DD216023A5 - PROCESS FOR THE PREPARATION OF A-RISTOMYCIN AND / OR RISTOMYCIN DERIVATIVES OF HIGH PURITY - Google Patents

Abstract

The invention relates to a process for the preparation of A-ristomycin and / or ristomycin derivatives for use in platelet aggregation experiments for the diagnosis of Willebrand disease. The aim of the invention is the preparation of A-ristomycin in suitable constitution and high purity. According to the invention, A-ristomycin and / or ristomycin derivatives of the general formula (I) are prepared in which, for example: R is a hydrogen atom, R 1 is an O-alpha-D-Araf (1 -> 2) -O-alpha-D -Manp (1 -> 2) -O-alpha-L-Rhap) - (1 -> 6) -O-beta-D-Glop group, R deep 2 a 2, 3, 6-Tridesoxy-3-amino - (NHY) -alpha-L-ribo-hexapyransyl group, R deep 3 an alpha-D-mannopyranosyl group, R deep 4 an alkyl group containing 1-4 carbon atoms, X the radical of a mineral acid, Y a hydrogen atom.

Description

-Y- Berlin, 9. 2. 1984 AP C Ο? D / 255 014/2 62 997 18

Process for the preparation of A-ristomycin and / or ristomycin derivatives;

Field of application of the invention

i '

The invention relates to a process for the preparation of A-ristomycin and / or ristomycin derivatives. The invention furthermore relates to a biological reagent for platelet aggregation experiments which comprises the A-ristomycin prepared according to the invention, which is used in particular for the diagnosis of Willebrand disease, and a method of making the same · .- ''

Characteristic of the known technical solutions

It is known that in the screening of hemophilic patients in the assessment of platelet functions the most valuable information is provided by the platelet aggregation experiments · lifting the aggregation agents used according to the routing (eg ADP, epinephrine, collagen) since 1971 called the ristocetin, substance previously used as an antibiotic * Based on the experiments of Howard and Firkin, ristocetin made it possible to quickly and easily detect hemophilia, Willebrand's disease (thrombosis and diathesis Haemorhagica 26, 362 (1971)). .

The essence of von Willebrand's disease is a defect of the VIII complex of coagulation. The most important biological role of the von Willebrand factor is the promotion of platelet adhesion to the sub endo the oral formulas. The deficiency or qualitative disorder of the von Willebrand factor calls

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    ; '' '..'. - 2 -. , , , ,

It is very important to separate the patient suffering from von Willebrand's disease (heredity, family experiments, therapy), which is called Willebrand disease, which is the most common form of congenital hemophilia. The elucidation and secretion of such a condition before surgery is particularly important. .

The invention is based on the finding that the eistocetin widely used in the laboratory practice can be completely replaced by another member of the vancomyein antibiotic family - the ristomycin. The ristomycin can be made simpler and cheaper and is more favorable in plasma protein precipitation. * Due to the above properties, ristomycin has been found to be more favorable in hemato-biological experiments. The ristomyein used hitherto for the treatment of diseases caused by antibacterial infections can also be used in this new field of application (for diagnostic purposes). , ,

Soviet researchers isolated the ristomycin antibiotic from cultures of Proactinomyces fruetiferi var. Ristomycini by adsorption (advantageously on a SzDSz-3 or SzD7-3 cation exchange column) and elution with an acetonic ammonia solution or an ammonium acetate buffer (pH = 9-10). The 'ristomycin was isolated in the form of the base. Later, ristomycin - more like ristocetin - was split into two biologically active components - the A- and B-ristomycin. The production of the antibiotic in high-level operation has also been disclosed. For the determination of the active substance content of the fermentation broth and the

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In addition to the biological value and e-mood, a number of intermediate intermediates have also been prepared by polarimetric and UV spectrophotometric methods (SU-PS 180 754; Antibiotics 8, 392 (1962); Antibiotics jj>, 884 (1964); Antibiotics 10, (1965); 12, 129 (19,67); Antibiotiki 16, 786 (1971)).

After determining the exact structure of A-ristomyeins (J. Org, Chem, 21 * 2971 (1974); J. Antibiot, ^ 2, 446 (1979); Tetrahedron Letters 21, 2983 (1980); JACS JKD2); 7093 (198O)), we have tried to find out to what extent and to what extent the change in the structure of the compound or the degree of purity of the compound influence the platelet aggregation.

Aim of the invention

The aim of the invention is the provision of ristomycin and / or ristomycin derivatives which are suitable for platelet aggregation experiments for the diagnosis of Willebrand's disease. , ·; ·.

Explanation of the essence of the invention .

The invention has for its object to provide a process for the preparation of A-ristomycin in high purity " , '

According to the invention, ristomycin and / or ristomycin derivatives of the general formula (I)

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R-I-O

IJHY

produced, in which

(I)

R is a hydrogen atom,

R _{1 is} an O-alpha-D-Araf (1- * 2) -O-alpha-D-Manp (1 " -2) -0-

- (alpha-L-Rhap) - (i- * 6) -O-beta-D-Qlop group,

, 62 997 18

R _{2 represents} a 2,3,6-trideoxy-3-amino- (HHY) -alpha-L-ribo-hexapyranosyl group, ', .

R, an alpha-D-mannopyranosyl group. is

R, an alkyl group containing 1 to 4 carbon atoms, means. ,

Σ represents the radical of a mineral acid.,;

Y represents a hydrogen atom or a group of the general formula C (Z), - (CH ₂₎ -CO-, wherein Z is a hydrogen atom or a halogen atom and η is an integer between 0 and 5.

It was found that the. partial or complete removal of R .., Rp, Ro and R occurring in the general formula (I), groups or the blocking of the hydroxyl functions of these groups and the R group (in the form of an ester or ether) undesirable biological effects The carboxy-A-ristomycin obtained by replacing the R 1 group by a hydrogen atom does not exert any aggregating effect on human platelet plasma and does not inhibit ristomycin-induced platelet aggregation The phenolic hydroxy groups blocked tetra-O-methyl-A-ristomycin derivative (R = R ₄ = CHo) also has unfavorable properties · The peracetylated A-ristomycin is insoluble under the conditions used in the experiments and for the purpose of experiments not suitable. Although the aglyco-A-ristomycin (R = R 1 = R ₂ = IRo = Y = H; R = CH-) obtained by the carbohydrate hydrolysis causes agglutination, this derivative is also not optimal because of the pH and solubility conditions our knowledge is also the B-Ristomycin

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(This compound differs from the other derivatives only in that in the general formula (Γ) in the heterotetrasaccharide lcette of R ... the 2-0-D-arabinofuranosylalpha-D-manno-pyranosyl portion is missing) to the desired diagnostic Purpose unsuitable.

It has surprisingly been found that the active ingredient used in the blood coagulation test is optimal if and only if it has a high purity and a precisely defined structure of the general formula (I). The compounds of the general formula (I) in which Y represents hydrogen or CF.sub.1, - CO-- are advantageously suitable in high-purity form for laboratory diagnostic purposes,

It has been found due to the obigen'Erkenntnisse that is to _v exercise of normal human iPhrombocytenaggregationerforderliche basic condition is that in the general formula I four free phenolic hydroxyl groups (E = H), the C-tenninale Methozycarbonylgruppe (R. = CHO and the untouched Ristotetro.syl (= R) side chain must be present.

The invention is based on the finding that A-Ristomyine of high purity and its derivatives falling within the general formula are well suited to carry out A-ristocetin induced platelet aggregation experiments (Figure 1). ,

In our experiments, using six different PRP (platelet-rich plasma) citrate and ED (TA- (ethylenediamine tetraacetic acid) PRP and mean treatment, the points illustrated in Figure 1 were used.

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The curves show a similar course and contain three sections that can be easily separated from one another. · At a lower concentration (0.8 mg / ml) no agglutination is detected. In the range of 0.8-2.0 mg / ml, a linear relationship can be observed. · At concentrations above 2.0 mg / ml, the dose curves run in the direction of the upper peak value. If the dose is further increased, plasma protein precipitation becomes an increasingly disturbing factor. Using citrate and SDTA-PRP as the substrate, identical results are obtained. ^Λ

It is shown in Figure 2 that ristomycin can be used very well for the quantitative determination of the VIII R: Ag cofactor. Die.sog. Ristocetin cofactor assay experiments are used to measure plasma factor deficiency resulting in von Willebrand disease (J. Clin. Invest. % 2, 2708 (1973); Thromb. Diath. Haemorrh., 2, 306 (1975)). Our results indicate that ristomycin is also excellently suited for quantitative testing and that the ristocetin cofactor and ristocetin cofactor assay experiment can be replaced by the ristomycin cofactor or ristomycin cofactor assay experiment, respectively. Determination of the ristomycin cofactor can be performed using both platelets washed (Figure 2, top curve) and formalin treated (Figure 2, below curve). "Ristocetin and ristomycin are called at higher concentra- '. tions a Plasmaproteinpräcipitation. The results obtained at the values between 1.5 and 3 »5 mg / l are summarized in Table 1. _x :

0.01 0.01 0.07 0.20 0.15 0.55 0.66 2.02

62 997 18 - 8 Table 1

Concentration of Liehtabsorption (measured at 600 mn) (mg / ml) Ristocetin Ristomycin

It can be seen in the above table that with the ristomycin used according to the invention always higher absorption values are obtained than with the ristocetin.

It has surprisingly been found that the basic condition for the reliability of the test method according to the invention is that the reagent used for the test should have a high purity and contain A-ristomycin or its constant-release derivative. This phenomenon is probably due to the fact that that, although ristocetin and ristomycin exert similar antibacterial activity, the microorganisms which produce these compounds have very different properties. Depending on the composition of the soil and the metabolic processes of the biosynthesis, quite different impurities can be formed even if the Work-up is identical. It is also known that in the culture broth of the microorganisms Nocardia lurida URRL 2430 and Proactinomyces fruetiferi varristomycini, two biologically active components are produced. Using the antibiotic as an antibacterial remedy interferes with the presence of the B component in A-ristomycin

~ c τ; o o

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not because both drugs are effective against pathogenic microorganisms. "The B component even has a significantly higher bactericidal effect than the Α component.

However, in contrast to the above, only the Α-component is valuable in the application of the two antibiotics to hematological experiments. The articles by Jenkins and coworkers (Thromb., P. 7, 531 (1975)) also point to this fact. "These authors have found that B-eistocetin does not aggregate the normal herbalocytes.

It is known that under the alkaline conditions of fermentation and ion exchange purification, both ristomycin molecules can undergo decomposition. To clarify these processes, the carbene-A-ristomycin is produced as a mode II compound. It has been found that this compound does not aggregate the normal human platelet plasma does not inhibit A-ristomycin-induced aggregation. This means that the taken place under the action of alkaline hydrolysis of the C-terminal methoxycarbonyl group of very sensitive Glykopeptidmoleküls (and possibly subsequent retroaldol cleavage at both beta-Hydroxyphenylserin units), the A-Ristomycin-preparative ¹ rat hematological purposes entirely worthless and can make it inapplicable. According to our experience, the A-. Ristomycin can not be freed from these undesirable accompanying substances with the aid of the aqueous-alcoholic recrystallization used in the known methods.

It should be noted that the -ristomycin base is extremely hygroscopic. "This compound may become more than a few minutes long on standing in air

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15% by weight of moisture (based on the weight of the base). Therefore, it is almost impossible to make an accurate measurement of this preparation. "It was also found, surprisingly, that the aggregation activity of the A-rystomycin moths during laboratories in the everyday Precise customary drying carried out with phosphorus pentoxide causes irreversible damage «

It is undoubtedly that in the isolation of the antibiotic. for the identification of the possible accompanying agents and impurities (namely the B component and the various products formed by alkaline decomposition) the measurement methods used hitherto alone are not sufficient (known biological method using test microorganisms, UY-spectrophotometry / mood detection; rotatory power). At 280 nm, the accompanying substances also have a UY light absorption and an optical rotation, similar direction »

In the process of the invention, A-ristomycin and / or ristomycin derivatives of the general formula (I) of high purity are prepared by preparing from crude ristomycin base a 30 to 50 % aqueous solution and the pH of this solution with a mineral acid with dilute sulfuric acid, between 6.7 and 7.0 .- advantageously to 6.8 - sets; or the pH of an aqueous solution, otherwise identical to the above solutions of contaminated A-ristomycin monosulfate with an alkali solution advantageously with ammonium hydroxide - between 6.7 and 7 sets. The solution is then at room temperature with an amount of 20 to 50% (Yol / Yol) of a mixable with? / Er low carbonitriles - suitable acetonitrile -

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treated. The A-ristomyein is crystallized at 4 ⁰ C, the crystals are separated, purified and optionally in an organic solvent - conveniently in a 1 to 5 carbon atoms containing alkanol - dissolved and subsequently at 0 to 20 ⁰ C "with an acylating agent of the acid anhydride type - advantageously acylated with trifluoroacetic anhydride - the product obtained or the products obtained are isolated.

The thus obtained A-ristomycin or its derivative is dissolved in an aqueous buffer solution, advantageously in an aqueous solution of tris-hydroxyaminomethane. The reagent thus prepared is suitable for the purpose of thrombocyte aggregation experiments, in particular for the diagnosis of von Willebrand disease.

According to an advantageous embodiment of the process according to the invention, a 30 to 50% strength solution is prepared from the crude ristomycin base prepared according to known fermentation process by dissolving in distilled water at room temperature. Thereafter, the pH of this solution is adjusted with a mineral acid - expediently with dilute sulfuric acid - between 6.7 and 7.0. .

As starting material it is also possible to use crude A-ristomycin sulfate. In this Palle after dissolution of the desired pH by addition of a dilute alkali solution, preferably ammonium hydroxide solution adjusted. Optimal pH adjustment may be carried out by instrument measurement or, advantageously, by turning a bromothymol blue indicator from yellow to sky blue. The solution thus prepared will contain from 20 to 50 % (v / v) of a water-miscible alkyl nitrile.

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advantageously acetonitrile added, whereupon the crystallization begins.

In the crystallization of a crude product of higher purity, it is also possible to proceed by first adding 15 to 50 % (v / v) of acetonitrile to the aqueous solution of A-ristomycin sulfate prepared in the manner indicated above, then the pH is between 6 * 7 and 7.0, heat the opalescent solution until dissolved and allow to cool slowly to room temperature. - '.

The desired crystals are washed on the filter with acetone and subsequently dried in a vacuum desiccator over heated potassium chloride and paraffin shavings to a dry weight. According to this method, the non-hygroscopic, crystalline A-ristomyein having excellent aggregation ability or its high-purity sulfate is obtained in a yield of 60 to 80% (depending on the active substance content of the starting material).

According to a further advantageous embodiment of the process according to the invention, the crude A-ristomycin is dissolved or suspended in an organic solvent, preferably in an alkanol, whereupon an acylating agent (eg trifluoroacetic anhydride) is added dropwise. The crude product may be purified by recrystallization if desired. , ,

The reagent according to the invention contains a standardized, high-purity, stable-weighted and stable ristomycin or ristomycin derivative prepared by the above process. The reagent according to the invention (AGGRISTEJT)

C? O ρ O "CO C " OQ * ·

, - 62 997 18

preferably keeps dose units of 15 mg · The reagent's free phenolic hydroxyl groups may, however, upon prolonged storage in the presence of atmospheric oxygen and exposure to sunlight, take up a chinoidal structure unfavorable to thrombocyte aggregation. This change is sustained by a long (40 hour) UY Confirmation of the Solid Specimen Confirmed In the light of the above, the hemophilia diagnostic kit reagent of the present invention is to be completed in hermetically sealed "hampers" made of brown glass.

The main advantages of the present invention can be summarized as follows:

1 According to the method of the invention can be used in blood coagulation tests excellent usable compounds of the 'general formula (I) high purity'

Ί -

2 "The reagent according to the invention can be used with great success in quantitative blood plasma experiments and ensures a more favorable measurement interval than the known means and methods."

3. The reagent according to the invention is relatively stable, its weight is constant, kai and is well storable.

its weight is constant, can be sterilized at 100 ⁰ C.

Our stability experiments undoubtedly showed that a solution of crystalline A-ristomycin monosulfate in distilled water. Water can be sterilized long 0.5 to 1.0 hour without substantial damage at 100 ^'0 C' It was also found that the present in AGGRISTHT kit solution (in TRIS

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Buffer, pH 7.4; Concentration 15 mg / ml) at +4 ⁰ C '3 months without decomposition can be stored.

Further details of the present invention can be found in the examples below, without limiting the scope of protection to these examples.

embodiment

The invention will be explained in more detail below with reference to some examples.

example 1 Preparation of the A-ristomycin sulfate

(R, R ^, R ₂ and R- are as indicated in point 1; R, = CH ^; X = HSO ₄ ; Y = H)

2 g of the crude ristomycin base prepared in a known manner are dissolved at room temperature in 5.0 ml of distilled water, whereupon the pH of the solution is adjusted to 6.8 with 1S sulfuric acid; the exact pH is measured instrumentally. · The yellow, slightly opalescent solution is clarified with a little bone char and filtered. 15 to 20 % (Yol./vol.) Of acetonitrile are added dropwise to turbidity until clear, and the mixture is allowed to stand at room temperature until crystallization occurs, if necessary by adding a further small amount of acetonitrile during 2 to 3 days so that the crystallization is terminated by means of a 24-hour cooling in the refrigerator at +4 ⁰ C.

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The formed square-shaped white crystals are filtered, first washed twice with 1.0 ml each of an ice-cold mixture of acetonitrile and water (1: 1) and then washed twice with 2 ml cold acetone, in a vacuum desiccator over heated calcium chloride and paraffin chips Room temperature under reduced pressure to constant weight dried. Yield 71 %.

rs

Example 2 . -

Preparation of the A-ristomycin sulfate

1.5 to 2.0 g of crude A-ristomycin sulfate used as starting material are dissolved with constant shaking in 4.0 ml of distilled water. The vigorously foaming solution is added to 1.0 ml of acetonitrile, the mixture is clarified with activated charcoal and The pH is neutralized with 2 IT ammonium hydroxide in the presence of a bromothymol blue indicator (yellow to sky blue). A further amount of acetonitrile is added to the solution until cloudy. The mixture is allowed to stand until crystallization occurs, the precipitated Crystals are redissolved by heating on the water bath and the solution is allowed to cool slowly to room temperature. The further work-up is carried out in the manner described in Example 1. Yield 65 %.

The A-ristomycin monosulfate isolated by both methods has excellent platelet aggregation properties and meets the following very high qualitative requirements: '.....

a) The decomposition of the product starts at 165 bis. 170

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(Photocell registration measured in a Mettler PP-5 melting point apparatus).

b) Using a high-pressure liquid chromatographic apparatus (Hewlett-Packard 1082, HPLC), the product is used to produce a chromatogram of excellent quality.

c) The product is uniform after layer chromatography. The log l value measured in aqueous solution at 280 nm is between 4.0 and 4.5, the sulphated ash content is not higher than 0.25 %. For a water jet pump (about 15 Hgmm) at the boiling point of. Acetone, continuously sucked for 4 hours, the weight loss of A-ristomycin monosulfate is at most 1 to 2.5 %. '.' .. '

The thin-layer chromatographic characterization of the crude product and of the A-ristomycin monosulfate prepared according to Examples 1 and 2 is carried out as follows:

The substances and reagents used in the determination:

I. "A freshly prepared 1-2% aqueous solution of the A-ristomycin sample"

II * 1 ./DC- roll cellulose (Merck 0.1 mm) thin film. 2. Fixion 50 ^: 8 plate (Reanal) ,.

III. Solvent mixtures as eluent:

a) n-butanol-pyridine-acetic acid-water (15: 10: 3: 12) or

b) n-butanol-pyridine-n-propanol-acetic acid-α / ater (20: 10: 5: 3: 32). Upper organic phase to II «1. Thin film. ,

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For equilibration and start of the II · 2. The following citrate buffer (pH 6) in ion-free water is used: 7.0 g of citric acid 4.0 g of sodium hydroxide 81.9 g of sodium chloride and 100 ml of methyl cellosolve

are dissolved in a 1000 ml volumetric flask and filled to the mark with distilled water.

IY, developer: Pauly's reagent

0.05 g of diazotised sulfanylic acid are dissolved in 10 ml of a 10% sodium carbonate solution and the freshly prepared solution is used.

The pure crude product shows on the celluloic layer in the solvent mixtures a) and b) R ^ values of 0.59 and 0.52, respectively. On a PircLon 50x8 plate, a RI value of 0.61 is measured in a citrate buffer (pH = 6). * The reagent is very sensitive, detecting virtually all decomposition products; the lower wedge limit is 0.5 to 1.0 / Ug #

High Pressure Liquid Chromatographic measurements are performed using a Hewlett-Packard 1082 Isochromatic HPLC Apparatus. In our experiments, an analytical column prepared in our laboratories is used (Cg-Lichrosorb, 10 μm). The active substance content of the A-ristomycin samples is determined using a UY detector operating at 254 nm. From the samples, 1 millimolar solutions are prepared, the 20 to 30 / Ul aliquots are sprayed onto the column. The chromatograms are developed as follows:

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(I) In a citrate buffer (pH 6.3) containing 12% methyl cellosolve, liquid flow 2.0 ml / min, paper speed 0.1 cm / min.

(II) In 10 % acetonitrile-containing (formic acid for egg), 0.1 molar Ammoiiiumformiat (pH, 7.4); Liquid flow 3> 0 ml / min *; Paper speed 0.3 cm / min,

The above conditions have proven to be most favorable »

The stability of the crystalline A-Bistomycin-sulfate prepared according to Examples 1 and 2, after UV-irradiation, the fixed pattern and after thermal sterilization of the aqueous solutions of various concentrations or * standing at +4 ⁰ G in a buffer THIS determined ·

a) 50 to 100 mg of A-ristomycin are irradiated in a small bottle with a stopper closed with UV light so that the distance between the bottle containing the sample and the lamp is about 20 cm, Hach 40-hour irradiation is the color the solution made a little deeper; At a concentration of 15 mg / ml, the aggregation of human shrombocytes is very slow.

b) Two distilled water solutions of A-ristamycin are made up of two solutions of similar dilution (0.39 mg / ml and 0.35 mg / ml, respectively) and one more concentrated solution (6.8 mg / ml). The solutions are kept for 3 hours in a water bath of 100 ^° C. * The evaporated water is occasionally replaced. The solutions are cooled to room temperature

1 % and the Si ^ values are determined on the basis of the intensity of the light absorption measured at 280 nm. It can be stated that the light absorption increase of the

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ι - 19 -

at 100 ⁰ C held solutions is not linear. In the range of 0 to 1 hour, the substances recovered by Liophylization show no decomposition after thin-layer chromatography and high-pressure liquid chromatography. The substances aggregate the normal human thrombocytes satisfactorily in the desired concentration.

c) From A-ristomycin, in a TRIS buffer (pH = 7.4) ', a solution with a concentration of 15 mg / ml is prepared, which is stored in a refrigerator at +4 ⁰ G for 12 weeks * from this The usual thf-platelet aggregation test is carried out biweekly or, after carrying out the appropriate buffer dilution (0.2 ml-1 ml), the S ^ '' ° value is determined.

x cm

During the experiment, the aggregating activity remains

1 % and the Et value of the A-ristomycin solution unchanged.

Example 3 , >

Preparation of the bis-IT ^ T'-trifluoroacetyl-A-ristöinycins

mg of A-ristomycin are suspended in 10 ml of anhydrous methanol, after which 0.28 ml of trifluoroacetic anhydride is added dropwise under ice cooling. The reaction mixture is allowed to stand at room temperature and concentrated to dryness under reduced pressure. The residue is redissolved in anhydrous methanol and concentrated again. This process is repeated three more times, after which the product is dried in a Vakuumessikkator over heated calcium chloride and paraffinic acid to constant weight. ¹ yield 90%.

Shows the bis-H, IT ^f -trifluoroacetyl-A-ristomycin thus obtained

  , 62 997 18

- 20 -,

no characteristic melting point (decomposition point). Standardization: on a cellulose layer in the solvent · · mixed HIa (see Example 2) is the. R ^ value 0.45 ·

Analysis: C ₉₉ H ₁₀₈ O ₄₆ H ₈ P ₆ (2258):

calculated: P 5.05 % H '4.96%

found: P 4.91 % Έ 4.86 % _a

4,88, 4,74

The compound thus obtained aggregates the normal platelets in the same manner as the A-Ristbmyein.

Example 4

Preparation of reagents ...

.15.00 g of an A-ristomyein monosulphate conforming to the abovementioned qualitative requirements are suspended in 100 ml of distilled water (pH 6.8) in a 1 1 Erlenmeyer flask *. The active substance excreted on the wall of the flask is shaken continuously with another amount of 400 to 500 ml of distilled water again brought into solution .. the thus obtained a homogeneous drug content solution exhibiting flask is fiber-free filtered into a 100 ml, the flask and used to dissolve the filters are washed in a quantitative manner and the measuring vessel is to filled to the mark "The A-Ristomaein-. mono sulfate concentration of the solution thus obtained is 15 mg / ml "(To prepare a sterile preparation, the solution is kept for 30 to 60 minutes at .100 ⁰ C and then cooled to" room temperature) From this solution using an automatic Püllvorrichtung under aseptic conditions exactly 1.00 ml volume in brown, so-called ..

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"- 21 -

Tear-off bottles filled · The bottles are lyophilised and sealed after rapid freezing.

As a check on the exact filling and the duration of the liophylation, the A-ristomycin content (15 mg) of some bottles per batch is checked by HPLC method and weight determination.

Claims (1)

62 997 18 Erfindungsanapruch 1. A process for the preparation of A-ristomycin and / or histomycin derivatives of the general formula (I) of high purity .'..-. ' OR (D NHY wherein,> R is a hydrogen atom; 62 997 18 - 23 - R _{1 is} an O-alpha-D-Araf (1 - ^ - 2) -O-alpha-D-Manp (1- ^ 2) -O- - (alpha-L-Rhap) - (i-5 * 6) -O-beta-D-Glop group; > Rp represents a 2,3,6-trideoxy-3-amino (ltHX) -alpha-L-ribo-hexapyranosyl group; , , Ro is an alpha-2-mannopyranose group; S, an alkyl group containing 1-4 carbon atoms means? '-. ' X represents the residue of a mineral acid; X represents a hydrogen atom or a group of the general formula C (Z) ₃ - (CH ₂ ) _n -GO, wherein Z is a hydrogen atom or a halogen atom and η is an integer - 'between O and. 5 stands, and optionally a reagent for use in thrombocyte aggregation preparations, in particular for the diagnosis of von Willebrand disease, characterized in that the pH of a 30 to 50% aqueous solution of the crude icedomycin base is adjusted to 6.8 by treatment with a mineral acid - preferably with dilute / sulfuric acid - between 6.7 and 7.0. or adjusting the pH of a 30 to 50% aqueous solution of a contaminated A-ristomycin sulfate with an alkali solution-advantageously with ammonium hydroxide-between 6.7 and 7.0, then with an amount of 20 to 50 % ./YoI.) Of a water-miscible lower aliphatic carboxylic acid nitrile, conveniently acetonitrile-treated at room temperature, subsequently crystallized at + 4 ° C, desirably dissolved in an organic solvent, preferably alkenol containing 1-5 carbon atoms. and then using an acylating agent of the acid anhydride type - advantageously with trifluoroacetic anhydride - at a / '- 2s ^. 62 997 18 - 24 - The temperature is from 0 to 20 C and subsequently isolating the product or products obtained, and optionally dissolving the finished ristomycin and / or estomycin derivative in a known aqueous buffer solution, preferably in an aqueous tris-hydrosilaminomethane solution, immediately prior to use. For this ... iL.Seiien drawings