WO2015004485A1

WO2015004485A1 - Therapeutic compounds

Info

Publication number: WO2015004485A1
Application number: PCT/GB2014/052143
Authority: WO
Inventors: Noelia ARAUJO; Ana FERREIRA DA SILVA; Yujia QING; Michele LUFINO; Angela Jane RUSSELL; Bethany SMALL; Richard Wade-Martins; Graham Michael Wynne
Original assignee: Isis Innovation Limited
Priority date: 2013-07-12
Filing date: 2014-07-14
Publication date: 2015-01-15
Also published as: GB201312499D0

Abstract

The present invention relates to therapeutic compounds useful for the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases (e.g. Friedreich's ataxia). The compounds have the structural formula I shown below: wherein Q, X, p, R₁, q, R₃ and R₄ are as defined herein. The present invention also relates to pharmaceutical compositions comprising the compounds defined herein, the use of these compositions for the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases (e.g. Friedreich's ataxia), and to processes for the preparation of the pharmaceutical compositions defined herein.

Description

THERAPEUTIC COMPOUNDS

FIELD OF THE INVENTION

[0001] The present invention relates to therapeutic compounds. More specifically, the present invention relates to compounds useful for the treatment of the diseases or conditions defined herein, including triplet repeat diseases (such as, for example, Friedreich' s ataxia). The present invention also relates to pharmaceutical compositions comprising the compounds defined herein, the use of these compositions for the treatment of the diseases or conditions defined herein (including triplet repeat diseases, such as, for example, Friedreich's ataxia), and to processes for the preparation of the pharmaceutical compositions defined herein.

BACKGROUND OF THE INVENTION

[0002] Friedreich's ataxia (FRDA; OMIM 229300) is a progressive neurodegenerative disorder and the most common form of recessive ataxia, affecting approximately 1-2 in 50,000 Caucasians (1). Patients present with progressive gait and limb ataxia, lower limb arefiexia, dysarthria, increased incidence of diabetes and hypertrophic cardiomyopathy, which subsequently leads to death in the fourth or fifth decade of life (2, 3). The neurological symptoms are mainly caused by degeneration of the large sensory neurons of the dorsal root ganglia (DRG), the spinocerebellar tracts and the dentate nucleus of the cerebellum (4, 5).

[0003] FRDA is caused by an abnormal expansion of GAA repeats in intron 1 of the frataxin gene (FXN) (1). Approximately 98% of FRDA patients are homozygous for a GAA repeat expansion and the remaining patients are compound heterozygotes with one expanded allele and a point mutation in the second allele (6, 7). Normal unaffected individuals have < 36 GAA repeats whereas FRDA patients present GAA expansions ranging from 70 to > 1000 GAA repeats which lead to reduced levels of frataxin, a nuclear-encoded mitochondrial protein essential for life (1, 7). The GAA size of the small allele has been shown to correlate with residual frataxin levels, earlier onset and increased severity of disease (8, 9). Frataxin deficiency leads to iron-sulfur cluster (ISC) deficiency, mitochondrial iron accumulation and increased susceptibility to oxidative stress (10-15).

[0004] The mechanism through which expanded GAA repeats silence XN expression still needs further elucidation. Two non-exclusive models have been proposed (10, 16). Initial evidence suggested that expanded GAA repeats in intron 1 of FXN form unusual DNA structures such as triplexes or sticky DNA and DNA/RNA hybrid structures, which impede the progress of the RNA polymerase and perturb transcription in a length-dependent manner (17-23). However, more recently a second model suggests that long GAA expansions can induce silencing of FXN expression via a heterochromatin-mediated mechanism of repression (24, 25). Epigenetic changes around expanded GAA repeats have been identified which include increased DNA methylation at specific CpG sites upstream of the GAA repeats (26-29) and reduced acetylation of histones H3 and H4 accompanied by increased levels of methylated histones H3K9me2 and H3K9me3 in regions flanking GAA repeats (24, 30). The FXN promoter in patient-derived cells and tissues shows a less permissive configuration for transcription initiation (26, 31). More recently a depletion of chromatin insulator protein CTCF was identified at the XN promoter of FRDA patient-derived cells and a correlation between CTCF depletion and increased levels of the frataxin antisense transcript-1 was suggested (32).

[0005] Currently there is no proven treatment for Friedreich's ataxia and, although there are promising therapies under development (24, 33-36), there remains a need to identify new drug molecules capable of treating this condition. SUMMARY OF THE INVENTION

[0006] The present invention resides in the recognition that the compounds of the invention defined herein are potentially useful agents for the treatment of various clinical conditions, including triplet repeat diseases, such as, for example, Friedreich' s ataxia.

[0007] Therefore, in a first aspect, the present invention provides a pharmaceutical composition comprising a compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutical excipients.

[0008] In a further aspect, the present invention provides a compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as defined herein, optionally for use in therapy (or for use as a medicament).

[0009] In a further aspect, the present invention provides a compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as defined herein, for use in the treatment of the diseases or conditions defined herein (including triplet repeat diseases such as, for example, Friedreich' s ataxia).

[0010] In a further aspect, the present invention provides the use of a compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for use in the treatment of the diseases or conditions defined herein (including triplet repeat diseases such as, for example, Friedreich' s ataxia).

[0011] In a further aspect, the present invention provides a method of treating the diseases or conditions defined herein (including triplet repeat diseases, such as, for example, Friedreich's ataxia), said method comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as defined herein. DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0012] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0013] It is to be appreciated that references to "treating" or "treatment" include prophylaxis as well as the alleviation of established symptoms of a disease or condition. "Treating" or "treatment" therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the disease or condition developing in a subject that may be afflicted with or predisposed to the disease or condition, but does not yet experience or display clinical or subclinical symptoms of the disease or condition, (2) inhibiting the disease or condition, i.e., arresting, reducing or delaying the development of the disease or condition or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease or condition, i.e., causing regression of the disease or condition or at least one of its clinical or subclinical symptoms.

[0014] A "therapeutically effective amount" means the amount of the compound that, when administered to a subject for treating a disease or condition referred to herein, is sufficient to effect such treatment for the disease or condition. The "therapeutically effective amount" will vary depending on the form of the compound (e.g. the salt form), the disease or condition concerned and its severity, as well as the age, weight, etc., of the subject to be treated.

[0015] The term "subject" is used herein to mean a warm blooded mammal. Thus, the compound of the present invention may be used for human and/or veterinary applications. In a particular embodiment, the subject is a human.

[0016] In this specification the term "alkyl" includes both straight and branched chain alkyl groups. References to individual alkyl groups such as "propyl" are specific for the straight chain version only and references to individual branched chain alkyl groups such as "isopropyl" are specific for the branched chain version only. For example, "(l-4C)alkyl" includes (l-2C)alkyl, (l-3C)alkyl, propyl, isopropyl and i-butyl.

[0017] The term "alkylene" includes both straight and branched chain alkylene groups.

[0018] The term "(m-nC)" or "(m-nC) group" used alone or as a prefix, refers to any group having m to n carbon atoms.

[0019] "(3-8C)cycloalkyl" means a hydrocarbon ring containing from 3 to 8 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or bicycle[2.2.2]octane, bicycle[2.1.1]hexane, bicycle [l.l. l]pentane and bicyclo[2.2.1]heptyl.

[0020] The term "halo" refers to fluoro, chloro, bromo and iodo. [0021] The term "heterocyclyl", "heterocyclic" or "heterocycle" means a non-aromatic saturated or partially saturated monocyclic, fused, bridged, or spiro bicyclic heterocyclic ring system(s). Monocyclic heterocyclic rings contain from about 3 to 12 (suitably from 3 to 7) ring atoms, with from 1 to 5 (suitably 1, 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur in the ring. Bicyclic heterocycles contain from 7 to 17 member atoms, suitably 7 to 12 member atoms, in the ring. Bicyclic heterocyclic(s) rings may be fused, spiro, or bridged ring systems. Examples of heterocyclic groups include cyclic ethers such as oxiranyl, oxetanyl, tetrahydrofuranyl, dioxanyl, and substituted cyclic ethers. Heterocycles containing nitrogen include, for example, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, tetrahydrotriazinyl, tetrahydropyrazolyl, and the like. Typical sulfur containing heterocycles include tetrahydrothienyl, dihydro-l,3-dithiol, tetrahydro- 2H-thiopyran, and hexahydrothiepine. Other heterocycles include dihydro-oxathiolyl, tetrahydro-oxazolyl, tetrahydro-oxadiazolyl, tetrahydrodioxazolyl, tetrahydro-oxathiazolyl, hexahydrotriazinyl, tetrahydro-oxazinyl, morpholinyl, thiomorpholinyl, tetrahydropyrimidinyl, dioxolinyl, octahydrobenzofuranyl, octahydrobenzimidazolyl, and octahydrobenzothiazolyl. For heterocycles containing sulfur, the oxidized sulfur heterocycles containing SO or S0₂ groups are also included. Examples include the sulfoxide and sulfone forms of tetrahydrothienyl and thiomorpholinyl such as tetrahydrothiene 1,1 -dioxide and thiomorpholinyl 1,1 -dioxide. A suitable value for a heterocyclyl group which bears 1 or 2 oxo (=0) or thioxo (=S) substituents is, for example, 2-oxopyrrolidinyl, 2-thioxopyrrolidinyl, 2-oxoimidazolidinyl, 2-thioxoimidazolidinyl, 2-oxopiperidinyl, 2,5-dioxopyrrolidinyl, 2,5-dioxoimidazolidinyl or 2,6-dioxopiperidinyl. Particular heterocyclyl groups are saturated monocyclic 3 to 7 membered heterocyclyls containing 1, 2 or 3 heteroatoms selected from nitrogen, oxygen or sulfur, for example azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, morpholinyl, tetrahydrothienyl, tetrahydrothienyl 1,1 -dioxide, thiomorpholinyl, thiomorpholinyl 1,1 -dioxide, piperidinyl, homopiperidinyl, piperazinyl or homopiperazinyl. As the skilled person would appreciate, any heterocycle may be linked to another group via any suitable atom, such as via a carbon or nitrogen atom. However, reference herein to piperidino or morpholino refers to a piperidin-l-yl or morpholin-4-yl ring that is linked via the ring nitrogen.

[0022] By "bridged ring systems" is meant ring systems in which two rings share more than two atoms, see for example Advanced Organic Chemistry, by Jerry March, 4^th Edition, Wiley Interscience, pages 131-133, 1992. Examples of bridged heterocyclyl ring systems include, aza- bicyclo[2.2.1]heptane, 2-oxa-5-azabicyclo[2.2.1]heptane, aza-bicyclo[2.2.2]octane, aza- bicyclo[3.2.1]octane and quinuclidine.

[0023] By "spiro bi-cyclic ring systems" we mean that the two ring systems share one common spiro carbon atom, i.e. the heterocyclic ring is linked to a further carbocyclic or heterocyclic ring through a single common spiro carbon atom. Examples of spiro ring systems include 6- azaspiro[3.4]octane, 2-oxa-6-azaspiro[3.4]octane, 2-azaspiro[3.3]heptanes and 2-oxa-6- azaspiro[3.3]heptanes.

[0024] The term "heteroaryl" or "heteroaromatic" means an aromatic mono-, bi-, or polycyclic ring incorporating one or more (for example 1-4, particularly 1, 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heteroaryl group can be, for example, a 5- or 6-membered monocyclic ring or a 9- or 10-membered bicyclic ring, for example a bicyclic structure formed from fused five and six membered rings or two fused six membered rings. Each ring may contain up to about four heteroatoms typically selected from nitrogen, sulfur and oxygen. Typically the heteroaryl ring will contain up to 3 heteroatoms, more usually up to 2, for example a single heteroatom. In one embodiment, the heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non- basic as in the case of an indole or pyrrole nitrogen. In general the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.

[0025] Examples of heteroaryl include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazenyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, carbazolyl, phenazinyl, benzisoquinolinyl, pyridopyrazinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]-pyranyl, 5H-pyrido[2,3-d]-o-oxazinyl, lH-pyrazolo[4,3-d]-oxazolyl,

4H-imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo [2, 1-b] thiazolyl, imidazo[l,2-b][l,2,4]triazinyl. "Heteroaryl" also covers partially aromatic bi- or polycyclic ring systems wherein at least one ring is an aromatic ring and one or more of the other ring(s) is a non-aromatic, saturated or partially saturated ring, provided at least one ring contains one or more heteroatoms selected from nitrogen, oxygen or sulfur. Examples of partially aromatic heteroaryl groups include for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo- 1,2,3,4-tetrahydroquinolinyl, dihydrobenzthienyl, dihydrobenzfuranyl, 2,3-dihydro- benzo[l,4]dioxinyl, benzo[l,3]dioxolyl, 2,2-dioxo-l,3-dihydro-2-benzothienyl, 4,5,6,7- tetrahydrobenzofuranyl, indolinyl, 1 ,2,3 ,4-tetrahydro- 1 ,8-naphthyridinyl, l,2,3,4-tetrahydropyrido[2,3-b]pyrazinyl and 3,4-dihydro-2H-pyrido[3,2-b][l,4]oxazinyl.

[0026] The term "carbocyclic ring" means a cyclic or polycyclic ring system comprising carbon atoms (typically from 5 to 12 carbon atoms). This terms encompasses saturated (cycloalkyl), partially saturated and unsaturated (aryl) ring systems.

[0027] The term "aryl" means a cyclic or polycyclic aromatic ring having from 5 to 12 carbon atoms. The term aryl includes both monovalent species and divalent species. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl and the like. In particular embodiment, an aryl is phenyl.

[0028] The term "optionally substituted" refers to either groups, structures, or molecules that are substituted and those that are not substituted.

[0029] Where optional substituents are chosen from "one or more" groups it is to be understood that this definition includes all substituents being chosen from one of the specified groups or the substituents being chosen from two or more of the specified groups.

[0030] The phrase "compounds of the invention" means those compounds which are disclosed herein, both generically and specifically.

The compounds of the invention

[0031] In one aspect, the compounds of the present invention have the structural formula la shown below:

la

wherein

Q is selected from a mono or bicyclic carbocyclic ring, a mono or bicyclic heteroaryl ring or a mono or bicyclic heterocyclic ring, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, ureido, (l-4C)alkyl, (l-4C)alkoxy, (2-4C)alkenyl, (2-4C)alkynyl, , (1- 4C)alkylthio, (l-4C)alkylsulphinyl, (l-4C)alkylsulphonyl, (l-4C)alkylamino, di-[(l- 4C)alkyl] amino, (l-4C)alkoxycarbonyl, N-(l-4C)alkylcarbamoyl, N,N-di-[(l- 4C)alkyl]carbamoyl, (2-4C)alkanoyl, (2-4C)alkanoyloxy, (2-4C)alkanoylamino, N-(l- 4C)alkylsulphamoyl and N,N-di-[(l-4C)alkyl]sulphamoyl;

X is -0-, a bond, -NR^a-, -CHR^a-, -S-, -SO-, -SO2-, -NR^a-C(0)-, -C(0)-NR^a-, or -NR^a-C(0)-NR^b-, wherein R^a and R^b are each independently selected from H or (1- 2C)alkyl;

p is 1 or 2;

q is 1 or 2;

Ri is hydroxyl or methoxy;

R₃ is (l-3C)alkyl and R₄ is a group -L1-X2-R5 wherein Li is (l-3C)alkylene, X₂ is O or S and R5 is (l-3C)alkyl, and wherein any alkylene or alkyl groups present in R₃ and and R₄ are optionally substituted with one or more halo groups (e.g fluoro);

or R₃ and R₄ are linked such that, together with the nitrogen atom to which they are attached, they form a ring of the formula:

wherein * donates the nitrogen to which the linked R₃ and R₄ group is attached;

Xi is O, S, S(O), S(0)₂, -NR^f or CR^hR\ wherein R^f is selected from H, methyl or (2- 4C)alkanoyl and R^h and R¹ are each independently selected from hydrogen, halo, methyl, methoxy or (2-4C)alkanoyl;

n is 0, 1, 2, 3 or 4;

R₂ is selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, ureido, (l-4C)alkyl, (l-4C)alkoxy, (2-4C)alkenyl, (2-4C)alkynyl, , (l-4C)alkylthio, (1- 4C)alkylsulphinyl, (l-4C)alkylsulphonyl, (l-4C)alkylamino, di-[(l-4C)alkyl] amino, (1- 4C)alkoxycarbonyl, N-(l-4C)alkylcarbamoyl, N,N-di-[(l-4C)alkyl]carbamoyl, (2- 4C)alkanoyl, (2-4C)alkanoyloxy, (2-4C)alkanoylamino, N-(l-4C)alkylsulphamoyl and N,N-di- [( 1 -4C)alkyl] sulphamoyl;

or when n is 2 or more, two of the R₂ groups may be linked to form a linear or branched (l-4C)alkylene bridge;

or a pharmaceutically acceptable salt or solvate thereof.

[0032] In a further aspect, the compounds of the present invention have the structural formula lb shown below:

lb wherein

X is -0-, a bond, -NR^a-, -CHR^a-, -S-, -SO-, -SO2-, -NR^a-C(0)-, -C(0)-NR^a-, or

-NR^a-C(0)-NR^b-, wherein R^a and R^b are each independently selected from H or (1- 2C)alkyl;

Ri is hydroxyl or methoxy;

Xi is O, S, S(O), S(0)₂, C(S)₂, -NR^c-C(0)-, -C(0)-NR^c-, or -NR^c-C(0)-NR^d-, wherein R^c and R^d are each independently selected from H or (l-2C)alkyl;

n is 0, 1, 2, 3 or 4;

or a pharmaceutically acceptable salt or solvate thereof.

[0033] Particular compounds of the invention include, for example, compounds of the formula la or lb, or pharmaceutically acceptable salts or solvates thereof, wherein, unless otherwise stated, each of Q, X, Ri, R₂, R3, R4, R5, Li, Xi, X₂, p, q and n has any of the meanings defined hereinbefore or in any one of paragraphs (1) to (35) hereinafter: -

(1) Q is selected from phenyl, naphthyl, a mono or bicyclic heteroaryl ring, or a mono or bicyclic heterocyclic ring, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, ureido, (l-4C)alkyl, (l-4C)alkoxy, (2-4C)alkenyl, (2-4C)alkynyl, , (1- 4C)alkylthio, (l-4C)alkylsulphinyl, (l-4C)alkylsulphonyl, (l-4C)alkylamino, di-[(l- 4C)alkyl] amino, (l-4C)alkoxycarbonyl, N-(l-4C)alkylcarbamoyl, N,N-di-[(l- 4C)alkyl]carbamoyl, (2-4C)alkanoyl, (2-4C)alkanoyloxy, (2-4C)alkanoylamino, N-(l- 4C)alkylsulphamoyl and N,N-di-[(l-4C)alkyl]sulphamoyl;

Q is selected from phenyl, naphthyl, a mono or bicyclic heteroaryl ring, or a mono or bicyclic heterocyclic ring, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl,

trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, ureido, (l-2C)alkyl, (l-2C)alkoxy, (2C)alkenyl, (2C)alkynyl, , (1- 2C)alkylthio, (l-2C)alkylsulphinyl, (l-2C)alkylsulphonyl, (l-2C)alkylamino, di-[(l- 2C)alkyl] amino, (l-2C)alkoxycarbonyl, N-(l-2C)alkylcarbamoyl, N,N-di-[(l- 2C)alkyl]carbamoyl, (2C)alkanoyl, (2C)alkanoyloxy, (2C)alkanoylamino, N-(l- 2C)alkylsulphamoyl and N,N-di-[(l-2C)alkyl]sulphamoyl;

Q is selected from phenyl, naphthyl, or a mono or bicyclic heteroaryl ring, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, ureido, (l-4C)alkyl, (l-4C)alkoxy, (2- 4C)alkenyl, (2-4C)alkynyl, , (l-4C)alkylthio, (l-4C)alkylsulphinyl, (1- 4C)alkylsulphonyl, (l-4C)alkylamino, di-[(l-4C)alkyl] amino, (l-4C)alkoxycarbonyl, N- (l-4C)alkylcarbamoyl, N,N-di-[(l-4C)alkyl]carbamoyl, (2-4C)alkanoyl, (2- 4C)alkanoyloxy, (2-4C)alkanoylamino, N-(l-4C)alkylsulphamoyl and N,N-di-[(l- 4C)alkyl] sulphamoyl;

Q is selected from phenyl, naphthyl, or a phenyl ring which is fused with 5 or 6- membered carbocyclic, heterocyclic or heteroaryl ring each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, ureido, (l-4C)alkyl, (l-4C)alkoxy, (2-4C)alkenyl, (2- 4C)alkynyl, , (l-4C)alkylthio, (l-4C)alkylsulphinyl, (l-4C)alkylsulphonyl, (1- 4C)alkylamino, di-[(l-4C)alkyl]amino, (l-4C)alkoxycarbonyl, N-(l-4C)alkylcarbamoyl, N,N-di-[(l-4C)alkyl]carbamoyl, (2-4C)alkanoyl, (2-4C)alkanoyloxy, (2- 4C)alkanoylamino, N-(l-4C)alkylsulphamoyl and N,N-di-[(l-4C)alkyl] sulphamoyl; (5) Q is selected from phenyl or naphthyl, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, (l-2C)alkyl, and (l-2C)alkoxy;

(6) Q is selected from phenyl or naphthyl, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, amino, carboxy, carbamoyl, sulphamoyl, (l-2C)alkyl, and (l-2C)alkoxy;

(7) Q is selected from phenyl or naphthyl, each of which is optionally substituted by a one or more substituents selected from the group consisting of (l-2C)alkyl and (l-2C)alkoxy;

(8) Q is selected from phenyl or naphthyl, each of which is optionally substituted by a one or more substituents selected from the group consisting of (l-2C)alkyl and (l-2C)alkoxy;

(9) X is -0-, -NR^a-, -S-, -SO-, or -SO2-;

(10) X is -O-, -S-, -SO-, or -SO2-;

(11) X is -O-;

(12) Ri is hydroxy;

(13) Ri is methoxy;

(14) Xi is O, S, S(O), S(0)₂, -NR^f or CR^hR\ wherein R^f is selected from H, methyl or (2C)alkanoyl, and R^h and R¹ are each independently selected from hydrogen, fluoro, chloro, bromo methyl, methoxy or (2C)alkanoyl;

(15) Xi is O, S, S(O), S(0)₂, -NR^f or CR^hR\ wherein R^f is selected from H, methyl or (2C)alkanoyl, and R^h and R¹ are each independently selected from hydrogen or fluoro;

(16) Xi is O, S, S(O), S(0)₂, or C(S)₂;

(17) Xi is O, S, S(O), or S(0)₂;

(18) Xi is O or S;

(19) Xi is O;

(20) Xi is S;

(21) n is 0, 1, 2 or 3;

(22) n is 0, 1, or 2;

(23) n is 0;

(24) R₂ is selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy,

cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, (1- 4C)alkyl, (l-4C)alkoxy, (l-4C)alkylthio, (l-4C)alkylsulphinyl, (l-4C)alkylsulphonyl, (l-4C)alkylamino, di-[(l-4C)alkyl] amino, (l-4C)alkoxycarbonyl, N-(l- 4C)alkylcarbamoyl, N,N-di-[(l-4C)alkyl]carbamoyl, (2-4C)alkanoyl, (2-4C)alkanoyloxy, (2-4C)alkanoylamino, N-(l-4C)alkylsulphamoyl and N,N-dH(l-4C)alkyl] sulphamoyl; or when n is 2 or more, two of the R₂ groups may be linked to form a linear or branched (1- 4C)alkylene bridge;

(25) R₂ is selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy,

cyano, nitro, hydroxyl, mercapto, amino, carboxy, carbamoyl, sulphamoyl, (l-2C)alkyl, (l-2C)alkoxy, (l-2C)alkylthio, (l-2C)alkylsulphinyl, (l-2C)alkylsulphonyl, (1- 2C)alkylamino, di-[(l-2C)alkyl]amino, (l-2C)alkoxycarbonyl, N-(l-2C)alkylcarbamoyl, N,N-di-[(l-2C)alkyl]carbamoyl, (2C)alkanoyl, (2C)alkanoyloxy, (2C)alkanoylamino, N- (l-2C)alkylsulphamoyl and N,N-di-[(l-2C)alkyl] sulphamoyl; or when n is 2 or more, two of the R₂ groups may be linked to form a linear or branched (l-3C)alkylene bridge;

(26) p is l;

(27) p is 2;

(28) q is l;

(29) q is 2;

(30) p is 1 and q is 1;

(31) p is 1 and q is 2;

(32) p is 2 and q is 1;

(33) R₃ is (l-2C)alkyl and R₄ is a group -L1-X2-R5 wherein Li is (l-3C)alkylene, X₂ is O or S and R5 is (l-2C)alkyl and wherein any alkylene or alkyl groups present in R₃ and and R₄ are optionally substituted with one or more halo groups (e.g fluoro);

wherein * donates the nitrogen to which the linked groups R₃ and R₄ are attached;

Xi is O, S, S(O), S(0)₂, -NR^f or CR^hR\ wherein R^f is selected from H, methyl or

(2C)alkanoyl, and R^h and R¹ are each independently selected from hydrogen, fluoro, chloro, bromo methyl, methoxy or (2C)alkanoyl;

n is 0, 1, 2 or 3;

R₂ is selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, amino, carboxy, carbamoyl, sulphamoyl, (l-3C)alkyl, (l-3C)alkoxy, (2-3C)alkenyl, (2-3C)alkynyl, (l-3C)alkylthio, (l-3C)alkylsulphinyl, (1- 3C)alkylsulphonyl, (l-3C)alkylamino, di-[(l-3C)alkyl]amino, (l-3C)alkoxycarbonyl, N- (l-3C)alkylcarbamoyl, N,N-di-[(l-3C)alkyl]carbamoyl, (2-3C)alkanoyl, (2- 3C)alkanoyloxy, (2-3C)alkanoylamino, N-(l-3C)alkylsulphamoyl and N,N-di-[(l- 3C)alkyl] sulphamoyl;

or when n is 2 or more, two of the R₂ groups may be linked to form a linear or branched (l-3C)alkylene bridge;

(34) R₃ is methyl and R₄ is a group -L1-X2-R5 wherein Li is (l-2C)alkylene, X₂ is O or S and R5 is (l-2C)alkyl and wherein any alkylene or alkyl groups present in R₃ and and R₄ are optionally substituted with one or more fluoro groups;

Xi is O, S, S(O), S(0)₂, -NR^f or CR^hR\ wherein R^f is selected from H, methyl or (2C)alkanoyl, and R^h and R¹ are each independently selected from hydrogen or fluoro; n is 0, 1 or 2;

R₂ is selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, amino, carbamoyl, sulphamoyl, (l-3C)alkyl, (l-3C)alkoxy, (2- 3C)alkenyl, (2-3C)alkynyl, (l-3C)alkylthio, (l-3C)alkylsulphinyl, (l-3C)alkylsulphonyl, (l-3C)alkylamino, and di-[(l-3C)alkyl]amino;

or when n is 2 or more, two of the R₂ groups may be linked to form a linear or branched (l-2C)alkylene bridge;

(35) R₃ is methyl and R₄ is a group -L1-X2-R5 wherein Li is (l-2C)alkylene, X₂ is O or S and R₅ is (l-2C)alkyl;

Xi is O, S, S(O) or S(0)₂; and n is 0.

[0034] Suitably, Q is a group as defined in any one of paragraphs (1) to (8) above. In a particular embodiment, Q is phenyl or naphthyl, each of which is optionally substituted with one or more of (l-2C)alkyl and (l-2C)alkoxy groups. In a further embodiment, Q is phenyl optionally substituted with one or more of (l-2C)alkyl and (l-2C)alkoxy groups. In a further embodiment, Q is naphthyl.

[0035] Suitably, X is a group as defined in any one of paragraphs (9) to (11) above. In a particular embodiment, X is O.

[0036] Suitably, Ri is a group as defined in any one of paragraphs (12) or (13) above. In a particular embodiment, Ri is hydroxy.

[0037] Suitably, Xi is a group as defined in any one of paragraphs (14) to (20) above. In a particular embodiment, Xi is O, S, S(O), or S(0)₂.

[0038] Suitably, n is a group as defined in any one of paragraphs (21) to (23) above. In a particular embodiment, n is 0.

[0039] Suitably, R₂ is a group as defined in any one of paragraphs (24) or (25) above.

[0040] Suitably, p and q are as defined in any one of paragraphs (30) to (32) above. Most suitably, one of p and q can only be 2 when the other is 1.

[0041] Suitably, R₃ and R₄ is a group as defined in any one of paragraphs (33) to (35) above.

[0042] In a particular group of compounds of formula la,

Q is as defined in any one of paragraphs (1) to (8) above;

X is a group as defined in any one of paragraphs (9) to (11) above;

Ri is a group as defined in any one of paragraphs (12) or (13) above;

p and q are as defined in any one of paragraphs (28) to (30) above; and

R₃ and R₄ is a group as defined in any one of paragraphs (33) to (35).

[0043] In a particular group of compounds of formula la,

Q is phenyl or naphthyl, each of which is optionally substituted with one or more of (1-

2C)alkyl and (l-2C)alkoxy groups;

X is O;

Ri is hydroxyl;

p and q are 1 or one of p and q is 2 and the other is 1 ; and

R₃ and R₄ is a group as defined in any one of paragraphs (33) to (35).

[0044] In a particular group of compounds of formula lb, Q is as defined in any one of paragraphs (1) to (8) above, X is -0-, Ri is hydroxy, Xi has any one of the definitions set out herein, R₂ has any one of the definitions set out herein and n is 0, 1 or 2. Such compounds have the structural formula Ic shown below:

IC

[0045] In a further group of compounds of formula lb, Q is as defined in any one of paragraphs (1) to (8) above, X is -0-, Ri is hydroxy, Xi is O, R₂ has any one of the definitions provided herein and n is 0, 1 or 2. Such compounds have the structural formula Id shown below:

Id

[0046] Particular com ounds of the invention include an one of the following:

or a pharmaceutically acceptable salt or solvate thereof.

[0047] Particular compounds of the invention include any one of the following:

or a pharmaceutically acceptable salt or solvate thereof.

[0048] Further compounds of the invention include any one of the following: l-(3,4-dimethylphenoxy)-3-(4-morpholinyl)-2-propanol;

l-(3,4-dimethylphenoxy)-3-morpholinopropan-2-ol (YQ01);

l-morpholino-3-(naphthalen-2-yloxy)propan-2-ol (YQ25);

4-(2-methoxy-3-(naphthalen-2-yloxy)propyl)morpholine (YQ26);

(S)- l-(3,4-dimethylphenoxy)-3-morpholinopropan-2-ol (YQ02); ( ?)-l-(3,4-dimethylphenoxy)-3-morpholinopropan-2-ol (YQ03);

l-morpholino-3-(o-tolyloxy)propan-2-ol (YQ04);

l-morpholino-3-(m-tolyloxy)propan-2-ol (YQ05);

l-morpholino-3-(/?-tolyloxy)propan-2-ol (YQ06);

l-(4-methoxyphenoxy)-3-morpholinopropan-2-ol (YQ07);

l-morpholino-3-((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)propan-2-ol (YQ16);

l-(3,4-dichlorophenoxy)-3-morpholinopropan-2-ol (YQ17);

l-(4-methylpiperazin-l-yl)-3-(naphthalen-2-yloxy)propan-2-ol (YQ22);

l-morpholino-3-(naphthalen-2-yloxy)propan-2-ol (Al / YQ25);

-(3,4-dimethylphenoxy)-3'-morpholinopropan-2'-ol (1 / C5);

1 '-(naphthalen-2-yloxy)-3 '-thiomorpholinopropan-2'-ol (22);

l '-(4"₅4"-difluoropiperidin-l "-yl)-3'-(naphthalen-2-yloxy)propan-2'-ol (23);

7"-(4"-(2'-hydroxy-3'-(naphthalen-2-yloxy)propyl)piperazin-l "-yl)ethanone (25);

1 '-((2"-methoxyethyl)(methyl)amino)-3 '-(naphthalen-2-yloxy)propan-2'-ol (27);

4"-(2'-hydroxy-3'-(naphthalen-2-yloxy)propyl)thiomorpholine 1 "-oxide (28);

4"-(2'-hydroxy-3 '-(naphthalen-2-yloxy)propyl)thiomorpholine 1 ", 1 "-dioxide (29);

4'-morpholino-l '-(naphthalen-2-yloxy)butan-2'-ol (37);

1 '-morpholino-4'-(naphthalen-2-yloxy)butan-2'-ol (36);

or a pharmaceutically acceptable salt or solvate thereof.

[0049] The various functional groups and substituents making up the compounds of the present invention are typically chosen such that the molecular weight of the compound does not exceed

1000. More usually, the molecular weight of the compound will be less than 750, for example less than 700, or less than 650, or less than 600, or less than 550. More preferably, the molecular weight is less than 525 and, for example, is 500 or less.

[0050] Suitable or preferred features of any compounds of the present invention may also be suitable features of any other aspect.

[0051] A suitable pharmaceutically acceptable salt of a compound of the invention is, for example, an acid-addition salt of a compound of the invention which is sufficiently basic, for example, an acid-addition salt with, for example, an inorganic or organic acid, for example hydrochloric, hydrobromic, sulfuric, phosphoric, trifluoroacetic, formic, citric or maleic acid. In addition, a suitable pharmaceutically acceptable salt of a compound of the invention which is sufficiently acidic is an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a physiologically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or

tris- (2-hydroxyethyl) amine .

[0052] Compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed "isomers". Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers".

Stereoisomers that are not mirror images of one another are termed "diastereomers" and those that are non-superimposable mirror images of each other are termed "enantiomers". When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. An enantiomer can be characterized by the absolute configuration of its asymmetric center and is described by the R- and S-sequencing rules of Cahn and Prelog, or by the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral compound can exist as either individual enantiomer or as a mixture thereof. A mixture containing equal proportions of the enantiomers is called a "racemic mixture".

[0053] The compounds of this invention may possess one or more asymmetric centers; such compounds can therefore be produced as individual (R)- or (S)-stereoisomers or as mixtures thereof. Unless indicated otherwise, the description or naming of a particular compound in the specification and claims is intended to include both individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well-known in the art (see discussion in Chapter 4 of "Advanced Organic Chemistry", 4th edition J. March, John Wiley and Sons, New York, 2001), for example by synthesis from optically active starting materials or by resolution of a racemic form. Some of the compounds of the invention may have geometric isomeric centres (E- and Z- isomers). It is to be understood that the present invention encompasses all optical, diastereoisomers and geometric isomers and mixtures thereof that possess the desired therapeutic activity.

[0054] The present invention also encompasses compounds of the invention as defined herein which comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including ¾ ²H(D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; and O may be in any isotopic form, including ¹⁶0 and¹⁸0; and the like.

[0055] It is also to be understood that certain compounds of the invention may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms that possess the desired therapeutic activity. [0056] It is also to be understood that certain compounds of the invention may exhibit polymorphism, and that the invention encompasses all such forms that possess the desired therapeutic activity.

[0057] Certain compounds of the invention may exist in a number of different tautomeric forms and references to compounds of the invention include all such forms. For the avoidance of doubt, where a compound can exist in one of several tautomeric forms, and only one is specifically described or shown, all others are nevertheless embraced by compounds of the invention. Examples of tautomeric forms include keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, and nitro/aci-nitro.

keto enol enolate

[0058] Certain compounds of the invention containing an amine function may also form N- oxides. A reference herein to a compound of the formula la or lb that contains an amine function also includes the N-oxide. Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle. N- Oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 4^th Edition, Wiley Interscience, pages. More particularly, N-oxides can be made by the procedure of L. W. Deady (Syn. Comm. 1977, 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.

[0059] The compounds of the invention may be administered in the form of a pro-drug which is broken down in the human or animal body to release a compound of the invention. A pro-drug may be used to alter the physical properties and/or the pharmacokinetic properties of a compound of the invention. A pro-drug can be formed when the compound of the invention contains a suitable group or substituent to which a property-modifying group can be attached. Examples of pro-drugs include in vivo cleavable ester derivatives that may be formed at a carboxy group or a hydroxy group in a compound of the invention and in-vivo cleavable amide derivatives that may be formed at a carboxy group or an amino group in a compound of the invention. [0060] Accordingly, the present invention includes those compounds of the invention as defined hereinbefore when made available by organic synthesis and when made available within the human or animal body by way of cleavage of a pro-drug thereof. Accordingly, the present invention includes those compounds of the formula I that are produced by organic synthetic means and also such compounds that are produced in the human or animal body by way of metabolism of a precursor compound, that is a compound of the formula la or lb may be a synthetically-produced compound or a metabolically-produced compound.

[0061] A suitable pharmaceutically acceptable pro-drug of a compound of the formula I is one that is based on reasonable medical judgement as being suitable for administration to the human or animal body without undesirable pharmacological activities and without undue toxicity.

[0062] Various forms of pro-drug have been described, for example in the following documents :- a) Methods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al. (Academic Press, 1985);

b) Design of Pro-drugs, edited by H. Bundgaard, (Elsevier, 1985);

c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen and

H. Bundgaard, Chapter 5 "Design and Application of Pro-drugs", by H. Bundgaard p. 113-191 (1991);

d) H. Bundgaard, Advanced Drug Delivery Reviews, 8, 1-38 (1992);

e) H. Bundgaard, et ah, Journal of Pharmaceutical Sciences, 77, 285 (1988);

f) N. Kakeya, et ah, Chem. Pharm. Bull., 32, 692 (1984);

g) T. Higuchi and V. Stella, "Pro-Drugs as Novel Delivery Systems", A.C.S. Symposium Series, Volume 14; and

h) E. Roche (editor), "Bioreversible Carriers in Drug Design", Pergamon Press, 1987.

[0063] The in vivo effects of a compound of the formula I may be exerted in part by one or more metabolites that are formed within the human or animal body after administration of a compound of the formula I. As stated hereinbefore, the in vivo effects of a compound of the formula I may also be exerted by way of metabolism of a precursor compound (a pro-drug).

[0064] It shall also be appreciated that compounds of formula I may also be covalently linked (at any suitable position) to other groups such as, for example, solubilising moieties (for example, PEG polymers), moieties that enable them to be bound to a solid support (such as, for example, biotin-containing moieties), and targeting ligands (such as antibodies or antibody fragments). Synthesis

[0065] The compounds of the present invention can be sourced commercially and/or prepared by synthetic techniques known in the art or those techniques described in the accompanying examples.

Pharmaceutical Compositions

[0066] According to a further aspect of the invention there is provided a pharmaceutical composition which comprises the compound of the invention as defined hereinbefore, or a pharmaceutically acceptable salt or solvate thereof, in association with a pharmaceutically acceptable diluent or carrier.

[0067] Suitably, the pharmaceutical composition is intended to be for use in the treatment of the diseases or conditions defined herein (including triplet repeat diseases such as, for example, Friedreich's ataxia).

[0068] The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular dosing or as a suppository for rectal dosing).

[0069] The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.

[0070] The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the individual treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 0.5 g of active agent (more suitably from 0.5 to 100 mg, for example from 1 to 30 mg) compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.

[0071] The size of the dose for therapeutic or prophylactic purposes of a compound of the invention will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.

[0072] In using a compound of the invention for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.1 mg/kg to 75 mg/kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous or intraperitoneal administration, a dose in the range, for example, 0.1 mg/kg to 30 mg/kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.05 mg/kg to 25 mg/kg body weight will be used. Oral administration may also be suitable, particularly in tablet form. Typically, unit dosage forms will contain about 0.5 mg to 0.5 g of a compound of this invention.

Therapeutic use

[0073] As previously stated, the present invention provides a compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as defined herein, for use in therapy (or for use as a medicament).

[0074] The biological assays described in the accompany examples demonstrate that the compounds of the invention are potentially useful therapeutic agents for the treatment of Friedreich's ataxia.

[0075] Based on the available data, it is anticipated that the compounds of the present invention are particularly useful agents for the treatment of range of diseases or conditions. Such diseases or conditions include:

• Neurodegenerative and neuromuscular diseases (for example, amyotrophic lateral sclerosis and frontotemporal dementia; Alzheimer's disease, Parkinson's disease, motor neuron disease. Kennedy's Disease, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy (PBP), amyotrophic lateral sclerosis and spinal and bulbar muscular atrophy)

• Triplet repeat diseases (including non-polyglutamine diseases (e.g. Friedreich's ataxia, Fragile X, Myotonic dystrophy (DM1), and certain spinocerebellar ataxias [e.g. SCA8 (Spinocerebellar ataxia Type 8) and SCA12 (Spinocerebellar ataxia Type 12)]; and polyglutamine (PolyQ) diseases (e.g. DRPLA (Dentatorubropallidoluysian atrophy), HD (Huntington's disease), SBMA (Spinobulbar muscular atrophy or Kennedy disease) and certain spinocerebellar ataxias [e.g. SCA1 (Spinocerebellar ataxia Type 1), SCA2 (Spinocerebellar ataxia Type 2), SCA3 (Spinocerebellar ataxia Type 3 or Machado- Joseph disease), SCA6 (Spinocerebellar ataxia Type 6), SCA7 (Spinocerebellar ataxia

Type 7), or SCA17 (Spinocerebellar ataxia Type 17)].

[0076] Suitably, the compounds of the present invention are useful for the treatment of triplet repeat diseases (including non-polyglutamine diseases (e.g. Friedreich's ataxia, Fragile X, Myotonic dystrophy (DM1), and certain spinocerebellar ataxias [e.g. SCA8 (Spinocerebellar ataxia Type 8) and SCA12 (Spinocerebellar ataxia Type 12)]; and polyglutamine (PolyQ) diseases (e.g. DRPLA (Dentatorubropallidoluysian atrophy), HD (Huntington's disease), SBMA (Spinobulbar muscular atrophy or Kennedy disease) and certain spinocerebellar ataxias [e.g. SCA1 (Spinocerebellar ataxia Type 1), SCA2 (Spinocerebellar ataxia Type 2), SCA3 (Spinocerebellar ataxia Type 3 or Machado-Joseph disease), SCA6 (Spinocerebellar ataxia Type 6), SCA7 (Spinocerebellar ataxia Type 7), or SCA17 (Spinocerebellar ataxia Type 17)]).

[0077] Most suitably, the compounds of the invention are suitable for the treatment (including prophylactic treatment) of Friedreich's ataxia.

[0078] The compounds of the invention are suitably administered in a therapeutically effective amount to a patient in need of treatment.

[0079] In a further aspect, the present invention provides a compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as defined herein, for use in the treatment of neurodegenerative and neuromuscular diseases as defined herein and/or triplet repeat diseases (e.g. Friedreich's ataxia) as defined herein.

[0080] In a further aspect, the present invention provides a method of treating neurodegenerative and neuromuscular diseases as defined herein and/or triplet repeat diseases (e.g. Friedreich's ataxia) as defined herein, said method comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound as defined herein, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as defined herein.

Routes of Administration

[0081] The compound of the invention or a pharmaceutical composition comprising this compound may be administered to a subject by any convenient route of administration. Routes of administration include, but are not limited to, oral (e.g, by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intra-arterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly. Combination Therapies

[0082] The compound of the invention may be used as a sole therapy or may involve, in addition to the compound of the invention, therapy with one or more additional therapeutic agents.

[0083] Thus, in another aspect, the present invention provides the compound of the invention as defined herein, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as defined herein, for use in the treatment of neurodegenerative and neuromuscular diseases as defined herein, and/or triplet repeat diseases (e.g. Friedreich's ataxia) as defined herein, in combination with one or more additional therapeutic agents.

[0084] In another aspect, the present invention provides the use of the compound of the invention as defined herein, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for use in the treatment of neurodegenerative and neuromuscular diseases as defined herein, and/or triplet repeat diseases (e.g. Friedreich's ataxia) as defined herein, in combination with one or more additional therapeutic agents.

[0085] In another aspect, the present invention provides a method of treating neurodegenerative and neuromuscular diseases as defined herein, and/or triplet repeat diseases (e.g. Friedreich's ataxia) as defined herein, said method comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of the invention as defined herein, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition as defined herein, and a therapeutically effective amount of one or more additional therapeutic agents.

[0086] Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compound of this invention within the dosage range described hereinbefore and the other pharmaceutically-active agent within its approved dosage range.

[0087] According to a further aspect of the invention there is provided a combination suitable for use in the treatment of neurodegenerative and neuromuscular diseases as defined herein, and/or triplet repeat diseases (e.g. Friedreich's ataxia) as defined herein, comprising a compound of the invention as defined hereinbefore, or a pharmaceutically acceptable salt or solvate thereof, and one or more additional therapeutic agents.

[0088] Herein, where the term "combination" is used, it is to be understood that this refers to simultaneous, separate or sequential administration. In one aspect of the invention "combination" refers to simultaneous administration. In another aspect of the invention "combination" refers to separate administration. In a further aspect of the invention "combination" refers to sequential administration. Where the administration is sequential or separate, the delay in administering the second component should not be such as to lose the beneficial effect of the combination.

[0089] According to a further aspect of the invention there is provided a pharmaceutical composition which comprises the compound of the invention, or a pharmaceutically acceptable salt or solvate thereof, one or more additional therapeutic agents, and a pharmaceutically acceptable diluent or carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] The invention is described further in reference to the accompanying Figures in which: Figure 1. Generation of pBAC- XN-Lwc and ^ AC-FXN-GAA-Luc genomic DNA reporter vectors. (A) Schematic representation of the construction of pBAC- XN-Lac and pBAC- XN- GAA-Luc vectors. Construction was achieved in two successive rounds of selection-counter selection homologous recombination. First a luciferase sequence preceded by a Gly-Ser-Gly-Ser- Gly (GSGSG) peptide linker was introduced at the 5' end of exon 5a, immediately before the stop codon, generating pBAC- XN-Lac vector. This vector expresses a FXN-luciferase fusion protein and carries 6 GAA repeats in intron 1. To generate pB AC -FXN-GAA -Luc vector, a second recombination was performed to replace the 6 GAA repeats present in intron 1 with -310 GAA repeats amplified from FRDA patient-derived cells. (B) Successful insertion of expanded GAA repeats was confirmed by Southern blot by using a DIG-labelled TTCio probe. Comparison to ladder allowed sizing of GAA repeats as reported in the table. (C) To assess whether expanded GAA repeats reduce FXN-luciferase expression, we delivered iBAC- XN-Lac and i AC-FXN- GAA-Luc vectors to the neuronal cell line SH-SY5Y using HSV-1 amplicon vectors. Luciferase assay showed a 70-75% reduction in FXN-luciferase levels in the three i AC-FXN-GAA-Luc clones. Error bars represent mean +/- SEM (n=3). ** P<0.01, *** P<0.001 as determined by oneway ANOVA compared to iBAC- XN-Lac, with Dunnett' s test. (D) LacZ staining of the experiment described in (C) shows high efficiency of vector delivery to SH-SY5Y cells.

Figure 2. Generation and characterisation of a GAA-expanded genomic DNA reporter model of Friedreich's ataxia. (A) Schematic representation of Cre-loxP retrofitting of pBAC- FXN-Luc and pB AC -FXN-GAA -Luc vectors with pH-FRT-Hy. pH-FRT-Hy carries a promoter- less hygromycin cassette preceded by an FRT site. (B) Site-specific vector integration in FXN- Luc and FXN-GAA-Luc cells is confirmed by PCR using primers targeting the Psv40 promoter and the hygromycin cassette. (C) FISH analysis was performed on a representative clonal cell line for each vector, using the unmodified FXN-BAC (red) and the retrofitting vector pH-FRT- Hy (green) as probes. Co-localisation of the two probes (arrows) shows vector integration in chromosome lp. Chromosome identity was confirmed using a chromosome 1 centromeric probe (data not shown). The cells analysed here carry three copies of the endogenous FXN locus, due to the hypotriploid nature of HEK cells. (D) Copy number was used to identify stable cell lines with one vector copy. Copy number was determined by real time PCR using UpGAA primers and data normalised by GAPDH. Data are expressed as relative to HEK FRT, which was set to 3 copies based on the identification in (C) of three endogenous FXN loci. (E) Western blot analysis of FXN-Luc and FXN-GAA-Luc clonal cell lines using an anti-luciferase antibody shows a -79 KDa band which corresponds to the expected size for the FXN-luciferase fusion protein. (F) qRT-PCR in FXN-Luc and FXN-GAA-Luc clonal cell lines using exon 5a-Luc primers shows a 37% reduction in FXN-luciferase mRNA levels in FXN-GAA-Luc cells. Exon 5a-Luc data are normalised to GAPDH and expressed as relative to FXN-Luc cells. Error bars represent mean +/- SEM (n=4). ** P<0.01, as determined by Student's t-test. (G) Lucif erase assay shows a 42% reduction in FXN-luciferase protein levels. Data expressed as relative light units (RLU) and normalised to total cell protein. Error bars represent mean +/- SEM (n=3). *** P<0.001, as determined by Student's t-test.

Figure 3. Expanded GAA repeats induce heterochromatin-mediated FXN silencing. (A)

Chromatin immunoprecipitation was performed on FXN-Luc and FXN-GAA-Luc cells using antibodies specific for the human acetylated histones H3K9 and H4K8 and di- and tri-methylated histone H3K9. The schematic diagram shows the position of the primers used. Error bars represent mean +/- SEM from two independent immunoprecipitations and each immunoprecipitation quantified in triplicate. * P<0.05, ** P<0.01, *** P<0.001 as determined by Student' s t-test. (B) CpG methylation was analysed upstream and downstream of GAA repeats using bisulfite sequencing. We found increased DNA methylation at CpG sites 4 and 5 upstream of GAA repeats and at CpG sites 1, 2 and 7 downstream of GAA repeats. A total of 10 colonies were sequenced per region and for each cell line.

Figure 4. Screening of 88 pre-selected small molecules. (A) FXN-GAA-Luc cells were incubated in a 96-well format for 48 hr with each compound at a final concentration of 20 μΜ. FXN-luciferase levels were quantified through luciferase assay. Each compound was tested in triplicate. (B) Four compounds identified from the screen were tested on HEK cells transfected with a CMV-luciferase plasmid to exclude molecules which directly affect the reporter assay or cause unspecific increase of expression. Transfected cells were incubated with each compound at a concentration of 20 μΜ for 48 hr. (C) C5 increases H3K9 and H4K8 histone acetylation in FRDA lymphoblastoid cells (GM15850) to the normal levels observed in control cells (GM15851). Cells were incubated with C5 at 20 μΜ for 48 hr and chromatin immunoprecipitation was performed using antibodies specific for the H3K9ac and H4K8ac residues. Error bars represent mean +/- SEM from three independent immunoprecipitations and each immunoprecipitation quantified in triplicate. (D) Structure of the amino alcohol C5.

Figure 5. Effect of C5 on FXN expression in HEK cells. (A) Dose response of C5 on FXN- GAA-Luc cells. FXN-GAA-Luc cells were incubated for 48 hr and luciferase assay was carried out. We identified 300 μΜ, 600 μΜ and 1000 μΜ as the doses achieving the highest increase in FXN-luciferase expression. Error bars represent mean +/- SEM (n=3). *** P<0.001 as determined by one-way ANOVA compared to DMSO, with Dunnett's test. (B) qRT-PCR on FXN-GAA-Luc cells incubated at the three concentrations for 48 hr shows a significant increase in FXN-GAA-Luc mRNA levels at 600 μΜ and 1000 μΜ. Data are normalised to GAPDH mRNA levels. (C) Luciferase assay on FXN-Luc and FXN-GAA-Luc cells after 48 hr incubation with C5 at 300 μΜ, 600 μΜ and 1000 μΜ. C5 shows a greater up-regulation on FXN-GAA-Luc cells at all three concentrations, although a smaller but significant increase in FXN-Luc cells is also observed. Data for each cell line are normalised by their respective DMSO. (D) qRT-PCR on HEK cells incubated with C5 for 48 hr shows that C5 is active on the FXN endogenous locus. (E) C5 significantly increases endogenous frataxin protein levels by 1.2-fold at 600 μΜ and by 1.7-fold at 1000 μΜ as determined by frataxin dipstick assay. In graphs B, C D and E error bars represent mean +/- SEM (n=4). * P<0.05, ** P<0.01, *** P<0.001 as determined by Student's t- test.

Figure 6. Validation of C5 on primary lymphocytes from FRDA patients. FRDA patient primary lymphocytes were extracted from blood using a Ficoll-Paque gradient and incubated with C5 for 72 hr at different concentrations. C5 significantly increases FXN expression in patient 1 and 2 and shows a dose-dependent trend in patient 3. FXN data are normalised by the geometric mean of three reference genes: GAPDH, HPRT and Beta-Actin. Error bars represent mean +/- SEM (n=4). * P<0.05, ** P<0.001 as determined by one-way ANOVA compared to FRDA DMSO, with Dunnett's test.

Figures 7 to 16. Effect of compounds shown in Example 4 on FXN-GAA-Luc cells.

Figures 17 to 23. Dose-response curves plotted using GraphPad Prism for the compounds of Example 5. ^*"^*P < 0.0001, "^*P < 0.001, "P < 0.01, ^*P < 0.05, as determined by the one-way ANOVA compared with DMSO, with Dunnett's test. EXAMPLES

[0091] The invention will now be illustrated in the following Examples.

Materials & Methods

Vector construction

The BAC clone RPl 1-265B8 carrying the whole 80 kb FXN locus with exons 1 to 5b of the FXN gene (41) was used to generate pBAC-FXN-Lac. To insert the GSGSG-lucif erase sequence in exon 5a, we used a selection/counter- selection homologous recombination protocol based on RedET recombination (GeneBridges, Dresden, Germany). The recombination using the RPSL- Neo and the pSClOl-BAD plasmids was carried out in two steps in E. Coli according to manufacturer's instructions. In the first step a PCR product containing the RPSL-Neo cassette flanked by 58 bp homology arms to either side of exon 5a was used to insert the cassette in exon 5a. In the second step the RPSL-Neo cassette was replaced with a PCR product containing the GSGSG-lucif erase sequence flanked by -155 bp homology arms. The luciferase sequence was obtained by PCR amplification on pGL3-promoter vector (Promega). Successful construction was confirmed by PCR and sequencing. To replace the ~6 GAA repeats present in pBAC- XN- Luc with -310 GAA repeats, the RPSL-Neo cassette was amplified with primers carrying 58 bp homology arms to sequences immediately upstream and downstream of GAA repeats and the product was inserted in intron 1 of pBAC- XN-Lac. Subsequently the RPSL-Neo was replaced with a PCR product containing 280 and 830 GAA repeats amplified from NA 16207 using GAA- F and GAA-R primers (1). Due to low recombination efficiency, successful recombinant bacterial colonies were identified by colony blot assay and later confirmed by Southern blot. Colony blot and Southern blot were carried out using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) according to manufacturer's instructions and using a digoxigenin (DIG)-labelled TTCio probe for detection. Sequencing of GAA repeats on the TTC strand was performed using 602R. Since sequencing of the GAA strand is blocked by the presence of a long stretch of A immediately upstream of the GAA repeats we designed a primer called LX-lst-GAA (5 ' -TACTAAAAAATAC AAAAAAAAAAAAAAAAGAAG-3 ' ) which overcomes this area and allows sequencing of the GAA strand. To test the purity of the GAA expansion, we performed a PCR of pBAC- XN-Lac and pB AC -FXN- GAA -Luc vectors using primers GAA-F and GAA-R (1) and digested the products with MboII according to a method described by Holloway et al (45). Cre-loxP mediated retrofitting of pBAC- XN-Lac and pBAC- FXN-GAA-Luc vectors to iBAC vector and to pH-FRT-Hy was performed as previously described (39). pH-FRT-Hy was obtained by modifying pcDNA5/FRT (Life Technologies) (61). Packaging into HSV-1 amplicons was carried out as previously described (47, 62). Cell culture and primary lymphocytes

[0092] HEK-293 cells were cultured in DMEM medium supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin. HEK FRT cells were generated by transfecting the pFRT/lacZeo plasmid (Life Technologies) followed by selection in medium containing 100 g/ml Zeocin (Life Technologies). FXN-GAA-Luc and FXN-Luc clonal cell lines were propagated in complete DMEM medium (see above) supplemented with 100 μg/ml Hygromycin B (Life Technologies). For the CMV-Luc experiment, HEK cells were seeded in a 24-well plate at a density of 2xl0⁵ cells/well and after 24h they were transfected with a CMV-Luc expressing plasmid using Lipofectamine (Life Technologies) and Plus Reagent (Life Technologies). Epstein Barr virus-transformed lymphoblastoid cell lines GM15850 (from individuals affected by FRDA, alleles with 1030 and 650 GAA repeats) and GM15851 (from an unaffected sibling with normal range of GAA repeats) were obtained from the Human Genetic Cell Repository of the Coriell Institute (USA) and propagated in RPMI1640 medium supplemented with 15% FBS and 2 mM L-glutamine. SH-SY5Y cells were cultured in DMEM/F-12 supplemented with 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin/streptomycin. SH-SY5Y cells were infected with iBAC -FXN-Luc and iBAC-FXN- GAA-Luc vectors packaged in HSV-1 amplicons as previously reported (39, 47). Blood was collected from anonymous individuals affected by FRDA using Vacutainer tubes. Only patients who were shown to have two expanded alleles using a PCR based assay were included in the study. The assays were performed by accredited molecular genetic testing laboratories in the UK. Primary lymphocytes were isolated from blood samples by centrifugation on Ficoll-Paque PLUS gradient (GE Healthcare) in Leucosep tubes (Greiner Bio-One), according to protocol described by Miltenyl Biotec (MACS). Cell viability was monitored using trypan blue exclusion and purified cells maintained in RPMI1640 medium supplemented with 15% FBS and 2 mM L- glutamine. Compounds were added to the purified cells at the concentrations indicated above and incubated for 72 hr. Cells were spun down at 200 g for 5 min at room temperature and lysed for RNA total extraction. All cells were grown at 37°C in 5% C0₂.

PCR, qPCR and copy number assay

[0093] Genomic DNA from FXN-GAA-Luc and FXN-Luc clonal cell lines was isolated by standard phenol/chloroform extraction and ethanol precipitation. PCR amplification of the GAA repeat sequence was carried out on 200 ng of genomic DNA using primers 147F and 602R (63) and Expand Long Template DNA Polymerase (Roche) as previously described (1, 44). Vector integration at the docking site was assessed by PCR analysis using primers pSV40-F (5'- CCAGTTCCGCCCATTCTC-3') and Hygro-R (5 ' -C AGCTATTTACCCGCAGGAC-3 ' ) using AmpliTaq Gold (Roche). For copy number determination, genomic DNA from FXN-Luc and FXN-GAA-Luc clonal cell lines was isolated using Illustra Tissue and Cells GenomicPrep Mini Spin Kit (GE Healthcare), according to the manufacturer's instructions. The number of transgene copies was determined by real time PCR, using the relative standard curve method. Five-fold dilution standards were prepared to generate a standard curve for each primer pair. The upstream region of the GAA repeats in intron 1 of the FXN gene was amplified using the primers UpGAA- F and UpGAA-R (24) and normalising data by GAPDH, using the primers GAPDH-F and GAPDH-R (24). Three independent genomic DNA samples per cell line were quantified in triplicate by real-time PCR using the SYBR Green PCR Master Mix (Applied Biosystems). Each 25 μΐ reaction contained 2 μΐ of genomic DNA dilution, IX SYBR Green PCR Master Mix and 70 nM of each primer. The assay was performed using the StepOnePlus Real-Time PCR system (Applied Biosystems) with the following protocol: 10 minutes at 95°C for enzyme activation, followed by 40 cycles of denaturation at 95 °C for 15 seconds and primer annealing and extension at 60°C for 1 minute. Specificity of amplification was monitored with a final dissociation stage which generates a melting curve. The number of transgene copies was determined by comparison with the acceptor cell line HEK FRT as reference sample, which carries three endogenous FXN loci as determined by FISH.

qRT-PCR

[0094] Total RNA from HEK FRT, FXN-Luc and FXN-GAA-Luc clonal cell lines was extracted using RNeasy Mini Kit (Qiagen) and treated with RNase-Free DNase (Qiagen). cDNA was synthesized from 1 μg of total RNA using random primers (Life Technologies) and Superscript III Reverse Transcriptase (Life Technologies) in a reaction volume of 20 μΐ. qPCR was carried out as described above, using qFXN-Luc-F (5 ' -CGGAAAAGATGCTGGAAGTG-3 ' ) and qFXN-Luc-R (5 ' - AACCAGGGCGTATCTCTTCA-3 ' ) for FXN-luciferase mRNA detection, FXN-F and FXN-R (24) for FXN mRNA detection and data was normalised to GAPDH. Total RNA from primary lymphocytes was extracted and treated with DNase using RNAqueous -Micro Kit (Life Technologies). RNA was reverse-transcribed as above. FXN mRNA was detected using FXN-F and FXN-R (24) and data normalised to GAPDH, HPRT and Beta-Actin using the following primers: GAPDH-2F (5 ' -GGTCTCCTCTGACTTCAAC A-3 ' ) and GAPDH-2R (5'- AGCCAAATTCGTTGTCATAC-3 ' ) (RTPrimerDB, ID: 912), HPRT-F (5'- GCCAGACTTTGTTGGATTTG-3 ' ) and HPRT-R (5 ' -CTCTC ATCTTAGGCTTTGT ATTTTG- 3') (RTPrimerDB, ID: 984), ACTB-F (5 ' - AGCGCGGCTAC AGCTTC A-3 ' ) and ACTB-R (5'- CGTAGC ACAGCTTCTCCTTAATGTC-3 ' ) (RTPrimerDB, ID: 2203).

Luciferase assay [0095] FXN-GAA-Luc and FXN-Luc clonal cell lines were counted with trypan blue or Scepter (Millipore) and seeded in 6-cm dishes (1.5xl0⁶ cells/dish), 24-well (lxlO⁵ cells/well) or 96-well (3xl0⁴ cells/well) format. When assaying luciferase expression, cells were washed with PBS and lysed in Lysis Buffer (25 mM TrisP0₄ pH7.8, 2 mM CDTA, 10% Glycerol and 1% Triton-X 100) for 20 minutes at 4°C. 75 μΐ of lysate were mixed with 100 μΐ of Luciferase Assay Buffer (15 mM MgS0₄, 15 mM KP0₄ pH 7.8, 4 mM EGTA pH 7.8, 2 mM ATP and 2mM DTT) and 50 μΐ of D-Luciferin (0.3mg/ml). The relative light units (R.L.U.) of luciferase of each cell line were determined using the Dynex MLX 96 Well Plate Luminometer and were normalised by total protein concentration, determined using Bicinchoninic acid solution (BCA, Sigma).

Compound library screening

[0096] The complete library is made of 25,000 compounds dissolved in dimethyl sulfoxide (DMSO) at a concentration of 2.5 mg/ml. For the primary screening, FXN-GAA-Luc cells were seeded in 96-well at a density of 3xl0⁴ cells/well and incubated in triplicate with the 88 preselected compounds at a final concentration of 20 μΜ for 48 hr. Luciferase assay was performed as described above. An authentic sample of C5 [l-(3,4-dimethylphenoxy)-3-(4-morpholinyl)-2- propanol hydrochloride] was purchased from ChemBridge Corporation (ID: 5358626); m/z (ESI+) 266 (100%, [M+H]⁺), 288 (35%, [M+Na]⁺).

Chromatin immunoprecipitation

[0097] Analysis of histone modifications in the FXN promoter and regions flanking GAA repeats in lymphoblastoid (GM15850 and GM15851), FXN-Luc and FXN-GAA-Luc cell lines was performed as previously described (64). Lymphoblastoid cell lines were treated with DMSO or C5 for 48 hours before crosslinking. Proteins were cross-linked to DNA by 1% formaldehyde treatment for 4 minutes (GM15850 and GM15851) or 7 minutes (FXN-Luc and FXN-GAA-Luc cells). Chromatin from lysed cells was sheared by sonication to obtain fragments from 100 to 800 bp using a Bioruptor (Diagenode). Immunoprecipitation experiments were performed using the Immunoprecipitation Kit with Dynabeads Protein G (Life Technologies), according to the manufacturer's instructions. Chromatin was incubated with one of the following antibodies at 4°C: (i) anti-H3K9ac (07-352), (ii) anti-H4K8ac (07-328), (iii) anti-H3K9me2 (07-441), (v) anti- H3K9me3 (07-442) and (vi) normal rabbit serum (12-370) used as a negative control (all antibodies were purchased from Millipore). Samples were treated with 500 mM NaCl, 0.25 mg/ml RNase A and 0.25 mg/ml proteinase K and incubated at 65°C overnight to reverse cross- linking. After reversing the crosslinking, the amount of FXN DNA immunoprecipitated was quantified in triplicate by real-time PCR using the SYBR Green PCR Master Mix (Applied Biosystems) and determined using the AACt method. The immunoprecipitated DNA was normalised to 10% of input and taking into consideration the background signal. For the analysis of the FXN promoter and the regions upstream and downstream of the GAA repeats, we used primers described in (24).

Bisulfite sequencing

[0098] Genomic DNA of the FXN-Luc and FXN-GAA-Luc cell lines was isolated using the Illustra Tissue and Cells GenomicPrep Mini Spin Kit (GE Healthcare). Two micrograms of DNA was used in the bisulfite conversion reaction using the EpiTect Bisulfite kit (Qiagen), according to the manufacturer's instructions. Nested PCR was performed on bisulfite-converted DNA using HotStart Taq DNA Polymerase (Qiagen) with the following primers, described in (26): FIG and RIG (first round PCR) and F2G and R2G (second round PCR) for the upstream region of the GAA repeats in intron 1 of the FXN gene; NH1F and SLGR2 (first round PCR) and NH2F and SLGRl (second round PCR) for the downstream region of the GAA repeats in intron 1 of the FXN gene. PCR products were cloned into pGEM-T easy vector. A total of 10 colonies were sequenced per region and for each cell line.

Analysis of frataxin protein

[0099] For western blot analysis of FXN-lucif erase protein, FXN-Luc and FXN-GAA-Luc cells were washed in PBS and lysed in RIPA buffer (50 mM Tris pH 8, 150 mM NaCl, 2 mM EGTA, 0.5 % sodium deoxycholate, 1 % Igepal 630, 0.1% SDS) with protease inhibitors (Complete Mini, EDTA-free, Roche). Cells disruption was performed by repeated pipetting followed by sonication on ice (1.5 sec for 10 times) (Misonix XL-2000 sonicator). Cell lysates were spun at 1,300 g for 15 min at 4 °C, supernatant transferred to a new tube and protein concentration was determined through BCA assay. Protein samples were reduced in Laemmli buffer and incubated for 5 min at 100 °C. 50 μg of protein was resolved on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Following transfer on a PVDF type membrane (Immobilon P, Millipore), protein samples were incubated with the following antibodies: mouse monoclonal anti-luciferase (Santa Cruz, sc-57604, 1/1000 dilution) and rabbit polyclonal anti- beta Actin (Abeam, ab8227, 1/1000 dilution). Analysis of frataxin protein in HEK cells was carried out using Frataxin Dipstick Assay Kit (Mitosciences) according to manufacturer's instructions. Protein concentration in cell lysates was quantified by BCA assay and 10 μg of total protein was loaded in each well. Dried dipsticks were imaged with Chemidoc XRS system (Biorad) and quantication was performed using Image J software.

Fluorescence in situ hybridisation (FISH)

[00100] Chromosome preparation and FISH analyses were carried out as previously described (65) using the unmodified FXN BAC and the plasmid pH-FRT-Hy as probes. Transgene integration on chromosome 1 was confirmed using a chromosome 1 centromeric probe.

Assay development

Construction of pBAC- XN-Lwc and ^BAC-FXN-GAA-Luc genomic DNA reporter vectors

[00101] A BAC clone (RP11-265B8) containing the whole FXN locus from human chromosome 9 was used to generate FXN-Luc BAC vectors. This vector contains approximately 38 kb of promoter region, the 80 kb FXN locus, and 17 kb of downstream sequence (41). Since the most abundant FXN transcript consists of exons l-5a (1, 42), we inserted the firefly luciferase sequence in exon 5a immediately prior to the stop codon, generating pBAC- XN-Lac fusion vector (Fig. 1A). We included a sequence encoding a five amino-acid Gly-Ser-Gly-Ser-Gly (GSGSG) peptide linker between the exon 5a and luciferase sequence (Fig. 1A), to allow correct folding of the frataxin and luciferase proteins; the choice of the composition of the linker was based on previous reports (43). Construction of pBAC- XN-Lac was achieved by a selection/counter- selection homologous recombination strategy using homology arms to exon 5a. To confirm correct vector recombination, successful colonies were analysed by junction PCR, by restriction enzyme digestion followed by pulsed-field gel electrophoresis (PFGE) and by sequencing, which confirmed correct insertion of the GSGSG-lucif erase sequence in exon 5a (data not shown). The resulting modified FXN gene expresses a FXN-luciferase fusion protein within the context of the FXN genomic DNA locus.

[00102] In order to insert a GAA expansion into intron 1 of the pBAC- XN-Lac vector, a

PCR product containing expanded GAA repeats was amplified from genomic DNA isolated from a FRDA patient-derived lymphoblastoid cell line (GM 16207, alleles with 280 and 830 GAA repeats in the FXN gene) using an established PCR protocol (1, 44). The smaller PCR product carrying -280 GAA repeats flanked by 195 bp and 255 bp of homology arms upstream and downstream of the GAA repeats, respectively, was preferentially amplified and used for recombination. We used the RPSL-Neo selection/counter- selection homologous recombination method to replace the 6 GAA repeats present in the FXN BAC in intron 1 of FXN-Luc gene with the expanded GAA repeats amplified by PCR, generating pB AC -FXN- GAA -Luc vector. Due to low efficiency of recombination, successful colonies were identified through colony blot using a TTCio probe. The GAA expansion of three independent colonies was sized through Southern blotting, which confirmed an insertion of up to -310 GAA repeats (Fig. IB and C). To assess the purity of the -310 GAA repeats, sequencing was performed from both sides. We confirmed the presence of 213 GAA repeats on the GAA strand and 125 GAA repeats on the TTC strand, therefore achieving an overlap of at least 28 GAA repeats (data not shown). The presence of such overlap ensured the whole GAA expansion was sequenced. We detected no interruptions in the expanded GAA sequence and this was further corroborated by MboII digest according to a method previously described (Fig. ID) (45). The GAA repeats were amplified from pBAC- XN- GAA-Luc cl 2 vector by PCR and digested with MboII. The complete digestion of the PCR product confirms the purity of the GAA expansion in the pB AC -FXN- GAA -Luc cl 2 vector (Fig. ID).

Validation of pBAC- XN-Lwc and ^BAC-FXN-GAA-Luc reporter constructs

[00103] To investigate the effect of -310 GAA repeats on FXN-luciferase expression in the human neuronal cell line SH-SY5Y, we used the herpes simplex virus type-1 (HSV-1) amplicon vector system. SH-SY5Y cells are characterised by low efficiency of transfection and HSV-1 vectors mediate intact delivery of BACs to infected cells at high efficiency (39, 46). To allow packaging into HSV-1 amplicons, we used Cre/loxP recombination (Fig. 2A) to incorporate into pBAC- XN-Lac and pBAC-FXN-GAA-Luc a retrofitting vector containing the HSV-1 oriS origin of replication and pac packaging signals, a lacZ cassette for titration and sequences derived from the Epstein-Barr virus for extra-chromosomal vector retention, generating iBAC- XN-Lac and i AC-FXN-GAA-Luc respectively. Correctly retrofitted vectors were identified using PFGE analysis and vectors were packaged into HSV-1 amplicons using an improved helper virus-free packaging protocol as described previously (data not shown) (47). Average titres of 1-2 X 10⁷ transducing units per ml (t.u./ml) were obtained for all vectors. SH- SY5Y cells were transduced with iBAC-FXN-Luc and iB AC -FXN- GAA -Luc cl 1, 2 and 3 amplicons at a multiplicity of infection (M.O.I.) of 4 and luciferase assay was performed 4 days after infection. High efficiency of transduction was achieved as determined by LacZ staining (Fig. IF). The presence of up to -310 GAA repeats in iB AC -FXN- GAA -Luc vectors causes a reduction in FXN-luciferase expression by approximately 75% when compared to iBAC- XN- Luc vector, recapitulating the effect of GAA repeats on FXN expression observed in FRDA patient cells (Fig. IE). pB AC -FXN- GAA -Luc cl 2 vector (referred to as pBAC-FXN-GAA-Luc) was chosen for the following experiments.

Generation and characterisation of a GAA-expanded genomic DNA reporter model of FRDA

[00104] In order to generate a cell model which allows the dissection of the effect of

GAA repeats on FXN expression we then generated stable clonal cell lines carrying pBAC- XN- Luc and pB AC -FXN- GAA -Luc vectors by using site-specific vector integration, since random vector integration in the genome can affect transgene expression levels. Site-specific integration allows precise comparison of the two vectors in the absence of confounding effects due to differential integration site, since the two vectors are integrated at the same genomic location. To generate these cell lines, we adapted the previously described Flp-In system (Life Technologies) to BAC vectors. We developed a Flp-In BAC integration system by generating the retrofitting vector pH-FRT-Hy (Fig. 2A), which contains the Flp-In promoter-less hygromycin cassette. pH- FRT-Hy was then retrofitted into the pBAC-FXN-Luc and pB AC- FXN-GAA-Luc vectors using the Cre/loxP retrofitting strategy previously described, generating pFRT -FXN-Luc and pFRT- FXN-GAA-Luc vectors (Fig. 2A). A stable FRT acceptor cell line was generated by transfecting HEK cells with the plasmid pFRT-LacZeo followed by zeocin selection and confirmation of positive LacZ staining (HEK FRT cells). These cells were transfected with pFRT- XN-Lac and pFRT -FXN-GAA -Luc vectors together with the Flp recombinase-encoding plasmid pOG44 (Life Technologies). Stable clonal cell lines (referred to as FXN-Luc and FXN-GAA-Luc) were isolated in the presence of hygromycin and PCR was used to confirm correct vector integration at the FRT acceptor site (Fig. 2B). Random integration events are prevented since the promoter which drives hygromycin is only present at the docking site and any integration at other genomic locations will not result in antibiotic resistance. The presence of GAA repeats in FXN-GAA-Luc cells was confirmed by PCR (data not shown). Clonal cell lines were expanded and characterized.

[00105] Fluorescence in situ hybridization (FISH) was performed on FXN-Luc and FXN-

GAA-Luc clonal cell lines using the unmodified FXN BAC and the pH-FRT-Hy retrofitting vector as probes which, when co-localizing, indicate the presence of the integrated vector (Fig. 2C). FXN-Luc and FXN-GAA-Luc cell lines which have been generated using different vectors both show vector integration at the same location, on chromosome lp as confirmed by the use of a chromosome 1 centromeric probe (data not shown), confirming the consistency of the site- specific integration for large genomic DNA vectors (Fig. 2C). We then assessed vector copy number in FXN-Luc and FXN-GAA-Luc cells by real time PCR, using as reference sample the acceptor cell line HEK FRT, which carries three endogenous FXN loci as determined by FISH (Fig. 2C). We used this assay to select FXN-Luc and FXN-GAA-Luc clonal cell lines carrying one copy of transgene (Fig. 2D).

[00106] We then determined the effect of the GAA repeat expansion on FXN-luciferase expression by reverse transcriptase real time PCR (qRT-PCR) and by luciferase assay. The presence of GAA repeats causes a reduction of 37% in FXN-luciferase mRNA levels (Fig. 2F) and 42% in FXN-luciferase protein levels (Fig. 2G), as determined by qRT-PCR and by luciferase assay, respectively. Western blot analysis shows that FXN-Luc and FXN-GAA-Luc cells express a FXN-luciferase fusion protein of the expected size of -79 KDa (Fig. 2E). Epigenetic characterization of FXN-Luc and FXN-GAA-Luc cell lines

[00107] It has been shown that the presence of GAA repeats in intron 1 of the FXN locus reduces FXN expression by heterochromatin formation and increased CpG methylation around GAA repeats (24, 26). To test for the presence of repressive epigenetic hallmarks in our cell model, we first analysed histone modifications by chromatin immunoprecipitation (ChIP) at three sites, at the promoter and the regions flanking GAA repeats, upstream and downstream of GAA repeats, using antibodies specific for the human acetylated histones H3K9 and H4K8 and di- and tri-methylated histone H3K9. This analysis revealed a decrease in histone acetylation and an increase in histone methylation across the three regions in the FXN-GAA-Luc cell line when compared to FXN-Luc cells (Fig. 3A), as previously reported in FRDA patient brain tissue and patient-derived cell lines (24, 26, 31).

[00108] We then analysed CpG methylation by bisulfite sequencing as reported in previous studies (26). In the region upstream of the expanded GAA repeats, we found increased DNA methylation in FXN-GAA-Luc cells at CpG sites 4 and 5 (Fig. 3B), in agreement with previously published methylation data from FRDA patients (26, 28). Our results for CpG 6 differ from the results previously found in patients (26, 27), but are in agreement with methylation data from brain of FRDA mice (26). In the region downstream of the GAA repeats, we observed increased DNA methylation at CpG sites 1, 2 and 7 (Fig. 3B).

[00109] Since FXN-Luc and FXN-GAA-Luc cell lines share the same integration site and only differ by the presence of a ~310 GAA repeats expansion, it is most likely that the expansion is causing the changes in histone acetylation/methylation and DNA methylation observed (Fig. 3).

Example 1 - Use of FXN-GAA-Luc cell line for the screening of chemical libraries - identification of C5 [l-(3,4-dimethylphenoxy)-3-(4-morpholinyl)-2-propanol hydrochloride]

[00110] We have shown above that FXN-GAA-Luc cells carry a GAA expansion which causes heterochromatin-mediated silencing of FXN-GAA-Luc expression. Since recent publications report the successful up-regulation of FXN expression with histone deacetylase (HDAC) inhibitors (24, 33), we applied our novel genomic DNA reporter model of FRDA to the screening of novel small molecules with potential HDAC inhibitor function. We screened in silico a library of 25,000 compounds to extract representative structures incorporating known pharmacophores associated with HDAC inhibitors and, more generally, with Zn(II) binding motifs. This library has been designed to cover a wide range of biological space, including pharmacophores with well-characterised biological mechanisms in addition to structural motifs which exhibit biological effects with an unknown mechanism (48, 49). Structures have been excluded which contain highly reactive functionalities (e.g. aldehydes, or Schiff bases) or known toxicophores (e.g. poly-halogenated species or poly-nitro aromatics) and selected structures are amenable to both resynthesis and rapid diversification. Compounds were selected from this library based on the presence of motifs likely to bind to Zn(II), such as hydroxamic acids, diamines and amino alcohols, which may plausibly inhibit zinc-dependent enzymes such as HDACs. However, alternative mechanims of action cannot be ruled out. We identified 88 potential Zn(II) -binding compounds and performed the screening of such molecules in a 96 well format in triplicate by incubating FXN-GAA-Luc cells at a standard concentration of 20 μΜ for 48 hr and assessing FXN-luciferase expression by luciferase assay (Fig. 4A). We identified four compounds which significantly increased FXN-GAA-Luc expression levels above DMSO levels. In order to exclude the possibility of interaction of compounds with the luciferase assay and to discard those that show a generalized unspecific increase of gene expression we tested the effect of the selected four compounds on luciferase expression driven by the ubiquitous cytomegalovirus (CMV) promoter (Fig. 4B). None of the four compounds shows an increase in luciferase expression, however, three compounds show a decrease in luciferase levels, which could indicate early signs of cell toxicity. To avoid potentially cytotoxic molecules, we focussed subsequent studies on compound C5 (C5), the only molecule which did not affect CMV- luciferase levels (Fig. 4B).

[00111] To assess whether C5 acts as a HDAC inhibitor on the FXN gene and is able to reverse the GAA-mediated FXN silencing we incubated FRDA patient lymphoblastoid cells with C5 at 20 μΜ for 48 hr and analysed H3K9 and H4K8 acetylation upstream and downstream of GAA repeats. The FRDA cell line GM15850 shows a reduction in histone acetylation at these areas when compared to the wild-type cell line GM15851. When FRDA patient cells are incubated with C5, the FRDA histone acetylation is restored to wild-type levels suggesting C5 acts either directly or indirectly in inhibiting HDAC activity (Fig. 4C). C5 is an amino alcohol and its structure is showed in Figure 4D. To confirm the activity of C5 we sourced an authentic sample and further corroborated its identity through mass spectrometry. Example 2 - Characterisation of the effect of C5 on FXN expression

[00112] In order to further characterise this compound, we then performed a dose- response assay by incubating FXN-GAA-Luc cells with C5 at concentrations ranging from 1 μΜ to 1 mM for 48 hr. A sigmoidal- shaped dose-dependent increase in FXN-GAA-Luc expression was observed, with a high up-regulating effect observed at 300, 600 and 1000 μΜ (Fig. 5A). These three doses were chosen for further studies.

[00113] In order to determine whether the increase in FXN-GAA-Luc expression is due to an increase in transcription, we incubated FXN-GAA-Luc cells with C5 at the three chosen concentrations and analysed FXN-GAA-Luc mRNA by qRT-PCR. We show that C5 significantly increases FXN-GAA-Luc mRNA levels at 600 and 1000 μΜ (Fig. 5B). We then compared the effect of C5 on normal and GAA-expanded FXN loci, by incubation of FXN-Luc and FXN-GAA- Luc cells with C5 for 48 hr followed by luciferase assay. We found that C5 induces a significantly greater up-regulating effect on the FXN-GAA-Luc cells, demonstrating a relative specificity for the expanded FXN locus (Fig. 5C). However, a small but significant increase was also observed on FXN-Luc cells.

[00114] Finally, to exclude a specific action of C5 on the transgene only, we incubated untransfected HEK cells with C5 at the three concentrations for 48 hr and quantified endogenous FXN mRNA levels. We observed a 1.5-2 fold increase in endogenous FXN mRNA at 600 and 1000 μΜ (Fig. 5D). Furthermore, C5 significantly increases endogenous frataxin protein levels at by 1.2-fold at 600 μΜ and by 1.7-fold at 1000 μΜ, as determined by frataxin dipstick assay (Fig. 5E).

Example 3 - Validation of C5 in primary cells from Friedreich's ataxia patients

[00115] To test the efficacy of C5 as a potential novel therapeutic molecule for

Friedreich's ataxia, we tested C5 on primary lymphocytes isolated from FRDA patients, as these cells provide a readily obtainable source of patient primary cells. Primary lymphocytes were isolated from blood samples from three patients, using the Ficoll-Paque gradient as previously described (50). We incubated isolated primary lymphocytes with C5 at concentrations ranging from 300 μΜ to 800 μΜ and after 72 hr analysed FXN mRNA levels by qRT-PCR. In order to reduce the influence of fluctuations of reference genes on the final data, we normalised the FXN mRNA levels by the geometric mean of three different reference genes. Incubation with C5 up- regulates FXN expression in all three patients by 1.5-2 fold, an increase similar to that observed in FXN-GAA-Luc cells. The identification of a new molecule through our cell model and the confirmation of its effect in primary lymphocytes from FRDA patients highlight the suitability of the FXN-GAA-Luc cells as a high-throughput expression model of Friedreich's ataxia. Example 4 - Assessment of further compounds

General Experimental

[00116] All reactions involving moisture-sensitive reagents were carried out under a nitrogen atmosphere using standard vacuum line techniques and glassware that was flame-dried before use. Solvents were dried following the procedure outlined by Grubbs and co-workers ⁶⁶. Water was purified by an Elix^® UV- 10 system. All other solvents and reagents were used as supplied (analytical or HPLC grade) without prior purification. Organic layers were dried over anhydrous MgS0₄. In vacuo refers to the use of a rotary evaporator attached to a diaphragm pump. Pet ether refers to the fraction of petroleum spirit boiling between 30 and 40 °C, unless otherwise stated.

[00117] Thin layer chromatography was performed on Merck aluminium plates coated with 60 F254 silica. Plates were visualised using UV light (254 nm) or 1 % aqueous KMn0₄. Flash column chromatography was performed on Kieselgel 60 silica in a glass column. Melting points were recorded on a Gallenkamp Hot Stage apparatus and are uncorrected. Infrared spectra were recorded on a Bruker Tensor 27 FT-IR spectrometer, as neat samples. Selected characteristic peaks are reported in wavenumbers (cm ¹). NMR spectra were recorded on Bruker Avance spectrometers (DPX200, DQX400, AVN500 or AVC500) in the deuterated solvent stated. The field was locked by external referencing to the relevant deuteron resonance. Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS) where 5H (TMS) = 0.00 and 5c (TMS) = 0.00. Coupling constants (J) are quoted in Hz and are reported to the nearest 0.1 Hz. The coupling constants were determined by analysis using ACD Labs software. Low-resolution mass spectra were recorded on either a VG MassLab 20-250 or a Micromass Platform 1 spectrometer from solutions of methanol. Accurate mass measurements were run on either a Bruker Micro TOF internally calibrated with polyalanine, or a Micromass GCT instrument fitted with a Scientific Glass Instruments BPX5 column (15 m x 0.25 mm) using amyl acetate as a lock mass, by the mass spectrometry department of the Chemistry Research Laboratory, University of Oxford, UK. m/z values are reported in Daltons and followed by their percentage abundance in parentheses. General Synthetic Procedures

General procedure 1: synthesis of glycidyl ethers (YQ01-25A) (Modified from: Connolly et a/., 200

[00118] A stirred solution of aryl alcohol (1.3- 1.5 mmol, 1 eq.), caesium carbonate

(CS2CO3) (5 eq.), and epichlorohydrin (5 eq.) in acetonitrile (10 mL) was heated under reflux (82 °C) for 3 hours. The reaction mixture was cooled to room temperature, filtered and concentrated in vacuo. The resultant oil was taken up in toluene and washed successively with water, 1 M sodium hydroxide and water. The organic phase was dried, filtered and concentrated in vacuo.

HNR₂

[00119] A stirred solution of glycidyl ether (0.8- 1.5 mmol, 1 eq.), amine (1 eq.), and catalytic amount of pyridine in ethanol (10 mL) was heated at reflux (78 °C) for 4 hours. The reaction mixture was concentrated in vacuo. Subsequent purification was achieved through flash column chromatography (solvent system specified for each compound).

Synthesis of intermediates

2-((3,4-dimethylphenoxy)methyl)oxirane (YQ01A)

10

[00120] According to General Procedure 1, using 3,4-dimethylphenol (0.156 g, 1.28 mmol), Cs₂C0₃ (2.08 g, 6.4 mmol), and epichlorohydrin (0.51 mL, 6.5 mmol), YQ01A was obtained as an orange oil (0.18 g, 80%). δ_Η (400MHz, CDCI3) 2.22 (3H, s, C(7)CH₃), 2.26 (3Η, s, C(8)CH₃), 2.77 (1Η, dd, J=4.9 Hz, 2.7 Hz, C(1)H"), 2.86-2.97 (1Η, m, C(l)H'), 3.29-3.44 (1Η, m, C(2)H), 3.96 (1Η, dd, 7=10.99, 5.68 Hz, C(3)H"), 4.20 (1Η, dd, 7=11.12, 3.28 Hz, C(3)H'), 6.69 (1Η, dd, 7=8.21, 2.65 Hz, C(5)H), 6.77 (1Η, d, 7=2.78 Η z, C(9)H), 7.06 (1Η, d, 7=8.34 Hz, C(6)H); 5_C (400MHz, CDCb) 18.84 (C(8)H₃), 20.06 (C(7)H₃), 44.77(C(11)H₂), 50.27 (C(10)O), 68.78 (C(9)H₂), 111.51 (C(6)), 116.27(C(2)), 129.20 (C(4)CH₃), 130.34 (C(5)H), 137.81 (C(3)CH₃), 156.64 (C(l)O); m/z (ESI⁺) 179 ([M+H]⁺, 100%); HRMS (ESI⁺) CnHi₄Na0₂ ([M+Na]⁺) requires 201.0891 found 201.0885.

2-((naphthalen-2-yloxy)methyl)oxirane (YQ18-25A) ⁶⁸

[00121] According to General Procedure 1 , using 2-naphthol (0.64 g, 4.47 mmol),

Cs₂C0₃ (7.33 g, 22.50 mmol), and epichlorohydrin (1.76 mL, 22.50 mmol), YQ18-25A was obtained as a brown oil (0.74 g, 83 %). δ_Η (400MHz, CDCb) 2.82 (IH, dd, 7=4.89, 2.69 Hz, C(1)H"), 2.90-3.00 (1Η, m, C(l)H'), 3.45 (1Η, ddt, 7=5.75, 4.03, 2.93, 2.93 Hz, C(2)H), 4.04 (1Η, dd, 7=11.00, 5.87 Hz, C(3)H"), 4.29-4.39 (1Η, m, C(3)H'), 7.20 (1Η, d, 7=2.45 Hz, C(13)H), 7.29 (1Η, dd, 7=8.90, 2.59 Hz, C(5)H), 7.41-7.49 (1Η, m, C(IO)H), 7.51-7.58 (1Η, m, C(9)H), 7.78-7.89 (3Η, m, C(8)H, C(6)H, C(l l)H); 5_C (400MHz, CDCb) 44.72 (C(1)H₂), 50.2 (C(2)H), 68.82(C(3)H₂), 106.97 (C(13)H), 118.92 (C(5)H), 123.99 (C(9)H), 126.6 (C(10)H), 126.95 (C(l l)H), 127.82 (C(8)H), 129.28 (C(7)), 129.66 (C(6)H), 134.57 (C(12)), 156.56 (C(4)0); m/z (ESI⁺) 201 ([M+H]⁺, 100%).

(S)-2-((3,4-dimethylphenoxy)methyl)oxirane (YQ02A)

1 1

[00122] According to General Procedure 1, using 3,4-dimethylphenol (0.16 g, 1.28 mmol), (tf)-epichlorohydrin (0.50 mL, 6.38 mmol), and Cs₂C0₃ (2.93 g, 9.00 mmol), YQ02A was obtained as an orange oil (0.1597 g, 70.28%). δ_Η (400MHz, CDCb) 2.23 (3H, s, C(7)CH ), 2.27 (3Η, s, C(6)CH ), 2.78 (1Η, dd, 7=4.93, 2.65 Hz, C(1)H"), 2.87-2.96 (1Η, m, C(l)H'), 3.32- 3.41 (1Η, m, C(2)H), 3.96 (1Η, dd, 7=11.12, 5.56 Hz, C(3)H"), 4.20 (1Η, dd, 7=10.86, 3.28 Hz, C(3)H'), 6.70 (1Η, dd, 7=8.21, 2.65 Hz, C(9)H), 6.78 (1Η, d, 7=2.78 Hz, C(5)H), 7.07 (1Η, d, 7=8.08 Hz, C(8)H); 5_C (400MHz, CDCb) 18.84 (C(11)H₃), 20.06 (C(10)H₃), 44.77 (C(1)H₂), 50.27 (C(2)H), 68.78 (C(3)H₂), 111.51 (C(9)H), 116.27 (C(5)H), 129.19 (C(8)H), 130.34 (C(7)H), 137.81 (C(6)CH₃), 156.67 (C(4)0); m/z (ESI⁺) 201 ([M+Na]⁺, 100%); HRMS (ESI⁺) CnHi₄Na0₂ ([M+Na]) requires 201.0891 found 201.0889. /?)-2-((3,4-dimethylphenoxy)methyl)oxirane (YQ03A)

[00123] According to General Procedure 1, using 3,4-dimethylphenol (0.16 g, 1.28 mmol), (S)-epichlorohydrin (0.50 mL, 6.38 mmol), and Cs₂C0₃ (2.93 g, 9.00 mmol), YQ03A was obtained as an orange oil (0.1813 g, 79.78%). δ_Η (400MHz, CDC1 ) 2.23 (3H, s, C(lO)Hj), 2.27 (3Η, s, C(l l)Hj), 2.77 (1Η, dd, 7=4.93, 2.65 Hz, C(1)H"), 2.92 (1Η, t, 7=4.55 Hz, C(l)H'), 3.37 (1Η, dq, 7=6.76, 2.88 Hz, C(2)H), 3.96 (1Η, dd, 7=11.12, 5.56 Hz, C(3)H"), 4.20 (1Η, dd, 7=11.12, 3.28 Hz, C(3)H'), 6.70 (1Η, dd, 7=8.21, 2.65 Hz, C(9)H), 6.78 (1Η, d, 7=2.53 Hz, C(5)H), 7.06 (1Η, d, 7=8.08 Hz, C(8)H); 5_C (400MHz, CDC1 ) 18.84 (C(11)H₃), 20.06 (C(10)H₃), 44.76 (C(1)H₂), 50.28 (C(2)H), 68.79 (C(3)H₂), 111.51 (C(9)H), 116.28 (C(5)H), 129.19 (C(8)H), 130.34 (C(7)H), 137.81(C(6)CH₃), 156.65 (C(4)0); m/z (ESI⁺) 201 ([M+Na]⁺, 100%); HRMS (ESI⁺) CnHi₅0₂ ([M+H]) requires 179.1072 found 179.1070.

-tolyloxy)methyl)oxirane (YQ04A)

7

[00124] According to General Procedure 1 , using o-cresol (0.16 g, 1.50 mmol), epichlorohydrin (0.59 mL, 7.50 mmol), and Cs₂C0₃ (2.40 g, 7.5 mmol), YQ04A was obtained as a light yellow oil (0.20 g, 82 %). δ_Η (400MHz, CDC1 ) 2.29-2.32 (3H, s, C(lO)Hj), 2.83 (1Η, dd, 7=5.01, 2.57 Hz, C(1)H₂ "), 2.92-2.97 (1Η, m, C(1)H₂ '), 3.41 (1Η, ddt, 7=5.50, 4.16, 2.87, 2.87 Hz, C(2)H), 4.02 (1Η, dd, 7=11.00, 5.38 Hz, C(3)H₂ "), 4.27 (1Η, dd, 7=11.13, 3.06 Hz, C(3)H₂ 'X 6.85 (1Η, d, 7=8.70 Hz, C(9)H), 6.93 (1Η, td, 7=7.34, 0.73 Hz, C(7)H), 7.14-7.23 (2Η, m, C(6)H, C(8)H); 5_C (400MHz, CDC1 ) 16.24 (C(10)H₃), 44.69 (C(1)H₂), 50.38 (C(2)H), 68.68 (C(3)H₂), 111.26 (C(9)H), 120.95 (C(7)H), 126.81 (C(8)H), 127.07 (C(5)), 130.84 (C(6)H), 156.65 (C(4)0); m/z (ESI⁺) 187 ([M+Na]⁺, 50%). -tolyloxy)methyl)oxirane (YQ05A)

[00125] According to General Procedure 1 , using m-cresol (0.16 g, 1.50 mmol), epichlorohydrin (0.59 mL, 7.50 mmol), and CS2CO3 (2.40 g, 7.50 mmol), YQ05A was obtained as a light yellow oil (0.2790 g, 98.72 %). δ_Η (400MHz, CDCI3) 2.36 (3H, s, C(6)CH₃), 2.77 (1Η, dd, 7=4.93, 2.65 Hz, C(l)H'), 2.92 (1Η, dd, 7=4.93, 4.17 Hz, C(1)H"), 3.30-3.43 (1Η, m, C(2)H), 3.96 (1Η, dd, 7=10.99, 5.68 Hz, C(3)H'), 4.22 (1Η, dd, 7=11.12, 3.28 Hz, C(3)H"), 6.68-6.88 (3Η, m, C(7)H, C(5)H, C(9)H), 7.20 (1Η, t, 7=7.71 Hz, C(8)H); 5_C (400MHz, CDC1₃) 21.53 (C(10)H₃), 44.73 (C(1)H₂), 50.22 (C(2)H), 68.62 (C(3)H₂), 111.47 (C(9)H), 115.49 (C(5)H), 122.06 (C(7)H), 129.26 (C(8)H), 139.59 (C(6)), 158.50 (C(4)0); m/z (ESI⁺) 187 ([M+Na]⁺, 40%). -((/?-tolyloxy)methyl)oxirane (YQ06A)

[00126] According to General Procedure 1, using /?-cresol (0.16 g, 1.50 mmol), Cs₂C0₃

(2.40 g, 7.50 mmol), and epichlorohydrin (0.59 mL, 7.50 mmol), YQ06A was obtained as a clear oil (0.17 g, 68 %). δ_Η (400MHz, CDC1 ) 2.33 (3H, s, C(6)CH ), 2.79 (1Η, dd, 7=5.01, 2.57 Hz, C(l)H'), 2.93 (1Η, t, 7=4.52 Hz, C(1)H"), 3.34-3.42 (1Η, m, C(2)H), 3.97 (1Η, dd, 7=11.00, 5.62 Hz, C(3)H'), 4.22 (1Η, dd, 7=11.13, 3.30 Hz, C(3)H"), 6.82-6.91 (2Η, m, C(5)H, C(9)H), 7.12 (2Η, d, 7=8.56 Hz, C(6)H, C(8)H); 5_C (400MHz, CDC1 ) 20.50 (C(10)H₃), 44.78 (C(1)H₂), 50.26 (C(2)H), 68.88 (C(3)H₂), 114.53 (C(5)H, C(9)H), 129.97 (C(6)H, C(8)H), 130.50 (C(6)), 156.42 (C(4)0); m/z (ESI⁺) 187 ([M+Na]⁺, 30%).

2-(((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)methyl)oxirane (YQ16A)

15

[00127] According to General Procedure 1, using Cs₂C0₃ (2.40 g, 7.50 mmol), 5,6,7,8- tetrahydro-2-naphthol (0.23 g, 1.50 mmol), and epichlorohydrin (0.59 mL, 7.50 mmol), YQ16A was obtained as a pink oil (0.29 g, 94 %). δ_Η (400MHz, CDC1₃) 1.72-1.86 (4H, m, C(8)H₂, C(9)H₂), 2.67- 2.78 (5Η, m, C(1)H\ C(7)H₂, C(10)H₂), 2.91 (1Η, dd, 7=4.80, 4.29 Hz, C(1)H"), 3.26-3.42 (1Η, m, C(2)H), 3.95 (1Η, dd, 7=11.12, 5.56 Hz, C(3)H'), 4.18 (1Η, dd, 7=11.12, 3.28 Hz, C(3)H"), 6.64 (1Η, d, 7=2.53 Hz, C(5)H), 6.70 (1Η, dd, 7=8.34, 2.53 Hz, C(13)H), 6.99 (1Η, d, 7=8.34 Hz, C(12)H); 5_C (400MHz, CDCI3) 23.12 (C(9)H₂), 23.38 (C(8)H₂), 28.57 (C(10)H₂), 29.68 (C(7)H₂), 44.80 (C(1)H₂), 50.27 (C(2)H), 68.78 (C(3)H₂), 112.40 (C(5)H), 114.59 (C(13)H), 129.88 (C(l l)), 129.98 (C(12)H), 138.25 (C(6)), 156.27 (C(4)0); m/z (ESI⁺) 227 ([M+Na]⁺, 60%). 2- 4-dichlorophenoxy)methyl)oxirane (YQ17A)

[00128] According to General Procedure 1, using 3,4-dichlorophenol (0.26 g, 1.60 mmol),

Cs₂C0₃ (2.40 g, 7.50 mmol), and (S)-epichlorohydrin (0.59 mL, 7.50 mmol), YQ17A was obtained as a yellow oil (0.33 g, 95 %). δ_Η (400MHz, CDCI3) 2.75 (IH, dd, J=4.80 Hz, 2.78 Hz, C(l)H), 2.92 (1Η, dd, J=4.80 Hz, 4.04 Hz, C(l)H), 3.34 (1Η, m, C(2)H), 3.89 (1Η, dd, J=10.99 Hz, 5.94 Hz, C(3)H), 4.24 (1Η, dd, J=10.86 Hz, 2.78 Hz, C(3)H), 6.75-6.84 (1Η, m, C(9)H), 6.96-7.08 (1Η, m, C(5)H), 7.30-7.37 (1Η, m, C(8)H); 5_C (400MHz, CDCI3) 44.49 (C(1)H₂), 49.86 (C(2)H), 69.3 (C(3)H₂), 114.67 (C(9)H), 116.53 (C(5)H), 124.52 (C(6)), 130.73 (C(8)H), 132.88 (C(7)), 157.46 (C(4)0).

Synthesis of Example compounds

-(3,4-dimethylphenoxy)-3-morpholinopropan-2-ol (YQ01)

[00129] According to General Procedure 2, YQ01A (0.13 g, 0.74 mmol) and morpholine (0.07 mL, 0.74 mmol) were used. Purification by chromatography (Methanol/DCM, 3: 100 to 1 :20) afforded YQ01 as a yellow oil (0.1371 g, 69 %). v_max (neat) 3433 (O-H); δ_Η (400MHz, CDCI3) 2.20 (3H, s, C(7)f¾), 2.24 (3H, s, C(8)f¾), 2.41-2.62 (4H, m, C(12)Hox, C(15)H^, C(11)H₂), 2.63-2.71 (2Η, m, C{\2)H_eq, C{\5)H_eq, 3.66-3.80 (4Η, m, C(13)H₂, C(14)H₂), 3.97 (2Η, d, 7=5.05 Hz, C(9)H₂), 4.06-4.17 (1Η, m, C(IO)H), 6.67 (1Η, dd, 7=8.08, 2.78 Hz, C(6)H), 6.75 (1Η, d, 7=2.53 Hz, C(2)H), 7.03 (1Η, d, 7=8.34 Hz, C(5)H); 5_C (400MHz, CDC1₃) 18.82 (C(7)H₃), 20.05 (C(8)H₃), 53.78 (C(12)H₂, C(15)H₂), 61.14 (C(11)H₂), 65.52 (C(IO)OH), 66.99 (C(13)H₂, C(14)H₂), 70.22 (C(9)H), 111.41 (C(5)H), 116.17 (C(2)H), 128.98 (C(4)), 130.29 (C(6)H), 137.74 (C(3)), 156.80 (C(l)O); m/z (ESI⁺) 266 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₅H₂₄N0 ⁺ ([M+H]⁺) requires 266.1756 found 266.1760.

(5)-l-(3,4-dimethylphenoxy)-3-morpholinopropan-2-ol (YQ02)

8

[00130] According to General Procedure 2, YQ02A (0.12 g, 0.67 mmol) and morpholine

(0.05 mL, 0.59 mmol) were used. Purification by chromatography (Methanol/DCM, 5: 100) afforded YQ02 as an orange oil (0.17 g, 95 %). [<x]∞ -13.10 (c 0.5 in CHC1 ); v_max (film) 3433 (O-H); δ_Η (400MHz, CDC1 ) 2.20 (3H, s, C(7)Hj), 2.24 (3Η, s, C(8)Hj), 2.41-2.62 (4Η, m, C(12)Hox, C(15)Hax, C(11)H₂), 2.63-2.72 (2Η, m, C(\2)H_eq, C(\5)H_eq, 3.46 (1Η, s, OH), 3.66- 3.81 (4Η, m, C(13)H₂, C(14)H₂), 3.97 (2Η, d, 7=5.05 Hz, C(9)H₂), 4.05-4.17 (1Η, m, C(IO)H), 6.67 (1Η, dd, 7=8.08, 2.53 Hz, C(6)H), 6.75 (1Η, , d, 7=2.27 Hz, C(2)H), 7.03 (1Η, d, 7=8.08 Hz, C(5)H); 5c (400MHz, CDC1 ) 18.82 (C(7)H₃), 20.05 (C(8)H₃), 53.78 (C(12)H₂, C(15)H₂), 61.15 (C(11)H₂), 65.51 (C(IO)OH), 66.98 (C(13)H₂, C(14)H₂), 70.22 (C(9)H), 111.41 (C(5)H), 116.17 (C(2)H), 128.98 (C(4)), 130.29 (C(6)H), 137.74 (C(3)), 156.80 (C(l)O); m/z (ESI⁺) 266 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₅H₂₄N0 ⁺ ([M+H]⁺) requires 266.1756 found 266.1761.

(R)-l-(3,4-dimethylphenoxy)-3-morpholinopropan-2-ol (YQ03)

8

[00131] According to General Procedure 2, YQ03A (0.13 g, 0.70 mmol) and morpholine

(0.06 mL, 0.67 mmol) were used. Purification by chromatography (Methanol/DCM, 5: 100) afforded YQ03 as a yellow oil (0.17 g, 92 %). [<x]∞ -13.10 (c 0.6 in CHC1 ); v_max (film) 3429 (O- H); δ_Η (400MHz, CDC1₃) 2.22 (3H, s, C(7)i¾), 2.26 (3H, s, C(8)Hj), 2.41-2.63 (4Η, m, C(12)Hox, C(15)Hax, C(11)H₂), 2.64-2.73 (2Η, m, C(l2)H_eq, C(\5)H_eq, 3.44 (1Η, s, OH), 3.66- 3.81 (4Η, m, C(13)H₂, C(14)H₂), 3.98 (2Η, d, 7=4.89 Hz, C(9)H₂), 4.05-4.17 (1Η, m, C(IO)H), 6.67 (1Η, dd, 7=8.31, 2.69 Hz, C(6)H), 6.77 (1Η, d, 7=2.45 Hz, C(2)H), 7.05 (1Η, d, 7=8.07 Hz, C(5)H); 5c (400MHz, CDCI3) 18.82 (C(7)H₃), 20.05 (C(8)H₃), 53.83 (C(12)H₂, C(15)H₂), 61.19 (C(11)H₂), 65.58 (C(IO)OH), 67.01 (C(13)H₂, C(14)H₂), 70.27 (C(9)H₂), 111.46 (C(5)H), 116.21 (C(2)H), 128.99 (C(4)), 130.31 (C(6)H), 137.75 (C(3)), 156.84 (C(l)O); m/z (ESI⁺) 266 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₅H₂₄N0 ⁺ ([M+H]⁺) requires 266.1756 found 266.1755. l-morpholino-3-(o-tolyloxy)propan-2-ol (YQ04)

[00132] According to General Procedure 2, YQ04A (0.11 g, 0.66 mmol) and morpholine

(0.06 mL, 0.66 mmol) were used. Purification by chromatography (Methanol/DCM, 3: 100) afforded YQ04 as a yellow oil (0.11 g, 56 %). v_max (film) 3200 (O-H); δ_Η (400MHz, CDC1 ) 2.16 (3H, s, C(7)Hj), 2.35-2.44 (2Η, m, C(11)H^, C(14)H^), 2.47-2.55 (2Η, m, C(10)H₂), 2.55-2.62 (2Η, m, C{\ \)H_eq, C(U)H_eq,), 3.28 (1Η, s, OH), 3.59-3.72 (4Η, m, C(12)H₂, C(13)H₂), 3.82-3.92 (2Η, m, C(8)H₂), 4.05-4.17 (1Η, m, C(9)H), 6.70-6.83 (2Η, m, C(4)H, C(6)H), 7.02-7.11 (2Η, m, C(3)H, C(5)H); 5c (400MHz, CDC1 ) 16.26 (C(7)H₃), 53.86 (C(11)H₂, C(14)H₂), 61.38 (C(10)H₂), 65.68 (C(9)OH), 67.01 (C(12)H₂, C(13)H₂), 70.28 (C(8)H₂), 111.16 (C(6)H), 120.75 (C(4)H), 126.82 (C(2)), 126.84 (C(3)H), 130.74 (C(5)H), 156.75 (C(l)O); m/z (ESI⁺) 252 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₅H₂₄N0 ⁺ ([M+H]⁺) requires 274.1419 found 274.1424. l-morpholino-3-(/n-tolyloxy)propan-2-ol (YQ05)

[00133] According to General Procedure 2, YQ05A (0.04 g, 0.27 mmol) and morpholine

(0.02 mL, 0.27 mmol) were used. Purification by chromatography (Methanol/DCM, 3: 100) afforded YQ05 as a yellow oil (0.14 g, 54 %). v_max (film) 3444 (O-H); δ_Η (400MHz, CDC1 ) 2.19 (3H, s, C(7)Hj), 2.32-2.63 (6Η, m, C(10)H₂ ,C(11)H₂, C(14)H₂), 3.01 (1Η, s, OH), 3.59-3.69 (4Η, m, C(12)H₂, C(13)H₂), 3.83-3.90 (2Η, m, C(8)H₂), 4.05-4.17 (1Η, m, C(9)H), 6.72-6.74 (2H, m, C(4)H, C(5)H), 6.99 (2Η, d, /=8.07 Hz, C(2)H, C(6)H); 5_C (400MHz, CDC1₃) 21.53 (C(7)H₃), 53.79 (C(11)H₂, C(14)H₂), 61.15 (C(10)H₂), 65.46 (C(9)OH), 66.99 (C(12)H₂, C(13)H₂), 70.05 (C(8)H₂), 111.40 (C(6)H), 115.43 (C(2)H), 121.89 (C(4)), 129.22 (C(5)H), 139.54 (C(3)H), 158.68 (C(l)O); m/z (ESI⁺) 252 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₅H₂₄N0₃ ⁺ ([M+H]⁺) requires 274.1419 found 274.1423. l-morpholino-3-(/?-tolyloxy)propan-2-ol (YQ06)

7

[00134] According to General Procedure 2, YQ06A (0.11 g, 0.67 mmol) and morpholine (0.06 mL, 0.67 mmol) were used. Purification by chromatography (Methanol/DCM, 3: 100) afforded YQ06 as a yellow oil (0.14 g, 54 %). v_max (film) 3423 (O-H); δ_Η (400MHz, CDC1 ) 2.19 (3H, s, C(7)Hj), 2.331-2.66 (6Η, m, C(10)H₂ ,C(11)H₂, C(14)H₂), 3.05 (1Η, s, OH), 3.57-3.71 (4Η, m, C(12)H₂, C(13)H₂), 3.81-3.91 (2Η, m, C(8)H₂), 3.97-4.07 (1Η, m, C(9)H), 6.67-6.80 (2Η, m, C(3)H, C(5)H), 6.99 (2Η, d, =8.07 Hz, C(2)H, C(6)H); 5_C (400MHz, CDC1 ) 20.49 (C(7)H₃), 53.82 (C(11)H₂, C(14)H₂), 61.18 (C(10)H₂), 65.60 (C(9)OH), 66.97 (C(12)H₂, C(13)H₂), 70.36 (C(8)H₂), 114.42 (C(6)H, (C(2)H), 129.92 (C(3)H, C(5)H), 130.25 (C(4)H), 156.59 (C(l)O); m/z (ESI⁺) 252 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₅H₂₄N0 ⁺ ([M+H]⁺) requires 274.1419 found 274.1425. l-(4-methoxyphenoxy)-3-morpholinopropan-2-ol (YQ07)

[00135] According to General Procedure 2, YQ07A (0.18 g, 1.02 mmol) and morpholine

(0.09 mL, 1.02 mmol) were used. Purification by chromatography (Methanol/DCM, 3: 100) afforded YQ07 as a yellow solid (0.18 g, 79 %). δ_Η (400MHz, CDC1 ) 2.41 - 2.73 (6H, m, C(10)H₂ ,C(11)H2, C(14)H₂), 3.65 - 3.79 (7Η, m, C(7)H₃, C(12)H₂, C(13)H₂), 3.87 - 4.00 (2Η, m, C(8)H₂), 4.05-4.14 (1Η, m, C(9)H), 6.79 - 6.90 (4Η, m, C(2)H, C(6)H, C(3)H, C(5)H); 5_C (400MHz, CDC1 ) 53.74 (C(7)H₃), 55.70 (C(11)H₂, C(14)H₂), 61.08 (C(10)H₂), 65.48 (C(9)OH), 66.96 (C(12)H₂, C(13)H₂), 70.87 (C(8)H₂), 114.61 (C(3)H, C(5)H), 115.50 (C(2)H, C(6)H), 152.82 (C(l)O), 154.00 (C(4)0); m/z (ESI⁺) 268 ([M+H]⁺, 100%); HRMS (ESI⁺) O₄H₂iN0₄ ⁺ ([M+]⁺) requires 268.1549 found 268.1538. l-morpholino-3-((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)propan-2-ol (YQ16)

[00136] According to General Procedure 2, YQ16A (0.24 g, 1.19 mmol) and morpholine

(0.10 g, 1.19 mmol) were used. Purification by chromatography (Methanol/DCM, 5: 100) afforded YQ16 as a yellow oil (0.16 g, 45 %). v_max (neat) 3426 (O-H); 1.72-1.85 (4H, m, C(6)H₂, C(7)H₂), 2.40-2.61 (4Η, m, C(13)H₂, C(14)Hax, C(17)Hax), 2.63-2.79 (6Η, m, C(5)H₂, C(8)H₂, C(14)H_eq, C(17)H_eq), 3.66-3.80 (4Η, m, C(15)H₂, C(16)H₂), 3.96 (2Η, d, =5.05 Hz, C(11)H₂), 4.04-4.17 (1Η, m, C(12)H), 6.64 (1Η, d, =2.78 Hz, C(IO)H), 6.69 (1Η, dd, =8.46, 2.65 Hz, C(2)H), 6.97 (1Η, d, =8.34 Hz, C(3)H); 5_C (400MHz, CDC1₃) 23.12 (C(6)H), 23.38 (C(7)H), 28.55 (C(9)H), 29.67 (C(8)H), 53.77 (C(14)H₂, C(17)H₂), 61.13 (C(13)H₂), 65.50 (C(12)H),66.98 (C(15)H₂, C(16)H₂), 70.20 (C(11)H₂), 112.32 (C(10)H), 114.48 (C(2)H), 129.69 (C(4)), 129.93 (C(3)H), 138.20 (C(9)), 156.45 (C(l)O); m/z (ESI⁺) 292 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₇H₂₆N03⁺ ([M+H]⁺) requires 292.1926 found 292.1910. l-(3,4-dichlorophenoxy)-3-morpholinopropan-2-ol (YQ17)

8

[00137] According to General Procedure 2, YQ17A (0.28 g, 1.27 mmol) and morpholine

(0.11 g, 1.27 mmol) were used. Purification by chromatography (Methanol/DCM, 5: 100) afforded YQ17 as a yellow solid (0.25 g, 64 %); v_max (neat) 3170 (O-H); δ_Η (400MHz, CDCI3) 2.41-2.60 (4H, m, C(14)Hax, C(17)Hax, C(7)H₂), 2.63-2.73 (2Η, m, C(14)H_eq, C(17)H_eq), 3.20- 3.65 (1Η, s, OH), 3.67-3.80 (4Η, m, C(15)H, C (16)H), 3.91-4.00 (2Η, m, C(IO)H), 4.05-4.15 (1Η, m, C(l l)H), 6.79 (1Η, dd, =8.84, 3.03 Hz, C(2)H), 7.03 (1Η, d, =3.03 Hz, C(6)H), 7.32 (1Η, d, =8.90 Hz, C(3)H); 5_C (400MHz, CDCI3) 157.72 (C(l)O), 132.84 (C(5)C1), 130.69 (C(2)H), 124.29 (C(4)C1), 116.43 (C(6)H), 114.6 (C(3)H), 70.68 (C(10)H₂), 66.94 (C(15)H₂, C(16)H₂), 65.15 (C(l l)OH), 60.78 (C(12)H₂), 53.69 (C(14)H₂, C(17)H₂); m/z (ESI⁺) 306 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci3Hi₈Cl₂N03⁺ ([M+H]⁺) requires 306.0664 found 306.0656. l-(4-methylpiperazin-l-yl)-3-(naphthalen-2-yloxy)propan-2-ol (YQ22) ⁶⁸

[00138] According to General Procedure 2, YQ18-25A (0.14 g, 1.37 mmol) and 1- methylpiperazine (0.15 g, 1.37 mmol) were used. Purification by chromatography (Ethyl acetate to Methanol, then filtering through Celite) afforded YQ22 was obtained as an yellow crystals (0.22 g, 52 %). Vmax (neat) 3345 (O-H); δ_Η (400MHz, CDC1₃) 2.31 (3H, s, C(18)H₃), 2.42 - 2.85 (9Η, m, C(13)H₂, C(14)H₂, C(15)H₂, C(16)H₂, C(17)H₂), 3.48 - 3.82 (1Η, s, OH), 4.08 - 4.13 (2Η, m, C(11)H₂), 4.14 - 4.22 (1Η, m, C(12)H), 7.15 (1Η, d, 7=2.53 Hz, C(7)H), 7.19 (1Η, dd, 7=8.84, 2.53 Hz, C(5)H), 7.34 (1Η, ddd, 7=8.08, 6.95, 1.14 Hz, C(l)H), 7.44 (1Η, ddd, 7=8.15, 6.88, 1.14 Hz, C(2)H), 7.69 - 7.81 (3Η, m, C(3)H, C(4)H ,C(8)H); 5_C (400MHz, CDC1₃) 46.02 (C(15)H₃), 55.16 (C(14)H₂, C(15)H₂, C(16)H₂, C(17)H₂), 60.43 (C(13)H₂), 65.45 (C(12)OH), 70.28 (C(11)H₂), 106.71 (C(10)H), 118.86 (C(2)H), 123.71 (C(6)H), 126.37 (C(7)H), 126.76 (C(5)H), 127.64 (C(8)H), 129.05 (C(4)), 129.41 (C(3)H), 134.45 (C(9)), 156.68 (C(l)O); m/z (ESI⁺) 301 ([M+H]⁺, 100%). l-morpholino-3-(naphthalen-2-yloxy)propan-2-ol (Al / YQ25)

5 3

[00139] According to General Procedure 2, YQ18-25A (0.13 g, 1.44 mmol) and aniline (0.13 g, 1.44 mmol) were used. YQ25 was obtained as an orange solid (1.7 g, 85 %). v_max (neat) 3429 (O-H); δ_Η (400MHz, CDC1 ) 2.42-2.52 (2H, m, C(14)Hax, C(17)H_ax), 2.53-2.63 (2Η, m, C(13)H₂), 2.63-2.71 (2Η, m, C(14)H_eq, C(17)H_eq), 3.71-3.79 (4Η, m, C(15)H₂, C(16)H₂), 4.06-4.14 (2Η, m, C(11)H₂), 4.14-4.22 (1Η, m, C(12)H), 7.16 (1Η, d, 7=2.53 Hz, C(IO)H), 7.20 (1Η, dd, 7=9.00, 2.50 Hz, C(2)H), 7.38 (1Η, ddd, 7=8.10, 6.88, 1.14 Hz, C(6)H), 7.45 (1Η, ddd, 7=8.15, 6.88, 1.14 Hz, C(7)H), 7.69-7.81 (3Η, m, C(5)H, C(3)H, C(8)H); 5_C (400MHz, CDC1 ) 53.77 (C(14)H₂, C(17)H₂), 61.12 (C(13)H₂), 65.44 (C(12)OH), 66.98 (C(15)H₂, C(16)H₂), 70.22 (C(11)H₂), 106.73 (C(10)H), 118.82 (C(2)H), 123.78 (C(6)H), 126.44 (C(7)H), 126.79 (C(8)H), 127.67 (C(5)), 129.07 (C(4)), 129.46 (C(3)H), 134.46 (C(9)), 156.63 (C(l)O); (ESI⁺) 288 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci7H₂₂N03⁺ ([M+Na]⁺) requires 310.1419 found 310.1412.

Methylation of the hydroxyl group (YQ26)

4- 2-methoxy-3-(naphthalen-2-yloxy)propyl)morpholine (YQ26)

[00140] A mixture of YQ25 (0.43 g, 1.51 mmol) and sodium hydride (0.16 g, 3.88 mmol) in dry THF (10 mL) was stirred under nitrogen protection. After 30 min, iodomethane (0.23 g, 3.69 mmol) was injected dropwise. The reaction mixture was left at room temperature overnight and quenched by water while the pH was maintained above 8. Resulting mixture was extracted with ethyl acetate, washed with water for three times, and concentrated in vacuo. Flash column chromatography (methanol/DCM, 1 : 100 to 3: 100) afforded YQ27 as a yellow oil (0.1514 g, 39.07%); Vmax (neat) 2853 (COC-H3); δ_Η (400MHz, CDCI3) 2.48-2.69 (6H, m, C(13)H₂, C(14)H₂, C(17)H₂), 3.54 (3Η, s, C(18)Hj), 3.69-3.83 (5Η, m, C(12)H, C(15)H₂, C(16)H₂), 4.10- 4.32 (2Η, m, C(11)H₂), 7.16-7.24 (2Η, m, C(10)H, C(22)H), 7.31-7.40 (1Η, m, C(6)H), 7.41- 7.49 (1Η, m, C(7)H), 7.68-7.89 (3Η, m, C(5)H, C(3)H, C(8)H); 5_C (400MHz, CDCI3) 54.43 (C(3)H₂, C(5)H₂), 57.97 (C(18)H₃), 59.62 (C(13)H₂), 67.02 (C(15)H₂, C(16)H₂), 68.67 (C(11)H₂), 77.35 (C(12)OH), 106.69 (C(10)H), 118.96 (C(2)H), 123.70 (C(6)H), 126.41 (C(7)H), 126.72 (C(5)H), 127.66 (C(8)H), 129.03 (C(4)), 129.42 (C(3)H), 134.48 (C(9)), 156.73 (C(l)O); m/z (ESI⁺) 302 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₈H₂₄N0₃ ⁺ ([M+H]⁺) requires 302.1756 found 302.1755.

Biological Assessment

a. Cell Culture

[00141] The FXN-GAA-Luc clonal cell line used was generated previously (Lufino et al. ,

2013). With the presence of -310 GAA repeats within intron 1 of the FXN gene, such cell line expresses the XN-Laciferase fusion protein at a relatively low level, recapitulating the gene silencing in FDRA. They were cultured in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum, 2 mM L-glutamine and 100 U/ml penicillin/streptomycin. Cells were first washed with phosphate buffered saline (PBS) and treated with trypsin to detach them from the flask. They were counted with Scepter (Millipore) and seeded afterwards in 96-well plates at a density of 25,000 cells/well in 200

The growing conditions were 37 °C and 5% CO2.

b. Luciferase Assay

[00142] The FXN-GAA-Luc clonal cells were seeded and cultured in 96-well plates. On occasions that the assay needed to be delayed, cells in plates would be parafilmed and snap froze on dry ice before storage under -80 °C. Prior to the measurement of luciferase expression, cells were washed with PBS and lysed in lysis buffer (25 mM TrisP0₄ pH 7.8, 2 mM CDTA, 10% Glycerol and 1% Triton-X 100) at 4 °C for 20 minutes. The plates were centrifuged at 4,500 rpm for 10 minutes. 75

luciferase assay buffer (15 mM MgS0₄, 15 mM KP0₄ pH 7.8, 4 mM EGTA, pH 7.8, 2 mM ATP and 2 mM DTT) and 50 μΐ. D- luciferin (0.3 mg/ml). The relative light units of luciferase were detected using Dynex MLX 96- Well Plate Luminometer and normalised by the total protein concentration which was determined using bicinchoninic acid solution.

c. Compound Screening

[00143] Fifteen compounds were selected based on the structural similarity with C5/YQ01 from the commercial libraries (Chembridge Corp.). They were obtained in the form of solutions in dimethyl sulfoxide (DMSO) with a uniform concentration of 2.5 mg/mL. Serial dilution was performed on them to prepare samples for luciferase assays. The final concentrations were 0.03 μΜ, 0.1 μΜ, 0.3 μΜ, 1 μΜ, 3 μΜ, 10 μΜ, 30 μΜ and -65 μΜ. Propranolol hydrochloride was purchased from Sigma Aldrich. Along with the compounds made in house, it was dissolved in DMSO and serially diluted to the following concentrations: 0.3 μΜ, 1 μΜ, 3 μΜ, 10 μΜ, 30 μΜ, 100 μΜ, 300 μΜ and 1000 μΜ. For the primary high-throughput screening, FXN-GAA-Luc cells were seeded as described above and incubated in triplicate with the compounds for 48 hr. Luciferase assay was performed following the aforementioned protocol.

Results:

Compound Biological Activity

YQ.02 See Figure 7

YQ.03 See Figure 8

YQ.04 See Figure 9

YQ.05 See Figure 10

YQ.06 See Figure 11

YQ.07 See Figure 12 Compound Biological Activity

Al (YQ.25) See Figure 13

YQ.16 See Figure 14

YQ22 See Figure 15

YQ26 See Figure 16

Example 5 - preparation of further example compounds

General Experimental

[00144] All solvents and reagents were used as supplied (analytical or HPLC grade) without prior purification. Water was purified by an Elix^® UV-10 system. Organic layers were dried over anhydrous MgS0₄. Brine refers to a sat. aq. solution of sodium chloride. In vacuo refers to the use of a rotary evaporator attached to a diaphragm pump. Pet ether refers to the fraction of petroleum spirit boiling between 30 and 40 °C. Thin layer chromatography was performed on aluminium plates coated with 60 F254 silica. Plates were visualised using UV light (254 nm) or 1% aq. KMn0₄. Flash column chromatography was performed on Kieselgel 60M silica in a glass column. Melting points were recorded on a Gallenkamp Hot Stage apparatus and are uncorrected. Infrared spectra were recorded on a Bruker Tensor 27 FT-IR spectrometer, as neat samples. Selected characteristic peaks are reported in wavenumbers (cm ¹). NMR spectra were recorded on Bruker Avance spectrometers (AVII400, AVIII 400 or AVII 500) in the deuterated solvent stated. The field was locked by external referencing to the relevant deuteron resonance. Chemical shifts (δ) are reported in parts per million (ppm) referenced to the solvent peak. ^lH spectra reported to two decimal places, and ¹³C spectra reported to one decimal place, and coupling constants (J) are quoted in Hz (reported to one decimal place). The coupling constants were determined by analysis using ACD Labs software. Low-resolution mass spectra were recorded on an Agilent 6120 spectrometer from solutions of MeOH, and high resolution mass spectra (HRMS) were recorded on a Bruker MicroTOF mass analyser using electrospray ionisation; m/z values are reported in Daltons and followed by their percentage abundance in parentheses. General Synthetic Procedures

General Procedure 1: Synthesis of glycidyl ethers (16 and 17).⁶⁷

[00145] A stirred solution of aryl alcohol (0.694 - 10.4 mmol, 1 eq.), CS2CO3 (5 eq.), and epichlorohydrin (5 eq.) in CH3CN (5 mL - 75 mL) was heated under reflux for 3 hours. The reaction mixture was cooled to room temperature, filtered and concentrated in vacuo. The concentrate was dissolved in toluene and washed with water, 1M NaOH, and water (10 mL 100 mL each). The organic phase was dried, filtered and concentrated in vacuo.

General Procedure 2: Synthesis of β-amino alcohol derivatives (1, 2, 18 - 27).

[00146] The glycidyl ether 16 or 17 (0.404 - 1.75 mmol, leq.), amine (1 eq.), and a catalytic amount of pyridine were stirred in EtOH (20 mL/mmol) and heated under reflux for 4 hours (unless otherwise stated). The reaction mixture was concentrated in vacuo and subsequently purified through flash column chromatography (eluent specified for each compound).

Preparation and Characterisation of Individual Compounds

Intermediate preparation

'- -dimethylphenoxy)methyl)oxirane (16)

8

[00147] Following General Procedure 1, using 3,4-dimethylphenol (107 mg, 0.873 mmol), epichlorohydrin (0.3 mL, 4.09 mmol) and Cs₂C0₃ (1.33 g, 4.09 mmol), in CH₃CN (10 mL), 16 was obtained as an orange oil (144 mg, 99%). v_max (neat) 2922 (C-H); δ_Η (400 MHz, CDC1 ) 2.22 (3H, s, C(7)H₃ or C(8)H₃), 2.26 (6H, s, C(7)H₃ or C(8)H₃), 2.77 (IH, dd, 4.9, 2.7, C(l ')H), 2.89 - 2.95 (IH, dd, 5.0, 4.2, C(l ')H'), 3.33 - 3.40 (IH, m, C(2')H), 3.96 (IH, dd, 11.0, 5.6 Hz, C(3')H), 4.20 (IH, dd, 11.1, 3.3 Hz, C(3')H"), 6.69 (IH, dd, 8.3, 2.7 Hz, C(6)H), 6.77 (IH, d, 2.7 Hz, C(2)H), 7.06 (IH, d, 8.3 Hz, C(5)H); 5c (100 MHz, CDC1 ) 18.7 (C(7) or C(8)), 19.9 (C(7) or C(8)), 44.6 (C(l l)). 50.1 (C(2')), 68.7 (C(3')), 111.4 (C(6)), 116.4 (C(2)), 129.1 (C(3) or C(4)), 130.2 (C(5)H), 137.7 (C(3) or C(4)), 156.5 (C(l)); m/z (ESI⁺) 201 ([M+Na]⁺, 100%); HRMS (ESI⁺) CnHi₄Na0₂ ⁺ ([M+Na]⁺) requires 201.0886, found 201.0895. '-((naphthalen-2-yloxy)methyl)oxirane (17)

[00148] Following General Procedure 1, using 2-naphthol (1.50 g, 10.4 mmol), epichlorohydrin (4.07 mL, 52.0 mmol) and Cs₂C0₃ (17.0 g, 52.0 mmol), in CH CN (75 mL), 17 was obtained as a pale brown solid (1.85 g, 89%). mp 53-55 °C; v_max (neat) 3059 (C-H); δ_Η (400 MHz, CDCb) 2.73 (1H, dd, J 4.9, 2.7, C(l ')H), 2.86 (1H, dd, J 4.9, 4.2, C(l ')H'), 3.28 - 3.40 (1H, m, C(2')H), 3.98 (1H, dd, J 11.0, 5.9, C(3')H), 4.26 (1H, dd, J 11.0, 3.2, C(3')H'), 7.06 (1H, d, J 2.5, C(l)H), 7.11 (1H, dd, J 8.9, 2.6, C(3)H), 7.23 - 7.32 (1H, ddd, J 8.1, 6.9, 1.2, C(6)H), 7.32 - 7.40 (1H, ddd, J 8.1, 6.9, 1.2, C(7)H), 7.59 - 7.73 (3H, m, C(4)H, C(5)H, C(8)H); 5c (100 MHz, CDCb) 44.7 (C(l ')), 50.1 (C(2')), 68.7 (C(3')), 106.8 (C(l)), 118.7 (C(3)), 123.8 (C(6)), 126.4 (C(7)), 126.8 (C(8)), 127.6 (C(5)), 129.1 (C(9)), 129.5 (C(4)), 134.4 (C(10)), 156.4 (C(2)); mJz (ESI⁺) 223 ([M+Na]⁺, 100%); HRMS (ESI⁺) Ci3Hi₂Na0₂ ⁺ ([M+Na]⁺) requires 223.0730, found 223.0738. -(bute-3'-en-1 '-yl)morpholine (42)

3' r 3

[00149] K₂C0₃ (384 mg, 2.78 mmol) was added to a stirred solution of morpholine (0.10 mL, 1.11 mmol) in CH₃CN 4 mL. 4-bromo-but-l-ene (0.11 mL, 1.11 mmol) was then added, and the reaction was heated to reflux for 4 hours. The solvent was then removed in vacuo, and the residue partitioned between CH₂C1₂ (20 mL) and water (20 mL). The aqueous layer was extracted with CH₂C1₂ (3 x 20 mL), and the combined organics were dried, filtered, and concentrated in vacuo to afford the title compound as a clear oil (149 mg, 98%). v_max (neat) 2854 (C-H); δ_Η (400 MHz, CDCb) 2.14 - 2.28 (2H, m, C(2')H₂), 2.33 - 2.52 (6H, m, C(1 ')H₂, C(3)H₂, C(5)H₂), 3.68 (4H, t, 4.7, C(2)H₂, C(6)H₂), 4.97 (1H, dd, 10.2, 1.4, C(4')H), 5.03 (1H, dd, 17.1, 1.4, C(4')H'), 5.77 (1H, ddt, 17.1, 10.2, 6.7, 6.7, C(3')H); 5c (100 MHz, CDCb) 31.0 (C(2')), 53.6 (C(3), C(5)), 58.3 (C(l ')), 66.9 (C(2), C(6)), 115.7 (C(4')), 136.3 (C(3')); m/z (ESI⁺) 142 ([M+H]⁺, 100%); HRMS (ESI⁺) C₈Hi₆NO⁺ ([M+H]⁺) requires 142.1226, found 142.1227. -(4-bromoethyl)oxirane

[00150] 44 was prepared following the literature procedure. mCPBA (479 mg, 2.70 mmol) was added potion- wise to a stirred solution of 4-bromo-but-l-ene (0.19 mL, 1.87 mmol) in CH₂C1₂ (2.5 mL) at 0°C. The reaction mixture was then allowed to stir overnight at room temperature. The reaction was then quenched with sat. aq. NaHC0₃, and washed with water (20 mL). The aqueous layer was then extracted with CH₂C1₂ (3 x 20 mL), and the combined organics were dried, filtered and concentrated in vacuo to afford the title compound as a colourless oil (283 mg, 98%). δ_Η (400 MHz, CDC1₃) 1.97 - 2.22 (2H, m, C(3)H₂), 2.58 (1H, dd, J 4.7, 2.8, C(l)H), 2.83 (1H, dd, J 4.7, 4.7, C(l)H'), 3.05 - 3.12 (1H, m, C(2)H), 3.46 - 3.56 (2H, m, C(4)H₂). 5c (100 MHz, CDCI3) 29.0 (C(4)), 35.7 (C(3)), 47.1 (C(l)), 50.8 (C(2)); m/z (FI⁺) 152 ([M(⁸¹Br)]⁺, 100%), 150 ([M(⁷⁹Br)]⁺, 97%).

-(oxiran-2-yl)ethyl)morpholine (43)

[00151] K2CO3 (328 mg, 2.38 mmol) was added to a stirred solution of 44 (276 mg, 1.83 mmol) in CH3CN (10 mL). Morpholine (0.16 mL, 1.83 mmol) was then added and the mixture was heated to reflux for 2 hours. The reaction mixture was then allowed to cool, and the solvent removed in vacuo. The residue was then partitioned between CH2CI2 (20 mL) and water (20 mL), and the aqueous layer extracted with CH2CI2 (2 x 20 mL). The combined organics were washed with water (2 x 20 mL) and then dried, filtered, and concentrated in vacuo, giving the title compound as a colourless oil (203 mg, 71%), which was used without further purification. Vmax (neat) 2809 (C-H); δ_Η (400 MHz, CDCI3) 1.60 - 1.85 (2H, m, C(3)H₂), 2.35 - 2.58 (7H, m, C(1)H, C(4)H₂, C(5')H₂, C(3')H₂), 2.78 (1H, dd, 4.4, 4.4, C(l)H'), 2.93 - 3.02 (1H, m, C(2)H), 3.64 - 3.77 (4H, m, C(6')H₂, C(2')H₂); 5c (100 MHz, CDCI3) 29.6 (C(3)), 47.0 (C(l)), 50.7 (C(2)), 53.5, 55.3 (C(4), C(3'), C(5')), 66.7 (C(6'), C(2')); m/z (ESI+) 158 ([M+H]⁺, 100%); HRMS (ESI⁺) C₈Hi₆N0₂ ⁺ ([M+H]⁺) requires 158.1176, found 158.1174.

-(but-3'-en-l'-yloxy) (45)

[00152] Intermediate 45 was prepared according to the literature procedure.⁴⁵ K2CO3 (384 mg, 2.70 mmol) was added to a stirred solution of 2-naphthol (160 mg, 1.11 mmol) in CH3CN (4 mL). 4-bromo-but-l-ene was then added and the reaction was heated to reflux for 7 hours. The reaction mixture was then allowed to cool, and the solvent was removed in vacuo. The residue was partitioned between CH2CI2 (10 mL) and water (10 mL), and the aqueous layer was extracted with CH2CI2 (2 x 10 mL). The combined organic layers were then washed with water (2 x 10 mL), and dried, filtered, and concentrated in vacuo. Purification via column chromatography (eluent pet ether) gave 45 as a colourless oil (60.4 mg, 27%). 5H (400 MHz, CDCI3) 2.54 (2H, app. q, 6.7, C(2')H₂), 4.06 (2H, t, 6.7, C(1 ')H₂), 5.06 (1H, dd, 10.3, 1.4, C(4')H), 5.13 (1H, dd, 17.2, 1.4, C(4')H'), 5.88 (1H, ddt, 17.2, 10.3, 6.7, 6.7, C(3')H), 7.04 - 7.10 (2H, m, C(1)H, C(3)H), 7.22 - 7.28 (1H, m, C(6)H), 7.33 - 7.38 (1H, m, C(7)H), 7.61 - 7.71 (3H, m, C(4)H, C(5)H, C(8)H); 5c (100 MHz, CDC1₃) 33.6 (C(2')), 67.2 (C(l ')), 106.6 (C(l)), 117.1 (C(4')), 119.0 (C(3)), 123.5 (C(6)), 126.3 (C(7)), 126.7 (C(8)), 127.6 (C(5)), 128.9 (C(9)), 129.3 (C(4)), 134.5 (C(10)), 156.8 (C(2)); m/z (FI⁺) 199 ([M]⁺, 100%). '-(4'-(naphthalen-2-yloxy)ethyl)oxirane (46)

[00153] K2CO3 (126 mg, 0.915 mmol) was added to a stirred solution of 44 (106 mg,

0.704 mmol) in CH3CN (5 mL). 2-naphthol (102 mg, 0.704 mmol) was then added and the reaction was heated to reflux for 18 hours. The mixture was then allowed to cool, and the solvent was removed in vacuo. The residue was then partitioned between CH2CI2 (20 mL) and water (20 mL), and the aqueous layer extracted with CH2CI2 (2 x 20 mL). The combined organics were washed with water (2 x 20 mL) and then dried, filtered, and concentrated in vacuo. Purification via column chromatography (eluent MeOH:CH2Cl2 1:200) afforded the title compound as a yellow oil (68.8 mg, 46%). v_max (neat) 2912 (C-H); δ_Η (400 MHz, CDCI3) 1.95 - 2.08 (1H, m, C(3')H), 2.13 - 2.25 (1H, m, C(3')H₂'), 2.63 (1H, dd, / 4.7, 2.7, C(l ')H), 2.87 (1H, dd, / 4.7, 4.7, C(l ')H'), 3.17 - 3.26 (1H, m, C(2')H), 4.20 - 4.31 (2H, m, C(4')H₂), 7.17 (2H, m, C(3)H, C(l)H), 7.31 - 7.40 (1H, m, C(6)H), 7.41 - 7.49 (1H, m, C(7)H), 7.70 - 7.82 (3H, m, C(4)H, C(5)H, C(8)H); 5c (100 MHz, CDCI3) 32.4 (C(3')), 47.2 (C(l ')), 49.8 (C(2')), 64.6 (C(4')), 107.6 (C(l)), 118.8 (C(3)), 123.6 (C(6)), 126.4 (C(7)), 126.7 (C(8)), 127.6 (C(5)), 129.0 (C(9)), 129.4 (C(4)), 134.5 (C(10)), 156.6 (C(2)); m/z (ESI⁺) 237 ([M+Na]⁺, 100%); HRMS (ESI⁺) Ci₄Hi₄Na0₂ ⁺ ([M+Na]⁺) requires 237.0886, found 237.0893.

Preparation of example compounds

'-(3,4-dimethylphenoxy)-3'-morpholinopropan-2'-ol (1 / C5)

[00154] Following General Procedure 2, using glycidyl ether 16 (125 mg, 0.700 mmol), morpholine (0.06 mL, 0.700 mmol), cat. pyridine in EtOH (10 mL). Purification via column chromatography (eluent MeOH:CH₂Cl2 3:100 to 5:100) afforded the title compound as a yellow oil (162 mg, 89%). v_max (neat) 3419 (O-H), 2921 (C-H); δ_Η (400 MHz, CDCI₃) 2.20 (3H, s, C(7)H₃ or C(8)H₃), 2.24 (3H, s, C(7)H₃ or C(8)H₃), 2.43 - 2.72 (6H, m, C(3')H₂, C(3")H₂, C(5")H₂), 3.69 - 3.80 (4H, m, C(2")H₂, C(6")H₂), 3.96 (2H, d, J 4.9, C(1 ')H₂), 4.06 - 4.15 (1 H, m, C(2')H), 6.67 (1 H, dd, J 8.3, 2.6, C(6)H), 6.75 (1 H, d, J 2.6, C(2)H), 7.03 (1 H, d, J 8.3, C(5)H); 5_C (100 MHz, CDCI₃) 18.8 (C(7) or C(8)), 20.0 (C(7) or C(8)), 53.7 (C(3"), C(5")), 61 .1 (C(3')), 65.5 (C(2')), 66.9 (C(2"), C(6")), 70.2 (C(1 ')), 1 1 1 .4 (C(6)), 1 16.1 (C(2)), 129.0 (C(3) or C(4)), 130.2 (C(5)), 137.7 (C(3) or C(4)), 156.8 (C(1 )); m/z (ESI⁺) 266 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₅H₂₄N0₃ ⁺ ([M+H]⁺) requires 266.1751 , found 266.1746.

1 '-(naphthalen-2-yloxy)-3'-thiomorpholinopropan-2'-ol (22)

[00155] Following General Procedure 2, using glycidyl ether 17 (103 mg 0.514 mmol), thiomorpholine (0.05 mL, 0.514 mmol), cat. pyridine in EtOH (10 mL). Purification via column chromatography (eluent MeOH:CH₂Cl₂ 0:100 to 1: 100) afforded the title compound as a brown solid (115 mg, 76%). mp 107 - 110 °C; v_max (neat) 3383 (O-H), 2945 (C-H); δ_Η (400 MHz, CDCI3) 2.55 - 2.83 (8H, m, C(3")H₂, C(2")H₂, C(6")H₂, C(5")H₂), 2.95 - 3.00 (2H, m, C(3')H₂), 4.07 - 4.13 (2H, m, C(1 ')H₂), 4.13 - 4.21 (1H, m, C(2')H), 7.16 (1H, d, 2.5, C(l)H), 7.19 (1H, dd, 8.9, 2.5, C(3)H), 7.31 - 7.40 (1H, ddd, 8.1, 6.9, 1.2, C(6)H), 7.43 - 7.47 (1H, ddd, 8.1, 6.9, 1.2, C(7)H), 7.65 - 7.82 (3H, m C(4)H, C(5)H, C(8)H); 5c (100 MHz, CDCI3) 28.0 (C(2"), C(6")), 55.4 (C(3')), 61.3 (C(3"), C(5")), 65.4 (C(2')H), 70.1 (C(l ')), 106.8 (C(l)), 118.8 (C(3)), 123.8 (C(6)), 126.5 (C(7)), 126.8 (C(8)), 127.7 (C(5)), 129.1 (C(9)), 129.5 (C(4)), 134.5 (C(10)), 156.6 (C(2)); m/z (ESI⁺) 304 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₇H22N0₂S⁺ ([M+H]⁺) requires 304.1366, found 304.1365.

' -difluoropiperidin- 1 " -yl)-3 '-(naphthalen-2-yloxy)propan-2 '-ol (23)

[00156] Following General Procedure 2, using glycidyl ether 17 (101 mg, 0.503 mmol),

4,4-difluoropiperidine HCl (79.0 mg, 0.503 mmol), cat. pyridine in EtOH (10 mL). Purification via column chromatography (eluent MeOH:CH₂Cl₂ 0: 100 to 2: 100) afforded the title compound as a brown solid (47.0 mg, 29%). mp 88 - 96 °C; v_max (neat) 3235 (O-H); δ_Η (400 MHz, CDCI3) 2.06 - 2.27 (4H, m, C(5")H₂, C(3")H₂), 2.69 - 2.85 (4H, m, C(6")H₂, C(2")H₂), 2.86 - 3.01 (2H, m, C(1 ')H₂), 3.77 (1H, br. s, OH), 4.07 - 4.19 (2H, m, C(3')H₂), 4.23 - 4.35 (1H, m, C(2')H), 7.11 - 7.20 (2H, m, C(1)H, C(3)H), 7.32 - 7.40 (1H, ddd, 8.1, 7.0, 1.2, C(6)H), 7.41 - 7.52 (1H, m, C(7)H), 7.67 - 7.83 (3H, m C(4)H, C(5)H, C(8)H); 5_F (376 MHz, CDC1₃) 100.4 (2F, s, C(4")F₂); m/z (ESI⁺) 322 ([M+H]⁺, 100%); 5c (100 MHz, CDCI3) 33.5 (t, ²J_FC 24.2, C(5"), C(3")), 50.6 (C(6"), C(2")), 60.1 (C(l')X 65.7 (C(2')), 69.8 (C(3')), 106.8 (C(l)), , 118.6 (C(3), C(15)), 123.8 (C(6)), 126.5 (C(7)), 126.8 (C(8)), 127.6 (C(5)), 129.1 (C(9)), 129.5 (C(4)), 134.4 (C(10)), 156.4 (C(2)); m/z (ESI⁺) 322 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₈H₂₂F₂N0₂ ⁺ ([M+H]⁺) requires 322.1613, found 322.1619 7"-(4"-(2'-hydroxy-3'-(naphthalen-2-yloxy)propyl)piperazin-l "-yl)ethanone (25)

[00157] Following General Procedure 2, using glycidyl ether 17 (105 mg, 0.522 mmol),

1-acetylpiperazine (64.0 mg, 0.522 mmol), cat. pyridine in EtOH (10 mL). Purification via column chromatography (eluent MeOH:CH₂Cl₂ 0: 100 to 3: 100) afford the title compound as a brown solid (70.9 mg, 43%). mp 110 - 113 °C; v_max (neat) 3386 (O-H), 2925 (C-H), 1627 (C=0); δ_Η (400 MHz, CDCI3) 2.11 (3H, s, C(8")H₃), 2.49 - 2.82 (6H, m, C(1 ')H₂, C(3")H₂, C(5")H₂), 3.50 - 3.84 (4H, m, C(2")H₂, C(6")H₂), 4.11 - 4.16 (2H, m, C(3')H₂), 4.23 - 4.30 (1H, m, C(2')H), 7.13 - 7.20 (2H, m, C(l)H), C(3)H), 7.32 - 7.40 (1H, m, C(6)H), 7.42 - 7.49 (1H, m, C(7)H), 7.70 - 7.82 (3H, m, C(4)H, C(5)H, C(8)H); 5c (100 MHz, CDC1₃) 21.3 (C(8")), 41.2 (C(2") or C(6")), 46.1 (C(2") or C(6")), 53.0 (C(3") or C(5")), 53.4 (C(3") or C(5")), 60.6 (C(l ')), 65.6 (C(2')), 70.0 (C(3')), 106.7 (C(l)), 118.7 (C(3)), 123.8 (C(6)), 126.4 (C(7)), 126.7 (C(8)), 127.6 (C(5)), 129.1 (C(9)), 129.5 (C(4)), 134.4 (C(10)), 156.5 (C(2)), 168.9 (C(7")); m/z (ESI⁺) 329 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₉H₂₆N₂0 ⁺ ([M+H]⁺) requires 329.1860, found 329.1858. l'-((2"-methoxyethyl)(methyl)amino)-3'-(naphthalen-2-yloxy)propan-2'-ol (27)

[00158] Following General Procedure 2, using glycidyl ether 17 (101 mg, 0.503 mmol),

(2-methoxyethyl)methylamine (0.05 mL, 0.503 mmol), cat. pyridine, in EtOH (10 mL). Purification via column chromatography (eluent MeOH:CH₂Cl₂ 1: 100 to 3: 100) gave the title compound as a yellow oil (76.3 mg, 57%). v_max (neat) 3416 (O-H); δ_Η (400 MHz, CDCh) 2.43 (3H, s, C(3")H₃), 2.62 - 2.85 (4H, m, C(1 ')H₂, C(1 ")H₂), 3.38 (3H, s, C(4")H₃), 3.46 - 3.58 (2H, m, C(2")H₂), 4.05 - 4.19 (3H, m, C(3')H₂, C(2')H), 7.16 (1H, d, 2.2, C(l)H), 7.20 (1H, dd, 8.8, 2.2, C(3)H), 7.29 - 7.37 (1H, ddd, 8.2, 6.9, 1.2, C(6)H), 7.40 - 7.47 (1H, ddd, 8.2, 6.9, 1.2, C(7)H), 7.70 - 7.79 (3H, m C(4)H, C(5)H, C(8)H); 5c (100 MHz, CDC1 ) 43.2 (C(3")), 57.1 (C(l")), 58.8 (C(4")), 60.2 (C(2')), 66.4 (C(l ')), 70.2 (C(2")), 70.6 (C(3')), 106.7 (C(l)), 118.9 (C(3)), 123.6 (C(6)), 126.3 (C(7)), 126.8 (C(8)), 127.6 (C(5)), 129.0 (C(9)), 129.3 (C(4)), 134.5 (C(10)), 156.7 (C(2)); m/z (ESI⁺) 290 ([M+H]⁺, 100%); HRMS (ESI⁺) O₇H₂₄N0 ⁺ ([M+H]⁺) requires 290.1754, found 290.1751. "-(2'-hydroxy-3'-(naphthalen-2-yloxy)propyl)thiomorpholine 1 "-oxide (28)

[00159] A stirred solution of 22 (42.4 mg, 0.140 mmol) in MeOH:H₂0 1: 1 (3 mL) was cooled to 0°C. Solid NaI0₄ (28.2 mg, 0.140 mmol) was then added portion-wise, and the suspension was left to stir for 3 hours. The reaction mixture was then allowed to warm up to room temperature and stirred overnight. The solids were then filtered, washing with MeOH, and the filtrate concentrated in vacuo to remove the MeOH. The residual water was then saturated with solid NaCl, and extracted with EtOAc (3 x 10 mL). The organic phase was dried, filtered and concentrated in vacuo. The resultant solid was then dissolved in CH₂C1₂ (10 mL), and filtered through a glass microfiber filter to remove remaining impurities. The filtrate was concentrated in vacuo to afford 28 as a yellow oil (24.8 mg, 59%). v_max (neat) 3367 (O-H), 2922 (C-H); δ_Η (400 MHz, CD₃OD) 2.58 - 2.73 (2H, m, C(1 ')H₂), 2.78 - 2.89 (4H, m, C(3")H₂, C(5")H₂ or C(2")H₂, C(6")H₂), 2.93 - 3.17 (4H, m, C(3")H₂, C(5")H₂ or C(2")H₂, C(6")H₂), 4.03 - 4.19 (3H, m, C(3')¾, C(2')H), 7.16 (1H, dd, 8.9, 2.5, C(3)H), 7.23 (1H, d, 2.5, C(l)H), 7.26 - 7.33 (1H, ddd, 8.2, 7.0, 1.2, C(6)H), 7.37 - 7.43 (1H, ddd, 8.2, 7.0, 1.2, C(7)H), 7.70 - 7.77 (3H, m, C(4)H, C(5)H, C(8)H); 5c (100 MHz, CD₃OD) 47.0, 49.4 (C(2"), C(6"), C(3"), C(5")), 61.7 (C(l ')), 68.8 (C(2')), 71.8 (C(3')), 107.9 (C(l)), 120.0 (C(3)), 124.8 (C(6)), 127.5 (C(7)), 128.0 (C(8)), 128.7 (C(5)), 130.5 (C(4)), 130.7 (C(9)), 136.3 (C(10)), 158.4 (C(2)); m/z (ESI⁺) 320 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₇H₂₂N0 S⁺ ([M+H]⁺) requires 320.1315, found 320.1315.

4"-(2'-hydroxy-3'-(naphthalen-2-yloxy)propyl)thiomorpholine 1",1 "-dioxide (29)

[00160] mCPBA (59.1 mg, 0.343 mmol) was added portion- wise to a stirred solution of

22 (39.5 mg, 0.130 mmol) in CH2CI2, (2 mL). The reaction mixture was then allowed to stir for 3 hours at room temperature, and then quenched with sat. aq. NaHC0₃. The mixture was then washed with water (10 mL), and the aqueous layer extracted with CH2CI2 (3 x 10 mL). The organic phase was dried and filtered through a glass microfiber filter, then concentrated in vacuo to afford the title compound as pale yellow solid (29.7 mg, 68%). mp 194 - 196 °C; v_max (neat) 3250 (O-H), 2935 (C-H); δ_Η (500 MHz, CD₃OD) 2.90 - 3.04 (2H, m, C(1 ')H₂), 3.41 - 3.73 (6H, m, C(1 ')H₂, C(3")H, C(5")H C(2")H, C(6")H), 3.90 - 4.27 (6H, m, C(3')H, C(3")H, C(5")H C(2")H, C(6")H), 4.80 (1H, m, C(2'), 7.20 (1H, dd, 8.9, 2.5, C(3)H), 7.28 (1H, d, 2.5, C(l)H), 7.24 (1H, ddd, 8.1, 7.0, 1.1, C(6)H), 7.39 - 7.46 (1H, ddd, 8.1, 7.0, 1.1, C(7)H), 7.65 - 7.72 (3H, m, C(4)H, C(5)H, C(8)H); 5c (125 MHz, CD₃OD) 55.5, 56.5 (C(3"), C(5"), C(2"), C(6")), 65.9 (C(2')), 71.7 (C(l ')), 75.0 (C(3')), 108.1 (C(l)), 119.8 (C(3)), 125.0 (C(6)), 127.6 (C(7)), 128.0 (C(8)), 128.8 (C(5)), 130.6 (C(4)), 130.9 (C(9)), 136.2 (C(10)), 158.0 (C(2)); m/z (ESI⁺) 336 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₇H₂iNNa0₄S⁺ ([M+Na]⁺) requires 358.1083, found 358.1087.

4 '-morpholino- 1 '-(naphthalen-2-yloxy)butan-2 '-ol (37)

[00161] K₂C0₃ (357 mg, 2.60 mmol) was added to a solution of 43 (203 mg, 1.29 mmol) in EtOH (10 mL). 2-naphthol was then added (186 mg, 1.29 mmol) and the reaction was heated to reflux overnight. The mixture was then allowed to cool, and the solvent was removed in vacuo. The residue was then partitioned between CH2CI2 (20 mL) and water (20 mL), and the aqueous layer extracted with CH2CI2 (2 x 20 mL). The combined organics were washed with water (2 x 20 mL), then dried, filtered, and concentrated in vacuo. Purification via column chromatography (eluent MeOH:CH₂Cl₂ 0:100 to 1: 100) afford 37 as a yellow oil (87.4 mg, 26%). Vmax (neat) 3399 (O-H), 2922 (C-H); δ_Η (400 MHz, (CD₃)₂CO) 1.69 - 1.90 (2H, m C(3')H₂), 2.33 - 2.68 (6H, m, C(4')H₂, C(5")H₂, C(3")H₂), 3.61 (4H, t, 4.6, C(6")H₂, C(2")H₂), 4.01 - 4.11 (2H, m, C(1 ')H₂), 4.11 - 4.19 (1H, m, C(2')H), 7.17 (1H, dd, / 9.0, 2.4, C(3)H), 7.27 - 7.36 (2H, m, C(1)H, C(6)H), 7.41 - 7.47 (1H, m, C(7)H), 7.75 - 7.84 (3H, m, C(4)H, C(5)H, C(8)H); 5c (100 MHz, (CD₃)₂CO) 30.5 (C(3')H), 54.7, 57.1 (C(4'), C(5"), C(3")), 67.5 (C(6"), C(2")), 70.4 (C(2')), 73.4 (C(l ')), 107.6 (C(l)), 119.8 (C(3)), 124.4 (C(6)), 127.2 (C(7)), 127.8 (C(8)), 128.5 (C(5)), 130.0 (C(9)), 130.2 (C(4)), 135.8 (C(10)), 158.0 (C(2)); m/z (ESI⁺) 302 ([M+H]⁺, 100%); HRMS (ESI⁺) O₈H₂₄N0 ⁺ ([M+H]⁺) requires 302.1751, found 302.1749.

1 '-morpholino-4 '-(naphthalen-2-yloxy)butan-2 '-ol (36)

[00162] Morpholine (0.03 mL, 0.320 mmol) was added to a stirred solution of 46 (68.8 mg, 0.320 mmol) in EtOH (8 mL), followed by cat. pyridine. The reaction was heated to reflux for 4 hours, and then allowed to cool. The mixture was concentrated in vacuo, and the residue purified via column chromatography (eluent MeOH:CH₂Cl₂ 0: 100 to 1: 100) to afford 36 as a yellow oil (60.3 mg, 63%). v_max (neat) 3442 (O-H), 2952 (C-H); δ_Η (400 MHz, CDC1 ) 1.84 - 2.08 (2H, m C(3')H₂), 2.33 - 2.52 (4H, m, C(5")H₂, C(3")H₂), 2.64 - 2.76 (2H, m, C(1 ')H₂), 3.65 - 3.81 (4H, m, C(6")H₂, C(2")H₂), 3.95 - 4.08 (1H, m, C(2')H), 4.22 - 4.33 (2H, m, C(4')H₂), 7.11 - 7.21 (2H, m, C(1)H, C(3)H), 7.30 - 7.37 (1H, m, C(6)H), 7.40 - 7.49 (1H, m, C(7)H), 7.68 - 7.80 (3H, m, C(4)H, C(5)H, C(8)H); 5c (100 MHz, CDC1 ) 34.3 (C(3')), 53.6 (C(l '), C(5"), C(3")), 63.4 (C(2')), 64.6 (C(4')), 66.9 (C(6"), C(2")), 106.7 (C(l)), 118.8 (C(3)), 123.5 (C(6)), 126.3 (C(7)), 126.7 (C(8)), 127.6 (C(5)), 129.0 (C(9)), 129.3 (C(4)), 134.5 (C(10)), 156.7 (C(2)); m/z (ESI⁺) 302 ([M+H]⁺, 100%); HRMS (ESI⁺) Ci₈H₂₄N0 ⁺ ([M+H]⁺) requires 302.1751, found 302.1749.

Biological Assessment

[00163] Using the process outlined in Example 4 above, dose response curves were prepared for the compounds of Example 5. Results:

Compound Activity

22 See Figure 17

23 See Figure 18

25 See Figure 19

27 See Figure 20

28 See Figure 21

36 See Figure 22

37 See Figure 23 References

1 Campuzano, V. et al, (1996), Science, 271, 1423-1427.

2 Schulz, J.B. et al, (2009) Nat Rev Neurol, 5, 222-234.

3 Tsou. A.Y. et al., (201 1) J Neurol Sci, 307, 46-49.

4 Koeppen, A.H. et al., (2007) Acta neuropathology, 114, 163-173.

5 Koeppen, A.H. et al, (2009) Acta neuropathologica, 118, 763-776.

6 Delatycki, M.B. et al, (2000) J Med Genet, 37, 1-8.

7 Cossee, M. et al., (1999) Ann A¾wroZ, 45, 200-206.

8 Filla, A. et al., (1996) Am J Hum Genet, 59, 554-560.

9 Montermini, L. ef a/., (1997) Annals of neurology, 41, 675-682.

10 Schmucker, S. and Puccio. H. (2010) Hum Mol Genet, 19, R103-110.

11 Puccio, H. et al, (2001) Nat Genet, 27, 181-186.

12 Martelli. A. et al., (2007) Hum Mol Genet, 16, 2651 -2658.

13 Babcock, M. et al., ( 1997) Science, 276, 1709-1712.

14 Rotig, A. et al, (1997) Nat Genet, 17, 215-217.

15 Wong, A. et al, (1999) Hum Mol Genet, 8, 425-430.

16 Wells, R.D. (2008) Faseb J, 22, 1625-1634.

17 Bidichandani, S.I. et al, (1998) Am J Hum Genet, 62, 111-121.

18 Sakamoto, N. et al, (1999) Mol Cell, 3, 465-475.

19 Sakamoto, N. et al, (2001) J Biol Chem, 276, 27171-27177.

20 Grabczyk. E. et al, (2007) Nucleic Acids Res, 35. 5351 -5359.

21 Vetcher, A.A. et al, (2002) 7 B/oZ Chem, 277, 39217-39227.

22 Ohshima, K. et al, ( [998) J Biol Chem, 273, 14588-14595.

23 Grabczyk, E. and Usdin, K. (2000) Nucleic Acids Res, 28, 2815-2822.

24 Herman, D. et al, (2006) Nat Chem Biol, 2, 551-558.

25 Saveliev, A. et al, (2003) Nature, 422, 909-913.

26 Al-Mahdawi, S. et al, (2008) Hum Mol Genet, 17, 735-746.

27 Greene. E. et al, (2007) Nucleic Acids Res, 35, 3383-3390.

28 Evans-Galea, M.V. et al, (2012) Annals of neurology, 71, 487-497.

29 Castaldo, I. et al, (2008) Journal of medical genetics, 45, 808-812.

30 Soragni, E. et al, (2008) Nucleic Acids Res, 36, 6056-6065.

31 Kumari, D. et al, (2011) / Biol Chem, 286, 4209-4215.

32 De Biase, I. et al, (2009) PLoS One, 4, e7914.

33 Rai. M. et al, (2008) PLoS One, 3, el958.

34 Sturm, B. et al, (2005) Eur J Clin Invest, 35, 71 1 -717.

35 Wilson. R.B. (2012) J Child Neurol, 21, 1212-1216.

36 Velasco-Sanchez, D. et al, (201 1) Cerebellum, 10, 1 -8.

37 Grant, L. et al, (2006) FEBS Lett, 580, 5399-5405.

38 Ditch, S. et al, (2009) PLoS Genet, 5, el000704.

39 Lufino, M.M. et al, (2007) Nucleic Acids Res, 35, e98.

40 Wade-Martins, R. et al, (2000) Nat Biotechnol, 18, 1311-1314.

41 Gomez-Sebastian, S. et al. (2007) Mol Ther, 15. 248-254;

42 Puccio, H. and Koenig, M. (2000) Hum Mol Genet. 9, 887-892.

43 Robinson. C.R. and Sauer. R.T. (1998) Proc Natl Acad Sci U S A, 95, 5929-5934. 44 Al-Mahdawi, S. et al, (2004) Genomics, 84, 301-310.

45 Holloway, T.P. et al, (201 1) Biotechniques, 50, 182- 186.

46 Wade-Martins, R. et al, (2001) Nat Biotechnol, 19, 1067-1070

47 Saeki, Y. et al, (2001) Mol Ther, 3, 591-601.

48 Westwood, I.M. et al, (2010) Protein & cell, 1, 82-95.

49 Russell, A.J. et al, (2009) Bioorganic & medicinal chemistry, 17, 905-918.

50 Rai. M. et al, (2010) PLoS One, 5, e8825.

51 Gimenez-Cassina, A. et al. (201 1) Gene Ther, 18. 1015-1019.

52 Zhang, Y. et al. , (2000) Nat Biotechnol, 18, 1314- 1317.

53 Sarsero, J.P. et al, (2003) / Gene Med, 5, 72-81.

54 Li, L. et al, (2013) PLoS One, 8, e55940.

55 Marmolino, D. et al, (2009) Cerebellum, 8, 98- 103.

56 Li, L. et al, (2013) PLoS One, 8, e55940.

57 Acquaviva, F. et al, (2008) Cerebellum, 7, 360-365.

58 Chan. P.K. et al, (2013) Hum Mol Genet, 22, 2662-2675.

59 Soragni. E et al, (2012) J Child Neurol, 27, 1164-1 173. Rai, M. et al, (2008) PLoS One, 3, el958.

Alegre-Abarrategui, J. et al, (2009) Human Molecular Genetics, 18, 4022-4034. Wade-Martins, R. et al, (2001) Nat Biotechnol, 19, 1067-1070.

Montermini, L. et al, (1997) Hum Mol Genet, 6, 1261-1266.

Carey, M.F. et al, (2009) Cold Spring Harb Protoc, 2009, pdb.prot5279.

Jefferson, A. and Volpi, E.V. (2010) Methods in molecular biology, 659, 409-426. Pangborn et al. (1996) Organomet 15, 1518-1520;

Fullam et al. (2011) Bioorganic & Medicinal Chemistry Letters, 21, 1185-1190; Tacon et al. (2012) Bioorganic & Medicinal Chemistry, 20, 893-902.

Claims

1. A pharmaceutical composition for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases (e.g. Friedreich's ataxia) comprising a compound of structural formula la:

la

wherein

Q is selected from a mono or bicyclic carbocyclic ring, a mono or bicyclic heteroaryl ring or a mono or bicyclic heterocyclic ring, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, ureido, (l-4C)alkyl, (l-4C)alkoxy, (2-4C)alkenyl, (2-4C)alkynyl, , (1- 4C)alkylthio, (l-4C)alkylsulphinyl, (l-4C)alkylsulphonyl, (l-4C)alkylamino, di-[(l- 4C)alkyl] amino, (l-4C)alkoxycarbonyl, N-(l-4C)alkylcarbamoyl, N,N-di-[(l-

4C)alkyl]carbamoyl, (2-4C)alkanoyl, (2-4C)alkanoyloxy, (2-4C)alkanoylamino, N-(l- 4C)alkylsulphamoyl and N,N-di-[(l-4C)alkyl]sulphamoyl;

p is 1 or 2;

q is 1 or 2;

Ri is hydroxyl or methoxy;

R3 is (l-3C)alkyl and R₄ is a group -L1-X2-R5 wherein Li is (l-3C)alkylene, X2 is O or S and R5 is (l-3C)alkyl and wherein any alkylene or alkyl groups present in R3 and and R₄ are optionally substituted with one or more halo groups (e.g fluoro);

or R3 and R₄ are linked such that, together with the nitrogen atom to which they are attached, they form a ring of the formula:

n is 0, 1, 2, 3 or 4;

or when n is 2 or more, two of the R₂ groups may be linked to form a linear or branched

(l-4C)alkylene bridge;

or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients.

2. A pharmaceutical composition for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases (e.g. Friedreich's ataxia) according to claim 1, wherein said composition comprises a compound of structural formula lb:

lb

wherein

Q is selected from a mono or bicyclic carbocyclic ring, a mono or bicyclic heteroaryl ring or a mono or bicyclic heterocyclic ring, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, ureido, (l-4C)alkyl, (l-4C)alkoxy; (2-4C)alkenyl, (2-4C)alkynyl, , (1- 4C)alkylthio, (l-4C)alkylsulphinyl, (l-4C)alkylsulphonyl, (l-4C)alkylamino, di-[(l- 4C)alkyl] amino, (l-4C)alkoxycarbonyl, N-(l-4C)alkylcarbamoyl, N,N-di-[(l- 4C)alkyl]carbamoyl, (2-4C)alkanoyl, (2-4C)alkanoyloxy, (2-4C)alkanoylamino, N-(l- 4C)alkylsulphamoyl and N,N-di-[(l-4C)alkyl]sulphamoyl; X is -0-, a bond, -NR^a-, -CHR^a-, -S-, -SO-, -SO2-, -NR^a-C(0)-, -C(0)-NR^a-, or

Ri is hydroxyl or methoxy;

Xi is O, S, S(O), or S(0)₂;

n is 0, 1, 2, 3 or 4;

or a pharmaceutically acceptable salt or solvate thereof, and one or more

pharmaceutically acceptable excipients.

3. A compound of structural formula la:

la

wherein

p is 1 or 2;

q is 1 or 2;

Ri is hydroxyl or methoxy;

n is 0, 1, 2, 3 or 4;

or a pharmaceutically acceptable salt or solvate thereof,

for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases (e.g. Friedreich's ataxia).

4. A compound according to claim 3, wherein said compound is of structural formula lb:

lb

wherein

Ri is hydroxyl or methoxy;

Xi is O, S, S(O), S(0)₂, -NR^c-C(0)-, -C(0)-NR^c-, or -NR^c-C(0)-NR^d-, wherein R^c and R^d are each independently selected from H or (l-2C)alkyl;

n is 0, 1, 2, 3 or 4;

or a pharmaceutically acceptable salt or solvate thereof,

5. A pharmaceutical composition according to claim 1 or claim 2, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases according to claim 3 or claim 4, wherein Q is selected from phenyl, naphthyl, or a mono or bicyclic heteroaryl ring, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, ureido, (l-4C)alkyl, (1- 4C)alkoxy, (2-4C)alkenyl, (2-4C)alkynyl, , (l-4C)alkylthio, (l-4C)alkylsulphinyl, (1- 4C)alkylsulphonyl, (l-4C)alkylamino, di-[(l-4C)alkyl] amino, (l-4C)alkoxycarbonyl, N-(l- 4C)alkylcarbamoyl, N,N-di-[(l-4C)alkyl]carbamoyl, (2-4C)alkanoyl, (2-4C)alkanoyloxy, (2- 4C)alkanoylamino, N-(l-4C)alkylsulphamoyl and N,N-di-[(l-4C)alkyl] sulphamoyl.

6. A pharmaceutical composition according to any one of claims 1, 2 or 5, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases according to claim 4 or claim 5, wherein Q is selected from phenyl or naphthyl, each of which is optionally substituted by a one or more substituents selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, hydroxyl, amino, carboxy, carbamoyl, sulphamoyl, (l-2C)alkyl, and (l-2C)alkoxy.

7. A pharmaceutical composition according to any one of claims 1, 2, 5 or 6, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases according to any one of claims 3 to 6, wherein X is -0-, -NR^a-, -S-, -SO-, or -SO2-.

8. A pharmaceutical composition according to any one of claims 1, 2, or 5 to 7, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases according to any one of claims 3 to 7, wherein X is -0-.

9. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 8, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases according to any one of claims 3 to 7, wherein Ri is hydroxy.

10. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 9, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases according to any one of claims 3 to 9, wherein Xi is O, S, S(O), S(0)₂, - NR^f or CR^hR\ wherein R^f is selected from H, methyl or (2C)alkanoyl, and R^h and R¹ are each independently selected from hydrogen, fluoro, chloro, bromo methyl, methoxy or (2C)alkanoyl.

11. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 10, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases according to any one of claims 3 to 10, wherein Xi is O.

12. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 11, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases and/or triplet repeat diseases according to any one of claims 3 to 11, wherein n is 0, 1, or 2.

13. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 12, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases as defined herein and/or triplet repeat diseases according to any one of claims 3 to 12, wherein R₂ is selected from the group consisting of halo, trifluoromethyl, trifluoromethoxy, cyano, nitro, hydroxyl, mercapto, amino, formyl, carboxy, carbamoyl, sulphamoyl, (l-4C)alkyl, (1- 4C)alkoxy, (l-4C)alkylthio, (l-4C)alkylsulphinyl, (l-4C)alkylsulphonyl, (l-4C)alkylamino, di- [(l-4C)alkyl] amino, (l-4C)alkoxycarbonyl, N-(l-4C)alkylcarbamoyl, N,N-di-[(l- 4C)alkyl]carbamoyl, (2-4C)alkanoyl, (2-4C)alkanoyloxy, (2-4C)alkanoylamino, N-(l- 4C)alkylsulphamoyl and N,N-di-[(l-4C)alkyl]sulphamoyl; or when n is 2 or more, two of the R₂ groups may be linked to form a linear or branched (l-4C)alkylene bridge.

14. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 13, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases as defined herein and/or triplet repeat diseases according to any one of claims 3 to 13, wherein the compound has the structural formula Ic shown below:

Ic

wherein Q, Xi, R₂ and n are as defined in any one of claims 1 to 13;

or a pharmaceutically acceptable salt or solvate thereof.

15. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 14, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases as defined herein and/or triplet repeat diseases according to any one of claims 3 to 14, wherein the

wherein Q, R₂ and n are as defined in any one of claims 1 to 13;

or a pharmaceutically acceptable salt or solvate thereof.

16. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 15, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases as defined herein and/or triplet repeat diseases according to any one of claims 3 to 15 wherein the compound is selected from any one of the following:

harmaceu tic ally acceptable salt or solvate thereof.

17. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 15, or a compound for use in the treatment of neurodegenerative and neuromuscular diseases as defined herein and/or triplet repeat diseases according to any one of claims 3 to 15 wherein the compound is selected from any one of the following:

l-(3,4-dimethylphenoxy)-3-(4-morpholinyl)-2-propanol;

l-(3,4-dimethylphenoxy)-3-morpholinopropan-2-ol;

l-morpholino-3-(naphthalen-2-yloxy)propan-2-ol;

4-(2-methoxy-3-(naphthalen-2-yloxy)propyl)morpholine;

(S)-l-(3,4-dimethylphenoxy)-3-morpholinopropan-2-ol;

( ?)-l-(3,4-dimethylphenoxy)-3-morpholinopropan-2-ol;

l-morpholino-3-(o-tolyloxy)propan-2-ol;

l-morpholino-3-(m-tolyloxy)propan-2-ol;

l-morpholino-3-( ?-tolyloxy)propan-2-ol;

l-(4-methoxyphenoxy)-3-morpholinopropan-2-ol;

1 - (4 -methylpiperazin- 1 -y 1) - 3 - (naphthalen-2-yloxy )propan-2- ol ;

l-morpholino-3-(naphthalen-2-yloxy)propan-2-ol;

1 '-(3,4-dimethylphenoxy)-3 '-morpholinopropan-2'-ol;

1 '-(naphthalen-2-yloxy)-3 '-thiomorpholinopropan-2'-ol;

l '-(4",4"-difluoropiperidin-l "-yl)-3'-(naphthalen-2-yloxy)propan-2'-ol;

7"-(4"-(2'-hydroxy-3'-(naphthalen-2-yloxy)propyl)piperazin-l "-yl)ethanone;

l '-((2"-methoxyethyl)(methyl)amino)-3'-(naphthalen-2-yloxy)propan-2'-ol;

4"-(2'-hydroxy-3 '-(naphthalen-2-yloxy)propyl)thiomorpholine 1 "-oxide;

4"-(2'-hydroxy-3 '-(naphthalen-2-yloxy)propyl)thiomorpholine 1 ", 1 "-dioxide;

4'-morpholino-l '-(naphthalen-2-yloxy)butan-2'-ol; -morpholino-4'-(naphthalen-2-yloxy)butan-2'-ol;

l-morpholino-3-((5,6,7,8-tetrahydronaphthalen-2-yl)oxy)propan-2-ol;

l-(3,4-dichlorophenoxy)-3-morpholinopropan-2-ol;

or a pharmaceutically acceptable salt or solvate thereof.

18. A pharmaceutical composition according to any one of claims 1, 2, or 4 to 17, or a compound according to any one of claims 3 to 17, for use in the treatment of triplet repeat diseases.

19. A pharmaceutical composition according to claims 18, or a compound for use according to claim 18, wherein the triplet repeat disease is selected from the group consisting of:

(i) non-polyglutamine diseases (e.g. Friedreich's ataxia, Fragile X, Myotonic dystrophy (DM1), and certain spinocerebellar ataxias [e.g. SCA8 (Spinocerebellar ataxia Type 8) and SCA12 (Spinocerebellar ataxia Type 12)]; and

(ii) polyglutamine (PolyQ) diseases (e.g. DRPLA (Dentatorubropallidoluysian atrophy), HD (Huntington's disease), SBMA (Spinobulbar muscular atrophy or Kennedy disease) and certain spinocerebellar ataxias [e.g. SCA1 (Spinocerebellar ataxia Type 1), SCA2

(Spinocerebellar ataxia Type 2), SCA3 (Spinocerebellar ataxia Type 3 or Machado-Joseph disease), SCA6 (Spinocerebellar ataxia Type 6), SCA7 (Spinocerebellar ataxia Type 7), or SCA17 (Spinocerebellar ataxia Type 17)]).

20. A pharmaceutical composition according to claims 18 or claim 19, or a compound for use according to claim 18 or claim 19, wherein the triplet repeat disease is Friedreich's ataxia.

21. A method of treating neurodegenerative and neuromuscular diseases and/or triplet repeat diseases (e.g. Friedreich's ataxia) as defined herein, said method comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound according to claims 3 to 17, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition according to claims 1, 2 or 4 to 17.

22. A method of treating triplet repeat diseases according to claim 21, wherein said triplet repeat disease selected from the group consisting of: (i) non-polyglutamine diseases (e.g. Friedreich's ataxia, Fragile X, Myotonic dystrophy (DM1), and certain spinocerebellar ataxias [e.g. SCA8 (Spinocerebellar ataxia Type 8) and SCA12 (Spinocerebellar ataxia Type 12)]; and

23. A method according to claim 19, wherein said triplet repeat disease is Friedreich's ataxia.