Iron binding dendrimers: A novel approach for the treatment of haemochromatosis

Article abstract of DOI:10.1021/jm0600949

A range of iron binding dendrimers terminated with hexadentate ligands formed from hydroxypyridinone, hydroxypyranone, and catechol moieties have been synthesized in order to investigate their potential as clinically useful iron(III)-selective chelators capable of removing dietary iron from the gastrointestinal tract and preventing the development of iron overload typical of haemochromatosis and thalassaemia intermedia. The iron chelating abilities of these molecules have been characterized by MALDI-TOF mass spectrometry and UV spectrometry. Hydroxypyridinone-terminated dendrimers were found to possess a high affinity and selectivity for iron(III). A hydroxypyridinone-based dendrimer was demonstrated to be highly efficient at reducing the absorption of iron(III) in rat intestine. This family of dendrimers may find an application in the treatment of iron overload.

Full text of DOI:10.1021/jm0600949

J. Med. Chem. 2006, 49, 4171-4182
4171
Iron Binding Dendrimers: A Novel Approach for the Treatment of Haemochromatosis
Tao Zhou,† Hendrik Neubert,‡ Ding Yong Liu,† Zu Dong Liu,† Yong Min Ma,† Xiao Le Kong,† Wei Luo,† Sykes Mark,† and
Robert C. Hider*,†
Department of Pharmacy and Drug Control Centre, King’s College London, Franklin-Wilkins Building, 150 Stamford Street,
London SE1 9NH, U.K.
ReceiVed January 27, 2006
A range of iron binding dendrimers terminated with hexadentate ligands formed from hydroxypyridinone,
hydroxypyranone, and catechol moieties have been synthesized in order to investigate their potential as
clinically useful iron(III)-selective chelators capable of removing dietary iron from the gastrointestinal tract
and preventing the development of iron overload typical of haemochromatosis and thalassaemia intermedia.
The iron chelating abilities of these molecules have been characterized by MALDI-TOF mass spectrometry
and UV spectrometry. Hydroxypyridinone-terminated dendrimers were found to possess a high affinity and
selectivity for iron(III). A hydroxypyridinone-based dendrimer was demonstrated to be highly efficient at
reducing the absorption of iron(III) in rat intestine. This family of dendrimers may find an application in
the treatment of iron overload.
Introduction
drimers, 1a-4a, containing 9, 12, 18, and 27 carboxyl groups,
respectively. Bidentate chelators, including hydroxypyridinones,
hydroxypyranones, and catechols, were then conjugated to the
carboxyl-terminated dendrimers via amide bonds using standard
peptide synthetic methods.13 Six dendritic chelators were
synthesized using this method (Chart 1).
Transfusion-dependent patients such as those suffering from
â-thalassaemia major develop a potentially fatal secondary
haemosiderosis, and consequently, a selective iron chelator must
be used to relieve such iron overload.1 For this purpose, the
design of an orally active iron chelator has been a major
objective, resulting in a large number of iron chelators being
synthesized. The orally active chelators deferiprone2 and de-
feritrim3 have been subjected to extensive clinical investigation.
A method of preventing iron overload, which has been less
extensively investigated, involves the reduction of iron absorp-
tion from the intestine by adminstering iron chelators which
bind dietary iron irreversibly to form nontoxic, kinetically inert
complexes that are not absorbed. Macromolecular chelators
could, in principle, find application as nonabsorbable iron-
selective additives. Such therapy is relevant for the treatment
of haemochromatosis and thalassaemia intermedia.
The synthetic route of the carboxyl-terminated dendrimers
(1a-4a) is presented in Scheme 1. When amine 514 is treated
with 1,3,5-benzenetricarbonyltrichloride in the presence of
triethylamine in dichloromethane, the three-directional nona-
ester 6 was generated in excellent yield. Hydrolysis of 6 in
formic acid afforded the first-generation carboxyl-terminated
dendrimer 1a. Coupling of the tetraacid 7 with amine 5 via
treatment with DCCI/HOBt in DMF at room temperature
provided dodeca-ester 9, which was hydrolyzed in formic acid
to give the four-directional first-generation carboxyl-terminated
dendrimer 2a. The second-generation 18-acid dendrimer 3a was
synthesized in four steps. The synthesis of second-generation
27-acid dendrimer 4a was achieved via coupling of 1a and
amine 5 in the presence of DCCI and HOBt, followed by
hydrolysis in formic acid. These carboxyl-terminated dendrimers
have been characterized in detail.
Coupling of the polyacid dendrimers 1a-4a with a protected
bidentate chelator containing a free amino group such as
2-(aminomethyl)-3-(benzyloxy)-1,6-dimethylpyridin-4(1H)-
one (13), 2-(aminomethyl)-3-(benzyloxy)-6-methyl-4H-pyran-
4-one (14), and 2,3-dimethoxyphenyl methylamine (15) was
carried out in the presence of DCCI and HOBt in DMF at room
temperature. This yielded the corresponding protected dendritic
chelators, which were isolated by chromatography on silica gel.
Deprotection of the benzyl or methyl functions on the protected
dendritic chelators was achieved by treatment with boron
trichloride. It was found that the conjugation of the protected
Dendrimers4 are unique synthetic macromolecules with highly
branched structures and possess a number of applications in
supramolecular chemistry,5 nanoscience,6 medicine,7 and ca-
talysis.8 Surprisingly, dendritic metal scavengers have rarely
been reported.9 Considering the disadvantages of bidentate
chelator-based macromolecules, we have designed a range of
hexadentate ligand-terminated dendrimers. Extending our previ-
ous reports on preliminary studies on iron(III)-selective dendritic
chelators,10 we herein report the detailed synthesis of hexaden-
tate-terminated dendrimers by adopting both divergent11 and
convergent synthetic approaches.12 We demonstrate the iron
chelating ability of these dendritic macromolecules by MALDI-
TOF MSa and UV/Vis spectrometry. We have evaluated the
iron(III) affinity of the hydroxypyridinone-based dendrimers by
competition with a fluorescence probe. The ability of one of
these hydropyridinone-based dendrimers to reduce intestinal
absorption of iron(III) is reported.
a Abbreviations: MALDI-TOF MS, matrix-assisted laser desorption/
ionization time-of-light mass spectrometry; DCCI, 1,3-dicyclohexylcarbo-
diimide; HOBt, 1-hydroxybenzotriazole; DMF, N,N-dimethylformamide;
MBT, 2-mercaptobenzothiazole; DCTB, trans-2-[3-{4-tert-butylphenyl}-
2-methyl-2- propenylidene]malononitrile; ICP-MS, inductively coupled
plasma mass spectrometry; TMS, tetramethylsilane; ESI MS, electrospray
ionization mass spectrometry; CHCA, 4-hydroxy-alpha-cyano-cinnamic
acid; THAP, trihydroxyacetophenone; NTA, nitrilotriacetic acid; MOPS,
3-(N-morpholino)propane sulphonic acid.
Results and Discussion
Synthetic Strategy. (a) Divergent Approach. Initially, we
synthesized four well-defined carboxyl group-terminated den-
* Corresponding author. Tel.: (+)44 207 848 6979. Fax: (+)44 207
848 6394. E-mail: robert.hider@kcl.ac.uk.
† Department of Pharmacy.
‡ Drug Control Center.
10.1021/jm0600949 CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/21/2006

4172 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Zhou et al.
Chart 1. Polyacid Dendrimers, Protected and Deprotected Dendritic Chelators
bidentate chelators 13-15 on the polyacid dendrimers 1a-3a
could be carried out smoothly and the anticipated fully coupled
products were obtained in good yield. Conjugation of the
protected bidentate chelators 13-15 with 27-acid dendrimer 4a
was also investigated. An acceptable result was obtained from
the reaction of 15 and 4a, and although the reaction of 14 and
4a produced the fully coupled product as a major product,
incompletely converted products were also formed. The reaction
of 13 and 4a led to appreciable amounts of incompletely
converted products, in addition to a small quantity of the desired
fully coupled product, as confirmed by the MALDI-TOF mass
spectrum. These differences in behavior probably result from a
gradation of steric congestion, which is minimal with 15 but
greater with 14 and 13.
2). Coupling of 17 with amine 5 occurred in the presence of
DCCI and HOBt in DMF to give a triester 18, which generated
the triacid 19 on hydrolysis in formic acid. Compound 20 was
prepared by conjugating the bidentate ligand 13 with the triacid
19. Deprotection of amino group in 20 by hydrazinelysis yielded
16. The polyacid core 23 was synthesized in four steps (Scheme
3). Treatment of crude 5-nitroisophthaloyl dichloride with ethyl
3-aminopropanoate hydrochloride in the presence of triethyl-
amine gave 24, which was subjected to hydrogenation in the
presence of T1-Raney Ni catalyst to produce 25. Reaction of
25 with 1,3,5-benzenetricarbonyltrichloride afforded 26, which
on hydrolysis with dilute sodium hydroxide in methanol
followed by acidification generated the corresponding 6-acid
core 23. Conjugation of 16 to the polyacid cores 21, nitrilotri-
acetic acid (22) and 23 gave the corresponding protected
dendritic chelators 27, 29, and 31 (Scheme 4). After the removal
of the protective benzyl groups, the dendritic chelators 28, 30,
and 32 were obtained in 93%, 89%, and 94% yields, respec-
tively.
(b) Convergent Approach. We first synthesized a benzyl
protected hexadentate chelator containing the free amino group
16 and the polyacid cores 2115 and 23. The protection of the
amino group of â-analine was achieved by treatment with ethyl
1-methylene-3-oxoisoindoline-2-carboxylate to give 17(Scheme

Iron Binding Dendrimers: Treatment of Haemochromatosis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4173
Scheme 1. Synthesis of the Carboxyl Group-Terminated Dendrimers 1a-4aa
a (a) Et3N, CH2Cl2, 95%; (b) 96% HCOOH, rt, nearly quantitative; (c) DCCI, HOBt, DMF, rt, 4d, 76%; (d) DCCI, HOBt, DMF, rt, 2d, 79%; (e) Et3N,
CH2Cl2, 92%; (f) H2, T-1 Raney Ni, EtOH, 95%; (g) NEt3, CH2Cl2, 65%.
All compounds synthesized were fully characterized using
1H and 13C NMR spectroscopy and mass spectrometry. The
purity of these dendrimers could not be consistently character-
ized by elemental analysis, as they were hygroscopic. However,
1H and 13C NMR spectra provided strong qualitative evidence
for the purity of these compounds.16 NMR spectra demonstrated
that the removal of benzyl and methyl protecting groups was
complete. MALDI-TOF MS has previously proven to be a
valuable technique for characterizing various dendrimers.17 The
efficiency of coupling reactions could be assessed by MALDI-
TOF MS, and normally signals, as proton, sodium, or potassium
adducts of the dendrimers were obtained. Occasionally, cesium
chloride was added to promote the formation of cesium adducts
of the dendrimers. A representative MALDI-TOF mass spectrum
of the alkali metal adducts of dendrimer 4g is presented in Figure
1. The peaks at m/z 6254.8, 6364.1, and 6497.3 correspond to
sodium adduct ([M + Na]+), cesium adduct ([M + Cs]+), and
dicesium adduct ([M - H + 2Cs]+) of dendrimer 4g, respec-
tively.
Iron Chelating Properties. Theoretically, three hydroxypy-
ridinone ligands with ortho position groups relative to one of
the chelating oxygen atoms attached to a suitable tripodal

4174 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Zhou et al.
Scheme 2. Synthesis of Protected Pyridinone Hexadentate Ligand Containing a Free Amino Group 16a
a (a) N-Ethyloxycarbonylphthalimide, Na2CO3, H2O, 72%; (b) HOBt, DCCI, DMF, 1d, 66%; (c) 96% HCOOH, rt, 97%; (d) HOBt, DCCI, DMF, 2d,
76%; (e) NH2NH2, EtOH, 96%.
Scheme 3. Synthesis of the [6]-Acid Core 23a
a (a) Ethyl â-analine hydrochloride, Et3N, CH2Cl2, 78%; (b) H2, T-1 Raney Ni, EtOH, 88%; (c) 1,3,5-benzenetricarbonyltrichlodide, Et3N, CH2Cl2, 89%;
(d) 2 M NaOH, MeOH and then 2 M HCl to pH 2, 84%.
-
molecule can form a high-affinity hexacoordination site.18
Likewise, three catechol units linked to a suitable tripodal
molecule can also form a hexadentate ligand.19 Thus, it was
reasoned that 1c, 1e, 1g, 28, and 30 effectively contain three
hexadentate ligands, 2c contains four hexadentate ligands, 3c
and 32 contain six hexadentate ligands, and 4g contains nine
hexadentate ligands. To evaluate the iron chelating properties
of these dendrimers, both MALDI-TOF mass spectrometry and
UV/Vis spectrometry were employed.
(a) Mass Spectrometry. These dendritic chelators were
investigated for their iron chelating abilities using MALDI-TOF
mass spectrometry. Direct evidence was obtained for the
formation of iron complexes of pyridinone- and pyranone-
terminated dendrimers (Table 1). The MALDI-TOF spectrum
obtained from a one-to-three mixture of dendritic chelator 1c
and iron contains only one signal at m/z 2408.7, which
corresponds to the proton adduct of one-to-three dendrimer-
iron complex also annotated as [M + (FeIII - 3H)3 + H]+.10a
Evidently, three protons are released upon complexation of each
iron(III). Similarly, the MALDI-TOF spectrum obtained from
a one-to-four mixture of dendritic chelator 2c and iron also
contains only one signal at m/z 3290.0, which corresponds to
the proton adduct of the one-to-four dendrimer-iron complex
([M + (FeIII
3H)4 + H]+). The MALDI-TOF spectrum
obtained from the one-to-three mixture of dendritic chelator 28
and iron contains only one signal at m/z 2833.0, which
corresponds to the proton adduct of the one-to-three dendrimer-
iron complex also annotated as [M + (FeIII - 3H)3 + H]+. The
MALDI-TOF spectrum obtained from the one-to-three mixture
of dendritic chelator 30 and iron contains two signals at m/z
2601.9 and 2623.9. These correspond to the proton adduct of
the one-to-three dendrimer-iron complex ([M + (FeIII - 3H)3
+ H]+) and the sodium adduct of the one-to-three dendrimer-
iron complex ([M + (FeIII - 3H)3 + Na]+), respectively. The
MALDI spectrum obtained from a one-to-six mixture of
dendritic chelator 32 and iron also demonstrates complete iron
binding; the signal at m/z 5949.6 corresponds to the proton
adduct of the one-to-six dendrimer-iron complex also annotated
as [M + (FeIII
3H)6 + H]+ (Figure 2). With these five
-
pyridinone-based dendrimers, no evidence for the existence of
iron-induced interdendrimer association was observed.
The MALDI-TOF spectrum obtained from a one-to-three
mixture of dendritic chelator 1e and iron contains a main signal
at m/z 2312.3, which corresponds to the sodium adduct of the
one-to-three dendrimer-iron complex also annotated as [M +

Iron Binding Dendrimers: Treatment of Haemochromatosis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4175
Scheme 4. Convergent Synthesis of the Dendritic Chelators 28, 30, and 32a
a (a) DCCI, HOBt, DMF; (b) BCl3, CH2Cl2.
(FeIII - 3H)3 + Na]+, a minor peak at m/z 2290.2, corresponding
to the proton adduct of the one-to-three dendrimer-iron complex
([M + (FeIII - 3H)3 + H]+). However, another minor peak at
m/z 2259.2, corresponding to the sodium adduct of the one-to-
two dendrimer-iron complex ([M + (FeIII - 3H)2 + Na]+, was
also observed (Figure 3), which indicates 1e did not chelate
iron(III) completely and that pyranone-terminated dendrimers
possibly possess a lower iron(III) affinity than the pyridinone-
terminated dendrimers. The identification of a sodium adduct
of the desired complex was due to the ubiquitous presence of

4176 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Zhou et al.
Figure 1. MALDI-TOF mass spectrum of dendrimer 4g. Cesium
chloride was added to the dendrimer preparation in order to encourage
adduct formation. The MALDI matrix was CHCA.
Figure 3. MALDI-TOF mass spectrum of a one-to-three mixture of
dendritic chelator 1e and iron. The peaks at m/z 2259.3, 2290.4, and
2312.3 correspond to the sodium adduct of a one-to-two dendrimer-
iron complex ([M + (FeIII - 3H)2 + Na]+, the proton adduct of a one-
to-three dendrimer-iron complex ([M + (FeIII - 3H)3 + H]+), and the
sodium adduct of a one-to-three dendrimer-iron complex ([M + (FeIII
- 3H)3 + Na]+), respectively. The spectrum was recorded from a DCTB
matrix.
Titrations with iron(III) were undertaken on samples that were
maintained at room temperature for at least 4 h in order to ensure
the binding process was complete. In fact, kinetic studies
indicated that pyridinone-terminated dendrimers bind iron(III)
relatively quickly and complete acquisition within a few minutes
(data not shown). The 3-hydroxypyridin-4-one-containing den-
drimers 1c, 2c, 3c, 28, 30, and 32 all displayed similar spectral
behavior. Addition of iron to hydroxypyridinone-terminated
dendrimer-containing solutions resulted in the formation of
iron-dendrimer complexes, which have a maximum absorbance
at approximately 460 nm (Figure 4). In the case of dendrimers
1c, 28, and 30, the intensity of absorbance at 460 nm increased
until a 3:1 ratio of iron(III):dendrimer was obtained, after which
the absorbance remained essentially constant, indicating the
formation of a one-to-three ligand-iron(III) complex. With the
iron(III) to dendrimer 1c, 28, and 30 ratios less than 3, the plot
is linear, suggesting that the chelators 1c, 28, and 30 each
possess a high affinity for iron(III) (as a sample, the data related
to 28 is shown in Figures 4a and 4e). Likewise, in the case of
dendrimer 2c, the intensity of absorbance at 460 nm increased
until a 4:1 ratio of iron(III):dendrimer was obtained (Figures
4b, 4f), indicating that 2c binds 4 iron ions. For 3c and 32, the
intensity of absorbance at 460 nm increased until a 6:1 ratio of
iron(III):dendrimer was obtained (as a sample, the data related
to 32 is shown in Figures 4c and 4g), indicating that both 3c
and 32 form 1:6 ligand-iron(III) complexes. These results
Figure 2. MALDI-TOF mass spectrum showing the proton adduct of
-
the 1:6 dendrimer 32-iron(III) complex [M + (FeIII 3H)6 + H]+.
The peak at m/z 5171.6 shows the 1:5 dendrimer-iron(III) complex
of the defect dendrimer, lacking one hexadentate branch. The spectrum
was recorded from a DCTB matrix.
sodium in this dendrimer preparation and should not be
misinterpreted as a specific chelation phenomenon.
The selection of the MALDI matrix was critical in ensuring
desorption of intact metal complexes and matrixes such as MBT
and DCTB often proved useful. Although these and several other
matrixes were investigated for suitability in confirming the
formation of iron-3c complex and iron-dendrimer function-
alized with catechol (1g, 4g) complexes, the corresponding peaks
were not observed.
(b) UV/Vis Spectrometry. Several dendrimers were inves-
tigated by employing UV/ Vis spectrophotometric titration in
order to evaluate the properties of the different binding groups
(3-hydroxypyridin-4-one, pyranone, and catechol), dendrimer
structures, and dendrimer generations that were synthesized.
Table 1. MALDI Mass Spectrometric Data Showing the Saturated Iron Complexation of Dendritic Chelators
molecular
formula
calculated MW
for iron(III)-dendrimer complex
MALDI-TOF MS
for iron(III)-dendrimer complex
dendrimers
matrix
DCTB
1c
1e
C
C
111H141N21O30
102H114N12O39
2406.7 [M + (Fe - 3H)3 + H]+a
2290.5 [M + (Fe - 3H)3 + H]+a
2312.5 [M + (Fe - 3H)3 + Na]+a
3290.2 [M + (Fe - 3H)4 + H]+a
5096.1[M + (Fe - 3H)6 + H]+b
2834.0 [M + (Fe - 3H)3 + H]+a
2601.9 [M + (Fe - 3H)3 + H]+a
2623.9 [M + (Fe - 3H)3 + Na]+a
5949.1 [M + (Fe - 3H)6 + H]+b
2407.6 [M + (Fe - 3H)3 + H]+
2290.2 [M + (Fe - 3H)3 + H]+
2312.3 [M + (Fe - 3H)3 + N a]+
3290.0 [M + (Fe - 3H)4 + H]+
not available
DCTB
2c
3c
28
30
C153H208N28O40
MBT
MBT, DCTB
MBT
C
C
C
237H291N45O63
129H171N27O36
117H159N25O33
2833.0 [M + (Fe - 3H)3 + H]+
2601.9 [M + (Fe - 3H)3 + H]+
2623.9 [M + (Fe - 3H)3 + Na]+
5949.6 [M + (Fe - 3H)6 + H]+
MBT
32
C273H351N57O75
DCTB
a Monoisotopic molecular weight. b Average molecular weight.

Iron Binding Dendrimers: Treatment of Haemochromatosis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4177
Figure 4. UV-Visible titration spectra of dendrimer 28 (a), 2c (b), 32 (c), and 1e (d) with iron(III) at pH 7.0. Plots of absorbance versus equivalent
ratios of iron(III) to dendrimer 28 (e), 2c (f), and 32 (g) at 460 nm and 1e (h) at 420 nm. The concentrations of dendrimers 28, 2c, 32, and 1e were
14.3, 12.5, 10.0, and 14.3 µM. The solvent system was water for dendrimers 28 and 2c, 5% methanol aqueous (v/v) for dendrimer 32, and 7.1%
methanol aqueous (v/v) for dendrimer 1e.
suggest that each hexadentate branch binds one Fe3+, as
predicted, which is in agreement with the MALDI-TOF MS
results.
the free, “nonchelated” dendrimer and a variety of signals from
numerous 1:1 complexes. This indicated that the dendrimers
bind these other metal ions only very weakly. The example
shown in Figure 5a illustrates the selectivity for iron(III) in
comparison to that for Zn(II), whereby no higher order Zn(II)
complexes were observed; in contrast, contaminating iron(III)
ions were chelated. No higher order complexes could be
observed other than mixed 1:1:1 iron(III)-zinc-1c complexes.
In contrast, spectra obtained from 3:3:1 mixtures of iron, any
of the above metal ions, and dendritic chelator 1c provided
evidence for the presence of only the 3:1 of iron and dendritic
chelator 1c complex. No other metal ion-dendrimer complexes
were observed, indicating that dendrimer 1c possesses a high
selectivity for iron(III). As an example, a MALDI-TOF mass
spectrum investigating the selectivity of 1c between iron and
zinc is presented in Figure 5b.
The iron-1e complex has a maximum absorbance at 420 nm.
However, the changes in the absorbance at 420 nm increased
linearly up to a iron(III):dendrimer 1e ratio of 2.5:1, at which
point the absorbance only marginally increased further with the
addition of iron (Figures 4d and 4h). This indicates that 1e failed
to become fully iron saturated. Surprisingly, with the catechol-
terminated dendrimers 1g and 4g, the addition of iron to the
chelator initially resulted in the formation of a purple-red
solution, which changed to dark-blue after a day and to brown
after a further 3 days. This phenomenon is attributed to the
catechol-mediated reduction of iron(III) to iron(II) (blue) which
is then gradually oxidized by oxygen.20
Selectivity for Iron. The selectivity of the hydroxypyridinone
dendrimers for Fe3+ compared with other metal ions, for
instance, Cu2+, Mg2+, Zn2+, and Ca2+, was investigated using
MALDI-TOF mass spectrometry and this analysis is illustrated
using the dendritic chelator 1c. The MALDI spectra recorded
from 3:1 mixtures of any of the above metal ions excluding
iron(III) and dendritic chelator 1c yielded a strong signal for
Affinity Constant of Iron Complex. To estimate the affinity
of the hydroxypyridinone-terminated dendritic chelators for iron-
(III), the pKa values of the analogous hexadentate ligand 33
(Chart 2) were investigated using an automated titration
system;21 the stability constant (log K1) of hexadentate ligand
33 was determined spectrophotometrically by competition with

4178 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Zhou et al.
Table 3. Affinity Constants of 3-Hydroxypyridin-4-one-Terminated
Dendrimers
1c
28
30
2c
3c
32
log K
32.60
28.55
32.74
28.69
32.58
28.53
32.28
28.24
32.52
28.47
32.60
28.55
pFe3+
affinities of the hexadentate pyridinone moieties on these
hydroxypyridinone-based dendrimers were estimated from the
pKa values and affinity constant of 33. They are presented in
Table 3. Essentially all the determined affinity constants are
not significantly different from each other or from that of 33.
These findings indicate that the length of arm, nature of core,
and generation of dendrimer do not greatly influence the iron
binding affinities of the chelating end groups. Thus, there is no
evidence for a dendritic effect typically associated with many
dendritic binding phenomena.23 This results from the maximum
binding enhancement of iron already having been achieved in
the design of the optimal hexadentate binding motifs.24
Figure 5. (a) MALDI mass spectrum obtained from the 3:1 mixture
of Zn2+ and dendritic chelator 1c; (b) MALDI mass spectrum obtained
from the 3:3:1 mixture of Fe3+, Zn2+, and dendritic chelator 1c.
In Vitro Intestinal Perfusion Study. To evaluate the ability
of dendrimer 1c to prevent gastointestinal iron(III) absorption,
an in vitro rat intestinal perfusion method described by
Schu¨mann et al.25 was adopted. 58Fe is utilized in these
experiments because 58Fe presented to samples can be selectively
measured by ICP-MS, thus avoiding interference from the
existing iron in lumen. Two 58Fe concentrations (50 and 10 µM)
were used in the perfusion studies. The ratios of the calculated
iron binding site numbers of dendrimer 1c to 58Fe were adjusted
to either 1:1 or 2:1. Thus, in the case of 50 µM iron
concentration, the concentration of dendrimer 1c in perfusate
was 16.7 or 33.3 µM, respectively, because 1 mol of dendrimer
1c is able to bind 3 mol of iron. Similarly, in the case of 10
µM 58Fe concentration, the concentration of dendrimer 1c in
perfusate was 3.33 or 6.67 µM, respectively. The samples
(absorbates) were collected at the same time intervals as the
control groups. The 58Fe content in the samples was determined
by ICP-MS. The results are presented in Figure 6. In the 50
µM 58Fe concentration perfusion experiments, the accumulated
iron contents in the absorbates of the 1:1 group and 2:1 group
at 120 min were 123.9 and 48.0 pmol/cm of tissue, respectively,
significantly less than that from the control groups (1826.5 pmol/
cm of tissue) (Figure 6a). In the 10 µM 58Fe concentration
perfusion experiments, the accumulated iron contents in the
absorbates of the 1:1 group and 2:1 group in 120 min are 68.5
and 31.5 pmol/cm of tissue, respectively (Figure 6b). Compared
with the control groups (249.2 pmol/cm of tissue), the absorption
of iron was again significantly reduced. Thus, dendritic chela-
tors, by reducing iron absorption, may find application in iron
overload therapy. Hydroxypyridinone-containing dendrimers
have the advantage over tannic acid,26 an iron chelator normally
present in the diet, of being highly specific for iron(III); thus,
they are predicted not to interfere with the gastrointesinal
absorption of zinc and copper. With molecular weights in the
range of 2000-6000, these iron-chelating dendrimers are
Chart 2. Hexadentate Ligand 33
EDTA. Hexadentate ligand 33 was found to possess a very high
affinity for iron(III), namely, log K1 ) 32.52; pFe3+ ) 28.47.
The pFe3+ value of 33 is over 8 log units higher than that of
the bidentate analogue 3-hydroxy-1,2-dimethylpyridin-4(1H)-
one (pFe3+ ) 19.7).
To evaluate the iron(III) affinity of hexadentate hydroxypy-
ridinone-based dendrimers, a study associated with competition
between a fluorescent chelator CP69122 and hexadentate 33 or
dendrimers (1c, 2c, 3c, 28, 30, and 32) was performed. When
CP691 is mixed with iron, fluorescence is quenched to a low
level, but is gradually increased on the addition of a competing
ligand (either hexadentate 33 or dendritic chelators). When the
competition reaches equilibrium, the relative fluorescence
intensity of the corresponding solutions were found to be similar
(Table 2), which indicates that the hexadentate moieties on the
dendrimers possess a similar affinity for iron(III) as hexadentate
33. As the hexadentate moieties on the dendrimers under
investigation utilize the same pyridinone bidentate ligands as
hexadentate 33, it is assumed that these hexadentate moieties
possess the same pKa values as those of 33. The iron(III)
Table 2. Comparison of the Iron(III) Affinities of the Hexadentate Ligand 33 and Dendritic Chelators Using the Fluorescence Methoda
relative fluorescence intensity (%)
CP691 +
Fe(III) + 33
CP691 +
Fe(III) + 1c
CP691 +
Fe(III) + 28
CP691 +
Fe(III) + 30
CP691 +
Fe(III) + 2c
CP691 +
Fe(III) + 3c
CP691 +
Fe(III) + 32
time
4 h
1 d
2 d
3 d
4 d
5 d
31.5
44.4
50.5
54.7
55.1
55.3
33.7
46.6
51.8
56.6
57.5
57.6
34.4
49.2
54.5
60.1
61.3
61.5
33.0
46.5
50.4
54.6
56.7
57.0
29.2
39.2
44.5
47.2
48.2
48.5
31.0
43.9
48.1
54.2
55.2
55.2
31.7
45.5
50.7
56.7
57.2
57.6
a Final concentration: [CP691] ) 6 µM, [Fe(III)] ) 6 µM, [33] ) 6 µM, [1c] ) [28] ) [30] ) 2 µM, [2c] ) 1.5 µM, [3c] ) [32] ) 1 µM.

Iron Binding Dendrimers: Treatment of Haemochromatosis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4179
Compound 8. A mixture of tetraacid 7 (0.72 g, 2 mmol), amine
5 (3.99 g, 9.6 mmol), DCCI (1.98 g, 9.6 mmol), HOBt (1.30 g, 9.6
mmol), and DMF (30 mL) was stirred at room temperature for 2
d. After filtration and removal of the solvent under reduced pressure,
the residue was chromatographed on silica gel using EtOAc/hexane
(2:1) as an eluent to afford 3.08 g (79%) of 8 as a white solid.
Compound 9. 5-Nitroisophthalic acid (3.16 g, 15 mmol) was
suspended in SOCl2 (30 mL); the mixture was refluxed for 24 h.
Excess of SOCl2 was removed in vacuo, dry benzene (3 × 5 mL)
was added, and the solution was concentrated in vacuo. The residue
was dissolved in dry CH2Cl2 (60 mL). After cooling to 0 °C, amine
5 (13.71 g, 33 mmol) and Et3N (3.33 g, 33 mmol) were added; the
mixture was stirred at room temperature overnight. The reaction
mixture was washed successively with aqueous sodium hydrogen
carbonate (10%), water, cold aqueous hydrochloric acid (10%), and
brine. The organic phase was dried over Na2SO4 and concentrated.
The residue was subjected to column chromatography on silica gel
(EtOAc/hexane 1:1) to give 9 (13.87 g, 92%) as a white solid.
Analogous procedures were used for the preparation of compound
24.
Amino Ester 10. A solution of nitro ester 9 (10.10 g, 10 mmol)
in anhydrous DMF (100 mL) with T-1 Raney Ni27 (5.0 g) was
hydrogenated at 50 psi and room temperature for 2 d. After the
catalyst was cautiously filtered, the solvent was removed in vacuo,
affording a solid, which was column-chromatographed on silica
gel, eluting with CHCl3/MeOH (9.5:0.5) to give 10 (8.68 g, 89%)
as a white solid.
Analogous procedures were used for the preparation of compound
25.
Compound 11. A solution of 1,3,5-benzenetricarbonyltrichloride
(0.53 g, 2 mmol) in DCM was slowly added to a stirred mixture of
amine 10 (7.03 g, 7.2 mmol) and triethylamine (7.27 g, 7.2 mmol)
in DCM cooled by an ice bath. Stirring was continued overnight.
With use of the workup procedure described above for 6, compound
11 (4.96 g, 65%) was isolated as a white solid. Analogous
procedures were used for the preparation of compound 26.
General Procedure for Coupling of Bidentate or Hexadentate
Ligand Containing a Free Amine Group with Polyacid. Il-
lustrated for the preparation of protected dendritic chelator
1b. A mixture of nonaacid dendrimer 1a (0.90 g, 1 mmol), 1,6-
dimethyl-2-aminomethyl-3-benzyloxy-pyridin-4(1H)-one 13 (2.79
g, 10.8 mmol), DCCI (2.22 g, 10.8 mmol), HOBt (1.46 g, 10.8
mmol), and DMF (30 mL) was stirred for 2 d. After filtration and
removal of solvent under reduced pressure, the residue was
chromatographed in a silica gel column using MeOH/CHCl3 (1:4)
as eluent, followed by MeOH/CHCl3/40% ammonium hydroxide
aqueous (10:40:1) to afford 1b (2.70 g, 88% yield) as a white solid.
Figure 6. In vitro intestinal perfusion investigation. (a) Initial
concentration of 58Fe is 50 µM; (b) initial concentration of 58Fe is 10
µM.
predicted to not be apprecially absorbed by the mammalian
intestine. This prediction is currently under investigation using
14C-labeled dendrimers.
Experimental Section
General. All chemicals were reagent grade and were used
without further purification. Melting points were determined on an
Electothermal IA 9100 Digital Melting Point Apparatus and were
1
uncorrected. H NMR spectra were recorded on a Bruker Avance
360 (360 MHz) or Bruker Avance 500 (500 MHz) spectrometer.
1
Chemical shifts (δ) for H spectra are given in parts per million
(ppm) downfield from the internal standard TMS. 13C NMR spectra
(90 MHz) were recorded on a Bruker Avance 360 spectrometer.
Chemical shifts (δ) for 13C spectra are given in ppm relative to the
signal of CDCl3 (δ 77.16) or DMSO (δ 39.52). MALDI-TOF mass
spectra were recorded on an Autoflex MALDI-TOF mass spec-
trometer (Brukes Daltonics, Coventry, UK). ESI mass spectra were
obtained by infusing samples into an LCQ Deca XP ion trap
instrument. HRMS were determined at the Strand campus, King’s
College London. UV spectra were recorded on a HP8453 UV
spectrometer.
Analogous procedures starting with other bidentate ligands or
hexadentate ligand and polyacid gave protected dendritic chelators
1d, 1f, 2b, 3b, 4f, 20, 27, 29, and 31.
General Procedure for the Deprotection of Benzyl Groups
and Methyl Groups on Protected Dendritic Chelators. Il-
lustrated for the Preparation of Dendritic Chelator 1b. In an
atmosphere of nitrogen, 1 M boron trichloride in dichloromethane
(18 mL, 18 mmol) was dropped slowly into an ice-bath-cooled
suspension of solution of 1b (2.45 g, 0.8 mmol) in CH2Cl2 (25
mL). The mixture was stirred at room temperature for 1 d. Methanol
(20 mL) was added to quench the reaction. After the removal of
the solvent, the residue was precipitated with methanol/acetone three
times to afford the hydrochloric acid salt of 1c as a white powder
Nonaester 6. A solution of 1,3,5-benzenetricarbonyltrichloride
(1.33 g, 5 mmol) in CH2Cl2 was slowly added to a stirred mixture
of amine 5 (7.48 g, 18 mmol) and triethylamine (1.82 g, 18 mmol)
in CH2Cl2 cooled by an ice bath. Stirring was continued overnight.
The reaction mixture was washed successively with cold aqueous
hydrochloric acid (10%), aqueous sodium hydrogen carbonate
(10%), and water, dried over Na2SO4, and concentrated. The residue
was subjected to column chromatography on silica gel (EtOAc/
hexane 1:1) to give 6.67 g (95%) of 6 as a white solid.
General Procedure for Preparation of Polyacid Dendrimers
(1a, 2a, 3a, 4a, and 19). A solution of polyester dendrimers (6, 8,
11, 12, and 18) in formic acid was stirred overnight. After
concentration and the removal of residual formic acid, the crude
product was obtained.
1
(1.88 g, 91%). H NMR (360 MHz, DMSO-d6): δ 2.01 (br, CH2,
18H), 2.16 (br, CH2, 18H), 2.56 (s, CH3, 27H), 3.88 (s, CH3, 27H),
4.55 (br, CH2, 18H), 7.25 (s, pyridinone C-5H, 9H), 8.06 (br, ArH,
3H), 8.41 (s, NH, 3H), 8.99 (br, NH, 3H); 13C NMR (90 MHz,
DMSO-d6) δ 21.0 (CH3), 30.1 (CCH2CH2), 35.1 (NHCH2), 39.4
(NCH3), 113.1 (pyridinone C-5H), 140.1 (pyridinone C-2), 143.1
(pyridinone C-3), 148.9 (pyridinone C-6), 160.1 (pyridinone C-4),
173.8 (CONH). MALDI-TOF MS: Calculated for C111H141N21O30,
2248.0 (monoisotopic molecular weight); found, 2248.7 [M + H]+
(CHCA as matrix).

4180 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
Zhou et al.
Analogous procedures for deprotection of other protected
chelators (1d, 1f, 2b, 3b, 4f, 27, 29, and 31) gave chelators 1e, 1g,
2c, 3c, 4g, 28, 30, and 32.
DMF (60 mL) was stirred at room temperature for 1 d. After
filtration and solvent removal under reduced pressure, the residue
was purified on a silica gel column using EtOAc/MeOH (9.5:0.5)
as an eluent to afford 18 (4.08 g, 66%). mp 144-145 °C.
Compound 16. To a solution of 20 (3.51 g, 3 mmol) in ethanol
(40 mL) was added 5.5% aqueous hydrazine (3 mL). After being
refluxed for 3 h, the reaction mixture was cooled to 0 °C, acidified
to pH 1 with concentrated HCl, and filtered. The filtrate was
concentrated in vacuo, and the residue was dissolved in distilled
water (40 mL), adjusted to pH 12 with 10 M NaOH, and extracted
with chloroform (5 × 100 mL). The combined organic extracts
were dried over anhydrous Na2SO4 under reduced pressure to
furnish 16 (2.99 g, 96%) as a white solid.
[6]-Acid Core 23. To an ice-bath-cooled solution of 26 (2.59 g,
2 mmol) in methanol (15 mL) was added 2 M NaOH (50 mL); the
mixture was left to stir for 3 h. Neutralized with 2 M HCl to pH 2
and the precipitate collected, washed with water several times, and
dried in air, compound 23 (1.89 g, 84%) was obtained as a white
solid.
Dendritic Chelator 1e. After quenching of the reaction and the
removal of solvent, the residue was precipitated with methanol/
diethyl ether three times to afford 1e (82%) as a white powder. 1H
NMR (DMDO-d6) δ 2.02 (br, CH2, 18H), 2.11 (br, CH2, 18H),
2.20 (s, CH3, 27H), 4.21 (d, J ) 5.0 Hz, CH2, 18H), 6.19 (s,
pyranone C-5H, 9H), 7.88 (m, ArH, 3H), 8.33-8.37 (m, NH, 12H);
13C NMR δ 19.6 (CH3), 29.8 (CCH2CH2), 29.3 (CCH2CH2), 35.8
(NHCH2), 111.6 (pyranone C-5H), 142.0 (pyranone C-2), 147.3
(pyranone C-3), 164.8 (pyranone C-6), 172.8 (CONH), 173.9
(pyranone C-4). MALDI-TOF MS: Calculated for C102H114N12O39,
2130.7 (monoisotopic molecular weight M); found, 2131.7 [M +
H]+ (CHCA as matrix).
Dendritic Chelator 1g. The reaction time was 2 d. 78% yield.
1H NMR (DMDO-d6) δ 2.02 (br, CH2, 18H), 2.13 (br, CH2, 18H),
4.13 (d, J ) 5.3 Hz, CH2, 18H), 6.53-6.58 (m, ArH, 18H), 6.66
(m, ArH, 9H), 7.88 (br, ArH, 3H), 8.39 (br, NH, 3H), 8.44 (m,
NH, 9H); 13C NMR δ 29.9 (CCH2CH2), 30.6 (CCH2CH2), 38.4
(NHCH2), 114.8 (CH in Ar), 119.2 (CH in Ar), 119.8 (CH in Ar),
126.3 (C in Ar), 143.3 (C in Ar), 145.7 (C in Ar), 173.5 (CONH).
MALDI-TOF MS: Calculated C102H114N12O30, 1986.8 (monoiso-
topic molecular weight); found, 1987.9 [M + H]+, 2009.9 [M +
Na]+ (CHCA as matrix).
Dendritic Chelator 28 Hydrochloride. 93% yield, a white
1
powder. H NMR (360 MHz, DMSO-d6) δ 1.81 (br, CH2, 18H),
2.09 (br, CH2, 18H), 2.25 (m, CH2, 6H), 2.41 (m, CH2, 6H), 2.56
(s, CH3, 27H), 3.22 (m, CH2, 6H), 3.45 (m, CH2, 6H), 3.89 (s,
CH3, 27H), 4.57 (d, J ) 3.9 Hz, CH2, 18H), 7.28 (s, pyridinone
C-5H, 9H), 7.35 (br, NH, 3H), 8.10 (m, NH, 3H), 8.49 (s, NH,
3H), 8.94 (m, NH, 9H; Ar, 3H); 13C NMR (90 MHz, DMSO-d6) δ
20.9 (CH3), 29.6 (CCH2CH2CO), 30.3 (CCH2CH2CO), 35.1
(NHCH2), 39.5 (NCH3), 57.2 (NHC), 113.1 (C-5H in pyridinone),
140.4 (C-2 in pyridinone), 143.0 (C-3 in pyridinone), 148.9 (C-6
in pyridinone), 159.8 (C-4 in pyridinone), 170.7 (CONH), 173.8
(CONH). MS: Calculated for C129H171N27O36, 2674.2 (monoisotopic
molecular weight M); found, MALDI-TOF MS 2674.7 [M + H]+
(MBT as matrix).
1
Dendritic Chelator 2c Hydrochloride. 91% yield. H NMR
(DMDO-d6) δ 0.99 (br, CH2, 8H), 1.22 (br, CH2, 8H), 1.79 (br,
CH2, 24H), 1.97 (br, CH2, 8H), 2.08 (br, CH2, 24H), 2.55 (s, CH3,
36H), 3.87 (s, CH3, 36H), 4.55 (br, CH2, 24H), 7.23 (pyridinone
C-5H, 12H), 8.94 (br, NH, 12H); 13C NMR δ 21.0 (CH3), 29.7
(CCH2CH2), 30.3 (CCH2CH2), 35.1 (NHCH2), 39.4 (NCH3), 113.1
(pyridinone C-5H), 140.0 (pyridinone C-2), 143.1 (pyridinone C-3),
149.0 (pyridinone C-6), 160.0 (pyridinone C-4), 174.0 (CONH).
MALDI-TOF MS (CHCA as matrix): Calculated for C153H208N28O40,
3077.5 (monoisotopic molecular weight M); found, 3078.6 [M +
H]+ (CHCA as matrix).
Dendritic Chelator 30 Hydrochloride. 89% yield, a white solid.
1H NMR (360 MHz, DMSO-d6) δ 1.79 (br, CH2, 18H), 2.08 (br,
CH2, 18H), 2.30 (br, CH2, 6H), 2.57 (s, CH3, 27H), 3.25 (br, CH2,
6H), 3.89 (s, CH3, 27H; and buried CH2, 6H), 4.56 (br, CH2, 18H),
7.30 (s, pyridinone C-5H, 9H), 7.43 (br, NH, 3H), 8.82 (br, NH,
3H), 9.02 (br, NH, 3H), 10.73 (br, HN+); 13C NMR (90 MHz,
DMSO-d6) δ 21.0 (CH3), 29.7 (CCH2CH2CO), 30.3 (CCH2CH2-
CO), 35.1 (NHCH2), 39.6 (NCH3), 57.3 (NCH2CO), (NHC), 113.1
(C-5H in pyradinone), 140.3 (C-2 in pyridinone), 143.0 (C-3 in
pyridinone), 148.9 (C-6 in pyridinone), 159.8 (C-4 in pyridinone),
170.2 (CONH), 173.8 (CONH). MS: Calculated for C117H159N25O33,
2442.15 (monoisotopic molecular weight M); found, MALDI-TOF
MS 2443.01 [M + H]+, 2465.02 [M + Na]+, 2481.02 [M + K ]
1
Dendritic Chelator 3c Hydrochloride. 88% yield. H NMR
(MeOD-d4) δ 2.20 (br, CH2, 36H), 2.43 (br, CH2, 36H), 2.63 (s,
CH3, 54H), 4.03 (s, CH3, 54H), 4.74 (br, CH2, 36H), 7.11 (s,
pyridinone C5-H, 18H), 7.99 (m, ArH, 3H), 8.52 (m, ArH, 6H),
9.01 (m, ArH, 3H); 13C NMR (MeOD-d4) δ 21.9 (CH3), 31.1
(CCH2CH2), 31.4 (CCH2CH2), 37.0 (NHCH2), 40.7 (NCH3), 114.7
(pyridinone C-5H), 141.3 (pyridinone C-2), 145.0 (pyridinone C-3),
151.4 (pyridinone C-6), 161.5 (pyridinone C-4), 177.2 (CONH).
MALDI-TOF MS: Calculated for C237H291N45O63, 4775.1 (monoiso-
topic molecular weight M); found, 4776.3 [M + H]+ (CHCA as
matrix).
+
(MBT as matrix).
Dendritic Chelator 4g. The reaction time was 4 d. White solid,
73% yield. 1H NMR (DMDO-d6) δ 1.85 (br, CH2, 72H), 2.10 (br,
CH2, 72H), 4.13 (br, CH2, 54H), 6.53 (m, ArH, 54H), 6.65 (m,
ArH, 27H), 7.34 (br, NH, 12H), 7.91 (br, ArH, 3H), 8.45 (br, NH,
27H); 13C NMR δ 29.7 (CCH2CH2), 30.3 (CCH2CH2), 38.5
(CONHCH2), 57.1 (CCH2CH2), 114.8 (CH in Ar), 119.3 (CH in
Ar), 120.0 (CH in Ar), 126.2 (C in Ar), 143.3 (C in Ar), 145.7 (C
in Ar), 173.9 (CONH). MALDI-TOF MS: Calculated for
Dendritic Chelator 32 Hydrochloride. 94% yield, a white solid.
1H NMR (500 MHz, CD3OD): δ 1.91 (br, CH2, 36H), 2.22 (br,
CH2, 36H), 2.41 (br, CH2, 12H), 2.57 (br, CH2, 12H), 2.60 (s, CH3,
54H), 3.44 (br, CH2, 12H), 3.66 (br, CH2, 12H), 4.00 (s, CH3, 54H),
4.71 (s, CH2, 36H), 4.91 (br, OH), 7.08 (s, pyridinone C-5H, 18H),
8.04 (s, ArH, 3H), 8.49 (s, ArH, 6H), 8.95 (s, ArH, 3H); 13C NMR
(90 MHz, CD3OD): δ 21.8 (CH3), 31.2 (CCH2CH2), 31.5
(CCH2CH2), 36.9 (NHCH2-pyridinone), 40.6 (NCH3), 59.6 (NHC),
114.6 (C-5H in pyridinone), 141.4 (C-2 in pyridinone), 145.1 (C-3
in pyridinone), 151.3 (C-6 in pyridinone), 161.4 (C-4 in pyridinone),
177.1 (CONH). MS: Calculated for C273H351N57O75, 5631.1 (aver-
age molecular weight); found, MALDI-TOF MS, 5631.5 for [M +
H]+ (DCTB as matrix).
UV/Vis Spectrophotometry for Iron Chelation. Batch spec-
trophotometric determinations were performed on a UV spectro-
photometer scanning from 250 to 600 nm. A range of solutions
with different ratios of dendrimer and iron were prepared by adding
iron solution (100 µM, 1 mM NTA, 50 mM NH4HCO3) to 0.5 mL
of dendrimer (100 µM in water or 50% methanol aqueous) followed
by the addition of water to yield a certain volume (3.5 mL for
dendrimer 1c, 28, 30, and 1e; 4 mL for dendrimer 2c, 5 mL for
dendrimer 3c and 32). The final pH of the solution was 7.0-7.2.
All samples were equilibrated at room temperature for at least 4 h
before spectral acquisition.
C
318H375N39O93, 6231.7 (average molecular weight M); found,
6354.8 [M + Na]+, 6364.1 [M + Cs]+, 6497.3 [M - H + 2Cs]+
(CHCA as matrix).
3-(1,3-Dioxo-1,2-dihydro-isoindol-2-yl)-propionic Acid 17. A
vigorously stirred solution of â-analine (4.46 g, 50 mmol) and
sodium carbonate (5.35 g, 50.5 mmol) was treated with N-
ethyloxycarbonylphthalimide (11.51 g, 52.5 mmol). After 30 min
almost all the reagent dissolved. The solution was filtered from a
small amount of unreacted starting material and acidified to pH
2-3 with 6 M HCl. Filtration was followed by washing with water
and drying in air. Compound 17 (7.89 g, 72% yield) was collected
on a filter as white crystals. mp 150-152 °C (lit:28 152-153 °C).
4-(2-tert-Butoxycarboxy-ethyl)-4-[3-(1,3-dioxo-1,3-dihydro-
isoindol-2-yl)-propionylamino]-heptanedioic Acid Di-tert-butyl
Ester (18). A mixture of 17 (2.19 g, 10 mmol), amine 5 (4.57 g,
11 mmol), DCCI (2.27 g, 11 mmol), HOBt (1.49 g, 11 mmol), and

Iron Binding Dendrimers: Treatment of Haemochromatosis
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14 4181
Sample Preparation for Iron Selectivity Studies. One hundred
microliters of dendrimer 1c (100 µM) was mixed with 300 µL of
metal salt such as CuSO4, MgSO4, ZnSO4, and CaCl2 solutions.
The final pH was about 6 and was not adjusted to pH 7.0 because
the dendrimer binds these metals only very weakly and the free
dendrimer tends to precipitate at pH 7.0. The mixture was used for
MALDI-TOF MS determination 4 h after preparation. To the
of 33. With this assumption, the stability constant K of hexadentate
ligand on the dendrimers could be calculated using eq 6. As an
example, K1c was calculated as follows:
(1 - 0.553)(1 - 0.553) 0.576‚0.576
0.553‚0.553(1 - 0.576)(1 - 0.576)
K1c
)
mixture of 300 µL of metal ion solution (Cu2+, Mg2+, Zn2+, Ca2+
)
log K1c ) log 1.2KHex ) 32.60
and 300 µL of iron solution (pH 3.0), 100 µL of dendrimer 1c
(100 µM) was added followed by 25 µL of NH4HCO3 (50 mM)
(final pH 7.2). After 4 h, the mixture was used for probing iron
selectivity by MALDI-TOF MS.
The pFe3+ value was calculated by HYSS29 based on the affinity
constants determined and assuming identical pKa values for the
hexadentate units. The conditions for the calculation were pH )
7.4, [Fe3+] ) 10-6 M, [Ligand] ) 10-5 M.
Determination of Iron Affinities of the Dendritic Chelators
Using the Fluorescence Method. The fluorescent probe CP691
was selected for this purpose. Fe(III) (12 µM) prepared from ferric
chloride in the presence of 4-fold NTA solution was added to MOPS
buffer (50 mM) containing CP691 (12 µM). After a few minutes,
the competing chelator at a concentration yielding an equal amount
number of iron binding units [33 (12 µM), 1c, 28, and 30 (4 µM),
2c (3 µM), 3c, and 32 (2 µM)] in DMSO was mixed with the iron-
probe complex at the same volume. The time course of the
fluorescence intensity of the samples was measured by a spectro-
fluorometer. The iron-free CP691 fluorescence was set at 100%
probe fluorescence intensity, and the fluorescence intensity of
CP691 in the presence of an equimolar amount of iron(III) was set
at 0%. The percentage of fluorescence intensity of the mixture of
the iron-probe complex with various competing ligands could be
thus calculated based on their measured arbitrary units of fluores-
cence.
Calculation of Iron Affinities of the Dendritic Chelators by
Competition with a Fluorescent Chelator, The fluorescent probe
CP691, the hexadentate ligand 33, and the hexadentate branches
on the dendritic chelator form 1:1 iron(III)-ligand complexes as
indicated above. The stability constants of CP691, 33, and the
hexadentate ligands on the dendritic structures can be represented
as follows:
In Vitro Intestinal Perfusion.58Fe was purchased from Chemgas
(Boulogne, France) in metal piece form. This product contains 56Fe
0.35%, 57Fe 6.50%, and 58Fe 93.13%. It was dissolved in nitric
acid (1 M) to contain 170 mM 58Fe and diluted to 25 mM or 5
mM 58Fe using NTA solution (pH 7.0). The molecular ratio of 58Fe:
NTA in these two 58Fe solutions was kept at 1:5. Male Wistar rats
(230-250 g) were purchased from Harlan UK Ltd. (Oxon, England)
and housed in the Biological Service Unit, King’s College London,
for at least 3 days before experiments. The animals were maintained
at a temperature between 20 and 23 °C, with food and water ad
libitum. The experiments carried out were authorized by the
Secretary of State (England) under Animals Act 1986.
The experiments were undertaken using an in vitro intestinal
perfusion model described by Schu¨mann et al.25 The animals were
terminally anaesthetized by a combined i.p. application of Hypnorm
and Hypnovel. Then 10-15 cm of the jejunal section was removed
from body and perfused for periods up to 2 h using 50 mL of Tyrode
solution at 37 °C, pH 7.0, saturated with carbogen (95% O2 and
5% CO2). Tyrode solution contains NaCl (137 mM), KCl (2.7 mM),
NaHCO3 (11.9 mM), NaH2PO4 (0.4 mM), CaCl2‚2H2O (0.14 mM),
MgCl2‚6H2O (0.05 mM), and d-glucose (15 mM).
For the control group, 10 min after the start of perfusion, 100
µL of 58Fe solution (25 or 5 mM) was added to perfusate (50 mL).
The perfusion time was reset and the samples (absorbates) were
collected over the periods 0-30, 30-60, and 60-120 min. For
the test groups, 5 min after the start of perfusion, the dendrimer 1c
(10 mM in water) was added to the perfusate. After 10 min of
perfusion, 100 µL of 58Fe solution (25 or 5 mM) was added to the
perfusate (50 mL). The ratios of the calculated iron binding site
numbers of dendrimer 1c to 58Fe were adjusted to either 1:1 or
2:1. The samples were collected in the same periods as the control
groups.
[FeFlu]
Fe + Flu h FeFlu; KFlu
Fe + Hex h FeHex; KHex
Fe + Den h FeDen; KDen
)
(1)
(2)
(3)
[Fe][Flu]
[FeHex]
)
[Fe][Hex]
[FeDen]
)
[Fe][Den]
58Fe Analysis. A quadrupole-based ICP-MS Spectrometer (Per-
kin-Elmer SCIEX ELAN 6100 DRC) with a dynamic reaction cell
(DRC) was used in the present study (PerkinElmer SCIEX,
Concord, Ontario, Canada). A quartz cyclonic spray chamber with
Meinhard nebulizer was used for sample introduction. The dynamic
reaction cell gas used was 5% hydrogen in argon (BOC Special
Gases, Guildford, Surrey). A summary of the analytical parameters
is shown in the Supporting Information. Samples (200 µL) were
diluted to 1.5 mL using 0.5% HNO3 containing indium (10 mg/L
as internal standard) before being subjected to ICP-MS analysis.
where Flu is the fluorescent probe CP691, Hex is the hexadentate
ligand 33, and Den is a hexadentate ligand on the dendrimer. For
the sake of brevity, all charges have been omitted. In the equilibrium
situation, the system can be represented in the following way:
FeFlu + Hex h FeHex + Flu
KHex
KFlu
[FeHex][Flu]
[FeFlu][Hex]
Kq1
)
)
(4)
Acknowledgment. The authors are thankful for financial
support from EC Framework 5 project QLK1-2001-00444.
FeFlu + Den h FeDen + Flu
KDen
KFlu
[FeDen][Flu]′
[FeFlu]′[Den]
Supporting Information Available: Routine spectroscopic data
and ICP-MS operating parameters for the analysis of 58Fe in the
perfusion samples. This material is available free of charge via the
Internet at http://pubs.acs.org.
Kq2
)
)
(5)
(6)
From eqs 4 and 5, the following equation can be obtained:
References
[FeFlu][Hex][FeDen][Flu]′
KDen
)
KHex
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4182 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 14
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