Amido-3-hydroxypyridin-4-ones as iron(III) ligands

Article abstract of DOI:10.1002/chem.200902455

The synthesis and physicochemical properties of a range of 2and 6-amido-3-hydroxypyridin-4-ones are described. All the amido-substituted 3-hydroxypyridin-4-ones have lower pK_a values than 1,2-dimethyl-3-hydr-oxypyridin-4-one (deferiprone). This

Full text of DOI:10.1002/chem.200902455

DOI: 10.1002/chem.200902455
Amido-3-hydroxypyridin-4-ones as IronACTHNUTRGENU(GN III) Ligands
Sirivipa Piyamongkol,[c] Yong M. Ma,[a] Xiao L. Kong,[a] Zu D. Liu,[a] Mutlu D. Aytemir,[a]
Dick van der Helm,[b] and Robert C. Hider*[a]
Abstract: The synthesis and physico-
chemical properties of a range of 2-
and 6-amido-3-hydroxypyridin-4-ones
are described. All the amido-substitut-
ed 3-hydroxypyridin-4-ones have lower
pKa values than 1,2-dimethyl-3-hydr-
ly lower than those of the correspond-
ing 1-alkyl analogues, indicating that a
strong hydrogen bond exists between
the 2-amido function and the 3-oxygen
anion, which stabilises the anion. As a
result of the decreased competition
with protons, the pFe3+ values of this
group of molecules are higher than
that of deferiprone. The distribution
coefficients of these molecules are also
increased despite the lack of a hydro-
phobic 1-alkyl substituent and this is
ascribed to the intramolecular hydro-
gen bond. X-ray diffraction studies
confirm the existence of the intramo-
lecular hydrogen bond.
ACHTUNGTRENNUNGoxypyridin-4-one (deferiprone). This is
due to the inductive effect of the
amido group. Furthermore, the pKa
values of the 3-hydroxy group in 1-non-
substituted pyridinones are dramatical-
Keywords: acidity
·
hydrogen
bonds · hydroxypyridinones · induc-
tive effects · iron
Introduction
vantage that it is not orally active and is rapidly cleared by
the kidneys.[5,6] Therefore, there is an urgent need to devel-
Although iron is an essential element for all living processes,
it is toxic when present in excess. In the presence of molecu-
lar oxygen, weakly coordinated iron can redox cycle be-
tween the two most stable oxidation states, iron(II) and
op an ironACTHNGUTERNN(UG III)-selective, orally active and non-toxic seques-
tering agent. 3-Hydroxypyridin-4-ones (HPOs) are currently
one of the main candidates for the development of such che-
lators.[7] Indeed, 1,2-dimethyl-3-hydroxypyridin-4-one (defer-
iprone; 2; Scheme 1) is one of two orally active iron chela-
tors currently available for clinical use. In an attempt to im-
prove the efficacy of this family of bidentate HPOs, metal
selectivity and ligand–metal complex stability are key pa-
rameters to be considered.[7,8] The pFe3+ value, defined as
the negative logarithm of the concentration of the free
ironACHTUNGTRENNUNG(III), thereby generating oxygen-derived free radicals
such as the toxic hydroxyl radical.[1,2] Iron overload is ob-
served in haemoglobinopathic disorders such as haemochro-
matosis, thalassaemia and sickle cell disease. These compli-
cations can be relieved by the use of selective iron
lators, which reduce the total body iron load. Desferriox-
amine B (DFO; 1; Scheme 1) is the most widely used
ACHTUNGTRENN(UNG III) che-
G
ironACHTNGUTERNNU(G III) in solution, is a more suitable comparator for mole-
A
E
cules of therapeutic potential because it takes into account
the effect of ligand basicity, denticity, degree of protonation
and difference in metal–ligand stoichiometries, factors which
do not influence the corresponding stability constants.[9] Typ-
ically, pFe3+ values are calculated for ligand[total] =10À5 m,
iron[total] =10À6 m and pH 7.4. The pFe3+ value of deferiprone
is 19.4.[9] To increase the iron-chelating ability further, we
have established that introduction of a 1’-hydroxyalkyl
group at the 2 position of HPOs leads to an appreciable en-
hancement of pFe3+ values.[10] Chelators with high pFe3+
values are predicted not only to be able to scavenge iron ef-
ficiently at a lower ligand concentration, but also to dissoci-
ate less readily and, therefore, are less likely to redistribute
iron to other parts of the body. With 3-hydroxypyridin-4-
ones, the increase of the pFe3+ value can result from a de-
over thirty years.[3,4] However, DFO suffers from the disad-
[a] Dr. Y. M. Ma, Dr. X. L. Kong, Dr. Z. D. Liu, Dr. M. D. Aytemir,
Prof. R. C. Hider
[b] Dr. D. van der Helm
[c] Dr. S. Piyamongkol
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Chem. Eur. J. 2010, 16, 6374 – 6381

FULL PAPER
solution of alkyl amine at 708C.
The 3-hydroxyl-protected group
was hydrogenated to yield the
corresponding bidentate ligands
3a–f as hydrochloride salts.
The synthetic pathway for
the 2-amido-3-hydroxypyridin-
4-ones
is
summarised
in
Scheme 3. Direct conversion of
the 2-hydroxymethyl derivative
7[11] to a carboxylic acid was ini-
tially attempted by using Jones
reagent.[14] However, this reac-
tion resulted in a poor yield.
Thus the primary alcohol 7 was
firstly oxidised to the inter-
mediate aldehyde 8 by using
PySO3/DMSO (Py=pyridine)
Scheme 1. Structures of DFO (1), deferiprone (2) and 3-hydroxypyridin-4-one amido derivatives (3).
crease of pKa values corresponding to either of the 3- and 4-
pyridinone oxygen atoms.[11,12] These differences may be as-
sociated with the enhanced stability of the ionised species.
Such enhanced stability can result from a combination of in-
tramolecular hydrogen bonding between the 2-(1’-hydr-
oxyalkyl) group and the adjacent 3-hydroxyl moiety, togeth-
er with an inductive effect.[10,13] In the present paper, we
demonstrate the influence of such factors on the iron-chelat-
ing properties of a range of amido HPOs (Scheme 1).
before further oxidation to the carboxylic acid 9. Activation
of 9 was achieved prior to reacting with ammonia or etha-
nolamine. As a result, 10a and 10b were obtained in reason-
able yields after purification by column chromatography.
The benzyl-protected groups were removed by hydrogena-
tion to obtain 3g and 3h.
The synthetic route employed for 6-amido-1-methyl-3-hy-
droxypyridin-4-one is summarised in Scheme 4. The carbox-
yl pyranone 13 was prepared by directly oxidising 6-hydr-
AHCTUNGTREGoNNNU xymethyl pyranone 12 by using Jones reagent where the 3-
hydroxy group is protected. The reaction proceeded rapidly
and in good yield with no indication of oxidative cleavage of
the pyran-4-one double bond. Similarly, the acid 13 was acti-
vated, coupled with methylamine, converted to the pyridi-
none, followed by deprotection to yield the final ligand 3i.
Results and Discussion
The general methodology adopted for the synthesis of 6-
methyl-2-amido-3-hydroxypyridin-4-ones is outlined in
Scheme 2. Active amide 4 was prepared from 5-hydroxy-2-
(hydroxymethyl)-4-pyrone (kojic acid) in several steps as de-
scribed previously.[13] Active amides were stirred with meth-
ylamine or dimethylamine at room temperature to produce
the desired 2-amido-substituted pyranones 5a–c, which were
then converted to the corresponding pyridinone derivatives
6a–f by reaction with an excess of ammonia or an aqueous
Physicochemical properties of 3-hydroxypyridin-4-ones: All
ligands were investigated by spectrophotometric titration.
The pH-dependent 2D and 3D UV/Vis spectra of 3b and 3 f
are presented in Figure 1 as examples. An incremental ab-
sorption at 278 nm is observed for 3b with increased pH
over the range of pH 1–6 (Figure 1a and b). With continu-
Scheme 2. Synthesis of iron chelators 3a–f (Bn=benzyl).
Scheme 3. Synthesis of 3g and h.
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6375

R. C. Hider et al.
Scheme 4. Synthesis of 3i from kojic acid.
Figure 1. The pH-dependent UV/Vis spectra of 3b and 3 f over the range pH 1–12: a) 2D spectra of 3b, b) 3D spectra of 3b, c) 2D spectra of 3 f and
d) 3D spectra of 3 f.
ous increase of pH (pH 6–12), the maximum absorption
wavelength is shifted to 315 nm. This phenomenon is similar
to that observed in our previous study on 2 and also for
other compounds 3a, 3c–e and 3i. The four protonation
states of the general 3-hydroxypyridin-4-one structure are
shown in Scheme 5. There are two monodeprotonated forms
that can exist depending on the location of the labile
proton, which can be bound to the oxygen atom of either
the 3- or 4-hydroxy group (HB1 and HB2). In the previous
study centred on 2, the pKa value of 3.56 was assigned to
the protonation of the 4-oxo group and the pKa value of
9.64 to the dissociation of the 3-hydroxy group.[15] Because
there is a similar pH dependence of the UV/Vis spectra of 2
and 3b, it is most probable that the 4-hydroxy group of 3b
is first ionised (route 1 of Scheme 5), that is, the pKa value
2.77 is assigned to the 4-oxo group and the pKa value 8.44 to
the 3-hydroxy group. Route 1 will be favoured by the exis-
tence of the two canonical forms of HB1.
The pH-dependent UV/Vis spectra of 3 f are quite differ-
ent to those of 2 and 3b (Figure 1c and d). Over the range
pH 2–6, there is one isosbestic point at 298 nm. Over the
range pH 6–12, the maximum absorption intensity is in-
creased and shifted to 330 nm with an isosbestic point at
313 nm. A similar phenomenon was also observed for 3g
and 3h. These distinct differences from 2 and 3b indicate
that the protonation route of 3 f may differ to that of 3b,
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Hydroxypyridinones as IronACHTNUTRGNE(NUG III) Ligands
FULL PAPER
ring. Among them, 3a–e and 3i have similar pKa values and
the pKa2 values fall in the range 8.05–8.45. This group, which
relates to the 3-hydroxy substituent, is thought to be ionised
after the 4-oxo group through route 1 (Scheme 5). In con-
trast, 3 f–h have similar pKa values and the pKa2 values fall
in the range 1.89–2.32, which relates to the 3-hydroxy group.
This group is believed to ionise before the 4-oxo group
through route 2. Compounds 3a–h contain an amido group
at the 2 position and 3i contains an amido group at the 6
position. The presence of an electron-withdrawing amido
group leads to the stabilisation of the anions (HB and BÀ)
and, therefore, to the lower pKa values. Thus, the introduc-
tion of a negative inductive effect plays an important role in
reducing the pKa values of this series of compounds and,
therefore, results in an improvement of the associated pFe3+
values (Table 1).
In principle, in the group of 1-alkyl-2-amido compounds
3a–d, compounds 3b–d could form an intramolecular hydro-
gen bond between the NH group of the 2-amido group and
the ionised 3-hydroxy group. However, there is no such op-
portunity for 3a owing to a tertiary amide group located at
the 2 position, and yet the pKa value of the 3-hydroxy group
in 3a was found to be even lower than those of 3b–d. In an
attempt to understand this effect, analogues 3e and 3i were
synthesised for the physicochemical analysis. Both com-
pounds are structurally incapable of forming an intramolec-
ular hydrogen bond. Again, the pKa values of the 3-hydroxy
group in both are very close to that of 3a. These combined
results suggested that the drop in the pKa2 value of com-
pounds 3b–d results from the inductive effect of the amide
substituent and that there is no intramolecular hydrogen
bond.
Scheme 5. Ionisation equilibrium of the general 3-hydroxypyridin-4-one.
that is, the 3-hydroxy group may be first ionised to form
HB2 (Scheme 5). A possible driving force for this difference
could be the existence of a strong intramolecular hydrogen
bond between substituents at the
2
and
3
positions
Very interestingly, a further remarkable reduction in the
pKa value of the 3-hydroxy group in 3 f was observed when
compared with the 3a–d group. That is, the pKa value of the
3-hydroxy group dramatically drops from 8.44 in 3b or 8.20
in 3e to 2.32 in 3 f. To expand the study, two analogues 3g
and 3h were synthesised and both were found to have simi-
lar pKa values to that of 3 f. A marked decrease in the affini-
(Scheme 5, R1 =H). Thus, the pKa value of 2.32 is assigned
to the 3-hydroxy group and 6.66 to the 4-oxo group.
The physicochemical properties of the entire 3-hydroxy-
pyridin-4-one group are presented in Table 1. All the com-
pounds (3a–i) possess lower pKa values and stability con-
stants for ironACHTUNGTRENNUNG(III) than those of 2, these changes being in-
duced by the introduction of the amido substituent on the
ty constant for ironACTHUNGTERN(NUG III) (logb3) of 3 f as compared to that of
Table 1. Physicochemical properties of the amido-3-hydroxypyridin-4-ones.
pKa
(4-oxo, 3-OH)
Affinity constants for FeIII[a]
D7.4
ACHTUNGTRENNUNG
(n=5)[c]
[d]
Compound
pFe3+[b]
Fn
logP[e]
logK1
logK2
logK3
logb3
2
3.56, 9.64
2.57, 8.14
2.77, 8.44
2.58, 8.45
2.64, 8.41
2.53, 8.20
6.66, 2.32
6.38, 2.19
6.27, 1.89
2.32, 8.05
14.56
12.48
13.41
12.84
12.96
12.85
14.50
12.60
11.67
11.77
12.19
10.81
11.47
11.88
11.27
11.18
10.49
10.18
10.22
10.52
9.69
8.43
9.43
8.70
9.18
9.19
7.47
8.28
8.53
8.79
36.44
31.72
34.31
33.42
33.41
33.22
32.46
31.06
30.42
31.08
19.4
20.0
21.7
20.9
21.0
20.4
22.8
21.5
20.9
19.5
0.17
0.16
0.04
0.12
0.08
0.09
0.17
0.01
0.15
0.03
0.99
0.86
0.92
0.92
0.92
0.86
0.16
0.09
0.07
0.83
À0.77
À0.73
À1.36
À0.90
À1.04
À0.98
0.03
3a
3b
3c
3d
3e
3 f
3g
3h
3i
À0.91
0.34
À1.44
[a] The cumulative affinity constants obtained by summation of the three stepwise equilibrium constants (K1, K2, K3); [b] pFe3+ is the negative logarithm
of the concentration of the free ironACTHNUTRGNEUNG
(III) in solution, calculated for ligand[total] =10À5 m, iron[total] =10À6 m at pH 7.4; [c] D7.4 is the distribution coefficient
(n-octanol/water) at pH 7.4; [d] Fn is the fraction of non-ionised species calculated at pH 7.4; [e] logP is the logarithm of the partition coefficient of the
neutral species (n-octanol/water).
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6377

R. C. Hider et al.
2 was also found. However, the pFe3+ value of 3 f was in-
creased to 22.8. Within the group 3 f–h, in which the pKa
values are similar, the logb3 value decreases from 32.46 to
30.42 and the pFe3+ value decreased correspondingly from
22.8 to 20.85 (Table 1). Surprisingly, 3 f was found to be
more hydrophobic (D7.4 =0.17, in which D7.4 is the distribu-
better, 3b, 3 f and their hydrochloride salts were investigat-
ed by X-ray diffraction. As examples, views of both neutral
ligands from the front of the plane of the pyridine molecules
are shown in Figure 2. Bond lengths, angles and selected tor-
sion angles of the 3 f and 3b neutral ligands are presented in
Table 3 and Table 4, respectively.
tion coefficient (n-octanol/water) at pH 7.4) than 3b (D7.4
=
The structure of 3 f in the neutral state clearly demon-
strates that the oxygen in the 3 position (O2) is ionised and
its bond length with C5 is 1.279 ꢂ, whereas the oxygen in
the 4 position (O1) is protonated and its bond length with
C4 is 1.336 ꢂ (Table 3). This finding confirms the existence
of HB2 as the monodeprotonated form and confirms the
route 2 deprotonation pathway (Scheme 5). Ligand 3 f is
almost planar because the torsion angles of any four atoms
in the molecule (except H) are close to 0 or 1808 (Figure 2a
0.04), whereas in normal circumstances it would be expected
to be more hydrophilic owing to the lack of the 1-methyl
substituent. These combined findings can best be explained
by the existence of a highly stable intramolecular hydrogen
bond between the substituents at the 2 and 3 positions
À
(Scheme 5) in 3 f–h. When the pair 3a and 3b (N methyl
compounds) are compared with the pair 3e and 3 f (NH
compounds; Table 2), the differences in physicochemical pa-
rameters, such as pKa, pFe3+ and logP values (logP is the
logarithm of the partition coefficient of the neutral species),
are less marked in the former pair. Initially, it was not clear
why such a similar structural alteration within each pair
could lead to such large changes in physicochemical parame-
ters. These results prompted us to undertake X-ray crystal
studies to investigate the conformation of both 3b and 3 f.
The logP values of the entire group of pyridinones
(Table 1) fit with the above proposed hydrogen-bonding
schemes. The three pyridinones (3a, 3c and 3d), which each
possess one additional alkyl carbon over that of 3b, are less
hydrophilic than 3b. In contrast, the logP value of 3 f is
much more hydrophobic than that of 3b despite 3 f contain-
ing one less alkyl function. This is readily explained by the
existence of a strong hydrogen bond between the 2-amido
and the 3-hydroxy functions in 3 f and the absence of such a
bond in 3b. The existence of strong intramolecular hydrogen
bonds reduces the ability of a molecule to form hydrogen
bonds with the solvent. The finding that 3e is more hydro-
philic than 3 f is in agreement with this proposal because an
analogous intramolecular hydrogen bond between the 2-
amido and 3-hydroxyl functions is not possible. The most
hydrophobic pyridinone of the series is 3h, which presum-
ACHTUNGTRENNUNGably, in addition to the 2,3-intramolecular hydrogen bond,
also possesses an intramolecular hydrogen bond between
the 2-amido oxygen and the aliphatic hydroxyl group.
Figure 2. Crystal structures of a) 3 f and b) 3b viewed from the front of
the pyridinone plane.
X-ray crystal structures of 3b and 3 f: To understand the
effect of the structures on their physicochemical properties
Table 2. Comparison of the influence of the inductive effect and the hydrogen-bond effect on the pKa, pFe3+ and logP values.
3b 3a 3 f
3e
DpKa1
DpKa2
DpFe3+
DlogP
0.20
0.30
1.7
À0.63
4.13
À5.88
2.4
1.01
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Hydroxypyridinones as IronACHTNUTRGNE(NUG III) Ligands
FULL PAPER
Table 3. Bond lengths [ꢂ], angles and selected torsion angles [8] of 3 f.
Bond length
Angle
Torsion angle
À
O1 C4
1.336 (3)
1.279 (3)
1.238 (3)
1.329 (3)
1.365 (3)
1.328 (3)
1.449 (3)
1.504 (3)
1.389 (3)
1.371 (3)
1.443 (3)
1.410 (3)
1.481 (3)
2.339 (3)
1.933 (3)
C2-N1-C6
C7-N2-C8
N1-C2-C3
N1-C2-C1
C3-C2-C1
C4-C3-C2
O1-C4-C3
O1-C4-C5
C3-C4-C5
O2-C5-C6
O2-C5-C4
C6-C5-C4
N1-C6-C5
N1-C6-C7
C5-C6-C7
O3-C7-N2
O3-C7-C6
N2-C7-C6
123.4 (2)
121.4 (2)
118.7 (2)
118.3 (2)
122.9 (2)
120.7 (2)
119.7 (2)
119.2 (2)
121.1 (2)
125.1 (2)
120.0 (2)
114.9 (2)
121.1 (2)
114.9 (2)
124.0 (2)
123.2 (2)
121.3 (2)
115.6 (2)
C6-N1-C2-C3
0.3(4)
À176.4(2)
À1.0(4)
175.5(2)
À179.6(2)
À0.2(4)
2.1(4)
À177.2(2)
À178.5(2)
2.1(3)
À
O2 C5
C6-N1-C2-C1
N1-C2-C3-C4
C1-C2-C3-C4
C2-C3-C4-O1
C2-C3-C4-C5
O1-C4-C5-O2
C3-C4-C5-O2
O1-C4-C5-C6
C3-C4-C5-C6
C2-N1-C6-C5
C2-N1-C6-C7
O2-C5-C6-N1
C4-C5-C6-N1
O2-C5-C6-C7
C4-C5-C6-C7
C8-N2-C7-O3
C8-N2-C7-C6
N1-C6-C7-O3
C5-C6-C7-O3
N1-C6-C7-N2
C5-C6-C7-N2
À
O3 C7
À
N1 C2
À
N1 C6
À
N2 C7
À
N2 C8
À
C1 C2
À
C2 C3
À
C3 C4
À
C4 C5
1.8(4)
À
C5 C6
À178.3(2)
176.4(2)
À2.9(3)
À3.4(4)
177.3(2)
À0.2(4)
À179.4(2)
4.0(3)
À
C6 C7
À
O1H O2
À
N2H O2
À176.2(2)
À176.8(2)
3.1(3)
Table 4. Bond lengths [ꢂ], angles and selected torsion angles [o] of 3b.
Bond length
Angle
Torsion angle
À
O1 C4
1.2731 (14)
1.3483 (14)
1.2268 (14)
1.359 (2)
1.374 (2)
1.4773 (14)
1.330 (2)
1.455 (2)
1.497 (2)
1.372 (2)
1.414 (2)
1.436 (2)
1.367 (2)
1.510 (2)
2.358 (2)
C2-N1-C6
C2-N1-C9
C6-N1-C9
C7-N2-C8
N1-C2-C3
N1-C2-C1
C3-C2-C1
C4-C3-C2
O1-C4-C3
O1-C4-C5
C3-C4-C5
O2-C5-C6
O2-C5-C4
C6-C5-C4
N1-C6-C5
N1-C6-C7
C5-C6-C7
O3-C7-N2
O3-C7-C6
N2-C7-C6
119.91 (9)
120.46 (9)
119.62 (9)
121.68 (10)
119.56 (10)
118.99 (10)
121.45 (10)
123.49 (10)
124.09 (10)
121.31 (10)
114.59 (10)
118.37 (10)
121.10 (10)
120.54 (10)
121.87 (10)
118.25 (9)
119.73 (10)
125.61 (11)
119.41 (10)
114.98 (10)
C6-N1-C2-C3
C9-N1-C2-C3
C6-N1-C2-C1
C9-N1-C2-C1
N1-C2-C3-C4
C1-C2-C3-C4
C2-C3-C4-O1
C2-C3-C4-C5
O1-C4-C5-O2
C3-C4-C5-O2
O1-C4-C5-C6
C3-C4-C5-C6
O2-C5-C6-N1
C4-C5-C6-N1
O2-C5-C6-C7
C4-C5-C6-C7
C2-N1-C6-C5
C9-N1-C6-C5
C2-N1-C6-C7
C9-N1-C6-C7
C8-N2-C7-O3
C8-N2-C7-C6
C5-C6-C7-O3
N1-C6-C7-O3
C5-C6-C7-N2
N1-C6-C7-N2
1.16(16)
À177.67(10)
À178.42(10)
2.74(16)
À0.18(18)
179.39(10)
179.07(11)
À1.52(17)
2.20(17)
À177.23(10)
À178.24(10)
2.33(16)
178.05(10)
À1.53(17)
2.65(16)
À176.92(9)
À0.31(17)
178.54(10)
175.15(9)
À6.00(15)
À1.58(18)
178.49(9)
95.95(14)
À79.61(14)
À84.12(13)
100.32(12)
À
O2 C5
À
O3 C7
À
N1 C2
N1-C6
À
N1 C9
À
N2 C7
À
N2 C8
À
C1 C2
À
C2 C3
À
C3 C4
À
C4 C5
À
C5 C6
À
C6 C7
À
O2H O1
and Table 3). Thus, the amide substituent on the 2 position
is coplanar with the pyridine ring. This structural arrange-
of an electronegative function at the 2 position and the in-
tramolecular hydrogen bond, which are associated with the
uniquely high pFe3+ value (Table 1). Interestingly, the ex-
tremely efficient intramolecular hydrogen bond in 3 f also
reduces its ability to form hydrogen bonds with water mole-
cules, resulting in the surprising hydrophobic properties.
In contrast, the structure of 3b in the neutral state shows
that the oxygen in the 3 position (O2) is protonated and its
bond length with C5 is 1.3483 ꢂ, whereas the oxygen in the
À
ment results in a N2 O2 bond length of only 2.657 ꢂ, a
value that is similar to that reported for N,N’,N’’-tris((1,2-di-
dehydro-1-hydroxy-2-thioxopyrid-6-yl)-carbonyl)-2,2’,2’’-tri-
aminotriethylamine, 2.62 ꢂ,[16] which is indicative of a very
strong hydrogen-bond interaction between the amide NH
and the adjacent oxygen atom. Thus, the pKa values of 3 f
are probably influenced by two factors, namely, the presence
Chem. Eur. J. 2010, 16, 6374 – 6381
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6379

R. C. Hider et al.
4 position (O1) is ionised and forms a double bond with C4
with a length of 1.2731 ꢂ (Figure 2b and Table 4). This find-
ing confirms our conclusion that 3b is deprotonated through
the route 1 pathway. The amide functional group is almost
perpendicular to the heterocyclic ring with the torsion
angles of C5-C6-C7-O3, N1-C6-C7-O3, C5-C6-C7-N2 and
N1-C6-C7-N2 at approximately 908 (Table 4). This is attrib-
uted to the steric constraint between the N-methyl group at
position 1 and the amide O or NH at the 2 position. The dis-
Ligand 3 f was also found to be inferior to 3b in its ability
to scavenge iron
ACHTUNGTRENNUNG
450 mmolkgÀ1
;
150 mmolkgÀ1 both 3b and 3 f were found to be truly re-
markable in removing more iron than 2 at a dose of
450 mmol kgÀ1, thus confirming that chelators with high
pFe3+ values have the potential to remove iron
ficiently at lower ligand concentrations.
ACHTUNGTREN(NUNG III) more ef-
À
tance between N2 H2 and O2 is 3.24 ꢂ, which is much
longer than the sum of van der Waals radii (2.6 ꢂ).[17] There-
Conclusion
À
fore, it is unlikely that a hydrogen bond forms between N2
H2···O2. This finding agrees well with the conclusion ob-
tained from the spectroscopic studies.
In contrast to the two neutral forms of 3b and 3 f, which
show marked differences in structure, the bond lengths in
the two corresponding HCl salts are similar although the
amide group is only coplanar with the aromatic ring in 3 f.
There are two effects influencing the pFe3+ value of 3-hy-
droxypyridin-4-one chelators, the inductive effect induced
from positions 2 and 6 and the intramolecular hydrogen-
bond effect centred at position 2 (3 f–h). Both the 2- and 6-
amido derivatives have been demonstrated to possess such
properties, which lead to high pFe3+ values at pH 7.4. How-
ever, X-ray crystal-structure analyses have demonstrated
that intramolecular hydrogen-bond formation is not possible
for the 2-amido derivatives with an alkyl function at position
1, as with 3b, because of the bulk associated with the N-
alkyl group. Such intramolecular hydrogen-bond formation
is only possible when there is a hydrogen atom at position 1,
as in 3 f. Thus, 3 f benefits from both effects and the pKa2
value is heavily influenced by the presence of the hydrogen
bond. The high pFe3+ value has an appreciable effect on the
Iron mobilisation efficacies: The in vivo iron mobilisation
abilities of 3a, 3b and 3 f were compared with that of 2 in
the 59Fe ferritin-labelled rat model (Table 5). All the amide
Table 5. Iron mobilisation efficacy studies of 3a, 3b and 3 f in the 59Fe
ferritin-loaded rat model. All chelating ligands were given orally at the
corresponding dose and control rats were administered with an equiva-
lent volume of water. Values are expressed as meansÆSD (n=5; SD=
standard deviation).
speciation plot of the ironACTHNURGTNE(NUG III) as illustrated in Figure 3.
Dose at
Dose at
Dose at
450 mmolkgÀ1
% Fe mobilisa-
tion
300 mmolkgÀ1
% Fe mobilisa-
tion
150 mmolkgÀ1
% Fe mobilisa-
tion
Chelator
D7.4
control
2
3a
3b
3 f
–
3.87Æ1.0
13.4Æ5.2
35.0Æ9.8
58.7Æ10.9
50.1Æ9.6
3.87Æ1.0
9.2Æ2.2
–
3.87Æ1.0
6.3Æ2.1
–
0.17
0.16
0.04
0.17
54.1Æ14.1
33.1Æ6.8
41.4Æ2.3
25.3Æ3.4
derivatives were found to be more potent ironACTHNURTGNEU(GN III) scaveng-
ers than 2 when given at the dose of 450 mmolkgÀ1. This is
due to their higher pFe values and lower rate of metabolism
(unpublished data). Based on the physicochemical character-
istics (Table 1), 3 f might have been expected to be superior
to 3b for removing iron in vivo because 3 f is more hydro-
phobic (D7.4 =0.17 vs. 0.04) and possesses a higher pFe3+
value (22.8 vs. 21.7) than 3b. However, another limiting
factor for chelating ligands is their net charge at physiologi-
cal pH. Normally only the neutral uncharged form crosses
biological membranes at an appreciable rate. With 3b, over
85% of the total drug is in the non-ionised form (Table 1),
enabling 3b to penetrate membranes readily. In contrast, for
3 f, both pKa values are below 7.4 and, therefore, it is largely
ionised at pH 7.4 and only 16% is present as the zwitterion
form (HB2 in Scheme 5). As a result cell penetration is pre-
dicted to be reduced, and this probably contributes to its
lower iron-mobilisation efficacy.
Figure 3. Comparison of the iron complex speciation plots between 3 f
(c) and 2 (a); [iron]total =10À6 m and [ligand]total =10À5 m.
Under the conditions of the speciation study, 2 begins to dis-
sociate at pH 7.0 and at pH 4.5 the plot is dominated by the
2:1 ironACTHNUGRTNENUG(III) complex. In contrast, the 3:1 ironACHTUTGNREN(NUGN III) complex
of 3 f does not begin to dissociate until pH 5.5, that is,
beyond the pH range experienced within mammalian cells,
namely, pH 5.5–8.0. The difference in their pFe3+ values in-
dicates that 3 f binds ironACHTNUGRTNEUNG
(III) 103 times more tightly than 2
at pH 7.4. The range of pFe3+ values for the corresponding
2-(1’-hydroxyalkyl) series of pyridinones was found to be
20.4–21.5[10] and so none of these compounds were found to
bind ironACTHNUTRGNEUG(N III) as tightly as 3 f. This information demon-
6380
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Chem. Eur. J. 2010, 16, 6374 – 6381

Hydroxypyridinones as IronACHTNUTRGNE(NUG III) Ligands
FULL PAPER
strates that a high pFe3+ value bidentate compound can
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behave like a hexadentate ligand in that the 3:1 iron
complex is strongly favoured.
ACHTUNGTNER(NUNG III)
Despite the high pFe3+ value and distribution coefficient
values of 3 f, the iron mobilisation efficacy of 3 f was found
to be lower than that of 3b. This is almost certainly a result
of the dominant ionised form at pH 7.4. In contrast to 3 f,
the excellent ability of 3b in removing excess iron from the
liver demonstrates that it penetrates biological membranes
in an efficient manner.
Experimental Section
For synthetic details, analytical data, procedures for the determination of
physicochemical properties and in vivo studies, see the Supporting Infor-
mation.
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Acknowledgements
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The authors would like to thank Apotex Research Canada for supporting
the research project and The Royal Thai Government and the Faculty of
Pharmacy, Chiang Mai University, Chiang Mai, Thailand for a student-
ship to S. Piyamongkol. Also thanks to Dr D. R. Powell for completing
the crystallographic results. D. van der Helm acknowledges support from
the National Institutes of Health, GM21822.
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[1] R. R. Crichton, Inorganic Biochemistry of Iron Metabolism, West
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Received: September 4, 2009
Published online: April 15, 2010
Chem. Eur. J. 2010, 16, 6374 – 6381
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6381