Design, synthesis, physicochemical properties, and evaluation of novel iron chelators with fluorescent sensors

Article abstract of DOI:10.1021/jm049751s

The synthesis of a range of novel 3-hydroxypyridin-4-ones and 3-hydroxypyran-4-ones linked with different coumarin substituents is described. These compounds have been developed in order to provide a series of molecular probes for the quantification of intracellular labile iron pools. An evaluation of the effect of iron(III) on fluorescence intensity was undertaken. Chelation of iron(III) causes quenching of fluorescence. The relationship between iron(III) concentration and the extent of fluorescence quenching indicates that the metal is chelated in a complex with a metal-to-ligand stoichiometry of 1:3. The fluorescence of hydroxypyridinone compounds was found to be more efficiently quenched by iron(III) than were the hydroxypyranones. The metal-to-ligand stoichiometry at which maximum quenching is observed was found to depend on the site at which coumarin is attached. The efficiency of fluorescence quenching by iron(III) is markedly influenced by solvent polarity and pH. The permeability of two representative fluorescent chelators across human erythrocyte ghost membranes was investigated. The rate of permeability for a series of probes was found to be related to the corresponding ClogP values.

Full text of DOI:10.1021/jm049751s

J. Med. Chem. 2004, 47, 6349-6362
6349
Design, Synthesis, Physicochemical Properties, and Evaluation of Novel Iron
Chelators with Fluorescent Sensors
Yongmin Ma,† Wei Luo,†,‡ Peter J. Quinn,‡ Zudong Liu,† and Robert C. Hider*,†
Departments of Pharmacy and Life Sciences, King’s College London, Franklin-Wilkins Building, Stamford Street,
London, SE1 9NN, UK
Received March 30, 2004
The synthesis of a range of novel 3-hydroxypyridin-4-ones and 3-hydroxypyran-4-ones linked
with different coumarin substituents is described. These compounds have been developed in
order to provide a series of molecular probes for the quantification of intracellular labile iron
pools. An evaluation of the effect of iron(III) on fluorescence intensity was undertaken. Chelation
of iron(III) causes quenching of fluorescence. The relationship between iron(III) concentration
and the extent of fluorescence quenching indicates that the metal is chelated in a complex
with a metal-to-ligand stoichiometry of 1:3. The fluorescence of hydroxypyridinone compounds
was found to be more efficiently quenched by iron(III) than were the hydroxypyranones. The
metal-to-ligand stoichiometry at which maximum quenching is observed was found to depend
on the site at which coumarin is attached. The efficiency of fluorescence quenching by iron(III)
is markedly influenced by solvent polarity and pH. The permeability of two representative
fluorescent chelators across human erythrocyte ghost membranes was investigated. The rate
of permeability for a series of probes was found to be related to the corresponding ClogP values.
Iron is a ubiquitous metal in cells, it being present in
the structure of many enzymes and proteins and
therefore essential for cellular metabolism.1 However,
the presence of iron can also be detrimental, as it may
facilitate the generation of highly reactive oxygen spe-
cies. In the presence of molecular oxygen, labile iron is
able to redox cycle between the two most stable oxida-
tion states iron(II) and iron(III).2 Hydroxyl radicals are
highly reactive and capable of interacting with most
types of biological molecule including sugars, lipids,
proteins and nucleic acids, resulting in peroxidative
tissue damage.3 Since man lacks a physiological mech-
anism for excreting iron, iron homeostasis is largely
achieved by the regulation of iron absorption.4 Despite
its high physiological and pathophysiological interest,
only a few methods of detecting the chelatable iron pool
in cells have been described.5-14 These methods either
require the destruction of the sample, lack the requisite
sensitivity, or are not easily adaptable for high-
throughput assays. Recently, fluorescent dyes in com-
bination with digital fluorescence microscopy have
become increasingly important for the detection of
intracellular ions. In 1995, Cabantchik and co-workers
introduced a fluorescent method for assessing the labile
iron pool in cultured erythroleukemia K562 cells, based
on the quenching of calcein fluorescence by metal ions.15
Unfortunately, this method is not well suited for analo-
gous measurements in hepatocytes, an important cell
type in mammalian iron metabolism. The in situ
calibration method is difficult to perform in a reliable
and reproduceable manner. To quantitatively monitor
the distribution of the labile iron pool in biological
systems, the design of alternate iron-specific fluorescent
probes is essential. Selective chelators are used to
remove excess iron in a number of clinical situations
and among a wide range of iron chelating compounds,
3-hydroxypyridin-4-ones (HPOs) are currently one of the
main candidates for development of orally active iron
chelators.16 On the basis of selectivity and affinity,
particularly considering the pFe3+ value, HPOs are the
optimal bidentate ligands for the chelation of iron(III)
over the pH range of 6.0-9.0. For this reason, we
considered it appropriate to investigate the possibility
of such molecules being converted into iron-specific
fluorescent probes. In the present study, we have
synthesized a series of coumarin-containing probes
combined with either a hydroxypyridinone or hydroxy-
pyranone moiety using a range of different links. The
physicochemical properties of this series of compounds
have been characterized. The permeability of represen-
tative probes across human erythrocyte membranes is
reported.
Results
Chemistry. The synthetic route employed for the
fluorescent moieties 4a,b and 5a-f is summarized in
Schemes 1 and 2. 4-Substituted coumarins were syn-
thesized by reaction of phenol derivatives with ethyl
acetonedicarboxylate or ethyl 4-chloroacetoacetate by
either use of Amberlyst-15 Cation-Exchange resin
(method A) or zinc chloride (method B). With 3-substi-
tuted coumarins, piperidine and glacial acetic acid were
used as catalyst in the reaction of substituted salicyla-
ldehydes with diethyl malonate. The activated mercap-
tothiazoline coumarin derivatives 5a-f were obtained
by hydrolysis of the corresponding coumarin esters,
followed by coupling with 2-mercaptothiazoline using
dicyclohexylcarbodiimide (DCCI) and 4-(dimethylami-
no)pyridine (DMAP). The quaternary phosphonium
* To whom correspondence should be addressed. Tel: 0207 848 4882,
Fax: 0207 848 4800, E-mail: robert.hider@kcl.ac.uk.
† Department of Pharmacy.
‡ Department of Life Sciences.
10.1021/jm049751s CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/30/2004

6350 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Scheme 1. Synthesis of Compounds 4a,b and 5a-d
Ma et al.
Scheme 2. Synthesis of Compounds 6e-f
Scheme 3. Synthesis of Compounds 10a-h
salts, 4a,b were formed by addition of triphenylphos-
phine to the 4-chloromethylcoumarins 2′a,b.
The general methodology adopted for the synthesis
of 1-amido-3-hydroxypyridin-4-ones 10a-g is summa-
rized in Scheme 3. The 3-hydroxyl group was protected
using the benzyl group, and the resulting protected
maltol was then reacted with the corresponding amines
to produce 8 and 11, respectively, under mild conditions.
The compounds 10a-g were then synthesized by cou-
pling the protected pyridinones 8 and 11 with the
activated mercaptothiazoline coumarin derivative 5a-
f, followed by catalytic hydrogenation (method C) or
BCl3 (method D) to remove the protecting benzyl group.
The 5-amido-hydroxypyridinone 10h can be further

Novel Iron Chelators with Fluorescent Sensors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6351
modified using the methodology illustrated in Scheme
3. Methylation of maltol was achieved using dimethyl
sulfate under basic conditions, 2-methyl-3-methoxy-
4(1H)-pyridinone was obtained by the reaction of the
protected maltol with ammonia. 5-Methylaminometh-
ylpyridinone 15 was then prepared by the reaction of
compound 13 with formaldehyde and benzylmethyl-
amine under Mannich reaction conditions, followed by
hydrogenation to remove the benzyl group.
The synthetic route for the synthesis of 2- or/and
6-aryl-substituted pyranones and pyridinones (10i-w)
is summarized in Scheme 4. 6-Formyl-3-benzyloxypy-
ran-4(1H)-one 18 was obtained by the benzylation of
kojic acid followed by selective oxidation with sulfur
trioxide pyridine complex in combination with dimethyl
sulfoxide. The double bond was formed by Wittig reac-
tion of aldehyde 18 with phosphonium salt 4a,b. The
deprotection of compound 9j by two different methods
(method c and method d) afforded two different products
10j and 10′j.
The conversion of 6-hydroxymethyl-3-benzyloxypyran-
4(1H)-one 17, 2-hydroxymethyl-6-methyl-3-benzyloxy-
pyran-4(1H)-one 27a, or 2-hydroxymethyl-6-methyl-3-
benzyloxypyridin-4(1H)-one 27b to the corresponding
phthalimido intermediates, respectively, 19, 28a, or
28b, was accomplished in good yield using phthal-
imide, triphenylphosphine, and diethyl azodicarboxylate
(DEAD). The reaction is believed to proceed through the
formation of a quaternary phosphonium salt by addition
of triphenylphosphine to DEAD, which reacts in situ
with 17, 27a, or 27b to form an alkoxyphosphonium
salt.17 The phosphonium moiety was then displaced by
the phthalimido anion to furnish the desired phthal-
imido derivatives 19, 28a, or 28b respectively, which
were subsequently reacted with hydrazine to afford the
corresponding amine derivatives 20, 29a, or 29b. The
method of synthesis of the phthalimido intermediate
from 21 with phthalimide according to the above
procedure only produced low yields (less than 20%)
Therefore, a different synthetic method for the amino
compound 23 was developed where the hydroxyl group
of 21 was converted to a chloride with thioyl chloride,
followed by the addition of ammonia. An improved yield
of over 80% was obtained. The direct conversion of the
benzylated 6-hydroxymethylpyranone 17 to the corre-
sponding N-methylpyridinone analogue 21 was success-
ful when water was used as a solvent. However, the
yield of the direct conversion of the benzylated 2-hy-
droxmethylpyranone 27a to the corresponding N-meth-
ylpyridinone analogue 27b was lower than that of the
conversion of 17 to 21. As a result of this finding, the
2-hydroxyl function of 27a was protected using 3,4-
dihydro-2H-pyran before reacting with methylamine.
The pyran protecting group was then selectively re-
moved under mild acidic conditions without deprotection
of the benzyl group, resulting in the desired protected
2-hydroxymethylpyridinone 27b in a much improved
yield.
and 6-position (10n, 10o). Similarly, the coumarin
substituent was linked to the 3-hydroxypyran-4-one
nucleus at the 2-position (10p-r) and 6-position (10i-
m). In addition, one probe (10g) was prepared contain-
ing two fluorescent groups linked to 3-hydroxypyridin-
4-one.
Six types of coumarin (Fla-Flf, see Table 1) were
selected as fluorescence units in this molecular series.
These fluorescent moieties were linked to the pyridinone
and pyranone moieties utilizing amido and amino
functions (10n), carbon-carbon single bonds (10′j) and
carbon-carbon double bonds (10i and 10j).
Fluorescent Properties. Coumarin itself is not
fluorescent; however, the addition of various electron
donating substituents at the 6- and 7-positions result
in a red shift and an enhanced fluorescence intensity.18
The emission wavelength (Em) of the methoxycoumarin
derivatives lies in the 380-423 nm range (Table 2),
dimethoxycoumarin possessing the highest Em, namely
423 nm. When the dimethylamino group is introduced
at the 7-position of coumarin, an increase in the Em
value was observed (473 nm). The effect was similar
with the introduction of the diethylamino group (470
nm). However, when the methoxy group was substituted
by the phenylamino moiety (10c), fluorescence was
abolished. When the iron chelator and fluorescent probe
components are conjugated (for example: 10i and 10j),
the fluorescence is abolished. A similar effect occurs
when the two moieties are connected by an amino link
(10n). The probable reason for these latter observations
is that the double bond or the amino link between the
coumarin and the chelator moiety changes the electron
distribution in the coumarin ring. A similar phenom-
enon occurs with the phenyl group of probe 10c. In
contrast, the presence of an amido link between the two
moieties does not appreciably influence the fluorescence
properties, probably because the amido group is able to
attract electrons from the electron donor group at the
7-position of coumarin.
Not surprisingly, the different chelator moieties did
not appreciably influence the fluorescent properties of
the free ligand as judged by excitation wavelength,
emission wavelength and extinction coefficient (see for
example: 10f, 10h, 10o, and 10v). However, the quan-
tum yield was found to be larger for the 3-hydroxypyran-
4-ones when compared with the 3-hydroxypyridin-4-
ones (10a to 10p, 10b to 10q, 10e to 10r). The probable
reason for the decrease of the quantum yield is that the
3-hydroxypyridin-4-one ring exists as a highly polarized
aromatic structure in which nitrogen is positively
charged and oxygen at the 4-position of 3-hydroxypyri-
din-4-one is negatively charged.19
Surprisingly, the fluorescent intensity of probe 10g
which contains two 7-diethylaminocoumarin groups is
weak in water, indeed virtually undetectable. Bond-
rotation in the molecule induced by the hydrophilic
solvent leads to an overlap of the two fluorescent probes,
which in turn causes self-quenching. In an attempt to
avoid this type of interaction, the molecule was inves-
tigated in methanol, whereupon the fluorescent inten-
sity increased although not reaching twice that of probe
10f.
Molecular Structures of Iron Probes. The struc-
tures of a range of 3-hydroxypyridin-4-ones and 3-hy-
droxypyran-4-ones linked with different coumarin sub-
stituents are presented in Table 1. The coumarin
substituent was linked to 3-hydroxypyridin-4-one at the
1-position (10a-f), 2-position (10s-v), 5-position (10h),
Fluorescence Quenching. The fluorescent emission
intensity of the pyridinone fluorescent probes was found

6352 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Ma et al.

Novel Iron Chelators with Fluorescent Sensors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6353
Table 1. Chemical Structure of Fluorescent Probes
a
compd
X
R1
R2
R5
R6
10a
10b
10c
10d
10e
10f
N
N
N
N
N
N
N
N
O
O
O
O
O
O
N
N
O
O
O
N
N
N
N
FlaCH2CONHCH2CH2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
FlbCH2CONHCH2CH2
FlcCH2CONHCH2CH2
FldCH2CONHCH2CH2
FleCONHCH2CH2
FlfCONHCH2CH2
10g
10h
10i
(FlfCONHCH2CH2)2NCH2CH2
H
FlfCON(CH3)CH2
-
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
FlaCHdCH
FlbCHdCH
FlbCH2CH2
FlaCH2CONHCH2
FlbCH2CONHCH2
FleCONHCH2
FlaCH2NHCH2
FlfCONHCH2
CH3
10j
-
H
10j′
10k
10l
-
H
-
H
-
H
10m
10n
10o
10p
10q
10r
10s
10t
10u
10v
-
H
CH3
CH3
-
H
H
FlaCH2CONHCH2
FlbCH2CONHCH2
FleCONHCH2
-
-
CH3
CH3
CH3
CH3
CH3
CH3
FlaCH2CONHCH2
FldCH2CONHCH2
FleCONHCH2
CH3
CH3
CH3
FlfCONHCH2
CH3
a
to be sensitive to the presence of metal ions, and a clear
quenching effect was observed on the addition of ferric
ions. In contrast, the iron(III)-induced quenching of the
fluorescent emission intensity of the pyranone fluores-
cent probes was found to be comparatively moderate.
The major reason for this difference is that the affinity
constants (â3) of the pyridinone fluorescent probes for
iron(III) are approximately 104-fold greater than those
of the pyranone probes.20 The specific quenching ratio
of these fluorescent probes is shown in Table 2. The
quenching ratios of pyridinone fluorescent probes fall
between 46.4% (10u) and 95.1% (10o) and those of the
pyranone fluorescent probes between 0% (10′j) and
46.4% (10p). A clear example for the difference of
quenching ratios between pyridinones and pyranones
is that the quenching ratio of the probe 10o, where the
fluorescent moiety is linked to the pyridinone site at the
6-position, is 95.1%; whereas, the quenching ratios of
the probes 10k, 10l, and 10m, where the fluorescent
moiety is linked at the same position on the pyranone
nucleus, are 18.5%, 15.9%, and 27.5%, respectively.
The mechanism of fluorescence quenching was as-
sessed for probe 10a. The relationship between iron
concentration and fluorescence quenching was deter-
mined, and the results are plotted in a Stern-Volmer
plot (Figure 1). The relationship between Fo/F vs [Fe]
is nonlinear, indicating that the mechanism of quench-
ing is static rather than dynamic.46 This is consistent
with the action of the probe as an iron chelator, the
result of which causes loss of fluorescence properties.
Prediction of Intracellular Distribution. By vir-
tue of Lipinski’s rule-of-five, four parameters namely
molecular weight, LogP, the number of H-bond donors,
and the number of H-bond acceptors can be used to
predict the likelihood of a molecule crossing biological
membranes by simple diffusion.21 The fluorescent probes
(10a-f, 10h-v) are relatively small in size, their
molecular weights being less than 500; they also possess
less than 5 H-bond donors and 10 H-bond acceptors. As
the ClogP values for this group of molecules are all less
than 5, it would appear likely that the entire group will
readily penetrate membranes and are therefore favor-
able for efficient intracellular distribution. Exceptionally
the molecular weight of probe 10g is rather more than
500, the number of H-bond acceptors is close to 10, and
the ClogP value is close to 5. Thus 10g is not predicted
to efficiently permeate cell membranes.
Permeability across Human Erythrocyte Mem-
branes. The permeability of two representative fluo-
rescent chelators, 10a and 10v, differing in ClogP values

6354 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Table 2. Physiochemical Properties of Probesa
Ma et al.
Ex,
λ (nm)
Em,
λ (nm)
ꢀ,b
quenchingd
c
× 103 M-1 cm-1
ΦF
(Fe3+) (%)
MWe
no. of H donors
no. of H acceptors
ClogP
10a
10b
10c
10d
10e
10f
325
349
380
387
351
431
412
416
314
314
342
324
346
349
322
432
325
345
349
325
387
349
431
380
423
NDf
473
397
468
ND
477
ND
ND
412
382
423
397
ND
470
380
420
397
380
474
397
470
12.2
10.4
- -
0.04
0.04
80.6
59.9
- -
50.6
49.4
93.9
- -
76.5
- -
- -
0
18.5
15.9
27.5
- -
95.1
46.4
32.8
9.8
65.4
77.3
46.4
79.2
420
450
481
433
406
447
813
447
312
342
344
357
387
343
415
433
371
401
357
420
433
406
447
2
2
3
2
2
2
3
2
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
5
6
5
5
5
5
9
5
4
5
5
5
6
5
5
5
5
6
5
5
5
5
5
-0.48
-0.80
1.56
- -
6.8
0.11
0.02
0.01
- -
0.02
- -
- -
0.30
-0.14
-0.02
1.55
25.2
31.7
- -
24.5
- -
- -
7.6
10g
10h
10i
4.43
0.43
1.55
10j
1.23
10j′
10k
10l
1.38
12.3
10.3
22.7
- -
0.14
0.14
0.51
-0.17
-0.49
0.39
10m
10n
10o
10p
10q
10r
10s
10t
10u
10v
- -
-1.22
0.69
0.28
-0.04
0.84
-0.72
-0.39
-0.17
1.14
38
0.01
0.18
0.24
0.12
0.06
0.11
0.01
0.01
12.7
11.4
24.9
11.4
14.3
20.6
42.1
a Conditions of analysis. [Fe3+] 0.5 µM; [probe] 3-15 µM; MOPS buffer pH 7.4. b Molar extinction coefficient. c Quantum yield.
d Quenching of fluorescence intensity (%) (metal-to-ligand molar ratio 1:3). e Molecular weight. f No detectable fluorescence.
Table 3. Permeability Parameters for Fluorescent Probes across Human Erythrocyte Ghost Membranes
10a
10v
A1
t1
A2
t2
A1
t1
A2
t2
- DTPA
+ DTPA
ClogP
37.4 ( 1.1
42.4 ( 1.0
-0.48
18.4 ( 0.9
84.7 ( 2.7
87.9 ( 0.4
-
221.7 ( 1.3
124.2 ( 0.9
137.5 ( 3.6
1.14
38.0 ( 0.5
61.0 ( 2.2
269.1 ( 1.0
212.3 ( 0.6
-
-
-
Figure 1. Stern-Volmer plot of fluorescence quenching of 15
µM 10a by Fe(III) in MOPS buffer (25 mM), pH 7.4.
(-0.45 and 1.14, respectively) across human erythrocyte
ghost membranes was determined (Figure 2). Red cell
ghosts were resealed in the presence of iron(II) am-
monium sulfate and ascorbic acid and washed to remove
untrapped iron. The fluorescence intensity of the probe
was monitored during a 30 min incubation at 20 °C with
washed erythrocyte ghost suspensions in the presence
and absence of the impermeant nonfluorescent iron
chelator, DTPA. Changes in fluorescence intensity as a
function of incubation time are presented in Figure 2.
The fluorescence was quenched to a greater extent in
the absence of DTPA because under these conditions
iron not only quenched the chelator that permeates into
the resealed ghosts, but also iron leaked from the ghosts
and quenched the fluorescence of the probe present in
Figure 2. Fluorescence intensity of A, 15 µM 10a (λex 325;
λem 380); B, 6 µM 10v (λex 431; λem 470) recorded during
incubation at 20 °C in the presence of suspensions of washed
human erythrocyte ghosts resealed in the presence of 100 µM
Fe2+, 1 mM ascorbic acid. The suspension medium also
contained 1 mM ascorbic acid plus 0.1 mM DTPA (a, c) and 1
mM ascorbic acid (b, d).
the suspension medium. The two processes are consis-
tent with a fit of the respective data to a double
exponential decay function as shown in Table 3. With
incubations performed in the presence of excess DTPA

Novel Iron Chelators with Fluorescent Sensors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6355
proximately 1 and 3, respectively. Thus the estimated
D value of compound 10f is 100 times larger than that
of compound 10o. Clearly the iron complex of 10f by
virtue of its enhanced lipophilicity penetrates the ghost
membrane more readily and therefore effluxes from the
cell (Figure 4) in marked contrast to the more hydro-
philic analogue, 10o.
Discussion
It has previously been established that the novel
fluorescent probes described in this paper are more
selective sensors of iron(III) than calcein.23 Further-
more, calcein suffers from a number of limitations,
namely it is not membrane permeable (and therefore,
even with the use of ester proprobe analogues, cannot
be used to monitor iron levels in intracellular organelle
matrixes), it possesses several different chelation modes
with iron(III), 24 and it is a substrate for multiple drug
resistance (MDR) receptor proteins. The probes de-
scribed in this work are based either on 3-hydroxypyr-
ridin-4-ones or 3-hydroxypyran-4-ones, and so they
possess similar iron(III) binding properties to those of
compounds 10a (CP600) and 10p (CP610).23 The high
affinity of these well characterized chelators of iron(III)
is known to facilitate the autoxidation of iron(II) under
aerobic conditions.20 Thus, the novel probes are pre-
dicted to be suitable to monitor combined levels of
iron(II) and iron(III).
Figure 3. Relationship between permeability across the
human erythrocyte ghost membrane and ClogP value of 10a
(λex 325; λem 380); 10d (λex 387; λem 473), 10f (λex 431; λem 468),
10h (λex 416; λem 477), 10o (λex 432; λem 470), 10v (λex 431; λem
470) in the presence of 100 µM Fe2+, 1 mM ascorbic acid and
0.1 mM DTPA. All probes were investigated at 15 µM.
The probes bind iron optimally in a 3:1 molar ratio;
thus with 10a the free ligand has a molecular weight
of 421, whereas the resulting iron complex has the much
higher molecular weight of 1,316. Thus although the
probes are able to enter cells by simple diffusion, once
coordinated to iron they efflux much more slowly. Thus
in principle it should be possible to monitor intracellular
iron pools, if the chelation by iron leads to a change in
fluorescent properties.
Figure 4. Fluorescence intensity of 10o (15 µM, λex 432; λem
470) and 10f (15 µM, λex 431; λem 468) recorded during
incubation at 20 °C in the presence of suspensions of washed
human erythrocyte ghosts resealed in the presence of 100 µM
Fe2+, 1 mM ascorbic acid. The suspension medium also
contained 1 mM ascorbic acid and 0.1 mM DTPA.
In this investigation we have demonstrated that the
bidentate hydroxypyridinone-based probes are associ-
ated with a greater iron-induced fluorescence quenching
effect than that of their hydroxypyranone analogues.
Moreover, the hydroxypyridinone probes are generally
more water soluble than their hydroxypyranone coun-
terparts. The probable reason for this latter observation
is that the charged mesomeric contribution associated
with the hydroxypyridinone molecules makes a greater
contribution than with the equivalent hydroxypyranone
mesomer. Potentially useful HPO probes (10a, 10d, 10h,
10o, 10v, and 10f) were selected on the basis of iron(III)-
induced fluorescence quenching (Table 2). They possess
a range of ClogP values from -0.48 to +1.55. To check
for their suitability for cell studies, we monitored the
ability of these selected probes to penetrate erythrocyte
ghost membranes. Probes with ClogP values >0 were
found to penetrate the membrane efficiently (Figure 3),
and we believe that these probes have potential for
imaging intracellular iron pools. Compound 10o would
appear to be optimal for monitoring intracellular iron
pools, as it has the largest fluorescence quenching and
weighting values of all the compounds presented in
Table 2 and when dissolved in water is extremely
sensitive to stoichiometric binding with iron(III). The
formation of the relatively hydrophilic 3:1 iron complex
associated with 10o within the erythrocyte leads to a
in the suspending medium, fluorescence quenching due
to the leakage of iron from the ghost membranes is
avoided. As expected, these data fit single-exponential
decay functions, representing permeation of the fluo-
rescent chelator into the resealed ghosts. A slight
increase in fluorescence intensity observed during the
incubation is due to the efflux of the fluorescent chelator
from the resealed ghosts into the suspension medium.
The comparison of the relaxation times and related
weighting functions of six fluorescent chelators demon-
strated a direct relationship between permeability
across the erythrocyte ghost membrane and ClogP
values (Figure 3).
The behavior of two representative probes (10o and
10f) with the ClogP values 0.69 and 1.55, respectively,
in the presence of human erythrocyte ghost membranes
was recorded over a 30 min period (Figure 4). The
fluorescence associated with 10o was found to be stable
in the presence of human erythrocyte ghosts. In contrast
with 10f the fluorescence failed to stabilize. This dif-
ference in behavior is readily explained by the estimated
LogD values of the corresponding 3:1 iron(III) complexes
22
of the two molecules which are calculated
as ap-

6356 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Ma et al.
1H NMR (CDCl3) δ: 1.22 (t, J ) 7.2 Hz, 3H), 3.02 (s, 6H), 3.61
(s, 2H), 4.12 (q, J ) 7.2 Hz, 2H), 5.94 (s, 1H), 6.35-6.60 (m,
2H), 7.28 (d, J ) 8.4 Hz, 1H).
stable fluorescence signal. Clearly the intracellular
levels of the iron complex remain constant for at least
30 min (Figure 4).
Ethyl 7-Methoxy-2-oxo-2H-chromene-3-carboxylate (2e).
A solution of 4-methoxysalicylaldehyde (7.6 g, 0.05 mol) and
diethyl malonate (9.6 g, 0.06 mol) in EtOH (60 mL) was treated
with piperidine (0.5 mL) and glacial acetic acid (2 drops) and
refluxed for 3 h. To the reaction mixture was added 100 mL
of H2O, and the mixture was cooled to 0 °C. The crystalline
solid was filtered and washed by 50% cold ethanol (20 mL).
Recrystallization from 50% EtOH gave colorless crystals (8.8
g, 71%); mp 135-137 °C (lit. value30 117 °C); 1H NMR (CDCl3)
δ: 1.39 (t, J ) 7.2 Hz, 3H), 3.84 (s, 3H), 4.32 (q, J ) 7.2 Hz,
2H), 6.70 (d, J ) 2.3 Hz, 1H), 6.84 (dd, J ) 2.3 8.4 Hz, 1H),
7.42(d, J ) 8.4 Hz, 1H), 8.37 (s, 1H).
Compound 10o possesses a fluorescence emission
maximum wavelength of 470 nm (Table 2). This excel-
lent signaling property of fluorescence is predicted to
facilitate the potential of probe 10o for application in
determining intracellular labile iron levels by confocal
microscopy techniques. Studies are currently in progress
with a range of 3-hydroxypyridin-4-one-based probes in
both hepatocytes and leucocytes.
Experimental Section
An analogous procedure starting with 1f gave ethyl 7-(di-
ethylamino)-2-oxo-2H-chromene-3-carboxylate (2f) as a
yellow crystalline solid : (7.1 g, 49%); mp 75-76 °C (lit. value31
77-78 °C); 1H NMR (CDCl3) δ: 1.22 (t, J ) 7.2 Hz, 6H), 1.37
(t, J ) 7.2 Hz, 3H), 3.42 (q, J ) 7.2 Hz, 4H), 4.34 (q, J ) 7.2
Hz, 2H), 6.40 (d, J ) 2.2 Hz, 1H), 6.56 (dd, J ) 2.2 9.0 Hz,
1H), 7.30(d, J ) 9.0 Hz, 1H), 8.45 (s, 1H).
General Procedure. Methyl maltol (6) was purchased from
Cultor Food Science. Kojic acid (16) was purchased from Fluka.
Sepharose 2B was obtained from Pharmacia Ltd. All other
chemicals were obtained from Sigma-Aldrich. Melting points
were determined using an Electrothermal IA 9100 Digital
Melting Point Apparatus and are uncorrected. 1H NMR spectra
were recorded using a Bruker (360 MHz) NMR spectrometer.
Chemical shifts (δ) are reported in ppm downfield from the
internal standard tetramethylsilane (TMS). Mass spectra
(FAB) analyses were carried out by the Mass Spectrometry
Facility, School of Health and Life Science, 150 Stamford St.,
London SE1 9NN, UK.
(7-Methoxy-2-oxo-2H-chromen-4-yl)acetic Acid (3a). To
a solution of ethyl (7-methoxy-2-oxo-2H-chromen-4-yl)acetate
(2a) (2.62 g, 10 mmol) in EtOH (50 mL) was added 250 mL of
0.5% NaOH, and the mixture was heated under reflux for 2
h. Acidification to pH 2 using concentrated hydrochloric acid
and cooling to 0 °C gave a crystalline deposit (1.87 g, 80%):
Ethyl
(7-methoxy-2-oxo-2H-chromen-4-yl)acetate
1
mp 166-167 °C (lit. value32 167-170 °C); H NMR (DMSO-
(2a): Method A. To a solution of 3-methoxyphenol (1a) (6.2
g, 0.05 mol) and ethyl acetonedicarboxylate (11.1 g, 0.055 mol)
in toluene (200 mL) was added Amberlyst-15 cation-exchange
resin (3 g). The reaction mixture was heated under reflux with
stirring using a Dean-Stark apparatus until no more water
was produced (about 10 h). The hot reaction mixture was
filtered to remove the catalyst, and upon cooling a yellow
precipitate was obtained (if no precipitate occurred, evapora-
tion of the solvent is necessary). Recrystallization from ethanol
produced the coumarin as pale yellow powder solid (8.1 g,
62%): mp 100-102 °C (lit. value25 101-102 °C);1H NMR
(DMSO-d6) δ: 1.30 (t, J ) 7.2 Hz, 3H), 3.70 (s, 2H), 3.75 (s,
3H), 4.20 (q, J ) 7.2 Hz, 2H), 6.10 (s, 1H), 6.70-6.90 (m, 2H),
7.42 (d, J ) 9.6 Hz, 1H).
d6) δ: 3.70 (s, 2H), 3.75 (s, 3H), 6.10 (s, 1H), 6.65-6.88 (m,
2H), 7.42 (d, J ) 9.6 Hz, 1H).
Analogous procedures starting with 2b-d gave compounds
3b-d.
(6,7-Dimethoxy-2-oxo-2H-chromen-4-yl)acetic
acid
1
(3b): (2.0 g, 78%); mp 174.5-175 °C; H NMR (DMSO-d6) δ:
3.76 (s, 3H), 3.81 (s, 2H), 3.85 (s, 3H), 6.04 (s, 1H), 6.80 (s,
1H), 6.88 (s, 1H).
(7-Anilino-2-oxo-2H-chromen-4-yl)acetic acid (3c): (2.21
g, 75%); mp 183.5-184.5 °C (lit. value28 186-189 °C); 1H NMR
(DMSO-d6) δ: 3.64 (s, 2H), 4.20 (br s, 1H), 5.93 (s, 1H), 6.65-
7.40 (m, 8H).
(7-Dimethylamino-2-oxo-2H-chromen-4-yl)acetic acid
(3d): (2.00 g, 81%); mp 163.5-164.5 °C (lit. value29 166-167
°C); 1H NMR (DMSO-d6) δ: 3.00 (s, 6H), 3.71 (s, 2H), 5.94 (s,
1H), 6.40-6.75 (m, 2H), 7.40 (d, J ) 10.2 Hz, 1H).
Analogous reactions of 1a or 1b with ethyl acetonedicar-
boxylate or ethyl 4-chloroacetoacetate gave compounds 2b, 2′a,
and 2′b.
Ethyl (6,7-dimethoxy-2-oxo-2H-chromen-4-yl)acetate
(2b): (9.9 g, 68%); mp 140-141 °C; 1H NMR (DMSO-d6) δ:
1.35 (t, J ) 7.2 Hz, 3H), 3.75 (s, 3H), 3.81 (s, 2H), 3.87 (s, 3H),
4.25 (q, J ) 7.2 Hz, 2H), 6.05 (s, 1H), 6.80 (s, 1H), 6.88 (s,
1H).
4-(Chloromethyl)-7-methoxy-2H-chromen-2-one (2′a):
(6.7 g, 60%); mp 194-195 °C (lit. value26 195 °C); 1H NMR
(DMSO-d6) δ: 3.84 (s, 3H), 4.55 (s, 2H), 6.30 (s, 1H), 6.72 (d,
J ) 2.0 Hz, 1H), 6.80 (dd, J ) 2.2, 8.4 Hz, 1H), 7.47 (d, J )
8.4 Hz, 1H).
4-(Chloromethyl)-6,7-dimethoxy-2H-chromen-2-one
(2′b): (7.8 g, 61%); mp 204.5-205.5 °C (lit. value27 194-196
°C); 1H NMR (DMSO-d6) δ: 3.80 (s, 3H), 3.85 (s, 3H), 5.01 (s,
2H), 6.45 (s, 1H), 7.08 (s, 1H), 7.22 (s, 1H).
Ethyl (7-Anilino-2-oxo-2H-chromen-4-yl)acetate (2c):
Method B. To a solution of 3-anilinophenol (9.3 g, 0.05 mol)
and ethyl acetonedicarboxylate (11.1 g, 0.055 mol) in anhy-
drous EtOH (30 mL) was added anhydrous ZnCl2 (8.2 g, 0.06
mol), and the reaction mixture was refluxed overnight. After
cooling, the reaction mixture was poured into ice-water under
stirring. The resulting separated dark oil slowly solidified on
being left in contact with cold EtOH. Recrystallization from
EtOH gave needles (6.8 g, 42%): mp 148-149.5 °C (lit. value28
150-151 °C); 1H NMR (CDCl3) δ: 1.30 (t, J ) 7.2 Hz, 3H),
3.69 (s, 2H), 4.20 (q, J ) 7.2 Hz, 2H), 6.10 (s, 1H), 6.80-7.50
(m, 8H).
7-Methoxy-2-oxo-2H-chromen-3-carboxylic Acid (3e).
To a solution of ethyl 7-methoxy-2-oxo-2H-chromene-3-car-
boxylate (2e) (2.48 g, 10 mmol) in EtOH (15 mL) was added
15 mL of 10% NaOH and heated under reflux for 15 min.
Acidification to pH 2 using concentrated hydrochloric acid and
cooling to 0 °C gave a crystalline deposit (1.87 g, 85%): mp
191.5-192.5 °C (lit. value33 176-177 °C); 1H NMR (DMSO-
d6) δ: 3.80 (s, 3H), 6.72 (d, J ) 2.3 Hz, 1H), 6.84 (dd, J ) 2.3,
8.4 Hz, 1H), 7.40 (d, J ) 8.4 Hz, 1H), 8.38 (s, 1H).
An analogous procedure starting with 2f gave 7-diethyl-
amino-2-oxo-2H-chromen-3-carboxylic acid (3f): (2.14 g,
82%); mp 224-225.5 °C (lit. value34 215-217 °C); 1H NMR
(DMSO-d6) δ: 1.22 (t, J ) 7.2 Hz, 6H), 3.42 (q, J ) 7.2 Hz,
4H), 6.40 (d, J ) 2.2 Hz, 1H), 6.55 (dd, J ) 2.2, 9.0 Hz, 1H),
7.30 (d, J ) 9.0 Hz, 1H), 8.49 (s, 1H).
2-Methyl-3-benzyloxypyran-4(1H)-one (7). To a solution
of methyl maltol 6 (63 g, 0.5 mol) in methanol (500 mL) was
added sodium hydroxide (22 g, 0.55 mol) dissolved in water
(50 mL), and the mixture was heated to reflux. Benzyl chloride
(70 g, 0.55 mol) was added dropwise over 30 min, and the
resulting mixture was refluxed overnight. After removal of
solvent by rotary evaporation, the residue was mixed with
water (200 mL) and extracted with dichloromethane (3 × 150
mL). The combined extracts were washed with 5% aqueous
sodium hydroxide (2 × 200 mL) followed by water (200 mL).
The organic fraction was then dried over anhydrous sodium
sulfate, filtered, and rotary evaporated to yield an orange oil
which solidified on cooling. Recrystallization from diethyl ether
gave the pure product 7 as colorless needles (87.5 g, 81%); mp
An analogous procedure starting with 1d gave ethyl
[7-(dimethylamino)-2-oxo-2H-chromen-4-yl]acetate
(2d): (4.1 g, 30%); mp 129-131 °C (lit. value29 131-132 °C);

Novel Iron Chelators with Fluorescent Sensors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6357
51-52 °C (lit. value35 53-55 °C); 1H NMR (CDCl3) δ: 2.12 (s,
3H), 5.11 (s, 2H), 6.25 (d, J ) 6 Hz, 1H), 7.28 (s, 5H), 7.47 (d,
J ) 6 Hz, 1H).
3.31 (q, J ) 6.6 Hz, 4H), 3.70 (s, 3H), 4.48 (s, 2H), 6.25-6.55
(m, 2H), 7.14 (d, J ) 7.2 Hz, 1H), 7.63 (s, 2H). m/z: 426 (M +
1)+.
N-[(5-Benzoxy-4-oxo-4H-pyran-2-yl)methyl]-2-(7-meth-
oxy-2-oxo-2H-chromen-4-yl)acetamide (9k): (2.23 g, 50%);
mp 206-207 °C; 1H NMR (CDCl3) δ: 3.77 (s, 2H), 3.80 (s, 3H),
4.09 (d, J ) 5.8 Hz, 2H), 4.82 (s, 2H), 6.12 (s, 1H), 6.15 (s,
1H), 6.70-6.90 (m, 2H), 7.25 (s, 5H), 7.55 (d, J ) 8.8 Hz, 1H),
8,00 (s, 1H), 8.68 (br s, 1H). m/z: 448 (M + 1)+.
N-[(5-Benzoxy-4-oxo-4H-pyran-2-yl)methyl]-2-(6,7-
dimethoxy-2-oxo-2H-chromen-4-yl)acetamide (9l): (2.62
g, 55%); mp 230-231 °C; 1H NMR (CDCl3) δ: 3.78 (s, 3H),
3.83 (s, 2H), 3.87 (s, 3H), 4.19 (d, J ) 5.8 Hz, 2H), 4.93 (s,
2H), 6.25 (s, 1H), 6.29 (s, 1H), 7.09 (s, 1H), 7.17 (s, 1H), 7.34-
7.42 (m, 5H), 8.13 (s, 1H), 8.82 (d,J ) 5.8 Hz, 2H). m/z: 478
(M + 1)+.
7-Methoxy-N-[(5-benzoxy-4-oxo-4H-pyran-2-yl)methyl]-
2-oxo-2H-chromene-3-carboxyamide (9m): (2.08 g, 48%);
mp 186-188 °C; 1H NMR (CDCl3) δ: 3.80 (s, 3H), 4.32 (d, J )
6.0 Hz, 2H), 4.90 (s, 2H), 6.22 (s, 1H), 6.60-6.85 (m, 2H), 7.20
(s, 5H), 7.34 (s, 1H), 7.55 (d, J ) 8.0 Hz, 1H), 8.64 (s, 1H),
9.00 (br s, 1H). m/z: 434 (M + 1)+.
7-Ddiethylamino)-N-[(5-benzoxy-1-methyl-4-oxo-1,4-di-
hydropyridin-2-yl)methyl]-2-oxo-2H-chromene-3-carbox-
amide (9o): (52%); mp 209-210 °C; 1H NMR (CDCl3) δ: 1.25
(t, J ) 7.1 Hz, 6H), 3.47 (q, J ) 7.1 Hz, 4H), 3.57 (s, 3H), 4.51
(d, J ) 6.0 Hz, 2H), 5.17 (s, 2H), 6.51 (d, J ) 2.4 Hz, 1H), 6.52
(s, 1H), 6.66 (dd, J ) 2.4, 9.0 Hz, 1H), 6.93 (s, 1H), 7.27-7.45
(m, 6H), 8.67 (s, 1H), 9.19 (t, J ) 6.0 Hz, 1H). m/z: 488 (M +
1)+.
N-[(3-Benzoxy-6-methyl-4-oxo-4H-pyran-2-yl)methyl]-
2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide (9p): (2.07
g, 45%); mp 183-184 °C; 1H NMR (CDCl3) δ: 2.16 (s, 3H),
3.73 (s, 2H), 3.85 (s, 3H), 4.20 (d, J ) 5.8 Hz, 2H), 4.99 (s,
2H), 6.19 (s, 1H), 6.23 (s, 1H), 6.23 (s, 1H), 6.80-6.94 (m, 2H),
7.30-7.70 (m, 6H), 8.59 (t, J ) 5.6 Hz, 1H). m/z: 462 (M +
1)+.
N-[(3-Benzoxy-6-methyl-4-oxo-4H-pyran-2-yl)methyl]-
2-(6,7-dimethoxy-2-oxo-2H-chromen-4-yl)acetamide
(9q): (2.41 g, 49%); mp 224-225 °C; 1H NMR (CDCl3) δ: 2.06
(s, 3H), 3.68 (s, 3H), 3.69 (s, 2H), 3.78 (s, 3H), 4.08-4.20 (m,
2H), 4.90 (s, 2H), 6.08 (s, 1H), 6.15 (s, 1H), 6.90-7.30 (m, 7H),
8.65 (brs, 1H). m/z: 492 (M + 1)+.
1-(2-Aminoethyl)-3-(benzyloxy)-2-methylpyridin-4(1H)-
one (8). To a solution of 7 (2.16 g, 10 mmol) in water (100
mL) was added ethylenediamine (1.2 g, 20 mmol). The mixture
was heated at 70 °C overnight and then extracted with
dichloromethane (4 × 100 mL). The combined organic layers
were dried over anhydrous sodium sulfate, filtered, and rotary
evaporated to give a brown oil (2.27 g, 88%). 1H NMR (CDCl3)
δ: 2.20 (s, 3H), 2.95 (t, J ) 6 Hz, 2H), 3.85 (t, J ) 6 Hz, 2H),
5.16 (s, 2H), 6.32 (d, J ) 7.8 Hz, 1H), 7.31 (d, J ) 7.8 Hz, 1H),
7.34 (s, 5H).
N-[2-(3-Benzoxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide (9a). To
a vigorously stirred suspension of 3a (2.34 g, 10 mmol) in
dichloromethane (100 mL) were added dicyclohexylcarbodi-
imide (DCCI) (2.3 g, 11 mmol), 2-mercaptothiazoline (1.32 g,
11 mmol), and a catalytic amount of 4-(dimethylamino)pyridine
(DMAP) (50 mg). The mixture was stirred for 24 h, the white
precipitate N,N′-dicyclohexylurea (DCU) filtered from the
yellow solution, the filtrate added to the amine 8 (2.58 g, 10
mmol), and the reaction mixture allowed to stir overnight. The
dichloromethane layer was washed with 0.1 N sodium hydrox-
ide solution (3 × 50 mL) and water (50 mL) and dried over
anhydrous sodium sulfate, and the solvent was removed in
vacuo. The crude product was further purified by column
chromatography on silica gel (eluant: methanol:chloroform,
1:9 v/v) to afford a white solid (2.32 g, 49%): mp 202-203 °C;
1H NMR (CDCl3) δ: 2.05 (s, 3H), 3.54 (s, 2H), 3.72-3.84 (m,
7H), 4.81 (s, 2H), 5.90 (d, J ) 7.8 Hz, 1H), 6.06 (s, 1H), 6.70-
6.85 (m, 2H), 7.18 (s, 5H), 7.38 (d, J ) 9.0 Hz, 1H), 8.10 (d, J
) 7.8 Hz, 1H). m/z: 475 (M + 1)+.
Analogous procedures starting with coumarin acids with
amino derivatives gave 9b-f, 9h, 9k-m, 9o-v.
N-[2-(3-Benzoxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
2-(6,7-dimethoxy-2-oxo-2H-chromen-4-yl)acetamide
(9b): (2.67 g, 53%); mp 164-166 °C; 1H NMR (CDCl3) δ: 2.15
(s, 3H), 3.65 (s, 2H), 3.70-3.90 (m, 10H), 4.90 (s, 2H), 5.95 (d,
J ) 7.8 Hz, 1H), 6.08 (s, 1H), 7.05 (s, 1H), 7.12 (s, 1H), 7.20 (s,
5H), 8.01 (d, J ) 7.8 Hz, 1H). m/z: 505 (M + 1)+.
N-[2-(3-Benzoxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
2-(7-anilino-2-oxo-2H-chromen-4-yl)acetamide (9c): (2.94
g, 55%); mp 146-148 °C; 1H NMR (CDCl3) δ: 2.15 (s, 3H),
2.90-3.25 (m, 4H), 4.55 (s, 2H), 5.24 (s, 2H), 6.16-8.58 (m,
16H). m/z: 536 (M + 1)+.
N-[2-(3-Benzoxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)acet-
amide (9d): (2.63 g, 54%); mp 210-211 °C; 1H NMR (CDCl3)
δ: 2.12 (s, 3H), 3.00 (s, 6H), 3.35-3.80 (m, 6H), 4.98 (s, 2H),
5.99 (s, 1H), 6.30 (d, J ) 7.8 Hz, 1H), 6.40-6.70 (m, 2H), 7.20
(s, 5H),7.30 (d, J ) 9.0 Hz, 1H), 8.05 (d, J ) 7.2 Hz, 1H). m/z:
488 (M + 1)+.
N-[2-(3-Benzoxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
7-methoxy-2-oxo-2H-chromen-3-carboxamide (9e): (2.61
g, 58%); mp 182-183 °C; 1H NMR (CDCl3) δ: 2.18 (s, 3H),
3.61 (q, J ) 6.5 Hz, 2H), 3.93 (s, 3H), 4.01 (t, J ) 6.8 Hz, 2H),
5.21 (s, 2H), 6.41 (d, J ) 7.5 Hz, 1H), 6.88 (d, J ) 2.3 Hz, 1H),
6.97 (dd, J ) 2.3, 8.7 Hz, 1H), 7.23 (d, J ) 7.5 Hz, 1H), 7.26-
7.43 (m, 5H), 7.60 (d, J ) 8.7 Hz, 1H), 8.80 (s, 1H), 8.95 (t, J
) 5.9 Hz, 1H). m/z: 461 (M + 1)+.
N-[2-(3-Benzoxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
7-diethylamino-2-oxo-2H-chromene-3-carboxamide
(9f): (2.45 g, 49%); mp 208-209 °C; 1H NMR (CDCl3) δ: 1.25
(t, J ) 7.1 Hz, 6H), 2.18 (s, 3H), 3.46 (q, J ) 7.1 Hz, 4H), 3.58
(q, J ) 6.6 Hz, 2H), 3.99 (t, J ) 6.8 Hz, 2H), 5.22 (s, 2H), 6.40
(d, J ) 7.5 Hz, 1H), 6.50 (d, J ) 2.3 Hz, 1H), 6.67 (dd, J ) 2.3,
9.0 Hz, 1H), 7.22 (d, J ) 7.5 Hz, 1H), 7.27-7.45 (m, 6H), 8.65
(s, 1H), 8.99 (t, J ) 5.9 Hz, 1H). m/z: 502 (M + 1)+.
7-Diethylamino-N-[(5-methoxy-6-methyl-4-oxo-1,4-di-
hydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromene-
3-carboxamide (9h): (2.72 g, 64%); mp 201-202 °C; 1H NMR
(CDCl3) δ: 1.12 (t, J ) 6.6 Hz, 6H), 2.20 (s, 3H), 3.00 (s, 3H),
N-[(3-Benzoxy-6-methyl-4-oxo-4H-pyran-2-yl)methyl]-
7-methoxy-2-oxo-2H-chromene-3-caroxamide (9r): (2.50
g, 56%); mp 185-186 °C; 1H NMR (CDCl3) δ: 2.20 (s, 3H),
3.84 (s, 3H), 4.40 (d, J ) 5.8 Hz, 2H), 5.14 (s, 2H), 6.09 (s,
1H), 6.65-7.65 (m, 9H), 8.70 (s, 1H). m/z: 448 (M + 1)+.
N-[(3-Benzoxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-
2-yl)methyl]-2-(7-methoxy-2-oxo-2H-chromen-4-yl)aceta-
1
mide (9s): (2.51 g, 53%); mp 244-245 °C; H NMR (CDCl3)
δ: 2.20 (s, 3H), 3.30 (s, 3H), 3.63 (s, 2H), 3.75 (s, 3H), 4.28 (d
J ) 4.8 Hz, 2H), 4.96 (s, 2H), 6.05 (s, 1H), 6.10 (s, 1H), 6.70-
6.90 (m, 2H), 7.18-7.40 (m, 6H), 8.35 (t, J ) 4.8 Hz, 1H). m/z:
475 (M + 1)+.
N-[(3-Benzoxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-
2-yl)methyl]-2-(7-(dimethylamino)-2-oxo-2H-chromen-4-
1
yl)acetamide (9t): (2.34 g, 48%); mp 278-279 °C; H NMR
(CDCl3) δ: 1.24 (t, J ) 7.1 Hz, 6H), 2.29 (s, 3H), 3.46(q, J )
7.1 Hz, 4H), 3.57 (s, 3H), 4.63 (d J ) 5.7 Hz, 2H), 5.36 (s, 2H),
6.38 (s, 2H), 6.43 (d, J ) 2.3 Hz, 1H), 6.49 (dd, J ) 2.3, 8.6
Hz, 1H), 7.24-7.50 (m, 6H), 8.64 (s, 1H), 8.78 (t, J ) 5.7 Hz,
1H). m/z: 488 (M + 1)+.
N-[(3-Benzoxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-
2-yl)methyl]-7-methoxy-2-oxo-2H-chromene-3-carboxam-
1
ide (9u): (2.34 g, 52%); mp 186-188 °C; H NMR (CDCl3) δ:
2.31 (s, 3H), 3.57 (s, 3H), 3.89 (s, 3H), 4.57 (d J ) 5.8 Hz, 2H),
5.31 (s, 2H), 6.29 (s, 1H), 6.70-6.92 (m, 2H), 7.10-7.50 (m,
6H), 8.68 (s, 1H). m/z: 461 (M + 1)+.
N-[(3-Benzoxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-
2-yl)methyl]-7-diethylamino-2-oxo-2H-chromene-3-car-
boxamide (9v): (2.90 g, 58%); mp 243-244 °C; 1H NMR
(CDCl3) δ: 1.13 (t, J ) 7.0 Hz, 6H), 2.19 (s, 3H), 3.39 (q, J )
7.0 Hz, 4H), 3.44 (s, 3H), 4.48 (d J ) 5.8 Hz, 2H), 5.20 (s, 2H),

6358 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Ma et al.
6.20 (s, 1H), 6.32-6.60 (m, 2H), 7.02-7.38 (m, 6H), 8.45 (s,
1H). m/z: 502 (M + 1)+.
Analogous hydrogenation procedures were used to the
preparation for 10b, 10d-10f, 10′j, 10k-10m, 10p-10v.
N-[2-(3-Hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
2-(6,7-dimethoxy-2-oxo-2H-chromen-4-yl)acetamide hy-
drochloride (10b): (88%); mp 261-262 °C; 1H NMR (DMSO-
d6) δ: 2.50 (s, 3H), 3.51 (q, J ) 5.8 Hz, 2H), 3.71 (s, 2H), 3.80
(s, 3H), 3.87 (s, 3H), 4.38 (t, J ) 5.8 Hz, 2H), 6.23 (s, 1H), 7.06
(s, 1H), 7.12 (d, J ) 7.0 Hz, 1H), 7.19 (s, 1H), 8.06 (d, J ) 7.0
Hz, 1H), 8.74 (t, J ) 5.8 Hz, 1H). m/z: 415 (M - Cl)+.
N-[2-(3-Hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
2-(7-(dimethylamino)-2-oxo-2H-chromen-4-yl)aceta-
mide hydrochloride (10d): (82%); mp 258-260 °C; 1H NMR
(DMSO-d6) δ: 2.52 (s, 3H), 3.03 (s, 6H), 3.49-3.53 (m, 2H),
3.61 (s, 2H), 4.40 (t, J ) 5.6 Hz, 2H), 5.98 (s, 1H), 6.55 (d, J )
2.5 Hz, 1H), 6.72 (dd, J ) 2.5, 9.0 Hz, 1H), 7.28 (d, J ) 6.9 Hz,
1H), 7.40 (d, J ) 9.0 Hz, 1H), 8.09 (d, J ) 6.9 Hz, 1H), 8.58 (t,
J ) 5.8 Hz, 1H). m/z: 398 (M - Cl)+.
N-[2-(3-Hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
7-methoxy-2-oxo-2H-chromene-3-carboxamide hydro-
chloride (10e): (85%); mp 263-264 °C; 1H NMR (DMSO-d6)
δ: 2.58 (s, 3H), 3.75 (m, 2H), 3.90 (s, 3H), 4.51 (t, J ) 5.7 Hz,
2H), 7.05 (dd, J ) 2.4, 8.7 Hz, 1H), 7.11 (d, J ) 2.4 Hz, 1H),
7.22 (d, J ) 6.9 Hz, 1H), 7.91 (d, J ) 8.7 Hz, 1H), 8.04 (d, J )
6.9 Hz, 1H), 8.78 (s, 1H), 8.87 (t, J ) 6.1 Hz, 1H). m/z: 370 (M
- Cl)+.
N-[2-(3-Hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
7-diethylamino-2-oxo-2H-chromene-3-carboxamide hy-
drochloride (10f): (75%); mp 249-251 °C; 1H NMR (DMSO-
d6) δ: 1.13 (t, J ) 7.0 Hz, 6H), 2.58 (s, 3H), 3.47 (q, J ) 7.0
Hz), 3.73 (q, J ) 5.7 Hz, 2H), 4.50 (t, J ) 5.7 Hz, 2H), 6.59 (d,
J ) 2.3 Hz, 1H), 6.79 (dd, J ) 2.3, 9.0 Hz, 1H), 7.31 (d, J )
7.0 Hz, 1H), 7.66 (d, J ) 9.0 Hz, 1H), 8.07 (d, J ) 7.0 Hz, 1H),
8.58 (s, 1H), 8.83 (t, J ) 5.7 Hz, 1H), 8.93 (brs, 1H). m/z: 412
(M - Cl)+.
4-[2-(5-Hydroxy-4-oxo-4H-pyran-2-yl)ethyl]-6,7-
dimethoxychroman-2-one (10′j): (32%); mp 285-287 °C; 1H
NMR (DMSO-d6) δ:2.70-3.00 (m, 4H), 3.87 (s, 3H), 3.89 (s,
3H), 6.05 (s, 1H), 6.58-6.94 (m, 3H), 7.19 (s, 1H). m/z: 345
(M + 1)+.
N-(2-{[2-(Acetylamino)ethyl][2-(3-benzoxy-2-methyl-4-
oxopyridin-1(4H)-yl)ethyl]amino}ethyl)- 7-diethylamino-
2-oxo-2H-chromene-3-carboxamide (9g). To a vigorously
stirred suspension of 3f (5.24 g, 20 mmol) in dichloromethane
(150 mL) were added dicyclohexylcarbodiimide (DCCI) (4.6 g,
22 mmol), 2-mercaptothiazoline (2.64 g, 22 mmol), and a
catalytic amount of 4-(dimethylamino)pyridine (DMAP) (100
mg). The mixture was stirred for 24 h, the white precipitate
N,N′-dicyclohexylurea (DCU) filtered from the yellow solution,
the filtrate added to the amine 11 (1.72 g, 5 mmol), and the
reaction mixture stirred overnight. The compound 9g was
purified following the representative procedure described for
9a to obtain a yellow solid in 55% yield (2.29 g); mp 189-191
°C; 1H NMR (CDCl3) δ: 1.13 (t, J ) 7.0 Hz, 12 H), 2.58 (s,
3H), 3.46 (q, J ) 7.0 Hz, 8 H), 3.71 (br s, 12H), 5.05 (s, 2H),
6.53 (s, 2H), 6.74 (dd, J ) 2.2, 9.1 Hz, 2H), 7.35-7.58 (m, 8H),
8.52-8.58 (m, 3H), 8.88 (br s, 2H). m/z: 831 (M + 1)+.
4-[2-(5-Benzyloxy-4-oxo-4H-pyran-2-yl)vinyl]-7-meth-
oxychroman-2-one (9i). To a solution of 4-(chloromethyl)-7-
methoxy-2H-chromen-2-one 2′a (4.49 g, 20 mmol) in CHCl3 (30
mL) was added triphenyl phosphine (5.29 g, 21 mmol), and
the mixture was refluxed for 2.5 h. Rotary evaporation to
remove the solvent, and the resulting precipitate was isolated
by filtration, washed with toluene (10 mL), and dried under
high vacuum to yield colorless amorphous powder. The mixture
of the former powder (4.86 g, 10 mmol) and 2-formyl-5-
benzyloxy-4H-pyran-4-one 18 (2.3 g, 10 mmol) in CH2Cl2 (10
mL) was added dropwise aqueous solution of sodium hydroxide
(50%, 5 mL) over 15 min and stirred for a further 0.5 h at
room temperature. Distilled water (30 mL) was added and
extracted with dichloromethane (3 × 50 mL). The combined
organic extracts were dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo to yield an brown oil.
Further purification by column chromatography on silica gel
(eluant: ethyl acetate) and followed by recrystallization from
methanol furnished a yellow crystalline solid (32%). mp 177-
179 °C; 1H NMR (CDCl3) δ: 3.75 (s, 3H), 5.00 (s, 2H), 6.20 (s,
1H), 6.32 (s, 1H), 6.63-7.50 (m, 11H). m/z: 403 (M + 1)+.
N-[(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl]-2-(7-meth-
oxy-2-oxo-2H-chromen-4-yl)acetamide (10k): (90%); mp
254-256 °C; 1H NMR (DMSO-d6) δ: 3.80 (s, 2H), 3.86 (s, 3H),
4.17 (d, J ) 5.8 Hz, 2H), 6.25 (s, 1H), 6.29 (s, 1H), 6.97 (dd, J
) 2.6, 8.9 Hz, 1H), 7.01 (d, J ) 2.6 Hz, 1H), 7.67 (d, J ) 8.9
Hz, 1H), 8,02 (s, 1H), 8.82 (t, J ) 5.8 Hz, 1H), 9.13 (s, 1H).
m/z: 358 (M + 1)+.
An analogous procedure starting with 2′b gave 4-[2-(5-
benzyloxy-4-oxo-4H-pyran-2-yl)vinyl]-6,7-dimethoxychro-
man-2-one (9j): (38%); mp 202-203 °C; 1H NMR (CDCl3) δ:
3.88 (s, 6H), 5.01 (s, 2H), 6.27 (s, 1H), 6.38 (s, 1H), 6.72 (s,
1H), 6.80-6.86 (m, 2H), 7.20-7.30 (m, 6H), 7.48 (s, 1H). m/z:
433 (M + 1)+.
N-[(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl]-2-(6,7-
5-Benzyloxy-2-({[(7-methoxy-2-oxo-2H-chromen-4-yl)-
methyl]amino}methyl)-1-methylpyridin-4(1H)-one (9n).
To a solution of 2′a (3.37 g, 15 mmol) and 23 (3.65 g, 15 mmol)
in ethanol (100 mL) was added triethylamine (1.6 g, 16 mmol),
and the mixture was heat under reflux for 2 days. After
evaporation to remove the solvent, the residue was added 100
mL of water and was extracted with dichloromethane (4 × 100
mL). The organic fraction was then dried over anhydrous
sodium sulfate, filtered, and rotary evaporated to yield a pale
dimethoxy-2-oxo-2H-chromen-4-yl)acetamide (10l): (88%);
1
mp 263-265 °C; H NMR (DMSO-d6) δ: 3.79 (s, 3H), 3.83 (s,
2H), 3.87 (s, 3H), 4.19 (d, J ) 5.8 Hz, 2H), 6.25 (s, 1H), 6.29
(s, 1H), 7.09 (s, 1H), 7.18 (s, 1H), 7.99 (s, 1H), 8.84 (t, J ) 5.8
Hz, 1H), 9.13 (br s, 1H). m/z: 388 (M + 1)+.
7-Methoxy-N-[(5-hydroxy-4-oxo-4H-pyran-2-yl)methyl]-
2-oxo-2H-chromene-3-carboxyamide (10m): (86%); mp
264-266 °C; 1H NMR (DMSO-d6) δ: 3.91 (s, 3H), 4.42 (d, J )
6.0 Hz, 2H), 6.30 (s, 1H), 7.05 (dd, J ) 2.4, 8.8 Hz, 1H), 7.13
(d, J ) 2.4 Hz, 1H), 7.92 (d, J ) 8.8 Hz, 1H), 8.06 (s, 1H), 8.84
(s, 1H), 9.13 (br s, 1H). m/z: 344 (M + 1)+.
1
yellow solid (2.92 g, 45%). mp 121-123 °C; H NMR (CDCl3)
δ: 3.54 (s, 3H), 3.65 (s, 2H), 3.78 (s, 3H), 3.80 (s, 2H), 4.98 (s,
2H), 6.20 (s, 1H), 6.32 (s, 1H), 6.58-6.74 (m, 2H), 6.90 (d, J )
2.5 Hz, 1H), 7.20 (s, 5H), 7.30 (s, 1H). m/z: 433 (M + 1)+.
N-[(3-Hydroxy-6-methyl-4-oxo-4H-pyran-2-yl)methyl]-
2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide(10p): (82%);
N-[2-(3-Hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
2-(7-methoxy-2-oxo-2H-chromen-4-yl)acetamide Hydro-
chloride (10a): Method i. Solutions of 9a in N,N-dimethyl-
formamide (DMF) were subjected to hydrogenolysis in the
presence of 5% Pd/C (w/w) catalyst for 5 h. 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 to afford a white solid (89%): mp 256-
1
mp 262-263 °C; H NMR (DMSO-d6) δ: 2.17 (s, 3H), 3.73 (s,
2H), 3.85 (s, 3H), 4.29 (d, J ) 5.5 Hz, 2H), 6.21 (s, 1H), 6.25
(s, 1H), 6.94 (dd, J ) 2.5, 8.8 Hz, 1H), 7.00 (d, J ) 2.5 Hz,
1H), 7.67 (d, J ) 8.8 Hz, 1H), 8.72 (t, J ) 5.6 Hz, 1H), 9.07 (s,
1H). m/z: 372 (M + 1)+.
N-[(3-Hydroxy-6-methyl-4-oxo-4H-pyran-2-yl)methyl]-
2-(6,7-dimethoxy-2-oxo-2H-chromen-4-yl)acetamide
1
(10q): (89%); mp 261-262 °C; H NMR (DMSO-d6) δ: 2.12
1
(s, 3H), 3.74 (s, 3H), 3.75 (s, 2H), 3.86 (s, 3H), 4.30 (d, J ) 5.5
Hz, 2H), 6.21 (s, 1H), 6.26 (s, 1H), 7.09 (s, 1H), 7.19 (s, 1H),
8.83 (t, J ) 5.5 Hz, 1H). m/z: 402 (M + 1)+.
258 °C; H NMR (DMSO-d6) δ: 2.51 (s, 3H), 3.51 (q, J ) 5.7
Hz, 2H), 3.69 (s, 2H), 3.87 (s, 3H), 4.39 (t, J ) 5.7 Hz, 2H),
6.23 (s, 1H), 6.96 (dd, J ) 2.5, 8.7 Hz, 1H), 6.98 (d, J ) 2.5
Hz, 1H), 7.19 (d, J ) 6.9 Hz, 1H), 7.56 (d, J ) 8.7 Hz, 1H),
8.07 (d, J ) 6.9 Hz, 1H), 8.61 (t, J ) 5.9 Hz, 1H). m/z: 385 (M
- Cl)+.
N-[(3-Hydroxy-6-methyl-4-oxo-4H-pyran-2-yl)methyl]-
7-methoxy-2-oxo-2H-chromene-3-caroxamide (10r): (78%);
1
mp 261-263 °C; H NMR (DMSO-d6) δ: 2.24 (s, 3H), 3.90 (s,

Novel Iron Chelators with Fluorescent Sensors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6359
3H), 4.54 (d, J ) 5.8 Hz, 2H), 6.24 (s, 1H), 7.06 (dd, J ) 2.4,
8.7 Hz, 1H), 7.13 (d, J ) 2.4 Hz, 1H), 7.92 (d, J ) 8.7 Hz, 1H),
8.86 (s, 1H), 9.02 (t, J ) 5.8 Hz, 1H), 9.14 (s, 1H). m/z: 358 (M
+ 1)+.
N-[(3-Hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-
2-yl)methyl]-2-(7-methoxy-2-oxo-2H-chromen-4-yl)aceta-
mide hydrochloride (10s): (82%); mp 250-251 °C; 1H NMR
(DMSO-d6) δ: 2.53 (s, 3H), 3.75 (s, 2H), 3.80 (s, 3H), 3.86 (s,
3H), 4.61 (d J ) 4.9 Hz, 2H), 6.25 (s, 1H), 6.96 (dd, J ) 2.2,
8.8 Hz, 1H), 7.01 (s, 1H), 7.02 (d, J ) 2.2 Hz, 1H), 7.64 (d, J
) 8.8 Hz, 1H), 8.91 (t, J ) 4.9 Hz, 1H). m/z: 385 (M - Cl)+.
N-[(3-Hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-
2-yl)methyl]-2-(7-(dimethylamino)-2-oxo-2H-chromen-4-
yl)acetamide hydrochloride (10t): (81%); mp 232-234 °C;
1H NMR (DMSO-d6) δ: 2.56 (s, 3H), 3.02 (s, 6H), 3.69 (s, 2H),
3.87 (s, 3H), 4.62 (d, J ) 5.1 Hz, 2H), 6.01 (s, 1H), 6.55 (d, J
) 2.5 Hz, 1H), 6.71 (dd, J ) 2.5, 9.0 Hz, 1H), 7.29 (s, 1H), 7.51
(d, J ) 9.0 Hz, 1H), 9.12 (t, J ) 5.1 Hz, 1H). m/z: 398 (M -
Cl)+.
5-Hydroxy-2-({[(7-methoxy-2-oxo-2H-chromen-4-yl)-
methyl]amino}methyl)-1-methylpyridin-4(1H)-one hy-
drochloride (10n): (60%); mp 234-236 °C; 1H NMR (DMSO-
d6) δ: 3.60 (br s, 1H), 3.88 (s, 3H), 4.20 (s, 3H), 4.55 (s, 2H),
4.61 (s, 2H), 6.66 (s, 1H), 7.03 (dd, J ) 2.5, 8.8 Hz, 1H), 7.07
(d, J ) 2.5 Hz, 1H), 7.72 (s, 1H), 7.80 (d, J ) 8.8 Hz, 1H), 8.39
(s, 1H). m/z: 343 (M - 2HCl + 1)+.
7-Diethylamino-N-[(5-hydroxy-1-methyl-4-oxo-1,4-di-
hydropyridin-2-yl)methyl]-2-oxo-2H-chromene-3-carbox-
amide hydrochloride (10o): (72%); mp 246-248 °C; 1H NMR
(DMSO-d6) δ: 1.15 (t, J ) 7.0 Hz, 6H), 3.50 (q, J ) 7.0 Hz,
4H), 4.07 (s, 3H), 4.71 (d, J ) 5.9 Hz, 2H), 6.65 (d, J ) 2.0 Hz,
1H), 6.83 (dd, J ) 2.0, 9.0 Hz, 1H), 7.22 (s, 1H), 7.71 (d, J )
9.0 Hz, 1H), 8.28 (s, 1H), 8.69 (s, 1H), 9.27 (t, J ) 5.9 Hz, 1H);
m/z: 398 (M - Cl)+.
An analogous reaction of 3-benzyloxy-2-methylpyran-4-one
with tris(2-aminoethyl)amine as described for 8 gave brown
oil
1-{2-[bis(2-Aminoethyl)amino]ethyl}-3-benzoxy-2-
methylpyridin-4(1H)-one (11): (2.92 g, 85%); 1H NMR
(CDCl3) δ: 2.05 (s, 3H), 2.35 (m, 10H),3.20 (br s, 4H), 3.70 (t,
J ) 7.2 Hz, 2H), 4.84 (s, 2H), 6.02 (d, J ) 7.2 Hz, 1H), 7.18 (s,
5H), 7.44 (d, J ) 7.2 Hz, 1H).
N-[(3-Hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-
2-yl)methyl]-7-methoxy-2-oxo-2H-chromene-3-carboxam-
1
ide hydrochloride (10u): (81%); mp 280-281 °C; H NMR
2-Methyl-3-methoxypyran-4(1H)-one (12). To a solution
of methyl maltol (6) (12.6 g, 0.1 mol) in 62 mL of 10%
potassium hydroxide was added redistilled dimethyl sulfate
(13.9 g, 0.11 mol) in portions over a period of 30 min. The
temperature was kept below 25 °C by occasional cooling in an
ice-bath. Stirring was continued for an additional 1 h, and the
mixture was extracted with dichloromethane (3 × 100 mL).
The combined extracts were washed with 5% aqueous potas-
sium hydroxide (2 × 100 mL) followed by water (3 × 100 mL).
The organic fraction was then dried over anhydrous sodium
sulfate, filtered, and rotary evaporated to yield an orange oil.
(DMSO-d6) δ: 2.56 (s, 3H), 3.90 (s, 3H), 4.00 (s, 3H), 4.88 (d J
) 5.6 Hz, 2H), 7.06 (dd, J ) 2.4, 8.7 Hz, 1H), 7.14 (d, J ) 2.4
Hz, 1H), 7.21 (s, 1H), 7.91 (d, J ) 8.7 Hz, 1H), 8.85 (s, 1H),
9.17 (t, J ) 5.6 Hz, 1H). m/z: 371 (M - Cl)+.
N-[(3-Hydroxy-1,6-dimethyl-4-oxo-1,4-dihydropyridin-
2-yl)methyl]-7-diethylamino-2-oxo-2H-chromene-3-car-
boxamide hydrochloride (10v): (85%); mp 276-277 °C; 1H
NMR (DMSO-d6) δ: 1.13 (t, J ) 7.0 Hz, 6H), 2.56 (s, 3H), 3.46
(q, J ) 7.0 Hz, 4H), 4.00 (s, 3H), 4.85 (d J ) 5.8 Hz, 2H), 6.62
(d, J ) 2.2 Hz, 1H), 6.81 (dd, J ) 2.2, 9.1 Hz, 1H), 7.20 (s,
1H), 7.68 (d, J ) 9.1 Hz, 1H), 8.67 (s, 1H), 9.20 (t, J ) 5.8 Hz,
1H). m/z: 412 (M - Cl)+.
1
(10.4 g, 74%); H NMR (CDCl3) δ: 2.10 (s, 3H), 3.80 (s, 3H),
6.22 (d, J ) 6.0 Hz, 1H), 7.60 (d, J ) 6.0 Hz, 1H).
N-[2-(3-Hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)ethyl]-
2-(7-anilino-2-oxo-2H-chromen-4-yl)acetamide Hydro-
chloride (10c): Method ii. 9c (1.34 g, 2.5 mmol) was
dissolved into CH2Cl2 (50 mL) and flushed with nitrogen. After
the flask was cooled to 0 °C, boron trichloride (1M in CH2Cl2,
10 mL) was slowly added, and the reaction mixture was
allowed to stir at room temperature for 5 h. The excess BCl3
was eliminated at the end of the reaction by the addition of
methanol (10 mL) and left to stir for another 0.5 h. After
removel of the solvents under reduced pressure, the residues
were purified by recrystallization from methanol/acetone to
An analogous reaction of 3-methoxy-2-methylpyran-4-one
with ammonia as described for 8 gave 3-methoxy-2-meth-
ylpyridin-4(1H)-one (13): (1.32 g, 95%); mp 153-154 °C (lit.
1
value36 155-156.5 °C); H NMR (CDCl3) δ: 2.35 (s, 3H), 3.76
(s, 3H), 6.30 (d, J ) 7.8 Hz, 1H), 7.34 (d, J ) 7.8 Hz, 1H).
5-{[Benzyl(methyl)amino]methyl}-3-methoxy-2-meth-
ylpyridin-4(1H)-one (14). Benzylmethylamine (4.84 g, 40
mmol) and 40% formaldehyde (1.5 g, 20 mmol) were added to
100 mL of ethanol, followed by 3-methoxy-2-methyl-4(1H)-
pyridinone (1.4 g, 10 mmol). The mixture was refluxed for 24
h and then rotary evaporated to an oil which crystallized on
standing. The crude product was recrystallized from acetone/
ethanol to afford colorless solid (2.67 g, 98%). mp 138-139 °C;
1H NMR (DMSO-d6) δ: 2.20 (s, 3H), 2.38 (s, 3H), 3.55 (s, 2H),
3.63 (s, 2H), 3.78 (s, 3H), 7.21 (s, 5H), 7.70 (s, 1H).
3-Methoxy-2-methyl-5-[(methylamino)methyl]pyridin-
4(1H)-one (15): Solutions of 14 in EtOH were subjected to
hydrogenolysis in the presence of 5% Pd/C (w/w) catalyst for
4 h. The catalysts were removed by filtration. After removal
of the solvents in vacuo, the residues were dissolved in
dichloromethane and cooled for 2 days to afford a white solid
(99%); mp 99-108 °C; 1H NMR (DMSO-d6) δ: 2.12 (s, 3H),
2.37 (s, 3H) 3.61 (s, 2H), 3.77 (s, 3H), 7.20 (br s, 1H), 7.46 (s,
1H).
5-(Benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one (17).
To a solution of kojic acid 16 (71 g, 0.5 mol) in methanol (500
mL) was added sodium hydroxide (22 g, 0.55 mol) dissolved
in water (50 mL), and the mixture was heated to reflux. Benzyl
chloride (70 g, 0.55 mol) was added dropwise over 30 min, and
the resulting mixture was refluxed overnight. After removal
of solvent by rotary evaporation, the brown solid was washed
with water (200 mL) followed by methanol (100 mL). Recrys-
tallization from methanol gave the pure product as colorless
needles (90.5 g, 78%); mp 132-133 °C (lit. value37 132 °C); 1H
NMR (DMSO-d6) δ: 4.17 (s, 2H), 4.80 (s, 2H), 5.52 (br s, 1H),
6.14 (s, 1H), 7.16 (s, 5H), 7.89 (s, 1H).
1
afford yellow solid (67%); mp 241-242 °C; H NMR (CDCl3)
δ: 2.51 (s, 3H), 2.95 (m, 2H), 4.13-4.44 (m, 4H), 5.95 (s, 1H),
6.82-8.54 (m, 12H). m/z: 446 (M - 2HCl + 1)+.
Analogous hydrogenation procedures were used to the
preparation for 10g-j, 10n-o.
N-(2-{[2-(Acetylamino)ethyl][2-(3-hydroxy-2-methyl-4-
oxopyridin-1(4H)-yl)ethyl]amino}ethyl)-7-diethylamino-
2-oxo-2H-chromene-3-carboxamide hydrochloride (10g):
1
(74%); mp 186-188 °C; H NMR (DMSO-d6) δ: 1.13 (t, J )
7.0 Hz, 12H), 2.56 (s, 3H), 3.47 (q, J ) 7.0 Hz, 8H), 3.50 (br s,
12H), 6.54 (s, 2H), 6.74 (dd, J ) 2.2, 9.1 Hz, 2H), 7.30 (d, J )
6.9 Hz, 1H), 7.57 (d, J ) 9.1 Hz, 2H), 8.31 (d, J ) 6.9 Hz, 1H),
8.53 (s, 2H), 8.91 (br s, 2H). m/z: 741 (M - 2HCl + 1)+.
7-Diethylamino-N-[(5-hydroxy-6-methyl-4-oxo-1,4-di-
hydropyridin-3-yl)methyl]-N-methyl-2-oxo-2H-chromene-
3-carboxamide hydrochloride (10h): (75%); mp 209-210
1
°C; H NMR (DMSO-d6) δ: 1.14 (t, J ) 6.9 Hz, 6H), 2.54 (s,
3H), 3.01 (s, 3H), 3.47 (q, J ) 6.9 Hz, 4H), 4.66 (s, 2H), 6.61
(d, J ) 2.0 Hz, 1H), 6.78 (dd, J ) 2.0, 8.9 Hz, 1H), 7.54 (d, J
) 8.9 Hz, 1H), 8.09 (s, 2H); m/z: 412 (M - Cl)+.
4-[2-(5-Hydroxy-4-oxo-4H-pyran-2-yl)vinyl]-7-methoxy-
chroman-2-one (10i): (67%); mp 268-269 °C; 1H NMR
(DMSO-d6) δ: 3.70 (s, 3H), 6.27-7.84 (m, 8H). m/z: 313 (M +
1)+.
4-[2-(5-Hydroxy-4-oxo-4H-pyran-2-yl)vinyl]-6,7-
dimethoxychroman-2-one (10j): (70%); mp 278-280 °C; 1H
NMR (DMSO-d6) δ: 3.88 (s, 3H), 3.89 (s, 3H), 6.51-8.13 (m,
7H). m/z: 343 (M + 1)+.
2-Formyl-5-benzyloxy-4H-pyran-4-one (18). To a solu-
tion of 17 (5.0 g, 21.5 mmol) in chloroform (100 mL) were added
dimethyl sulfoxide (27 mL) and triethylamine (18.5 mL), and

6360 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Ma et al.
the reaction mixture was cooled with an ice-bath to an internal
temperature of 3-5 °C. Then sulfur trioxide pyridine complex
(17.1 g, 107 mmol) was added, and the mixture was allowed
to thaw to room temperature. After stirring for 12 h at room
temperature, the reaction mixture was washed with water (2
× 50 mL), and the organic phase was dried over anhydrous
sodium sulfate, filtered, and concentrated in vacuo to yield a
yellow solid. Further purification by column chromatography
(eluent: EtOAc) furnished a pale yellow solid (3.9 g, 79%). mp
130-132 °C (lit. value38 133-135 °C); 1H NMR (CDCl3) δ: 5.00
(s, 2H), 6.85 (s, 1H), 7.25 (s, 5H), 7.57 (s, 1H), 9.53 (s, 1H).
2-Phthalimidomethyl-5-benzyloxy-pyran-4-one (19). To
a solution of triphenylphosphine (6.3 g, 24 mmol) and phthal-
imide (3.5 g, 24 mmol) in THF (100 mL) was added 2-hy-
droxymethyl-5-benzyloxypyran-4-one 17 (4.64 g, 20 mmol), and
the mixture was cooled to 0 °C in an ice bath. Diethyl
azodicarboxylate (4.2 g, 24 mmol) was added dropwise with
stirring over 30 min, after which the reaction mixture was
allowed to warm slowly to room temperature and to stand
overnight. The resulting precipitate was isolated by filtration,
washed with THF (5 mL), and dried under high vacuum to
yield a white amorphous powder (4.8 g, 66%); mp 192-193
excess zinc was removed by hot filtration, and the filtrate was
extracted with dichloromethane (5 × 200 mL). The combined
organic extracts were dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo to yield the crude product.
Recrystallization from 2-propanol afforded colorless plates
(17.4 g, 74%). mp 149-150 °C (lit. value41 152-153 °C); 1H
NMR (DMSO-d6) δ: 2.25 (s, 3H), 6.14 (s, 1H), 6.70 (br s, 1H),
7.63 (s, 1H).
2-Hydroxymethyl-3-hydroxy-6-methyl-pyran-4(1H)-
one (26). Allomaltol 25 (12.6 g, 100 mmol) was added to an
aqueous solution of sodium hydroxide (4.4 g, 110 mmol) in
distilled water (100 mL) and stirred at room temperature for
5 min. 35% Formaldehyde solution (9 mL) was added dropwise
over 10 min and the solution allowed stirred for 12 h.
Acidification to pH 1 using concentrated hydrochloric acid and
cooling to 3-5 °C for 12 h gave a crystalline deposit (14.4 g,
1
92%); mp 157 °C (lit. value41 159-161 °C); H NMR (DMSO-
d6) δ: 2.23 (s, 3H), 4.38 (s, 2H), 4.90 (br s, 1H), 6.20 (s, 1H),
8.80 (br s, 1H).
An analogous procedure starting with 26 as described for 7
gave 2-(hydroxymethyl)-3-benzyloxy-6-methyl-4H-pyran-
4-one (27a): (80%); mp 113-114 °C (lit. value42 115-116 °C).
1H NMR (CDCl3) δ: 2.20 (s, 3H), 2.63 (br s, 1H), 4.28 (s, 2H),
5.15 (s, 2H), 6.15 (s, 1H), 7.36 (s, 5H).
1
°C; H NMR (DMSO-d6) δ: 4.26 (s, 2H), 4.55 (s, 2H), 6.14 (s,
1H), 6.96 (s, 5H), 7.33 (s, 4H), 7.84 (s, 1H).
2-Aminomethyl-5-benzyloxy-pyran-4-one (20). To a so-
lution of 19 (5.4 g, 15 mmol) in ethanol (96% 50 mL) was added
hydrazine monohydrate (0.8 g, 16 mmol). After being refluxed
for 3 h, the reaction mixture was chilled to 0 °C and acidified
to pH 1 with concentrated in vacuo, and the residue was
redissolved in distilled water (50 mL), adjusted to pH 12 with
10 N sodium hydroxide, and extracted with dichloromethane
(3 × 100 mL). The combined organic extracts were dried over
anhydrous sodium sulfate, and the solvent was removed under
reduced pressure to yield brown oil. Purification by column
chromatography on silica gel furnished orange oil (2.32 g, 67%);
1,6-Dimethyl-2-hydroxymethyl-3-benzyloxypyridin-
4(1H)-one (27b). To a solution of 27a (2.46 g, 10 mmol) in
dichloromethane (50 mL) was added 3,4-dihydro-2H-pyran (1.7
g, 20 mmol) followed by p-toluenesulfonic acid monhydrate (30
mg). After being stirred at room temperature for 3 h, the
reaction mixture was washed with 5% aqueous sodium car-
bonate (20 mL) followed by water (2 × 20 mL). The organic
fraction was then rotary evaporated to yield light yellow oil,
which was then dissolved in water (50 mL)/40% aqueous
methylamine (10 mL). The reaction mixture was heated at 70
°C overnight. After the solution was concentrated ∼2-fold by
rotary evaporation, the residue was redissolved in 20 mL of
water and was adjusted pH 1 with concentrated hydrochloric
acid and refluxed for 4 h, followed by extraction into dichlo-
romethane (4 × 50 mL), and the combined organic layers were
then dried over anhydrous sodium sulfate, filtered, and rotary
evaporate to give a light brown solid. Recrystallization from
methanol/diethyl ether afforded the pure product as a white
crystalline solid (2.02 g, 79%); mp 182.5-184 °C (lit. value42
1
mp 81-83 °C (lit. value39 84.5-86.5 °C); H NMR (CDCl3) δ:
2.98 (br s 2H), 3.50 (s 2H), 4.90 (s, 2H), 6.20 (s, 1H), 7.18 (s,
5H), 7.33 (s, 1H).
An analogous reaction of protected kojic acid with methyl-
amine as described for 8 gave 5-(benzyloxy)-2-(hydroxy-
methyl)-1-methylpyridin-4(1H)-one (21): (1.76 g, 72%); mp
1
215.5-217.5 °C (lit. value40 213-217 °C); H NMR (CDCl3) δ:
3.39 (s, 3H), 4.09 (s, 2H), 4.71 (s, 2H), 5.93 (s, 1H), 7.08 (s,
5H), 7.24 (s, 1H).
1
181-183 °C); H NMR (CDCl3) δ: 2.13 (s, 3H), 3.42 (s, 3H),
1-Methyl-2-chloromethyl-5-benzyloxypyridin-4(1H)-
one hydrochloride (22). 1-Methyl-2-hydroxymethyl-5-ben-
zyloxypyridin-4(1H)-one 21 (8.54 g, 35 mmol) was dissolved
in distilled thionyl chloride (20 mL) and stirred for 1 h, after
which time a pale yellow crystalline mass formed. The product
was collected by filtration and washed with petroleum ether
to afford colorless amorphous powder (9.4 g, 90%). mp 165.5-
166 °C; 1H NMR (DMSO-d6) δ: 4.10 (s, 3H), 4.82 (s, 2H), 4.95
(s, 2H), 7.12 (s, 5H), 7.40 (s, 1H), 8.66 (s, 1H).
4.42 (s, 2H), 4.83 (s, 2H), 5.95 (s, 1H), 7.18 (s, 5H).
An analogous procedure starting with 27a as described for
19 gave 6-methyl-2-phthalimidomethyl-3-benzyloxypy-
ran-4-one (28a): (53%); mp 223-225 °C; 1H NMR (CDCl3) δ:
2.10 (s, 3H), 4.64 (s, 2H), 5.16 (s, 2H), 6.03 (s, 1H), 7.10-7.70
(m, 9H).
An analogous procedure starting with 27b as described for
19 gave 1,6-dimethyl-2-phthalimidomethyl-3-benzyloxy-
pyridin-4(1H)-one (28b): (80%); mp 250.5-252.5 °C (lit.
1-Methyl-2-aminomethyl-5-benzyloxypyridin-4(1H)-
one (23). To a solution of 22 (6.0 g, 20 mmol) in distilled water
(100 mL) was added ammonia (40%, 10 mL) and was stirred
overnight at room temperature. After removal of water by
rotary evaporation, the residue was extracted with hot 2-pro-
panol (100 mL) and was filtered. The filtrate was concentrated
in vacuo to yield pale yellow solid (3.94 g, 81%). mp 184-187
1
value42 250 °C); H NMR (DMSO-d6) δ: 2.20 (s, 3H), 3.43 (s,
3H), 4.80 (s, 2H), 5.08 (s, 2H), 6.08 (s, 1H), 7.23 (s, 5H), 7.71
(s, 4H).
An analogous procedure starting with 28a as described for
20 gave 2-aminomethyl-6-methyl-3-benzyloxy-pyran-4-
one (29a) as a brown oil: (65%); 1H NMR (CDCl3) δ: 2.17 (s,
3H), 3.41 (s, 2H), 5.05 (s, 2H), 6.02 (s, 1H), 7.20 (s, 5H).
1
°C; H NMR (DMSO-d6) δ: 2.50 (br s, 2H), 3.42 (s, 2H), 3.48
(s, 3H), 4.78 (s, 2H), 6.04 (s, 1H), 7.14 (s, 5H), 7.25 (s, 1H).
An analogous procedure starting with 16 as described for
22 gave 2-chloromethyl-5-hydroxypyran-4(1H)-one (24):
An analogous procedure starting with 28b as described for
20 gave 1,6-dimethyl-2-aminomethyl-3-benzyloxypyridin-
4(1H)-one (29b): (88%); mp 140-142 °C (lit. value42 143-
144 °C); C. 1H NMR (CDCl3) δ: 2.20 (s, 3H), 3.54 (s, 3H), 3.78
(s, 2H), 5.26 (s, 2H), 6.24 (s, 1H), 7.30 (s, 5H).
Fluorescence Methods. Optical Characterization. The
UV/vis spectra of the two probes were recorded using a Perkin-
Elmer spectrophotometer (type UV/VIS Lambda 2S), and a
Perkin-Elmer spectrofluorometer (type LS 50B) was used to
record fluorescence emission spectra operating at a scan rate
of 120 nm/min. Spectra were not corrected for light intensity
or detector sensitivity. Data were recorded on-line and ana-
lyzed by Excel software on a PC computer. Fluorescence
1
(80%); mp 164-164.5 °C (lit. value41 166-168 °C); H NMR
(DMSO-d6) δ: 4.65 (s, 2H), 6.62 (s, 1H), 8.23 (s, 1H), 8.80 (br
s, 1H).
2-Methyl-5-hydroxypyran-4(1H)-one (25). Chlorokojic
acid 24 (30 g, 0.187 mol) was added to 100 mL of distilled water
and heated to 50 °C with stirring. Zinc dust (24.4 g, 0.375 mol)
was added followed by the dropwise addition of concentrated
hydrochloric acid (56.1 mL) over 1 h with vigorous stirring
maintaining the temperature between 70 and 80 °C. The
reaction mixture was stirred for a further 3 h at 70 °C. The

Novel Iron Chelators with Fluorescent Sensors
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6361
quenching measurements were performed in MOPS buffer (pH
7.4) at 22 °C.
7.4), 1 mM ascorbic acid ,and 100 µM Fe2+, at 0 °C. The
resuspended membranes were placed on ice for 10 min and
then incubated at 37 °C for 30 min to reseal the lysed ghost
cells. After resealing, the ghost cells were washed three times
in MOPS buffer (25 mM MOPS, 1 mM ascorbic acid) by
successive centrifugation (10 000g, 5 min) and resuspension
in fresh buffer.
(c) Permeability Measurements. Resealed ghost cells
were resuspended in 1 mL of MOPS buffer (25 mM MOPS, 1
mM ascorbic acid) containing either 15 µM 10a or 6 µM 10v,
without and with 0.1mM DTPA. Fluorescence intensity was
recorded during 30 min incubation at 20 °C using a Perkin-
Elmer luminescence spectrometer. The results were digitized
and subjected to analysis using MicroCal v6 software.
Recording of Fluorescence Quantum Yield. The fluo-
rescence quantum yield (ΦF), defined as the ratio of photons
absorbed to photons emitted by fluorescence, gives the prob-
ability of the excited-state being deactivated by fluorescence
rather than by another, nonradiative mechanism. The most
reliable method for recording ΦF is the comparative method
of Williams et al. 1983,43 which involves the use of well
characterized standard samples with known ΦF values. For
these measurements, quinine and harmane were adopted as
standards because they absorb at the excitation wavelength
appropriate to the samples and emit in similar spectral
regions. To minimize reabsorption effects,44 absorbances in the
10 mm fluorescence cuvette should never exceed 0.1 at and
above the excitation wavelength. Above this level, nonlinear
effects may be observed due to inner filter effects. Spectroscopic
grade solvents were used and checked for background fluo-
rescence. The fluorescence of each sample was recorded in a
range of five concentrations (Absorbance values in the range
0.02-0.1), and plots of integrated fluorescence intensity (IF)
vs absorbance (A) were recorded.
Acknowledgment. This investigation was partially
supported from funds from Apotex, Canada. Y. M. Ma
and W. Luo were supported by ORS awards.
Note Added after ASAP Posting. A revised Figure
3 and caption, with 10h and 10v transposed, were added
to the version of the manuscript posted October 30,
2004. The new version was posted November 8, 2004.
The gradient of each plot (IF vs A) is proportional to the
quantum yield of the sample. Absolute values of quantum yield
were calculated using standard samples which have a known
fluorescence quantum yield, given by the following equation:
Supporting Information Available: Table of elemental
analysis data. This material is available free of charge via the
Internet at http://pubs.acs.org.
Gradx η2
x
Φx ) ΦST
2
(
)(η
)
GradST
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