CS268796B2 - Method of ristomycine a and/or high-purity ristomycine's derivatives production - Google Patents

Description

The invention relates to a process for the preparation of ritomycin and / or ristomycin derivatives of the general formula I

(I)>

where

R is hydrogen,

R _? means 0- <tf-D-araf / 1 ---> 2 / -0-4-D-manp / 1-> 2 / -0- / ^ - L-rhap / - / 1-> 6 / -O -beta-D-glop * -group

R ₂ represents 2,3,6-tridesoxy-3-amino- / KHY / -cÍMlf-TÍbohexapyrano8ylovou group,

R 6 is (H - D-mannopyronosyl group,

R 4 represents an alkyl group having 1 to 4 carbon atoms,

X is a pharmaceutically acceptable residue of an inorganic acid,

Ϊ represents a hydrogen atom or a group of the general formula

C (Z) ₃ - (CH) _and -CO-, wherein

Z is hydrogen or halogen and n is an integer between 0 and 5 ·

CS 268 796 B2

It is known that in a number of examinations of haemophilia patients (a disease manifested by a blood coagulation disorder), the most important data are obtained in assessing platelet function by detecting platelet aggregation. In addition to routinely used aggregating agents (eg ADP, adrenaline, collagen), since 1971 a substance called ristocetin, which has previously been used as an antibiotic, has played an important role. Based on the experiments of Howard and Firkin, ristocetin enabled rapid and simple recognition of congenital haemophilia, Willebrand's disease [Thrombosis und Diathesis Haemorhagica 26, 362 (1971)] ♦

The essence of Willebrand's disease is the absence of VIII. clotting factor complex. The most important biological role of the Willebrand factor is to promote the adhesion of platelets to subendothelial forms. The absence or quality disorder of Willebrand factor induces a clinical picture called Willebrand disease, which is the most widespread form of congenital haemophilia. It is very important to separate patients suffering from Willebrand's disease (heredity, family history, treatment). The clarification and differentiation of such a clinical picture is of paramount importance, in particular, prior to surgery.

The invention is based on the discovery that ristocetin, widespread in laboratory practice, can be perfectly replaced by another member of the family of vancomycin antibiotics - ristomycin. Ristomycin is easier and cheaper to produce and behaves more favorably in protein plasma clotting. Based on the above properties, ristomycin has been shown to be better in hematological experiments. Ristomycin, used hitherto for the treatment of diseases caused by bacterial infections, can also be successfully used in this new field of application (for diagnostic purposes).

Soviet researchers isolated riatomycin antibiotic from cultures of Proactinomycetes fructiferi var. ristomycin! adsorption (preferably on a SzDSz -3 or SzDV -3 cation exchanger column) and eluting with an ammonia solution in acetone or an ammonium acetate buffer (pH = 9-10). Ristomycin was isolated as a base. Later, ristomycin - like ristocetin - was divided into two biologically active ingredients - ristomycin A and ristomycin B. The antibiotic was produced on a large scale in operation. In addition to biological evaluation, a polarimetric and UV-spectrophotometric method has been developed to determine the active ingredient content of the fermentation broth and the intermediate product (Soviet Patent No. 180,754; Antibiotik! 8, 392 (1962); Antibiotik! 9, 884 (1964); Antibiotik! 110, 217 (1965); Antibiotik! 12, 129 (1967); Antibiotik! 6, 786 (1971)]. ~~

After determining the exact structure of rietomycin A [J. Org. Chem. 39, 2971 (1974); J. Antibiot. 32, 446 (1979); Tetrahaedron Letters 2983 (1980); JACS Д02, 7093 (1980)] was attempted to explain to what extent and how far changes in the structure of a compound or the degree of purity of a compound affect platelet aggregation.

It is an object of the present invention to provide a structure useful in biological experiments and to develop a process for the preparation of an active substance of suitable and very high purity.

It has been found that the partial or total removal of the R ^, Rg, R ^ and R ^ groups present in formula (I) or blocking of the functional hydroxyls of these groups and the R group (ester or ether) results in undesirable biological effects of the antibiotic. in many cases, an adverse change in solubility. The carboxyristomycin A, obtained by replacing the group R1 with a hydrogen atom, shows no effect of clotting plasma of human platelets and does not prevent the platelet aggregation induced by ristomycin. The tetra-O-methylristomycin A derivative (R = R ^ = CH ^) blocked on phenolic hydroxy groups also has unfavorable properties. Peracetylated ristomycin A is insoluble under the conditions used in the experiment and is unsuitable for the purposes of the experiment. Although aglycoristomycin A obtained by elimination of carbohydrates (R = R ₁ = R 8 = R 6 = Y = H; Η = ΟΗβ) induces agglutination, this derivative is also not optimal with respect to pH and solubility ratios. To the knowledge of the authors, ristomycin V is not (this compound differs from other derivatives in that in the general formula I in the heterotetrasaccharide chain R 2 -O-arabinofuranosyl-alpha-D-mannopyranosyl moiety is absent (suitable for the desired diagnostic purpose).

Surprisingly, it has been found that the active substance used in the blood coagulation test is optimal if and only if it has a high purity and a well-defined

The compounds of formula I in which Y is hydrogen or CF3-CO- are in highly pure form suitable for laboratory diagnostic purposes.

Based on the above findings, it has been found that the basic condition necessary for inducing aggregation of normal human platelets is that there must be four free hydroxy groups (R = H), a C-terminal methoxycarbonyl group (R R + CHp and a side chain intact ristotetrosyl (= R1).

The invention is based on the discovery that high purity ristomycin A and its derivatives, which fall within the general formula I, are well suited for carrying out assays of platelet aggregation induced by ristomycin A. (Fig. 1).

In the experiments, using six different PRPs (platelet-rich plasma) and EMA (ethylenediaminetetraacetic acid) PRP and averaging, points on the curve shown in Fig. 1 were obtained. The abscissa axis shows the concentration of substances in mg / ml; density in%. Data obtained for ristocetin are represented by dots, data for ristomycin squares. The curves show a similar pattern and comprise three easily separable sections. No agglutination was observed at a lower concentration (0.8 mg / ml). In the range of 0.8 to 2.0 mg / ml, linear continuity can be observed. At concentrations above 2.0 mg / ml, dose curves run towards the upper maximum value. At a further dose increase and the plasma protein coagulation becomes an increasingly disturbing factor. Using citrate and EMA-PRP as substrate gave identical results.

Figure 2 shows that ristomycin can be used very well for the quantitative determination of VIII R: Ag cofactor. The abscissa axis shows the number of units of ristomycin-cofactor per ml, the ordinate of the change in optical density in%. The so-called ristomycin-cofactor-assay experiments serve to measure the plasma factor deficiency resulting from Willebrand's disease [j. Clin. Invest. 52, 2708 (1973); Thromb. Diath. Haemorrh. 306 (1975)]. The results show that ristomycin is also well suited for quantitative purposes and that the ristocetin cofactor and ristocetin co-factor may be replaced by ristomycin co-factor or ristomycin factor-as say experiment. The ristomycin cofactor assay can be performed using both washed (Fig. 2, upper curve) and formalin-treated platelets (Fig. 2, lower curve). Ristocetin and ristomycin induce plasma protein clotting at higher concentrations. The results obtained at values between 1.5 and 3> 5 mg / ml are summarized in Table 1.

Table 1

concentration (ng / ml) light absorption (measured with ristocetin 600 nm) ristomycin 45 0.01 0.01 2.7 0.07 0.20 3.0 0.15 0.55 3.5 0.66 2.02

From the above table, it is clear that the ristomycin used according to the invention always obtains higher absorption values than with ristocetin.

Surprisingly, it has 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 is of high purity and that ristomycin A or a derivative thereof has a constant active substance content. This phenomenon is likely to be derived from the fact that although ristocetin and ristomycin have similar antibacterial effects, the microorganisms producing these compounds have very different properties · Depending on the composition of the nutrient medium and the metabolism processes in biosynthesis, completely different contaminants may arise, although the processing is identical · It is further known that in the slurry of a culture of microorganisms Nocardia lurida NRRL 2430 and Proactinomycee fructiferi var. ristomycini vzni4

CS 268 796 B2 lacks two biologically active ingredients. When the antibiotic is used as an antibacterial drug, the presence of component V in ristomycin A does not guarantee that both active substances are effective against pathogenic microorganisms. Component В exhibits a substantially higher bactericidal effect than component A.

In contrast to the above, however, only component A is valuable when using both antibiotics for haematological experiments. Article by Jenkins and colleagues [Thromb. Res. 7, 531 (1975)] also points to this fact. These authors found that ristocetin V did not aggregate normal human platelets.

It is known that under alkaline fermentation and ion exchange purification conditions, both ristomycin molecules may undergo decomposition. To explain these processes, it was made as a model compound carboxyristomycin A. It was found that this compound did not aggregate normal human thrombocyte plasma and did not prevent aggregation induced by ristomycin A. This means that hydrolysis of the C-terminal methoxycarbonyl group of very sensitive glycopeptide molecule (and possibly the subsequent cleavage of retroaldol on both beta-hydroxyphenylserine units), will render the ristomycin A preparation completely useless and unusable for hematological purposes. Experience has shown that ristomycin A cannot be depleted by recrystallization from water and alcohol used in known methods of these unwanted accompanying substances.

It should be noted that the base of ristomycin is extremely hygroscopic. This compound can, if left in air, absorb more than 15% by weight of moisture (based on the weight of the base) in minutes. Therefore, it is almost impossible to make an accurate measurement of this preparation. At the same time, it has surprisingly been found that the activity of aggregation, a sample of ristomycin, during phosphorus pentoxide drying usually performed in laboratory practice, is subject to irreversible damage.

There is no doubt that the isolation of the antibiotic methods used hitherto to prove possible accompanying agents and contaminants (namely components V and various products resulting from alkaline decomposition) alone are not sufficient (known biological methods performed by test microorganisms; UV spectrophotometric determination of the active substance) optical rotation measurement). In fact, the accompanying substances also exhibit UV absorption at 280 nm and have an optical rotation in a similar direction.

According to the process of the invention, ristomycin A and / or high purity ristomycin derivatives of the formula I are prepared by adjusting the pH of 30 to 50% of an aqueous solution of crude ristomycin base with an inorganic acid, preferably dilute sulfuric acid, between 6.7 to 7.0, preferably 6.8, or a pH of 30 to 50% of an aqueous solution of contaminated ristomycin A sulphate is adjusted to 6.7 to 7.0 with an alkali solution, preferably ammonium hydroxide, and then treated with 20 up to 50% by volume of a water-miscible, preferably acetonitrile, water-miscible nitrile at room temperature, then allowed to crystallize at +4 ° C and optionally dissolved in an organic solvent, preferably a C 1 -C 5 alkanol carbon and then acylated with an acylating agent of the type of carboxylic anhydride of the formula 0 (Ζ) ^ - ₍₂ 0Η) _{η -0Ο0Η} wherein Z is hydrogen or halogen n ing An integer of from 0 to 5, preferably trifluoroacetic anhydride at 0 to 20 ° C, and then recovering the product or products obtained.

The ristomycin or derivative thereof thus obtained is dissolved in an aqueous buffer solution, preferably in an aqueous solution of tris-hydroxyaminomethane. The solution thus prepared is suitable for the purpose of platelet aggregation experiments and in particular for the diagnosis of Willebrand's disease.

According to a preferred embodiment of the process according to the invention, a 30-50% solution is prepared from the crude ristomycin base produced by the known fermentation process by dissolving in distilled water at room temperature. The pH of the solution is then adjusted to between 6.7 and 7.0 with an inorganic acid, preferably dilute sulfuric acid.

Crude ristomycin sulfate A may also be used as starting material. In this case, after dissolution, the desired pH is adjusted by adding a dilute alkali solution,

CS 268 796 B2 preferably with ammonium hydroxide solution. The adjustment of the optimum pH can be done by means of a pH meter or preferably by changing the indicator of bromine thymol blue from yellow to sky blue. 20 to 50% by volume of a miscible, water-miscible alkylnitrile, preferably acetonitrile, is added to the solution thus prepared, and crystallization occurs.

The crude product of higher purity can also be crystallized by first adding 15 to 50% by volume of acetonitrile to the aqueous solution of ristomycin sulphate A prepared as described above, and then adjusting the pH to between 6.7 and 7.0. The opalescent solution is heated to dissolution and slowly allowed to cool to room temperature.

The desired crystals are washed on the filter with acetone and then dried in a vacuum desiccator over heated calcium chloride and paraffin clips to constant weight. In this way, tin A or sulphate thereof having an excellent aggregation capacity, non-hygroscopic, of high purity, is obtained in a yield of 60 to 80% (depending on the active substance content of the starting product).

According to another preferred embodiment of the process according to the invention, the crude ristomycin A is dissolved or suspended in an organic solvent, preferably an alkanol, and then an acylating agent (e.g. trifluoroacetic anhydride) is added dropwise. The crude product can, if desired, be purified by recrystallization.

The solution contains a stable rostimycin or ristomycin derivative produced by the process of the invention, standardized, highly pure, and of constant weight. However, the free phenolic hydroxyl groups of the reagent may adopt an unfavorable quinoid structure for platelet aggregation upon prolonged storage, in the presence of atmospheric oxygen and under the influence of sunlight. This change is confirmed by long-term (40-hour) illumination of the solid preparation by ultraviolet radiation. In view of the above, the ristomycin solution for instrumental diagnosis of hernophilia should be stored in bottles made of brown glass and hermetically sealed. The single dose of the solution is preferably 15 mg.

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

1. The process according to the invention can be used to produce compounds of the formula I of high purity and excellent in blood clotting tests.

2. A solution of the compounds of formula I can be used with great success in quantitative blood plasma experiments, which provides a more favorable measurement interval than known means and methods.

3. The solution is relatively stable, its weight is constant, can be sterilized at 100 ° C and can be stored well.

Stability experiments have clearly shown that a solution of crystalline ristomycin A mono sulphate in distilled water can be sterilized without disturbance for 0.5 to 1.0 hours at 100 ° C. It was also found that in TRIS buffer, pH ³ 7.4; a concentration of 15 mg / ml can be stored without degradation at * 4 ° C for 3 months.

Further details of the present invention can be seen. of the following examples, without limiting the scope of protection to these examples.

Example 1

The production of ristomycin A sulphate (R, R ^, Rg and R ^ are the same as above; R ^ = CH4; X = HSO4;

Y 1 H) g of crude ristomycin base prepared in a manner known per se is dissolved in 5 ml of distilled water at room temperature, then the pH of the solution is adjusted to 6.8 with 1 N sulfuric acid; the exact pH is measured with a pH meter. The yellow, slightly opalescent solution is clarified with a little bone charcoal and filtered. 15 to 20% (v / v) acetonitrile is added dropwise to the clear solution obtained until turbidity. The mixture was allowed to stand at room temperature until it began to crystallize. This measure is repeated, if necessary, over a period of 2 to 3 days by the addition of an additional small amount of acetonitrile so that crystallization is complete.

CS 268 796 B2 chilled by 24-hour refrigeration at 4 ° C.

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

Example 2

Production of ristomycin sulphate

Dissolve 1.5 to 2.0 g of crude ristomycin A sulphate used as starting material in 4.0 ml of distilled water, with shaking. The strong foaming solution is added

I. 0 ml of acetonitrile, the mixture is clarified with activated carbon and filtered. The pH was neutralized with a 2 N ammonium hydroxide solution in the presence of a bromothymol blue indicator (transition from yellow to sky blue). An additional amount of acetonitrile was added to the solution until turbidity occurred. The mixture was allowed to stand until crystallization began, the precipitated crystals were redissolved by heating in a water bath, and the solution was allowed to slowly cool to room temperature. Further work-up was carried out as described in Example 1.

Yield 65%.

Ristomycin A mono sulphate, isolated according to both methods, has excellent platelet aggregation capabilities and conforms to the following very high quality standards:

(a) Product decomposition begins at 165 to 170 ° C (measured in Mettler FP-5 melting point apparatus with photocell registration).

b) Using a high pressure liquid chromatograph (Hewlett-Packard 1082), a chromatogram of excellent quality is prepared from the product.

c) The product is uniform according to layer chromatography. The log hodnota value measured in the aqueous solution at 280 nm is between 4.0 and 4.5, the sulfate content being not more than 25%. When aspirated by a water pump at a boiling point of acetone continuously for 4 hours, the weight loss of ristomycin A monosulfate is at most 1 to 2.5 $ ·

The characterization of the crude product by thin layer chromatography and ristomycin A monosulfate produced according to Examples 1 and 2 was performed as follows:

Substances and reagents used in the determination:

1. a freshly prepared 1 to 2% aqueous solution of a sample of ristomycin A.

II. 1. DC-Alurolle cellulose (Merck 0.1 mm) thin layer.

2. Fixion 50x8 Plate (Reanal)

III. solvent mixtures As eluents:

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

b) n-butanol-pyridine-n-propanol-acetic acid-water (20: 10: 5: 3: 32). Upper organic phase to II. 1. thin layer.

For equilibration and start II. 2. The following citrate buffer (pH 6) in deionized water is used:

7.0 g citric acid,

4.0 g sodium hydroxide,

81.9 g of sodium chloride and

Dissolve 100 ml of methylcellosolv (2-methoxyethanol) in a 1000 ml graduated flask and make up to the mark with distilled water.

IV. Developing agent: Pauly reagent

Dissolve 0,05 β diazotized sulfanylic acid in 10 ml of 10% sodium carbonate solution and use the freshly prepared solution.

CS 268 796 B2 fresh crude product has, on the cellulosic layer in the solvent mixtures of a) and b) of R _f = 0.59, or 0.52. On a Fixion 50x8 plate R _f = 0.61 is measured in citrate buffer (pH = 6). Reagent It is very sensitive and detects virtually all degradation products; the lower detection limit is 0.5 to 1.0 µg.

High pressure liquid chromatography measurements are performed using a Hewlett-Packard 1082 HPLC instrument with isochronous operation. A laboratory-made analytical column (Cg-Lichrosorb, 10 µm) was used in the experiments. The active substance content of the ristomycin A sample is determined using a UV detector operating at 254 nm. 1 millimolar solutions are prepared from the samples and sprayed onto the column with a 20-30 µm aliquot. Chromatograms are developed as follows:

(I) in citrate buffer (pH 6.3) containing 12% 2-methoxyethanol, liquid flow rate 2.0 ml / min, paper feed rate 0.1 cm / min.

(II) in a 0.1 molar solution of ammonium formate (free of formic acid) containing 10% acetonitrile (pH 7.4); liquid flow rate 3.0 ml / mln; paper feed speed 0.3 cm / min.

The above conditions and e proved to be the best.

The stability of the crystalline ristomycin A sulphate produced according to Examples 1 and 2 is determined after irradiation of the solid sample with ultraviolet radiation and after heat sterilization of aqueous solutions of various concentrations, or left to stand at 4 ° C in TRIS buffer.

(a) 50 to 100 mg of ristomycin A is irradiated in a small vial closed by a stopper with ultraviolet light such that the distance between the vial containing the rf sample and the lamp is about 20 cm. After 40 hours of irradiation, the color of the solution is slightly darker; at a concentration of mg / ml, the aggregation of human platelets is very slow,

b) Two solutions of similar dilution (0.39 mg / ml and 0.35 mg / ml, respectively) and one more concentrated solution (6.8 mg / ml) are prepared from ristomycin A in distilled water. The solutions were kept in a 100 ° C water bath for three hours. Evaporated water is occasionally replenished. The solutions were cooled to room temperature and the values ^are determined based on the intensity of the light absorption measured at 280 nm. It can be found that the increase in light absorption of solutions maintained at 100 ° C is not linear. Over a time period of 0 to 1 hour, the recovered substances by lyophilization by thin layer chromatography and high pressure liquid chromatography showed no decomposition. The substances aggregate satisfactorily at the desired concentrations of normal human platelets.

The results are graphically depicted in Figure 3, where the abscissa axis shows the time in minutes, the extinction ordinate line Curve 1 is 3.99 mg ristomycin A / 10 ml, Curve 2 is 3.56 mg / ml, and Curve 3 contains 68.77 mg / 10 ml of the same substance. Obviously, there is no decomposition of the substance within 1 hour,

(c) A solution of β at a concentration of 15 mg / ml is prepared from ristomycin A in TRIS buffer (pH = 7> 4) and stored for 12 weeks at 4 ° C in a refrigerator. From this stock solution, a conventional platelet aggregation experiment is performed after 2 weeks, or the value is determined after an appropriate buffer dilution (0.2 mL -> 10 mL). During the experiment, the aggregation activity and ristomycin A solution remain unchanged.

Example 3

Preparation of bis-Ν, Ν * - trifluoroacetylristcmyoin A

206 mg of ristomycin A are suspended in 10 ml of anhydrous methanol, and 0.28 ml of trifluoroacetic anhydride are added dropwise under ice-cooling. The reaction mixture was allowed to stand at room temperature and evaporated to dryness under reduced pressure. The residue was redissolved in anhydrous methanol and concentrated again. This process is repeated three more times and then dried in a vacuum desiccator over heated calcium chloride and pafafinic acid to constant weight.

The bis-Ν, Ν * - trifluoroacetylristomycin A thus obtained shows no characteristic

CS 268 796 B2 melting point (decomposition temperature). Standardization: On the cellulose layer in the solvent mixture IIIa (see Example 2) the value is 0.45

Analysis: ^C 99 ^H 108 ° 46 ^N 8 ^F 6 (2258):

calculated: F 5.05% N 4.96% found; F 4.91% N 4.86%

4.88 4.74

The compound thus obtained aggregates normal platelets as well as ristomycin A.

Example 4

Production of reagents

15.00 g of ristomycin A monosulphate corresponding to the above quality standards are suspended in a 1 liter Erlenmeyer flask in 100 ml of distilled water (pH 6.8). The active substance deposited on the wall of the flask is again brought into solution by shaking with an additional quantity of 400 to 500 ml of distilled water. The solution thus obtained, showing a homogeneous active substance, is filtered without fibers into a 100 ml volumetric flask, the flask used for dissolution and the filter is washed quantitatively and the volumetric flask is filled up to the mark. The concentration of ristomycin A monosulfate in the solution thus obtained is 15 mg / ml (for the preparation of a sterile preparation, the solution is kept at 100 ° C for 30 to 60 minutes and then cooled to room temperature). From this solution, aseptic conditions are filled into brown so-called tear-cap bottles of 1.00 ml. The vials are lyophilized and sealed after rapid freezing.

To check the exact filling and perfection of lyophilization, the ristomycin A content (15mg) was tested by HPLC and weight determination for several vials of a particular lot.

Claims (1)

Process for the preparation of ristomycin A and / or the purity of the ristomycin derivatives of the general formula I, high. HX where R is hydrogen, R ₁ represents O-alpha-D-araf (1 ---> 2) -O-alpha-D-manp (1 ---> 2 / -0- / alpha-L-rhaf) - / 1 --- > 6 / -O-beta-D-glop- group, _R2 is 2,3, 6-tridesoxy-3-amino- / NHY / -alpha-L-ribo-hexapyr yes sylovou group, R, alpha-D-mannopyranosyl, R 1 is C 1 -C 4 alkyl, X is a pharmaceutically acceptable inorganic acid residue; γ is a hydrogen atom or a group of formula C (Z) ₃ - (CH ₂ ) _and -CO-, wherein CS 2-8 796 B2 Z is hydrogen or halogen and n is an integer between 0-5 characterized in that the pH of the 30 to 50% aqueous solution of the crude ristomycin base is adjusted with an inorganic acid, preferably dilute sulfuric acid, between 6.7 to 7.0, preferably to 6.8, or the pH is 30 to 50% The 50% aqueous solution of impure ristomycin sulfate is adjusted to 6.7-7.0 with an alkali solution, preferably ammonium hydroxide, and then treated with 20-50% by volume of a low aliphatic carboxylic acid nitrile, preferably water-miscible, preferably acetonitrile, at room temperature, then allowed to crystallize at + 4 ° C and optionally dissolved in an organic solvent, preferably a C 1 -C 5 alkanol, and then acylated with a carboxylic acid anhydride c) reagent (z ) ₃ - ₍₂ сн) _{п -соон} wherein Z is hydrogen or halogen and n is an integer from 0 to 5, preferably trifluoroacetic anhydride at 0 to 20 ° C, and then recovering the product or products obtained.