DK157302B - ESTERS OF CLAVULANIC ACID USED AS INTERMEDIATES IN THE PRODUCTION OF CLAVULANIC ACID OR PHARMACEUTICAL TOLERABLE SALTS THEREOF - Google Patents

Description

DK 157302 B

The present invention relates to novel esters of clavulanic acid for use as intermediates in the preparation of clavulanic acid or pharmaceutically tolerable salts thereof, which esters are peculiar in that they have the general formula I

. CH-OH

n ^ W

COOR

wherein R represents a hydrocarbon group having a maximum of 16 carbon atoms optionally substituted by halogen, C 1-6 alkoxy or hydroxy, or R represents a group of formula 10 I

- CH - O - CO - A

yLX ,.

represents hydrogen or methyl, methyl, tert-butyl or phenyl, and A 2 represents hydrogen or methoxy, or R represents a hydrocarbon group of 1-9 carbon atoms substituted with optionally converted salts with salts. the general formula NR ^R ^ wherein R ^ represents hydrogen or C ^g alkyl alkyl, R ^ represents hydrogen or C ^gg alkyl or is attached to R ^ such that NR NRR R is a pyrrolidine, piperidine - or morpholine ring.

The stereochemistry at the carbon atoms 5 and 2 of the clavulanic acid is the same as that found in naturally occurring penicillins and cephalosporins, so that clavulanic acid can be illustrated by the structural formula

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2

CH OH

Γ / "Ν '\

CO OH

Thus, a more satisfactory chemical name for clavulanic acid is Z- (2R, 5R) -3- (3-hydroxyethylidene) -7-oxo-4-oxa-1-azabicyclo [3,2,0] heptane-5-2-carboxylic acid .

The effect of clavulanic acid as a β-lactamase inhibitor is illustrated by showing strains of Klebsiella aerogenes A whose growth is not inhibited in the presence of 125 µg / ml ampicillin, amoxycillin, carbenicillin or benzylpenicillin or in the presence of 10 µg / ml. 10 ml of clavulanic acid is inhibited in the presence of less than 12.5 µg / ml of the penicillins listed above when 5 µg / ml of clavulanic acid is present. Similar results have been observed for combinations containing different esters of clavulanic acid. For example, strains of Klebsiella aerogenes A whose growth is not inhibited by 125 µg / ml ampicillin or 10 µg / ml clavulanic acid methyl ester are inhibited by less than 12.5 µg / ml ampicillin in the presence of 5 µg / ml clavulanic acid methyl ester. It has also been found that strains of Staphylococcus aureus Russell, whose growth is not inhibited in the presence of 100 µg / ml ampicillin or 5 µg / ml clavulanic acid, are inhibited by the presence of less than 10 µg / ml ampicillin in the presence of 1 µg / ml clavulanic acid. In female mice, it has been found that blood and tissue levels of clavulanic acid at significantly greater than 5 µg / ml can be easily reached by subcutaneous administration of 100 mg / kg of the sodium salt of clavulanic acid, and that valuable levels of clavulanic acid can be reached after Oral administration of 100 mg / kg of the sodium salt of clavulanic acid.

Clavulanic acid and its salts appear to be somewhat more useful synergistic agents than the ester itself.

Particularly suitable pharmaceutically tolerable salts of clavulanic acid are, for example, sodium, potassium, calcium, magnesium, aluminum, ammonium and substituted ammonium salts such as trimethylammonium, benzathine,

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3 procaine and similar salts, soin usually formed with penicillins or cephalosporins.

Salts of clavulanic acid appear to be more stable than the acid itself.

Particularly suitable salts of clavulanic acid are the sodium and potassium salts,

5 having the following formulas III and IV

. CH90H. CH / H

/ 2 -_ / 2 \ \

COONa COOK

III IV

crystalline forms of such salts may contain hydrate water.

The esters referred to in this specification are theoretically derived from an alcohol of the general formula ROH, where R has the meaning set forth above. Suitable groups R are, for example, alkyl, alkenyl, alkynyl, aryl, arylalkyl or other similar groups which, if desired, may be substituted as indicated above. The groups R 15 do not include more than 16 carbon atoms, in particular not more than 12 carbon atoms and, in particular, not more than 8 carbon atoms.

The group R is preferably, purely theoretical, derived from an alcohol ROH which is pharmaceutically tolerable.

Suitable esters of clavulanic acid are those which are theoretically derived from alcohols such as methanol, ethanol, propanol, butanol, 2,2,2-trichloroethanol, 2,2,2-trifluoroethanol, benzyl alcohol, phenol and 2-dimethylaminoethanol.

Suitable substituents which may be included in the group R are, for example, halogen atoms and alkoxy, hydroxy, alkylamino and dialkylamino groups 25 as defined above. Thus, for example, R may be methyl, ethyl, n-propyl, isopropyl, straight or branched butyl, pentyl, heptyl, octyl,

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4 nonyl, decyl, undecyl, dodecyl, vinyl, allyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexenyl, cyclohexadienyl, methylcyclopentyl, methylcyclohexyl, benzyl, benzhydryl, phenylethyl, phenylethyl, phenylethyl, phenylethyl, phenylethyl 2-chloroethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, 2-methoxyethyl, 2-dimethylaminoethyl, 2-diethylaminoethyl, 2-piperidinoethyl, 2-morpholholinoethyl 3-dimethylaminopropyl, p-chlorobenzyl, p-methoxobenzyl , p-bromobenzyl, m-chlorobenzyl, 6-methoxynaphthyl-2-methyl, p-chlorophenyl or p-methoxyphenyl.

Particularly suitable esters of formula I are those having general formulas VII and VIII

now CHOOH

—R \ / 2/2 0 \ f 5 ° X ^ / A6 "y-Uv ο

VIII

where A ^ and A ^ have the meaning given above.

Particularly good esters of clavulanic acid for use as intermediates are intestines having the general formula IX

CH0OH

nV

XCOORX

represents a hydrocarbon group of 1-9 carbon atoms optionally substituted with halogen, C1-6 alkoxy, hydroxy or optionally salted basic groups of the general formula NR% ^ where hydrogen or C1 represents hydrogen or C 1-6 alkyl or is attached to R 2, such that NR 1 R 2 is a pyrrolidine, piperidine or morpholine ring.

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Suitable groups rA are alkyl and aralkyl, soin is optionally substituted with halogen, methoxy, hydroxy, or NR1 R3 groups which are converted, where R4 represents methyl or ethyl and R4 is methyl or ethyl.

Particularly suitable alkyl groups R · are straight-chain groups having a maximum of 6 carbon atoms optionally substituted with a methoxy, hydroxy or a NR NRR ^ group converted or with a chloro, bromo or iodo atom or with a CCI3 or CF3 group.

Clavulanic acid esters of formula I can be converted to clavulanic acid or 10 pharmaceutically tolerable salts thereof with various forms of hydrolysis.

A further group of particularly suitable esters according to the invention are such valuable intermediates which are readily converted to clavulanic acid or a site thereof by chemical or biological techniques which, from the penicillin or cephalosporin technique, are known to be sufficiently mild to not degrade reactive acid labile γ9. lactam rings.

A suitable group of esters for use as intermediates in the preparation of clavulanic acid or pharmaceutically tolerable salts thereof is one which has the general formula X

20

^ H20H

0S ~ ^ <\ 7

FCO-O-CH-A

i8 where ellerJ represents a hydrogen atom or an optionally chloro, bromo or methoxy substituted phenyl group and represents an optionally chloro, bromo or methoxy substituted phenyl group.

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Particularly conveniently, one is a hydrogen atom or phenyl, tolyl, chlorophenyl or methoxyphenyl, and is phenyl, tolyl, chlorophenyl or methoxyphenyl.

is preferably a hydrogen atom and is preferably a phenyl group.

The ester of formula X can be cleaved by hydrogenolysis to give clavulanic acid or a site thereof.

Such reactions usually take place in the presence of a transition metal catalyst using low or medium hydrogen pressure. The reaction can be carried out at high temperature, room temperature or low temperature. for example at 0-100 ° C. Particularly suitable reaction conditions for such hydrogenations are to use hydrogen pressure slightly above atmospheric pressure and an approximate room temperature (12-20 ° C).

The reaction can be carried out in conventional solvents such as lower alkanols, eg ethanol ·. It has been found that a particularly suitable catalyst is palladium on coal.

If the hydrogenation is carried out in the presence of a base, a site of clavulanic acid is obtained, for example, the sodium or potassium salt is obtained if the reaction is carried out in the presence of sodium or potassium hydrogen carbonate.

The clavulanic acid or salt thereof obtained by such reactions is usually of good purity.

Particularly suitable esters are benzyl clavulanate, p-chlorobenzyl clavulanate, p-methoxybenzylclavulanate, p-bromobenzylclavulanate and m-chlorobenzylclavulanate.

As previously claimed, clavulanic acid and its salts have valuable therapeutic properties. Therefore, a pharmaceutical composition containing clavulanic acid or a site thereof can be prepared together with a pharmaceutically tolerable carrier.

Such preparations are in a form suitable for oral, two-

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7 whip or parental use, and they can be used to treat infections in mammals, including humans.

Suitable formulations are, for example, tablets, capsules, creams, syrups, suspensions, solutions, reconstitutable powders and sterile formulations suitable for injection or infusion. Such compositions may contain conventional pharmaceutically tolerable materials such as diluents, binders, dyes, flavors, preservatives and disintegrants in accordance with conventional pharmaceutical practice in the manner known to those skilled in the art of formulating antibiotics.

Injectable or infusible preparations of clavulanic acid or salts thereof are particularly suitable, as high tissue levels of the compound of clavulanic acid may occur after administration by injection or infusion. A preferred composition contains clavulanic acid or a site thereof in sterile form.

Under certain conditions, the effectiveness of oral preparations of clavulanic acid and its salts can be improved if such preparations contain a buffering agent or an enteric coating agent so that the compounds of the present invention do not prolong contact with highly acidic gastric juice. Such buffered or enteric coated preparations may be prepared in accordance with conventional pharmaceutical practice.

The clavulanic acid or salts thereof may be present in the composition as the sole therapeutic agent or may be present with other therapeutic agents such as a β-lactam antibiotic. Suitable β-lactara antibiotics for use in such synergistic preparations include not only those known to be highly susceptible to β-lactamases but also those known to have a good degree of resistance in themselves to β-lactamases. . Such suitable β-lactam antibiotics for use in these preparations include benzylpenicillin, phenoxymethylpenicillin, carbenicillin, methicillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, 6-aminopicillinic acid, 7 aminodesacetoxycephalo-sporanoic acid, cephaloridine, cephalothin, cefazoline, cephalexin, cephoxitin, cephacetril, cephamandol, cephapirin, cephradine, cephaloglycine

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s and other well-known penicillins and cephalosporins or "pro-drugs" therefor, e.g., hetacillin, metampicillin, acetoxymethyl, pivaloyloxy-methyl or phthalidyl esters of benzylpenicillin, ampicillin, amoxycillin or cephaloglycine or phenyl, tolyl or of carbenicillin or ticarcillin.

Obviously, if the penicillin or cephalosporin present in the preparation is not suitable for oral administration, the preparation should be adapted for parenteral administration.

When the clavulanic acid or a site thereof is present in a pharmaceutical composition together with an / 8-lactam antibiotic, the ratio of the clavulanic acid or a site thereof to the / S-lactam antibiotic may be, for example, 20: 1-1: 12, especially 10: 1-1: 10 and especially 3: 1-1: 3.

The total amount of antibacterial agents in any unit dosage form will usually be between 50 and 1500 mg and will usually be between 100 and 1000 mg.

The said compositions can be used to treat infections in, inter alia, the respiratory tract, urinary tract and bleed tissues bos people.

The compositions may also be used for the treatment of infections in domestic animals, for example in the treatment of mastitis in cattle.

The penicillin or cephalosporin in said synergistic preparations will usually be present in up to or almost the amount usually used when the penicillin or cephalosporin in question is the only therapeutic agent used to treat the infection.

Particularly preferred compositions contain 150-1000 mg of amoxycillin, ampicillin or a pro-drug thereto and 50-500 mg of clavulanic acid or a site thereof, especially 200-500 mg of amoxycillin, ampicillin, or a "prodrug" thereof and 50-250 mg of clavulanic acid or a site thereof.

The substances present in such preparations may, if necessary, be hydrated. The weight of the substances present in such preparations

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9 tibiotics are expressed on the basis of the antibiotic available theoretically in the composition and not on the weight of "pro-drug".

Esters of clavulanic acid may be prepared by converting clavulanic acid or a site thereof to an ester of clavulanic acid in the usual manner.

Preparation of clavulanic acid or a site thereof which can be used in the preparation of the esters according to the invention is described in Danish Patent No. 141,254.

The impure form of clavulanic acid or a site thereof used as starting material for the preparation of esters I may be in the form of a solid or a solution which will usually also contain considerable amounts of organic or inorganic impurities.

The clavulanic acid or a site thereof can be converted to an ester by esterification reactions described below. The preferred method for preparing the desired ester of clavulanic acid is reacting a site of clavulanic acid with an esterifying agent, for example a reactive halide or a sulfonate ester. Such reactions are frequently carried out in an organic solvent of high dielectric constant, for example, dimethylformamide, dimethylformamide / acetone, dimethylsulfoxide, N-methylacetamide or hexamethylphosphoramide.

If desired, the salt of clavulanic acid may be dissolved in solvents in the usual manner or it may be bound to a polymeric carrier. Suitable carriers for use in this process are strong base anion exchange resins, especially those which have a macroreticular nature which permit the use of non-aqueous solvent systems.

It has been found that "Amberlyst A26" is applicable for this purpose. The clavulanic acid salt can be adsorbed on the resin from the culture filtrate and the resin can then be suspended in dimethylformamide containing sodium iodide, or it may be eluted as a column with a solution of sodium iodide in dimethylformamide or in a mixture of dimethylformamide and acetone.

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When the crude ester is formed, it is normally purified chromatographically.

In such processes, the ester is normally dissolved in an organic solvent such as ethyl acetate, methylene chloride, chloroform or cyclohexane. The solid phase used in the chromatographic process is usually an inert material such as silica gel or chromatographically similar materials.

The fractions leaving the column can be examined for the presence of clavulanic acid by making use of its synergistic properties. Active fractions are normally coalesced and the organic solvent is evaporated under reduced pressure.

The ester obtained by this process is usually of acceptable purity, but the material can, if desired, be re-chromatographed.

Suitable methods of ester formation include

a) reacting a site of clavulanic acid with a compound of the general formula Q - R, wherein Q represents an easily interchangeable group, and R

has the meaning given above; b) reaction of clavulanic acid with diazoalkane; and c) reaction of clavulanic acid with an alcohol ROH in the presence of a condensation promoting agent such as carbodiimide.

Suitable salts of clavulanic acid which can be reacted with compounds R-Q are, for example, alkali metal salts such as sodium or potassium salts or other usual salts, for example the solvate salt.

Suitable groups Q include such atoms or groups known to be interchangeable with carboxylate anions and include chlorine, bromine and iodine atoms, sulfonic acid esters such as OSO2CH3 or OSO2C6H4CH3 groups, active ester groups such as OCOH or OCOCF3 groups, and other usual which can be replaced by nucleophilic groups.

The above reaction is usually carried out in an organic solvent having a relatively high dielectric constant such as dimethylformamide, acetone, dioxane or tetrahydrofuran and at non-extreme temperatures, for example between -5 and + 100 ° C, usually between +5 and + 30 ° C, for example at room temperature.

U

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The reaction of clavulanic acid with a diazo compound is a mild process for the preparation of alkyl or aralkyl esters or similar esters. The diazotation reaction can be carried out under usual reaction conditions, for example at non-extreme temperatures and in a usual solvent. Such reactions are usually carried out at a temperature between -5 and + 100 ° C, especially between +5 and + 30 ° C, for example at room temperature. Suitable solvents for this reaction include lower alkanols such as methanol and ethanol and solvents such as tetrahydrofuran or dioxane. Ethanol has been found to be a particularly useful solvent in this reaction.

The reaction between clavulanic acid and an alcohol in the presence of a condensation promoter usually takes place in an inert organic solvent having a relatively high dielectric constant, for example acetonitrile. The reaction is usually carried out at room temperature or lowered temperature, for example at a temperature between -10 and + 22 ° C, especially between -5 and + 18 ° C, for example initially at 0 ° C and then gradually heating to approx. 15 ° C. The condensation promoting agent used is usually an agent which removes water from the reaction mixture. Suitable agents are, for example, carbodiimides, carbodiimidazoles or equivalent reagents. Dicyclohexylcarbodiimide has been found to be a particularly suitable condensation enhancer for use in this process. In order to suppress the internal condensation of the clavulanic acid, this reaction is usually carried out in the presence of a significant excess of the alcohol.

The esters of clavulanic acid and their use as intermediates are elucidated by Examples 1-11 below, preparation of some esters is further elucidated by Examples 12-18, and some by the use of esters of Formula I as intermediate-produced acids and salts antibacterial / synergistic effect is described in Examples 19-21.

30 Methods for demonstrating antibacterial and synergistic effect are described in Danish patent specification 141,254.

Preparation of Clavulanic Acid Sodium Sait.

Example 1.

12

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ÎHOH. yCü0OE

rfy = / 2 - r-rw 2

<T N 0 ^ ~ NV

282 mg of substantially pure benzyl clavulanate in 25 ml of ethanol containing 82 mg of sodium bicarbonate is hydrogenated over 90 mg of 10% palladium / carbon for 25 minutes at room temperature and under atmospheric pressure. The catalyst is filtered off and washed with water and ethanol and the combined filtrates are evaporated under reduced pressure at room temperature. The semi-solid obtained as the residue is triturated with acetone, filtered and washed with ether to give 135 mg of sodium clavulanate.

Example 2.

Hydrolysis of clavulanic acid methyl esters to form clavulanic acid.

Dissolve 2.17 mg of clavulanic acid ester in 0.1 ml of methanol and treat with 0.208 ml of O, G482N sodium hydroxide solution. After 1 hour at room temperature, the reaction mixture contains several products. Thin layer chromatographic analysis shows that one of the predominant components has an Rf value which is identical to the value for the sodium salt of clavulanic acid; color reactions and biological examination are consistent with this substance, which is the sodium salt of clavulanic acid.

Slow conversion of the ester to clavulanic acid is seen when 1 mg / ml of the compound is incubated at 37 ° C in 0.05M phosphate buffer at pH 13

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7. The reaction is precipitated by paper chromatography (Bioautographic System).

Using the butanol / ethanol / water system to monitor the reaction over a period of 2 hours, the methyl ester zone at a Rf value of 0.79 decreases in size, while the zone of clavulanic acid at Rf-5 value increases 0.12.

Example 3

Preparation of clavulanic acid.

100 mg of benzylclavulanate in 5 ml of ethanol is hydrogenated over 30 mg of 10% palladium / coal for 45 minutes at room temperature and atmospheric pressure. The catalyst is filtered off and washed with ethanol and the combined filtrates are evaporated in vacuo to give 58 mg of clavulanic acid in the form of an unstable viscous oil.

NMR Spectrum (C 5 D 5 N): 3.05 (d, 1, J = 18 Hz), 3.60 (dd, 1, - 18

Hz, J 2 = 2.5 Hz), 4.75 (d, 2, J = 7.5 Hz), 5.58 (t, 1, J - 7.5 Hz), 5.66 (S, 1 ), 6.0 δ (d, 1, J - 2.5 Hz).

Example 4

Preparation of sodium clavulanate.

840 mg of benzylclavulanate in 30 ml of ethanol and 5 ml of water are hydrogenated over 267 mg of 10% palladium / carbon and 244 mg of sodium bicarbonate for 20 minutes at room temperature and atmospheric pressure. The catalyst is filtered off and washed with water and ethanol and the combined filtrates are evaporated in vacuo. The product is recrystallized from a 565 micron needle water-acetone mixture. Upon recrystallization of water-acetone, needles are obtained, which after curing over P2®5 * vacuum for 24 hours 25 show the following analysis: C 41.01, 40.86; H, 3.77, 3.64; N, 5.68, 5.51.

IR spectrum (KBr disk): 1785, 1700 and 1620 cm

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14 NMR spectrum (D 2 O>: 3.06, (d, 1, J - 18.5 Hz), 3.57 (dd, 1, J - 18.5 Hz, J 2 15 (d, 2, J = 8 Hz), 5.3 (HOD), 4.9 (m), 5.71 (d, 1, J = 2.5 Hz).

Example 5

5 Preparation of lithium salt of clavulanic acid.

A solution of 0.84 g of benzyl clavulanate in 30 ml of ethanol is mixed with a solution of lithium hydrogen carbonate (prepared as described below) in 5 ml of water. This solution is hydrogenated over 0.27 g of 10% palladium / carbon for 25 minutes at room temperature (~ 20 ° C), after which no starting material can be detected after thin layer chromatography (SiO 2 ethyl acetate: KMnO 4 spray). The catalyst is filtered off and washed with 5 ml of water and 5 ml of ethanol and the combined filtrates are evaporated in a trader-reduced manner. pressure. The crystalline residue is triturated with 25 ml of acetone and the solid is filtered off, washed with 10 ml of acetone and terres over P2Û5 to give 0.41 g of product.

The product is taken up in 3 ml of water and slowly acetone is added until crystallization begins. After cooling for 20 minutes to 2 - 3 ° C, a crystal yield can be obtained which is washed with crystal. a small amount of acetone and terres in vacuo (80 mg). The filtrate 20 is diluted with an equal volume of acetone and cooled again to give an additional 80 mg of yield.

Analysis: first yield = 84.3% pure lithium clavulanate second yield = 85.1% pure lithium clavulanate.

(The lithium hydrogen carbonate solution is prepared by suspending 25 g of lithium carbonate in 480 ml of water at 5-10 ° C and passing a constant stream of carbon dioxide gas through the suspension for 8 hours, after which a clear solution is added. The volume of the solution is filled up to 500.0 ml with water which is then saturated with 5.15% w / v LiHCO 3).

Preparation of potassium salt of clavulanic acid.

Example 6

15

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A solution of 2.89 g of benzyl clavulanate (with good purity) in 100 ml of ethanol and 17 ml of water is hydrogenated at room temperature and atmospheric pressure over 0.92 g of 10% palladized coal (Engelhard 4505) in the presence of 1.0 g of finely ground potassium hydrogen carbonate. After 30 minutes, thin layer chromatography shows only traces of residual benzyl ester.

The catalyst is removed by filtration and washed with 10 ml of water and 10 ml of ethanol. The combined filtrates and washings (neutral, pH 7) are evaporated under reduced pressure at room temperature to give a pale yellow syrup. This is dissolved in 17 ml of water and diluted with 450 ml of acetone. The cloudy mixture is filtered through diatomaceous earth and the filtrate is diluted to 1.0 liter with acetone. The compound crystallizes and after 30 minutes at room temperature it is isolated by filtration, washed with 50 ml of acetone and terresh in vacuum to give 0.75 g of the individual potassium clavulanate as a colorless crystalline solid which is 98.5% pure potassium clavulanate anhydrate (containing about 0.2 - 0.3% water).

Example 7.

Trimethylammoniumclavulanat.

A trimethylammonium hydrogen carbonate solution is prepared by dissolving 5 g of trimethylamine in 50 ml of ice-cold water and passing CO2 through the solution in a rapid stream until the pH of the solution is approx.

8.5. This solution is analyzed by titration with 1.0M hydrochloric acid using sereened methyl orange as indicator and turns out to be 0.4M.

To a solution of 2.89 g of benzyl clavulanate in 100 ml of ethanol is added 0.9 g of 10% palladized carbon and 25 ml of 0.4 M trimethylammonium hydrogen carbonate. The mixture is hydrogenated for 30 minutes at room temperature.

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16 temperature and atmospheric pressure; thin layer chromatography shows that no starting material is left. The catalyst is removed by filtration and washed with 30 ml of ethanol and 10 ml of water. The filtrate is evaporated in vacuo to give a brown oil. The oil is triturated with 50 ml of 5 acetone and then crystallized. The crystals are isolated by filtration, washed with 10 ml of acetone and 10 ml of ether, and triturated in vacuo to give 0.84 g of light brown trimethylammonium clavulanate.

Analysis (by high-pressure liquid chromatography) shows 68.7% as free acid; 89.1% as trimethylammonium clavulanate.

IR Spectrum (KBr): υ = 3320 (wide, with sharp cut), 1775, 1690 and 1590 - 1620 cm -1 (wide).

No deoxy visible in the NMR spectrum.

Example 8.

Preparation of Aluminum Clavulanate. A solution of 2.89 g of benzyl clavulanate and 0.68 g of aluminum isopropoxide in 150 ml of tort, restored distilled tetrahydrofuran is hydrogenated over 0.86 g of 10% palladized charcoal for 40 minutes (thin layer chromatography shows no unreacted benzyl chloride after this time). The catalyst is filtered off and washed with 20 ml of tetrahydrofuran and the filtrate is evaporated to dryness in vacuo. The solid residue is triturated with 100 ml of acetone, and the resulting solid is filtered off and triturated in desiccator over phosphorus pentoxide. In this way, the aluminum salt is obtained in the form of 0.3 g white solid.

Analysis by hay pressure liquid chromatography shows 48% clavulanate; The aluminum content of EDTA titration is 5.76%; Karl Fischer determination shows 5.4% H2O.

IR Spectrum (KBr): umax = 1790, 1695 and 1615 cm

Example 9

17

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Magnesum clavulanate.

A solution of 4.34 g of benzyl clavulanate and 1.61 g of magnesium acetate tetrahydrate in 100 ml of ethanol is hydrogenated over 1.45 g of 10% palladium charcoal until no further hydrogen is absorbed (about 20 minutes). At this time, thin layer chromatography shows no unreacted benzyl clavulanate. The catalyst is removed by filtration and washed with 30 ml of ethanol and 10 ml of water. The filtrate is evaporated in vacuo to give 5.2 g of pale yellow gum containing some solvent. This is triturated with 50 ml of acetone and the resulting solid is filtered off, washed with 5 ml of acetone and aerated to give 2.4 g of colorless solid. High Pressure Liquid Chromatograph i-sub-sample shows 75% pure magnesium salt.

Example 10.

Preparation of Ammonium Clavulanate.

4.34 g of benzylclavulanate in 50 ml of ethanol are treated with 1.115 g of ammonium acetate (1 equivalent) in 50 ml of ethanol and hydrogenated over 1.4 g of 10% palladium / carbon for 10 minutes. The catalyst is filtered off and the solvent removed in vacuo to give a semi-solid. The residue is dissolved in 5 ml of water and treated with 75 ml of acetone.

The remaining liquid is decanted from the initially formed oil and set aside to give 0.348 g of needles. High-pressure liquid chromatography examination shows that the ammonium clavulanate content is 97.4%.

Karl Fischer analysis shows 0.44% water.

IR Spectrum (KBr): rmax = 1785, 1700 and 1595 cm-1.

Preparation of crystalline potassium clavulanate.

Example 11.

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A solution of 2.89 g of benzyl clavulanate in 100 ml of ethanol containing 1.0 g of potassium hydrogen carbonate is hydrogenated over 0.93 g of 10% palladium / coal for 25 minutes at room temperature and atmospheric pressure. At this time, thin layer chromatography shows no unreduced benzyl clavulanate. The catalyst is removed by filtration and washed with 10 ml of ethanol and then with 150 ml of water. The filtrate is evaporated to a semi-solid under reduced pressure on the rotary evaporator. To this residue is added 800 ml of acetone. After stirring, crystals are obtained. The crystalline product is filtered off, washed with ether (20 ml) and dried in vacuo to give 1.75 g of crystalline potassium clavulanate.

Analysis (by high-pressure liquid chromatography): 89.1 w / w in the form of potassium clavulanate.

NMR and IR Spectra: Essentially the same as previously prepared pure crystalline potassium clavulanate). (The NMR spectrum shows the presence of <3% deoxy, <0.5% acetone).

Example 12.

Preparation of an ester of clavulanic acid (methyl ester).

20

^, CH OH. CH20H

19.8 mg of the sodium salt of clavulanic acid are dissolved in 0.5 ml of tert dimethylformarnide and treated with 0.25 ml of methyl iodide. After standing at room temperature for 1 1/2 hours under anhydrous conditions, remove

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19 of the solvents in vacuo. The residue is purified by P.L.C. on silica gel (Kieselgel 60F254, marketed by E.Merck, Darmstadt,

West Germany) and elution with ethyl acetate to give clavulanic acid methyl ester in the form of a colorless oil (Rf value 0.38; root 5 color with triphenyltetrazolium chloride spray) and the oil having the following properties:

Analysis:

Calculated for C CHH NO NOOg: C, 50.70; H, 5.20; N, 6.57 Found: C, 50.49; H, 5.43; N, 6.29.

UV spectrum (methanol: Amax: no absorption> 215 nm; IR spectrum: umax (film): 3300 - 3600 (wide), 1800, 1750, 1695 cm -1 - * -.

Approximate first-order NMR spectrum (CDCl3): 2.49 (broad S, 1, replaced by D 2 O), 3.05 (d, 1, J = 17.5 Hz), 3.54 (dd, 1, J = 17.5 Hz, J 2 = 2.5 Hz), 3.84 (S, 3) 4.24 (d, 2, J = 7 Hz), 4.93 (dt, 1, J = 7 Hz, J2 = 1.5 Hz), 5.07 (d, 1, J »1.5 Hz), 5.72 (d, 1, J = 2.5 Hz).

Molecular Weight (Mass Spectrum): 213.0635.

Calcd for C9H1JNO5: 213.0637.

Thin layer chromatography on the methyl ester shows a single zone in each of the following solvent systems: butanol / ethanol / water in volume / volume ratio 4: 1: 5, peak phase Rf value 0.75; isoprop-anol / water in volume / volume ratio 7: 3, Rf value 0.95; ethyl acetate / ethyl alcohol in volume / volume ratio 8: 2, Rf value 0.87.

These zones are determined by bioautography using

Klebsiella aerogenes with added benzylpenicillin (synergism system).

Preparation of an ester of clavulanic acid (benzyl ester).

20

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Example 13

CH-OH

z ch2oh I-Κ ° Υ ^ Β

J — n — L * ___I

<f COONa / TOOCHO-Ph 5 Urent 3 - (5-hydroxyethylidine) - 7-oxo-4-oxa-1-azabicyclo [3.2.0] hep tan-2-carboxylic acid sodium salt (assumed to be about 55 mg of pure substance) in 0.64 ml of tort dimethylformamide treated with 0.18 ml of benzyl bromide. The solution is kept at room temperature (about 17 - 18 ° C) for 3 hours under anhydrous conditions. The reaction mixture is fractionated on silica gel 10 and eluted with ethyl acetate to give 63 mg of a substantially pure form of the benzyl ester of 3 - (β-hydroxyethylxdine) -7-oxo-4-oxa-1-azabicyclo [3, 2,0] heptane-2-carboxylic acid in the form of a colorless oil.

IR spectrum (film): 1800, 1745 and 1695 cm cm "; NMR spectrum (CDCl3): 2.25 (s, 1, interchangeable with D₂O), 3.05 (d, 1, J = 17 Hz ), 3.51 (dd, 1, J - 17 Hz, J 2 = 2.5 Hz), 4.24 (d, 2, J = 7.5 Hz), 4.92 (dt, 1, J = 7 , 5 Hz, J 2 - 1.5 Hz), 5.15 (d, 1, J = 1.5 Hz), 5.24 (s, 2), 5.71 (d, 1, J = 2.5 Hz), 7.45 S (s, 5).

Example 14.

Preparation of the benzyl ester of clavulanic acid from crude extracts of the culture filtrate of Streptomyces clavuligerus.

1. Cultivation of Streptomyces clavuligerus.

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21

A "Yeatex" / glucose strain agar culture is used to seed a "yeatex" / glucose agar culture in a Roux bottle by preparing a mycelium / spore suspension in sterile water. The slant culture of the Roux flask is incubated at 26 ° C for 10 days. To this slant culture is added 100 ml of sterile water and a mycelial suspension is prepared. This is used to graft 50 liters of steam sterilized germ medium into the following composition in tap water: "Oxoid" malt extract 1% w / v "Oxoid" bacteriological peptone 1% w / v 10 Glycerol 1% w / v 10% "Pluronic L81" antifoam in soybean oil 0 , 05% w / v.

Pluronic is marketed by Jacobs and Van den Berg UK Ltd., 231 The Vale, London, W3, and contains a polypropylene-polyethylene block-polymer, and soybean oil is marketed by British Oil and Cake Mills Ltd., Stoneferry Road, Hull , United Kingdom).

The medium is in a 90 liter stainless steel fermentation vessel with baffle plates and stirred by a 12.5 cm blade disk rotor at 240 rpm. Sterile air is introduced at a rate of 50 to 20 liters / minute and the tank is incubated at 26 ° C.

After 72 hours, the germination is used to seed 150 liters of the same medium using a 5% v / v addition by sterile transfer. This pxoduction step medium is in a 30 liter stainless steel fermentation vessel, fully provided with 25 baffles, and agitated by a 21.6 cm blade disk rotor at 210 rpm. Sterile air is blown in at a rate of 150 liters / minute. The fermentation is maintained at 26 ° C. Whenever necessary, anti-foaming agent is added in 10 ml portions (10% "Pluronic L81" in soybean oil). At regular intervals, samples are taken to determine β-lactamase inhibition. The fermenter is hosted between day 4 and day 5 at the optimal level of β-lactamase inhibitory activity, and the results are presented in Table II.

Table II.

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β-Lactamase inhibitory effect in culture filtrate samples taken from a 300-liter fermentation vessel with Streptomyces clavuligerus.

Fermentation time% iribration by β-lactamase inhibition (days) attempted at a final dilution of 1/2500 1.0 12 1.5 20 10 2.0 31 2.5 36 3.0 50 3.5 54 4.0 51 15 4.5 56 5.0 55.

2) 20 liters of culture filtrate freely set as described above are evaporated 20 in vacuo to 5 liters using an "ascending film" evaporator. The concentrate is freeze-dried using an "Edwards E.F.6" shell freezer manufactured by Edwards High Vacuum Ltd. 300 g of the solid thus prepared contains 3 g of sodium clavulanic acid as determined by the enzyme inhibition test. The solid is suspended in 900 ml of tort dimethylformamide and 150 ml of benzyl bromide is added. The mixture is stirred for 2 hours at room temperature and then diluted with 1 liter of ethyl acetate. The reaction mixture is filtered and the filtrate is evaporated to the minimum volume. The oily residue is extracted with an additional 1 liter of ethyl acetate, and the extract is filtered. The filtrate is concentrated again and the resulting oily residue is poured onto a 7.62 cm x 35.56 cm silica gel column ("Biogel Biosil A" 100 mesh) in cyclohexane. The column is eluted with cyclohexane to remove benzyl bromide and the solvent is then changed to ethyl acetate and 35 fractions are collected in 20 ml. The fractions were examined for the presence of the benzyl ester of clavulanic acid by staining them on the glass bars

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23 silica gel thin layer chromatography plates (Merck precoated silica gel 60 F 254) and spraying of 2,3,5-triphenyltetrazolium chloride (TTC) spray reagent. Fractions showing intense red spots with this reagent were further investigated by thin layer chromatography on silica gel plates using 8: 2 chloroform ethyl acetate as solvent and by spraying the developed plates with TTC spray. The benzyl ester of clavulanic acid runs with an Rf of 0.31 at 22 ° C. Fractions containing this ester are combined and concentrated to 15 ml, and this solution is further chromatographed on a 3.81 cm x 40.64 cm silica gel column (Merck silica gel H, type 60) with chloroform / ethyl acetate 8: 2 as solvent. Fractions of 15 ml are collected and examined for benzyl ester in the manner described above. The fractions containing the ester are concentrated to 8 ml and finally purified by column chromatography on a 2.54 cm x 40.64 cm silica gel column (Merck silica gel H, type 60) with 8: 2 ethyl acetate / cyclohexane as solvent. Selected fractions were combined and evaporated in vacuo to give 160 mg of the pure benzyl ester in the form of an oil.

Example 15

Preparation of Clavulanic Acid Benzyl Ester ..

150 liters of culture filtrate with pH 7.0 contains 16.2 g of clavulanic acid (sodium salt) according to enzyme inhibition experiments. This filtrate is stirred with 5 kg of "Amberlyst A.26" anion exchange resin in Cl 'form (Rohm & Hass Company, Philadelphia, U.S.A.) for 1 hour at room temperature.

The resin is then filtered and the filtrate re-examined to show that 6.4 g of clavulanic acid is removed. The resin is washed with 20 liters of deionized water followed by 20 liters of acetone and 10 liters of dimethylformamide. After re-filtration, the resin is suspended in 2.3 liters of dimethylformamide / 0.2M sodium iodide. To this is added 200 ml of benzyl bromide and the suspension is thoroughly stirred. After standing at room temperature for 16 hours, 2 liters of ethyl acetate are added, after which the resin is filtered and additional ethyl acetate wash liquids from the resin are combined with the filtrate. The extract is evaporated to a small volume and chromatographed on a 7.62 cm x 45.72 cm silica gel.

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24 silica gel column (Merck silica gel H type 60) with 8: 2 ethyl acetate / cyclohexane as solvent. Fractions containing benzyl ylcavulanate are selected by spot samples on silica gel thin layer chromatography plates and sprayed with TTC reagent as described in Example 24. The selected fractions are evaporated to 20 ml and then chromatographed on a 3.81 cm x 45.72 cm silica gel column. (Merck silica gel H type 60) with chloroform / ethyl acetate in the ratio of 8: 2 as solvent. Selected fractions are then poured and evaporated to give 440 mg of a goodbye's oil, which according to NMR spectroscopy is 90% benzyl clavulanate.

Example 16.

Preparation of methyl clavulanate.

130 mg of clavulanic acid in 10 ml of ethanol are treated with an excess amount of diazomethane in ether. After 2 minutes at room temperature, the reaction by thin layer chromatography is shown to be complete. The solution is evaporated in vacuo and the residue is purified by chromatography on silica gel and eluted with ethyl acetate. The fractions containing methyl clavulanate are combined and evaporated to give 104 mg of a clear oil.

Example 17.

Preparation of methyl clavulanate.

Cool 200 mg of clavulanic acid in 5 ml of acetonitrile and stir at 0 ° C.

0.5 ml of methanol and then 206 mg of dicyclohexylcarbodiimide are added and the reaction mixture is stirred at room temperature overnight.

The suspension is filtered and the filtrate is evaporated in vacuo to give crude methyl clavulanate. The crude product is purified by chromatography on silica gel eluting with ethyl acetate to give 140 mg of a clear oil.

Example 18.

Preparation of phenylclavulanate.

25

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100 mg of clavulanic acid in 5 ml of acetonitrile are cooled and stirred at 0 ° C.

To the solution is added 0.94 g of phenol and 100 mg of dicyclohexylcarbodiimide and the reaction mixture is stirred at room temperature overnight. The suspension is filtered and the filtrate is evaporated. The residue is fractionated on silica gel and eluted with ethyl acetate-hexane in the 1: 1 ratio to give 70 mg of phenylclavulanate.

IR spectrum (film): 1800, 1770 and 1690 cm -1 NMR spectrum (CDCl3): 2.18 (broad s, 1), 3.06 (dd, 1, J = 17 Hz, J 2 = 0.9 Hz), 3.54 (dd, 1, J 1 = 17 Hz, J 2 = 2.6 Hz), 4.29 (d, 2, J = 7.5 Hz), 5.1 (dt, 1, = 7 , 5 Hz, J 2 = 1.5 Hz), 5.29 (d, 1, J - 1.5 Hz), 5.76 (dd, 1, = 2.6 Hz, J 2 = 0.9 Hz), 7.35 δ (m, 5).

Molecular weight (mass spectrometry) = 275.0777, which corresponds to Cj.4H₂.3NO5 (calculated 275.0794).

Example 19.

Antibacterial spectrum of clavulanic acid sodium salt.

The antibacterial action of clavulanic acid sodium salt against a variety of bacteria is determined using the microtiter method. Series 20 dilutions of the clavulanic acid sodium salt in "Oxoid" sensitivity test liquid in a microtiter plastic tray are seeded with an overnight culture culture of each organism such that the final inoculum dilution is <0.5 x The cultures are incubated overnight and the points of bacterial growth are determined the next 25 morning by observing the turbidity of the culture. The results, expressed as approximate MIC values (minimum inhibition concentration / µg / ml) are given in Table I below, which shows , that the compound has broad spectrum antibacterial effect.

Table I.

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Antibacterial spectrum of clavulanic acid sodium salt.

Bacterial strains Minimum inhibitory concentration / µg / ml 5

Staphylococcus aureus (Oxford H) 7.5

Staphylococcus aureus (Russell) 7.5

Bacillus subtilis 62

Streptococcus faecalis> 500 10 Streptococcus pyogenes CN 10 125

Escherichia coli NCTG 10418 31

Klebsiella aerogenes 31 - 62

Klebsiella oxytocum 62

Enterobacter aerogenes T 624 31 15 Enterobacter cloacae 62

Acinetobacter anitratus 125

Providentia stuartii 125

Serratia marcescens 125

Proteus mirabilis C977 62 20 Proteus vulgaris W090 31

Salmonella typhimurium 31

Shigella sonnei 62

Pseudomonas aeruginosa A 500 Example 20.

Examples of β-lactamase inhibition with clavulanic acid sodium salt.

Clavulanic acid progressively and irreversibly inhibits the β-lactamase of Escherichia coli. The method described previously in "Description 1" shows that the other β-lactamases shown in Table II are also inhibited by 30 clavulanic acid.

Inhibition of β-lactamases by clavulanic acid.

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Table II.

Source of / î-lactamase Approximately Iejg value in relation to Escherichia coli JT 4 - 1

Staphylococcus aureus (Russell) 1.0

Escherichia coli JT4 1.0 10 Escherichia coli Bll 2.0

Klebsiella aerogenes A 0.6

Pseudomonas aeruginosa 1822 (R factor) 5.0

Pseudomonas dalgleish 0.1 With penicillin G as a substrate, the Igg value of the clavulanic acid sodium salt against the β-lactamase of Staphylococcus aureus (Russell) is approx. 0.06 pg / ml.

Example 21.

Antibacterial synergism between ampicillin and clavulanic acid sodium salt.

The minimum inhibitory concentration (MIC value) of ampicillin, clavulanic acid sodium salt and ampicillin 1 presence of 1 / zg / ml clavulanic acid sodium salt is determined for a variety of β-lactamase-producing bacteria. The organisms are inoculated into "Oxoid" sensitivity test culture broth placed in small trays in a plastic tray and containing various concentration gradients of ampicillin, clavulanic acid sodium salt or ampicillin plus 1 µg / ml clavulanic acid sodium salt (microtiter method). The final dilution of the inoculum in culture medium

ISLAND

The overnight liquid is 0.5 x 10. The tray is incubated at

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28 37 ° C overnight, and the next morning the bacterial growth endpoints are determined. The MIG values in µg / ml are given in Table V, which shows that, at the low concentration of 1 µg / ml, the synergist enhances the antibacterial effect of ampicillin 5 against certain gram-positive and gram-negative bacteria. The mechanism of this synergism is believed to consist in inhibition of ampicillin-degrading β-lactamase enzymes, but the existence of other mechanisms cannot be ruled out.

Similar results are obtained as those shown in Table III when 10 ampicillin is replaced by amoxycillin or with the phthalidyl ester of ampicillin.

Table III.

Antibacterial synergism between ampicillin and clavulanic acid sodium salt Bacterial strain Minimum inhibitory concentration / tg / ml

Clavulanic Acid Ampicillin Ampicillin in Sodium Salt Presence of 1 / zg / ml 20 Clavulanic Acid Sodium Salt

Escherichia coli NCTC 10481 31 1.8 <0.4 25 Escherichia coli B 11 62> 500 125

Klebsiella aerogenes A 31 125 <0.4

Klebsiella sp 62 31 125 <0.4

Enterobacter cloacae 62 250 62

Serratia marcescens 125> 500 62 30 Staphylococcus aureus (Russell) 15 500 <0.4

Staphylococcus aureus 62 250 7.5 (a methicillin resistant strain) 35 Antibacterial synergism between cephaloridine and clavulanic acid sodium salt.

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29

The minimum inhibitory concentration of cephaloridine, clavulanic acid sodium salt and cephaloridine in the presence of 5 μg / ml clavulanic acid sodium salt is determined in the manner described above. The results are listed in Table IV and show that synergism can be achieved between 5 clavulanic acid sodium salt and cephaloridine, especially for the / 9-lactamase-producing strain of Staphylococcus aureus (Russell).

Table IV.

Antibacterial synergism between cephaloridine and clavulanic acid sodium salt.

10 Bacterial strain Minimum inhibition concentration µg / ml

Clavulanic acid Cephaloridine Cephaloridine in sodium salt in the presence of 5

Mg / ml clavulanic acid sodium salt

Proteus mirabilis 889> 500 * 62 7.5 20 Staphylococcus aureus (Russell) 15 3.1 <0.03+

Staphylococcus aureus (a methicillin resistance 62 15 3.7 stent strain) 25 * "Tailing point" + Same value obtained when the synergist is added at 1 µg / ml instead of 5 µg / ml.

Antibacterial synergism between clavulanic acid sodium salt and 30 different penicillins.

The results shown in Table V are obtained by the method described above.

Claims (5)

15 PATENT REQUIREMENTS 1. Esters of clavulanic acid for use as intermediates in the preparation of clavulanic acid or pharmaceutically tolerable salts thereof, characterized in that they have the general formula I 20 CH , G1-6 alkoxy or hydroxy, or R represents a group of formula DK 157302 B A4! 5 - CH - O - CO - A {"XX where hydrogen or methyl, represents methyl, tert-butyl or phenyl, and A® represents hydrogen or methoxy, or 5 R represents a hydrocarbon group of 1-9 carbon atoms which is substituted with optionally saline converted basic groups of the general formula NR ^ rX wherein R ^ represents hydrogen or C1-6 alkyl, R ^ represents hydrogen or C1-6 alkyl or is attached to R ^, such that NR ^ R 1 is a pyrrolidine, piperidine or morpholine ring. Esters according to claim 1, characterized in that they have the general formula IX CH-OH l / ix 0 ncoor wherein R 1 - represents a hydrocarbon group of 1-9 carbon atoms optionally substituted with halogen, C alkoxy, hydroxy, or optionally converted to salts, basic groups of the general formula NR 1 -RX wherein R 1 represents hydrogen or C 1-6 alkyl and R 2 represents hydrogen or C 1-6 alkyl or is attached to R 2, is a pyrrolidine, piperidine or morpholine ring. 1 Esters according to claim 1, characterized in that they have the general formula X where 157 represents a hydrogen atom or optionally substituted with chlorine, bromine or methoxy. phenyl group, and optionally represents a phenyl group optionally chloro, bromo or methoxy. Ester according to claim 3, characterized in that it is benzyl clavulanate. Ester according to claim 3, characterized in that it is p-chlorobenzylclavulanate, p-10 methoxybenzylclavulanate, p-bromobenzylclavulanate or m-chlorobenzylclavulanate.