GB2345058A - Hydroxypyridone compounds useful in the treatment of oxidative damage to the central nervous system - Google Patents

Abstract

A compound of the formula (1): <EMI ID=1.1 HE=30 WI=48 LX=816 LY=400 TI=CF> <PC>wherein A is <EMI ID=1.2 HE=46 WI=140 LX=288 LY=772 TI=CF> <PC>wherein R<SP>1</SP>, R<SP>2</SP> and R<SP>3</SP> are independently selected from H and alkyl;<BR> wherein X is O, S, NR<SP>4</SP> or a direct bond, wherein R<SP>4</SP> is H or alkyl;<BR> wherein Z is a hydrocarbyl chain comprising from 1 to 10 carbon atoms optionally interrupted by one or more heteroatom(s) and optionally substituted by one or more oxo substituent(s);<BR> wherein q is 1, 2 or 3;<BR> wherein if q is 2 or 3, then each A can be the same or different;<BR> wherein the or each R<SP>5</SP> is independently selected from H and alkyl;<BR> wherein the or each R<SP>6</SP> is independently selected from alkyl;<BR> wherein n is 1 to 5;<BR> wherein p is 0 to 4; and<BR> wherein the sum of n and p is less than 6,<BR> or a pharmaceutically acceptable salt thereof, and the use thereof in therapy, particularly for the treatment of a condition associated with oxidative stress, such as oxidative damage of the central nervous system or an acute or chronic neurological disorder such as traumatic brain injury, spinal cord injury, cerebral tumour, subharrachnoid haemorrage/cerebral vasospasm, cerebral ischaemia, stroke (ischaemic or haemorragic), Alzheimer's disease, Huntington's Disease, Parkinson's Disease, Friedrich's ataxia, motor neuron disease or multiple sclerosis.

Description

CHEMICAL COMPOUNDS m The present invention relates to compounds containing both ortho-hydroxypyridone and oxygenated aryl (including heteroaryl) functionalities, which possess the dual ability to chelate iron and scavenge reactive oxygen species (ROS). In particular, the invention relates to specific compounds containing a 3-hydroxy-4 (lh)-pyridinone or 3-hydroxy2 (1H)-pyridinone or N-hydroxy-2 (lH)-pyridinone iron chelating moiety as well as an oxysubstituted aryl (including heteroaryl) antioxidant moiety. The present invention also relates to the synthesis of such compounds, to pharmaceutical preparations comprising such compounds and to the use of such compounds in the treatment and prophylaxis of conditions associated with oxidative stress, particularly oxidative damage to the central nervous system.

One particularly relevant example of a condition involving oxidative damage to the central nervous system, which can be treated with compounds of the present invention, is stroke. Stroke is the third leading cause of death in major industrialised countries and the commonest cause of permanent disability (Hunter et al., Trends in Pharmacological Sciences, 1995,16,123-128). Each year, in the US and Europe, approximately 1 million people suffer acute stroke (Dorman et al., CNS Drugs, 1996,5,457-474). Between 25% and 35% of these patients die within the first three weeks, and of the survivors 25% to 50% will be totally dependant on family or institutional care for the rest of their lives.

The incidence of stroke increases with age, roughly doubling with each passing decade, with 30% of persons aged over 65 years being affected (Babikian et al., Cerebrovascular disease in the elderly. In Clinical Neurology of Aging, Eds Albert M. L. and Knoefel J. E., OUP, New York, 1994). These statistics translate into an annual incidence of 0.1 to 0.2% in the US and Europe, with the world-wide market for stroke estimated to be worth $3 billion in 1995 and projected to rise to $10 billion in 2005.

Stroke is defined as an interruption of the blood flow to the brain or leakage of blood out of the brain resulting in oxygen deprivation (ischaemia) and subsequent neuronal cell death. Strokes can be divided into two classes, ischaemic and haemorragic. The former accounts for approximately 83% of all strokes and is caused by thrombosis (65%) and/or detachment of a previously formed clot (embolus, 18%). Haemorrhagic strokes, which account for the remaining 17% of all strokes, can be subdivided into subarachnoid haemorrhage (7%) and cerebral haemorrhage (10%).

Markers of oxidative damage have been detected in the brains of ischaemic animals, and a variety of antioxidant molecules have been demonstrated to be neuroprotective in ischaemic stroke models. This provides conclusive evidence that cerebral ischaemia leads to the production of reactive free radicals. Defined as a chemical species containing one or more unpaired electrons, and capable of independent existence, free radicals are highly destructive towards cellular membrane lipids, DNA and proteins.

This"oxidative-modification"of cellular components ultimately leads to a loss of cell function. One example of this oxidative process is lipid peroxidation (LP), a process which increases membrane fluidity leading to failure of normal membrane potential, disturbance of calcium homeostasis and transmembrane signalling, with the ultimate result of neuronal cell death.

A combination of the brains high-energy demand and subsequent high rate of oxygen consumption, with its limited endogenous antioxidant defences (superoxide dismutase (SOD), glutathione peroxidase (GSPx), and catalase) makes the brain very susceptible to free radical attack. Additionally, neuronal cell membranes, being rich in polyunsaturated fatty acids, are especially vulnerable to oxidative modification. The brains vulnerability to free radical attack is further exacerbated by relatively high levels of iron in the brain. Iron is the fundamental catalyst in the production of the hydroxyl radical ('OH) (Fenton and Haber-Weiss Reactions), reportedly the most destructive of all free radicals (Palmer, C., Metals and Oxidative Damage in Neurological Disorders, Ed.

Connor, Plenum Press, New York, 1997, pp 205-236).

Exposure to free radicals is a natural consequence of aerobic respiration, to which the human body possesses a variety of natural antioxidant mechanisms. However, during pathological conditions such as stroke homeostatic mechanisms break down, and the balance between the generation of free radicals and natural antioxidant defences is shifted, resulting in a state of oxidative stress (Beal M. F., Ann. Neurol., 1995,31,119130; Gerlach et al., J. Neurochem., 1994,63,793-807).

In animal models of stroke, supplementation of antioxidant defences with exogenous antioxidant molecules has resulted in neuroprotection, as assessed both histologically and behaviourally (Hall E. H., Metals and Oxidative Damage in Neurological Disorders, supra, pp 325-339). Furthermore, transgenic animals overexpressing SOD have been demonstrated to be more resistant to cerebral ischaemia than their wild type littermates (Chan et al., Ann. N. Y. Acad. Sci., 1994,738,93-103).

The iron chelator deferiprone (1, 2-dimethyl-3-hydroxy-4 (1H)-pyridinone) has also been shown to possess antioxidant properties. For example the use of deferiprone to inhibit free radical formation has been disclosed by Kontochiorghes et al. (Free Rad. Res. Comms., 1986,2,115-124) and by Mostert et al. (Free Rad. Res. Comms., 1987,3,379-388). The use of deferiprone in conjunction with an antioxidant is also disclosed in WO 94/03169 for use in the treatment of sickle cell disease, and by Antonius et al. (Circulation, 1989,80, 158-164) for use in the prevention of postischemic cardiac injury. Deferiprone has recently been in clinical trials as a potential treatment for iron overload in thalassemia major, and has also been disclosed for the treatment of parasitic infections,-anemia and Alzheimer's disease.

There are many cellular systems known to be inappropriately activated or regulated as a result of oxygen starvation to the brain (e. g. glutamate receptors, voltage dependent ion channels). A major consequence of this is a loss of calcium homeostasis and inappropriate enzyme activation via several routes. Generation of free radicals is a common biochemical end point to many of the processes that are inappropriately regulated following cerebral ischaemia (Dorman et al., supra, Hall E. H. supra ; Patt et al., J. Pediatric Surg., 1990,25,224-227). Hence intervention"down stream"with an antioxidant molecule at a point where many of these processes converge, is considered to be strategically sound owing to a universal applicability to many intracellular processes.

Based on the above rationale, a low molecular weight molecule designed to simultaneously trap radicals and chelate iron is a novel, scientifically relevant approach towards the treatment of conditions associated with oxidative stress, in particular cerebral ischaemia/stroke.

There have been three related reports describing molecular entities with dual iron chelating and anti-oxidant capabilities. The first report, Sato et al (Bioconjugate Chem., 1995,6, 249-54), describes Cu, Zn-superoxide dismutase and desferrioxamine conjugated via polyoxyethylene. This high molecular weight conjugate was not used to show protection against oxidative stress in vitro, nor was it investigated for its effectiveness in in vivo models of oxidative stress. The second report, Rojanasakul et al (Biochim.

Biophys. Acta, 1996,1315 (1), 21-8), describes a transferrin-catalase conjugate which gave increased protection to cells from oxidative stress as a result of its increased uptake in to cells via the transferrin receptor. The paper suggests the potential therapeutic use of the"... conjugate for the treatment of pathological processes in the lung". This high molecular weight conjugate was not investigated for its effectiveness in in vivo models of oxidative stress. The third report, Tilbrook et al (WO 9825905), claims compounds containing iron chelator units linked to a group containing reducing-SH groups for the treatment of Alzheimer's disease and related neurodegenerative diseases. The compounds were not investigated for their effectiveness in in vivo models of oxidative stress.

Currently there are only two recognised forms of treatment available for stroke victims.

The first, Altepase (recombinant tissue plasminogen activator, rTPA) is a clot busting drug only suitable for cerebral thrombosis. Therapeutic thrombolysis can, however, be complicated by a) systemic haemorrhage, b) intracerebral haemorrhage, c) distal embolism of a partially digested clot leading to secondary infarction and d) cerebral oedema secondary to reperfusion injury. It is, therefore, necessary to exclude the possibility of haemorrhagic stroke by computerised tomographic (CT) scanning of patients before administering Alteplase. The second recognised treatment, carotid endarterectomy is a surgical procedure for unblocking the carotid artery. However, both treatments have the potential to exacerbate and complicate the original injury, and neither treatment is neuroprotective nor universal for all types of stroke. A third treatment, the antioxidant idebenone is licensed for the treatment of stroke in Japan.

There is therefore a large umnet medical need for an effective neuroprotective compound for the treatment of stroke.

It is an object of this invention to provide compounds which, unlike the current therapies used for the treatment of stroke, protect against damage due to reperfusion injury, and are neuroprotective. Such compounds are potentially useful for all types of stroke. It is a further object of this invention to provide compounds which may be used in the treatment of oxidative stress generally and particularly oxidative damage to the central nervous system.

According to the present invention there is provided a compound of the formula (1) :

wherein A is

wherein R', W and R3 are independently selected from H and alkyl ; wherein X is O, S, Nu4 or a direct bond, wherein g4 iS H or alkyl ; wherein Z is a hydrocarbyl chain comprising from 1 to 10 carbon atoms optionally interrupted by one or more heteroatom (s) and optionally substituted by one or more oxo substituent (s) wherein q is 1,2 or 3; wherein if q is 2 or 3, then each A can be the same or different; wherein the or each Rs is independently selected from H and alkyl ; wherein the or each R6 is independently selected from alkyl ; wherein n is 1 to 5 ; wherein p is 0 to 4; and wherein the sum of n and p is less than 6, or a pharmaceutically acceptable salt thereof.

The compounds of the present invention which have a combined antioxidant and iron chelating activity can be used for treating oxidative stress, particularly oxidative damage to the central nervous system. The compounds of the present invention are surprisingly more effective, especially at low concentrations in vitro, than the simultaneous use of the separate ortho-hydroxypyridone iron chelating compound and the oxygenated aryl (including heteroaryl) antioxidant compound.

As used herein, the term"alkyl"means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e. g. alkenyl or alkynyl) hydrocarbyl radical which may be substituted or unsubstituted. Where cyclic, the alkyl group is preferably C3 to C, 2, more preferably Cs to Cil, more preferably Cs to C7. Where acyclic, the alkyl group is preferably C, to C, o, more preferably C, to C6, more preferably methyl, ethyl, propyl (n-propyl or isopropyl), butyl (n-butyl, isobutyl or tertiary-butyl) or pentyl (including n-pentyl and isopentyl), more preferably methyl. It will be appreciated therefore that the term"alkyl"as used herein includes alkyl (branched or unbranched), substituted alkyl (branched or unbranched), alkenyl (branched or unbranched), substituted alkenyl (branched or unbranched), alkynyl (branched or unbranched), substituted alkynyl (branched or unbranched), cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, cycloalkynyl and substituted cycloalkynyl.

As used herein, the term"aryl"means a substituted or unsubstituted carbocyclic aromatic group, such as phenyl or naphthyl, or a substituted or unsubstituted heteroaromatic group containing one or more, preferably one, heteroatom, such as pyridyl, pyrrolyl, furanyl, thienyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl pyrazolyl, imidazolyl, triazolyl, pyrimidinyl pyridazinyl, pyrazinyl, triazinyl, indolyl, indazolyl, quinolyl, quinazolyl, benzimidazolyl, benzothiazolyl, benzisoxazolyl and benzisothiazolyl.

As used herein, the term"carbocyclic"refers to a ring system in which all the ring atoms are carbon atoms.

The alkyl and aryl groups may be substituted or unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent. Substituents may include: carbon-containing groups such as alkyl, aryl, arylalkyl (e. g. substituted and unsubstituted phenyl, substituted and unsubstituted benzyl); halogen atoms and halogen-containing groups such as haloalkyl (e. g. trifluoromethyl); oxygen-containing groups such as alcohols (e. g. hydroxy, hydroxyalkyl, aryl (hydroxy) alkyl), ethers (e. g. alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl), aldehydes (e. g. carboxaldehyde), ketones (e. g. alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arylcarbonylalkyl), acids (e. g. carboxy, carboxyalkyl), acid derivatives such as esters (e. g. alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl), amides (e. g. aminocarbonyl, mono-or di-alkylaminocarbonyl, aminocarbonylalkyl, mono-or di alkylaminocarbonylalkyl, arylaminocarbonyl), carbamates (e. g. alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyloxy, mono-or di alkylaminocarbonyloxy, arylaminocarbonyloxy) and ureas (e. g. mono-or di-alkylaminocarbonylamino or arylaminocarbonylamino) ; and nitrogen-containing groups such as amines (e. g. amino, mono-or di-alkylamino, aminoalkyl, mono-or di-alkylaminoalkyl).

As used herein, the term"alkoxy"means alkyl-O-. Alkoxy substituent groups or alkoxycontaining substituent groups may be substituted by one or more alkyl groups.

As used herein, the term"halogen"means a fluorine, chlorine, bromine or iodine radical, preferably a fluorine or chlorine radical.

In a preferred embodiment, the alkyl groups are either unsubstituted or substituted by substitution of a hydrogen atom with a group selected from oR7, OCoR7, COOR7, NHR7, NHCOR'and CONHR'wherein R'is H or alkyl.

As used herein, the term"oxy-substituted"is a general term used to describe substitution by a hydroxy and/or an alkoxy group.

As used herein, the term"heteroatom"means an atom other than a carbon atom, such as nitrogen, oxygen or sulphur.

As used herein, the term"oxo substituent"means an oxygen atom joined to a carbon atom or to a heteroatom, such as sulphur, in the hydrocarbyl chain by a double bond.

As used herein, the term"interrupted by one or more heteroatom (s)" means that the or each heteroatom may be positioned at any position along the hydrocarbyl chain including at either end of the chain.

As used herein, the term"interrupted by one or more group (s)", the group (s) being selected from O, S, SO, SO2, SO2NH, NHSO2, NH, NHCO, CONH, NHCONH, NHCOO, OCONH, OCO and COO, means that the or each group may be positioned at any position along the hydrocarbyl chain including at either end of the chain.

As used herein, the term"pharmaceutically acceptable salt"means any pharmaceutically acceptable salt of the compound of formula (1). Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, dichloroacetic, fumaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic and the like. Particularly preferred are hydrochloric, hydrobromic, phosphoric, sulfuric and methanesulfonic acids, and most particularly preferred is the methanesulfonate salt.

Acceptable base salts include alkali metal (e. g. sodium, potassium), alkaline earth metal (e. g. calcium, magnesium) and aluminium salts.

According to the present invention, the or each A may be independently linked to the chain Z at any atom of the chain Z. It is preferred that the or each A is independently linked to the chain Z at a carbon, nitrogen or oxygen atom of the chain Z.

According to the present invention, when A is AI, preferably R'and R'are independently selected from H, unsubstituted alkyl, CH20R7, CH2OCOR', COOR', CH2NHR', CH2NHCOR'and CONHR'wherein R'is H or alkyl. Preferably, R', R'and R'are independently selected from H and unsubstituted alkyl. More preferably R'and R2 are H and R3 is unsubstituted alkyl. It is further preferred that R'is methyl.

In an embodiment of the invention, when A is AI, it is preferred that AI is either AIa :

According to the present invention, when A is AII, preferably R'and W are independently selected from H, unsubstituted alkyl, CH2OR', CH2OCOR', COOR', CH2NHR7, CH2NHCOR7 and CONHR7 wherein R7 is H or alkyl. Preferably, R', R2 and R3 are independently selected from H and unsubstituted alkyl. More preferably R'and R ? are H and R3 is unsubstituted alkyl. It is further preferred that R3 is methyl.

In an embodiment of the invention, when A is AH, it is preferred that AD is AIIa :

According to the present invention, when A is AIE, preferably R, R2 and R3 are independently selected from H, unsubstituted alkyl, CH2OR7, CH2OCOR7, COOR', CH2NHR', CH2NHCOR'and CONHR'wherein R'is H or alkyl. Preferably, R', R2 and R3 are independently selected from H and unsubstituted alkyl. More preferably R', R2 and R3 are H.

In an embodiment of the invention, when A is AIII, it is preferred that AIII is AIIIa :

Preferably the present invention provides a compound wherein q=l.

In a preferred embodiment, Z is a hydrocarbyl chain comprising from 1 to 10 carbon atoms optionally interrupted by one or more group (s) selected from O, S, SO, SO2, SO2NH, NHS02, NH, NHCO, CONH, NHCONH, NHCOO, OCONH, OCO and COO.

Preferably, the present invention provides compounds wherein Z comprises from 1 to 8 carbon atoms.

In a further preferred embodiment, Z is (CH2) kWY wherein k is 0 to 10; wherein W is selected from 0, S, SO, SO2, SO2NH,. NHSO2, NH, NHCO, CONH, NHCONH, NHCOO, OCONH, OCO, COO and a direct bond; wherein Y is a hydrocarbyl chain comprising from 2 to 10 carbon atoms and one or more carbon-carbon double bond (s); and wherein the chain (CH2) comprises from 1 to 10 carbon atoms. In this embodiment, it is preferred that when k is 0, W is selected from O, NH, NHCO, NHCONH, NHCOO, OCONH and a direct bond; and when k is 1, W is selected from SO, SO2, SONH, CONH, COO and a direct bond.

According to the invention, k is preferably 1,2,3 or 4. Preferably, the hydrocarbyl chain Y comprises 2,3,4 or 5 carbon atoms, more preferably 3,4 or 5 carbon atoms.

Preferably, the present invention provides compounds wherein W is selected from O, NH, CONH, NHCO and a direct bond.

According to the present invention, the chain Z may be branched or unbranched, optionally substituted by one or more alkyl groups, and may be cyclic. Preferably, Z is an unbranched chain. Where Z contains from 2 to 10 carbon atoms, the chain may comprise one or more carbon-carbon double bonds. It will be appreciated that where q=1, Z is a di-valent hydrocarbyl chain; where q=2, Z is a tri-valent hydrocarbyl chain; and where q=3, Z is a tetra-valent hydrocarbyl chain.

As used herein to describe the hydrocarbyl chain Z, the term"cyclic"means either that Z may comprise a cyclic hydrocarbyl group of from 3 to 10 carbon atoms, preferably 5,6 or 7 carbon atoms; or that where R'or R'is alkyl, a cyclic group is present as a result of cyclisation of the alkyl group of W or R6 onto Z; or that, where X is NR4 and V is alkyl, a cyclic group is present as a result of cyclisation of W onto Z; or that a cyclic group is present as a result of cyclisation of Z onto the phenyl ring shown in formula (1) to form a fused ring system. It is preferred that a cyclic group formed as a result of cyclisation of R4, R5 or R6 onto Z, or as a result of cyclisation of Z onto the phenyl ring shown in formula (1), is a 5,6 or 7-membered ring.

In an embodiment of the invention, there is provided compounds wherein Z contains one carbon-carbon double bond.

According to the present invention, X is preferably NR4 or a direct bond. Most preferably, X is NR4.

In an embodiment of the present invention, where X is N R4 and the chain Z contains a carbon-carbon double bond, the carbon-carbon double bond is immediately adjacent the N R4 group in the chain Z-X. In this preferred embodiment, preferably R4is H.

According to the present invention, where X is NR'and W is alkyl, R'may be cyclized onto the chain Z.

According to the present invention, R5is preferably selected from H and C,, 0 alkyl. In one embodiment of the invention at least one Rs is H. When X is not NH, it is preferred that at least one R5is H.

Preferably, the present invention provides compounds wherein n is 1 to 3, more preferably nis 1 or2.

According to the present invention, when R5is H, preferably Or$ ils positioned in the ortho or para position in the ring with respect to X. More preferably, ORs is positioned in the para position with respect to X.

According to the present invention, the or each R6 is preferably independently selected from C"0 aLkyl, more preferably from Cl, alkyl, more preferably from methyl, isopropyl and t-butyl and most preferably t-butyl.

Preferably, the present invention provides compounds wherein p is 2,3 or 4.

According to the present invention, when p is 1 or 2, alkyl groups represented by R6 is/are preferably positioned ortho to oR5, preferably in the meta-position of the ring with respect to X to give, for example, a compound of formula :

According to the present invention, when p is 3, alkyl groups represented by R6 are preferably positioned to give, for example where three R6 are methyl, a compound of formula :

In an embodiment of the present invention, where W or R6 are alkyl, the alkyl group ofR or R6 can be cyclized onto a carbon atom or a nitrogen atom of the chain defined as Z to form a ring.

In a preferred embodiment of the invention, Z contains at least one carbon-carbon double bond, X is O, S or NR4, preferably NR4, and more preferably NH, a carbon-carbon double bond of the chain Z is immediately adjacent the group X in the chain Z-X, and Z is cyclized onto the phenyl ring shown in formula (1) to give a 5 or 6 membered heterocyclic ring, preferably a 5-membered ring, containing a carbon-carbon double bond.

According to a further aspect of the present invention there is provided a method of preparing the compounds of the present invention. The compounds of the present invention may be prepared using standard synthetic chemistry.

A method for the synthesis of compounds where q = 1, A is a AIb moiety and Z is (CH2) where W is a CONH, COO, SO2NH or SO20 unit (for example, the compound of Example 1) comprises coupling the acid derivative (carboxylic or sulfonic) of the protected hydroxy pyridinone (2) with the primary amine or alcohol of the respective antioxidant unit (3) using standard coupling techniques (for example via acid activation as a succinimdyl ester, or with coupling reagents such as EDCI), followed by removal of the benzyl protecting group, as illustrated in Reaction Scheme 1.

Reaction Scheme 1

R' Oss+QOH BnO R3 Ri (2) 1)"acid activation" (OR.

2)"coupling"with Hg'y X \/ (3) (R*), R'3)"deprotectionr (ORS), 0 k Q, B-Y-x HO 4N'-R3 (R-) p HO t oR3 (Rsp RI Q=CO, or 5 2 B=NH,or O and Q-B = W The acid derivatives (carboxylic or sulfonic) of the protected hydroxy pyridinone (2) can be prepared using standard synthetic chemistry (for the synthesis of a compound of the type (2) see EP281289).

The primary amine or alcohol of the respective antioxidants (3) can be prepared using standard synthetic chemistry.

A method for the synthesis of compounds where q = 1, A is a AIb moiety and Z is (CH2) kWY where W is a CONH, COO, SONH or SO20 unit comprises coupling the acid derivative (carboxylic or sulfonic) of the protected hydroxy pyridinone (4) with the primary amine or alcohol of the respective antioxidant unit (3) using standard coupling techniques (for example via acid activation as a succinimdyl ester, or with coupling reagents such as EDCI), followed by removal of the benzyl protecting group, as illustrated in Reaction Scheme 2.

Reaction Scheme 2

OBn out OH Ri N"'R3 R2 (4) I)"acidactivation" 2)"coupling"with I. g'Y X \/ (3) tw, 3)"deprotection" OH (OR5) n k Q, BY-X ) NRa RI Q=CO,or SO2 B=NH,or O andQ-B=W The acid derivatives (carboxylic or sulfonic) of the protected hydroxy pyridinone (4) can be prepared using standard synthetic chemistry.

Compounds of the present invention which contain more than one hydroxy-pyridinone Fechelating unit (where q = 2 or 3) may be prepared via a general method, using standard synthetic chemistry. For example, compounds where q = 2, A is a AIa moiety and Z is (CH2) where W is a CONH, COO, S02NH or SO20 unit (for example, the compound of Example 2) can be prepared by coupling two molecules of the acid derivative (carboxylic or sulfonic) of the protected hydroxy pyridinone (2) with the bis-primary amine or alcohol of the respective antioxidant unit (5) using standard coupling techniques (for example via acid activation as a succinimdyl ester, or with coupling reagents such as EDCI), followed by removal of the benzyl protecting groups, as illustrated in Reaction Scheme 3.

Reaction Scheme 3

Ri 2x t OH -nu BnO \ Rs R2 (2) 1)"acid activation" (OR, 2)"coupling"with HB'Y X (5) 3)"deprotection^ O HO RI Ruz I N R B/y (OR5) ; W q < Q (R6) p HOt uR3 R2 Q=CO,or SO2 B=NH, or O andQ-B=W The bis-primary amine or alcohol (5), (and tris-primary amines) may be prepared using standard synthetic chemistry.

A method for the synthesis of compounds where q = 1, A is a AIIa moiety and Z is (CH2) where W is a CONH, COO, SONH or SO, O unit (for example, the compound of Example 3) comprises coupling the acid derivative (carboxylic or sulfonic) of the protected hydroxy pyridinone (6) with the primary amine or alcohol of the respective antioxidant unit (3) using standard coupling techniques (for example via acid activation as a succinimdyl ester, or with coupling reagents such as EDCI), followed by removal of the benzyl protecting group, as illustrated in Reaction Scheme 4.

Reaction Scheme 4

OBn OBn R3 RI R2 (e 1)"acid activation" (OR,).

2)"coupling"with HB (3) (R6) r 3)"deprotection" OH (OR5) n O+QxB, YXX t W $il'~X R2 Ruz R Q=CO, or SO2 B=NH, or O andQ-B=W The acid derivatives (carboxylic or sulfonic) of the protected hydroxy pyridinone (6) can be prepared using standard synthetic chemistry (for the synthesis of a compound of the type (6) see Xu et al. J. Am. Chem. Soc. (1995), 117 (27), 7245-7246).

A method for the synthesis of compounds where q = 1, A is a AIIIa moiety and Z is (CH2) where W is a CONH, COO, SO2NH or SO20 unit (for example, the compound of Example 4) comprises reacting the pyridinone ester polymer (carboxylic or sulfonic) (7) with the primary amine or alcohol of the respective antioxidant unit (3), as illustrated in Reaction Scheme 5.

Reaction Scheme 5

R2 Ri R3 O I O n (OR, "coupling"with H 2 (3) (RX R2 RI R3 / W O N-' (-Q Y-X (rot (ruz Q=CO,orSO2 B=NH,or O andQ-B=W The pyridinone ester polymer (carboxylic or sulfonic) (7) can be prepared using standard synthetic chemistry (for the synthesis of a compound of the type (7) see Scarrow et al.

Inorg. Chem., (1985), 24 (6), 954-967).

A method for the synthesis of compounds where q = 1, A is a AIa moiety and Z is hydrocarbyl chain (for example, the compound of Example 5) comprises condensation of the aldehyde derivative of the respective antioxidant unit (9) with the hydroxy pyrone (8), followed by reduction (with for example zinc dust and concentrated hydrochloric acid) and finally condensation with the primary amine (10), as illustrated in Reaction Scheme 6.

Reaction Scheme 6

OH O Ri R R2 (8) I)"condensation"with HCOZ'-X HCOZ'= aldehyde (9)precursor to Z 2)"reduction" 3)"condensarion"with HZNRs (10) OH (OR5) oq4fZ-xw 0 Z-X R. Y R2 The hydroxy pyrones (8) can be prepared using standard synthetic chemistry (for the synthesis of a compound of the type (8) see Ellis et al., J. Med. Chem., (1996), 39,36593670).

The aldehyde derivative of the respective antioxidant units (9) can be prepared using standard synthetic chemistry (for the synthesis of a compound of the type (9) see GB2026479).

Phenolic-OH groups of the antioxidant unit starting materials/precursors, described above, can be optionally protected during synthetic manipulation (for example, as a benzyl ether).

Deprotection to reveal the phenolic-OH groups of the antioxidant unit can be carried out simultaneously with removal of the protecting group of the hydroxy pyridinone unit, in the last step of the sequences.

The compounds of the present invention may contain one or more asymmetric carbon atoms, so that the compounds exist in different stereoisomeric forms. The compounds can be, for example, racemates or optically active forms. The optically active forms can be obtained by resolution of the racemates or by asymmetric syntheses.

The compounds of the present invention may also be in a prodrug form wherein some or all of the free-OH groups of the preferred compounds are derivatised (for example, via an ester, amide or phosphate bond) with a suitable group (the group may contain, for example, an alkyl, aryl, phosphate, sugar, amine, glycol, sulfonate or acid function) which is suitably labile so as it will be removed/cleaved (eg. by hydrolysis) to reveal the preferred compound sometime after administration or when exposed to the desired biological environment. Prodrug forms of the compounds of the present invention can be used to increase compound solubility and stability for formulation, increase efficacy and decrease metabolism.

Preferably the prodrug forms of the compounds of the present invention are ester linked alkyl or aryl amines. For example, esters derived from glycine, N-methyl glycine, N, Ndimethyl glycine, (3-alanine, y-aminobutyric acid, valine, or aminobenzoic acid.

Preferably the ester linked amine prodrug is derived from N, N-dimethyl glycine.

The prodrugs of the compounds of the present invention may be prepared using conventional synthetic methods. For example, a general method for synthesis of the preferred prodrugs of the compounds of the present invention comprises coupling a Nprotected amino acid derivative with the hydroxy group of the antioxidant unit (using for example potassium carbonate in acetone), followed by deprotection. This is illustrated in Scheme 7 using a N-hydroxysuccinimide ester or an acid chloride as an example of the N-protected amino acid derivative.

Reaction Scheme 7

pi (oRf) r R2.-X R (R") p 1)"coupling"with 0 R O /where There R is the same or different, o ; ; a ! /NR and can be alkyl, H or a protecting group o 2)"deprotection" | 3) salt fomiation O 0 HORt, p R II re R is the same or different -x N"R and can be alkyl or H.

Ruz Ly R (RI), 2HCI G = a bivalent hydrocarbyl chain, substituted or unsubstituted. such as an alkyene or aryiene group Alternatively, the N-protected amino acid derivative can be coupled to the hydroxy group of the pyridone unit, followed by deprotection, as illustrated in Scheme 8.

Reaction Scheme 8

0 Ho pu RI (OR5L R3 (R5 ( 1)"coupling"with o R where R is the same or different eoaf or a < /NsR and can be alkyl, H or a protecting group 0 o 2)"deprotection" 3)"sah formation" 0 R-, R I II1 (O R3 (R-), R (R*') where R is the same or different, and can be alkyl or H.

G = a bivalent hydrocarbyl chain, substituted or unsubstituted, such as an alkyene or arytene group Schemes 7 and 8 illustrate the preparation of prodrugs of the compounds of the invention where A = AI, q = 1 and A is bound to the chain Z at the 2-position of the 4-pyridinone ring. However, the synthetic routes shown in Schemes 7 and 8 are generally applicable to the preparation of prodrugs of any of the compounds of the present invention.

To further increase their efficacy, compounds of the present invention may also contain additional non-covalently linked components such as dextrans or cyclodextrins, which aid stability and dispersion, and decrease metabolism of the active ingredient.

According to a further aspect of the present invention there is provided a compound of the present invention for use in therapy.

According to a further aspect of the present invention there is provided use of a compound of the present invention in the manufacture of a medicament for the treatment of a condition associated with oxidative stress, particularly oxidative damage of the central nervous system.

The term"treatment"as used herein includes prophylaxis.

Diseases, disorders and medical treatments/procedures associated with oxidative stress that can be treated with the compounds of the present invention include: aging; acute intermittent porphyria ; adriamycin-induced cardiomyopathy; AIDS dementia and HIV-1 induced neurotoxicity; Alzheimer's disease; atherosclerosis; cateract; cerebral ischaemia; cerebral palsy; cerebral tumour ; chemotherapy-induced organ damage; cisplatin-induced nephrotoxicity; coronary artery bypass surgery; diabetic neuropathy; Down's syndrome; drowning ; epilepsy and post-traumatic epilepsy; Friedrich's ataxia; frontotemporal dementia; glaucoma; glomerulopathy; haemochromatosis; haemodialysis; haemolysis; haemolytic uraemic syndrome (Weil's disease); haemorrhagic stroke; heart attack and reperfusion injury; Huntington's disease; Lewy body disease; intermittent claudication; ischaemic stroke; inflammatory bowel disease; macular degeneration; malaria; methanolinduced toxicity; meningitis (aseptic and tuberculous); motor neuron disease; multiple sclerosis; multiple system atrophy; myocardial ischaemia; neoplasia; Parkinson's disease; peri-natal asphyxia; Pick's disease; progressive supra-nuclear palsy; radiotherapy-induced organ damage; restenosis after angioplasty; retinopathy; senile dementia; schizophrenia; sepsis; septic shock; spongiform encephalopathies; subharrachnoid haemorrage/cerebral vasospasm; subdural haematoma ; surgical trauma, including neurosurgery; thalassemia; transient ischaemic attack (TIA) ; traumatic brain injury (TBI) ; traumatic spinal injury; transplantation; vascular dementia; viral meningitis; and viral encephalitis.

Additionally, compounds of the present invention may also be used to potentiate the effects of other treatments, for example to potentiate the neuroprotective effects of brain derived nerve growth factor.

The invention is particularly directed to conditions which induce oxidative damage of the central nervous system, including acute and chronic neurological disorders such as traumatic brain injury, spinal cord injury, cerebral ischaemia, stroke (ischaemic and haemorragic), subharrachnoid haemorrage/cerebral vasospasm, cerebral tumour, Alzheimer's disease, Huntington's disease, Parkinson's disease, Friedrich's ataxia, motor neuron disease and multiple sclerosis.

The invention further provides a method of treating a condition associated with oxidative stress, particularly oxidative damage of the central nervous system, comprising administering to a patient in need of such treatment an effective dose of a compound of the present invention.

The invention further provides a pharmaceutical composition comprising a compound of the present invention in combination with a pharmaceutically acceptable carrier or excipient and a method of making such a composition comprising combining a compound of the present invention with a pharmaceutically acceptable carrier or excipient.

Compounds of the present invention may be administered in a form suitable for oral use, for example a tablet, capsule, granule, powder, aqueous or oily solution, suspension or emulsion; for topical use including transmucosal and transdermal use, for example a cream, ointment, gel, aqueous or oil solution or suspension, salve, patch or plaster; for nasal use, for a example a snuff, nasal spray or nasal drops; for vaginal or rectal use, for example a suppository; for administration by inhalation, for example a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, for example a tablet or capsule; or for parenteral use (including intravenous, subcutaneous, intramuscular, intravascular, intrathecal or infusion), for example a sterile aqueous or oil solution or suspension. In general the above compositions may be prepared in a conventional manner using conventional excipients, using standard techniques well known to those skilled in the art of pharmacy. Preferably, the compound is administered orally for chronic disorders such as Alzheimer's and Parkinson's disease, and intravenously for acute disorders such as stroke and TBI.

For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, cyclodextrin, and lactose, while com starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, lipids, sodium alginate, polyvinylpyrrolidone, cyclodextrins, gum tragacanth, polyethylene glycol, propylene glycol, N, N- dimethylacetamide, cremophors, polysorbates, liposomes and wetting agents such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl phydroxybenzoate.

It will be appreciated that the dosage levels used may vary over quite a wide range depending upon the compound used, the severity of the symptoms exhibited by the patient and the patient's body weight.

The invention will now be described in detail with reference to the following examples. It will be appreciated that the invention is described by way of example only and modification of detail may be made without departing from the scope of the invention.

EXPERIMENTAL S~Xnthetic Examples Example 1 N- (3- (3, 5-Di-tert-butyl-4-hydroxyphenyl) propyl)-1, 4-dihydro-5-hydroxy-1-methyl-4- oxo-2-pyridinylcarboxamide

5-Benzyloxy-N- (3- (3, 5-di-tert-butyl-4-hydroxyphenyl) propyl)-l, 4-dihydro-I-methyl-4-oxo- 2-pyridinylcarboxamide

A mixture of 5-benzyloxy-1, 4-dihydro-1-methyl-4-oxo-2-pyridinylcarboxylic acid [EP281289] (7.9 mmol), 3- (3, 5-di-tert-butyl-4-hydroxyphenyl) propylamine [NL7905000] (7.9 mmol), 1-ethyl-3- [3- (dimethylamino) propyl] carbodiimide hydrochloride (1.53g, 7.9 mmol) and DMAP (0.98 g, 7.9 mmol) in THF (50 mL) and DMF (50 mL) is stirred at room temperature for 17 h. H20 (20 mL) is added, and the mixture is concentrated in vacuo. The residue is extracted with CH2Cl2 (2 x 20 mL), dried (MgSO4), concentrated in vacuo and purified by chromatography [SiO2 ; CHzCIz-MeOH] to give the product.

N- (3- (3, 5-Di-tert-butyl-4-hydroxyphenyl) propyl)-1, 4-dihydro-5-hydroxy-1-methyl-4-oxo-2pyridinylcarboxamide

5-Benzyloxy-N-(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propyl)-1,4-dihydro-1-methyl-4oxo-2-pyridinylcarboxamide (1.0 mmol) and 10 % Pd/C (0. 3 g) in EtOH (16 mL) is stirred under a H2 atmosphere for 20 h. The mixture is filtered through Celite#, concentrated in vacuo, and purified by chromatography [Sephedexs LH-20 ; CH2CI2-MeOH] to give the title compound.

Example 2 N-(3-(3,5-Di-tert-butyl-4-hydroxyphenyl)-2-(1,4-dihydro-5-hydroxy-1-methyl-4-oxo 2-pyridinylcarboxamidomethyl) propyl)-1, 4-dihydro-5-hydroxy-1-methyl-4-oxo-2- pyridinylcarboxamide

Dimethyl 2- (3, 5-di-tert-butyl-4-hydroxybenzyl) malonate

A suspension of hexane-washed NaH (2.91g of 60 % dispersion in mineral oil, 73 mmol) in a mixture of THF (80 mL) and DMF (30 mL) is cooled to 0 C under Ar and treated dropwise with dimethyl malonate (7.9 mL, 69 mmol). After a further 10 min at 20 C, a solution of 3,5-di-tert-butyl-4-hydroxybenzyl bromide (69 mmol) in a mixture of THF (20 mL) and DMF (20 mL) is added, and the mixture is heated under gentle reflux for 1 h. The reaction is then poured into brine, extracted with EtOAc and concentrated in vacuo.

Kugelrohr distillation at 130 C (20mm Hg) is used to remove unreacted dimethyl malonate. The residue is purified by chromatography [SiO2 ; EtOAc-Hexane] to give the product.

2-3, 5-Di-tert-butyl-4-hydroxybenzyl) propane-1, 3-d iol

BH3-Me2S (130 mmol) is added to a mixture of dimethyl 2- (3, di-tert-butyl-4hydroxybenzyl) malonate (40 mmol) and THF (100 mL) under Ar. The resulting solution is heated under reflux for 40 h. MeOH is added slowly to the cooled solution to destroy excess reagent. The mixture is diluted with brine, extracted with EtOAc and concentrated in vacuo. The residue is purified by chromatography [SiO2 ; EtOAc-Hexane] to give the product.

2- (3, 5-Di-tert-butyl-4-hydroxybenzyl) propane-1, 3-diamine dihydrochloride

A solution of 2- (3, 5-di-tert-butyl-4-hydroxybenzyl) propane-1, 3-diol (37.8 mmol) in CH2Cl2 (150 mL) and Et3N (15.8 mL, 113 mmol) is treated dropwise at 0 C with methanesulphonyl chloride (6.5 mL, 83. 3 mmol). After 15 min the solution is washed with water, NaHC03 and brine, and concentrated in vacuo. The crude mesylate is then immediately dissolved in DMF (30 mL), NaN3 (15 g, 230 mmol) is added, and the suspension is stirred at 120 C for 1 h. The cooled mixture is diluted with brine, extracted with EtOAc and concentrated in vacuo. The residue is purified by chromatography [SiO2 ; EtOAc-Hexane] to give the bisdiazide. This is immediately dissolved in EtOH (100 mL) and hydrogenated over 10 % Pd/C (500 mg) at 60 psi for 20 h. The catalyst is removed by filtration and washed well with EtOH. The filtrate is immediately saturated with HCl gas and concentrated in vacuo. The residue is crystallised from MeOH: Et2O to give the product.

5-Benzyloxy-N-(3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(5-benzyloxy-1, 4-dihydro-1methyl-4-oxo-2-pyridinylcarboxamidomethyl)propyl)-1,4-dihydro-1-methyl-4-oxo-2 pyridinylcarboxamide

A solution of 5-benzyloxy-1, 4-dihydro-l-methyl-4-oxo-2-pyridinylcarboxylic acid [EP281289] (8.0 mmol), 2- (3, 5-di-tert-butyl-4-hydroxybenzyl) propane-1, 3-diamine dihydrochloride (4.0 mmol), 1-ethyl-3[3-(dimethylamino) propyl] carbodiimide hydrochloride (8.0 mmol) and DMAP (16 mmol) in THF (50 mL) is stirred at room temperature for 17 h. H20 (20 mL) is added, and the mixture is concentrated in vacuo.

The residue is extracted with CH, CL, (2 x 20 mL), dried (MgSO4) concentrated in vacuo and purified by chromatography [SiO2 ; CH2Cl2-MeOH] to give the product.

N- 3- (3, 5-Di-tert-butyl-4-hydroxyphenyl)-2- (l, 4-dihydro-5-hydroxy-1-methyl-4-oxo-2- pyridinylcarboxamidomethyl)propyl)-1,4-dihydro-5-hydroxy-1-methyl-4-oxo-2 pyridinylcarboxamide

This is prepared from 5-benzyloxy-N- (3- (3, 5-di-tert-butyl-4-hydroxyphenyl)-2- (5benzyloxy-1,4-dihydro-1-methyl-4-oxo-2-pyridinylcarboxamidomethyl) propyl)-1,4 dihydro-1-methyl-4-oxo-2-pyridinylcarboxamide according to the method described in Example 1 to give the title compound.

Example 3 1, 2-Dihydro-3-hydroxy-N- (2-(5-methoxy-3-indolyl) ethylfil-methyl-2-oxow pyridinylcarboxamide

3-Benzyloxy-1,2-dihydro-N-(2-(5-methoxy-3-indolyl~ethyl)-1-methyl-2-oxo-4-pyridinyl carboxamide

This is prepared from and 3-benzyloxy-1, 2-dihydro-l-methyl-2-oxo4-pyridinyl carboxylic acid [Xu et al. J. Am. Chem. Soc. (1995), 117 (27), 7245-7246] and 5- methoxytryptamine according to the method described in Example 1 to give the product.

1, 2-Dihydro-3-hydroxy-N (2- (5-methoxy-3-indolyl) ethyl)-I-methyl-2-oxo-4-pyridinyl carboxamide

This is prepared from 3-benzyloxy-1, 2-dihydro-N- (2- (5-methoxy-3-indolyl) ethyl)-l- methyl-2-oxo-4-pyridinylcarboxamide according to the method described in Example 1 to give the title compound.

Example 4 N-(2-(2,3-Dihydro-5-hydroxy-4, 6,7-trimethylbenzofuran-2-yl) ethyl)-1, 6-dihydro-l hydroxy-6-oxo-2-pyridinylcarboxamide

N- (2-62, 3-Dihydro-5-hydroxy4, 6, 7-trimethylbenzofieran-2-yl) ethyl)-I, 6-dihydro-Ihydroxy-6-oxo-2-pyridinylcarboxamide

A mixture of 1, 6-dihydro-1-hydroxy-6-oxo-2-pyridinecarboxylic acid homopolymer [Scarrow et al. Inorg. Chem. (1985), 24 (6), 954-967] (7.9 mmol) and 2- (2, 3-dihydro-5hydroxy-4, 6,7-trimethylbenzofuran-2-yl) ethylamine [Ceccarelli et al. J. Heterocycl.

Chem. (1993), 30 (3), 679-90] (7.9 mmol) in THF (50 mL) is stirred at room temperature for 17 h. The mixture is concentrated in vacuo. The residue is purified by chromatography [SiO2 ; CH2C'2-MeOI-1] to give the title compound.

Example 5 2-(3-(3,5-Di-tert-butyl-4-hydroxyphenyl)propyl)-3-hydroxy-6-methyl-4(1H)pyridinone mesylate

2-(3-(3,5-Di-tert-butyl-4-hydroxyphenyl)-1-hydroxypropyl)-3-hydroxy-6-methyl-4H-pyran4-one

A mixture of 5-hydroxy-2-methyl-4H-pyran-4-one [Ellis et al. J. Med. Chem. (1996), 39, 3659-3670] (74 mmol), and water (100 mL) is adjusted to pH 10.5 with 10.0-M NaOH.

A solution of 3- (3, 5-di-tert-butyl-4-hydroxyphenyl) propanal [GB2026479] (74 mmol) in CH2C12 (50 mL) and tetrabutylammonium iodide (15 mmol) is added, and the solution is stirred at room temperature for 24 h. The reaction mixture is acidifie to pH 1 using conc. HCl, then concentrated in vacuo. The residue is extracted with CH2C12 (2 x 20 mL), dried (MgSO4), concentrated in vacuo and purified by chromatography [SiO2 ; CH2Cl2-MeOH] to give the product.

2- (3- (3, 5-Di-tert-butyl-4-hydroxyphenyl) propyl)-3-hydroxy-6-methyl-4H-pyran-4-one

To a mixture of 2- (3- (3, 5-di-tert-butyl-4-hydroxyphenyl)-1-hydroxypropyl)-3-hydroxy- 6-methyl-4H-pyran-4-one (42.0 mmol) and water (100 mL) is added activated zinc dust (210 mmol). The mixture is stirred vigorously at 60 C. Conc. HC1 (40 mL) is added dropwise to the mixture over a period of 50 min, the mixture is then stirred at 60 C for 24 h. The excess zinc is removed by filtration, and the filtrate is adjusted to pH 1 and extracted with CH2C12 (2 x 20 mL), dried (MgSO4), concentrated in vacuo and purified by chromatography [SiO2, CH2CI2-MeOH] to give the product.

2- (3-(3, 5-Di-tert-butyl-4-hydroxyphenylkropyl)-3-hydroGy-6-methyl-4 (1H)-pyridinone

Methylamine (40 wt. % solution in water, 60 mmol), 2- (3- (3, 5-di-tert-butyl-4hydroxyphenyl) propyl)-3-hydroxy-6-methyl-4H-pyran-4-one (15 mmol) and 5.0-M NaOH (30 mmol) in EtOH (40 mL) and water (20 mL) is refluxed for 5 h. The reaction is cooled, adjusted to pH 4-5 with 1.0-M HCI, concentrated in vacuo, extracted with CHCl3, dried (MgSO4), concentrated in vacuo and purified by chromatography SiO2 ; CH2CI2- MeOH] to give the product.

2- (3-(3, 5-Di-tert-butyl-4-hydroxyphenyl) propyl)-3-hydroxy-6-methyl-4 (1H)-pyridinone mesylate

Methanesulphonic acid (3.0 mmol) is added dropwise to 2- (3- (3, 5-di-tert-butyl-4 hydroxyphenyl) propyl)-3-hydroxy-6-methyl-4 (lI)-pyridinone (3.0 mmol) in Et2O (50 mL) and CH2CI2 (50 mL). The mixture is stirred for 1.5 h, concentrated in vacuo, suspended in CHCI3 and the solid collected by filtration to give the title compound.

Biological Testing Procedures The models to demonstrate the ability of the compounds of the present invention to protect against oxidative damage are discussed below.

Lipid Peroxidation assay Lipid peroxidation in rat brain homogenates is a general procedure used to measure the antioxidant capacity of molecules in a biological environment (Das N. P. and Ratty A. K., Biochem. Med. Metab. Biol., 1987,37,256-264).

Iodoacetate induced cell toxicity in culture Iodoacetate, via its alkylation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is a potent inhibitor of glycolysis and hence energy production in cells. This form of chemical hypoxia has been demonstrated to result in a state of oxidative stress, from which the cells can be rescued by anti-oxidant molecules (Uto A. et al., J. Neurochem.

1995,64,2185-2192; Malcolm C. S. et al., Soc. Neurosci. Abstr. 1998).

Malonic Acid lesion model Malonic acid is a competitive inhibitor of succinate dehydrogenase. It depletes extracellular ATP production, and intrastriatal injection has previously been demonstrated to cause a NMDA receptor mediated lesion (Greene et al., J. Neurochem., 1993,61,11511154).

1. In Vitro Assays la. Lipid Peroxidation Procedure Rat cortex was homogenised in 20 vols. of ice cold 0.32-M sucrose and centrifuged at 1,000 g for 10 min. The pellet was discarded whilst the resulting supernatant was centrifuged at 15,000 g for 20 min at 4 C to yield p2 pellets. The pellet was re-suspended in ice-cold 50.0-mM phosphate buffer and centrifuged at 30,000 g for 20 min at 4 C.

The pellet was re-suspended in 30 vols. of 50.0-mM phosphate buffer and used immediately in the assay.

Assays contained 500-uM L-ascorbic acid to induce lipid peroxidation, plus various concentrations of test compound, with the tissue preparation in a total volume of 500 pL.

This was incubated at 37 C for 30 min. Lipid peroxidation was measured by adding to the reaction mixture, 300 uL 40 % (w/v) trichloroacetic acid, 150 RL 5. 0-M HC1 and 300 gL 2 % (w/v) 2-thiobarbituric acid (TBA). Samples were then heated at 90 C in a water bath for 15 min, and centrifuged at 15,000 g for 10 min. The pink colour of the supernatant was assessed spectrophotometrically at a wavelength of 532 nm. The amount of malondialdehyde (MDA) in the samples was calculated using a standard curve prepared with malondialdehyde tetrabutylammonium salt (Das, N. P. and Ratty, A. K.

Biochem. Med. Metab. Biol. 1987,37,256-264). lb. Cell Death Procedure After 6-8 days in culture, cerebellar granule cell (CGC) cultures in 96-well plates (250,000 cells per well) were prepared for hypoglycaemia. Iodoacetate (IAA) was made up in a balanced salt solution (BSS) (NaCI, 154.0-mM; KCI, 5.6-mM; MgCl2, 1.0-mM; CaCI2, 2.5-mM ; N- [2-hydroxyethyl] piperazine-N'- [2-ethanesulfonic acid] (HEPES), 10.0-mM; D-glucose, 5.6-mM; pH 7.4) and any neuroprotective agents were made up in pre-warmed tissue culture media and allowed to equilibrate in a controlled environment (5 % CO2, 95 % air). The assay was initiated by aspiration of the maintenance media, which was replaced by either BSS (control) or 30-wu IAA in BSS, both solutions containing 10-1lu of the NMDA receptor antagonist MK-801. The exposure to IAA was for 30 min only at 37 C, after which time the BSS was aspirated and replaced with fresh, pre-equilibriated maintenance media containing various concentrations of test compound. All conditions were performed at least in duplicate in each 96 well plate.

The final volume for each well was always 200 uL. Following a 24 hour incubation, visual inspection of the cells was followed by quantification of neuronal cell death by measuring 3- [4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)reductase activity as described previously (Malcolm et al., J. Neurochem. 1996,66 2350-2360).

2. In Vivo Assay Malonic Acid Lesion Model Procedure Malonic acid is a competitive inhibitor of succinate dehydrogenase, a key enzyme in both the tricarboxylic acid cycle and oxidative phosphorylation. Injection of malonic acid into the striatum causes ATP depletion, resulting in an excitotoxic lesion (Greene et al., J. Neurochem., 1993,61,1151-1154). 2uLofa 0.5-M malonic acid solution is injected into the right striatum of rats, with or without test compounds. 24 hours after surgery the animals are sacrificed and the size of the lesion measured using TTC histochemistry.

Claims (39)

1. CLAIMS 1. A compound of the formula (1):

   wherein A is

   wherein R', R2 and R3 are independently selected from H and alkyl ; wherein X is O, S, NR4 or a direct bond, wherein R4 is H or alkyl ; wherein Z is a hydrocarbyl chain comprising from 1 to 10 carbon atoms optionally interrupted by one or more heteroatom (s) and optionally substituted by one or more oxo substituent (s) ; wherein q is 1,2 or 3; wherein if q is 2 or 3, then each A can be the same or different; wherein the or each V is independently selected from H and alkyl ; wherein the or each R6 is independently selected from alkyl ; wherein n is 1 to 5; wherein p is 0 to 4 ; and wherein the sum of n and p is less than 6, or a pharmaceutically acceptable salt thereof.
2. 2. A compound according to claim 1 wherein A is AI or AII and R'and W are independently selected from H, unsubstituted alkyl, CHOR', CH20COR', COOR', CH2NHR', CH2NHCOR'and CONHR'wherein R'is H or alkyl.
3. 3. A compound according to claim 2 wherein R', R2 and R3 are independently selected from H and unsubstituted alkyl.
4. 4. A compound according to claim 2 wherein R'and R2 are H and R3 is unsubstituted alkyl.
5. 5. A compound according to claim 2,3 or 4 wherein R3 is methyl.
6. 6. A compound according to claim 1,2,3,4 or 5 wherein A is AI and AI is either AIa or AIb :
7. 7. A compound according to claim 1,2,3,4 or 5 wherein A is AII and AII is AIIa :
8. 8. A compound according to claim 1 wherein A is AIII and R', R'and R'are independently selected from H, unsubstituted alkyl, CH2OR', CH2OCOR', COOR7, CH2NHR7, CH2NHCOR7 and CONHR7 wherein R7 is H or alkyl.
9. 9. A compound according to claim 8 wherein R1, R'and R'are independently selected from H and unsubstituted alkyl.
10. 10. A compound according to claim 8 wherein R', R2 and R3 are H.
11. 11. A compound according to claim 1,8 or 9 wherein A is AMI and AIM is AIIIa :
12. 12. A compound according to any one of claims 1 to 11 wherein q = 1.
13. 13. A compound according to any one of claims 1 to 12 wherein Z contains one or more carbon-carbon double bond (s).
14. 14. A compound according to any one of claims 1 to 13 wherein X is NR4 or a direct bond.
15. 15. A compound according to claim 13 where X is NR4, the chain Z contains a carbon carbon double bond and said carbon-carbon double bond is immediately adjacent the NR4 group in the chain defined as Z-X.
16. 16. A compound according to any one of claims 1 to 15 wherein X is NR4 and R4 is H.
17. 17. A compound according to claim 14 or 15 wherein R4 as alkyl and is cyclised on to the chain Z.
18. 18. A compound according to any one of the previous claims wherein the or each R5 is independently selected from H and C,, 0 alkyl.
19. 19. A compound according to any one of the previous claims wherein at least one R5 is H.
20. 20. A compound according to any one of the previous claims wherein the or each R6 is independently selected from t-butyl.
21. 21. A compound according to any one of claims 1 to 16 wherein RS is alkyl and is cyclised onto the chain defined as Z.
22. 22. A compound according to any one of claims 1 to 16 wherein R6 is alkyl and is cyclised onto the chain defined as Z.
23. 23. A compound according to any one of claims 1 to 16 wherein X is NR4, Z contains at least one carbon-carbon double bond, and Z is cyclised onto the phenyl ring shown in formula (1) to give a 5 or 6 membered heterocyclic ring containing a carbon-carbon double bond.
24. 24. A compound according to any preceding claim where Z is a hydrocarbyl chain comprising from 1 to 10 carbon atoms optionally interrupted by one or more group (s) selected from O, S, SO, SO2, SO2NH, NHSO2, NH, NHCO, CONH, NHCONH, NHCOO, OCONH, OCO and COO.
25. 25. A compound according to any preceding claim wherein Z is (CH, kWY wherein k is 0 to 10; wherein W is selected from O, S, SO, SO2, SO2NH, NHSO2, NH, NHCO, CONH, NHCONH, NHCOO, OCONH, OCO, COO and a direct bond; wherein Y is a hydrocarbyl chain comprising from 2 to 10 carbon atoms and one or more carbon-carbon double bond (s); and wherein the chain (CH2) comprises from 1 to 10 carbon atoms.
26. 26. A compound according to claim 25 wherein when k is 0, W is selected from O, NH, NHCO, NHCONH, NHCOO, OCONH and a direct bond
27. 27. A compound according to claim 25 wherein when k is 1, W is selected from SO, SO2, SO2NH, CONH, COO and a direct bond.
28. 28. A compound according to claim 25, 26 or 27 wherein Y comprises 3,4 or 5 carbon atoms.
29. 29. A compound according to claim 25,26,27 or 28 wherein k is 1,2,3 or 4.
30. 30. A compound according to any of claims 1 to 24 wherein Z is interrupted by O, NH, CONH, NHCO.
31. 31. A compound according to any of claims 25 to 29 wherein W is selected from O, NH, CONH, NHCO and a direct bond.
32. 32. A compound according to any preceding claim wherein Z contains one carbon carbon double bond.
33. 33. A compound according to any one of claims 1 to 32 for use in therapy.
34. 34. Use of a compound according to any one claims 1 to 32 in the manufacture of a medicament for the treatment of a condition associated with oxidative stress.
35. 35. A method of treating a condition associated with oxidative stress comprising administering to a patient in need of such treatment an effective dose of a compound according to any one of claims 1 to 32.
36. 36. A use or method according to claim 34 or 35 wherein said oxidative stress is oxidative damage of the central nervous system.
37. 37. A use or method according to claim 34 or 35 wherein said condition is an acute or chronic neurological disorder.
38. 38. A use or method according to claim 37 wherein said neurological disorder is traumatic brain injury, spinal cord injury, cerebral tumour, subharrachnoid haemorrage/cerebral vasospasm, cerebral ischaemia, stroke (ischaemic or haemorragic), Alzheimer's disease, Huntington's Disease, Parkinson's Disease, Friedrich's ataxia, motor neuron disease or multiple sclerosis.
39. 39. A pharmaceutical composition comprising a compound according to any one of claims 1 to 32 in combination with a pharmaceutically acceptable carrier or excipient.