IMPROVED PROCESS FOR THE PREPARATION OF SALTS AND ESTERS OF CLAVULANIC ACID

Abstract: A process for the production of an amine salt of clavulanic acid and the conversion thereof into a pharmaceutically acceptable salt or ester thereof has been provided. During the crystallisation phase of the amine salt of clavulanic acid either a clavulanic acid containing solution, optionally with a cosolvent, is added to the amine or both reactants are added together to a cristallisation vessel. Also the application of a temperature profile (decreasing in time), profile of speed of addition (increasing in time) and a profile of the ratio amine/clavulanic acid (increasing in time) for this cristallisation process has been provided for.

Description: IMPROVED PROCESS FOR THE PREPARATION OF SALTS AND ESTERS OF CLAVULANIC ACID Background and field of the invention The present invention relates to a process for the preparation of an amine salt of clavulanic acid and to the conversion thereof into pharmaceutically acceptable alkali metal salts and esters of clavulanic acid, especially potassium clavulanate. Clavulanic acid and its alkali metal salts and esters are β- lactamase inhibitors, able to enhance the effectiveness of penicillins and cephalosporins. Usually, clavulanic acid is prepared by the fermentation of a microorganism which produces clavulanic acid as for instance Streptomvces strains such as Streptomyces clavuligerus. The resulting aqueous broth is normally subjected to conventional purification and concentration processes, such as disclosed in British patent GB 1 508977. Techniques described to remove the biomass are filtration of the broth using e.g. rolling sieves (EP 0 387 1 78), rotary drum filtration or membrane filtration (EP 0 748 387), optionally after the addition of flocculants or a water miscible solvent. Centrifugation may also be used to separate the biomass. After filtration, purification may be applied e.g. by adsorption and the filtrate may be concentrated by reverse osmosis or evaporation, after which at low pH extraction into an organic solvent occurs using e.g. an extraction column or centrifugal extractors. Also whole broth extraction can be applied (WO 98/21 21 2) . Then, usually an amine crystallisation is performed, optionally after a purification step. Subsequently, a recrystallisation might be performed and finally a crystallisation to prepare a pharmaceutically acceptable salt of clavulanic acid, such as potassium clavulanate. Also, processes in which back extraction is applied are described (WO 96/22296) . European patent application EP-A-26044 discloses the use of the tertiary butyl amine salt of clavulanic acid as an useful intermediate in the preparation of clavulanic acid. Other amine salts of clavulanic acid are disclosed in the patent documents EP-A-3871 78, EP-A-562583, WO 93/25557, WO 94/22873, WO 96/201 99 and WO 96/26944. Most procedures to recover clavulanic acid suffer of bad crystallisation behaviour when an amine is contacted with clavulanic acid to form an amine clavulanate. Usually, material sticks to the walls in the normally applied solvent solution, resulting in a yield loss. Therefore a water miscible organic solvent (i.e. cosolvent) like a ketone or an alcohol is often added before or during the crystallisation which prevents the tendency of the amine crystals to stick to the walls. This is described for example in the international patent applications WO 93/25557, WO 96/201 99, WO 96/26944, WO 96/331 97 and WO 98/21 21 2. However, a disadvantage of using a cosolvent is the fact that the resulting mother liquid consists of a mixture of solvents. Therefore, an additional separation step is needed in order to recycle the solvents. In case of azeotrope formation, separation will be very difficult. Also, raw material costs increase due to the use of a cosolvent. The aim of the present invention is to prepare clavulanic acid and its pharmaceutically acceptable salts, such as potassium clavulanate with the desired substance obtained in a high yield and of high purity, while using a minimal amount of solvents. Surprisingly the volume of cosolvent to be added to ensure proper crystallisation can be significantly reduced or avoided completely, if the order of addition of the clavulanic acid solution and the amine is reversed, i.e. upon addition of the clavulanic acid solution to the amine solution instead of the usual addition of the amine solution to the clavulanic acid solution or in case the clavulanic acid solution and the amine solution are added simultaneously to the crystallisation vessel. The crystallisation can further be improved by applying a temperature profile, a variable speed of addition of reactants and/or a variable ratio of reactants during addition. This method, which has not been described earlier, results in a more economical attractive process as the use of a cosolvent has been minimized or even avoided completely. Summary of the invention The present invention provides a process for the crystallisation of an amine salt of clavulanic acid involving the addition of clavulanic acid in an organic solvent to a solution of amine preferably in the same organic solvent. According to another embodiment, also a process containing simultaneous addition of a solution containing clavulanic acid and a solution of the amine, is provided. Both processes optionally include the application of a temperature profile (decreasing in time), profile of speed of addition (increasing in time) and/or profile of ratio amine/clavulanic acid (increasing in time). According to another aspect of the invention the advantageous conversion of the amine clavulanate formed according to anyone of these processes into pure clavulanic acid or a pharmaceutically acceptable salt or ester thereof, has been provided. Description of the invention The essence of the crystallisation process according to the present invention comprises of placing a solution of amine in a solvent, optionally containing a small volume of cosolvent in a crystallisation vessel. Subsequently, a solution of clavulanic acid in a solvent, preferably the same solvent, optionally containing a small volume of cosolvent is added to the vessel. By applying this method the use of a cosolvent has been minimized. This results in a more economical attractive process as the volume of solvents decreases significantly. Also, if no cosolvent is used at all, recycling of the mother liquid is possible and/or facilitated because no additional process is needed to separate a mixture of solvents. The 5 invention decreases also pollution effects, as currently mixtures of solvents might be disposed off. Also in case a cosolvent is needed, normally the volume thereof can be reduced significantly compared to the process in which the amine solution is added to the extract containing clavulanic acid instead of the other way around. Recycling of the mother ιo liquid wil be economically attractive, even in case of azeotropic mixtures. The volumes used depend on the solvent and cosolvent applied. For instance, in case ethylacetate is used as solvent, it is preferred to use ethanol in such a volume that V ethanol /V ethyl acetate is less than 0. 1 5, preferably less than 0.1 0. i s A further embodiment of the invention is the simultaneous and separate addition of a solution of clavulanic acid in a solvent and an amine optionally dissolved in a solvent, preferably the same solvent, optionally containing a small amount of cosolvent. Optionally, before starting the addition, a small quantity of either the solvent, the cosolvent, the 20 clavulanic acid solution in the solvent, the amine or a combination of the solutions can be placed in the crystallisation vessel. Also, the ratio of the amount of clavulanic acid added to the amount of amine added can be varied in time. For example, the concentration of amine relative to that of clavulanic acid can be increased in time in this way, which might improve 25 the crystal quality. Both in case of the addition of a solution of clavulanic acid to the amine and in case of the simultaneous addition of both reactants to a crystallisation vessel, optionally a cosolvent may be added to one or both of the reactant solutions. It is of course in both cases also within the 30 scope of the invention that this cosolvent is added as a third stream, besides the reactants, optionally also in case cosolvent has been added to one or both of the reactants. Crystallisation occurs upon stirring and controlling the temperature between - 50 and + 50 °C, and more specifically between 0 and 40 °C. It is also possible to create a temperature profile, i.e. for instance a higher temperature (T = 20-40 °C) during the addition of clavulanic acid or during the simultaneous addition of clavulanic acid and amine solution followed by cooling down directly after the first crystal formation or during the ageing of the crystals (T = 0-1 0 ° C) . In this way, the quality of the crystals can be improved. The period of addition may vary between 2 and 1 20 minutes, preferably between 30 and 60 minutes. According to another aspect of the invention, a profile of addition velocity may advantageously be applied, i.e. slowly until the first crystals are formed after which the addition velocity may be increased. For instance, 1 -1 0% of the volume may be added in about 50% of the addition time, while the rest of the volume may be added during the later 50%. In this way, formation of lumps is prevented. Furthermore, seeding material may be added to the vessel before addition of the clavulanic acid solution. The concentration of clavulanic acid in the solvent may vary between 1 0 and 1 00 g/l, preferably between 1 5 and 50 g/l. The overall molar excess of amine related to clavulanic acid should be between 1 .2 and 2.4. Preferably, the organic solvent comprises an aliphatic carboxylic ester or a substantially water-immiscible aliphatic ketone, such as an acetate as ethyl acetate, methyl acetate, propyl acetate, or butyl acetate, or a methyl ethyl ketone, methyl isobutyl ketone or an n-butyl alcohol. Suitable co-solvents are alcohols such as methanol, ethanol, propanol, isopropanol, butanol and isobutanol and ketones such as acetone, propanone, etc. Suitable amines are for instance N,N,N,N- tetramethylethylenediamine, 1 ,3-bis(di-methylamino)-2-propanol, N,N'- diisopropylethylenediamine, t-butylamine, t-octylamine, benzhydrylamine and bis (2-(dimethyl-amino)ethyl)ether. Optionally, a purification step can be applied before or after the amine crystallisation, such as an adsorption. Another advantage of the process of the invention which contributes to an increased economically process involves the recovery of the mother liquid, which can be reintroduced in the process, e.g. during the extraction phase. Optionally, the liquid is purified before reintroduction, e.g. by adsorption or evaporation. In case a small cosolvent volume is used, which formes an azeotrope with the solvent, the azeotropic mixture can either be discharged, after which the solvent is reused, or the azeotropic mixture is reused as such in the subsequent crystallisation. Preferably, the amine and clavulanic acid present in the solvent are recovered prior to recycling the solvent. This can be done for example by extraction with water at low or high pH, by ion exchange or by crystallisation. Thus, according to the present invention, a possible process for the preparation of a pharmaceutically acceptable salt or ester of clavulanic acid can be fermentation of a clavulanic acid producing micro-organism followed by separation of the biomass by (membrane) filtration, optionally after addition of a water miscible solvent or a flocculant. Then, optionally the filtrate can be concentrated, e.g. by reverse osmosis or evaporation. Subsequently, the clavulanic acid is extracted to an organic solvent at low pH. Another possibility of simultaneous biomass removal and extraction is whole broth extraction after acidification. After the extraction, the solution can be purified e.g. by adsorption and coal treatment. Optionally, the extract can be concentrated before or after purification, e.g. by evaporation. Then the amine crystallisation can take place as mentioned above, followed by conversion into purified clavulanic acid by acidifying or into a pharmaceutically acceptable salt or ester clavulanate by adding a source of the corresponding salt or ester. Optionally, before this final crystallisation, a recrystallisation of the amine clavulanate can be performed. Finally, purified clavulanic acid or the pharmaceutically acceptable ester or salt clavulanate is separated from the solution. The following examples will illustrate the invention further. Example 1 Crystallisation of bis(2-(dimethylamino)ethyl)ether clavulanate from a clavulanic acid solution in ethyl acetate and conversion thereof into potassium clavulanate The following experiment was carried out in nitrogen atmosphere. A volume of 1 000 ml ethyl acetate containing clavulanic acid, was mixed during 2 hours with 1 55 gram Norit SX ultra at T = 0-2 ° C. Then the solution was filtrated and washed with 400 ml of ethyl acetate. The wash solution was added to the filtrate, leading to a clavulanic acid concentration of 40 g/l. 500 ml of the carbon treated solution was added to 350 ml of ethyl acetate which contained 1 1 .2 gram of 2-(dimethylamino)ethyl)ether. The solution was stirred during 1 hour at T = 0 °C. The crystals were separated by filtration and washed with 400 ml of ethyl acetate. After drying the wet cake volume in nitrogen atmosphere at room temperature, 32.2 grams of product was obtained, with a purity of 90.5 %. Purification of bis(2-(dimethylamino)ethyl)ether clavulanate by recrystallisation 20 Grams of these crystals were dissolved in 1 40 ml of a mixture of ethanol (87%) and water ( 1 3%) . 2 Grams of Norit SX Ultra were added at T = 0-2 ° C and the solution was stirred during 1 5 minutes. After filtration, the cake was washed twice with 50 ml of the ethanol-water mixture. Then during 30 minutes the filtrate was added to 1 500 ml of acetone and the solution was stirred during 1 hour at 1 0 ° C. The crystals were separated using filtration and the cake was washed twice with 50 ml of acetone. After drying the crystals in nitrogen atmosphere at room temperature, 1 5.73 g of product was obtained, with a purity of 96.3%. Crystallisation of potassium clavulanate using recrystallised bis(2- (dimethylamino)ethyDether clavulanate 1 5 Grams of these crystals were suspended in 375 ml of acetone and 3.75 ml of water, at a temperature of 1 0 °C. Then, during circa 25 minutes 220 ml of a 0.34 M potassium 2-ethyl hexanoate solution in acetone was added. The suspension was stirred for an hour at 1 0 ° C after which the crystals were separated by filtration. The cake was washed using 3 cake volumes of acetone. Then the crystals were dried in nitrogen atmosphere at room temperature. In this way, 1 2.32 g of potassium clavulanate was produced, with a purity of 96.4 %. Example 2 Crystallisation of bis(2-(dimethylamino)ethyl)ether clavulanate from a clavulanic acid solution in ethyl acetate at different temperatures The following experiment was carried out under nitrogen atmosphere. A volume of 700 ml of ethyl acetate containing circa 60 g/l of clavulanic acid, was mixed during 2 hours with 1 06 g Norit SX ultra at a temperature of 0-2 °C. Then the solution was filtrated and washed with 265 ml of ethyl acetate. The wash solution was added to the filtrate, leading to a clavulanic acid concentration of 40 g/l. This solution was divided into 5 portions of 1 00 ml each. Each portion was added in circa 20 minutes to 70 ml of ethyl acetate containing 2.25 g 2- (dimethylamino)ethyl)ether. The solutions were stirred during 1 hour at a temperature of -40 °C, -20 °C, 0 ° C, 20 ° C and 40 ° C, respectively. The crystals were separated by filtration and washed with circa 1 00 ml of ethyl acetate. After drying the wet cake in nitrogen atmosphere at room temperature, the mass of the product was 7.83, 7.97, 7.79, 7.64 and 7.44 g, respectively. The purities were 86.7, 86.3, 87.7, 87.7 and 89.6%, respectively. Example 3 Crystallisation of bis(2-(dimethylamino)ethyl)ether clavulanate from a clavulanic acid solution by simultaneous addition of clavulanic acid and amine The following experiment was carried out under nitrogen atmosphere. A volume of 1 00 ml of ethyl acetate was added to an empty crystallisation vessel at a temperature of 1 0 °C. Then in circa 20 minutes simultaneously a solution of 1 1 .2 g of 2-(dimethylamino)ethyl)ether in 350 ml of ethyl acetate and a 500 ml solution of ethyl acetate containing clavulanic acid (ca 40 g/l) were added to the crystallisation vessel. The temperature was decreased to 5 °C and the solution was stirred during 1 .5 hours. The crystals were separated by filtration and washed with circa 500 ml of ethyl acetate. After drying the wet cake in nitrogen atmosphere at room temperature, the mass of the product was 28.04 g. Example 4 Crystallisation of bis(2-(dimethylamino)ethyl)ether clavulanate from a clavulanic acid solution by simultaneous addition of clavulanic acid and amine using a profile for both speed of addition and temperature. The following experiment was carried out under nitrogen atmosphere. A volume of 250 ml of dry ethyl acetate was added to an empty vessel. Then, in approximately 5 minutes under vigorous stirring simultaneously 2 vol-% of 1 000 ml of a solution of clavulanic acid in ethyl acetate, with a concentration of ca 40 g/l of clavulanic acid, and 2 vol-% of a solution of 22.9 grams of amine in 250 ml of ethyl acetate are added. The temperature during addition was 37° C. After crystallisation the remaining 98 voi-% of both clavulanic acid solution and amine solution were added in ratio in approximately 5 minutes. In the meantime, cooling was started to reach 1 8 °C at the time the addition of the solutions was finished. Cooling was proceeded until 0 - 5 °C and the crystal slurry was stirred at this temperature for one hour. The crystals were separated by filtration and washed twice with 1 00 ml of ethyl acetate. The crystals were dried under vacuum atmosphere at room temperature to yield 64.32 grams. The purity of the crystals was 87.9% and the yield was 99.5%. Example 5 Crystallisation of bis(2-(dimethylamino)ethylether clavulanate from a clavulanic acid solution by simultaneous addition of clavulanic acid and a solution of amine using 8% ethanol as a co-solvent and conversion thereof into potassium clavulanate. The following experiment was carried out under Nitrogen atmosphere. A volume of 1 50 ml of dry ethyl acetate was added to an empty vessel. 0.6 grams of bis(2-(dimethylamino)ethylether were added as seeding crystals. Then, in approximately 60 minutes 1 200 ml of a solution of clavulanic acid in ethyl acetate (ca 40 g/l), and a solution of 29.4 g of amine in 1 50 ml of ethanol (99.5%) and 300 ml of ethyl acetate were added to the stirred vessel. The temperature during the addition was 20 °C. The crystal slurry was cooled to 1 0° C and immediately after reaching this temperature the crystals were collected by filtration. The crystals were washed with ethyl acetate. The crystals were dried under a vacuum atmosphere at room temperature to yield 76.32 grams. The purity of the crystals was 95.7 % and and the yield was 1 00%. Purification of bis(2-(dimethylamino)ethyl)ether clavulanate by recrystallisation 65 Grams of these crystals were dissolved in 433 ml of a mixture of ethanol (87%) and water ( 1 3%) . The solution was filtrated through an active carbon containing filter plate followed by a 0.45 μm filter. During filtration the temperature was kept a 0 - 5 ° C. The filters were rinsed with 81 ml of the ethanol/water mixture. The filtrates were collected in an empty vessel. Then, during one hour, 3088 ml " of acetone was added while the temperature was kept at 1 0 ° C. After addition the temperature was decreased to 5 °C and the crystal suspension was stirred during one hour at this temperature. The crystals were collected by filtration, the crystals were washed with acetone. Crystallisation of potassium clavulanate using recrvstallised bis(2- (dimethylamino)ethyDether clavulanate The wet recrystallised bis(2-(dimethylamino)ethylether clavulanate crystals were suspended in 1 437 ml of acetone and 1 4.4 grams of water. Then, during one hour, 847 ml of 0.34M potassium 2-ethyl hexanoate in acetone was added. The temperature during the addition was 1 0°C. The suspension was stirred for one hour at 1 0°C after which the crystals were collected by filtration. The crystals were washed with acetone. The crystals were dried under a vacuum atmosphere to yield 47.29 grams of potassium clavulanate with a purity of 96.0%. Example 6 Crystallisation of bis(2-(dimethylamino)ethylether clavulanate from a clavulanic acid solution by simultaneous addition of clavulanic acid and a solution of amine at different temperatures using 8% ethanol as a co- solvent. The following experiments were carried out under Nitrogen atmosphere. A volume of 100 ml of dry ethyl acetate was added to an empty vessel and 0.4 grams of bis(2-(dimethylamino)ethylether were added as seeding crystals. Then, in approximately 60 minutes 800 ml of a solution of clavulanic acid in ethyl acetate, containing ca 40 g/l, and a solution of 1 9.8 g of amine dissolved in 100 ml of ethanol (99.5%) together with 200 ml of ethyl acetate were added to the stirred vessel. The temperatures during the addition were, respectively 40, 30 and 20°C. The crystal suspensions were cooled to 1 0°C and immediately after reaching this temperature the crystals were collected by filtration. The crystals were washed with ethyl acetate. The crystals were dried under a vacuum atmosphere at room temperature to yield 53.59 , 53.96 and 54.87 grams, respectively. The purities of the crystals were 91 .7%, 90.5% and 90.6%, respectively. Example 7 Crystallisation of bis(2-(dimethylamino)ethylether clavulanate from a clavulanic acid solution by simultaneous addition of clavulanic acid and a solution of amine using different amounts of ethanol as co-solvent. The following experiments were carried out under Nitrogen atmosphere. A volume of 1 00 ml of dry ethyl acetate was added to an empty vessel and 0.4 grams of bis(2-(dimethylamino)ethylether were added as seeding crystals. Then, in approximately 60 minutes 800 ml of a solution of clavulanic acid in ethyl acetate, containing ca 40 g/l, and a solution of 1 7.5 g amine dissolved in, respectively 1 00, 75, 50, 25 and 0 ml ethanol (99.5%) together with, respectively 200, 225, 250, 275 and 300 ml of ethyl acetate were added to the stirred vessel. The temperature during the addition was 20°C. The crystal suspensions were cooled to 1 0°C and immediately after reaching this temperature the crystals were collected by filtration. The crystals were washed with ethyl acetate. The crystals were dried under a vacuum atmosphere at room temperature to yield 47.1 7 , 47.94 , 48.37, 48.99 and 49.73 grams, respectively. The purities of the crystals were 90.0%, 89.9%, 89.7%, 88.3% and 87.0%, respectively.

Claims: CLAIMS 1 . A process for the preparation of an amine salt of clavulanic acid by: • fermentation of a clavulanic acid producing microorganism; • extraction of the clavulanic acid containing fermentation broth with a water immiscible organic solvent resulting in an extracted solution; • optionally purification by adsorption; • conversion of clavulanic acid into an amine salt thereof; • separation of the amine salt of clavulanic acid from the solution, characterized by addition of the clavulanic acid containing extracted solution to the amine optionally dissolved in a water immiscible organic solvent, with one or both of the streams optionally containing a small volume of cosolvent which is miscible with the solvent, or simultaneous addition of the clavulanic acid containing extracted solution and the amine optionally dissolved in a water immiscible organic solvent, with one or both of the streams optionally containing a small volume of cosolvent which is miscible with the solvent, to a crystallisation vessel to prepare the amine salt of clavulanic acid. 2. A process according to claim 1 , characterized by varying the temperature from 20-40 °C during the addition of the clavulanic acid containing extracted solution and optionally the amine to the crystallisation vessel and lowering the temperature to 0-1 0 °C upon ageing of the crystals formed. 3. A process according to claim 1 or 2, characterized by starting with a slow addition of the clavulanic acid containing extracted solution and optionally slow addition of the amine to the crystallisation vessel until the first crystals are formed followed by increasing the addition velocity. 4. A process according anyone of the claims 1 , 2 or 3, characterized in that upon simultaneous addition of clavulanic acid and amine in the beginning a low amount of amine compared to a higher amount of clavulanic acid is added which is changed to a higher amount of amine compared to clavulanic acid during the addition. 5. A process according to anyone of the claims 1 - 4 wherein as amine anyone of the amines N,N,N',N'-tetramethylethylenediamine, 1 ,3-bis (dimethylamino)-2-propanol, N,N'-diisopropylethylenediamine, t- butylamine, t-octylamine, benzhydrylamine and bis (2- (dimethylamino)ethyl) ether is applied. 6. A process according to anyone of the claims 1 - 5 wherein solids are removed from the clavulanic acid containing fermentation broth before extraction, preferably by filtration or centrifugation, optionally upon adding a flocculation agent to the fermentation broth. 7. A process according to anyone of the claims 1 - 6, wherein an alcohol or ketone acts as cosolvent. 8. A process according to claim 7, wherein methanol, ethanol, propanol, isopropanol, butanol, isobutanol, acetone or propanone has been used. 9. A process according to anyone of the claims 1 - 8, characterized by extraction of the clavulanic acid containing broth with an acetate. 10. A process according to claim 9 wherein the amine has been dissolved in the same acetate. 1 1 . A process according to claim 9 or 1 0 wherein the acetate is preferably ethyl acetate. 1 2. A process for the preparation of isolated clavulanic acid or a pharmaceutically acceptable salt or ester of clavulanic acid by conversion of an amine clavulanate solution formed according to the process of anyone of the claims 1 - 1 1 , optionally after recrystallisation, into purified clavulanic acid by acidifying or by conversion into a pharmaceutically acceptable salt or ester clavulanate by adding a source of the corresponding salt or ester, followed by separation of the clavulanic acid or pharmaceutically acceptable salt or ester clavulanate formed. 1 3. A process for the preparation of potassium clavulanate according to claim 1 2 characterized by simultaneous addition of the extracted solution of clavulanic acid in ethyl acetate and an amine, preferably bis (2-(dimethylamino)ethyl)ether or benzhydrylamine, dissolved in ethyl acetate optionally containing ethanol in such a volume that V ethano i ethy i acetate is ,ess tnan 0.1 5%, to a crystallisation vessel, optionally recrystallisation and conversion of the clavulanate salt of said amine with potassium ethylhexanoate into potassium clavulanate.

Inventors: KERKHOF PIETER THEODORUS, LEFFERTS AGEETH GEERTRUIDA, KERKHOF, PIETER, THEODORUS, LEFFERTS, AGEETH, GEERTRUIDA
Applicants: DSM NV, KERKHOF PIETER THEODORUS, LEFFERTS AGEETH GEERTRUIDA, DSM N.V., KERKHOF, PIETER, THEODORUS, LEFFERTS, AGEETH, GEERTRUIDA
Application year: 1999
Main classification: C07D503
IPC codes: C07D503, C12P17