WO2000035932A1 - Process for the preparation of ganglioside gm3 by acid hydrolysis of ganglioside inner esters and its use in the pharmaceutical field - Google Patents
Process for the preparation of ganglioside gm3 by acid hydrolysis of ganglioside inner esters and its use in the pharmaceutical field Download PDFInfo
- Publication number
- WO2000035932A1 WO2000035932A1 PCT/EP1999/009958 EP9909958W WO0035932A1 WO 2000035932 A1 WO2000035932 A1 WO 2000035932A1 EP 9909958 W EP9909958 W EP 9909958W WO 0035932 A1 WO0035932 A1 WO 0035932A1
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- WO
- WIPO (PCT)
- Prior art keywords
- ganglioside
- derivatives
- preparation
- process according
- gml
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
- C07H15/02—Acyclic radicals, not substituted by cyclic structures
- C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
- C07H15/10—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical containing unsaturated carbon-to-carbon bonds
Definitions
- the present invention describes a process for the preparation, by semisynthetic means, of the ganglioside GM3, ⁇ -Neu5Ac-(2-3)- ⁇ -Gal-(l- 4)- ⁇ -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.
- 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
- 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.
- ganglioside GM3 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.
- ganglioside GM3 modulates specific membrane receptors by modification of proteinkinase activity of EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) receptors
- 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.
- 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.
- 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)].
- gangliosides 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.
- 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.
- ganglioside GM3 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, Kunststoffe (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 ⁇ -sialyl glycoside.
- ganglioside GM3 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.
- the present invention describes a new process for the preparation of ganglioside GM3 starting from gangliosides with more complex structures.
- 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.
- 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 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 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.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002355106A CA2355106A1 (en) | 1998-12-16 | 1999-12-15 | Process for the preparation of ganglioside gm3 by acid hydrolysis of ganglioside inner esters and its use in the pharmaceutical field |
AU19798/00A AU1979800A (en) | 1998-12-16 | 1999-12-15 | Process for the preparation of ganglioside gm3 by acid hydrolysis of gangliosideinner esters and its use in the pharmaceutical field |
EP99963541A EP1140961A1 (en) | 1998-12-16 | 1999-12-15 | Process for the preparation of ganglioside gm3 by acid hydrolysis of ganglioside inner esters and its use in the pharmaceutical field |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ITPD98A000293 | 1998-12-16 | ||
IT000293 IT1302530B1 (en) | 1998-12-16 | 1998-12-16 | PREPARATION PROCESS FOR GANGLIOSIDE GM3 AND ITS LYSODERIVATES TO START FROM GANGLIOSIDE GM1. |
Publications (1)
Publication Number | Publication Date |
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WO2000035932A1 true WO2000035932A1 (en) | 2000-06-22 |
Family
ID=11392394
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1999/009958 WO2000035932A1 (en) | 1998-12-16 | 1999-12-15 | Process for the preparation of ganglioside gm3 by acid hydrolysis of ganglioside inner esters and its use in the pharmaceutical field |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1140961A1 (en) |
AU (1) | AU1979800A (en) |
CA (1) | CA2355106A1 (en) |
IT (1) | IT1302530B1 (en) |
WO (1) | WO2000035932A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110507663A (en) * | 2019-08-07 | 2019-11-29 | 南昌大学 | Application of the Ganglioside GM3 in preparation treatment atherosclerosis drug |
IT201800007093A1 (en) * | 2018-07-11 | 2020-01-11 | Oligosaccharides for use in the treatment of Parkinson's disease |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0072722A2 (en) * | 1981-08-04 | 1983-02-23 | FIDIA S.p.A. | A method for preparing ganglioside derivatives and use thereof in pharmaceutical compositions |
US4713374A (en) * | 1984-06-27 | 1987-12-15 | Fidia, S.P.A. | Ganglioside derivatives |
EP0711782A1 (en) * | 1994-09-30 | 1996-05-15 | Snow Brand Milk Products Co., Ltd. | A method of preparing ganglioside |
WO1999041264A1 (en) * | 1998-02-17 | 1999-08-19 | Ludwig Institute For Cancer Research | Methods of synthesizing gm3 |
-
1998
- 1998-12-16 IT IT000293 patent/IT1302530B1/en active IP Right Grant
-
1999
- 1999-12-15 AU AU19798/00A patent/AU1979800A/en not_active Abandoned
- 1999-12-15 WO PCT/EP1999/009958 patent/WO2000035932A1/en not_active Application Discontinuation
- 1999-12-15 EP EP99963541A patent/EP1140961A1/en not_active Withdrawn
- 1999-12-15 CA CA002355106A patent/CA2355106A1/en not_active Abandoned
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0072722A2 (en) * | 1981-08-04 | 1983-02-23 | FIDIA S.p.A. | A method for preparing ganglioside derivatives and use thereof in pharmaceutical compositions |
US4713374A (en) * | 1984-06-27 | 1987-12-15 | Fidia, S.P.A. | Ganglioside derivatives |
EP0711782A1 (en) * | 1994-09-30 | 1996-05-15 | Snow Brand Milk Products Co., Ltd. | A method of preparing ganglioside |
WO1999041264A1 (en) * | 1998-02-17 | 1999-08-19 | Ludwig Institute For Cancer Research | Methods of synthesizing gm3 |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IT201800007093A1 (en) * | 2018-07-11 | 2020-01-11 | Oligosaccharides for use in the treatment of Parkinson's disease | |
WO2020012400A1 (en) * | 2018-07-11 | 2020-01-16 | Universita' Degli Studi Di Milano | Oligosaccharides for use in the treatment of parkinson's disease |
CN110507663A (en) * | 2019-08-07 | 2019-11-29 | 南昌大学 | Application of the Ganglioside GM3 in preparation treatment atherosclerosis drug |
Also Published As
Publication number | Publication date |
---|---|
AU1979800A (en) | 2000-07-03 |
EP1140961A1 (en) | 2001-10-10 |
CA2355106A1 (en) | 2000-06-22 |
ITPD980293A1 (en) | 2000-06-16 |
IT1302530B1 (en) | 2000-09-05 |
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