GB2467562A - Dual calpain-ROS inhibitors - Google Patents

Abstract

Described are compounds of Formula (1)or a pharmaceutically acceptable derivative, N-oxide, salt, hydrate, solvate, complex, bioisostere, metabolite or prodrug thereof, wherein Ar2, L2, Ar1, L1, Raa, R3, R5and Y are as defined therein, the compound being a dual calpain-ROS inhibitor for the treatment of a disease mediated by ROS and/or calpain hyperactivity.

Description

DUAL CALPAIN-ROS INHIBITORS

Field of the Invention

This invention relates to dual calpain-ROS inhibitors and their use in medicine.

Background of the Invention

Calpain Calpain defines a ubiquitous class of calcium-dependent cysteine proteases which cleave many cytoskeletal and myelin proteins. Thus, calpain defines a family of Ca2 activated intracellular proteases the activity of which is upregulated when abnormal amounts of Ca2 enter the cell by virtue of increased membrane permeability (e.g. as a result of traumata, ischaemic event and/or a genetic defect). Increase in concentration of calcium in the cell results in calpain activation, which leads to unregulated proteolysis of both target and non-target proteins and consequent irreversible tissue damage. For example, calpain hyperactivity results in the destruction of cytoskeletal components such as spectrin, microtubule subunits, microtubule-associated proteins and neurofilaments. It may also damage ion channels, other enzymes, cell adhesion molecules, and cell surface receptors.

This can lead to degradation of both the cytoskeleton and plasma membranes.

Calpain may also break down sodium channels that have been damaged due to axonal stretch injury, leading to an influx of sodium into the cell. This leads to depolarization and further Ca2 influx and can thereby lead to cardiac contractile dysfunction that follows ischemic insult to the heart which upon reperfusion can lead to the development of calcium overload and further activation of calpain.

Calpains also mediate apoptosis and have also been implicated in the initiation of necrotic cell death. When calpain activity is abnormally upregulated (calpain hyperactivity), cellular and tissue degradation may outstrip regeneration. Thus, calpain hyperactivity can cause a wide variety of serious neuromuscular and neurodegenerative diseases: for example, calpain has been implicated in the muscle wasting associated with muscular dystrophies (including DMD).

At least two genetic disorders and one form of cancer have been linked to tissue-specific calpains. When defective, the mammalian calpain 3 is responsible for limb-girdle muscular dystrophy type 2A, calpain 10 appears to be involved in type II diabetes mellitus and calpain 9 has been identified as a tumor suppressor for gastric cancer. Moreover, the hyperactivation of calpains is implicated in a number of pathologies associated with altered calcium homeostasis such as Alzheimer's disease and cataract formation, as well as secondary degeneration resulting from acute cellular stress following myocardial ischemia, cerebral (neuronal) ischemia, traumatic brain injury and spinal cord injury.

Excessive amounts of calpain can be activated due to Ca2 influx after cerebrovascular accident (e.g. during the ischaemic cascade) or some types of traumatic brain injury (such as diffuse axonal injury).

Calpain may be released in the brain for up to a month after a head injury and is thought to be responsible for a shrinkage of the brain sometimes found after such injuries.

Reactive oxygen species (ROS) Aerobic metabolism in living organisms can lead to generation of reactive oxygen species (ROS). These include hydroxyl radicals, superoxide anion, hydrogen peroxide and nitric oxide. Production of ROS can be due to various enzymatic and non-enzymatic processes.

In aerobic organisms, ROS are formed from the partial reduction of molecular oxygen to water during oxidative metabolism.

Under normal conditions, ROS may play an important role in different biological processes.

However, when ROS are excessively produced under certain unusual conditions (leading to ROS hyperactivity), they can cause oxidative damage to DNA, proteins, lipids and enzymes (e.g. via oxidative inactivation of co-factors).

Thus, ROS are implicated in the pathogenesis of many different diseases, disorders and conditions. These include aging, AIDS, atherosclerosis, cancer, cataracts, hearing impairment (via cochlea damage induced by elevated sound levels and in certain congenital forms of deafness), congestive heart failure, diabetes, inflammatory disorders, cardiovascular diseases, rheumatoid arthritis and neuro-degenerative diseases such as Alzheimer's, Parkinson's, multiple sclerosis and Down's syndrome. Hyperactivity of ROS may also mediate the cytotoxic effects of drugs such as cisplatin.

There is therefore a need for pharmaceutical compounds which inhibit calpain and ROS activity in vivo.

Prior art

WO01/32654 describes chimaeric calpain-ROS inhibitors of general formula: Hc**i for the treatment of pathologies where calpains and/or reactive oxygen species are involved, including muscular dystrophies.

W02002/4001 6 describes pharmaceutical compositions comprising combinations of a calpain inhibiting substance and a substance trapping reactive oxygen species (ROS), the active principles being separate.

W02005/056551 describes chimaeric calpain-ROS inhibitors being derivatives of 2-hydroxytetrahydrofuran corresponding to the general formula described in WO 0 1/32654 (and shown above) for the treatment of inflammatory and immunological diseases, cardio-vascular and cerebro-vascular diseases, disorders of the central or peripheral nervous system, cachexia, osteoporosis, muscular dystrophy, proliferative diseases, cataract, rejection reactions following organ transplants and autoimmune and viral diseases.

W02005/092345 describes chimaeric calpain-ROS inhibitors being phenothiazine derivative general formula: o - -- ,Q N i

I N 0 OR 1*

for preventing and/or treating hearing loss.

W02007/045761 describes chimaeric calpain-ROS inhibitors of general formula: R 1 *V kY > N 0 R -i'--

-I

R R' H OR for the treatment of pathologies where calpains and/or reactive oxygen species are involved, including muscular dystrophies.

W02007/1 01937 describes chimaeric calpain-ROS inhibitors of the general formula described in WO 2007/045761 (and shown above) in combination with steroids, corticoids or corticosteroids for the treatment of pathologies where calpains and/or reactive oxygen species are involved, including muscular dystrophies.

Auvin eta!. (2004) Bioorganic & Medicinal Chemistry Letters 14(14):3825-8 describe a series of molecules with dual inhibitory activities on calpain and lipid peroxidation built on the calpain pharmacophore 2-hydroxytetrahydrofuran linked to a set of antioxidants via a I-leucine linker.

Pignol eta!. (2006) Journal of Neurochemistry 98(4):1217 describe a synergistic action of calpain inhibitors and antioxidants in the prevention of cell necrosis.

Auvin eta!. (2006) Bioorganic & Medicinal Chemistry Letters 16(6):1586-9 describe a series of dipeptides with dual inhibitory activities on calpain and lipid peroxidation.

Summary of the Invention

According to a first aspect of the present invention there is provided a compound of Formula (1) Ar2LArLL1 0 H in which Y represents an amino acid residue optionally substituted by one or more halo, OR7, S(O)R7, ON orC15alkyl R7 represents independently H; 01-5 alkyl; O38cycloalkyl and optionally substituted aryl n represents an integer from 0 to 2 R3 represents H or Oi5alkyl, optionally substituted by halo R5 represents H; 015a1ky1, optionally substituted by one or more halo, OH, 0-alkyl; aryl, optionally substituted by one or more halo, OH, 0-alkyl; aralkyl, optionally substituted by one or more halo, OH, 0-alkyl; glycosyl or uronyl Raa represents the side chain of a natural amino acid L1 represents a bond; (CR3R3); 0(0); C(O)NR3; C(S); C(S)NR3; C(NR3); C(NR3)NR3; 0(0)0; SO2 or C(O)(0H2)mO L2 is absent or represents a bond; (CR3R3); 0(0); 0, S(O), NR3 or P(O)0R3 Ar1 represents aryl or aralkyl, optionally containing one or more heteroatoms selected from 0, N and S and optionally substituted by one or more R6 groups Ar2 is absent or represents aryl or aralkyl, optionally containing one or more heteroatoms selected from 0, N and S and optionally substituted by one or more R6 g rou PS R6 represents one or more substituents attached to either or both Ar1 and Ar2 and is independently selected from H; halo; ON, NO2, OR7; S(O)R7; NR3R7; 01-06 alkyl; 02-06 alkenyl; 02-06 alkynyl; 03-08 cycloalkyl; aryl; aralkyl; 038 heterocyclyl, OO(O)R7; O(O)0R7; NR3O(O)R7; O(O)NR3R7; NR3O(S)R7; O(S)NR3R7; NR3O(NR3)R7; O(NR3)NR3R7; NR3SO2R7; SO2NR3R7 or P(O)R70R7, wherein where R3 and R7 are attached to the same nitrogen atom they may form an optionally substituted heterocyclic ring containing one additional atom or group selected from 0, NR7 or S and wherein where two R6 substituents are present on the same ring system they may form an optionally substituted fused ring m represents an integer from 1 to 2 p represents an integer from 0 to 2 or a pharmaceutically acceptable derivative, N-oxide, salt, hydrate, solvate, complex, bioisostere, metabolite or prodrug thereof, the compound being a dual calpain-ROS inhibitor for the treatment of a disease mediated by ROS and/or calpain hyperactivity.

Y may represent an amino acid in the natural L form. In preferred embodiments, Y represents a lipophilic amino acid residue, for example a lipophilic amino acid residue in the natural L form. In preferred embodiments, Y is an amino acid residue selected from phenylalanine, valine, leucine and isoleucine residues.

Other aspects and embodiments of the invention are defined and described in the claims set out below.

Detailed Description of the Invention

All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

Definitions and general preferences Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art: Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

The phrase "consisting essentially of" is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention.

As used herein, the term "consisting" is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) alone.

As used herein, the term "disease" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses diseases mediated by infection, trauma, injury, surgery, radiological ablation, poisoning or nutritional deficiencies.

As used herein, the term "disease mediated by" as applied to various agents/mechanisms/processes or events (e.g. ROS and/or calpain hyperactivity) defines a disease (as defined above) which is mediated by the referenced agents/mechanisms/processes or events (i.e. a disease in which the referenced agents/mechanisms/processes or events (e.g. ROS and/or calpain hyperactivity) form at least part of the aetiology). The term "mediated" in this context defines diseases in which referenced agents/mechanisms/processes or events (e.g. ROS and/or calpain activity are pathologically elevated, dysregulated or active, act directly or indirectly and which may be necessary and/or sufficient for the manifestation of the symptoms of the disease (or its progression). Thus, diseases mediated by ROS and/or calpain (and in particular aberrant levels of ROS and/or calpain activity, e.g. ROS and/or calpain hyperactivity) need not necessarily be the proximal cause of the disease: rather, it is contemplated that the ROS and/or calpain mediated diseases include those having multifactorial aetiologies and complex progressions in which the ROS and/or calpain activity forms a component, other aspects of the disease and/or its progression arising from other aetiologies.

As used herein, the term "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s). In this case, the term is used synonymously with the term "therapy".

Additionally, the terms "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term "prophylaxis".

In this context "subject" (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In preferred embodiments, the subject is a human.

As used herein, an effective amount or a therapeutically effective amount of a compound defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure.

As used herein, a "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.

Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

As used herein, the term "calpain" defines any protease which is a member of a family of calcium-dependent, non-lysosomal cysteine proteases constituting the 02 family of protease clan CA in the MEROPS database (see Rawlings et al. ,(2008) MEROPS: the peptidase database. Nucleic Acids Res 36, D320-D325). The term therefore includes calpain-1.

The term "calpain inhibitor" is used herein to define any agent capable of inhibiting the activity of calpain (e.g. calpain-1). The term "calpain inhibiting pharmacophore" is used herein to define the chemical group(s) responsible for the inhibitory activity of a calpain inhibitor. Suitable calpain inhibitors and pharmacophores can be identified using commercially available assay kits (e.g. the calpain activity kit based on a fluorogenic substrate from Oncogene Research Products, San Diego, CA). This assay measures the ability of calpain to digest the synthetic substrate Suc-LLVY-AMC: free AMC can be measured fluorometrically at an excitation of 360-380 nm and an emission of 440-460 nm.

As used herein, the term "reactive oxygen species" (ROS) include without limitation hydroxyl radicals, superoxide anion, hydrogen peroxide and nitric oxide and the term "ROS inhibiting moiety"/"ROS inhibitor" is used herein to define any agent, moiety or chemical group(s) capable of suppressing the activity or removing ROS in tissues (e.g. by breaking them down chemically or by sequestering them).

The term "dual calpain-ROS inhibitor" is used herein to define compounds which are calpain inhibitors and which also act as ROS inhibitors by virtue of the presence of a calpain inhibiting pharmacophore and a ROS inhibiting moiety.

As used herein, the terms mobilizing agent and mobilization are terms of art referring to agents and treatments which serve to promote the migration of CD34, stem, progenitor and/or precursor cells from the marrow to the peripheral blood (for a review, see e.g. Cottler-Fox etal. (2003) Stem cell mobilization Hematology: 419-437). Current standard agents for mobilization suitable for use according to the invention include G-CSF (FilgrastimTM, Amgen), GM-CSF (SargramostimTM, Berlex, Richmond, CA) and erythropoietin (which has some mobilizing activity w.r.t. CD34 cells). Alternative agents include stem cell factor (SCF) (which is particularly effective when used in combination with G-CSF) and various derivatives of G-CSF (PegfilgrastimTM, Amgen) and erythropoietin (Darbopoietin�, Amgen). The latter agents benefit from extended half-lives and so increase the temporal window available for collection. AMD3100 (AnorMedTM, Vancouver, Canada), which is a reversible inhibitor of the binding of stromal derived factor (SDF-1 a) to its cognate receptor CXCR4, is currently in clinical trials as a mobilizing agent. Other agents include docetaxel (see e.g. Prince et al. (2000) Bone Marrow Transplantation 26: 483-487).

The term "upregulation of utrophin" as used herein includes elevated expression or over-expression of utrophin, including gene amplification (i.e. multiple gene copies) and increased expression by a transcriptional effect, and hyperactivity and activation of utrophin, including activation by mutations. The term "utrophin upregulating agent" is to be interpreted accordingly. Thus, upregulation of utrophin covers increasing utrophin activity at the level of the encoding DNA as well as the transcriptional, translational or post-translational level.

As used herein, the term "antibody" defines whole antibodies (including polyclonal antibodies and monoclonal antibodies (Mabs)). The term is also used herein to refer to antibody fragments, including F(ab), F(ab'), F(ab')2, Fv, Fc3 and single chain antibodies (and combinations thereof), which may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. The term "antibody" is also used herein to cover bispecific or bifunctional antibodies which are synthetic hybrid antibodies having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. Also covered by the term "antibody" are chimaeric antibodies (antibodies having a human constant antibody immunoglobulin domain coupled to one or more non-human variable antibody immunoglobulin domain, or fragments thereof). Such chimaeric antibodies therefore include "humanized" antibodies. Also covered by the term "antibody" are minibodies (see WO 94/09817), single chain Fv-Fc fusions and human antibodies antibodies produced by transgenic animals The term "antibody' also includes multimeric antibodies and higher-order complexes of proteins (e.g. heterodimeric antibodies).

The term "adjunctive" as applied to the use of the compounds of the invention in therapy or prophylaxis defines uses in which the compound is administered together with one or more other drugs, interventions, regimens or treatments (such as surgery and/or irradiation).

Such adjunctive therapies may comprise the concurrent, separate or sequential administration/application of the materials of the invention and the other treatment(s).

Thus, in some embodiments, adjunctive use of the materials of the invention is reflected in the formulation of the pharmaceutical compositions of the invention. For example, adjunctive use may be reflected in a specific unit dosage, or in formulations in which the compound of the invention is present in admixture with the other drug(s) with which it is to be used adjunctively (or else physically associated with the other drug(s) within a single unit dose). In other embodiments, adjunctive use of the compounds or compositions of the invention may be reflected in the composition of the pharmaceutical kits of the invention, wherein the compound of the invention is co-packaged (e.g. as part of an array of unit doses) with the other drug(s) with which it is to be used adjunctively. In yet other embodiments, adjunctive use of the compounds of the invention may be reflected in the content of the information and/or instructions co-packaged with the compound relating to formulation and/or posology.

As used herein, the term "combination", as applied to two or more compounds and/or agents (also referred to herein as the components), is intended to define material in which the two or more compounds/agents are associated. The terms "combined" and "combining" in this context are to be interpreted accordingly.

The association of the two or more compounds/agents in a combination may be physical or non-physical. Examples of physically associated combined compounds/agents include: * compositions (e.g. unitary formulations) comprising the two or more compounds/agents in admixture (for example within the same unit dose); * compositions comprising material in which the two or more compounds/agents are chemically/physicochemically linked (for example by crosslinking, molecular agglomeration or binding to a common vehicle moiety); * compositions comprising material in which the two or more compounds/agents are chemically/physicochemically co-packaged (for example, disposed on or within lipid vesicles, particles (e.g. micro-or nanoparticles) or emulsion droplets); * pharmaceutical kits, pharmaceutical packs or patient packs in which the two or more compounds/agents are co-packaged or co-presented (e.g. as part of an array of unit doses); Examples of non-physically associated combined compounds/agents include: * material (e.g. a non-unitary formulation) comprising at least one of the two or more compounds/agents together with instructions for the extemporaneous association of the at least one compound/agent to form a physical association of the two or more compounds/agents; * material (e.g. a non-unitary formulation) comprising at least one of the two or more compounds/agents together with instructions for combination therapy with the two or more compounds/agents; * material comprising at least one of the two or more compounds/agents together with instructions for administration to a patient population in which the other(s) of the two or more compounds/agents have been (or are being) administered; * material comprising at least one of the two or more compounds/agents in an amount or in a form which is specifically adapted for use in combination with the other(s) of the two or more compounds/agents.

As used herein, the term "combination therapy" is intended to define therapies which comprise the use of a combination of two or more compounds/agents (as defined above).

Thus, references to "combination therapy", "combinations" and the use of compounds/agents "in combination" in this application may refer to compounds/agents that are administered as part of the same overall treatment regimen. As such, the posology of each of the two or more compounds/agents may differ: each may be administered at the same time or at different times. It will therefore be appreciated that the compounds/agents of the combination may be administered sequentially (e.g. before or after) or simultaneously, either in the same pharmaceutical formulation (i.e. together), or in different pharmaceutical formulations (i.e. separately). Simultaneously in the same formulation is as a unitary formulation whereas simultaneously in different pharmaceutical formulations is non-unitary. The posologies of each of the two or more compounds/agents in a combination therapy may also differ with respect to the route of administration.

As used herein, the term "pharmaceutical kit" defines an array of one or more unit doses of a pharmaceutical composition together with dosing means (e.g. measuring device) and/or delivery means (e.g. inhaler or syringe), optionally all contained within common outer packaging. In pharmaceutical kits comprising a combination of two or more compounds/agents, the individual compounds/agents may unitary or non-unitary formulations. The unit dose(s) may be contained within a blister pack. The pharmaceutical kit may optionally further comprise instructions for use.

As used herein, the term "pharmaceutical pack" defines an array of one or more unit doses of a pharmaceutical composition, optionally contained within common outer packaging. In pharmaceutical packs comprising a combination of two or more compounds/agents, the individual compounds/agents may unitary or non-unitary formulations. The unit dose(s) may be contained within a blister pack. The pharmaceutical pack may optionally further comprise instructions for use.

As used herein, the term "patient pack" defines a package, prescribed to a patient, which contains pharmaceutical compositions for the whole course of treatment. Patient packs usually contain one or more blister pack(s). Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions.

The combinations of the invention may produce a therapeutically efficacious effect relative to the therapeutic effect of the individual compounds/agents when administered separately.

The term pharmaceutically acceptable solvate as applied to the compounds of the invention defines any pharmaceutically acceptable solvate form of a specified compound that retains the biological effectiveness of such compound. Examples of solvates include compounds of the invention in combination with water (hydrates), isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine, or acetone. Also included are miscible formulations of solvate mixtures such as a compound of the invention in combination with an acetone and ethanol mixture. In a preferred embodiment, the solvate includes a compound of the invention in combination with about 20% ethanol and about 80% acetone. Thus, the structural formulae include compounds having the indicated structure, including the hydrated as well as the non-hydrated forms.

The term pharmaceutically acceptable prodrug as applied to the compounds of the invention defines any pharmaceutically acceptable compound that may be converted under physiological conditions or by solvolysis to the specified compound, to a pharmaceutically acceptable salt of such compound or to a compound that shares at least some of the activity of the specified compound.

The term pharmaceutically acceptable metabolite as applied to the compounds of the invention defines a pharmacologically active product produced through metabolism in the body of the specified compound or salt thereof.

Prodrugs and active metabolites of the compounds of the invention may be identified using routine techniques known in the art (see for example, Bertolini et al., J. Med. Chem., 1997, 40, 2011-2016).

The term pharmaceutically acceptable complex as applied to the compounds of the invention defines compounds or compositions in which the compound of the invention forms a component part. Thus, the complexes of the invention include derivatives in which the compound of the invention is physically associated (e.g. by covalent or non-covalent bonding) to another moiety or moieties. The term therefore includes multimeric forms of the compounds of the invention. Such multimers may be generated by linking or placing multiple copies of a compound of the invention in close proximity to each other (e.g. via a scaffolding or carrier moiety).

The term bioisostere (or simply isostere) is a term of art used to define drug analogues in which one or more atoms (or groups of atoms) have been substituted with replacement atoms (or groups of atoms) having similar steric and/or electronic features to those atoms which they replace. The substitution of a hydrogen atom or a hydroxyl group with a fluorine atom is a commonly employed bioisosteric replacement. Sila-substitution (C/Si-exchange) is a relatively recent technique for producing isosteres. This approach involves the replacement of one or more specific carbon atoms in a compound with silicon (for a review, see article by Tacke and Zilch in Endeavour, New Series, 1986, 10, 191-197). The sila-substituted isosteres (silicon isosteres) may exhibit improved pharmacological properties, and may for example be better tolerated, have a longer half-life or exhibit increased potency (see for example article by Englebienne in Med. Chem., 2005, 1(3), 215-226). In its broadest aspect, the present invention contemplates all bioisosteres (and specifically, all silicon bioisosteres) of the compounds of the invention.

In its broadest aspect, the present invention contemplates all optical isomers, racemic forms and diastereoisomers of the compounds described herein. Those skilled in the art will appreciate that, owing to the asymmetrically substituted carbon atoms present in the compounds of the invention, the compounds may be produced in optically active and racemic forms. If a chiral centre or another form of isomeric centre is present in a compound of the present invention, all forms of such isomer or isomers, including enantiomers and diastereoisomers, are intended to be covered herein. Compounds of the invention containing a chiral centre (or multiple chiral centres) may be used as a racemic mixture, an enantiomerically enriched mixture, or the racemic mixture may be separated using well-known techniques and an individual enantiomer may be used alone. Thus, references to the compounds of the present invention encompass the products as a mixture of diastereoisomers, as individual diastereoisomers, as a mixture of enantiomers as well as in the form of individual enantiomers.

Therefore, the present invention contemplates all optical isomers and racemic forms thereof of the compounds of the invention, and unless indicated otherwise (e.g. by use of dash-wedge structural formulae) the compounds shown herein are intended to encompass all possible optical isomers of the compounds so depicted. In cases where the stereochemical form of the compound is important for pharmaceutical utility, the invention contemplates use of an isolated eutomer.

The terms derivative and pharmaceutically acceptable derivative as applied to the compounds of the invention define compounds which are obtained (or obtainable) by chemical derivatization of the parent compound of the invention. The pharmaceutically acceptable derivatives are therefore suitable for administration to or use in contact with the tissues of humans without undue toxicity, irritation or allergic response (i.e. commensurate with a reasonable benefit/risk ratio). Preferred derivatives are those obtained (or obtainable) by alkylation, esterification or acylation of the parent compounds.

The derivatives may be active per Se, or may be inactive until processed in vivo. In the latter case, the derivatives of the invention act as prodrugs. Particularly preferred prodrugs are ester derivatives which are esterified at one or more of the free hydroxyls and which are activated by hydrolysis in vivo. Other preferred prodrugs are covalently bonded compounds which release the active parent drug according to general formula (I) after cleavage of the covalent bond(s) in vivo.

The pharmaceutically acceptable derivatives of the invention may retain some or all of the biological activities described herein. In some cases, the biological activity is increased by derivatization. The derivatives may act as pro-drugs, and one or more of the biological activities described herein may arise only after in vivo processing. Particularly preferred pro-drugs are ester derivatives which are esterified at one or more of the free hydroxyls and which are activated by hydrolysis in vivo. Derivatization may also augment other biological activities of the compound, for example bioavailability and/or calpain inhibitory activity and/or ROS scavenging activity. For example, derivatization may increase calpain inhibitory potency and/or specificity and/or CNS penetration (e.g. penetration of the blood-brain barrier).

The term pharmaceutically acceptable salt as applied to the compounds of the invention defines any non-toxic organic or inorganic acid addition salt of the free base which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and which are commensurate with a reasonable benefit/risk ratio. Suitable pharmaceutically acceptable salts are well known in the art.

Examples are the salts with inorganic acids (for example hydrochloric, hydrobromic, sulphuric and phosphoric acids), organic carboxylic acids (for example acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, dihydroxymaleic, benzoic, phenylacetic, 4-aminobenzoic, 4-hydroxybenzoic, anthranilic, cinnamic, salicylic, 2-phenoxybenzoic, 2-acetoxybenzoic and mandelic acid) and organic sulfonic acids (for example methanesulfonic acid and p-toluenesulfonic acid).

These salts and the free base compounds can exist in either a hydrated or a substantially anhydrous form. Crystalline forms, including all polymorphic forms, of the compounds of the invention are also contemplated and in general the acid addition salts of the compounds are crystalline materials which are soluble in water and various hydrophilic organic solvents and which in comparison to their free base forms, demonstrate higher melting points and an increased solubility.

The term "amino acid" is used herein to cover any naturally-occurring amino acid.

Preferred are natural amino acids in the L form. The term "amino acid residue" is to be interpreted accordingly.

The term "lipophilic amino acid" as used herein defines an optionally substituted amino acid group bearing an alkyl, aryl, alkaryl or carbocyclic substituent with no ionisable groups, for example phenylalanine, valine, leucine and isoleucine.

In the present specification the term "alkyl" defines a straight or branched saturated hydrocarbon chain. The term "C1-C5alkyl" refers to a straight or branched saturated hydrocarbon chain having one to five carbon atoms. The term "C1-C10 alkyl" refers to a straight or branched saturated hydrocarbon chain having one to ten carbon atoms.

Examples include methyl, ethyl, n-propyl, isopropyl and t-butyl. The alkyl groups of the invention may be optionally substituted by one or more halogen atoms.

In the present specification the term "alkenyl" defines a straight or branched hydrocarbon chain having containing at least one carbon-carbon double bond. The term "C1-C6 alkenyl" refers to a straight or branched unsaturated hydrocarbon chain having one to six carbon atoms. The term "01-09 alkenyl" refers to a straight or branched unsaturated hydrocarbon chain having one to nine carbon atoms. The term "01-015 alkenyl" refers to a straight or branched unsaturated hydrocarbon chain having one to fifteen carbon atoms. Preferred is C-C alkenyl. Examples include ethenyl, 2-propenyl, and 3-hexenyl. The alkenyl groups of the invention may be optionally substituted by one or more halogen atoms.

In the present specification the term "alkynyl" defines a straight or branched hydrocarbon chain having containing at least one carbon-carbon triple bond. The term 01-06 alkynyl" refers to a straight or branched unsaturated hydrocarbon chain having one to six carbon atoms. The term "01-09 alkynyl" refers to a straight or branched unsaturated hydrocarbon chain having one to nine carbon atoms. The term "01-015 alkynyl" refers to a straight or branched unsaturated hydrocarbon chain having one to fifteen carbon atoms. Preferred is 01-06 alkynyl. Examples include ethynyl, 2-propynyl, and 3-hexynyl. The alkynyl groups of the invention may be optionally substituted by one or more halogen atoms.

As used herein, the term "cycloalkyl" defines a saturated 3 to 8 membered carbocyclic ring including fused bicyclic or tricyclic systems. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and also bridged systems such as norbornyl and adamantyl. The cycloalkyl residues of the invention may be optionally substituted by one or more halogen atoms.

The term "heterocyclyl" defines a saturated or partially saturated 3 to 14 membered ring system (except when alternative numbers of ring atoms are specified) similar to cycloalkyl but in which at least one of the carbon atoms has been replaced by N, 0, S, SO or SO2.

Examples include azetidinyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, morpholinyl and may be optionally substituted by one or more halogen atoms.

In the present specification the term "aryl" defines a 5-10 membered mono-or bicyclic group defining a ring system at least one ring of which is aromatic. Thus, bicyclic aryl groups may contain only one aromatic ring. Examples include tetrahydronaphthyl, tetrahydroisoquinolyl and benzodioxyl. Thus, as used herein, the term "aryl" includes heteroaryls containing heteroatoms (e.g. nitrogen, sulphur and/or oxygen) being otherwise as defined above. The aryl groups of the invention may optionally be substituted by one or more halogen atoms. Examples of aromatic moieties are benzene, naphthalene, imidazole and pyridine. Monocyclic aryls include furanyl, thienyl, pyrrolyl, pyrazolyl, oxazolyl, thiazolyl, phenyl, pyridyl, pyrimidinyl, pyridizinyl and pyrazinyl, while bicyclic aryls include naphthyl, quinolyl, benzofuranyl, benzothienyl, indolyl and indazonyl.

The term "aralkyl" as used herein defines an alkyl substituent substituted by an aryl group as defined above.

The term "glycosyl" as used herein defines a monosaccharide motif or mimetic.

The term "uronyl" as used herein defines a monosaccharide uronic acid motif or mimetic.

In the present specification, "halo" refers to fluoro, chloro, bromo or iodo.

In the general formulae of the present invention (and in particular in general formula (1) as described below), the bond orders of the specified rings may vary when the various possible heteroatom(s) imply specific requirements in order to satisfy aromaticity, prevent antiaromaticity and stabilize tautomeric forms due to localization. In such cases, the appropriate bond orders of the ring structures in the structural formulae of the present invention are contemplated herein.

Compounds for use according to the invention

Specific examples

Particular examples of compounds suitable for use according to the invention are listed in

Table 1 (below):

In situ Cpd Calpain-1 ROS IUPAC calpain Comments Number activity activity activity (S)-N-(4-amino-3,4-d ioxo-1 -phenylbutan-2-yl)-2-I A C B A = �=luM (phenylamino)benzamide N-((S)-1 -((S)-4-(benzylamino)- 3,4-d ioxo-i -phenylbutan-2-B= ylamino)-4-methyl-i-oxopentan-2-2 A IA NT �=iOuM yl)-2-(2-methoxyphenylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-3 A B B C = methyl-i -oxopentan-2-yl)-2-�=5OuM (phenylamino)benzamide N-((S)-i -((S)-4-(ethylamino)-3,4-dioxo-i-phenylbutan-2-ylamino)-4 A C D = 4-methyl-i-oxopentan-2-yl)-2-(2-<i OOuM methoxyphenylamino)benzamide N-((S)-i -((S)-4-(ethylamino)-3,4-dioxo-i-phenylbutan-2-ylamino)-5 A IA B IA = 4-methyl-i-oxopentan-2-yl)-2-�=i OOuM (phenylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-NT = not methyl-i-oxopentan-2-yl)-2-(2-6 A B B tested chlorophenylamino)-5-methoxybenzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-7 A D NT methyl-i -oxopentan-2-yl)-2-(2-fluorophenylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-8 A IA NT methyl-i -oxopentan-2-yl)-2-(p-tolylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-9 A IA NT methyl-i -oxopentan-2-yl)-2-(3-chlorophenylamino)benzamide N-((S)-1 -((S)-4-(tert-butylamino)- 3,4-dioxo-1-phenylbutan-2-10 B B B ylamino)-4-methyl-1 -oxopentan-2-yl)-2-(phenylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo- 1 -phenylbutan-2-ylamino)-4-methyl-i-oxopentan-2-yl)-2-11 A B C (benzo[d][i,3]dioxol-5-ylamino)nicotinamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-3-12 A IA NT methyl-i -oxobutan-2-yl)-2- (phenylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-13 A D NT methyl-i -oxopentan-2-yl)-2-(2-methoxyphenylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-14 A IA NT methyl-i -oxopentan-2-yl)-2-(3-fluorophenylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-15 A IA NT methyl-i -oxopentan-2-yl)-2-(4-fluorophenylamino)benzamide N-((S)-i -((S)-4-(ethylamino)-3,4-dioxo-i-phenylbutan-2-ylamino)-16 A C NT 3-methyl-i -oxobutan-2-yl)-2-(2-methoxyphenylamino)benzamide N-((S)-i -((S)-4-(ethylamino)-3,4-dioxo-i-phenylbutan-2-ylamino)-17 A C NT 3-methyl-i -oxobutan-2-yl)-2- (phenylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-18 A C NT methyl-i -oxopentan-2-yl)-2- (phenylam ino)nicotinamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-19 A IA NT methyl-i -oxopentan-2-yl)-2-(o-tolylamino)benzamide N-((S)-i -((S)-4-amino-3,4-d ioxo-i-phenylbutan-2-ylamino)-4-20 A B NT methyl-i -oxopentan-2-yl)-2-(p-tolylamino)nicotinamide N-((S)-i -((S)-4-amino-3,4-d ioxo- i -phenylbutan-2-ylamino)-4-methyl-i-oxopentan-2-yl)-2-(3-21 A IA NT (trifluoromethyl)phenylamino)benz am ide N-((S)-i -((S)-4-amino-3,4-d ioxo- i -phenylbutan-2-ylamino)-4-methyl-i-oxopentan-2-yl)-2-(4-22 A B NT methoxyphenylamino)nicotinamid e (S)-N-((S)-i -(4-bromophenyl)-4- (ethylamino)-3,4-dioxobutan-2-yl)-N-(2-(2-chlorophenylamino)-5-23 A C NT methoxyphenyl)-2-isobutyl malonam ide N-((S)-i -((S)-1 -(4-bromophenyl)- 4-(ethylamino)-3,4-dioxobutan-2-24 A C NT ylamino)-3-methyl-i -oxobutan-2-yl)-2-(phenylamino)benzamide N-((S)-i -((S)-1 -(4-bromophenyl)- 4-(ethylamino)-3,4-dioxobutan-2-25 A D NT ylamino)-4-methyl-i -oxopentane- 2-yl)-2-(phenylamino)benzamide N-((S)-1 -((S)-1 -(4-bromophenyl)- 4-(ethylamino)-3,3-dioxobutan-2-ylamino)-3-methyl-1-oxobutan-2-26 B B NT yl)-2-(2-chlorophenylamino)-5-methoxybenzamide N-((S)-1 -((S)-4-(benzylamino)- 3,4-dioxo-1-phenylbutan-2-27 A C NT ylamino)-4-methyl-1 -oxopentan-2-yl)-2-(phenylamino)benzamide N-((S)-1 -((S)-4-(benzylamino)- 3,4-d ioxo-1 -phenylbutan-2-ylamino)-4-methyl-1-oxopentan-2-28 NT C NT yl)-2-(2-chlorophenylamino)-5-methoxybenzamide Other examples include pharmaceutically acceptable derivatives, N-oxides, salts, hydrates, solvates, complexes, bioisosteres, metabolites and prodrugs of each of the compounds

listedinTablel.

Certain compounds as described above are novel. According to the invention, those compounds which are novel are claimed as compounds per Se, together with processes for their preparation, compositions containing them, as well as their use as pharmaceuticals (for example in any of the particular medical uses described herein).

Moreover, to the extent that certain of the compounds as described below are known as such but not as pharmaceuticals, those compounds are claimed for use as pharmaceuticals (for example in any of the particular medical uses described herein).

References herein to particular compound numbers herein refer to the numbers in Table 1.

Assay for calpain inhibitory activity I. Recombinant Human Calpain-1 enzymatic assay Sum mary The capacity of test compounds to inhibit recombinant human calpain-1 activity was evaluated by determining the ability of recombinant human calpain-1 to cleave a synthetic peptidic fluorescent substrate. The substrate bears a covalently bound fluorochrome (aminomethyl coumarin, AMC), whose fluorescence is enhanced after proteolytic cleavage.

Inhibitory responses at each concentration of reference and test compound were used to determine 1050 values.

Experimental procedure Suc-Leu-Tyr-AMC (Calbiochem) was used as a substrate to measure Calpain-1 activity from Human Erythrocytes (Calbiochem). Enzyme assays were performed in 96-well microtitre plates (NUNC). lOul of test compound was mixed at room temperature with 45u1 assay buffer containing human calpain-1 at a final concentration of lOU/mI and Suc-Leu-Tyr-AMC at a final concentration of 0.5mM, the buffer contained 110mM Tris-HCI pH7.5, 110mM NaCI, 2.2mM EDTA, 2.2mM EGTA and 1.1mM mercaptoethanol. The reaction was initiated using 45u1 0a012 at a final concentration of 10mM. The activity was measured by determining the rate of product (AMC) released using a fluorometer (OPTIMA, BMG) using excitation 380nm, emission 460nm filters. Each reference/test compound was tested in triplicate at final concentrations ranging from 0.lnM-lOuM.

II. In situ calpain activity Sum mary The capacity of test compounds to inhibit in situ calpain activity in 06 glial cells was determined by incubating cells with the AMC fluorescent substrate of the calpain, as above.

In the presence of A23187 (a calcium ionophore) calpain is activated in cells allowing proteolysis of the AMC coupled substrate. Activity can be evaluated by counting the fluorescence of AMC released by activated calpain. The reference molecule, Calpeptin, used exhibits an 1050 in the order of 8.5 +/-2.4 pM.

Experimental procedure 06 rat glioma cells (ATCC No. CCL-1 07) were cultured in DMEM (PAA) supplemented with 10% FBS (PAA) and 2mM L-glutamine (PAA). The day before the experiment, cells were seeded (2.5 x i04 cells/well) in complete culture medium in white 96-well plates (NUNC) to a final volume of 200pL, and incubated at 37°C in 5% 002 incubator. Serial dilution of test compounds between 6uM -2mM were made in 40 mM HEPES pH.7.5 (20x final concentration). Cells were rinsed twice with 200 p1 / well of serum-free medium supplemented with 40 mM HEPES pH.7.5 and then pre-incubated for 1 hour with compounds at 37°C, 5% 002 in serum-free medium supplemented with 40mM HEPES pH.7.5. Calpain substrate was dissolved in the same medium at 10 mM (20x of the final concentration) and the reaction initiated with A231 87 diluted in the substrate mixture at 300 uM (20x the final concentration). Plates were read using a fluorometer (OPTIMA, BMG) over a 3hr period at 37°C using excitation 360nm, emission 450nm filters. All experiments were performed in triplicates. For each concentration of compound tested, the percents of in situ calpain activity inhibition were calculated and an 1050 value determined.

Cell-based assay for ROS inhibitory activity ROS inhibitory activity can be determined by assaying 8-isoprostane levels in rat C6 cells.

8-isoprostane (8-iso PGF2) is a by-product of lipid peroxidation. Elevated levels of 8 isoprostane are therefore an indication of reactive oxygen species (ROS) and oxidative stress. Compounds can therefore be tested for their ability to prevent oxidative stress induced by the marine toxin maitotoxin (MTX).

For measuring the amount of 8-isoprostane in the cell supernatants, the 8-isoprostane EIA (enzyme immunoassay) kit (Cayman) was used. Briefly, acetylcholinesterase-linked 8-isoprostane (Tracer, kit) competes with the 8-isoprostane from cell supernatants (samples) for binding to mouse anti-8-isoprostane (kit). The antigen-antibody complex binds to anti-mouse lgG with which the EIA plate (kit) wells are coated. Acetylcholinesterase catalyses hydrolization of ElIman's reagent, resulting in a colour reaction. Thus, a decrease in colour reaction signifies increased 8-isoprostane levels in the samples. Total levels of 8-isoprostane can be deduced from 8-isoprostane standards.

Maintenance and seeding of C6 cells Rat 06 cells (glioma) were kept in cell culture medium (DMEM, 10% FBS Gold, 2mM L-gluthamine, penicillin/streptomycin) and kept at 37°C and 5% 002. Cells were seeded in white 96 well plates at 25.000/well in 85u1 cell culture medium and incubated for 24h.

Dosing of cells The cells were dosed with 5u1 compound (final concentrations were 0.05-l5OuM).

Compounds dissolved in DMSO were diluted in cell culture medium (final DMSO concentration in well: 0.1%). Each compound was dosed in triplicates. The dual calpain-ROS inhibitor (3S)-3-(4-methyl-2-(1 OH-phenothiazine-2-carboxamido)pentanamido) tetrahydrofuran-2-yl acetate was used as an internal control.

Two triplicates of wells were dosed with DMSO. The cells were incubated for 1 h and then dosed with 1 Oul of 1 OnM MTX for a final concentration of 1 nM (1 uM MTX in ethanol was diluted 1:100 with cell culture medium to make up lOnM). One DMSO triplicate was dosed with MTX, the other with ethanol (lOul of 1:100 ethanol in cell culture medium). Final ethanol concentration in well was 0.1%. Cells were incubated for 3h.

Cell proliferation assay After taking off some of the supernatant for the 8-isoprostane assay (see 4.), cell viability was measured using the CellTiter-Glo� luminescent cell viability assay (Promega). ATP standards were diluted in cell culture medium.

8-isoprostane assay 8-isoprostane standards (kit) were diluted in cell culture medium. The following controls were added to the 96 well EIA plate provided with the kit, according to protocol: Blank, Non-specific Binding, Maximum Binding, Total Activity, 8-isoprostane standards.

The base level of 8-isoprostane of C6 cells increases with passaging. For the oxidative stress-induced and -uninduced (DMSO + MTX and DMSO -ethanol) values to be in the linear part of the standard curve, the cell supernatant may need to be diluted with cell culture medium. The following dilutions have been appropriate: P4-P6: undiluted, P7-PlO: 1:2 dilution, P11-16 1:4 dilution, >P17: 1:10 dilution.

Supernatants of dosed cells were added to the previously prepared EIA plate in the appropriate dilution. Anti-8-isoprostane and Tracer were added according to protocol. The final volume was 150u1/well. The plate was sealed and incubated at 4°C over night.

Supernatants were taken off (MTX waste) and the wells were washed 5x with 200u1 wash buffer (kit) kept at 4°C. 200u1/well ElIman's reagent (kit) was added. The plate was sealed, wrapped in foil and shaken horizontally at 300/mm. The colour reaction was followed by measuring the absorbance with a FLUOstar OPTIMA (BMG) plate reader using a 405nm filter, until the maximum binding minus blanks was at 0.85-0.95 (after 4-6 hours).

Data analysis The readings were analysed according to protocol, using Excel (Microsoft) and XL fit (IDBS). Percentage of free 8-isoprostane (in sample) versus Tracer was calculated, as well as total amount of 8-isoprostane in the samples using the standard curve. For dose response, the 8-isoprostane levels of the DMSO + MTX and DMSO + ethanol samples were translated as 100% and 0% 8-isoprostane and the sample data was fitted between these values to calculate the IC50.

Other assays for ROS inhibitory activity ROS inhibitory activity may also be determined in an assay for the inhibition of Fe2 induced lipid peroxidation (LPO) in rat brain microsomes as described in Esterbauer et a!.

(1991) Free Radic. Biol. Med. 11,81-128. Othersuitabletechniquesforidentifyingsuitable ROS inhibiting moieties are described in US2004/0072218A1 (the teachings of which relating to the bidentification of ROS inhibiting moieties is hereby incorporated herein by reference).

Medical uses of the compounds of the invention The invention finds general application in the treatment of any disease mediated by ROS and/or calpain hyperactivity, including in particular the diseases described below: Muscular dystrophy The invention finds application in the treatment of all muscular dystrophies, including cardiac myopathies arising from any of the various forms of muscular dystrophy. Particular examples include but are not limited to: Limb girdle muscular dystrophy Limb girdle muscular dystrophies (LGMD5) are a clinically and genetically heterogeneous group of disorders. However, all involve progressive weakening of the proximal limb girdle muscles. At least seven different forms of autosomal dominant LGMD (LGMD1) have been described and at least 11 autosomal recessive (LGMD2) forms.

Becker's muscular dystrophy Becker muscular dystrophy (BMD) is a less severe variant of Duchenne muscular dystrophy (see below) and is caused by the production of a truncated, but partially functional form of dystrophin.

Congenital muscular dystrophy Congenital muscular dystrophy includes several disorders with a range of symptoms.

Muscle degeneration may be mild or severe. Problems may be restricted to skeletal muscle, or muscle degeneration may be paired with effects on the brain and other organ systems. A number of the forms of the congenital muscular dystrophies are caused by defects in proteins that are thought to have some relationship to the dystrophin-glycoprotein complex and to the connections between muscle cells and their surrounding cellular structure. Some forms of congenital muscular dystrophy show severe brain malformations, such as lissencephaly and hydrocephalus.

Distal muscular dystrophy Symptoms include weakness and wasting of muscles of the hands, forearms and lower legs. The progress of the disease is slow and it is not life-threatening.

Emery-Dreifuss muscular dystrophy Symptoms include weakness and wasting of shoulder, upper arm and shin muscles. Joint deformities are common. The progress is slow and death may occur from cardiac problems.

Facioscapulohumeral muscular dystrophy Facioscapulohumeral muscular dystrophy (FSHD) initially affects muscles of the face, shoulders, and upper arms with progressive weakness. Symptoms usually develop in the teenage years. Some affected individuals become severely disabled.

Myotonic muscular dystrophy Myotonic muscular dystrophy is the most common adult form of muscular dystrophy. It is marked by myotonia as well as muscle wasting and weakness. Myotonic dystrophy varies in severity and manifestations and affects many body systems in addition to skeletal muscles, including the heart, endocrine organs, eyes and gastrointestinal tract.

Oculopharynqeal muscular dystrophy This form of muscular dystrophy affects the muscles of the eyelids, face and throat. The involvement of pelvic and shoulder muscles may follow.

In preferred embodiments, the muscular dystrophy treated according to the invention is Duchenne muscular dystrophy (DMD), discussed in more detail below: Duchenne muscular dystrophy Duchenne muscular dystrophy (DMD) is a common, genetic neuromuscular disease associated with the progressive deterioration of muscle function, first described over 150 years ago by the French neurologist, Duchenne de Boulogne, after whom the disease is named. DMD has been characterized as an X-linked recessive disorder that affects 1 in 3,500 males caused by mutations in the dystrophin gene. The gene is the largest in the human genome, encompassing 2.6 million base pairs of DNA and containing 79 exons.

Approximately 60% of dystrophin mutations are large insertion or deletions that lead to frameshift errors downstream, whereas approximately 40% are point mutations or small frameshift rearrangements. The vast majority of DMD patients lack the dystrophin protein.

Becker muscular dystrophy is a much milder form of DMD caused by reduction in the amount, or alteration in the size, of the dystrophin protein. The high incidence of DMD (1 in 10,000 sperm or eggs) means that genetic screening will never eliminate the disease, so an effective therapy is highly desirable.

A number of natural and engineered animal models of DM0 exist, and provide a mainstay for preclinical studies (Allamand, V. & Campbell, K. P. Animal models for muscular dystrophy: valuable tools for the development of therapies. Hum. Mo!. Genet. 9, 2459-2467 (2000).) Although the mouse, cat and dog models all have mutations in the DMD gene and exhibit a biochemical dystrophinopathy similar to that seen in humans, they show surprising and considerable variation in terms of their phenotype. Like humans, the canine (Golden retriever muscular dystrophy and German short-haired pointer) models have a severe phenotype; these dogs typically die of cardiac failure. Dogs offer the best phenocopy for human disease, and are considered a high benchmark for preclinical studies. Unfortunately, breeding these animals is expensive and difficult, and the clinical time course can be variable among litters.

The mdx mouse is the most widely used model due to availability, short gestation time, time to mature and relatively low cost (Bulfield, G., Siller, W. G., Wight, P. A. & Moore, K. J. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Nat!Acad. Sd. USA 81, 1189-1192 (1984)).

Since the discovery of the DMD gene about 20 years ago, varying degrees of success in the treatment of DMD have been achieved in preclinical animal studies, some of which are being followed up in humans. Present therapeutic strategies can be broadly divided into three groups: first, gene therapy approaches; second, cell therapy; and last, pharmacological therapy. Gene-and cell-based therapies offer the fundamental advantage of obviating the need to separately correct secondary defects! pathology (for example, contractures), especially if initiated early in the course of the disease. Unfortunately, these approaches face a number of technical hurdles. Immunological responses against viral vectors, myoblasts and newly synthesized dystrophin have been reported, in addition to toxicity, lack of stable expression and difficulty in delivery.

Pharmacological approaches for the treatment of muscular dystrophy differ from gene-and cell-based approaches in not being designed to deliver either the missing gene and/or protein. In general, the pharmacological strategies use drugs/molecules in an attempt to improve the phenotype by means such as decreasing inflammation, improving calcium homeostasis and increasing muscle progenitor proliferation or commitment. These strategies offer the advantage that they are easy to deliver systemically and can circumvent many of the immunological and/or toxicity issues that are related to vectors and cell-based therapies. Although investigations with corticosteroids and sodium cromoglycate, to reduce inflammation, dantrolene to maintain calcium homeostasis and clenbuterol to increase muscle strength, have produced promising results none of these potential therapies alone has yet been shown to be effective in treating DMD.

An alternative pharmacological approach is upregulation therapy. Upregulation therapy is based on increasing the expression of alternative genes to replace a defective gene and is particularly beneficial when an immune response is mounted against a previously absent protein. Upregulation of utrophin, an autosomal paralogue of dystrophin has been proposed as a potential therapy for DMD (Perkins & Davies, Neuromuscul Disord, Si: S78-S89 (2002), Khurana & Davies, Nat Rev Drug Discov 2:379-390 (2003)). When utrophin is overexpressed in transgenic mdx mice it localizes to the sarcolemma of muscle cells and restores the components of the dystrophin-associated protein complex (DAPC), which prevents the dystrophic development and in turn leads to functional improvement of skeletal muscle. Adenoviral delivery of utrophin in the dog has been shown to prevent pathology. Commencement of increased utrophin expression shortly after birth in the mouse model can be effective and no toxicity is observed when utrophin is ubiquitously expressed, which is promising for the translation of this therapy to humans. Upregulation of endogenous utrophin to sufficient levels to decrease pathology might be achieved by the delivery of small diffusible compounds.

Neurological disorders Many neurological disorders, neurological injury and also neurodegenerative diseases involve loss of calcium homeostasis and upregulation of intracellular calcium levels. Such diseases include Alzheimer's, Parkinson's, Huntington's and Multiple Sclerosis.

Thus, the compounds of the invention find application in the treatment of neurological disorders, including neurological injury, neurodegenerative disease, excitotoxicity and ischaemia. Particular examples are described in more detail below.

Neuroprotection The compounds of the invention may prevent or slow cell death (e.g. by inhibiting apoptotic and/or necrotic cell death) and so may be used for neuroprotection (i.e. in neuroprotective therapy).

For example, the compounds of the invention may be used to prevent or slow calpain-mediated cell apoptosis, and in particular neuronal cell apoptosis (e.g. following spinal cord injury).

Thus, the invention finds application in the treatment of neurodegenerative disorders, spinal cord injury and brain injury (e.g. following stroke or ischaemia).

lschaemia/reperfusion iniury Brain ischaemia (e.g. following stroke or traumatic brain injury) produces the accumulation of excess glutamate ultimately resulting in a sustained cellular influx of Na and Ca2 which activates calpain and so contributes to neuronal death (e.g. via apoptosis or necrosis).

Such processes also occur in excitotoxic diseases (e.g. Huntington's Disease).

lschaemic injury to the CNS, heart and kidneys often arises from stroke or physical injury and involves excitotoxic cell damage. Spinal cord injury (SCI) often arises as a result of ischaemic injury resulting from damage to local vasculature. It often results in neuronal cell death and often produces severe neurological dysfunction and paralysis.

Thus, the compounds of the invention find application in the treatment of ischaemia/reperfusion injury, CNS damage, heart damage caused by myocardial infarction, renal dysfunction (and failure) and spinal cord injury.

Amyloidoses Certain proteins can assume a non-native, misfolded R-pleated sheet conformation which accumulates as amyloid fibrils (sometimes referred to as amyloid deposits or plaques) in organs and/or tissues, causing disease. These diseases are collectively known as amyloidoses.

Amyloidoses can be classified clinically as primary, secondary, familial, systemic or isolated. Primary amyloidosis appears without any preceding disease, for example mediated by immune cell dysfunction such as multiple myeloma and other immunocyte dyscrasias. Secondary amyloidosis is a sequela of an existing disorder (typically a chronic inflammatory disease). Familial amyloidosis (which includes neuropathic, cardiopathic and or nephropathic forms) arises from an inherited mutation and can now be identified by DNA tests. Systemic forms involve amyloid deposition in plural tissues and/or organs (although the brain is almost never directly involved in systemic amyloidosis), while isolated (or localized) amyloidosis involves a single organ, tissue type or system. Thus, recognized clinical forms include ocular amyloidosis and central nervous system amyloidosis.

Amyloidoses can also be classified according to the chemical type of the amyloid protein.

The amyloidoses are referred to with a capital A (for amyloid) followed by an abbreviation for the fibril protein. Thus, AA amyloidosis is characterized by extracellular deposition of fibrils that are composed of fragments of serum amyloid A (SAA) protein, a major acute-phase reactant protein, produced predominantly by hepatocytes. Similarly, AL amyloidosis (also called primary amyloidosis or light chain amyloidosis) is characterized by extracellular deposition of fibrils that are composed of an immunoglobulin light chain or light chain fragment, while ATTR amyloidosis is characterized by extracellular deposition of fibrils consisting of the transport protein transthyretin (TTR). AR amyloidosis (which appears in Alzheimer's disease) is characterized by extracellular deposition of fibrils that are composed of R-protein precursor.

Symptoms, prognosis and clinical setting differ greatly between amyloid types. Although about 23 different proteins are known to form amyloid in humans, only a few are associated with clinically significant amyloidosis. The various amyloid proteins and the type of amyloidosis and clinical setting in which they are involved are shown in the table below: Amyloidosis Amyloid protein type Principal Clinical Setting(s) Immunoglobulin light Plasma cell disorders chains Familial amyloid polyneuropathies; senile Transthyretin (TTR) cardiac amyloidosis Systemic Amyloid A (AA) amyloidosis; Inflammation-Serum amyloid A associated amyloidosis; familial mediterranean fever R2-microglobulin Dialysis-associated amyloidosis Immunoglobulin heavy chains Systemic amyloidosis Fibrinogen alpha chain Familial systemic amyloidosis Apolipoprotein Al Familial systemic amyloidosis Hereditary Apolipoprotein All Familial systemic amyloidosis Lysozyme Familial systemic amyloidosis Alzheimer's disease; Down's syndrome; R-protein precursor hereditary cerebral hemorrhage with amyloidosis (Dutch) Creutzfeldt-Jakob disease; Gerstmann-Central Prion protein (AScr or PrP-Sträussler-Scheinker disease; fatal familial nervous 27) insomnia system hereditary cerebral hemorrhage with Cystatin C amyloidosis (Icelandic) ABri precursor protein Familial dementia (British) ADan precursor protein Familial dementia (Danish) Gelsolin Familial amyloidosis (Finnish) Ocular Lactoferrin Familial corneal amyloidosis Keratoepithelin Familial corneal dystrophies Calcitonin Med ullary thyroid carcinoma Amylin Insulinoma; type 2 diabetes Atrial natriuretic factor Atrial amyloidosis Localized Prolactin Pituitary amyloid Keratin Cutaneous amyloidosis Medin Senile aortic amyloidosis Amyloid A (AA) amyloidosis is the most common form of systemic amyloidosis worldwide.

It occurs in the course of a chronic inflammatory disease of either infectious or noninfectious aetiology, hereditary periodic fevers and with certain neoplasms such as Hodgkin disease and renal cell carcinoma.

Calpain activation and/or ROS overproduction may be triggered by the accumulation of amyloid fibrils. Thus, the compounds of the invention find application in the treatment of any of the various amyloidoses described above (and in particular those listed in the above

table).

Synucleinopathies Synucleinopathies comprise a diverse group of neurodegenerative diseases characterized by the presence of lesions composed of aggregates of conformational and posttranslational modifications of a-synuclein in certain populations of neurons and glia. Abnormal filamentous aggregates of misfolded a-synuclein protein are the major components of Lewy bodies, dystrophic (Lewy) neurites, and the Papp-Lantos filaments in oligodendroglia and neurons in multiple system atrophy linked to degeneration of affected brain regions. In contrast to the extracellular amyloid plaques found in the brains of Alzheimer's patients, Lewy bodies are intracellular.

The synucleinopathies include Lewy body diseases (LBD5), dementia with Lewy bodies, multiple system atrophy (MSA), Hallervorden-Spatz disease, Parkinson's disease (PD), the Lewy body variant of Alzheimer's disease (LBVAD), neurodegeneration with brain iron accumulation type-i (NBIA-i), pure autonomic failure, neuroaxonal dystrophy, amytrophic lateral sclerosis and Pick disease and various tauopathies.

Thus, the compounds of the invention find application in the treatment of Lewy body diseases (LBD5), dementia with Lewy bodies, multiple system atrophy (MSA), Hallervorden-Spatz disease, Parkinson's disease (PD), the Lewy body variant of Alzheimer's disease (LBVAD), neurodegeneration with brain iron accumulation type-i (NBIA-i), pure autonomic failure, neuroaxonal dystrophy, amytrophic lateral sclerosis and Pick disease and various tauopathies.

Calpain activation and/or ROS overproduction may be triggered by the accumulation of aggregates of conformational and posttranslational modifications of ct-synuclein. Thus, the compounds of the invention find application in the treatment of any of the various synucleinopathies described above.

Expanded CAG repeat diseases Certain protein aggregation diseases stem from the expansion of CAG repeats in particular genes with the encoded proteins having corresponding polyglutamine tracts which lead to aggregation and accumulation in the nuclei and cytoplasm of neurons. Aggregated amino-terminal fragments of mutant huntingtin are toxic to neuronal cells and are thought to mediate neurodegeneration.

An example is Huntington's disease (HD). Huntington's disease (HD) is characterized by selective neuronal cell death primarily in the cortex and striatum. It is caused by a CAG repeat expansion in the first exon of the huntingtin gene, which encodes a large protein of unknown function. The CAG repeat is highly polymorphic and varies from 6 to 39 repeats in normal individuals and from 35 to 180 repeats in HD cases.

In addition to HD, CAG expansions have been found in at least seven other inherited neurodegenerative disorders, including for example spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, some forms of amyotrophic lateral sclerosis (ALS), dentatorubral pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia (SCA) types 1, 2, 3, 6 and 7.

Calpain activation and/or ROS overproduction may be triggered by protein aggregation arising from the expansion of CAG repeats. Thus, the compounds of the invention find application in the treatment of HD, SBMA, Kennedy's disease, ALS, DRPLA and SCA (e.g. types 1,2,3,6 and 7).

Tauopathies The tauopathies are a group of diverse dementias and movement disorders which have as a common pathological feature the presence of intracellular accumulations of abnormal filaments of tau protein. Examples include Down's Syndrome (DS), Corticobasal Degeneration (CBD), Frontotemporal Dementia with Parkinsonism linked to Chromosome 17 (FTDP17), Pick Disease (PiD) and Progressive Supranuclear Palsy (PSP).

Calpain activation and/or ROS overproduction may be triggered by intracellular accumulations of abnormal filaments of tau protein. Thus, the compounds of the invention find application in the treatment of Down's Syndrome (DS), Corticobasal Degeneration (CBD), Frontotemporal Dementia with Parkinsonism linked to Chromosome 17 (FTDP17), Pick Disease (PiD) and Progressive Supranuclear Palsy (PSP).

Other aggregation diseases Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (fALS). A growing body of evidence suggests that the familial form of ALS (fALS) is caused by destabilization of the native structure of SOD1 leading to aggregation. Calpain activation and/or ROS overproduction may be triggered by the aggregation of SOD1 and so the compounds of the invention find application in the treatment of fALS.

Dementias and memory loss Many dementias are mediated by calpain activation and/or ROS overproduction. For example, memory loss associated with AD is associated with abnormal phosphorylation levels of the transcription factor CREB and attendant redistribution of the synaptic protein synapsin I. Calpain inhibition has been shown to restore normal phosphorylation in tehse circumstances (see Trinchese et al. (2008) The Journal of Clinical Investigation 118(8): 2796-2807) and so the compounds of the invention find application in the treatment of memory loss and dementia and may be used to improve synaptic transmission.

Any form of dementia or memory loss may be treated according to the invention, including cortical and subcortical dementias. In preferred embodiments, the invention finds application in the treatment of a dementia selected from: (a) vascular dementia; (b) dementia associated with Alzheimer's disease; (c) dementia associated with Parkinson's disease; (d) dementia with Lewy bodies; (e) dementia induced by demyelinating disease; (f) toxin-induced dementia, for example alcohol-induced dementia; (g) dementia associated with vitamin deficiency; (h) frontotemporal dementia; (i) dementia associated with Huntington's disease; and (j) dementia associated with amyotrophic lateral sclerosis.

Myelination disease Calpain can cleave myelin basic protein and myelin and calpain hyperactivity is associated with age-dependent myelin degeneration.

Thus, the compounds of the invention finds broad application in the treatment of diseases in which the stimulation of myelination and/or oligodendrocyte progenitor cell proliferation, migration, differentiation and/or promyelinative activity is indicated.

This may be the case, for example, in patients exhibiting T2 hyperintensity of white matter tracts on brain MRI. It may also be the case in patients having a peripheral conduction velocity of the median nerve of less than 35 m/s.

Preferred is the treatment of multiple sclerosis, including: (a) secondary progressive multiple sclerosis; (b) primary progressive multiple sclerosis; and (c) progressive relapsing multiple sclerosis. Particularly preferred is the treatment of secondary progressive multiple sclerosis.

Further exemplary and non-limiting examples of the various medical uses to which the compounds and combinations of the invention may be put are described below: Demyelinatinq diseases The invention finds broad application in the treatment of all demyelinative (sometimes referred to as demyelinative) diseases, either of the CNS or PNS alone or (less commonly) demyelinating diseases in which both the CNS and PNS are affected.

Suitable subjects for treatment may be identified by known diagnostic criteria.

Morphologically, neuronal demyelination can be characterized by a loss of oligodendrocytes in the central nervous system or Schwann cells in the peripheral nervous system. It can also be detected (e.g. diagnosed) by a decrease in myelinated axons in the nervous system or by a reduction in the levels of oligodendrocyte or Schwann cell markers.

In the latter case, exemplary marker proteins of oligodendrocytes or Schwann cells include (but are not limited to): 001; myelin basic protein (MBP); ceramide galactosyltransferase (CGT); myelin associated glycoprotein (MAG); myelin oligodendrocyte glycoprotein (MOG); oligodendrocyte-myelin glycoprotein (0MG); cyclic nucleotide phosphodiesterase (CNP); NOGO; myelin protein zero (MPZ); peripheral myelin protein 22 (PMP22); protein 2 (P2); galactocerebroside (GaIC); sulfatide and proteolipid protein (PLP).

Other methods of diagnosing a disorder of central myelin include MRI of brain and spinal cord to look for altered signal arising from white matter tracts, or the measurement of evoked potentials to look for slower conduction of nerve impulses along white matter tracts.

Methods of diagnosing a peripheral disorder of myelin include nerve conduction studies to detect slowed nerve conduction velocities.

Thus, the invention finds particular application the treatment of subjects having T2 hyperintensity of white matter tracts as revealed by brain MRI and subjects having a peripheral conduction velocity of the median nerve of less than 35 m/s. In both of these subject groups, myelin dysfunction/abnormality is implied and stimulation of myelination and/or oligodendrocyte progenitor cell proliferation, migration, differentiation and/or promyelinative activity by administration of the compounds of the invention may be indicated.

Demyelinating diseases are sporadic or acquired (although genetic factors may play some role) and are characterized by the destruction of biochemically normal myelin, or of the cells that produce it, by inflammatory processes. For example, destruction or functional impairment of myelin-producing Schwann/oligodendrocytes leads to loss of biochemically normal myelin, an example being progressive multifocal leukoencephalopathy (PML) (caused by JC papovavirus infection of oligodendrocytes). The involvement of inflammatory cells directed against specific antigenic myelin components (which may be partitioned between CNS and PNS) means that most demyelinating diseases (unlike the dysmyelinating diseases -see below) involve CNS or PNS exclusively.

The present invention finds broad application in the treatment of all demyelinating diseases irrespective of aetiology, including for example disease arising from pathogens (such as bacteria, viruses and prions); toxic substances or the accumulation of toxic metabolites in the body (e.g. central pontine myclinolysis and vitamin deficiencies); physical trauma (such as spinal cord injury); and genetic disorders (including the various leukodystrophies discussed in detail below), tumours in the central nervous system and multiple sclerosis (including in particular the demyelinating component of multiple sclerosis associated with secondary progressive, primary progressive and progressive-relapsing multiple sclerosis).

Exemplary demyelinating diseases which may be treated according to the invention include multiple sclerosis (MS), progressive multifocal leukoencephalopathy (PML), optic neuritis, demyelinating encephalomyelitis (post viral and post vaccinal), central pontine myelolysis (CPM), leukodystrophies; Cockayne syndrome, Van der Knapp syndrome; Guillain-Barre Syndrome (GBS); chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy (MMN), optic neuritis, transverse myelitis, HMSN types 1 and 3, neuromyelitis optica (NMO), Balo concentric sclerosis, Schilder's diffuse sclerosis and Marburg multiple sclerosis.

Multiple sclerosis By far the most important of the myelination diseases is multiple sclerosis (MS). Multiple sclerosis is the most common demyelinating disease of the central nervous system, affecting over 1,000,000 people worldwide and about 500,000 people in the United States.

It is an immune-mediated inflammatory disease that attacks myelinated axons in the CNS, destroying both the myelin and the axon in variable degrees. The aetiology of the disease has not been fully elucidated, but it appears to involve a combination of genetic susceptibility and an environmental trigger which resulting in a self-sustaining autoimmune/inflammatory disorder that leads to recurrent immune attacks on the CNS.

The disease is usually characterized by relapses and remissions with accumulating neurological deficit leading eventually to chronic disability. The initial phases of the disease appear to involve an autoimmune inflammatory attack on the myelin sheath, producing symptoms ranging from skin tingling to paralysis, lack of coordination, sensory disturbances and visual impairment. The subsequent chronic progressive phase of the disease is associated with the loss of axons.

Several distinct subclasses of MS are recognized and are used as a basis for both prognosis and therapeutic decisions. Relapsing-remitting MS defines the initial course in 85% to 90% of patients. This subclass is characterized by unpredictable attacks (relapses) followed by periods of months to years of relative freedom from symptoms (remission).

Deficits arising during relapse may either resolve or remain. When deficits always completely resolve between attacks with no or only minor accumulation of disability after decades, the disease may be classed as benign MS.

Secondary progressive MS defines a condition to which about 80% of patients with initial relapsing-remitting MS progress. It is characterized by neurological decline between relapses without any periods of remission and appears to be associated with progressive axonal loss. Secondary progressive MS is the most common subclass of MS and causes the greatest amount of disability. Its impact is exacerbated by the fact that it does not seem to be responsive to the available treatments for relapsing-remitting MS (see further discussion below).

Of all multiple sclerosis patients, 10-15% never have a relapsing phase. In these patients decline is continuous with no clear attacks or periods of remission. This subclass is called primary progressive MS. Little or no inflammation is detectable in these patients on neuroimaging and indeed it is debated whether this is a distinct condition. A fourth subclass of MS, progressive relapsing MS, defines a disease course in which a continuous neurological decline is superimposed with relapses. It is the least common of all MS subclasses.

Rare cases of the disease with non-standard behaviour have also been described although they are generally regarded as different diseases. These diseases may be referred to as borderline forms of multiple sclerosis and include Balo concentric sclerosis, Schilder's diffuse sclerosis and Marburg multiple sclerosis.

There is also a distinct condition known as neuromyelitis optica, associated with antibodies to the aquaporin 4 antigen.

The compounds of the invention find application in the treatment of all of the above forms of MS.

Dysmyelinatinq diseases The invention finds broad application in the treatment of all dysmyelinating (sometimes referred to as dysmyelinative) diseases, either of the CNS or PNS alone or (more commonly) dysmyelinating diseases in which both the CNS and PNS are affected.

The dysmyelinating diseases which may be treated according to the invention are hereditary and characterized by the presence of biochemically abnormal myelin or an abnormality in the myelin-forming Schwann/oligodendrocytes that produce it.

The invention finds application in the treatment of all leukodystrophies. Leukodystrophy defines a class of dysmyelinating diseases characterized by progressive degeneration of the white matter of the brain as a result of a defect in a gene involved in the production or metabolism of a specific myelin component. This class includes diseases such as Pelizaeus-Merzbacher Disease (PMD) and Alexander disease.

Specific leukodystrophies that may be treated according to the invention include: 18q syndrome with deficiency of myelin basic protein; adrenoleukodystrophy (ALD); adrenomyeloneuropathy (AMN); Aicardi-Goutiere's syndrome; Alexander disease; autosomal dominant diffuse leukoencephalopathy with neuroaxonal spheroids; autosomal dominant late-onset leukoencephalopathy; childhood ataxia with diffuse CNS hypomyelination (CACH or Vanishing White Matter Disease); Canavan disease; cerebrotendinousxanthomatosis (CTX); craniometaphysical dysplasia with leukoencephalopathy; extensive cerebral white matter abnormality without clinical symptoms; familial leukodystrophy with adult onset dementia and abnormal glycolipid storage; globoid cell leukodystrophy (also known as Krabbe disease); hereditary adult onset leukodystrophy simulating chronic progressive multiple sclerosis; lipomembranous osteodysplasia with leukodystrophy (Nasu Disease); metachromatic leukodystrophy (MLD); megalencephalic leukodystrophy with subcortal cysts (MLC); neonatal ALD; neuroaxonal leukoencephalopathy with axonal spheroids; oculodetatoldigital dysplasia with cerebral white matter abnormalities; orthochormatic leukodystrophy with pigmented glia; ovarioleukodystrophy syndrome; Pelizaeus-Merzbacher Disease (PMD); phenylketonuria (PKU); Refsum disease; Sjogren-Larssen syndrome; sudanophilic leukodystrophy; Van der Knaap Syndrome (vacuolating leukodystrophy with subcortal cysts or MLC); vanishing white matter disease (childhood ataxia with diffuse CNS hypomyelination or CACH); X-linked ALD; Zellweger spectrum and Zellweger syndrome.

Other myelination associated indications The invention finds broad application in all indications in which remyelination or neuroregeneration/neuroprotection is indicated, and so finds application in the treatment of spinal cord injury, traumatic brain injury, post radiation injury, neurological complications of chemotherapy, stroke, acute ischaemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome.

Treatment of neoplasia Calpain can promote cell proliferation: sequential progression through Gi, S, G2 and M phases of the cell cycle is required for mitosis and cell proliferation and calpain can cleave several cell-cycle control proteins (including cyclin A, cyclin D, cyclin E and p27kIPl) as well as downstream regulators (including p53 and p107).

Thus, the compounds of the invention find general application in the treatment of any proliferative disease, including neoplasia, benign, pre-cancerous and malignant neoplasia, hyperlasia, metaplasia and dysplasia.

The invention therefore finds application in the treatment of proliferative disorders which include, but are not limited to cancer, cancer metastasis, smooth muscle cell proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy (e.g. diabetic retinopathy), cardiac hyperplasia, benign prostatic hyperplasia, ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, harmatomas, lymphangiomatosis, sarcoidosis and desmoid tumours.

Neoplasia involving smooth muscle cell proliferation include hyperproliferation of cells in the vasculature (e.g. intimal smooth muscle cell hyperplasia, restenosis and vascular occlusion, including in particular stenosis following biologically-or mechanically-mediated vascular injury, such as angioplasty). Moreover, intimal smooth muscle cell hyperplasia can include hyperplasia in smooth muscle other than the vasculature (e.g. blockage of the bile duct, bronchial airways and in the kidneys of patients with renal interstitial fibrosis). Non-cancerous proliferative disorders also include hyperproliferation of cells in the skin such as psoriasis and its varied clinical forms, Reiter's syndrome, pityriasis rubra pilaris and hyperproliferative variants of disorders of keratinization (including actinic keratosis, senile keratosis and scleroderma).

Particularly preferred is the treatment of malignant neoplasia (cancer).

The cancers to be treated according to the invention may be of any stage or grade. For example, the compounds of the invention may be used to treat cancers staged by reference to tumour size (T), the degree of regional spread or node involvement (N) and distant metastasis (M) as follows: Carcinoma in situ (limited to surface cells), Ti, T2, T3 and T4 (increasing tumour size and involvement, from very localised tumour with no remote metastases, to locally limited extension with or without minimal node satellite extension and with no remote metastases to locally advanced extension with or without major node satellite extension and with no remote metastases); NO (no lymph node involvement), Ni, N2, N3 and N4 (increasing degrees of lymph node involvement); MO (no evidence of distant metastases) and Mi (evidence of distant metastases).

The compounds of the invention may be used to treat cancers of any grade. Grading involves examining tumour cells that have been obtained through biopsy under a microscope. The abnormality of the cells determines the grade of the cancer. Increasing i 0 abnormality increases the grade (from i to 4). Cells that are well differentiated closely resemble mature, specialized cells. Cells that are undifferentiated are highly abnormal, that is, immature and primitive.

The compounds of the invention may be used to treat cancers selected from: (a) Grade i iS (cells slightly abnormal and well differentiated); (b) Grade 2 (cells more abnormal and moderately differentiated); (c) Grade 3 (cells very abnormal and poorly differentiated and (d) Grade 4 (cells immature and undifferentiated).

The compounds of the invention may be used to treat cancers in adults, children or infants.

They also find application in the treatment of drug-resistant cancers.

The invention finds application in the treatment of any cancer, including those selected from the following major groupings: (a) carcinoma; (b) blastoma; (c) leukemia; (d) lymphoma; (e) myeloma; (f) sarcoma and (g) cancers of mixed type. These are discussed in more detail below.

Carcinoma Carcinoma refers to a malignant neoplasm of epithelial origin or cancer of the internal or external lining of the body. Carcinomas, malignancies of epithelial tissue, account for 80 to percent of all cancer cases. Epithelial tissue is found throughout the body. It is present in the skin, as well as the covering and lining of organs and internal passageways, such as the gastrointestinal tract.

Carcinomas are divided into two major subtypes: adenocarcinoma, which develops in an organ or gland, and squamous cell carcinoma, which originates in the squamous epithelium. The invention finds application in the treatment of all carcinomas, including adenocarcinomas and squamous cell carcinomas, as described below.

Adenocarcinomas generally occur in mucus membranes and are first seen as a thickened plaque-like white mucosa. They often spread easily through the soft tissue where they occur. Squamous cell carcinomas occur in many areas of the body.

Most carcinomas affect organs or glands capable of secretion, such as the breasts, which produce milk, or the lungs, which secrete mucus, or colon or prostate or bladder.

The invention therefore finds application in the treatment of various carcinomas, for example a carcinoma of the bladder, breast (e.g. primary breast tumours, node-negative breast cancer, invasive duct adenocarcinomas of the breast and non-endometrioid breast cancers), colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermis (e.g. malignant melanoma), liver, lung (e.g. adenocarcinoma, adrenocortical, nasopharyngeal, small cell lung cancer and non-small cell lung carcinomas), oesophagus, gall bladder, ovary, pancreas (e.g. exocrine pancreatic carcinoma), stomach, cervix, thyroid, prostate, gastrointestinal system (e.g. gastrointestinal stromal tumours) or skin (e.g. squamous cell carcinoma).

In preferred embodiments the carcinoma treated according to the invention is selected from carcinomas of: salivary glands; colon; rectum; appendix; lung; thymus; breast; cervix uteri; bladder and eye.

Blastoma The invention finds application in the treatment of all blastomas, including hepatoblastomas (e.g. nephroblastomas, nonepithelial renal tumours, rhabdoid renal tumour, kidney sarcomas and pPNET of the kidney), medulloblastomas, pancreatoblastomas, pulmonary blastoma, pleuropulmonary blastoma, neuroblastomas (including peripheral nervous cell tumours in general as well as ganglioneuroblastoma and retinoblastomas).

Leukemias, myeloproliferative diseases, and myelodysplastic diseases The invention finds application in the treatment of all leukemias, myeloproliferative diseases and myelodysplastic diseases, including: lymphoid leukemias (for example precursor cell leukemias, mature B-cell leukemias, mature T-cell leukemias and NK cell leukemias); acute myeloid leukemias; chronic myeloproliferative diseases; myelodysplastic syndrome and other myeloproliferative diseases.

The invention therefore finds application in the treatment of various leukemias, including lymphatic, lymphocytic, or lymphoblastic leukemia (malignancy of the lymphoid and lymphocytic blood cell series) and polycythemia vera or erythremia (malignancy of various blood cell products, but with red cells predominating).

Lymphomas and reticuloendothelial neoplasms Lymphomas develop in the glands or nodes of the lymphatic system, a network of vessels, nodes, and organs (specifically the spleen, tonsils, and thymus) that purify bodily fluids and produce infection-fighting white blood cells, or lymphocytes. Unlike the leukemias which are sometimes called "liquid cancers," lymphomas are "solid cancers" Lymphomas may also occur in specific organs such as the stomach, breast or brain. These lymphomas are referred to as extranodal lymphomas. The lymphomas are subclassified into two categories: Hodgkin lymphoma and Non-Hodgkin lymphoma. The presence of Reed-Sternberg cells in Hodgkin lymphoma diagnostically distinguishes Hodgkin lymphoma from Non-Hodgkin lymphoma.

The invention finds application in the treatment of all such lymphomas and reticuloendothelial neoplasms, including: (a) Hodgkin lymphomas; (b) Non-Hodgkin lymphomas (for example precursor cell lymphomas, mature B-cell lymphomas, mature T-cell lymphomas and N K-cell lymphomas; (c) Burkitt lymphoma and (d) other lymphoreticular neoplasms, including mantle cell lymphoma.

The invention therefore finds application in the treatment of a wide range of lymphomas, including for example tumours of the glands or nodes of the lymphatic system (including the spleen, tonsils, and thymus) and extranodal lymphomas of the stomach, breast and brain.

Myeloma (multiple myeloma or myelomatosis) Myeloma is cancer that originates in the plasma cells of bone marrow.

The invention therefore finds application in the treatment of hematopoieitic tumours and haematological malignancies, including those of lymphoid lineage (e.g. leukaemia, acute lymphocytic leukaemia, chronic lymphocytic leukaemia, B-cell lymphoma (such as diffuse large B cell lymphoma), T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (the presence of Reed-Sternberg cells in Hodgkin lymphoma distinguishes Hodgkin lymphoma from Non-Hodgkin lymphoma), hairy cell lymphoma and Burkitt's lymphoma) as well as hematopoieitic tumours of myeloid lineage (for example acute myeloid leukaemia, chronic myeloid leukaemias, myelogenous leukaemias and Imatinib sensitive and refractory chronic myelogenous leukaemias, myelodysplastic syndrome, Bortezomib sensitive and refractory multiple myeloma, myeloproliferative disease or promyelocytic leukaemia and thyroid follicular cancer).

Sarcoma The invention finds application in the treatment of all sarcomas. Sarcoma refers to cancer that originates in supportive and connective tissues such as bones, tendons, cartilage, muscle and fat. Generally occurring in young adults, the most common sarcoma often develops as a painful mass on the bone. Sarcoma tumours usually resemble the tissue in which they grow.

Exemplary sarcomas for treatment according to the invention include osteosarcoma (or osteogenic sarcoma); chondrosarcoma; leiomyosarcoma (smooth muscle); rhabdomyosarcoma (skeletal muscle); mesothelial sarcoma or mesothelioma (membranous lining of body cavities); fibrosarcoma (fibrous tissue); angiosarcoma or hemangioendothelioma (blood vessels); liposarcoma; glioma; astrocytoma; myxosarcoma (primitive embryonic connective tissue) and mesenchymous or mixed mesodermal tumour (mixed connective tissue types).

Fibrosarcomas include peripheral nerve sheath tumours and other fibrous neoplasms, for example fibroblastic and myofibroblastic tumours, nerve sheath tumours and other fibromatous neoplasms. Also included is Kaposi sarcoma.

Also included are soft tissue sarcomas, for example Ewing tumour and Askin tumour of soft tissue, pPNET of soft tissue, extrarenal rhabdoid tumour; fibrohistiocytic tumours; synovial sarcomas; osseous and chondromatous neoplasms of soft tissue and alveolar soft parts sarcoma.

Osteosarcomas (malignant bone tumours) include: malignant fibrous neoplasms of bone; malignant chordomas and odontogenic malignant tumours. Gliomas include oligodendrogliomas, mixed and unspecified gliomas and neuroepithelial glial tumours.

Mixed types The invention finds application in the treatment of cancers of the mixed type, including for example adenosquamous carcinoma, mixed mesodermal tumour, carcinosarcoma and teratocarcinoma.

The invention therefore finds application in the treatment of various CNS, PNS and miscellaneous intracranial and intraspinal neoplasms, including: astrocytoma, neuroblastoma, glioma, schwannoma, ependymomas and choroid plexus tumour (for example ependymomas and choroid plexus tumours); intracranial and intraspinal embryonal tumours (for example medulloblastomas, primitive neuroectodermal tumour (PNET), medulloepithelioma, atypical teratoid/rhabdoid tumour and other intracranial and intraspinal neoplasms (for example pituitary adenomas and carcinomas, tumours of the sellar region (craniopharyngiomas), pineal parenchymal tumours, neuronal and mixed neuronal-glial tumours, meningiomas and intracranial and intraspinal neoplasms in general).

Thus, the invention finds particular application in the treatment of: intracranial and intraspinal germ cell tumours; intracranial and intraspinal germinomas; intracranial and intraspinal teratomas; intracranial and intraspinal embryonal carcinomas; intracranial and intraspinal yolk sac tumour; intracranial and intraspinal choriocarcinoma and intracranial and intraspinal tumours of mixed forms.

The invention also finds application in the treatment of various germ cell tumours, trophoblastic tumours and neoplasms of the gonads. Thus, the invention finds application in the treatment of malignant extracranial and extragonadal germ cell tumours include, for example, malignant germinomas of extracranial and extragonadal sites, malignant teratomas of extracranial and extragonadal sites, embryonal carcinomas of extracranial and extragonadal sites, yolk sac tumour of extracranial and extragonadal sites; choriocarcinomas of extracranial and extragonadal sites and malignant mixed germ cell tumours of extracranial and extragonadal sites in general. The invention also finds application in the treatment of malignant gonadal germ cell tumours, including for example malignant gonadal germinomas, seminomas, malignant gonadal teratomas, gonadal embryonal carcinomas, gonadal yolk sac tumour, gonadal choriocarcinoma, malignant gonadal tumours of mixed forms and malignant gonadal gonadoblastoma.

The invention also finds application in the treatment of various cancers associated with neoplastic cells having a particular gene expression profile/genotype, as described below: c-Kit associated neoplasia OD1 17, also called KIT, C-kit receptor or c-Kit, is a cytokine receptor expressed on the surface of hematopoietic stem cells. Altered forms of this receptor are associated with certain types of neoplastic cells and the invention finds application in the treatment of neoplasia involving neoplastic cells having an altered c-kit receptor.

Expression of c-kit is frequently observed in acute myelocytic leukemia (AML), acute lymphocytic leukemia (ALL) and chronic myelogenous leukemia (CML). Constitutive c-kit activation also appears to be important for gastrointestinal stromal tumours (GIST5). GISTs are the most common mesenchymal tumours of the digestive system.

Male germ cell tumours have been histologically categorized into seminomas, which retain germ cell characteristics, and nonseminomas which can display characteristics of embryonal differentiation. These tumours express both c-kit and SCF and an autocrine loop may contribute to the tumourigenesis.

Glioblastoma and astrocytoma arise from neoplastic transformation of astrocytes and expression of c-kit has been observed in glioblastoma cell lines and tissues.

Bcr-Abl associated neoplasia The Philadelphia chromosome which generates the fusion protein Bcr-Abl is associated with the bulk of chronic myelogenous leukemia (CML) patients (more than 95%), 10-25% of acute lymphocytic leukemia (ALL) patients and 2-3% of acute myelogenous leukemias (AML). In addition, Bcr-Abl is a factor in a variety of other hematological malignancies, including granulocytic hyperplasia resembling CML, myelomonocytic leukemia, lymphomas, and erythroid leukemia.

FLT3 associated neoplasia FLT3 associated neoplasias are cancers in which inappropriate FLT3 activity has been implicated as a contributing factor. FLT3 associated cancers include hematologic malignancies such as leukemia and lymphoma. In some embodiments FLT3 associated cancers include acute myelogenous leukemia (AML), B-precursor cell acute lymphoblastic leukemia, myelodysplastic leukemia, T-cell acute lymphoblastic leukemia, mixed lineage leukemia (MLL) and chronic myelogenous leukemia (CML).

EGFR associated neoplasia EGFR associated cancers are cancers in which inappropriate EGFR activity (e.g. overexpression of EGFR or mutation of EGFR which causes constitutive tyrosine kinase activity) has been implicated as a contributing factor.

The invention therefore finds application in the treatment of neoplasia associated with neoplasms exhibiting inappropriate EGFR activity, including EGFR associated neuroblastoma, intestine carcinoma (for example rectum carcinoma, colon carcinoma, familiary adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer), oesophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tong carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroidea carcinoma, papillary thyroidea carcinoma, renal carcinoma, kidney parenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma, testis carcinoma, breast carcinoma, urinary carcinoma, melanoma, brain tumours such as glioblastoma, astrocytoma, meningioma, medulloblastoma and peripheral neuroectodermal tumours, Hodgkin lymphoma, non-Hodgkin lymphoma, Burkitt lymphoma, acute lymphatic leukemia (ALL), chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), adult T-cell leukemia lymphoma, hepatocellular carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyo sarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmocytoma.

EGFR appears to have an important role in the development of human brain tumours: the amplification of the EGFR gene in glioblastoma multiforme tumours is one of the most consistent genetic alterations known, with EGFR being overexpressed in approximately 40% of malignant gliomas and EGFRvIII mutation being found in about 50% of all glioblastomas.

In addition to gliomas, abnormal EGFR expression has also been reported in a number of squamous epidermoid cancers and breast cancers. Over-expression of EGFR may be associated with a poorer prognosis relative to tumours that do not over-express EGFR.

Atherosclerosis Atherosclerosis can arise from intimal hyperplasia which produce occlusive atherosclerotic lesions. These can cause stroke, myocardial infarction and other peripheral vascular diseases.

Calpain is involved in many of the key stages in the pathogenesis of atherosclerosis, including initial endothelial cell damage, subsequent inflammatory response and smooth muscle cell migration and proliferation.

Thus, the compounds of the invention find application in the treatment of atherosclerosis and other diseases associated with vascular occlusion and intimal hyperplasia, including in particular diseases involving intimal hyperplasia of blood vessels as well as intimal smooth muscle cell hyperplasia in outside of the vasculature (e.g. bile duct, bronchial airways and the kidneys, for example in the treatment of renal interstitial fibrosis).

Other applications of the compounds of the invention include the treatment of restenosis and vascular occlusion, including in particular stenosis following biologically-or mechanically-mediated vascular injury, such as angioplasty.

Hearing loss The compounds of the invention find application in the treatment of hearing loss or impairment caused by ROS and/or calpain hyperactivity.

Thus, the invention finds application in the treatment of deafness and hearing impairment, including presbycusis (age-related deafness) and hearing loss arising as a consequence of meningitis or otitis, genetic causes, injuries, tumours, drugs, the administration of medicaments such as certain antibiotics (e.g. gentamicin and tobramycin), anti-cancer drugs, non-steroidal anti-inflammatory agents, diuretics, ulcer drugs or anticonvulsants, prolonged exposure to aromatic organic solvents such as toluene or xylene, ageing and exposure to noise.

The invention finds aparticular application in the treatment of hearing loss attendant on the administration of aminoglycoside antibiotics (including but not limited to amikacin, dibekacin, gentamicin, isepamicin, netilmicin, spectinomycin and tobramycin).

Thus, the invention also finds particular application in the treatment of hearing loss caused by ototoxicity.

The invention also finds particular application in the treatment of hearing loss caused by noise. This occurs when the acoustic hair cells which convey sound towards the inner ear are damaged and can no longer stimulate the auditory nerve. It has been estimated that 8 to 10% of the population in America and Europe suffer from cochlear pathologies mediated by sound exposure.

The invention also finds application in the treatment of tinnitus.

Ocular disorders The compounds of the invention find application in the treatment of ocular disease, including the diseases and disorders described in more detail below.

Ocular amyloidosis As described above, the compounds of the invention find application in the treatment of ocular amyloidosis. Vitreous involvement in amyloidosis seems to be especially linked to some of the hereditary neuropathies associated with the amyloid protein transthyretin (see e.g. Sandgren (1995) Surv Ophthalmol. 40(3):173-96).

Thus, the compounds of the invention find application in the treatment of familial and non-familial ocular amyloidosis.

Age-related macular degeneration (AMD) Oxidative stress and cellular damage caused by ROS has been implicated in AMD (see e.g. Petrukhin (2007) Exp Opin Ther Targets 11: 625-639). Moreover, Drusen plaques associated with AMD contain amyloid fibrils and so can trigger calpain activation and/or ROS overproduction (see e.g. Mullins eta!. (2000) The FASEB Journal 14: 836-846).

Thus, the compounds of the invention find application in the treatment of AMD, including dry (a.k.a. atrophic) and wet AMD.

Ocular neural pathology There is increasing evidence for the presence of calpain in the retina (see e.g. Karlsson (1992) Neuroscience Letters 141: 127-129). Thus, the compounds of the invention may be used to protect ocular neural tissue.

The compounds of the invention therefore find application in the treatment or prophylaxis of ocular cell damage and neurodegenerative diseases of the retina, including in particular those caused by ischaemia, hypoxia, oedema, oxidative stress, metabolic insufficiency, excitotoxicity, trauma and apoptosis.

Thus, the compounds of the invention find application in the treatment of ischaemic retinopathies, optic neuropathies (including anterior ischemic optic neuropathy (AION)), commotio retinae, glaucoma, macular degeneration, retinitis pigmentosa, retinal detachment, retinal tears or holes, diabetic retinopathy and iatrogenic retinopathy.

Cataract Calpain cleaves a-and 13-crystallin proteins in the lens of the eye, promoting precipitation and leading to light scattering characteristic of lens opacity and cataract formation.

Thus, the compounds of the invention find application in the treatment of cataract formation.

Blood clotting diseases Some calpain substrates (including all-spectrin, talin, ezrin, FAK, paxillin, vimentin and Desmin) are structural components of the cellular cytoskeleton. Calpain-mediated cleavage of these components plays an important role in the cytoskeletal remodelling and integrin-signalling during platelet adhesion, aggregation and post-aggregation events (including pro-coagulant release and platelet-mediated retraction of fibrin clots).

The compounds of the invention therefore find application in the treatment of blood clotting diseases and diseases mediated by platelet dysfunction (and in particular diseases mediated by platelet aggregation).

Cell migration mediated diseases Many of the cytoskeletal calpain substrates described above are also components of integrin-linked focal adhesions in many other adhesive cell types. The regulation of focal adhesion assembly and disassembly is critical for the control of cell migration. Moreover, anchorage-independent growth is a hallmark of oncogenic transformation. Thus, calpain may modulate cell morphology, adhesion, spreading and motility by cleavage of structural proteins that regulate the actin cytoskeleton and play a role in the progression and spread of various cancers.

The compounds of the invention therefore find application in the treatment of diseases mediated by cell migration dysfunction, including for example cancer metastasis, inflammatory diseases and immune diseases. The compounds also find application in wound healing.

Cachexia Cachexia involves dramatic weight loss, muscle atrophy, fatigue, weakness and significant loss of appetite. It typically arises as a complication of various underlying disorders, including cancer, metabolic acidosis, infectious diseases (such as tuberculosis and AIDS) and various autoimmune disorders.

Calpain is involved in mediated tissue loss (including muscle wasting) associated with cachexia, and thus, the compounds of the invention therefore find application in the treatment of cachexia.

Sum mary As explained above, the compounds of the invention find application in the treatment of a wide variety of diseases including inflammatory and immunological diseases, cardiovascular and cerebrovascular diseases, disorders of the central or peripheral nervous system, cachexia, osteoporosis, muscular dystrophy, proliferative diseases, cataract, rejection reactions following organ transplants and autoimmune and infectious (e.g. viral) diseases.

The diseases treated according to the invention include rheumatoid arthritis, pancreatitis, inflammation of the gastrointestinal system (including ulcerative or non-ulcerative colitis and Crohn's disease), cardiovascular and cerebrovascular diseases (including for example arterial hypertension, septic shock, cardiac or cerebral infarction, ischaemic or haemorrhagic trauma, cerebral or spinal cord trauma, subarachnoid haemorrhage, epilepsy, ageing, senile dementia, peripheral neuropathies, cachexia, osteoporosis and organ and tissue transplant rejection.

Adjunctive agents for use with the compounds of the invention Any of a wide variety of adjunctive agents may be used in the combinations of the invention. Preferably, the adjunctive agents for use in the combinations of the invention as described herein are selected from the following classes: 1. Antiinflammatory agents; 2. Protease inhibitors; 3. Myostatin antagonists; 4. Cytokines and mobilizing agents; 5. Corticosteroids; 6. Anabolic steroids; 7. TGF-3 antagonists; 8. Antioxidants and mitochondrial supporting agents; 9. Dystrophin expression enhancing agents; 10. Gene replacement/repair agents; 11. Cell-based compositions; 12. Creatine; 13. anti-osteoporotic agents; 14. utrophin upregulating agents; 15. cGMP signalling modulators; and 16. a combination of two or more of the foregoing classes.

A reference to a particular adjunctive agent herein is intended to include ionic, salt, solvate, isomers, tautomers, N-oxides, ester, prodrugs, isotopes and protected forms thereof (preferably the salts or tautomers or isomers or N-oxides or solvates thereof, and more preferably, the salts or tautomers or N-oxides or solvates thereof).

1. Antinflammatory agents Disease mediated by ROS and/or calpain hyperactivity typically involve an inflammatory component. For example, muscles affected by DMD show signs of inflammation, including an abundance of macrophages. Thus, a wide range of antiinflammatory agents can be used as adjunctive agents, as discussed below.

1.1 Beta2-adrenergic receptor agonists In one embodiment of the invention, the adjunctive agent is a beta2-adrenergic receptor agonist (e.g. albuterol).

Definitions and technical background: The term beta2-adrenergic receptor agonist is used herein to define a class of drugs which act on the 132-adrenergic receptor, thereby causing smooth muscle relaxation resulting in dilation of bronchial passages, vasodilation in muscle and liver, relaxation of uterine muscle and release of insulin. A preferred beta2-adrenergic receptor agonist for use according to the invention is albuterol, an immunosuppressant drug that is widely used in inhalant form for asthmatics. Albuterol is thought to slow disease progression by suppressing the infiltration of macrophages and other immune cells that contribute to inflammatory tissue loss. Albuterol also appears to have some anabolic effects and promotes the growth of muscle tissue. Albuterol may also suppress protein degradation (possibly via calpain inhibition).

1.2 nNOS stimulators Disease mediated by ROS and/or calpain hyperactivity may involve destabilization of neuronal nitric oxide synthase (nNOS). For example, in DMD, the loss of dystrophin leads to breaks in the membrane, and destabilizes neuronal nitric oxide synthase (nNOS), a protein which normally generates nitric oxide (NO). It is thought that at least part of the muscle degeneration observed in DMD patients may result from the reduced production of muscle membrane-associated neuronal nitric oxide synthase. This reduction may lead to impaired regulation of the vasoconstrictor response and eventual muscle damage.

1.3 Nuclear Factor Kappa-B inhibitors A preferred class of antiinflammatory agent suitable for use in the combinations of the invention are Nuclear Factor Kappa-B (NF-kB) inhibitors. NF-kB is a major transcription factor modulating the cellular immune, inflammatory and proliferative responses. NF-kB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells.

The activation of this factor in DMD contributes to diseases pathology. Thus, NF-kB plays an important role in the progression of muscular dystrophy and the IKK/NF-B signaling pathway is a potential therapeutic target for the treatment of DMD. Inhibitors of NF-kB (for example, IRFI 042, a vitamin E analogue) ameliorate muscle function, decrease serum OK level and muscle necrosis and enhance muscle regeneration. Furthermore, specific inhibition of N F-kB/IKK-mediated signalling has similar benefits.

1.4 TN F-a antagonists TNFa is one of the key cytokines that triggers and sustains the inflammation response. In one embodiment of the invention, the adjunctive agent is a TNF-a antagonist (e.g. i nfl ixi ma b).

Preferences and specific embodiments: Preferred TN F-a antagonists for use according to the invention include infliximab (RemicadeTM), a chimeric monoclonal antibody comprising murine VK and VH domains and human constant Fc domains. The drug blocks the action of TNFa by binding to it and preventing it from signaling the receptors for TNFa on the surface of cells. Another preferred TNF-a antagonists for use according to the invention is adalimumab (HumiraTM). Adalimumab is a fully human monoclonal antibody. Another preferred TNF-a antagonists for use according to the invention is etanercept (EnbrelTM).

Etanercept is a dimeric fusion protein comprising soluble human TNF receptor linked to an Fc portion of an IgGi. It is a large molecule that binds to and so blocks the action of TNFa.

Etanercept mimics the inhibitory effects of naturally occurring soluble TNF receptors, but as a fusion protein it has a greatly extended half-life in the bloodstream and therefore a more profound and long-lasting inhibitory effect. Enbrel is marketed as a lyophylized powder in 25mg vials which must be reconstituted with a diluent and then injected subcutaneously, typically by the patient at home.

Another preferred TNF-a antagonist for use according to the invention is pentoxifylline (TrentalTM), chemical name 1-(5-oxohexyl)-3, 7-dimethylxanthine. The usual dosage in controlled-release tablet form is one tablet (400 mg) three times a day with meals.

Posology: Remicade is administered by intravenous infusion, typically at 2-month intervals. The recommended dose is 3 mg/kg given as an intravenous infusion followed with additional similar doses at 2 and 6 weeks after the first infusion then every 8 weeks thereafter. For patients who have an incomplete response, consideration may be given to adjusting the dose up to 10 mg/kg or treating as often as every 4 weeks. Humira is marketed in both preloaded 0.8 ml syringes and also in preloaded pen devices, both injected subcutaneously, typically by the patient at home. Etanercept can be administered at a dose of 25 mg (twice weekly) or 50 mg (once weekly).

1.5 Ciclosporin In one embodiment of the invention, the antinflammatory agent is ciclosporin. Ciclosporin A, the main form of the drug, is a cyclic nonribosomal peptide of 11 amino acids produced by the fungus Tolypocladium inflatum. Ciclosporin is thought to bind to the cytosolic protein cyclophilin (immunophilin) of immunocompetent lymphocytes (especially T-lymphocytes). This complex of ciclosporin and cyclophylin inhibits calcineurin, which under normal circumstances is responsible for activating the transcription of interleukin-2. It also inhibits lymphokine production and interleukin release and therefore leads to a reduced function of effector T-cells. It does not affect cytostatic activity. It has also an effect on mitochondria, preventing the mitochondrial PT pore from opening, thus inhibiting cytochrome c release (a potent apoptotic stimulation factor). Ciclosporin may be administered at a dose of 1-10 mg/kg/day.

2. Protease inhibitors Proteins in skeletal muscle are degraded by at least three different proteolytic pathways: (a) lysosomal proteases (e.g. the cathepsins); (b) non-lysosomal Ca2 -dependent proteases (e.g. calpain); and (c) non-lysosomal ATP-ubiquitin-dependent proteases (e.g. the multicatalytic protease complex or proteasome). Several lines of evidence have suggested that enhanced activation of proteolytic degradation pathways underlies the pathogenesis of muscular dystrophy. Thus, protease inhibitors can be used in the treatment of muscular dystrophies, as discussed below.

Preferred protease inhibitors for use according to the invention may specifically target one of the three degradtion pathways described above. Particularly preferred are protease inhibitors which target the non-lysosomal Ca2 -dependent pathway (calpain inhibitors) or the non-lysosomal ATP-ubiquitin-dependent pathway (proteasome inhibitors), as described below: 2.1 Auxiliary calpain inhibitors In one embodiment of the invention, the adjunctive agent is an auxiliary calpain inhibitor.

The term "auxiliary calpain inhibitor" is used herein to define calpain inhibitors which do not conform to the structure of formula (I) as defined herein, including the ionic, salt, solvate, isomers, tautomers, N-oxides, ester, prodrugs, isotopes and protected forms thereof (preferably the salts or tautomers or isomers or N-oxides or solvates thereof, and more preferably, the salts or tautomers or N-oxides or solvates thereof), as described above.

Preferences and specific embodiments: Auxiliary calpain inhibitors for use according to the invention preferably comprise a calpain inhibiting moiety linked to (or associated with) a carrier (which acts to facilitate targeting of the calpain inhibiting moiety to muscle tissue).

The targeting moiety may be chemically linked to the calpain inhibiting moiety, or may be physically associated therewith (a liposome carrier). Preferred targeting moieties include carnitine or aminocarnitine. The calpain inhibiting moiety may be leupeptin. Particularly preferred may be Ceptor's MyodurTM. Other such calpain inhibitors are described in W02005124563 (the contents of which are incorporated herein by reference). Other suitable calpain inhibitors are the a-ketocarbonyl calpain inhibitors disclosed in WO 2004/078908 (the contents of which are incorporated herein by reference) and BMCL 2005p5176 (Santhera). Of the calpain inhibitors described in WO 2004/078908, preferred may be those which target both calpain and the proteasome.

The auxiliary calpain inhibitors for use according to the invention may be chimaeric compounds or combinations in which the calpain inhibiting moiety is associated (e.g. combined with, co-administered with or covalently linked) to a ROS inhibitor. Such agents combine relief of oxidative stress with a reduction in calpain-mediated muscle tissue breakdown. Suitable dual action calpain/ROS inhibitors are described for example in WO01/32654, W02007/045761, W02005/056551 and WO 2002/40016 (the contents of which are incorporated herein by reference).

Other suitable auxiliary calpain inhibitors can be identified using commercially available assay kits (e.g. the calpain activity kit based on a fluorogenic substrate from Oncogene Research Products, San Diego, CA). This assay measures the ability of calpain to digest the synthetic substrate Suc-LLVY-AMC: free AMC can be measured fluorometrically at an excitation of 360-380 nm and an emission of 440-460 nm.

2.2 Proteasome inhibitors Definitions and technical background: Another class of adjunctive agents suitable for use in the combinations of the invention are proteasome inhibitors. Proteasomes control the half-life of many short-lived biological processes. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers and inhibition of the proteasomal degradation pathway rescues the expression and subcellular localization of dystrophin-associated proteins. Thus, proteasome inhibitors have recently been identified as potential therapeutics for the treatment of DMD (see Bonuccelli et al. (2003) Am J Pathol. October; 163(4): 1663-1 675). The term "proteasome inhibitor" as used herein refers to compounds which directly or indirectly perturb, disrupt, block, modulate or inhibit the action of proteasomes (large protein complexes that are involved in the turnover of other cellular proteins). The term also embraces the ionic, salt, solvate, isomers, tautomers, N-oxides, ester, prodrugs, isotopes and protected forms thereof (preferably the salts or tautomers or isomers or N-oxides or solvates thereof, and more preferably, the salts or tautomers or N-oxides or solvates thereof), as described above.

Preferences and specific embodiments: There are several classes of proteasome Inhibitors suitable for us em the combinations of the invention, including peptide aldehydes (such as MG-I 32) and the dipeptidyl boronic acid bortezimib (VelcadeTM; formerly known as PS-341) which is a more specific inhibitor of the proteasome. Thus, preferred proteasome inhibitors for use in accordance with the invention include bortezimib ([(1 R)-3-methyl-i -[(2S)-1 -oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]-boronic acid). Bortezimib is commercially available for example from Millennium Pharmaceuticals Inc under the trade name Velcade, or may be prepared for example as described in POT patent specification No. WO 96/1 3266, or by processes analogous thereto. Bortezimib specifically interacts with a key amino acid, namely threonine, within the catalytic site of the proteasome. Another preferred proteasome inhibitor for use in the combinations of the invention is the cell-permeable proteasomal inhibitor OBZ-leucyl-leucyl-leucinal (MG-i 32) (as described in Bonuccelli et al. (2003) Am J Pathol. October; 163(4): 1663-1675, the content of which relating to this compound is incorporated herein by reference). Other inhibitors include those structurally related to MG-i 32, including MG-i 15 (CBZ-leucyl-leucyl-norvalinal) and ALLN (N-acetyl-leucyl-leucyl-norleucinal) (as also described in Bonuccelli et a!. (2003) Am J Pathol. October; 163(4): 1663-1675, the content of which relating to this compound is incorporated herein by reference).

Posology: The proteasome inhibitor (such as bortezimib) can be administered in a dosage such as 100 to 200 mg/m2. These dosages may be administered for example once, twice or more per course of treatment, which may be repeated for example every 7, 14, 21 or 28 days. MG-132 can be administered at a dose of 10 pg/kg/day.

3. Myostatin antagonists Definitions and technical background: Another class of adjunctive agents suitable for use in the combinations of the invention are myostatin antagonists. Myostatin, also known as growth/differentiation factor 8 (GDF-8) is a transforming growth factor-R (TGF-R) family member involved in the regulation of skeletal muscle mass. Most members of the TGF-R-GDF family are widely expressed and are pleiotropic: however, myostatin is primarily expressed in skeletal muscle tissue where it negatively controls skeletal muscle growth.

Myostatin is synthesized as an inactive preproprotein which is activated by proteolyic cleavage. The precurser protein is cleaved to produce an approximately 109 amino acid 000H-terminal protein which, in the form of a homodimer of about 25 kDa, is the mature, active form. The mature dimer appears to circulate in the blood as an inactive latent complex bound to the propeptide. As used herein the term "myostatin antagonist" defines a class of agents which inhibit or block at least one activity of myostatin, or alternatively, blocks or reduces the expression of myostatin or its receptor (for example, by interference with the binding of myostatin to its receptor and/or blocking signal transduction resulting from the binding of myostatin to its receptor). Such agents therefore include agents which bind to myostatin itself or to its receptor.

Preferences and specific embodiments: Myostatin antagonists for use according to the invention include antibodies to GDF-8; antibodies to GDF-8 receptors; soluble GDF-8 receptors and fragments thereof (e. g. the ActRIIB fusion polypeptides as described in US10/689,677, including soluble ActRIIB receptors in whichActRllB is joined to the Fc portion of an immunoglobulin); GDF-8 propeptide and modified forms thereof (e. g. as described in WO 02/068650 or US10/071499, including forms in which GDF-8 propeptide is joined to the Fc portion of an immunoglobulin and/or form in which GDF-8 is mutated at an aspartate (asp) residue, e. g. , asp-99 in murine GDF-8 propeptide and asp-i 00 in human GDF-8 propeptide); a small molecule inhibitor of GDF-8; follistatin (e. g. as described in US6,004, 937) or follistatin-domain-containing proteins (e. g. GASP-i or other proteins as described in USiO/369, 736 and USiO/369, 738); and modulators of metalloprotease activity that affect GDF-8 activation, as described in USiO/662,438.

Preferred myostatin antagonists include myostatin antibodies which bind to and inhibit or neutralize myostatin (including the myostatin proprotein and/or mature protein, in monomeric or dimeric form). Myostatin antibodies are preferably mammalian or non-mammalian derived antibodies, for example an IgNAR antibody derived from sharks, or i 0 humanised antibodies (or comprise a functional fragment derived from antibodie. Such antibodies are described, for example, in US 2004/0i42383, US 2003/i 038422, WO 2005/094446 and WO 2006/li 6269 (the content of which is incorporated herein by reference). Myostatin antibodies also include those which bind to the myostatin proprotein and prevent cleavage into the mature active form. A particularly preferred myostatin iS antibody for use in the combinations of the invention is Wyeth's Stamulumab (MYO-029).

MYO-029 is a recombinant human antibody which binds to and inhibits the activity of myostatin. Other preferred antibody antagonists include the antibodies described in U56096506 and U56468535 (incorporated herein by reference). In some embodiments, the GDF-8 inhibitor is a monoclonal antibody or a fragment thereof that blocks GDF-8 binding to its receptor. Other illustrative embodiments include murine monoclonal antibody JA-i6 (as described in U52003/0i38422 (ATCC Deposit No. PTA-4236); humanized derivatives thereof and fully human monoclonal anti-GDF-8 antibodies (e. g. , Myo-29, Myo-28 and Myo-22, ATCC Deposit Nos. PTA-474i, PTA-4740, and PTA-4739, respectively, or derivatives thereof) as described in U52004/0i42382 and incorporated herein by reference.

Other preferred myostatin antagonists include soluble receptors which bind to myostatin and inhibit at least one activity thereof. The term "soluble receptor" here includes truncated versions or fragments of the myostatin receptor which specifically bind myostatin thereby blocking or inhibiting myostatin signal transduction. Truncated versions of the myostatin receptor, for example, include the naturally-occurring soluble domains, as well as variations elaborated by proteolysis of the N-or C-termini. The soluble domain includes all or part of the extracellular domain of the receptor, either alone or attached to additional peptides or other moieties. Since myostatin binds activin receptors (including activin type IEB receptor (ActRHB) and activin type HA receptor (ActRHA), activin receptors can form the basis of soluble receptor antagonists. Soluble receptor fusion proteins can also be used, including soluble receptor Fc (see US2004/0223966 and W02006/012627, both of which are incorporated herein by reference).

Other preferred myostatin antagonists based on the myostatin receptors are ALK-5 and/or ALK-7 inhibitors (see for example W02006025988 and W02005084699, the disclosure of which is incorporated herein by reference). As a TGF-R cytokine, myostatin signals through a family of single transmembrane serine/threonine kinase receptors. These receptors can be divided in two classes, the type I or activin like kinase (ALK) receptors and type II receptors. The ALK receptors are distinguished from the Type II receptors in that the ALK receptors (a) lack the serine/threonine rich intracellular tail, (b) possess serine/threonine kinase domains that are very homologous between Type I receptors, and (c) share a common sequence motif called the GS domain, consisting of a region rich in glycine and serine residues. The GS domain is at the amino terminal end of the intracellular kinase domain and is believed to be critical for activation by the Type II receptor. Several studies have shown that TGF-R signaling requires both the ALK (Type I) and Type II receptors. Specifically, the Type II receptor phosphorylates the GS domain of the Type I receptor for TGF-[beta] ALK5, in the presence of TGF-[beta]. The ALK5, in turn, phosphorylates the cytoplasmic proteins smad2 and smad3 at two carboxy terminal serines. Generally, it is believed that in many species, the Type II receptors regulate cell proliferation and the Type I receptors regulate matrix production. Various ALK5 receptor inhibitors have been described (see, for example, US 6,465,493, U52003/0149277, U52003/0166633, U520040063745, and US2004/0039198, the disclosure of which is incoprorated herein by reference). Thus, the myostatin antagonists for use according to the invention may comprise the myostatin binding domain of an ALK5 and/or ALK7 receptor.

Other preferred myostatin antagonists include soluble ligand antagonists which compete with myostatin for binding to myostatin receptors. The term "soluble ligand antagonist" here refers to soluble peptides, polypeptides or peptidomimetics capable of non-productively binding the myostatin receptor(s) (e.g. the activin type HB receptor (ActRHA)) and thereby competitively blocking myostatin-receptor signal transduction. Soluble ligand antagonists include variants of myostatin, also referred to as "myostatin analogues" that have homology with but not the activity of myostatin. Such analogues include truncates (such an N-or C-terminal truncations, substitutions, deletions, and other alterations in the amino acid sequence, such as variants having non-amino acid substitutions).

Other preferred myostatin antagonists further include polynucleotide antagonists. These antagonists include antisense or sense oligonucleotides comprising a single-stranded polynucleotide sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences. Antisense or sense oligonucleotides for use according to the invention comprise fragments of the targeted polynucleotide sequence encoding myostatin or its receptor, transcription factors, or other polynucleotides involved in the expression of myostatin or its receptor. Such a fragment generally comprises at least about 14 nucleotides, typically from about 14 to about 30 nucleotides. Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/1 0448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L)-lysine and morpholinos. Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence. Thus, RNA interference (RNAi) produced by the introduction of specific small interfering RNA (5iRNA), may also be used to inhibit or eliminate the activity of myostatin.

Particularly preferred myostatin antagonists include but are not limited to follistatin, the myostatin prodomain, growth and differentiation factor 11 (GDF-1 1) prodomain, prodomain fusion proteins, antagonistic antibodies that bind to myostatin, antagonistic antibodies or antibody fragments that bind to the activin type IEB receptor, soluble activin type IHB receptor, soluble activin type IEB receptor fusion proteins, soluble myostatin analogs (soluble ligands), oligonucleotides, small molecules, peptidomimetics, and myostatin binding agents. disclose anti-myostatin antibodies. Other preferred antagonists include the peptide immunogens described in U56369201 and WO 0 1/05820 (incorporated herein by reference) and myostatin multimers and immunoconjugates capable of eliciting an immune response and thereby blocking myostatin activity. Other preferred antagonists include the protein inhibitors of myostatin described in W002/085306 (and incorporated herein by reference), which include the truncated Activin type II receptor, the myostatin pro-domain, and follistatin. Other myostatin inhibitors include those released into culture from cells overexpressing myostatin (see W000/43781), dominant negatives of myostatin (see WO 01/53350) including the Piedmontese allele, and mature myostatin peptides having a C-terminal truncation at a position either at or between amino acid positions 335 to 375. The small peptides described in US2004/0181033 (incorporated herein by reference) which comprise the amino acid sequence WMCPP, are also suitable for use in the combinations of the invention.

4. Cytokines and mobilizing agents Definitions and technical background: Another class of adjunctive agents suitable for use in the combinations of the invention are cytokines, and in particular anabolic cytokines and insulin-like growth factors (such as IGF-1 or IGF-2). The anabolic effect of IGF-1 on muscle is very well established. In muscular dystrophies, a progressive reduction in the proliferative capacity of satellite cells occures and this loss of proliferative capacity may be ameliorated by treatment with IGF-1. Thus, IGF-1 (and other members of this class of cytokine) may help to slow the progress of the dystrophinopathies by enhancing activation of dormant satellite cells. Insulin-like growth factors (IGF5) are members of the highly diverse insulin gene family that includes insulin, IGF-l, IGF-ll, relaxin, prothoraciotropic hormone (PTTH), and molluscan insulin-related peptide. The IGFs are circulating, mitogenic peptide hormones that have an important role in stimulating growth, differentiation, metabolism and regeneration both in vitro and in vivo.

Preferences and specific embodiments: Preferred cytokines for use according to the invention include IGF-1 and IGF-2. Approximately 99% of IGF-1 in healthy individuals circulates in the blood stream bound to IGFBP-3 where it forms a large ternary l5OkD complex after association with acid-labile subunit protein (ALS). The ternary complex is restricted to the circulation by the capillary endothelium and thus serves as a circulatory reservoir of IGF-1. Thus, for therapeutic applications according to the invention IGF-1 is preferably administered in the form of a complex. For example, a preferred cytokine for use in the combinations of the invention is IPLEXTM (recombinant protein complex of insulin-like growth factor-I (IGF-1) and its most abundant binding protein, insulin-like growth factor binding protein-3 (IGFBP-3)). Another suitable cytokine is G-CSF (or other mobilizing agents as herein defined, e.g. GM-CSF), which can support muscle regeneration by mobilizing stem cells from the marrow. Other preferred cytokines include IGF-1 derivatives (IGF-1 E peptides) as described in W02006056885 (the content of which is incorporated herein by reference) which have the appropriate subsets of the function of the full-length IGF-1 and, in particular, its regenerative capacity. Thus, in a preferred embodiment the combinations of the invention comprise the IGF-l Ea peptide (i.e. the 35 amino acid C terminal peptide translated from part of exons 4 and 5 of the IGF-l gene as part of the IGF-l propeptide and which is cleaved off during post-translational processing) and/or the IGF-l Eb peptide (i.e. the 41 amino acid C terminal peptide translated from parts of exons 4, 5 and 6 of the IGF-l gene as part of the IGF-l propeptide and which is cleaved off during post-translational processing).

Posology: IPLEXTM can be administered via subcutaneous injection at an initial dose of 0.5 mg/kg, to be increased into the therapeutic dose range of ito 2 mg/kg, given once daily.

IPLEXTM can be given in the morning or in the evening but should be administered at approximately the same time every day. In order to establish tolerability to IPLEXTM, glucose monitoring should be considered at treatment initiation or when a dose has been increased. If frequent symptoms of hypoglycemia or severe hypoglycemia occur, preprandial glucose monitoring should continue. Glucose monitoring is also advised for patients with recent occurrences of asymptomatic or symptomatic hypoglycemia. If evidence of hypoglycemia is present at the time of dosing, the dose should be withheld.

Dosage can be titrated up to a maximum of 2 mg/kg daily based on measurement of IGF-1 levels obtained 8-18 hours after the previous dose. Dosage should be adjusted downward in the event of adverse effects (including hypoglycemia) and/or IGF-i levels that are greater than or equal to 3 standard deviations above the normal reference range for IGF-i.

5. Corticosteroids In one embodiment of the invention, the adjunctive agent is a corticosteroid.

Definition and biological activities: The term "corticosteroid" as used herein refers to any of several steroid hormones secreted by the cortex of the adrenal glands and which are involved in one or more of the following physiological processes: stress response, immune response and regulation of inflammation, carbohydrate metabolism, protein catabolism and blood electrolyte levels. The term also includes synthetic analogues which share the aforementioned properties. Corticosteroids include glucocorticoids and mineralocorticoids.

Glucocorticoids control carbohydrate, fat and protein metabolism and are anti-inflammatory. Mineralocorticoids control electrolyte and water levels, mainly by promoting sodium retention in the kidney. Some corticosteroids have dual glucocorticoid and mineralocorticoid activities. For example, prednisone (see below) and its derivatives have some mineralocorticoid action in addition to a glucocorticoid effect. The precise cellular mechanism(s) by which corticosteroids produce antidystrophic effects are not yet known.

A multifactorial mechanism is likely and the effects of corticosteroids probably involve a reduction of inflammation, suppression of the immune system, improvement in calcium homeostasis, upregulation of the expression of compensatory proteins and an increase in myoblast proliferation.

Problems: The use of corticosteroids is associated with side effects which vary from person to person and on the dosage of the regime used, but they can be severe. The most common side effects are weight gain and mood changes. Weight gain (and attendant changes in muscle activity and use) can abrogate some of the benefits of treatment. Long-term use may lead to growth suppression, cataracts, osteoporosis and muscle atrophy (affecting the same proximal muscles affected in DMD and BMD). These side effects may limit the long-term effectiveness of corticosteroid therapy. Other side effects include hypertension, diabetes, skin atrophy, poor wound healing and immunosuppression.

Deflazacort was evaluated in the hope that it would have fewer side effects than pred n ison e.

Preferences and Specific embodiments: Preferred are glucocorticoids (or corticosteroids having dual glucocorticoid/minerlocorticoid activity). Synthetic corticosteroids are preferred. In one embodiment, the corticosteroid is prednisone (prodrug) or prednisolone (liver metabolite of prednisone and active drug). In another embodiment, the corticosteroid is deflazacort. Deflazacort is an oxazoline analogue of prednisone. Other synthetic corticosteroids suitable for use in the combinations of the invention include one or more corticosteroids selected from: alclometasone, amcinonide, beclomethasone (including beclomethasone dipropionate), betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortivazol, deoxycorticosterone, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone, fluprednidene, fluticasone, formocortal, halcinonide, halometasone, hydrocortisone aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol, medrysone, meprednisone, methylprednisolone, methyiprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednylidene, rimexolone, tixocortol, triamcinolone and ulobetasol (or combinations and/or derivatives (e.g. pharmaceutically acceptable salts) of one or more of the foregoing). Suitable endogenous corticosteroids for use in the combinations of the invention include include one or more corticosteroids selected from aldosterone, cortisone, hydrocortisone/cortisol and desoxycortone (or combinations and/or derivatives (e.g. pharmaceutically acceptable salts) of one or more of the foregoing).

Posology: Prednisone may be administered daily in dosages ranging from 0.3 to 1.5 mg/kg (typically 0.7 mg/kg). Some patienmts respond better to �=2.5 mg/kg every other day. Deflazacort has an estimated dosage equivalency of 1:1.3 compared with prednisone, though biological equivalence between deflazacort and prednisone also depends on the specific actions under examination. Corticosterods (including delazacort and prednisone) are usually taken orally but can be delivered by intramuscular injection.

6. Anabolic steroids In one embodiment of the invention, the adjunctive agent is an anabolic steroid.

Definition and biological activities: The term "anabolic steroid" as used herein refers to any of several steroid hormones related to the male hormone testosterone and synthetic analogues thereof. Such steroids are may also be referred to as "anabolic-androgenic steroids" or "AAS". Anabolic steroids increase protein synthesis within cells, promoting anabolism (especially in muscles). The precise cellular mechanism(s) by which anabolic steroids produce antidystrophic effects are not yet known, but it seems that their anabolic effects in muscles effectively compensates for muscle loss. Oxandrolone has been shown to have anabolic effects on DMD muscle as well as decreasing muscle degeneration and so easing the demands for muscle regeneration. By conserving regenerative capacity, anabolic steroids such as oxandrolone may prolong muscle function.

Problems: The use of anabolic steroids is associated with severe side effects. The most common side effects are liver and kidney damage, sterility, stunting of growth and severe mood swings. Anabolic steroids also also tend to be androgenizing and can promote growth of beard and body hair, maturation of genitalia and development of acne.

Withdrawal can lead to rapid and severe deterioration in muscle mass and function.

Preferences and Specific embodiments: Preferred are synthetic anabolic steroids such as oxandrolone (Anavar), norethandrolone and methandrostenolone (Dianabol). Oxandrolone (an oral synthetic analog of testosterone) may be particularly preferred because in addition to its anabolic properties it also blocks the binding of cortisol to glucocorticoid receptors on muscle, thus preventing muscle breakdown. Other anabolic steroids suitable for use in the combinations of the invention include one or more anabolic steroids selected from: DHEA, DHT, methenolone, oxymetholone, quinbolone, stanozolol, ethylestrenol, nandrolone (Deca Durabolin), oxabolone cipionate, boldenone undecylenate (Equipoise), stanozolol (Winstrol), oxymetholone (Anadrol-50), fluoxymesterone (Halotestin), trenbolone (Fina), methenolone enanthate (Primobolan), 4-chlordehydromethyltestosterone (Turinabol), mesterolone (Proviron), mibolerone (Cheque Drops), tetrahydrogestrinone and testosterone (or combinations and/or derivatives (e.g. pharmaceutically acceptable salts) of one or more of the foregoing).

Posology: Anabolic steroids may be administered orally in the form of pills, by injection or via skin patches. Oral administration is most convenient, but since the steroid must be chemically modified so that the liver cannot break it down before it reaches the blood stream these formulations can cause liver damage in high doses. Injectable steroids are typically administered intramuscularly. Transdermal patches can be used to deliver a steady dose through the skin and into the bloodstream. Oxandrolone may be administered orally at a daily dosage of 0.1 mg/kg.

7. TGF-13 antagonists Definitions and technical background: Transforming growth factor beta (TGF-13) promotes fibrosis in response to muscle tissue damage associated with DMD that can contribute to disease pathology. In one embodiment of the invention, the adjunctive agent is a TGF-13 antagonist.

The term TGF-13 antagonist is used herein to refer to compounds which directly or indirectly perturb, disrupt, block, modulate or inhibit the action of TGF-13. The term also embraces the ionic, salt, solvate, isomers, tautomers, N-oxides, ester, prodrugs, isotopes and protected forms thereof (preferably the salts or tautomers or isomers or N-oxides or solvates thereof, and more preferably, the salts or tautomers or N-oxides or solvates thereof).

Preferences and specific embodiments: Preferred TGF-13 antagonists for use according to the invention include anti-TGF-13 antibodies, tamoxifen, losartan and pirfenidone.

Pirfenodone is an orally active synthetic antifibrotic agent structurally similar to pyridine 2,4-dicarboxylate. Pirfenidone inhibits fibroblast, epidermal, platelet-derived, and TGF-13 -1 growth factors and also inhibits DNA synthesis and the production of mRNA for collagen types I and Ill, resulting in a reduction in radiation-induced fibrosis. Losartan is an angiotensin II receptor antagonist drug used mainly to treat hypertension currently marketed by Merck & Co. under the trade name CozaarTM. However, losartan also downregulates the expression of (TGF-13 types I and II receptors. Tamoxifen is an orally active selective estrogen receptor modulator (SERM) which is used in the treatment of breast cancer and is currently the world's largest selling drug for this indication. Tamoxifen is sold under the trade names NolvadexTM, lstubalTM and ValodexTM. Tamoxifen may be administered at a dose of 10-1 00 mg per day (e.g. 20-40 mg/day).

8. Antioxidants and mitochondrial supporting agents Disease mediated by ROS and/or calpain hyperactivity may be ameliorated by antioxidants and/or mitochondrial support agents. For example, in DMD, the cytoskeletal protein dystrophin is absent leading to numerous cellular dysfunctions that culminate in muscle cell necrosis. Subsequently, an inflammatory response develops in the necrotic muscle tissue, resulting in increased oxidative stress, responsible for further tissue damage. In the mdx dystrophic mouse, both inflammation and oxidative stress have been identified as aggravating factors for the course of the disease.

GTE and EGCG also display pro-myogenic properties. Primary cultures of skeletal muscle cells were established from both normal and dystrophic mice and treated with GTE and EGCG for 1-7 days. As judged by in situ staining of myosin heavy chains (MyHC), it has been shown that GTE and EGCG concentration-dependently stimulated the rate of formation of myotubes within the first 2-4 days of application. The amount of myotubes reached similar level with both agents compared to control thereafter. Western-blot analysis was performed on myotube cultures treated for 7 days. GTE and EGCG promoted the expression of several muscle-specific proteins, such as dystrophin (in control cultures), sarcomeric alpha actinin, and MyHC, while myogenin was unchanged. By contrast, the expression of desmin was down-regulated and redistributed to Z discs. The results suggest that green tea polyphenols display pro-myogenic properties by acting directly on skeletal muscle cells. These findings suggest a beneficial action for muscle regeneration and strengthening in dystrophic condition.

Green tea polyphenols, such as epigallocatechin gallate (EGCG), are known to be powerful antioxidants. Because inflammation is involved in the degradation of muscle tissue in MD, oxidative stress is believed to play a role in this process. Thus, green tea and its active constituents (including EGCG and other polyphenols) may improve MD prognosis by reducing this oxidative stress. Feeding studies with mdx mice have shown a protective effect of EGCG against the first massive wave of necrosis. It also stimulated muscle adaptation toward a stronger and more resistant phenotype. The effective dosage corresponds to about seven cups of brewed green tea per day in humans Coenzyme Q10 (C0Q1O; also called ubiquitin) is a powerful antioxidant and mitochondrial respiratory chain cofactor. It possesses membrane-stabilizing properties and is capable of penetrating cell membranes and mitochondria. Dosages of 100 mg CoQ1 0 daily for three months have been shown to be beneficial in human trials, though higher dosages are likely to yield better results.

Idebenone is a synthetic analog of Coenzyme Q10 and is thought to perform the same functions as CoQ1 0 without the risk of auto-oxidation. Like CoQ1 0, idebenone can therefore contribute to maintaining correct electron balance, which is necessary for the production of cellular energy. Since muscle cells are particularly energy-demanding idebenone and CoQ1 0 can preserve mitochondrial function and protect cells from oxidative stress.

Glutamine is an important energy source and acute oral glutamine administration appears to have a protein-sparing effect. Arginine (and other pharmacological activators of the NO pathway) may enhance the production of utrophin in MDX mice. The increase is likely to be mediated by arginine-fueled production of nitric oxide (NO), which plays an important role in blood vessel function and is generally lower in people with MD. Studies with MDX mice have also shown that a combination of arginine and deflazacort may be more beneficial than deflazacort alone.

Other antioxidants suitable for use according to the invention are the chimaeric compounds or combinations in which the a ROS inhibitor is associated (e.g. combined with, co-administered with or covalently linked) to calpain inhibiting moiety. Such agents combine relief of oxidative stress with a reduction in calpain-mediated muscle tissue breakdown.

Suitable dual action calpain/ROS inhibitors are described for example in WOO 1/32654, W02007/045761, W02005/056551 and WO 2002/40016 (the contents of which are incorporated herein by reference).

9. Dystrophin expression enhancing agents In embodiments where the disease mediated by ROS and/or calpain hyperactivity is DMD, the adjunctive agent may comprise dystrophin expression enhancing agents, as described below: 9.1 Read-through agents A subset of DMD patients (around 15%) have a nonsense mutation that produces a premature stop signal in their RNA, resulting in abnormal truncation of protein translation.

In one embodiment of the invention, the adjunctive agent is an agent which promotes readthrough of premature stop codons ("read-through agent"), thereby bypassing the premature stop codon and restoring the expression of full-length, functional dystrophin.

Suitable read-through agents for use according to the invention are 1,2,4-oxadiazole compounds as described in U56992096 (which is incorporated herein by reference): è One such compound is 3-[5-(2-fluoro-phenyl)-[l,2,4]oxadiazol-3-yl] -benzoic acid. A preferred readthrough agent is PTC124. PTC124 is a 284-Dalton 1,2,4-oxadiazole that promotes ribosomal readthrough of premature stop codons in mRNA. Thus, the combinations of the invention may comprise 1,2,4-oxadiazole benzoic acid compounds (including 3-[5-(2-fluoro-phenyl)-[l,2,4]oxadiazol-3-yl]-benzoic acid) (see e.g. W020061 10483, the content of which is incorporated herein by reference).

PTC124, 3-[5-(2-fluoro-phenyl)-[l,2,4]oxadiazol-3-yl]-benzoic acid or a pharmaceutically acceptable salt, solvate or hydrate thereof can be administered in single or divided (e.g., three times daily) doses between 0.1 mg/kg and 500 mg/kg, 1 mg/kg and 250 mg/kg, 1 mg/kg and 150 mg/kg, 1 mg/kg and 100 mg/kg, 1 mg/kg and 50 mg/kg, 1 mg/kg and 25 mg/kg, 1 mg/kg and 10mg/kg or2 mg/kg and 10mg/kg to a patent in need thereof. In a particular embodiment, the 3-[5-(2-fluoro-phenyl)-[l,2,4]oxadiazol-3-yl]-benzoic acid or a pharmaceutically acceptable salt, solvate or hydrate thereof is administered in a dose of about 4 mg/kg, about 7 mg/kg, about 8 mg/kg, about 10 mg/kg, about 14 mg/kg or about 20 mg/kg.

Other readthrough agents for use according to the invention include aminoglycoside antibiotics, including gentamicin. Particularly preferred may be aminoglycosides that contain a 6' hydroxyl group (e.g. paromomycin), which may be effective at lower doses and may display less toxicity than compounds such as gentamicin.

9.2 Exon skipping Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene mutations that disrupt the mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional dystrophin protein (so called quasi-dystrophin). Antisense oligonucleotides (AON5) designed to cause exon skipping can target a broader range of mutations than can compounds that cause cells to ignore premature stop codons by inducing cells to leave out sections of genetic instructions that contain mistakes and join together the surrounding, correct instructions. However, since AONs are not self-renewed, they cannot achieve long-term correction. To overcome this limitation, antisense sequences can be introduced into small nuclear RNAs (5nRNA) and vectorized in AAV and lentiviral vectors.

10. Gene replacement/repair agents In one embodiment of the invention, the adjunctive agent is a nucleic acid construct adapted to replace or repair non-functional endogenous genetic material. Gene therapy may be adeno-associated virus (AAV) vector-mediated gene therapy, preferably using the microdystrophin gene. Highly abbreviated microdystrophin cDNAs have been developed for adeno-associated virus (AAV)-mediated DMD gene therapy. Among these, a C-terminal-truncated R4-R23/C microgene (AR4/AC) is a very promising therapeutic candidate gene.

Targeted correction of mutations in the genome holds great promise for the repair/treatment of disease causing mutations either on their own applied directly to the affected tissue, or in combination with other techniques such as stem cell transplantation.

Various DNA or RNNDNA based Corrective Nucleic Acid (CNA) molecules such as chimeraplasts, single stranded oligonucleotides, triplex forming oligonucleotides and SFHR have been used to change specific mutant loci. MyoDys� is comprised of plasmid DNA encoding the full-length human dystrophin gene. Mirus' Pathway IV TM delivery technology is used to administer the pDNA to a patient's limb skeletal muscles.

11. Cell-based therapies In one embodiment of the invention, the adjunctive agent is a myogenic cell or tissue composition. Various types of myogenic cell have been shown to have potential in the treatment of DMD, including stem cells from umbilical cord, mesenchymal stem cells and muscle-derived stem cells.

12. Creatine Definition and biological activities: Creatine is an energy precursor that is naturally produced by the body. Creatine kinase (OK) phosphorylates creatine for later donation to contractile muscle filaments: phosphocreatine enters muscle cells and promotes protein synthesis while reducing protein breakdown. In healthy individuals, creatine has been shown to enhance endurance and increase energy levels by preventing depletion of adenosine triphosphate. Among MD patients, studies have suggested that supplemental creatine can improve muscle performance and strength, decrease fatigue, and slightly improve bone mineral density.

Problems: High doses of creatine can cause kidney damage and requires cohydration.

Behavioral changes have been recorded.

Posology: Creatine can be administered as a powdered nutritional supplement. In recent trials with DMD patients, slight increases in muscle strength on administration of low levels (ito 10 g/day) of creatine monohydrate have been recorded. Intermittent administration (involving a break of one to several weeks) may mitigate side effects whilst providing the same benefits as constant use. Dosages in the region of 100mg/kg/day are well-tolerated and have been found to decrease bone degradation and increase strength and fat-free mass. Benefits have been reported for the co-administration of creatine with conjugated linoleic acid (alpha-lipoic acid), hydroxyl-beta-methylbutyrate and prednisolone.

13. Anti-osteoporotic agents Disease mediated by ROS and/or calpain hyperactivity may involve an osteoporotic component. Combined therapy to inhibit bone resorption, prevent osteoporosis, reduce skeletal fracture, enhance the healing of bone fractures, stimulate bone formation and increase bone mineral density can be effectuated by combinations comprising various anti-osteoporotic agents. Preferred are bisphosphonates including alendronate, tiludronate, dimethyl-APD, risedronate, etidronate, YM-175, clodronate, pamidronate and BM-210995 (ibandronate). Others include oestrogen agonist/antagonists. The term oestrogen agonist/antagonists refers to compounds which bind with the estrogen receptor, inhibit bone turnover and prevent bone loss. In particular, oestrogen agonists are herein defined as chemical compounds capable of binding to the estrogen receptor sites in mammalian tissue, and mimicking the actions of estrogen in one or more tissue. Exemplary oestrogen agonist/antagonists include droloxifene and associated compounds (see US 5047431), tamoxifen and associated compounds (see U54536516), 4-hydroxy tamoxifen (see U54623660), raloxifene and associated compounds (see U54418068 and idoxifene and associated compounds (see U54839155).

14. Utrophin upregulating agents In embodiments where the disease mediated by ROS and/or calpain hyperactivity is DMD, the adjunctive agent may include one or more utrophin upregulating agents. Such auxiliary utrophin upregulating agents are compounds that upregulate (i.e. increase the expression or activity of utrophin). Examples include the compounds described in W02007/091106, W02007/091 107, W02008/0291 52 and W02008/0291 68.

15. cGMP signalling modulators It has recently been shown (Khairallah et a!. (2008) PNAS 105(19): 7028-7033) that enhancement of cGMP signaling by administration of the phosphodiesterase 5 (PDE5) inhibitor sildenafil prevents deterioration of myocardial contractile peformance in mdx hearts.

Thus, cGMP signaling enhancers, including in particular selective PDE5 inhibitors (including for example sildenafil, tadalafil, vardenafil, udenafil and avanafil) may be used in combination with the compounds of the invention to treat DMD or BMD. Such combinations find particular application in the treatment of dystrophic cardiopmyopathies and may be used to prevent or delay the onset of dystrophin-related cardiomyopathies as the clinical course of DMD/BMD progresses.

Thus, the invention contemplates combinations of the compounds of the invention with cGMP signaling enhancers, including in particular selective PDE5 inhibitors. Preferred combinations are comprise a compound of the invention and a PDE5 inhibitor selected from sildenafil, tadalafil, vardenafil, udenafil and avanafil. Particularly preferred is a combination comprising a compound of the invention and sildenafil.

Posoloqy The compounds of the present invention can be administered by oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.

The amount administered can vary widely according to the particular dosage unit employed, the period of treatment, the age and sex of the patient treated, the nature and extent of the disorder treated, and the particular compound selected.

Moreover, the compounds of the invention can be used in conjunction with other agents known to be useful in the treatment of diseases or disorders mediated by protein folding abnormalities (as described infra) and in such embodiments the dose may be adjusted accordingly.

In general, the effective amount of the compound administered will generally range from about 0.01 mg/kg to 500 mg/kg daily. A unit dosage may contain from 0.05 to 500 mg of the compound, and can be taken one or more times per day. The compound can be administered with a pharmaceutical carrier using conventional dosage unit forms either orally, parenterally, or topically, as described below.

The preferred route of administration is oral administration. In general a suitable dose will be in the range of 0.01 to 500 mg per kilogram body weight of the recipient per day, preferably in the range of 0.1 to 50 mg per kilogram body weight per day and most preferably in the range 1 to 5 mg per kilogram body weight per day.

The desired dose is preferably presented as a single dose for daily administration.

However, two, three, four, five or six or more sub-doses administered at appropriate intervals throughout the day may also be employed. These sub-doses may be administered in unit dosage forms, for example, containing 0.001 to 100 mg, preferably 0.01 to 10 mg. and most preferably 0.5 to 1.0 mg of active ingredient per unit dosage form.

Formulation The compound for use according to the invention may take any form. It may be synthetic, purified or isolated from natural sources.

When isolated from a natural source, the compound may be purified. In embodiments where the compound is formulated together with a pharmaceutically acceptable excipient, any suitable excipient may be used, including for example inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc.

The pharmaceutical compositions may take any suitable form, and include for example tablets, elixirs, capsules, solutions, suspensions, powders, granules and aerosols.

The pharmaceutical composition may take the form of a kit of parts, which kit may comprise the composition of the invention together with instructions for use and/or a plurality of different components in unit dosage form.

Tablets for oral use may include the compound for use according to the invention, mixed with pharmaceutically acceptable excipients, such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Capsules for oral use include hard gelatin capsules in which the compound for use according to the invention is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.

Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

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

The compounds of the invention may also be presented as liposome formulations.

For oral administration the compound can be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, granules, solutions, suspensions, dispersions or emulsions (which solutions, suspensions dispersions or emulsions may be aqueous or non-aqueous). The solid unit dosage forms can be a capsule which can be of the ordinary hard-or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and cornstarch.

In another embodiment, the compounds of the invention are tableted with conventional tablet bases such as lactose, sucrose, and cornstarch in combination with binders such as acacia, cornstarch, or gelatin, disintegrating agents intended to assist the break-up and dissolution of the tablet following administration such as potato starch, alginic acid, corn starch, and guar gum, lubricants intended to improve the flow of tablet granulations and to prevent the adhesion of tablet material to the surfaces of the tablet dies and punches, for example, talc, stearic acid, or magnesium, calcium, or zinc stearate, dyes, coloring agents, and flavoring agents intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient.

Suitable excipients for use in oral liquid dosage forms include diluents such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptably surfactant, suspending agent or emulsifying agent.

The compounds of the invention may also be administered parenterally, that is, subcutaneously, intravenously, intramuscularly, or interperitoneally.

In such embodiments, the compound is provided as injectable doses in a physiologically acceptable diluent together with a pharmaceutical carrier (which can be a sterile liquid or mixture of liquids). Suitable liquids include water, saline, aqueous dextrose and related sugar solutions, an alcohol (such as ethanol, isopropanol, or hexadecyl alcohol), glycols (such as propylene glycol or polyethylene glycol), glycerol ketals (such as 2,2-dimethyl-1,3-dioxolane-4-methanol), ethers (such as poly(ethylene-glycol) 400), an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant (such as a soap or a detergent), suspending agent (such as pectin, carhomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose), or emulsifying agent and other pharmaceutically adjuvants.

Suitable oils which can be used in the parenteral formulations of this invention are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum, and mineral oil. Suitable fatty acids include oleic acid, stearic acid, and isostearic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate.

Suitable soaps include fatty alkali metal, ammonium, and triethanolamine salts and suitable detergents include cationic detergents, for example, dimethyl dialkyl ammonium halides, alkyl pyridinium halides, and alkylamines acetates; anionic detergents, for example, alkyl, aryl, and olefin sulphonates, alkyl, olefin, ether, and monoglyceride sulphates, and sulphosuccinates; nonionic detergents, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers; and amphoteric detergents, for example, alkyl-beta-aminopropionates, and 2-alkylimidazoline quarternary ammonium salts, as well as mixtures.

The parenteral compositions of this invention will typically contain from about 0.5 to about 25% by weight of the compound for use according to the invention in solution.

Preservatives and buffers may also be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain a non-ionic surfactant having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations ranges from about 5 to about 15% by weight. The surfactant can be a single component having the above HLB or can be a mixture of two or more components having the desired HLB. Illustrative of surfactants used in parenteral formulations are the class of polyethylene sorbitan fatty acid esters, for example, sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

The compound for use according to the invention may also be administered topically, and when done so the carrier may suitably comprise a solution, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Topical formulations may contain a concentration of the compound from about 0.1 to about 10% w/v (weight per unit volume).

The compound for use according to the invention may also be administered to the eye (e.g. by dropper). Ophthalmic formulations are typically packaged in multidose form and include preservatives to prevent microbial contamination during use. Suitable preservatives include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl paraben, phenylethyl alcohol, edetate disodium and sorbic acid. Such preservatives may be present from 0.00 1 to 1.0% weight/volume ("% w/v").

When used adjunctively, the compound for use according to the invention may be formulated for use with one or more other drug(s). In particular, the compounds may be used in combination with lysosomal enzymes adjunctive to enzyme replacement therapy.

Thus, adjunctive use may be reflected in a specific unit dosage designed to be compatible (or to synergize) with the other drug(s), or in formulations in which the compound is admixed with one or more enzymes. Adjunctive uses may also be reflected in the composition of the pharmaceutical kits of the invention, in which the compounds of the invention is co-packaged (e.g. as part of an array of unit doses) with the enzymes.

Adjunctive use may also be reflected in information and/or instructions relating to the co-administration of the compound and/or enzyme.

Exemplification The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

General HPLC-UV-MS was performed on a Gilson 321 HPLC with detection performed by a Gilson DAD and a Finnigan AQA mass spectrometer operating in electrospray ionisation mode. The HPLC column used is a Phenomenex Gemini 018 150x4.6mm. Preparative HPLC was performed on a Gilson 321 with detection performed by a Gilson 170 DAD.

Fractions were collected using a Gilson 215 fraction collector. The preparative HPLC column used is a Phenomenex Gemini 018 l5OxlOmm and the mobile phase is aceton itrile/water.

1H NMR spectra were recorded on a Bruker instrument operating at 300 MHz. NMR spectra were obtained as CDCI3 solutions (reported in ppm), using chloroform as the reference standard (7.25 ppm) or DMSO-D6 (2.50 ppm). When peak multiplicities are reported, the following abbreviations are used s (singlet), d (doublet), t (triplet), m (multiplet), br (broadened), dd (doublet of doublets), dt (doublet of triplets), td (triplet of doublets). Coupling constants, when given, are reported in Hertz (Hz).

Column chromatography was performed either by flash chromatography (40-65pm silica gel) or using an automated purification system (CompanionTM Purification System from ISCO�).

The abbreviations used are DMSO (dimethylsulfoxide), HCI (hydrochloric acid), Mg504 (magnesium sulfate), NaOH (sodium hydroxide), Na2003 (sodium carbonate), NaHCO3 (sodium bicarbonate), THF (tetrahydrofuran), K2003 (potassium carbonate).

Scheme XX: Procedure A 0 Procedure B N H OH Procedure c N H 0

N NH N NH

HN T OH B0cNH NH OH OH 0 C Procedure D X=H x x x ProcedureE ProcedureC o HN NHR Ri N N NHR Ri N N NHR R OH 0 R OH 0 R 0 Procedure F BOcNH COH x

LCHO

Scheme 7. Reagents & conditions: (i) HCI in dioxane, lh, rt, Quant; (ii) Boc-L-leucine, EDO, HOBt, DIPEA, DMF, 18 h; (iii) HCI in dioxane, 40-54% over 3 steps; (iv) EDO, HOBt, DIPEA, DMF, 18 h, rt; (v) Dess-Martin periodinane, 0H2012, RT, 24h.

Procedure A Tert-butyl (2S, 3S)-4-amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylcarbamate A solution of (Boc)20 (2.07g, 9.5 mmol) in dioxane (lOmL) was added to a stirred solution of (2S, 3S)-3-amino-2-hydroxy-4-phenylbutanoic acid (2.OOg, 8.6 mmol) in aq. NaOH (1M, 1 OmL) and the resulting reaction mixture was stirred at room temperature for 4h, maintaining the pH between 10-11 with 1 N NaOH. The reaction mixture was diluted with H20 and washed with Et20 (2x). The aqueous phase was cooled to 0 °O, acidified to pH 2 with 1 N HCI and extracted with Et20 (3x). The combined organic phases were dried over MgSO4 and concentrated under reduced pressure. The residue was dissolved in DMF (2OmL) and HOBt (1.03g, 7.6 mmol), EDCI (2.92g, 15.2 mmol) and NH3 in dioxane (0.5M, 55mL, 27.6 mmol) were added followed by DIPEA (4mL, 22.8mmol). The reaction mixture was stirred at room temperature for 16h, diluted with EtOAc, washed with brine (2x), dried over MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography eluting using a gradient (petroleum ether 40/60 / ethyl acetate 1:0 v/v 0:1) to give 1.13g (44%) of tert-butyl (2S, 3S)-4-amino-3-hydroxy-4-oxo-1-phenylbutan- 2-ylcarbamate. LCMS RT= 1.59mm, MH 295.2 Procedure B N-((2S,3S)-4-Amino-3-hydroxy-4-oxo-1 -phenylbutan-2-yI)-2-(phenylami no)benzamide A solution of tert-butyl (2S, 3S)-4-amino-3-hyd roxy-4-oxo-1 -phenylbutan-2-ylcarbamate (22mg, 0.075 mmol) and HCI in dioxane (4M, 2mL) was stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure and the residue was dissolved in DMF (5mL). N-phenylanthranilic acid (18mg, 0.083 mmol), HOBt (11.2mg, 0.083 mmol), EDCI (16mg, 0.083 mmol) and DIPEA (26mL, 0.15 mmol) were added and the resulting reaction mixture was stirred at room temperature for 16h. The solution was diluted with EtOAc, washed with aq. NH4CI, aq. NaHCO3 and brine, dried over MgSO4 and concentrated under reduced pressure to give 29mg (98%) of N-((2S,3S)-4-amino-3-hydroxy-4-oxo-1 -phenylbutan-2-yl)-2-(phenylamino)benzamide. LCMS RT= 1.93mm, MH 390.3 Procedure C (S)-N-(4-Amino-3, 4-dioxo-1 -phenylbutan-2-yl)-2-(phenylamino)benzamide (Cpd 1) Dess Martin reagent (40mg, 0.096 mmol) was added to a solution of N-((2S,3S)-4-amino- 3-hydroxy-4-oxo-1 -phenylbutan-2-yl)-2-(phenylamino)benzamide (30mg, 0.077 mmol) in DCM (5mL) and the resulting reaction mixture was stirred at room temperature for 2.5h.

The solvent was evaporated in vacuo, aq. Na2S2O3 (1 M), was added and the aqueous phase was extracted with EtOAc. The organic phase was washed with aq. NaHCO3 and brine, dried over MgSO4 and concentrated under reduced pressure. The residue was purified by column chromatography eluting using a gradient (petroleum ether 40/60 / ethyl acetate 1:1 v/v), followed by more column chromatography eluting using a gradient (DCM/EtOAc 95/5 v/v) gave 5mg (17%) of (S)-N-(4-amino-3, 4-dioxo-1-phenylbutan-2-yl)- 2-(phenylamino)benzamide. LCMS RT= 1.81mm, 2.11mm, MH388.3, 406.2; ; 1H NMR (DMSO): 9.31 (1 H, bs), 8.93 (1 H, d, J 7.2 Hz), 8.11 (1 H, bs), 7.82 (1 H, bs), 7.62 (1 H, dd, J 1.4 Hz, J7.9 Hz), 7.35-7.16 (1OH, m), ,6.98 (1H, t, J7.3 Hz), 6.82 (1H, t, J7.4 Hz), 5.33 (1H, m), 3.22 (1H, dd, J3.4 Hz, J 13.7 Hz), 2.94 (1H, dd, J 10.4, J 13.7 Hz).

All compounds below were synthesized using Procedure C N-((S)-1 -((S)-4-(Benzylamino)-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(2-methoxyphenylamino)benzamide (Cpd 2) LCMS RT= 2.37mm, 2.67mm, MH 639.9, 621.6; 1H NMR (DMSO): 9.51 (1 H, bs), 9.22 (1H, t, J6.1 Hz), 8.43 (2H, d, J6.5 Hz), 7.68 (1H, d, J7.6 Hz), 7.33-7.14 (12H, m), 7.10- 7.03 (2H, m), 6.98-6.87 (2H, m), 6.80 (1H, t, J7.0 Hz), 5.24-5.18 (1H, m), 4.60-4.52 (1H, m), 4.29 (2H, d, J6.1 Hz), 3.80 (3H, s), 3.12 (1H, dd, J4.0 Hz, J 14.2 Hz), 2.87 (1H, dd, J 9.0 Hz, J 14.1 Hz), 1.69-1.56 (2H, m), 1.51-10.41 (1H, m), 0.89 (3H, d, J6.4 Hz), 0.86 (3H, d, J6.4 Hz).

N-((S)-1 -((S)-4-Amino-3,4dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)- 2-(phenylamino)benzamide (Cpd 3) LCMS RT= 2.04mm, 2.28mm, MH 501.3; 1H NMR (DMSO): 9.56 (1 H, bs), 8.48-8.34 (2H, m), 8.13-8.03 (1H, m), 7.83 (1H, bs), 7.72-7.68 (1H, m), 7.34-7.09 (12H, m), 6.97 (1H, t, J 7.2 Hz), 6.84 (1H, J6.8 Hz), 5.25-5.19 (1H, m), 4.57-4.48 (1H, m), 3.21-3.10 (2H, m), 1.67- 1.56 (1H, m), 1.52-1.42 (2H, m), 0.90-0.79 (6H, m).

N-((S)-1 -((S)-4-(Ethylamino)-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(2-methoxyphenylamino)benzamide (Cpd 4) LCMS RT= 2.17, 2.45mm, MH559.4; 1H NMR(DMSO): 9.50 (1H, s), 8.68 (1H, t, J5.9 Hz), 8.40 (2H, t, J7.3 Hz), 7.68 (1H, d, J7.5 Hz), 7.35-7.15 (8H, m), 7.10-7.03 (2H, m), 6.95 (1 H, dt, J 1.7 Hz, J 7.4 Hz), 6.89 (1 H, dt, J 1.6 Hz, J 7.5 Hz), 6.80 (1 H, ddd, J 1.5 Hz, J6.8, J8.0 Hz), 5.21-5.14 (1H, m), 4.59-4.51 (1H, m), 3.16-3.07 (3H, m), 2.84 (1H, dd, J 9.3 Hz, J14.0 Hz), 1.66-1.56 (2H, m), 1.51-1.45(1H, m), 1.01 (3H, t, J7.2 Hz), 0.90 (3H, d, J6.2 Hz), 0.86 (3H, d, J6.3 Hz).

N-((S)-1 -((S)-4-(Ethylamino)-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(phenylamino)benzamide (Cpd 5) LCMS RT= 2.17, 2.46mm, MH529.1, 547.0; 1H NMR(DMSO): 9.55 (1H, s), 8.68 (1H, t, J 5.9 Hz), 8.460 (2H, d, J 8.5 Hz), 8.39 (1 H, d, J 7.4 Hz), 7.70 (1 H, d, J 6.6 Hz), 7.36-7.07 (12H, m), 6.97 (1H, t, J7.4 Hz), 6.83 (1H, t, J8.0 Hz), 5.21-5.14 (1H, m), 4.59-4.48 (1H, m), 3.16-3.06 (3H, m), 2.83 (1H, dd, J9.3 Hz, J 13.9 Hz), 1.63-1.55 (2H, m), 1.51-1.42 (1H, m), 1.01 (3H, t, J7.2 Hz), 0.88 (3H, d, J6.2 Hz), 0.85 (3H, d, J6.3 Hz).

N-((S)-1 -((S)-4-Amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(2-chlorophenylamino)-5-methoxybenzamide (Cpd 6) LCMS RT= 2.12, 2.35; MH565.3, 583.2; 1H NMR(CDCI3): 7.30-6.90 (13H, m), 6.77-6.56 (3H, m), 5.43-5.36 (2H, m), 456-4.44 (1H, m), 3.75 (3H, s), 3.24 (1H, dd, J5.2 Hz, J 14.1 Hz), 2.96 (1H, dd, J7.9 Hz, J 14.1 Hz), 1.60-1.44 (2H, m), 1.38-1.29 (1H, m), 0.77-0.72 (6H, m).

N-((S)-1 -((S)-4-Amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(2-fluorophenylamino)benzamide (Cpd 7) LCMS RT= 2.07, 2.31; MH 519.1, 537.2; 1H NMR(DMSO): 9.73 (1H, s), 850 (1H, d, J8.6 Hz), 8.41 (1H, d, J7.8 Hz), 8.07 (1H, s), 7.76 (1H, d, J7.1 Hz), 7.46-7.34 (2H, m), 7.25- 7.12 (8H, m), 7.03 (1H, m), 6.87 (1H, t, J7.8 Hz), 5.23-5.18 (1H, m), 5.54-4.49 (1H, m), 3.14 (1H, dd, J3.5 Hz, J13.6 Hz), 2.73 (1H, m), 1.53-1.44 (2H, m), 1.35-1.23 (1H, m), 0.82 (6H, m).

N-((S)-1 -((S)-4-Amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(p-tolylamino)benzamide (Cpd 8) LCMS RT= 2.17, 2.43; MH 515.3, 533.2; 1H NMR (DMSO): 9.52 (1 H, s), 841 (1 H, d, J 8.7 Hz), 8.38 (1H, d, J8.1 Hz), 8.07 (1H, s), 7.83 (1H, s), 7.70 (1H, d, J6.7 Hz), 7.30 (1H, t, J 8.2 Hz), 7.19-7.11 (9H, m), 7.04 (2H, d, J8.3 Hz), 6.78 (1H, t, J6.8 Hz), 5.25-5.18 (1H, m), 4.54-4.47 (1H, m), 3.13 (1H, dd, J3.9 Hz, J 13.7 Hz), 2.74 (1H, dd, J 10.1 Hz, J 13.7 Hz), 2.26 (3H, s), 1.52-1.44 (2H, m), 1.34-1.24 (1H, m), 0.86-0.79 (6H, m).

N-((S)-1 -((S)-4-Amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(3-chlorophenylamino)benzamide (Cpd 9) LCMS RT= 2.16, 2.42; MH535.23, 553.72; 1H NMR(DMSO): 9.46 (1H, s), 8.47 (1H, d, J 8.0 Hz), 8.41 (1H, d, J7.7 Hz), 8.08 (1H, s), 7.85 (1H, s), 7.72 (1H, d, J6.5 Hz), 7.44-6.94 (12H, m), 5.26-5.19 (1H, m), 4.54-4.46 (1H, m), 3.14 (1H, dd, J3.2 Hz, J 13.1 Hz), 2.74 (1H, m), 1.49-1.38 (2H, m), t33-1.24 (1H, m), 0.81 (3H, d, J6.0 Hz), 0.79 (3H, d, J6.1 Hz).

N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(p-tolylamino)nicotinamide (Cpd 20) LCMS RT= 1.87mm, 2.13mm; MH516.2, 534.2; 1H NMR(DMSO): 10.62 (1H, s), 8.64 (1H, d, J8.6 Hz), 8.44 (1H, d, J8.1 Hz), 8.31-8.27 (1H, m), 8.17-8.07 (2H, m), 7.86-7.79 (1H, m), 7.55 (2H, d, J8.6 Hz), 7.23-7.08 (7H, m), 6.85-6.80 (1H, m), 5.28-5.17 (1H, m), 4.58-4.47 (1H, m), 3.18-3.10 (1H, m), 2.79-2.68 (1H, m), 2.26 (3H, s), 1.55-1.31 (3H, m), 0.86-0.80 (6H, m) N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(3-(trifluoromethyl)phenylamino)benzamide (Cpd 21) LCMS RT= 2.23mm, 2.44mm; MH 569.0; 1H NMR (DMSO): 9.47 (1H, s), 8.48-8.38 (2H, m), 8.07 (1H, s), 7.84 (1H, s), 7.72 (1H, dd, J8.0 1.4 Hz), 7.50-7.39 (2H, m), 7.37-7.31 (3H, m), 7.23-7.13 (6H, m), 7.02-6.97 (1H, m), 5.26-5.18 (1H, m), 4.52-4.44 (1H, m), 3.13 (1H, dd, J 13.7 4.0 Hz), 2.79-2.69 (1H, m), 1.45-1.22 (3H, m), 0.79-0.75 (6H, m).

N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(4-methoxyphenylami no)nicotinamide (Cpd 22) LCMS RT= 1.65mm, 1.86mm; MH532.1, 550.6; 1H NMR(DMSO): 10.50 (1H, s), 8.61 (1H, d, J8.1 Hz), 8.43 (1H, d, J8.1 Hz), 8.27-8.24 (1H, m), 8.16-8.08 (2H, m), 7.86-7.83 (1H, m), 7.54 (2H, d, J9.1 Hz), 7.24-7.13 (6H, m), 6.89 (2H, d, J9.1 Hz), 6.81-6.76 (1H, m), 5.27-5.18 (1H, m), 4.56-4.45 (1H, m), 3.73 (3H, s), 3.18-3.10 (1H, m), 2.82-2.68 (1H, m), 1.60-1.28 (3H, m), 0.86-0.80 (6H, m) (S)-N-((S)-1 -(4-bromophenyl)-4-(ethylamino)-3,4-dioxobutan-2-yI)-N-(2- (2-chlorophenylamino)-5-methoxyphenyl)-2-isobutylmalonamide (Cpd 23) LCMS RT= 2.38mm, 2.73mm; MH 673.4; 1H NMR(DMSO): 9.19 (1H, s), 8.75-8.65 (1H, m), 8.61-8.47 (2H, m), 7.45-7.03 (1OH, m), 6.89-6.82 (1H, m), 5.17-5.07 (1H, m), 4.58-4.47 (1H, m), 3.79 (3H, s), 3.16-3.05 (3H, m), 2.85-2.76 (1H, m), 1.60-1.17 (3H, m), 1.05-0.98 (3H, m), 0.88-0.74 (6H, m) N-((S)-1 -((S)-1 -(4-bromophenyl)-4-(ethylamino)-3,4-dioxobutan-2-ylami no)-3-methyl-1 -oxobutan-2-yI)-2-(phenylamino)benzamide (Cpd 24) LCMS RT= 2.25mm, 2.30mm and 2.54mm; MH 594.6; 1H NMR (DMSO): 9.36-9.31 (1 H, m), 8.67-8.56 (1H, m), 8.40 (1H, d, J7.5 Hz), 8.25-8.18 (1H, m), 7.64-7.57 (1H, m), 7.38- 6.99 (1OH, m), 6.90-6.83 (1H, m), 6.80-6.73 (1H, m), 5.13-5.05 (1H, m), 4.23-4.03 (1H, m), 3.09-2.93 (3H, m), 2.72-2.61 (1H, m), 2.00-1.81 (1H, m), 0.96-0.88 (3H, m), 0.76 (3H, d, J 7.0 Hz), 0.66-0.58 (3H, m) N-((S)-1 -((S)-1 -(4-bromophenyl)-4-(ethylamino)-3,4-dioxobutan-2-ylami no)-4-methyl-1 -oxopentane-2-yI)-2-(phenylamino)benzamide (Cpd 25) LCMS RT= 2.35mm, 2.66mm; MH 608.7; 1H NMR (DMSO): 9.66-9.47 (1H, m), 8.78-8.37 (2H, m), 7.73-7.68 (1H, m), 7.49-7.11 (11H, m), 7.02-6.94 (1H, m), 6.87-6.80 (1H, m), 5.21- 5.09 (1H, m), 4.55-4.37 (1H, m), 3.17-3.05 (3H, m), 2.87-2.71 (1H, m), 1.65-1.39 (3H, m), 1.07-0.97 (3H, m), 0.92-0.80 (6H, m) N-((S)-1 -((S)-1 -(4-bromophenyl)-4-(ethylamino)-3,3-dioxobutan-2-ylami no)-3-methyl-1 -oxobutan-2-yI)-2-(2-chlorophenylamino)-5-methoxybenzamide (Cpd 26) LCMS RT= 2.30mm, 2.35mm and 2.61 mm; MH 659.0; 1H NMR (DMSO): 9.05 (1 H, d, J 17.3 Hz), 8.75-8.68 (1H, m), 8.58-8.45 (2H, m), 7.44-7.28 (5H, m), 7.22-7.15 (4H, m), 7.09- 7.04 (1H, m), 6.88-6.81 (1H, m), 5.23-5.13 (IH, m), 4.33 (1H, t, J8.4 Hz), 3.80 (3H, s), 3.16-3.03 (3H, m), 2.77-2.71 (1H, m), 2.06-1.87 (1H, m), 1.01 (3H, d, J7.0 Hz), 0.86-0.82 (3H, m), 0.72-0.64 (3H. m) N-((S)-1 -((S)-4-(benzylami no)-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(phenylamino)benzamide (Cpd 27) LCMS RT=2.44min, 2.75mm; MH591.5; 1H NMR(DMSO): 9.56 (1H, s), 9.24-9.19(1H, m), 8.49-8.41 (2H, m), 7.73-7.69 (1H, m), 7.34-7.09 (16H, m), 7.00-6.80 (2H, m), 5.24-5.16 (1H, m), 4.58-4.49 (1H, m), 430 (1H, d, J6.O Hz), 3.19-3.07 (1H, m), 2.93-2.80 (1H, m), 1.67-1.42 (3H, m), 0.91-0.84 (6H, m) N-((S)-1 -((S)-4-(benzylamino)-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(2-chlorophenylamino)-5-methoxybenzamide (Cpd 28) LCMS RT=2.46min, 2.80mm; MH655.3; 1H NMR(DMSO): 9.25-9.20 (1H, m), 9.14(1H, s), 8.59 (1H, d, J8.7 Hz), 8.48 (1H, d, J6.8 Hz), 7.48-7.01 (16H, m), 6.88-6.82 (1H, m), 5.24-5.16 (1H, m), 4.62-4.53 (1H, m), 4.29 (2H, d, J6.2 Hz), 3.79 (3H, s), 3.16-3.07 (1H, m), 2.88-2.79 (1H, m), 1.61-1.43 (3H, m), 0.87-0.80 (6H, m) N-((S)-1 -((S)-4-(tert-butylamino)-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(phenylamino)benzamide (Cpd 10) LCMS RT=2.44min, 2.88mm, MH557.5, MH+18 575.6; 1H NMR(DMSO): 9.56 (1H, s), 8.46 (1 H, d, J 8.40 Hz), 8.38 (1 H, d, J 7.6 Hz), 7.86 (1 H, s), 7.70 (1 H, dd, J 1.4, 8.0 Hz), 7.38-7.05 (11H, m), 7.01-6.94 (1H, m), 6.87-6.80 (1H, m), 5.22-5.11 (1H, m), 4.59-4.47 (1H, m), 3.08 (1H, dd, J4.4, 14.2 Hz), 2.83 (1H, dd, J9.4, 14.0 Hz), 1.69-1.38 (3H, m), 1.27 (9H, s), 0.88 (3H, d, J6.3 Hz), 0.85 (3H, d, J6.3 Hz).

N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(benzo[d][1,3]dioxol-5-ylamino)nicotinamide (Cpd 11) LCMS RT= 1.65mm, 1.89mm, MH 546.2, MH+18 564.3; 1H NMR(DMSO): 10.86 (0.28H, s), 10.57 (0.72H, s), 8.71-7.64 (7H, m), 7.48 (1H, d, J2.1 Hz), 7.27-7.09 (5H, m), 6.94-6.77 (2H, m), 6.05 (0.62H, s), 5.98 (1.38H, s), 5.29-5.16 (1H, m), 4.62-4.44 (1H, m), 3.49-3.10 (2H, m), 1.58-1.21 (3H, m), 0.84 (3H, d, J7.3 Hz), 0.82 (3H, d, J7.0 Hz).

N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylamino)-3-methyl-1 -oxobutan-2-yI)- 2-(phenylamino)benzamide (Cpd 12) LCMS RT= 1.89mm, 1.95mm, 2.15 mm MH487.6; 1H NMR(DMSO): 9.39(1H, s), 8.45 (1H, d, J7.5 Hz), 8.38-8.25(1H, m), 8.08 (1H, d, J10.4 Hz), 7.82 (1H, d, J12.0 Hz), 7.75- 7.66 (1H, m), 7.41-6.81 (13H, m), 5.32-5.20 (1H, m), 4.39-4.27 (1H, m), 3.20-3.05 (1H, m), 2.86-2.66 (1H, m), 2.14-1.89 (1H, m), 0.87 (3H, dd, J3.0, 6.6 Hz), 0.72 (1.5H, d, J7.1 Hz), 0.70 (1.5H, d, J7.1 Hz).

N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(2-methoxyphenylamino)benzamide (Cpd 13) LCMS RT= 2.06mm, 2.28mm MH 531.2; 1H NMR (DMSO): 9.52 (1 H, s), 8.40 (2H, d, J 8.1 Hz), 8.15-7.98 (2H, m), 7.91-7.75 (2H, m), 7.70 (1H, d, J7.8 Hz), 7.38-7.01 (1OH, m), 7.0- 6.76 (3H, m), 5.27-5.15 (1H, m), 4.60-4.48 (IH, m), 3.82 (3H, s), 3.13 (1H, dd, J3.7, 13.8 Hz), 2.80-2.69 (1H, m), 1.57-1.41 (2H, m), 1.37-1.21 (1H, m), 0.83 (3H, d, J6.4 Hz), 0.81 (3H, d, J6.4 Hz).

N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(3-fluorophenylamino)benzamide (Cpd 14) LCMS RT=2.O7min, 2.29mm MH519.2; 1H NMR(DMSO): 9.52 (1H, s), 8.46 (1H, d, J8.3 Hz), 8.41(1H, d, J8.0 Hz), 8.12-8.0 (1H, m), 7.91-7.75 (1H, m), 7.75-7.68 (1H, m), 7.44- 7.09 (8H, m), 6.99-6.86 (3H, m), 6.77-6.66 (IH, m), 5.28-5.17 (1H, m), 4.56-4.44 (1H, m), 3.14 (1H, dd, J3.7, 13.7 Hz), 2.80-2.67 (1H, m), 1.53-1.22 (3H, m), 0.91-0.73 (6H, m).

N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(4-fluorophenylamino)benzamide (Cpd 15) LCMS RT=2.O7min, 2.29mm MH519.2; 1H NMR(DMSO): 9.50 (1H, s), 8.43 (1H, d, J 8.40 Hz), 8.39 (1H, d, J7.7 Hz), 8.12-8.02 (1H, m), 7.88-7.78 (1H, m), 7.76-7.68 (1H, m), 7.37-7.27 (1H, m), 7.26-7.10 (1OH, m), 6.86-6.77 (1H, m), 5.29-5.17 (1H, m), 4.57-4.43 (1H, m), 3.14 (1H, dd, J3.8, 13.9 Hz), 2.80-2.69 (1H, m), 1.55-1.39 (2H, m), 1.39-1.22 (1H, m), 0.92-0.76 (6H, m).

N-((S)-1 -((S)-4-(ethylamino)-3,4-dioxo-1 -phenylbutan-2-ylami no)-3-methyl-1 -oxobutan-2-yI)-2-(2-methoxyphenylamino)benzamide (Cpd 16) LCMS RT= 2.05mm, 2.32mm MH 545.3; 1H NMR (DMSO): 9.38 (1 H, s), 8.77-8.66 (1 H, m), 8.53-8.44 (1H, m), 8.29-8.20 (1H, m), 7.72-7.62 (1H, m), 7.38-6.76 (12H, m), 5.29-5.18 (1H, m), 4.39-4.29 (1H, m), 381 (3H, s), 3.19-3.05 (3H, m), 2.87-2.70 (1H, m), 2.12-2.0 (1H, m), 1.01 (3H, t J7.1 Hz), 0.89 (2.55H, d, J2.7 Hz), 0.87 (2.55H, d, J2.7Hz), 0.77 (0.45H, d J6.8 Hz), 0.70 (0.45H, d, J6.8Hz).

N-((S)-1 -((S)-4-(ethylamino)-3,4-dioxo-1 -phenylbutan-2-ylami no)-3-methyl-1 -oxobutan-2-yI)-2-(phenylamino)benzamide (Cpd 17) LCMS RT=2.l0min, 2.36mm MH515.3; 1H NMR(DMSO): 9.39 (1H, s), 8.78-8.67(1H, m), 8.52-8.44 (1H, m), 8.36-8.26 (1H, m), 7.74-7.65 (1H, m), 7.40-6.80 (13H, m), 5.29-5.17 (1H, m), 4.37-4.26 (1H, m), 3i9-3.04 (3H, m), 2.87-2.68 (1H, m), 2.13-1.94 (1H, m), 1.08- 0.97 (3H, m), 0.87 (4.62H, d, J6.8 Hz), 0.73 (0.69H, d, J6.8 Hz), 0.69 (0.69H, 6.8 Hz).

N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(phenylamino)nicotinamide (Cpd 18) LCMS RT= 1.82mm, 2.07mm MH502.3, MH+18 520.3; 1H NMR(DMSO): 10.71 (1H, s), 8.74-8.63 (1H, m), 8.50-8.40 (1H, m), 8.35-8.28 (1H, m), 8.21-8.02 (2H, m), 7.88-7.78 (1H, m), 7.67 (2H, d, J8.1 Hz), 7.36-7.08 (7H, m), 7.02-6.93 (1H, m), 6.86 (1H, dd, J4.9, 7.7 Hz), 5.28-5.18 (1H, m), 4.62-4.47 (1H, m), 3.21-3.08 (1H, m), 2.91-2.69 (1H, m), 1.76-1.27 (3H, m), 0.97-0.77 (6H, m).

N-((S)-1 -((S)-4-amino-3,4-dioxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(o-tolylamino)benzamide (Cpd 19) LCMS RT= 2.15mm, 2.38mm MH515.3, MH+18 532.9; 1H NMR (DMSO): 9.52 (0.67H, s), 9.49 (0.33H, s), 8.52-8.34 (2H, m), 8.10-8.02 (1H, m), 7.86-7.69 (2H, m), 7.35-7.11 (9H, m), 7.11-7.02 (1H, m), 7.01-6.92 (1H, m), 6.85-6.74 (1H, m), 5.29-5.16 (1H, m), 4.61-4.48 (1H, m), 3.19-3.06 (1H, m), 288-2.68 (1H, m), 2.20 (2.O1H, s), 2.19 (0.99H, s), 1.72-1.41 (2H, m), 1.37-1.22 (1H, m), 090 (0.94H, d, J6.2 Hz), 0.87 (0.94H, d, J6.3 Hz) 0.83 ( 2.06H, d, J6.lHz), 0.81 (2.06H, d, J6.lHz).

Procedure D (S)-2-amino-N-((2S,3R)-4-amino-3-hydroxy-4-oxo-1 -phenylbutan-2-yI)-4-methylpentanamide hydrochloride A solution of tert-butyl (2S, 3S)-4-amino-3-hydroxy-4-oxo-1-phenylbutan-2-ylcarbamate (1.13g, 3.84mmol) and HCI in dioxane (4M, lOmL) was stirred at room temperature forlh.

The solvent was evaporated under reduced pressure and the residue was dissolved in DMF (2OmL). (S)-2-(tert-butoxycarbonylamino)-4-methylpentanoic acid (888mg, 3.84mmol), HOBt (571mg, 4.22mmol), EDCI (1.62g, 8.44mmol) and DIPEA (2.2mL, 12.66mmol) were added and the resulting reaction mixture was stirred at room temperature for 16h. The solution was diluted with EtOAc, washed with aq. NH4CI, aq. NaHCO3 and brine, dried over MgSO4 and concentrated under reduced pressure. The resulting compound was purified by column chromatography eluting using a gradient (ethyl acetate/hexanes 0:100 v/v to ethyl acetate/hexanes 100:0 v/v). The obtained white solid was stirred for 1 h at room temperature in HCI in dioxane (4M, 1 OmL). The solvent was evaporated in vacuo to afford 1.08g (82%) of the title compound.

Procedure E N-((S)-1 -((S)-4-(Benzylamino)-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yl)-2-(2-methoxyphenylami no)benzamide 2-(2-Methoxyphenylamino)benzoic acid (0.20g, 0.83 mmol), HOBt (0.1 ig, 0.83 mmol)and EDCI (0.16g, 0.83 mmol) were added to a solution of (S)-2-amino-N-((S)-4-(benzylamino)- 3-hydroxy-4-oxo-1 -phenylbutan-2-yl)-4-methylpentanamide (0.30g, 0.75 mmol) in DMF (lOmL), followed by DIPEA(0.26mL, 1.50 mmol). The resulting reaction mixture was stirred at room temperature for 16h, diluted with EtOAc, washed aq. NH4CI, aq HCI (1M), aq.

NaHCO3 (3x) and brine, dried over MgSO4 and concentrated in vacuo to give 0.48g, (98%) of N-((S)-1 -((S)-4-(benzylamino)-3-hyd roxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yl)-2-(2-methoxyphenylamino)benzamide. LCMS RT= 2.45mm, MH 623.3 All compounds below were synthesized using Procedure E N-((S)-1 -((S)-4-(Ethylamino)-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yl)-2-(2-methoxyphenylami no)benzamide LCMS RT= 2.25mm, MH 561.4 N-((S)-1 -((S)-4-(Ethylamino)-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(phenylami no)benzamide LCMS RT= 2.24mm, MH 531.3 N-((S)-1 -((S)-4-Amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(2-chlorophenylamino)-5-methoxybenzamide LCMS RT= 4.56; MH 567.5 N-((S)-1 -((S)-4-Amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(2-fluorophenylamino)benzamide LCMS RT= 2.12; MH 521.5 N-((S)-1 -((S)-4-Amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(p-tolylamino)benzamide LCMS RT= 2.23; MH 517.1 N-((S)-1 -((S)-4-Amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(3-chlorophenylamino)benzamide LCMS RT= 2.25; MH 537.6 N-((S)-1 -((2S,3R)-4-amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylamino)-4-methyl-1 -oxopentan-2-yI)-2-(p-tolylamino)nicotinamide (0455-068-01) LCMS RT= 1.90mm; MH 518.2 N-((S)-1 -((2S,3R)-4-amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(3-(trifl uoromethyl)phenylamino)benzamide LCMS RT= 2.25mm; MH 571.3 N-((S)-1 -((2S,3R)-4-amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(4-methoxyphenylamino)nicotinamide LCMS RT= 1.69mm; MH 534.1 (S)-N I -((2S,3R)-1 -(4-bromophenyl)-4-(ethylamino)-3-hydroxy-4-oxobutan-2-yI)-N3-(2- (2-chlorophenylamino)-5-methoxyphenyl)-2-isobutyl malonamide LCMS RT= 2.43mm; MH 675.6 N-((S)-I -((2S,3R)-I -(4-bromophenyl)-4-(ethylamino)-3-hydroxy-4-oxobutan-2-ylami no)-3-methyl-I -oxobutan-2-yI)-2-(phenylami no)benzamide LCMS RT= 2.27mm; MH 596.0 N-((S)-I -((2S,3R)-I -(4-bromophenyl)-4-(ethylamino)-3-hydroxy-4-oxobutan-2-ylami no)-4-methyl-I -oxopentan-2-yI)-2-(phenylami no)benzamide LCMS RT= 2.38mm; MH 609.3 N-((S)-I -((2S,3R)-I -(4-bromophenyl)-4-(ethylamino)-3-hydroxy-4-oxobutan-2-ylami no)-3-methyl-I -oxobutan-2-yI)-2-(2-chlorophenylamino)-5-methoxybenzamide LCMS RT= 2.31 mm; MH 661.0 N-((S)-I -((2S,3R)-4-(benzylamino)-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-yI)-2-(phenylami no)benzamide LCMS RT= 2.46mm; MH 593.4 N-((S)-I -((2S,3R)-4-(benzylamino)-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-yl)-2-(2-chlorophenylamino)-5-methoxybenzamide LCMS RT= 2.52mm; MH 657.0 Benzyl (S)-I -((S)-4-(ethylamino)-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-ylcarbamate LCMS RT= 1.93mm, MH 470.3 N-((2S)-I -((2S)-4-amino-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-yl)-2-(benzo[d][I,3]dioxol-5-ylamino)nicotinamide LCMS RT= 1.73mm, MH 548.2 N-((2S)-I -((2S)-4-amino-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-3-methyl-I -oxobutan-2-yl)-2-(phenylami no)benzamide LCMS RT=2.O0min, MH489.2; 1H NMR(DMSO): 9.36 (1H, s), 8.34 (1H, d, J9.1 Hz), 7.76-7.67 (2H, m), 7.40-7.23 (5H, m), 7.23-7.04 (7H, m), 7.00-6.84 (2H, m), 5.78 (1H, d, J 5.6 Hz), 4.29-4.16 (2H, m), 3.80 (1H, dd, J2.8, 5.6 Hz), 2.83 (1H, dd, J6.0, 13.6 Hz), 2.61 (1H, dd, J9.1, 13.4 Hz), 2.07-1.91 (1H, m), 0.75 (3H, d, J, 4.0 Hz), 0.73 (3H, d, J4.0 Hz).

N-((2S)-I -((2S)-4-amino-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-yI)-2-(2-methoxyphenylami no)benzamide LCMS RT= 2.09mm, MH 533.3 N-((2S)-I -((2S)-4-amino-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-yI)-2-(3-fluorophenylamino)benzamide LCMS RT= 2.11 mm, MH 521.1 N-((2S)-I -((2S)-4-amino-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-yI)-2-(4-fluorophenylamino)benzamide LCMS RT= 2.12mm, MH 521.1 N-((2S)-I -((2S)-4-amino-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-yI)-2-(phenylamino)nicotinamide LCMS RT= 1.67mm, MH 504.3 N-((2S)-I -((2S)-4-amino-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-yI)-2-(o-tolylami no)benzamide LCMS RT= 2.19mm, MH 517.3 N-((2S)-I -((2S)-4-(tert-butylamino)-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-4-methyl-I -oxopentan-2-yI)-2-(phenylami no)benzamide LCMS RT= 2.52mm, MH 559.5 N-((2S)-I -((2S)-4-(ethylamino)-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-3-methyl-I -oxobutan-2-yI)-2-(2-methoxyphenylami no)benzamide LCMS RT= 2.11 mm, MH 547.0 N-((2S)-I -((2S)-4-(ethylami no)-3-hydroxy-4-oxo-I -phenylbutan-2-ylami no)-3-methyl-I -oxobutan-2-yI)-2-(phenylami no)benzamide LCMS RT= 2.14mm, MH 517.0 Procedure F (S)-2-Amino-N-((S)-4-(benzylamino)-3-hydroxy-4-oxo-1 -phenylbutan-2-yI)-4-methyl pentanamide Benzyl isocyanide (21 6mL, 1.78 mmol) was added to a solution of S-phenylalaninal (0.37g, 1.48 mmol) in DCM (1 5mL), followed by Boc-L-Leucine (0.36g, 1.55 mmol). After stirring at room temperature for 64h, TFA (5mL) was added and stirring was continued for a further 3h. The solvents were evaporated under reduced pressure and the residue dissolved in EtOAc, washed with aq. NaOH (1 M), water and brine, dried over MgSO4 and concentrated in vacuo. The resulting solid was trituated with Et20 to give 0.47g (80%) of (S)-2-amino-N- ((S)-4-(benzylamino)-3-hydroxy-4-oxo-1 -phenylbutan-2-yl)-4-methylpentanamide. LCMS RT= 1.24mm, MH398.2; 1H NMR(DMSO): 8.45 (0.4H, t, J6.1 Hz), 8.31 (0.6H, t, J6.2 Hz), 7.84 (0.6H, d, J9.5 Hz), 7.78 (0.4H, d, J9.4 Hz), 7.31-7.08 (1OH, m), 6.19 (0.6H, d, J 5.6 Hz), 5.99 (0.4H, d, J5.7 Hz),4.37-4.17 (3H, m), 3.99 (0.4H, dd, J3.5 Hz, J5.6 Hz), 3.88 (0.6H, dd, J2.2 Hz, J5.5 Hz), 3.07-3.00 (1H, m), 2.83 (0.6H. dd, J7.4 Hz, J13.3 Hz), 2.73-2.55 (1.4H, m), 1.69-1.51 (2.6H, m), 1.31-0.94 (2.4H, m), 0.84-0.75 (6H, m).

The compound below was prepared using tert-butyl isocyanide instead of benzyl isocyanide: (2S)-2-amino-N-((2S)-4-(tert-butylamino)-3-hydroxy-4-oxo-1 -phenylbutan-2-yl)-4-methyl pentanamide LCMS RT= 1.23mm, MH 364.3 The compound below was prepared using ethyl isocyanide instead of benzyl isocyanide and Boc valine in place of Boc leucine: (3S)-3-((S)-2-amino-3-methyl butanamido)-N-ethyl-2-hydroxy-4-phenylbutanamide LCMS RT= 0.48mm, 1.0mm, 1.07mm, MH 322.2 The compound below was prepared using ethyl isocyanide instead of benzyl isocyanide; Boc valine in place of Boc leucine and (S)-tert-butyl 1-(4-bromophenyl)-3-oxopropan-2-ylcarbamate in place of (S)-(-)-2-(tert-butoxycarbonylamino)-3-phenylpropanal.

(3S)-3-((S)-2-amino-3-methyl butanamido)-4-(4-bromophenyl)-N-ethyl-2-hydroxybutanamide LCMS RT= 1.21mm, MH 400.0 The compound below was prepared using ethyl isocyanide instead of benzyl isocyanide and (S)-tert-butyl 1 -(4-bromophenyl)-3-oxopropan-2-ylcarbamate in place of (S)-(-)-2-(tert-butoxycarbonylamino)-3-phenylpropanal.

(2S)-2-amino-N-((2S)-1 -(4-bromophenyl)-4-(ethylamino)-3-hydroxy-4-oxobutan-2-yI)- 4-methylpentanamide LCMS RT= 1.25mm, MH415.0 Procedure G _ p 90 C'OH + H2N cç o R=Me R = H Procedure G (S)-Methyl 4-methyl-2-(2-(phenylami no)benzamide)pentanoate DIPEA(0.26mL, 1.50 mmol) was added to a solution of N-Phenylanthranilic acid (ig, 4.69 mmol), L-Leucine methyl ester hydrochlodride (0.85g, 4.69 mmol), HOBt (O.63g, 4.69 mmol) and EDCI (1.6g, 10.32 mmol) in DMF (2OmL). The resulting reaction mixture was stirred at room temperature for 16h, diluted with EtOAc, washed H20 (2x) and brine, dried over MgSO4 and concentrated in vacuo to give 1.87g, (qunatitative) of (S)-methyl 4-methyl- 2-(2-(phenylamino)benzamide)pentanoate. LCMS RT= 2.45mm, MH 341.2 (S)-4-Methyl-2-(2-(phenylamino)benzamido)pentanoic acid LiOH (0.59g, 14.07 mmol) was added to a solution of (S)-methyl 4-methyl-2-(2- (phenylamino)benzamide)pentanoate (1.59g, 4.69 mmol) in THF/H20 (1/1, v/v, 5OmL) and the resulting reaction mixture was stirred at room temperature for 16h. The THF was evaporated under reduced pressure,the residue poured into H20 and extracted with EtOAc (2x). The combined organic phases were washed with brine, dried over MgSO4 and concentrated in vacuo to give 0.55g (36%) of (S)-4-methyl-2-(2- (phenylamino)benzamido)pentanoic acid. LCMS RT= 2.18mm, MH 327.2; 1H NMR (DMSO): 9.52 (1H, bs), 7.76 (1H, bs), 7.28 (IH, d, J 6.7 Hz), 7.03-6.90 (6H, m), 6.66 (1H, t, J7.2 Hz), 6.58-6.53 (1H, m), 3.88 (1H, m), 1.43-1.28 (3H, m), 0.63 (3H, d, J5.8 Hz), 0.61 (3H, d, J5.9 Hz).

N-((S)-1 -((S)-4-amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylami no)-4-methyl-1 -oxopentan-2-yI)-2-(phenylami no)benzamide To tert-butyl (2S)-4-amino-3-hydroxy-4-oxo-1 -phenylbutan-2-ylcarbamate (390mg, 1.33mmol) was added 6M HCI in dioxane (3.OmL) and the reaction mixture was stirred at rt for 1 h. The reaction mixture was evaporated in-vacuo to give (3S)-3-amino-2-hydroxy-4- phenylbutanamide hydrochloride (486mg) as a white solid. (3S)-3-amino-2-hydroxy-4-phenylbutanamide hydrochloride (153mg, 0.67mmol) was dissolved in dry DMF (lOmL) and (S)-4-methyl-2-(2-(phenylamino)benzamido)pentanoic acid (217mg, O.67mmol), along with HOBt (98.8mg, 0.73mmol) and EDCI (281mg, 1.46mmol). DIPEA (347pL, 2.Ommol) was added dropwise and the reaction mixture was stirred at rt for 18h. The reaction mixture was diluted with ethyl acetate and washed with water, brine (2x), dried over anhydrous magnesium sulfate, filtered and concentrated in-vacuo. Purification by column chromatography eluting using a gradient (petroleum ether 40/60 / ethyl acetate 1:0 v/v 0:1, followed by DCM/MeOH 1:0 v/v 9:1)gave 0.14g (21%) of N-((S)-1-((S)-4-amino-3- hydroxy-4-oxo-1 -phenylbutan-2-ylamino)-4-methyl-1 -oxopentan-2-yl)-2- (phenylamino)benzamide. LCMS RT= 2.09mm, MH 503.3 Br B0cHN OH BocHN 0 0 0 Reagents & conditions: (i) CDI, N,O-dimethyIhydroxylamineHCl, DIPEA, THF, DMF, rt, 42.5h, 89%; (ii) LAH, Ether, 35 mins, 5 °C, 76%.

(S)-tert-butyl 3-(4-bromophenyl)-1 -(methoxy(methyl)amino)-1 -oxopropan-2-ylcarbamate To a stirred solution of (S)-3-(4-bromophenyl)-2-(tert-butoxycarbonylamino)propanoic acid (1.Og, 2.9lmmol) in dry THF (l5mL) under Ar, was added carbonyldiimidazole(0.56g, 3.49mmol) portionwise over a 10 minute period. The solution was stirred for 30 minutes under Ar at room temperature. N,O-dimethylhydroxylamine hydrochloride (0.31 g, 3.2Ommol) was dissolved in DMF (5.OmL) with DIPEA (0.53mL, 3.O6mmol) and added to the amino acid solution. The reaction mixture was stirred at rt for 18h and then concentrated in-vacuo. Ethyl acetate (1 5OmL) was added to the residue and it was then washed with 1M HCI solution (3 x 6OmL), saturated aq. sodium hydrogen carbonate solution (3 x 6OmL) and brine (5OmL). The aqueous washings were back extracted with ethyl acetate (2 x 5OmL). The combined organic layers were dried over anhydrous magnesium sulfate, filtered and evaporated. The residue was purified by column chromatography (0-35% ethyl acetate I petroleum ether) to give 1.Og (89%) of the title compound.

LCMS RT=2.l3min; MH387.1; 1H NMR(DMSO): 7.48 (2H, d, J8.4 Hz), 7.20 (2H, d, J 8.4 Hz), 4.52 (1H, brs), 3.73 (3H, s), 3.11 (3H, s), 2.82 (1H, dd, J4.4, 13.7 Hz), 2.76-2.62 (1H, m), 1.31 (9H, s).

(S)-tert-butyl I -(4-bromophenyl)-3-oxopropan-2-ylcarbamate To a solution of (S)-tert-butyl 3-(4-bromophenyl)-1 -(methoxy(methyl)amino)-1 -oxopropan-2-ylcarbamate (1.Og, 2.S8mmol) in dry ether (10.OmL) at 5°C under Ar, was added a solution of 1M lithium aluminium hydride in ether (2.8lmL, 2.8lmmol) over 15 minutes. The solution was allowed to stir for a further 35 minutes following completion of addition. Ethyl acetate (1.5mL) was added dropwise to destroy any excess lithium aluminium hydride, followed by 5% aq. solution of KHSO4 (1.5mL) added drop wise to destroy the complex whilst the temperature was maintained at less than 5 °C. Ethyl acetate (lOOmL) was added to the solid and it was washed with 1 M HCI (3 x 5OmL), sat. aq. sodium hydrogen carbonate solution (3 x 5OmL), brine (6OmL), dried over anhydrous magnesium sulfate, filtered and concentrated in-vacuo. The residue was purified by column chromatography (0-40% ethyl acetate! petroleum ether) to give 0.61g (72%) of the title compound.

LCMS RT=2.l5min, 2.38mm MH515.3, MH+18 532.9; 1H NMR(DMSO): 9.51 (1H, s), 7.48 (2H, d, J8.5 Hz), 7.35 (1H, d, J7.7 Hz), 7.18 (2H, d, J8.5 Hz),4.12-3.99 (1H, m), 3.07 (1H, dd, J4.6, 13.8 Hz), 2.65 (1H, dd, J 10.5, 14.1 Hz), 1.33 (9H, s).

Eciu ivalents The foregoing description details presently preferred embodiments of the present invention.

Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims (24)

1. CLAIMS1. A compound of Formula (1) R3 0 0 Ar2LArLL1 NYNR 0 H in which Y represents an amino acid residue optionally substituted by one or more halo, OR7, S(O)R7, ON orC15alkyl R7 represents independently H; 01-5 alkyl; O38cycloalkyl and optionally substituted aryl n represents an integer from 0 to 2 R3 represents H or Oi5alkyl, optionally substituted by halo R5 represents H; 015a1ky1, optionally substituted by one or more halo, OH, 0-alkyl; aryl, optionally substituted by one or more halo, OH, 0-alkyl; aralkyl, optionally substituted by one or more halo, OH, 0-alkyl; glycosyl or uronyl Raa represents the side chain of a natural amino acid L1 represents a bond; (CR3R3); 0(0); C(O)NR3; C(S); C(S)NR3; C(NR3); C(NR3)NR3; 0(0)0; SO2 or C(O)(0H2)mO L2 is absent or represents a bond; (CR3R3); 0(0); 0, S(O), NR3 or P(O)0R3 Ar1 represents aryl or aralkyl, optionally containing one or more heteroatoms selected from 0, N and S and optionally substituted by one or more R6 groups Ar2 is absent or represents aryl or aralkyl, optionally containing one or more heteroatoms selected from 0, N and S and optionally substituted by one or more R6 g rou PS R6 represents one or more substituents attached to either or both Ar1 and Ar2 and is independently selected from H; halo; ON, NO2, OR7; S(O)R7; NR3R7; 01-06 alkyl; 02-06 alkenyl; 02-06 alkynyl; 03-08 cycloalkyl; aryl; aralkyl; 038 heterocyclyl, OO(O)R7; O(O)0R7; NR3O(O)R7; O(O)NR3R7; NR3O(S)R7; O(S)NR3R7; NR3O(NR3)R7; O(NR3)NR3R7; NR3SO2R7; SO2NR3R7 or P(O)R70R7, wherein where R3 and R7 are attached to the same nitrogen atom they may form an optionally substituted heterocyclic ring containing one additional atom or group selected from 0, NR7 or S and wherein where two R6 substituents are present on the same ring system they may form an optionally substituted fused ring m represents an integer from 1 to 2 p represents an integer from 0 to 2 or a pharmaceutically acceptable derivative, N-oxide, salt, hydrate, solvate, complex, bioisostere, metabolite or prodrug thereof, the compound being a dual calpain-ROS inhibitor for the treatment of a disease mediated by ROS and/or calpain hyperactivity.
2. 2. The compound of claim 1 wherein Raa represents the side chain of leucine, being C HC H (C H3)2.
3. 3. The compound of claim 1 wherein Raa represents the side chain of valine, being OH (C H3)2.
4. 4. The compound of any one of the preceding claims wherein Y is an amino acid residue in natural L form.
5. 5. The compound of any one of the preceding claims wherein Y is a lipophilic amino acid residue, for example a lipophilic amino acid residue substituted by one or more halo, OR7, S(O)R7, ON orC15alkyl.
6. 6. The compound of claim 4 wherein Y is selected from phenylalanine, valine, leucine and isoleucine residues.
7. 7. The compound of claim 6 wherein Y is selected from phenylalanine, valine, leucine and isoleucine residues substituted by one or more halo, OR7, S(O)R7, ON or C15a1ky1.
8. 8. The compound of claim 6 wherein Y is phenylalanine, optionally substituted by one or more halo, OR7, S(O)R7, ON or 015a1ky1.
9. 9. The compound of any one of the preceding claims wherein R5 represents H or 01-5 alkyl, optionally methyl or ethyl.
10. 10. The compound of claim 9 wherein R5 represents glycosyl or uronyl.
11. 11. The compound of any one of the preceding claims in which R3 represents H.
12. 12. The compound of any one of the preceding claims wherein L1 represents 0(0) or O(O)N R3.
13. 13. The compound of any one of the preceding claims wherein Ar1 and Ar2 independently represent aryl (for example pyridyl), optionally containing one or more heteroatoms selected from 0, N and S, and optionally substituted by one or more R6 groups.
14. 14. The compound of claim 13 wherein Ar1 and Ar2 represent aryl.
15. 15. The compound of claim 14 wherein Ar1 and Ar2 represent phenyl.
16. 16. The compound of any one of the preceding claims wherein L2 represents NH.
17. 17. The compound of claim 16 wherein L1, L2, Ar1 and Ar2 together represent:CRB_{. J1 or a pyridine analogue thereof in which one or both ring systems contains an N heteroatom, wherein R6 represents one or more substituents attached to either or both ring systems, optionally being selected from H, alkyl, halo and 0-alkyl.
18. 18. The compound of claim 16 wherein L1, L2, Ar1 and Ar2 together represent: or a pyridine analogue thereof in which one or both ring systems contains an N heteroatom.
19. 19. The compound of any one of claims 1 to 15 wherein L2 represents S.
20. 20. The compound of claim 19 wherein L' , L2, Ar1 and Ar2 together represent: or a pyridine analogue thereof in which one or both ring systems contains an N heteroatom, wherein R6 represents one or more substituents attached to either or both ring systems, optionally being selected from H, alkyl, halo and 0-alkyl.
21. 21. The compound of claim 20 wherein L1, L2, Ar1 and Ar2 together represent: or a pyridine analogue thereof in which one or both ring systems contains an N heteroatom.
22. 22. The compound of any one of claims 1 to 15 wherein L2 represents 0.
23. 23. The compound of claim 22 wherein L' , L2, Ar1 and Ar2 together represent:R BCor a pyridine analogue thereof in which one or both ring systems contains an N heteroatom, wherein R6 represents one or more substituents attached to either or both ring systems, optionally being selected from H, alkyl, halo and 0-alkyl.
24. 24. The compound of claim 23 wherein L' , L2, Ar1 and Ar2 together represent: HN$ or a pyridine analogue thereof in which one or both ring systems contains an N heteroatom.