MXPA97006373A - Conjugated bioactive compounds of carbohydr - Google Patents

Abstract

Methods and compositions are provided that increase the cellular uptake of bioactive materials by covalently linking such compounds to carbohydrate moieties or moieties through chemical linkers using different linkages to the cyclic groups. Numerous carbohydrates, linkers and bioactive materials can be joined in this form to form new compositions, to which reference is made here collectively, glycosides. The glycinates are preferentially taken by the glucose receptor and / or other cellular receptors and once inside the cells, the glycinates are divided into a sugar, a binder or binding fragments and a biologically active compound. Various aspects of this invention include processes for the synthesis of glycinates, glycinate compositions and methods of treating diseases using glycine.

Description

BIOACTIVE COMPOUNDS CONJUGATED WITH CARBOHYDRATES 1. FIELD OF THE INVENTION The field of the invention is the distribution and choice of targets of bioactive materials, including especially the use of increased bioavailability and increased cellular absorption to influence this distribution and choice of targets. 2- BACKGROUND OF THE INVENTION A significant problem in clinical pharmacology is the selective distribution of specific bioactive compounds to target cells of an organism. In many cases, the desirable compounds only passively and partially diffuse into the target cells, and the plasma concentrations required to achieve significant intracellular levels are difficult to achieve due to toxicity, evacuation and degradation by the liver, kidneys and other organs or body fluids. In conditions of the central nervous system, the problem is often aggravated by the blood REF: 25549 brain (BBB), and in neoplasms, the problem can be further aggravated due to poor or inefficient vascularization. Additional difficulties may result from the digestion or degradation of the bioactive compounds within the intestines, and / or the poor transport of many of these compounds through the intestinal wall. Still further difficulties arise from the inadvertent distribution of the bioactive compounds to the non-target cells. Problems related to the specific distribution have been found with many bioactive compounds, including highly toxic anti-thyrous drugs such as doxorubicin and methotrexate, antiviral drugs such as arabinosil cytosine and arabinosil adenosine, and antiparasitic drugs such as chloroquine and pyrimethamine in which it is Particularly important is the specificity of the site. Distribution problems are also encountered with respect to substances that are not technically considered to be drugs, such as radioactive labels and contrast substances, all of which are designed to be considered to be bioactive compounds because they have an effect or application on or within a living organism.

It is known that carbohydrate portions of glycoproteins play an important role in the absorption, transport and subsequent distribution in tissue in the body, and several investigators have previously faced the problem of distribution by administering sugars in conjunction with bioactive materials. Significant results have already been achieved in this area. One study showed that intestinal absorption of poorly absorbed drugs was greatly increased by glycosylating the drug with a sugar. Mizuma, T., et al., Intestinal Active Absorption of Sugar-Conjugated Compounds by Glucose Transport System: Implication of Improvement of Poorly Absorbable Drugs, Biochem, Pharma, 43 (9) 2037-2039 (1992). Another study identified the differential choice of targets of different conjugates of saccharide-poly-L-lysine, reporting that galactose conjugates target the liver as the target, conjugates of mannose and fructose target the reticuloendothelial system as the target, and conjugates of xylose target the liver and lungs preferably. Gonsho, A., et al., Tissue-Targeting Ability of Saccharide-Poly (L-Lysine) Conjugates, Biol. Pharm Bull., 17 (2) 275-282 (1994).

Research in this area is divided into three general categories: (1) therapy in which sugar does not bind in any way to the bioactive compound; (2) compositions in which the sugar is covalently linked either directly or indirectly through a glycosidic linkage (which couples the Ci carbon atom of the sugar) to the bioactive compound; and (3) compositions in which the drug is associated with an amphiphile, such as a closure within a liposome coated with sugar. The development in the first category has • greatly understood the use of mannitol to overcome the blood barrier of the brain. Concentrated solutions of mannitol, for example, have been poured into the carotid arteries to break the blood barrier of the brain in a manner sufficient to allow methotrexate and other potent chemotherapeutic agents to penetrate brain tumors. Angier, N., Discover, May 199, 67-72. While significant results have been reported with this approach, it suffers from several disadvantages. In particular, the opening of the blood barrier of the brain is clearly non-specific, and allows numerous dangerous compounds to enter the brain together with the desired compound. In addition, the technique chooses its purpose mainly the central nervous system, and is largely inapplicable to other systems. The developments in the second category, the direct or indirect glycosidic binding of sugars to drugs, are of much greater importance. In this way, the cytotoxic drug, methotrexate (MTC) has been conjugated to bovine serum albumin (BSA), with the resulting compound that is recognized by the mannose receptors present on the surface of the macrophages (Chakraborty, P. and collaborators, Sugar Receptor Mediated Drug Delivery to Macrophages in the Therapy Experimental Visceral Leishmaniasis, Biochem, Biophys, Res. Co m., 166 (1) 404-410 (1990)). Insulin has been glycosylated to take advantage of competitive binding between glucose and glycosylated insulin (Seminoff, LA et al, A. Self-regulating insulin delivery system I. Characterization of a synthetic glycosylated insulin derivative, Int '1. J Pharm., 54 (1989) 241-249). The anti-HIV agent, 3 '-azido-3'-deoxythymidine (AZT) has also been glycosidically coupled to human serum albumin (HSA) and several portions of sugar to produce mañosa-, fucosa- , galactose- and glucose-neoglycoproteins (Molema, G., Targeting of Antiviral Druqs to T4-Lymphocytes, Biochem, Pharm, 40 (12) 2603-2610 (1990)). The anti-inflammatory agent, naproxin, has also been glycosidically bound to the sugar-terminated HSA (Franssen, EJF, Hepatic and Intrahepatic Targeting of an Anti-inflame matory Agent With Human Serum Albumin and Neoglycoproteins as Carrier Molecules, Biochem, Pharm., 45 (6) 1215-1226 (1993)). In these last two cases, the glycosidic union is formed to HSA, and in this way only indirectly to the drugs, AZT or naproxin. In 1994, a Japanese group reported a method of radioiodinating digoxin using glycosides (Takemuru, Y., et al., Development of Glycoside-Bound Radiopharmaceuticals: Novel Radioiodination Method for Digoxin, Biol. Pharm, Bul!, 17 (1) 97-101 (1974)). Sugars have also been covalently linked to pol-L-lysine by a glycosidic linkage (Monsigny, M., et al., Sugar Specific Delivery of Drugs, Oligonucleotides and Genes, Targeting of Drugs, 431-50 (Ed. By G. Gregoriadis et al., Putnam Press, N.Y., 1994)). In addition to these synthetic sugar conjugates, there are numerous examples in nature of the covalent attachment of a sugar to a biologically active portion. The glycosides, for example, are products of condensation of sugars with various kinds of hydroxy (or occasionally amino or thiol) organic compounds, in which the OH of the healcetal portion of the carbohydrate participates in the condensation (Remington, The Science and Practice of Pharmacy, 19th edition 386-387 (Mack Publ., Co. Easton, PA., 1995)). In fact, many well-known biologically active compounds are glycosides, including amygdalin, cyimarine, digitoxin, ouabain, rutin, and salicin. There are also endogenous glycosides that include gangliosides, sugar nucleotides and neural cell addition molecules. Other compounds of natural origin not usually classified as glycosides actually contain glycosidic bonds in their structures. Examples include antibiotics, gentamicin, amikacin, netilmicin, tobramycin, novobiocin and streptomycin, glucoalkaloids such as solanine, and nucleosides, which consist of a purine base or pyrimidine linked to D-ribose or D-2-deoxyribose. As demonstrated by the utility of both natural and synthetic compounds, direct or indirect, covalent glycosidic binding of sugars to drugs is a successful strategy for the distribution of drugs. In spite of the extensive occurrence and the knowledge that considers the bioactive compounds coupled to sugars, however, compounds that can be developed through the use of normal glycosidic binding are limited. For example, in some cases this linkage is completely inadequate because the size and steric hindrance of the resulting conjugate compound can not be recognized any longer or transported appropriately by the respective cellular transporters. In other cases, the glycosidic linkages are too labile and are easily hydrolyzed by glycosidases. With respect to the third category, it is known that liposomes having glycosides can be used in vivo to deliver drugs specifically to macrophages (Medda, S. et al., Sugar-coated Liposomes: a Novel Delivery System for Increasing Drug Efficacy and Reduced Drug Toxicity, Biotechnol, Appl. Biochem, 17 37-47 (1993)). Anionic amphiphiles with a phosphate ester linkage between a fluorophilic-lipophilic extremity and a sugar-based hydrophilic head have also been synthesized (Guillod, F., et al., Amphiphilic Sugar Phosphates with Single or Double Perfluoro-alkylated Hydrophobic Chains for Use in Oxygen and Drug Delivery Systems, Art. Cells, Blood Subs., And I mob, Biotech., 22 (4) 1273-1279 (1994)). In 1995, a scholarly analysis of the function of liposomes with surface glycolipids was published (Jones, M. The Surface Properties of Phospholipid Liposome Systems and Their Characterization, Advances in Colloid and Interface Science, 54 93-128 (1995)). However, this third category of compounds, while promising, suffers from the same disadvantages as the glycosides and neoglycoproteins discussed above. The normal glycosidic bond from which all these compounds are made is too much • limiting. In addition, there are synthetic and / or purification problems, constant in the creation of steriochemically pure α- or β-glycosidic linkages. Given the inconveniences of currently available methods and compositions for improving cellular absorption and the choice of targets such as absorption to specific tissues, it is of interest to provide new methods and compositions for linking sugars to biologically active compounds. 3. BRIEF DESCRIPTION OF THE INVENTION Methods and compositions that increase the cellular absorption of materials are provided bioactives by covalently linking these compounds to carbohydrate moieties through chemical linkers using non-glycosidic linkages. Numerous carbohydrates, linkers and bioactive materials can be joined in this manner to form new compositions, collectively referred to herein as "glycosides". The preferred glycosides are preferably taken by the glucose receptors and / or other monosaccharide cell receptors, and once inside the cells, the glycosides are cleaved into a sugar derivative, a linker or linker fragments, and a biologically active compound. . Various aspects of the invention include glycoside compositions, processes for synthesizing glycosides, and methods for treating diseases using glycosides. 4. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing an example synthesis of derivatized linkers.

Figure 2 is a diagram showing an example synthesis of intermediate compounds of 3-0- and 6-0-D-Galactose-Linker.

Figure 3 is a diagram showing an example synthesis of the reactive 3-0- and 6-0-D-Galactose-Linker intermediates.

Figure 4 is a diagram showing an example synthesis of thymidine conjugates with LG-5 via an ester linkage.

Figure 5 is a diagram showing a conjugation of exemplary Virazole ™ with LG-5 via an ester linkage.

Figure 6 is a diagram showing a conjugation of exemplary Virazole ™ with LG-6 via an ether linkage.

Figure 7 is a diagram showing an example synthesis of the intermediate compounds of 6-O-D-Galactose-Linker.

Figure 8 is a diagram showing an example synthesis of the 4-0- and 6-0-D-Mannose-Linker intermediates.

Figure 9 is a diagram showing an example synthesis of the 3-0-, 4-0- and 5-0-D-Fructose-Binding intermediates.

Figure 10 is a diagram showing an exemplary synthesis of glucose-linker phosphoramidate intermediates for the automated synthesis of oligodeoxynucleotides.

Figure 11 is a diagram showing an exemplary synthesis of derivatized linker phosphoramidate for the automated synthesis of oligodeoxynucleotides.

Figure 12 is a diagram showing an alternative synthesis of 1: 2: 5,6-di-0-isopropylidene-3-0- (6'-hydroxy) -hexyl-a-D-glucofuranose (LG-3).

Figure 13 is a diagram showing an example synthesis of modified oligodeoxynucleotides (ODN) at the 3 'and 5' positions (ICN 16967).

Figure 14 contains the UV and TM 'absorption curves for modified oligonucleotides (14a, c) (ICN 16967) and unmodified (14b, d).

. DETAILED DESCRIPTION According to the new terminology employed herein, the glycosides have the general structure of MS-LINK-BAM, where MS is a monosaccharide, LINK is a linker, BAM is a bioactive material, MS is covalently linked to LINK in a position different from the Ci carbon atom of MS, and LINK is covalently linked to BAM. There is a large number of permutations of glycosides. For example, some monosaccharide can be used in a glycoside, including the monosaccharide having from three to eight or more carbon atoms. The LINK portion of a glycoside is also subject to great variation and may be straight or branched chain alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl. The BAM portion may consist of any bioactive molecule, including the following: gastrointestinal and liver drugs hematological drugs, cardiovascular drugs, respiratory drugs, sympathomimetic drugs, cholinomimetic drugs, adrenergic and adrenergic drugs blockers of neurons antimuscarinic and antispasmodic drugs relaxants of the skeletal muscle uterine and antimigraine drugs hormones general drugs and local anesthetics sedative and hypnotic drugs antiepileptic drugs psychopharmacological agents anti-inflrmator stimulants of the CNS (central nervous system) The MS-LINK and Suitable LINK-BAMs also vary widely, and by way of example may independently consist of ether, ester, amide, disulfide, hemiacetal, hemicetal, acetal, and ketal linkages. The presently preferred glycosides use naturally occurring ribose (5 carbon atoms) or hexose (6 carbon atoms) sugars, straight chain linkers of four to sixteen carbon atoms, linkers a,? bifunctional such as hexane, and known bioactive drugs. The MS-LINK and LINK-BAM junctions can preferably be excised under physiological conditions such that a glycoside acts as a prodrug, distributing an active or activatable drug to a target position in or within a target cell. The presently preferred embodiments comprise glycosides in which the LINK-BAM binding is stronger than the MS-LINK binding. Glucosides have numerous advantages over glycosides, which include the following: (1) Monosaccharide can be chemically linked through a bifunctional linker a,? specifically designated a BAM in any of the different OH substituents (except Ci) found in the monosaccharide. (2) The use of a linker to eliminate synthetic stereochemical problems. (3) In relation to the glycosides, and particularly glycosides in which the BAM binds directly to the monosaccharide, the steric hindrance is largely or completely eliminated. This facilitates the passage of the glycoside through specific membrane protein transporters such as one of the glucose transporter systems. The glutamine transporter system (GLUT-1) is an example of this system. (4) The relative lengths of the monosaccharide-linker and linker-BAM linkages can be vary so that the BAM can be released at an appropriate point in transport. (5) Glucosides also offer improved passage through the blood barrier of the brain.

Synthesis of Glucosides In order to chemically connect the carbohydrates to the bioactive compound (s), selected functional groups in both chemical entities can be used to covalently link them through a specific chemical linker. Frequently, completely functional groups in the carbohydrates and bioactive materials comprise: hydroxy, amino, mercapto, carbonyl, carboxyl, and amido functions. The chemical linkers, in most cases, comprise alpha, omega difunctional alkyl chains. Obviously there are many other functional groups and chemical bonds available for this particular purpose. In general, the glycosides can be prepared using the following steps: (1) prepare an appropriate linker. In the case of an a, bifunctional linker, the end a would be reactive and the end? I would be protected; (2) select a monosaccharide and join chemically one of its groups -OH not in Ci to the linker; and (3) chemically linking the linker to a bioactive material. Exemplary synthesis methods and glycoside compounds are described in the following examples. Of course, it should be appreciated that the examples presented are illustrative only, and are not intended to limit the scope of the invention.

EXAMPLES 1-3 - SYNTHESIS OF DERIVATIZED LINKERS Figure 1, the first row shows an example synthesis of 1-O-p-toluenesulfonyl-l, 6-hexanediol (L-1) using the following reagents: 1,6-hexanediol = 1,6-HD; p-toluenesulfonyl chloride = TsCl, pyridine = Py; and dichloromethane = CH2C12. In this procedure, 6-HD (44 g, 0.372 mol) was dissolved in a mixture of Py / CH2C12 (1: 1) (80 ml). The resulting solution was cooled to 0 ° C, and TsCl (20 g, 0.105 mol) was added. The obtained reaction mixture was kept at 4 ° C for 5 hours. The reaction solution was then neutralized with 10% aqueous HCl and the product was extracted by CH2C12 (3 x 100 mL). The organic layer was washed with brine, dried and evaporated. The crude product was purified by flash chromatography on column of silica gel (using the TLC solvent system). L-1 (oil); Yield: 20.6 g (72%). TLC: Ethyl acetate / hexane (1: 1); Rf: 0.56. The structure and purity of L-1 was verified by the XH NMR spectrum. The second row shows an example synthesis of l-O-p-toluenesulfonyl-6-O- (2'-tetrahydropyranyl) -1,6-hexanediol (L-2) using the following reagents: L-1; dihydropyran = DHP; pyridinium p-toluenesulfonate = PPTS (prepared from Py and p-toluenesulfonic acid = TsOH); and CH2C12. In this procedure, L-1 (10 g, 36.7 mM), DHP (3.4 g, 40.3 mM) and PPTS (1.0 g, 3.7 mM) were added one by one to anhydrous CH 2 C 12 (40 ml). The reaction mixture was stirred at room temperature until the reaction was finished (6-8 hours). The reaction mixture was washed with brine (3 x 20 ml), dried and evaporated. The crude product was purified by column chromatography on silica gel (using TLC solvent system). (L-2 (oil) Yield: 9.42 g (72%) TLC, 15% ethyl acetate, 1% triethylamine in hexane, Rf = 0.9, The structure and purity of L-2 was verified by the spectrum of 1N-NMR The third row shows an example synthesis of 1-0- (2'-tetrahydropyranyl) -6-iodo-1-hexanol (L-3) using the following reagents: L-2, Nal; and acetone. In this procedure, a mixture of L-2 (9.0 g, 25.2 mM), Nal (11.3 g, 75 mM) and dry acetone (100 ml) at 50 ° C until the reaction was finished (8-12 hours). The acetone was evaporated and the residue was treated with water (100 ml). The product was extracted with CH2C12 (3 x 50 ml). The organic layer was washed with brine (3 x 50 ml), dried and evaporated. The product was collected via column chromatography on silica gel (TLC solvent system). L-3 (oil); Yield: 5.0 g (63%). TLC: 5% triethylamine in • hexane; Rf = 0.50; 2H NMR spectrum (CDC13) confirms the structure and purity of L-3.

EXAMPLE 4 - SYNTHESIS OF THE INTERMEDIATE COMPOUNDS 3-0- AND 6-0-D-GLUCOSE-LINKER Figure 2 shows an example synthesis of 1,2-O-isopropylidene-3-0- (6'-tetrahydropyranyloxy) -hexyl-alpha-D-glucofuranose (LG-la) and 1,2-0-isopropylidene-6-O- (6'-tetrahydropyranyloxy) - hexyl-alpha-D-glucofuranose (LG-lb) using the following reagents: L-3; 1,2-O-isopropylidene-alpha-D-glucofuranose = IPGF; NaH; and dimethylsulfoxide = DMSO. In this procedure, the IPGF was dissolved (2.0 g, 9.08 mM) in anhydrous DMSO (7 ml) and NaH (total of 545 mg, 13.6 mM) was added in several small portions at room temperature. The reaction mixture was maintained at 40 ° C for 0.5 hours and L-3 (2.27 g, 7.26 mM) was added. The resulting mixture was kept at room temperature overnight then poured into ethyl acetate (200 ml). The organic layer was washed with brine (3 x 50 ml), dried (anhydrous Na 2 SO 4) and evaporated. The crude product mixture was purified and the isomers were separated by flash column chromatography on silica gel (TLC solvent system). LG-la (oil); Performance: 1.06 g, (36%), and LG-lb (oil); Yield: 0.94 g (32%). TLC: 50% ethyl acetate, 2.5% triethylamine, 47.5% hexane; Rf (LG-la): 0.34; Rf (LG-lb): 0.47. XH NMR spectra of both isomers in CDC13 are in agreement with the proposed structures (the samples were chemically pure).

EXAMPLE 5-1, 2: 5, 6-DI-0-ISOPROPILIDEN-3-O- (6'-TETRAHYDROPYRANYLOXY) -HEXIL-ALPHA-D-GLUCOFURANOSA (LG-2) Figure 2 also shows an example synthesis of 1: 2: 5,6-di-O-isopropylidene-3-0- (6 '- tetrahydropyranyloxy) -hexyl-alpha-D-glucofuranose (LG-2) using the following reagents: LG-la; acetone and PPTS (prepared from TsOH and Py). In this procedure, a mixture of LG-la (500 mg, 1.233 mM), PPTS (67 mg, 0.247 mM) and acetone was stirred at room temperature until the reaction was finished (6 hours). The acetone was evaporated and the residual material was chromatographed. LG-2 (oil). Yield: 490 mg (91%) TLC: 15% ethyl acetate, 5% triethylamine, 80% hexane. Rf :. 0.66. XH NMR spectrum (CDC13) confirms the purity and structure of LG-2.

EXAMPLE 6 - ALTERNATIVE SYNTHESIS OF 1, 2: 5, 6-DI-O-ISOPROPYLIDEN-3-0- (6 '-TETRAHYDROPYRANYLOXY) -HEXIL-ALPHA-D-GLUCOFURANOSA An alternative direct synthesis of LG-2 can be performed using the following reagents: 1, 2: 5, 6-di-O-isopropylidene-alpha-D-glucofuranose = DIPGF L-3; NaH and DMSO. In this procedure, DIPGF (2.5 g, 9.6 mM) was dissolved in anhydrous DMSO (7 ml) and NaH (576 mg, 14.40 mM) was dissolved in small portions at room temperature. The reaction mixture was maintained at 40 ° C for 30 minutes and L-3 (3.6 g, 11.54 mM) was added. The reaction mixture was kept overnight at room temperature and then poured into ethyl acetate (200 ml). The organic layer was washed with brine (3 x 50 ml), dried (Na 2 SO 4) and evaporated. The product was purified by column chromatography on silica gel. LG-2 (oil); Yield: 2.42 g (58%). TLC: 15% ethyl acetate, 5% triethylamine, 80% hexane. Rf: 0.66.

EXAMPLE 7 - SYNTHESIS OF REAGENT INTERMEDIATE COMPOUNDS OF 3-O-D-GLUCOSE-LINKER Figure 3 shows an example synthesis of 1,2: 5,6-di-O-isopropylidene-3-0- (6'-hydroxy) -hexyl-alpha-D-glucofuranose (LG-3) using the following reagents: LG-2; Ethanol (96%); PPTS and acetone. In this procedure, LG-2 (2.0 g, 4.49 mM) and PPTS (245 mg, 0.90 mM) were dissolved in ethanol (100 ml) and the reaction mixture was heated at 55 ° C for 5 hours. Then the ethanol was evaporated. The residual material was dissolved in acetone (100 ml) and stirred at room temperature for the next 4 hours. After removal of the acetone in vacuo, the residue was dissolved in ethyl acetate (50 ml), washed with brine (3 x 50 ml), dried (Na 2 SO 4) and evaporated. The product was finally purified by flash chromatography on silica gel. LG-3 (oil); Yield: 1.52 g (94%). TLC: ethyl acetate / hexane (1: 1); Rf: 0.48; the XH NMR spectrum confirms the purity and structure of LG-3.

EXAMPLE 8 - 1,2: 5, 6-DI-0-ISOPROPILIDEN-3-0- (5'-HYDROXYCARBONYL) -PENTIL-ALPHA-D-GLUCOFURANOSA (LG-5) Figure 3 also shows an example synthesis of 1: 2: 5-6-Di-0-isopropylidene-3-O- (5'-hydroxycarbonyl) -pentyl-alpha-D-GLUCOFURANOSA (LG-5) using the following Reagents: LG-3; Py2H2Cr207 (pyridinium dichromate) = PDCh; KMn04 and acetone. In this procedure, a mixture of LG-3 (1.0 g, 2.77 mM), PDCh (2.13 g, 5.65 mM), KMn04 (0.89 g, 5.65 mM) and acetone (50 ml) was stirred at 50 ° C until the reaction was finished (7-8 hours). Upon inspecting the reaction by TLC, almost complete transformation of the starting LG-3 to the intermediate aldehyde (LG-4) was observed after only 5 minutes (in a separate experiment LG-4 was isolated and its purity and structure was tested by the corresponding XH NMR spectrum). After the oxidation to LG-5 acid was finished, the reaction mixture was filtered and the filtrate was evaporated. The product was purified by flash chromatography (TLC solvent system). LG-5 (oil); Yield: 700 mg (67%); TLC: ethyl acetate / hexane (1: 1); Rf: 0.23; 1K NMR spectrum (CDC13) confirms the purity and structure of LG-5.

EXAMPLE 9-1.2: 5, 6-DI-0-ISOPROPYLIDEN-3-0- (6'-P-TOLUENSULFONYLOXY) -HEXIL-ALPHA-D-GLUCOFURANOSA (LG-6) Figure 3 also shows an example synthesis of 1, 2: 5, 6-di-0-isopropylidene-3-0 ^ (6'-p-toluenesulfonyloxy) -hexyl-alpha-D-glucofuranose (LG-6) using the following reagents: LG-3, TsCl; Py, and CH2C12. In this procedure LG-3 (1.40 g, 3.884 mM) was dissolved in a mixture of pyridine (5 ml) and CH2C12, (10 ml). The reaction mixture was cooled to 0 ° C and TsCl (0.89 g, 4.668 mM) was added. The reaction mixture was maintained for 5 hours at 4 ° C and then neutralized with 10% aqueous HCl. The organic layer was washed with brine, dried and evaporated. The product was purified by column chromatography (TLC solvent system). LG-6 (oil); Yield: 1.8 g (90%); TLC: 33% ethyl acetate, 67% hexane, the spectrum XH NMR (CDC13) confirms the purity and structure of LG-6.

EXAMPLE 10 - CONJUGATES OF TIMIDINE WITH LG-5 (VIA AN UNION OF ESTER).

Figure 4 shows an example synthesis of the 3'-O- and 5'-0-thymidine esters with LG-5 using the following reagents: thymidine; LG-5; dicyclohexylcarbodiimide = DCC; and dimethylamino pyridine = DMAP. In this procedure a mixture of thymidine (100 mg, 0.413 mM), LG-5 (233 mg, 0.620 mM), DCC (128 mg, 0.620 mM), DMAP (15.1 mg, 0.124 mM) and Py (3 ml) were maintained at 60 ° C during the night which was enough for the reaction to go to completion. The Py was evaporated in vacuo and the residual material was chromatographed on a column of silica gel (TLC solvent system). 5'-0- and 3'-0-thymidine esters (oils); yield: 68.7 mg (28%) and 59.0 mg (24%). TLC: 25% hexane, 75% ethyl acetate; Rf. (5): 0.40 and Rf (3): 0.25. NMR spectra: H (CDC13) of both isomers are in agreement with the proposed structures.

EXAMPLE 11 - DES-I? OPROPILIDENATION (DESPROTECTION) OF SEPARATED TIMIDINE ESTERS Figure 4 also shows an exemplary de-isopropylidenation (deprotection) of separate thymidine esters using trifluoroacetic acid = TFA. In this procedure, the separate (individual) thymidine ester (from a previous experiment), 30 mg, 0.05 mM, with TFA / water (9/1, v / v) was treated at room temperature for 5 minutes. The reaction mixture was then evaporated in vacuo, redissolved in 1 ml of water, filtered and evaporated once more. Unprotected esters (oil); Yields: 81-89%; TLC: 10% methanol, 2% acetic acid, 88% ethyl acetate; Rf. 0.17 (5'-0H free) and 0.20 (3 '-OH free). The H-NMR spectra are in agreement with the proposed structures.

EXAMPLE 12 - CONJUGATION OF VIRAZOL WITH LG-5 (VIA AN UNION OF ESTER) Figure 5 shows an example synthesis of 2, 3-O-Isopropylidene-virazole (V-1) using the following reagents: Virazole ™ (trade name of ICN for Ribavirin USP); acetone; 2, 2-dimethoxypropane = DMP; perchloric acid (70%). In this procedure, Virazole ™ (3.0 g, 12.28 mM) was dispersed in a mixture of acetone (40 ml) and DMP (20 ml). This mixture was cooled in an ice bath and perchloric acid (600 μl) was added. The mixture was kept at room temperature for 3 hours and then at 5 ° C overnight. The resulting orange solution was neutralized with 2M aqueous KOH, filtered and evaporated to dryness. The solid residue was treated with methanol (5 ml) and the insoluble product was removed by filtration. The methanolic filtrate was evaporated to dryness and the solid residue was recrystallized from acetone. Yield: 2.95 g (81%); TLC: 15% methanol, 85% ethyl acetate; Rf: 0.54. The H-NMR spectrum (acetone-d6) confirms the purity and structure of V-1.

EXAMPLE 13-2 ', 3'-O-ISOPROPILIDEN-VIRAZOL-5' -0-ESTER.

Figure 5 also shows an example synthesis of 2'-3 '-0-Isopropylidene-virazole-5'-O-ester with LG-5 (Vl-5' -O-LG-5) using the following reagents: Vl; LG-5; DCC; DMAP; Pyridine In this procedure, a mixture of Vl (236 mg, 0.796 mM), LG-5 (200 mg, 0.531 mM), DCC (108 mg, 0.531 mM), DMAP (13 mg, 0.106 mM) and Py (5 ml) it was maintained at 60 ° C overnight. The pyridine was removed in vacuo and the residue was purified by column chromatography on silica gel. Yield: 160 mg (46%) TLC: 25% hexane, 75% ethyl acetate; RF: 0.21. The XH NMR spectrum (acetone-d6) confirms the purity and structure of V-l-5'-O-LG-5.

EXAMPLE 14 - DEPROTECTION OF V-l-5 '-O-LG-5 AND SYNTHESIS OF V-5'-0-LG-7 Figure 5 also shows an exemplary deprotection of Vl-5 '-O-LG-5 and the synthesis of V-5'-0-LG-7 using the VirazoleMR ester from TFA of the previous experiment (V-1 -ester). In this procedure, the V-1 ester (50 mg, 0.076 mM) was treated with TFA / water (9/1, v / v) at room temperature for 5 minutes. The reaction mixture was evaporated to dryness, redissolved in 1 ml of water, filtered and evaporated once more. Yield: 35 mg (87%); TLC: 10% methanol, 2% acetic acid, 88% ethyl acetate. Rf. 0.19. the 2H NMR spectrum (D20) confirms the purity and structure of V-5'-0-LG-7.

EXAMPLE 15 - CONJUGATION OF VIRAZOLEM WITH LG-6 (VIA UNIÓN DE ESTER) Figure 6 shows an example synthesis of V-l-ETH-5 '-O-: LG-8 and LG-9 using the following reagents: V-l; LG-6; NaH; and DMSO. In this procedure, V-1 (250 mg, 0.843 mM) was dissolved in anhydrous DMSO (5 ml) and NaH (118 mg, 2.95 mM) was added in small portions over 30 minutes. The reaction mixture was heated for the next 30 minutes to 40 ° C and then LG-6 was added. The mixture was maintained at • room temperature overnight and then diluted with 100 ml of ethyl acetate. The solution was washed with brine (3 x 30 ml), dried (Na 2 SO 4), evaporated and purified by column chromatography on silica gel. Yield: 220 mg (49%); TLC: 10% hexane, 90% ethyl acetate; Rf: 0.53; the 1E NMR spectrum (acetone-d6) supports the proposed structure.

EXAMPLE 16 - CONJUGATION OF VIRAZOLEMR WITH LG-6 (VIA A UNION OF ESTER) In another example synthesis, the previous compound (50 mg, 0.078 mM) was treated with TFA / water (9/1, v / v) at room temperature for 5 minutes. The The reaction mixture was evaporated to dryness, redissolved in 1 ml of water, filtered and evaporated again. The yield of V-ETH-5 '-O-LG-: 36 MG (88%); TLC: 10% methanol, 2% acetic acid, 88% ethyl acetate.

EXAMPLE 17 - SYNTHESIS OF THE INTERMEDIATE COMPOUNDS OF 6-O-D-GALACTOSA-LINKER Figure 7 shows an example synthesis of 1,2: 3, 4-Di-0-isopropylidene-6-0- (6'-tetrahydropyranyloxy) -hexyl-alpha-D-galactopyranose (LGa-2) using the following reagents: L-3; 1,2: 3,4-Di-O-isopropylidene-alpha-D-galactopyranose = DIPGap; NaH; and DMSO. In this procedure, DIPGap (500 mg, 1.92 mM) was dissolved in anhydrous DMSO (5 ml) and NaH (96 mg, 2.40 mM) was added in small portions at room temperature. The reaction mixture was heated at 40 ° C for 0.5 hours and L-3 (660 mg, 2.11 mM) was added at room temperature. The mixture was kept at room temperature overnight and then poured into 100 ml of ethyl acetate. The organic layer was washed with brine (3 x 25 ml), dried (Na2SO4) and evaporated. The product was purified by flash chromatography on silica (solvent system: 10% ethyl acetate). %, 2.5% triethylamine, 87.5% hexane). Yield: 500 mg (62%); TLC: 15% ethyl acetate, 2.5% triethylamine, 82.5% hexane; Rf: 0.44. The XH NMR spectrum (CDC13) confirms the purity and structure of Lga-2. LGa-1 was obtained in a similar procedure as LG-3 (See Figure 3).

EXAMPLE 18 - SYNTHESIS OF THE INTERMEDIATE COMPOUNDS OF 4-0- AND 6-O-D-MANOSA-LINKER Figure 8 shows the synthesis of the intermediate compounds of 4-0- and 6-0-D-mannose-linker. The synthesis is carried out using chemistry similar to that previously described for galactose and glucose.

EXAMPLE 19 - SYNTHESIS OF THE INTERMEDIATE COMPOUNDS OF 3-0-, 4-0- AND 5-O-D-FRUCTOSA LINKER Figure 9 shows the synthesis of the intermediate compounds of 4-0- and 6-0-D-mannose-linker. The synthesis is carried out using chemistry similar to that previously described for galactose and glucose.

EXAMPLE 20 - INTERMEDIATE GLUCOSE-LINKER PHOSPHORAMIDATE COMPOUNDS FOR AUTOMATIC SYNTHESIS OF OLIGODESOXINUCLEÓTIDOS (ODN) Figure 10 shows an example synthesis of LG-3, but using the following reagents: LG-3; benzyl bromide = BnBr; Sodium hydride = NaH; and tetrahydrofuran = THF. In this procedure, a mixture of LG-3 (1.00 g, 2.77 mM) and NaH (150 mg, 3.75 M) in THF (10 ml) was stirred at 40 ° C for 1 hour followed by the addition of BnBr (1.3 ml). , 11.08 mM). The reaction mixture was further stirred at 40 ° C until the reaction was finished (6 hours). Then ethyl acetate (100 ml) was added and the obtained solution was washed with brine (3 x 50 ml). The organic layer was dried (Na2SO) and evaporated. The product was purified by flash chromatography on silica gel (TLC solvent system). Yield: 1.25 g (94%); TLC: 15% ethyl acetate, 85% hexane; Rf: 0.45. The NMR spectrum * H (acetone-d6) is in agreement with the composite structure.

EXAMPLE 21 - LG-3BnpTA Figure 10 also shows an example synthesis of LG-3BnpTA using the following reagents: LG-3 Bnf; trifluoroacetic acid = TFA; Acetic anhydride - Ac20; and Pyridine = Py. In this procedure the LG-3Bnf (1.00 g, 2.22 mM) was treated with a mixture of 4 ml of TFA / water (9: 1, v / v) at room temperature for 7 minutes. The reaction mixture was evaporated in vacuo to dryness, a mixture of Ac20 / Py (10 ml, 1: 1, v / v) and left at temperature • atmosphere during the night. After the neutralization (Aqueous 5% HCl, 50 ml) The product was extracted with ethyl acetate (3 x 50 ml). The organic layer was washed with brine (3 x 50 ml), and dried (Na 2 SO 4) and evaporated. The product was purified by flash chromatography on a silica gel column (TLC solvent system). Yield: 1.15 g (96%); TLC: 33% ethyl acetate, 67% hexane; Rf: 0.44. the H-NMR spectrum (acetone-d6) confirms the purity and structure of LG-3BnpTA.

EXAMPLE 22 - LG-3pTA Figure 10 also shows an example synthesis of LG-3pTA using the following reagents. LG-3BnpTA; Pd at 10% / C; in methanol. In this procedure, the LG-3BnpTA (1.00 g, 1.86 mM) was dissolved in methanol (50 ml) and then added to the catalyst (Pd / C, 200 mg). The reaction mixture was stirred under an atmosphere of H2 until the reaction was finished (18-24 hours). The catalyst was then removed by filtration and the methanolic solution was evaporated to dryness. The product was purified by flash chromatography on silica. Yield: 815 mg (98.1%); TLC: ethyl acetate / hexane (1/1), Rf: 0.21; the XH NMR spectrum (CDC13) is in agreement with the composite structure.

EXAMPLE 23 - LG-3pTAPA Figure 10 also shows an example synthesis of the phosphoramidate intermediate, LG-3pTAPA, using the following reagents: LG-3pTA, chloro-cyanoethyloxy-diisopropylamino-phosphine = PAP (Phosphoramidate Precursor); diisopropylethylamine = DIPEA; and CH2C12. In this procedure, LG-3pTA (300 mg, 0.669 mM) was dissolved in CH2C12, (3 ml) under argon. The resulting solution was cooled to 0 ° C and DIPEA (450 μL, 2.62 mM) was added. After 15 minutes PAP was also added (315 μl, 1,408 mM, 0 ° C). The reaction mixture obtained was kept at room temperature for the next two hours, then cooled (0 ° C), diluted with CH2C12 (100 ml), washed with NaHCO3, aqueous, dried (Na2SO4) and evaporated in vacuo. empty. The crude product was purified by flash chromatography on silica. The pure product was kept under argon before its use in an automatic synthesis of oligodeoxynucleotides. For the automatic synthesis of ODN, LG-3pTAPA was used as a 0.1 M solution in anhydrous acetonitrile. Yield: 257 mg (62.2%); TLC: 32% ethyl acetate, 5% triethylamine, 63% hexane; Rf: 0.53. The 31P NMR spectrum was in agreement with the proposed structure.

EXAMPLE 24 - PHOSPHOROAMIDATES OF THE DERIVATIZED LINKER FOR THE AUTOMATIC SYNTHESIS OF OLIGODESOXINUCLEÓTIDOS.

Figure 11 shows an example synthesis of L-4 using the following reagents: 1,6-hexanediol = HD; and dimethoxytrityl chloride = DMT-C1. In this procedure, the HD (4.00 g, 29.52 mM) was dissolved in a mixture of Py / CH2C12 (1: 1, v / v) (8.0 ml). The resulting solution was cooled to 0 ° C and DMT-C1 (2.50 g, 7.38 mM) was added. The obtained reaction mixture was kept at 4 ° C for 6 hours. The reaction solution was then carefully neutralized (pH8) with 5% HCl, aqueous, and the product was extracted with CH2C12 (3 x 50 ml). The organic layer was washed with brine (3 x 30 ml), dried (Na2SO4) and evaporated. The product was purified by flash chromatography on silica. Yield: 2.53 g (81.5%); TLC; 32% ethyl acetate, 3% triethylamine, 65% hexane; Rf: 0.38; The XH NMR spectrum is in accordance with the proposed structure.

EXAMPLE 25 - L-5 Figure 11 also shows an example synthesis of L-5 using the following reagents: L-4; PAP; and DIPEA. In this procedure, L-4 (500 mg, 1.19 mM) was dissolved in CH2C12 (5 ml) under argon. The resulting solution was cooled to 0 ° C and DIPEA (800 μl, 4.65 mM) was added. After 15 minutes PAP was also added (550 μl, 2.46 mM). The obtained reaction mixture was kept at room temperature for the next two hours, then cooled (0 ° C), diluted with cold CH2C12 (100 ml), washed with cold 5% aqueous NaHCO3, dried (Na2SO4, NaHCO3) and evaporated in vacuo. The crude product was purified by flash chromatography on silica. The pure product (colorless oil) was kept in argon at -18 ° C before use. L-5 of phosphoroamidate was used as a 0.1 M solution in anhydrous acetonitrile. Yield: 430 mg (58.3%); TLC: 16% ethyl acetate, 4% triethylamine, 80% hexane; Rf: 0.37. The XH and 31P NMR spectra were in agreement with the proposed structure.

EXAMPLE 26 - L-6 In Figure 12, the first row shows an example synthesis of 1,6-O-p-ditoluesulfonyl-l, 6-hexanediol (L-6) using the following reagents: 1,6-hexanediol = 1,6-HD; p-toluenesulfonyl chloride = TsCl; Pyridine = Py; and dichloromethane = CH2C12. In that procedure, 6-HD (70.9 g, 0.60 mol) was dissolved in 150 ml of Py and 250 ml of CH2C12. The resulting solution was cooled to 0 ° C and TsCl (251.65 g, 1.32 mol) was added. The obtained reaction mixture was kept at 4 ° C for 5 hours. The reaction solution was then neutralized with aqueous 10% HCl and the product was extracted by CH2C12 (3 x 250 ml). The organic layer is washed with brine, dried and evaporated. The crude product was dispersed in ethanol (300 ml), filtered and washed with cold ethanol. L-6 (white crystals); Yield: 219.1 g (85.6%). TLC: ethyl acetate / hexane (1: 2); Rf: 0.48. The structure and purity of L-6 were verified and confirmed by the XH NMR spectrum and high resolution mass spectroscopy of FAB.

EXAMPLES 27 AND 28 - ALTERNATIVE SYNTHESIS OF LG-6 and LG-3 Figure 12 in a second row also shows the alternative direct synthesis of LG-6 that can be performed using the following reagents: 1, 2: 5, 6-di-O-isopropylidene-a-D-glucofuranose = DIPGF, L-6; NaH; DMSO. In this procedure, DIPGF (11.68 g, 44.88 mM) was dissolved in anhydrous DMSO (50 ml) and NaH (2.334 g, 58.34 mM) was added in small portions at room temperature. The reaction mixture was maintained at 40 ° C for 30 minutes and L-6 (67.0 g, 157.0 mM) dissolved in 150 ml of hot DMSO (40 ° C) was added. After 30 minutes, the reaction mixture was poured into ethyl acetate (800 ml). The organic layer was washed with brine (3 x 50 ml), dried (Na 2 SO 4) and evaporated. The product was purified by chromatography on silica gel column (TLC solvent system). LG-6 (colorless oil); Yield: 16.0 g (69.0%). TLC: 33% ethyl acetate, 67% hexane; Rf: 0.53. The structure and purity of LG-6 were verified and confirmed by the XH-NMR spectrum and FAB high-resolution mass spectroscopy. In addition, Figure 12 shows an alternative synthesis of 1, 2: 5, 6-di-0-isopropylidene-3-0- (6'-hydroxy) -hexyl-aD-glucofuranose (LG-3) using the following reagents: LG-6; DMF; K2C03; H20 In this procedure, LG-6 (16.0 g, 31.03 mM) and K2C03 were dissolved in DMF (200 ml) and water (20 ml) and the reaction mixture was heated for 5 hours at 90 ° C. After removal of the solvent (evaporation under high vacuum), the residual material was dissolved in ethyl acetate (600 ml), washed with brine (3 x 200 ml), dried (Na 2 SO 4) and evaporated. The product was finally purified by flash chromatography on silica. LG-3 (colorless oil); Yield: 7.85 g (70.2%); TLC: ethyl acetate / hexane; Rf: 0.48; the structure and purity of LG-3 were verified and confirmed by the H-NMR spectrum and the high-resolution mass spectroscopy of FAB.

EXAMPLE 29 - SYNTHESIS OF MODIFIED OLIGODESOXINUCLEOTIDES (ODN) IN THE 3 'AND 5' POSITIONS AND THEIR PROPERTIES BIOPHYSICS.

Figure 13 shows an example synthesis of modified oligodeoxynucleotides (ODN) at the 3 'and 5' positions (ICN 16967) using an automated DNA synthesizer (Applied Biosystems, model 394) and the normal phosphoramidite chemistry; β-cyano-ethyl-phosphoramidites, synthesis reagents and CPG polystyrene columns (1 μM and 10 μM T, pore size 500 A, ABI). After the synthesis, the oligonucleotide was deprotected with concentrated ammonium hydroxide for 8 hours at 55 ° C. Crude and final analysis was carried out using the C8 HPLC column (Beckman, octyl in ultra-sphere). Purification was done on an inverted phase semi-prep C8 HPLC column (Beckman C8 ODS ultrasphere, 5 μM, 10 mm x 25 cm) with a flow rate of 3 ml / min using 0.1 M TEAA and 5% acetonitrile . After purification and conditioning, the crude product was precipitated with 2-propanol, dissolved in water and lyophilized. Yield: 2.23 mg (36% for 1 μmol synthesis). The structure and purity of ICN 16967 was verified and confirmed by HPLC analysis, capillary electrophoresis and mass spectroscopy by electroroad.

BIOPHYSICAL PROPERTIES OF ICN 16967 The hybridization properties of these 3 ', 5' -modified oligonucleotides were studied by thermodynamic fusion (Tm) experiments. (See methods in Fuglisi, 3. D., Tinoco, I. Jr. Methods EnzymoL 1989, 180, 304). The experiments were carried out on a Varian UV spectrometer equipped with an electronic temperature controller and a Cary hybridization program. As shown by Figure 14 (a), the modified sequence exhibits almost identical hybridization to the complementary RNA as compared to the unmodified sequence (b). Samples for Tm measurements contain 2 μM of modified oligos and 2.0 μM of complementary RNA in a buffer (10 mM sodium phosphate, 0.1 M EDTA, and 0.1 M sodium chloride, pH = 7.0). The stability and half-lives of the modified oligonucleotide (Figure 14, c) and unmodified oligonucleotide (d) were calculated from UV absorbance curves of oligonucleotide samples during degradation by phosphodiesterase of venom. snake at 37 ° C using a similar procedure as described by Wenel et al. (See methods in Svendsen, M. L .; Wengel, 3; Dahl, 0; Kirpekar, F .; Roepstorff, P. Tetrahedron 1993, 49, 11341). Oligonucleotides (0.75 OD) were incubated with snake venom phosphodiesterase (1.2 units) in 1.5 ml of buffer (0.1 M Tris-HCl, pH 7.5, 0.1 M NaCl, 14 mM MgCl2) in a specimen on a UV Varian spectrometer. 25 ° C, and the increase in absorbance at 260 nm was recorded during degradation against time. From these absorbance curves, the half-lives of the oligonucleotides were calculated. Under these conditions, the oligonucleotide (glycoside) ICN 16976 was approximately 20 times more stable than the corresponding unmodified oligonucleotide.

EVIDENCE OF INCREASED BIO-AVAILABILITY The bioavailability of an example glycoside, tritiated thymidine with 5'-0-glucose (D-G-L-Thy) was tested by comparing the incorporation of free thymidine with the incorporation of D-G-L-Thy in a simple in vitro assay. In that assay, cells from lung cancer cell line 177 (non-small cell lung carcinoma, human) were used that grow in a medium supplemented with 10% fetal calf serum (SSM) and in a serum-free medium (SFM, for its acronym in English). The cells were treated with free thymidine and with conjugated thymidine (D-G-L-Thy) for 4 and 24 hours. The D-G-L- * Thy was synthesized using the isotope dilution technique (1 part of hot Thy: 100 parts of cold Thy). The concentrated solution was prepared having an average value of cpm: Thy: 481.660 and 5'-0-conjugate: 467.242 (CORRELATION FACTOR: 1.03). The following results were obtained (average values).

Under all conditions, the thymidine incorporation released from the D-G-L-Thy compound was improved over the control. These data indicate that thymidine can be released from the D-G-L-Thy complex in cells due to the presence of phosphodiesterases, and that the thymidine released could easily be incorporated into the cell.

DNA It is more unlikely that thymidine has been enzymatically released from the compound extracellularly because the medium is free of phosphodiesterases. Thus, the present data show that the compound glycoside D-G-L-Thy is taken up by the cells as a prodrug, and is changed intracellularly to free thymidine and free glucose. Since the absorption together with the hydrolysis of the prodrug, parallel and subsequent (outside and inside the cells) is kinetically complex, the experiment, at this stage, can be interpreted simply as follows: • Absorption of the glycoside is very high, and more likely much higher than the absorption of thymidine itself. • The intracellular hydrolysis of the prodrug is fast enough to release a sufficient amount of the free drug intracellularly. • This represents a good sign for a possible absorption of the glycoside via the GLUT system.

EVIDENCE OF INCREASED BIO-AVAILABILITY Human melanoma cells ATTC HTB140 (HS294T) were cultured in DMEM supplemented with 10% bovine serum (culture medium) for the experiment, these cells were trypsinized and collected in suspension in the culture medium. After plating in 96-well plates at a density of approximately 10,000 cells / well / 200 μl of the culture medium, the cells were treated with 0.5 μM of oligonucleotides for 72 hours. After that time, the cells were counted following the trypsinization using a haemocytometer. Each count was repeated three times for each experiment. The list below is calculated as the average inhibition of cell growth against untreated cells (control) and is based on three independent experiments.

Oligonucleotide $ of Growth Deviation Standard of control Unchanged 64.59 18.80 Modified 54.32 11.10 Experiments show that the unmodified oligonucleotide is effective at concentrations of 0.5 μM for 72 hours of cell growth, and that the modified oligonucleotide is more effective than the unmodified oligonucleotide at the same concentration and experimental conditions.

TREATMENT USING GLINCÓSIDOS The glycosides can be used to improve the absorption, distribution and cellular absorption of the pharmaceutical formulations both existing and those yet to be developed. For example, the glycosides can be used for the control and in vitro treatment, in vivo, and ex vivo of the pathological and senescent cell growth, for the induction / prevention of apoptosis, for the induction / inhibition of cell differentiation, for the control of cellular metabolism and behavior, for the propagation and activity of viruses in infected cells in operation and cells infected by viruses, and for the treatment of neurodegenerative diseases. The invention has particular relevance for transporting several monosaccharides and specifically for the functioning of glucose transporters expressed in various tissues in cells. The Glucose transporters are directly involved in the transport of several monosaccharides in cells (glucose, mannose, galactose and others). Some of these glucose transporters are characteristic of certain tissues (for example) or cell type (for example), or biological functioning (for example, normal and rapidly growing cancer cells by GLUT-1). The fact that different types of glucose transporters are differentially expressed in different cells offers a possibility of absorption or specific distribution in the cells of the therapeutically conjugated bioactive compounds with saccharide are described herein. 1. Glucose transporters in fast-growing cells. Normal cells that do not grow (quiescent) activated by growth factors enter the cell cycle and eventually divide. Growth factors cause the cascade of events observed in the gene, MRNA, protein expression, and the level of cell structure organization. For example, the short term (0.5-1 hour) expression of GLUT-1 mRNA and the protein has been observed in the activated cells of growth factors (Endocrinology, 1990, 127, 2025). Sodium vanadate which is known as a specific growth stimulant has been able to induce the expression of GLUT-1 in normal 3T3 fibroblasts and also extend the half-life of GLUT-1 mRNA by 2 to 3 times (Endocrinology, 1990, 126, 2778). An autocrine mechanism of uncontrolled proliferation of cancer cells has been well established. The cancer cell grows rapidly and expresses a very high level of GLUT-1. In this way, cancer cells absorb much more glucose than normal cells and use it for glycolysis. I also know • has proven that the high level of functional GLUT-1 protein expression is required for cancer growth. Any inhibition of the functioning of GLUT-1 in cancer cells caused by the binding of specific antibodies to GLUT-1 or by the binding of suramin significantly limits the absorption of glucose and the growth rate of the treated cells. These data indicate the need for high glucose uptake to maintain a high proliferation rate of cancer cells, specifically mediated by GLUT dependent on the growth factor. In this way, cancer cells of the breast, prostate, lung, glioma and others show significantly increased levels of GLUT. 2. Glucose transporters in the blood barrier of the brain. In order to cross the blood-brain barrier (BBB), drugs must penetrate the luminal and basal membranes of endothelial cells. This is possible by taking advantage of receptor-mediated absorption, such as, for example, in the case of the drug L-Dopa anti-Parkinson. Dopamine itself can not penetrate the blood barrier of the brain. However, its natural precursor L-Dopa is transported through the blood barrier of the brain via an amino acid carrier system. Antisense oligonucleotides are highly charged molecules and exhibit very little transport through the capillary walls of the brain. By incorporating the oligonucleotides into the glycosides, as described herein, the transport of these potentially valuable anti-sense drugs should be greatly facilitated by absorption via the glucose transporters of the endothelial cells constituting the BBB. The transport of glucose from the blood in the neural and equal cells of the brain was measured by two specific transporters located in the capillary endothelium of the brain and constitutes the BBB (M.

Brightman 1977, "Morphology of Blood-brain Interfaces" Exp. Eye Res. 25, 1-25). These two glucose transporters were identified as two different members of the family of sodium-independent glucose transporter super-genes. While the type 3 isoform of glucose transporter (GLUT-3) is located in the neuronal cell membrane (S. Naga Atsu et al., J. Biol. Chem. 267, 467-472), isoform type 1 glucose transporter (GLUT-1) is located in the BBB (RJ Boado and WM Partridge, Biochem. Biophys., Res. Commun. 166, 174-179 (1990)). A glincoside may be able to penetrate the BBB due to the abundance of GLU-1 transporter systems. This would greatly improve the potential applications of anti-sense oligonucleotides for neurodegenerative diseases of the CNS, such as, for example, Parkinson's disease and Alzheimer's disease. The drug could be administered orally or by intravenous injection, and then distributed via the bloodstream to the brain, or alternatively it could be injected into the carotid artery to improve local distribution to the brain. 3. Other objectives that are particularly suitable for the application of glycosides due to the presence of specific glucose transporters, They include neural cells, liver, intestine, adipose tissue, muscle and senescent cells.

PHARMACEUTICAL FORMULATIONS USING GLINCOIDS As with other pharmaceutical formulations, the glycosides can be adapted for administration to the body in a number of suitable ways by the selected method of administration, which include orally, intravenously, intramuscularly, intraperitoneally, topically, and the like. In addition to comprising one or more different glycosides, the pharmaceutical formulations present may comprise one or more biologically inactive compounds, i.e., excipients, such as stabilizers (to promote long-term storage), emulsifiers, binding agents, thickening agents, salts, preservatives, and the like. The glycosides can be employed in doses and amounts that are conventional in the art for the underlying bioactive compound, but are adjusted for more efficient absorption, transport and cellular uptake. In this way, for Ribavirin, which can be given orally at 1200 mg / day, the dose of the glycoside corresponding will include approximately 600 mg / day of Ribavirin, or less. The doses can be administered all at once, or they can be divided into a number of small doses that are then administered at varying intervals of time. The dose regimen can be adapted to provide the optical therapeutic response. For example, the most preferred dose will vary with the particular agent chosen, and during the course of administration, the dose may be increased proportionally or reduced as indicated by the exigencies of the therapeutic situation. The glycosides can be administered in any convenient manner, such as by the oral, intravenous, intraperitoneal, intramuscular or subcutaneous routes or other known routes. For oral administration, the glycosides can be administered in an inert diluent or with an edible assimilable carrier, the glycosides can be incorporated directly with the diet food. Orally administered glycosides can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspension syrups, wafers, and the like.

The tablets, pills, pills, capsules and the like may contain the following: a binder, such as gum tragacanth, gum arabic, corn starch or gelatin; excipients, such as dicalcium phosphate, a disintegrating agent such as corn starch, potato starch, sodium acid and the like; a lubricant such as magnesium extrude; and flavoring agents, such as sucrose, lactose and saccharin; a flavoring agent such as peppermint, oil of wintergreen or cherry flavor. When the dosage unit is a capsule, it may contain, in addition to the materials of the type • previous, a liquid carrier. Various other materials may also be present as coatings, or to otherwise modify the physical form of the dose unit. For example, tablets, pills or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain sucrose as a flavoring agent, methyl and propylparabens as preservatives, a dye and flavor such as cherry or natural flavor. These additional materials must be substantially non-toxic in the amounts employed. Additionally, the glycosides can be incorporated into preparations, formulations and sustained vibration.

Formulations for parenteral administration may include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile, injectable solutions or dispersions. The solutions or dispersions may also contain buffers, diluents and other suitable additives, and may be designed to promote cellular uptake of the glycosides in the composition, for example, the glycosides may be encapsulated in suitable liposomes. Preferably, the solutions and dispersions for parenteral administration are sterile and sufficiently fluid for proper administration, sufficiently stable from manufacture and use, and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with one or more of the various other ingredients described above, followed by sterilization. Dispersions are generally prepared by incorporating the various sterile active ingredients into the sterile vehicle containing the basic expression medium and the other ingredients required from those of the above list. In the case of sterile powders used to prepare sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques which produce a powder of active ingredient plus any desired ingredient, additional to previously sterile filtered solutions. Pharmaceutical formulations for topical administration may be especially useful with certain bioactive compounds for localized treatment. Formulations for topical treatment include ointments, sprays, gels, suspensions, lotions, creams and the like. Formulations for topical administration may include, in addition to the glycosides present, known carrier materials such as isopropanol, glycerol, paraffin, stearyl alcohol, polyethylene glycol, etc. The pharmaceutically acceptable carrier can also include a known chemical absorption promoter. Examples of absorption promoters are, for example, dimethylacetamide (U.S. Patent No. 3,472,931), trichloro-ethanol or trifluoroethanol (U.S. Patent No. 3,891,757), certain alcohols and mixtures thereof (British Patent No. 1,001,949), and British Patent Specification No. 1,464,975. The solutions of the glycosides can be stored and / or administered as free base or pharmacologically acceptable salts, and can be advantageously prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. These compositions and preparations may advantageously contain a preservative to prevent the growth of micro-organisms. The prevention of the action of microorganisms can be caused by various antibacterial and anti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isothermal agents such as sodium chloride. The prolonged distribution of injectable compositions can be caused by the use of agents that delay absorption, such as aluminum monostearate and gelatin. The compositions and preparations described preferably contain at least 0.1% active glycoside. The percentage of the compositions and preparations can be varied, of course, and may contain between about 2% and 60% of the weight of the amount administered. The amount of the active compounds in these therapeutically useful compositions and preparations is such that an adequate dose will be obtained. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating, antibacterial and antifungal agents, isotonic and delaying absorption agents, and the like. The use of these agent media for active, pharmaceutical substances is well known in the art. Except as regards any conventional medium or agent that is incompatible with the therapeutic active ingredients, its use in the compositions and therapeutic preparations is contemplated. The Complementary active ingredients can also be incorporated into the compositions and preparations. In addition to the therapeutic uses of the glycosides present, the glycosides can also be used as a laboratory tool for the study of absorption, distribution, cellular absorption and efficiency.

EQUIVALENTS The above-described specification is considered to be sufficient to enable one skilled in the art to practice the invention. In fact, the various modifications of the forms described above to carry out the invention that are obvious to those skilled in the field of organic clinical or related fields are proposed to be within the scope of the following claims: It is noted that with respect to this date, the best method - known by the applicant to carry out the present invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (15)

1. A compound having the general formula MS-LINK-BAM, characterized in that MS is a monosaccharide, LINK is a linker, BAM is a bioactive material, MS is covalently linked to LINK at a position different from the Ci carbon atom of MS, and LINK joins covalently to BAM.

2. The compound according to claim 1, characterized in that MS comprises a hexose.

3. The compound according to claim 1, characterized in that MS comprises a ribose.

4. The compound according to claim 1, characterized in that MS comprises a pentose.

5. The compound according to claim 1, characterized in that LINK comprises a straight chain alkane having between 4 and 16 carbon atoms, inclusive.

6. The compound according to claim 1, characterized in that BAM comprises Ribavirin.

7. The compound according to claim 1, characterized in that the MS-LINK junction is differentially cleavable or segmentable from the LINK-BAM binding.

8. The compound according to claim 7, characterized in that the MS-LINK binding is stronger than the LINK-BAM binding under physiological conditions.

9. A method for synthesizing a glycoside, characterized in that it comprises the following steps: providing a monosaccharide, a linker having an α and an α position, and a bioactive material; protect the position? of the linker; form a non-glycosidic chemical bond between the monosaccharide and the a-position of the linker; Check out a functionality in the position? of the linker; form a chemical bond between the position? of the linker and the bioactive material.

10. A method for increasing the bioavailability of a bioactive material by covalently attaching the bioactive material to a linker, and covalently linking the linker to a sugar at one of the C2 carbon atoms. C3, C4 and C6 of sugar.

11. A method for treating a disease of a patient, characterized in that it comprises the steps of: providing a glycoside in a pharmaceutically acceptable form; administer an effective amount of the glycoside to the patient.

12. A compound having the general form MS-LINK-BAM, characterized in that MS is a monosaccharide, LINK is a linker, BAM is a bioactive material, MS is covalently linked to LINK at a position different from the Ci carbon atom of MS, and LINK binds covalently to BAM, where the compound is transported more actively through a target cell membrane than BAM alone.

13. The compound according to claim 11, characterized in that BAM can be easily cleaved by the target cell.

14. The compound according to claim 11, characterized in that BAM can not be easily cleaved by the target cell.

15. The compound according to claim 2, characterized in that the hexose is glucose.