WO2013020909A1

WO2013020909A1 - Materials and methods for the treatment of tauopathies

Info

Publication number: WO2013020909A1
Application number: PCT/EP2012/065235
Authority: WO
Inventors: Seyedeh Maryam ALAVI NAINI; Nadia Soussi-Yanicostas; Constantin YANICOSTAS
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2011-08-05
Filing date: 2012-08-03
Publication date: 2013-02-14

Abstract

The present invention relates to surfen and derivatives and analogues thereofand their use in prevention and treatment of tauopathies, including Alzheimer's disease. Derivatives of surfen and novel medical uses and methods relating thereto are also provided.

Description

MATERIALS AND METHODS FOR THE TREATMENT OF TAUOPATHIES

Related Application

The present application claims priority to European Patent Application No. 1 1306017.2, which was filed on August 5, 201 1 . The European patent application is incorporated herein by reference in its entirety.

Field of the invention

The present invention relates to surfen and derivatives and analogues thereof, and their use in prevention and treatment of tauopathies including Alzheimer's disease.

Derivatives of surfen and novel medical uses and methods relating thereto are also provided.

Background of the invention Tauopathies are a family of neurodegenerative disorders that includes Alzheimer's disease (AD), a major health problem in countries with increasing aging population. These disorders are characterized by neuronal inclusions of fibrillary tangles, which are proteinaceous aggregates comprising hyperphosphorylated isoforms of the microtubule- associated protein tau. In the brain of AD patients, the mean content of abnormally hyperphosphorylated tau is significantly correlated with the severity of the disease.

Tau protein is phosphorylated at several sites under normal physiological conditions, and indeed phosphorylation appears to be involved in controlling its association with microtubules. However, hyperphosphorylation of tau is associated with abnormal pathology. Hyperphosphorylation of tau appears as a key initiator event for the detachment of this protein from microtubules and its subsequent oligomerization and aggregation. Hyperphosphorylation of tau precedes the appearance of neuofibrillary tangles, and animal studies suggest that neurodegeneration is caused by tau hyperphosphorylation and not by the presence of the fibrillary tangles.

In this context, tau hyperphosphorylation is now recognized as an important therapeutic target for the treatment of tauopathies, including AD. However, while efforts have been made to identify agents that could prevent or reverse tau hyperphosphorylation, few molecules with therapeutic activities have yet been identified. In addition, as the known tau kinases have a large number of cellular targets, the use of kinase inhibitors as therapeutic molecules is hampered by the low selectivity of these molecules and thus by their detrimental side effects. The requirement for specific agents which target the underlying physiology, rather than just the symptoms, is particularly acute in neurodegenerative diseases due to the distressing side effects which are often seen with nonspecific neuroactive agents.

Tau hyperphosphorylation is a complex phenomenon which is still poorly understood. Several factors play important roles in tau hyperphosphorylation. One factor which appears to be involved is heparan sulphate (HS), a sulphated polysaccharide found in the extracellular matrix and at the surface of most cells as part of proteoglycans. Studies have shown that HS can stimulate tau phosphorylation by various kinases and cause tau to aggregate into filaments, in a manner which appears to be related to its degree of sulphation. The presence and density of tau aggregates is correlated with the degree of neurological impairment, suggesting a causal relationship between tau hyperphosphorylation, tangle formation and neurological disease. Several approaches have been thus explored to target tau pathology in tauopathies, and decreasing tau phosphorylation appears a promising approach. These strategies are in the early stages of development, and though a few molecules have already been identified that modify tau phosphorylation, no molecule has so far emerged as an efficient therapeutic agent (Panza et al., 2009). An effective inhibitor of tau phosphorylation, in particular a small molecule inhibitor which is well tolerated and shows low toxicity, would thus have considerable therapeutic potential in the treatment and prevention of tauopathies.

Description of the invention

The inventors have shown for the first time that surfen and derivatives thereof can inhibit tau hyperphosphorylation. Using a zebrafish transgenic line that expresses human mutant tau-P301 L protein, the inventors demonstrated that treatment of transgenic embryos with surfen, or the surfen derivatives surfen HCI, oxalyl surfen HCI and monomeric urea HCL surfen, induces a marked decrease in the accumulation of hyperphosphorylated tau isoforms in vivo, indicating that these molecules may be valuable therapeutic agents for the treatment of tauopathies. As tau hyperphosphorylation is an early step in the development of neurodegenerative disease, they may be used even before the development of detectable pathology to prevent the development of disease.

In its broadest aspect, therefore, the present invention relates to a method of treatment or prevention of a tauopathy using surfen (bis-2-methyl-4-amino-quinolyl-6- carbamide), or an analogue or derivative thereof. The method may comprise administering said compound to an individual in need thereof, such as an individual suffering from a tauopathy. Alternatively, the method may comprise prophylactic administration to an individual not yet suffering from a tauopathy.

The invention also relates to surfen, an analogue or derivative thereof for use in treatment or prevention of tauopathy.

In another aspect, the invention relates to the use of surfen, an analogue or derivative thereof in the manufacture of a medicament for use in the prevention or treatment of tauopathy.

In another aspect, the invention relates to a method of inhibiting or reversing tau hyperphosphorylation in a cell, the method comprising contacting said cell with surfen, an analogue or derivative thereof. In one embodiment, the method is an in vitro or ex vivo method. In another embodiment, the method is an in vivo method. Where the method is an in vivo method, said cell may be a brain cell of a subject, preferably a human subject. Preferably, said cell is a neuronal or glial cell, for example an astrocye, oligodendrocyte or Schwann cell.

In one embodiment, the invention provides a pharmaceutical composition comprising surfen, an analogue or derivative thereof in combination with a pharmaceutically acceptable carrier. Another aspect of the invention relates to a kit for the prevention or treatment of a tauopathy, the kit comprising surfen, an analogue or derivative thereof, preferably provided as a pharmaceutical composition and in a suitable container and/or with suitable packaging, and optionally instructions for use, for example, written instructions on how to administer the compound in the treatment of a tauopathy. In certain embodiments, the tauopathy is Alzheimer's disease. Other tauopathies are known to the skilled person, examples of which are described herein.

The surfen derivatives referred to herein may include In another aspect, the invention provides compounds with the formula I, 1-1 or I-2 as defined below.

In another aspect, the invention provides the use of heparan sulphate inhibitors in the prevention of treatment of tauopathies is also provided, as are methods of prevention and treatment of tauopathy using said HS inhibitors. Said methods may comprise administering said HS inhibitor to an individual in need thereof, such as an individual suffering from a tauopathy. Alternatively, the methods may comprise prophylactic administration to an individual not yet suffering from a tauopathy.

Surfen, analogues and derivatives thereof

The inventors have shown that surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) and derivatives thereof can inhibit tau hyperphosphorylation and thus have potential as a therapeutic agent in the treatment and prevention of tauopathies. Surfe, has the structure shown in Fig 2. Surfen is a small molecule antagonist of HS, which was initially used for the production of depot insulin for diabetic patients. Importantly, surfen displays low toxicity in diabetic patients and is well tolerated in mice, and is thus suitable for therapeutic use.

Derivatives and analogues of surfen are also encompassed by the present invention. Thus, the present invention relates inter alia to a compound of formula (I):

(I)

wherein :

Ri , R₂, R₃, R₄, R5 and R₆ are independently selected from the group consisting of H, optionally substituted C C₆ alkyl, optionally substituted C3-C12 cycloalkyl, optionally substituted C C₆ heterocycloalkyl, optionally substituted C₆-Ci₀ aryl, optionally substituted C₁-C₁₀ heteroaryl, hydroxyl, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted alkylthio, optionally substituted arylthio, C C₆ ester, C₁-C₁₀ acyl, optionally substituted alkylsulfone, optionally substituted arylsulfone, cyano, halo, nitro, haloalkyl, haloalkoxy, fluoroalkyl, amino, alkylamino, dialkylamino and amido;

R_a and R_b are independently selected from the group consisting of H and optionally substituted C C₆ alkyl;

- is a group selected from the group consisting of optionally substituted Ci-C₆ alkylene, optionally substituted C₃-Ci₂ cycloalkylene, optionally substituted C C₆ heterocycloalkylene, optionally substituted C₆-Ci₀ arylene, optionally substituted C₁-C₁₀ heteroarylene, -S0₂- and -[C(0)]_n- wherein n is 1 or 2;

- A₂ is a group selected from the group consisting of H and optionally substituted CrC₆ alkyl, optionally substituted C3-C₁₂ cycloalkyi, optionally substituted CrC₆ heterocycloalkyl, optionally substituted C₆-Ci₀ aryl, optionally substituted C₁-C₁₀ heteroaryl, optionally substituted C C₆ acyl and optionally substituted C C₆ ester.

The term "alkyl" means a saturated or unsaturated aliphatic hydrocarbon group which may be straight or branched having 1 to 6 carbon atoms in the chain. "Branched" means that one or lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. «Lower alkyl» means 1 to 4 carbon atoms in the chain which may be straight or branched. The alkyl may be substituted with one or more «alkyl group substituents» which may be the same or different, and include for instance halo, cycloalkyi, hydroxy (OH), alkoxy, amino (NH₂), acylamino (NHCOAlk), aroylamino (NHCOAr), carboxy (COOH). The term "alkoxy" refers to an -O-alkyl radical.

The term "cycloalkyi" as employed herein includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom capable of substitution may be substituted by a substituent. Examples of cycloalkyi moieties include, but are not limited to, cyclohexyl and adamantyl.

The term "halo" refers to the atoms of the group 17 of the periodic table (halogens) and includes in particular fluorine, chlorine, bromine, and iodine atom. The term "aryl" (or Ar) refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution may be substituted by a substituent. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl.

The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 1 1 -14 membered tricyclic ring system having 1 -4 heteroatoms if monocyclic, 1 -6 heteroatoms if bicyclic, or 1 -9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e. g. , carbon atoms and 1 -4, 1 -6, or 1 -9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution may be substituted by a substituent.

The term "heterocycloalkyl" refers to a nonaromatic 5-7 membered monocyclic, ring system having 1 -3 heteroatoms, said heteroatoms being selected from O, N, or S (e. g. , carbon atoms and 1 -3 heteroatoms of N, O, or S), wherein any ring atom capable of substitution may be substituted by a substituent.

The term "substituents" refers to a group "substituted" on an alkyl, heterocyclyl or aryl group at any atom of that group. Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, S0₃H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(0)_n alkyl (where n' is 0-2), S(0)_n aryl (where n' is 0-2), S(0)_n heteroaryl (where n' is 0-2), S(0)_n heterocyclyl (where n' is 0- 2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heterocyclyl, and unsubstituted cycloalkyl.

The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.

The term "alkylene" refers to a divalent alkyl radical.

The term "cycloalkylene" refers to a divalent cycloalkyl radical.

The term "heterocycloalkylene" refers to a divalent heterocycloalkyl radical.

The term "arylene" refers to a divalent aryl radical.

The term "heteroarylene" refers to a divalent heteroaryl radical.

In formula (I) as defined above, preferably, R₂ is H. Preferably, R₄, R₅ and R₆ are H.

Preferably, Ri is optionally substituted C C₆ alkyl, advantageously methyl.

Preferably, R₃ is amino, alkylamino or dialkylamino, advantageously -NH₂.

Preferably, R_a and R_b are H.

Preferably, is -[C(0)]_n- and n is 1 or 2.

Preferably, A₂ is an optionally substituted C Ci ₀ heteroaryl, comprising at least one nitrogen atom.

Preferably, A₂ is a 6-quinolinyl ring, optionally substituted.

Preferably, A₂ is:

wherein R_{1 ;} R₂, R₃, R₄, R₅ and R₆ are as defined in above formula (I).

Preferably, A₂ is:

As preferred compounds of formula (I), one may cite the compounds wherein:

- Ri , R₂, R₃, R₄, R5 and R₆ are independently selected from the group consisting of

H, optionally substituted C C₆ alkyl, amino, alkylamino and dialkylamino;

- R_a and R_b are independently selected from the group consisting of H and optionally substituted C C₆ alkyl;

- Ai is a group -[C(0)]_n- wherein n is 1 or 2; and

- A₂ is a group selected from the group consisting of H and optionally substituted

C1 -C10 heteroaryl.

As preferred compounds of formula (I), one may cite the compounds having the following formula (1-1 ):

(1-1 )

wherein R_{1 ;} R₂, R₃, R₄, R₅, R₆, A₂ and n are as defined above in formula (I).

Preferably, in formula (1-1 ), Ri is an optionally substituted C C₆ alkyl, advantageously methyl.

Preferably, in formula (1-1 ), R₂, R₄, R₅ and R₆ are H.

Preferably, in formula (1-1 ), R₃ is amino, alkylamino or dialkylamino, advantageously -NH₂. Preferably, in formula (1-1 ), A₂ is a group selected from the group consisting of H and optionally substituted C Ci₀ heteroaryl.

As preferred compounds of formula (1-1 ), one may cite the compounds wherein:

Ri is an optionally substituted C C₆ alkyl;

- R₂, R₄, R₅ and R₆ are H;

- R₃ is selected from the group consisting of amino, alkylamino and dialkylamino; and

- A₂ is a group selected from the group consisting of H and optionally substituted C₁-C₁0 heteroaryl.

As preferred compounds of formula (I), one may cite the compounds having the following formula (I-2):

(I-2)

wherein and A₂ are as defined above in formula (I).

Preferably, in formula (I-2), is is a group -[C(0)]_n- and n is 1 or 2.

Preferably, in formula (I-2), A₂ is H. Preferably, in formula (1-2), A₂ is an optionally substituted C Ci₀ heteroaryl, advantageously comprising at least one nitrogen atom.

As preferred compounds of formula (I-2), one may cite the compounds wherein:

- is a group -[C(0)]_n- wherein n is 1 or 2; and

Preferred derivatives of surfen are shown in Figures 3-5. Characteristics of said derivatives include:

Surfen HCL (shown in Figure 3): molecular weight 408.88; soluble in DMSO, DMF and MeOH; extinction coefficients: 10500 (220nm), 74400 (260nm), 14500 (340nm); preferably used or stored as a 30mM stock solution in DMSO.

Oxalyl Surfen HCL (shown in Figure 4): molecular weight 436.89; soluble in DMSO/H₂0 mixture, DMF and MeOH; preferably used or stored as a 21 .74mM stock solution in 70:30 DMSO/H₂0.

Monomeric Urea Surfen HCL (shown in Figure 5): molecular weight 252.70; soluble in DMSO, DMF and MeOH, preferably used or stored as a 30mM stock solution in DMSO. The synthesis and characterisation of surfen and its derivatives are described in Peng, C- T. and Dasiel, T.C. (1956): The synthesis of some 6-N-substituted amido derivatives of 4,6-diaminoquinaldine and a study of their in vitro antibacterial activity, J. Amer.

Chem.Soc. 78: 3703-3708 and Lanza TJ et al (1992) Substituted 4,6-diaminoquinolines as inhibitors of C5a receptor binding. J Med Chem. 35:252-8.

The present invention also relates to the following preferred compounds:

The compounds herein described may have asymmetric centres. Compounds of the present invention containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well-known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a compound are intended, unless the stereochemistry or the isomeric form is specifically indicated. Also included within the scope of the invention are pharmaceutically acceptable salts, hydrates and solvates of the compounds disclosed herein. "Pharmaceutically acceptable" means it is, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable salt" refers to salts which retain the biological effectiveness and properties of the compounds of the invention and which are not biologically or otherwise undesirable. In many cases, the compounds of the invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids, while pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. For a review of pharmaceutically acceptable salts see Berge, et al. ((1977) J. Pharm. Sd, vol. 66, 1 ). The expression "non-toxic pharmaceutically acceptable salts" refers to non-toxic salts formed with nontoxic, pharmaceutically acceptable inorganic or organic acids or inorganic or organic bases. For example, the salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like, as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, fumaric, methanesulfonic, and toluenesulfonic acid and the like.

A preferred salt of compound of formula (I) is a monohydrochloride salt of compound of formula (I). A preferred salt of compound of formula (1-1 ) is a monohydrochloride salt of compound of formula (1-1 ).

A preferred salt of compound of formula (I-2) is a monohydrochloride salt of compound of formula (I-2).

It should be mentioned here that the monohydrochloride salt of a compound of formula (I-2) may also be present under its corresponding tautomeric form, as follows:

Heparan sulphate inhibitors

Surfen has been characterized as an antagonist of HS, which is able both to antagonize the activity of HS and neutralize anticoagulant activity of heparin (Schuksz et al., 2008). This activity is retained by certain derivatives of surfen. However, it is thought that other derivatives, including monomeric urea surfen, do not have such activity.

A HS inhibitor may be an agent that inhibits HS biosynthesis. For example, a HS inhibitor may be an agent that inhibits sulphation of heparin sulphate, an agent that inhibits deacetylation of heparan sulphate amino groups, for example glucosamine groups, or an agent that inhibits epimerisation of heparan sulphate. Such agents include sodium chlorate, a sulphate donor inhibitor, and and beta-D-xylosides, which act as artificial primers of GAG biosynthesis.

An inhibitor of heparan sulphate may block the interaction of HS with its binding partners, which include lipases, apolipoproteins, lectins, chemokines, cytokines and growth factors such as FGF and VEGF .

Such HS inhibitors include heparin mimics, such as sucrose octasulphate, suramin, pentosan polysulphate and dextran sulphates, as well as proteins and polypeptides containing clusters of positively charged amino acid residues that bind to the negatively charged sulphate and carboxyl groups on HS, such as protamine and lactoferrin. Preferably, a heparin sulphate inhibitor antagonises one or more in vivo activities of heparin sulphate. Functional assays may be used to identify inhibitors of heparan sulphate activity. For example, assays may be used to determine the ability of a candidate agent to block the ability of heparin to activate antithrombin and inactivate Factor Xa, to block heparan sulphate-mediated cell adhesion to the Hep-ll domain of fibrinectin, or to prevent infection by HSV-1 that depends on glycoprotein D interaction with heparan sulphate. Assays for inhibitors of HS are described in, for example, Schuksz et al, 2008, PNAS 105 13075-13080.

Heparan sulphate inhibitors are known in the art, such as, for example, those described in WO/2010/003023. The skilled person will understand that other inhibitors of heparan sulphate may be used to the same end. Other inhibitors of heparan sulphate may thus be encompassed within the scope of the present invention.

Tauopathy

Tauopathies are a class of neurodegenerative diseases characterised by the pathological aggregation of tau protein in the brain. Tau is a microtubule-associated protein which are involved in regulating neuronal mictotubule assembly and stability. The brains of tauopathy patients exhibit neuofibrillary tangles (NFTs) formed by hyperphosphorylation of tau, which causes it to form insoluble aggregates. NFTs consist of tau filaments containing hyperpohosphorylated tau, either in paired helical filaments (PHFs) or straight filaments (SFs). Hyperphosphorylation of tau and the formation of NFTs appear to lead to the death of neurons and degeneration in brain function.

Tau is a phosphoprotein, the phosphorylation of which appears to be involved in regulating its association with microtubules. Increased phosphorylation of tau on multiple serine and threonine residues reduces the ability of tau to promote microtubule assembly and to stabilise assembled microtubules. It is now well established that PHF-tau from AD brain is more heavily phosphorylated on serine and threonine than tau from control brain. The pathological tau from most other cases of other tauopathies seems to be similarly hyperphosphorylated to PHF-tau. These findings strongly imply that similar abnormalities in regulating phosphorylation of tau are shared by all the tauopathies.

'Hyperphosphorylation' of tau may be phosphorylation of an abnormally large number of physiological phosphorylation sites, and/or phosphorylation at sites which are not phosphorylated under normal physiological conditions or in normal subjects. Such abnormal phosphorylation may be detected using specific antibodies AT100, AP422, 988, PHF-27, CP-3, PG-5 et TG3 (Matsuo et al., 1994 ; Hasegawa et al., 1996 ; Hoffman et al., 1997 ; Jicha et al., 1997 ; Zheng-Fischhofer et al., 1998 ; Bussiere et al., 1999 ; Jicha et al., 1999).

The most common and best known tauopathy is Alzheimer's disease (AD), in which tau protein is deposited within neuofibrillary tangles in neurons. In other tauopathies, such tau deposits may be found in both neurons and glial cells. AD, a debilitating neurodegenerative disease for which there is currently no cure, is the most common cause of dementia in older individuals. It destroys neurons in parts of the brain, chiefly the hippocampus, which is a region involved in coding memories. Alzheimer's disease gives rise to an irreversible progressive loss of cognitive functions and of functional autonomy. The earliest signs of AD may be mistaken for simple forgetfulness, but in those who are eventually diagnosed with the disease, these initial signs inexorably progress to more severe symptoms of mental deterioration. While the time it takes for AD to develop will vary from person to person, advanced signs include severe memory impairment, confusion, language disturbances, personality and behaviour changes, and impaired judgement. Persons with AD may become non-communicative and hostile. As the disease ends its course in profound dementia, patients are unable to care for themselves and often require institutionalisation or professional care in the home setting. While some patients may live for years after being diagnosed with AD, the average life expectancy after diagnosis is eight years .

Other tauopathies include progressive supranuclear palsy; dementia pugilistica (chronic traumatic encephalopathy); multiple system atrophy (MSA); Hallervorden-Spatz disease; frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17); Lytico-Bodig disease (Parkinson-dementia complex of Guam); tangle-predominant dementia; ganglioglioma; gangliocytoma; meningioangiomatosis; subacute sclerosing panencephalitis; lead encephalopathy; tuberous sclerosis; Hallervorden-Spatz disease; lipofuscinosis; Pick's disease; corticobasal degeneration; argyrophilic grain dementia (AGD); proteopathy; frontotemporal dementia; frontotemporal lobar degeneration. The non-AD tauopathies are often referred to collectively as 'Pick's complex'.

Therapy

'Prevention' includes stopping or reducing the appearance of, or decreasing the occurrence of, tau hyperphosphorylation or neurodegenerative symptoms in an animal. The prevention may be complete, for example manifest by the total absence of tau hyperphosphorylation or neurodegenerative symptoms. Prevention may also be partial, such that the occurrence of tau hyperphosphorylation or neurodegenerative symptoms in a subject is less than that which would have occurred without the present invention.

'Treatment' includes both therapeutic treatment and prophylactic or preventative treatment, wherein the object is to prevent or slow down the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. The terms 'therapy', 'therapeutic', 'treatment' or 'treating' include reducing, alleviating or inhibiting or eliminating the symptoms or progress of a disease, as well as treatment intended to reduce, alleviate, inhibit or eliminate said symptoms or progress. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, methods and compositions of the invention are used to delay development of a disease or disorder or to slow the progression of a disease or disorder.

Treatment in accordance with the invention includes a method of treating a tauopathy which comprises administering to a patient in need of treatment surfen or a derivative or analogue thereof. The treatment may further comprises administering to said patient an additional agent, as described below. The different components may be administered together, for example in the form of a combined pill, or separately. Administration may be sequential or simultaneous. 'Sequential' administration indicates that the components are administered at different times or time points, which may nonetheless be overlapping. Simultaneous administration indicates that the components are administered at the same time.

Preferably, an effective amount, preferably a therapeutically effective amount of the protein or vector of the invention is administered. An 'effective amount' refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. The effective amount may vary according to the drug or prodrug with which the protein or vector is co-administered.

A 'therapeutically effective amount' of a protein or vector of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein, to elicit a desired therapeutic result. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the protein are outweighed by the therapeutically beneficial effects. A therapeutically effective amount also encompasses an amount sufficient to confer benefit, e.g., clinical benefit.

Pharmaceutical compositions

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more carriers or excipients, preferable a pharmaceutically acceptable carrier or excipient. "Carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions, as detailed below. The phrase "pharmaceutically-acceptable" or "pharmacologically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral and rectal administration .

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pre-gelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methyl-cellulose); fillers (e.g. lactose, microcrystaklline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium, stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycollate) ; or wetting agents (e.g. sodium lauryl sulphate) . The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); and preservatives (e.g. methyl or propyl-p- hydroxybenzoates or sorbic acid) . The preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound . For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurised packs or a nebuliser, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, carbon dioxide or other suitable gas . In the case of a pressurised aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g. gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g. by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides .

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation, for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenser device, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as blister pack. The pack or dispenser device may be accompanied by instructions for administration.

The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.

Administration

The skilled person will appreciate that specific formulations of compositions and precise therapeutic regimes (such as daily doses of the compounds and the frequency of administration) can be determined using known procedures. Such procedures conventionally employed by the pharmaceutical industry include in vivo experimentation and clinical trials.

Generally, a daily dose of between 0.01 g/kg of body weight and 1 .0 g/kg of body weight of the active agent can be used with the methods disclosed herein. In one embodiment, the daily dose is between 0.01 mg/kg of body weight and 100 mg/kg of body weight, or any milligram or half-milligram quantity in this disclosed range, e.g., 1 .5, 2, 2.5, etc.

Daily doses may be given as a single administration (e.g. a daily tablet for oral consumption or as a single daily injection). Alternatively the compound used may require administration twice or more times during a day, depending on the kinetics of the drug associated with the individual patient. Alternatively a slow release device may be used to provide optimal doses to a patient without the need to administer repeated doses

The compounds of the invention may be administered with one or more other therapeutic agent, either sequentially or simultaneously. Where the compound is comprised within a pharmaceutical composition, said composition may further comprise one or more additional therapeutic agents, for example a kinase inhibitor. The invention will now be described in more detail with reference to the following figures. All literature and patent references cited herein are hereby incorporated by reference.

Description of the Figures

Figure 1. Accumulation of hyperphosphorylated Tau isoforms in dissected brains of 5 day old transgenic HuC::TauP301 L and wild-type zebrafish embryos. Quantification of hyperphosphorylated Tau accumulation in transgenic HucC::TauP301 L and wild-type zebrafish embryos, as visualized by AT 180 antibody.

Figures 2-5. Chemical structure of surfen and surfen derivatives. Schematic representation of the chemical structure of surfen (Fig 1 ), surfen HCI (Fig 3), oxalyl surfen HCI (Fig 4 and monomeric urea surfen HCL (Fig 5).

Figure 6. Treatment with surfen and surfen derivatives decreases tau hyperphosphorylation in transgenic HuC::TauP301 L zebrafish embryos. Quantification of hyperphosphorylated tau accumulation transgenic HuC::TauP301 L zebrafish embryos after 48 hours treatment with a negative control (FW+1 %DMSO), a positive control (LiCI, 80 mM), and surfen and surfen derivatives (3 μΜ each) using solid phase sandwich enzyme-linked immunosorbent assays (ELISA).

Experiments

In order to study the effect of surfen treatment on the accumulation of pathologic hyperphosphorylated tau isoforms, the inventors made use of a zebrafish transgenic line, the Tg[Huc::TauP301 L] line, that expresses human mutant tau-P301 L protein and displays the main characteristic features of tauopathies (neuronal loss, accumulation of hyperphosphorylated tau isoforms, tau aggregation, tangles formation and behavioural abnormalities) (Paquet et al., 2009). Using this zebrafish line, the inventors demonstrated here that treatment of transgenic embryos with surfen or two surfen derivatives, surfen HCI and oxalyl surfen HCI, induces in vivo a marked decrease in the accumulation of hyperphosphorylated tau isoforms, indicating that these molecules, may constitute in a near future a valuable therapeutic agent for the treatment of AD and related tauopathies. Hyperphosphorylated isoforms of the microtubule-associated tau protein are the major proteinaceous components of the paired helical and straight filaments, which define a major neuropathological feature of tauopathies, a family of neurodegenerative diseases that comrprises Alzheimer's disease (AD). Previous works have demonstrated that heparin stimulates tau phosphorylation, prevents its binding to taxol-stabilized microtubules, and also promotes tau assembly into Alzheimer-like filaments, thus suggesting that interaction between sulphated heparan and tau is a crucial event in the neuropathology of AD. The inventors made use of a transgenic zebrafish model of tauopathies, the Tg[Huc::TauP301 L] line, which recapitulates key pathological features of the disease, including hyperphosphorylation and conformational changes of the Tau protein, to study whether treatment with surfen and the surfen derivatives surfen HCI, oxalyl surfen HCI and monomeric urea surfen modifies accumulation of hyperphosphorylated tau isoforms. Results show that treatment with surfen, surfen HCI, oxalyl surfen HCI and monomeric urea surfen markedly decreases the accumulation of hyperphosphorylated Tau in embryos of the Tg[Huc::TauP301 L] transgenic line. These data thus show that treatment with surfen and/or surfen derivatives could be an efficient therapeutic strategy for the treatment of tauopathies, including AD. Materials and Methods

Animals

Zebrafish were maintained at 28^ under standard conditions as described by Westerfield (1995). Developmental stages were determined as hour post fertilization (hpf) as described by Kimmel et al. (1995). Wild-type embryos were from the AB and TL strains. The zebrafish transgenic line stably expressing the human mutant Tau-P301 L protein, which is associated with FTD, has been previously described (Paquet et al., 2009). All experiments were performed in accordance with ethical policies for the care and use of laboratory vertebrate animals and were approved by the government (Direction departementale des services veterinaries de Paris).

Compound treatments

Surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) was obtained from the Open Chemical Repository in the Developmental Therapeutic Program at the National Cancer Institute (NSC12155). The synthesis and characterization of surfen and its derivatives) was essentially as described (Peng, C.-T. and Dasiel, T.C. (1956) J. Amer. Chem. Soc. 78: 3703-3708; Lanza TJ et al (1992) J Med Chem. 35:252-8).

As surfen and surfen derivatives bind avidly to plastic, all plasticware was precoated with serum containing medium or glass vessels were used. Stock solutions of surfen and surfen derivatives were prepared as 30 mM stock solutions in dimethyl sulfoxide (DMSO) and stored at -20 ^ in the absence of light and in glass containers under argon. Fresh aqueous solutions were prepared as needed. Stock solutions were diluted with embryo medium E3 also containing 1 % DMSO, which was added as solvent to allow penetration of the compounds in all tissues. Final concentrations were 3 μΜ for surfen and 80 mM for LiCI. 24 hpf embryos were manually dechorionated and then incubated in 1 or 2 ml of the tested compounds in BSA-coated 6-well microtiter plates. All solutions were changed daily. Embryos were treated from either 24 to 72 hpf or 24 to 168 hpf.

ELISA

Following removal of the vitellus, embryos were snap frozen on dry ice and homogenized by sonication in lysis buffer (50 mM Tris HCI pH 8, 150 mM NaCI, 10%

Triton X100, 1 mM EDTA, 1 * Protease Inhibitor Cocktail (Roche)). Insoluble material was removed by a 30 min centrifugation (10,000* g) at 4°C. Protein concentration was determined using a BioRad total protein assay system (Bradford).

Phosphorylated Tau was determined with the INNOTEST™ Phospho-Tau (181 P) ELISA (Innogenetics, Gent Belgium), using mAb HT7 for coating, a phospho-dependent mAb AT270 (specific for phospho-threonine-181 ) as detector antibody, and a synthetic phosphopeptide for standardization. Total tau was measured by INNOTEST hTau-Ag ELISA, (Innogenetics, Gent, Belgium). The T-tau assay utilizes monoclonal antibody (AT120) for capture, and biotinylated monoclonal antibodies (HT7 and BT2) for detection.

Immunocytochemistry.

Whole embryos or dissected brains were fixed in 4% Paraformaldehyde in phosphate buffer saline and preserved in MeOH 100%. Fixed and acetone cracked embryos were blocked and permeabilized for 1 hour at room temperature in a solution containing 10% NGS, 1 % DMSO and 0.1 % Tween 20 in 1 X PBS (or 0.5% Tween 20 for embryos older than 120 hpf). Embryos were then incubated overnight at 4^<Ό with the primary Anti-Human total Tau antibody (Rabbit Polyclonal Antibody, DakoCytomation) diluted at 1 :300 and Anti-PHF-Tau Antibody PHF1 (Mouse Monoclonal Antibody) diluted at 1 :100, a gift from D. Paquet (Adolf Butenandt-lnstitute, Germany), in the same buffer. After several washes, embryos were blocked as previously described and incubated overnight at 4°C with a solution containing goat anti-rabbit CY3 (1 :500) and Alexa Fluor 488-coupled goat anti-mouse antibody (1 :500). Embryos were mounted in 1 % of agarose (low melting, Biorad) in PBS buffer. Image analysis

Bright field images of embryos were captured using a stereomicroscope (SteREO Lumar. V12, Zeiss) equipped with a digital camera (DXM 1200F, Nikon) controlled by the ACT-1 software (Version 2.63 Nikon). Fluorescently labelled embryos were imaged using a microscope equipped with an ApoTome system (Zeiss) equipped with an AxioCam MRm camera (Zeiss) controlled by the Axiovision software. The thickness of the z stacks was always comprised between 2.5 and 3 μηι. All images were processed with Adobe Photoshop 7.0 (Adobe System, San Jose, CA) software. Brightness, contrast, and colour balance were optimized uniformly, if necessary. Results

Zebrafish Tg[Huc::TauP301L] transgenic embryos accumulate hyperphosphorylated tau isoforms in several populations of brain neurons

The two characteristic features of PHFs are tau aggregation and abnormal tau hyperphosphorylation. However, the cellular and biochemical mechanisms underlying these two events remain poorly understood.

The inventors investigated whether treatment with surfen and its derivatives might modulate tau hyperphosphorylation. They made use of the zebrafish Tg[Huc::TauP301 L] transgenic line that expresses human mutant Tau-P301 L protein and displays all the biochemical features of tauopathies, including tau hyperphosphorylation and aggregation, tangle formation, neuronal loss and behavioral abnormalities. In this model, a Gal4/UAS- based vector system was designed to optimize expression of the human mutant Tau- P301 L protein (a mutation genetically linked to FTD) in a large number of zebrafish neurons. In this line, the identification of neurons accumulating the Tau-P301 L protein is greatly facilitated by the simultaneous expression of the fluorescent DsRed protein . In embryos of the Tg[Huc::TauP301 L] line, abnormal disease-related tau hyperphosphorylation was detected in spinal cord neurons as early as 32 hours post fertilization (hpf).

To investigate whether accumulation of pathologic hyperphosphorylated tau also takes place in brain neurons, dissected brains of 5 day post-fertilizatoin (5 dpf) Huc::TauP301 L transgenic embryos were assessed for immunoreactivity with a set of antibodies specific for different hyperphosphorylated tau epitopes corresponding to potentially phosphorylated serine and threonine residues scattered throughout the protein. Interestingly, phosphorylation of these amino acids is specific of either the early or late stages of the disease, thus making it possible to follow a possible evolution of the disease in zebrafish transgenic embryos. In agreement with previous observations in spinal cord neurons (Paquet et al., 2009), in brain neurons of 5 dpf Tg[Huc::TauP301 L] embryos, we detected high levels of accumulation of early stage isoforms when compared to that of isoforms specific of the late stages of the disease, suggesting a progression to advanced pathology in brain neurons. In particular, we detected a strong immunoreactivity with AT180, AT270, and PHF1 , which recognize early stage-epitopes and also AT8, which is specific for a late-stage phosphorylated residue, corresponding to serine and threonine residues T231/S235, T181 , S396/S404 and S202/T205, respectively. We also observed, that accumulation of hyperphosphorylated tau is more prominent in a few brain areas, such as the telencephalon and cerebellum. Quantification of hyperphosphorylated Tau accumulation in transgenic versus wild-type zebrafish embryos is shown in Table 1 and Figure 1 .

Table 1 : Degree of tau hyperphosphorylation in HuC::TauP301 L zebrafish embryos, expressed as hyperphosphorylated tau/micrograms protein

In current diagnostic assays, accumulation of phospho-Tau is used as a biomarker for tauopathies, and a sandwich enzyme-linked immunosorbent assay (ELISA) specific for the proline-directed phosphorylation site, Thr181 , is frequently used in diagnosis (Vanmechelen et al. 2000). In particular, simultaneous determination of total tau levels by ELISA allows us to determine phospho-tau/total tau ratio, thus providing a powerful and sensitive test, which is frequently used in patients.

Analysis of protein extracts from 48 hpf wild-type and Tg[Huc::TauP301 L] transgenic zebrafish embryos showed a very large and significant 90 fold increase in phosphor-tau/ total tau ratio in Tg[Huc::TauP301 L] transgenic zebrafish embryos when compared to that observed in wild-type embryos (Figure 2). Beta amyloid peptide level was also assessed in both samples using a specific sandwich ELISA for beta amyloid peptide (1 -42) and beta amyloid peptide was not detected in either sample (Data not shown).

Surfen and derivatives thereof reduce abnormal hyperphosphorylation of tau

Tau hyperphosphorylation, which is a crucial event in neurofibrillary pathologies, such as tauopathies including AD, has long been considered as a potentially key therapeutic target. The development of inhibitors of tau kinases bears several significant challenges such as achieving selective kinase inhibition and bypassing the essential requirements for these enzymes in several key physiological processes (Klein and Melton, 1996; Brunden et al., 2009; Morris et al., 201 1 ).

The inventors have made use of embryos of the Tg[Huc::TauP301 L] transgenic line to assess whether bis-2-methyl-5-amino-quinolyl-6-carbamide, also named surfen (Figure 2) and derivatives thereof (Figures 3-5) could mitigate tau hyperphosphorylation. In order to determine whether surfen administration could impair, at least partly, tau hyperphosphorylation, 24 hpf Tg[Huc::TauP301 L] embryos were dechorionated and subsequently incubated for 2 days in 3 μΜ surfen in E3 medium also containing 1 % DMSO (see materials and methods). As negative control, age-matched transgenic embryos were incubated for 2 days in E3 medium containing 1 % DMSO. Because lithium chloride has long been shown to decrease tau hyperphosphorylation through inhibition of GSK3B, age-matched transgenic embryos were also incubated for 2 days in 80 mM lithium chloride dissolved in E3 medium and 1 % DMSO as positive control (Perez et al., 2003;Noble et al., 2005;Nakashima et al., 2005;Engel et al., 2006; Caccamo et al. 2007; Leroy et al., 2010). ELISA assays for phospho-tau and total tau revealed that while treatment with lithium chloride caused a 50% decrease in the amount of pathologically phosphorylated T181 tau residue, embryos treated with surfen, surfen HCI and oxalyl surfen HCL embryos showed 35%, and 37% and 39% decrease in the amount of T181 phosphorylated tau, respectively (Figure 6). Importantly, careful examination of these five sets of embryos showed that no identifiable abnormalities could be detected following a 4 day treatment with either surfen, surfen HCI, oxalyl surfen HCI, monomeric urea surfen or lithium chloride treatments.

Immunohistochemical studies using PHF1 antibody that recognizes phosphorylated S396/S404 tau residues, also revealed a marked decreased accumulation of these pathological epitopes in motor neuron axons and neurites of embryos treated with either LiCI or surfen when compared to that observed in motor neuron axons and neurites of untreated embryos.

The inventors have thus demonstrated that treatment with surfen, surfen HCI, oxalyl surfen HCI and monomeric urea surfen markedly reduces in vivo accumulation of hyperphosphorylated tau in a zebrafish transgenic model that displays all biochemical tau characteristics observed in tauopathies.

Claims

1 . A compound of general formula (I) for use in the treatment or prevention of a tauopathy, wherein general formula (I) is:

wherein

Ri , R₂, R3, R₄, R5 and R₆ are independently selected from the group consisting of H, optionally substituted C C₆ alkyl, optionally substituted C₃- C₁₂ cycloalkyl, optionally substituted C C₆ heterocycloalkyl, optionally substituted C₆-Ci₀ aryl, optionally substituted C₁-C₁0 heteroaryl, hydroxyl, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted alkylthio, optionally substituted arylthio, C C₆ ester, C₁-C₁₀ acyl, optionally substituted alkylsulfone, optionally substituted arylsulfone, cyano, halo, nitro, haloalkyl, haloalkoxy, fluoroalkyl, amino, alkylamino, dialkylamino and amido;

- is a group selected from the group consisting of optionally substituted Ci-C₆ alkylene, optionally substituted C₃-Ci₂ cycloalkylene, optionally substituted C C₆ heterocycloalkylene, optionally substituted C₆-Ci₀ arylene, optionally substituted C₁-C₁₀ heteroarylene, -S0₂- and -[C(0)]_n- wherein n is 1 or 2; and

- A₂ is a group selected from the group consisting of H and optionally substituted C C₆ alkyl, optionally substituted C₃-Ci₂ cycloalkyl, optionally substituted C C₆ heterocycloalkyl, optionally substituted C₆-Ci₀ aryl, optionally substituted C₁-C₁₀ heteroaryl, optionally substituted C C₆ acyl and optionally substituted C C₆ ester; or a pharmaceutically acceptable salt, hydrate or solvate thereof; wherein optionally:

Ri , R₂, R3, R₄, R5 and R₆ are independently selected from the group consisting of H, optionally substituted C C₆ alkyl, amino, alkylamino and dialkylamino;

- is a group -[C(0)]_n- wherein n is 1 or 2; and

- A₂ is a group selected from the group consisting of H and optionally substituted C₁-C₁₀ heteroaryl.

2. A compound of general formula (I) for use according to claim 1 , wherein said compound is a compound of general formula (1-1 ):

(1-1 )

wherein Ri , R₂, R₃, R₄, R5, Re, A₂ and n are as defined in claim 1 ;

wherein optionally:

Ri is an optionally substituted C C₆ alkyl;

R₂, R₄, R₅ and R₆ are H;

R₃ is selected from the group consisting of amino, alkylamino and dialkylamino; and

3. A compound of general formula (I) for use according to claim 1 , wherein said compound is a compound of general formula (I-2):

(I-2)

wherein Ai and A₂ are as defined in claim 1 ; wherein optionally:

- Ai is a group -[C(0)]_n- wherein n is 1 or 2; and

- A₂ is a group selected from the group consisting of H and optionally substituted C Ci₀ heteroaryl.

4. A compound of general formula (I) for use according to claim 1 , wherein

(i) A₂ is an optionally substituted C Ci₀ heteroaryl, comprising at least one nitrogen atom; or

(ii) A₂ is H.

5. A monohydrochloride salt of a compound of formula (I) for use according to claim 1 .

6. A compound of general formula (I) for use according to claim 1 , wherein said compound of general formula (I) is a compound selected from:

7. A compound of general formula (I) for use according to any one of claims 1 to 6, wherein the tauopathy is Alzheimer's disease; progressive supranuclear palsy; dementia pugilistica; multiple system atrophy; Hallervorden-Spatz disease; frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17); Lytico-Bodig disease (Parkinson- dementia complex of Guam); tangle-predominant dementia; ganglioglioma; gangliocytoma; meningioangiomatosis; subacute sclerosing panencephalitis; lead encephalopathy; tuberous sclerosis; Hallervorden-Spatz disease; lipofuscinosis; Pick's disease; corticobasal degeneration; argyrophilic grain dementia ; frontotemporal dementia or frontotemporal lobar degeneration.

8. A compound of formula I, 1-1 or I-2 as defined in any one of claims 1 to 6 for use in the prevention of treatment of a tauopathy.

9. A pharmaceutical composition comprising compound according to any one of claims 1 to 8 in combination with a pharmaceutically acceptable carrier, for use in treatment or prevention of tauopathy.

10. A pharmaceutical composition according to claim 9, wherein the compound of general formula (I) is as defined in any one of claims 1 to 6 and/or the tauopathy is as defined in claim 7.

1 1 . An in vitro method of inhibiting or reversing tau hyperphosphorylation in a cell, the method comprising contacting said cell with a compound according to any one of claims 1 to 8.

12. A method according to claim 1 1 , wherein the compound is as defined in any one of claims 1 to 6.

13. A kit for the prevention or treatment of a tauopathy, the kit comprising a pharmaceutical composition according to claim 10 or claim 1 1 , and instructions on how to administer the compound in the treatment of a tauopathy.

14. A kit according to claim 13, wherein the tauopathy is as defined in claim 7.