FI76090C - Process for isolating suitable A-ristomycin suitable for diagnostic purposes and its high purity derivatives from crude ristomycin or crude ristomycin - Google Patents

Description

76090

Method for isolation of high purity A-ristomycin and its derivatives from impure ristomycin or crude Risto-mycin for diagnostic purposes 5 OR 4

/> OR

0 = \> - / 10 Γ ~ β) V ^ 0R5

15 O o <. A

/ - ') - o o / "/ N — f j — l HO U \ Λ

H / - \ (Jr0R

NHY

(i) for the preparation of ristomycin and / or ristomycin derivatives according to 35 2 76090, wherein R is a hydrogen atom; 5 denotes O-alpha-D-Araf (1—) 2) -O-alpha-D-Manp (1—) 2) -O- (alpha-L-Rhap) - (1-—) 6) -O-beta -D-Glop group; R 2 is a 2,3,6-tridesoxy-3-amino- (NHY) -alpha-L-ribohexa-pyranosyl group; R 2 is an alpha-D-mannopyranosyl group; R 1 represents an alkyl group having 1 to 4 carbon atoms; X is a mineral acid residue; Y represents a hydrogen atom or a group of the general formula C (Z) ^ - (CH2) n -CO, where Z is a hydrogen atom or a halogen atom and 15 n represents an integer from 0 to 5.

The invention further relates to a biological reagent for platelet aggregation experiments, in particular for the diagnosis of Wille brand disease, and to a method for its preparation.

20 It is known that mass screening of hemophilic patients for the evaluation of platelet function provides the most valuable information through platelet aggregation tests. In addition to the aggregating agents routinely used (e.g., ADP, adrenaline, collagen), the substance previously used as an antibiotic, ristocetin, has been important since 1971. Based on experiments by Howard and Firkin, ristocetin has allowed the rapid and simple detection of inherited hemophilia, Willebrand's disease (Thrombosis und 30 Diathesis Haemorhagica 26, 362 (1971)) ·

Willebrand's disease lacks the factor VIII complex of blood clotting. The main biological function of Willebrand factor is to promote platelet adhees.ion to subendo-telial formulas. Lack of Willebrand factor or a qualitative disorder produces a picture of 3,76090, termed Willebrand disease, which is the most common form of congenital hemophilia. It is very important to differentiate between patients with V / illebrand disease (heredity, family trials, therapy). Clarification and separation of such a disease 5 before surgery is very important.

The invention is based on the knowledge that ristocetin, which is widely used in laboratory practice, can be completely replaced by another member of vancomycin antibiotics - ristomycin. Ristomycin is simpler and cheaper to formulate and behaves more favorably in plasma protein precipitation. Due to the above properties, ristomycin has been shown to be more advantageous in haematological experiments. Until now, ristomycin, used to treat diseases caused by antibacterial infections, can also be used in this new field of application (for diagnostic purposes).

Soviet researchers isolated the antibiotic ristomycin Proactinomyces fructiferi var. Risto-20 from mycin cultures by adsorption (preferably on a SzDSz-3 or SzDV-3 cation exchange column) and elution with acetone ammonia solution or ammonium acetate buffer (pH = 9-10). Ristomycin was isolated in the form of a base. Later, ristomycin was separated - as with ristocetin - into two biologically active components - A- and B-ristomycin. The manufacture of antibiotics for large-scale use has also been published. In addition to biological determination, a polarimetric and UV spectrophotometric method was also prepared to determine the active ingredient content of the fermentation broth and intermediate intermediates (SU Patent Publication 180804; Antibiotics 8, 392 (1962); Antibiotics 9, 884 (1964); Antibiotics). 786 (1971)).

The determination of the exact structure of 35 A-ristomycin remained "76090 (J. Organ. Chem. 39, 2971 (197 * 0; J. Antibiot. 32, 446 (1979)); Tetrahedron Letters 21, 2983 (1980); JACS 102; 7093 (1980) we have tried to determine to what extent and to what extent a change in the structure of a compound or the degree of purity of a compound affects platelet aggregation.

The object of the invention is to determine the structure used in biological experiments and to provide a method for preparing a suitable and highly pure active substance.

It has been found that the partial or complete removal of the Rg, Rj and Rjj groups present in the general formula (I) or the inhibition of the hydroxy functions and the R group (in the form of an ester or ether) of these groups, respectively, causes undesired biological effects of the antibiotic and in many cases also an undesirable change in solubility. Carboxy-A-ristomycin obtained by hydrogen atom substitution at the R 1 group does not act in an aggregating manner on human platelet plasma and does not inhibit ristomycin-induced platelet aggregation. The tetra-O-methyl-A-ristomycin derivative (Ρ = Ρ ^ = 0Η ^) blocked in the penolic hydroxy groups also has unfavorable properties. Peracetylated A-ristomycin A is insoluble under the conditions used in the experiments and is not suitable for the purpose of the experiments. Although aglyco-A-ristomycin (R = R1 = R2 = R1 = Y = H; R = CH2) obtained by elimination of carbohydrates causes agglutination, this derivative is also not optimal due to pH and Liu hardness ratios. To our knowledge, B-ristomycin is also not different from this compound (this compound differs from other derivatives only in that the general formula (I) in the heterotretasaccharide chain of R 1 lacks a 2-O-D-arabinofuranosyl-alpha-D-mannopyranosyl moiety) , not suitable for the desired diagnostic purpose.

Surprisingly, it was found that the active substance used in the coagulation test 35 is then and only then n 5 76090 optimal if it is very pure and has a well-defined structure of general formula (I). The compounds of general formula (I) in which Y represents hydrogen or the group CF 2 -CO- are preferably suitable in highly pure form for laboratory diagnostic purposes.

Based on the above data, it was concluded that the basic condition required to perform normal human platelet aggregation is the presence of four free phenolic hydroxy groups (R = H), a C-terminal methoxycarbonyl group (R 2 = CH 2) and an intact formula in General Formula I. ristosetrosyl (= R 2) side chain.

The invention is based on the knowledge that high-purity A-ristomycin and its derivatives are excellent for performing A-ristocetin A-induced platelet aggregation experiments (Figure 1).

In our experiments, using six different PRP (platelet-rich plasma) citrate and 20 EDTA (ethylenediaminetetraacetic acid) PRP and averaging, scores on the curve shown in Figure 1 were obtained. The curves show a similar course and contain three well-separable parts. No agglutination is observed at low concentration (0.8 mg / ml).

In the range of 0.8 to 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. As the dose continues to increase, plasma protein precipitation becomes an increasingly disturbing factor. When citrate and EDTA-30 PRPr are used as substrate, identical results are obtained.

Figure 2 shows that ristomycin can be very well used for the quantitative determination of cofactor VIII R: Ag. So-called ristocetin-cofactor assays serve to measure plasma factor deficiency in Willebrand's disease 35 (J. Cl.

Invest. 52, 2708 (1973); Thromb. Diath. Haemorrh. 2ii> 306 (1975)) · Our results show that ristomycin 6 76090 is equally well suited for quantitative experiments and that ristocetin cofactor and ristocetin cofactor assay can be replaced by ristomycin cofactor or ristomycin cofactor assay, respectively. TYS-test. The assay for ristomycin cofactor can be performed using both washed (Figure 2, upper curve) and formalin-treated (Figure 2, lower curve) platelets. Ristocetin and ristomycin cause plasma protein precipitation at higher concentrations. The results obtained at 1.5 to 3.5 mg / ml are shown in Table 1.

Table 1 15 __

Concentration Light absorption (measured at 600 nm) (mg / ml) _

Ristocetin Ristomycin 20 1.5 0.01 0.01 2.7 0.07 0.20 3.0 0.15 0.55 3.5 0.66 2.02 25

It can be seen from the table above that ristomycin used according to the invention always gives higher absorption values than ristocetin.

Surprisingly, it was found that the basic condition for the reliability of the test method according to the invention is that the reagent used for the test must be very pure and contain ristomycin A or a derivative thereof, respectively, at a constant concentration of active substance. This phenomenon can probably be attributed to the fact that although ristocetin and ristomycin have a similar antibacterial effect, the microorganisms producing these compounds have very different properties. Depending on the composition of the medium and the metabolic events of the biosynthesis, completely different impurities can be formed, even if the treatment is identical. It is further known that the microorganisms Nocardia lurida NRRL 2 * 130 and Proactino-myces fructiferi var. ristomycin broth produces two biologically active components in each case. If an antibiotic is used as an antibacterial drug, the presence of component B in A-ristomycin does not interfere, since both active substances act against pathogenic microorganisms. Component B even has a substantially higher bactericidal effect than component A.

However, in contrast to the above, only component A is valuable when using both antibiotics for hematological tests. An article by Jenkins and Contributors (Thromb. Res. 7, 531 (1975)) 20 also refers to this fact. These factors have shown that β-ristocetin does not aggregate normal human platelets.

It is known that under temporal conditions of fermentation and ion exchange purification, both Risto-25 mycine molecules can degrade. To elucidate these events, carboxy-A-ristomycin is prepared as a model compound. This compound was found not to aggregate normal human platelet plasma and did not inhibit A-Risto-mycin-induced aggregation. This means that alkali hydrolysis of the C-terminal methoxycarbonyl group of a highly sensitive glycopeptide molecule (and possibly subsequent retroaldol cleavage in beta-hydroxyphenylserine units) can render the A-ristomycin preparation completely worthless and unusable for hematological purposes. In our experience, A-ristomycin cannot be released from these unwanted by-products by recrystallization with water and alcohol used in known methods.

It should also be noted that ristomycin base 5 is highly hygroscopic. This compound can absorb more than 15% by weight of moisture (relative to the weight of the base) when left to stand in air for a few minutes. For this reason, it is almost impossible to perform an accurate measurement of this preparation. At the same time, it was surprisingly found that the aggregation activity of the A-ristomycin model is irreversibly impaired in daily laboratory practice during normal drying with phosphorus pentoxide.

It is certain that the measurement methods used so far for isolating an antibiotic to detect possible by-product and impurities (namely various products formed by component B and alkaline degradation) alone are not sufficient (biological method using known test micro-organisms; effects-20 UV spectrophotometric determination of the analyte; measurement of optical rotation). Namely, the excipients also have UV light absorption at 280 nm and have a parallel optical rotation.

According to the process of the invention, A-ristomycin A and of the general formula (I) and / or ristomycin derivatives are prepared by preparing an aqueous solution of 30-50% from the crude ristomycin base and adjusting the pH of this solution to that of a mineral acid, preferably dilute sulfuric acid. 6.7-7.0, preferably 6.8; or adjusting the pH of an aqueous solution of impure A-Risto-mycine monosulfate, otherwise identical to the above solutions, with an alkaline solution, preferably ammonium hydroxide, to 6.7 to 7 · The solution is then treated at room temperature with water-miscible lower carboxylic acids. -ril - preferably acetonitrile - 20-50 # with an amount of 9,76090 (v / v). A-ristomycin A is crystallized at 4 ° C, the crystals are separated, purified and optionally dissolved in an organic solvent - preferably an alkanol having 1 to 5 carbon atoms - and then acylated at 0 to 20 ° C with an acid anhydride-type acylating agent - preferably with trifluoroacetic anhydride. The product - or products obtained - is isolated.

The ristomycin A or its derivative thus obtained is dissolved in an aqueous buffer solution - preferably an aqueous solution of tris-hydroxyaminomethane.

The reagent thus prepared is evaporated for platelet aggregation experiments - especially to diagnose Willebrand's disease.

According to a preferred embodiment of the process of the invention, a solution of crude histomycin base prepared at $ 30-50 is prepared by dissolving it in distilled water at room temperature. The pH of this solution is then adjusted to 6.7 to 7.0 with mineral acid - preferably dilute sulfuric acid.

Crude A-ristomycin sulfate can also be used as a starting material. In this case, after dissolution, the desired pH is adjusted by adding a dilute alkali solution - preferably ammonium hydroxide solution. The adjustment of the optimal pH can be performed by instrument measurement or when the bromothymolysin indicator changes from yellow to sky blue. To the solution thus prepared, a 20-50% (v / v) amount of water-miscible alkyl nitrile - preferably acetonitrile - is added, after which crystallization begins.

The crystallization of the higher purity crude product can also be carried out by first adding 15-50¾ (v / v) acetonitrile to the aqueous solution of A-rhistomycin sulfate prepared as described above, then adjusting the pH to 6.7-35. 7.0, warm the opalescent solution until dissolved and allow to slowly cool to room temperature.

10 76090

The desired crystals are washed as a filter with acetone and then dried in a vacuum desiccator over heated calcium chloride and paraffin chips to constant weight. According to this method, non-hygroscopic, crystalline, highly pure A-ristomycin or its sulfate with excellent aggregation capacity is obtained in a yield of 60-80¾ (depending on the active substance content of the starting material).

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

The reagent of the invention contains a standardized, high purity, constant weight and stable ristomycin or ristomycin derivative prepared according to the above method. The reagent of the invention (AGGRISTINR) preferably contains 15 mg dosage units. However, the free phenolic hydroxy groups of the reagent can have an unfavorable quinoidal structure for platelet aggregation under prolonged storage, in the presence of oxygen in the air, and under the influence of sunlight.

25 This change is confirmed by long-term (40 hours) UV illumination of the solid preparation. In view of the above, the reagent of the invention should be placed in ampoules made of amber glass and hermetically sealed for hemophilia diagnostic devices.

The main advantages of the present invention can be summarized as follows: 1. According to the process of the invention, highly pure compounds of general formula (I) useful in blood coagulation tests can be prepared.

76090 2. The reagent of the invention can be used very successfully in quantitative plasma experiments and provides a more favorable measuring range than known instruments and methods.

3. The reagent of the invention is relatively stable, has a constant weight, can be sterilized at 100 ° C and is highly storable.

Our stability experiments have shown doubts that a solution of crystalline A-ristomycin monosulfate in distilled water can be sterilized at 100 ° C for 0.5 to 1.0 hours without substantial damage. It has also been found that the solution in AGGRISTIN media (in TRIS buffer, pH = 7.1; concentration 15 mg / ml) can be stored at 15 + 4 ° C for 3 months without decomposition.

Other details of the present invention will become apparent from the following examples without limiting the scope of protection to these examples.

Example 1 Preparation of A-ristomycin sulfate (R 1, R 2, and R 2 are as set forth in claim 1; R 2 = CH 2; X = HSO 4; Y = H) 2 g of crude Risto-25 mysin base prepared in a known manner are dissolved at room temperature. To 5.0 ml of distilled water, after which the pH of the solution is adjusted to 6.8 with 1 N sulfuric acid; the exact pH is measured with a device. The yellow, slightly opalescent solution is clarified with a small amount of charcoal and filtered. To the resulting clear solution is added dropwise 15-20¾ (v / v) acetonitrile until cloudy. The mixture is allowed to stand at room temperature until crystallization occurs. This procedure is repeated, if necessary, by adding a further small amount of aceto-35 nitrile for 2-3 days, so that crystallization is stopped by cooling for 2 H in a refrigerator at + 14 ° C.

12,760 90

The square white crystals formed are filtered off, washed twice with 1.0 ml of an ice-cold mixture of acetonitrile and water (1: 1) and then twice with 2 ml of cold 5 acetone each time, dried in a vacuum desiccator over heated calcium chloride and paraffin chips at room temperature. under reduced pressure to constant weight. Yield Ί1% ο.

Example 2 Preparation of A-ristomycin sulphate 1.5 to 2.0 g of crude A-Ristomycin sulphate used as starting material are dissolved in 4.0 ml of distilled water by constant shaking. To the strongly foaming solution is added 1.0 ml of acetonitrile, the mixture is clarified with activated carbon and filtered. The pH is neutralized with 2 N ammonium hydroxide in the presence of the bromothymolysin indicator (change from yellow to sky blue). An additional amount of acetonitrile is added to the solvent until cloudy. The mixture is allowed to stand until crystallization begins, the separated crystals are redissolved by heating on a water bath and the solution is allowed to cool slowly to room temperature. Further processing is performed as described in Example 1. Yield 65¾.

25 A-ristomycin monosulphate isolated according to both methods has excellent thrombocyte aggregation properties and meets the following very good qualitative requirements: a) The decomposition of the product starts at 165-170 ° C (measured in a Mettler FP-5 melting point apparatus - lokennorekisteröinti).

b) A high quality liquid chromatographic apparatus (Hewett-Packard 1082, HPLC) is used to prepare a chromatogram of excellent quality from the product.

35 c) The product is uniform according to layer chromatography «76090. The log e value measured in an aqueous solution at 280 nm is 4,0 to 4,5, the sulphate ash content is not higher than 0,255 · At a boiling point of acetone with a water jet pump (approximately 15 mmHg), suction for 4 consecutive hours is A -ristomycin monosulphate weight loss up to 1 - 2.5¾.

Thin layer chromatographic characterization of the crude product and A-ristomycin + monosulfate prepared according to Examples 1 and 2 is performed as follows: Materials and reagents used in the assay: I. Freshly prepared aqueous solution of 1-2% of A-ristomycin model.

II. 1. DC bottom roll of cellulose (Merck 0.1 mm) thin-15 layer.

2. Pixion 50x8 disc (Reanal).

III. Solvent mixture as eluent: a) n-butanol-pyridine-acetic acid-water (15: 10: 3: 12) or 20 b) n-butanol-pyridine-n-propanol-acetic acid-water (20: 10: 5: 3: 32). Upper organic phase II. 1 thin layer.

II. To equilibrate and start layer 2, use the following citrate buffer 25 (pH 6) in deionized water: 7.0 g of citric acid 4.0 g of sodium hydroxide 8l, 0 g of sodium chloride and 100 ml of methylcellosolve 30 are dissolved in a 1000 ml volumetric flask and made up to the mark with distilled water.

IV. Developer: Pauly's reagent Dissolve 0.05 g of diazotized sul'1anylic acid in 10 ml of 10 [mu] l of sodium carbonate solution and use the freshly prepared solution.

m 76090

The pure crude product in the cellulose layer in the solvent mixtures a) and b) has Rf values of 0.59 or 0.52, respectively. On a Pixion 50x8 plate, an Rf value of 0.6 L is measured in citrate buffer (pH = 6). The reagent is very sensitive, 5 it shows practically all decomposition products; the lower display limit is 0.5 to 1.0 yg.

High performance liquid chromatographic measurement is performed using a Hewlett-Packard 1082 HPLC instrument equipped with an isocratically operated pump. Our experiments use 10 analytical columns prepared in our laboratories (Cg-Lichrosorb, 10, etc.). The active substance content of the A-ristomycin models is determined with a UV detector operating at 254 nm. 1 milli-molar solutions are prepared from the models, and 20-30 μl samples are sprayed onto the column. The chromatograms are developed as follows: (I) in a citrate droplet buffer (pH 6.3) containing 12% methylcellosolve, liquid flow 2.0 ml / min, paper speed 0.1 cm / min.

20 (II) in 0.1 M ammonium formate (pH 7.4) containing $ 10 acetonitrile (formic acid free); fluid flow 3.0 ml / min; paper speed 0.3 cm / min.

The above conditions have proved to be the most suitable.

The stability of crystalline A-ristomycin sulfate prepared according to Examples 1 and 2 is determined after thermal sterilization of the UV irradiation extension of the solid models and aqueous solutions of various concentrations, or by standing at + 4 ° C in TRIS buffer, respectively.

(a) 50 to 100 mg of ristomycin A is irradiated in a small stoppered bottle with UV light so that the distance between the bottle containing the model 35 and the lamp is about 20 cm. 40-hour

II

15 76090 after irradiation, the color of the prepared solution deepens slightly; At a concentration of 15 mg / ml, the aggregation of human platelets is rather slow.

5 b) Two solutions of similar dilution (0.39 mg / ml or 0.35 mg / ml, respectively) and a more concentrated solution (6.8 mg / ml) are prepared from distilled water A-ristomycin. The solutions are kept for 3 hours in a water bath at 100 ° C. Evaporated water is replaced from time to time.

The solutions are cooled to room temperature and 1%

The Elcm value is determined from the light absorption intensity measured at 280 nm. It can be seen that the increase in light absorption of the solutions kept at 100 ° C is not linear.

In the range of 0-1 hours, the substances recovered by lyophilization do not show decomposition according to thin layer chromatography and high pressure liquid chromatography. The substances aggregate satisfactorily with normal human platelets at the desired concentration.

c) A solution of 15 mg / ml in TRIS buffer (pH 7.4) is prepared from ristomycin A and stored in a refrigerator at + 4 ° C for 12 weeks. This stock solution is subjected to a standard platelet aggregation test, which is determined after two weeks or after a corresponding dilution of buffer (0.2 ml) (10 ml). During experiment 30, the aggre- Λ 'of the A-ristomycin solution remained. the activity to be measured and the E „value change- ° lcm as a tower.

i6 76090

Example 3 Preparation of bis-Ν, Ν * -trifluoroacetyl-A-ristomycin 206 mg of A-ristomycin A are suspended in 10 ml of anhydrous methanol, followed by dropwise addition of 0.28 ml of trifluoroacetic anhydride under ice-cooling. The reaction mixture is left to stand at room temperature and evaporated to dryness under reduced pressure. The residue is redissolved in anhydrous methanol and re-evaporated. This operation is repeated three more times, after which the product is dried in a vacuum desiccator, over heated calcium chloride and paraffinic acid to constant weight. Yield 90%.

The bis-Ν, Ν'-trifluoroacetyl-A-Risto-15 mycine thus obtained does not have a characteristic melting point (decomposition point). Conditioning: the cellulose layer in the solvent mixture purple (see Example 2) has an Rf value of 0.45.

Analysis: C99H108 ° 46N8P6 (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 in the same manner as A-ristomycin A.

25

Example 4

Preparation of reagents 15.00 g of A-ristomycin monosulphate meeting the above qualitative requirements are suspended in 100 ml of distilled water (pH 6.8) in a 1 l Erlenmeyer flask. The active substance separating into the wall of the flask is again reconstituted with 400 to 500 ml of distilled water. The solution thus obtained, having a homogeneous concentration of active substance, is filtered defiberless in a 100 ml volumetric flask, the flask used for dissolution and the filter are washed quantitatively and the measuring vessel is filled to the 17 76090 mark. The concentration of A-ristomycin monosulfate A thus obtained is 15 mg / ml. (To prepare a sterile preparation, the solution is kept at 100 ° C for 30 to 60 minutes and then cooled to room temperature.) From this solution, use an automatic filling device under aseptic conditions to fill exactly 1.00 ml each time into brown, so-called re-opening bottles. After quick-freezing, the flasks are lyophilized and sealed.

To check the accuracy of the accurate filling and lyophilization, check the A-risomycin content (15 mg) per dose in a few vials according to the HPLC method and weight determination.

Claims (3)

18 76090 Claim: A method for isolating high-purity A-ristomycin 5 of formula I and its derivatives from impure ristomycin or crude ristomycin, OR * / .OR HV o V <0R 3 <nHQ> -0 “ ”" -0-p-r0 / - NH O 0 ^ ^ 0R K »J 51 K 'r ^ irm N HY, s (O II 19 76090 in which R is a hydrogen atom, denotes O-alpha-D-Araf (1- * 2) -O-alpha-D-Manp (1- * 2) - O- (alpha-L-Rhap) - (1- * 6) -O-beta-D-Glop; R 2 represents 2,3,6-tridesoxy-3-amino- (NHY) -alpha-L-ribohexapyranosyl; R 2 is an alpha-F-mannopyranosyl group; Rjj represents an alkyl group having 1 to 4 carbon atoms, X represents a mineral acid residue; 10. represents a hydrogen atom or a group of the general formula C (Z) 3- (CH2) n-CO-, in which Z is a hydrogen atom or a halogen atom and n represents an integer from 0 to 3, characterized in that the crude ristomycin base has 30-50 5 & the pH of the aqueous solution is adjusted to 6.7 to 7.0, preferably 6.8, by treatment with mineral acid - preferably dilute sulfuric acid; or a 30-50% aqueous solution of crude A-ristomycin sulphate is adjusted to pH 6.7 to 20 7SO with an alkaline solution - preferably ammonium hydroxide - and then treated with a water-miscible lower aliphatic carboxylic acid nitrile - preferably acetonitrile - 20- 50% (v / v) at room temperature, then crystallized at + 4 ° C, if desired dissolved in an organic solvent - preferably an alkanol having 1 to 5 carbon atoms - and then acylated with an acid anhydride-type acylating agent - preferably trifluoroacetic anhydride at a temperature of - 0-20 ° C and then isolating the product or products obtained. 76090 20 For the isolation of the diagnostic assay A-cross-cyclized and derived from the formulation of 5 or more cross-sectional forms of the formulation (I), OR δ / .OR 0 = (/ - (10 / o or j /? = $. NH \ 15 (o> ° <. A y_ ^ n> -0R 20 - \ 'HX' / NH OR NHY 35 (i)