22 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 1
Bergeron et al.
(5.78 g, 58%) as a yellow oil: 1H NMR δ 1.84 (m, 6 H), 2.67 (t,
4 H, J ) 7.5), 3.41 (t, 4 H, J ) 6.6), 3.46 (t, 4 H, J ) 6.3), 4.99
(s, 4 H), 5.01 (s, 4 H), 6.49 (dd, 2 H, J ) 8.1, 2.4), 6.58 (d, 2 H,
J ) 2.4), 7.04 (d, 2 H, J ) 8.1), 7.39 (m, 20 H); 13C NMR δ
26.31, 29.85, 30.20, 67.71, 69.75, 70.13, 70.42, 100.51, 105.16,
123.35, 127.00, 127.54, 127.69, 127.91, 128.48, 128.54, 130.18,
137.09, 137.23, 157.35, 158.18; HRMS m/z calcd for C49H53O6
737.3842 (M + H), found 737.3819.
1,11-Bis(2,4-d ib en zyloxy-5-for m ylp h en yl)-4,8-d ioxa -
u n d eca n e (9). Phosphorus oxychloride (5.808 g, 37.88 mmol)
in CH3CN (80 mL) was added dropwise to DMF (3.251 g, 44.47
mmol) and CH3CN (16 mL), and the mixture was stirred at
room temperature for 1 h. Compound 8 (12.14 g, 16.47 mmol)
in CH3CN (80 mL) was slowly added. The reaction mixture
was stirred at room temperature for 1 h, refluxed overnight,
and concentrated under reduced pressure. The residue was
treated with H2O (100 mL) and 1,4-dioxane (100 mL), heated
at 50 °C for 2 h, and concentrated in vacuo. The residue was
dissolved in EtOAc (500 mL), washed with brine (500 mL),
and concentrated by rotary evaporation. Chromatography (2:1
hexanes/EtOAc) gave 9 (7.96 g, 61%) as a white solid: 1H NMR
δ 1.82 (m, 6 H), 2.65 (t, 4 H, J ) 7.2), 3.40 (t, 4 H, J ) 6.3),
3.44 (t, 4 H, J ) 6.3), 5.09 (s, 4 H), 5.10 (s, 4 H), 6.49 (s, 2 H),
7.38 (m, 20 H), 7.65 (s, 2 H), 10.36 (s, 2 H); 13C NMR δ 26.16,
29.46, 30.18, 67.75, 70.18, 70.32, 70.79, 97.20, 118.56, 124.08,
126.98, 127.21, 128.14, 128.24, 128.71, 129.48, 136.14, 161.59,
162.78, 188.23; HRMS m/z calcd for C51H53O8 793.3740 (M +
H), found 793.3815.
1,11-Bis(5-cya n o-2,4-d ib e n zyloxyp h e n yl)-4,8-d ioxa -
u n d eca n e (10). A solution of 9 (20.42 g, 25.8 mmol), hydroxyl-
amine hydrochloride (3.95 g, 56.8 mmol), and Et3N (6.26 g,
61.9 mmol) in CH3CN (500 mL) was stirred at 45 °C overnight.
Phthalic anhydride (11.5 g, 77.4 mmol) was added, and the
mixture was heated at reflux overnight. After the solution was
concentrated under reduced pressure, the residue was diluted
with CH2Cl2 (600 mL) and washed with aqueous NaHCO3 (600
mL) and brine (600 mL). Solvent removal and chromatography
(3:1 hexanes/EtOAc) afforded 10 (15.64 g, 77%) as a white
solid: 1H NMR δ 1.80 (m, 6 H), 2.62 (t, 4 H, J ) 7.5), 3.38 (t,
4 H, J ) 6.3), 3.45 (t, 4 H, J ) 6.3), 5.03 (s, 4 H), 5.12 (s, 4 H),
6.47 (s, 2 H), 7.28 (s, 2 H), 7.36 (m, 20 H); 13C NMR δ 26.04,
29.24, 30.12, 67.71, 70.01, 70.14, 70.87, 93.46, 97.96, 117.09,
124.18, 126.95, 128.17, 128.20, 128.71, 133.98, 135.80, 135.88,
160.58, 160.93; HRMS m/z calcd for C51H51N2O6 787.3747 (M
+ H), found 787.3745.
1,11-Bis(5-cya n o-2,4-d ih yd r oxyp h en yl)-4,8-d ioxa u n d e-
ca n e (11). Palladium on activated carbon (10%, 3.14 g) was
added to a solution of 10 (5.23 g, 6.65 mmol) in EtOAc (500
mL) and iron-free EtOH (100 mL), and the suspension was
stirred under H2 (1 atm) at room temperature for 5.5 h. The
reaction mixture was heated on a steam bath and was filtered
through Celite. The filtrate was concentrated in vacuo; chro-
matography (20:3 CHCl3/CH3OH) gave 11 (2.55 g, 90%) as a
white solid: 1H NMR (DMSO-d6, 2.49) δ 1.69 (m, 6 H), 2.42
(t, 4 H, J ) 7.5), 3.30 (t, 4 H, J ) 6.6), 3.38 (t, 4 H, J ) 6.6),
6.47 (s, 2 H), 7.17 (s, 2 H), 10.30 (s, 2 H), 10.54 (s, 2 H); 13C
NMR (DMSO-d6, 39.50): δ 25.34, 28.99, 29.71, 67.06, 69.50,
88.82, 102.11, 117.97, 120.72, 133.40, 159.94, 160.60; HRMS
m/z calcd for C23H27N2O6 427.1869 (M + H), found 427.1845.
(dd, 1 H, J ) 9.3, 11.1), 5.34 (dd, 2 H, J ) 7.2, 9.3), 6.36 (s, 2
H), 7.03 (s, 2 H), 10.24 (br s, 2 H), 12.45 (br s, 2 H), 13.04 (br
s, 2 H); 13C NMR (DMSO-d6, 39.50) δ 25.50, 29.19, 29.78, 33.15,
67.17, 69.25, 75.91, 102.00, 107.64, 120.11, 131.49, 158.55,
160.17, 171.57, 171.95; HRMS m/z calcd for C29H35N2O10S2
635.1733 (M + H), found 635.1696. Anal. (C29H34N2O10S2) H,
N. C: calcd, 54.88; found, 54.17.
P r even tion of Ir on -Med ia ted Oxid a tion of Ascor ba te.
The iron chelators (NTA, L1, DFO, 1, and 2) were tested for
their ability to diminish the iron-mediated oxidation of ascor-
bate by the method of Dean and Nicholson.27 Briefly, a solution
of freshly prepared ascorbate (100 µM) in sodium phosphate
buffer (5 mM, pH 7.4) was incubated in the presence of FeCl3
(30 µM) and chelator (ligand/Fe ratios varied from 0 to 3) for
40 min. The A265 was read at 10 and 40 min; the ∆A265 in the
presence of ligand was compared to that in its absence.
Qu en ch in g of th e ABTS Ra d ica l Ca tion . The iron
chelators were tested for their ability to quench the radical
cation formed from 2,2′-azinobis(3-ethylbenzothiazoline-6-sul-
fonic acid) (ABTS) by a published method.47 Briefly, a stock
solution of ABTS radical cation was generated by mixing ABTS
(10 mM, 2.10 mL) with K2S2O8 (8.17 mM, 0.90 mL) in H2O
and allowing the solution to sit in the dark at room temper-
ature for 18 h. This stock solution of deep blue-green ABTS
radical cation was diluted in sufficient sodium phosphate (10
mM, pH 7.4) to give an A734 of about 0.900. Test compounds
were added to a final concentration ranging from 1.25 to 15
µM, and the decrease in A734 was read after 1, 2, 4, and 6 min.
Assays were performed in triplicate at each concentration. The
reaction was largely complete by 1 min, but the data presented
are based on a 6 min reaction time.
Stoich iom etr y of th e Liga n d /F e(III) Com p lex. The
stoichiometry of the complex was determined spectrophoto-
metrically for 2 at the λmax (529 nm) of the visible absorption
band of the ferric complex by the method given in detail in an
earlier publication.35 The J ob’s plot for mixtures containing
various ratios of ligand to Fe(III) NTA ([ligand] + [Fe] ) 1.00
mM constant) was then derived.
Ca n n u la tion of Bile Du ct in Ra ts. The cannulation has
been described previously.48,53,54 Briefly, male Sprague-Dawley
rats averaging 450 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) administered ip. The bile duct was can-
nulated using 22 gauge polyethylene tubing. The cannula was
inserted into the duct about 1 cm from the duodenum and tied
snugly in place. 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. The urine
sample was taken at 24 h. Sample collection and handling were
as previously described.48
Ir on Loa d in g of C. a pella Mon k eys. The monkeys were
iron-overloaded with iv iron dextran as previously described
to provide about 500 mg of iron per kg of body weight;49 the
serum transferrin iron saturation rose to 70-80%. We waited
at least 20 half-lives, 60 days,55 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 at 24 h intervals and processed as given
in detail in earlier publications.51,53,56 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 atomic absorp-
tion spectroscopy as accounted previously.54,56
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/water (v/v) and
given po and sc to the rats at the doses shown in Table 2. In
the primates, DFO was dissolved in sterile H2O at a concen-
tration of 50 or 100 mg/mL and given po or sc at a volume of
1 mL/kg. The desferrithiocin analogues were solubilized in 40%
(S,S)-1,11-Bis[5-(4-ca r boxy-4,5-d ih yd r oth ia zol-2-yl)-2,4-
d ih yd r oxyp h en yl]-4,8-d ioxa u n d eca n e (2). Distilled sol-
vents and glassware that had been presoaked in 3 N HCl for
15 min were employed. D-Cysteine hydrochloride monohydrate
(1.23 g, 7.02 mmol) was added to 11 (1.00 g, 2.34 mmol) in
degassed CH3OH (20 mL) and 0.1 M phosphate buffer at pH
6.052 (15 mL). Sodium bicarbonate (0.590 g, 7.02 mmol) was
carefully added, and the mixture was stirred at reflux for 2
days. The reaction mixture was concentrated under reduced
pressure, H2O was added, and the pH was adjusted to 2 by
addition of 10% citric acid solution. Solid was filtered and
recrystallized from aqueous EtOH to furnish 2 (0.81 g, 55%)
as a beige powder: [R]24D +3.1 (c 1.06, DMF); 1H NMR (DMSO-
d6, 2.49) δ 1.72 (m, 6 H), 2.48 (t, 4 H, J ) 7.2), 3.32 (t, 4 H, J
) 6.3), 3.41 (t, 4 H, J ) 6.3), 3.54 (dd, 1 H, J ) 7.2, 11.1), 3.61