RELATED APPLICATIONS

[0001] This is a Patent Cooperation Treaty Application which claims the benefit of 35 USC 1 19 based on the priority of corresponding U.S. provisional application No. 61/563,681 filed November 25, 2011 , the contents of which are incorporated herein by reference in their entirety

FIELD

[0002] This application relates to compounds of the formula (I) which are pharmacological chaperones for the enzyme β-glucocerebrosidase and mutant forms thereof, and for the treatment of diseases in which β-glucocerebrosidase, or its mutant form, is implicated.

BACKGROUND

[0003] The presence of a mutation in one of the glucocerebrosidase genes is the single most common risk factor associated with Parkinsons disease (PD), accounting for 4% of all PD patients in the UK (Lees et al, 2009— A.J. Lees, J. Hardy, T. Revesz, Parkinson's disease Lancet 373 (2009) 2055-2066.). Gaucher patients with two mutant alleles (prevalence of 1/150,000) have a 13 fold increased chance of developing PD. The N370S missense mutation is the most common mutation associated with Gaucher disease. According to one model, the decreased levels of glucocerebrosidase (GCase) (either as a result of a mutant form of the enzyme, or simply reduced activity or levels of the wild type enzyme) activity in these patients results in increased levels of glucocerebroside, which in turn leads to increased levels of the neurotoxic form of a- synuclein, leading to Parkinson's disease. Lewy Body disease is also associated with increased levels of the neurotoxic form of a-synuclein and thus may also benefit from increasing the levels of GCase activity. An example of the benefits of increased GCase activity is that the overexpression of the WT GCase using an adenovirus construct, has been reported to decrease the levels of this neurotoxic form of a-synuclein, with a corresponding improvement in neurophysiological parameters in mouse models of PD.

SUMMARY

[0004] The present disclosure relates to pharmacological chaperones for the enzyme β-glucocerebrosidase, and for the treatment of medical disorders or diseases in which β-glucocerebrosidase (wild type or a mutant form), is implicated. In one embodiment, the compounds of the disclosure chaperone the β-glucocerebrosidase (wild type or a mutant form) to the lysosome through the endoplasmic reticulum.

[0005] Accordingly, the present disclosure includes compounds of the Formula (I), or pharmaceutically acceptable salts thereof:

wherein,

R ¹ and R ² are independently H, (C C ₂ o)-alkyl, or -R ^a -S-R ^b , wherein

R ^a is (CrCi ₂ )-alkylene, (C ₂ -Ci ₂ )-alkenylene, (C ₂ -Ci ₂ )-alkynylene, or (C ₃ - Cio)-cycloalkylene, each group optionally substituted one to five times with halo, OH, (C C ₆ )-alkyl or fluoro-substituted-(C C ₆ )-alkyl;

R ^b is H, (d-C^-alkyl, (C ₂ -Ci ₂ )-alkenyl, (C ₂ -Ci ₂ )-alkynyl,

(C ₃ -Cio)-cycloalkyl, (C Ci ₂ )-alkylene-(C ₆ -Cio)-aryl, (d-C^-alkylene-NH- C(=O)-R', (C Ci ₂ )-alkylene-NH-C(=O)-NH-R'R" or (C-,-Ci ₂ )-alkylene- C(=O)-N-R'R", wherein the latter eight groups are optionally substituted one to five times with halo, OH, (Ci-Ce)-alkyl or fluoro-substituted-(Ci-C6)- alkyl;

R' and R" are independently or simultaneously H, (CrC ₆ )-alkyl, (C ₆ -Ci ₀ )- aryl or (Ci-C ₆ )-alkylene-(C6-C ₁₀ )-aryl, or R' and R" are joined together to optionally form, including the nitrogen atom to which they are attached, a 5-10-membered heterocyclic ring, and wherein one of R ¹ or R ² is R ^a -S-R ^b ;

R ³ is H, (C C ₆ )-alkyl, (C ₂ -C ₆ )-alkenyl, (C ₂ -C ₆ )-alkynyl, or

(C3-C6)-cycloalkyl, the latter four groups optionally substituted one to five times with halo, OH, (CrC6)-alkyl or fluoro-substituted-(CrC ₆ )-alkyl; R ⁴ - R ⁶ are each simultaneously or independently H, OH or -OR ^c , wherein

R ^c is (C C ₆ )-alkyl, (C ₂ -C ₆ )-alkenyl, (C ₂ -C ₆ )-alkynyl, or

(C3-C6)-cycloalkyl, each group optionally substituted one to five times with halo, OH, (C C ₆ )-alkyl or fluoro-substituted-(C C ₆ )-alkyl,

and all of the stereoisomers, prodrugs or solvates thereof.

In one embodiment of the disclosure, the compound of the formula (I) is

[0007] The present disclosure also includes pharmaceutical compositions comprising a compound of the formula (I) and a pharmaceutically acceptable excipient. [0008] The present disclosure also includes the use of a therapeutically effective amount of a compound of the formula (I), or pharmaceutical composition comprising a compound of the formula (I), for the treatment of any disease in which β- glucocerebrosidase (wild type or a mutant form) is implicated. In one embodiment, the disease associated with β-glucocerebrosidase (wild type or a mutant form) is Parkinson's or Lewy body disease. In another embodiment, the disease is Gaucher disease, which include types I, II or III.

[0009] The present disclosure also includes a method for treating a patient with a disease in which β-glucocerebrosidase (wild type or a mutant form) is implicated, comprising administering a therapeutically active dose of a compound of the formula (I), or pharmaceutical composition comprising a compound of the formula (I). In one embodiment, the disease in which β-glucocerebrosidase (wild type or a mutant form) is implicated is Parkinson's disease, Lewy body disease or Gaucher disease.

[0010] Further aspects and advantages of the embodiments described herein will appear from the following description taken together with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

[0011] For a better understanding of the embodiments described herein and to show more clearly how they may be carried into effect, reference will now be made, by way of example only, to the accompanying drawing which shows at least one exemplary embodiment, and in which:

[00 2] Figure 1 is a graph showing the effects on GBA activity in fibroblasts of compounds of the formula (I) of the present disclosure;

[0013] Figure 2 is a graph showing the effects on GBA activity in fibroblasts of compounds of the formula (I) of the present disclosure;

[0014] Figure 3 is a graph showing the effects on GBA activity in fibroblasts from patients with Gaucher disease of compounds of the formula (I) of the present disclosure; [0015] Figure 4 is a graph demonstrating the correlation of AC50 values and K, values of compounds of the disclosure;

[0016] Figure 5 is a graph demonstrating the correlation between K, and AT _m of compounds of the present disclosure;

[0017] Figure 6 is a graph showing the effects on the GCase activity in mouse neurospheres of a compound of the formula (I) of the present disclosure; and

[0018] Figure 7 is a graph showing the effects on the GCase activity in wild type fibroblasts of a compound of the formula (I) of the present disclosure.

DETAILED DESCRIPTION a) Definitions

[0019] When describing the compounds, pharmaceutical compositions containing such compounds and methods of using such compounds and compositions, the following terms have the following meanings unless otherwise indicated.

[0020] The term "Ci _-n alkyl" as used herein means straight and/or branched chain, saturated alkyl radicals containing from one to "n" carbon atoms and includes (depending on the identity of n) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n- hexyl and the like, where the variable n is an integer representing the largest number of carbon atoms in the alkyl radical.

[0021] The term "C-2-nalkenyl" as used herein means straight and/or branched chain, unsaturated alkyl radicals containing at least one double bond and containing from two to "n" carbon atoms and includes (depending on the identity of n) ethenyl, propenyl, isopropenyl, n-butenyl, s-butenyl, isobutenyl, n-pentenyl, 2-methylpentenyl, n- hexenyl and the like, where the variable n is an integer representing the largest number of carbon atoms in the alkyl radical.

[0022] The term "C _2-n alkynyl" as used herein means straight and/or branched chain, unsaturated alkyl radicals containing at least one triple bond and containing from two to "n" carbon atoms and includes (depending on the identity of n) ethynyl, propynyl, n-butynyl, s-butynyl, n-pentynyl, 2-methylpentynyl, n-hexynyl and the like, where the variable n is an integer representing the largest number of carbon atoms in the alkyl radical.

[0023] The term "C _3- mCycloalkyl" as used herein means a monocyclic or bicyclic saturated or partially unsaturated carbocylic group containing from 3 to "m" carbon atoms and includes (depending on the identity of m) cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyi, cyciohexenyl, and the like, where the variable m is an integer representing the highest number of carbon atoms in the cycloalkyl radical.

[0024] The term "aryl" as used herein means a monocyclic or polycyclic aromatic ring system containing from 6 to 10 carbon atoms and at least one aromatic group and includes phenyl, naphthyl, anthracenyl, 1 ,2-dihydronaphthyl, 1 ,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl, ferrocenyl and the like.

[0025] The term "heterocyclic" as used herein means heterocyclic rings containing between 5 and 10 atoms together with 1 , 2 or 3 heteroatoms which are selected from nitrogen, oxygen, and sulfur atoms, and includes piperidinyl, piperazinyl, morpholinyl, pyrrolidinyl, and the like.

[0026] The term "halo" as used herein means halogen and includes chloro, fluoro, bromo, iodo and the like.

[0027] The suffix "ene" added on to any of the above groups means that the group is divalent, i.e. inserted between two other groups.

[0028] The term "fluoro-substituted" as used herein means that at least one, including all, of the hydrogens on the referenced group is replaced with fluorine.

[0029] The term "stereoisomer" as used herein means an isomer that possesses identical constitution as a corresponding stereoisomer, but which differs in the arrangement of its atoms in space from the corresponding stereoisomer. For example, stereoisomers may be enantiomers, diastereomers and/or cis-trans (E/Z) isomers. It should be understood that a composition comprising a compound of the Formula (I) may comprise single enantiomers, single diastereomers as well as mixtures thereof at any ratio (for example racemic mixtures, non-racemic mixtures, mixtures of at least two diastereomers and so forth). [0030] The term "solvate" as used herein means a pharmaceutically acceptable solvate form of a specified compound of the Formula (I) that retains the biological effectiveness of such compound, for example, resulting from a physical association of the compound with one or more solvent molecules. Examples of solvates, without limitation, include compounds of the invention in combination with water, 1-propanol, 2- propanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine. When the solvent is water, the form is known as a "hydrate".

[0031] The term "pharmaceutically acceptable salt" refers to salts that retain the biological effectiveness and properties of the compounds of the Formula (I) and which are not biologically or otherwise undesirable. In many cases, the disclosed compounds are capable of forming acid or base salts by virtue of the presence of acidic or basic moieties. The preparation of the salts and suitable acids or bases is known in the art.

[0032] The term "therapeutically effective amount" as used herein means a dosage to produce a selected effect. For example, an effective amount of a compound or composition of the disclosure is an amount which is sufficient for the amelioration or improvement of diseases in which the enzyme β-glucocerebrosidase (wild type or a mutant form) is implicated, or the symptoms thereof, the diseases including Parkinson's and Lewy body disease, and Gaucher disease.

[0033] The phrase "a disease in which the wild type or a mutant form of the enzyme β-glucocerebrosidase is implicated" as used herein refers to any disease or disorder which arises as a result of decreased levels of glucocerebrosidase activity, and consequently, increased levels of glucocerebroside in the endoplasmic reticulum and/or lysosome. In one embodiment, the decreased activity of glucocerebrosidase arises as a result of decreased levels of wild type glucocerebrosidase, or of the presence of the mutant form of glucocerebrosidase. Diseases which are associated with increased levels of glucocerebroside include, but are not limited to, Parkinson's disease, Lewy Body disease, Gaucher disease etc.

b) Compounds of the Formula (I)

[0034] The present disclosure includes compounds of the Formula (I) which are useful for the treatment of diseases in which decreased activity of the enzyme β- glucocerebrosidase is implicated. In one embodiment, decreased levels of activity of β-glucocerebrosidase include the variation in expression levels of the wild type enzyme in the general population, 100 ± 50%, and/or the presence of a single mutant GBA allele. In one embodiment, the compounds of the Formula (I) are pharmacological chaperone compounds for the enzyme β-glucocerebrosidase (wild type or a mutant form). Mutant forms of the β-glucocerebrosidase enzyme most often arise as a result of point mutations in the gene encoding the enzyme and include the p.N370S, or the L444P, missense mutations. These point mutations affect the folding of the enzyme, which results in increased retention of the mutant in the endoplasmic reticulum, and accordingly, decreased levels of the enzyme being transported to the lysosome. Decreased levels of β-glucocerebrosidase activity in patients (as a result of low levels or low activity of the wild type glucocerebrosidase or the presence of the mutant form) results in increased levels of glucocerebroside, which consequently leads to increased levels of the neurotoxic form of a-synuclein. In one embodiment, the compounds of the Formula (I) of the present disclosure bind to and stabilize the folded form of the enzyme (wild-type or mutant) that produces functional β-glucocerebrosidase (i.e. its functional- fold), which subsequently results in increased trafficking of the enzyme to the lysosomes. In one embodiment, the compounds of the Formula (I) bind to and stabilize the functional-fold of glucocerebrosidase (wild-type or mutant) increasing the percentage of newly synthesized enzymes that are able to obtain and retain their functional-fold. This results in an increase in the steady-state cellular activity of both wild-type and mutant enzymes.

[0035] Accordingly, the present disclosure includes compounds of the Formula (I) or a pharmaceutically acceptable salt thereof:

wherein,

wherein,

R ¹ and R ² are independently H, (C C ₂ o)-alkyl, or -R ^a -S-R ^b , wherein

R ^a is (CrCi ₂ )-alkylene, (C ₂ -C ₂ )-alkenylene, (C ₂ -C ₁₂ )-alkynylene, or (C ₃ - Cio)-cycloalkylene, each group optionally substituted one to five times with halo, OH, (C C ₆ )-alkyl or fluoro-substituted-(d-C ₆ )-alkyl;

R is H, (C C ₂ )-alkyl, (C ₂ -C ₁₂ )-alkenyl, (C ₂ -C ₁₂ )-alkynyl,

(C ₃ -Ci ₀ )-cycloalkyl, (C Ci ₂ )-alkylene-(C ₆ -Ci ₀ )-aryl, (Ci-C ₂ )-alkylene-NH- C(=0)-R', (C C ₁₂ )-alkylene-NH-C(=0)-NH-R'R" or (C C ₁₂ )-alkylene- C(=0)-N-R'R", wherein the latter eight groups are optionally substituted one to five times with halo, OH, (C-i-C ₆ )-alkyl or fluoro-substituted-(C C-6)- alkyl;

R' and R" are independently or simultaneously H, (C-i-C-6)-alkyl, (C6-C10)- aryl or (Ci-C6)-alkylene-(C ₆ -Ci ₀ )-aryl, or

R' and R" are joined together to optionally form, including the nitrogen atom to which they are attached, a 5-10-membered heterocyclic ring, and wherein one (optionally only one, or only one) of R or R ² is R ^a -S-R ^b ; R ³ is H, (C C ₆ )-alkyl, (C ₂ -C ₆ )-alkenyl, (C ₂ -C ₆ )-alkynyl, or

(C ₃ -C ₆ )-cycloalkyl, the latter four groups optionally substituted one to five times with halo, OH, (CrC6)-alkyl or fluoro-substituted-(CrC6)-alkyl; R ⁴ - R ⁶ are each simultaneously or independently H, OH or -OR ^c , wherein R° is (CrC ₆ )-alkyl, (C ₂ -C ₆ )-alkenyl, (C ₂ -C ₆ )-alkynyl, or

(C3-C6)-cycloalkyl, each group optionally substituted one to five times with halo, OH, (CrC ₆ )-alkyl or fluoro-substituted-(CrC ₆ )-alkyl,

and all of the stereoisomers, prodrugs or solvates thereof.

[0036] In one embodiment, R ^a is (C C ₆ )-alkylene, (C ₂ -C ₆ )-alkenylene, (C ₂ -C ₆ )- alkynylene, or (C ₃ -C6)-cycloalkylene. In another embodiment, R ^a is (C C ₃ )-alkylene, for example C ₂ -alkylene. [0037] In another embodiment of the disclosure, R ^b is (Ci-C ₈ )-alkyl, (C ₂ -C ₈ )- alkenyl, (C ₂ -C ₈ )-alkynyl, (C3-C ₆ )-cycloalkyl, (CrC6)-alkylene-phenyl, (CrC ₆ )-alkylene- NH-C(=0)-R', (CrC ₆ )-alkylene-NH-C(=0)-NH-R'R" or (CrC ₆ )-alkylene-C(=0)-N-R'R". In a further embodiment, R is (C-i-C ₈ )-alkyl, for example, ethyl, isopropyl, butyl, t-butyl, iso-pentyl, hexyl, or octyl. In another embodiment, R ^b is (C3-C6)-cycloalkyl, for example, cyclopentyl or cyclohexyl. In another embodiment, R ^b is (CrC ₆ )-alkylene-phenyl, for example, -CH2-phenyl or -CH2-CH2-phenyl. In another embodiment, R ^b is (C ₁ -C3)- alkylene-NH-C(=0)-R', for example -CH ₂ -CH ₂ - NH-C(=0)-R'. In another embodiment, R is (CrC ₃ )-alkylene-NH-C(=0)-NR'R", for example, -CH ₂ -CH ₂ -NH-C(=0)-NH-R'R". In another embodiment, R is (CrC ₃ )-alkylene-C(=0)-N-R'R", for example, -CH ₂ -C(=0)-N- R'R", -CH ₂ -CH ₂ -C(=0)-N-R'R", -CH ₂ -CH ₂ -CH ₂ -C(=0)-N-R'R". In another embodiment of the disclosure, R' and R" are independently or simultaneously H, (CrC3)-alkyl, phenyl or (C<i-C3)-alkylene-phenyl, or R' and R" are joined together to optionally form, including the nitrogen atom to which they are attached, a 5-6-membered heterocyclic ring. In another embodiment, R' and R" are independently or simultaneously H, methyl, ethyl or propyl, phenyl or -CH ₂ -phenyl, or R' and R" are joined together to optionally form, including the nitrogen atom to which they are attached, a 6-membered heterocyclic ring, for example morpholinyl.

[0038] In another embodiment of the disclosure, R' and R" are independently or simultaneously H, (Ci-C3)-alkyl, phenyl or (CrC ₃ )-alkylene-phenyl, or R' and R" are joined together to optionally form, including the nitrogen atom to which they are attached, a 5-6-membered heterocyclic ring. In another embodiment, R' and R" are independently or simultaneously H, methyl, ethyl or propyl, phenyl or -CH ₂ -CH ₂ -phenyl, or R' and R" are joined together to optionally form, including the nitrogen atom to which they are attached, a 6-membered heterocyclic ring, for example morpholinyl.

[0039] In an embodiment of the disclosure, R ¹ is -R ^a -S-R ^b , wherein R ^a is C ₂ - alkylene and R ^c is isopropyl, -CH ₂ -CH ₂ -NH-C(=0)-NR'R", where R' is H and R" is phenyl, or R ^c is -CH ₂ -C(=0)-NR'R", where R' is H and R" is -CH ₂ -phenyl.

[0040] In another embodiment, R ² is H. [0041] In a further embodiment of the disclosure, R ³ is H or (Ci-C3)-alkyl. In another embodiment, R ³ is H.

[0042] In one embodiment, R ⁴ - R ⁶ are each simultaneously or independently OH or -OR ^c , wherein R ^c is (C C ₃ )-alkyl, (C ₂ -C ₃ )-alkenyl, (C ₂ -C ₃ )-alkynyl, or (C ₃ -C ₆ )- cycloalkyl. In a further embodiment, R ⁴ - R ⁶ are each simultaneously or independently H, OH or -OCH3. In one embodiment, R ⁴ - R ⁶ are each OH.

[0043] In another embodiment, the optional substituents are F, CI, Br, I, OH, (d- C ₃ )-alkyl or fluoro-substituted-(CrC ₃ )-alkyl.

[0044] In another embodiment of the disclosure, the compound of the formula (I) has the following structure, wherein R ¹ -R ⁶ are as defined above:

[0045] In a further embodiment of the disclosure, the compound of the formula (I) has the following structure wherein R -R ⁶ are as defined above:

[0046] In one embodiment, the compound of the formula (I) is:

0047] In one embodiment, the compound of the formula

[0048] In one embodiment, the compounds of the Formula (I) as defined above are inhibitors of β-glucocerebrosidase. It will be understood by a person skilled in the art that binding of inhibitors to the active site of an enzyme, in one embodiment, is typically associated with stabilization of that enzyme against denaturation or proteolysis. Therefore, in one embodiment, compounds demonstrating inhibition of an enzyme also demonstrate the ability to stabilize the same enzyme.

[0049] In another embodiment, the compounds of the Formula (I) have high specificity for β-glucocerebrosidase (β-glucocidase), in that they do not affect the activities of other lysosomal glycosidases, such as a-glucosidase, β-galactosidase or a- galactosidase, either in vitro or in treated fibroblasts. In one embodiment, the compound of the Formula (I) having the following structure

has an inhibition constant Ki value of 76 ± 12 nM for wild-type glucocerebrosidase; similarly the compound of the Formula (I) having the following structure has an inhibition constant K, value of 12 ± 2 nM and

the compound of the Formula (I) having the following structure has an inhibition constant K, value of 30 ± 2 nM,

[0050] In one embodiment, the compounds of the formula (I) are formulated into pharmaceutical compositions. The production of pharmaceutical compositions is effected in a manner which will be familiar to any person skilled in the art by bringing a compound of the Formula (I), together with suitable, non-toxic, inert, therapeutically compatible solid, liquid or aerosol carrier materials, pharmaceutically acceptable excipients and, if desired, usual pharmaceutical adjuvants.

[0051] Suitable carrier materials are not only inorganic carrier materials, but also organic carrier materials. Suitable carrier materials for topical preparations are glycerides, semi-synthetic and synthetic glycerides, hydrogenated oils, liquid waxes, liquid paraffins, liquid fatty alcohols, sterols, polyethylene glycols and cellulose derivatives.

[0052] Usual stabilizers, preservatives, wetting and emulsifying agents, consistency-improving agents, salts for varying the osmotic pressure, buffer substances, solubilizers, colorants and antioxidants come into consideration as pharmaceutical adjuvants.

c) Uses

[0053] In one embodiment of the disclosure, the aforementioned compounds of the Formula (I), or pharmaceutical composition comprising a compound of the Formula (I), in a therapeutically effective amount thereof, are used for the treatment of diseases in which the enzyme β-glucocerebrosidase (wild type or a mutant form) is implicated. In one embodiment, the disease is Parkinson's disease or Lewy body disease, optionally involving -synuclein. In another embodiment, the disease is Gaucher disease.

[0054] In another embodiment of the disclosure, the aforementioned compounds of the Formula (I), or pharmaceutical composition comprising a compound of the Formula (I), in a therapeutically effective amount thereof, are used in the treatment of diseases in which the enzyme β-glucocerebrosidase (wild type or a mutant form) is implicated. In one embodiment, the mutant form of the enzyme arises as a result of the p.N470S or L444P missense mutations. In one embodiment, patients who suffer from diseases associated with the mutant form of β-glucocerebrosidase as a result of the p.N470S mutation, in one embodiment, display visceral symptoms of Gaucher disease (Type I). Patients who suffer from diseases associated with the mutant form of β- glucocerebrosidase as a result of the L444P mutation, in one embodiment, display neurological as well as visceral symptoms of Gaucher disease (Type II and III). Other less common mutations resulting in Gaucher disease that also may benefit from this treatment include F213I, R353W

[0055] In another embodiment, the compounds of the Formula (I) are useful to prevent the buildup of the neurotoxin a-synuclein in the brain of a subject.

[0056] The present disclosure also includes methods of medical treatment comprising the administration of a compound of the Formula (I), or pharmaceutical composition comprising a compound of the Formula (I), to a mammal.

[0057] Accordingly, in one embodiment, the present disclosure includes a method of treating a disease in which the enzyme β-glucocerebrosidase (wild type or a mutant form) is implicated, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the Formula (I), or pharmaceutical composition comprising a compound of the Formula (I). In one embodiment, the disease is Parkinson's or Lewy body disease. In another embodiment, the disease is Gaucher disease.

[0058] In another embodiment, the present disclosure includes a method of treating a disease in which the enzyme β-glucocerebrosidase (wild type or a mutant form), is implicated, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the Formula (I), or pharmaceutical composition comprising a compound of the Formula (I), in which the mutation arises as a result of the p.N470S, L444P, F213I or R353W, missense mutations. In one embodiment, the disease is Parkinson's or Lewy body disease. In another embodiment, the disease is Gaucher disease.

[0059] The dosage of a compound of the Formula I or a pharmaceutical composition comprising a compound of the Formula (I), varies within wide limits depending on the disease to be controlled, the age and the individual condition of the patient and the mode of administration, and will, of course, be fitted to the individual requirements in each particular case. For adult patients a daily dosage of about 1 mg to about 1000 mg, especially about 1 mg to about 100 mg, comes into consideration. Depending on the dosage it is convenient to administer the daily dosage in several dosage units.

[0060] Although the disclosure has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present disclosure. [0061] The operation of the disclosure is illustrated by the following representative examples. As is apparent to those skilled in the art, many of the details of the examples may be changed while still practicing the disclosure described herein.

EXAMPLES

[0062] General materials and methods

[0063] All reagents for synthesis were obtained from Sigma-Aldrich (USA) and were of reagent grade. All reagents were used without further purification except for the commercially obtained thiols, which were redistilled just prior to use. Thin layer chromatography (t.l.c.) was performed on Merck silica gel 60 F ₂ 5 ₄ plates. Flash chromatography was performed with Merck silica gel 60 (230-400 mesh). H and ³ C NMR (referenced using the residual solvent peak, or an internal MeOH standard in the case of ¹³ C for D ₂ 0) spectra were recorded on a Bruker AV-300 or AV-400 instrument. High-resolution mass spectra of all compounds were obtained in the mass spectrometry laboratory of the Chemistry Department at U.B.C. Pure GBA was obtained as Cerezyme ^® from Genzyme (USA). Purified human cytosolic neutral β-glucosidase was kindly provided by Dr. N. Juge (Institue of Food Research, Norwich , UK) [Tribolo, S., J. G. Berrin, et al. (2007). "The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase." J Mol Biol 370(5): 964-975.]. A lysosomal enriched concanavalin A-fraction from human placenta was used a source of human 3-galactosidase, r-galactosidase and ar-glucosidase [Mahuran, D. and J. A. Lowden (1980). "The subunit and polypeptide structure of hexosaminidases from human placenta." Can J Biochem 58(4): 287-294.]. Concanavalin A conjugated to agarose beads was obtained from GE Healthcare (USA). 2,4-Dinitrophenyl /3-D-glucopyranoside was prepared according to the procedure of Sharma et al.,[Sharma, S.K.; Corrales, G.; Penades, S. Tetrahedron Lett. 1995, 36, 5627-5630] while the fluorogenic enzyme substrates (4-methylumbelliferyl β-D- galactopyranoside, 4-methylumbelliferyl a-D-galactopyranoside, 4-methylumbelliferyl β- D-glucopyranoside, 4-methylumbelliferyl a-D-glucopyranoside and 4-methylumbelliferyl A/-acetyl-/?-D-glucosaminide) were obtained commercially from Sigma-Aldrich (USA) or Toronto Research Chemicals (Canada). NanoOrange ^® was obtained from Invitrogen (USA). Gaucher fibroblast cell lines homozygous for the p.N370S and p.L444P mutations in GBA were obtained from the cell line repositories at the Toronto Hospital for Sick Kids and the Coriell Institute for Medical Research, respectively.

Enzymology

[0064] All GBA kinetics were performed at 37 °C using a buffer at pH 5.5 or 7.0 that contained: 20 mM citric acid, 50 mM Na ₂ HP0 ₄ , 1 .0 mM tetrasodium EDTA, 0.25 % v/v Triton X-100 ^® and 0.25 % w/v taurocholic acid. To determine the K _t value of each inhibitor for GBA, initial reaction rates were measured at three substrate concentrations for each of a range of inhibitor concentrations (typically seven concentrations bracketing the K value ultimately determined such that 0.2 , < [I] < 5K). These data were fit to a competitive inhibition model using nonlinear regression analysis, as performed by the GraFit 5.0.13 program, to provide the K value. Dixon and Lineweaver-Burke plots of each data set validated the use of a competitive inhibition model. For inhibitors with K values greater than 20 nM, this value was determined using a continuous UV spectrophotometric assay where the final GBA concentration was 4.0 nM and 2,4- dinitrophenyl 3-D-glucopyranoside served as the substrate. Reactions were initiated by the addition of GBA, and the time-dependent increase in absorption at 400 nm, as determined using a Varian Cary 4000 or Varian Cary 300 UV-Vis spectrophotometer, gave the initial reaction rate. This increase in absorbance was linear for all measurements over a period of three minutes. For inhibitors with K values less than 20 nM, this value was determined using a discontinuous fluorimetric assay where the final GBA concentration was 10 pM and 4-methylumbelliferyl 3-D-glucopyranoside served as the substrate. Reactions were initiated by the addition of GBA and, after 4, 10 and 15 min, 150 μΙ aliquots of the reaction mixture were removed and diluted into a cuvette containing 450 μΙ of glycine buffer (1.0 M, pH 10.8). The fluorescence of each sample (A _ex 365 nm, A _em 460 nm) was determined using a Varian Cary Eclipse fluorimeter. The three time points were fit by linear regression to provide the initial reaction rate. In each case, an excellent linear fit was obtained over this fifteen minute interval.

Example 1: Inhibitory Activity of Compounds Against Lysosomal Enzymes [0065] The inhibitory activities of compounds against selected human lysosomal enzymes (a-glucosidase, a-galactosidase and β-galactosidase) were evaluated at a single, high concentration of 200 μΜ, in triplicate. All reactions were performed using 100 mM citrate-phosphate buffer at pH 4.5 supplemented with 0.025 % w/v human serum albumin. The activities of human lysosomal α-glucosidase, α-galactosidase and β-galactosidase in a lysosomal enzyme enriched ConA-bound fraction from human placenta were monitored using an equal volume of the fluorogenic substrates [4- methylumbelliferyl a-D-glucopyranoside (3.0 mM), 4-methylumbelliferyl a-D- galactopyranoside (1 .0 mM) and 4-methylumbelliferyl /?-D-galactopyranoside (0.5 mM), respectively] at 37 °C. All reactions were terminated by the addition of a four-fold excess of 100 mM 2-amino-2-methyl-propanol (MAP) and fluorescence measured using a Molecular Devices M2 fluorimeter (A _ex 365 nm, A _em 460 nm).

Example 2: Inhibitory Activity of Compounds Against β-glucocerebrosidase

[0066] Inhibitory activities against purified cytosolic neutral human β-glucosidase were determined by incubating a three-fold dilution series of the compound in the presence of the enzyme for 15 min. at room temperature, followed by the addition of an equal volume of substrate (10 mM 4-methylumbelliferyl β-D-glucopyranoside) in 100 mM citrate-phosphate buffer at pH 7.0. Each reaction was incubated at 37 °C for 30 min, terminated by the addition of a four-fold excess of 00 mM MAP and the fluorescence determined as described above. IC ₅ o values were deduced by non-linear fitting of the data to the appropriate model using Prism Graphpad v5.2.

Example 3: Differential scanning fluorimetry

The melting temperatures of human GBA (1 μg) in the presence of compounds 2, 3 and 9-25 (200 μΜ) were determined by scanning differential fluorimetry, using NanoOrange ^® (1/100 dilution as per the manufacturers protocol) as the fluorescent reporter, in accordance with established protocols [Kornhaber, G. J., M. B. Tropak, et al. (2008). "Isofagomine induced stabilization of glucocerebrosidase." Chembiochem 9(16): 2643- 2649]. Example 4: Cell-based assay for GBA chaperone activity

[0067] Patient fibroblasts homozygous for the p.N370S mutation or p.L444P mutation or lacking any mutations in GBA were grown in 96 well tissue culture plate (10- 20,000 cells/well) in alpha-MEM media supplemented with 10 % v/v fetal calf serum at 37 °C in a CO2 humidified \ncubator. Chaperoning efficiencies of the compounds were evaluated by preparing a series of three-fold dilutions starting at 10 mM in water. Compounds were directly added to the media at a 1/100 dilution. Following incubation of the cells for five days in the compound-supplemented media, lysates in 100 mM citrate-phosphate buffer at pH 5.5 containing 0.4 % v/v Triton X-100 ^® and 0.2 % w/v taurocholic acid were prepared as described [Hill, T., M. B. Tropak, et al. (201 1 ). "Synthesis, kinetic evaluation and cell-based analysis of C-alkylated isofagomines as chaperones of beta-glucocerebrosidase." Chembiochem 12(14): 2151-2154.] GBA and lysosomal hexosaminidase (Hex) present in the lysate from treated patient cells was captured on ConA-conjugated agarose beads and washed three times with lysis buffer to remove residuals amounts of the compounds [Hill, T., M. B. Tropak, et al. (201 1 ). "Synthesis, kinetic evaluation and cell-based analysis of C-alkylated isofagomines as chaperones of beta-glucocerebrosidase." Chembiochem 12(14): 2151 -2154.]. GBA activity was measured using 10 mM 4-methylumbelliferyl ?-D-glucopyranoside and, for compound treated cells, this value was normalized against the GBA activity measured in mock-treated control cells (n = 3). Similar activity measurements were also performed for lysosomal hexosaminidase (Hex) using 3.2 mM 4-methylumbelliferyl /V-acetyl-/?-D- glucosaminide as the substrate.

Example 5: Synthesis. Thiol-ene reaction protocol.

[0068] A solution of AIBN (3.3 mg, 20 pmol) in degassed MeOH (1.0 ml) was added drop-wise over 8 h. to a solution of thiol (0.50 mmol) and alkene 3 (16 mg, 0.10 mmol) in degassed MeOH (2.0 ml) refluxing under an atmosphere of N2. The solution was refluxed for a further 16 h., concentrated to dryness and then subjected to flash chromatography. The product was dissolved in HCI (2.0 ml, 0.1 M) and the solution concentrated to dryness. The residue was dissolved in H ₂ 0 and passed through a Waters tCi ₃ Sep-Pak ^® (2 g, H ₂ 0/MeOH, 1 :0- 1 :4). Fractions containing product were combined and lyophilized.

(i) N,N'-Dibutyrylcystamine

[0069] Butyryl chloride (0.91 ml, 8.8 mmol) was added drop-wise to a suspension of cystamine dihydrochloride (0.90 g, 4.0 mmol) and pyridine (1.3 ml) in CH ₂ CI ₂ (20 ml) at 0 °C and the mixture stirred (r.t., 4 h). Methanol (1 .0 ml) was added to the mixture at 0 °C and stirring continued (r.t., 30 min). The mixture was diluted with CH ₂ CI ₂ (50 ml) and the organic phase collected, then washed with HCI (30 ml, 1 M), dried (MgS0 ₄ ), filtered and evaporated to dryness. The residue was recrystallised from EtOAc/hexanes to give Λ/,/V'-dibutyrylcystamine as a colorless powder (0.84 g, 72 %). ¹ H NMR (300.1 MHz, CDCI ₃ ) δ = 0.91 (t, 3 H, J = 7.5 Hz, CH ₃ ), 1 .63 (tq, 2 H, J = 7.5, 7.5 Hz, CH ₂ CH ₃ ), 2.17 (t, 2 H, J = 7.5 Hz, CH ₂ CH ₂ CH ₃ ), 2.79 (t, 2 H, J = 6.7 Hz, SCH ₂ ), 3.53 (dt, 2 H, J = 6.7, 6.7 Hz, NCH ₂ ), 6.66 (bt, 1 H, J = 6.7 Hz, NH); ¹³ C NMR (75.5 MHz, CDCI ₃ ) δ = 13.9 (CH ₃ ), 19.3, 38.0, 38.5, 38.6 (CH ₂ ), 173.9 (C=0). HRMS (ES): m/z = 293.1354; [M + H] ⁺ requires 293.1357.

(ii) N-(2-Mercaptoethyl)-butyramide

[0070] 1 ,3-Propanedithiol (0.40 ml, 4.0 mmol) was added to a suspension of /V./V-dibutyrylcystamine (0.58 g, 2.0 mmol) and Et ₃ N (40 μΙ) in degassed MeOH (15 ml) and the mixture stirred (2 h). The solution was concentrated to dryness and the residue subjected to flash chromatography (EtOAc/hexanes, 1 :1 ) to give A/-(2-mercaptoethyl)- butyramide as a colorless solid (0.49 g, 83 %). ¹ H NMR (400.2 MHz, CD ₃ OD) δ = 1 .00 (t, 3 H, J = 7.2 Hz, CH ₃ ), 1.69 (tq, 2 H, J = 7.2, 7.2 Hz, CH ₂ CH ₃ ), 2.23 (t, 2 H, J = 7.2 Hz, CH2CH2CH3), 2.65 (t, 2 H, J = 6.8 Hz, CH ₂ ), 3.38 (t, 2 H, J = 6.8 Hz, CH ₂ ); ¹³ C NMR (100.6 MHz, CD3OD) δ = 14.1 (CH ₃ ), 20.5, 24.6, 39.1 , 44.0 (CH ₂ ), 176.3 (C=O). HRMS (ES): m/z = 148.0798; [M + H] ⁺ requires 148.0796.

(Hi) 1 -(2-Mercaptoethyl)-3-phenyl-urea

[0071] 1 ,3-Propanedithiol (0.40 ml, 4.0 mmol) was added to a suspension of bis- [2-(3-phenylureido)ethyl] disulfide [Tovilla, J.A.; Vilar, R.; White, A.J. P. Chem. Commun. 2005, 4839-4841]] (0.78 g, 2.0 mmol) and Et ₃ N (40 μΙ) in degassed MeOH (15 ml) and the mixture stirred (2 h). The solution was concentrated to dryness and the residue subjected to flash chromatography (Me2CO/hexanes, 1 :3) to give 1 -(2-mercaptoethyl)-3- phenyl-urea as colorless needles (0.60 g, 77 %). ¹ H NMR (400.2 MHz, CD ₃ OD) δ = 2.62 (t, 2 H, J = 6.8 Hz, CH ₂ ), 3.35 (t, 2 H, J = 6.8 Hz, CH ₂ ), 6.93-7.38 (m, 5 H, Ph); ¹³ C NMR (100.6 MHz, CD ₃ OD) δ = 25.6, 44.4 (CH ₂ ), 120.4, 123.7, 130.0, 141.0 (Ph), 158.3 (C=0). HRMS (ES): m/z = 197.0748; [M + H] ⁺ requires 197.0749.

(iv) 2,2'-Dithiobis(N-benzyl-acetamide)

[0072] Λ/,/V-Dimethylformamide (20 μΙ) was added to 2,2'-dithiobis(acetic acid) (1.8 g, 10 mmol) and SOCI ₂ (2.9 ml, 40 mmol) in CH ₂ CI ₂ (25 ml) and the solution refluxed (2 h). The solution was concentrated to dryness and the residue dissolved in CH ₂ CI ₂ (50 ml). Benzyl amine (4.8 ml, 44 mmol) was added drop-wise at 0 ^° C and the mixture vigorously stirred (r.t., 4 h). Water (10 ml) was added to the mixture at 5 ^° C and stirring continued (r.t., 30 min). The mixture was filtered and the residue washed with H ₂ 0 (2 x 30 ml) then cold EtOH (2 10 ml). Recrystallisation of the residue from Me ₂ CO/hexanes gave 2,2'-dithiobis(/V-benzyl-acetamide) as colorless needles (1.9 g, 52 %). H NMR [400.2 MHz, (CD ₃ ) ₂ SO] δ = 3.55 (s, 2 H, SCH ₂ ), 4.30 (d, 2 H, J = 6.0 Hz, NCH ₂ ), 7.20-7.56 (m, 5 H, Ph), 8.59 (bt, 1 H, J = 6.0 Hz, NH); ¹³ C NMR [100.6 MHz, (CD ₃ ) ₂ SO] δ = 41 .9, 42.5 (CH ₂ ), 126.9, 127.3, 128.3, 139.0 (Ph), 167.8 (C=0). HRMS (ES): m/z = 359.0884; [M + H] ⁺ requires 359.0888.

( v) N-Benzyl-2-mercapto-acetamide

[0073] 1 ,3-Propanedithiol (0.40 ml, 4.0 mmol) was added to a suspension of 2,2'- dithiobis(/V-benzyl-acetamide) (0.72 g, 2.0 mmol) and Et ₃ N (40 μΙ) in degassed MeOH (15 ml) and the mixture stirred (2 h). The solution was concentrated to dryness and the residue subjected to flash chromatography (EtOAc/hexanes, 7:13) to give A/-benzyl-2- mercapto-acetamide as a colorless needles (0.52 g, 72 %). H NMR (300.1 MHz, CD ₃ OD) δ = 3.18 (s, 2 H, SCH ₂ ), 4.37 (s, 2 H, NCH ₂ Ph), 7.20-7.36 (m, 5 H, Ph); ¹³ C NMR (75.5 MHz, CD ₃ OD) δ = 28.4, 44.5 (CH ₂ ), 128.4, 128.7, 129.7, 139.8 (Ph), 173.4 (C=0). HRMS (ES): m/z = 182.0643; [M + H] ⁺ requires 182.0640.

( vi) N, N-Diethyl-4-mercapto-butyramide [0074] Diethylamine (1 .6 ml, 15 mmol) was added to a solution of 4- thiobuyrolactone (0.87 ml, 10 mmol) in MeCN and the mixture refluxed (24 h). The solution was concentrated to dryness and the residue subjected to flash chromatography (EtOAc/hexanes, 3:2-7:3) to give A/,A/-diethyl-4-mercapto-butyramide as a colorless oil (1.3 g, 74 %). ¹ H NMR (300.1 MHz, CD ₃ OD) δ = 1.1 1 (t, 3 H, J = 6.9 Hz, CH ₃ ), 1.22 (t, 3 H, J = 6.9 Hz, CH ₃ ), 2.01 (tt, 2 H, J = 6.9 Hz, CH ₂ CH ₂ CH ₂ ), 2.50 (t, 2 H, J = 6.9 Hz, CH ₂ ), 2.76 (t, 2 H, J = 6.9 Hz, CH ₂ ), 3.30-3.46 (m, 4 H, NCH ₂ CH ₃ ); ³ C NMR (75.5 MHz, CD ₃ OD) δ = 13.5, 14.7 (CH ₃ ), 25.9, 32.3, 38.9 [(CH ₂ ) ₃ ], 41 .6, 43.6 (NCH ₂ ), 173.8 (C=0). HRMS (ES): m/z = 176.1 1 1 1 ; [M + H] ⁺ requires 176.1 109.

( vii) 4-Mercapto- 1 -(morpholin-4-yl)-butan- 1 -one

[0075] Morpholine (1.3 ml, 15 mmol) was added to a solution of 4- thiobuyrolactone (0.87 ml, 10 mmol) in MeCN and the mixture refluxed (24 h). The solution was concentrated to dryness and the residue subjected to flash chromatography (EtOAc/MeOH, 1 :0-19:1 ) to give 4-mercapto-1 -(morpholin-4-yl)-butan- 1 -one as a colorless solid (1 .2 g, 63 %). ¹ H NMR (300.1 MHz, CD ₃ OD) δ = 2.00 (tt, 2 H, J = 7.2, 7.2 Hz, CH ₂ CH ₂ CH ₂ ), 2.53 (t, 2 H, J = 7.2 Hz, CH ₂ ), 2.76 (t, 2 H, J = 7.2 Hz, CH ₂ ), 3.50-3.72 (m, 8 H, NCH ₂ CH ₂ 0); ³ C NMR (75.5 MHz, CD ₃ OD) δ = 25.8, 32.2, 38.8 [(CH ₂ ) ₃ ], 43.4, 47.4 (NCH ₂ ), 67.9 (OCH ₂ ), 173.4 (C=0). HRMS (ES): m/z = 190.0905; [M + H] ⁺ requires 190.0902.

(viii) (2R,3R S,5R)-2,3 -Tris(benzyloxy)-5-(4-methoxybenzylamino)-hept-6-en- 1-ol (5)

[0076] Vinylmagnesium bromide (70 ml 1 .0 M in THF, 70 mmol) was added drop- wise to a well-stirred suspension of 2,3,4-tri-0-benzyl-1 -deoxy-1 -(4- methoxybenzylamino)-D-xylose [Wennekes, T.; van den Berg, R.J.B.H.N.; Boltje, T.J.; Donker-Koopman, W.E.; Kuijper, B.; van der Marel, G.A.; Strijland, A.; Verhagen, CP.; Aerts, J.M.F.G.; Overkleeft, H.S. Eur. J. Org. Chem. 2010, 1258-1283.] (9.40 g, 17.4 mmol) in anhydrous Et ₂ 0 (500 ml) at 0 ^° C and the mixture stirred (r.t., 2 h). The mixture was cooled to 0 ^° C and quenched by the careful addition of aqueous NH ₄ CI (100 ml, 1.0 M). The organic phase was collected and the aqueous layer extracted with EtOAc (2 ^χ 100 ml). The combined organic phases were washed with sat. NaHC0 ₃ (300 ml), sat. NaCI (300 ml), dried (MgS0 ₄ ), filtered and evaporated to dryness. The residue was subjected to flash chromatography (EtOAc/hexanes/Et ₃ N, 39:60:1-49:50:1 ) to give the amine 5 as a colorless oil (8.49 g, 86 %). ¹ H NMR (400.2 MHz, CDCI ₃ ) δ = 1 ,92 (bs, 1 H, NH), 3.1 1 (dd, 1 H, J = 4.0, 8.4 Hz, H-5), 3.39-3.52 (m, 2 H, NCH ₂ Ar, H-2), 3.67 (dd, 1 H, J = 4.0, 1.6 Hz, H-1 ), 3.73-3.83 (m, 6 H, OCH ₃ , NCH ₂ Ar, H-1 ,4), 4.04 (dd, 1 H, J = 4.4, 6.4 Hz, H-3), 4.37-4.84 (OCH ₂ Ph), 5.04 (dd, 1 H, J = 1 .6, 17.2 Hz, =CH ₂ ), 5.21 (dd, 1 H, = 1.6, 10.4 Hz, =CH ₂ ), 5.76 (ddd, 1 H, J = 8.4, 10.4, 17.2 Hz, =CH), 6.84, 7.18 (ΑΑ'ΒΒ', 4 H, ArH), 7.22-7.42 (m, 15 H, Ph); ³ C NMR (100.6 MHz, CDCI ₃ ) <5 = 50.0 (NCH ₂ Ar), 55.4 (OCH ₃ ), 60.6 (C-5), 61 .9 (C-1 ), 72.2, 74.6, 74.9 (OCH ₂ Ph), 78.6 (C-2), 80.2 (C-3), 82.7 (C-4), 1 13.8 (Ar), 1 17.3 (=CH ₂ ), 127.5-138.7 (Ar, =CH). HRMS (ES): m/z = 568.3063; [M + H] ⁺ requires 568.3063.

(ix) ( 2R, 3S, 4S, 5R)-3, 4, 5- Tris(benzyloxy)-1-(4-methoxybenzyl)-2-vinyl-piperidine (6)

[0077] Methanesulfonyl chloride (1 .02 ml, 13.2 mmol) was added drop-wise to a solution of amine 5 (6.81 g, 12.0 mmol) in pyridine (13.0 ml) at 0 °C and the mixture stirred (r.t., 16 h). Water (1 .0 ml) was added to the mixture, it was stirred for 15 min. and then concentrated to dryness. The residue was partitioned between EtOAc (200 ml) and HCI (200 ml, 0.1 M). The organic phase was collected and the aqueous layer extracted with EtOAc (2 x 100 ml). The combined organic phases were washed with sat. NaHCO ₃ (300 ml), sat. NaCI (300 ml), dried (MgSO ₄ ), filtered and evaporated to dryness. The residue was subjected to flash chromatography (EtOAc/hexanes/Et ₃ N, 9:90:1 -14:85:1 ) to give the amine 6 as a colorless oil (5.41 g, 82 %). H NMR (400.2 MHz, CDCI ₃ ) δ = 2.59 (dd, 1 H, J = 10.8, 1 1.6 Hz, H-6), 2.87 (dd, 1 H, J = 5.6, 1 1 .6 Hz, H-6), 3.48-3.57 (m, 2 H, NC/- ₂ Ar, H-2), 3.61 -3.82 (m, 4 H, NCH ₂ Ar, H-3,4,5), 3.86 (s, 3 H, OCH ₃ ), 4.55-5.00 (m, 6 H, OCH ₂ Ph), 5.23 (dd, 1 H, J = 2.0, 17.2 Hz, =CH ₂ ), 5.50 (dd, 1 H, J = 2.0, 10.4 Hz, =CH ₂ ), 6.13 (ddd, 1 H, J = 10.4, 10.4, 17.2 Hz, =CH), 6.89, 7.21 (ΑΑΊΒΒ', 4 H, ArH), 7.28-7.44 (m, 15 H, Ph); ³ C NMR (100.6 MHz, CDCI ₃ ) δ = 49.7 (C-6), 55.4 (OCH ₃ ), 57.8 (NCH ₂ Ar), 62.9 (C-2), 72.3, 73.1 , 75.7 (OCH ₂ Ph), 79.3, 80.9, 83.1 (C- 3,4,5), 1 13.9 (Ar), 121.6 (=CH ₂ ), 127.5-129.7 (Ar), 130.5 (=CH), 158.8 (Ar). HRMS (ES): m/z = 550.2949; [M + H] ⁺ requires 550.2957. (x) (2R, 3S, 4S, 5R)-3, 4, 5-Tris(benzyloxy)-2-vinyl-piperidine (7)

[0078] A solution of the amine 6 (1 .1 g, 2.0 mmol) in THF (2.0 ml) was added to a solution of CAN (5.5 g, 10 mmol) in THF (12 ml) and H ₂ 0 (7.0 ml) at 0 °C and the mixture stirred (0 °C, 1 h). Saturated NaHC0 ₃ (25 ml) and EtOAc (25 ml) were added and the mixture was stirred well before being filtered through Celite ^® . The organic phase of the filtrate was collected and the aqueous layer extracted with EtOAc (2 χ 25 ml). The combined organic phases were dried (MgS0 ₄ ), filtered and evaporated to dryness. The residue was subjected to flash chromatography (EtOAc/hexanes/Et ₃ N, 19:80:1 ) to give the amine 7 as a pale yellow oil (0.45 g, 53 %). H NMR (400.2 MHz, CDCI ₃ ) δ = 1 .89 (bs, 1 H, NH), 2.96 (dd, 1 H, J = 7.2, 13.2 Hz, H-6), 3.04 (dd, 1 H, J = 4.4, 13.2 Hz, H-6), 3.47 (ddd, 1 H, J = 4.4, 7.2, 7.2 Hz, H-5), 3.56 (dd, 1 H, J = 4.8, 6.8 Hz, H-3), 3.67-3.75 (m, 2 H, H-2,4), 4.59-4.79 (m, 6 H, CH ₂ Ph), 5.24-5.38 (m, 2 H, =CH ₂ ), 6.13 (ddd, 1 H, J = 6.0, 10.8, 17.2 Hz, =CH), 7.25-7.39 (m, 15 H, Ph); ¹³ C NMR (100.6 MHz, CDCI ₃ ) δ = 44.6 (C-6), 57.3 (C-2), 72.4, 72.7, 74.6 (CH ₂ Ph), 77.7 q1 (C-5), 79.1 (C-4), 79.5 (C-3), 1 16.9 (=CH ₂ ), 127.7-128.6 (Ph), 136.0 (=CH), 138.7-138.9 (Ph). HRMS (ES): m/z = 430.2373; [M + H] ⁺ requires 430.2382.

(xi) (2R,3S,4S,5R)-3,4,5-Tris(benzyloxy)-1-benzyloxycarbonyl-2-vi nyl-piperidine (8)

[0079] Benzyl chloroformate (7.1 ml, 50 mmol) was added to a solution of the amine 6 (4.4 g, 8.0 mmol) in PhMe (80 ml) and the mixture refluxed (48 h). The mixture was concentrated to dryness and the residue was subjected to flash chromatography (EtOAc/hexanes, 1 :19-1 :9) to give the carbamate 8 as a colourless oil (3.5 g, 78 %). H NMR (400.2 MHz, CDCI ₃ , two rotamers observed) δ = 2.87-3.02 (m, 1 H, H-6), 3.42-3.70 (m, 3 H, H-3,4,5), 4.13-4.46 (m, 1 H, H-6), 4.62-5.44 (m, 1 1 H, CH ₂ Ph, =CH ₂ , H-2), 6.05-6.19 (m, 1 H, =CH), 7.25-7.48 (m, 20 H, Ph); ³ C NMR (100.6 MHz, CDCI ₃ , two rotamers observed) δ = 41 .98, 42.20 (C-6), 54.5, 55.1 (C-2), 67.7, 67.8, 72.8, 73.20, 73.23, 73.4, 75.86, 75.88 (CH ₂ Ph), 78.3, 78.4, 79.6, 79.7, 82.6, 82.7 (C- 3,4,5), 1 18.1 , 1 18.4 (=CH ₂ ), 127.7-128.8 (Ph), 130.8, 131 .0 (=CH), 136.5-139.0 (Ph), 155.7, 155.8 (NC0 ₂ ). HRMS (ES): m/z = 564.2748; [M + H] ⁺ requires 564.2750.

(xii) (2R,3S,4S,5R)-3,4,5-Trihydroxy-2-vinyl-piperidine hydrochloride (3.HCI) [0080] i) Sodium (0.23 g, 10 mmol) was dissolved in liquid NH ₃ (7 ml) at -78 ^° C. A solution of the amine 7 (0.29 g, 0.67 mmol) and allylamine (0.50 ml, 6.7 mmol) in anhydrous THF (2.0 ml) was chilled to -78 ^° C, and then rapidly added via a cannula to the blue NH ₃ solution at the same temperature. As soon as the addition was complete, H ₂ 0 (1.0 ml) was added to quench the reaction. The mixture was left to warm to room temperature, and, once the NH ₃ had evaporated, the residue was partitioned between H ₂ 0 (5.0 ml) and Et ₂ 0 (3.0 ml). The aqueous layer was collected, concentrated to dryness, redissolved in H ₂ 0 (2 ml) and applied to a column of cation-exchange resin (Dowex 50W X 8, 200-400 mesh, H ⁺ form). The column was washed with H ₂ 0, then eluted with NH OH (2.0 M). Fractions containing product were combined and concentrated to dryness. The residue was dissolved in H ₂ 0 and passed through a Waters tCi ₈ Sep-Pak ^® (2.0 g, H ₂ 0). Fractions containing product were combined and lyophilized to give a pale yellow foam consisting of a mixture of amines 3 and 9 in a ratio of 93:7, respectively (80 mg, 75 %). This sample was sufficiently pure for subsequent thiol-ene reactions. A small quantity was purified by h.p.l.c. (TSKgel Amide- 80, 5 ym, MeCN/H ₂ 0 gradient) for analytical purposes. This pure sample was lyophilized from HCI (0.1 M) to provide the corresponding hydrochloride, which crystallised from MeOH/Et ₂ 0 as colourless plates. H NMR (400.2 MHz, D ₂ 0) δ = 3.33-3.41 (m, 1 H, H-6), 3.48-3.56 (m, 1 H, H-6), 3.98-4.07 (m, 3 H, H-3,4,5), 4.15-4.21 (m, 1 H, H-2), 5.56-5.66 (m, 2 H, =CH ₂ ), 6.06 (ddd, 1 H, J = 7.6, 10.8, 17.2 Hz, =CH); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 45.7 (C-6), 58.5 (C-2), 67.1 , 69.1 , 70.3 (C- 3,4,5), 124.0 (=CH ₂ ), 130.1 (=CH). HRMS (ES): m/z = 160.0965; [M + H] ⁺ requires 160.0974.

[0081 ] ii) The carbamate 8 (2.82 g, 5.00 mmol) was subjected to the above procedure, with the quantity of all reagents scaled linearly, to give a pale yellow foam consisting of a mixture of the amines 3 and 9, in a ratio of 19:1 , respectively (732 mg, 92 %).

(xiii) (2R,3S,4S,5R)-2-Ethyl-3,4,5-trihydroxy-piperidine hydrochloride (9.HCI)

[0082] Palladium-on-carbon (20 mg, 10 wt. %) was added to a solution of the carbamate 8 (0.1 1 g, 0.20 mmol) in degassed MeOH (2.0 ml). Hydrochloric acid (200 μΙ, 6.0 M) was added and the mixture stirred under H ₂ (1 atm, 24 h). The mixture was filtered through Celite ^® , concentrated to dryness and the residue recrystallised (MeOH/Et ₂ 0) to give the hydrochloride 9.HCI as colourless needles (34 mg, 85 %). ¹ H NMR (400.2 MHz, D ₂ 0) δ 1.06 (at, 3 H, J = 7.2 Hz, CH ₃ ), 1 .73-1.95 (m, 2 H, CH ₂ CH ₃ ), 3.33-3.40 (m, 1 H, H-6), 3.41 -3.53 (m, 2 H, H-2,6), 4.02-4.15 (m, 3 H, H-3,4,5); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 8.8 (CH ₃ ), 21 .1 (CH ₂ CH ₃ ), 45.4 (C-6), 56.6 (C-2), 66.2, 67.1 , 67.3 (C-3,4,5). HRMS (ES): m/z = 162.1 122; [M + H] ⁺ requires 162.1 130.

(xiv) (2R, 3S, 4S, 5R)-2-(2-Ethylsulfanyl-ethyl)-3, 4, 5-trihydroxy-piperidine

hydrochloride (10.HCI)

[0083] The thiol-ene reaction protocol utilizing ethanethiol provided the thioether 10.HCI as a colorless foam (16 mg, 62 %). H NMR (400.2 MHz, D ₂ 0) <5 = 1.29 (at, 3 H, J = 7.6 Hz, CH ₃ ), 1 .97-2.09 (m, 1 H, CH ₂ CH ₂ S), 2.13-2.25 (m, 1 H, CH ₂ CH ₂ S), 2.66 (aq, 2 H, J = 7.6 Hz, SCH ₂ CH ₃ ), 2.67-2.89 (m, 2 H, CH ₂ CH ₂ S), 3.30-3.41 (m, 1 H, H-6), 3.46-3.54 (m, 1 H, H-6), 3.71 (ddd, 1 H, J = 2.4, 6.4, 8.4 Hz, H-2), 4.01 -4.13 (m, 3 H, H-3,4,5); ³ C NMR (100.6 MHz, D ₂ 0) δ = 14.9 (CH ₃ ), 26.0 (SCH ₂ CH ₃ ), 26.9 (CH ₂ CH ₂ S), 28.4 (CH ₂ CH ₂ S), 46.5 (C-6), 55.1 (C-2), 67.0, 68.0, 68.5 (C-3,4,5). HRMS (ES): m/z = 222.1 166; [M + H] ⁺ requires 222.1 164.

(xv) (2R, 3S, 4S, 5R)-2-(2-Butylsulfanyl-ethyl)-3, 4, 5-trihydroxy-piperidine hydrochloride (11.HCI)

[0084] The thiol-ene reaction protocol utilizing 1-butanethiol provided the thioether 11.HCI as a colorless foam (15 mg, 52 %). ¹ H NMR (400.2 MHz, D ₂ 0) δ = 0.89 (at, 3 H, J = 7.2 Hz, CH ₃ ), 1 .33- 1 .45 (m, 2 H, CH ₂ CH ₃ ), 1.53-1.63 (m, 2 H, CH ₂ CH ₂ CH ₃ ), 1 .92-2.04 (m, 1 H, CHCH ₂ CH ₂ S), 2.08-2.20 (m, 1 H, CHCH ₂ CH ₂ S), 2.58-2.78 (m, 4 H, CH ₂ SCH ₂ ), 3.29-3.36 (m, 1 H, H-6), 3.42-3.49 (m, 1 H, H-6), 3.63- 3.70 (m, 1 H, H-2), 3.95-4.09 (m, 3 H, H-3,4,5); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 13.0 (CH ₃ ), 21 .4 (CH ₂ CH ₃ ), 26.3 (CHCH ₂ CH ₂ S), 27.4 (CHCH ₂ CH ₂ S), 30.8, 30.9 (SCH ₂ CH ₂ CH ₂ CH ₃ ), 45.5 (C-6), 54.1 (C-2), 66.0, 67.0, 67.5 (C-3,4,5). HRMS (ES): m/z = 250.1472; [M + H] ⁺ requires 250.1477.

(xvi) (2R, 3S, 4S, 5R)-2-(2-Hexylsulfanyl-ethyl)-3, 4, 5-trihydroxy-piperidine hydrochloride (12.HCI) [0085] The thiol-ene reaction protocol utilizing 1-hexanethiol provided the thioether 12.HCI as a colorless foam (18 mg, 57 %). ¹ H NMR (400.2 MHz, D ₂ 0) δ = 0.87-0.97 (m, 3 H, CH ₃ ), 1.28-1 .50 (m, 6 H, (CH ₂ ) ₃ CH ₃ ), 1 .58-1.71 (m, 2 H, CH ₂ (CH ₂ ) ₃ CH ₃ ), 1 .96-2.09 (m, 1 H, CHCtf ₂ CH ₂ S), 2.13-2.25 (m, 1 H, CHCH ₂ CH ₂ S), 2.62-2.82 (m, 4 H, CH ₂ SCH ₂ ), 3.34-3.42 (m, 1 H, H-6), 3.46-3.54 (m, 1 H, H-6), 3.67-3.74 (m, 1 H, H-2), 4.01 -4.13 (m, 3 H, H-3,4,5); ¹³ C NMR (100.6 MHz, D ₂ 0) δ =

14.4 (CH ₃ ), 23.0 (CH ₂ ), 27.4 (CHCH ₂ CH ₂ S), 28.4 (CHCH ₂ CH ₂ S), 28.7, 29.6, 31 .7, 32.1 (4C, CH ₂ ), 46.5 (C-6), 55.1 (C-2), 67.0, 68.0, 68.5 (C-3,4,5). HRMS (ES): m/z = 278.1792; [M + H] ⁺ requires 278.1790.

(xvii) (2R,3S,4S,5R)-3,4,5-Trihydroxy-2-(2-octylsulfanyl-ethyl)-pip eridine hydrochloride (13.HCI)

[0086] The thiol-ene reaction protocol utilizing 1 -octanethiol provided the thioether 13.HCI as a colorless foam (16 mg, 47 %). H NMR (400.2 MHz, D ₂ 0) δ = 0.85-0.95 (m, 3 H, CH ₃ ), 1.25-1 .48 (m, 10 H, (CH ₂ ) ₅ CH ₃ ), 1 .57-1 .70 (m, 2 H, CH ₂ (CH ₂ ) ₅ CH ₃ ), 1.95-2.08 (m, 1 H, CHCH ₂ CH ₂ S), 2.12-2.24 (m, 1 H, CHCH ₂ CH ₂ S), 2.60-2.82 (m, 4 H, CH ₂ SCH ₂ ), 3.32-3.40 (m, 1 H, H-6), 3.44-3.54 (m, 1 H, H-6), 3.65-3.74 (m, 1 H, H-2), 3.98-4.13 (m, 3 H, H-3,4,5); ³ C NMR (100.6 MHz, D ₂ 0) δ =

14.5 (CH ₃ ), 23.1 (CH ₂ ), 27.4 (CHCH ₂ CH ₂ S), 28.4 (CHCH ₂ CH ₂ S), 29.0, 29.3, 29.4, 29.7, 32.1 , 32.2 (6C, CH ₂ ), 46.5 (C-6), 55.1 (C-2), 67.1 , 68.0, 68.6 (C-3,4,5). HRMS (ES): m/z = 306.2101 ; [M + H] ⁺ requires 306.2103

(xviii) (2R, 3S, 4S, 5R)-3, 4, 5-Trihydroxy-2-(2-isopropylsulfanyl-ethyl)-piperidine hydrochloride (14.HCI)

[0087] The thiol-ene reaction protocol utilizing 2-propanethiol provided the thioether 14.HCI as a colorless foam (18 mg, 67 %). H NMR (400.2 MHz, D ₂ 0) δ = 1 .28 (ad, 6 H, J = 6.8 Hz, CH ₃ ), 1 .93-2.05 (m, 1 H, CH ₂ CH ₂ S), 2.1 1 -2.23 (m, 1 H, CH ₂ CH ₂ S), 2.65-2.83 (m, 2 H, CH ₂ CH ₂ S), 3.07 (a. sept., 1 H, J = 6.8 Hz, SCH), 3.30- 3.38 (m, 1 H, H-6), 3.43-3.51 (m, 1 H, H-6), 3.63-3.71 (m, 1 H, H-2), 3.98-4.1 1 (m, 3 H, H-3,4,5); ³ C NMR (100.6 MHz, D ₂ 0) δ = 22.46, 22.48 (CH ₃ ), 24.9 (CH ₂ CH ₂ S), 27.7 (CH ₂ CH ₂ S), 34.6 (SCH), 45.5 (C-6), 54.1 (C-2), 66.0, 67.0, 67.5 (C-3,4,5). HRMS (ES): m/z = 236.1327; [M + H] ⁺ requires 236.1320. (xix) (2R, 3S, 4S, 5R)-2-(2-tert-Butylsulfanyl-ethyl)-3, 4, 5-trihydroxy-piperidine hydrochloride (15.HCI)

[0088] The thiol-ene reaction protocol utilizing terf-butanethiol provided the thioether 15.HCI as a colorless foam (11 mg, 38 %). ¹ H NMR (400.2 MHz, D ₂ 0) δ = 1 .38 [s, 9 H, C(CH ₃ ) ₃ ], 1.92-2.04 (m, 1 H, CH ₂ CH ₂ S), 2.13-2.25 (m, 1 H, CH ₂ CH ₂ S), 2.67-2.85 (m, 2 H, CH ₂ CH ₂ S), 3.33-3.40 (m, 1 H, H-6), 3.45-3.52 (m, 1 H, H-6), 3.63- 3.69 (m, 1 H, H-2), 4.01 -4.12 (m, 3 H, H-3,4,5); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 24.1 (CH ₂ CH ₂ S), 29.2 (CH ₂ CH ₂ S), 31.0 (CH ₃ ), 44.0 [C(CH ₃ ) ₃ ], 46.5 (C-6), 55.3 (C-2), 67.0, 68.0, 68.4 (C-3,4,5). HRMS (ES): m/z = 250.1472; [M + H] ⁺ requires 250.1477.

(xx) (2R, 3S,4S, 5R)-3, 4, 5- Trihydroxy-2-[2-(3-methyl-butylsulfanyl)-ethyl]- piperidine hydrochloride (16.HCI)

[0089] The thiol-ene reaction protocol utilizing 3-methyl-1-butanethiol provided the thioether 16.HCI as a colorless foam (17 mg, 56 %). ¹ H NMR (400.2 MHz, D ₂ 0) δ = 0.91 (ad, 6 H, J = 6.4 Hz, CH ₃ ), 1 .45- 1 .54 [m, 2 H, CH ₂ CH(CH ₃ ) ₂ ], 1.67 [a. sept., 1 H, J = 6.4 Hz, CH(CH ₃ ) ₂ ], 1.94-2.06 (m, 1 H, NCHCH ₂ CH ₂ S), 2.10-2.22 (m, 1 H, NCHCH ₂ CH ₂ S), 2.60-2.80 (m, 4 H, CH ₂ SCH ₂ ), 3.30-3.38 (m, 1 H, H-6), 3.43-3.50 (m,

1 H, H-6), 3.64-3.71 (m, 1 H, H-2), 3.97-4.10 (m, 3 H, H-3,4,5); ³ C NMR (100.6 MHz, D ₂ 0) δ = 21.55, 21.57 (CH ₃ ), 26.3 (NCHCH ₂ CH ₂ S), 26.9 [CH(CH ₃ ) ₂ ], 27.4 (NCHCH ₂ CH ₂ S), 29.1 [SCH ₂ CH ₂ CH(CH ₃ )2], 37.8 [CH ₂ CH(CH ₃ ) ₂ ], 45.5 (C-6), 54.1 (C-2), 66.0, 67.0, 67.5 (C-3,4,5). HRMS (ES): m/z = 264.1628; [M + H] ⁺ requires 264.1633.

(xxi) (2R, 3S, 4S, 5R)-2-(2-Cyclopentylsulfanyl-ethyl)-3, 4, 5-trihydroxy-piperidine hydrochloride (17.HCI)

[0090] The thiol-ene reaction protocol utilizing cyclopentanethiol provided the thioether 17.HCI as a colorless foam (19 mg, 63 %). ¹ H NMR (400.2 MHz, D ₂ 0) δ = 1 .43- 1.79 (m, 6 H, SCHCH ₂ CH ₂ ), 1.95-2.24 (m, 4 H, CH ₂ CH ₂ SCHCH ₂ ), 2.66-2.83 (m,

2 H, CH ₂ S), 3.23 (a. quin., 1 H, J = 6.8 Hz, SCH), 3.30-3.38 (m, 1 H, H-6), 3.43-3.50 (m, 1 H, H-6), 3.63-3.71 (m, 1 H, H-2), 3.97-4.09 (m, 3 H, H-3,4,5); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 24.3, 24.4 (SCHCH ₂ CH ₂ ), 26.3 (CH ₂ S), 27.7 (CH ₂ CH ₂ S), 33.26, 33.30 (SCHCH ₂ ), 43.3 (SCH), 45.5 (C-6), 54.2 (C-2), 66.0, 67.0, 67.5 (C-3,4,5). HRMS (ES): m/z = 262.1482; [M + H] ⁺ requires 262.1477. (xxii) (2R, 3S, 4S, 5R)-2-(2-Cyclohexylsulfanyl-ethyl)-3, 4, 5-trihydroxy-piperidine hydrochloride (18.HCI)

[0091] The thiol-ene reaction protocol utilizing cyclohexanethiol provided the thioether 18.HCI as a colorless foam (18 mg, 59 %). ¹ H NMR (400.2 MHz, D ₂ 0) δ = 1.19-1 .41 (m, 5 H, SCHCH ₂ CH ₂ CH ₂ ), 1.56- 1.66 (m, 1 H, SCHCH ₂ CH ₂ CH ₂ ), 1 .69- 1 .81 (m, 2 H, SCHCH ₂ CH ₂ ), 1.92-2.04 (m, 3 H, CH ₂ CH ₂ SCHCH ₂ ), 2.09-2.22 (m, 1 H, CH ₂ CH ₂ S), 2.65-2.89 (m, 3 H, CH ₂ SCH), 3.30-3.38 (m, 1 H, H-6), 3.43-3.50 (m, 1 H, H-6), 3.63-3.70 (m, 1 H, H-2), 3.97-4.10 (m, 3 H, H-3,4,5); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 24.5 (CH ₂ S), 25.4 (SCHCH ₂ CH ₂ CH ₂ ), 25.62, 25.63 (SCHCH ₂ CH ₂ ), 27.9 (CH ₂ CH ₂ S), 33.1 , 33.2 (SCHCH ₂ ), 43.1 (SCH), 45.5 (C-6), 54.1 (C-2), 66.0, 67.0, 67.5 (C-3,4,5). HRMS (ES): m/z = 276.1627; [M + H] ⁺ requires 276.1633.

(xxiii) (2R, 3S, 4S, 5R)-2-(2-Benzylsulfanyl-ethyl)-3, 4, 5-trihydroxy-piperidine hydrochloride (19.HCI)

[0092] The thiol-ene reaction protocol utilizing benzylmercaptan provided the thioether 19.HCI as a colorless foam (16 mg, 50 %). H NMR (400.2 MHz, D ₂ 0) δ = 1 .92-2.03 (m, 1 H, CH ₂ CH ₂ S), 2.05-2.16 (m, 1 H, CH ₂ CH ₂ S), 2.56-2.72 (m, 2 H, CH ₂ CH ₂ S), 3.29-3.36 (m, 1 H, H-6), 3.41 -3.48 (m, 1 H, H-6), 3.57-3.65 (m, 1 H, H-2), 3.84-3.87 (m, 2 H, SCH ₂ Ph), 3.88-4.09 (m, 3 H, H-3,4,5), 7.34-7.68 (m, 5 H, Ph); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 26.0 (CH ₂ CH ₂ S), 27.2 (CH ₂ CH ₂ S), 35.1 (SCH ₂ Ph), 45.5 (C- 6), 54.1 (C-2), 66.0, 67.0, 67.6 (C-3,4,5), 127.5, 129.0, 129.1 , 138.5 (Ph). HRMS (ES): m/z = 284.1315; [M + H] ⁺ requires 284.1320.

(xxiv) (2R, 3S, 4S, 5R)-3, 4, 5-trihydroxy-2-(2-phenethylsulfanyl-ethyl)-piperidine hydrochloride (20.HCI)

[0093] The thiol-ene reaction protocol utilizing phenethylmercaptan provided the thioether 20.HCI as a colorless foam (19 mg, 56 %). ¹ H NMR (400.2 MHz, D ₂ 0) δ = 1 .92-2.04 (m, 1 H, CHCH ₂ CH ₂ S), 2.08-2.20 (m, 1 H, CHCr7 ₂ CH ₂ S), 2.61 -2.78 (m, 2 H, CHCH ₂ Ctf ₂ S), 2.90-3.03 [m, 4 H, S(CH ₂ ) ₂ Ph], 3.31 -3.38 (m, 1 H, H-6), 3.42-3.49 (m, 1 H, H-6), 3.58-3.66 (m, 1 H, H-2), 3.94-4.10 (m, 3 H, H-3,4,5), 7.30-7.47 (m, 5 H, Ph); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 26.4 (CHCH ₂ CH ₂ S), 27.3 (CHCH ₂ CH ₂ S), 32.5, 34.8 (S(CH ₂ ) ₂ P ), 45.5 (C-6), 54.0 (C-2), 66.0, 67.0, 67.5 (C-3,4,5), 126.7, 128.8, 128.9, 140.7 (Ph). HRMS (ES): m/z = 298.1480; [M + H] ⁺ requires 298.1477.

(xxv) (2R, 3S, 4S, 5R)-2-[2-(2-butyrylamino-ethylsulfanyl)-ethyl]-3, 4, 5-trihydroxy- piperidine hydrochloride (21.HCI)

[0094] The thiol-ene reaction protocol utilizing A/-(2-mercaptoethyl)-butyramide provided the thioether 21.HCI as a colorless foam (15 mg, 43 %). ¹ H NMR (400.2 MHz, D ₂ 0) δ = 0.96 (t, 3 H, J = 7.2 Hz, CH ₃ ), 1 .66 (tq, 2 H, J = 7.2, 7.2 Hz, CH ₂ CH ₃ ), 1 .98- 2.09 (m, 1 H, CHCH ₂ CH ₂ S), 2.14-2.25 (m, 1 H, CHCH ₂ CH ₂ S), 2.28 (t, 2 H, J = 7.2 Hz, CH ₂ CH ₂ CH ₃ ), 2.68-2.86 (m, 4 H, CH ₂ SCH ₂ ), 3.34-3.42 (m, 1 H, H-6), 3.44-3.54 (m, 3 H, CH ₂ CH ₂ N, H-6), 3.67-3.74 (m, 1 H, H-2), 4.01 -4.15 (m, 3 H, H-3,4,5); ³ C NMR (100.6 MHz, D ₂ 0) δ = 12.8 (CH ₃ ), 19.1 (CH ₂ CH ₃ ), 26.3 (CHCH ₂ CH ₂ S), 27.4 (CHCH ₂ CH ₂ S), 30.5 (SCH ₂ CH ₂ N), 37.8 (CH ₂ CH ₂ CH ₃ ), 38.5 (SCH ₂ CH ₂ N), 45.6 (C-6), 54.0 (C-2), 66.0, 67.0, 67.5 (C-3,4,5), 177.4 (C=0). HRMS (ES): m/z = 307.1685; [M + H] ⁺ requires 307.1692.

(xxvi) (2R, 3S, 4S, 5R)-3, 4, 5-trihydroxy-2-{2-[2-(phenylaminocarbonyl)amino- ethylsulfanyl]-ethyl}-piperidine hydrochloride (22.HCI)

[0095] The thiol-ene reaction protocol utilizing 1-(2-mercaptoethyl)-3-phenyl-urea provided the thioether 22.HCI as a colorless foam (19 mg, 48 %). H NMR (400.2 MHz, D ₂ 0) δ = 1 .96-2.08 (m, 1 H, CHCH ₂ CH ₂ S), 2.1 1 -2.23 (m, 1 H, CHCH ₂ CH ₂ S), 2.67- 2.84 (m, 4 H, CH ₂ SCH ₂ ), 3.28-3.36 (m, 1 H, H-6), 3.40-3.49 (m, 3 H, CH ₂ CH ₂ N, H-6), 3.64-3.72 (m, 1 H, H-2), 3.97-4.09 (m, 3 H, H-3,4,5), 7.17-7.46 (m, 5 H, Ph); ³ C NMR (100.6 MHz, D ₂ 0) δ = 26.3 (CHCH ₂ CH ₂ S), 27.4 (CHCH ₂ CH ₂ S), 31 .3 (SCH ₂ CH ₂ N), 39.0 (SCH ₂ CH ₂ N), 45.5 (C-6), 54.0 (C-2), 66.0, 67.0, 67.5 (C-3,4,5), 121 .7, 124.3, 129.4, 138.0 (Ph), 158.4 (C=0). HRMS (ES): m/z = 356.1639; [M + H] ⁺ requires 356.1644.

(xxvii) (2R, 3S, 4S, 5R)-2-[2-(N-benzyl-2-acetamidyl)sulfanyl-ethyl]-3, 4, 5- trihydroxy-piperidine hydrochloride (23.HCI)

[0096] The thiol-ene reaction protocol utilizing give A/-benzyl-2-mercapto- acetamide provided the thioether 23.HCI as a colorless foam (15 mg, 41 %). H NMR (400.2 MHz, D ₂ 0) δ = 1.91 -2.03 (m, 1 H, CH ₂ CH ₂ S), 2.04-2.18 (m, 1 H, CH ₂ CH ₂ S), 2.61 -2.78 (m, 2 H, CH ₂ CH ₂ S), 3.27-3.35 (m, 1 H, H-6), 3.37 (SCH ₂ CO), 3.40-3.47 (m, 1 H, H-6), 3.56-3.63 (m, 1 H, H-2), 3.87-4.07 (m, 3 H, H-3,4,5), 4.44 (NCH ₂ Ph), 7.34- 7.48 (m, 5 H, Ph); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 27.1 , 27.2 [(CH ₂ ) ₂ S], 34.8 (SCH ₂ CO), 43.4 (NCH ₂ Ph), 45.5 (C-6), 53.9 (C-2), 66.0, 66.9, 67.4 (C-3,4,5), 127.5, 127.7, 128.9, 137.9 (Ph), 172.5 (C=0). HRMS (ES): m/z = 341.1526; [M + H] ⁺ requires 341.1535.

(xxviii) (2R, 3S, 4S, 5R)-2-[2-(N, N-Diethyl-4-butyramidyl)sulfanyl-ethyl]-3, 4, 5- trihydroxy-piperidine hydrochloride (24.HCI)

[0097] The thiol-ene reaction protocol utilizing A/,A/-diethyl-4-mercapto- butyramide provided the thioether 24.HCI as a colorless foam (18 mg, 47 %). H NMR (400.2 MHz, D ₂ 0) δ = .10 (t, 3 H, J = 7.2 Hz, CH ₃ ), .20 (t, 3 H, J = 7.2 Hz, CH ₃ ), 1 .85- 2.06 (m, 3 H, CHCH ₂ CH ₂ SCH ₂ CH ₂ ), 2.10-2.21 (m, 1 H, CHCH ₂ CH ₂ S), 2.50-2.79 (m, 6 H, CH ₂ SCH ₂ CH ₂ CH ₂ ), 3.29-3.50 (m, 6 H, NCH ₂ CH ₃ , H-6,6), 3.64-3.71 (m, 1 H, H-2), 3.97-4.09 (m, 3 H, H-3,4,5); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 12.2, 13.3 (CH ₃ ), 24.9 (SCH ₂ CH ₂ CH ₂ ), 26.2 (CHCH ₂ CH ₂ S), 27.4 (CHCH ₂ CH ₂ S), 30.4, 31.5 (SCH ₂ CH ₂ CH ₂ ), 40.9, 43.1 (NCH ₂ CH ₃ ), 45.5 (C-6), 54.0 (C-2), 66.0, 67.0, 67.5 (C-3,4,5), 174.7 (C=0). HRMS (ES): m/z = 335.2010; [M + H] ⁺ requires 335.2005.

(xxix) (2R, 3S, 4S, 5R)-3, 4, 5-trihydroxy-2-[2-(4-Morpholin-4-yl-4-oxo-butyl)sulfanyl- ethyl]- piperidine hydrochloride (25.HCI)

[0098] The thiol-ene reaction protocol utilizing 4-mercapto-1 -(morpholin-4-yl)- butan-1-one provided the thioether 25.HCI as a colorless foam (15 mg, 39 %). H NMR (400.2 MHz, D ₂ 0) δ = 1.88-2.08 (m, 3 H, CHCH ₂ CH ₂ SCH ₂ CH ₂ ), 2.12-2.24 (m, 1 H, CHCH ₂ CH ₂ S), 2.57-2.82 (m, 6 H, CH ₂ SCH ₂ CH ₂ CH ₂ ), 3.33-3.40 (m, 1 H, H-6), 3.46- 3.53 (m, 1 H, H-6), 3.61 -3.73 (m, 5 H, NCH ₂ CH ₂ 0, H-2), 3.74-3.82 (m, 4 H, NCH ₂ CH ₂ 0), 4.00-4.13 (m, 3 H, H-3,4,5); ¹³ C NMR (100.6 MHz, D ₂ 0) δ = 24.6 (SCH ₂ CH ₂ CH ₂ ), 26.3 (CHCH ₂ CH ₂ S), 27.4 (CHCH ₂ CH ₂ S), 30.4, 31.4 (SCH ₂ CH ₂ CH ₂ ), 42.3 (NCH ₂ CH ₂ 0), 45.5 (C-6), 46.3 (NCH ₂ CH ₂ 0), 54.1 (C-2), 66.1 , 67.0 (C-4,5), 66.5 (NCH ₂ CH ₂ 0), 67.6 (C-3), 174.3 (C=0). HRMS (ES): m/z = 349.1802; [M + H] ⁺ requires 349.1797.

Discussion [0099] The inhibition constants of compounds 10-25, against GBA were determined at a pH of 5.5 (see Table 1 ). All of these compounds were found to inhibit GBA in a competitive manner. Every thiol-ene product had a sub-micromolar K, value, with one, compound 13, having a sub-nanomolar K _{ value.

[00100] The specificity of this collection of inhibitors for GBA over other cellular glycosidases was subsequently evaluated. None of the compounds inhibited human o glucosidase, a-galactosidase or β-galactosidase even at concentrations as high as 200 μΜ, with the one exception being compound 13, which inhibited human neutral cytosolic β-glucosidase [Tribolo, S., J. G. Berrin, et al. (2007). "The crystal structure of human cytosolic beta-glucosidase unravels the substrate aglycone specificity of a family 1 glycoside hydrolase." J Mol Biol 370(5): 964-975.] with an IC ₅₀ of 2 μΜ.

Cell-based Assay for Pharmacological Chaperone Activity

GD patient fibroblasts homozygous for the p.N370S or p.L444P mutation were treated with each of the compounds in Table 1 to determine if any could behave as pharmacological chaperones for GBA in cellulo. The simple vinyl and ethyl derivatives 3 and 9, low micromolar inhibitors of GBA showed minimal levels of enzyme enhancement (see Figures 1 and 2) (Figure 1 shows the Increased GBA activity in compound-treated fibroblasts derived from a GD patient with the p.N370S GBA mutation. Patient fibroblasts were grown in the presence of each of the compounds for five days. GBA activity in the lysates from treated cells was normalized to activity in untreated cells; Figure 2 shows the increased GBA activity in compound-treated fibroblasts derived from a GD patient with the p.L444P GBA mutation. Patient fibroblasts were grown in the presence of each of the compounds for five days. GBA activity in the lysates from treated cells was normalized to activity in untreated cells.) All of the other compounds were capable of increasing GBA activity at one concentration or another in fibroblasts homozygous for the p.N370S or p.L444P mutations in GBA (see Figures 1 and 2, Table 1 , where compounds 2, 3 and 9 have the following structure):

2 3 9

Improvements in GBA activity were observed for cells harbouring the p.N370S mutation than for those with the p.L444P mutation. The amide/urea-containing compounds 21-25 elicited the highest GBA activity, managing a 2.4-3.1 fold increase in GBA activity in the p.N370S cell line and a 1 .4 fold increase in the p.L444P cell line.

A subset of four compounds (13, 14, 20 and 23), were picked from each of the structural subclasses on the basis of their appended ligature (alkyl, branched, cyclic and amide/urea) and were evaluated over a wider range of concentrations. This revealed that, above a certain concentration for each compound, the GBA activity in the cell lysate decreased (see Figure 3 which shows GBA activity in GD patient fibroblasts homozygous for the p.N370S GBA mutation when treated with representative compounds from each subclass. Patient fibroblasts were grown in the presence of each of the compounds for five days. GBA activity in lysates from treated cells was normalized to activity in untreated cells).

[00101] There is a direct linear correlation between K, values and the concentration at which a half maximal increase in GBA activity (AC ₅₀ ) is observed (see Figure 4). Compounds 21 -25 promote the greatest enhancement in GBA activity for both cell lines. Table 2 provides tabulated data for the Figures 4 and 5.

[00102] Compounds shown to act as pharmacological chaperones for GBA (or other lysosomal enzymes for that matter) also stabilize the enzyme against thermal denaturation. Since the ability of a pharmacological chaperone to stabilise GBA is most relevant in the ER, the melting temperature of this protein was determined at pH 7.0 in the absence and presence of saturating concentrations of each inhibitor. The difference between the melting temperatures of free and inhibitor-bound GBA for each compound is presented in Table 1 . An inverse linear relationship was observed between the , values of each compound and the increase in melting temperature (Figure 5). [00103] On average there is a 20-35% increase wild type GCase activity levels when either mouse neuronal precursor cells (see Figure 6 which shows Compound 14 increases GCase Activity levels in mouse neurospheres overexpressing WT SNCA. A. Wild type Neural Precursor Cells (NPC) were isolated from mice brains and cultured in vitro. NPC were treated for five days with Compound 14, subsequently lysed and lysosomal enzymes were bound to concanavilin A conjugated beads and subsequently evaluated in terms of GCase activity in treated cells compared to untreated cells, ^* p < 0.05, ^*** p <0.001 ) or human fibroblasts (see Figure 7 which shows compound 14 increases GCase Activity levels in wild type fibroblasts. A. Wild fibroblasts were treated for five days with Compound 14 subsequently lysed and lysosomal enzymes were bound to concanavilin A conjugated beads and evaluated in terms of GCase and beta- hexosaminidase activity in treated cells compared to untreated cells. To control for cell number GCase activity was first normalized to beta-hexosaminidase activity. Normalized GCase activity relative to the activity in untreated cells in shown) are treated for five days with iminoxylitol compound 14. This increase is less than the 2-3 fold increase that results from treatment of Gaucher patient fibroblasts expressing the mutant GCase bearing the N370S (see Figure 1 ). However, when one takes into consideration the specific activity of the WT enzyme, the absolute magnitude of the activity increase associated with WT GCase is greater than that observed with the mutant enzyme. For example, the average specific activity of WT human Gcase from skin fibroblasts for GC is 463 nmoles/hr/mg [Grabowski, G.A., Goldblatt, J., Dinur, T., Kruse, J., Svennerholm, L, Gatt, S., and Desnick, R.J. (1985). Genetic heterogeneity in Gaucher disease: physicokinetic and immunologic studies of the residual enzyme in cultured fibroblasts from non-neuronopathic and neuronopathic patients. Am J Med Genet 21 , 529-549]. An increase of 20-35% following treatment with CMPD 4 (see Figures 5,6) corresponds to an increase of 93-162 nmoles/h/mg. In contrast GCase in Type I patients has an average specific activity of 62 nmoles/h/mg (13.4% residual) [Grabowski, G.A., Goldblatt, J., Dinur, T., Kruse, J., Svennerholm, L, Gatt, S., and Desnick, R.J. (1985). Genetic heterogeneity in Gaucher disease: physicokinetic and immunologic studies of the residual enzyme in cultured fibroblasts from non- neuronopathic and neuronopathic patients. Am J Med Genet 21 , 529-549], if this activity is increased by 2-3 fold (see Figure 1 ) this represents an absolute increase of 62-124 nmoles/h/mg. The L444P allele, more common in the non-Ashkenazi Jewish population and associated with type 2 patients, has an average specific activity of 14.9 (3.4% of Normal values). Treatment of this allele with the iminoxylitol compounds, typically results in a 20-30% fold increase which corresponds to an additional increase of 2-3 nmoles/h/mg (see Figure 2). Although these examples rely of the specific activity of GCase from skin fibroblasts, the variation in GCase levels in different tissue is estimated from mouse studies to be no more than two-fold [Liu, Y., Suzuki, K., Reed, J.D., Grinberg, A., Westphal, H., Hoffmann, A., Doring, T., Sandhoff, K., and Proia, R.L. (1998). Mice with type 2 and 3 Gaucher disease point mutations generated by a single insertion mutagenesis procedure. Proc Natl Acad Sci U S A 95, 2503-2508]. Based on the distribution of GCase activities in the normal population, there is an approximately two-fold difference between quartiles, e.g. a normal range of 460± 230 nmoles/h/mg [Li, Y., Scott, C.R., Chamoles, N.A., Ghavami, A., Pinto, B.M., Turecek, F., and Gelb, M.H. (2004). Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening. Clin Chem 50, 1785-1796]. Since heterozygotes bearing one of the Gaucher alleles have an increased risk of developing PD [Lesage, S., Anheim, M., Condroyer, C, Pollak, P., Durif, F., Dupuits, C, Viallet, F., Lohmann, E., Corvol, J.C., Honore, A., et al. (201 1 ). Large-scale screening of the Gaucher's disease-related glucocerebrosidase gene in Europeans with Parkinson's disease. Hum Mol Genet 20, 202-210 Sidransky, E., Nails, M.A., Aasly, J.O., Aharon-Peretz, J., Annesi, G., Barbosa, E.R., Bar-Shira, A., Berg, D., Bras, J., Brice, A., et al. (2009). Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease. N Engl J Med 361 , 1651 -1661] it can be assumed that the minimal threshold for GCase activity associated with the increased risk is between 25-75% of the average normal GCase specificity activity, which overlaps with the range of activities seen in individuals that do not carry a Gaucher allele, i.e. 150-50% of the normal average. Given the most recent model describing the connection between decreased GCase activities levels, leading to increased steady-state levels of its GC substrate, which in turn promotes synuclein aggregation [Mazzulli, J.R., Xu, Y.H., Sun, Y., Knight, A.L., McLean, P. J., Caldwell, G.A., Sidransky, E., Grabowski, G.A., and Krainc, D. (201 1 ). Gaucher disease glucocerebrosidase and alpha-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell 146, 37-52.] there would be a presumed therapeutic benefit associated with raising WT GCase activity even in the population of PD patients lacking a Gaucher allele.

Table 1. A comparison of kinetic, biophysical and biological parameters for each of the dideoxyiminoxylitol derivatives.

A actvty was ac eve n auc er patent i ro asts omozygous or t e 70S mutaton.

Table 2: Summary of Biochemical properties of compounds