NZ270557A

NZ270557A - Process for the preparation/purification of clavulanic acid or salts thereof using tertiary octylamine

Info

Publication number: NZ270557A
Application number: NZ270557A
Authority: NZ
Inventors: Michael Allen Cook; Robert Bennett Wilkins
Original assignee: Smithkline Beecham Plc
Priority date: 1992-06-11
Filing date: 1993-06-07
Publication date: 1996-04-26
Also published as: NZ280385A; NZ270989A

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £70557 270557 Priority Date(s): Complete Specification Filed: .1.:.$'..%%.. ' Class: (?).. I Publication Date: P.O Journal Ho: NO thm provUKoM oI Mton as (1) th® N.Z. PATENT OFFICE 2 3 FEB 1995 RECEIVED E-XrJfcrrtOfi, —— NEW ZEALAND PATENTS ACT, 1953 COMPLETE SPECIFICATION Divided out of No.: 253248 Dated: 7 June 1993 "PROCESS FOR PREPARATION OF CLAVULANIC ACID" We, SMITHKLINE BEECHAM PLC a British Company of New Horizons Court, Brentford, Middlesex, England, hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- (followed by page la) PROCESS FOR THE PREPARATION OF CLA This invention relates to a novel process for the preparation and/or purification of clavulanic acid and pharmaceutical ly acceptable salts and esters 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 5. clavuligerus NRRL 3585,5. jumoninensis 10 NRRL 5741, S. katsurahamanus 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 15 organic solvent to yield a solution of crude clavulanic acid in the organic solvent GB 1508977 discloses inter alia that salts of clavulanic acid can be obtained by absorbing the clavulanate anion in filtered broth on to an anion exchange resin, eluting therefrom with an electrolyte, desalting the resulting solution, applying the desalted solution to a further anion exchange resin, chromatographically eluting 20 therefrom with an electrolyte, desalting the resulting solution and thereafter removing the solvent This process can be used to give acceptable yields of pure material but the use of resin columns involves significant investment and they can introduce limitations in large scale production operations. It would therefore be desirable to have an alternative procedure available that involved few resin utilizing stages. GB 1543563 discloses a process for the preparation of clavulanic acid salts via precipitation of lithium clavulanate. GB 1578739 describes various amine salts of clavulanic acid as pharmaceutical compounds. EP 0026044 discloses the use of the tertiary-butylamine salt of clavulanic acid as a useful intermediate in the preparation 30 of clavulanic acid. The salt has been disclosed in BE 862211, but only as a suitable ingredient for pharmaceutical formulations. PT.94.908 describes the use of tri-(lowcr alkyl)amine salts and the dimethylaniline salts of clavulanic acid in a purification process for clavulanic acid in which the triethylamine salt of clavulanic acid is formed and is then convened into a silyl diester of clavulanic acid. EP 0887178A discloses a 35 process for the purification of clavulanic acid in which organic amines may be used to (I): H 5 CQjH 25 - la- 270557 form an intermediate amine salt with clavulanic acid in an impure solution. The present invention provides the use of the salts of clavulanic acid with an amine of formula (II): where m is zero or an integer 1 to 5, R is an optionally substituted aliphatic hydrocarbon ring system containing from 3 to 8 ring carbon atoms, R4 is hydrogen or alkyi, amino- or hydroxy- substituted aikyl or substituted amino- substituted aikyl, or a group of the same general formula or Rl above:, and R^ may be selected from the same groups from which R* is selected, or from hydrogen, alkyi, alkenyl, amino-or hydroxy-substituted aikyl or alkeny'., or substituted amino-substituied alkyi or alkenyl; but with the exception of cyclohexylamine: or (2) each of Rl, r2 and R3 are the same or different and are independently selected from hydrogen, alkyi, alkenyl, amino - or hydroxy- or alkoxy- substituted aikyl or alkenyl, or substituted amino- substituted alkyi or alkenyl, but with the exception of t-butylamine, s-butylamine, N,N-dimethylethylamine, 1,2-dirnethylpropylamine, neopentylamine and 2-amino-3,3-dimethylbutane; and provided that if the amine (II) is trimethylamine or triethylamine the salt of clavulanic acid with the amine (II) is formed by readton of clavulanic acid or a labile derivative thereof in solution in an organic solvent with the amine (II) or a labile derivative thereof, and the salt is then isolated as or in a separate phase from the organic solvent: or (3) R1 being an optionally substituted aryl group of general formula: (II) as an intermediate in a process for the preparation of clavulanic acid or pharmaceutically acceptable salts and esters thereof, wherein Rl, and R^ are selected according to the following options: (1) R1 being an optionally substituted cyclic group of general formula: R-(CHR")m. 4 where R4 is hydrogen or one or more substituents, and m is zero or an integer 1 to 5, and R^ and R3 are independently selected from hydrogen, alkyi, substituted aikyl or substituted - amino- substituted alkyi or grou general formula as R1, provided that if R4 is hydrogen and m is -2 270557 are not both methyl; but with the exception of benzylterbutylnmine: or (4) Rl and R2, and optionally R^« together with the nitrogen atom shown being the residue of an optionally substituted heterocyclic ring system including the nitrogen atom as a ring member, and optionally including one or more additional ring 5 hetero atoms, and if R^ is not part of the ring system it is independently selected from hydrogen, aikyl, amino- or hydroxy- substituted aikyl or substituted amino-substituted aikyl; but with the exception of piperidine: or (5) Rl being a group of general formula: s R \ N -"PcHjCHaNHT—CHJCHJ 4 / ^ ™ R 10 where R4 and are independently hydrogen, aikyl, amino- substituted aikyl or substituted amino- substituted alkyi, and R2 and r3 are independently selected from hydrogen, alkyi, amino- or hydroxy- substituted aikyl or substituted amino-subsritutcd alkyi, and m is zero or an integer 1 to 5: or (6) One or both of R1 and R2 are hydrogen and represents the residue of an 15 amino acid in which the carboxylate group of the amino acid may be esterified or in the form of an amide. When alkyi groups or substituted alkyi groups are referred to herein unless otherwise defined herein they may suitably contain 1 to 6 carbon atoms in the aikyl system. Suitable substituents on amino groups include alkyi. 20 In option (1) above the amine (II) is suitably other than an amine in which R* is a cycloalkyl group and m is zero and R^ and R^ are both selected from cycloalkyl or from hydrogen or CnH2n+i where n is 1 to 7. In option (1) above the cyclic group R may suitably be saturated, with m being suitably zero. The group R may be monocyclic or polycyclic, and each ring 25 may suitably contain 5,6 or 7 ring carbon atoms including atoms shared between rings in fused or bridged ring systems. Suitably the cyclic group R may be unsubsdtuted. Suitably the amine (II) may include two or more cyclic groups R* or a fused ring system R* or a substituted ring system R for example having one or more alkyi 30 substituents such as methyl. Suitably R2 and R^ may be other than hydrogen, eg one or both may be aikyl or substituted aikyl. Examples of such amines include cyclopentylamine, cycloheptylamine, NN-dimethylcyclohexylamine, dicyclohexylamine, adamantylamine, NN-diethylcyclohexylamine, N-isopropylcyclohexylamine, N-methylcvclohexylamine, 35 cyclopropylamine, cyclobutylamine, norbornylamine, and dehydroabietylamine. In option (2) above the amine (II) is suitably other than an amine in which R* - 3 - 270557 is hydrogen or CnH2n+| where n is 1 to 7 and and R-* are also both selected from hydrogen or CnH2n+l where n is I to 7. Suitably in option (2) above, R' may be an alkyi or substituted alkyi group of general formula: 4 R r6-c-ls 5 R where R4, R^ and R^ independently represent Cj.^q alkyi, or amino- or hydroxy- substituted aikyl or substituted amino- substituted aikyl. R4, r5 and R^ may suitably all be alkyi, suitably two of R4, R^ or R*> being methyl. Examples of such amines include t-octylamine, (ie 2-amino-2,4,4-10 trimethylpentane) and t-amylamine. Alternatively two of R4, R^ or R^ may be alkyi and one may be hydroxy- substituted alkyi. Examples of such amines include 1-hydroxy-2-methyl-2-propylamine. In option (2) above, R* may alternatively suitably be Cj.20 alkyi, e.g. Cg_20 alkl, Cj.20 alkenyl, Cj.20 hydroxyalkyl, or Cj_20 aminoalkyl. IS Examples of stjeh amines include tri-n-propylamine, tri-n-octylamine, tri-n- butylamine, dimethylamine, i-propylamine, di-n-hexylamine, di-n-butylamine, diethylamine, 2-aminoethanol, NN-diethylethanolamine, NN-dimethylethanolamine, ethanolamine, n-butylamine, n-hexylamine, n-octadecylamine. N-ethylethanolamine, 1-hydroxy ethylamine, diethanolamine, NN-dimethylethanolamine, N-ethyl 20 diethanolamine, 1,6-diamino hexane, triethanolamine, diisobutylamine, diisopropylamine, 2-methoxyethylamine, hydroxylamine, ammonia, methylamine, ethylamine, n-piopylamine, n-butylamine, n-pentylamine, n-hexylamine, n-heptylamine, n-octylamine, n-nonylamine, n-decylamine, n-undecylamine, n-dodecylamine, n-prop-2-ylamine, n-but-2-ylamine, n-pent-2-ylamine, n-hex-2-yl-25 amine, n-hept-2-ylamine, n-oct-2-ylamine, n-non-2-ylamine, n-dec-2-ylamine, n-undec-2-ylamine, n-dodec-2-ylamine, n-hex-3-ylamine, n-hept-3-ylamine, n-oct-3-ylamine, n-non-3-ylamine, n-dec-3-yl-amine, n-undec-3-ylamine, n-dodec-3-ylamine, n-oct-4-ylamine, n-non-4-ylamine, n-dec-4-ylamine, n-undec-4-ylamine, n-dodec-4-ylamine, n-non-5-ylamine, n-undec-5-ylamine, n-dodec-5-ylamine, and n-30 octadecylamine. In option (3) above the amine (II) is suitably not an amine in which R2 and are selected from hydrogen or CnH2n+l where n is 1 to 7 or benzyl or substituted benzyl. In option (3) above suitable substituent groups R4 include Cj.g alkyi such as 35 methyl, phenyl or optionally substituted phenyl, carboxylic or sulphonic acid groups and derivatives of such acid groups such as esters (eg C i .5 aikyl esters) and amides; -4 - 27055 nitro. and halogen such as bromine. Suitably m may be zero. I or 2, and may be hydrogen or methyl. Suitably R- and may be hydrogen, or one of R- and R^ may be hydrogen and the other may be an aromatic group of the same general formula as Rl. 5 Examples of such amines include I phenylethylamine. p-toluidine. p- aminobenzoic acid, p-bromoaniline, ethyl-4-aminobenzoate (ie benzocaine), benzylamine, diphenylaniine, p-methylaminobenzene sulphonamide, m-nitroaniline, N,N'-dibenzylethylenediamine (ie benzathine), diphenylmethylamine, 4-methylbenzylamine and 4-phenylbutylamine. 10 In option (4) above the ring system may be aromatic or aliphatic, and may be monocyclic or polycyclic. Suitably each ring in the system may contain 5 or 6 ring atoms, including the ring nitrogen atoms, and including atoms which are shared between rings. Suitable optional substituents on the ring system include alkyi, amino, substituted amino, oxo and halogen. If the ring system includes additional ring 15 hetero atoms to the nitrogen atom shown, such hetero atoms may suitably be selected from nitrogen and oxygen. Examples of classes of such amines include substituted piperidines and optionally substituted piperidines, for example where the substituents are selected firom alkyi, hydroxyalkyl, halogen, amino, substituted amino and amino-substituted 20 alkyi. Specific examples of such amines include N-ethyl piperidine, 2, 6-dimethyl piperidine, 2-methyl-N-hydroxypropyl piperidine (ie cyclo- methycane), 4-methyl piperazine, l-methyI-4-phenyl piperazine, N-ethyl morpholamine, hexamethylenimine, pyridine, 2-propylpyridine, 3-chloro-2-aminopyridine, morpholamine, 1,5-diazabicyclo [4,3,0] non-5-ene, 1,4-diazabicyclo [2, 2,2] 25 octane, pyrrolidone, quinuclidine and xanthinol. In option (5) above R4 and R^ may suitably both be hydrogen, or one may be hydrogen and the other alkyi. R2 and R^ may suitably be hydrogen or aikyl. / Examples of such amines include ethylene diamine, NN-diethylethylene diamine, NN'-diisopropylethylenediamine and methylene terramine. 30 In option (6) above the amino acid may be a naturally occurring amino acid. Amino acid esters may be with aikyl groups or substituted alkyi groups such as benzyl. Examples of such amines include arginine, ornithine, histidine, lysine, benzyiglycine, 3-amino-3-methylbutanoic acid, L-ethyl lysinate, L-methyl histidinate, 35 methyl N-carbobenzyloxy-L- lysinate, methyl L-phenylalanate. ethyl glycyl glycinate, ethyl p-hydroxy phenyl glycinate, ethyl p-hydroxy phenyl glycinate, ethyl glycinate, ethyl L-tyrosinate, p-methoxybenzyl a-aminophenylacetate, n-butyl a-aminophenylacetate, methyl arginate, benzyiglycine, benzyl phenylglycine, l-nitrobenzyl phenyl glycine, n-butyl phenylglycine, p-methoxybenzy! phenylglycine, - 5 - 10 27 0 55 7 ethyl phenyl glycine, p-nitrobenzyl p-hydroxy phenyl-glycine, p-nitrobenzylserine, n-butyl serine, methyl arginine, dimethyl glutamate, p-nitrobenzyl tyrosinate, p-nitrobenzyl glycinate, benzylglycinate, p-nitrobenzyl a-amino-p-hydroxy-phenyl acetate, p-nitrobenzyl a-aminophenylacetate, ethyl a-amino-p-hydroxy phenyl acetate, ethyl L-tyrosinate. When the amine (II) contains more than one nitrogen aton the clavulanic acid may form a salt with one or more of the nitrogen atoms, for example as in NN'-diisopropylethylenediamine diclavulanate. Of the amines mentioned above, preferred amines are: phenylethylamine, t-amylamine, t-octylamine, l-hydroxy-2-tnethyl-2-propylamine, cyclopentylamine, cycloheptylamine, 1-adamantanamine, N-ethylpiperidine, N'N'-diisopropylethylenediamine and N N-dimethylcyclohexylamine. 15 The present invention provides a process for the preparation and/or purification of clavulanic arid or a pharmaceutically acceptable salti or ester thereof which process comprises: i) contacting impure clavulanic acid pr a labile derivative thereof in an organic solvent, with tertiary octylamine; 20 ii) isolating the tertiary-octylamine salt of clavulanic acid formed; and, iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof. In the process of the present invention die salt of clavulanic acid with tertiary 25 octylamine may be use l to purify impure clavulanic acid during its preparation. Therefore the salt is may be formed in a solution of clavulanic acid or a labile derivative thereof containing impurities, isolating the salt as a separate phase, eg as a solid precipitate, from the solution containing residual impurities, then reforming clavulanic acid or forming a pharmaceutically acceptable salt or ester thereof. 30 Suitable labile derivatives of clavulanic acid include salts, eg an alkali metal salt such as lithium or sodium clavulanate or esters, such as silyl esters. Conveniently the tertiary octylamine itself is contacted with impure clavulanic acid itself in solution in an organic solvent The above process is suitably carried out in an organic solvent, which although preferably substantially dry, for example containing less than 6 g/L, eg 0.25-0-6 g/L of water, may contain some water, as a solvent for the clavulanic add and the tertiary octylamine. A suitabW^i«£!£S^Jf»^fyness may be achieved by conventional^ewatering -6- - 35 270557 processes such as centrifuging. Water present in the solvent may be dissolved or in the form of droplets of a separate phase. The solution of clavulanic acid in organic solvent may be obtained by extraction of an acidified aqueous solution of clavulanic acid such as the fermentation liquor referred to above. 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 may also be desirable in addition 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 - 100 mg/ml. e.g 10 - 40 mg/ml, such as 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 desalting. It is also prefened 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 with tertiary octylamine, e.g. to less than 6g/L of water. Preferably the extraction is carried out at a temperature from 5 to 15°C. Suitable organic solvents in which impure clavulanic acid may be contacted with the tertiary octylamine include hydrocarbon solvents such as toluene and hexane, ethers such as tetrahydrofuran, dioxan, diethyl ether, halogenated solvents such as dichloromethane and chloroform, ketones such as acetone and methyl isobutyl ketone, and psters such as ethyl acetate. Solvents which include a carbonyl group, eg those of the formula (III): O J' . ' 8 9 R -C-R (III) wherein R8 is a Cj.g aikyl group or a alkoxy group and is a Cj.g aikyl group are examples of a sub-class of suitable solvents, for example organic ketones or organic alkanoate esters. The present invention also encompasses the use of mixtures of such solvents. More suitably the organic solvent is one which can be used directly to extract the acidified aqueous for example organic aikyl alkanoate esters, ketones and certain aliphatic alcohols, or mixtures thereof, such as ethyl acetate, methyl acetate, propyl -7 - z/0557 acetate, n-butyl acetate, methyl ethyl ketone, methyl isobuiyl ketone, tetrahydrofuran, butanol and mixtures of such solvents. Of these the most suitable appear to be methyl isobutyl ketone, methyl ethyl ketone, and ethyl acetate. Suitable solvent mixtures include methyl ethyl ketone/methyl isobutyl ketone and teirahydrofunin/meihyl 5 isobutyl ketone. A preferred solvent is ethyl acetate Suitable solvents for tertiaiy octylamine include those referred to above in which the clavulanic acid may be dissolved, or extracted, for example acetone, ethyl acetate, methyl isobutyl ketone, and methyl ethyl ketone. It appears to be particularly desirable to include ketones such as acetone in the 10 solvent system, as these appear to inhibit the formation of the salt of clavulanic acid with tertiary octylamine as an oil. In general one equivalent of the tertiary octylamine or a slight excess thereof per mole of clavulanic acid is used to produce the salt of clavulanic acid. Solutions of clavulanic acid and tertiary octylamine may for example be mixed slowly with stirring and 15 the mixture stirred for some time after addition is complete. The reaction between the clavulanic acid or its labile derivative is suitably carried out at a temperature below ambient, for example 0 to 15°C, eg 0 to 10°C, eg 0 to S°C. A suitable concentration for the clavulanic acid or its labile deriytive in the solution 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 20 further concentrate the solvent extract to a concentration in excess of this eg greater than 20g/L. For example in another procedure the tertiary octylamine may be introduced by mixing it into a stream of a solution of the clavulanic acid in the solvent, so that the salt is formed in the stream, either in solution or as particles or suspended droplets of the 25 dissolved salt in suspension. The tertiary octylamine introduced in this way may be introduced neat, or may be introduced as a solution in a solvent, for example the same organic solvent as the clavulanic acid is dissolved in. • The desired salt of clavulanic acid with tertiaiy octylamine may then be isolated. In this way, the salt of clavulanic acid with tertiary octylamine is separated from most or 30 all of the impurities. Isolation may be effected in a conventional manner, for example by centrifugation or filtration. In an alternative isolation procedure the salt of clavulanic acid with tertiary-octylamine may be isolated from the organic solvent, if the solvent is wholly or partly immiscible with water, by contacting the solvent with water so as to extract the salt, 35 which may be in solution or suspension, from the organic solvent and to form an aqueous solution of the salt. As salts of clavulanic acid with tertiary octylamine are fairly soluble water, such an aqueous solution may be very concentrated, eg around 20-30% w:w. In this way the bulk of organic impurities in the organic solvent solution of - 8 - 270557 clavulanic acid remain in the organic solvent whilst the clavulanic acid, in the form of its salt with tertiary octylamine, in a relatively pure state is obtained in the aqueous solution. The aqueous solution of the clavulanic acid salt so formed may be subjected to conventional further treatment, eg purification, or conversion into clavulanic acid 5 or a pharmaceutically acceptable salt or ester as described below. In another alternative or additional procedure the clavulanic acid and the amine may be mixed in solution in a First organic solvent, then the salt may be caused to separate from solution by addition of a second organic solvent. Suitably the first organic solvent may be an organic ester such as ethyl acetate, and the second solvent 10 may for example be a halogenated solvent such as chloroform, an ether such as diethyl ether, a hydrocarbon such as toluene, an alcohol such as ethanol, or a solvent of formula (III) above such as acetone or methyl isobutyl ketone. In one embodiment of this invention, the salt of clavulanic acid with tertiary octylamine may be employed as an acetone solvate. Acetone solvates in some cases have 15 advantageous stability and purity characteristics compared to salts of clavulanic acid with amines themselves. Such solvates are particularly useful in the present invention because they can often be isolated as a highly pure and stable crystalline compound. Accordingly the present invention also provides the salt of clavulanic acid with tertiary octylamine in the form of an acetone solvate. During isolation and/or drying, 20 sotnc acetone may be lost since the strength of solvation may not be high, but die amount of acetone in the product is not critical. The acetone solvate may be formed by contacting clavulanic acid with tertiaiy octylamine in the presence of acetone. In general, a solution containing clavulanic acid may be mixed with at least the same volume of acetone together with tertiaiy 2g octylamine, when the salt is precipitated. The tertiaiy octylamine may be dissolved in acetone and mixed with a solution of clavulanic acid in an organic solvent. Favoured organic solvents include ethyl acetate, tetrahydrofuran, methyl ethyl ketone, methyl isobutyl ketone and mixtures of such solvents, of which ethyl acetate is preferred. Alternatively an aqueous solution of the salt of clavulanic acid with tertiaiy octylamine. obtained by water extraction as oudined above, may be mixed with acetone to form the solvate. Suitably a concentrated aqueous solution of the salt may be mixed with excess acetone to form the soivate. oc Recrystallisation of the salt of clavulanic acid or the accione solvate may be -9- 270557 advantageous to further reduce the level of impurities. A convenient solvent for the recystallisation is aqueous acetone: Such recrystallisation is performed in a conventional manner, for example the salt or solvate is dissolved in water, treated with a small amount of acetone, filtered, and then treated with larger volumes of acetone optionally with stirring and/or cooling to afford the recrystallised product. In another aspect the present invention provides a process for the preparation of clavulanic acid or a pharmaceutically acceptable salt or ester thereof which process comprises converting the salt of clavulanic acid with tertiary octylamine into clavulanic acid or a pharmaceutically acceptable salt or ester thereof. The pharmaceutically acceptable salts and esters of clavulanic acid prepared by the processes of this invention include those described in GB 7508977 and 1S08978 which are herein incorporated by reference. Particularly suitable pharmaceutically acceptable salts include the pharmaceutically acceptable alkali and alkaline earth metal salts, for example the sodium, potassium, calcium and magnesium salts. Of these salts the sodium and potassium are most suitable and the potassium is preferred. Suitable esters include those cleavable to provide clavulanic acid or a salt thereof, by chemical methods such as hydrogenolysis or by biological methods. The salt of clavulanic acid with tertiaiy octylamine optionally in the form of its acetone solvate may be converted into clavulanic acid or a pharmaceutically acceptable salt or ester thereof by for example ion-replacement in the case of the free acid or salts, or by esterification. Ion-replacement may be performed using ion-exchange resins, for example by passing a solution of the salt through a bed of a cation exchange resin in sodium, potassium or calcium form. Suitable cation exchange resins include Amberlite IR 120 and equivalent resins. Alternatively ion-replacement may be effected by reaction of the protonated amine cation with a salt-precursor compound, which may be a base, for example a carbonate, bicarbonate or hydroxide of a pharmaceutically acceptable alkali or alkaline earth metal, or a salt of an organic carboxylic acid with a pharmaceutically acceptable cation such as an alkali or alkaline earth metal, for example a salt of an alkanoic acid of formula (IV): 10 R -O^H (IV) wherein R*0 is an alkyi group, containing for example from 1 to preferably from 1 to 8 carbon atoms. Examples of suitable salts propionate or ethylhexanoate salts, potassium 2-ethylhexanoate z ethylhdxanoate being preferred. Typically the salt of clavulanic in solution may be reacted with a salt of an alkali metal with aci< - 10- 270557 suspension in a suitable solvent, which may for example be an organic solvent, water, or a mixture of water and an organic solvent such as isopropanol. Suitably solutions of the salt of clavulanic acid with tertiary octylamine and of the salt-precursor compound may be mixed, and the pharmaceutically acceptable salt allowed to crystallise. 5 Suitably the reaction may be carried out of a temperature below ambient, e.g. 0 to 15° C. e.g. 0 to 10°C. suitably 0 to l).5°C. Suitably if the salt of clavulanic acid with tertiaiy octyl - amine is formed in aqueous solution it may be precipitated out by admixing the aqueous solution with excess acetone. Suitable methods of esterification include: 10 a) the reaction of the salt of clavulanic acid with tertiary octylamine with a compound of the formula Q-R^ wherein Q is a readily displaceable group and R^ is an organic group; b) the reaction of the salt of clavulanic acid with tertiaiy octylamine with an alcohol or thiol in the presence of a condensation promoting agent such as carbodiimide; and 15 c) the reaction of the salt of clavulanic acid with tertiary octylamine with a diazo compound. The foregoing processes extend to cover those aspects wherein the salt of clavulanic acid with tertiaiy octylamine is first converted to clavulanic acid or another salt thereof and subsequently is converted to the desired ester. Further details of 20 esterification methods are disclosed in GB 1S08977 and 1508978. Use of the present invention enables salts and esters of clavulanic acid to be more readily obtained in pure form than operation of the processes of GB 1508977 and 1543563. The invention will now be described by way of example only. 25 Example 1 In the following procedure, the amine was mixed with clavulanic acid in solution in THF and rapid crystallisation to foim a solid salt was observed. - n - 270557 Amine Comments Stability of Salt t-octylamine crystalline solid Stable >70h,20°C 50% RH Example 2 In the following a salt of clavulanic acid with an amine was formed in solution in a first organic solvent, and was then caused to separate out from solution as a solid precipitate by mixing with a second organic solvent. 2a. t-Octylamine A stock solution of clavulanic acid in ethyl acetate was prepared containing ca. 28g/L of clavulanic acid in an impure form, by extraction of an impure 5. clavuligerus fermentation broth with ethyl acetate. To 46 ml of this was added t-octylamine (0.84g). After 10 minutes the mixture became cloudy and fine crystals of the salt crystallised out. To an aliquot of the solution was added chloroform, resulting in the formation of larger needles. To a further aliquot was added acetone, again resulting in the formation of needle crystals, but smaller and less quickly than with chloroform. To the remainder of the solution was added ca. 20 ml chloroform then a volume of toluene approximately equal to the initial bulk of the solution, which resulted in precipitation of a substantial quantity of needle crystals. 2b. t-Octylamine - To 500 ml of the stock solution prepared as for 2a above was added t-octylamine (6.7g). The solution became slightly hazy. Acetone (20 ml) was added, which cleared the solution. To an aliquot of the solution was added diethyl ether, resulting in immediate crystallisation. Diethyl ether (55 ml) was added to the main bulk of the solution, resulting in crystallisation. The crystals were filtered and washed with acetone. The yield (I2.9g) represented a 77% recovery of the clavulanic acid from the solution. - 12 - 270557 Example 3 A solution of clavulanic acid in ethyl acetate (ca 20ng/ml) was diluted with an equal volume of acetone. An acetone solution of t-octylamine (1.25 mole equivalents) was then added dropwise over Vi hours at 10°C. After further stirring for 5 1 hour the precipitated crystals were collected, washed with acetone and dried under vacuum. A precipitate formed quite readily and was white in colour. Yield (corrected for purity)« 76%. 10 Example 4 To an impure solution of clavulanic acid, being a crude extract from an S. clavuligerus fermentation broth after some pre-purilication by ion-exchange (500 ml. 21iig/ml in ethyl acetate), additionally containing acetone (20 ml) was added 15 t-octylamine (7.6g 1.0 e.g.) which produced a slight haziness. Addition of diethyl ether (55 ml) caused the separation of the amine salt as fine white needles which were filtered off and washed with acetone. Yield 12.9g, 77.2% recovery, 62.8% pfa (theoretical = 60.6%). 20 Example 5 A impure aqueous solution of clavulanic acid, being obtianed from an S.clavuligerus fermentation broth by pre-purification involving ion-exchange chromatography or generally oudined above, containing ca. 17.1 g/L was acidified to pH 2.0 with 25% v/vsulphuric acid, and was then continuously extracted into ethyl 25 acetate. The ethyl acetate extract was cooled to 2°C, dewatered by centrifuging then dried with MgS(>4 then passed down a CPG carbon column. At this stage the ethyl acetate extract contained 6.02 g/L of clavulanic acid, and it was then concentrated by evaporation to a concentration of a 25.7 g/L of clavulanic acid, and was used at this concentration. The moisture level of the concentrate was ca. 026% v/v. 30 7.8 ml of t-octylamine was mixed with 25 ml of fresh ethyl acetate. This mixture was slowly added to 2 litres of the clavulanate rich ethyl acetate extract diluted back to a titre of 23.0 g/L with fresh ethyl acetate, with rapid stirring. The slurry was stirred for a further hour at 5°C. The t-octylamine clavulanate was subsequently isolated by filtration, and wasshed with ethyl acetate. Final drying was 35 carried out overnight in a vacuum oven at 20°C, with a nitrogen bleed. Product weight = 6.13 g. -13- </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 270557 Example 6 5g of the amine salt produced in example 5 was dissolved in 97 ml of isopropanol. The product did not dissolve readily, and it was found necessary to 5 apply gentle warming, to 24° C, in order to effect total dissolution. 14 ml of 1.5 N potassium 2-ethylhexanoate in isopropanol were added over 10 minutes. The slurry was subsequently stirred for 1.5 hours, at 5°C. The product potassium clavulanate was then filtered off, washed with small amounts of isopropanol then acetone, and $ dried under vcacuum overnight, with a nitrogen bleed, at 20°C. Product weight = 10 3.16g. Example 7 Filtered (RVF) S. clavuligerus fermentation broth containing 2 g/L at clavulanic acid was acidified to pH 1.6 with 25% v/v sulphuric acid and continuously 15 extracted into ethyl acetate. The solvent extract was cooled to 3°C then dewatered by centrifuging, then dried with MgS04, then passed down a CPG carbon column. The carbon-treated extract was then concentrated by evaporation to a concentration of clavulanic acid of ca. 20 g/L, with a moisture content of ca. 0.06% v/v. 13.5ml of t-octylamine was mixed with 43 mis of fresh ethyl acetate. This 20 mixture was slowly added to 400 ml of clavulanate rich ethyl acetate extract at a titre of 20 g/L clavulanic acid, with rapid stirring. The slurry was stirred for a further hour at 5°C The t-octylamine clavulanate was subsequently isolated by filtration, and washed with ethyl acetate. Final drying was carried out overnight in a vacuum oven at 20°C, with a nitrogen bleed. Produa weight = 12.44 g. 25 Example 8 1 lg of the amine salt produced in example 7 was dissolved in 213 mis of isopropanol. The product did not dissolve readily, even after applying gentle wanning, to 24°C. Water (3.75ml) was added to enable dissolution. Once a solution 30 was obtained additional isopropanol (100ml) was added to reduce the water content prior to crystallisation. 30ml of 1.5 N. potassium 2-ethyl hexanoate in isopropanol was added over 15 minutes. The slurry was subsequently stirred for 1.5 hours at 5°C. The potassium ciavulanatc produa was then filtered off, washed with small amounts of isopropanol then acetone, and dried under vacuum overnight, with a nitrogen 35 bleed, at 20°C. Product weight = 7.29 g. - 14- - 15- 27 0 5 5 7 WHAT WE CLAIM IS:

1. A process for the preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt or ester thereof which process comprises i) contacting impure clavulanic acid or an alkali metal salt thereof in solution in an organic solvent, with tertiary octylamine; ii) isolating the tertiary-octylamine salt of clavulanic acid formed; iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof.

2. A process according to claim 1 in which the alkali metal salt is the sodium or lithium salt.

3. A process for the preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt or ester thereof which process comprises i) contacting impure clavulanic acid or a labile derivative thereof in solution in an organic solvent which is dried by dewatering the solvent by centrifuging, with tertiary octylamine; ii) isolating the tertiary-octylamine salt of clavulanic acid formed; iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof.

4. A process for the preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt or ester thereof which process comprises i) contacting impure clavulanic acid (obtained by fermentation of a clavulanic acid producing micro-organism in which at least some of the suspended solids in the impure clavulanic acid broth are removed) in solution in an organic solvent, with tertiary-octylamine; .MM*1*'*" 27 0 55 7 -16- iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof.

5. A process according to claim 4 in which the solids are removed by nitration.

6. A process for the preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt or ester thereof which process comprises i) contacting impure clavulanic acid obtained by fermentation of a clavulanic acid-producing micro-organism in which the aqueous solution of clavulanic acid obtained in the fermentation is preconcentrated before extraction in an organic solvent, with tertiary octylamine; ii) isolating the tertiary-octylamine salt of clavulanic acid formed; iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof.

7. A process according to claim 6 in which the aqueous solution of clavulanic acid is preconcentrated to a concentration of lO-lOOmg/ml.

8. A process according to claim 6 in which the aqueous solution of clavulanic acid is preconcentrated to a concentration of 10-40mg/ml.

9. A process according to claim 6 in which the aqueous solution of clavulanic acid is preconcentrated to a concentration of 10-25mg/ml.

10. A process according to any one of claims 6 to 9 in which the preconcentration process is by 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.

11. A process according to claim 10 in which the electrolyte is sodium chloride.

12. A process according to claim 10 or 11 in which the resulting concentrate is de-salted.

13. A process for the preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt or ester thereof which process comprises -17- 27 0 5 57 i) contacting impure clavulanic acid or a labile derivative thereof in an organic solvent, with tertiary octylamine in which the tertiary octylamine is introduced by mixing it into a stream of the clavulanic acid in the organic solvent; ii) isolating the tertiary-octylamine salt of clavulanic acid formed; iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof. A process for the preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt or ester thereof which process comprises i) contacting impure clavulanic acid or a labile derivative thereof in an organic solvent selected from the group consisting of hydrocarbon solvents, ether solvents, halogenated solvents and ketone solvents, with tertiaiy octylamine; ii) isolating the tertiary-octylamine salt of clavulanic acid formed; iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof. A process according to claim 14, in which the solvents are selected from the group consisting of toluene, hexane, tetrahydrofuran, dioxan, diethyl ether, dichloromethane, chloroform, acetone and methyl isobutyl ketone. A process for the preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt: or ester thereof which process comprises i) contacting impure clavulanic acid or a labile derivative thereof in an organic solvent, with tertiaiy octylamine; ii) isolating the tertiary-octylamine salt of clavulanic acid formed in which the isolation of the tertiary-octylamine salt of clavulanic acid is carried out by extrr.;^lng the organic solvent containing the tertiary octylamine salt with water to extract the salt; iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof. ^ / *■ O .j ' , 7 JMI >sf f c -18- 27 0 5 57 17. 18. 19. 20. A process for the preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt or ester thereof, which process comprises i) contacting impure clavulanic acid or a labile derivative thereof in an organic solvent, with tertiary-octylamine; ii) isolating the tertiary-octylamine salt of clavulanic acid formed and recrystallising it; iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof. A process according to claim 17 in which the recrystallisation of the tertiary-octylamine salt is carried out by aqueous acetone. A process for the preparation and/or purification of clavulanic acid or a pharmaceutically acceptable salt; or ester thereof which process comprises i) contacting impure clavulanic acid or a labile derivative thereof in an organic solvent, with tertiaiy octylamine; ii) isolating the tertiary-octylamine salt of clavulanic acid formed; iii) converting the thus formed salt into clavulanic acid or a pharmaceutically acceptable salt or ester thereof in which the conversion is by ion-replacement and the ion replacement is performed by passing a solution of tertiary octylamine salt through a bed of a cation exchange resin in sodium, potassium or calcium form. Clavulanic acid or a pharmaceutically acceptable salt or ester thereof whenever prepared and/or purified by a process according to any one of the preceding claims. </div>