Process for obtaining clavulinic acid esthers

© 4) Process for purifying clavulanic acid salts

The invention relates to a process for purifying clavulanic acid salts from impurities, antibiotics with a broad spectrum of action.

Sc knows the use of some penicillins and 5 semi-synthetic cephalosporins, resistant to betalactamase, with synergistic betalactamase inhibitors for penicillins and cephalosporins. Certain actinomycete cultures have been described as producing inhibitory substances of betalactamases, substances that act synergistically with penicillins or cephalosporins, for example, the cultures described in Patent, England no. 1363075, as well as those described by Hata et al. in 15 The Journal of Antibiotics, 25,473 (1972) and by Umezawa et al. In Journal of Antibiotics, 26.51 (1973).

Also known are processes for obtaining antibiotics using 20 strains of the species Sireplomt / ces clavuligerus, for example, Streplomyces clavuligeriis NRRL 3585, which produces deacetoxycephalosporin C (Patent, RSB, No. 65113). 25

With these known antibiotics numerous attempts have been made for use in Lera potica, either as such or in the form of salts or other derivatives, more or less purified, in various forms of administration, the efficacy of which is conditioned. and the presence of antibiotic impurities.

These beta-lactamase inhibiting antibiotics have disadvantages, either because they do not have an obvious effect on the spectrum of the other antibiotic associated. mixed, either they have no practical utility in the clinic.

The present invention removes the disadvantages shown by separating the impurities from the clavulanic acid salts, contacting a clavulanic acid salt solution with an anion exchange resin, in the form of CP, until saturation, then washing with water, and then eluting with a solution. 0.2 M sodium chloride, the combined fractions are concentrated in vacuo, and the excess NaCl solution is removed chromatographically, then again eluted with a 1% solution of n-butauol in water, the active fractions thus obtained it had concentrated and dried by freezing.

Examples of carrying out the process according to the invention are given below.

Example 1. One liter of filtrate of. culture, which gives a 53% inhibition at the 1/2500 dilution in the beta-lactamase inhibition sample, is percolated downstream by a column with a diameter of 2.5 cm x 15.2

LAW PRICE 84.20

; cm u plated with resin Permitted orc hyssop, FF IP ISRA 62) in CP form. After | passage of the culture filtrate through the column

300 300 ml dc of distilled water is passed to wash the column. The active beta-lactamase inhibitor is eluted with a 0.2 M sodium chloride solution. Collect fractions (20 ml), which are analyzed at a final dilution of 1/2500 based on the sample dc inhi | betalactamazone. Active fractions! Gather and concentrate in vacuo to a final volume of 20 ml. From this solution, the exclusion salt by gel chromatography is eliminated, on a column with a diameter of 3.8 cm loaded with a bed of

Biorad 'Biogel P 2 on a height of 24! cm, after which the column is eluted with a solution of 1% normalbutanol in water. The active actions determined by

J sample for beta-lactamase inhibition. Sodium chloride, eluted after clavulanic acid,

Ί is determined using a silver nitrate solution <j. The combined active fractions are concentrated and dried by freezing.

One liter of culture filtrate, treated as shown above, from 0.45 g of a crude solid preparation of clavulanic acid, | who has a f_SFL 0.92 pg / ml. This solid preparation is stored at -20 ° C while waiting for further purification.

The crude solid preparation of clavulanic acid can also be obtained in the following way:

I The culture liquid is collected and clarified by continuous centrifugation, the mycelium being removed. Out of 150 1 fermentation liquid, 120 1 clarified culture liquid is obtained. This filtrate; gives a 58% inhibition in the beta-lactamase inhibition sample at a dilution of 1/2500. The filtrate is cooled to 5 ° C, after which 40 L of normalbutanol is added.

; The resulting mixture is stirred and 25% sulfuric acid is added to it until the pH reaches 2.0. The mixture / acidified is stirred for a further 10 min.

χ before the phases are separated by centers; fleeting. The aqueous phase is removed. 0.5% charcoal is added to normalbutanol extract

GSX clouds, and the mixture is stirred for 15 minutes. They are removed and removed

I filter the coal using a diatomite soil as a filtering aid.

An equal volume of deionized water is added to the normalbutanol extract and the resulting mixture is stirred, adding a solution of 20% sodium hydroxide at this time. until the pH equals value; 7.0. The phases are separated by centrifugation, the normalbutanolic phase being removed. The aqueous phase is concentrated under reduced vacuum to a volume of 800 ml, then dried, 1 by freezing. Thus 35 g of 1 tr is obtained from a crude solid product of clavulanic acid ί with one I_6o of 1.3 pg / ml in the beta-lactamase inhibition sample. This solid product is stored in a dry state at - 20 ° C, awaiting further purification.

For partial purification, the procedure is as follows:

A culture filtrate was extracted with solvent to give a solid preparation which was further purified by chromatography on dc ion exchangers using diethylaminoctylcellulose. This solid preparation (10 g) is dissolved in 20 ml of distilled water and the solution obtained is passed through a cellulose column DE 52 with the dimensions of

3.8 x 50.8 cm, previously equilibrated with a 0.01 M phosphate buffer with pH = 7.5. The column is eluted with a 0.1 M sodium chloride gradient, sodium chloride in 0.01 M phosphate buffer with pH = 7.5 is fed into a mixing chamber containing 1 1 of 0.0 phosphate buffer solution , M with pH = 7.5 - chamber which, in turn, is linked to the column. Collect fractions (10 ml), which are analyzed for the activity of beta-lactamase inhibition at a dilution of 1/2500. The fractions are also examined in terms of antibacterial activity, by the method of plaque cavities, using nutrient agar plates seeded with Klebsiella aerogenes. The fractions having the highest inhibition activity of betalactamase and which have inhibition zones in the sc cavity sample are combined and concentrated, after which the salt is removed from them on a Biorad Biogel P 2 column. These fractions prove that they contain ciavulanic acid for analysis by paper chromatography and thin layer chromatography.

Example 2. A culture filtrate containing 300 µg / ml of clavulanic acid is acidified using a flow mixing system, after which it is extracted with normalbutanol, the clavulanic acid is re-extracted into water at neutral pH. The cooled culture filtrate (5 ... 10 ° C) is pumped into a flow-mounted mixer, to which is added a sufficient amount of 6% (v / v) nitric acid, to maintain a pH at value of 2.0 ± 0, l. The acidified filtrate is passed at a flow rate of 41 / min. by a glycol-cooled plate heat exchanger to maintain a temperature in the range of 2-5 ° C. The pH is controlled and adjusted in a continuous cell, before the filtrate passes through a 3-stage counter current separator. Normal saturated butane (at about 5 ° C) is pumped at 3 1 / min. in the counter current separator. The aqueous solution coming out of the counter current spectrum is eliminated at the channel. The entrained water is removed from the butanol stream coming out of the counter current separator, using a liquid / liquid centrifugal separator. Bu'5

7Ț320 the tanol is collected in a stainless steel vessel, provided with a cooling jacket and kept at a temperature of about 5 ° C. From this vessel, remove 40 1 aliquots, which are intensively mixed with 2 L of chilled water (5 ° C), saturated with normalbutanol. The pH of this mixture is adjusted to 6.8 ± 0, l, using a 20% sodium hydroxide solution. This mixture of aqueous extract and butanol sc is introduced into a liquid / liquid centrifugal separator with a pumping rate of 2 1 / min. From 1800 1 culture filtrate a 90 1 volume aqueous phase is recovered, which contains 39% of the clavulanic acid present in the culture filtrate. 15 1 of the aqueous extract is adjusted from a concentration in total solid products of 2% to 8%, by adding 60 g of sodium chloride / liter, then dried by atomization. The operating conditions are as follows: power supply 2 1 / h, atomizer voltage 170 v; heating regulation Take values ​​to be ... 7; inlet temperature 150 ° C; outlet temperature 80 ° C. The dry product, with a total weight of 1 kg, contains fi2% of the clavulanic acid present in the feed solution. The other 75 l of aqueous extract are concentrated by ultrafiltration.

The operating mode provides for the recirculation of the retention of a stainless steel tank, provided with a cooling system, the cooling valve being adjusted so as to obtain a pressure drop along the 40 at 25 membranes. The temperature is maintained at 2 ... 5 ° C, and the pl-l at 6.8 ± 0.1, by adding 2 N hydrochloric acid to the extent necessary. The volume is reduced to 34 1, they contain 72% of the clavulanic acid present in the feed liquid. The aqueous concentrate is stored at about 5 ° C, adjusted to a concentration of 8% solid products, then dried by atomization, as shown above. The dry material contains 75% of the clavulanic acid present in the atomizer supply. The total dry product by atomization, obtained from the 90 1 aqueous extract, contains 69.4 g of clavulanic acid, which represents 72% of the clavulanic acid present in the atomizer feed and 21% of the clavulanic acid present in the 1800 1 filtrate. culture.

A concentrated leextract (fi 1 from ultrafiltration) containing 10 g of clavulanic acid, determined by the beta-lactamase inhibition sample, is flowed at a flow rate of 1 1 / h through a column with dimensions of 5,1x61 cm, filled with changing resin. of anions, in the form of chlorine.

After that, the column was washed with 21 deionized water, before being eluted with a sodium chloride gradient. The gradient contains 41 of 1.4 M sodium chloride, which supplies a tank with stirring, containing 4 1 of a 0.7 M solution of sodium chloride, a tank which, in turn, is linked to a tank with stirring, containing 41 deionized water, the latter tank being connected by means of a pump to the column above. The column is eluted with a flow rate of 2.5 ml / min., Collecting fractions of 25 ml. The collected fractions are analyzed by the beta-lactamase inhibition method. The active fractions (bearing the numbers 140 ... 230) come together, and then evaporate under vacuum until close to dryness. Thereafter, ethanol (500 ml) was added and the solid product was filtered off after vigorous shaking. The ethanolic extract was evaporated in vacuo to dryness on a rotary evaporator, then redissolved in deionized water (40 ml). The formed solution is passed on a column with dimensions 10.1 cmxfil cm filled with Biorad Biogel P 2, after which the column is eluted with a 1% normalbutanol solution. Collect fractions (two to 5 ml) that are analyzed for the activity of beta-lactamase inhibition, at a final dilution of 1/2500. Tests are performed to determine the sodium chloride dc content of the fractions at a dilution of 1/25, using a silver nitrate solution. Those fractions containing clavulanic acid free of sodium chloride are combined, then concentrated, by evaporation under reduced pressure, to a volume of 20 ml and finally dried by freezing. 4.8 g of sodium clavulanic acid salt are obtained (Ι_5θ about 0.06 pg / ml).

Also in solid form, the sodium salt of clavulanic acid can be further separated as follows:

A solid preparation, partially purified from clavulanic acid (500 mg), is loaded onto a microcrystalline cellulose column with dimensions of 2.5 cm χ 50.8 cm. The effusion phase is a 4: 1: 5 v / v normalbutanol / water solvent mixture. The column is operated at 4 ° C, collecting fractions of 4 ml. The fractions are tested for the presence of clavulanic acid by pipetting on filter paper and spraying with Ehrlich reagent (bright red spot) or with triphenyltrazole reagent (red blade). These stain tests confirm by analysis of the inhibition of betalactamase at the dilution of 1/250. The active fractions are combined and dried under vacuum on a rotary evaporator. The solid product obtained is dissolved in a small volume of distilled water, after which it is dried by freezing. A white solid preparation of clavulanic acid sodium salt (40 mg) is obtained, which has a_6o with a value of 0.08 g / ml in the beta-lactamase inhibition sample.

Esters can be obtained from the sodium salt of clavulanic acid in different ways, as follows:

Preparation of methyl ester 5

19.8 mg of the sodium salt of clavulanic acid (I) is dissolved in 0.5 ml of dried dimethylformamide, the solution obtained being treated with 0.25 ml of methyl iodide. After being allowed to stand at room temperature for an hour and a half under anhydrous conditions, the solvents are removed in vacuo. The residue is purified by liquid phase chromatography on silica gel:

<img file="RO77320A_D0001.tif" />

eluting with ethyl acetate to obtain clavulanic acid methyl ester (II) as a colorless oil (Rf = 0.38; red color with spray reagent, triphenyltetrazole broth), which has the following properties:

Analysis: Found: C, 50.49; H, 5.43; N, = 6.29; theoretical formula C₉H_{and i}N0₆ - ask for: C = 50.70; H, 5.20; Ν = 6.57 / λ max (methanol): no absorption at 215 mm. max. (film): 3300 - 3600 (wide), 1800, 1750, 1695 cm^-1. Approximate first-order NMR (CDCfj): 2.49 (wide S, 1 changed to D₂O), 3.05. (d, 1, J = 17.5Hz), 3.54 (dd, 1 J = 17.5 Hz, J_of= 2.5 Hz), 3.84 (S, 3); 4.24 (d, 2 J = 7 Hz), 4.93 (dt, 1,.] =

<img file="RO77320A_D0002.tif" />

f ^X\_=/ch₃oh co₂ch₃ (Π) = 7 Hz, J_of= l, 5 Hz) 5.07 (d, 1 J = l, 5 Hz), 5.72 (d, 1, J = 2.5 Hz). Molecular weight (determined by mass spectrography): 213.0635. Molecular weight calculated on the basis of formula C₉tI₁₁NO₅= 213,0637.

Thin layer chromatographic analysis of methyl ester shows a single zone in each of the following solvent systems: butanol / ethanol / water elution phase 4: 1: 5 v / v R / = 0.75; isopropanol / water 7: 3 v / v R / = 0.95; ethyl acetate / ethyl alcohol 8: 2 v / v R / = 0.87. The zones are detected by bio-autography, using Klebsieila aerogenes with the addition of benzylpenicillin (synergism system).

Preparation of p-nitrobenzyl ester

XfLOH

-N

-N

CO₂Na \ C (b, CH, -S -NO, (I)

By treating the sodium salt of clavulanic acid (I) with p-nitrobenzyl bromide in dry dimethylformamide, a colorless oil is obtained, after liquid chromatography, which crystallizes from a mixture of 25 chloroform-ether to obtain the ester. -nitroPreparation of benzyl (III) benzyl ester of clavulanic acid (III) in the form of fluffy white needles, having a melting point in the range 111 ... 112 ° C, which, after recrystallization, has a melting point in the range 117.5 ... 118 ° C.

<img file="RO77320A_D0003.tif" />

(I)

An impure sodium salt of 3- (beta-hydroxyethylidene) -7-oxo-4-oxo-1- azaCH acid ₍CH_ofOH_ / Y (h) bicyclo [3, 2, 0] beptan-2-carboxylic (I) (considered to contain at most 55 mg of material

In dry dimethylformamide (0.64 ml), treat with benzyl bromide (0.18 ml). The solution is kept at room temperature (approximately 17, 18 ° C) for 3 hours under anhydrous conditions. The reaction mixture is fractionated on silica gel, eluting with ethyl acetate, to obtain, practically in pure form, the benzyl ester of 3- (betahydroxyethi li den) -7 οχο-4-οχο-1-azabic clo [3 2,0] heptane-2-earboxylic (IV) (63 mg) as a colorless oil. Ir spectrum (film)

1800, 1745, 1695 cm NMR spectrum

(CDC 1₃), 2.25 (s, 1, being changeable with D₂C), 3.05 (d, 1, J = 17 Hz), 3.51 (dd 1, J = 17 Hz), 4.24 (d, 2, J = 7.5 Hz), 4.92 15 (dt, 1, j = 7.5 Hz, J₃= l, 5 Hz) 5.15 (d, 1, J = l, 5 Hz), 5.24 (s, 2), 5.71 (d, 1, .1 = 2.5 Hz), 7, 45 (s, 5).

The benzyl ester can also be obtained as follows: 20

1 The culture filtrate was evaporated in vacuo, using an evaporator in a rising film, to a volume of 5! The concentrated solution is dried by freezing. The 300 g of solid product thus obtained contains 25 3 g of sodium salt of clavulanic acid, determined by the enzyme inhibition sample. The solid product is suspended in 900 ml of dried dimethylformamide, followed by the addition of 150 ml of benzyl bromide. The resulting mixture was stirred for 2 h at room temperature, then diluted with 1 L of ethyl acetate. The reaction mixture is filtered and the filtrate is concentrated to the smallest possible volume. The oily residue is extracted with another liter of ethyl acetate and the extract filtered. The filtrate is concentrated again and the resulting oily residue is passed on a silica gel column immersed in cyclohexane, with dimensions of 7.6 cm X 35.5 cm. The column was eluted with cyclohexane to remove the benzyl bromide, after which the elution solvent was changed with ethyl acetate, collecting fractions of about 20 ml. Fractions- 45 are tested for the presence of benzyl ester of clavulanic acid, by pipetting pc plates of glass coated with silica gel, for thin layer chromatography and spraying and the chlorinated spray reagent of 2, 3, 5-thiophenyltetrazole (TTC) . The fractions having intense red spots with this reagent are further analyzed by thin layer chromatography on silica gel plates, using as 55 solvent a mixture of chloroform-ethyl acetate in 8: 2 ratio and developing the plates by spraying. with the TTC reagent. Clavulanic acid benzyl ester appears at R / = 0.31 at 22 ° C. Fractions containing_θθthis ester is combined and concentrated to a volume of 15 ml, after which this concentrated solution is further chromatographed on a silica gel column with dimensions 3.8x40.6 cm with a solvent ₆₅ consisting of a mixture of 8: 2 chloroform / ethyl acetate. Collections of 15 ml fractions are collected, which are tested for the presence of benzyl ester, as shown above. Those fractions containing the ester are concentrated to a final volume of 8 ml, after which this concentrated solution is purified by column chromatography, on a silica gel bed, with dimensions of 2.5x40.6 cm, using as a solvent u a mixture of ethyl acetate / cyclohexane 8: 2. The collected fractions are combined and evaporated in vacuo to give pure benzyl ester in the form of an oil, in a quantity of 160 mg.

A solid product, dried by atomization (3.3 kg), containing 69.4 g of clavulanic acid, determined by the enzyme inhibition sample obtained as above, is slaked in 5.5 1 dimethylformamide, after which 500 is added. ml benzyl bromide. After the resultant mixture was stirred for. 2 h, at room temperature, add 12 1 of ethyl acetate, then remove the solids by filtration. The filtrate was separated in vacuo to give an oily residue (212 g). The residue sc passes on a column loaded with a silica gel bed with dimensions dc 10.1 x33 cm in cyclohexane. The column was eluted with 12 L of cyclohexane to remove excess bromide from the bands. After this, the wort is changed, eluting with ethyl acetate and collecting fractions of about 500 ml. These fractions are tested for clavulanate of the bands, by pipetting on silica gel plates, for thin-layer chromatography, the plates being sprayed with 2, 3, 5-triphenyltetrazole (TTC) chloride spray reagent. The fractions giving intense red spots are further examined by thin layer chromatography on silica gel, by eluting a solvent with 8: 2 chloroform / ethyl acetate mixture and developing the plates by sputtering with TTC spray reagent, Fractions 5, 13 contains the main part of the ester and they are combined and evaporated in vacuo to an oily residue (79.3 g). This preparation is chromatographed on a silica gel column, dc dimensions 10.1x45.7 cm, using a solvent of 8: 2 chloroform / ethyl acetate as the solvent, as described above, and, after concentration , results in an oil in quantity of

45.9 g, which has a purity of 62%, estimated based on the MRI spectrum. This product is finally chromatographed on a column with Sephadex LH 20, with dimensions of 7x45.7 cm, using as a solvent a mixture of 1: 1 skyloxane / chloroform. After selecting and concentrating the fractions, a core oil is obtained (27, 6 g), which proved to be the benzyl ester of clavulanic acid, with a purity of 95%, determined by spec.

MRI troscopy. (Sephadex LII 20 assigns a hydroxypropyl derivative of Sephadex Q 25).

The culture filtrate (150 1) with pH = 7.0 containing 16.2 g clavulanic acid (as dc sodium salt), determined, by the enzyme inhibition sample, is stirred with 5 kg anion exchange resin in the form chlorine, Amberlite type A.26, for one hour, at room temperature. After that, the resin is filtered and the filter is reanalyzed, indicating that 6.4 g of clavulanic acid were removed. The resin is washed with 20 1 deionized water, after which. followed by a wash with 201 acetone and 10 1 dimethylformamide (DMF). After filtration, the resin is suspended in 2.3 1 dc mixture of DMF / 0.2 M sodium iodide. To this suspension is added 200 ml of benzyl bromide, then stirred vigorously. After your room temperature has been clear of 16 b, ethyl acetate (2 L) is added to this suspension, then the resin is filtered and the washing water (ethyl acetate) of the resin is combined with the filtrate. The extract is concentrated to a small volume and chromatographed on a column of silica gel with dimensions of 7.6x45.7 cm, using a solvent of a mixture of acetyl acetate / cyclohexane 8: 2. The fractions containing clavulanate of bands are collected by pipetarc on silica gel plates, for thin layer chromatography, is developed by spraying with TTC reagent. The selected fractions are concentrated to a volume of 20 ml, which is chromatographed on a silica gel column with dimensions of 3.8x45.7 cm, using as a solvent a mixture of chloroform / ethyl acetate 8: 2. The collected fractions are combined, after which evaporates to give a colorless oil (440 mg), which is clavulanated by 90% purity bands, determined by NMR spectroscopy.

An aliquot part of the aqueous re-extract to the butanolic extract of the culture filter, obtained as in Example 1, is dried by freezing. A portion of dc 24 g of the solid product obtained contains 0.96 g of sodium salt of clavulanic acid, determined by enzyme inhibition samples. This solid product is suspended in 75 ml of dry dimethylformamide, 75 ml of bromide dc is added to the suspension. The mixture was stirred for 2 h at room temperature.

Thereafter, the suspension is diluted with 500 ml of ethyl acetate and the resulting mixture. sc filters. The filtrate obtained is concentrated to an oily residue on a rotary evaporator, under vacuum. This oily residue is passed on a column of silica gel, with dimensions of 5.1 x35.6 cm, in cyclohexane, the benzyl bromide is eluted from the column, after which the solvent is changed, eluting with ethyl acetate and colec30 continuing. - fractions of 10 ml each. Fractions containing benzyl ester of clavulanic acid are collected. Further purification is performed by column chromatography. Finally, 220 mg of pure benzyl ester is obtained.

Preparation of the sodium salt of clavulanic acid

-N \ Z \

but it₂oh / ~ '11 cO, CH, C₆H_r>

-N \ / (IV)

-&lt;

'COssNa (O cii₂Oh

II

Benzyl clavulanate (IV), practically pure (281 mg), in ethanol (25 ml), containing sodium hydrogen carbonate (82 mg), is hydrogenated in the presence of 10% Pd / C (90 mg) for 25 min, at room temperature and atmospheric pressure. The catalyst is removed by filtration, washed with water and ethanol, and the combined washing water and filtrate are evaporated, under reduced pressure, at room temperature. The obtained semi-solid residue is triturated with acetone, then filtered and washed with ether to give sodium clavulant (135 mg).

Hydrolysis of clavulanic acid methyl ester to clavulanic acid 2.17 mg of clavulanic acid ester is dissolved in 0.1 ml of methanol and treated with 0.208 ml of sodium hydroxide solution (0.0482 N). After one hour at room temperature, the reaction mixture contains several products. Thin layer chromatographic analysis shows that one of the major components has an R / identical with that of the sodium salt of clavulanic acid, the color reactions and the biological analysis confirming that this component is the sodium salt of clavulanic acid. Reduced ester conversion to clavulanic acid is observed when 1 mg / ml of the compound is incubated at 37 ° C in a 0.05 phosphate buffer at pH = 7. The reaction is trace⁵⁰ rite by paper chromatography (bioautography system). Using the butanol / ethanol / water system to track the reaction progress over a period of 2 h, it is observed that

.13

-.·!

-1 the methyl ester area at R / 0.79 decreases in size, while the clavulanic acid area at R 0.12 increases in size.

Preparation of the clavulanal of pivalouloximelil 5 in a solution of pivalate dc brommethyl (0.37 g) in dry dimethylformamide (5 ml), with stirring, sodium clavulanate (0.49 g) is added. After 2 li at room temperature, the reaction mixture is treated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (20 ml). The mixture was separated into two layers: the aqueous layer was separated, washed with water (20 ml) and dried over sodium sulfate. The dried solution was evaporated, leaving product 15 followed as a pale yellow oil (500 mg). NMR spectrum (CDC1₃),

1.26 (s, 9). 3.13 (d, 1, .1 = 17 Hz), 3.62 (dd,

1, J = 17 Hz). ^ = 2.5 Hz) 4.3 (d, 2, J = = 7.5 Hz), 5.0 (d, t, 1, J = 7.5 Hz, J₀= 20 = 1.5 Hz), 5.16 (d, 1, J = l, 5 Hz), 5.79 (d, 1, J = 2.5 Hz), 5.92 (s, 2); infrared spectrum (liquid film), / Mn-lactam CO 1808 cm^-1, ester C = 0 1760 cm^-1.

The preparation of il-phallidic esters and clavulanic acid in a solution of 3-bromphthalide (0.43 g) in dry dimethylformamide (5 ml), with stirring, sodium clavulanate (0.5 g) is added, followed by the solution. leave at room temperature for 2 h. After that, the solution is treated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (30 ml), being shaken intensely by shaking. The unpaved layer se. I wash water (20 ml), dry (on sodium sulfate) and evaporate to give a pale yellow gum. The two diastereomeric esters are separated using liquid chromatography, under high pressure, on a silica gel column with dimensions 40 cm χ 10 mm; the column is eluted with ethyl acetate with a flow rate of. 3 ml / min. The first phthalide ester (retention time 7.15 min.) Crystallizes from ethyl acetate in the form of needles with a melting point 102 ° C and has the following ir spectrum (mule sludge): thyme-lactam C = O 1790 cm^-1, ester C = O 1755 cm¹, NMR spectrum (CD3COCD3); 3.14 (d, 1, J = 17.5 Hz) 3.76 (dd, 1, J = 7.5 Hz, 52 = 2.5 Hz), 4.25 (d, 2, .1 = 7 , 5 IIz), 5.0 (d, t, 1,. ^ = 7.5 IIz, J2 = l, 5 Hz), 5.4 (s, 1, .J = l, 5 Hz), 5, 82 (d, 1, J = 2.5 Hz), 7.6 (s, 1), 8.06 (m, 4); molecular weight (by mass spectrometry): 331.0696 corresponds to the formula CloH13NO7 (calculated 331.0692), the second diastereoisomer (retention time 8.85 min.) has the following spectrum ir (methylene chloride solution): beige -lactam C = 1800 c^-1, C = O ester 1780 cm^-1, NMR spectrum (CDClg) 2.42 (wide s, 1, possible to change with D2O). 3.12 (d, 1, .1 = 18 Hz), 3.60 (dd, 1, J₁= 18 Hz, J₂= 2.5 IIz) 4.30 (d, 2, J = 7.5 Hz), 5; 0 (dr, 1, ^ = 7.5 Hz, J₂= l, 5 Hz), 5.12 (d, 1, .1 = 1.5 Hz), 5.76 (d, 1, J = 2.5 Hz), 7.52 (S, 1), 7 , 85 (m, 4).

<img file="RO77320A_D0004.tif" />

-N \ z

V

Z = &lt;

ch₂Oh

H ·>

C0₂N / A

A

-/ \

-N +

<img file="RO77320A_D0005.tif" />

CH, OH \₌&lt;.com

Ζ π ¹ ζθ \

O = C0 —— Z 2 = 0. It was born on silica gel, eluting with ethyl acetate / lixane (2: 1); a product is obtained in the form of an oil with an ir spectrum (film) 1800, 1690 cm '¹. Molecular weight, (mass spectrometry)

Preparation of the nonyl clavulanate Sodium clavulanate (44 mg) in dry dimethylformamide (2 ml) is treated with nonyl iodide (76 mg), then left at room temperature for two hours.

The solution sc evaporates, and the residue)? E .fracțio

ί] j

j ί

ΐ ΐ

?

i = 325 1890, which corresponds to formula C₁₇I1₂₇NO₅ (calculated value = 325 1889).

Preparation of clavulanic acid

The benzyl clavulanate (100 mg) in ethanol (5 ml) is hydrogenated in the presence of 10% Pd / C (30 mg) for 45 minutes at ambient temperature and atmospheric pressure. The catalyst is filtered off, washed with ethanol, after which the combined washing water and filtrate are evaporated in vacuo to give clavulanic acid as an unstable viscous oil (58 mg). NMR spectrum (C₅D_SN): 3.05 (d, 1, J = 18 Hz), 3.60 (dd, 1, Jj = 18 Hz, J₂= 2.5 Hz), 4.75 (d, 2, J = 7.5 IIz), 5.58 (6t, 1, J = 7.5 Hz), 5.66 (S, 1), 6, 0 (d, l, J = 2.5 Hz),

Preparation of melil clavulanal

Clavulanic acid (130 mg) in ethanol (10 ml) is treated with excess diazomethane in ether. After 2 min at room temperature, the reaction is complete (analysis by thin layer chromatography). The solution was evaporated in vacuo, and the residue was purified by chromatography on silica gel, eluting with ethyl acetate. The fractions containing methyl clavulanate are combined and evaporated to give a clear oil (104 mg).

Preparation of methyl clavulanal

The clavulanic acid (200 mg) has kidney ions (5 ml) sc cool and is stirred at 0 ° C. Methanol (0.5 ml) was added and thereafter dicycloliexyldicarbodiimide (206 mg), after which the reaction mixture was stirred at room temperature overnight. The suspension is filtered and the filtrate is evaporated in vacuo to give crude methyl clavulanate. The crude product is purified by chromatography on silica gel, eluting with ethyl acetate, to obtain a clear oil (140 mg).

Preparation of phenyl clavulanate

The clavulanic acid (100 mg) in acetonitrile (5 ml) sc cools and is stirred at 0 ° C. Sc adds phenol (0.94 g) and dicyclohexyldicarvodiimide (100 mg) to the solution, after which the reaction mixture is stirred at room temperature overnight. The suspension is filtered and the treatment is evaporated. The residue was fractionated on silica gel, eluting with a mixture of ethyl acetate-hexane (1: t) to give phenyl clavulanate (70 mg). Ir spectrum (film): 1800, 1770, 1690 cm^-1. NMR spectrum (CDCI₃): 2.18 (wide s, 1), 3.06 (dd, 1, J = 17 Hz), 3.54 (dd, 1, Jj = 17 Hz, J₂= 2.6 Hz), 4.29 (d, 2, J = 7.5 Hz), 5.1 (dt, 1, .1 = 7.5 Hz, J₂= l, 5 Hz) 5.29 (d, ί, -J-1.5 Hz), 5.76 (dd, 1, Jj = 2.6 Hz, J₂= = 0.9 Hz), 7.35 (m, 5). Molecular weight (mass spectrometry) = 275 0777, which corresponds to formula C_l4H₁₃NO₅ (result from calculation 275 0794).

Preparation of the clavulanal dc 2, 2, 2-trichlorel l

Sodium clavulanate (221 mg) is suspended in dry tetrahydrofuran (5 in), with stirring, at 0 ° C. The suspension above²⁵ add, for 20 minutes, trichlorelil chloro-formoformate (211 mg) in dry tetrahydrofuran. (1 ml). The mixture is allowed to reach room temperature, with stirring, during the night. The suspension is filtered and the filtrate³⁰ evaporate under vacuum. The residue was chromatographed on silica gel, eluting with a mixture of ethyl hexane (2: 1) to give the desired product as an oil. Ir spectrum (film) 1800, 1760, 1690 ctn¹. NMR spectrum (CDC1₃): 1.56 (wide S, 1), 3.07 (dd, 1, J_IT= 17.5 Hz, J₂= 0.7 Hz), 3.56 (dd, 1, J₄= 17.5 Hz, J_z= 2.5 Hz), 4.24 (d, 2,, 1 = 7.5 IIz), 4.69 (d, 1, J = 12 IIz), 4.92 (d, 1, 1- = 12 Hz), 5.02 (dt, 1,. ^ = 7.5 Hz, J₂= 1.3 Hz), 5.19 (d, 1, .J = = 1.3 Hz), 5.73 (dd, 1,. ^ = 2.5 Hz, J₂= = 0.7 Hz). Molecular weight (mass spectrometry) = 328 9621, which corresponds₄₅ of formula C₁₀H₁₀NO₅Cl₃ (calculation result 328 9625).

ί

-N

A \ _yCH₂0H / “\ z

I

CO₂N / A

Cico

- * / o

CH₂0H / - &lt;11.

—^N\ / / I / 1 / CO-O-CO-OR

<img file="RO77320A_D0006.tif" />

I

CORI

Preparation of sodium clavulanate

Benzyl clavulanate (840 mg) in ethanol (30 ml) and water (5 ml) is hydrogenated in the presence of 10% Pd / C (267 mg) and

I ϊ!

and

Ί bicarbonate, sodium (244 mg), for 25 minutes, at room temperature and atmospheric pressure. The catalyst is filtered, washed with water and ethanol, after which the washing water and filtrate are combined and evaporated as microcrystalline needles (565 mg). Recrystallized from water-acetone gives a needle-like product which, after drying on phosphorus pentoxide, in vacuo for 24 hours, presents the following analysis: C = = 41.01; 40.86; H, 3.77; 3.64; N, 5.68; 5.51, ir spectrum (IiBr disk): 1785, 1700, 1620 cm-¹. NMR spectrum (D₂O): 3.06 (d, 1, J = 18.5 Hz), 3.57 (dd, 1, J = 18.5 Hz, J₂= 2.5 Hz), 4ml5 (d, 2, J = 8 Hz), 5.3 (HOD), 4.9 (in), 5.71 (d, 1,, 1 = 2.5 Hz).

Determination of the sodium salt of clavulanic acid by thin layer chromatography

Pipette (5 µl of a 1 mg / ml solution) solutions of preparations dc salt. clavulanic acid sodium on glass plates, coated with a 0.25 mm silica gel layer. Chromatograms are performed at 22 ° C, using as the elution phase the normalbutanol / ethanol / water mixture 4: 1: 5 v / v. The chromatographic plates are dried at 40 ° C, after which the sodium salt of clavulanic acid is localized by bioautography on agar plates containing 6 mg / ml benzylpenicillin and seeded with Klebsiella aerogenes (synergy system). The surface of the agar is covered with a fine filter cloth, before the plates with the thin layer chromatogram are placed on this surface. After being allowed to stand for 15 ... 30 minutes for humidification and diffusion, the plate with the thin layer chromatogram is lifted using the filter cloth and the plate. The agar is incubated overnight at 30 ° C to develop inhibited growth areas. The R / value of the sodium salt of clavulanic acid in the above solvent is about 0.38. Two spray reagents, Ehrlich reagent and triphenyltelrazole chloride, are also used to develop the sodium salt area of ​​clavulanic acid. The first reagent consists of 300 mg of paradiinetylaniinobeuzalde.idide dissolved in 9 ml of. ethyl alcohol, 54 ml of normalbutanol and 9 ml of concentrated hydrochloric acid. By heating the plate with the chromatogram in the layer, thin at 120 ° C, for about 2 minutes, the sodium salt of the clavulanic acid appears as a bright red color. The triphenyltetrazole chloride reagent consists of a mixture of 1 vol of a 4% solution of this compound in methanol and 1 vol of methanolic sodium hydroxide solution. After spraying this reagent, the plates with the thin layer chromatograms are heated to 80 ° C. The sodium salt of clavulanic acid appears in the form of a red spot on a white background.

Antibacterial synergism between ampicillin and clavulanic acid esters

The results presented in table 1 demonstrate this synergism.

Table I

Antibacterial synergism between ampicillin and clavulanic acid esters against Klebsiella aerogencs rays

<td> 15</td><td>Pasha</td><td>Ampici- smooth one</td><td>Ampicillin + 5 µg / ml methyl acid ester clavulanic</td><td>Ampicillin + 5 µg / nyl benzyl acid ester clavulanic</td>

<td rowspan="2"> 20</td><td>A</td><td> .500</td><td> 1,9</td><td> 1,9</td>

<td>I 70s</td><td> 500</td><td> 3,9</td><td> 3,9</td>

<td></td><td> 62</td><td> 500</td><td> 3,9</td><td> 3,9</td>

²⁵ Both clavulanic acid methyl ester and clavulanic acid benzyl ester do not inhibit the development of the tested organisms, at a concentration of 100, µg / ml.

Antibacterial activity of clavulanic acid esters

At a 1/100 dilution of the broth left overnight, the CMI values ​​from Table 2 are obtained for some acid esters.

3·-¹ clavulanic against a number of organisms.

Table: t

Antibacterial aclivitcilca of some clavulanic acid slugs

<td rowspan="2">body</td><td colspan="4">CMI of some esters of clavulanic acid</td><td rowspan="2">| CMI *) of the sodium salt^! of clavulanic acid</td>

<td>.2 c CJ Λ u CJ UJ W</td><td>1 ; Nonyl ester j</td><td>Pivaloyl ester methyl oxide</td><td>11 tn CS H</td>

<td>Baciltae sublilis A Siupiuccocus aureus, Oxford Slafiloccocus aureus, Bussel fischerichia coli 10408</td><td> 250 62 125 125</td><td> 31 31 31 .250</td><td> 62 31 62 125</td><td> 125 31 15 125</td><td> 62 15 15 125</td>

*) The CMI of the sodium salt of clavulanic acid is closed for comparison; CMl rnaTÎ values ​​are due to the powerful ittueulâTi uîilifalc

Most desirable is that the clavulanic acid salts are pharmaceutically acceptable salts, such as ₆₅ they are the salts of sodium, potassium, calcium, mag19 nonzium, aluminum, ammonium and substituted ammonium, such as trimethylammonium, salts of benzatine, procaine and other similar salts, which are usually formed with penicillins or cephalosporins. The pharmaceutically unacceptable clavulanic acid salts are also included in the provisions of the present invention, as they are useful intermediates in the preparation of clavulanic acid esters, for example, lithium or silver salts of clavulanic acid may be reacted. with benzyl bromide, to obtain clavulanic acid benzyl ester, a useful substance.

The salts of clavulanic acid tend to be more stable than the parent acid per se, and thereby form a preferred aspect of the present invention. Particularly suitable salts of clavulanic acid include the sodium and potassium salts having the formulas I and Ia respectively:

-N (I) \ CI-IgOH /^=&lt;H

CO₂N / A

<img file="RO77320A_D0007.tif" />

\

CO₂K (Ia)

J s

The crystalline forms of such salts may comprise hydrating water.

Suitable esters of clavulanic acid include those esters which theoretically derive from alcohols, such as: methanol, ethanol, propanol, butanol, 2, 2, 2-trichlorethanol,

2, 2, 2-Lrifluorethanol, benzyl alcohol, p-nitrobenzyl alcohol, phenol, acetoxymethanol, pivaloyloxymethanol, 2-dimethylaminoethanol, as well as other conventional alcohols. Various esters of clavulanic acid are useful intermediates in certain processes for purifying clavulanic acid. Many esters of clavulanic acid are useful compounds due to their synergism. The activity of such esters could be due to the hydrolysis of the ester to the mother acid.

When used in the present invention, the term "ester" includes esters derived, theoretically, from an alcohol or thiol of the formulas ROH or RSII, wherein R is an organic residue. Suitable R groups comprise alkyl, alkenyl, alkynyl, aryl, arylalkyl, or other similar groups, each of which may be substituted; tweet if this is desired. In order not to increase the molecular weight beyond the national limits, the R groups should not normally contain more than 16 carbon atoms, more suitable at no more than 12 carbon atoms, and more suitable at most no more of 8 carbon atoms.

It is preferable that the R group is theoretically derived dc from a ROH alcohol or (more 4; less favorable) dc to thiol RSH, which are pharmaceutically acceptable. Suitable substituents that may be included in the R groups comprise halogen atoms, as well as alkoxy, hydroxy, g &lt;lower acyloxy groups, lower alkylamino groups, lower dialkylamino groups and the like. The term "lower means that the group contains up to 4 carbon atoms and preferably up to 4 dc carbon atoms. For urination, for example, R may be a methyl, ethyl, n-propyl, iso-propyl, linear or branched butyl group, pentyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, vinyl, allyl, butcnyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, heptyl cyclo, cyclohexyl, cyclohexadienyl, methylcyclopentyl, methylcyclohexyl, benzyl, benzhydryl, phenylethyl, naphthylethyl, phenyl, naphthyl, propynyl, tolyl, 2-chloroethyl, 2,2,2-trichloroethyl, 2,2,2-trichloroethyl , 2-trifluoroethyl, acetylmethyl, benzoylmethyl, 2-methoxyethyl, 2-dimethylaminoethyl, 2-diethylaninoethyl, 2-piperidinoethyl, 2-morpholinoethyl, 3-dimethylaniinopropyl, p-chlorobenzyl, p-methyloxibenzyl, p-niLobenzyl-p-brombenzyl, m-chlorobenzyl, 6-methoxynyl-p-2-methyl p-methoxyphenyl or any similar group, as well as those groups that are known from the penicillin or cefaiosporin technology to produce esters, which are known to be directly hydrolyzed in vivo to the parent antibiotic.

Esters that are directly hydrolyzed include esters of formulas V and VI, without being limited to the latter esters, wherein Aj is a hydrogen atom, an alkyl, aryl or aralkyl group, A₂ is a hydrogen atom or a methyl group, A. is an alkyl, aryl or aralkyl group, X is an oxygen or sulfur atom, Y is an oxygen or sulfur atom, and Z assigns a bivalent organic group. The esters of formulas V and VI, which release the clavulanic acid directly into the bloodstream after administration, include those esters, where Aj is an atom of. hydrogen, A₂ is a hydrogen atom or a methyl group, and A₃ is a methyl, ethyl, propyl, benzyl or phenyl group, as well as those esters in which X is an oxygen atom, Y is an oxygen atom, and Z is - CH_ZCH₂ -. CH: CH them,

OCH \ x \ (V)

Z 'ti z \ / or ^N\ Z \

z \ z \

OCH, ch₂Oh

Z^==&lt;H

CO.

It

OC X-CO-A, \ o-

<img file="RO77320A_D0008.tif" />

(VI)

XC = Y

When used in combination with the preceding formulas, the term "alkyl" comprises an alkyl group with up to 6 carbon atoms; Similarly, the term "aryl includes a phenyl, naphthyl, or phenyl group substituted with a substituent, such as a fluorine or chlorine atom, or a methyl or methoxy group, or a like group; when used in the present invention, the term "aralkyl" means an alkyl group substituted with an aryl group.

Particularly suitable esters of formulas V and VI include those esters having formulas VII and VIII below:

\ _Υ; Η₂οπ

Z \

-N \ I

CO-O-CH-O-CO-A,

<img file="RO77320A_D0009.tif" />

wherein A₄ is a hydrogen atom or a methyl group, A₅ is a methyl, hieributyl or ienyl group, and A_e is a hydrogen atom or a methoxy group. Many esters of clavulanic acid differ from the analogous esters of penicillins or cephalosporins, in that they exhibit a more pronounced tendency to hydrolyze to clavulanic acid under mild conditions. Thus, for example, simple alkyl esters, such as methyl ester, slowly hydrolyze to clavulanic acid in buffered water at pH = 7. Esters undergoing some hydrolysis under mild conditions are included in the formula IX:

A

<img file="RO77320A_D0010.tif" />

Z '

CH₂0H

H ilu who R₃ is a hydrocarbon group with 1 to 9 carbon atoms, optionally substituted by halogens, a lower alkoxy group, a hydroxyl, or basic groups in the form of salt, optionally, of formula NR₂R₃, wherein R_of is a hydrogen atom or a lower alkyl group, Il₃ is a hydrogen atom or a lower alkyl group, or is bonded to R_of so that NR_ofR₃forms a ring with 5 or 6 atoms. When used with reference to formula IX, the term "lower means that the group contains 1 ... 4 carbon atoms. Suitable R 1 groups include alkyl and aralkyl groups, optionally substituted by halogens, methoxy, hydroxy or NR groups₂R₃ in the form of a salt, wherein R₃ is a methyl or ethyl group, and R₃ is a methyl or ethyl group or is linked to R₂, in such a way that NR₂R₃is a pyrrolidinic, piperidine or morpholinic group. The alkyl groups Π, the most suitable are the groups with a linear chain with up to 6 carbon atoms, optionally substituted with a methoxy, hydroxy, (VII) group

<td>or</td><td>a group NR₂R₃ as dc salt,</td>

<td> 45 ^or</td><td>with a dc chlorine, bromine or iodine atom,</td>

<td>or</td><td>with an OC1 group₃ or CF₃.</td>

<td colspan="2">Esters of clavulanic acid that are de-</td>

Although useful as energetics, they are those esters that hydrolyze in mammalian tissues, especially in human blood, to give clavulanic acid or a salt thereof, because it is considered that clavulanic acid and its salts tend to be, to some extent, more useful as energetic agents than esters per sc. Many of the esters of formulas V ... IX are useful from this point of view.

Another particularly suitable group of esters made available by the present invention are those useful intermediates, which are directly transformed into clavulanic acid or a salt thereof, by chemical or biochemical methods, which are known from penicillin or cephalosporin technology. gentle enough not to degrade r ».isnr23 ί

£ Χ 'J reagent rings fccin-Iactamic labile to acids. The most suitable ester is that ester which can be decomposed by hydrogenolysis. Esters suitable for such a process include benzyl esters, 5 substituted benzyl esters, benzhydryl, substituted benzhydryl, trityl esters, and other similar esters. Benzyl ester has been found to be particularly useful in this respect, in general, the nature of any substituent in the esthetic half is unimportant, as long as it does not interfere with the hydrogenolysis reaction.

Esters of clavulanic acid can be prepared by esterification of clavulanic acid 15 or a salt thereof by conventional methods. Methods suitable for the formation of esters include the following methods: a) the reaction from a clavulanic acid salt and a compound with the forinula Q - R, 20 in which Q is an easily displaceable group and R is an organic group; b) the reaction between clavulanic acid and a diazocyan and c) the reaction between clavulanic acid and a ROH alcohol, in the presence of a condensing motor agent, such as carbodiimide or another similar agent.

Suitable clavulanic acid salts which can be reacted with compounds R - Q comprise alkali metal salts, such as sodium or potassium salts, or other conventional salts, such as silver salt. Suitable Q groups include those atoms or those groups known to be displaceable by carboxylic anions and these groups include chlorine, bromine atoms. and iodine, sulfonic acid esters such as OS, O groups₂CH₃ or O.SO₂C₆H₄CH₃, active ester groups, such as O.CO.H or O.CO.CF groups₃, such as 40 and other conventional groups that can be displaced with the help of nucleophile substances. The preceding reaction is normally carried out in an organic solvent with a relatively high dielectric constant, such as di-melylformamide, acetone, dioxane. tetrahydrofuran or other similar solvents, and at a moderate temperature, with a range from -5 to 100 ° C, usually between +5 and 3Q ° C, for example, at ambient temperature. Reaction of clavulanic acid with a diazocompound is a gentle method of forming alkyl esters, chili aryl or similar esters. The diazotation reaction can be carried out under ordinary reaction conditions, for example, at a moderate temperature in a conventional solvent. Thus dc. reactions are normally carried out in a temperature range between about -5 and 100 ° C, usually in_(I0range from 5 to 30 ° C, for example, takes the ambient temperature, Suitable solvents for this reaction are lower alkanols, such as, for example, methanol and ethanol, as well as solvents, such as tetrahydrofuran, dioxane and other similar solvents. Ethanol has proven to be a particularly useful solvent for this reaction.

The reaction of clavulanic acid with an alcohol or thiol, in the presence of a condensing promoter, typically has IOC in an inert organic solvent with a relatively high dielectric constant, such as acetonitrile. This reaction is usually carried out at room temperature or at a low temperature, for example, in the range of -10 to + 22 ° C, preferably in the range of -5 to + 18 ° C, for example, initially Take 0 ° C and then, by gradually heating up to about 15 ° C. The condensing promoter used is normally an agent that removes water from the reaction mixture. Suitable agents include carbodiimides, carbodiimidazoles, or other equivalent reactants. Dicyclohexylcarbodiimides have been shown to be particularly suitable as condensing promoter agents used in this process. To minimize the self-condensation of clavulanic acid, this reaction is usually performed. in the presence of a considerable excess of alcohol or thiol.

Other suitable methods of ester formation also include d) removing carbon dioxide atoms from a compound of formula X:

\ _ / CH₃OH / ^H ^ CO-O-CO-OR, (X) wherein R₄ is an inert organic group, and e) the reaction of a compound of formula X with an ROH alcohol (or less favorable with a thiol RSH). The carbon dioxide atoms can be removed from the compound of formula X spontaneously, during its preparation, or alternatively, by heating the compound of formula X in an inert solvent. Inert solvents suitable for this include ethereal solvents, such as diethyl ether, tetrahydrofuran, dioxane, and the like. In many cases, the compound of formula X decomposes spontaneously, even at low temperatures, for example at -5 ° C, to give an ester of formula:

=&lt;

-NCH_ofOH

II ^ COnRj wherein R₄ is an inert group within the definition of R. When the compound with t

ΐ

<img file="RO77320A_D0011.tif" />

formula X is to be reacted with an alcohol (or less favorable with a thiol), then this reaction is normally carried out in an inert solvent, such as an ethereal solvent, in the presence of an excess of alcohol ( or thiol), to prevent self-condensation of the clavulanic acid derivative. Such esterification methods are generally not as useful as those involving the reaction of a salt of clavulanic acid ro with a compound R-Q, as described above.

The compound of formula X may be prepared by the reaction between a clavulanic acid salt with the compound C1.CO.OR₄ or with a chemical equivalent equivalent to the latter. Normally, this reaction is carried out at a low temperature, for example, at a temperature no higher than 5^AC, and in an inert solvent, for example, ²⁰in diethyl ether, tetrahydrofuran, dioxane or other similar solvent. Most suitable is that the clavulanic acid salt used in this reaction is a lipophilic salt, so that it dissolves in the solvent, although less favorable sodium salt may be used, by suspension, but in the reaction medium.

The advantages of the present invention are that it allows efficient separation of impurities from clavulanic acid salts, valuable antibiotics, with a broad spectrum of action, with a synergistic capacity accentuated by penicillins and cephalosporins.