SU738517A3 - Method of preparing antibiotic - Google Patents

Abstract

PURPOSE:Antibiotic PS-5 and its derivatives, particularly a trityl derivative. Characteristics thereof are as follows: soluble in water of pH6-9; thin layer chromatography: shows Rf=0.94, in an example, using Avicel SF cellulose thin-layer plate made by Funakoshi Yakuhin and a solvent of n-butanol/ethanol/water (7/7/6); paper chromatography: shows Rf=0.68, using Toyo filter paper No.50 and, in an example, a solvent of n-propanol/water (7/3); etc.

Description

(54) METHOD FOR OBTAINING ANTIBIOTICS

This invention relates to the field of microbiology for the production of tibiotics with inhibitory effects. The proposed antibiotic is new and the method for its preparation in the scientific, technical and patent literature has not been described. The aim of the invention is to obtain an antibiotic of the general formula 5-CH2-CH, -NH-CO-CH Yes OI where R is hydrogen, metal, alkyl or triphenylmethyl. This is achieved by the fact that the Streptomyces A 271 strain is grown under aerobic conditions in a nutrient medium containing carbon and nitrogen sources and mineral salts, at pH 4.0–9.0 and 20–40 ° C, followed by isolation of the desired product. in the form of acid or ester or salt. The Streptomyces strain was isolated from a soil sample collected near the Eiheiji temple in the Voshida area of the Fukui prefecture in Japan, designated A 271. The culture is deposited with Fermentation Research 3nstitute, Agency o JndustriaE Science and Techno ogie, where it is located under HOiepOM PERM - P.. 3984. A sample of the organism Streptomyces A 271 is also deposited by the ATCC Institute under No. 31358. Morphological features. Razvet-g. The appearance of the obtained mycelium is simple; its upper part forms hooks, loops or incomplete spirals. These forms are observed when cultured on a medium of oat flour and glycerin-aspartic agar medium, while straight or curved forms are usually formed on a medium of yeast and malt extracts, the shape of an oval or cylindrical / and the spores form a chain, consisting of more than 10 (usually 10-50) spores. Size 0.8-1.0 x 1; 0-1.8 microns, the surface is smooth, no flagella or sporangia are observed. Hyphae form on the aerial mycelium. The cultural characteristics of the Stap.ma Streptomyces A 271 are shown in Table 1,

Air color

Growth

Wednesday mycelium

Bold orange-strong-yellow

Pale orangeToo-yellow to light orange-yellow

Pale orange yellow to light orange yellow

- -

Pale orange yellow to light orange yellow

. Pale orange yellow to light orange yellow is almost undefined

Pale orangeFrom yellow to light orange yellow

Pale ora nzhevy

nd to light orange hf addition Physiological characteristics. It grows and develops at 10-40 seconds, the optimum is 20-30 ° C. Gelatino liquefies at 20 ° C; starch hydrolyzes, milk peptonizes, but does not coagulate. Forms melanoid pigments on pyrosine agar, on peptone yeast glandular agar, and in broth from tryptone-yeast extract. It absorbs arabinose, xylose, glucose, rhamnose, and poorly absorbs sucrose. It does not absorb fructose, inositol, raffinose, mannitol. A new antibiotic is obtained by inoculating spore strain of the mycelium. Streptomyces A 271 in an aerobic nutrient medium. As a nutrient medium using sources of carbon, nitrogen, inorganic salts. As carbon sources, glucose, glycerin, maltose, sucrose, molasses, dextrin, starch, low carbon or fatty carbon sources such as peanut butter are taken. When carried, nitrogen sources, peptone, meat extract, soybean flour, cotton seed oil, dried yeast, corn maize liquid, yeast extract, casein from off milk, sodium nitrate, ammonium nitrate, sulfate are added. ammonium, inorganic salts; Dickers Table 1

The color of the mycelium Soluble - substrate pigment

Light yellow light orange yellow

Light yellow

Light yellow to slightly yellow-pink

Light orange yellow to light yellow

Light orange yellow to brownish yellow pale yellow

Light orange yellow to pale brown

Light yellow lipophosphate, sodium chloride, calcium carbonate, magnesium sulfate, as well as traces of metals, such as cobalt, manganese. To prevent foaming, fermentation is added, silicone foam, silicone and vegetable oil are added. The medium can be sterilized before cultivation, the pH is adjusted to 4-9, preferably 6-8, before or after sterilization. The cultivation is carried out under aerobic conditions. Shake techniques in flasks or growing are carried out in a submerged state. The fermentation temperature ranges from 20 to 40 ° C, preferably from 25 to. The pH of the culture broth during fermentation is adjusted to 4-9, preferably 6-8. Fermentation is carried out until a sufficient amount of antibiotic is concentrated. Fermentation usually lasts from 30 to 90 hours, although this period varies depending on the composition of the medium, the temperature of the fermentation, the strain. The amount of accumulated antibiotic in the Fermentation Bouillon is determined by the bioassay method and bioautography.

The accumulated antibiotic is soluble in water and is mostly outside the mycelium; therefore, the mycelium is removed after fermentation by known methods, filtration by centrifugation, extraction, followed by isolation of α-5 tibiotic from the filtrate.

To regenerate and isolate the antibiotic, extraction is applied at low pH using a solvent, ethyl acetate, N-butanol or with back extraction from a layer of solvent into an aqueous layer at a higher pH, extraction at neutral pH using solvents; methylene chloride, chloroform or in the presence of LII lipophilic quaternary ammonium salt, such as benzalkonium chloride tetra-n-butyl ammonium hydrogen sulfate with reverse extraction from a layer of a solvent containing sodium iodide, potassium iodide.20

Adsorption is carried out on activated carbon or highly porous styrene-divinyl benzene resins, such as amberlite and

elution with aqueous methanol, aqueous acetone, gel filtration using Sephadex, column chromatography or thin layer chromatography using cellulose, silica gel, alumina, and precipitation by the addition of solvent with acetone. thirty

The antibiotic has a broad spectrum of antibiotic action, having a strong effect on various protib. bacteria, for example gram-positive, such as StaphyEococcus, DipEo-35coccus. Streptococcus, Sarcina, BaciEpus, and gAm-negative, belonging to the genera ACca Cygenes, Comamonas, Escherichia, KEebsieEBa, Proteus, Citrobacter, Enterobacter, Serratia. 4Q Antibiotic has the ability to increase the antibiotic effect of other antibiotics, such as penicillin ,, and cephalosporins, as well as Citrobacter, frendii, Proteus vu garis, Entero-5 bacter aerogenes, Serratia marcescens.

When administered to mice infected with pathogenic Gram-positive bacteria, the antibiotic has a significant therapeutic effect.

With the introduction of myoam by the intraperitoneal route in the amount of 500 mg / kg, the antibiotic does not have a lethal effect in experimental animals.

Since the antibiotic is more stable in the form of salt than in the form of free acid, it is preferably used for all pharmaceutical purposes.

As salts of antibiotic is

use alkali metal salts (sodium, 6, potassium, lithium), alkaline earth

(calcium and magnesium), as well as salts of other metals - aluminum, ammonium salt, primary, secondary or tertiary; -mines amines (monoethylamine, dimethylamine, 65

trimethylamine, monoethanolamine, diethanolamine), salts with organic bases, such as benzathine, procaine.

Suitable salts for pharmaceutical purposes are alkali metal, sodium, potassium salts.

The antibiotic is a monobasic acid containing a carboxyl group in the molecule. Therefore, from an antibiotic it is possible to obtain various esters with various alcohols, mercaptans, or their derivatives; This is done in a manner similar to the method used for known clavulanic acid or thienamycin-based antibiotics.

According to the invention, the corresponding antibiotic ester is an ester of the general structural formula

S - CH J-CH J-NH -CO-CH3

CHj-CHj.

(II

-N

COOR

one

where R is a low alkyl group. or triphenyl group.

In the above structural formula (II), a lower alkyl group is designated by a branched or unbranched chain group, in particular an alkyl group with C atoms less than b, namely a group with C atoms from 1 to 4. These include methyl, ethyl , n-propyl, isopropyl, and-butyl, iso-butyl, n-pentyl, iso-pen, tyl, n-hexyl, etc.

According to the invention, the esters of the given structural formula (II) can be obtained by the interaction of the antibiotic of the structural formula (1) or its salts with compounds of the general structural formula

(Iii)

CU

where R has the same meaning as above, and Y is an atom or group that. can split.

As for the atoms or groups expressed by the symbol Y in formula (III), any kind of atoms or groups that can be split when contacted with the carboxyl group of the antibiotic, for example halogen atoms, chlorine, bromine or iodine, sulfonyloxy groups, carbonyloxy groups, are applicable. reactive, such as -O-CO-CF, j, etc. The halogen atoms are preferred.

. Examples of the compound of structural formula (III) are: methyl alcohol, methyl iodide, dimethyl sulfate, methyl merc: ptan, ethanol, ethyl bromide ,. ethyl iodide, ethyl mercaptan, n-propyl chloride ,.  n-propyl iodide, propyl alcohol, iso-propyl alcohol, iso-propyl bromide, -butyl alcohol, n-butyl bromide-, n-butyl iodide, i-pentyl alcohol, n-pentyl-1 chloride, n-penyl albromide, n-pentyl iodide, hexyl alcohol, n-hexyl bromide, H-hexyl iodide, trityl alcohol, trityl mercaptan, trityl chloride, trityl bromide, and the like.  P.  One example of lower diaoalkanes suitable for producing antibiotic esters is diazomethane.  The interaction of the antibiotic with compounds of the general structural formula (III) or with lower diazoalkv-namnollet should be carried out by known methods used in the esterification of an ester.  For example, the interaction of an antibiotic with compounds of the general structural formula (III or lower diaoalkanes is preferably carried out in an inert liquid medium, although it can be carried on without it.  As such a medium can be used hydrocarbons such as benzene, toluene, n-hexane, cyclohexane and halogenated hydrocarbons such as chloroform, methylene chloride or Dru Gia amidyr as dimethylformamide, hexamethylphosphoric triamide or other ,, dimethyl sulfoxide, ethers such as diethyl, diisopropyl, di -and-butyl tetrahydrofuran, dioxane or others.  esters, such as ethyl acetate, N -but acetate or others, ketones, such as methyl ethyl keto acetone. n or t.  P.  These solution 1e. Whether used alone or as a mixture of two or more of them.  The reaction temperature can vary from within the range depending on the type of compounds of the general structural formula (III), the type of lower diazoalkane of the kind of liquid medium or the products used.  The reaction temperature can be selected in the range in which the antibiotic does not decompose, however, preferably, a temperature in the order of less than 60 ° C, preferably 0-40 ° C, more preferably, 5 ° C and room temperature is used.  When this reaction is carried out, a stimulating reaction can be added, such as trimethylamine, triethylamine, pyridine, dicyclohexylcarbodiimide, or others.  Under these conditions, the reaction can be completed in 1-24 hours, usually 3-12 hours.  According to a preferred embodiment of the invention, an antibiotic used in Mbij to interact with a reactive derivative of structural formula III, where R is triphenylmethyl ,.  it is not necessary to be the isolated purified product.  For the reaction can be used.  cultivated product or filtered broth after removing the mycelium from the cultured product and a crude antibiotic preparation partially purified by regeneration and seeding partially purified preparations include, for example, concentrated activated carbon syrup which is treated with the filtered broth; a concentrated styrene-divinylbenzene resin eluate, such as Diaion HP-20, with which the shaped broth was treated, a desalted concentrate with activated carbon of the eluate, prepared by a stepwise concentration of sodium chloride in phosphate buffer from an ion-exchange resin, for example, QAE-Sephadex, which adsorbed concentrate. NR-20, concentrated methylene chloride extract in the presence of benzalkonium chloride, concentrated extract with chloroform in the presence of CRO 7P-compounds; concentrated butanol extract with low pH (3.5) and low temperature.  The antibiotic-trityl ester thus obtained is isolated from the reaction mixture and purified by a number of known methods.  Upon completion of the reaction, the reaction mixture is first poured into an aqueous medium to remove aqueous impurities, as by-products, and the like.  d.  Preferably a neutral buffer is used as the aqueous medium so that the pH value approaches neutral.  The antibiotic trityl ester is extracted with a low-polar organic solvent that is generally not miscible with water, such as ethyl acetate, benzene, chloroform, etc.  P.  during the extraction step, in order to improve it, the salt can be added: sodium chloride, ammonium sulfate or the like. P. , By drying the organic ext. Anhydrous sodium sulphate, trityl ester is isolated from the solvent by known methods, for example, gel filtration using products like Bio-Beads S-X3, Sephadex LH-20 or adsorption chromatography using carriers such as silica gel, alumina, Fuller's earth, Crityl The ester is further purified by crystallization from a solvent or solvent mixture, such as benzene, toluene, xylene, ethyl acetate, diethyl ether, methylene chloride, chloroform, hexane, petroleum ether (boiling in the range of 30-60 ° C).  Among the esters of general structural formula (III), which can be described by the above methods, the trityl ester antibiotic of the structural formula (P-a) S-cHi-cHj-iH-co-cHj Hj-CHj is one of the most useful Hbik. it is more stable compared to the antibiotic of structural form (I), it is easier to isolate, and it retains a strong antibiotic effect and p) lactamase inhibitory action, while the trityl ester of known penicillins does not have any tangible antibiotic effect .  Further, this trityl ester of structural formula (ia) is very important as one of the active esters and useful intermediate for the synthesis of other pharmaceutical useful products since the trityl group can be easily removed.  Bios antibiotics antibiotics, tritylchases esycrypsycids antibiotics esters have a broad spectrum of antibiotic effects, in particular, against a number of different gram-positive bacteria from the genera DipCococcus, Streptococcus, Staphy6ococcus, Sarcza, sycros, Sarcza, Sycchaus, Staphy6ococcus, Sarcza, Syccha, Stapcocccus, Streptococcus, Staphy6ococcus, Syctocrype , Comamona to t.  P.  The trityl ester of the antibiotic also has a good, action against gram-negative bacteria of the genera Escherichia, KEebsieta, Proteus.  An essential distinguishing feature of the antibiotic trityl ester is its strong action against temple-negative bacteria, resistant to antibiotics, having a 1-lactam ring structure and belonging to the genus Citrobacter, Proteus, Enterobacter, Cebsiet6a, Serratia.  The antibiotic trityl ester also has the property of increasing. The antibiotic effects of other antibiotics, in particular (i-lactam antibiotics like penicillins and cephalosporins, against Becteria forming fr-lactamase, such as Citrobacte f eundii, Proteus vuEgaris, Enterobacter aerogenes. , Serratia marcescen trityl complex. Antibiotic ester when administered to mice infected with pathogenic Gram-positive bacteria (MI) has a significant therapeutic effect.  The trityl ester, when administered intraperitoneally by mice in an amount of 500 mg / kg, does not cause the death of experimental animals.  Antibiotic and its derivatives, in particular trityl complex eff-ir, have an effect on gram-negative and gram-positive microorganisms in a test tube and in a living organism, and as a result, they are useful in combating and preventing bacterial infections in experimental animals.  The antibiotic or its trityl ester and the corresponding formulations can be administered orally, topically or parenterally (intravenously, intramuscularly, intraperitoneally, etc.  d. ) and can be used in a whole range of various pharmaceutical preparations depending. from the mode of administration.  For example, an antibiotic or its tritrosh ester is possible.  , combined with a pharmacologically compatible carrier / sbiM carrier, a diluent in solid form (for example, tablets, powders, granules, coated sugars, rum tablets, pellets, powders for pulverization, suppositories, etc.  d. ), in a semi-solid form (for example, in the form of ointments, creams, semi-solid capsules, and so on), in a liquid form (for example, in the form of liquid solutions, emulsions,, suspensions, lotions, syrups, solution. injection solutions, liquid solutions for spraying, and the like.  d. ).  A single dose containing an antibiotic or its trityl ester may contain 0.1-99% by weight of the active ingredient in any form; - liquid, semi-liquid and solid, i Tablets and capsules for oral.  administrations can be obtained in unit doses and contain binding agents, e.g., syrup, gum arabic, gelatin, sorbitol, trigacanth, polyvinylpyrrolidone, or fillers, e.g. lactose, sucrose, starch, calcium phosphate, sorbitol, glycine or lubricating agents, e.g. magnesium stearate, polyethylene glycol, talc, silica or others; disintegrating agents, for example potato starch; wetting agents — sodium lauryl sulfate or others.  - Tablets can be coated according to well-known methods.   Liquid preparations can be formulated for oral administration in the form of oily or aqueous suspensions, solutions, emulsions, syrups, etc., or they can be prepared in the form of spicy ingredients that are diluted with one or the other appropriate carriers before administration.  The above liquid preparations for oral administration. may contain the following pharmaceutically compatible.  Ingredients; suspending ingredients (e.g., methyl cellulose, sorbitol syrup, sugar syrup, hydroxyethyl cellulose, gelatin, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible fats and oils), non-aqueous vehicles (e.g., ethyl alcohol, propylene glycol, oily esters, fractionated coconut oil, almond oil); emulsifying agents (for example, lecithin based on gum arabic, sorbitan monooleate); preservatives (for example, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid).  Suppositories-may contain common basic products for these media as cocoa butter and various glycerides. Injectable formulations can be obtained as single doses in ampoules or multi-dose containers with a preservative agent.  They may be in the form of suspensions, solutions, and emulsions in oily or aqueous vehicles, and, if appropriate, may contain suspending or dispersing agents and stabilizers.  The antibiotic can be obtained in powder form, which can be combined with non-pyrogenic sterile water before use.  Compositions containing an antibiotic or its trityl ester can be obtained in various forms suitable for adsorption through the mucous membrane of the nose, throat, and bronchi.  For example, for this purpose, powders or liquid solutions for spraying, inhalation preparations, lozenges, gargles, etc. are preferable. For the treatment of the eyes and ears, the antibiotic is made as separate capsules for administration dropwise in liquid or semi-liquid the form.  For topical use, preparations based on hydrophobic or hydrophilic bases, such as powders, lotions, creams, ointments, are suitable.  The compositions may contain ingredients, for example, preservative, anti-oxidative, lubricating, viscosity, flavoring, suspending, binding, stabilizing products.  If an antibiotic and / or trityl ester is used to treat infections in an orgus of pigs, cows, sheep, chickens, preptsfatz can be obtained as regs located within the mammary gland based on substances of slow or fast action, for example, in the form of concentrates for the addition of in feed, the pharmaceutical compositions according to the invention may contain an antibiotic or its trityl ester as a single active ingredient or in combination with other therapeutically effective ingredients.  Since the antibiotic and its trityl ester have a synergistic effect on various bacteria forming fb-lactamase in combination with ib-lactam compounds, it is advisable to combine them with.  pharmaceutical formulations with L-lactam compounds.  Benzylpenicillin, phenoxypenicillin, carbenicillin, ampicillin and amoxicillin are used as the suitable β-lactam compound; as well as cefalosporin derivatives such as cefaloridine, cephalotin, cefazolin, cefalexin, cefoxitin, cefacetryl, cefamondol, cefapirin, cefradine and cefaloglycine.  An antibiotic and known fl-lactam compounds are used in ratios from 20: 1 to 1: 150, preferably from 10: 1 to 1: 100.  When treating bacterial infections in mammals, the amount of its trityl ester antibiotic can vary depending on the treatment object, body weight, type, severity and symptoms of the infection, the method and number of injections.  For ordinary oral OR parenteral administration, a daily dose of 0.05-500 mg / kg, preferably 0.5-200 mg / kg, more preferably in partial doses, is advisable.  Doses outside the given range can also be applied depending on the individual conditions of the body being treated.  An antibiotic or its trityl ester can be used not only in pharmaceuticals. compositions, and can also be added in the form of a concentrate or directly to the feed.  Additionally, it can be used as an active ingredient for preserving or disinfecting feeds.  In all examples, qualitative and quantitative experiments on antimicrobial activity are based on the following method.  Bioassay for antimicrobial activity.  The Comainonas terrigena B-996 culture obtained overnight on oblique nutrient agar was weighed in nutrient broth to achieve an optical density of cells of the order of 0.040 at 610 nm.  The ground slurry is added in an amount of 1% to molten agar medium containing 0.8% Kyokuto Nutrient Broth Powder and 1% Bacto-Agar.  7 ml of the seeded molten agar medium are distributed in a 9 cm diameter Petri dish and allowed to solidify.  Shepherds of the Comamonas type are obtained.  StaphyEococcus aureus FDA 209 P is cultured overnight in shaking broth, diluted 50 times with nutrient broth to obtain a seed suspension, 1% by volume of this suspension is thoroughly mixed with molten agar medium containing 1% Kyokuto Nutrie Broth Powder and 1% Bacto-Agar.  7 ml of each of the inoculated molten agar medium is infused with gelation in.  Petri dish with a diameter of 9 cm.  E. This plate is designated a bioplate of the StaphyEococcus type.  Similarly receive bioplastin type ACcaligenes.  The nutrient culture obtained overnight on oblique agar of the body AEca igenes faecaCis B-326 is weighed in nutrient broth to obtain a suspension of seeds, the cell concentration of which is adjusted to an optical density of 0.020 at 610 nm.  Agar media consisting of 0.5% Kyokuto Nutrient Broth Powder and 1% Bacto-Agar are melted at an acceptable temperature and inoculated with 1.0% of the inoculum of the seed suspension.  7 ml of the seeded molten agar medium is spread in a petri dish 9 cm in diameter and allowed to form a gel.  This plate is referred to as AEcaEigenes type bioplate.  The pulp circle (8 mm) is usually impregnated with a solution of the test sample, kept on a clean sheet of filter paper for a time sufficient to remove the excess solution, and then transferred onto a bioassay plate.  After incubation at 35 ° C for 20 h, the diameter of the formed zone of inhibition is measured and compared with standard solutions of ts: phaloridine.  The antimicrobial effect of the antibiotic and related compounds is expressed as units of cephaloridin equivalent per ml.  In particular, a solution of antibiotic and related compounds according to the invention, showing the same inhibition zone diameter as 100 mg / defaloridine, is expressed as 100 cef.  loridine units / ml.  Similarly, if a solid sample of an antibiotic and related compounds of the invention gives, at a concentration of 1 mg / ml, the same diameter as 1 mg / ml cephalori din, the specific activity of the solid sample will be expressed as 1 Cephaloridicin unit per mg.  The standard on the curve of biological experience varies slightly depending on the type of the test organism.  For experimental microorganisms, the following names are abbreviated as abbreviations: Comamonas cephaloridinic unit JCCV, Staphygococcus — ce. Faloridin unit (SCV); AEcaEigenes - cefaloridicin unit (ACV).  Bioautography.  A large bioassay plate is made, except that 100 ml of the sown molten agar medium is poured into a rectangular cup 32 x 24 cm and not into a Petri dish (9 cm).  The bioassay paper chromatogram is placed for 15 minutes on this large test plate.  After removing the paper, the test plate is incubated for 20 hours at 35 ° C in order to detect inhibition zones.  This technique allows not only to read the value of () Rf (qualitative test), it also determines an. thymicrobial action based on zone-inhibition sizes.  In the case of using a thin layer chromatography plate, thin paper is placed between it ii on the surface of the test plate.  A similar method is used for qualitative and semi-quantitative biological tests.  Example 1  A 500 ml Erlenmeyer flask containing seeding culture medium is sterilized at 120 s for 15 min.  To the spore-rich oblique culture of Streptoravces A 271, 10 ml of 0.02% iween-SO is added, after which the mixture is slightly stirred to obtain a spore suspension.  A 500 ml Erlenmeyer flask is inoculated with 1 iJm spore suspension, then shaken for 48 hours of cultivation at 28 ° C on a rotary vibrator.  Then, 2 ml of the seed culture is inoculated into each of 6 Erlenmeyer flasks, each containing 100 M. P described medium, after which 48-96 h cultured, shaking with on a rotary vibrator.  Wednesday crop culture, wt. % Mactic extract 0.3 Bacto-tryptone 0.5 Glucose 0.1 Soluble starch 2.4 Yeast extract 0-, 5 Calcium carbonate 0.4 Fat-free soybean flour 0.5 pH before sterilization 7.5; after sterilization 7.1; after 2 days of fermentation - 7.4.  Production environment, weight. % Medium AG-1 Glucose 1/5.  Corn starch 2.5 Liquid from.  corn locks 2.0 Dry yeast 1.0 Methionine 0.1 0.00013 soe2- pH before sterilization 7.2; after sterilization - 6.1; after 4 days of fermentation | 7,8.  Continued table.  Eluates Nos. 8-14 are combined, concentrated to 100 ml at a temperature below 30 ° C on a rotary evaporator and then freeze dried to obtain 2.6 g of a yellowish brown powder.  This powder is dissolved in distilled water with a concentration of 500 ecu / ml.  Phosphate buffer solutions of predetermined pH are obtained by adjusting 15 M of dipotassium phosphate to the desired value using 5 and.  sodium hydroxide or 5 n.  phosphoric acid.  In 1 ml of the antibiotic solution is added 1. ml of phosphate buffer and the pH value was adjusted to the initial.  Antibiotic solutions with a change in pH are kept for 30 minutes under an aqueous bath, cooled under running water and neutralized to pH 7.0 with a small amount of 5N.  sodium hydroxide or 5 n.  phosphoric acid.  The control tube, containing a PS-5 antibiotic solution with a pH of 7.0, is placed in an ice bath for half an hour.  Residual antimicrobial activity was measured by the described method using Comamonas terrigena B-996 as the experimental microorganism.  Residual activity,% These results show that the yellow brown powder obtained in the present example is noticeably stable at a PX range of 6.9-9.0 for 30 minutes at 60 ° C. The stability of aytibiotic increases at low temperature even at acidic pH.  For example, after treatment of the antibiotic at pH 3.0 for 5 minutes at -17 ° C, at least 30% of the initial antibiotic effect can still be established.  Example 3 , Extraction at low temperature and -butanol.  A large test unit: A thin tube containing 20 ml of distilled water, 12 ml of n-butanol and 7 g of sodium chloride is cooled to -17 s without freezing.  The yellowish-brown antibiotic powder obtained in Example 2 is diluted to a concentration of 200 mg / ml in distilled water and pre-frozen, 1 ml of cold antibiotic solution is added to this test tube, quickly adjusted to pH 2.75, 3.0 or 3.25 with sulfuric acid and keeping the temperature of the mixture below - Yu-C.  After thorough mixing, the n-butanol layer is regenerated and mixed thoroughly with 5 ml of 0.5 M (pH 6.8), transferring the active ingredient to the aqueous layer.  ; Example 4  Preparation of a powder, similar to Example 2, out of 100 l of broth filtrate after freeze-drying, 27.7 g of a yellowish-brown antibiotic powder are obtained.  This powder (activity 13.2 CCU / mg) was dissolved in 30 ml of 25 M phosphate buffer (pH 6.8) and the basic anion exchange resin was transferred to a QAE-Sephadex A-25 column, completely transferred: to the quaternary compound, obtained introducing diethyl 2-hydroxypropylammonium groups into a dextran gel (3.3 X 25.0 cm).  After washing with a small amount of the same phosphate buffer, the active fraction is eluted with a buffer containing sodium chloride at a concentration of 0.5 M during the elution.  The active fraction (300 ml) is cooled to 0 ° C and treated with b g of active carbon.  The active carbon is collected, washed with water, and eluted with 50% by volume of acetone.  Upon removal of acetone by evaporation at a temperature below 30 ° C, 727 mg of brownish-yellow poroapse of the antibiotic sodium salt are regenerated by lyophilization.  The specific activity of the drug - 264 ssi / mg.  Example 5  Obtaining powder deae cellulose.  727 g of the brownish-yellow antibiotic powder obtained in Example 4 was dissolved in 1 ml of 25 M phosphate buffer (pH 6.8) and loaded onto a column (1.5 X 27.0 cm from BiO-Gee P-2 (cross-linked synthetic copolymer composition in the form of a methylene-based catalytic layer. -bis-acrylamide copolymer balanced by the same phosphate buffer.  The same phosphate buffer showed 15 ml of the active fraction.  The obtained active fraction is placed on a column (2.5 x 28.0 cm) of diethylene-minoethyl (DEAE) DE 32 cellulose, equilibrated with the same phosphate buffer.  The elution is carried out with a linear gradient of sodium chloride in the same phosphate buffer (0-0.5 M).  The resulting active fraction (240 ml) is adsorbed onto 4.5 g of active carbon, at.  Active carbon with: peeled off by filtration, washed with water and eluted with 50% by volume of acetone.  The acetone eluate is evaporated at a temperature below until complete removal of the ac tone, then lyophilized to obtain 120 mg of a brownish-white powder of antibiotic sodium salt PS-5.  The specific activity of this drug is 60. 0 ssi / mg  Example 6  Highly purified antibiotic drug.  Analogously to example 1, Streptomyces A 271 is cultured in a 500 ml Erlenmeyer flask containing 100 ml of SE-4 medium inoculated then into a 30-liter fermenter containing 15 liters of SE-4 medium.  Cultivation was continued for 24 hours at 28 ° C and 200 rpm with forced aeration in the amount of 7.5 l / min, obtaining a seed culture.  2 fermentation tanks with a capacity of 200 liters, made of stainless steel, are filled every 200 liters of ML-19 medium containing 2.5% defatted soy flour, then treated in an autoclave, inoculated with 1 liter of the Streptomyces microorganism and 271 obtained as described and fermented for 12 hours with aeration and stirring.  The contents of both vessels of the compound OT are mixed with 5% by weight Torsr Pertite, Torso N 34 (Torso Pereite Kogyo K. TO. ) and filtered through a filter press, yielding 160 liters of boule filtrate.  The antibiotic titer of this filtrate is 235 ssi / ml.  This broth filtrate is passed through a DIAION PA-306 ion exchange resin column (strongly based on bottom-exchange resin, with cross-linked polystyrene matrix and three methyl ammonium chloride units) 10 X 52 cm, 4.5 ml of wet volume without residue.  Then, the active product passed through this column was adsorbed on a column of DIAION HP-20 (14 x X 97 cm; 15 l) and eluted with 75% methanol.  The collected active eluate (2 L, 9,854 ssi / ml) was diluted three times with 4 L of the feed and loaded onto a 2 liter column (5.5 x 84 cm) of DIAION PA-306S.  After washing with 500 ml of water, the antibiotic material is eluted with 8 liters of a linear sodium chloride gradient of 0–3% and collected in 200 ml fractions.  Active fractions Nos. 17-31 are collected by taking 2.8 liters of active eluate (4.120 si / ml).  This eluate is again loaded onto a 1-liter column from DIAION HP-20 (4.5 X 63 cm) and eluted with a 3-liter linear gradient of acetone from 0 to 25%.  The volume of each fraction is 30 ml.  The active eluate (390 ml 27.220 axis / ml) was isolated, the antimicrobially active fractions Nos. 54-66 were combined.  After removal of acetone by distillation under reduced pressure, the eluate DIAIOl HP-20 is passed through a 200 ml column from QAE-Sephadex A-25 (2.5 X 41 cm).  The antimicrobial active substance is collected in fractions in 10 ml by elution with a 2-liter linear sodium chloride gradient (0-1.5%).  Fractions Nos. 68-81 are combined as active eluate (140 ml; 42, 120 Cs / ml).  The pH of this QAE-Sephadex A-25 eluate is carefully adjusted to 8.3 using 1% sodium hydroxide and loaded onto a column of 200 ml of DIAION HP-20AG (2.5 x 41 cm).  Elution with a linear gradient is carried out using 1 l of aqueous acetone from 0 to 10%. The volume of each fraction is 10 ml.  Fractions Nos. 48-53 were combined to give 60 ml of active eluate (45,000 CCU / ml).  Dry by freezing to obtain 249 mg of a yellow powder (8,000 ssi / mg).  : For the purpose of further purification, 150 mg of this yellow powder is dissolved in a small amount of 0.01 M sodium sulfate buffer (pH 8.0) by passing through a column (130 ml; 1.5 x X 73 cm) from Sephadex G-10 (dextran gel in the form of a catalytic layer, obtained by crosslinking the dextran fractions with epirchlorohydran).  The antibiotic is developed with 0.01 M sodium phosphate buffer (pH 8.0-), fractionator eluate, 36 ml of active eluate is obtained from fractions nos. 38-55 (65,000 Csi / ml).  The Sephadex G-10 eluate is chromatographed on a QAE-Sephadex A-25 column (100 ml; 2.0 X 32 cm), followed by elution using a linear assay. o gradient through 1 liter solution of sodium chloride from 0 to 1.5%.  The volume of fractions - 10 ml.  As active eluate, 5 active fractions Nos. 48-52 (50 ml; 22,000 Cs / ml) are collected.  The pH of this active eluate is carefully adjusted to 8.3 with 1% sodium hydroxide and loading onto a column of 50 ml of DIAION HP-20.  (1.2 x x 44 cm).  The antibiotic sodium salt is separated from the fractions with 5 ml by elution with a linear gradient with 400 ml of aqueous acetone from 0 to 10%.  Active fractions Nos. 39-41 are collected and lyophilized to obtain 51 ml of white powder (21,000 CCU / mg).  Such a preparation based on the sodium salt of the antibiotic is a white-colored substance, soluble in water and insoluble in acetone.  When measured in a Kofler VU micrometer for measuring the melting point, when the temperature rises at a rate of l C / MHH, this preparation does not show a melting melting point.  It turns yellow around 148C and gradually softens to 1bO ° C.  At about 203C, the shade of the drug slowly changes from yellow to brown.  At 220 ° C - the resin is brown.  UV absorption spectrum.  60 µg of the antibiotic sodium salt was dissolved in 3.0 ml of water, measured in a spectrophotometer.  The measured results / obtained by calculation are as follows. 246 nm (82.0 301 nm (11 ° 267.5).  A hydroxylamine solution (pH 7.5) is added to an aqueous solution of this preparation (21,000 CCi / mg) in distilled water to obtain a reaction mixture containing 21.3 µg / ml of the antibiotic sodium salt and 10 mM hydroxylamine.  After half an hour at 22 ° C, the reaction mixture loses about 94% of the initial optical density at 301 nm.  IR absorption spectrum.  Characteristic absorption maxima are observed at specified wavelengths; about 1750 cm (-CO- in the p-lacto moss ring); about 1650 cm- (-CO-B agishdnoy communication); about 1640 - 1540 cm- (-COCf).  PMR spectrum.  The following characteristic signals are obtained as a measurement: a triple with a center of about 1.06. parts per million (J is about 7.5 Hz) (CH —CHg-); multiplet in the region of 1.7 2.00 parts per million); sharp synglet about 2.65 parts / million (); multiplets in the region of 2.88-3.58 p / m (-CH-, -CH-); multiplet in the region of 3.9-A, 20 p / m (-CH-) Color reaction.  Ehrlich's reagent is a positive reaction. Iodoplatinate Same Ninhydrin Negative reaction.  Specific rotation.  -Tts- + 1.23 (1.59, 0.01 M, pH 8) tri-phosphate buffer.  Thin layer chromatography (TLC).  The antibiotic sodium salt is subjected to thin layer chromatography under an inoculated condition.  Rf values are determined by bioautography.  Cellulose TLC-plate.  Solvent system R (f) n-Butanol (ethanol) water -7 / 7/6 / by volume.  0.94 Isopropanol / water -7/3 v / v 0.96 Silica gel TLC plate pre-coated with Silica gel plumna 60 F. - ,.  ).  Solvent system Ethanol / water - 7/3 by volume n-Propanol / water - 7/3 by volume proper chromatography.  The antibiotic sodium salt gives the blowing R (f) values on the filter paper Touo Fitter Paper N 50 according to the conditions indicated Solvent n-Propanol / water -7/3 by volume system, 0.68 n-Prothanol / isopropanol / water - 7/7/6 by volume volume 0.70 Acetonitrile / water - 8/2 v / v 0.36 Acetonitrile / tris buffer / EDTA0.34 (Solvent mixture consisting of 120 ml of acetonitrile, 30 ml of M / 10 tris (hydroxymethyl) aminomethane hydrochloric acid based buffer pH 7.5 and 1 ml M / 10 ethylenediaminetetraacetate pH 7.5) Ethanol-water - 7/3 by volume.  0.63 High-voltage paper electricity.  The antibiotic sodium salt is analyzed by a high-voltage paper electrosis under the indicated conditions.  Filter paper for analysis - Tooo Fitter Paper N 50.  The results are as follows: conducting electrophoresis for 30 minutes with cooling below f With a potential of 42 V / cm in buffer (pH), containing 3.3 g of barbital and 5 g of medinal in 3000 ml of water, the antitum migrates to the anode (28 mm).   - Magnetic resonance spectrum. Applying dioxane C as an internal grade magnetic rezosny spectrum of the sodium salt of antibic in heavy water is measured with a spectrometer.  The following actor signals are obtained: (1) 184.04 ppm (2) 174.96

169.29 141.11 130.40

(dioxane) 67.39 60.19 55.63 40.41 40.00 31.49 22.62 22.46 11.36

Antimicrobial spectrum of action. The values of the minimum inhibitory concentration (MIC) of the sodium salt of the antibiotic are determined on various pathogenic microorganisms, including resistant strains, using the diluted broth method,

The antibiotic sodium salt is dissolved in Brain Heart infusion Broth. Eiken broth (pH 7.0) at a concentration of about 5-50 µg / ml, from which a number of diluted products are made in the same liquid medium. Microorganisms are cultivated for 18 hours in a boulevard. Note:

Brain Heart jnfiision Broth EiKen at 28 C with the final size of the inoculum 1x10 cells / ml. The culture is allowed to stand for 20 hours, after which the growth of the microorganism of each sample of the diluted antibiotic is recorded. The MIC shows the minimum concentration of the antibiotic (its sodium salt), where it is not possible to visually determine the multiplication of the main microorganism. As a control, 2 known b-lactam antibiotics, cephaloridin and cefoxitin, are dissolved in brain heart infusion broth broth (pH 7.0) at concentrations in the order of 1-100 µg / ml and then diluted to obtain a variety of diluted cultures. in the above liquid medium. These samples are treated in the manner described for the MIC.

In tab. 4 shows the results obtained (in addition to the MIC values of the antibiotic, for comparison, data for cephaloridine and cefoxitin are included).

Table 4 + organism-lactamase; 2+ resistant to anamycin / gentamicin and tobramycin; 3 resistant to gentamicin and tobramycin, 4+ - 10% of horse blood added to the medium.

Effort of antimicrobial action of known (L-lactam compounds against microorganisms resistant to / i-lactam.

10 ml of molten nutrient agar containing 50 μg / ml penicillin G or cephaloridine, 0.8% Kyoto Nutrient Broth Powder broth powder and 1.0% Difco Bacto-Agar agar (pH 7.0) are seeded with organisms resistant to {L- lactam and generators (5-lactamase, then pouring into the bowl; pet kiree (9 cm) in order to obtain a bioassay agar plate.

Steady cr-lactam

Pulp circles (8 mm) are placed on this agar plate, each containing 25 µl of antibiotic PS-5 solutions at a given concentration, and then incubated for 18 hours before recording the zones of inhibition. A control test plate is made similarly, but without penicillin G or cephaloridin. , In the control plates experience: In the same conditions, penicillin G, am: picillin, oxacillin and cefazolin.

In tab. 5 and 6 shows the results obtained.

Table 5

27

73851728

Strong inhibition of the penicillinase of the microorganism Proteus vuCgaris due to the action of the antibiotic is observed. In the presence of 1/42 equivalent of antibiotic in penicillin G as the substrate quality, more than 50% of the activity

Table 6

Proteus P-5-p1-lactamase is inhibited.

Example 7. Method of preparation (Antibiotic trityl ester preparation 65. 30.0 g of the yellowish-brown lyophilized powder of the antibiotic sodium salt (specific activity 27 CCi / mg) is dissolved in 100 ml of dimethylformamide with the addition of 3.0 g of tritylamine, After cooling with ice, 9.0 g of trityl chloride was added to the reaction mixture with the solution temperature below. After stirring for 12 hours at 5 ° C, all the active material of the antibiotic was converted into tritylation product. The reaction solution was poured into 1000 ml of 0.5 M phosphate puff (extract pH 6.8), the extractor is then thrice with 1000 ml of ethyl acetate in each case. The ethyl acetate extracts are combined, dehydrated with anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The residue from the residue is dissolved in 20 m of benzene and passed through a Bio-Beads S-ChS column (porous layer of styrene-divinylbenzene copolymer for gel-permeation; 4.5 x 60.0 cm), which was pre-equilibrated with benzene. The column was developed with benzene. dry under reduced pressure. The resulting residue is dissolved in 1 ml of benzene and loaded onto a silica gel column (25 g; 1.5 x 24.0 cm). After washing with a benzol ethyl acetate mixture (10: 1) to remove residual reagents, the active material is eluted with a benz ethyl acetate mixture (1: 2). The active eluate is evaporated to dryness under reduced pressure and again dissolved in 0.5 ml of benzene. The benzene solution is placed on a neutral alumina column and is developed with a beneol ethyl acetate mixture at various ratios (10: 1, 6: 1, 4: 1, 3: 1, 2: 1 and 1: 1). The active eluates are combined and evaporated to dryness under reduced pressure. The residue from the residue is dissolved in 0.3 ml of benzene, loaded onto a silica gel column (10 g, 0.9 x 22.0 s). After removing the impurities with a benzo-ethyl acetate mixture (10: 1), the active material is eluted with a benzol ethyl acetate mixture (1: 2) and concentrate under reduced pressure to obtain a solid powder. The resulting powder is dissolved in 0.5 ml of benzene and purified on a Bio-Beads S-Chr column (1.2 x 96 cm) using benzene as a developing agent. The active eluate is evaporated to dryness under vacuum.The resulting dry powder is dissolved in 0.5 ml of acetone and subjected to gel. filtration using a Sephadex LH-20 column, a dextran gel in the form of a layer, obtained by crosslinking the dextran chains by hydroxypropylation (1.2 x 96.0 cm), which is pre-filled with acetone before swelling. The active fraction is evaporated to dryness, to obtain 23 mg a white powder. By recrystallizing this powder from a benzene-hexane mixture, 12 mg of a colorless crystalline powder-product (10,800 Cs / ml) is obtained. The colorless crystalline product is an antibiotic trityl ester. The solubility of the product of the tritylation of the antibiotic is determined by dissolving with shaking 5 mg in each case of the product of the tritylation of the antibiotic PS-5 in 0.1 ml of each of the tested solvents at 20 s. The results are as follows: mostly insoluble in water, AND-hexane, and petroleum ether (m.p. SO-BC). Highly soluble in benzene, ethyl acetate, chloroform, methanol, ethanol, acetone, dimethylformamne (DMF) and dimethyl sulfoxide (DMSO). Sustainability. The antibiotic trityl ester is dissolved in 1 unit by volume of methanol, then diluted with 9 units by volume of distilled water, as soon as it is possible to obtain a solution of 125 CCi / ml of 10 M solution of dipotassium phosphate is treated with 5N. phosphoric acid or 5 n. sodium hydroxide until a pH of 3-9 is reached. 1 ml of each of the mentioned solutions of the antibiotic trityl ester is added to 1 CH1 of each of the phosphate buffers; if appropriate, the pH of the mixture is adjusted to the desired value using phosphoric acid or sodium hydroxide. The solution mixture was held in a water bath for 30 minutes, cooled with running water and neutralized to pH 7.0 with a small amount of phosphoric acid or sodium hydroxide. The control solution (pH 7.0) was kept in an ice bath for the duration of the experiment. Residual antibiotic material is tested by an explosive bio-test method using Comaraonas terrigena B-996 as an experimental microorganism. The product of tritylation of the antibiotic melts at 83.5-85, gradually becoming brown at a temperature above 140 ° C. The UV absorption spectrum of the antibiotic tritylination product according to isobetics is recorded with a spectrophotometer at a concentration of 128 µg / 3 ml of methanol. C ° 315.5 (E 156) Characteristic absorption peaks occur at the following wavelength ranges: 3430, 3100-3000 (C — H phenyl group); 2990.2950.1770 (С-О р-lactam ring); 1695 (-CO-amide bond 1665 (-CO-0, ester bond); 1445, 1340, 1275, FROM sph., PMR spectrum. The most characteristic signals are as follows: a triplet of about 1.10 h (3 - about 7.0 Hz; singlet at 1.86 ppm; multiplet in the region of 7.107 56 ppm (protons in the benzene ring of the trityl group). Elementary analysis. 3.5 Ml of the antibiotic tritylation product dried at room temperature H. under reduced pressure of 1 X 10 mm Hg and subjected to elemental analysis. Found: C, 61.89%; H, 5.78%; N, 4.48%; S, 4.62%. Color reaction. Reagent Erlich Positively Triphenyltetrazoliumnegatively chlori e Negative iron chloride ferric chloride negative iodine B-996 Silica gel TLC: plate, preliminarily coated, silica gel 60 R 54 Solvent system: benzene / acetone (2: 1) R: 0.29. Cell TLC: Eastman Chromagran Sheet 13254 Ce & EuE-ose plate with a fluorescent reagent., (NO 6065). SolventR (f) Upper phase, and-butanol / / ethanol water (4: 1: 5) 0.56 m-Butanol, 1.0

StaphyEOCOCCUS aureus FDA 209 p StaphyCococcus aureus Smith Dip ococcus pneumoniae Type III Streptococcus pyogenes NY-5 Sacrina Eutea S-19 Escherichia co2i K 12

Claims (1)

0.04 0.04 0.01 0.01 0.05 2.5 Isopropanol / water (8: 7) 0.91 Isopropanol / water (1: 4) 1.0 Molecular structure of the antibiotic and reduced physical and chemical properties confirm structural pho. the antibiotic trityl ester mule. Biological properties of tritylovogs .. ester. Antimicrobial spectrum of action. MIC values of the compound are determined on various pathogens, including certain resistant strains, using the dilution method. no broth. In particular, the antibiotic trityl ester is dissolved in a small amount of methanol and diluted as soon as it becomes possible in Brain Heart Dnfusion Broth Eiken broth to a concentration of the antibiotic trityl ester in the order of 40-20 µg / ml. The methanol concentration in the target solution does not exceed 10%. This solution is diluted according to a double geometric row. The microorganisms listed in Table 7 are cultured for 18 hours at 28 ° C in broth and inoculated to obtain a series of diluted product from windows with an inoculum size of 1 X 10 cells / ml. The results are recorded after incubation for 20 hours. The MIC values indicate the lowest unit concentration of the tritylating product of the antibiotic when no growth of the corresponding microorganisms is observed under the conditions described. As control antibiotics, solutions of ampicillin and. Cephaloridine are made at a concentration of 100 µg / ml with a treatment similar to that of the compound according to the invention. In tab. 7 summarizes the MIC values of an antibiotic trityl ester along with those of ampicillin and cephaloridine as controls. Table 7 As can be seen from the table, the antibiotic trityl ester has a wide antimicrobial effect, in particular, a strong antibiotic effect on various strains of microorganisms resistant to β-lactam {forming fb-lactamase). STRENGTHENING OF THE STATE } -lactamase), on a solid base layer of nutrient agar (pH 7.0, 15 ml) containing 50 μg / ml penicillin G or cephaloridin. Test Continuation of the table. Seven antibiotic solutions are placed on a pulp circle (8 mm) in a volume of 25 μl and then on test plates. After incubation at. 35 ° C for 18 hours is measured for inhibition, and compared with the results on the corresponding plates containing neither penicillin G nor cephaloridin. in tab. 8 shows the results. From the data table. 8 it is quite clear that a combination of the product of the antibiotic tritylation at a concentration below the lower limit of biological experience with plates with penicillin G or defaloridine at a concentration below this limit gives a significant zone of inhibition. This fact clearly confirms the enhancement of the antibiotic effect of penicillin G or cefalori (Qin is the product of tritylating of the antibiotic. Confirmation of the antimicrobial synergism of the product of tritylation of the antibiotic and cephaloridin by the method of diluting the broth on Proteus vufigaris, which is resistant to –– lactams, which is resistant to –– lactams, which is resistant to –– lactams, which is resistant to anti-infective antibacterial symme- β-lactam compounds due to the formation of p) -lactamases, therefore a combination of 7.5% MIC cephaloridine with 12.5% MIC of trityl antibiotic ester is used, which inhibits the growth of experimental microorganisms. -organisms. The activity in vivo of the product of the tritylation of an antibiotic is measured on myoshes infected intraperitoneally with the organism StaphyEococcu aureus Smith in an amount of 5 x 10 cells per mouse. The antibiotic trityl ester is administered subcutaneously immediately after infection. The 50% therapeutic dose in male mice is 2.55 mg / kg (27.540 CCU / kg). The inhibitory effect of p-lactamase on 50% of cephaloridine hydrolyzate is inhibited by 0.60 and 0.80 µg / mg of the tritylation product of the antibiotic PS-5 during the course of the experiment (Proteus vuEgaris P-5 lactamase and Citrobacter freundii E-9, respectively. Toxicity. Trityl). the antibiotic ester PS-5 does not cause lethal effects in male mice after intraperitoneal administration in an amount of 500 mg / kg. Example 8. The method of obtaining the tritylation product of antibiotic Brownish yellow powder of sodium salt of the antibiotic PS-5 (715 mg, 235 CCi / mg), obtained as in example 4, solution in 3.5 ml of hexamethylphosphorotriamide.About 70 mg of triethylamine with ice-cooling, 250 mg of tri a b l and 1.1 are added to the mixture, and tilbromide at a temperature below 10 ° C. The mixture is stirred for 7 hours to complete Tritylation reactions. The reaction mixture is poured into 50 ml of ice water and diluted to solid ice melting. The antibiotic trityl ester is extracted three times using 15 ml of benzene (each time) and worked up as in Example 7, to obtain 9.4 mg of a colorless crystalline powder. The physicochemical properties of this preparation are similar to those in Example 7. Example 9. Preparation of an antibiotic tritylation product in the presence of CROWN ether. The crude brownish-white powder (10.1 g) of lyophilization, prepared as in Example 2, is weighed into 20-0 ml of methylene chloride and dissolved with stirring and the addition of 1 ml of 15 - Crown - 5. Keeping the solution temperature below 5 ° C with ice, 8.5 g of trityl chloride are added. The reaction mixture is warmed to room temperature and stirred for 3 hours. After filtration, the filtrate is evaporated to dryness under reduced pressure. The residue is dissolved in. 10 ml of benzene in one portion, filtered and purified as in Example 7, to obtain 7.3 mg of a colorless crystalline powder. Example 10. A method for the preparation of the product tritylated or antibiotic in the presence of a quaternary ammonium salt. The antibiotic is extracted from 48 l of broth filtrate (11.7 CCU / ml) twice using 15 l in each case of dichloromethane containing. , 0.05% cetyl dimethyl benzyl ammonium chloride (dichloromethane / extract activity 24.0 CCU / mp; activity of the broth filtrate used - 5.7 Csi / ml). Dichloromethane extras are dried with 4UU of anhydrous sodium sulfate and evaporated to 500 ml under reduced pressure on a rotary evaporator. The concentrate (450 ssi / ml) is first dehydrated with 10 g of anhydrous sodium sulphate, and then 5 g of molecules, 4 S of the sieve by filtration. 4.5 g of trityl chloride are added to this dry solution at a temperature below 5c while stirring the mixture for 5 hours 5c. After dichloromethane was removed by evaporation at room temperature, the residue was dissolved in 15 ml of benzene and purified as in Example 7, to obtain 8 mg of anhydrous crystalline powder of trityl ester of the antibiotic. Physico-chemical properties of this drug are almost identical with those of Example 7. Example 11. Methyl ester of antibiotic. 90 mg of the sodium salt of the antibiotic, prepared analogously to Example 6 (8,000 CCi / mg), are weighed in 3 ml of dry dimethylformamide, then 50 mg of triethylamine and 0.3 ml of methyl iodide is added. After stirring for 2.5 hours at room temperature, 100 ml of benzene is added, and the mixture is thoroughly stirred for extraction. The benzoic layer was separated, washed with 100 ml of 0.1 M (pH 6.8) and then dried with anhydrous sodium sulfate. The dry benzene solution was concentrated to a small volume under reduced pressure and loaded onto a column (1.2 x 90 cm) from Bio-Beads S-XZ. Methyl ester exhibit benzene. The eluate fractions containing the ester are combined and evaporated to dryness under reduced pressure, semi-colored oil. The oily product is dissolved in a small amount of acetone and chromatographed on a column (1.2 x 90 cm) of Sephadex LH-20 with acetone as a developer. The eluate fractions containing the antibiotic methyl ester are collected and evaporated to dryness to give 11.2 mg of the antibiotic methyl ester. The methyl ester is distinguished by the following physical and chemical properties. . , Thin layer chromatography. Rj 0.45 (silica gel plate 60 P benzene: acetone 1: 1). UV absorption spectrum: maximum in methanol CHjOH L 315.5 nm max IR absorption spectrum: maximum in shoroform 3430, 1766, 1660 cm. Signals in PMR in deuterium chloroform (&) 1.05 (3 N, t, 3 7.5 Hz); 1.7-2.0 (2H, m); 2.00 (3 H, S); 2.83, 65 (7 H, t); 3.83 (3 H, S); 3,844, 06 (1 N, t). Molecular weight (high resolution mass spectrometry). Find nos: 312.1131. CH4H S Calculated; 312, 1143. Based on the above data, the antibiotic metal ester does correspond to the structural formula II indicated above, where R is methyl. Receiving described in Example 2 was repeated using ethyl iodide, isopropyl iodide, izobutilyodida, n-pentilyodida or n-hexyl iodide instead of methyl iodide to give the antibiotic complex esters of ethyl, isopropyl, isobutyl, n-pentyl, "-geksilovy ester ,. The formation of these esters is confirmed by thin layer chromatography, IR absorption spectrometry, PMR spectrometry and mass spectrometry. Formulations containing an antibiotic and / or trityl, an antibiotic ester, can be administered in different unit doses, as solid or liquid, oral forms for ingestion. These formulations contain a single dose, liquid or solid, active maggerial in amounts from -0.1 to 99%, preferably 10-60%. The amount of active ingredient in the formulation can vary depending on the form administered and the total weight of the formulation, and usually ranges from 10-1000 mg, preferably 100-1000 mg. In parenteral formulations, the unit dose is usually a pure or highly purified antibiotic, its pharmaceutically compatible salt and / or product of its tritylation in solution in sterile water, or in the form of a soluble soluble powder, intended for dissolution. The active product can also be administered in combination with compounds sensitive to 3-lactamase. The proposed method allows a new antibiotic to be administered. The formula of the invention The method of obtaining an antibiotic of the formulas tej-CHjS - CHj - CKj-NH- CO - CHj 0 where Rj is hydrogen, metal, ashyl or triphenylmethyl, 397385174 u and u with the fact that the Strepto strain is a neutral salt, at pH 4.0–9.0 and temperature A 271 is grown in an aerobic setting of 20–40 ° C, followed by excretion in a nutrient medium, containing the target product in the form of acid sources of carbon and nitrogen, and mints or in the form of an ester or salt.