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Title:
ADENINE DERIVATIVES SUITABLE FOR THE TREATMENT OF (INTER ALIA) MUSCULAR DYSTROPHY
Document Type and Number:
WIPO Patent Application WO/2014/083327
Kind Code:
A1
Abstract:
The invention provides a compound of formula (I), wherein R1, R2, R3, R4, R5, Ra and Rb have the meanings given in the description, with the proviso that the compound is not a compound selected from the group given in the description. The invention also provides the defined compounds (with or without the proviso) for use in the treatment of muscular dystrophy.

Inventors:
LEHMANN FREDRIK (SE)
MALMSTRÖM JONAS (SE)
EVENÄS LARS JOHAN (SE)
BRIMERT LARS THOMAS (SE)
SVENSSON BO ROGER (SE)
WALSE BJÖRN ULRIK (SE)
Application Number:
PCT/GB2013/053121
Publication Date:
June 05, 2014
Filing Date:
November 26, 2013
Export Citation:
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Assignee:
MD PHARMA AB (SE)
SMITH STEPHEN EDWARD (GB)
International Classes:
C07D473/34; A61K31/52; A61P25/00
Domestic Patent References:
WO2010034707A12010-04-01
WO2010100144A12010-09-10
Attorney, Agent or Firm:
SMITH, Stephen Edward (Talbot StreetNottingham, Nottinghamshire NG1 5GG, GB)
Download PDF:
Claims:
Claims

1. A compound of formula I,

wherein: when R2 is present, then the C1-C2 and C3-N2 bonds are double bonds, the N1-C1 and C2-C3 bonds are single bonds, and one of Ra and Rb is absent; when R2 is absent, then the C1-C2 and C3-N2 bonds are single bonds, and the N1-C1 and C2-C3 bonds are double bonds; when R3 is present, then R5 is absent, the C4-N3 bond is a single bond and the C4- N4 bond is a double bond; when R3 is absent, then R5 is present, the C4-N3 bond is a double bond and the C4- N4 bond is a single bond;

C\ C2, C3 and C4 all represent C;

N1, N2, N3 and N4 all represent N;

Ra and R independently represent hydrogen or a C1-12 alkyl group optionally substituted by one or more substituents selected from E1; or

Ra and Rb are linked together, along with the requisite nitrogen atom to which they are necessarily attached, to form a 3- to 7-membered heterocycloalkyl group, optionally containing one further heteroatom selected from nitrogen, sulfur and oxygen, and which cyclic group is optionally substituted by one or more substituents selected from =0 and E2; R represents hydrogen, halo, -OR10, -SR11 , -N(R30)R31 , Cm alkyl, C2.6 alkenyl, C2-6 alkynyl, aryl or heteroaryl, which latter five groups are optionally substituted by one or more substituents selected from E3;

R2 may be present or absent and, when present, represents Ci-e alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from E4;

R3 may be present or absent and, when present, represents hydrogen or Ci-4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more halo atoms;

R4 represents hydrogen, -N(R32)R33, aryl or heteroaryl, which latter two groups are optionally substituted by one or more substituents selected from E5;

R5 may be present or absent and, when present, represents hydrogen or Ci-4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from E6;

E1 represents -OR20 or aryl, which latter group is optionally substituted by one or more substituents selected from Q1;

E2 represents halo, -OR21 or -N(R34)R35;

E3 represents halo, -OR22, -SR23, -N(R36)R37, Ci-6 alkyl or aryl, which latter two groups are optionally substituted by one or more halo atoms;

E4 represents halo, -OR24, -N(R38)R39, or aryl, which latter group is optionally substituted by one or more halo atoms;

E5 represents halo, -OR25, -N(R40)R41 or Ci-6 alkyl which latter group is optionally substituted by one or more halo atoms;

E6 represents halo or C1.4 alkyl optionally substituted by one or more halo atoms; Q1 represents halo, -OR26 or C1.4 alkyl, which latter group is optionally substituted by one or more halo atoms;

R10 and R11 independently represent hydrogen, C1-4 alkyl or aryl, which latter two groups are optionally substituted by one or more substituents selected from halo and -OR27;

R20 to R27 independently represent hydrogen or C1-4 alkyl optionally substituted by one or more halo atoms; and

R30 to R4 independently represent hydrogen or C1-4 alkyl optionally substituted by one or more aryl substituents, which aryl group may in turn be optionally substituted by one or more halo atoms; or a pharmaceutically acceptable ester, amide, solvate or salt thereof; provided that:

(i) when R2 is absent, at least one of Ra, Rb, R1, R3, R4 and R5 is present and is not hydrogen; and

(ii) the compound is not a compound selected from the group consisting of:

3-methyladenine; Phosphatidylinositol 3-Kinase a Inhibitor 2;2-(6-Aminopurin- 9-yl)ethanol; 7-methyladenine; 9H-Purin-6-amine,9-(phenylmethyl)-; 2- Fluroadenine; 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine; 9-THF-Ade; 2,4- Difluoro-N-[2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3- pyridinyl]benzenesulfonamide; 6-Fluoro-N-[(4-methoxyphenyl)ethyl]-4- quinazolinamine; 6-Fluoro-N-[(4-fluorophenyl)methyl]-4-quinazolinamine; Hydrochloroquine sulphate; SCHISTOSOMICIDE (lucanthone hydrochloride); Bortezomib (Velcade); CAA0225; LY294002 (2-morpholin-4-yl-8- phenylchromen-4-one); Wortmannin; (3-Pyridinecarboxamide,N-(2,3-dihydro- 7,8-dimethoxyimidazo[1 ,2-c]quinazolin-5-yl)-; N-[5-[4-Chloro-3-[(2- hydroxyethyl)sulfamoyl]phenyl]-4-methylthiazol-2-yl]acetamide; and PI 103 hydrochloride.

2. A compound as claimed in Claim 1 , wherein, when R2 is absent, at least one of Ra, R , R1, R3, R4 and R5 is present and is not hydrogen.

3. A compound as claimed in Claim 1 or Claim 2, wherein R4 represents hydrogen and one of R3 and R5 represents hydrogen while the other one of R3 and R5 is absent.

4. A compound as claimed in Claim 1 or Claim 2, wherein at least one of R3 to R5 is present and is not hydrogen.

5. A compound as claimed in any one of the preceding claims, wherein at least one of Ra, Rb, R1 and R2 is present and is not hydrogen.

6. A compound as claimed in any one of the preceding claims, wherein:

Ra and Rb independently represent hydrogen or a C1-12 alkyl group optionally substituted by one or more substituents selected from E1 , provided that at least one of Ra and Rb is present and represents a group other than hydrogen; or

Ra and Rb are linked together, along with the requisite nitrogen atom to which they are necessarily attached, to form a 3- to 7-membered heterocycloalkyl group, optionally containing one further heteroatom selected from nitrogen, sulfur and oxygen, and which cyclic group is optionally substituted by one or more substituents selected from halo and -OR21.

7. A compound as claimed in any one of the preceding claims, wherein R1 represents -OR10, -N(R30)R31, Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, aryl or heteroaryl, which latter five groups are optionally substituted by one or more substituents selected from E3.

8. A compound as claimed in any one of the preceding claims, wherein R2 is present and represents C1-6 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from halo or -OR24.

9. A compound as claimed in any one of Claims 1 , 2 and 4 to 8, wherein R3 is present and represents C1-4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more halo atoms.

10. A compound as claimed in any one of Claims 1 , 2 and 4 to 9, wherein R4 represents -N(R32)R33, aryl or heteroaryl, which latter two groups are optionally substituted by one or more substituents selected from E5.

1 1. A compound as claimed in any one of Claims 1 , 2 and 4 to 10, wherein R5 is present and represents Ci-4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from E6.

12. A Class III PI 3-kinase inhibitor comprising or consisting of:

a. a compound as defined in any one of Claims 1 to 1 1 ; or b. a compound selected from the group defined in Table 1 , for use in the treatment or prevention of muscular dystrophy.

13. The Class III PI 3-kinase inhibitor for use according to Claim 12 wherein the Class III PI 3-kinase inhibitor comprises or consists of:

a. Table 1 , compound 1 ;

b. Table 1 , compound 2;

c. Table 1 , compound 3;

d. Table 1 , compound 4;

e. Table 1 , compound 5;

f. Table 1 , compound 6;

g- Table 1 , compound 7;

h. Table 1 , compound 8;

Ί. Table 1 , compound 9;

j- Table 1 , compound 10

k. Table 1 , compound 1 1

I. Table 1 , compound 12

m. Table 1 , compound 13

n. Table 1 , compound 14

o. Table 1 , compound 15

P- Table 1 , compound 16

q- Table 1 , compound 17 r. Table 1 , compound 18.

14. The Class III PI 3-kinase inhibitor for use according to Claim 12 wherein the Class III PI 3-kinase inhibitor does not comprise or consist of:

a. Table 1 , compound 1 ;

b. Table 1 , compound 2;

c. Table 1 , compound 3;

d. Table 1 , compound 4;

e. Table 1 , compound 5;

f. Table 1 , compound 6;

g- Table 1 , compound 7;

h. Table 1 , compound 8;

i. Table 1 , compound 9;

j- Table 1 , compound 10

k. Table 1 , compound 11

I. Table 1 , compound 12

m. Table 1 , compound 13

n. Table 1 , compound 14

o. Table 1 , compound 15

P- Table 1 , compound 16

q- Table 1 , compound 17

r. Table 1 , compound 18

15. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to 14 wherein the Class III PI 3-kinase is VPS34.

16. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

15 wherein the Class III PI 3-kinase is hVPS34.

17. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

16 wherein the VPS34 comprises or consists of the amino acid sequence according to SEQ ID NO: 1 or variants thereof.

18. The Class III PI 3-kinase inhibitor for use according to Claim 17 wherein the VPS34 is a variant of SEQ ID NO: 1 comprising or consisting of a sequence with at least 60% identity to the amino acid sequence of SEQ ID NO: 1 , for example, at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID ΝΟ. .

19. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

18 wherein the Class III PI 3-kinase inhibitor is a selective inhibitor of Class III PI 3-kinase.

20. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

19 wherein the Class III PI 3-kinase inhibitor is not an inhibitor of Class I PI 3-kinase and/or Class II PI 3-kinase.

21. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

20 wherein the Class III PI 3-kinase inhibitor has an IC50 of 250 μιη or lower, for example, 200 μιη or lower, 150 m or lower, 100 μηι or lower, 90 μιη or lower, 80 μητι or lower, 70 μητι or lower, 60 μηη or lower, 55 μηη or lower, 54 μηη or lower, 53 μιτι or lower, 52 μιη or lower, 51 μιη or lower, 50 μιη or lower, 49 μιη or lower, 48 μιη or lower, 47 μηι or lower, 46 μηη or lower, 45 μιη or lower, 44 μιη or lower, 43 μηη or lower, 42 μηι or lower, 41 μηι or lower, 40 μιη or lower, 39 μπι or lower, 38 μιτι or lower, 37 μηι or lower, 36 μπι or lower, 35 μιη or lower, 34 μηι or lower, 33 μιτι or lower, 32 μιτι or lower, 31 μηι or lower, 30 μηη or lower, 29 μιτι or lower, 28 μιη or lower, 27 μηι or lower, 26 μιτΊ or lower, 25 μιη or lower, 24 μιη or lower, 23 μηη or lower, 22 μιτι or lower, 21 μιτι or lower, 20 μιη or lower, 19 μιτι or lower, 18 μπι or lower, 17 μιη or lower, 16 μιτι or lower, 15 μηι or lower, 14 μηη or lower, 13 μηη or lower, 12 μηι or lower, 11 μιη or lower, 10 μιτι or lower, 9 μηι or lower, 8 μηη or lower, 7 μηη or lower, 6 μηη or lower, 5 μιτι or lower, 4 μιη or lower, 3 μπι or lower, 2 μιτι or lower, 1 μηι or lower, 950 nm or lower, 900 nm or lower, 850 nm or lower, 800 nm or lower, 750 nm or lower, 700 nm or lower, 650 nm or lower, 600 nm or lower, 550 nm or lower, 500 nm or lower, 450 nm or lower, 400 nm or lower, 350 nm or lower, 300 nm or lower, 250 nm or lower, 200 nm or lower, 150 nm or lower, 100 nm or lower, 50 nm or lower, 45 nm or lower, 40 nm or lower, 30 nm or lower, 35 nm or lower, 30 nm or lower, 25 nm or lower, 20 nm or lower, 15 nm or lower, 10 nm or lower, 5 nm or lower, 4 nm or lower, 3 nm or lower, 2 nm or lower, or 1 nm or lower.

22. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

21 wherein the Class III PI 3-kinase inhibitor has a highest thermal shift of at least 0.2 °C, for example, a highest thermal shift of at least 0.4 °C, at least 0.6 °C, at least 0.8 °C, at least 1 °C, at least 1.5 °C, at least 2 °C, at least 3 °C, at least 4 °C, at least 5 °C, at least 10 °C, at least 15 °C, at least 20 °C, at least 25 °C, , at least 30 °C, at least 35 °C, at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 70 °C, at least 80 °C, at least 90 °C or at least 100 °C.

23. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to 22 wherein the muscular dystrophy is selected from the group consisting of congenital muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker's muscular dystrophy (BMD, Benign pseudohypertrophic muscular dystrophy), distal muscular dystrophy (distal myopathy), Emery-Dreifuss muscular dystrophy (EDMD), facioscapulohumeral muscular dystrophy (FSHMD, FSHD or FSH), limb-girdle muscular dystrophy (LGMD), myotonic muscular dystrophy, centronuclear myopathies and oculopharyngeal muscular dystrophy.

24. The Class III PI 3-kinase inhibitor for use according to Claim 23 wherein the muscular dystrophy is a congenital muscular dystrophy, for example selected from the group consisting of:

(a) Congenital muscular dystrophy with abnormalities in the extracellular matrix, such as Merosin (laminin a2) deficient CMD (MDC1A) and Collagen VI deficient CMD (Ullrich CMD and Bethlem myopathy);

(b) Dystroglycanopathies (abnormalities of a -dystroglycan), such as Fukuyama-type CMD, Variants of muscle-eye brain disease, Walker-Warburg syndrome, Congenital muscular dystrophy type 1C, Congenital muscular dystrophy type 1 D and Limb-girdle muscular dystrophy 2I;

(c) Defects in the integrin a7 subunit, such as Congenital myopathy with integrin a7 deficiency;

(d) Abnormalities of nuclear envelope proteins, such as L-CMD;

(e) Abnormalities in ER, such as SEPN1 related myopathy (formerly known as Rigid Spine Muscular Dystrophy);

(f) Undiagnosed CMD, including merosin positive; and

(g) Ryanodine receptor gene (RYR1) CMD

25. The Class III PI 3-kinase inhibitor for use according to Claim 24 wherein the muscular dystrophy is Iaminin-a2-deficient congenital muscular dystrophy (Muscular Dystrophy, Congenital Merosin-Deficient, 1a / MDC1A).

26. The Class III PI 3-kinase inhibitor for use according to Claim 24 wherein the muscular dystrophy is not Iaminin-a2-deficient congenital muscular dystrophy (Muscular Dystrophy, Congenital Merosin-Deficient, 1a / MDC1A.

27. The Class III PI 3-kinase inhibitor for use according to Claim 23 wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD).

28. The Class III PI 3-kinase inhibitor for use according to Claim 23 wherein the muscular dystrophy is a distal muscular dystrophy (distal myopathy), for example selected from the group consisting of Miyoshi myopathy, distal myopathy with anterior tibial onset, and Welander distal myopathy.

29. The Class III PI 3-kinase inhibitor for use according to Claim 23 wherein the muscular dystrophy is an Emery-Dreifuss muscular dystrophy (EDMD), for example selected from the group consisting of EDMD1 , EDMD2, EDMD3, EDMD4, EDMD5 and EDMD6.

30. The Class III PI 3-kinase inhibitor for use according to Claim 23 wherein the muscular dystrophy is a facioscapulohumeral muscular dystrophy (FSHMD, FSHD or FSH), for example selected from the group consisting of FSHMD1A (4q35 deletion) and FSHMD1 B.

31. The Class III PI 3-kinase inhibitor for use according to Claim 23 wherein the muscular dystrophy is a Limb-girdle muscular dystrophy or (Erb's muscular dystrophy), for example selected from the group consisting of LGMD1A, LGMD1 B, LGMD1 C, LGMD1 D, LGMD1 E, LGMD1 F, LGMD1 G, LGMD2A, LGMD2B, LGMD2C, LGMD2D, LGMD2E, LGMD2F, LGMD2G, LGMD2H, LGMD2I, LGMD2J, LGMD2K, LGMD2L, LGMD2M, LGMD2N and LGMD20.

32. The Class III PI 3-kinase inhibitor for use according to Claim 23 wherein the muscular dystrophy is a myotonic dystrophy, for example selected from the group consisting of DM1 (also called Steinert's disease) severe congenital form, DM1 childhood-onset form and DM2 (also called proximal myotonic myopathy or PROMM).

33. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to 32 wherein the muscular dystrophy is associated with excessive autophagy (for example excessive macroautophagy, excessive microautophagy and/or excessive chaperone-associated autophagy).

34. The Class III PI 3-kinase inhibitor for use according to Claim 33 wherein the muscular dystrophy is associated with excessive macroautophagy.

35. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

34 wherein the muscular dystrophy is not associated with macroautophagy dysregulation.

36. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

35 wherein the muscular dystrophy is not associated with reduced macroautophagy.

37. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

36 wherein treatment or prevention of muscular dystrophy results in one or more of the following parameters being reduced in the mammal:

i. muscle fibrosis;

ii. muscle atrophy;

iii. muscular apoptosis (caspase-3 positive muscle fibres); iv. collagen III expression;

v. tenascin-C expression;

vi. proportion of muscle fibre cells with centrally locate nuclei and/or vii. laminin alpha-4 expression.

38. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to

37 wherein the one or more parameters are reduced by at least 10%, for example, by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or by 100% relative to the level prior to treatment with the Class III PI 3-kinase inhibitor.

39. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to 38 wherein treatment or prevention of muscular dystrophy results in one or more of the following parameters being increased:

a. muscle regeneration;

b. muscle weight;

c. average muscle fibre diameter;

d. ratio of quadriceps muscle wet weight per body weight

e. lifespan;

f. locomotive function;

g. laminin beta-2 expression;

h. proportion of muscle fibre cells with centrally locate nuclei; i. expression of MyoD1 in satellite cells; and/or

j. expression of eMHC in regenerating muscle fibres.

40. The Class III PI 3-kinase inhibitor for use according to Claim 39 wherein the one or more parameters are increased by at least 10%, for example, by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 450%, 500%, 600%, 700%, 800%, 900% or 1000% relative to the level prior to treatment with the Class III PI 3-kinase inhibitor.

41. The Class III PI 3-kinase inhibitor for use according any one of Claims 12 to 40 wherein treatment or prevention of muscular dystrophy results in Akt phosphorylation at threonine 308 and/or 473 being restored to wild type or near wild type levels, for example, within 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, 0.05% of wild type levels.

42. The Class III PI 3-kinase inhibitor for use according any one of Claims 12 to 41 wherein the inhibitor is for use in combination with a second therapeutic agent or treatment for muscular dystrophy.

43. The Class III PI 3-kinase inhibitor for use according any Claim 42 wherein the second therapeutic agent or treatment comprises: physical therapy, corrective orthopedic surgery and/or steroids; and/or gene replacement, cell therapy and/or anti-apoptosis therapy.

44. The Class III PI 3-kinase inhibitor for use according any Claim 42 wherein the second therapeutic agent or treatment is an inhibitor of the ubiquitin- proteasome system (for example, a proteasome inhibitor).

45. The Class III PI 3-kinase inhibitor for use according any Claim 42 wherein the second therapeutic agent or treatment is an autophagy inhibitor.

46. The Class III PI 3-kinase inhibitor for use according to any one of Claims 12 to 45 wherein the Class III PI 3-kinase inhibitor is for use in the treatment or prevention of muscular dystrophy in a mammal.

47. The Class III PI 3-kinase inhibitor for use according to Claim 46 wherein the mammal is a human.

48. Use of a Class III PI 3-kinase inhibitor comprising or consisting of a compound as defined in any one of Claims 1 to 47 without proviso (ii).

49. A method for treating or preventing muscular dystrophy comprising Class III PI 3-kinase inhibitor comprising or consisting of a compound as defined in any one of Claims 1 to 47 without proviso (ii).

50. A compound as defined in any one of Claims 1 to 11 for use in medicine.

51. A compound as defined in any one of Claims 1 to 11, without proviso (ii), for use treating a disease or condition requiring inhibition of a Class III PI 3-kinase.

52. A compound for use according to Claim 51 wherein the disease or condition requiring inhibition of a Class III PI 3-kinase is selected from the group consisting of muscular dystrophy wherein the disease is cancer, an immune disorder, a cardiovascular disease, a viral infection, inflammation, a metabolism/endocrine function disorder, a neurological disorder, an obstructive airways disease, an allergic disease, an inflammatory disease, immunosuppression, a disorder commonly connected with organ transplantation, an AIDS-related disease, benign prostate hyperplasia, familial adenomatosis, polyposis, neuro-fibromatosis, psoriasis, a bone disorder, atherosclerosis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post- surgical stenosis, restenosis, stroke, diabetes, hepatomegaly, Alzheimer's disease, cystic fibrosis, a hormone-related disease, an immunodeficiency disorder, a destructive bone disorder, an infectious disease, a condition associated with cell death, thrombin-induced platelet aggregation, chronic myelogenous leukaemia, liver disease, a pathologic immune condition involving T cell activation, CNS disorders, and other associated diseases.

A process for the preparation of a compound of formula I as defined in any one of Claims 1 to 11 , which process comprises:

(i) for compounds of formula I in which R2 is present, reaction of a corresponding compound of formula I in which R2 is absent, with a compound of formula II, wherein R2x represents R2 as defined in Claim 1 , and La represents a suitable leaving group;

(ii) for compounds of formula I in which R represents an aryl or heteroaryl group, reaction of a compound of formula III,

in which Lb represents a suitable leaving group, and Ra, Rb, R2 to R5, and C1 to C4 are as defined in Claim 1 , with a compound of formula IV,

IV wherein L° represents a suitable group, and R1w represents R1 is as defined in Claim 1 ; (iii) for compounds of formula I in which R1 represents -N(R30)R31, reaction of a compound of formula III with a compound of formula V,

R1x-H V wherein R x represents R1 as defined in Claim 1 ;

(iv) for compounds of formula I in which R1 represents -OR10 or -SR1 , reaction of a compound of formula III with a compound of formula VI,

R1y-BH VI wherein B represents O or S, and R1y represents R10 or R11, as appropriate, is as defined in Claim 1 ;

(v) for compounds of formula I in which R1 represents a C2-6 alkynyl group, reaction of a compound of formula III with a compound of formula VII,

R1z-C≡CH VII in which R1z represents H or an optionally substituted C1.4 alkyl group, as appropriate;

(vi) for compounds of formula I in which R2 is absent, reaction of a compound of formula VIII,

in which Ld represents a suitable leaving group, and R\ R3, R4, R5, C4, N3 and N4 are as defined in Claim 1 , with a compound of formula IX, in which Ra1 and Rb1 represent Ra and Rb, respectively, as defined in Claim 1 ;

(vii) for compounds of formula I in which R5 is present and is not hydrogen, reaction of a corresponding compound of formula I in which R5 represents hydrogen with a compound of formula X, in which R5x represents R5, as defined in Claim 1 , and Le represents a suitable leaving group;

(viii) for compounds of formula I in which R4 is not hydrogen, reaction of a corresponding compound of formula XI,

in which Lf represents a suitable leaving group, and Ra, Rb, R1 to R3, R5, N4 and C1 to C4 are as defined in Claim 1 , with a compound of formula XII, wherein L9 represents a suitable group, and R4x represents R4 is as defined in Claim 1 ;

(ix) for compounds of formula I in which R2 is absent, and R3 is present and is not hydrogen, reaction of a compound of formula I in which R2 is absent, and R3 is present and is hydrogen with a compound of formula XIII R3x_[_h XIII in which Lh represents a suitable leaving group and R3x represents R3 is as defined in Claim 1.

54. A pharmaceutical formulation including a compound of formula I, as defined in any one of Claims 1 to 11 , or a pharmaceutically acceptable ester, amide, solvate or salt thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

55. A compound substantially as described herein with reference to the description.

56. A Class III PI 3-kinase inhibitor for use substantially as described herein with reference to the description.

57. A use of a Class III PI 3-kinase inhibitor substantially as described herein with reference to the description.

58. A method for treating or preventing muscular dystrophy substantially as described herein with reference to the description.

59. A process for the preparation of a compound of formula I substantially as described herein with reference to the description.

60. A pharmaceutical formulation including a compound of formula I substantially as described herein with reference to the description.

Description:
NOVEL TREATMENTS

Field of Invention

The present invention relates to agents and methods for the treatment and prevention of muscular dystrophy. In particular, the invention provides Class III PI 3-kinase inhibitor compounds of formula I for use in the treatment and prevention of muscular dystrophies, including but not limited to Iaminin-a2-deficient congenital muscular dystrophy and Duchenne muscular dystrophy.

Background

Muscular dystrophy (MD) refers to a group of hereditary muscle diseases that weakens the muscles that move the human body. MDs are characterised by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue. Nine diseases including Duchenne, Becker, limb girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreifuss are always classified as MD but there are more than one hundred diseases in total with similarities to MD.

Most types of MD are multi-system disorders with manifestations in body systems including the heart, gastrointestinal and nervous systems, endocrine glands, skin, eyes and even brain. The condition may also lead to mood swings and learning difficulties. There is no specific treatment for any of the forms of MD. MD may lead to a decline in lung function and therefore assisted ventilation may confer significant clinical benefits in MD patients. Physical therapy to prevent contractures and maintain muscle tone, orthoses (orthopedic appliances used for support) and corrective orthopedic surgery may be needed to improve the quality of life in some cases. The cardiac problems that occur with Emery- Dreifuss muscular dystrophy and myotonic muscular dystrophy may require a pacemaker. The myotonia (delayed relaxation of a muscle after a strong contraction) occurring in myotonic muscular dystrophy may be treated with medications such as quinine, phenytoin, or mexiletine, but no actual long term treatment has been found.

Occupational therapy assists the individual with MD in engaging in his/her activities of daily living (self-feeding, self-care activities, etc.) and leisure activities at the most independent level possible. This may be achieved with use of adaptive equipment or the use of energy conservation techniques. Occupational therapy may implement changes to a person's environment, both at home or work, to increase the individual's function and accessibility. Occupational therapists also address psychosocial changes and cognitive decline which may accompany MD, as well as provide support and education about the disease to the family and individual.

New gene-based therapies for MD are emerging with particular noted advances in using conventional gene replacement strategies, RNA-based technology and pharmacological approaches. However, while the proof of principle has been demonstrated in animal models, success in clinical trials has yet to be demonstrated.

Using the dy3K/dy3K mouse model of laminin a2 chain deficiency and MDC1A patient muscle cells Carmignac et al., 2011 showed that that expression of autophagy-related genes is upregulated in laminin a2 chain deficient muscle and that autophagy inhibition improves the dystrophic dy3K/dy3K phenotype. Systemic injection of 3-methyladenine (3-MA) was shown to reduce muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and weight. Lifespan and locomotive behaviour were also improved. 3-MA as an autophagy inhibitor was first discovered via screening of purine-related substances using isolated hepatocytes from starved rats (Seglen and Gordon (1982) PNAS. 79:1889-1892). Subsequent studies confirmed that 3-MA, together with wortmannin and LY294002, are inhibitors of both class I and Class III PI3K (Blommaart et al., (1997) Eur. J. Biochem. 243, 240-246; Petiot et al., (2000) J. Biol. Chem. 275, 992-998). However, there are several concerns regarding the role of 3-MA as an autophagy inhibitor. For instance, 3- MA, which is usually used at very high concentrations to inhibit autophagy, can target other kinases and affect other cellular processes, such as glycogen metabolism, lysosomal acidification, endocytosis, and mitochondrial permeability transition (Caro et al., (1988) Eur. J. Biochem. 175, 325-329; Punnonen Marjomaki and Reunanen (1994) Eur. J. Cell Biol. 65, 14-25; Xue, Borutaite and Tolkovsky. (2002) Biochem. Pharmacol. 64, 441-449). Moreover, it has been reported earlier that 3-MA can suppress proteolysis even in Atg5-deficient cells, suggesting that its effects on protein degradation extend beyond its role in autophagy inhibition (Mizushimaet al., (2001) J. Cell Biol. 152:657-668). What is more, there currently exits a very limited range of alternative autophagy inhibitors, few (if any) of which are more suitable candidate drugs compared to 3-MA. Accordingly, there exists a need for potent, Class III PI3-kinase-specific and non-transient autophagy inhibitors as effective agents for the treatment and prevention of MD and other diseases.

Summary of Invention

The first aspect of the invention provides a compound of formula I

wherein: when R 2 is present, then the C 1 -C 2 and C 3 -N 2 bonds are double bonds, the N 1 -C 1 and C 2 -C 3 bonds are single bonds, and one of R a and R b is absent; when R 2 is absent, then the C 1 -C 2 and C 3 -N 2 bonds are single bonds, and the N 1 -C 1 and C 2 -C 3 bonds are double bonds; when R 3 is present, then R 5 is absent, the C 4 -N 3 bond is a single bond and the C 4 -N 4 bond is a double bond; when R 3 is absent, then R 5 is present, the C 4 -N 3 bond is a double bond and the C -N 4 bond is a single bond;

C 1 , C 2 , C 3 and C 4 all represent C; N 1 , N 2 , N 3 and N 4 all represent N;

R a and R b independently represent hydrogen or a Ci-12 alkyl group optionally substituted by one or more substituents selected from E 1 ; or

R a and R b are linked together, along with the requisite nitrogen atom to which they are necessarily attached, to form a 3- to 7-membered heterocycloalkyl group, optionally containing one further heteroatom selected from nitrogen, sulfur and oxygen, and which cyclic group is optionally substituted by one or more substituents selected from =0 and E 2 ;

R 1 represents hydrogen, halo, -OR 10 , -SR 1 , -N(R 30 )R 31 , Ci -6 alkyl, C 2 . 6 alkenyl, C 2 - 6 alkynyl, aryl or heteroaryl, which latter five groups are optionally substituted by one or more substituents selected from E 3 ; R 2 may be present or absent and, when present, represents Ci- 6 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from E 4 ;

R 3 may be present or absent and, when present, represents hydrogen or Ci -4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more halo atoms;

R 4 represents hydrogen, -N(R 32 )R 33 , aryl or heteroaryl, which latter two groups are optionally substituted by one or more substituents selected from E 5 ;

R 5 may be present or absent and, when present, represents hydrogen or Ci- 4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from E 6 ; E 1 represents -OR 20 or aryl, which latter group is optionally substituted by one or more substituents selected from Q 1 ;

E 2 represents halo, -OR 21 or -N(R 34 )R 35 ; E 3 represents halo, -OR 22 , -SR 23 , -N(R 36 )R 37 , Ci -6 alkyl or aryl, which latter two groups are optionally substituted by one or more halo atoms; E 4 represents halo, -OR 24 , -N(R 38 )R 39 , or aryl, which latter group is optionally substituted by one or more halo atoms;

E 5 represents halo, -OR 25 , -N(R 40 )R 41 or Ci -6 alkyl which latter group is optionally substituted by one or more halo atoms;

E 6 represents halo or Ci-4 alkyl optionally substituted by one or more halo atoms;

Q 1 represents halo, -OR 26 or Ci-4 alkyl, which latter group is optionally substituted by one or more halo atoms;

R 10 and R 1 independently represent hydrogen, Ci -4 alkyl or aryl, which latter two groups are optionally substituted by one or more substituents selected from halo and -OR 27 ; R 20 to R 27 independently represent hydrogen or Ci -4 alkyl optionally substituted by one or more halo atoms; and

R 30 to R 41 independently represent hydrogen or Ci -4 alkyl optionally substituted by one or more aryl substituents, which aryl group may in turn be optionally substituted by one or more halo atoms; or a pharmaceutically acceptable ester, amide, solvate or salt thereof, provided that:

(i) when R 2 is absent, at least one of R a , R , R 1 , R 3 , R 4 and R 5 is present and is not hydrogen; and

(ii) the compound is not a compound selected from the group consisting of:

3-methyladenine; Phosphatidylinositol 3-Kinase a Inhibitor 2;2-(6-Aminopurin-9- yl)ethanol; 7-methyladenine; 9H-Purin-6-amine,9-(phenylmethyl)- ; 2-Fluoroadenine; 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine; 9-THF-Ade; 2,4-Difluoro-N-[2-methoxy-5- [4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl]benzenesulfonam ide; 6-Fluoro-N-[(4- methoxyphenyl)ethyl]-4-quinazolinamine; 6-Fluoro-N-[(4-fluorophenyl)methyl]-4- quinazolinamine; Hydrochloroquine sulphate; SCHISTOSOMICIDE (lucanthone hydrochloride); Bortezomib (Velcade); CAA0225; LY294002 (2-morpholin-4-yl-8- phenylchromen-4-one); Wortmannin; (3-Pyridinecarboxamide,N-(2,3-dihydro-7,8- dimethoxyimidazo[1 ,2-c]quinazolin-5-yl)-; N-[5-[4-Chloro-3-[(2- hydroxyethyl)sulfamoyl]phenyl]-4-methylthiazol-2-yl]acetamid e; and PI 103 hydrochloride; which compounds, esters, amides, solvates and salts are referred to hereinafter as "the compounds of the invention".

Pharmaceutically-acceptable salts include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze- drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of the invention in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

By "pharmaceutically acceptable ester, amide, solvate or salt thereof, we include salts of pharmaceutically acceptable esters or amides, and solvates of pharmaceutically acceptable esters, amides or salts. For instance, pharmaceutically acceptable esters and amides such as those defined herein may be mentioned, as well as pharmaceutically acceptable solvates or salts. Specific salts that may be mentioned include HCOOH and HCI salts. Oxide salts, such as N-oxides (e.g. in which there is a "N + -0 " " moiety present) may also be mentioned (for instance, when the nitrogen atom is an integral part of the compound of the invention).

Pharmaceutically acceptable esters and amides of the compounds of the invention are also included within the scope of the invention. Pharmaceutically acceptable esters and amides of compounds of the invention may be formed from corresponding compounds that have an appropriate group, for example an acid group, converted to the appropriate ester or amide. For example, pharmaceutically acceptable esters (of hydroxyl-containing compounds of the invention) that may be mentioned include those of the formula R x1 -C(0)0-, in which R x represents optionally substituted Ci -6 alkyl, C 5- io aryl and/or C5-10 aryl-Ci- 6 -alkyl-. Pharmaceutically acceptable amides (of amine-containing compounds of the invention) that may be mentioned include those of the formula R y1 -C(0)N(R z1 )-, in which R y1 represents optionally substituted C1-6 alkyl, C5-10 aryl and/or C5-10 aryl-Ci-e-alkyl-, and R z1 represents an existing substituent (including a hydrogen atom) on the amino portion of the compound of formula I. Particular C1-6 alkyl groups that may be mentioned in the context of such pharmaceutically acceptable esters and amides are non-cyclic Ci -6 alkyl groups, e.g. linear and/or branched Ci-e alkyl groups.

Further compounds of the invention that may be mentioned include carbamate, carboxamido or ureido derivatives, e.g. such derivatives of existing amino functional groups.

For the purposes of this invention, therefore, prodrugs of compounds of the invention are also included within the scope of the invention. The term "prodrug" of a relevant compound of the invention includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)). For the avoidance of doubt, the term "parenteral" administration includes all forms of administration other than oral administration.

Prodrugs of compounds of the invention may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesising the parent compound with a prodrug substituent. Prodrugs include compounds of the invention wherein a hydroxyl, amino, sulfhydryl, carboxy or carbonyl group in a compound of the invention is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxy or carbonyl group, respectively. Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxy functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. "Design of Prodrugs" p. I-92, Elesevier, New York-Oxford (1985). Compounds of the invention may contain double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. Positional isomers may also be embraced by the compounds of the invention. All such isomers (e.g. if a compound of the invention incorporates a double bond or a fused ring, the cis- and transforms, are embraced) and mixtures thereof are included within the scope of the invention (e.g. single positional isomers and mixtures of positional isomers may be included within the scope of the invention). Compounds of the invention may also exhibit tautomerism. All tautomeric forms (or tautomers) and mixtures thereof are included within the scope of the invention. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerisations. Valence tautomers include interconversions by reorganisation of some of the bonding electrons. For example, the following tautomers are included within the sco e of the invention:

Compounds of the invention may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers 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 or epimerisation (i.e. a 'chiral pool' method), by reaction of the appropriate starting material with a 'chiral auxiliary' which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person.

All stereoisomers (including but not limited to diastereoisomers, enantiomers and atropisomers) and mixtures thereof (e.g. racemic mixtures) are included within the scope of the invention.

In the structures shown herein, where the stereochemistry of any particular chiral atom is not specified, then all stereoisomers are contemplated and included as the compounds of the invention. Where stereochemistry is specified by a solid wedge or dashed line representing a particular configuration, then that stereoisomer is so specified and defined. The compounds of the present invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms.

Unless otherwise specified, Ci. q alkyl groups (where q is the upper limit of the range) defined herein may be straight-chain or, when there is a sufficient number (i.e. a minimum of two or three, as appropriate) of carbon atoms, be branched-chain, and/or cyclic (so forming a C 3 -q-cycloalkyl group). Such cycloalkyl groups may be monocyclic or bicyclic and may further be bridged. Further, when there is a sufficient number (i.e. a minimum of four) of carbon atoms, such groups may also be part cyclic.

Unless otherwise stated, the term Ci. q alkylene (where q is the upper limit of the range) defined herein may be straight-chain or, when there is a sufficient number of carbon atoms, be saturated or unsaturated (so forming, for example, an alkenylene or alkynylene linker group). Such Ci -q alkylene groups may be branched (if sufficient number of atoms), but are preferably straight-chained.

C3- q cycloalkyl groups (where q is the upper limit of the range) that may be specifically mentioned may be monocyclic or bicyclic alkyl groups, which cycloalkyl groups may further be bridged (so forming, for example, fused ring systems such as three fused cycloalkyl groups). Substituents may be attached at any point on the cycloalkyl group. Further, where there is a sufficient number (i.e. a minimum of four) such cycloalkyl groups may also be part cyclic.

The term "halo", when used herein, includes fluoro, chloro, bromo and iodo.

Heterocycloalkyl groups that may be mentioned include non-aromatic monocyclic and bicyclic heterocycloalkyl groups in which at least one (e.g. one to four) of the atoms in the ring system is other than carbon (i.e. a heteroatom), and in which the total number of atoms in the ring system is between 3 and 20 (e.g. between three and ten, e.g between 3 and 8, such as 5- to 8-). Such heterocycloalkyl groups may also be bridged. Further, such heterocycloalkyl groups may be saturated or unsaturated containing one or more double and/or triple bonds, forming for example a C 2 - q heterocycloalkenyl (where q is the upper limit of the range) group. C2- q heterocycloalkyl groups that may be mentioned include 7- azabicyclo[2.2.1]heptanyl, 6-azabicyclo[3.1.1]heptanyl, 6-azabicyclo[3.2.1]-octanyl, 8- azabicyclo-[3.2.1]octanyl, aziridinyl, azetidinyl, dihydropyranyl, dihydropyridyl, dihydropyrrolyl (including 2,5-dihydropyrrolyl), dioxolanyl (including 1 ,3-dioxolanyl), dioxanyl (including 1 ,3- dioxanyl and 1 ,4-dioxanyl), dithianyl (including 1 ,4-dithianyl), dithiolanyl (including 1 ,3- dithiolanyl), imidazolidinyl, imidazolinyl, morpholinyl, 7-oxabicyclo[2.2.1]heptanyl, 6- oxabicyclo-[3.2.1]octanyl, oxetanyl, oxiranyl, piperazinyl, piperidinyl, non-aromatic pyranyl, pyrazolidinyl, pyrrolidinonyl, pyrrolidinyl, pyrrolinyl, quinuclidinyl, sulfolanyl, 3-sulfolenyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydropyridyl (such as 1 ,2,3,4-tetrahydropyridyl and 1 ,2,3,6-tetrahydropyridyl), thietanyl, thiiranyl, thiolanyl, thiomorpholinyl, trithianyl (including 1 ,3,5-trithianyl), tropanyl and the like. Particular heterocycloalkyi groups that may be mentioned include morpholinyl (such as 4-morpholinyl) groups. Substituents on heterocycloalkyi groups may, where appropriate, be located on any atom in the ring system including a heteroatom. The point of attachment of heterocycloalkyi groups may be via any atom in the ring system including (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbocyclic ring that may be present as part of the ring system. Heterocycloalkyi groups may also be in the N- or S- oxidised form. Heterocycloalkyi mentioned herein may be stated to be specifically monocyclic or bicyclic.

For the avoidance of doubt, the term "bicyclic" (e.g. when employed in the context of heterocycloalkyi groups) refers to groups in which the second ring of a two-ring system is formed between two adjacent atoms of the first ring. The term "bridged" (e.g. when employed in the context of cycloalkyi or heterocycloalkyi groups) refers to monocyclic or bicyclic groups in which two non-adjacent atoms are linked by either an alkylene or heteroalkylene chain (as appropriate).

Aryl groups that may be mentioned include Ce-20, such as Ce-12 (e.g. Ce-io) aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 12 (e.g. 6 and 10) ring carbon atoms, in which at least one ring is aromatic. Ce-io aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl. The point of attachment of aryl groups may be via any atom of the ring system. For example, when the aryl group is polycyclic the point of attachment may be via atom including an atom of a non-aromatic ring. However, when aryl groups are polycyclic (e.g. bicyclic or tricyclic), they are preferably linked to the rest of the molecule via an aromatic ring.

Unless otherwise specified, the term "heteroaryl" when used herein refers to an aromatic group containing one or more heteroatom(s) (e.g. one to four heteroatoms) preferably selected from N, O and S. Heteroaryl groups include those which have between 5 and 20 members (e.g. between 5 and 10) and may be monocyclic, bicyclic or tricyclic, provided that at least one of the rings is aromatic (so forming, for example, a mono-, bi-, or tricyclic heteroaromatic group). When the heteroaryl group is polycyclic the point of attachment may be via any atom including an atom of a non-aromatic ring. However, when heteroaryl groups are polycyclic (e.g. bicyclic or tricyclic), they are preferably linked to the rest of the molecule via an aromatic ring. Heteroaryl groups that may be mentioned include 3,4-dihydro-1H- isoquinolinyl, 1 ,3-dihydroisoindolyl, 1,3-dihydroisoindolyl (e.g. 3,4-dihydro-1W-isoquinolin-2- yl, 1 ,3-dihydroisoindol-2-yl, 1 ,3-dihydroisoindol-2-yl; i.e. heteroaryl groups that are linked via a non-aromatic ring), or, preferably, acridinyl, benzimidazolyl, benzodioxanyl, benzodioxepinyl, benzofuranyl, benzofurazanyl, benzothiadiazolyl (including 2,1 ,3- benzothiadiazolyl), benzothiazolyl, benzoxadiazolyl (including 2,1 ,3-benzoxadiazolyl), benzoxazinyl (including 3,4-dihydro-2H-1 ,4-benzoxazinyl), benzoxazolyl, benzomorpholinyl, benzoselenadiazolyl (including 2,1,3-benzoselenadiazolyl), benzothienyl, carbazolyl, chromanyl, cinnolinyl, furanyl, imidazolyl, imidazo[1 ,2-a]pyridyl, indazolyl, indolinyl, indolyl, isobenzofuranyl, isochromanyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiaziolyl, isothiochromanyl, isoxazolyl, naphthyridinyl (including 1 ,6-naphthyridinyl or, particularly, benzodioxolyl (including 1 ,3-benzodioxolyl), 1 ,5-naphthyridinyl and 1 ,8-naphthyridinyl), oxadiazolyl (including 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl and 1 ,3,4-oxadiazolyl), oxazolyl, phenazinyl, phenothiazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolizinyl, quinoxalinyl, tetrahydroisoquinolinyl (including 1 ,2,3,4-tetrahydroisoquinolinyl and 5,6,7,8- tetrahydroisoquinolinyl), tetrahydroquinolinyl (including 1 ,2,3,4-tetrahydroquinolinyl and 5,6,7,8-tetrahydroquinolinyl), tetrazolyl, thiadiazolyl (including 1 ,2,3-thiadiazolyl, 1 ,2,4- thiadiazolyl and 1 ,3,4-thiadiazolyl), thiazolyl, thiochromanyl, thiophenetyl, thienyl, triazolyl (including 1 ,2,3-triazolyl, 1 ,2,4-triazolyl and 1 ,3,4-triazolyl) and the like. Substituents on heteroaryl groups may, where appropriate, be located on any atom in the ring system including a heteroatom. Heteroaryl groups may also be in the N- or S- oxidised form. Heteroaryl groups mentioned herein may be stated to be specifically monocyclic or bicyclic.

It may be specifically stated that the heteroaryl group is monocyclic or bicyclic. In the case where it is specified that the heteroaryl is bicyclic, then it may consist of a five-, six- or seven- membered monocyclic ring (e.g. a monocyclic heteroaryl ring) fused with another a five-, six- or seven-membered ring (e.g. a monocyclic aryl or heteroaryl ring).

Heteroatoms that may be mentioned include phosphorus, silicon, boron and, particularly, oxygen, nitrogen and sulfur.

For the avoidance of doubt, where it is stated herein that a group (e.g. a C1-12 alkyl group) may be substituted by one or more substituents (e.g. selected from E 1 ), then those substituents (e.g. defined by E 1 ) are independent of one another. That is, such groups may be substituted with the same substituent (e.g. defined by E 1 ) or different substituents (defined by E 1 ). For the avoidance of doubt, in cases in which the identity of two or more substituents in a compound of the invention may be the same, the actual identities of the respective substituents are not in any way interdependent. For example, in the situation in which there is more than one e.g. E 1 to E 5 (such as E 3 ) substituent present, then those E 1 to E 5 (e.g. E 3 ) substituents may be the same or different. Also, when e.g. there are two -OR 20 substituents present, then those -OR 20 groups may be the same or different (i.e. each R 20 group may be the same or different).

For the avoidance of doubt, when a term such as "E 1 to E 5 " is employed herein, this will be understood by the skilled person to mean E 1 , E 2 , E 3 , E 4 , and E 5 , inclusively.

All individual features (e.g. preferred features) mentioned herein may be taken in isolation or in combination with any other feature (including preferred feature) mentioned herein (hence, preferred features may be taken in conjunction with other preferred features, or independently of them).

The skilled person will appreciate that compounds of the invention that are the subject of this invention include those that are stable. That is, compounds of the invention include those that are sufficiently robust to survive isolation from e.g. a reaction mixture to a useful degree of purity.

In an embodiment of the invention, there is provided a compound of formula I wherein, when R 2 is absent, at least one of R a , R b , R\ R 3 , R 4 and R 5 is not hydrogen.

In particular embodiments of the invention, R 4 represents hydrogen and one of R 3 and R 5 represents hydrogen while the other one of R 3 and R 5 is absent.

In a further embodiment of the invention, at least one of R a , R , R and R 2 is present and is not hydrogen. In a yet further embodiment of the invention at least one of R 3 to R 5 is present and is not hydrogen. In certain embodiments of the invention, at least one of R A and R B is present and represents a group other than hydrogen.

In particular embodiments in which at least one of R A and R B is present and represents a group other than hydrogen:

R A and R B independently represent hydrogen or a C M2 alkyl group optionally substituted by one or more substituents selected from E\ provided that at least one of R A and R B is present and represents a group other than hydrogen; or

R A and R B are linked together, along with the requisite nitrogen atom to which they are necessarily attached, to form a 3- to 7-membered heterocycloalkyl group, optionally containing one further heteroatom selected from nitrogen, sulfur and oxygen, and which cyclic group is optionally substituted by one or more substituents selected from halo and -OR 21 .

For example, in particular embodiments of the invention in which at least one of R A and R B is present and represents a group other than hydrogen, the compound of formula I may be represented as a compound of formula IA,

wherein: when R 2 is present, then the C 1 -C 2 and C 3 -N 2 bonds are double bonds, the N 1 -C 1 and C -C 3 bonds are single bonds, and one of R A and R B is absent; when R 2 is absent, then the C -C 2 and C 3 -N 2 bonds are single bonds, and the N -C 1 and C 2 -C 3 bonds are double bonds; C\ C 2 and C 3 all represent C;

N and N 2 represent N; R a and R independently represent hydrogen or a C1-12 alkyl group optionally substituted by one or more substituents selected from E\ provided that at least one of R a and R b is present and represents a group other than hydrogen; or

R a and R are linked together, along with the requisite nitrogen atom to which they are necessarily attached, to form a 3- to 7-membered heterocycloalkyl group, optionally containing one further heteroatom selected from nitrogen, sulfur and oxygen, and which cyclic group is optionally substituted by one or more substituents selected from halo and

-OR 21 ; R 1 represents hydrogen or halo;

R 2 may be present or absent and, when present, represents C1-6 alkyl optionally substituted by one or more halo atoms; E represents -OR 20 or aryl, which latter group is optionally substituted by one or more substituents selected from Q 1 ;

Q represents halo, -OR 26 or C1-4 alkyl, which latter group is optionally substituted by one or more halo atoms; and

R 20 , R 21 and R 26 independently represent hydrogen or Ci-4 alkyl optionally substituted by one or more halo atoms.

Particular compounds of formulae IA that may be mentioned include those in which R 2 is absent.

In other embodiments of the invention, R 1 represents a group other than hydrogen.

In particular embodiments in which R 1 represents a group other than hydrogen, R represents -OR 10 , -N(R 30 )R 31 , Ci -6 alkyl, C2-6 alkenyl, C 2 -e alkynyl, aryl or heteroaryl, which latter five groups are optionally substituted by one or more substituents selected from E 3 , wherein R 10 , R 30 , R 31 and E 3 are as defined above in respect of compounds of formula I. For example, in particular embodiments of the invention in which R 1 represents a group other than hydrogen, the compound of formula I may be represented as a compound of formula IB,

wherein: when R 2 is present, then the C 1 -C 2 and C 3 -N 2 bonds are double bonds, the N -C 1 and C 2 -C 3 bonds are single bonds, and R b is absent; when R 2 is absent, then the C 1 -C 2 and C 3 -N 2 bonds are single bonds, and the N 1 -C 1 and C 2 -C 3 bonds are double bonds; C 1 , C 2 and C 3 all represent C;

N 1 and N 2 represent N;

R b , if present, represents hydrogen;

R 1 represents -OR 10 , -SR 11 , -N(R 30 )R 31 , Ci -6 alkyl, C 2 - 6 alkenyl, C 2 - 6 alkynyl, aryl or heteroaryl, which latter five groups are optionally substituted by one or more substituents selected from

E 3 ; R 2 may be present or absent and, when present, represents Ci-e alkyl optionally substituted by one or more halo atoms;

E 3 represents halo, -OR 22 , -SR 23 , -N(R 36 )R 37 , Ci -6 alkyl or aryl, which latter two groups are optionally substituted by one or more halo atoms; R 10 and R 11 independently represent aryl optionally substituted by one or more substituents selected from halo and -OR 27 ;

R 22 , R 23 and R 27 independently represent hydrogen or C1- alkyl optionally substituted by one or more halo atoms; and

R 30 , R 3 , R 36 and R 37 independently represent hydrogen or C1-4 alkyl optionally substituted by one or more aryl substituents. Particular compounds of formulae IB that may be mentioned include those in which R 2 is absent.

In other embodiments of the invention, R 2 is present. In particular embodiments in which R 2 is present, R 2 represents C1-6 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from halo or -OR 24 , wherein R 24 is as defined above in respect of compounds of formula I. In other particular embodiments in which R 2 is present, R 1 represents hydrogen.

For example, in particular embodiments of the invention in which R 2 is present, the compound of formula I may be represented as a compound of formula IC,

wherein:

R 1 represents hydrogen, halo; R 2 represents d -6 alkyl (e.g. C2-6 alkyl) optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from halo or -OR 24 ; and R 24 represents hydrogen or C1-4 alkyl optionally substituted by one or more halo atoms.

Particular compounds of formula IC that may be mentioned include those in which R represents hydrogen. In other embodiments of the invention, R 3 is present and represents a group other than hydrogen.

In particular embodiments in which R 3 is present and represents a group other than hydrogen, R 3 represents C1-4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more halo atoms.

For example, in particular embodiments of the invention in which R 3 is present and represents a group other than hydrogen, the compound of formula I may be represented as a compound of formula ID,

wherein:

R 2 represents C1.6 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more halo atoms; and

R 3 represents Ci -4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more halo atoms. In other embodiments of the invention, R 4 represents a group other than hydrogen. In particular embodiments in which R 4 represents a group other than hydrogen, R 4 represents -N(R 32 )R 33 , aryl or heteroaryl, which latter two groups are optionally substituted by one or more substituents selected from E 5 , wherein E 5 is as defined above in respect of compounds of formula I. For example, in particular embodiments of the invention in which R 4 represents a group other than hydrogen, the compound of formula I may be represented as a compound of formula IE,

wherein: when R 2 is present, then the C 1 -C 2 and C 3 -N 2 bonds are double bonds, the N 1 -C 1 and C 2 -C 3 bonds are single bonds, and one of R a and R is absent; when R 2 is absent, then the C 1 -C 2 and C 3 -N 2 bonds are single bonds, and the N 1 -C 1 and C 2 -C 3 bonds are double bonds;

C 1 , C 2 and C 3 all represent C; N 1 and N 2 represent N;

R , if present, represents hydrogen;

R 2 may be present or absent and, when present, represents Ci-e alkyl optionally substituted by one or more halo atoms; R 4 represents -N(R 32 )R 33 , aryl or heteroaryl, which latter two groups are optionally substituted by one or more substituents selected from E 5 ; E 5 represents halo, -OR 25 or Ci -4 alkyl, which latter group is optionally substituted by one or more halo atoms;

R 25 represents hydrogen or Ci -4 alkyl optionally substituted by one or more halo atoms; and R 32 and R 33 independently represent hydrogen or Ci- alkyl.

In other embodiments of the invention, R 5 is present and represents a group other than hydrogen.

In particular embodiments in which R 5 is present and represents a group other than hydrogen, R 5 represents Ci- 4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from E 6 , wherein E 6 is as defined above in respect of compounds of formula I.

For example, in particular embodiments of the invention in which R 5 is present and represents a group other than hydrogen, the compound of formula I may be represented as a compound of formula IC,

wherein: R 1 represents hydrogen or halo; R 2 represents Ci-e alkyl optionally substituted by one or more substituents selected from halo and aryl;

R 5 represents C1.4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more substituents selected from E 6 ; and

E 6 represents halo or C1-4 alkyl optionally substituted by one or more halo atoms. In other particular embodiments of the invention in which at least one of R A and R B is present and represents a group other than hydrogen, the compound of formula I may be represented as a compound of formula IG,

wherein:

R A and R B independently represent hydrogen or a CM 2 alkyl group optionally substituted by one or more substituents selected from E 1 , provided that at least one of R A and R is present and represents a group other than hydrogen; or

R A and R are linked together, along with the requisite nitrogen atom to which they are necessarily attached, to form a 3- to 7-membered heterocycloalkyl group, optionally containing one further heteroatom selected from nitrogen, sulfur and oxygen, and which cyclic group is optionally substituted by one or more substituents selected from halo and

-OR 21 ;

R represents halo; R 3 represents C1-4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more halo atoms;

E 1 represents -OR 20 or aryl, which latter group is optionally substituted by one or more substituents selected from Q 1 ;

Q 1 represents halo, -OR 26 or C1-4 alkyl, which latter group is optionally substituted by one or more halo atoms; and R 20 , R 21 and R 26 independently represent hydrogen or C1-4 alkyl optionally substituted by one or more halo atoms.

In other particular embodiments of the invention in which R 1 represents a group other than hydrogen, the compound of formula I may be represented as a compound of formula IH,

wherein:

R 1 represents halo, -OR 10 , -SR 1 , -N(R 30 )R 31 , Ci -6 alkyl, C 2-6 alkenyl, C 2 - 6 alkynyl, aryl or heteroaryl, which latter five groups are optionally substituted by one or more substituents selected from E 3 ;

R 3 represents C1.4 alkyl optionally substituted by one or more substituents selected from halo and aryl, which aryl group may in turn be optionally substituted by one or more halo atoms;

E 3 represents halo, -OR 22 , -SR 23 , -N(R 36 )R 37 , Ci- 6 alkyl or aryl, which latter two groups are optionally substituted by one or more halo atoms; R 10 and R 11 independently represent aryl optionally substituted by one or more substituents selected from halo and -OR 27 ;

R 22 , R 23 and R 27 independently represent hydrogen or C1-4 alkyl optionally substituted by one or more halo atoms; and

R 30 , R 31 , R 36 and R 37 independently represent hydrogen or C1-4 alkyl optionally substituted by one or more aryl substituents.

More particular compounds of the invention that may be mentioned include compounds of formulae IA, IB, IC, ID, IE and IF.

Other particular compounds of the invention that may be mentioned include compounds of formulae IA, IB, IC, ID, IE, IF, IG and IH.

In addition, other particular compounds of the invention that may be mentioned include compounds of formulae IA (in which R 2 is absent), IB (in which R 2 is absent), IC (in which R 1 represents hydrogen), ID, IE, IF, IG and IH.

Still more particular compounds of the invention include those of the examples described hereinafter.

MDP-027 2-Chloro-N-ethyl-9-methyl-9H-purin-6-amine (TFA salt)

MDP-028 2-Chloro-N-[(2-methoxyphenyl)methyl]-9-methyl-9H-purin-6-ami ne

MDP-029 2-Chloro-N-(2-methoxyethyl)-9-methyl-9H-purin-6-amine (TFA salt)

MDP-030 2-Chloro-9-methyl-N-propyl-9H-purin-6-amine (TFA salt)

MDP-031 9-Benzyl-3-methyl-6,9-dihydro-3H-purin-6-imine

MDP-032 2-(2H-1 ,3-benzodioxol-5-yl)-9-methyl-9H-purin-6-amine

MDP-033 2-(4- ethoxyphenyl)-9H-purin-6-amine

MDP-034 2-(4-Fluorophenyl)-9H-purin-6-amine

MDP-035 2-Chloro-9-methyl-9H-purin-6-amine (TFA salt)

MDP-040 3-Ethyl-8-(4-methoxyphenyl)-3H-purin-6-amine

MDP-041 8-(4-Methoxyphenyl)-3-methyl-3H-purin-6-amine

MDP-042 8-(4-methoxyp enyl)-9H-purin-6-amine

MDP-043 8-(4-Ethylphenyl)-3-methyl-3H-purin-6-amine

MDP-044 8-(4-ethylphenyl)-9H-purin-6-amine

MDP-045 8-[3-(Trifluoromethyl)phenyl]-9H-purin-6-amine

MDP-046 3-Ethyl-3H-purin-6-amine

MDP-047 3-(Propan-2-yl)-3H-puriri-6-amine

MDP-048 3-Benzyl-3H-purin-6-amine

MDP-049 3,9-Dibenzyl-6,9-dihydro-3H-purin-6-imine

MDP-050 9-Methyl-2-(4-methylphenyl)-9H-purin-6-amine

MDP-051 3-Propyl-3H-purin-6-amine

MDP-052 9-Benzyl-3-propyl-6,9-dihydro-3H-purin-6-imine

MDP-053 9-Methyl-2-(2-phenylethynyl)-9H-purin-6-amine

MDP-054 2-(4-Ethylphenyl)-9-methyl-9H-purin-6-amine

MDP-055 2-(3,5-Difluorophenyl)-9-methyl-9H-purin-6-amine

MDP-056 9-Methyl-2-[4-(methylsulfanyl)phenyl]-9H-purin-6-amine

MDP-057 2-(4-tert-Butylphenyl)-9-methyl-9H-purin-6-amine

MDP-058 9-Methyl-2-[3-(trifluoromethyl)phenyl]-9H-purin-6-amine

MDP-059 2-(4-fluorophenyl)-9-methyl-9H-purin-6-amine

MDP-060 9-Benzyl-3-ethyl-6,9-dihydro-3H-purin-6-imine

MDP-061 2-N-Benzyl-9-methyl-9H-purine-2,6-diamine

MDP-062 2-(6-Amino-9-methyl-9H-purin-2-yl)phenol

MDP-063 9-Methyl-2-phenoxy-9H-purin-6-amine

MDP-064 6-amino-2-chloro-9-ethyl-7-{[4-(trifluoromethyl)phenyl]methy l}-9H-purin-7-ium

MDP-065 2-(4-Methoxyphenoxy)-9-methyl-9H-purin-6-amine MDP-066 2-(3,5-Difluorophenyl)-9-ethyl-9H-purin-6-amine

MDP-067 8-(2H-1 ,3-Benzodioxol-5-yl)-3-methyl-3H-purin-6-amine

MDP-068 8-(4-tert-Butylphenyl)-9H-purin-6-amine

MDP-069 8-Phenyl-9H-purin-6-amine

MDP-070 8-(4-tert-Butylphenyl)-3-methyl-3H-purin-6-amine

MDP-071 3-Methyl-8-phenyl-3H-purin-6-amine

MDP-072 8-N-Ethyl-3,8-N-dimethyl-3H-purine-6,8-diamine

2-Chloro-N-propyl-9H-purin-6-amine

2-Chloro-N-[(2-methoxyphenyl)methyl]-9H-purin-6-amine

8-(2H-1 ,3-Benzodioxol-5-yl)-9H-purin-6-amine

The following compounds may be commercially available.

Compound Name (CAS name in parenthesis)

MDP-001 3-methyladenine (5142-23-4)

MDP-002 Phosphatidylinositol 3-Kinase a Inhibitor 2 (371943-05-4)

MDP-003 2-(6-Aminopurin-9-yl)ethanol (707-99-3)

MDP-004 7-methyladenine (935-69-3)

MDP-005 9H-Purin-6-amine,9-(phenylmethyl)- (4261-14-7)

MDP-006 2-Fluoroadenine (700-49-2)

MDP-007 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine; 9-THF-Ade (17318-31-9)

2,4-Difluoro-N-[2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl] -3-

MDP-008 pyridinyljbenzenesulfonamide (1086062-66-9)

MDP-009 6-Fluoro-N-[(4-methoxyphenyl)ethyl]-4-quinazolinamine (1262888-42-5)

MDP-010 6-Fluoro-N-[(4-fluorophenyl)methyl]-4-quinazolinamine (1262888-28-7)

MDP-011 Hydrochloroquine sulphate (747-36-4)

MDP-012 SCHISTOSOMICIDE (lucanthone hydrochloride) (548-57-2)

MDP-013 Bortezomib (Velcade) (179324-69-7)

MDP-014 CAA0225 (244072-26-2)

MDP-036 Wortmannin (19545-26-7, 1405-03-4)

(3-Pyridinecarboxamide,N-(2,3-dihydro-7,8-dimethoxyimidazo[1 ,2-c]quinazolin- 5-yl)-

MDP-037 (677338-12-4)

N-[5-[4-Chloro-3-[(2-hydroxyethyl)sulfamoyl]phenyl]-4-methyl thiazol-2-

MDP-038 yljacetamide (593960-11-3)

MDP-039 PI-103 hydrochloride (371935-74-9)

Adenine In particular embodiments, the compounds of the invention do not include any commercially available compounds and, in particular, do not include any of the compounds listed above.

Throughout this specification, structures may or may not be presented with chemical names. Where any question arises as to nomenclature, the structure prevails. Where it is possible for a compound to exist as a tautomer the depicted structure represents one of the possible tautomeric forms, wherein the actual tautomeric form(s) observed may vary depending on environmental factors such as solvent, temperature or pH. Compounds of formula I may be prepared in accordance with techniques that are well known to those skilled in the art, for example as described hereinafter.

According to a further embodiment of the invention there is provided a process for the preparation of a compound of formula I, which process comprises:

(i) for compounds of formula I in which R 2 is present, and particularly for compounds in which R 2 is present and R 1 represents hydrogen, reaction of a corresponding compound of formula I in which R 2 is absent (or a compound of formula I in which R 2 is present and R 1 represents hydrogen, as appropriate), with a compound of formula II,

R 2 *-L a II wherein R represents R 2 as defined above, and L a represents a suitable leaving group such as chloro, bromo, iodo, a sulfonate group (e.g. -OS(0) 2 CF 3 , -OS(0) 2 CH 3 , -OS(0) 2 PhMe or a nonaflate), or a similar group known to the skilled person, under reaction conditions known to those skilled in the art, for example optionally in the presence of an appropriate metal catalyst (or a salt or complex thereof) such as Cu, Cu(OAc)2, Cul (or Cul/diamine complex), copper tris(triphenyl-phosphine)bromide, Pd(OAc)2, tris(dibenzylideneacetone)- dipalladium(O) (Pd 2 (dba) 3 ) or NiCI 2 and an optional additive such as Ph 3 P, 2,2'- bis(diphenylphosphino)-1 ,1'-binaphthyl, xantphos, Nal or an appropriate crown ether such as 18-crown-6-benzene, in the presence of an appropriate base such as NaH, Et 3 N, pyridine, Ν,Ν'-dimethylethylenediamine, Na 2 C0 3 , K 2 C0 3 , K 3 P0 4 , Cs 2 C0 3 , t-BuONa or t-BuOK (or a mixture thereof, optionally in the presence of 4A molecular sieves), in a suitable solvent (e.g. dichloromethane, dioxane, toluene, ethanol, isopropanol, dimethylformamide, ethylene glycol, ethylene glycol dimethyl ether, water, dimethylsulfoxide, acetonitrile, dimethylacetamide, N-methylpyrrolidinone, tetrahydrofuran or a mixture thereof) or in the absence of an additional solvent when the reagent may itself act as a solvent. This reaction may be carried out at room temperature or above (e.g. at a high temperature, such as the reflux temperature of the solvent system that is employed) or using microwave irradiation;

(ii) for compounds of formula I in which R 1 represents an aryl or heteroaryl group, reaction of a compound of formula III,

in which L b represents a suitable leaving group such as chloro, bromo, iodo, or a sulfonate group (e.g. -OS(0) 2 CF 3 , -OS(0) 2 CH 3 or -OS(0) 2 PhMe), and R a , R b , R 2 to R 5 , N 1 to N 4 and C to C 4 are as hereinbefore defined, with a compound of formula IV,

R 1w. L c IV wherein L c represents a suitable group, such as -B(OH) 2 , -B(OR x ) 2 , -BF 3 K or -Sn(R x ) 3 , in which each R x independently represents a Ci-e alkyl group, or, in the case of -B(OR x ) 2 , the respective R groups may be linked together to form a 4- to 6-membered cyclic group (such as a 4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl group), thereby forming e.g. a pinacolato boronate ester group, (or L c may represent iodo, bromo or chloro, provided that L b and L c are mutually compatible) and R 1w represents R 1 is as hereinbefore defined. The reaction may be performed, for example in the presence of a suitable catalyst system, e.g. a metal (or a salt or complex thereof) such as Pd, Cul, Pd/C, PdCI 2 , Pd(OAc) 2 , Pd(Ph 3 P) 2 CI 2 , Pd(Ph 3 P) 4 (i.e. palladium tetrakistriphenylphosphine), Pd 2 (dba) 3 and/or NiCI 2 (preferred catalysts include palladium) and a ligand such as PdCI 2 (dppf).DCM, i-Bu 3 P, (C 6 Hn) 3 P, Ph 3 P, AsPh 3 , P(o-Tol) 3 , 1 ,2-bis(diphenylphosphino)ethane, 2,2'-bis(di-tert-butylphosphino)-1 , 1 '-biphenyl,

2,2'-bis(diphenylphosphino)-1 , 1 '-bi-naphthyl, 1 , 1 '-bis(diphenyl-phosphino-ferrocene),

1 ,3-bis(diphenylphosphino)propane, xantphos, or a mixture thereof (preferred ligands include PdCI 2 (dppf).DCM), together with a suitable base such as, Na 2 C0 3 , K 3 P0 4 , Cs 2 C0 3 , NaOH, KOH, K 2 C0 3 , CsF, Et 3 N, (/-Pr) 2 NEt, f-BuONa or f-BuOK (or mixtures thereof; preferred bases include Na 2 C0 3 and K 2 C0 3 ) in a suitable solvent such as dioxane, toluene, ethanol, dimethylformamide, dimethoxyethane, ethylene glycol dimethyl ether, water, dimethylsulfoxide, acetonitrile, dimethylacetamide, A/-methylpyrrolidinone, tetrahydrofuran or mixtures thereof (preferred solvents include dimethylformamide and dimethoxyethane). The reaction may be carried out for example at room temperature or above (e.g. at a high temperature such as at about the reflux temperature of the solvent system). Alternative reaction conditions include microwave irradiation conditions, for example at elevated temperature of about 130°C;

(iii) for compounds of formula I in which R 1 represents -N(R 30 )R 31 , reaction of a compound of formula III, as defined above, with a compound of formula V,

R x -H V wherein R 1x represents R 1 as hereinbefore defined, under reaction conditions known to those skilled in the art, such as those described hereinbefore in respect of process step (i) above;

(iv) for compounds of formula I in which R 1 represents -OR 10 or -SR 11 , reaction of a compound of formula III, as defined above, with a compound of formula VI, R 1y -BH VI wherein B represents O or S, and R y represents R 10 or R 11 , as appropriate, is as hereinbefore defined, under reaction conditions known to those skilled in the art, such as those described hereinbefore in respect of process step (i) above;

(v) for compounds of formula I in which R 1 represents a C2-6 alkynyl group, reaction of a compound of formula III, as defined above, with a compound of formula VII,

R 1z -C≡CH VII in which R 1z represents H or a C1-4 alkyl group (optionally substituted as hereinbefore defined with respect to C2-6 alkynyl groups of R 1 ), as appropriate, optionally in the presence of an appropriate catalyst system (e.g. a palladium catalyst, such as PdC , Pd(OAc)2, Pd(Ph 3 P)2CI 2 , Pd(Ph 3 P)4, Pd 2 (dba) 3 or the like; and a metal halide, for example copper iodide) and a suitable base (for example sodium hydride, sodium bicarbonate, potassium carbonate, pyrrolidinopyridine, pyridine, triethylamine, tributylamine, trimethylamine, dimethylaminopyridine, diisopropylamine, diisopropylethylamine, 1 ,8- diazabicyclo[5.4.0]undec-7-ene, sodium hydroxide, A/-ethyldiisopropylamine, N- (methylpolystyrene)-4-(methylamino)pyridine, potassium bis(trimethylsilyl)amide, sodium bis(trimethylsilyl)amide, potassium tert-butoxide, lithium diisopropylamide, lithium 2,2,6,6- tetramethylpiperidine or mixtures thereof, and an appropriate solvent (e.g. tetrahydrofuran, pyridine, toluene, dichloromethane, chloroform, acetonitrile, dimethylformamide, dimethylsulfoxide, trifluoromethylbenzene, dioxane or triethylamine), performed at around room temperature or above (e.g. up to 40-180°C), under conditions known to those skilled in the art. The skilled person will appreciate that the compound so formed may be isolated by precipitation or crystallisation (from e.g. n-hexane) and purified by recrystallisation techniques (e.g. from a suitable solvent such as THF, hexane (e.g. n-hexane), methanol, dioxane, water, or mixtures thereof);

(vi) for compounds of formula I in which R 2 is absent, reaction of a compound of formula VIII,

in which L d represents a suitable leaving group such as chloro, bromo, iodo, a sulfonate group (e.g. -OS(0) 2 CF 3 , -OS(0) 2 CH 3 , -OS(0) 2 PhMe or a nonaflate), -B(OH) 2 , -B(OR y ) 2l -Sn(R y )3, -BF 3 K or diazonium salts, in which each R y independently represents a Ci-e alkyl group, or, in the case of -B(OR y ) 2 , the respective R y groups may be linked together to form a 4- to 6-membered cyclic group (such as a 4,4,5,5-tetramethyl-1 ,3,2-dioxaborolan-2-yl group), and R 1 , R 3 , R 4 , R 5 , C 4 , N 3 and N 4 are as defined above, with a compound of formula IX,

HN(R a1 )R' IX in which R a1 and R b1 represent R a and R b as defined above, respectively, under reaction conditions known to those skilled in the art, such as those described hereinbefore in respect of process step (i) above; (vii) for compounds of formula I in which R 5 is present and is not hydrogen, reaction of a corresponding compound of formula I in which R 5 represents hydrogen with a compound of formula X,

R s *-U in which R 5x represents R 5 , as defined above, and L e represents a suitable leaving group such as chloro, bromo, iodo, or a sulfonate group (e.g. -OS(0)2CF3, -OS(0) 2 CH3 or -OS(0) 2 PhMe), under reaction conditions known to those skilled in the art, such as those described hereinbefore in respect of process step (i) above;

(viii) for compounds of formula I in which R 4 is not hydrogen, reaction of a corresponding compound of formula XI,

in which L f represents a suitable leaving group such as that defined above in respect of L b , and R a , R b , R 1 to R 3 , R 5 , N 1 to N 4 and C 1 to C 4 are as hereinbefore defined, with a compound of formula XII

R 4x_ L g XII wherein L 9 represents a suitable group, such as -B(OH) 2 , -B(OR x ) 2 , -BF 3 K or -Sn(R x ) 3 , in which R is as defined above, (or L 9 may represent iodo, bromo or chloro, provided that L f and L 9 are mutually compatible), or L 9 represents H in the case where R 4 represents -N(R 32 )R 33 , and R 4x represents R 4 is as hereinbefore defined. The reaction may be performed under reaction conditions known to those skilled in the art, such as those described hereinbefore in respect of process step (ii) above; (ix) for compounds of formula I in which R 2 is absent, and R 3 is present and is not hydrogen, reaction of a compound of formula I in which R 2 is absent, and R 3 is present and is hydrogen with a compound of formula XIII

R 3x. L h XIII in which L h represents a suitable leaving group such as that defined above in respect of L b , and R 3 represents R 3 is as hereinbefore defined. The reaction may be performed under reaction conditions known to those skilled in the art, such as those described hereinbefore in respect of process step (ii) above.

Compounds of formula XI in which L f represents a halogen may be prepared by reacting a compound of formula XIV,

which R a , R b , R 1 to R 3 , R 5 , N 1 to N 4 and C 1 to C 4 are as hereinbefore defined, with either: an electrophile that provides a source of the halogen atoms (for example, for bromine atoms, reagents include /V-bromosuccinimide, bromine and 1 ,2- dibromotetrachloroethane; for chlorine atoms reagents include /V-chlorosuccinimide, chlorine, iodine monochloride and hexachloroethane; for iodine atoms, appropriate reagents include iodine, diiodoethane and diiodotetrachloroethane; and for fluorine atoms reagents include xenon difluoride, SELECTFLUOR® ([1-(chloromethyl)-4-fluoro- 1 ,4-diazonia-bicyclo[2.2.2]octane bis(tetrafluoroborate)]), CF3OF, perchloryl fluoride, F2 and acetylhypofluoride) optionally in the presence of an appropriate catalyst system (e.g. a palladium catalyst, such as PdCI 2l Pd(OAc) 2 , Pd(Ph 3 P) 2 CI 2 , Pd(Ph 3 P)4, Pd 2 (dba)3 or the like, or AuCI 3 ) and an appropriate solvent performed at around room temperature or above (e.g. up to 40-180°C), under conditions known to those skilled in the art; or a source of halide ions (such as ammonium halide or a metal halide (e.g. LiBr)) in the presence of an oxidant (such as a peroxide-based compound (e.g. oxone, H2O2, m- CPBA or ammonium persulfate)) optionally in the presence of an appropriate catalyst system (such as iodobenzene) and an appropriate solvent (such as ethanol, isopropanol, dimethylformamide, ethylene glycol, ethylene glycol dimethyl ether, water, dimethylsulfoxide, acetonitrile, dimethylacetamide or a mixture thereof) performed at around room temperature or above (e.g. up to 40-180°C), under conditions known to those skilled in the art. Compounds other than compounds of formula I may be commercially available, known in the literature, or may be obtained either by analogy with the processes described herein, or by conventional synthetic procedures, in accordance with standard techniques, from available starting materials using appropriate reagents and reaction conditions. In this respect, the skilled person may refer to inter alia "Comprehensive Organic Synthesis" by B. M. Trost and I. Fleming, Pergamon Press, 1991.

The substituents R a , R b and R 1 to R 5 in final compounds of the invention or relevant intermediates may be modified one or more times, after or during the processes described above by way of methods that are well known to those skilled in the art. Examples of such methods include substitutions, reductions, oxidations, alkylations, acylations, sulfonylations, hydrolyses, esterifications, etherifications, halogenations or nitrations. The precursor groups can be changed to a different such group, or to the groups defined in formula I, at any time during the reaction sequence. In this respect, the skilled person may also refer to "Comprehensive Organic Functional Group Transformations" by A. R. Katritzky, O. Meth- Cohn and C. W. Rees, Pergamon Press, 1995. Other specific transformation steps standard nucleophilic aromatic substitution reactions, for example in which a fluoro- or bromo-phenyl group is converted into a cyanophenyl group by employing a source of cyanide ions (e.g. KCN) as a reagent (alternatively, in this case, palladium catalysed cyanation reaction conditions may also be employed).

Further, the skilled person will appreciate that the bicyclic core may be prepared with reference to a standard heterocyclic chemistry textbook (e.g. "Heterocyclic Chemistry" by J. A. Joule, K. Mills and G. F. Smith, 3 rd edition, published by Chapman & Hall, "Comprehensive Heterocyclic Chemistry //" by A. R. Katritzky, C. W. Rees and E. F. V. Scriven, Pergamon Press, 1996 or "Science of Synthesis", Volumes 9-17 (Hetarenes and Related Ring Systems), Georg Thieme Verlag, 2006). Hence, the reactions disclosed herein that relate to compounds containing the bicyclic (purine) core may also be performed with compounds that are pre-cursors to the bicyclic core, and which pre-cursors may be converted to have the purine core at a later stage in the synthesis.

Compounds of the invention may be isolated from their reaction mixtures using conventional techniques (e.g. recrystallisations).

It will be appreciated by those skilled in the art that, in the processes described above and hereinafter, the functional groups of intermediate compounds may need to be protected by protecting groups.

The protection and deprotection of functional groups may take place before or after a reaction in the above-mentioned schemes.

Protecting groups may be removed in accordance with techniques that are well known to those skilled in the art and as described hereinafter. For example, protected compounds/intermediates described herein may be converted chemically to unprotected compounds using standard deprotection techniques. By 'protecting group' we also include suitable alternative groups that are precursors to the actual group that it is desired to protect.

For example, instead of a 'standard' amino protecting group, a nitro or azido group may be employed to effectively serve as an amino protecting group, which groups may be later converted (having served the purpose of acting as a protecting group) to the amino group, for example under standard reduction conditions described herein.

The type of chemistry involved will dictate the need, and type, of protecting groups as well as the sequence for accomplishing the synthesis.

The use of protecting groups is fully described in ''Protective Groups in Organic Chemistry, edited by J W F McOmie, Plenum Press (1973), and "Protective Groups in Organic Synthesis", 3 rd edition, T.W. Greene & P.G.M. Wutz, Wiley-lnterscience (1999).

As used herein, the term "functional groups" means, in the case of unprotected functional groups, hydroxy-, thiolo-, am inof unction, carboxylic acid and, in the case of protected functional groups, lower alkoxy, N-, 0-, S- acetyl, carboxylic acid ester. The invention is illustrated by way of the following examples.

General methods Preparative column chromatography was performed on Merck silica gel 60 (230-400 mesh) or Carlo Erba silica gel 60A (40-63 μητι). Preparative HPLC was performed on a Gilson system equipped with a UV detector in accordance to the experimental details specified below. The mobile phases used were either acidic (0.1 % TFA / MeOH) or basic (50 mM ammonium bicarbonate/ammonia (aq) / MeOH). The purest fractions were collected, concentrated and dried under vacuum. All compounds were analyzed by analytical HPLC/LCMS. The analysis was performed using an Agilent 1 100 Series Liquid Chromatograph/Mass Selective Detector (MSD) (Single Quadrupole) equipped with an electrospray interface and a UV diode array detector. NMR spectra were recorded on a Varian Mercury plus at 25 °C and 400 MHz for H and 100 MHz for 13 C. Chemical shifts (δ) are reported in ppm. The compounds prepared were given lUPAC names obtained from the software Marvin Sketch 5.6.0.1. In addition, the commercial or trivial names were used for many of the commercial available starting materials and reagents. Preparation of intermediates and final compounds

2-Chloro-9-methyl-9H-purin-6-amine

To a suspension of 6-amino-2-chloro-7H-purine (5.0 g, 29.5 mmol, 1 eq.) in N,N-dimethylformamide (50 mL) was added iodomethane (5.51 mL, 88.5 mmol, 3 eq.). The flask was equipped with a condenser and the reaction mixture was heated at 130 °C in an oilbath for 30 minutes. The mixture was allowed to attain room temperature and was concentrated at a rotavapor connected to a high-vacuum pump. The residue (some solvent left) was added brine (100 mL) and extracted with ethyl acetate (5x200 mL). The combined organic phase was dried (Na 2 S0 4 ), filtered and concentrated at vacuo. The residue was recrystallized in methanol (30 mL) under heat. The mixture was allowed to attain rt. After 2 h, an orange solid could be collected with filtration. The solid was washed with MeOH (3x2 mL) and dried over night at a high vacuum pump. Yield 414 mg (8%). Purity determined by HPLC: 95.6% (acidic), 97.2% (basic). 1 H NMR (DMSO-d6) δ 3.68 (s, 3H), 7.71 (br. s, 2H), 8.09 (s, H). MS ESI* m/z 184; 186 [M+H; M+2+H] + .

2-Chloro-9-ethyl-9-H-purin-6-amine

To a suspension of 6-amino-2-chloro-7H-purine (2.0 g, 11.8 mmol, 1 eq.) in N,N- dimethylformamide (20 mL) was added iodoethane (2.85 mL, 35.4 mmol, 3 eq.). The flask was equipped with a condenser and the reaction mixture was heated at 130 °C in an oilbath for 3 h and thereafter allowed to attain room temperature. The mixture was concentrated to dryness at a rotavapor connected to a high-vacuum pump. The black residue was added brine (100 mL) extracted with ethyl acetate (200+100 mL). The combined extracts were pooled, dried (Na 2 S0 4 ), filtered and concentrated in vacuo. The residue was recrystallized in methanol (15 mL). The mixture was allowed to attain rt and was left over night. The product was collected by filtration, washed with MeOH (3x2 mL) and dried over night at a high vacuum pump. Yield 452 mg (19%). Purity determined by HPLC: 94.3% (acidic), 95.3% (basic). MS ESI + m/z 198; 200 [M+H; M+2+H] + .

8-Bromo-9H-purin-6-amine

To bromine (1.0 mL, 20.0 mmol)) in water (80 mL) was added adenine (1.35 g, 10.0 mmol) and the reaction stirred at rt overnight. The product precipitated and was filtered off and washed with a small portion of water and dried to give 0.543 g (25%) of the title compound as an orange solid. The product was taken to the next step without further purification. MS ESI + m/z 214, 216 [M+H; M+2+H] + .

N-Benzyl-2-chloro-9H-purin-6-amine

To a suspension of 2,6-dichloro-9H-purine (1.00 g, 5.3 mmol) in EtOH (25 mL) was added benzylamine (0.58 mL, 5.3 mmol) and triethylamine (0.74 mL, 5.3 mmol) and the reaction stirred at 80 °C overnight. The ethanol was removed and EtOAc and water were added and the phases separated. The organic phase was washed with citric acid (10% aq) and brine, dried (MgS0 4 ), filtered and evaporated to give 0.580 g (42%) of the product. 1 H NMR (DMSO-d6) 54.63 (s, 2H), 7.19-7.38 (m, 5H), 8.12 (s, 1H), 8.72 (br. s, 1H), 13.1 (br. s, 1H). MS ESI + m/z 260 [M+H] + .

General method for SNAr on 2, 6-dichloro-9H-purine

To a suspension of 2,6-dichloro-9H-purine (0.085 mmol) in EtOH (0.7 mL) was added amine (0.085 mmol) and DIPEA (0.085 mmol) and the reaction stirred at 80 °C overnight. The ethanol was removed and EtOAc and water were added and the phases separated. The organic phase was washed with citric acid (10% aq) and brine, dried (MgS04), filtered and evaporated. The product was then filtered through a short plug of silica with EtOAc as eluent and dried. The product was taken to the next step without further purification.

General method; Suzuki-Miyaura coupling in the 8-position

To 8-bromo-9H-purin-6-amine were added the boronic acid, potassium carbonate and Pd(dppf)Cl2 x DCM and the reaction heated at 120 °C in a microwave oven for 15-25 min. The product precipitated and was filtered off, washed with water and dried to give the product. The product was taken to the next step without further purification.

Preparation of end products General method for alkylation (position 3) of 7H-purin-6-amine

To a suspension of 7H-purin-6-amine (15 mg, 0.1 mmol, 1 eq.) in N,N-dimethylformamide (0.64 mL), in a reaction vial with screw cap, was added iodoalkane (3 eq.) and the reaction was heated at 130 °C in a heating block for 15-40 minutes and thereafter allowed to attain room temperature. HPLC-MS indicated a mixture of regioisomers. The product was isolated with preparative HPLC (XBridge column, MeOH in 50 mM NH 4 HC0 3 -buffer). The purest fractions were pooled and concentrated in vacuo to yield the product which was dried at a high vacuum pump over night.

signal which sometimes is observe . General method for alkylation (position 3) of 9-benzyl-9H-purin-6-amine

To a suspension of 9-benzyl-9H-purin-6-amine (15 mg, 0.07 mmol, 1 eq.) in N,N- dimethylformamide (0.64 mL), in a reaction vial with screw cap, was added haloalkane (3 eq.) and the reaction was heated at 130 °C in a heating block for 15 minutes to 3 h and thereafter allowed to attain room temperature. HPLC-MS indicated a mixture of regioisomers. The product was isolated with preparative HPLC (XBridge column, MeOH in 50 mM NH 4 HC03-buffer). The purest fractions were pooled and concentrated in vacuo to yield the product which was dried at a high vacuum pump.

General method for Suzuki-Miyaura cross-coupling reactions from 6-amino-2-chloro-7H- purine

A microwave vial was charged with 6-amino-2-chloro-7H-purine, boronic acid (1.25 eq.), cesium carbonate (3 eq.) and PEPPSI™-IPr (0.5-1 mol%) followed by 2:1 water-acetonitrile. The vial was flushed with N 2 (g), capped and heated at 130-150 °C in microwave oven for 45 minutes to 2 h. The mixture was allowed to attain room temperature and was thereafter cooled to 0 °C on an ice-waterbath and filtered. The solid was purified with preparative HPLC (Xbridge column, MeOH in 50 mM NH 4 HC0 3 -buffer). The cleanest fractions were pooled and concentrated in vacuo to afford the product. The product was dried at high vacuum over night.

General method for Suzuki-Miyaura cross-coupling reactions from 2-chloro-9-methyl-9H- purin-6-amine

A microwave vial was charged with 2-chloro-9-methyl-9H-purin-6-amine and boronic acid (1.4 equiv). Then water and acetonitrile were added followed by cesium carbonate (3 equiv) and PEPPSI™-IPr (0.01 equiv). The reaction mixture was heated in a microwave oven at 150°C for 45 min under nitrogen. The product was purified by preparative HPLC (basic method 50mM NH 4 HCC>3 buffer and MeOH). The combined fractions were evaporated and dried to give the product.

General method for Suzuki-Miyaura cross-coupling reactions from 2-chloro-9-methyl-9H- purin-6-amine

A microwave vial was charged with 2-chloro-9-methyl-9H-purin-6-amine (30 mg, 0.16 mmol, 1 eq.), boronic acid (1.5 eq.), Pd(dppf)CI 2 x DCM (13 mg, 0.02 mmol, 10 mol%) and potassium carbonate (50 mg, 0.36 mmol, 2.2 eq.) followed by 3:1 DCE/water (3 ml_). The vial was flushed with N2(g), capped and heated at 120 °C in microwave oven for 15 minutes to 1.5 h. The reaction mixture was concentrated and the residue was purified with preparative HPLC (XBridge column, methanol in 50 mM NH 4 HC03-buffer). The purest fractions were pooled and concentrated in vacuo to yield the product. Compounds of insufficient purity were treated with a small amount of acetonitrile with 2-3 drops of methanol to dissolve impurities/discolorations. The solid was allowed to settle and the liquid was pipetted off. The product was dried at high vacuum over night.

- -Difluorophenyl)-9-ethyl-9H-purin-6-amine (MDP-066)

A microwave vial was charged with 2-chloro-9-ethyl-9H-purin-6-amine (30 mg, 0.15 mmol, 1 eq.), boronic acid (1.5 eq.), Pd(dppf)CI 2 x DCM (12 mg, 0.02 mmol, 10 mol%) and potassium carbonate (46 mg, 0.33 mmol, 2.2 eq.) following by 3:1 DCE/water (3 ml_). The vial was flushed with nitrogen gas, capped and heated at 120 °C in microwave oven for 30 minutes. The aqueous phase was decanted. The organic phase was concentrated and purified with preparative HPLC (XBridge column, methanol in 50 mM NH 4 HC0 3 -buffer). The purest fractions were pooled and concentrated in vacuo. The product was dried at high vacuum over night to yield 25.4 mg (61 %) of the title compound. (MS ESI + m/z 276 [M+H] + .

General method for synthesis of diaryl ethers from 2-chloro-9-methyl-9H-purin-6-amine A microwave vial was charged with 2-chloro-9-methyl-9H-purin-6-amine (30 mg, 0.16 mmol, 1 eq.), cesium carbonate (160 mg, 0.49 mmol, 3 eq.) and Cul (3 mg, 0.02 mmol, 10 mol%) followed by N.N-dimethylformamide (1.5 mL) and phenol (2 eq.). The vial was flushed with nitrogen, capped and heated to 195 °C in microwave oven for 1-2 h. The reaction mixture was purified with preparative HPLC (XBridge column, methanol in 50 mM NH 4 HC0 3 -buffer). The cleanest fractions were pooled and concentrated at vacuo to yield the product. The product was dried at high vacuum over night.

2-Chloro-9-ethyl-7-{[4-(trifluoromethyl)phenyl]methyl}-6,7-d ihydro-9H-purin-6-imine 6-amino-2-chloro-9-ethyl-7-{[4-(trifluoromethyl)phenyl]methy l}-9H-purin-7-ium (MDP- 064)

To a suspension of 2-chloro-9-ethyl-9H-purin-6-amine (30 mg, 0.15 mmol, 1 eq.) in N,N-dimethylacetamide (3 mL) was added potassium carbonate (21 mg, 0.15 mmol, 1.0 eq.) followed by 1-(bromomethyl)-4-(trifluoromethyl)benzene (36 mg, 0.15 mg, 1 eq.). The reaction was heated at 110 °C in a heating block over night. A single regio isomer was obtained as product. The alkylation is hypothesized to take place in position 7 due to steric effects. An example in literature supports the regiochemistry (Fujii, Tozo ei a/., Chemical & Pharmaceutical Bulletin, 34(4), 1821-5; 1986). The mixture was diluted with methanol (6 mL) and water (1.5 mL) and purified with preparative HPLC (XBridge column, MeOH in 50 mM NH 4 HC03-buffer). The purest fractions were pooled and concentrated in vacuo to afford the product. The product was dried at high vacuum to give 6.1 mg (11%) of the title compound. MS ESI + m/z 356; 358 [M+H; M+2+H] + .

9-Methyl-2-(2-phenylethynyl)-9H-purin-6-amine (MDP-053)

To a suspension of 2-chloro-9-methyl-9H-purin-6-amine (30 mg, 0.16 mmol, 1 eq.) in acetonitrile (2.5 mL), in a microwave vial, was added phenylacetylene (23 μί, 0.21 mmol, 1.3 eq.), DIPEA (85 μί, 0.49 mmol, 3 eq.), Cul (2 mg, 0.01 mmol, 5 mol%) and PdCI 2 (PPh 3 ) 2 (6 mg, 0.01 mmol, 5 mol%). The vial was flushed with N2(g), capped and heated at 100 °C in microwave oven for 30 minutes. The vial was recapped and heated in MW to 100 °C for another 60 minutes. The mixture was added more phenylacetylene (23 μί, 0.21 mmol, 1.3 eq) and PdCI 2 (PPh 3 ) 2 (12 mg, 0.02 mmol, 10 mol%) and heated at 130 °C in microwave oven for 30 minutes. The mixture was purified with preparative HPLC (Xbridge column, methanol in 50 mM NH 4 HC0 3 -buffer). The fractions were pooled and concentrated in vacuo to afford the product as a white solid with yellow discolorations. Yield 1.5 mg (4%). MS ESI + m/z 250 [M+H] + . N-Benzyl-2-chloro-9-methyl-9H-purin-6-amine

To a suspension of N-benzyl-2-chloro-9H-purin-6-amine (100.0 mg, 0.385 mmol) in DMF (3.5 mL) was added iodomethane (71.9 μΙ_, 1.16 mmol) and the reaction was heated at 130 °C in a microwave oven for 15 min. The reaction mixture was purified by preparative HPLC (acidic method; 0.1% TFA /MeOH) to give 16 mg (15%) of the title compound. Ή NMR (DMSO-d6) δ 4.00 (s, 3H), 4.83-4.86 (m (a singlet and a doublet, 2H)), 7.26-7.36 (m, 4H), 7.44-7.46 (m, 2H), 8.96 (br. s, 1 H), 9.57 (br. s, 1 H) (tautomers). MS ESI + m/z 21A [M+H] + .

General method Methylation

To a suspension of the purine in DMF was added iodomethane and the reaction was heated at 130-150 °C in a microwave for 15 min. The reaction mixture was purified by preparative HPLC (acidic, 0.1 % TFA / MeOH or basic, 50 mM NH 4 HC0 3 buffer / MeOH) to yield the product.

Methylation of Suzuki-Miyaura products

To a suspension of the purine in DMF was added iodomethane and the reaction was heated at 130-150 °C in a microwave for 15 min. The reaction mixture was purified by preparative HPLC (acidic, TFA / MeOH or basic, 50 mM NH 4 HC0 3 (aq) / MeOH) to yield the product. If the product was unpure after preparative HPLC, it was triturated with acetonitrile/MeOH and dried.

3-Ethyl-8-(4-methoxyphenyl)-3H-purin-6-amine

To 8-(4-methoxyphenyl)-9H-purin-6-amine (22.4 mg, 0.093 mmol) were added DMF (0.7 mL) and iodoethane (22.3 pL, 0.279 mmol) and the reaction heated at 150 °C for 15 min. The crude was purified by preparative HPLC (basic method; 50 mM NH 4 HCO 3 buffer and MeOH). The combined fractions were evaporated and dried to give 5.1 mg (20%) of the product as a pale yellow solid. 1 H NMR (DMSO-c/6) δ 1.50 (t, 3H), 3.83 (s, 3H), 4.41 (q, 2H), 7.10 (d, 2H), 8.07-8.10 (m, 2H), 8.11 (s, 1H), 8.58 (br. S, 1H). MS ESI + m/z 270 [M+H] + .

8-N-Ethyi-3,8-N-dimethyl-3H-purine-6,8-diamine

To 8-bromo-9H-purin-6-amine (30.0 mg, 0.140 mmol) in EtOH (1.5 mL) was added N-ethylmethylamine (0.120 mL 1.40 mmol) and the reaction was stirred for 48 h at 100 °C in a sealed vial. The solvent was evaporated to give crude 8-N-ethyl-8-N-methyl-9H-purine-6,8- diamine (x) , MS ESI + m/z 193 [M+H] + . The crude material was used in the next step without purification. To the crude amine (x) were added DMF (0.7 mL) and iodomethane (26.1 pL, 0.420 mmol) and the reaction heated at 130 °C for 15 min. The crude material was purified by preparative HPLC (basic method; 50 mM NH4HCO3 buffer and MeOH) to give 2.8 mg (9.7%, two steps) of the title compound. MS ESI + m/z 207 [M+H] + . -N-Benzyl-9-methyl-9H-purine-2,6-diamine

To a suspension of 2-fluoro-1 H-purin-6-amine (19.9 mg, 0.130 mmol) in DMF (0.7 mL) were added triethylamine (23.1 pL, 0.130 mmol) and benzylamine (14.2 pL, 0.130 mmol), and the reaction heated at 150 °C for 20 min to give crude 2-N-benzyl-9H-purine-2,6-diamine The crude reaction was taken to the next step. Iodomethane (24.3 pL, 0.390 mmol) was added and the reaction heated at 130 °C for 15 min. The crude material was purified by prep HPLC to give 0.5 mg (1.5%, two steps) of title compound. MS ESI + m/z 255 [M+H] + .

Abbreviations

DIPEA N,N-Diisopropylethylamine

PdCI 2 (PPh 3 )2 Bis(triphenylphosphine)palladium(ll) dichloride Pd(dppf)Cl2 x DCM [l .l '-BisCdiphenylphosphinoJferroceneldichloropalladiumill), complex with dichloromethane

PEPPSI™-IPr [1 ,3-Bis(2,6-Diisopropylphenyl)imidazol-2-ylidene](3- chloropyridyl)palladium(ll) dichloride

The second aspect of the invention provides a class III PI 3-kinase inhibitor comprising or consisting of:

a. a compound as defined in the first aspect of the invention; or

b. a compound selected from the group defined in Table 1 ,

for use in the treatment or prevention of muscular dystrophy.

Table 1

The term "inhibit" may refer to any measurable reduction and/or prevention of enzyme activity or catalytic kinase (e.g., class III PI3-kinase) activity. The reduction and/or prevention of enzyme/kinase activity may be measured by comparing the kinase activity in a sample containing a compound of the invention and an equivalent sample of enzyme/kinase in the absence of a compound of the invention, as would be apparent to those skilled in the art. The measurable change may be objective (e.g. measurable by some test or marker, for example in an in vitro or in vivo assay or test, such as one described hereinafter, or otherwise another suitable assay or test known to those skilled in the art) or subjective (e.g. the subject gives an indication of or feels an effect).

Compounds of the invention may be found to exhibit 50% inhibition of enzyme activity or catalytic kinase (e.g., class III PI3-kinase) activity at a concentration of 100 μΜ or below (for example at a concentration of below 50 μΜ, or even below 10 μΜ, such as below 1 μΜ), when tested in an assay (or other test), for example as described hereinafter, or otherwise another suitable assay or test known to the skilled person.

In one embodiment, the class III PI 3-kinase inhibitor is an autophagy inhibitor.

By "autophagy inhibitor" we include any agent which is capable of inhibiting, at least in part, the autophagy-lysosome pathway in mammals. It will be appreciated that the agent may inhibit such autophagocytosis either directly (by acting on a component of the autophagy-lysosome pathway) or indirectly (by acting on another cell component or factor that itself inhibits, directly or indirectly, the autophagy-lysosome pathway).

Regulation of the autophagy-lysosome pathway in mammals is discussed in detail in the scientific literature (for example, see Mehrpour et a/., 2010, Cell Res. 20(7)748- 62 and Mehrpour et a/., 2010, Am J Physiol Cell Physiol. 298(4):C776-85, the disclosures of which are incorporated herein by reference).

Examples of autophagy inhibitors are well-known in the art, in part through their suggested use in the treatment of cancer (for example, see Livesey et a/., 2009, Curr Opin Investig Drugs. 10(12): 1269-79, the disclosures of which are incorporated herein by reference). Typically, autophagy levels/rates are determined by measuring one or more autophagy-associated marker using, for example, immunohistochemical methods. For example, the presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form LC3II have been used as indicators of autophagy (Levine, B., and Kroemer, G. 2008. Autophagy in the pathogenesis of disease. Cell 132:27-42; Maiuri, M.C., Zalckvar, E., Kimchi, A., and Kroemer, G. 2007. Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8:741-752). Other autophagy markers include Vps34, Cathepsin L and Beclin.

Hence, although autophagy may be measured using any suitable method known in the art, the methods used in the foregoing examples are used by preference. In one embodiment, autophagy levels are determined through measuring the presence of LC3 in autophagosomes and/or the conversion of LC3 to the lower migrating form LC3II (preferably using immunofluorescence). Alternatively or additionally autophagy levels may be determined by measuring the expression levels of one or more of GabarapH , Beclin, Vps34, Atg4B, Cathepsin L and Lamp2a, on the mRNA and/or protein level.

It will be appreciated by skilled persons that the autophagy inhibitor may be capable of inhibiting, in whole or in part, macroautophagy, m/ ' croautophagy and/or chaperone-mediated autophagy.

Hence, the class III PI 3-kinase inhibitor may be a macroautophagy inhibitor. The class III PI 3-kinase inhibitor may be a microautophagy inhibitor. The class III PI 3- kinase inhibitor may be a chaperone-mediated autophagy inhibitor.

In one embodiment, the class III PI 3-kinase inhibitor is not one or more of the autophagy inhibitors selected from the group consisting of 3-methyladenine, wortmannin, bafilomycins (such as bafilomycin A1), chloroquine, hydroxychloroquine, PI3K class III inhibitors (such as LY294002), L-asparagine, catalase, E64D, leupeptin, N-acetyl-L-cysteine, pepstatin A, propylamine,

4- aminoquionolines, 3-methyl adenosine, adenosine, okadaic acid, N6-mercaptopurine riboside (N6-MPR), an aminothiolated adenosine analogue and

5- amino-4-imidazole carboxamide riboside (AICAR).

In one embodiment, the class III PI 3-kinase inhibitor is an inhibitor of the ubiquitin- proteasome system.

By "inhibitor of the ubiquitin-proteasome system" we mean an agent (e.g. small chemical entity, polypeptide or the like) which is capable of inhibiting, at least in part, a function of the ubiquitin-proteasome system (preferably in vivo in humans). Such an inhibitor may act at any point along the ubiquitin-proteasome protein degradation pathway, for example by inhibiting (at least, in part) the marking of proteins for degradation by modulating ubiquitination or deubiquitination, by inhibiting the ability of the proteasome to recognize or bind proteins to be degraded, and/or by inhibiting the ability of the proteasome to degrade proteins.

The ubiquitin-proteasome system, and components thereof, are described in detail in the scientific literature, for example see Ciechanover, 1998, The EMBO Journal 17, 7151-7160 (see Figures 1 and 2 therein) and Bedford et a/., 201 1 , Nat Rev Drug Discov 10, 29-46; the disclosures of which are incorporated herein by reference.

In one embodiment, the inhibitor of the ubiquitin-proteasome system is a proteasome inhibitor acting directly upon the proteasome to inhibit its function. For example, the proteasome inhibitor may inhibit (at least, in part) the ability of the human proteasome to degrade proteins. Examples of proteasome inhibitors are well known in the art (for example, see de Bettignies & Coux, 2010, Biochimie. 92(1 1): 1530-45, Kling et a/., 2010, Nature Biotechnology, 28(12): 1236-1238, the disclosures of which are incorporated herein by reference).

Proteasome inhibition may be measured using any suitable method known in the art (for a review see, for example, Novel Anticancer Drug Protocols; "Assays for Proteasome Inhibition"; Methods in Molecular Medicine Volume 85, 2003, pp 163- 172). A preferred method is described in Lam et a/., 2000, PNAS, 97(18):9902-9906 (which is incorporated herein by reference).

In one embodiment, the class III PI 3-kinase inhibitor is not selected from the group consisting of LY294002, bortezomib (PS-341 , MG-341 , Velcade®), PI-083, MLN 9708, MLN 4924, MLN 519, carfilzomib, ONX 0912, CEP-1877, NPI-0047, NPI-0052, BU-32 (NSC D750499-S), PR-171 , IPSI-001 , disulfiram, epigallocatechin-3-gallate, MG-132, MG-262, salinosporamide A, leupeptin, calpain inhibitor I, calpain inhibitor II, MG-1 15, PSI (Z-lle-Glu(OtBu)-Ala-Leu-H (aldehyde)), peptide glyoxal, peptide alpha-ketoamide, peptide boronic ester, peptide benzamide, P'-extended peptide alpha-ketoamide, lactacystin, clastro-lactacystin β-lactone, epoxomicin, eponemycin, TCM-86A, TCM-86B, TCM 89, TCM-96, YU101 , TCM-95, gliotoxin, the T-L activity specific aldehyde developed by Loidl et al., (Chem. Biol., (1999) 6:197-204), HNE (4- hydroxy-2-nonenal), YU102 and natural products with proteasome-inhibitory effects, such as green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG), soy isoflavone genistein, and the spice turmeric compound curcumin.

The Class III PI 3-kinase inhibitor of the invention may comprise or consist of a compound selected from the group consisting of:

a. Table 1 , compound 1 ;

b. Table 1 , compound 2;

c. Table 1 , compound 3;

d. Table 1 , compound 4;

e. Table 1 , compound 5;

f. Table 1 , compound 6;

g- Table 1 , compound 7;

h. Table 1 , compound 8;

i. Table 1 , compound 9;

j- Table 1 , compound 10

k. Table 1 , compound 11

1. Table 1 , compound 12"

m. Table 1 , compound 13

n. Table 1 , compound 14

o. Table 1 , compound 15

P- Table 1 , compound 16

q- Table 1 , compound 17

r. Table 1 , compound 18

However, The Class III PI 3-kinase inhibitor of the invention may not comprise or consist of one or more compound selected from the group consisting of:

a. Table 1 , compound 1 ;

b. Table 1 , compound 2;

c. Table 1 , compound 3;

d. Table 1 , compound 4;

e. Table 1 , compound 5;

f. Table 1 , compound 6;

g- Table 1 , compound 7;

h. Table 1 , compound 8;

i. Table 1 , compound 9;

j- Table 1 , compound 10;

k. Table 1 , compound 11 ; I. Table 1 , compound 12;

m. Table 1 , compound 13;

n. Table 1 , compound 14;

o. Table 1 , compound 15;

p. Table 1, compound 16;

q. Table 1 , compound 17; and

r. Table 1 , compound 18.

The malfunctioning of protein kinases (PKs) is the hallmark of numerous diseases. A large share of the oncogenes and proto-oncogenes involved in human cancers code for PKs. The enhanced activities of PKs are also implicated in many non-malignant diseases, such as benign prostate hyperplasia, familial adenomatosis, polyposis, neurofibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis. PKs are also implicated in inflammatory conditions and in the multiplication of viruses and parasites. PKs may also play a major role in the pathogenesis and development of neurodegenerative disorders.

For a general reference to PKs malfunctioning or disregulation see, for instance, Current Opinion in Chemical Biology 1999, 3, 459 - 465.

Phosphatidylinositol 3-kinases (PI3Ks) are a family of lipid and serine/threonine kinases that catalyze the phosphorylation of the membrane lipid phosphatidylinositol (PI) on the 3'-OH of the inositol ring to produce phosphoinositol-3-phosphate (PIP), phosphoinositol-3,4-diphosphate (PIP2) and phosphoinositol-3,4,5-triphosphate (PIP 3 ), which act as recruitment sites for various intracellular signalling proteins, which in turn form signalling complexes to relay extracellular signals to the cytoplasmic face of the plasma membrane. These 3'-phosphoinositide subtypes function as second messengers in intra-cellular signal transduction pathways (see e.g. Trends Biochem. Sci 22 87,267-72 (1997) by Vanhaesebroeck et al.; Chem. Rev. 101 (8), 2365-80 (2001) by Leslie et al (2001); Annu. Rev. Cell. Dev. Boil. 17, 615-75 (2001) by Katso et al; and Cell. Mol. Life Sci. 59 (5), 761-79 (2002) by Toker et al).

Multiple PI3K isoforms categorized by their catalytic subunits, their regulation by corresponding regulatory subunits, expression patterns and signalling specific functions (ρ1 10α, β, δ, γ) perform this enzymatic reaction (Exp. Cell. Res. 25 (1),. 239-54 (1999) by Vanhaesebroeck and Katso et al., 2001 , above).

The closely related isoforms p1 10a and β are ubiquitously expressed, while δ and γ are more specifically expressed in the haematopoietic cell system, smooth muscle cells, myocytes and endothelial cells (see e.g. Trends Biochem. Sci. 22 (7),. 267-72 (1997) by Vanhaesebroeck et al). Their expression might also be regulated in an inducible manner depending on the cellular, tissue type and stimuli as well as disease context. Inductibility of protein expression includes synthesis of protein as well as protein stabilization that is in part regulated by association with regulatory subunits.

Eight mammalian PI3Ks have been identified so far, including one class III PI3K - VPS34 (vacuolar protein sorting 34). VPS was first described as a component of the vacuolar sorting system in Saccharomyces cerevisiae and is the sole PI3K in yeast. The homologue in mammalian cells, hVps34, has been studied extensively in the context of endocytic sorting. However, hVps34 also plays an important role in the ability of cells to respond to changes in nutrient conditions. Recent studies have shown that mammalian hVps34 is required for the activation of the mTOR (mammalian target of rapamycin)/S6K1 (S6 kinase 1 ) pathway, which regulates protein synthesis in response to nutrient availability. In both yeast and mammalian cells, Class III PI3Ks are also required for the induction of autophagy during nutrient deprivation. Finally, mammalian hVps34 is itself regulated by nutrients. Thus Class III PI3Ks are implicated in the regulation of both autophagy and, through the mTOR pathway, protein synthesis, and thus contribute to the integration of cellular responses to changing nutritional status.

The substrate specificity of Vps34 is limited to phosphatidylinositol. Thus its product in cells is Ptdlns3P. This distinguishes it from the more numerous and better studied Class I and Class II enzymes, which can produce Ptdlns3P, Ptdlns P2 or Ptdlns P3, depending on the isoform (Fruman, Meyers and Cantley (1998) Phosphoinositide kinases. Annu. Rev. Biochem. 67, 481-507; Vanhaesebroeck et al., (2001) Synthesis and function of 3-phosphorylated inositol lipids. Annu. Rev. Biochem. 70, 535-602; Odorizzi, Babst and Emr (2000) Phosphoinositide signaling and the regulation of membrane trafficking in yeast. Trends Biochem. Sci. 25, 229-235; Lindmo and Stenmark (2006) Regulation of membrane traffic by phosphoinositide 3-kinases. J. Cell Sci. 119, 605-614). For a review on this topic, see Backer (2008) The regulation and function of Class III PI3Ks: novel roles for Vps34. Biochem. J. 410, 1-17 which is incorporated herein by reference.

The Vps34 may comprise or consist of an amino acid sequence according to accession sequence NP_852079 (in particular NP_852079.2), the murine Vps34 or active fragments thereof. Protein accession sequences may be viewed at the NCBI online protein database (http://www.ncbi.nlm.nih.gov/protein).

In humans Vps34 is encoded by the PIK3C3 gene. Hence, the Class III PI 3-kinase inhibitor for use may be an inhibitor of VPS34, for example, hVPS34. The hVPS34 may comprise or consist of an amino acid sequence according to accession sequence NP_002638 (in particular NP_002638.2) or active fragments thereof.

By "active fragments thereof we mean fragments of the protein or polypeptide that retain the core biochemical function of the parent protein or polypeptide. For example, in the case of Vps34, we include truncated versions that retain full or partial ability to catalyze the phosphorylation of the substrate phosphatidylinositol. For example, the fragment 282-877 relative to SEQ ID NO: 1.

Hence, the VPS34 may comprise or consist of an amino acid sequence according to SEQ ID NO: 1 or variants thereof:

MGEAEKFHYIYSCDLDINVQLKIGSLEGKREQKSYKAVLEDPMLKFSGLYQETCSDL YVTCQVFAEGKPLALPVRTSYKAFSTRW W EWLKLPVKYPDLPR AQVALTIWDVY GPGKAVPVGGTTVSLFGKYG FRQGMHDLKVWP VEADGSEPTKTPGRTSSTLSEDQ MSRLAKLTKAHRQGHMVKVD LDRLTFREIEMINESEKRSSNFMYL VEFRCVKCDD KEYGIVYYEKDGDESSPILTSFELVKVPDPQ S ENLVESKHHKLARSLRSGPSDHD LKPNAATRDQLNIIVSYPPTKQLTYEEQDLV KFRYYLTNQEKALTKFLKCVNWDLP QEAKQALELLGKWKPMDVEDSLELLSSHYTNPTVRRYAVARLRQADDEDLLMYLLQL VQALKYENFDDIKNGLEPTKKDSQSSVSE VSNSGINSAEIDSSQIITSPLPSVSSP PPASKTKEVPDGENLEQDLCTFLISRACKNSTLA YLY YVIVECEDQDTQQRDPKT HEMYLNVMRRFSQALLKGDKSVRVMRSLLAAQQTFVDRLVHLMKAVQRESGNRKKKN ERLQALLGDNEKMNLSDVELIPLPLEPQVKIRGIIPETATLFKSALMPAQLFFKTED GGKYPVIFKHGDDLRQDQLILQIISLMDKLLRKENLDLKLTPYKVLATSTKHGFMQF IQSVPVAEVLDTEGSIQNFFRKYAPSENGPNGISAEVMDTYVKSCAGYCVI YILGV GDRHLDNLLLTKTGKLFHIDFGYILGRDPKPLPPP KLNKEMVEGMGGTQSEQYQEF RKQCYTAFLHL RYSNLILNLFSLMVDA I PDIALEPDKTVKKVQDKF LDLSDEEA VH YMQ SLIDES VHAL F AAWE Q I HKF AQ Y WRK

[SEQ ID NO: 1]

By "variant" we include polypeptides having insertions, deletions and/or substitutions, either conservative or non-conservative, as compared to the amino acid sequence of a reference amino acid sequence (for example, SEQ ID NO: 1).

Thus, in one embodiment the variant is or comprises a fragment of the amino acid sequence of a reference amino acid sequence (for example, SEQ ID NO: 1 ). In a further embodiment, the polypeptide comprises or consists of at least 50 contiguous amino acids of a reference sequence (for example, SEQ ID NO: 1 ) for example at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or 877 contiguous amino acids of a reference sequence (for example, SEQ ID NO: 1 ).

The variant may have an amino acid sequence which has at least 50% identity with a reference amino acid sequence (for example, SEQ ID N0.1 ), more preferably at least 90%, and most preferably 99.9% identity with a reference amino acid sequence (for example, SEQ ID N0.1), for example at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 99.9%.

The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (as described in Thompson er a/., 1994, Nuc. Acid Res. 22:4673-4680).

The parameters used may be as follows:

Fast pairwise alignment parameters: K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.

Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine local sequence alignments.

Preferably, the Class III PI 3-kinase inhibitor of the invention is a selective inhibitor of Class III PI 3-kinase.

Class III PI 3-kinase inhibition can be determined using any suitable method known in the art. However, it is preferred that the method described in the foregoing examples (below) is used.

By "selective inhibitor of Class III PI 3-kinase" we include compounds which inhibit Class III PI 3-kinase more strongly than other classes of PI3-kinase (for example, Class I or Class II PI 3-kinases); preferably at least 1.2-fold more strongly, at least 1.4-fold more strongly, at least 1.6-fold more strongly, at least 1.8-fold more strongly, at least 2-fold more strongly, at least 5-fold more strongly, at least 10-fold more strongly, at least 50-fold more strongly or at least 100-fold more strongly.

Preferably, the selective inhibitors of Class III PI 3-kinase of the invention inhibit Class III PI 3-kinase more strongly than they inhibit any other enzyme. Preferably, at least 1.2-fold more strongly, at least 1.4-fold more strongly, at least 1.6-fold more strongly, at least 1.8-fold more strongly, at least 2-fold more strongly, at least 5-fold more strongly, at least 10-fold more strongly, at least 50-fold more strongly or at least 100-fold more strongly.

Preferably, the Class III PI 3-kinase inhibitor for use of the invention is not an inhibitor of Class I PI 3-kinase and/or Class II PI 3-kinase.

Compounds of the invention that exhibit single selectivity for a target may have the additional benefit that they exhibit less side effects. Hence, the compounds of the invention may not target other kinases (i.e., they may be selective for Class III).

In a further or additional embodiment, the Class III PI 3-kinase inhibitor for use of the invention has an IC50 of 250 μιη or lower, for example, 200 μητι or lower, 150 μηη or lower, 100 μη or lower, 90 μηι or lower, 80 μητι or lower, 70 μηπ or lower, 60 μητι or lower, 55 μιη or lower, 54 μιη or lower, 53 μιη or lower, 52 μηι or lower, 51 μηη or lower, 50 μητι or lower, 49 μιη or lower, 48 μηι or lower, 47 μιη or lower, 46 μηη or lower, 45 μιη or lower, 44 μηι or lower, 43 μηι or lower, 42 μητι or lower, 41 μηι or lower, 40 pm or lower, 39 μητι or lower, 38 μητ» or lower, 37 μηι or lower, 36 μιη or lower, 35 μιη or lower, 34 μιη or lower, 33 μηι or lower, 32 μηι or lower, 31 μιη or lower, 30 μιη or lower, 29 μιη or lower, 28 μπι or lower, 27 μηι or lower, 26 μηι or lower, 25 μΓη or lower, 24 μιη or lower, 23 μιη or lower, 22 μιτι or lower, 21 μιη or lower, 20 pm or lower, 19 μιη or lower, 18 μιη or lower, 17 μηη or lower, 16 μιη or lower, 15 μιτι or lower, 14 μηι or lower, 13 μηη or lower, 12 μητι or lower, 11 μηι or lower, 10 μηι or lower, 9 μιη or lower, 8 μηι or lower, 7 μιη or lower, 6 μηι or lower, 5 μηη or lower, 4 μιη or lower, 3 μηι or lower, 2 μιη or lower, 1 μηη or lower, 950 nm or lower, 900 nm or lower, 850 nm or lower, 800 nm or lower, 750 nm or lower, 700 nm or lower, 650 nm or lower, 600 nm or lower, 550 nm or lower, 500 nm or lower, 450 nm or lower, 400 nm or lower, 350 nm or lower, 300 nm or lower, 250 nm or lower, 200 nm or lower, 150 nm or lower, 100 nm or lower, 50 nm or lower, 45 nm or lower, 40 nm or lower, 30 nm or lower, 35 nm or lower, 30 nm or lower, 25 nm or lower, 20 nm or lower, 15 nm or lower, 10 nm or lower, 5 nm or lower, 4 nm or lower, 3 nm or lower, 2 nm or lower, or 1 nm or lower.

In a further or additional embodiment, the Class III PI 3-kinase inhibitor for use of the invention has a highest thermal shift of at least 0.2 °C, for example, a highest thermal shift of at least 0.4 °C, at least 0.6 °C, at least 0.8 °C, at least 1 °C, at least 1.5 °C, at least 2 °C, at least 3 °C, at least 4 °C, at least 5 °C, at least 10 °C, at least 15 °C, at least 20 °C, at least 25 °C, , at least 30 °C, at least 35 °C, at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 70 °C, at least 80 °C, at least 90 °C or at least 100 °C.

By "highest thermal shift" we include the highest melting temperature of Vps34 protein or active fragment thereof bound by a test compound in comparison to the unbound Vps34 protein or active fragment thereof. Preferably, "highest thermal shift" is determined using the method described in the Examples section, below.

It will be further appreciated by persons skilled in the art that the present invention provides agents suitable for the treatment and prevention of several different forms of muscular dystrophy and muscular dystrophy-like indications, such as related myopathies.

In one embodiment, the muscular dystrophy is selected from the group consisting of congenital muscular dystrophy, Duchenne muscular dystrophy (DMD), Becker's muscular dystrophy (BMD, Benign pseudohypertrophic muscular dystrophy), distal muscular dystrophy (distal myopathy), Emery-Dreifuss muscular dystrophy (EDMD), facioscapulohumeral muscular dystrophy (FSHMD, FSHD or FSH), limb-girdle muscular dystrophy (LGMD), myotonic muscular dystrophy, centronuclear myopathies and oculopharyngeal muscular dystrophy.

Thus, the muscular dystrophy may be a congenital muscular dystrophy, for example selected from the group consisting of

(a) Congenital muscular dystrophy with abnormalities in the extracellular

matrix, such as Merosin (laminin a2) deficient CMD (MDC1A) and Collagen VI deficient CMD (Ullrich CMD and Bethlem myopathy);

(b) Dystroglycanopathies (abnormalities of a -dystroglycan), such as

Fukuyama-type CMD, Variants of muscle-eye brain disease, Walker- Warburg syndrome, Congenital muscular dystrophy type 1C, Congenital muscular dystrophy type 1 D and Limb-girdle muscular dystrophy 2I;

(c) Defects in the integrin a7 subunit, such as Congenital myopathy with

integrin a7 deficiency;

(d) Abnormalities of nuclear envelope proteins, such as L-CMD;

(e) Abnormalities in ER, such as SEPN1 related myopathy (formerly known as Rigid Spine Muscular Dystrophy);

(f) Undiagnosed CMD, including merosin positive; and

(g) Ryanodine receptor gene (RYR1) CMD

In one preferred embodiment, the muscular dystrophy is Iaminin-a2-deficient congenital muscular dystrophy (Muscular Dystrophy, Congenital Merosin-Deficient, 1a / MDC1A).

However, in another embodiment the muscular dystrophy is not Iaminin-a2-deficient congenital muscular dystrophy (Muscular Dystrophy, Congenital Merosin-Deficient, 1a / MDC1A).

In an alternative embodiment, the muscular dystrophy is the muscular dystrophy is Duchenne muscular dystrophy (DMD).

In a further embodiment, the muscular dystrophy is a distal muscular dystrophy (distal myopathy), for example selected from the group consisting of Miyoshi myopathy, distal myopathy with anterior tibial onset, and Welander distal myopathy.

In a further embodiment, the muscular dystrophy is an Emery-Dreifuss muscular dystrophy (EDMD), for example selected from the group consisting of EDMD1 , EDMD2, EDMD3, EDMD4, EDMD5 and EDMD6.

In a further embodiment, the muscular dystrophy is a facioscapulohumeral muscular dystrophy (FSHMD, FSHD or FSH), for example selected from the group consisting of FSHMD1A (4q35 deletion) and FSHMD1 B.

In a further embodiment, the muscular dystrophy is a Limb-girdle muscular dystrophy or (Erb's muscular dystrophy), for example selected from the group consisting of LGMD1A, LGMD1 B, LGMD1 C, LGMD1 D, LGMD1 E, LGMD1 F, LGMD1 G, LGMD2A, LGMD2B, LGMD2C, LGMD2D, LGMD2E, LGMD2F, LGMD2G, LGMD2H, LGMD2I, LGMD2J, LGMD2K, LGMD2L, LGMD2M, LGMD2N and LGMD20.

In a still further embodiment, the muscular dystrophy is a myotonic dystrophy, for example selected from the group consisting of DM1 (also called Steinert's disease) severe congenital form, DM1 childhood-onset form and DM2 (also called proximal myotonic myopathy or PROMM).

As discussed above, the term "muscular dystrophy" encompasses a number of related hereditary diseases associated with weakening of the muscles that move the body.

In one embodiment, the muscular dystrophy is associated with excessive autophagy (i.e. excessive macroautophagy, microautophagy and/or chaperone-associated autophagy).

Thus, the muscular dystrophy may be associated with excessive macroautophagy. For example, the muscular dystrophy may be Iaminin-a2-deficient congenital muscular dystrophy, MDC1A).

In an alternative embodiment, the muscular dystrophy is not associated with macroautophagy dysregulation. For example, the muscular dystrophy may be Duchenne muscular dystrophy). In a further alternative embodiment, the muscular dystrophy is not associated with reduced macroautophagy.

The inhibitors of intracellular protein degradation of the invention are for use in the treatment and/or prevention of muscular dystrophy.

By "treatment and/or prevention" we mean that the inhibitor of intracellular protein degradation is used to prevent, reduce and/or eliminate one or more symptoms or parameters associated with muscular dystrophy.

In one embodiment, the treatment or prevention of muscular dystrophy results in one or more of the following parameters being reduced in the mammal:

(i) muscle fibrosis;

(ii) muscle atrophy;

(iii) muscular apoptosis (caspase-3 positive muscle fibres);

(iv) collagen III expression;

(v) tenascin-C expression,

(vi) proportion of muscle fibre cells with centrally locate nuclei; and/or

(vii) laminin alpha-4 expression.

Methods for the assessment of these parameters are well known in the art (for example, see Gawlik et al., 2010, PLoS ONE 5(7):e11549 and Meinen et al., 2007, J Cell Biol. 176(7):979-93).

Said reduction in the parameter(s) may be in whole or in part. For example, the one or more parameters may be reduced by at least 10%, for example, by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or by 100% relative to the level prior to treatment with the inhibitor.

Alternatively, or additionally, the treatment or prevention of muscular dystrophy may result in one or more of the following parameters being increased in the mammal:

(a) muscle regeneration;

(b) muscle weight;

(c) average muscle fibre diameter; (d) ratio of quadriceps muscle wet weight per body weight

(e) lifespan;

(f) locomotive function;

(g) laminin beta-2 expression;

(h) proportion of muscle fibre cells with centrally locate nuclei;

(i) expression of MyoD1 in satellite cells; and/or

(j) expression of eMHC in regenerating muscle fibres.

Methods for the assessment of these parameters are well known in the art (for example, see Gawlik et al., 2010, PLoS ONE 5(7):e11549 and Meinen et al., 2007, J Cell Biol. 176(7):979-93, the disclosures of which are incorporated herein by reference).

Said increase in the parameter(s) may be in whole or in part. For example, the one or more parameters may be increased by at least 10%, for example, by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 450%, 500%, 600%, 700%, 800%, 900% or 1000% relative to the level prior to treatment with the inhibitor.

Alternatively, or additionally, the treatment or prevention of muscular dystrophy may result in Akt phosphorylation at threonine 308 and/or 473 being restored to wild type or near wild type levels, for example, within 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, 0.05% of wild type levels.

It will be appreciated by persons skilled in the art that the inhibitors of intracellular protein degradation of the invention may be for use in combination with a second therapeutic agent or treatment for muscular dystrophy.

For example, the second therapeutic agent or treatment may comprise or consist of physical therapy, corrective orthopedic surgery and/or steroids.

Alternatively, or in addition, the second therapeutic agent or treatment may comprise or consist of gene replacement, cell therapy and/or anti-apoptosis therapy (for example, see Gawlik et al., 2004, Hum Mol Genet. 13(16): 1775-84, Hagiwara et al., 2006, FEBS Lett. 580(18):4463-8, Meinen er al., 2007, J Cell Biol. 176(7):979-93 and Girgenrath et al., 2009, Ann Neurol. 65(1) 47-56, the disclosures of which are incorporated herein by reference).

In one embodiment, the inhibitor of intracellular protein degradation is an autophagy inhibitor for use in combination with a proteasome inhibitor, or vice-versa. Such combination therapies thus seek to inhibit both of the main pathways of protein degradation in mammalian cells.

It will be appreciated by persons skilled in the art that the inhibitors of the invention may be for use in any mammal.

In one embodiment, the mammal is a human.

Alternatively, the mammal may be a dog, cat, horse, or other domestic or farm mammalian animal.

A third embodiment of the invention provides the use of a Class III PI 3-kinase inhibitor as defined in the first aspect or second aspect of the invention for use in treating muscular dystrophy.

A fourth embodiment of the invention provides the use of a Class III PI 3-kinase inhibitor as defined in the first aspect or second aspect of the invention in the manufacture of a medicament for the treatment of muscular dystrophy.

A fifth embodiment of the invention provides a method for treating or preventing muscular dystrophy comprising administering to a patient in need thereof an effective amount of a Class III PI 3-kinase inhibitor as defined in the first aspect or second aspect of the invention.

"Patients" include mammalian (including human) patients. Hence, the method of treatment discussed above may include the treatment of a human or animal body.

The term "effective amount" refers to an amount of a compound, which confers a therapeutic effect on the treated patient. The effect may be objective (e.g. measurable by some test or marker) or subjective (e.g. the subject gives an indication of or feels an effect). Compounds of the invention may be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, sublingually, by any other parenteral route or via inhalation, in a pharmaceutically acceptable dosage form.

Compounds of the invention may be administered alone, but are preferably administered by way of known pharmaceutical formulations, including tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, and the like. The type of pharmaceutical formulation may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.

Such formulations may be prepared in accordance with standard and/or accepted pharmaceutical practice. Otherwise, the preparation of suitable formulations may be achieved non-inventively by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.

According to a further aspect of the invention there is thus provided a pharmaceutical formulation including a compound of the invention, as hereinbefore defined, in admixture with a pharmaceutically acceptable adjuvant, diluent and/or carrier.

Depending on e.g. potency and physical characteristics of the compound of the invention (i.e. active ingredient), pharmaceutical formulations that may be mentioned include those in which the active ingredient is present in at least 1% (or at least 10%, at least 30% or at least 50%) by weight. That is, the ratio of active ingredient to the other components (i.e. the addition of adjuvant, diluent and carrier) of the pharmaceutical composition is at least 1 :99 (or at least 10:90, at least 30:70 or at least 50:50) by weight.

The amount of compound of the invention in the formulation will depend on the severity of the condition, and on the patient, to be treated, as well as the compound(s) which is/are employed, but may be determined non-inventively by the skilled person. The invention further provides a process for the preparation of a pharmaceutical formulation, as hereinbefore defined, which process comprises bringing into association a compound of the invention, as hereinbefore defined, or a pharmaceutically acceptable ester, amide, solvate or salt thereof with a pharmaceutically-acceptable adjuvant, diluent or carrier. By "bringing into association", we mean that the two components are rendered suitable for administration in conjunction with each other.

Compounds of the invention may also be combined with other therapeutic agents that are inhibitors of protein or lipid kinases (e.g. PI3K, such as class I PI3K, a PIM family kinase (e.g. PIM-1 , PIM-2- and/or PIM-3) and/or mTOR), inhibitors of other enzymes such as HDAC and/or useful in the treatment of a muscular dystrophy, a cancer and/or a proliferative disease. Compounds of the invention may also be combined with other therapies (e.g., physiotherapy).

For instance, compounds of the invention may be combined with one or more treatments independently selected from surgery, one or more therapeutic agent for treating muscular dystrophy, one or more hormone therapies, one or more antibodies, and one or more immunotherapies.

A sixth aspect of the invention provides a Class III PI 3-kinase inhibitor as defined in the first aspect or second aspect of the invention for use in medicine. Hence, the Class III PI 3-kinase inhibitor may be for use in methods for treatment of the human or animal body by surgery or therapy and diagnostic methods practiced on the human or animal body.

The compounds of the invention may be useful in the treatment of those disorders in an individual in which the inhibition of Class III PI 3-kinase is desired and/or required.

According to a further aspect of the invention, there is provided a combination product comprising:

(A) a compound of the invention, as hereinbefore defined; and

(B) another therapeutic agent that is useful in the treatment of those disorders in an individual in which the inhibition of Class III PI 3-kinase is desired and/or required (e.g., muscular dystrophy),

wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier. Such combination products provide for the administration of a compound of the invention in conjunction with the other therapeutic agent, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises a compound of the invention, and at least one comprises the other therapeutic agent, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including a compound of the invention and the other therapeutic agent).

Thus, there is further provided:

(1) a pharmaceutical formulation including a compound of the invention, as hereinbefore defined, another therapeutic agent that is useful in the treatment of those disorders in an individual in which the inhibition of Class III PI 3-kinase is desired and/or required (e.g., muscular dystrophy), and a pharmaceutically- acceptable adjuvant, diluent or carrier; and

(2) a kit of parts comprising components:

(a) a pharmaceutical formulation including a compound of the invention, as hereinbefore defined, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier; and

(b) a pharmaceutical formulation including another therapeutic agent that is useful in the treatment of the treatment of those disorders in an individual in which the inhibition of Class III PI 3-kinase is desired and/or required (e.g., muscular dystrophy) in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier,

which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.

Such active ingredients in combinations may act in synergy.

Thus, in relation to the process for the preparation of a kit of parts as hereinbefore defined, by bringing the two components "into association with" each other, we include that the two components of the kit of parts may be:

(i) provided as separate formulations (i.e. independently of one another), which are subsequently brought together for use in conjunction with each other in combination therapy; or (ii) packaged and presented together as separate components of a "combination pack" for use in conjunction with each other in combination therapy.

Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of the invention may be administered at varying therapeutically effective doses to a patient in need thereof. However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of the invention.

In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Compounds of the invention may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the above-stated indications or otherwise.

The compounds of the invention may be useful in the treatment of a disease/disorder arising from abnormal cell growth, function or behaviour associated with the protein or lipid kinase. Such conditions/disorders include cancer, immune disorders, cardiovascular diseases, viral infections, inflammation, metabolism/endocrine function disorders and neurological disorders.

The disorders/conditions that the compounds of the invention may be useful in treating include cancer (such as lymphomas, solid tumours or a cancer as described hereinafter), obstructive airways diseases, allergic diseases, inflammatory diseases (such as asthma, allergy and Chrohn's disease), immunosuppression (such as transplantation rejection and autoimmune diseases), disorders commonly connected with organ transplantation, AIDS-related diseases and other associated diseases. Other associated diseases that may be mentioned (particularly due to the key role of kinases in the regulation of cellular proliferation) include other cell proliferative disorders and/or non-malignant diseases, such as benign prostate hyperplasia, familial adenomatosis, polyposis, neuro-fibromatosis, psoriasis, bone disorders, atherosclerosis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis. Other disease states that may be mentioned include cardiovascular disease, stroke, diabetes, hepatomegaly, Alzheimer's disease, cystic fibrosis, hormone-related diseases, immunodeficiency disorders, destructive bone disorders, infectious diseases, Muscular dystrophies, conditions associated with cell death, thrombin-induced platelet aggregation, chronic myelogenous leukaemia, liver disease, pathologic immune conditions involving T cell activation and CNS disorders.

As stated above, the compounds of the invention may be useful in the treatment of cancer. More, specifically, the compounds of the invention may therefore be useful in the treatment of a variety of cancer including, but not limited to: carcinoma such as cancer of the bladder, breast, colon, kidney, liver, lung (including non-small cell cancer and small cell lung cancer), esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, skin, squamous cell carcinoma, testis, genitourinary tract, larynx, glioblastoma, neuroblastoma, keratoacanthoma, epidermoid carcinoma, large cell carcinoma, non-small cell lung carcinoma, small cell lung carcinoma, lung adenocarcinoma, bone, adenoma, adenocarcinoma, follicular carcinoma, undifferentiated carcinoma, papilliary carcinoma, seminona, melanoma, sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidney carcinoma, myeloid disorders, lymphoid disorders, hairy cells, buccal cavity and pharynx (oral), lip, tongue, mouth, pharynx, small intestine, colon-rectum, large intestine, rectum, brain and central nervous system, Hodgkin's and leukaemia; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocitic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplasia syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; and other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoxanthoma, thyroid follicular cancer and Kaposi's sarcoma.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

Figure 1 : Autophagy is increased in skeletal muscle from dy3K/dy3K mice. (A)

Relative amounts of LC3B, GabarapH, Atg4b, Vps34, Beclin, Cathepsin L and Lamp2a mRNAs in 3.5-week-old wild-type, dy3K/dy3K mice and in 4.5-week-old 3- MA injected wild-type and dy3K/dy3K mice (n=6 for each group). The GAPDH gene expression served as a reference. (B) Left panel: Co-immunostaining on cross sections of quadriceps muscles from uninjected wild-type (a, n=5) and dy3K/dy3K (b, n=5) mice and 3-MA wild type (c, n=6) and dy3K/dy3K (d and e, n=6) mice. LC3B (in red) is present in autophagosomes and laminin y1 chain (in green) serves as delineating fibre boundaries. Bar = 40 μιη. (C) Densitometry analysis of LC3B and Vps34 Western blot analysis in quadriceps muscle from wild-type and dy3K/dy3K mice (3.5-week-old; n=6 per group). Results are expressed in arbitrary units (AU). Labeling of tubulin served as internal loading control. (D) Densitometric analysis of LC3B, Vps34, Cathepsin L and Beclin-1 in human primary myoblasts and myotubes from a control and a laminin a2 chain deficient patient. Data represent mean of 4 different culture wells are expressed in arbitrary units (AU). *, p<0.05; **, p<0.001.

Figure 2: Muscle morphology is improved and fibrosis is reduced in skeletal muscle with systemic injection of 3-MA. (A) Hematoxylin-eosin staining of cross- sections of quadriceps (a-d) and tibialis anterior (e-h) muscles from wild-type (a, e), non-injected dy3K/dy3K (b, f), injected wild-type and dy3K/dy3K at 2.5 and 3.5 weeks of age (c, d and g, h respectively). Fourteen days later, muscles were isolated and stained. (B) Densitometric quantification of fibrosis in quadriceps muscles from 3-MA wild-type and dy3K/dy3K injected mice (n=6 for each genotype) or non-injected dy3K/dy3K (n=7) and wild-type mice (n=9). Left panel: Percentage of collagen III positive labeling of the total area of the section. Right panel: Percentage of tenascin- C positive labeling of the total area of the cross-section. *, p<0.05; **, p<0.001.

Figure 3: Atrophy is prevented in quadriceps muscle from 3-MA treated dy3K/dy3K mice. (A) Determination of fibre diameter repartition (in percentage of total number of fibres) in 3-MA injected (green and orange) and non-injected (blue and red) wild-type and dy3K/dy3K mice respectively. A significant difference exists between the curves (p<0.0001) (B) Average of fibre diameter in m. (C) Proportion (in percentage) of quadriceps muscle wet weight per body weight (wild-type, n=5; dy3K/dy3K, n=4; injected mice, n=5 respectively). *, p<0.05; **, p<0.001.

Figure 4: Systemic injection of 3-MA stimulates regeneration in quadriceps of dy3K/dy3K mice. (A) Proportion of fibres with centrally located nuclei in 3-MA injected dy3K/dy3K and wild-type mice, non-injected dy3K/dy3K and wild-type mice (n=6 for each group). (B) Upper part: Co-immunolabeling on cross sections of quadriceps muscles from uninjected wild-type (n=5) and dy3K/dy3K (n=5) mice and 3-MA injected wild-type (n=6) and dy3K/dy3K (n=6) mice. Laminin γ1 chain (red) delineates fibre boundaries and embryonic myosin heavy chain (eMHC) (green) is expressed only by regenerative fibres. DAPI (in blue) denotes nuclei. Bar = 40 μηι. Lower part: Percentage of fibres expressing eMHC. (C) Co-immunostaining using antibodies against laminin γ1 chain (red), MyoD (green) and DAPI (blue). Arrows denote MyoD positive nuclei in the interstitial space between myofibres *, p<0.05; **, p<0.001.

Figure 5: Apoptosis is diminished after systemic injection of 3-MA in dy3K/dy3K mice. (A) Co-immunostaining using antibodies against pro-caspase 3 and caspase 3 isoforms (green) and laminin y1 chain (red) in 3-MA injected wild-type and dy3K/dy3K quadriceps muscle (a, b). Fourteen days after 3-MA systemic injection, green positive fibres were found in restricted areas of dy3K/dy3K quadriceps muscle, (b) but most parts of the muscle are marked by the absence of apoptotic fibres (a). Scale bar = 40 μηι. (B) Percentage of caspase 3 positive fibres in whole quadriceps muscle sections from 3-MA injected dy3K/dy3K and wild-type mice (n=6 for both). (C) Percentage of TUNEL-positive myonuclei in whole quadriceps sections from 3-MA injected dy3K/dy3K and wild-type mice (n=6 for each group). *, p<0.05; ***; pO.001. Figure 6: Akt signaling is restored upon autophagy inhibition. Densitometric analysis and representative Western blot images of phospho-Akt308/Akt and phospho-Akt473/Akt in quadriceps muscle from 3-MA injected wild-type and dy3K/dy3K after 48 hours (A) (n=3 for each genotype) and after 14 days (B) (n=4 and 6 respectively). Data are expressed in arbitrary units (AU) as phospho-Akt is normalized to Akt. Tubulin is used as an internal loading control.

Figure 7. Systemic injection of 3-MA improves dy3K/dy3K mice locomotion, body weight and survival. (A) Exploratory locomotion of approximately 4-week-old mice in an open field test (n=14 for each group). (B) Body weight measurement of 4- week-old non-injected and 3-MA treated wild type (n=7 respectively) and dy3K/dy3K mice (n=12 respectively) (C) Survival curves of dy3K/dy3K ± two systemic injections of 3-MA. The median survival for non-injected dy3K/dy3K mice is 22 days (15) whereas it is 37 days for the treated animals. *p<0.05; **, p<0.001.

Figure 8. Autophagy is not upregulated in quadriceps muscle from mdx mice.

Relative mRNA expression of LC3B, Cathepsin L, Lamp2a, GabarapH, Atg4b, Vps34 and Beclin mRNAs in 5-week-old wild-type and mdx mice (n=6 for each genotype) (upper part) and 3-month-old wild-type and mdx mice (n=3 for each genotype). The GAPDH gene expression served as a reference. *, p< 0.01 ; **, p<0.001 ; ***, p<0.0001.

Figure 9. Systemic injection of 3-MA normalizes laminin a4 and a2 chain expression. (A) Immunofluorescence experiments using antibodies against laminin a2 (a, b) and laminin a4 (c, d) chain on cross-sections of quadriceps muscle from wild-type (a, c) and d ^/dy 3 mice (b, d) treated with systemic injection of 3-MA (n=6 for each genotype). Scale bar = 40 Mm. (B) Densitometric analysis of laminin a4 chain in quadriceps muscle from wild-type, non-treated d^/dy 3 and 3-MA injected after 14 days (n=6, 4 and 5, respectively). Data are expressed in arbitrary units (AU) as laminin Π4 is normalized to a-actinin. *, p<0.05.

Figure 10. Atrogene expression is not significantly modified in laminin a2 deficient peripheral nerve. Relative amounts of LC3B, Cathepsin L, Lamp2a, GabarapH, Atg4b, Vps34 and Beclin mRNAs in 3.5-week-old wild-type (n=5) and mice (n=5). The GAPDH gene expression served as a reference. Figure 11. (A) Graphs showing Vps34 inhibition as a function of concentration for eight compounds. MDP-number is given in upper left corner in each figure (with coloured background) and IC50 value is given in the lower left of each figure. For example, uppermost figure to the left corresponds to MDP-001 with IC50 equal to 51 μΜ. (B) Graphs showing Vps34 inhibition as a function of concentration for eight compounds. See figure 1A for explanation. (C) Graphs showing Vps34 inhibition as a function of concentration for eight compounds. See figure 1A for explanation. (D) Graphs showing Vps34 inhibition as a function of concentration for eight compounds. See figure 1 A for explanation.

Figure 12. (A) Compound structures and their assayed IC50 data. (B) Compound structures and assayed IC50 data. The last four compounds are known potent Vps34 inhibitors.

Figure 13. (A) Graphs showing Vps34 inhibition as a function of concentration for eight compounds. MDP-number is given in upper left corner in each figure and on top of each graph. (B) Graphs showing Vps34 inhibition as a function of concentration for eight compounds. MDP-number is given in upper left corner in each figure and on top of each graph. (C) Graphs showing Vps34 inhibition as a function of concentration for eight compounds. MDP-number is given in upper left corner in each figure and on top of each graph. (D) Graphs showing Vps34 inhibition as a function of concentration for eight compounds. MDP-number is given in upper left corner in each figure and on top of each graph.

Figure 1 . (A) AT m for the concentration dependent stabilization for MDP-008 in duplicate. (B) AT m for the concentration dependent stabilization for MDP-040 in duplicate.

EXAMPLES

Example A - Biological examples

Abstract

Congenital muscular dystrophy with laminin a2 chain deficiency (also known as MDC1A) is a severe and incapacitating disease. It has recently been shown that increased proteasomal activity is a feature of this disorder. The autophagy-lysosome pathway is the other major system involved in degradation of proteins and organelles within the muscle cell. However, it remains to be determined if the autophagy- lysosome pathway is overactive in muscular dystrophies including MDC1A. Using the dy3K/dy3K mouse model of laminin a2 chain deficiency and MDC1A patient muscle cells, it is now shown that expression of autophagy-related genes is upregulated in laminin a2 chain deficient muscle. Moreover, it is found that autophagy inhibition significantly improves the dystrophic dy3K/dy3K phenotype. In particular, it is shown that systemic injection of 3-methyladenine (3-MA) reduces muscle fibrosis, atrophy, apoptosis and increases muscle regeneration and weight. Importantly, lifespan and locomotive behaviour were also greatly improved. These findings demonstrate that enhanced autophagic activity is pathogenic and that autophagy inhibition has therapeutic potential in the treatment of MDC1A.

Introduction

Macroautophagy (hereafter referred to as autophagy or autophagocytosis) is a multi- step catabolic process involving the sequestration of bulk cytoplasm, long-lived proteins and cellular organelles in autophagosomes, which are subsequently fused with lysosomes and content is digested by lysosomal hydrolases (1 , 2). Autophagy is generally activated by conditions of nutrient or growth factor deprivation as well as endoplasmic reticulum stress. In addition, autophagy has also been associated with a number of physiological processes including development, differentiation, or pathologies like neurodegenerative diseases, lysosomal storage diseases, infection, or cancer (1 ,3). However, very little is known about autophagy and muscular dystrophy. The role and regulation of the autophagic pathway in skeletal muscle is still largely unknown but it is believed that excessive autophagy activation contributes to muscle loss in different catabolic conditions (4). Interestingly, inhibition of the autophagic flow may also result in muscle atrophy (5). In yeast, autophagy is controlled by more than 30 autophagy-related genes and many of them have mammalian homologues (6). Notably, through inhibition of Akt, Fox03 controls the transcription of autophagy-related genes (e.g. LC3, Cathepsin L, Lamp2a, GabarapH, Vps34, Atg4b and Beclin) and therefore the autophagic-lysosomal pathway during muscle atrophy (7-9).

Recently, it was demonstrated that autophagy is impaired in collagen VI deficient muscular dystrophy and that its reactivation ameliorated the dystrophic phenotype in a mouse model of the disease (10). Another type of congenital muscular dystrophy is MDC1A (OMIM #607855), which is caused by autosomal recessive mutations in the human LAMA2 gene, encoding the a2 subunit of the extracellular basement membrane protein laminin-211. MDC1A is characterized by severe generalized muscle weakness, joint contractures and peripheral neuropathy. Around 30% of the patients die within their first decade of life (11 ,12). The generated null mutant dy3K/dy3K mouse model for laminin a2 chain deficiency recapitulates human disease and presents severe muscular dystrophy and dy3K/dy3K mice also display peripheral neuropathy (13, 14). Histological features of laminin a2 chain deficient muscles include degeneration/regeneration cycles, fibre size variability, apoptosis and marked connective tissue proliferation. Also, skeletal muscle atrophy is a prevalent feature of MDC 1 A (11 , 12, 15).

In the following study it is shown that expression of autophagy-related genes is upregulated in laminin a2 chain deficient muscle and that inhibition of the autophagy process significantly improves the dystrophic phenotype in the dy3K/dy3K mouse model.

Materials & Methods Transgenic animals

Laminin a2 chain deficient mice {dy3K/dy3K), which lack laminin a2 chain completely, were used and previously described (13, 20). These mice develop severe muscular dystrophy and peripheral neuropathy and the median survival is around 22 days. For all experiments, dy3K/dy3K mice were compared with their wild- type (WT) littermates. Animals were maintained in the animal facilities of Biomedical Center (Lund) according to animal care guidelines, and permission was given by the regional ethical board.

Primary muscle cell culture and differentiation

Primary myoblasts were obtained from a control fetus (12 weeks of gestation) and a MDC1A fetus (15 weeks of gestation), presenting a homozygous nonsense mutation in exon 31 of the LAMA2 gene. Muscle cells were obtained in accordance with the French legislation on ethical rules. Cells were cultivated in 6-well plates with growth medium (F10-Ham medium, Gibco) containing 20% foetal bovine serum (Gibco) at 37°C, 5% C02. At about 70% confluency, differentiation into myotubes was initiated by switching to fusion medium (DMEM, Gibco) containing 2% horse serum (Gibco), 10-6 M insulin (Sigma) and 2.5x10-6 M dexamethasone (Sigma). Protein lysates were obtained by scrapping the cells directly into the lysis buffer (50mM Tris-HCI, pH 6.8, 10% β-mercaptoethanol, 4% SDS, 0.03% bromophenol blue and 20% glycerol).

Systemic injections of 3-methyladenine

Systemic administration was performed by intraperitoneal injection of 3-MA (15 mg/kg) into dy3K/dy3K mice and control littermates at the age of 2.5 weeks and 3.5 weeks. Mice were sacrificed 14 days after injection and quadriceps and tibialis anterior muscles were processed for morphometric analysis, immunofluorescence experiments, qRT-PCR or Western blot analysis. Prior to the euthanasia, an exploratory locomotion test was performed.

RNA extraction, reverse transcription and quantitative real-time PCR

Total RNA was extracted from 10 mg quadriceps muscle of 6 dy3K/dy3K mice (3.5- week-old) and 6 WT littermates and 5 WT and 5 dy3K/dy3K mice treated with 3-MA using RNeasy mini kit (Qiagen) including an initial step of proteinase K digestion (Fermentas, 240 ng/μΙ). Complementary DNA was synthesized from I ig of total RNA with random primers and SuperScriptlll reverse transcriptase (Invitrogen) following manufacturer's instructions. Quantitative PCRs were performed in triplicate with the Maxima SYBR Green qPCR Master Mix (Fermentas). Expression of target and reference genes was monitored using a real-time qRT-PCR method (Light Cycler, Roche) with the previously described primers for the autophagic genes LC3B, Cathepsin L, Lamp2a, GabarapH, Atg4b, Vps34 and Beclin (8). The amplification efficiency for each primer pair was evaluated by amplification of serially diluted template cDNAs (E=10 "r/Slope ). Efficiency corrected RNA levels (in arbitrary units) were calculated by using the formula E ct . Expression levels were then calculated relative to the endogenous control gene GAPDH and relative to wild-type quadriceps.

Protein extraction and Western blot analyses Isolated quadriceps muscles were obtained from 6 wild-type, 6 dy3K/dy3K mice (3.5 weeks of age) and 6 dy3K/dy3K mice 48h or 14 days after 3-MA injection. Each sample was immediately frozen in liquid nitrogen and reduced to powder using a mortar. Protein extracts were obtained as previously described (16). A total of 30 g of denaturated protein was loaded on 10-20% acrylamide SDS-gels (Clearpage, CBS Scientific) and blotted onto nitrocellulose membranes (Hybond-C, Amersham) during 1.5 hour (Biorad). The membranes were blocked for one hour at RT in PBS, 0.01% Tween-20, 5% milk and incubated overnight at 4°C with rabbit polyclonal antibodies directed against pAkt (Ser 473, 1/2000, #4060 or Thr 308, 1/1000, #2965, Cell Signaling Technology), Akt (1/1000, #4685, Cell Signaling Technology), Vps34 (1/200, V9764, Sigma) or LC3B (1/250, #2775, Cell Signaling Technology). Blots were then washed 3 times 10 minutes with PBS, 0.05% Tween 20, incubated with horseradish peroxidase-conjugated polyclonal goat anti-rabbit (1/4000, sc-2004, Santa Cruz Biotechnology) or goat anti-mouse (1/4000, sc-2005, Santa Cruz Biotechnology) antibody for 1 hour. Membranes were incubated in ECL (Amersham Biosciences), exposed on Hyperfilm (Amersham Biosciences) and developed (AGFA, Curix 60). Each membrane was rehybridized with mouse monoclonal anti-tubulin (1/4000, clone DM 1A, Sigma) for loading normalization. The quantifications were performed using ImageJ 1.40 (http://rsb.info.nih.gov/ij/download.html).

Histology and immunofluorescence experiments

Quadriceps and tibialis anterior muscles from wild-type, dy3K/dy3K and injected mice (n=6 for each group) were rapidly dissected after euthanasia and frozen in OCT (Tissue Tek) in liquid nitrogen. Serial sections of 7 were either stained with hematoxylin and eosin or processed for immunofluorescence experiments following standard procedures (20) with rabbit polyclonal antibodies directed against LC3B (1/100, #3868, Cell Signaling Technology), laminin γ1 chain (1/1000, #1083), laminin a4 chain (1/400, #1100) and laminin β2 chain (1/400, #1117) generously provided by Dr. T. Sasaki, rat monoclonal antibodies against laminin γ1 chain (1/200, MAB 1914, Chemicon) and tenascin-C (undiluted, MTn15), goat polyclonal antibody against collagen III (1/100, #1330, SouthernBiotech) and mouse monoclonal antibodies against caspase-3 (1/100, CPP32, BD Transduction Laboratory), embryonic myosin heavy chain (F1.652, Developmental Studies Hybridoma Bank) and MyoD (1/100, clone 5.8A Dako). For apoptotic myofibre detection, a TUNEL detection kit was used following instructions of the manufacturer (GenScript). Sections were analyzed using a Zeiss Axioplan fluorescence microscope. Images were captured using an ORCA 1394 ER digital camera with the Openlab 3 software.

Exploratory locomotion test

Exploratory locomotion was examined in an open field test. In each experiment, the mouse 14 days after 3-MA injection (n=11 for dy3K/dy3K and wild-type, respectively) was placed into a new cage and allowed to explore the cage for 5 min. The time that the mouse spent moving around was measured manually.

Survival curves

Death was monitored in 3-MA injected dy3K/dy3K mice (n=9). A survival curve was constructed using the GraphPad Prism 4 software.

Morphometric analysis

Measurements were performed on whole quadriceps or tibialis anterior muscle sections from untreated wild-type and dy3K/dy3K, wild-type and dy3K/dy3K 3-MA injected animals (n=6 for each group). Tenascin-C and collagen III positive areas, eMHC positive fibres, caspase-3 positive fibres, TUNEL-positive myonuclei and fibre diameters were measured using the ImageJ software. Minimal Feret's diameter was measured (41) for at least 1500 fibres for each mouse. The same number of fibres was used for quantification of fibres with centrally located nuclei. Wet quadriceps muscle weights were determined from 7 uninjected wild-type or dy3K/dy3K and 3-MA treated wild-type (n=4) and dy3K/dy3K (n=6) animals and correlated to body weight.

Statistical analysis

All tests for analysis of significance were done using the GraphPad Prism 4 software. For quantitative PCR experiments, protein quantifications, morphometric analysis and exploratory locomotion test, one way ANOVA followed by a Bonferroni's post multiple comparison test was performed. Regarding fibre size distribution, a x2-test was calculated and paired comparison of distribution was estimated related for a p- value inferior to 0.0001. Finally, statistic LogRank test was used for analysis of significance of survival curves. Data always represent mean ± SEM. Results

Increased expression of autophagy related genes in laminin a2 chain-deficient muscle

To determine whether the activity of the autophagy lysosome pathway is increased in laminin a2 chain deficient muscle, we first analyzed the expression of members of this pathway in dy3K/dy3K animals and in particular those controlled by the transcription Fox03, whose expression is increased approximately 2-fold in dy3K/dy3K animals (16). We detected significantly increased mRNA levels of the microtubule-associated protein-1 light chain 3B (LC3B) in quadriceps muscles from dy3K/dy3K mice (Fig. 1A). LC3B is one of the three (human) LC3 isoforms that undergoes post-translational modifications during autophagy. The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form LC3II have been used as indicators of autophagy (17, 18). By immunofluorescence analysis we detected accumulated LC3B in dy3K/dy3K quadriceps muscle fibres (Fig. 1 B) and Western blot analysis revealed an approximate 2-fold increase of LC3BII expression in dy3K/dy3K quadriceps muscle (Fig. 1C). Similarly, we noted an increased mRNA expression of the autophagosome membrane markers GabarapH, Beclin and Vps34 as well as the cysteine protease Atg4B in dy3K/dy3K quadriceps muscle (Fig. 1A). Finally, mRNA expression of lysosomal markers Cathepsin L and Lamp2a was also significantly increased in dy3K/dy3K quadriceps muscle.

To determine if enhanced expression of autophagy-related genes also is seen in human laminin a2 chain deficient muscle, we analyzed primary myoblasts and myotubes from a control and a laminin a2 chain deficient patient. Increased protein expression of LC3BII, Vps34, Cathepsin L and Beclin was noted in the MDC1A myotubes but not in corresponding myoblasts (Fig. 1 D).

Laminin a2 chain interacts with the dystrophin-glycoprotein and mutations in several of its components lead to various forms of muscular dystrophy (19). To investigate if autophagy is modified when dystrophin is absent and other members of the dystrophin-glycoprotein complex are reduced, we quantified the expression level of autophagy related genes in quadriceps from mdx mice (a Duchenne muscular dystrophy mouse model). We found no major modification in the expression of LC3B, GabarapH, Beclin, Vps34 and Atg4B mRNAs in 5-week- or 3-month-old mice. Only Cathepsin L mRNA expression was elevated in 5-week- and 3-month-old mdx muscle and Lamp-2 mRNA expression was also increased in 3-month- old mdx mice (Figure 8), suggesting that microautophagy followed by chaperone-mediated autophagy could be modified in this disease.

Systemic injection of 3-methyladenine (3-MA) restores autophagic gene expression in laminin a2 chain-deficient muscle

Since the autophagy-lysosome pathway system seemed to be overactive in dy3K/dy3K muscle, we envisaged that inhibition of the autophagy pathway could improve muscle shape and mouse physiology. Thus, we administered the autophagy inhibitor 3-MA into the peritoneum of 2.5-week-old dy3K/dy3K mice. At this age, the dy3K/dy3K mice start to be distinguishable from their littermates. We repeated the injection at 3.5 weeks of age. The median survival of dy3K/dy3K mice is around 22 days and most if not all dy3K/dy3K are dead by 4 weeks of age (16). We analyzed mice and muscles 14 days post-injection (a time point when dy3K/dy3K mice should be dead). Notably, we found that the systemic injection of 3-MA restored the expression of the autophagy-related genes to the basal level (Fig. 1A-C).

Systemic injection of 3-MA improves muscle morphology in laminin a2 chain-deficient muscle

Remarkably, the 3-MA injections resulted in considerably improved muscle morphology. We first evaluated the main histological hallmarks of the dystrophic process (pathological fibrosis and muscle fibre diameter) by morphometric measurements. Collagen III expression, which previously has been shown to be increased in dy3K/dy3K muscle (16), was reduced in 3-MA injected mice compared with non-injected dy3K/dy3K mice (Fig. 2B). To further confirm the reduction of fibrosis in 3-MA-treated animals, we analyzed tenascin-C expression, which also has been demonstrated to be increased in dy3K/dy3K muscle (16, 20). Similarly, tenascin-C expression was reduced in 3-MA injected mice compared with non- injected dy3K/dy3K mice (Fig. 2B).

We also investigated the expression of laminin a4 and β2 chains in 3-MA treated dy3K/dy3K mice. It has previously been shown that the expression of laminin a4 chain is increased at the dy3K/dy3K sarcolemma whereas the laminin β2 chain expression is reduced (20, 21 ). Expression of both proteins was near normal in injected mice (Figure 9). It is well established that the average fibre diameter is significantly reduced in dy3K/dy3K muscle (16, 22, 23). Notably, the average fibre diameter was increased upon 3-MA injection and fibre size distribution in quadriceps muscle was significantly shifted towards larger fibres for both wild-type and dy3K/dy3K injected animals (Fig. 3A, B). We observed that 25% of dy3K/dy3K quadriceps fibres have a diameter inferior to 26 μητι, whereas the number is about 15% in wild-type and dy3K/dy3K injected animals, respectively. Furthermore, the ratio of quadriceps muscle wet weight per body weight was normalized in 3-MA injected dy3K/dy3K mice, compared to age-matched non-injected dy3K/dy3K mice (Fig. 3C).

Systemic injection of 3-MA stimulates muscle regeneration in laminin a2 chain- deficient muscle

The proportion of centrally-located nuclei is one of the main features of the degeneration-regeneration process. The number of cells with centrally located nuclei was slightly but significantly elevated in 3-MA injected dy3K/dy3K mice (Fig. 4A). We additionally performed immunofluorescence experiments analyzing the expression of regeneration markers embryonic myosin heavy chain (a specific marker of newly regenerated fibres) and MyoD1 (present in activated satellite cells and myoblasts). Indeed, the proportion of fibres expressing eMHC significantly increased with the 3- MA injection of dy3K/dy3K mice (Fig. 4B). Also, the amount of MyoD1 positive nuclei was increased in 3-MA injected dy3K/dy3K mice (Fig. 4C).

Apoptosis is decreased after systemic injection of 3-MA

As apoptosis contributes to the disease progression, we analyzed the apoptosis rate occurring in skeletal muscle of systemically injected mice. As previously described, the number of caspase-3 positive fibres (containing caspase-3 and pro-caspase 3 proteins) in dy3K/dy3K mice, was significantly increased when compared to controls (16). Forty-eight hours after 3-MA injection, we were able to find caspase-3 positive fibres in the same proportion as in non-injected dy3K/dy3K mice (data not shown). However, 14 days after injection the proportion of caspase 3 positive fibres was significantly decreased in 3-MA injected dy3K/dy3K quadriceps (Fig. 5A-B). These results were further confirmed using the TUNEL enzymatic labeling assay. We found that the proportion of TUNEL-positive myonuclei was significantly reduced in 3-MA treated dy3K/dy3K animals (Fig. 5C). Systemic injection of 3-MA restores Akt phosphorylation

We have recently demonstrated that Akt phosphorylation on both threonine 308 and serine 473 is diminished in dy3K/dy3K quadriceps muscle, whereas the total level of Akt is unchanged (16). To investigate whether injection of 3-MA could restore Akt activity, we sacrificed mice 48 h and 14 days after injection and learned that Akt phosphorylation on both sites was restored to wild-type levels at both time points (Fig. 6A-B).

Systemic injection of 3-MA increases survival and locomotive behaviour, but does not significantly improve peripheral neuropathy

Dy3K/dy3K mice are significantly less active in an open field test (16). Remarkably, 3-MA injected dy3K/dy3K mice displayed the same level of activity as wild-type animals (Fig. 7A). Also, 3-MA treated dy3K/dy3K mice weighed significantly more than non-injected dy3K/dy3K mice, although they never reached the weight of wild- type mice (Fig. 7B). Moreover, the median survival of 3-MA injected dy3K/dy3K mice was 37 days (Fig. 7C), whereas it has been shown to be 22 days for non-treated dy3K/dy3K mice (16). Finally, although survival and muscle morphology was significantly improved, transient hind leg paralysis often occurred in one leg of 3-MA treated dy3K/dy3K mice and similar paralysis occurred in non-treated dy3K/dy3K mice but not in 3-MA injected wild-type mice (16) (data not shown). Yet, this transient paralysis of had no obvious effect on the locomotive behavior. Nevertheless, it is clear that 3-MA did not appreciably improve the pathology of the peripheral nerve. In agreement with this observation, we found no increased mRNA levels of autophagy- related genes in laminin a2 chain deficient sciatic nerve (Figure 10).

Discussion

MDC1A is a debilitating muscle disease for which there currently is no cure. Several approaches to prevent disease in MDC1A mouse models have been explored and they include gene replacement- (20, 24, 25), anti-apoptosis- (26-28), proteasome inhibition- (16), cell- (29) and improved regeneration therapy (30). While the transgenic strategies (e.g. over-expression of laminin a2 chain, mini-agrin and in particular laminin a1 chain) may have offered the most complete muscle restoration they are not yet clinically feasible and the pharmacological inhibition of apoptosis and proteasome, respectively, have only resulted in partial recovery. Hence, other potential therapeutic targets should be explored. Here we present data indicating that increased autophagy is pathogenic in MDC1A. We found increased expression of several autophagy-related genes in laminin a2 chain deficient mouse and human muscle cells. We have shown that autophagy inhibition, using 3-MA in the dy3K/dy3K mouse model for MDC1A, significantly reduces many of the pathological symptoms in the dystrophic mice.

Apoptosis has been described as a major feature in MDC1A and its inhibition by genetic or pharmacological therapy ameliorated several pathological symptoms in the dyW/dyW mouse model of MDC1A (26-28, 31 ). Autophagy and apoptosis are interconnected by common proteins and functions. First, autophagy is a basal mechanism for elimination of damaged protein or organelles. Therefore accumulation of mitochondria or misfolded proteins could initiate oxidative stress and cell death. Second, it has recently been described that the regulation of autophagy by survival signals in skeletal muscle is controlled by a rapid transcription-independent mechanism through mTOR, and a long term but more effective transcription program requiring Fox03 (7-9, 32). Finally, anti-apoptotic proteins, such as Bcl-2 family members, inhibit Beclin-1 and induction of autophagy proteins could enhance cell death (33, 34). Consequently, it would be interesting to test whether the combined inhibition of apoptosis and autophagy would further restore the phenotype of laminin a2 chain deficient mice. Furthermore, we recently showed that global ubiquitination of proteins is raised in dy3K/dy3K muscles and that proteasome inhibition improves the dystrophic phenotype (16). In addition, it has been demonstrated that ubiquitinated proteins also can be delivered to the autophagosomes through the p62/SQSTM1 complex that is able to bind LC3 (35-39). Hence, we would like to evaluate combinatorial treatment of autophagy and proteasome inhibition.

Interestingly, together with the data we presented here, incorrect function of autophagy has been discovered to be pathogenic in the two most common forms of congenital muscular dystrophy and both are linked to deficiency of extracellular matrix proteins (10). We therefore hypothesize that an extracellular matrix unbalance affects the autophagy pathway. The additional data that we provide on the Duchenne mouse model mdx, showing that autophagy is not modified, reinforces this hypothesis. In this model, it seems that microautophagy followed by chaperone- mediated autophagy (dependant of Lamp2) could be stimulated with the progression of the disease. This should be further clarified as well as the potential primary or secondary contribution of autophagy in other congenital muscle diseases (dystroglycanopathies or congenital myopathies). Autophagosomes are present in many myopathies and are the major features of a group of muscle disorders named autophagic vacuolar myopathies. This group is composed by the late-onset Pompe disease, caused by a defect in lysosomal acid maltase (MIM ID #232300), Danon disease that primarily affects the heart, due to a defect in the LAMP2 gene (MIM ID #300257), and X-linked myopathy with excessive autophagy (XMEA), associated with mutations in the VMA21 gene (40). Therefore, autophagy related genes could be potential candidate genes mutated in genetically irresolute muscle diseases.

In summary, our study demonstrates for the first time that autophagy can be overactive in a congenital muscular dystrophy condition. In addition, its inhibition improves the muscle phenotype of laminin a2 chain deficient mice.

The results provide compelling evidence in support of the efficacy of autophagy inhibitors in the treatment and prevention of muscular dystrophies, such as MDC1A.

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Example B - Enzymatic assays of test compounds

Introduction Lipid kinase activity of human VPS34 was determined using a ADP-Glo™ luminescent ADP detection kinase assay (Promega) according to the manufacturer's protocol. In addition, binding of the compounds has also been monitored by concentration dependent stabilization using differential scanning fluorimetry.

Human VPS34 enzyme assay method

0.5μΙ compounds in 10 % DMSO (5-fold dilution series from 50 mM DMSO stock solutions, followed by 10-fold dilution in water) were added to wells of a 384 well microtitre plate (Greiner #784080). 2.5 μΙ VPS34 (20 nM, ProQinase product no. 1160-0000- 1)/ATP (20 μΜ, from Promega ADP-Glo™ kit #V9101) mix in kinase buffer (50 mM Hepes, pH 7.5, 1 mM EGTA, 0,1 % CHAPS, 3 mM MnCI 2 , 0.5 mM MgCfe, 100 mM NaCI and 2 mM DTT) was added and pre-incubated 5 min before adding 2.5 μΙ of a mix of PI substrate (Avanti Polar Lipids #840044) in kinase buffer (as above) and the plate was incubated after shaking at room temperature for 1 hour. Then 5 μΙ ADP-Glo™ reagent from ADP-Glo™ kit (as above) was added and incubated 40 min to stop reaction and consume remaining ATP, before adding 10 μΙ kinase detection substrate (dissolved in kinase detection buffer, both from ADP- Glo™ kit as above) to convert ADP to ATP and detect ATP with luciferase/luciferin. The plates were incubated for 30 minutes at room temperature before reading luminescence on an Envision (PerkinElmer) reader.

Vps34 inhibition Screening: Round 1

The Vps34 inhibition activity was determined for various test compounds using the above assay.

First screening round with estimated IC50 values are given in Table 2A and 2B. The comments are subjective interpretations added by person performing the measurements. Notations are as follows, P="Precipitation", AI="Assay Interference", NV="No Value".

Graphs with more detailed results are given in figures 11 A to 11 D.

The chemical structures of the tested compounds are given in Figures 12A and 12B (some compound structures correspond to ionized states). Table 2A. Kinase inhibition results for 3-MA analogues

Table 2B. Kinase inhibition results for known Vps34 inhibitors

Vps34 inhibition Screening: Round 2 All compounds studied in screening round 2 are MDP-001 analogues, except for the reference compound MDP-008, which is an unrelated known potent Vps34 inhibitor.

Estimations of IC50 values are given in Table 3. The comments are subjective interpretations added by person at company performing the measurements. P="Precipitation".

Graphs with more detailed results are given in figures 13 A to 13D.

Table 3. Kinase inhibition results for 3-MA analogues and for the known inhibitor

MDP-008.

VPS34 - Concentration dependent stabilization

The method Differential Scanning Fluorimetry (DSF) has been used to identify binding of compounds to a protein. Upon unfolding of a protein hydrophobic parts of the protein are exposed. By adding a dye with affinity for hydrophobic parts of the protein there is possible to measure the unfolding of a protein when the temperature increases. Adding a compound that binds to the protein usually increases the stability and thus a higher melting temperature is observed in comparison with the apo protein (Niesen FH et. al. Nat Protoc. 2007;2(9):2212-21). Here we have used DSF to test 58 different compounds for concentration dependent stabilization of VPS34.

In this assay a truncated version of VPS34 was used containing the kinase domain (amino acids 282-879). The VPS34 was purchased from Protein Science Facility, Karolinska Insitutet, Stockholm, Sweden, that followed the SGC protocol for protein purification (http://www.thesgc.org/structures/details?pdbid=3IHY). Prior to measuring protein melting temperature using DSF, VPS34 were centrifuged for 5 min in order to remove possible protein aggregates. 1mM compound in DMSO was added to wells containing the highest concentration. Protein was added to each well to a final concentration of 0.2 mg/ml in a buffer containing 20mM Hepes pH 7.5, 300mM NaCI, 10% glycerol and 2mM tris(2-carboxyethyl)phosphine with the addition of 5X SYPRO orange (Invitrogen, cat. no. S6650). For each compound a series of dilutions were made from the well containing the highest concentration 1mM down to 0.0625 mM in five steps. The experiment was performed in 96-well plate (# AB-0700/W, Thermo SCIENTIFIC). All compounds were run in duplicates together with controls with no compound added. Before running the experiment the plate was centrifuged for 1 min at 200 g at 20°C. The temperature scan was run from 25 - 95°C, at 1°C min 1 using a real time PCR instrument (Stratagene, Mx3005p, AH Diagnostics).

Table 4 Indicates the largest shift for each compound and at what concentration it was obtained at.

Compound Highest Concentration where thermal shift the highest thermal (°C) shift is observed

MDP-020 7.5 0.5mM

MDP-021 4.2 0.5mM DP-022 5.4 0.5mM

MDP-023 4.2 0.5mM

MDP-024 5.2 1mM

MDP-025 5.9 1mM

MDP-026 4.4 1 mM

MDP-027 9.5 1mM

MDP-028 2.7 1 M

MDP-029 5.9 1mM

MDP-030 6.8 1 mM

MDP-031 3.2 1mM

MDP-032 8.13 1mM

MDP-033 5.5 0.5mM

MDP-034 5 1 mM

MDP-035 10.3 1mM

MDP-036 21.7 0.0625mM

MDP-038 12.6 0.25mM

MDP-039 5.7 0.25mM

MDP-040 5.4 1mM

MDP-041 6 0.5mM

MDP-042 9.5 1mM

MDP-043 4.4 1 mM

MDP-044 6.6 1mM

MDP-045 5.4 1 mM

MDP-046 3.4 1mM

MDP-047 2.2 0.5mM

MDP-048 1.6 0.0625mM

MDP-049 1.2 0.5mM

MDP-050 6.6 0.5mM

MDP-051 2.4 1 mM

MDP-052 3.4 1mM

MDP-053 9.1 1 mM

MDP-054 3.7 0.5mM

MDP-055 8 1 mM

MDP-056 5.6 0.0625mM

MDP-057 4.4 0.0625mM

MDP-058 7.7 1mM

MDP-059 5.8 1mM

MDP-060 1.5 1mM

MDP-061 6 1 mM DP-062 6.8 0.5mM Compound Highest Concentration where

thermal shift the highest thermal

(°C) shift is observed

MDP-063 9.3 1mM

MDP-064 4.9 0.5mM

MDP-065 5.4 0.5mM

MDP-066 3.8 0.062mM

MDP-067 6.5 0.5mM

MDP-068 7.4 1mM

MDP-069 7 1mM

MDP-070 4.2 0.5mM

MDP-071 7.5 1 mM

MDP-072 6 1 mM

Figure 14 (A) shows AT m for the concentration dependent stabilization for MDP-008 in duplicate. Figure 14 (B) shows AJ m for the concentration dependent stabilization for MDP-040 in duplicate.