WO2016079502A1

WO2016079502A1 - Metal chelating compounds for use in treating diseases

Info

Publication number: WO2016079502A1
Application number: PCT/GB2015/053492
Authority: WO
Inventors: David Tetard; Francis William LEWIS
Original assignee: University Of Northumbria
Priority date: 2014-11-17
Filing date: 2015-11-17
Publication date: 2016-05-26
Also published as: GB201420348D0

Abstract

The present invention relates to heterocyclic compounds for use in the treatment of diseases. In particular, the invention relates to heterocyclic compounds for use in the treatment of neurodegenerative diseases, such as Parkinson's or Alzheimer's disease, and methods of making and using the same. The heterocycle compounds contain at least one electronegative atom and comprise a hydroxyl group or substituted hydroxyl group at the 1-position and the carbon at the 2-position being a carbonyl group.

Description

METAL CHELATING COMPOUNDS FOR USE IN TREATING DISEASES

The present invention relates to heterocyclic compounds for use in the treatment of diseases. In particular, the invention relates to heterocyclic compounds for use in the treatment of neurodegenerative diseases, such as Parkinson's or Alzheimer's disease.

Neurodegeneration is the progressive loss of structure or function, and in some cases death, of neurons responsible for processing and transmitting information in the human body through chemical or electrical signals. As a consequence of such progressive loss and death of neurons, diseases such as Parkinson's and Alzheimer's may result.

A common cause of neurodegenerative diseases is disregulation and subsequent accumulation of metals in certain regions of the brain, in particular iron(III) but also copper and zinc, which in excess cause oxidative damage to neurons. While the origins are not fully understood, metal accumulation is due to metal dyshomeostasis, i.e. a breakdown in the mechanism through which the body regulates metal ion concentrations. As a downstream consequence of metal disregulation, displaced metals detrimentally interact with proteins present in the brain, for example, beta amyloid in the case of Alzheimer's disease and tau protein and alpha-synuclein in the case of Parkinson's disease. Accordingly, excessive metal accumulation causes increased interactions with these proteins, leading to accumulation of protein-metal aggregates which are one of the causes of nerve cell death and, therefore, neurodegeneration.

Following the onset of neurodegenerative diseases such as Parkinson's or Alzheimer's, steps are taken to manage the disease, thereby providing symptomatic relief through the use of pharmaceutical drugs. Alternatively but less commonly, surgical procedures may be offered to Parkinson patients including deep brain stimulation, in which a brain pacemaker is implanted to send electrical impulses to specific parts of the brain to control symptoms, or lesional surgery, which involves the intentional creation of lesions to suppress over activity of specific subcortical areas. However, symptomatic treatment is largely ineffective for Alzheimer's patients and so in most situations palliative care is provided.

At present, there are no widely used measures effective in delaying or preventing the progression of neurodegenerative diseases such as Parkinson's or Alzheimer's. Further, no measure has been proposed which has the potential to reverse neuro degeneration, thus providing the possibility of an effective cure for such diseases.

It has previously been proposed that metal chelation may be used to reduce excessive metal accumulation in regions of the brain, thereby reducing neurodegeneration and so delaying or halting the progression of neurodegenerative diseases. For example, the use of 3-hydroxypyridin-4-one based compounds has been proposed for use as iron, copper and/or zinc chelators. Additionally, 5-chloro- 7-iodo-quinolin-8-ol, the antifungal and antiprotozoal drug clioquinol, has been proposed for use in treating neurodegenerative diseases. However, the majority of the metal chelators proposed to date suffer serious drawbacks, such as poor target specificity, poor penetration of the blood-brain barrier and clinical safety, limiting their widespread use in the treatment and prevention of such neurodegenerative diseases. Further, the use of clioquinol is controversial since 10,000 people in Japan developed subacute myelo-optico neuropathy when using it. In addition, known metal chelators such as clioquinol and related 8-hydroxyquinolines may not fully stabilise iron in the 3⁺ oxidation state, thus providing a risk of Fenton chemistry and associated oxidative stress. As such, there is no commercially available metal chelator drug used to treat neurodegenerative diseases.

To date, the only commercially available drug based on 3-hydroxypyridin-4-one is deferiprone, a drug used to treat thalassaemia major, a blood disorder caused by the weakening and destruction of red blood cells. Deferiprone chelates iron, reducing iron overload, a complication caused by thalassaemia major, thereby preventing the patient from accumulating potentially fatal iron levels. Deferiprone is the subject of ongoing clinical trials for the treatment of neurodegenerative diseases; it has also been commercially available for use in treatment of iron overload for some time now, buy typically has a 85% efficacy at treating iron overload. Other drugs used in medical applications as chelating agents to remove excess iron from the body include desferrioxamine, a hexadentate ligand, which has the drawback of being orally inactive as it is too hydrophilic and so must be injected to cross the blood- brain barrier, thus leading to poor patient compliance.

There exists a need to provide an effective treatment for neurodegenerative diseases based on metal chelation which effectively reduces metal accumulation, in particular iron accumulation, and restores metal ion homeostasis in the brain to delay or halt the progression of neurodegenerative diseases, which does not suffer the drawbacks of previously proposed metal chelators.

Accordingly, in a first aspect of the present invention, there is provided a heterocycle containing at least one electronegative atom and comprising a hydroxyl group or substituted hydroxyl group at the 1-position and the carbon at the 2- position being a carbonyl group for use as a medicament.

Without being bound to a specific mechanism of action, it is believed that the heterocycles of the present invention (hereafter called "1,2 -substituted heterocycles") strongly chelate to metals present in the brain, including metals in the 3⁺ oxidation state, thus rendering the metals redox inactive, preventing oxidative damage to neurons through the production of free radicals via the Fenton reaction and/or Haber-Weiss reaction and, therefore preventing neuro degeneration. Additionally, chelation with the heterocycles of the present invention increases the solubility of the metal, thus increasing distribution of the metal away from the brain. These surprising effects also render the heterocycles of the present invention suitable for use in a wide range of medical applications, for example, as antimicrobial agents, antifungal agents, biostatic agents, drugs against iron overload, cancer and malaria. This is surprising despite previous proposals to use 3-hydroxypyridin-4-one (hereafter called "3,4-substituted heterocycles") as it is appreciated that 1,2- substituted heterocycles have different properties, for example pKa, to 3,4- substituted heterocycles and so it is impossible to predict the effect of moving groups within a heterocycle. In addition, it has been found that the 1,2 -substituted heterocycles of the present invention are non-toxic to nerve cells, thus suggesting that they may be clinically safe, and provide effective chelation to metals present in the brain, thus delaying, halting and, in some cases, potentially reversing the damage cause by excess accumulation of the metals. Accordingly, the heterocycles of the present invention offer an effective treatment for neurodegenerative diseases such as Parkinson's and Alzheimer's.

The presence of a hydroxyl group or substituted hydroxyl group and the carbonyl group provide a bidentate ligand for chelation to metal ions such as iron, copper and zinc present in the brain. In particular, the 1,2 -substituted heterocycles of the present invention have lower pKa's than other derivatives of the substituted heterocycles, such as 3,4-substituted heterocycles, due to the alpha effect of the hydroxyl group or substituted hydroxyl group being attached directly to the electronegative atom of the heterocyclic ring. Therefore, at physiological pH the hydrogen or cation of the hydroxyl group or substituted hydroxyl group is more easily lost for efficient chelation. Further, as a consequence of the lower pKa, the metal complexes once formed between the 1,2 -substituted heterocycles of the present invention and the metal ions present in the brain are thermodynamically stronger complexes and so are less likely to dissociate.

Additionally, the presence of a hydroxyl or substituted hydroxyl group and a carbonyl group at adjacent positions on the heterocyclic ring provide effective chelation of metal ions present in the brain as they provide two hard Lewis bases for chelation to a hard Lewis acid (i.e. metal ions such as Fe(III)) and due to the enhanced entropy of binding. In one or more embodiments of the present invention, the heterocycle of the present invention provides a bidentate ligand for chelation to Fe(III), thereby providing an effective treatment for diseases caused by excess Fe(III) accumulation in the human and animal body.

The substituted form of the hydroxyl group may either be an ester or comprise an alkali metal such as sodium, potassium or lithium, an alkaline earth metal such as magnesium or calcium, an ammonium salt, a phosphonium salt or a sulfonium salt. The substituted form dissociates in situ to enable coordinate bonds to form between the heterocycle ligand and the metal ion. Alternatively or additionally, the substituted form of the hydroxyl group may be a protecting group which can be easily cleaved in the body by enzymatic action or other means to reveal the oxygen of the hydroxyl group. Examples of such protecting groups include an acetate, ether, silyl ether, carbonate ester, carbamate ester, phosphate ester or sulfonate ester. Where the protecting group is a carbamate ester, this may provide the additional benefit in use of inhibiting the acetylcholinesterase (AChE) enzyme which converts the neurotransmitter acetylcholine to choline and which is overexpressed in Alzheimer's disease. In particular, the ester or protecting group form of the heterocycle may be favoured to assist the heterocycle of the present invention to cross the barrier between the blood and the brain following administration, where the heterocycle is required as a treatment for neurodegenerative diseases. However, the heterocycle may further comprise at least one additional substituent selected to provide this functionality.

Preferably, the heterocycle contains at least one oxygen atom, at least one nitrogen atom or a combination thereof. More preferably, the heterocycle contains at least one nitrogen atom.

Preferably the heterocycle is a 4-, 5-, 6-, or 7-membered ring. In one preferred embodiment of the present invention, the heterocycle is a 5- or 6-membered ring. More preferably the heterocycle is a 6-membered ring. The heterocycle may be saturated or unsaturated. Alternatively and/or additionally, the heterocycle may be substituted with or fused to one or more cyclic compounds or two or more heterocycles of the present invention may be linked together to form a bis, tris, tetrakis, etc. heterocycle linked by any suitable linking group, as will be appreciated in the art, for example ^"OCH2CH2O, thus forming tetradentate, hexadentate, etc chelators. In a preferred embodiment, the heterocycle is unsaturated.

The 1,2 -substituted heterocycles in accordance with the preferred embodiments of the present invention chelate strongly to iron(III) and selectively in the presence of other metals, such as copper(II) and zinc(II). This is particularly beneficial where the heterocycles are intended for use in treating neurodegenerative diseases as iron(III) is redox active and is believed to play a significant role in neuro degeneration. Accordingly, these heterocycles may further overcome the problem of poor target specificity.

Examples of the heterocycles in accordance with the first aspect of the present invention include:

As will be appreciated, each of the structures exemplified above provide a hydroxyl group, which may be provided in a substituted form, directly attached to the electronegative atom of the heterocyclic ring and located adjacent to a carbonyl group, thus providing effective bidentate chelation to metal ions in the brain. Alternatively, and/or additionally, the heterocycle of the present invention may be a hydroxypyridinone, hydroxypyranone, hydroxypyrazinone, hydroxypyridazinone or hydroxypyrimidinone.

In a preferred embodiment of the present invention, the heterocycle is a nitrogen containing six membered heterocycle having the general formula:

(I) wherein X, Y, Z and X' are independently selected from the group consisting of C, N, 0 or S;

R¹ is selected from the group comprising H, an alkyl, an aryl, a benzyl an ester, an amide, a carbamate, a carbonate, a sulfinate, a sulfonate, a phosphonate, an alkali metal, an alkaline earth metal, an ammonium salt, a phosphonium salt, a sulfonium salt, an O-glycoside, an antioxidant, a protecting group or a group capable of being cleaved enzymatically in the cell to reveal the oxygen for chelation; and

R² and/or R³ are absent or comprises at least one additional substituent selected to provide additional functionality to the heterocycle or a pharmaceutically acceptable salt and/or an isomer thereof.

The protecting group may be any of the types described above. The group capable of being cleaved enzymatically in the cell to reveal the oxygen for chelation may be selected from carbonates, carbamates, glycosides, benzyl ethers or one or more protecting group as defined above. Alternatively and/or additionally, R¹ may also be substituent selected to provide additional functionality to the heterocycle, as discussed in further detail below. Preferably, at least one of X, Y, Z and X' are carbon atoms, more preferably at least Z and X' is a carbon atom. In one embodiment, X and/or Y is a nitrogen atom. In an alternative embodiment, all of X, Y, Z and X' are carbon atoms.

At least one additional substituent may be present or absent depending upon whether the compound requires modifications to be suitable for use in the human body or further reduce toxicity, if necessary. As will be appreciated, the selection of a suitable substituent requires a simple design modification rather than any inventive skill, depending on the intended use of the compound.

The nitrogen containing six membered heterocycle may comprise from 1 to 4 additional substituents at the 3-, 4-, 5- and/or 6- positions.

For example, R² or R³, i.e. the from 1 to 4 additional substituents, may be independently selected from the groups comprising:

F, CI, Br or I;

OH, OR, OCOR, NH₂, NHR⁴, NR⁴R⁵, ⁺NR⁴R⁵R⁶, C0₂R⁴, C0NHR⁴, CONR⁴R⁵ or

C=0R⁴;

R⁴OR⁵;

a substituted or unsubstituted alkyl, heteroalkyl, alkenyl, heteroalkenyl or alkynyl, which may be linear, branched or cyclic;

a substituted or unsubstituted aryl or heteroaryl;

S0₃-, X⁺, SO3H, SH or SR⁴;

P=0(OH)₂ or salts P=0(OR⁴)₂ or P=0(OR⁴)(OR⁵);

S=0R⁴ or 0=S=0R⁴;

B(OH)₂ or B(OR⁴)(OR⁵);

CN;

N0₂; or

C(=NH)(NH₂), C=SR⁴;

wherein R⁴, R⁵ and R⁶ may be independently selected from the group consisting of: H; OH; 0(CH₂)_XCH3; (CH₂)_XCH3; a alkyl, heteroalkyl, alkenyl, heteroalkenyl or alkynyl, which may be linear, branched or cyclic; a methylene moiety; an acyl; a sulfanyl; an aryl; a heteroaryl; or an antioxidant moiety; where x = 0 to 12, and X⁺ may be independently selected from the group consisting of Na⁺, K⁺, Li⁺, Mg²⁺, Ca²⁺ or an organic cation.

In one or more embodiments, where at least two additional substituents are present, the additional substituents may be linked to form a cyclic structure. Alternatively and/or additionally, the additional substituents may be linked together via a linking group, such as ^"OCH2CH2O, to form a bis-, tris- or tetrakis structure.

The heterocycle of the present invention may be a pharmaceutically acceptable salt. Pharmaceutically or physiologically acceptable salts are particularly suitable for medical applications because of their greater aqueous solubility relative to the parent compounds. Such salts must clearly have a physiologically acceptable anion or cation. Suitably physiologically acceptable salts of the compounds of the present invention include acid addition salts formed with inorganic acids such as hydrochloric, hydrobromic, hydroiodic, phosphoric, metaphosphoric, nitric and sulfuric acids, and with organic acids, such as tartaric, acetic, trifluoroacetic, citric, malic, lactic, fumaric, benzoic, formic, propionic, glycolic, gluconic, maleic, succinic, camphorsulfonic, isothionic, mucic, gentisic, isonicotinic, saccharic, glucuronic, furoic, glutamic, ascorbic, anthranilic, salicylic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, pantothenic, stearic, sulfinilic, alginic, galacturonic and arylsulfonic, for example benzenesulfonic and p-toluenesulfonic, acids; base addition salts formed with alkali metals and alkaline earth metals and organic bases such as Ν,Ν-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), lysine and procaine; and internally formed salts. Salts having a non-physiologically acceptable anion or cation are within the scope of the invention as useful intermediates for the preparation of physiologically acceptable salts and/or for use in non-therapeutic, for example, in vitro, situations. As will be appreciated, the heterocycle may exist as an isomer of formula I, preferably a stereoisomer thereof including but not limited to diastereomeric, geometric and optical isomers, such as cis- and trans- forms, £-and Z- forms, D- and L- forms, (+) and (-) forms, and combinations thereof.

In one or more preferred embodiments, the nitrogen containing six membered heterocycle has the formula:

wherein X and Y are independently selected from C or N;

R¹ is selected from the group comprising H, an alkyl, an aryl, a benzyl an ester, an amide, a carbamate, a carbonate, a sulfinate, a sulfonate, a phosphonate, an alkali metal, an alkaline earth metal, an ammonium salt, a phosphonium salt, a sulfonium salt, an O-glycoside, an antioxidant, a protecting group, a group capable of being cleaved enzymatically in the cell to reveal the oxygen for chelation or a substituent selected to provide additional functionality to the heterocycle, as defined above;

R² is an alkyl, an aryl, an heteroaryl, OH, OR', SH, SR', S(0)R', S0₂R', NH, NR', sulfinyl, sulfonyl, F, CI, Br, I, C=0, COOR', C=NR' or R⁵OR⁶, wherein R' is H, alkyl, cycloalkyl, heterocycloalkyl, acyl, aryl or heteroaryl, R⁵ is (CH2)_X wherein x = 0 to 100 and R⁶ is H, an alkyl, an acyl, an aryl, a sulfinyl, a sulfonyl, an antioxidant moiety or any other functional groups, such as groups which help deliver the nitrogen containing six membered heterocycle to a certain region of the nerve cell or assist in crossing the blood-brain barrier; and

R³ and/or R⁴ are each independently absent or present on the ring and comprise at least one additional substituent selected to provide additional functionality to the heterocycle, as defined above; or a pharmaceutically acceptable salt and/or an isomer thereof.

The additional substituent may be any of the groups described above. Further, the nitrogen containing six membered heterocycle may comprise from 1 to 3 additional substituents at the 3-, 4-, and/or 5- position, independently selected from the aforementioned groups.

At least X may be N. Preferably R² and/or R³ are at the 3-, 5- or 6- position in the nitrogen containing six membered ring. In a preferred embodiment, R² and/or R³ are selected from the group consisting of: H, an alkyl, such as CH3,; cycloalkyl, such as R²-(CH₂)x-R³ where x = 0 to 6, COOR', such as COOH; and R⁶OR⁷, such as CH₂OH.

In a preferred embodiment of the present invention, the heterocycle of the present invention is intended for use in the treatment of neurodegenerative diseases, preferably Parkinson's disease or Alzheimer's disease.

In a second aspect of the present invention, there is provided a method for treating a neurodegenerative disease comprising the steps of:

providing a composition comprising a heterocycle containing at least one electronegative atom and comprising a hydroxyl group or salt derived therefrom at the 1-position and a carbonyl group at the 2-position; and administering an effective dose of the composition to a patient.

The "effective dose" of the composition is an amount sufficient to provide therapeutic relief or efficacy without any toxic side-effects. This is dependent upon the 1,2 -substituted heterocycle provided and may be readily calculated.

As will be appreciated, the preferred features and embodiments of the first aspect of the present invention apply mutatis mutandis to the second aspect of the present invention. The composition may further comprise a pharmaceutically acceptable excipient. For example, the composition may comprise one or more pharmaceutically acceptable excipients well known in the art. Additionally or alternatively, the composition may comprise one or more known active pharmaceutical ingredient used for the treatment of symptoms of neurodegenerative disease, such as Alzheimer's or Parkinson's, which complement the activity of the 1,2 -substituted heterocycles of the present invention. Examples of such known active pharmaceutical ingredients include additional chelating compounds or antioxidants.

In a third aspect of the present invention, there is provided a nitrogen containing six membered heterocycle having the formula:

(III) wherein R is selected from the group comprising H, an alkyl, an aryl, a benzyl an ester, an amide, a carbamate, a carbonate, a sulfinate, a sulfonate, a phosphonate, an alkali metal, an alkaline earth metal, an ammonium salt, a phosphonium salt, a sulfonium salt, an O-glycoside, an antioxidant, a protecting group, a group capable of being cleaved enzymatically in the cell to reveal the oxygen for chelation or a substituent selected to provide additional functionality to the heterocycle, as defined above in relation to the first aspect of the present invention;

R¹ is (CH₂)x, wherein x = 0 to 100;

R² is H, an alkyl, an acyl, an aryl, a sulfanyl, an antioxidant moiety or any other functional groups, such as groups which help deliver the nitrogen containing six membered heterocycle to a certain region of the nerve cell or assist in crossing the blood-brain barrier; and

R³ is absent or present at at least one of the 3-, 4- or 5 -positions on the ring and comprises at least one additional substituent selected to provide additional functionality to the heterocycle or a pharmaceutically acceptable salt and/or an isomer thereof.

The additional substituent may be any of the groups described above in relation to the preferred embodiment of the first aspect of the present invention. Further, the nitrogen containing six membered heterocycle may comprise from 1 to 3 additional substituents at the 3-, 4-, and/or 5- position, independently selected from the aforementioned groups.

It has been found that the heterocycle in accordance with the third aspect of the present invention, which is a previously unknown heterocycle, is particularly effective at chelating to excess metals in the brain and so reducing oxidative damage to neurons. In particular, testing has shown that this heterocycle provides a 5% cell viability compared to a control at concentrations of ΙΟΟμΜ, in the presence of 6- hydroxydopamine (6-OHDA), a synthetic compound used to selectively destroy neurons and induce Parkinsonism. This is an improvement of over 111% when compared to the results achieved with commercially available drugs (such as deferiprone) at the same concentration. Accordingly, the heterocycle of the third aspect of the present invention provides a more effective treatment for neurodegenerative diseases.

In a preferred embodiment of the present invention, R is H and so the nitrogen containing six membered heterocycle is l-hydroxy-6-(hydroxymethyl)pyridin-2- one.

In one or more embodiments of the present invention, the nitrogen containing six membered heterocycle has the formula:

(IV) wherein R is selected from the group comprising H, an alkyl, an aryl, a benzyl an ester, an amide, a carbamate, a carbonate, a sulfinate, a sulfonate, a phosphonate, an alkali metal, an alkaline earth metal, an ammonium salt, a phosphonium salt, a sulfonium salt, an O-glycoside, an antioxidant, a protecting group, a group capable of being cleaved enzymatically in the cell to reveal the oxygen for chelation or a substituent selected to provide additional functionality to the heterocycle, as defined above in relation to the first aspect of the present invention;

or a pharmaceutically acceptable salt and/or an isomer thereof.

The nitrogen containing six membered heterocycle of formula (IV) may further comprise one or more additional substituents at the 3-, 4- or 5- position independently selected from the groups described in relation to the first aspect of the present invention to provide functionality to the heterocycle.

In a fourth aspect of the present invention, there is provide a method of forming the nitrogen containing six membered heterocycle of the third aspect comprising the steps of:

providing a l-hydroxypyridin-2-one-6-carboxylic acid methyl ester; reacting the l-hydroxypyridin-2-one-6-carboxylic acid methyl ester with a protecting group to protect the N-hydroxyl group, thereby forming a protected l-hydroxypyridin-2-one-6-carboxylic acid methyl ester;

reduction of the methyl ester group of the protected 1-hydroxypyridin- 2-one-6-carboxylic acid methyl ester to form protected l-hydroxy-6- hydroxymethyl-pyridin-2-one; and

deprotecting of the N-hydroxyl group of the protected l-hydroxy-6- hydroxymethyl-pyridin-2-one by removal of the protecting group to form the nitrogen containing heterocycle of the third aspect of the present invention.

In one embodiment, the l-hydroxypyridin-2-one-6-carboxylic acid methyl ester is provided by reaction of a l-hydroxypyridin-2-one-6-carboxylic acid with thionyl chloride in methanol. Further, the l-hydroxypyridin-2-one-6-carboxylic acid may be synthesised by treatment of 6-hydroxypicolinic acid with peracetic acid in acetic acid.

The protecting group may be an allyl, benzyl, silyl or sulfonate ester group, or any other protecting group known in the art. In a preferred embodiment, the protecting group is an allyl group for ease of subsequent removal and also to avoid the risk of cleavage of the benzylic C-0 bond (i.e., the C-0 bond of the 6-hydroxymethyl group) under standard hydrogenation conditions.

Reduction of the ester group of the l-hydroxypyridin-2-one-6-carboxylic acid methyl ester may be accomplished by reaction with a reducing agent, for example, a reducing agent comprising potassium, calcium, barium, sodium or magnesium and/or borohydride. Preferably, the reducing agent is sodium borohydride.

Deprotection of the N-hydroxyl group of the protected l-hydroxy-6-hydroxymethyl- pyridin-2-one by removal of the protecting group may be accomplished in the presence of palladium on activated carbon and trifluoroacetic acid.

In a fifth aspect of the present invention, there is provided the nitrogen containing six membered heterocycle of the third aspect for use as a medicament.

Preferably, the nitrogen containing six membered heterocycle is intended for use in the treatment of neurodegenerative diseases, more preferably Parkinson's disease or Alzheimer's disease.

In a sixth aspect of the present invention, there is provided a method for treating a neurodegenerative disease comprising the steps of:

providing a composition comprising the nitrogen containing six membered heterocycle of the third aspect of the present invention; and administering an effective dose of the composition to a patient. As will be appreciated, the preferred features and embodiments of the various aspects of the present invention apply mutatis mutandis to the other aspects of the present invention.

Various embodiments of the present invention will now be described more particularly by way of examples, with reference to the accompanying figures in which:

Figures 1 to 12 graphically show the results of the analysis of the neuroprotection of the tested compounds, as detailed below, to 6-OHDA toxicity;

Figures 13 to 16 graphically show data obtained via analysis of the neuroprotection of the l-hydroxypyridin-2-one compound in accordance with the fifth embodiment of the present invention and its analogues;

Figure 17 shows a comparison of the metal chelation ability of the compound of the fifth embodiment of the present invention, its analogues and the known 3- hydroxypyridin-4-one compound, Deferiprone (DFP); and

Figure 18 illustrates the changes to known markers of Parkinson's disease toxicity with deferiprone (DFP), the compound of the fifth embodiment of the present invention and its analogues.

Compounds of the Present Invention

l-hydroxy-2(i/f)-pyrazinone compound in accordance with a first embodiment synthesised by the reaction of glycine hydroxyamic acid and pyruvaldehyde in water in at an alkaline pH.

[15] l-hydroxypyridin-2-one compound in accordance with a second embodiment, synthesised in accordance with the method disclosed in /. Med. Chem. 2002, 45, 3963-3971, in which acetic acid was employed as the solvent instead of trifluoroacetic acid/trifluoroacetic anh dride for safety reasons.

l-hydroxypyridin-2-one compound in accordance with a third embodiment, synthesised in accordance with the method disclosed in /. Labelled Cpd. Radiopharm. 2001, 44, 13-19.

l-hydroxypyridin-2-one compound in accordance with a fourth embodiment.

[3]

This compound was synthesised via the procedure described in detail below. l-hydroxypyridin-2-one compound in accordance with a fifth embodiment

[9] This compound, which is in accordance with a third aspect of the present invention, was synthesised via the procedure described in detail below.

Prior art compounds

Compounds known in the art were provided for comparison to the compounds in accordance with the present invention. The prior art compounds are commercially available or were synthesised in accordance with techniques described in the art, having the following formulae: l-hydroxy-2(iH)-pyrazinone compound, synthesised in accordance with the method disclosed in Chem. Phar 36, 2323-2330.

[16]

3-hydroxypyridin-2-one compound, synthesised in accordance with the method disclosed in Tetrahedron, 1995, 55, 1129-1142.

3-hydroxypyran-4-one compound (maltol, commercially available from Sigma- Aldrich).

3 -hydroxypyridin-4-one compound (deferiprone, commercially available from Sigma-Aldrich).

In addition, an N-oxide [11] and pyridone [12] were synthesised, as described in detail below, having the following formulae:

These compounds are considered structural analogues of the l-hydroxypyridin-2 - one compound in accordance with the fifth embodiment of the present invention but lack the cyclic hydroxamic acid metal chelating functionality. Accordingly, the screening results for these compounds provide information on the extent to which drug efficacy in l-hydroxypyridin-2 -one compounds is due to their metal chelating ability. Experimental

General Procedures. All solvents and reagents were purchased from Sigma-Aldrich, Acros Organics or Alfa-Aesar and used without further purification unless otherwise specified. Reactions were monitored by TLC using silica gel with UV254 fluorescent indicator. Organic extracts of reaction products were dried over anhydrous magnesium sulfate.

Synthesis of l-hydroxypyridin-2-one in accordance with the fourth embodiment

Formation of 2-chloropyridine-3-carboxylic acid N-oxide:

To a solution of 2-chloronicotinic acid [1] (12.00 g, 76 mmol), having the formula:

in glacial acetic acid (90 mL) was added peroxyacetic acid (36-40 %, 35 mL). The solution was carefully raised to 80 °C and stirring was continued for 10 hours. The solution was cooled to room temperature and the resulting precipitate was filtered and washed with diethyl ether. The solid was allowed to dry in air to afford 2-chloropyridine-3-carboxylic acid N-oxide [2] as a white powder (5.56 g, 42 %), having the formula:

[2]

Formation of l-hydroxy-2-oxo-l,2-dihydropyridine-3-carboxylic acid:

A solution of N-oxide [2] (7.85 g, 45 mmol) in aqueous potassium hydroxide (10 %, 125 mL) was stirred at 70 °C for 3 days. The solution was then cooled to 0 °C and concentrated hydrochloric acid was added to pH 1. The precipitated solid was collected by filtration and washed with water. The crude solid was then recrystallised from methanol to afford l-hydroxy-2-oxo- l,2-dihydropyridine-3-carboxylic acid [3] as white needles (6.51 g, 92 %), having the formula:

[3]

Synthesis of l-hydroxypyridin-2-one in accordance with the fifth embodiment

Formation of l-hydroxy-6-oxo-l,6-dihydropyridine-2-carboxylic acid:

To a suspension of 6-hydroxypicolinic acid [4] (26.20 g, 188 mmol) having the formula:

in glacial acetic acid (160 mL) was carefully added peroxyacetic acid (36-40 %, 80 mL). The temperature was carefully raised to 80 °C and stirring was continued for 12 hours. The flask was allowed to cool to room temperature and the resulting solid precipitate was collected by filtration and washed with diethyl ether, affording l-Hydroxy-6-oxo-l,6-dihydropyridine-2-carboxylic acid [5] as a cream solid (18.17 g, 77 %), having the formula:

Formation of methyl l-hydroxy-6-oxo-l,6-dihydropyridine-2-carboxylate: To a suspension of acid [5] (15.73 g, 101 mmol) in methanol (200 mL) at 0 °C was added thionyl chloride (31.00 g, 426 mmol) dropwise. The mixture was heated under reflux for 4 hours. The solution was then allowed to cool to room temperature and the solvent was removed in vacuo to afford methyl 1- hydroxy-6-oxo-l,6-dihydropyridine-2-carboxylate [6] as a cream solid (16.47 g, 96 %), having the formula:

Formation of methyl 6-oxo-l-(allyloxy)-l,6-dihydropyridine-2-carboxylate: To a solution of [6] (16.47 g, 97 mmol) in acetonitrile (200 mL) was added potassium carbonate (32.11 g, 232 mmol), followed by allyl bromide (28.10 g, 232 mmol). The flask was heated under reflux for 4 hours before the reaction mixture was filtered and the solvent removed under high vacuum. The residue was dissolved in toluene (100 mL) and the solvent was evaporated in vacuo to afford methyl 6-oxo-l-(allyloxy)-l,6-dihydropyridine-2-carboxylate

[7] as a white crystalline solid (19.12 g, 94 %), having the formula:

Formation of 6-(hydroxymethyl)-l-(allyloxy)pyridin-2 (lH)-one:

To a suspension of [7] (19.12 g, 92 mmol) in tetrahydrofuran (200 mL) was added solid sodium borohydride (25.07 g, 663 mmol) in small portions. The solution was heated under reflux for 15 minutes. Methanol (14 mL) was then added dropwise at reflux over 2 hours. The solution was then cooled to 0 °C, quenched by careful addition of saturated aqueous ammonium chloride (25 mL) and stirring was continued for 15 minutes. The solvents were removed in vacuo and the residue was extracted with dichloromethane (3 25 mL). The combined organic extracts were dried and evaporated to afford 6- (hydroxymethyl)-l-(allyloxy)pyridin-2(lH)-one [8] an off-white solid (10.83 g, 65 %), having the formula:

[8] Formation of l-hydroxy-6-(hydroxymethyl)pyridin-2 (lH)-one:

To a solution of compound [8] (0.21 g, 1.06 mmol) in 20 % water in dioxane (10 mL) was added trifluoroacetic acid (0.11 mL). The solution was heated under reflux for 1 hour. Palladium on activated carbon (10% Pd/C, 0.02 g) was then added and the mixture was heated under reflux for a further 20 hours. The mixture was filtered and the filtrate was evaporated in vacuo to afford l-hydroxy-6-(hydroxymethyl)pyridin-2 (lH)-one [9] as a brown solid (0.08 g, 60 %), having the formula:

Synthesis of 2- (hydroxymethyl) pyridine TV-oxide

To a solution of m-CPBA (14.67 g, 50 wt % in Η2Ο, 42.5 mmol) in chloroform (30 mL) was slowly added a solution of 2-(hydroxymethyl)pyridine [10] (3.85 g, 35.25 mmol) in chloroform (15 mL), having the formula:

The solution was heated at 65 °C for 20 hours. The solution was then allowed to cool to room temperature and was dried and evaporated. The resulting residue was triturated with diethyl ether (150 mL) and the suspension was heated under reflux for 30 minutes. The insoluble solid was filtered and washed with diethyl ether (20 mL) and allowed to dry in air to afford 2- (hydroxymethyl)pyridine N-oxide [11] as a white solid (2.77 g, 63 %), having the formula:

[11]

Synthesis of 6-(hydroxymethyl)pyridin-2(lH)-one

To a suspension of acid [4] (1.00 g, 7.19 mmol) in dry tetrahydrofuran (35 mL) was added a solution of borane in tetrahydrofuran (35.9 mL, 1 M, 5 eq) dropwise. After the addition was complete, the flask was heated to 65 °C for 24 hours. The solution was then cooled to 0 °C and methanol (30 mL) was added. The flask was heated at 65 ° overnight and the solvent was evaporated. Methanol (50 mL) was added and the solvent was again evaporated. The resulting solid was triturated with acetone (30 mL), and then filtered and washed with methanol (5 mL) and acetone (40 mL) to afford 6- (hydroxymethyl)pyridin-2 (lH)-one [12] as a white solid (0.47 g, 52 %), having the formula:

Η [12]

Synthesis of 6-methyl-l-hydroxy-2(lH)-pyrazinone

Formation of glycine hydroxamic acid:

To a solution of glycine ethyl ester hydrochloride [13] (6.02 g, 43.129 mmol), having the formula:

in water (4 mL) was added hydroxylamine hydrochloride (2.997 g, 43.129 mmol). The solution was cooled to 0 °C and aqueous sodium hydroxide (11.8 mL, 12 M, 3.3 equiv) was added dropwise over 10 minutes. The solution was stirred at 0 °C for 30 minutes and was then allowed to warm to room temperature and stirring was continued for 24 hours. Aqueous hydrochloric acid (37 %, 4.3 mL, 10 M, 1 equiv) was then added to bring the solution to pH 2 and the solution was cooled to 0 °C. The precipitated solid was filtered and washed with cold water (10 mL) and was allowed to dry in air to afford glycine hydroxamic acid [14] as a white solid (2.49 g, 64 %), having the formula:

[14]

Formation of 6-methyl-l-hydroxy-2(i//)-pyrazinone:

To a solution of glycine hydroxamic acid [14] (0.50 g, 5.555 mmol) in methanol (40 mL) and water (20 mL) at 0 °C was added a solution of pyruvaldehyde (1.15 g, 40 wt. % in water, 1.15 equiv) in methanol (15 mL). Aqueous sodium hydroxide (2.0 mL, 2 M) was added dropwise over 15 minutes and the solution was stirred at 0 °C for 1 hour. The solution was then allowed to warm to room temperature and stirring was continued for 24 hours. Aqueous hydrochloric acid (37 %, 10 M) was then added dropwise to bring the solution to pH 3 and the solution was evaporated to a small volume. The solution was diluted with water (40 mL) and extracted with chloroform (3 x 100 mL). The combined organic extracts were dried and evaporated to afford a light brown solid. The solid was triturated with ether (50 mL) and filtered and washed with ether (50 mL) to afford 6-methyl-l-hydroxy-2(i//)- pyrazinone [15] as a light brow g, 11 %), having the formula:

[15] Synthesis of 5,6-dimethyl-l-hydroxy-2(lH)-pyrazinone

To a solution of glycine hydroxamic acid [14] (0.70 g, 7.777 mmol) in methanol (50 mL) and water (30 mL) at 0 °C was added a solution of butane-2,3-dione (o.78 mL, 1.15 equiv) in methanol (10 mL). Aqueous sodium hydroxide (1.0 mL, 2 M) was added dropwise over 5 minutes and the solution was stirred at 0 °C for 1 hour. The solution was then allowed to warm to room temperature and stirring was continued for 24 hours. Aqueous hydrochloric acid (37 %, 10 M) was then added dropwise to bring the solution to pH 3 and the solution was evaporated to a small volume. The solution was diluted with water (20 mL) and extracted with chloroform (5 x 50 mL). The combined organic extracts were dried and evaporated to afford a light brown solid. The solid was triturated with acetone (10 mL) and filtered and washed with acetone to afford 5,6-dimethyl-l-hydroxy-2(i//)-pyrazinone [16] as a light brown solid (0.26 g, 24 %), having the formula:

[16]

Testing Methodology

Analysis of tested compound toxicity in SH-SY-5Y neuroblastoma cells

SH-SY-5Y neuroblastoma cells were plated at 20,000 cells/well in a 96 well plate and left to adhere to the well surface and culture overnight in 50% advanced minimum essential medium (MEM), 50% Ham's F12 medium, 1% L-Glutamine and 2% fetal bovine serum (FBS). The media was then replaced with 100 μί^εΐΐ of FBS free MEM/Ham's F12 containing varying concentrations of the tested compound ranging from 0 to 300 μΜ. After a further 24 hour incubation at 37 °C in a 5% C0₂ environment cell viability was measured using the tetrazolium dye 3-(4,5- dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT). For this colorimetric assay, 11 μΐ. of MTT (5 mg/mL) was added to the 100 μΐ. media in the well and incubated for 3 hours at 37 °C. After this time an equal volume of solubilizing solution (24 mL isopropyl alcohol, 1 mL HC1) was added to each well and thoroughly mixed to lyse the cells. Absorption was measured at 570 nm using a Tecan Sunrise spectrophotometer; model Sunrise-basic Tecan.

The cell toxicity of the tested compound on normal healthy neurons was calculated to obtain a non-toxic concentration. This was done by adding increasing concentrations of the tested compound to the cells to calculate the concentration which may be applied before the cells die due to toxicity, if any, of the tested compounds.

Cell viability was calculated as a % compared to the untreated control, i.e. in the absence of the tested compound. In particular, the cell viability was measured in the presence of 6-hydroxydopamine hydrobromide (6-OHDA), which synthetically induces Parkinson's disease in nerve cells, and the tested compound at the non-toxic concentration found in order to assess the efficacy in preventing neuro degeneration. The results for the cell viability of the tested compounds are as follows:

Analysis of tested compound neuroprotection to 6-OHDA toxicity in SH-SY-5Y neuroblastoma cells.

SH-SY-5Y neuroblastoma cells were plated at 20,000 cells/well in a 96 well plate and left to adhere to the well surface and culture overnight in 50% advanced minimum essential medium (MEM), 50% Ham's F12 medium, 1% L-Glutamine and 2% fetal bovine serum (FBS). Just before addition to cells, 6-hydroxydopamine hydrobromide (6-OHDA) was made to a stock concentration of 10 mM and stored in the dark at 4 °C. Media was then replaced with 100 μΙ/weW FBS free MEM/Ham's F12 containing 50 μΜ 6-OHDA and varying concentration of tested compound ranging from 0 to 300 μΜ. After a further 24 hour incubation at 37 °C in a 5% C0₂ environment cell viability was measured using the tetrazolium dye 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). For this colorimetric assay, 11 of MTT (5 mg/mL) was added to the 100 media in the well and incubated for 3 hours at 37 °C. After this time an equal volume of solubilizing solution (24 mL isopropyl alcohol, 1 mL HC1) was added to each well and thoroughly mixed to lyse the cells. Absorption was measured at 570 nm using a spectrophotometer. Cell viability was calculated as a % compared to the untreated control, i.e. in the absence of 6-OHDA or the tested compound.

Measurement of the neuronal cytoplasmic labile iron pool using Calcein-AM assay.

The use of Calcein-AM to measure neuronal cytoplasmic labile iron pool was adapted from a previously reported procedure disclosed in Free Radic. Biol. Med. 2002, 33, 1037-1046. In brief, SH-SY5Y cells plated at 20,000 cells/well in a black 96-well microplate were treated with ferric ammonium citrate (FAC; 50 μΜ) for 6 hrs in serum-free media. Cells were washed twice with phosphate buffered saline (PBS) after which Calcein-AM (60 nM) was added. Fluorescence at an excitation of 485 nm and emission of 535 nm measurements were started immediately and taken every minute using a Biotek Fluorescence microplate reader for 10 min or until a consistent minimum reading had been reached. In triplicate, the compounds of interest (100 μΜ) were rapidly added and fluorescence measurements started again immediately. Readings were continued for a further 5 min or until a plateau had been reached. The percentage labile iron pool chelated was calculated as the AF (the difference in Fluorescence from before and after compound addition) compared to deferiprone (DFP) as a known lipid permeable chelator.

Analysis of iron and 6-OHDA changes to iron response proteins.

For investigating compound effect on iron response protein expression after iron loading, SH-SY5Y's prepared 6-well plates, were incubated in serum-free media with FAC (50 μΜ) for 24 hrs. After iron loading, iron containing media was replaced with fresh serum-free media alone or in the presence of the compound of interest (100 μΜ) and incubated for a further 12 hrs. To evaluate iron response protein expression to each compound on 6-OHDA, a similar procedure to the MTT assay was followed. Therefore, SH-SY5Y's prepared in 6-well plates, were incubated in serum- free media with 6-OHDA (50 μΜ) with or without the compound (100 μΜ) for 24 hrs. After each experimental condition, cells were washed twice with cold PBS, before collection and homogenizing in RIPA buffer (150 mM NaCl, 1% (v/v) Nonidet P-40, 1% (w/v) sodium deoxycholate, 0.1% (v/v) SDS, 25 mM Tris-HCl, pH 7.6) with EDTA-free protease inhibitor cocktail (Complete; Roche). Lysates were clarified by centrifugation at 14,000 x g for 15 min.

Western blot analysis.

As determined by BCA assay, 10 μg of the protein from each experimental condition was separated either on 10% PAGE (Tris-Glycine, BioRad) for 22C11 and Transferrin (TfR) or 4-20% PAGE (Tris-Glycine, BioRad) for Ferritin (FT), Tyrosine Hydroxylase (TH) and a-Synuclein (Syn). Resolved proteins transferred to polyvinylidene difluoride membranes (Hybond-P, Pierce) were probed with mouse anti-APP (1:1000, 22C11, in house), mouse anti-TfR (1:2000, H68.4, Invitrogen), rabbit anti-Ferritin (1:1000, Cell Signaling Technology), mouse anti-TH (1:1000, AB152, Millipore) or mouse anti-Syn (1:1000, D35E4, Cell Signaling Technology) and the appropriate secondary antibodies. The load control was mouse anti- -actin (1:5,000, AC15, Sigma). Proteins of interest were visualized with ECL (Pierce) and a LAS-3000 Imaging suite, and analyzed using Multi Gauge (Fuji). Densitometry using Image J (NIH) was performed in triplicate on 3 separate experiments. All quantitation was standardized against β-actin levels.

Results

The data tables below show the results of the analysis of the neuroprotection of the tested compounds to 6-OHDA toxicity. The compounds of the present invention provide comparable cell survival in the presence of 6-OHDA compared to the known compounds and higher non-toxic concentrations in the control. These results show that the treatment of healthy neuroblastoma cells (i.e. the control) with 50 μΜ 6-OHDA typically results in the death of approximately 50% of the cells. This is due to the oxidative stress and metal dysregulation, etc. initiated by 6-OHDA. Further, at relatively low concentrations, i.e. 3 μΜ to 30 μΜ, the known 3- hydroxypyridin-4-one compound (Deferiprone) and the compound of the present invention exhibit similar % cell viability in the 6-OHDA model of Parkinson's disease when compared to the control. At a concentration of 100 μΜ, the compound of the present invention exhibits cell viability comparable to the known 3-hydroxypyridin- 4-one. This reduction in cell death for the present invention is believed to occur as a result of the compound of the present invention binding to metal ions present, particularly iron(III), which prevents Fenton chemistry, stops the onset of oxidative stress and prevents metal-protein interactions.

Figures 1 to 12 graphically show the data in relation to the tables given below, and the compound identification numbers relate to the identifying numbers described below in relation to the data tables. The y-axis indicates percentage cell viability as compared to the control.

Figures 13 to 16 show pooled data obtained via analysis of the neuroprotection of the l-hydroxypyridin-2-one compound in accordance with the fifth embodiment of the present invention, and its analogues which lack the cyclic hydroxamic acid moiety and thus do not have the ability to chelate metals.

The results shown in Figures 13 to 16 show that, while the structural analogues may show a slight initial improvement on cell viability compared to the cells in the presence of 50 μΜ 6-OHDA, this improvement does not increase with concentration of the tested compound. Therefore, these compounds do not demonstrate a cell rescue effect, presumably as these compounds are unable to effectively chelate the free metal ions present, such as iron. In contrast, the results shown in Figure 13 show that the compound in accordance with the fifth embodiment of the present invention provides a greater improvement on cell viability, which increases as the concentration of the compound is increased. Therefore, these results demonstrate that the compounds of the present invention provide a cell rescue effect as a result of the strong chelation between the compound and the free metal ions.

Figure 17 shows that l-hydroxypyridin-2-one compound in accordance with the fifth embodiment of the present invention (Compound 17; Compound [9]) has comparable ability to bind the labile pool of iron (LIP) in the cell as the known 3- hydroxypyridin-4-one compound, Deferiprone (DFP). With reference to Figure 17A Calcein-AM is a fluorescent dye whose fluorescence is quenched by excess labile iron in the cell. The Calcein-AM assay indicates that the compound of the present invention is as efficient as Deferiprone (DFP) at causing Calcein fluorescence (which was originally quenched by the LIP upon incubation of the cells with 6-OHDA or iron), whereas the non-chelating compounds [8] (Compound 17A), [11] (Compound FL8) and [12] (Compound FL9) are unable to reduce LIP quenched calcein fluorescence. With reference to Figure 17B, the expression of the iron responsive proteins ferritin and transferrin receptor changes upon incubation of the cells with iron. Corresponding to Figure 17A, ferritin and transferrin receptor expression is restored to levels seen in a healthy neuron following incubation with iron in the presence of DFP and compound [9], while compound [12] (Compound FL9) has no effect. With reference to Figure 17C, ferritin and transferrin receptor expression are altered by 6-OHDA in a similar way as iron incubation and accordingly, both DFP and compound [9] reduce this response back to control levels whereas compound [12] (Compound FL9) is unable to do so.

These results show that the neuroprotective mode of action of the compounds of the present invention is chelation of the labile iron pool inside the cell.

Figure 18 illustrates the changes to known markers of Parkinson's disease toxicity with deferiprone (DFP) and compound [9] (Compound 17). Tyrosine hydroxylase and Synaptophysin levels are both reduced in Parkinson's disease and are similarly reduced in neurons after incubation with Iron (Figure 18A) and 6-OHDA (Figure 18B). While DFP and compound [9] are able to negate the reduced expression of both proteins to levels seen in a healthy neuron after incubation with iron (Figure 18A) or 6-OHDA (Figure 18B), the non-chelating compound [12] (Compound FL9) is unable to do so.

These results further show that the neuroprotective mode of action of the compounds of the present invention is chelation of the labile iron pool inside the cell.

Accordingly, in the 6-hydroxydopamine hydrobromide (6-OHDA) cellular model of Parkinson's disease, it has been shown that the compounds of the present invention offer an effective treatment for neurodegenerative disease based on strong metal chelation. This effectively stops the Fenton chemistry that leads to the formation of free-radical species and reactive oxygen species which lead to oxidative stress, reduces metal accumulation in the neuroblastoma cells and reduces metal interactions with alpha-synuclein and/or tau protein, which in turn reduces the accumulation of protein-metal aggregates capable of causing cell death. Therefore, the compounds of the present invention have been shown to be effective in delaying or halting the progression of Parkinson's disease to a similar extent to previously proposed metal chelators, thus providing an alternative. Further, in some situations, the compounds have been shown to reverse the damage caused by excess accumulation of metals. It follows that the compounds of the present invention will also be an effective treatment for other neurodegenerative diseases caused by disregulation/accumulation of metals in certain regions of the brain and central nervous system, such as Alzheimer's disease, Huntington's disease, macular degeneration, Friedreich's ataxia, amyotrophic lateral sclerosis and neurodegeneration with brain iron accumulation. Data Tables

(Key: Abs. = absorbance, AVG = average, StDev = standard deviation, 6-OHDA = 6- hydroxydopamine, n/a = not measured)

Table 1 shows compound toxicity in absence of 6-OHDA for compound [9],

Table 2 shows cell rescue effect in presence of 6-OHDA for compound [9].

Compound [9] is also identified as Compound 17 in the Tables and Figures.

[9]

Table 3 shows compound toxicity in absence of 6-OHDA for compound [16], Table 4 shows cell rescue effect in presence of 6-OHDA for compound [16].

Compound [16] is also identified as Compound FLl in the Tables and Figures.

[16]

Table 5 shows compound toxicity in absence of 6-OHDA for compound [15], Table 6 shows cell rescue effect in presence of 6-OHDA for compound [15].

Compound [15] is identified as Compound FL2 in the Tables and Figures.

[15]

Table 7 shows compound toxicity in absence of 6-OHDA for compound [5], Table 8 shows cell rescue effect in presence of 6-OHDA for compound [5] . Compound [5] is also identified as Compound 7 in the Tables and Figures.

Table 9 shows compound toxicity in absence of 6-OHDA for compound [6], Table 10 shows cell rescue effect in presence of 6-OHDA for compound [6]. Compound [6] is also identified as Compound 8 in the Tables and Figures.

Table 11 shows compound toxicity in absence of 6-OHDA for compound [3], Table 12 shows cell rescue effect in presence of 6-OHDA for compound [3]. Compound [3] is also identified as Compound 14 in the Tables and Figures.

Table 13 shows compound toxicity in absence of 6-OHDA for compound [8], Table 14 shows cell rescue effect in presence of 6-OHDA for compound [8]. Compound [8] is also identified as Compound 17A in the Tables and Figures.

Table 15 shows compound toxicity in absence of 6-OHDA for compound [11], Table 16 shows cell rescue effect in presence of 6-OHDA for compound [11]. Compound [11] is also identified as Compound FL8 in the Tables and Figures.

[11]

Table 17 shows compound toxicity in absence of 6-OHDA for compound [12], Table 18 shows cell rescue effect in presence of 6-OHDA for compound [12]. Compound [12] is also identified as Compound FL9 in the Tables and Figures.

Table 19 shows compound toxicity in absence of 6-OHDA for 3-hydroxypyridin-4- one (Deferiprone), Table 20 shows cell rescue effect in presence of 6-OHDA for Deferiprone. This compound is also identified as Compound 11 in the Tables and Figures, and is shown below.

Table 21 shows compound toxicity in absence of 6-OHDA for compound [17], Table 22 shows cell rescue effect in presence of 6-OHDA for compound [17]. This compound is also identified as Compound 2 in the Tables and Figures, and is shown below.

[17]

Table 23 shows compound toxicity in absence of 6-OHDA for 3-hydroxypyran-4-one (Maltol), Table 24 shows cell rescue effect in presence of 6-OHDA for Maltol. This compound is also identified as Compound 10 in the Tables and Figures, and is shown below.

Table 4.

Table 6.

Table 8.

Table 10.

Table 12.

Table 14.

Table 16.

Table 18.

Table 20.

Table 22.

Table 24.

Claims

1. A heterocycle compound containing at least one electronegative atom and comprising a hydroxyl group or substituted hydroxyl group at the 1 -position and the carbon at the 2 -position being a carbonyl group for use as a medicament.

2. The heterocycle of claim 1 wherein the compound contains at least one oxygen atom, at least one nitrogen atom or a combination thereof.

3. The heterocycle of claim 1 or 2 wherein the compound contains at least one nitrogen atom.

4. The heterocycle of any preceding claim, wherein the compound is a nitrogen containing six membered heterocycle having the general formula:

R¹ is selected from the group comprising H, an alkyl, an aryl, a benzyl, an ester, an amide, a carbamate, a carbonate, a sulfinate, a sulfonate, a phosphonate, an alkali metal, an alkaline earth metal, an ammonium salt, a phosphonium salt, a sulfonium salt, an O-glycoside, an antioxidant, a protecting group, a group capable of being cleaved enzymatically in the cell to reveal the oxygen for chelation or a substituent selected to provide additional functionality to the heterocycle; and

5. The heterocycle of claim 4, wherein at least one of X, Y, Z and X' are carbon atoms.

6. The heterocycle of claim 4 or 5, which further comprises from 1 to 4 additional substituents, which are independently selected from the groups comprising:

F, CI, Br or I;

OH, OR, OCOR, NH₂, NHR⁴, NR⁴R⁵, ⁺NR⁴R⁵R⁶, C0₂R⁴, C0NHR⁴, CONR⁴R⁵ or C=0R⁴;

R⁴OR⁵;

a substituted or unsubstituted aryl or heteroaryl;

S0₃-, X⁺, SO3H, SH or SR⁴;

P=0(OH)₂ or salts P=0(OR⁴)₂ or P=0(OR⁴)(OR⁵);

S=0R⁴ or 0=S=0R⁴;

B(OH)₂ or B(OR⁴)(OR⁵);

CN;

N0₂; or

C(=NH)(NH₂), C=SR⁴;

7. The heterocycle of any one of claims 4 to 6, wherein at least two additional substituents are present, and the additional substituents may be linked to form a cyclic structure. The heterocycle of claim any of claim 4 to 7, wherein the additional substituents may be linked together via a linking group, such as ^"OCH2CH2O, to form a bis-, tris- or tetrakis structure.

The heterocycle of any one of claims 1 to 8, wherein the heterocycle is a nitrogen containing six membered heterocycle having the formula:

wherein X and Y are independently selected from C or N;

R¹ is selected from the group comprising H, an alkyl, an aryl, a benzyl an ester, an amide, a carbamate, a carbonate, a sulfinate, a sulfonate, a phosphonate, an alkali metal, an alkaline earth metal, an ammonium salt, a phosphonium salt, a sulfonium salt, an O-glycoside, an antioxidant, a protecting group, a group capable of being cleaved enzymatically in the cell to reveal the oxygen for chelation or a substituent selected to provide additional functionality to the heterocycle;

R³ and/or R⁴ are each independently absent or present on the ring and comprise at least one additional substituent selected to provide additional functionality to the heterocycle; or a pharmaceutically acceptable salt and/or an isomer thereof.

10. The heterocycle of any one of claims 1 to 9, wherein the heterocycle a nitrogen containing six membered heterocycle having the formula:

(III) wherein R is selected from the group comprising H, an alkyl, an aryl, a benzyl an ester, an amide, a carbamate, a carbonate, a sulfinate, a sulfonate, a phosphonate, an alkali metal, an alkaline earth metal, an ammonium salt, a phosphonium salt, a sulfonium salt, an O-glycoside, an antioxidant, a protecting group, a group capable of being cleaved enzymatically in the cell to reveal the oxygen for chelation or a substituent selected to provide additional functionality to the heterocycle;

R¹ is (CH₂)x, wherein x = 0 to 100;

R³ is absent or present at at least one of the 3-, 4- or 5 -positions on the ring and comprises at least one additional substituent selected to provide additional functionality to the heterocycle

or a pharmaceutically acceptable salt and/or an isomer thereof.

11. The heterocycle according to claim 10, wherein R is H and so the nitrogen containing six membered heterocycle is l-hydroxy-6- (hydroxymethyl)pyridin-2-one or a pharmaceutically acceptable salt and/or an isomer thereof.

12. The heterocycle according to any one of claims 1 to 10, wherein the heterocycle is a nitrogen containing six membered heterocycle having the formula:

(IV) wherein R is selected from the group comprising H, an alkyl, an aryl, a benzyl an ester, an amide, a carbamate, a carbonate, a sulfinate, a sulfonate, a phosphonate, an alkali metal, an alkaline earth metal, an ammonium salt, a phosphonium salt, a sulfonium salt, an O-glycoside, an antioxidant, a protecting group, a group capable of being cleaved enzymatically in the cell to reveal the oxygen for chelation or a substituent selected to provide additional functionality to the heterocycle;

or a pharmaceutically acceptable salt and/or an isomer thereof.

13. A method of forming the nitrogen containing six membered heterocycle according to claim 10 to 12 comprising the steps of:

deprotecting of the N-hydroxyl group of the protected l-hydroxy-6- hydroxymethyl-pyridin-2-one by removal of the protecting group to form the nitrogen containing heterocycle of any of claims 10 to 12.

14. The method of claim 13 wherein the l-hydroxypyridin-2-one-6-carboxylic acid methyl ester is provided by reaction of a l-hydroxypyridin-2-one-6- carboxylic acid with thionyl chloride in methanol, or the l-hydroxypyridin-2- one-6-carboxylic acid is synthesised by treatment of 6-hydroxypicolinic acid with peracetic acid in acetic acid.

15. The method of claim 13 or 14 wherein, the protecting group is an allyl, benzyl, silyl or sulfonate ester group, or any other protecting group known in the art.

16. The method of claim 15, wherein the protecting group is an allyl.

17. The method of any one of claims 13 to 16, wherein reduction of the ester group of the l-hydroxypyridin-2-one-6-carboxylic acid methyl ester is accomplished by reaction with a reducing agent, such as a reducing agent comprising potassium, calcium, barium, sodium or magnesium and/or borohydride.

18. The method of any one of claims 13 to 17 wherein the deprotection of the N- hydroxyl group of the protected l-hydroxy-6-hydroxymethyl-pyridin-2-one by removal of the protecting group is accomplished in the presence of palladium on activated carbon and trifluoroacetic acid.

19. The heterocycle of any preceding claim for use in the treatment of a neurodegenerative disease.

20. The heterocycle of any preceding claim for use in treatment of Parkinson's disease or Alzheimer's disease.

21. A method for treating a neurodegenerative disease comprising the steps of: providing a composition comprising a heterocycle compound according to any one of claim 1 to 12; and

administering an effective dose of the composition to a patient.

22. The method of claim 21, wherein the composition further comprises a pharmaceutically acceptable excipient.

23. The method of claim 22 or 23, wherein the composition comprises one or more known active pharmaceutical ingredient used in the treatment of symptoms of neurodegenerative disease, such as known active pharmaceutical ingredients including additional chelating compounds or antioxidants.

24. A composition for the treatment of a neurodegenerative disease comprising a heterocycle in accordance with any one of claims 1 to 12, optionally a pharmaceutically acceptable excipient and optionally one or more known pharmaceutical ingredients.