There are a number of inherited disease
states which are associated with the gradual accumulation of
and thalassaemia intermedia being particularly well
characterized (1). In some regions of the world genes are
relatively common; For instance in South East Asia,
approximately 100000 children are born each year suffering from
thalassaemia. The normal total body iron is 4-5 g per adult,
whereas some thalassaemic patients may accumulate 50-70 g.
Iron, by virtue of its facile redox chemistry, is toxic when
present in excess (2). Desferrioxamine (DFO) (Scheme 1, l) a natural
siderophore, has been used for the treatment of iron overload
for over 30 years (3), and currently it is the only clinically
useful drug available for this purpose. However, DFO suffers
from the disadvantage that it is inactive when administered
orally, and only causes sufficient iron excretion to keep pace
with the transfusion regimes when given either subcutaneously
or intravenously over 12-18 h several times per week. For this
reason, many patients find it difficult to comply with the
treatment, and some even stop taking the drug altogether,
subsequently developing the complications of iron overload.
There is, therefore, no doubt that an orally active chelating
agent is needed to treat patients on lifelong transfusion
programs. The development of an oral iron chelator might also
allow the extension of the therapeutic use of red-cell
transfusions in sickle-cell anemia.
3-Hydroxypyridin-4-ones (HPOs) are
currently one of the main candidates for the development of
orally active iron chelators, being alternative to DFO (4).
Indeed, the 1,2-dimethyl derivatives CP2O (Deferiprone, L1) (5a) is currently
in clinical trials. Unfortunately, the dose required to keep a
previously well-chelated patient in negative iron balance
appears to be relatively high. Not surprisingly, side effects
have been observed in some patients receiving L1 (3).
One of the major reasons for the limited efficacy of L1 in
clinical use is that it undergoes extensive phase II metabolism
in the liver (5).
In order to investigate further ligands,
which are able to scavenge iron effectively at low ligand
concentrations, it was decided to synthesize another analogue
of this type, namely 2-methyl-3-hydroxypyridin-4-one (L2) (5b).
In this work, we describe the synthesis,
partition coefficients (Kpart) and the intestinal absorption (I.A) of both
ligands L1 and L2.
All the chemicals used in this work were
obtained from Aldrich (Gillingham, UK). Melting points are
uncorrected. IR spectra were recorded on a Perkin-Elmer 1420.
Proton NMR spectra were determined with EM-390 (80 MHz). Mass
spectra were take using a Vacuum Generaters 16F (35ev).
Elemental analyses were performed by micro analytical
laboratories, University of Manchester, Manchester. The optical
absorbance spectra of the hydroxypyridine ligands were measured
with a Perkin-Elmer model 551 UV/Vis spectrophotometer.
2-Alkyl-3-hydroxypyridinenes (L1 and L2) in
this study were synthesized utilizing the methodology of Harris
(6) (Schema 2).
2-methyl-3-benzyloxypran-4-one (Benzyl maltol) (3)
To a solution of maltol (2) (12 g, 0.1
mol) in methanol (100 ml) 10 ml of an aqueous solution of
sodium hydroxide (4.4 g, 0.11 mol) was added dissolved in water
(10 ml) followed by benzyl chloride (13.9 g, 0.11 mol) and the
mixture was refluxed for 6 h. After removal of the solvent by
rotary evaporation, the residue was mixed with water (50 ml)
and extracted into dichloromethane (3×50 ml). The
combined extracts were washed with 5% sodium hydroxide
(3×150 ml) and then with water (2× 150 ml). The
organic fraction was dried over anhydrous sodium sulphate,
filtered and rotary-evaporated to yield an orange-colored oil,
which solidified on cooling. Recrystallization form diethyl
ether gave the pure product, as colorless needles. 17.7 g
(82%). mp 52-53°C, 1H NMR (DMSO-d6): 2.10
(s, 3H, 2-CH3), 5.10 (s, 2H, O-CH2-Ph), 7.94(d, 1H, 6-H): MS
(EI): m/z=216 (M), IR (KBr): 1640 (C=O) Cm-1. Anal. CalCd. for
C13H12O3: C, 72.21; H, 5.59%. Found: C, 72.31; H, 5.65%.
1,2-dimethyl-3-benzyloxypyridin-4-one hydrochloride (4a)
To a solution of compound 3 (25 g, 0.12
mol) in ethanol (200 ml)/water (200 ml) a 40% aqueous
methylamine (14 g, o.18 mol) was added, followed by 2N sodium
hydroxide solution (10 ml), and mixture refluxed for 12 h.
After pH adjustment with HCl, the volume was reduced to 200 ml
by rotary evaporation prior to addition of water (200 ml) and
washing with diethyl ether (400 ml). Subsequent adjustment of
the aqueous fraction to pH 7 with 10 N NaOH solution was
followed by extraction into dichloromethane (3×400 ml);
the organic layers were then dried over anhydrous sodium
sulphate, filtered and rotary-evaporated to give an orange oil.
This oil was dissolved in ethanol/hydrochloric acid and
rotary-evaporated, the resulting white solid was recrystallized
from ethanol/diethyl ether to give a white powder (24.2 g, 76%)
mp 206-207°C. 1H NMR (DMSO-d6): 2.21 (s, 3H, 2-CH3), 3.94 (s, 3H,
N-CH3), 5.03 (s, 2H, O-CH2-Ph), 6.18 (d, 1H, 5-H) 7.25-7.52 (m,
5H, Ph), 7.58 (d, 1H, 6-H): MS (EI)): m/z=265 (M-HCl). Anal.
Calcd. for C14H16NO2Cl: C, 63.27; H, 6.08; N,5.27; Cl,13.34%. Found:
C, 63.15; H, 6.11; N, 5.21; Cl 13.43%.
1,2-dimethyl-3-hydroxypyridin-4-one hydrochloride (5a)
Compound 4a (20 g, 0.075 mol) was dissolved in ethanol (270
ml)/water (30 ml) and subjected to hydrogenolysis in the
presence of Pd/C catalyst. Filtration, followed by rotary
evaporation gave a white solid; Recrystallization from
ethanol/diethyl ether yielded a white powder (11.6 g, 88%); mp
190-191°C, 1H NMR (DMSO-d6): 2.55 (s, 3H, 2-CH3), 4.05 (s, 3H,
N-CH3), 7.4(d, 1H, 5-H) 8.25 (d, 1H, 6-H): MS (EI): m/z=139
(M), IR (KBr): 3120 (OH), 1632 (C=O, for free base) Cm-1. Anal.
Calcd. for C7H10NO2Cl.H2O: C, 43.42; H, 6.26; N, 7.24; Cl, 18.31%.
Found: C, 43.58; H, 6.18; N, 7.31; Cl, 18.22%.
To a solution of 3 (25 g, 0.12 mol) in
ethanol (200 ml) a 35% aqueous ammonia solution (400 ml) was
added and the mixture was refluxed for 18 h. Removal of the
solvent by rotary evaporation gave an oil which solidified on
addition of acetone and cooling; recrystallization from ethanol
yielded colorless prisms (22 g, 85%); mp 164-165°C, 1H NMR
(DMSO-d6): 2.10 (s, 3H, 2-CH3), 5.01 (s, 2H,
O-CH2-Ph), 6.1(d, 1H, 5-H), 7.20-7.41 (m, 5H, Ph), 7.45 (d, 1H,
6-H): MS (EI): m/z=215 (M), IR (KBR): 1635 (C=O) Cm-1. Anal.
Calcd. for C13H13NO2: C, 72.53; H, 6.01; N, 6.51%. Found: C, 72.41;
H, 6.09 N, 6.43%.
2-methyl-3-hydroxypyridin-4-one hydrochloride (5b)
A solution of 4b (20 g, 0.093 mol) in
ethanol (270 ml)/H2O (30 ml) was adjusted to pH 1 with HCl prior to
hydrogenolysis in the presence of Pd/C catalyst. Filtration,
followed by rotary evaporation gave a white solid;
recystallization from ethanol/diethyl ether yielded a white
powder (13.5 g, 90%): mp 173-174°C, 1H NMR (DMSO-d6):
2.4 (s, 3H, 2-CH3), 7.3 (d, 1H, 5-H), 8.0 (d, 1H, 6-H).
MS (EI): m/z=125 (M-HCl), IR (KBr): 3255 (OH), 1640 (C=O, for
free base) Cm-1. Anal. Calcd. for C6H8NO2Cl: C, 42.24; H, 4.74; N, 8.21; Cl, 20.78%.
Found: C, 42.14; H, 4.69; N, 8.29; Cl, 20.85%.
Determination of partition coefficients
using the shake flask method
A solution of ligands with a concentration
of 10-4 M was prepared in tris buffer (pH 7.4) and the
absorbance of solution was measured in the ultraviolet region
at a wavelength of approximately 280 nm, using the buffer as
the blank. A 50 ml sample of the solution was stirred
vigorously with 50 ml of 1-octanol in a glass vessel for 1 h.
The two layers were separated by centrifugation for 5 min. An
aliquot of the aqueous layer was then carefully removed, using
a glass Pasteur pipette, ensuring that the sample was not
contaminated with 1-octanol. The absorbance of the sample was
measured as above and the partition coefficient, Kpart, was
then calculated using the following formula:
A1=Absorbance reading in the aqueous layer before
A2=Absorbance reading in the aqueous layer after
VW=Volume of aqueous layer used in partitioning
V0=Volume of 1-octanol layer used in partitioning
For each sample, the experiment was
repeated at least four times which led to calculation of a mean
Kpart value and standard deviation (Table 1).
Intestinal absorption (Everted Gut Sac) In this study, the intestinal absorption
(I.A) of bidentate ligands was determined using Everted Gut Sac
(E.G.S) method (7). For this purpose, male wistar rats were
purchased from Tehran Pasteur Institute and kept in the animal
house under standard conditions and fed until their weights
reached 250-300 grams.
Animals were killed by cervical
dislocation. Small intestine was removed, cleaned from debris,
washed, bottle-dried and weighed. The intestine was cut into
small pieces (between 7-8 cm) and the segments were everted.
The everted gut sacs were filled with 200 ml tris buffer (pH
7.4) and suspended in the tris buffer medium with a ligand
concentration of 0-100 mg/lit. The incubated mixture was capped
and gassed with 0.2/CO2=95/5 on water bath shaker at 37°C.
At set time intervals, absorbance of the ligands present within
the sacs was measured using spectrophotometrically at maximum
wavelength (lmax) of 280 nm;
the corresponding concentrations were then determined by
elaborating the Beer-Lambert standard curves.
Results and Discussion
The general methodology (6) adopted for
the synthesis of L1 and L2, is summarized in Schema 1. The commercially
available maltol 2 was benzylated to give 3. Reaction of 3 with
methylamine or ammonia, gave the benzylated pyridinenes 4a and
4b, which were subsequently subjected to catalytic
hydrogenation to remove the protecting group, yielding the
corresponding bidentate chelaters L1 and L2 as hydrochloride salts.
Conversion of maltol to the corresponding
pyridinones can be achieved without the protection of the
3-hydroxyl group (8). However, the yield of this synthetic
route is less than 40% (9).
The partition coefficients of ligands
between 1-octanol and tris buffer (pH 7.4) were determined
using the shake flask method (10). The partition coefficients
of their Fe-complexes (Kpart Fe-complex) can be calculated from
equation 2 (11). The resulting values are presented in table 1.
Surprisingly, the unsubstituted pyridine L2
possesses a higher Kpart value than the corresponding N-methyl
pyridinene L1. This trend also holds for their iron (III)
complexes. A likely explanation for this observation is the
change in the balance of the relative contribution of the
canonical forms (Figure 1, 6 and 7).
When R1 is a methyl group, the resonance
from 7 is stabilized due to the electron donation of methyl
group. Such stabilization is not possible with the
Thus, the dipole of the N-methyl
pyridinone (L1) is predicted to be larger and hence more
hydrophilic than that of the non methylated pyridinone (L2).
In order to investigate the intestinal
absorption of both ligands, by the E.G.S method, the effect of
incubation time on this process was studied first. To follow
this, E.G.S was prepared and incubated in two series of conical
flasks in the tris medium containing 100 mg/lit L1 and/or
L2. At set intervals, E.G.S was removed from the medium
and the concentrations of both ligands within the sacs were
determined. The results showed that maximum L1 and L2 uptake
occurred after 45 min of incubation time (Figures 4 and 5). The
level of ligands uptake was then decreased, suggesting that the
mucosal cells gradually loose their ability to take up ligands
(Fig 4 and 5).
In order to determine whether ligand
uptake by E.G.S was dependent on the concentration of these
iron chelators, various concentrations of ligands (0-100
mg/lit) were added to two series of conical flasks. The sample
solutions were incubated for 45 min under the same conditions
mentioned above. At the end of the incubation time, E.G.S from
each flask was removed and ligand concentration within the sacs
was determined. The data presented in figures 6 and 7 showed
that there was a gradual increase in L1 and L2 uptake
by E.G.S up to a value of 60 mg/lit and thereafter the level
In order for a chelating agent to exert
its pharmacological effect, a drug must be able to reach the
target sites at a sufficient concentration. Hence, the key
property for an orally active iron chelator is its ability to
be efficiently absorbed from the gastrointestinal tract and to
cross biological membranes, thereby gaining access to the
desired target sites, such as the liver. There are several
factors which influence the ability of a compound to freely
permeate a lipid membrane, three of which are, lipophilicity,
ionization state and molecular size.
In order to achieve efficient oral
absorption, the chelator should possess appreciable lipid
solubility, which could facilitate the molecule to penetrate
the gastrointestinal tract (octanol/water partition coefficient
greater than 0.1) (12). Membrane permeability can also be
affected by the ionic state of the compound. Uncharged
molecules penetrate cell membranes more rapidly than charged
molecules. As the pKa values for hydroxypyridinones are in the
region of 3.6 and 9.9, they are neutral over a wide range of
physiological pH values (12). Molecular size is another factor
which influences the rate of drug absorption. Generally,
molecules with molecular masses>400 Da only poorly penetrate
the biological membranes by simple diffusion (12). Bidentate
ligands typically fall within the molecular-mass range of
100-250 Da. Thus, by virtue of their lower molecular masses,
bidentate ligands are likely to have a sufficient
Data which has been reported in table 1
show that the Kpart value of L2 is much higher than the Kpart value of L1. As
mentioned above, this factor plays an important role in drug
absorption. Thus, we expected L2 to exhibit a higher intestinal absorption. The
results presented in figures 4-7, showed that the rate and
extent of intestinal absorption of L2 are not
statistically different from those of L1. It seems
that drug absorption by E.G.S depends on some other factors
such as the number of hydrogen bonds between the drug and
It is possible that for N-methyl
pyridinone (L1), intramolecular hydrogen bonding (Fig 8)
restricts the number of hydrogen-bonding sites, which in turn
aids the transfer of such molecules across the cell membranes,
with H-N derivative (L2). Although the same intramolecular hydrogen
bonding will occur, the hydrogen bonding ability of the H-N
group is unaffected by such interactions and clearly this
functional group has a dominant influence on the penetration of
In support of this hypothesis, the
introduction of a hydroxylalkyl group at the N-position of
hydroxypyridinenes is associated with a decrease in blood brain
barrier penetration compared with N-alkylated
hydroxypyridinones (14). This is due to the formation a
hydrogen bond between the hydroxyalkyl group (at N-position)
and the adjacent molecules.
In conclusion, it can be said that
although from the point of intestinal absorption, the drug L2 has no
advantage over L1, but ammonia in comparison to methylamine,
which is used as a starting material for the synthesis of L2 is more
available than methylamine. The overall yield of the synthesis
of L2 is marginally higher than that of L1; this is
possibly another advantage of L2 over L1.