Basic 3-hydroxypyridin-4-ones: Potential antimalarial agents

Article abstract of DOI:10.1016/j.ejmech.2007.07.011

3-Hydroxypyridin-4-ones selectively bind iron under biological conditions and one such compound has found application in the treatment of thalassaemia-linked iron overload. Related molecules have also been demonstrated to possess an antimalarial effect at levels which are non-toxic to mammalian cells. In an attempt to improve the efficiency of such molecules we have investigated the effect of introducing basic nitrogen centres into 3-hydroxypyridin-4-ones in an attempt to achieve targeting to lysosomes and other intracellular acidic vacuoles. Several of the compounds reported in this communication possess enhanced antimalarial activity over that of the simple hydroxypyridinone class.

Full text of DOI:10.1016/j.ejmech.2007.07.011

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1035e1047
http://www.elsevier.com/locate/ejmech
Original article
Basic 3-hydroxypyridin-4-ones: Potential antimalarial agents
Lotfollah S. Dehkordi a, Zu D. Liu b, Robert C. Hider b,
*
a School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran
b Department of Pharmacy, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
Received 7 December 2006; received in revised form 11 July 2007; accepted 12 July 2007
Available online 2 August 2007
Abstract
3-Hydroxypyridin-4-ones selectively bind iron under biological conditions and one such compound has found application in the treatment of
thalassaemia-linked iron overload. Related molecules have also been demonstrated to possess an antimalarial effect at levels which are non-toxic
to mammalian cells. In an attempt to improve the efficiency of such molecules we have investigated the effect of introducing basic nitrogen
centres into 3-hydroxypyridin-4-ones in an attempt to achieve targeting to lysosomes and other intracellular acidic vacuoles. Several of the com-
pounds reported in this communication possess enhanced antimalarial activity over that of the simple hydroxypyridinone class.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Hydroxypyridinones; Iron chelators; Malaria; Lysosomotrophic
1. Introduction
extracellular origin [6]. In fact the growth rate of the parasite
is independent of host iron status. When parasites invade eryth-
rocytes they generate a low molecular weight iron pool which
originates from haemoglobin digestion. The use of powerful
iron chelators could, in principle, limit the size of this pool
thereby reducing the rate of incorporation of iron into critical
metalloenzymes which can lead to parasite death [7]. It is clear
from previous studies that iron chelators have the capacity to
clear parasites [8,9].
3-Hydroxypyridin-4-ones(HPOs)havebeenextensivelystud-
ied as orally active iron(III) chelating agents [10] and have been
investigated for antimalarial activity [11,12]. Unfortunately,
many bidentate 3-hydroxypyridin-4-ones possessing good anti-
malarial activity are also toxic to mammalian cells due to their
relatively high lipophilicity [13]. In order to improve drug effi-
cacy and/or decrease drug toxicity, it will be necessary to design
chelators which can be efficiently and selectively delivered to the
malaria-infected erythrocyte.
Malaria is one of the most prevalent and devastating human
parasitic diseases. It is estimated that over 2000 million people,
living in more than 100 countries, are exposed to the risk of ma-
laria and that some 270 million of these are infected with malar-
ial parasites [1,2]. Annual deaths are estimated to approximately
2 million, the majority being children under the age of five. Due
to the increasing resistance of Plasmodium falciparum to cur-
rent antimalarial drugs such as the antifolate pyrimethamine
and the quinine family [3,4], the search for new chemotherapeu-
tic agents has become highly demanding. Iron chelation offers
one possible approach to control malaria infections [5].
Iron is an essential micronutrient required by the malaria
parasite for many biochemical reactions involved in growth
and multiplication. The iron source for P. falciparum is not of
Abbreviations: HPO, 3-hydroxypyridin-4-one; DR, distribution ratio;
MOPS, morpholinopropane sulphonic acid.
Studies by several investigators have demonstrated that in-
tracellular vesicles of the malaria parasite reach pH values in
the vicinity of 5.0 [14]. Estimates of red cell cytoplasmic and
parasite cytoplasmic pH are 7.0e7.2 and 6.8e7.0, respectively
[15]. 3-Hydroxypyridin-4-ones containing basic side chains are
* Corresponding author. Department of Pharmacy, King’s College London,
Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, UK.
Tel.: þ44 20 7848 6979; fax: þ44 20 7848 6394.
E-mail address: robert.hider@kcl.ac.uk (R.C. Hider).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.07.011

1036
L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
Table 1
The structure of 3-hydroxypyridin-4-ones
predicted to accumulate in such acidic organelles. It is assumed
that only the neutral form of weak base (L) can readily diffuse
across both plasma and vesicle membranes and that their con-
centrations are equal in all three compartments. In contrast
the protonated forms (LHþ) will not readily cross membranes
and so basic hydroxypyridinones will be accumulated in acidic
vesicles. Such an elevated concentration of chelator would be
predicted to increase the efficiency of iron scavenging, particu-
larly with bidentate ligands. Furthermore, the marked pH de-
pendence of haemoglobin digestion may provide an additive
inhibitory effect for basic iron chelators, as an accumulation
of such molecules will influence the vesicle pH value.
This concept led to the design and synthesis of a series of
basic hydroxypyridinones with pKa values covering the range
4.9e7.5 (4ae18) (Table 1). It was anticipated that an ideal
chelator for this purpose should have a pKa value in the range
of 6.0e8.0. Such molecules are almost fully protonated in acid
intracellular compartment (pH 5.5), whereas at the cytoplas-
mic pH value of 7 the neutral species make an appreciable
contribution. Thus, it would be expected that these ligands ac-
cumulate within acid intracellular compartments.
O
OH
R2
R6
N
R1
Compd.
R1
R2
R6
H
N
4a
CH3
N
N
CH2CH2CH2
CH2CH2CH2
N
4b
C2H5
H
4c
4d
4e
CH2CH2N(CH3)2
CH2CH2N(CH3)2
CH2CH2N(C2H5)2
CH3
C2H5
CH3
H
H
H
4f
CH2CH2
CH2CH2
CH2CH2
CH3
C2H5
CH3
H
N
N
N
4g
4h
9a
H
In this work we describe the synthesis, physico-chemical
properties and iron mobilization efficacy of a wide range of
novel bidentate 3-hydroxypyridin-4-ones containing different
basic substituents at pyridinone ring positions 1, 2 and 6. Pre-
liminarily in vitro antimalarial activities are also reported.
H
O
CH3
CH3
N
N
N
N
CH2
CH2
CH2
CH2
9b
CH3
CH3
CH3
CH3
O
CH3
2. Synthesis of basic hydroxypyridin-4-ones
9c
CH2OH
CH2OH
N
The 3-hydroxypyridin-4-one derivatives are conveniently
prepared from the corresponding 3-hydroxypyran-4-ones in
three steps through protection of hydroxyl group. The protected
compoundisthenreactedwitha primaryamine R1NH2, in which
R1 represents the group on the nitrogen atom of the desired
3-hydroxypyridin-4-ones, in the presence of base (Scheme 1)
[16,17]. Although the 3-hydroxy substituents of 3-hydroxy-
pyran-4-ones can also be protected by methyl ether formation
[18,19,20,21], the corresponding 2-alkyl-3-methoxy-4-pyrones
are oils which are less convenient to work with than the crystal-
line 2-alkyl-3-benzyloxy-4-pyrones [10,22,23]. Furthermore,
the benzyl protecting group can be removed by hydrogenation
under acidic, neutral or basic conditions. For these reasons the
benzyl group was selected for the work described in this paper.
The conversion of 4-pyrone to 4-pyridinone involves an ini-
tial Michael reaction followed by ring opening and ring clo-
sure. Mesomerisation of a,b-unsaturated carbonyl compound
causes the b-carbon to be electron deficient and therefore sus-
ceptible to nucleophilic attack. When the nucleophile is a pri-
mary amine, double attack at both a,b-unsaturated functions
of the 4(1H )-pyranone leads to the formation of 4H-pyridin-
4-one with the loss of a water molecule [24,25]. Conversion
of 3-hydroxypyran-4-one derivatives to the corresponding pyr-
idine-4-one analogues can be achieved without protection of
the 3-hydroxyl group [26]. However, the synthetic utility of
this reaction is limited to small primary amines since larger
amines result in yields less than 10% [27].
9d
O
H
H
13a
13b
18a
18b
CH2
N
CH2CH2
N CH2
CH2OH
H
H
N CH2CH2
CH(OH)CH3
N
CH2CH2
19a
19b
20
CH3
C2H5
C2H5
CH3
C2H5
H
H
H
CH(OH)CH3
The general methodology adopted for the synthesis of N-
basic substituted 3-hydroxypyridin-4-ones 4aeh is summarised
in Scheme 1 [10,28]. The commercially available maltol 1a or
ethyl maltol 1b was benzylated to give 2a and 2b. Reaction of
2a or 2b with primary amines gave the benzylated pyridinones
3aeh, which were subsequently subjected to catalytic hydroge-
nation to remove the protecting group, yielding the correspond-
ing bidentate chelators 4aeh as hydrochloride salts.

L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
1037
O
O
O
O
O
O
OH
R2
OBn
R2
OBn
R2
OH
R2
R1NH2
BnBr
H2 / Pd-C
N
N
R1
R1
1a: R2 = CH3
1b: R2 = C2H5
2a: R2 = CH3
2b: R2 = C2H5
3a-h
4a-h
Scheme 1. Synthesis of N-substituted 3-hydroxypyridin-4-ones.
The basic substituent can also be introduced at the 2-position
of allomaltol 5a or kojic acid 5b via a Mannich-type reaction,
using formaldehyde and a secondary amine in 95% aqueous eth-
anol at room temperature (Scheme 2). The desired 2-basic
substituted 3-hydroxypyridin-4-ones 9aed were then prepared
by the reaction of the corresponding benzylated pyranone 7aed
with methylamine, followed by acidic hydrolysis to remove the
protecting group (Scheme 2). The methodology adopted for in-
troduction of basic substituents at the 6-position (2-position of
the 5-hydroxypyridin-4-ones) is presented in Scheme 3. Chlori-
nation of protected kojic acid 10 in neat thionyl chloride below
0 ꢀC afforded benzylated chlorokojic acid 11. The chlorine was
then substituted by reaction with a secondary amine (piperi-
dine) in the presence of tributylamine in DMF. The reactions
of the protected pyranone 12 with an excess of primary amines
were performed by refluxing in 50% aqueous ethanol in the
presence of a catalytic amount of sodium hydroxide. After
acidic reflux the bidentate chelators 13a and 13b were isolated
as hydrochloride salts.
Two 2-(10-hydroxyalkyl)-N-basic substituted 3-hydroxypyr-
idin-4-ones 18a and 18b were also synthesised (Scheme 4). The
2-position of pyromeconic acid 14 can be functionalised in an
analogous fashion to the aldol condensation whereby the pyrone
anion aldehyde, under alkaline aqueous conditions, furnishes,
on acidic work up, the corresponding 2-(10-hydroxyalkyl)-3-
hydroxypyranone 15 in high yields (80e90%). The pH of the
reaction solution was found to be critical, since highly alkaline
conditions resulted in an extensive aldehyde polymerisation.
The optimal pH for this reaction, as adopted in this study, was
found to be 10.5. The protection of the 2-(10-hydroxyl) function
proved to be essential since without protection marked decom-
position was observed, which consequently resulted in low
yield (<10%). In this work, both the 3-hydroxyl and the
2-(10-hydroxyl) groups were protected in one step by reacting
the corresponding pyran-4-ones 15 with benzaldehyde dimethyl
acetal in N,N-dimethylformamide in the presence of a catalytic
amount of toluene-p-sulphonic acid. The desired pyridinone
products 18 were then prepared by the reaction of the protected
pyranone 16 with the 2-piperidinoethylamine, followed by hy-
drogenation to remove the protecting group. The introduction of
a 10-hydroxyalkyl group at the 2-position has been previously
found to produce appreciable increases in the pFe3þ values [29].
3. Results
3.1. Physico-chemical characterisation
3.1.1. pKa values of basic hydroxypyridinones and
their corresponding iron(III) complexes
All ligands were investigated by simultaneous spectrophoto-
metric and potentiometric titrations. Most of the basic 3-hydrox-
ypyridin-4-ones in this current work possess three pKa values.
The pKa1 values correspond to the protonation of the 4-hydroxy
group, the pKa2 values to the deprotonation of the basic substit-
uent and pKa3 to the dissociation of the 3-hydroxyl group
(Scheme 5). The only exception is 13b which has an additional
0
pKa2 associated with the second basic substituent (Scheme 5).
The optimized pKa values obtained from non-linear least-square
regression analysis [30] are shown in Table 2. The pKa values ob-
tained from spectrophotometric titration were in good agree-
ment with those calculated from the potentiometric titration.
The observed pKa values of the basic side chain cover the range
of 4.8e7.6.
3.1.2. Distribution and partition coefficients of
basic hydroxypyridinones
The distribution coefficients (D7.4) between 1-octanol and
MOPS (3-(N-morpholino)propanesulphonic acid) buffer (pH
7.4) were determined via the automated filter-probe system
[28]. As a result of ionization at pH 7.4, the fraction of the
O
O
O
O
O
O
O
O
OBn
N
OH
OH
OBn
N
OH
CH3NH2
6N HCl
HCHO
piperidine or
morpholine
BnBr
X
X
X
X
N
N
R6
N
CH3
R6
R6
N
CH3
R6
R6
5a: R6 = CH3
5b: R6 = CH2OH
6a: R6 = CH3; X = CH2
6b: R6 = CH3; X = O
7a-d
8a-d
9a-d
6c: R6 = CH2OH; X = CH2
6d: R6 = CH2OH; X = O
Scheme 2. Synthesis of 2-basic substituted 3-hydroxypyridin-4-ones (9aed).

1038
L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
O
O
O
O
O
SOCl2
1: R1NH2
2: 6N HCl
BnBr
piperidine
n-Bu3N
OH
OBn
OBn
OBn
OH
HO
HO
Cl
N
N
O
O
O
O
N
R1
5d
10
11
12
13a: R1 = CH3
N
13b: R1 = CH2CH2
Scheme 3. Synthesis of mono- and di-basic substituted 3-hydroxypyridin-4-ones (13a and 13b).
neutral species (Fn) can be calculated by employing the pKa
values determined above (Eq. (1)). The partition coefficient
(P) of each compound can then be calculated from Eq. (2)
and the resulting values are given in Table 3. The distribution
coefficients of the Fe-complexes (D7.4 Fe-complex) can be cal-
culated from Eq. (3) [28]. This equation holds for ligand
log D7.4 values > 0.1. For those ligands which possess
log D7.4 values < 0.1 (the more hydrophilic ligands), Eq. (4)
provides a superior estimate [28].
The cumulative constant b3 is the multiple of these three con-
stants. The optimized b3 values are presented in Table 4.
The pKa values of two selected Fe-complexes (9aeiron(III)
and 9beiron(III)) were also determined. Bidentate 3-hydroxy-
pyridin-4-ones form 3:1 iron(III) complexes at neutral and alka-
line pH values. Thus these 3:1 complexes possess three pKa
values, corresponding to the protonation of the basic side chain
at 2-position of each of the ligands bound to iron(III). The deter-
mined pKa values of 9aeiron(III) complex are 8.49, 7.14 and
5.21 whereas for the morpholine-containing 9beiron(III) com-
plex the corresponding values are 5.02, 4.47 and 4.01.
Neutral fractionðFnÞ
1
¼
ð1Þ
þ 10pK
þ 10pHꢁpK
a1þpKa2ꢁ2pH
1 þ 10pK
a2ꢁpH
a3
3.1.3. Biological evaluation
3.1.3.1. Iron mobilization efficacy of basic hydroxypyridinones.
The in vivo iron scavenging ability of the basic hydroxypyridi-
nones is compared with those of the dialkylpyridinones (Table
5). Several hydroxypyridinone derivatives such as 4a and 4b
were found to be relatively poor scavengers of iron under in
vivo conditions. However, the substituted 2-(10-hydroxyalkyl)
derivatives, 18a and 18b were found to be superior to the dia-
lkylpyridinones 19a and 19b, with efficacies close to 20%.
The ligands 4c and 4e were found to be as equally effective
as 19a. The most effective basic hydroxypyridinone was 4g
with an associated efficacy of 26.5%. All the basic compounds
were found to be less effective than the non-basic ligands 19b
and 20.
D7:4
Fn
a1þpKa2ꢁ2pH
¼ D7:4 ꢂ ð1 þ 10pK
a2ꢁpH
þ 10pK
þ 10pHꢁpK
Þ
a3
P ¼
ð2Þ
log D7:4 Fe-complex ¼ 2:53 log D7:4 ligand ꢁ 0:80
log D7:4 Fe-complex ¼ 0:49 log D7:4 ligand ꢁ 2:45
ð3Þ
ð4Þ
3.1.2.1. Stability constants of ligandeiron(III) complexes. Bi-
dentate 3-hydroxypyridin-ones are capable offorming a number
of complexes with iron(III) so that aqueous solutions equili-
brate to give a mixture of species, the composition of which de-
pends on the metal ion, ligand and hydrogen ion concentrations.
In this current work, the stepwise equilibrium constants (K1, K2
and K3) of selected basic 3-hydroxypyridinones were evaluated
using an automated spectrophotometric titration system [30].
3.1.3.2. In vitro antimalarial activity. The in vitro antimalarial
activity of the basic HPOs was compared with that associated
with pyridinones 19a, 19b and 20 (Table 5). All iron chelators
inhibited the growth of P. falciparum. The basic hydroxypyridi-
nones, 4aed with a mean IC50 around 80, were found to have the
O
O
O
O
O
O
N
O
N
OH
OH
O
R
Ph
OH
O
R
Ph
OH
OH
PhCH(OCH3)2
2-piperidinoethylamine
H2 / Pd-C
RCHO
O
O
O
R
R
15a: R = H
15b: R = CH3
N
N
16a: R = H
16b: R = CH3
14
17a: R = H
17b: R = CH3
18a-b
Scheme 4. Synthesis of 1-(20-piperidinoethyl)-2-(10-hydroxyalkyl)-3-hydroxypyridin-4(1H )-ones (18a and 18b).

L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
1039
OH
O
N
O
N
O
N
O
N
OH
OH
OH
OH
O-
pKa1
pKa2
pKa2
’
pKa3
+
+
+
NH
NH
N
N
N
N
NH
NH
NH
N
N
+
+
+
Scheme 5. Protonation equilibria of 1-(20-piperidinoethyl)-2-piperidinomethyl-5-hydroxypyridin-4(1H )-one (13b).
highest IC50 values in comparison with 19b and 20. Signifi-
cantly, several hydroxypyridinones such as 4e and 4h exhibited
similar activity to that of 19b and 20. The IC50 values of 18a and
18b were close to 10 mM, the most potent of the group and sig-
nificantly lower than the value associated with the analogous
non-basic ‘‘high pFe3þ’’compound 20.
at a sufficiently high rate. It is predicted that an ideal hydrox-
ypyridin-4-one chelator for this purpose should have a pKa2
value in the range of 6.0e8.0 (Fig. 1).
Distribution RatioðDRÞ
À
Á
Á
1 þ 10pK
þ 10pKa2ꢁpHB þ 10pHBꢁpKa3
þ 10pKa2ꢁpHA þ 10pHAꢁpKa3
a1þpKa2ꢁ2pHB
À
¼
ð5Þ
a1þpKa2ꢁ2pHA
1 þ 10pK
4. Discussion
In the current study, a wide range of bidentate hydroxypyridi-
none iron chelators with different basic substituents have been
synthesised. The pKa value of the basic function in the mole-
cule (pKa2) varies not only with the nature of the substituent
but also with the position of the substitution (Table 2). A par-
ticularly marked influence was noted between the pairs 9a and
13a, with pKa1 values of 2.73 and 2.16 and the pKa3 values of
10.28 and 8.86, respectively. Interestingly, the pKa3 values fall
either side of the typical values obtained with the dialkylpyr-
idinones 19a and 19b. The reason for these differences is
the existence of the electron withdrawal influence by the pos-
itive moiety at the 6-position in 13a which assists in the stabi-
lisation of the phenoxide anion, whereas with 9a there is
4.1. Physico-chemical properties of hydroxypyridinones
In order to design a basic compound with a useful acid
compartment accumulation effect, it is necessary to select op-
timal substituents. The distribution ratio (DR) (Table 3) of ba-
sic hydroxypyridinones between cytoplasm (pHA) and parasite
lysosome (pHB) can be calculated from Eq. (5). Although
compounds with pKa values above 8.0 have a greater chance
of accumulating efficiently within the organelle, the penetra-
tion rate of such compounds will be slower due to the high
proportion of ionized species present at pH 7.0. Ideally, drugs
designed for accumulating in acidic intracellular vesicles re-
lated to the parasite should not only possess an appreciable
distribution ratio but must also be able to penetrate the cell
Table 3
Distribution ratio, distribution coefficient values of ligands and their corre-
sponding iron(III) complexes between an aqueous phase buffered at pH 7.4
and 1-octanol
Table 2
Spectrophotometric and potentiometric determined pKa values for ligands
Compd.
pKas (potentiometric)
pKas (spectrophotometric)
Compd.
D7.4 ligand
(n ¼ 5)
Fn
(pH 7.4)
log P
ligand
D7.4
Fe-complex
DR
pKa1
pKa2
pKa3
pKa1
pKa2
pKa3
4a
4b
0.344 ꢃ 0.005
0.420 ꢃ 0.019
0.154 ꢃ 0.005
0.538 ꢃ 0.047
0.493 ꢃ 0.005
0.960 ꢃ 0.029
4.591 ꢃ 0.031
0.122 ꢃ 0.001
2.536 ꢃ 0.012
0.364 ꢃ 0.003
1.636 ꢃ 0.059
0.170 ꢃ 0.001
3.493 ꢃ 0.056
26.11 ꢃ 0.450
0.501 ꢃ 0.018
0.908 ꢃ 0.023
0.170 ꢃ 0.010
1.790 ꢃ 0.010
0.266 ꢃ 0.002
0.85
0.89
0.70
0.70
0.43
0.47
0.52
0.99
0.70
0.99
0.83
0.99
0.80
0.38
0.48
0.52
0.99
0.99
0.96
ꢁ0.392
ꢁ0.326
ꢁ0.657
ꢁ0.114
0.059
0.012
0.018
0.001
0.033
0.027
0.143
7.493
0.001
1.670
0.012
0.551
0.002
3.752
608.8
0.028
0.124
0.003
0.240
0.006
31.8
4a
4b
3.08
3.15
2.86
2.68
2.66
2.88
2.74
2.64
2.73
2.43
2.41
2.14
2.16
2.04
2.20
2.14
3.68
3.81
3.03
6.71
6.47
7.02
7.16
7.50
7.47
7.38
4.91
6.98
4.82
6.67
4.99
6.72
5.57; 7.60
7.43
7.35
e
9.82
9.66
9.58
9.58
9.60
9.71
9.70
9.68
10.28
9.72
10.38
9.75
8.86
8.95
9.24
8.73
9.77
9.93
8.77
3.13
3.09
2.74
2.62
2.63
2.77
2.73
2.67
2.50
2.38
2.14
1.98
2.15
1.16
2.25
2.17
3.56
6.60
6.55
7.05
6.88
7.53
7.42
7.34
4.89
7.07
4.89
6.74
5.10
6.74
5.66; 7.58
7.35
7.37
e
9.92
9.71
9.75
9.66
9.65
9.63
9.67
9.71
10.81
10.07
10.51
9.77
8.85
8.83
9.28
8.70
9.64
25.2
52.5
51.6
76.8
73.9
69.9
4c
4d
4c
4d
4e
4e
4f
4g
0.310
0.946
4f
4g
4h
9a
ꢁ0.909
0.559
1.92
51.8
1.76
36.2
1.95
4h
9a
9b
9c
ꢁ0.435
0.295
9b
9c
9d
ꢁ0.765
0.640
1.837
9d
13a
13b
18a
18b
19a
19b
20
34.2
424.7
71.3
69.9
1.0
13a
13b
18a
18b
19a
19b
20
0.019
0.242
ꢁ0.770
0.257
1.0
1.0
e
e
ꢁ0.557
3.11
e
8.74

1040
L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
Table 4
Hydroxypyridinone affinity constants for Fe(III)
pH 5/pH 7
100
80
60
40
20
0
1
0.8
0.6
0.4
0.2
0
Compd.
log b3
pFe3þ
(at pH 7.45)
9a
9b
38.0
38.0
38.6
38.6
35.7
35.7
35.3
36.5
36.7
35.4
19.0
19.0
20.0
19.9
21.9
22.5
20.4
19.4
19.7
21.3
9c
9d
13a
13b
18a
19a
19b
20
strong hydrogen bonding possible with the 2-substituent which
has the opposite influence of stabilising the protonated pheno-
lic form 21. In supporting this hypothesis, 9c has a similar
pKa3 value to that of 9a and 13b has a similar pKa3 value to
that of 13a. The pKa2 values for the aliphatic amine side chains
at positions 1, 2 and 6 are all markedly lower than that of the
corresponding free amines. This results from the electron de-
ficient pyridinone ring which exerts an electron withdrawing
effect on the side chains, irrespective of the position. As a re-
sult of electron withdrawal associated with the morpholine
ring oxygen, the pKa2 values of the compounds 4h, 9b and
9d are lower than that of the corresponding piperidine-contain-
ing compounds, 4g, 9a and 9c.
0
1
2
3
4
5
6
7
8
9
10 11 12 13
pKa (Basic group)
Fig. 1. The relationship between the pKa value of hydroxypyridinones and (B)
distribution ration (DR) between two phases at pH values 5 and 7 and (C) the
fraction of non-ionized species present at pH 7.0.
substitution, the pKa2 values for 18a and 4g being 7.35 and
7.38, respectively.
O
-
O
H
O
O
The introduction of a hydroxyl function on the 2-substitu-
ent (18b) is associated with a decrease in the pKa3 value;
8.73 as compared with 9.70 of 4g. This is due to the favourable
hydrogen bonding with the phenoxide anion 22 [29]. Not sur-
prisingly, the pKa2 value is largely unaffected by this
O
N
H
N
CH3
N
H3C
N
CH3
21
22
Table 5
In vitro antimalarial activities and iron mobilization efficacy of basic
hydroxypyridinones
The ligands reported in this study cover a wide range of dis-
tribution coefficients (0.1e26.0, Table 3). As all the D7.4 values
exceeded 0.1, it was anticipated that the compounds would be
orally active and some (D7.4 > 1.0) may well be susceptible
to efficient first-pass extraction by the liver. The calculated
D7.4 values of the (3:1) iron(III) complexes covered the range
0.001e26 (Table 3). In most cases the iron(III) complexes are
predicted to be more hydrophilic than their corresponding
free ligands. However, this trend does not hold for compounds
which have D7.4 values greater than 3 (4g, 13a and 13b). In
these cases the iron(III) complexes were predicted to be more
hydrophobic than their corresponding free ligands [28].
With regards to chelation ofiron(III), a suitable comparator is
the pFe3þ value, defined as the negativelogarithm of the concen-
tration of the free iron(III) in solution under defined conditions.
Typically pFe3þ values are calculated for [ligand]total ¼ 10ꢁ5 M
and [iron]total ¼ 10ꢁ6 M at pH 7.4. Comparison of ligands using
this parameter is useful, since the pFe3þ value, unlike the corre-
sponding stability constant, takes into account the effects of li-
gand basicity, denticity and degree of protonation, as well as
differences in metaleligand stoichiometries [29]. Chelators
with high pFe3þ values are predicted not only to scavenge iron
Compd.
IC50 (mM)
Iron
mobilization (%)
Efficacy of iron
removal (%)
Control
4a
4b
e
3.9 ꢃ 1.0
5.3 ꢃ 1.9
6.8 ꢃ 1.5
13.0 ꢃ 2.5
20.3 ꢃ 6.8
12.4 ꢃ 1.9
22.7 ꢃ 3.3
30.4 ꢃ 5.3
e
0.0
1.4
2.9
9.1
16.4
8.5
18.8
26.5
e
85
65
80
85
50
35
25
50
23
50
29
35
31
21.5
10
8
4c
4d
4e
4f
4g
4h
9a
e
e
9b
9c
e
e
e
e
9d
e
e
13a
13b
18a
18b
19a
19b
20
e
e
e
e
23.3 ꢃ 3.4
23.1 ꢃ 3.9
13.4 ꢃ 5.2
58.3 ꢃ 9.4
48.4 ꢃ 7.2
19.4
19.2
9.5
54.4
44.5
67.5
55
45

L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
1041
more effectively at low ligand concentrations, but also to disso-
ciate less readily and therefore form lower concentrations of the
partially coordinated complexes. The compounds of this study
(Table 4) fall into two broad classes: one with pFe3þ values < 20
(which are similar to the parent 19a) and the other group, with
pFe3þ values > 20. It has been previously established that the in-
troduction of an 1-hydroxyalkyl group at the 2-position of 3-hy-
droxypyridinones reduces the pKa values of the ligand and hence
increases the pFe3þ values, due to the formation of a stable intra-
molecular hydrogen bond between the 2-(10-hydroxyl) group
and the adjacent 3-hydroxyl function, as indicated in 22 [29].
The introduction of an aliphatic amine group at the 6-position
also leads to a marked reduction of pKa1 and pKa3 values and
therefore to a significant improvement in the pFe3þ values, for
example 13a and 13b (Table 4). This effect was found to be
marginally greater with the di-substituted compound (13b,
pFe3þ ¼ 22.5) than with the mono-substituted compound
(13a, pFe3þ ¼ 21.9). The compound 13a has a pFe3þ value
which is in the region of the three orders magnitude larger
than that of 19a (deferiprone). For the strongly basic chelators
such as 9a the 3:1 neutral complex was found to be the dominant
species over the pH range 6.0e10.0. Thus at the cytoplasmic pH
value of 7.0, the 3:1 iron(III) complex of 9a forms a mixture of
the monopositive and dipositive charged species. In contrast at
pH 5.5 the dominant species is the dipositive cation. Thus al-
though the free ligand is predicted to be accumulated in acidic
intracellular compartments (Fig. 1), if it chelates iron(III) within
such a compartment it will form a highly charged iron complex
which is unlikely to partition through membranes. In contrast,
the pKa values of the morpholine-containing iron complex
(9b)3$Fe(III) remain largely uncharged at both pH 5.5 and 7.0.
enhance the ability of a pyridinone to scavenge iron under in
vivo conditions. This finding is likely to be associated with
the formation of the highly charged iron complexes in acidic in-
tracellular compartments, such as the lysosome.
The data obtained, from the in vitro antimalarial study, in-
dicate that all of the tested iron chelators inhibit the growth of
the parasites (Table 5). The hydrophobic basic chelators, such
as 4g, 9a, 13a and 13b, were more active against P. falciparum
than the dialkyl hydroxypyridinones (19a and 19b). In con-
trast, the hydrophilic and imidazole-containing molecules
(4a, 4b, 4c and 4d) demonstrated a weak inhibitory action.
In addition to lipophilicity, it is clear that the basic strength of
3-hydroxypyridinones is an important factor for antimalarial ac-
tivity. For instance, the IC50 values of the morpholine-containing
ring compounds, 4h and 9b, are close to those corresponding to
theneutral 3-hydroxypyridinoneswhencomparedwiththecorre-
sponding piperidine-containing ring ligands 4g and 9a (Table 5).
Interestingly, the basic 2-(10-hydroxyethyl)-3-hydroxypyridi-
none18bismoreactiveagainstmalarialparasitesthantheneutral
analogues such as 20, although it is not as effective in iron re-
moval. Compound 18a is predicted to be strongly accumulated
in acidic organelles, but will form highly charged iron complexes
which will not readily escape from the organelle. It appears that
this accumulation effect has a dominant influence on antimalarial
properties. The iron chelator 13b which possesses two basic side
chains has the highest pFe3þ value and log P value of the entire
group, and yet is not as effective as an antimalarial when com-
pared with the monobasic hydrophilic chelators, 18a and 18b.
This is probably due to its high net charge which renders it less
able to penetrate membranes by non-facilitated diffusion.
The comparison of the basic pyridinones highlights 18a and
18b as the most promising candidates for further investigation.
These two compounds apparently possess suitable partition co-
efficients to facilitate their penetration of intracellular organelles
and yet are sufficiently hydrophilic to lack the general toxicity
associated with more lipophilic hydroxypyridinones [13].
4.2. Biological properties of hydroxypyridinones
The in vivo iron mobilization efficacy of selected ligands was
evaluated in a non-iron overloaded rat model [31]. [59Fe]ferritin
was used to label the liver iron pool, and this was followed by
a challenge with a test chelator at a time when the iron released
by lysosomal degradation of ferritin was maximally available
[31,32]. This [59Fe]ferritin-loaded rat model can be used to as-
sess oral bioavailability and to compare the ability of chelators
to remove iron from liver. Many of the basic hydroxypyridinones
led to superior iron excretion via the bile as compared to deferi-
prone (19a) (Table 5). There was no obvious correlation between
efficacy and the pKa2 value of the side chain, although those with
pKa2 values > 7.0 tended to be more efficient than those with
pKa2 values < 7.0. The imidazole-containing molecules were
much less effective than the tertiary amine derivatives. Compar-
ison of the tertiary amino-containing pyridinones indicates that
4g is worthy of further investigation. It is likely that this high rel-
ative efficacy is associated with the high log P value (0.943),
which would be predicted to favour efficient extraction by the
liver.
5. Experimental section
5.1. Materials and general procedures
Maltol and ethyl maltol were purchased from Pfizer (Widnes,
UK). All other chemicals were obtained from Aldrich Chemical
Co. (Gillingham, UK). Melting points were determined using an
Electrothermal IA 9100 Digital Melting Point Apparatus and
1
are uncorrected. H NMR spectra were recorded using a Per-
kineElmer (60 MHz) NMR spectrometer. Chemical shifts (d)
are reported in parts per million downfield from the internal
standard tetramethylsilane (TMS). Mass spectra (EI) were re-
corded on a Joel AX505W. Elemental analyses were performed
by Micro Analytical Laboratories, Department of Chemistry,
The University of Manchester, Manchester M13 9PL.
Surprisingly thebasic pyridinones which also possessed a hy-
droxylated side chain on the 2-substituent (18a and 18b) were
found to be less effective than the non-basic analogue 20.
Clearly the introduction of a basic function does not necessarily
Compounds 19a (1,2-dimethyl-3-hydroxypyridin-4-one),
19b (1,2-diethyl-3-hydroxypyridin-4-one) and 20 (1-ethyl-2-
(10-hydroxyethyl)-3-hydroxypyridin-4-one) were synthesised
from maltol 1a, ethyl maltol 1a and pyromeconic acid 14,

1042
L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
respectively, as previously described [10,29]. 3-Benzyloxy-2-
methylpyran-4(1H )-one (2a) and 3-benzyloxy-2-ethylpyran-
4(1H )-one (2b) were synthesised from maltol 1a and ethyl
maltol 1b, respectively, following the methodology as de-
scribed by Dobbin et al. [10].
C10H18N2O2Cl2 (C, H, N were within 0.4% of the theoretical
values).
5.2.4. 1-[20-(Dimethylamino)ethyl]-2-ethyl-3-
hydroxypyridin-4(1H )-one dihydrochloride (4d)
Yield 52%; mp 234e236 ꢀC; dH (60 MHz; D2O; Me4Si) 1.28
(3H, t, 2-CH2CH3), 2.75e3.3 (2H, q, 2-CH2CH3), 3.05 (6H, s,
N(CH3)2), 3.5e3.9 (2H, m, NeCH2CH2N(CH3)2), 4.74 (t, Ne
CH2CH2N(CH3)2), 7.15 (2H, d, 1H, 5-H(pyridinone)), 8.1 (d,
6-H(pyridinone)). Analysis C11H20N2O2Cl2$H2O (C, H, N
were within 0.4% of the theoretical values).
5.2. General procedure for the preparation of N-basic
substituted 3-hydroxypyridin-4-ones 4aeh
To a solution of (2a) or (2b) (20 mmol, 1 eq.) in ethanol (50
mL)/water (50 mL) was added the selected amine (30 mmol,
1.5 eq.) followed by 2 N sodium hydroxide solution until pH
13.5 was obtained. The mixture was then refluxed for 12 h. After
adjustment to pH 1 with concentrated hydrochloric acid, the sol-
ventwasremovedbyrotaryevaporationpriortoadditionofwater
(50 mL) and washing with diethyl ether (2 ꢂ 50 mL). Subse-
quent adjustment of the aqueous fraction to pH 9 with 10 N
sodium hydroxide solution was followed by extraction into
dichloromethane (4 ꢂ 50 mL). The combined organic layers
were dried over anhydrous sodium sulphate, filtered, and rotary
evaporated to give the benzylated hydroxypyridinones 3aeh
(Scheme 1) as a yellow oil, which were subsequently dissolved
in ethanol (90 mL)/water (10 mL) and subjected to hydrogenol-
ysis in the presence of 5% Pd/C (5e10% w/w of the compound)
catalyst for 2 h. Following the filtration, the pH of the solution
was adjusted to 1 using concentrated hydrochloric acid and the
solvent was removed in vacuo to yield the crude product. Recrys-
tallization from methanol/diethyl ether gave 4aeh as a white or
yellow solid.
5.2.5. 1-[20-(Diethylamino)ethyl]-3-hydroxy-2-
methylpyridin-4(1H )-one dihydrochloride (4e)
Yield 85.5%; mp 244e247 ꢀC (dec.); dH (360 MHz; DMSO-
d6; Me4Si) 1.26 (6H, t, J ¼ 7.2 Hz, N(CH2CH3)2), 2.6 (3H, s, 2-
CH3), 2.9e3.8 (6H, m, N(CH2CH3)2 and NeCH2CH2N(C2H5)2),
4.9 (2H, t, J ¼ 7.9 Hz, NeCH2CH2N(C2H5)2), 7.4 (1H, d, J ¼
7.0 Hz, 5-H(pyridinone)), 8.45 (1H, d, J ¼ 7.0 Hz, 6-H(pyridi-
none)), 9.1e10.4 (3H, br, OH and NH ). Analysis C12H22N2O2Cl2
(C, H, N were within 0.4% of the theoretical values).
5.2.6. 3-Hydroxy-2-methyl-1-(20-piperidinoethyl)-
pyridin-4(1H )-one dihydrochloride (4f)
Yield 75.6%; mp 213e215 ꢀC; dH (60 MHz; D2O; Me4Si)
1.3e2.4 (6H, m, br, eCH2CH2CH2e(piperidine ring)), 2.64
(3H, s, 2-CH3), 2.7e4.2 (6H, m, eCH2eNeCH2ꢁ(piperidine
ring) and (pyridinone)NeCH2CH2N(piperidine)), 4.8 (2H, t,
(pyridinone)NeCH2CH2Ne(piperidine)), 7.18 (1H, d, 5-H(pyr-
idinone)), 8.18 (1H, d, 6-H(pyridinone)). Analysis C13H22N2O2
Cl2$H2O (C, H, N were within 0.4% of the theoretical values).
5.2.1. 3-Hydroxy-1-[30-(imidazol-1-yl)propyl]-2-
methylpyridin-4(1H )-one dihydrochloride (4a)
Yield 69%; mp 160e161 ꢀC; dH (360 MHz; DMSO-d6;
Me4Si) 2.2e2.75 (2H, m, CH2CH2CH2), 2.6 (3H, s, 2-CH3),
4.1e4.8 (4H, m, CH2CH2CH2), 7.4 (1H, dd, J ¼ 2.6, 6.7 Hz, 5-
H(pyridinone)), 7.7 (1H, t, J ¼ 1.6 Hz, 5-H(imidazole)), 7.92
(1H, d, J ¼ 1.5 Hz, 6-H(imidazole)), 8.4 (1H, dd, J ¼ 2.4,
6.8 Hz, 6-H(pyridinone)), 9.38 (1H, s, 2-H(imidazole)), 6.1e
8.8 (3H, br, OH and NH ). Analysis C12H17N3O2Cl2$H2O (C,
H, N were within 0.4% of the theoretical values).
5.2.7. 2-Ethyl-3-hydroxy-1-(20-piperidinoethyl)-
pyridin-4(1H )-one dihydrochloride (4g)
Yield 65%; mp 207e209 ꢀC; dH (60 MHz; D2O; Me4Si)
1.22 (3H, t, 2-CH2CH3), 1.4e2.5 (6H, m, br, eCH2CH2
CH2e(piperidine ring)), 3.0 (2H, q, 2-CH2CH3), 3.1e3.9
(6H, m, eCH2eNeCH2-(piperidine ring) and (pyridinone)N
eCH2CH2N(piperidine)), 4.8 (2H, t, (pyridinone)Ne
CH2CH2N(piperidine)), 7.18 (1H, d, 5-H(pyridinone)), 8.18
(1H, d, 6-H(pyridinone)). Analysis C14H24N2O2Cl2$H2O (C,
H, N were within 0.4% of the theoretical values).
5.2.2. 2-Ethyl-3-hydroxy-1-[30-(imidazol-1-yl)propyl]-
pyridin-4(1H )-one dihydrochloride (4b)
Yield59%; mp189e191 ꢀC;dH (60 MHz;DMSO-d6; Me4Si)
1.03 (3H, t, 2-CH2CH3), 1.85e2.55 (2H, m, CH2CH2CH2), 2.8
(2H, q, 2-CH2CH3), 3.9e4.8 (4H, m, CH2CH2CH2), 7.4 (1H,
d, 5-H(pyridinone)), 7.65 (1H, d, 5-H(imidazole)), 7.9 (1H, d,
6-H(imidazole)), 8.35 (1H, d, 6-H(pyridinone)), 9.35 (1H, s, 2-
H(imidazole)). Analysis C13H19N3O2Cl2 (C, H, N were within
0.4% of the theoretical values).
5.2.8. 3-Hydroxy-2-methyl-1-(20-morpholinoethyl)-
pyridin-4(1H )-one dihydrochloride (4h)
Yield 69.7%; mp 214e216 ꢀC; dH (360 MHz; D2O; Me4Si)
2.65 (3H, s, 2-CH3), 3.4e4.2 (12H, m, eCH2OCH2e and
eCH2eNeCH2e(morpholine ring) and CH2CH2), 7.1 (1H,
d, J ¼ 7.1 Hz, 5-H(pyridinone)), 8.1 (1H, d, J ¼ 7.1 Hz, 6-
H(pyridinone)). Analysis C12H20N2O3Cl2 (C, H, N were
within 0.4% of the theoretical values).
5.2.3. 1-[20-(Dimethylamino)ethyl]-3-hydroxy-2-
methylpyridin-4(1H )-one dihydrochloride (4c)
Yield 65.7%; mp 251e252 ꢀC; dH (60 MHz; D2O; Me4Si)
2.58(3H, s, 2-CH3), 3.0 (6H, s, N(CH3)2), 3.4e3.9 (2H, m, Ne
CH2CH2N(CH3)2), 4.75 (2H, t, NeCH2CH2N(CH3)2), 7.1 (1H,
d, 5-H(pyridinone)), 8.1 (1H, d, 6-H(pyridinone)). Analysis
5-Hydroxy-2-methylpyran-4(1H )-one (allomaltol) (5a) was
synthesised from kojic acid (5b) in two steps using the estab-
lished procedure [28].

L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
1043
5.3. General procedure for the preparation of 2-basic
substituted 3-hydroxypyran-4(1H )-one 6aed
rotary evaporation prior to addition of water (50 mL) and washed
with diethyl ether (2 ꢂ 50 mL). Subsequent adjustment of the
aqueous fraction to pH 9 with 10 N sodium hydroxide solution
was followed by extraction into dichloromethane (4 ꢂ 50 mL),
the combined organic layers then being dried over anhydrous so-
dium sulphate, filtered, and rotary evaporated to give the benzy-
lated pyridinones 8aed (Scheme 2), which were then dissolved
in 6 N hydrochloric acid (40 mL) and refluxed for 4 h. After
removal of the solvent by rotary evaporation, the residue was
purified by recrystallization from methanol/diethyl ether to
afford 9aed as a white solid.
To a solution of piperidine or morpholine (40 mmol) and
40% aqueous formaldehyde (3 mL) in 95% ethanol (40 mL)
was slowly added 5a or 5b (30 mmol). The mixture was stirred
for 1 h and then cooled and refrigerated. The resulting crystals
were collected by suction filtration and washed with ethanol
and diethyl ether to give 6aed as a white or yellow crystalline
solid.
5.3.1. 3-Hydroxy-6-methyl-2-piperidinomethylpyran-4(1H )-
one (6a)
Yield 74%; mp 146e148 ꢀC; dH (60 MHz; DMSO-d6;
Me4Si) 1.1e1.6 (6H, s, br, eCH2CH2CH2e(piperidine ring)),
2.1e2.4 (7H, m, 6-CH3 and eCH2eNeCH2e(piperidine
ring)), 3.3 (2H, s, 2-CH2), 6.1 (1H, s, 5-H(pyranone)).
5.4.1. 1,6-Dimethyl-3-hydroxy-2-piperidinomethylpyridin-
4(1H )-one dihydrochloride (9a)
Yield 54%; mp 186e188 ꢀC; dH (60 MHz; D2O; Me4Si)
1.2e2.2 (6H, m, br, eCH2CH2CH2e(piperidine ring)), 2.5
(3H, s, 6-CH3), 3.0e3.5 (6H, m, eCH2eNeCH2e(piperidine
ring) and 2-CH2), 3.95 (3H, s, NeCH3), 7.2 (1H, s, 5-H(pyridi-
none)). Analysis C13H22N2O2Cl2$H2O (C, H, N were within
0.4% of the theoretical values).
5.3.2. 3-Hydroxy-6-methyl-2-morpholinomethylpyran-4(1H )-
one (6b)
Yield 79%; mp 132e136 ꢀC; dH (60 MHz; DMSO-d6;
Me4Si) 2.2e3.0 (7H, m, eCH2eNeCH2e(morpholine ring)
and 6-CH3), 3.5e4.0 (6H, m, eCH2OCH2e(morpholine ring)
and 2-CH2), 6.4 (1H, s, 5-H(pyranone)).
5.4.2. 1,6-Dimethyl-3-hydroxy-2-morpholinomethylpyridin-
4(1H )-one dihydrochloride (9b)
Yield 47%; mp 196e200 ꢀC; dH (60 MHz; D2O; Me4Si) 2.5
(3H, s, 6-CH3), 3.1e3.6 (4H, m, eCH2eNeCH2e(morpholine
ring)), 3.7e4.0 (7H, m, eCH2OCH2e(morpholine ring) and
NeCH3), 4.7 (2H, s, 2-CH2), 6.95 (1H, s, 5-H(pyridinone)).
Analysis C12H20N2O3Cl2 (C, H, N were within 0.4% of the the-
oretical values).
5.3.3. 3-Hydroxy-6-hydroxymethyl-2-
piperidinomethylpyran-4(1H )-one (6c)
Yield82%; mp163e165 ꢀC;dH (60 MHz;DMSO-d6; Me4Si)
1.3e1.7 (6H, m, br, eCH2CH2CH2e(piperidine ring)), 2.2e2.6
(4H, m, br, eCH2eNeCH2e(piperidine ring)), 3.4 (2H, s,
2-CH2), 4.2 (2H, s, 6-CH2OH), 6.2 (1H, s, 5-H(pyranone)).
5.4.3. 3-Hydroxy-6-hydroxymethyl-1-methyl-2-
piperidinomethylpyridin-4(1H )-one dihydrochloride (9c)
Yield 34%; mp 194e197 ꢀC; dH (60 MHz; D2O; Me4Si) 1.5e
2.1 (6H, m, br, eCH2CH2CH2e(piperidine ring)), 3.1e3.75 (4H,
m, eCH2eNeCH2e(piperidine ring)), 3.9 (3H, s, NeCH3), 4.65
(2H, s, 2-CH2), 4.75 (2H, s, 6-CH2OH), 7.2 (1H, s, 5-H(pyridi-
none)). Analysis C13H22N2O3Cl2$1/2H2O (C, H, N were within
0.4% of the theoretical values).
5.3.4. 3-Hydroxy-6-hydroxymethyl-2-
morpholinomethylpyran-4(1H )-one (6d)
Yield88%; mp156e158 ꢀC;dH (60 MHz;DMSO-d6; Me4Si)
2.6e32.8 (4H, m, eCH2eNeCH2e(morpholine ring)), 3.6e3.9
(6H, m, eCH2OCH2e(morpholine ring) and 2-CH2), 4.5 (2H, s,
6-CH2OH), 6.5 (1H, s, 5-H(pyranone)).
5.4. General procedure for the preparation of 2-basic
5.4.4. 3-Hydroxy-6-hydroxymethyl-1-methyl-2-
substituted 3-hydroxypyridin-4-ones 9aed
morpholinomethylpyridin-4(1H )-one dihydrochloride (9d)
Yield 37%; mp 190e1940 ꢀC; dH (60 MHz; D2O; Me4Si)
3.2e3.7 (4H, m, eCH2eNeCH2e(morpholine ring)), 3.75e
4.1 (7H, m, eCH2OCH2e(morpholine ring) and NeCH3), 4.7
(4H, s, 2-CH2 and 6-CH2OH), 7.15 (1H, s, 5-H(pyridinone)).
Analysis C12H20N2O4Cl2 (C, H, N were within 0.4% of the
theoretical values).
To a solution of 6aed (20 mmol, 1 eq.) in methanol (50 mL)
was added sodium hydroxide (25 mol, 1.25 eq.) dissolved in wa-
ter (5 mL) and the mixture was heated to reflux. Benzyl bromide
(25 mol, 1.25 eq.) was added dropwise over 30 min and then re-
fluxed for6 h. After removal of solventbyrotary evaporation, the
residue was mixed with water (50 mL) and extracted with di-
chloromethane (3 ꢂ 50 mL). The combined organic fraction
was washed with 5% aqueous sodium hydroxide (2 ꢂ 50 mL)
and water (2 ꢂ 50 mL), dried over anhydrous sodium sulphate,
filtered and rotary evaporated to yield the benzylated pyranones
7aed (Scheme 2), which were subsequently dissolved in ethanol
(10 mL) followed by addition of 40% aqueous methylamine
(10 mL). The reaction mixturewas sealed in a thick-walled glass
tube and stirred at 70 ꢀC for 12 h. After adjustment to pH 1 with
concentrated hydrochloric acid, the solvent was removed by
5.4.5. 2-Chloromethyl-5-benzyloxypyran-4(1H )-one (11)
To a well stirred thionyl chloride (50 mL) at 0 ꢀC was added
4.92 g (20 mmol) of benzylated kojic acid 10 and the reaction
was stirred at 0 ꢀC for 2 h. The mixture was then poured into
ice and extracted with chloroform (3 ꢂ 100 mL). The extracts
were combined and washed successively with saturated sodium
hydrogen carbonate solution and brine. After drying over anhy-
drous sodium sulphate, the solvent was removed to give the crude
product. Recrystallizationfromdichloromethane/petroleumether

1044
L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
(40e60 ꢀC) afforded a white solid (4.13 g, 77%); mp 97e99 ꢀC;
dH (60 MHz; DMSO-d6; Me4Si) 4.3 (2H, d, 2-CH2Cl), 5.1 (2H, s,
CH2Ph), 6.4 (1H, s, 3-H(pyranone)), 7.3 (5H, s, Ph) 7.5 (1H, s,
6-H(pyranone)). Analysis C13H9O4Cl (C, H, Cl were within
0.4% of the theoretical values).
6 N hydrochloric acid (40 mL) and refluxed for 4 h. After re-
moval of the solvent by rotary evaporation, the residue was puri-
fied by recrystallization from methanol/diethyl ether to afford
13b as a yellow solid (1.2 g, 28%); mp 190e194 ꢀC; dH (60
MHz; D2O; Me4Si) 1.4e2.0 (14H, m, br, 2 ꢂ eCH2CH2CH2e
(piperidine ring) and (pyridinone)NeCH2CH2N(piperidine)),
3.0e3.5 (10H, br, 2 ꢂ eCH2eNeCH2e(piperidine ring) and
(pyridinone)NeCH2CH2N(piperidine)), 4.34 (2H, s, 2-CH2N),
6.9 (s, 1H, 3-H(pyridinone)),7.8 (1H, s, 6-H(pyridinone)). Anal-
ysis C18H32N3O2Cl3$3H2O (C, H, N were within 0.4% of the
theoretical values).
5.4.6. 5-Benzyloxy-2-piperidinomethylpyran-4(1H )-one (12)
To a solution of piperidine and tributylamine in N,N-dime-
thylformamide (50 mL) was added 4.4 g (17.7 mmol) of 11
and the reaction mixture was allowed to stir at room temperature
overnight. The mixture was then diluted with water (250 mL)
and extracted with dichloromethane (3 ꢂ 150 mL). The extracts
were combined, washed with water, dried over anhydrous so-
dium sulphate, filtered and the solvent removed in vacuo. The
residue was purified by column chromatography on silica gel
(eluant: methanol/chloroform; 10:90 v/v) to afford a light
yellow oil (4.9 g, 93%); dH (60 MHz; CDCl3; Me4Si) 1.4e1.9
(6H, m, br, eCH2CH2CH2e(piperidine ring)), 2.3e2.6 (4H,
m, br, eCH2eNeCH2e(piperidine ring)), 3.25 (2H, s, 2-
CH2N), 5.0 (2H, s, CH2Ph), 6.35 (1H, s, 3-H(pyranone)), 7.3
(5H, s, Ph) 7.45 (1H, s, 6-H(pyranone)). Analysis C18H19NO4
(C, H, N were within 0.4% of the theoretical values).
5.4.9. 2-Hydroxymethyl-3-hydroxypyran-4(1H )-one (15a)
Sodium hydroxide (4 g, 100 mmol) dissolved in distilled wa-
ter (10 mL) was added to a solution of 14 (8.96 g, 80 mL, 1 eq.)
in methanol (50 mL) and allowed to stir at room temperature for
5 min; 35% formaldehyde solution (16 mL) was added drop-
wise over 15 min and the solution was stirred overnight. After
adjustment to pH 1 with 37% hydrochloric acid, the reaction
mixture was concentrated in vacuo to dryness and the resulting
solid was extracted with isopropanol (2 ꢂ 100 mL) at 90 ꢀC.
The isopropanol extracts were concentrated to yield the crude
products. Recrystallization from isopropanol afforded a white
crystalline solid (9.7 g, 85.4%); mp 154e156 ꢀC (lit. value
[33]: 154e155 ꢀC); dH (60 MHz; DMSO-d6; Me4Si) 4.4 (2H,
s, 2-CH2OH), 4.6e5.7 (1H, br, 2-CH2OH ), 6.34 (1H, d, 5-H ),
8.1 (1H, d, 6-H ), 9.0 (1H, br, s, 3-OH ).
5.4.7. 5-Hydroxy-1-methyl-2-piperidinomethylpyridin-4(1H)-
one dihydrochloride (13a)
To a solution of 12 (1.6 g, 5 mmol, 1 eq.) in ethanol (10 mL)
was added 10 mL of 40% aqueous methylamine and the mixture
was sealed in a thick-walled glass tube and stirred at 70 ꢀC for
6 h. After removal of the solvent, the residue was purified by col-
umn chromatography on silica gel (eluant: methanol/chloro-
form; 15:85 v/v) to afford a yellow oil, which was then
dissolved in 6 N hydrochloric acid (40 mL) and refluxed for
4 h. After removal of the solvent by rotary evaporation, the resi-
duewaspurifiedbyrecrystallizationfrom methanol/diethylether
to afford 13a as a white solid (0.92 g, 62%); mp 225e227 ꢀC; dH
(60 MHz; D2O; Me4Si) 1.4e2.0 (6H, s, br, eCH2CH2CH2e
(piperidinering)), 3.0e3.5(4H, br, eCH2eNeCH2e(piperidine
ring)), 3.95 (3H, s, NeCH3), 4.4 (2H, s, 2-CH2N), 7.2 (1H, s, 3-
H(pyridinone)), 8.0 (1H, s, 6-H(pyridinone)). Analysis C12H20
N2O2Cl2 (C, H, N were within 0.4% of the theoretical values).
5.4.10. 2-(10-Hydroxyethyl)-3-hydroxypyran-4(1H)-one (15b)
Pyromeconic acid (12) (5.6 g, 50 mmol) was added to water
(50 mL) and the pH of the solution was adjusted to 10.5 using
50% aqueous sodium hydroxide. Acetaldehyde (2.64 g, 60
mmol) dissolved in water (20 mL) was slowly added dropwise
over 1 h and the solution allowed to stir overnight. The reaction
mixturewasacidified to pH 1 with 37%hydrochloric acid and con-
centrated in vacuo to dryness. The residue was extracted with iso-
propanol (2 ꢂ 70 mL) at 90 ꢀC. The isopropanol extracts were
combined and concentrated to yield, after recrystallization from
toluene, a pale yellow crystalline solid (3.7 g, 47.4%); mp 131e
132 ꢀC (lit. value [33]: 130e131 ꢀC); dH (60 MHz; DMSO-d6;
Me4Si) 1.3 (3H, d, 2-CH2CH3), 5.03 (1H, q, 2-CHCH3), 6.38
(1H, d, 5-H ), 8.2 (1H, d, 6-H ).
5.4.8. 5-Hydroxy-1-(20-piperidinoethyl)-2-
piperidinomethylpyridin-4(1H )-one trihydrochloride (13b)
To a solution of 12 (3.2 g, 10 mmol, 1 eq.) in ethanol (50 mL)/
water (50 mL) was added 2-piperidinoethylamine (2.56 g, 20
mmol, 2 eq.) followed by 2 N sodium hydroxide solution until
pH 12.5 was obtained. The mixture was then refluxed for 12 h.
After adjustment to pH 1 with concentrated hydrochloric acid,
the solvent was removed by rotary evaporation prior to addition
of water (50 mL) and washed with diethyl ether (2 ꢂ 50 mL).
Subsequent adjustment of the aqueous fraction to pH 9 with
10 N sodium hydroxide solution was followed by extraction
into dichloromethane (4 ꢂ 50 mL). The combined organic layers
were then dried over anhydrous sodium sulphate, filtered, and
concentrated to yield a light brown oil which was purified by col-
umn chromatography on silica gel (eluant: methanol/chloroform;
20:80 v/v). The resulting light yellow oil was then dissolved in
5.4.11. 8-Oxo-4,8-dihydro-2-phenyl-4H-pyrano
[3,2-d]-m-dioxin (16a)
A solution of 15a (2.84 g, 20 mmol), benzaldehyde dimethyl
acetal (6.08 g, 40 mmol), and toluene-p-sulphonic acid mono-
hydrate (0.04 g, cat) in N,N-dimethylformamide (50 mL) was
rotated under aspirator pressure at 80 ꢀC for 3 h. The solvent
was removed under high vacuum and the residue taken into di-
chloromethane (100 mL). The organic solution was washed suc-
cessively with 5% sodium hydrogen carbonate solution and
brine. After drying over magnesium sulphate, the solventwas re-
moved to give the crude product. Recrystallization from di-
chloromethane/petroleum ether (40e60 ꢀC) afforded a white
crystalline solid (3.77 g, 82%); mp 141e143 ꢀC; dH (60 MHz;
CDCl3; Me4Si) 4.7 (2H, d, CH2O), 5.88 (1H, s, 2-CHPh), 6.35

L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
1045
(1H, d, 7-H(pyranone)), 7.2e7.9 (6H, m, Ar and 6-H(pyra-
none)); m/z, 230 (Mþ).
6-H(pyridinone)). Analysis C13H22N2O3Cl2 (C, H, N were
within 0.4% of the theoretical values).
Analogous synthesis starting with 15b gave compound 16b
as shown in Scheme 4.
5.4.16. 2-(10-Hydroxyethyl)-3-hydroxy-1-(20-piperidinoethyl)-
pyridin-4(1H )-one dihydrochloride (18b)
An analogous hydrogenation procedure to the preparation of
18a using 17b (1.24 g, 3.5 mmol) and 5% Pd/C catalyst (0.3 g)
yielded 0.92 g of the title compound (77.5%) after recrystalliza-
tion from methanol/diethyl ether, as a white solid; mp 191e
193 ꢀC; dH (360 MHz; DMSO-d6; Me4Si) 1.55 (3H, d,
J ¼ 6.9 Hz, CHCH3), 1.3e2.4 (6H, m, br, eCH2CH2CH2e(pi-
peridine ring)), 2.7e4.1 (6H, m, eCH2eNeCH2e(piperidine
ring) and (pyridinone)NeCH2CH2N(piperidine)), 4.5e5.4 (2H,
m, (pyridinone)NeCH2CH2N(piperidine)), 5.6 (1H, q, J ¼
6.9 Hz, CHCH3), 7.4 (1H, d, J ¼ 7.0 Hz, 5-H(pyridinone)), 8.5
(1H, d, J ¼ 7.0 Hz, 6-H(pyridinone)), 7.1e9.0 (2H, br, OH and
NH ). Analysis C14H24N2O3Cl2$1/2H2O (C, H, N were within
0.4% of the theoretical values).
5.4.12. 8-Oxo-4,8-dihydro-4-methyl-2-phenyl-4H-pyrano-
[3,2-d]-m-dioxin (16b)
Mp 112e113 ꢀC; dH (60 MHz; CDCl3; Me4Si) 1.55 (3H, d,
CHCH3), 5.0 (1H, q, CHCH3), 5.8 (1H, s, CHPh), 6.25 (1H, d,
7-H(pyranone)), 7.10e7.75 (6H, m, Ph and 6-H(pyranone));
m/z, 244 (Mþ).
5.4.13. 8-Oxo-4,8-dihydro-2-phenyl-5-(20-piperidinoethyl)-
4H-pyridino[3,2-d]-m-dioxin (17a)
To a solution of 16a (3.45 g, 15 mmol, 1 eq.) in ethanol
(50 mL)/water (50 mL) was added 2-piperidinoethylamine
(3.84 g, 30 mmol, 2 eq.) followed by 2 N sodium hydroxide so-
lution until pH 12.5 was obtained. The mixturewas then refluxed
for 3 h. TLC analysis (eluant: methanol/chloroform; 10:90 v/v)
showed that no starting material was present. After removal of
solventbyrotary evaporation, theresiduewas purified bycolumn
chromatography on silica gel (eluant: methanol/chloroform;
20:80 v/v) to afford a yellow crystalline solid (2.6 g, 51%); mp
137e140 ꢀC; dH (60 MHz; CDCl3; Me4Si) 1.2e1.9 (6H, br, m,
eCH2CH2CH2e(piperidine ring)), 2.0e2.9 (6H, m, eCH2e
NeCH2e(piperidine ring) and (pyridinone)NeCH2CH2N(pi-
peridine)), 3.65 (2H, t, (pyridinone)NeCH2CH2N(piperidine)),
4.9 (2H, s, CH2O), 5.85 (1H, s, CHPh), 6.3 (1H, d, 7-H(pyridi-
none)), 7.0e7.8 (6H, m, Ph and 6-H(pyridinone)).
5.5. Determination of physico-chemical properties
5.5.1. pKa determination of ligands
Equilibrium constants of protonated ligands were deter-
mined using an established automated titration system [30].
The pKa values were obtained to an accuracy of ꢃ0.02 pH unit.
5.5.2. Iron(III) affinity constant determination
The cumulative iron(III) stability constants (b3) for the
ligands were determined by spectrophotometric titration of
iron(III)eligand complexes using an automated system [30].
The iron(III) complexes of the ligand were prepared in 5:1 molar
ratio in 0.1 M KCl. This solution was acidified to pH 1.5 using
0.2 M HCl and then titrated against 0.2 M KOH. The resulting
spectrophotometric data were analysed using COMPT1 pro-
gram and the cumulative stability constant was obtained to an
accuracy of ꢃ0.2 log unit.
5.4.14. 8-Oxo-4,8-dihydro-2-phenyl-4-methyl-5-
(20-piperidinoethyl)-4H-pyridino[3,2-d]-m-dioxin (17b)
An analogous procedure to the preparation of 17a using 7b
(1.83 g, 7.5 mmol, 1 eq.) afforded 17b (1.24 g, 46.7%) as a light
brown oil; dH (60 MHz; CDCl3; Me4Si) 1.0e1.8 (9H, m, CHCH3
and eCH2CH2CH2e(piperidine ring)), 1.9e2.8 (6H, m, eCH2
NCH2e(piperidine ring) and (pyridinone)NeCH2CH2N(piperi-
dine)), 3.4e4.0(2H, m, (pyridinone)NeCH2CH2N(piperidine)),
5.1 (1H, q, CHCH3), 5.5 (1H, s, CHPh), 6.2 (1H, d, 7-H(pyridi-
none)), 7.0e7.8 (6H, m, Ph and 6-H(pyridinone)).
The stepwise stability constants(K1, K2 and K3) of the ligands
were optimized from the spectrophotometric titration of the
iron(III)eligand [30].
5.5.3. Distribution coefficient determination
Distribution coefficients were determined using an auto-
mated continuous flow technique [28]. The aqueous and octa-
nol phases were presaturated with respect to each other before
use. The aqueous phase (50 mM MOPS buffer, pH 7.4) was
separated from the two phase system (1-octanol/MOPS buffer,
pH 7.4) by means of a hydrophilic cellulose mounted in the
gel-filtration column adjuster. A known volume of MOPS
buffer is taken in the flat base mixing chamber. After a base
line was obtained the solution was used for reference absor-
bance. The compound to be examined was dissolved in buffer
so as to give an absorbance between 0.5e1.5 absorbance units
at the preselected wavelength.
5.4.15. 2-(10-Hydroxymethyl)-3-hydroxy-1-
(20-piperidinoethyl)-pyridin-4(1H )-one
dihydrochloride (18a)
A solution of 17a (2.55 g, 7.5 mmol) in ethanol (30 mL)
was adjusted to pH 1 with concentrated hydrochloric acid
prior to hydrogenolysis for 3 h in the presence of 5% Pd/C cat-
alyst (0.5 g). Filtration followed by rotary evaporation gave
the crude product as a white solid. Recrystallization from
methanol/diethyl ether gave a white crystalline solid (1.98 g,
81.2%); mp 278e280 ꢀC (dec.); dH (360 MHz; D2O; Me4Si)
1.0e2.4 (6H, m, br, eCH2CH2CH2e(piperidine ring)), 2.7e
3.9 (6H, m, eCH2NCH2e(piperidine ring) and (pyridino-
ne)NeCH2CH2N(piperidine)), 4.8 (2H, t, J ¼ 7.7 Hz, (pyridi-
none)NeCH2CH2N(piperidine)), 4.95 (2H, s, CH2O), 7.18
(1H, d, J ¼ 6.9 Hz, 5-H(pyridinone)), 8.2 (1H, d, J ¼ 7.0 Hz,
5.5.4. pKa determination of iron(III) complexes
The pKa values of the iron(III) complexes were determined
by spectrophotometric titration using an automated system

1046
L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
3
[30]. The iron(III) complexes of the ligand (10ꢁ5 M) were pre-
pared in a 5-fold molar excess of ligand in 0.1 M KCl. This
solution was acidified to pH 1.5 using 0.2 M HCl and then ti-
trated against 0.2 M KOH. The resulting spectrophotometric
data were analysed using the STABOPT program [34]. The
pKa values were obtained to an accuracy of ꢃ0.2 log unit.
cultures were pulsed with H-hypoxanthine (specific activity
1 Ci/mL by adding 0.6 mCi of isotope to each well). Microtiter
plates were kept in the incubation chamber for an additional
18 h. Then, each plate was harvested onto glass fiber discs us-
ing a TOMTEC MASH II cell harvester. Scintillation cocktail
(Omnifluor, New England Nuclear Research Products, Boston)
was added and radioactivity was determined by a Betaplate
liquid scintillation counter (Wallac, Finland). The IC50 values
of individually tested agents were obtained from doseeresponse
curves generated from serial dilutions conducted in triplicate by
a computerized, non-linear regression analysis.
5.6. Biological experiments
5.6.1. Animals
Male Wistar rats were purchased from Tuck & Son (Bat-
tlesbridge, Essex SSI, UK) and housed in the Biological Ser-
vice Unit, King’s College London. The animals were
maintained at 20e23 ꢀC with food and water freely available.
All animal experiments performed were specified in project
licence PPL 70/4561, authorized by the Secretary of State
(England) under Animals Act 1986.
Acknowledgements
The authors would like to thank Biomed EC grant BMH4-
CT97-2149 for supporting this research project, Apotex Re-
search Inc., the Ministry of Health and Medical Education
of Islamic Republic of Iran and Isfahan University of Medical
Science for funding.
5.6.2. Iron mobilization efficacy study in [59Fe]ferritin
loaded rat
Hepatocytes of normal fasted rats (190e230 g) were la-
belled with 59Fe by administration of [59Fe]ferritin to the tail
vein [31]. One hour later, each rat was administered orally
with chelator. Control rats were administered with an equiva-
lent volume of water. The rats were placed in individual met-
abolic cages, and urine and faeces collected. Rats were
allowed access to food 1 h after oral administration of chela-
tor. There was no restriction of water throughout the study pe-
riod. The investigation was terminated 24 h after the
[59Fe]ferritin administration, rats were killed, and the liver
and gastrointestinal tract (including its content and faeces)
were removed for gamma counting. The ‘‘iron mobilization’’
and ‘‘efficacy’’ were calculated according to Eqs. (6) and (7).
Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.ejmech.2007.07.011.
References
[1] Practical Chemotherapy of Malaria, In: World Health Organisation
Technical Report Series, vol. 805, World Health Organization, 1990.
[2] G.F. Mabeza, G. Biemba, V.C. Gordeuk, Acta Haematol. 95 (1996) 78e86.
[3] W. Peters, Recent Prog. Med. 81 (1990) 749e753.
[4] D.F. Clyde, Epidemiol. Rev. 9 (1987) 219e243.
[5] R.C. Hider, Exp. Opin. Ther. Patents 4 (1994) 931e940.
[6] T.E.A. Peto, J.L. Thompson, Br. J. Haematol. 63 (1986) 273e280.
[7] C. Hershko, T.E.A. Peto, J. Exp. Med. 168 (1988) 375e387.
[8] C. Raventos-Suarez, S. Pollack, R.L. Nagel, Am. J. Trop. Med. Hyg. 31
(1982) 919e929.
Iron mobilizationð%Þ
59Fe-activity
59Fe-activityðgut and faecesÞ þ 59Fe-activity
[9] A.M. Yinnon, E.N. Theanacho, R.W. Grady, D.T. Spira, C. Hershko,
Blood 74 (1989) 2166e2171.
[10] P.S. Dobbin, R.C. Hider, A.D. Hall, P.D. Taylor, P. Sarpong, J.B. Porter,
G. Xiao, D. van der Helm, J. Med. Chem. 36 (1993) 2448e2458.
[11] C. Hershko, E.N. Theanacho, D.T. Spira, H.H. Peter, P. Dobbin,
R.C. Hider, Blood 77 (1991) 637e643.
ðgut and faecesÞ
¼
ꢂ 100 ð6Þ
ð7Þ
ðliverÞ
Efficacyð%Þ ¼ iron mobilizationð%Þ ꢁ controlð%Þ
[12] C. Hershko, V.R. Gordeuk, P.E. Thuma, E.N. Theanacho, D.T. Spira,
R.C. Hider, T.E.A. Peto, G.M. Brittenham, J. Inorg. Biochem. 47
(1992) 267e277.
[13] R.C. Hider, Z.D. Liu, J. Pharm. Pharmacol. 49 (Suppl. 2) (1997) 59e64.
[14] C. de Duve, T. de Barsy, B. Pool, A. Trouet, P. Tulkens, F. van Hoof,
Biochem. Pharmacol. 23 (1974) 2495e2531.
5.6.3. In vitro antimalarial activity
A chloroquine-resistant clone of P. falciparum (TM267TR)
was obtained from a stock of a continuous line maintained in
human red blood cells in RPMI 1640 medium (Gibco) contain-
ing 10% human serum, 25 mM HEPES (Sigma), and 25 mM
NaHCO3. Parasite growth was synchronized by a sorbitol lysis
method [35]. Before use, the parasites were washed twice with
warm RPMI 1640 medium, and diluted with normal red blood
cells to final hematocrit and parasitemia of 1% and 0.5%, re-
spectively. Tests for drug susceptibility were performed by an
established method [36], whereby 200 ml of a parasitized red
blood cell suspension was incubated with 50 ml of the iron
chelator alone or in combination with pyridines at various con-
centrations in 96-well microtiter plates. The plates were incu-
bated at 37 ꢀC in 5% O2, 5% CO2 and 90% N2. After 24 h, the
[15] D.J. Krogstad, P.H. Schlesinger, Biochem. Pharmacol. 35 (1986) 547e552.
[16] R.L.N. Harris, Aust. J. Chem. 29 (1976) 1329e1334.
[17] H. Stunzi, R.L.N. Harris, D.D. Perrin, T. Teitei, Aust. J. Chem. 33 (1980)
2207e2220.
[18] J.W. Armit, T.J. Nolan, J. Chem. Soc. (1932) 3023e3031.
[19] A.F. Bickel, J. Am. Chem. Soc. 69 (1947) 1801e1803.
[20] I.D. Spenser, A.D. Notation, Can. J. Chem. 40 (1962) 1374e1379.
[21] M. Yasue, N. Kawamua, J. Sakakibara, Yakugaku Zasshi 90 (1970)
1222e1225.
[22] M. Mintas, Z. Orhanovic, K. Jakopcic, H. Koller, G. Stuhler,
A. Mannschreck, Tetrahedron 41 (1985) 229e233.
[23] P. Sarpong, The Design of Pyridinone Iron(III) Chelators. Ph.D. Thesis,
King’s College, London, 1992.

L.S. Dehkordi et al. / European Journal of Medicinal Chemistry 43 (2008) 1035e1047
1047
[24] F. Brody, P. Ruby, in: E. Klinsburg (Ed.), The Chemistry of Heterocyclic
Compounds, Interscience, New York, 1960.
[29] Z.D. Liu, H. Khodr, D.Y. Liu, S.L. Lu, R.C. Hider, J. Med. Chem. 42
(1999) 4814e4823.
[25] A.R. Katritzky, C.W. Rees (Eds.), Comprehensive Heterocyclic Chemis-
try, vol. 3, Pergamon, Oxford, 1984.
[30] P.D. Taylor, I.E.G. Morrison, R.C. Hider, Talanta 35 (1988) 507e512.
[31] Z.D. Liu, S.L. Lu, R.C. Hider, Biochem. Pharmacol. 57 (1999) 559e566.
[32] M.J. Pippard, D.K. Johnson, C.A. Finch, Blood 58 (1981) 685e692.
[33] B.E. Tate, R.L. Miller. U.S.A. Patent 3,130,204, 1964.
[34] P.D. Taylor. TITRFIT, STABOPT, University of Essex, Colchester, UK.
[35] C. Lambros, J.P. Vanderberg, J. Parasitol. 65 (1979) 418e420.
[36] H.K. Webster, E.F. Boudreau, K. Pavanand, K. Yongvanitchit, L.W. Pang,
Am. J. Trop. Med. Hyg. 34 (1985) 228e235.
[26] K. Imafuku, K. Takahashi, H. Matsumura, Bull. Chem. Soc. Jpn. 52
(1979) 111e113.
[27] B.L. Rai. Design and Synthesis of Iron(III) Chelators for Clinical Use.
Ph.D. Thesis, King’s College, London, 1996.
[28] B.L. Rai, L.S. Dekhordi, H. Khodr, Y. Jin, Z.D. Liu, R.C. Hider, J. Med.
Chem. 41 (1998) 3347e3359.