WO1994021647A1

WO1994021647A1 - Diclavulanate salt with a diamine and process of preparation

Info

Publication number: WO1994021647A1
Application number: PCT/GB1994/000482
Authority: WO
Inventors: Michael Allen Cook; Julian Stanley Harber; Victor Witold Jacewicz; Neal Ward
Original assignee: Smithkline Beecham Plc
Priority date: 1993-03-18
Filing date: 1994-03-11
Publication date: 1994-09-29
Also published as: AU6212194A; GB9305565D0

Abstract

A diclavulanate salt with a diamine, of formula (I), wherein R1, R2, R3, R4 and R5 are substituents; and n denotes an integer from 1 to 3 in the form of solvate of the salt with an organic solvent of with water. The invention also provides a process for the use of such salts in the purification of clavulanic acid.

Description

DI.CU_.VULANATE SALT WITH A DIAMINE AND PROCESS OF PREPARATION

The present invention relates to novel alkylenediammonium diclavulanate derivatives, to processes for their preparation and to their use as intermediate compounds for the preparation of clavulanic acid and of pharmaceutically acceptable alkali salts thereof, such as potassium clavulanate.

Clavulanic acid is a known compound of the following structure:

This compound as well as its salts and esters are active as inhibitors of β- lactamase enzymes produced by gram-positive and gram-negative microorganisms which degrade β-lactam antibiotics and confer antibiotic resistance on the organism. Hence clavulanic acid and its salts are used in pharmaceutical preparations in order to inhibit the inactivation of b-lactam antibiotics. Commercial preparations contain a stable potassium salt of clavulanic acid (clavulanic acid itself being rather unstable) in combination with amoxycillin trihydrate.

Clavulanic acid and its salts were first disclosed in GB 1,508,977. However, the processes described therein for the preparation of clavulanic acid described therein are time-consuming and is based on exacting purifications by means of various chromatographic methods. The salts of clavulanic acid are obtained by binding the clavulanic anion present in the filtrate of the fermentation broth on an anionic exchange resin, subsequent elution of the clavulanate anion therefrom by means of an electrolyte, desalting the obtained eluate, passing the latter through another anionic exchange resin and a subsequent chromatographic elution therefrom by means of an electrolyte, repeatedly desalting the obtained eluate and removing the solvent. The process requires the use of preparative chromatographic columns, which represents a considerable investment expenditure; in addition, the applicability on a large scale is limited. A further drawback of this process is the fact that many of its stages are carried out in an aqueous medium in which clavulanic acid is very unstable. GB 1,543,563 discloses a modified fermentation process, wherein the pH of the medium is maintained within the range from 6.3 to 6.7, which results in an increased yield of the desired compound. The salts of clavulanic acid such as potassium clavulanate are obtained from lithium clavulanate by double exchange. In the process for the preparation of clavulanic acid and its pharmaceutically acceptable salts disclosed in EP-0-026044. the methods of purification by means of exchange resins are largely avoided. The process describe therein is based on the preparation of the t-butylamine salt of clavulanic acid which is prepared by treating the extract, preferably the ethyl acetate extract, containing crude clavulanic acid, which was prepared according to the process as described in GB 1,508,977, with t- butylamine in an organic solvent such as a ketone, followed by the conversion of the isolated t-butylamine salt of clavulanic acid to clavulanic acid or a pharmaceutically acceptable salt thereof.

Clavulanic acid is normally prepared by the fermentation of a microorganism which produces clavulanic acid, such as various microorganisms belonging to various Streptomyces strains such as S. clavuligerus NRRL 3585, S. jumoninensis NRRL 5741, S. katsurahaman s IFO 13716 and Streptomyces sp. P 6621 FERM P2804 e.g. as described in JP Kokai 80-162993. The resulting aqueous broth may be subjected to conventional purification and concentration processes, for example involving filtration and chromatographic purification, such as disclosed in GB 1508977 and JP Kokai 80-62993, before extraction of the aqueous solution with an organic solvent to yield a solution of crude clavulanic acid in the organic solvent. Solvents suitable for the extraction are organic alkyl alkanoyl esters such as ethyl acetate, ketones such as methyl isobutyl ketone or acetone, aliphatic alcohols such as butanol, or mixtures thereof, a preferred solvent being ethyl acetate. The obtained extract in the organic solvent, such as the ethyl acetate extract, may also be further subjected to purification such as de-watering and/or a treatment with activated charcoal to eliminate a further quantity of impurities.

Novel compounds of clavulanic acid have been discovered which are useful as intermediates in processes for purification of the above-described clavulanic acid extracts. Also improved processes for isolating clavulanic acid from such extracts have been discovered.

According to a first aspect of this invention, a diclavulanate salt with a diamine, of formula (I) is provided:

(I)

wherein the substituents R\, R2, R3 and R4 denote; a hydrogen atom, a straight chain or a branched chain Cj.g alkyl group, an arylalkyl group wherein the alkyl group is a methyl or ethyl group and the aryl group is a phenyl group, which is optionally substituted by an N-alkyl or N,N-dialkyl group wherein the alkyl groups are Cj .4 alkyl, or; R , R2, R3 and R4 jointly independently denote a cyclic alkylene ring having 3 to 6 methylene groups, one of these groups being optionally substituted by an oxygen or a sulphur atom or by an amino group and R5 denotes a hydrogen atom, or a methyl group; and n denotes an integer from 1 to 3; in the form of solvate of the salt with an organic solvent or with water. The solvates of the salts (I) may be solvates in which the solvating solvent is an organic solvent such as an organic alkyl alkanoate ester such as ethyl acetate, ketones such as acetone etc, or hydrates in which the solvating solvent is water. Such solvates are found to be stable forms of the salts (I), that in these salts may. be readily isolated as these solvates and kept for a convenient time, or handled, without excessive decomposition of the salt molecule itself or loss of solvating solvent.

The solvates may be of various stoichiometries of clavulanic acid : solvent, but ceπain solvates have a ratio clavulanic acid : solvent of >1 : 1, for example approximately 2 : 1. The overall stoichiometry of such solvates is consequently clavulanic acid : diamine : solvent 2 : 1 : ca. 1. Preferred solvates, of which the stability can be demonstrated by inter alia easy formation, are solvates with organic alkyl esters such as ethyl acetate, ketones such as acetone, and hydrates. Examples of such solvates include those formed between up to one molecule of ethyl acetate or acetone, and one molecule of the salt

(I), i.e including two molecules of clavulanic acid. Suitable salts are those in which the diamine is an N,N'-disubstituted symmetric ethylenediamine having an alkyl chain of medium length, e.g. Cj.g, for example, wherein R] and R3 denote an ethyl or an isopropyl group, R2 and R4 denote a hydrogen atom and n denotes 1. A preferred diamine is N,N'- diisopropylethylenediamine. Suitable solvates of salts (I) are:

NN'-diisopropylethylenediamine diclavulanate ethyl acetate solvate,

NN'-diisopropylethylenediamine diclavulanate acetone solvate, and

NN'-diisopropylethylenediamine diclavulanate hydrate.

According to a further aspect of this invention a process is provided in which a solution of clavulanic acid or a labile derivative thereof, in an organic solvent, is reacted with a diamine of formula (II):

(II) wherein R\, R2, R3 R4 R5 and n are as defined above with reference to formula (I) to form a salt of formula (I) in the form of a solvate as described above.

Preferably the clavulanic acid is used in the form of the free acid, but suitable labile derivatives include silyl esters.

The organic solvent in which the reaction takes place may suitably be a solvent used to extract clavulanic acid from the aqueous solution obtained from the fermentation process as described above. A preferred solvent is ethyl acetate.

If the initial source of the clavulanic acid is a broth resulting from ^• fermentation of a clavulanic acid-producing microorganism, such as those mentioned above, then to obtain a solvent extract of a suitable concentration of clavulanic acid for use in this process it may be desirable not to extract the broth itself, but to at least remove some of the suspended solids in the broth, e.g by filtration prior to extraction. It is also desirable to pre-concentrate the aqueous solution of clavulanic acid obtained in fermentation, so that for example the aqueous solution of clavulanic acid is several times more concentrated in clavulanic acid than the starting broth, for example pre- concentrated to a concentration of ca. 10 - 25 g/L clavulanic acid.

Suitable pre-concentration processes include absorption of the clavulanic acid onto an anion exchange resin, followed by elution of the clavulanic acid therefrom with an aqueous solution of an electrolyte such as sodium chloride, and optionally de¬ salting. It is also preferred to acidify the aqueous solution, e.g the broth or the pre- concentrated aqueous solution prior to solvent extraction, e.g to pH 1 to 3, e.g around pH 1.5 to 2.5. It is also preferred to dry or de-water the organic solvent extract prior to formation of the salt (I), e.g to less than 6g/L of water, although as discussed below, the presence of small quantities of water in the solvent may be advantageous in achieving crystallisation of the solvate. A suitable concentration for the clavulanic acid or its labile derivative in the organic solvent is at least 1.0 g/L, for example in the range 1.0 to 4.0 g/L of clavulanic acid. It may be advantageous to further concentrate the solvent extract, for example by evaporation, to a concentration of clavulanic acid in excess of this, e.g greater than 10 g/L, e.g. greater than 20 g/L. The solvates of salts (I) may be formed in the process of the invention by solvation of the salts of formula (I), formed by reaction between clavulanic acid or the labile derivative thereof and the diamine (II), with the organic solvent in which the reaction is carried out, or as a hydrate with water present as an impurity in this solvent. Alternatively the process may be carried out in a non-solvating solvent, which is admixed with a solvating solvent.

Suitable and preferred diamines (II) are those described above, especially N,N'-diisopropylethylenediamine. The diamine (II) can be used either as such or in the form of a solution in an organic solvent, such as the above-described extraction solvents, for example acetone or ethyl acetate. For the preparation of the salt (I) of the clavulanic acid preferably at least one equivalent of the selected diamine (II) per mole of clavulanic acid should be used. The solvates of the salt (I) are generally insoluble in organic solvents, and if they are formed by a reaction which takes place in an organic solvent they generally precipitate out. The presence of a small quantity of water in the organic solvent in which the salt (I) is formed may be advantageous in achieving crystallisation of the solvate of the salt (I), for example the solvent may suitably contain around 0.25-0.6 g/L of water as it is believed that such a quantity of water may assist in crystallisation. If the solvate of the salt (I) is to be isolated in a solid form it may suitably be filtered off, and is preferably then washed with an organic solvent, which may be the same organic solvent that the reaction to prepare salt (I) is carried out in, for example ethyl acetate. Alternatively the solvate may be washed with a different solvent to that in which the salt is prepared, for example acetone in the case of an ethyl acetate solvate, although this may lead to formation of a solvate of the solvent used for washing. The solvent-wet solvate may then be dried, e,g in vacuo or may be further used wet.

Additionally or alternatively the solid solvate as filtered off may be redissolved in a solvent, such as water, or an alcohol such as methanol, or a water: acetone mixture, and then reprecipitated by admixing the solution with an organic solvent, for example acetone (thereby increasing the proportion of acetone if the solvate has been redissolved in water : acetone) or an alkyl alkanoate ester such as ethyl acetate. It is preferred to redissolve the solvate in the minimum of solvent and then admix the solution with an excess of the organic solvent. The reprecipitated solvate may then be collected, e.g. by filtration and optionally washed and dried as above.

In the process of the invention, the reaction between the clavulanic acid or its labile derivative and the diamine (II) may be carried out at around ambient temperature, e.g. ca. 20°C, but preferably the reaction is carried out at a temperature lower than ambient, e.g.-15 to +15°C, e.g 0 to 15°C, e.g. 0 to 10°C suitably 0 to 5°C. The use of such lower temperatures may advantageously result in a higher yield of the solvate. The use of temperatures lower than ambient appears to be of advantage generally in improving yield in processes in which salts (I) are prepared, whether these salts are obtained in a solvated or non-solvated form.

Therefore in a further aspect of this invention, an improved process for the preparation of a salt of formula (I) as defined above in a non-solvated form is provided, in which a solution of clavulanic acid or a labile derivative thereof in an organic solvent, such as the solvent extract obtained in the above-described manner, is reacted with a substituted diamine of formula (II) as defined above to yield a substituted diammonium diclavulanate salt of general formula (I) as defined above, wherein the reaction is carried out at a temperature lower than ambient.

Suitably this last-described process is carried out at a temperature of 0 to 15° C, e.g. 0 to 10°C, e.g. 0 to 5°C.

The solvated salts of formula (I) may be used as intermediates in the preparation of substantially pure clavulanic acid or pharmaceutically acceptable salts of clavulanic acid, because these solvates can be obtained in a substantially pure form, from which impurities in the clavulanic acid solution, e.g. in the above- mentioned extracts, have been substantially removed. Such pharmaceutically acceptable salts may be prepared from these solvates of salts (I) or the non-solvated salts (I) by double decomposition. By "pharmaceutically acceptable salts of clavulanic acid" herein is meant salts of clavulanic acid with a pharmaceutically acceptable cation.

Therefore according to a further process of this invention, a pharmaceutically acceptable salt of clavulanic acid is prepared by reacting a solvate of a clavulanic acid salt of formula (I), as defined above, with a salt precursor compound of a pharmaceutically acceptable cation.

Preferably the precursor compound is a compound of the cation with a counter anion which can exchange with clavulanate anion to form a pharmaceutically acceptable clavulanate salt of the cation.

Suitably the cation is a metal cation. Suitable metals include alkali metals, particularly potassium so that potassium clavulanate is formed which is preferred for its stability.

Suitable counter anions include carbonate, hydrogen carbonate, hydroxide or C\.\Q alkanoate, such as butanoates, pentanoates, hexanoates, heptanoates, octanoates, and alkyl-substituted forms of these particularly 2-ethylhexanoate, a preferred salt precursor compound being potassium 2-ethylhexanoate. Alternatively the counter anion may be present in the form of an anionic resin.

The reaction between the solvate of the salt (I) and the salt precursor may be carried out in solution or suspension, for example in an organic solvent or organic solvent: water mixture. Typically the solvate and the precursor may be respectively made up in separate solutions or suspension and mixed. A suitable solvent for the solvate of salt (I), particularly for ethyl acetate solvates, is an isopropanol: water mixture, for example having an isopropanol: water ratio between the inclusive ranges 50:1 and 1:50.

Suitably the solvent: water mixture may be made up first, and the solvate of salt (I) then dissolved in the mixture.

A preferred solvent for the salt precursor, particularly for potassium 2- ethylhexanoate, is isopropanol. Preferably a stoichiometric excess of the salt precursor, e.g. potassium 2-ethylhexanoate, over the salt (I) or solvate is used.

The product pharmaceutically acceptable salt of clavulanic acid may then be obtained in the form of crystals separating from the reaction mixture and may be collected by, conventional procedures.

In this last-describes further process of the invention, the reaction between the solvate and the salt precursor may be carried out ambient temperature, e.g. ca 20°C, but preferably the reaction is carried out at a temperature lower than ambient, e.g. 0 to 15°C, e.g. 0 to 10°C, suitably 0 to 5°C. The use of such lower temperatures may advantageously result in a higher yield of the salt product.

The use of temperatures lower than ambient appears to be of advantage generally in improving yield in processes in which pharmaceutically acceptable salts of clavulanic acid are prepared by reaction between a salt (I) and a salt precursor, whether the salt (I) is in a solvated or non-solvated form.

Therefore in a further aspect of this invention a pharmaceutically acceptable salt of clavulanic acid is prepared by reacting a clavulanic acid salt of formula (I) as defined above in a non-solvated form, with a salt precursor compound of a pharmaceutically acceptable cation at a temperature below ambient.

Suitable and preferred precursors and reaction conditions are analogous to those described above. A suitable temperature is between 0 to 15°C, e.g. 0 to 10°C, e.g. 0 to 5°C. Although in the processes described above the salt (I) or the solvate of the salt

(I) may be isolated in a separate solid form, (I) e.g. by filtration, it is also possible to extract the salt (I) or solvate from the organic solvent in which it is formed, it this solvent is substantially imiscible with water, into a separate aqueous phase of water or a water : organic solvent mixture to form a concentrated aqueous solution of the solvate in the phase. The solvate may then for example be reprecipitated from this concentrated solution analogously as described above by admixing this concentrated solution with a organic solvent such as acetone.

The invention will now be described by way of non-limiting examples. Example 1: Solvent Extraction.

An ion-exchange eluate (70L), ex-clavulanate production, was acidified inline to pH 2.0 with 25% v/v sulphuric acid and continuously extracted into ethyl acetate (85L). A solvent to aqueous feed ratio of 1.2: 1.0 was used to maximise the solvent titre. The resulting rich solvent extract was chilled to 2°C and dewatered via a dewatering centrifuge and then dried with magnesium sulphate (200g per 15L rich ethyl acetate). The dried rich ethyl acetate was then passed down a CPG carbon column (5.0L) at a flow rate of 600 ml min. The carbon treated rich solvent was immediately concentrated from 85L to approximately 20L on a cyclic evaporator.

Solvent extract moisture levels (%v:v):

post dewatering centrifuge: 2.86 post magnesium sulphate: 1.86 concentrate: 0.26

Solvent extract clavulanic acid concentration:

rich solvent assay: 6,020 μg/ml

Concentrate assay: 25,700 μg/ml

Solvent extraction yield = 42.2%

Example 2: Solvent Extraction.

Rotary vacuum filter filtrate (150L), ex-clavulanate production, was acidified inline to pH 1.6 with 25% v/v sulphuric acid and continuously extracted into ethyl acetate (160L). A solvent to aqueous feed ratio of approximately 1:1 in conjunction with the lower pH (cf pH 2.0) was employed to maximise the solvent titre. The resulting rich solvent extract was chilled to 3°C and dewatered via a dewatering centrifuge and then dried with magnesium sulphate (200g per 15L rich ethyl acetate). The dried rich ethyl acetate was then passed down a CPG carbon column (5.0L) at a flow rate of 600 ml/min.

The carbon treated rich solvent was immediately preconcentrated from 150L to 1 IL on a cyclic evaporator, and yielded a potency of 9.815 μg/ml. Preconcentrate (5.0L) was further concentrated on a rotary evaporator to give a final concentrate titre of 20,007 μg/ml.

Solvent extract moisture levels (% v/v): post dewatering centrifuge: 2.86 post magnesium sulphate: 2.24 preconcentrate: 0.06

Solvent extract clavulanic acid concentration:

rich solvent assay: 750 μg/ml concentrate assay: 20,007 mg/ml

Solvent extraction yield = 40%

Example 3: N,N'-Diisopropylethylenediammonium diclavulanate.

1.0 litre of concentrated clavulanate rich ethyl acetate extract from Example 2, at a titre of 20,000μg/ml was adjusted to 6 g/L water by addition of de-ionised water.

N,N'-Diisopropylethylenediamine was added to the extract, maintained at 5°C, with rapid stirring, at the rate of 9.0 ml over ten minutes. The resulting slurry was stirred, at 5°C for a further 30 minutes, then filtered, using standard Whatman filter paper. The wet cake was washed with acetone, in order to remove the mother liquors. The wet cake was redissolved, using 20mls water. Acetone at the rate of 400 mis over 5 minutes was added, and thus the N,N'-diisopropylethylenediammonium diclavulanate was precipitated. The product was filtered off and washed with acetone, then dried under vacuum at 20°C overnight with a nitrogen bleed. The product was obtained as fine needles which were clumped and granular.

Product weight = 29.4 lg. Assay = 65% as parent free acid ("pfa"). Yield = 95.6%

Example 4: N,N'-Diisopropylethylenediammonium diclavulanate. 1 litre of concentrated clavulanate rich ethyl acetate extract from Example 2, at a titre of 20,000 μg/ml was adjusted to 6g/L water by addition of de-ionised water. N,N'-Diisopropylethylenediamine (9.0ml) was added over a period of ten minutes, maintaining the temperature at 5°C, while stirring rapidly. The resulting slurry was stirred, at 5°C for a further 30 minutes, then filtered, using standard Whatman filter paper. The product was filtered off and washed with fresh ethyl acetate (50ml), then dried under vacuum at 20°C overnight with a nitrogen bleed. The product was obtained as fine needles which were clumped and granular. Product weight = 30.46g. Assay = 65.0% as pfa. Yield = 99.0%

Example 5: N,N'-Di_sopropylethylenediammonium diclavulanate.

2.1 ml of the diamine were mixed with 6.8 ml fresh ethyl acetate. This mixture was slowly added to 2 litres of concentrated clavulanic acid rich ethyl acetate extract preconcentrate from Example 2 previously diluted back to a titre of 2300μ g/ml with fresh ethyl acetate, maintaining the temperature at 5°C, with rapid stirring. The slurry was stirred for a further hour with chilling. The wet cake was redissolved, using 4.6ml water. Acetone, 92ml over 5 minutes, was added and thus the N,N'- diisopropylethylenediammonium diclavulanate was precipitated. The product was filtered off and washed with acetone, then dried under vacuum at 20°C overnight with a nitrogen bleed. The product was obtained as very fine needles.

Product weight = 3.59g. Assay = 70%. Yield = 68.2%

Example 6: N,N'-Diisopropyiethylenediamπ_onium diclavulanate conversion to potassium clavulanate.

25g of the diamine salt produced in Example 3 was dissolved in 25ml of water and 475 ml isopropyl alcohol ("IPA") was added, with stirring. To this was added 2N potassium 2-ethyl hexanoate, at the rate of 38.0ml over 15 minutes, maintaining the temperature at 5°C. The slurry was stirred, at 5°C for a further 30 minutes. The product was filtered off and washed with 2 x 20mls IPA then 1 x 50 mis acetone. The wet cake was dried under vacuum overnight with a nitrogen bleed, at 20°C. The product was obtained as large needles. Product weight = 11.2g. Assay = 83%. Yield = 57%

Yield, Extract to potassium salt = 54.7%

Example 7: N,N'-Diisopropylethylenediammonium Diclavulanate conversion to Potassium Clavulanate 25g of the diamine salt produced in Example 4 was dissolved in 25ml of water and 475ml IPA, with stirring. To this was added 2N potassium 2-ethyl hexanoate, at the rate of 38.0ml over 15 minutes. The slurry was stirred, at 5°C for a further 30 minutes. The product was filtered off and washed with 2 x 20mls IPA then 1 x 50 mis acetone. The wet cake was dried under vacuum overnight with a nitrogen bleed, at 20°C. The product was obtained as large needles.

Product weight = 11.6g. Assay = 83%. Yield = 59.2% Yield, Extract to potassium salt = 58.6%

Example 8: N,N'-Diisopropylethylenediammonium Diclavulanate conversion to Potassium Clavulanate

3.0g of the diamine salt produced in Example 5 was added to 3ml of water and 57ml of IPA, with stirring. In order to fully dissolve the amine salt, it proved necessary to add a further 0.5ml of water. To this was added 2N potassium ethyl hexanoate, at the rate of 4.3mls over 10 minutes. The slurry was stirred, at 5°C for a further 30 minutes. The product was filtered off and washed with 2 x 10ml IPA then

1 x 20 ml acetone. The wet cake was dried under vacuum overnight with a nitrogen bleed, at 20°C. The product was obtained as nucleated rods. Product weight = 1.17g. Assay = 83%. Yield = 46.2%

Yield, Extract to potassium salt = 31.5%

Example 9: N,N'-diisopropylethylene diamine salt of clavulanic acid - ethyl acetate solvate. An ethyl acetate extract of clavulanic acid was obtained as above and dried over magnesium sulphate. A portion of the dried extract (360ml, clavulanic acid content ca. 12,800 μg/ml) was cooled in an ice bath to 0 to 5°C and treated with a mixture of N,N'-diisopropylethylene diamine (3.6ml) and ethyl acetate (50ml) over ca. 10 minutes with stirring. The mixture was stirred at 0 to 5°C for a further 30 minutes, the needle crystals collected by filtration, washed with ethyl acetate (2 x 50ml) and dried in vacuum. (Yield 7.29g)

The infra-red spectrum (nujol mull) showed strong bands for bound ethyl acetate at 1725 and 1253 cm" , and the nmr spectrum (in deuterated dimethyl sulphoxide) indicated the presence of ca. 0.5 mole ethyl acetate.

Example 10: Recrystallisation as the ethyl acetate solvate: i) The ethyl acetate solvate of example 9 (l.Og) was dissolved in methanol (1.0ml) and cooled in ice. The solution was diluted with ethyl acetate (20ml) to give needle crystals. These were collected, washed with ethyl acetate and dried. (Yield 0.9g)

The infra-red spectrum showed the presence of strong bands at 1725 and 1253 cm^~l, confirming that the salt had been recovered as the ethyl acetate solvate. ii) The ethyl acetate solvate of example 9 (l.Og) was suspended in acetone (10ml) and treated with water, dropwise, until a clear solution was obtained (0.77ml was required). The solution was cooled in ice and treated with ethyl acetate (10ml) to give needle crystals. These crystals were collected, washed with ethyl acetate and dried. (Yield 0.9g)

The infra-red spectrum showed the presence of strong bands at 1725 and 1253 cm^"-, confirming that the salt had been recovered as the ethyl acetate solvate.

Example 11: Preparation of the acetone solvate.

The ethyl acetate solvate of example 9 (4.0g) was dissolved in water (6.0ml) at room temperature and diluted with acetone (120ml). The crystals were collected, washed with acetone, and dried in high vacuum. The nmr spectrum of the product showed the presence of ca. 0.5 mole of acetone. This was confirmed by elemental analysis:

Found C 53.95 H 7.39 N 9.43%

Requires for 0.5 mole acetone C 53.99 H 7.38 N 9.33%

Requires for non-solvated salt C 53.13 H 7.06 N 10.33

Example 12: Conversion of ethyl acetate solvate to potassium clavulanate The ethyl acetate solvate (2.70g) was dissolved in a mixture of IPA (5.0ml) and water (2.15ml) at ca. 5°C. The solution was diluted with IPA (25ml), and treated with an excess of potassium-2-ethyl hexanoate (6.0ml of a 2.16M solution in IPA) with cooling in ice. The mixture was stirred at 0 to 5°C for 30 minutes, the crystalline product collected, washed with IPA (2 x 10ml), then with acetone (20ml) and dried. (Yield 1.50g, 74%).

Example 13: Conversion of acetone solvate of N,N'-diisopropyI- ethylenediamine diclavulanate to hydrate.

Exposure of the acetone solvate obtained in Example 11 to a moist atmosphere appeared to result in conversion to a hydrate containing 0.5 moles of solvated water per molecule, shown by elemental analysis and manifested by a change in the X-ray diffractogram.

Elemental analysis:

Found: C 52.06, H 7.24, N 10.07% Requires for non-hydrated salt: C 53.13, H 7.06, N 10.33% Requires for 0.5 moles water: C 52.26, H 7.13, N 10.16%

X-ray diffractogram.

The following peaks in the X-ray diffractogram (expressed in degrees 2 Q) are characteristic of the solvates, the ethyl acetate solvate being included for comparison:

w = weak m = moderate s = strong

Ethyl acetate solvate: 6.37m 8.48s

Acetone solvate: 8.72s 9.77m

HHvvddrraattee:: 66..8899ww 88..7700mm 99..8833ss 10.45s

Claims

Claims:

1. A diclavulanate salt with a diamine, of formula (I):

(I) wherein the substituents R , R2, R3 and R4 denote a hydrogen atom, a straight chain or a branched chain C_j.g alkyl group, an arylalkyl group wherein the alkyl group is a methyl or ethyl group and the aryl group is a phenyl group, which is optionally substituted by an N-alkyl or N,N-dialkyl group wherein the alkyl groups are C\ . 4 alkyl, or; Rj, R2, R3 and R4 jointly independently denote a cyclic alkylene ring having 3 to 6 methylene groups, one of these groups being optionally substituted by an oxygen or a sulphur atom or by an amino group and R5 denotes a hydrogen atom, or a methyl group; and n denotes an integer from 1 to 3; in the form of solvate of the salt with water or an organic solvent.

2. A solvate of a salt (I) according to claim 1, which is a hydrate in which the solvating solvent is water, or in which the solvating solvent is an organic solvent.

3. A solvate according to claim 1 or 2 in which the solvating solvent is an alkyl alkanoate ester or a ketone.

4. A solvate according to claim 3 in which the solvating organic solvent is ethyl acetate or acetone.

5. A solvate according to any one of the preceding claims in which the salt (I) is one in which the diamine is an N,N'-disubstituted symmetric ethylenediamine having an alkyl chain of length C\.(..

6. A solvate according to claim 5 in which R\ and R3 denote an ethyl or an isopropyl group, R2 and R4 denote a hydrogen atom and n denotes 1.

7. A solvate according to claim 6 in which the diamine is N,N'- diisopropylethylenediamine.

8. A solvate according to claim 7 being selected from: NN'-diisopropylethylenediamine diclavulanate ethyl acetate solvate, NN'-diisopropylethylenediamine diclavulanate acetone solvate, or NN'-diisopropylethylenediamine diclavulanate hydrate.

9. A process in which a solution of clavulanic acid or a labile derivative thereof, in an organic solvent, is reacted with a diamine of formula (II):

(ID wherein R\, Ri, R3, R4, R5 and n are as defined above with reference to formula (I) to form a salt of formula (I) in the form of a solvate as claimed in any one of claims 1 to 8.

10. A process according to claim 9 in which the clavulanic acid is used in the form of the free acid and the solvent is ethyl acetate.

11. A process according to claim 9 or 10 in which the initial source of the clavulanic acid is a broth resulting from fermentation of a clavulanic acid-producing microorganism from which at least some of the suspended solids in the broth have been removed prior to extraction.

12. A process according to claim 11 in which the aqueous solution of clavulanic acid obtained in fermentation is pre-concentrated prior to extraction to a concentration of ca. 10 - 25 g L clavulanic acid.

13. A process according to claim 1 1 or 12 in which the aqueous solution of clavulanic acid is acidified to pH 1 to 3 prior to solvent extraction.

14. A process according to any one of claims 9 to 13 in which the concentration of the clavulanic acid or its labile derivative in the organic solvent is at least 1.0 g/L.

15. A process according to claim 14 in which the concentration of clavulanic acid or its labile derivative is in the range 10 to 25 g/L.

16. A process according to any one of claims 9 to 15 in which the diamine is N,N'-diisopropylethylenedιamιne.

17. A process according to any one of claims 9 to 16 in which the organic solvent contains about 0.25-0.6 g/L of water.

18. A process according to any one of claims 9 to 17 in which the solid solvate is redissolved in a solvent and then reprecipitated by admixing the solution with an organic solvent.

19. A process according to any one of claims 9 to 18 in which the reaction between the clavulanic acid or its labile derivative and the diamine (II) is carried out at a temperature lower than ambient.

20. A process for the preparation of a salt of formula (I) as claimed in any one of claims 1 to 8 in a non-solvated form, in which a solution of clavulanic acid or a labile derivative thereof in an organic solvent, is reacted with a substituted diamine of formula (II) as defined above to yield a substituted diammonium diclavulanate salt of general formula (I) as defined above, wherein the reaction is carried out at a temperature lower than ambient.

21. A process according to any one of claims 9 to 20 which is a purification process for crude clavulanic acid.

22. A process in which a pharmaceutically acceptable salt of clavulanic acid is prepared by reacting a solvate of a clavulanic acid salt of formula (I), as claimed in any one of claims 1 to 8, with a salt precursor compound of a pharmaceutically acceptable cation.

23. A process according to claim 22 in which the salt precursor compound is potassium 2-ethylhexanoate.

24. A process according to claim 23 in which the reaction between the solvate and the salt precursor is carried out at a temperature lower than ambient.

25. A process in which a pharmaceutically acceptable salt of clavulanic acid is prepared by reacting a clavulanic acid salt of formula (I) as claimed in any one of claims 1 to 8 in a non-solvated form, with a salt precursor compound of a pharmaceutically acceptable cation at a temperature below ambient.

26. A process according to claim 25 which is carried out at a temperature between 0 to 15°C.

27. A process according to any one of claims 9 to 26 in which the salt (I) or solvate is extracted from the organic solvent in which it is formed, into a separate aqueous phase of water or a water : organic solvent mixture to form a concentrated aqueous solution of the solvate in the phase, from which the solvate is then reprecipitated by admixing this concentrated solution with a organic solvent.

28. A pharmaceutically acceptable salt of clavulanic acid when prepared by a process as claimed in any one of claims 22 to 27.