Evaluation of the desferrithiocin pharmacophore as a vector for hydroxamates

Article abstract of DOI:10.1021/jm980611q

A series of (S)-desmethyldesferrithiocin (DMDFT, 1) hydroxamates and a bis-salicyl polyether hydroxamate are evaluated for their iron-clearing properties in rodents; some of these are further assessed in primates. These hydroxamates include (S)-desmethyld

Full text of DOI:10.1021/jm980611q

J . Med. Chem. 1999, 42, 2881-2886
2881
Eva lu a tion of th e Desfer r ith iocin P h a r m a cop h or e a s a Vector for
Hyd r oxa m a tes
Raymond J . Bergeron,* J ames S. McManis, J o¨rg Bussenius, Gary M. Brittenham,† and J an Wiegand
Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610-0485, and Department of Pediatrics,
Division of Hematology, Columbia University College of Physicians and Surgeons, New York, New York 10032
Received October 30, 1998
A series of (S)-desmethyldesferrithiocin (DMDFT, 1) hydroxamates and a bis-salicyl polyether
hydroxamate are evaluated for their iron-clearing properties in rodents; some of these are
further assessed in primates. These hydroxamates include (S)-desmethyldesferrithiocin,
N-methylhydroxamate (2); (S)-desmethyldesferrithiocin, N-[5-(acetylhydroxyamino)pentyl]-
hydroxamate (3); desmethyldesferrithiocin, N-benzylhydroxamate (4); (S,S)-N1,N8-bis[4,5-
dihydro-2-(3-hydroxy-2-pyridinyl)-4-thiazoyl]-N1,N8-dihydroxy-3,6-dioxa-1,8-octanediamine (5);
and N1,N8-bis(2-hydroxybenzoyl)-N1,N8-dihydroxy-3,6-dioxa-1,8-octanediamine (6). The ligands
are evaluated when given both orally (po) and subcutaneously (sc) in the bile-duct-cannulated
rodent model. In iron-overloaded primates, ligands 1-4 are assessed when administered po
and sc. The efficiencies of the hydroxamates are shown to vary considerably; giving the
compounds sc consistently resulted in greater chelating efficiency in vivo. After oral administra-
tion in the primate, compound 3, a pentacoordinate unsymmetrical dihydroxamate, produces
iron excretion sufficient to warrant further preclinical evaluation both as a potential orally
active iron-chelating agent and as a parenteral iron chelator. The increased iron clearance of
several of these ligands when administered sc versus po also underscores the idea that
parenteral administration is a reasonable alternative to a less efficient, orally active device
which would require large and frequent doses.
Ch a r t 1. Naturally Occurring Siderophore
(S)-Desferrithiocin (DFT) and Its Analogue
(S)-Desmethyldesferrithiocin (DMDFT, 1)
In tr od u ction
Desferrioxamine B (DFO) as its methanesulfonate
salt, Desferal, is the drug of choice for the treatment of
transfusional iron overload. This hexacoordinate iron
chelator, a hydroxamate bacterial siderophore isolated
from Streptomyces pilosus,1 forms a very tight 1:1
complex with Fe(III). Unfortunately, desferrioxamine is
not well-absorbed from the gastrointestinal tract and
is rapidly eliminated from the circulation; consequently,
prolonged parenteral infusion is needed.2-4 Effective
therapy usually requires subcutaneous (sc) or intrave-
nous (iv) administration by a portable infusion pump
for 9-12 h daily, 5-6 days a week; patient compliance
is poor. Because transfusion-dependent patients require
life-long therapy, considerable effort has been directed
at a search for an iron chelator that would be easier for
patients to use.
Desferrithiocin (DFT; Chart 1), a tridentate sidero-
phore which forms 2:1 complexes with Fe(III),5,6 is
bioavailable after oral administration. Although the
naturally occurring siderophore is nephrotoxic,7 struc-
ture-activity studies have made it possible to construct
DFT analogues which are substantially less toxic than
the parent ligand and still orally active.8,9 We have
previously assessed two hydroxamate derivatives of the
DFT analogue (S)-desmethyldesferrithiocin (DMDFT, 1;
Chart 1), the N-methylhydroxamate and a pentacoor-
dinate dihydroxamate ligand, in the bile-duct-cannu-
lated rat model.9 In the current article, we describe the
further evaluation of these DMDFT hydroxamates in
the primate model as well as the assessment of several
additional DMDFT hydroxamate ligands.
Design Con cep ts
Our earlier work demonstrated that conversion of the
carboxylic acid group of DMDFT (1; Table 1) to an
N-methylhydroxamate (2) or to the pentacoordinate
dihydroxamate ligand (3) resulted in compounds that
were effective iron chelators in the bile-duct-cannulated
rat model when given orally (po).9 To determine whether
DMDFT hydroxamates are effective iron-clearing agents
as a class of compounds when given po or sc, two
additonal DMDFT hydroxamate systems were evalu-
ated: the tricoordinate N-benzylhydroxamate of DM-
DFT, 4, a more lipophilic version of the corresponding
N-methylhydroxamate 2, and (S,S)-N1,N8-bis[4,5-dihy-
dro-2-(3-hydroxy-2-pyridinyl)-4-thiazoyl]-N1,N8-dihydroxy-
3,6-dioxa-1,8-octanediamine (5), in which two (S)-
DMDFTs are joined by an eight-membered dioxa tether.
The latter compound, a hexacoordinate ligand, facili-
tates formation of a 1:1 complex with Fe(III). Thus, all
of the metal’s coordination sites are occupied. This offers
an entropic advantage over the other Fe(III) hydrox-
* To whom correspondence should be addressed.
† Columbia University College of Physicians and Surgeons.
10.1021/jm980611q CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/02/1999

2882 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Bergeron et al.
Sch em e 1. Synthesis of 4,5-Dihydro-N-hydroxy-
2-(3-hydroxy-2-pyridinyl)-N-(phenylmethyl)-4-
thiazolecarboxamide (4)a
Sch em e 2. Synthesis of (S,S)-N1,N8-Bis[4,5-dihydro-
2-(3-hydroxy-2-pyridinyl)-4-thiazoyl]-N1,N8-dihydroxy-
3,6-dioxa-1,8-octanediamine (5)a
a
Reagents: (a) BOP reagent/DIEA (3 equiv)/DMF.
amate complexes described. In addition, it renders
reduction of Fe(III) to Fe(II) by biological reductants
such as superoxide anion or ascorbate unlikely. Thus,
participation of Fe(II) in the reduction of hydrogen
peroxide to the highly reactive and destructive hydroxyl
radical (Fenton chemistry) is minimized. N1,N8-Bis(2-
hydroxybenzoyl)-N1,N8-dihydroxy-3,6-dioxa-1,8-octanedi-
amine (6), a hexacoordinate ligand in which the DMDFT
donor groups are substituted with salicyl donors, was
synthesized in an effort to determine whether the
DMDFT donors were the source of toxicity associated
with the hexacoordinate ligand 5. The iron-clearing
properties and toxicities in vivo of the hydroxamate
chelators in Table 1 were evaluated in comparison to
the most effective, least toxic DFT derivative to date,
1.
a
Reagents: (a) NaH/DMF; (b) H2/Pd-C/MeOH; (c) HCl/MeOH;
(d) 1/BOP reagent/DIEA/DMF.
Sch em e 3. Synthesis of N1,N8-Bis(2-hydroxybenzoyl)-
N1,N8-dihydroxy-3,6-dioxa-1,8-octanediamine (6)a
Syn th etic Meth od s
The N-benzylhydroxamate of (S)-DMDFT (4) was
synthesized by N-acylation of N-benzylhydroxylamine
hydrochloride with (S)-DMDFT (1) activated by
(benzot ria zol-1-yloxy)t ris(dim et h yla m ino)ph ospho-
nium hexafluorophosphate (BOP reagent) in N,N-diiso-
propylethylamine (DIEA; 3 equiv) and DMF (Scheme
1). Although it was possible to isolate an optically active
hydroxamate via Sephadex LH-20 chromatography, the
compound epimerized on recrystallization. The magni-
tude of optical rotation decreased from [R]D ) -16.4°
to essentially zero. Because of the spontaneous epimer-
ization, no attempt was made to evaluate the biological
properties of optically active material. The racemic
product was utilized in all of the studies.
Hexacoordinate ligand 5 can be visualized as the
joining of two (S)-DMDFT hydroxamic acids through
their nitrogens by an eight-membered diether chain.
N-(tert-Butoxycarbonyl)-O-benzylhydroxylamine10 was
alkylated (NaH/DMF) with 1,2-bis(2-chloroethoxy)-
ethane (0.5 equiv) to give masked dihydroxamate 7.
Hydrogenolysis of the benzyl groups of 7 afforded
tetracoordinate chelator 8. The BOC protecting groups
were removed with 3 M methanolic hydrochloric acid,
providing dihydroxylamine dihydrochloride 9. Bis-N-
acylation of 9 with 1 using BOP (excess DIEA, DMF)
completed the synthesis of hexadentate chelator 5
(Scheme 2).
a
Reagents: (a) HCl/CH3OH; (b) acetylsalicyloyl chloride/DIEA
(2.2 equiv)/CH2Cl2; (c) CH3ONa/CH3OH; (d) H2/10% Pd-C/CH3OH.
zoyl)-N1,N8-dihydroxy-3,6-dioxa-1,8-octanediamine (6),
a bis-salicyl analogue of 5, also hexacoordinate, in which
the thiazoline groups have been removed (Scheme 3).
Selective deprotection of 7 with hydrogen chloride in
methanol yielded bis(benzyloxyamine) 10. Acylation of
10 with acetylsalicyloyl chloride (DIEA/CH2Cl2) afforded
the fully protected chelator 11. Next, deacetylation of
11 with sodium methoxide in methanol gave the diphe-
nol 12, followed by hydrogenolysis (1 atm, 10% Pd/C,
MeOH) to afford hexacoordinate chelator 6.
Fully masked dihydroxamate 7 (Scheme 2) was also
utilized for the preparation of N1,N8-bis(2-hydroxyben-

DFT Pharmacophore as a Vector for Hydroxamates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2883
Ta ble 1. Summary of Evaluations of the Desferrithiocin-Based Hydroxamates
a
In the rats, doses were 150 µmol/kg via the route indicated. The net iron excretion was calculated by subtracting the iron excretion
of control animals from the iron excretion of treated animals. Efficiency of chelation is defined as net iron excretion/total iron-binding
capacity of chelator administered, expressed as a percent. Directly beneath the efficiency calculation is the percentage breakdown of iron
b
excretion in the bile and urine, respectively. In the monkeys, the doses and routes were as shown. 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. Directly
beneath the efficiency calculation is the percentage breakdown of iron excretion in the stool and urine, respectively. c This compound was
d
first dissolved in hot ethanol and then in 40% Cremophor. All of the rats died during sc evaluation of this compound. e N.D., not determined.
Biologica l An a lysis
efficient as 1; 98% of the iron was excreted in the bile
and 2% in the urine. However, in the current experi-
ments this drug was substantially better when given
sc, but much less of the iron excretion was biliary (64%,
Table 1). This suggests that the oral bioavailability of
the hydroxamate is less than that of 1. The N-benzyl-
hydroxamate 4 was less effective than 1 or 2 when
administered either po or sc (Table 1). Once again, po
dosing led to more biliary iron clearance (93%) than did
administration by the sc route (68%). However, in the
case of the unsymmetrical dihydroxamate 3, the differ-
ence between the efficiencies of the drug when given
po or sc became more pronounced, 2.8 ( 0.8% and 8.5
( 0.4%, respectively (Table 1). In both instances most
of the iron was excreted in the bile, ∼86%. Furthermore,
the difference in iron excretion after po and sc admin-
istration was even more exaggerated with the sym-
In previous studies, DMDFT (1), which forms a 2:1
ligand:metal complex with Fe(III), had an iron-clearing
efficiency of 2.4 ( 0.6% with 82% of the iron excreted
in the bile and 18% in the urine when administered to
rodents at a dose of 150 µmol/kg po.7 When administered
sc at the same dose, the efficiency and excretion patterns
were nearly identical to what was found when the ligand
was given po (Table 1). This strongly suggests that the
oral bioavailability of the parent ligand is very high. The
corresponding N-methylhydroxamate 2 was also previ-
ously shown to form a 2:1 ligand:metal complex with
Fe(III). Although addition of the hydroxamate group to
DMDFT results in 2 having an additional donor site,
because of steric constraints, only three of the four donor
atoms of 2 are actually involved in ligation.9 When given
to rodents at a dose of 150 µmol/kg po, it was about as

2884 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Bergeron et al.
metrical dihydroxamate 5. When bis-DMDFT ligand 5
was administered sc to the rodents, the efficiency was
the highest observed in the rodents for any DFT
analogue, 20.6 ( 1.3% (Table 1), almost 10-fold greater
than that of the parent ligand, 1. Interestingly, sc
administration of this ligand elicited overwhelmingly
biliary iron excretion, 92%. However, this standard dose
of 150 µmol/kg proved to be lethal during sc evaluation
of this compound. When administered po, this ligand
was no more effective than administration of twice the
molar equivalent of DMDFT itself, 4.6%. This suggests
possible hydrolysis of this ligand to the parent com-
pound 1 in the gut. The origin of the toxicity of 5 when
administered sc remains uncertain.
intestines, stomach, spleen, pancreas, and kidney, was
performed. The only notable effects were on the kidneys;
in the opinion of the pathologist the drug was a “renal
tubular epithelial toxin.” Due to the renal toxicity of the
compound given po, the sc studies were not carried out.
When 4 was given to the primates either po or sc at
a dose of 300 µmol/kg, the performance was similar to
that in rodents (Table 1). Neither 5 nor 6 was taken
forward into the primates. Symmetrical dihydroxamate
5 was simply too toxic in the rodents; the poor perfor-
mance of 6 did not justify further studies in primates.
However, unsymmetrical dihydroxamate 3 was some-
what effective when given to primates po; this ligand
was very effective when given sc. Orally at 225 µmol/
kg this dihydroxamate had an efficiency of 3.2 ( 2.0%.
In contrast to po administration of 2, most of the iron,
61%, was cleared in the urine and 39% was in the stool.
Interestingly, its effectiveness climbed to 12.8 ( 3.4%
when given sc; 90% of the iron was cleared in the stool
and 10% in the urine (Table 1). Furthermore, 3 did not
elicit any deleterious effects in either rodents or pri-
mates.
In an attempt to determine whether the toxicity of
hexacoordinate ligand 5 was implicit in the diether
linkage, compound 6, containing salicyl donors in place
of the DMDFT moieties, was synthesized and tested.
This chelator was certainly less toxic than was 5 as no
lethalities were observed during evaluation. However,
for compound 6, this loss of toxicity was associated with
a loss of iron-chelating activity (Table 1).
We next evaluated compounds 1-4 in primates. When
1 was given to iron-overloaded primates at a dose of 150
or 300 µmol/kg po, it was more efficient than it was in
rodents, with about an equal distribution of the iron in
stool and urine (Table 1).7 When administered sc, the
efficiency was essentially the same as when adminis-
tered po. The pattern of iron excretion was somewhat
different; 75% was in the stool. The N-methylhydrox-
amate 2 was also substantially more active in primates
than in rodents at a po dose of 150 µmol/kg, but less of
the iron was excreted through the stool, 58% (Table 1).
However, at a po dose of 300 µmol/kg, the efficiency was
higher, and a greater percentage of the iron, 83%, was
found in the stool (Table 1). Compound 2 was well-
tolerated when administered either po or sc to the
rodents at a dose of 150 µmol/kg. When the drug was
initially given sc to two primates at this dose, it was
similarly well-tolerated. However, in a subsequent
experiment in which two additional monkeys were given
the drug sc at 150 µmol/kg and four primates were given
it sc at 300 µmol/kg, one of the animals in the low-dose
group and three of the four monkeys in the high-dose
group had to be dropped from the study on day +1 due
to development of clinically significant tremors and an
abnormally low serum potassium level. One of the
animals (300 µmol/kg) died approximately 25 h post-
drug. Postmortem examination was unable to unequivo-
cally identify a cause of death. The iron-clearing effi-
ciency of the ligand when administered at 150 µmol/kg
was about twice that seen in the rodents at the same sc
dose, 11.7 ( 5.1%, with 86% of the iron excreted in the
stool and 14% excreted in the urine.
Discu ssion
The current study brings several issues to light. Of
the ligands (1-3 and 5) that had good iron-clearing
efficiencies in rodents when given po, 2, 3, and 5 were
even more efficient when given sc. This is consistent
with the idea that 1 has the greatest oral bioavailability
of the ligands. Bis analogue 5 perfomed exceptionally
well when administered sc to the rodents; however, it
remains to be seen whether the toxicity of this com-
pound is due to its astounding iron-clearing efficiency.
The same scenario holds for the primate evaluations:
Of the three chelators that were active in this model
(1-3), only 1 was equally effective when given po or sc.
Both ligands 2 and 3 performed far better when given
sc. The other notable observation is that all three of
these chelators were more active in primates than in
rodents whether administered po or sc. Of the hydrox-
amate derivatives of DMDFT assembled and evaluated,
the unsymmetrical dihydroxamate compound 3, the
N-[5-(acetylhydroxyamino)pentyl]hydroxamate of DM-
DFT, seems the best candidate for further preclinical
evaluation, and additional studies are underway.
Exp er im en ta l Section
Chelators 1,8 2,9 and 39 were previously prepared in these
laboratories. All other reagents were purchased from Aldrich
Chemical Co. (Milwaukee, WI). Fisher Optima grade solvents
were routinely used, and DMF was distilled. Organic extracts
were dried with sodium sulfate. Glassware that was presoaked
in 3 N HCl for 15 min and distilled solvents were employed
for reactions involving chelators. Silica gel 32-63 (40 µm
“flash”) from Selecto, Inc. (Kennesaw, GA) or Lipophilic
Sephadex LH-20 from Sigma Chemical Co. (St. Louis, MO) was
used for column chromatography. Melting points are uncor-
rected. Proton NMR spectra were run at 300 MHz in deuter-
ated organic solvents (CDCl3 not indicated) or in D2O with
chemical shifts in parts per million downfield from tetram-
ethylsilane or sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4,
respectively. Coupling constants (J ) are in hertz (Hz). Optical
rotations were determined at 589 nm (sodium D line) with c
as g of compound/100 mL of solution. Elemental analyses were
performed by Atlantic Microlabs (Norcross, GA). Cremophor
RH-40 was obtained from BASF (Parsippany, NJ ). Sprague-
Dawley rats were purchased from Charles River (Wilmington,
The apparent toxicity of this compound in the pri-
mates was unexpected based on the absence of any
deleterious effects when it was administered acutely to
rodents. Thus, we were compelled to conduct a chronic
toxicity evaluation in rats. Ten-day po7 and sc dosing
regimens were planned. When the rats were given 2 po
at a dose of 384 µmol/kg/day for 10 days, all the animals
survived the exposure to the drug; no tremors or any
other behavioral abnormalities were noted. Histopatho-
logical examination of numerous tissues, including

DFT Pharmacophore as a Vector for Hydroxamates
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15 2885
MA). Nalgene metabolic cages, rat jackets, and fluid swivels
were obtained from Harvard Bioscience (South Natick, MA).
Intramedic polyethylene tubing PE 50 was obtained from
Fisher Scientific (Pittsburgh, PA). Cebus apella monkeys were
purchased from World Wide Primates (Miami, FL). Ultrapure
salts were obtained from J ohnson Matthey Electronics (Roys-
ton, U.K.). Imferon, an iron dextran solution, was obtained
from Fisons (Bedford, MA).
was purified by silica gel flash column chromatography using
24% EtOAc/petroleum ether providing 13.16 g (73%) of 7 as
an oil: NMR δ 1.49 (s, 18 H), 3.57-3.64 (m, 12 H), 4.84 (s, 4
H), 7.30-7.42 (m, 10 H). Anal. (C30H44N2O8) C, H, N.
N1,N8-Bis(ter t-b u t oxyca r b on yl)-N1,N8-d ih yd r oxy-3,6-
d ioxa -1,8-octa n ed ia m in e (8). Degassed CH3OH (400 mL)
was added to 7 (10.2 g, 18.1 mmol), and 10% Pd-C (1.44 g)
was carefully introduced. The mixture was stirred under H2
at 1 atm for 7.5 h. Solids were filtered using Celite, which was
washed with CH3OH (400 mL). Solvent was removed in vacuo.
Purification of the concentrate on a Sephadex LH-20 column,
eluting with 5% EtOH/toluene, produced 3.67 g (53%) of 8 as
a colorless oil: NMR δ 1.50 (s, 18 H), 3.63 (s, 4 H), 3.70 (s, 8
H), 7.70 (s, 2 H). Anal. (C16H32N2O8) C, H, N.
N1,N8-Dih yd r oxy-3,6-d ioxa -1,8-oct a n ed ia m in e Dih y-
d r och lor id e (9). Methanolic HCl (3 N, from concentrated HCl,
100 mL) was added to 8 (3.66 g, 9.62 mmol) at 0 °C, and the
reaction mixture was stirred at room temperature for 1 day.
Solvent was removed under high vacuum. The residue was
dissolved in EtOH and concentrated. Toluene was added and
removed in vacuo to give 2.34 g (96%) of 9 as a hygroscopic
white solid: NMR (D2O) δ 3.47-3.52 (m, 4 H), 3.75 (s, 4 H),
3.85-3.90 (m, 4 H). Anal. (C6H18Cl2N2O4) C, H, N.
N1,N8-Bis(ben zyloxy)-3,6-d ioxa -1,8-octa n ed ia m in e (10).
A solution of 7 (6.00 g, 10.7 mmol) in CH3OH (144 mL) and
concentrated HCl (56 mL) was stirred at room temperature
for 20 h. The mixture was concentrated to dryness, H2O (50
mL) was added to the residue, the pH was adjusted to 9 with
saturated Na2CO3, and the aqueous layer was extracted with
EtOAc (3 × 100 mL). The combined organic extracts were
washed with H2O (2 × 50 mL), and the solvent was removed
in vacuo. Silica gel flash chromatography (1:1 cyclohexane/
EtOAc) afforded 3.25 g (84%) of 10 as a colorless oil: NMR δ
3.09 (t, 4 H, J ) 5.1), 3.58 (s, 4 H), 3.60 (t, 4 H, J ) 5.1), 4.70
(s, 4 H), 5.93 (br s, 2 H), 7.26-7.37 (m, 10 H). Anal.
(C20H28N2O4) C, H, N.
N1,N8-Bis(2-a cetoxyben zoyl)-N1,N8-bis(ben zyloxy)-3,6-
d ioxa -1,8-octa n ed ia m in e (11). A solution of acetylsalicyloyl
chloride (1.10 g, 5.54 mmol) in CH2Cl2 (7 mL) was added
dropwise under Ar to a solution of 10 (0.91 g, 2.52 mmol) and
DIEA (0.72 g, 5.57 mmol) in CH2Cl2 (30 mL). The mixture was
stirred at room temperature under Ar for 4 h, and CH2Cl2 (100
mL) was added. The organic layer was washed with 10% citric
acid (3 × 30 mL), saturated NaHCO3 (3 × 30 mL), H2O (2 ×
30 mL), and brine (30 mL), and the solvent was removed in
vacuo. Silica gel flash chromatography (5% then 10% acetone
in CH2Cl2) gave 1.54 g (89%) of 11 as a colorless oil: NMR δ
2.24 (s, 6 H), 3.62 (s, 4 H), 3.71 (m, 4 H), 3.84 (br s, 4 H), 4.69
(br s, 4 H), 6.95-7.29 (m, 14 H), 7.37-7.46 (m, 4 H). Anal.
(C38H40N2O10) C, H, N.
(S)-4,5-Dih yd r o-N-h yd r oxy-2-(3-h yd r oxy-2-p yr id in yl)-
N-(p h en ylm eth yl)-4-th ia zoleca r boxa m id e (4). BOP re-
agent (6.13 g, 13.9 mmol) was added to a solution of 1 (3.16 g,
14.1 mmol) and N-benzylhydroxylamine hydrochloride (2.01
g, 12.6 mmol) in DMF (130 mL). Diisopropylethylamine (DIEA;
7.0 mL, 40 mmol) in DMF (17 mL) was added dropwise at 0
°C to the reaction solution, which was stirred as it warmed to
room temperature for 1 day. Solvents were removed under
high vacuum, and the residue was treated with 0.4 M citric
acid (200 mL) and extracted with EtOAc (3 × 100 mL). The
organic extracts were washed with 100 mL: 1:1 saturated
NaHCO3/brine (2×), H2O (3×), and brine, and solvent was
removed by rotary evaporation. Purification of the residue on
a Sephadex LH-20 column, eluting with 4% EtOH/toluene,
produced 1.72 g (41%) of 4 as a yellow solid: mp 171-173 °C;
[R]25 -16.4° (c 0.52, CH3OH). Anal. (C16H15N3O3S) C, H, N.
D
(()-4. Reaction of 1 (7.03 g, 31.4 mmol) and N-benzylhy-
droxylamine hydrochloride (4.55 g, 28.5 mmol) under the above
conditions and recrystallization from aqueous EtOH gave 4.83
g (51%) of (()-4 as light-green needles: mp 175-177 °C; [R]21
D
-0.39° (c 0.52, CH3OH); NMR (CD3OD) δ 3.47 (dd, 1 H, J )
11, 9), 3.59-3.71 (m, 1 H), 4.84 (d, 1 H, J ) 2, second N-CH2-
Ph under OH), 6.01 (t, 1 H, J ) 9), 7.26-7.42 (m, 7 H), 8.13
(t, 1 H, J ) 3).
(S,S)-N1,N8-Bis[4,5-d ih yd r o-2-(3-h yd r oxy-2-p yr id in yl)-
4-t h ia zo y l]-N 1,N 8-d ih y d r o x y -3,6-d io x a -1,8-o c t a n e d i-
a m in e (5). BOP reagent (8.59 g, 19.4 mmol) was added to a
solution of 1 (4.26 g, 19.0 mmol) and 9 (2.23 g, 8.81 mmol) in
DMF (130 mL). DIEA (9.6 mL, 55 mmol) in DMF (23 mL) was
added dropwise at 0 °C to the reaction solution, which was
stirred as it warmed to room temperature for 1 day. Solvents
were removed under high vacuum, and the residue was treated
with 0.5 M citric acid (150 mL) and brine (50 mL) and was
extracted with EtOAc (4 × 100 mL). The organic extracts were
washed with 100 mL: 1:1 saturated NaHCO3/brine (2×), H2O
(4×), and brine; solvent was removed by rotary evaporation.
Purification of the residue on a Sephadex LH-20 column,
eluting with 4% EtOH/toluene, furnished 0.59 g (11%) of 5 as
a pale-green foam: [R]22 -8.7° (c 0.28, CH3OH); NMR (CD3-
D
OD) δ 3.45-3.98 (m, 16 H), 6.00 (t, 2 H, J ) 9), 7.38 (d, 4 H,
J ) 3), 8.09-8.15 (m, 2 H). Anal. (C24H28N6O8S2) C, H, N.
N1,N8-Bis(2-h ydr oxyben zoyl)-N1,N8-dih ydr oxy-3,6-dioxa-
1,8-octa n ed ia m in e (6). To a solution of 12 (455 mg, 0.76
mmol) in CH3OH (30 mL) was added 10% Pd-C (160 mg). The
mixture was stirred under H2 (1 atm) at room temperature
for 1 h. The catalyst was filtered, and the solvent was removed
in vacuo. Purification of the residue on a Sephadex LH-20
column, eluting with 4% EtOH/toluene, followed by lyophiliza-
tion gave 254 mg (81%) of 6 as a pale-yellow oil: NMR (CD3-
OD) δ 3.57 (s, 4 H), 3.73 (m, 4 H), 3.81 (m, 4 H), 6.86 (m, 4 H),
7.27 (m, 2 H), 7.44 (m, 2 H). Anal. (C20H24N2O8) C, H, N.
N1,N8-Bis(ben zyloxy)-N1,N8-bis(ter t-bu toxyca r bon yl)-
3,6-d ioxa -1,8-octa n ed ia m in e (7). NaH (60%, 3.46 g, 86.5
mmol) was added to N-(tert-butoxycarbonyl)-O-benzylhydroxy-
lamine (15.08 g, 67.5 mmol) in DMF (160 mL) at 0 °C. The
reaction mixture was stirred for 40 min at room temperature,
and 1,2-bis(2-chloroethoxy)ethane (5.0 mL, 32 mmol) was
introduced by syringe. After the flask was heated for 21 h at
53 °C, it was cooled in ice water; the reaction was quenched
with H2O (20 mL). Solvents were removed in vacuo, and the
residue was treated with dilute brine (200 mL) and EtOAc (250
mL). The aqueous portion was further extracted with EtOAc
(2 × 100 mL). The organic extracts were washed with H2O (2
× 100 mL) and brine (100 mL). After solvent removal, the solid
N1,N8-Bis(ben zyloxy)-N1,N8-bis(2-h yd r oxyben zoyl)-3,6-
d ioxa -1,8-octa n ed ia m in e (12). Sodium methoxide (0.5 M,
18.0 mL, 9.0 mmol) was added at 0 °C to a solution of 11 (1.45
g, 2.12 mmol) in CH3OH (50 mL). The mixture was stirred at
0 °C for 1 h. A solution of concentrated HCl (0.8 mL) in EtOH
(19.2 mL) was added, and the solvent was removed in vacuo.
Water (100 mL) was added to the residue, and the aqueous
layer was extracted with Et2O (4 × 80 mL). The combined
organic extracts were washed with H2O (2 × 50 mL) and brine
(50 mL), and the solvent was removed by rotary evaporation.
Silica gel flash chromatography (5% acetone in CH2Cl2) yielded
1.15 g (91%) of 12 as a colorless oil: NMR δ 3.61 (s, 4 H), 3.73
(t, 4 H, J ) 5.3), 3.89 (t, 4 H, J ) 5.3), 4.76 (s, 4 H), 6.80 (m,
2 H), 6.95 (m, 2 H), 7.18-7.38 (m, 12 H), 7.83 (m, 2 H). Anal.
(C34H36N2O8) C, H, N.
Ca n n u la tion of Bile Du ct in Ra ts. The cannulation has
been described previously.8,11,12 Briefly, male Sprague-Dawley
rats averaging 400 g were housed in Nalgene plastic metabolic
cages during the experimental period and given free access to
water. The animals were anesthetized using sodium pento-
barbital (55 mg/kg) given ip. The bile duct was cannulated
using 22-gauge polyethylene tubing. The cannula was inserted
into the duct about 1 cm from the duodenum and tied in place.

2886 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 15
Bergeron et al.
After threading through the shoulder, the cannula was passed
from the rat to the swivel inside a metal torque-transmitting
tether, which was attached to a rodent jacket around the
animal’s chest. The cannula was directed from the rat to a
Gilson microfraction collector (Middleton, WI) by a fluid swivel
mounted above the metabolic cage. Bile samples were collected
at 3-h intervals for 24 h. Urine samples were taken every 24
h. Sample collection and handling were as previously de-
scribed.8
Ack n ow led gm en t. Financial support was provided
by National Institutes of Health Grant No. RO1DK49108.
We thank Katie Ratliff-Thompson, Brian Raisler, Curt
Zimmerman, and Elizabeth Nelson for their technical
assistance and Dr. Eileen Eiler-Hughes for her editorial
comments.
Refer en ces
Ir on Loa d in g of C. a pella Mon k eys. C. apella monkeys
were given iron dextran iv as presented in earlier publica-
tions.7,11,13 Briefly, the iron dextran was administered to the
animals by slow infusion at a dose of 200-300 mg of iron/kg
of body weight over 45-60 min. Infusions were performed as
necessary to provide about 500 mg of iron/kg of body weight.
After administration of iron dextran, we waited at least 60
days before using any of the animals in experiments evaluating
iron-chelating agents.
P r im a te F eca l a n d Ur in e Sa m p les. Fecal and urine
samples were collected and processed as previously de-
scribed.7,11,13 The collections began 4 days prior to the admin-
istration of the test drug and continued for an additional 5
days after the drug was given. Iron concentrations were
determined by flame atomic absorption spectroscopy as de-
scribed in earlier publications.12,13
Dr u g P r ep a r a tion a n d Ad m in istr a tion . The iron chela-
tors were solubilized in 40% Cremophor RH-40/H2O (v/v) and
given po or sc to the rats at a dose of 150 µmol/kg. In the
primates, the compounds were solubilized in 40% Cremophor
RH-40/H2O (v/v) and given po or sc at a dose of 150, 225, or
300 µmol/kg as indicated in Table 1.
Ca lcu la tion of Ir on Ch ela tor Efficien cy. The efficiency
of each chelator was calculated on the basis of a 2:1 (com-
pounds 1, 2,9 and 4), 3:2 (3),9 or 1:1 (5 and 6) ligand:iron
complex. The efficiencies in the rodent model were calculated
by subtracting the iron excretion of control animals from the
iron excretion of treated animals. This number was then
divided by the theoretical output; the result is expressed as a
percentage. In the monkeys the numbers were generated 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 percentage.
(1) Bickel, H.; Hall, G. E.; Keller-Schierlein, W.; Prelog, V.; Vischer,
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(2) Lee, P.; Mohammed, N.; Marshall, L.; Abeysinghe, R. D.; Hider,
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(7) Bergeron, R. J .; Streiff, R. R.; Creary, E. A.; Daniels, R. D., J r.;
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A Comparative Study of the Iron-Clearing Properties of Desfer-
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(8) Bergeron, R. J .; Wiegand, J .; Dionis, J . B.; Egli-Karmakka, M.;
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(9) Bergeron, R. J .; Liu, C. Z.; McManis, J . S.; Xia, M. X. B.; Algee,
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(10) Ramasamy, K.; Olsen, R. K.; Emery, T. N-Methylation of
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rivatives. J . Org. Chem. 1981, 46, 5438-5441.
(11) Bergeron, R. J .; Wiegand, J .; Brittenham, G. M. HBED:
A
Potential Alternative to Deferoxamine for Iron-Chelating Therapy.
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(12) Bergeron, R. J .; Wiegand, J .; Wollenweber, M.; McManis, J . S.;
Algee, S. E.; Ratliff-Thompson, K. Synthesis and Biological
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(13) Bergeron, R. J .; Streiff, R. R.; Wiegand, J .; Vinson, J . R. T.;
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Toxicity in Rod en ts. Compound 2 was evaluated in a
chronic toxicity study in rodents.7 The compound was given
po at a dose of 384 µmol/kg/day for 10 days; 24 h after the
final dose, histopathological analysis of tissues (spleen, kidney,
liver, lung, esophagus, small intestine, stomach, large intes-
tine) was carried out by an outside pathologist.
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