PL193017B1 - Method of obtaining a gamma-lactamic compound, a gamma-lactamic compound and their application - Google Patents

Abstract

1. A method of producing a γ-lactam compound of general formula I, wherein R represents a hydrogen atom or an acceptable group in the pharmacy of a cationic salt, characterized in that the strain Streptomyces rimosus B is grown PZH, showing the ability to produce the compound γ-lactam in a culture medium containing available sources of carbon and nitrogen and inorganic salts under subsurface conditions fermentation with aeration, pending obtaining separable the amount of γ-lactam compound that followed carried out into its pharmaceutically acceptable cationic salt.

Description

Description of the invention

The invention relates to a process for the preparation of a γ-lactam compound of general formula I wherein R is a hydrogen atom or a pharmaceutically acceptable cationic salt group. The obtained γ-lactam compound, a new inhibitor of DD-peptidase 64-575, isolated from the fermentation broth of the Streptomyces rimosus strain, exhibits antibacterial properties.

Actinomycetes are known to be a rich source of antibiotics, as well as extracellular DD-carboxypeptidases / transpeptidases (DD-peptidases) classified as PBPs (penicyllin binding proteins), which are in turn binding sites for β-lactam antibiotics.

DD-peptidase inhibitors inhibit bacterial cell wall peptidoglycan biosynthesis, which in turn leads to the death of the bacterial cell. Known natural, semi-synthetic and synthetic DD-peptidase inhibitors are β-lactam compounds (Frere et al. In The Chemistry of β-lactams, 148-197, 1992) and lacticin - the only natural γ-lactam analog (Harada et al. In Tetrahedron Lett., 27, 6229-6232, 1986; Nozaki et al. In Nature, 325, 179-180, 1987; Okazaki in Frontiers of Antibiotic Resarch, 303-311, 1987). Β-lactam antibiotics are inhibitors of DD-peptidases and constitute an important group of antibacterial drugs. Due to the wide use of these antibiotics, the number of bacterial strains resistant to their action is constantly increasing. Bacterial resistance arises through the mutation of genes encoding DD-peptidases and β-lactamases and the transfer of genes encoding modified β-lactamases. β-lactamases inactivate β-lactam antibiotics. One of the possibilities of avoiding inactivation by β-lactamases is the modification of β-lactam antibiotics to obtain compounds not recognized by these enzymes, but binding with DD-peptidases. The growing resistance of pathogenic microorganisms to the antibiotics used makes it necessary to introduce new compounds of therapeutic importance.

The discovery of lacticin in 1986 and the study of its mechanism of action initiated a series of studies on non-β-lactam compounds. It is known that lacticin (LTW), non-β-lactam DD-peptidase inhibitors and β-lactam antibiotics have the same mechanism of action. LTW shows inhibition of PBPs activity in Escherichia coli in analogy to β-lactam antibiotics (Nozaki et al. In J. Antibiot. 42, 84-93, 1989). Lacticin is hydrolyzed by various cephalosporinases and penicillinases, induces the production of β-lactamases, shows much higher antibiotic activity against susceptible mutants of Escherichia coli and Pseudomonas aeruginosa strains, inhibits the growth of anaerobic bacteria, and does not inhibit the growth of mycoplasmas and fungi (Baldwin and fungi). J. Antibiot. 44, 1-24, 1991). In addition to affinity for PBPs, LTW blocks the -SH (thiol) groups of biologically active proteins in cell membranes. Due to the high toxicity of LTW with parenteral administration, it cannot be introduced into therapy at the current stage of research. The discovery of lacticin and the study of its mechanism of action allowed the conclusion that the presence of a β-lactam ring is not necessary for the compound to have an affinity for PBPs and to have antibacterial properties.

So far, a certain amount of γ-lactam compounds, penem analogs, carbapenems and monobactams has been obtained by chemical synthesis, which in vitro showed very little or no antibacterial activity. The formation of γ-lactam compounds in bacteria is unknown (Henriksen et al. In J. Antibiot., 51, 99-106, 1998).

Several methods of searching for microorganisms producing β-lactam antibiotics have been described. One of them is the use of bacterial strains hypersensitive to β-lactam antibiotics, e.g. mutants of Pseudomonas aeruginosa (Kitano et al. In J. Ferment. Technol., 53, 327-338, 1975) and mutants of the Escherichia coli strain (Aoki et al., in J. Antibiot. 29, 492-500, 1976). This method was used to search for penicillins and cephalosporins in mushroom cultures. A specific and sensitive method is also used to detect β-lactam compounds that induce β-lactamase production by standard strains, e.g. Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa (Sykes and Wells in J. Antibiot, 38, 119-121, 1985). A method for the detection of β-lactam antibiotics using DD-carboxypeptidase has also been described (Frere et al. In Methods Enzymol., 45, 610-636, 1976). A modified version of this method is used in the present invention. Β- and γ-lactam antibiotics and the strains that produce them are the subject of many publications and patents, for example: Streptomyces clavuligerus - U. K. Patent 1,508,977; Streptomyces jumonijnensis - U. S. Patent 3,865,693; Empedobacter lactamgenus, Lysobacter albus (Harada et al. 1986, Nozaki et al. 1987).

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In view of the growing resistance of bacteria to the action of β-lactam antibiotics, such as penicillins or cephalosporins, DD-carboxypeptidases are of particular importance as models for the search and study of new antibiotics.

The problem that the invention was supposed to solve was:

Preparation of the γ-lactam compound of the new inhibitor DD-peptidase 64-575 by culturing the strain Streptomyces rimosus B PZH, purification of the obtained inhibitor produced by this strain, determination of the chemical structure and determination of its bactericidal properties.

According to the invention, the method for producing a γ-lactam compound of general formula I, wherein R is a hydrogen atom or a pharmaceutically acceptable cationic salt group, comprises culturing a Streptomyces rimosus B PZH strain having the ability to produce a γ-lactam compound in a culture medium containing digestible carbon, nitrogen and inorganic salt sources under subsurface aerated fermentation conditions until an extractable amount of the γ-lactam compound is obtained, which is then converted to its pharmaceutically acceptable cationic salt. The Streptomyces rimosus B strain PZH 64575 was obtained from the Streptomyces rimosus TZF 0-29 strain from the Collection of the Independent Laboratory of Actinomycetes and Imperfect Fungi of the National Institute of Hygiene by mutation and selection among regenerated protoplast strains (genetically diverse population). Strain

Streptomyces rimosus B PZH differs from the starting strain in morphological and physiological properties, and also shows the ability to produce a DD-peptidase inhibitor. This strain was identified as a new strain of Streptomyces rimosus B of the National Institute of Hygiene, deposited under the number 001/99 at the Central Laboratory of Serum and Vaccines in Warsaw Poland.

The environment and procedures used to study the taxonomy of the strain were as described by Shiring and Gottlieb in "Methods for characterization of Streptomyces Species", Int. J. Syst. Bact., 1966, 16: 313-340; by Waksman in The Actinomycetes, vol. III, "Classification, Identyfication and Description of Genera and Species", 328-334, published by The Williams and Wilkins Co., Baltimor, 1961; and by Arai in Culture Media for Actinomyces, published by The Society for Actinomycetes Japan, 1975. The strain was incubated at 28 ° C for 7, 14, 21 days. The colors were described using common terminology except that the exact color was determined by comparison with the color standards "The ISCC-NBS Method of Designating Colors and a dictionary of Colors Names". In addition, the strain was grown at the following temperatures: 4 ° C, 20 ° C, 32 ° C and 37 ° C for 7.14, 21 days.

The microorganism showed the following characteristics:

Morphological features.

The strain producing the DD-peptidase inhibitor allows the formation of a single branched superficial mycelium in various environments. Morphological properties were observed on yeast-malt agar and starch agar with inorganic salts on which the strain spore best. Spore weights were observed after 14 and 21 days under light and electron microscopy. Color of spores on yeast-malt medium gray-yellow (2db), light straw-colored (2ca) and yellow-pink (3ca), color of spores on starch agar with inorganic pearly yellow (2ba) and white series (a). Spire type branched sporophores (S), spiral spore chains with an average of 5-6 turns. Spores of cylindrical shape 1.0 nm long and 0.45 μm wide, arranged in chains of 10 or more spores. The surface of the spores is smooth.

Characteristics of breeding in different environments:

1. Liquid tryptone-yeast medium - inoculated with spores from slants in the amount of 4 · 10 ⁴ · cm ^-3 . After 48 hours of incubation, the mycelium is very well developed in the form of a mesh, few branches visible under the light microscope. The culture was centrifuged for 20 minutes, washed three times with sterile distilled water, and transplanted into further media. The culture on the following media was incubated at 28 ° C and the results were observed after 7, 14 and 21 days.

2. Yeast-malt agar - very good growth, the surface of the colony raised, visible furrows, air mycelium forming centrifugally. Color of air mycelium gray-yellow (2db), bamboo-colored (2fb) and light straw-colored (2ca), basic mycelium color bamboo (2fb), no soluble pigment. Plates with the strain growing on this medium were additionally incubated at the following temperatures: 4 ° C, 20 ° C, 32 ° C and 37 ° C.

3. Oat agar - moderate to good growth, flat colony surface, strain spores poorly on this medium. Color of air mycelium pearly-yellowish (2ba), gray-yellow (2db), bar4

The base mycelium is pistachio-yellowish (11/2 fb), and bamboo-colored (2fb). No soluble pigment.

4. Starch agar with inorganic salts - good growth, the surface of the colony slightly raised, the base mycelium slightly wrinkled, the strain spores very well. The color of the air mycelium is pearly-yellow (2ba) and from the white series (a), the color of the base mycelium is bamboo (2fb) and yellow-orange-coffee (3ea, 4gc).

5. Glycerin-aspartate agar - good growth, leathery, visible furrows, slightly raised mycelium. The strain spores very poorly. Pearly yellowish (2ba) and pink yellow (3ca) aerial mycelium, bamboo-colored (2fb) and light straw-colored (2ca) basal mycelium, no soluble pigment.

6. Iron, yeast-peptone agar - poor growth, slightly leathery, non-sporulating mycelium. The color of the aerial mycelium is gray-yellow (2db), the color of the base mycelium is bamboo (2fb) and pistachio-yellowish (11 / 2fb).

7. Tyrosine agar - good growth, mycelium slightly raised, visible furrows. The color of the aerial mycelium is light straw (2ca), the color of the base mycelium is bamboo (2fb) and gray-yellow (2db).

Temperature dependencies

4 ° C 20 ° C 28 ° C 32 ° C 37 ° C Growth poor Growth good Growth excellent Growth good Growth good

The use of carbon carriers in the Pridham and Gottlieb agar environment was investigated with the following results presented in Table 1.

T a b e l a 1.

Coal sources used:

Carbon source Usage D-glucose ++ L-arabinose + _- Saccharose + _- D-xylose - Inositol ++ D-mannitol ++ D-fructose ++ L-rhamnose - Raffinose + _- Cellulose -

Nitrate reduction (Difco, nitrate broth) was positive, the strain has the ability to reduce nitrate to nitrite. The strain does not produce hydrogen sulfide. The stability of the deposited culture No. 001/99 at the Central Laboratory of Serum and Vaccines (CLSiS) in Warsaw, Poland is guaranteed for the duration of each patent granted on the basis of this application. Any restriction on availability to the deposited culture will be removed upon grant of a patent to this application.

The resulting γ-lactam compound of general formula I, wherein R is a hydrogen atom or a pharmaceutically acceptable cationic salt group, is produced by the fermentation of a new strain

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Streptomyces rimosus B PZH, showing the ability to produce γ-lactam compound-DD-peptidase inhibitor.

The inoculum is prepared by collecting the top layer of spores from 10-day-old slants from a yeast-malt medium to obtain a suspension containing 10 ⁹ spores in 1.0 cm ³ of distilled water. The inoculum prepared in this way is inoculated into the prepared liquid medium.

The Streptomyces rimosus B strain of the National Institute of Hygiene was grown under favorable conditions at a temperature of 24 ° C to 36 ° C under subsurface aeration fermentation conditions on a liquid medium containing carbohydrate sources such as sugars (lactose, glucose), organic nitrogen substances such as tryptone, peptone, yeast extract, growth substances such as corn steep extract, minerals containing trace elements such as iron, magnesium, zinc, and calcium carbonate or phosphates as buffering agents. From culture grown up to 96 hours, samples were regularly withdrawn, centrifuged and tested for inhibition of DD-peptidase 64-575 activity in the supernatant.

The activity of the obtained γ-lactam in terms of DD-peptidase inhibition is determined as follows: 0.01 cm ^{3 of the} supernatant of liquid culture or samples after another purification stage are added with 0.01 cm ³ of the enzyme solution with the activity of 45 U mg ^{- 1} (1 mg of DD-peptidase lyophilisate is dissolved in 1 cm ^{3 of} 0.1 M phosphate buffer pH 8.0). Highly pure DD-peptidase was obtained from the Saccharopolyspora erythraea strain 64-575 in the Independent Laboratory of Actinomycetes and Imperfect Fungi of the National Institute of Hygiene. The sample is incubated in a 37 ° C water bath for 30 minutes (to bind the inhibitor to the enzyme). ^{Then 0.02 cm 3 of a} solution containing 4.52 mg · cm ^{3 of} N “N ⁱ tripeptide” are added to the sample! Ac2-L-Lys-D-Ala-D-Ala (enzyme substrate) in 0.1M phosphate buffer, pH 8.0. Instead of the enzyme substrate, 0.02 cm ³ of pH 8.0 phosphate buffer is added to the controls. The standard sample contained 0.02 cm ^{3 of a} solution of 0.089 mg · cm ^-3 D-alanine in distilled water instead of the substrate solution. All samples prepared at 4 ° C are incubated for 30 minutes at 37 ° C. The enzymatic reaction is stopped by placing the samples in a water bath at 100 ° C for 2 minutes. After cooling, 0.077 cm ^{3 of} the reaction mixture containing 0.06 cm ^{3 of a} solution containing 0.05 mg · cm ³ of flavin adenine dinucleotide (FAD) in 0.1 M phosphate buffer at pH 8.0 are added to the samples; 0.01 cm ^{3 of a} solution containing 0.05 mg · cm ³ of horseradish peroxidase with an activity of 1230 U · mg ^-1 in distilled water; 0.005 cm ^{3 of a} solution containing 5.0 mg · cm ³ of o-dianizidine in methanol; 0.002 cm ^{3 of a} solution containing 11.77 mg · cm ³ of D-amino acid oxidase isolated from pig kidneys with an activity of 6.7 U · mg ^-1 in 0.1 M. phosphate buffer pH 8.0. The samples with the reaction mixture are incubated for 10 minutes at 37 ° C, and then 0.35 cm ^{3 of the} mixture containing methanol, distilled water and concentrated sulfuric acid in the ratio 5: 5: 6 are added. Extinction is measured spectrophotometrically at 540 nm.

The highest level of γ-lactam compound is obtained in the range of 24-96 hours. At 48 hours, the supernatant inhibits approximately 100% of DD-peptidase activity (Table 2).

T a b e l a 2.

Inhibition of DD-peptidase 64-575 activity by the culture supernatant of the test strain.

Breeding time [hours] Inhibition of enzyme activity 64-575 [%] Deviation standard 24 76 1.5 Strain 48 96 2.6 Streptomyces rimosus B PZH 72 87 0.2 96 75 0.3

The compound can be isolated by extraction from the deproteinized supernatant of the strain culture with organic solvents, e.g., ethyl acetate or diethyl ether at pH 2.0. In order to obtain a pure compound, the extract is chromatographed on an ion exchange resin with an anion exchanger. An acetic acid solution is used as the mobile phase. The next purification step is performed by a gradient method using HPLC.

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The fraction of a small amount of crude product as an amorphous precipitate obtained by the gradient method after being dissolved in D2O was analyzed by NMR. On the basis of proton chemical shifts obtained in the spectrum ¹ H-NMR (Fig. 1) of the fraction (4,42d-d; 4,28d-q, d 4.21;

1.18d; 2.1-2.6m), the structure of the obtained compound was established as a γ-lactam compound - pyroglutamyltreonine. Molecular weight was measured by mass spectrometry using the Liquid SIMS technique (Fig. 2). A value of 231 u corresponds to the molecular weight of the inhibitor and one proton (M + H) ⁺ , and 253 u corresponds to the molecular weight of the inhibitor and the sodium cation (M + Na) ⁺ . The above study was confirmed by the Peak matching technique (Fig. 3) as the standard ion mass 207u (a cluster of two molecules of glycerol and Na ⁺ cation) was used. The solid line in the graph corresponds to the standard, dashed line to the DD-peptidase inhibitor 64-575 - γ-lactam obtained by the method of the invention. The molecular weight of the protonated inhibitor is 231 µ, minus one proton, the molecular weight of the resulting inhibitor is 230 µ.

The obtained γ-lactam inhibitor of DD-peptidase 64-575 of the strain Saccharopolyspora erythraea PZH TZ 64-575 is a dipeptide and has the chemical structure of pyroglutamyltreonine, specifically 3-hydroxy-2 [(5-oxo-pyrrolidine-2-carbonyl) -amino] - butyrate of the formula C9H14N2O5 shown in formula 1.

The γ-lactam compound of general formula I obtained by the process of the invention is acidic in nature and may form cationic salts by reaction with basic agents. Such salts may be prepared by the process of the invention. They are obtained using methods typical for the compounds - γ-lactam antibiotics. For example, a solution of a compound of Formula 1 in a water-immiscible organic solvent after washing with an aqueous solution may be treated with at least one equivalent, and preferably an excess, of a suitable basic agent. The organic solution is dried or evaporated in vacuo to give the correct cationic salt. Typical basic agents used for this purpose are alkali metal hydroxides such as sodium or potassium hydroxide, calcium or ammonium hydroxide.

Only limited data on pyroglutamyltreonine have been found in the literature. Pyroglutamyltreonine has been used as an ingredient in clear soaps (Japanese Patent No. 59204698, Onodera et al. 665-670, 1984). It was found that the addition of pyroglutamic acid dipeptides shortens the production time, the soaps harden faster and are more transparent, and the skin washed with them maintains moisture for longer.

No data identifying pyroglutamyltreonine as a separate, selected chemical compound with specific physico-chemical properties have been found.

No data were found on the study of the antimicrobial properties of pyroglutamyltreonine or its sodium or potassium salt. Our research carried out by the plate method showed that the γ-lactam - DD-peptidase inhibitor 64-575 obtained with the method according to the invention inhibits the growth of Escherichia coli and Staphylococcus aureus (Table 3).

T a b e l a 3.

Antimicrobial properties of the DD-peptidase inhibitor 64-575.

Concentration of the inhibitor lyophilisate [mgcm ' ³ ] Bacterial growth inhibition zone [mm] Escherichia coli x 580 (+) Escherichia coli x 580 (-) Staphylococcus aureus DS 267 0.01 thirty 29 26 0.03 32 32 28 0.05 34 33 29 0.07 36 36 thirty 0.10 36 36 thirty

(-) penicillinase free medium (+) penicillinase medium

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The MIC and therapeutic doses of the compound according to the invention were established in mice experimentally infected with Escherichia coli and Staphylococcus aureus (Table 4).

T a b e l a 4.

Protective effect of DD-peptidase inhibitor in experimentally infected mice.

Examined MIC ^ gcm ' ³ ) ED50 (mg / kg) bacteria DD-peptidase inhibitor DD-peptidase inhibitor Staphylococcus aureus 5.8 24.5 (sc) Escherichia coli 38.0 56.0 (sc)

The minimum inhibitory concentration (MIC) was determined using the standard dilution method. The bacterial inoculum was prepared according to the Mc Farland scale.

The inhibitor inhibits the activity of DD-peptidases included in PBPs, which are involved in the synthesis (cross-linking) of bacterial peptidoglycan. DD-peptidase inhibitors - known β-lactam antibiotics show low toxicity towards human and animal cells that do not have specific chemoreceptors (PBPs). Accordingly, the invention can be used for the development of new antimicrobial compounds. As already mentioned, due to the constant increase in resistance of bacterial strains to known antibiotics, it is necessary to search for and introduce new antibacterial compounds.

As shown by the data in Table 3, the γ-lactam compound of general formula I, obtained by the process of the invention, shows antibacterial activity against Staphylococcus aureus and Escherichia coli. Therefore, the compound according to the invention and its salts are useful for sanitary-hygienic purposes and for sterilization of hospital utility surfaces, for example operating tables. The usefulness of γ-lactam - an inhibitor of DD-peptidase 64-575 obtained by the method of the invention in the treatment of cutaneous purulent processes and otitis media caused by Staphylococcus aureus bacteria in dogs.

At the same time, it is useful in the experimentally induced infection of the urinary tract with the bacterium Escherichia coli in mice. The compound may be administered alone or in the form of a pharmaceutical composition in which it is mixed with a pharmaceutically acceptable carrier and / or other acceptable excipients.

The following examples serve to further illustrate the process of the invention without in any way limiting it.

P r z x l a d I.

The Streptomyces rimosus B strain of PZH was grown under favorable conditions on a liquid medium containing tryptone (Difco, USA) 17 g, peptone (Difco, USA) 4 g, yeast extract (Difco, USA) 5 g, corn steep extract 10 g, lactose 10 g , CaCO ₃ 3 g, K ₂ HPO ₄ 4 g, KH ₂ PO ₄ 2 g, MgSO ₄ 7H ₂ O

0.5 g, glycine 2 g in 1 dm ^{3 of} tap water was boiled for 10 minutes, and then the pH was adjusted to 6.7 _{with 10% NaOH 3 solution.} Said conical fermentation flasks containing 500 ^cm3 poured over 45 cm ^{3 of} medium and sterilized. The medium was inoculated with 1.0 cm ^{3 of a} suspension containing 10 ⁹ spores from a yeast-malt slant. Cultures were grown up to 120 hours, aliquots were collected every 24 hours, centrifuged and the supernatant tested for inhibition of DD-peptidase 64-575 activity. The results are presented in Table 2. The highest level of inhibitor is obtained in the range of 24-96 hours. At 48 hours, the supernatant inhibited 96% of the activity of DD-peptidase.

P r z x l a d II

The supernatant of Example 1 obtained from 48 hours of culture was subjected to protein precipitation by adding acetone until 70% acetone was obtained, centrifuged at 7000 g for 10 minutes, the pellet was discarded. The acetone was distilled off on a vacuum evaporator (Vinipan, Poland). The supernatant, deproteinized with acetone, was heated to 50 ° C and the precipitate was centrifuged at 7000 g for 10 minutes. The pellet was discarded and the culture supernatant after acetone evaporation was frozen at -70 ° C and subjected to a lyophilization process using a Beta 1-8 apparatus (Christ, RFN).

The lyophilisate was dissolved in 20 cm ^{3 of} phosphate buffer pH 8.0 and the solution was transferred to a 50 cm ³ separating funnel. Then 20 cm ³ of ethyl acetate were added. It was shaken in the funnel for 5 minutes. Then the aqueous layer and the organic layer were separated. ^{20 cm 3} of ethyl acetate was again added to the aqueous layer and the operation was repeated three times. The organic layer was discarded. The aqueous layer was adjusted to pH 1.0 with 1M HCl solution. Extraction was performed on 8

The mixture was added three times with 20 cm ³ of ethyl acetate. The aqueous layer was adjusted to pH 7.0 with use

M NaOH solution. Ethyl acetate was distilled off using a vacuum evaporator. The residue was dissolved in 1 cm ^{3 of} 0.1 M phosphate buffer pH 8.0 and adjusted to pH 7.0 with 1 M NaOH solution.

The preparation was purified on an AG 1-X8 anion exchange resin column (Resin 200-400 Mesh, Acetate Form, Bio-Rad, RFN). A column (Pharmacia, Sweden) with a diameter of 5 cm was used which was filled with 150 cm ^{3 of} resin suspended in a 2 M acetic acid solution. The bed was rinsed with deionized water until equilibrated. A P-1 peristaltic pump (Pharmacia, Sweden) was used. The flow rate during the elution was 5 cm ³ / min. (one column volume in a 30 min period). The pH of the eluate was measured every 30 minutes, and after reaching pH 4.9, 150 cm ^{3 of} dissolved deproteinate lyophilisate was introduced on the column and successively washed with 450 cm ^{3 of} : deionization water, 1 M acetic acid solution (conductivity 1.375 mS) and 2 M acetic acid solution ( conductivity 1.685 mS). Successive fractions were collected that were frozen at -70 ° C and lyophilized in a Beta 1-8 apparatus.

In 300 mm ^{3 of} 0.1% trifluoroacetic acid (TFA) solution 0.1 g of lyophilisate of the Streptomyces rimosus B PZH strain fraction eluted from the AG 1-X8 resin with 2 M acetic acid solution was injected and 10 mm ³ and then 50 mm ^{3 of the} solution were injected for HPLC apparatus loop (Shimadzu LC-6A System, Japan). Spherisorb 5 ODS 2 (C 18) columns with dimensions of 250 mm · 4.6 mm were used. The mobile phase flow was 0.5 cm / min ^-1 . A gradient was applied using two starting solutions: A (acetonitrile for HPLC) and B (0.1% TFA solution). The following gradient program was used: 10 min. 100% B, 25 min. 80% B and 35 min. 100% B. Absorption reading was at 218 nm. The injections were repeated and peak fractions with the same retention time were collected into one vessel. They were frozen and freeze-dried. 0.02 g of a white, amorphous, highly water-soluble solid is obtained. A small sample of the pure product was analyzed by NMR. On the basis of proton chemical shifts obtained in the spectrum ¹ H-NMR of this fraction (4,42d-d; 4,28d-q; 4,21d; 1,18d; 2,1-2,6m) (Fig. 1) is fixed structure of the obtained compound as a γ-lactam compound -3-hydroxy-2 [(5-oxo-pyrrolidine-2-carbonyl) -amino] -butyric acid. The measurement of the molecular weight, which is 230 u, was performed by mass spectrometry using the Liquid SIMS technique and the Peak matching technique (Fig. 2 and Fig. 3).

Elemental analysis for C9H14N2O5

Calculated: C 46.93 H 6.13 N 12.17 O 34.76

Found: C 46.95 H 6.09 N 12.17 O 34.78

P r z x l a d III

Investigation of the bactericidal properties of the γ-lactam compound of the DD-peptidase inhibitor 64-575

The following substrates were used for the tests:

Growth medium A

37 g of cardiac-cerebral extract (Oxoid CM 225, Great Britain), 10 g of agar No. 1 (Oxoid L II, WB) were dissolved in 1 dm ^{3 of deionized water, then the solution was boiled.} The medium was adjusted to a pH of 7.4; poured into test tubes and sterilized at 121 ° C for 15 minutes. After sterilization, the tubes were arranged to obtain the substrate in the form of slants.

Poor soil B.

18.5 g of cardiac-cerebral extract and 10 g of agar No. 1 were dissolved in 1 dm ^{3 of deionized water, and the procedure was continued as above.} The finished medium was poured into bottles and sterilized for 15 minutes at 121 ° C.

C propagation medium for Escherichia coli strain

In one dm ³ of deionized water was dissolved 13 g of nutrient broth # 1 (Oxoid L 29, UK. UK), after complete dissolution pH was adjusted to 7.4. ^{5 cm 3 of the} medium was poured into test tubes and sterilized for 15 minutes at 121 ° C.

D propagation medium for Staphylococcus aureus strain

10 g of Lab-Lemco agar (Oxoid L 29, WB), 10 g of peptone (Oxoid L 37, Great Britain), 5 g of NaCl, 4.54 g of KH2PO4, 5.94 g of Na2HPO4 were dissolved in 1 dm ^{3 of deionized water,} 10 g of No.1 agar (Oxoid L II, UK). The medium was boiled until the ingredients were completely dissolved, and then the pH was adjusted to 6.9. Sterilization was performed for 15 minutes at 121 ° C.

Two standard strains were used for the research: Escherichia coli X 580 and Staphylococcus aureus DS 267.

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E. coli strain X 580 was plated on growth medium A and incubated for 16 hours at 37 ° C. The propagation medium C was then inoculated with the bacteria harvested from the growth medium A and incubated for 16 hours at 37 ° C.

Medium B was liquefied and transferred to a water bath at 44 ° C. To 100 cm ^{3 of the} liquid medium, 1 cm ^{3 of the} inoculum from the propagation medium C was added. The whole was mixed slightly so that the bacterial culture was evenly distributed in the medium. Using sterile pipettes a volume of 25 ^cm3 were loaded at 18 cm ^{3 of} the substrate with the bacteria on sterile Petri plates. The plates were stored at 4 ° C for up to 14 days.

Solid medium culture of the E. coli penicillinase and X-580 were prepared in the same manner as described above for the 100 cm ^{3 of} the inoculated substrate temp. 44 ° C was added 100 mm ³ penicillinase solution (Bacto-Penas Concentrate, Penicillinase 10,000,000 j. Cm ^-3 , Difco, USA).

The medium was gently mixed to distribute the β-lactamase evenly.

A culture of S. aureus DS 267 was seeded on slants with growth medium A and incubated for 24 hours. at 37 ° C. After incubation, a thick suspension of bacteria was prepared in deionized water, which was then diluted 50 times.

The solid medium with the culture of the S. aureus DS 267 strain was prepared in such a way that the required amount of medium D was brought to a liquid state, and then it was transferred to a water bath at 44 ° C. 100 cm ^{3 of} medium D was added 1 ^cm3 of the diluted bacterial suspension of S. aureus and gently stirred. 18 cm ^{3 of} substrate were poured onto Petri dishes. After the agar coagulated, the plates were placed at 4 ° C.

₃

^{Five 100 mm 3} cavities were cut in the media on Petri dishes.

Five dilutions were prepared (0.01; 0.03; 0.05; 0.07; 0.1 mg of the γ-lactam compound of the DD-peptidase inhibitor in 1 cm ^{3 of} 1 M phosphate buffer at pH 8.0. the wells were poured with 100 mm of ³ consecutive dilutions of the neutralized solution of the DD-peptidase inhibitor 64-575. The plates were incubated for 16 hours at 37 ° C. The zones of inhibition of bacterial growth were then read. The lowest concentration of the inhibitor, i.e. 0.01 mg · cm ^-3 shows large zones of inhibition of bacterial growth: 29 mm for Escherichia coli x 580, 30 mm for Escherichia coli x 580 in penicillinase medium and 26 mm for Staphylococcus aureus DS 267.

Claims (7)

Patent claims A method for producing a γ-lactam compound of general formula I, wherein R is a hydrogen atom or a pharmaceutically acceptable cationic salt group, characterized in that a Streptomyces rimosus B PZH strain is cultivated, showing the ability to produce a γ-lactam compound in a culture medium containing available sources of carbon, nitrogen and inorganic salts under subsurface aeration fermentation conditions, until an extractable amount of a γ-lactam compound is obtained, which is then converted to its pharmaceutically acceptable cationic salt. 2. The method according to p. A method according to claim 1, characterized in that the γ-lactam compound of general formula I is produced by the Streptomyces rimosus B strain of the National Institute of Hygiene deposited under No. 001/99 at CLSiS Warsaw, Poland. 3. The method according to p. The method of claim 1, wherein the strain was cultivated from 24 to 96 hours. 4. The method according to p. The process of claim 1, wherein the γ-lactam compound of general formula I is converted to its pharmaceutically acceptable sodium or potassium salt. 5. A γ-lactam compound of general formula I wherein R is hydrogen or a pharmaceutically acceptable cationic salt. 6. The compound according to claim 5, produced in the culture of the strain Streptomyces rimosus B PZH. 7. Use of a compound of general formula I, wherein R is hydrogen or a pharmaceutically acceptable cationic salt as bacterial growth inhibitory agent.