Design, synthesis and evaluation of N-basic substituted 3-hydroxypyridin-4-ones: Orally active iron chelators with lysosomotrophic potential

Article abstract of DOI:10.1211/0022357001773940

To investigate the possibility of targeting chelators into the lysosomal iron pool, nine bidentate 3-hydroxypyridin-4-ones with basic chains have been synthesized. As the turnover of ferritin iron is centred in the lysosome, such strategy is predicted to increase chelator efficacy of bidentate ligands. The pK(a) values of the ligands together with their distribution coefficients between 1-octanol and 4-morpholinepropane sulphonic acid (MOPS) buffer pH 7.4 have been determined. The in-vivo iron mobilization efficacy of these basic 3-hydroxypyridin-4-ones has been investigated in a ⁵⁹Fe-ferritin-loaded rat model. No obvious correlation was observed between efficacy and the pK(a) value of the side chain, although those with pK(a) > 7.0 tended to be more efficient than those with pK(a) < 7.0. The imidazole-containing molecules are much less effective than the tertiary amine derivatives. A dose-response study suggested that basic pyridinones are relatively more effective at lower doses when compared with N-alkyl hydroxypyridinones. Optimal effects were observed with the piperidine derivatives 4h and 4i. The derivative 4i at a dose of 150 μmol kg^-1 was more effective than 450 μmol kg^-1 deferiprone, the widely adopted clinical dose.

Full text of DOI:10.1211/0022357001773940

J. Pharm. Pharmacol. 2000, 52: 263±272
Received September 3, 1999
Accepted October 20, 1999
# 2000 J. Pharm. Pharmacol.
Design, Synthesis and Evaluation of N-Basic Substituted
3-Hydroxypyridin-4-ones: Orally Active Iron Chelators
with Lysosomotrophic Potential
ZU D. LIU, HICHAM H. KHODR, SHU L. LU AND ROBERT C. HIDER
Department of Pharmacy, King's College London, Franklin-Wilkins Building, 150 Stamford Street,
London SE1 8WA, UK
Abstract
To investigate the possibility of targeting chelators into the lysosomal iron pool, nine
bidentate 3-hydroxypyridin-4-ones with basic chains have been synthesized. As the
turnover of ferritin iron is centred in the lysosome, such strategy is predicted to increase
chelator ef®cacy of bidentate ligands.
The pKa values of the ligands together with their distribution coef®cients between 1-
octanol and 4-morpholinepropane sulphonic acid (MOPS) buffer pH 7Á4 have been
determined. The in-vivo iron mobilization ef®cacy of these basic 3-hydroxypyridin-4-
ones has been investigated in a 59Fe-ferritin-loaded rat model. No obvious correlation was
observed between ef®cacy and the pKa value of the side chain, although those with
pKa > 7Á0 tended to be more ef®cient than those with pKa < 7Á0. The imidazole-containing
molecules are much less effective than the tertiary amine derivatives. A dose±response
study suggested that basic pyridinones are relatively more effective at lower doses when
compared with N-alkyl hydroxypyridinones.
Optimal effects were observed with the piperidine derivatives 4h and 4i. The derivative
1
1
4i at a dose of 150 mmol kg was more effective than 450 mmol kg deferiprone, the
widely adopted clinical dose.
1
Frequent blood transfusion remains the basic thera-
peutic treatment for the survival of b-thalasaemia
major patients. Such long-term blood transfusion
leads to the accumulation of excess iron and
the associated toxic consequences. Complications
associated with elevated iron levels can be largely
avoided by the use of iron-speci®c chelating agents
and in particular desferrioxamine. Unfortunately,
desferrioxamine lacks oral activity and this has a
dramatic in¯uence on patient compliance (Hershko
et al 1998).
3-Hydroxypyridin-4-ones are one of the main
candidates for the development of orally active iron
chelating alternatives to desferrioxamine (Tilbrook
& Hider 1998). Indeed, the 1,2-dimethyl derivative
(deferiprone) is the only orally active iron chelator
currently available for clinical use. Unfortunately,
the dose required to keep a previously well-chel-
ated patient in negative iron balance appears to be
relatively high (450 mmol kg ). Not surprisingly
therefore, side effects have been observed in some
patients receiving deferiprone (Hershko et al 1998).
In order to improve drug ef®cacy or decrease
drug toxicity, it is necessary to design a chelator
that can be ef®ciently and selectively delivered to
its site of action such as the liver, the major iron
storage organ. The possibility of using an ester
prodrug strategy to increase drug absorption and
hepatic extraction, thereby increasing the drug
ef®cacy has been previously investigated (Liu et al
1999a; Rai et al 1999). Results demonstrated that
selective targeting of a chelator to the liver is
possible (Choudhury et al 1997; Liu et al 1999a).
This strategy can be further re®ned by targeting the
drug not only to the hepatocyte, but to lysosomes
located in the hepatocyte.
Lysosomes are intracellular organelles that con-
tain over 40 hydrolytic enzymes each possessing
acid pH optima. The major functions of lysosomes
include intracellular digestion, transcellular trans-
port, extracellular release or secretion of their
contents, and the sequestration and storage of
Correspondence: R. C. Hider, Department of Pharmacy,
King's College London, Franklin-Wilkins Building, 150 Stam-
ford Street, London SE1 8WA, UK.
E-Mail: robert.hider@kcl.ac.uk

264
ZU D. LIU ET AL
endogenous or exogenous cell components (de
Duve 1963). As can be seen from the lysosomal
catabolism of ferritin (Figure 1), ferritin is con-
stantly broken down in cells within lysosomes and
much of the liberated iron is reincorporated into
new ferritin molecules (Pippard et al 1986; Radisky
& Kaplan 1998). Therefore, it is expected that the
acidic lysosomal compartment possesses a rela-
tively high concentration of iron in a chemical form
suited to ef®cient chelation. This conclusion is
further endorsed by the results of Laub et al (1985)
who, by using radiolabelled desferrioxamine,
showed that the likely source of chelatable iron in
the hepatocyte is the lysosomal pool. Thus, the
selective delivery of chelating agents to lysosomes
may greatly improve their ef®ciency in iron
removal from iron overloaded patients.
3-Hydroxypyridin-4-ones with basic side chains
might be expected to accumulate in acidic organ-
elles such as lysosomes more ef®ciently than neu-
tral compounds (de Duve et al 1974). Such a
strategy is demonstrated in the two compartment
model represented in Figure 2. If it is assumed that
only neutral molecules are able to cross lysosomal
membranes, then at equilibrium the concentration
of the neutral form will be the same in both com-
partments. Thus, the distribution ratio (DR) can be
calculated from equation 1:
Figure 2. Distribution of a monobasic drug between the
cytoplasm (pH 7Á0) and the lumen of the lysosome (pH 5Á0±
5Á5).
librium accumulation of drug in the acidic intrave-
sicular compartment B (pH 5Á0) is calculated as 10. If
it is assumed that the drug is maintained at a constant
concentration in the cytoplasm (pH 7) (for example
25 mM), then the total drug concentration (neutral
form and protonated form) in the acidic lysosomes
(pH 5Á0) will be 250 mM. Such an elevated lysosomal
concentration of chelator would be predicted to
increase the chelator ef®cacy of bidentate ligands.
Normally only the neutral uncharged form can
cross biological membranes at an appreciable rate.
Therefore, when attempting to design a basic
compound with a useful accumulation effect, the
proportion of non-ionized species present at pH 7Á0
must also be considered since this may greatly
in¯uence the penetration rate of such drugs into the
cell. The fraction of non-ionized species (Fn) can
be calculated from equation 2:
Distribution ratio ꢀDR† ˆ ‰LtotalŠB=‰Ltotal A
Š
pHB
pHB
ˆ ‰LHŠBꢀ1 ‡ 10pKa
‰LHŠAꢀ1 ‡ 10pKa
†=
Š
If ‰LHŠ ˆ ‰LHŠ then
B
A
pHB
DR ˆ ꢀ1 ‡ 10pKa
†=1 ‡ 10pKa pHA
†
ꢀ1†
For a compound with a pKa value of 6Á0, over 90%
will be neutral at pH 7Á0, whereas at pH 5Á0 over 90%
of the compound will be protonated. Thus the equi-
‡
Neutral fraction ꢀFn† ˆ ‰LHŠ=ꢀ‰LHŠ ‡ ‰LH2 Š†
pH
ˆ 1=ꢀ1 ‡ 10pKa
†
ꢀ2†
For compounds with a pKa value less than 6Á0, the
fraction of non-ionized species (which can cross the
cell membrane) at pH 7Á0 is relatively high but the
equilibrium distribution ratio is relatively low
(Figure 3). In contrast, drugs with pKa values
greater than 8Á0 have a greater chance of accumu-
Figure 1. Schematic representation of the major intracellular
iron ¯uxes in the hepatocyte.

BASIC HYDROXYPYRIDINONES
265
lating ef®ciently within the cell. However, the
penetration rate of such compounds into the cell
will be much slower due to the high proportion of
ionized species present at pH 7Á0 (Figure 3).
Ideally, drugs for accumulation in the lysosome
should not only possess an appreciable distribution
ratio but must also be able to penetrate the cell at a
suf®ciently high rate. It is predicted that an ideal
chelator for this purpose should have a pKa value in
the range of 6Á0±8Á0, although it has been sug-
gested in the literature that the optimum pKa value
for lysosomotropic agents is around 8 (de Duve
et al 1974). Thus, it is expected that chelators
possessing such properties would scavenge iron
more effectively at lower doses than the corre-
sponding neutral ligands.
Figure 4. The mesomeric effect of the pyridin-4-one ring. 3-
Hydroxypyridin-4-ones have two resonance forms (a and b)
and the major resonance form (b) possesses an appreciable
positive charge located on the ring nitrogen atom.
(Dehkordi 1996). It is therefore possible to predict
the pKa value of the basic group on hypothetical
3-hydroxypyridin-4-ones if the pKa values of the
corresponding amine are known. Adopting this
approach, a range of N-basic substituted 3-hy-
droxypyridin-4-ones have been synthesized by sel-
ecting amines such as histamine, 1-(amino-propyl)
imidazole, N,N-dimethyl-ethylenediamine, N,N-
diethylethylenediamine, 1-(20-aminoethyl)-pyrroli-
dine and 2-piperidinoethylamine.
In order to achieve the optimum pKa value (pKa
6±8) for the lysosomotrophic chelation approach,
it is necessary to select suitable basic side chains
for hydroxypyridinone ligands. 3-Hydroxypyridin-
4-ones have two resonance forms (a and b) and the
major resonance form (b) possesses an appreciable
positive charge located on the ring nitrogen atom
(Figure 4). Due to this positive charge on the
nitrogen, the pyridinone ring will exert an electron
withdrawing effect on the side chain. Since the net
charge on the hydroxypyridinone is less than unity
‡
( ꢁ 0Á7 ) (Xiao et al 1992), it is expected that the
Materials and Methods
pyridinone ring will have a somewhat reduced
in¯uence on the basic function than, for example, a
protonated amino group. Indeed preliminary studies
indicate that the basic strength of the 3-hydroxy-
pyridin-4-one side chain is increased compared
with the corresponding unsubstituted amine, the
pKa values being approximately 10% greater
Chemistry
Maltol and ethyl maltol were purchased from P®zer
Ltd (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 (Southend, UK) and are uncorrected. 1H
NMR spectra were recorded using a Perkin-Elmer
(60 MHz) NMR spectrometer (Tokyo, Japan).
Chemical shifts (d) are reported in ppm down®eld
from the internal standard tetramethylsilane (TMS).
Elemental analyses were performed by Micro
analytical laboratories, Department of Chemistry,
The University of Manchester (Manchester M13
9PL, UK). The synthetic pathway of the basic 3-
hydroxypyridin-4-ones is summarized in Figure 5.
2-Methyl-3-benzyloxypyran-4(1H)-one (2a). Sodium
hydroxide (22 g, 0Á55 mol, 1Á1 eq.) dissolved in
water (50 mL) was added to a solution of maltol
(1a) (63 g, 0Á5 mol, 1 eq.) in methanol (500 mL)
followed by benzyl chloride (69Á6 g, 0Á55 mol,
1Á1 eq.). The resulting mixture was re¯uxed for 6 h.
After removal of solvent by rotary evaporation, the
residue was mixed with water (250 mL) and
extracted with dichloromethane (3 6 150 mL). The
Figure 3. The relationship between pKa value of a monobasic
drug with equilibrium distribution ratio between two compart-
ments (pH 5Á0 and pH 7Á0) (s), and the noncharged fraction of
the drug at pH 7Á0 (r).

266
ZU D. LIU ET AL
Figure 5. Synthesis of basic 3-hydroxypyridin-4-ones.
combined extracts were washed with 5% aqueous
sodium hydroxide (2 6 250 mL) followed by water
(2 6 250 mL). The organic fraction was then dried
over anhydrous sodium sulphate, ®ltered and rotary
evaporated. This yielded an orange oil which solid-
i®ed on cooling. Recrystallization from diethyl
ether gave the pure product (2a) as colourless
2 h. After ®ltration, the pH of the solution was
adjusted to 1 using conc. hydrochloride acid and
the solvent was then removed in-vacuo to yield the
crude product. Re-crystallization from metha-
nol=diethyl ether gave the pure compound (4a±i)
as a white or yellow solid.
1
needles (92Á5 g, 85Á6%); mp 52±53ꢂC; H NMR
(CDCl3) d: 2Á10 (s, 3H, 2-CH3), 5Á10 (s, 2H,
CH2Ph), 6Á3 (d, 1H, 5-H(pyranone)), 7Á32 (s, 5H,
Ar), 7Á54 (d, 1H, 6-H(pyranone)).
1-[20-(Imidazol-4-yl)ethyl]-2-methyl-3-hydroxypyri-
din-4(1H)-one dihydrochloride (4a). Yield 52%;
1
mp 239±240ꢂC; H NMR (DMSO-d6) d: 2Á54 (s,
An analogous procedure using ethyl maltol (1b)
gave 2-ethyl-3-benzyloxypyran-4(1H)-one (2b)
3H, 2-CH3), 3Á2 (t, 2H, NCH2CH2), 4Á65 (t, 2H,
NCH2CH2), 7Á3 (d, 1H, 5-H(pyridinone)), 7Á44 (s,
1H, 5-H (imidazole)), 8Á18 (d, 1H, 6-H(pyridi-
none)), 9Á08 (s, 1H, 2-H(imidazole)). Anal. Calc.
for C11H15N3O2Cl2: C, 45Á21; H, 5Á14; N, 14Á38%.
Found: C, 45Á57; H, 5Á07; N, 13Á98%.
1
(84Á8%). mp 33±34ꢂC; H NMR (CDCl3) d: 1Á0
(t, 3H, 2-CH2 CH3), 2Á55 (q, 2H, 2-CH2CH3), 5Á13
(s, 2H, CH2Ph), 6Á3 (d, 1H, 5-H(pyranone)), 7Á35 (s,
5H, Ar), 7Á6 (d, 1H, 6-H(pyranone)).
General method for the preparation of basic 3-
hydroxypyridin-4-ones (4a±i). Selected amine
(30 mmol, 1Á5 eq.) was added to a solution of 2a
or 2b (20 mmol, 1 eq.) in ethanol (50 mL)=water
(50 mL) followed by 2 M sodium hydroxide solu-
tion until pH 13Á5 was obtained. The mixture was
then re¯uxed for 12 h. After adjustment to pH 1
with conc. hydrochloride acid, the solvent was
removed by rotary evaporation before addition of
water (50 mL) and washing with diethyl ether
(2 6 50 mL). Subsequent adjustment of the aque-
ous fraction to pH 9 with 10 M sodium hydroxide
solution was followed by extraction into dichloro-
methane (4 6 50 mL). The combined organic
layers were then dried over anhydrous sodium
sulphate, ®ltered, and then rotary evaporated to
give a brown oil. Puri®cation by column chroma-
tography on silica gel (eluant: 12% CH3OH=88%
CHCl3) gave the benzylated hydroxypyridinones
3a±i as a yellow oil, which was subsequently
dissolved in ethanol (90 mL)=water (10 mL) and
subjected to hydrogenolysis in presence of 5%
Pd=C (5±10% w=w of the compound) catalyst for
1-[30-(Imidazol-1-yl)propyl]-2-methyl-3-hydroxypyri-
din-4(1H)-one dihydrochloride (4b). Yield 69%;
mp 160±161ꢂC;1H NMR (DMSO-d6) d: 2Á2±2Á75
(m, 2H, CH2CH2CH2), 2Á6 (s, 3H, 2-CH3), 4Á1±4Á8
(m, 4H, CH2CH2CH2), 7Á4 (d, 1H, 5-H (pyridi-
none)), 7Á7 (d, 1H, 5-H(imidazole)), 7Á92 (d, 1H, 6-
H(imidazole)), 8Á4 (d, 1H, 6-H(pyridinone)), 9Á38
(s, 1H, 2-H(imidazole)), 6Á1±8Á8 (br., 3H, OH and
NH). Anal. Calc. for C12H17N3O2Cl2ÁH2O: C,
44Á46; H, 5Á91; N, 12Á96%. Found: C, 44Á43; H,
5Á79; N, 12Á72%.
1-[30-(Imidazol-1-yl)propyl]-2-ethyl-3-hydroxypyri-
din-4(1H)-one dihydrochloride (4c). Yield 59%;
1
mp 189±191ꢂC; H NMR (DMSO-d6) d: 1Á03 (t,
3H, 2-CH2CH3), 1Á85±2Á55 (m, 2H, CH2CH2CH2),
2Á8 (q, 2H, 2-CH2CH3), 3Á9±4Á8 (m, 4H,
CH2CH2CH2), 7Á4 (d, 1H, 5-H(pyridinone)), 7Á65
(d, 1H, 5-H(imidazole)), 7Á9 (d, 1H, 6-H-
(imidazole)), 8Á35 (d, 1H, 6-H(pyridinone)),
9Á35 (s, 1H, 2-H(imidazole)). Anal. Calc. for

BASIC HYDROXYPYRIDINONES
267
C13H19N3O2Cl2: C, 48Á76; H, 5Á98; N, 13Á12%.
Found: C, 48Á64; H, 5Á82; N, 12Á94%.
idinone)). Anal. Calc. for C13H22N2O2Cl2ÁH2O:
C, 47Á71; H, 7Á39; N, 8Á56%. Found: C, 47Á94; H,
7Á32; N, 8Á42%.
1-[20-(Dimethylamino)ethyl]-2-methyl-3-hydroxy-
1 - (20 - Piperidinoethyl)- 2- ethyl- 3- hydroxypyridin-
pyridin-4(1H)-one dihydrochloride (4d). Yield
1
65Á7%; mp 251±252ꢂC; H NMR (D2O) d: 2Á58
4(1H)-one dihydrochloride (4i). Yield 65%; mp
1
207±209ꢂC; H NMR (D2O) d: 1Á22 (t, 3H, 2-
(s, 3H, 2-CH3), 3Á0 (s, 6H, N(CH3)2), 3Á4±3Á9 (m,
2H, N-CH2CH2N(CH3)2), 4Á75 (t, 2H, N-
CH2CH2N(CH3)2), 7Á1 (d, 1H, 5-H(pyridinone)),
8Á1 (d, 1H, 6-H(pyridinone)). Anal. Calc. for
C10H18N2O2Cl2: C, 44Á61; H, 6Á69; N, 10Á41%.
Found: C, 44Á26; H, 6Á85; N, 10Á04%.
CH2 CH3), 1Á4±2Á5 (m, br., 6H, -CH2CH2CH2-
(piperidine ring)), 3Á0 (q, 2H, 2-CH2CH3), 3Á1±
3Á9 (m, 6H, -CH2-N-CH2-(piperidine ring) and
(pyridinone)N-CH2 CH2N(piperidine)), 4Á8 (t, 2H,
(pyridinone)N-CH2CH2N (piperidine)), 7Á18 (d,
1H, 5-H(pyridinone)), 8Á18 (d, 1H, 6-H(pyridi-
none)). Anal. Calc. for C14H24N2O2Cl2ÁH2O: C,
49Á27; H, 7Á68; N, 8Á21%. Found: C, 49Á01; H,
7Á49; N, 8Á04%.
1-[20-(Dimethylamino)ethyl]-2-ethyl-3-hydroxypyri-
din-4(1H)-one dihydrochloride (4e). Yield 52%;
1
mp 234±236ꢂC; H NMR (D2O) d: 1Á28 (t, 3H,
2-CH2CH3), 2Á75±3Á3 (q, 2H, 2-CH2CH3), 3Á05 (s,
6H, N(CH3)2), 3Á5±3Á9 (m, 2H, N-CH2
CH2N(CH3)2), 4Á74 (t, 2H, N-CH2CH2N(CH3)2),
7Á15 (d, 1H, 5-H(pyridinone)), 8Á1 (d, 1H, 6-H(pyri-
dinone)). Anal. Calc. for C11H20N2O2Cl2ÁH2O: C,
43Á86; H, 7Á36; N, 9Á30%. Found: C, 43Á73; H, 7Á24;
N, 9Á21%.
Determination of physicochemical properties of
ligandsÐpKa determination. Equilibrium constants
of protonated ligands were determined using an
automated computerized system (Taylor et al
1988). This consisted of a Metrohm 665 dosimat
(Herisau, Switzerland), a Perkin-Elmer Lambda 5
UV=vis Spectrophotometer (Beacons®eld, UK), a
Corning Delta 225 pH meter, and an IBM compat-
ible PC (Opus Technology, Taiwan) to control the
integrated system. A combined Sirius electrode was
used to calibrate the electrode zero and to measure
pH values. This system is capable of undertaking
simultaneous potentiometric and spectrophoto-
metric measurements. A blank titration of 0Á1 M
KCl (25 mL) was carried out to determine the
electrode zero using Gran's plot method (Gran
1952). The solution (0Á1 M KCl, 25 mL), contained
in a jacketed titration cell, was acidi®ed by 0Á15 mL
0Á2 M HCl. Titration was carried out against 0Á3 mL
0Á2 M KOH using 0Á01 mL increments dispensed
from the dosimat. Solutions were maintained at
25Æ 0Á5ꢂC under an argon atmosphere. The above
titration was repeated in the presence of ligand. The
data obtained from titration were subjected to non-
linear least-square regression analysis using the
NONLINM1 program (Taylor et al 1988). The
pKa values were obtained to an accuracy of
Æ 0Á02 pH unit.
1-[20-(Diethylamino)ethyl]-2-methyl-3-hydroxypyri-
din-4(1H)-one dihydrochloride (4f). Yield 85Á5%;
mp 244±247ꢂC (dec); 1H NMR (DMSO-d6) d: 1Á26
(t, 6H, N(CH2CH3)2), 2Á6 (s, 3H, 2-CH3), 2Á9±3Á8
(m, 6H, N(CH2CH3)2 and N-CH2CH2N(C2H5)2),
4Á9 (t, 2H, N-CH2CH2N(C2H5)2), 7Á4 (d, 1H, 5-
H(pyridinone)), 8Á45 (d, 1H, 6-H(pyridinone)), 9Á1±
10Á4 (br., 3H, OH and NH). Anal. Calc. for
C12H22N2O2Cl2: C, 48Á49; H, 7Á46; N, 9Á42%.
Found: C, 48Á29; H, 7Á35; N, 9Á28%.
1-[20-(1-Pyrrolidinyl)ethyl]-2-methyl-3-hydroxypyr-
idin-4(1H)-one dihydrochloride (4g). Yield 71%;
mp 234±236ꢂC;1H NMR (D2O) d: 1Á9±2Á5 (m, br.,
4H, - CH2CH2-(pyrrolidine ring)), 2Á64 (s, 3H, 2-
CH3), 2Á8±3Á8 (m, br., 4H, -CH2-N-CH2-(pyrroli-
dine ring)), 3Á75 (t, 2H, (pyridinone)N-CH2
CH2N(pyrrolidine)), 4Á8 (t, 2H, (pyridinone)N-
CH2CH2N(pyrrolidine)), 7Á2 (d, 1H, 5-H(pyridi-
none)), 8Á21 (d, 1H, 6-H(pyridinone)). Anal. Calc.
for C12H20N2O2Cl2ÁH2O: C, 46Á02; H, 7Á08; N,
8Á94%. Found: C, 46Á17; H, 6Á82; N, 8Á68%.
Determination of distribution coef®cients. Distribu-
tion coef®cients were determined using an auto-
mated continuous ¯ow technique (Rai et al 1998).
The system comprised an IBM compatible PC
running the Omniferous Personal Computer Auto-
Titrator ``TOPCAT'' program, which controlled a
Metrohm 665 Dosimat autoburette and a Pye-Uni-
cam Lambda 5 UV=vis spectrophotometer, as well
as performing all calculations of distribution coef-
1- (20- Piperidinoethyl)- 2-methyl- 3-hydroxypyridin-
4(1H)-one dihydrochloride (4h). Yield 75Á6%; mp
1
213±215ꢂC; H NMR (D2O) d: 1Á3±2Á4 (m, br.,
6H, -CH2CH2CH2-(piperidine ring)), 2Á64 (s, 3H,
2-CH3), 2Á7±4Á2 (m, 6H, -CH2-N-CH2-(piperidine
ring) and (pyridinone)N-CH2CH2N(piperidine)),
4Á8 (t, 2H, (pyridinone)N-CH2CH2N-(piperidine)),
7Á18 (d, 1H, 5-H(pyridinone)), 8Á18 (d, 1H, 6-H(pyr-

268
ZU D. LIU ET AL
®cients. Determinations were performed using
AnalaR grade reagents under a nitrogen atmosphere
using a ¯at-based glass vessel equipped with a
sealable lid at 25ꢂC. The aqueous and octanol
phases were presaturated with respect to each
other before use. The ®lter probe consisted of a
polytetra¯uoroethylene plunger associated with a
gel-®ltration column. The aqueous phase (50 mM
MOPS buffer, pH 7Á4, prepared using Milli-Q
water) was separated from the two-phase system
(1-octanol=4-morpholinepropane sulphonic acid
(MOPS) buffer, pH 7Á4) by means of a hydrophilic
cellulose ®lter 5-mm diameter, 589=3 Blauband
®lter paper, Schleicher and Schuell) mounted in
the gel-®ltration column adjuster (SR 25=50, Phar-
macia). MOPS was adopted as a buffer for this
study because of its low af®nity for metal cations.
A known volume (normally 25±50 mL) of MOPS
buffer (saturated with octanol) is taken in the ¯at
base mixing chamber. After a base line was
obtained the solution was used for reference absor-
bance. The compound to be examined was dis-
solved in buffer (saturated with octanol) so as to
give an absorbance of between 0Á5±1Á5 absorbance
units at the preselected wavelength ( ꢁ 280 nm).
The ¯ow rate of the aqueous circuit was limited
to 1 mL. The computer program calculates the
distribution coef®cient (D7Á4) for each octanol
addition.
the liver and gastrointestinal tract (including its
content and faeces) were removed for gamma
counting. The ``iron mobilization'' and ``ef®cacy''
were calculated according to equations 3 and 4:
59
Iron mobilization ꢀ%† ˆ ꢀ Fe-activityꢀgut and faeces†
=
59
ꢀ Fe-activityꢀgut and faeces†
‡
59 Fe-activityꢀliver†††
 100%
ꢀ3†
Efficacy ꢀ%† ˆ iron mobilization ꢀ%† control ꢀ%†
ꢀ4†
Results
Chemistry
The basic 3-hydroxypyridin-4-ones 4a±i were
conveniently prepared by utilizing the methodology
of Harris and co-workers (Figure 5) (Harris 1976).
Commercially available 3-hydroxypyran-4-ones 1a
and 1b were benzylated in aqueous methanol. The
reactions of the protected pyranones (2a and 2b)
with an excess of primary amines were generally
performed by re¯uxing in 50% aqueous ethanol in
the presence of a catalytic amount of sodium
hydroxide. Removal of the protecting benzyl group
was achieved by catalytic hydrogenation to yield
the corresponding bidentate chelators, which were
®nally isolated as the hydrochloride salts. Nine
substituted 3-hydroxypyridin-4-ones (4a±i) were
prepared by using selected amines (Table 1).
Biological experiment
Animals. Male Wistar rats were purchased (local
breed) from A. Tuck & Son (Battlesbridge, Essex
SS1, UK) and housed in the Biological Service
Unit, King's College London. The animals were
maintained at 20±23ꢂC, with food and water freely
available. All animal experiments performed were
speci®ed in project licence PPL 70=4561, author-
ized by the Secretary of State (England) under
Animals Act 1986.
pKa values of basic hydroxypyridinones
All ligands were investigated by simultaneous
spectrophotometric and potentiometric titration.
The pH dependence of the UV spectra of 4i (Figure
6) is presented as an example. A clear shift in lmax
was observed in the full speciation spectra of ligand
4i over pH range 2Á05±11Á98 (Figure 6) which
displays the pH dependence of the ligand ionization
equilibrium. The optimized pKa values obtained
from non-linear least-square regression analysis are
shown in Table 1. All the basic 3-hydroxypyridin-
4-ones in this study possess three pKa values. The
pKa1 values correspond to the protonation of the 4-
oxo group, the pKa2 values to the deprotonation of
the basic function at the 1-position and pKa3 to the
dissociation of the 3-hydroxyl group (Figure 7).
The pKa values obtained from spectrophotometric
titration were in good agreement with those cal-
culated from the potentiometric titration (Table 1).
The speciation plot of 4i (Figure 8) provides
information on the distribution of the species in the
Iron mobilization ef®cacy study in 59Fe-ferritin-
loaded rat. Hepatocytes of normal fasted rats
(190±230 g) were labelled with 59Fe by admin-
istration of [59Fe]ferritin to the tail vein (Liu et al
1999b). One hour later, each rat was administered
orally with chelator. Control rats were administered
with an equivalent volume of water. The rats were
placed in individual metabolic cages and urine and
faeces collected. Rats were allowed access to food
1 h after oral administration of chelator. There was
no restriction of water throughout the study period.
The investigation was terminated 24 h after the
[59Fe]ferritin administration, rats were killed and

BASIC HYDROXYPYRIDINONES
269
Table 1. Basic hydroxypyridinones (4a±i) and their measured physicochemical properties.
Compound
Rbasic
n
R2
pKa (poteniometric) pKa (spectrophotometric) D7Á4 (n ˆ 5) Fn (pH 7Á4) Log P DR
pKa1 pKa2 pKa3
pKa1
pKa2
pKa3
4a
4b
4c
2
3
CH3 3Á01 6Á25
CH3 3Á08 6Á71
9Á80
9Á82
9Á66
2Á99
6Á23
9Á81 0Á293Æ 0Á006
0Á935
0Á848
0Á886
0Á504 15Á7
0Á382 31Á8
0Á324 25Á2
3Á13
3Á09
6Á60
6Á55
9Á92 0Á344Æ 0Á005
9Á71 0Á420Æ 0Á019
3 C2H5 3Á15 6Á47
CH3 2Á86 7Á02
4d
4e
4f
(CH3)2N
2
9Á58
9Á58
9Á60
2Á74
2Á62
2Á63
7Á05
6Á88
7Á53
9Á75 0Á154Æ 0Á005
9Á66 0Á538Æ 0Á047
9Á65 0Á493Æ 0Á005
0Á699
0Á706
0Á434
0Á657 52Á5
0Á118 51Á6
0Á055 76Á8
(CH3)2N 2 C2H5 2Á68 7Á16
(C2H5)2N
2
CH3 2Á66 7Á50
4g
4h
4i
2
CH3 2Á64 7Á69
9Á61
9Á71
9Á70
2Á70
2Á77
2Á73
7Á60
7Á42
7Á34
9Á61 0Á285Æ 0Á011
9Á63 0Á960Æ 0Á029
9Á67 4Á591Æ 0Á031
0Á363
0Á474
0Á523
0Á105 81Á7
0Á307 73Á9
0Á943 69Á9
2
CH3 2Á88 7Á47
2 C2H5 2Á74 7Á38
Figure 6. pH dependence of the UV spectrum of 4i over the
pH range 2Á05±11Á98.
Figure 8. The pH dependence of the speciation plot of 4i.
Figure 7. Protonation equilibria of basic 3-hydroxypyridin-4-ones.

270
ZU D. LIU ET AL
1
pH range of 0±12. Therefore, the predominant
species can be predicted at speci®c pH values.
at 450 mmol kg
experiments have also been undertaken with the
of 26Á5%. Dose±response
following compounds, deferiprone, 4g, 4h and 4i at
1
three different doses (150, 300 and 450 mmol kg ).
All three basic compounds, 4g, 4h and 4i, per-
Distribution and partition coef®cients of basic
hydroxypyridinones
formed better than deferiprone at low doses. In
particular 4i was more effective at 150 mmol kg
1
The distribution coef®cients (D7Á4) between 1-
octanol and MOPS buffer (pH 7Á4) were determined
via the automated ®lter-probe system. As a result of
the degree of ionization of the two functional
groups on the pyridinone ring (pKa1 and pKa3)
being relatively small at pH 7Á4, the neutral fraction
(Fn) of the basic hydroxypyridinones can be cal-
culated by employing the pKa2 values determined
above. The partition coef®cient (P) of each com-
pound can then be calculated from equation 5. The
resulting values are given in the Table 1.
1
than deferiprone at 450 mmol kg .
Discussion
To investigate the possibility of achieving lyso-
somotrophic strategy, a range of bidentate 3-hy-
droxypyridin-4-ones with basic side chains was
synthesized. The compounds fell into two classes,
the imidazoles (4a±c) and tertiary amines (4d±i).
The log P values covered the range 0Á12±0Á943
(Table 1). Therefore, it was anticipated that all
compounds would be orally active and some may
well be susceptible to ef®cient ®rst-pass extraction
by the liver. By incorporating selected basic func-
tions into the pyridinone ring, the pKa values of
the basic side chain covered the desired range
(6Á24±7Á64).
pH
Fn ˆ 1=ꢀ1 ‡ 10pKa2
†
P ˆ D7Á4=Fn ˆ D7Á4  ꢀ1 ‡ 10pKa2
†
ꢀ5†
pH
Iron mobilization ef®cacy of basic hydroxy-
pyridinones
In-vivo iron scavenging ability of basic hydroxy-
pyridinones was compared with deferiprone. Sev-
eral compounds such as 4e, 4g, 4h and 4i were
found to be superior to deferiprone (Table 2). The
imidazole-containing ligands 4a, 4b and 4c were
found to be much less effective than the tertiary
amine derivatives. The most effective basic
hydroxypyridinone was 4i, which possesses a
piperidine substitutent, with an associated ef®cacy
In-vivo iron mobilization ef®cacy of all ligands
was evaluated in a non-iron overloaded rat model.
[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 lyso-
somal degradation of ferritin was maximally
available (Pippard et al 1981; Liu et al 1999b). This
[59Fe]ferritin-loaded rat model can be used to
assess oral bioavailability and to compare the
ability of chelators to remove iron from liver, the
major iron storage organ in iron-overloaded con-
ditions. Several basic hydroxypyridinones led to
superior iron excretion via the bile compared with
deferiprone (Table 2). There was no obvious cor-
relation between ef®cacy and the pKa value of the
side chain, although those with pKa > 7Á0 tended to
be more ef®cient than those with pKa < 7Á0. The
imidazole-containing molecules were much less
effective than the tertiary amine derivatives. This
may result from unfavourable metabolism of the
imidazole function. Comparison of the tertiary
amino-containing pyridinones highlights 4i as the
most promising candidate. It is likely that 4i was
the most ef®cient at facilitating the excretion of
iron because of its relatively high log P value
(0Á943) and hence more ef®cient extraction by the
liver.
Table 2. Iron mobilization ef®cacy studies of basic hydroxy-
pyridinones (4a±i) in the [59Fe]ferritin-loaded rat model.
Chelator
Dose
(mmol kg
Iron
mobilization (%)
Ef®cacy
(%)
1
)
Control
Deferiprone
±
3Á9Æ 1Á0
13Á4Æ 5Á2
9Á2Æ 2Á2
6Á3Æ 2Á1
5Á5Æ 2Á5
5Á3Æ 1Á9
6Á8Æ 3Á5
13Á0Æ 2Á5
20Á3Æ 6Á8
12Á4Æ 1Á9
18Á8Æ 3Á6
13Á9Æ 3Á5
9Á5Æ 1Á8
22Á7Æ 3Á3
15Á2Æ 1Á8
11Á5Æ 1Á6
30Á4Æ 5Á3
17Á9Æ 2Á3
14Á2Æ 3Á2
0Á0
9Á5
450
300
150
450
450
450
450
450
450
450
300
150
450
300
150
450
300
150
5Á4
2Á4
4a
4b
4c
4d
4e
4f
1Á6
1Á4
2Á9
9Á1
16Á4
8Á5
4g
14Á9
10Á0
5Á6
4h
4i
18Á8
11Á3
7Á6
26Á5
14Á0
10Á3
One of the reasons for investigating basic
hydroxypyridinones is the belief that they will be
relatively more effective at lower concentrations
than neutral hydroxypyridinones due to their
Values are expressed as meansÆ s.d. (n ˆ 5).

BASIC HYDROXYPYRIDINONES
271
predicted accumulation by hepatocyte lyso-
somes. Consequently, the dose±response experi-
ment was undertaken in order to investigate the
predicted lysosomotrophic effect of basic hydroxy-
pyridinones. Detailed comparisons of ef®cacy
between different doses are made in Table 3.
Generally, the ef®cacy of basic hydroxypyridinones
decreased more slowly than that of deferiprone.
The ef®cacy ratios of 4g, 4h and 4i (0Á376, 0Á404
and 0Á387, respectively) were greater than that
for deferiprone (0Á253) between 150 and
1.
450 mmol kg
over the more dilute dose range 150±
The largest differences occurred
1
300 mmol kg , where the ratios were larger for the
basic hydroxypyridinones, particularly 4h and 4i.
There are, however, potential disadvantages
associated with bidentate iron ligands in thera-
peutic applications such as kinetic lability, con-
centration dependence on iron speciation and the
possible toxicity of 2 : 1 (ligand : iron) complexes
(Tilbrook & Hider 1998). For bidentate 3-hydroxy-
pyridin-4-ones such as 4i, the 3 : 1 complex was the
dominant species at pH 7Á0 (Figure 9A), due to the
high af®nity for iron(III). However, such 3 : 1
complexes tend to dissociate under acidic condi-
tions, for instance those found in lysosomes
(pH 5Á0±5Á5). With a ligand (4i) concentration of
1 6 10 M ([FeIII] ˆ 1 6 10 M) at pH 5Á0, the
2 : 1 complex will be the major species (Figure 9A).
With the 2 : 1 complexes, the incompletely envel-
oped iron atom is able to interact with hydrogen
peroxide or oxygen to generate free radicals (Til-
brook & Hider 1998). However, if such basic
bidentate chelators can be ef®ciently accumulated
in the lysosomal compartments, then the ligand-
metal complex will dissociate less readily in the
presence of the resulting higher concentration of
ligand. Thus any toxicity associated with the pre-
5
6
Figure 9. Comparison of the speciation plots of 4i-iron(III)
complex at different ligand concentrations. [FeIII]total
ˆ
6
5
4
1 6 10 M; A, [4i] ˆ 1 6 10 M; B, [4i] ˆ 7 6 10 M. The
following measured parameters were incorporated into the
model for the system: logb1(ML) ˆ 14Á40, logb2(ML2) ˆ
26Á49, logb3(ML3) ˆ 36Á38, pKa1 ˆ 2Á73, pKa2 ˆ 7Á34, pKa3 ˆ
9Á67.
these conditions, the concentration of the 2 : 1
complex is negligible.
‡
sence of FeIIIL2 species would be reduced. As
The precise pH value between the lysosome and
the cytoplasm in the hepatocyte is unknown. If the
pH of the lysosome is 5Á0, then the introduction of
one basic group on the molecule can lead to a
maximum accumulation of approximately 100
shown in Figure 9B, at the higher ligand con-
4
centration of 7 6 10 M, which is the predicted
lysosomal concentration of 4i, the 3 : 1 complex
will be the dominant species even at pH 5Á0. Under
Table 3. Ef®cacy ratio of hydroxypyridinones between different doses.
Chelator
Ef®cacy ratio between different doses
Ef®cacy(300 mmol kg
Ef®cacy(450 mmol kg
1
=
Ef®cacy(150 mmol kg
Ef®cacy(300 mmol kg
1
=
)
Ef®cacy(150 mmol kg
Ef®cacy(450 mmol kg
1
=
)
)
)
)
1
1
1
)
Deferiprone
0Á568
0Á671
0Á601
0Á528
0Á444
0Á560
0Á673
0Á736
0Á253
0Á376
0Á404
0Á389
4g
4h
4i

272
ZU D. LIU ET AL
Dehkordi, L. S. (1996) Design Of Orally Active Pyridinone
Iron(III) Selective Ligands. Ph.D. Thesis. King's College
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Gran, G. (1952) Determination of the equivalence point in
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Harris, R. L. N. (1976) Potential wool growth inhibitors.
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between those two phases (Figure 3). If however
the intralysosomal pH is 5Á5, the maximum accu-
mulation effect drops to 32. In contrast, the intro-
duction of two basic groups leads to a maximum
accumulation of 104 between two phases differing
by 2 pH units and 103 for 1Á5 pH units. Thus, a
more dramatic concentration dependence of ef®-
cacy might be predicted for dibasic chelators.
In conclusion, the fundamental concept of basic
hydroxypyridinones being relatively more effective
at lower doses has been con®rmed in this study.
Selection of the basic substitutent is critical, a pKa
value57Á0 being necessary to provide enhanced
iron scavenging ability. To improve the ef®cacy of
this chelator class, it will be necessary to design
monobasic and dibasic molecules that experience
ef®cient liver extraction.
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R. R. (1985) Cellular pharmacology of desferrioxamine B.
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matic ester prodrugs of 1-(20-hydroxyethyl)-2-ethyl-3-hy-
droxypyridin-4-one (CP102): orally active iron chelators
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Pippard, M. J., Johnson, D. K., Finch, C. A. (1981) A rapid
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Acknowledgements
The research described in this manuscript was
supported by Apotex Research Inc. Canada and
Biomed EC grant BMH4-CT97-2149. Z. D. Liu
would like to thank the KC Wong Education
Foundation (Hong Kong) and the Committee of
Vice-Chancellors and Principals of the Universities
of the United Kingdom for the joint scholarships
that enabled him to carry out the studies.
Pippard, M. J., Tikerpae, J., Peters, T. J. (1986) Ferritin iron
metabolism in the rat liver. Br. J. Haematol. 64: 839
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be recycled through lysosomal degradation in human ®bro-
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