the DADFT
sets [1 vs 2] and [3 vs 4], the less lipophilic ligand was also the
less toxic. When rodents were treated with DADFT chelators
with log Papp values greater than -1.05 at 384 µmol/kg/d, all
of the animals were dead before the end of the planned 10-day
dosing regimen. This same relationship also held up with
dimethoxy analogue (5) and the polyether (6). The polyether
(log Papp ) -1.10) was profoundly less toxic than 5 (Table 3).
In each instance of mortality (2, 4, 5), the rats presented with
nephrotoxicity, which was acute, diffuse, and severe and charac-
terized by proximal tubular epithelial necrosis and sloughing.
Chelator Tissue Distribution in Rodents. The tissue
distribution description will focus first on the DADFT ligands
[1, 2], [3, 4], [5] (Figure 1) and next on the DADMDFT ana-
logues [7, 8], [9, 10], [11] (Figure 2). In each instance, we will
consider plasma, kidney, liver, heart, and pancreas. In these
studies, the rats were given a single 300 µmol/kg dose of the
drug sc. Subcutaneous administration was chosen to minimize
possible, but unlikely, complications surrounding differences
in oral absorption.
Desazadesferrithiocin Analogues. In the case of the DADFT
analogues in the plasma (Figure 1), the more lipophilic systems
achieve much higher concentrations and maintain them for a
longer period of time. The parent drug (S)-4′-(HO)-DADFT (1)
reaches a level of 92 µM at 0.5 h and drops to 13 µM at 2 h.
The methoxy analogue 2 (plus its metabolite 1) reaches nearly
eight times the level of the parent 1 (p < 0.002) at 0.5 h. Even
at 8 h the concentration of the methoxy 2 is still as high as the
initial level of the parent 1. A similar scenario is true for (S)-
5′-(HO)-DADFT (3) and its methylated analogue (S)-5′-(CH3O)-
DADFT (4). At 0.5 h, the more lipophilic 4, at a concentration
of 803 µM, reaches more than three times the concentration of
3 (p < 0.001). It is noteworthy that the 5′-substituted ligands
achieve higher plasma levels than the 4′-substituted ligands
(Figure 1). The (S)-3′,4′-(CH3O)2-DADFT analogue (5) achieves
the highest initial plasma concentration of all of the DADFT
analogues (907 µM, p < 0.001 for 1 and 3 and p < 0.05 for 2
and 4), although it is essentially gone by 8 h.
In the kidney, the situation is a little different (Figure 1).
The parent 1 and its methoxy analogue 2 achieve similar initial
concentrations at 0.5 h (361 vs 282 nmol/g wet weight, respec-
tively, p > 0.05). However, at 1 h postdrug, 2 (plus its
metabolite 1) is nearly three times the concentration of the parent
1 (518 vs 179 nmol/g wet weight, respectively, p < 0.002),
then decreasing, although always at higher levels than the parent

7038 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Bergeron et al.
Figure 2. Tissue distribution in plasma, kidney, liver, heart, and pancreas of rats treated with DADMDFT analogues 7-11 given sc at a dose of
300 µmol/kg. The concentrations (y-axis) are reported as µM (plasma) or as nmol compound per g wet weight of tissue. For all time points, n )
3. The demethylation in the liver of 8 f 7 is also shown.
Scheme 1. Synthesis of DADFTs 3-5 and DADMDFTs 9-11a
Scheme 2. Preparation of 2,5-Dihydroxybenzonitrile (12)a
a Reagents: (a) BBr3, CH2Cl2 (80%).
analogue 4 at 0.5 h (p < 0.002), although the parent drug 3
levels drop very quickly, by 98% at 2 h. Finally, the (S)-3′,4′-
dimethoxy chelator (5) achieves the highest initial renal tissue
concentration of all the DADFT analogues (1375 nmol/g wet
weight, p < 0.001 for 1, 2, and 4), although it drops quickly,
almost mirroring the plasma levels.
The liver tissue concentrations for ligands 1-4 (Figure 1)
display a trend similar to plasma at each time point; the more
lipophilic methoxylated ligands achieve and maintain greater
levels than the hydroxylated parent drugs. The parent 1 reaches
124 nmol/g wet weight at 0.5 h, while its methoxy analogue 2
(plus its metabolite 1) rises to 275 nmol/g wet weight (p < 0.05).
The 5′-(HO) analogue 3 at 0.5 h is very similar to 1, while its
5′-(CH3O) analogue 4 achieves 382 nmol/g wet weight, p
< 0.001 versus 3 (Figure 1). Again, the (S)-3′,4′-dimethoxy
a Reagents: (a) CH3OH (aq), pH 6, heat, g1 d.
1. A similar scenario holds for 5′-substituted ligands (Figure
1). The 5′-hydroxy parent ligand 3 (1216 nmol/g wet weight)
achieves over twice the concentration of the 5′-methoxy

Organ-Specific Iron Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7039
Scheme 3. Preparation of 3,4-Dimethoxy-2-hydroxybenzonitrile (14)a
a Reagents: (a) H2NOH‚HCl, NaOAc, CH3OH (84%); (b) Ac2O, reflux; (c) NaOH, CH3OH (aq, 82%).
parts 8 and 10, respectively, though this difference was only
significant with 9 vs 10, p < 0.05. However, the parent ligands
7 and 9 diminish far more quickly than the methoxy analogues
(Figure 2). There is not nearly as great an initial difference in
concentration between the dimethoxy analogue 11 and 7-10
as seen in the DADFT series (Figure 1). It is notable that at 0.5
h, (S)-4′-(HO)-DADMDFT (7) and (S)-4′-(CH3O)-DADMDFT
(8) both reach over twice the level in kidney tissue as their
DADFT counterparts 1 and 2 (Figures 1 and 2).
In the liver (Figure 2), the more lipophilic ligands achieve
higher concentrations (8 > 7 and 10 > 9). The difference in
concentration between the ligands is greater than in the renal
tissue. The 4′-(CH3O) analogue 8 (plus its metabolite 7) rises
to a level of 615 nmol/g wet weight, nearly three times that of
the parent (7) 0.5 h postdrug (p < 0.001). In the case of the
5′-(CH3O) analogue, the concentration of 10, 564 nmol/g wet
weight, reaches over three times that of the parent 9 (p < 0.01).
The dimethoxy analogue 11 is greater in concentration than
7-10 (p < 0.001 for 7-9 and p < 0.02 vs 10) but drops off
quickly. However, ligand 10 has the longest residence time
(Figure 2).
The most notable observation in the DADMDFT series relates
to cardiac drug levels (Figure 2). The cardiac tissue presents
with the largest differences in concentration between the parent
ligands and the methoxylated counterparts (8 . 7, p < 0.002,
and 10 . 9, p < 0.002). It is particularly interesting that (S)-
5′-(CH3O)-DADMDFT (10) achieves a level of 380 nmol/g wet
weight at 0.5 h and remains at 61 nmol/g wet weight even at 4
h. Its parent 9 never rises above 42 nmol/g wet weight
Furthermore, ligand 10 also reaches significantly higher con-
centrations than the (S)-3′,4′-dimethoxy chelator 11 (185 nmol/g
wet weight, p < 0.001).
Figure 3. Iron-clearing efficiency of DADFT analogues 1-5 plotted
vs the liver chelator concentration. The rats were given a single 300
µmol/kg dose of the compounds po for the ICE determinations and sc
for the tissue distribution studies.
analogue (5) reaches the highest liver concentration (924 nmol/g
wet weight, p < 0.001 vs 1-4).
Cardiac ligand concentrations are lower than those seen in
the plasma, kidney, or liver (Figure 1). However, they again
mirror both the plasma and the liver concentrations with the
more lipophilic ligands at higher levels. The highest cardiac
levels are achieved by the (S)-3′,4′-dimethoxy analogue (5), 259
nmol/g wet weight at 0.5 h (p < 0.003 for 1-3 and p < 0.03
vs 4), dropping to 3 nmol/g wet weight at 8 h. The next most
effectively concentrated chelator was (S)-5′-(CH3O)-DADFT (4).
Although its initial 0.5 h level, 208 nmol/g wet weight, was
lower than 5, it surpassed 5 at 1 h and remained higher for 8 h.
Of all the tissues, the pancreas presented with the least amount
of ligand accumulation (Figure 1). In this organ (S)-5′-(CH3O)-
DADFT (4) achieved the highest tissue levels at all time points,
although still reaching only 153 nmol/g wet weight at 1 h.
Desazadesmethyldesferrithiocin Analogues. The DADM-
DFT analogues in the plasma (Figure 2) are similar to their
DADFT counterparts (Figure 1); the more lipophilic systems
achieve much higher concentrations and maintain them for a
longer period of time. The less lipophilic parent drug (S)-4′-
(HO)-DADMDFT (7) reaches a level of 209 µM at 0.5 h and
drops to 5 µM at 2 h. The more lipophilic (S)-4′-(CH3O)-
DADMDFT (8; plus its metabolite 7) achieves a concentration
of 774 µM at 0.5 h postdrug (p < 0.001). At 2 h, the plasma
concentration is still greater than 200 µM (Figure 2). A similar
scenario is true for (S)-5′-(HO)-DADMDFT (9) and its methoxy
analogue (S)-5′-(CH3O)-DADMDFT (10; p < 0.001). It is
noteworthy that, while the 5′-substituted parent ligand 9 achieves
a higher plasma level than its 4′-substituted counterpart 7, the
difference between the 5′-substituted compounds (9 vs 10) is
not as great as that seen with the 4′-substituted drugs (7 vs 8).
The (S)-3′,4′-(CH3O)2-DADMDFT analogue (11) also achieves
a high initial plasma concentration (695 µM), although it is
essentially gone by 4 h.
The scenario is slightly different with the pancreas (Figure
2). The concentration of the 4′-methoxylated ligand 8 (102
nmol/g wet weight) is slightly greater than its hydroxylated
counterpart 7 (p < 0.05). However, analogue 10 reaches the
highest concentration of any of the DADFT or DADMDFT
analogues, 198 nmol/g wet weight and is nearly five times
greater than its hydroxylated counterpart 9 (p < 0.02). Even
4 h postdrug, the tissue concentration is 45 nmol/g wet weight.
Conclusion
There is a pressing need for the continued development of
new iron-chelating agents that are either more effective than
currently available therapies or can selectively remove iron from
organs and tissues especially vulnerable to iron-induced toxicity.
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. This is especially true with respect to heart disease,
still the leading cause of death in thalassemia major and related
forms of transfusional iron overload; the development of such
agents could be life-saving.
The renal data for the DADMDFT series (Figure 2) is similar
to the DADFT findings (Figure 1). Initially, at 0.5 h, the less
lipophilic parent ligands (S)-4′-(HO)-DADMDFT (7; 667 nmol/g
wet weight) and (S)-5′-(HO)-DADMDFT (9; 629 nmol/g wet
weight) are at levels slightly higher than their methoxy counter-
With early onset of iron overload in which there is no cardiac
involvement, the simplest solution is global iron chelation, for
example, treatment with DFO or the triazole deferasirox. While

7040 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Bergeron et al.
hematological and biochemical studies35 were performed by Antech
Diagnostics (Tampa, FL). Atomic absorption (AA) measurements
were made on a Perkin-Elmer model 5100 PC (Norwalk, CT).
Histopathological 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.34,35 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.34,35
Iron Loading of C. apella Monkeys. The monkeys (3.5-4 kg)
were iron overloaded with intravenous iron dextran as specified in
earlier publications to provide about 500 mg of iron per kg of body
weight;44 the serum transferrin iron saturation rose to between 70
and 80%. At least 20 half-lives, 60 d,45 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.34,35,46 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.34,47
Drug Preparation and Administration. In the iron-clearing
experiments, the rats were given a single 150 µmol/kg (9) or 300
µmol/kg (1-8, 10, 11) dose of the drugs orally (po). The com-
pounds were administered as (1) a solution in water (6); (2)
solubilized in 40% Cremophor RH-40/water (7, 9); or (3) the
monosodium salt of the compound of interest (prepared by the
addition of 1 equiv of NaOH to a suspension of the free acid in
distilled water; 1-5, 8, 10, 11).
it is true that neither of these ligands achieves notable levels in
cardiac tissue, iron contained in all organ systems ultimately
equilibrates as the metal is chelated and removed. Thus, cardiac
iron is slowly removed but is accessible. However, in a scenario
in which iron overload has achieved critical levels in cardiac
tissue, ligands that can selectively access this compartment
become particularly attractive. The current study is focused on
identifying chelators with such organ-specific access. A group
of tricoordinate desferrithiocin analogues were assembled
(Tables 1 and 2). The ligands belonged to one of two different
families, (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thia-
zolecarboxylic acid (DADFT) or (S)-4,5-dihydro-2-(2-hydroxy-
phenyl)-4-thiazolecarboxylic acid (DADMDFT) analogues. The
chelators were easily assembled by cyclization of (S)-R-methyl
cysteine or (S)-cysteine with the appropriate aromatic nitrile to
provide the DADFTs or the DADMDFTs, respectively (Scheme
1). Both the physicochemical properties (log Papp) and biological
propertiessiron-clearing efficiency (ICE) in rodents and pri-
mates, toxicity in rodents, and organ distribution in rodentss
were measured. The outcome of these measurements was
compared and integrated with previous data on related desferri-
thiocin chelators. The results have led to a number of findings
that are critical to future design strategies aimed at the devel-
opment of organ-specific desferrithiocin ligands belonging to
the DADFT or DADMDFT families.
First of all, in rodents, while lipophilicity is a good indicator
of how a ligand will perform as an iron chelator, it now seems
that the level of the drug achieved in liver tissue (Figure 3) is
better. Studies are underway in rodents that will hopefully
demonstrate that cardiac ligand tissue levels (Figures 1 and 2)
are also a credible indicator for how well a ligand will remove
iron from the heart. Although log Papp is a good diagnostic tool
for ICE in primates, it is only of value when comparing
structurally very similar chelators.
Second, there are profound differences in the toxicity between
the two families: the DADMDFTs are less toxic than the
DADFTs. However, it is possible to impact the toxicity of the
DADFTs by reducing their lipophilicity; the less lipophilic the
ligand, the less toxic it is (1, 3, and 6 vs 2, 4, and 5, Table 3).
The same changes in lipophilicity can have rather profound
effects on organ distribution and residence time.
Finally, in the case of the DADFT set, the more lipophilic
ligands (1 vs 2 and 3 vs 4) achieve and maintain the highest
tissue levels, with the exception of the 0.5 h time point in the
kidneys. The dimethoxy analogue 5 achieves the highest tissue
levels in plasma, kidney, liver, and heart. With the DADMDFT
series (7 vs 8 and 9 vs 10), the situation is very similar.
However, the dimethoxy ligand 11 no longer consistently
achieves the highest tissue concentrations. What is most notable,
however, is the level of chelator in the heart tissue in this family;
the methoxy analogues 8 and 10 achieve and maintain much
higher levels in cardiac tissue than their DADFT counterparts
2 and 4, respectively. The tissue distribution data of (S)-5′-
(CH3O)-DADMDFT (10), coupled with its iron-clearing effi-
ciency and toxicity profile, is consistent with the idea that 10
may well serve as an excellent ligand for removing iron from
the liver, heart, and pancreas, the most important organs in iron
overload.
The drugs were given to the monkeys po at a dose of 150 µmol/
kg. The drugs were prepared as for the rats, except that 2 and 8
were solubilized in 40% Cremophor RH-40/water, and compound
9 was administered as its monosodium salt.
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.37 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 DADFT and DADMDFT Analogues
in Rodents. Male Sprague-Dawley rats (250-300 g) were fasted
overnight and were given the DADFT ligands 3-5 and the
DADMDFT compounds 9-11 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. Additional animals served as age-matched controls. The
animals were monitored at least three times daily, and those found
in moribund condition were euthanized. Surviving rodents were
sacrificed 1 day after the last dose, and extensive tissues were sent
out for histopathological analysis.
Collection of Tissue Distribution Samples from Rodents. Male
Sprague-Dawley rats (250-350 g) were given a single sc injection
of the monosodium salts of 1-5 and 7-11 prepared as described
above at a dose of 300 µmol/kg. At times 0.5, 1, 2, 4, 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 was separated for analysis. The liver, heart, kidneys,
and pancreas were then removed from the animals.
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, and 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
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
acquired from BASF (Parsippany, NJ). Ultrapure salts were
purchased from Johnson Matthey Electronics (Royston, U.K.). All

Organ-Specific Iron Chelators
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7041
mM KH2PO4, pH 3.0]/5% CH3CN), vortexed, and filtered as above
prior to injection.
3.74 (s, 3 H), 3.83 (d, 1 H, J ) 12.0), 6.88 (d, 1 H, J ) 3.2), 6.94
(d, 1 H, J ) 8.8), 7.09 (dd, 1 H, J ) 9.2, 3.2), 11.97 (s, 1 H),
13.23 (s, 1 H); 13C NMR (CD3OD) δ 24.65, 40.79, 56.30, 84.86,
114.63, 116.91, 118.86, 121.64, 153.57, 154.39, 172.56, 175.80;
HRMS m/z calcd for C12H14NO4S, 268.0644 (M + H); found,
268.0630. Anal. (C12H13NO4S) C, H, N.
Analytical separation was performed on a Discovery RP Amide
C16 HPLC system with UV detection at 310 nm, as described
previously.48,49 Mobile phase and chromatographic conditions were
as follows: solvent A, 5% CH3CN/95% buffer; solvent B, 60%
CH3CN/40% buffer.
(S)-4,5-Dihydro-2-(3,4-dimethoxy-2-hydroxyphenyl)-4-meth-
yl-4-thiazolecarboxylic Acid (5). Sodium bicarbonate (3.50 g, 41.7
mmol) was added to a mixture of 14 (4.855 g, 27.09 mmol) and
15 (6.120 g, 35.66 mmol) in degassed CH3OH (180 mL) and
phosphate buffer (180 mL) to a pH of 6, and the reactants were
heated at 66 °C for 2 d 20 h with stirring under nitrogen. The
reaction mixture was cooled to room temperature, and the volume
of the reaction mixture was reduced by rotary evaporation. Cold 1
M HCl (70 mL) and saturated NaCl (20 mL) were added to the
residue, which was extracted with EtOAc (150 mL, 2 × 50 mL).
The EtOAc layers were washed with saturated NaCl (70 mL) and
were concentrated in vacuo to crude 5, which was dissolved in
DMF (135 mL). Iodoethane (3.0 mL, 38 mmol) and N,N-
diisopropylethylamine (6.5 mL, 37 mmol) were introduced, and
the solution was stirred for 1 d. After solvent removal under high
vacuum, the residue was combined with 1:1 0.5 M citric acid/
saturated NaCl (200 mL) and was extracted with EtOAc (200 mL,
3 × 50 mL). The combined extracts were washed with 80-mL
portions of 0.25 M citric acid, 1% NaHSO3, H2O, and saturated
NaCl, and the solvent was evaporated. Purification by flash column
chromatography using 24% EtOAc/pet ether gave the ethyl ester
of 5 as a yellow oil: 1H NMR (CDCl3) δ 1.30 (t, 3 H, J ) 7.0),
1.66 (s, 3 H), 3.20 (d, 1 H, J ) 11.4), 3.86 (d, 1 H, J ) 11.4), 3.90
and 3.91 (2 s, 6 H), 4.19-4.30 (m, 2 H), 6.48 (d, 1 H, J ) 9.0),
7.14 (d, 1 H, J ) 9.0), 12.72 (s, 1 H). The oil was treated with a
solution of 50% NaOH (18.64 g, 0.233 mol) and CH3OH (350 mL)
with brief ice bath cooling. After stirring for 1 d at room
temperature, solvents were removed by rotary evaporation. Dilute
NaCl (210 mL) was added, followed by extraction with Et2O (3 ×
60 mL). Cold 2 N HCl (130 mL) was added to the aqueous layer,
which was extracted with EtOAc (170 mL, 3 × 80 mL). The EtOAc
layers were washed with saturated NaCl (60 mL) and were
concentrated in vacuo, furnishing 4.79 g (60%) of 5 as a light green
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.
Tissue distribution data are presented as 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.
Synthetic Methods. Compounds 1, 2, and 6-8 were synthesized
using methods published by this laboratory.36-39 Amino acid 16
was obtained from Advanced ChemTech, Louisville, KY. Reagents
were purchased from Aldrich Chemical Co. (Milwaukee, WI) and
Fisher Optima-grade solvents were routinely used. Phosphate buffer
was made up to a concentration of 0.1 M at a pH of 6.50 Reactions
were run under a nitrogen atmosphere, and organic extracts were
dried with sodium sulfate, which was filtered. Silica gel 40-63
from Silicycle, Inc. (Quebec City, QC, Canada) was used for flash
column chromatography. Distilled solvents were employed for
reactions involving chelators. Glassware that was presoaked in 3 N
HCl for 15 min was used for the isolation of chelators. Melting
points are uncorrected. Optical rotations were run at 589 nm
(sodium D line) utilizing a Perkin-Elmer 341 polarimeter with c
as g of compound per 100 mL of DMF (distilled) solution. 1H NMR
spectra were measured at 400 MHz in DMSO-d6 (not indicated) or
other deuterated solvents, and chemical shifts (δ) are presented in
parts per million downfield from tetramethylsilane. Coupling
constants (J) are in hertz. 13C NMR spectra were run at 100 MHz,
and chemical shifts (δ) are given in parts per million referenced to
the residual solvent resonance in DMSO-d6 (δ 39.52) or CD3OD
(δ 49.00). Elemental analyses were performed by Atlantic Microlabs
(Norcross, GA).
(S)-2-(2,5-Dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazole-
carboxylic Acid (3). Compound 15 (12.2 g, 64.8 mmol) was added
to a solution of 12 (5.40 g, 40.0 mmol) in degassed CH3OH (225
mL). Phosphate buffer (150 mL) and NaHCO3 (6.72 g, 80.0 mmol)
were added, and the reactants were heated at 70 °C for 40 h with
stirring under nitrogen. The reaction mixture was cooled to room
temperature, and the solvents were removed by rotary evaporation.
The residue was dissolved in saturated NaHCO3 (600 mL) and was
washed with EtOAc (3 × 60 mL). The aqueous portion was
acidified to pH 2 with cold concentrated HCl. Extraction with
EtOAc (4 × 125 mL), solvent removal in vacuo, and recrystalli-
zation from EtOAc/hexanes gave 9.83 g (97%) of 3 as brownish
yellow crystals, mp 223-224.5 °C: [R]24 +28.7° (c 1.01); 1H NMR
δ 1.56 (s, 3 H), 3.36 (d, 1 H, J ) 11.6), 3.79 (d, 1 H, J ) 11.6),
6.80 (m, 2 H), 6.88 (m, 1 H), 9.17 (br s, 1 H), 11.72 (br s, 1 H),
13.21 (br s, 1 H); 13C NMR δ 24.04, 39.48, 83.01, 114.69, 115.23,
117.59, 121.54, 149.51, 151.29, 170.27, 173.61; HRMS m/z calcd
for C11H12NO4S, 254.0487 (M + H); found, 254.0479. Anal.
(C11H11NO4S) C, H, N.
1
solid: [R]24 +45.7° (c 1.16); H NMR δ 1.59 (s, 3 H), 3.37 (d,
1 H, J ) 11.2), 3.71 (s, 3 H), 3.79 (d, 1 H, J ) 11.6), 3.84 (s, 3
H), 6.67 (d, 1 H, J ) 8.8), 7.12 (d, 1 H, J ) 8.8), 12.72 (s, 1 H),
13.21 (br s, 1 H); 13C NMR (CD3OD) δ 24.70, 40.57, 56.53, 60.89,
84.26, 104.43, 112.34, 127.37, 137.46, 154.49, 157.98, 172.63,
175.87; HRMS m/z calcd for C13H16NO5S, 298.0749 (M + H);
found, 298.0735.
(S)-2-(2,5-Dihydroxyphenyl)-4,5-dihydro-4-thiazolecarbox-
ylic Acid (9). Sodium bicarbonate (4.37 g, 52.0 mmol) was added
to a mixture of 12 (4.088 g, 30.26 mmol) and 16 (8.19 g, 46.6
mmol) in degassed CH3OH (220 mL) and phosphate buffer (220
mL) to a pH of 6, and the reactants were heated at 65 °C for 2 d
with stirring under nitrogen. The reaction mixture was cooled to
room temperature, and the volume of the reaction mixture was
reduced by rotary evaporation. Cold 0.5 M HCl (150 mL) was
added, followed by extraction with EtOAc (150 mL, 2 × 100 mL).
The EtOAc layers were washed with saturated NaCl (80 mL) and
were concentrated in vacuo. Recrystallization from aqueous EtOH
gave 5.395 g (75%) of 9 as yellow crystals, mp 233-234.5 °C
1
(dec): [R]23 -8.5° (c 1.18); H NMR δ 3.62 (dd, 1 H, J ) 11.4,
(S)-4,5-Dihydro-2-(2-hydroxy-5-methoxyphenyl)-4-methyl-4-
thiazolecarboxylic Acid (4). Sodium bicarbonate (3.88 g, 46.2
mmol) was added to a mixture of 1340 (4.607 g, 30.90 mmol) and
15 (6.96 g, 40.6 mmol) in degassed CH3OH (200 mL) and
phosphate buffer (200 mL) to a pH of 6, and the reactants were
heated at 60 °C for 2 d with stirring under nitrogen. The reaction
mixture was cooled to room temperature, and the volume of the
reaction mixture was reduced by rotary evaporation. Cold 0.2 M
HCl (280 mL) was added to the residue, which was extracted with
EtOAc (150 mL, 2 × 100 mL). The EtOAc layers were washed
with saturated NaCl and were concentrated in vacuo to generate
8.21 g (99%) of 4 as yellow solid, mp 126.5-127.5 °C: [R]24
+25.9° (c 1.06); 1H NMR δ 1.59 (s, 3 H), 3.41 (d, 1 H, J ) 11.6),
7.4), 3.70 (dd, 1 H, J ) 11, 9.4), 5.46 (dd, 1 H, J ) 9.4, 7.4),
6.81-6.85 (m, 2 H), 6.89 (dd, 1 H, J ) 9, 2.6), 9.20 (s, 1 H),
11.74 (s, 1 H), 13.22 (s, 1 H); 13C NMR δ 33.45, 76.56, 114.93,
115.29, 117.65, 121.59, 149.53, 151.30, 171.37, 172.31; HRMS
m/z calcd for C10H10NO4S, 240.0330 (M + H); found, 240.0262.
Anal. (C10H9NO4S) C, H, N.
(S)-4,5-Dihydro-2-(2-hydroxy-5-methoxyphenyl)-4-thiazole-
carboxylic Acid (10). Sodium bicarbonate (3.83 g, 45.6 mmol)
was added to a mixture of 1340 (4.600 g, 30.85 mmol) and 16 (7.136
g, 40.64 mmol) in degassed CH3OH (200 mL) and phosphate buffer
(200 mL) to a pH of 6, and the reactants were heated at 60 °C for
3 d 17 h with stirring under nitrogen. The reaction mixture was

7042 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24
Bergeron et al.
cooled to room temperature, and the volume of the reaction mixture
was reduced by rotary evaporation. Cold 0.3 M HCl (200 mL) was
added, followed by extraction with EtOAc (2 × 150 mL, 3 × 50
mL). The EtOAc layers were washed with saturated NaCl (50 mL)
and were concentrated in vacuo. Recrystallization from EtOAc/
hexanes afforded 5.999 g (77%) of 10 as a pale green solid, mp
0.1921 mol) were added to a warmed solution of 1841,42 (23.29 g,
0.1278 mol) in CH3OH (150 mL). The reaction mixture was heated
at reflux for 1 h under a Drierite tube. The reaction mixture was
concentrated by rotary evaporation, and the residue was diluted
with saturated NaCl (200 mL) and 0.5 M citric acid (100 mL) and
was extracted with EtOAc (300 mL, 2 × 100 mL). The EtOAc
layers were washed with H2O (100 mL) and saturated NaCl (100
mL) and were concentrated in vacuo. Recrystallization from EtOAc/
hexanes afforded 21.22 g (84%) of 19 as pale yellow crystals, mp
111.5-112 °C: 1H NMR δ 3.69 (s, 3 H), 3.80 (s, 3 H), 6.60 (d,
1 H, J ) 8.6), 7.17 (d, 1 H, J ) 9.0), 8.25 (s, 1 H), 9.98 (s, 1 H),
11.18 (s, 1 H); 13C NMR δ 55.78, 59.98, 103.94, 112.62, 123.42,
136.10, 148.28, 150.05, 154.12; HRMS m/z calcd for C9H12NO4,
198.0766 (M + H); found, 198.0759. Anal. (C9H11NO4) C, H, N.
1
139.5-142 °C: [R]23 -7.1° (c 1.20); H NMR δ 3.64 (dd, 1 H,
J ) 11.2, 7.6), 3.69-3.76 (m, 1 H), 3.74 (s, 3 H), 5.48 (dd, 1 H,
J ) 9.2, 7.6), 6.90 (d, 1 H, J ) 3), 6.95 (d, 1 H, J ) 9), 7.10 (dd,
1 H, J ) 9, 3), 11.97 (s, 1 H), 13.23 (s, 1 H); 13C NMR δ 33.60,
55.67, 76.46, 113.42, 115.36, 117.91, 120.74, 151.68, 152.54,
171.32, 172.35; HRMS m/z calcd for C11H12NO4S, 254.0487
(M + H); found, 254.0516. Anal. (C11H11NO4S) C, H, N.
(S)-4,5-Dihydro-2-(3,4-dimethoxy-2-hydroxyphenyl)-4-thia-
zolecarboxylic Acid (11). Sodium bicarbonate (4.24 g, 50.5 mmol)
was added to a mixture of 14 (6.05 g, 33.8 mmol) and 16 (7.95 g,
45.3 mmol) in degassed CH3OH (225 mL) and phosphate buffer
(225 mL) to a pH of 6, and the reactants were heated at 65 °C for
2 d 19 h with stirring under nitrogen. The reaction mixture was
cooled to room temperature, and the volume of the reaction mixture
was reduced by rotary evaporation. Cold 1 M HCl (67 mL) was
added to the chilled mixture, which was extracted with EtOAc (200
mL, 4 × 50 mL) while adding salt. The EtOAc layers were washed
with saturated NaCl (50 mL) and were concentrated in vacuo.
Recrystallization from EtOAc/hexanes afforded 7.317 g (76%) of
11 as pale yellow crystals, mp 124.5-127.5 °C: [R]24 +13.5°
Acknowledgment. Funding was provided by the National
Institutes of Health Grant No. R37-DK49108. We thank
Elizabeth M. Nelson, Tanaya Lindstrom, and Katie Ratliff-
Thompson for their technical assistance and Carrie A. Blaustein
for her editorial and organizational support. We acknowledge
the spectroscopy services in the Chemistry Department, Uni-
versity of Florida, for the mass spectrometry analyses.
Supporting Information Available: Elemental analytical data
for synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
(c 0.92); H NMR δ 3.61 (dd, 1 H, J ) 11.2, 7.2), 3.66-3.72
(m + s, 4 H), 3.84 (s, 3 H), 5.44 (dd, 1 H, J ) 9.4, 7.4), 6.68 (d,
1 H, J ) 8.8), 7.18 (d, 1 H, J ) 8.8), 12.71 (s, 1 H), 13.19 (br s,
1 H); 13C NMR δ 33.27, 55.95, 59.82, 75.99, 103.80, 110.54,
126.11, 135.90, 152.94, 156.32, 171.43, 172.46; HRMS m/z calcd
for C12H14NO5S, 284.0593 (M + H); found, 284.0606. Anal.
(C12H13NO5S) C, H, N.
2,5-Dihydroxybenzonitrile (12). Boron tribromide (1 M in CH2-
Cl2, 300 mL, 0.3 mol) was added dropwise to 17 (12.24 g, 75.00
mmol) in CH2Cl2 (30 mL) with dry ice/acetone cooling. The
reaction solution was allowed to warm to room temperature and
was stirred for 18 h. After quenching with H2O (100 mL) with
ice-bath cooling, solids were filtered, and the layers of the filtrate
were separated. The aqueous phase was diluted with H2O (200 mL),
was adjusted to pH 2 with 1 N HCl, and was extracted with EtOAc
(3 × 150 mL). The extracts were concentrated in vacuo, and the
residue was recrystallized from EtOAc/hexanes, producing 8.5 g
(80%) of 12 as a pale brown solid, mp 167-169 °C (lit51 163-
165 °C): 1H NMR (CD3OD) δ 6.78 (d, 1 H, J ) 8.8), 6.85 (d,
1 H, J ) 2.8), 6.92 (dd, 1 H, J ) 3.2, 2.8); 13C NMR (CD3OD) δ
100.27, 117.82, 118.12, 118.71, 123.48, 151.29, 154.70.
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