Metal delivery agents and therapeutic uses of the same   

Abstract: The present invention relates to metal complexes, processes for their preparation and their use as pharmaceutical or veterinary agents, in particular for the treatment of conditions in which metal delivery can prevent, alleviate or ameliorate the condition. There are a number of clinical conditions which are caused by or associated with abnormal levels of metals (typically low metal levels). Conditions in of this type include cancer and conditions characterised by or associated with oxidative damage, more specifically neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease or Huntington's disease. The invention also relates to ligands useful in the preparation of metal complexes of this type.

FIELD OF THE INVENTION

The present invention relates to the use of metal complexes as pharmaceutical or veterinary agents, in particular for the treatment of conditions in which metal delivery can prevent, alleviate or ameliorate the condition. There are a number of clinical conditions which are caused by or associated with abnormal levels of metals (typically low metal levels). Conditions in of this type include cancer and conditions characterised by or associated with oxidative damage, more specifically neurodegenerative conditions such as Alzheimer\'s disease, Parkinson\'s disease or Huntington\'s disease.

BACKGROUND OF THE INVENTION

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 metalloproteins (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. In addition even if there are adequate levels of bio-available metal in a biological system it is important that its distribution in the biological system be such that the biological processes that rely on the presence of the metal function appropriately.

Whilst there is a wide range of ways in which bio-available metals impact on biological systems, two of the better known would be 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 R-amyloid plaques of Alzheimer\'s disease; 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 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, BRCA1 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 either too high a level or too low a level the normal biological 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 abnormal levels of metal in the biological environment. In respect of these conditions they are all typically ones in which metal delivery can prevent, alleviate or ameliorate the condition. An example of a condition of this type is oxidative stress which is related to abnormal metal levels as many of the protective enzymes responsible for alleviating oxidative stress are deactivated if biological metal levels are too low.

Research in the last few decades has identified that there are a number of conditions that are caused by or associated with oxidative stress placed on the body. For example a number of cardiovascular conditions have been identified that are the result of oxidative stress (OS). Other conditions associated with OS include cancer, cataracts, neurodegenerative disorders such as Alzheimer\'s disease and heart diseases. In addition, there\'s 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 Alzheimer\'s disease (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 April; 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.

A number of therapeutic agents have been developed in an attempt to provide therapeutic solutions to the conditions caused by or associated with OS as discussed above with varied results. In general, in order to lower OS levels, various antioxidants are being used. The most common are vitamin E and vitamin C. However, vitamin E was found to be ineffective at decreasing the oxidative stress at the substantia nigra (The Parkinson Study Group, 1993, Offen et al., 1996) since this compound, although capable of crossing the blood brain barrier, is trapped in the cell membrane and therefore does not reach the cytoplasm where its antioxidant properties are needed. Vitamin C also does not cross the blood brain barrier and therefore, cannot be used effectively for neurodegenerative diseases of central origin.

There is thus still a need for, and it would be highly advantageous to have novel antioxidant compounds and methods for use of antioxidants in treatment of disease associated with oxidative damage and particularly central nervous system neurodegenerative disorders such as PD, AD and CJD. Treatment is further desirable for and in treating conditions of peripheral tissues, such as acute respiratory distress syndrome, ALS, atherosclerotic cardiovascular disease and multiple organ dysfunction. During such treatment the complexes can act as oxygen scavengers to lower the OS within and in the vicinity of affected cells and this treatment eventually stops cell death which is associated with OS in the brain and/or peripheral tissues.

The present invention is therefore based on the finding that certain metal complexes are effective in delivering bio-available metal and could thus be used in the treatment of conditions which can be prevented, treated or ameliorated by metal delivery. In certain conditions it is desirable that the metal be released in the cell such that after metal delivery the metal is present in the form of the free cation and it is the free cation that leads to the observed biological activity. In respect of other conditions it is desirable that the metal stay in the form of the bound complex even after metal delivery and with these conditions it is the bound form of the metal (the metal complex) that is biologically active in the cell.

In particular these complexes were found to be effective in delivering metal to the cells in a form which lead to a significant anti-oxidant effect being observed in the cell. Thus, certain metal complexes demonstrated an ability to mediate OS.

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.

SUMMARY

In one aspect the invention provides a method of treatment or prophylaxis of a condition in a subject in which metal delivery can prevent, alleviate or ameliorate the condition, the method including administration of a therapeutically effective amount of a metal complex of Formula (I).

wherein M is a divalent metal;

R1 and R2 are each independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkoxy, —N═R7, —NH(R7), —N(R7)2, —COOH, —COR7, —COOR7, —CONHR7, —CSNHR7, —S(O)R7, —S(O)2R7, —C(O)N(R7)2, —SO2N(R7)2, —(CH2)mR8 and acyl, each of which may be optionally substituted; or

R1 and R2 when taken together to the nitrogen atom to which they are attached form an optionally substituted heterocycloalkyl or heteroaryl group;

R3 and R4 are each independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, each of which may be optionally substituted;

or R3 and R4 when taken together with the carbon atoms to which they are attached form an optionally substituted cycloalkyl group;

R5 and R6 are each independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkoxy, —N═R7, —NH(R7), —N(R7)2, —COOH, —COR7, —COOR7, —CONHR7, —CSNHR7, —S(O)R7, —S(O)2R7, —C(O)N(R7)2, —SO2N(R7)2, —(CH2)mR8 and acyl, each of which may be optionally substituted; or

R5 and R6 when taken together to the nitrogen atom to which they are attached form an optionally substituted heterocycloalkyl or heteroaryl group;

each R7 is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, and acyl, each of which may be optionally substituted;

each R8 is independently selected from the group consisting of cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, each of which may be optionally substituted;

m is an integer selected from the group consisting of 1, 2, 3, 4, 5 and 6.

In one embodiment the condition is selected from the group consisting of tau related disorders, disorders caused by or associated with oxidative stress in a subject, and Abeta related disorders. In one specific embodiment the condition is caused by or associated with oxidative stress in the subject. In another specific embodiment the condition is a tau related disorder, particularly a tau related neurodegenerative disorder. In another aspect the condition is an Abeta related disorder.

In a further aspect the invention provides the use of a metal complex of formula (I) in the preparation of a medicament for the treatment or prophylaxis of a condition in which metal delivery can prevent, alleviate or ameliorate the condition. In one embodiment the condition is selected from the group consisting of tau related disorders, disorders caused by or associated with oxidative stress in a subject, and Abeta related disorders. In one specific embodiment the condition is caused by or associated with oxidative stress in the subject. In another specific embodiment the condition is a tau related disorder, particularly a tau related neurodegenerative disorder. In another aspect the condition is an Abeta related disorder.

In one form of each of these two aspects the condition is selected from the group consisting of cardiovascular disease, central nervous system disorders, cancers and neurological disorders.

Examples of conditions of this type include conditions selected from the group consisting of acute intermittent porphyria; adriamycin-induced cardiomyopathy; AIDS dementia and HIV-1 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; diabetic neuropathy; Down\'s syndrome; near drowning; epilepsy and post-traumatic epilepsy; Friedrich\'s ataxia; frontotemporal dementia; glaucoma; glomerulopathy; haemochromatosis; haemodialysis; haemolysis; haemolytic uraemic syndrome (Weil\'s disease); Menke\'s disease; haemorrhagic stroke; Hallerboden-Spatz disease; heart attack and reperfusion injury; Huntington\'s disease; Lewy body disease; intermittent claudication; ischaemic stroke; inflammatory bowel disease; macular degeneration; malaria; methanol-induced toxicity; meningitis (aseptic and tuberculous); motor neuron disease; multiple sclerosis; multiple system atrophy; myocardial ischaemia; neoplasia; Parkinson\'s disease; peri-natal asphyxia; Pick\'s disease; progressive supra-nuclear palsy; radiotherapy-induced organ damage; restenosis after angioplasty; retinopathy; senile dementia; schizophrenia; sepsis; septic shock; spongiform encephalopathies; subharrachnoid haemorrage/cerebral vasospasm; subdural haematoma; surgical trauma, including neurosurgery; thalassemia; transient ischaemic attack (TIA); transplantation; vascular dementia; viral meningitis; and viral encephalitis.

In one specific embodiment the condition is a neurological disorder. In one form of this embodiment the neurological disorder is selected from the group consisting of Parkinson\'s disease, Alzheimer\'s disease, Multiple sclerosis, Neuropathies, Huntington\'s disease, Prion disease, motor neurone disease, Amyotrophic lateral sclerosis (ALS) and Menke\'s disease. In a specific embodiment the disorder is Alzheimer\'s disease. In a specific embodiment the disorder is Parkinson\'s disease. In a specific embodiment the disorder is Amyotrophic lateral sclerosis (ALS).

In a further aspect the invention provides a method of prophylaxis or treatment of oxidative stress including administering a therapeutically effective amount of a metal complex of formula (I) to the subject.

In a further aspect the invention provides the use of a metal complex of formula (I) in the preparation of a medicament for the treatment or prophylaxis of OS.

In an even further aspect the invention provides a method of protecting a cell from OS the method including exposing the cell to an effective amount of a metal complex of formula (I). In one embodiment the cell is a cell in a subject and exposing the cell to the metal complex includes administering the metal complex to the subject.

In an even further aspect the invention provides a method of prophylaxis or treatment of a tau related disorder the method including administering a therapeutically effective amount of a metal complex of formula (I) to the subject. In one embodiment the tau related disorder is a neurodegenerative disorder.

In a further aspect the invention provides the use of a metal complex of formula (I) in the preparation of a medicament for the treatment or prophylaxis of a tau related disorder.

In yet an even further aspect the invention provides a method of reducing or preventing the effects of Abeta on a cell the method including exposing the cell to an effective amount of a metal complex of the formula (I). In one embodiment the cell is a cell in a subject and exposing the cell to the metal complex includes administering the metal complex to the subject.

In yet an even further aspect the invention provides a method of prophylaxis or treatment of an Abeta related disorder the method including administering a therapeutically effective amount of a metal complex of formula (I) to the subject.

In yet a further aspect the present invention provides a method of phosphorylation of a kinase in a cell, the method including exposing the cell to a metal complex of Formula (I) as described above. In one embodiment the kinase is a receptor tyrosine kinase. In a specific embodiment the receptor tyrosine kinase is epidermal growth factor receptor (EGFR). In another specific embodiment the kinase is selected from the group consisting of ERK, PI3K, Akt, GSK3 and JNK.

A common feature of the methods and uses as outlined above is the use of a metal complex of formula (I). In one embodiment of the aspects described above the metal complex is sufficiently stable that upon administration to the subject the metal is not released in the extracellular environment but rather is released in the cells of the subject. This is preferable as it ensures that the metal is delivered to the cells of the subject rather than being released prior to delivery to the cells. In embodiments where the metal is released from the complex in the cell it is therefore present in the cell as the free cation and it is the free cation that is responsible for the biological activity in the subject. In another embodiment the metal complex does not release the metal in the extracellular matrix nor does it release the metal in the cell rather it is the metal complex that leads to the observed biological activity. Modifications to the metal complex either through changes in the nature of the metal or changes in the nature of the ligand may be made to obtain the desired delivery of the metal to the cells of the subject.

In one embodiment of the complex used in the aspects of the invention described above the metal is selected from the group consisting of Iron, Nickel, Palladium, Cadmium, Manganese, Cobalt, Copper and Zinc. In another embodiment the metal is Copper or Zinc. In one specific embodiment the metal is Copper. In another specific embodiment the metal is Zinc.

In one embodiment of the complex used in the aspects of the invention described above the complex is symmetrical. In another embodiment of the complex used in the aspects of the invention described above the complex is asymmetrical.

In one embodiment of the complex used in the aspects of the invention described above R1 is selected from the group consisting of H, alkyl and aryl, each of which may be substituted. In another embodiment R1 is selected from the group consisting of H, methyl, ethyl and phenyl. In one specific embodiment R1 is H.

In one embodiment of the complex used in the aspects of the invention described above R2 is selected from the group consisting of H, alkyl, aryl, and —(CH2)mR8, each of which may be optionally substituted. In one embodiment m is 1 or 2. In one embodiment R8 is aryl or heterocycloalkyl, each of which may be optionally substituted.

In a specific embodiment R8 is phenyl, or morpholin-4-yl. In further specific embodiment R2 is selected from the group consisting of H, methyl, ethyl, phenyl-methyl, 2-morpholin-4-yl-ethyl, phenyl, 4-chloro-phenyl and 4-methoxy-phenyl.

In one embodiment of the complex used in the aspects of the invention described above R3 is selected from the group consisting of H, methyl, ethyl and phenyl. In one specific embodiment R3 is H. In another specific embodiment R3 is methyl.

In one embodiment of the complex used in the aspects of the invention described above R4 is selected from the group consisting of H, methyl, ethyl and phenyl. In one specific embodiment R4 is H. In another specific embodiment R4 is methyl.

In one embodiment of the complex used in the aspects of the invention described above R3 and R4, when taken together with the carbon atoms to which they are attached form an optionally substituted cycloalkyl group. In one specific embodiment the cycloalkyl group is a cyclohexyl group. In another specific embodiment of the complex used in the invention R3 and R4 are both H.

In one embodiment of the complex used in the aspects of the invention described above R5 is selected from the group consisting of H, alkyl and aryl, each of which may be substituted. In another embodiment R5 is selected from the group consisting of H, methyl, ethyl and phenyl. In one specific embodiment R5 is H.

In one embodiment of the complex used in the aspects of the invention described above R6 is selected from the group consisting of H, alkyl, aryl, and —(CH2)mR8, each of which may be optionally substituted. In one embodiment m is 1 or 2. In one embodiment R8 is aryl or heterocycloalkyl, each of which may be optionally substituted.

In a specific embodiment R8 is phenyl, or morpholin-4-yl. In further specific embodiment R6 is selected from the group consisting of H, methyl, ethyl, phenyl-methyl, 2-morpholin-4-yl-ethyl, phenyl, 4-chloro-phenyl and 4-methoxy-phenyl.

In one preferred embodiment of the complex used in the aspects of the invention described above the metal complex increases phosphoinositol-3-kinase (PI3K)-Akt activity in the subject. In another preferred embodiment the metal complex decreases glycogen synthase kinase 3 (GSK3) activity in the subject. In another preferred embodiment the metal complex increases JNK activity in the subject. In another embodiment of the invention the metal complex leads to activation of one or more anti-oxidant enzymes. In one embodiment the anti-oxidant enzyme is superoxide dismutase (SOD).

Specific examples of complexes that are useful in the methods of the invention include the following:

FIG. 1: illustrates cellular copper levels when cells were treated with a variety of free ligand and copper ligand complexes.

FIG. 2: illustrates cellular zinc levels when cells were treated with a variety of free ligand and zinc ligand complexes.

FIG. 3: illustrates the different effect of Cu-GTSM versus Cu-ATSM on extracellular amyloid Seta levels.

FIG. 4: illustrates the effect of various zinc complexes on extracellular amyloid Seta levels

FIG. 5: illustrates how Copper inhibits uptake of Zinc in cells treated with Zn-BSTC and inhibits effect of Zn-BTSC on amyloid Seta.

FIG. 6: illustrates the effect of temperature on metal uptake.

FIG. 7: illustrates the effect of temperature effect on BTSC—amyloid Seta and metal uptake.

FIG. 8A: illustrates that ZnBTSC induces activation of phosphoinositol-3-kinase (phosphorylation of Akt, p-Akt) and activates JNK (resulting in JNK phosphorylation, p-JNK).

FIG. 8B: illustrates that ZnATSE inhibits activation of GSK3 by inducing its phosphorylation (p-GSK3).

FIG. 8C: illustrates that Cu-GTSM induces activation of phosphoinositol-3-kinase (phosphorylation of Akt, p-Akt), activation of JNK (p-JNK) and inhibition of GSK3 (p-GSK3).

FIG. 8D: illustrates that inhibition of amyloid βeta in cultures by Zn-BTSC is dependent on activation of JNK and phosphoinositol-3-kinase. Inhibition of JNK by SP600125 prevents the loss of amyloid βeta. Inhibition of phosphoinositol-3-kinase by LY294002 prevents loss of amyloid βeta. SB203580 (p38 inhibitor) has no effect).

FIG. 9: illustrates the results of the Oxyblot™ assay for the insoluble mouse brain fraction versus control for complex A8.

FIG. 10: illustrates the results of the Oxyblot™ assay for the soluble mouse brain fraction versus control for complex A8.

FIG. 11: illustrates the results of the Oxyblot™ assay for the insoluble mouse brain fraction versus control for complex CuGTSM.

FIG. 12: illustrates the results of the Oxyblot™ assay for the soluble mouse brain fraction versus control for complex CuGTSM.

FIG. 13: illustrates the results of a tau phosphorylation assay for the insoluble mouse brain fraction versus control for complex CuGTSM.

FIG. 14: illustrates the results of a tau phosphorylation assay for the soluble mouse brain fraction versus control for complex CuGTSM.

FIG. 15: Shows GSK3β and phosphorylated GSK3β (p-GSK3β) levels in M17 and N2a cells treated with CuGTSM or vehicle control for 24 hours. GSK3β in inhibited when phosphorylated, therefore treatment with CuGTSM is shown to inhibit GSK33 activity in M17 and N2a cells. B-actin is shown as a control.

FIG. 16: Shows the effect of various BSTC\'s on Dopamine induced WT cell death.

FIG. 17: Shows the effect of various BSTC\'s on Dopamine induced A30P cell death.

FIG. 18: Graphically illustrates the total rotations of control mice and mice treated with metal complexes in Parkinsons disease model.

FIG. 19: Shows cell counts on post-mortem examination of the brains in the mice used in example 27

FIG. 20: Graphically illustrates chemical depletion of PrPC in GT1-7 cells following 6 hour treatment with increasing concentrations of Cu (GTSM).

FIG. 21: Graphically illustrates chemical depletion of PrPC in Hela cells following 6 hour treatment with increasing concentrations of Cu (GTSM).

FIG. 22: Shows rotarod data indicating the effectiveness of CuATSM in treating ALS mice.

FIG. 23: Shows the onset of hind limb paralysis in ALS mice treated with CuATSM

FIG. 24A: Shows Western blotting of cell lysates. CuGTSM (25 μM) activated EGFR (tyr1068) in U87MG-EGFR cells compared to vehicle control. The addition of PD153035 to CuGTSM-treated cells inhibited activation of EGFR (A).

FIGS. 24 B to 24D: Show similar experiments as for FIG. 24A but for JNK (B), GSK3 (C) and ERK (D) respectively.

DETAILED DESCRIPTION

In this specification a number of terms are used which are well known to a skilled addressee. Nevertheless for the purposes of clarity a number of terms will be defined.

As used herein, the term “unsubstituted” means that there is no substituent or that the only substituents are hydrogen.

The term “optionally substituted” as used throughout the specification denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more substituent groups. Preferably the substituent groups are one or more groups independently selected from the group consisting of halogen, ═O, ═S, —CN, —NO2, —CF3, —OCF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxycycloalkyl, alkoxyheterocycloalkyl, alkoxyaryl, alkoxyheteroaryl, alkoxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, —COOH, —COR9, —C(O)OR9, —CONHR9, —CSNHR9, —NHCOR9, —NHCOOR9, NHCONHR9, C(═NOH)R9, —SH, —SR9, —OR9, acyl, a group of formula —NR9R10 or a group of formula —CONR9R10 or a group of formula —NHCONR9R10 wherein R10 is a protein, hormone, antibody or carbohydrate moiety.

“Alkyl” as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C1-C14 alkyl, more preferably C1-C10 alkyl, most preferably C1-C6 unless otherwise noted. Examples of suitable straight and branched C1-C6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, and the like. When alkyl is used as a bridging group it is typically (but not exclusively) referred to as alkylene. A similar convention applies to other bridging groups.

“Acyl” means an alkyl-CO— group in which the alkyl group is as described herein.

Examples of acyl include acetyl and benzoyl. The alkyl group is preferably a C1-C6 alkyl group.

“Alkenyl” as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-14 carbon atoms, more preferably 2-12 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain. The group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl.

“Alkoxy” refers to an —O-alkyl group in which alkyl is defined herein. Preferably the alkoxy is a C1-C6alkoxy. Examples include, but are not limited to, methoxy and ethoxy.

“Alkynyl” as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-14 carbon atoms, more preferably 2-12 carbon atoms, more preferably 2-6 carbon atoms in the normal chain. Exemplary structures include, but are not limited to, ethynyl and propynyl.

“Cycloalkyl” refers to a saturated or partially saturated, monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane.

“Heterocycloalkyl” refers to a saturated or partially saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1,3-diazapane, 1,4-diazapane, 1,4-oxazepane, and 1,4-oxathiapane.

“Heteroalkyl” refers to a straight- or branched-chain alkyl group preferably having from 2 to 14 carbons, more preferably 2 to 10 atoms in the chain, one or more of which is a heteroatom selected from S, O, and N. Exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, alkyl sulfides, and the like.

“Aryl” as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring. Examples of aryl groups include phenyl, naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C5-7 cycloalkyl or C5-7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl.

“Heteroaryl” either alone or part of a group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, isoindole, 1H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-,3- or 4-pyridyl, 2-, 3-, 4-, 5-, or 8-quinolyl, 1-, 3-, 4-, or 5-isoquinolinyl 1-, 2-, or 3-indolyl, and 2-, or 3-thienyl.

The term “therapeutically effective amount” or “effective amount” is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.

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 biologically-active agent. The subject may be a mammal, typically 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 present invention is based on the observation that metal plays an important role in a wide number of biological processes and adequate metal levels are especially important in the efficient functioning of a wide range of biologically important enzymes and cellular signaling processes. Enzymes that involve metal activation include many of the enzymes related to oxidation at the cellular level. Accordingly it was felt that selective metal delivery to subjects with conditions relating to abnormal levels of metals could provide a useful therapeutic outcome for a number of biological applications. In particular it was felt that this could be useful in respect of conditions caused by or associated with oxidative stress as this is a condition where many of the protective mechanisms or enzymes that protect the body from oxidative stress involve metal catalysis and so the provision of bio-available metal may be a useful therapeutic in treating these conditions.

As such, investigations were based on identifying appropriate metal complexes that would be able to deliver metal to sites wherein metal is depleted in a subject. Investigations were particularly based on complexes that would be able to deliver metal to the cells of a subject. A number of important biological processes that are mediated by metal, such as metal mediated enzymes, occur in the cells rather than in the extra-cellular matrix. The present applicants therefore decided that it was preferable that the metal be delivered in the form of cell permeable metal complex in order to ensure that the metal acted on the cell rather than in the extra-cellular environment. In addition, it was found that in order to ensure that the metal was delivered to the cell it was preferable that the cell permeable metal complex be sufficiently stable such that upon administration to a subject the metal is not released in the extra-cellular environment. A further advantage of the use of metal complexes over the “naked” metal ion is that delivery of the metal can be targeted which reduces the chance that unwanted side effects will be observed (for example copper toxicity). A number of metal complexes meet these criteria.

One attractive group of metal complexes for use in the methods of the present invention are metal complexes of bis(thiosemicarbazone) (BTSC) ligands which have been investigated as metallodrugs and have proven to have a broad range of pharmacological activity. In particular, recent interest has focused on the use of BTSC ligands as vehicles for the selective delivery of radioactive copper isotopes to hypoxic tissue and leucocytes in the development of radiopharmaceuticals. Copper(II)-BTSC complexes are stable (log K=1018) neutral, low molecular weight complexes capable of crossing cell membranes. In some cases, once inside cells the copper(II) is reduced by intracellular reductants to Cu(I) which subsequently dissociates from the ligand. Other Cu(II)-BTSC complexes are more resistant to reduction and disassociation, and are only trapped in hypoxic cells. This selectivity is remarkably sensitive to the number of alkyl groups attached to the diimine backbone of the ligand. For example, copper(II)diacetylbis(N(4)-methylthiosemicarbazone) [Cu(ATSM)] with two methyl substituents on the backbone, is selective for hypoxic cells whereas the copper of [Cu(GTSM)] is trapped in all cells. The hypoxic cell selectivity has been correlated with the Cu(II)/Cu(I) reduction potential, [Cu(ATSM)] is some 160 mV harder to reduce than [Cu(GTSM)], but differences in pKa and the stability of the reduced state may also be important.

[Zinc(BTSC)] complexes are also capable of transporting zinc into cells and a recent report used the intrinsic fluorescence of certain [Zn(BTSC)] complexes to probe the intracellular distribution of the complexes via fluorescence microscopy in several cancer-cell lines. Sub-cellular localization was a sensitive function of terminal nitrogen substituents on the complexes and cell type, varying from predominantly nucleolar to lysosomal. Zinc is central to a number of cell signal pathways including modulation of NMDA receptor activity expression of metallothienein and activation of mitogen activated protein kinase (MAPK)-mediated signal transduction pathways and therefore, Zn-BTSC uptake could have complex effects on downstream metal-mediated cell signalling.

It was therefore felt that BTSC metal complexes of this type were attractive as potential cell permeable metal complexes for use in the methods of the invention. BTSC-metal complexes have several properties that make them worthy of investigation as potential therapeutic agents for the treatment of conditions related to oxidative stress including neurodegenerative disorders such as Alzheimer\'s disease. Organ and tissue distribution of these types of materials are well characterized, several BTSC complexes are known to be capable of crossing the blood brain barrier and there is no inherent class toxicity with these complexes. Importantly, the ligands can be readily modified by varying the nature and number of alkyl substituents on the ligand and these modifications can allow subtle control of subcellular targeting and metal release/retention properties.

In addition, the complexes are attractive as different metal complexes have different modes of metal release in the cell, potentially opening the way for the selective use of different metal complexes for different applications. For example, the zinc and copper complexes increase the bio-available metal via different mechanisms.

Without wishing to be bound by theory it is felt that in the case of the zinc complexes the associations constants have been measured to be of the order of 107-8. As such, these cell permeable complexes are stable enough to effectively enter the cell as they are sufficiently stable in the extracellular matrix. Once inside the cell it is thought that the zinc is released from the ligand due to an increased competition from intracellular ligands (and perhaps decreased [Zn2+]). This therefore ultimately leads to bio-available metal in the cell.

In contrast, it is felt that copper complexes release their metal via a different mechanism. The stability constants of copper BSTC complexes have been measured to be of the order of 1018. Both [Cu(ATSM)] and [Cu(GTSM)] have similar stability constants for Cu(II). Where they differ is in their reduction potentials. For [Cu(ATSM)] E112=−0.59 V whereas for [Cu(GTSM)] E1/2=0.43V, this means it is easier to reduce Cu(II) to Cu(I) in [Cu(GTSM)]. This results from the modification of the backbone of the ligand (R3 and R4). The methyl groups of ATSM are electron donating and make it harder to reduce to Cu(I). Once [Cu(GTSM)] enters the cell it is reduced to Cu(I) via cell reducing agents. The Cu(I) complex is less stable than the Cu(II) complex and the metal disassociates from the ligand making the copper bio-available as either (Cu(I) or Cu(II)). In the case of [Cu(II)ATSM] the copper is not released due to its increased resistance to reduction and trans-metallation. In addition complexes of this type were attractive as not only do they have different mechanisms of metal release depending upon the metal ion chosen some of the complexes are such that they do not release the metal at all and so they may be used in circumstances where it is desirable not to deliver the metal in the form of a metal cation but rather it is desirable to deliver the metal in the form of a bound metal still in complex with the ligand.

A number of metal complexes of this type were therefore initially synthesised to examine their behavior.

The ligands and complexes selected for the intitial study were as follows:

It was found that treatment of APP-transfected Chinese Hamster Ovary (APP-CHO) cells with both Cu (BTSC) and Zn (BTSC) increased cellular metal levels demonstrating uptake of the BTSC-metal complexes. This supports the proposition that the complexes are sufficiently stable in the extracellular matrix to allow the metal to be delivered to the cell. As such, it was possible to demonstrate that complexes of this type were candidate complexes that could be used to deliver metal to the cell without the risk of releasing the metal from the complex in the extracellular matrix leading to the adverse affects noted by others who have taught that metals such as zinc should be reduced in the extracellular matrix.

Treatment of (APP-CHO) cells with a range of [Cu(BTSC] complexes with di-alkyl backbones at 1-50 μM for 6 hr resulted in significant increases in intracellular copper levels when compared to treatment with free ligands or Copper alone and the results are shown in FIG. 1. This suggests that the complex is important in the transportation of the metal across the cell membrane. The highest levels of copper were induced by treatment with [Cu(ATSM)] which resulted in a 177±9 fold increase in cellular copper levels when compared to untreated control cells. This corresponded to a cellular Copper level of 4.5 ng/mg protein and 796 ng/mg protein for control and [Cu(ATSM)] treated cells respectively. The other three [Cu(BTSC)] complexes resulted in 90-115 fold increases in cellular copper levels.

Zn-BTSC complexes are less stable than their copper complexes (related derivatives having association constants of the order of log K=7 but are still capable of effectively transporting Zinc into the cell. Treatment of cells with [Zn(BTSC)] complexes resulted in significant increases in the intracellular Zinc levels as measured by ICP-MS (FIG. 2.). [Zn(ATSM)] and [Zn(ATSE)] induced 8.2±0.25 and 9.8±0.9 fold increases in cellular Zinc levels respectively (FIG. 2). The data obtained suggested that the complexes were capable of delivering metals to cells and so attention was turned to probing a number of biological systems where it was envisaged that metal delivery could be useful

Treatment of APP-CHO cells with [Cu(GTSM)] resulted in an increase in the intracellular copper levels as expected of the cell permeable Cu-ligand. There also was a dose-dependent reduction in the extracellular levels of Aβ1-40 (amyloid βeta). The concentration of Aβ1-40 was 0.70 ng mL−1 in untreated cells. Treatment with 1 μM [Cu(GTSM)] reduced this to 0.43 ng mL−1 (FIG. 3). Aβ1-40 levels were further reduced to negligible levels following treatment with 50 μM [Cu(GTSM)]. The very small reduction in the levels of Aβ1-40 that were evident following administration of the ligand (GTSMH2) to the cells was most likely due to the formation of either [Cu(GTSM)] or [Zn(ATSM)] from trace metals in the culture medium.

The lower stability of the Zn-BTSC complexes means they are more susceptible to intracellular transchelation than their Copper analogues and therefore, could elevate levels of bio-available Zinc within the cells. The elevated Zinc levels in the cells treated with [Zn(BTSC)] complexes correlated with a reduction in the extracellular levels of Aβ1-40. The concentration of Aβ1-40 in the medium of untreated cells was 0.6-0.8 ng mL−1 and was reduced to less than 0.2 ng mL−1 following treatment with 25 μM [Zn(BTSC)]. The different [Zn(BTSC)] complexes exhibited some detectable differences in the dose dependent reduction of Aβ1-40. Treatment with [Zn(ATSE)] and [Zn(ChexTSE)] resulted in greater reductions at a lower dose (1 μM) when compared to the other two complexes, [Zn(ATSM)] and [Zn(ATSP)]) (FIG. 4). This could reflect different binding affinities or alternative subcellular localisation and subsequently initiate different metal-mediated cell signalling pathways. The results clearly demonstrated that the zinc complexes were highly effective in reducing the extra-cellular concentration of amyloid βeta.

It was known that copper can transmetallate [Zn(BTSC)] complexes. Therefore, if [Zn(BTSC)] complexes were administered to the culture medium in the presence of exogenous Cu2+, we would expect [Cu(BTSC)] complexes to form. To examine this, cells were exposed to 10 μM [Zn(ATSE)] or [Zn(ATSP)] with or without 5-50 μM Cu2+ for 6 hr. Treatment of cells with 10 μM [Zn(ATSE)] alone resulted in a 9.7±0.7 fold increase the cellular zinc levels compared to untreated cultures (FIG. 5). In comparison, treatment with 10 μM [Zn(ATSE)] in the presence of 10 μM Cu2+ only resulted in a 2.9±0.3 fold increase in intracellular zinc (FIG. 5). Similar effects were seen for [Zn(ATSP)] in the presence of Cu2+. These data strongly suggest that transmetallation of a proportion of the zinc complexes to give the analogous Cu2+ complexes diminished the amount of zinc transported into the cell.

A study was conducted in which cells were exposed to differing concentrations of one of the complexes of the invention at a number of various concentrations and at either 4 or 37° C. The results as shown in FIGS. 6 and 7 which clearly indicate that cellular uptake is dependent upon temperature and can have a significant effect upon the level of amyloid Seta. Treatment of cells at 37° C. results in a high level of metal uptake which results in loss of extracellular Amyloid Seta. Incubation of cells at 4° C. results in substantially lower metal uptake and therefore, reduced effects on extracellular Amyloid Seta. The results indicate that metal uptake is therefore likely to be an active, rather than passive process and could provide the opportunity to target specific metal-BTSC receptors to improve efficacy of the complexes.

As the initial complexes showed promise a number of additional complexes were synthesized in order to probe the activity of the family of complexes. These complexes were synthesized and then subjected to in vivo mouse assays to determine a number of biological properties of the complexes.

With the results clearly indicating that there was metal uptake in the cells an investigation was conducted to determine the relevant pathways leading to the observed result. APP-CHO cells were treated with 10 μM metal-BTSC complexes for 6 hr and cell lysates examined for activation of PI3K and MAPK signal pathways. CuATSP and CuATSE did not induce activation of PI3K (Akt phosphorylation) or JNK (FIG. 8A). In contrast, [Zn(ATSE)] and [Zn(ATSM)] both induced activated Akt and JNK (FIG. 8A). Activation of PI3K-Akt by [Zn(ATSE)] also induced down-stream phosphorylation (de-activation) of GSK3 as well as increased GSK3 expression (FIG. 8B).

Interestingly, [Zn(ATSP)] did not induce activation of Akt or JNK (FIG. 8A), although a small increase in GSK3 expression was observed (FIG. 8B).

The complexes of the invention have been shown to be effective as metal delivery agents, particularly agents for the delivery of metals to cells. According the complexes of the invention may be used in the treatment or prophylaxis of a number of conditions in which metal delivery can prevent, alleviate or ameliorate the condition.

There are a number of conditions of this type. An example of conditions of this type is conditions associated with or caused by oxidative stress. It is known that many of the protective biological anti-oxidant mechanisms involve metal catalysed enzymes and thus metal delivery can serve to stimulate or re-start the activity of the biological anti-oxidant mechanisms leading to an overall anti-oxidant effect being achieved. In one embodiment the condition associated with or caused by oxidative stress is selected from the group consisting of cardiovascular conditions, cancers, cataracts, neurological disorders such as Alzheimer\'s disease, prion diseases—including Creutzfeldt-Jakob Disease (CJD), and heart diseases, amyloidogenic amylotrophic lateral sclerosis (ALS), prion transmissible spongioform encephalopathies (TSE), cataracts, mitochondrial disorders, Menke\'s disease, Parkinson\'s disease and Huntington\'s disease.

In another embodiment the disorder is a neuromuscular disorder selected from the group consisting of amyotrophic lateral sclerosis (ALS), mitochondrial/metabolic disease and Friedreich\'s ataxia.

In one embodiment of the invention the condition is a neurological condition or a neurodegenerative disorder.

The term “neurological condition” is used herein in its broadest sense and refers to conditions in which various cell types of the nervous system are degenerated and/or have been damaged as a result of neurodegenerative disorders or injuries or exposures. In particular, complexes 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 complex 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, 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 disorder” 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 complexes of the present invention include acute intermittent porphyria; adriamycin-induced cardiomyopathy; AIDS dementia and HIV-1 induced neurotoxicity; AD; ALS; atherosclerosis; cataract; cerebral ischaemia; cerebral palsy; cerebral tumour; chemotherapy-induced organ damage; cisplatin-induced nephrotoxicity; coronary artery bypass surgery; CJD and its new variant associated with “mad cow” disease; diabetic neuropathy; Down\'s syndrome; drowning; epilepsy and post-traumatic epilepsy; Friedrich\'s ataxia; frontotemporal dementia; glaucoma; glomerulopathy; haemochromatosis; haemodialysis; haemolysis; haemolytic uraemic syndrome (Weil\'s disease); Menke\'s disease; haemorrhagic stroke; Hallerboden-Spatz disease; heart attack and reperfusion injury; HD; Lewy body disease; intermittent claudication; ischaemic stroke; inflammatory bowel disease; macular degeneration; malaria; methanol-induced toxicity; meningitis (aseptic and tuberculous); motor neuron disease; multiple sclerosis; multiple system atrophy; myocardial ischaemia; neoplasia; Parkinson\'s disease; peri-natal asphyxia; Pick\'s disease; progressive supra-nuclear palsy; radiotherapy-induced organ damage; restenosis after angioplasty; retinopathy; senile dementia; schizophrenia; sepsis; septic shock; spongiform encephalopathies; subharrachnoid haemorrage/cerebral vasospasm; subdural haematoma; surgical trauma, including neurosurgery; thalassemia; transient ischaemic attack (TIA); transplantation; vascular dementia; viral meningitis; and viral encephalitis.

Additionally, the complexes 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 complexes of the invention may also be used to treat Anemia, Neutropenia, Copper deficiency Myelopathy, Copper deficiency Syndrome and Hyperzincaemia.

The invention is particularly directed to conditions which induce oxidative damage of the central nervous system, including acute and chronic neurological disorders such as, cerebral ischaemia, stroke (ischaemic and haemorragic), subharrachnoid haemorrage/cerebral vasospasm, cerebral tumour, AD, CJD and its new variant associated with “mad cow” disease, HD, PD, 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 in one embodiment is selected from the group consisting of sporadic or familial AD, ALS, motor neuron disease, cataract, PD, Creutzfeldt-Jacob disease and its new variant associated with “mad cow” disease, HD, dementia with Lewy body formation, multiple system atrophy, Hallerboden-Spatz disease, and diffuse Lewy body disease.

In one specific embodiment the neurodegenerative amyloidosis is an Aβ-related condition, such as AD or dementia associated with Down syndrome or one of several forms of autosomal dominant forms of familial AD (reviewed in St George-Hyslop, 2000). Most preferably the Aβ-related condition is AD.

In a specific aspect of the invention, prior to treatment the subject has moderately or severely impaired cognitive function, as assessed by the AD Assessment Scale (ADAS)-cog test, for example an ADAS-cog value of 25 or greater.

In addition to slowing or arresting the cognitive decline of a subject, the complex 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. 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 AD 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.

Another condition that may be able to be treated by metal delivery is cancer. 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 tumor. A brain cancer or tumor may be a glioma or non-glioma brain tumor. The term “cancer” and “tumor” 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 Tumors. Reference to a “glioma” includes GMB, astrocytoma and anaplastic astrocytoma or related brain cancers.

The complexes of the present invention may also be able to be used to treat tau related disorders. Tau protein is an important protein as it is the protein expressed in the central nervous system and plays a critical role in the neuronal architecture by stabilizing intracellular microtubule network. Accordingly any impairment of the physiological role of the tau protein either by truncation, hyper-phosphorylation or by disturbing the balance between the six naturally occurring tau isoforms is detrimental to the subject and leads to the formation of neurofibrillary tangles (NFT), dystrophic neurites and neuropil threads. The major protein subunit of these structures is microtubule associated protein tau. The amount of NFT found in autopsies of AD patients correlates with clinical symptoms including intellectual decline. Accordingly tau protein plays a critical role in AD pathology. The recent discovery of cosegregation of specific mutations in the tau gene with the disease frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) has confirmed that certain abnormalities in the tau protein can be a primary cause of neurodegeneration and dementia in affected individuals.

Without wishing to be bound by theory it is felt that the activity of the complexes of the present invention to reduce levels of tau phosphorylation is as a result of their ability to deliver metal to cells and hence their anti-oxidant activity. It is felt that the ability of the complexes to act as anti-oxidants mean that they provide protection from OS which is desirable as OS can lead to hyper-phosphorylation of tau and cell dysfunction. As a consequence the ability of these complexes to deliver biologically important metals to cells allows them to function as anti-oxidants (especially where the oxidative stress is caused by metal deficiency) which in turn means the metal complexes may have the ability to prevent (or treat) tau-opathies.

There are a number of disorders or conditions that are recognized as being tau disorders or more colloquially Tauopathies. Disorders of this type include Richardson\'s syndrome, Progressive Supranuclear Palsy, Agryrophilic grain disease, corticobasal degeneration, Pick\'s disease, frontotemporar dementia linked with parkinsonism linked to chromosome 17 9FTDP-17), post-encephalitic parkinsonism (PEP), dementia pugilistica, Down\'s syndrome, Alzheimers disease, Familial British dementia, Familial Danish dementia, Parkinsons\' disease, Parkinsons Disease complex of Guam (PDC), myotonic dystrophy, Hallevorden-Spatz disease, and Niemann-Pick type C.

The complexes may also be used in the treatment of an Abeta related disorder. A number of Abeta disorders are known including disorders selected from the group consisting of Parkinson\'s disease, Alzheimer\'s disease, Multiple sclerosis, Neuropathies, Huntington\'s disease, Prion disease, motor neurone disease, Amyotrophic lateral sclerosis (ALS), Menke\'s disease, and amyloidoses.

As the complexes of the invention have also been shown to be able to deliver metal to cells they have the ability to influence matrix metallo-proteinases (MMP\'s). Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent secreted or membrane anchored endopeptidases which play a number of important biological functions. MMPs are involved in many physiological processes but also take part in the pathophysiological mechanisms responsible for a wide range of diseases. Pathological expression and activation of MMPs are associated with cancer, atherosclerosis, stroke, arthritis, periodontal disease, multiple sclerosis and liver fibrosis. Accordingly the complexes of the invention have the potential to influence these conditions.

Administration of complexes within Formula (I) to humans can be by any of the accepted modes of administration well known in the art. For example they may be administered by enteral administration such as oral or rectal, or by parenteral administration such as subcutaneous, intramuscular, intravenous and intradermal routes. Injection can be bolus or via constant or intermittent infusion. The active complex is typically included in a pharmaceutically acceptable carrier or diluent and in an amount sufficient to deliver to the subject a therapeutically effective dose.

In using the complexes of the invention they can be administered in any form or mode which makes the complex bio-available. One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the complex selected, the condition to be treated, the stage of the condition to be treated and other relevant circumstances. We refer the reader to Remingtons Pharmaceutical Sciences, 19th edition, Mach Publishing Co. (1995) for further information.

The complexes of the present invention can be administered alone or in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier, diluent or excipient.

The complexes are, however, typically used in the form of pharmaceutical compositions which are formulated depending on the desired mode of administration. As such, in a further embodiment the present invention provides a pharmaceutical composition including a complex of Formula (I) and a pharmaceutically acceptable carrier, diluent or excipient. The compositions are prepared in manners well known in the art.

The invention in other embodiments provides a pharmaceutical pack or kit including one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. In such a pack or kit can be found a container having a unit dosage of the agent (s). The kits can include a composition including an effective agent either as concentrates (including lyophilized compositions), which can be diluted further prior to use or they can be provided at the concentration of use, where the vials may include one or more dosages. Conveniently, in the kits, single dosages can be provided in sterile vials so that the physician can employ the vials directly, where the vials will have the desired amount and concentration of agent(s). Associated with such container(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The complexes of the invention may be used or administered in combination with one or more additional drug (s) that are useful for the treatment of the disorder/diseases mentioned. The components can be administered in the same formulation or in separate formulations. If administered in separate formulations the complexes of the invention may be administered sequentially or simultaneously with the other drug(s).

Pharmaceutical compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of micro-organisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin.

If desired, and for more effective distribution, the complexes can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active complex is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

If desired, and for more effective distribution, the complexes can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.

The active complexes can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active complexes, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring, and perfuming agents.

Suspensions, in addition to the active complexes, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the complexes of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active complex.

Dosage forms for topical administration of a complex of this invention include powders, patches, sprays, ointments and inhalants. The active complex is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required.

The amount of complex administered will preferably treat and reduce or alleviate the condition. A therapeutically effective amount can be readily determined by an attending diagnostician by the use of conventional techniques and by observing results obtained under analogous circumstances. In determining the therapeutically effective amount a number of factors are to be considered including but not limited to, the species of animal, its size, age and general health, the specific condition involved, the severity of the condition, the response of the subject to treatment, the particular complex administered, the mode of administration, the bioavailability of the preparation administered, the dose regime selected, the use of other medications and other relevant circumstances.

A preferred dosage will be a range from about 0.01 to 300 mg per kilogram of body weight per day. A more preferred dosage will be in the range from 0.1 to 100 mg per kilogram of body weight per day, more preferably from 0.2 to 80 mg per kilogram of body weight per day, even more preferably 0.2 to 50 mg per kilogram of body weight per day. A suitable dose can be administered in multiple sub-doses per day.

The complexes of the various embodiments may be prepared using the reaction routes and synthesis schemes as described below, employing the techniques available in the art for each of the individual step/reactions and using starting materials that are readily available. The preparation of particular complexes of the embodiments is described in detail in the following examples, but the artisan will recognize that the chemical reactions described may be readily adapted to prepare a number of other agents of the various embodiments. For example, the synthesis of non-exemplified complexes may be successfully performed by modifications apparent to those skilled in the art, e.g. by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by making routine modifications of reaction conditions. A list of suitable protecting groups in organic synthesis can be found in T. W. Greene\'s Protective Groups in Organic Synthesis, John Wiley & Sons, 1981. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other complexes of the various embodiments.

A suitable scheme for the production of some of the complexes of the invention is shown below in Scheme 1.

Thus condensation of dione (X) with two equivalents of a suitably functionalised thio semicarbazide (XI) under acidic conditions leads to the formation of the bis (thiosemicarbazone) (XIII). Using the reaction scheme outlined the resultant semi carbazide (XIII) will be symmetrical as the same thio semicarbazide will condense with both aldehyde moieties. The bis(thiosemicarbazone) can then be reacted with a suitable metal salt such as the metal acetate to produce the desired metal complex (XIV) and acetic acid. A wide variety of thiosemicarbazones may be produced by varying the substituents on either the aldehyde moiety or on the semicarbazide.

An alternative procedure which is particularly applicable to non-symmetrical (bis semicarbazones) is shown in Scheme 2.

Thus as before reaction of a dione (X) with one equivalent of thio semi-carbazide (XI) under acidic conditions leads to formation of the mono thio semicarbazione derivative (XV). This can then be subjected to condensation with a second thiosemicarbazide moiety (XVI) to produce a bis(thiosemicarbazone) (XVII) which can again be reacted with a metal salt such as the metal acetate to produce the desired unsymmetrical complex (XVIII). Once again by judicious choice of starting materials (X), (XI) and (XVI) a wide variety of materials can be synthesised.

With certain bis thiosemicarbazones it is difficult to stop the formation reaction at the mono-addition step leading to the formation of the bis adduct as well as starting material. Whilst this is desired when the final product is a symmettrical adduct it is undesirable in circumstances where an asymmetrical adduct is desired. Whilst this was not an issue with all backbones it was certainly observed with a number of the products desired to be produced and so an alternative procedure was developed for adducts of this type. An alternative procedure that avoided the formation of this bis adduct in a single step was therefore developed and is shown in scheme 3.

Thus molecule (XIX) was reacted with a thiosemicarbazides to afford the mono-adduct, acetal (XX). The acetal can be oxidatively cleaved to give the aldehyde (XXI) using lithium tetrafluoroboracete, a mild Lewis acid. Reaction of the aldehyde (XXI) with a different thiosemicarbazide, gave the desired asymmetric ligand (XXII) which could then be converted into the metal complex (XXIII) using the standard conditions.

Reagents useful for synthesizing compounds may be obtained or prepared according to techniques known in the art.

In the examples described below, unless otherwise indicated, all temperatures in the following description are in degrees Celsius and all parts and percentages are by weight, unless indicated otherwise.

Various starting materials and other reagents were purchased from commercial suppliers, such as Aldrich Chemical Company or Lancaster Synthesis Ltd., and used without further purification, unless otherwise indicated. Tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) were purchased from Aldrich in SureSeal bottles and used as received. All solvents were purified by using standard methods in the art, unless otherwise indicated.

Nuclear magnetic resonance spectra (NMR) spectra were acquired with either a Varian 400 MHz spectrometer (1H at 400 MHz) or a Varian Inova 500NMR (1H at 500 MHz). All chemical shifts were referenced to residual solvent peaks and are quoted in ppm relative to TMS. All spectra were recorded in d6-DMSO. Mass spectra were recorded using the electrospray technique (positive ion) VG BioQ Triple Quadrupole Mass Spectrometer. All reagents and other solvents were obtained from standard commercial sources and were used as received. ATSMH2, [Cu(ATSM)], [Zn(ATSM)], ATSPH2, [Cu(ATSP], [Zn(ATSP)]. ATSEH2, [Cu(ATSE)], GTSMH2 and [Cu(GTSM)] were prepared by variations of reported procedures, see: 1) P. J. Blower, T. C. Castle, A. R. Cowley, J. R. Dilworth, P. S. Donnelly, E. Labisbal, F. E. Sowrey, S. J. Teat and M. J. Went, Dalton Trans., 2003, 4416-4425 and references therein; 2) J. L. J. Dearling, J. S. Lewis, G. D. Mullen, M. J. Welch, and P. J. Blower, J. Biol. Inorg. Chem., 2002, 7, 249 and references therein; 3) P. McQuade, K. E. Martin, T. C. Castle, M. J. Went, P. J. Blower, M. J. Welch and J. S. Lewis, Nucl. Med. Biol., 2005, 32, 147. All 1H NMR spectra and ES MS were as expected.

ATSEH2 (0.134 g, 0.46 mmol) and Zn(CH3CO2)2.2H2O (0.102 g, 0.46 mmol) were added to ethanol (5 mL). The mixture was heated at reflux for 2 hours under an atmosphere of nitrogen and then allowed to cool to room temperature. The bright yellow solid that formed was collected by filtration and washed with ethanol, and diethyl ether to give [Zn(ATSE)] as a yellow powder (0.122 g, 0.35 mmol, 76%). 1H NMR (400 MHz): δ 1.08, 6H, t, 3JHH=7 Hz, CH2CH3; 2.18 ppm, 6H, s, 2×CH3; 3.30, 4H, m partially obscured by H-OD peak from solvent, CH2CH3. ES MS (+ve ion): m/z=351=[Zn(C10H18N6S2)+H+]+.