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WO2006058943A1 - Procede d'obtention de pravastatine sodique purifiee - Google Patents

Procede d'obtention de pravastatine sodique purifiee Download PDF

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Publication number
WO2006058943A1
WO2006058943A1 PCT/ES2005/000645 ES2005000645W WO2006058943A1 WO 2006058943 A1 WO2006058943 A1 WO 2006058943A1 ES 2005000645 W ES2005000645 W ES 2005000645W WO 2006058943 A1 WO2006058943 A1 WO 2006058943A1
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Prior art keywords
pravastatin
sodium
purified
solution
obtaining
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PCT/ES2005/000645
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English (en)
Spanish (es)
Inventor
Felipe Requena Perez
Luis Angel Diaz Tejo
Ramón ASENSIO DOMINGUEZ
Fernando Garcia Chapinal
Mª Carmen CRUZADO RODRIGUEZ
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Ercros Industrial SA
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Ercros Industrial SA
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Publication of WO2006058943A1 publication Critical patent/WO2006058943A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/48—Separation; Purification; Stabilisation; Use of additives

Definitions

  • the present invention relates to a new method for the isolation and purification of pravastatin sodium, from a fermentation broth.
  • the object of the invention is to provide a process that allows obtaining pravastatin sodium, from a fermentation broth, in a simple, fast and at the same time economical way, thereby obtaining a high quality final product. Procedure that is easy to scale industrial and very clean from an environmental point of view.
  • the present invention falls within the scope of the pharmaceutical industry.
  • Pravastatin is a well known compound, belonging to the family of so-called statins, which are compounds that inhibit the enzyme HMG-CoA reductase. This enzyme acts as a catalyst for the mevalonic acid formation reaction, which is the limiting stage of cholesterol biosynthesis.
  • Pravastatin chemically referred to as 3,5-dihydroxy-7- [6-hydroxy-2-methyl-8- (2-methylbutyryloxy) -1, 2,6,7,8,8a-hexahydro-l acid -naphthyl] heptanoic, is used as an antihypercholesterolemic agent in the form of sodium salt, and has the following formula:
  • Pravastatin was initially isolated from canine urine as a compactin metabolite during a study of compactin metabolism (Tanaka and cois .; unpublished results), and subsequently structurally elucidated. (Haruyama.H and cois; Chemical & Pharmaceutical
  • This compound can also be obtained by enzymatic hydroxylation of the compactin, through the use of strains of various microorganisms, as shown in US Patent 4346227 of the Sankyo company, in which, in addition, its properties are described as inhibitor of the enzyme HMG-CoA reductase .
  • pravastatin is extracted from the fermentation broth at acidic pH with ethyl acetate, previously separating the biomass by filtration.
  • a catalytic amount of trifluoro acetic acid is added to the Pravastatin extract obtained to the Pravastatin extract obtained to the Pravastatin extract obtained to the Pravastatin extract obtained to the Pravastatin extract obtained to the Pravastatin extract obtained to the Pravastatin extract obtained to the Pravastatin extract obtained is added a catalytic amount of trifluoro acetic acid, neutralized with sodium bicarbonate and concentrated to obtain a residue of pravastatin in its lactone form, which is purified by HPLC.
  • a method of purifying a crude HMG-CoA reductase inhibitor by displacement chromatography is described in US Patent 6695969.
  • the HMG-CoA reductase inhibitor is dissolved in a mobile phase and seeded on a column. Said column is washed with a second mobile phase that displaces the HMG-CoA reductase inhibitor through the column. Fractions that are analyzed by HPLC are collected and the HMG-CoA reductase inhibitor is obtained by lyophilization or recrystallization.
  • US 5616595 provides a process for the isolation of different water-insoluble compounds, which are present in a fermentation broth, by tangential filtration.
  • the above method is applicable to a wide variety of - A - compounds, among which is pravastatin.
  • the fermentation broth is passed through a filter cyclically, so that it loses water, until the desired concentration is achieved.
  • the desired compound is isolated from the filtrate by extraction with a solvent, subsequently subjecting it to a purification process.
  • pravastatin sodium is obtained from the tert-butylamine salt, by dissolving said salt in ethanolic sodium hydroxide solution or in aqueous sodium carbonate solution.
  • pravastatin can be converted into its corresponding secondary amine salt by the addition of a suitable amine. After recrystallization, this pravastatin amine salt is transformed into sodium salt by the addition of sodium ethoxide. By recrystallization or lyophilization, pravastatin sodium is obtained.
  • pravastatin sodium after the extraction of the broth at acidic pH, consists in re-extracting the pravastatin contained in the organic phase of ethyl acetate or butyl acetate, in the form of sodium salt by contact with a solution aqueous alkaline
  • a subsequent purification by chromatography on non-ionic adsorbent resin allows the sodium pravastatin obtained to be of high purity.
  • EPI 190087 describes pravastatin isolation methods similar to those of the previous patent, with the difference that they refer to fermentation broths of the Micromonospora strain.
  • Pravastatin is separated from the fermentation broth by adsorption in an anionic resin or by extraction with an organic solvent immiscible in water, followed by its conversion into its lactone form or into a secondary amine salt or by purification of the aqueous alkaline extract obtained from the organic extract from the fermentation broth, by chromatography on a non-ionic resin.
  • pravastatin sodium is removed by treating the aqueous solution of pravastatin sodium with a water-insoluble cation exchange resin, for example, with a weak acid cationic resin, Amberlite IRC-50. Finally, the isolation of pravastatin sodium is produced by lyophilization or recrystallization.
  • Patent application WO 0232847 describes a method of purification of an aqueous solution of pravastatin sodium, called “ salting out. " The fermentation broth is extracted at acidic pH with ethyl acetate and re-extracted by contact with an aqueous solution of sodium hydroxide.
  • pravastatin sodium To the aqueous solution of pravastatin sodium thus obtained, various inorganic salts are added, for example, sulfate, nitrate, or ammonium acetate or sodium chloride and the resulting mixture is heated to 20-60 ° C. After allowing to cool to 0 or C precipitation of pravastatin sodium is achieved with a purity of about 90%.
  • inorganic salts for example, sulfate, nitrate, or ammonium acetate or sodium chloride and the resulting mixture is heated to 20-60 ° C. After allowing to cool to 0 or C precipitation of pravastatin sodium is achieved with a purity of about 90%.
  • patent application WO 0232848 provides another method of isolation and purification of pravastatin sodium from a fermentation broth.
  • the procedure consists in separating the mycelium and adding an inorganic base to the filtered broth, for example sodium hydroxide, and refluxing at basic pH.
  • an extraction of pravastatin at acidic pH is carried out using propyl acetate or butyl acetate.
  • the enriched organic phase is re-extracted with an aqueous solution of sodium hydroxide at pH 8-9.
  • To this aqueous phase of pravastatin sodium is added an inorganic acid and heated at a temperature of between 20 and 50 ° C and at pH between 2 and 5.
  • High purity pravastatin is isolated by subsequent crystallization.
  • fermentation broths contain a large number of substances, necessary for the fermentation process, which, however, constitute potential impurities of the active ingredients.
  • fermentation broths generally have very low concentrations of the active substance, which forces the treatment of large volumes of broth and hinders the subsequent isolation of the active substances.
  • Part of the problem is solved through the application of successive extraction and re-extraction operations, to obtain organic or aqueous solutions enriched with the active ingredients, while achieving partial removal of impurities contained in the fermentation broth.
  • subsequent purification operations are usually required by chromatographic methods or isolation of intermediate products to obtain an active principle of the quality required in the pharmaceutical industry.
  • the process object of the present invention comprises the formation of an enriched aqueous solution of pravastatin from a fermentation broth, the obtaining and isolation of raw sodium pravastatin or calcium pravastatin, and its conversion into pravastatin sodium purified, as can be seen in the following scheme:
  • the process that the invention provides begins with the formation of an enriched aqueous solution of pravastatin from a fermentation broth.
  • the fermentation broth of pravastatin is adjusted to basic pH, preferably between 8.5 and 9.5, by using an aqueous solution of an inorganic base, for example sodium hydroxide.
  • a polyamine type coagulant preferably cationic and a cationic or anionic polyacrylamide type flocculant, is added. Coagulant addition is optional and can also be done prior to the pH adjustment of the fermentation broth.
  • the separation of the fermentation broth is achieved, by centrifugation.
  • the clarification of the fermentation broth through the use of coagulants and flocculants and the consequent centrifugation has important competitive advantages over other methods of separating the mycelium, such as conventional filtration, for example, by a filter press. These advantages are: reduction in solid-liquid separation times, with a broth flow rate of 10,000 1 / h, higher separation yields, greater than 97%, and obtaining a cleaner filtered broth, which favors extraction later of the broth with an organic solvent, since the formation of emulsions in the liquid-liquid interface is avoided.
  • the broth is adjusted to an acidic pH, preferably between 3.5 and 5.5, by using inorganic or organic acids, preferably inorganic, and more preferably hydrochloric acid, sulfuric acid or phosphoric acid.
  • Pravastatin is extracted from the acidified broth "by ü ⁇ ⁇ solvent with a high yield, so that an organic solution of the compound.
  • the solvent used is an organic solvent immiscible in water, such as alkyl carboxylic esters, halogenated hydrocarbons and ketones, such as ethyl acetate, propyl acetate, butyl acetate, methylene chloride and methyl isobutyl ketone Preferred solvents are ethyl acetate and methyl isobutyl ketone.
  • the next operation to achieve the formation of the enriched aqueous solution of pravastatin is the re-extraction of the organic solution by contact with a basic aqueous solution, in the form of alkaline or ammonium salt.
  • the base used is an alkali hydroxide or ammonium hydroxide, preferably sodium hydroxide.
  • the pH of the basic aqueous solution must be between 8 and 13, preferably between 9 and 11.
  • Pravastatin is preferably extracted in the form of sodium salt, while achieving the elimination of potential non-polar organic impurities.
  • the aqueous extract is concentrated to obtain an enriched aqueous solution of pravastatin.
  • This concentration of the aqueous extract is carried out by partial distillation under vacuum of water, so that the removal of a residual volume of organic solvent is also achieved.
  • the procedure proposed by the invention continues with a step for obtaining and isolating raw sodium pravastatin or calcium pravastatin from the previously enriched aqueous pravastatin solution obtained.
  • a carboxylic acid group capable of forming salts such as pravastatin
  • the process for the isolation of a given salt of said compound, with good purity is difficult.
  • the isolation "of the" active ingredient in aqueous medium since, usually alkali metal salts and ammonium are water soluble. Therefore, the formation and isolation of ammonium salts or amine salts, commonly used to purify pravastatin, is carried out from an enriched organic phase of the compound.
  • the enriched aqueous pravastatin extract of the present invention obtained from a fermentation broth, can be extracted with a water immiscible organic solvent, to obtain an enriched pravastatin organic solution.
  • the solvents used for extraction are alkyl esters of carboxylic acid, halogenated hydrocarbons and ketones, such as ethyl acetate, propyl acetate, butyl acetate, methylene chloride and methyl isobutyl ketone.
  • Preferred solvents are ethyl acetate and methyl isobutyl ketone.
  • the extraction is carried out after an adjustment at acidic pH, preferably between 3.5 and 5.5, by using inorganic or organic acids, preferably inorganic acids, and more preferably hydrochloric acid, sulfuric acid or phosphoric acid.
  • raw sodium pravastatin of good purity can be obtained by adding a solution of sodium salt of carboxylic acid in low molecular weight alcohol, preferably a solution of sodium octanoate in methanol. After several hours of maturation at low temperature, preferably between 0 o C and 10 ° C, filtered raw pravastatin sodium is obtained using a filter press, for example.
  • calcium pravastatin can be obtained and isolated directly from the enriched pravastatin aqueous solution.
  • an aqueous solution of calcium chloride to the enriched aqueous solution of pravastatin, it is possible to isolate calcium pravastatin of good purity.
  • the method of obtaining pravastatin calcium salt is simple, since it precipitates in aqueous medium and is easily isolated by filtration, for example by means of a filter press.
  • the calcium salt of pravastatin can be used as an intermediate for obtaining pravastatin sodium of adequate purity.
  • the procedure described in the invention ends with obtaining of purified pravastatin sodium from raw pravastatin sodium or calcium pravastatin obtained.
  • raw pravastatin sodium or calcium pravastatin is suspended in water and, after adjusting the solution to acidic pH, pravastatin is extracted with an organic solvent immiscible in water, preferably with ethyl acetate or methyl isobutyl ketone.
  • the adjustment of the solution to acidic pH is carried out by using inorganic or organic acids, preferably inorganic acids, and more preferably hydrochloric acid, sulfuric acid or phosphoric acid.
  • the organic pravastatin extract may, optionally be discolored with activated carbon and washed with an aqueous saline solution, preferably with sodium chloride.
  • the process is continued with the addition of a solution of sodium salt of carboxylic acid in low molecular weight alcohol, preferably a solution of sodium octanoate in methanol, the precipitation of sodium pravastatin being observed.
  • a solution of sodium salt of carboxylic acid in low molecular weight alcohol preferably a solution of sodium octanoate in methanol
  • purified pravastatin is isolated by filtration.
  • Pravastatin sodium purified of the present invention may be recrystallized to obtain pravastatin pharmaceutical grade, by any of the methods known in the art, such as recrystallization from a mixture of ethanol and "acetate” I acetate, or "récristalizaciórT by addition of ethyl acetate to a solution of pravastatin sodium in ethanol and water.
  • Example 1 Obtaining raw pravastatin sodium from a fermentation broth.
  • pravastatin fermentation broth From an initial volume of 7 m 3 of pravastatin fermentation broth, it is diluted with 4 m 3 of water and 105 1 of coagulant is added. The broth is adjusted to pH 9.5 by the addition of 10% sodium hydroxide, and then a flocculant solution is added and the biomass is removed by centrifugation, obtaining 14 m 3 of broth.
  • the broth is adjusted to pH 3.5 with 10% hydrochloric acid and extracted with 3500 1 of methyl isobutyl ketone.
  • the organic pravastatin extract is re-extracted by contact with 1050 1 of an aqueous solution of sodium hydroxide at pH 9.5.
  • the aqueous extract is distilled under vacuum until an enriched aqueous solution of pravastatin is obtained.
  • the enriched aqueous solution is adjusted to pH 4. with hydrochloric acid and extracted with 600 1 of methyl isobutyl ketone.
  • the organic phase is washed with a saturated solution of sodium chloride and decanted.
  • a solution of 11.5 kg of sodium octanoate dissolved in 100 1 of methanol is added to the organic phase and the mixture is completed with the addition of 500 1 of methyl isobutyl ketone.
  • the mixture is cooled to 5 o C and kept ripening for 5 hours. After this period, the mixture is filtered and the product obtained is washed with 50 1 of methyl isobutyl ketone. After drying under vacuum at 50 ° C, 21.5 kg of raw sodium pravastatin are obtained.
  • Example 2 Obtaining calcium pravastatin from a fermentation broth.
  • the broth obtained is adjusted to pH 4 with hydrochloric acid and extracted with 2,000 ml of ethyl acetate.
  • the organic pravastatin extract is re-extracted by contact with 350 ml of aqueous sodium hydroxide solution at pH 9.5.
  • the aqueous extract is distilled under vacuum until an enriched pravastatin solution is obtained.
  • Example 3 Obtaining purified pravastatin sodium from calcium pravastatin. This process starts from 50 g of pravastatin calcium obtained previously. 1000 ml of ethyl acetate and 250 ml of water are added and the pH of the mixture is adjusted to 3.5 with phosphoric acid. After an hour of stirring, the organic phase, which contains pravastatin, is separated by decantation.
  • the mixture is allowed to cool to 5 o C and is kept ripening for a period of approximately 4 hours. After this time, the solid obtained is filtered and dried under vacuum. Finally, 40 g of purified pravastatin sodium are obtained.
  • Example 4 Obtaining raw sodium pravastatin from a fermentation broth.
  • the process begins with adjusting an initial volume of 5,000 ml of pravastatin fermentation broth to pH 9.5 by adding sodium hydroxide. Next, a polyamine type coagulant and a polyacrylamine type flocculant are added and the biomass is removed by centrifugation.
  • the broth obtained is filtered, adjusted to pH 4 with phosphoric acid and extracted with 2,000 ml of ethyl acetate.
  • This organic pravastatin extract is re-extracted by contact with 350 ml of aqueous sodium hydroxide solution at pH 9.5.
  • the new aqueous extract is distilled under vacuum until an enriched pravastatin solution is obtained.
  • To the enriched aqueous solution is added 350 ml of ethyl acetate and adjusted to pH 4 with hydrochloric acid.
  • the organic phase is then separated, decolorized with activated carbon and filtered.
  • 8 g of sodium octanoate dissolved in 80 ml of methanol are added and the mixture is completed with the addition of 500 ml of ethyl acetate.
  • the mixture is cooled to
  • Example 5 Obtaining purified pravastatin sodium from raw pravastatin sodium.
  • a sample of 15O g of raw pravastatin sodium is taken to which 2250 ml of ethyl acetate and 450 ml of water are added, subsequently the pH of the solution is adjusted to 3.4 with hydrochloric acid. After 15 minutes of stirring, the organic phase, which contains pravastatin, is separated by decantation.
  • the solution of pravastatin in ethyl acetate obtained is decolorized with 15 g of activated carbon, filtered and washed with 375 ml of 25% sodium chloride. Next, 67.5 g of sodium octanaoate dissolved in 675 ml of methanol are added on the organic phase and the solution is diluted with the addition of 4500 ml more of ethyl acetate.
  • the mixture is allowed to cool to 10 ° C and is maintained in maturation for a time of approximately 8 hours. After this period, the solid obtained is filtered and dried under vacuum. 100 g of purified pravastatin sodium are thus obtained.
  • Example 6 Obtaining pharmaceutical grade sodium pravastatin from purified sodium pravastatin. Once the purified sodium pravastatin has been obtained, according to the previous example, 10 g of it are dissolved in 20 ml of ethanol and 10 ml of water, and the resulting solution is decolorized with activated carbon, then washed with 5 ml of a mixture of ethanol and water, and filtered.
  • the clarified solution is heated to 50 ° C and 420 ml of ethyl acetate is added, keeping the mixture under stirring for a period of approximately one hour.
  • the solution is cooled by maintaining it at a temperature of 10 ° C and is subjected to a maturation period for 2 hours. Then, the solid obtained is filtered and finally dried under vacuum at 50 ° C. As a final result, 6.7 g of pharmaceutical grade sodium pravastatin are obtained.
  • the pravastatin sodium obtained is subjected to a spectroscopic analysis, whose X-ray diffraction spectrum is represented in the figure that accompanies this description.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

L'invention concerne un procédé simple, rapide, économique et à simple échelonnement industriel, permettant l'obtention de pravastatine sodique purifiée à partir d'un bouillon de fermentation, consistant à former une solution aqueuse enrichie en pravastatine, à obtenir et isoler une pravastatine sodique brute ou une pravastatine calcique, et à la convertir en pravastatine sodique purifiée par suspension dans l'eau, à ajuster son pH de manière à obtenir un pH acide, à procéder à son extraction à l'aide d'un dissolvant organique comme l'acétate d'éthyle ou le méthyle isobutyle cétone, à ajouter une solution de sel sodique d'acide carboxylique dans de l'alcool de faible poids moléculaire, comme l'octanoate sodique dans du méthanol, et à isoler la pravastatine sodique purifiée par filtration, afin d'obtenir un produit final de qualité pharmaceutique, tout en procédant à une recristallisation éventuelle de la pravastatine sodique purifiée obtenue.
PCT/ES2005/000645 2004-11-29 2005-11-25 Procede d'obtention de pravastatine sodique purifiee Ceased WO2006058943A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200402866A ES2254028B1 (es) 2004-11-29 2004-11-29 Procedimiento para la obtencion de pravastatina sodica purificada.
ESP200402866 2004-11-29

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002030415A1 (fr) * 2000-10-05 2002-04-18 Biogal Gyogyszergyar Rt Pravastatine sel de sodium ne contenant sensiblement pas de pravastatine sous forme lactone ni d'epi-pravastatine, et compositions correspondantes
EP1452519A1 (fr) * 2003-02-25 2004-09-01 Balkanpharma-Razgrad AD Procédé d'isolation et purification de pravastatine sodique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002030415A1 (fr) * 2000-10-05 2002-04-18 Biogal Gyogyszergyar Rt Pravastatine sel de sodium ne contenant sensiblement pas de pravastatine sous forme lactone ni d'epi-pravastatine, et compositions correspondantes
EP1452519A1 (fr) * 2003-02-25 2004-09-01 Balkanpharma-Razgrad AD Procédé d'isolation et purification de pravastatine sodique

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ES2254028B1 (es) 2007-03-01
ES2254028A1 (es) 2006-06-01

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