112i UK Patent 11s1 GB 111i 2 118 176 B
`
`(54) Title ofinvention
`Pharmaceutically active 3- hydroxypyrid- 2- and -
`4-gnes
`
`(73) Proprietors
`National Research Development
`Corporation
`(United Kingdom)
`101 Newington Causeway
`London SE1 6BU
`
`(72)
`
`Inventors
`Robert Charles Hider
`George Kontoghiorghes
`Jack Silver
`
`(74) Agent and/or
`Address for Service
`DrG: F. Stephenson,
`Patent Department,
`National Research Development
`Corporation,
`101 Newington Causeway,
`London SE1 6BU
`
`(51)
`
`INTCL4 ; C070213/69
`A61K 31144
`
`(21) Application No
`8308056
`
`(22) Date offillng
`24Mar1983
`
`(30) Priority data
`
`(31) 8208608
`
`(32) 24Mar1982
`
`(33) United Kingdom (GB)
`
`(43) Application published
`260C11983
`
`(45) Patent published
`4Dec1985
`
`(52) Domestic classification
`C2C1531167021522022622Y
`247 250 251 253 25Y 305 30Y 351
`35236036436536Y38843X500
`509 SOY 623 624 625 672 760 761
`762 80280Y AA TTTYTZ
`U1S1317C2C
`
`(56) Documents cited
`Yakugaku Zasshi (1970) 90(10)
`pp1222..S
`Rec. Trav. Chim. Vol69pp 1041·7
`(1950)
`J. Med. Chem. (1973) 16(5} pp
`581·3
`Toxicol. Appl. Pharmacol. (1969)
`Vol.14Part2pp249-258
`
`(58) Field of search
`C2C
`LONDON THE PATENT OFFICE
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`1 of 22
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`Taro Pharmaceuticals, Ltd.
`Exhibit 1021
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`JI 1.g I 7~
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`PHARMACEUTICALLY ACTIVE 3-HYDROXYPYRID-2- AND-4-0NES
`This invention relates to compounds for use in pharmaceutical
`compositions.
`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 condi(cid:173)
`tion 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 produc't obtained by the culture
`ot 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 desferri(cid:173)
`oxamine 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 fro~ 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 toxi~ity
`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 involv(cid:173)
`ing iron overload .
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`Exhibit 1021
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`Accordingly the present invention comprises a compound
`being a 3-hydroxypyrid-2-one or 3-hydroxypyrid- 4-one in which
`the hydrogen atom attached to the nitrogen atom is replaced by an
`aliphatic hydrocarbon group of 1 to 6 carbon atoms and, optionally,
`in which one or more of the hydrogen atoms attached to ring carbon
`atoms are also replaced by an aliphatic hydrocarbon group of 1
`to 6 ~arbon atoms, or a salt thereof containing a physiologically
`acceptable cation, for use in medicine.
`The 3-hydroxypyrid-2- and -4-ones may carry more than one
`type of aliphatic hydrocarbon group and, in particular, the group
`attached to the nitrogen atom may be different from any aliphatic
`hydrocarbon group or groups attached to ring carbon atoms. Groups
`attached to carbon atoms are, however, more often the same when
`more than one is present. The aliphatic hydrocarbon groups, whether
`attached to a nitrogen or a carbon atom, may be cyclic or acyclic,
`having a branched chain or especially a straight chain in the lauer
`case, and may ~e unsaturated or especially saturated. Groups of
`from 1 to 4 carbon atoms and particularly of 1 to 3 carbon atoms
`are of most interest. Alkyl groups are preferred, for example
`cyclic groups such a cyclopropyl and especially cyclohexyl but,
`more particularly preferred are acyclic alkyl groups such as methyl,
`ethyl, n-propyl and isopropyl. Where the riog·carbon atoms are
`substituted by an aliphatic hydrocarbon group or groups, these groups
`are preferably methyl but in the case of the gro~p substituting the
`nitrogen atom larger groups may more often be utilised with parti(cid:173)
`cular advantage. Substitution of the ring carbon atoms, which is
`preferably by one rather than two or three aliphatic hydrocarbon
`groups, is of particular interest in the case of the 3- hydroxy(cid:173)
`pyrid- 4-ones, for example at t he 6- or particularly the 2-position,
`whilst the 3-hydroxypyrid-2-ones may more often be used without
`any additional aliphatic hydrocarbon group substitutent on
`the ring carbon atoms . Particularly if tha ring carbon atoms
`are substituted by the larger aliphatic hydrocarbon groups,
`however, there may be ·an advantage in avoiding substitution on a
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`carbon atom alpha to the -ft-~( system. This system is involved
`0 OH
`in the complexing with iron and the close proximity of one of the
`larger aliphatic hydrocarbon groups may lead to steric effects
`whi~h inhibit complex formation.
`The compounds may, if desired, be used in the form of salts
`thereof containing a physiologically acceptable cation, for example
`the cation of an alkali metal such as sodium, quaternary ammoniu~
`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.
`Examples of specific compounds which may be used in composi(cid:173)
`tions according to the present invention are shown by the following
`formulae (I), (II) and (III):-
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`4 5a3 OH
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`2
`
`0
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`61
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`I
`N
`R
`
`(I)
`
`(II)
`
`(III)
`
`in which R is an alkyl group, ~or example methyl , ethyl , n-propyl
`or isopropyl, and Rl is hydrogen or an alkyl group, for example
`methyl. Among these compounds and others of use in compositions
`· according to the present invention, the 3-hydroxypyrid-4-ones are
`of particular interest.
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`Certain of the compou~ds described herein are novel and
`the present invention thus also includes as compounds, per se,
`(a) a 3-hydroxypyrid-2-one in which the hydrogen atom attached to
`the nitrogen atom is replaced by an aliphatic hydrocarbon group of
`1 to 6 carbon atoms and, optionally, in which one or more of the
`hydrogen atoms attached to ring carbon atoms are also replaced by
`an aliphatic hydrocarbon group of 1 to 6 carbon atoms,and (b) a
`3-hydroxypyrid-4-one in which the hydrogen atom attached to the
`nitrogen atom is replaced ~Y an aliphatic hydrocarbon group of
`1 to 6 carbon atoms and in which one or more of the hydrogen atoms
`attached to ring carbon atoms are also replaced by an aliphatic
`hydrocarbon group of 1 to 6 carbon atoms, or a salt of such a
`3-hydroxypyrid-2-one or 3-hydroxypyrid-4-one containing a
`physiologically acceptable cation, but excluding specifically
`3-hydroxy- 1- methylpyrid-2-one, 3-hydroxy-1,6-dimethylpyrid-4-one
`and 3- hydroxypyrid-4-ones in whi~h the only ring carbon a t om
`substituent is a methyl group at the 2- position, and salts thereof .
`The 3-hydroxy-pyrid- 2-one compounds may conveniently be
`prepared by nucleophilic substitution at the nitrogen atom of the
`corresponding 2, 3-dihydroxypyridine, for example using an organic
`halide R'X in which R' represents the aliphatic hydrocarbon group
`present on the nitrogen atom of the desired 3- hydroxypyrid - 2-one
`and X represents an iodo group. The 3-hydroxypyrid- 4- one compounds
`may conveniently be prepared ~imilarly or preferably from the more
`readily accessible corresponding 3-hydroxy-4-pyrone. Thus, the
`3-hydroxy-4-pyrone may conveniently be converted to the 3-hydroxy(cid:173)
`pyrid-4-one through protection of the hydroxy group, for example
`as an ether group such as a benzyloxy group, reaction of the
`protected compound with a compound R'NH 2, in which R' represents
`the aliphatic hydrocarbon group present on the nitrogen atom of
`the desired 3- hydroxypyrid- 4-one, in the presence of a base, for
`example an alkali metal hydroxide such a sodium hydroxide . The
`protecting group may then be removed. The cqmpounds may be con(cid:173)
`verted to .salts formed at the hydroxy group thereof through its
`conversion to the anion (OH ~-> 0- ) by reaction with the
`appropriate base according to standard procedu~es .
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`It will be appreciated that these are not the only routes
`available to thes~ compounds and that various alternatives may be
`In general,
`useJ as will be apparent to those skilled in the art.
`ho~ever, it is preferred that the compounds are isolated in sub-
`stantially pure fonn, Le .• substantially free from by-products of
`
`manuf ac tu re.
`The compounds may be formulated for use as pharmaceuticals
`for veterinary or particularly human use by a variety of methods.
`For instance~ they may be applied as an aqueous, oily or emulsified
`co·mposition 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, for example compositions containing
`water and/or an organic solvent and having the form of a solution,
`suspension or emulsion, may be used for oral as well as parenteral
`administration, it is preferred for oral administration to use
`compositions incorporating a solid carrier. This may, for example,
`be a conventional solid carrier material such as starch, lactose,
`dextrin or magnesium stearate, and the composition may, for example,
`
`take the form of tablets.
`Other forms of administration than by injection or through
`the oral route may also be considered in both human and veterinary
`contexts, for example the use of suppositories for human adminis(cid:173)
`
`t'I'ation.
`Compositions may be formulatetl 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 whi~h 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 O.l g to S g, particularly of
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`Exhibit 1021
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`about 0.5 g to 2 g, veterinary doses bei~g 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.
`The present invention thus includes a pharmaceutical composition
`comprising as an active component thereof a 3-hydroxypyrid-2- one or
`3- hydroxypyrid-4-one in which the hydrogen atom attached to the
`nitrogen atom is replaced by an aliphatic hydrocarbon group of 1 to 6
`carbon atoms and , optionally, in which one or more of the hydrogen
`atoms attached to ring carbon atoms are also replaced by an aliphatic
`hydrocarbon group of 1 to 6 carbon atoms , or a salt thereof containing
`a physiologically acceptable cation, together with a physiologically
`acceptable diluent or carrier but excluding any diluent which is a
`non-sterile and non-pyrogen free aqueous and/or organic solvent.
`Although 3- hydroxy- l- metbylpyrid-4-one has previously been
`recognised as a siderophore, it has never before been appreciated
`that compounds such as this might be used in a pharmaceutical
`context, and with real advantage. We have found that the
`
`3-hydroxypyrid- 2- and - 4- ones described above are particularly
`suited to the removal of iron from patients having an iron over(cid:173)
`load. The compounds form neutr al 3:1 iron complexes at most
`physiol~gical pH values, and have the advantage that they do not
`co-ordinate cal cium or magnesium. Both the compounds and their
`complexes will partition into n- octanol indicating t hat they will
`permeate biological membr~nes , this property being confirmed in
`59
`practice by tests of the ability of the
`Fe labelled iron com-
`plexes to permeate erythrocytes. The measured coefficients
`) for p~rtition of various of the compounds and their
`(K
`part
`3~1 hYdroxypyridone :iron(III) complexes are presented in Table 1
`of Example 5 hereinafter. Although the ability of both the free
`compound and its iron complex to permeate membranes is important,
`it is also desirable for both to possess some degree of water solubility.
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`- 6a-
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`05
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`Preferred compounds show a value of Kpart for the free compound
`of above 0 . 05 but less than 3.0, especially of above 0.2 but less
`than 1.0, together with a value of K
`t for the 3:1 hydroxy-
`par
`pyridone:iron(III) complex of above 0.02 but less than 6.0,
`especially of above 0.2 but less than 1. 0. Reference to Table 1
`will show that the preferences as to the structure of the compounds
`in compositions according to the present invention which are
`expressed hereinbef ore lead to compounds which have K
`t values
`par
`both in the free state and as the 3:1 iron(III) complexes which
`are broadl y in line with the ranges indicated above.
`Both the 3-hydroxypyrid- 2-ones and the 3- hydroxypyrid- 4-ones
`possess. a high affinity for iron (III), as evidenced by log K501
`values {log 1<
`is defined as being equal to log B Fe(L)n + 21 -
`501
`(pK
`p + n log aL(H+) + 111 l og aL(Ca++)] where log SFe(L)n is the
`5
`cu1!llllative affinity constant of the ligand in question for
`iron (III), pK
`is the negative logarithm of the solubility
`sp
`and has a value of 39 , n and mare the number
`product for Fe(OH)
`3
`of hydrogen and calcium ions, respectively, which are bound to the
`
`(Ca++) are the affinities of the ligand
`ligand, and 8J,(H+) and a
`1
`for hydrogen ions and calcium ions, respectively} .
`In order to
`solubilise iron (III) hydroxide, log Ksol must be greater than 0
`and in order to remove i r on f r om transferrin, log K 1 should be
`so
`in excess of 6.0. The log K 1 values for 3- hydroxy- 1- methylpyrid(cid:173)
`so
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`8 of 22
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`2-one and l, 2-dimethyl-3-hyd roxypyrid-4-one, by way of example,
`are 10.0 and 9.S, respectively, thus comparing favourabley with
`those of the bidentate hydroxamates at ahout 4. 0, of catechols at
`about 8.0, of desferrioxamine at 6.0, and of die t hylenetriamine
`penta-acetic a~id (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~ vivo tests in mice. It is particu(cid:173)
`larly significant tha t these la tter tes t s are successful whether
`the compound is given intraperitoneally or orally by stomach tube,
`the compounds being stable under acidic conditions. Oral activity
`is not generally present among the other types of compound previously
`sugges ted for use as iron co-ordinating drugs and although certain
`EDTA analogues do show such activity, they possess drawbacks for
`
`phar maceutical use .
`Although the major use of the compounds is in the removal or
`iron, they are also of potential interest for the removal of some
`other metals present in the boJy in deleterious amounts . The
`present invention thus includes the use of a 3-hyd roxypyrid-2-
`or -4-one or salt thereof as described above for the manufacture
`of a medicament for use in effecting a reduction in toxic levels
`of a metal in a patient' s body.
`This invention is illustrated by the following Examples.
`EXAMPLES
`Example 1 : The preparation of 3-hydroxy-l-methylpyrid-2-one
`2,3-Dihydroxypyridine (5.55 g) is suspended in methyl iodide
`0
`(2r0 ml) in a sealed tube and heated for 24 hours at 140 C. The
`reaction is taken to be complete when a dark brown residue forms
`as a separate phase from the methyl iodide and the tube is then
`cooled in solid carbon dioxide and opened. The excess methyl
`iodide is poured off, distil led water (10 ml) is added to the
`brown residue, and sulphur dioxide gas is bubbled through the
`
`OS
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`-'
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`mixture until the aqueous phase becomes clear. The pH of the
`reaction mixture is adjusted to a value of 6 with 1 M aqueous
`sodium carbonate and the resulting solution then saturated with
`ammonium sulphate and extracted with chloroform until the chloro-
`form layer no longer gives a blue colouration when added to ferric
`chloride solution . The chloroform extracts are combined and dried
`over sodium sulphate. The solvent is then evaporated under vacuum
`and the resulting residue is crystallised from petroleum ether
`0
`0
`- 120 C) using activated charco.al to give 3-hydroxy-1-
`(b.p. 100
`-1
`o
`0
`- 131 C; v
`(nujol) 1660, 3100 cm
`methylpyrid-2-one, m. p. 129
`max
`+
`o(d 6DMSO) 3. 6(s,3H), 6.l(t , lH), 6.8(m,2H), 7.3(s,IH); M 125.
`Example 2 : The preparation of other 3- bydroxyPyrid-2- ones
`2,3- Dihyroxypyridine is reacted with ethyl iodide, n-propyl
`iodide and isopropyl iodide under similar conditions to those
`described in Example I for methyl iod i de. The reaction mixtures
`are worked up as described in Example 1 to give the following
`compounds:-
`0
`0
`- 132 C; v
`(nujol)
`l-Ethyl-3- hydroxypyrid- 2-one: m. p. 130
`max
`-1
`; o(d 6DMSO) l.2(t,3H) 3.8(m,2H), 6.0(t,2H),
`1620, 3100 cm
`6.8(m,2H) , 8.9(s , 1H); M+ 139 .
`0
`3- Hydroxy-l- propylpyrid-2- one : m. p. 148 C;
`- 1
`; 6 (d 6DMSO) O. 7(t,3H), l.5(m,2H),
`3150 cm
`+
`6. 5 - 7,0(m,2H), 8 , 7(s,1H); M 153.
`3-Hydroxy-1-(2'-methylethyl)pyrid-2- one: m.p.
`- 1
`; 6(d6DMSO) l.O(d,6H),
`(nujol) 1660, 3200 C1ll
`+
`6.S(t,lH), 6.7(m,2H); M 153.
`Example 3 : The preparation of 3-hydroxy- l,2-dimethylpyrid-4- one
`3-Benzyloxy- 2- methyl-4-pyrone
`3-Hydroxy- 2-methyl- 4- pyrone (22. 2 g) in methanol 225 ml) is
`added to aqueous sodium hydroxide (25 ml containing 7.5 g NaOH) .
`Benzyl chloride (25.5 g) is added and the mixture is refluxed
`for 6 hours and is then allowed to cool overnight. The bulk of
`the methanol is removed under vacuum and the residue is treated
`with water (50 ml). The mixture i s extracted into dichloromethane
`(3 x 25 ml) . The extracts are combined, washed with 5% w/v NaOH
`(2 x 25 ml), then water (2 x 25 ml) and dried over magnesium
`
`vmax (nujol) 1620,
`3. 7(t,2H) , 5.8(t,1H)
`
`190°C;
`v
`max
`6.0(m,lH) ,
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`sulphate. Evaporation of the solvent gives crude 3-benzyloxy- 2-
`methyl-4- pyrone (35 g, 92%) which is purified by distillation in
`nitrogen under reduced pressure to yield a colourless oil (28 g)
`of b.p. 148°C/0.2 mm.
`1,2-Dimethyl- 3-benzyloxypyrid- 4- one
`3- Benzyloxy- 2- methyl-4-pyrone (4.8 g) and methyl amine hydro(cid:173)
`chloride (1 .56 g) are dissolved in water (200 ml) and ethanol
`(100 ml) containing sodium hydroxide (2 g) is added . The mixture
`is stirred at room temperature for 6 days and is then acidfied
`10 with concentrated hydrochloric acid to pH 2, and evaporated to
`dryness. The resulting colourless solid is washed with water and
`extracted into chl oroform (2 x 50 ml). The chloroform extracts
`are combined , dried over magnes.ium sulpha'te , and evaporated to
`yield 1,2-dimethyl-3-benzyloxypyrid-4- one (3.2 g) .
`1, 2-Dimethyl- 3- hydroxypyrid- 4- one
`1, 2- Dimetbyl- 3- benzyloxypyrid- 4-one (2 g) is added to concen(cid:173)
`trated hydrobromic acid (10 ml) and heated in a steam bath for 30
`minutes. The resulting mixture is then recrystallised from water
`to yield l , 2-dimethyl- 3- hydroxypyrid-4-one (1 g), m.p . 230°c (with
`decomposition);
`"
`(nujol) '1620, 3150 cm- l; o(d6DMSO) 2.3(s,3H),
`max
`+
`3. 8(s, 3H) , 6.9(d,1H), 7.8(d , 1H); M 139.
`Example 4 : The preparation of other 3- hydroxypyrid - 4- ones
`3- Benzyloxy-2-methyl-4- pyrone is prepared as described in
`Example
`and is reacted with ethylamine, n-propylamine, iso-
`propylamine, n-butylamine and n-hexylamine hydrochloride under
`similar conditions to those described in Example 3 for methylamine
`hydrochloride. The reaction mixture is worked up and the hydroxy
`group deprotected as described in Example 3 to give the foloowing
`compounds:
`l - Ethyl- 3- hydroxy- 2- methylpyrid - 4- one : m.p . 190° - 195°C; v
`.
`_
`- 1
`max
`(nuJol) 1620, 31::>0 cm
`;
`DMSO) 1.1(t,3H) , 2. 6(s, 3H), 3. 5(m, 2H),
`o(d
`6
`7. 3(d,1H), 8 . 5(d.1H) ; M+ 153.
`0
`0
`3-Hydroxy-2-methyl- l - propylpyrid-4-one: . m. p. 182
`- 183 C;
`vmax
`.
`-1
`(nuJol) 1630 , 3200 cm
`o(d6DMS0) 0.9(t,3H) , l. 6(m,2H) , 2. 43(s,3H),
`;
`+
`4.2(t,2H), 7.l(d,lH), 8.15(d,1H); M 167.
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`05
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`v max
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`15
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`20
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`v max
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`3-Hydroxy-2-methyl-1-(1 1-methylethyl)pyrid-4-one: m.p. 198°· - 200°c ;
`-l
`(nujol) 1630, 3150 cm
`; 6{d6DMSO) l.28(d,6H) , 2.43(s,3H},
`v
`max
`+
`4. 8(m,1H). 7, 15(d,1H), 8 . lS(d , lH); M 167.
`0
`0
`- 190 C;
`l-Butyl-3-hydroxy-2-methylpyrid-4-one: m. p. 188
`- 1
`(nujol) 1630, 3200 cm
`; O(d6DMSO) 0. 9(t,3H) , l.3(m,4H),
`+
`2.4l(s , 3H), 4.2(t ,2H}, 7.2(d,1H), 8 . 3(d,1H); M 181.
`0
`0
`- 168 c;
`l-Hexyl-3-hydroxy-2- methylpxrid-4-one: m.p . 166
`-1
`(nuj ol) 1630, 3200 cm
`; 6(d6DMSO) 0.8(t, 3H), l,3 (m,8H) ,
`+
`2.5(s ,3H), 4.2(t,2H), 7.4(d,1H), 8.J(d ,lH); M 209.
`Example S: Partition data on 3-hydroxypyrid-2-and-4-ones and
`their iron complexes
`The partition coefficient K
`, being the ratio (concentra-
`part
`tion of compound in n-octanol)/(concent~ation of compound in
`aqueous phase) on par tition between n- octanol and aqueous tris
`hydrochloride (20 mM, pH 7.4), is measured at 20°c for various of
`the compounds of Examples 1 to 4 and for their 3:1 hydroxy(cid:173)
`pyridone:iron(III) complexes (at l0-4M) by spectrophotometry.
`Acid washed glassware is used throughout and, following mixing
`of 5 ml of the 10- 4M 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 run is used for
`co~centration determinations whilst for the iron complexes,
`2~ the range 340-640 nm· i s usea.
`Values typical of those obtained are shown in Table 1 where
`it will be seen that quite small changes in structure such as the
`replacement of a 1-propyl group by a 1-(1'-methylethyl) group can
`produce quite large d~fferences in Kpart values.
`
`
`12 of 22
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1021
`
`

`

`Table 1: Partition coefficients
`
`- 11 -
`
`Compound
`
`Partition Coefficient, K part
`
`Free
`Compound
`
`Iron complex
`III
`[Fe
`- (compound) 3]
`
`3-hydroxy-l-methylpyrid-2-one
`l-ethyl-3-hydroxypyrid-2-one
`3-hydroxy-l-propylpyrid-2-one
`3-hydroxy-1-(1'-methylethyl)-
`pyrid-2-one
`3- hydroxy- l,2- dimethylpyrid- 4- one
`l-ethyl-3-hydroxy-2-methylpyrid - 4- one
`3-hydroxy-2-methyl-l-propylpyrid-4-one
`3- hydroxy-l- (l'- methylethyl)- 2-methyl-
`pyrid-4- one
`l - butyl- 3- hydroxy- 2- methylpyrid- 4- one
`
`0.44
`0.52
`0.78
`3.10
`
`0.21
`0.40
`0.67
`0. 95
`
`5.30
`
`0.10
`1.06
`6.20
`13.50
`
`0.05
`0.03
`0.53
`0.20
`
`7.70
`
`05
`
`Example 6:
`In vitro tests of an iron binding capacity
`The 3- hydroxypyridones used in this Example were prepared as
`described in Examples l to 4.
`(1) Mobilisation of iron from ferr itin
`Horse spleen ferritin (Sigma) was used without fu rther purifi-
`ca~ion and its iron content was estimated spectrophotometrically
`at 420 nm. The ferritin solution in phosphate buffered saline
`.
`6
`(Dulbecco-OXOID (Trade Mark), io- M, pH 7.4) was enclosed in a
`Visking (Trade Mark) dialysis tube and dialysed against a
`10 3 x 10-3 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
`~~P.eated 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 x 10- 3 M desferrioxamine (Crichton et al,
`J. Inorganic Biochem., 1980, 13 , 305) and with 6 x 10-3 M LICAMS
`(LICAMS is N,N',N"-tris {2,3-dihydroxy-5-sulphonatobenzoyl)-
`1, 5,10-triazadecane;
`
`15
`
`20
`
`
`13 of 22
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1021
`
`

`

`- 12 -
`
`05
`
`Tufano!!.~· 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 have been
`confirmed by separating apoferritin and the 3-hydroxypyridone iron
`complex f;om tQ.e reaction prod11ct in each case by chromatography
`\.~"T"M}
`on Sephadex~GlO.
`Table 2: Rem~val of iron from ferritin
`
`Compound
`
`Percentage of iron removed
`
`6 hours
`
`24 hours
`
`Control
`3-hydroxy-l-methylpyrid-2-one
`l-ethyl-3-hydroxypyrid-2-one
`3-hydroxy-l-propylpyrid-2-one
`3-hydroxy-1-(1'-methylethyl)-
`pyrid-2-one
`3-hydroxy-l , 2-dimethylpyrid-4-one
`l-ethyl-3-hydroxy-2-methylpyrid-4-one
`3-hydroxy-2-methyl-l-propylpyrid-4-one
`3-hydroxy-2-methyl-1-(1 1-methylethyl)-
`pyrid-4-one
`l-butyl-3-hydroxy-2-methylpyrid-4-one
`Desferrioxam.ine (lmM)
`LI CAMS (6mM)
`LI CAMS (6mM+l2 mH ascorbic acid)
`
`0
`11
`14
`11
`
`11
`
`14
`19
`15
`17
`
`6
`1.5
`0
`7
`
`0
`22
`24
`21
`20
`
`31
`34
`26
`24
`
`7
`-
`-
`-
`
`10
`
`15
`
`(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.
`59
`-5
`-
`-3
`Iron (III) transferrin (10 M) was incubated with a 4 x 10 M
`solution in tris HCl (0.1 M, pH 7.4) of one of various pyridones
`as indicated in Table 2 for periods of 4 hours and 18 hours. The
`
`
`14 of 22
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1021
`
`

`

`- 13 -
`
`05
`
`IO
`
`15
`
`s~lution was then dialysed against phosphate buffered saline for
`24 hours. The 59Fe reraaining in the dialysis tube was then recorded.
`For comparative purposes, this procedure was repeated with desforri(cid:173)
`oxallli.ne using incubation for both 4 hours and 18 hours and with
`EDTA using incubation for 4 hours only.
`The results are shown in Table 3 in terms of the percentage
`of transferrin bound iron removed by the compound under t est. It
`will be seen that the pyrid-4-one compounds are very effective at
`iron removal, as compared with desferrioxamine or EDTA, aft er only
`4 hours. Although t he efficiency at iron removal of the pyrid-2-
`ooe compounds is only at a similar level to that of desferrioxamine
`and EDTA after 4 hours, it increases markedly aft er 18 hours
`whereas the level for desferrioxamine at 18 hours is substantially
`similar to that at 4 hours .
`Similar relative levels of efficiency were observed when the
`iron was measured spectrophotometrically. Moreover, the results
`shown in Table 3 have been confirmed by separating apotransferrin
`and the 3-hydroxypyridone iron complex from the reaction product
`in each case by chromatography on Sephadex GlO.
`Table 3: Removal of iron from transferrin
`
`Compound
`
`Percentage of iron removed
`
`4 hours
`
`18 hours
`
`Control
`3-hydroxy- l-methylpyrid- 2- one
`l-ethyl-3- hydroxypyr id-2-one
`3- hy,droxy-l-p r opylpyrid-2-one
`3-hydroxy- 1- (1 '-methylethyl) -
`pyrid-2-one
`3-hydroxy-1,2-dimethylpyrid-4-one
`l-ethyl- 3- hydroxy- 2- roethylpyrid-4- one
`3- hydroxy-2-methyl- l - propylpyrid-4- one
`
`0
`11
`12
`15
`17
`
`90
`88
`90
`
`0
`62
`52
`45
`57
`
`91
`90
`92
`
`continued
`
`
`15 of 22
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1021
`
`

`

`Table 3: Removal of iron from transferrin (continued)
`
`- 14 -
`
`Compound
`
`Percentage of iron removed
`
`6 hours
`
`24 hours
`
`3-hydroxy-2-methyl-l-(l'-methylethyl)-
`pyrid-4-one
`Desferrioxatnine
`EDTA
`
`94
`
`17
`27
`
`94
`
`22
`-
`
`10
`
`In vivo tests of iron binding capacity
`Example 7~
`The 3- hydroxypyridones used in this Example were prepared as
`described in Examples 1, 3 and 4 .
`Mice were injected intraperitoneally with iron dextran (2 mg)
`05 at weekly intervals over a four week period. Two weeks after the
`final injection, the mice were injected via the tail vein with
`59Fe lactoferrin (human lactoferrin, 1 mg per injection 2 Ci).
`The mice were then caged individually. After a ten day period,
`one of the various pyridones listed in Table 4 was administered to
`groups of mice at 10 mg per mous~ either intraperitoneally or
`intragastrically. 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
`desferrioxam.ine, also at 10 mg per mouse.
`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
`20 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 (the range of
`25 values obtained is given in the Table for each compound) .
`
`15
`
`
`16 of 22
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1021
`
`

`

`:
`
`Table 4: Excretion of iron in vivo
`
`- 15 -
`
`Compound
`
`Intraperitoneal
`Administration
`
`Intragastric
`Administration
`
`Exc:retion of
`59Fe!so
`Number (Range of
`values)
`of
`percent
`Mice
`
`Excretion of
`59Fe!so
`Number (Range of
`values)
`of
`percent
`
`Mice
`
`Control
`3-hydroxy-l-methyl-
`pyrid-2-one
`l-ethyl-3-hydroxypyrid-
`2-one
`3-hydroxy-l-propylpyrid-
`2-one
`3-hyd roxy-1, 2-
`dimethylpyrid-4-one
`Desf errioxamine
`
`12
`7
`
`13
`
`13
`
`7
`
`7
`
`100 :!: 10
`150 .:!: 30
`(107 - 192)
`223 .:!: 117
`(133 - 590)
`169 .:!: 49
`(112 - 280)
`-
`265 + 70
`(181 - 401)
`340 .:!: 90
`(172 - 472)
`
`-
`
`3
`
`13
`
`13
`
`3
`
`3
`
`-
`235 + 30
`-
`(222 - 240)
`188 .:!: 66
`(95 - 303)
`149 .:!: 56
`(53 - 260)
`320 : 90
`(242 - 425)
`90 .:!: 20
`(80 - 107)
`
`
`17 of 22
`
`Taro Pharmaceuticals, Ltd.
`Exhibit 1021
`
`

`

`- 16 -
`CLAIMS
`· A compound being a 3-hydroxypyrid-2-one or 3-hydroxypyrid-4-
`l.
`one in which the hydrogen atom attached to the nitrogen atom is
`replaced by an aliphatic hydrocarbon group of l to 6 carbon atoms
`and, optionally, in which one or more of the hydrogen atoms attached
`to ring carbon atoms are also replaced by an aliphatic hydrocarbon
`group of 1 to 6 carbon a