PROCESS FOR THE PREPARATION OF GANGLIOSIDE GM3 BY ACID HYDROLYSIS OF GANGLIOSIDE INNER ESTERS AND ITS USE IN THE PHARMACEUTICAL FIELD

Abstract: The present invention describes a process for the preparation, by semisynthetic means, of the ganglioside GM3, alpha -Neu5Ac-(2-3)- beta -Gal-(1-4)- beta -Glc-(1-1')-Cer, and of its derivatives, starting with the ganglioside GM1, GD1a, GD1b, or GT1b, etc.

Description: PROCESS FOR THE PREPARATION OF GANGLIOSIDE GM3 BY ACID HYDROLYSIS OF GANGLIOSIDE INNER ESTERS AND ITS USE IN THE PHARMACEUTICAL FIELD SUBJECT OF THE INVENTION The present invention describes a process for the preparation, by semisynthetic means, of the ganglioside GM3, α-Neu5Ac-(2-3)-β-Gal-(l- 4)-β-Glc-(l-l ')-Cer, starting with the ganglioside GMl, GDla, Gdlb or GTlb etc.. The compounds thus obtained can be used to advantage in the preparation of pharmaceutical compositions for the treatment of pathologies correlated with the death of neuronal cells, such as stroke, traumatic insult and neurodegenerative diseases, in the fields of oncology and immunology. FIELD OF THE INVENTION The molecule of natural GM3 ganglioside is formed by a lipid component constituted by sphingosine and a fatty acid and by a saccharide component constituted by glucose, galactose and sialic acid. Sphingosine is generally constituted by a carbon chain formed by 18 or 20 atoms, while the fatty acid is generally constituted by a carbon chain formed by 12 to 24 atoms, possibly hydroxylated in 1. Sialic acid, or neuraminic acid, a fundamental element of the oligosaccharide component, may be constituted by the N-acetyl or N-glycol derivative. The nomenclature used to identify GMl (β-Gal-(l-3)-β-GalNAc-(l- 4)-[α-Neu5Ac-(2-3)]-β-Gal-(l-4)-β-Glc-(l-l ')-Cer) and gangliosides in general was Svennerholm's [J. Neurochem., 10, 613 (1963)] or that recommended by IUPAC-IUB [Lipids, 12, 455 (1977) and J. Biol. Chem., 257. 3347 (1982)]. The physiological and pharmacological role of gangliosides and their derivatives is amply described in the scientific and patent literature, and a particularly important role is played by ganglioside GM3. For example, ganglioside GM3 modulates specific membrane receptors by modification of proteinkinase activity of EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) receptors [Bremer et al., J. Biol. Chem. 259, 6818 (1984), Bremer et al., J. Biol. Chem. 261, 2434 (1986)]. It is also known that some gangliosides in particular are expressed in a specific manner during malign alterations, causing significant changes in the pattern of ganglioside expression. The fact that these differences can be immunologically recognised has stimulated considerable interest in these compounds as potential targets in immunotherapy for tumours, using monoclonal antibodies or by active immunotherapy with vaccines. GM3 is one of the gangliosides expressed from some tumoral forms. In particular, studies on immunisation with monoclonal antibodies have shown that the antigen associated with the tumour in general is not directly GM3, but its corresponding lactone [Harada et al., Jpn. J. Cancer Res. 81, 383 (1990); Nores et al., J. Immunol. 139. 379 (1991)]. Again, by using monoclonal antibodies it has been shown, in the case of anti-melanoma antibodies, that the lipid component of GM3, and the fatty acid in particular, plays a crucial role in the formation or maintenance of the antigen structure of the saccharide component of the molecule [Itonori et al., Glycoconjugate J. 6, 551 (1989)]. For the preparation of the vaccine, the gangliosides are conjugated with specific vectors so as to stimulate the antibody response [Livingston et al, J. Clin. Oncol. 12, 1036 (1994)]. Numerous in vitro studies have shown, moreover, GM3's ability to inhibit the proliferation of T cells by the inactivation of interleukin 2 [Parker et al., FEBS Lett. 170, 391 (1984); Robb, J. Immunol. 36, 971 (1986)]. It has also been reported that the administration of GM3 in rats which have received allografts has a positive effect on take rate by inhibiting the accumulation of CD4 + ' CD8 + cells and the macrophages of the allograft [Hachida et al., Transplant. Proc. 22, 1663 (1990)], likewise the administration of GM3 and immunosuppressor agents has a combined effect on the immune response in vivo [Matsuo and Okamoto, Life Sci. 57, 165 (1995)]. The greatest interest in gangliosides, however, stems from the neurological field, since there is consistent evidence that exogenously administered gangliosides have neuritogenic and/or neurotrophic properties, able to influence neuronal differentiation in vitro and functional neuronal maintenance or recovery in vivo [Ledeen, J. Neurosci. Res., 1 , 147 (1984)] These particular properties may also have evident clinical consequences, as demonstrated by the fact that numerous studies have been and are being carried out to assess the potential of gangliosides as therapeutic agents in the treatment of pathologies correlated with neuronal cell death, such as stroke, traumatic insults and neurodegenerative diseases [Schneider, CNS Drugs 3, 213 (1994); Nobile- Orazio et al., Drugs 47, 576 (1994): Geisler et al., Ann. Emerg. Med. 22, 1041 (1993)]. Studies performed in this field have shown that GM3 too, and its derivatives, share these properties with the other gangliosides [Cannella et al., New Trends Ganglioside Research, Fidia Research Series 14 . 379 (1988)]. The study of these diverse functions of GM3 is, however, limited by the difficulty of obtaining adequate quantities of pure, homogeneous preparations and, therefore, the preparation of its derivatives too is severely compromised, whereas these substances are necessary for a complete understanding of the role of this ganglioside. Generally, ganglioside GM3 is prepared by extraction from organic matrices such as erythrocytes [Yu and Ledeen, J. Lipid Res. 13, 680 (1972), Watanabe and Arao, J. Lipid Res. 22, 1020 (1981)], bovine brain [Sonnino et al., Chem. Phys. Lipids 52, 231 (1990)] or hybridoma cells [Heitmann et al., J. Chromatogr. B. 710. 1 (1988)]. These procedures are, however, extremely lengthy and complex because of the difficulty of isolating the compound in a pure form and separating the N- glycolineuraminic derivative from the N-acetylneuraminic derivative. Yields of this ganglioside are also very small because of its low concentrations in the biological material of origin. Another possibility of obtaining ganglioside GM3 is by its synthetic or semisynthetic preparation. Indeed, it is possibly to synthesise chemically both its lipid component [Devant, Kontakte (Merck) 11 (1992) and recently Dondoni et al., J. Chem. Soc. Perkin Trans. 1 , 2389 (1997)], and its saccharide component [Martin et al., Glycoconjugate J. 10, 16 (1993) and recently Martichonok and Whitesides, Carbohydr. Res. 302. 123 (1997)]. These synthetic methods too are very lengthy and complex as they are hampered not only by poor yields but also the specificity of the anomers of the bond between the saccharide components, with particular reference to α-sialyl glycoside. Another approach towards the preparation of ganglioside GM3 is represented by the possibility, theoretical at least, of hydrolysis of the more complex gangliosides such as GMl, GDla, GDlb, GTlb etc., that are more abundant in biological materials. This possibility is not practicable, however, because in the conditions of hydrolysis of glycoside bonds, pH lower than 3, the bond that is initially cleaved in a selective matter is the one between sialic acid and the saccharide (galactose) to which it is bound. DETAILED DESCRIPTION OF THE INVENTION The present invention describes a new process for the preparation of ganglioside GM3 starting from gangliosides with more complex structures. It has now been discovered, surprisingly, that complex gangliosides such as GMl , GDla, GDlb, GTlb etc, chemically modified in a suitable manner, can undergo selective hydrolysis of the bond between the galactose (II), to which the sialic acid is bound, and the vicinal N- acetylgalactosamine (III), without the ketose bond between sialic acid and galactose being hydrolysed at the same time. In this way, therefore, it is possible to obtain GM3 starting from a relatively abundant raw material such as that reported above, with high yields and anomeric purity characteristic of the substance of natural origin. Indeed, it has been observed that the esterification of sialic acid, and in particular the formation of an inner ester between the carboxyl of sialic acid and the hydroxyl in 2 of the vicinal galactose destabilises, surprisingly, the glycoside bond between the galactose (II) and the galactosamine (III), rendering the bond more labile that that between sialic acid and galactose (II), in certain reaction conditions. It is possible in this way to prepare adequate quantities of the ganglioside GM3 to be used both as a final product and as an intermediate product for the synthesis of derivatives such as those described, for example, in EP 0 328 420, or in patents No.s EP 0 373 039, EP 0 410 881, EP 0 410 883, EP 0 433 112, EP 0 688 330 and EP 0 688 331 by the Applicant, belonging to the groups of lysoderivatives and dilysoderivatives such as N-acyl, N'-acyl, N,N'-diacyl- derivatives of the N,N'-dilysoderivatives of GM3 and the N-acyl-derivatives of the N- lysoderivatives of GM3. The present invention is described hereafter by means of specific examples, but is not limited to the same. PREPARATION EXAMPLES Example 1 : Preparation of GMl inner ester Ten grams of GMl sodium salt is dissolved in 100 ml of anhydrous dimethylsulfoxide and transformed into acid form by passing the solution through a chromatographic column loaded with AG 50 resin (Bio-Rad, Richmond, USA) in H + form, with a ratio of resin/ganglioside of 4: 1, and eluting with dimethylsulfoxide. The elution mixture is treated directly with dicyclohexylcarbodiimide (1.42 gr per 10 gr of GMl) at room temperature for one hour under continuous agitation. The dicyclohexylurea that is formed during the reaction is removed by filtration and the solution is treated with acetone (500 ml per 10 gr of GMl) so as to obtain precipitation of the inner ester of GMl . The precipitate thus obtained is redissolved in a mixture of chloroform/isopropyl alcohol 1 : 1 (50 ml per 10 gr of starting GMl), reprecipitated with acetone and then vacuum-dried. Yield of the reaction: 9.7 gr of GMl inner ester. Example 2: Preparation of GDla inner ester Following the same procedure as described in Example 1, starting from ten grams of GDla sodium salt, using a resin/ganglioside ratio of 8: 1 , and 2.84 gr of dicyclohexylcarbodiimide per 10 gr of GDla, 9.6 gr of GDla inner ester are obtained. Example 3: Preparation of GDlb inner ester Following the same procedure as described in Example 1, but dissolving two grams of GDlb sodium salt in 20 ml of anhydrous dimethylsulfoxide, using a resin/ganglioside ratio of 8: 1, 0.57 gr of dicyclohexylcarbodiimide per 2 gr of GDlb, 100 ml of acetone per 2 gr of GDlb, and 10 ml of the mixture of chloroform/isopropyl alcohol 1 : 1 per 2 gr of starting GDlb, 1.7 gr of GDlb inner ester are obtained. Example 4: Preparation of GM3 from GMl inner ester A 1 -litre two-spouted flask connected to a cooler and fitted with a magnetic stirrer is heated to 70°C in a bath of oil. It is thoroughly treated with anhydrous nitrogen. Once the flow of nitrogen has been shut off, 10 gr of GMl inner ester is rapidly added and 2.8 ml of a 0.25M solution of sulfuric acid in anhydrous dimethylsulfoxide, treated with nitrogen and the temperature adjusted to 70°C. The GMl inner ester is vigorously stirred and dissolves in 5 minutes, after which the nitrogen flow is resumed and the reaction conducted at a temperature of 70°C for 30 minutes. The flask is then cooled by placing it in an ice bath and the solution is brought to pH 9 by the addition of IN NaOH. The solution is decanted into a 2-litre Erlenmeyer flask and the reaction products are precipitated by the addition of 1 litre of cold acetonitrile. The precipitate is separated on a Buchner filter in a cold chamber, dried then dialysed in dialysis bags against distilled water until the solution is clear. The clear solution is concentrated with a rotating evaporator, mixed with 20 gr of silica gel, after which 200 ml of chloroform/methanol 2: 1 is added. The solvent is evaporated with a rotating evaporator and the dry residue is loaded on a column (10x130 cm) containing 100 silica gel. It is eluted with a mixture of chloroform/methanol/water 60:35:5. The pure fractions are harvested, concentrated in a rotating evaporator, the concentrated product is precipitated in acetone and vacuum-dried. The product obtained is 3.5 gr of 98% GM3. Its structural characterisation is reported in Figure 1 (FAB-MS spectrum) and Figures 2 and 3 (ID 'H-NMR and 2D COSY Η-NMR spectra). Example 5: Preparation of GM3 from GDla inner ester Following the same procedure as described in Example 4, starting from 10 gr of GDla inner ester, 3.1 gr of 98% GM3 are obtained. Example 6: Preparation of GM3 from GDlb inner ester A 250-ml two-spouted flask connected to a cooler and fitted with a magnetic stirrer is heated to 70°C in an oil bath. It is thoroughly treated with anhydrous nitrogen. Once the flow of nitrogen has been cut off, 1 gr of GDlb inner ester is rapidly added with 0.28 ml of a 0.25M solution of sulfuric acid in anhydrous dimethylsulfoxide, treated with nitrogen and heated to 70°C. When vigorously stirred, the GDlb dissolves within 5 minutes, after which the flow of anhydrous nitrogen is resumed and the reaction is conducted at a temperature of 70°C for 30 minutes. The flask is then cooled by placing it in ice and the pH of the solution is brought to 9 by adding IN NaOH. The solution is decanted into a 500-ml Erlenmeyer flask and the reaction products are precipitated by the addition of 100 ml of cold acetonitrile. The precipitate is separated on a Buchner filter in a cold chamber, dried and then dialysed in dialysis bags against distilled water until the solution is clear. The clear solution is concentrated with a rotating evaporator, mixed with 2 gr of silica gel, after which 20 ml of chloroform/methanol 2: 1 is added. The solvent is evaporated with a rotating evaporator, then the dry residue is loaded on a column (2x75 cm) containing silica gel 100. It is eluted with a mixture of chloroform/methanol/water 60:35:5. The pure fractions are harvested, concentrated with a rotating evaporator, the concentrated product is precipitated in acetone and vacuum-dried. Product obtained: 0.45 gr of 98% GM3. Example 7: Preparation of GM3 from GMl inner ester A 5-litre, two-spouted flask, connected with a cooler and fitted with a magnetic stirrer, is heated to 70°C in an oil bath. It is thoroughly treated with anhydrous nitrogen. Once the flow of nitrogen has been cut off, 50 gr of GMl inner ester is rapidly added with 14 ml of a 0.25M solution of sulfuric acid in anhydrous dimethylsulfoxide, treated with nitrogen and heated to 70° C. Under vigorous stirring the GMl inner ester dissolves within 5 minutes, the flow of anhydrous nitrogen is resumed and the reaction is conducted at a temperature of 70°C for 30 minutes. The flask is cooled by placing it in ice and the pH of the solution is adjusted to 9 by adding IN NaOH, checking that the temperature does not exceed 35°C. The solution thus obtained is supplemented with 1500 ml of a saturated aqueous solution of NaCl while stirring. This is filtered and the precipitate is dissolved, while still wet, in 2000 ml of water. Partition is achieved with 10 litres of a mixture of chloroform/methanol 2: 1. The subnatant is separated and concentrated and chromatography is performed five times on a column (10x130 cm) containing silica gel 100. It is eluted with a mixture of chloroform/methanol/ammonia 30%, 60:35:8. The pure fractions are harvested, concentrated with a rotating evaporator, precipitated in acetone and vacuum dried. Product obtained: 18 gr of 98% GM3. Example 8: Preparation of lyso-GM3 In a reactor set at a temperature of 90°C, connected to a cooler and fitted with a magnetic stirrer, 15.2 gr of sodium bisulfate is solubilised in 160 ml of 8M KOH. 8 gr of GM3 is then added and left to react at a temperature of 90°C for 120 hours. After this, it is neutralised with concentrated HCl and the mixture thus obtained is loaded on a chromatographic column containing RP8 silica gel. The salts are eluted with water and then the lyso-GM3 is eluted with a solvent constituted by a mixture of methanol/water at a ratio of between 20:80 to 80:20. The pure fractions, analysed by TLC, and the solvent of chloroform/methanol/CaC12 0.3% 60:40:9, are mixed together, concentrated in a rotating evaporator and dried. Product obtained: 5.4 gr of lyso-GM3, 95%. Example 9 Following the same procedure as described in Example 6, starting from 1 g of GTlb inner ester, 0,35 g of 98% GM3 are obtained. The invention being thus described in detail, it is clear that it can be modified in various ways. Such modifications are not to be considered as variations from the spirit and purpose of the present invention and any such modification which would be obvious to an expert in the sector is to be considered as coming within the scope of the following claims.

Claims: WO 00/35932 -, , PCT/EP99/09958 CLAIMS I . Process for the preparation of ganglioside GM3 by acid hydrolysis of the inner ester of GMl, GDla or GDlb, GTlb. 2. Process according to claim 1, wherein the hydrolysis reaction is conducted in aprotic solvent. 3. Process according to claim 2, wherein the aprotic solvent is anhydrous. 4. Process according to claim 2, wherein the aprotic solvent is dimethylsulfoxide. 5. Process according to claim 1, wherein the concentration of acid used ranges between 0.25 and 2 N. 6. Process according to claim 5, wherein the acid used is sulfuric acid. 7. Process according to claims 1 to 6, wherein the temperature of the reaction is between 50 and 100°C. 8. Process according to claims 1 to 7, wherein the reaction time is between 10 and 60 minutes. 9. Process according to claims 1 to 8, wherein the reaction is conducted in an inert environment. 10. Ganglioside GM3 obtained from a ganglioside selected from the group consisting of GMl, GDla, GDlb and GTlb. I I . Derivatives of GM3 prepared from GM3 obtained according to claims 1 to 9. 12. Pharmaceutical compositions containing GM3 obtained according to claims 1 to 9 and/or its derivatives for the treatment of pathologies correlated with neuronal cell death, such as stroke, traumatic insults and neurodegenerative diseases. 13. Pharmaceutical compositions containing GM3 obtained according to claims 1 to 9 and/or its derivatives in the treatment for tumours and in immunology. 14. Use of ganglioside GM3 obtained according to claims 1 to 9 for the preparation of its derivatives. 15. Use of ganglioside GM3 according to claim 14 wherein the derivatives are chosen from the group including lysoderivatives and dilysoderivatives such as N-acyl-, N'acyl-, N,N'-diacyl-derivatives of the N,N'-dilysoderivatives of GM3, N-acyl-derivatives of the N- lysoderivatives of GM3. 16. Use of ganglioside GM3 and the derivatives thereof, according to claims 14 and 15 for the preparation of pharmaceutical compositions for the treatment of pathologies correlated with neuronal cell death, such as stroke, traumatic insults and neurodegenerative diseases. 17. Use of ganglioside GM3 and the derivatives thereof according to claims 14 and 15 for the preparation of pharmaceutical compositions to be used in the field of oncology and in immunology.

Inventors: KIRSCHNER GUENTER, SONNINO SANDRO, KIRSCHNER, GUENTER, SONNINO, SANDRO
Applicants: FIDIA SPA, KIRSCHNER GUENTER, SONNINO SANDRO, FIDIA S.P.A., KIRSCHNER, GUENTER, SONNINO, SANDRO
Application year: 1999
Main classification: C07H15
IPC codes: C07H15