DE2523432A1 - PROCESS FOR THE PREPARATION OF SULFOGLYCOPEPTIDES FROM GLYCOPEPTIDES - Google Patents

Description

FROM KREISLER SCHONWALD MEYER EiSHOLD FUES FROM KREISLER KELLER SELTING

PATENT LAWYERS Dr.-Ing. by Kreisler f 1973

Dr.-Ing. K. Schönwa'd, Cologne Dr.-Ing. Th. Meyer, Cologne Dr.-Ing. K. W. Eishold, Bad Soden Dr. J. F. Fues, Cologne Dipl.-Chem. AIeIc from Kreisler, Cologne Dipl.-Chem. Carola Keller, Cologne Dipl.-Ing. G. Selting, Cologne

AvK / Ax

5 Cologne ι May 26th in 37a

DEICHMANNHAUS AT THE MAIN RAILWAY STATION

Crinos Industria Farmacobiologica S.p.A., Villa Guardia / Italy

Process for the production of sulfoglycopeptides from glycopeptides

The invention relates to a method for the preparation of sulfoglycopeptides from glycopeptides obtained from milk or • the corresponding gas units are extracted. According to a further feature, the invention is based on ammonium and metal salts of the sulfoglycopeptides prepared according to the invention directed the valuable medicines for the treatment of osteoarthritis and gastric ulcer as well are valuable as anti-scarring agents and fibrinolytic agents.

The extraction of glycopeptides from milk of various mammals or from the corresponding caseins has been made already described in numerous publications. (C.Alais and P.Jolles, Biochim. Biophys. Acta 51 (1961) 315, J. Carbulis and C.A. Zittle, Arch. Biochem. Biophys.

81 (1959) 418, T. A. J. Payens, Biochem. Biophys. Acta 46 (1961) - 441, H.A.McKenzie and R.G. Wake, Biochem. Biophys. Acta 47 (1961) 240, C.A. Zittle - J.Dairy Sci. 45 (1962) 650, R.D. Hill - J.Dairy Res. 30 (1963) 101, K.K. Tripatbi and C.W. Gehrke - J.Chromatog. 46: 280 (1970).

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Telephone: (02 21) 23 45 41 - 4 - Telex : 888 2307 dopa d ■ Telegram : Dompatent Cologne

US Pat. No. 3,518,243 also discloses a process for the preparation of sulfoglycopeptides from glycopeptides known from animal organs, in particular from the gastric mucosa or the duodenum of the pig have been extracted. According to this US patent, the sulfonation of glycoproteins is animal Origin using the adducts of a heterocyclic tertiary base with sulfonating agents, e.g. Chlorosulfonic acid and oleum.

It has now been found that the in of the US patent mentioned sulfoglycopeptides applied Working conditions are not effective when they act on the sulfonation of glycoproteins that come from milk or caseins extracted can be applied.

The impossibility of transmission of the working conditions under which the glycoproteins nen from animal organisms [, especially of the gastric mucosa or; Duodenum of the pig are extracted, effectively sulfo-; as such, to the process of sulphonation of glycopro- extracted from milk or caseins; due to the different chemical compositions \

composition of the two types of glycoproteins. j The chemical composition of glycoproteins that

obtained from milk or caseins is in use;

equal to the glycosides extracted from animal organs

proteins due to a higher ratio of amino acids to:

Carbohydrate as well as a larger amount of phos-!

labeled phorylated amino acids and neuraminic acid, net, as the following table shows.

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Glycopeptides from milk from animal Organs 0.5 phosphorus - 16.9. Hexoses as a whole 23.1 7.7 Total hexosamines 45.1 . 64.7 Total amino acids 21.6 8.6 N-acyl neuraminic acid 4.8

These differences in chemical composition,

. to which there are also notable differences in ^:

physical properties (solubility, molecular:

weight etc.) require the implementation of the ■

Sulfonation reaction under different conditions. ■

In particular, it is necessary to check the solubility of these;

.Glycoproteins to be consumed during the sulfonation stage!

better to a satisfactory yield of sulfonated! ■

Obtain product of the desired quality. ■

The invention accordingly relates to the production of sulfoglycopeptides starting from milk or ' Caseins extracted glycopeptides. according to a procedure ^ whose work stages the special properties of this Glycopeptides are adapted. - J

The invention also includes ammonium and metal salts of those extracted from milk or caseins by sulfonation Glycopeptides obtained sulfoglycopeptides. These ; Salts have valuable therapeutic properties. !

The method according to the invention for sulfonating glycopeptides extracted from milk or caseins comprises the following stages:

. a) The glycopeptides extracted from milk or casein are dissolved in an aprotic, non-aromatic one water-miscible solvents.

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for the solution (a) ti) Place a tertiary base with a boiling point of about 100 ° to 250 ⁰ C.

the product obtained in step (b) c) One sulfonated by treatment with a sulfonating agent from the group of sulfuric acid, oleum, chlorosulfonic acid, and adducts of sulfuric anhydride with an organic compound existing first at a temperature of about -20 ° C to +20 ⁰ and then at a temperature of about 60 to about 90 ^° C. and isolates the sulfoglycopeptide formed.

The glycopeptide can optionally after the addition of the tertiary base or even after the addition of the Sulphonating agent added instead of in step (a) will. I.

'While the optimal sulfonation conditions in the case of glycopeptides' extracted from animal organs are achieved when the glycopeptide is suspended in a reaction medium consisting of a heterocyclic tertiary base in such a way that the sulfonation reaction takes place in a heterogeneous phase, it has been found that in the case of sulfoglycopeptides extracted from milk or caseins, the optimal sulfonation conditions are achieved when the glycopeptide is in a solvent phase consisting of an aprotic, non-aromatic, water-miscible compound and a tertiary base, preferably an aromatic base with a boiling point of about 100 to 250 ⁰ C exists, is completely dissolved.

To dissolve the extracted from milk or caseins To achieve glycopeptide under conditions that are suitable for carrying out the method according to the invention are most suitable, it has been found to be advantageous in a mixture with the tertiary base, which is preferably a heterocyclic tertiary base from the pyridine,

Methylpyridine, dimethylpyridine and Cbinolin is to use an aprotic, non-aromatic, water-miscible solvent, such as dimethylformamide, dimethyl sulfoxide, hexamethylphosphoramide and dimethylacetamide. '.

The main stages of the procedure according to of the invention are described in detail below.

Resolution i

The glycopeptide which has been extracted from milk or casein as described in the above-identified publications manner is first well dried and ground and then, in an aprotic non-flavored [tables, with V / ater-miscible solvent, eg dimethylformamide Dimethyl sulfoxide, hexamethyl phosphoramide and dimethylacetamide, dissolved. A tertiary base, preferably an aromatic base with a boiling point of about 100 to 250 °, is added to the solution obtained. As examples of typical tertiary heterocyclic bases are pyridine, methylpyridines, ethylpyridines,! Dimethylpyridine and ghinoline should be mentioned. Anhydrous pyridine is particularly preferred.

Sulfonation I

Sulfuric acid, oleum, Chlorosulfonic acid and the adducts of sulfuric acid an-; hydride with organic compounds, e.g. pyridine, | Dioxane and tertiary amines (e.g. triethylamine). if Oleum or chlorosulfonic acids can be used, it can; be expedient to separate the sulfonant with ί to live together of the heterocyclic base and get the i

i tene adduct to use in the sulfonation reaction.

The sulfonation reaction is carried out. ■; ■ leads by the the mixed solvent containing the glycopeptide with the;

i sulfonating agent at an initial temperature between ι

5 0 9 8 ~ 5ΊΓ / Ί) 9 81

about -20 ° and about 20 C and then at an elevated temperature between about 60 and about 90 G, preferably at about 80 ⁰ G, 4 to 8 hours, preferably 5 hours.

After the sulfonation reaction has ended, this is called The sulfonate formed is filtered off, whereupon the impurities removed with the solvent phase by dialysis or by treatment with ion exchange resins will. ":

Sulfoglycopeptide salts

The sulfoglycopeptide prepared in the manner described is then converted into the corresponding sodium salt with sodium hydroxide. The sodium salt is made by diluting it its aqueous solution with a suitable solvent, e.g. acetone or methanol.

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

These salts can be obtained from the same sulfoglycopeptide that has been obtained from the sulfonation reaction, by neutralization with alkali metals, alkaline earth metals or ammonium hydroxide, and from the sodium salt by a double exchange reaction with an alkaline earth metal or heavy metal salt. Salts which are obtained by neutralizing the sulfoglycopeptide with metal hydroxides are isolated by treating their aqueous solutions with a precipitant, for example acetone or methanol. It has been found that particularly good results are obtained if a small amount of about 3 to 7 wt. - ^c / o, based on the weight of the hydroxide, of one before the precipitation

Acetate or halide of the same metal whose Hydroxide used for the neutralization reaction is added. For this embodiment "especially suitable cations 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 also be carried out by adding an aqueous solution the Sulfoglykopeptid-ilatriumsalzes perko through a column of strong cationic exchange resin - can ', lose that has been previously transferred to the metal cation, its SuIfoglykopeptidsalz to be produced in the salt the percolated solution is with a precipitating agent, for example acetone or methanol. , treated after preferably adding a minor amount of an acetate or halide of the same metal whose salt is desired. Particularly suitable for the production due to this double design! exchange reaction are the salts of the sulfoglycopeptide with! the following metals: zinc, calcium, barium, strontium, copper, nickel, cobalt, magnesium, bismuth, gold and:

Aluminum. ^!

In the following table some analytical values are given, which are used in the manner described above produced sodium salts of sulfoglycopeptides characteristic are. These values apply to the connections with the lowest and the highest degree of suIfonung.

Pp. 5-1196

N 5-3 %

P. '0.2-0.5 $

Hexosamine 6 -

Hexoses 9 -

Proteins 45-50 %

N-acetylneuraminic acid $ 5 - $ 8 "

Viscosity 1.15-1.30 cps

pH 5.5 - 7

In animal studies, the following Properties for the sulfoglycopeptide salts prepared by the process according to the invention were found:

Pharmacological activity of sulfoglycopeptide, the obtained from the glycopeptide extracted from acid casein by the method according to the invention is. "·

The sulfoglycopeptide salts prepared by the process according to the invention (the sodium salt of Example 1 and the aluminum salt of example 5c) were tested. The results were matched with those with the appropriate results obtained from unsulfonated glycopeptide were compared.

From the pharmacological point of view, those resulted from sulfonation of the glycopeptide extracted from casein achieved benefits through new anti-ulcer, antipeptic and antisecretory properties. the Pharmacological studies were carried out on rats performed the following methods: j

a) Gastric ulcers caused by ligation of the pylorus (Shay and coworkers, Gastroenterology 5 (1945) 43 under the conditions of the test described by Prino et al. (Eur.J.Pharmac.15 (1971) 119.

b) Inhibition of the digestive effect of gastric juice according to the method of Prino and co-workers (Eur.J.Pharmac. 15 (1971) 119, Am.J.Dig.Dis. 17 (1972) 863.

c) Secretion of gastric acid according to the method of the perfused "Stilach" (Ghosh and Schild, Br_.J.Pharmac. 13 (1958) 54, Niada and Prino, Br.J. Pharmac. 48 (1973) 550. j

The results showed that the sulfoglycopeptide salts according to the invention are not toxic even at doses of 400 mg / kg endoperitoneai and 1 g / kg orally. If you administered orally at doses of 40, 64, 102.4 and 163.8 mg / kg, the sulfoglycopeptides' caused one Inhibition of the degree of ulceration by 15 $ · (N.S.), 465ε

(P. 0.01), 70? Ύ or 76%. The antipeptic effect was studied both in vivo and in vitro. The applied concentration (0.5 to 4 mg / ml) and the administered I The doses reached (40 to 160 mg / kg) were comparable to those already used for other sulfated macromolecules küle have been described (Lietti and coworkers, BoIl.. Soc.It. Biol. Sper. 47 (1971) 493). !

The antisecretory effect was shown when the acid secretion was stimulated by histamine and pentagastrin

became. j

The method according to the invention is carried out by the following j

the examples further explained «

example 1

A mixture of 170 ml of dimethylformamide (DMP) and 230 ml Pyridine was cooled to -20 ° C. 60 ml of chlorosulphonic acid were added to this solution, whereby on it care was taken that the temperature of -15 0 was not exceeded. Under these conditions a Adduct of pyridine and chlorosulfonic acid formed. Of the the adduct-containing phase were 20 g of glycopeptide, which was carefully dried and in the Ball mill treated milk had been extracted at

0 ^to

20 C given under guidance. After the addition was complete

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- ίο -

the temperature increased to 80 G and 3 hours at 80 C held. The heating and stirring were then discontinued and the mixture was left for 2 hours at room temperature was left standing.

A gel mass formed which was decanted by isolation. A yellowish pulper was precipitated from this mass by adding 150 ml of methanol, which was filtered off and dissolved in 250 ml of water. The aqueous solution was treated with 200 ml of cation exchange resin (Amberlite IR-120). After 10 minutes the resin was filtered off and the filtrate was treated with 200 ml of anion exchange resin (Amberlite IR-4-10). Au-h this resin was filtered off after 10 minutes. The filtrate was adjusted to pH 12 with sodium hydroxide solution and then to pH 6.5 with acetic acid. The solution was concentrated to a volume of 200 ml under reduced pressure and then treated with 4 g of sodium iodide and diluted with 1.2 times the volume of acetone, a precipitate! development was formed. The precipitation was carried out with methanol up to; . for the disappearance of the iodide ions and then with ethyl ether _:

washed and finally dried. Here j

i 19 g of an ivory-colored powder that isolates the ^!

had the following composition:

Moisture $ 4.9

S 8.35 '/ *

'■ N $ 7.05

P. $ 0.3

Hexosamine $ 7.71

Hexoses $ 11.6

Proteins $ 46.44

N-acetylneuraminic acid $ 5.9

Viscosity 1.237 cPs

pH value 5 * 8 (1 $ in 0 _i 5 $ NaCl)

Well $ 6

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Example 2

To a cooled to -20 ⁰ C mixture of 1J30 mL of dimethylformamide and 180 ml of dry pyridine in a reaction vessel, a mixture of 16 ml of fuming sulfuric acid (65 $ SO ·) and, 16 ml of concentrated sulfuric acid was being careful that the Temperature -15 ⁰ C did not exceed. To the suspension thus obtained, 15 g of a glycopeptide obtained from cow's milk casein, dried well and ground in a ball mill, were added with stirring at 20 ° C. After the addition, the temperature of the mixture was raised to 75 ⁰ C and held for 2.5 hours at 75 ° C. The stirrer was then switched off and the mixture was ^{kept at 75 °} C. for 90 minutes.

An oily mass settled on the bottom of the reaction vessel and was separated off by decanting. By Addition of 120 ml of methyl alcohol formed a sticky precipitate with methanol and then with ethyl ether was washed to obtain 23 g of light brown powder. The raw product was in Dissolved 250 ml of distilled water and purified in the manner described in Example 1. Here were 16 g of an ivory colored powder of the following composition ■ received:

humidity 509850/0981 $ 5.45 - 6 ($ 1 in $ 0.5 NaCl) S. $ 8.48 $ 6.1 N $ 6.96 P. $ 0.24 Hexosamines $ 7.82 Hexoses $ 11.53 Proteins $ 47.15 N-acetyl neuraminic acid $ 6.1 viscosity 1.195 cps PH value N / A

Example 3 <

A chilled to -2O ⁰ C mixture of 200 g of pyridine and 300 g -trockenem Methylsulfoxyd was prepared. 50 ml of chlorosulfonic acid were added dropwise to this mixture, care being taken that the temperature did not exceed -20.degree. After the chlorosulfonic acid had been added, the temperature of the mixture was brought to 20 ° C. Then 15 pounds of glycopeptide, extracted from milk and previously dried well and finely ground in a ball mill, was added to the mixture.

After the addition of the glycopeptide, the temperature of the mixture was increased to 85 ° C. and 4 hours at 85 C held. The mixture was left to stand at room temperature for 3 hours. At the bottom of the reaction vessel:

a clear gelatinous mass settled, which was separated from the supernatant by .1 decanting. By adding 120 ml of methanol to the gelatinous mass;

• Formed a light ivory-colored powder that comes in 200 ml ^!

Dissolved distilled water and purified in the manner described in Example 1. The end product was 18 g of a white powder of the following composition _; (based on the dry product) received: -!

N 6.7 *

P- 0.2 *

Hexosamine 7.6 *

Hexoses 11

N-acetylneuraminic acid 5 *

Viscosity. - 1.2 cPa

pH 6

Na 6.3 *

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Example 4

10Og de3 sodium salt of the extracted from cub milk and glycopeptide sulfonated in the manner described in Example 1 were dissolved in 1000 ml of water and dissolved in 1OC0ml of a 0.3 molar solution of bismuth nitrate in Poured glacial acetic acid.

By adding 2000 ml of methanol to the solution, a precipitate formed, which was separated by centrifugation and washed three times by trituration with a methanol-acetic acid mixture (3: 1) and then three times with acetone, 110 g of a powdery product being obtained. The powder was min-; dertem print is dried and then distilled in the 15-fold volume \ V / ater dissolved. By adding an aqueous; The solution was adjusted to pH 6.5 i in sodium hydroxide solution. After dilution with 1.5 times the volume of acetone, a precipitate formed. The precipitate became

• separated by centrifugation, three times with a mixture of acetone and water (2: 1) and then three times with _; Acetone washed. The end product was reduced under-! dried under pressure to give 105 g of a product containing 7.8 $ sulfur and 17.1 $ bismuth.

Example 5

In the manner described in Example 4, the following salts of the sulfonated glycopeptide were prepared:

a) Magnesium salt of the sulfonated glycopeptide Mg 5.3 $ S 9.1 $

b) Calcium salt of the sulfonated glycopeptide

Approx $ 5.6. S a, 9 $ c) Aluminum salt of the sulfonated glycopeptide Al $ 8.6 S $ 8.6

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Claims (16)

-H- claims 1) Process for the production of sulfoglycopeptides from glycopeptides extracted from milk or casein have been, characterized in that nia'n A) a mixture of a) a glycated extract extracted from milk or casein peptide,
Td) an aprotic, non-aromatic, water-miscible solvent, c) a tertiary base with a boiling point of about 100 to 25O ⁰ C and d) a sulfonating agent made from the sulfuric acid, Oleum, chlorosulfonic acid and adducts of sulfuric anhydride and an organic compound forms, B) first obtains a contact temperature between the glycopeptide and the SuIfonierungsmittel of about -20 ° to 20 ⁰ C upright, C) raising the temperature to a value in the range of about 60 ° to 90 ⁰ C and D) the "sulfoglycopeptide formed" is isolated. 2) Method according to claim 1, characterized in that the aprotic solvent is dimethylformamide, Dimethyl sulfoxide, hexamethyl phosphoramide or Dimetbylacetamide used. 3) Process according to claim 1 and 2, characterized in that the tertiary base is a heterocyclic Base from the pyridine, methylpyridine, dimethyl . pyridine and ghinolin are used. 1TÜ 9ΊΓ5ΊΓ7ΤΠΓΒΎ 4) The method according to claim 1 to 3, characterized in that the duration of contact with the Sölfonierungsmittel in steps (B) and (C) is about 4 to 8 hours. | 5) Method according to claim 1 to 4, characterized by the following further stages:. j E) the sulfoglycopeptide formed in step (D) is set with an alkali, alkaline earth or ammonium hydroxide and thereby converts the sulfoglycopeptide into its corresponding alkali, alkaline earth or Ammonium salt around and F) treats the product obtained in step (E) with Acetone or methyl alcohol and this causes the salt to precipitate. 6) Process according to claims 1 to 5 » characterized in that sodium hydroxide is used as the hydroxide and the sodium salt of the sulfoglycopeptide is obtained. 7) Method according to claim 1 to 6, characterized in that that an aqueous solution of the sodium salt through a cation exchange resin, which is called the cation a metal made from zinc, calcium, barium, strontium, copper, nickel, cobalt, magnesium, bismuth, ■ Gold and aluminum existing group contains, percolate leaves. ^{8) 1} sulfoglycopeptide salts prepared by the process according to claims 1 to 7, in which the cation of the salt is an alkali metal, an alkaline earth metal, a heavy metal or ammonium. 9) salt according to claim 8 ,. characterized in that the cation is sodium. 5 0 9 8 5 0/0 ΪΒΤ 10) salt according to claim 8, characterized in that the cation is calcium. 11) salt according to claim 8, characterized in that the cation is barium. 12) salt according to claim 8, characterized in that the cation is magnesium. 13) salt according to claim 8, characterized in that the cation is aluminum. 14-) salt according to claim 8, characterized in that the cation is zinc. 15) salt according to claim 8, characterized in that the cation is copper. - 16) salt according to claim 8, characterized in that the cation is nickel. 5098 50/0981