NZ270989A

NZ270989A - Process for the preparation and/or purification of clavulanic acid or a salt or ester thereof using amines

Info

Publication number: NZ270989A
Application number: NZ270989A
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: 1996-02-27
Also published as: NZ280385A; NZ270557A

Description

<div class="application article clearfix" id="description"> New Zealand Paient Spedficaiion for Paient Number £70989 270 lirtdw ttwi provisions of fcjlof) 33 tfti? ii#»«> . i SIpvdficfltkxi has been arfte-<lirt«i Priority Oate{s): ..].Vt.:.^:3.^...!.l:..\7U:.5^..( Complete Specification Filed-: Class: B.SSJlSJS&SSiaSU Publication Date: 2-.7-..K8..-19SB— P.O. Journal No: .LJrfcS)! NO DRAWINGS NEW ZEALAND PATENTS ACT, 1953 Divided out of No.: 253248 Date: 7 June 1993 2 6 APR 1995 COMPLETE SPECIFICATION "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 W£ 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:- -1 - (followed by page la) PROCESS FOR THE PREPARATION OF CLAVULANIC ACID This invention relates to a novel process for the preparation and/or purification of clavulanic acid (I): H and pharmaceutically 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 Strepiomyces strains such as S. clavuligenis NRRL 3585, S. jumoninensis NRRL 5741, S. katsurahamanus IFO 13716 and Strepiomyces sp. P 6621 FERM P2804 e.g. as described in JP Kokai 80-162993. The resulting aqueous broth may be subjected to convenrional 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. 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, eludng therefrom with an electrolyte, desalting the resulting solution, applying the desalted solution to a further anion exchange resin, chromatographically eluting 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 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-(rower 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 process for the purification of clavulanic acid in which"organic amines may be used to C02H - la - 10 270989 form an intermediate amine salt with clavulanic acid in an impure solution. The present invention provides the use of the salts of clavulanic add with an amine of formula (II): \—R r2/ (ID as an intermediate in a process for the preparation of clavulanic acid or phaimaceutically acceptable salts and esters thereof, wherein R1, R2 and R^ are selected according to the following options: (1) R* being an optionally substituted cyclic group of general formula: R-(CHR4) m' 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 15 alkyl, amino- or hydroxy- substituted alkyl or substituted amino- substituted alkyl, or a group of the same general formula or R* above:, R2 and R3 may be selected from the same groups from which R* is selected, or from hydrogen, alkyl, alkenyl, amino-. or hydroxy-substituted alkyl or alkenyl, or substituted amino-substituted alkyl or alkenyl; but with the exception of cyclohexylamine: or 20 (2) each of R*, R2 and R^ are the same or different and are independendy selected from hydrogen, alkyl, alkenyl, amino - or hydroxy- or alkoxy- substituted alkyl or alkenyl, or substituted amino- substituted alkyl or alkenyl, but with the excepdon of t-butylamine, s-butylamine, N,N-dimethylethylamine, 1,2-dimethylpropylamine, neopentylamine and 2-amino-3,3-dimethylbutane; and 25 provided that if the amine (II) is trimethylamine or triethylamine the salt of clavulanic acid with the amine (II) is formed by reaciton 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 30 (3) R* being an optionally substituted aryl group of general fonmuj where R4 is hydrogen or one or more substituents, and m is zero on and R2 and R^ are independently selected from hydrogen, alkyl, amint substituted alkyl or substituted - amino- substituted alkyl or groups of the same. 35 general formula as R1, provided that if R4 is hydrogen and m is zero, then R2 and R3 -2- are not both methyl; but with the exception of benzylterbutylamine: or (4) Rl and R2, and optionally R3' 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 hetero atoms, and if R3 is not part of the ring system it is independently selected from hydrogen, alkyl, amino- or hydroxy- substituted alkyl or substituted arnino-substituted alkyl; but with the exception of piperidine: or (5) R* being a group of general formula: s R \ r ~] M Lpu J MU 1 N —f-CH2CH,NH A— CH,CH, 4/ L Jm R 10 where R4 and R5 are independently hydrogen, alkyl, amino- substituted alkyl or substituted amino- substituted alkyl, and R- and R3 arc independently selected from hydrogen, alkyl, amino- or hydroxy- substituted alkyl or substituted amino-substituted alkyl, and m is zero or an integer 1 to 5: or (6) One or both of R* and R2 arc hydrogen and R3 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 alkyl groups or substituted alkyl groups are referred to herein unless otherwise defined herein they may suitably contain 1 to 6 carbon atoms in the alkyl system. Suitable substituents on amino groups include alkyl. 20 In option (I) above the amine (II) is suitably other than an amine in which Rl is a cycloalkyl group and m is zero and R2 and R3 are both selected from cycloalkyl or from hydrogen or CnH2n+l 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 unsubstituted. 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 alkyl 30 substituents such as methyl. Suitably R2 and R3 may be other than hydrogen, eg one or both may be alkyl or substituted alkyl. Examples of such amines include cyclopentylamine, cycloheptylamine, NN- 4 dimethylcyclohexylamine, dicyclohexylamine, adamantylamine, NN-diethylcyclohexylamine, N-isopropylcvclohexylamine, N-methylcyclohexylamine, 35 cyclopropylamine, cyclobutylamine, norbornylamine, and dehydroabietylamine. In option (2) above the amine (II) is suitably other than an amine in which R^ *\ - j - z Gsrp. is hydrogen or C„H2n+| where n is I to 7 and R2 and R-1 are also both selected from hydrogen or Cnli2n+i where n is 1 to 7. Suitably in option (2) above, R1 may be an alkyl or substituted alkyl group of general formula: 4 R R6-C- U 5 R where R4, and R*> independently represent Ci_jo alkyl. or amino- or hydroxy-substituted alkyl or substituted amino- substituted alkyl. R4, r5 and R^ may suitably all be alkyl, 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, or R^ may be alkyl and one may be hydroxy- substituted alkyl. Examples of such amines include 1-hydroxy-2-methyl-2-propylamine. In option (2) above, R* may alternatively suitably be Cj_20 alkyl, e.g. Cg_20 alkl, C^_20 alkenyl, Ci_20 hydroxyalkyl, or C1.20 aminoalkyl.^^ 15 Examples of such amines include tri-n-propylamire, tri-ffoctylamine, tri-n- butylamine, dimethylamine, i-propylamine, di-n-hexylamine, di-n-butylamine, diethylaminc, 2-aminoethanol, NN-diethylethanolamine, NN-dimethylethanolamine, ethanolamine, n-butylamine, n-hexylamine, n-octadecylamine, N-ethylethanolamine, 1-hydroxyethylamine, diethanolamine, NN-dimethylethanolamine, N-ethyl 20 diethanolamine, 1,6-diamino hexane, triethanolamine, diisobutylamine, diisopropylamine, 2-methoxyethylamine, hydroxylamine, ammonia, methylamine, ethylamine, n-propylamine, 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-ylatnine, 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 arc 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.6 alkyl 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 alkyl esters) and amides; -4- 27 nitro. and halogen such as bromine. Suitably m may be zero. I or 2. and R5 may be hydrogen or methyl. Suitably R- ;md R-* may be hydrogen, or one of R- and RJ may be hydrogen and the other may be an aromatic group of the same general formula as Rl. Examples of such amines include l-phcnyletliylantine. p-ioluidine, p-aminobenzoic acid, p-bromoaniline, ethyl-4-aminobenzoate (ie benzocaine). benzylamine, diphenylamine, p-methylaminobenzene sulphonamide, m-nitroaniline, N,N'-dibenzylethylenediamine (ie benzathine), diphenylmethylamine, 4-methylbenzylamine and 4-phenylbutylamine. 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 alkyl, amino, substituted amino, oxo and halogen. If the ring system includes additional ring 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 from alkyl, hydroxyalkyl, halogen, amino, substituted amino and amino-substituted alkyl. Specific examples of such amines include N-ethyl piperidine, 2, 6-dimethyl piperidine, 2-methyl-N-hydroxypropyl piperidine (ie cyclo- methycane), 4-methyl piperazine, 1 -methy 1-4-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] octane, pyrrolidone, quinuclidine and xanthinol. In option (5) above R4 and RD may suitably both be hydrogen, or one may be hydrogen and the other alkyl. R2 and R^ may suitably be hydrogen or alkyl. Examples of such amines include ethylene diamine, NN-diethylethylene diamine, NN'-diisopropylethylenediamine and methylene tetramine. In option (6) above the amino acid may be a naturally occurring amino acid. Amino acid esters may be with alkyl groups or substituted alkyl groups such as benzyl. Examples of such amines include arginine, ornithine, histidine, lysine, benzylglycine, 3-amino-3-methylbutanoic acid, L-ethyl lysinate, L-methyl histidinate, 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 ot-aminophenylacetate, n-butyl a-aminophenylacetate, methyl arginate, benzylglycine. benzyl phenylglycine, 1-nicrobenzyl phenyl glycine, n-butyl phenylglycine, p-methoxybenzyl phenylglycine. 270989 ethyl phenyl glycinc, p-nitrobenzyl p-hydroxyphenyl-glycine, p-nitrobenzylserine, n-butyl serine, methyl arginine, dimethyl gluiamate. p-nitrobenzyl tyrosinate, p-nitrobenzyl glycinate, bcniylglycinate, 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 (11) 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-methyl-2-propylamine, cyclopentylamine, cycloheptylamine, 1-adamantanamine, N-ethylpiperidine, N'N'-diisopropylethylcnediamine and N N-dimcthylcyclohexylamine. The present invention provides 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, with an amine of formula (II) where R4 and R5 are independently hydrogen, alkyl, amino-substituted alkyl or substituted amino-substituted alkyl, and R2 and R3 are independently selected from hydrogen, alkyl, amino- or hydroxy-substituted alkyl or substituted amino-substituted alkyl, and m is zero or an integer 1 to 5; where R* is a group of general formula R5 N -f CH2CH2NH-JhCH2CH2- iii) converting the thus formed salt into clavulanic acid or acceptable salt or ester thereof. ii) isolating the amine salt of clavulanic acid formed; -6- 27 \J Q In the process of the present invention the salt of clavulanic acid with the amine (II) may be used 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 esteir thereof. 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. Suitable labile derivatives of the amine (U) include salts such as the phosphate, borate, chloride, chlorate, perchlorate, bromide, toluene sulphonate or alkanoates, such as the acetate or ethylhexonoate. Conveniently the amine (II) 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 acid and the amine (II). A suitable degree of dryness may be achieved by conventional dewatering -7- processes such as cemrifuging. W;uer present in the solvent may be dissolved or in ihe 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 5 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 10 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 15 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 preferred to acidify the aqueous solution, e.g the broth or the pre-20 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 the amine (II), 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 25 with the amine (II) 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 esters such as ethyl acetate. Solvents which include a carbonyl group, eg those of the formula (III): O . 11 . 30 R -C-R (Itt) wherein R** is a Ci_$ alkyl group or a Cj.g alkoxy group and is a Ci_6 alkyl 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 35 of such solvents. More suitably the organic solvent is one which can be used directly to extract the acidified aqueous for example organic alkyl alkanoate esters, ketones and certain aliphatic alcohols, or mixtures thereof, such as ethyl acetate, methyl acetate, propyl - 8.- 270S0 acetate, n-butyl acetate, methyl ethyl ketone, methyl isobutyl 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 ketonc/methyl isobutyl ketone and tetrahydrofuran/methyl 5 isobutyl ketone. A preferred solvent is ethyl acetate. Suitable solvents for the amine (II) 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 the amine (II) as an oil. In general one equivalent of the amine (II) or a slight excess thereof per mole of clavulanic acid is used to produce the salt of clavulanic acid. Solutions of clavulanic acid and amine (II) may for example be mixed slowly with stirring and the 15 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 I0°C, eg 0 to 5°C. A suitable concentration for the clavulanic acid or its labile derivtive 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 auvantageous 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 amine (II) 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 amine (II) 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 the amine (II) may then be isolated. In this way, the salt of clavulanic acid with the amine (II) 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 the amine (II) 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 the amine (II) are fairly soluble in 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 - 9- 270989 clavulanic acid remain in ihc organic solvent whilst the clavulanic acid, in the form of its salt with the amine (II). 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 pharmaceutical^ 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. Some of the above-mentioned salts of clavulanic acid are believed to be novel compounds and as such are a further aspect of this invention, in particular the salts of 15 clavulanic acid with the amines (II) where R1 is a group of general formula D4 20 25 30 35 R4 as hereinbefore defined, for example, the salts of clavulanic add with triethylene tetramine and NN'-diisopropylethylenediamine. In one embodiment of this invention, the salt of clavulanic acid with the amine (II) may be employed as an acetone solvate. Acetone solvates in some cases have 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 the amine (II) in the form of an acetone solvate. During isolation and/or drying, some acetone may be lost since the strength of solvation may not be high, but the amount of acetone in the product is not critical. The acetone solvate may be formed by contacting clavulanic acid with the amine (II) 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 the amine (II), when the salt is precipitated. The amine (II) 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 rp$cture$ 'eff 'V^ such solvents, of which ethyl acetate is preferred. Alternatively an aqueous solution of the salt of clavulanic acid with the amine (II), obtained by water extraction-at outlined above, may be mixed with acetone to form the solvate. Suitabl]^, ^ concentrated aqueous solution of the salt may be mixed with excess aceton^njj^ftyipp the solvate; Rectystallisation of the salt of clavulanic acid or the acetone solvate may be -10- 270989 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 pharmaceutical^ acceptablc salt or ester thereof which process comprises convening the salt of clavulanic acid with an amine of formula (II) into clavulanic acid or a pharmaceutical!/ acceptable salt or ester thereof. The pharmaceutical^ acceptable salts and esters of clavulanic acid prepared by the processes of this invention include those described in GB 1508977 and 1508978 which are herein incorporated by reference. Particularly suitable pharmaceutical^ acceptable salts include the pharmaceutical^ 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 amine (II) optionally in the form of its acetone solvate may be convened into clavulanic acid or a pharmaceutical^ 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 eanh metal, or a salt of an organic carboxylic acid with a phanmaceutically acceptable cation such as an alkali or alkaline earth metal, for example a alkanoic acid of formula (IV): 10 R -CQjH wherein R ^ is an alkyl group, containing for example from 1 to 20 ca^j^toms?" preferably from 1 to 8 carbon atoms- Examples of suitable salts include propionate or ethylhexanoate salts, potassium 2-ethylhexanoate and sodium 2-ethylhexanoate being preferred. Typically the salt of clavulanic acid with amine (II) in solution may be reacted with a salt of an alkali metal with acid (IV) in solution or -11 - £7 U 98 9 suspension in ;i 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 the amine (11) and of ihe sait-precurssor compound may be mixed, and the pharmaccuticallv 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 the amine (II) is formed in aqueous solution it may be precipitated out by admixing the aqueous solution with excess acetone. Suitable methods of esterification include: jO a) the reaction of the salt of clavulanic acid with the amine (II) with a compound of the formula Q-R* 1 wherein Q is a readily displaccable group and is an organic group; b) the reaction of the salt of clavulanic acid with the amine (II) with an alcohol or thiol in the presence of a condensation promoting agent such as carbodiimide; and c) the reaction of the salt of clavulanic acid with amine (II) with a diazo compound. The foregoing processes extend to cover those aspects wherein the salt of clavulanic acid with aniine (II) is first converted to clavulanic acid or another salt thereof and subsequently is convened to the desired ester. Further details of esterification methods are disclosed in GB 1508977 and 1508978. Use of the present 20 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. Example 1 25 In the following procedure, the amine was mixed with clavulanic acid in solution in THF and rapid crystallisation to form a solid salt was observed. Amine Comments Stability of Salt triethylene tetramine pale yellow solid low assay </div>

Claims

<div class="application article clearfix printTableText" id="claims"> Example 2 27 0 9 e 9 An impure, wet (ca. 1% water), solution of clavulanic acid in ethyl acetate (1L, 10.14/zg/ml) was obtained by extraction of an S. clavuligerus 5 fermentation broth with ethyl acetate and some pre-purification by ion-exchange. To separate 1L batches of this solution was added with stirring an excess of neat ethylene diamine, NN'-diethylethylene diamine, and NN'-diisopropylethylenediamine, the quantity of each amine being in excess of the amount needed to form a diammonium salt with the clavulanic acid. 10 After continued stirring a precipitate of the salt formed. A further crop of crystals was obtained by addition of excess acetone. The precipitated diammonium salt was filtered off and washed with acetone. The crystals of each of these diammonium salts so formed were 15 converted to potassium clavulanate by dissolution of the salt in the minimum quantity of water, followed by the addition of a solution of an excess of potassium 2-ethylhexanoate in isopropyl alcohol. After continued stirring a precipitate of potassium clavulanate formed and was filtered, washed with isopropyl alcohol and dried. 20 WHAT WE CLAIM IS: -14- 270989

1. A process for the preparation and/or purification of clavulanic acid or a pharmaceutical^ acceptable salt or ester thereof which process comprises i) contacting impure clavulanic acid or a labile derivative thereof in solution in an organic solvent, with an amine of formula (II) R1 N—R r2 (II) where R* is a group of general formula R® Yl -f CH,CH2NH-}- ch,CH, R« z m 2 2 where R4 and R^ are independently hydrogen, alkvl, amino-substituted alkyl or substituted amino-substituted alkyl, and R^ and R^ are independently selected from hydrogen, alkyl, amino- orhydroxy-substituted alkyl or substituted amino-substituted alkyl, and m is zero or an integer 1 to 5; ii) isolating the amine 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 labile derivative of clavulanic acid is an alkali metal salt.

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

4. A process according to any one of claims 1 to 3 in which the organic solvent is substantially dry.

5. A process according to claim 4 in which the organic solvent contains less than 6g per litre of water.

6. A process according to claim 4 in which the organic solvent contains between 0.25 to 0.6g per litre of water.

7. A process according to any one of claims 4 to 6 in which the organic solvent is dried by dewatering the solvent by centrifuging.

8. A process according to any one of claims 1 to 7 in which the impure clavulanic acid results from the fermentation of a clavulanic acid-producing micro-organism and is extracted from the fermentation broth. 10 20 -15- 270989

9. A process according to claim 8 in which at least some of the suspended solids in the impure clavulanic acid broth are removed.

10. A process according to claim 9 in which the solids are removed by filtration.

11. A process according to any one of claims 8 to 10 in which the aqueous solution of clavulanic acid obtained in the fermentation is preconcentrated before extraction.

12. A process according to claim 11 in which the aqueous solution of clavulanic acid is preconcentrated to a concentration of 10- lOOmg/ml.

13. A process according to claim 11 in which the aqueous solution of clavulanic acid is preconcentrated to a concentration of 10-40mg/ml.

14. A process according to claim 11 in which the aqueous solution of clavulanic acid is 15 preconcentrated to a concentration of 10-25mg/ml.

15. A process according to any one of claims 11 to 14 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.

16. A process according to claim 15 in which the electrolyte is sodium chloride.

17. A process according to claim 15 or 16 in which the resulting concentrate is de-salted. 25

18. A process according to any one of claims 8 to 17 in which the broth or aqueous solution of clavulanic acid is acidified prior to solvent extraction.

19. A process according to claim 18 in which the broth or aqueous solution of clavulanic acid is acidified to a pH of 1 to 3. 30

20. A process according to claim 18 in which the broth or aqueous solution of clavulanic acid is acidified to a pH of 1.5 to 2.5.

21. A process according to any one of claims 1 to 20 in which extraction of impure clavulanic acid or a labile derivative thereof into an organic solvent is carried out at 2^ a temperature from 5 to 15°C.

22. A process according to any one of claims 1 to 21 in which the organic solvent used to extract the impure clavulanic acid or labile derivative thereof is selected from the group consisting of hydrocarbon solvents, ether solvents, halogenated solvents, r ketone solvents and ester solvents.

23. A process according to claim 22 in which the solvents are selected from the consisting of toluene, hexane, tetrahydrofuran, dioxane, diethyl ether, vf? dichloromethane, chloroform, acetone, methyl isobutyl ketone and etnyfacetate. it ^ro 40 45 Jt V E O 16- 270989

24. A process according to any one of claims 1 to 23 in which the amine salt of clavulanic acid is formed by treating the clavulanic acid or labile derivative thereof in an organic solvent with one equivalent per mole or a slight excess of the amine of formula II as defined in claim 1. 5

25. A process according to claim 24 in which the amine salt < formed at 0 to 15°C.

26. A process according to claim 24 or 25 in which the amirfe is /ntfi0pc^bymixing it into a stream of the clavulanic acid in the organic solvmrfU J 10

27. A process according to any one of claims 1 to 26 in which the isolation of the amine salt of clavulanic acid is carried out by extracting the organic solvent containing the amine salt with water to extract the salt and thereafter converting the amine salt into clavulanic acid or pharmaceutical^ acceptable salt or ester thereof. ^

28. A process according to any one of claims 1 to 27 in which the amine salt of clavulanic acid is recrystallised.

29. A process according to claim 28 in which the amine salt of clavulanic acid is recrystallised using aqueous acetone. 20

30. A process according to any one of claims 1 to 29 in which the amine salt of clavulanic acid is converted into a pharmaceutically acceptable salt thereof by ion-replacement

31. A process according to claim 30 in which ion replacement is performed by passing a solution of the amine salt through a bed of a cation exchange resin in sodium, potassium or calcium form.

32. A process according to claim 30 in which ion replacement is carried out by protonating the amine cation with a salt precursor compound. 30

33. A process according to claim 32 in which the salt precursor compound is potassium ethyl hexanoate.

34. A process according to any one of claims 1 to 33 in which in the amine of formula (II) one of R4 and R5 is hydrogen and the other is alkyl 35 and R2 and R3 are independently hydrogen or alkyl.

35. A process according to any one of claims 1 to 34 in which the amine of formula (II) is triethylene tetramine. 40

36. A salt of clavulanic acid with an amine of formula (II) as defined in claim 1.

37. A salt of clavulanic acid with an amine of formula (II) as defined in claim 1 in which one of R4 and R5 is hydrogen and the other is alkyl and R2 and R3 are independently hydrogen or alkyl. - 17- 270989 A salt of clavulanic acid as defined in claim 36 or 37 in which the amine is triethylene tetramine. Clavulanic acid or a pharmaceutical^ acceptable salt or ester thereof, whenever prepared and/or purified by a process according to any one of claims 1 to 35. DATED THIS- $ DAY A. J. PARK & SON </div>