WO2008029168A2

WO2008029168A2 - Treatment of duchenne muscular dystrophy

Info

Publication number: WO2008029168A2
Application number: PCT/GB2007/003403
Authority: WO
Inventors: Graham Michael Wynne; Stephen Paul Wren; Cristine Lecci; Storer Richard
Original assignee: Summit Corporation Plc
Priority date: 2006-09-08
Filing date: 2007-09-07
Publication date: 2008-03-13
Also published as: WO2008029168A3

Abstract

There are discloses compounds of formula I wherein A1, A2, A3 and A4, which may be the same or different, represent N or CR1, X, Y and Z, which may be the same or different, represent O, S(O)n, C=W, NR4, NC(=O)R5 and CR6R7, W is O, S, NR20, R1, R4, R5, R6, R7, R20 are according to the present specification, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.

Description

Treatment of Duchenne muscular dystrophy

The present invention relates to a method of treatment of 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 DMD 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. MoI. 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. Natl Acad. ScL 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 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, Sl : 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. We have now found a group of compounds which upregulate endogenous utrophin in predictive screens and, thus, may be useful in the treatment of DMD.

According to the invention, we provide use of a compound of formula I,

wherein

A¹, A², A³ and A⁴, which may be the same or different;, represent N or CR ,

X, Y and Z, which may be the same or different, represent O, S(O)_n, C=W, NR⁴, NC(=O)R⁵ and CR⁶R⁷, W is O, S, NR²⁰, one of R⁴, R⁵, R⁶, and R²⁰ represents - L -R³, in which L is a single bond or a linker group, additionally, R³, R ⁴, R ⁵, R⁶, and R⁷, which may be the same or different, independently represent hydrogen or a substituent and R₂₀ represents hydrogen, aryl, hydroxyl, alkyl optionally substituted by aryl, alkoxy optionally substituted by aryl, CN, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted alkanoyl, optionally substituted aroyl, NR³⁰R³¹, in which R³⁰ and R³¹, which may be the same or different, represent hydrogen, optionally substituted alkyl or optionally substituted aryl; additionally, one of R³⁰ and R³¹ may represent optionally substituted alkanoyl or optionally substituted aroyl, n represents an integer from 0 to 2, in addition, an adjacent pair of X, Y and Z cannot both represent O, or S or together represent O - S, any pair of R⁶, R⁷ and R⁴ may when attached to adjacent atoms together represent a single bond between the adjacent atoms to which they are attached, when an adjacent pair of A₁ — A₄ each represent CR¹, then the adjacent carbon atoms, together with their substituents may form a ring B, when X, Y or Z is CR⁶R⁷, R⁶ and R⁷, together with the carbon atom to which they are attached may form a ring C, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.

Compounds of formula I may exist in tautomeric, enantiomeric and diastereomeric forms, all of which are included within the scope of the invention.

Certain compounds of formula I are novel. According to the invention, we also provide those compounds of formula I which are novel, together with processes for their preparation, compositions containing them, as well as their use as pharmaceuticals.

Some of the compounds falling within the scope of formula I are known, as such, but not as pharmaceuticals. According to the invention, we claim compounds known in the art as such, but not previously described for use as pharmaceuticals, as pharmaceuticals.

All of the compounds of formula I may be made by conventional methods. Methods of making heteroaromatic ring systems are well known in the art. In particular, methods of synthesis are discussed in Comprehensive Heterocyclic Chemistry, Vol. 1 (Eds.: AR Katritzky, CW Rees), Pergamon Press, Oxford, 1984 and Comprehensive Heterocyclic Chemistry II: A Review of the Literature 1982-1995 The Structure, Reactions, Synthesis, and Uses of Heterocyclic Compounds, Alan R. Katritzky (Editor), Charles W. Rees (Editor), E.F.V. Scriven (Editor), Pergamon Pr, June 1996. Other general resources which would aid synthesis of the compounds of interest include March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley-Interscience; 5th edition (January 15, 2001).

Certain benzodioxoles of formula 1 can be made as shown below.

III

R = H

Nitration

Coupling of a diol II with a benzaldehyde III gives a benzodioxole IV. This can be achieved in the presence of an acid catalyst e.g. p-ΥsOH or the heterogeneous solid acid catalyst, montmorillonite, and in an appropriate solvent, such as toluene, benzene or xylenes (see Cole, E. R.; Crank, G.; Minh, H. T. H. Aust. J. Chem. 1980, 33, 675 and Li, T. -S.; Li, L. -J.; Lu, B.; Yang, F. J. Chem. Soc. Perkin Trans. 1 1998, 3561).

The benzodioxole product IV can be manipulated, for example by nitration.

The skilled person is well aware of the wide variety of methods available for nitration, the most simple of which is by use of HNO₃; however, if other functionality within the molecule requires, a wide variety of milder conditions can also be used to achieve nitration.

Reduction of nitro compound V using one of the wide variety of reducing conditions known to bring about this transformation for example H₂ZPt gives an amine VI which can be further manipulated in a variety of standard ways - such as acylation to give an amide. Certain isoindoles derivatives of formula I can be made as illustrated below (see Suizu, M. et al. Chem. Pharm. Bull. 2003, 57, 1098-1102).

succimide reduction

XII

Commercially available anhydrides VII can be converted to succinimides VIII by treatment with an appropriate aniline. Varying reduction strategies can then be used to produce a variety of compounds of formula I.

Reduction of the succinimide core of succinimide VIII gives perhydroisoindole IX, and subsequent reduction of the nitro functionality gives amine X, which can be further manipulated using straightforward methods to produce amides and secondary amines.

Amine X can also be synthesised by initial reduction of the nitro group of compound VIII giving amine XI (commercially available for R = 4-Me, 4-COOMe). Subsequent complete reduction of the succinimide core gives amine X. Alternatively, partial reduction of the succinimide core of amine XI gives lactone XII. The amine functionality of lactone XII can be manipulated as before to give, for example, amides.

Cyclic sulfonamides of formula I may be synthesised as below (see

Kamogawa, H. et al. Bull Chem. Soc. Jpn. 1982, 55, 3824-3827 and Katoghi, M. et al. Tetrahedron 1999, 55, 14885-14900).

commercially available

commercially available

Cyclic sulfonamides XIII can be synthesised by cyclisation of bromide XIV, which can in turn be synthesised in two steps via bromination of phenyl derivative XV, and reaction of the subsequently formed sulfonyl chloride XVI with an aniline derivative. The nitro functionality of compound XIII can be manipulated as required to give amino and amide compounds.

Alternatively, cyclic sulfonamides XVIII can be synthesised from commercially available open chain sulfonamides XIX. Cromate mediated ring closure gives sulphonamide XX, which can be exhaustively reduced, and then selectively oxidised to give the cylic sulfonamide XVIII.

In the above processes it may be necessary for any functional groups, e.g. hydroxy or amino groups, present in the starting materials to be protected, thus it may be necessary to remove one or more protective groups to generate the compound of formula I.

Suitable protecting groups and methods for their removal are, for example, those described in "Protective Groups in Organic Synthesis" by T. Greene and P.G.M.

Wutts, John Wiley and Sons Inc., 1991. Hydroxy groups may, for example, be protected by arylmethyl groups such as phenylmethyl, diphenylmethyl or triphenylmethyl; acyl groups such as acetyl, trichloroacetyl or trifluoroacetyl; or as tetrahydropyranyl derivatives. Suitable amino protecting groups include arylmethyl groups such as benzyl, (R,S)-α-phenylethyl, diphenylmethyl or triphenylmethyl, and acyl groups such as acetyl, trichloroacetyl or trifluoroacetyl. Conventional methods of deprotection may be used including hydrogenolysis, acid or base hydrolysis, or photolysis. Arylmethyl groups may, for example, be removed by hydrogenolysis in the presence of a metal catalyst e.g. palladium on charcoal. Tetrahydropyranyl groups may be cleaved by hydrolysis under acidic conditions. Acyl groups may be removed by hydrolysis with a base such as sodium hydroxide or potassium carbonate, or a group such as trichloroacetyl may be removed by reduction with, for example, zinc and acetic acid.

The compounds of formula I, and salts thereof, may be isolated from their reaction mixtures using conventional techniques.

Salts of the compounds of formula I may be formed by reacting the free acid, or a salt thereof, or the free base, or a salt or derivative thereof, with one or more equivalents of the appropriate base or acid. The reaction may be carried out in a solvent or medium in which the salt is insoluble or in a solvent in which the salt is soluble, e.g. ethanol, tetrahydrofuran or diethyl ether, which may be removed in vacuo, or by freeze drying. The reaction may also be a metathetical process or it may be carried out on an ion exchange resin.

Pharmaceutically acceptable salts of the compounds of formula I include alkali metal salts, e.g. sodium and potassium salts; alkaline earth metal salts, e.g. calcium and magnesium salts; salts of the Group III elements, e.g. aluminium salts; and ammonium salts. Salts with suitable organic bases, for example, salts with hydroxylamine; lower alkylamines, e.g. methylamine or ethylamine; with substituted lower alkylamines, e.g. hydroxy substituted alkylamines; or with monocyclic nitrogen heterocyclic compounds, e.g. piperidine or morpholine; and salts with amino acids, e.g. with arginine, lysine etc, or an N-alkyl derivative thereof; or with an aminosugar, e.g. N-methyl-D-glucamine or glucosamine. The non-toxic physiologically acceptable salts are preferred, although other salts are also useful, e.g. in isolating or purifying the product.

Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various optical isomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation.

Substituents that alkyl may represent include methyl, ethyl, butyl, eg sec butyl.

Halogen may represent F, Cl, Br and I, especially Cl.

Examples of substituents that R³ in the compound of formula 1 may represent include alkyl, alkoxy or aryl, each optionally substituted by one or more, preferably one to three substituents, R², which may be the same or different.

In one group of compounds A¹, A², A³ and A⁴ _, which may be the same or different, represent N or CR¹ _, X, Y and Z, which may be the same or different, represent O, S(O)_n, C=W, NR⁴, NC(=O)R⁵ and CR⁶R⁷,

W is O, S, NR²⁰, '^■ one of R⁴, R⁵, R⁶, and R²⁰ represents - L -R³, in which L is a single bond or a linker group, additionally, R³, R ⁴, R ^s, R⁶, and R⁷, which may be the same or different, independently represent hydrogen or a substituent and

R²⁰ represents hydrogen, aryl, hydroxyl or alkyl, n represents an integer from 0 to 2, in addition, an adjacent pair of X, Y and Z cannot both represent O, or S or together represent O - S, any pair of R , R⁷ and R⁴ may when attached to adjacent atoms together represent a single bond between the adjacent atoms to which they are attached, when an adjacent pair of Aj - A₄ each represent CR¹, then the adjacent carbon atoms, together with their substituents may form a ring B, and when X, Y or Z is CR⁶R⁷, R⁶ and R⁷, together with the carbon atom to which they are attached may form a ring C.

In addition, compounds that may be mentioned include those wherein either: one of R⁵ or R⁶ represents L - R³, and wherein L is single bond and R³ represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl,

NO₂, ' CN,

NR¹⁰R", halogen,

SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶, OC(=W)NR^I0Rⁿ

NR¹⁵C(=W)R¹⁷,

R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,

R¹⁰ and R^π together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹ ', R¹⁶ and R¹⁷, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl or NR¹⁰R¹¹, and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R¹¹, may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxyl;

or wherein one of R⁴ or R²⁰ represents L - R³, and wherein L is single bond and R³ represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl, NR¹⁰R",

SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶,

NR¹⁵C(=W)R¹⁷, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R¹⁰ and R¹ ' together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹ ',

R¹⁶ and R¹⁷, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl OrNR¹⁰R¹¹ _, and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R¹¹, may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxy 1.

In addition, compounds that may be mentioned include those wherein either:

one of R⁵ or R⁶ represents L - R³, and wherein L is single bond and R³ represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, NR¹⁰R¹¹,

SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶,

OC(=W)NR¹⁰Rⁿ NR¹⁵C(=W)R¹⁷,

R¹⁰, R", R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R¹⁰ and R¹¹ together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹ ',

R and R , which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy,

and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R¹¹, may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxyl;

or wherein one of R⁴ or R²⁰ represents L - R³, and wherein L is single bond and R³ represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl,

O-aryl or thioaryl, in which the aryl is optionally substituted, optionally substituted aryl,

NR¹⁰R¹¹, SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶,

NR^{I 5}C(=W)R¹⁷,

R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,

R¹⁰ and R¹ ' together with the nitrogen to which they are attached may form a ring, R¹² may have the same meaning as NR¹⁰R¹ ',

R¹⁶ and R¹⁷, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl Or NR¹⁰R¹¹, and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R¹¹, may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxyl. Examples of substituents that R¹ and R², which may be the same or different, may represent include: alkyl optionally substituted by one or more halogen, alkoxy or optionally substituted aryl, thioaryl or aryloxy, alkoxy optionally substituted by optionally by alkyl or optionally substituted aryl, hydroxyl, OCC=W)NR¹⁰R¹ ' aryl, thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, NO₂, CN, NR¹⁰R¹¹, halogen, SO₂R¹², NR¹³SO₂R¹⁴, C(=W)R¹⁶, NR¹⁵C(^W)R¹⁷,

R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, NR¹⁰R¹ * together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹ ', when R¹⁷ represents NR¹⁰R¹¹, that NR¹⁰R¹¹ may represent hydrogen, COalkyl and CO optionally substituted aryl,

R¹⁶ may represent hydroxy, alkoxy, or NR¹⁰R¹ ', and R¹⁷ may represent alkyl substituted by one or more of halogen, alkoxy, optionally substituted aryl or NR¹⁰R¹ '.

Compounds that may be mentioned include those wherein either: one of R⁵ or R⁶ represents L - R³, in which L represents a linker group which is: O, S, NR¹⁸, alkylene, alkenylene,. alkynylene, each of which may be optionally interrupted by one or more of O, S, NR¹⁸, or one or more C-C single, double or triple bonds, a -N-N- single or double bond, and R¹⁸ represents hydrogen, alkyl, COR¹⁶;

or one of R⁴ or R²⁰ represents L - R³, in which L represents a linker group which is: O₅ S or NR¹⁸, alkylene, alkenylene, alkynylene, each of which may be optionally interrupted by one or more of O, S, NR¹⁸, or one or more C-C single, double or triple bonds, and R¹⁸ represents hydrogen, alkyl, COR¹⁶.

Although the scope for variation of R⁴, R⁵, R⁶ and R⁷ is large, preferably R⁴, R⁵,

R⁶ and R⁷ represent hydrogen, alkyl or optionally substituted aryl.

Alkyl may represent any alkyl chain. Alkyl includes straight and branched, saturated and unsaturated alkyl, as well as cyclic alkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. However, preferably, when any of the substituents represents alkyl, alkyl is saturated, linear or branched and has from 1 to 10 carbon atoms, preferably from 1 to 8 carbon atoms and more preferably from 1 to 6 carbon atoms. When any of the substituents represents alkyl, a particularly preferred group is cycloalkyl, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

Aryl may represent any aromatic system. Preferably, in the compounds of formula I, aryl is an aromatic hydrocarbon or a 5 to 10 membered aromatic heterocycle containing 1 to 4 hetero atoms selected from an oxygen atom, a sulfur atom and a nitrogen atom as a ring constituent besides- carbon. We prefer heterocycles which contain one or two heteroatoms. Aromatic heterocycles that may be mentioned include fiiran, thiophene, pyrrole, and pyridine. Particularly preferably, when aryl is an aromatic hydrocarbon, aryl represents a 6 to 10 membered monocyclic or bicyclic system, for example phenyl or naphthalene.

Saturated and unsaturated heterocycles that may be mentioned include those containing 4 to 7 ring atoms, preferably 5 or 6 ring atoms, preferably containing one to two heteroatoms selected from N, S and O. Heterocycles that may be mentioned include pyrrolidine, piperidine, tetrahydrofuran, piperazine and morpholine. N- containing heterocycles are particularly preferred, eg when NR¹⁰R¹¹ forms a heterocyclic ring.

As detailed above, when an adjacent pair of A¹ — A⁴ each represent CRi, the adjacent carbon atoms, together with their substituents may form a ring B. Also, when

X is CR⁶R⁷, R⁶ and R⁷, together with the carbon to which they are attached may form a ring C. Preferably ring B and/or ring C is a saturated or unsaturated 3 to 10 membered carbocylic or heterocyclic ring.

Particularly preferably ring B is benzene ring.

Particularly preferably ring C is a 3- 10 membered saturated or unsaturated carbocylic ring.

We particularly prefer compounds in which at least one R¹ represents NR^{I 5}C(=W)R¹⁷, more particularly the group NR¹⁵COR¹⁷.

We also prefer compounds in which at least one R¹ represents CONR¹⁰R¹ '.

For one group of particularly preferred compounds at least one R¹ represents an amide group NHCOR¹⁷ wherein R¹⁷ is selected from: alkyl Ci- C₆, alkyl Ci - C₆ substituted by phenyl, alkyl Cj - C₆ substituted by alkoxy Ci - C₆, haloalkyl Cj - C₆, perfluoroalkyl Ci - C₆, phenyl optionally substituted by one or more of halogen, alkyl Ci - C₆, alkoxy Ci - C₆, amino,, (alkyl C₁ - C₆)amino, di(alkyl Ci - C₆) amino or phenyl, CH:CH phenyl, naphthyl, pyridinyl, thiophenyl and furanyl.

We prefer compounds in which one or both of R and R² are other than -COOH.

For another group of particularly preferred compounds at least one R¹ represents a group NR¹⁵CONR¹⁰R", in which R¹⁰ and R^{1 1}, which may be the same or different, are selected from optionally substituted aryl, alkyl and COaryl optionally substituted.

A particularly preferred group which at least one of R¹ may represent is NHCONHR¹⁵ wherein R ¹⁵ is selected from phenyl, alkyl Ci to C₆ and COphenyl optionally substituted by one or more halogen.

For another group of particularly preferred compounds at least one R¹ represents alkyl Ci to C₆, optionally substituted by phenyl or a 5 or 6- membered saturated or unsaturated heterocycle containing one to two heteroatoms selected from N, S and O.

Another group of compounds that may be mentioned are those in which at least one of Ri represents SO_nR¹² wherein n represent O, 1 or 2, R¹² represents alkyl Ci - C₆ or NR⁹⁸R⁹⁹ wherein R⁹⁸ and R⁹⁹, which may be the same or different, represent hydrogen or alkyl Cj - C₆.

For another group of particularly preferred compounds at least one R¹ represents COR¹⁶ and R¹⁶ is alkoxy Ci - C₆, amino, (alkyl Ci - C₆)amino or di(alkyl Cj - C₆) amino.

For another group of particularly preferred compounds at least one R¹ represents: NO₂, halogen, amino or (alkyl Ci - C₆)amino or di(alkyl Ci - C₆) amino in which the alkyl Ci to C₆ is optionally substituted by phenyl or a 5 or 6 membered saturated or unsaturated heterocycle,

NHSO₂alkyl C₁ - C₆, NHSO₂phenyl, SO₂alkyl Ci - C₆, phenyl optionally substituted by Ci to C₆ alkoxy Cl - C6, a 5 — 10 membered, saturated or unsaturated, mono- or bi-cyclic heterocycle containing from 1 - 3 heteroatoms selected from N, S and O.

There is also wide scope for variation of the group R³. Preferably R³ represents aryl and is optionally substituted by one to three substituents, R², which may be the same or different.

Particularly preferably, R³ is a 5 - 10 membered aromatic mono- or bi-cyclic system, especially a hydrocarbon 5 - 10 membered aromatic mono- or bi-cyclic system, for example benzene or naphthalene.

Alternatively, the 5 - 10 membered aromatic mono- or bi-cyclic system, may be a heterocyclic system containing up to three heteroatoms selected from N, O and S, for example a thiophene, furan, pyridine or pyrrole.

Preferably the substituent(s) R² is/are selected from: alkyl Ci - C₆, optionally substituted by thiophenyl or phenoxy, each optionally substituted by halogen, alkoxy Ci - C₆ phenyl, thioalkyl C) - C₆ thiophenyl, optionally substituted by halogen,

NO₂, CN

NR¹⁰R¹¹, in which R¹⁰ and R¹¹, which may be the same or different represent hydrogen, alkyl C) - C₆, or together with the nitrogen to which they are attached form a 5 to 7 membered ring which may contain one or more additional heteroatoms selected

halogen

SO₂R¹², in which R¹² represents a 5 to 7 membered ring which may contain one or more additional heteroatoms selected from N, O and S NHCOR¹ \ in which R¹⁷ represents alkyl Ci - C₆, optionally substituted by: phenyl or halogen, or phenyl optionally substituted by alkoxy Cj - C₆, carboxy or halogen, or a 5 or 6 membered saturated or unsaturated heterocycle, phenyl or a 5 or 6 membered saturated or unsaturated heterocycle optionally substituted by halogen, alkoxy Ci to C₆, carboxy or a group SO₂NR¹⁰R¹¹,

_ Particularly preferably when R₂ represents NR¹⁰R¹¹, NR¹⁰ R¹¹ represents N- pyrrole, N-piperidine, N'(Ci - C₆) alkyl N piperazine or N-morpholine.

One group of compounds that may be mentioned include those wherein R⁵ or R⁶ represent L - R³ and the group L represents: -NH.NH-

-CH=CH-, -C≡C-, or

-NCOR¹⁶ in which R¹⁶ represents phenyl or a 5 or 6 membered saturated or unsaturated heterocycle optionally substituted by halogen, alkoxy Ci to C₆, carboxy.

A¹ - A⁴ may represent N or CR¹. Consequently, the six membered ring may contain 1, 2, 3 or 4 nitrogen atoms. Embodiments of the invention exist in which two of A¹ - A⁴ represent nitrogen, one of A¹ - A⁴ represents nitrogen and in which all of A¹ - A⁴ represents CR¹.

In one preferred group of compounds: X and Z represent O, and Y represents CR⁶R⁷.

In another preferred group of compounds: X and Z represent C=O, Y represents NR⁴.

In another preferred group of compounds: X represents CR⁶R⁷,

Y represents NR⁴ and Z represent C=O.

In another preferred group of compounds: X and Z represent CR⁶R⁷ and

Y represents NR⁴.

In another preferred group of compounds: X represents CR⁶R⁷,

Y represents NR⁴ and Z represents SO₂.

In a particularly preferred group of compounds: A¹, A², A³ and A⁴ which may be the same or different, represent N or CR¹, and either

X and Z represent O, and Y represents CR⁶R⁷ or

X and Z represent C=O,

Y represents NR⁴ or

X represents CR⁶R⁷,

Y represents NR⁴ an Z represent C=O or X and Z represent CR⁶R⁷ and Y represents NR⁴ or

or X represents CR⁶R⁷, Y represents NR⁴ and Z represents SO₂,

R⁴, R⁶ and R⁷, which may be the same or different, independently represent hydrogen or a substituent and one of R⁴ or R⁶ represents - L -R³, in which L is a single bond or a linker group, additionally,

R³ represents alkyl, alkoxy or aryl, each optionally substituted by one to three subsitutuents, R², which may be the same or different and either: R⁶ represents L - R³, and wherein L is single bond and R³ represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl,

NO₂,

CN,

NR¹⁰R¹¹, halogen, SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶,

OCC=W)NR¹⁰R¹ '

NR¹⁵C(=W)R¹⁷, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R¹⁰ and R¹¹ together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹¹,

R¹⁶ and R¹⁷, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl Or NR¹⁰R¹¹, and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R¹¹, may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxyl;

or wherein R⁴ represents L - R³, and wherein L is single bond and R³ represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl,

NR¹⁰R¹¹, SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶,

NR¹⁵C(=W)R¹⁷,

R¹⁰, R^π, R¹², R¹³, R¹⁴, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,

R¹⁰ and R^H together with the nitrogen to which they are attached may form a ring, R¹² may have the same meaning as NR¹⁰R¹ ' ,

R¹⁶ and R¹⁷, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted axyloxy, aryl Or NR¹⁰R¹¹, and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R¹¹, may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxyl.

R and R , which may be the same or different, represent: alkyl optionally substituted by one or more halogen, alkoxy or optionally substituted aryl, thioaryl or aryloxy, alkoxy optionally substituted by optionally by alkyl or optionally substituted aryl, hydroxyl,

OC(^W)NR¹⁰R¹¹ optionally substituted aryl, thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted,

NO₂,

CN,

NR¹⁰R¹¹, halogen, SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶,

NR¹⁵C(=W)R¹⁷,

R¹⁰, R^u, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,

NR¹⁰R¹ ' together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹¹, when R¹⁷ represents NR¹⁰R^{1 1}, that NR¹⁰R^{1 1} may represent hydrogen, COalkyl and CO optionally substituted aryl, <

R¹⁶ may represent hydroxy, alkoxy, or NR¹⁰R^{1 1}, R¹⁷ may represent alkyl substituted by one or more of halogen, alkoxy, optionally substituted aryl OrNR¹⁰R¹¹ _, and when R¹⁷ represents NR¹⁰R¹ \ that NR¹⁰R may represent hydrogen, COalkyl and CO optionally substituted aryl, and in addition,

R¹ and R², which may be the same or different, may represent: alkyl optionally substituted by one or more halogen, alkoxy or optionally substituted aryl, thioaryl or aryloxy, alkoxy optionally substituted by optionally by alkyl or optionally substituted aryl, hydroxyl,

OC(=W)NR¹⁰R^U aryl, thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted,

NO₂,

CN,

NR¹⁰R¹¹, halogen, SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶,

NR¹⁵C(=W)R¹⁷,

NR¹⁰R¹¹ together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹¹, when R¹⁷ represents NR¹⁰R¹¹, that NR¹⁰R¹¹ may represent hydrogen, COalkyl and CO optionally substituted aryl,

R¹⁶ may represent hydroxy, alkoxy, or NR¹⁰R¹ ', and R¹⁷ may represent alkyl substituted by one or more of halogen, alkoxy, optionally substituted aryl or NR¹⁰R¹¹.

when an adjacent pair of A¹ - A⁴ each represent CR¹, then the adjacent carbon atoms, together with their substituents may form a ring B, when X, Y or Z is CR⁶R⁷, R⁶ and R⁷, together with the carbon atom to which they are attached may form a ring C, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.

The compounds of formula I for use in the treatment of DMD will generally be administered in the form of a pharmaceutical composition.

Thus, according to a further aspect of the invention there is provided a pharmaceutical composition including preferably less than 80% w/w, more preferably less than 50% w/w, e.g. 0.1 to 20%, of a compound of formula I, or a pharmaceutically acceptable salt thereof, as defined above, in admixture with a pharmaceutically acceptable diluent or carrier.

We also provide a process for the production of such a pharmaceutical composition which comprises mixing the ingredients. Examples of pharmaceutical formulations which may be used, and suitable diluents or carriers, are as follows: for intravenous injection or infusion - purified water or saline solution; for inhalation compositions - coarse lactose; for tablets, capsules and dragees - microcrystalline cellulose, calcium phosphate, diatomaceous earth, a sugar such as lactose, dextrose or mannitol, talc, stearic acid, starch, sodium bicarbonate and/or gelatin; for suppositories - natural or hardened oils or waxes.

When the compound is to be used in aqueous solution, e.g. for infusion, it may be necessary to incorporate other excipients. In particular there may be mentioned chelating or, sequestering agents, antioxidants, tonicity adjusting agents, pH-modifying agents and buffering agents. Solutions containing a compound of formula I may, if desired, be evaporated, e.g. by freeze drying or spray drying, to give a solid composition, which may be reconstituted prior to use.

When not in solution, the compound of formula I preferably is in a form having a mass median diameter of from 0.01 to lOμm. The compositions may also contain suitable preserving, stabilising and wetting agents, solubilisers, e.g. a water-soluble cellulose polymer such as hydrσxypropyl methylcellulose, or a water-soluble glycol such as propylene glycol, sweetening and colouring agents and flavourings. Where appropriate, the compositions may be formulated in sustained release form. The content of compound formula I in a pharmaceutical composition is generally about 0.01-about 99.9wt%, preferably about 0.1-about 50wt%, relative to the entire preparation.

The dose of the compound of formula I is determined in consideration of age, body weight, general health condition, diet, administration time, administration method, clearance rate, combination of drugs, the level of disease for which the patient is under treatment then, and other factors.

While the dose varies depending on the target disease, condition, subject of administration, administration method and the like, for oral administration as a therapeutic agent for the treatment of Duchenne muscular dystrophy in a patient suffering from such a disease is from 0.01 mg - 1O g, preferably 0.1 - 100 mg, is preferably administered in a single dose or in 2 or 3 portions per day.

The potential activity of the compounds of formula I for use in the treatment of DMD may be demonstrated in the following predictive assay and screens.

1. Luciferase reporter assay Cmurine H2K cells)

The cell line used for the screen is an immortalized mdx mouse H2K cell line that has been stably transfected with a plasmid containing ~5kb fragment of the Utrophin A promoter including the first untranslated exon linked to a luciferase reporter gene (see figure 1). Under conditions of low temperature and interferon containing media, the cells remain as myoblasts. These are plated into 96 well plates and cultured in the presence of compound for three days. The level of luciferase is then determined by cell lysis and reading of the light output from the expressed luciferase gene utilising a plate luminometer.

Example of pharmacological dose response of compounds in the assay is shown in figure 2.

2. mdx mouse

10 Data obtained from the ADMET data was prioritised and the compounds with the best in vitro luciferase activity and reasonable ADMET data were prioritised for testing in the mdx proof of concept study where the outcome was to identify whether any of the compounds had the ability to increase the levels of utrophin protein in dystrophin deficient muscle when compared to vehicle only dosed control animals.

15 There were two animals injected with 10mg/kg of compound administered ip daily for 28 days plus age matched controls. Muscle samples were taken and processed for sectioning (to identify increases in sarcolemmal staining of utrophin) and Western blotting (to identify overall increases in utrophin levels).

Figure 3 shows an example of TA muscle sections stained with antibody specific for 10 mouse utrophin. Comparison to the mdx muscle only injected with vehicle shows an increase in the amount of sarcolemmal bound utrophin.

Muscles from the above treated mice were also excised and processed for Western blotting and stained with specific antibodies (figure 4). Again using muscle dosed with \5 CPD-A shows a significant increase in the overall levels of utrophin present in both the TA leg muscle and the diaphragm. Both mice exposed to CPD-A (V2 and V3) showed increased levels of utrophin expression compared to control. Positive upregulation data from the first 28 day study were then repeated in a further two mouse 28 day study. A total of three different compounds have shown in duplicate the ability to increase the level of utrophin expression in the mdx mouse when delivered daily by ip for 28 days. This data demonstrates the ability of the compound when delivered ip causes a significant increase in the levels of utrophin found in the mdx muscle and therefore gives us the confidence that this approach will ameliorate the disease as all the published data to date demonstrates that any increase of utrophin levels over three fold has significant functional effects on dystrophin deficient muscle.

The H2K/mdx/Utro A reporter cell line maintenance

The H2K/mdx/Utro A reporter cell line was passaged twice a week until <30% confluent . The cells were grown at 33⁰C in the presence of 10% CO₂

To remove the myoblasts for platting, they were incubated with Trypsin / EDTA until the monolayer started to detach. Growth Medium

DMEM Gibco 41966

20% FCS

1% Pen/Strep l% glutamine lOmls Chick embryo extract

Interferon(1276 905 Roche) Add fresh lOμl / 5OmIs medium

Luciferase Assay for 96 Well Plates

The H2K/mdx/Utro A reporter cell line cells were plated out into 96 well plates (Falcon 353296, white opaque) at a density of approximately 5000 cells/well in 190μl normal growth medium. The plates were then incubated at 33⁰C in the presence of 10% CO₂ for 24 hrs. Compounds were dosed by adding lOμl of diluted compound to each well giving a final concentration of lOμM. The plates were then incubated for a further 48hrs

Cells were then lysed in situ following the manufacture's protocols (Promega Steady-Glo Luciferase Assay System(E2520). Then counted for 10 seconds using a plate luminometer (Victor 1420).

Compound Storage

Compounds for screening were stored at -2O⁰C as 1OmM stocks in 100% DMSO until required.

Injection of mdx mice with compounds

Mdx from a breeding colony were selected for testing. Mice were injected daily with either vehicle or 10mg/kg of compound using the intraperitoneal route (ip). Mice were weighed and compounds diluted in 5% DMSO, 0.1 % tween in PBS.

Mice were sacrificed by cervical dislocation at desired time points, and muscles excised for analysis

Muscle Analysis Immunohistochemistry

Tissues for sectioning were dissected, immersed in OCT (Bright Cryo-M-Bed) and frozen on liquid nitrogen cooled isopentane. Unfixed 8μM cryosections were cut on a Bright Cryostat, and stored at -8O⁰C

In readiness for staining, sections were blocked in 5% foetal calf serum in PBS for 30 mins. The primary antibodies were diluted in blocking reagent and incubated on sections for 1.5 hrs in a humid chamber then washed three times for 5 mins in PBS. Secondary antibodies also diluted in blocking reagent, were incubated for lhr in the dark in a humid chamber. Finally sections were washed three times 5mins in PBS and coverslip mounted with hydromount. Slides were analysed using a Leica fluorescent microscope.

Results

Biological activity as assessed using the luciferase reporter assay in murine H2K cells, and is classified as follows:

+ Up to 200% relative to control

-H- Between 201 % and 300% relative to control

+++ Between 301 % and 400% relative to control

++++ Above 401 % relative to control

Table 1 :

Example Chemical Name Activity number

1. 2-((3-chloro-4-fluorophenylamino)methylene)-1 H-indene-1 ,3(2H)- + dione

2. 2-(3-methoxyphenyl)isoindoline-1,3-dione +

3. I.S-dioxo^-o-tolylisoindoline-δ-carboxylic acid +

4. 2-(2,3-dimethylphenyl)-1,3-dioxoisoindoline-5-carboxylic acid +

5. 2-(3,4-dimethylphenyl)-5-(4-oxo-4H-benzo[d][1 ,3]oxazin-2- + yl)isoindoline-1 ,3-dione

6. 2-(2-methoxypheny!)-5-(4-oxo-4H-benzo[d][1,3]oxazin-2- + yl)isoindoline-1 ,3-dione

7. ethyl 4-(4-(1,3-dioxoisoindolin-2-yl)benzamido)benzoate +

8. 2-(4-chlorophenyl)-1,3-dioxoisoindoline-5-carboxylic acid +

9. N-(4-(6,7-dimethoxy-1-oxoisoindolin-2-yl)phenyl)acetamide +

10. 2-(3,4-dimethylphenyl)-N-(4-hydroxyphenyl)-1 ,3-dioxoisoindoliπe-5- + carbσxamide

11. 5-bromo-2-(5-chloro-2-hydroxyphenyl)isoiπdoline-1 ,3-dione +

12. 2-(4-chlorc-2,5-dimethσxyphenyl)-1 ,3-dioxoisoindoline-5-carboxylic + acid

13. 5-(benzo[d]oxazol-2-yl)-2-m-tolylisoindoline-1,3-dione +

14. 2-(4-fluorophenyl)isoiπdoline-5-carbohydrazide ++

15. 2-(4-methoxyphenyl)isoindoline-5-carbohydrazide ++

16. 5-(3-aminophenoxy)-2-(2,4-dimethy!phenyl)isoindoline-1,3-dione +

17. 2-(3-fluorophenyl)-6,7-dimethoxyisoindolin-1-one +

18. N-(2,4-dimethylphenyl)-4-(1,3-dioxoisoindoiin-2- + y I) benzenesulfonamide

19. methyl 2-(4-(1,3-dioxoisoindolin-2-yl)benzamido)benzoate +

20. • 2-(4-(isopentyloxy)phenyl)isoindoline-1 ,3-dione +

21. 2-{4-(1 ,3-dioxoisoindolin-2-yl)benzamido)-5-hydroxybenzoic acid +

22. N-cyclohexyi-4-(1 ,3-dioxoisoindoliπ-2-yl)-N-methylbenzamide + 23.. 3-(1,3-dioxo-5-(4-oxo-4H-benzo[d][1,3]oxazin-2-yl)isoindolin-2-yl)-2- + methylbenzoic acid

24. 2-(4-chlorophenyl)-6,7-dimethoxyisoindolin-1-one +

25. 4-(N,N-dimethylsulfanπoyl)-N-(3-(isoindolin-2-yl)phenyi)benzamide +

26. 2-(4-(1-oxoisoindolin-2-yl)phenyl)propanoic acid +

27. 5-amiπo-2-phenylisoindoline-1,3-dione + 28. N-(1,3-dioxo-2-phenylisoindo!in-5-yl)isobutyramide +

29. 2-pheny!-1H-indene-1 ,3(2H)-dione +

30. 2-p-tolylisoindolin-5-amine ++

31. 2-(1-oxoisoindolin-2-yl)benzoic acid +

32. N-(2-p-tolylisoindolin-5-yl)isobutyramide +

33. 3-oxo-2M-tolyl-2,3-dihydro-1 H-isoindole-4-carboxylic acid +

34. 1 H-isoindole-4-carboxylic acid, 2-(2-chlorophenyl)-2,3-dihydro-3-oxo- +

35. 1 H-isoindole-4-carboxylic acid, 2,3-dihydro-3-oxo-2-[3- +

(trifluoromethyl)phenyl]-

36. 1 H-isoindole-4-carboxylic acid, 2,3-dihydro-2-(3-methoxyphenyl)-3- +

OXO-

37. 3-oxo-2-phenyl-2,3-dihydro-1 H-isoindole-4-carboxylic acid +

38. 1 H-isoindole-4-carboxylic acid, 2,3-dihydro-2-(2-methoxyphenyl)-3- + oxo-

39. 2-(4-(1-oxoisoindolin-2-yl)phenyl)butanoic acid +

40. 3,3,5,7-tetramethyl-2-phenylindoline +

Experimental

HPLC-UV-MS was performed on a Gilson 321 HPLC with detection performed by a Gilson 170 DAD and a Finnigan AQA mass spectrometer operating in electrospray ionisation mode. The HPLC column used is a Phenomenex Gemini C18 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 Cl 8 15 Ox 10mm and the mobile phase is acetonitrile/water. 1H NMR spectra were recorded on a Bruker instrument operating at 300 MHz. NMR spectra were obtained as CDCl₃ 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 (40-65 μm silica gel) or using an automated purification system (SP1™ Purification System from Biotage^®). Reactions in the microwave were done in an Initiator 8™ (Biotage). The abbreviations used are DMSO (dimethylsulfoxide), HCl (hydrochloric acid), MgSO₄ (magnesium sulfate), NaOH (sodium hydroxide), Na₂CO₃ (sodium carbonate), NaHCO₃ (sodium bicarbonate), THF (tetrahydrofuran).

H₂N .

Acetic acid reflux, 2 days

Method 1

Ammonium formate Pd/C, ethaπol rt, 2days

Method 2

lsobutyryl chloride, Pyridine, 3h, rt

Method 5

Method 1 : Compounds I

2-(4-ChIorophenyl)-5-nitroisoindoIine-l,3-dione

A mixture of 5-nitroisobenzofuran-l ,3-dione (4g, 21mmol) and 4-chloroaniline (2.62g, 21mmol) in acetic acid was refluxed overnight. After cooling, the mixture was poured onto ice/water to give a yellow solid, which was recovered by filtration to afford 5.2g (90%) of the title compound. Method 2 : Compounds II

5-Amino-2-phenylisoindoline-l,3-dione

To a stirred suspension of 2-(4-chlorophenyl)-5-nitroisoindoline-l,3-dione (500mg, 1.65mmol) in ethanol (10OmL) was added ammonium formate (470mg, 7.45mmol) in one portion followed by palladium on carbon (450mg). The resulting suspension was stirred at room temperature for 2 days. The mixture was then filtered through a pad of Celite^® and washed, first with ethanol and then with tetrahydrofuran. After removal of the solvents, the yellow solid was taken up in ethyl acetate and washed with brine. The combined organic layers were dried over anhydrous MgSO₄ and evaporated to afford 150mg (38%) of the title compound (LCMS RT= 5.65min, MH⁺ 238.8) 1H NMR (DMSO): 7.58 (IH₅ d, J 8.1 Hz), 1.53-1 Al (2H, m), 7.42-7.37 (3H, m), 7.01 (IH, d_? J2.0 Hz), 6.87 (IH, dd, J8.22.0 Hz), 6.55 (2H, s)

Method 3 : Compounds HI

N-(l,3-Dioxo-2-phenylisoindolin-5-yl)isobutyramide

To a solution of 5-amino-2-phenylisoindoline-l,3-dione (lOOmg, 0.42mmol) in pyridine (1OmL) was added isobutyryl chloride (42μL, 0.43mmol). The resulting mixture was stirred at room temperature for 3h. Ethyl acetate was added and the organic layer was washed twice with aqueous copper sulfate solution. The combined organic layers were dried over anhydrous MgSO₄ and evaporated. The resulting solid was triturated with diethyl ether to afford 33.9mg (26%) of the title compound (LCMS RT= 6.03min, MH⁺ 309.0) 1H NMR (DMSO): 10.50 (IH, s), 8.31 (IH, d, J 1.5 Hz), 7.97 (IH, d, J 8.2 1.8 Hz), 7.91 (IH, d, J8.2 Hz)₅ 7.56-7.43 (5H, m), 2.72-2.62 (IH, m), 1.15 (6H, d, J6.8 Hz)

Method 4 : Compounds III

2-p-ToIylisoindoIin-5-amine

To a solution of 5-amino-2-p-tolylisoindoline-l,3-dione (230mg, 0.91mmol) in tetrahydrofuran (2OmL) was added dropwise a IM solution of borane in tetrahydrofuran (5.49mL, 5.49mmol). The resulting mixture was refluxed for 18h. After cooling, 2M aqueous hydrochloric acid solution was added to reach pH 2. The mixture was then transferred to a separating runnel and the aqueous layer neutralized with saturated aqueous sodium bicarbonate and then basified with a few pellets of sodium hydroxide to reach pH 14. The aqueous layer was then extracted with dichloromethane (3x10OmL), and the combined extracts were washed with brine until pH 7 was reached. The combined organic layers were dried over anhydrous MgSO₄ and evaporated. The resulting solid was dissolved in dichloromethane and filtrated through a short column of silica to afford 94mg (46%) of the title compound (LCMS RT= 6.85min, MH⁺ 225.1) 1H NMR (CDCl₃): 7.05-7.01 (3H, m), 6.60-6.49 (4H₅ m), 4.45 (4H, s), 3.62 (2H, br), 2.21 (3H, s)

Method 5 : Compounds V

N-(2-p-Tolylisoindolin-5-yl)isobutyramide To a solution of 2-p-tolylisoindolin-5- amine (247mg, l.lOmmol) in pyridine (2OmL) was added isobutyryl chloride (0.14mL, 1.32mmol). The resulting mixture was stirred at room temperature for 48h. Ethyl acetate was then added and the organic layer was washed three times with aqueous copper sulfate solution. The combined organic layers were dried over anhydrous MgSO₄ and evaporated. The solid was then purified by column chromatography on silica gel eluting using a gradient (dichloromethane to dichloromethane/ethyl acetate 90:10 v/v), and then reverse phase HPLC to afford 8.2mg (3%) of the title product (LCMS RT= 9.70min, MH⁺ 295.1) 1H NMR (DMSO): 9.87 (IH, s), 7.75 (IH, s), 7.46 (IH, dd, J8.3 1.8 Hz), 7.30 (IH, d, J 8.3 Hz), 7.06 (2H, d, J 8.3 Hz), 6.58 (2H, d, J 8.4 Hz), 4.51 (4H, d, J 7.3 Hz), 2.67- 2.56 (IH, m), 1.11 (6H, d, J6.8 Hz)

Claims

Claims:

1. Use of a compound of formula I,

wherein

A¹, A², A³ and A⁴ _, which may be the same or different, represent N or CR¹, X, Y and Z, which may be the same or different, represent O, S(O)_n, C=W, NR⁴, NC(=O)R⁵ and CR⁶R⁷, W is O, S, NR²⁰, one of R⁴, R⁵, R⁶, and R²⁰ represents - L -R³, in which L is a single bond or a linker group, additionally, R³, R ⁴, R ⁵, R⁶, and R⁷, which may be the same or different, independently represent hydrogen or a substituent and

R²⁰ represents hydrogen, aryl, hydroxyl, alkyl optionally substituted by aryl, alkoxy optionally substituted by aryl, CN, optionally substituted alkoxy, optionally substituted aryloxy, optionally substituted alkanoyl, optionally substituted aroyl, NR³⁰R³¹, in which R³⁰ and R³¹, which may be the same or different, represent hydrogen, optionally substituted alkyl or optionally substituted aryl; additionally, one of R³⁰ and R³¹ may represent optionally substituted alkanoyl or optionally substituted aroyl,

¹ n represents an integer from 0 to 2, in addition, an adjacent pair of X, Y and Z cannot both represent O, or S or together represent O - S, any pair of R⁶, R⁷ and R⁴ may when attached to adjacent atoms together represent a single bond between the adjacent atoms to which they are attached, when an adjacent pair of Ai - A₄ each represent CR¹, then the adjacent carbon atoms, together with their substituents may form a ring B, when X, Y or Z is CR⁶R⁷, R⁶ and R⁷, together with the carbon atom to which they are attached may form a ring C, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the therapeutic and/or prophylactic treatment of Duchenne muscular dystrophy, Becker muscular dystrophy or cachexia.

2. Use according to claim 1, wherein A¹, A², A³ and A⁴, which may be the same or different, represent N or CR¹ _,

X, Y and Z, which may be the same or different, represent O, S(O)_n, C=W, NR⁴, NC(=0)R⁵ and CR⁶R⁷, W is O, S, NR²⁰, one of R⁴, R⁵, R⁶, and R²⁰ represents - L -R³, in which L is a single bond or a linker group, additionally, R³, R ⁴, R ⁵, R⁶, and R⁷, which may be the same or different, independently represent hydrogen or a substituent and R represents hydrogen, aryl, hydroxyl or alkyl, n represents an integer from 0 to 2, in addition, an adjacent pair of X, Y and Z cannot both represent O, or S or together represent O - S, any pair of R⁶, R⁷ and R⁴ may when attached to adjacent atoms together represent a single bond between the adjacent atoms to which they are attached, when an adjacent pair of Ai - A₄ each represent CR¹, then the adjacent carbon atoms, together with their substituents may form a ring B, and when X, Y or Z is CR⁶R⁷, R⁶ and R⁷, together with the carbon atom to which they are attached may form a ring C.

3. Use according to claim 1 or 2, wherein R³ in the compound of formula 1 represents alkyl, alkoxy or aryl, each optionally substituted by one to three subsitutuents, R₂, which may be the same or different.

4. Use according to claim 1 or 2, wherein either: one of R or R⁶ represents L - R , and wherein L is single bond and R³ represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl,

NO₂,

CN,

NR¹⁰R¹¹, halogen,

SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶,

OC(=W)NR¹⁰Rⁿ NR¹⁵C(=W)R¹⁷,

R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R¹⁰ and R¹ ' together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹¹,

R¹⁶ and R¹⁷, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl orNR'V¹, and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R¹¹, may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxyl;

O-aryl or thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, hydroxyl, NR¹⁰R¹¹,

SO₂R¹²,

NR¹³SO₂R¹⁴,

C(=W)R¹⁶,

NR^I5C(=W)R¹⁷, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition,

R¹⁰ and R¹¹ together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹¹,

R¹⁶ and R¹⁷, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl Or NR¹⁰R¹¹, and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R^{1 1}, may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxyl.

5. Use according to claim 1 or 2, wherein either:

one of R⁵ or R⁶ represents L - R³, and wherein L is single bond and R³ represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted, optionally substituted aryl, NR¹⁰R", SO₂R¹², NR¹³SO₂R¹⁴, C(=W)R¹⁶, OCC=W)NR¹⁰R¹¹ NR¹⁵C(=W)R¹⁷,

R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R¹⁰ and R¹¹ together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹ ', R¹ and R¹⁷, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl OrNR¹⁰R¹¹ _, and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R¹¹, may additionally . represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxyl;

or wherein one of R⁴ or R²⁰ represents L - R³, and wherein L is single bond and R represents: thioalkyl optionally substituted by alkyl or optionally substituted aryl, O-aryl or thioaryl, in which the aryl is optionally substituted, optionally substituted aryl,

NR¹⁰R¹¹,

SO₂R¹²,

NR¹³SO₂R¹⁴, CC=W)R¹⁶,

NR¹⁵C(=W)R¹⁷, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁶ and R¹⁷, which may be the same or different, represent hydrogen, alkyl optionally substituted by optionally substituted aryl, optionally substituted aryl, in addition, R¹⁰ and R¹¹ together with the nitrogen to which they are attached may form a ring,

R¹² may have the same meaning as NR¹⁰R¹¹,

R¹⁶ and R¹⁷, which may be the same or different, may each represent alkyl substituted by one or more of halogen, alkoxy optionally substituted aryl or optionally substituted aryl, optionally substituted aryloxy, aryl or NR¹⁰R", and when R¹⁶ or R¹⁷ represents NR¹⁰R¹¹, one of R¹⁰ and R¹¹, may additionally represent CO alkyl optionally substituted or COaryl optionally substituted, and in addition to the definitions shared with R¹⁷, R¹⁶ may represent hydroxyl.

6. Use according to claim 3, in which R¹ and R², which may be the same or different, may represent include: alkyl optionally substituted by one or more halogen, alkoxy or optionally substituted aryl, thioaryl or aryloxy, alkoxy optionally substituted by optionally by alkyl or optionally substituted aryl, hydroxyl,

OC(=W)NR¹⁰R^π aryl, thioalkyl optionally substituted by alkyl or optionally substituted aryl, thioaryl, in which the aryl is optionally substituted,

NO₂,

CN, NR¹⁰R^{1 1}, halogen,

SO₂R¹²,

NR¹³SO₂R¹⁴, C(=W)R¹⁶,

NR¹⁵C(=W)R¹⁷,

R¹² may have the same meaning as NR¹⁰R¹ ', when R¹⁷ represents NR¹⁰R¹', that NR¹⁰R¹¹ may represent hydrogen, COalkyl and CO optionally substituted aryl,

R¹⁶ may represent hydroxy, alkoxy, or NR¹⁰R¹¹, and R¹⁷ may represent alkyl substituted by one or more of halogen, alkoxy, optionally substituted aryl or NR¹⁰R¹¹.

7. Use according to claim 1 or 2, wherein either:

one of R⁵ or R⁶ represents L - R³, in which L represents a linker group which is: O, S, NR¹⁸, alkylene, alkenylene, alkynylene, each of which may be optionally interrupted by one or more of O, S, NR¹⁸, or one or more C-C single, double or triple bonds, a -N-N- single or double bond, and R¹⁸ represents hydrogen, alkyl, COR¹⁶;

or one of R⁴ or R²⁰ represents L - R³, in which L represents a linker group which is:

O, S or NR¹⁸, alkylene, alkenylene, alkynylene, each of which may be optionally interrupted by one or more of O, S, NR¹⁸, or one or more C-C single, double or triple bonds, and R¹⁸ represents hydrogen, alkyl, COR¹⁶.

8. Use of a compound according to claim 1 or 2, in which R⁴, R⁵, R⁶, and R⁷ represent hydrogen, alkyl or optionally substituted aryl.

9 Use of a compound according to claim 1 or 2 in which when any of the substituents represents alkyl, alkyl is saturated and has from 1 to 10 carbon atoms.

10. Use of a compound according to claim 1 or 2, in which aryl is an aromatic hydrocarbon or a 5 to 10 membered aromatic heterocyle containing 1 to 4 hetero atoms selected from an oxygen atom, a sulphur atom and a nitrogen atom as a ring constituent besides carbon.

11. Use of a compound according to claim 1 or 2, in which aryl is phenyl or naphthalene.

12. Use if a compound according to claim 1 or 2, in which aryl is furan, thiophene, pyrrole or pyridine.

13. Use of a compound according to claim 1 or 2, in which ring B or ring C is a saturated or unsaturated 3 to 10 membered carbocylic or heterocyclic ring.

14. Use of a compound according to claim 1 or 2, in which ring B is benzene ring.

15. Use of a compound according to claim 1 or 2, in which ring C is a 3- 10 membered saturated or unsaturated carbocylic ring.

16. Use of a compound according to claim 1 or 2, in which at least one R¹ represents NR¹⁵C(=W)R¹⁷.

17. Use of a compound according to claim 1 or 2, in which at least one R¹ represents NR¹⁵C(K))R¹⁷.

18. Use of a compound according to claim 1 or 2, in which at least one R¹ represents CONR¹⁰R¹¹.

19. Use of a compound according to claim 1 or 2, in which at least one R¹ represents NHCOR¹⁷ _, wherein R¹⁷ is selected from: alkyl Ci- C₆, alkyl Ci - C₆ substituted by phenyl, alkyl Ci - C₆ substituted by alkoxy Ci - C₆, haloalkyl Ci - Ce, perflouroalkyl Ci - C₆, phenyl optionally substituted by one or more of halogen, alkyl Ci - C₆, alkoxy Ci - C₆, amino, (alkyl Ci - C₆)amino, di(alkyl Ci - C₆) amino or phenyl, CH:CH phenyl, naphthyl, pyridinyl, thiophenyl and furanyl.

20. Use of a compound according to claim 1 or 2 in which one or both of R¹ and R² is other than -COOH.

21. Use of a compound according to claim 1 or 2, in which at least one of R^! represents NR¹⁵CONR¹⁰R¹ ', wherein R¹⁰ and R¹¹, which may be the same or different, are selected from optionally substituted aryl, alkyl and COaryl optionally substituted.

22. Use of a compound according to claim 1 or 2, in which at least one of R represents NHCONHR¹⁵ and R¹⁵ is selected from phenyl, alkyl C₁ to C₆ and COphenyl optionally substituted by one or more halogen.

23. Use of a compound according to claim 1 or 2, in which at least one of R¹ represents alkyl Ci to C₆, optionally substituted by phenyl or a 4 to 7- membered, preferably 5 or 6-membered saturated or unsaturated heterocycle preferably containing one to two heteroatoms selected from N, S and O.

24. Use of a compound according to claim 1 or 2 in which at least one of R¹ represents COR¹⁶ and R¹⁶ is alkoxy Ci - C₆, amino, (alkyl Cj - C₆)amino or di(alkyl Ci - C₆) amino.

25. Use of a compound according to claim 1 or 2, in which at least one of Rj represents:

NO₂, halogen, amino or (alkyl Ci - C₆)amino or di(alkyl Cj - C₆) amino in which the alkyl Ci to C₆ is optionally substituted by phenyl or a 5 or 6 membered saturated or unsaturated heterocycle, NHSO₂alkyl C i - C₆, NHSO₂phenyl,

S0₂alkyl C₁ - C₆, phenyl optionally substituted by Cj to C₆ alkoxy Ci - C₆, a 5 - 10 membered, saturated or unsaturated, mono- or bi-cyclic heterocycle containing from 1 - 3 heteroatoms selected from N₅ S and O.

26. Use of a compound according to claim 1 or 2, in which at least one of R¹ represents SO_nR¹² wherein n represents 0, 1 or 2 and R¹² represents alkyl Ci - C₆ or NR⁹⁸R⁹⁹ wherein R⁹⁸ and R⁹⁹, which may be the same or different, represent hydrogen or alkyl Ci - C₆.

27, Use of a compound according to claim 1 or 2, in which R³ represent aryl and is optionally substituted by one to three substituents, R², which may be the same or different.

28. Use of a compound according to claim 27 in which R³ is a 5 - 10 membered aromatic mono- or bi-cyclic system.

29. Use of a compound according to claim 28, in which the aromatic system is a hydrocarbon.

30. Use of a compound according to claim 29, in which the aromatic hydrocarbon is benzene or naphthalene.

31. Use of a compound according to claim 28, in which the aromatic system is a heterocyclic system containing up to three heteroatoms, which may be the same or different, selected from N, O and S.

32. Use of a compound according to claim 31, in which the heterocyclic system is thiophene, furan, pyridine or pyrrole.

33. Use of a compound according to claim 3, in which the substituent(s) R² is/are selected from is: alkyl C₁ - C₆, optionally substituted by thiophenyl or phenoxy, each optionally substituted by halogen, alkoxy Cj - C₆, phenyl, thioalkyl Ci - C₆, thiophenyl, optionally substituted by halogen, NO₂, CN

NR¹⁰R¹¹, in which R¹⁰ and R¹¹, which may be the same or different represent hydrogen, alkyl Ci - C₆, or together with the nitrogen to which they are attached form a 5 to 7 membered ring which may contain one or more additional heteroatoms selected from N, O and S, > halogen,

SO₂R¹², in which R₁₂ represents a 5 to 7 membered ring which may contain one or more additional heteroatoms selected from N, O and S, NHCOR¹⁷, in which R¹⁷ represents alkyl Ci - C₆, optionally substituted by: phenyl or halogen, or phenyl optionally substituted by alkoxy Cj - C₆, carboxy, or halogen, or a 5 or 6 membered saturated or unsaturated heterocycle, phenyl or a 5 or 6 membered saturated or unsaturated heterocycle optionally substituted by halogen, alkoxy Ci to C₆, carboxy or a group SO₂N NR¹⁰R¹¹.

34. Use of a compound according to claim 33 in which NR¹⁰R¹¹ represents N- pyrrole, N-piperidine, N' (Ci - C₆) alkyl N piperazine or N-morpholine.

35. Use of a compound according to claim 7 in which R⁵ or R⁶ represents L - R³, in which L represents a linker group which is: -NH.NH-, -CH=CH, -C≡C-, or

36. Use of a compound according to claim 1 or 2 in which two of A¹ - A⁴ represent nitrogen.

37. Use of a compound according to claim 1 or 2 in which one of A¹ - A⁴ represents nitrogen.

38. Use of a compound according to claim 1 or 2 in which all of A¹ - A⁴ represents CR¹.

39. Use according to any preceding claim, wherein X and Z represent O, and Y represents CR⁶R⁷.

40. Use according to any preceding claim, wherein X and Z represent C=O, Y represents NR⁴.

41. Use according to any preceding claim, wherein X represents CR⁶R⁷,

Y represents NR⁴ and Z represent C=O.

42. Use according to any preceding claim, wherein X and Z represent CR⁶R⁷ and Y represents NR⁴.

43. Use according to any preceding claim, wherein X represents CR⁶R⁷,

Y represents NR⁴ and Z represents SO₂.

44. Use of a compound as listed in table 1 according to claim 1.