THE TREATMENT OF LYSOSOMAL STORAGE DISEASES

TECHNICAL FIELD The present invention relates to 4-epi-isofagomine enantionier derivatives,

pharmaceutically acceptable salts thereof, and said 4-epi-isofagomine enantionier derivatives for use in the treatment and prevention of lysosomal storage diseases. The present invention is furthermore related to processes for their preparation.

BACKGROUND OF THE INVENTION

Lysosomal storage diseases constitute a group of approximately 50 rare inherited metabolic disorders that result from defects in lysosomal function, usually as a consequence of deficiency of a single enzyme required for the metabolism of lipids, glycoproteins or

mucopolysaccharides. Morquio disease type B, also called mucopolysaccharidosis IV typ PSIVB), and

GM 1 -gangliosidosis are lysosomal storage diseases. They are caused by deficiencies of the beta- galactosidase (Brunetti-Pierri and Scaglia 2008, Caciotti et al. 201 1, Sandhoff and Harzer 2013). In the absence of beta-galactosidase activity, keratan sulfate and GMl-ganglioside are not properly degraded and accumulate in the lysosomes, causing cell dysfunction and, ultimately, the diseases. In most cases, these two diseases appear early in life and in many cases the patients succumb before reaching adulthood, Morquio disease type B affects mainly the development of peripheral organs, such as skeleton and heart, with a limited involvement of the central pervous system, and GM 1 -gangliosidosis mainly affects brain development.

Different therapies have been exploited for the treatment of lysosomal storage diseases. The enzyme replacement therapy has been successfully exploited for the treatment of lysosomal storage diseases affecting mainly peripheral organs readily accessible to protein administered in the circulation. Notable examples of successful enzyme replacement drugs are Fabrazyme for the treatment of Fabry disease, Cerezyme, for the treatment of Gaucher Disease and Naglazyme for the treatment of mucopolysaccharidosis VI.

The hematopoietic stem cell transplantation has been successful in severe cases of Hurler syndrome. This medical procedure, however, has not been found to alleviate symptoms in other mucopolysaccharidoses and, in addition, constitutes a high-risk medical procedure (Noh and Lee 2014).

The substrate reduction therapy has been exploited to treat Gaucher disease with the development of the drugs iniglustat and eliglustat. The therapy consists in limiting the synthesis of the cell constituent, which is no longer degraded due to an enzymatic deficiency. It follows that accumulation of the un-degraded cell constituent is slowed down, delaying onset and severity of the disease.

Imi no sugars constitute a class of compounds, which has been extensively and successfully exploited for the development of potent and specific glycosidase inhibitors (Nash et al 2011). Attempts to develop iminosugars for the treatment of certain forms of Gaucher disease met insurmountable difficulties during clinical development. In 2009, Phase II clinical trials with the experimental drag afegostat, also known as isofagomine, failed to demonstrate sufficient therapeutic efficacy, and led to the termination of its development.

As of today, no curative treatment is available to fight lysosomal storage diseases related to beta-galactosidase enzyme dysfunction, such as Morquio disease type B and GM1- gangliosodosis. Patients are only offered palliative care. This situation means that there is a significant unmet medical need for efficient and selective compounds for the treatment of lysosomal storage diseases related to beta-galactosidase enzyme dysfunction. In particular, an efficient and selective drug having limited toxic effects and capable to pass the blood brain barrier is still awaited. SUMMARY OF THE INVENTION

The present invention provides a 4-epi-isofagomine cnantiomer derivative of general formula (I):

and pharmaceutically acceptable salts thereof wherein:

Ro is selected from the group comprising hydrogen, C1-C3 alkyl, Ci-C alkenyl, C2-C6 alkynyl, C \- C ₆ cycloalkyl, C3-C18 heterocycle, Ce aryl, and C?-€g aryl alkyl;

R; is selected from the group comprising hydrogen, Ci-C ₆ alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3- Ce cycloalkyl, C3-C is heterocycle, Ce aryl, and C?-Cg arylalkyl;

R2, R3 and R ₄ are each independently selected from the group comprising hydrogen, C1-C4 alkyl, saturated or non-saturated C1-C4 acyl, C2-C4 alkenyl, and C2-C4 alkynyl;

R5 is selected from the group comprising substituted or unsubstituted C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C 12 cycloalkyl, C3-C18 heterocycle, Ce-Cis aryl, C7-C18 arylalkyl, with the proviso that R5 is not methyl; and wherein the 4-epi-isofagomine enantiomer derivative of general formula (I) is a (3R, 4S, 5R) enantiomer. The invention also provides a 4-epi-isofagomine enantiomer derivative of general formula (I), for use as a medicament. It further provides a 4-epi-isofagomine enantiomer derivative of general formula (I), for use in the treatment and/or prevention of diseases caused by dysfunctions in beta-galactosidase. Also contemplated is a 4~epi-i sofagomine enantiomer derivative of general formula (I), for use in the treatment and/or prevention of Morquio disease type B, GMl -gangliosodosis, Krabbc disease, galactosemia, galactosialidosis, sialidosis, Tay- Sachs disease, S andhoff- J atzkewitz's disease, Fabry disease, metachromatic leukodystrophy, and/or Morquio disease type A. Another object of the invention is to provide a 4-epi-isofagomine enantiomer derivative of general formula (I), for use in the treatment and/or prevention of lysosomal storage diseases, wherein said lysosomal storage diseases are selected from the group of pathologies caused by dysfunctions in beta-galactosidase, alpha-galactosidase, galactocerebrosidase, alpha-glucosidase, alpha-mannosidase, beta-mannosidase, alpha-fucosidase galactose- 1 -phosphate

uridylyltransferase, galactosylceramide beta-galactosidase, galactosyltransferases, neuraminidase and galactosamine 6-sulfate sulfatase. Furthermore, the invention provides pharmaceutical compositions comprising a 4-cpi- isofagomine enantiomer derivative f general formula (I) and a pharmaceutically acceptable carrier, diluent or excipicnt.

The present invention also provides a process for preparing and isolating the 4-epi-isofagomine enantiomer derivatives of general formula (I) comprising the step of reacting (2S, 3S, 4S, 5R) 2- benzyloxy-5-fonnyl-3.4-( -isopropylidene-tetrahydr()turan-3,4-dic)l with the Crignard reagent pentyimagnesium bromide in tetrahydrofuran to produce (2S, 3S, 4S, 5S) 2-benzyloxy-5-( 1 S- i - liydroxyhexyl)-3,4-0-isopropylidene-tetrahydiOfuran-3,4-diol ,

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : The beta-galactosidase activity in human peripheral blood mononuclear cell lysate was measured in the presence of increasing concentrations of N-nonyl-4-epi-isofagomine or 4-epi- isofagomine. Fluorescence measurements at 445 nm, using an excitation wavelength of 365 nm, are correlated to the inhibition of beta-galactosidase obtained with N-nonyl-4-epi-isofagomine (squares), 4-epi-isofagomine (triangles) and DMSO vehicle (circles).

Figure 2; The beta-galactosidase activity in human peripheral blood mononuclear cell lysate is inhibited in the presence of increasing concentrations of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2- pentylpipcridine-4,5-diol, (2S, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-dioI, or 4- epi-isofagomine. Fluorescence measurements at 445 nm, using an excitation wavelength of 365 nm, are correlated to the inhibition of beta-galactosidase by (2R, 3R, 45, 5 R)-3-hydroxymethyl- 2-pentylpiperidine-4,5-diol (squares), (2S, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpipcridine-4,5- diol (circles) and 4-epi-isofagomine (triangles).

Figure 3: The human beta-galactosidase activity is stabilized in the presence of (2R, 3R, 4S, 5R)- 34iydroxymethyl-2-pentylpiperidine-4,5-diol following heat denaturation.

Samples of toman peripheral blood mononuclear cell lysate were incubated at 48°C in the presence of graded concentrations of (2R, 3R, 4S, 5R)-3-hydroxyniethyl-2-pentylpiperidine-4,5- dio . Fluorescence was determined at 445 nm, using an excitation wavelength at 365 ran. and is correlated to the remaining beta-galactosidase activity. The tested concentrations of (2R, 3R, 4S, 5 )-3-hydroxymethyl-2-penty]piperidine-4,5-diol were 20 μΜ (square), 2 μΜ (triangle), 0.2 μΜ (diamond), 0.02 μΜ (circle) and 0 μΜ (line).

Figure 4: The beta-galactosidase and beta-hexosaminidase activity in fibroblasts of G i - gangliosidosis patients is induced in the presence of (2R, 3R, 4S, 5 R ) -3 - hy d ro x ymet h yl -2 - pcntylpipcridine-4,5-diol. GM02439 and GM05335 fibroblasts (Coriell Institute) were cultured in the presence or the absence of 2 μΜ of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpipericline-

4,5-diol. Beta-galactosidase and beta-hexosaminidase activities were determined in the cell lysates using fluorescence and colorimetric assays, respectively. Results of figure 4 represent the fold increase of beta-galactosidase or beta-hexosaminidase activities in (2R, 3R, 4S, 5R)-3- hydroxymethyl-2-pentylpiperidine-4,5-diol-treated cells relative to untreated cells. Results are means of 4 values obtained from independent cultures. Percent of deviation of each value is <3()% from the mean. Hatched and empty bars: beta-galactosidase and beta-hexosaminidase activities, respectively. Figure 5A and B: Beta-galactosidase (Fig. 5A) and beta-glucosidase (Fig. zyrnatic activities were determined in the presence of graded concentrations of compounds 4-epi- isofagomine (squares), (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentyIpiperidine-4,5-diol

(diamonds), isofagomine (triangles) and vehicle (circles). The stock solutions of the compounds were at 20 mM in water. The fluorescence was measured at 445 rim using 365 nm as the excitation wavelength.

Figure 6: Recombinant human galactocerebrosidase (from R&D Systems; 9 ng/assay) was incubated with 4-methylumbelliferyl β-D-galactopyranoside at 1 mM in 50 mM sodium phosphate pH 4.3, 125 inM NaCl and 0.5% Triton X- 1 00 in the presence of graded concentrations of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidiiie-4,5-diol. After 2 hours at 37°C, an equal volume of 0.4 M sodium carbonate pH 1 1.6 was added to the enzymatic reaction samples and fluorescence was measured at 445 nm using an excitation wavelength of 365 mn.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a 4-epi-isofagomine enantiomer derivative of general formula (I):

and pharmaceutically acceptable salts thereof wherein:

Ro is selected from the group comprising hydrogen, C1-C3 alkyl, Ci-Cf, alkenyl, Ci-Ce alkynyl,€3-

Ce cycloalkyl, C3--C18 heterocycle, C aryl, and C7-C8 aryl alkyl; Preferably, Ro is hydrogen;

Ri is selected from the group comprising hydrogen, Ci-C ₆ alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-

C ₆ cycloalkyl, C3-C18 heterocycle, C ₆ aryl, and C7-C8 arylalkyl; Preferably, R i is hydrogen;

R2, R3 and R4 are each independently selected from the group comprising hydrogen, C1-C4 alkyl, saturated or non-saturated C1-C4 acyl, C2-C4 alkenyl, and C2-C4 alkynyl; Preferably, R2, R3 and R ₄ are hydrogen;

Ks is selected from the group comprising substituted or unsubstituted C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl, C3-C18 heterocycle, C ₆ -Ci 8 aryl, C7-C18 arylalkyl, with the proviso that R5 is not methyl and wherein the 4-epi-isofagomine enantiomer derivative of general formula (I) is a (3R, 4S, 5R) enantiomer. Preferably, Rs is selected from the group comprising pentyl, hexyl, heptyl, octyl, nonyl, 2- phenylethyl, 2-(4~trifluoromethylphenyl)ethyl, 2-ethylhexyl, cyclopropylmethyl,

cyclohexylmethyl, cycloheptylmethyl, 5-hexenyl, 5-hexynyl, 5-acetamido- 1 -hydroxypent-2-yl, and ό-acetamido-hexyl.

More preferably, Rs is pentyl, hexyl. heptyl, or octyl. Even more preferably, Rs is pentyl.

The following definitions are supplied in order to facilitate the understanding of the present invention.

As used herein, the term "comprise" is generally used in the sense of include, that is to say permitting the presence o one or more features or components.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

The following paragraphs provide definitions of the various chemical moieties that make up the compounds according to the invention and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

"C1-C12 alkyl" refers to monovalent straight-chained and branched alkyl groups having 1 to 12 carbon atoms, such as C 1 -C3 alkyl, C i ~C ₄ alkyl or Ci -C ₆ alkyl. Examples of straight chain alkyl groups include, but are not limited to, those with from 1 to 12 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl groups, n-heptyl, n-octyl, n-nonyl and n-decyl groups. Examples of branched alkyl groups include, but are not limited to, i so propyl, iso-butyl, sec- butyl, tert-hutyl, isopentyl, and 2,2-dimethylpropyl groups. Alkyl groups may be substituted or unsubstituted. Representative substituted alkyl groups may be substituted one or more times with for example, amino, oxo, hydroxy, cyano, carboxy, nitro, hio, alkoxy, F, CI, Br, I, cycloalkyl, aryl, heterocyclyl and heteroaryl groups.

"C2-C12 alkenyl" refers to straight and branched chain alkyl and cycloalkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 12 carbon atoms, such as C2-C alkenyl, C2-C4 alkenyl or C2-C 10 alkenyl. Examples include, but are not limited to, vinyl, -CH=CH(CH ₃ ), -CH=C(CH ₃ )2, -C(CH ₃ )=CH ₂ , -

- 1 ~ C(CE >- ^' TI(CH ₃ ), -C(CH ₂ CF ^· ri , ₂ , cyclohexenyl, cyclopen.ten.yl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl, among others. Alkenyl groups may be substituted or unsubstituted.

"C2-C12 alkynyl" refers to straight and branched chain alkyl groups, comprising at least one triple bond between, two carbon atoms. Thus, alkynyl groups have from 2 to 12 carbon atoms, such as C2-O, alkynyl, C2-C4 alkynyl, or C2-C12 alkynyl. Examples include, but are not limited to, 1- ethynyl, 1 -propynyl, 2-propynyl, 1 -butynyl, 2-butynyl, 1 -pentynyl or 2 -pentynyl radical, among others. Alkynyl groups may be substituted or unsubstituted.

"C3-C12 cycloalkyl" refers to cyclic alkyl groups having from 3 to 12 carbon atoms such, as, but not limited to Cs-Ce cycloalkyl (cyclopropyl, cyclobuty!, cyclopentyl, cyclohexyl), cycloheptyl, cyclooctyl groups, and C ₃ -Cio cycloalkyl. Cycloalkyl groups further include mono-, bicyclic and polycyclic ring systems, such as, for example bridged cycloalkyl groups as described below, such as, but not limited to, adamantyl, and fused rings, such as, but not limited to, decalinyl, and the like. Cycloalkyl groups may be substituted or unsubstituted. Cycloalkyl groups may be substituted one or more times with non-hydrogen groups as defined above (substituents).

However, substituted cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups, which may be substituted with any of the groups listed above, for example, methyl, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, F, CI, Br and I groups.

"C3-C18 heterocycle" refers to non-aromatic ring groups containing 3 or more ring members, of which one or more is a hetcroatom such as, but not limited to, N, O and S. In some

embodiments, the heterocyclyl group contains 1, 2, 3, or 4 heteroatoms. In some embodiments, heterocyclyl groups include 3 to 6, 10, 12, or 15 ring atoms. Heterocyclyl groups encompass unsaturated, partially saturated and saturated ring systems, such as, for example, imidazolinyl and imidazolidinyl groups. The phrase "heterocyclyl group" includes fused ring species

including those comprising fused aromatic and non-aromatic groups, such as, for example, 2,3- dihydrobenzo [1,4] dioxinyl . The phrase also includes bridged polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups may have other groups, such as alkyl, oxo or halo groups, bonded to one of the ring members. These are referred to as "substituted heterocyclyl groups." Heterocyclyl groups may be substituted or unsubstituted. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, pyrrol inyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrazolidinyl, tetrahydropyranyl, fhiomorphoiinyl, pyranyl, tetrahydrofuranyl , dihydrobenzofuranyl, dihydroindolyl,

azabenzimidazolyl, benzothiadiazolyl, imidazopyridinyl, thianaphthalenyl, xanthinyl, guaninyl, tetrahydroquinolinyl, and 2,3 -dihydrobenzo [1,4] dioxinyl . Representative substituted heterocyclyl groups may be mono-substituted or substituted more than once, such as, but not limited to, triazolyl, pyridinyl or morpholinyl groups, which are 1-, 2-, 3-, 4-, 5-, or 6- substituted, or disubstituted with various groups as defined above, including, but not limited to, alkyl, xo, carbon yl, amino, alkoxy, cyano, and/or halo. The term "aryl" refers to cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups include monocyclic, bicyclic and polycyclic ring systems. Thus, aryl groups include, but are not limited to Ce aryl (such as phenyl, benzyl, tolyl. xylyl, benzyl iden, benzoyl), Cc-Ci x aryl such as azulenyl, heptalenyl, biphenylenyl, indacenyl, fluorenyl, phenanthrcnyl, tri phenyl enyl, pyrcnyl, naphthacenyl, chrysenyi, biphenyl, anthracenyl, indenyl, indanyl, pcntaienyl. naphthyl groups. The phenyl groups substituted or not, are particularly preferred. The term "aryl" includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g. indanyl.

tetrahydronaphthyl, and the like). Aryl groups may be substituted or unsubstituted. Groups such as tolyl are referred to as substituted aryl groups. Representative substituted aryl groups may be mono-substituted or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with groups such as those listed above.

"C7-C18 arylalkyi" refers to a radical of the alkyl type as defined above substituted by an aryl group as defined above, such as C7-C8 arylalkyi. The benzyl and phenylethyl groups are particularly preferred.

"C1 -C4 acyl" refers to the group -C(0)R where R includes C1-C0 alkyl, aryl, and heteroaryl.

According to the invention, the groups identified above may be substituted or unsubstituted. In general, the term "substituted" refers to a functional group, as defined below, in which one or more bonds to a hydrogen atom are replaced by a bond to a non-hydrogen atom. Substituted groups also include groups, in which one or more bonds to a hydrogen atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. In some embodiments, substituted groups have 1 , 2, 3, 4, 5, or 6 substituents. Examples of substituent groups include, but are not limited to, halogens (i.e. F, CI, Br and I), hydroxy! s, alkoxy, alkenoxy, alkynoxy, aryloxy, aralkyloxy, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo);

carboxyls; esters; ethers; urethanes; oximes; hydroxylarnines; alkoxyamines; thiols; sulfides such as alkyl, alkenyl, alkynyl, aryl, ar alkyl, heterocyclyl and heterocyclylalkyl sulfide groups;

sulfoxides; sulfon.es; sulfonyls; sulfonamides; amines; N-oxides; hydrazines; hydrazides;

hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides; isocyanates;

isothiocyanates; cyanates; thiocyanates; imines; nitriles; alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocyclyl, heterocyclylalkyl and cycloalkyl groups.

Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and fused ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with alkoxy, alkyl, alkenyl, and alkynyl groups as defined below.

Thus, the present invention relates to 4-epi-isofagomine enantiorner derivatives of general fomiula (I) and pharmaceutically acceptable salts thereof that contain at least four to five asymmetric chiral carbon atoms in the subtituted piperidine cycle.

According to nomenclature 1, enantiomers of 4-epi-isofagomine derivatives of general formula (I) arc selected from the group comprising (2R or 2S, 3R, 4S, 5R, 5aR or 5aS) 4-epi-isofagomine derivatives, and (3R, 4S, 5R, 5aR or 5aS) 4-epi-isofagomine derivatives. Preferably, 4-epi- isofagomine enantiorner derivatives of general formula (I) are selected from the group comprising (3R, 4S, 5R) 4-epi-isofagomine enantiorner derivatives. More preferably, 4-epi- isofagomine derivatives of general formula (I) are selected from the group comprising (3R, 4S, 5R, 5aR) 4-epi-isofagomine derivatives:

Systematic nomenclature can be used to define the 4-epi-isofagomine enantiomer derivatives of general formula (I) according to the invention as follows:

Nomenclature 2 (IUPAC nomenclature):

According to nomenclature 2, cnantiomers of 4-epi-isofagomine derivatives of general formula (1) are selected from the group comprising (2R or 2$, 3R, 4S, 5R, 6R or 6S) 4-epi-isofagomine enantiomer derivatives, and (2R or 2S, 3R, 4S, 5R) 4-epi-isofagomine enantiomer derivatives. Preferably, 4-epi-isofagomine enantiomer derivatives of general formula (I) are selected from the group comprising (3R, 4S, 5R) 4-epi-isofagomine enantiomer derivatives. More preferably, 4- epi-is fagorninc enantiomer derivatives of general formula (I) are selected from the group comprising (2R, 3R, 4S, 5R) 4-epi-isofagomine enantiomer derivatives.

The invention also relates to salts of the 4-epi-isofagomine enantiomer derivatives of general formula (I), pure or mixed, stereoisomers, for example C2-epimers or CSa-cpimers, hydrates, solvates, solid forms, chemical modified compounds, and/or mixtures thereof.

Preferably, these salts are pharmaceutically acceptable. According to the present invention, pharmaceutically acceptable salts are produced from acidic inorganic or organic compounds. As used herein, the phrase ""pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness of the free acids and bases of a specified compound and that is not biologically or otherwise undesirable.

4-epi-isofagomine enantiomer derivatives of general formula (I) and pharmaceutically acceptable salts thereof are stable in several solid forms. The present invention includes all the solid forms of the compounds according to the invention which includes amorphous, polymorphous, mono- and polycrystalline forms. 4-epi-isofagomine enantiomer derivatives of general formul d pharmaceutically acceptable salts thereof can also exist in non-solvated or solvated form, for example with pharmaceutically acceptable solvents such as water (hydrates) or ethanol.

In a preferred embodiment, the present invention relates to a 4-epi-isofagornine enantiomer derivative of general formula (I) and pharmaceuticall y acceptable salts thereof wherein Ro is hydrogen. More preferably, R i is hydrogen. Even more preferably, Ro and Ri are hydrogen. In another preferred embodiment, the invention relates to 4-epi-isofagomine enantiomer derivatives of general formula (I), and pharmaceutically acceptable salts thereof, wherein Ro, R i , R.2, R and 4 are hydrogen; and R5 is selected from the group comprising substituted or unsubstituted O-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C12 cycloalkyl, C3-C18 heterocycle, Ce-Cig aryl, C7-C18 arylalkyl, with the proviso that R5 is not methyl and wherein the 4-epi- isofagomine enantiomer derivative of general formula (I) is a (3R, 4S, 5R) enantiomer.

Preferably, R5 is selected from the group comprising substituted or unsubstituted C2-C12 alkyl, C2- C12 alkenyl, C2-C 12 alkynyl, C3-C12 cycloalkyl, C3-C18 heterocycle, Ce-Cig aryl, C7-C18 arylalkyl. More preferably, R5 is selected from the group comprising pentyl, hexyl, heptyl, octyl, nonyl, 2- phenylethyl, 2-(4-trifluoromethylphenyl)ethyl, 2-ethylhexyl, cyclopropylmethyl,

cyclohexylmethyl, cycloheptylmethyl, 5-hexenyl, 5-hexynyl, 5-acetamido- 1 -hydroxypent-2-yh and 6-acetamido-hexyl. Even more preferably, the 4-epi-isofagomine enantiomer derivatives of general formula (I), and pharmaceutically acceptable salts thereof, are selected from the group comprising;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-pentylpiperidine-4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-hexylpiperidine-4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-heptylpiperidine-4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-octylpiperidine-4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-nonylpiperidine-4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-(2-phenylethyl)-piperidine-4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-[2-(4-trifluoromethylphenyl)ethyl]pipe ridine-4,5-diol; (2 , 3R, 45, 5R)-3-FIydrox}TOcthyl-2--(2-ethylhexyl)piperidinc-4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-cyclopropylmethylpiperidine~4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-cyclohexylmethylpiperidine-4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-cycloheptylmethylpiperidine-4,5-diol;

(2R, 3R, 45, 5R)-3 Iydroxymcthyl-2-(5-hexenyl)pipcridine-4,5-diol;

(2R, 3R, 4S, 5R)4^Hydrox>TOethyl-2-(5 iex Tiyl)piperidine-4,5-diol;

(2R, 3R, 4S, 5R)-3-Hydroxymethyl-2-(5-acctamido- l -hydroxy^ and (2R, 3R, 4S, 5R)-3-Hydroxyrncthyl-2-(6-acetan ido-hexyl)pipcridine-4,5-diol.

Yet more preferably, the 4-epi-isofagomine enantiomer derivative of general formula (I), and pharmaceutically acceptable salts thereof, is (2R, 3R, 4S, 5 )- 3 - hy d ro ym eth yl-2- pentylpiperidine-4,5-diol of formula:

It is one object of the present invention to provide for 4-epi-isofagomine enaiitiomer derivatives of general formula (I) and pharmaceutically acceptable salts thereof, wherein said 4-epi- isofagomine derivatives are inhibitor of wild type beta-gal aetosidase activity and have an IC50 of less than about 50 μΜ, preferably of less than about 10 μΜ, more preferably of less than about 5 μΜ.

As shown in the examples, (2R, 3R, 4S, 5R)-3-hydroxymcthyi-2-pcntylpiperidine-4,5-diol has an IC50 of Ο.ΟΙ μΜ, and (2S, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol has an IC50 of 1.5 μΜ (Example 3).

Generally, the beta-galactosidase is a hydrolase enzyme that cleaves beta-linked terminal galactosyl residues from different substrates, including gangliosides, glycoproteins and glycosaminoglycans. Preferably, the beta-galactosidase is a lysosomal beta-galactosidase also called acid beta-galactosidase, as it refers to a protein located in the acidic environment of the lysosomes, while other beta-galactosidase activities are found in neutral environments (e.g. cytoplasm). The lysosomal beta-galactosidase is the product of the GLB1 gene.

As used herein, the term "beta-galactosidase" refers to the enzyme from mammalian origin, preferably human beta-galactosidase.

As used herein, the term "inhibitor" refers to compounds that block or partially block, directly or indirectly the activity of an enzyme. As used herein, the term "IC50" represents the concentration of a drug that is required for 50% inhibition of the enzyme activity.

As used herein, the term "about" applies to numeric values and refers to a range of numbers that one of skill in the art would consider equivalent to the recited values. For example, "about 10 μΜ" refers to 10 μΜ ± 10%.

Advantageously, the 4-epi-isofagomine enaiitiomer derivatives of general formula (I) are highly potent specific inhibitors of beta-galactosidase activity. As shown in the examples, (2R, 3R, 4S, 5R)-3~hydrox Tnethyl-2-pentylpiperidine-4,5-diol is a highly specific beta-galactosidase inhibitor (IC50 = 0.02 μ,Μ), whereas it considerably less potently inhibits the human beta-glucosidase and the human alpha-galactosidase (IC50 = 60 μΜ and 150 μΜ, respectively), and does not inhibit any of the other four human glycosidases tested (IC50 > 160 μΜ) (Example 4).

Interestingly, the present invention demonstrated that the 4-epi-isofagomine enantiomer derivatives of general formula (I) are highly potent specific inhibitors of beta-galactosidase activity in contrast to the isofagomine compound. As shown in the example 7, 4-epi-isofagomine and its (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol derivative are potent inhibitors of human lysosomal beta-galactosidase (Fig. 5 A; 1C50 = 0.5 and 0.01 μΜ, respectively), while they are weakly active on human beta-glucosidase (Fig. 5B; 1C50 > 800 pM for both compounds). By contrast, isofagomine is devoid of any activity on the beta- galactosidase (1C50 > 800 μΜ), while it is a moderately active beta-glucosidase inhibitor (10 μΜ).

Remarquably, the present invention demonstrated that a (2R, 3R, 4S, 5R)-3-hydroxymcthyl-2- mcthy]pipcridine-4.5-diol derivative is at least 10-fold less potent as beta-galactosidase inhibitor than (2R, 3R, 4S, 5R)-3-hydroxymcthyl-2-pentylpiperidinc-4 _; 5-diol derivative, (2R, 3R, 4S, 5R)-3-h>xh'oxvTOCthyl-2-phcnylethylpiperidine-4,5-diol or (2R, 3R, 4S, 5R)-3 -h ydroxymcthyl -2 - nonylpiperidine-4,5-diol derivative (Example 8).

In addition, the present invention demonstrated that 4-epi-isofagomine enantiomer derivatives of general formula (I) are also inhibitors of human gal actoccrcbrosid ase activity.

As shown in the example 15, the 4-epi-isofagomine enantiomer derivatives of general formula (I), such as (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-dio) is a potent inhibitor of recombinant human ga!actocerebrosidasc (IC50 = 0.04 μΜ).

The present invention also relates to 4-epi-isofagomine enantiomer derivatives of general formula (I) as chaperonc molecules. Generally, pharmacological chaperones are small molecular weight compounds which tightly bind in the endoplasmic reticulum to newly synthesized proteins and favor them to adopt a native conformation. Once this status is attained, the protein is no longer retained in the endoplasmic reticulum and traffics to its normal intracellular or extracellular destination. In the case of mutated proteins which fail to adopt a native

conformation, the quality control system of the endoplasmic reticulum clear them, leading to their early degradation. The binding of chaperone compounds is believed to support the correct folding of the proteins and promote further maturation, avoiding the polypeptide to undergo premature degradation. According to this model, the mutated protein reaches its final targeted cellular compartment (e.g. lysosomes, plasma membranes and secretion in the extracellular space), where it can perform its functions. This mechanism of action relies on the fact that in many situations, mutations do not affect the catalytic activity of the protein, meaning that if the pharmacological chaperone compounds allow the transport of the mutated proteins to its normal cellular destination, it would partly restore the physiological enzymatic activity (Fan et al 1 99, Boyd et al 2013). In the context of the present invention, in addition to be potent specific inhibitors of wild type beta-galactosidase activity, the 4-epi-isofagomine enantiomer derivatives of general formula (I) are chapcrone molecules of deficient mutated versions of the beta-galactosidase protein. The present invention also relates to 4-epi-isofagomine enantiomer derivatives of general formula (I) as ehaperone molecules of deficient mutated versions of the galactocerebrosidase protein.

As used herein, the term "ehaperone molecule" refers to small molecular weight compounds which bind to proteins to support their folding and/or stabilization in their native conformation, or favor them to adopt or restore a conformation ressembling the physiologically native one.

As used herein, the term "deficient" refers to totally or partially inactivated enzymes resulting from mutations, or denaturation through exposure to chemicals (e.g. urea and guanidine hydrochloride) or physical factors (e.g. heat and freeze and thaw cycles).

Thus, the present invention provides 4-epi-isofagomine enantiomer derivatives of general formula (I) and pharmaceutically acceptable salt thereof, wherein said 4-epi-isofagomine enantiomer derivatives prevent denaturation of beta-galactosidase.

As used herein, the term "denaturation" refers to a process in which the folding conformation of a protein is altered due to exposure to certain chemical or physical factors (e.g. heat, acid, solvents, etc), causing the protein to become biologically inactive. As shown in the examples, following heat denaturation of peripheral blood mononuclear cell lysate at 48°C for 10 minutes, 84% of the beta-galactosidase activity was spared by the presence of 2 μ ^' Μ of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pcntylpiperidine-4,5-diol, while this percentage was reduced to 13% in the absence of compound (Example 5, Figure 3). The present invention also relates to 4-epi-isofagomine enantiomer derivatives of general formula (I) and pharmaceutically acceptable salt thereof, for use as a medicament.

It further relates to 4-epi-isofagomine enantiomer derivatives of general formula (I) and pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of Morquio

- - disease type B, GM1 -gangliosidosis, Krab e disease, galactosialidosis, siaiidosis, Tay-Sachs disease, Sandlioff-Jatzkewitz's disease, Fabry disease, metachromatic leukodystrophy, Morquio disease type A and/or galactosemia, Thus, the present invention provides for 4-cpi-isofagomine cnantiomer derivatives for use in the treatment and/or prevention of diseases caused by dysfunctions in beta-galactosidase.

Morquio disease type B and GM1 -gangliosidosis, are genetic lysosomal storage diseases associated with beta-galactosidase enzyme dysfunctions. Human beta-galactosidase gene mutations have been found in patients with Morquio disease type B and GM1 -gangliosidosis.

Krabbe disease, Tay-Sachs disease, Sandhoff-Jatzkewitz's disease, Fabry disease, metachromatic leukodystrophy, Morquio disease type A and galactosemia are metabolic diseases caused by deficiencies of enzymatic activities necessary for the disposal of cell constituents containing at least at one point in their catabolic pathways terminal galactosyl residues.

In particular, Krabbe disease, also called globoid cell leukodystrophy or galactosylceramide lipidosis, is caused by mutations in the gene coding the galactocerebrosidase, an enzyme removing galactose residues from galactocerebrosides. This disease is characterized by rapidly progressing neurologic symptoms resulting in the death of the children in most of the cases.

Galactosialidosis and siaiidosis are metabolic diseases caused by dysfunctional cathepsin A, which normally forms complexes with other proteins, such as beta-galactosidase, neuraminidase, galactosamine 6-suifate sulfatase and elastin binding protein.

Another object of the present invention relates to 4-epi-isofagomine enantiomer derivatives of general formula (I) and pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of lysosomal storage diseases, wherein said lysosomal storage diseases are selected from the group of pathologies caused by deficiencies in beta-galactosidase, alpha-galactosidase, gal actocerebrosidase, alpha-glueosidase, alpha-mannosidase, beta- mannosidase, alpha-fucosidase galactose-1 -phosphate uridylyltransferase, galactosylceramide beta-galactosidase, galactosyltransferases, neuraminidase and galactosamine 6-sulfate sulfatase. Remarquably, all these enzymes either use galactose as substrate or are able to form complexes with beta-galactosidase. Preferably, the lysosomal storage diseases are related to beta- galactosida.se enzyme dysfunctions.

Surprisingly, the 4-epi-isofagomine enaiitiomer deri vatives of general formula (I), for example (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-dioi induces increase of mutated beta- galactosidase activity in cell lines of patients suffering from lysosomal storage diseases associated with beta-galactosidase enzyme dysfunctions, such as GMl -gangliosidosis. The beta- galactosidase activity following treatment with 4-epi-i.sofagomine enantiomer derivatives of general formula (I) is at least 2-fold, preferably 5-fold higher than in the absence of treatment.

As shown in the example, the 4-epi-isofagomiiie enantiomer derivative, (2R, 3R, 4S, 5 )-3- hydroxymethyl-2-pentylpiperidine-4,5-diol, induces increase of the mutated beta-galactosidase activity by 6.5- and 19-fold in GMl -gangliosidosis cell lines GM02439 and GM05335, respectively (Example 6 and Fig, 4). In contrast, there is no increase of the beta-hexosaminidase activity at all.

Interestingly, the 4-epi-isofagomine enantiomer derivative of general formula (I), such as (2R, 3R, 4S, 5R)-3-hydroxyrnethyl-2-pentylpiperidine-4,5-diol, has the ability of preventing early degradation of mutated forms of newly synthesized beta-galactosidase polypeptide in GM1- gangliosidosis and Morquio disease cell lines.

The 4-epi-isofagomine enantiomer derivative of general formula (I), such as (2R, 3R, 4S. 5R)-3- hydroxymethyl-2-pentylpiperidine-4,5-diol, induces increase of the mutated beta-galactosidase activity in different cell lines of patients suffering from lysosomal storage diseases associated with beta-galactosidase enzyme dysfunctions, such as infantile, juvenile and adult GM1- gangliosidosis, as well as Morquio disease type B patients (Examples 11 -13).

GMl -gangliosidosis and Morquio disease type B are caused by the pathological accumulation of GMl -gangliosides and keratan sulfate in cells, due to the deficiency in beta-galactosidase, which is one of the enzymes participating in the degradation of these two cell constituents.

Furthermore, the 4-epi-isofagomine enantiomer derivative of general formula (I), (2R, 3R, 4S, 5R)-3~hydroxymethyl-2-pcntylpiperidine-4,5-diol induces the reduction of keratan sulfate level in GMl -gangliosidosis patient cells (Example 12). The present invention further relates to a pharmaceutical composition comprising 4-cpi- isofagomine enantiomer derivatives of general formula (I) and pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

The pharmaceutical composition of the invention is formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th cd.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art. The excipient of the composition can be any pharmaceutically acceptable excipient, including specific carriers able to target specific cells or tissues. As stated earlier, possible pharmaceutical compositions include those suitable for oral, rectal, topical, transdermal, buccal, sublingual, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. For these formulations, conventional excipieiits can be used according to techniques well known by those skilled in the art. The compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions which can optionally be prepared immediately before use from solid or lyophilized form. For oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations, such as syrups, elixirs, and concentrated drops. Non-toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. For compressed tablets, binders, which are agents which impart cohesive qualities to powdered materials, are also necessary. For example, starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also necessary in the tablets to facilitate break-up of the tablets. Disintegrants include starches, clays, celluloses, algins, gums and cross-linked polymers. Moreover, lubricants and glidants are also included in the tablets to prevent adhesion to the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture. Colloidal silicon dioxide is most commonly used as a glidant and compounds, such as talc or stearic acids, arc most commonly used as lubricants. For transdermal administration, the composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and

dimethylformamide. For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate. In a preferred embodiment, the pharmaceutical composition of the invention i s suitable for parenteral administration.

As shown in the examples 9 and 10, the 4-epi -i so fagomine enantiomer derivatives of general formula (I), such as (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-per)tylpiperidine-4,5-diol are found in the plasma, brain and bones tissues following their intravenous or oral administration .

Therefore, these derivatives can be useful for the treatment and/or prevention of lysosomal storage diseases, when incorporated in a pharmaceutical composition.

Pharmaceutical composition according to the invention may be formulated to substantially release the active drag immediately upon administration or at any predetermined time or a time period after administration.

In a particular embodiment, the pharmaceutical composition according to the invention comprises 0.001 mg to 5 g of the compound of the invention. Preferably, pharmaceutical composition according to the invention comprises 0.01 mg to 1 g of the compound of the invention.

Pharmaceutical compositions according to the invention can comprise one or more compounds of the invention in association with pharmaceutically acceptable excipieiits and/or carriers. These excipieiits and/or carriers are chosen according to the form of administration as described above.

The invention also relates to a pharmaceutical composition comprising 4-epi-isofagomine enantiomer derivatives of general formula (I) and pharmaceutically acceptable salts thereof, and further comprising at least one or more additional agents selected from the group comprising non-competitive chaperone compounds and GMl-ganglioside synthesis inhibitors.

Non-competitive chaperone compounds are selected from the group comprising substances selected for their capacity to promote rescue of mutated forms of beta-galactosidase without inhibiting their enzymatic activity, a feature explained by the binding of these substances to the beta- gal acto sidase outside its catalytic site.

GMl -ganglioside synthesis inhibitors are selected from the group comprising for instance d- threo- 1 -phenyl-2-palmitoylarnino~3 -pyrrolidine- 1 -propanol (Tifft & Proia 2000) and N- butyldeoxygal actonoj irimyc in (Back et al 2008).

As shown in the example 14, the combined treatment of GMl -gangliosidosis cells with (2R, 3 , 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-dio! and pharmacological agents, such as the proteasome inhibitor MG-132 or the calcium channel blocker diltiazem, interfering with proteostasis resulted in a recovery of beta-gal actosidase activity higher than the levels obtained following treatments with the agents used separately.

The invention further relates to a process for preparing and isolating a 4-epi-isofagomine enantiomer derivative of general formula (I) comprising the steps described in the scheme below starting from D-lyxose (Synthesis 1):

+13, epimer 6S (major) 31 %

In particular, the invention relates to a process for preparing and isolating a 4-epi- i sofagomine enantiomer derivative comprising the steps of:

1 ) modification of D-lyxose (1) with benzyl alcohol to afford benzyl a-D-lyxopyranoside (2), 2) modification of 2 with 2,2-dimethoxypropane to afford benzyl 2,3-C)-isopropylidcnc-a-[>- lyxopyranoside (3),

3) reaction of 3 with Dess-Martin. pcriodinane, resulting in benzyl 2,3-0-isopropylidene-p-L- er //iro-pent-4-uloside (4),

4) reaction between 4 and 1 -nitrohexane to produce benzyl 2,3-Oisopropylidene-4-C- [(! '/?, 1 i -nitrohcx- i -yl)]-[i-L-ribopyranosidcs (5),

5) reduction of 5 with Raney nickel to afford benzyl 2.3-O-isopropylidene— [( ΓΛ, 1 ^' S){ I - aminohex- 1 -yl)]-p-L-ribopyranosides (6),

6) rearrangement of 6 catalyzed by palladium hydroxide in the presence of hydrogen, affording (2R/S, 35, 411, 5/i)-3-hydiOxymcthyl-4,5-0-i;sopropylidene-2-pentyl-piptTidi ne-3,4,5-triol (7),

7) reaction of 7 with benzyl chloroformate to obtain (2R, 35, 4R, 5i?)-l-benzyloxycarbonyl-3- hydix)xyiTiethyl-4,5-0-isopropylidene-2-peiityl-piperidine-3 ,4,5-triol (8),

8) modification of 8 with thiocarbonyidiimidazolc to afford (2R, 35, 4R, Ji?)-1- benzyloxycarboEyl-3-hydroxyinethyh4,5- dsopropylidene-2-pentyl-piperidine-3,4,5"triol, 3,3'- thiocarbonyl derivative (9),

9) modification of 9 by reaction with tributyltin hydride to obtain (2R, 3R, 4S, 5R)-1 - bcn/.y]oxycarbonyl-3-hydroxyTnethyl-4,5-^isopropylidenc-2-pe ntyl-p (10),

10) deprotection of 10 with palladium in the presence of hydrogen to produce (2R, 3R, 4S, 5R)~ 3-hydroxyiiiethyl-4,5-0-isopropylidene-2-pentyhpiperid!ne-4, 5-diol (11), and

1 1) treatment of 11 with an acidic ion-exchange resin in dioxane and H ₂ 0 to obtain (2R, 3R, 4S, 5R)~3diy(lroxymcthyl-2-pcntyl-pipcridine-4.5-diol (12), in particular, important steps in the process for preparing and isolating a 4-epi-isofagomine derivative from -lyxosc are the modification of 2,3-0~isopropylidene-P-L-eryrAro~pent-4- uloskle with 1 -nitroalkane to produce benzyl 2,3-0-isopropylidene~4-C-[(l -nitroalk- i - yl)]-P-L-ribopyranosides and, ultimately, production of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-C- alkyl-piperidine-4,5-diol.

Alternatively, the invention relates to another process for preparing and isolating 4-epi- isofagomine e antiomer derivatives of general formula (I) comprising the steps described in the scheme below starting from L-ribose:

The invention relates to a process for preparing and isolating 4-epi-isofagomine enantiomer derivatives of general formula (I) comprising the step of:

Reacting (2S, 3S, 45, 5R) 2-benzyloxy-5-formyl-3,4-( -isopropylidene-tetrahydrofuran-3,4-diol (2) with the Grignard reagent pentylmagnesium bromide in tetrahydrofuran to produce, following purification, (2S, 3S, 4S, 5S) 2-benzyloxy-5 -( 1 S - 1 -hydroxyhexyl)-3 ,4-0

isopropylidene-tetrahydrofuran-3,4-diol (3).

The invention also relates to a process for preparing and isolating 4-epi-isofagomine eHantiomer derivatives of general formula (I) comprising the step of:

Reacting (2S, 3S, 4S, 5S) 5-(lR-l-azidohexyl)-2-benzyloxy-3,4-0-isopropylidcne- tetrahydrofuran-3 ,4-diol (5) in. anhydrous methanol with palladium on carbon, followed by a catalytic amount of acetic acid, to produce a crude preparation of (2R, 3S, 4S, 5R)- 4,5-0- isopropylidene-2-pentyl-piperidinc-3,4,5-triol (6).

The invention further relates to a process for preparing and isolating 4-epi-isofagomine enantiomer derivatives of general formula (I) comprising the step of:

Reacting 1 -tert-butoxycarbonyl (2R, 4S, 5R)-4,5-0-isopropylidene-3-methylene-2-pentyl- piperidine-4,5-diol (9) in anhydrous tetrahydrofuran with 9-borabicyclo[3.3.1 Jnonanc to produce, following solvent extraction and purification, 1-tert-butoxycarbonyl (2R, 3R, 4S, 5i?)-3- hydroxvTnethyl-4,5-0-isopropylidene-2-pentylpiperidme-4,5-di

Thus, the invention relates to a process for preparing and isolating 4-epi-isofagomine enantiomer derivatives of general formula (I) comprising the steps of:

ction of L-ribose with concentrated sulfuric acid in benzyl alcohol and acetone to afford 1 - C?-benzyl-2,3-0-isopropylidene-L-ribofuranose (1);

2) reaction of 1 with Dess-Martin reagent in anhydrous CH2CI2 to afford crude (25, 35, 4S, 5R) 2-benzyloxy-5-fomiyl-3,4-0-isopropylidene~tetrahydrofuran-3, 4-diol (2);

3) direct modification of 2, due to its instability, with the Grignard reagent pentylmagnesium bromide in tetraliydrofuran to afford, following purification, (25, 3S, 4S, 5S) 2-benzyloxy-5-( I S- 1 -hydroxyhexyl)-3.4-0-isopropylidene-tetrahydrofuran-3 ,4-diol (3);

4) reaction of 3 in anhydrous CH2CI2 with methanesulfonyl chloride to afford, following purification, (2S, 3S, 4S, 511) 2-ben/,yloxy-3,4-0-isopropylidene-5-( 1 R- ! - methanesullbnyloxyhexyi)-tetrahydrofuran-3 _; 4-diol (4);

5) reaction of 4 with sodium azide to afford, following solvent extraction, (2S, 3S, 4S, 5S) 5- (1 R-l -azidohexyl)-2-benzyloxy-3,4-0-isopropylidene-tetrahydrofura n-3,4-diol (5);

6) reaction of 5 in anhydrous methanol with palladium on carbon, followed by a catalytic amount of acetic acid, to afford a crude preparation of (2R, 3S, 4S, 5R)~ 4,5- >-isopropylidene-2-pentyl- piperidine-3,4 _< 5-triol (6);

7) reaction of 6 with di-tert-buty! dicarbonate to afford, following purification, 1 -tert- butoxycarbonyl (2R, 3S, 4S, 5R)-4,5-0-isopropylidene-2-pentyl-piperidine-3,4,5-triol (7);

8) reaction of 7 i anhydrous CH2CI2 with Dess-Martin reagent to afford crude 1 -tert- butoxycarbonyl (2R, 4R, 5R)-4,5-0-isopropylidene-2-pentyl-3-oxo-piperidine-4,5-diol (8);

9) reaction of crude compound 8, in anhydrous tetrahydrofuran, with the Wittig reagent, prepared by mixing methyltriphenylphosphonium bromide in anhydrous tetrahydrofuran with n- butylithium in hexane, to afford, following solvent extraction and purification, 1 -tert- butoxycarbonyl (2R, 45, 5R)-4,5-0-isopropylidene-3-methylene-2-pentyl-piperidine~4,5 -diol (9);

10) reaction of 9 in anhydrous tetrahydrofuran with 9-borabicyclo[3.3.1 jnonane to afford, following solvent extraction and purification, 1 -teri-butoxycarbonyl (2R, 3R, 45, 5 ?)-3- hydroxymethyl-4,5-0-isopropylidene-2-pentylpiperidine-4,5-di ol (10); and 1 1) reaction of 10 in tetrahyclrofuran with aqueous solution of HCl to afford, following evaporation, solubilization in water and lyophilization, (2R, 3 R, 45, 5R)-3-hydroxymethyl-2- pentylpiperidine-4,5-diol hydrochloride salt (11).

EXAMPLES

Example l in hibition of wild type beta-galactosida _. se activity jn jiii|ii§p peripheral Mood mononuclear cell lysate by N-nonyl-epi-isofagoniine and 4-epi-isofagomine

Experimental Protocol

The Applicant has synthesized a compound of formula - n o n y 1 -4 - e i - i s o f ago m i n e wherein the nitrogen atom of 4-epi-isofagomine was derivatized with a nonyl chain.

4-epi-i so fagom i ne (3R, 45, 5/ -3-(hydroxymethyl)-piperidine-4,5-diol)

N-nonyl-4-epi-isofagomine

Thus, these compounds do not belong to the group of derivatives comprising 4-epi-isofagomine enantiomer derivatives of general formula (I).

Samples of human peripheral blood mononuclear cell lysate (2 x If) ⁶ cells per assay) were incubated with graded concentrations of compound N-nonyl -4-epi-i sofagomine or 4-epi- isofagomine in 50 mM sodium phosphate, pH 7.3, supplemented with 1% Triton X-100 and protease inhibitors, and 0,8 mM of 4-methylumbelliferyl β-D-galactopyranoside. After an incubation of 2 hours at 37°C, the enzymatic reactions were stopped by the addition of 0,15 M glycine, pH 10.2, and fluorescence was measured at 445 nm using an excitation wavelength of 365 nm. The stock solutions of compound were at 20 mM in DMSO, The results obtained with N-nonyl-4-epi-isofagomine (squares), 4-epi-isofagomine (triangles) and DM SO vehicle (circles) are presented on Figure 1.

Results

The N-nonyl-4-epi-isofagomine derivative that is not a 4-epi-isofagomine enantiomer derivative of general formula (I) is 140-fold less inhibitory on human beta-galactosidase than 4-epi- isofagomine (Fig. 1). In this experiment, the 1C50 of 4-epi-isofagomine is 0.07 μΜ and the 1C50 N-nonyl-4-epi-isofagomine is 10 μΜ.

Example 2: Syntlics I <>l ^' -epi-IsofagQ.iii¾ae en^ _. tjomer derivatives of general formula (I) Extensive medicinal chemistry efforts around the 4-epi-isofagomine scaffold resulted in the identification of (2R, 3R, 45, 5R)-3-hydrox>TOethyl-2-pentylpiperidine-4,5-diol as a promising compound.

The process for preparing and isolating a 4-epi-isofagomine derivative according to the invention starts from D-lyxose, and comprises the 11 steps described in details below.

Benzyl a-D-lvxopyrajioside .2

To a solution of D-lyxose (20.0 g, 0.13 mol) in benzyl alcohol (37 raL) was added p- toluenesulfonic acid (201 mg, 1.10 mmol, 0.008 eq). The reaction was left stirring for 78 h at 60°C. Then the mixture was cooled down to RT, concentrated under vacuum and co-evaporated with toluene (3 x 30 mL) to give a soft white solid. This residue was slurred into a mixture of hexane/CrhCh (2:1) (180 inL). The solid was filtered, then stirred with cold Et ₂ 0 (25 mL), filtered again and dried under vacuum. The filtrate was concentrated and the procedure was repeated three times to give the desired compound 2 (21.6 g, 69%) as a white solid. The obtained NMR-spectrum is consistent with literature data (Keck, G. E.; Kachensky, I). F.; Enholm, E. J. J Org Chem 1985, 50, 4317).

Benzyl 2,3-O-isopropylidene- -i lyxop 3

The reaction was conducted under Ar. To a solution of compound 2 (9.62 g, 0.04 mol) in acetone (128 HiL) were added dimethoxypropane (16.7 mL, 0.14 mol, 3.5 eq) and / oluencsullbnic acid

(150 mg, 0.8 mmol, 0.02 eq). The reaction was left stirring overnight at RT. The mixture was concentrated under vacuum and the residue was diluted with EtOAc (120 mL). The solution was washed with saturated aqueous NaHCCh (40 mL), water (2 x 40 mL) and dried over MgS€ ⁾ ₄ . Concentration under vacuum gave crude 3 as a colorless oil. Flash column chromatography of the crude product on silica gel (PE/EtOAc 85:15/5:5) afforded compound 3 (6.46 g, 57%) as a white solid. The obtained N R-spectrum is consistent with literature data (same ref. as 2).

Benzyl 2 _< 3-0-isopi:opylidjM 4

The reaction was conducted under Ar. To a solution of alcohol 3 (1.0 g, 3.57 mmol) in anhydrous CH2CI2 (36 mL) was added Dess-Martin periodinane (1.82 g, 4.28 mmol, 1.2 eq). The reaction was left stirring oveniiglit at RT. The solvent was then evaporated under vacuum. The residue was suspended in the minimal volume of cold Et ₂ 0 and the solids were removed by filtration through a membrane. The filtrate was concentrated under vacuum to give the crude product as a colorless oil with traces of a white solid. Rapid filtration of the product through silica gel (PE/EtOAc 8:2) gave the desired ketone 4 (0.98 g, 97%) as a colorless oil. The NMR spectrum of 4 is consistent with literature data (Anastasi, C; Buchet, F. F.; Crowe, M. A.;

Helliwell, M.; Raftery, J.; Sutherland, J. D. Chem. Eur. J. 2008, 14, 2375).

Benzyl 2,3-O-isopropylidene-4^C )(l -nito 5a and 5b

To a solution of ketone 4 (1.98 g, 7.1 1 mmol) in 1 -nitrohexane (5.5 mL) was added tri ethyl amine

(2.8 mL, 20.2 mmol, 2.8 eq). The mixture was left stirring for 4 d at RT. The reaction was quenched with saturated aqueous NH4CI (40 mL). The aqueous phase was separated and extracted with EtOAc (80 mL). The organic phase was then washed with brine (40 mL) and dried over gSCXi. Concentration of the organic phase under vacuum gave the crade product. Flash column chromatography on silica gel (PE/EtOAc, 92:8) gave an inseparable mixture of the two diastereoisomers at 4'-position in a 7:3 ratio, 5a and 5b (1.5 g, 55%), as a colorless oil. Numbering of compounds orj^MR data

VI (400 MHz, isomers labelled as MAJ and min, CDCI3), δ 7.47-4.28 (m, SH, H _AROM ), 5.05 (br s, lH, HI), 4.75 (d, 1H, CH ₂ Ph, J= 1 1.6 Hz), 4.60-4.51 (m, 1 H, H6), 4.54 (d, 1H, CH2PI1, J = 1 1.6 Hz), 4.26 (d, 0.7H, H3MAJ, J = 6.4 Hz), 4.17-4.09 (m, 1.3H, H2MAJ, H3 _min , H2 _mm ), 3.88 (d, 0.3H, HSbmir,, J = 12.4 Hz), 3.81 (d, 0.7H, H5 _B MAJ, J= 12.0 Hz), 3.73 (d, 0.7H, H5 _aM AJ, J= 12.0 Hz), 3.58 (d, 0.3H, H5 _ami „ ₃ J= 12.4 Hz), 3.04 (s, 0.7H, OHMAJ), 2.96 (s, 0.3H, OH _nii „), 2.16-1.95 (m, 1.3H, H7bMAJ, 2H7 _ram ), 1.72- 1.65 (m, 0.7H, H7 _aMA j), 1.55 (s, 0.9H, CH ₃ iPr _mm ), 1 .54 (s, 2.1H, CH ₃ iPr _MA j), 1 .37 (s, 0.9H, CH ₃ iPr _min ), 1.36 (s, 2. ΓΗ, CH ₃ iPr _M Aj), 1.30- 1.29 (m, 6H, CH ₂ - hexyl), 0.95-0.82 (m, 3H, CH ₃ -hexyl).

¹³ C NM R (100 MHz, CDCI3), δ 136.65 (C ^Iv _arom ), 128.69- 128.28 (CH _arom ), 1 10.24 (C ^Iv iPr _M Aj), 1 10.15 (C ^lv iPr _mi „), 96.60 (CI MAJ), 96.40 (Cl _min ), 92.71 (C6 _min ), 92.68 (C6MAJ), 74.35 (C2MAJ), 74.32 (C2 _mm ), 72.52 (C3 _iran ), 72.03 (C3 _MA J), 69.74 (CH ₂ Ph _M Aj), 69.66 (CH ₂ Ph _mi „), 69.49

(C4MAJ), 69.05 (C4 _min ), 61.26 (C5 _mm ), 60.79 (C5 _MA J), 31 .12, 28.02, 25.99, 22.41 (CH ₂ -hexyl _M Aj), 31.17, 27.23, 25.93, 22.45 (CH ₂ -hexyl _min ), 26.17, 25.36 (CHiiPr _M Aj), 26.13, 2536 (CH ₃ iPr _min ), 14.00 (CH ₃ -hexy n), fI ₃ -hexyl _M Aj).

MS (ESI) [M+NH ₄ ] ⁺ cald for C21H35NO7 m/z 427.243878; found m/z 427.243845; [M+Na] ⁺ cald for C2iH ₃ iNNa0 ₇ m/z 432.199273; found m/z 432.199176.

Benzyl 2,3-0-isopropylidene-r( 6a and 6b

To a solution of the mixture of isomers 5a and 5b (289 mg, 0.71 mmol) in isopropanol (7.1 mL) were added glacial acetic acid (0.71 mL) and Raney Ni (106 mg). The mixture was left stirring under H ₂ atmosphere for 5 days at RT. Then, the mixture was filtered through a membrane, the retained catalyst was washed with MeOH and the solvents were evaporated to give the crude product as a thick, green oil. Flash column chromatography on silica gel (CH ₂ Cl ₂ /MeOH 9:1 ) gave an inseparable mixture of epimers 6a and 6b (228 mg, 85%) as a slightly green, oil.

Ή NMR (400 MHz, isomers labelled as MAJ and min, CD3OD), δ 7.43-7.28 (m, 5H, H _arom ), 4.90 (d, 1H, HI, J _u = 3.3 Hz); 4.79 (d, IB, CH ₂ Ph, J= 1 1.90 Hz), 4.61 (d, 1H, CH ₂ Ph, J =

1 1.90 Hz), 4.36 (d, 1 H, H3, J ₃ j = 5.8 Hz), 4.13 (dd, 1H, H2, J _2J = 3.3 Hz, J ₂ = 5.8 Hz), 3.78 (d, 1H, H5 _b , J5 ^' b,5a = Γ2.0 Hz), 3.57 (d, ΓΗ, H5 _a , J _5a j _b = 12.0 Hz), 2,96 (dd, 0.7H, H6MAJ, J = 2.4 Hz, ,1= 9.6 Hz), 2.81-2.79 (m, 0.3H, H6 _min ), 1.694.26 (m, 14H, CH ₂ -hexyl, CH ₃ iPr), 0.95 (t, 3Η, CH ₃ -hexyl, J= 6.6 Hz).

1 ³ C NMR (100 MHz, CD3OD), δ 138.63 (C ^iV _arom ), 129.41 , 129.19, 128.90 (CH _arom ), 1 10.96 (C ^,v iPr _MA j), 1 10.63 (C ^lv iPr _min ), 99.73 (Cl _min ), 99.56 (CI MAJ), 76.33 (C2), 76.09 (C3), 71.20 (C4), 70.76 (CH ₂ Ph), 64.84 (C5 _min ), 63.52 (C5 _M AJ), 58.39 (C6 _min ), 57.59 (C6MAJ), 32.95, 30.66, 27.24, 23.60 (CH ₂ -hexyl _M Aj), 3 1.66, 27.48 (CH ₂ ~hexyl _mi n), 26.92 (CH ₃ iPr _m ,n), 26.84, 25.96 (CH ₃ iPr _M Aj), 14.39 (CH ₃ -hexyl).

HMMS (ESI) [M+H] ⁺ cald for C21H34NO5 m/z 380.243150; found m/z 380.243272. (2M/S _% 3£ 41?, 5R)-3-[-lydroxymetliyl-4.5-C>-ispprppy

7a and 7b

To a solution of the mixture of compounds 6a and 6b (367 nig, 0,968 mraol) in isopropanol (10 mL) were added glacial acetic acid (1 ml.) and the catalyst 20% Pd(OH)2 on C (150 mg). The mixture was left stirring under ¾ atmosphere for 2 d at RT. The mixture was then filtered through a membrane, the retained catalyst was washed with isopropanol and the filtrate was concentrated to give the cmde product as a clean mixture of epimers at C-6 (7:3), 7a and 7b (264 mg, quant.), as a colorless oil.

¾ NMR (600 MHz, isomers labelled as MAJ and min, CD ₃ OD), δ 4.55 (dt. 0.7H, H2MAJ, J2,/¾ = J ₂ , ia = 3.9 Hz, J ₂ = 12 Hz), 4.49-4.46 (m, 0.3H, H2 _min ), 4.29 (d, 0.7H, H3MAJ, J= 7.2 Hz), 4.21 (dd, 0.3H, H3 _m i„, J.u = 1 . Hz, Ju = 6.6 Hz), 3.72 (d, 0.3H, H6 _bm in, J= 1 1.4 Hz), 3.66 (d, 0.3H, H6amin, J = 1 1.4 Hz), 3.64 (d, 0.7H, H6 _B MAJ, J«»,& = 1 1.4 Hz), 3.61 (d, 0.7H, H6 _aMA j, J ₆ aM = 1 1 .4 Hz ), 3.51 (dd, 0.7H, HUMAJ, J 1,2 = 3.9 Hz, J _lh ,, _a = 13.5 Hz), 3.34 (dd, 0.3H, Hlbmin, J/,2 = 5.4 Hz, J _{! b} a = 13.2 Hz), 3.23 (dd, 0.7H, H5MA.F, J= 3.6 Hz, J= 13.2 Hz), 3.21 -3.1 8 (m, 0.3H, H5 _min ), 3. 1 8 (dd, 0.7Η, Hl _aMA j, Jia,2 = 3.9 Hz, Jj„,, _b = 13.5 Hz), 2.92 (dd, 0.3H. Hl _amin , Ji _a = 6.0 Hz, Jia,ib = 13.2 Hz), 1.93-1 .83 (m, 1H, CH ₂ -pcntyl), 1.76-1.60 (m, 2H, CH ₂ -pcntyl), 1.51 (s, 3H, C¾iP ), 1.404.27 (m, 5H, CH ₂ -pentyl ), 1.38 (s, 3H, CH ₃ iPr), 0.94 (t, 3H, CH ₃ -pentyl, J= 6.6 Hz).

¹³ C NMR (62.5 MHz, CD3OD):

MAJOR epimcr: δ 1 10.68 (C ^IY iPr), 75.20 (C3), 72.74 (C4), 70.79 (C2), 64.70 (C6), 54.83 (C5), 42.43 (CI ), 32.84, 28.41 , 26.53, 23.45 (CH ₂ -pcntyl), 27.08, 24.58 (CH ₃ iPr), 14.35 (CH ₃ -pentyl).

MINOR cpimer: δ 1 1 1.20 (C ^lv iPr), 75.52 (C3), 72.82 (C4), 70.82 (C2), 65.27 (C6), 60.01 (C5), 41.61 (CI), 32.67, 28.60, 27.40 (CH ₂ -pentyl), 27.18, 24.86 (CH ₃ iPr), 14.35 (CH ₃ -pentyl).

HRMS (ESI) [M+Hf cald for Ci4H28N0 ₄ m/z 274.201285; found m/z 274.201459.

SyjTthggjs of com pound 8 and epinier 13 Piperidine 7 (500 mg, 1.83 miiiol) as a mixture of epimers 7a and 7b was dissolved in EtOH (1 5 iiiL). The solution was cooled down to 0°C, N,N-diisopropyle1hylamine (956 μΐ,, 5.49 mmol, 3 eq) and benzyl cMoroforaiate (392 μΐ,, 2.75 mmol, 1.5 eq) were added. The mixture was stirred for 30 mill at 0°C and t RT. The solvent was removed under vacuum and the residue was purified by flash column chromatography on silica gel (toluene/acetone 7/1) to give the separated epimers, major compound 13 (228 mg, 31 %) and epimer 8 (132 mg, 18%) as mixtures of retainers (65:35). fgg, gj ^

l t PlBgMine-3 ,4.5-triol. 8

MM (400 MHz, rotamers labelled as A J and min, CDCh), δ 7.44-7.22 (m, 5H, H _arom ), 5.25-5.03 (m, 2H, CH2PI1), 4.40-4.23 (m, 2H, Hlbmin, H2, H5MAJ) _» 4.22 -4.16 (m, 0.35H, H5 _min ), 4.12 (dd, 0.65H, HlbMAJ, Jib = 7.1 Hz, Ji _{h a} = 13.8 Hz), 4.04-3.97 (m, 1H, H3), 3.83-3.64 (m, 2H, H6), 3.22 (s, 0.65H, OHMAJ), 2.81 -2.58 (m, 1.65H, Hl _a , OHMAJ), 2.58 (s, 0.35H, OH _min ), 2.17 (br s, 0.35H, OH _MI „), 2.10-1.93 (m, 1H, H7 _B ), 1.47 (s, 3H, CH ₃ iPr), 1.31 (s, 3H, CH ₃ iPr), 1.41 -1.02 (m, 7H, 3CH2-pentyl, H7 _a ), 0.95-0.78 (m, 3H, CH ₃ -pentyl).

¹³ C NMR (100 MHz, CDCI3), δ 157.29 (COMAJ), 156.64 (CO _mi „), 136.67 (C ^LV _AROMMIN ), 136.56 (C ^Iv _aro mMAj), 129.10427.70 (CH _arom ), 110.04 (C ^lv iPr _min ), 109.95 (C ^lv iPr _MA j), 7634 (C3), 7431 (C4 _MA j), 73.57 (C4 _min ), 70.03 (C2MAJ), 69.85 (C2 _rain ), 67.70 (CH ₂ P1IMAJ), 67.62 (CH ₂ Ph _ni i _rl ),

65 SMAJ), 65.06 (C6 _MIN ), 57.45 (C5 _min ), 57.10 (C5MAJ), 40.27 (CI MAJ), 39.87 (Cl _min ), 31.59 (CH ₂ -pentylmin), 31.54 (CH2-pentyl _M Aj), 28.53, 26.20 (CH ₃ iPr _M Aj), 28.48, 26.27 (CH ₃ IP ^' r _mm ), 25.99-25. h-pentyl, C7), 22.63 (Ofc-pentyl), 14.14 (CH ₃ -pentyl).

|α|ΐ) = + 6.4 (20°C, c = 1.01, CHCI3).

HEMS (ESI) [M+H] ⁺ cald for C22H34NO6 m/z 408.238064; found m/z 408.237733; [M+Na] ⁺ cald for CaiHssNNaOe m/z 430.220008; found m/z 430.220916.

pentylpiperidine-3,4,5-triol 13

Ή NMR (400 MHz, retainers labelled as MAJ and mln, CDCb), δ 7.42-7.22 (m, 5H, H _AROM ),

5.21-5.04 (m, 2H, CH ₂ Ph), 4.40-4.25 (m, 2H, 1 12, H3), 4.25-4.09 (m, IH, Hl _b ), 4.09-4.00 (m, 0.65H, H5MAJ), 3.97-3.87 (m, 0.35H, H5 _min ), 3.78 (br d, IH, H6 _b , J<sb,6a = 1 1.2 Hz), 3.57-3.44 (m, IH, H6a), 3.37-3.18 (m, I H, Hl _a ), 2.85-2.68 (m, IH, OH), 2.58 (br s, 0.65H, OHMAJ), 2.41 (br s, 0.35H, ΟΗ,,,ίπ), 1 .69- 1 . 1 1 (m, 14H, 3CH?-pcntyl, H7, 2CH ₃ iPr) ₅ 0.97-0.76 (m, 31 1, Cf fc-pentyl).

¹³ C NMR (100 MHz, CDCI3), 6 156.92 (COMAJ), 156.58 (CO _min ), 138.00 136.92 129.17-127.90 (CH _arom ), 109.1 1 (C ^Iv iPr _min ) ₅ 109.00 (C ^lv iPr _M Aj), 75.47 (C2 _MI!! ), 75.27 (C2MAJ), 73.46 (C3 _MIN ), 73.15 (C3MAJ), 72.84 (C4MAJ), 72.42 (C4 _min ), 67.58 (CH ₂ Ph _mm ), 67.24 (CH ₂ Ph _M Aj), 65.59 (C6 _min ), 65.22 (C6MAJ), 54.38 (C5 _min ), 54.06 (C5MAJ), 41.90 (Cl _min ), 41.80 (CIMAJ), 32.13, 26.08 (CH ₂ -pentylMAj), 28.76, 26.24 (CH ₂ -pcntyl _n;i!l ), 28.09 (C7), 26.01

(CHsiPrMAj), 25.88 (CH ₃ iPr _min ), 24.53 (CH ₃ iPr), 22.72 (CH ₂ -pentyl), 14.19 (CHs-peiityl).

[α|ι> = + 52.2 (20°C, c = 1.38, MeOH).

HUMS (ESI) [M+H] ⁺ cald for C22H34NO6 m/z 408.238064; found m/z 408.237695; [M+Na] ⁺ cald for C ₂₂ H ₃ 3NNa0 ₆ m/z 430.220008; found m/z 430.220420.

(2g, 3S, 4^^

Bgll y|piperit!|ne-3,4,5-triol ,3^†hiocarbonate

derivative 9

Diol 8 (78 mg, 0.1 1 mmol) was dissolved in anhydrous THF (2 mL) under argon.

Thiocarbonyldiimidazole was adc nig, 0.288 mmol, 1.5 eq). The mixture was stirred under reflux until disappearance of the starting material on TLC (10 h), then cooled down to RT. The solvent was removed under vacuum and the residue was purified by flash column chromatography on silica gel (PE/EtOAc 7,5/1) to give 9 (74 mg, 86%) as a mixture of rotamers (6:4).

Ή NMR (400 MHz, rotamers labelled as MAJ and mm, CDCb), d 7.46-7.28 (m, 5H, H _arom ), 5.25-5.07 (m, 2H, CH2PI1), 4.85-4.72 (m, 1H, H6 _b ), 4.66-4.54 (m, 0.4H, H5 _min ), 4.50-4.32 (m, 3.2H, H6 _A , H3, H5MAJ, HlbMAj), 4.24-4.14 (m, 1.4H, H2, Hlhmin), 2.94-2.82 (m, 0.4H, Hl _amin ), 2.75 (t, 0.6H, Hl aMAJ, Jia.ib = Jiaj = 1 1.2 Hz), 2.074.91 (m, 1H, H7 _b ), 1.51 (s, 3H, CH ₃ iPr), 1.36 (s, 3H, CH ₃ iPr), 1 .404.08 (m, 7H, 3CH ₂ -pentyl, H7 _a ), 0.95-0.77 (m, 311, CHj-pcntyl).

1 ³ C NMR (100 MHz, CDCb), δ 189.92 (CS _n ii„), 189.78 (CSMAJ), 155.52 (COMAJ), 155.47 (CO _mi „), 136.31 (C ^iv _arommi „), 136.20 (C ^Iv _aron ,MAj), 128.70427.92 (CH _mm ), 111.12 (C¾), 87.71 (C4 _MIN ), 87.52 (C4MAJ) ₅ 74.60 (C2MAJ), 74.35 (C2 _rain ), 72.95 (C6), 69.83 (C3mi„), 69.55 (C3MAJ), 68.08 (CHbPhMAj), 67.89 (CH ₂ Pl½m), 55.62 (C5 _M AJ), 55.17 (C5 _min ), 39.73 (Cl _min ), 39.07 (CIMAJ), 31.45, 26.32 (CH ₂ -pentyl _mi „), 31.32, 25.45 (CH ₂ -pentyl _M Aj), 28.01 (CH ₃ iPr _M Aj), 27.81 (CH ₃ iPr _min ), 26.14 (ClfciPr), 25.96 (C7), 22.48 (CH ₂ -pcntyl), 14.03 (CH ₃ -pcntyl).

HHMS (ESI) [M+Hf cald for C23H32NO6S m/z 450.194485; found m/z 450.194331 ; [M+NH ₄ ] ⁺ cald for C23H35N2O6S m/z 467.221034; found m/z 467.221007; [M+Na cald for C ₂₃ H ₃ iNNa06S m/z 472.176429; found m/z 472.176275.

(2R. 3R. 4S. 5j? l-B ¾zyloxycarbonyl-3-hvdroxymethyl^ -0-isopropylidene-2- pentylpiperidine-4.5-diol 10

A solution of cyclic thioearbonate 9 (72 mg, 0.160 miiiol) and Bi¾SnH (86 μί, 0.320 mmol, 2 eq) was prepared in anhydrous and degassed toluene (3 mL). The solution was degassed 3 times by alternating vacuum and Ar bubbling. A solution of AIBN (3.2 mg, 0.019 mmol, 0.12 eq) in anhydrous and degassed toluene (4 mL) was then prepared. The solution was also degassed 3 times. Half of the AIBN solution was added to the solution of 9, the mixture was stirred for 2 hours at RT, and then the second half of the solution was added. The mixture was stirred for 16 h under reflux. After cooling down to RT, the reaction mixture was washed twice with 10% aqueous KF (10 mL) and saturated aqueous NaHC0 ₃ (10 mL). The aqueous phases were collected and extracted 3 times with EtOAc (30 mL). The organic phases were combined and dried over MgS04. The solvent was removed under vacuum and the residue was purified by flash column chromatography on silica gel (PE/EtOAc 7/3) to give compound 10 (38 mg, 60%) as a colorless syrup containing traces of impurities from Bu ₃ SnH and as a mixture of rotamers

(1 : 1).

¾ N.MR (400 MHz, CDCI3), δ 7.43-7.20 (m, 511, H _aro m), 5.20 (d, 0.5H, CH ₂ Ph, J = 12.4 Hz),

5.12 (s, 1H, CH2PI1), 5.05 (d, 0.5H, CH2PI1, J = 12.4 Hz), 4.42-4.26 (m, 2H, H3, HS), 4.22 (dd, 0.5H, 0.5 ^' Hl b, Jib,2 = 7.2 Hz, Jib,ia = 13.6 Hz), 4.17-4.00 (m, 1.5H, H2, O.SHlb), 3.87-3.73 (m, 2H, 2H6), 2.82-2.64 (m, 1H, Hl _a ), 2.26-2.13 (m, 1H, H4), 2.034.81 (m, 2H, H7 _b , OH), 1.49 (s, 3H, CH ₃ iPr), 1.33 (s, 3H, CH ₃ iPr), 1.43-1.05 (m, 711, 3CH ₂ -pentyl. H7 _a ), 0.90-0.78 (m, 3H, CH ₃ - pentyl).

¹³ C NMR (100 MHz, CDCI3), δ 155.76, 1 55.48 (CO), 136.78, 136 'aiom), 128.61-127.76

(CHarom), 109.54 (C ^lv iPr), 73.41 , 73.31 (C3), 71.22, 70.98 (C2), 67.45, 67.31 (CH ₂ Ph), 61.82, 61.75 (C6), 51.17, 50.99 (C5), 42.24, 41.86 (C4), 39.84, 39.49 (CI), 31.70, 25.90, 25.81 , 22.66 (CPl2-pentyi), 28.77, 26.29, 26.24 (CH ₃ iPr), 26.93, 26.78 (C7), 14.15, 14.14 (CH ₃ -pentyl).

HRMS (ESI) [M+H] ⁺ cald for C22H34NO5 m/z 392.243150; found m/z 392.2431 60; [M+Na] ⁺ cald for C22H ₃ 3NNaOs m/z 414.225094; found m/z 414.224975.

The N-protected compound 10 (38 mg, 0.097 mmol) was dissolved in isopropanol (6 mL). 10%

Pd on C (80 mg) was added. The reaction was stirred at RT under 1 12 atmosphere until completion and the mixture was filtered through a membrane. The retained catalyst was washed with MeOH and the filtrate was concentrated under high vacuum to give the deprotected compound 11 (24 mg, 96%).

' I I NMR (250 MHz, CD3OD), 5 4.38 (dd, 1H, H3, J ₃ , ₄ = 3.3 Hz, J ₃ , ₂ = 5.8 Hz), 4. 14 (dt, 1H, H2, J2.3 = J2,i = 5.8, J2,ia = 7-0 Hz), 3.68 (d, 2H, 2H6, J ₆ ,4 = 7.5 Hz), 2.99 (ddd, 1 H, H5, J _{5i 7} = 2.8 Hz, Js,4 = 6.3 Hz, Jj = 9.2 Hz), 2.79 (dd, 1 H, H l _b , J/6,2 = 5.8 Hz, J, _b ja = 13.0 Hz), 2.68 (dd, 1 H, HI Jia,2 = 7.0 Hz, Jiajb = 13.0 Hz), 2.1 6 (ddt, I H, H4, J ₄ j = 3.3 Hz, J ₄ j = 6.3 Hz, J ₄ ,e = 7.5 Hz), 1.924.74 (ni, 1 H, H7 _b ), 1.46 (s, 31 i, CH ₃ iPr), 1.31 (s, 3H, CH ₃ iPr), 1.56- 1.14 (m, 7H, 3CH ₂ - pentyl, H7 _a ), 0.97-0.87 (m, 3H, CHs^pentyl).

¹³ C NME (100 MHz, CD ₃ OD), δ 109.81 (C ^Iv iPr), 73.90 (C3), T. 2), 61.76 (C6), 52.70 (C5), 42.84 (CI), 42.46 (C4), 33.09, 27.46, 23.62 (CH ₂ ~pentyl), 29.26 (C7), 28.06, 25.45 (CH ₃ iPr), 14.40 (CH ₃ -pentyl).

[a]» = + 31.7 (20°C, c = \ , MeOH).

HEMS (ESI) [M+H] ⁺ cald for C14H28NO3 m/z 258.206370; found m/z 258.206607.

isofagornine 12

The protected compound 11 (23 mg, 0.089 mmol) was dissolved in a mixture of dioxane/HiO

(5/3; 8 111L) and Dowex ion-exchange resin (H ⁺ form, 50WX8, 1 mL) was added. The reaction was stirred for 18 fa at RT. The mixture of resin and solvents was poured into a column and washed with dioxane/tfcO (1/1 , 50 mL), then with H mL). Elution with aqueous 0.5 N

NH ₄ OH (80 mL) and evaporation of solvent under hig vacuum gave the expected pure compound 12 (16 mg, 84%).

VI R (250 MHz, CD3OD), δ 3.90 (dd, 1H, H6 _b , ,4 = 3-5 Hz, J¾ _6fl = 11.5 Hz), 3.87-3.84

(m, 1H, H3), 3.80 (dd, 1 J _6a ,4 = 4.5 Hz, J _6a ,6b = 1 1.5 Hz), 3.75^3,68 (m, 1H, H2), 3.00 (dd, 1H, Hlb, Jib,2 = 2.8 Hz, Ji _{b a} = 13.: L73 (d, 1H, Hl _a , J _{!a b} = 13.3 Hz), 2.68-2.64 (m, 1H,

H5), 1.86 (quint, 1H, H4, J4 ^' ,3 = Ju = = 4.3 Hz), 1.72-1.53 (111, 2H, 2H7), 1.49-1.26 (m, 6H,

CH ₂ -pentyl), 0.97-0.87 (m, 3H, CH ₃ ).

^I3 C NMR (100 MHz, CD3OD), δ 72.04 (C3), 68.92 (C2), 58.63 (C5), 58.15 (C6), 50.76 (CI), 44.08 (C4), 33.1 1 , 27.39, 23.6: pentyl), 32.96 (C7), 14.39 (CH ₃ ).

|a|» = - 9.5 (20°C, c = 1 , McOH).

HUMS (ESI) [M+H] ⁺ cald for C1 1H24NO3 m/z 218.175070; found m/z 218.175272. Example 3: Inhibition of J in human peripheral blood n.on nu ear ceil lysate .by 4-epi-isofagomine eiiaiit{omcrj.l.crivative.s of general formula (t), an c¾ 4~epi-i$cifa.goiyiine

4-epi - i sofagomi n e enantiomcr derivatives of general formula (I) were synthesized according to the protocol disclosed on Example 2. Two isomeric forms were synthesized, one with a pentyl group in the configuration, (2R, 3R, 4S, 5R)-3-liydroxyiiiethyl-2-peiitylpiperiiiine-4,5~dioI, and another one with a pentyl group in the S configuration, (2S, 3R, 4S, 5R)-3-hydroxymethyl-2- penty]piperidinc-4,5- diol. Both 4-epi-isofagomine enantiomcr derivatives of general formula (I) were tested for their beta-galactosidase inhibitory activity,

Experimental Protocol

Samples of human peripheral blood mononuclear cell lysate (2 x 10 ⁶ cells per assay) were incubated with graded concentrations of compound in 25 mM sodium phosphate, pH 7.3, supplemented with. 0.5% Triton X-100 and protease inhibitors, and 0.8 mM of 4- m eth y 1 umbel 1 i feryl β-D-galactopyranoside. After an. incubation of 2 hours at 37°C, the enzymatic reactions were stopped by the addition of 0, 15 M glycine, pH 10.2, and fluorescence was measured at 445 nm using an excitation wavelength of 365 run. The relative fluorescence corresponding to (2R, 3R, 4S, 5R)-3-hydrox Tnethyl-2-pentylpiperidine-4,5-diol, (squares), (2S, 3R, 4S, 5 )-3-hydH)xyrnethyl-2-pentylpiperidine-4,5-diol (circles) and 4-epi-isofagomine (triangles) is shown on Figure 2.

Results

The 4-epi-isofagomine enantiomer derivative of general formula (I), (2R, 3R, 4S, 5R)-3- hydroxymethyl-2-pentylpiperidine-4,5-diol is a strong inhibitor of human beta-galactosidase activity, while the S isomer, (2S, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidinc-4,5-diol. is

150-fold less potent. This latter is however still a potent inhibitor of human beta-galactosidase activity, as well as the compound 4-epi-isofagomine (Fig. 2).

IC50 of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol, (2S, 3R, 4S, 5R)-3- hydroxymethyl-2 -pentyl piperidine-4, 5 -diol , and 4-epi-isofagomine are 0.01 μΜ, 1.5 μΜ and 0.8 μΜ, respectively. Example 4: (2R, ML, 4S, IQ - ftydroxymeth is a potent and specific inhibitor of wild type human beta-galactosidase activity

Experimental Protocol

Human peripheral blood mononuclear cell lysate was used as a source of various human glycosidases. The activities of the different enzymes were revealed with a corresponding coiorimetric or fliiorogeiiic substrate. IC50 values indicated in Table 1 are the concentrations of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol compound required to inhibit by half the different enzymatic activities.

Table 1 :

Results

The 4-epi-isofagomine enantiomer derivative of general formula (I), (2R, 3R, 4S, 5 )-3- hydroxymethyl-2-pentylpiperidine~4,5~diol is a strong beta-galactosidase inhibitor (IC50 = 20 iiM; Table 1). The specificity of said compound for other closely related glycosidases was evaluated and revealed that it weakly inhibits the activity of human beta-glucosidase and alpha- galactosidase (1C50 = 60 and 150 μΜ, respectively; Table 1 ) and any of the other four human glycosidases tested (IC50 > 160 μΜ; Table 1). These results indicated that (2R, 311, 4S, 5R)-3- hydroxvmethyl-2-pentylpiperidine-4,5-diol is highly specific for human beta-galactosidase. The exquisite specificity of said compound for beta-galactosidase would thus limit the in vivo side effects due to the blockade of other glycosidases.

Example 5: (2R, 3R, 4S, S ^ prevents heat denaturation of human beta-galactosidase The capacity of a compound of protecting an enzyme from denaturation predicts to some extent its pharmacological chaperone activity, i.e. its capacity of restoring enzymatic activity in patient cells exposed to the compound. The Applicant has evaluated whether (2R, 3R, 4S, 5R)-3- hydrox\Tncthyl-2-pentylpipcridine-4,5-diol can prevent heat denaturation of human beta- galactosidase following a procedure described by Suzuki et al 2014.

Experimental _. Protocol

Samples of human peripheral blood mononuclear cell lysate (2 x 10 ⁶ cells) in phosphate buffer, pH 7.3, 0.5% Triton X- 100 and protease inhibitors were incubated at 48°C for 0, 5, 10, 20, and 40 minutes in the presence of graded concentrations of (2R, 3R, 45, 5R)-3-hydroxymethyl-2- pentylpiperidine-4,5-diol. The samples were then diluted in citrate buffer, pH 4.3, and 0.25 mM of 4 - methy 1 umb el 1 i fer y 1 β-D-galactopyranoside. After an incubation of 17 hours at 37°C, the reaction was stopped by the addition of glycine 0.15 M, pH 10.2. Fluorescence was determined at 445 mm using an excitation wavelength at 365 nm. The tested concentrations of (2R, 3R, 4S, 5R)-3diydroxymethyl-2-pentylpiperidine-4.5-diol, are 20 μΜ (square), 2 μΜ (triangle), 0.2 μΜ (diamond), 0.02 μΜ (circle) and 0 pM (line).

Results

Beta-galactosidase activity in peripheral blood mononuclear cell lysate treated at 48°C in absence of compound was reduced by 50% in 3 minutes (Fig. 3). Addition of increasing concentrations of (2R. 3R, 4S, 5R)-3-hydroxytncthyl-2-pentylpiperidine-4.5-diol progressively reduced the loss of beta-galactosidase activity. In the presence of 0.02, 0.2, 2 and 20 uM of said compound, loss of 50% of the beta-galactosidase activity was attained in 10, 25 and >40 minutes, respectively (Table 2 and Fig. 3).

Table 2:

20 100 93 95 85 87

Upon treatment of peripheral blood mononuclear cell lysate at 48°C for 10 minutes, 84% and 95% of beta-gal actosidase activity was spared by the presence of 2μΜ and 20 μΜ of (2R, 3R, 4S, 5R)-3-hydrox\ ncthyl-2-pentylpiperidine-4,5--dio], respectively. These results indicate that this compound prevents heat dcnaturation of beta-galactosidase activity. These results, by showing that (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-penty]piperidine-4,5-diol prevents heat denaturation of human beta-galactosidase, predict the compound is endowed with beta-galactosidase chaperone activity.

Example 6: (2R, 3R^ 4S., 5R -3 _: ~^

increase of mutated beta-galactosidase activity in fibroblasts of _. GMl-gangliosidosis patients

(2R, 3R, 4S, 5R)-3-hydrox nethyl-2-pcntylpiperidine-4,5-diol was tested for its capacity to increase mutated beta-galactosidase activity levels in fibroblasts from Morquio disease type B and GM1 -gangliosidosis patients.

Experimental Protoco I

G I 02439 and GM05335 fibroblasts (Coricll Institute) were cultured for 4 days in the presence or the absence of 2 μΜ of (2R, 3R, 4S, 5R)-3-hydrox>Tnethyl-2-pentylpipcridine-4,5-diol. Cells were lysed in 100 m.Vl sodium citrate, pH 4.8, supplemented with 1 % Triton X-100 and protease inhibitors. Beta-galactosidase and bcta-hexosaminidase activities were then determined in the cell lysates using fluorescence and colorimetric assays, respectively. Results presented are means of 4 values obtained from independent cultures. Percent of deviation of each value is <30% from, the mean. Hatched and empty bars: beta-galactosidase and beta-hexosaminidase activities, respectively.

Results

This treatment results in an increase of mutated beta-galactosidase activity of 6.5- and 1 -fold in the juvenile GM1 -gangliosidosis cell line GM02439 and the infantile GM1 -gangliosidosis cell line GMGST^, respectively (Fig. 4). In contrast, there is no increase of the beta-hexosaminidase activity at all.

Example 7: Specificity of the 4-epi-isofagpmine core structure and (2R, 3R, 4S, 5R)-3- The 4-epi-isofagomine and isofagomine differ only by the stereochemistry of the hydroxyl group at the C4 position, an S configuration in 4-epi-isofagomine and an R configuration in

isofagomine.

Experimental Protocol

Samples of human peripheral blood mononuclear cell lysate (equivalent of 2 x 10 ⁶ cells/assay) in beta-galactosidase buffer (50 mM sodium phosphate pH 7.3, supplemented with 1% Triton X- 100 and protease inhibitors) with 4-in ethyl umbel 1 i fery 1 [1- D-gal actopyr an o i de at 0.8 mM or in beta-glucosidase buffer (50 mM sodium citrate pH 6.0, supplemented with 1% Triton X-100, 10 mM taurocholie acid, 5 mM 2-mereaptoethanol and protease inhibitors) with 4- methyl umbel hfery I (5 - D - gl u co r an o s i d at 1 mM were incubated at 37°C in the presence of vehicule or of graded concentrations of compound 4-epi-isofagomine, isofagomine, or (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpipaidine-4,5-diol.

After 1 hour (beta-galactosidase) or 2 hours (beta-glucosidase), the enzymatic reactions were stopped by the addition of 0.4 M sodium carbonate pH 11.6, and fluorescence was measured at 445 mil using 365 ran as the excitation wavelength. The stock solutions of the compounds were at 20 mM in water. The compounds were as follows: 4-epi-isofagomine (squares), (2R, 3R, 4S, 5R)-3-hydroxyniethvT2-pentylpiperidine-4,5-diol (diamonds), isofagomine (triangles) and vehicle (circles).

Results

As illustrated in Fig. 5A and 5B, 4-epi-isofagomine and its (2R, 3R, 4S, 5R)-3-hydroxyrnethyl-2- pentylpiperidine-4,5-diol derivative are potent inhibitors of human lysosomal beta-galactosidase

(Fig. 5A; IC50 = 0.5 and 0.01 μ ^' Μ, respectively), while they are weakly active on human beta- glucosidase (Fig. 5B; IC50 > 800 μΜ for both compounds). By contrast, isofagomine is devoid of activity on the beta-galactosidase (IC50 > 800 μΜ), while it is a moderately active beta- glucosidase inhibitor (10 μΜ). Altogether, these observations unambiguously establish that 4- epi-isofagornine and its (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol derivative are highly specific for human beta-galactosidase, while isofagomine shows a distinct glycosidase preference. Example 8: Activities of (2R, 3R, 4S, 5R)-3-hycli : xyniethyl-2-niethylpipeiid

OR, 3R, 4S, SRVa-hydroxymethyl- -nonyipi eridine-^-diol and (2R. 3R, 4S. 5R)-3- hydro ymethyl-2-ethy _. lphcnyl^ _^ ^ s ⁷ R. m. 4 s , ^* >R )-3 hyclroxyiMethyl-

2-pentylpiper.duie-4,5-diol Experimental Pmtocol

(2R, 3R, 4S, 5R)-3-hydroxynicthyl-2-mctliy!piperidinc-4,5-diol and (2R, 3R, 4S, 5R)-3- hydroxyiiiethyl-2-nonylpipcridine-4,5-diol were synthesized according to Synthesis 2 (Example 16) and (2R, 3R, 4S, 5R)-3-hydroxymctl!yl-2-phenylethylpipcridinc-4,5-diol was synthesized according to Synthesis 1. These compounds were then characterized.

Results

Comparison of the characteristics of the four compounds reported in the table below indicates that the nonyl derivative of 4-epi-isofagomine has a potent beta-galactosidase inhibitory activity (IC50 = 0.001 μΜ). However, it lacks specificity for beta-galactosidase (e.g. beta-glueosidasc inhibitory activity) and does not have chaperone activity at high concentrations.

The methyl derivative was found to be 10-fold less potent than the pentyl derivative, as a beta- galactosidase inhibitor.

The 2-phenylethyl derivative was found to be similarly potent to the pentyl derivative, as a beta- galactosidase inhibitor. These results confirm that 4-epi-isofagomine of formula (I) according to the present invention such as pentyl derivative or 2-phenylethyl derivative of 4-epi-iso fagomine of formula (I) constitute optimal compounds, as a pharmacological chaperone-based drug candidates, for the treatment of GM1 -gangliosidosis, Morquio disease type B and other diseases controlled by enzymes specific for structures containing galactose-derived moieties or by enzymes associated with beta-galactosidase.

Table 3 :

IC50: dose of compound required to reduce by half the maximal enzymatic activity

^2/ ED50: dose of compound eliciting half-maximal enhancement of beta-galactosidase activity in compound-treated cells from a GMl -gangliosidosis patient, as compared with beta-galactosidase activity measured in untreated cells ^3/ Not applicable due to a bell-shape response with maximal enhancement of beta-galactosida.se activity in compound-treated cells from a GMl -gangliosidosis patient obtained with 0.01-0.1 uM of compound, and decrease of half-maximal enhancement of beta-galactosidase activity attained with 1 μΜ of compound.

Example 9: Intravenous administration of (2R. 3R, 4S, 5R)-3-liydroxymethyi-2- pentv¾pipe>- iiie-4,5-diol in mice

Experimental Protocol

Male C57BL6/J mice were intravenously administered three times one hour apart with (2R, 3R, 4S, 5R)-3-hydroxymethyI-2-pentyipiperidine-4,5-dicd in phosphate buffer saline at 50 mg per kg, and one hour following the last injection, animals were sacrificed. Compound was determined in plasma, brain and bones using a liquid ehromatography-coupled mass spectrometry method. Results

The compound was detected in plasma, brain and bones tissues, indicating that (2R, 31 , 45, 5R)~ 3-hydroxymethyl-2-pentylpiperidine-4,5-diol, following its intravenous administration, pass the blood brain barrier and reaches the organs undergoing severe damages in GMl -gangliosidosis and Morquio disease type B. This feature is a necessary requirement for the development of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol as a drug.

Example 10: Oral administration in mice of (2R, 3R. 4S, 5R)-3-hydroxymethyI-2- penty.piperidme-4,5-diol

.ElBilii l I to/ Protocol

Male C57BL6/J mice were orally administered with (2R, 3R, 4S, 5R)-3-hydroxymethyl-2- pentylpipcridine-4,5-diol in phosphate buffer saline at 50 mg per kg, and at different time points (0.25, 0.5, 1, 3, 7 and 24 hours) groups of animals were sacrificed. Compound was determined in plasma and brain using a liquid ehromatography-coupled mass spectrometry method.

Results The compound was detected in plasma, as well as in the brain tisssue, indicating that (2R, 3R, 45, 5R)-3-hydroxv-rnethyl-2-pentylpiperidine-4.5-diol is orally available, a desirable feature for a medicine. Example I i : ( l^ 3R, S early. ciegraclatioB of .mutated forms o newly synthesized beta-galactosidase polypeptide

Experimental Pro tocol

An appropriate anti-beta-galactosidase antibody was used with the Western blotting technique as a probe to detect the precursor and mature forms of beta-galactosidase.

Detection of the precursor and mature forms of beta-galactosidase by the Western blotting technique was done as essentially described in Rigat et al 2012 Mol Genet etab. Briefly, patient cells, treated or not for 5 days with (2R, 3R, 45, 5R)-3-hydroxymcthyl-2- pentylpiperidine-4,5-diol at 1-5 μ. , were lysed with 0, 1 % Triton X-100. The cell lysates were electrophoresed on 8% polyacrylamide gels and then blotted onto nitrocellulose membranes. The beta-galactosidase polypeptides were detected with the anti-beta-galactosidase antibody AF6464 (R&D Systems), followed by the anti-sheep IgG antibody HAF016 (R&D Systems). The membranes were finally revealed by ehemi luminescence. The precursor and mature forms of beta-galactosidase were detected as bands with apparent molecular weights of 84Ό00 and 64' 000, respectively. Detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using the anti-GAPDH antibody TAB 1001 (Thermo Fisher Scientific), was used to control that even quantities of each sample were loaded on. the gels.

Results

It appears that the beta-galactosidase mature form is not detected, or to low levels, in cells of GM1 -gangliosidosis and Morquio disease type B patients. This situation reflects the diminished beta-galactosidase activity in these cells, as compared to the levels detected in cells from healthy subjects.

Treatment of patient cells with (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol results in the appearance of the mature form of beta-galactosidase in two thirds of the patient cells, as detected by Western blotting.

These observations indicate that the compound has the ability of preventing early degradation of mutated forms of newly synthesized beta-galactosidase polypeptide in the endoplasmic reticulum, and of inducing transport and maturation of these mutated forms of the beta- galactosidase precursors to the lysosomes, as revealed in Western blotting by a beta- galactosidase form of smaller size. Example 12: ReclEetigf of kerataii siiifate ievel in GM 1 -gangliosidosis patient cells treated with (2R, 3R, 4S, 5RV3-hvdroxvniethvl-2-pentylpipcridine-4.5-diol

In the present experiment, it was investigated whether treatment of GM1 -gangliosidosis patient cells with (2R, 3R, 4S, 5R)-3-hydroxymethy!-2-pentylpipcridine-4,5-diol decreased the level of keratan sulfate, one of the substrates of the beta-galactosidase.

Ex rm ^' mt l Protocol

The dosage of keratan sulfate in patient cells, treated or not with (2R, 3R, 4S, 5R)-3- hydroxymethyl-2-pentylpiperidine-4,5-diol, was conducted as described in Oguma et al 2007 Anal Biochem. Briefly, cells from a GM1 -gangliosidosis patient were cultured for one week with or without (2R, 3R, 45, 5R)-3-hydroxymethyl-2-pentylpipcridinc-4,5-diol at 2 μΜ. At the end of the treatment, the cells were harvested, resuspended in acetate buffer, sonicated and digested with keratan as e II. The resulting samples were clarified and analysed by liquid chromatography- coupled mass spectrometry to detect keratan fragments.

Results

It was found that following this treatment, the keratan sulfate content was reduced by 40% as compared with cells cultured in the absence of compound. These results constitute a direct in vitro evidence of the therapeutic potential of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2- pentylpiperidine-4,5-diol for the treatment of GM1 -gangliosidosis and Morquio disease type B.

Example 13: Increased betargalacto dase activity induced 3R, 4S, 5.Ϊ

hydroxymethyl^ in cells from infantile, juvenile and adults

GM l-gangliosidosis, as well as _. Morquio disease type B patients

Expetimeni l Protocol Patient cells obtained from different institutions (Coriell Institute, Camden, NJ; University of Graz, Austria; SickKids Hospital, Toronto, Canada; Tottori University, Japan) were seeded (2 x 10 ⁴ cells) in wells of 24 well tissue culture plates in DMEM medium containing 10% foetal calf serum. The cells were treated with graded concentrations (0, 0.08, 0.4, 2 and 10 μΜ) of (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol for 5 days. Cells were then lysed in 50 mM sodium citrate pH 4.3 and 1 % Triton X-100. The activity of beta-galactosidase in the lysates was finally determined using a fluorogenic beta-galactosidase substrate.

Results Table 4 illustrates that (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol has the capacity of inducing the recovery of beta-galactosidase activity in cells derived from patients suffering from the various forms of GM1 -gangliosidosis and Morquio disease type B. These results indicate that (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-4,5-diol might constitute a potential treatment for many patients suffering from the different pathologies caused by mutations in the lysosomal beta-galactosidase gene.

Table 4:

/Fold enhancement is the ratio between the highest beta-galactosidase activity obtained over the range of compound concentrations tested (0 to 10 μΜ) and the beta-galactosidase activity in untreated cells

Exampit qmbinations of (2R, 3R, 4S, 5R)-3 jiy^

with other pharmacological age i » i

Experimental Protocol

GM1 -gangliosidosis cells (Zurich #4 fibroblasts, from a GM1 -gangliosidosis patient bearing the G76E and R201H mutations, from the Zurich University Children's Hospital) were seeded (2 x 10 ⁴ cells) in wells of 24 well tissue culture plates in DMEM medium containing 10% foetal calf serum. The cells were cultured for 5 days with either (2R, 3R, 4S, 5R)-3-hydroxymethyl-2- pentylpiperidine-4,5-diol at 0 or 0.2 μΜ, the proteasome inhibitor MG- 132 at 0, 0.07 or 0.2 μΜ, or a combination of these two substances. Cells were then lysed in 50 mM sodium citrate pH 4,3 and 1% Triton X- 100. The activity o f beta-gal acto i dase in the lysates was finally determined using a fluorogenic beta-galactosidase substrate.

Results Treatments with (2 , 3R, 4S, 5R)-3-hydroxymethyl-2-pcnty!piperidine-4,5-diol and with MG-132 used separately resulted in an increase in beta-galactosidase activity of 4.4- and 3.0-fold, respectively. Remarkably, when the agents were used in combination, the increase in beta- galactosidase activity was of 7.1 -fold. Similar results were obtained with the calcium channel blocker diltiazem. These results illustrate that the beta-galactosidase chaperone activity of (2R, 3R, 4S, 5R)-3- hydroxymethyl-2-pentylpiperidine-4,5-diol can be further enhanced by combining it with clinically approved drugs (e.g. Velcade, Cardiz m and Tiazac). These findings are of particular importance for the treatment of disease forms caused by mutations responding only weakly to (2R, 3R, 4S, 5R)-3-hydroxynaethyl-2-peiitylpiperidine-4,5-diol (e.g. some of the mutations associated with Morquio disease type B).

Example 15: Inhibition of fecoii.il3inai.it human.. gi;ilacttcerebrftsiflise by (|IL 3R, 4S, 5RV3- hydLroxymethyl^-pentylpiperid

Experimental Protocol Recombinant human galactocerebrosidase (from R&D Systems; 9 ng/assay) was incubated with 4-methylumbellifer galactopyranoside at 1 mM in 50 inM sodium phosphate pH 4.3, 125 mM NaCl and 0.5% Triton X-100 in the presence of graded concentrations of (2R, 3R, 4S, 5R)-3- hydroxymethyl-2-pcntylpiperidine-4,5-diol. After 2 hours at 37°C, an equal volume of 0.4 M sodium carbonate pH 1 1.6 was added to the enzymatic reaction samples and fluorescence was measured at 445 nm using an excitation wavelength of 365 nm.

Results

As shown in Fig. 6, it was observed that (2R, 3R, 4S, 5R)-3-hydroxymethyl-2-pentylpiperidine-

4,5-diol is a potent inhibitor of recombinant human galactocerebrosidase (1C50 = ^■ 0.04 μΜ), indicating that this compound might act as a pharmacological chaperone in rabbe disease. Example 16: _;:; ¾ i-isofagominc enantiomer derivatives of general formula (I)

The invention further relates to an alternative process for preparing and isolating 4-epi- isofagomine enantiomer derivatives of general formula (I), comprising the steps described in the scheme below starting from L-ribose;

An alternative process for preparing and isolating 4-epi-isofagomine enantiomer derivatives of general formula (I) comprises the steps of:

1) reaction of L-ribose with concentrated sulfuric acid in benzyl alcohol and acetone to afford 1-

0- benzyl-2,3-0-isopropylidene-L-ribofuranose (i);

2) reaction of 1 with Dess-Martiii reagent in anhydrous CH2CI2 to afford crude (2S, 3S, 4S, 5R) 2- benzyloxy-5-formyl-3,4-0-isopropylidene-tetrahydrofuran-3,4- diol (2);

3) direct modification of 2, due to its instability, with the Grignard reagent pentylmagnesium bromide in tetrahydrofuran to afford, following purification, (2S, 3S, 4S, 5S) 2-benzyloxy-5-( 1 S-

1- hydroxyhexyl)-3,4-0-isopropylidene-tetrahydrofuran~3,4-diol (3),

4) reaction of 3 in anhydrous CH2CI2 with methanesulfonyl chloride to afford, following purification, (2S, 3S, 4S, 5R) 2-benzyloxy-3,4-0-isopropylidene-5-(lR-l- methanesulfonyloxyhexyl)-tetrahydrofuran-3,4-diol (4); 5) reaction of 4 with sodium azide to afford, following solvent extraction, (2$, 3S, 4S, SS) 5-(lR- l -a/idohexyl)-2-bcnzyloxy-3,4-0-isopropylidci-ic-tetrahydrofu ran-3,4-diol (5);

6) reaction of 5 in anhydrous methanol with, palladium on carbon, followed by a catalytic amount of acetic acid, to afford a crude preparation of (2R, 3S, 4S, 5 )- 4,5-0-isopropylitlene-2-pcntyl- piperidine-3,4,5-triol (6),

7) reaction of 6 with di-/erf-buty] dicarbonate to afford, following purification, 1 -tert- butoxycarbonyl (2R, 3S, 4S, 5R)-4,5-0-isopropylidciie-2-pcnty]-piperidine-3,4,5-triol (7),

8) reaction of 7 in anhydrous CH2CI2 with Dess-Martin reagent to afford crude 1 -tert- butoxycarbonyl (2R, 4R, 5R)-4,5-0-isopropylidene-2-pcinyl-3-oxo-piperidine-4,5-diol (8), 9) reaction of erode compound 8, in anhydrous tetrahydrofuran, with the Wittig reagent, prepared by mixing methyltriphcnylphosphonium bromide in anhydrous tetrahydrofuran with n- butylithium in hexane, to afford, following solvent extraction and purification, 1 -tert- butoxycarbonyl (2R,4S,5R)-4,5-0-isopropylidene-3-methylene-2-perityhpiperid ine-4,5-diol (9),

10) reaction of 9 in anhydrous tetrahydrofuran with 9-borabicyclo[3.3.1 Jnonane to afford, following solvent extraction and purification, 1 -/m-butoxyearbonyl (2R, 3R, 45, 5/0-3- hydroxyTOcthyl-4,5- -isopropylidcnc-2-pentylpipcridinc-4,5-diol (10),

11) reaction of 10 in tetrahydrofuran with aqueous solution of HC1 to afford, following evaporation, solubilization in water and lyophilization, (2R, 3R, 45, 5/^)-3-hydroxymethyl-2- pcntylpipcridine-4,5-diol hydrochloride salt (11).

The advantages of this process are several:

1) It offers a better overall yield in the range of 12%, than the initial process,

2) It is substantially faster than the initial process,

3) It is cheaper than the initial process as it does not rely on. the use of nitrohexane, and

4) It is easier to conduct as it does not require the separation of diastereoisomers.