Substituted quinoline derivatives as antiamyloidogeneic agents

The present invention relates to__heterocyclic__ compounds, processes for their preparation and their use as pharmaceutical or veterinary agents, in particular for the treatment, amelioration and/or prophylaxis of conditions caused by or associated with unbalanced metal levels and/or oxidative stress, such as neurological conditions and cellular proliferative disorders, for example Alzheimer's disease, Parkinson's disease, Huntington' s disease or brain cancer or tumours .

BACKGROUND

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that , although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

The life span is thought to be biologically fixed for each species, and the length of the human life span is uncertain, but may be up to 120 years. Since life expectancy has risen significantly in this century, the elderly are an increasing segment of our population, and their health care needs will continue to grow for decades .

Bio-available metal ions play crucial roles in a number of important biological processes. It is estimated that one-third of all proteins are rαetalloproteins (proteins containing a tightly bound metal ion) and therefore a number of biologically important processes are impaired if bio-available metal levels are either elevated or suppressed. Furthermore, the actual distribution of

normal metal levels in the biological system is critical to processes that rely on metals to function appropriately.

While there is a wide range of ways in which bio- available metals impact on biological systems, two of the better known are the role of metals in enzyme systems and the role of metals in signaling mechanisms within biological systems. Examples of the role of metals in biological processes include the potential importance of Zn in the β-amyloid (Aβ) plaques of Alzheimer's disease

(AD) ; the effect of the (Cu, Zn) superoxide dismutase enzyme in mediating reactive oxygen species damage associated with amyotrophic lateral sclerosis,- the participation of the heme enzymes Nitric oxide (NO) synthase and guanylyl cyclase in the production and sensing, respectively, of nitric oxide (NO) , and the discovery of a "zinc-finger" motif in the breast and ovarian cancer susceptibility gene, BRCAl merely by way of example . It is also known that Cu plays a role in XIAP activity which modulates caspase activity which in turn controls apoptosis. Apopotosis is a process of controlled cell death and dysregulation of this process has been implicated in many disease states.

A large percentage of newly discovered enzymes and proteins also contain metal ions at their active sites and variations in metal levels can significantly interfere with the functioning of these enzymes and proteins . Metalloenzymes of this type are involved in a number of important bio catalytic processes including reduction of excess oxygen species. Accordingly whenever there is either too high or too low a level of metals present in a biological system, normal processes are interrupted, typically leading to undesirable consequences. This typically occurs as many of the crucial enzymatic processes that provide protection in the biological system are suppressed or inactivated leading to undesirable consequences .

As a result of the importance of metals in the

biological environment, research conducted into the roles of metals in biological systems have identified a number of conditions which are caused by or associated with unbalanced levels of metal or site specific concentrations of metals in the biological environment . In respect of these conditions they are all typically those in which metal delivery or metal attenuation can prevent, alleviate or ameliorate the condition. A common result of unbalanced levels of metal is oxidative stress (OS) . This can be principally due to the fact that many of the protective enzymes responsible for alleviating OS are deactivated if biological metal levels are too low. It is further recognised that a number of conditions involving OS do not necessarily involve the imbalance of metals. Research in the last few decades has identified that there are a number of conditions that are caused by or associated with OS placed on the body. For example a number of cardiovascular conditions have been identified that are the result of OS. Other conditions associated with OS include cancer, cataracts, neurodegenerative disorders such as Alzheimer's disease and heart diseases. In addition, there is evidence that OS plays a prominent role in three types of neuromuscular disorders: amyotrophic lateral sclerosis (ALS) , mitochondrial/metabolic disease and Friedreich's ataxia. The effect of OS is not limited to any one part of the human body, with examples of the negative effects of OS being observed for almost all organs. For example, the human brain is an organ that concentrates metal ions and recent evidence suggests that a breakdown in metal homeostasis plays a critical role in a variety of age- related neurodegenerative diseases. Common features of these diseases include the deposition of misfolded protein (each disease can have its own specific amyloid protein) and substantial cellular damage as a result of OS.

Significant data suggests that OS is the primary cause of physical damage in a wide range of disease states, including amyloidogenic neurological disorders such as AD,

amylotrophic lateral sclerosis (ALS) , prion diseases - including Creutzfeldt-Jakob Disease (CJD) , transmissible spongioform encephalopathies (TSE), cataracts, mitochondrial disorders, Menke's disease, Parkinson's disease (PD) and Huntington's disease (HD). [Bush, 2000 (Curr Opin Chem Biol. 2000 Apr,- 4(2) : 184-91)] .

In this regard it is notable that copper metal ion deficiency has been reported as a condition associated with AD. Copper is an essential element that is required for many enzymes to function properly, particularly those enzymes that maintain a balance in antioxidant/pro-oxidant homeostasis such as superoxide dismutase and cytochrome C oxidase. One consequence of copper deficiency is that the protective enzymes responsible for detoxifying reactive oxygen species (ROS) are inadequately loaded with copper and therefore do not effectively carry out normal enzyme function. The inadequate loading of such protective enzymes, for example in the brain, leads to a general increase in OS (as is observed in AD) which will be reflected in increased protein oxidation, such as increased protein carbonyls .

The brains of AD patients display abnormal deposits which include β-amyloid protein (Aβ) . A hypothesis is that AD is caused by the toxic accumulation of Aβ amyloid, due in part to excess binding of copper and zinc, metal ions which are abundant in the regions most affected. Moreover, it has been suggested that when Zn ²⁺ and Cu ²⁺ ions interact with Aβ, aggregation of Aβ into fibrils and plaques occurs (Atwood et al . , 1998) ; confirmed by recent data from animals deficient in synaptic Zn ²⁺ (Lee et al . , 2002) . It has also been suggested that redox-active Cu ²⁺ -Aβ interactions can generate H ₂ O ₂ from O ₂ (Huang et al . , 1999) . Both Cu ²⁺ and Zn ²⁺ have been shown to affect Aβ- lipid membrane interactions (Curtain et al . , 2001). It has also been suggested that deposition of amyloid-like fibrils may also be important in other neurodegenerative diseases. These include PD, dementia

with Lewy body formation, multiple system atrophy, Hallerboden-Spatz disease, and diffuse Lewy body disease.

SUMMiVRY The present invention provides a method for the treatment, amelioration and/or prophylaxis of conditions caused by or associated with unbalanced metal levels and/or oxidative stress, in particular conditions in which metal delivery to or from a target biological site or metal attenuation can treat, ameliorate or prevent the condition such as neurological conditions or cellular proliferative disorders including those characterised by an abnormal reaction between proteins and metals such as conditions associated with a toxic gain of function of a protein.

We have now developed heterocyclic compounds having two fused 6-membered aromatic rings with a nitrogen atom at position 1 and a group at position 8 that provides a lone pair of electrons such as N, S, Se or P. The lone pair of electrons at position 8 is believed to assist with the attenuation of metals by the compound. The compounds have been prepared through the collective optimization of one or more of the following properties : (a) aqueous solubility; (b) reduced cell toxicity;

(c) amyloid dispersion properties;

(d) membrane permeability appropriate for CNS penetration;

(e) metabolic stability; and (f) capable of oral administration.

While not wishing to be bound by any theory, it is believed that the nature of the substituent R may further be important in enhancing plaque disaggregation. It is preferable that these substituents are planar in 3D terms . Planar substituents on the ring system allow both the free ligand and the metal attenuation component to more effectively interact with, and disaggregate, protein

aggregates such as amyloid and in particular neurotic plaques .

It is also presently understood that these compounds can act as ionophores . An ionophore is a lipid-soluble molecule that can transportions across the lipid bilayer of the cell membrane. Such small molecules bind to a particular ion, shielding its charge from the surrounding environment, and thus facilitating its crossing of the hydrophobic interior of the lipid membrane. Ionophores disrupt transmembrane ion concentration gradients, required for the proper functioning and indicate an ability to specifically target the death of cancer type cells .

Thus, the compounds may be used in treating cellular proliferative disorders such as cancer or tumours, in particular brain cancers or tumours .

In a first aspect, there is provided a compound of formula I

in which

R ² is H; optionally substituted C _1-6 alkyl; optionally substituted C ₂ -e alkenyl; optionally substituted C ₂ -e alkynyl; optionally substituted C _3-6 cycloalkyl; optionally substituted aryl; optionally substituted heterocyclyl; OR ⁶ , SR ⁶ , COR ⁶ , CSR ⁶ , HCNOR ⁶ or HCNNR ⁶ in which R ⁶ is H, optionally substituted C _1-6 alkyl, optionally substituted C ₂ -S alkenyl, optionally substituted C ₂ - ₆ alkynyl, optionally substituted Ci- _S alkoxy, optionally substituted

C _3-5 cycloalkyl, optionally substituted aryl or optionally substituted heterocyclyl; NR ⁸ R ⁹ , CONR ⁸ R ⁹ or SO ₂ NR ⁸ R ⁹ in which R ⁸ and R ⁹ are independently selected from H, optionalIy substituted C ₁ -S alkyl, optionally substituted C _2-e alkenyl, optionally substituted C ₂ - ₆ alkynyl, optionally substituted C _3-5 cycloalkyl, optionally substituted aryl and optionally substituted heterocyclyl; NR ⁹ COR ¹⁰ in which R ⁹ is as defined above and R ¹⁰ is optionally substituted Ci- ₆ alkyl, optionally substituted C ₂ -s alkenyl, optionally substituted C _2-S alkynyl, optionally substituted C ₃ _ _ε cycloalkyl, optionally substituted aryl or optionally substituted heterocyclyl; CH ₂ CONR ⁸ R ⁹ in which R ⁸ and R ⁹ are as defined above; and (CH ₂ ) _n NR ⁹ R ^i:L in which R ⁹ is as defined above and R ¹¹ is selected from optionally substituted Ci_ _s alkyl, optionally substituted C _2-5 alkenyl, optionally substituted alkynyl, optionally substituted C _3-5 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl and SO ₂ R ¹² in which R ¹² is optionally substituted C _1-6 alkyl, optionally substituted C _2-ε alkenyl, optionally substituted C ₂ -6 alkynyl, optionally substituted C _3-6 cycloalkyl, optionally substituted aryl or optionally substituted heterocycyl and n is 1 to 6 ;

R ⁵ is selected from H and halo;

R ⁷ is selected from H, halo, CO ₂ R ¹³ , CONR ¹³ R ¹⁴ , optionally substituted Ci- ₄ alkyl, optionally substituted C _3- gcycloalkyl, optionally substituted aryl and optionally substituted heterocyclyl;

R ⁸ is selected from amines, (RNR'), amides (RNC=OR'), sulfonamides (RNSO ₂ R'), ureas (RNC=ONHR'), thioureas (RNC=SNHR'), hydroxylamines (RNOH), nitrogen oxides (RNNO or RNNO ₂ ), amine salts (NH ₂ ⁺ R ^" ), hydrazines (RN-NHR'), silylamines (RNSiR ₃ ) , carbamates (RNC=OOR' ) , enamines (RN- CR'=CR ₂ ^; ), thiols (SH), thioethers (SR), sulfoxides (S-OR), sulfones (SO ₂ R) , sulfates (SO ₄ ^2" ) , sulfites (SO ₃ ^2" ) , disulfides (S-SR'), thioester (SC=OR'), sulfenic acids (S- OH) , sulfinic acids (S=OOH) , sulfonic acids (SO ₂ H) , sulfonate esters (SO ₂ -OR') selenols (SeH), selenides (SeR), diselenides (Se-SeR) , selenoxides (Se=OR) , selenones

(SeO ₂ R) , selenoesters (SeC=OR) , phosphanes (PH ₂ ) , phosphines (PRR'), phosphine oxides (P=OR), phosphates (P=O(OR) ₂ , phosphoalkanes (P=CR ₂ ), phosphoalkynes diphosphines (P=PR) in which R and R' are independently selected from H, optionally substituted C _3. _ _e alkyl, optionally interrupted by an NH or CO group, optionally substituted C ₂ -e alkenyl, optionally substituted alkynyl, optionally substituted C ₃ . _e cycloalkyl, optionally substituted aryl and optionally substituted heterocyclyl; R ¹³ and R ¹⁴ are independently selected from H, optionally substituted C ₁ . ₄ alkyl and optionally substituted heterocyclyl ;

W and X are independently selected from CH and N;

Y is CH, CO or CS; Z is C or N; and q is 1, 2 or 3 , salts, hydrates, solvates, derivatives, pro-drugs, tautomers and/or isomers thereof.

In a second aspect, there is provided a process for the preparation of the compound of formula I substantially as described hereinafter.

In a third aspect, there is provided use of the compound of formula I as a pharmaceutical or antioxidant, preferably a neurotherapeutic or neuroprotective agent such as an antiamyloidogenic agent or an anti-cancer agent .

It will be appreciated that the term "anti-cancer" includes "anti-tumour" .

The compound of formula I is advantageously administered in the form of a pharmaceutical or veterinary composition together with a pharmaceutically or veterinarily acceptable carrier.

In a fourth aspect, there is provided a pharmaceutical or veterinary composition comprising the compound of formula I and a pharmaceutically or veterinarily acceptable carrier.

In a fifth aspect, there is provided a method for the treatment, amelioration and/or prophylaxis of a condition

caused by or associated with unbalanced metal levels and/or oxidative stress which comprises the administration of an effective amount of the compound of formula I_ _. _to a ^~ subj ect in need thereof . There is also provided use of the compound of formula I in the manufacture of a medicament for the treatment, amelioration and/or prophylaxis of a condition caused by or associated with unbalanced metal levels and/or oxidative stress. There is further provided use of the compound of formula I for the treatment, amelioration and/or prophylaxis of a condition caused by or associated with unbalanced metal levels.

There is still further provided the compound of formula I for use in the treatment, amelioration and/or prophylaxis of a condition caused by or associated with unbalanced metal levels .

Preferably, the condition caused by or associated with unbalanced metal levels and/or oxidative stress is a neurological condition or a cellular proliferative disorder.

The neurological condition is preferably a neurodegenerative condition, more preferably a neurodegenerative amyloidosis such as Alzheimer's disease, Parkinson's disease or Huntington's disease.

The cellular proliferative disorder is preferably a cancer or a tumour such as a brain cancer or tumour.

DETAILED DESCRIPTION

Compounds

The present invention relates to compounds of formula I, salts, solvates, derivatives, prodrugs and/or isomers thereof . Preferably both X and Y in the compound of formula I are CH or X is N and Y is CO.

Preferably, the compound of formula I has the formula

in which

R ² and R ⁸ are as defined above;

R ⁵ and R ⁷ are independently selected from H and halo;

X is CH or N;

Y is CH, CO or CS; and q is 1, 2 or 3 , salts, hydrates, solvates, derivatives, pro-drugs, tautomers and/or isomers thereof.

In one embodiment, the compound of formula I ¹ has the

formula IA in which

R ² , R ⁵ , R ⁷ , R ⁸ and q are as defined above.

R ⁸ is preferably selected from sufonamides (RNSO ₂ R'), amides (RNC=OR'), ureas (RNC=ONHR), thioureas (RNC=SNHR'), amines (RNR'), thioethers (SR), thiols (SH), sulfoxides (S=OR), sulfones (SO ₂ R), selenols, (SeH), selenides (SeR), selenoxides (Se=OR) , selenones (SeO ₂ R) , phosphanes (PH ₂ ) and phosphines (PRR ¹ ) in which R and R ¹ are as defined above .

A subclass of compounds of formula IA have the formula Ia

in which

R ² , R ⁵ , R ⁷ and q are as defined above,- and R ⁸ _a is optionally substituted Ci_ ₆ alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C ₂ . _s alkynyl, optionally substituted C ₃ _ _ε cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl, SO ₂ R', C=OR', C=ONHR' or C=SNHR ¹ in which R' is as defined above .

Preferably R ⁵ and R ⁷ are both halo, more preferably both are chloro or one is chloro and the other is iodo.

R ² is preferably located at either the 2 or 3 positions or both and is selected from H; optionally substituted Ci_ ₄ alkyl; optionally substituted Ci _-4 alkenyl; optionally substituted C _3-5 cycloalkyl; optionally substituted 6-membered aryl optionally condensed with an optionally substituted 6 membered aryl or heteroaryl; optionally substituted saturated or unsaturated 5- or 6- membered N-containing heterocyclyl optionally condensed with an optionally substituted 6-membered aryl or heteroaryl; (CH ₂ ) _n R ¹³ in which n is as defined above and R ¹³ is optionally substituted Ci_ ₄ alkyl, optionally substituted C _3-5 cycloalkyl, optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl or optionally substituted 6-membered aryl; NR ¹⁴ R ¹⁵ in which R ¹⁴ and R ¹⁵ are independently selected from H, optionally substituted Ci _-4 alkyl, optionally

substituted C ₃ - ₆ cycloalkyl, optionally substituted saturated or unsaturated 5-or 6-membered N-containing heterocyclyl and optionally substituted 6-membered aryl;____ NHCOR ¹⁶ in which R ^lε is optionally substituted C _1-4 alkyl, optionally substituted C _3-5 cycloalkyl, optionally substituted membered saturated or unsaturated 5- or 6- N- containing heterocyclyl or optionally substituted 6- membered aryl; CH ₂ CONR ¹⁷ R ¹⁸ in which R ¹⁷ and R ¹⁸ are independently selected from H, optionally substituted Ci_ _ε alkyl, optionally substituted C ₂ -6 alkynyl and optionally substituted 5 or 6-membered N-containing heterocyclyl optionally condensed with optionally substituted 6- membered aryl; and (CH ₂ ) _n NR ¹⁹ R ²⁰ in which R ¹⁹ and R ²⁰ are independently selected from optionally substituted Ci_ _s alkyl, optionally substituted C ₃ _ _s cycloalkyl and SO ₂ R ²¹ in which R ²¹ is selected from optionally substituted C _1-6 alkyl and optionally substituted 6-membered aryl and n is as defined above,- more preferably optionally substituted C _1-4 alkyl or (CH ₂ ) _n NR ¹⁹ R ²⁰ in which n, R ¹⁹ and R ²⁰ are as defined above .

Subclasses of compounds of the formula Ia are as follows

in which R ¹ , R ² , R ⁵ , R ⁷ and q are as defined above. Representative examples are shown below:

in which R, R ² , R ⁵ , R ⁷ and q are as defined above. Specific examples are shown below.

31 34 35

36 39

40

41

43

in which R', R ² , R ⁵ , R ⁷ and q are as defined above, Representative examples are shown below:

(iii)

in which R' , R ² , R ⁵ , R ⁷ and q are as defined above. Representative examples are shown below.

in which R' , R ² , R ⁵ , R ⁷ and q are as defined above, Representative examples are shown below:

in which R' , R ² , R ⁵ , R ⁷ and q are as defined above, Representative examples are shown below.

Specific examples are shown below.

in which R ¹ , R ² , R ⁵ , R ⁷ and q are as defined above. A representative example is shown below.

Another subclass of compounds of formula IA have the formula Ia '

Ia ¹

in which R, R , R , R and q are as defined above.

Representative examples are shown below.

in which R ² , R ⁵ , R ⁷ and p are as defined above. In another embodiment, the compound of formula I ¹ has the formula IB

IB

in which R ² , R ⁵ , R ⁷ and R ⁸ are as defined above; and r is 1 or 2.

R ⁸ is preferably selected from sufonamides (RNSO ₂ R'), amides (RNC=OR ¹ ) , ureas (RNC=ONHR'), thioureas (RNC=SNHR' )m amines (RNR'), thioethers (SR), thiols (SH), sulfoxides (S=OR), sulfones (SO ₂ R), selenols, (SeH), selenides (SeR), selenoxides (Se=OR) , selenones (SeO ₂ R) , phosphanes (PH ₂ ) and phosphines (PRR ¹ ) in which R and R' are as defined above .

A subclass of compounds of formula IB have the formula Ib

Ib in which

R ² , R ⁵ , R ⁷ and r are as defined above,- and R ⁸ _b is optionally substituted Ci _-6 alkyl, optionally substituted C _2-6 alkenyl, optionally substituted C ₂ _ _s alkynyl, optionally substituted C _3-e cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl,

SO ₂ R ¹ , C=OR', C=ONHR' or C=SNHR ¹ in which R' is as defined above .

Preferably R ⁵ and R ⁷ are both halo, more preferably chloro . R ² is preferably located at either the 2 or 3 positions or both and is selected from H; optionally substituted C _1-4 alkyl; optionally substituted C _1-4 alkenyl; optionally substituted C ₃ _ _s cycloalkyl; optionally substituted 6-membered aryl optionally condensed with an optionally substituted 6 membered aryl or heteroaryl; optionally substituted saturated or unsaturated 5- or 6- membered N-containing heterocyclyl optionally condensed with an optionally substituted 6-membered aryl or heteroaryl; (CH ₂ ) _n R ¹³ in which n is as defined above and R ¹³ is optionally substituted Ci _-4 alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl or optionally substituted 6-membered aryl; NR ¹⁴ R ¹⁵ in which R ¹⁴ and R ¹⁵ are independently selected from

H, optionally substituted Ci_ ₄ alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted saturated or unsaturated 5-or 6-membered N-containing heterocyclyl and optionally substituted 6-τnembered aryl; NHCOR ¹⁵ in which R ¹⁶ is optionally substituted C _x-4 alkyl, optionally substituted C _3-5 cycloalkyl, optionally substituted membered saturated or unsaturated 5- or 6- N- containing heterocyclyl or optionally substituted 6- membered aryl; CH ₂ CONR ¹⁷ R ¹⁸ in which R ¹⁷ and R ¹⁸ are independently selected from H, optionally substituted C _1-6 alkyl, optionally substituted C ₂ - ₆ alkynyl and optionally substituted 5 or 6-membered N-containing heterocyclyl optionally condensed with optionally substituted 6- membered aryl; and (CH ₂ ) _n NR ¹⁹ R ^2Q in which R ¹³ and R ²⁰ are independently selected from optionally substituted C _λ . ₆ alkyl, optionally substituted C ₃ -6 cycloalkyl and SO ₂ R ²¹ in which R ²¹ is selected from optionally substituted C _1-6 alkyl and optionally substituted 6-membered aryl and n is as defined above, More preferably R ² is selected from optionally substituted Ci_ ₄ alkyl; optionally substituted Ci _-4 alkenyl; an optionally substituted saturated or unsaturated 5- or 6-membered N-containing heterocyclyl optionally condensed with an optionally substituted 6-membered aryl or heteroaryl; (CH ₂ ) _π R ¹³ in which n is 1 to 3 and R ¹³ is optionally substituted C _3-6 cycloalkyl or an optionally substituted saturated or unsaturated 5- or 6-membered N- containing heterocyclyl; NR ¹⁴ R ¹⁵ in which R ¹⁴ is H and R ¹⁵ is H or optionally substituted C _1-4 alkyl or optionally substituted 6-membered aryl ; NHCOR ¹⁵ in which. R ¹⁵ is optionally substituted Ci_ ₄ alkyl or optionally substituted 6 -membered aryl .

Representative examples of compounds of formula IB are shown below.

in which R ⁸ is as defined above.

While not wishing to be bound by theory, it is believed that substituent R ² at position 3 and R ⁵ generally have a limited effect, electronically or sterically, in the metal attenuating properties of the compounds of the present invention. Substitution can therefore be used to modulate other parameters such as cytotoxicity and physicochemical properties including the number of hydrogen bond donors and acceptors, lipophilicity (ClogP, ElogP and LogD) , solubility and polar surface area. Modulation of these parameters contribute to the optimisation of the pharmacokinetic profile of the compounds. It is also postulated that when substituents R ² and R ⁷ in addition to modulating cytotoxicity and physicochemical properties could also affect activity if the substituent provides metal attenuating properties.

The compound of formula I may have the formula I ' '

in which

R ² , R ⁵ , R ⁷ , R ⁸ , Y and q are as defined above.

Preferably R ² is located at position 2 and is selected from H, optionally substituted C _α - _ε alkyl, optionally substituted aryl, COR ⁶ and CONR ⁸ R ⁹ in which R ^s , R ⁸ and R ⁹ are as defined above .

Preferably Y is CO or CS.

Preferably R ⁵ is H and R ⁷ is H, halo, optionally substituted aryl or optionally substituted heterocyclyl .

Representative examples are shown below.

10

20

The compound of formula I may have the formula I '

in which

R ² , R ⁵ , R ⁷ , R ⁸ and q are as defined above.

Preferably R ² is located at positions 2 or 3 and is selected from H, (CH ₂ ) _n NR ⁹ R ^i:L , COR ^S and CONR ⁸ R ⁹ in which R ^s , R ⁸ , R ⁹ , R ¹¹ and n are as defined above.

Preferably R ⁷ is CO ₂ R ¹³ , CONR ¹³ R ¹⁴ , C _1-4 alkyl, cyclopropyl, optionally substituted aryl or optionally substituted heterocyclyl .

Representative examples are shown below.

The terms "Ci- _S alkyl" or "C _3. -4 alkyl" used either alone or in compound words such as "optionally substituted Ci-4 alkyl" refers to straight chain or branched chain hydrocarbon groups having from 1 to 6 and 1 to 4 carbon atoms, respectively. Illustrative of such alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl or hexyl, preferably methyl , ethyl or propyl . The terms "CH ₂ ) _n " or "(CH ₂ ) _ra " as used herein include both linear and branched chains.

The term "C ₂ _ ₆ alkenyl" refers to straight chain or branched chain hydrocarbon groups having at least one double bond of either E or Z stereochemistry where applicable and 2 to 6 carbon atoms. Examples include vinyl, l-propenyl, 1- and 2-butenyl and 2-methyl-2- propenyl .

The term ^Xλ C ₂ . ₆ alkynyl" used either alone or in compound words such as "optionally substituted C ₂ -s alkynyl" refers to straight chain or branched chain hydrocarbon groups having from 2 to 6 carbon atoms and having in addition one triple bond. Illustrative of such groups are ethynyl, 1-propynyl, 1- and 2-butynyl, 2- methyl-2-propynyl , 2-pentynyl, 3-pentynyl, 4-pentynyl, 2- hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl.

The term "C _3-5 cycloalkyl" used either alone or in compound words such as "optionally substituted C _3-6 cycloalkyl" refers to saturated carbocyclic groups having 3 to 6 carbon atoms. Illustrative of such groups are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl, preferably cyclopropyl .

The term "C ₁ - _G aIkOXy" refers to an oxy-containing radical having C;i _. - ₆ alkyl as defined above. Examples include methoxy, ethoxy, propoxy, butoxy, tert-butoxy and pentoxy. The term "heterocyclyl" refers to saturated or unsaturated, monocyclic or polycyclic hydrocarbon groups containing at least one heteroatom atom selected from the group consisting of nitrogen, sulphur and oxygen. Suitable heterocyclyls include N-containing heterocyclic groups, such as, unsaturated 3 to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl; saturated 3 to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, such as, pyrrolidinyl, imidazolidinyl, piperidino or piperazinyl; unsaturated condensed heterocyclic groups containing 1 to 5 nitrogen atoms, such as indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl or tetrazolopyridazinyl; unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom, such as, pyranyl or furyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms, such as, thienyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms , such as, oxazolyl, isoxazolyl or oxadiazolyl; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, morpholinyl; unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, benzoxazolyl or benzoxadiazolyl ; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolyl or thiadiazolyl; saturated 3 to 6-membered heteromonocyclic group

containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as , thiazolidinyl ; and unsaturated condensed heterocyclic group containing_JL to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, benzothiazolyl or benzothiadiazolyl.

The term "unsaturated or saturated 5- or 6-membered N-containing heterocyclyl group optionally condensed with an optionally substituted 6-membered aryl" used either alone or in compound words such as "optionally substituted unsaturated or saturated 5- or 6-membered N-containing heterocyclyl group optionally condensed with an optionally substituted 6-merabered aryl" refers to monocyclic or polycyclic heterocyclic groups containing at least one nitrogen atom and optionally other heteroatoms selected from sulphur and oxygen.

Suitable heterocyclic groups include N-containing heterocyclic groups, such as, unsaturated 5- or β-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl; saturated 5- or 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, such as, pyrrolidinyl, imidazolidinyl, piperidino or piperazinyl; unsaturated condensed heterocyclic groups containing 1 to 5 nitrogen atoms, such as indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl or tetrazolopyridazinyl; unsaturated 5- or 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl or oxadiazolyl; saturated 5- or 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, morpholinyl; unsaturated 5- or 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolyl or thiadiazolyl; and

saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolidinyl .

Preferably the heterocyclyl is an unsaturated 5 or 6- membered heteromonocyclic group containing 1 to 3 nitrogen atoms such as pyrazolyl, pyridinyl or pyrimidinyl; a saturated 5 or 6-membered heteromonocyclic group containing 1 to 4 nitrogen atoms such as pyrrolidinyl or piperazinyl; an unsaturated condensed heterocyclic group containing 1 to 5 nitrogen atoms such as benzimidazolyl; a saturated 5 or 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as morpholinyl; or an unsaturated 5- or 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 oxygen atoms, such as thiazolyl .

The term "aryl" refers to single, polynuclear, conjugated or fused residues of aromatic hydrocarbons. Examples include phenyl, biphenyl, terphenyl, quaterphenyl , naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenxanthracenyl and phenanthrenyl . A preferred aryl is phenyl.

The term "6-membered aryl" used either alone or in compound words such as "optionally substituted 6-membered aryl" denotes a 6-membered carbocyclic aromatic group. Illustrative of such aryl groups are phenyl. Preferably, the aryl is optionally substituted phenyl such as 4- halophenyl, more preferably 4-fluorophenyl .

The term ^W 6-membered heteroaryl" used either alone or in compound words such as "optionally substituted 6- membered hetroaryl" denotes a 6-membered aromatic heterocycle containing one or more heteroatoms . Examples include pyridyl pyrazinyl, pyrimidinyl and pyridazinyl, each of which may be optionally substituted by methyl or methoxy. The term "halo" refers to fluorine, chlorine, bromine or iodine, preferably fluorine, iodine or chlorine, more preferably chlorine or iodine .

The term "optionally substituted" refers to a group

that may or may not be further substituted with one or more groups selected from Ci_ ₆ alkyl, C _3-6 cycloalkyl, C ₂ _ _ε alkenyl, C _2-6 alkynyl, aryl, heterocycylyl, halo, _ haloCi- ₆ alkyl, haloCs-scycloalkyl, haloC ₂ -salkenyl, haloC ₂ . ₆ alkynyl, haloaryl, haloheterocycylyl, hydroxy, Cχ_ _e alkoxy, C ₂ . _e alkenyloxy, C ₂ _ _s alkynyloxy, aryloxy, heterocyclyloxy, carboxy, haloCi- ₆ alkoxy, haloC ₂ . ₆ alkenyloxy, haloC ₂ - _e alkynyloxy, haloaryloxy, nitro, nitroCα-s, alkyl, nitroC ₂ _ _ε alkenyl, nitroaryl, nitroheterocyclyl, azido, amino, Ci- ₆ alkylamino, C ₂ - ₆ alkenylamino, C ₂ . _s alkynylamino, arylamino, heterocyclamino acyl, Ci_ _s alkylacyl, C ₂ - ₆ alkenylacyl, C ₂ - ₆ alkynylacyl, arylacyl, heterocycylylacyl, acylamino, acyloxy, aldehydo, C ₁ . ₆ alkylsulphonyl, arylsulphonyl, Ci_ ₆ alkylsulphonylamino, arylsulphonylamino,

Ci- ₆ alkylsulphonyloxy, arylsulphonyloxy, Ci_ _e alkylsulphenyl, C ₂ - ₆ alklysulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto, Ci _-e alkylthio, arylthio, acylthio, cyano and the like. Preferably, the optional substituent is Ci- ₄ alkyl, halo C ₁ . ₄ alkyl, hydroxy, halo, C _1-4 alkoxy or

Cχ_ ₄ alkylacyl .

The term "protecting group" refers to an introduced functionality which renders a particular functional groups, such as a hydroxy, amino, carbonyl or carboxy group, unreactive under selected conditions and which may later be optionally removed to unmask the functional group. A hydroxy protecting group is one which can temporarily render a hydroxy group unreactive. A hydroxy protecting group refers to a hydroxy group which has temporarily been rendered unreactive by a hydroxy protecting group. A protected phenyl group is taken to be one in which attached reactive substituents, such as OH, NH ₂ , are protected by a protecting group. Suitable protecting groups are known in the art and are described in Protective Groups in Organic Synthesis, Third Edition, T.W. Greene and P. G. White, John Wiley & Sons, Inc., 1999, (the contents of which are incorporated herein by reference) as are methods for their installation and

removal . Examples of protecting groups which may be used to protect a hydroxy group include, but are not limited to, silyl groups (eg trimethylsilyl, t-butyldimethylsj-JlyJL _t __ t-butyldiphenylsilyl) , benzyl groups (eg benzyl, methoxybenzyl , nitrobenzyl) , alkyl groups (eg methyl, ethyl, n- and i-propyl, and n- , sec- and t- butyl) and acyl groups (eg acetyl and benzoyl) .

The leaving group may be of any suitable known type , such as, for example, those leaving groups disclosed in J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure" 4 ^th Edition, pp 352-357, John Wiley & Sons, New York, 1992 which is incorporated herein by reference. Preferably, the leaving group is halogen.

The use of the present compounds as metal delivery agents or metal attenuators is distinguished from alternative concepts of "chelation therapy"

"Chelation therapy" is a term associated clinically with the removal of bulk metals such as in Wilson's disease, β-thallesemia and haemochromatosis . The breakdown in metal homeostasis in these diseases can be described as a catastrophic event much like a dam bursting leading to overwhelming flooding of the problem metal. The mechanism of action of such compounds is that bulk metal is sequestered by the chelators and cleared by excretion. By way of comparison the breakdown in metal homeostasis associated with neurological conditions of the present invention is more akin to the constant drip of a leaky tap, which if left long enough will eventually cause local damage over a long period of time . Compounds of the present invention do not treat conditions associated with systemic metal overload, such as haemochromatosis, rather they function in the attenuation or redistribution of metals closer to normal biological levels of concentration, albeit in a diseased state. Preferably the derivative is a "pharmaceutically acceptable derivative" . By "pharmaceutically acceptable derivative" is meant any pharmaceutically acceptable salt, hydrate, ester, ether, amide, active metabolite, analogue,

residue or any other compound which is not biologically or otherwise undesirable and induces the desired pharmacological and/or physiological effect. ... .

The salts of the compound of formula I are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and aIky1ammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, trihalomethanesulphonic, toluenesulphonic , benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Salts of amine groups may also comprise quaternary ammonium salts in which the amino nitrogen atom carries a suitable organic group such as an alkyl, alkenyl, alkynyl or aralkyl moiety.

The salts may be formed by conventional means, such as by reacting the free base form of the compound with one or more equivalents of the appropriate acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is removed in vacuo or by freeze drying or by exchanging the anions of an existing salt for another anion on a suitable ion exchange resin.

In addition, some of the compounds of the present invention may form solvates with water or common organic

solvents. Such solvates are encompassed within the scope of the invention.

The term "pro-drug" refers to functional derivatiγes_ of the compound of formula I which are readily convertible in vivo into the required compound of formula I .

Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985. A prodrug may be a pharmacologically inactive derivative of the active compound that requires transformation within the body in order to release the active compound, and that has improved delivery properties over the active compound. The transformation in vivo may be, for example, as the result of some metabolic process, such as chemical or enzymatic hydrolysis of a carboxylic, phosphoric or sulphate ester, or reduction or oxidation of a susceptible functionality. In one embodiment, the term refers to the attachment at position 2 of an antioxidant group, in particular the 3 , 4, 5-trimethoxyphenyl moiety or derivatives thereof. Exposure to the prooxidative environment of the brain will then lead to hydroxylation of the 3 , 4 , 5-trimethoxyphenyl group to give a 2-hydroxy- 3 , 4, 5-trimethoxyphenyl substituent, the hydroxyl group of which acts to enhance the metal attenuation properties of the compound of formula I .

The term "antioxidant" is used herein in its broadest sense and refers to a group which has the capacity to react with a reactive oxygen species such as a hydroxyl radical in such a way as to generate a non toxic product.

Examples include phenols such as 3, 4, 5-trimethoxyphenyl and 3 , 5-di-t-butyl-4-hydroxyphenyl, indole amines such as melatonin and flavonoids . Other examples may be found the literature (Wright, 2001; Karbownik, 2001; Gilgun-Sherki, 2001) .

The term "tautomer" is used herein in its broadest sense to include compounds of formula I which are capable of existing in a state of equilibrium between two isomeric

forms. Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound.

The term "isomer" is used herein in its broadest sense and includes structural, geometric and stereo isomers. As the compound of formula I may have one or more chiral centres, it is capable of existing in enantiomeric forms .

Included within the scope of this invention are compounds of the formula I to which at least one of a detectable label, an affinity tag and a photoreactive group is linked.

Pharmaceutical Compositions

The administration of the compound of the present invention may be any suitable means that results in a concentration of the compound that is effective to yield the desired therapeutic or prophylactic response. The compound may be contained in any appropriate amount in any suitable carrier and is generally present in an amount of 1-95% by weight of the total weight of the composition. The carrier must be "pharmaceutically acceptable" in the sense of being compatible with other ingredients of the composition and not injurious to the subject.

The compound of the present invention may additionally be combined with other medicaments to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically- active agents, as long as the combination does not eliminate the activity of the compound of formula I or II. It will be appreciated that the compound of the invention and that other medicament may be administered separately, sequentially or simultaneously. Other medicaments may include, for example, where the condition is a β-amyloid related condition, particularly Alzheimer's disease, an inhibitor of the acetylcholinesterase active site, for example phenserine,

galantamine, or tacrine; an antioxidant, such as Vitamin E or Vitamin C; an anti-inflammatory agent such as flurobiprofen or ibuprofen optionally modified to release nitric oxide (for example NCX-2216, produced by NicOx) or an oestrogenic agent such as 17-β-oestradiol . Where the condition is a cellular proliferative disease, other medicaments may include, for example, anti-angiogenesis agents (drugs that interfere with growth of blood vessels that feed the tumor) , immunotoxins (a toxin is attached to an antibody that hones in on tumor cells) , and differentiating agents.

The composition may be provided in a dosage form that is suitable for oral, parenteral (including intravenous, intramuscular, subcutaneous and intradermal) , rectal, vaginal, nasal, inhalation, topical or ocular administration routes. Thus, the composition may be in form of tablets, capsules, pills, powders, granulates, suspensions, emulsions, liquids, gels including hydrogels, pastes, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays or aerosols. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see , e.g., Remington: The Science and Practice of Pharmacy, (19 ^th ed.). A. R. Gennaro, 1995, Mack Publishing Company, Easton, PA. and Encyclopedia of

Pharmaceutical Technology, eds . J.Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) .

Pharmaceutical compositions may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the active compound within the body over an extended period of time,- (ii) formulations that after a predetermined lay time create a substantially constant concentration of the active compound within the body over an extended period of time,- (iii) formulations

that sustain active compound action during a predetermined time period by maintaining a relatively, constant, effective active compound level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active compound (sawtooth kinetic pattern) ; (iv) formulations that localise active compound action by, e.g. , special placement of a controlled release composition adjacent to or in the diseased tissue or organ; and (v) formulations that target active compound action by using carriers or chemical derivatives to deliver the active compound to a particular target cell type.

Administration of compounds in the form of a controlled release formulation is especially preferred in cases in which the compound has (i) a narrow therapeutic index (i.e., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; in general, the therapeutic index, TI, is defined as the ratio of median lethal dose (LD ₅₀ ) to median effective dose (ED ₅₀ ) ) ; (ii) a narrow absorption window in the gastro-intestinal tract; or (iii) a very short biological half-like so that frequent dosing during a day is required in order to sustain the plasma level at a therapeutic level.

Any number of strategies can be applied in order to obtain a controlled release formulation in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the active compound is formulated with appropriate excipients into a pharmaceutical composition, that, upon administration to the subject, releases the active compound in a controlled manner. Examples include single or multiple unit tablet or capsule compositions,

oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches and liposomes.

Solid Dosage Forms For Oral Use Formulations for oral use include tablets containing the active compound in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, mirocrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate) ; granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminium silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone or polyethylene glycol) ; and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils or talc) . Other pharmaceutically acceptable excipients can be colourants, flavouring agents, plasticisers, humectants, buffering agents and the like.

The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active compound in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active compound until after passage of the stomach (enteric coating) . The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl

hydroxyethylcellulose, hydroxypropylcellulose, carboxyπietϊiylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone) , or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac and/or ethylcellulose) . Furthermore, a time delay material such as, glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, {e.g., chemical degradation prior to the release of the active compound) . The coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of Pharmaceutical Technology, supra.

Formulations for oral use may also be presented as chewing tablets or as hard gelatin capsules wherein the active compound is mixed with an inert solid diluent {e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active compound is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil . Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g, a mixer, a fluid bed apparatus or a spray drying equipment .

Liquids for Oral Administration

Powders, dispersible powders, or granules suitable for preparation of an aqueous suspension by addition of water are convenient dosage forms for oral administration. Formulation as a suspension provides the active compound in a mixture with a dispersing or wetting agent, suspending agent, and one or more preservatives. Suitable dispersing or wetting agents are, for example, naturally- occurring phosphatides {e.g., lecithin or condensation

products of ethylene oxide with a fatty acid, a long chain aliphatic alcohol or a partial ester derived from fatty acids) and a hexitol or a hexitol anhydride [e _^ g. , __ polyoxyethylene stearate, polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitan monooleate and the like) . Suitable suspending agents are, for example, sodium carboxymethylcellulose, methylcellulose, sodium alginate and the like .

Parenteral Compositions

The compound may be administered parenterally by injection, infusion or implantation (intravenous, intramuscular, subcutaneous or the like) in dosage forms, formulations or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers . The formulation and preparation of such compositions is well known to those skilled in the art of pharmaceutical formulation. Specific formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

Compositions for parenteral use may be presented in unit dosage forms (e.g., in single-dose ampoules) or in vials containing several doses and in which a suitable preservative may be added (see below) . The composition may be in form of a solution, a suspension, an emulsion, an infusion device or a delivery device for implantation or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the compound, the composition may include suitable parenterally acceptable carriers. The active compound may be incorporated into microspheres, microcapsules, nanoparticles, liposomes or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents and/or dispersing agents.

As indicated above, the pharmaceutical compositions may be in the form suitable for sterile injection. To prepare such a composition, the suitable active compound

is dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1, 3-butanediol, Ringer's solution and isotonic sodium chloride solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate) . In cases where the compound is only sparingly or slightly soluble in water, a dissolution enhancing or solubilising agent can be added or the solvent may include 10- 60% w/w of propylene glycol or the like.

Rectal Compositions

For rectal application, suitable dosage forms for a composition include suppositories (emulsion or suspension type) and rectal gelatin capsules (solutions or suspensions) . In a typical suppository formulation, the active compound is combined with an appropriate pharmaceutically acceptable suppository base such as cocoa butter, esterified fatty acids, glycerinated gelatin and various water-soluble or dispersible bases like polyethylene glycols and polyoxyethylene sorbitan fatty acid esters. Various additives, enhancers or surfactants may be incorporated.

Vaginal Compositions

Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate .

Nasal and Inhalation Compostions

For administration to the respiratory tract, including intranasal administration, the active compound may be administered by any of the methods and formulations

employed in the art for administration to the respiratory tract.

Thus in general the active compound may be administered in the form of a solution or a suspension or as a dry powder .

Solutions and suspensions will generally be aqueous, for example prepared from water alone (for example sterile or pyrogen-free water) or water and a physiologically acceptable co-solvent (for example ethanol, propylene glycol or polyethylene glycols such as PEG 400) .

Such solutions or suspensions may additionally contain other excipients for example preservatives (such as benzalkonium chloride) , solubilising agents/surfactants such as polysorbates {eg. Tween 80, Span 80, benzalkonium chloride) , buffering agents, isotonicity-adjusting agents (for example sodium chloride) , absorption enhancers and viscosity enhancers. Suspensions may additionally contain suspending agents (for example microcrystalline cellulose and carboxymethyl cellulose sodium) . Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray. The formulations may be provided in single or multidose form. In the latter case a means of dose metering is desirably provided. In the case of a dropper or pipette this may be achieved by the subject administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray this may be achieved for example by means of a metering atomising spray pump. Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the compound is provided in a pressurised pack with a suitable propellant, such as a chlorofluorocarbon (CFC) , for example dichlorodifluoromethane , trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of active compound may be controlled by provision of a metered valve.

Alternatively the active compound may be provided in the form of a dry powder, for example a powder mix of the

compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidine (PVP) . Conveniently the powder carrier will form a gel in the nasal cavity. The powder composition may be presented in unit dose form, for example in capsules or cartridges of eg. gelatin, or blister packs from which the powder may be administered by means of an inhaler.

In formulations intended for administration to the respiratory tract, including intranasal formulations, the active compound will generally have a small particle size, for example of the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronisation. When desired, formulations adapted to give sustained release of the active compound may be employed.

The active compound may be administered by oral inhalation as a free-flow powder via a "Diskhaler" (trade mark of Glaxo Wellcome pic or a meter dose aerosol inhaler.

Topical Compositions

The pharmaceutical compositions may also be administered topically on the skin for percutaneous absorption in dosage forms or formulations containing conventionally non-toxic pharmaceutical acceptable carriers and excipients including microspheres and liposomes. The formulations include creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters and other kinds of transdermal drug delivery systems. The pharmaceutically acceptable carriers may include emulsifying agents, antioxidants, buffering agents, preservatives, humectants, penetration enhancers, chelating agents, gel forming agents, ointment bases, perfumes and skin protective agents.

Examples of emulsifying agents are naturally occurring gums (e.g., gum acacia or gum tragacanth) and naturally occurring phosphatides (e.g., soybean lecithin

and sorbitan monooleate derivatives) . Examples of antioxidants are butylated hydroxy anisole (BHA) , ascorbic acid and derivatives thereof, tocopherol and derivatives -_ thereof, butylated hydroxy anisole and cysteine. Examples of preservatives are parabens, such as methyl or propyl p- hydroxybenzoate and benzalonium chloride. Examples of humectants are glycerin, propylene glycol, sorbitol and urea . Examples of penetration enhancers are propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide, N,N- dimethylformamide, 2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol and Azone .RTM. Examples of chelating agents are sodium EDTA, citric acid and phosphoric acid. Examples of gel forming agents are Carbopol, cellulose derivatives, bentonite, alginates, gelatin and polyvinylpyrrolidone. Examples of ointment bases are beeswax, paraffin, cetyl palmitate, vegetable oils, sorbitan esters of fatty acids (Span) , polyethylene glycols and condensation products between sorbitan esters of fatty acids and ethylene oxide (e.g., polyoxyethylene sorbitan monooleate (Tween) ) .

The pharmaceutical compositions described above for topical administration on the skin may also be used in connection with topical administration onto or close to the part of the body that is to be treated. The compositions may be adapted for direct application or for introduction into relevant orifice(s) of the body (e.g. , rectal, urethral, vaginal or oral orifices) . The composition may be applied by means of special delivery devices such as dressings or alternatively plasters, pads, sponges, strips or other forms of suitable flexible material .

Ocular Compositions

For application to the eye, the active compound may be in the form of a solution or suspension in a suitable sterile aqueous or non-aqueous vehicle. Additives, for instance buffers, preservatives including bactericidal and fungicidal agents, such as phenyl mercuric acetate or

nitrate, benzalkonium chloride, or chlorohexidine and thickening agents such as hypromellose may also be included. _ _ ... . _

Veterinary Compositions

The active compounds may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for:

(a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses,- powders, granules or pellets for admixture with feed stuffs,- pastes for application to the tongue;

(b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced in the udder via the teat;

(c) topical applications, e.g. as a cream, ointment or spray applied to the skin,- or

(d) rectally or intravaginally, e.g. as a pessary, cream or foam.

Methods of treatment, amelioration and/or prophylaxis

The compounds of formula I may be used in the treatment, amelioration and/or prophylaxis of a condition caused by or associated with unbalanced metal levels and/or oxidative stress for example a neurological condition or a cellular proliferative disorder.

Generally, the terms "treatment" and "prophylaxis" mean affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect and include: (a) preventing the condition from occurring in a subject that may be predisposed to the condition, but has not yet been diagnosed as having it; (b) inhibiting the condition, i.e., arresting its development; or (c)

relieving or ameliorating the effects of the condition, i.e., cause regression of the effects of the condition.

The term "subject" as used herein refers_ to_any animal having a disease or condition which requires treatment or prophylaxis with a pharmaceutically-active agent. The subject may be a mammal, preferably a human, or may be a non-human primate or non-primates such as used in animal model testing. While it is particularly contemplated that the compounds are suitable for use in medical treatment of humans, it is also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, ponies, donkeys, mules, llama, alpaca, pigs, cattle and sheep, or zoo animals such as primates, felids, canids, bovids and ungulates.

The term "condition caused by or associated with unbalanced metal levels" refers to a condition whereby a subject has either a too high or too low total amount of metal. This term also refers to a subject with a normal total amount of metal, but the metal is not correctly or is abnormally distributed.

The term "condition caused by or associated with oxidative stress" refers to a condition whereby biological constituents of a subject are damaged by reactive oxygen species. It is particularly contemplated that such constituents are damaged by reactive oxygen species such as the hydroxyl radical, hydrogen peroxide and superoxide produced in Fenton's and similar reactions. In particular it is understood that metals such as iron, copper, zinc chromium, vanadium and cobalt are capable of redox cycling in which a single electron may be accepted or donated by the metal, facilitating oxidative reactions. Actual damage results when the oxidative species causes modifications of amino acids (e.g. meta-tyrosine and ortho-tyrosine formation from phenylalanine) , carbohydrates and lipids (inducing peroxidation) . In some cases such modification may cause a toxic gain of function or corruption of the biological constituent substrate.

The term "neurological condition" is used herein in its broadest sense and refers to conditions in which various cell types of the nervous sγstem_are degenerated __ and/or have been damaged as a result of neurodegenerative disorders or injuries or exposures. In particular, compound of formula I can be used for the treatment of resulting conditions, in which damage to cells of the nervous system has occurred due to surgical interventions, infections, exposure to toxic agents, tumours, nutritional deficits or metabolic disorders. In addition, the compound of formula I can be used for the treatment of the sequelae of neurodegenerative disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amylotrophic lateral sclerosis, epilepsy, drug abuse or drug addiction (alcohol, cocaine, heroin, amphetamine or the like) , spinal cord disorders and/or injuries, dystrophy or degeneration of the neural retina (retinopathies) and peripheral neuropathies, such as diabetic neuropathy and/or the peripheral neuropathies induced by toxins.

The term "neurodegenerative condition" as used herein refers to an abnormality in which neuronal integrity is threatened. Neuronal integrity can be threatened when neuronal cells display decreased survival or when the neurons can no longer propagate a signal.

Neurological conditions that can be treated with the compound of the present invention include acute intermittent porphyria; adriamycin-induced cardiomyopathy; AIDS dementia and HIV-I induced neurotoxicity; Alzheimer's disease; amylotrophic lateral sclerosis; atherosclerosis; cataract; cerebral ischaemia; cerebral palsy,- cerebral tumour; chemotherapy-induced organ damage; cisplatin- induced nephrotoxicity; coronary artery bypass surgery; Creutzfeldt-Jacob disease and its new variant associated with "mad cow" disease; dementia; diabetic neuropathy; Down's syndrome; drowning; epilepsy and post-traumatic epilepsy; fatal familial insomnia; Friedrich's ataxia; frontotemporal dementia; Gertsmann Straussler Sheinker

disease,- glaucoma; glomerulopathy; haemochromatosis,- haemodialysis; haemolysis; haemolytic uraemic syndrome (Weil's disease); haemorrhagic stroke; Hallerboden-Spatz disease; heart attack and reperfusion injury; hereditary cerebral haemorrhage with amyloidois-Dutch type;

Huntington's disease; Lewy body disease; intermittent claudication; ischaemic stroke,- inflammatory bowel disease; macular degeneration; malaria,- methanol-induced toxicity; meningitis (aseptic and tuberculous) ; Mild Cognitive Impairment (MCI) ,- 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,- spinal cord injury,- 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, the compound 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 conditions such as traumatic brain injury, spinal cord injury, cerebral ischaemia, stroke (ischaemic and haemorragic) , subharrachnoid haemorrage/cerebral vasospasm, cerebral tumour, Alzheimer's disease, Creutzfeldt-Jacob disease and its new variant associated with "mad cow" disease, Huntington's disease, Parkinson's disease, Friedrich's ataxia, cataract, dementia with Lewy body formation, multiple system atrophy, Hallerboden-Spatz disease, diffuse Lewy body disease, amylotrophic lateral

sclerosis, motor neuron disease, multiple sclerosis, fatal familial insomnia, Gertsmann Straussler Sheinker disease and hereditary cerebral haemorrhage with amyoidoisis-Dutch- _, ^" type ^" . More particularly, the invention is directed to the treatment of neurodegenerative amyloidosis . The neurodegenerative amyloidosis may be any condition in which neurological damage results from the deposition of amyloid. The amyloid may be formed from a variety of protein or polypeptide precursors, including but not limited to Aβ, synuclein, huntingtin, or prion protein. Thus the condition is preferably selected from the group consisting of sporadic or familial Alzheimer's disease, amyotrophic lateral sclerosis, motor neuron disease, cataract, Parkinson's disease, Creutzfeldt-Jacob disease and its new variant associated with "mad cow" disease, Huntington's disease, dementia with Lewy body formation, multiple system atrophy, Hallerboden-Spatz disease, and diffuse Lewy body disease. More preferably the neurodegenerative amyloidosis is an Aβ-related condition, such as Alzheimer's disease or dementia associated with Down syndrome or one of several forms of autosomal dominant forms of familial Alzheimer's disease (reviewed in St George-Hyslop, 2000) . Most preferably the Aβ-related condition is Alzheimer's disease.

In addition to slowing or arresting the cognitive decline of a subject, the compound and methods of the invention may also be suitable for use in the treatment or prevention of neurodegenerative conditions, or may be suitable for use in alleviating the symptoms of neurodegenerative conditions . The compound may be able to provide at least a partial reversal of the cognitive decline experienced by patients . If administered to a subject who has been identified as having an increased risk of a predisposition to neurodegenerative conditions, or to a subject exhibiting pre-clinical manifestations of cognitive decline, such as Mild Cognitive Impairment or

minimal progressive cognitive impairment, these methods and compounds may be able to prevent or delay the onset of clinical symptoms, in addition to the effect of slowing or reducing the rate of cognitive decline. Currently Alzheimer's disease and other dementias are usually not diagnosed until one or more warning symptoms have appeared. These symptoms constitute a syndrome known as Mild Cognitive Impairment (MCI) , which was recently defined by the American Academy of Neurology, and refers to the clinical state of individuals who have memory impairment, but who are otherwise functioning well, and who do not meet clinical criteria for dementia (Petersen et al . , 2001) . Symptoms of MCI include:

(1) Memory loss which affects job skills (2) Difficulty performing familiar tasks

(3) Problems with language

(4) Disorientation as to time and place (getting lost)

(5) Poor or decreased judgement (6) Problems with abstract thinking

(7) Misplacing things

(8) Changes in mood or behaviour

(9) Changes in personality

(10) Loss of initiative MCI can be detected using conventional cognitive screening tests, such as the Mini Mental Status Exam, and the Memory Impairment Screen, and neuropsychological screening batteries.

The term "cellular proliferative disorder" refers to any cellular disorder in which the cells proliferate more rapidly than normal tissue growth. Cellular proliferative disorder, includes but is not limited to neoplasms. A neoplasm is an abnormal tissue growth, generally forming a distinct mass, that grows by cellular proliferation more rapidly than normal tissue growth. Neoplasms show partial or total lack of structural organisation and functional coordination with normal tissue. These can be broadly classified into three major types. Malignant neoplasms

arising from epithelial structures called carcinomas, malignant neoplasms that originate from connective tissues such as muscle, cartilage, fat or bone are called sarcomas and malignant tumours affecting hematopoietic structures (structures pertaining to the formation of blood cells) including components of the immune system called leukaemias and lymphomas. A tumour is the neoplastic growth of the disease cancer. As used herein, a "neoplasm" , also referred to as a "tumour" is intended to encompass hematopoitic neoplasms as well as solid neoplasms . Other cellular proliferative disorders include, but are not limited to arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like.

The compounds of formula I are particularly useful for the treatment or prophylaxis of cancer including solid tumours. The term "cancer" describes any array of different diseases linked by cumulative multiple genetic mutations, which result in the activation of oncogenes and/or the inactivation of tumor suppressor genes and/or linked by uncontrolled cellular proliferation. The cause and source of these mutations differs between different cancers of human body organs . The invention is particularly directed to brain cancer, which includes a brain tumour. A brain cancer or tumour may be a glioma or non-glioma brain tumour. The term "cancer" and "tumour" may be used interchangeably herein. "Cancer" may include any one of the following states: glioma, adenoma, blastoma, carcinoma, sarcoma and inclusive of any one of Medulloblastoma, Ependymoma, Astrocytoma, Optical nerve glioma, Brain stem glioma, Oligodendroglioma, Gangliogliomas, Craniopharyngioma or Pineal Region Tumours. Reference to a "glioma" includes astrocytoma, glioblastoma multiforme (GBM) , anaplastic astrocytoma, mixed glioma or related brain cancers.

Dosages

The term "therapeutically effective amount" means an

^{' ~} amount of the compound of formula I to yield the desired 5 therapeutic response.

Dosage levels of the compound of formula I of the present invention are of the order of about 0.1 mg to about 20 mg per kilogram body weight, with a preferred dosage range between about 0.1 mg to about 10 mg per 0 kilogram body weight per day (from about 0.1 gms to about 3 gms per patient per day) . The amount of active ingredient that may be combined with the carrier materials to produce a single dosage will vary depending upon the host treated and the particular mode of administration. 5 For example, a formulation intended for oral administration to humans may contain about 5 mg to 1.5g of an active compound with an appropriate and convenient amount of carrier material which may vary from about 5 to 95 percent of the total composition. Dosage unit forms 0 will generally contain between from about 5 mg to 500 mg of active ingredient .

Optionally the compounds of the invention are administered in a divided dose schedule, such that there are at least two administrations in total in the schedule . 5 Administrations are given preferably at least every two hours for up to four hours or longer,- for example the compound may be administered every hour or every half hour. In one preferred embodiment, the divided-dose regimen comprises a second administration of the compound 0 of the invention after an interval from the first administration sufficiently long that the level of active compound in the blood has decreased to approximately from 5-30% of the maximum plasma level reached after the first administration, so as to maintain an effective content of 5 active agent in the blood. Optionally one or more subsequent administrations may be given at a corresponding interval from each preceding administration, preferably

when the plasma level has decreased to approximately from 10-50% of the immediately-preceding maximum.

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy. In the subject specification, except where the context requires otherwise due to express language or necessary implication, the words "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

EXAMPLES The invention will now be described in detail by way of reference only to the following non-limiting examples.

Example 1

Synthetic Scheme

Following a modified literature procedure (Peet et. al . 1997) aromatic hydroxy compounds were converted to heteroarylamines using a Smiles rearrangement.

39 R1=4MeOPh

40 R1=4FPh 43 R1=CF3

Reaction Conditions i) NaH, Cs ₂ CO ₃ , 2-bromo-2-methylpropanamide, 110°C, 20 h. ii) NMP, HMPT, NaH, 150 "C, 3 days, iii) cone. HCl reflux, 6 h. iv) benzene sulfonyl chloride, pyridine, cat. DMAP, 50-60 ⁰ C. v) SeO ₂ , 1,4-dioxane, 75°C. vi) NMe ₂ -HCl, NaBH(OAc) ₃ , 1,2-DCE, DIEA or 2.0M NMe ₂ in MeOH, NaBH ₄ , CH ₂ Cl ₂ 2-Bromo-2-methyl-propanamide

To a IL conical flask containing hexane (500 τtιL) was added bromoisobutyryl bromide and the solution was cooled to 0°C. Concentrated aqueous ammonia (80 mL) was added dropwise to the reaction via dropping funnel over 20 min and the resulting white suspension was stirred at 0°C for a further 30 min. The white solid was filtered and washed with ice cold H2O several times. Recrystallisation from CHCl ₃ (200 mL) / petroleum ether 60-80 °C (85 mL) provided

the propanamide as shiny white plates (24.8 g, 79%) . ¹ H NMR (400 MHz, d6-DMSO) δ (s, 6H, 2xCH ₃ )

N- (5 _/ 7-Dichloro-2-methylquinolin-8-yl) -2-hydroxy-2- methylpropionamide

To a solution of 5, 7-Dichloro-2-methyl-quinolin-8-ol (2.95 g, 12.93 mmol) in 1,4-dioxane (65 mL) was added dry NaH (1.02 g, 42.5 mmol) with the evolution of H ₂ gas. Cesium carbonate (13.9 g, 42. 7 mmol) was added and the yellow suspension was stirred at rt for 30 min then 2-bromo-2- methylpropanamide (7.1 g, 42.7 mmol) was added and the reaction was heated to 110° C under argon for 20 h. After removing from the heat, NMP (70 mL) , 1, 3 -dimethyl 3,4,5,6- tetrahydro-lH-pyrimidone (7.6 mL) were added followed by the addition of dry NaH (380 mg, 15 .8 mmol) and the reaction was heated to 150° C for 3 days. Cooled and partitioned between EtOAc and H ₂ O the mixture was extracted into EtOAc (x3) . The combined organic layers were washed with H ₂ O, brine, dried Na ₂ SO ₄ , filtered, concentrated and purified by flash chromatography eluting with 10%- 20%EtOAc/petroleum ether 40-60°C to provide the 5,7- Dichloro-2-methyl-quinolin-8-yl amine 3 as a tan solid (941 mg, 32%). ^x H NMR (400 MHz, d6-DMSO) δ.2.68 (s, 3H, CH ₃ ), 6.07 (s, 2H, NH ₂ ), 7.57 (s, IH, ArH), 7.59 (d,

J-=8.8Hz, IH, ArH), 8.33 (d, J= 8.8 Hz, IH, ArH). MS: m/z 227.2 [M+H] ⁺ .

Further elution of the column with 30% then 40% EtOAc/ petroleum ether 40-60°C afforded the amide 2 (1.95 g, 48%) as a crystalline white solid. ¹ H NMR (400 MHz, d6-DMSO) δ.1.43 (s, 6H, 2xCH ₃ ) , 2.69 (s, 3H, CH ₃ ), 5.83 (s, IH, OH), 7.64 (d, J=8.8Hz, IH, ArH), 7.91 (s, IH, ArH), 8.45 (d, <J=8.8Hz, IH, ArH), 9.61 (s, IH, NH). MS: m/z 313.3 [MH-H] ⁺ .

5,7-Dichloro-2-methyl-quinolin-8-yl amine

N- (5, 7-Dichloro-2-methylquinolin-8-yl) -2-hydroxy-2- methylpropionamide (1.65 g, 5.27 mmol) was heated to

reflux in concentrated HCl (12 tnL) for 5 h. The resulting orange solution was cooled then concentrated on a rotary- evaporator. The orange solid was then taken up in 2 N NaOH and extracted with EtOAc (x3) . The combined organic layers were washed with brine, dried Na ₂ SO ₄ , filtered and concentrated to afford the amine 3 as a yellow solid (1.01 g, 84%) .

General procedure for the synthesis of sulfonamides

N- (5, 7-Dichloro-2-methyl-quinolin-8-yl) -4- methylbenzenesulfonamide

5, 7-Dichloro-2-methyl-quinolin-8-yl amine was dissolved in pyridine (10 mL) and then treated with p-toluenesulfonyl chloride (561 mg, 2.94 mmol) and DMAP (18 mg) . The reaction was heated to 50-60° C under N ₂ overnight. After diluting with H ₂ O and EtOAc the mixture was extracted into EtOAc (x4) and the organic layers were washed with saturated aqueous NaHCO ₃ , brine, dried Na ₂ SO ₄ , filtered, concentrated and purified by flash chromatography eluting with 10% EtOAc/petrol then increasing to EtOAc to provide the desired sulfonamide compound 31 (431 mg, 57%) as a tan solid. ¹ H NMR (400 MHz, d6-DMSO) δ.2.23 (s, 3H, CH ₃ ), 2.33 (s, 3H, CH ₃ ), 7.23 (d, J=8.0Hz, 2H, ArH), 7.45(d, J=8.8Hz, IH), 7.51 (d, J=8.0Hz, 2H), 7.95 (s, IH, ArH), 8.34 (d, J=8.0Hz, IH, ArH). MS: m/z 381.3 (100) [M+H] ⁺ .

N- (5, 7-Dichloro-2-methyl-quinolin-8-yl) -4- methoxybenzenesulfonamide

¹ H NMR (400 MHz, d6-DMSO) δ2.33 (s, 3H, CH ₃ ) , 3.78 (s, 3H, OCH ₃ ) , 6.94 (d, IH, ArH) , 7.56 (d, <J=8.0Hz, 2H, ArH) , 7.98 (s, IH, ArH) , 8.36 (d, J-=8.8Hz, IH, ArH) , 9.83 (s, IH, NH) . MS: m/z 397.3 (100) [M+H] ⁺ .

N- (5,7-Dichloro-2-methyl-quinolin-8-yl) -4- fluorobenzenesulfonamide

¹ H NMR (400 MHz, d6-DMSO) .2.24 (s, 3H, CH ₃ ), 7.30 (dd, J=8.4Hz, 1.8Hz, 2H, ArH), 7.48 (d, J=8.8Hz, IH, ArH), 7.70 (dd, J ^" =8.4 Hz, 1.8Hz, 2H, ArH), 7.91 (s, IH, ArH), _. 8.35 IH, ArH), 10.2 (s, IH, NH). MS: m/z 385.3 (100) [M+H] ⁺ .

N- (5 _/ 7-Dichloro-2-methyl-quinolin-8-yl) - trifluoromethanesulfonamide

Amine (579 mg, 2.55 mmol) was dissolved in pyridine (20 mL) then treated with triflic anhydride (773 μL, 4.59 mmol) at 0°C. It was then warmed to rt, heated at 80° C for 6 h, cooled and then diluted with H ₂ O and EtOAc. The mixture was extracted into EtOAc (x3) and the organic layers were washed with saturated aqueous NaHCO ₃ , dried Na ₂ SO ₄ , filtered, concentrated and purified by FC eluting with 50%-60%-100% EtOAc/petroleum ether 40-60° C to furnish the triflamide as a brown solid (541 mg, 59%) . ¹ H NMR (400 MHz, d6-DMSO) δ 2.69 (s, 3H, CH ₃ ), 7.S5 (d, .7=8.8Hz, IH, ArH), 7.99 (S, IH, ArH), 8.44 (d, <J=8.8Hz, IH, ArH). MS: m/z 359.3 (30) [M+H] ⁺ , 130.2(100).

General procedure for the SeO ₂ oxidation

N- (5, 7-Dichloro-2-formylquinolin-8-yl) -4- methylbenzenesulfonamide

N- (5, 7-Dichloro-2-methyl-quinolin-8-yl) -4- methylbenzenesulfonamide (295 mg, 0.774 mmol) was dissolved in 1,4-dioxane (9 mL) then treated with SeO ₂

(130 mg, 1.17 mmol) . The reaction was heated to 75 ⁰ C for 5 h under N ₂ . The reaction was cooled then filtered through celite washing with EtOAc. The filtrate was concentrated on a rotary evaporator then purified by FC eluting with 20% EtOAc/petroleum ether 40-60 °C to afford the aldehyde (168 mg, 55%) as an orange solid. ¹ H NMR (400 MHz, d6-DMS0) δ.2.23(s, 3H, CH ₃ ), 7.18 (d, J=8.0Hz, 2H, ArH), 7.41 (d, J=8.0Hz, 2H, ArH), 7.98 (d, J=8.8Hz, IH,

ArH) , 8.24 (s, IH, ArH) , 8.65 (d, J ^" =8.8Hz, IH, ArH) , 9.22 (s, IH, NH) , 10.31 (S, IH, CHO) .

N- (5,7-Dichloro-2-formylquinolin-8-yl) -4- methoxybenzenesulfonamide

¹ H NMR (400 MHz, d6-DMSO) δ 3.75 (s, 3H, OCH ₃ ), 6.82 (d, J=8.0Hz, 2H, ArH), 7.41 (d, J=8.0Hz, 2H, ArH), 7.97 (d, J = 8.8 Hz, IH, ArH), ), 8.23 (s, IH, ArH), 8.69 (d, J=8.4 Hz, IH, ArH), 9.42 (s, IH, NH), 10.17 (s, IH, CHO).

N- (5,7-Dichloro-2-formylquinolin-8-yl) -4- fluorobenzenesulfonamide

¹ H NMR (400 MHz, d6-DMSO) δ 7.22 (app t, J= 7.2 Hz, 2H), 7.61-7.78 (m, 2H, ArH), 7.99 (d, J ^" =8.8Hz, IH, ArH), 8.31

(s, IH, ArH), 8.75 (d, J ^" =8.8Hz, IH, ArH), 8.96 (s, IH, NH), 9.39 (S, IH, NH), 10.46 (s, IH, CHO)

N- (5,7-Dichloro-2-formylquinolin-8-yl) -4- trifluoromethanesulfonamide

¹ H NMR (400 MHz, dδ-DMSO) δ 7.95 (s, IH, ArH), 8.00 (d, J=8.4Hz, IH, ArH), 8.58 (d, <J=8.4Hz, IH, ArH), 8.96 (s, IH, NH) , 10.07 (S, IH, CHO) .

General procedure for the reductive amination

Method A

N- (5, 7-Dichloro-2-dimethylaminomethyl-quinolin-8-yl) -4- methylbenzenesulfonamide hydrochloride

N- (5, 7-Dichloro-2-formylquinolin-8-yl) -4- methylbenzenesulfonamide (100 mg, 0.253 mmol) in anhydrous CH ₂ Cl ₂ (3 mL) was treated with 2.0M NMe ₂ solution in MeOH (800μL, 1.6 mmol) . After stirring for 10 min, NaBH ₄ (11 mg, 0.29 mmol) was added and the reaction was stirred for 24 h. Note: additional NaBH ₄ (13 mg) was added after 5 h and a further 60mg was added after 20 h. The reaction

was diluted with CH ₂ Cl ₂ and H ₂ O then extracted into CH ₂ Cl ₂ (x3) . The combined organic extracts were washed with brine, dried Na ₂ SO ₄ , filtered, concentrated and purified by FC eluting with 5% MeOH/CH ₂ Cl ₂ to afford (60mg, 56%) . This material was dissolved in MeOH (2 mL) then treated with cone. HCl (3 drops), solvent was removed on a rotary evaporator and the residue was redissolved in MeOH (1 mL) and ether (25 mL) was added to precipitate the dimethylamine hydrochloride 36 (48 mg) as pinkish red solid that was collected by filtration, washed with ether several times then dried at the pump. ¹ H NMR (400 MHz, d6- DMSO) δ 2.30 (s, 3H, CH ₃ ), 2.77 (s, 6H, N(CH ₃ ) ₂ ), 4.34 (s, 2H, CH ₂ N), 7.25 (d, J=8.0 Hz, 2H ₇ ArH), 7.48 (d, J= 8.0Hz, 2H, ArH), 7.70 (d, J ^" =8.8Hz, IH, ArH), 8.09 (s, IH, ArH), 10.19 (s, IH, NH). MS: m/z 424.4 (100) [M+H] ⁺ .

N- (5, 7-Dichloro-2-dimethylaminomethyl-quinolin-8-yl) -4- methoxybenzenesulfonamide hydrochloride and JV- (5,7- Dichloro-2-hydroxymethyl-quinolin-8-yl) -4- methoxybenzenesulfonamide

N- (5 , 7-Dichloro-2-formylquinolin-8-yl) -4- methoxybenzenesulfonamide (217 mg, 0.528 mmol) was dissolved in anhydrous CH ₂ Cl ₂ (5 mL) treated with 2.0 M NMe ₂ in MeOH (700μL, 1.4 mmol) then NaBH ₄ (60 mg, 1.59 mmol) was added and the reaction was stirred overnight at rt . TLC indicated the presence of starting material so an additional portion of 2.0 M NMe ₂ (500. μL, 1.0 mmol) and NaBH ₄ (20 mg, 0.529 mmol) was added to the reaction with stirring continuing for a further Ih. The reaction was quenched with H ₂ O, extracted into CH ₂ Cl ₂ (x3) and the organic layers washed with brine, dried Na ₂ SO ₄ , filtered, concentrated and purified by FC eluting with 50% EtOAc/petroleum ether 40-60 ⁰ C to afford the alcohol compound 41 (60 mg, 27%) as a tan solid. ¹ H NMR (400 MHz, d6-DMSO) δ 3.78 (s, 3H, OCH ₃ ), 4.34 (d, J=6.8Hz, 2H, CH ₂ OH), 5.42 (t, <J=6.8Hz, OH), 6.96 (d, J=8.0Hz, 2H, ArH), 7.56 (d, J=8.0Hz, 2H, ArH), 7.67(d, J=8.8 Hz ₇ IH, ArH),

8.00 (s, IH, ArH), 8.48 (d, J-=8.8Hz, IH, ArH), 9.98 (s, IH, NH). MS: m/z 413.4 (100) [M+H] ⁺ .

Further elution of the column with 5% MeOH/CH ₂ Cl ₂ provided a mixture of the alcohol and desired dimethylamine . This mixture was further purified by FC eluting with 5%

MeOH/CH ₂ Cl ₂ to afford the dimethylamine (52 mg, 22%) . The residue was dissolved in MeOH (2 mL) then treated with concentrated HCl (3 drops) and sonicated. Solvent was removed in vacuo and the residue was redissolved in MeOH (0.5 mL) then ether (2OmL) was added to precipitate the dimethylamine hydrochloride compound 39 as an off-white solid (39 mg) . ¹ H NMR (400 MHz, d6-DMSO) δ 2.81(s, 6H, N(CH ₃ ) ₂ ), 3.78 (s, 3H, OCH ₃ ), 4.41 (s, 2H, CH ₂ N(CH ₃ ) ₂ ), 6.88 (d, J=8.8Hz, 2H, ArH), 7.61 (d, J =Q-QRz ₁ 2H, ArH), 7.73 (d, J ^" =8.4Hz, IH, ArH), 8.11 (s, IH, ArH), 8.27 (d,

J-=8.4Hz, IH, ArH), 10.10 (s, IH, NH). MS: m/z 440.4(100) [M+H] ⁺ .

N- (5, 7-Dichloro-2-dimethylaminomethyl-quinolin-8-yl) -4- fluorobenzenesulfonamide hydrochloride

¹ H NMR (400 MHz, d6-DMSO) δ 2.81 (s, 6H, N(CH ₃ J ₂ ), 4.41 (s, 2H, CH ₂ W(CH ₃ ) ₂ ), 7.34 (t, J=8.8Hz, 2H, ArH), 7.45 (d, J=8.4Hz, IH, ArH), 7.78-7.81 (m, 2H, ArH), 8.12 (s, IH, ArH), 8.32 (d, J ^" =8.4Hz, IH, ArH), 10.44 (s, IH, NH)-MS: m/z 428.4 (100) [M+H] ⁺ .

Method B

N- (5,7-Dichloro-2-dimethylaminomethyl-quinolin-8-yl) -4- trifluoromethylsulfonamide hydrochloride -V- (5, 7-Dichloro-2-formylquinolin-8-yl) -4- trifluoromethanesulfonamide (90mg, 0.241 mmol) was suspended in 1, 2-dichlorethane (6 mL) and treated with a solution of dimethylamine hydrogen chloride (85mg, ) and DIEA (21OmM, 1.21 mmol) in 1,2-DCE (6 mL) . The reaction was cooled to 0°C then sodium triacetoxyborohydride (66 mg, 0.313 mmol) was added and the reaction warmed to rt for 6h. The reaction was diluted with CH ₂ Cl ₂ and H ₂ O and extracted into CH ₂ Cl ₂ (x3) . The combined organic layers

were washed with brine, H ₂ O, saturated aqueous sodium bicarbonate, dried Na ₂ SO ₄ , filtered concentrated and purified by flash chromatography eluting with 5% MeOH/CH ₂ Cl ₂ . Obtained a mixture of starting aldehyde and product. The mixture was re-purified with EtOAc to elute the aldehyde and then 5% MeOH/CH ₂ Cl ₂ to elute the dimethylamine free base as a white powder (17mg) . This material was taken up methanol (2mL) and treated with four drops of concentrated HCl . After removal of the solvent the residue was treated with MeOH (0.5 mL) and ether 20 mL to precipitate the desired methylenedimethyalmine 43 (llmg) as an off-white powder. ¹ H NMR (400 MHz, d6 -DMSO) δ 3.02 (s, 6H, N(CH ₃ ) ₂ ), 4.73 (s, 2H, CH ₂ N (CH ₃ ) ₃ ), 7.75 (d, J=8.4 Hz, IH, ArH), 7.92 (s, IH, ArH), 8.59 (d, J-=8.4 Hz, IH), 9.89 (s, IH, NH). MS: m/z 402.4 (100) [M+H] ⁺ .

Example 2 - Synthesis of quinoline thioureas

R-isothiocyanate, acetone

R= phenyl, 1282 R= propyl, 1283

8-Amxnoquinoline

8-Nitroquinoline (10.0 g, 60.0 mmol) was dissolved in acetic acid (150 mL) to which was added iron powder (22.1 g, 400 mmol) . The mixture was stirred under an argon atmosphere at 65 ⁰ C for 2 h. In this time a thick orange precipitate was evident in the reaction mixture. The reaction was filtered hot through a pad of celite, which was washed with 1:1 mixture of acetic acid and ethyl

acetate. The filtrate was concentrated under reduced pressure to afford a brown gum, which was dissolved in a mixture of ethyl acetate and a saturated aqueous solution of sodium hydrogen carbonate (300 mL of each) . The mixture was filtered thorough a pad of celite to remove the emulsion, the ethyl acetate layer was separated and the aqueous solution extracted with, more ethyl acetate

(150 mL) . The combined organic layers were washed with water (150 mL) , brine solution (150 mL) , and then dried over sodium sulphate. Concentration under reduced pressure afforded a brown oil, that solidified to a grey solid on standing at room temperature.

The solid was washed with hexane (10 mL x 2) to afford the title compound as a cream solid (7.2 g, 83%) . ¹ H NMR (CDCl ₃ ) :δ 5.11 (bs, 2H, NH ₂ ), 6.87 (d, IH, J= 7.6 Hz), 7.07 (d, IH, J= 8.0 Hz), 7.23-7.32 (m, 2H), 8.03 (d, IH, J= 8.4 Hz), 8.68 (dd, IH, J= 1.6, 4.4 Hz).

1-Phenyl-3-qμinolin-8-yl-thiourea

Phenyl isothiocyanate (0.56 g, 4.20 mmol) was added drop wise to a solution of 8-aminoquinoline (0.50 g, 3.50 mmol) in acetone (3.5 mL) at room temperature. The reaction was heated at reflux in an argon atmosphere for 48 h. The reaction was concentrated under reduced pressure and purified using flash chromatography on silica (30 g) eluting with a 40% solution of ethyl acetate in hexane

(500 mL) followed by a 40% solution of hexane in ethyl acetate (250 mL) . The desired product was isolated as pale yellow solid (0.42 g, 43%) .

¹ H NMR (DMSO-d _s ) :δ 7.20 (dd, IH, J= 6.4, 7.2 Hz, Ar-H), 7.42 (dd, IH, J= 7.6, 8.0 Hz, Ar-H), 7.58-7.70 (m, 5H, 2 x Ar-H, 3 X Q-H), 8.42 (dd, IH, J= 1.2, 8.0 Hz, Q-H), 8.87 (d, IH, J= 4.0 Hz, Q-H), 9.22 (d, IH, J= 8.0 Hz, Q-H), 10.60 (bs, IH, NH), 10.74 (bs, IH, NH).

1 -Propyl -3-quxnolin-8-yl -thiourea

Propyl isothiocyanate (0.73 g, 7.20 mmol) was added drop wise to a solution of 8-aminoquinoline (0.80 g, 5.50 mmol) in acetone (5.5 ttiL) at room temperature. The reaction was heated at reflux in an argon atmosphere for 48 h. Additional propyl isothiocyanate (3 mL) , was added after 24 h. The reaction was concentrated under reduced pressure and purified using flash chromatography on silica

(10 g) eluting with a 40% solution of ethyl acetate in hexane (400 mL) . The desired product was isolated as pale yellow viscous oil that slowly crystallised on standing

(1.09 g, 81%) .

¹ H NMR (DMSO-d ₆ ) :δ 0.93 (t, 3H, CH ₃ ), 1.59 (m, 2H, CH ₂ ), 3.45 (q, 2H, CH ₂ ), 7.52-7.63 (m, 3H, Q-H), 8.30 (d, IH, J= 8.0 Hz, Q-H), 8.89 (d, IH, J= 4.0 Hz, Q-H), 8.99 (bs, IH, NH), 9.13 (d, IH, J= 8.0 Hz, Q-H), 10.23 (bs, IH, NH).

Example 3 - Assessment of Compounds of Formula I

The following Assays were used in the assessment of the compounds of formula I for suitability for use in the methods of the invention.

Assay 1. Fluorometric H ₂ O ₂ Assay

A fluorometric assay was used to test the ability of a test compound to inhibit hydrogen peroxide generation by AS in the presence of copper based on dichlorofluoroscein diacetate (DCF; Molecular Probes, Eugene OR) . The DCF solution (5mM) in 100% dimethyl sulphoxide (previously purged with argon for lhr at 20 ⁰ C) was deacetylated in the presence of 0.025M NaOH for 30min and neutralised at pH 7.4 to a final concentration of ImM. Horseradish peroxidase (HRP) stock solution was prepared to lμM at pH

7.4. The reactions were carried out in PBS, pH 7.4 in a 96 well plate (total volume =250μl/well) . The reaction solutions contained AS 1-42 at concentrations in the range

of 5OnM to lμM, copper-glycine chelate (Cu-GIy) , was prepared by adding CuCl ₂ to glycine in the ratio of 1:6 and added to the Aβ in the proportion 2Cu-GIy : IAS ) , reducing agents including dopamine (5μM) or ascorbic acid, deacetylated DCF lOOμM, and HRP, O.lμM. l-10μM EDTA or another chelator may also be present as a control for free copper, but was not required for the assay to function. The reaction mixture was incubated at 37C for 60 min. Catalase (4000 units/ml) and H ₂ O ₂ (1-2.5μM) standards in PBS pH 7.4 may be included as positive controls.

Fluorescence was recorded using a plate reader with excitation and emission filters at 485nM and 53OnM respectively. H ₂ O ₂ concentration may be established by comparing fluorescence with the H ₂ O ₂ standards. Inhibition of Aβ H ₂ O ₂ production was assayed by including a given concentration of test compound (s) in the test wells.

Assay 2. Neurotoxicity Assays

Primary cortical neuronal cultures Cortical cultures were prepared as previously described (White et al., 1998). Embryonic day 14 BL6Jxl29sv mouse cortices were removed, dissected free of meninges and dissociated in 0.025% (wt/vol) trypsin. Dissociated cells were plated in 48 well culture plates at a density of 2 x 10 ⁶ cells/mL in MEM with 25% (vol/vol) FCS and 5% (vol/vol) HS and incubated at 37°C, 2hrs . Media was then replaced with Neurobasal media (Invitrogen Life Technologies) and B27 supplements (Invitrogen Life Technologies) . Cultures were maintained at 37°C in 5% CO ₂ . Prior to experimentation, the culture medium was replaced with Neurobasal media and B27 minus antioxidants (Invitrogen Life Technologies) .

Assay 3. Assays for Cell Viability

(a) MTS Assay for Cell Viability

Cell viability is determined using the MTS assay. Culture medium is replaced with fresh neurobasal medium

plus B27 supplements minus antioxidants. 1/lOth volume MTS solution (Cell Titre 36 Aqueous One, Promega Corporation) and incubated at at 37°C, 2hrs . 200 microlitre aliquots .are measured with a spectrophotometer at 560 nm.

(b) LDB Assay for Cell Viability

Cell death is determined from culture supernatants free of serum and cell debris using the lactate dehydrogenase (LDH) Cytotoxicity Detection Kit (Boehringer Ingelheim) according to the manufacturer's instructions.

(c) Assay for Aβ Neurotoxicity and Aβ Neuroprotection

Neuronal cortical cells were cultured for five days as per Assay 2. On day six the neurobasal (NB) media (Invitrogen Life Technologies) and B27 supplement

(Invitrogen Life Technologies) were replaced with NB media and B27 supplement (no antioxidants) . On day six, test compounds were individually added to the neuronal cell cultures : The test compounds were dissolved in 100% DMSO to a concentration of 2.5 mM (1OmM if excess compound was weighed out per vial - then diluted to 2.5mM) . 2.5mM stock solution was serially diluted 1 in 10 to give working solutions of 25OuM, 25uM, 2.5uM.

Aβ preparation:

Aβ was initially dissolved in 2OmM NaOH to a concentration of ImM and sonicated for 5 minutes . The peptide was then diluted in H ₂ O and 10 X PBS to a final concentration of 20OuM Aβ in IX PBS. The peptide was again sonicated for 5 minutes and then spun at 14000 rpm for 5 min and transferred to a fresh tube.

The test compounds were dissolved in 100% DMSO to a concentration of 2.5 mM (1OmM if excess compound was weighed out per vial - then diluted to 2.5mM) . 2.5mM stock solution was serially diluted 1 in 10 [in NB media and B27 (no antioxidants)] to give working solutions of 25OuM, 25uM, 2.5uM. Test compounds were not added

directly to cells, instead they were added to a 48 well ^λ Drug Plate' as comprised below:

Preparation of "Drug Plate" :

To a 48 well plate add: Well 1: 515 ul NB+B27(no antioxidant)* + 24 ul 25uM test compound + 60ul Aβ diluent**

Well 2 : 515 ul NB+B27(no antioxidant) + 24 ul 25OuM test compound + 60ul Aβ diluent

Well 3 : 515 ul NB+B27 (no antioxidant) + 24 ul test compound diluent*** + 60ul Aβl-42

Well 4 : 515 ul NB+B27 (no antioxidant) + 24 ul 2.5uM test compound + 60ul Aβl-42

Well 5 : 515 ul NB+B27 (no antioxidant) + 24 ul 25uM test compound + 60ul Aβl-42 Well 6 : 515 ul NB+B27(no antioxidant) + 24 ul 250 uM test compound + 60ul Aβl-42 diluent

Well 7 : 515 ul NB+B27 (no antioxidant) + 24 ul test compound diluent + 60ul Aβl-42 diluent

Well 8 : 600 ul NB+B27 (no antioxidant) N. B. 60ul Aβl-42 equals 20ul Aβl-42 per well equals 20 UM Aβl-42

The Drug Plate was incubated at 37° C for 15 mins . 200 ul of each well was added in triplicate to the corresponding cell plate. The cell plate was incubated at

37 C, for 4 days.

* NB media + B27 (no antioxidants) ,

** Aβ diluent 2mM NaOH, 1 X PBS

*** pBT diluent 10% DMSO in NB+B27(no antioxidant)

Completion of the assay:

On the 4 ^th day after treating the cells the assay is completed by adding MTS to the cells.

(d) Assay for Test Compound Cytoxicxty

Neuronal cortical cells were cultured for five days as per Assay 2 in NB media and B27 supplement.

On day six the test compounds were added to the neuronal cell cultures in NB media and B27 supplement minus antioxidants.

Test compounds were dissolved in 100% DMSO to a concentration of 2.5 mM (1OmM if excess compound was weighed out per vial - then diluted to 2.5mM) . 2.5mM stock solution was serially diluted 1 in 10 to give working solutions of 25OuM, 25uM, 2.5uM. Test compounds were not added directly to cells, instead they were added to a 48 well 'Drug Plate' as comprised below:

Preparation of "Drug Plate" : To a 48 well plate add:

Well 1: 576 ul NB+B27 (no antioxidant)* + 24 ul 2.5uM test compound Well 2 : 576 ul NB+B27 (no antioxidant) + 24 ul 25uM test compound

Well 3 : 576 ul NB+B27 (no antioxidant) + 24 ul 25OuM test compound Well 4 : 576 ul NB+B27(no antioxidant) + 24 ul 2.5uM test compound

Well 5 : 576 ul NB+B27 (no antioxidant) + 24 ul 25uM test compound

Well 6 : 576 ul NB+B27(no antioxidant) + 24 ul 25OuM test compound Well 7 : 576 ul NB+B27(no antioxidant) + 24 ul test compound diluent** Well 8 : 600 ul NB+B27 (no antioxidant)

The Drug Plate was incubated at 37°C for 15 mins . 200 ul of each well was added in triplicate to the corresponding cell plate. The cell plate was incubated at

37 ⁰ C, for 4 days, (2 compounds are tested on each plate of cells) .

* NB media and B27 (no antioxidants) , ** PBT diluent 10% DMSO in NB+B27 (no antioxidants)

On completion of the assay, l/lθ volume MTS was added per well of plate (ie 25ul/ 250 ul) . The plates were

incubated at 37°C for 2hrs, and then absorbance was read at 560nm.

Assay 4. Lipid Peroxidation Assay Two different assays of metal-mediated lipid peroxidation can be utilized. The first assay involves measuring the oxidative activity of metallated proteins. This is determined by mixing dialyzed metallated or native protein (at designated concentrations) with 0.5 mg/mL LDL for 24 hr (37°C) . Lipid peroxidation (LPO) is measured using a lipid peroxidation assay kit (LPO 486, Oxis International Inc. Portland, OR) as per kit instructions. The level of LPO is determined by comparing absorbance (486 nm) with LDL alone (100% LPO) . The second assay is used to measure the LPO activity of native proteins in the presence of free, non-protein-bound Cu. This involves adding non-metallated peptides (140 μM) to 0.5 mg/mL LDL together with 20 μM Cu-gly and assaying for LPO as for the metallated proteins . The level of LPO is determined by comparing the absorbance (486 nm) with LDL + Cu-gly (100% LPO) . As a negative control, LDL is also exposed to dialysed Cu-gly solutions comparable to those used to Cu- metallate the proteins.

Assay 5. Metal Partitioning

To assay effects upon the partitioning of various metals, including zinc and copper, following extraction of brain tissue in the presence of a test compound, soluble and insoluble fractions from an extract of human brain tissue are prepared as for the amyloid solubilisation assay. Metals in the two fractions are analysed by inductively-coupled plasma mass spectrometry, following appropriate pretreatment with nitric acid and/or hydrogen peroxide where necessary.

Assay 6. Ionophore

Ml7 human neuroblastoma cells are plated out on 6 well plates and left overnight. Enough cells are added to

give approximately 70 % confluent the following day of the experiment. Test compounds are added to media and mixed with equi-molar amounts of CuCl2 solution. A=IO μM Cu + 10 μM MPAC; B=IO μM Cu + 10 μM MPAC. Cells are incubated in 1 ml of media/MPAC/Cu mix for 5 hours at 37°C. At the end of the incubation the media is removed with a vacuum aspirator and 1 ml of PBS added to dislodge the cells. Cells are then put into Eppendorf tubes and pelleted. The PBS is removed and the remaining cell pellets are frozen at -20 C.

The cell pellets are prepared as follows: Received cell pellets of similar levels in 1.5 ml microfuge tubes. Added 50 μl of concentrated Nitric Acid (Aristar, BDH) to each cell pellet and allowed them to digest over night. Heated the samples for 20 min at 90 ⁰ C to complete the digestion. The volume of each sample was reduced to -45 ul after digestion. Added 1 ml of the 1% Nitric Acid diluent to each sample. (referred to as the "preparation solution" samples) . Measurements were made using a Varian UltraMass ICPMS instrument under operating conditions suitable for routine multi-element analysis.

The instrument was calibrated using Blank, 10, 50 and 100 ppb of a certified multi-element ICPMS standard solution (ICP-MS- CA12-1, Accustandard) for Fe, Cu and Zn in 1% nitric acid. Used an certified internal standard solution containing 100 ppb Yttrium (Y 89) as an internal control (ICP-MS- IS-MIXl-I, Accustandard) .

Assay 7. Effect of Administration of Test Compounds on Aβ deposits in Transgenic Animals

Transgenic mouse models are available for a number of neurological disorders, including Alzheimer's disease (Games et al., 1995; Hsiao et al . , 1996); Parkinson's disease (Masliah et al . , 2000); familial amyotrophic lateral sclerosis (ALS) (Gurney et al . , 1994); Huntington's disease (Reddy et al . , 1998); and Creutzfeld- Jakob disease (CJD) (Telling et al . , 1994) . We have found

that one of the transgenic models for Alzheimer's disease, the APP2576 transgenic mouse (Hsiao et al . , 1996) also has a high incidence of cataract. These animal models are _ suitable for testing the methods of the invention. Transgenic mice of the strain APP2576 (Hsiao et al 1996) are used. Eight to nine month old female mice are selected and divided into groups for treatment .

Mice are sacrificed at intervals, and their brains examined to determine whether the treatment with test compounds decreased brain amyloid formation, and the identification of the most effective administration protocol. The levels of soluble and insoluble Aβ in the brain and serum are determined using standard calibrated Western blots .

Assay 8. Cognition Assay- Mice are tested over a period of up to eight months for cognitive performance, using a Morris water maze according to standard methods. The general health and well-being of the animals is also measured every day by a blinded operator, using a five point integer scale which subjectively rates a combination of features, including motor activity, alertness and general health signs.

Assay 9. Physiochemical Properties Polar Surface Area Calculations (PSA)

Polar surface area values were calculated using the web-based program available through "Molinspiration" , a package for calculation of molecular properties.

Turbidimetric Solubility Measurements

The solubility estimate was measured at both pH 2.0 and pH 6.5. This is within the pH range that can be anticipated along the proximal gastrointestinal tract in humans .

The compounds are dissolved in DMSO to appropriate concentrations and then spiked into either 0.01M HCl

(approx. pH = 2.0) or pH 6.5 isotonic phosphate buffer, the final DMSO concentration being 1%. Samples are then analysed via Nephelometry to determine a solubility range . _.. [as per D. Bevan and R. S. Lloyd, Anal. Chem. 2000, 72, 1781-1787] .

cLog P values

Theoretical Log P values are determined using the ACD Log P software . The values quoted have been calculated from an untrained database and refer to the unionised species .

Assay 10. Metal-mediated Aβ oligomerisation - ^λ CuTy' assay

The assay (CuTy) is a Western blot assay which evaluates the ability of a test compound to inhibit the metal-mediated cross-linking reaction which leads to the formation of dimeric and higher order oligomers of Aβ . The assay models the process by which a putative agent acts either to compete with Aβ for redox active metals or alternatively to displace such metals by binding competitively at the Aβ metal binding site.

Protocol for Oligomeriszation Materials'. Aβ 1-40/1-42 (from Keck Laboratory) ; HFIP (from Sigma- Aldrich, Cat#105228) ; CuCl ₂ (from BDH laboratory supplies, Cat# 100884E; Gylcine (from Ajax Finechem, Cat# A1083; L- ASCORBIC ACID (Sigma Cat# A-0278; PBS (from Sigma, Cat# D8662) ; DMSO (from Ajax Finechem Cat# A2225; NaOH (from Sigma Cat#S-5851; 2-Mercaptoethanol (Sigma, Cat# M-7154; Tris (from BDH Cat# 103157P)

Tricine (SigmaCat#T-5816) ; SDS (from Bio-Rad Labororatorias Cat#161-0302) ; ECL (from Amersham, Cat# RPN2106V1) ; Rabbit anti mouse-Immunoglobulin HRP (from DAKO Cat# P0260) ;

10-20% Tricine Gel 10 wells (from Invitrogen Cat# EC6625

Box)

Stock Solutions: Cu (II) (ImM) -GyIcine (6mM) solution in PBS; L- ASCORBIC ACID (5 mM) solution in PBS; NaOH (0.02M) solution in MQ H ₂ O; TBST: Tris buffer saline 0.1 % Tween 20.

Method for metal-mediated generation of Aβ oligomers

Treat Abeta 1-40/1-42 in 100ug/l00ul HFIP, air dry, and store at -80C. Dissolve the HFIP treated Abeta in 0.02 M NaOH 100μg/20μl, vortex for about 10 seconds. Add PBS (sigma D8662) 100ug/80ul, vortex 10 seconds again. Sonicate in water bath, (add ice from time to time to keep water cool) for 15-20 min. Centrifuge for 20 min, collect the supernatant and determine the concentration by measuring the absorbance at 214nτn {Concentration (M) =absorbance reading *dilution factor/55771 (for 1-40)}. Compounds to be assayed are dissolved in 100% DMSO and made up to a 5 mM stock solution. Serial dilutions of 4000 μM, 2000 μM, 1000 μM, 500 μM, 100 μM are prepared. Set up reaction in 2 ml microcentrifuge tube with the following components: Abeta 10 μM; CuCl ₂ 25 μM; ASC250 μM; DMSO or test compounds 1%.

PBS to make up 700 μl . Incubate the tube at 37 ⁰ C rotating for one hour. Stop the reaction by adding EDTA to 250 μM-final concentration.

Analysis by Western Blot

Run samples on 10-20 % gradient precast Tricine gel (4-12%Tris Glycine for A-Il antibody) , (Novex) . Running Buffer-Tris-Tricine (0.1 M Tris pH 8.25, 0.1 M Tricine,

0.1% SDS) .Sample Buffer-4x (red); SDS 16%; Glycerol 60%;

Tris 200 mM; Tricine 20OmM; Phenol Red 0.02%; 10% β- mercaptoethanol ; Mix 30 ul of sample and 10 ul of 4x SB and boil for 5 min. ; Load samples onto gel and run; Transfer onto nitrocellulose membrane; Transfer Buffer-25 mM Tris, 192 mM glycine, 20% methanol; After transfer, Boil membrane for 5mins then block in 10% skim milk in TBST (0.01% Tween 20) for 1 hour at RT; Probe with primary antibodies* in 3%BSA/TBST (0.01% Tween20) at 4°C O/N or 1 hour at RT; Wash three times 15 min with TBST; Probe with secondary antibody- Goat anti- Rabbit IgG HRP (DAKO) 1 hour at RT; Wash three time 15 min in TBST; Develop blot using chemiluminescence reagents.

* the primary antibody for detecting dityrosine is the antidityrosine monoclonal antibody 1C3 [Kato, Y. et al (2000), Biochem. Biophys. Res. Coimun. 275, 11-5.] Oligomerisation can also be detected using generic antibodies which recognise full length Abeta and specific antibodies which are claimed only to detect soluble oligomeric forms of the peptide. (Lesne, S. et al (2006) Nature VoI 44θ]l6 March 2006 | doi : 10.1038/Nature04533) .

Assay 11. Transgenic Mouse Brain Ixnmunohistochemistry The APP2576 transgenic mouse (Hsiao et al . , 1996) as referred to in Assay 7 is utilized in this assay. The contralateral formalin-fixed mouse brain tissue is coronally cut. Sections (10 μm) are taken from the corresponding sites and treated with 80% formic acid for antigen retrieval. The primary antibody used is monoclonal antibody 1E8 , which recognizes epitopes between residues 18 and 22 of Aβ (SmithKline Beecham, UK) . Immunoreactivity is developed with secondary antibody linked to horseradish peroxidase (using a 3,39- diaminobenzidinechromagen) (Dako) and alkaline phosphatase

(using 5-bromo-4-chloro 3-indoxyl phosphate and nitroblue tetrazolium chloride chromagen) (Dako) . Plaque abundance per section is assessed by two operators blinded to ._ _. _. treatment according to the following scale: 0 = no plaques apparent

1 = plaques present but very sparse

2 = several plaques present

3 = numerous plaques visible in restricted areas

4 = plaques abundant and not restricted to any particular area.

Intermediate values eg 2.5 are assigned where applicable. Students 't' test is used for comparisons between groups.

Assay 12. Pharmacokinetic Profile The pharmacokinetic profile of test compounds is determined by the following assay:

• Intravenous infusion of test compound; 2 mg/Kg in a suitable vehicle is administered to 2 rats and arterial blood is sampled up to 24 hours.

• Oral administration of test compound; 30 mg/Kg in a suitable vehicle is administered via oral gavage to 2 rats and arterial blood is sampled up to 24 hours.

• Plasma concentrations of test compound are determined by suitable analytical method.

Calculations:

^LLtotal AUC ₁ , ^iβ β AUC _!V *Dose _ϋml

CL _t o _ta i = total plasma clearance after IV administration V _d p = volume of distribution during the elimination phase after IV administration BA =oral bioavailability AUCiv = area under the plasma concentration versus time profile from time zero to infinity after IV administration

AUCorai = area under the plasma concentration versus time profile from time zero to infinity after oral administration

^~~~ β = terminal elimination rate constant after IV

5 administration

Assay 13. Determination of mouse plasma levels of test compounds

Oral administration of a test compound at 30mg/kg, as 10 a suspension in Na-Carboxymethyl Cellulose (CMC) is administered by oral gavage to four mice. Mice are sacrificed at intervals after administration in groups of 2. Blood is obtained by cardiac puncture and plasma separated by centrifugation.

15 The concentration of a test compound is determined by LC/MS using the triple quadrupole instrument. The mobile phase consisted of an acetonitrile (ACN) /water gradient (containing 0.05% Formic acid) and the column is a Phenomenex Lunea 5μm C8 (50 x 2mm) column.

20 The supplied acute toxicity mouse plasma samples are directly injected following a protein precipitation with ACN. The analytical method in plasma is linear in the range of 10 to 10,000 ng/ml (R ² = 0.994) . Recovery of a test compound from plasma is ~ 100%. 25

Assay 14. Testing cancer agents for in vitro and in vivo efficacy

Emulsion carrier is used as a control for the in vitro and in vivo test compounds. All the test compounds

30 are tested initially via in vitro testing to determine an efficacy profile with three glioma cell lines and a control cell line .

Jn vitro efficacy protocol

The in vitro efficacy of the test compounds are analyzed via the MTT cell viability assay.

The following cell lines at least are used to determine cell viability on exposure to the test compounds : C6 - rat glioma cell line, VMDK - mouse glioma cell line, U87MG - human glioma cell line, 3T3 - Control cell line. Cells are plated in 96 well plates with 100 μl of cell culture medium and be allowed to adhere over 24 hours allowing for approximately 50 % confluence. At 24 hours, the cell medium is replaced with fresh cell culture medium containing test compounds or the carrier emulsions. The cells are then incubated and grown for a designated period (72 hours) after which the MTT solution are added to the wells and incubated at 37°C for 1-2 hours. The absorbance of each well are then be measured with a plate reader at 570 nm. The efficacy profiles are calculated relevant to the cells incubated in the absence of the test compounds over the course of the experiment .

In vivo efficacy protocol

2 doses are used: at the maximum tolerated dose and one level below the maximum tolerated dose.

3 mouse models are employed:

C6 - CBA Xenograft model (ATCC Number: CCL-107) SMA560 - VMDK mouse model (ATCC Number: CCL-163) U87MG Nude mouse model (ATCC Number: CRL-9589) Initially, CBA mice are used to receive an intracranial inoculation of the Cβ glioma cells. Briefly, IxIO ⁵ cells are inoculated into the left hemisphere via at day 5 post CS cell inoculation. The mice receive daily intraperitoneal (ip) administration of test compounds in a

carrier emulsion or carrier emulsion alone as a control for 8 days until day 12. At day 14, the mice are euthanised via CO ₂ inhalation and the brain rejnpved _ fox. histological processing.

The VMDK mouse strain is then used to screen the identical test compounds and carrier emulsions as per the C6 xenograft model in the CBA mice. The VMDK mice received an inoculation of IxIO ⁵ SMA560 cells into the left hemisphere via standard methods. At day 5 post SMA560 cell inoculation, the mice receive daily ip administration of test compounds in a carrier emulsion or carrier emulsion alone as a control for 12 days until day 16. Identical doses of test compounds and carrier emulsions as used in the C6 xenograft model are used with the SMA560 model. At day 18, the mice are euthanized via CO ₂ inhalation and the brain removed for histological processing.

A nude mouse model utilizing the U87MG human glioma cell line is used to screen compounds . The nude mouse Nu/nu strain receives an inoculation of IxIO ⁵ U87MG cells into the left hemisphere. At day 5 post U87MG cell inoculation, the mice receive daily ip administration of the test compound or carrier emulsion alone as a control for 12 days until day 16. At day 18, the mice are euthanized via CO ₂ inhalation and the brain removed for histological processing.

Haematoxylin and eosin stained sections are used to measure tumour dimensions in order to determine the efficacy of the test compounds on tumour growth relative to the control mice.

Assay 15. Huntington' s Disease (HD) - in vitro protocol

PC12 or HEK cells are transfected with 'long' and "short' CAG repeat constructs , to determine effects of a

panel of test compounds on production and aggregation of htt protein, by microscopy and to determine cell survival/apoptotic indicators. PC12 cells are cultured and transfected. Cells cultured in Optimem media in 24-well plates are pretreated 30 minutes prior to transfection with each of the test compounds in DMSO, initially at concentrations of 0.05, 0.1, 0.5, 1, 2, 5 and 10 μM, or vehicle alone and transfected, using Lipofectamine 2000, with Q103HDexonl/GFP, Q25HDexonI or GFP alone vectors. Cells are then digitally imaged (Nikon inverted fluorescence microscope with Magnafire digital imager) under phase contrast and for GFP fluorescence at -20 and 40 hours post transfection. At 48 hours they are stained with propidium iodide (PI) and Hoechst 33342 and imaged again with filters appropriate for those dyes . The percentage of cells exhibiting detectable GFP fluorescence or PI staining is determined by semi-automated counting (using NIH image) of the same field under fluorescent and phase imaging. Apoptotic nuclear fragmentation of PI negative cells is assessed by manual visual counting using overlapped Pl/Hoechst images. This, plus PI positive cells are considered to represent the total number of dead cells. At least 3 fields are counted per condition, and the experiment repeated at least 4 times after the initial concentration screen. Should a compound prove toxic or ineffective but not toxic in the initial concentration range, the range is extended accordingly.

Test compounds showing demonstrable positive results in the above are tested for effects on: RNA and protein expression, by standard art methods of Northern and Western blotting; proteosomal degradation/inhibitor studies; polyQ construct degradation/pulse-chase experiments .

Statistical significance for between group comparisons are then determined by AMOVA, with differences between individual values determined by post hoc testing,-., with Dunnet's test. Any compound and concentration that produces a substantial (>25%) drop in percentages of Q103 expressing cells, with at most a 3-fold lesser drop in Q25 and GFP-only expression, are considered for further evaluation, including analysis of mRNA expression by Northern blotting or reverse transcription quantitative PCR, Western blotting and densitometric quantitation with anti-polyQ and antiGFP, proteasome inhibitor treatment and pulse-chase analysis.

Assay 16. Huntington' s Disease - in vivo protocol R6/2 mouse models

Breeding: 3 to 5 months to first experimental cohorts, then ongoing. Strategy: Breeding pairs of R6/2 mice are obtained. Hemizygous ovary transplanted wild-type females crossed with wild type males are used to generate Fl. Approximately one-half of the Fl males are expected to be fertile; these are bred with wildtype females to generate litter cohorts for experiments. Selective trialing (PCR of sampled mice from each litter) of mice to ensure relatively low degree of genotypic variation. Animals are bred and genotyped with the addition of a strategy to minimize the tendency of CAG repeat length to lengthen, especially when transmitted by males, as suggested by Hockly, et al . (Hockly E, Woodman B, Mahal A, Lewis CM, Bates G. Standardization and statistical approaches to therapeutic trials in the R6/2 mouse. Brain Res Bull 61: 469-79 (2003)). This involves sequencing the genotyping PCR products to determine CAG repeat length prior to breeding and only breeding animals at the lower end of the length distribution. Comparing weight data by

sex and adjusting for weight at weaning, ensuring a standardized environment, randomizing based on litter origin, and housing animals with the equal numbers of wild type and transgenics in each cage . Conduct study 5-7 months: 20 mice per arm: 0, 15 mg per kg dosing of test compound, with compounds adminsitered by daily gavage. Dosing will depend on pharmacokinetic and toxicity data available for the MPAC chosen. Assess : Rotorod performance once per week; Clasping behavior once per week; Effects on accumulation of brain htt protein. Ventricular size and; Life-span.

Rotarod motor performance testing: Beginning at 5 weeks of age, animals are tested by an experimenter blinded to genotype on a rotarod (Rotamex 4/8, Columbus instruments) . For training and establishment of baselines, animals are placed on the rod at 24 rpm and the latency to falling (to a maximum test length of 60 seconds) measured, in 4 sessions on each of 4 consecutive days. Subsequent testing is performed at intervals at rotor speeds of 15 and 44 rpm (2 trials at each speed) and continue until animals are unable to maintain balance for more than a few seconds .

For testing clasping, mice are suspended by the tail for 30 s and the time of tonic spasm of the lower extremities (foot-clasping) is scored such that a 0-5 s clasping duration is given a score of 1, 5-10 s a score of 2, and greater than 10 s a score of 3.

Effects on accumulation of brain htt protein: All antibodies necessary for these studies are commercially available. Immunohistochemistry are performed on paraformaldehyde fixed tissues, using standard techniques and visualization with HRP/diaminobenzidine or fluorescent secondary. Western blotting is performed by standard

methods, with chemifluorescent detection, and stripped and reprobing for controls such as GAPDH. Bands are quantitated using a Typhoon Multimodal imager. Band intensities on blots reprobed for control proteins such as actin can then be normalized and compared.

Ventricular size: ImageJ 1.32j (National Institutes of Health, USA) is used to measure the size of the lateral ventricles in standardized Nissl stained brain sections.

Assay 17. Parkinsons Disease (PD) 6-OHDA Assay

The 6-OHDA assay investigates if a test compound is effective at reducing 6-hydroxydopamine (6-OHDA) induced lesions and whether there is a common pathway for propagation of neuronal cell death in a mouse model of PD. Mice are anaethetised and secured in a stereotaxic frame . A single injection of 6-OHDA (2.5 μg, 1.5 μg/μl) is injected slowly (0.8 μl/min) into the right SN (AP 3 mm, L 1.1 mm, DV 4.7mm with respect to bregma) this produces lesions of between 60-70% (Parish et al . , 2001) .The test compound is delivered by oral gavage at a daily dosage of 5 or 30 mg/ml from the day of lesion for 14 consecutive days .

Rotational Behavioural monitoring: The lesioned mice are placed in bowls and videotaped for one hour, then injected with 5 mg/kg amphetamine by intraperitoneal injection and videotaped for another hour. The animals rotate towards the lesioned side. The number of rotations is proportional to the loss of cells from the SN. Barnham et al . , (2004) Faseb J. 18 (12) 1427-1429; Kaur et al., (2003) Neuron 37(6) 899-909; Parish et al . , (2001) J. Neurosci. 21(14) 5147-5157.

MPTP Assay

MPTP (1-methyl 4-phenyl 1, 2, 3, 6-tetrahydropyridine) is a chemical that is related to the opioid analgesic drugs. MPTP itself does not have any opioid effect, but it may be produced accidentally during illicit manufacture of MPPP and MPTP causes Parkinsonian side-effects. This happens when MPTP is metabolized into MPP+, which kills neurons in a part of the brain called the substantia nigra. MPP+ interferes with mitochondria metabolism which leads to cell death and causes the buildup of free radicals, toxic molecules that contribute further to cell destruction. MPTP has quite selective abilities to effect neuronal death in dopaminergic cells, apparently through a high- affinity uptake process in nerve terminals normally used to reuptake dopamine after it has been released into the synaptic cleft. Such effects lead to gross depletion of dopaminergic neurons which has severe implications on cortical control of complex movements .

Experiment Ia Does MPTP cause continued cell death

Control (non tg) and transgenic mice receive five intraperitoneal injections of MPTP-HCl (23 gauge needle,

20 mg\kg of free base; Sigma, St. Louis, MO, USA) dissolved in sterile 0.9% saline at 2-h interval in 1 day.

Control animals receive five intraperitoneal injections of

0.9% saline. This results in a « 60% lesion of the SN. 20 C57BL6, 10 control and 10 hA53T tg are be used to establish the precise dose of toxin at 2 weeks.

Animals are allowed to recover for 1 week, 2 weeks, 1 month, 12, months and 18 months. The animals are then

killed and the brains removed for histological

(stereological) and biochemical analysis.

Experiment 2 Do test compounds or analogues reduce MPTP induced cell death.

Animals are treated as described above. They are treated with the test compound by oral gavage at a daily dosage of 30 mg/kg 2 days after MPTP injection until death. Test compound and analogues are be given by oral gavage at a daily dosage of 30 mg/kg. The time of killing the animals depends on the analysis of Experiment 1. However, it is one month or less.

Control (non tg) mice receive five intraperitoneal injections of MPTP-HCl (23 gauge needle, 20 mg/kg of free base; Sigma, St. Louis, MO, USA) dissolved in sterile 0.9% saline at 2-h interval in 1 day. Control animals receive five intraperitoneal injections of 0.9% saline.

Behavioral Monitoring; Mice are assessed at prior to killing the mice for histological analysis.

Rotarod. Motor coordination and strength are assessed using the rotarod. The rotarod consists of a plastic rotating rod of 3.6 cm axial diameter partitioned by metal disks into five sections to allow the testing of multiple mice simultaneously. Mice are trained on two sessions where the rotation speed is ramped from 0-30 rpm over 5 min and one training session where rotation is a constant 16 rpm for 5min. Within two days of training, animals are formally assessed on the rotarod rotating at 16 rpm for a maximum of 3 min: the time to fall on this single test is the recorded data point .

Pole test. This consists of a 700 mm long, 5 mm diameter, wooden rod. The rod is supported at its base and held vertical. The total walking distance for the mice is 550., mm. The time taken for the mouse to descend the pole is measured with a maximum time of 120 s. If a mouse falls, the time is scored as 120 s.

Fe H ₂ O ₂ inhibition assay

This fluoresence assay evaluates the ability of a test compound to inhibit the generation of hydrogen peroxide

(H ₂ O ₂ ) by iron in the presence of a reducing substrate such as ascorbic acid. In the assay, iron in the form of FeCl ₃ is allowed to react with ascorbic acid by incubating for lhr at 37°C in the presence of the fluorescing compound DCF and horseradish peroxidase. H ₂ O ₂ generated by the system is assessed by measuring the specific fluorescence profile at the excitation and emission wavelengths of 485 and 530nm respectively, in the presence of increasing concentrations of test compound. Test compounds are ranked according to their capacity to inhibit H2O2 generated by the system where lower values in Mean Fluorescence Units (mfu) reflect greater ability to inhibit H2O2 production.

Assay 18. Blood Brain Barrier Penetration

Each compound tested demonstrates a permeability across a healthy BBB.

A bolus injection of each of the test compound (50 μL of a 3 mg/mL aqueous solution containing 40% propylene glycol and 10% ethanol) was administered by tail vein injection to male Swiss Outbred mice (5-7 weeks of age) . At 5 and 60 min post-dose (n=3 mice at each time point) , blood was collected by cardiac puncture and the whole brain was removed by making an incision through the back

of the skull. Mice were anaesthetised approximately 3-4 min prior to blood and brain harvest with an intraperitoneal injection of ketamine and xylazine (133 nag/kg and 10 τng/kg, respectively) . The whole brain was placed into preweighed polypropylene vials and stored at -20 ⁰ C until analysis. On the day of analysis, the whole brain was homogenised in 3 parts of water (on ice to reduce the potential for ex vivo brain degradation) and an aliquot of the brain homogenate and plasma was analysed for compound concentration by LCMS. Standards were prepared by spiking blank brain homogenate and both samples and standards were processed by adding acetonitrile to the tissue homogenate, centrifuging and injecting an aliquot of the supernatant onto the LCMS. To ensure complete recovery of compound from the brain, brain homogenate was spiked with compound (in 50% acetonitrile: 50% water) to a nominal concentration of 500 ng/mL. The concentration of compound in the supernatant was then determined by LCMS and compared to the supernatant concentration when compound was added following precipitation with acetonitrile.

Calculations

'-'brain ⁼ ^ brain homogenate " ¹ ^ bran vasculature '-"brain vasculature ⁼ ^plasma * ^p

B p ₌ ^brain ^plasma

P _app (c^s) ₌ ⁰ ^

JfjCplasma * ^* A

C _bra i _n = concentration of compound in brain parenchyma (ng/g)

Cbrai _{n h} o _m ogenate = concentration of compound in brain homogenate (ng/g)

Cbrain vasculature = concentration of compound in__ brain vasculature (ng/g) Cpias _r aa = concentration of compound in plasma (ng/mL)

Vp = brain plasma volume (26 μL/g for male Swiss Outbred mice)

B: P = brain-to-plasma ratio p _app = apparent permeability coefficient of compound permeating the blood-brain barrier

1 Cp| _asma dt = concentration of compound in plasma from time zero to 5 min post-dose (equivalent to the 5 min post-dose plasma concentration, assuming no back diffusion from brain to plasma within this time period) A = surface area of capillaries forming the blood-brain barrier (240 cm ² /g brain weight for mouse) .

Table 1

References cited in the description and examples are listed on the following pages, and are incorporated herein by this reference. - ^~

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It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.