Pharmaceutical compositions

Compounds which are a 1-hydroxypyrid-2-one in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by a substituent selected from aliphatic acyl, aliphatic amide, aliphatic amine, carboxy, cyano, aliphatic ester, halogen, hydroxy and sulpho groups, alkoxy groups and alkoxy groups substituted by an alkoxy, aliphatic amide, aliphatic amine, aliphatic ester, halogen or hydroxy group, aliphatic hydrocarbon groups and aliphatic hydrocarbon groups substituted by an alkoxy, aliphatic ester, halogen or hydroxy group, but excluding compounds in which said replacement of hydrogen atoms in the compound is effected only by substituents selected from aliphatic hydrocarbon groups, halogen groups and aliphatic hydrocarbon groups substituted by a halogen group, or a salt thereof containing a physiolgically acceptable ion or ions, are of value in the treatment of patients having a toxic concentration of a metal, particularly iron, in the body whilst the iron complexes of such compounds are of value in the treatment of iron deficiency anaemia.

This invention relates to compounds for use in medicine, particularly in 
the treatment of iron overload. 
Certain pathological conditions such as thalassaemia, sickle cell anaemia, 
idiopathic haemochromatosis and aplastic anaemia are treated by regular 
blood transfusions. It is commonly found that such transfusions lead to a 
widespread iron overload, which condition can also arise through increased 
iron absorption by the body in certain other circumstances. Iron overload 
is most undesirable since, following saturation of the ferritin and 
transferrin in the body, deposition of iron can occur and many tissues can 
be adversely affected, particular toxic effects being degenerative changes 
in the myocardium, liver and endocrine organs. Such iron overload is most 
often treated by the use of desferrioxamine. However, this compound is an 
expensive natural product obtained by the culture of Streptomyces and, as 
it is susceptible to acid hydrolysis, it cannot be given orally to the 
patient and has to be given by a parenteral route. Since relatively large 
amounts of desferrioxamine may be required daily over an extended period, 
these disadvantages are particularly relevant and an extensive amount of 
research has been directed towards the development of alternative drugs. 
However, work has been concentrated on three major classes of iron 
chelating agents or siderophores, namely hydroxamates, ethylenediamine 
tetra-acetic acid (EDTA) analogues and catechols. The hydroxamates 
generally suffer from the same defects as desferrioxamine, being expensive 
and acid labile, whilst the other two classes are ineffective at removing 
iron from intracellular sites. Moreover, some cathechol derivatives are 
retained by the liver and spleen and EDTA analogues possess a high 
affinity for calcium and so are also likely to have associated toxicity 
problems. 
We have accordingly studied the iron chelating ability of a wide range of 
compounds and have identified a group of compounds as being of particular 
use for the treatment of conditions involving iron overload. These 
compounds consist of a 1-hydroxypyrid-2-one in which one or more of the 
hydrogen atoms attached to ring carbon atoms are replaced by one of a 
carefully selected group of substituents. None of these compounds has 
previously been used therapeutically. Thus, although certain of the 
substituted compounds described herein have been suggested as potential 
anti-microbial agents, subsequent tests reported from the same source 
(Nishimura et al, Ann. Rept. Shionogi Res. Lab., 1966, 16, 37) showed the 
compounds to have negligible activity. In vitro tests illustrated the lack 
of anti-bacterial and anti-fungal activity and, although some compounds 
showed some anti-protozoal activity in vitro, when tested in mice against 
the fungus Trichomonas vaginalis all of the compounds tested proved to be 
inactive. Moreover, although it has been reported that 
1-hydroxypyrid-2-one will form metal complexes, including an iron complex, 
it has never before been appreciated that certain substituted derivatives 
of this compound might be used with great advantage in a pharmaceutical 
context for the treatment of conditions producing toxic concentrations of 
iron in the body. 
Accordingly the present invention comprises a compound being a 
1-hydroxypyrid-2-one in which one or more of the hydrogen atoms attached 
to ring carbon atoms are replaced by a substituent selected from aliphatic 
acyl, aliphatic amide, aliphatic amine, carboxy, cyano, aliphatic ester, 
halogen, hydroxy and sulpho groups, alkoxy groups and alkoxy groups 
substituted by an alkoxy, aliphatic amide, aliphatic amine, aliphatic 
ester, halogen or hydroxy group, aliphatic hydrocarbon groups and 
aliphatic hydrocarbon groups substituted by an alkoxy, aliphatic ester, 
halogen or hydroxy group, but excluding compounds in which said 
replacement of hydrogen atoms in the compound is effected only by 
substituents selected from aliphatic hydrocarbon groups, halogen groups 
and aliphatic hydrocarbon groups substituted by a halogen group, or a salt 
thereof containing a physiologically acceptable ion or ions, for use in 
medicine. 
Such compounds may be used in both human and veterinary treatment but are 
of particular interest for the treatment of the human body by therapy, 
especially in the context of the treatment of iron overload. 
The 1-hydroxypyrid-2-ones are tautomeric compounds, being alternatively 
named as 2-hydroxypyridine 1-oxides, the two tautomeric structures being 
shown below for the unsubstituted parent compound. 
##STR1## 
The ability of both the free compound and its iron complex to permeate 
membranes is important in the context of the treatment of iron overload, 
and it is also desirable for both to possess some degree of water 
solubility. A good indication of the physical properties of a compound and 
its iron complex in this respect is provided by the value of the partition 
coefficient (K.sub.part) obtained on partition between n-octanol and tris 
hydrochloride (20 mM, pH 7.4; tris representing 
2-amino-2-hydroxymethylpropane 1,3-diol) at 20.degree. C. and expressed as 
the ratio (concentration of compound in organic phase)/(concentration of 
compound in aqueous phase). Preferred compounds show a value of K.sub.part 
for the free compound of above 0.02 but less than 3.0, especially of above 
0.2 but less than 1.0, together with a value of K.sub.part for the neutral 
3:1 hydroxypyridone:iron(III) complex of above 0.02 but less than 6.0, 
especially of above 0.2 but less than 1.0. The following comments upon 
preferences among the groups used for replacement of hydrogen atoms 
attached to carbon atoms of the pyridone ring are directed towards the use 
of compounds having partition coefficients in the free and complexed state 
which lie in these preferred ranges. For examples of measured partition 
coefficients of specific compounds reference should be made to Table 1 of 
Example 2. 
More than one of the ring carbon atoms may be substituted, for example two 
of such atoms, either by the same substituent group or by different 
substituent groups, for example by halogen or especially by an aliphatic 
hydrocarbon group together with another type of substituent, although 
compounds in which only one of the ring carbon atoms is substituted are 
preferred. Substitution may occur at any of the 3-, 4-, 5-and 6-positions 
or at a combination of two or more of these positions. Particularly when 
the ring carbon atoms are substituted by the larger groups, however, there 
may be an advantage in avoiding substitution on a carbon alpha to the 
##STR2## 
system. The system is involved in the complexing with iron and the close 
proximity of one of the larger aliphatic hydrocarbon groups may lead to 
steric effects which inhibit complex formation. Substitution at the 5- and 
particularly the 4-position is thus of some especial interest. 
Where a ring carbon atom is substituted by an aliphatic hydrocarbon group, 
this group may be cyclic or acyclic, having a branched chain or especially 
a straight chain in the latter case, and may be unsaturated or especially 
saturated. Groups of from 1 to 6 carbon atoms, particularly of 1 to 4 and 
especially of 1 to 3 carbon atoms, are of most interest. Alkyl groups are 
preferred, for example cyclic groups such as cyclopropyl and especially 
cyclohexyl but, more particularly preferred are acyclic groups such as 
isopropyl, n-propyl, ethyl and especially methyl. However, although 
substitution by an aliphatic hydrocarbon group, for example methyl, in 
addition to another substituent as specified above is quite acceptable, it 
will not generally contribute with any particular advantage to the 
properties of the compound and is thus not of especial interest. 
In the case of substituted aliphatic hydrocarbon groups, the preferences as 
to the nature of these groups are broadly as expressed above with regard 
to the hydrocarbon group and hereinafter with regard to the substituent, 
for example these groups conveniently being substituted alkyl groups of 1 
to 3 carbon atoms and particularly substituted methyl groups such as 
chloromethyl, ethoxymethyl, and especially hydroxymethyl. In general, 
however, substitutents as defined hereinbefore other than aliphatic 
hydrocarbon groups and substituted aliphatic hydrocarbon groups are of the 
most interest. Various preferences may be expressed among such other 
substituent groups, the following comments applying equally to these 
groups when substituted on the ring directly and, where appropriate, also 
to the groups when substituted on an aliphatic hydrocarbon or alkoxy group 
which is itself substituted on the ring. 
An aliphatic acyl group may contain a sulphonyl or carbonyl group. The 
latter type are however preferred and although the acyl group may be a 
formyl group, alkylcarbonyl groups are of most interest. Such acyl groups 
may, for example, be of 2 to 4 or 5 carbon atoms, and particularly may 
contain alkyl groups of the type described above as being preferred as an 
aliphatic hydrocarbon group substituent on the ring, being, for example, 
--COCH.sub.2 CH.sub.3 or especially --COCH.sub.3. Alkoxy groups may 
conveniently be of 1 to 4 carbon atoms and contain similar alkyl groups to 
those which are preferred in the alkylcarbonyl groups, examples of such 
substituents being ethoxy and particularly methoxy. Alkoxy groups which 
are substituted, however, may often conveniently contain 2 or more carbon 
atoms in view of the relative instability of groups such as 
##STR3## 
etc., so that a particular substituted alkoxy group of interest is 
--OCH.sub.2 CH.sub.2 OCH.sub.3. Moreover, the presence of a hydrophilic 
substituent on an alkoxy group will tend to offset the hydrophobic effect 
of the aliphatic hydrocarbon group which that alkoxy group contains, 
thereby sometimes favouring the use of slightly larger alkoxy groups when 
these are substituted. Substituent alkoxy groups are of particular 
interest in the context of the present invention and are discussed in more 
detail hereinafter. 
Amine substituents may consist of a group --NH.sub.2 or its charged 
equivalent, a group --NH.sub.3, which will be associated with a 
physiologically acceptable anion, for example a chloride or other halide 
ion, a solubilising ion such as that from methane sulphonic or isethionic 
acid, or an anion derived from the hydroxy group of the ring 
(OH.fwdarw.O.sup.-), or such a --NH.sub.2 or NH.sub.3 group in which one 
or more of the hydrogen atoms is replaced by an aliphatic hydrocarbon 
group, for example such a group as is described above as a substitutent. 
Amide substituents may contain a sulphonyl or a carbonyl group. The latter 
type are, however, of most interest and the further discussion will 
therefore refer to them although it applies equally to the sulphonyl type. 
The amide substituent may be of the unsubstituted form --CONH.sub.2, i.e. 
being a carbamoyl group, or may contain a nitrogen atom which is mono- or 
di-substituted as just described for the amine substituents, for example 
being a group --CONHCH.sub.3, etc. Alternatively, the 
##STR4## 
grouping of the amide substituent may be arranged in the opposite sense so 
that the nitrogen atom of the amide grouping is attached to the ring, the 
carbonyl group being attached to an aliphatic hydrocarbon group, for 
example an alkyl group such as is described above as a substituent, or in 
the case of a carboxylic acid amide but not in that of a sulphonic acid 
amide, to hydrogen. In the case of an amide group arranged in this 
opposite sense, the nitrogen atom may carry a hydrogen atom or be 
mono-substituted as discussed for amide substituents of the first 
mentioned form, that form of amide substituent being the one of particular 
interest. 
Carboxy and sulpho substituents, the former of which are preferred, may be 
present as the group --CO.sub.2 H or --SO.sub.3 H, or as the anion derived 
therefrom in combination with a physiologically acceptable cation, for 
example as described hereinafter. Ester substituents may contain a 
sulphonyloxy or preferably a carbonyloxy group and this may be arranged in 
either sense, i.e. with a carboxylic acid ester the group --CO.O-- may 
have either the carbonyl group or the oxy group linked to the carbon atom 
of the ring (through an aliphatic hydrocarbon group on which the ester 
group is substituted, where appropriate). The other group of oxy and 
carbonyl will be linked to an aliphatic hydrocarbon group forming part of 
the ester group or, in the case where this is a carbonyl group may 
alternatively be linked to hydrogen (this latter possibility does not 
apply in the case of sulphonic acid esters). Once again, preferred 
aliphatic hydrocarbon groups contained by the ester group are those 
described above as substituents. Ester groups in which the oxy group is 
linked to the ring are preferred, for example the groups --O.COCH.sub.3 
and --O.COC.sub.2 H.sub.5 rather than --CO.sub.2 CH.sub.3 and --CO.sub.2 
CH.sub.2 CH.sub.3. With aliphatic hydrocarbon groups or alkoxy groups 
substituted by an ester group there is a particularly strong preference 
for the oxy group to be attached to this aliphatic hydrocarbon group or 
alkoxy group, groups such --CH.sub.2 O.COCH.sub.3 therefore being of 
interest. Halogen substituents may conveniently be iodo, fluoro, bromo or 
especially chloro. 
Among preferred substituents are the hydroxy group, and also alkoxy groups, 
for example ethoxy and particularly methoxy, and, more particularly, 
substituted alkoxy groups, especially those substituted by a hydroxy group 
or another alkoxy group, for example the substituted ethoxy groups such as 
--OCH.sub.2 CH.sub.2 OCOCH.sub.3, --OCH.sub.2 CH.sub.2 NHCOCH.sub.3, 
--OCH.sub.2 CH.sub.2 NH.sub.2 and especially --OCH.sub.2 CH.sub.2 OH and 
--OCH.sub.2 CH.sub.2 OCH.sub.3. Hydroxy substituted aliphatic hydrocarbon 
groups, for example hydroxymethyl, are also of generally greater interest 
than other substituted aliphatic hydrocarbon groups. 
Although simple alkoxy substituents, the alkoxy groups of hydroxyalkoxy 
substituents and both components of alkoxyalkoxy substituents may, as 
indicated previously, be of a range of sizes, for example 1 to 6 carbon 
atoms, certain factors result in a preference for groups of a particular 
size. Thus, the hydrophilic/hydrophobic balance in a compound, which is 
indicated by its K.sub.part value, may be adjusted to a value in the 
preferred range quoted hereinbefore by the use of additional ring 
substituents, so that the hydrophobic effect of a large unsubstituted 
alkoxy group can be offset by the presence of a further hydrophilic 
substituent, such as a hydroxy group, on another carbon atom of the ring. 
However, it is generally preferable to use a single substituent which 
itself confers the appropriate degree of balance. Accordingly, 
unsubstituted alkoxy group substituents of 1 to 3 or 4, preferably 1 or 2 
carbon atoms, and hydroxy substituted alkoxy group substituents of 2 to 4, 
preferably 2 or 3 carbon atoms (substituted methoxy groups being of less 
interest in view of the instability of the 
##STR5## 
linkage referred to previously), are of particular interest. For similar 
reasons there is particular interest in alkoxy substituted alkoxy group 
substituents of 2 to 4, preferably 2 or 3 carbon atoms, in the first 
alkoxy group substituted onto the ring and of 1 to 4, preferably 1 to 3 
carbon atoms in the second alkoxy group which is substituted onto the 
first alkoxy group, with the proviso that the overall number of carbon 
atoms is preferably no greater than 6, and especially no greater than 3 or 
4 carbon atoms. 
Although the hydroxy, methoxy, hydroxymethoxy and methoxyethoxy groups 
already referred to are of particular interest as substitutents, other 
specific examples of alkoxy and substituted alkoxy groups, in addition to 
those specifically mentioned previously, are 3-hydroxypropoxy, 
2-hydroxy-1-methylethoxy and 3-methoxypropoxy. 
Hydroxy, alkoxy, substituted alkoxy and other groups may conveniently be 
substituted at the 4-position of a 1-hydroxypyrid-2-one, for example at 
the 4-position of 1-hydroxy-6-methylpyrid-2-one or other C-methyl 
substituted 1-hydroxypyrid-2-one or, more especially, at the 4-position of 
otherwise unsubstituted 1-hydroxypyrid-2-one. Specific examples of 
compounds according to the present invention are thus as follows: 
##STR6## 
wherein R is a substituent group as defined hereinbefore, for example 
methyl and especially 6-methyl, hydroxy, etc., x is 0, 1, 2 or 3 (the ring 
not containing any further substituent R when x is 0), n is 0, 1, 2, 3 or 
4, m is 1, 2, 3 or 4 and R' is hydrogen or --(CH.sub.2)nCH.sub.3, 
preferences among the groups at the 4-position being as described 
hereinbefore. 
The compounds may, if desired, contain substituent groups, particularly an 
aliphatic amine, carboxy or sulpho group, in the salt form. Alternatively, 
a salt may be formed with the 
##STR7## 
system produced by the loss of a proton from the hydroxy group 
N-substituted at the 1-position of the ring (or C-substituted at the 
2-position of the ring in the tautomeric form). Such salts contain a 
physiologically acceptable cation, for example the cation of an alkali 
metal such as sodium, quaternary ammonium ions or protonated amines such 
as the cation derived from tris (tris represents 2-amino-2-hydroxymethyl 
propane 1,3-diol). Salt formation may be advantageous in increasing the 
water solubility of a compound but, in general, the use of the compounds 
themselves rather than their salts, is preferred. 
Certain of the substituted 1-hydroxypyrid-2-ones described herein are known 
compounds, in particular the compounds having a single substituent at the 
4-position which is an acetamido, amino, butoxy, carbamyl, carboxy, cyano, 
ethoxy, ethoxycarbonyl, methoxy or propoxy group, but all the other 
compounds described above are believed to be novel, including the 
particularly interesting compounds which are substituted by an additional 
hydroxy group, for example 1,4-dihydroxypyrid-2-one. The present invention 
thus includes, per se, the compounds described hereinbefore but excluding 
these known compounds. 
The substituted 1-hydroxypyrid-2-ones (or 2-hydroxypyridine N-oxides) for 
use in the present invention may be synthesised by various routes applying 
standard reactions for the introduction of the substituent groups within 
the art of pyridine chemistry. In particular, substituents may be 
introduced either by replacement of a hydrogen atom or of an existing 
substituent at the appropriate position or positions in a pyridine or 
pyridine 1-oxide ring system. Pyridine compounds may conveniently be 
converted to the corresponding pyridine 1-oxide by the use of an oxidizing 
agent such as peracetic or perbenzoic acid. The oxygen atom at the 
2-position of compounds according to the present invention may 
conveniently be introduced by the basic hydrolysis of a halogen group or 
the acidic hydrolysis of an alkoxy group, for example a methoxy group, at 
that position, preferably in a pyridine 1-oxide rather than a pyridine and 
conveniently following introduction of the other substituent groups or 
groups. Such a procedure will introduce a hydroxy group at the 2-position 
as in the 2-hydroxypyridine N-oxide tautomeric form shown hereinbefore. 
Such procedures and the preparation of various suitable intermediates are 
described in the art, for example by Shaw et al, J. Amer. Chem. Soc., 
1949, 71, 70 and ibid, 1950, 72, 4362, and particularly by Mizukami et al, 
Ann. Rept. Shionogi Res. Lab., 1966, 16, 29. A particularly useful type of 
intermediate for the preparation of the compounds described herein is a 
nitro substituted 2-chloro-pyridine N-oxide, 4-nitro, 5-nitro and 
3,5-dinitro substituted compounds all being reported in the literature. 
Thus, 2-chloro-4-nitropyridine-1-oxide, for example, may be subjected to 
nucleophilic substitution to replace the nitro group by an alkoxy group or 
alkoxy substituted alkoxy group, for example --OCH.sub.3 or --OCH.sub.2 
CH.sub.2 OCH.sub.3, the chloro group then being converted to a hydroxy 
group by basic hydrolysis. Alternatively, a nitro group substituent may be 
reduced to give an amino group which may in turn be acylated. 
The compounds may be converted to salts formed with the anion produced by 
the loss of the hydroxy group proton or with a substituent such as a 
carboxy, sulpho or amino group by reaction with the appropriate base or 
acid according to standard procedures (amino substituted compounds of a 
zwitterion type containing a cation from the amino group and such a 
hydroxy group-derived anion may be prepared by crystallisation from 
aqueous media at a pH of about 9). 
In general, it is preferred that the compounds are isolated in 
substantially pure form, i.e. substantially free from by-products of 
manufacture. 
It will be appreciated that these are not the only routes available to 
these compounds and that various alternatives may be used as will be 
apparent to those skilled in the art, as will be the routes to the various 
intermediates required. 
Moreover, it will be appreciated that certain of the compounds may be 
converted in vivo to other compounds which will be involved in the metal 
binding activity observed in vivo. This will be true, for example, of 
compounds containing ester groups which are likely to be converted to 
carboxy groups when the compounds are administered orally. 
The compounds may be formulated for use as pharmaceuticals for veterinary, 
for example in an avian or particularly a mammalian context, or 
particularly human use by a variety of methods. For instance, they may be 
applied as an aqueous, oily or emulsified composition incorporating a 
liquid diluent which most usually will be employed for parenteral 
administration and therefore will be sterile and pyrogen free. However, it 
will be appreciated from the foregoing discussion in relation to 
desferrioxamine that oral administration is to be preferred and the 
compounds of the present invention may be given by such a route. Although 
compositions incorporating a liquid diluent may be used for oral 
administration, it is preferred to use compositions incorporating a solid 
carrier, for example a conventional solid carrier material such as starch, 
lactose, dextrin or magnesium stearate, the oral composition then 
conveniently being of a formed type, for example as tablets, capsules 
(including spansules), etc. 
The present invention accordingly further comprises a pharmaceutical 
composition containing a compound being a a 1-hydroxypyrid-2-one in which 
one or more of the hydrogen atoms attached to ring carbon atoms are 
replaced by a substituent selected from aliphatic acyl, aliphatic amide, 
aliphatic amine, carboxy, cyano, aliphatic ester, halogen, hydroxy and 
sulpho groups, alkoxy groups and alkoxy groups substituted by an alkoxy, 
aliphatic amide, aliphatic amine, aliphatic ester, halogen or hydroxy 
group, aliphatic hydrocarbon groups and aliphatic hydrocarbon groups 
substituted by an alkoxy, aliphatic ester, halogen or hydroxy group, but 
excluding compounds in which said replacement of hydrogen atoms in the 
compound is effected only by substituents selected from aliphatic 
hydrocarbon groups, halogen groups and aliphatic hydrocarbon groups 
substituted by a halogen group, or a salt thereof formed between the anion 
produced by the loss of the hydroxy group proton and a physiologically 
acceptable cation, together with a physiologically acceptable solid 
carrier. 
Other forms of administration than by injection or through the oral route 
may also be considered in both human and veterinary contexts, for example 
other forms known in the art such as the use of suppositories or 
pessaries, particularly for human administration. 
Compositions may be formulated in unit dosage form, i.e. in the form of 
discrete portions each comprising a unit dose, or a multiple or 
sub-multiple of a unit dose. Whilst the dosage of active compound given 
will depend on various factors, including the particular compound which is 
employed in the composition, it may be stated by way of guidance that 
satisfactory control of the amount of iron present in the human body will 
often be achieved using a daily dosage of about 0.1 g to 5 g, particularly 
of about 0.5 g to 2 g, veterinary doses being on a similar g/Kg body 
weight ratio. However, it will be appreciated that it may be appropriate 
under certain circumstances to give daily dosages either below or above 
these levels. Where desired, more than one compound according to the 
present invention may be administered in the pharmaceutical composition 
or, indeed, other active compounds may be included in the composition. 
Although suggestions have previoulsy been made concerning use of certain of 
the compounds described herein in a pharmaceutical context as 
anti-microbials, these suggestions did not lead to a therapeutic use for 
the compounds. We have found that the 1-hydroxypyrid-2- ones described 
herein are particularly suited to the removal of iron from patients having 
an iron overload. The compounds form neutral 3:1 iron complexes at most 
physiological pH values, and have the advantage that they do not 
co-ordinate calcium or magnesium. Both the compounds and their complexes 
will partition into n-octanol indicating that they will permeate 
biological membranes, this property being confirmed in practice by tests 
of the ability of the .sup.59 Fe labelled iron complexes to permeate 
erythrocytes. 
The 1-hydroxypyrid-2-ones possess a high affinity for iron(III), as 
evidenced by log K.sub.sol values (log K.sub.sol is defined as being equal 
to log .beta..sub.Fe(L)n +21-[pK.sub.sp +n log a.sub.L(H+) +m log a.sub.L 
(Ca++)] where log .beta..sub.Fe(L)n is the cumulative affinity constant of 
the ligand in question for iron(III), pK.sub.sp is the negative logarithm 
of the solubility product for Fe(OH).sub.3 and has a value of 39, n and m 
are the number of hydrogen and calcium ions, respectively, which are bound 
to the ligand, and a.sub.L(H+) and a.sub.L (Ca++) are the affinities of 
the ligand for hydrogen ions and calcium ions, respectively). In order to 
solubilise iron(III) hydroxide, log K.sub.sol must be greater than 0 and 
in order to remove iron from transferrin, log K.sub.sol should be in 
excess of 6.0. The log K.sub.sol values for 1,4-dihydroxypyrid-2-one and 
1-hydroxy-4-methoxypyrid-2-one by way of example, are 9.9 and 11.3, 
respectively, thus comparing favourably with those of the bidentate 
hydroxamates at about 4.0, of catechols at about 8.0, of desferrioxamine 
at 6.0, and of diethylenetriamine penta-acetic acid (DTPA) at 2.0. 
Moreover, the ability of the compounds to remove iron efficiently has been 
confirmed both by in vitro tests and also by in vivo tests in mice. It is 
particularly significant that these latter tests are successful whether 
the compound is given intraperitoneally or orally by stomach tube, the 
compounds generally either being stable under acidic conditions or being 
converted thereby to acid stable active compounds. Oral activity is not 
generally present among the other types of compound previously suggested 
for use as iron co-ordinating drugs and although certain EDTA analogues do 
show such activity, they possess drawbacks for pharmaceutical use. 
In addition to the use described hereinbefore for the treatment of general 
iron overload, the hydroxypyridones described herein are also of interest 
for use in certain pathological conditions where there may be an excess of 
iron deposited at certain sites even though the patient does not exhibit a 
general iron overload, this being the case, for example, in certain 
arthritic and cancerous conditions. Indeed in some patients having such 
conditions, the patient may exhibit an overall aneamia and the metal-free 
1-hydroxypyrid-2-ones may then be used in conjunction with an iron 
complex, for example an iron complex of the same or another of these 
1-hydroxypyrid-2-ones, the iron complex acting to correct the overall 
anaemia whilst the metal-free compound acts to remove iron from 
pathological to physiological sites. Such iron complexes of the 
1-hydroxypyrid-2-ones and their use in this context are discussed in 
detail hereinafter. 
Uses of the compounds of the present invention for combination with metals 
other than iron may extend to the treatment of body fluids outside the 
body or even to quite other contexts than the treatment of patients. One 
particular area of some interest involves the treatment of patients on 
haemodialysis who may show a dangerous build up of aluminium in the body. 
For the treatment of such patients the compounds of the present invention 
may be insolubilised through attachment to a support material and then 
contacted with the patient's blood to remove aluminium therefrom. The 
support material may conveniently be one of various types of polymer 
described in the art for use in similar contexts, for example a 
carbohydrate material which may be of an agarose, dextran or other type, 
or a polystyrene or other material such as is used in ion-exchange resins. 
Various approaches known in the art may be used for effecting attachment of 
the compounds to such support materials but one convenient approach is to 
use an acidic or basic group on the support material to provide an amide 
type linkage through reaction with the hydroxypyridone. Hydroxypyridones 
of particular interest in this context are those containing acidic or 
basic substituents on a ring carbon atom, i.e. those containing an 
aliphatic amine or a sulpho or especially a carboxy group substituent. 
(Substituted hydroxypyridones containing such a substituent which is an 
ionisable group are in fact generally of rather lesser interest for use in 
the pharmaceutical compositions of the present invention in view of their 
less effective membrane permeating properties.) 
Just as iron overload can pose problems in some patients, iron deficiency 
anaemia can pose problems in others. As well as being of value as the 
metal-free compounds for the treatment of conditions involving iron 
overload, the substituted 1-hydroxypyrid-2-ones described hereinbefore are 
of interest in the iron complex form for the treatment of iron deficiency 
anaemia. 
An adequate supply of iron to the body is an essential requirement for 
tissue growth in both man and animals. Although there is normally an ample 
amount of iron in the diet, the level of absorption of iron from food is 
generally low so that the supply of iron to the body can easily become 
critical under a variety of conditions. Iron deficiency anaemia is 
commonly encountered in pregnancy and may also present a problem in the 
newly born, particularly in certain animal species such as the pig. 
Moreover, in certain pathological conditions there is a mal distribution 
of body iron leading to a state of chronic anaemia. This is seen in 
chronic diseases such as rheumatoid arthritis, certain haemolytic diseases 
and cancer. 
Although a wide range of iron compounds is already marketed for the 
treatment of iron deficiency anaemia, the level of iron uptake by the body 
from these compounds is often quite low, necessitating the administration 
of relatively high dosage levels of the compound. The administration of 
high dose, poorly absorbed, iron complexes may cause siderosis of the gut 
wall and a variety of side effects such as nausea, vomiting, constipation 
and heavy malodorous stools. We have now found that the iron complexes of 
the substituted 1-hydroxypyrid-2-ones described hereinbefore, none of 
which are believed to have been previously prepared, are of particular 
value in the treatment of such conditions. 
Accordingly the present invention further comprises an iron complex of a 
1-hydroxypyrid-2-one in which one or more of the hydrogen atoms attached 
to ring carbon atoms are replaced by a substituent selected from aliphatic 
acyl, aliphatic amide, aliphatic amine, carboxy, cyano, aliphatic ester, 
halogen, hydroxy and sulpho groups, alkoxy groups and alkoxy groups 
substituted by an alkoxy, aliphatic amide, aliphatic amine, aliphatic 
ester, halogen or hydroxy group, aliphatic hydrocarbon groups and 
aliphatic hydrocarbon groups substituted by an alkoxy, aliphatic ester, 
halogen or hydroxy group, but excluding compounds in which said 
replacement of hydrogen atoms in the compound is effected only by 
substituents selected from aliphatic hydrocarbon groups, halogen groups 
and aliphatic hydrocarbon groups substituted by a halogen group. 
The comments made hereinbefore in relation to K.sub.part values for the 
metal-free compounds and their corresponding iron complexes in the case of 
preferred compounds apply equally to the selection of preferred metal-free 
compounds and of preferred iron complexes. The comments made hereinbefore 
with regard to preferences as to the nature and position of substituents 
thus apply equally in relation to the iron complexes. 
The iron complexes present in the pharmaceutical compositions according to 
the present invention preferably contain iron in the ferric state. 
Although the use of complexes containing iron in the ferrous state may be 
considered, such complexes tend to be less stable and are thus of less 
interest. The iron complexes are preferably neutral, i.e. there being an 
internal balance of charges between the metal cation and the ligand(s) 
bound covalently thereto without the necessity for the presence of a 
non-covalently bound ion or ions, for example a chloride ion, to achieve 
balance. Moreover, the use of hydroxypyridones containing ionisable 
substituent groups is of less interest and it is preferred that this 
internal balance of charges is achieved by complexing with the iron cation 
the appropriate number of anions derived from a hydroxypyridone by the 
loss of a hydroxy proton which are necessary to produce neutrality. 
Preferred iron complexes of use in the present invention are thus of the 
3:1 form, containing three hydroxypyridone anions complexed with a ferric 
cation. It will be appreciated, however, that the invention does not 
exclude the use of complexes of the 1:1 or particularly the 2:1 form, 
usually in association with a physiologically acceptable anion or anions 
to achieve neutrality, for example the chloride ion. It will be 
appreciated, therefore, that the invention particularly includes as 
compounds, per se, a neutral iron complex containing 1 molar proportion of 
iron(III) and 3 molar proportions of a hydroxypyridone as defined 
hereinbefore. 
The iron complexes are conveniently prepared by the reaction of the 
hydroxypyridone and iron ions, the latter conveniently being derived from 
an iron salt, particularly a ferric halide and especially ferric chloride. 
The reaction is conveniently effected in a suitable mutual solvent and 
water may often be used for this purpose. If desired, however, an 
aqueous/organic solvent mixture may be used or an organic solvent, for 
example ethanol, methanol, chloroform and mixtures of these solvents 
together and/or with water where appropriate. In particular, methanol or 
especially ethanol may be used as the solvent where it is desired to 
effect the separation of at least a major part of a by-product such as 
sodium chloride by precipitation whilst the iron complex is retained in 
solution. Alternative procedures may, however, be used and will be 
apparent to those skilled in the art. 
It will be appreciated that the nature of the iron complex obtained by the 
reaction of a hydroxypyridone and iron ions will depend both on the 
proportion of these two reactants and upon the pH of the reaction medium. 
Thus, for the preparation of the 3:1 ferric complex, for example, the 
hydroxypyridone and the ferric salt are conveniently mixed in solution in 
a 3:1 molar proportion and the pH adjusted to a value in the range of 6 to 
9, for example 7 or 8. If a similar excess of hydroxypyridone:iron is 
employed, but no adjustment is made of the acidic pH which results on the 
admixture of the hydroxypyridone and an iron salt such as ferric chloride, 
then a mixture of the 2:1 and 1:1 complex will instead be obtained. 
Adjustment of the pH may conveniently be effected by the addition either 
of sodium carbonate or of a hydroxide base such as sodium or ammonium 
hydroxide, the use of a hydroxide base being or particular interest when 
preparing the iron complexes in batches of 20 g or more. When using a 
hydroxide base, the reaction may conveniently be carried out in a medium 
containing water as the solvent, for example in water or an ethanol:water 
mixture, and the pH adjusted by the addition of a 2 molar aqueous solution 
of the base. It will be appreciated that the presence of water in the 
reaction mixture will lead to the retention of a by-product in the iron 
complex on evaporation of the solvent (a chloride where the iron salt is 
ferric chloride). However, this can be removed, if desired, by procedures 
such as crystallisation from a suitable solvent system or sublimation in 
the particular case of ammonium chloride. 
Reaction to form the iron complex is generally rapid and will usually have 
proceeded substantially to completion after 5 minutes at about 20.degree. 
C., although a longer reaction time may be used if necessary. Following 
separation of any precipitated by-product, such as sodium chloride in the 
case of certain solvent systems, the reaction mixture may conveniently be 
evaporated on a rotary evaporator or freeze dried to yield the solid iron 
complex. This may, if desired, be crystallised from a suitable solvent, 
for example water, an alcohol such as ethanol, or a solvent mixture, 
including mixtures containing an ether. The present invention thus further 
includes a process for the preparation of an iron complex of a 
1-hydroxypyrid-2-one as defined hereinbefore which comprises reacting said 
hydroxypyridone with iron ions and isolating the resultant complex. 
Whilst for some uses it may be appropriate to prepare the iron complex in 
substantially pure form, i.e. substantially free from by-products of 
manufacture, in other cases, for example with a solid oral formulation as 
described hereinafter, the presence of by-products such as sodium chloride 
may be quite acceptable. In general, however, the neutral 3:1 
[hydroxypyridone:iron(III)]complex is of particular interest in a form 
free from by-products which are complexes containing different proportions 
of hydroxypyridone and iron, in particular the 2:1 and 1:1 complexes. 
Accordingly the present invention includes an iron complex, for example 
the 3:1 hydroxypyridone:iron(III) complex, of a 1-hydroxypyrid-2-one as 
defined hereinbefore, when in a form substantially free from iron 
complexes of the hydroxypyridone containing other proportions of iron. As 
indicated hereinafter, it may be advantageous under some circumstances for 
the iron complex to be used in admixture with the free hydroxypyridone 
and, if desired, such a mixture may be obtained directly by reacting a 
molar proportion of the hydroxypyridone and iron ions of greater than 3:1. 
The iron complexes may be formulated as pharmaceuticals for veterinary, for 
example in an avian or particularly a mammalian context, or human use by a 
variety of methods and the invention includes a pharmaceutical composition 
comprising an iron complex as hereinbefore defined together with a 
physiologically acceptable diluent or carrier. The comments made 
hereinbefore with regard to the formulation of the metal-free compounds 
apply equally to the iron complexes, although in this instance 
compositions for parenteral administration are of greater interest 
particularly in the context of animal treatment. The problems of iron 
deficiency anaemia in newly born pigs arise primarily during the first 
three weeks or so of their life when a very rapid weight gain takes place. 
The iron complexes of the present invention may be used to treat piglets 
directly by a parenteral route, such as intramuscular or oral, for example 
as a liquid preparation "injected into the mouth". However, an alternative 
approach is to enhance the iron content of the milk on which the piglets 
are feeding by treating the mother pig using oral or parenteral 
administration, for example an injectable slow release preparation (such 
an approach may also be an interest in a human context). When it is 
applicable to feed piglets on foodstuffs other than the milk of the mother 
pig, it may also be possible to effect the pharmaceutical administration 
of the iron complex in this other foodstuff. 
As with the metal-free compounds, the dosage of the hydroxypyridone iron 
complex which is given will depend on various factors, including the 
particular compound which is employed in the composition. It may be stated 
by way of guidance, however, that maintenance of the amount of iron 
present in the human body at a satisfactory level will often be achieved 
using a daily dosage, in terms of the iron content of the compound, which 
lies in a range from about 0.1to 100 mg and often in a range from 0.5 to 
10 mg, for example 1 or 2 mg, veterinary doses being on a similar g/Kg 
body weight ratio. However, it will be appreciated that it may be 
appropriate under certain circumstances to give daily dosages either below 
or above these levels. In general, the aim should be to provide the amount 
of iron required by the patient without administering any undue excess and 
the properties of the pharmaceutical compositions according to the present 
invention are particularly suited to the achievement of this aim. 
Where desired, an iron complex of more than one hydroxypyridone as 
described above may be present in the pharmaceutical composition or indeed 
other active compounds may be included in the composition, for example 
compounds having the ability to facilitate the treatment of anaemia, such 
as folic acid. Another additional component which may be included in the 
composition, if desired, is a source of zinc. Iron compounds used in the 
treatment of iron deficiency anaemia can inhibit the mechanism of zinc 
uptake in the body and this can cause serious side effects in the foetus 
when treating anaemia in a pregnant female. It is believed, however, that 
the iron complexes of the present ivnention have a further advantage in 
that they either do not have this effect or exhibit the effect at a lower 
level than the compounds at present used in the treatment of anaemia. 
Accordingly, it may often be the case that the level of zinc providing 
compound added to the composition may not require to be high or, with 
preferred formulations of the iron complexes, may be dispensed with 
altogether. 
It has never before been appreciated that the iron complexes such as those 
described herein might be used, and with great advantage, in a 
pharmaceutical context. Accordingly the present invention includes an iron 
complex of a 1-hydroxypyrid-2-one as defined hereinbefore for use in 
medicine, particularly in the treatment of iron deficiency anaemia (in the 
broad sense of this term). 
We have found that the iron complexes described herein are of value in the 
treatement of iron deficiency anaemia both in humans and also in a 
veterinary context, particularly for the treatment of various mammalian 
species and especially pigs. The complexes will partition into n-octanol 
indicating that they are able to permeate biological membranes, this 
property being confirmed in practice by tests of the ability of the 
.sup.59 Fe labelled iron complexes to permeate erythrocytes. The ability 
of the compounds in this respect will depend on the nature of the 
substituent(s) present therein and the reflection of this ability in the 
K.sub.part values of various compounds has been referred to hereinbefore. 
The ability of the iron complexes of the present invention to promote iron 
uptake with a high level of efficiency, as compared with a range of other 
iron complexes currently marketed for the treatment of iron deficiency 
anaemia, has been confirmed by measurements in the rat small intestine. 
Once present in the bloodstream, the complexes will donate iron to 
transferrin, a position of equilibrium being set up between the complexes 
and transferrin. It is because of the existence of this equilibrium that 
the corresponding free hydroxypyridones may equally be used in the 
treatment of iron overload, although certain of these compounds may be of 
particular value for use in the free state for iron removal and others may 
be of particular value for use as iron complexes for iron supply. 
Certain aspects of their formulation may enhance the activity of the 
complexes in particular contexts. Thus, although the neutral 3:1 ferric 
complexes are of particular value as being stable over a wide pH range 
from about 4 or 5 up to 10, they will dissociate at the pH values of less 
than 4 prevailing in the stomach to form a mixture of the 2:1 and 1:1 
complex together with the free hydroxypyridone. If these complexes and the 
free hydroxypyridone are cleared simultaneously from the stomach, when 
they reach the small intestine a large proportion of the 3:1 complex 
should reform under the alkaline conditions present therein. However, in 
the event that this dissociation under acid conditions leads to a 
significant reduction in the uptake of iron by the body, due for instance 
to absorption of the free hydroxypyridone through the stomach wall, the 
uptake may be improved by using one or more of the following procedures in 
the formulation of the iron complex. 
Firstly, one of several variations may be employed which avoid or reduce 
exposure of the iron complex to the acidic conditions of the stomach. Such 
approaches may involve various types of controlled release system, ranging 
from one, which may for example be based on a polymer, which simply 
provides a delayed release of the complex with time, through a system 
which is resistant to dissociation under acidic conditions, for example by 
the use of buffering, to a system which and is biased towards release 
under conditions such as prevail in the small intestine, for example a pH 
sensitive system which is stabilised towards a pH of 1 to 3 such as 
prevails in the stomach but not one of 7 to 9 such as prevails in the 
small intestine. Since the pH of the stomach is higher after a meal, it 
may be advantageous, whatever method of formulation is used, to administer 
the iron complexes at such a time. 
A particularly convenient approach to a controlled release composition 
involves encapsulating the iron complex by a material which is resistant 
to dissociation in the stomach but which is adapted towards dissociation 
in the small intestine (or possibly, if the dissociation is slow, in the 
large intestine). Such encapsulation may be achieved with liposomes, 
phospholipids generally being resistant to dissociation under acidic 
conditions. The liposomally entrapped 3:1 iron(III) complexes can 
therefore survive the acid environment of the stomach without dissociating 
to the 2:1 and 1:1 complexes, and the free hydroxypyridone. On entry into 
the small intestine, the pancreatic enzymes rapidly destroy the 
phospholipid-dependent structure of the liposomes thereby releasing the 
3:1 complex. Liposome disruption is further facilitated by the presence of 
bile salts. However, it is usually more convenient to effect the 
encapsulation, including microencapsulation, by the use of a solid 
composition of a pH sensitive nature. 
The preparation of solid compositions adapted to resist dissociation under 
acidic conditions but adapted towards dissociation under non-acidic 
conditions is well known in the art and most often involves the use of 
enteric coating, whereby tablets, capsules, etc, or the inidividual 
particles or granules contained therein, are coated with a suitable 
material. Such procedures are described, for example, in the article 
entitled "Production of enteric coated capsules" by Jones in Manufacturing 
Chemist and Aerosol News, May 1970, and in such standard reference books 
as "Pharmaceutical Dosage Forms, Volume III by Liebermann and Lackmann 
(published by Marcel Decker). One particular method of encapsulation 
involves the use of gelatine capsules coated with a cellulose acetate 
phthalate/diethylphthalate layer. This coating protects the gelatin 
capsule from the action of water under the acid conditions of the stomach 
where the coating is protonated and therefore stable. The coating is 
however destabilised under the neutral/alkaline conditions of the 
intestine where it is not protonated, thereby allowing water to act on the 
gelatin. Once released in the intestine the rate of permeation of the 
intestine wall by the water soluble 3:1 iron(III) complex is relatively 
constant irrespective of the position within the intestine, i.e. whether 
in the jejunum, ileum or large intestine. Other examples of methods of 
formulation which may be used include the use of polymeric hydrogel 
formulations which do not actually encapsulate the iron complex but which 
are resistant to dissociation under acidic conditions. 
A second approach to countering the effect of the acidic conditions 
prevailing in the stomach is to formulate the iron complex in the 
pharmaceutical composition together with the metal-free hydroxypyridone 
from which it is derived. The dissociation of the neutral 3:1 ferric 
complex, for example, involves various equilibria between this complex, 
the 2:1 and 1:1 complexes, and the metal-free compound, so that the 
presence of the latter will inhibit this dissociation. Any proportion of 
the free compound can be advantageous in this context but little further 
advantage accrues from increasing the proportion beyond a certain level. A 
preferred range for the molar proportion of the free compound present in 
compositions according to the present invention is thus from 0 to 100 
moles free hydroxypyridone:1 mole of iron complex, particularly the 
neutral 3:1 iron(III) complex. Conveniently, a proportion of up to no more 
than 20, 30 or 50 moles:1 mole is used with a lower level of 0.5, 1 or 2 
moles:1 mole. Although to obtain a marked effect upon dissociation of the 
iron complex a proportion of at least 5 or 10 moles:1 mole is usually 
employed it should be emphasised that even a molar ratio such as 1:1 will 
achieve a noticeable degree of acid stabilisation of the iron complex. 
Thus, although a range of, for example, from 10 moles:1 to 20 moles:1 mole 
of metal-free hydroxypyridone:iron complex will often be suitable to 
produce a marked effect, a range of, for example, 3 or even 1 mole:1 mole 
to 10 moles:1 mole will still produce a worthwhile effect without 
requiring administration of the larger amounts of the hydroxypyridone. The 
use of such a mixture is an important feature of the present invention 
since it can enable one to obtain almost quantitative uptake of iron from 
the complex. It should be appreciated, however, that the equilibrium 
between the complexes of various types and the metal-free compound will be 
effected by any take up of the latter in the body and the degree of such 
uptake from the stomach, for example, will depend on the particular 
metal-free compound. 
A further advantage than prevention of dissociation of the iron complex 
under acidic conditions may accrue from the use of a free hydroxypyridone 
in admixture with its iron complex. Thus, as referred to hereinbefore, in 
certain pathological conditions there may be an excess of iron deposited 
at certain sites even though the patient exhibits an overall anaemia. In 
patients having such conditions the use of such a mixture has the 
advantage that the iron complex will remedy the overall anaemia whilst the 
free hydroxypyridone will act to remove iron from pathological to 
physiological sites. Moreover, there may be an advantage in formulating 
the iron complex of one hydroxypyridone as described herein with another 
one of such hydroxypyridones in free form or with a mixture of the 
corresponding free hydroxypyridone, present primarily to prevent 
dissociation of the iron complex, and of another such hydroxypyridone in 
free form, present primarily to effect iron transfer. Thus, it is 
preferable for the hydroxypyridone present in an iron donor to be rapidly 
metabolized so as to effect its removal from the system once it has given 
up its iron at an appropriate site in the system, whilst it is preferable 
for a hydroxypyridone being used as an iron remover not to be rapidly 
metabolized so that it remains in the system, taking up iron, for an 
extended period. For this reason the use of different hydroxypyridones in 
the free form and as the iron complex has certain advantages. Moreover, 
different hydroxypyridones may, for other reasons, function more 
efficiently either in the free form as an iron remover or in complex form 
as an iron donor. If desired, the free hydroxypyridone may alternatively 
be used in the form of a salt formed with the anion produced by the loss 
of a hydroxy proton and containing a physiologically acceptable cation, 
for example as described hereinbefore. 
It will be appreciated that, as an alternative to combination with a 
different free hydroxypyridone of the same type, the iron complex may be 
used in combination with another iron chelating agent, for example an 
alternative form of hydroxypyridone such as is described in UK patent 
application Nos. 8308056, (published under the number GB 211876A (U.S. 
application Ser. No. 478,493, filed Mar. 24, 1983), and 8407181 (published 
under the number GB 2136807A (U.S. application Ser. No. 592,271, filed 
Mar. 22, 1984, now U.S. Pat. No. 4,585,780 issued Apr. 29, 1986). 
When a free 1-hydroxypyrid-2-one is present in admixture with an iron 
complex of the same or a different 1-hydroxypyrid-2-one for the purpose of 
acting as an iron remover, then the amount of the metal-free compound may 
be different than when the free hydroxypyridone necessarily corresponds to 
that present in the iron complex and is present primarily to prevent 
dissociation. Thus the daily dosage of the iron complex may be as above 
and the daily dosage of the free hydroxypyridone may be that described in 
relation to the use of such compounds in iron overload conditions. Thus, 
it will be seen that the proportion of iron complex and free 
hydroxypyridone used in such a context may extend across a wide range but 
preferred amounts of the free compound tend to be higher than in the other 
instance involving the prevention of dissociation of the complex. 
It will be appreciated that the present invention also includes a method 
for the treatment of a patient which comprises administering to said 
patient an amount of an iron complex of a 1-hydroxypyrid-2-one as 
described hereinbefore in order to effect an increase in the levels of 
iron in the patient's blood stream. 
In addition to the pharmaceutical uses of the iron complexes discussed 
above they are also of potential interest as a source of iron in various 
other contexts including in cell and bacterial growth, in plant growth, as 
a colouring agent and in the control of iron transport across membranes.

This invention is illustrated by the following Examples. 
EXAMPLES 
EXAMPLE 1 
The preparation of 1,4-dihydroxypyrid-2-one 
(1) 2-Chloro-4-nitropyridine-1-oxide 
2-Chloro-pyridine-1-oxide (10 g) is cooled in an ice bath and treated with 
concentrated H.sub.2 SO.sub.4 (15 ml), followed by the dropwise addition 
of a mixture of concentrated H.sub.2 SO.sub.4 (15 ml) and fuming HNO.sub.3 
(27 ml, s.g. 1.5) over a 70 minute period. The acidic solution is heated 
in a steam bath for 2.5 hours, then allowed to reach room temperature and 
poured onto ice water (600 ml), stirring being continued until all the ice 
has melted. The resultant solid is filtered off and dissolved in hot 
chloroform, the solution being dried and the solvent evaporated in vacuo 
to give a yellow solid. The aqueous filtrate obtained after the removal of 
the original solid is neutralised with saturated aqueous Na.sub.2 CO.sub.3 
and extracted continuously with chloroform, the extract being dried and 
evaporated in vacuo to yield a yellow solid. The two yellow solids are 
combined and recrystallised from ethanol to give 
2-chloro-4-nitro-pyridine-1-oxide as yellow crystals (7.46 g, 56%). 
(2) 2,4-Dimethoxypyridine-1-oxide 
Sodium methoxide is prepared by dissolving sodium metal (0.66 g) in 
methanol (33 ml). This solution is mixed with 2-chloro-4-nitro- 
pyridine-1-oxide (2.3 g) in methanol (20 ml) and the mixture is refluxed 
for 6 hours, then filtered and the solvent evaporated in vacuo. The 
resultant solid is extracted with chloroform, the chloroform solution then 
being reduced in volume and left to crystallise, yielding 
2,4-dimethoxypyridine-1-oxide in 54% yield. 
(3) 1,4-Dihydroxypyrid-2-one 
2,4-Dimethoxypyrid-1-oxide is refluxed together with 20% w/v HCl for 13 
hours. On cooling the solution 2,4-dihydroxypyridine-1-oxide is obtained 
as an orange-white solid (0.42 g, 30%), .delta.(d.sub.6 DMSO+trace of 
D.sub.2 O), 6.08 (s, 1H), 6.12 (q, 1H), 7.88 (d, 1H). 
EXAMPLE 2 
The preparation of 1-hydroxy-4-methoxypyrid-2-one 
(1) 2-Chloro-4-methoxypyridine-1-oxide 
Sodium (0.46 g) is dissolved in absolute methanol (50 ml) and the resultant 
solution of sodium methoxide is added to a solution of 
2-chloro-4-nitropyridine-1-oxide (3.5 g, prepared as described in Example 
1) in methanol (10 ml). The reaction mixture is allowed to stand at room 
temperature for 50 hours and is then subjected to rotary evaporation to 
give 2-chloro-4-methoxypyridine-1-oxide. 
(2) 1-Hydroxy-4-methoxypyrid-2-one 
2-Chloro-4-methoxypyridine-1-oxide (3.3 g) is dissolved in 10% w/v aqueous 
NaOH (33 ml) and the mixture is heated on a steam bath for 3.5 hours when 
it is cooled and acidified with concentrated HCl to a pH of 2.5 to yield 
white crystals. Recrystallisation of these from water gives 
1-hydroxy-4-methoxypyrid-2-one (0.75 g, 20%), m.p. 174.degree.-175.degree. 
C., .delta.(D.sub.2 O) 5.9 (s, 1H), 6.00 (q, 1H), 7.5 (d, 1H). 
EXAMPLE 3 
Preparation of 1-hydroxy-4-(2'-methoxyethoxy)-pyrid-2-one 
(1) 2-Chloro-4-(2'-methoxyethoxy)-pyridine-1-oxide 
Sodium metal (0.23 g) is dissolved in redistilled methoxyethanol (30 ml). 
The resulting solution is added to 2-chloro-4-nitro-pyridine-1-oxide (1.75 
g, prepared as described in Example 1) and stirred for 28 hours at 
20.degree. C. The methoxy-ethanol is removed by distillation under reduced 
pressure leaving an oily brown solid which is washed with diethyl either 
(25 ml) and then dissolved in water (25 ml). The aqueous solution is 
extracted into chloroform (3.times.25 ml) and the extracts are then 
evaporated in vacuo to give 2-chloro-4-(2'-methoxyethoxy)-pyridine-1-oxide 
as a yellow solid. 
(2) 1-Hydroxy-4-(2'-methoxyethoxy)-pyrid-2-one 
2-Chloro-4-(2'-methoxyethoxy)-pyridine-1-oxide is treated with 10% w/v 
aqueous NaOH and the mixture is heated on a steam bath for 3 hours. The 
resulting solution is acidified to pH 2 with concentrated HCl, then 
reduced in volume by evaporating in vacuo and left to crystallise. The 
resultant white solid is recrystallised from ethanol to give 
1-hydroxy-4-(2'-methoxyethoxy)-pyrid-2-one (0.58 g, 29%), m.p. 134.degree. 
C., .delta.(CDCl.sub.3) 3.42 (s, 3H), 3.7 (t, 1H), 4.08 (t, 1H), 6.05 (d, 
1H), 6.05 (q, 1H), 7.62 (t, 1H). 
EXAMPLE 4 
Partition data on 1-hydroxypyrid-2-ones and their iron complexes 
The partition coefficient K.sub.part, being the ratio (concentration of 
compound in n-octanol)/(concentration of compound in aqueous phase) on 
partition between n-octanol and aqueous tris hydrochloride (20 mM, pH 
7.4), is measured at 20.degree. C. the compounds of Examples 1 to 3 and 
1-hydroxypyrid-2-one by way of comparison, and for their iron complexes 
(at 10.sup.-4 M) by spectrophotometry. Acid washed glassware is used 
throughout and, following mixing of 5 ml of the 10.sup.-4 M aqueous 
solution with 5 ml n-octanol for 1 minute, the aqueous n-octanol mixture 
is centrifuged at 1,000 g for 30 seconds. The two resulting phases are 
separated for a concentration determination by spectrophotometry on each. 
For the free hydroxypyridones, the range 220-340 nm is used for 
concentration determinations whilst for the iron complexes, the range 
340-640 nm is used. 
Values typical of those obtained are shown in Table 1. 
TABLE 1 
______________________________________ 
Partition coefficients 
Partition coefficient K.sub.part 
Iron complex 
[Fe.sup.III -- 
Compound Free Compound 
(compound).sub.3 ] 
______________________________________ 
1-hydroxypyrid-2-one 
0.3 0.95 
1,4-dihydroxypyrid-2-one 
0.04 0.04 
1-hydroxy-4-methoxypyrid-2-one 
0.15 4.85 
1-hydroxy-4-(2'-methoxyethoxy)- 
0.14 0.6 
pyrid-2-one 
______________________________________ 
EXAMPLE 5 
In vitro tests of iron binding capacity 
The 1-hydroxypyrid-2-ones used in this Example were prepared as described 
in Examples 1, 2 and 3, and 1-hydroxypyrid-2-one was also used for 
comparative purposes. 
(1) Mobilisation of iron from ferritin 
Horse spleen ferritin (Sigma) was used without further purification and its 
iron content was estimated spectrophotometrically at 420 nm. The ferritin 
solution in phosphate buffered saline (Dulbecco-OXOID, 10.sup.-6 M, pH 
7.4) was enclosed in a Visking dialysis tube and dialysed against a 
3.times.10.sup.-3 M buffered solution of one of various pyridones as 
indicated in Table 2. The absorption spectrum of the resulting iron(III) 
complex in the dialysis solution was recorded after 6 and 24 hours. For 
comparative purposes, the procedure was repeated using a blank control. 
The results are shown in Table 2 where the percentage of ferritin-bound 
iron removed by the compound under test is shown. For comparative 
purposes, results reported in the literature for similar tests with 
1.times.10.sup.-3 M desferrioxamine (Crichton et al, J. Inorganic 
Biochem., 1980, 13, 305) and with 6.times.10.sup.-3 M LICAMS (Tufano et 
al, Biochem. Biophys. Acta, 1981, 668, 420) are also given in the Table. 
It will be seen that the pyridone compounds are able to remove iron 
effectively from ferritin in contrast with desferrioxamine and LICAMS 
(although the latter will remove iron in the presence of ascorbic acid 
such a mixture is very difficult to manage clinically). These results 
shown in Table 2 may be confirmed by separating apoferritin (in admixture 
with ferritin) and the particular hydroxypyridone iron(III) complex from 
the reaction product in each case by chromatography on Sephadex G10. 
TABLE 2 
______________________________________ 
Removal of iron from ferritin 
Percentage of iron removed 
Compound 6 hours 24 hours 
______________________________________ 
Control 0 0 
1-hydroxypyrid-2-one 
.sup. 34.sup.(1) 
-- 
1,4-dihydroxypyrid-2-one 
22 54 
1-hydroxy-2-methoxypyrid-2-one 
13 46 
1-hydroxy-4-(2'-methoxyethoxy)- 
2 8 
pyrid-2-one 
Desferrioxamine (1 mM) 
1.5 -- 
LICAMS (6 mM + 12 mM 
7 -- 
ascorbic acid) 
______________________________________ 
.sup.(1) 1hydroxypyrid-2-one iron complex precipitated from incubation 
medium. 
(2) Mobilisation of iron from transferrin 
Human transferrin (Sigma) was loaded with iron(III) by the method of Bates 
and Schlaback, J. Biol. Chem. (1973) 248, 3228. .sup.59 Iron(III) 
transferrin (10.sup.-5 M) was incubated with a 4.times.10.sup.-3 M 
solution in tris HCl (0.1 M, pH 7.4) of one of various pyridones as 
indicated in Table 3 for periods of 6 hours and 24 hours. The solution was 
then dialysed against phosphate buffered saline for 24 hours. The .sup.59 
Fe remaining in the dialysis tube was then recorded. For comparative 
purposes, this procedure was repeated with desferrioxamine and EDTA. 
The results are shown in Table 3 in terms of the percentage of transferrin 
bound iron removed by the compound under test. illustrate the efficiency 
of the compounds at iron removal. The results shown in Table 3 may be 
confirmed by separating apotransferrin (in admixture with transferrin) and 
the particular hydroxypyridone iron complex from the reaction product in 
each case by chromatography on Sephadex G10. 
TABLE 3 
______________________________________ 
Removal of iron from transferrin 
Percentage of iron removed 
Compound 6 hours 24 hours 
______________________________________ 
1-hydroxypyrid-2-one 
60 73 
1,4-dihydroxypyrid-2-one 
80 91 
1-hydroxy-4-methoxypyrid-2-one 
70 71 
1-hydroxy-4-(2'-methoxyethoxy)- 
72 75 
pyrid-2-one 
Desferrioxamine 17 22 
EDTA 27 67 
______________________________________ 
EXAMPLE 6 
In vivo tests of iron binding capacity 
The 1-hydroxypyrid-2-one used in this Example was prepared as described in 
Example 1. 
Mice were injected intraperitoneally with iron dextran (2 mg) at weekly 
intervals over a four week period. Two weeks after the final injection, 
the mice were injected via the tail vein with .sup.59 Fe lactoferrin 
(human lactoferrin, 1 mg per injection 2 .mu.Ci). The mice were then caged 
individually. After a ten day period, 1,4-hydroxypyrid-2-one was 
administered to groups of 8 mice at 10 mg per mouse either 
intraperitoneally or intragastrically (in each case 3 of the mice received 
only one dose whilst 5 received 2 doses at a 24 hour interval). The 
excretion of iron was recorded at either 12 or 24 hourly intervals over a 
three day period before and a two day period after administration of the 
compound. For comparative purposes, the procedure was repeated with a 
blank control and with desferrioxamine, also at 10 mg per mouse (the 
intraperitoneally treated mice receving one dose of desferrioxamine and 
the intragastrically treated mice two doses at a 24 hour interval). 
The results are shown in Table 4, being given on the basis of the control 
representing 100% excretion, and illustrate the particular advantage of 
the pyridones as compared with desferrioxamine for oral administration. It 
should be mentioned that the large standard deviation (SD) values are 
somewhat misleading as uniformly positive results can yield high SDs which 
might be taken to suggest that the results are not significantly different 
from zero. However, this is not the case here, the large SD values being a 
consequence of the large range among the positive responses. 
TABLE 4 
______________________________________ 
Excretion of iron in vivo 
Intraperitoneal 
Intragastric 
Administration 
Administration 
Number Excretion Number Excretion 
of of .sup.59 Fe .+-. SD 
of of .sup.59 Fe .+-. SD 
Compound Mice percent Mice percent 
______________________________________ 
Control 12 100 .+-. 10 
-- -- 
1,4-dihydroxy- 
11 195 .+-. 57 
6 166 .+-. 40 
pyrid-2-one 
______________________________________ 
EXAMPLE 7 
Preparation of iron complexes 
The iron complex of 1-hydroxy-4-methoxypyrid-2-one is prepared by either 
procedure (a) or procedure (b). (a) An aqueous solution of ferric chloride 
is reacted for 5 minutes at room temperature with an aqueous solution 
containing 3 molar equivalents.sup.(1) of 1-hydroxy-4-methoxypyrid-2-one. 
The resultant solution is adjusted to pH 7.0 using 2 molar aqueous sodium 
hydroxide and is then freeze dried. The resulting powder is extracted with 
chloroform, filtered and the filtrate subjected to rotary evaporation to 
give an essentially quantitative yield of the neutral complex containing 
the 1-hydroxy-4-methoxypyrid-2-one anion and the ferric cation in 3:1 
proportion. Recrystallisation of the 3:1 complex from ethanol gives orange 
crystals, m.p. 103.degree.-106.degree. C. 
FNT .sup.(1) The concentration of the hydroxypyridone is 0.1 M although this 
figure may be varied, for example in a range of 0.01 to 0.5 M, being 
constrained at the upper end of the range by the solubility of the 
compound in the reactions solvent. 
(b) An ethanolic solution of ferric chloride is reacted for 5 minutes at 
room temperature with a chloroform solution containing 3 molar equivalents 
of 1-hydroxy-4-methoxypyrid-2-one. The resultant solution is neutralised 
by the addition of solid sodium carbonate, the precipitated sodium 
chloride removed by filtration and the filtrate evaporated to give an 
essentially quantitative yield of the 3:1 complex, m.p. 
103.degree.-106.degree. C. 
The 3:1 iron(III) complexes of 1,4-dihydroxypyrid-2-one and 
1-hydroxy-4-(2'-methoxyethoxy)-pyrid-2-one may be prepared in an exactly 
similar manner. 
When an excess (5 to 50 molar equivalents) of any pyridone is used, both 
procedure (a) and procedure (b) lead to an essentially quantitative yield 
of the excess pyridone in free form in admixture with the 3:1 complex. 
EXAMPLE 8 
The ability of iron complexes to donate iron to apotransferrin 
Apotransferrin (10.sup.-4 M) and the iron complex of 1-hydroxy-4- 
methoxypyrid-4-one (10.sup.-4 M; prepared as described in Example 7) were 
incubated together in tris hydrochloride (50 mM, buffered to pH 7.4) at 
37.degree. C. for 10 minutes when a 1 ml aliquot was removed from the 
medium and added to a PD10 colum. 0.5 ml fractions were collected directly 
into scintillation vials for counting. The .sup.59 Fe associated with both 
the apotransferrin and the ligand was estimated and it was found that over 
90% of the iron was removed from the iron complex. 
EXAMPLE 9 
In vitro tests on permeation of rat jejunal sac by iron complexes 
The iron uptake into the serosal space of the inverted rat jejunal sac was 
compared for various iron compounds. Rats (male Sprague Dawley, 60 g) were 
killed and the jejunum removed, everted and cut into three segments (4 cm 
length). The segments were tied at both ends and filled with Krebs Ringer 
buffer (0.2 ml) and incubated in Krebs Ringer buffer containing .sup.59 Fe 
complexes at 37.degree. C. for periods up to 1 hour. The contents of the 
sac were counted for .sup.59 Fe and measured spectrophotometrically. 
The results obtained for the three iron complexes described in Example 7 
and for seven other iron compounds which are each contained in 
preparations marketed for the treatment of iron deficiency anaemia are 
shown in Table 5, the iron uptake for each compound being shown relative 
to that for ferric chloride as 1. It will be seen that the complexes of 
Example 7 each provide a level of iron uptake which is significantly 
higher than the levels observed for any of the 7 compounds in current use 
for the treatment or iron deficiency anaemia. 
TABLE 5 
______________________________________ 
Relative Relative 
Iron Iron 
Compound Uptake Compound Uptake 
______________________________________ 
FeCl.sub.3 1 FeCl.sub.3 1 
Fe.sup.III complex of: Fe.sup.II sulphate 
2.4 
1,4-dihydroxypyrid-2-one 
9.4 Fe.sup.II fumarate 
4.0 
1-hydroxy-4-methoxy- 
12.3 Fe.sup.II gluconate 
1.6 
pyrid-2-one 
1-hydroxy-4-(2'-methoxy- 
11.4 Fe.sup.II succinate 
2.0 
ethoxy)-pyrid-2-one Fe.sup.III EDTA 
3.6 
Fe.sup.III ascorbate 
0.4 
Fe.sup.III citrate 
2.0 
______________________________________