Pharmacological Chaperones for Enzyme Treatment Therapy

FIELD OF THE INVENTION

The present invention relates generally to the field of therapeutics for lysosomal storage diseases and glycosidase deficiency related diseases. More specifically, the invention relates to various combinations of enzyme replacement therapy or gene therapy, for the treatment of lysosomal storage diseases. It further relates to cyclophellitol cyclosulfamidates as pharmacological chaperones of glycosidases, compositions comprising these compounds, and their therapeutic uses, as well as pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof.

BACKGROUND OF THE INVENTION

Lysosomal storage disorders (LSDs) are diseases typically involving a compromised enzyme, in particular a lysosomal hydrolase. Typically, the activity of a single lysosomal hydrolytic enzyme is compromised, either reduced, or lacking altogether, usually due to the inheritance of an autosomal recessive mutation. As a consequence, the nominal substrates of the compromised enzyme accumulate in the lysosomes, producing a severe disruption of the cellular architecture, and resulting in various manifestations of the disease. Lysosomal storage disorders include sphingolipidoses and other complex carbohydrate metabolism disorders, such as those include diseases known as Gaucher, Niemann-Pick, Farber, GMl-gangliosidosis, GM2-gangliosidosis, Tay-Sachs, Krabbe, Fabry, Schindler, Pompe, Fucosidosis, Mannosidosis, and Wolman's disease, as well as disorders that show aberrant accumulation of a-synuclein (a-syn) or such as Parkinson's disease (PD) or Lewy-body dementia.

A somewhat successful approach to treating many of the acute LSDs is the so-called Enzyme Replacement Therapy (ERT). This therapy involves intravenous administration of an exogenously- produced natural or recombinant, replacement enzyme to a patient.

As an example, Fabry disease is characterized by the toxic accumulation of globotriaosylceramide (Gb3) in lysosomes and globotriaosyl-sphingosine (Lyso-Gb3) in a patient's plasma and tissue, due to a deficiency in a patient's a-galactosidase A (a-gal A).

In particular for this disease, a recombinant human a-galactosidase, such as Fabrazyme ^® from Genzyme, see also US patent 7,011,831, has been found to be particularly useful for treating a deficiency in a-galactosidase A (a-gal A). This ERT usually involves bi-weekly infusions of Fabrazyme ^® at a dose of 1.0 and 0.2 mg/kg body weight. A similar approach has recently been applied for treating Pompe disease, by an ERT based on recombinant GAA (Myozyme; Genzyme, Inc.), see also US-A- 6,537,785. An unsolved problem with ERTs for treating LSDs has remained in the development of immunity by patients to the enzymes of the ERTs upon repeated treatment. Antibodies developed by patients against ERT enzymes not only reduce the enzymes' efficacy but also put patients at risk of anaphylactic shock. This is particularly pronounced in hemizygous Fabry male patients, which produce no a-Gal A at all.

As set out above, the elicited antibody response to the therapeutic protein also usually results in a decreased effect of the ERT during further treatment of Fabry patients.

Another problem with ERTs for treating LSDs remains the lack of stability of the ERT enzymes in the human body. As a result, only a small amount of the injected enzymes reaches in active form the target lysosome of the patients.

Another problem with conventional ERT treatments for treating LSDs is the stressful high frequency of required infusions, typically involving intravenous injections on a weekly or bi-weekly basis, with comparatively high amounts of enzymes.

An alternative therapy approach for lysosomal storage disease has focussed on the use of small molecules for treatment, whereby a class of molecules was found to inhibit upstream generation of lysosomal hydrolase substrates to relieve the input burden to the defective enzyme, in the so-called "substrate reduction therapy" (SRT). An example of this class of molecules are deoxynojirimycin (DNJ) and derivatives thereof, that inhibit glucosylceramide synthase (GCS). Certain uses of GCS inhibitors DNJ type either alone (WO 00/62780) or in combination with a glycolipid degrading enzyme (WO 00/62779) have been described. Another example of the substrate deprivation class of molecules are the amino ceramide-like small molecules which have been developed for glucosylceramide synthase inhibition. Glucosylceramide synthase catalyzes the first glycosylation step in the synthesis of glucosylceramide-based glycosphingolipids. Glucosylceramide itself is the precursor of hundreds of different glycosphingolipids. Also, amino ceramide-like compounds have been developed for use in Fabry and Gaucher's disease, see for instance US-B-5,916,911 and US6,051,598. However, these are directed towards the defective enzyme, and hence will disturb the molecular pathways whereby patients are effectively deprived of the products of normal metabolism.

Generally, ERT methods are known, as set out for instance in AU2017268649, which discloses a method to predict response to pharmacological chaperone treatment of diseases; US2011152319, which discloses Methods for Treatment of Fabry Disease; US2010113517, which discloses a method for the treatment of Fabry disease using pharmacological chaperones; JP2018027945, which discloses high concentration oc-glucosidase compositions for the treatment of Pompe disease; US2016051528 which discloses a method for the Treatment of Pompe Disease Using 1-Deoxynojirimycin Derivatives; US2011136151 , which discloses assays for diagnosing and evaluating treatment options for Pompe disease; CN103373955, which discloses multivalent azasugar derivatives and synthetic method thereof; JP2009137933 disclosing a method for treating Gaucher's disease; W02004037373 (A2) : Chemical chaperones and their effect upon the cellular activity of beta-glucosidase; US5242942 (A): Anticonvulsant fructopyranose cyclic sulfites and sulfates and W02016162588 (Al): Compositions for treating diseases related to lysosomal storage disorders.

However, none of the disclosed methods results in an in-situ reversible composition that maintains the activity of the enzyme sufficiently high.

An approach to stabilize the enzyme is disclosed in EP2874648 which suggests an a- galactosidase A and 1-deoxygalactonojirimycin (also known as "migalastat" or galacto-DNJ) coformulation for the ERT treatment of Fabry disease. Herein, the 1-deoxygalactonojirimycin is disclosed as to act acts as a pharmacological chaperone for a specific mutant a-galactosidase A, by selectively binding to the enzyme, thereby increasing the amount of active enzyme in a patient's lysosomes. However, this composition is limited to specific conformations and mutations of this particular enzyme.

Accordingly, there has remained a need to improve ERTs for treating LSDs to overcome significant limitations associated with these treatment modalities when used alone or with existing combination regimens. A further need to improve ERTs has involved reducing the frequency and/or size of doses of ERTs.

A multigene therapy is under phase 2 clinical trials for Fabry disease (AVR-RD-01), for the endogenous expression of a modified lysosomal a-A/-acetyl-galactosaminidase (ot-NAGAL) with increased a-galactosidase activity (ot-NAGAL ^EL ). This is however still in the early stages. It is considered that also modified lysosomal a-NAGAL with increased a-galactosidase activity (a-NAGAL ) may require stabilisation.

WO-A-2015/123385 discloses a number of different compounds that may act as reversible glycosidase inhibitors for treatment of Sanfilippo type C disease. WO-A-2004/06919 discloses various imino sugars, while WO-A-2010/015816, other than a very large list of various imino sugars also discloses (l/?,2S,3S,4S,5/?,6S)-5-amino-6-hydroxymethyl)cyclohexane-l, 2,3,4-tetraol, which showed some inhibition effect on a-mannosidase. Zhaozhong Jia et al: "Ready routes to key myo-inositol component of GPis employing microbial arene oxidation or Ferrier reaction", Journal of the Chemical Society, Perkin Transactions 1, 1998-01-01, p. 631-632, discloses a preparation route for myo-inositol derivatives. Marta Artola, et al, entitled "1,6-Cyclophellitol Cyclosulfates: A New Class of Irreversible Glycosidase Inhibitor", ACS Central Science, part 3, No. 7, (2017-07-13), pages 784-793, discloses cyclic sulfates which are irreversible inhibitors for glycosidase enzymes and hence not at all useful for reversibly stabilizing enzymes. Accordingly, there emerges a need to stabilize endogenously expressed modified lysosomal enzymes for treating LSDs. The present invention meets this need by providing improved approaches utilizing a combination of enzyme replacement therapy and a chaperone therapy for treating LSDs. In this chaperone therapy; the compounds according to the invention aim to pharmacological chaperone, i.e. stabilize a therapeutic recombinant enzyme.

SUMMARY OF THE INVENTION

This invention provides various combinations of enzyme replacement therapy or (multi)gene therapies for the treatment of lysosomal storage diseases and glycosidase deficiency related diseases. Several general approaches are provided. Each general approach involves enzyme replacement therapy (ERT) or gene therapy with an enzyme chaperone in a manner which optimizes the clinical benefit, i.e., treatment, while minimizing disadvantages associated with ERT.

An embodiment of the invention provides for a composition for treating a lysosomal storage disease and/or glycosidase and/or a-glucosidase deficiency related diseases, particularly Fabry, Pompe, or Gaucher disease, comprising:

a) an exogenously produced, natural or recombinant lysosomal hydrolase, or an endogenous produced natural mutant lysosomal hydrolase by (multi)gene therapy; and

b) one or more reversible cyclophellitol-derived glycosidase inhibitors capable of competitively blocking the active site of the lysosomal hydrolase.

Preferably, the composition concerns treatments where the lysosomal hydrolase is an a- galactosidase, an a-glucosidase, or a b-glucosidase.

More preferably, the reversible glycosidase inhibitor is a reversible glycosidase inhibitor based on a cyclic sulfamidate functional group, more preferably a reversible glycosidase inhibitor comprising a cyclic sulfamidate functional group.

In an embodiment, the reversible glycosidase inhibitor has a structure according to formula I:

wherein each of R ² to R ⁴ individually equals H, a lower alkyl, a lower alkenyl, and/or a lower alkynyl group, and wherein X represents O, S or NfR ¹ ), wherein R ¹ each individually represent H, a lower substituted alkyl such as (CH ₂ ) _n X wherein X is CH ₃ , OH, NH ₂ , 1, Br, Cl or CF ₃ , and n ranges of from 0 to 5; wherein R ⁵ represents -OH, -CH ₂ OH, -CH ₂ 0-alkyl, or -CH ₂ 0-glycoside. Preferably, the present invention also relates to a compound (I) wherein X represents 0 or NfR ¹ ),, and wherein R ⁵ represents -OH, -CH ₂ OH, -CH ₂ 0-alkyl, or -CH ₂ 0-glycoside; and/or pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures, including both R and 5 enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof.

Preferably, the reversible glycosidase inhibitor has the structure according to formula la:

wherein X represents -I^R ¹ )- or oxygen, preferably wherein one of X represents -NlR ¹ )-, the other X represents oxygen, more preferably wherein the X at the exposition represents-NiR ¹ )- and the X at the exposition represents oxygen,

wherein R ¹ each individually represent H, a lower substituted alkyl such as (CH ₂ ) _n X wherein X is CH ₃ , OH, f\IH ₂ , 1, Br, Cl or CF ₃ , and n ranges of from 0 to 5;

an optionally substituted (hetero)aryl group; or a carboxy group (CO)Z wherein Z represents (CH2) _n CH3, -OH, or -NH ₂ ;

wherein each of R ² to R ⁴ individually represents H, a lower substituted alkyl such as (CH ₂ ) _n Y wherein Y represents -CH ₃ , -OH, -f\IH ₂ , -I, -Br, -Cl or -CF ₃ , and n ranges of from 0 to 5 and/or an optionally substituted (hetero)aryl group; or a carboxy group (CO)X, wherein X represents (CH ₂ ) _n CH ₃ , OH, NH ₂ , or CF ₃ and n = 0-5, and/or an optionally substituted (hetero)aryl group;

wherein R ⁵ represents -OH, -CH ₂ OH, -CH ₂ 0-alkyl, or -CH ₂ 0-glycoside. Preferably the present invention also relates to pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof.

Preferably for Fabry disease, the a-galactosidase is a recombinant human a-galactosidase or an endogenous produced natural mutant a-galactosidase by (multi)gene therapy, preferably Fabrazyme ^® , and preferably, the reversible glycosidase inhibitor comprises an a-gal-cyclic sulfamidate, or a form suitable for administration and for forming the inhibitor in situ. A particularly preferred a-gal-cyclic sulfamidate is an a-gal 1,6 epi-cyclophellitol cyclosulfamidate of the formula I, wherein each of R ¹ to R ⁵ individually equals H, a lower alkyl, a lower alkenyl, a lower alkynyl group and/or an optionally substituted (hetero)aryl group.

Alternatively, in the case of patients suffering from Pompe disease, the a-glucosidase is a recombinant acid human a-glucosidase or an endogenous produced natural mutant a-glucosidase by (multi)gene therapy, preferably Myozyme ^® , and preferably, the reversible glycosidase inhibitor comprises an a-g/c-cyclic sulfamidate, or a form suitable for administration and for forming the inhibitor in situ.

A particularly preferred a-glc-c yclic sulfamidate is an a-glc l,6-ep/-cyclophellitol cyclosulfamidate of the formula I, wherein each of R ¹ to R ⁵ individually equals H, a lower alkyl, a lower alkenyl, a lower alkynyl group and/or an optionally substituted (hetero)aryl group.

The present invention also relates to the composition according to the invention for administering to a patient at substantially the same time the lysosomal hydrolase and the reversible glycosidase inhibitor.

Another embodiment of the invention provides a composition useful for treating a lysosomal storage disease, particularly Fabry or Pompe disease, respectively, comprising: a) the exogenously produced, natural or recombinant lysosomal hydrolase; b) the reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase.

Another embodiment of the invention provides a kit useful for treating a lysosomal storage disease, particularly Fabry or Pompe disease, respectively, comprising: a) the exogenously produced, natural or recombinant lysosomal hydrolase; (b) the reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase.

Another embodiment of the invention provides the compounds of structure according to formula II:

wherein each of R ¹ to R ⁵ individually equals H, a lower alkyl, a lower alkenyl, a lower alkynyl group and/or an optionally substituted (hetero)aryl group, preferably wherein all rests R ¹ to R ⁵ are H; and more preferably wherein the compounds are in a chair conformation. Another embodiment of the invention provides for a composition, wherein the reversible glycosidase inhibitor has the structure according to formula II, wherein each of R ¹ to R ⁴ individually represents H, a lower substituted alkyl such as (CH2) _n X wherein X is -OH ₃ , -OH, -NH ₂ , -I, -Br, -Cl or -CF3 and n ranges of from 0 to 5; a lower alkyl, a lower alkenyl, and a lower alkynyl group, and wherein R ⁵ represents -OH, -CH2OH, -CH ₂ 0-alkyl, preferably a Ci to C5 alkyl, or a -CH ₂ 0-glycoside. Preferably, each of R ¹ to R ⁴ in (II) individually represents H, a lower substituted alkyl such as -(CH ₂ ) _n X wherein X is -CH3, -OH, -NH ₂ , -I, -Br, -Cl or -CF ₃ , and n ranges of from 0 to 5; and wherein R ⁵ represents H, CH2OH, CH ₂ 0- alkyl, or CH ₂ 0-glycoside. More preferably, R ¹ to R ⁵ represent H. Yet more preferably, the hexyl ring is predominantly in a ⁴ Ci conformation for Fabry or Pompe disease.

Another preferred embodiment of the invention provides a composition according to formula I or II, wherein R ¹ equals a substituted alkyl such as (CH ₂ ) _n Y wherein Y represents CH3, OH, NH ₂ , 1, Br, Cl or CF3 and ranges from 0 to 5; wherein R ¹ is H, or a carboxy group (CO)Z wherein Z represents (CH ₂ ) _n CH ₃ , OH, NH ₂ , or CF3 and n ranges from 0 to 5.

Another preferred embodiment of the invention provides a composition according to formula I or II herein-below, wherein R ¹ equals a substituted alkyl such as (CH ₂ ) _n X, wherein X is CH3, OH, NH ₂ , I, Br, Cl or CF3 and n = 0-5; and wherein R ² to R ⁴ represent H or a carboxy group (CO)X, wherein X represents (CH ₂ ) _n CH3, OH, NH ₂ , or CF3 and n = 0-5, and/or an optionally substituted (hetero)aryl group.

Preferably, the a-galactosidase is a recombinant human a-galactosidase, more preferably Fabrazyme ^® . Preferably, the reversible galactosidase inhibitor is an a-ga/-cyclic sulfamidate.

Preferably, the human a-glucosidase inhibitor is a recombinant human acid a-glucosidase, more preferably Myozyme ^® . Preferably, the reversible glycosidase inhibitor is an ot-g/c-cyclic sulfamidate.

A particularly preferred embodiment of the invention provides for a composition, wherein the a-gal-cyclic sulfamidate is an a-gal 1,6 cyclophellitol cyclosulfamidate of the formula III:

wherein R ¹ is H or lower substituted alkyl (CH ₂ ) _n X, wherein X is CH3, OH, NH ₂ , I, Br, Cl or CF3 and n = 0-5; a lower alkyl, preferably a Ci to Cs-alkyl; or an optionally substituted carboxyl group (CO)X wherein X is (CH ₂ ) _n CH3, OH, NH ₂ , or CF3 _, and n = 0-5. More preferably, R ¹ represents H.

The present invention preferably also relates to a-gal 1,6 cyclophellitol cyclosulfamidate of the formula III wherein R ¹ is H or lower, optionally substituted alkyl, such as (CH ₂ ) _n X where X is CH3, OH, f\IH ₂ , 1, Br, Cl or CF ₃ and n = 0-5; wherein R ¹ is H or lower alkyl such as (CO)X where X is (CH ₂ ) _n CH ₃ , OH, NH ₂ , or CF ₃ and n = 0-5; preferably H.

Another embodiment of the invention provides for a composition as set out herein above, for administering to a patient at substantially the same time with the lysosomal hydrolase and the reversible glycosidase inhibitor, and/or wherein the components are administered such that they form a reversible complex in situ.

Advantageously, a composition as set out herein above, may also be employed for administering to a patient having been treated with gene therapy and endogenously expressing an a- galactosidase formed by a recombinant mutated a-NAGAL , or expressing an a-glucosidase formed by a recombinant mutated a-glucosidase GAA,

Another embodiment of the invention provides for a kit useful for treating a lysosomal storage disease, particularly Fabry or Pompe disease, comprising: a) the lysosomal hydrolase; and b) the reversible glycosidase as set out above, pharmaceutically acceptable salts, solvates, chelates, non- covalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof; wherein the components are administered such that they form a reversible complex in situ.

Preferred compounds include a-gal-1,6 cyclophellitol cyclosulfamidate of the formula III wherein R ¹ is H or lower alkyl, preferably H.

Another preferred embodiment of the present invention relates to a method to prepare the compounds according to formula I, in line with the reaction scheme disclosed in Figure 2. Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

Another preferred embodiment relates to a method of treating a patient diagnosed as having a lysosomal storage disease and/or a glycosidase deficiency related diseases, particularly Fabry or Pompe disease, comprising:

a) subjecting the patient to a gene therapy to endogenously express a natural or recombinant lysosomal hydrolase; and

b) administering to the patient a reversible glycosidase inhibitor according to the invention capable of competitively blocking the active site of the lysosomal hydrolase.

It is to be understood that both the foregoing Summary of the Invention and the following Description of Preferred Embodiments are exemplary and explanatory only and are not restrictive of the invention(s) herein. The present invention also relates to novel 1,6-cyclophellitol cyclosulfamidates and related compounds of formula (I):

wherein each of R ² to R ⁴ individually equals H, a lower alkyl, a lower alkenyl, and/or a lower alkynyl group, and wherein X represents O, S or NR', wherein R' represents H, or an alkyl substituent. Preferably, X represents O or NR', wherein R' represents H, and wherein R ⁵ represents H, CH2OH, ChhO-alkyl, or ChhO-glycoside.

Preferably the present invention also relates to novel 1,6-cyclophellitol cyclosulfamidates of the formula (IA), as reversible glycosidase inhibitors:

wherein X represents -NfR ¹ )- or oxygen, preferably wherein one of X represents -NfR ¹ )-, the other X represents oxygen, more preferably wherein the X at the exposition represents-NfR ¹ )- and the X at the (^-position represents oxygen, wherein R ¹ each individually represent H, a lower substituted alkyl such as (ChhJ _n X wherein X is CH ₃ , OH, NH ₂ , I, Br, Cl or CF ₃ , and n ranges of from 0 to 5; an optionally substituted (hetero)aryl group; or a carboxy group (CO)Z wherein Z represents (CH ₂ ) _n CH ₃ , -OH, or - NH ₂ ; wherein each of R ² to R ⁴ individually represents H, a lower substituted alkyl such as (CH ₂ ) _n Y wherein Y represents -CH ₃ , -OH, -NH ₂ , -I, -Br, -Cl or -CF ₃ , and n ranges of from 0 to 5 and/or an optionally substituted (hetero)aryl group; or a carboxy group (CO)X, wherein X represents (CH ₂ ) _n CH ₃ , OH, NH ₂ , or CFs and n = 0-5, and/or an optionally substituted (hetero)aryl group; wherein R ⁵ represents -OH, -CH ₂ OH, -CH ₂ 0-alkyl, or -CH ₂ 0-glycoside; and/or pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof. More specifically, the present invention relates to the 1,6 cyclophellitol cyclosulfamidate of the formula I, wherein R ¹ is H.

Another embodiment of the invention provides for 1,6 cyclophellitol cyclosulfamidates according to formula II, wherein each of R ¹ to R ⁴ individually represents H, a lower substituted alkyl such as (CH ₂ ) _n X wherein X is -CH ₃ , -OH, -NH ₂ , -I, -Br, -Cl or -CF ₃ and n ranges of from 0 to 5; a lower alkyl, a lower alkenyl, and a lower alkynyl group, and wherein R ⁵ represents -OH, -CH ₂ OH, -CH ₂ 0-alkyl, preferably a Ci to Cs alkyl, or a -CH ₂ 0-glycoside. Preferably, each of R ¹ to R ⁴ in (II) individually represents H, a lower substituted alkyl such as -(CH ₂ ) _n X wherein X is -CH3, -OH, -NH ₂ , -I, -Br, -Cl or -CF ₃ , and n ranges of from 0 to 5; and wherein R ⁵ represents OH, CH ₂ OH, CH ₂ 0-alkyl, or CH ₂ 0-glycoside. More preferably, R ¹ to R ⁵ represent H. Yet more preferably, the hexyl ring is predominantly in a ⁴ Ci conformation.

The present invention also relates to 1,6-cyclophellitol cyclosulfamidates according to formula III, wherein R ¹ is H, a lower alkyl, a lower substituted alkyl (CH ₂ ) _n X, wherein X is CH ₃ , OH, NH ₂ , 1, Br, Cl or CF ₃ and n = 0-5; or an optionally substituted carboxyl group (CO)X wherein X is (CH ₂ ) _n CH ₃ , OH, NH ₂ , or CF _{3 ,} and n = 0-5.

The present invention also relates to a method of treating a patient diagnosed as having a lysosomal storage disease and/or a glycosidase deficiency related diseases, particularly Fabry, Pompe or Gaucher disease, comprising administering to the patient:

a) an exogenously produced, natural or recombinant lysosomal hydrolase, or an endogenous produced natural mutant glycosidase by (multi)gene therapy; and

b) a reversible cyclophellitol-derived glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase as set out herein above, more preferably wherein the lysosomal hydrolase is an a-galactosidase or a-glucosidase, such as wherein the a-galactosidase is a recombinant human a-galactosidase, preferably Fabrazyme ^® . Preferably, the reversible glycosidase inhibitor is a reversible glycosidase inhibitor based on a cyclic sulfamidate functional group.

Another preferred embodiment of the present invention relates to a method as described above, herein the lysosomal hydrolase is a recombinant ot-glucosidase, preferably Myozyme ^® , and wherein the reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase is an a-glucose -configured cyclosulfamidate.

Preferably the lysosomal hydrolase and the reversible glycosidase inhibitor are administered together, at substantially the same time, to the patient, and/or wherein the components are administered, to the patient, such that they form a reversible complex in situ.

The present invention also relates to the use of a composition and/or a compound according to the invention, and pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof capable of competitively blocking the active site of a lysosomal hydrolase, for treating a lysosomal storage disease and/or glycosidase deficiency related diseases, particularly Fabry, Pompe or Gaucher disease.

The present invention also relates to a pharmaceutical composition comprising a co formulation of between about 0.5 and about 20 mM a-galactosidase A; and between about 50 and about 20,000 mM a compound according to the invention, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is formulated for parenteral administration to a subject.

The present invention also relates to a method of treating a patient diagnosed as having a lysosomal storage disease and/or a glycosidase deficiency related diseases, particularly Fabry or Pompe disease, comprising:

a) subjecting the patient to a gene therapy to endogenously express a natural or recombinant lysosomal hydrolase; and

b) administering to the patient a reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 demonstrates the reaction coordinates of a-galactosidases and inhibitors. A. Reaction itinerary of retaining a-galactosidase, showing conformations of the Michaelis complex, transition state, and covalent intermediates. B. Glucose configured cyclosulfates 1 and 2 irreversibly inhibits a- and b-glucosidases respectively. New galactose configured cyclosulfates 3 and 4, and galactose configured cyclosulfates biosiosters 5-9 mimic the Michaelis complex ⁴ Ci conformation. New galactose configured cyclosulfates 3 and 4, and a-Ga/-cyclophellitol 10 and a-Ga/-cyclophellitol aziridine 11 inhibit irreversibly a-gal A and do not stabilize the enzyme.

Figure 2A discloses a preferred reaction scheme for obtaining compounds according to the invention: Reagents and conditions: a) (i) BCI3, DCM, -78 °C, 3.5 h; (ii) BzCI, Pyr, rt, 18 h, 79%; b) RuCl ₃ -3H ₂ 0, Nal0 ₄ , EtOAc, ACN, 0 °C, 2 h, 15: 0% and 16: 78%; c) Os0 ₄ , NMO, H ₂ 0, acetone, rt, 3 days, 17: 44% and 18: 34%; d) (i) SOCI ₂ , Et ₃ N, imidazole, DCM, 0 °C; (ii) RuCI ₃ , Nal0 ₄ , CCI ₄ , ACN, 0°C, 3 h, 19: 56%, 20: 69%, 21:82%, 30: 62% over 3 steps and 39: 59% over 3 steps; e) NH ₃ , MeOH, rt, 3 h, 34%; f) H ₂ , Pd(OH) ₂ , MeOH, rt, 18 h, 4: 90%, 5: 92%, 6: 90%, 7: 57%, 8: 28% and 9: 94%; g) m-cpba, DCM, rt, 18 h, 23: 76%, a-epoxide: 19%; h) NaN ₃ , LiCI0 ₄ , DMF, 80-100 °C, 18 h, 24: 40%, 25: 38% and 42: 69%; i) MsCI, Et ₃ N, DCM, rt, 4 h, 26: 92% and 35: quant; j) H ₂ 0, DMF, 140“C, 3 days, 67%; k) Pt0 ₂ , H ₂ , THF, rt, 4 h, 28: 80%, 33: 99%, 43: 88%; I) Boc ₂ 0, Et ₃ N, DCM, rt, 18 h, 29: 99%, 34: 93%; m) TFA, DCM, rt, 8 h, 31: 99% and 40: 71%; n) 1-iodooctane, K2CO3, TBAI, DMF, 18 h, 32: 62% and 41: 66%; o) DMF, 140 °C, 3 days, 47% over 2 steps; p) 1 M NaOH, EtOH, 70 °C, 2 h, 86%; q) S0 ₂ (NH ₂ ) ₂ , Pyr, reflux, 18 h, 61%.

Figure 2B shows compounds 45 to 47.

Figure 2C shows the synthesis of amines 51 and 52. Reagents and conditions: a) NaN ₃ , DMF, overnight, 120 °C; b) Pt0 ₂ , H ₂ , THF, overnight, rt.

Figure 2D shows a scheme for the synthesis of a-glc-cyclosulfamidate 45. Reagents and conditions: a) B0C2O, Et ₃ N, DCM, overnight, rt; b) MsCI, Me-imidazole, Et ₃ N, CHCI ₃ , overnight, rt; c) DMF, overnight, 120 °C; d) 1 M NaOH, EtOH, 3 h at 70 °C followed by overnight stirring at rt; e) B0C2O, Et ₃ N, DCM, overnight, rt; f) (i) SOCI2, imidazole, Et ₃ N, DCM, 15 min, 0 °C; (ii) NalC , RuCI ₃ , 1:1:1 EtOAc, H20, MeCN, 1 h, 0°C; g) TFA, DCM, overnight, rt; h) Pd/C (10 wt %), H2, MeOH, overnight, rt.

Figure 2E then shows the synthesis of a-gf/c-cyclosulfamidate 46. Reagents and conditions: a) B0C2O, Et ₃ N, DCM, overnight, rt; b) MsCI, Me-imidazole, Et ₃ N, overnight, rt; c) DMF, overnight, 120°C; d) 1M NaOH, EtOH, 3 h at 70 °C, followed by overnight stirring at rt; e) B0C2O, Et ₃ N, DCM, overnight, rt; f) (i) SOC , imidazole, Et ₃ N, DCM, 15 min, 0 °C; (ii) Nal0 ₄ , RuCI ₃ , 1:1:1 EtOAc, H ₂ 0, MeCN, 1 h, 0°C; g) TFA, DCM, overnight, rt; h) Pd/C (10 wt %), H2, MeOH, overnight, rt.

Figure 3A discloses crystal structures of a-ga/-cyclosulfate 3 and a-ga/-cyclosulfamidate 7 in Fabrazyme. (A) a-ga/-cyclosulfate 3 reacts with Aspl70 nucleophile and adopts a ¹ S ₃ covalent intermediate conformation in complex with Fabrazyme ^® . (B) Unreacted 7 in complex with Fabrazyme ^® adopts a ⁴ Ci Michaelis complex conformation in the active site.

Figure 3B discloses the crystal structures of cyclosulfamidates 45 and 46 in complex with bacterial a-glucosidase CjAgd31B: (A) Cyclosulfamidate 45 reacts with the catalytic amino acid residue in the active site of Q ^' Agd31B, forming a covalent intermediate in a 1S3 conformation. (B): Cyclosulfamidate 46 does not react with the active site of the enzyme and adopts a 4C1 conformation, mimicking a Michaelis complex.

Figure 4 discloses the effect of comparative compounds a-gal-cyclosulfate 3 and Gal-DNJ, and a-gal-cyclosulfamidate 7 on thermal stability and cell culture medium stability of Fabrazyme ^® . A. Heat- induced melting profiles of lysosomal Fabrazyme ^® recorded by thermal shift. Temperature melting profiles of Fabrazyme ^® were recorded at optimum pH 4.5 in the presence of a-gal-cyclosulfamidate 7 and Gal-DNJ. The protein (0.5 mg/mL) was heated from 25 to 95° at l°C/min in the presence of Sypro Orange. Data were shown as normalized curves. B. Schematic representation of stabilization effect assay. Fabrazyme ^® was incubated with inhibitor for 15 min in DMEM/F-12 (Ham) medium and subsequently incubated with ConA sepharose beads for 1 h at 4 C and washed (x3) to remove bound inhibitor. a-Gal activity was finally determined by 4-MU-a-Gal assays. C. Percentage of Fabrazyme ^® residual activity after 15 min incubation in DMEM/F-12 (Ham) medium in the presence of inhibitors 3 and 7 (at 0, 100, 200, 500 mM) and Gal-DNJ (0, 1, 10 and 50 mM), followed by final ConA purification. Percentages are calculated considering the 100% activity of Fabrazyme ^® observed at 0 min incubation time (n=2, error bars indicate mean ± standard deviation).

Figure 5 demonstrates the effect of a-gal-cyclosulfamidate 7 and Gal-DNJ in cultured fibroblasts from Fabry disease. A. FD fibroblasts of WT (cl04, classic Fabry (R301X and D136Y) and variant Fabry (A143T and R112H) were incubated with a-cyclosulfamidate 7, Gal-DNJ, Fabrazyme ^® or the combination of enzyme and chaperone for 24 h. Then, the medium was collected, cells were harvested and a-Gal A activity was measured in the cell homogenates by 4-MU-a-Gal assay. In all cell lines co-administration of a-cyclosulfamidate 7 or Gal-DNJ with Fabrazyme ^® increased intracellular a- Gal A activity when compared to cells treated Fabrazyme alone. B. a-Gal A activity in cell culture medium samples was measured after ConA purification. a-Gal A activity is at least two times higher in all the cell lines treated with a-gal-cyclosulfamidate 7 (200 mM) or Gal-DNJ (at 20 mM). Reported activities are mean ± standard deviation from two biological replicates, each with two technical replicates.

Figure 6 discloses Gb3 and Lyso-Gb3 quantification after a-cyclosulfamidate 7 and Gal-DNJ cotreatment with Fabrazyme ^® in cultured fibroblasts. Gb3 (A) and lysoGb3 (B) levels measured by LC- MS/ MS, in Fabry fibroblasts from WT (cl04), 2 different classic Fabry patient cell lines (R301X and D136Y) and 2 different variant Fabry patient cell lines (A143T and R112H), with 24 h treatment of Fabrazyme ^® (400 ng/mL of culture medium) with or without a-gal-cyclosulfamidate 7 (200 mM) and Gal-DNJ (20mM). Reported values are mean ± standard deviation from at least three biological replicates.

Figure 7 illustrates the time dependent inhibition of acid a-glucosidase (GAA) by a-glc- cyclosulfamidates 45 and 46. Herein, the residual activity of recombinant human acid a-glucosidase (GAA, myozyme) with different pre-incubation times (15, 30, 45, 60, 120, 180 and 240 min) in the presence of 45 and 46 at their in vitro apparent ICso concentrations (66 mM and 5.1mM respectively) is shown as follows: Fig. 7 A: Time-dependent inhibition curve of 46; Fig 7 B: Time dependent inhibition curve of 45.

Figure 8 shows the effect of 45 and 46 on the thermostability of C/Agd31B. Graph shows the heat-induced melting profiles of C/Agd31B in complex with 46 (upper line, grey) and 45 (lower line, black). It is noted that a-glc-cyclosulfamidate 46 showed stabilitzation of bacterial a-glucosidase Q ^' Agd31B and a better inhibition for recombinant a-glucosidase GAA than IMB-DNJ (Miglustat), which is currently in clinical trials

DESCRIPTION OF PREFERRED EMBODIMENTS

The therapeutic methods of the invention described herein provide treatment options for the management of various lysosomal storage diseases and glycosidase deficiency related diseases. More specifically, the therapeutic methods of the invention involve various combinations of enzyme replacement therapy for the treatment of lysosomal storage diseases and glycosidase deficiency related diseases. The therapeutic methods involve the use of combinations of:

a) an exogenously produced, natural or recombinant lysosomal hydrolase, preferably an a- galactosidase, more preferably a human a-galactosidase, more preferably a recombinant human a- galactosidase, particularly Fabrazyme ^® (registered trademark of Genzyme Therapeutic Products Ltd. Partnership);

b) a reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase, preferably a reversible glycosidase inhibitor based on a cyclic sulfamidate functional group and derived from a cyclophellitol.

The present invention preferably relates to a composition wherein the reversible glycosidase inhibitor has the structure according to formula I wherein X represents -NfR ¹ )- or oxygen, preferably wherein one of X represents -NiR ¹ )-, the other X represents oxygen, more preferably wherein the X at the exposition represents-NiR ¹ )- and the X at the Opposition represents oxygen, wherein R ¹ each individually represent H, a lower substituted alkyl such as (CFhl _n X wherein X is CH _B , OH, IMH2, I, Br, Cl or CF3 , and n ranges of from 0 to 5; an optionally substituted (hetero)aryl group; or a carboxy group (CO)Z wherein Z represents (CH2) _n CH3, -OH, or -NH2; wherein each of R ² to R ⁴ individually represents H, a lower substituted alkyl such as (CH2) _n Y wherein Y represents -CH3, -OH, -NH2, -I, -Br, -Cl or -CF3 , and n ranges of from 0 to 5 and/or an optionally substituted (hetero)aryl group; or a carboxy group (CO)X, wherein X represents (CH2) _n CH3, OH, NH2, or CF3 and n = 0-5, and/or an optionally substituted (hetero)aryl group; wherein R ⁵ represents -OH, -CH2OH, -CH ₂ 0-alkyl, or -CH ₂ 0-glycoside; and/or pharmaceutically acceptable salts, solvates, chelates, non-covalent complexes, prodrugs, mixtures, including both R and S enantiomeric forms and racemic mixtures thereof, and pharmaceutical formulations thereof.

Preferably, the present invention relates also to the use of kits comprising a) the exogenously produced, natural or recombinant lysosomal hydrolase or endogenously produced by (multi)gene therapy, natural or recombinant lysosomal hydrolase; b) the reversible glycosidase inhibitor capable of reversibly blocking the active site of the lysosomal hydrolase.

More preferably, the present invention furthermore preferably relates to a composition more preferably an a-gal-cyclic sulfamidate, still more preferably a a-gal 1,6 cyclophellitol cyclosulfamidate of the formula III wherein R ¹ is H or lower, optionally substituted, alkyl, including but not limited to (CH2) _n X where X is CH ₃ , OH, NH2, I, Br, Cl or CF ₃ and n = 0-5; a carboxyl such as (CO)X wherein X is (CH ₂ )„CH ₃ , OH, NH ₂ , or CF ₃ and n = 0-5; preferably H.

It is believed that co-administration of a) the exogenously produced, natural or recombinant lysosomal hydrolase or endogenously produced by (multi)gene therapy, natural or recombinant lysosomal hydrolase with b) the reversible glycosidase inhibitor capable of competitively blocking the active site of the lysosomal hydrolase can be effective in making it possible to treat a larger number of patient mutations with reduced a-Gal A activity with the lysosomal hydrolase than the pharmacological chaperone alone which would just stabilize specific mutations. Secondly, the stabilization of the lysosomal hydrolase by the reversible glycosidase inhibitor and preferably also 1- deoxygalactonojirimycin may reduce the required enzyme dosages or extend IV injections intervals, and therefore reduce side effects and treatment costs.

Herein, the term "effective amount", in relation to delivery of an enzyme to a subject in a combination therapy of the invention, preferably means an amount sufficient to improve the clinical course of a lysosomal storage disease.

According to the application, a "subject" or "patient" is a human or non-human animal. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. Although the animal subject is preferably a human, the compounds and compositions of the application have application in veterinary medicine as well, e.g., for the treatment of domesticated species such as canine, feline, and various other pets; farm animal species such as bovine, equine, ovine, caprine, porcine, etc.; wild animals, e.g., in the wild or in a zoological garden; and avian species, such as chickens, turkeys, quail, songbirds, etc.

The term "cyclophellitol" relates to a compound with the IUPAC nomenclature name (l/?,2R,3 ?,45,5/?,6S)-2-(hydroxymethyl)-7-oxabicyclo[4.1.0]heptane-3, 4,5-triol.

The term "cyclophellitol-derived" implies compounds with a cyclohexyl ring and various substituents which may be derived chemically from the cyclophellitol, or synthesized differently, whereby the stereochemistry of the substituents at the carbon atoms of the cyclohexene ring may be varied in line with the required or desired conformation.

The term "enzyme replacement therapy" or "ERT" refers to refers to the introduction of a nonnative, purified enzyme into an individual having a deficiency in such enzyme. The administered enzyme can be obtained from natural sources or by recombinant expression. The term also refers to the introduction of a purified enzyme in an individual otherwise requiring or benefiting from administration of a purified enzyme, e.g., suffering from protein insufficiency. The introduced enzyme can be a purified, recombinant enzyme produced in vitro, or enzyme purified from isolated tissue or fluid, such as, e.g., placenta or animal milk, or from plants.

The term "adjuvant" or "adjuvant therapy" refers to any additional substance, treatment, or procedure used for increasing the efficacy, safety, or otherwise facilitating or enhancing the performance of a primary substance, treatment, or procedure

The term "co-formulation" refers to a composition comprising an enzyme, such as an enzyme used for ERT, for example, an ot-Gal A or GAA enzyme (e.g., a human recombinant a-Gal A or GAA enzyme that is formulated together with a chaperone for the enzyme.

The term "gene therapy" herein relates to using genetically engineered cells with DNA (RNA) encoding a marker or therapeutic which is expressed to be expressed in vivo. In certain embodiments the chaperone is a compound according to structure (I), or a pharmaceutically acceptable salt, ester or pro-drug thereof. In certain embodiments, treating a subject with the co-formulation comprises administering the co-formulation to the subject such that the a-Gal A or GAA enzyme and chaperone are administered concurrently at the same time as part of the co-formulation.

As used herein, the term "glycoside" relates to a material containing a saccharide structure which may be represented by the formula (IV)

-(OG)z (IV)

wherein O represents an oxygen atom and (G) is a saccharide structure (backbone) of the glycoside. The value z represents the number of monosaccharide units in the glycoside. The oxygen atom is attached to the saccharide in an ether linkage. Where the aldehyde or ketone structure of the saccharide is involved in the glycoside formation the product may be termed an acetal or ketal respectively. The favoured reaction is the acetal or ketal formation to give the glycoside with the oxygen being attached to the carbon in the one position. It is less likely that the hydrophobic moiety will be attached through one of the remaining hydroxyl groups present on the starting saccharide. The glycosides which are suggested for use in the present invention include those selected from the group consisting of fructoside, glucoside, mannoside, galactoside, taloside, aldoside, altroside, idoside, arabinoside, xyloside, lyxoside, iduronide, glucuronide and riboside and mixtures thereof. Preferably the glycoside is a fructoside and most preferably a galactoside or a glucoside. All of the aforementioned glycosides may be obtained from sugars (saccharides) with the preferential fructose and glucose starting materials being obtained from corn syrup. Complex glycosides, those containing one or more different saccharide units, may also be used as starting materials.

The glycoside has the ability to utilize the monomeric saccharide unit to promote chain growth of the glycoside. Thus z in the above formula may vary between 1 and 10, preferably from about 1.2 to about 5, and most preferably from about 1.3 to about 3.5. The value z, may also be referred to as the degree of polymerization (D.P.) of the glycoside. This number is an average degree of polymerization. In obtaining a glycoside, polymerization of the monosaccharide units occurs to some extent usually through a 1,6 linkage. The saccharide portion of the glycoside molecule enhances water solubility and thus for detergent purposes it is advantageous to have the D.P. somewhat greater than 1. It is also desirable for detergents that the hydrophobic moiety have sufficient length to give a proper HLB e.g. C4 and above.

As used herein, the term "active site" refers to the region of a protein that has some specific biological activity. For example, it can be a site that binds a substrate or other binding partner and contributes the amino acid residues that directly participate in the making and breaking of chemical bonds. Active sites in this application can encompass catalytic sites of enzymes, antigen biding sites of antibodies, ligand binding domains of receptors, binding domains of regulators, or receptor binding domains of secreted proteins. The active sites can also encompass transactivation, protein-protein interaction, or DNA binding domains of transcription factors and regulators.

As used herein, the term " chaperone" refers to a compound that specifically interacts reversibly with an active site of a protein and enhances formation of a stable molecular conformation. As used herein, "chaperone" does not include endogenous general chaperones present in the ER of cells, or general, non-specific chemical chaperones such as water

The term "combination therapy" refers to any therapy wherein the results are enhanced as compared to the effect of each therapy when it is performed individually. The individual therapies in a combination therapy can be administered concurrently or consecutively.

The term "modified lysosomal enzyme" may also comprise endogenously expressed modified naturally occurring enzymes, such as a-/V-acetyl-galactosaminidase (a-NAGAL) with increased a-galactosidase activity (ot-NAGAL ^EL ).AIso herein, the term "lysosomal storage disease" or "glycosidase deficiency related enzymes" preferably means any disease that can be treated in accordance with the invention with an exogenously produced natural or recombinant lysosomal hydrolase. Lysosomal storage diseases include the following diseases: Fabry, Gaucher, Pompe, Schindler, Krabbe, mucolipidosis I, Niemann-Pick, Farber, GMl-gangliosidosis, GM2-gangliosidosis (Sandhoff), Tay-Sachs, Krabbe, Schindler, sialic acid storage, fucosidosis, mannosidosis, aspartylglucosaminuria, Wolman, and neuronal ceroid lipofuscinoses. Glycosidase deficiency related diseases include pathological conditions driven by the deficiency or malfunction of glycosidases, including Parkinson's Disease or Alzheimer related to glucocerebrosidase (GBA) or a- galactosidase malfunction or mutations.

Inherited lysosomal storage disorders (LSDs) in humans are caused by the deficiency in different lysosomal enzymes: Gaucher disease (GD, deficiency in glucocerebrosidase, (GBA), Pompe disease (defiency in acid alpha-glucosidase) and Fabry disease (FD, deficiency in alpha-galactosidase A, a-GAL A), Krabbe disease (deficiency in beta-glucosidase), Schindler (deficiency in a-N- acetylgalactosaminidase (a-NAGAL)), mucopolisacaridosis I (deficiency in a-L-iduronidase) among others. Enzyme replacement therapies (ERT) are in use with variable success depending on the disease.

The deficiency in a-galactosidase A (a-gal A) generates a LSD known as Fabry disease which is characterized by the toxic accumulation of glycosphingolipid globotriaosylceramide (Gb3) in lysosomes and globotriaosylsphingosine (Lyso-Gb3) in plasma. Similarly, glycogen storage disease type II, also called Pompe disease, is an autosomal recessive metabolic disorder which damages muscle and nerve cells throughout the body. It is caused by an accumulation of glycogen in the lysosome due to deficiency of the lysosomal acid alpha- glucosidase (GAA) enzyme. It is the only glycogen storage disease with a defect in lysosomal metabolism, and the first glycogen storage disease to be identified, in 1932 by the Dutch pathologist J. C. Pompe. The build-up of glycogen causes progressive muscle weakness (myopathy) throughout the body and affects various body tissues, particularly in the heart, skeletal muscles, liver and the nervous system. Similar to Fabry disease, Pompe disease is the result of an inactive Human acid a- glucosidase (GAA, EC 3.2.1.20). This enzyme is a member of glycosyl hydrolase family GH31 and responsible for the lysosomal degradation of glycogen into glucose. Based on its cDNA GAA is synthesized as a 105 kDa precursor. This precursor contains a signal peptide which initiates its co- translational transport to the endoplasmic reticulum (ER). Within the ER the precursor is N- glycosylated on multiple glycosylation sites, which results in the formation of a 110 kDa glycoprotein. This 110 kDa glycoprotein, is then transported towards the late endosome/lysosome via mannose-6- phosphate receptor mediated transport. Multiple proteolytic cleavages and N-glycan processing of the precursor result in four tightly bound peptide regions forming the active enzyme. Mutations in the gene encoding for GAA can result in deficient or malfunctioning enzyme leading to the toxic accumulation of glycogen in the lysosomes. These mutations resulting in the deficiency of GAA are the cause of the lysosomal storage disorder (LSD) Pompe disease (also known as glycogen storage disorder 2). Pompe disease is therefore characterized by a lysosomal accumulation of glycogen which causes cellular disfunction and apoptosis, primarily in cardiac and skeletal muscles.

This accumulation of lysosomal glycogen in muscle tissue can result in loss of respiratory, motor and cardiac functions.

Pompe disease patients are generally categorized into two main groups; the classic infantile early onset Pompe disease and the late-onset Pompe disease. For infants suffering from the classical early onset form of Pompe disease, lysosomal accumulation of glycogen in heart, lung and skeletal muscle tissue results in severe cardiomyopathy and respiratory failure. These severe medical problems often lead to death of the infant in the first year of its life. In contrast, the late onset form observed in children and adults often show different rates of disease progression. In these late onset forms the lysosomal accumulation of glycogen is usually confined to skeletal and respiratory tissue. The accumulation of glycogen in these tissues can respectively lead to progressive limb-girdle myopathy and respiratory deficiency, of which the latter is one of the main causes of death. Current treatment options for Pompe disease primarily consist of enzyme replacement therapy (ERT), in which recombinant human GAA (Myozyme) is intravenously administered to the patients. This ERT was approved in 2006 and has shown the ability to improve cardiac, motor and respiratory functions thereby being beneficial for patient survival. However, in spite of these clinical benefits, the efficacy of the ERT is variable among patients. One of the reasons for this variability is the limited targeting and uptake of the recombinant human enzyme in muscle tissue. In addition, the formation of host antibodies against the recombinant enzyme and the accumulation of autophagic compartments in myocytes also severely affect the efficacy of the treatment. Therefore additional therapeutic interventions are sought. One of the current approaches for treatment of LSDs is the administration of small-molecule chaperones named pharmacological chaperone therapy (PCT). These chaperones have the task of stabilizing or refolding the enzyme to help them pass the quality control machinery of the ER. Patients who contain missense mutations in their GAA gene are most likely amenable for PCT since these mutations often lead to improper folding of the enzyme. However, it has been shown that only a small group of Pompe patients (10-15%) have mutations that are susceptible for PCT. Another treatment strategy that has been proposed is the combinatorial administration of ERT and a PC. PCT with the iminosugar deoxynojirimycin 1 (DNJ, Duvoglustat) or W-butyldeoxynojirimycin 2 (NB- DNJ, Miglustat) is now being evaluated in the first clinical trials (Phase 2), either as monotherapy (DNJ) or in combination (DNJ and NB-DNJ) with enzyme replacement therapy. Research towards such a combinatorial therapeutic intervention has provided evidence that NB-DNJ is able to improve the stability, delivery and maturation of recombinant human GAA. NB-DNJ has previously been approved as a small-molecule for substrate reduction therapy in type 1 Gaucher disease in which it functions as a ceramide mimic, thereby inhibiting glucosylceramide synthase and reducing the formation of glycosphingolipids.

The term "Fabry disease" refers to the classical Fabry disease, late-onset. Fabry disease, and hemizygous females having mutations in the gene encoding a- galactosidase A (a-gal A). The term "Fabry disease," as used herein, further includes any condition in which a subject exhibits lower than normal endogenous a-gal A activity.

Th term "Pompe disease" herein refers to the classical Pompe disease, and further includes any condition in which a subject exhibits lower than normal endogenous ot-glucosidase GAA activity.

Various a-Gal A mutations lead to a wide range of different phenotypes, classified as classic and variant Fabry patients, with diverse enzyme and metabolite levels. The accumulation of glycosphingolipid metabolites is thought to be the basis of progressive renal and cardiac insufficiency, and CNS pathology in Fabry patients.

Although intravenous enzyme replacement therapy (ERT) ameliorates Gb3 and lyso-Gb3 metabolite levels, its long term clinical efficacy is relatively reduced and patients develop neutralizing antibodies upon the recombinant enzyme that can result in inhibition of the ERT. In addition, Fabrazyme ^® biweekly intravenous (IV) dosage of Fabrazyme ^® can result in intermittent efficacy of the treatment, and high rate of infusion is associated with stroke incidents.

Recently, a pharmacological chaperone, 1-deoxygalactonojirimycin (Gal-DNJ, Migalastat ^® ) which binds to some specific mutant forms of a-gal A and restores some lysosomal activity, has been approved as a new treatment for Fabry patients with amenable mutations in United States of America, Europe, Australia and Canada among others.

This alternative to intravenous enzyme replacement therapy (ERT) is, however, limited to certain specific mutations and its approval by the FDA is still under consideration.

In addition, a-Gal A deficiency has recently has been detected in late-stage Parkinson's disease (PD) brains and associated to the aberrant accumulation of ot-synuclein (a-syn).

On the other hand, carriers of a GBA defect or mutation do not develop its corresponding lysosomal storage disorder (Gaucher Disease, GD) but show a markedly increased risk for PD and Lewy-body dementia. It is still a matter of debate the interconnection between increased levels of Gb3 isoforms cause by deficiency in a-gal A and/or GBA and the pathological accumulation of a-syn. Importantly, a-Gal A positive modulators or PCs may be important therapeutic and biological tools not only for Fabry disease but also for Parkinson's disease.

Carbohydrates are metabolized by their processing enzymes such glycosidases (GHs, enzymes that hydrolize a glycosidic bond) or glycosyltransferases (GTs, enzymes that generate a glycosidic linkage). Glycosidase inhibition or stimulation with pharmacological chaperones (PCs) is the basis of many therapeutic strategies for the treatment of different diseases such as diabetes type II, viral infections, cancer or lysosomal storage disorders among others.

Most glycosidases hydrolyse their substrates through an overall configurational retaining reaction known as Koshland double displacement mechanism.

However, although different GHs and GTs process their substrate through the same mechanistic reaction, many accomplish this by following different conformational itineraries. This is the case for example of a- and b-retaining glucosidases which hydrolyse their substrates through the same Koshland double displacement mechanism but follow opposite conformational reaction itineraries and for which the Michaelis complex -> TS -> intermediate enzymatic half reactions are ⁴ Ci -> ⁴ H ₃ ^F -> ¹ S ₃ in a-glucosidases and ¾ _¾ - ⁴ H ₃ ^F -> ⁴ Ci in b-glucosidase.

Transition state analogues mimicking the oxocarbenium ion transition state by their half chair conformations have proved to be an important class of glycosidase inhibitors. This transition state mimicry exerted by for example cyclophellitol (aziridines) has also turned into promiscuous glycosidase inhibition, an advantage for the development of broad spectrum glycosidase activity- based probes. Applicants have now found a novel class of selective and potent glucosidase irreversible inhibitors which favors ⁴ Ci chair conformations are a- and b-cyclophellitol cyciosulfates 1 and 2, respectively (see Figure IB).

However, these compounds which are mimicking both the "anomeric" stereochemistry and the initial Michaelis complex conformation of the target enzyme, a- and b-glucosidase respectively, have a far higher selectivity than cyclophellitol or cyclophellitol aziridine irreversible inhibitors, and thus a-cyclosulfates 1 exhibits exquisite selective inhibition of gastrointestinal a-glucosidase specially relevant for the treatment of type II diabetes. b-Cyclosulfate 2, which also adopts a ⁴ Ci different from the ¾ ₃ conformation of typical b-glucosidase Michaelis complexes, reacts much slower than its congener 1, but still inhibits b-glucosidases in the micromolar range. However, the inhibition was surprisingly found to be permanent, at least under the conditions that one expected in a patient's body.

Surprisingly, it was found that in kinetic and activity in vitro studies that whereas a-gal- cyclosulfate 3 irreversibly inhibits a-Gal A, a-ga/-cyclosulfamidate 7 according to the invention reversibly binds the enzyme and thereby, as demonstrated by in vitro and in situ cell experiments, a- ga/-cyclosulfamidate 7 promotes the stabilization of the human ot-gal A in cell culture and consequently increases the lysosomal uptake of a-Gal A.

Accordingly, a composition comprising a-ga/-cyclosulfamidate 7 appears a novel and entirely superior combination ERT therapy for Fabry disease, as well as for the study of a-gal A deficiency linked to PD. Furthermore, the same principle applies to all lysosomal disorders disclosed herein, whereby the chaperone preferably is chosen to mimic the substrate of the respective enzyme.

Similarly, a composition comprising a-g/c-cyclosulfamidate 46 appears a novel and entirely superior combination ERT therapy for Pompe disease. Furthermore, the same principle applies to all lysosomal disorders disclosed herein, whereby the chaperone preferably is chosen to mimic the substrate of the respective enzyme.

Yet further, the present compounds such as a-ga/-cyclosulfamidate 7 or a-g/c- cyclosulfamidate 46 may also be employed to stabilize mutant endogenously produced enzymes, such as modified lysosomal a-A/-acetyl-galactosaminidase (a-NAGAL) with increased a-galactosidase activity (a-NAGAL ), or GAA expressed after gene therapy, or by cells introduced to a patient and capable of expressing such enzyme.

According to the present application, compounds according to the invention can be administered as the free base or as a pharmacologically acceptable salt form. It can be administered in a form suitable for parenteral administration, including e.g., in a sterile aqueous solution for intravenous administration. The compounds and compositions of the application can be formulated as pharmaceutical compositions by admixture with a pharmaceutically acceptable carrier or excipient.

In certain embodiments, compounds according to the invent and the enzyme, in particular ot- Gal A or GAA are formulated together in a single composition, i.e., co-formulated together. Such a composition enhances stability of the enzyme both during storage (i.e., in vitro) and in vivo after administration to a subject, thereby increasing circulating half-life, tissue uptake, and resulting in increased therapeutic efficacy of the enzyme. The co-formulation is preferably suitable for intravenous administration.

The present application may feature liquid pharmaceutical co-formulations (e.g., formulations comprising a-Gal A and a compound according to the invention, having improved properties as compared to art-recognized formulations.

In certain embodiments, co-formulations of the application include an enzyme and compounds according to the invention that are suitable for intravenous administration.

In certain embodiments, the co-formulation composition comprises a lysosomal enzyme at a concentration of between about 0.05 and about 100 mM, or between about 0.1 and about 75 mM, or between about 0.2 and about 50 mM, or between about 0.3 and about 40 mM, or between about 0.4 and about 30 mM, or between about 0.5 and about 20 mM, or between about 0.6 and about 15 mM, or between about 0.7 and about 10 mM, or between about 0.8 and about 9 mM, or between about 0.9 and about 8 mM, or between about 1 and about 7 mM, or between about 2 and about 6 mM, or between about 3 and about 5 mM.

In certain embodiments, the co-formulation composition comprises compounds according to the invention at a concentration of between about 1 and about 25,000 mM, or between about 10 and about 20,000 mM, or between about 100 and about 15,000 mM, or between about 150 and about 10,000 mM, or between about 200 and about 5,000 mM, or between about 250 and about 1,500 mM, or between about 300 and about 1 ,000 mM, or between about 350 and about 550 mM, or between about 400 and about 500 mM.

Concentrations and ranges intermediate to the above recited concentrations are also intended to be part of this application.

In certain embodiments, the co-formulation composition comprises compounds according to the invention at a concentration of between about 0.002 and about 5 mg/mL, or between about 0.005 and about 4.5 mg/mL, or between about 0.02 and about 4 mg/mL, or between about 0.05 and about 3.5 mg/mL, or between about 0.2 and about 3 mg/mL, or between about 0.5, and about 2.5 mg/mL, or between about 1 and about 2 mg/mL. Design and synthesis of comparative a-D-galactosidase cyclosulfates 3 and 4 was investigated (Figure IB). Based on results with a-glu and b-g/u-cyclosulfates 1 and 2, applicants investigated if an a-galactose configured cyclosulfate 3 would be an effective inhibitor of a-galactosidases which follow the same ⁴ Ci -> ⁴ H ₃ -> % conformational trajectories by mimicking the initial Michaelis complex conformation. It was found that -go/-cyclosulfate 4 would be conformationally excluded from both a-galactosidase reaction itineraries and therefore a poor inhibitor (Figure 1A).

Additionally, applicants synthesized the novel compounds according to the invention, namely cyclosulfate bioisosteres a-gal-cyclosulfamidates 5-8, as well as cyclosulfamide 9.

The present invention also preferably relates to a process for the preparation of inhibitor compound. In a preferred embodiment first, perbenzylation of starting cyclohexene 14 and subsequent oxidation with OsC gave diols 15 and 16 as a non-separable mixture of a/b with a 1:4 ratio (Figure 2A). In parallel, oxidation with RUCI ₃ and sodium periodate by in situ formation of RUO ₄ exclusively afforded b-diol 16. Oxidation reactions on acetylated cyclohexene (following either OSO ₄ or RUO ₄ protocol) yielded a mixture of multiple compounds probably due to acetyl migration.

Interestingly, stereoselective oxidation was achieved on perbenzoylated cyclohexene 17. Thus, oxidation with OSO ₄ afforded a separable mixture of diols 17 and 18 in a 1:0.6 ratio, whereas RUCI ₃ and sodium periodate conditions yielded a 0.5:1 ratio of pure a- and b- diols after column chromatography. Benzoylated diols 17 and 18 were then treated with thionyl chloride and subsequently oxidized to the cyclic sulfate to afford the protected cyclosulfates 19 and 21. Deprotection of b-analogue 21 with different mild/basic conditions (i.e. NH ₃ in MeOH, KCN or Et ₃ N/H ₂ 0 under reflux) resulted in the E2-elimination product 22.

a-ga/-cyclosulfate 3 was obtained after benzoyls removal in methanolic ammonia. b-Gal-cis- cyclosulfate 4 was alternatively synthesized from the perbenzylated b-cyclosulfate 20 after hydrogenation in the presence of Pearlman's catalyst.

Synthesis of q-D-galactosidase cvclosulfate bioisosteres 5-9 according to the invention

Applicants also explored the alkylation of cyclosulfamidates 5 and 7 with an octyl chain: analogues 6 and 8.

In a preferred embodiment, synthesis of cyclosulfamidates 5 and 6 started from cis-l-hydroxy- 6-azido cyclohexene 27, which was obtained by first nucleophilic addition of sodium azide to b- cyclophellitol 23 followed by mesylation and subsequent inversion of the stereochemistry of the secondary alcohol with optimal yields (Figure 2B). Azido intermediate 27 was reduced and ensuing Boc protection afforded intermediate 29, which was treated with thionyl chloride under basic conditions to form a mixture of sulfite enantiomers that were further oxidized to the boc-protected cyclosulfamidate 30. Cyclosulfamidate 30 was deprotected with TFA and intermediate 31 was either alkylated with 1-iodooctane and/or directly deprotected by hydrogenolysis to afford the desired final cyclosulfamidates 5 and 6.

In a preferred embodiment, synthesis of sulfamidate 7 and 8 was performed using a strategy involving intramolecular N- Boc participation to facilitate oxazolidinone 36 formation with simultaneous hydroxyl inversion, and subsequent deprotection yielded the desired cis-l-amino-6- hydroxy cyclohexene 37. Then, cyclic sulfamidate synthesis from boc-protected analogue 38, followed by boc deprotection with TFA and either alkylation with 1-iodooctane and/or directly deprotection by hydrogenation in the presence of Pd(OH)2 catalyst afforded desired final cyclosulfamidates 7 and 8.

In a preferred embodiment, cis-diamino 43 was synthesized by nucleophilic addition of sodium azide to mesylated intermediate 26 and subsequent reduction with H2/RΪ20 with excellent yields. Then, treatment with SC^NFh in pyridine under reflux to create the cyclic sulfate and benzyl removal by hydrogenation afforded final cyclosulfamide 9.

a-D-Galactosidase cyclosulfate 3 and bioisosteres 7 and 9 inhibit ot-gal A in vitro

Inhibition and selectivity in pure a- and b-galactosidases (human recombinant a-galactosidase, Fabrazyme ^® from Genzyme, GH27 and purified bacterial b-galactosidase homologue from Cellvibrio japonicus C/GH35A, GH35) was investigated. As initial screening, we determined their apparent ICso values by using commercial 4-methylumbelliferyl (4MU) -a- or b-galactose as substrate at the optimal pH 4.5 (Table 1).

It was found that a-<?a/-cyclosulfate 3 effectively inhibited a-galactosidase on a par with a-gal- cyclophellitol 10 (IC50 = 25 vs 11 mM respectively in Fabrazyme), although resulting in a much weaker inhibitor than a-gal-cyclophellitol aziridine 11 (IC50 = 40 nM). b-cyclosulfate 4 was inactive against b- or a-galactosidase up to 1 mM.

Interestingly, the replacement of the electrophilic cyclic sulfate by a cyclic sulfamidate moiety (compounds 5 and 7) was just tolerated when the NH group was placed close to the anomeric position, suggesting that H bonding may probably be taking place in the neighboring of the acid/base amino acid. The instalment of a NH moiety on the catalytic amino acid site indeed seems detrimental for activity, whereby sulfamidate 5 and sulfamide 9 inactive or massively weak a-galactosidase inhibitors. Remarkably, whereas introduction of an alkyl chain in the aziridine scaffold results in an increase in a- gal A activity, alkylation of sulfamidates 5 and 7 was detrimental for a-galactosidase inhibition (IC50 of 6 and 8 > 1 mM).

To study the selectivity of these compounds apparent IC50 values were measured in b- galactosidase QGH35A. Remarkably, following the same trend as glucose configured cyclosulfates, cyclosulfate 3 present an advantage in specificity when compared to aziridine or cyclophellitol type inhibitors, by virtue of mimicking a reactive ⁴ Ci conformation not shared between a- and b- galactosidases (compound 3 inactive up to 1 mM whereas 10, 11 and Gal-DNJ present apparent IC50 values of 18, 0.57 and 331 mM respectively in b-galactosidase C/GH35A). Additionally, aziridine 11 and Gal-DNJ showed off-target inhibition of a-A/-acetylgalactosaminidase (ot-NAGAL) in plasma samples with apparent IC50S of 137 and 15.2 mM respectively, as disclosed in Table 1.

Table 1 below shows the apparent IC50 values for in vitro inhibition of human recombinant a- galactosidase A (Fabrazyme), a-A/-acetylgalactosaminidase (a-NAGAL) in human plasma and bacterial b-galactosidase homologue (C GH35A). Inactivation rates and inhibition constants [k _ma a and K _t ) in human recombinant a-galactosidase (Fabrazyme ^® ); N.D., not determined; ^a due to weak inhibition; ^b due to fast inhibition; N.I., no inhibition observed; *reversible inhibition observed. Reported values are mean ± standard deviation from 3 technical replicates.

Table 1 : Apparent IC50 values for in vitro inhibition of human recombinant a-galactosidase A (Fabrazyme), a-A/-acetylgalactosaminidase (a-NAGAL) in human plasma and bacterial b-galactosidase homologue (QGH35A).

The reversibility character of the inhibition of this family of compounds was studied, first by time dependent inhibition studies and afterwards determining the kinetic parameters of the inhibition in human a-galactosidase. Thus, compounds at concentrations above their corresponding apparent IC ₅₀ values were incubated for different times (30, 60, 120, 240 min) and subsequent fluorescent readout with 4-methylumbelliferyl-a-galactopyranose allowed the quantification of the residual enzyme activity. Interestingly, whilst irreversible inhibition (a decrease in a-galactosidase activity with longer incubations) was clearly observed with cyclosulfate 3, cyclosulfamide 9, epoxide 10 and aziridine 11; cyclosulfamidate 7 showed a constant residual activity with different incubation times, pointing to a non-covalent inhibition of Fabrazyme. This was demonstrated by kinetic studies monitoring the absorbance generated by the hydrolysis of 2,4-dinitrophenyl-a-D-galactopyranoside substrate (2,4-DNP-a-Gal) at pH 4.5 (Table 1). Irreversible cyclosulfate 3, epoxide 10 and aziridine 11 follow pseudo-first order kinetics and kinetics parameters of cyclosulfamide 9 could not be measured due to very slow/weak inhibition. Although ot-cyclosulfate 3 presents a similar k _maci /K _\ ratio than ot- cyclophellitol 10 {k _mact /Ki = 0.25 vs 0.55 min ½M ^_1 , respectively), inhibitor 3 shows a stronger initial binding constant (k _| ) and a slower inactivation rate constant (k _mact ) than 10 (3: K _\ - 237 mM and k _\mA = 0.06 min ¹ vs 10: K _\ - 430 mM and k _{ma rt} = 0.24 min ¹ ), and only a k _\m a/k _\ ratio could be measured for a- aziridine 11 due to fast inhibition (k _nact /K _\ = 16.4 min^mM ¹ ). Reversible kinetics with increasing 2,4- DNP-a-Gal concentrations demonstrated that cyclosulfamidate 7 reversibly inhibits a-galactosidase with a K _\ = 110 mM.

Structural analysis of a- o/-cyclosulfate 3 and a-Qfa/-cyclosulfamite 7 in complex to Fabrazyme.

Crystal structures of a-ga/-cyclosulfate 3 and a-ga/-cyclosulfamidate 7 in complex with the recombinant human a-gal A (Fabrazyme) were obtained (Figure 3A). It was observed that a-gal- cyclosulfate 3 (Figure 3AA) reacts with Aspl70 nucleophile and adopts a % covalent intermediate conformation in complex with Fabrazyme ^® . On the other hand, unreacted (Figure 3AB) binds reversibly and adopts a ⁴ Ci Michaelis conformation in the active site.

Furthermore, a-g/c-cyclosulfamidates were also developed as competitive reversible enzyme stabilizers for Pompe disease.

The present invention also relates to a-g/c-cyclosulfamidate analogues 45-47 according to the invention, and to their use in treating patients suffering from Pompe disease. The inhibitory potential and selectivity of these compounds was assessed by ICso determination in recombinant human GAA and competitive activity-based protein profiling (cABPP) in fibroblast lysates. For the determination of the compounds reversibility, time-dependent inhibition assays were performed, to show the binding and the activity of these inhibitors to function as stabilizers of a-glucosidases was supported by crystallographic and thermostability assays.

With respect to the formulae expressed herein, the following terms apply: the term "lower alkyl" preferably means a saturated, branched or straight chain hydrocarbon group of 1 to 6 carbon atoms, preferably one to three carbon atoms, such as a methyl, isopropyl or n-pentyl group.

"Substituted lower alkyl" preferably means substitute lower alkyl groups, such as (CH ) _n X, wherein X is CH _B , OH, H , I, Br, Cl or CF ₃ and n = 0-5. Particularly preferred compound are those wherein R ¹ is H or an optionally substituted lower alkyl group such as (CO)X where X is (CH2 _n CH ₃ , OH, NH ₂ , or CF ₃ and n = 0-5. "Lower alkyls" herein in includes straight or branched chains. Examples of such alkyls include alkyl chains containing from 1 to about 30, preferably from about 1 to about 20, and most preferably from about 2 to about 10 carbon atoms. Specific alkyls which may be utilized herein include methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonal, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl.

Any conventional method can be used to monitor the status of lysosomal storage disease of a patient being treated in accordance with this invention and the effectiveness of a combination therapy of the invention on the disease. Clinical monitors of disease status can include but are not limited to organ volume (e.g. liver, spleen), hemoglobin, erythrocyte count, haematocrit, thrombocytopenia, cachexia (wasting), and plasma chitinase levels (e.g. chitotriosidase). Chitotriosidase, an enzyme of the chitinase family, is known to be produced by macrophages in high levels in subjects with lysosomal storage diseases (see: Guo et al., 1995, J. Inherit. Metab. Dis. 18, 717- 722; den Tandt et al., 1996, J. Inherit. Metab. Dis. 19, 344-350; Dodelson de Kremer et al., 1997, Medicina (Buenos Aires) 57, 677-684; Czartoryska et al., 2000, Clin. Biochem. 33, 147 149; Czartoryska et al., 1998, Clin. Biochem. 31, 417-420; Mistry et al., 1997, Baillieres Clin. Haematol. 10, 817-838; Young et al., 1997, J. Inherit. Metab. Dis. 20, 595-602; Hollak et al., 1994, J. Clin. Invest. 93, 1288-1292).

Methods and formulations for administering a combination therapy of the invention to a patient include all conventional methods and formulations (see: e.g. Remington's Pharmaceutical Sciences, 1980 and subsequent years, 16 ^th ed. and subsequent editions, A. Oslo editor, Easton Pa.; Controlled Drug Delivery, 1987, 2 ^nd rev., Joseph R. Robinson & Vincent H. L. Lee, eds., Marcel Dekker, ISBN: 0824775880; Encyclopedia of Controlled Drug Delivery, 1999, Edith Mathiowitz, John Wiley & Sons, ISBN: 0471148288; U.S. Pat. No. 6,066,626 and references cited therein).

In the combination therapy of the invention, the lysosomal hydrolase is administered intravenously to a patient to initiate treatment (i.e. to de-bulk the subject). The initial treatment can include administration also of the reversible glycosidase inhibitor. Thereafter, treatment with the lysosomal hydrolase, the reversible glycosidase inhibitor can be administered to the patient over a one to two-hour period on a weekly or bi-weekly basis for one to several weeks or months, or longer.

In certain embodiments, the foregoing combination therapy may also provide an effective reversible inhibition of an enzyme selected from the group consisting of glucocerebrosidase, sphingomyelinase, ceramidase, GMl-ganglioside-P-galactosidase, hexosaminidase A, hexosamimidase B, b-galactocerebrosidase, ot-L-iduronidase, iduronate sulfatase, heparan-/V- sulfatase, /V-acetyl-a-glucosaminidase, acetyl CoA: a-glucosaminide acetyl-transferase, a- galactosidase A, a-/V-acetylgalactosaminidase, a-glucosidase, a-fucosidase, a-mannosidase, aspartylglucosamine amidase, acid lipase, and palmitoyl-protein thioesterase (CLN-1).

Still further, in certain embodiments, the foregoing composition, and combination therapy method may produce a diminution in at least one stored material selected from the group consisting of glucocerebroside, sphingomyelin, ceramide, GMl-ganglioside, GM2-ganglioside, globoside, galactosylceramide, glycolipids, glycosphingolipids, globotriaosylceramide, O-linked glycopeptides, glycogen, free sialic acid, fucoglycolipids, fucosyloligosaccharides, mannosyloligosaccharides, aspartylglucosamine, cholesteryl esters, triglycerides, and ceroid lipofuscin pigments.

In certain embodiments, the present specification also relates to a process or method for preparing the compounds (I) to (III) via the reaction pathway as set out below.

The following examples illustrate the present invention further.

EXAMPLES

Example 1: Synthesis of compound 7 according to the invention

Synthesis of compound 7, herein below, i.e., the a-gal-l,6-cyclophellitol cyclosulfamidate of formula I wherein R is H, via compounds 24 and 38 is disclosed in the flow chart below and starting from compound 14 (see: Y. Harrak Chem. Soc.2011, 133, 12079-12084 and L. I. Willems et al, European J. Org. Chem.2014, 2014, 6044-6056) in the flow chart below (and Figure 2):

7

38 40

Example 2: Thermostability of Fabrazyme ^® in the presence of compound 7 (as compared to Migalastat) Fabrazyme ^® usually presents poor stability in plasma (pH 7.4) and only ~25% of the hydrolytic activity remains after incubation of 1 pg/mL pure enzyme in human plasma for 15 minutes. As result, a very small amount of the injected Fabrazyme ^® dosage actually reaches the intracellular targeted lysosome. The enormous quantity of required recombinant enzyme is a major drawback of ERT which can be reflected in increased development of neutralizing antibodies in treated patients and elevated costs of the required treatment.

Appropriately configured iminosugars are known to stabilize diverse lysosomal glycosidases with so called pharmacological chaperone (PC) effect.

Ideally, a PC for glycosidases should reversibly bind its enzyme in plasma circulation and in the endoplasmic reticulum at neutral pH, stabilizing it during its transport to the lysosomes and upon dissociation in the acidic compartment aided by the excess of substrate, the enzyme hydrolyzes its natural substrate Gb3.

Thermal shift assays (TSAs) can be used for high-trough put screening of PCs and it has been suggested that the recently approved 2-O-a-D-Galactopyranosyl-l-deoxynojirimycin (Migalastat) is not the ideal PC therapy for Fabry disease, as it stabilizes the enzyme under neutral and acidic conditions. However, the high concentrations of substrate accumulated in the lysosomes aid the displacement of the PC by the natural substrate.

Heat-induced melting profiles of lysosomal Fabrazyme ^® recorded by thermal shift at pH 4.5, 5.5 and 7.4 showed that Fabrazyme ^® is more thermostable at pH 5.5.

A comparison whether compound 7, as compared to Migalastat had a stabilization effect on Fabrazyme ^® was investigated by measuring the heat-induced melting profiles of lysosomal a-gal A at different pHs 4.5, 5.5 and 7.4.

TSAs showed that compound 7 stabilizes Fabrazyme ^® at pH 7.4 with a ATm _m3x of 17.4 °C whereas Migalastat presented a slightly higherthermal stability effect with a ATm _m3x of 34.3 °C (Figure 4A, Figure S4).

Thermostability was also measure at acid pH mimicking the lysosomal environment and the stabilization of Fabrazyme ^® was slightly lower in both cases with ATm _max of 9.3 and 22.3 °C at pH 5.5 for compound 7 and Migalastat, respectively.

It was then investigated whether compound 7 or Migalastat had stabilization effect on Fabrazyme in Dulbecco's Modified Eagles Medium/Nutrient Mixture F-12 (DMEM/F12, Sigma-Aldrich) medium, supplemented with 10 % fetal calf serum and 1 % penicillin/streptomycin at (pH 7.2), mimicking fibroblasts cell culture conditions. After 15 min incubation of Fabrazyme ^® (5 mg/mL) in cell culture medium, 80% degradation was observed and this time point was used for further analysis. Thus, compound 7 and Migalastat were incubated with Fabrazyme ^® (5 mg/mL) at increasing concentrations (starting around the ICso value) of the corresponding inhibitor in cell medium conditions at pH 7.2 for 15 min and 0 min (for degradation quantification). Samples were then incubated for 1 h at 4 °C with Concanavalin A (ConA) sepharose beads which are known to bind a-Gal A, and washed (3x) to remove the inhibitor bound to a-Gal A. Quantification of a-galactosidase activity was then performed via 4MU-a-Gal assays.

In line with TSAs results, compound 7 and Migalastat reversibly bind a-Gal A, preventing its degradation under cell culture conditions at pH 7.2. A comparable value of about 75% stabilized residual a-gal-A activity was observed after 15 min incubation with compound 7 (500 mM), or with Migalastat (50 mM), whereas just 20% activity remained with no inhibitor.

Example 3

In situ treatment of cultured fibroblasts from patients with Fabry disease.

It was investigated if compound 7 or Migalastat would stabilize a-Gal A activity in situ. In situ studies were carried out in 5 different male cell lines: wild type fibroblast (WT, cl04) which present normal a-Gal A activity, 2 classic Fabry patients fibroblasts (R301X and D136Y mutations) with no a- Gal A activity and 2 variant Fabry patients fibroblasts in latter onset disease (A143T and R112H mutations) with substantially lowered, yet high, enzyme activity that exhibit almost normal plasma Lyso-Gb3 concentration.

Of note, the R112H mutation present a large structural change on the molecular surface but its active site remains intact and the a-Gal A activity and Gb3 and lysoGb3 levels may vary within patients. On the other hand, A143T mutation within the same family also presents diverse activity and metabolites levels.

Fabry disease fibroblasts were incubated with compound 7 (200 mM) or Migalastat (at 20 mM), and Fabrazyme ^® (400 ng/mL), or with a combination of all three. After 24 h the cells were harvested, homogenized and the intracellular activity of a-Gal A was measured of the corresponding cell lysates. As expected WT cell line (cl04) presented normal a-Gal A activity whereas untreated classic Fabry patients (R301X and D136Y) and variant mutation samples (A143T and R112H) showed reduced enzymatic activity. None of the cell lines, not classical Fabry fibroblasts R301X and D136Y, showed significant increase in a-Gal A activity when incubated with the inhibitors alone for 24 h, indicating that longer incubation times or higher concentrations may be required to observe this effect (3-5 days for Migalastat at 200 mM, see Asano et a I, 1995, Eur. J. Biochem. 2000,267, 4179-4186).

Treatment with Fabrazyme ^® showed a considerable increase in a-Gal A activity in all the studied cell lines. Interestingly, this effect was improved in most cases with the combinatory treatment of Fabrazyme ^® and PCs after 24 h incubation. In order to investigate whether this increase in a-Gal A activity is due to a stabilization effect on Fabrazyme ^® , we also measured a-Gal A activity in the medium. Thus, the culture medium was collected before harvesting the cells and a-Gal A activity was measured after ConA purification to remove the bound inhibitor and subsequent 4-MU a-Gal assay. a-Gal A activity was at least two times higher in all the cell lines treated with compound 7 (200 mM) or Migalastat (at 20 mM), demonstrating that compound 7 and Migalastat prevent Fabrazyme ^® degradation in cell culture medium.

Gb3 metabolites correction

Normally, Fabry patients present elevated Gb3 levels which can be further metabolized by acid ceramidase into lysoGb3 in lysosomes. These metabolites, which increase with the age of the patient, constitute a distinguishing feature of Fabry disease diagnosis and progression. These metabolites are known to be responsible for the disease manifestations such as neuronophatic pain and renal failure by affecting nociceptive neurons and podocytes.

It was therefore further investigated whether the co-administration of compound 7 and Migalastat with Fabrazyme ^® would also have a positive effect on the correction of these toxic metabolite levels.

Thus, Gb3 and lysoGb3 levels were measured in the in situ treated cell lysates by LC-MS/MS. Normal physiologic Gb3 and lysoGb3 levels are observed in wild type samples (around 1000 pmol/mL and 1 pmol/mL of Gb3 and lysoGb3, respectively). In line with the ERT efficacy, cultured fibroblasts from classic Fabry patients (A143T and D136Y) treated with Fabrazyme ^® resulted in a reduction of the accumulating Gb3 and is deacylated metabolite lysoGb3.

Interestingly, this reduction was similar when fibroblasts were treated with the combination of a-cyclosulfamidate 7 (200 mM) and Fabrazyme ^® , or Gal-DNJ and Fabrazyme ^® co-treatment. Variant Fabry cells lines, A143T and R112H, showed instead different patterns. A143T cell line presents normal Gb3 and lysoGb3 basal levels, whereas in R112H fibroblasts these metabolites are increased and they are not corrected by Fabrazyme ^® itself nor by chaperone-Fabrazyme co treatment.

It has, therefore, been found by applicants that cyclosulfamidates such as compound 7 react reversibly with a-gal A and stabilized the enzyme in vitro and in situ. The in vitro activity and selectivity (IC ₅₀ values and kinetic parameters) and the thermostability results show that compound 7 and Migalastat are able to stabilize Fabrazyme ^® .

In vitro incubation of Fabrazyme ^® in fibroblast culture medium at neutral pH with further purification with ConA beads also showed that compound 7 and Migalastat stabilize Fabrazyme ^® .

This effect also occurred in in situ cell experiments where increased a-gal A activity was observed in the medium of the cells co-treated with Fabrazyme ^® and with compound 7 and Migalastat, and this is translated into at least double amount of active Fabrazyme remaining in the cell culture medium, and slightly higher activity in the cell lysates after 24 h incubation. Regarding toxic accumulation of Gb3 and lysoGb3 metabolites during treatments with Fabrazyme ^® , we found that the combination of Fabrazyme ^® and compound 7 treatment in culture fibroblasts has a similar effect when compared to Fabrazyme ^® alone or Fabrazyme ^® /Migalastat cotreatment after 24 h incubation.

Example 4; Synthesis of Compounds 46 according to the invention, and comparative compounds 45 and 47.

Epoxide 48 was then opened with NaN ₃ to obtain a mixture of regioisomers 49 and 50 (Fig. 2C). These regioisomers proved to be difficult to separate by silica gel column chromatography. Therefore, the mixture of azides was reduced with Pt ₂ 0 to obtain its corresponding 1:1 mixture of amines 51 and 52 which could at this stage be separated by column chromatography. Theses amines were used in separate synthetic routes to obtain cyclosulfamidates 45 and 46.

For the synthesis of cyclosulfamidate 45, amine 51 was-protected with a Boc group under basic conditions to obtain 53 (Fig. 2D). The Boc-protected compound 53 was mesylated under basic conditions to obtain intermediate 54 in 80% yield. Mesylate 54 was converted to cyclic carbamate 55 via neighboring group participation of the Boc protecting group in 85% yield. The cyclic carbamate, with inverted stereochemistry at C-l, was hydrolyzed to obtain amino-alcohol 56, which was subsequently protected with a Boc-group resulting in the formation of 57. The protected amino- alcohol 57 was converted by SOCh into cyclosulfamidate 58 via double nucleophilic displacement at the sulfur center followed and Nal catalyzed oxidation of the intermediate sulfite mixture. The Boc-protecting group was removed by trifluoroacetic acid (TFA) to obtain 59. Removal of the Boc-group was followed by global deprotection with Pd/C to obtain fully deprotected cyclosulfamidate 45 in a 87% yield.

For the synthesis of cyclosulfamidate 46 a similar synthetic route was applied as with 45 (Fig. 2E). Amino-alcohol 52 was Boc-protected to obtain 60. The Boc-protected amino-alcohol 60 was mesylated to obtain the fully protected mesylate 61 without the formation of any side product. The mesylate was heated to promote neighboring group participation, which resulted in the formation of cyclic carbamate 62 with inverted stereochemistry at C-6 in a 64% yield. The carbamate was hydrolyzed with 1 M NaOH to amino-alcohol 63. Amino-alcohol 63 was Boc-protected to obtain 64, which was treated with SOCI ₂ and subsequently oxidized with RuCI ₃ and NalC to obtain fully protected cyclosulfamidate 65 in a 77% yield over 3 steps. The Boc-group was removed with TFA and the purified compound 66 was completely deprotected using Pd/C to obtain a-g/c-cyclosulfamidate 46 in a quantitative yield (see reaction scheme flow chart in Figures 2C to 2E).

Figure 3B shows a-glc-yclosulfamidates 45 and 46 in complex with bacterial a-glucosidase C/Agd31B. 3BA: Cyclosulfamidate 45 reacts with the catalytic amino acid residue in the active site of Q ^' Agd31B, forming a covalent intermediate in a S3 conformation. 3BB: Cyclosulfamidate 46 does not react with the active site of the enzyme and adopts a ⁴ Ci conformation, mimicking a Michaelis complex. Figure 7 illustrates time dependent inhibition of acid a-glucosidase (GAA) by cyclosulfamidates 45 and 46. Herein, the residual activity of recombinant human acid a-glucosidase (GAA, myozyme) with different pre-incubation times (15, 30, 45, 60, 120, 180 and 240 min) in the presence of 45 and 46 at their in vitro apparent ICso concentrations (66 mM and 5.1 mM respectively) is shown as follows: Fig. 7A: Time-dependent inhibition curve of 45 showing irreversible inhibition; Fig 7B: Time dependent inhibition curve of 46 showing reversible inhibition.

Figure 8 then shows the thermostability data of cyclosulfamidates 45 and 46 , which indicates that irreversible inhibitor 45 does not stabilize CjAgdl31B.

In contrast, reversible inhibitor 46 showed the ability to stabilize the bacterial enzyme with a

DTM of 6.29 °C.

This shows conclusively that 46 can stabilize the folding of the enzyme up to 6°C above the temperature at which the enzyme itself would normally become unstable and start to unfold.

This finding is also illustrated by a comparison of apparent IC ₅₀ values for the in vitro inhibition of a-glucosidases GAA (Myozyme) and GANAB (obtaiend from Pompe disease fibroblast lysates) and GBA1 (Cerezyme).

This table clearly shows the efficacy of in particular compound 46, as chaperon for GAA:

Table 2: Apparent ICso values for in vitro inhibition of a-glucosidases GAA (Myozyme) and GANAB (from Pompe disease fibroblast lysates) and GBA1 (Cerezyme). Reported values are mean ± standard deviation from 3 technical triplicates. N.D.: not determined. Compounds listed are 45 to 47.

Structures Compounds In vitro GAA In vitro GANAB In vitro GBA1 apparent ICso apparent ICso apparent ICso

Below, the synthesis and characterization data of the compounds disclosed herein is listed in detail:

Synthesis and Characterization Data of Compounds 3 and 4:

Synthesis of Galactose Configured Cis Diols 16-18.

(l/?,2/?,35,45,5S,6S)-3,4,5-Tris(benzyloxy)-6-((benzyloxy )methyl)cyclohexane-l,2-diol (16) Compound 14 (507 mg, 0.97 mmol) was dissolved in EtOAcMeCN (1:1, 30 mL) and cooled to 0°C. A solution of Nal0 (302 mg, 1.4 mmol) and a catalytic amount of RuCl ₃ -3H ₂ 0 (0.027 mg, 0.13 mmol) in H ₂ 0 (8 mL) was added and the reaction mixture was stirred for 2 h at 0°C. The reaction was then quenched with saturated aqueous Na ₂ S ₂ 03 (10 mL) and the different phases were separated. The aqueous phase was extracted with EtOAc (3 x 30 mL) and the combined organic fractions were washed with brine (100 mL), dried over MgS0 ₄ , filtered, and concentrated in vacuo. Purification by column chromatography (EtOAc from 10% to 70% in pentane) gave pure b-diol 16 (420 mg, 0.76 mmol, 78%). [a] _D ²⁰ = +16.8 (c = 1, MeOH). IR (neat, cm ¹ ) 3446, 3030, 2914, 2862, 1452, 1084, 1059. ² H-NMR (400 MHz, CDCI3): <5 7.42 - 7.20 (m, 20H, CH Ar), 4.99 - 4.88 (m, 3H, CH ₂ Ph, CHHPh), 4.81 - 4.72 (m, 2H, CH ₂ Ph), 4.52 - 4.41 (m, 3H, CH ₂ Ph, CHHPh), 4.26 (s, 1H, CH-4), 3.99 - 3.87 (m, 2H, CH-2, CH-6), 3.77 (t, J = 9.1 Hz, 1H, CHHOBn), 3.66 (dd, J = 9.2, 5.5 Hz, 1H, CHHOBn), 3.54 (d, J = 10.3 Hz, 1H, CH-1), 3.48 - 3.43 (m, 1H, CH-3), 2.71 (s, 1H, OH-1), 1.82 - 1.75 (m, 1H, CH-5), 1.62 (br s, 1H, OH-6). ¹³ C-NMR (100 MHz, CDCI3): <5 138.9, 138.5, 138.0, 137.9 (4 C _P Ph), 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5 (20 CH Ar) 83.9 (C-3), 80.9 (C-2), 77.6 (C-4), 76.0, 75.9 (2 CH ₂ Ph), 75.7 (C-l),

73.6, 73.3 (2 CH ₂ Ph), 72.1 (C-6), 68.1 (C-7), 40.9 (C-5). HRMS: calcd. for [CasHsgOe] ^* 555.27466; found 555.27412. HRMS: calcd for [C ₃ sH ₃ sNa0 ₆ ] ⁺ 577.25661; found 577.25569; in agreement with literature. (lS,2S,3S,4S,5 ?,6/?)-4-((Benzoyloxy)methyl)-5,6-dihydroxycyclohexane-l,2,3 -triyl tribenzoate (17) and (lS,2S,35,45,5S,6S)-4-((benzoyloxy)methyl)-5,6-dihydroxycycl ohexane-l,2,3-triyl tribenzoate (18) Compound 13 (3.77 g, 6.54 mmol) was dissolved in acetone:H ₂ 0 (6:1, 58 mL) and NMO (1.53 g, 13.1 mmol) and a catalytic amount of 0s04 (2.5 wt% in H ₂ 0, 4.99 g, 6.16 mL) were added. The reaction mixture was stirred for 72 h at room temperature. The reaction was then quenched with saturated aqueous [\la2SO3 (50 mL), diluted with H ₂ 0 (20 mL), and extracted with EtOAc (3 x 50 mL). The combined organic phases were washed with brine (100 mL), dried over MgS0 ₄ , filtered, and concentrated in vacuo. The crude mixture gave a ratio of a/b: 1/0.6. Purification by column chromatography with silica gel from Fisher Scientific (60 A, particle size 20-45 micron, toluene to 15% EtOAc in toluene) gave b-diol 18 as a white foam (1.36 g, 2.23 mmol, 34%), a mixture of b-diol 18 and a-diol 17 as a white foam (0.95 g, 1.55 mmol, 24%, ratio a/b: 1/0.1), and a-diol 17 as a white foam (0.81 g, 1.33 mmol, 20%).

ot-Diol 17: [a] _D ²⁰ = +65.2 (c = 1, MeOH). IR (neat, cm ¹ ) 1720, 1601, 1450, 1265, 1109, 1094, 1069, 1026. ¹ H-NMR (400 MHz, CDCI ₃ ): <5 8.01 - 7.89 (m, 6H, CH Ar), 7.71 (dd, J = 8.3, 1.2 Hz, 2H, CH Ar), 7.61 - 7.55 (m, 1H, CH Ar 7.51 - 7.36 (m, 5H,

CH Ar), 7.33 - 7.15 (m, 6H, CH Ar), 6.16 (t, J = 2.7 Hz, 1H, CH-4), 5.99 (dd, J = 10.6,

3.2 Hz, 1H, CH-3), 5.75 (dd, J = 10.6, 2.7 Hz, 1H, CH-2), 4.85 (dd, J = 11.5, 3.9 Hz, 1H, CHHOBz), 4.66 (d, J = 2.3 Hz, 1H, CH-1), 4.47 (dd, J = 11.5, 8.6 Hz, 1H, CHHOBz), 4.27 - 4.19 (m, 1H, CH-6), 3.45 (d, J = 9.2 Hz, 1H, OH-6), 3.37 (d, J = 1.9 Hz, 1H, OH-1), 3.02 - 2.94 (m, 1H, CH-5). ¹³ C-NMR (100 MHz, CDCI ₃ ): d 167.3, 166.1, 165.9, 165.5, (4 C=0), 133.5, 133.2, 129.9, 129.8, 129.2, 128.8, 128.5, 128.4, 128.3 (20 CH Ar), 129.6, 129.5, 129.3, 129.2 (4 C _q Bz), 71.6 (C-l/C-2), 71.3 (C-l/C-2), 70.5 (C-3), 69.1 (C-4), 67.8 (C-6), 63.0 (C-7), 40.0 (C-5). HRMS: calcd. for [CB _S HB _I OI _O ]' 611.19172; found 611.19139.

b-Diol 18: [a] _D ²⁰ = +51.4 (c = 1, MeOH). IR (neat, cm ¹ ) 1720, 1601, 1450, 1267, 1109, 1096, 1070, 1026. ¹ H-NMR (400 MHz, CDCI3): d 8.14 - 8.09 (m, 2H, CH Ar), 8.06 - 7.99 (m, 2H, CH Ar), 7.97 - 7.92 (m, 2H, CH Ar), 7.79 - 7.74 (m, 2H, CH Ar), 7.59 - 7.50 (m, 2H, CH Ar), 7.48 - 7.36 (m, 7H, CH Ar), 7.34 - 7.12 (m, 3H, CH Ar), 6.12 - 6.05 (m, 2H, CH-2, CH-4), 5.52 (dd, J = 10.5, 3.3 Hz, 1H, CH-3), 4.79 (dd, J = 11.3, 7.6 Hz, 1H, CHHOBz), 4.48 (dd, J = 11.3, 7.4 Hz, 1H, CHWOBz), 4.30 (dd, J = 6.4, 3.4 Hz, 1H, CH-6), 3.96 - 3.89 (m, 1H, CH-1), 3.56 (d, J = 7.4 Hz, 1H, OH-1), 3.43 (d, J = 4.2 Hz, 1H, OH-6), 2.49 - 2.42 (m, 1H, CH-5). ¹³ C- IMMR (100 MHz, CDCI3): 5 167.3, 166.8, 165.9, 165.8 (4 C=0), 133.5, 133.4, 133.3, 130.1, 129.8, 129.1, 129.0, 128.6, 128.4, 128.3 (20 CH Ar), 129.5, 129.4 129.3, 128.7 (4 C _q Bz), 73.3 (C-l), 72.1 (C-3), 71.8 (C-2/C-4), 69.5 (C-6), 68.93(C-2/C-4), 61.9 (C-7), 39.7 (C-5). HRMS: calcd. for [CB _S H _SI OI _O F 611.19172; found 611.19127.

General Procedure for the Synthesis of Cyclosulfates 19-21.

Thionyl chloride (3.5 eq. for cis and 7 eq. for trans diol) was added over 5 min to a solution of diol (1 eq.) and triethylamine (4 eq. for cis diols) in DCM (50 mL/mmol) at 0 °C and keeping the reaction at neutral pH. The reaction mixture was then diluted with cold diethyl ether and washed with cold water and brine. The organic phase was dried (MgS04), filtered, concentrated under reduced pressure, coevaporated with toluene (3 x 10 mL) and the residual triethylamine was removed under high vacuum (1 h). The resulting oil was dissolved in CCI ₄ (40 mL/mmol) and MeCN (40 mL/mmol), and the solution was cooled to 0 °C in an ice-bath. A solution of catalytic amount of RuCl ₃ -3H ₂ 0 (0.1 eq.) and Nal0 ₄ (2 eq.) in water (40 mL/mmol) was added and the reaction mixture was stirred at 0 °C for 3 h. Diethyl ether was added and the two layers were separated. The aqueous phase was extracted again with diethyl ether and the combined organic extracts were washed with brine and dried over MgSC> ₄ . The crude was concentrated under reduced pressure and purified by silica column chromatography (from Pentane to Pentane/EtOAc to 8:2) to afford the desired intermediates.

(3a/?,4/?,5S.6S.7/?,7aS)-7-((Benzoyloxy)methyl)-2,2-dioxidoh exahvdrobenzofc/lfl,3,2] dioxathiole-4.5,6-triyl tribenzoate (19)

Obtained as a white solid from 17 (100 mg, 0.16 mmol) in 56% yield (62 mg, 0.09 mmol). [a] _D ²⁰ = +86.5 (c = 1, MeOH). ³ H NMR (400 MHz, CDCI ₃ ): <5 8.05 - 7.97 (m, 2H, CH Ar), 7.97 - 7.87 (m, 4H, CH Ar), 7.80 (d, 2H, CH Ar), 7.66 - 7.51 (m, 2H, CH Ar), 7.51 - 7.35 (m, 6H, CH Ar), 7.35 - 7.23 (m, 4H, CH Ar), 6.27 (t, J = 2.5 Hz, 1H, CH-4), 6.07 (dd, J = 10.6, 3.8 Hz, 1H, CH-2), 5.93 (dd, J = 10.6, 2.7 Hz, 1H, CH-3), 5.80 (t, J = 4.1 Hz, 1H, CH-1), 5.34 (dd, J = 10.4, 4.4 Hz, 1H, CH-6), 4.72 (dd, J = 11.6, 4.5 Hz, 1H, CHHOBz), 4.53 (dd, J = 11.6, 7.4 Hz, 1H, CHHOBz), 3.31 - 3.23 (m, 1H, CH-5). ¹³ C NMR (101 MHz, CDCI ): <5 166.1, 165.6, 165.4, 164.9 (4 C=0), 134.2, 134.1, 133.7, 133.4, 130.2, 129.9, 129.8 129.7 (12 CH Ar), 129.1 (C _q Bz), 128.9, 128.8 (4 CH Ar), 128.6 (C _q -Bz), 128.54, 128.51 (4 CH Ar), 128.1 (C _q -Bz), 81.3 (C-6), 80.4 (C-l), 69.6 (C3), 68.5 (C- 4), 67.0 (C-2), 61.1 (CH ₂ ), 39.8 (C-5). HRM5: calcd. for [C ₃₅ H ₂₉ 0i ₂ S] ⁺ 673.13797; found 673.13794; HRMS: calcd. for [C ₃₅ H ₃ 2NO _I2 S] ⁺ 690.16452; found 690.16436; HRMS: calcd. for [CasHasNaOiaSr 695.11992; found 695.11936.

(3a5,4/?,55,65,7 ?,7a ?)-4,5,6-Tris(benzyloxy)-7-((benzyloxy)methyl)hexahvdrobenzo fc/1 fl,2,31dioxa thiole 2,2-dioxide (20)

Obtained as an oil from 16 (200 mg, 0.36 mmol) in 69% yield (153 mg, 0.25 mmol). [a] _D ²⁰ = -1.7 (c = 1, MeOH). IR (neat, cm ¹ ) 1724, 1454, 1384, 1207, 1091. 2H-NMR (400 MHz, CDCIa): <5 7.36 - 7.23 (m, 20H, CH Ar), 5.21 - 5.18 (m, 1H, CH- 6), 5.03 (d, J = 12.1 Hz, 1H, CHHPh), 4.87 - 4.62 (m, 6H, 2 CH ₂ Ph, CH-1, CH-2), 4.59 - 4.53 (m, 1H, CHHPh), 4.45 - 4.37 (m, 2H, CH ₂ Ph), 4.06 (t, J = 2.2 Hz, 1H,

CH-4), 3.69 - 3.54 (m, 2H, CH ₂ OBn), 3.36 (dd, J = 10.3, 2.1 Hz, 1H, CH-3), 2.07 - 2.00 (m, 1H, CH-5). ¹³ C- NMR (100 MHz, CDCIa): d 138.6, 138.0, 137.9, 137.6 (4 C _q Bn), 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.7, 127.5, 127.3 (20 CH Ar), 87.3 (C-3), 81.5 (C-l), 81.1 (C-4), 77.8 (C-2), 76.1, 74.7 (2 CH ₂ Ph), 73.9 (C-6), 73.8, 73.5 (2 CH ₂ Ph), 67.1 (C-7), 40.7 (C-5). HRMS: calcd. for [C ₃ 5H ₃₇ 0 ₈ S] ⁺ 617.22091; found 617.22083. HRMS: calcd. for [CasHaeNaOgS] ¹ 639.20286; found 639.20223.

(3a5.4/?,5S,6S.7/?,7a/?)-7-((Benzoyloxy)methyl)-2,2-dioxidoh exahvdrobenzofc/lfl,3,2] dioxathiole-4,5,6-triyl tribenzoate (21)

Obtained as a white solid from 18 (45 mg, 0.08 mmol) in 82% yield (41 mg, 0.07 mmol). [ct] _D ²⁰ = +56.1 (c = 1, MeOH). IR (neat, crn ¹ ) 1722, 1601, 1450, 1397, 1258, 1211, 1090, 1069. ³ H-NMR (400 MHz, CDCIa): <58.18 (d , J = 7.3 Hz, 2H, CH Ar), 8.03 (d, J = 7.3 Hz, 2H, CH Ar), 7.94 (d, J = 7.3 Hz, 2H, CH Ar), 7.76 (d, J = 7.3 Hz, 2H, CH

Ar), 7.68 - 7.41 (m, 8H, CH Ar), 7.36 (t, J = 7.8 Hz, 2H, CH Ar), 7.29 - 7.23 (m, 2H, CH Ar), 6.62 (dd, J = 11.0, 8.6 Hz, 1H, CH-2), 6.19 (t, J = 2.4 Hz, 1H, CH-4), 5.56 (t, J = 4.3 Hz, 1H, CH-6), 5.50 (dd, J = 11.1, 2.5 Hz, 1H, CH-3), 5.28 (dd, J = 8.6, 5.1 Hz, 1H, CH-1), 4.68 - 4.55 (m, 2H, CH ₂ OBz), 3.04 - 2.97 (m, 1H, CH-5). ¹³ C-NMR (100 MHz, CDCIa): <5 166.2, 165.6, 165.5, 165.0 (4 C=0), 134.1,

133.8, 130.4, 130.0, 129.9, 129.0, 128.8, 128.6 (20 CH Ar), 128.5, 128.3 (4 C _q Bz), 83.7 (C-l), 79.8 (C- 6), 70.8 (C-3), 68.8 (C-2), 67.0 (C-4), 60.6 (C-7), 38.5 (C-5), 29.9. HRMS: calcd. for [C ₃₅ H ₂₉ 0i ₂ S] ⁺ 673.13797; found 673.13784. HRMS: calcd. for [C ₃₅ H ₂₈ IMaOi ₂ S] ⁺ 695.11992; found 695.11973.

General Procedure for the Deprotection of Cvclosulfates 19 and 21. 7N methanolic ammonia (0.4 eq.) was added to a solution of intermediates 19 and 21 (1 eq.) in MeOH (40 mL/mmol) under argon atmosphere. The reaction mixture was stirred at room temperature and under argon atmosphere for 2 h. Then, the reaction mixture was concentrated under reduced pressure and the crude was purified by chromatography (from DCM to DCM/MeOH 9:1) to afford the desired final products 3 and side product 30.

(3a ?,4/?,5S,6S,7/?.7aS)-4.5,6-Trihvdroxy-7-(hvdroxymethyl)hexah vdrobenzofdUl,3,21 dioxathiole 2,2-dioxide (3)

Obtained as a white solid from 19 (46 mg, 0.07 mmol) in 34% yield (5.9 mg, 0.02 mmol). [a] _D ²⁰ = -9.0 (c 0.5, MeOH). ² H NMR (400 MHz, Methanol-d ₄ ): <5 5.32 (t, J = 4.1 Hz, 1H, CH-1), 4.97 (dd, J = 10.6, 4.4 Hz, 1H, CH-6), 4.18 (t, J = 2.4 Hz, 1H, CH- 4), 4.14 (dd, J = 10.0, 3.9 Hz, 1H, CH-2), 3.88 - 3.75 (m, 2H, CH ₂ ), 3.69 (dd, J = 10.0,

2.5 Hz, 1H, CH-3), 2.31 - 2.23 (m, 1H, CH-5). ¹³ C NMR (101 MHz, Methanol-c/ ₄ ): <5 86.3 (C-l), 85.2 (C-6), 72.6 (C-3), 70.3 (C-4), 68.5 (C-2), 60.2 (CH ₂ ), 44.9 (C-5). HRMS: calcd. for [C ₇ Hi ₂ Na0 ₈ S] 279.01506; found 279.01436.

Deprotection of Cvclosulfate 20

(3a5,4 ?,55,65,7/?,7af?)-4.5,6-Trihvdroxy-7-(hvdroxymethyl)hexahydr obenzofc/]fl,3,2] dioxathiole 2,2-dioxide (4)

Compound 20 (133 mg, 0.216 mmol) was dissolved in dry MeOH (10 mL). The solution was bubbled through with argon and catalytic amount of palladium hydroxide on carbon (20%, 61 mg, 0.086 mmol, 0.4 eq.) was added. The reaction mixture was hydrogenated with a hydrogen balloon and stirred under hydrogen

atmosphere overnight at room temperature. The reaction mixture was filtered over Celite ^® and concentrated in vacuo. Purification by column chromatography (DCM to 20% MeOH in DCM) gave deprotected b-cyclosulfate 4 as a colorless oil in 63% yield (35 mg, 0.137 mmol). [OC]D ²⁰ = -17.7 (c = 1, MeOH). IR (neat, cm ¹ ) 3350, 1373, 1206, 1049. ² H-NMR (400 MHz, Methanol-c/ ₄ ): <5 5.328 (t, J = 4.3 Hz, 1H, CH-6), 4.78 (dd, J = 8.6, 5.0 Hz, 1H, CH-1), 4.31 (dd, J = 10.3, 8.6 Hz, 1H, CH-2), 4.01 (t, J = 2.7 Hz, 1H, CH-4), 3.87 - 3.78 (m, 2H, CH ₂ ), 3.35 (dd, J = 10.4, 2.7 Hz, 1H, CH-3), 2.11 - 2.04 (m, 1H, CH-5). ¹³ C-NMR (100 MHz, Methanol-d ₄ ): <589.7 (C-l), 83.5 (C-6), 73.7 (C-3), 70.4 (C-2), 70.0 (C-4), 60.3 (CH ₂ ), 43.0 (C-5). HRMS: calcd. for [C ₇ Hi ₃ 0 ₈ S] ⁺ 257.03311; found 257.03254. HRMS: calcd. for [C ₇ Hi ₂ Na0 ₈ S] ⁺ 279.01506; found 279.01475.

Synthesis and Characterization Data of Compounds 5 and 6.

(l/?.2/?,3S,4S,5/?,6 ?)-2,3.4-Tris(benzyloxy)-5-((benzyloxy)methyl)-7-oxabicvcloi 4.1.0] heptane

(23) and (lS,2/?,35.4S.5/?,6S)-2,3.4-tris(benzyloxy)-5-((benzyloxy)me thyl)-7-oxabicvcloi4.1.0] heptane

(ot-epoxide) The perbenzylated cyclohexene 14 (0.517 g, 0.99 mmol) was dissolved in dry DCM (10 mL). m-CPBA (<77 %, 0.565 g, 2.5 mmol, 2.5 eq.) was added at 0 °C.

The reaction mixture was stirred at rt overnight. The reaction was quenched

with sat. aq. Na ₂ S0 ₃ . The aqueous phase was extracted with DCM (x3) and the resulting organic phase was washed with sat. aq. NaHC0 ₃ , dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (Pentane/EtOAc 50:1 6:1) to give a mixture of enantiomers (0.509 g, 0.95 mmol, 95 %, 1:4 mixture of ot-epoxide and 23 (b-epoxide) as colorless oils. The isomers were almost completely separated.

23: R _f 0.31 (Pentane/EtOAc 5:1). ¹ H NMR (400 MHz, CDCh): 57.41-7.21 (m, 20H, CH Ar), 4.86 (d, J _gem = 12.0 Hz, 1H, C/JHPh), 4.81 (d, J _{ge m} = 11.4 Hz, 1H, C/JHPh), 4.76 (d, J _gem = 11.4 Hz, 1H, CHHPh), 4.72 (d, Jg _e m = 12.0 Hz, 1H, C/JHPh), 4.70 (d, J _gem = 12.0 Hz, 1H, CHHPh), 4.56 (d, J _gem = 12.0 Hz, 1H, CHHPh), 4.45 (s, 2H, CH ₂ Ph), 4.14 (d, J _2,3 = 8.7 Hz, 1H, CH-2), 3.94 (dd, J _4,5 = 4.5 Hz, J _4,3 = 2.1 Hz, 1H, CH-4), 3.72 (dd, Jg _e m = 8.9 6.9 Hz, 1H, CWHOBn), 3.64 ( 7.5 Hz, 1H, CHHOBn), 3.46 (dd, 1H, CH-3), 3.24 (d, Ji _,6 - 3.8 Hz, 1H, CH-1), 3.17 (t, J _6,5 = 2.9 Hz, 1H, CH-6), 2.32 (tdd, 1H, CH-5). The spectroscopic data are in agreement with those previously reported.

a-epoxide: R _f 0.48 (Pentane/EtOAc 5:1). ^: H NMR (400 MHz, CDCb): 57.43-7.21 (m, 20H, CH Ar), 4.89 (d, J _gem - 11.4 Hz, 1H, CWHPh), 4.85 (d, J _{ge m} = 11.9 Hz, 1H, C/JHPh), 4.81 (d, J _ge m = 11.9 Hz, 1H, CHHPh), 4.72 (d, J _gem - 11.9 Hz, 1H, CHHPh),

4.68 (d, Jg _e m = 11.4 Hz, 1H, CHHPh), 4.51 (d app, 2H, CHHPh, CHHPh), 4.47 (d, J _gem - 11.9 Hz, 1H, CHHPh), 4.25 (dd, J ₂ , ₃ = 8.5 Hz, J _2,i = 2.4 Hz, 1H, CH-2), 3.93-3.91 (m, 1H, CH-4), 3.62 (dd, J _{3, 4} = 1.3 Hz, 1H, CH-3), 3.59 (d, J _C H ₂ , ₅ = 8.0 Hz, 2H. CH ₂ OBn), 3.37 (dd, Ji, ₆ = 3.9 Hz, 1H, CH-1), 2.96 (dd, J _6,5 = 1.5 Hz, 1H, CH-6), 2.30 (td, J ₅ , ₄ = 3.5 Hz, 1H, CH-5).

(1/?, 25,35,45,55, 65)-2-Azido-3.4,5-tris(benzyloxy)-6-((benzyloxy)methyl)cvclo hexan-l-ol (24) and (l/?,25,3/?,45,55,65)-2-azido-4.5,6-tris(benzyloxy)-3-((benz yloxy)methyl)cvclo-hexan-l-ol 25)

Epoxide 23 (0.960 g, 1.8 mmol) was dissolved in dry DMF (50 mL). LiCI0 ₄ (3.81 g, 36 mmol, 20 eq.) and NaN ₃ (1.61 g, 25 mmol, 14 eq.) were added. The reaction mixture was stirred at 80 °C overnight. H ₂ 0 was added and the aqueous phase was extracted with EtOAc (x3). The resulting organic phase was washed with H ₂ 0 (x3) and brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (Pentane/EtOAc 20:1 7:1) to give 24 (0.413 g, 0.71 mmol, 40 %) and 25 (0.397 g, 0.69 mmol, 38 %) as colorless oils.

Cyclohexene 24: R _f 0.42 (Pentane/EtOAc 9:1). [a]r ²⁰ +16.1 (c = 1, CHCI ₃ ). IR (neat, cm ¹ ) 3474, 3030, 2918, 2102, 1454, 1273, 1055, 735, 696. ^X H NMR (400 MHz,

CDCI ₃ ): <5 7.41-7.24 (m, 18H, CH Ar), 7.21-7.15 (m, 2H, CH Ar), 4.94 (d, J _gem = 11.0

Hz, 1H, CHHPh), 4.87-4.69 (m, 4H, 2 x C/JHPh, 2 x CHHPh) 4.46 (d, J _gem = 12.1 Hz, 1H, CHHPh), 4.44 (d, 7 _ge m = 12.1 Hz, 1H, CHHPh), 4.40 (d, 7 _gem = 12.1 Hz, 1H, CHHPh), 4.29 (dd, 7 _2,3 = 9.9 Hz, J _2,i = 3.0 Hz, 1H, CH-2), 4.25 (bs, 1H, CH-4), 4.09 (t, 7I, _2/6 = 3.0 Hz, 1H, CH-1), 3.89 (d, J ₀ = 10.1 Hz,

1H, OH), 3.82-3.75 (m, 1H, CH-6), 3.77 (dd, 1H, CH-3), 3.67 (t, CHH.S = 9.2 Hz, 1H, CHHOBn), 3.54 (dd, 7CHH,5 = 5.4 Hz, 1H, CHHOBn), 2.06-1.98 (m, 1H, CH-5). ¹³ C NMR (100 MHz, CDCI ₃ ) <5 138.7, 138.2, 138.1, 137.9 (C _q Ar), 128.5-127.5 (CH Ar), 80.8 (C-3), 78.4 (C-4), 76.7 (C-2), 75.9, 73.6, 73.4, 73.4 (CH ₂ Ph), 71.6 (C-6), 67.8 (CH ₂ OBn), 64.9 (C-l), 38.7 (C-5). HRMS: Calcd. for CasHsgNaOs ¹ m/z 580.28060, found m/z 580.28048.

Cyclohexene 25: R _f 0.26 (Pentane/EtOAc 9:1). [a]o ²⁰ -29.7 (c = 1, CHCh). IR

N ₃

►OH (neat, cm ³ ) 3431, 3030, 2916, 2102, 1454, 1057, 733, 696. ³ H NMR (400 MHz,

10 CDCIB): d 7.39-7.24 (m, 20H, CH Ar), 5.00 (d, J _ge m 11.1 Hz, 1H, CWHPh), 4.98

BhO^g 'OBn

OBn (<Ugem ^{= 11 L H z} ' ^1H ' ^{CWH Ph} )' ⁴ · ⁷⁷ ( ^d ' ^J gem = ll· ⁷ Hz, 1H, CHHPh), 4.69 (d, 7 _gem

= 11.1 Hz, 1H, CHHPh), 4.68 (d, J _gem = 11.7 Hz, 1H, CHHPh), 4.49 (d app, 2H, CHHPh, CHWPh), 4.41 (d,

./g _e m = 11.7 Hz, 1H, CHHPh), 4.22 (t, y _4,3/5 = 2.3 Hz, 1H, CH-4), 3.89 (t, J _2,i / ₃ = 9.3 Hz, 1H, CH-2), 3.68 (dd, y _gem = 8.8 Hz, Jcm.5 = 4.1 Hz, 1H, CHHOBn), 3.60 (dd, JCHH,S = 10.2 Hz, 1H, CHHOBn), 3.52 (bt, L,b = 9.3

Hz, 1H, CH-1), 3.45 (dd, h _, s = 11.5 Hz, 1H, CH-6), 3.42 (dd, 1H, CH-3), 2.59 (d, J ₀ H,I = 1.5 Hz, 1H, OH), 1.66 (m, 1H, CH-5). ¹³ C NMR (100 MHz, CDCI ₃ ): <5 138.9, 138.6, 138.3, 138.1 (C _q Ar), 128.7-127.6 (CH Ar), 83.7 (C-3), 81.4 (C-2), 77.1 (C-l), 75.7, 75.2 (CH ₂ Ph), 73.6 (C-4), 73.5, 72.6 (CH ₂ Ph), 67.9 (CH ₂ OBn),

61.7 (C-6), 42.5 (C-5). HRMS: Calcd. for C ₃₅ H ₃₈ N ₃ 0 ₅ ⁺ m/z 580.28060, found m/z 580.28030.

(l ?.2S.3/?,45,5S,6/?)-2-Azido-4,5,6-tris(benzyloxy)-3-((benzyl oxy)methyl)cvclohexyl methanesulfonate (26)

The azido alcohol 25 (0.390 g, 0.67 mmol) was dissolved in dry DCM (7 mL). The solution was cooled to 0 °C and Et ₃ N (0.47 mL, 3.4 mmol, 5 eq.) and MsCI (0.26 mL, 3.4 mmol, 5 eq.) were added. The reaction mixture was stirred at rt for 4 h.

The reaction was quenched by the addition of 1 M aq. HCI. The aqueous phase was then extracted with DCM (x3) and the resulting organic phase was dried over MgS0 , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (Pentane/EtOAc 20:1 3:1) to give 26 (0.407 g, 0.62 mmol, 92 %) as a colorless oil. R _f 0.48

(Pentane/EtOAc 5:1). [a] _D ²⁰ -21.4 (c = 1, CHCI ₃ ). IR (neat, cm ¹ ): 3030, 2868, 2106, 1454, 1350, 1175, 735, 696. *H NMR (400 MHz, CDCl ₃ ): <5 7.39-7.24 (m, 20H, CH Ar), 4.97 (d, J _gem = 11.0 Hz, 1H, CHHPh), 4.95 (d, 7 _ge m = 10.6 Hz, 1H, CHHPh), 4.76 (d, 7 _gem = 10.6 Hz, 1H, CHHPh), 4.74 (d, 7 _gem = 11.6 Hz, 1H, CHHPh), 4.68 (d, 7 _gem = 11.6 Hz, 1H, CHHPh), 4.53-4.47 (m, 3H, CH-1, CHHPh, CHHPh), 4.44 (d, 7 _gem = 11.7 Hz, 1H, CHHPh), 4.21 (t, 7 _4,3 /s = 1.8 Hz, 1H, CH-4), 4.06 (t, 7 ₂ I/ ₃ = 9.6 Hz, 1H, CH-2), 3.70 (dd, 7 _gem =

8.8 Hz, 7CHH, ₅ = 4.2 Hz, 1H, CHHOBn), 3.63 (t, 7CHH,S = 9.7 Hz, 1H, CHHOBn), 3.58 (dd, _{, 5} = 11.9 Hz, h _{, i} =

9.9 Hz, 1H, CH-6), 3.47 (dd, 1H, CH-3), 2.96 (s, 3H, CH ₃ ), 1.77 (m, 1H, H5). ¹³ C NMR (100 MHz, CDCI ₃ ): 5 138.6, 137.9, 137.8 (C _q Ar), 128.6-127.7 (CH Ar), 84.5 (C-l), 83.8 (C-3), 78.6 (C-2), 75.6, 75.3, 73.6 (CH ₂ Ph), 73.3 (C-4), 73.2 (CH ₂ Ph), 67.7 (CH ₂ OBn), 61.2 (C-6), 42.4 (C-5), 39.1 (CH ₃ ). HRMS: Calcd. for

C ₃₆ H ₃₉ N ₃ O ₇ SNH m/z 675.28470, found m/z 675.28452.

(15,25.3 ?,45,55,65)-2-Azido-4.5,6-tris(benzyloxy)-3-((benzyloxy)meth v0cvclohexan-l-ol (27)

Mesylate 26 (0.618 g, 0.94 mmol) was dissolved in dry DMF (12 mL) and H ₂ O (3 mL) was added. The reaction mixture was stirred at 140 °C for 3 days in a sealed microwave vial. The reaction mixture was allowed to reach rt and diluted with

EtOAc. The solution was washed with water (x2) and brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (Pentane/EtOAc 20:1 8:1) to give 27 (0.363 g, 0.63 mmol, 67 %) as a colorless oil. R _f 0.57

(Pentane/EtOAc 5:1). [a] _D ²⁰ -17.7 (c = 1, CHCI ₃ ). IR (neat, cm ¹ ): 3422, 3030, 2866, 2097, 1454, 1086, 1074, 733, 696. ³ H NMR (400 MHz, CDCI ₃ ): <5 7.40-7.20 (m, 20H, CH Ar), 4.95 (d, J _gem = 10.8 Hz, 1H, CHHPh), 4.77 (m, 3H, CHHPh, CH ₂ Ph), 4.69 (d, J _gem = 11.5 Hz, 1H, CHHPh), 4.52 (d, J _gem = 11.7 Hz, 1H, CHHPh), 4.47 (d, J _gem = 10.8 Hz, 1H, CHWPh), 4.44 (d, J _gem = 11.7 Hz, 1H, CHHPh), 4.28 (t, 7I _,2/6 = 2.8 Hz,

1H, CH-1), 4.24 (t, J43/5 = 2.2 Hz, 1H, CH-4), 3.89 (dd, J _2,3 = 9.7 Hz, 1H, CH-2), 3.80 (dd, 1H, CH-3), 3.63

(d, J _C H _2,5 7.3 Hz, 2H, CH ₂ OBn), 3.29 (dd, J _{e, 5} 12.0 Hz, 1H, C-H6), 2.63 (s, 1H, OH), 2.46 (dtd, 1H, CH-5). ¹³ C NMR (100 MHz, CDCI ₃ ): <5 139.1, 138.8, 138.2, 138.2 (C _q Ar), 128.6-127.6 (CH Ar), 80.5 (C-3), 78.6 (C-2), 75.3 (CH ₂ Ph), 74.5 (C-4), 73.5, 73.2, 73.2 (CH ₂ Ph), 70.7 (C-l), 68.1 (CH ₂ OBn), 58.6 (C-6), 39.1 (C- 5). HRMS: Calcd. for Css^g sOs* m/z 580.28060, found m/z 580.28029.

(15.25,3/?,45,55,65)-2-Amino-4,5,6-tris(benzyloxy)-3-((benzy loxy)methyl)cvclohexan-l-ol (28)

The azide 27 (0.316 g, 0.54 mmol) was dissolved in dryTHF (20 mL). Pt0 ₂ (0.039 g, 0.17 mmol, 0.3 eq.) was added. The reaction was stirred under an H ₂ atmosphere for 4 h at rt. The mixture wasfiltered through celite and the solvent was removed in vacuo. The crude residue was purified by flash column chromatography (DCM/MeOH 10:1) to give 28 (0.242 g, 0.44 mmol, 80 %) as a colorless oil.

R _t 0.46 (DCM/MeOH 10:1). [a] _D ²⁰ -1.7 (c = 1, CHC ). IR (neat, crrT ¹ ): 3400, 3030, 2918, 1452, 1063, 731, 694. ² H NMR (400 MHz, CDCI ₃ ): d 7.41-7.21 (m, 20H, CH Ar), 4.96 (d, J _gem = 11.2 Hz, 1H, CHHPh), 4.78 (d, Jgem = 11.8 Hz, 1H, CHHPh), 4.77 (d, 7 _gem = 11.5 Hz, 1H, CHHPh), 4.75 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.70 (d, J _gem = 11.5 Hz, 1H, CHWPh), 4.48 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.47 (d, J _gem = 11.2 Hz, 1H, CHHPh), 4.42 (d, 7 _gem = 11.8 Hz, 1H, CHHPh), 4.10 (t, 4, ₃ / ₅ = 2.4 Hz, 1H, CH-4), 3.99 (t, 4, ₂ / ₆ = 3.0 Hz, 1H, CH-1), 3.92 (dd, 4 _,3 9.8 Hz, 1H, CH-2), 3.80 (dd, 1H, CH-3), 3.73 (dd, J _sem 9.0 Hz, 4HH,S = 5.5 Hz, 1H, CWHOBn), 3.57 (t, 4HH, ₅ = 9.0 Hz, 1H, CHHOBn), 2.99 (dd, s = 11.3 Hz, 1H, CH-6), 2.22 (br s, 3H, OH, NH ₂ ), 2.12-2.02 (m, 1H, CH-5). ¹³ C NMR (100 MHz, CDCI ₃ ): <5 139.4, 139.1, 138.6, 138.3 (C„ Ar), 128.5- 127.4 (CH Ar), 80.9 (C-3), 79.1 (C-2), 75.6 (C-4), 74.9, 73.4, 73.1, 72.9 (4 x CH ₂ Ph), 72.0 (C-l), 70.2 (CH ₂ Ph), 50.1 (C-6), 41.3 (C-5). HRM5: Calcd. for C ₃₅ H ₄ oN0 ₅ ⁺ m/z 554.29010, found m/z 554.28981.

ferf-Butyl-(3a5.4/?,5S,6S.7/?.7aS)-5.6.7-tris(benzyloxy)-4-( (benzyloxy)methyl)hexahvdro-3/-/- benzofcilfl.2,3loxathiazole-3-carboxylate 2, 2-dioxide (30)

₀ 5 28 (0.122 g, 0.22 mmol) was dissolved in dry DCM (4 mL). Et ₃ N (0.15 mL, 1.08

I' -O

BocN-Sy mmol, 5 eq.) and Boc ₂ 0 (0.059 g, 0.27 mmol, 1.2 eq.) were added at 0 °C. The

BnCr 'y y ^' reaction mixture was stirred at rt overnight before the addition of sat. aq. NH CI.

BnO 'OB ^h The aqueous phase was extracted with DCM (x3). The resulting organic phase

OBn

was dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was dissolved in dry DCM (5 mL). Imidazole (0.078 g, 1.15 mmol, 5 eq.), Et ₃ N (0.15 mL, 1.08 mmol, 5 eq.) and SOCI ₂ (0.16 mL, 2.19 mmol, 10 eq.) were added at 0 °C. The reaction mixture was stirred at this temperature for 1 h before the addition of H ₂ 0. The aqueous phase was extracted with DCM (x3) and the combined organic phases were dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was dissolved in a 1:1:1 mixture of MeCN, CCI ₄ , and H ₂ 0 (12 mL). RuCI ₃ -3H ₂ 0 (0.010 g, 0.048 mmol, 0.2 eq.) and Nal0 ₄ (0.114 g, 0.53 mmol, 2.4 eq.) were added at 0 °C. The reaction mixture was stirred at this temperature for 1.5 h. H ₂ 0 and sat. aq. Na ₂ S ₂ 0 ₃ were added and the aqueous phase was extracted with EtOAc (x3). The resulting organic phase was washed with brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography

1H, CH-5). ¹³ C NMR (100 MHz, CDCI ₃ ): d 149.0 (C=0), 138.5, 138.4, 137.9, 137.5 (C _q Ar), 128.7-127.6 (CH Ar), 85.7 (C(CHB) ₃ ), 81.0 (C-l), 80.5 (C-3), 75.2 (CH ₂ Ph), 74.4 (C-2), 74.2 (C-4), 73.6, 73.6, 73.4 (CH ₂ Ph), 67.4 (CH ₂ OBn), 57.4 (C-6), 43.2 (C-5), 27.8 (C(CH ₃ ) ₃ ). HRMS: Calcd. for C ₀ H ₄₅ NO ₉ SNa ⁺ m/z 738.27072, found m/z 738.27015.

(3a5,4/?.55.6S.7/?.7a5)-5.6,7-Tris(benzyloxy)-4-((benzyloxy) methyl)hexahvdro-3/-/- benzoidHl,2.3loxathiazole 2,2-dioxide (31) Boc-protected sulfamidate 30 (0.098 g, 0.14 mmol) was dissolved in DCM (2 mL) and TFA (0.2 mL) was added. The reaction mixture was stirred at rt for 8 h. The mixture was then concentrated in vacuo and remaining volatiles were coevaporated with toluene (x3) to give 31 (0.087 g, 0.14 mmol, 100 %) as a

colorless oil. R _f 0.41 (Pentane/EtOAc 3:1). [a]o ²⁰ -2.1 (c = 1, CHCI ₃ ). IR (neat, cm ¹ ): 3030, 2868, 1454, 1339, 1190, 1083, 737, 696. *H NMR (400 MHz, CDCI ₃ ): d 739-7.22 (m, 18H, CH Ar), 7.19-7.11 (m, 2H, CH Ar), 5.36 (d, , ₆ = 3.0 Hz, 1H, NH), 5.04 (t, L _, 2/6 = 4.0 Hz, 1H, CH-1), 4.88 (d, J _gem = 11.1 = Hz, 1H, CHHPh), 4.82 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.77 (s, 2H, CH ₂ Ph), 4.71 (d, J _{ge m} = 11.8 Hz, 1H, CHHPh), 4.45 (s, 2H, CH ₂ Ph), 4.43 (d, 7 _gem = 11.1 Hz, 1H, CHHPh), 4.16 (dd, h _,i = 9.4, 1H, CH-2), 3.96 (t s/s = 2.1 Hz, 1H, CH-4), 3.86 (dd, 1H, CH-3), 3.68 (dt, J _6, s = 10.6 Hz, 1H, CH-6), 3.61 (dd, y _gem = 8.8 HZ, LΉH,B = 8.0 Hz, 1H, CHHOBn), 3.49 (dd, 7 _C HH,5 = 6.5 Hz, 1H, CHHOBn), 2.44-2.36 (m, 1H, CH-5). ¹³ C NMR (100 MHz, CDCIs): d 138.3, 138.2, 137.6, 137.4 (C _q Ar), 128.7-127.6 (CH Ar), 83.0 (C-l), 80.4 (C-3), 75.1 (C-4), 75.0 (C-2), 74.9, 73.7, 73.6, 73.5 (CH ₂ Ph), 70.4 (CH ₂ OBn), 58.6 (C-6), 41.0 (C-5). HRMS: Calcd. for C ₃₅ H ₃₈ N0 ₇ S ⁺ m/z 616.23635, found m/z 616.23619.

(3a5,4 ?.55,65,7 ?,7a5)-5,6,7-tris(benzyloxy)-4-((benzyloxy)methyl)-3-octylhe xahydro-3H- benzofdlfl,2,31oxathiazole 2,2-dioxide (32)

8-lodooctane (24 mΐ, 0.14 mmol), K ₂ C0 ₃ (11 mg, 0.08 mmol), and a catalytic amount of TBAI (3 mg, 6.8 mitioI) were added at 0 °C to a solution of 30 (42 mg, 0.068 mmol) in DMF (2 mL) and the mixture was stirred at rt for 18 h. The reaction mixture was then diluted with ethyl acetate (30 mL) and H ₂ 0 (15 mL)

and the biphasic solution was extracted with ethyl acetate (x2). The combined organic layer was washed successively with water and brine, dried over anhydrous MgSO-i, and concentrated in vacuo to give a residue that was purified by flash column chromatography (from Pentane to Pentane/EtOAc 9:1) to afford the desired product 32 (0.031 g, 0.043 mmol, 62 %) as a colorless oil. [a] _D ²⁰ -24.8 (c = 1, CHCI ₃ ). *H NMR (400 MHz, CDCI ₃ ): <5 7.38 - 7.26 (m, 18H, CH Ar), 7.20 (dd, J = 6.7, 2.9 Hz, 2H, CH Ar), 4.92 - 4.82 (m, 2H, CH-1, CHHPh), 4.82 - 4.72 (m, 3H, CHHPh, Ctf ₂ Ph), 4.57 - 4.37 (m, 3H, CHWPh, CW ₂ Ph), 4.23 (t, J = 2.1 Hz, 1H, CH-4), 4.12 (dd, J = 10.0, 3.8 Hz, 1H, CH-2), 3.86 (dd, J = 10.0, 2.0 Hz, 1H, CH-3), 3.62 - 3.50 (m, 2H, CH ₂ OBn), 3.33 - 3.22 (m, 2H, CHHN, CH-6), 2.84 - 2.74 (m, 1H, CHHN), 2.51 - 2.40 (m, 1H, CH-5), 1.55 - 1.42 (m, 2H, CH ₂ ), 1.33 - 1.19 (m, 10H, 5 x CH ₂ ), 0.88 (t, J = 6.8 Hz, 3H, CH ₃ ). ¹³ C NMR (100 MHz, CDCI ₃ ): <5 138.6, 138.5, 137.9, 137.8 (C _q Ar), 128.6, 128.5, 128.5, 128.4, 128.3, 128.3, 128.2, 128.04, 128.01, 127.96, 127.95, 127.92, 127.8, 127.6, 127.5 (20 x CH Ar), 81.7 (C-l), 80.5 (C-3), 75.2 (CH ₂ Ph), 74.7, 74.6 (C-3, C-4), 73.6 73.4, 73.3 (3 x CH ₂ Ph), 67.2 (CH ₂ OBn), 62.3 (C-6), 51.3 (CH ₂ N), 42.2 (C-5), 31.9, 29.3, 29.2, 29.1, 26.7, 22.8 (6 x CH ₂ ), 14.2 (CH ₃ ). HRMS: Calcd. for C ₄₃ H ₅₄ 0 ₇ S ⁺ m/z 728.36210, found m/z 728.36182; Calcd. for C ₄₃ Hs ₃ N a ₂ 0 ₇ S ^,· m/z 773.33381, found m/z 773.41962.

(3aS,4/?.5S.65.7 ?.7aS)-5.6,7-Trihvdroxy-4-(hvdroxymethyl)hexahvdro-3H-benzof Gilfl.2,3] oxathiazole

2, 2-dioxide (5)

Perbenzylated 31 (0.042 g, 0.068 mmol) was dissolved in MeOH (3 mL), purged with Argon and Pd(OH) ₂ on carbon (20 wt. %, 0.032 g, 0.046 mmol, 0.7 eq.) was added. The reaction mixture was stirred vigorously at rt under a H ₂ atmosphere for 8 h. The mixture was filtered through a celite plug and concentrated in vacuo.

The crude residue was purified by flash column chromatography (from DCM to DCM/MeOH 9:1) to give final compound 7 (0.016 g, 0.063 mmol, 92 %) as a colorless oil. R; 0.55 (DCM/MeOH 7:3 + 1% Et ₃ N). [a] _D ²⁰ -10.7 (c = 0.5, MeOH). IR (neat, cm ¹ ): 3271, 2970, 1327, 1180, 1080,

1028. ³ H NMR (400 MHz, CD ₃ OD): d 4.98 (t, L _,z /b = 3.9 Hz, 1H, CH-1), 4.17 (t, J _4,3 /s = 2.3 Hz, 1H, CH-4),

4.06 (dd, J _2.3 = 10.1 Hz, 1H, CH-2), 3.79-3.70 (m, 2H, CH ₂ OH), 3.68 (dd, J _6, s = 11.5 Hz, 1H, CH-6), 3.67 (dd, 1H, CH-3), 2.16-2.07 (m, 1H, CH-5). ¹³ C NMR (100 MHz, CD ₃ OD): d 88.8 (C-l), 72.7 (C-3), 70.7 (C- 4), 69.2 (C-2), 61.0 (CH ₂ ), 57.2 (C-6), 44.0 (C-5).

(3a5.4/?.55,65,7/?,7a5)-5,6.7-Trihvdroxy-4-(hvdroxymethyl)-3 -octylhexahvdro-3/-/-

fl,2,3]oxathiazole 2,2-dioxide (6)

Perbenzylated 32 (0.022 g, 0.03 mmol) was dissolved in MeOH (5 mL), purged with Argon and Pd(OH) ₂ on carbon (20 wt. %, 0.009 g, 0.012 mmol, 0.4 eq.) was added. The reaction mixture was stirred vigorously at rt under a H ₂ atmosphere for 18 h. The mixture was filtered through a celite plug and concentrated in vacuo. The crude residue was purified by flash column chromatography (from DCM to DCM/MeOH 9:1) to give final compound 8 (0.010 g, 0.027 mmol, 90 %) as a colorless oil. [a] _D ²⁰ -3.0 (c = 0.2, MeOH). ² H NMR (400

MHz, CD ₃ OD): <5 5.01 (t, L _, 2/6 = 3.8 Hz, 1H, CH-1), 4.18 (t, 43/5 = 2.3 Hz, 1H, CH-4), 4.04 (dd, h, ₃ = 10.1,

3.8 Hz, 1H, CH-2), 3.88 - 3.75 (m, 2H, CH ₂ OH), 3.72 - 3.60 (m, 2H, CH-3, CH-6), 3.47 - 3.36 (m, 1H, CHHN), 2.21 - 2.09 (m, 1H, CHHN), 1.71 - 1.51 (m, 2H, CH ₂ ), 1.45 - 1.23 (m, 10H, 5 x CH ₂ ), 0.91 (d, J =

6.8 Hz, 3H, CH ₃ ). ¹³ C NMR (100 MHz, CD ₃ OD): 585.7 (C-l), 72.6 (C-3), 71.2 (C-4), 69.0 (C-2), 63.4 (C-6),

60.9 (CHzOH), 52.3 (CHzN), 45.0 (C-5), 33.0, 30.3, 30.30, 30.2, 27.7, 23.7 (6 x CH ₂ ), 14.4 (CH ₃ ). Calcd. for CI ₅ H ₃ ON0 ₇ S ⁺ m/z 368.17430, found m/z 368.17376

Synthesis and Characterization Data of 7 and 8

ferf-Butyl-((15.2S.35,4S,5S,6/?)-2.3,4-tris(benzyloxy)-5- ((benzyloxy)methyl)-6-hvdroxy- cvclohexyDcarbamate (34) OH Azide 24 (1.475 g, 2.54 mmol) was dissolved in dry THF (25 mL) and Pt0 ₂ 0.175 g, 0.77 mmol, 0.3 eq.) was added. The reaction was stirred vigorously or 1.5 h while being bubbled through with H ₂ . The mixture was then filtered

over celite and concentrated in vacuo. The crude residue was redissolved in dry DCM (25 mL) and Et ₃ N (1.8 mL, 12.9 mmol, 5 eq.) and B0C2O (0.623 g, 2.86 mmol, 1.1 eq.) were added at 0 °C. The reaction mixture was stirred overnight at rt before the addition of sat. aq. NH ₄ CI. The aqueous phase was extracted with DCM (x3) and the resulting organic phase was dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (Pentane/EtOAc 9:1 5:1 -^3:1) to give boc-protected 34 (1.521 g, 2.33 mmol, 91

%) as a colorless oil. R _f 0.64 (Pentane/EtOAc 3:1). [a]o ²⁰ +15.1 (c = 1, CHCI3). IR (neat, cm ¹ ): 3485, 3340, 3030, 2922, 1713, 1497, 1454, 1366, 1169, 1098, 735, 696. ^X H NMR (500 MHz, DMSO -d _6, 373 K): <5 7.37-7.19 (m, 20H, CH Ar), 6.13 (s, 1H, NH), 4.74 (d, J _sem = 11.5 Hz, 1H, CHHPh), 4.70 (d, J _gem = 12.1 Hz, 1H, CHHPh), 4.67 (d, J _gem = 12.1 Hz, 1H, CHHPh), 4.60 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.52 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.48 (d, J _gem = 11.5 Hz, 1H, CHWPh), 4.46 (d, J _gem = 12.1 Hz, 1H, CHHPh), 4.41 (d, J _gem =

12.1 Hz, 1H, CHHPh), 4.13-4.02 (m, 3H, CH-1, CH-2, CH-4), 3.90 (dd, h _,i = 8.6 Hz, J _3,4 2.9 Hz, 1H, CH-3), 3.83 (bs, 1H, OH), 3.73 (dd, 7 _gem = 9.4 Hz, J _C H H,S - 6.9 Hz, 1H, CHHOBn), 3.68 (br s, 1H, CH-6), 3.65 (dd, JCHH,5 = 6.4 Hz, 1H, CHHOBn), 2.28 (br s, 1H, CH-5), 1.38 (s, 9H, C(CH ₃ ) ₃ ). ¹³ C NMR (125 MHz, DMSO-de _, 373 K): <5 155.1 (C=0), 138.7, 138.3, 138.2 (C _q Ar), 127.6-126.5 (20 x CH Ar), 78.6 (C-3), 77.5 (C(CH ₃ ) ₃ ),

77.1 (C-4), 74.5 (C-2), 73.3, 72.0, 71.8, 71.0 (4 x CH ₂ Ph), 69.9 (C-6), 67.5 (CH ₂ OBn), 52.9 (C-l), 40.0 (C- 5), 27.8 (C(CH ₃ ) ₃ ). Calcd. for C ₄ oH ₄₈ 07 ⁺ m/z 654.34308, found m/z 654.34265.

(3aS,4S,55.65,75,7aS)-4,5.6-Tris(benzyloxy)-7-((benzyloxy )methyl)hexahvdrobenzofd]-oxazol-2(3H)- one (36)

Boc-protected 34 (1.488 g, 2.28 mmol) was dissolved in dry CHCI ₃ (20 mL) and Et ₃ (1.6 mL, 11.5 mmol, 5 eq.), 1-methylimidazole (1.8 mL, 22.6 mmol, 10 eq.) and MsCI (0.9 mL, 11.6 mmol, 5 eq.) were added at 0 °C. The reaction mixture was stirred at rt overnight. EtOAc was added and the mixture was washed with 1 M aq. HCI (x3), H ₂ 0 and brine. The organic phase was dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude mesylated residue was dissolved in dry DMF and stirred at 120 °C for 2 days. The mixture was then diluted with EtOAc and washed with H ₂ 0 (x2) and brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography 3:1) to give 36 (0.616 g, 1.1 mmol, 47 % over 2 steps) as a colorless oil.

Rf 0.51 (Pentane/EtOAc 2:1). [a] _D ²⁰ -13.1 (c = 1, CHCI ₃ ). IR (neat, cm ¹ ): 3267, 3030, 2866, 1759, 1452, 1074, 1016, 732, 694. ^X H IMMR (400 MHz, CDCl ₃ ): 57.41-7.27 (m, 18H, CH Ar), 7.23-7.18 (m, 2H, CH Ar), 5.23 (s, 1H, NH), 4.90 (d, J _gem = 11.2 Hz, 1H, CHHPh), 4.86 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.79 (d, 7 _gem = 11.7 Hz, 1H, CWHPh), 4.74 (d, J _{ge m} = 11.7 Hz, 1H, CHHPh), 4.67 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.49 (d, J _ge m = 11.7 Hz, 1H, CHHPh), 4.47 (d, J _gem = 11.2 Hz, 1H, CHHPh), 4.40 (d, J _gem = 11.7 Hz, 1H, CHWPh),

4.27-4.20 (m, 3H, CH-1, CH-4, CH-6), 4.08 (dd, J ₂ , ₃ = 9.6 Hz, J _{2,i mA) = 4.7 Hz, 1H, CH-2/CH-3), 3.75 (dd, J _{ 2 _{, 1} / _{3, 4)} 1.8 Hz, 1H, CH-2/CH-3), 3.66-3.59 (m, 2H, CH ₂ OBn), 2.17-2.07 (m, 1H, CH-5). ¹³ C NMR (100 MHz, CDCI ): <5 158.4 (C=0), 138.6, 138.4, 138.1, 138.0 (C„ Ar), 128.7-127.6 (CH Ar), 81.5 (C-2/C-3), 76.0 (C- 2/C-3), 75.7 (C-4/C-6), 74.9, 74.3, 73.5 (CH ₂ Ph), 73.5 (C-4/C-6), 72.9 (CH ₂ Ph), 67.5 (CH ₂ OBn), 55.5 (C-

1), 43.5 (C-5). Calcd. for C ₃₆ H ₃₈ N0 ₆ ⁺ m/z 580.26994, found m/z 580.26959; Calcd. for C ₃₆ H _4i N ₂ 0 ₆ ⁺ m/z 597.29646, found m/z 59729573; Calcd. for C ₃₆ H ₃₇ NaN0 ₆ ⁺ m/z 602.25186, found m/z 602.25074. (15,25, 35, 45.55.65)-2-Amino-3.4,5-tris(benzyloxy)-6-((benzyloxy)methyl )cyclohexan-l-ol (37)

Intermediate 36 (0.589 g, 1.0 mmol) was dissolved in EtOH (60 mL) and 1 M aq.

NaOH (15 mL) was added. The reaction mixture was stirred at 70 °C for 3 h and then ^{at rt} overnight. EtOH was removed under reduced pressure. Afterwards,

H ₂ 0 was added and the aqueous mixture was extracted with EtOAc (x3). The resulting organic phase was washed with H ₂ 0 and brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (DCM/MeOH 100:1 - 25:1) to give 37 (0.461 g, 0.83 mmol, 82 %) as a white solid. R _f 0.66 (DCM/MeOH 10:1). [a]o ²⁰ +5.5 (c = 1, CHCI ₃ ). IR (neat, cm ¹ ): 3331, 3026, 2920, 1450, 1092, 1022, 719, 696. ³ H NMR (400 MHz, CDCI ₃ ): d

7.39-7.21 (m, 20H, CH Ar), 4.93 (d, J _gem = 11.4 Hz, 1H, CHHPh), 4.80 (d, J _gem = 11.9 Hz, 1H, CHHPh), 4.74 (d, 7 _ge m = 11.7 Hz, 1H, CHHPh), 4.73 (d, J _gem = 11.9 Hz, 1H, CHHPh),4.67 (d, J _gem = 11.7 Hz, 1H, CHHPh), 4.49 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.45 (d, J _gem = 11.4 Hz, 1H, CHHPh), 4.44 (d, J _{ge m} = 11.8 Hz, 1H, CHWPh), 3.96 (t, y _4, 3/s = 2.5 Hz, 1H, CH-4), 3.96-3.92 (m, 1H, CH-2), 3.90-3.85 (m, 2H, CH-3, CH-6), 3.80

(dd, J _gem = 9.0 Hz, Jc HH,5 = 7.6 Hz, 1H, CHHOBn), 3.60 (dd, J _c , ₅ = 6.7 Hz, 1H, CHWOBn), 3.53 (t, Ji, _2/6 = 3.7 Hz, 1H, CH-1), 2.30-2.19 (m, 1H, CH-5), 2.13-1.40 (br s, 2H, IMH ₂ ). ¹³ C NMR (100 MHz, CDCI ₃ ): <5 139.2, 139.1, 138.9, 137.9 (C„ Ar), 128.5-127.5 (20 x CH Ar), 80.5 (C-3), 78.6 (C-2), 75.7 (C-4), 74.9, 73.5, 73.3, 72.7 (4 x CH ₂ Ph), 71.2 (CH ₂ OBn), 70.0 (C-6), 53.2 (C-l), 41.2 (C-5). HRMS: Calcd. for C ₃₅ H ₄ O 0 ₅ ⁺ m/z 554.29010, found m/z 554.28981.

ferf-Butyl-(3a/?, 45,55,65, 7/?, 7a5)-4, 5.6-tris(benzyloxy)-7-((benzyloxy)methyl)hexahvdro-3/-/- benzofdHl,2,3]oxathiazole-3-carboxylate 2, 2-dioxide (39)

The amino alcohol 38 (0.151 g, 0.27 mmol) was dissolved in dry DCM (5 mL). Et ₃ N (0.19 mL, 1.4 mmol, 5 eq.) and Boc ₂ 0 (0.074 g, 0.34 mmol, 1.2 eq.) were added at 0 °C and the reaction mixture was stirred at rt overnight. The reaction was quenched with sat. aq. NH ₄ CI and the aqueous phase was extracted with DCM (x3). The resulting organic phase was dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude mixture was dissolved in dry DCM (5 mL) and Et ₃ N (0.4 mL, 2.9 mmol, 10.5 eq.), imidazole (0.102 g, 1.5 mmol, 5.5 eq.) and SOCI ₂ (0.2 mL, 2.7 mmol, 10 eq.) were added at 0 °C. The reaction mixture was stirred at this temperature for 30 min before the addition of H ₂ 0. The aqueous phase was extracted with DCM (x3). The resulting organic phase was dried over MgS04, filtered and concentrated in vacuo. The crude mixture was dissolved in MeCN (4 mL), CCU (4 mL) and H ₂ 0 (4 mL). RuCI ₃ -xH ₂ 0 (0.014 g, 0.067 mmol, 0.25 eq.) and Nal0 ₄ (0.146 g, 0.68 mmol, 2.5 eq.) were added at 0 °C and the reaction mixture was stirred at this temperature for 1 h. Afterwards, sat. aq. Na ₂ S ₂ C>3 was added and the aqueous phase was extracted with EtOAc (x3). The resulting organic phase was washed with brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was finally purified by flash column chromatography (Pentane/EtOAc 50:1 15:1) to give 39 (0.115 g, 0.16 mmol, 59 % over 3 steps) as a colorless oil. R _f 0.44 (Pentane/EtOAc 10:1). [CX]D ²⁰ +8.8 (c = 1, CHCb). IR (neat, cm ¹ ): 3049, 2976, 1732, 1379, 1329, 1194, 1150, 837. ^X H NMR (400 MHz, CDCIs): <5 7.35-7.25 (m, 16H, CH Ar), 7.22- 7.16 (m, 4H, CH Ar), 4.94 (t, 1/5 = 5.1 Hz, 1H, CH-6), 4.65 (d, J _gei „ = 12.1 Hz, 1H, CHHPh), 4.58 (d, J _gem =

11.5 Hz, 1H, CHHPh), 4.51 (dd, J = 4.0 Hz, 1H, CH-1) 4.44 (d, J _gem = 12.0 Hz, 1H, CHHPh), 4.44-4.37 (m, 4H, CHHPh, 3 x CHHPh), 4.35 (d, J _sem = 12.1 Hz, 1H, CHHPh), 4.19 (t, J _4, 3/5 = 3.8 Hz, 1H, CH-4), 4.09 (t, h _, 3 = 3.0 Hz, 1H, CH-2), 3.76 (dd, J _gem = 9.9 Hz, J _C HH,S = 6.2 Hz, 1H, CHHOBn), 3.69 (t, 1H, CH-3), 3.61 (dd, y _CHH ,5 = 8.1 Hz, 1H, CHHOBn), 2.73 (ddt, 1H, CH-5), 1.55 (s, 9H, (CH ₃ )s). ¹³ C NMR (100 MHz, CDCI3): <5 148.9 (C=0), 138.3, 137.9, 137.8, 137.5 (Cq Ar), 128.6-127.7 (20 x CH Ar), 85.3 (C(CH ₃ ) ₃ ), 79.1 (C-6),

77.5 (C-3), 73.9 (CH ₂ Ph), 73.8 (C-2), 73.3 (2 x CH ₂ Ph), 72.4 (CH ₂ Ph), 72.2 (C-4), 66.6 (CH ₂ OBn), 56.7 (C- 1), 39.8 (C-5), 28.1 ((CH ₃ ) ₃ ). HRMS: Calcd. for C ₄₀ H ₄ 9N ₂ O ₉ S ⁺ m/z 733.31588, found m/z 733.31588; Calcd. for C ₄ oH ₄₅ N0 ₉ SNa ⁺ m/z 738.27072, found m/z 738.27063.

(3a/?, 45,55,65, 7/?, 7a5)-4, 5.6-Tris(benzyloxy)-7-((benzyloxy)methyl)hexahvdro-3/-/- benzofdHl,2,3]oxathiazole 2,2-dioxide (40)

Boc-protected 39 (0.092 g, 0.13 mmol) was dissolved in DCM (5 mL), TFA (0.5 mL) was added and the reaction was stirred at rt for 16 h. The reaction mixture was concentrated and the remaining volatiles were coevaporated with toluene (x3). The crude residue was purified by flash column chromatography to give 40

(0.057 g, 0.092 mmol, 71 %) as a colorless oil. R _f 0.40 (Pentane/EtOAc 9:1). [CX]D ²⁰

-19.8 (c = 1, CHC ). IR (neat, cm ¹ ): 3030, 2866, 1497, 1454, 1339, 1184, 1069, 748, 735, 696. ³ H NMR (400 MHz, CDCIs): d 7.38 - 7.25 (m, 18H, CH Ar), 7.17 (dd, J = 7.1, 2.5 Hz, 2H, CH Ar), 4.96 (d, J = 3.5 Hz, 1H, NH), 4.87 (d, J = 11.0 Hz, 1H, CHHPh), 4.81 (d, J = 11.6 Hz, 1H, CHHPh), 4.76 - 4.65 (m, 3H CH ₂ Ph, CHHPh), 4.55 - 4.47 (m, 2H, CWHPh, CH-6), 4.47 - 4.36 (m, 3H, CW ₂ Ph, CH-1), 4.24 (t, J = 2.1 Hz, 1H, CH-4), 4.11 (dd, J = 9.5, 4.8 Hz, 1H, CH-2), 3.84 (dd, J = 9.6, 2.0 Hz, 1H, CH-3), 3.66 - 3.56 (m, 2H, CH ₂ OBn), 2.61 (tdd, J = 9.3, 4.8, 2.0 Hz, 1H, CH-5). ¹³ C NMR (100 MHz, CDCIs): <5 138.4, 138.3, 137.9, 137.7 (4 x C _q Ar), 128.7, 128.6, 128.4, 128.2, 128.1, 128.1, 128.1, 128.0, 127.9, 127.8, 127.6 (20 x CH Ar.), 82.4 (C-6), 80.8 (C-3), 75.4 (C-2), 75.2, 74.1 (2 x CH ₂ Ph), 73.9 (C-4), 73.4, 73.1 (2 x CH ₂ Ph), 66.8 (CH ₂ OBn), 58.2 (C-l), 41.9 (C-5). HRMS: Calcd. for CB ₅ H _4I N ₂ 0 ₇ S ⁺ m/z 633.26345, found m/z 633.26306; Calcd. for C3sH ₃ 7NNaC>7S ⁺ m/z 638.21584, found m/z 638.21823.

(3a/?, 45,55,65,7/?, 7a5)-4, 5, 6-Tris(benzyloxy)-7-((benzyloxy)methyl)-3-octylhexahvdro-3/- /- benzofdUl,2,3]oxathiazole 2,2-dioxide (41)

8-lodooctane (29 mί, 0.16 mmol, 2 eq.), K2CO3 (13 mg, 0.095 mmol, 1.2 eq.), and a catalytic amount of TBAI (3 mg, 8.0 pmol) were added at 0 °C to a solution of 40 (49 mg, 0.08 mmol) in DMF (2 mL) and the mixture was stirred at rt for 18 h. The reaction mixture was then diluted with ethyl acetate (30 mL) and H ₂ 0

(15 mL) and the biphasic solution was extracted with ethyl acetate (x2). The combined organic layer was washed successively with water and brine, dried over anhydrous MgS04, and concentrated in vacuo to give a residue that was purified by flash column chromatography (from Pentane to Pentane/EtOAc 9:1) to afford the desired product 41 (0.038 g, 0.052 mmol, 66 %) as a colorless oil. [a] _D ²⁰ -21.3 (c = 0.2, CHCI ₃ ). ² H NMR (400 MHz, CDCI ₃ ): <5 7.42 - 7.25 (m, 18H, CH Ar), 7.25 - 7.17 (m, 2H, CH Ar), 4.90 (d, J = 10.9 Hz, 1H, CHHPh), 4.83 (d, J = 11.5 Hz, 1H, CHHPh), 4.75 (s, 2H, CW ₂ Ph), 4.67 (d, J = 11.5 Hz, 1H, CHHPh), 4.53 (d, J = 11.8 Hz, 1H, CHHPh), 4.48 (d, J = 10.9 Hz, 1H, CHHPh), 4.45 - 4.38 (m, 2H, CHHPh, CH-6), 4.25 (t, J = 2.4 Hz, 1H, CH-4), 4.13 (dd, J = 9.6, 3.6 Hz, 1H, CH-2), 4.07 (dd, J = 5.2, 3.6 Hz, 1H, CH-1), 3.88 (dd, J = 9.6, 2.3 Hz, 1H, CH-3), 3.72 - 3.59 (m, 2H, CH ₂ OBn), 3.59 - 3.47 (m, 1H, CHHN), 3.32 - 3.16 (m, 1H, CH/-/N), 2.71 (tdd, J = 9.9, 4.4, 2.5 Hz, 1H, CH-5), 1.72 - 1.59 (m, 2H, CH ₂ ), 1.32 - 1.15 (m, 10H, 5 x CH ₂ ), 0.88 (t, J = 6.9 Hz, 3H, CH ₃ ). ¹³ C NMR (100 MHz, CDCI3): d 138.6, 138.3, 138.0, 137.7 (C _q Ar), 128.6, 128.5, 128.1, 128.0, 127.9, 127.6 (20 x CH Ar), 79.9 (C-3), 79.1 (C-6), 76.3 (C-2), 75.3, 74.9 (2 x CH ₂ Ph), 73.9 (C-4), 73.4, 73.1 (2 x CH ₂ Ph), 66.9 (CH ₂ OBn), 61.4 (C-l), 47.7

(CH ₂ N), 41.9 (C-5), 32.0, 29.4, 29.3, 27.9, 27.0, 22.8 (6 x CH ₂ ), 14.3 (CH ₃ ).

(3a/?.45.55.65.7/?,7a5)-4,5.6-trihvdroxy-7-(hvdroxymethyl)he xahvdro- 2,3]oxathiazole

2, 2-dioxide (7)

Perbenzylated 40 (0.049 g, 0.08 mmol) was dissolved in MeOH (3 mL), purged with Argon and Pd(OH) ₂ on carbon (20 wt. %, 0.022 g, 0.032 mmol, 0.4 eq.) was added. The reaction mixture was stirred vigorously at rt under a H ₂ atmosphere for 18 h. The mixture was filtered through a celite plug and concentrated in vacuo. The

crude residue was purified by flash column chromatography (from DCM to DCM/MeOH 9:1) to give final compound 7 (0.009 g, 0.035 mmol, 44 %) as a colorless oil. R _f 0.45 (DCM/MeOH 8:2 + 1% Ets ).

[oc] _D ²⁰ -14.5 (c = 0.2, MeOH). ³ H NMR (400 MHz, CD ₃ OD): <54.73 (dd, J = 10.6, 5.3 Hz, 1H, CH-6), 4.40 (t,

J = 5.1 Hz, 1H, CH-1), 4.16 (t, J = 2.4 Hz, 1H, CH-4), 4.06 (dd, J = 9.8, 4.9 Hz, 1H, CH-2), 3.85 - 3.76 (m, 2H, CW2OH), 3.72 (dd, J = 9.8, 2.5 Hz, 1H, CH-3), 2.28 (dddd, J = 10.4, 8.0, 4.3, 2.2 Hz, 1H, CH-5). ¹³ C NMR (100 MHz, CD ₃ OD): d 83.8 (C-6), 72.6 (C-3), 70.6 (C-4), 69.0 (C-2), 61.2 (C-l), 60.4 (CH ₂ ), 44.8 (C- 5).

(3a/?.4S.5S.65.7 ?.7aS)-4.5,6-Trihvdroxy-7-(hvdroxymethyl)-3-octylhexahvdro-3 H- benzofcilfl,2,3loxathiazole 2,2-dioxide (8)

O 5 Perbenzylated 41 (0.035 g, 0.048 mmol) was dissolved in MeOH (5 mL), purged

0-S*°

' with Argon and Pd(OH) ₂ on carbon (20 wt. %, 0.014 g, 0.019 mmol, 0.4 eq.) was

HO^V · ' ^N ~(

added. The reaction mixture was stirred vigorously at rt under a H ₂ atmosphere

Hcr y ^' ΌH

QI_I for 3 days. The mixture was filtered through a celite plug and concentrated in vacuo to give final compound 8 (0.010 g, 0.027 mmol, 57 %) as a colorless oil. ¹ H NMR (400 MHz, CD ₃ OD): 54.76 - 4.66 (m, 1H, CH-6), 4.19 (t, / = 2.7 Hz, 1H, CH-4), 4.14 - 4.03 (m, 2H, CH-1, CH-2), 3.88

- 3.78 (m, 2H, CH ₂ OH), 3.78 - 3.74 (m, 1H, CH-3), 3.71 - 3.60 (m, 1H, CHHN), 3.22 (ddd, J = 13.6, 7.8,

6.1 Hz, 1H, CHHN), 2.43 (dddd, J = 10.4, 7.9, 4.4, 2.5 Hz, 1H, CH-5), 1.79 - 1.68 (m, 2H, CH ₂ ), 1.36 - 1.26 (m, 10H, 5 x CH ₂ ), 0.90 (d, J = 6.9 Hz, 3H, CH ₃ ). ¹³ C NMR (101 MHz, MeOD) d 80.8 (C-6), 72.3 (C-3), 70.0

(C-2), 69.8 (C-4), 64.0 (C-l), 60.5 (CH ₂ OH), 49.0 (CH ₂ N ₃ , under MeOD signal), 44.8 (C-5), 33.0, 30.4, 30.3, 28.8, 28.0, 23.7 (6x CH ₂ ), 14.5 (CH ₃ ).

Synthesis and Characterization Data of Compound 9

((((15,25, 35, 45,55, -4,5-Diazido-6-((benzyloxy)methyl)cvclohexane-l,2,3-triyl)-

tris(oxy))tris(methylene))tribenzene (42)

NaN ₃ (0.297 g, 4.5 mmol, 10 eq.) was added to a solution of mesylate 26 (0.289 g, 0.44 mmol) in dry DMF (7 mL) and the reaction mixture was stirred at 100 °C overnight. The mixture was allowed to cool to rt and diluted with EtOAc and H ₂ 0. ^T ^ ^{e a} d ^ueous phase was extracted with EtOAc (x2), and the combined organic phases were washed with H ₂ 0 (x2) and brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (Pentane/EtOAc 50:1 25:1) to give 42 (0.184 g, 0.31 mmol, 69 %) as a colorless oil. Rt 0.44 (Pentane/EtOAc 20:1). [CX]D ²⁰ -1.7 (C = 1, CHC ). IR (neat, cm ¹ ): 3030, 2920, 2099, 1454, 1086, 733, 696. H NMR (400 MHz, CDCI ₃ ): 5 7.39-7.19 (m, 20H, CH Ar), 4.95 (d, J _ge m = 10.9 Hz, 1H, CHHPh), 4.82 (d app, 2H, 2 x CHHPh), 4.74 (d, J _gem = 11.7 Hz, 1H, CHHPh), 4.73 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.50 (d, J _gem = 11.8 Hz, 1H, CWHPh), 4.45 (d, J _gem = 11.8 Hz, 1H, CHHPh), 4.43 (d, 7 _gem = 11.8 Hz, 1H, CHWPh), 4.18 (t, ₄ , ₃ /s = 2.3 Hz, 1H, CH-4), 4.10 (t, Vi, _2/6

= 3.2 Hz, 1H, CH-1), 4.03 (dd, 1 ₂ , ₃ = 9.8 Hz, 1H, CH-2), 3.78 (dd, 1H, CH-3), 3.61-3.55 (m, 2H, CH ₂ OBn), 3.38 (dd, Je _,5 = 11.8 Hz, 1H, CH-6), 2.28-2.18 (m, 1H, CH-5). ¹³ C NMR (100 MHz, CDCI ₃ ): 5 138.9, 138.7, 138.1, 138.0 (4 x C _q Ar), 128.6-127.6 (20 x CH Ar), 80.8 (C-3), 78.1 (C-2), 75.5 (CH ₂ Ph), 74.6 (C-4), 73.6, 73.5, 73.5 (3 x CH ₂ Ph), 67.8 (CH ₂ OBn), 64.9 (C-l), 57.8 (C-6), 40.1 (C-5). HRMS: Calcd. for C ₃₅ H _{37 6} C m/z 605.28708, found m/z 605.28670. (lS.25,3S,4S.5S.6/?)-3.4,5-Tris(benzyloxy)-6-((benzyloxy)met hyl)cvclohexane-l.2-diamine (43)

Pt ₂ 0 (0.005 g, 0.022 mmol, 0.4 eq.) was added to a solution of 42 (0.035 g, 0.058 mmol) in dry THF (2 mL) and the reaction mixture was stirred vigorously under an atmosphere of H ₂ for 2 h. The reaction mixture was then filtered through celite and concentrated in vacuo. The crude residue was purified by

flash column chromatography (DCM/MeOH 100:1 20:1) to give 43 (0.028 g, 0.051 mmol, 88 %) as a colorless oil. R _f 0.56 (DCM/MeOH 9:1 + 1% Et ₃ N). [a] ₀ ²⁰ + 5.3 (c = 1, CHCI ₃ ). IR (neat, cm ¹ ): 3300, 3030, 2922, 1452, 1362, 1090, 733, 696. ^X H NMR (400 MHz, CDCI ₃ ): <57.41-7.22 (m, 20H, CH Ar), 4.96 (d, J _gem - 10.8 Hz, 1H, CHHPh), 4.80 (d, J _gem = 11.7 Hz, 1H, CHHPh), 4.74 (d, J _gem = 11.7 Hz, 1H, CHHPh), 4.72 (d, 7 _gem = 11.7 Hz, 1H, CHHPh), 4.67 (d, 7 _gem = 11.7 Hz, 1H, CHHPh), 4.48 (d, 7 _gem = 11.7 Hz, 1H, CHHPh), 4.47 (d,7 _gem = 10.7 Hz, 1H, CHHPh), 4.42 (d, y _gem = 11.9 Hz, 1H, CHHPh), 4.07 (t, V _4, 3/s = 2.4 Hz, 1H, CH-4), 3.97 (dd, J ₂ ,3 = 9.7 Hz, J _{2, i} = 4.0 Hz, 1H, CH-2), 3.83 (dd, 1H, CH-3), 3.69 (dd, J _gem = 9.0 Hz, J _C HH.S = 5.9 Hz, 1H, CHHOBn), 3.56 (t, 7 _C HH, ₅ = 8.5 Hz, 1H, CHHOBn), 3.44 (t, L _, e = 3.3 Hz, 1H, CH-1), 3.07 (dd, J _6,5 = 11.1 Hz, 1H, CH-6), 2.25-2.04 (m, 5H, CH-5, 2 x NH ₂ ). ¹³ C NMR (100 MHz, CDCI ₃ ): <5 139.4, 139.1, 138.9, 138.2 (4 x C _P Ar), 128.5-127.4 (20 x CH Ar), 80.3 (C-3), 78.8 (C-2), 75.9 (C-4), 74.9, 73.4, 73.2, 72.5 (4 x CH ₂ Ph), 70.3 (CH ₂ OBn), 54.0 (C-l), 50.2 (C-6), 40.9 (C-5). Note: broad ¹³ C NMR signals are observed. HRMS: Calcd. for C35H4o ₂ 04H ⁺ m/z 553.30608, found m/z 553.30592.

(3a5,45,55,65,7 ?,7a5)-4,5,6-Tris(benzyloxy)-7-((benzyloxy)methyl)octahydrob enzofcl-fl,2,5l thiadiazole 2,2-dioxide (44)

Sulfamide (0.106 g, 1.1 mmol, 20 eq.) was added to a solution of 44 (0.030 g,

0.054 mmol) in dry pyridine (5 mL). The reaction mixture was stirred at reflux temperature overnight before it was concentrated in vacuo and the remaining s°l ^{vent was} coevaporated with toluene (x3). Water was added, and the aqueous solution was extracted with DCM (x3). The resulting organic phase was dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (Pentane/EtOAc 9:1 1:1 + 1% Et ₃ N) to give 44 (0.033 g, 0.054 mmol, 99 %) as a colorless oil. [aj _D ²⁰ +9.0 (c = 1, CHCI ₃ ). IR (neat, cm ¹ ): 3254, 3030, 2922, 1454, 1304, 1260, 1111, 737, 698. ^J H NMR (400 MHz, CDCI ₃ ): <5 7.40 - 7.23 (m, 18H, CH Ar), 7.19 - 7.09 (m, 2H, CH Ar), 5.39 (d, J = 1.9 Hz, 1H, NH), 4.89 (d, J = 11.3 Hz, 1H, CHHPh), 4.81 (d, J = 11.6 Hz, 1H, CHHPh), 4.79 - 4.67 (m, 3H, CHHPh, CH ₂ Ph), 4.54 (d, J = 3.5 Hz, 1H, NH), 4.50 - 4.40 (m, 3H, CHHPh, CH ₂ Ph), 4.33 (q, J = 4.4 Hz,

1H, CH-1), 4.11 (dd, J = 9.4, 4.5 Hz, 1H, CH-2), 3.92 - 3.82 (m, 2H, CH-3, CH-4), 3.69 - 3.60 (m, 2H, CH- 6, CWHOBn), 3.49 (dd, J = 9.0, 5.5 Hz, 1H, CHHOBn), 2.54 - 2.43 (m, 1H, CH-5). ¹³ C NMR (100 MHz, CDCIs): <5 138.4, 138.4, 137.9, 137.4 (4 x C _q Ar), 128.7, 128.7, 128.6, 128.5, 128.2, 128.1, 128.1, 128.0, 127.98, 127.95, 127.88, 127.6 (20 x CH Ar), 80.9 (C-3), 76.2 (C-2), 75.8 (C-4), 74.8, 73.9, 73.7, 73.4 (4 x CH ₂ Ph), 71.6 (CH ₂ OBn), 58.5 (C-l), 57.7 (C-6), 41.1 (C-5). HRMS: Calcd. for C ₃₅ H ₃₉ N ₂ 0 ₆ S ⁺ m/z 615.25288, found m/z 615.25222; Calcd. for C ₃₅ H ₄₂ N ₃ 0 ₆ S ⁺ m/z 632.27943, found m/z 632.27814; Calcd. for CasHsgNjNaOeS ^·1· m/z 637.23346, found m/z 637.23483.

(3a5.45.55.65,7/?,7a5)-4,5,6-Trihvdroxy-7-(hvdroxymethvnocta hvdrobenzofcl-fl,2,5l thiadiazole 2,2- dioxide (9)

Pd(OH) ₂ on carbon (20 wt. %, 0.013 g, 0.019 mmol, 0.6 eq.) was added to a solution of 44 (0.018 g, 0.029 mmol) in MeOH (3 mL). The reaction mixture was stirred vigorously at rt under an atmosphere of H ₂ for 6 h. Extra Pd(OH) ₂ on carbon (0.015 g, 0.021 mmol, 0.7 eq.) was added and the reaction mixture was stirred at rt overnight. The mixture was filtered through celite and concentrated in vacuo to give final compound 9 (0.007 g, 0.028 mmol, 94 %) as a colorless oil. R _f 0.31 (DCM/MeOH 7:3 + 1 % Et ₃ N). [a] _D ²⁰ -8.7 (c = 0.5, MeOH). IR (neat, cm ¹ ): 3262, 2926, 1728, 1271, 1130. ² H NMR (400 MHz, CD ₃ OD): 5 4.17 (t, L _,2 / ₆ = 4.8 Hz, 1H, CH-1), 4.11 (t, J _4, 3/s = 2.2. Hz, 1H, CH-4), 4.01 (dd, 7 _2,3 9.9 Hz, 1H, CH-2), 3.78 (dd, J _gem 10.5 Hz, J _C HH. _S = 5.4 Hz, 1H, CHHOH), 3.76-3.68 (m, 2H, CHHOH, CH-3), 3.55 (dd, h _, s = 11.3 Hz, 1H, CH-6), 2.18-2.08 (m, 1H, CH-5). ¹³ C-NMR (100 MHz, CD ₃ OD): 5 11.1 (C-3), 71.1 (C-4), 69.6 (C-2), 63.7 (C-l), 61.7 (CH ₂ OH), 56.7 (C-6), 44.1 (C-5).

Synthesis and Characterization Data of Compounds 45-47

(l/?.2/?,3/?,4S,5/?.6/?)-4.5-Bis(benzyloxy)-2-(hvdroxymet hyl)-7-oxabicvcloi4.1.0)heptan-3-ol (48) and

(lS.2 ?,3/?,4S,5 ?.6S)-4,5-bis(benzyloxy)-2-(hvdroxymethyl)-7-oxabicvclo[4.1. 01heptan-3-ol _ ( O epoxide).

Cyclohexene precursor (2.50 g, 7.34 mmol) was dissolved in anhydrous 73.4 mL DCM, m-CPBA (<77 %, 3.17 g, 18.4 mmol, 2.5 eq) was added at 0 °C and the reaction was for 48 h at 4 °C. The reaction was quenched with sat. aq. ISIa ₂ S0 ₃ and the aqueous layer was extracted with DCM (3x). The combined organic layers were washed with sat. aq. NaHC0 ₃ , dried over MgSC> ₄ , filtered and concentrated in vacuo. The crude mixture was purified by silica column chromatography (DCM/MeOH 100:1 -> 90:10) to obtain 48 (b-epoxide 1.88 g, 5.27 mmol, 72%) and a-epoxide (0.25 g, 0.47 mmol, 6.4 %) as white solids.

-epoxide (15): ² H NMR (400 MHz, CDCI ₃ ) 5 7.41 - 7.27 (m, 10H, CH Ar), 4.95 (d, = 11.3 Hz, 1H, CHHPh), 4.82 (d, J = 11.3 Hz, 1H, CHHPh), 4.69 (d, J = 7.8 Hz, 1H, HHPh), 4.66 (d, J = 7.8 Hz, 1H, CHHPh), 4.01 (dd, J = 10.8, 6.7 Hz, 1H, CHHPh),

3.90 (dd, J = 10.9, 5.3 Hz, 1H, CH-7b), 3.82 (dd, J = 8.0, 0.7 Hz, 1H, CH-7a), 3.49 (t, J = 9.7 Hz, 1H, CH-4), 3.40 (dd, J = 10.0, 8.0 Hz, 1H, CH-3), 3.28 (dd, J = 4.0, 1.7 Hz, 1H, CH-6), 3.17 (d, J = 3.7 Hz, 1H, CH-1), 2.70 (s, 2H, 20H), 2.20 - 2.13 (m, 1H, CH-5). ¹³ C NMR (101 MHz, CDCI ₃ ) d 138.3, 137.5 (2C _q Ar), 128.7, 128.7, 128.2, 128.1, 128.1, 128.00 (10CH Ar), 83.6 (C-3), 79.5 (C-2), 75.0, 72.7 (2CH ₂ Ph), 68.7 (C-4),

64.1 (C-7), 55.0 (C-6), 53.1 (C-l), 43.4 (C-5). LC-MS: 379.01 [M+Na ⁺ ].

a-epoxide: ⁴ H NMR (400 MHz, CDCI3) <5 7.43 - 7.27 (m, 10H, CH Ar), 4.99 (d, J = 11.2 Hz, 1H, CHHPh), 4.83 (d, J = 11.8 Hz, 1H, CHHPh), 4.78 (d, J = 12.0 Hz, 1H, CHHPh), 4.66 (d, J = 11.2 Hz, 1H, CHHPh), 3.89 - 3.80 (m, 3H, CH-2, CH ₂ OH), 3.58

(dd, J = 9.9, 8.1 Hz, 1H, CH-3), 3.44 (t, 1H, CH-4), 3.36 (dd, J = 4.0, 1.9 Hz, 1H,

CH-6), 3.12 (d, V = 4.0 Hz, 1H, CH-1), 2.72 (s, 2H, 20H), 2.15 (dt, J = 9.9, 5.1 Hz, 1H, CH-5). ¹³ C NMR (101

MHz, CDCI3) 5 138.3, 138.1 (2C _q Ar), 128.7, 128.7, 128.6, 128.1, 128.1, 128.0, 127.9 (10CH Ar), 80.9 (C- 3), 79.6 (C-2), 75.6, 72.2 (2CH ₂ Ph), 70.4 (C-4), 62.7 (C-7), 54.6 (C-6), 54.2 (C-l), 43.7 (C-5).

(l/?,25,3 ?.4/?,55,65)-2-Amino-4,5,6-tris(benzyloxy)-3-((benzyloxy)met hyl)cyclohexan-l-ol (51) and (l/?,25,35.45.5/?,65)-2-amino-3,4,5-tris(benzyloxy)-6-((benz yloxy)methyl)cvclohexan-l-ol (52).

Epoxide 48 (0.57 g, 1.06 mmol) was dissolved in anhydrous DMF (22.3 mL) and NaN3 (1.38 g, 21.2 mmol, 20 eq) was added to the mixture. The reaction was stirred overnight at 120 °C. The reaction mixture was allowed to cool to rt, diluted with H ₂ 0 and the aqueous phase was extracted with EtOAc (2x). The combined organic layers were washed with H ₂ 0 and brine, dried over MgSC>4, filtered and concentrated in vacuo. The reaction crude was purified by silica gel chromatography (Pentane/EtOAc 90:10 -> 70:30) to obtain an inseperable mixture of azides. This mixture was dissolved in anhydrous THF (25 mL) and the solution was purged with f\l ₂ . Pt0 ₂ (66 mg, 0.29 mmol, 0.4 eq) was added and the reaction mixture was purged again with N ₂ . The reaction was then stirred under H ₂ atmosphere, overnight at rt. The reaction was flushed with N ₂ , filtered over a celite plug and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (Pentane/Acetone 100:1 -> 40:60) to afford pure 51 (0.16 g, 0.29 mmol, 27 % over 2 steps) and 52 (0.19 g, 0.33 mmol, 33% over 2 steps).

51: [a] _D ²⁰ = +13.1 (c = 1, CHCI ₃ ) IR (neat cm ⁴ ) 3123, 2359, 1452, 1357, 1063, 732, 695. ⁴ H NMR (400 MHz, CDCI ₃ ) <5 7.36 - 7.24 (m, 18H, CH Ar), 7.22 - 7.19 (m, 2H, CH Ar), 4.94 (d, J = 11.2 Hz, 1H, CHHPh), 4.91 - 4.86 (m, 3H, CHHPh), 4.79 (d, J = 11.3 Hz, 1H, CHHPh), 4.53 (d, J = 10.8 Hz, 1H, CHHPh), 4.45 (d, J = 12.0 Hz, 1H, CHHPh), 4.41 (d, J = 12.0 Hz, 1H, CHHPh), 3.76 (dd, J = 9.3, 2.4 Hz, 1H, CH-7a), 3.68 - 3.62

(m, 2H, CH-6, CH-7b), 3.57 (t, J = 9.2 Hz, 1H, CH-1), 3.38 (t, J = 9.2 Hz, 1H, CH-2), 3.24 (t, J = 9.5 Hz, 1H, CH-3), 2.79 (t, J = 10.4 Hz, 1H, CH-4), 2.13 (s, 3H, OH, NH ₂ ), 1.41 (tt, J = 10.9, 2.5 Hz, 1H, CH-5). ¹³ C NMR

(101 MHz, CDCI3) <5 140.4, 140.3, 140.2, 139.9 (4C _q Ar), 130.3, 130.1, 130.1, 129.7, 129.6, 129.5, 129.4, 129.4, 129.3 (20CH Ar), 87.8 (C-l), 85.5 (C-2), 80.3 (C-6), 78.3 (C-3), 77.3, 77.2, 77.1, 74.7 (4CH ₂ Ph), 66.6 (C-7), 52.6 (C-4), 48.40 (C-5). LC-MS: 554.28 [M+H ⁺ ]. 52: [a] _D ²⁰ = +33.5 (c = 1, CHCI ₃ ). IR (neat crn ¹ ) 3123, 2861, 1356, 1090, 731,

695. ⁴ H NMR (400 MHz, CDCI ₃ ) d 7.40 - 7.19 (m, 20H, CH Ar), 4.95 (d, J = 1 ^{02 Hz} ' ^2H ' ^CHWPh )> 4.83 ( ^d > ^J = ¹⁰ · ^{8 Hz} ' ^1H ' CHHPh), 4.73 (d, J = 11.5 Hz, 1H, CHHPh), 4.69 (d, J = 11.5 Hz, 1H, CHHPh), 4.55 (d, J = 11.0 Hz, 1H,

CHHPh), 4.51 (d, J = 11.7 Hz, 1H, CHHPh), 4.44 (d, 7 = 11.7 Hz, 1H, CHHPh), 4.13 (dd, 7 = 9.1, 3.1 Hz, 1H, CH-7a), 4.05 (dd, J = 3.5, 2.1 Hz, 1H, CH-2), 4.04 - 3.90 (m, 3H, CH-1, CH-4, CH-6), 3.73 (dd, J = 9.1, 2.4 Hz, 1H, CH-7b), 3.50 (t, J = 3.6 Hz, 1H, CH-3), 2.30 - 2.23 (m, 1H, CH-5). ¹³ C NMR (101 MHz, CDC ) <5 139.2, 139.0, 138.9, 137.4 (4C _q Ar), 128.7, 128.5, 128.5, 128.5, 128.4, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7, 127.6, 127.5 (20CH Ar), 83.8 (C-l/C-6), 80.7 (C-l/C-6), 77.2 (C-4), 75.6, 75.5 (2CH ₂ Ph), 75.4 (C- 2), 73.8, 72.7 (2CH ₂ Ph), 70.7 (C-7), 52.7 (C-3), 40.9 (C-5). LC-MS: 554.31 [M+H ⁺ ],

7~erf-butyl-((15.2/?,3/?.45.5S.6/?)-3,4.5-tris(benzyloxy) -2-((benzyloxy)methyl)-6

hydroxycyclohexyDcarbamate (53).

Amino alcohol 51 (74 mg, 0.13 mmol) was dissolved in anhydrous DCM (2.6 mL) and Et ₃ N (93 pL, 0.7 mmol, 5 eq) and Boc ₂ 0 (37 pL, 0.16 mmol, 1.2 eq) were added at 0 °C. The reaction was stirred overnight at rt. Then, the reaction was quenched with sat. aq. NH4CI and the aqueous phase was extracted with DCM (3x). The combined organic layers were dried over MgSC , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pentane/EtOAc 100:1 -> 6:4) to obtain 53 (66 mg, 0.1 mmol, 78%) as a colorless oil. [a] _ϋ ²⁰ = + 5.5 (c = 1, CHCI3). IR (neat cm ⁴ ) 2911, 2869, 2359, 1694, 1525, 1365, 1157, 1061, 696. ⁴ H NMR (500 MHz, DMSO-d ₆ ) d 7.36 - 7.22 (m, 20H, CH Ar), 5.33 (dd, J = 11.9, 4.9 Hz, 1H, CHHPh), 4.67 (d, J = 11.9 Hz, 1H, CHHPh), 4.62 (d, J = 12.1 Hz, 1H, CHHPh), 4.56 (td, J = 12.6, 11.9, 2.6 Hz, 4H, CHWPh), 4.44 (d, J = 1.1 Hz, 2H, CHWPh), 4.41 - 4.38 (m, 1H, OH), 3.99 (dd, J = 10.6, 2.9 Hz, 1H, CH-4), 3.95 (dd, J = 5.5, 2.9 Hz, 1H, CH-3), 3.92 (t, J = 3.3 Hz, 1H, CH-7b), 3.82 (dd, J = 11.9, 2.6 Hz, 1H, CH-6), 3.78 (dd, J = 10.7, 6.6 Hz, 1H, CH-7a), 3.15 (q, J = 3.7, 2.5 Hz, 1H, CH-5), 1.46 (s, 9H, (CH ₃ ) ₃ ). ¹³ C NMR (126 MHz, DMSO) d 128.8, 128.7, 128.7, 128.5, 128.1, 128.00, 127.9, 127.9, 127.9, 127.8, 127.7 (CH Ar), 83.8 (C-3), 81.8 (C(CH ₃ ) ₃ ), 77.0, 75.6, 73.5, 73.0, 72.9, 72.6 (4CH ₂ Ph), 71.2 (C-6), 65.4 (C-7), 57.7 (C-l), 41.8 (C-5), 28.4 ((CH ₃ ) ₃ ). TLC-MS: 654.3 [M+H ⁺

(l/?,2/?,35.4/?.5/?,65)-2,3,4-Tris(benzyloxy)-5-((benzylo xy)methyl)-6-((ferf- butoxycarbonyl)amino)cvclohexyl ethanesulfonate (54).

Intermediate 53 (40 mg, 61 pmol) was dissolved in anhydrous DCM (0.6 mL) and Et ₃ N (42 pL, 0.3 mmol, 5 eq), Me-imidazole (50 pL, 0.61 mmol, 10 eq) and MsCI (23 pL, 0.30 mmol, 5 eq) were added to the reaction at 0 °C. The reaction was stirred at rt overnight. The reaction was diluted with

EtOAc and washed with 1M HCI (3x), H ₂ 0 and brine. The organic layer was dried over MgSC , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pentane/EtOAc 100:1 - 7:3) to obtain 54 (35,5 mg, 49 mitioI, 80%) as a yellow oil. [a] _D ²⁰ = +9.2 (c = 1, CHCh). IR (neat cm ¹ ) 3342, 2906, 2358, 1689, 1532, 1349, 1179, 1160 ,960. ^C H NMR (400 MHz, CDCI ) <5 7.41 - 7.25 (m, 18H, CH Ar), 7.24 - 7.20 (m, 2H, CH Ar), 4.97 (d, J = 10.9 Hz, 1H, CHHPh), 4.94 - 4.87 (m, 3H, CHHPh), 4.81 (d, J = 10.9 Hz, 1H, CHHPh), 4.68 (d, J = 9.4 Hz, 1H, CHHPh), 4.61 (d, J = 10.7 Hz, 2H, CHHPh, CH-1), 4.51 (d, J = 11.5 Hz, 1H, CHHPh), 4.41 (d, J = 11.5 Hz, 1H, CHHPh), 3.93 (dd, 1H, CH-6), 3.81 (dd, J = 9.3, 2.6 Hz, 1H, CH-7a), 3.76 (ddd, J = 10.3, 6.5, 2.7 Hz, 1H, CH-4), 3.67 - 3.60 (m, 2H, CH-3, CH-7b), 3.55 (dd, J = 9.4, 2.1 Hz, 1H, CH-2), 2.89 (s, 3H, CH ₃ ), 1.88 - 1.76 (m, 1H, CH-5), 1.48 (s, 9H, (CH ₃ ) ₃ ). ¹³ C NMR (101 MHz, CDCIa) <5 155.2 (C=0), 138.3, 138.2, 138.1, 137.8 (4C _q Ar), 128.5, 128.5, 128.4, 128.0, 127.9, 127.8, 127.7, 127.7, 127.7 (20CH Ar), 85.6 (C- 2/C-3), 83.8 (C-l), 80.8 (C-2/C-3), 77.8 (C-4), 75.7, 75.6, 75.5, 73.5 (4CH ₂ Ph), 64.9 (C-7), 50.0 (C-6), 44.2 (C-5), 38.8 (CH ₃ ), 28.4 ((CH ₃ )a). LC-MS: 754.28 [M+Na ⁺ ].

7erf-butyl-((lS,2S,35,4/?,5S,6/?)-2,3,4-tris(benzyloxy)-5 -((benzyloxy)methyl)-6-

was extracted with DCM (3x). The combined organic layers were dried over MgS04, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pentane/EtOAc 9:1 - 7:3) to obtain 60 (113 mg, 0.17 mmol, 82%) as a colorless oil. [a]o ²⁰ = + 35.2 (c = 1, CHCh). IR (neat cm ¹ ) 3428, 2924, 1698, 1496, 1365, 1159, 1067, 734. ^J H NMR (400 MHz, CDCIa) <5 7.39 - 7.28 (m, 18H, CH Ar), 7.24 - 7.21 (m, 2H, CH Ar), 4.99 (d, J = 10.8 Hz, 1H, CHHPh), 4.95 (d, J = 10.8 Hz, 1H, CHHPh), 4.83 - 4.76 (d, 1H, CHHPh)), 4.70 (d, J = 11.1 Hz, 1H, CHHPh)), 4.59 (d, J = 11.1 Hz, 1H, CHWPh), 4.52 (d, J = 11.0 Hz, 1H, CHHPh), 4.49 - 4.44 (m, 3H, 2CHHPh, CH-6), 4.18 (m, 2H, CH-1, CH-2), 4.09 (dd, J = 9.1, 3.2 Hz, 1H, CH-7a), 4.04 (dd , J = 11.4, 8.9 Hz, 1H, CH-4), 3.80

(s, 1H, OH), 3.75 (dd, J = 9.1, 2.5 Hz, 1H, CH-7b), 3.59 (t, 7 = 9.1 Hz, 1H, CH-3), 1.92 (dd, J = 11.4, 2.5 Hz, 1H, CH-5), 1.47 (s, 9H, (CH ₃ ) ₃ ). ¹³ C NMR (101 MHz, CDCl ₃ ) <5 156.0 (C=0), 138.9, 138.6, 138.1, 137.2 (4C _q Ar), 128.6, 128.4, 128.4, 128.4, 128.1, 128.1, 127.9, 127.8, 127.8, 127.8, 127.7, 127.5 (20CH Ar), 84.3, (C-3) 78.1 (C-l), 76.4 (C-4), 75.6, 75.5, 73.7 (3CH ₂ Ph), 72.1 (C-6), 71.9 (CH ₂ Ph), 70.1 (C-7), 51.8 (C-2), 41.2 (C-5), 28.4 ((CH ₃ )a). LC-MS 654.28 [M+H ⁺ ],

(l/?,2/?,3/?,45,5S,6/?)-3,4,5-Tris(benzyloxy)-2-((benzylo xy)methyl)-6-

((tertbutoxycarbqnyl)aminq)cyclohexyl methanesulfonate (61). Intermediate 60 (146 mg, 0.22 mmol) was dissolved in anhydrous CHCh

(2.20 mL) and Et3lM (0.16 mL, 1.11 mmol, 5 eq), Me-imidazole (0.18 mL,

2.23 mmol, 10 eq) and MsCI (86 pL, 1.11 mmol, 5 eq) were added to the

solution at 0 °C. The reaction was stirred overnight at rt. The reaction mixture was diluted with EtOAc and the organic layer was washed with 1M HCI (3x), H ₂ 0 and brine.

The washed organic layer was dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pentane/EtoAc 9:1 -> 7:3) to obtain 61 (146 mg, 0.19 mmol, 91%) as a yellow oil. [ot] _D ²⁰ = + 2.1 (c = 1, CHCI3). IR (neat cm ¹ ) 3368, 2858, 1703,

1342, 1082. ² H NMR (500 MHz, CDCI ₃ ) d 7.34 - 7.24 (m, 18H, CH Ar), 7.18 - 7.14 (m, 2H ,CH Ar), 5.39 (t, 1H, CH-6), 4.91 (d, 7 = 10.8 Hz, 1H, CHHPh), 4.85 - 4.81 (m, 2H, CHHPh, NH), 4.74 (d, 7 = 10.8 Hz, 1H, CHHPh), 4.64 (d, J = 11.3 Hz, 1H, CHHPh), 4.59 (d, J = 11.3 Hz, 1H, CHHPh), 4.46 (d, J = 12.7 Hz, 3H CHHPh), 4.33 (s, 1H, CH-1), 4.00 (dd, J = 9.4, 4.7 Hz, 1H, CH-2), 3.78 (dd, J = 9.4, 4.3 Hz, 1H, CH-7a),

3.63 (t, J = 8.9 Hz, 1H, CH-3), 3.56 (t, J = 9.5 Hz, 1H, CH-4), 3.50 (s, 1H, CH-7b), 2.91 (s, 3H, CH ₃ ), 2.42 (t, J = 8.4 Hz, 1H, CH-5), 1.44 (s, 9H, (CH ₃ ) ₃ ). ¹³ C NMR (126 MHz, CDCI3) d 155.7 (C=0), 138.6, 138.2,

137.9, 137.6 (4C _q Ar), 128.7, 128.6, 128.6, 128.4, 128.2, 128.0, 127.9, 127.9, 127.8 (CH Ar), 83.0 (C-3),

77.7 (C-6), 77.2 (C-4), 77.1 (C-2), 75.8, 75.4, 73.1, 72.4 (4CH ₂ Ph), 66.4 (C-7), 50.8 (C-l), 40.5 (C-5), 37.6 (CHB), 28.4 (CH ₃ ) ₃ ). LC-MS: 754.3 [M+Na ⁺ ],

(3a5.45,55,6 ?,75,7a5)-4,5,6-Tris(benzyloxy)-7-((benzyloxy)methyl)hexahvd robenzofd)oxazol-2(3/7)-one (62).

Intermediate 61 (140 mg, 0.19 mmol) was dissolved in anhydrous DMF (8.7 mL) and the reaction mixture was stirred for 24 h at 120 °C. The reaction was then cooled to rt and diluted with H ₂ 0. The aqueous phase was extracted with EtOAc (3x) and the combined organic layers were washed

with H ₂ 0 (2X) and brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pent/EtOAc 100:1 -> 50:50) to obtain 62 (70 mg, 0.12 mmol, 64%) as an orange oil. [a] _D ²⁰ = - 1.4 (c = 1, CHCU). IR (neat cm ¹ ) 2923, 2361, 1755, 1453, 1093,

1027. ² H NMR (500 MHz, CDCI ₃ ) 6 7.40 - 7.27 (m, 18H, CH Ar), 7.26 - 7.22 (m, 2H, CH Ar), 5.35 (s, 1H,

NH), 4.83 (d, 1H, CHWPh), 4.80 (dd, J = 7.1, 2.1 Hz, 1H, CH-6), 4.78 (d, 7 = 1.2 Hz, 3H, CHHPh), 4.61 (d , 7 = 11.9 Hz, 1H, CHHPh), 4.57 (d, J = 10.9 Hz, 1H, CHtfPh), 4.50 (d, J = 11.7 Hz, 1H, CHHPh), 4.47 (d, J =

11.7 Hz, 1H, CHHPh), 4.08 (dd, J = 7.4, 4.3 Hz, 1H, CH-1), 3.89 (dd, J = 9.3, 2.4 Hz, 1H, CH-7a), 3.86 (dd,

J = 8.2, 7.3 Hz, 1H, CH-3), 3.65 (dd, J = 9.3, 2.2 Hz, 1H, CH-7b), 3.60 (dd, J = 7.3, 4.3 Hz, 1H, CH-2), 3.56 (dd, J = 11.6, 8.2 Hz, 1H, CH-4), 2.24 - 2.17 (m, 1H, CH-5). ¹³ C NMR (126 MHz, CDCI3) <5 158.9 (C=0),

138.4, 138.3, 138.1, 137.7 (4C _q Ar), 128.8, 128.6, 128.6, 128.6, 128.3, 128.1, 128.0, 128.0, 127.9, 127.8 (20CH Ar), 82.5 (C-3), 77.4 (C-2), 76.2 (C-4), 75.1, 74.8 (2CH ₂ Ph), 74.2 (C-6), 73.6, 73.5 (2CH ₂ Ph), 65.5 (C-7), 54.5 (C-l), 44.6 (C-5). LC-MS: 580.00 [M+H ⁺ ], (3a5,4/?.5 ?.65.75.7a5)-5.6,7-Tris(benzyloxy)-4-((benzyloxy)methyl)hexa hvdrobenzofGiloxazol-2(3/-/)-one

Intermediate 54 (89 mg, 0.12 mmol) was dissolved in anhydrous DMF (5.6 mL). The reaction mixture was stirred for 24 h at 120 °C. The reaction was cooled to rt and diluted with H2O. The aqueous phase was extracted with EtOAc (3x) and the combined organic layers were washed with H2O (2x) and brine, dried over MgS0 ₄ ,

filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pent/EtOAc 100:1 -> 50:50) to obtain 25 (60 mg, 0.11 mmol, 85%) as a colorless oil. [a] _D ²⁰ = +29.6 (c - 0.5, CHC ). IR (neat cm ¹ ) 2926, 2359, 1762, 1264, 732. ⁴ H NMR (400 MHz, CDCI3) <5 7.41 - 7.24 (m, 16H, CH Ar), 7.24 - 7.20 (m, 2H CH Ar), 7.20 - 7.14 (m, 2H, CH Ar), 5.67 (s, 1H, NH), 4.79 (d, J = 12.0 Hz, 1H, CHHPh), 4.71 (dd, J = 8.7, 3.7 Hz, 1H, CH-1), 4.64 (d, J = 12.1 Hz, 1H, CHHPh), 4.60 (d, J = 11.5 Hz, 1H, CHHPh), 4.48 (dd, J = 11.6, 4.4 Hz, 2H, CHHPh), 4.43 - 4.34 (m, 3H, CHHPh), 3.87 (t, J = 3.7 Hz, 1H, CH- 2), 3.82 - 3.75 (m, 3H, CH-3, CH-6, CH-7a), 3.29 (t, J = 9.0 Hz, 1H, CH-4), 3.24 (dd, J = 12.0, 5.5 Hz, 1H, CH-7b), 2.61 (dtd, J = 12.8, 9.1, 3.8 Hz, 1H, CH-5). ¹³ C NMR (101 MHz, CDCI3) d 158.7 (C=0), 138.0, 137.8, 137.7, 137.6 (4C _q Ar), 128.7, 128.7, 128.6, 128.5, 128.3, 128.2, 128.1, 128.1, 128.0, 127.9 (20CH Ar), 81.9 (C-3/C-6), 78.3 (C-4), 75.7 (C-2), 74.8 (C-l), 73.7, 73.0, 72.6 (3CH ₂ Ph), 71.5 (C-7), 54.4 (C-3/C-6), 42.5 (C-5). LC-MS 580.06 [M+H ⁺ ],

(15,25,3/?, 4/?, 55, 65)-2-Amino-4.5,6-tris(benzyloxy)-3-((benzyloxy)methyl)cvclo hexan-l-ol (56) .

Cyclic carbamate 55 (60 mg, 0.11 mmol) was dissolved in EtOH (6.6 mL) and NaOH (1M, 1.65 mL, 1.65 mmol, 15 eq) was added to the solution. The reaction mixture was stirred at 70 °C for 3 h and was subsequently stirred overnight at

rt. After 24 h extra NaOH (1M, 1.65 mL, 1.65 mmol, 15 eq) was added and the reaction was again heated to 70 °C and stirred for 3 h followed by overnight stirring at rt.. The reaction mixture was concentrated and the crude residue was diluted with H ₂ 0. The aqueous phase was extracted with EtOAc (3x) and the combined organic layers were washed with H ₂ 0 and brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (DCM/MeOH 100:1 - 85:15) to obtain 56 (50 mg, 90 pmol, 82%) as a colorless oil. [a] _D ²⁰ = + 42.8 (c = 0.3, CHCI3). IR (neat cm ⁴ ) 2923, 1735, 1367, 1143, 1096, 1046, 734, 695. ⁴ H NMR

(500 MHz, CDCI3) <5 7.37 - 7.24 (m, 18H, CH Ar), 7.22 - 7.18 (m, 2H, CH Ar), 4.91 (d, J = 10.8 Hz, 1H, CHHPh), 4.88 (d, J = 10.9 Hz, 1H, CHHPh), 4.81 (d, J = 10.8 Hz, 1H, CHWPh), 4.71 (d, 1H, CHHPh), 4.70 (d, 1H, CHHPh), 4.48 (d, J = 10.9 Hz, 1H, CHHPh), 4.43 (s, 2H, CHHPh), 4.12 (t, J = 2.7 Hz, 1H, CH-1), 3.90

(t, J = 9.4 Hz, 1H, CH-3), 3.75 (dd, J = 9.5, 3.2 Hz, 1H, CH-7a), 3.67 (dd, J = 9.5, 2.6 Hz, 1H, CH-7b), 3.49

(dd, J = 10.8, 9.3 Hz, 1H, CH-4), 3.43 (dd, J = 9.5, 2.7 Hz, 1H, CH-2), 2.92 (dd, J = 10.9, 2.5 Hz, 1H, CH-6), 2.64 (s, 3H, NH ₂ , OH), 1.97 (tt, J = 11.0, 2.9 Hz, 1H, CH-5). ¹³ C NMR (126 MHz, CDCI ₃ ) d 139.0, 138.7, 138.3, 138.1 (4C _q Ar), 128.6, 128.6, 128.5, 128.5, 128.1, 128.1, 128.0, 127.9, 127.7, 127.6 (20CH Ar), 83.3 (C-3), 81.3 (C-2), 78.7 (C-4), 75.8, 75.5, 73.2, 72.6 (4CH ₂ Ph), 70.9 (C-l), 66.3 (C-7), 49.9 (C-6), 43.4 (C-5). LC-MS: 554.21 [M+H ⁺ ],

( 15,25,35,45, 5/?,65)-2-Amino-3,4.5-tris(benzyloxy)-6-((benzyloxy)methyl)c vclohexan-l-ol (63).

Cyclic carbamate 62 (70 mg, 120 pmol) was dissolved in EtOH (7.2 mL) and NaOH (1M, 1.8 mL, 1.8 mmol, 15 eq) was added to the solution. The reaction was stirred at 70 °C for 3 h and was subsequently stirred overnight

at rt. The reaction mixture was concentrated and the crude residue was diluted with H ₂ 0. The aqueous phase was extracted with EtOAc (3x) and the combined organic layers were washed with H ₂ 0 and brine, dried over MgSC , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (DCM/MeOH 100:l-> 80:20) to obtain 63 (64 mg, 115 pmol, 96%) as a colorless oil. IR (clean cm ⁴ ) 2915, 1723, 1298, 1121, 1032, 736, 696. ³ H NMR (400 MHz, CDCI ₅ ) <5 7.38 - 7.23 (m, 18H, CH Ar), 7.22 - 7.18 (m, 2H, CH Ar), 4.93 (d, J = 10.7 Hz, 1H, CHWPh), 4.87 (d, J = 10.9 Hz, 1H, CHHPh)), 4.79 (d, J = 10.8 Hz, 1H, CHHPh), 4.67 (s, 2H, CHHPh)), 4.50 (d, J = 11.6 Hz, 2H, CHHPh), 4.45 (d, J = 12.0 Hz, 2H, CH-6), 3.98 (t, J = 9.3 Hz, 1H, CH-3), 3.85 (dd,

J = 9.0, 2.8 Hz, 1H, CH-7a), 3.69 - 3.60 (m, 2H, CH-6, CH-7b), 3.52 (t, J = 3.7 Hz, 1H, CH-1), 3.43 (dd, J = 9.6, 3.7 Hz, 1H, CH-2), 3.35 (dd, J = 11.0, 9.1 Hz, 1H, CH-4), 2.32 - 2.05 (m, 4H, CH-5, NH ₂ , OH). ¹³ C NMR (101 MHz, CDCIs) d 139.0, 138.7, 138.5, 138.1 (4C _q Ar), 128.6, 128.6, 128.5, 128.5, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6 (20CH Ar), 82.8 (C-3), 80.7 (C-2), 75.7, 75.2, 73.4, 72.4 (4CH ₂ Ph), 69.9 (C-6), 68.7 (C-7), 52.8 (C-l), 42.9 (C-5). LC-MS: 554.18 [M+H ⁺ ],

butyl-(3a5.4/?,5/?,65,7/?,7a5)-5.6,7-tris(benzyloxy)-4-((ben zyloxy)methyl)hexahvdro-3H- benzofdHl,2,31oxathiazole-3-carboxylate 2, 2-dioxide (58).

The amino alcohol 56 (50 mg, 90 mihoI) was dissolved in anhydrous DCM (1.67 mL). Ets (63 mί, 0.45 mmol, 5 eq) and Boc ₂ 0 (25 pL, 0.11 mmol, 1.2 eq) were added at 0 °C and the reaction was stirred overnight at rt. The reaction was then quenched with sat. aq. NH ₄ CI and the aqueous phase was

extracted with DCM (3x). The combined organic layers were dried over MgS0 ₄ , filtered and concentrated in vacuo. The Boc proctected intermediate was dissolved in anhydrous DCM (1.67 mL) and Ets (132 pL, 0.95 mmol, 10.5 eq), imidazole (34 mg, 0.50 mmol, 5.5 eq) and SOCI ₂ (66pL, 0.9 mmol, 10 eq) were added at 0 °C and the reaction was stirred at rt for 20 min. H ₂ 0 was added to the reaction mixture and the aqueous phase was extracted with DCM (3x). The combined organic layers were dried over MgSC> ₄ , filtered and concentrated in vacuo. The crude mixture of sulfites was dissolved in a 1:1:1 mixture of H ₂ 0 (1.3 mL), EtOAc (1.3 mL) and MeCN (1.3 mL), and Nal0 ₄ (48 mg, 0.23 mmol, 2.5 eq) and RuCI ₃ -H ₂ 0 (4.7 mg, 23 pmol, 0.25 eq) were added at 0 °C and the reaction was stirred at this temperature for lh. The reaction was quenched with sat. aq. Na2S2C>3 and the aqueous phase was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over MgS04, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pentane/EtOAc 100:1 - 90:10) to obtain 58 (21 mg, 30 pmol, 33% over 3 steps) as a colorless oil. [ot] _D ²⁰ = + 15.2 (c = 0.5, CHCI3). IR (neat cm ¹ ) 2923, 1735, 1453, 1379, 1319, 1260, 1027, 842, 735, 695. NMR (400 MHz, CDCI3) <57.37 - 7.23 (m, 18H, CH Ar), 7.19 - 7.14 (m, 2H, CH Ar), 4.99 (t, J = 3.6 Hz, 1H, CH-1), 4.92 (d, J = 10.7 Hz, 1H, CHHPh), 4.91 - 4.84 (m, 2H, CHHPh), 4.79 (d, J = 11.9 Hz, 1H, CHHPh), 4.74 (d, J = 11.9 Hz, 1H, CHHPh), 4.66 (dd, J = 10.4, 3.9 Hz, 1H, CH-6), d 4.52 (d, J = 11.3 Hz, 1H, CHHPh), 4.35 (d, J = 11.3 Hz, 1H, CHHPh), 3.94 (t, 1H, CH-3), 3.91 - 3.88 (m, 1H, CH-7a), 3.63 (dd, J = 7.0, 2.9 Hz, 1H, CH-4), 3.59 (dd, J = 1.8 Hz 1H, CH-2), 3.34 (dd, J = 9.5, 1.9 Hz, 1H, CH-7b), 2.22 (tt, 1H, CH-5), 1.55 (s, 9H (CH ₃ ) ₃ ). ¹³ C NMR (101 MHz, CDCI3) d 149.0 (C=0), 138.5, 138.4, 138.2, 137.2 (4C _q Ar), 128.8, 128.6, 128.5, 128.5, 128.4, 128.2, 128.2, 128.0, 127.9, 127.8 (20CH Ar), 85.7 (CH ₂ Ph), 81.9 (C-3), 80.7 (C-l), 76.8 (C-2/C-4) 76.5 (C-2/C-4), 76.1, 75.8, 73.6 (3CH ₂ Ph), 64.7 (C-7), 56.1 (C-6), 45.4 (C-5), 28.0 ((CH ₃ ) ₃ ). LC-MS: 716.14 [M+H ⁺ ],

7 ^~ erf-butyl-(3a/?.4S,5S,6 ?.7/?.7a5)-4,5.6-tris(benzyloxy)-7-((benzyloxy)methyl)hexahv dro-3f-/- benzofdlfl,2,31oxathiazole-3-carboxylate 2, 2-dioxide .

The amino alcohol 63 (63 mg, 0.12 mmol) was dissolved in anhydrous DCM (2.22 mL). Et ₃ N (84 pL, 0.6 mmol, 5 eq) and B0C2O (32pL, 0.14 mmol, 1.2 eq) were added to the solution at 0 °C and the reaction was stirred overnight at rt. The reaction was quenched with sat. aq. NH4CI and the

aqueous phase was extracted with DCM (3x). The combined organic layers were dried over MgSC , filtered and concentrated in vacuo. The Boc protected intermediate was dissolved in anhydrous DCM (2.22 mL) and Et ₃ N (0.18 mL, 1.26 mmol, 10.5 eq), imidazole (45 mg, 0.66 mmol, 5.5 eq) and SOC (88 pL, 1.2 mmol, 10 eq) were added at 0 °C. The reaction was stirred at this temperature for 20 min. H2O was added to the reaction mixture and the aqueous phase was extracted with DCM (3x). The combined organic layers were dried over MgSC , filtered and concentrated in vacuo. The crude mixture of sulfites was dissolved in a 1:1:1 mixture of H2O (0.6 mL), EtOAc (0.6 mL) and MeCN (0.6 mL), and NalC> ₄ (64 mg, 0.30 mmol, 2.5 eq) and RuCl ₃ -H ₂ 0 (6.2 mg, 30 pmol, 0.25 eq) were added at 0 °C and the reaction was stirred at this temperature for 1 h. The reaction was quenched with sat. aq. Na ₂ S ₂ C> ₃ and the aqueous phase was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over MgS04, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pentane/EtOAc 100:1 -> 90:10) to obtain 65 (66 mg, 92 pmol, 77% over 3 steps) as a colorless oil. [a]o ²⁰ = -14.4 (c = 1, CHCI3). IR (neat cm ¹ ) 2923, 1732, 1453, 1379, 1325, 1090, 865, 735, 696. NMR (500 MHz, CDCI3) d 7.40 - 7.23 (m, 18H, CH Ar), 7.18 - 7.15 (m, 2H, CH Ar), 5.24 (t, J = 8.4, 7.3, 0.9 Hz, 1H, CH-6), 4.65 (d, J = 12.2 Hz, 1H, CHHPh), 4.61 (d, J = 11.1 Hz, 1H, CHHPh), 4.58 - 4.50 (m, 3H, CHHPh), 4.48 - 4.42 (m, 2H, CHHPh), 4.33 - 4.27 (m, 2H, CHHPh, CH-1), 4.10 (s, 1H, CH-2), 3.87 (dd, J = 9.4, 2.4 Hz, 1H, CH-7a), 3.82 (dd, J = 5.6,

1.4 Hz, 1H, CH-3), 3.64 - 3.58 (m, 2H, CH-4, CH-7b), 2.73 (ddt, J = 13.0, 8.8, 2.3 Hz, 1H, CH-5), 1.51 (s, 9H, (CH ₃ ) ₃ ). ¹³ C NMR (126 MHz, CDCh) d 148.7 (0=0), 138.2, 138.0, 137.4, 137.4 (4C _q Ar), 128.7, 128.7,

128.6, 128.5, 128.5, 128.3, 128.2, 128.1, 127.9, 127.9, 127.8 (20CH Ar), 82.6 (C-3), 76.2 (C-4) , 76.1 (C- 6) , 74.0 (CH ₂ Ph), 73.6 (C-2), 73.5, 73.0, 71.6 (3CH ₂ Ph), 64.9 (C-7), 56.8 (C-l), 41.1 (C-5), 28.1 ((CH ₃ )B). LC-MS: 716.17 [M+H ⁺ ],

(3a5,4 ?.5/?.65.7/?,7a5)-5,6.7-Tris(benzyloxy)-4-((benzyloxy)methyl )hexahydro-3H

benzofdlfl.2,31oxathiazole 2,2-dioxide (59) .

Cyclosulfamidate 58 (20 mg, 30 mitioI) was dissolved in anhydrous DCM (1.2 mL) and TFA (0.11 mL) was added to the reaction mixture. The reaction was stirred overnight at rt. Then, the reaction mixture was diluted with H ₂ 0 and the aqueous phase was extracted with DCM (3x). The combined organic layers were dried over MgS0 ₄ , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pent/EtOAc 95:5 - 70:30) to obtain 59 (13 mg, 21 pmol, 65%) as a colorless oil. [a] _D ²⁰ = -25.8 (c = 0.5, CHCI ₃ ). 2923, 1453, 1362, 1190, 1098, 837, 735, 696. ² H NMR (400 MHz, CDCI ₃ ) d 7.40 - 7.22 (m, 18H, CH Ar), 7.21 - 7.14 (m, 2H, CH Ar), 4.85 (dd, J = 5.6, 3.1 Hz, 1H, CH-1), 4.77 (d, J = 11.0 Hz, 1H, CHHPh), 4.73 (d, J = 11.7 Hz, 1H, CHHPh), 4.71 - 4.60 (m, 3H, CHHPh), 4.59 (d, J = 11.1 Hz, 1H, CHHPh), 4.48 (dd, J = 20.9, 9.7 Hz, 3H, CHHPh),

4.07 (ddd, J = 10.0, 8.2, 5.5 Hz, 1H, CH-6), 3.88 (dd, J = 7.6, 5.5 Hz, 1H, CH-3), 3.77 (dd, J = 5.6, 3.0 Hz,

1H, CH-2), 3.73 (dd, J = 9.3, 3.9 Hz, 1H, CH-7a), 3.61 (dd, J = 9.2, 2.5 Hz, 1H, CH-7b), 3.52 (dd, J = 11.8,

7.6 Hz, 1H, CH-4), 2.33 (tt, J = 12.2, 3.2 Hz, 1H, CH-5), 1.56 (s, 1H, NH). ¹³ C NMR (101 MHz, CDCI ₃ ) <5 138.2, 137.8, 137.7, 137.1, (4C _q Ar) 128.8, 128.7, 128.7, 128.6, 128.5, 128.5, 128.1, 128.1, 128.1, 128.0, 128.0, 127.9 (20CH Ar), 82.4 (C-l), 82.1 (C-3), 77.2 (C-2), 76.9 (C-4), 74.9, 74.1, 73.7, 73.5 (4CH ₂ Ph),

66.6 (C-7), 55.8 (C-6), 42.8 (C-5). TLC-MS: 616.2 [M+H ⁺ ],

(3a/?.45,55,6/?.7/?,7a5)-4,5.6-Tris(benzyloxy)-7-((benzyloxy )methyl)hexahvdro-3H- benzofdHl,2,3]oxathiazole 2, 2-dioxide (66).

Cyclosulfamidate 65 (60 mg, 84 pmol) was dissolved in anhydrous DCM (4 mL) and TFA (0.38 mL) was added to the reaction mixture. The reaction was stirred overnight at rt. The reaction mixture was diluted with H ₂ 0 and the aqueous phase was extracted with DCM (3x). The combined organic layers were dried

over MgS0 ₄ , filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pentane/EtOAc 95:5 - 80:20) to obtain 66 (32 mg, 52 pmol, 62%) as a colorless oil. [a] _D ²⁰ = -3.4 (c = 1, CHCI ₃ ). IR (neat cm ¹ ) 2919, 1453, 1359, 1067, 734, 696. ³ H NMR (400 MHz, CDC ) 5 7.36 - 7.23 (m, 18H, CH Ar), 7.21 (m, 2H, CH Ar), 4.96 (dd, J = 10.9, 6.5 Hz, 1H, CH-6), 4.86 (d, J = 7.1 Hz, 1H, NH), 4.67 (m, J = 23.4, 11.7 Hz, 4H, CHWPh), 4.52 (d, J = 11.5 Hz, 1H, CHHPh), 4.47 (d, J = 11.8 Hz, 1H, CHHPh), 4.43 (d, J = 11.1 Hz, 1H, CHHPh), 4.38 (d, J = 11.8 Hz, 1H, CHHPh), 4.28 (td, J = 6.8, 4.7 Hz, 1H, CH-1), 3.92 (t, 1H, CH-3), 3.80 (dd, J = 6.5, 4.8 Hz, 1H, CH-2), 3.77 (dd, J = 9.4, 2.1 Hz, 1H,

CH-7a), 3.65 (dd, J = 10.8, 5.4 Hz, 1H, CH-4), 3.59 (dd, J = 9.5, 2.2 Hz, 1H, CH-7b), 2.46 (tt, J = 10.8, 2.0 Hz, 1H, CH-5). ¹³ C NMR (101 MHz, CDCh) <5 138.1, 137.8, 136.8 (4C _q Ar), 128.9, 128.7, 128.6, 128.6, 128.3, 128.2, 128.0, 127.9, 127.9 (20CH Ar), 79.7 (C-6), 78.7 (C-3), 76.0 (C-4), 74.8 (C-2), 74.1, 73.9, 73.3, 73.3 (4CH ₂ Ph), 64.6 (C-7), 55.7 (C-l), 43.1 (C-5). LC-MS: 616.14 [M+H ⁺ ]

(3a5,4 ?.5/?,65,7/?,7a5)-5,6.7-Trihvdroxy-4-(hvdroxymethyl)hexahydr o-3/-/-benzoid1il.2,31oxathiazole

2,2-dioxide (45).

Perbenzylated 59 (13 mg, 21 mitioI) was dissolved in MeOH (1 mL) and the solution was purged with N ₂ . Pd/C (10 wt %, 16 mg, 15 mitioI, 0.7 eq) was added , the solution was purged with N ₂ and the reaction was stirred overnight under H ₂ atmosphere at rt. The reaction was flushed with N ₂ ,

filtered over a celite plug and concentrated in vacuo. The crude product was purified by silica gel column chromatography (DCM/MeOH 100:1 -> 80:20) to obtain 45 (4.66 mg, 18.27 mitioI, 87%) as a white solid. ³ H NMR (500 MHz, MeOD) <5 4.95 (t, 1H, CH-1), 3.98 (dd, J = 11.0, 2.6 Hz, 1H, CH-7a), 3.87 (dd, J = 11.1, 4.0 Hz, 1H, CH-6), 3.67 (dd, J = 11.0, 2.9 Hz, 1H, CH-7b), 3.61 - 3.55 (m,

2H, CH-2, CH-3), 3.39 - 3.35 (m, 1H, CH-4), 1.96 (tt, J = 11.2, 8.4, 2.7 Hz, 1H, CH-5). ¹³ C NMR (126 MHz, MeOD) 588.5 (C-l), 74.9 (C-2/C-3), 71.7 (C-2/C-3), 70.3 (C-4), 58.3 (C-7), 56.5 (C-6), 46.2 (C-5).

(3a/?,45,55,6/?.7/?,7a5)-4,5,6-Trihvdroxy-7-(hvdroxymethy l)hexahvdro-3/-/-benzoidlil,2,31oxathiazole

2,2-dioxide (46).

Perbenzylated 66 (21 mg, 31 pmol) was dissolved in MeOH (1.16 mL), purged with N ₂ and Pd/C (10 wt %, 13 mg, 12 pmol, 0.4 eq) was added and the reaction mixture was purged again with N ₂ . The reaction was then stirred overnight under H ₂ atmosphere at rt. The reaction was flushed with N ₂ ,

filtered over a celite plug and concentrated in vacuo. The crude product was purified by silica gel column chromatography (DCM/MeOH 100:1 - 80:20) to obtain 46 (8.23 mg, 31 pmol, 99%) as a clear oil. ² H NMR (400 MHz, MeOD) 5 4.93 - 4.90 (1H, CH-6), 4.37 (t, J = 4.8 Hz, 1H, CH-1), 4.01 (dd, J = 11.2, 2.3 Hz, 1H, CH-7a), 3.70 - 3.62 (m, 2H, CH-3, CH-7b), 3.59 (dd, J = 9.5, 4.3 Hz, 1H, CH-2), 3.36 - 3.33 (m, 1H, CH-4), 2.17 - 2.09 (m, 1H, CH-5). ¹³ C NMR (101 MHz, MeOD) 5 81.7 (C- 6), 74.9 (C-3), 71.0 (C-2), 69.3 (C-4), 61.0 (C-l), 57.6 (C-7), 46.6 (C-5). (3a ?.45.55.6 ?.7/?,7a5)-4,5.6-Tris(benzyloxy)-7-((benzyloxy)methyl)-3-met hylhexahvdro-

benzofcilfl.2,3loxathiazole 2,2-dioxide (67).

K2CO3 (4.1 mg, 30 mhioI 1.2 eq), TBAI (1 mg 0.1 eq) and iodomethane (3.12 pL, 49 mitioI, 2 eq) were added to a solution of 66 (15 mg, 24 mitioI) in anhydrous DMF (1 mL) and the reaction was stirred at rt overnight. The reaction mixture was diluted with EtOAc and H ₂ 0 and the aqueous phase was extracted with EtOAc (3x). The

combined organic layers were washed with water and brine, dried over MgSCU, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (Pent/EtOAc 100:1 -> 85:15) to afford 67 (10 mg, 16 mitioI, 65%) as a purple oil. IR (neat cm ¹ ) 2923, 1728, 1453, 1157, 1066, 735, 696. ³ H NMR (500 MHz, CDCb) <5 7.37 - 7.25 (m, 18H, CH Ar), 7.21 - 7.18 (m, 2H, CH Ar), 4.88 (dd, J = 10.0, 5.7 Hz, 1H, CH-6), 4.84 (d, J = 11.0 Hz, 1H, CHHPh), 4.76 (d, J = 12.2 Hz, 3H, CHHPh), 4.66 (d, J = 11.7 Hz, 1H, CHHPh), 4.51 (d, 7 = 11.0 Hz, 1H, CHHPh), 4.46 (d, J = 11.7 Hz, 1H, CHHPh), 4.40 {d, J = 11.7 Hz, 1H, CHHPh), 3.95 (t, J = 8.3 Hz, 1H, CH-3), 3.85 - 3.81 (m, 2H, CH-1, CH-7a), 3.61 - 3.54 (m, 3H, CH-2, CH-4, CH-7b), 2.90 (s, 3H, CW ₃ ), 2.56 (ddt, J = 11.8, 9.9, 2.0 Hz, 1H, CH-5). ¹³ C NMR (126 MHz, CDCI ₃ ) d 138.4, 138.2, 138.0, 137.4 (4C _q Ar), 128.8, 128.6, 128.6, 128.3, 128.1, 128.1, 128.0, 128.0, 127.9, 127.8 (20CH Ar), 81.9 (C-3), 77.6 (C-2/C-4), 77.3 (C-6), 76.4 (C-2/C-4), 75.2, 75.1, 74.3, 73.5 (4CH ₂ Ph), 64.5 (C-7), 61.3 (C-l), 44.1 (C-5), 33.4 (CH ₃ ). LC-MS: 652.08 [M+H ⁺ ],

(3aR, 45,55,6/?, 7/?,7a5)-4,5, 6-Trihvdroxy-7-(hvdroxymethyl)-3-methylhexahydro-

benzofdUl,2,3]oxathiazole 2, 2-dioxide (47).

Perbenzylated 67 (10 mg, 16 pmol) was dissolved in a 4:1 mixture of

MeOH (1 mL) and DCM (0.25 mL), purged with N ₂ and Pd/C (10 wt %, 6.8 mg, 6.4 pmol, 0.4 eq) was added and the reaction mixture was purged again with N ₂ . The reaction was stirred overnight under H ₂ atmosphere at rt. The reaction mixture was flushed with N ₂ , filtered over a celite plug acuo. The crude product was purified by silica gel column chromatography 0:20) to obtain 47 (2.3 mg, 8.8 pmol, 55%) as a white solid. ³ H NMR (500 MHz,

MeOD) d 4.89 (dd, J = 10.2, 5.3 Hz, 1H, CH-6), 3.98 (dd, J = 11.2, 2.3 Hz, 1H, CH-7a), 3.93 (dd, J = 5.3,

3.4 Hz, 1H, CH-1), 3.67 (dd, J = 8.5, 2.7 Hz, 1H, CH-7b), 3.66 - 3.63 (m, 1H, CH-3), 3.59 (dd, J = 9.8, 3.4

Hz, 1H, CH-2), 3.36 (dd, J = 11.2, 8.7 Hz, 1H, CH-4), 2.93 (s, 3H, CH ₃ ), 2.19 (tt, J = 10.3, 2.5 Hz, 1H, CH- 5). ¹³ C NMR (126 MHz, MeOD) <579.4 (C-6), 74.7 (C-3), 72.1 (C-2), 69.7 (C-4), 64.8 (C-l), 57.5 (C-7), 47.5

(C-5), 34.5 (CH ₃ ).

The above data shows that the present invention not only may provide novel compounds of formula I to III, but also provides for a versatile platform to design and synthesise various enzyme chaperons for both ERT and in support of Gene-therapy on lysosomal storage disease and/or glycosidase and/or a-glucosidase deficiency related diseases, particularly Fabry, Gaucher or Pompe disease.