WO2010046517A1

WO2010046517A1 - Compounds promoting the activity of mutant glycosidases

Info

Publication number: WO2010046517A1
Application number: PCT/ES2009/070449
Authority: WO
Inventors: José Manuel GARCÍA FERNÁNDEZ; Carmen Ortiz Mellet; M. Isabel GARCÍA MONCAYO; Yoshiyuki Suzuki; Kousaku Ohno; Matilde Aguilar Moreno
Original assignee: Consejo Superior De Investigaciones Científicas (Csic); Universidad De Sevilla; International University Of Health And Welfare; Tottori University
Priority date: 2008-10-22
Filing date: 2009-10-21
Publication date: 2010-04-29
Also published as: ES2337435B8; ES2337435A1; ES2337435B2

Abstract

Compounds promoting the activity of mutant glycosidases having inhibitory activity for glycosidase enzymes, furthermore relating to a procedure for activation of mutant β-glycosidase (β-glucocerebrosidase) and mutant β-galactosidase in patients suffering lysosomal storage disorders through administration of said enzyme competitor inhibitor compounds, characterised by very high bonding specificity and a favourable ratio between the concentration thereof for pharmacological chaperone activity and the concentration thereof for inhibitor activity.

Description

POTENTIATING COMPOUNDS OF THE ACTIVITY OF MUTANT GLYCOSIDASES

The present invention relates to compounds with inhibitory activity of glycosidases enzymes and a method for the activation of mutant β-glucosidase (β-glucocerebrosidase) and mutant β-galactosidase in patients suffering from lysosomal storage environments by administering said inhibitory compounds. enzymes, characterized by a very specific binding specificity and a favorable relationship between its concentration for pharmacological chaperone activity and its concentration for inhibitory activity. The invention also relates to these compounds, their method of production and therapeutic compositions containing said compounds.

STATE OF THE PREVIOUS TECHNIQUE

Lysosomal storage disorders are a group of diseases resulting from the abnormal metabolism of several substrates that do not degrade and accumulate in lysosomes, leading to a series of phenotypes that include megalovisceralia, neurological pathologies, skeletal injuries and premature death (AH Futerman, G. van Meer, N ature Rev. Mol. CeII Biol. 2004, 554-565). These diseases are the result of mutations in the genes that encode enzymes involved in the degradation process. Current therapeutic strategies include the inhibition of substrate production using inhibitors of the enzymes involved in its biosynthesis (US2005 / 0075305). The increase of the defective enzyme could be achieved clinically by enzymatic replacement (R. J. Desnick, E. H. Schuchman, Nature Rev. Genet. 2002, 954-966). For the most predominant lysosomal storage disorder, Gaucher disease, this therapy costs between $ 100,000 and $ 750,000 a year, and is not very effective for cases that show involvement of the central nervous system. Bone marrow transplantation can also reverse the disease, but until now gene therapy strategies have not been successful.

Some of these pernicious mutations are manifested by proteins mutants that retain catalytic activity but that present folding defects and experience degradation mediated by the cellular quality control machinery. The use of functional compounds that can serve as a template for proper protein folding with a tendency to misfold is well documented. Thus, it is known that some enzyme inhibitors are capable of binding to the active site and stabilizing the appropriate folding of the catalytic form of the enzyme. Said compounds can act as "pharmacological chaperones" to stabilize the mutant protein in a conformation suitable for transport to lysosomes, where it remains stable due to the high concentration of substrate and the low pH environment.

Gaucher disease is the most predominant lysosomal storage disorder, with an estimated incidence of approximately 1: 60,000 in the general population and 1: 100 in the population of Ashkenazi Jews (TD Butters, Curr. Opin. Chem. Biol. 2007 , 11, 412-418). It is the result of mutations in β-glucosidase acid (β-glucocerebrosidase), a lysosomal membrane-associated glycoprotein of 497 residues that have as a consequence the accumulation of the corresponding substrate (glucosylceramide). More than 200 different point mutations have been identified in the gene encoding β-gluocerebrosidase (E. Sidransky, Mol. Genet. Metab. 2004, 83, 6-15). The mutations lead to significant defects in the folding of the protein during translation in the endoplasmic reticulum, resulting in a reduction of the transport of the enzyme to the lysosome.

Another pathology, the hereditary deficiency of lysosomal acid β-galactosidase (β-galactosidosis), causes two clinically distinct diseases in humans, G _MI gangliosidosis and Morquio B disease (Y. Suzuki, J¿ Inherit. Metab. Dis. 2006, 29, 471-476). Both are the result of mutations in the GLB1 gene that lead to an erroneous protein folding (S. Zhang, R. Bagshow, W. Hilson, Y. Oho, A. Hinek, JTR Clarke, A. Hinek, JW Callahan, Biochem. J. 2000, 348, 621-632). The mode of inheritance is autosomal recessive. G _M i gangliosidosis is a generalized neurosomatic disease that appears mainly in early childhood, and rarely in childhood or in young adults. Disease Morquio B is a rare bone disease without involvement of the central nervous system. In patients with these clinical phenotypes, glucoconjugates accumulate with terminal β-galactose residues in tissues and urine. The ganglioside G _M i and its Asian derivative G _A i accumulate in the brain in the case of G _MI gangliosidosis - In both patients with G _MI gangliosidosis and in patients suffering from Morquio B disease high amounts of oligosaccharides derived from keratane sulfate or glycoproteins in visceral organs and urine. Currently, only symptomatic therapy is available for patients with β-galactosidosis.

On the other hand, some natural and synthetic polyhydroxylated alkaloids structurally related to sugars (glycemic) that incorporate an amine-type endocyclic nitrogen (sp ³ hybridization) exhibit significant inhibitory activity against glycosidases. Similarly, it has been shown that carbocycles structurally related to sugars that carry amine substituents behave as glycosidases inhibitors (S. Ogawa, M. Kanto, Y. Suzuki, Mini-Rev. Med. Chem. 2007, 7, 679-691). In some cases, these compounds used at subinhibitory concentrations have been shown to act as mutant β-glucocerebrosidase and mutant β-galactosidase chaperones (US 2006/0100241; WO2004 / 037373). However, these types of compounds generally behave as broad-type glucosidase inhibitors, simultaneously inhibiting several glycosidases, which represented a serious inconvenience for clinical applications. Simultaneous inhibition of β and α-glucosidases is particularly problematic. Therefore, there is a need to develop sugar-related molecules with a high ratio of chaperone to inhibitor activity that, at the same time, exhibit high anomeric selectivity for the corresponding β-glycosidases enzymes.

DESCRIPTION OF THE INVENTION

The present invention provides bicyclic compounds structurally related to sugars that incorporate at least one endocyclic nitrogen atom with a substantially sp ² character (as of now, azazúcar sp ² ) and behave as highly selective competitive inhibitors of the lysosomal β-glucocerebrosidase and β-galactosidase acid associated with Gaucher's disease and β-galactosidosis, respectively. These compounds enhance the activity of said enzymes when they are administered at concentrations lower than those necessary to inhibit the intracellular enzymatic activity, acting as well as pharmacological chaperones. The effect is particularly significant in certain mutant enzymes, but it also appears in cells that contain the normal enzyme.

Consequently, the present invention also provides a method for activating β-glucosidase or β-galactosidase and a method for treating patients suffering from Gaucher disease or β-galactosidosis, by using the compounds of the invention.

This method of the invention, in addition to being useful in mammalian cells, is also useful in cells of another origin, such as, for example, insect cells and cultured CHO cells, used in the production of recombinant enzymes for enzyme replacement therapy.

In accordance with the above, a first aspect of the present invention refers to a compound of general formula (I) or any of its salts:

where: R is a substituent, the same or different in each case, and is selected from an H atom, a hydroxyl group (-OH) or an OR ² group; wherein R ² is selected from an acyl group, substituted or unsubstituted, a (C1-C18) alkyl group, substituted or unsubstituted, an aryl group, substituted or unsubstituted, an aralkyl group, substituted or unsubstituted, a group amino (NH ₂ ), an acetamide group (NHAc), or an NHR ³ group, where R ³ is selected from an acyl group, substituted or unsubstituted, an alkyl group (CrCi ₈ ), substituted or unsubstituted, an aryl group, substituted or unsubstituted, or an aralkyl group, substituted or unsubstituted.

Preferably R is the same in all cases and is OH;

And it is selected from an O atom, an NH group or an S atom; Y

X is the group -NR ¹ ; wherein R ¹ is selected from an H atom, an alkyl group (CrCi ₈ ), substituted or unsubstituted, an aryl group, substituted or unsubstituted or an aralkyl group, substituted or unsubstituted.

The term "alkyl" refers, in the present invention, to aliphatic, linear or branched chains, having 1 to 18 carbon atoms, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl, tere-butyl, sec-butyl, n-pentyl, n-hexyl, etc. Preferably the alkyl group has between 3 and 9 carbon atoms. More preferably n-butyl, n-pentyl, n-hexyl, n-heptyl or n-octyl. The alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amido, carboxylic ester, ether, thiol, acylamino or carboxamido. When the alkyl group is substituted, it is preferably one or more amine, amide or ether groups, which in turn may or may not be substituted by alkyl, amide, cycloalkyl or ethers groups and these, in turn, may also be substituted or no.

"Cycloalkyl" refers to a stable monocyclic or bicyclic radical of 3 to 10 members, which is saturated or partially saturated, and which only consists of carbon and hydrogen atoms, such as cyclohexyl or adamantyl.

The term "aryl" refers, in the present invention, to single or multiple aromatic rings, which have between 5 and 18 links in which a proton has been removed from the ring. Preferably the aryl group has 5 to 7 carbon atoms. The aryl groups are for example, but not limited to phenyl, naphthyl, diphenyl, indenyl, phenanthryl or anthracil. The aryl radicals may be optionally substituted by one or more substituents. The term "aralkyl" refers, in the present invention, to an aliphatic chain in which at least one of the hydrogens has been substituted by an aryl group, with the above meanings. As for example, but not limited to, a benzyl or phenethyl group.

The term "acyl" refers, in the present invention, to a carboxylic acid derivative by elimination of a hydroxyl group. The carboxylic acid derivatives have as general formula R ⁴ -CO-, where R ⁴ is an alkyl group with the above meanings and preferably refers to (C ₃ -Ci ₄ ) alkyl groups, linear or branched. As for example, but not limited to propionyl, butanoyl, hexanoyl, pivaloyl, octanoyl or myristoyl.

The sugar mimetic compounds of the invention, as represented in the general formula (I), include condensed bicycles of six members / five members that have a bridgehead nitrogen atom that is part of a cyclic amide or pseudoamide functionality. By "pseudoamide functionality", a group of general formula N- C (= X) Y is defined, in which X represents a heteroatom that carries, if necessary, substituents of varied nature, Y is a heteroatom, which carries given the case substituents of varied nature. Preferred heteroatoms are nitrogen (N), oxygen (O) or sulfur (S). Non-limiting examples of pseudoamide groups, according to the present invention are urea, thiourea, isourea, isothiourea, guanidine or amidine. Hereinafter, the term azaazúcares sp ^{2 will be used} to refer to the compounds of the invention.

The carbon atoms of the bicyclic nucleus of the azaazúcar sp ² may be substituted with hydroxyls to closely mimic a sugar structure or they may be devoid of hydroxyls. Preferred hydroxylation profiles are those that coincide with those of D-glucose and D-galactose in positions equivalent to C-2, C-3 and C-4 in the monosaccharide after superimposition of the six-membered ring of the bicycles with the ring of pyranose of the monosaccharide, with the bridgehead nitrogen atom in the position of the sugar oxygen atom (hereinafter, glucomimetics and galactomimetics, respectively).

Interestingly, the compounds of the invention can carry a hydroxyl group in the position equivalent to the anomeric position. The presence of the neighboring nitrogen atom of the pseudoamide type with substantially sp ² character favors the axial orientation of this hydroxyl and provides high stability to the reducing sugar mimetic. Consequently, the preferred pharmacological chaperones according to this invention respond to formulas Ia and Ib.

Ia Ib

Where Y and X are defined above.

Other compounds additional to formulas (Ia) and (Ib) include those in which one or more of the hydroxyl groups are added, alkylated or replaced by an H atom or other functional groups, preferably nitrogen functionalities, such and as stated in the description of the general formula (I). Therefore, another preferred embodiment comprises the compounds of general formula (Ia ') and (Ib'):

'a' Ib ¹

Where: R, X and Y are defined above.

When Y is oxygen the compound of the invention is of general formula (Ic):

When Y is a nitrogen atom, the compound of the invention is of general formula (Id):

(Id)

When Y is a sulfur atom, the compound of the invention is of general formula (Ie):

(Ie)

Additionally, the invention provides a process for the chemical synthesis of the bicyclic core of the aforementioned compounds from readily available commercial carbohydrates and is particularly well adapted to generate molecular diversity, allowing to synthesize compounds with substitution patterns of structural complementarity with D-glucose and D-galactose In addition, a battery of substituents can be introduced in different positions along the synthetic route, which is of great importance to optimize the activity Biological and pharmacodynamic properties.

Thus, another aspect of the present invention relates to the process for obtaining the compounds of general formula (I) comprising: (i) the introduction of an amino group in C-5 position of the corresponding sugar in the form of furanose; (ii) the closure of a 5-member ring between positions C-5 and C-6 by a segment of the pseudoamide type; Y

(iii) the transposition of the furanose ring to a piperidine cycle fused with the five-membered cyclic pseudoamide ring previously constructed.

The characteristics of the process for preparing the invention will be more clearly reflected in the examples provided below.

Another aspect of the present invention relates to the use of the compounds of general formula (I) for the preparation of a pharmaceutical composition.

Another aspect of the present invention relates to the use of the compound of general formula (I) for the preparation of a pharmaceutical composition for the treatment of a disease related to human mutated β-glucosidase and / or β-galactosidase. Preferably the diseases are Gaucher's disease, G _M gangliosidosis, or Morquio B disease.

Another aspect of the present invention relates to a method of enhancing the bioavailability of chemical chaperones using therapeutic compositions containing a pharmacologically acceptable vehicle, preferably a commercial cyclodextrin such as α-, β- or y-cyclodextrin (αCD, βCD or yCD ), a methylated β-cyclodextrin (TRIMEB, DIMEB or RAMEB), a hydroxypropylated β-cyclodextrin (HP-βCD) or a sulfobutylated βCD (Captisol®), or a chemically modified cyclodextrin such as a biorecognizable cyclodextrin derivative that carries oligosaccharides.

The present invention involves the use of bicyclic compounds that mimic D-glucose (glucomimetics) of the azazúcar sp ² type, which carry hydrophobic substituents, such as chemical chaperones to enhance the residual activity in a fibroblast culture medium for several mutations that cause Gaucher disease. This therapeutic strategy could be applied to G _MI gangliosidosis using compounds that mimic D-galactose (galactomimetics) of the azazúcar sp ² type that carry hydrophobic substituents.

Thus, a method of this invention can be used to treat a patient who has Gaucher disease or a patient suffering from β-galactosidosis. According to the methods of the invention, an effective activating amount of β-glucocerebrosidase or β-galactosidase of a suitable sp ² azazúcar or of a therapeutic composition of a suitable sp ² azazúcar is administered to the patient.

In particular, the present invention implies that the compound that mimics the azazúcar sp ² sugar mentioned above has, in addition to a high ratio of chaperone activity to inhibitory activity, a very high selectivity towards the target enzyme. This represents a significant improvement compared, for example, with the use of conventional type azazúcares in which a nitrogen atom with sp ³ hybridization replaces the endocyclic oxygen of a sugar, such as for example 1-deoxinojirimycin, which behave in general as broad spectrum glycosidase inhibitors. Significantly, the glycemic compounds of the azazúcar sp ² type of the invention exhibit a very high β-anomeric selectivity when compared with the inhibitory activity towards α- and β-glucosidases or α- and β-galactosidases human.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of azazúcar sp ² glycemic compound and a pharmaceutically acceptable carrier. The term "vehicle" designates a diluent, adjuvant, excipient or carrier with which the therapeutic agent is administered. Various administration systems are known and can be used to administer a compound of the invention, for example, liposomes, microparticles or microcapsules. In a specific embodiment, the administration system is a cycloomaltooligosaccharide (cyclodextrin, CD). The inventors discovered that the use of native CDs or chemically modified CDs allows the hydrophobic moiety of the inhibitor to be masked, thereby increasing its solubility in aqueous media. The resulting inclusion complexes maintain or even enhance the inhibitory and chaperone activities of the active agent.

Therefore, another aspect of the present invention refers to a pharmaceutical composition comprising at least one of the compounds of general formula (I) and a pharmaceutically acceptable carrier, as defined above.

In addition, another aspect of the present invention relates to an in vitro process to enhance the activity of a glycosidase enzyme, which comprises contacting the protein with an effective amount of a compound of general formula (I).

Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES

The inventors tested a series of novel sp ² azazúcar inhibitors chemically synthesized against a panel of commercially available glycosidases of plant, microorganism and animal origin. Some compounds of the invention were evaluated in the in vitro inhibition of human β-glucocerebrosidase and β-galactosidase and the ex vivo intracellular potentiation of mutant enzymes with Gaucher fibroblasts and G _M i - gangliosidosis.

EXAMPLE 1 1.1. Materials and procedures

The azazúcar sp ^2- type glycemimetic compounds of the invention, of general formula (I), include six-member / five-member condensed bicycles having a bridgehead nitrogen atom that is part of a cyclic amide or pseudoamide functionality.

Preferred pharmacological chaperones in the context of this invention respond to formulas Ia (glucomimetics) and Ib (galactomimetics).

To obtain the compounds having formula Ia, when Y is oxygen and X is NR ¹ (see formula Vl) the following synthesis was followed: the starting monosaccharide is D-glucose or a commercially available D-glucose derivative . The introduction of a nitrogen group in position C-5 can then be achieved via the corresponding 5-azide-5- deoxy-1, 2-O-isopropylidene-α-D-glucofuranose (II). For the preparation of this synthetic intermediate, the procedure outlined by K. Dax et al. (J. Carbohvdr. Chem. 1990, 9, 479-499), starting from commercial D-glucufuranurono-6,3-lactone. A synthetic strategy involving urea type intermediates of general formula (IV) was found advantageous. The ureas can be prepared, for example, by reaction of 5-amino-5- deoxy-1, 2-O-isopropylidene-α-D-gluco-furanose (III), which can be obtained by reducing the IL as described (VM Díaz Pérez et al., J. Org. Chem. 2000, 65, 136-143), by coupling reaction with the appropriate isocyanate of general formula R ¹ NCO. The ureas IV can be transformed into the corresponding cyclic isoureas V by treatment with a sulfonic acid anhydride or a sulfonyl chloride such as trifluoromethanesulfonic anhydride, methanesulfonic anhydride or p-toluenesulfonyl chloride. Acid catalyzed hydrolysis of the acetal protecting group in compounds V provides compounds Vl (scheme 1).

Scheme 1

For the preparation of compounds having the formula Ia in which Y is an atom of S and X represents NR ¹ (see formula IX), the reaction sequence represented in Scheme 2 can be followed. The reaction of the amine III with R ¹ NCS isothiocyanates provides thiourea VII adducts. The treatment of VII with a sulfonant reactive derivative, as described above for the preparation of cyclic isoureas V, provides the corresponding cyclic isothiourea precursors VIII, which after acid catalyzed deacetylation provide the bicyclic glucomimetics IX.

Scheme 2

For the preparation of the compounds having formula Ia, in which Y represents NH and X represents NR ¹ (see formula XIII), the reaction sequence represented in scheme 3 can be followed. The cyclic thiourea precursor X can be obtained from the azide derivative Il following the methodology outlined above (VM Díaz Pérez et al., J. Orq. Chem. 2000, 65, 136-143). The reaction of X with an alkylating reagent, such as methyl iodide, provides the corresponding S-alkylisothiouronium salt Xl. The nucleophilic displacement of the alkylthio group by an amine of the general formula R ¹ NH ₂ provides cyclic guanidines of the general formula XII. The bicyclic sp ² azazúcar glucomimetic XIII can be obtained by acid catalyzed hydrolysis of the acetal protecting group followed by treatment with a base such as, for example, sodium hydroxide. The compounds of general formula XIII can be stored and used in the context of the present invention as a free base or in the form of a guanidinium salt such as, for example, the corresponding guanidinium chloride.

Scheme 3

In another additional preferred embodiment, the starting monosaccharide is D-galactose or a commercially available D-galactose derivative. In this case, the final compound responds to the general formula Ib.

For the preparation of galactomimetics Ib in which Y represents O or S and X represents NR ¹ (see formulas XV and XVI, respectively), a synthetic strategy identical to that described above for the preparation of analog Vl and IX glucomimetics can be followed , using 5-amino-5- deoxy-1,2-O-isopropylidene-α-D-galactofuranose XIV as the precursor amino sugar (scheme 4). Compound XIV can be obtained from commercial D-galactose by known methodologies such as, for example, those reviewed by P. Díaz Pérez et al. in Eur. J. Org. Chem. 2005, 2903-2913.

Scheme 4

For the preparation of the galactomimetic Ib in which Y represents NH and X represents NR ¹ (see formula XVIII), a synthetic strategy identical to that described above for the preparation of the analogous glucomimetic XII can be followed, starting from the cyclic thiourea XVII (scheme 5). Compound XVII can be obtained from commercial D-galactose via the known derivative 3-O-benzoyl-1,2-O-isopropylidene-α-D-galactofuranose (H. Wang and J. Ning, J. Ora. Chem. 2003, 68, 2521-2524) through a reaction sequence that involves: (i) triphenylmethylation in the primary hydroxyl group, (ii) introduction of an azide group in C-5 by double inversion of the configuration in this center, ( iii) acid catalyzed removal of the triphenylmethyl group, (iv) introduction of a second C-6 azide group, (v) catalyzed removal based on the benzoyl group followed by simultaneous reduction of the two azide groups, giving 5,6-diamino-5,6-dideoxy-1, 2-O-isopropylidene-α-D-galactofuranose, and (vi) thiocarbonylation of the diamine to the cyclic target thiourea XVII. All the mentioned transformations are standard. As an example, the procedures outlined in P. Díaz Pérez et al. in Eur. J. Orq. Chem. 2005, 2903-2913 and VM Díaz Pérez et al., J. Orq.

Chem. 2000, 65, 136-143.

XVII XVIII

Scheme 5

In schemes 1-5, the substituent R ¹ in the formulas has the meaning indicated above for Ia and Ib.

Some examples of inhibitors of the glucomimetic (Ia) (compounds 1-14) and galactomimetic (Ib) (compounds 15-23) families chemically synthesized and used in this invention are shown below:

1.2. Structural characterization of some of the compounds.

The structural characterization of the compounds prepared and used in this invention was carried out by mass spectrometry, optical rotation, ¹ H and ¹³ C NMR spectroscopy and elemental analysis. Some of the results are presented below.

(5 / ?, 6 / ?, 7S, 8 / ?, 8a /?) - 5,6,7,8-Tetrahydroxy-3-phenylimino-2-oxaindolizidine (1). [α] _D -26.7 (c 0.75, pyridine). ¹ H NMR (500 MHz, D ₂ O) δ 7.44-7.27 (m, 5H, Ph), 5.73 (sa, 1 H, H-5), 4.96 (t, 1 H , J _{1a> 1b} = J _8aM = 8.8 Hz, H-1a), 4.69 (t, 1 H, Jβa.ib = 8.8 Hz, H-1b), 4.28 (td, 1 H , J ₈ , _8a = 9.4 Hz, H-8a), 3.81 (t, 1 H, J ₆₇ = J ₇ , ₈ = 9.4 Hz, H-7), 3.73 (dd, 1 H, J ₅ , ₆ = 3.7 Hz, H-6), 3.66 (t, 1 H, H-8); NMR- ¹³ C (75.5 MHz, D ₂ O) δ 157.4 (CN), 129.7-123.9 (Ph), 75.3 (C-5), 74.1 (C-6) , 73.0 (C-8), 71, 9 (C-7), 70.9 (C-1), 56.1 (C-8a). Anal. cale, for Ci ₃ Hi ₆ N ₂ O ₅ : C, 55.71; H, 5.75; N, 9.99. Found: C, 55.70; H, 5.81; N, 9.97.

(5 / ?, 6 / ?, 7S, 8 / ?, 8a /?) - 5,6,7,8-Tetrahydroxy-3-butylimino-2-oxaindolizidine

(2). [α] _D +5.9 (c 1.0, H ₂ O). ¹ H NMR (300 MHz, D ₂ O) δ 5.53 (d, 1 H, J ₅ , ₆ = 3.9 Hz, H-5), 5.01 (t, 1 H, J _{1a> 1b} = J _8a, i _a = 8.8 Hz, H-1a), 4.67 (t, 1 H, J _8a, i _b = 8.8 Hz, H-1 b), 4.24 (td, 1 H, J _8.8a = 9.4 Hz, H-8a), 3.74 (t, 1 H, J _6.7 = J _7.8 = 9.4 Hz, H-7), 3.62 (dd, 1 H, H-6), 3.61 (t, 1 H, H-8), 3.36 (dt, 2H, ² J _H , _H = 7.1 Hz, ³ J _H , _H = 2.1 Hz, CH ₂ N), 1.55 (c, 2 H, CH ₂ CH ₂ N), 1, 30 (m, 2H, CH ₂ CH ₃ ), 0.87 (t, 3H, CH ₃ ); NMR- ¹³ C (75.5 MHz, D ₂ O) δ 158.7 (CN), 74.9 (C-5), 73.9 (C-6), 73.0 (C-8), 71 , 9 (C-7), 70.9 (C-1), 56.2 (C-8a), 42.9 (CH ₂ N), 30.4 (CH ₂ CH ₂ N), 19.2 ( CH ₂ CH ₃ ), 12.9 (CH ₃ ). Anal. cale, for Ci ₃ H ₂₂ N ₂ Oi ₀ : C, 42.62; H, 6.05; N, 7.65. Found: C, 42.75; H, 6.13; N, 7.69.

(5 / ?, 6S, 7 / ?, 8 / ?, 8a /?) - 5,6,7,8-Tetrahydroxy-2-oxa-3-octylimino-indolizidine (3) _: [α] _D +5, 9 (c 1, 0, H ₂ O). ¹ H NMR (500 MHz, D ₂ O) δ 5.74 (d, 1 H, J _5.6 = 3.8 Hz, H-5), 5.22 (t, 1 H, J _{1a, 1b} = J _8a, i _a = 8.9 Hz, H-1a), 4.90 (t, 1 H, J _8a, i _b = 8.9 Hz, H-1 b), 4.45 (td, 1 H, J _8.8a = 9.5 Hz, H-8a), 3.95 (t, 1 H, J _6.7 = J _7.8 = 9.5 Hz, H-7), 3.83 ( dd, 1 H, H-6), 3.80 (t, 1 H, H-8), 3.57 (dt, 2H, ² J _H , _H = 7.1 Hz, ³ J _H , _H = 4 , 2 Hz, CH ₂ N), 1, 53 (m, 2H, CH ₂ CH ₂ N), 1.24 (m, 1OH, 5 CH ₂ ), 0.87 (t, 3H, CH ₃ ); NMR- ¹³ C (75.5 MHz, D ₂ O) δ 158.7 (CN), 74.9 (C-5), 73.9 (C-6), 73.0 (C-8), 71 , 9 (C-7), 70.9 (C-1), 56.2 (C-8a), 43.2 (CH ₂ N), 31, 2, 28.4, 28.2, 25.8 , (5 CH ₂ ), 28.3 (CH ₂ CH ₂ N), 22.1 (CH ₂ CH ₃ ), 13.5 (CH ₃ ). Anal. cale, for Ci ₃ H ₂₂ N ₂ Oi ₀ : C, 42.62; H, 6.05; N, 7.65. Found: C, 42.75; H, 6.13; N, 7.69.

(5 / ?, 6 / ?, 7S, 8 / ?, 8a /?) - 5,6,7,8-Tetrahydroxy-3-phenylimino-2-thianedolizidine (4). [α] _D -38.0 (c 0.5, H ₂ O). ¹ H NMR (500 MHz, CD ₃ OD) δ 7.22 (t, 2H, Ph), 7.00 (t, 1 H, Ph), 6.88 (d, 2H, Ph), 5.72 (d, 1 H, J _5.6 = 3.7 Hz, H-5), 3.82 (td, 1 H, Ja, aa = Jβa.ib = 9.5 Hz, J _8a , i _a = 6 , 5 Hz, H-8a), 3.71 (t, 1 H, J ₆ , ₇ = J ₇ , ₈ = 9.5 Hz, H-7), 3.45 (dd, 1 H, H-6 ), 3.35 (dd, 1 H, J _{1a> 1b} = 10.9 Hz, H-1a), 3.29 (t, 1 H, H- 8), 3.00 (dd, 1 H, H -1 B). NMR- ¹³ C (125.7 MHz, CD ₃ OD) δ 162.3 (CN), 152.6- 123.0 (Ph), 77.8 (C-5), 76.3 (C-8) , 74.8 (C-7), 73.4 (C-6), 61, 1 (C-8a), 31, 8 (C-1). FABMS: m / z 319 (75, [M + Na] ⁺ ). Anal. cale, for Ci ₃ Hi ₆ N ₂ O ₄ S: C, 52.69; H, 5.44; N, 9.45. Found: C, 52.50; H, 5.13; N, 9.29.

(5R, 6R, 7S, 8R, 8aR) -3-Butylimino-5,6,7,8-tetrahydroxy-2-thianedolizidine (5). [α] _D -22.9 (c 0.7, H ₂ O). ¹ H NMR (500 MHz, D ₂ O) δ 5.52 (d, 1 H, J _5.6 = 3.7 Hz, H-5), 3.75 (dt, 1 H, J _8.8a = J _8a, i _b = 9.5 Hz, J _8a, i _a = 6.4 Hz, H-8a), 3.71 (t, 1 H, Jβj = Jτ _{, 8} = 9.5 Hz, H- 7), 3.55 (dd, 1 H, H-6), 3.45 (dd, 1 H, J _{1a, 1b} = 10.5 Hz, H-1a), 3.40 (t, 1 H, H-8), 3.18 (m, 2H, CH ₂ N), 3.07 (dd, 1 H, H-1 b), 1.52 (m, 2H, CH ₂ CH ₂ N), 1, 30 (m, 2H, CH ₂ CH ₃ ), 0.87 (t, 3H, ³ J _{H, H} = 7.4 Hz, CH ₃ ). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 162.9 (CN), 76.0 (C-5), 74.3 (C-8), 72.8 (C-7), 71, 4 (C - 6), 59.8 (C-8a), 53.9 (CH ₂ N), 32.0 (C-1), 30.4 (CH ₂ CH ₂ N), 19.8 (CH ₂ CH ₃ ), 13.2 (CH ₃ ). FABMS: m / z 299 (40, [M + Na] ⁺ ), 277 (100, [M + H] ⁺ ). Anal. cale, for CnH ₂₀ N ₂ O ₄ S: C, 47.81; H, 7.29; N, 10.14. Found: C, 47.65; H, 7.14; N, 9.99.

(5R, 6R, 7S, 8R, 8aR) -5,6,7,8-Tetrahydroxy-3-octylimino-2-thianedolizidine (6).

[α] _D -24.5 (c 0.5, MeOH). ¹ H NMR (300 MHz, D ₂ O) δ 5.50 (d, 1 H, J _5.6 = 3.7 Hz, H-5), 4.20 (m, 1 H, H-8a) , 3.66 (m, 2H, H-7, H-1a), 3.52 (dd, 1 H, J ₆₇ = 10.0, H-6), 3.45 (t, 1 H, J _{7 , 8} = J _8.8a = 9.5 Hz, H-8), 3.32 (m, 3H, H-1 b, CH ₂ N), 1.56 (m, 2H, CH ₂ CH ₂ N) , 1, 18 (m, 1OH, CH ₂ ), 0.74 (t, 3H, ³ J _{H, H} = 6.9 Hz, CH ₃ ). NMR- ¹³ C (75.5 MHz, D ₂ O) δ 162.5 (CN), 76.6 (C-5), 73.5 (C-8), 72.0 (C-7), 70 , 9 (C-6), 63.6 (C-8a), 49.4 (CH ₂ N), 31, 5 (C-1), 31, 3, 28.6, 28.5, 28.3 , 26.0 (CH ₂ ), 22.3 (CH ₂ CH ₃ ), 13.7 (CH ₃ ). FABMS: m / z 355 (30, [M + Na] ⁺ ), 333 (100, [M + H] ⁺ ). Anal. cale, for Ci ₅ H ₂₈ N ₂ O ₄ S: C, 54.19; H, 8.49; N, 8.43. Found: C, 53.94; H, 8.42; N, 8.29.

(5 / ?, 6 / ?, 7S, 8 / ?, 8a /?) - 3- [2 '- (Λ /, Λ / -Bis- (2-hexanamidoethyl) aminoethyl) -imino] - 5,6, 7,8-tetrahydroxy-2-thianedolizidine (7). [α] _D -5.0 (c 1.0, H ₂ O). F _R 0.41 (CH ₂ CI ₂ -MeOH-H ₂ O 40: 10: 1). ¹ H NMR (500 MHz, D ₂ O) δ 5.62 (d, 1 H, J _5.6 = 3.8 Hz, H-5), 3.85 (m, 1 H, H-8a) , 3.74 (t, 1 H, J _6.7 = J _7, β = 9.5 Hz, H-7), 3.57 (dd, 1 H, H-6), 3.56 (dd, 1 H, J _{1a, 1 b} = 10.5 Hz, J _{8a, 1a} = 6.5 Hz, H-1a), 3.45 (m, 6H, CH ₂ NCS, CH ₂ NHCO), 3.44 ( t, 1 H, J _88a = 9.5 Hz, H-8), 3.25 (m, 2H, CH ₂ NCS), 3.18 (m, 4H, CH ₂ CH ₂ NHCO), 3.17 ( t, 1 H, J _{8a 1 b} = 10.5 Hz, H-1 b), 2.16 (t, 4H, ³ J _{H, H} = 7.5 Hz, CH ₂ CONH), 1.47 (m , 4H, CH ₂ CH ₂ CONH), 1, 18 (m, 8H, CH ₂ ), 0.76 (t, 6H, ³ J _{H, H} = 7.0 Hz, CH ₃ ). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 178.3 (CO), 162.9 (CN), 75.9 (C-5), 74.3 (C-8), 72.6 (C-7), 71, 3 (C-6), 60.5 (C-8a), 54.4 (CH ₂ CH ₂ NCS), 54.0 (CH ₂ CH ₂ NHCO), 48.0 ( CH ₂ NCS), 35.7 (CH ₂ NHCO), 35.6 (CH ₂ CONH), 31, 1 (C-1), 30.6 (CH ₂ ), 25.0 (CH ₂ CH ₂ CONH) , 21, 7 (CH ₂ CH ₃ ), 13.2 (CH ₃ ). FABMS: m / z 568 (30, [M + Na] ⁺ ), 546 (40, [M + H] ⁺ ). Anal. cale, for C ₂₅ H ₄₇ N ₅ O ₆ S: C, 55.02; H, 8.68; N, 12.83. Found: C, 55.09; H, 8.66; N, 12.86.

Hydrochloride (5R, 6R, 7S, 8R, 8aR) -3- (8-aminooctyl) imino-5,6,7,8-tetrahydroxy-2-thianedolizidine (8). [α] _D -7.0 (c 1.0, H ₂ O). ¹ H NMR (500 MHz,

D ₂ O, 313 K) δ 5.76 (d, 1 H, J ₅₆ = 4.0 Hz, H-5), 4.46 (m, 1 H, H-8a), 3.92 (m, 2H, H-7, H-1a), 3.78 (dd, 1 H, J _6.7 = 9.5 Hz, H-6), 3.71 (t, 1 H, J _7.8 = J _8.8a = 9.5 Hz, H-8), 3.61 (t, 1 H, J _{1a, 1b} = J _{8a, 1 b} = 10.0 Hz, H-1b), 3.57 (t, 2H, ³ J _{H, H} = 7.0 Hz, CH ₂ N), 3.12 (t, 2H, ³ J _{H, H} = 7.5 Hz, CH ₂ NH ₂ ), 1.79 (m, 4H , CH ₂ ), 1, 48 (m, 8H, CH ₂ ). NMR- ¹³ C (125.7 MHz, D ₂ O, 313 K) δ 173.0 (CN), 76.6 (C-5), 73.3 (C-8), 71, 9 (C-7 ), 70.8 (C-6), 63.6 (C-8a), 49.2 (CH ₂ N), 39.8 (CH ₂ NH ₂ ), 31, 4 (C-1), 28, 2, 28.1, 28.0, 26.9, 27.8, 25.7 (CH ₂ ). FABMS: m / z 348 (30, [M + H] ⁺ ). HRFABMS: m / z 348.194640; cale, for Ci ₅ H ₃₀ N ₃ O ₄ S: 348,195704. Anal. cale, for Ci ₅ H ₃₀ CIN ₃ O ₄ S: C, 46.92; H, 7.88; N, 10.94; S, 8.35. Found: C, 46.57; H, 7.69; N, 10.61; S, 8.04.

(5R, 6R, 7S, 8R, 8aR) -3- (4-Adamantane-1-ylcarboxamidobutyl) imino-5,6,7,8-tetrahydroxy-2-thianedolizidine (9). [α] _D -7.1 (c 1.0, H ₂ O). F _R 0.28 (CH ₂ CI ₂ - H ₂ O-MeOH 40: 10: 1). ¹ H NMR (500 MHz, D ₂ O, 313 K) δ 5.73 (d, 1 H, J ₅₆ = 3.7 Hz, H-5), 4.38 (m, 1 H, H-8a ), 3.91 (t, 1 H, J _6.7 = J _7, β = 9.5 Hz, H-7), 3.87 (dd, 1 H, Ji _a, i _b = 11, 3 Hz , J _8a, i _a = 7.5 Hz, H-1a), 3.74 (dd, 1 H, J _6.7 = 9.5 Hz, H- 6), 3.67 (t, 1 H, J _8.8a = 9.5 Hz, H-8), 3.53 (m, 3H, H-1 b, CH ₂ N), 3.33 (t, 2H, ³ J _{H, H} = 6.7 Hz, CH ₂ NHCO), 2.14 (sa, 3H, CH), 1.92 (m, 6H, CCH ₂ ), 1.83 (m, 6H, CHCH ₂ ), 1.67 (m, 2H, CH ₂ ). NMR- ¹³ C (125.7 MHz, D ₂ O, 313 K) δ 182.0 (CO), 173.0 (CN), 76.6 (C-5), 73.5 (C-8), 72.0 (C-7), 70.9 (C-6), 63.1 (C-8a), 49.3 (CH ₂ N), 40.8 (CCONH), 38.8 (CH), 38.6 (CH ₂ NHCO), 36.1 (CCH ₂ ), 31, 4 (C-1), 28.0 (CHCH ₂ ), 25.9, 25.6 (CH ₂ ). FABMS: m / z 476 (20, [M + Na] ⁺ ), 454 (10, [M + H] ⁺ ). Anal. cale, for C ₂₂ H ₃₅ CIN ₃ O ₅ S: C, 58.25; H, 7.78; N, 9.26; S, 7.07.

(5R, 6R, 7S, 8R, 8aR) -3- (11-Azide-3,6,9-trioxaundecyl) imino-5,6,7,8-tetrahydroxy-2-thianedolizidine (10). [α] _D -13.0 (c 1.0, H ₂ O). ¹ H NMR (500 MHz, D ₂ O) δ 5.55 (d, 1 H, J _{5 6} = 3.7 Hz, H-5), 3.73 (m, 14H, H-7, H- 8a, OCH ₂ ), 3.54 (dd, 1 H, J _{6 7} = 9.8 Hz, H-6), 3.45 (m, 2H, CH ₂ N), 3.45 (dd, 1 H , Ji _a .i _b = 10.7 Hz, J _8a, i _a = 6.3 Hz, H-1a), 3.39 (t, 1 H, J _8.8a = 9.7 Hz, H-8 ), 3.35 (m, 2H, CH ₂ N ₃ ), 3.05 (t, 1 H, J _8a, i _b = 10.7 Hz, H-1b). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 166.1 (CN), 78.3 (C-5), 77.0 (C-8), 75.3 (C-7), 73 , 9 (C-6), 73.4, 73.1, 72.1, 72.0, 71, 8 (OCH ₂ ), 62.1 (C-8a), 56.0 (CH ₂ N ₃ ) , 52.7 (CH ₂ N), 45.7 (CH ₂ CH ₂ N ₃ ), 33.1 (C-1). FABMS: m / z 444 (15, [M + Na] ⁺ ). HRFABMS: m / z 444.154344; cale, for Ci ₅ H ₂₇ N ₅ O ₇ NaS: 444.152890. Anal. cale, for Ci ₅ H ₂₇ N ₅ O ₇ S: C, 42.75; H, 6.46; N, 16.62; S, 7.61. Found: C, 42.51; H, 6.55; N, 16.46; S, 7.39. (5R, 6R, 7S, 8R, 8aR) -3- (A 1 -Adamantane-1-ylcarboxamido-3,6,9-trioxaundecyl) imino-5,6,7,8-tetrahydroxy-2-thianedolizidine (11) . [α] _D -6.2 (c 1.0, H ₂ O). F _R 0.51 (CH ₂ CI ₂ -H ₂ O-MeOH 40: 10: 1). ¹ H NMR (500 MHz, D ₂ O, 313 K) δ 5.76 (d, 1 H, J _5.6 = 3.7 Hz, H-5), 4.21 (m, 1 H, H -8a), 3.90 (t, 1 H, J ₆₇ = J _7.8 = 9.5 Hz, H-7), 3.84 (m, 12H, OCH ₂ ), 3.79 (dd, 1 H, J _{1a, 1b} = 11, 2 Hz, J _{8a, 1a} = 7.1 Hz, H-1a), 3.73 (dd, 1 H, H-6), 3.67 (m, 2H, CH ₂ N), 3.63 (t, 1 H, J _8.8a = 9.5 Hz, H-8), 3.52 (t, 2H, ³ J _{H, H} = 5.5 Hz, CH ₂ NHCO ), 3.42 (t, 1 H, J _{8a, 1 b} = 11, 2 Hz, H-1 b), 2.15 (sa, 3H, CH), 1.94 (m, 6H, CCH ₂ ) 1.85 (m, 6H, CH ₂ ). NMR- ¹³ C (125.7 MHz, D ₂ O, 313 K) δ 182.0 (CO), 163.0 (CN), 76.4 (C-5), 74.0 (C-8), 72.5 (C-7), 71, 2 (C-6), 69.9, 69.8, 69.7, 69.3, 69.2 (OCH ₂ ), 61, 9 (C-8a) , 51, 1 (CH ₂ N), 40.8 (CCONH), 39.1 (CH ₂ NHCO), 38.7 (CH), 36.1 (CCH ₂ ), 31, 2 (C-1), 27.9 (CH ₂ ). FABMS: m / z 580 (100, [M + Na] ⁺ ), 558 (5, [M + H] ⁺ ). Anal. cale, for C ₂₆ H ₄₃ N ₃ O ₈ S: C, 55.99; H, 7.77; N, 7.53; S, 5.75. Found: C, 55.68; H, 7.58; N, 7.32; S, 5.39.

Hydrochloride (5R, 6R, 7S, 8R, 8aR) -2-aza-3-benzylimino-5,6,7,8-tetrahydroxindolizidine (12). [α] _D +4.0 (c 1.0, H ₂ O). ¹ H NMR (400 MHz, D ₂ O) δ 7.34 (m, 5H, Ph), 5.40 (d, 1 H, J _5.6 = 3.8 Hz, H-5), 4, 40 (s, 2H, CH ₂ Ph), 3.93 (m, 1 H, H-8a), 3.76 (t, 1 H, J _{1a, 1b} = J _{8a, 1a} = 9.5 Hz, H -1a), 3.66 (t, 1 H, J ₆ j = J _7.8 = 9.5 Hz, H-7), 3.57 (dd, 1 H, H-6), 3.46 ( t, 1 H, J _8.8a = 9.5 Hz, H- 8), 3.42 (dd, 1 H, J _{8a, 1 b} = 9.5 Hz, H-1 b). NMR- ¹³ C (75.5 MHz, D ₂ O) δ 156.5 (CN), 136.3-127.0 (Ph), 74.4 (C-5), 73.3 (C-8) , 72.4 (C-7), 71, 4 (C-6), 56.2 (C-8a), 46.1 (C-1, CH ₂ Ph). FABMS: m / z 294 (60, [M - Cl] ⁺ ). Anal. cale, for Ci ₄ H ₂₂ CIN ₃ O ₅ : C, 48.35; H, 6.38; N, 12.08. Found: C, 48.19; H, 6.20; N, 11, 92.

Hydrochloride (5R, 6R, 7S, 8R, 8aR) -2-aza-3-butylimino-5,6,7,8-tetrahydroxindolizidine (13). [α] _D +7.7 (c 0.8, H ₂ O). F _R 0.53 (CH ₃ CN-H ₂ O- AcOH6: 3: 1). ¹ H NMR (300 MHz, D ₂ O) δ 5.42 (d, 1 H, J _5.6 = 3.7 Hz, H-5), 3.95 (m, 1 H, H-8a) , 3.86 (t, 1 H, J _{1a, 1b} = J _{8a, 1a} = 9.5 Hz, H-1a), 3.72 (t, 1 H, J _6.7 = J _7.8 = 9 , 5 Hz, H-7), 3.60 (dd, 1 H, H-6), 3.52 (t, 1 H, J _88a = 9.5 Hz, H-8), 3.50 (t , 1 H, J _{8a, 1 b} = 9.5 Hz, H-1 b), 3.23 (t, 2H, ³ J _{H, H} = 7.1 Hz, CH ₂ N), 1.57 (m , 2H, CH ₂ CH ₂ N), 1.35 (m, 2H, CH ₂ CH ₃ ), 0.92 (t, 3H, ³ J _{H, H} = 7.3 Hz, CH ₃ ). NMR- ¹³ C (75.5 MHz, D ₂ O) δ 156.6 (CN), 74.6 (C-5), 73.5 (C-8), 72.6 (C-7), 71 , 7 (C-6), 56.3 (C-8a), 46.3 (C-1), 42.8 (CH ₂ N), 30.3 (CH ₂ CH ₂ N), 19.3 ( CH ₂ CH ₃ ), 12.9 (CH ₃ ). FABMS: m / z 260 (100, [M - Cl] ⁺ ). Anal. cale, for CnH ₂₄ CIN ₃ O ₅ : C, 42.11; H, 7.71; N, 13.39. Found: C, 42.21; H, 7.81; N, 13.31. Hydrochloride (5R, 6R, 7S, 8R, 8aR) -2-aza-5,6,7,8-tetrahydroxy-3- octyliminoindolizidine (14). [α] _D +6.6 (c 0.97, H ₂ O). ¹ H NMR (300 MHz, D ₂ O) δ 5.31 (d, 1 H, J _5.6 = 3.8 Hz, H-5), 3.85 (m, 1 H, H-8a) , 3.75 (t, 1 H, J _{1a, 1b} = J _8a , ia = 9.7 Hz, H-1a), 3.64 (t, 1 H, J ₆ , ₇ = J ₇ , ₈ = 9 , 7 Hz, H-7), 3.49 (dd, 1 H, H- 6), 3.42 (t, 1 H, J _8.8a = 9.7 Hz, H-8), 3.39 (t, 1 H, J _{8a, 1b} = 9.7 Hz, H-1 b), 3.21 (t, 2H, ³ J _{H, H} = 7.1 Hz, CH ₂ N), 1.50 ( m, 2H, CH ₂ CH ₂ N), 1, 23 (m, 1OH, CH ₂ ), 0.79 (t, 3H, ³ J _{H, H} = 6.9 Hz, CH ₃ ). NMR- ¹³ C (75.5 MHz, D ₂ O) δ 156.6 (CN), 74.7 (C-5), 73.5 (C-8), 72.6 (C-7), 71 , 8 (C-6), 56.3 (C-8a), 46.3 (C-1), 43.0 (CH ₂ N), 31, 0, 28.3, 28.2, 25.8 (CH ₂ ), 22.0 (CH ₂ CH ₃ ), 13.4 (CH ₃ ). FABMS: m / z 316 (100, [M-Cl] ⁺ ). Anal. cale, for Ci ₅ H ₃₂ CIN ₃ O ₅ : C, 48.71; H, 8.72; N, 11, 36. Found: C, 48.58; H, 8.61; N, 11, 23.

(5 / ?, 6 / ?, 7S, 8S, 8a /?) - 5,6,7,8-Tetrahydroxy-3-phenylimino-2-oxaindolizidine (15). [α] _D -5.2 (c 0.58, H ₂ O). ¹ H NMR (500 MHz, D ₂ O) δ 7.23-6.91 (m, 5H, Ph), 5.44 (d, 1 H, J _5i6 = 4.0 Hz, H-5), 4.36 (t, 1 H, J _{8a, 1a} = J _{1a, 1b} = 8.3 Hz, H-1a), 4.20 (t, 1 H, J _{8a 1 b} = 7.8 Hz, H- 1 b), 4.11 (m, 1 H, H-8a), 3.93 (m, 1 H, H-8), 3.82 (dd, 1 H, J _6.7 = 10.2 Hz , J _7.8 = 2.6 Hz, H-7), 3.77 (dd, 1 H, H-6). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 153.5 (CN), 146.1-123.4 (Ph), 78.3 (C-5), 69.5 (C-7) , 68.2 (C-8), 67.7 (C-6), 66.4 (C-1), 53.6 (C-8a). FABMS: m / z 303 (60, [M + Na] ⁺ ), 281 (30, [M + H] ⁺ ). Anal. cale, for Ci ₃ H ₁₆ N ₂ O ₅ : C, 55.71; H, 5.75; N, 9.99. Found: C, 55.74; H, 5.64; N, 9.87.

(5R, 6R, 7S, 8S, 8aR) -3-Butylimino-5,6,7,8-tetrahydroxy-2-oxaindolizidine (16). [α] _D -4.1 (c 1.0, H ₂ O). ¹ H NMR (500 MHz, D ₂ O) δ 5.52 (d, 1 H, J _{5 6} = 4.0 Hz, H-5), 4.82 (t, 1 H, J _{8a, 1a} = J _{1a, 1b} = 9.0 Hz, H-1a), 4.69 (dd, 1 H, J _{8a, 1b} = 7.0 Hz, H-1 b), 4.49 (ddd, 1 H, J _88a = 2.5 Hz, H-8a), 4.02 (t, 1 H, J ₇₈ = 2.5 Hz, H- 8), 3.87 (dd, 1 H, J ₆₇ = 10.5, H-7), 3.77 (dd, 1 H, H-6), 3.30 (t, 2H, ³ J _{H, H} = 7.0 Hz, CH ₂ N), 1.48 (m, 2H , CH ₂ CH ₂ N), 1.25 (m, 2H, CH ₂ CH ₃ ), 0.81 (t, 3H, ³ J _{H, H} = 7.0 Hz, CH ₃ ). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 158.5 (CN), 74.9 (C-5), 70.6 (C-1), 68.8 (C-7), 68 , 0 (C-8), 67.3 (C-6), 56.2 (C-8a), 42.3 (CH ₂ N), 30.3 (CH ₂ CH ₂ N), 19.1 ( CH ₂ CH ₃ ), 12.8 (CH ₃ ). FABMS: m / z 283 (20, [M + Na] ⁺ ), 261 (100, [M + H] ⁺ ). Anal. cale, for CnH ₂₀ N ₂ O ₅ : C, 50.78; H, 7.75; N, 10.77. Found: C, 50.66; H, 8.04; N, 10.71.

(5 / ?, 6 / ?, 7S, 8S, 8a /?) - 5,6,7,8-Tetrahydroxy-3-octylimino-2-oxaindolizidine (17). [α] _D .9.8 (c 0.5, H ₂ O). ¹ H NMR (500 MHz, D ₂ O) δ 5.52 (d, 1 H, J _5.6 = 4.0 Hz, H-5), 4.83 (t, 1 H, J _{8a, 1a} = J _{1a, 1b} = 8.5 Hz, H-1a), 4.71 (dd, 1 H, J _{8a, 1b} = 8.5 Hz, H-1 b), 4.50 (t, 1 H, H-8a), 4.02 (m, 1 H, H-8), 3.87 (dd, 1 H, J ₆₇ = 10.0 Hz, J ₇ , ₈ = 2.5 Hz, H-7) , 3.78 (dd, 1 H, H-6), 3.30 (t, 2H, ³ J _H , _H = 6.5 Hz, CH ₂ N), 1.52 (m, 2H, CH ₂ CH ₂ N), 1, 21 (m, 1OH, CH ₂ ), 0.78 (t, 3H, ³ J _{H, H} = 6.5 Hz, CH ₃ ). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 158.5 (CN), 74.9 (C-5), 70.6 (C-1), 68.8 (C-7), 68 , 1 (C-8), 67.4 (C-6), 56.2 (C-8a), 43.0 (CH ₂ N), 31, 1, 28.5, 28.3, 28.2 , 25.7 (CH ₂ ), 22.0 (CH ₂ CH ₃ ), 13.4 (CH ₃ ). FABMS: m / z 317 (100, [M + H] ⁺ ). Anal. cale, for Ci ₅ H ₂₈ N ₂ O ₅ : C, 56.94; H, 8.92; N, 8.85. Found: C, 56.78; H, 8.56; N, 8.76.

(5 / ?, 6 / ?, 7S, 8S, 8a /?) - 5,6,7,8-Tetrahydroxy-3-phenylimino-2-thianedolizidine (18). [α] _D -25.5 (c 0.5, H ₂ O). ¹ H NMR (500 MHz, D ₂ O) δ 7.38-7.01 (m, 5H, Ph), 5.74 (sa, 1 H, H-5), 4.23 (ta, 1 H , J _{8a, 1a} = J _{8a, 1 b} = 9.0 Hz, 6.5 Hz, H-8a), 4.04 (sa, 1 H, H-8), 3.95 (da, 1 H, J _6.7 = 9.3 Hz, H-7), 3.88 (da, 1 H, H-6), 3.30 (ta, 1 H, J _{1a, 1b} = 9.0 Hz, H- 1a), 3.20 (ta, 1 H, H-1b). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 173.9 (CN), 149.0-122.5 (Ph), 75.8 (C-5), 69.7 (C-7) , 68.8 (C-8), 67.9 (C-6), 59.2 (C-8a), 26.7 (C-1). FABMS: m / z 322 (100, [M + Na + 3H] ⁺ ), 297 (10, [M + H] ⁺ ). Anal. cale, for Ci ₃ H ₁₆ N ₂ O ₄ S: C, 52.69; H, 5.44; N, 9.45. Found: C, 52.69; H, 5.38; N, 9.38.

(5 / ?, 6 / ?, 7S, 8S, 8a /?) - 3-Butylimino-5,6,7,8-tetrahydroxy-2-thianedolizidine (19).

[α] _D -5.5 (c 0.6, H ₂ O). [α] ₅₄₆ -7.3 (c 0.6, H ₂ O). ¹ H NMR (500 MHz, D ₂ O) δ 5.61 (d, 1 H, J _5.6 = 3.2 Hz, H-5), 4.51 (t, 1 H, J ₈ ^ = 9.0 Hz, H-8a), 4.05 (sa, 1 H, H- 8), 3.93 (dd, 1 H, J _6.7 = 10.2 Hz, J ₇₈ = 1, 8 Hz , H-7), 3.83 (dd, 1 H, H-6), 3.49 (d, 2H, H-1a, H-1 b), 3.34 (t, 2H, ³ J _{H, H} = 6.8 Hz, CH ₂ N), 1.61 (m, 2H, CH ₂ CH ₂ N), 1.33 (m, 2H, CH ₂ CH ₃ ), 0.89 (t, 3H, ³ J _{H, H} = 7.3 Hz, CH ₃ ). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 170.4 (CN), 76.0 (C-5), 69.1 (C-7), 68.9 (C-8), 67 , 4 (C- 6), 61, 8 (C-8a), 50.0 (CH ₂ N), 30.6 (CH ₂ CH ₂ N), 27.4 (C-1), 19.3 ( CH ₂ CH ₃ ), 12.9 (CH ₃ ). FABMS: m / z 299 (55, [M + Na] ⁺ ), 277 (100, [M + H] ⁺ ). Anal. cale, for CnH ₂₀ N ₂ O ₄ S: C, 47.81; H, 7.29; N, 10.14. Found: C, 47.69; H, 7.17; N, 9.94.

(5 / ?, 6 / ?, 7S, 8S, 8a /?) - 5,6,7,8-Tetrahydroxy-3-octylimino-2-thianedolizidine (20). [α] _D -14.3 (c 0.6, MeOH). ¹ H NMR (300 MHz, DMSO) δ 5.44 (d, 1 H, J _{5 6} = 3.4 Hz, H-5), 4.67 (ta, J _{8a, 1a} = J _{8a, 1 b} = 8.5 Hz, 1 H, H-8a), 4.03 (ta, 1 H, J _{1a, 1 b} = 8.5 Hz, H-1a), 3.69 (sa, 1 H, H-8), 3.64 (da, 1 H, J ₆₇ = 9.6, H-7), 3.52 ( dd, 1 H, H-6), 3.25 (m, 1 H, H-1 b), 3.09 (m, 2H, CH ₂ N), 1.49 (m, 2H, CH ₂ CH ₂ N), 1.24 (m, 1OH, CH ₂ ), 0.84 (t, 3H, ³ J _{H, H} = 6.9 Hz, CH ₃ ). NMR- ¹³ C (75.5 MHz, DMSO) δ 170.9 (CN), 76.7 (C-5), 70.0 (C-7), 69.4 (C-8), 68.3 (C-6), 60.2 (C- 8a), 52.6 (CH ₂ N), 31, 7, 30.5, 29.2, 29.1 (CH ₂ ), 27.4 (C- 1), 22.5 (CH ₂ CH ₃ ), 14.4 (CH ₃ ). FABMS: m / z 355 (65, [M + Na] ⁺ ), 333 (100, [M + H] ⁺ ). Anal. cale, for Ci ₅ H ₂₈ N ₂ O ₄ S: C, 54.19; H, 8.49; N, 8.43. Found: C, 53.83; H, 8.39; N, 8.30.

Hydrochloride (5R, 6R, 7S, 8S, 8aR) -2-aza-3-benzylimino-5,6,7,8-tetrahydroxindolizidine (21). [α] _D +7.0 (c 0.8, H ₂ O). F _R 0.32 (CH ₃ CN-H ₂ O- AcOH 20: 2: 1). ¹ H NMR (300 MHz, D ₂ O) δ 7.39-7.27 (m, 5H, Ph), 5.45 (d, 1 H, J _5.6 = 3.8 Hz, H-5 ), 4.41 (s, 2H, CH ₂ Ph), 4.28 (td, 1 H, J _{8a, 1a} = J _{8a, 1b} = 9.8 Hz, J _8.8a = 1, 4 Hz, H -8a), 3.99 (dd, 1 H, J _7.8 = 2.7 Hz, H-8), 3.84 (dd, 1 H, J _6.7 = 10.2 Hz, H-7 ), 3.77 (dd, 1 H, H-6), 3.68 (t, 1 H, J _{1a 1b} = 9.8 Hz, H-1a), 3.55 (t, 1 H, H- 1 B). NMR- ¹³ C (75.5 MHz, D ₂ O) δ 156.7 (CN), 136.1-127.6 (Ph), 74.8 (C-5), 69.7 (C-7) , 68.7 (C-8), 68.4 (C-6), 56.4 (C-8a), 46.4 (CH ₂ Ph), 42.5 (C-1). FABMS: m / z 294 (100, [M - Cl] ⁺ ). HRFABMS: m / z 294,144663; cale, for Ci ₄ H ₂₀ N ₃ O ₄ : 294,145381.

Hydrochloride (5R, 6R, 7S, 8S, 8aR) -2-aza-3-butylimino-5,6,7,8-tetrahydroxindolizidine (22). [α] _D +8.0 (c 1.0, H ₂ O). ¹ H NMR (500 MHz, D ₂ O) δ 5.40 (d, 1 H, J _5.6 = 4.0 Hz, H-5), 4.25 (t, 1 H, J _8a, i _a = J _8a, i _b = 9.7 Hz, H- 8a), 3.98 (m, 1 H, H-8), 3.82 (dd, 1 H, J _6.7 = 9.8 Hz , J ₇₈ = 2.5 Hz, H-7), 3.74 (dd, 1 H, H-6), 3.69 (t, 1 H, J _{1a> 1 b} = 9.7 Hz, H- 1a), 3.56 (t, 1 H, H-1 b), 3.15 (t, 2H, ³ J _{H, H} = 7.0 Hz, CH ₂ N), 1.48 (m, 2H, CH ₂ CH ₂ N), 1, 26 (m, 2H, CH ₂ CH ₃ ), 0.81 (t, 3H, ³ J _{H, H} = 7.3 Hz, CH ₃ ). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 155.9 (CN), 74.0 (C-5), 69.2 (C-7), 68.1 (C-8), 67 , 8 (C-6), 55.7 (C-8a), 42.7 (CH ₂ N), 41, 7 (C-1), 30.1 (CH ₂ CH ₂ N), 19.2 ( CH ₂ CH ₃ ), 12.9 (CH ₃ ). FABMS: m / z 260 (100, [M - Cl] ⁺ ). HRFABMS: m / z 260.160864; cale, for CnH ₂₂ N ₃ O ₄ : 260.161031. Anal. cale, for CnH ₂₂ CIN ₃ O ₄ : C, 44.67; H, 7.50; N, 14.21. Found: C, 44.29; H, 7.35; N, 13.93.

Hydrochloride (5R, 6R, 7S, 8S, 8aR) -2-aza-5,6,7,8-tetrahydroxy-3- octyliminoindolizidine (23). [α] _D -1, 9 (c 1, 0, H ₂ O), [α] ₅₇ 8 -3.9 (c 1, 0, H ₂ O).

¹ H NMR (500 MHz, D ₂ O) δ 5.42 (d, 1 H, J _5.6 = 4.0 Hz, H-5), 4.28 (t, 1 H, J _8a, i _to = J _{8a, 1b} = 9.8 Hz, H-8a), 4.01 (sa, 1 H, H-8), 3.85 (dd, 1 H, J _6.7 = 10.3 Hz, J _7.8 = 2.8 Hz, H-7), 3.77 (dd, 1 H, H-6), 3.72 (t, 1 H, J _{1a, 1b} = 9.8 Hz, H-1a ), 3.59 (t, 1 H, H-1 b), 3.18 (t, 2H, ³ J _{H, H} = 7.0 Hz, CH ₂ N), 1.53 (m, 2H, CH ₂ CH ₂ N), 1, 23 (m, 10H, CH ₂ ), 0.80 (t, 3H, ³ J _{H, H} = 7.0 Hz, CH ₃ ). NMR- ¹³ C (125.7 MHz, D ₂ O) δ 155.9 (CN), 74.1 (C-5), 69.2 (C-7), 68.1 (C-8), 67 , 8 (C-6), 55.7 (C-8a), 42.9 (CH ₂ N), 41, 7 (C-1), 31, 0, 28.3, 28.2, 28.0 , 25.7 (CH ₂ ), 22.0 (CH ₂ CH ₃ ), 13.4 (CH ₃ ). FABMS: m / z 316 (100, [M + H - Cl] ⁺ ). Anal. cale, for Ci ₅ H ₃₀ CIN ₃ O ₄ H ₂ O: C, 48.71; H, 8.72; N, 11, 36. Found: C, 48.84; H, 8.72; N, 11, 25.

EXAMPLE 2

2.1. Preparation of inclusion complexes between azazúcares sp ² and cyclodextrins. The presence of a hydrophobic moiety in the azazúcar sp ² inhibitors of the invention results in, in some cases, limited solubilities in aqueous media. To overcome this limitation, applicants found the preparation of inclusion complexes with chemically modified cyclodextrins or cyclodextrins particularly useful. Cyclodextrins are cyclic oligosaccharides that make up a cavity that can accept complementary host molecules of appropriate size. The preparation of said inclusion complexes involves the preparation of a solution in water of both the cyclodextrin and the inhibitor, in a relative proportion that can vary from 1: 9 to 9: 1, followed by lyophilization. As the cyclodextrin vehicle, the native α, β or yCD, the commercially available methylated, hydroxypropylated or sulfobutylated derivatives (DIMEB, TRIMEB, RAMEB, HPBCD, Captisol®) or other chemically modified CDs such as those described in patent applications WO 9733919 can be used and WO2004087768. The preferred cyclodextrin carrier according to this invention is βCD. After the formation of the corresponding inclusion complexes, the aqueous solubility of the inhibitors of the invention increased significantly, with solubility enhancements of up to 100 times.

2.2. Cell culture.

Dermal fibroblasts derived from a healthy person and from Gaucher disease patients (N370S / N370S, F213I / F213I, N188S / G193W and F213I / L444P mutations) at 37 ⁰ C in 5% CO ₂ atmosphere using Dulbecco-modified Eagle medium supplemented with antibiotics (streptomycin and penicillin) and 10% fetal bovine serum. When cells became confluent at 80%, they were scraped in H ₂ O cooled with ice (10 ⁶ / ml) and used by sonication. Insoluble materials were removed by centrifugation at 12,000 g for 10 min at 4 ⁰ C. Protein concentrations were determined in those used with a BCA microprotein assay kit (Pierce).

2.3. Enzymatic assay in used.

Β-Glucocerebrosidase activities in cell phones were determined using β-D-glucopyranoside conjugated with 4-methylumbelliferone as substrate (AM Vaccaro, M. Muschilli, M. Tatti, R. Salvioli, E. Gallozzi, K. Suzuki. Clin. Biochem. 20: 429-43, 1987). Briefly, 4 ul of cell lysates were incubated at 37 ⁰ C with 8 .mu.l of substrate solution in 0.1 M citrate buffer, pH 5.2, supplemented with sodium taurocholate (0.8% w / v). The reaction was terminated by adding 1.0 ml of 0.2 M sodium glycine-hydroxide buffer (pH 10.7). One unit of enzymatic activity was defined as nmoles of A-methylumbelliferone released per hour.

2.4. Enzymatic assay in used for the determination of inhibitory activities against human β-glucocerebrosidase.

To explore the effects of the compounds on mutant β-glucocerebrosidase, healthy control cells and patients with Gaucher disease were cultured for 4 days in the absence or presence of increasing concentrations of compounds. After exposure, they were scratched in ice cold H ₂ O (10 ⁶ / ml) and used by sonication. Insoluble materials were removed by centrifugation at 12,000 g for 10 min at 4 ⁰ C. The β-glucocerebrosidase activities in cellular used were measured.

2.5 Intact β-glucocerebrosidase assay for the determination of inhibitory activities against human β-glucocerebrosidase. The ex vivo enzyme assay did not indicate whether the lysosomal enzyme activity was enhanced by these compounds. To compensate for this, an "intact enzyme assay" described by Sawkar et al. (AR Sawkar, WC Cheng, E. Beutler, CH. Wong, WE Balch, JW Kelly. Proc. Nati. Acad. Sci. USA 99: 15428-15433, 2002) in which the cellular enzymatic activity was estimated by the application from the substrate (β-D-glucopyranoside conjugated with 4-methylumbelliferone) to intact cells followed by quantification of the released 4-methylumbelliferone. In this assay, cells were pretreated with or without a high concentration (0.5 mM) of chonduritol-B-epoxide (CBE), an irreversible inhibitor of lysosomal β-glucosidase, before exposure to the substrate.

Briefly, 24-well plates were treated with active ingredients of the present invention for 4 days. After washing with phosphate buffered saline (PBS), the cells were incubated in 80 μl of PBS and 80 μl of 0.2 M acetate buffer (pH 4.0). Ia reaction was initiated by the addition of 100 .mu.l of β-D-glucopyranoside 4-methylumbelliferone conjugated (5 mM), followed by incubation at 37 ⁰ C for 1 h. The reaction was stopped using the cells by the addition of 2 ml of 0.2 M glycine buffer (pH 10.7) and the release of 4-methylumbelliferone was quantified. Each experiment was performed in parallel with pre-incubated cells with or without 0.5 mM CBE for 1 h. The CBE sensitive component was assigned to lysosomal β-glucosidase, while the CBE insensitive component was assigned to non-lysosomal β-glucosidase.

2.6 Intracellular β-galactosidase assay. Fibroblasts were cultured in Dulbecco-modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics, and collected by scratching. They were combined by centrifugation, washed once with phosphate buffered saline and suspended in water. The cell suspension was sonicated and used for enzymatic assay (enzymatic solution). The β-galactosidase assay was performed in 96-well plates. The enzyme test mixture consisted of 10 μl of enzymatic solution, with or without active ingredient of the invention at a final concentration of up to 5 μM, and 10 μl of substrate solution containing 1 mM 4-methylumbelliferyl-β-galactoside (Sigma , St Louis, MO) in 0.1 M (pH 4.5) and 0.1M NaCI After the incubation citrate buffer for 1 h at 37 ⁰ C, the enzymatic reaction was terminated by adding buffer 0.2 M glycine-NaOH (pH 10.7) and the 4-methylumbelliferone released by fluorometry (355 nm excitation; 460 nm emission) was measured as described above (Sakuraba, Aoyagi T, Suzuki Y. Clin. Chim. Acta 1982; 125: 275-282). The protein was determined with the BCA protein assay kit (Pierce, Rockford, IL).

EXAMPLE 3

In vitro inhibition of human β-glucosidase and β-galactosidase by azazúcares sp ² .

The summary of the IC ₅₀ values selected for the inhibition of human β-glucosidase and β-galactosidase is shown in Table 1. In general, the sp ² azazúcares of the invention that have a hydroxylation profile of structural complementarity with D- Glucose (glucomimetics; for example, 3, 6 and 14) are very potent inhibitors of human β-glucosidase. Those who have a hydroxylation profile of structural complementarity with D-galactose (galactomimetics; for example 17, 20 and 23) are potent inhibitors of both β-glucosidase and β-galactosidase. It is remarkable that the sp ² azazúcares of the invention did not significantly inhibit either α-glucosidase or α-galactosidase. The inhibition of β-glucosidases was at least 1,000 times more effective, in terms of Cl ₅₀ values, compared to the inhibition of α-glucosidases. The results suggest to the applicants that the use of sp ² azazúcares of the invention as pharmacological chaperones for the treatment of diseases related to the malfunction of human β-glucosidase and / or β-galactosidase, such as Gaucher's disease or G _MI gangliosidosis , avoid or minimize the side effects associated with interference in the functioning of α-glucosidase and / or α-galactosidase.

Table 1. Inhibitory activity of some of the inhibitors on human β-glucosidase and β-galactosidase in fibroblasts

aThe data represents average values from three independent control cell lines

EXAMPLE 4

In vitro potentiation of the activity of mutant β-glucosidase by azazúcares sp ² .

The β-glucosidase activity in used obtained from Gaucher fibroblasts with different mutations was increased by increasing the concentration of inhibitor in a certain range. The summary of the data selected for some of the inhibitors is shown in Table 2. Both the glucomimetics and the galactomimetics of the invention are active as pharmacological chaperones against mutants N370S / N370S, F213I / F213I and N188S / G193W in this test, with relative activity enhancements that go up to 3.4 times in the most favorable case. The previous results support the therapeutic concept of the applicants that potent azazúcar sp ² inhibitors can serve as effective pharmacological chaperones to enhance the mutant enzymatic activity of patients with Gaucher disease.

Table 2. Activity enhancements on β-glucosidase activity in Gaucher fibroblasts (used) induced in the presence of some pharmacological chaperones of the invention 3 µM (above) and 30 µM (below) for different mutations. SP indicates no enhancement

³ Residual activity refers to wild control cells.

EXAMPLE 5

Intracellular potentiation of the activity of β-qlucosidase in fibroblasts of patients with Gaucher by azazúcares sp ² .

The intracellular potentiation activity of the azazúcar sp ² inhibitors of the invention with fibroblasts from patients with Gaucher with phenotypes N370S / N370S, F213I / F213I, N188S / G193W, N370S / 84GG, L444P / RecNcil and F213I was investigated ex vivo / L444P. The data selected for some glucomimetics (compounds 3, 6 and 14) are collected in Table 3, which were found to be much more active than the galactomimetics in this assay.

Compounds 3 and 6 at low concentration (3 μM) enhanced the activity of wild-type fibroblasts (control) by 23.6 and 6.5%, respectively. Concentrations greater than 30 μM nullified the effect of control enhancement. In contrast, increasing the concentration from 3 to 30 mM generally caused a greater potentiation of the activity in the case of mutant β-glucosidases, supporting a high ratio of chaperone to inhibitor activity for this family of derivatives.

The azazúcar sp ² glucomimetics of the invention demonstrated a significant impact on the potentiation of intracellular enzymatic activity in Gaucher fibroblasts. The residual enzyme activity in the mutants increased up to 3.5 times in the most favorable case. The magnitude of the potentiation depended on the phenotype and the structure of the compound. Compound 3, which has a cyclic isourea in the structure, was particularly effective against N370S / N370S and F213I / F213I mutations, with elevations of residual activity of 2.7 and 3.5 times, respectively, at a concentration of 30 μM. Compound 6, which has a cyclic isourea in the structure, was particularly effective against mutations N370S / N370S, F213I / F213I and L444P / RecNcil, with elevations of residual activity of 2.7, 3.5 and 1, 3 times , respectively, at a concentration of 30 μM. Compound 6, which has a cyclic isothiourea in the structure, was the most effective of the series against the N370S / 84GG mutation (potentiation of residual activity elevated at 1.8 times at a concentration of 30 μM), while compound 14 , which has a cyclic guanidine in the structure, was the most effective against the N188S / G193W and F213I / L444P mutations (enhancements of the residual activity increased 2.2 times at a concentration of 30 μM in both cases). It is believed that the bioavailability of these compounds is significantly improved by the amphiphilic nature of the molecules, which possibly facilitates their passage through the cell membranes and ER, thus increasing the intracellular concentration of these compounds.

Table 3. Enhancements of the activity on the activity of β-glucosidase in intact Gaucher fibroblasts induced in the presence of certain pharmacological chaperones of the invention at 3 μM (above) and 30 μM (below) for different mutations. SP indicates no enhancement.

EXAMPLE 6

Intracellular potentiation of β-galactosidase activity in fibroblasts of patients with GM ₁ ganqliosidosis by azazúcares sp ²

The azazúcares sp ² inhibitors of the invention that have a hydroxylation profile of structural complementarity with D-galactose (galactomimetics; for example, compounds 15-23) enhance the β-galactosidase activity in control cell fibroblasts as well as in fibroblasts of patients with G _M i gangliosidosis - As an example, Table 4 collects the corresponding data of compound 17, which has a cyclic isourea functionality in its structure. The intracellular β-galactosidase activity in the control was raised by 46% using a 50 μM concentration of 17. The intracellular β-galactosidase activity in G _M gangliosidosis fibroblasts was raised 17% using a 50 μM concentration of 17. Table 4. Enhancements of the activity on β-galactosidase activity in wild (control) fibroblasts and intact G _M i gangliosidosis induced by the presence of compound 17 at different concentrations.

EXAMPLE 7

In vitro and ex vivo potentiation of the activity of mutant β-qlucosidase by inclusion complexes of azazúcares sp ² : βCD.

The azazúcares sp ² inhibitors of the invention easily formed inclusion complexes with βCD in water at different relative proportions, as observed from the NMR experiments. The resulting complexes allowed increasing the solubility of the inhibitor in water without observing harmful effects on the inhibitory activity against human glucosidases.

The enzymatic activity was elevated in Gaucher fibroblasts, both used as in intact cells, increasing the concentration of inclusion complexes azazúcares sp ^2: βCD. Selected data of some complexes are collected in Table 5. Applicants found this formulation particularly suitable for compounds that have low aqueous solubility, as is the case of compound 6. By using relative ratios 1: 1 of 6 and βCD at a concentration of 30 μM, increases of up to 3 times were achieved. of the residual activity of the mutant β-glucosidase for the N370S / N370S phenotype.

Table 5. Enhancements of β-glucosidase activity in Gaucher fibroblasts (used) or intact cells induced in the presence of some complexes of sp ² -azazúcares: βCD for different mutations. SP indicates without potentiation

aThe concentrations refer to the active sp azazúcar component. In used. ^c In intact cells

Claims

1. Compound of general formula (I), or any of its salts,

where: R is a substituent, the same or different, and which is selected from an H atom, a hydroxyl group (-OH) or an OR ² group; where

R ² is a group selected from acyl, substituted or unsubstituted, (C1-C18) alkyl, substituted or unsubstituted, aryl, substituted or unsubstituted, aralkyl, substituted or unsubstituted, amino (NH ₂ ), acetamido (NHAc ), or an NHR ³ ; where R ³ is a group selected from acyl, substituted or unsubstituted, (C1-C18) alkyl, substituted or unsubstituted, aryl, substituted or unsubstituted or aralkyl, substituted or unsubstituted.

And it is selected from an O, NH or S; Y

X is the group -NR ¹ . Where R ¹ is selected from a (C1-C18) alkyl group, substituted or unsubstituted, an aryl (C5-C18) group, substituted or unsubstituted, or an aralkyl group, substituted or unsubstituted.

2. Compound according to claim 1, wherein Y is an oxygen atom.

3. Compound according to claim 1 wherein Y is NH.

4. Compound according to claim 1, wherein Y is a sulfur atom.

5. Compound according to any of claims 1 to 4, of general formula (Ia '):

6. Compound according to any of claims 1 to 4, of general formula (Ib '):

Ib ¹

7. Compound according to any of claims 1 to 6, wherein R is OH.

8. A compound according to any one of claims 1 to 7, wherein R ¹ is a (C ₃ -C ₉ ) alkyl group.

9. A compound according to any one of claims 1 to 7, wherein R ¹ is an aryl group (C ₅ -C ₇ ).

10. A compound according to any one of claims 1 to 9, wherein said compound is a salt of a hydrochloride.

11. Compound according to claim 1, of formula: a. (5R, 6R, 7S, 8R, 8aR) -5,6,7,8-Tetrahydroxy-3-phenylimino-2-oxaindolizidine b. (5R, 6R, 7S, 8R, 8aR) -5,6,7,8-Tetrahydroxy-3-butylimino-2-oxaindolizidine c. (5R, 6S, 7R, 8R, 8aR) -5,6,7,8-Tetrahydroxy-2-oxa-3-octylimino-indolizidine. d. (5R, 6R, 7S, 8R, 8aR) -5,6,7,8-Tetrahydroxy-3-phenylimino-2-thianedolizidine. and. (5R, 6R, 7S, 8R, 8aR) -3-Butylimino-5,6,7,8-tetrahydroxy-2-thianedolizidine F. (5R, 6R, 7S, 8R, 8aR) -5,6,7,8-Tetrahydroxy-3-octylimino-2-thianedolizidine g. (5R, 6R, 7S, 8R, 8aR) -3- [2 '- (Λ /, Λ / -Bis- (2-hexanamidoethyl) aminoethyl) -imino] -5,6,7,8-tetrahydroxy-2- thiaindolizidine h. (5R, 6R, 7S, 8R, 8aR) -3- (8-aminooctyl) imino-5,6,7,8-tetrahydroxy-2-thianedolizidine hydrochloride i. (5R, 6R, 7S, 8R, 8aR) -3- (4-Adamantane-1-ylcarboxamidobutyl) imino-

5,6,7,8-tetrahydroxy-2-thianedolizidine. j. (5R, 6R, 7S, 8R, 8aR) -3- (11-Azide-3,6,9-trioxaundecyl) imino-5,6,7,8-tetrahydroxy-2-thianedolizidine k. (5R, 6R, 7S, 8R, 8aR) -3- (11-Adamantane-1-ylcarboxamido-3,6,9-trioxaundecyl) imino-5,6,7,8-tetrahydroxy-2-thianedolizidine. I. (5R, 6R, 7S, 8R, 8aR) -2-aza-3-benzylimino-5,6,7,8-tetrahydroxindolizidine hydrochloride. m. Hydrochloride (5R, 6R, 7S, 8R, 8aR) -2-aza-3-butylimino-5,6,7,8-tetrahydroxindolizidine. n. Hydrochloride (5R, 6R, 7S, 8R, 8aR) -2-aza-5,6,7,8-tetrahydroxy-3- octyliminoindolizidine. or. (5R, 6R, 7S, 8S, 8aR) -5,6,7,8-Tetrahydroxy-3-phenylimino-2-oxaindolizidine. p. (5R, 6R, 7S, 8S, 8aR) -3-Butylimino-5,6,7,8-tetrahydroxy-2-oxaindolizidine. q. (5R, 6R, 7S, 8S, 8aR) -5,6,7,8-Tetrahydroxy-3-octylimino-2-oxaindolizidine. r. (5R, 6R, 7S, 8S, 8aR) -5,6,7,8-Tetrahydroxy-3-phenylimino-2-thianedolizidine. s. (5R, 6R, 7S, 8S, 8aR) -3-Butylimino-5,6,7,8-tetrahydroxy-2-thianedolizidine. t. (5R, 6R, 7S, 8S, 8aR) -5,6,7,8-Tetrahydroxy-3-octylimino-2-thianedolizidine. or. Hydrochloride (5R, 6R, 7S, 8S, 8aR) -2-aza-3-benzylimino-5,6,7,8-tetrahydroxindolizidine. v. (5R, 6R, 7S, 8S, 8aR) -2-aza-3-butylimino-5,6,7,8-tetrahydroxindolizidine hydrochloride; or w. Hydrochloride (5R, 6R, 7S, 8S, 8aR) -2-aza-5,6,7,8-tetrahydroxy-3- octyliminoindolizidine.

12. Method of obtaining the compounds of general formula (I) comprising: a. introducing an amino group in the C-5 position of the corresponding furanosa sugar; b. close a five-member ring between positions C-5 and C-6 using a pseudoamide type segment of the compound obtained in step (a); and c. Transposition of the furan ring to a piperidine cycle fused with the five-membered cyclic pseudoamide ring obtained in step (b).

13. Use of the compound of general formula (I) for the preparation of a pharmaceutical composition.

14. Use of the compound of general formula (I) for the preparation of a pharmaceutical composition for the treatment of a disease related to human mutated β-glucosidase and / or β-galactosidase.

15. Use of the compound according to claim 14, wherein the diseases are Gaucher disease, GM1 gangliosidosis or Morquio B disease.

16. Pharmaceutical composition comprising at least one of the compounds of general formula (I).

17. Pharmaceutical composition according to claim 16, which further comprises another active component and / or a pharmaceutically acceptable carrier.

18. Pharmaceutical composition according to claim 17, wherein the vehicle is a cyclodextrin or any of its derivatives.

19. Pharmaceutical composition according to claim 18, wherein the vehicle is β-cyclodextrin.

20. In vitro method to enhance the activity of a glycosidase enzyme, which comprises contacting the protein with an effective amount of a compound of general formula (I).