FI83423C - FOERFARANDE FOER FRAMSTAELLNING AV GANGLIOSIDDERIVAT. - Google Patents

Description

83423

Method for preparing ganglioside derivatives

The present invention relates to a new process for the preparation of functional ganglioside derivatives, in particular their esters, according to the preamble of claim 1.

Gangliosides are natural products contained in various animal tissues and organs, primarily the central and peripheral nervous system, but also the adrenal nucleus, erythrocytes, spleen, and the rest, from which they can be extracted in purified form. It has been possible to elucidate the basic structure of most of the gangliosides thus obtained. They are glycosphingolipids, i. compounds formed by the association of a specified amount of oligosaccharides and sphingosines and sialic acids, in which the units are linked by glucoside or ketoside bonds. The gangliosides obtained in pure form so far in the literature do not represent homogeneous chemical compounds, with the possible exception of their saccharide moiety (oligosaccharide), because the ceramide and sialic components are quite variable within certain limits. Thus, although reference is made to "pure" gangliosides, this term must be interpreted broadly to include ganglios species in which at least one moiety, e.g., the saccharide moiety, is uniform and chemically characteristic. With this in mind, before explaining the background of the present invention in more detail, it is useful to consider the following general formula, which contains the structures of all gangliosides obtained in purified form so far and which represents the functional groups functionally modified according to the invention ( formula I).

2 83423 HOOCmmm »/ siali— N miiiimiiOH / limuniOH acid, | | ........ OH ceramide HOOC "Il Ö δ OH OH δ (X)

OH OLIGOSACCHARIDE OH

In this formula, an oligosaccharide residue of up to 5 monosaccharides is linked by a glucosidic bond to a ceramic residue and one or more sialic acid residues, each with an equal number of direct glucosidic bonds and one or more bonds such that the remaining sialic acid bonds are linked together. The formula shows the hydroxyl groups of the saccharide moiety, sialic acids and ceramide as well as the above-mentioned glucoside bonds with sia acids and ceramide and the carboxyl groups of sialic acids. Sialic acids, which form part of the gangliosides of formula I, have the general formula II

B

λ- ° CH3OCNH / H — C — OH \ 1

/% \ \, COOH

/ H — C-OH

x I jX di) JX 'CH 2 OH / Xh * 1 3

OH

wherein one or more primary and secondary hydroxyl groups may also be acylated and wherein the acyl groups are derived from acetic or glycolic acid. The amount of sialic acids present in the gangliosides is usually 1-5.

Il 3 83423

The sia residues are linked to the oligosaccharide by a ketoside bond formed on the 2-hydroxyl with the oligosaccharide hydroxyl. When several sialic acids are joined together, their molecules are joined by ketoside bonds formed from the hydroxyls of the 2- and 8-positions of the two sialic acid molecules. The sialic acids of gangliosides, including the purified gangliosides described above, are various chemically homogeneous acids, such as N-acetylneuraminic and N-glycolylneuraminic acids, the former being predominant, and one or more of their O-acyl derivatives, such as 8-O-acyl derivatives.

Gangliosides are found in nature as metal salts, such as sodium salts, and therefore the carboxyl function (s) of sialic acids are in the form of a salt. The free forms of gangliosides can be readily obtained by treating salts, such as sodium salts, with an acid type ion exchanger, for example using a resin such as Dowex AG 50X8, H + form.

The ceramide residue in the gangliosides of the formula I generally denotes a specific N-acyl sphingosine of the formula ch2-o-ch2-o-CH-NH-acyl CH-NH-acyl

I I

CH-OH CH-OH

I I

CH CH2% \ CH CH2

I I

(CH2) n-CH3 (CH2) n-CH} where n = 10 to 16 and the acyl is derived from a saturated or unsaturated fatty acid having 16 to 22 carbon atoms or the corresponding hydroxy acid.

The oligosaccharide consists of up to 5 monosaccharides or derivatives containing an acylamine group thereof, especially hexoses and their derivatives of the above-mentioned type. However, at least one glucose or galactose molecule is always present in the oligosaccharide. The most common residue present as an acylamine derivative of the above sugars is N-acetylgalactosamine or N-acetylglucosamine.

To better illustrate the structure of the gangliosides of formula I, and in particular the nature of the bonds between the saccharide moieties, sialic acids and ceramide, the formula below is a pure Gi ganglioside containing only one sialic acid (represented by N-acetylamine or N-glycolylneuraminic acid).

B

T v Jh- \

CM-CO-W / CX> \ COOM Y chqh N

h A

HO H /

CHpH OH J CHPH

\ p-o-Jj / ”\\ y * ° \ T ° —kerid h \ oh h / ih h \ m / h h \ J h / h> sj \ oh h / h

m OH H M-CO-CH, H oh H OH

M V. M OH

It is well known that gangliosides are functionally important in the central nervous system and it has recently been shown that gangliosides are useful in the therapy of peripheral nervous system pathologies. The therapeutic action of gangliosides appears to consist primarily of stimulation of the neuronal development phenomenon and activation of membrane enzymes involved in the transmission of nerve stimuli, such as the enzyme + K +) -ATPase. Ganglioside-stimulated nerve development promotes functional recovery of damaged nerve tissue.

Further studies have been conducted to find compounds that could prove more effective than gangliosides in the therapy of nervous system pathologies. These studies have led to the finding that ganglios

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5,8423 Intra-esters in which one or more hydroxyls of the saccharide moiety are esterified with one or more carboxyl groups of sialic acids (intramolecular reaction) to form the same number of lactone rings are more active than the gangliosides themselves in promoting nerve development and activating membrane enzymes in transfer, such as the enzyme Na + K + -ATPase (see U.S. Patent 4,476,119).

A second class of ganglioside derivatives has now been discovered which offers advantages over the gangliosides themselves in that they have a prolonged activity time ("retard" effect "). These compounds are derivatives in which the carboxyl group of sialic acids is functionally modified by esterification or conversion to amides or derivatives of these esters and amides, in which the hydroxyl groups of the saccharide moiety, sialic acids and ceramide are also esterified with organic acids, or more acylates. and in particular "acetylates, pripionylates, etc."). These derivatives are described in more detail in the parent application (FI patent application 852515) and also include ganglioside compounds in which only the hydroxyl groups are esterified with organic acids, i.e. they contain free carboxyl groups.

The two methyl esters of the sialic acid carboxyl of gangliosides are described in "Notes on improved procedures for the chemical modification and degradation of glycosphyngolipids" in Journal of Lipid Research 21, 642-645 (1980), MacDonald et al. These compounds are in the ganglia and

Gm3 methyl esters [(Abbreviations used herein to identify gangliosides are those proposed by Svenernholm in J. Neurochem. 1 (), 613 (1963)] .However, MacDonald et al., Do not mention anything about the biological activity of the compounds. The Gm3 methyl ester of ganglioside is used in the preparation of one peracylate derivative thereof for use in a particular cleavage or combination of reactions [Methods of Enzymology, 50, 137-140 (1978)].

6 83423

In the above article, Journal of Lipid Research MacDonald et al. also show the acetylation of the methyl esters of the gangliosides G1 and G43, but without isolating the acylated compounds.

Acetylation by lipid acetic anhydride-pyridine extracted from the spleen, liver and kidney, Morris hepatoma and fibroblast cells of Buffalo rats has been reported by Terunobo Saito and Sen-Itiroh Hako-Mari in Journal of Lipid Research, 12, 257- 259 (1971). The authors have isolated by chromatography the acetylated mixture of gangliosides contained in these lipids from the acylated product, together with other glycolipids, but without identifying any specific ganglioside. Of the amides, the Gm3 unsubstituted amide of ganglioside is shown in [Ad.Exp. Med. Biol. 19_, 95 (1972)], but also in this case no biological properties are mentioned.

It is an object of the present invention to provide a new process for the novel functional ganglioside derivatives disclosed above and in strain application 852515, i. to prepare esters of carboxyl groups of gangliosides as defined in formula I or mixtures thereof.

A. Ganglioside Starting Compounds

The present invention provides useful ganglioside derivatives. These new derivatives are derived by functionally modifying the carboxyl groups present in the basic ganglioside structure. The gangliosides obtained so far in pure form can be represented by the following formula, which also emphasizes the functional groups modified according to the present invention.

Il 7 83423 HOOCmmim / siali- \ uumuuiOH / muimiOH acid, 1 I ............ (ceramide

HOOC’iniiiiii Ö Ö OH OH Ö (D

v_ s s = -OH OLIGOSACCHARIDE OH

These gangliosides, which form the starting materials for the functional derivatives according to the invention, are all those which can be extracted from various animal organs and tissues, in particular central and peripheral nervous system tissues, e.g. brain, spinal fluid, muscles, plasma and blood serum, kidneys, kidneys, liver, and erythrocytes or leukocytes. The starting angliosides may also be purified as described in the literature, such as those extracted from tissues and organs or microorganisms of vertebrates, especially mammals such as humans, cattle, calves, rats, mice.

According to the present invention, these ganglioside compounds are modified by treating the internal ganglioside ester with an alcohol to form the corresponding ester in the carboxyl groups of the sialic acid residues of the internal ganglioside ester or a mixture thereof, wherein the treatment is performed in the presence of an alcohol-corresponding metal alcoholate.

The esters to be prepared are in particular monoesters for monosialogangliosides and polyesters for polysialogangliosides, having as many ester groups as carboxyl groups present in the molecule and therefore as many sialic acid groups as present.

According to the invention, ganglioside derivatives are prepared by modifying "purified" individually characterized gangliosides or by modifying a mixture of gangliosides, such as a mixture of monosialo-gangliosides and polysialogangliosides. The mixtures used for the esterification according to the invention, for example for the preparation of the mixture shown in Example 3 below, contain both monosialogangliosides and polysialogangliosides, and in these mixtures all carboxyl groups are esterified to give ganglioside derivatives which are completely esterified. The term "esters" used in this specification, as well as in parent application 852515, should therefore be interpreted as meaning fully esterified derivatives. This also applies in particular to the derivatives of the illustrative examples below, which are referred to as "esters". These representations refer to mixtures containing polysialogangliosides that are fully esterified at all carboxyl groups.

Thus, the present invention includes derivatives of gangliosides that are either derived from a simple "pure" gan glioside or a mixture of gangliosides. The invention also encompasses derivatives obtained from different ganglioside structures, especially since the structure of the ganglioside may vary with respect to the amount and quality of the sialic acid residue, ceramide residues or oligosaccharide residue.

With respect to the structure of gangliosides, basic gangliosides can be monosialo, disialo, trisialo, tetrasialo or

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9,8423 pentasialogangliosides, wherein the preferred sialic acids are N-acetylneuraminic acid and N-glycolineuramic acid. Sialic acids may also be acylated at one of their lateral chain hydroxyls, such as at the hydroxyl at position 8, if this position is not already occupied by a ketoside bond that binds it to another adjacent sial residue.

The ceramide moiety may vary, as described above, and also with respect to the length of the carbon atom chain of the sphingosines forming the ceramide moiety, which may be from 16 to 22 carbon atoms. In addition, the length of any acyl residue can also vary, especially within the same range, i.e. it can be 16 to 22 carbon atoms. In contrast, the ceramide residue may vary so that a double sphingosine bond may be absent or present, and usually this residue is composed primarily of unsaturated N-acylated sphingosine and a low percentage of the corresponding saturated compound (which may, however, be about 10%). The acyl group may also be derived from aliphatic hydroxy acids having the above-mentioned number of carbon atoms, i.e. 16 to 20. One very important group of gangliosides contains sphingosines acylated at the ceramide residue having 18 or 20 carbon atoms in the chain and corresponding saturated compounds, while their hydroxyl-unsubstituted the unsaturated acyl group contains the same number of carbon atoms, i.e. 18 or 20.

As described above, the present invention provides, on the one hand, esters of "pure" gangliosides of formula I, i.e. derivatives having the uniform composition described above, and, on the other hand, esters of ganglioside mixtures obtained in various forms from various animal tissues. In the first case, the basic gangliosides are preferably prepared from those in which the oligosaccharide consists of up to 4 hexose residues or N-acetylhexoamine, since at least one hexose residue is present and in which this saccharide moiety is chemically homogeneous. The hexoses are preferably selected from the group consisting of glucose and galactose, and the N-acetylhexosamines are preferably selected from the group consisting of N-acetylglucosamine and N-acetylgalactosamine (ganglioside group A). Gangliosides in this group include, for example, those extracted from vertebrate brains, such as those described in "Gangliosides of the Nervous System" in Glycolipid Methodology, Lloyd A. Witting Fd., American Oil Chemists' Society, Champaign, 111. 187- 214 (1976) (see in particular Appendix 1), for example the gangliosides Gm4 »GM3, gM2 'gM1" g1cNAc' gD2 'GDla "Ga ^ NAc'GTlc' gQ 'GT1 3a, especially those in which the oligosaccharide contains at least one glucose or galactose residue and one N-acetylglucosamine or N-acetylgalactosamine residue, in particular the following (ganglioside group B) GM1

Gal (l-? 3) GalNACd -> 4) Gal (1 - »4) Glc (1 1) ceramides ®

NANA

GDla

Ga 1 (1 - "f 3) Ga 1NAC (1 * - * 4) Ga 1 (1? 4) G1 c (1 ——> 1) ceramides & Φ

NANA NANA

GDlb

Gal (1 3) Ga lNAC (1 4) Gal (1 4) Glc (1 «> 1) ceramides ©

NANA

(?)

NANA

II

11 83423 GTlb

Gal (1 - ^ 3) GalNAC (1 4) Gal (1 -> 4) Glc (1 - ♦ 1) ceramides Φ · (?)

NANA NANA

©

NANA

where Glc represents glucose, GalNAc represents N-acetyl-galactosamine, Gal represents galactose and NANA represents N-acetylneuraminic acid.

If ganglioside mixtures are used as starting material, the mixtures may consist of those obtained by direct extraction of gangliosides from various animal tissues as "total ganglioside extracts or their various fractions. Such extracts are described in the literature, for example in the above-mentioned papers or in Extraction and i2 83423 analysis of materials containing lipidbound. in Glycolipid Methodology, Lloyd A. Wittig Ed., American Oil Chemists' Society, Champaign, 111, 159-186 (1976) and in the same book, Gangliosides of the Nervous System, pp. 187-214. Some of the major mixtures, used according to the present invention are ganglioside extracts obtained from nervous system tissues, in particular the brain, which contain the gangliosides GM1, GD1a, GDib and GT1 / / already mentioned above, mixtures of this type being, for example, those shown in Example 2.

B. Esters

The ester groups of the novel ganglioside derivatives are derived from aliphatic alcohols, especially those having up to 12 and especially 6 carbon atoms, or araliphatic alcohols, preferably having only one benzene ring optionally substituted with 1-3 lower alkyl groups (C1-4), e.g. and having up to 4 carbon atoms in the aliphatic chain, or alicyclic or aliphatic-alicyclic alcohols having only one cycloaliphatic ring and up to 14 carbon atoms, or heterocyclic alcohols having up to 12 and in particular 6 carbon atoms and having only one heterocyclic ring containing nitrogen, oxygen or sulfur heteroatom.

The above alcohols may be substituted or unsubstituted, in particular by functions selected from the group consisting of hydroxyl, amine, alkoxy groups having up to 4 carbon atoms, alkyl, carboxyl or carbyloxy (such as carbonylmethoxy or carbonylethoxy) groups having up to 4 atoms in an alkyl, alkylamine or dialkylamine residue having up to 4 carbon atoms in the alkyls, which may be saturated or unsaturated, in particular having only one double bond. Alcohols which esterify the carboxyl functions of the gangliosides of the present invention may be monovalent or polyvalent, especially bivalent. Of the aliphatic alcohols, preference is given to lower alcohols having up to 6 carbon atoms, such as methyl alcohol, ethyl alcohol, propyl and isopropyl alcohol, n-butyl alcohol, isobutyl alcohol, tert-butyl alcohol and, of the dihydric alcohols, ethylene glycol and propylene. Of the araliphatic alcohols, those having only one benzene residue, such as benzyl alcohol and phenethyl alcohol, are preferred; of the alicyclic alcohols, those having only one cycloaliphatic ring are preferred, such as cyclohexyl alcohol (cyclohexanol) or terpineols or piperitols. Of the heterocyclic alcohols, tetrahydrofuranol and tetrahydropyranol are preferred. substituted aliphatic alcohols can also be used for the esterification of carboxylic ganglioside groups, for example those having amine functions, such as amino alcohols, such as those having up to 4 carbon atoms, and especially amino alcohols having a dialkyl (C 1-4) amine group, such as diethylamino.

Among the novel ester derivatives to be prepared according to the present invention, e.g. the following: - Ganglioside Gmi ethyl ester - Ganglioside Gmi propyl ester - Ganglioside Gmi isopropyl ester - Ganglioside Gmi normal butyl ester - Ganglioside Gmi isobutyl ester - Ganglioside Gmi tert-butyl ester - Ganglioside Gmi - esters of the above containing ganglioside Gpia instead of ganglioside Gmi, - esters presented here containing ganglioside G-rib instead of ganglioside Gmi, and i4 83423 as basic gangliosides G ^ i ^, Gpiita, G ^ ii and G <pib: ^ Methyl, ethyl, propyl, isopropyl, tert-butyl, benzyl, allyl, ethoxycarbonylmethyl esters of the ganglioside mixtures containing ^, especially the mixture shown in Example 2.

With regard to the properties of the compounds to be prepared, reference should be made to parent application 852515, in which they are described in more detail.

C. Preparation of esters

The novel process, which is characteristic of the present invention for the preparation of ganglioside esters and which can be used for the preparation of both the novel derivatives and already known esters in strain application 852515, treats an internal Gang lioside ester with a mixture of the desired alcohol and its corresponding alcoholate as mentioned above. The reaction can be carried out at a temperature corresponding to the boiling point of the alcohol. Lower temperatures can also be used, but in that case the reaction times are longer. It is also possible, however, which is detrimental to very good yields and short reaction times, to treat the internal ester only with the corresponding alcohol, preferably at a temperature corresponding to its boiling point.

Internal esters are described, for example, in BE Patent 894025 and U.S. Patent 4,476,119.

As the alcoholates, alkali metal alcoholates, in particular sodium alcoholate, are used in the process according to the invention.

Il 15 83423

The invention will now be examined in more detail by means of non-limiting embodiments.

Example 1: Ganglioside G1 methyl ester 5 g of ganglioside G1 internal ester (3.27 mmol) are dissolved in 200 ml of an anhydrous mixture of methylene chloride and methanol in a ratio of 4: 1. 176 mg (3.27 mmol) of sodium methylate dissolved in 50 ml of anhydrous methanol are added and the mixture is heated under reflux for 2 hours. After completion of the reaction, the mixture is neutralized with anhydrous Dowex AG 50x8 resin (H + form), the resin is filtered off and washed with methanol, and the solution is evaporated to dryness. The residue is taken up in 50 ml of methylene chloride / methanol 1: 1 and the reaction product is precipitated by pouring into 250 ml of acetone. The crude product (4.9 g) is purified by preparative high pressure chromatography on 60 H Merck silica gel using chloroform / methanol / isopropanol / ammonium carbonate (2%) 1140: 820: 180: 140 as solvent. The pure fractions are collected, evaporated to dryness, redissolved in 15 ml of chloroform / methanol 1: 1 and the product is precipitated with 75 ml of acetone. This product is the methyl ester of ganglioside Gm. Yield 4.2 g.

IR spectroscopy on KBr tablets shows a typical ester bond at 1750 cm -1. Chromatography on silica gel plates in chloroform / methanol / CaCl 2 J Ha at 0.3% 55:45:10 and determined with Ehrlich's reagent (Rf 0.72) showed the product to be a homogeneous product and free from the internal ester used as starting material (Rf 0.75). and ganglioside GMi (Rf 0.65). Treatment with a 0.1N solution of Na 2 CO 3 at 60 ° for 1 hour cleaves the ester bond to give the starting product, Gjji.

* ♦ · I * · * ie 83423

Example 2: Preparation of a ganglioside mixture (GA mixture) by extraction from bovine brain tissue and preparation of a corresponding mixture of internal esters (used in several of the following examples

Bovine cortex removed from the animal is homogenized in phosphate buffer, pH 6.8; 6 volumes of tetrahydrofuran are added and the resulting mixture is centrifuged. The mother liquor is re-extracted twice with tetrahydrofuran. After centrifugation, the non-polar substances are removed by separation with ethyl ether and the aqueous tetrahydrofuran layer is applied to an ion exchange column equilibrated with 50% ethanol. Barium hydroxide and four volumes of ice-cold ethanol are added to the effluent from the column.

After 18 hours under cold conditions, the precipitate is collected and then slightly acidified with hydrochloric acid after dissolving in water. The solution thus obtained is dialyzed and lyophilized. The yield at this point is about 0.6 mg of crude ganglioside mixture / g of nerve tissue used. The lyophilized powder is dispersed in 20 volumes of a 2: 1 mixture of chloroform and methanol, the resulting solution is filtered until completely clear, and then separated by adding 0.2 volumes of a solution of potassium chloride in water at 0.88%.

The upper layer is separated, dialyzed and lyophilized. The final yield is 0.3 mg of purified ganglioside salt mixture / g of brain tissue.

5 g of the mixture obtained according to the above procedure are dissolved in 50 ml of DMSO. 4 g of a styrene-type anhydrous resin (sulfonic acid) (50-100 mesh H + form) are added to the mixture, and the resulting system is stirred for 30 minutes at room temperature. This treatment with an ion exchange resin converts all carboxyl groups formed into a salt. Complete conversion is performed by a suitable physical analysis method, such as atomic absorption. The resin is filtered

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n 83423 is then treated with suction and the solution is treated with 1.5 g of dicyclohexylcarbodiimide and left to stand for one hour. The precipitated dicyclohexylurea is removed by filtration and the resulting solution is treated with 100 ml of 0, which precipitates the internal ganglioside esters prepared.

The yield is 4.6 g of a mixture of internal esters (about 90-95% of theory). The presence of internal ester derivatives is confirmed by infrared spectroscopy and thin layer chromatography. IR spectrometry on KBr tablets: an esterifying lactone bond gives a band at 1750 cm-1. Thin layer chromatography: silica gel plate, developing solvent: CHCl 3 / MeOH / CaCl 2 0.3% (55:45:10, v / v / v), the internal ester mixture has an Rf of 0.7-0.85. The Rf of the final products is higher than the Rf of the starting material mixture; thus, chromatography indicates the absence of starting material. By treatment with a 0.1 N solution of Na 2 CO 3 at 60 ° for 1 hour, the ester bonds are cleaved and it is possible to obtain an initial mixture of starting angliosides.

The resulting ganglioside mixture can be fractionated into separate portions representing substantially pure gangliosides (as used in the general description), using silica columns, and eluting with a methanol-chloroform mixture. In this way, a composition is obtained which contains on average about 40% ganglioside GD, 21% ganglioside GM1 / 19% ganglioside G <pib and ig% ganglioside Gon.

Example 3: Mixture of methyl esters of a mixture of gangliosides 5 g of a mixture of internal esters of a mixture of gangliosides (obtained by extracting bovine brain tissue as described in Example 3) are dissolved in 200 ml of a 4: 1 mixture of anhydrous methylene chloride and methanol. 318 mg (5.86 mmol) of sodium methylate dissolved in 50 ml of anhydrous methanol are added and the mixture is heated under reflux for 2 hours. The crude reaction product is then isolated as in Example 1 (4.9 g). The crude product is then purified by chromatography on Sephadex-DEAE acetate 83423 in A-25 using a 30: 60: 8 mixture of chloroform, methanol and water as solvent. The stirred neutral fractions are evaporated, dialyzed against water, evaporated to dryness again, the residue is dissolved in 15 ml of a 1: 1 mixture of chloroform and methanol and the product is precipitated with 75 ml of acetone. Yield: 4.3 g. IR spectroscopy on KBr tablets shows a typical ester bond at 1750 cm-1. Chromatography performed as described in Example 1 showed the product to be a mixture of methyl esters of gangliosides with an Rf of 0.72 to 0.85 (Rf 0. 2 to 0.70 for a mixture of gangliosides).

Complete transesterification can be performed by determining the molecular proportions between the alkoxy and sia groups obtained by quantitative head space gas chromatography of methyl alcohol after treatment with 0.1 N Na 2 CO 3 for 1 hour, during which time all ester bonds are cleaved, and Svennerholm. method for the determination of N-acetylneuraminic acid.

Example 4: Ganglioside G ^ i ethyl ester

The ethyl ester of the ganglioside Gjyji is prepared and isolated in the same manner as the methyl ester of Example 1 using ethyl alcohol and sodium ethylate instead of methyl alcohol and sodium methylate and using the same molar amounts of internal ester and ethylate as in Example 1. The Dowex resin is washed To 50 ml of a 1: 1 mixture of methylene chloride and ethanol. The yield of crude product is 4.9 g. The purification is carried out in the same manner as in Example 1. Yield of purified Gm-ganglioside ethyl ester: 4.3 g. Chromatographic analysis of the product, carried out in the same manner as in Example 1, shows the presence of a uniform compound with an Rf of 0.80 and the absence of a Gjii ganglioside and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with NaCO 3 as described in Example 1 yields the ganglioside Gjm.

Il 19 83423 The IR spectrum performed on KBr tablets shows a typical ester bond at 1750 cm -1.

Example 5: Mixture of ethyl esters of a mixture of gangliosides

A mixture of ethyl esters is prepared and isolated in the same manner as for the mixture of methyl esters of Example 3 using ethyl alcohol and sodium ethylate instead of methyl alcohol and sodium methylate and using 5 g of a mixture of internal esters and 318 mg (5.86 mmol) of sodium ethylate.

The Dowex resin is washed with ethyl alcohol and the residue obtained by evaporation of the filtrate is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol.

The yield of crude product is 4.9 g. The purification is carried out in the same manner as in Example 3. Yield of purified ethyl ester mixture: 4.5 g. The IR spectrum performed on KBr tablets shows a typical ester bond at 1750 emes. When chromatographed on silica gel plates with a freshly prepared solution of chloroform, methanol and tetramethylammonium hydroxide IM 55:45:10 and determined with Ehrlich's reagent, the product has an Rf value of 0.50 to 0.75 (ganglioside mixture 0.20 to 0.60). ).

Complete conversion esterification is performed in the same manner as in Example 3.

Example 6: Isopropyl ester of ganglioside Gjm This derivative is prepared and isolated in the same manner as the methyl ester of Example 1, but using isopropyl alcohol and sodium isopropylate instead of methyl alcohol and sodium methylate and using the same molar amounts of internal ester and isopropylate as in Example 1. is obtained by evaporation of the filtrate, is dissolved in 50 ml of a 1: 1 to 20 83423 mixture of methylene chloride and isopropanol. The yield of crude product is 4.9 g. The purification is carried out in the same manner as in Example 1. Yield of isopropyl ester of purified G 1 i ganglioside: 4.2 g.

Chromatographic analysis of the product, carried out in the same manner as in Example 1, shows the presence of a homogeneous compound having an Rf of 0.85 and the absence of a Gm ganglioside or an internal ester thereof. Hydrolysis with Na 2 O 3 as described in Example 1 affords the ganglioside GmI. The IR spectrum performed on KBr tablets shows a typical ester bond at 1750 cm -1.

Example 7: Mixture of isopropyl esters of a ganglioside mixture This derivative mixture is prepared and isolated in the same manner as for the mixture of methyl esters of Example 3 using isopropyl alcohol and sodium propylate instead of methyl alcohol and sodium methylate and using 5 g of a mixture of internal esters and 537 mg (6.52 mmol). The Dowex resin is washed with isopropyl alcohol and the residue obtained by evaporation of the filtrate is dissolved in 50 ml of a 1: 1 mixture of chloroform and isopropanol. The yield of crude product is 4.9 g. Purification is carried out in the same manner as in Example 3, but using a 20:60: 8 mixture of chloroform, isopropyl alcohol and water as the chromatographic eluent. Yield of a mixture of purified isopropyl esters: 4.3 g.

By chromatography as described in Example 5, the Rf of the product is 0.40-0.80 (0.20-0.60 of the ganlioside mixture). The IR spectrum performed on KBr tablets shows a typical barrier band at 1750 cm -1. Complete conversion esterification is performed as described in Example 3.

li

Example 8: Ganglioside G 1 tert-butyl ester 21 83423 This derivative is prepared and isolated in the same manner as the methyl ester of Example 1 using tert-butyl alcohol and sodium tert-butylate instead of methyl alcohol and sodium methylate and using the same molar amounts of internal ester and tert-butylate. in Example 1. The Dowex resin is washed with tert-butyl alcohol and the residue obtained by evaporating the filtered substance is dissolved in 50 ml of a 1: 1 mixture of methylene chloride and tert-butanol. The yield of crude product is 4.9 g. Yield of purified G 2 -gang lioside tert-butyl ester: 4.1 g.

Chromatographic analysis of the product, carried out in the same manner as in Example 1, shows the presence of a homogeneous compound having an Rf of 0.71 and the absence of Gwi ganglioside and its internal ester. Hydrolysis with Na 2 CO 3 as described in Example 1 affords the ganglioside GmI. The IR spectrum performed on KBr tablets shows a typical ester band at 1750 cm-1.

Example 9: Mixture of tang-butyl esters of a mixture of ganliosides This mixture is prepared and isolated in the same manner as for the mixture of methyl esters of Example 3 using tert-butyl alcohol and sodium tert-butylate instead of methyl alcohol and sodium methylate and using a mixture of 5 g of internal esters and 628.6 mg of sodium tert-butoxide are. The Dowex resin is washed with tert-butyl alcohol and the residue obtained by evaporation of the filtrate is dissolved in 50 ml of a 1: 1 mixture of chloroform and tert-butanol. The yield of crude product is 4.9 g. Purification is carried out in the same manner as in Example 3, but using a 1: 1 mixture of chloroform and tert-butyl alcohol as the chromatographic eluent. Yield of purified tert-butyl esters: 4.1 g. The IR spectrum performed on KBr tablets shows a typical ester bond at 1750 cm-1. Chromatographed as described in Example 5, the Rf of the product is 0.25-0.70. The complete reaction is carried out as described in Example 3.

Example 10: Benzyl ester of ganglioside G ^ i 5 g of the potassium salt of ganglioside G ^ i (3.14 mmol) are dissolved in 50 ml of DMSO and 1.58 g (12.5 mmol) of benzyl chloride and 2.08 g (12 .5 mmol) KI. This is allowed to react under nitrogen for 24 hours at 25 ° C. After completion of the reaction, the solution is partitioned between n-butanol and water (2: 1) to eliminate DMSO and salts. The butanol solution is evaporated to dryness and the residue is taken up in 50 ml of a 1: 1 mixture of chloroform and benzyl alcohol and the reaction product is precipitated with 250 ml of acetone.

The crude product thus obtained (5.3 g) is then purified by preparative chromatography on silica gel plates using a 65:32: 7 mixture of chloroform, methanol and water as solvent.

The pure fractions are stirred, evaporated, redissolved in 15 ml of a 1: 1 mixture of chloroform and isopropanol and the product is precipitated with 75 ml of acetone. Yield of pure benzyl ester: 4.8 g.

The IR spectrum performed on KBr tablets shows a typical ester band at 1750 cm-1 and the UV spectroscopy performed in absolute ethyl alcohol shows three maxima at 250, 255 and 261 nm.

Chromatography on silica gel plates with a 60: 35: 8 mixture of chloroform, methanol and CaCl2 (0.3%) and a 2.5 N 55:45:12 mixture of chloroform / methanol and ammonia and determined with Ehrlich's reagent shows that the product is uniform and has an Rf is 0.65 and 0.53, respectively, and is free of GM1 "starting material (Rf 0.40 and 0.45, respectively).

Treatment with a 0.1 N solution of Na 2 CO 3 at 60 ° for 1 hour causes cleavage of the ester bond to give the starting materials (GM 2 and benzyl alcohol).

Example 11: Mixture of benzyl esters of a mixture of gangliosides 23 83423 5 g of a mixture of salted gangliosides (potassium salts) obtained by extracting bovine brain tissue as described in Example 2, then exchanging with sodium potassium (ion exchange), are dissolved in 100 ml of DMSO and dissolved in 3 ml of DMSO. g (26.0 mmol) of benzyl chloride and 216 g (13.0 mmol) of KI. The mixture is left to react under nitrogen for 48 hours at 25 °. At the end of the reaction, the solution is partitioned between n-butanol / H 2 O (2: 1) to eliminate DMSO and salts. The butanol solution is evaporated to dryness and the residue is taken up in 50 ml of a 1: 1 mixture of chloroform and benzyl alcohol and the product is precipitated with 250 ml of acetone. The crude product thus obtained is purified by chromatography on Sephadex-DEAE in acetate form using a 30: 60: 8 mixture of chloroform, methanol and water as solvent.

The eluted neutral fractions are mixed, freed from the Liu by evaporation and the residue is taken up in 15 ml of a 1: 1 mixture of chloroform and isopropanol and the mixture of purified benzyl esters is precipitated with 75 ml of acetone. Yield: 4.9 g.

IR spectrometry on KBr tablets shows a typical ester bond at 1750 cm -1 and UV spectroscopy performed in absolute ethyl alcohol shows three maxima at 250, 255 and 261 nm.

Chromatography on silica gel plates with a mixture of chloroform, methanol and CaCl3 (0.3%, 60: 25: 8) and a mixture of chloroform, methanol and ammonia 2.5N 55:45:10 and determined with Ehlich's reagent gives an Rf value of 0, 40 -0.70 and 0.29-0.53, respectively (for the original ganglioside mixture 0.05-0.40 and 0.12-0.46, respectively).

The complete reaction is carried out as described in Example 3.

Example 12: Ganglioside G ^ i allyl ester 24 83423 5 g of the ganglioside G ^ i potassium salt (3.14 mmol) are dissolved in 50 ml of DMSO and 453.7 g (3.75 mmol) of allyl bromide and 625 mg (3 , 75 mmol) Thank you. This is allowed to react for 48 hours at 25 ° C.

After completion of the reaction, the solution is partitioned between n-butanol / t 2 O (2: 1) to eliminate DMSO and salts. The butanol solution is evaporated to dryness and the residue is taken up in 50 ml of a 1: 1 mixture of chloroform and methanol and the reaction product is precipitated with 250 ml of acetone.

The crude product thus obtained (5.1 g) is then purified by preparative chromatography on silica gel plates using a 60: 35: 8 mixture of chloroform, methanol and water as solvent.

The pure fractions are stirred, evaporated, redissolved in 15 ml of a 1: 1 mixture of chloroform and isopropanol and the product is precipitated with 75 ml of acetone. Yield of pure allyl ester: 4.5 g.

IR spectrometry on KBr tablets shows a typical ester band at 1750 cm -1.

Chromatography on silica gel plates with a 60:40: 9 mixture of chloroform, methanol and CaCl2 (0.3%) and a 2.5N 55:45:10 mixture of chloroform / methanol and ammonia and determined with Ehrlich's reagent gives the product as a uniform compound with Rf is 0.56 and 0.39, respectively, and is free of Gf4i starting material (Rf 0.40 and 0.42, respectively).

Treatment with a 0.1 N solution of Na 2 CO 3 in O 2 O for 1 hour causes cleavage of the ester bond to give the starting products ganglioside G 1 and allyl alcohol.

Il

Example 13: Ganglioside Gmi ethoxycarbonylmethyl ester 83423 5 g of the ganglioside Gmi potassium salt (3.14 mmol) are dissolved in 50 ml of DMSO and 2.12 g (12.5 mmol) of ethyl monobromoacetate and 2.08 g (12.5 mmol) of mmol) of KI. It is left to react under nitrogen for 24 hours at 25 ° C. The solution is partitioned between n-butanol and water (2: 1) to eliminate DMSO and salts. The butanol solution is evaporated to dryness and the residue is taken up in 50 ml of a 1: 1 mixture of chloroform and methanol and the product is precipitated with 250 ml of acetone. Yield 4.8 g.

The crude product is then purified by preparative column chromatography on silica gel plates using a 60: 32: 7 mixture of chloroform, methanol and water as solvent. The pure fractions are stirred, evaporated, dissolved in 15 ml of a 1: 1 mixture of chloroform and methanol and the product is precipitated with 75 ml of acetone. Yield: 2.4 g.

IR spectrometry on KBr tablets shows a typical ester band at 1750 cm-1.

Chromatography on silica gel plates with a 60:35: 8 mixture of chloroform, methanol and CaCl 2 (0.3%) and determined with Ehrlich's reagent gives the product as a homogeneous compound with an Rf of 0.64 and is free of the starting material (Rf 0.40 ).

Treatment with 0.1 N Na 2 CO 3 solution at 60 ° for 1 hour causes cleavage of the ester bond and yields the original ganglioside Gmi ·

Example 14: Ganglioside Gmi amide 5 g of ganglioside Gmi internal ester (3.27 mmol) are suspended in 100 ml of anhydrous isopropyl alcohol. The suspension is stirred at low temperature (-5 °) and dry ammonia is then bubbled through it under anhydrous conditions for 3 hours.

26 83 423

At the end of the reaction, the solvent is removed by evaporation and the residue is taken up in 50: 1: 1 mixture of chloroform and methanol and the product is precipitated with 250 ml of acetone.

The crude product (4.9 g) is treated with 100 ml of Na 2 CO 3 (1%) for 30 minutes at 25 ° C to hydrolyze the residual ester groups, dialyzed against water, evaporated to dryness in vacuo and then purified by preparative column chromatography on silica gel. using first a 60:40: 9 mixture of chloroform, methanol and H 2 O as the solvent and a 55:45:10 mixture of chloroform, methanol and H 2 O * as the second solvent. The pure, eluted fractions are evaporated off, the residue is dissolved in 15 ml of chloroform / methanol 1: 1 and the amide is precipitated with 75 ml of acetone. Yield: 4.8 g.

Chromatography on silica gel plates with a 4N 55:45:10 mixture of chloroform, methanol and ammonia and determined with a resorcinol reagent gives the product as a homogeneous compound (Rf 0.10 and 0.32, respectively) and free of Gf (Rf 0.35 and respectively 0.65).

Il

Example 15: Ganglioside Phenylethyl Ester 27 83423 The phenylethyl ester of GM1 is prepared and isolated in the same manner as the methyl ester of Example 1 using phenethyl alcohol and sodium ethylate instead of methyl alcohol and sodium methylate and heating on a water bath over Example D and the residue obtained by evaporating the filtered substance is dissolved in 50 ml of a 1: 1 mixture of methylene chloride and phenyl alcohol. The yield of crude product is 5.1 g. The purification is carried out in the same manner as in Example 1. Yield of phenylethyl ester of purified GMi-ganglioside: 4.3 g.

IR spectroscopic examination on KBr tablets shows a typical ester bond at 1750 cm -1. Chromatographic analysis of the product, performed as described in Example 1, shows the presence of a uniform compound with an Rf of 0.90 and the absence of GMi-ganglioside and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na 2 CO 3 as described in Example 1 to give the ganglioside GmI-28 83423

Example T6; A mixture of phenylethyl esters of a ganglioside mixture

A mixture of phenylethyl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using phenethyl alcohol and sodium phenylethylate instead of methyl alcohol and sodium methylate and heating on a water bath using 5 g of a mixture of internal esters and 844.8 mg (5.86 mmol) of sodium ethylate. The Dowex resin is washed with phenylethyl alcohol and the residue obtained by evaporation of the filtrate is dissolved in 50 ml of a 1: 1 mixture of chloroform and phenylethyl alcohol. The yield of crude product is 5.3 g. The purification is carried out in the same manner as in Example 3. Yield of purified ester mixture: 4.4 g.

IR spectroscopy on KBr tablets shows a typical ester band at 1750 cm -1. When chromatographed on silica gel plates with a freshly prepared mixture of chloroform, methanol and tetramethylammonium hydroxide IM 55:45:10 and determined with Ehrlich's reagent, the product has Rf values of 0.65 to 0.887 (ganglioside mixture 0.2 to 0.60). . Complete conversion esterification is performed as described in Example 2.

Example 17; Ganglioside G 2 i 2-cyclohexylethyl ester G 1 i 2-cyclohexyl ester is prepared and isolated in the same manner as the methyl ester of Example 1 using 2-cyclohexylethanol and sodium 2-cyclohexylethyl ester instead of methyl alcohol and sodium methylate and stirring with water and heating with water. 2-Cyclohexylethylate as in Example 1. The Dowex resin is washed with 2-cyclohexylethyl alcohol and the residue obtained by evaporating the filtered substance is dissolved in 50 ml of a 1: 1 mixture of methylene chloride and 2-cyclohexylethanol. The yield of crude product is 5.1 g. The purification is carried out as described in Example 1. Yield of purified 2 H-ganglioside 2-cyclohexylethyl ester: 4.3 g.

IR spectroscopic examination with KBr tablets shows a typical ester band at 1750 cm-1. Chromatographic analysis of the product, carried out as described in Example 1, shows the presence of a uniform compound, Rf 0.92, and the absence of G 1 i ganglioside and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na 2 O 3 as described in Example 1 affords the ganglioside GmI.

Example Ί8: Mixture of 2-cyclohexylethyl esters of a mixture of gangliosides A mixture of 2-cyclohexylethyl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using 2-cyclohexylethanol and sodium 2-cyclohexylethylate over methyl alcohol and sodium methylate a mixture of esters and 880.3 mg (5.86 mmol) of sodium 2-cyclohexylethylate. The Dowex resin is washed with 2-cyclohexylethyl alcohol and the residue obtained by evaporating the filtrate is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol. The yield of crude product is 5.3 g. The purification is carried out in the same manner as in Example 3. Yield of purified ester mixture: 4.3 g.

IR spectroscopy on KBr tablets shows a typical ester band at 1750 cm -1. When chromatographed on silica gel plates with freshly prepared IM 55: 45:10 solution of chloroform, methanol and tetramethylammonium and determined with Ehrlich's reagent, the product has Rf values of 0.67 to 0.90 (ganglioside mixture 0.20 to 0.60). Complete conversion esterification is performed as described in Example 2.

Example 19: Ganglioside G1 menthyl ester 30 83423

Gmijo menthyl ester is prepared and isolated in the same manner as the methyl ester of Example 1, but using menthol and sodium methylate instead of methyl alcohol and sodium methylate and heating on a water bath and using similar molar amounts of internal ester and sodium menthylate as in Example 1. Washing of Dowex resin with methylene alcohol 1 and the residue obtained by evaporation of the filtrate is dissolved in 50 ml of a 1: 1 mixture of methylene chloride and menthol. The yield of crude product is 3.9 g. The purification is carried out in the same manner as in Example 1. Yield of purified GM1.9919 ° menthyl ester: 4.2 g.

IR spectroscopic examination with KBr tablets shows a typical ester band at 1750 cm -1. Chromatographic analysis of the product carried out as described in Example 1 shows the presence of a homogeneous compound with an Rf of 0.93 and the absence of Gfli ganglioside and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na 2 CO 3 as described in Example 1 yields the ganglioside Gmi ·

Example 20. Mixture of menthyl esters of a ganglioside mixture

The mixture of menthyl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using menthol and sodium menthylate instead of methyl alcohol and sodium methylate and heating on a water bath using 5 g of a mixture of internal esters and 1038.8 mg (5.86 mmol) of sodium menthylate. The Dowex resin is washed with a 1: 1 mixture of menthyl alcohol and methylene chloride, and the residue obtained by evaporating the filtered substance is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol. The yield of crude product is 5.2 g. The purification is carried out in the same manner as in Example 3. Yield of purified ester mixture: 4.3 g.

IR 31 Spectroscopy with KBr tablets shows a typical ester band at 1750 cm-1. When chromatographed on silica gel plates with a freshly prepared mixture of chloroform, methanol and tetramethylammonium hydroxide IM 55:45:10 and determined with Ehrlich's reagent, the product has Rf values of 0.70 to 0.93 (ganglioside mixture 0.20 to 0.60). . Complete conversion esterification is performed as described in Example 2.

Example 21: Tetrahydrofurfuryl ester of the ganglioside G 1 The tetrahydrofurfuryl ester of the ganglioside is prepared and isolated in the same manner as the methyl ester of Example 1 using tetrahydrofurfuryl and sodium tetrahydrofurate instead of methyl alcohol and sodium methylate and exemplified by The Dowex resin is washed with tetrahydrofurfuryl alcohol and the residue obtained by evaporating the filtrate is dissolved in 50 ml of a 1: 1 mixture of methylene chloride and tetrahydrofurfuryl alcohol. The yield of crude product is 5.0 g. The purification is carried out as described in Example 1. Yield of tetrahydrofurfuryl ester of purified Gni-ganglioside: 4.2 g.

IR spectroscopic examination with KBr tablets shows a typical ester band at 1750 cm -1. Chromatographic analysis of the product carried out as described in Example 1 shows the presence of a homogeneous compound, Rf 0.72, and the absence of G 1 - ganglioside and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na 2 O 3 as described in Example 1 yields the gmlioside GmI.

Example 22: Tetrahydrofurfuryl ester mixture of a mixture of gangliosides

A mixture of tetrahydrofurfuryl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using tetrahydrofurfuryl alcohol and sodium tetrahydrofurfurylate instead of methyl alcohol and sodium methylate and heating over a water bath and using 5 g of a mixture of internal esters and . The Dowex resin is washed with tetrahydrofurfuryl alcohol and the residue obtained by evaporating the filtrate is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol. The yield of crude product is 5.2 g. The purification is carried out in the same manner as in Example 2. Yield of purified ester mixture: 4.2 g.

IR spectroscopy on KBr tablets shows a typical ester band at 1750 cm-1. When chromatographed on silica gel plates with freshly prepared IM 55: 45:10 solution of chloroform, methanol and tetramethylammonium and determined with Ehrlich's reagent, the product has Rf values of 0.45 to 0.68 (ganglioside mixture 0.20 to 0.60). Complete conversion inhibition is performed as described in Example 2.

Example 23; Ganglioside Gmi tetrahydro-2H-pyran-4-yl ester The tetrahydro-2H-pyran-4-yl ester of GM1 is prepared and isolated in the same manner as the methyl ester of Example 1 using tetrahydro-2H-pyran-4-ol and sodium tetrahydro- 2H-pyran-4-ylate instead of methyl alcohol and sodium methylate and by heating on a water bath and using similar molar amounts of internal ester and sodium tetrahydro-2H-pyran-4-ylate as in Example 1. The Dowex resin is washed with tetrahydro-2H-pyran-4-ylate. With 4-ol and the residue obtained by evaporation of the filtrate, 50 g of any methylene chloride alcohol are dissolved 1: 1. The yield of the crude product is 5.1. The purification is carried out in the same manner as in Example 1. Yield of purified tetrahydro-2H-pyran-4-yl ester of Gm ganglioside: 4.3 g.

IR spectroscopic examination with KBr tablets shows a typical ester band at 1750 cm -1. The product

II

33 83423 chromatographic analysis carried out as described in Example 1 shows the presence of a homogeneous compound with an Rf of 0.73 and the absence of a G 1 i ganglioside and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na 2 CO 3 as described in Example 1 yields the ganglioside Gmi ·

Example 24; A mixture of tetrahydro-2H-pyran-4-yl esters of a mixture of gangliosides

A mixture of tetrahydro-2H-pyran-4-yl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using tetrahydro-2H-pyran-4-ol and sodium tetrahydro-2H-pyran-4-ylate instead of methyl alcohol and sodium methylate. and heating on a water bath using a mixture of 5 g of internal esters and 727.5 mg (5.86 mmol) of sodium tetrahydro-2H-pyran-4-ylate. The Dowex resin is washed with tetrahydro-2H-pyran-4-yl and the residue obtained by evaporating the filtered substance is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol. The yield of crude product is 5.3 g. The purification is carried out in the same manner as in Example 3. Yield of purified crude product: 5.3 g. The purification is carried out in the same manner as in Example 3. Yield of purified ester mixture: 4.5 g.

IR spectroscopy on KBr tablets shows a typical ester band at 1750 cm -1. When chromatographed on silica gel plates with a freshly prepared mixture of chloroform, methanol and tetramethylammonium hydroxide IM 55:45:10 and determined with Ehrlich's reagent, the product has R 0.48 to 0.72 (ganglioside mixture 0.20 to 0.60) Complete transesterification is performed as described in Example 2.

Example 25: Ganglioside Gmi 1-heptyl ester 34 83423

The 1-heptyl ester of Gmi is prepared and isolated in the same manner as the methyl ester of Example 1, but using 1-heptanol and sodium 1-heptylate instead of methyl alcohol and sodium methylate and heating on a water bath and using the same molar amounts of internal ester and sodium 1-heptylate as in Example 1. The Dowex resin is washed with 1-heptyl alcohol and the residue obtained by evaporation of the filtrate is dissolved in 50 ml of a 1: 1 mixture of methylene chloride and 1-heptanol. The yield of crude product is 3.9 g. The purification is carried out as described in Example 1. Yield of purified Gwi ganglioside 1-heptyl ester: 4.3 g.

IR spectroscopic examination with KBr tablets shows a typical ester band at 1750 cm -1. Chromatographic analysis of the product carried out as described in Example 1 shows the presence of a homogeneous compound, Rf 0.89, and the absence of G 1 Ganglioside and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis of Na 2 O 3 * Ha as described in Example 1 yields the ganglioside Gmi.

Example 26; Mixture of 1-heptyl esters of a mixture of gangliosides A mixture of 1-heptyl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using 1-heptanol and sodium 1-heptylate instead of methyl alcohol and sodium methylate and heating on a water bath and using 5 g of internal esters. , 8 mg (5.86 mmol) of sodium 1-heptylate. The Dowex resin is washed with 1-heptyl alcohol and the residue obtained by evaporating the filtered substance is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol. The yield of crude product is 5.2 g. The purification is carried out in the same manner as in Example 2. Yield of purified ester mixture: 4.3 g.

IR spectroscopy with KBr tablets shows a typical ester band at 1750 cm-3. When chromatographed on silica gel plates with freshly prepared IM 55: 45: 10 solution of chloroform, methanol and tetramethylammonium and determined with Ehrlich's reagent, the product has Rf values of 0.68 to 0.90 (ganglioside mixture 0.20 to 0.60). Complete conversion inhibition is performed as described in Example 2.

Example 27. Ganglioside G 2 i-2-methyl-1-pentyl ester

The 2-methyl-1-pentyl ester of GmI is prepared and isolated in the same manner as the methyl ester of Example 1 using 2-methyl-1-pentanol and sodium 2-methyl-1-pentylate instead of methyl alcohol and sodium methylate and heating on a water bath and using similar molar amounts of internal ester and sodium 2-methyl-1-pentylate as in Example 1. The Dowex resin is washed with 2-methyl-1-pentyl alcohol and the residue obtained by evaporation of the filtered substance is dissolved in 50 ml of methyl. 1: 1 mixture of -leneyl chloride and 2-methyl-1-pentanol. The yield of crude product is 5.1 g. The purification is carried out in the same manner as in Example 1. Yield of purified Gfli ganglioside 2-methyl-1-pentyl ester: 4.4 g.

IR spectroscopic examination with KBr tablets shows a typical ester band at 1750 cm -1. Chromatographic analysis of the product, performed as described in Example 1, shows the presence of a uniform compound with an Rf of 0.90 and the absence of Gwi ganglioside and its internal ester. (Rf 0.65 and 0.75, respectively) Hydrolysis with Na 2 CO 3 as described in Example 1 yields the ganglioside Gjkji ·

Example 28; Mixture of 2-methyl-1-pentyl esters of a mixture of gangliosides A mixture of 2-methyl-1-pentyl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using 2-methyl-1-pentanol and 36 83423 sodium 2-methyl -1-pentylate instead of methyl alcohol and sodium methylate and heating on a water bath using 5 g of a mixture of internal esters and 727.7 mg (5.86 mmol) of sodium 2-methyl-1-pentylate. The Dowex resin is washed with 2-methyl-1-pentyl alcohol and the residue obtained by evaporating the filtered substance is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol. The yield of crude product is 5.3 g. The purification is carried out in the same manner as in Example 2. Yield of purified ester mixture: 4.4 g.

IR spectroscopy on KBr tablets shows a typical ester band at 1750 cm -1. When chromatographed on silica gel plates with a freshly prepared mixture of chloroform, methanol and tetramethylammonium hydroxide IM 55:45:10 and determined with Ehrlich's reagent, the product has Rf values of 0.70 to 0.93 (ganglioside mixture 0.20 to 0, 60). Complete conversion esterification is performed as described in Example 2.

Example 29: Ganglioside Gmi 3-methyl-2-pentyl ester The 3-methyl-2-pentyl ester of GM1 is prepared and isolated in the same manner as the methyl ester of Example 1 using 3-methyl-2-pentanol and sodium 3-methyl-2-pentanol. ethylate instead of methyl alcohol and sodium methylate and by heating on a water bath and using the same molar amounts of internal ester and sodium 3-methyl-2-pentylate as in Example 1. The Dowex resin is washed with 3-methyl-2-pentyl alcohol and the residue obtained by evaporation of the filtered substance, is dissolved in 50 ml of a 1: 1 mixture of methylene chloride and 3-methyl-2-pentanol. The yield of crude product is 5.1 g. The purification is carried out as described in Example 1. Yield of purified Gjm ganglioside 3-methyl-2-pentyl ester: 4.2 g.

IR spectroscopic examination with KBr tablets shows a typical ester band at 1750 cm -1. Chromatographic analysis of Kro 37 83423, performed as described in Example 1, shows the presence of a uniform compound, Rf 0.92, and the absence of GMi ganglioside and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with 203 as described in Example 1 yields the ganglioside Gmi.

Example 30:: Mixture of 3-methyl-2-pentyl esters of a mixture of gangliosides A mixture of 3-methyl-2-pentyl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using 3-methyl-2-pentanol and sodium 3-methyl -2-pentanol and sodium 3-methyl-2-pentylate instead of methyl alcohol and sodium methylate and heating on a water bath using a mixture of 5 g of internal esters and 727.7 mg (5.86 mmol) of sodium 3-methyl-2-pentylate. The Dowex resin is washed with 3-methyl-2-pentyl alcohol and the residue obtained by evaporating the filtrate is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol. The yield of crude product is 5.3 g. The purification is carried out in the same manner as in Example 2. Yield of purified ester mixture: 4.4 g.

IR spectroscopy on KBr tablets shows a typical ester band at 1750 cm-1. When chromatographed on silica gel plates with a freshly prepared IM 55: 45: 10 solution of chloroform, methanol and tetramethylammonium hydroxide and determined with Ehrlich's reagent, the product has Rf values of 0.72 to 0.95 (ganglioside mixture 0.20 to 0, 60). Complete conversion esterification is performed as described in Example 2.

Example 31: Ganglioside Gjh 3-methoxyethyl ester

Gulin's 2-methoxyethyl ester is prepared and isolated in the same manner as the methyl ester of Example 1 using 2-methoxyethanol and sodium 2-methoxyethylate instead of methyl alcohol and sodium methylate and heating on a water bath and using similar molar amounts of internal ester and sodium 2-methoxyethylate in Example 1. The Dowex resin is washed with 2-methoxyethyl alcohol and the residue obtained by evaporation of the filtered substance is dissolved in 50 ml of a 1: 1 mixture of methylene chloride and 2-methoxyethanol. The yield of crude product is 4.9 g. The purification is carried out in the same manner as in Example 1. Yield of purified 2 H-ganglioside 2-methoxyethyl ester: 4.3 g.

IR spectroscopic examination with KBr tablets shows a typical ester band at 1750 cm -1. Chromatographic analysis of the product, carried out as described in Example 1, shows the presence of a homogeneous compound with an Rf of 0.77 and the absence of GMi? <? Carbohydride and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na 2 CO 3 in Example 1 1 produces the ganglioside GmI ·

Example 32; Mixture of 2-methoxyethyl esters of a mixture of ganliosides A mixture of 2-methoxyethyl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using 2-methoxyethanol and sodium 2-methoxyethylate instead of methyl alcohol and sodium methylate and heating over an internal bath and using 5 g of 574.9 mg (5.86 mmol) of sodium 2-methoxyethylate. The Dowex resin is washed with 2-methoxyethyl alcohol and the residue obtained by evaporating the filtrate is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol. The yield of crude product is 5.2 g. Purification is similarly performed on KBr pellets and shows a typical ester band at 1750 cm -1. When chromatographed on silica gel plates with freshly prepared mixture of chloroform, methanol and tetramethylammonium hydroxide IM 55:45:10 and determined with Ehrlich's reagent, the product has Rf values of 0.51 to 0.78 (ganglioside mixture 0.20 to 0.60). . Complete conversion esterification is performed as described in Example 2.

li

Example 33: Ganglioside Gjm 1-methoxy-2-propyl ester 39 8 3 4 2 3

The 1-methoxy-2-propyl ester of Gmi is prepared and isolated in the same manner as the methyl ester of Example 1 using but 1-methoxy-2-propanol and sodium 1-methoxy-2-propylate instead of methyl alcohol and sodium methylate and heating on a water bath and using the same molar amounts of internal ester and sodium 1-methoxy-2-propylate as in Example 1. The Dowex resin is washed with 1-methoxy-2-propyl alcohol and the residue obtained by evaporation of the filtrate is dissolved in 50 ml of methylene chloride. and a 1: 1 mixture of 1-methoxy-2-propanol. The yield of crude product is 4.9 g. The purification is carried out as described in Example 1. Yield of 2-methoxyethyl ester of purified Gmm ganglioside: 4.2 g.

IR spectroscopic examination with KBr tablets shows a typical ester band at 1750 cm -1. Chromatographic analysis of the product carried out as described in Example 1 shows the presence of a uniform compound, Rf 0.81, and the absence of GjKj_ganglioside and its internal ester (Rf 0.65 and 0.75, respectively). Hydrolysis with Na 2 O 3 as described in Example 1 yields the ganglioside Gmi.

Example 34. Mixture of 1-methoxy-2-propyl esters of a mixture of gangliosides A mixture of 1-methoxy-2-propyl esters is prepared and isolated in the same manner as the mixture of methyl esters of Example 2 using 1-methoxy-2-propanol and sodium 1-methoxy-2 propylate instead of methyl alcohol and sodium methylate and by heating on a water bath using a mixture of 5 g of internal esters and 657.0 mg (5.86 mmol) of sodium 1-methoxy-2-propylate. The Dowex resin is washed with 1-methoxy-2-propyl alcohol and the residue obtained by evaporating the filtered substance is dissolved in 50 ml of a 1: 1 mixture of chloroform and ethanol. The yield of crude product is 5.2 g. The purification is carried out in the same manner as in Example 2. Yield of purified ester mixture: 4.3 g.

IR spectroscopy with KBr tablets shows a typical ester band at 1750 cm -1. When chromatographed on silica gel plates with freshly prepared IM 55:45:10 solution of chloroform, methanol and tetramethylammonium and determined with Ehrlich's reagent, the product has Rf values of 0.52-0.80 (ganglioside mixture 0.20-0.60). Complete conversion inhibition is performed as described in Example 2.

Example 35: Ganglioside G1 cyclohexyl ester 5 g of the ganglioside potassium salt (3.4 mmol) are dissolved in 50 ml of DMSO and 661.5 mg (3.75 mmol) of bromocyclohexane and 625 mg (3.75 mmol) of Ki are added. This is allowed to react for 48 hours at 25 ° C. At the end of the reaction, the solution is partitioned between n-butanol and N 2 O 3 (2: 1) to eliminate DMSO and salts. The butanol solution is evaporated to dryness and the residue is taken up in 50 ml of a mixture of chloroform and methanol and the product is precipitated with 250 ml of acetone. The crude product thus obtained (5.2 g) is then purified by preparative column chromatography on silica gel using a 60-35 s8 mixture of chloroform, methanol and water as a solvent. The pure fractions are stirred, evaporated, redissolved in 15 ml of a mixture of chloroform and isopropanol and the product is precipitated with 75 ml of acetone. Yield of pure cyclohexyl ester: 4.4 g.

IR spectroscopy on KBr tablets shows a typical ester band at 1750 cm-1. Chromatography on silica gel plates with a 60:40: 9 mixture of chloroform, methanol and CaCl 2 (0.3%) and a 2.5 N 55:45:10 mixture of chloroform, methanol and ammonia and determined with Ehrlich's reagent gives the product as a uniform compound and it has an Rf of 0.70 and 0.58, respectively, and is free of Gf4i® starting product (Rf 0.40 and 0.45, respectively). Treatment with a 0.1 N solution of Na 2 CO 3 at 60 ° for 1 hour causes cleavage of the ester bond and yields the starting anglioside G ^ i ·

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Example 36: Ganglioside G ^ i undecyl ester 4i 83423 5 g of the potassium salt of ganglioside G ^ i (3.14 mmol) are dissolved in 50 ml of DMSO and 882.1 mg (3.75 mmol) of 1-bromodecane and 625 mg (3 , 75 mmol) KI. This is allowed to react for 48 hours at 25 ° C. After completion of the reaction, the solution is partitioned between n-butanol / H 2 O (2: 1) to eliminate DMSO and salts. The butanol solution is evaporated to dryness and the residue is taken up in 50 ml of a mixture of chloroform and methanol and the reaction product is precipitated with 250 ml of acetone. The crude product thus obtained (5.3 g) is then purified by preparative flash chromatography on silica gel using a 60:35: 8 mixture of chloroform, methanol and water as solvent. The pure fractions are stirred, evaporated, redissolved in 15 ml of a 1: 1 mixture of chloroform and isopropanol and the product is precipitated with 75 ml of acetone. Yield of pure undecyl ester: 4.9 g.

IR spectroscopy on KBr tablets shows a typical ester band at 1750 cm-1. Chromatography on silica gel plates with a 60:40: 9 mixture of chloroform, methanol and CaCl 2 (0.3%) and a 2.5N 55:45:10 mixture of chloroform / methanol and ammonia and determined with Ehrlich's reagent shows that the product is uniform to a compound with an Rf of 0.68 and 0.56, respectively, and is free of GMi "from the starting material (Rf 0.40 and 0.45, respectively). Treatment with 0.1 N Na causes cleavage of the ester bond and produces the starting anglioside Gmi.

Example 37: Ganglioside Gmi 1-hydroxyundecan-11-yl ester 59 The potassium salt of ganglioside Gmi (3.14 mmol) is dissolved in 50 ml of DMSO and 0.42 mg (3.75 mmol) of 11- bromo-1-undecanol and 625 mg (3.75 mmol) of KI. This is allowed to react for 48 hours at 25 ° C. After completion of the reaction, the solution is partitioned between n-butanol / H 2 O (2: 1) to eliminate DMSO and salts. The butanol solution is evaporated to dryness and the residue is taken up in 50 ml of a 1: 1 mixture of chloroform and methanol and the reaction product is precipitated with 250 ml of acetone. The crude product thus obtained (5.3 g) is then purified by preparative column chromatography on silica gel using a 60: 35: 8 mixture of chloroform, methanol and water as a solvent. The pure fractions are stirred, evaporated, redissolved in 15 ml of a 1: 1 mixture of chloroform and isopropanol and the product is precipitated with 75 ml of acetone. Yield of pure 11-hydroxyundecan-11-yl ester: 4.8 g.

IR spectroscopy on KBr tablets shows a typical ester band at 1750 cm -1. Chromatography on silica gel plates with a 60:40: 9 mixture of chloroform, methanol and CaCl2 (0.3%) and a 2.5N 55:45:10 mixture of chloroform / methanol and ammonia and determined with Ehrlich's reagent shows that the product is uniform to a compound having an Rf of 0.65 and 0.52, respectively, and is free of Gjnn starting angioside (Rf 0.40 and 0.45, respectively). Treatment with a 0.1 N solution of Na 2 CO 3 at 60 ° for 1 hour causes cleavage of the ester bond and yields the initial ganglioside G 1.

Example 38: Ganglioside G ^ cyanobutyr-4-yl ester 5 g of the ganglioside G ^ i potassium salt (3.14 mmol) are dissolved in 50 ml of DMSO and 550 mg (3.75 mmol) of 4-bromobutyronitrile and 625 mg are added to the solution. (3.75 mmol) KI. Leave to react for 48 hours at 25 ° C. After completion of the reaction, the solution is partitioned between n-butanol and H 2 O (2: 1) to eliminate DMSO and salts. The butanol solution is evaporated to dryness and the residue is taken up in 50 ml of a 1: 1 mixture of chloroform and methanol and the product is precipitated with 250 ml of acetone. The crude product thus obtained (5.1 g) is then purified by preparative column chromatography on silica gel using a 60: 35: 8 mixture of chloroform, methanol and water as a solvent.

The pure fractions are stirred, evaporated, redissolved in 15 ml of a 1: 1 mixture of chloroform and isopropanol and the product is precipitated with 75 ml of acetone. Yield 4.6 g of pure cyanobutyr-4-yl ester.

IR 43 IR spectroscopy with KBr tablets shows a typical ester band at 1750 cm -1. Chromatography on silica gel plates with a 60:40: 9 mixture of chloroform, methanol and CaCl2 (0.3%) and a 2.5N 55:45:10 mixture of chloroform, methanol and ammonia and determined with Ehrlich's reagent gives the product as a homogeneous compound and it has an Rf of 0.42 and 0.45, respectively, and is free of the G 1 H starting anglioside (Rf 0.40 and 0.45, respectively). Treatment with a 0.1 N solution of Na 2 CO 3 at 60 ° for 1 hour causes cleavage of the ester bond and yields the starting anglioside Gmj_.

Example 39: Ganglioside GmI pyrrolidinamine This derivative is prepared from 5 g of ganglioside GmI internal ester (3.27 mmol) and 25 ml of pyrrolidine in the same manner as in Example 16 and following the same purification procedure. 5.1 g of pure ganglioside Gmi pyrrolidinamine are obtained.

The IR spectroscopic values are the same as for the methylamide of Example 16 and chromatographic analysis under the same conditions as in that example shows that the product is uniform and free of Gni and has an Rf of 0.29 and 0.46, respectively (GMi Rf 0.35 and 0.40, respectively).

Example 40; Mixture of pyrrolidinamides in a ganglioside mixture This derivative mixture is prepared from a mixture of 5 g of the internal esters of gangliosides used in Example 17 and 25 ml of pyrrolidine in the same manner as in Example 17, using the same purification method. 4.9 g of a mixture of pyrrolidine amides of a ganglioside mixture are obtained.

Rf values determined by chromatography on silica gel plates with a 4N mixture of chloroform, methanol and ammonia 55:45:10 and a mixture of chloroform, methanol and CaCl 2 (0.3%) 60:35: 8 and determined with a resorcinol reagent showed values of 0 .08 to 0.40 and 0.35 to 0.48, respectively (Rf of the original ganglioside mixture 0.15 to 0.70 and 0.05 to 0.40, respectively).

Example 41: Ganglioside G1 piperidinamide 44 83423 This derivative is prepared from 5 g of ganglioside G1 internal ester (3.27 mmol) and 25 ml of piperidine in the same manner as in Example 16 and the same purification procedure is also followed. 5.2 g of pure piperidinamide of the ganglioside G1 are obtained.

The IR spectroscopic values are the same as for the methylamide of Example 16 and chromatographic examination under the same conditions as in that example shows that the product is uniform and free of GI and has an Rf of 0.30 and 0.50, respectively (for GM1 0.35 and respectively 0.40).

Example 42: Mixture of piperidine amides of a ganglioside mixture This derivative mixture is prepared from a mixture of 5 g of the internal esters of gangliosides used in Example 17 and 25 ml of piperidine in the same manner as in Example 17, using the same purification method. 5.1 g of a mixture of piperidinamides of the ganglioside mixture are obtained. Rf values, determined by chromatography on silica gel plates with a 4N mixture of chloroform, methanol and ammonia 55:45:10 and a 60:35: 8 mixture of chloroform, methanol and CaCl 2 (0.3%) and determined with resorcinol reagent, showed values of 0 , 10 to 0.42 and 0.37 to 0.50, respectively (Rf of the original ganglioside mixture 0.15 to 0.70 and 0.05 to 0.40, respectively).

Example 43. Tetrahydrofurfurylamide of ganglioside G1 This derivative is prepared from 5 g of internal ester of ganglioside Gmi (3.27 mmol) and 25 ml of tetrahydrofurfurylamine in the same manner as in Example 16 and the same purification procedure is also followed. 5.3 g of pure ganglioside Gmi tetrahydrofurfurylamide are obtained.

The IR spectroscopic values are the same as for the methylamide of Example 16 and the chromatographic analysis in the same

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45 83423 under the conditions as in that example shows that the product is uniform and free of G 2 and has an Rf of 0.33 and 0.52, respectively (GM 1 Rf 0.35 and 0.40, respectively).

Example 44: Mixture of tetrahydrofurfurylamides of a ganglioside mixture This derivative mixture is prepared from a mixture of 5 g of the internal esters of gangliosides used in Example 17 and 25 ml of tetrahydrofurfurylamine in the same manner as in Example 17, and using the same purification method. 5.2 g of a mixture of tetrahydrofurfurylamides of the ganglioside mixture are obtained.

Rf values determined by chromatography on silica gel plates with a 4N mixture of chloroform, methanol and ammonia 55:45:10 and a mixture of chloroform, methanol and CaCl 2 (0.3%) 60:35: 8 and determined with a resorcinol reagent showed values of 0 , 12 to 0.45 and 0.40 to 0.57, respectively (Rf of the original ganglioside mixture 0.15 to 0.70 and 0.05 to 0.40, respectively).

Example 45: Ganglioside G 2 i-2-methylpiperidinamide This derivative is prepared from 5 g of ganglioside G 1 i internal ester (3.27 mmol) and 25 ml of 2-methylpiperidinamine in a sauna as in Example 16 and the same purification procedure is also followed. 5.2 g of 1 g of pure ganglioside 2-methylpiperidinamide are obtained.

The IR spectroscopic values are saunas as for the methylamide of Example 16 and chromatographic examination under the same conditions as in that example shows that the product is uniform and free of G 2 and has an Rf of 0.33 and 0.54 (Rf of 4i, respectively). , 35 and 0.40, respectively).

«83423

Example 46: Mixture of 2-methylpiperidinamides of a ganglioside mixture This derivative mixture is prepared from a mixture of 5 g of the internal esters of gangliosides used in Example 17 and 25 ml of 2-methylpiperidine in the same manner as in Example 17, using the same purification method. 5.4 g of a mixture of 2-methylpiperidinamides of a ganglioside mixture are obtained.

Rf values determined by chromatography on silica gel plates with a 4N 55:45:10 mixture of chloroform, methanol and ammonia and a 60:35: 8 mixture of chloroform, methanol and CaCl 2 (0.3%) and determined with a resorcinol reagent showed values of 0.14 to 0.46 and 0.39 to 0.59, respectively (Rf of the original ganglioside mixture 0.15 to 0.70 and 0.05 to 0.40, respectively).

Example 47: Ganglioside G1-1-methylpiperazinamide This derivative is prepared from 5 g of the internal ester of ganglioside G1 (3.27 mmol) and 25 ml of 1-methylpiperazine in the same manner as in Example 16 and following the same purification procedure. . 5.1 g of pure ganglioside G 1 -methylpiperazinamide are obtained. The IR spectroscopic values are the same as for the methylamide of Example 16 and chromatographic examination under the same conditions as in that example shows that the product is uniform and free of GI and has an Rf of 0.04 and 0.08, respectively (for GM1 0.35 and respectively 0.40).

Example 48; Mixture of 1-methylpiperazine amides of a ganglioside mixture This derivative mixture is prepared from a mixture of 5 g of the internal esters of gangliosides used in Example 17 and 25 ml of 1-methylpiperazine in the same manner as in Example 17, using the same purification method. 5.5 g of a mixture of 1-methylpiperazine amides in a ganglioside mixture are obtained.

47 83423

Rf values, determined by chromatography on silica gel plates with a 60: 35: 8 mixture of chloroform, methanol and CaCl 2 (0.3%) and determined with resorcinol reagent, showed values of 0.01 to 0.06 and 0.01 to 0, respectively. 12 (Rf of the initial mixture of gangliosides 0.15 to 0.70 and 0.05 to 0.40, respectively).

Example 49: Ganglioside 2-phenylethylamide This is prepared from 5 g of internal ganglioside ester (3.27 mmol) and 25 ml of 2-phenylethylamine in the same manner as in Example 16 and the same purification procedure is also followed. 5.3 g of pure ganglioside Gjuji 2-phenylethylamide are obtained.

The IR spectroscopic values are the same as for the methylamide of Example 16 and chromatographic examination under the same conditions as in that example shows that the product is uniform and free of Gm and has an Rf of 0.33 and 0.73, respectively (GmI 0.35 and respectively 0.40).

Example 50: Mixture of 2-phenylethylamides of a ganglioside mixture This derivative mixture is prepared from a mixture of 5 g of the internal esters of gangliosides used in Example 17 and 25 ml of 2-phenylethylamine in the same manner as in Example 17, using the same purification method. 5.5 g of a mixture of 2-phenylethylamides of a ganglioside mixture are obtained.

Rf values, determined by chromatography on silica gel plates with a 4N 55:45:10 mixture of chloroform, methanol and ammonia and a 60:35: 8 mixture of chloroform, methanol and CaCl 2 (0.3%) and determined with a resorcinol reagent, showed values of 0.12 to 0.44 and 0.60 to 0.78, respectively (Rf of the initial mixture of gangliosides 0.15 to 0.70 and 0.05 to 0.40, respectively).

Claims (3)

48 83423 A process for the preparation of ester derivatives of gangliosides of the general formula (ia) ROOCiime / 'siali-vitmiiiOH /' imimiiOH acid E e plum OH ceramide ..... '«H 5 Ö OH OH Ö (J) OH OLIGOSACCHARIDE OH, wherein R is a residue of an aliphatic alcohol having up to 12 carbon atoms, a residue of an araliphatic alcohol having up to 12 carbon atoms with only one benzene ring, a residue of a heterocyclic alcohol having up to 12 carbon atoms with a nitrogen, sulfur or oxygen heteroatom heterocyclic ring having up to 14 carbon atoms a residue, or a residue of an aliphatic-alicyclic alcohol having up to 14 carbon atoms and containing only one cyclo-aliphatic ring, the abovementioned residues being optionally substituted, characterized in that the internal ester of the ganglioside is treated with an alcohol to form the corresponding ganglios in the carboxyl groups of the sialic acid residues of the internal ester of the idi or a mixture thereof, the treatment being carried out in the presence of a metal alcoholate corresponding to the alcohol. Process according to Claim 1, characterized in that the alcohol used is an aliphatic alcohol having up to 12 carbon atoms; an araliphatic alcohol having only one benzene ring optionally substituted with 1 to 3 C 1-4 alkyl groups and having up to 4 carbon atoms in the aliphatic chain; a heterocyclic alcohol having up to 12 carbon atoms and only one heterocyclic ring containing a heteroatom which is N, S, or O; or a C 1-4 alicyclic or aliphatic alicyclic alcohol. Process according to Claim 1, characterized in that the alcohol used is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, undecyl, hydroxydecyl, heptyl, 2-methyl-1-pentyl, allyl, ethoxycarbonylmethyl, methoxyethyl, 1-methoxy-2-propyl, benzyl, phenethyl, cyclohexyl, menthyl, tetrahydrofurfuryl, tetrahydropyranyl or cyanobutyryl alcohol. so 83423