Design, synthesis, and testing of polyamine vectored iron chelators

Article abstract of DOI:10.1055/s-0030-1258245

Iron chelators have been shown to control the growth of cancer cells in culture by sequestering exogenous iron in the media. Thus, the ligands prevent cellular access to the metal. However, because transferrin provides iron to tumor cells in animals, chelators have not been effective antitumor agents. Polyamine chelator conjugates in which the polyamine vectored ligands into cells were far more active than the free chelators themselves. However, the free ligands were not released from the vector once in the cell. The current study focuses on the synthesis and preliminary evaluation of a polyamine chelator conjugate capable of releasing the free ligand intracellularly via a nonspecific esterase.

Full text of DOI:10.1055/s-0030-1258245

PAPER
3631
Design, Synthesis, and Testing of Polyamine Vectored Iron Chelators
P
olyamine Vecto
a
redIron
C
h
y
elators mond J. Bergeron,* Shailendra Singh, Neelam Bharti, Yi Jiang
Department of Medicinal Chemistry, University of Florida, Box 100485 JHMHC, Gainesville, FL, 32610-0485, USA
Fax +1(352)3928406; E-mail: rayb@ufl.edu
Received 7 May 2010; revised 22 July 2010
consistent with the idea that charge is critical to transport-
er recognition of the polyamine analogues. We have in
fact been able to successfully exploit the polyamine trans-
port apparatus in delivering iron chelators intracellularly.
Abstract: Iron chelators have been shown to control the growth of
cancer cells in culture by sequestering exogenous iron in the media.
Thus, the ligands prevent cellular access to the metal. However, be-
cause transferrin provides iron to tumor cells in animals, chelators
have not been effective antitumor agents. Polyamine chelator con-
jugates in which the polyamine vectored ligands into cells were far
more active than the free chelators themselves. However, the free
ligands were not released from the vector once in the cell. The cur-
rent study focuses on the synthesis and preliminary evaluation of a
polyamine chelator conjugate capable of releasing the free ligand
intracellularly via a nonspecific esterase.
Initial experiments focused on the delivery of a simple bi-
dentate ligand, 1,2-dimethyl-3-hydroxypyridin-4-one
(L1, 2, Figure 1, Table 1) using polyamines as vectors.
The chelator itself does not achieve a high concentration
(~1 mM) in cultured cells10 (L1, 2). L1 was linked to sper-
midine via a propyl tether to generate 1-(12-amino-4,9-di-
azadodecyl)-2-methyl-3-hydroxy-4(1H)-pyridinone (3,
Figure 1). Job’s plots revealed the expected 3:1 ligand/
Fe(III) complex. In conjugate 3, the hydroxypyridone, the
chelating cargo fragment covalently bonded to the
polyamine, is neutral at physiological pH. All parameters
measured in murine leukemia L1210 cells treated with
conjugate 3, including (1) the effect on cell proliferation
(IC50 values), (2) the ability to compete with radiolabeled
spermidine for the polyamine transport apparatus (Ki), (3)
impact on polyamine pools, and (4) effects on the
polyamine enzymes L-ornithine decarboxylase (ODC), S-
adenosyl-L-methionine decarboxylase (AdoMetDC), and
spermidine/spermine/N1-acetyltransferase (SSAT), sug-
gested that this L1 analogue both competed well for the
polyamine transport apparatus and indeed gained intracel-
lular access (390 mM) in contrast to L1 (2) itself. This
finding encouraged us to explore additional iron chelator
polyamine adducts. The norspermidine adducts, (S)-4,5-
dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyl-
oxy)phenyl]-4-methyl-4-thiazolecarboxylic acid (4) and
its ester 5 (Table 1) were thus assembled and evaluated.9
Key words: iron, chelator, conjugate, polyamine vector, intracellu-
lar esterase
It is well established that iron chelators can significantly
reduce the growth of tumor cells in vitro.1 It has been
demonstrated that this reduction could be accounted for
by the inhibition of ribonucleotide reductase, as evidenced
by thymidine uptake studies and arrest of cell division at
the G1-S border.2 One major shortcoming with iron che-
lators as antineoplastics is that most of these ligands [e.g.,
desferrioxamine (DFO)] do not cross the cell membrane
particularly well.3 Thus, while chelators are effective as
growth inhibitors of tumor cells in culture, where the iron
sources are limited, this has not been the case in tumor xe-
nografts in whole animals.4 In the former instance, extra-
cellular iron in the culture medium is ligated, preventing
uptake of the metal and transformation of apo-ribonucle-
otide reductase to its active form. In the latter instance,
there is a continual and ample supply of protein-bound
iron, for example, as transferrin.
Again, the IC50 of 4 and 5 were measured in L1210 cells
and compared with the parent drug, (S)-2-(2,4-dihydroxy-
phenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid
(6, Figure 1). The parent acid 6 presented with IC50 values
of 20 mM at 96 hours, the polyamine adduct ethyl ester 5
had a 96-hour IC50 of 1.5 mM, and the corresponding
polyamine free acid conjugate 4 had a 40 mM IC50 at this
time point (Table 1). The parent acid 6 did not effectively
accumulate in L1210 cells (<50 mM). The acid polyamine
adduct 4 achieved levels of <95 mM. However, the ester
adduct 5 reached intracellular levels of ~1000 mM. Fur-
thermore, concentrations of ~3000 mM of the free acid ad-
duct 4 were seen in these same cells, obviously deriving
from nonspecific serum esterase hydrolysis of the parent
ester 5.9 Interestingly, the polyamine ester adduct 5 com-
peted well for the polyamine transporter, Ki 5.7 mM, while
the acid conjugate 4 did not, Ki 73 mM. In keeping with the
idea, negatively charged cargo fragments fixed to
For intracellular chelation to be a therapeutically signifi-
cant antineoplastic strategy, higher levels of chelator must
be transported into the cell. We elected to exploit
polyamines as vectors to achieve this goal. However, in
each instance, the polyamine vector was linked to the
chelator such that the free ligand could not be liberated in-
tracellularly. The sustained increases in polyamine bio-
synthesis in preneoplastic and neoplastic tissues led to a
great deal of attention focused on the polyamine biosyn-
thetic network as a target in antineoplastics therapy.5 In
the course of developing N-alkylated analogues of the nat-
ural polyamines as anticancer drugs,6,7 the structural
boundary conditions set by the polyamine transporter on
these substrates were defined. All of the findings8,9 are
SYNTHESIS 2010, No. 21, pp 3631–3636
x
x.
x
x
.
2
0
1
0
Advanced online publication: 03.09.2010
DOI: 10.1055/s-0030-1258245; Art ID: M03410SS
© Georg Thieme Verlag Stuttgart · New York

3632
R. J. Bergeron et al.
PAPER
O
OH
H2N
N
H
N
H
N
N
N
H
N
H
H
H
N
N
1
COOH
S
4
6
O
OH
S
HO
OH
H2N
N
H
N
H
Me
N
O
N
OH
COOEt
COOH
S
2
5
H2N
N
N
H
H
O
OH
S
H
N
N
O
•3HCl
H2N
N
O
N
H
OH
COOEt
3
7
Figure 1 Polyamine analogue DENSPM (1), chelators 2 and 6, and polyamine analogue chelator conjugates 3, 4, 5, 7
Table 1 L1210 Cell Growth Inhibition and Transport for Selected
Polyamines, Iron Chelators, and Polyamine Conjugate Iron Chelators
er, the conjugates studied do not allow for the release of
the cargo molecule. They remain attached to the
polyamine vector and/or one of the vector’s metabolites.9
This encouraged the design of a system in which the
polyamine cargo linkage would allow for intracellular re-
lease of an active fragment. The simplest concept revolves
around the idea that intracellular esterase can be exploited
to cleave cargo molecules fixed to polyamine vectors as
esters. The first element of the program was to explore the
accessibility and stability of such molecules. It is not the
purpose of the study to develop a ‘more active’ chelator
conjugate, but rather, to assess whether or not a labile
polyamine conjugate ester can be assembled that utilizes
the polyamine transporter. The ultimate goal is to articu-
late a polyamine iron chelator conjugate that will release
the free chelator once in the cell.
Compound structure/abbreviation
IC50 (mM)
Ki (mM)
48 h
96 h
2
DENSPM (1)
20
46
0.2
40
1.5
19
17
L1 (2)
55
>500
3.7
L1-SPM-conjugate (3)
NSPD-(S)-4¢-(HO)-DADFT (4)
NSPD-(S)-4¢-(HO)-DADFT-EE (5)
(S)-4¢-(HO)-DADFT (6)
0.2
40
73
1.5
20
5.7
>500
4.0
NSPDBn-(S)-4¢-(HO)-DADFT-EE (7) 1.8
2.1
One of the concerns was, of course, that a polyamine car-
rier with a labile ester linkage could, by its very nature,
cyclize to amides. In order to overcome this, the molecule
was designed to render cyclization difficult because of
steric hindrance. Thus the target molecule, ethyl (S)-4,5-
dihydro-2-{2-hydroxy-4-[4-(9-amino-2,6-diazanonyl)ben-
zoyloxy]}-4-methyl-4-thiazolecarboxylate (7, Table 1,
Scheme 1), consists of iron chelator 6, masked as its ethyl
ester, and norspermidine covalently linked by a spacer.
Moreover, the cargo molecule is neutral.
polyamine vectors, for example, the carboxylate of 4 at
physiologic pH, are not well transported. It was clear that
cargo molecules needed to be neutral or positively
charged. In the same set of experiments, we were also able
to demonstrate that the norspermidine backbones of the
chelator conjugates were metabolized. Specifically, the 3-
aminopropyl segments were successively removed, re-
sulting in a diamine and a monoamine. Also the primary
amines of the parent conjugates and the diamine metabo-
lite were oxidized, resulting in dicarboxylic acids. How-
ever we were unable to demonstrate any transformation of
the final 4-aminobutyl metabolite.9 These findings forced
the question of whether a polyamine chelator conjugate
could be assembled such that the chelating unit could be
released from the cargo intracellularly. The current study
focuses on the synthesis of such a conjugate with a brief
description of the ligand’s biological properties. The vec-
tored molecule is designed for nonspecific esterase re-
lease of the iron ligand from the cargo.
The para-disubstituted aromatic ring fixed to the
polyamine vector of ‘the presumed esterase labile’
polyamine chelator conjugate sets significant restrictions
on potential intramolecular cyclization. The benzylic ni-
trogen cannot close onto the benzoate ester for steric rea-
sons. Similar constraints exist for cyclocondensation of
the internal norspermidine nitrogen with the benzoate es-
ter. While it is true in principle that the primary amine of
7 could form a 15-membered ring by displacing a mole-
cule of 13 (Scheme 1), the ethyl ester of chelator 6
(Figure 1), the ensuing decrease in entropy would be un-
favorable. Similar arguments can be made against nitro-
gen closures onto the thiazoline ester.
Labile Polyamine Vectors
The above findings support the idea that polyamines can
be utilized to vector various molecules into cells. Howev-
Synthesis 2010, No. 21, 3631–3636 © Thieme Stuttgart · New York

PAPER
Polyamine Vectored Iron Chelators
3633
O
O
b
a
S
HN
N
S
N
O
N
N
NH2
O
H
H
H
O
O
O
O
8
9
O
O
S
c, d
HN
N
R
N
O
N
N
N
O
H
R
R
O
S O
COOH
11 R = H⋅HBr
12 R = Boc
O
10
O
HO
OH
S
N
HN
R
N
R
N
COOEt
13
R
O
OH
S
e
f
C
O
N
14 R = Boc
7 R = H⋅HCl
COOEt
Scheme 1 Reagents and conditions: (a) mesitylenesulfonyl chloride, aq 1 N NaOH, CH2Cl2, 67%; (b) 4-(tert-butoxycarbonyl)benzyl bromide,
60% NaH, DMF, 70 °C, 70%; (c) 30% HBr in AcOH, PhOH, CH2Cl2, 84%; (d) Boc2O, aq THF, Et3N, 85%; (e) EDAC, DMAP, CH2Cl2, 55%;
(f) TFA, CH2Cl2, ion exchange, aq 1 N HCl, quant.
N1-(tert-Butoxycarbonyl)norspermidine (8)11 was reacted provide NSPDBn-(S)-4¢-(HO)-DADFT-EE (7) as its tri-
with mesitylenesulfonyl chloride (2 equiv) under biphasic hydrochloride salt in quantitative yield (Scheme 1).
conditions to produce N1,N4-bis(mesitylenesulfonyl)-N7-
(tert-butoxycarbonyl)norspermidine (9) in 67% yield
Preliminary Bioassays
Two pieces of data are consistent with the idea that the
polyamine vector is indeed working as planned; the 48-
hour and 96-hour IC50 values are 1.8 and 2.1 mM, respec-
tively, for conjugate 7. Recall that the parent ligand 6 had
19 and 20 mM IC50s at 48 hours and 96 hours. There is a
notable and consistent observation regarding IC50 values
of polyamine analogues, for example, DENSPM (1), ver-
sus chelators 2 and 6, and polyamine analogue chelator
conjugates 3, 4, 5, 7 (Table 1). Comparing the 48- versus
96-hour IC50 values for the polyamine analogues, for ex-
ample, 1, the 96-hour values are always much lower. We
have seen this consistently over the years with nearly all
polyamine analogues.7f However, L1210 cells exposed to
chelators or polyamine chelator conjugates always
present with very similar 48- and 96-hour IC50 values.9
This is consistent with the idea that the ethyl ester chelator
conjugate 7 is gaining intracellular access and the chelator
is being released. Finally, further underscoring this idea,
while the parent ligand 6 did not compete well for the
polyamine transporter Ki >500 mM, the conjugate 7 did,
Ki = 4.0 mM.
(Scheme 1). The difference in acidic strength12 between
the sulfonamide and carbamate hydrogens on 9 allowed
monoalkylation of this norspermidine system. Specific
deprotonation of 9 with NaH (1.1 equiv) and treatment
with 4-(tert-butyloxycarbonyl)benzyl bromide13 in DMF
at 70 °C provided tert-butyl ester 10 in 70% yield. The
four protecting groups of 10 were removed with 30% HBr
in acetic acid and phenol in CH2Cl2, giving N1-(4-carbox-
ybenzyl)norspermidine trihydrobromide (11) in 84%
yield. The amino groups were masked as their tert-butyl
carbamates utilizing di-tert-butyl dicarbonate and Et3N in
aqueous THF, affording N1,N4,N7-tris(tert-butoxycarbon-
yl)-N1-(4-carboxybenzyl)norspermidine (12) in 85%
yield. Ethyl (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-
methyl-4-thiazolecarboxylate (13),9 in which the carbox-
ylate group of (S)-4¢-(HO)-DADFT (6) is protected, has
been alkylated at the less hindered 4¢-hydroxy in the pres-
ence of the 2¢-hydroxy, an iron chelating site, providing
numerous DFT analogues.14 A corresponding selective
acylation of 13 with substituted benzoic acid 12 using N-
(3-dimethylaminopropyl)-N¢-ethylcarbodiimide hydro-
chloride (EDAC) with 4-(dimethylamino)pyridine
(DMAP) in CH2Cl2 furnished diester 14 in 55% yield. The
Boc protecting groups of 14 were removed with trifluoro-
acetic acid (TFA) in CH2Cl2, followed by ion exchange to
Studies now underway include: 1) assessment of the intra-
cellular concentration of free ligand 6 as a function of ex-
ternal adduct 7 concentration, 2) evaluation the
intracellular metabolites, 3) an assessment of the impact
of the conjugate 7 on polyamine pools, and 4) a study the
Synthesis 2010, No. 21, 3631–3636 © Thieme Stuttgart · New York

3634
R. J. Bergeron et al.
PAPER
1H NMR: d = 1.42 [s, 9 H, (CH3)3C], 1.51–1.56 (m, 2 H,
CH2CH2CH2), 1.59 [s, 9 H, (CH3)3C], 1.61–1.65 (m, 2 H,
CH2CH2CH2), 2.29 (s, 3 H, CH3Ar), 2.32 (s, 3 H, CH3Ar), 2.51 (s,
6 H, 2 CH3Ar), 2.59 (s, 6 H, 2 CH3Ar), 2.87 (t, J = 7.2 Hz, 2 H,
CH2NH), 2.91–2.99 (m, 4 H, 2 CH2N), 3.07 (t, 6.8 Hz, 2 H, CH2N),
4.26 (s, 2 H, NCH2Ar), 4.48 (br s, 1 H, NHCO), 6.91 (s, 2 H, Ar),
6.97 (s, 2 H, Ar), 7.09 (d, J = 8.4 Hz, 2 H, Ar), 7.88 (d, J = 8.4 Hz,
2 H, Ar).
13C NMR: d = 21.04, 21.07, 22.88, 22.99, 24.68, 27.48, 28.27,
28.50, 37.43, 42.89, 43.00, 43.04, 49.38, 79.79, 81.31, 128.42,
129.85, 131.75, 132.13, 132.23, 132.93, 133.12, 140.10, 140.24,
140.32, 142.69, 142.93, 155.97, 165.39.
impact of adduct 7 on key polyamine biosynthetic en-
zymes. This protracted biological screen is the subject of
another paper.
Reagents were purchased from Aldrich Chemical Co. (Milwaukee,
WI, USA). Fisher Optima grade solvents were routinely used, and
DMF was distilled. Reactions were run under a N2 atmosphere, and
organic extracts were dried with Na2SO4. Silica gel 40–63 from
SiliCycle, Inc. (Quebec City, Quebec, Canada) was used for column
chromatography. Glassware that was presoaked in aq 3 N HCl for
15 min, washed with distilled H2O and distilled EtOH, and oven-
dried was used during the isolation of 7. Sephadex LH-20 was ob-
tained from Amersham Biosciences (Piscataway, NJ, USA) and
AG1-X8 (hydroxide form) anion exchange resin from Bio-Rad
Laboratoties, Inc. (Hercules, CA, USA) was used for ion exchange
chromatography. Optical rotations were run at 589 nm (sodium D
line) and 20 °C on a Perkin-Elmer 341 polarimeter, with c being
concentration in grams of compound per 100 mL of solution. NMR
spectra were obtained at 400 MHz (1H) or 100 MHz (13C) on a Vari-
an Mercury 400BB. Chemical shifts (d) for 1H spectra are given in
parts per million downfield from TMS for CDCl3 (not indicated) or
sodium 3-(trimethylsilyl)propionate-2,2,3,3-d4 for D2O. Chemical
shifts (d) for 13C spectra are given in parts per million referenced to
the residual solvent resonance (d = 77.16) in CDCl3 (not indicated)
or to 1,4-dioxane (d = 67.19) in D2O. The base peaks are reported
for the ESI-FTICR mass spectra.
HRMS: m/z calcd for C41H60N3O8S2: 786.3777 (M + H)+; found:
786.3795.
N1,N4,N7-Tris(tert-butoxycarbonyl)-N1-(4-carboxybenzyl)nor-
spermidine (12)
Formation of 11: HBr (30% in HOAc, 100 mL) was added dropwise
to a solution of 10 (5.0 g, 6.4 mmol) and phenol (24.1 g, 0.256 mol)
in CH2Cl2 (200 mL) with ice bath cooling. The reaction mixture was
stirred for 96 h at r.t. and was quenched with dropwise addition of
H2O (100 mL) under ice bath cooling. The layers were separated
and the aqueous layer was further extracted with CH2Cl2 (3 × 100
mL). The aqueous portion was evaporated under high vacuum to
give 2.70 g (84%) of 11 as a light colored solid; mp 280–282 °C.
1H NMR (D2O): d = 2.05–2.18 (s, 4 H, 2 CH2CH2CH2), 3.11 (t,
J = 8.0 Hz, 2 H, CH2ND), 3.16–3.25 (m, 6 H, 2 CH2N + CH2ND),
4.35 (s, 2 H, NDCH2Ar), 7.59 (d, J = 7.6 Hz, 2 H, Ar), 8.06 (m,
J = 7.6 Hz, 2 H, Ar).
N1,N4-Bis(mesitylenesulfonyl)-N7-(tert-butoxycarbonyl)nor-
spermidine (9)
A solution of mesitylenesulfonyl chloride (2.19 g, 10.0 mmol) in
CH2Cl2 (30 mL) was added dropwise to a mixture of 811 (1.16 g, 5.0
mmol), aq 1 N NaOH (43 mL), and CH2Cl2 (20 mL) with ice bath
cooling. The mixture was stirred at r.t. for 16 h. The layers were sep-
arated, and the aqueous phase was further extracted with CH2Cl2
(2 × 50 mL). The combined organic extracts were washed with aq 1
N NaOH (50 mL), H2O (50 mL), and brine (50 mL). After solvent
removal in vacuo, flash chromatography, eluting with 33% EtOAc
in CHCl3 furnished 2.0 g (67%) of 9 as a colorless oil.
1H NMR: d = 1.41 [s, 9 H, (CH3)3C], 1.54–1.60 (m, 2 H,
CH2CH2CH2), 1.64–1.71 (m, 2 H, CH2CH2CH2), 2.30 (s, 6 H, 2
CH3Ar), 2.56 (s, 6 H, 2 CH3Ar), 2.61 (s, 6 H, 2 CH3Ar), 2.88 (q,
J = 6.4 Hz, 2 H, CH2NH), 2.94 (q, J = 6.4 Hz, 2 H, CH2N), 3.10 (t,
J = 6.8 Hz, 2 H, CH2N), 3.28 (q, J = 6.8 Hz, 2 H, CH2NH), 4.33 (br
s, 1 H), 4.92 (t, J = 6.8 Hz, 1 H), 6.95 (s, 4 H, Ar).
HRMS: m/z calcd for C14H23N3O2: 266.1863 (M + H)+ (free amine);
found: 266.1876.
Conversion of 11 to the Boc Derivative 12: Et3N (1.1 mL, 7.9 mmol)
and di-tert-butyl dicarbonate (0.78 g, 3.6 mmol) in THF (60 mL)
were added successively to 11 (0.54 g, 1.06 mmol) in 5% aq THF
(15 mL) with ice bath cooling, and the reaction mixture was stirred
for 16 h at r.t. Following solvent removal by rotary evaporation, the
residue was dissolved in EtOAc (75 mL) and washed with aq 0.25
M citric acid (50 mL), H2O (50 mL) ,and brine (50 mL). After sol-
vent removal in vacuo, flash chromatography eluting with 5%
MeOH–CHCl3 gave 0.51 g (85%) of 12 as a colorless oil.
1H NMR: d = 1.43 [s, 27 H, 3 (CH3)3C], 1.58–1.78 (m, 4 H,
CH2CH2CH2), 3.08–3.20 (m, 8 H, CH2NH + 3 CH2N), 4.47 (s, 2 H,
NCH2Ar), 7.29 (br s, 2 H, Ar), 8.04 (d, J = 7.2 Hz, 2 H, Ar).
13C NMR: d = 27.55, 28.41, 37.42, 43.82, 44.45, 44.91, 49.99,
50.58, 79.06, 79.85, 80.26, 125.49, 127.03, 128.42, 129.22, 130.57,
144.50, 155.75, 156.18, 166.61.
HRMS: m/z calcd for C29H48N3O8: 566.3436 (M + H)+; found:
566.3444.
13C NMR: d = 21.01, 21.05, 22.96, 22.98, 23.04, 27.57, 27.75,
28.38, 28.74, 37.56, 39.48, 42.97, 43.08, 79.30, 80.52, 125.39,
128.31, 129.12, 132.06, 132.23, 132.26, 133.10, 134.00, 139.00,
140.07, 142.16, 142.90, 155.97.
HRMS: m/z calcd for C29H46N3O6S2: 596.2783 (M + H)+; found:
596.2744.
Ethyl (S)-4,5-Dihydro-2-(2-hydroxy-4-{4-[(9-tert-butoxycarbo-
nylamino)-2,6-bis(tert-butoxycarbonyl)-2,6-diazanonyl]ben-
zoyloxy})-4-methyl-4-thiazolecarboxylate (14)
N1,N4-Bis(mesitylenesulfonyl)-N7-(tert-butoxycarbonyl)-N1-[4-
(tert-butoxycarbonyl)benzyl]norspermidine (10)
Compounds 12 (0.27 g, 0.48 mmol), 13 (0.141 g, 0.50 mmol), and
DMAP (0.0122 g, 0.11 mmol) were dissolved in anhyd CH2Cl2 (50
mL). EDAC (0.10 g, 0.52 mmol) was added and the mixture was
stirred at r.t. for 20 h. After solvent was removed by rotary evapo-
ration, the residue was combined with aq 0.25 M citric acid–brine
(1:1, 50 mL) and extracted with EtOAc (3 × 50 mL). The organic
extracts were washed with H2O (50 mL) and brine (50 mL). Solvent
removal and column chromatography on silica gel eluting with 10%
EtOAc in CH2Cl2 gave 220 mg (55%) of 14 as a viscous pale oil;
[a]D20 +15.2 (c 0.25, CHCl3).
NaH (60%, 0.44 g, 11.0 mmol) was added in portions to a solution
of 9 (5.9 g, 9.9 mmol) in DMF (100 mL) with ice bath cooling, and
the mixture was stirred at 0 °C for 20 min. A solution of 4-(tert-bu-
tyloxycarbonyl)benzyl bromide13 (2.82 g, 10.4 mmol) in DMF (50
mL) was added to the reaction mixture dropwise over 30 min, and
the mixture was stirred for 12 h at 70 °C. After solvent removal by
rotary evaporation, H2O (100 mL) was added to the residue and ex-
tracted with CHCl3 (4 × 75 mL). The organic phase was concentrat-
ed in vacuo, and column chromatography, eluting with 3:3:1
hexanes–CHCl3–EtOAc, gave 5.44 g (70%) of 10 as a white solid;
mp 65–66 °C.
1H NMR: d = 1.31 (t, J = 7.2 Hz, 3 H, CH3CH2), 1.44 [s, 27 H, 3
(CH3)3C], 1.61–1.82 (s + m, 7 H, 4-CH3 + 2 CH2CH2CH2), 3.05–
Synthesis 2010, No. 21, 3631–3636 © Thieme Stuttgart · New York

PAPER
Polyamine Vectored Iron Chelators
3635
3.21 (m, 8 H, CH2NH + 3 CH2N), 3.24 (d, J = 11.2 Hz, 1 H, H-5),
3.89 (d, J = 11.2 Hz, 1 H, H-5), 4.26 (dq, J = 4.0, 7.2 Hz, 2 H,
CH2CH3), 4.51 (s, 2 H, NCH2Ar), 6.77 (dd, J = 2.4, 8.4 Hz, 1 H,
Ar), 6.88 (d, J = 2.0 Hz, 1 H, Ar), 7.36 (br s, 2 H, Ar), 7.45 (d,
J = 8.8 Hz, 1 H, Ar), 8.14 (d, J = 8.0 Hz, 2 H, Ar), 12.72 (s, 1 H,
ArOH).
13C NMR: d = 14.29, 21.24, 24.61, 28.63, 37.62, 40.12, 44.01,
44.50, 45.19, 50.79, 62.22, 79.09, 79.91, 80.38, 83.58, 110.63,
112.96, 114.34, 127.22, 127.85, 128.34, 130.76, 131.67, 145.17,
154.88, 155.61, 156.05, 160.79, 164.49, 171.21, 172.72.
and neutralized prior to scintillation counting. Lineweaver–Burk
plots indicated simple competitive inhibition with respect to SPD.
Acknowledgment
Funding was provided by the National Institutes of Health Grant
No. R37DK49108. We thank Hua Yao and Elizabeth M. Nelson for
their technical assistance. We also thank Dr. James S. McManis and
Miranda Coger for their editorial and organizational support. We
acknowledge the spectroscopy services in the Chemistry Depart-
ment, University of Florida, for the mass spectrometry analyses.
HRMS: m/z calcd for C42H61N4O11S: 829.4052 (M + H)+; found:
829.4037.
References
Ethyl (S)-4,5-Dihydro-2-{2-hydroxy-4-[4-(9-amino-2,6-diaza-
nonyl)benzoyloxy]}-4-methyl-4-thiazolecarboxylate Trihydro-
chloride (7)
(1) (a) Bergeron, R. J. Trends Biochem. Sci. 1986, 11, 133.
(b) Bergeron, R. J.; Braylan, R.; Goldey, S.; Ingeno, M. J.
Biochem. Biophys. Res. Commun. 1986, 136, 273.
(c) Hershko, C. Bailliere’s Clin. Haematol. 1994, 7, 965.
(d) Chenoufi, N.; Drenou, B.; Loreal, O.; Pigeon, C.; Brissot,
P.; Lescoat, G. Biochem. Pharmacol. 1998, 56, 431.
(2) (a) Bergeron, R. J.; Ingeno, M. J. Cancer Res. 1987, 47,
6010. (b) Taetle, R.; Honeysett, J. M.; Bergeron, R. J.
J. Natl. Cancer Inst. 1989, 81, 1229. (c) Nyholm, S.; Mann,
G. J.; Johannson, A. G.; Bergeron, R. J.; Graslund, A.;
Thelander, L. J. Biol. Chem. 1993, 268, 26200.
(3) Porter, J. B.; Gyparaki, M.; Burke, L. C.; Huehns, E. R.;
Sarpong, P.; Saez, V.; Hider, R. C. Blood 1988, 72, 1497.
(4) Selig, R. A.; White, L.; Gramacho, C.; Sterling-Levis, K.;
Fraser, I. W.; Naidoo, D. Cancer Res. 1998, 58, 473.
(5) (a) Marton, L. J.; Pegg, A. E. Annu. Rev. Pharm. Toxicol.
1995, 35, 55. (b) Thomas, T.; Balabhadrapathruni, S.; Gallo,
M. A.; Thomas, T. J. Oncol. Res. 2002, 13, 123.
Freshly distilled TFA (3 mL) in CH2Cl2 (5 mL) was added dropwise
to 14 (0.157 g, 0.189 mmol) in CH2Cl2 (10 mL) with ice bath cool-
ing. The mixture was stirred at 0 °C for 1 h and at r.t. for 1 h, and
volatiles were removed by rotary evaporation. The residue was
passed through a short Sephadex LH-20 column, eluting with 2% aq
EtOH. The iron active band was lyophilized and passed through an
ion exchange column (AG1-X8, hydroxide form) eluting with aq 1
N HCl to yield 125 mg (quant) of 7 as a white solid; mp 267–269
°C; [a]D20 +57.5 (c 0.12, H2O).
1H NMR (D2O): d = 1.33 (t, J = 6.8 Hz, 3 H, CH3CH2), 1.74 (m, 3
H, 4 CH3), 2.08–2.22 (m, 4 H, 2 CH2CH2CH2), 3.12–3.27 (m, 8 H,
4 CH2N), 3.49 (d, J = 11.6 Hz, 1 H, H-5), 3.92 (d, J = 12 Hz, 1 H,
H-5), 4.32 (q, J = 6.8 Hz, 2 H, CH2CH3), 4.40 (s, 2 H, NCH2Ar),
6.91–6.93 (d + s, J = 9.2 Hz, 2 H, Ar), 7.66 (d, J = 8.4 Hz, 2 H, Ar),
7.70 (d, J = 8.4 Hz, 1 H, Ar), 8.28 (d, J = 7.6 Hz, 2 H, Ar).
13C NMR (D2O): d = 13.90, 23.33, 24.03, 24.38, 37.13, 40.13,
44.96, 45.25, 45.36, 51.35, 63.74, 83.35, 110.71, 113.85, 114.81,
130.14, 130.79, 131.39, 132.57, 137.23, 154.82, 159.90, 166.43,
172.85, 175.12.
HRMS: m/z calcd for C27H37N4O5S: 529.2479 (M + H)+ (free
amine); found: 529.2489.
(c) Wallace, H. M.; Fraser, A. V. Biochem. Soc. Trans. 2003,
31, 393.
(6) (a) Porter, C. W.; McManis, J.; Casero, R. A.; Bergeron, R.
J. Cancer Res. 1987, 47, 2821. (b) Bergeron, R. J.; Neims,
A. H.; McManis, J. S.; Hawthorne, T. R.; Vinson, J. R. T.;
Bortell, R.; Ingeno, M. J. J. Med. Chem. 1988, 31, 1183.
(c) Pegg, A. E.; Madhubala, R.; Kameji, T.; Bergeron, R. J.
J. Biol. Chem. 1988, 263, 11008. (d) Porter, C. W.;
Bergeron, R. J. Adv. Enzyme Regul. 1988, 27, 57.
Preparation of Cell Culture
Murine L1210 leukemia cells were maintained in logarithmic
growth as a suspension culture in RPMI-1640 medium (Gibco,
Grand Island, NY, USA) containing 10% fetal bovine serum
(Gibco), 2% HEPES-MOPS buffer, 1 mM L-glutamine (Gibco), and
1 mM aminoguanidine at 37 °C in a water-jacketed 5% CO2 incu-
bator.
(e) Bergeron, R. J.; Hawthorne, T. R.; Vinson, J. R. T.; Beck,
D. E. Jr.; Ingeno, M. J. Cancer Res. 1989, 49, 2959.
(7) (a) Libby, P. R.; Bergeron, R. J.; Porter, C. W. Biochem.
Pharmacol. 1989, 38, 1435. (b) Porter, C. W.; Pegg, A. E.;
Ganis, B.; Madhabala, R.; Bergeron, R. J. Biochem. J. 1990,
268, 207. (c) Bernacki, R. J.; Bergeron, R. J.; Porter, C. W.
Cancer Res. 1992, 52, 2424. (d) Bergeron, R. J.; McManis,
J. S.; Liu, C. Z.; Feng, Y.; Weimar, W. R.; Luchetta, G. R.;
Wu, Q.; Ortiz-Ocasio, J.; Vinson, J. R. T.; Kramer, D.;
Porter, C. J. Med. Chem. 1994, 37, 3464. (e) Bergeron, R.
J.; McManis, J. S.; Weimar, W. R.; Schreier, K. M.; Gao, F.;
Wu, Q.; Ortiz-Ocasio, J.; Luchetta, G. R.; Porter, C.; Vinson,
J. R. T. J. Med. Chem. 1995, 38, 2278. (f) Bergeron, R. J.;
Feng, Y.; Weimar, W. R.; McManis, J. S.; Dimova, H.;
Porter, C.; Raisler, B.; Phanstiel, O. J. Med. Chem. 1997, 40,
1475. (g) Bergeron, R. J.; Müller, R.; Bussenius, J.;
McManis, J. S.; Merriman, R. L.; Smith, R. E.; Yao, H.;
Weimar, W. R. J. Med. Chem. 2000, 43, 224. (h) Bergeron,
R. J.; Müller, R.; Huang, G.; McManis, J. S.; Algee, S. E.;
Yao, H.; Weimar, W. R.; Wiegand, J. J. Med. Chem. 2001,
44, 2451.
IC50 Determination
Cells were grown in 25 cm2 tissue culture flasks in a total volume of
10 mL. Culture were treated during logarithmic growth (0.5–
1.0 × 105 cells/mL) with the compounds of interest, reseeded, and
incubated as described previously.7g Cell counting and calculation
of percent of control growth were also carried out as given in an ear-
lier publication.7g The IC50 is defined as the concentration of com-
pound necessary to reduce cell growth to 50% of control growth
after defined intervals of exposure.
Uptake Determination; General Procedure
The molecules of interest were studied for their ability to compete
with [3H]SPD for uptake into L1210 leukemia cell suspension in
vitro as given in details in previous publications.6e,7d,g Briefly, cell
suspension were incubated in 1 mL of culture medium containing
radiolabeled SPD alone or radiolabeled SPD in the presence of
graduated concentration of a molecule. At the end of the incubation
period the tubes were centrifuged; the pellets was washed, digested,
(8) Delcros, J.-G.; Tomasi, S.; Carrington, S.; Martin, B.;
Renault, J.; Blagbrough, I. S.; Uriac, P. J. Med. Chem. 2002,
45, 5098.
Synthesis 2010, No. 21, 3631–3636 © Thieme Stuttgart · New York

3636
R. J. Bergeron et al.
PAPER
(9) Bergeron, R. J.; Bharti, N.; Wiegand, J.; McManis, J. S.;
Yao, H.; Prokai, L. J. Med. Chem. 2005, 48, 4120.
(10) Bergeron, R. J.; McManis, J. S.; Franklin, A. M.; Yao, H.;
Weimar, W. R. J. Med. Chem. 2003, 46, 5478.
(11) Krapcho, A. P.; Kuell, C. S. Synth. Commun. 1990, 20, 2559.
(12) (a) Bordwell, F. G. Acc. Chem. Res. 1988, 21, 456.
(b) Nyasse, B.; Grehn, L.; Ragnarsson, U.; Maia, H. L. S.;
Monteiro, L. S.; Leito, I.; Koppel, I.; Koppel, J. J. Chem.
Soc., Perkin Trans. 1 1995, 2025.
(13) Liu, W.-Q.; Vidal, M.; Gresh, N.; Roques, B. P.; Garbay, C.
J. Med. Chem. 1999, 42, 3737.
(14) (a) Bergeron, R. J.; Wiegand, J.; McManis, J. S.; Bussenius,
J.; Smith, R. E.; Weimar, W. R. J. Med. Chem. 2003, 46,
1470. (b) Bergeron, R. J.; Wiegand, J.; McManis, J. S.;
Vinson, J. R. T.; Yao, H.; Bharti, N.; Rocca, J. R.
J. Med.Chem. 2006, 49, 2772. (c) Bergeron, R. J.; Bharti,
N.; Wiegand, J.; McManis, J. S.; Singh, S.; Abboud, K. A.
J. Med. Chem. 2010, 53, 2843.
Synthesis 2010, No. 21, 3631–3636 © Thieme Stuttgart · New York