NO751908L - - Google Patents

Description

"Procedure for manufacturing

ling of Bulfoglycopeptides"

The present invention relates to a process for the production of aulfoglycopeptides by starting from glycopeptides extracted from milk or similar cateins. Ammonium and metal salts of the thus obtained aulfoglycopeptides can be used as therapeutic agents in the treatment of osteoarthritis and stomach ulcers as well as as scar preventing and fibrinolytic agents.

Extraction of glycopeptides from the milk of various mammals or from the corresponding caseins is already well known. A method is also known for the production of sulfoglyco-peptides by starting from glycopeptides extracted from animal organs, in particular from the mucous membrane of the stomach or the duodenum of pigs.

It has now been found that the working conditions used to prepare the known sulfoglycopeptides are not effective when used for sulfonation of glycoproteins extracted from milk or caseins. The impossibility of transferring the working conditions that effectively sulfonate glycoproteins extracted from animal organs, especially from the gastric mucosa or duodenum of pigs, to the method for sulfonating glycol proteins extracted from milk or caseins, is due to the different chemical composition of the two types of glycol proteins . The chemical composition of glycoproteins obtained from milk or caseins is namely, compared to glycoproteins extracted from animal organs, characterized by a higher ratio between the amino acid and carbohydrate content, as well as by a greater amount of phosphorylated amino acids and neuraminic acid.

The purpose of the present invention is therefore to provide a method for the production of sulfoglycopeptides by starting from glycopeptides extracted from milk or cain, in which the working steps are of such a nature that they correspond to the special properties of these glycopeptides.

The process for sulfonation of glycopeptides extracted from milk or caseins Inconsistency with the invention, comprises the following steps

a) dissolution of a glycopeptide extracted from milk or casein

in an aprotic, non-aromatic, water-miscible solvent, b) addition to the solution of a) of a tertiary ba©aom having a boiling point from approx. 100°C to ce. 250°c, c) sulfonating the product of b) by bringing it into contact with a sulfonating agent selected from the group consisting of sulfuric acid,

oleoresin, chlorosulfonic acid or an adduct of sulfuric anhydride and an organic compound, initially at a temperature of

from etc. -20°c to + 20°c, and then at a temperature from approx.

60°C to approx. 90°C, and recovery of the resulting sulfonyl glycopeptide. The glycopeptide can be added, if desired, after the addition of the tertiary base, even after the addition of the sulfonating agent, instead of in step a).

It has now been found that although the optimal sulfonation conditions, in the case of glycopeptides extracted from animal organs, are obtained when the glycopeptide is suspended in a reaction medium consisting of a heterocyclic tertiary base in a manner similar to the sulfonation reaction place in a heterogeneous phase, the optimal sulfonation conditions, in the case of sulfoglycopeptides extracted from milk or caseins, are achieved when. The glycopeptide is completely dissolved in a solvent phase consisting of an aprotic, non-aromatic, water-miscible compound and a tertiary base, preferably an aromatic base, which has a boiling point of from approx. 1C0°C to 250°C

In order to achieve dissolution of the glycopeptide extracted from QBlk or caseins under conditions that are best suited for carrying out the method according to the invention, it is advantageous to use in mixture with the tertiary base, which is preferably a

heterocyclic tertiary base selected from the group consisting of pyridine, methylpyridine, dimethylpyridine and quinoline, an aprotic, non-aromatic water-miscible solvent, such as e.g. dimstylformamide, dimethylsulfoxide, hexamethylphosphoramide, dimethylacetamide and the like. The method according to the invention will now be described in detail with reference to the main working steps.

Resolution

Glycopeptide extracted from milk or casein is first thoroughly dried and ground and then dissolved in an aprotic, non-aromatic, water-miscible solvent, e.g. dimethylformamide, dimethylsulfoxide, hexamethylformamide, dlmethylacetamide and the like. A tertiary base, preferably an aromatic base, which has a boiling point from approx. 100°C to 250°C. Typical close heterocyclic bases include pyridine, methylpyridines, ethylpyridines, dimethylpyridines and quinoline. Anhydrous pyridine is particularly preferred.

Sulfonation

Suitable sulfonating agents include sulfuric acid, oleum, chlorosulfonic acid and adducts of sulfuric anhydride with organic compounds, such as pyridine, dioxane and tertiary amines (eg triethylamine). When using oleum or chlorosulfonic acids, it may be advantageous to separately combine the sulfonating agent with the heterocyclic base, and use the resulting adduct in the sulfonation reaction.

The sulfonation reaction is carried out by bringing the glycopeptide-containing solvent mixture into contact with the sulfonating agent at an initial temperature between approx. -20°c and approx. +20°C, and then at a higher temperature between approx. 60°c and approx. 90°C, preferably at approx. 80°c, for from 4 to 8 and preferably 5 hours. The quantity of the sulfonating agent is between approx. 400 and 600%, preferably 500%, calculated on the weight of the glycopeptide.

At the end of the sulphonation reaction, the sulphonate product is removed by filtration, and the impurities are removed with the dissolution phase by dialysis or by treatment with ion exchange resins.

Sulfoglycopeptide salts

The sulfoglycopeptide thus obtained is then converted to the corresponding sodium salt with sodium hydroxide, and the sodium salt is precipitated by diluting its aqueous solutions with suitable solvents, such as acetone or methanol.

The same procedure can generally be used to convert the sulfoglycopeptide into corresponding salts with other alkali, alkaline earth and heavy metals, or into corresponding ammonium salts.

These salts can be obtained from the same sulfoglycopeptld formed by the sulfonation reaction, by means of neutralization with alkali, alkaline earth or ammonium hydroxide as well as from the sodium salt by double exchange reaction with an alkaline earth metal or heavy metal salt. Salts obtained by neutralization of sulfoglycopeptid with metal hydroxide are isolated by treating their aqueous solutions with a precipitating agent such as acetone or methanol. It has been found that particularly favorable results are obtained when, before precipitation, a small amount of from approx. 3 to approx. 7% by weight based on the weight of the hydroxide, of an acetate or halide of the same metal if the hydroxide is used for the neutralization reaction. Cations most suitable for this embodiment are potassium, lithium, calcium, barium, strontium and ammonium.

The double exchange reaction between the sulfoglycopeptide sodium salt and salts of other metals is carried out in aqueous solution, the anion of the salt in question preferably consisting of an acetate or halide. The double exchange reaction can be carried out by percolating an aqueous solution of the sodium salt of sulfoglycopeptide through a column of strong cation exchange resin which has previously been presalted with the metal cation whose sulfoglycopeptide salt is desired to be prepared. The percolated solution is then treated with a precipitating agent, such as acetone or methanol, preferably after the addition of a small amount of an acetate or halide of the same metal whose salt is desired. Salts of sulfoglycopeptid with the following metals zinc, calcium, barium, strontium, copper, nickel, cobalt, magnesium, bismuth, gold and aluminum can be prepared particularly advantageously by this double exchange reaction.

The following table shows some analytical values which are characteristic of sodium salts of sulfoglycopeptid prepared as indicated above, corresponding to materials with the lowest and highest degrees of sulfonation

From a pharmacological point of view, the advantages obtained by sulphonation of glycopeptide extracted from keeein consist in the presence of new anti-ulcer, anti-peptic and anti-excretion properties.

The pharmacological results showed that ulfoglycopeptide salts according to the invention were not toxic even at doses of

400 mg/kg endoperitonea.lt, and 1 g/kg orally. When administered orally at doses of 40, 64, 102.4 and 163.8 mg Ag, sulfoglycopeptid produced inhibition of the gastric ulcer grade of '&i^^Mc^fc""a£p"(N.S.), ~ 0^~ §®7^ fWe~^&&©976%, ' The antipeptic activity was investigated both in vivo and in vitro. The concentrations used (from 0.5 to 4 mg/ral) and the doses administered (from 40 to 160 mg Ag) could be compared with those already described for other sulfonates

macromolecules (Lietti et al., Boll. Soc. It.Biol. Sper. 47,493, 1971). ■*■ -

The secretion-inhibiting activity could be demonstrated when the uric acid secretion was stimulated with the help of histamine and pantagatrih. The following, non-limiting examples illustrate the method according to the invention.

Example 1

A mixture of 170 ml of dimethylformamide (DMF) and 230 ml of pyridine was cooled to -20°C. 60 ml of carbon sulfonic acid was added to this solution, taking care not to exceed a temperature of -15°C. Under these conditions, a pyridine/chlorosulfonic acid adduct was formed. During this adduct-containing phase, 20 grams of glycopeptide extracted from milk which had previously been thoroughly dried and ball milled were added while stirring at 20°c. After the addition was complete, the temperature was raised to 80°C and held at 80°C for 3 hours. The heating and stirring were then interrupted and the mixture was allowed to stand at room temperature for 2 hours. A gel mass was formed which was isolated by decantation. From this mass, a yellowish powder was precipitated by the addition of 150 ral of methanol, which was collected by filtration and dissolved in 250 ml of water. The aqueous solution was treated with 200 ml of a cation-exchange resin ("Araberlite" (^R-120) • After 10 minutes, the resin was removed by filtration, and the filtrate was treated with 200 ml of anion-exchange resin ("Amberlite" XR- 410). This resin was also removed by filtration after 10 minutes, and the pH of the filtrate was adjusted to 12 by means of a sodium hydroxide solution and then to 6.5 by the addition of acetic acid. The solution was evaporated under reduced pressure to one vol. of 200 ml and then treated with 4 grams of sodium iodide and diluted with 1.2 volumes of acetone to obtain a precipitate.The precipitate was washed with ethanol until iodide ions disappeared, then with ethyl ether and finally dried.

19 grams of an ivory colored powder which had the following composition was collected

Example 2

To a mixture of dimethylformamide (180 ml) and dry pyridine (180 ml) cooled to -20°C in a reaction vessel was added a mixture of 16 ml of fuming sulfuric acid (65% SO 4 ) and 16 ml of concentrated sulfuric acid, made sure not to exceed -15°c. To the thus obtained suspension was added 15 g of a glycopeptide obtained from cow's milk casein, thoroughly dried and ground in a ball mill, with stirring at 20°c. After the addition was complete, the temperature of the mixture was raised to 75°C and held at this value for 2.5 hours. Stirring was then stopped and the mixture held at 75°C for 90 minutes.

An oily mass settled at the bottom of the reaction vessel and was separated by decantation. Addition of ethyl alcohol (120 ml) gave a sticky precipitate which was washed with tsad methanol, then with ethyl ether to give 23 g of a light brown powder. The crude product was dissolved in 250 ml of distilled water and purified as described in the previous example.

16 g of an ivory colored powder with the following composition was obtained

Example 3

A mixture of 200 grams of dry pyridine and 300 g of methyl sulfoxide cooled to -20°C was prepared. 30 ml of chlorosulfonic acid was added dropwise to this mixture, taking care not to exceed the temperature of 20°c. After the completion of the addition of chlorosulfonic acid, the temperature of the mixture was brought to 20°C. Then 15 grams of glycopeptide extracted from milk and which had previously been thoroughly dried and finally ground in a ball mill were added to the mixture. After the glycopeptide addition was completed, the temperature of the mixture was raised to 85°C and held at this value for 4 hours. The mixture was allowed to stand at room temperature for 3 hours. A clear, gelatinous mass settled at the bottom of the reaction vessel, which was separated by decantation from the overlying phase. Addition of 120 ml of methanol to the gelatinous mass resulted in the formation of a pale ivory colored powder which was dissolved in 200 ml of distilled water and subjected to the same purification process as in the previous examples. In the end, 18 grams of a white powder was obtained which had the following composition (based on the dry product):

Example 4''

100 g of the sodium salt of glycopeptide extracted from cow's milk and sulfonated as described in Example 1 was dissolved in 1000 ml of water and poured into 1000 ml of a 0.3 m solution of bismuth nitrate in glacial acetic acid.

About 2000 ml of methanol was added to the solution, formed

there was a precipitate which was collected by centrifugation and washed three times by trituration with a 3 s methanol-acetic acid mixture and then three times with acetone, giving 1 gram of a powdered product. The product was dried under vacuum and then dissolved in 15 parts by volume of distilled water. A 1 N solution of aqueous sodium hydroxide was added to this solution to adjust its pB to 6.5. After dilution with 1.5 parts by volume of acetone, a precipitate formed.

This precipitate was collected by centrifugation, washed three times with one 2 tl acetone-water mixture and then three times with acetone. The final product was dried under vacuum to give 105 g of a product having the following percentages of sulfur and bismuth S «-7.8%:, • •;Bi:« 17.1%' -,'

Example 5

By working analogously to the method illustrated in example 4, the following salts of sulfonated glycopeptide were obtained"

aj the magnesium salt of sulfonated glycopeptide

b) the calcium salt of sulfonated glycopeptide c) the aluminum salt of sulfonated glycopeptide

Claims (7)

1. Process for the production of sulfoglycopeptides starting from glycopeptides extracted from milk or casein, characterized by the following steps" 1) one provides a mixture of a) a glycopeptide extracted from milk or casein, b) an aprotic, non-aromatic, water-miscible solvent, c) a tertiary base with a boiling point from approx. 100°C to approx. 250°C, d) a sulfonating agent selected from the group consisting of sulfuric acid, d oleum, chlorosulfonic acid and an adduct of sulfuric anhydride and an organic compound, 2) maintains the initial temperature at the contact between glycopeptide r» sulfrwsering&^iddelat at from approx. -20°C.to4 ;2 <0> oC, 3) raises the temperature to a range from approx. 60°C to approx. 90°c and 4) recovering the resulting sulfoglycopeptid. 2. Method as stated in claim 1, character!-s is t in that the aprotic solvent is dimethylformamide, dimethylsulfoxide, hexamethylphosphoramide or immethylacetamide. 3.. Method as set forth in claim 2, characterized in that the tertiary Bese is a heterocyclic base selected from the group consisting of pyridine, methylpyridine, dimethylpyrlidine and chloline. 4. Method as stated in claim 1, character!-s is t in that the contact time with the sulfonating agent in steps 2) and 3) is from approx. 4 to approx. 8 hours. 5. Method as stated in claim 1, k & r& k t e r i-s e r t in the following further step 5) one reacts the sulfoglycopeptide of 4) with a hydroxide selected from the group consisting of alkali, alkaline earth metal and ammonium hydroxide to convert the sulfoglycopeptide into its corresponding alkali -, earth-alkalimetsTl or ammonium øalt» and 6) bringing the resulting product of 5) into contact with acetone or methyl alcohol to precipitate said salt. 6. Process as stated in claim 5, characterized in that the hydroxide is sodium hydroxide and the sulfoglycopeptide is the sodium salt. 7. Method as stated in claim 6, characterized in that an aqueous solution of the sodium salt percoleroa through a cation-exchange resin which has as cation a metal selected from the group consisting of zinc, calcium, barium, strontium, copper, nickel, cobalt, magnesium, bismuth, gold and aluminium.