DK145543B - PROCEDURE FOR THE PREPARATION OF HYDRAULIC EASTERS OF CLAVULANIC ACID - Google Patents

Description

1 U5543

The present invention relates to a particular process for the preparation of novel hydrolyzable esters of clavulanic acid of the general formula

CH 'OH

j, __ N ^ y 0 ^

COOR

wherein R represents such a group that the ester is hydrolyzable, which process is characterized in that clavulanic acid of formula I

-r> * a Pe20e λ'Ύ \

COOH

or a salt thereof is esterified in a manner known per se for introduction of the group R. In the process of the invention, novel compounds of a whole new class of compounds are provided and these novel compounds exhibit useful biological effect due to their ability to hydrolyzed to clavulanic acid.

The process of the present invention is of a kind that cannot be termed an "analogous process" in that the compounds of the invention are prepared by esterifying a novel compound belonging to a new compound class. Although the structure of the desired end products had been given, the compounds could not have been prepared by one of ordinary skill in the art by any known method.

2 145543

Regarding the preparation of clavulanic acid in the cultivation of Strep-tomyces clavuligerus and the properties of this compound, reference is made to Danish Patent Publication No. 141,254 (Danish Patent Application No. 1672/75).

The stereochemistry at 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 structural formula I

CH-OH

\

COOH

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

The effect of clavulanic acid is illustrated by the fact that certain strains of Klebsiella aerogenes A, whose growth is not inhibited in the presence of 125 µg / ml ampicillin, amoxycillin, carbicillin or benzylpenicillin or in the presence of 10 µg / ml clavulanic acid, are inhibited in the presence of less than 12 5 µg / ml of the penicillins listed above, when 5 µg / ml clavulanic acid is also present. Similar results have been observed for combinations containing different esters of clavulanic acid. Eg. strains are inhibited by Klebsiella aerogenes A, whose growth is not inhibited by 125 µg / ml ampicillin or 10 µg / ml clavulanic acid methyl ester, of less than 12.5 µg / ml ampicillin in the presence of S 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 of 5 µg / ml clavulanic acid, are inhibited in the presence of less than 10 µg / ml ampicillin in the presence of lyug / ml clavulanic acid. In female mice, it has been found that blood and tissue levels of clavulanic acid of significantly more than 5 µg / ml can easily be 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. administration of 100 mg / kg of the sodium salt of clavulanic acid.

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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, p-nitrobenzyl alcohol, phenol, acetoxymethanol , pivaloyloxymethanol and 2-dimethylaminoethanol.

Various esters of clavulanic acid are valuable intermediates in certain methods of purifying clavulanic acid. Many cla-vulanoic acid esters are valuable synergistic compounds. The activity of such esters may be due to the hydrolysis of the ester to form the original acid.

In this specification, the term "ester" refers to esters which are theoretically derived from an alcohol or thiol of the general formula ROH or RSH, where R has the meaning given above. Suitable groups R are e.g. alkyl, alkenyl, alkynyl, aryl, arylalkyl or other similar groups which, if desired, may be substituted. In order not to increase the molecular weight to an unreasonable extent, the groups R usually do not comprise more than 16 carbon atoms, especially not more than 12 carbon atoms and in particular not more than 8 carbon atoms.

The group R is preferably, purely theoretically, derived from an alcohol ROH or (less advantageously) a thiol RSH which is pharmaceutically tolerable.

Suitable substituents which may be included in the group R are e.g. halogen atoms, and lower alkoxy, hydroxy, lower acyloxy, lower alkylamino and lower dialkylamino groups. The term "lower" means that the group contains at most 6 carbon atoms, preferably at most 4 carbon atoms. Thus, for example, R be methyl, ethyl, n-propyl, isopropyl, straight or branched butyl, pentyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, vinyl, allyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexenyl, cyclohexadienyl, methylcyclopentyl, methylcyclohexyl, benzyl, benzhydryl, phenylethyl, naphthylmethyl, phenyl, naphthyl, propynyl, tolyl, 2-chloroethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, acetylmethyl, benzoyl 2-methoxyethyl, 2-dimethylaminoethyl, 2-diethylaminoethyl, 2-piperidinoethyl, 2-morpholinoethyl, 3-dimethylaminopropyl, p-chlorobenzyl / p-methoxybenzyl, p-nitrobenzyl, p-bromo-4-benzyl, m-chlorobenzyl , 6-inethoxynaphthy1-2-methyl, p-chlorophenyl or p-methoxyphenyl1, as well as other such groups known from the penicillin or cephalosporin technique for the formation of esters which are known to be readily hydrolyzed in vivo to form the original antibiotic.

Easily hydrolysable esters include, but are not limited to, esters of general formulas V and VI

/ CHOH 0 / H2OH

Γ) = 7

CiWx-CO-A3 C® ^ o-CH-Z

2 II

AZ X C = Y

V VI

Wherein A represents hydrogen, alkyl, aryl or aralkyl, A represents hydrogen or methyl, A represents alkyl, aryl or aralkyl, X represents oxygen or sulfur, Y represents oxygen or sulfur, and Z represents a divalent organic group. Esters of the general formulas V and VI which readily release the clavulanic acid in the blood stream after administration include those wherein A is hydrogen, A is hydrogen or 3 methyl, and A is methyl, ethyl, propyl, butyl, benzyl or phenyl, and those wherein X is oxygen, Y is oxygen, and Z is -CH 2 CH 2 -, -CH.CH-, now X 6 carbon atoms; the term "aryl" means phenyl, naphthyl or phenyl which is substituted by an inert substituent, e.g. fluorine, chlorine, methyl or methoxy, and the term "aralkyl" means an alkyl group substituted by an aryl group.

Particularly suitable esters of formulas V and VI are those having general formulas VII and VIII

5 145543

CHo0H

ΓΤ ^ ν / 0 ”2 ™ ri ^ V ^ CO-O-CH-O-CO-A5 VI1 o

VIII

Where A represents hydrogen or methyl, A represents methyl, tert; butyl or phenyl, and A 6 represents hydrogen or methoxy.

Many esters of clavulanic acid differ from analogous esters of penicillins or cephalosporins in that they show increased tendency to hydrolyze to clavulanic acid under mild conditions. Eg. simple alkyl esters such as the methyl ester are slowly hydrolyzed to clavulanic acid in water buffered to pH 7. Esters which undergo some hydrolysis under mild conditions are included in general formula IX

CH-OH

wherein R · represents a hydrocarbon group of 1-9 carbon atoms optionally substituted with halogen, lower alkoxy, hydroxy or optionally converted salts of the basic formula NR2R4.

2 ...... 3 wherein R represents hydrogen or lower alkyl, R represents hydrogen 2 2 3 or lower alkyl or is attached to R such that NR R is a 5- or 6-membered ring.

For the purposes of the general formula IX, the term "lower" means that the group contains 1-4 carbon atoms.

Suitable groups R 1 are alkyl and aralkyl optionally substituted with halo, methoxy, hydroxy or salt converted NR R groups, where R represents methyl or ethyl and R represents methyl or 2 3 is ethyl or one attached to R such that NR R is a pyrrolidine, piperidine or morpholine group.

Particularly suitable alkyl groups are straight-chain groups having a maximum of 6 carbon atoms optionally substituted with a methoxy, hydroxy-2 3 or a salt converted NR R group or with a chlorine, bromine or iodine atom or with a CC j or CF3 group.

The esters of clavulanic acid which are particularly valuable as synergists are those which are hydrolyzed in mammalian tissues, especially in human blood, to form clavulanic acid or a salt thereof, assuming that clavulanic acid and its salts appear to be more useful synergistic agents. than the esters per se. Many of the esters of general formulas V - IX are valuable for this purpose.

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

Particularly suitable is an ester which can be cleaved by hydrogenolysis.

Usual esters for such a process are e.g. benzyl, substituted benzyl, benzhydryl, substituted benzhydryl and trityl esters. The benzyl ester has been found to be particularly valuable for this purpose.

By and large, the nature of any substituent in the ester group is insignificant as long as it does not exert an influence on the hydrogenolysis reaction.

As previously stated, clavulanic acid esters have valuable therapeutic properties. Therefore, a pharmaceutical composition can be prepared which contains a clavulanic acid ester together with a pharmaceutically tolerable carrier.

Such preparations are in a form suitable for oral, topical or parenteral use and can be used to treat infections in mammals, including humans.

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Under certain conditions, the effectiveness of oral preparations of clavulanic acid esters can be improved if such preparations contain a buffer or 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.

Clavulanic acid esters may be present in the preparation as the sole therapeutic agent or may occur with other therapeutic agents such as a β-lactam antibiotic. Suitable β-lactam 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 good resistance in themselves to β-lactamases. Such suitable β-lactam antibiotics for use in these compositions include benzylpenicillin, phenoxymethylpenicillin, carbenicillin, methicillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, 6-aminopenicillanic acid, 6-aminopenicillanic acid, , cephaloridine, cephalothin, cefazpline, cephalexin, cefoxitin, cephacetril, cephamandol, cephapirin, cephradine, cephaloglycine and other well-known penicillins and cephalosporins or "pro-drugs" therefor, e.g. hetacillin, metampicillin, acetoxymethyl, pivaloyloxymethyl or phthalidyl esters of benzylpenicillin, ampicillin, amoxycillin or cephaloglycine or phenyl, tolyl or indanyl α esters of carbenicillin or ticarcillin.

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

When the clavulanic acid ester is present in a pharmaceutical composition together with a β-lactam antibiotic, the ratio of the clavulanic acid ester to the β-lactam antibiotic may be e.g. 20: 1 - 1:12, especially 10: 1 - 1:10 and especially 3: 1 - 1: 3.

8 145563

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

The present compositions can be used to treat infections in, inter alia, the respiratory, urinary and soft tissues of humans.

The compositions may also be used to treat infections in domestic animals, e.g. for the treatment of mastitis in cattle.

Particularly preferred compositions contain 150-1000 mg of amoxycillin, ampicillin or a "pro-drug" thereof and 50-500 mg of in vivo hydrolysable ester of clavulanic acid, especially 200-500 mg of amoxycillin, ampicillin, or a "pro-drug" thereof and 50 - 250 mg in vivo hydrolysable ester of clavulanic acid.

The clavulanic acid is prepared by growing a strain of Streptomyces clavu ligerus and isolating clavulanic acid or a salt thereof from the culture medium. The cultivation is described in more detail in Danish publication no. 141,254.

In a process for preparing a pure form of clavulanic acid or its salts, an impure form of clavulanic acid or a salt thereof is isolated, converted into an ester of clavulanic acid in the usual manner, after which the ester is purified, and clavulanic acid or a salt thereof is recovered from the ester.

The impure clavulanic acid or a salt thereof used in this process will normally contain at least 1% by weight of antibiotic.

Suitable esters for use in this process include those which can be cleaved by hydrogenolysis, enzymatic methods or by hydrolysis under very mild conditions.

A suitable group of esters for use in this process is one having the general formula X;

Ao _ Pa2 ° a o CO-O-CH-A7 A8

7 A

wherein A represents a hydrogen atom or an optionally substituted one

ISLAND

phenyl group, and A represents an optionally substituted phenyl group.

Particularly conveniently, A is a hydrogen atom or phenyl, tolyl,

ISLAND

chlorophenyl or methoxyphenyl, and A is phenyl, tolyl, chlorophenyl; nyl or methoxyphenyl.

Preferably, A is a hydrogen atom and A is preferably a phenyl-R group.

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

Other groups of esters which may be used in this process are those having the general formulas V and VI as described above. Such esters can be converted to salts of clavulanic acid by mild basic hydrolysis, e.g. at pH 7.5.

The impure form of clavulanic acid or a salt thereof to be purified by this process may be in the form of a solid or solution which will usually also contain significant amounts of organic or inorganic impurities.

The clavulanic acid or a salt thereof can be converted to an ester by known esterification reactions as described below. The preferred method for preparing the desired ester of clavulanic acid is reacting a salt of clavulanic acid with a 1C 145543 esterifying agent, e.g. a reactive halide or a sulfonate ter. Such reactions are frequently carried out in an organic solvent with a high dielectric constant, e.g. 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 "Amerblyst A26" is useful for this purpose. The clavulanic acid salt may be adsorbed on the resin from the culture filtrate and the resin may 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.

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

In such processes, the ester is usually 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 using 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.

The purified ester of clavulanic acid can be used to prepare clavulanic acid or a salt thereof by the methods described above.

A particularly suitable method for preparing clavulanic acid or a salt thereof is hydrogenation of a compound of general formula 11 as described above. 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, e.g. 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, e.g. 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 salt of clavulanic acid, e.g. 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.

f. 4 Particularly preferred embodiments of the process according to the invention are that the compound of formula I or a salt thereof is reacted with a diazoalkane corresponding to the desired ester or with an alcohol or thipline corresponding to the desired ester in the presence of a condensation promoting agent. According to the invention, it is particularly desirable that a salt of the compound of formula I be reacted with a compound R-Q, wherein R is as defined in claim 1, and Q represents an easily interchangeable group.

As a condensation enhancer, carbodiimide may be mentioned.

Suitable salts of clavulanic acid which can be reacted with compounds R r-Q are e.g. alkali metal salts such as sodium or potassium salts or other common salts, e.g. silver salt.

Suitable groups Q include such atoms or groups which are known to be interchangeable with carboxylate anions, and include chlorine, bromine and iodine atoms, sulfonic acid esters such as OSC2 CH2 or OSc2 CgH2 CH2 groups, active ester groups such as OCOH- or OCOCF3 groups and other usual groups which can be replaced by nucleophilic groups.

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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, e.g. between -5 and + 100 ° C, usually between +5 and + 30 ° C, e.g. at room temperature.

The reaction of clavulanic acid with a diazo compound is a mild process for preparing alkyl or aralkyl esters or similar esters. The diazotation reaction can be carried out under usual reaction conditions, e.g. 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, e.g. 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 or thiol in the presence of a condensation promoter usually takes place in an inert organic solvent with a relatively high dielectric constant, e.g. acetonitrile. The reaction is usually carried out at room temperature or lowered temperature, e.g. at a temperature between -10 and + 22 ° C, especially between -5 and + 18 ° C, e.g. initially at 0 ° C and then gradually heating to approx. 15 ° C. The condensation promoting agent used is usually one agent which removes water from the reaction mixture. Suitable agents are e.g. 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 substantial excess of the alcohol or thiol.

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Description 1.

Samples that can be used to detect clavulanic acid. Study Principle.

Solutions containing clavulanic acid are incubated for 15 minutes with a lactamase preparation in 0.05M phosphate buffer at pH 7 and at 37 ° C. During this time, enzyme inhibition or inactivation occurs. Substrate (benzylpenicillin) is then added and incubation is continued for 30 minutes at 37 ° C. The extent of enzymatic degradation in the penicillic acid (penicillpic acid) substrate is determined by the hydroxylamine test for penicillin. The amount of β-lactamase used is such that it gives 75% hydrolysis of the benzyl penicillin in 30 minutes at 37 ° C.

The degree of hydrolysis is a reflection of the amount of enzyme that remains uninhibited. The results are expressed as percent inhibition of enzyme activity by a given dilution of the clavulanic acid solution (e.g., culture filtrate) or the concentration of clavulanic acid (yug / ml) which gives 50% inhibition of the enzyme under the above conditions (I5q) · Β-Lactamase enzyme.

As enzyme, β-lactamase produced by Escherichia coli JT4 is used: This culture is an ampicillin-resistant strain and its resistance is due to the production of an R-factor-determined β-lactamase. Other similar R-factor-determined β-lactamases may be used, if desired.

The culture is maintained on nutrient agar culture and inoculated into 400 ml of sterile tryptone medium in a 2-liter conical flask. This medium has the following composition: Η 145543

Tryptone (oxoid) 32 g / liter yeast extract (oxoid) 20 g / liter

NaCl 5 g / liter.

CaCl26H2O 2.2 g / L.

The final pH is adjusted to 7.4 by means of dilute sodium hydroxide. The flask is shaken at 25 ° C for 20 hours on a rotary shaker at 240 rpm. minute.

The bacterial cells are harvested by centrifugation, washed with 0.05 M phosphate buffer pH 7 (resuspended and centrifuged) and resuspended in water to give a cell concentration 25 times as high as in the culture medium. This cell suspension is then ejected in an MSE ultrasonic disintegrator at 4 ° C. The cell debris is removed by centrifugation and aliquots of the supernatant are stored in deep freeze. For use in the study technique, the supernatant is diluted in 0.005M phosphate buffer until approx. 75% hydrolysis of a 1 mg / ml solution of benzylpenicillin in 30 minutes at 37 ° C.

Experimental Technique.

Appropriate dilutions of inhibitor preparations and β-lactamase solution are mixed and incubated at 37 ° C for 15 minutes (test). A buffer sample with buffer instead of inhibitor preparation is also incubated. The benzyl penicillin solution (substrate) is then added to the test and control mixtures and the incubation is continued for an additional 30 minutes at 37 ° C. The residual benzylpenicillin in each mixture is then determined using the hydroxylamine assay as described by Batchelor et al., Proc. Roy. Soc., B 154, 498 (1961).

6 ml of hydroxylamine reagent is added to all test samples, control samples and blank samples and they are allowed to react for 10 minutes at room temperature before adding 2 ml of ferric ammonium sulfate reagent. Absorption of the final solution is measured in an EEL colorimeter or spectrophotometer at 490 nanometers against the reagent blank. The composition of the reaction mixtures, test mixtures and blank samples prior to the hydroxylamine test is given in the table below:

Components Test Benzylpeni-Control, Reagent, (all dissolved in or forcillin, ml blind, ml diluted with 0.005M phos- blind, ml phate buffer, pH 7)

Escherichia coli β-lactamase solution 1.9 0.0 1.9 1.9

Inhibitor Solution 0.1 0.0 0.0 0.0 .Benzylpenicillin 5 mg / ml 0.5 0.5 0.5 0.0 0, Q05M Phosphate Buffer, pH 7 0.0 2.0 0.1 0 , 6th

Calculation of results. '

The percent inhibition of β-lactamase is calculated as follows

Absorption of benzylpenicillin blind minus absorption of control sample (uninhibited reaction) = X

absorption of test (inhibited reaction) minus absorption of the control sample (non-inhibited reaction) = Y

Y

percent inhibition = - 100

X

To obtain the I50 value, the inhibitor preparation is diluted until 50% inhibition of the β-lactamase inactivation of benzylpenicillin is achieved by the procedure described above.

Description 2.

Paper chromatographic detection of clavulanic acid.

Culture filtrate and a reference solution of clavulanic acid (250 µg / ml partially purified preparation) are added as samples (20 µg / origin) to Whatman No. 1 paper strips, 1 cm. wide. The chromatograms are made by descending chromatography for 16 hours at 5 ° C using n-butanol / isopropanol / water in v / v ratio 7: 7: 6 as solvent. The strips are dried at 40 ° C and loaded onto agar plates containing 6 µg / ml benzylpenicillin and seeded with a β-lactamase-producing strain of Klebsiella aerogenes (synergism system). The plates are incubated overnight at 30 ° C and clavulanic acid appears as a 16 1455A3 zone of inhibited growth. The Rf value for the zone is 0.46. 6 µg / ml benzylpenicillin alone is below the concentration that is. necessary to kill Klebsiella aerogenes, but in the presence of a β-lactamase inhibitor this concentration becomes toxic, that is, there is synergism.

Use of the above synergism system makes it possible to determine clavulanic acid at concentrations below the concentrations where it shows antibacterial action.

The process of the invention is further elucidated by Examples 1-32 below, the preparation of clavulanic acid by growing and isolating the acid or a salt thereof is illustrated in Examples 33 - 41, and the use of the esters is illustrated in Examples 42 - 50. The antibacterial / synergistic effect of the esters produced are described in Examples 51 - 54.

Example 1.

Preparation of the methyl ester of clavulanic acid.

CH 2 OH 2 CH 2 OH

Dissolve 19.8 mg of the sodium salt of clavulanic acid in 0.5 ml of dry dimethylformamide and treat with 0.25 ml of methyl iodide. After standing at room temperature for 1 1/2 hours under anhydrous conditions, the solvents are removed in vacuo. The residue is purified by P.L.C. on silica gel (Kieselgel 60F254, marketed by E. Merck, Darmstadt, West Germany) and eluted with ethyl acetate to give clavulanic acid methyl ester in the form of a colorless oil (R 2 value 0.38; red color with triphenyl tetrazolium chloride spray) and the oil has the following properties: 17 145543

Analysis:

Calculated father C CHH-NO · C 50.70 Η 5.20 N 6.57 Found: C 50.49 H 5.43 N 6.29.

UV spectrum (methanol: λ: no absorption> 215 nm; max IR spectrum: vm (film): 3300 - 3600 (wide), 1800, 1750, 1695 cnT1. Max "Approximate first order NMR spectrum (CDCl : 2.49 (wide S, 1, replaced by D 2 O), 3.05 (d, 1, J = 17.5 Hz), 3.54 (dd, 1, J = 17.5 Hz, J 7 = 2, 5 Hz), 3.84 (S, 3) 4.24 (d, 2, J = 7 Hz), 4.93 (dt, 1, J = 7 Hz, J 2 = 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.

Calculated for C CHH NO NOO:: 213.0637.

Thin tlagschrojna tograf i on the methyl ester shows a single zone in each of the solvent systems below: butanol / etariol / water in volume / volume ratio 4: 1: 5, peak phase R 2 value 0.75; iso-propanol / water in volume / volume ratio 7: 3, R 2 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).

Example 2.

Preparation of the p-nitrobenzyl ester of clavulanic acid.

^ ΓΗ now, CH „OH

rfx = y rT \ = S

Λν - * · J-γ C00Na ^ COOCH - ^ \ ~ ϋΙ02 18 145543

Treatment of the sodium salt of clavulanic acid with p-nitrobenzyl bromide in dry dimethylformamide gives, after PLC, a colorless oil which crystallizes from chloroform ether to give the p-nitrobenzyl ester of clavulanic acid in the form of white feathery needles, m.p. 112 ° C which, after recrystallization, has a melting point of 117.5 - 118 ° C.

Example 3

Preparation of the benzyl ester of clavulanic acid.

CHo0H

I 1 ch2oh ^ - / όγ ** \ Η _ COONa x H '^' 'COOCH.-Ph u o 1

Impure 3- (β-hydroxyethylidene-7-oxo-4-oxa-1-azabicyclo [3.2.0] heptane-2-carboxylic acid sodium salt (assumed to be about 55 mg of pure dust) in 0.64 ml Dry dimethylformamide is treated with 0.18 ml of benzyl bromicl. 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 and eluted with ethyl acetate to give 63 mg of a substantially pure form the benzyl ester of 3- (β-hydroxyethylidene) -7-oxo-4-6xa-1-azabicyclo [3,2,0] heptane-2-carboxylic acid in the form of a colorless oil.

IR spectrum (film): 1800, 1745 and 1695 cm NMR spectrum (CDCl3): 2.25 (s, 1, interchangeable with D 2 O), 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, J2 = 1.5 Hz), 5.15 (d, 1, J - 1.5 Hz), 5.24 (s, 2), 5.71 (d, 1, J = 2.5 Hz), 7.445 (s, 5).

Example 4

19 145543

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

20 liters of culture filtrate prepared as described in Example 34 is evaporated under vacuum to 5 liters using a "ascending film" evaporator. The concentrate is freeze-dried using an "Edwards E.F.6" shell freeze dryer 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 dry 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 20 ml fractions are collected. The fractions are assayed for the presence of the benzyl ester of clavulanic acid by staining them on glass-borne silica gel thin layer chromatography plates (Merck precoated silica gel 60 F 254) and spraying 2,3,5-triphenyltetrazolium chloride (TTC) spray reagent. Fractions showing intense red spots with this reagent are 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 R ^ value 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. 15 ml fractions are collected and assayed 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 cagel H, type 60) with ethyl acetate / cyclohexane ratio 8: 2 as a solvent. Selected fractions are combined and evaporated in vacuo to give 160 mg of the pure benzyl ester in the form of an oil.

Example 5

Preparation of clavulanic acid benzyl ester.

3.3 kg of spray-dried solid containing 69.4 g of clavulanic acid is slurried in 5.5 liters of dimethylformamide and 500 ml of benzyl bromide is added. After stirring at room temperature for 2 hours, 12 liters of ethyl acetate are added and the solids removed by filtration. The filtrate is evaporated in vacuo to give 212 g of an oily residue. The residue is poured onto a column with a bed of 10.16 cm x 33.02 cm silica gel (Hopkins & Williams MFC) in cyclohexane. The column is eluted with 12 liters of cyclohexane to remove excess benzyl bromide. The eluent is then changed to ethyl acetate and fractions of 500 ml are collected. These are examined for benzyl clavulanate content by staining on silica gel thin layer chromatography plates (Merck precoated silica gel 60 F 254) and spraying 2,3,5-triphenyltetrazolium chloride (TTC) spray reagent. Fractions giving intense red spots are further investigated by thin layer chromatography on silica gel with chloroform / ethyl acetate in the ratio of 8: 2 as solvent and spraying the developed plates with TTC spray. Fractions 5-13 contain most of the ester and these are combined and evaporated in vacuo to give 79.3 g of oil. This preparation is then chromatographed on a 10.16 cm x 45.72 cm column of silica gel (Merck silica gel H type 60) with chloroform / ethyl acetate in the ratio of 8: 2 as solvent. Fractions are selected as described above and by evaporation 45.9 g of an oil which is of 62% 1 s purity as determined by NMR spectroscopy are obtained.

This product is finally chromatographed on a 6.99 cm x 45.72 cm column of "Sephadex LH 20" in cyclohexane / chloroform in a 1: 1 ratio. After selection of fractions and evaporation, 27.6 g of a colorless oil is obtained, which is found to be 95% pure benzyl ester of clavulanic acid determined by NMR spectroscopic examination. ("Sephadex LH 20" is a hydroxypropyl derivative of "Sephadex Q 25" marketed by Pharmacia Great Britain, 75 Uxbridge Road, London W5, United Kingdom),

Example 6

Preparation of clavulanic acid benzyl ester.

150 liters of culture filtrate with pH 7.0 contains 16.2 g of clavular 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 see that 6.4 g of clavulanic acid has been 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 stirred well. 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 column (Merck silica gel H type 60) with 8: 2 ethyl acetate / cyclohexane as solvent. Fractions containing benzylclavulanate are selected by staining on silica gel thin layer chromatography plates and spraying with TTC reagent as described in Example 4. 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 combined and evaporated to give 440 mg of a colorless oil, which according to NMR spectroscopy is 90% benzyl clavulanate.

Example 7

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

22 U5543

An aliquot of an aqueous extract of the butanol extract from a culture filtrate is lyophilized using an "Edwards" chamber drying apparatus. A portion of 24 g of the solid obtained contains 0/96 g of sodium clavulanic acid according to the enzyme inhibition test. This solid is suspended in 75 ml of dry dimethylformamide and 75 ml of benzyl bromide is added. The mixture is stirred for 2 hours at room temperature. The suspension is then diluted with 500 ml of ethyl acetate and the mixture is filtered. The filtrate is evaporated on a rotary vacuum evaporator to give an oily residue. This residue is poured onto a 5.08 cm x 35.56 cm silica gel column ("Biogel Biosil A" 100 mesh) in cyclohexane. Benzyl bromide is eluted from the column / and then the solvent is changed to ethyl acetate and fractions of 1-0 ml are collected. Fractions containing the benzyl ester of clavulanic acid are selected as described in Example 4. Further purification is performed as described in Example 4 by column chromatography.

By this process 220 mg of pure benzyl ester is obtained.

Example 8.

Preparation of pivaloyloxymethylclavulanate.

To a stirred solution of 0.37 g of bromomethyl pivalate in 5 ml of dry di-τ methylformamide is added 0.49 g of sodium clavulanate. After standing for 2 hours at room temperature, the reaction mixture is treated with 20 ml of ethyl acetate, 10 ml of cyclohexane and 20 ml of water. The mixture is separated into two phases and the non-aqueous phase is separated, washed with 20 ml of water and dried over sodium sulfate. The dried solution is evaporated to give the desired product in the form of 500 mg of a light yellow oil.

NMR Spectrum (CDCl 3): 1.26 (s, 9), 3.13 (d, 1, J = 17 Hz), 3.62 (dd, 1, J - 17 Hz, J 1 = 2.5 Hz) , 4.3 (d, 2, J = 7.5 Hz), 5.0 (dt, 1, J = 7.5 Hz, J 2 = 1.5 Hz), 5.16 (d, 1, J = 1.5 Hz), 5.79 (d, 1, J = 2.5 Hz), 5.926 (s, 2).

IR spectrum (liquid film): v β-lactam C.O 1800 cm "^, ester C = 0 1760 cm-1.

Example 9

23 145543

Preparation of clavulanic acid phthalide esters.

To a stirred solution of 0.43 g of 3-bromophthalide in 5 ml of dry dimethylformamide is added 0.5 g of sodium clavulanate and the solution is allowed to stand at room temperature for 2 hours. The solution is treated with 20 ml of ethyl acetate, 10 ml of cyclohexane and 30 ml of water and shaken thoroughly.

The non-aqueous phase is washed with 20 ml of water, dried over sodium sulfate and evaporated to give a pale yellow gum. The two diastereomeric esters are separated using high-pressure liquid chromatography on a 40 cm x 10 mm silica gel column (Merckosorb SI 60, 5 µ u) and eluted with ethyl acetate at a flow rate of 3 ml / minute.

The first phthalide ester (retention time 7.15 minutes) crystallizes from ethyl acetate in the form of needles with melting point 102 ° C and has the following spectra: IR spectrum (nujol mull) v S-lactam C = 0 1790 cm \ ester C = 0 1755 cm * ".

NMR Spectrum (CD 3 COCD 3): 3.14 (d, 1, J = 17.5 Hz), 3.76 (dd, 1, J = 17.5 Hz, J 2 = 2.5 Hz), 4.25 (d, 2, J = 7.5 Hz), 5.0 (dt, 1, = 7.5 Hz, J 2 = 1.5 Hz), 5.4 (s, 1, J = 1.5 Hz), 5.82 ( d, 1, J = 2.5 Hz), 7.7 (s, 1), 8.06 δ (m, 4).

Molecular Weight (Mass Spectrometry): 331.0696 corresponds to cigHi3N07 (calculated 331.0692).

The second diastereoisomer (retention time 8.85 minutes) has the following spectra: IR spectrum: (CH 2 Cl 2 solution): v β-lactam C = 0 1800 cm 1, ester C = 0 1780 cm -1.

NMR Spectrum (CDCl3): 2.42 (broad S, 1, interchangeable with D2 °), 3.12 (d, 1, J = 18. Hz), 3.60 (dd, 1, Hz, J 2 = 2.5 Hz), 4.30 (d, 2, J = 7.5 Hz), 5.0 (dt, 1, = 7.5 Hz, J 2 = 1.5 Hz), 5, 12 (d, 1, J 1 1.5 Hz), 5.76 (d, 1, J = 2.5 Hz), 7.52 (S, 1), 7.856 (m, 4).

CH-OH

py 'i + COONa

__yCH2OH

0 1 ° f0 ° - ^^ === - 0

Example 10.

Preparation of Nonyl Clavulanate.

44 mg of sodium clavulanate in 2 ml of dry dimethylformamide are treated with 76 mg of nonyl iodide and allowed to stand at room temperature for 2 hours. The solution is evaporated and the residue is fractionated on silica gel and eluted with 2: 1 ethyl acetate-hexane to give the product as an oil.

IR spectrum (film): 1800, 1745 and 1690 cm

Molecular weight (mass spectrometry) = 325.1890, which corresponds to C17H27NO5 (calculated 3-25.1889).

Example H · 255543

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 12.

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 and eluted with ethyl acetate to give 140 mg of a clear oil.

Example 13

Preparation of phenylclavulanate.

Cool 100 mg of clavulanic acid in 5 ml of acetonitrile and stir at 0 ° C.

To the solution are 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 1: 1 ethyl acetate-hexane to give 70 mg of phenylclavulanate.

IR spectrum (film): 1800, 1770, and 1690 cm 2 NMR spectrum (CDCl3): 2.18 (broad s, 1), 3.06 (dd, 1, J = 17 Hz, J 2 = 0.9 Hz), 3.54 (dd, 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 6 (m, 5).

Molecular weight (mass spectrometry) = 275.0777, which corresponds to C ^H- ^NOg (calculated 275.0794).

Example 14.

Preparation of 2,2,2-trichloroethyl clavulanate.

221 mg of sodium clavulanate is suspended in 5 ml of dry tetrahydrofuran and stirred at 0 ° C. 211 mg of chloroformic acid trichloroethyl ester in 1 ml of dry tetrahydrofuran are added to the above suspension over 20 minutes. The mixture is allowed to reach room temperature and stirred overnight. The suspension is filtered and the filtrate is evaporated in vacuo. The residue is chromatographed on silica gel and eluted with 2: 1 ethyl acetate-hexane to give the desired product as an oil.

IR spectrum (film): 1800, 1760 and 1690 cm

NMR Spectrum (CDCl3): 1.56 (broad S, 1), 3.07 (dd, 1, = 17.5 Hz, J2 = 0.7 Hz), 3.56 (dd, 1, 17.5 Hz, J 2 = 2.5 Hz), 4.24 (d, 2, J = 7.5 Hz), 4.69 (d, 1, J = 12 Hz), 4.92 (d, 1 , J = 12 Hz), 5.02 (dt, 1, J2 = 7.5 Hz, J2 = 1.3 Hz), 5.19 (d, 1, J = 1.3 Hz), 5.73 δ (dd, l, = 2.5 Hz, J 2 = 0.7 Hz).

Molecular weight (mass spectrometry) = 328.9621, which corresponds to C ^ qH q qNO₂jCl. (calculated 328.9625).

27 145543 / ° Η50Η γη ωη -r *) ^ y C1C0.0R -Γ ^ ° \ / 2 '0! COONa / j

/ CO-O-CO-O-R

> 0 Fl ™ R = CH, C1, j / - 6 J Nv. /

CT

l

COOR

Example 15

Preparation of benzyl ester of clavulanic acid.

A solution of 0.55 g of pure lithium clavulanate in 3 ml of warm water is added dropwise over 5 minutes to a stirred solution of 1.8 ml of benzyl bromide in 8 ml of dimethylformamide. The mixture is stirred at room temperature (20 ° C) for 2 hours. Thin layer chromatography (ethyl acetate-SiC> 2) then shows that a large proportion of the clavulanate salt has been esterified. The reaction mixture is poured on top of a column of 200 g of silica gel made in cyclohexane-ethyl acetate in a ratio of 10: 1 and eluted with cyclohexane and ethyl acetate, graduated from a ratio of 10: 1 to 1: 1, then with pure ethyl acetate. Fractions containing benzyl clavulanate are combined and evaporated under reduced pressure to give an oil from which the remaining solvents are evaporated under high vacuum to give 0.37 g (47% of theory) of pure benzyl clavulanate.

Examples 16-28 285543

Benzyloxycarbonylmethylclavulanat.

CH 2 OH

_ / CHOOH

ΓΤ) = / -> ΓΤ w t \ C00Na C00CH_C00CHoCrHc 2 2 6 5 0.6 g of sodium clavulanate and 0.46 ml of benzyl bromoacetate are dissolved in 7 ml of Ν, Ν-dimethylformamide. After standing for 2 hours at room temperature, the solvent is evaporated in vacuo. 50 ml of diethyl ether, 25 ml of ethyl acetate and 50 ml of ice water are added and the mixture is shaken and separated.

The aqueous phase is washed with an additional 50 ml of the solvent mixture. The combined solvent phases are washed with 10 ml of ice water, dried over sodium sulfate and evaporated to an oil which is subjected to chromatography on silica gel using cyclohexane and ethyl acetate as eluents. The fractions containing benzyloxycarbonylmethylclavulanate are isolated and evaporated to give a colorless oil.

IR spectrum (film): 3510 (b, OH), 1805 (β-lactam C = 0), 1760 (broad, ester C = 0's) and 1700 cm @ 1 (C = C).

NMR Spectrum (CDCl3): 1.86 (1H, bs, disappearing by deuteration, C ^OH), 3.04 (1H, d, J 18Hz, 6β-Η), 3.46 (1H, dd, J 18Hz) , J 2.5Hz, 6a-H), 4.19 (2H, d, J 8Hz, CH 2 OH), 4.71 (2H, s, OCH 2 CO), 4.97 (1H, t, J 8Hz, = CHCH2OH), 5.14 (1H, s, 3-CH), 5.19 (2H, s, PhCH2), 5.66 (1H, d, J 2.5Hz, 5-CH), 7.34 (5H , s, PHCH2).

Example 17

Natriumcarboxymethylclavulanat.

29 145543

CH-OH

/ 2 CHOOH

COOCH2COOCH2CgH5 COOCH2COONa 0.2 g of benzyloxycarbonylmethylclavulanate in 5 ml of re-distilled tetrahydrofuran is hydrogenated at 22 ° C over 0.2 g of 5% palladized barium sulfate for 30 minutes. The catalyst is removed by filtration.

To the filtrate is then added 1.0 ml of water and 0.11 g of solid sodium hydrogen carbonate. The mixture is cooled to 10 ° C and stirred for 5 minutes. Excess NaHCO 3 is filtered off and the filtrate is evaporated in vacuo to give a brittle foam.

IR Spectrum (Concentrated Dimethyl Sulfoxide Solution): 1801 (β-lactam OO), 1753 (ester C = 0), 1700 (C = C) and 1630 cm -1 (COO ~).

Example 18.

Benzhydrylclavulanat.

n CH90H

/ CHOOH

r ~ y COOH COOCH (C6H5) 2 112 mg diphenyldiazomethane in petroleum ether (boiling range 40 - 60 ° C) is added to a solution of 38 mg clavulanic acid in 4 ml of tetrahydrofuran.

The resulting solution is stirred overnight at room temperature, after which the solvent is removed under reduced pressure to give an oily residue which is chromatographed on silica gel and eluted with ethyl acetate-hexane mixture to give 135 mg of the title compound.

IR Spectrum (CHCl3): 3400 (OH), 1805 (β-lactam C = 0), 1755 cm 2 (ester C = 0) and 1700 (C = C).

NMR Spectrum (CDCl3): δ = 2.05 (1H, s, OH), 3.03 (1H, d, J 17Hz, ββ-CH), 3.53 (1H, dd, J 17Hz, J 2.5Hz, 6a-CH), 4.22 (2H, d, J 7Hz, = CHCH 2), 4.94 (1H, t, J 7Hz, = CHCH 2), 5.27 (1H, s, 3 CH), 5.76 (1H, d, J 2.5Hz, 5-CH), 7.03 (1H, s, CHPh 2), 7.46 (10H, s, aromatic protons).

Example 19.

2-benzyloxycarbonylamino-2-methoxycarbonylethylclavulanat.

CH-OH

- / H2OH -> I

500 mg of benzyloxycarbonyl-L-serine methyl ester and 143 mg of dicyclohexylcarbodiimide are added to a cooled and stirred solution of 138 mg of clavulanic acid in 4 ml of tetrahydro acid. The reaction mixture is allowed to warm to room temperature and stirred overnight. The reaction mixture is filtered, washed and dried and the solvent removed under reduced pressure to give a residue which is chromatographed on silica gel and eluted with ethyl acetate-hexane to give 103 mg of the title compound as a gum.

IR Spectrum (CHCl3): 3400, (broad, OH and NH), 1805 (β-lactam C = 0), 1760 and 1725 (C = 0 x 3), and 1700 cm -1 (C = C).

NMR Spectrum (CDCl3): d = 2.42 (1H, s, OH), 3.08 (1H, d, J 17H?, 6β-OΗ), 3.58 (1H, d, J 17Hz, J 2.5Hz, 6ct-CH), 3.88 (3H, s, C00CH3), 3.98 -5.15 (6H, m, = CHCH2 and C00CH2CH), 5.2 (1H, m, 3-CH) , 5.25 (2H, s, CH 2 Ph), 5.73 (1H, d, J 2.5Hz), 6.01 (1H, m, NH), 7.53 (5H, s, aromatic protons).

Example 20

2-Amino-2-methoxycarbonylethylclavulanat hydrochloride.

3ί 145543

CHOH OH CHOH

n> = / row w J-V / * / -K \ / <f \ o \

'V

COOCHOCH-COOCH, COOCH-CHCOOCH-2 j 3 2 in 3 NHCOOCH2C6H5 NH2, HCl 85 mg 2-benzyloxycarbonylamino-2-methoxycarbonylethylclavulanate in 2 ml distilled tetrahydrofuran containing 10 mg of acetic acid is hydrogenated over 40 mg 10% palladium. After 60 minutes, thin layer chromatography (solvent: ethyl acetate) shows formation of a product and some unreacted starting material. The catalyst is filtered off and the filtrate is evaporated to give an oil which is chromatographed on one ion exchange column ("Amber-lite" ® IRA 401 in Cl form) (eluting with ethyl acetate-hexane), evaporating the resulting solution fabric in the form of a viscous rubber.

IR Spectrum (liquid film): 1805 (β-lactam), 1760 (ester CO) and 1695 cm -1 (C = C).

NMR Spectrum (D20): δ = 3.13 (1H, d, J 17Hz, δβ-CH), 3.57 (1H, dd, J 17Hz, J 3Hz, 6a-CH), 3.8 (3H, s, OCH-j), 4.14 (2H, d, J 7Hz, = CHCH 2), 5.70 (1H, d, J 3Hz, 5-CH), residual peaks interfered with by HOD.

Example 21.

2- (2-phenylsulfonyl) ethylclavulanat.

f ^ ° ch2oh Ao ch2oh

/ "V / -" Y

\ \ C00H COOCH «CH0SO_C H_ 2 2 2 6 5 32 145543

A solution of 199 mg of clavulanic acid in 8 ml of tetrahydrofuran is cooled to 0 ° C and treated with 206 mg of dicyclohexylcarbodiimide, 79 mg of pyridine and 1.0 g of β- (phenylsulfonyl) ethanol. The stirred solution is allowed to warm to room temperature and stirred at room temperature overnight. The suspension is filtered and the filtrate is evaporated under reduced pressure. The residue obtained is chromatographed on silica gel and eluted with ethyl acetate-hexane mixtures to give 155 mg of the title compound in the form of an oil.

IR Spectrum (CHCl3): 3400 (broad OH), 1800 (β-lactam C = 0), 1750 (ester C = 0), 1695 (C = C), 1310 and 1150 cm -1 (SO2).

NMR Spectrum (CDCl3): δ = 2.18 (1H, s, OH), 3.08 (1H, d, J 17Hz, 6β-O), 3.52 (1H, dd, J 17Hz, J '2 , 5Hz, 6a-CH), 3.59 (2H, t, J 6Hz, CH 2 SO 2), 4.25 (2H, d, J 7.5Hz, = CHCH 2), 4.57 (2H, t, J 6Hz, CH 2 CH 2 SO 2), 4.7 - 5.01 (2H, m, = CHCH 2 and 3-CH), 5.7 (1H, d, J 2.5Hz, 5-CH), 7.5 - 8.2 (5H , m, Ph).

Example 22.

2-Methylthioethylclavulanat.

CHo0H

COOH \ lCOOCH2CH2SCH3

A solution of 199 mg of clavulanic acid in 8 ml of tetrahydrofuran is cooled to 0 ° C and treated with 200 mg of dicyclohexylcarbodiimide and 960 mg of β- (methylthio) ethanol. The stirred solution is allowed to warm to room temperature and stirred at this temperature overnight. The suspension is filtered and the filtrate is evaporated under reduced pressure. The residue obtained is chromatographed on silica gel and eluted with ethyl acetate-hexane mixtures to give 145 mg of the title compound as an oil.

IR Spectrum (CHCl3): 3400 (OH), 1805 (β-lactam C = 0), 1750 (ester C = 0), 1695 cm -1 (C = C).

NMR Spectrum (CDCl3): δ = 1.72 (1H, s, OH), 2.15 (3H, s, SCH-j), 2.85 (2H, t, J 7Hz, CH 2 SCH 3), 3.05 (1H, dd, J 17Hz, J '1Hz, ββ-CH), 3.55 (1H, dd, J 17Hz, J' 2.5Hz, 6a-CH), 4.32 (2H, t, J 7Hz, CH2CH2SCH3), 4.30 (2H, d, J 7.5Hz, = CHCH2), 4.97 (1H, dt, J 7.5Hz, J '1.5Hz, = CHCH2), 5.09 (1H, m , 3-CH), 5.73 (1H, dd, J 2.5Hz, J '1Hz, 5-CH).

Example 23

2-Pyridylethylclavulanat.

0CH „OH CH_OH

= / -> ΓΤ> = / * 'cooh cooch2ch2- ^ h

A solution of 135 mg of clavulanic acid in 5 ml of tetrahydrofuran is cooled to 0 ° C and treated with 200 mg (2.5 equivalents) of 2- (2-hydroxyethyl) pyridine and 143 mg (1 equivalent) of dicyclocarbodiimide. The stirred solution is allowed to warm to room temperature and stirred at this temperature overnight. The suspension is filtered and the filtrate is evaporated under reduced pressure. The residue obtained is chromatographed on silica gel and eluted with ethyl acetate containing 5% methanol to give 100 mg of the title compound as an oil.

IR Spectrum (CHCl3): 3400 (OH), 1805 (β-lactam OO), 1750 (ester C = 0) and 1695 cm -1 (C = c).

NMR Spectrum (CDCl3): δ = 2.20 (1 * 1, s, OH), 2.97 (1H, d, J 17Hz, ββ-CH), 3.11 (2H, t, J 7Hz, CH 2 CH 2 Py ), 3.42 (1H, dd, J 17Hz, J * 2.5Hz, 6a-CH), 4.15 (2H, d, J 7Hz, = CHCH 2), 4.45 - 4.9 (3H, m , CH2CH2Py and = CHCH2), 4.97 (1H, m, 3-CH), 5.51 (1H, d, J 2.5Hz, 5-CH), 7.0 - 7.4 (2H, m, pyridyl-3-CH and 5-CH), 7.45 - 7.85 (1H, m, pyridyl-4-CH), 8.35 - 8.70 (1H, m, pyridyl-6- CH).

Example 24.

Phthalimidomethylclavulanat.

Eo CH

-r Λ / λ CHo0H

PO1 COONa COOCH2N \ 1.95 g of phthalimidomethyl chloride is added to a stirred solution of 221 mg of sodium clavulanate in 2 ml of dry dimethylformamide and the solution is allowed to stand at room temperature for 4 hours. The solution is treated with ethyl acetate and water and shaken thoroughly. The non-aqueous phase is separated, dried and evaporated under reduced pressure to give a residue which is crystallized from ethyl acetate-petroleum ether (boiling range 40 - 60 ° C) to give 130 mg of the title compound, mp 163 ° C.

IR Spectrum (CHCl3: 3400 (broad, OH), 1805 (β-lactam C = 0), 1785, 1760 and 1735 cm 2 (phthalimidyl C = 0, ester C-O)).

NMR Spectrum: δ = 1.7 (1H, s, OH), 3.08 (1H, d, J 17Hz, ββ-CH), 3.56 (1H, dd, J 17HZ, Jr 2.5Hz, δ <x -CH), 4.25 (2H, d, J 7.0Hz, = CHCH2), 4.95 (1H, dt, J 7.5Hz, J '1Hz, * CHCH2), 5.11 (1H, d, J 1Hz, -3-CH), 5.75 (1H, d, J 2.5Hz, 5-CH), 5.87 (2H, d, CH 2 Phth), 7.75 - 8.2 ( 4H, m, Phth).

Example 25

Allylclavulanat.

35 U5543

ro CH2 ° h CH-OH

W L, rTw 2 rf r ^ i COONa COOCH2CH = CH2

To a stirred solution of 2.05 g of sodium clavulanate tetrahydrate in 9 ml of dry dimethylformamide is added 2.44 ml of allyl bromide. The mixture is stirred at room temperature for 3 hours, after which the solvent is removed under vacuum at below 30 ° C. The residue is partitioned between water and ethyl acetate. The ethyl acetate phase is washed with water, dried over magnesium sulfate and evaporated. The residue is purified by column chromatography (Kieselgel 60 / ethyl acetate, R f value 0.53) to give 1.31 g of the title compound as an oil.

XR Spectrum (film): 3400, 1805, 1745 and 1695 cm NMR Spectrum (CDCl3, 90MHg): δ = 1.98 (1H, singlet interchangeable), 3.02 (1H, doublet, J 17Hz), 3 , 46 (1H, double doubled, J 17Hz x 3Hz), 4.19 (2H, doubled, J 7Hz), 4.61 (2H, doubled with finer coupling J 6Hz), 4.88 (1H, triplet with fine coupling) , J 7Hz), 5.02 (1H, fine coupling singlet), 5.29 (2H, fine coupling triplet, J 8Hz), 5.64 (1H, doublet, J 3Hz), 5.7 - 6, 1 (1H, multiplet).

Example 26

Acetonylclavulanat.

£ H20H ^ o CH20H

-> in C.

/ x / —V

\ ° \ c0ONa COOCH2COCH3 36 145543

To a stirred solution of 2.05 g of sodium clavulanate tetrahydrate in 9 ml of dry dimethylformamide is added 2.35 ml of bromoacetone. The mixture is stirred overnight at room temperature, after which the solvent is removed in vacuo at below 30 ° C. The residue is partitioned between water and ethyl acetate. The ethyl acetate solution is washed with water, dried over magnesium sulfate and evaporated. The ester is isolated by column chromatography (Kieselgel 60 / ethyl acetate eluent, Rf value 0.36) to give 565 mg of the title compound.

IR Spectrum (film): 3400, 1800, 1755 and 1730 cm NMR Spectrum (CDCl 3 90 90 MHg): δ = 2.14 (3H, singlet), 2.20 (1H, broad interchangeable), 3.04 (1H , doubled, 17Hz), 3.46 (1H, double doubled, J 17Hz x 3Hz), 4.20 (2H, fine coupled doublet, J 8Hz), 4.7 (2H, singlet), 4.97 (1H , double triplet, Jtr ^ p 8Hz, JdQub'x «1.5Hz), 5.12 (1H, finely coupled singlet), 5.67 (1H, doubled J 3Hz).

Example 27

Cyclohexylclavulanat.

n CH, OH H CHOH

COOH COO

A solution of N-ethoxycarbonyl-N-nitrosocyclohexylamine (1.4 g prepared by reaction of nitrogen dioxide on N-ethoxycarbonyl - cyclohexylamine in CC14 in the presence of anhydrous sodium acetate) in 25 ml of dichloromethane at 0 ° C is treated with 0.62 ml pyrrolidine and stirred at 0 ° C for 30 minutes. To this solution, a solution of clavulanic acid in tetrahydrofuran (prepared by hydrogenation of 1 g of benzylclavulanate in 30 ml of tetrahydrofuran over 10% palladium / carbon) is added over 5 minutes. The resulting solution is treated with an excess amount of sodium hydrogen carbonate solution. The resulting mixture is extracted with several portions of chloroform and the chloroform solution is dried over magnesium sulfate and evaporated. By repeated chromatography (twice with 1: 1 ethyl acetate: cyclohexane and once with chloroform on Kieselgel H) 46 mg of the title compound are obtained.

IR Spectrum (film): 3400, 18Q0, 1740 and 1695 cm @ 1 NMR Spectrum (CDCl3): δ = 1.1 - 2.0 (11H, m 1H interchangeable), 3.01 (1H, d, J = 16Hz), 3.45 (1H, dd, J = 16 and 3Hz), 4.17 (2H, d, J = 7Hz), 4.5 - 5.0 (1H, m), 4.87 (1H, t, J = 7Hz), 4.96 (1H, s), 5.63 (1H, d, J - 3Hz).

Example 28.

Propargyl.

CH 0 OH

- ^ \ / 2 ___> ^ 0 CH2 ° H

Dissolve 4.4 g (0.015 mole) of sodium clavulanate tetrahydrate in 25 ml of dry dimethylformamide and with stirring 1.6 ml (2.53 g, 0.0212 mol) of propargyl bromide are slowly added. The mixture is stirred at room temperature for 2 hours and then poured onto 250 ml of water and the product is extracted three times with 100 ml of ethyl acetate each time. The combined ethyl acetate extracts are washed four times with 100 ml of water each time and dried over magnesium sulfate. The clear, dry ethyl acetate solution is evaporated to dryness in vacuo to give a thick oil which, according to thin layer chromatography (silica gel plates / ethyl acetate developer), contains two components.

(S)

The oil is dissolved in 2 ml of chloroform and chromatographed on "Sephadex" LH 2 O using a 1: 1 mixture of chloroform and cyclohexane as eluent. The fractions (25 ml) are examined by thin-layer chromatography and the fractions containing the fast-flowing component in admissible amounts are combined and evaporated in vacuo to give 2.2 g of a thick oil. This oil solidifies on standing overnight. Thin-layer chromatographic analysis shows only one component. NMR Spectrum shows presence of some chloroform.

The product is washed with ether to give 700 mg of solid (mp 65-66 ° C). The low recovery is due to the apparent solubility of the substance in this solvent. The test results listed below are available:

Analysis:

Calculated for C, 55.69; H, 4.68; N, 5.91

Found: C, 55.55; H, 4.68; N, 6.12.

NMR Spectrum (CDCl3): δ = 2.01 (s 1H), 2.6 (t 1H), 2.95 - 3.75 (m 2H), 4.29 (d 2H), 4.79 - 5.00 (m 3H), 5.13 (d 1H), 5.75 (d 1H).

Example 29.

4-methoxybenzyl ester.

CH20H ^ 2 ^ II ✓As —r ^ ° \ 9H 2oh _L_ ♦ O - »i

\ V / - * V

C00Na OCH, ° 1 3 cooch2c6h4och3 18.2 g of sodium clavulanate (about 0.07 mole of anhydrous sodium clavulanate, water content of 22.8%) are dissolved in 100 ml of dimethylformamide and treated with a solution of 13.5 g (0.07 mole) 4 -methoxybenzyl bromide in 25 ml of dimethylformamide. The clear solution is stirred at room temperature for 80 minutes and then diluted with 300 ml of ethyl acetate and 200 ml of water. The aqueous phase is separated and re-extracted with 300 ml of ethyl acetate. The ethyl acetate extracts are combined and washed six times with 200 ml of water each time and dried over magnesium sulfate.

By evaporation 19.5 g of an oil is obtained.

59 145543

The oil is dissolved in chloroform / cyclohexane in a 1: 1 ratio and chromatographed on a 65 x 400 mm column of "Sephadex" ® (500 g, LH 2 O, prepared in a 1: 1 mixture of chloroform and cyclohexane). The fractions containing the desired ester are combined and evaporated in vacuo to give a pale yellow oil. Trituration with n-hexane gives a solid powdered product which is filtered off, washed on the filter cake with fresh n-hexane and dried in vacuo to give 12.6 g (57% of theory), mp 54.5-55, 5 C.

IR Spectrum (KBr plate): max = 3400, 2950, 2840, 1795, 1735, 1690, 1615, 1515, 1305, 1250, 1175, 1005, 890 and 825 cm -1.

The same product can be prepared from clavulanic acid in tetrahydrofuran solution and 4-methoxybenzyl alcohol and dicyclohexylcarbodiimide.

Yield (on 0.0052 mill scale) 0.3 g (18% of theory).

Example 30.

4-nitrobenzyl ester.

CELBr I — X f., O.

ΓΓ r7, π _ nV

COONa NO2O in: 00CH2C6H4NO2 (p) 2.4 g of sodium clavulanate (about 0.01 mole, water content 10%) is dissolved in 1 ml of dimethylformamide and treated with 2.2 g (about 0.01 mole) of nitrobenzyl bromide at. room temperature for 40 minutes. The mixture is diluted with 100 ml of ethyl acetate, washed five times with 60 ml of water each time and with 50 ml of brine, dried over magnesium sulfate, filtered and evaporated to give an oil. The oil is triturated with n-hexane to give a crystalline product quickly. The solidified product is crushed in n-hexane, filtered, washed on the filter cake with fresh solvent and dried in vacuo.

40 145543

Yield 2.7 g (81% of theory), mp 110 - 112 ° C.

Analysis:

Calcd. For C 15 H 24 N 2 O 7: C, 53.89; H, 4.22; N, 8.38

Found ':. C, 54.07; H, 4.30; N, 8.32.

NMR Spectrum (CDCl3): 8.25, 8.10, 7.55, 7.40 (ABq, 4H, - + -) -, 5.70 (d, 1H, C5H), 5.25 (sec ), 5.05 (m, 1H, C-jH), 4.75 (m, 1H, H), 4.10 (d, 2H, -CH 2 O), 3.2 (m, 2H, CH 2), 1.95 (s, 1H, OH).

J- '

Example 31.

6-Me thoxynaphth-2-yl methyl ter.

CH9OH

-_ / visysiHj51 ^> 0 CH2 OH

0JX / COONa ^ xa

A solution of 2.2 g of anhydrous sodium clavulanate (10 millimoles) of OCH3 and 2.51 g of 6-methoxy-2-bromomethyl naphthalene (10 millimoles) in 80 ml of dry dimethylformamide is stirred at room temperature. After 3 hours, thin layer chromatographic analysis shows that bromomethyl naphthalene is no longer present in the reaction mixture. The reaction solvent is removed in vacuo at 30 ° C and the residue is treated with 100 ml of ethyl acetate. The suspension is filtered and the filtrate is washed twice with 25 ml of water each time, dried over magnesium sulfate, filtered and evaporated to give 3.2 g of an orange oil. By chromatography of this product on "Sephadex" ® LH 2 O (100 g) (eluent chloroform: cyclohexane 50:50 ratio, 10 ml fractions are collected; fractions 12-20 contain the desired ester as determined by thin layer chromatography and visualized with TTC spray *) is obtained a clear oil which solidifies 41. 145543 on standstill. This material is dissolved in dichloromethane and petroleum ether (boiling range 60 - 80 ° C) is added until the solution becomes cloudy. When left in an open beaker, a rubber is formed.

The supernatant is further evaporated and 2.52 g of a white crystalline solid are formed, mp 89-90 ° C (68% of theory).

Analysis:

Calculated for C 20 H 19 NO 65 C 65.04 H 5.15 N 3.79 Found: C 64.91 H 5.00 N 3.77.

NMR Spectrum (CDCl3): δ = 1.45 (1H, bs, CH 2 OH), 2.9 (1H, d, 60-H), 3.36 (1H, dd, 6a-H), 3.8 ( 3H, s, OCH 3), 4.03 (2H, d, CH 2 OH), 4.7 (1H, t * -H), 4.93 (1H, s, CHCOO-), 5.27 (2H, s, -CH₂COC), 5.65 (1H, d, 5β-Η), 7.37 (6H, m, aromatic H's).

* 4% triphenyl tetrazolium chloride in methanol + an equal volume of methanolic IN NaOH.

Example 32.

2-Hethoxyethylester.

1) soci2 g: h2oh ch3och2ch2oh ch3och2ch2i> ΓΤ ^ = / 000CH2CH2OCH3 43 g (0.455 mol) of 2-methoxyethyl chloride (JACS 1930, 5 * 2, 653) are added to a solution of 75 g (0.50 mol) of sodium iodide in 500 ml acetone and the reaction mixture is heated to reflux for 13-4 hours. The majority of the acetone is distilled off at atmospheric pressure and the residue is cooled and diluted with 200 ml of ether. The ethereal solution is filtered free of inorganic salts and then washed with 100 ml of brine, 100 ml of 5% aqueous sodium thiosulfate and again with 100 ml of brine in the order indicated and dried over magnesium sulfate. After filtration, the ether is removed by distillation under atmospheric pressure, and the product is isolated by distillation, collecting the fraction boiling between 130 and 140 ° C (atmospheric pressure). The product, a heavy light yellow liquid, weighs 20 g (23% of theory in the case of 100% purity).

To a solution of 6 g (0.0205 mol) of sodium clavulanate tetrahydrate in 100 ml of dimethylformamide is added 5.6 g (0.03 mol) of 2-methoxyethyl iodide at room temperature. The reaction is stirred for 5 hours, after which thin layer chromatography (silica plates developed in ethyl acetate, spots visualized with alkaline TTC spray, R R value 0.0 for sodium clavulanate, R The reaction mixture is diluted with 200 ml of ethyl acetate and 500 ml of water, the aqueous phase is re-extracted with 100 ml of ethyl acetate and the combined organic extracts are washed four times with 100 ml of water each time, with 100 ml of 1 N sodium hydrogencarbonate solution and three times with 100 ml of water again. and dried over magnesium sulfate. After filtration, the solvent is evaporated in vacuo and the ester thus obtained is obtained in the form of a light yellow oil which is finally dried under strong vacuum to remove the last traces of solvent and unreacted iodine compound. According to the PMR spectrum, the product obtained is essentially pure. Yield: less than 5% of theory.

PMR Spectrum (CDCl3): <$ - 5.65 (1H d), 5.05 (1H, s) and 4.9 (1H t) overlapping, 4.2 (4H m), 3.6 ( 2H m), 3.35 (3H, s) are above 3.3 (1H m), and 2.9 (1H s).

Example 33

Cultivation of Streptomyces clavuligerus. .

Streptomyces clavuligerus is grown at 26 ° C on agar slant culture containing 1% "Yeatex" (yeast extract), 1% glucose and 2% "oxoid" agar # 3, pH 6.8. A sterile eye is used to transfer mycelium and spores from the slant culture into 100 ml of a liquid medium in a 500 ml Erlenmeyer flask. The liquid medium has the following composition: r "Oxoid" malt extract 10 g / liter "Oxoid" bacteriological peptone 10 g / liter

Glycerol 20 g / liter

Tap water 1 liter.

145543 43

The medium is adjusted to pH 7 by addition of sodium hydroxide solution and the volume of 100 ml is distributed in the flasks, which are closed with 2 foam plugs before autoclaving at 1.0545 kg / cm for 20 minutes. One

ISLAND

grafted germ flask is shaken for 3 days at 26 ° C on a rotary shaker with a stroke of 5 cm. and a speed of 240 rpm. Production stage flasks containing the liquid medium as described above are seeded with 5% vegetative inoculum and grown under the same conditions as the germ flask. Samples of culture filtrate are examined for inhibitory action against β-lactamase from Escherichia coli JT4. Optimal activity is obtained after 3 days. The results are set forth in Table I. A zone of clavulanic acid at R ^ value 0.46 is revealed when the culture filtrate is examined by the paper chromatographic method described above. The growth in zone size corresponds to the growth of β-lactamase inhibitor potency determination.

Streptomyces clavuligerus is also grown in 2-liter shake flasks containing 400 ml of medium (production stage) using the same medium and culture conditions as described previously in this example. In these larger vessels, the growth of the organism is slower and the optimal β-lactamase inhibitory effect is obtained 7-9 days after inoculation with the vegetative germ. The results are also listed in Table I.

· In Table.

β-Lactamase inhibitory effect of Streptomyces clavuligerus.

Growth in 500 ml and 2000 ml flasks.

% inhibition of Escherichia coli β-lactamase by a final dilution on Fermentation time (days) 1/2500 of the culture filtrate _500 ml shake flask 2000 ml shake flask 1 15 - 2 30 - 3 55 - 4 50 10 5 51 21 6 57 36 7 - 51 8-53 9 - 50.

145543 44

Example 34.

Cultivation of Streptomyces clavuligerus.

A "Yeatex" / glucose strain agar slurry culture is used to seed a "yeatex" / glucose agar slurry culture into a Roux flask 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 inoculate 50 liters of steam sterilized germ medium with the following composition in tap water: "Oxoid" malt extract 1% w / v "Oxoid" bacteriological peptone 1% w / v

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 plate with baffle plates and is stirred by a 12.5 cm blade disc rotor at 240 rpm. Sterile air is introduced at a rate of 50 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 a sterile transfer. This production step medium. is in a 300 liter stainless steel fermentation vessel, completely fitted with baffles, and stirred by a 21.6 cm blade disc rotor at 210 rpm. Sterile air is blown in at a rate of 150 liters / minute. The fermentation is maintained at 26 ° C. When needed, anti-foaming agent is added in 10 ral portions (10% "Pluronic L81" in soybean oil). At regular intervals, samples are taken to determine β-lactamase inhibition. The fermenter is harvested between the 4th and 5th day at the optimum level of β-lactamase inhibitory action and the results are listed in Table II.

45 145543

Table II.

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

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

Example 35.

Isolation of raw clavulanic acid sodium salt.

Harvested culture fluid prepared as described in Example 34 is cleared by continuous liquid centrifugation and the mycelium discarded. Of 150 liters of fermentation liquid, 120 liters of clarified culture liquid are obtained. This filtrate shows 58% inhibition in the β-lactamase inhibition test at a dilution of 1/2500. The filtrate is cooled to 5 ° C and 40 liters of n-butanol are added. The mixture is stirred and 25% I 2 SO 2 is added until the pH is 2.0. The acidified mixture is stirred for a further 10 minutes before the phases are separated by centrifugation. The aqueous phase is discarded. To the n-butanol extract is added 0.5% "Norit GSX" and the mixture is stirred for 15 minutes. The charcoal is discarded after removal by filtration using diatomaceous earth as a filter aid. To the n-butanol is added a quarter volume of deionized water and the mixture is stirred while adding 20% NaOH solution until the pH has reached 7.0. The phases are separated by centrifugation and the n-butanol phase discarded. The aqueous phase is evaporated under reduced pressure to 800 ml and then lyophilized. Thereby, 35 g of a crude solid clavulanic acid product having an IQ of 1.3 µg / ml is obtained by the β-lactamase inhibition test. This solid composition is stored dry at -20 ° C before being further purified.

46 145543

Example 36.

- Isolation of raw clavulanic acid sodium salt.

1 liter culture filtrate with 53% inhibition at 1/2500 in the β-lactamase inhibition experiment is percolated down a 2.54 cm x 15.24 cm column of "Permutit Isopore resin FF IP (SRA 62)" in Cl form ( marketed by Permutit Co. Ltd., 632 - 652 London Road, Isleworth,

Middlesex, UK). The culture filtrate is followed by 300 ml of distilled water to wash the column. Elution of the active β-lactamase inhibitor is performed with 0.2M NaCl solution. Fractions of 20 ml are collected and assayed at a final dilution of 1/2500 by β-lactamase inhibition assay. Active fractions are combined and evaporated in vacuo to 20 ml. This solution is desalted by gel exclusion chromatography on a 3.81 cm (3.81 cm) diameter 40.44 cm Biorad Biogel column and eluted with 1% n-butanol in water ("Biogel P2" marketed by Biorad Laboratories, 32nd and Griffin Avenue, Richmond, California, USA). The active fractions determined by the β-lactamase inhibition assay are pooled. Sodium chloride is eluted after clavulanic acid and determined using silver nitrate solution. The combined active fractions are evaporated and lyophilized.

0.45 g of crude solid clavulanic acid product having an I5g value of 0.92 µg / ml is obtained from the .1 liter culture filtrate. This solid is stored at -20 ° C before further purification.

Example 37.

Isolation of raw clavulanic acid sodium salt.

Culture filtrate containing 300 µg / ml clavulanic acid is acidified using an "in-line" mixer system and extracted with n-butanol, and clavulanic acid is back-extracted in water at neutral pH.

Cooled culture filtrate (5 - 10 ° C) is pumped to an "in-line" mixer, by the addition of which a sufficient amount of 6 volume / volume 47 145543 percent nitric acid is added to maintain an initial pH of 2.0 - 0.1.

The acidified filtrate is passed at a rate of 4 liters / minute through a glycol cooled plate heat exchanger (A.P.V. Ltd.) to maintain a temperature between 2 and 5 ° C. The pH is measured in a flow cell before the liquid is fed into a three stage counter flow separator (Westfalia Separator Ltd., model EG 1006).

With cooled water saturated n-butanol (at about 5 ° C) is pumped into the countercurrent separator at a rate of 3 liters / minute.

The aqueous outlet from the countercurrent separator is directed away. Introduced water is removed from the butanol effluent from the countercurrent separator using a liquid / liquid centrifugal separator (Alfa Laval Ltd., model 3024X - G). The butanol is collected in a stainless steel tank fitted with a cooling jacket in which it is stored at approx.

5 ° C.

40 liters of sample are removed from the tank and thoroughly mixed with 2 liters of cooled water (5 ° C) saturated with n-butanol. The pH of this mixture is adjusted to 6.8 - 0.1 using 20% sodium hydroxide solution.

This aqueous extract / butanol mixture is fed into a liquid / liquid centrifugal separator (Sharpies Centrifuge Ltd., model M35PY - 5 PH) at a pumping rate of 2 liters / minute.

Of 1800 liters of culture filtrate, 90 liters of aqueous phase containing 39% of the clavulanic acid contained in the culture filtrate is isolated.

15 liters of the aqueous extract is adjusted from 2% to 8% total solids by the addition of 60 g of sodium chloride / liter and dried (Anhydro, Copenhagen, type Lab. S 1). The conditions are: feed rate 2 liters / hour; nebulizer voltage 170 volts; heater position 6-7; input temperature 150 ° C; outlet temperature 80 ° C.

The dried product with a total weight of 1 kg contains 62% of the clavulanic acid which is in the dissolved solution.

The remaining 75 liters of aqueous extract is concentrated by ultrafiltration (De Danske Sukkerfabrikker, laboratory module, membrane type 48 145543 900). The operating technique is to recycle the retentate from a stainless steel tank fitted with a cooling system and with an output valve set in such a way that differential pressure is obtained across 40 membranes in 25 atmospheres. The temperature is maintained at 2 - 5 ° C and the pH is maintained at 6/8 - 0.1 by addition of 2N hydrochloric acid as needed. The volume is reduced to 34 liters containing 72% of the clavulanic acid contained in the solution dissolved.

The aqueous concentrate is stored at ca. 5 ° C, adjust to a solids content of 8% and spray dry as described above. The dried material contains 75% of the clavulanic acid in the solution led to the spray drying apparatus.

The total spray-dried product from 90 liters of aqueous extract contains 69.4 g of clavulanic acid, which is 72% of the clavulanic acid in the spray drying access liquid and 21% of the clavulanic acid in the 1800 liter culture filtrate.

Example 38

Partial purification of crude clavulanic acid.

Crude clavulanic acid preparations prepared as described in Example 3g are purified by ion exchange chromatography. 18 g of material prepared as described in Example 36 having an IQQ value of 1.3 µg / ml (final concentration) is dissolved in 25 ml of distilled water and poured onto a 3.81 cm x 40.64 cm bed of Permutit FF IP (SRA 62) "resin in Cl form. The column is eluted with a sodium chloride gradient formed by gravity addition of 0.5M sodium chloride to a mixing vessel containing 1 liter of distilled water which is again fed to the chromatography column. 10 ml fractions are collected and the β-lactamase inhibitory effect is examined using a 1/2500 dilution of the fractions. The activity is eluted following a main color band between fractions 24 and 30. The active fractions are combined and concentrated to 30 ml.

This solution was desalted using a 5.08 cm x 45.72 cm bed of "Biorad Biogel P2" and eluted with 1% n-butanol in water.

20 ml fractions are examined for clavulanic acid content using β-lactamase inhibition experiments. The fractions are also loaded onto paper strips and atomized with either "Ehrlich" or triphenyltetrazolium nebulizing reagents as described in Example 4. The β-lactamase inhibitory effect is correlated with pink and red spots induced by these reagents, respectively. Active fractions are combined, with the exception of those containing sodium chloride, and evaporated to dryness under vacuum. Thereby, 520 mg of partially purified clavulanic acid sodium salt with an IQ value of 0.2 µg / ml is obtained by the standard β-lactamase inhibition test.

Thin layer chromatography (silica gel) of this clavulanic acid preparation gives the following R R values: n-butanol / ethanol / water in a 4: 1: 5 v / v, peak, Rf value 0.37; n- * butanol / acetic acid / water ratio 12: 3: 5 v / v, R 2 value 0.44; isopropanol / water 7: 3 v / v, R 2 value 0.78.

These zones are determined by spraying "EhrlichH reagent. 6-Ami-nopenicillanic acid is allowed to run as a marker and determined with the same spraying agent and has R 2 values of 0.38, 0.39 and 0.77 respectively. .:

Example 39

Partial purification of clavulanic acid sodium salt.

Culture filtrate prepared as described in Example 34 solvent is extracted as described in Example 35 to give a solid preparation which is further purified by ion exchange chromatography using "Whatman" diethylaminoethyl cellulose DE 52.

This solid (10 g) is dissolved in 20 ml of distilled water and poured onto a 3.81 cm x 50.80 cm column of DE 52 cellulose which is already equilibrated with 0.1 M sodium phosphate buffer of pH 7.5 .

The column is eluted with a NaCl gradient. 0.1M NaCl in 0.01M sodium phosphate buffer pH 7.5 is fed into a mixer chamber containing 1 liter of pHM 7.5 0.01M phosphate buffer which is again passed onto the column. 10 ml fractions are collected and assayed for β-lactamase inhibitory effect by dilution of 1/2500. The fractions are also examined for antibacterial action by the hollow plate test method using nutrient agar plates on which Klebsiella aerogenes have been grafted. The fractions with the highest β-lactamase inhibitory effect and with inhibition zones in the hollow plate experiment are pooled, evaporated and desalted on a "Biorad Biogel P2" column. These fractions are shown to contain clavulanic acid using paper chromatography and thin layer chromatography.

Example 40.

Isolation of solid clavulanic acid sodium salt.

A partially purified solid form of clavulanic acid (500 mg) prepared as described in Example 38 is poured onto a "Whatman" microcrystalline CC 31 cellulose column having a bed size of 2.54 cm x 50.8 cm. The chromatographic solvent is n-butanol / ethanol / water in volume / volume ratio 4: 1: 5, top phase. The column is operated at 4 ° C and fractions of 4 ml are collected. The fractions are assayed for the presence of clavulanic acid by being placed on filter paper and sprayed with "Ehrlich" (pink samples) or triphenyl tetrazolium (red samples) nebulizing reagent. These stain samples are confirmed by β-lactamase inhibition experiments at a dilution of 1/1250. Active fractions are combined and dried under vacuum on a rotary evaporator. The solid is dissolved in a small volume of distilled water and lyophilized. 40 mg of a white solid preparation of the sodium salt of clavulanic acid with an I, JQ value of 0.08 µg / ml is obtained in the β-lactamase inhibition test.

Example 41.

Isolation of solid clavulanic acid sodium salt.

Concentrated extract (6 liters) (from ultrafiltration of Example 37) containing 10 g of clavulanic acid according to the β-lacta-mase inhibition test as described above. This is percolated at a rate of 1 liter / hour on a 5.08 cm x 60.96 cm column of "Permutit Zerolit FF 1 P SRA 62" anion exchange resin in chloride form. The column is then washed with 2 liters of deionized water before eluting with a sodium chloride gradient. The gradient is formed by a reservoir containing 4 liters of 1.4M NaCl, which is led to a stirred reservoir inside 5Ί 145543 containing 4 liters of 0.7M NaCl, which in turn is fed to a stirred reservoir containing 4 liters of deionized water, which is connected via a pump to column. The column is eluted at a rate of 2.5 ml / minute and fractions of 25 ml are collected. The fractions are assayed by the β-lactamase inhibition assay.

The active fractions (numbers 140 - 230) are combined and evaporated in vacuo to near dryness. 500 ml of ethanol are added and the solid is filtered off after vigorous shaking. The ethanol extract is then evaporated in vacuo to dryness on a rotary evaporator and redissolved in 40 ml of deionized water. This is poured onto a 10.16 cm x 60.96 cm column with "Biorad Biogel P2" and eluted with a 1% n-butanol solution. Fractions of 25 ml are collected and tested for β-lactamase inhibitory activity at a final dilution of 1/2500. Sodium content studies in 1/25 dilutions of the fractions are performed using silver nitrate solution. The fractions containing clavulanic acid without sodium chloride are combined, concentrated to 20 ml by evaporation of the solvent under reduced pressure and lyophilized. Thereby 4.8 g of the sodium salt of clavulanic acid is obtained (IQ value of about 0.06 µg / ml).

Example 42

Hydrolysis of clavulanic acid methyl ester 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 0.0482N 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 Ivær 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 7. The reaction is followed by paper chromatography (bioautographic system). Using the butanol / ethanol / water system to monitor the reaction for a period of 2 hours, the methyl ester zone decreases at a R 2 value of 0.79 in size, while the zone of clavulanic acid at R 2 value increases 0.12.

52 145543

Example 43

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 by thin layer chromatography (SiO2-ethyl acetate: KMnO4 spray). The catalyst is filtered off and washed with 5 ml of water and 5 ml of ethanol and the combined filtrates are evaporated under reduced pressure. The crystalline residue is triturated with 25 ml of acetone and the solid is filtered off, washed with 10 ml of acetone and dried over P 0.41 g of product is obtained. 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 filtrate can be obtained by filtration, which is washed with a small amount of acetone and dried in vacuo (80 mg). The filtrate is diluted with an equal volume of acetone and cooled again to give an additional 80 mg of yield.

Analysis:, first yield = 84.3¾ pure lithium clavulanate second yield = 85.1% pure lithium clavulanate.

(The lithium hydrogen carbonate solution is prepared by suspending 14 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 formed. The volume of the solution is made up to 500.0 ml with water saturated with CO 2 · This solution then contains 5.15% w / v LiHCQ).

Example 44

Preparation of potassium salt of clavulanic acid.

A solution of 2.89 g of benzyl clavulanate (of 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 (which are 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 dried in vacuo to give 0.75 g of the desired potassium clavulanate as a colorless crystalline solid which is 98.5% pure potassium clavulanate anhydrate (containing about 0.2 - 0.3% water).

Example 45

Trimethylammoniumclavulanat.

A trimethylammonium hydrogen carbonate solution is prepared at. dissolve 5 g of trimethylamine in 50 ml of ice-cold water and pass CO 2 through the solution in a rapid stream until the pH of the solution is about 8.5. This solution is analyzed by titration with 1.0M hydrochloric acid using screened methyl orange as indicator and turns out to be 0.4M.

To a solution of 2.89 g of benzylclavulanate in 100 ml of ethanol is added 0.9 g of 10% palladized charcoal and 25 ml of 0.4M trimethylammo-. hydrogen carbonate. The mixture is hydrogenated for 30 minutes at room 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 acetone and crystallized. The crystals are isolated by filtration, washed with 10 ml of acetone and 10 ml of ether and dried in vacuo to give 0.84 g of light brown trimethylammonium clavulanate. Analysis (at high pressure wall chromatography) shows 68.7% as free acid? 89.1% as trimethylammonium clavulanate.

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

No deoxy visible in the NMR spectrum.

Example 46

Preparation of aluminum clavulanate.

A solution of 2.89 g of benzylclavulanate and 0.68 g of aluminum isopropoxide in 150 ml of dry, recovered tetrahydrofuran is hydrogenated over 0.86 g of 10% palladized charcoal for 40 minutes (thin layer chromatography shows no unreacted benzylclavulanate 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 dried in desiccator over phosphorus pentoxide. The aluminum salt is thus obtained in the form of 0.3 g of white solid. Analysis by high pressure liquid chromatography shows 48% clavulanate; the aluminum content of EDTA titration is 5.76%; Karl Fischer determination shows 5.4% H2 O.

IR Spectrum (KBr): v = 1790, 1695 and 1615 cm cm \ max

Example 47.

Preparation of magnesium clavulanate.

A solution containing sodium clavulanate (rated as approximately 4.4 g) in 15 ml of water is poured onto an ion exchange column, Zerolit FF (1P), SRA62, and the column is washed with one volume of water. The column is eluted with 0.2 M magnesium chloride solution and the collected fractions are examined by paper chromatography (detection with Ehrlich's reagent) and the most intense fractions are combined and evaporated under vacuum to a small volume.

55 U55A3

This crude preparation of the magnesium salt is desalted by percolation through a column of "Biogel" P2 (paper chromatography-Ehrlich's spray).

The ephalted material is then evaporated, mixed with ethanol and chromatographed on a cellulose column using butanol / ethanol / water in a 4: 1: 5 v / v top phase ratio as eluant. Fractions containing most of the material are combined and evaporated to dryness in vacuo. Trituration with acetone followed by filtration gives the desired salt in the form of 0.58 g white solid. High-pressure liquid chromatography study shows .j ^ j.

91% pure magnesium clavulanate. Atomic absorption shows 5.13% Mg Karl Fischer analysis shows 6.8% water.

IR Spectrum (KBr): max = 1780, 1693 and 1610 cm

Example 48.

Magnesiumclavulanat.

A solution of 4.34 g of benzyl clavulanate and 1.61 g of magnesium acetate trahydrate in 100 ml of ethanol is hydrogenated over 1.45 g of 10% palladized 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 rubber 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 air dried to give 2.4 g of colorless solid. High pressure liquid chromatography study shows 75% pure magnesium salt.

. '56 145543

Example 49.

Preparation of ammonium clavulanate.

4734 g of benzyl clavulanate 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 supernatant is decanted from the originally formed oil and set aside to give 0.348 g of needles. High pressure liquid chromatography study shows that the ammonium clavulan lanate content is 97.4%. Karl Fischer analysis shows 0.44% water.

IR Spectrum (KBr) TV = 1785, 1700 and 1595 cm c max 3

Example 50

Preparation of crystalline potassium clavulanate.

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 / carbon 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).

57 145543

Example 51.

Examples of activity of clavulanic acid methyl ester.

Studies for antibacterial action in culture fluid show that clavulanic acid methyl ester has broad-spectrum effect but of lower order than clavulanic acid. It is not clear whether this effect is the effect of the compound itself or of clavulanic acid which is released by slow aqueous hydrolysis of the ester. Clavulanic acid methyl ester shows significant antibacterial synergism in combination with ampicillin or cephaloridine against bacteria resistant to these antibiotics. Thus, the minimum inhibitory concentration (MIC) of ampicillin against Staphylococcus aureus (Russell) is reduced from SOO µg / ral to <0.4 in the presence of 1.0 µg / ml clavulanic acid methyl ester. The MIC value for cephaloridine is reduced from 1.5 µg / ml to <0.03 µg / ml in the presence of 1 µg / ml clavulanic acid methyl ester. The MIC value of ampicillin against Proteus mirabilis C889 is reduced from 500 µg / ml to 31 µg / ml in the presence of 5 µg / ml clavulanic acid methyl ester.

Example 52

Antibacterial synergism between ampicillin and esters of clavulanic acid.

The results set out in Table VIII were obtained by the method described below.

The minimum inhibition concentration (MIC value) of ampicillin, clavulanic acid derivative and ampicillin in the presence of 1 µg / ml of clavulanic acid derivative is determined for a variety of β-lactamase-producing bacteria. The organisms are inoculated into "Oxoid" sensitivity test culture liquid placed in small shelves in a plastic tray and containing various concentration gradients of ampicillin, clavulanic acid derivative or ampicillin plus 1 µg / ml clavulanic acid derivative (necrotiter method). The final dilution of the inoculum in the culture liquid, grown overnight, is 0.5 x 10.. The tray is incubated at 37 ° C overnight, and almost the endpoints are determined for the baking growth. The MIC values in µg / ml are tabulated, showing that at low concentrations, the synergist significantly improves the antibacterial action of ampicillin against certain gram-positive and gram-negative bacteria. The mechanism of this synergism is thought to consist in inhibition of ampicillin-degrading β-lactamase enzymes, but the existence of other mechanisms cannot be ruled out.

Table VIII.

Antibacterial synergism between ampicillin and esters of clavulanic acid against strains of Klebsiella aerogenes.

Strain Ampicillin Ampicillin + 5 / µg / ml of me- Ampcillin +5, µg / ml alone. the thyl ester of clavulanic acid by the benzyl ester of clavulanic acid A '500 1.9 1.9 E 70 500 3.9 3.9 62 500 3.9 3.9

Neither clavulanic acid methyl ester nor clavulanic acid benzyl ester inhibit the growth of the test organism at a concentration of 100 µg / ml.

Example 53.

Antibacterial action of clavulanic acid ester.

Using the microtiter method described below, but using a dilution of 1/100 of the overnight liquid, the MIC values listed in Table IX for certain esters of clavuric acid are obtained against a variety of organisms.

Mikrotitermetode:

Serial dilutions of the clavulanic acid derivative in "Oxoid" sensitivity test liquid in a microtiter plastic tray are seeded with an overnight culture culture of each organism such that the final dilution of the inoculum is 0.5 x 10-4. The cultures are incubated overnight, and the points of bacterial growth are determined the next morning by observing the turbidity of the culture. The results are expressed as approximate MIC values (minimum inhibition concentration / ml).

Table IX.

Antibacterial action of clavulanic acid esters.

Organic MIC values for esters of clavulanic acid MIC * value

Benzyl Nonyl Pivaloyl Phthalidyl For Clavulan Ester Ester Oxymethyl Ester Acid Sodium Ester Salt Sum

Bacillus subtilis A 250 31 62 125 62

Staphylococcus aureus Oxford 62 31 31 31 15

Staphylococcus. 125 31 62 15 15

Russell

Escherichia coli 10418 125 - ^ 500 125 125 125 * The MIC value for the clavulanic acid sodium salt is included for comparison purposes; the high MIC values are due to the kraftigo inocula used.

Example 54

Detection of synergism between ampicillin and esters of clavulanic acid against Staphylococcus aureus Russell.

Table XII

MIC (yug / ml) R Ester Ampicillin Ampicillin alone + 1 µg / ml ester + 5 µg / ml ester CH3 62 125 0.06 0.03 CH2CgH5 16 250 0.4 0.08 CH2CO06H5 62.5 500 0.9 <0.1 phthalidyl 62.5 500 4.0 1.6 CH 2 COCH 3 31 250 0.8 <0.1 60 145543

Table XII continued CH 2 CH 2 O CO 3 125 500 1.25 0.08 CH 2 CH 2 Br 125 500 1.25 0.3 cyclohexyl 500 62.5 2.5 2.5 p-CH 2 Cl 2 H 5 CH 3 62 500 1.25 0.3 CH 2 CCsCH 62 500 0.6 0 , 16 CH2CH2OCH3 1000 2.5 0.6

CgHg 62.5 500 0.9 <0.1 6-methoxy 62 250 2.5 0.3 naph thylmethyl1 Detection of synergism between sodium carboxymethylclavulanate and cephalosporins against Staphylococcus aureus Russell.

Table XIII

Cephalosporin MIC (, µg / ml)

Cephalosporin Cephalosporin alone + 1 µg / ml sodium carboxymethylclar, ________ vulanate

Cephaloridine> 0.156

Cephalothin 2.5 0.31

Cefazoline> 0.6

Cephalexin> 10 2.5

Cephradine> 10 2.5

Cefuroxime 2.5 2.5

Cephamandol> 10 2.5 Detection of Synergism with Sodium Carboxymethyl Clavulanate and Penicillin against Staphylococcus aureus Russell.

Table XIV

Penicillin MIC (µg / ml)

Penicillin Peniqillin + 10 µg / ml ε16Ω6 * sodium carboxymethyl ____clavulanate

Ampicillin> 500 0.6

Amoxycillin 500 0.6 61 145543

Table XIV continued

Carbenicillin 250 5

Ticarcillin 250 5

Sulbenicillin 62.5 5

Epicillin> 500 1.25

Cyclocillin> 500 2.5

Benzylpenicillin> 500 2.5 Detection of synergism between ampicillin and the benzyl ester of clavulanic acid against various bacteria.

Table XV

MIC (µg / ml)

Ampicillin Benzyl Ester Ampicillin + 5, ug / ral Benzyl Ester Only '

Staphylococcus aureus Russell 125 62 <0.03

Klebsiella A 500 250 0.23

Escherichia coli JT4> 4000 250 4000 * * MIC = 250 µg with 20 mg / ml synergist.

Detection of synergism between ampicillin and the methyl ester of clavulanic acid against various bacteria.

Table XVI

...... '' I - ". -'Ll · ·· I M - MIC (yug / ml)

Ampicillin Methyl Ester Ampicillin + 5 µg / ml Methyl Ester Alone alone S taphylococcus aureus Russell 250 31-62 <0.5

Proteus C889> 500 125 8

Escherichia coli JT 39> 500 125 16 62 145643 Detection of synergism between ampicillin and the phthalidyl ester against various bacteria.

Table XVII

MIC (yug / ml)

Ampicillin Phthalidyl x Ampicillin + 5 µg / ester alone ml phthalidyl ester

Staphylococcus aureus Russel 500 62 1/6

Klebsiella E70> 500 125 3-6.2

Escherichia coli JT4 62> 4000 2000 * ............. I "-" '.M' * ·· "'*' —- 1 1 ii ... if i Μ--. - 1 · * any of the isomers.

Detection of synergism between ampicillin and the phenyl ester against various bacteria.

Table XVIII MIC (µg / ml)

Ampicillin Phenyl Ester Ampicillin + 5, µg / __alene alone ml of phenyl ester / S taphylococcus aureus Russell 62.5 62 <0.1

Klebsiella E7Q 500 62 6

Escherichia coli JT4> 4000 62 250