The selective quantification of iron by hexadentate fluorescent probes

Article abstract of DOI:10.1016/j.bmc.2009.09.052

The synthesis of four hexadentate fluorescent probes is described, where the fluorescent moiety is based on either coumarin or fluorescein and the chelating moiety is based on either 3-hydroxypyridin-4-one or 3-hydroxypyran-4-one. The fluorescence is quenched when the probe chelating moieties bind iron. The probes were found to be selective for iron over other metals such as Cu, Zn, Ni, Mn and Co. The effect of Cu on fluorescence quenching can be eliminated in the presence of N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine. Competition studies demonstrate that the exchange of iron between pyridinone-based probes and apotransferrin is very slow. The ability to scavenge iron from oligomeric iron(III) citrate complexes demonstrate that the pyridinone probes scavenges iron faster than deferiprone and desferrioxamine. The fluorescence intensity of the fluorescein-based probe is quantitatively related to the iron concentration with the limit of detection being 10^-8 M.

Full text of DOI:10.1016/j.bmc.2009.09.052

Bioorganic & Medicinal Chemistry 17 (2009) 8093–8101
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The selective quantification of iron by hexadentate fluorescent probes
*
Yong Min Ma, Robert C. Hider
Department of Pharmacy, King’s College London, Franklin–Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of four hexadentate fluorescent probes is described, where the fluorescent moiety is based
on either coumarin or fluorescein and the chelating moiety is based on either 3-hydroxypyridin-4-one or
3-hydroxypyran-4-one. The fluorescence is quenched when the probe chelating moieties bind iron. The
probes were found to be selective for iron over other metals such as Cu, Zn, Ni, Mn and Co. The effect of Cu
on fluorescence quenching can be eliminated in the presence of N,N,N’,N’-tetrakis(2-pyridylmethyl)-eth-
ylenediamine. Competition studies demonstrate that the exchange of iron between pyridinone-based
probes and apotransferrin is very slow. The ability to scavenge iron from oligomeric iron(III) citrate com-
plexes demonstrate that the pyridinone probes scavenges iron faster than deferiprone and desferriox-
amine. The fluorescence intensity of the fluorescein-based probe is quantitatively related to the iron
concentration with the limit of detection being 10À8 M.
Received 1 September 2009
Revised 24 September 2009
Accepted 30 September 2009
Available online 8 October 2009
Keywords:
Fluorescence
Iron chelator
Non-transferrin-bound iron (NTBI)
Hexadentate
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussions
Iron is one of the most important transition trace metals of all
living cells. Iron overload, as well as iron deficiency, causes
numerous health problems such as iron deficiency anemia, sickle
cell anemia, thalassemia and hemochromatosis.1,2 Such iron disor-
ders can result from genetic defects, insufficient or excessive
absorption of dietary iron or environmental factors.3 Chemother-
apy, exposure to toxic chemicals and natural body processes such
as menstruation can all result in inappropriate distribution of
body iron.4 One parameter which changes with different body
iron levels is non-transferrin bound iron (NTBI), the concentration
of iron in the plasma which is not bound to transferrin.5,6 At the
present time there is no generally accepted method for the reli-
able measurement of the iron pool.7,8 A wide range of methods
have been investigated as possible means of making reliable NTBI
measurements, such as the bleomycin assay,9,10 inductively cou-
pled plasma (ICP),11,12 HPLC,13 atomic absorption spectros-
copy,14,15 electrospray ionisation (ESI)16 and fluorescence-based
methods.17–19 In this study, a fluorescent-based assay utilising
hexadentate chelators is presented. The method is highly sensitive
and relatively easy to use. The application to the measurement of
NTBI is, however, limited by the variable autofluorescence of the
serum samples.
2.1. Design of the hexadentate fluorescent probes
In order to adopt the correct geometry of aromatic ligands for
iron binding, it is essential that the backbone should be connected
to the ring at the ortho position relative to one of the chelating
oxygen atoms.20 In this work, approaches to the design were
made using an amide linkage located at either the 2- or 5-position
of the 3-hydroxypyridin-4-one or the 2-position of the 3-hydrox-
ypyran-4-one rings in order to direct the chelating oxygens to
provide a suitable octahedral field to bind iron(III). In such exam-
ples, the amide group which links the ligands to the backbone
structure is capable of forming an intramolecular hydrogen bond,
resulting in a modified affinity of the corresponding iron complex
(Fig. 1).21
O
O
H
R
N
O
O
H
N
O
R
N
H
N
H
O
2-Substituent
5-Substituent
* Corresponding author. Tel.: +44 20 7848 4882; fax: +44 20 7848 4800.
E-mail address: robert.hider@kcl.ac.uk (R.C. Hider).
Figure 1. Intramolecular H-bond formation in ortho amido-3-hydrogypyridin-4-
ones.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.052

8094
Y. M. Ma, R. C. Hider / Bioorg. Med. Chem. 17 (2009) 8093–8101
2.2. Synthesis of the hexadentate fluorescent probes
nine was achieved by treatment with N-ethyloxycarbonylphthali-
mide and sodium carbonate to give 14, followed by coupling
with amine 1 in DMF in the presence of DCC/HOBt to give a triester
15. Deprotection of amino group in 15 by hydrazinolysis yielded
amine 16, which on coupling with fluorescein isothiocyanate pro-
duces the triester 17. The triester was hydrolysed by formic acid to
generate the triacid 18. This acid was coupled to aminopyridinone
6 and de-protected using BCl3 to yield the hexadentate probe 20.
The synthetic route of the three coumarin-based hexadentate
probes is presented in Scheme 1. When amine 122 was treated with
coumarin 223 in DMF in the presence of DCC/HOBt, the desired cou-
marin-linked triester 3 was furnished in good yield (65%). Hydroly-
sis of ester 3 to the triacid 4 was accomplished in 95% yield by
treatment with formic acid. The coumarin-labelled triacid 4 was
activated by reaction with DCC/NHS, then coupled with the amin-
opyridinones 5 or 6 or aminopyranone 723 to furnish the corre-
sponding protected fluorescent hexadentate molecules 8–10. The
protecting methyl or benzyl groups were removed by either treat-
ment with BCl3 or by hydrogenation to produce the desired iron-
chelating probes 11–13.
2.3. Fluorescence properties of the probes
The introduction of chelating moieties did not influence the
kmax of both the absorption and emission of the fluorescent hexa-
dentate ligands. As shown in Table 1, the hexadentate probes have
the same maximum absorption wavelength (kabs) and the same
maximum emission wavelength (kem) as the parent molecule.
The synthesis of the fluorescein-labelled hexadentate ligand is
outlined in Scheme 2. The protection of the amino group of b-ala-
COOR
COOR
O
O
COOBu-t
COOH
N
H
a
H N
2
COOBu-t
COOBu-t
N
O
N
O
O
COOR
3, R=Bu-t
4, R=H
2
b
1
OH
O
OCH3
NH
O
O
N
NH
O
O
N
H
d
N
c / 5
O
N
H
3
N
O
O
O
3
N
O
11
8
O
HO
O
N
O
H
BnO
H
N
N
O
O
N
H
N
e
c / 6
O
N
H
3
N
O
O
O
3
N
O
12
9
O
O
O
HO
O
O
H
BnO
H
N
O
O
N
N
H
O
e
c / 7
N
H
O
3
N
O
O
3
N
O
13
10
O
O
O
OBn
NH
OMe
OBn
NH
N
H
2
N
2
N
H
O
7
6
5
Scheme 1. Reagents and conditions: (a) DCC/HOBt, DMF, rt (65%); (b) HCOOH, rt 95%; (c) DCC/NHS, DMF, after 1 h followed by 3-methoxy-2-methyl-5-methylaminomethyl-
1H-pyridin-4-one (5), 2-aminomethyl-3-benzyloxy-1,6-dimethyl-1H-pyridin-4-one (6) or 2-aminomethyl-3-benzyloxy-6-methyl-pyran-4-one (7), rt; (d) BCl3, CH2Cl2; (e)
Pd/C (cat.), H2 2 bars, rt.

Y. M. Ma, R. C. Hider / Bioorg. Med. Chem. 17 (2009) 8093–8101
8095
COOBu-t
COOBu-t
O
O
HN
O
COOH
COOH
a
b
N
H2N
N
COOBu-t
O
O
15
14
COOBu-t
COOBu-t
COOBu-t
COOBu-t
HN
O
c
HN
O
d
HN
Fl
H2N
COOBu-t
COOBu-t
17
16
BnO
N
O
O
COOH
COOH
COOH
N
H
HN
O
HN
O
f
e
OBn
O
HN
Fl
HN
Fl
O
N
O
H
N
HN
N
18
OBn
O
HO
N
O
O
O
19
N
H
HN
O
g
OH
HO
O
O
HN
Fl
O
N
H
O
N
HN
N
COOH
Fl =
OH
HN
S
O
20
Scheme 2. Reagents and conditions: (a) N-ethyloxycarbonylphthalimide, Na2CO3, 72%; (b) DCC/HOBt, DMF, overnight, 66%; (c) NH2NH2, EtOH, 95%; (d) fluorescein
isothiocyanate/DMF; (e) HCOOH, rt, 98%; (f) DCC/HOBt, 2-aminomethyl-3-benzyloxy-1,6-dimethyl-1H-pyridin-4-one (6), DMF; (g) BCl3, CH2Cl2.
Table 1
300
Fluorescence characteristics of hexadentate probes
250
Code
kabs (nm)
kem (nm)
200
150
100
50
7-Diethylamino-coumarin-3-carboxylic acid
11
12
13
Fluorescein-5-isothiocyanate
20
435
435
435
435
494
494
470
474
474
474
519
513
0
0
1
2
3
4
5
Iron(II) (μM)
Figure 2. Fluorescence quenching of 11 by iron. The fluorescence intensity of 11
(6 M) in MOPS (50 mM, pH 7.4) was measured by a Perkin–Elmer spectrofluo-
rometer and recorded in arbitrary units (a.u.). The fluorescence intensity was then
quenched by successive additions of iron using ferrous ammonium sulfate
(100 M) stock solution.
The addition of iron does not result in either a red or blue shift of
wavelength but does quench the fluorescence. Under ambient con-
ditions, the hexadentate probe:iron-binding stoichiometry is pre-
dicted to be 1:1. In practice, the probes exceed the sensitivity
predicted by their binding stoichiometry. Fluorimetric titrations
of 11 demonstrate that the maximal quenching is reached at an ir-
on:probe ratio of 0.67 (Fig. 2). Equivalent curves were obtained for
each of the four probe molecules.
l
a
l
immediately oxidised after combining with the probes to form ir-
on(III) complexes, due to its very high affinity for iron(III) (pFeIII–
pFeII >15; pFe is defined as the negative logarithm of the free iron
concentration, calculated for a total ligand concentration of 10À5 M
and a total iron concentration of 10À6 M at pH 7.4). Not only iro-
n(II) and iron(III) but also other metals such as Cu(II), Co(II), Zn(II)
were found to quench probe fluorescence. The relative quenching
2.4. The Influence of different metals on the probe fluorescence
Although the hexadentate probes are selective for iron(III), they
are also highly responsive to iron(II). This is because iron(II) is

8096
Y. M. Ma, R. C. Hider / Bioorg. Med. Chem. 17 (2009) 8093–8101
potency of metals to 12 was found to be in the sequence
Fe(III) ꢀ Fe(II) > Cu(II) > Zn(II) > Ni(II) ꢀ Mn(II) > Co(II) at me-
100
80
60
40
20
0
a
tal:probe ratio of 5:3 (Fig. 3). No quenching effect was observed
with the following metal cations: Na, K, Ca and Mg (data not
shown). All four compounds were found to behave in the same
manner. The effect of Cu(II) on the quenching can be eliminated
in the presence of the Cu(II) selective chelator N,N,N’,N’-tetra-
kis(2-pyridylmethyl)-ethylenediamine (TPEN) (Fig. 3). The slope
of fluorescence quenching by Zn(II) is higher than that of others,
which demonstrates that although Zn(II) only partly quenches
the probe fluorescence, it does bind to the probe and diminishes
fluorescence quenching by iron(III).
11
12
13
Figure 4. The effect of concentration of NTA on the fluorescence quenching of
hexadentate fluorescent probes. The fluorescence of 11–13 (6 M) in MOPS
(50 mM, pH 7.4) in the presence of different concentrations of NTA was recorded
l
2.5. Binding affinity of coumarin-labelled hexadentate probes
at kex = 435 nm and kem = 477 nm and set at 100% relative fluorescence. After the
addition of iron(II) (6 lM), the fluorescence of the mixture was measured using the
same instrument settings. The black, white and grey bars are expressed in the
presence of NTA at 0, 8 and 80 mM, respectively.
In order to investigate the relative iron affinities of the hexaden-
tate probes, we studied their competition with nitrolotriacetic acid
(NTA). NTA (0, 8 and 80 mM) was mixed with the coumarin-la-
belled hexadentate probes (6
and the fluorescence quenching induced by the addition of iron(II)
(6 M) was recorded. The presence of NTA did not influence the
lM) in tris-buffer (50 mM, pH 7.4)
concentration, the lower the iron concentration which can be
quantified. However, due to the low molar extinction coefficient
and low quantum yield, coumarin-labelled probes have limited
sensitivity in such measurements. In order to enhance the sensitiv-
ity, a fluorescein derivative was also investigated, as fluorescein
has a much higher molar extinction coefficient and quantum yield.
Furthermore, fluorescein has a higher emission kmax value and
therefore experiences a reduced interference from biological sam-
ples. Thus the probe 20, at a concentration of 50 nM, was used to
investigate the quenching by iron(III). The fluorescence decreased
correspondingly and a linear curve was obtained (Fig 5). Using this
system, iron can be reliably quantified down to levels of 10 nM.
l
fluorescence quenching of 12 on addition of iron (Fig. 4). In contrast,
the fluorescence quenching of 13 by iron was markedly affected in
the presence of 8 mM NTA. The higher the NTA concentration, the
less probe quenching occurs. This finding is consistent with the pre-
diction that a pyridinone hexadentate chelator will possess a higher
affinity for iron than a pyranone counterpart.24 Surprisingly, the
fluorescence quenching of 11, another pyridinone hexadentate
probe, was also found to be dependent on the NTA concentration
(Fig. 4). This may result from a lack of an intramolecular hydrogen
bond with 3-hydroxy group, which leads to a higher pKa value (3-
2
hydroxy group) and correspondingly a lower iron affinity compared
to that of 12. Based on these competition studies, the metal affinity
sequence of the three probes is: 12 > 11 > 13. This result was con-
firmed in other competition studies using non-fluorescent
chelators such as maltol (1 mM), deferiprone (1 mM) and desferri-
2.7. Competition between the hexadentate probes and
chelators present in serum
Solutions of monoiron-transferrin were mixed with partially
iron-chelated fluorescent probes and the fluorescence of the result-
ing solution was monitored every few hours. The fluorescence of
the solutions containing transferrin/11/iron and transferrin/12/
iron were found to gradually decrease with time (Fig. 6). The oppo-
site effect occurred with transferrin/13/iron (Fig. 6). This is due to
higher metal affinities of 11 and 12 than that of transferrin, which
leads to iron redistribution from transferrin to the probe. In con-
trast, 13, with the hydroxypyranone chelating unit, has a weaker
iron affinity constant and thus it transfers iron to transferrin. The
oxamine (DFO; 6
lM) (data not shown).
2.6. Iron quantification
Using 6
lM coumarin-labelled probes, the iron concentration
can be determined over the range 0.5–3
l
M. The lower the probe
14000
12000
10000
8000
6000
4000
2000
0
Figure 3. Fluorescence quenching of 12 by iron(III) in the presence of various
metals. The initial fluorescence of 12 (6 M) in MOPS (50 mM, pH 7.4) was set at
100% relative fluorescence. The fluorescence of probe in the presence of various
metals (10 M) was expressed as percentage of initial fluorescence. The fluores-
cence intensity was then decreased by successive additions of iron(III) (100
0
10
20
30
40
50
l
iron concentration (nM)
l
lM
Figure 5. Fluorescence quenching of 20 by iron(III). The fluorescence intensity of 20
(50 nM) and various concentrations of iron(III) in MOPS (50 mM, pH 7.4) was
measured at 494/513 nm excitation/emission and presented in arbitrary units.
stock solution) prepared from FeCl3 and NTA (1:2.2 molar ratio). The metal ions
were prepared from their corresponding salts (CuCl2, NiCl2, ZnCl2, CoCl2 and MnCl2).

Y. M. Ma, R. C. Hider / Bioorg. Med. Chem. 17 (2009) 8093–8101
8097
promising probes 12 and 20. Iron (0–1
was added to a MOPS buffer containing 2
10 min, followed by an addition of serum (10% for final percent-
age). The fluorescence of the mixture was then measured and cal-
culated as a percentage of the initial fluorescence in which no iron
was added. The difference of the fluorescence between probe only
l
M final concentration)
140
120
100
80
lM 12 at pH7.4 after
13
11
60
12
and probe with various iron additions is calculated as
increase of iron added, F increases correspondingly. A linear stan-
dard curve was obtained (Fig. 8a). Referring to the standard curves,
‘unknown’ samples (0–10 M, prepared blind by other member of
DF. With the
40
D
20
l
0
the research group) were performed under the same experimental
conditions and the iron concentrations were calculated. If neces-
sary, the ‘unknown’ iron solution is diluted to allow the
0
10
20
30
40
50
60
Time (h)
DF value
Figure 6. Competition of iron(III) between transferrin and hexadentate probes 11,
12 and 13. Monoferric transferrin (65 M) was prepared from apotransferrin and
Fe(NTA)2, both the final concentration at 65 M, in NaHCO3 (50 mM, pH 8.0) and
the solution was incubated at 37 °C for 1 h before use. Half-full iron-chelated probes
(12 M) was prepared from fluorescent probes (12 M) and Fe(NTA)2 (6 M) in
MOPS (50 mM, pH 7.4). Equal volumes of mono-transferrin and half-full iron-
chelated probes were mixed and the fluorescence was measured immediately and
continually at 435/474 nm excitation/emission using a spectrofluorometer for 53 h.
to fall in the range 0–0.9. The measured iron concentrations of
the ‘unknown’ samples using the probe 12 demonstrated good
agreement with their known iron content (Table 2).
However, when the probe 12 was applied for the determination
of NTBI in iron-overloaded patients using a multiwell plate reader,
it was found to be difficult to quantify the NTBI concentration in
l
l
l
l
l
the range of 0–10
excitation/emission filters which matched the maximum excita-
tion/emission wavelength of the probe (kex = 435 nm,
lM for two reasons. One, there were no suitable
rates of iron exchange under these conditions were relatively slow,
relatively little exchange occurring after 30 min.
kem = 474 nm). This decreased the sensitivity of the fluorescence
method. The second reason is that due to the comparatively low
excitation/emission wavelengths, the autofluorescence from the
serum influences the sample fluorescence determination to an
appreciable extent, which leads to false readings. In principle the
Citrate, a potential iron-binding ligand, combines with iron to
form many complexes including oligomeric complexes with
molecular masses in the range 1.5–5 kDa.25 It is important to
investigate the competition of the probes with iron-citrate com-
plexes as they are major components of NTBI.26 Iron citrate solu-
tion was prepared at pH 7.4 to final concentrations of 10
lM iron
1
a
and 100 M citrate. Four iron ligands, CP20, DFO, 12 and 13, were
l
selected to investigate the rate of scavenging oligomeric iron. DFO,
a highly hydrophilic hexadentate ligand, has been reported to che-
late oligomeric iron citrate by a relatively slow process.27 This re-
port is consistent with our own observations; the observed T1/2
value of this competition process was 92 min (Fig. 7). Compared
to DFO, CP20 was found to have a shorter T1/2 value (15 min).
Not surprisingly, 12, the hexadentate pyridinone ligand, was found
to exchange iron at the fastest rate (T1/2 = 9 min). The hexadentate
pyranone (13) was associated with the slow exchange rate of
46 min (Fig. 7). Moreover, the pyranone ligand failed to scavenge
all iron from this iron citrate solution, even after 24 h.
0.8
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
2.8. NTBI quantification
iron concentration(μM)
NTBI levels fall into the range of 0–15 l
M.19,28 In order to devel-
op a reliable quantitative method, we have investigated the most
1
0.8
0.6
0.4
0.2
0
b
120
CP20
DFO
100
80
60
40
20
0
12
13
0
100
200
Time (min)
300
400
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Figure 7. The comparison of several iron ligands chelating oligomeric iron. The
absorption of CP20 (30 M) and Fe(NTA)2 (10 M) was measured at 460 nm and set
at 100% relative absorption. The absorption of CP20 (30 M) in the presence of
oligomeric iron (10 M) was measured at same wavelength and given as
percentage of that of CP20–Fe(NTA)2 complex. The absorption of DFO, 12 and 13
(10 M each) in the presence of oligomeric iron (10 M) was measured at 430, 475
and 475 nm, respectively in a time-course and expressed as a percentage of the
absorption of these ligands with Fe(NTA)2 (10 M) at each respective wavelength.
iron concentration(μM)
l
l
l
Figure 8. Standard curves of the fluorescence quenching of 12 (a) and 20 (b) by
additions of various concentrations of iron. The initial fluorescence of 12 (2 M) or
20 (0.5 M) in MOPS (50 mM) at pH 7.4 in the presence of 10% serum was set at
100% and the relative fluorescence of the mixtures of the probe and various
concentrations of iron was calculated and the difference of the fluorescence
l
a
l
l
l
l
l
between the probe–iron mixtures and probe only is presented as DF.

8098
Y. M. Ma, R. C. Hider / Bioorg. Med. Chem. 17 (2009) 8093–8101
Table 2
3. Conclusion
Comparison of ‘unknown’ iron concentrations with measured concentrations in 10%
serum determined by 12 and 20 (n = 4)
Four hexadentate fluorescent probes have been described, three
based on the coumarin moiety and one on fluorescein. These
probes were found to be more selective for iron than other bivalent
metals such as Cu, Zn, Ni, Mn and Co. Although Cu has a marked
effect on the fluorescence quenching, the contribution of this metal
in our study is likely to be negligible due to its relatively low con-
centration in serum. Furthermore, should competition by copper
be a problem it can be abolished by the inclusion of TPEN in the as-
say. Iron affinity studies show that the pyridinone-based hexaden-
tates are capable of competing all iron from weak ligands such as
NTA and citrate. By increasing the molar extinction coefficient
and quantum yield of the fluorophore, the detection limit of iron
concentration has been decreased to 10 nM. However, neither cou-
marin- nor fluorescein-based probes are suitable for the direct
determination of NTBI, due to the variable color and autofluores-
cence of sera. We continue to seek a reliable fluorescence-based
method for the quantification of NTBI.
‘Unknown’
[iron]
Measured [iron]
with 12
‘Unknown’
[iron]
Measured [iron]
with 20
4
0
6
1
2
10
5
8
7
3
3.9 0.2
0.3 0.2
5.7 0.3
1.4 0.2
2.3 0.1
9.6 0.7
5.1 0.3
7.9 0.6
7.4 0.4
3.4 0.2
9.0 0.5
9.5
8.2
7
6.8
4.9
3.2
2.5
1.4
0
9.05 0.18
8.66 0.08
8.22 0.08
7.63 0.11
5.63 0.22
3.32 0.18
2.92 0.24
1.79 0.50
À0.02 0.13
9
use of the fluorescein-based hexadentate probe 20 could minimise
these problems. The fluorescence of 0.5 M 20 was measured by a
l
96-well plate reader (Ex: 485 20 nm; Em: 530 25 nm) and a
standard curve was constructed in similar fashion to that described
for 12 (Fig. 8b). The iron concentrations of ‘unknown’ samples were
then calculated according to the standard curve. Again, the mea-
sured iron concentrations are comparable to the known iron con-
tent (Table 2).
4. Experimental
4.1. General
All chemicals were purchased from commercial sources and
used without further purification. Normal mixed pool serum was
purchased from First Link (UK) Ltd. Melting points were deter-
mined using an Electrothermal IA 9100 Digital Melting Point Appa-
ratus and are uncorrected. 1H NMR spectra were recorded using a
Bruker (360 MHz) NMR spectrometer. Chemical shifts (d) are re-
ported in ppm downfield from the internal standard tetramethyl-
silane (TMS). Mass spectra (FAB) analyses were carried out by
the Mass Spectrometry Facility, School of Health and Life Science,
King’s College London. Elemental analyses were performed by Mi-
cro-analytical laboratories, Department of Chemistry, The Univer-
sity of Manchester.
Interestingly, when serum (10% dilution) was incubated with
various iron levels for 30 min before adding the probe 20, a linear
relationship was not obtained between the ‘unknown’ iron level
and measured iron (Fig. 9). Over the range 0–4
centration was recorded at the same level, that is, approximately
M. This is a result of the presence of apotransferrin in the serum
lM, the iron con-
0
l
sample which chelates added iron until saturated. The probe is not
able to scavenge iron from transferrin in the adopted experimental
time. When all the apotransferrin is saturated with iron, additional
iron is available to be measured by the probe. Therefore, a linear
curve is observed over the range of 4–8 lM added iron concentra-
tion (Fig. 9). Using this method, the percentage saturation of trans-
ferrin can be calculated.
4.1.1. 7-Diethylamino-N-[(tri-tert-
butoxycarbonylethyl)methyl]-2-oxo-2H-chromen-3-
carboxamide (3)
Unfortunately, when this direct method was applied to various
serum samples, the resulting NTBI concentrations were found to
vary and were not comparable to those determined by the HPLC
method.13 The colour and autofluorescence varies between differ-
ent serum samples and this has a major influence on the results.
A
mixture of di-tert-butyl 4-amino-4-[2-(tert-butoxycar-
bonyl)ethyl] heptanedioate 1 (10 mmol), 7-diethylamino-2-oxo-
2H-chromen-3-carboxylic acid 2 (10 mmol), dicyclohexylcarbodi-
imide (DCC, 12 mmol), 1-hydroxybenzotriazole (HOBt, 10 mmol),
and DMF (100 mL) was stirred at room temperature overnight.
After filtration and removal of the solvent under reduced pressure,
the residue was chromatographed on silica gel using MeOH/CHCl3
(1:9) as an eluent to afford a yellow solid (65%). Mp 196–197 °C; 1H
NMR (CDCl3) d: 1.24 (t, J = 7.1 Hz, CH3CH2N, 6H), 1.43 (s, C(CH3)3,
27H), 2.06–2.10 (m, CH2CH2CO, 6H), 2.23–2.28 (m, CH2CH2CO,
6H), 3.46 (q, J = 7.1 Hz, CH3CH2N, 4H), 6.50 (d, J = 2.3 Hz, courmain
C-8H, 1H), 6.64 (dd, J = 2.4, 9.0 Hz, courmain C-6H, 1H), 7.39 (d,
J = 9.0 Hz, courmain C-5H, 1H), 8.65 (s, courmain C-4H, 1H). ESI-
MS: 659 (M+1)+.
As this fluorescence method is so sensitive (only 0.5 lM of probe
is used in this study), any small change of fluorescence including
autofluorescence from the serum leads to a relatively large change
in fluorescence emission.
4
3.5
3
2.5
2
1.5
1
4.1.2. 7-Diethylamino-N-[(tri-(2-carboxylethyl)methyl]-2-oxo-
2H-chromen-3-carboxamide (4)
0.5
0
A solution of the tert-butyl ester 3 (10 mmol) in formic acid
(96%, 10 mL) was stirred at 25 °C overnight. After concentration,
toluene (5 mL) was added and the solution was again evaporated
in vacuo to remove azeotropically any residual formic acid. The
residue was then precipitated in acetone (50 mL) to afford a yellow
solid (95%), which can be used for the next step without further
purification. Mp 144–146 °C; 1H NMR (CDCl3) d: 1.24 (t,
J = 7.1 Hz, CH3CH2N, 6H), 1.94–1.98 (m, CH2CH2CO, 6H), 2.21–2.26
(m, CH2CH2CO, 6H), 3.45 (q, J = 7.1 Hz, CH3CH2N, 4H), 6.51 (d,
0
1
2
3
4
5
6
7
8
added iron (uM)
Figure 9. The relationship of concentrations between added iron and measured
iron by 20. The fluorescence of 20 (0.5 M) in a mixture of 10% serum with various
concentrations of iron was measured by a 96-well plate reader. The difference of
the fluorescence between probe-serum-iron complex and probe-serum only was
calculated and converted to iron concentration according to the standard curve of
20.
l

Y. M. Ma, R. C. Hider / Bioorg. Med. Chem. 17 (2009) 8093–8101
8099
J = 2.3 Hz, courmain C-8H, 1H), 6.63 (dd, J = 2.4, 9.0 Hz, courmain C-
6H, 1H), 7.39 (d, J = 9.0 Hz, courmain C-5H, 1H), 8.64 (s, courmain
C-4H, 1H). ESI-MS: 491 (M+1)+.
4.1.6. 7-Diethylamino-N-{[tri-(2-{N-[(5-hydroxy-6-methyl-4-
oxo-1,4-dihydropyridin-3-yl)methyl]-N-methylcarbamoyl}eth-
yl)]methyl}-2-oxo-2H-chromen-3-carboxamide (11)
A solution of compound 8 (3 mmol) in CH2Cl2 (50 ml) was
flushed with nitrogen. After the flask was cooled to 0 °C, boron
trichloride (1 M in CH2Cl2, 20 ml) was added dropwise and the
reaction mixture was allowed to stir at room temperature for
1 d. The excess BCl3 was eliminated at the end of the reaction
by the addition of methanol (10 ml) and left to stir for another
10 min. After removal of the solvents under reduced pressure,
the residue were purified by recrystallization from methanol/
acetone to afford yellow solid (77%). Mp 173–175 °C; 1H NMR
(400 MHz, CD3OD) d: 1.26 (t, J = 7.0 Hz, CH3CH2N, 6H), 2.16–
2.22 (m, CH2CH2CO, 6H), 2.35–2.61 (2 m, CH2CH2CO, 6H), 2.53
and 2.55 (2s, CCH3, 9H), 2.78 and 3.20 (2s, NCH3, 9H), 3.55 (q,
J = 7.0 Hz, CH3CH2N, 4H), 4.27 and 4.57 (2s, CCH2N, 6H), 6.56
(s, courmain C-8H, 1H), 6.85 (d, J = 8.3 Hz, courmain C-6H, 1H),
7.53 (d, J = 8.3 Hz, courmain C-5H, 1H), 8.15 and 8.16 (2s, pyrid-
inone C-6H, 3H), 8.51 (s, courmain C-4H, 1H). 13C NMR
(100 MHz, CD3OD) d: 12.73 (CH3), 14.30 and 14.51 (CH3), 28.46
(CH2), 29.28 (CH2), 31.06 (CH2), 33.54 (CH3), 36.78 (CH3), 46.10
(CH2), 46.39 and 46.65 (CH2), 59.15 (C), 97.34 (CH), 111.86
(CH), 121.99 (C), 132.72 (CH), 135.05 and 136.64 (CH), 141.41
(C), 144.27 (C), 148.94 (CH), 154.65 (C), 159.20 (C), 160.35 (C),
164.48 (C), 165.50 (C), 176.86 (C), 177.88 (C). ESI-MS: 941
(M+1)+. Elemental Anal. Calcd for C49H71N8O15Cl5: C, 49.48; H,
6.02; N, 9.42. Found: C, 49.94; H, 6.18; N, 9.55.
4.1.3. 7-Diethylamino-N-{[tri-(2-{N-[(5-methoxy-6-methyl-4-oxo
-1,4-dihydropyridin-3-yl)methyl]-N-methylcarbamoyl}ethyl)]
methyl}-2-oxo-2H-chromen-3-carboxamide (8)
To a solution of compound 4 (10 mmol) in dry DMF (100 mL),
DCC (12 mmol) and N-hydroxysuccinimide (NHS) (12 mmol) were
added. The mixture was allowed to stir for 2 h before 3-methoxy-
2-methyl-5-methylaminomethyl-1H-pyridin-4-one
5 (10 mmol)
was added, and the reaction was left to stir at room temperature
overnight. The precipitation was removed by filtration, and the
solvent was evaporated. The residue was dissolved in dichloro-
methane and washed with 0.1 N NaOH (3Â) and brine, dried over
anhydrous Na2SO4 and concentrated under reduced pressure, The
obtained residue was purified by column chromatography
(MeOH/CHCl3, 1:9), affording the title compound as a yellow solid
(Yield 59%). Mp 206–209 °C; 1H NMR (CDCl3) d: 1.14 (t, J = 7.1 Hz,
CH3CH2N, 6H), 1.92–2.09 (m, CH2CH2CO, 6H), 2.18 (s, CCH3, 9H),
2.32–2.42 (m, CH2CH2CO, 6H), 3.06 (s, NCH3, 9H), 3.47 (q,
J = 7.4 Hz, CH3CH2N, 4H), 3.79 (s, OCH3, 9H), 4.46 (s, CCH2N,
6H), 6.50 (d, J = 2.0 Hz, courmain C-8H, 1H), 6.67 (dd, J = 2.0,
9.0 Hz, courmain C-6H, 1H), 7.42 (d, J = 9.1 Hz, courmain C-5H,
1H), 8.57 (s, courmain C-4H, 1H), 8.61 (s, pyridinone C-6H, 3H).
ESI-MS: 983 (M+1)+. Elemental Anal. Calcd for C51H66N8O12ÁCHCl3
Á7H2O: C, 50.84; H, 6.65; N, 9.12. Found: C, 50.70; H, 6.44; N, 9.31.
Analogous procedures starting with triacid 4 with 2-amino-
methyl-3-benzyloxy-1,6-dimethyl-1H-pyrindin-4-one 6 or 2-ami-
nomethyl-3-benzyloxy-6-methyl-pyran-4-one 7 gave compounds
9 and 10, respectively.
4.1.7. 7-Diethylamino-N-{[tri-(2-{N-[(3-hydroxy-1,6-dimethyl-
4-oxo-1,4-dihydropyridin-2-yl)methyl]carbamoyl}ethyl)]meth-
yl}-2-oxo-2H-chromen-3-carboxamide (12)
A solution of compound 9 (5 mmol) in ethanol (30 ml) was sub-
jected to hydrogenolysis in presence of 5% Pd/C (w/w) catalyst over-
night. The catalysts were removed by filtration and the filtrates were
acidified to pH 1 with concentrated hydrochloric acid. After removal
of the solvents in vacuo, the residues were purified by recrystalliza-
tion from methanol/acetone to afford a yellow solid (87%). Mp 195–
196 °C; 1H NMR (360 MHz, CD3OD) d: 1.26 (t, J = 6.4 Hz, CH3CH2N,
6H), 2.12 (s, CH2CH2CO, 6H), 2.33 (s, CH2CH2CO, 6H), 2.65 (s, CCH3,
9H), 3.56 (q, J = 6.4 Hz, CH3CH2N, 4H), 4.02 (s, NCH3, 9H), 4.72 (s,
CCH2N, 6H), 6.56 (s, courmain C-8H, 1H), 6.86 (d, J = 8.6 Hz, cour-
main C-6H, 1H), 7.07 (s, pyridinone C-5H, 3H), 7.53 (d, J = 8.6 Hz,
courmain C-5H, 1H), 8.48 (s, courmain C-4H, 1H). 13C NMR
(100 MHz, CD3OD) d: 12.74 (CH3), 21.25 (CH3), 30.70 (CH2), 31.35
(CH2), 36.56 (CH2), 39.92 (CH3), 46.06 (CH2), 59.25 (C), 97.22 (CH),
109.44 (C), 110.38 (C), 111.90 (CH), 114.23 (CH), 132.74 (CH),
140.93 (C), 144.82 (C), 148.90 (CH), 150.81 (C), 154.72 (C), 159.10
(C), 161.36 (C), 164.21 (C), 164.37 (C), 176.50 (C). ESI-MS: 941
(M+1)+. Elemental Anal. Calcd for C48H73N8O17Cl3: C, 50.55; H,
6.45; N, 9.82. Found: C, 50.95; H, 6.49; N, 9.62.
4.1.4. 7-Diethylamino-N-{[tri-(2-{N-[(3-benzoxy-1,6-dimethyl-
4-oxo-1,4-dihydropyridin-2-yl)methyl]carbamoyl}ethyl)]meth-
yl}-2-oxo-2H-chromen-3-carboxamide (9)
Mp 231–232 °C; 1H NMR (CDCl3) d: 1.25 (t, J = 7.1 Hz, CH3CH2N,
6H), 2.05–2.10 (m, CH2CH2CO, 6H), 2.13 (s, CCH3, 9H), 2.15–2.21
(m, CH2CH2CO, 6H), 3.36 (s, NCH3, 9H), 3.44 (q, J = 7.1 Hz, CH3CH2N,
4H), 4.29 (d, J = 5.3 Hz, CCH2N, 6H), 4.95 (s, PhCH2O, 6H), 6.14 (s,
pyridinone C-5H, 3H), 6.47 (d, J = 2.2 Hz, courmain C-8H, 1H),
6.65 (dd, J = 2.3, 9.1 Hz, courmain C-6H, 1H), 6.73 (br s, NH, 3H),
7.22–7.30 (m, PhH, 15H), 7.39 (d, J = 9.0 Hz, courmain C-5H, 1H),
8.60 (s, courmain C-4H, 1H). ESI-MS: 1211 (M+1)+. Elemental Anal.
Calcd for C69H78N8O12: C, 68.41; H, 6.49; N, 9.25. Found: C, 68.08;
H, 6.43; N, 9.46.
4.1.5. 7-Diethylamino-N-{[tri-(2-{N-[(3-benzoxy-6-dimethyl-4-
oxo-4H-pyran-2-yl)methyl]carbamoyl}ethyl)]methyl}-2-oxo-
2H-chromen-3-carboxamide (10)
Mp 169–171 °C; 1H NMR (CDCl3) d: 1.25 (t, J = 7.0 Hz,
CH3CH2N, 6H), 2.06 (br s, CH2CH2CO, 12H), 2.20 (s, CCH3, 9H),
3.46 (q, J = 7.0 Hz, CH3CH2N, 4H), 4.13 (d, J = 5.9 Hz, CCH2N, 6H),
5.16 (s, PhCH2O, 6H), 5.63 (q, J = 5.9 Hz, NH, 3H), 6.15 (s, pyridi-
none C-5H, 3H), 6.49 (s, courmain C-8H, 1H), 6.66 (d, J = 9.0 Hz,
courmain C-6H, 1H), 7.31–7.35 (m, PhH, 15H), 7.40 (d,
J = 9.0 Hz, courmain C-5H, 1H), 8.60 (s, courmain C-4H, 1H),
8.63 (s, NH, 1H). 13C NMR (100 MHz, CDCl3) d: 12.42 (CH3),
19.66 (CH3), 30.55 (CH2), 30.76 (CH2), 36.24 (CH2), 45.15 (CH2),
58.17 (C), 73.58 (CH2), 96.50 (CH), 108.28 (C), 109.78 (C),
110.30 (CH), 114.93 (CH), 128.69 (CH), 129.50 (CH), 131.31
(CH), 136.58 (C), 142.43 (C), 148.01 (CH), 152.92 (C), 156.48 (C),
157.74 (C), 162.37 (C), 163.12 (C), 164.80 (C), 172.42 (C), 175.82
(C). ESI-MS: 1172 (M+1)+. Elemental Anal. Calcd for
C66H69N5O15Á0.5CHCl3: C, 64.83; H, 5.69; N, 5.68. Found: C,
64.45; H, 5.84; N, 5.65.
4.1.8. 7-Diethylamino-N-{[tri-(2-{N-[(3-hydroxy-6-dimethyl-4-
oxo-4H-pyran-2-yl)methyl]carbamoyl}ethyl)]methyl}-2-oxo-2H-
chromen-3-carboxamide (13)
An analogous procedure starting with compound 10 gave 7-
diethylamino-N-{[tri-(2-{N-[(3-hydroxy-6-dimethyl-4-oxo-4H-py-
ran-2-yl)methyl]carbamoyl}ethyl)]methyl}-2-oxo-2H-chromen-3-
carboxamide (13). Mp 139–141 °C; 1H NMR (DMSO-d6) d: 1.16 (t,
J = 7.0 Hz, CH3CH2N, 6H), 1.93–1.99 (m, CH2CH2CO, 6H), 2.16–2.19
(m, CH2CH2CO, 6H), 2.27 (s, CCH3, 9H), 3.46 (q, J = 7.1 Hz, CH3CH2N,
4H), 4.54 (d, J = 5.8 Hz, CCH2N, 6H), 6.24 (s, pyridinone C-5H, 3H),
6.64 (d, J = 1.9 Hz, courmain C-8H, 1H), 6.85 (dd, J = 2.0, 9.1 Hz,
courmain C-6H, 1H), 7.76 (d, J = 9.1 Hz, courmain C-5H, 1H), 8.60
(s, courmain C-4H, 1H), 8.99 (t, J = 5.8 Hz, NH, 3H). ESI-MS: 902
(M+1)+.

8100
Y. M. Ma, R. C. Hider / Bioorg. Med. Chem. 17 (2009) 8093–8101
4.1.9. 3-(1,3-Dioxoisoindolin-2-yl)propanoic acid (14)
A vigorously stirred solution of b-analine (4.46 g, 50 mmol) and
sodium carbonate (5.35 g, 50.5 mmol) in water (100 mL) was trea-
ted with N-ethyloxycarbonylphthalimide (11.51 g, 52.5 mmol) and
stirred for 1 h. The solution was filtered and the residue was acid-
ified to pH 2–3 with 6 N HCl, which resulted in a white precipitate.
The solution was filtered and the precipitate was washed with
water and drying in air to obtain a white crystal (7.89 g, 72% yield).
Mp 150–152 °C (lit29 152–153 °C). 1H NMR (DMSO-d6) d: 2.61 (t,
J = 7.3 Hz, CH2CO, 2H), 3.80 (t, J = 7.3 Hz, NCH2, 2H), 7.82–7.89
(m, ArH, 4H).
Calcd for C79H81N9O15SÁCHCl3: C, 62.07; H, 5.34; N, 8.14. Found: C,
62.32; H, 5.58; N, 8.25.
4.1.15. Fluorescein-5-thiocarbamoyl tri-(3-hydroxy)pyridin-4-
one 20
An analogous procedure described for 11 gave fluorescein-5-
thiocarbamoyl tri-(3-hydroxy)pyridin-4-one 20. Mp 202–205 °C.
1H NMR (400 MHz, CD3OD) d: 2.03 (m, CH2CH2CO, 6H), 2.30 (m,
CH2CH2CO, 6H), 2.42 (m, CH2CO, 2H), 2.64 (br s, CCH3 + CH2CO,
11H), 3.89 (m, NCH2, 2H), 4.02 (s, NCH3, 9H), 4.74 (s, CCH2N, 6H),
6.38–8.83 (m, ArH + pyridinone C-5H, 12H), 10.75 (br s, NH, 1H),
10.95 (br s, NH, 1H). 13C NMR (100 MHz, CD3OD) d: 21.35
(3CCH3), 30.85 (3CCH2CH2), 31.13 (CH2CH2CO), 31.26 (3CCH2CH2),
36.50 (3NHCH2-pyridinone), 40.13 (3NCH3), 41.85 (NHCH2CH2),
59.28 (C), 103.46 (CH), 103.57 (CH), 114.26 (3CH in pyridinone),
116.04 (C), 118.62 (CH), 129.77 (C), 130.29 (CH), 132.24 (C),
133.01 (CH), 135.27 (C), 140.99 (3C in pyridinone), 144.77 (3C in
pyridinone), 150.88 (3C in pyridinone), 158.44 (C), 160.81 (C),
161.19 (3CO in pyridinone), 168.85 (C), 173.58 (C), 176.65 (C),
176.68 (3CONH), 182.54 (C). m/z: 1158 (M+1)+. Elemental Anal.
Calcd for C59H72N9O17SCl5: C, 51.03; H, 5.23; N, 9.08. Found: C,
51.18; H, 5.48; N, 8.96.
4.1.10. Di-tert-butyl 4-(2-(tert-butoxycarbonyl)ethyl)-4-(3-(1,3-
dioxoisoindolin-2-yl)propanamido)heptanedioate (15)
A mixture of 14 (2.19 g, 10 mmol), amine 1 (4.57 g, 11 mmol),
DCC (2.27 g, 11 mmol), HOBt (1.49 g, 11 mmol) in DMF (60 mL)
was stirred at room temperature for 1 d. After filtration, the solvent
was removed under reduced pressure, the residue was purified on
a silica gel column using EtOAc/MeOH (9.5:0.5) as an eluent to af-
ford a white solid (4.08 g, 66%). Mp 144–145 °C. 1H NMR (CDCl3) d:
1.43 (s, C(CH3)3, 27H), 1.93–1.98 (m, CH2CH2CO, 6H), 2.18–2.23 (m,
CH2CH2CO, 6H), 2.56 (t, J = 7.5 Hz, CH2CO, 2H), 3.97 (t, J = 7.5 Hz,
NCH2, 2H), 6.13 (s, NH, 1H), 7.71 (m, ArH, 2H), 7.85 (m, ArH, 2H).
ESI-MS: 617 (M+1)+.
4.2. Fluorescence measurements
4.1.11. Di-tert-butyl 4-(2-(tert-butoxycarbonyl)ethyl)-4-(3-
amino)propanamido)heptanedioate (16)
The fluorescent intensity was scanned with either a Perkin–El-
mer spectrofluorometer (type LS 50B) or a CytoFluor 4000 Fluores-
cence Multi-Well Plate Reader. Spectra were not corrected for light
intensity or detector sensitivity. Data were recorded on-line and
analysed by excel software on a PC. Fluorescence measurements
were performed twice in MOPS buffer (50 mM, pH 7.4) at 22 °C
and carried out independently at least three times on different
days. The results are expressed as means SD.
To a solution of 15 (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 N NaOH, and extracted with chloroform (5 Â 100 mL).
The combined organic extracts were dried over anhydrous Na2SO4
under reduced pressure to furnish 16 (85%) as pale brown oil. 1H
NMR (CDCl3) d: 1.43 (s, C(CH3)3, 27H), 1.94–1.99 (m, CH2CH2CO,
6H), 2.19–2.24 (m, CH2CH2CO, 6H), 2.29 (t, J = 5.9 Hz, NCH2, 2H),
2.45 (br s, NH2, 2H), 3.00 (t, J = 5.9 Hz, CH2CO, 2H), 7.34 (br s,
NH, 1H). ESI-MS: 487 (M+1)+.
Acknowledgement
Authors would like to thank Dr. Dingyong Liu for preparing the
‘unknown’ concentrations of iron samples.
Supplementary data
4.1.12. Fluorescein-5-thiocarbamoyl triester 17
Fluorescein isothiocyanate (2 mmol) was added to a solution of
16 (2 mmol) in DMF (20 mL). The reaction mixture was stirred for
24 h at room temperature. After evaporation, the residue was puri-
fied by column chromatography (eluent: MeOH/CHCl3 = 1:9) to ob-
tain an orange foam (78%). 1H NMR (CDCl3) d: 1.40 (s, C(CH3)3,
27H), 1.95 (m, CH2CH2CO, 6H), 2.21 (m, CH2CH2CO, 6H), 2.55 (t,
CH2CO, J = 7.5 Hz, 2H), 3.93 (t, J = 7.5 Hz, NCH2, 2H), 6.53–8.30
(m, ArH, 9H). ESI-MS: 876 (M+1)+.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.09.052.
References and notes
1. Bothwell, T. H.; Charlton, R. W.; Cook, J. D.; Finch, C. A. Iron Metabolism in Man;
Blackwell Scientific: Oxford, 1979.
2. Crichton, R. R. Inorganic Biochemistry of Iron Metabolism; John Wiley & Sons:
West Sussex, 2001.
3. Conrad, M. E.; Barton, J. C. Am. J. Hematol. 1981, 10, 199.
4. Chung, J.; Wessling-Resnick, M. Crit. Rev. Clin. Lab. Sci. 2003, 40, 151.
5. Anderson, G. J. J. Gastroenterol. Hepatol. 1999, 14, 105.
6. Breuer, W.; Hershko, C.; Cabantchik, Z. I. Transfus. Sci. 2000, 23, 185.
7. Jacobs, E. M. G.; Hendriks, J. C. M.; van Tits, B. L. J. H.; Evans, P. J.; Breuer, W.;
Liu, D. Y.; Jansen, E. H. J. M.; Jauhiainen, K.; Sturm, B.; Porter, J. B.; Scheiber-
Mojdehkar, B.; von Bonsdorff, L.; Cabantchik, Z. I.; Hider, R. C.; Swinkels, D. W.
Anal. Biochem. 2005, 341, 241.
4.1.13. Fluorescein-5-thiocarbamoyl triacid 18
An analogous procedure described for 4 gave fluorescein-5-thi-
ocarbamoyl triacid 18. Mp 195–198 °C. 1H NMR (DMSO-d6) d:
1.84–1.88 (m, CH2CH2CO, 6H), 2.11–2.16 (m, CH2CH2CO, 6H), 2.46
(t, J = 6.4 Hz, CH2CO, 2H), 3.44 (q, J = 7.0 Hz, NCH2, 2H), 6.53–8.32
(m, ArH, 9H), 10.01 (br s, NH, 2H). ESI-MS: 708 (M+1)+.
8. Kolb, A. M.; Smit, N. P.; Lentz-Ljuboje, R.; Osanto, S.; van, P. J. Anal. Biochem.
2009, 385, 13.
9. Sahlstedt, L.; Ebeling, F.; von, B. L.; Parkkinen, J.; Ruutu, T. Br. J. Haematol. 2001,
113, 836.
10. von Bonsdorff, L.; Lindeberg, E.; Sahlstedt, L.; Lehto, J.; Parkkinen, J. Clin. Chem.
2002, 48, 307.
11. Nuttall, K. L.; Gordon, W. H.; Ash, K. O. Ann. Clin. Lab. Sci. 1995, 25, 264.
12. Yokoi, K.; Kimura, M.; Itokawa, Y. Biol. Trace Elem. Res. 1991, 31, 265.
13. Gosriwatana, I.; Loreal, O.; Lu, S.; Brissot, P.; Porter, J.; Hider, R. C. Anal. Biochem.
1999, 273, 212.
14. Jakeman, A.; Thompson, T.; McHattie, J.; Lehotay, D. C. Clin. Biochem. 2001, 34,
43.
4.1.14. Fluorescein-5-thiocarbamoyl tri-(3-benzoxy)pyridine-4-
one 19
An analogous procedure described for 9 gave fluorescein-5-thi-
ocarbamoyl tri-(3-benzoxy)pyridine-4-one 19. Mp 198–200 °C. 1H
NMR (DMSO-d6) d: 1.81 (m, CH2CH2CO, 6H), 2.03 (m, CH2CH2CO,
6H), 2.24 (s, CCH3, 9H), 2.41 (m, CH2CO, 2H), 3.38 (s, NCH3, 9H),
3.68 (m, NCH2, 2H), 4.33 (s, CCH2N, 6H), 5.06 (s, PhCH2O, 6H),
6.18 (s, pyridinone C-5H, 3H), 6.54–8.38 (m, ArH, 24H), 10.15 (br
s, NH, 1H), 10.27 (br s, NH, 1H). m/z: 1428 (M+1)+. Elemental Anal.
15. Jittangprasert, P.; Wilairat, P.; Pootrakul, P. Southeast Asian J. Trop. Med. Public
Health 2004, 35, 1039.

Y. M. Ma, R. C. Hider / Bioorg. Med. Chem. 17 (2009) 8093–8101
8101
16. von, B. L.; Tolo, H.; Lindeberg, E.; Nyman, T.; Harju, A.; Parkkinen, J. Biologicals
2001, 29, 27.
22. Newkome, G. R.; Behera, R. K.; Moorefield, C. N.; Baker, G. R. J. Org. Chem. 1991,
56, 7162.
17. Breuer, W.; Cabantchik, Z. I. Anal. Biochem. 2001, 299, 194.
18. Breuer, W.; Ermers, M. J.; Pootrakul, P.; Abramov, A.; Hershko, C.; Cabantchik, Z.
I. Blood 2001, 97, 792.
19. Breuer, W.; Ronson, A.; Slotki, I. N.; Abramov, A.; Hershko, C.; Cabantchik, Z. I.
Blood 2000, 95, 2975.
20. O’Sullivan, B.; Xu, J.; Raymond, K. N. In Iron chelators: new development
strategies, Badman, D. G., Bergeron, R. J., Brittenham, G. M., Eds.; Saratoga
Publishing Group: Ponte Vedra, 2000, pp 177–208.
21. Liu, Z. D.; Piyamongkol, S.; Liu, D. Y.; Khodr, H. H.; Lu, S. L.; Hider, R. C. Bioorg.
Med. Chem. 2001, 9, 563.
23. Ma, Y.; Luo, W.; Quinn, P. J.; Liu, Z.; Hider, R. C. J. Med. Chem. 2004, 47, 6349.
24. Liu, Z. D.; Hider, R. C. Med. Res. Rev. 2002, 22, 26.
25. Bino, A.; Shweky, I.; Cohen, S.; Bauminger, E. R.; Lippard, S. J. Inorg. Chem. 1998,
37, 5168.
26. Evans, R. W.; Rafique, R.; Zarea, A.; Rapisarda, C.; Cammack, R.; Evans, P. J.;
Porter, J. B.; Hider, R. C. J. Biol. Inorg. Chem. 2008, 13, 57.
27. Faller, B.; Nick, H. J. Am. Chem. Soc. 1994, 116, 3860.
28. al-Refaie, F. N.; Wickens, D. G.; Wonke, B.; Kontoghiorghes, G. J.; Hoffbrand, A.
V. Br. J. Haematol. 1992, 82, 431.
29. Chapman, J. M., Jr.; Cocolas, G. H.; Hall, I. H. J. Med. Chem. 1983, 26, 243.