JP2005013238A - Method for producing pravastatin sodium substantially free of pravastatin lactone and epi-pravastatin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel method for purifying pravastatin. <P>SOLUTION: The method for isolation and purification of pravastatin or pharmaceutically permissible salts thereof comprises salting out with an ammonium salt. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to statins and more particularly pravastatin sodium and a method for isolating it as an enzymatic hydroxylation product of compactin from culture broth.

  Statins are currently the most therapeutically effective drugs available to reduce LDL levels in the bloodstream of patients at risk for cardiovascular disease. This class of drugs specifically includes compactin, lovastatin, simvastatin, fluvastatin and atorvastatin.

Pravastatin is a compound [1S- [1α (β ^* , δ ^* ) 2α, 6α, 8β (R ^* ), 8aα]]-1,2,6,7,8,8a-hexahydro-β, δ, 6- Trihydroxy-2-methyl-8- (2-methyl-1-oxobutoxy) -1-naphthalene-heptanoic acid (CAS Registry Number 81093-370) is the common pharmaceutical name. The molecular structure of pravastatin is represented by the formula (Ia) (where R = OH). The form of the lactone is represented by formula (Ib) (having atoms labeled to indicate the order of the atoms).

Pravastatin, compactin (formula Ia, R = H), lovastatin (formula Ia, R = CH ₃ ), simvastatin, fluvastatin and atorvastatin are each terminated by a carboxylic acid and have two hydroxyls in the β and δ positions relative to the carboxylic acid Has an alkyl chain with groups. The carboxylic acid group and hydroxyl group at the δ position tend to lactonize as shown in formula (Ib). Lactonizing compounds such as statins may exist in free acid form or lactone form, or as an equilibrium mixture of both forms. Lactonization results in difficulty in the production of statin drugs because the free acid and lactone forms of the compound have different polarities. The method of producing one mold is easy to remove the other mold together with impurities, which results in a low yield. Therefore, in order to isolate them in high yield, they must generally be carried out very carefully when dealing with compounds that are lactonized.

  Currently, the most economically available method for producing pravastatin is hydroxylation by the C-6 position microorganism of compactin. Enzymatic methods are very stereoselective, but are common for pravastatin sodium obtained after isolation from cultures that may incorporate significant amounts of pravastatin C-6 epimer (“epiprava”) It is. Since the C-6 position is a bis-allyl position, the C-6 atom is easily epimerized. Careful adjustment of the pH and other conditions during the isolation of pravastatin are required to minimize epimerization.

  None of the known methods of isolating pravastatin from the culture medium is unsuitable for isolating pravastatin as its sodium salt, or produces pravastatin sodium contaminated with significant amounts of pravastatin lactone and / or epiprava . The present invention meets the need in the art for an efficient method of isolating pravastatin sodium from culture broth in high purity, high yield, preliminary scale and without chromatographic purification.

SUMMARY OF THE INVENTION The present invention provides pravastatin sodium substantially free of pravastatin lactone and epiprava, the C-6 epimer of pravastatin. The present invention further provides a process that can be carried out on an industrial scale for producing such substantially pure pravastatin sodium.

  Preferred embodiments of the method include extraction of pravastatin from an aqueous culture medium into an organic solvent, back extraction of pravastatin into a basic aqueous solution, and re-extraction into an organic solvent, so that the initial concentration of pravastatin in the culture medium In comparison results in an organic solution rich in pravastatin. Pravastatin can be obtained from a solution enriched by precipitation as its ammonium salt and subsequent purification by recrystallization of the ammonium salt. The recrystallized salt is subsequently transposed to form pravastatin sodium salt, and excess sodium ions are trapped by the ion exchange resin. The sodium salt of pravastatin can then be isolated in highly pure form from solution by recrystallization, lyophilization or other means.

  The present invention provides pravastatin sodium substantially free of pravastatin lactone and epiprava, and a method for isolating pravastatin sodium salt from culture broth in high purity.

Enzymatic hydroxylation of compactin The enzymatic hydroxylation medium from which pravastatin is isolated may be any aqueous medium known for the cultivation of compactin on an industrial scale, and such methods include: U.S. Pat. Nos. 5,942,423 and 4,346,227. Preferably, the enzymatic hydroxylation is performed using a living Steptomyces broth comprising a nutrient mixture of compactin and dextrose.

  If the culture is neutral or basic at the completion of the fermentation, an acid is added thereto to bring the culture to a pH of about 1-6, preferably 1-5.5, and more preferably 2-4. Acids that can be used include hydrochloric acid, sulfuric acid, trifluoroacetic acid or any other protic acid, preferably those having a pH of less than 1 as a 1 M solution in water. Acidification of the culture medium converts any pravastatin carboxylate in the culture medium to the free acid and / or lactone.

Isolation of substantially pure water pravastatin sodium Pravastatin is first isolated from an aqueous culture in a relatively highly concentrated organic solution by a series of extraction and back-extraction steps.

In the first stage, pravastatin is extracted from the culture. C ₁ -C ₄ alkyl esters of C ₂ -C ₄ formic acid salt and C ₂ -C ₄ carboxylic acid alkyl can perform pravastatin efficient extraction from an aqueous solvent solution. The alkyl group may be linear, branched or cyclic. Preferred esters are ethyl formate, n-propyl formate, i-propyl formate, n-butyl formate, s-butyl formate, i-butyl formate, t-butyl formate, methyl acetate, ethyl acetate, n-propyl acetate, i-acetate Propyl, n-butyl acetate, s-butyl acetate, i-butyl acetate, t-butyl acetate, methyl propionate, ethyl propionate, n-propyl propionate, i-propyl propionate, i-propyl propionate, propionic acid Includes butyl, methyl butyrate, ethyl butyrate, n-propyl butyrate, i-propyl butyrate, butyl butyrate, methyl isobutyrate, ethyl isobutyrate, propyl isobutyrate and butyl isobutyrate.

  Among these preferred organic solvents, we have found that ethyl acetate, i-butyl acetate, propyl acetate and ethyl formate are particularly well suited. The most preferred extraction solvent is i-butyl acetate. Other organic solvents may also be exchanged with the ester. Halogenated halocarbons, aromatics, ketones and ethers such as dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, butyl methyl ketone, diethyl ether and methyl t-butyl ether can also be used.

Pravastatin is optionally back extracted into a basic solution with a pH of about 8.0 to about 9.5. The base is preferably NaOH, NH ₄ OH or KOH, most preferably NaOH. The extraction solvent is preferably determined by thin-layer chromatography or any other method that involves subjective determination that sufficient contact has occurred for complete extraction, in the amount of pravastatin in the organic layer. In some cases it is contacted with a basic aqueous solution until it is substantially depleted. Multiple back extractions can be performed for optimal recovery. However, when the organic layer is i-butyl acetate, one back extraction is sufficient. Back extraction can be used to concentrate pravastatin by using an amount of aqueous base that is less than the amount of the organic extract. Preferably, the back extraction is contacted with a basic aqueous solution in an amount less than 1/3 of the amount of organic extract, more preferably less than 1/4, and most preferably less than about 1/5 of the amount of organic extract. Is done.

  The aqueous solution is preferably acidic to a pH of about 1.0 to about 6.5, more preferably about 2.0 to about 3.7, using an acid, preferably trifluoroacetic acid, hydrochloric acid, sulfuric acid, acetic acid, or phosphoric acid, more preferably sulfuric acid. It becomes.

  Pravastatin is preferably re-extracted into one of the organic solvents already described as suitable for extracting pravastatin from the culture. The organic solvent may be, but is not necessarily, the same solvent used to extract pravastatin from the culture medium. In this re-extraction, further concentration of pravastatin is preferably about 50% (v / v), more preferably about 33% (v / v) to about 20% (v / v) of the aqueous extract, and more More preferably, it can be achieved by re-extraction to a smaller amount of organic solvent than an aqueous extract of about 25% (v / v). Pravastatin can be concentrated from 100 L culture to 8 L concentrated organic solution in 89% yield from the original organic extract.

Those skilled in the art will appreciate that a high yield of purified pravastatin, which describes only one extraction in a preferred embodiment for the practice of the present invention, can be achieved by performing multiple extractions. This preferred embodiment provides a balance between solvent economy and high product yield. Deviations from this preferred form, which are described here only once, which further enhances the yield by repeated extraction, do not necessarily depart from the spirit of the present invention. Prior to the procedure of obtaining pravastatin from the concentrated organic solution by “salt folding”, the concentrated organic solution is preferably dried, which is a conventional desiccant such as MgSO ₄ , Na ₂ SO ₄ , CaSO ₄ , silica, perlite. Etc., and is optionally decolorized using activated carbon. The dried and / or decolorized concentrated organic solution is preferably subsequently separated in a conventional manner, for example by filtration or decantation.

In the next step, pravastatin can be salted out of the concentrated organic solution using ammonia or amine. The amine may be a primary, secondary or quaternary amine. Any alkyl or amine that is not hindered to prevent ionic interactions between the amine nitrogen and the carboxyl group of pravastatin may be used. The amines include, but are not limited to, methyl, dimethyl, trimethyl, ethyl, diethyl, triethyl and other C ₁ -C ₆ primary, secondary, and includes a quaternary amine; and further morpholino, N- methyl Contains morpholine, isopropylcyclohexylamine, piperidine and the like. Salts formed by reaction of ammonia or amines, whether with or without substitution on nitrogen, are hereinafter referred to as ammonium salts. This meaning is intended to include salts of amines and salts of ammonia.

Precipitation of the ammonium salt of pravastatin can also be induced by addition of the ammonium salt alone or in combination with ammonia or an amine. Preferred ammonium salts are the following ammonia salts: NH ₄ Cl, NH ₄ Br, NH ₄ I, (NH ₄ ) ₂ SO ₄ , NH ₄ NO ₃ , (NH ₄ ) ₃ PO ₄ , (NH ₄ ) ₂ S ₂ O ₄ and NH ₄ OAc, most preferred is NH ₄ Cl. Ammonium salts and high boiling liquids and solid amines can be added by conventional means, preferably in well ventilated areas, as solids, neat liquids or solutions in aqueous or organic solvents. The addition of gaseous ammonia requires special equipment to handle caustic gases. Such equipment, such as pressure vessels, regulator valves, valves and lines are widely available. In a particularly preferred embodiment, pravastatin is obtained from the concentrated organic solution as a pravastatin salt of ammonia by addition of gaseous ammonia and NH ₄ Cl to the concentrated organic solution.

  The temperature at which the ammonia, amine and / or ammonium salt is to be added can be determined by routine experimentation by conducting small scale experiments and monitoring the exothermic nature of the reaction. Preferably, the temperature of the solution is not allowed to exceed 40 ° C. Although temperatures on the order of 80 ° C. can be experienced without significant degradation of pravastatin, many organic solvents of the present invention boil at lower temperatures. When using ammonia, the preferred temperature is from about -10 ° C to about 40 ° C.

  Preferably, the addition should be terminated once it appears that the precipitation is complete, or that the consumption of some pravastatin has been substantially completed by other means. If ammonia or volatile amines are used, the container is preferably ventilated to disperse excess odor. The crystals can then be isolated by filtration, solvent decantation, solvent evaporation or other such method, preferably filtration. The crystals can then be washed, preferably with i-butyl acetate and acetone.

  After optionally washing the precipitated crystals, the pravastatin ammonium salt is preferably purified by one or more, most preferably three recrystallizations. In order to purify pravastatin ammonium salt, the salt is preferably dissolved in water. The polarity of the solution is preferably reduced by the addition of an antisolvent. The antisolvent is preferably a water-soluble organic solvent or a mixture of solvents in which pravastatin salt is hardly dissolved, and i-butyl acetate and acetone are preferred.

The pravastatin salt may be left to recrystallize spontaneously, or recrystallization may be introduced by performing further steps of adding common ions. According to a preferred method in which pravastatin is purified as its ammonia salt, NH ₄ Cl is added to induce recrystallization of the ammonium salt.

  The recrystallization can be carried out at about −10 ° C. to about 40 ° C., preferably about 0 ° C. to about 40 ° C. After the pravastatin salt is substantially recrystallized from solution, the crystals are isolated and may be washed, for example, with a 1: 1 mixture of i-butyl acetate and acetone and dried. Drying may be carried out at ambient temperature, but is demonstrated by raising the temperature preferably to a temperature below 45 ° C, preferably about 40 ° C. Recrystallization may optionally be repeated until good results are produced as shown in Examples 3 and 4. Each repetition occurs with a yield of about 92%.

  After purification of pravastatin ammonium salt, pravastatin ammonium salt is preferably replaced with pravastatin sodium. Pravastatin is preferably dissolved in an aqueous solvent and acidified to a pH of about 2 to about 4, more preferably about 3.1, using any protic acid, but preferably sulfuric acid, and using an organic solvent Is extracted from the ammonium salt by extracting pravastatin. Any of the organic solvents listed above, but preferably an organic solvent, preferably i-butyl acetate, is optionally contacted with the acidified solution until pravastatin is substantially completely transferred to the organic layer. After the organic layer is preferably separated from the aqueous layer and optionally washed with water to remove ammonium residues, pravastatin preferably uses an aqueous sodium hydroxide solution with a pH of about 7.4 to about 13.0. And back-extracted. The back extraction is preferably performed at a low temperature of about 8 to about 10 ° C.

  After extraction into aqueous sodium hydroxide, excess sodium cations are trapped to achieve approximately 1: 1 sodium cation and pravastatin equilibrium using a water-insoluble ion exchange resin. Suitable ion exchange resins are cationic and chelate type resins, preferred are strong and weak acid exchange resins.

Among strong acid cation exchange resins, those having a sulfonic acid (SO ₃ .H ⁺ ) group can be used. These are commercially available Amberlite ™ IR-118, IR-120 252H; Amberlyst ™ 15, 36; Amberjet 1200 (H) (Rohm and Haas), Dowex ™ 50WX series, Dowex HCR-W2, Dowex 650C , Dowex Marathon C, Dowex DR-2030, and Dowex HCR-S, ion exchange resins (Dow Chemical Co.); DIAION SK 102 to DIAION SK 116 resin series and Lewatit SP 120 (Bayer). Preferred strong acid cation exchange resins are Amberlite ™ 120, Dowex 50WX and DIAION SK series.

  Weak acid cation exchange resins include those having pendant carboxylic acid groups. Weak acid cation exchange resins include commercially available Amberlite CG-50, IRP-64, IRC50 and C67, Dowex CCR series, Lewatit CNP series (Bayer) and DIAION WK series (Mitsubishi), of which the most preferred Are Amberlite ™ IRC50, Lewatit CNP 80 and DIAION WK 10. Less preferred are chelate type exchange resins. Some of the many commercially available ones include Amberlite ™ IRC-718 and IRC-467.

The solution containing pravastatin sodium salt and excess sodium cation can be obtained by any method known to those skilled in the art, for example by passing the solution through a column or bed of resin or in a flask containing the solution with a sufficient amount of resin. By stirring, it can be brought into contact with the ion exchange resin. The form of contact is not essential. After capture of excess sodium ions, the pH of the pravastatin sodium solution will vary with dilution, but should be in the range of about 7 to about 10, preferably about 7.4 to about 7.8. A decrease in the pH of the pravastatin sodium solution from a higher pH to a lower pH, followed by a lower level of pH stability, indicates substantial completion of excess Na ⁺ ion capture. After capture is substantially complete, the pravastatin sodium solution is preferably separated from the resin in a conventional manner. It may be recovered as eluent from the column or bed, or may be separated by filtration, decantation, and the like.

Pravastatin sodium may be isolated from pravastatin sodium solution by crystallization. Efficient crystallization may initially require partial removal of water, which may be performed by vacuum distillation or nanofiltration. Preferably, the aqueous solution of pravastatin sodium is concentrated to about 20 to about 50 w / v% prior to crystallization. If necessary, after concentration, the aqueous pravastatin sodium solution can be adjusted to a pH of about 7 to about 10 using an ion exchange resin of the H ⁺ form.

Addition of a water-soluble organic solvent or mixture of organic solvents to the pravastatin sodium solution aids crystallization. In particular, mention may be made of acetone and acetone / acetonitrile, ethanol / acetonitrile and ethanol / ethyl acetate mixtures. One of the most preferred solvent systems for crystallizing pravastatin sodium is formed by concentrating pravastatin sodium solution to about 30 w / v% and then adding the appropriate amount of 1/4 acetone / acetonitrile mixture. 1/3/12 water / acetone / acetonitrile mixture. The most preferred crystallization solvent mixture is water-acetone (1:15).
Pravastatin sodium can also be isolated by lyophilization of an aqueous solution of pravastatin sodium.

  Whether isolated by lyophilization or crystallization or other means that does not impair the purity of the product, pravastatin sodium isolated in the practice of the method of the present invention is substantially free of pravastatin lactone and epiprava. As shown in the examples below, pravastatin sodium can be isolated with less than 0.5% (w / w) pravastatin lactone contamination and less than 0.2% (w / w) epiprava contamination. Pravastatin sodium is further reduced to less than 0.2% (w / w) pravastatin lactone and less than 0.1% (w / w) epiprastatin by complying with preferred embodiments of the invention, two exemplified in Examples 1 and 3. Can be isolated.

  The highly pure pravastatin sodium produced by the method of the present invention is preferably useful for the treatment of hypercholesterolemia and can be administered to mammalian patients by any route of administration for this purpose. A regimen of daily oral administration is the most preferred method of administration. In human patients with normal liver function and moderate body weight, a decrease in serum cholesterol levels is typically observed with an oral daily dose of 10 mg or more pravastatin sodium. The highly pure pravastatin sodium administered may optionally be an effective amount. Preferred oral dosage forms of the present invention comprise about 10 mg to about 40 mg pravastatin sodium. Oral dosage forms include tablets, pills, capsules, troches, sachets, suspensions, powders, lozenges, elixirs and the like. Although substantially pure pravastatin sodium can be administered by any route, the most preferred route of administration is oral.

  Highly pure pravastatin can be administered either alone or in combination with pharmaceutical excipients. Whether given alone or in the composition, the highly pure pravastatin sodium of the present invention may be in the form of a solution or solid, such as a powder, granule, aggregate or any other solid.

  The composition of the present invention includes a composition for tableting. A tableting composition may have a few or many excipients, depending on the tableting method used, the desired release rate and other factors. For example, the composition of the present invention can be prepared by using a diluent such as cellulose-derived material, such as powdered cellulose, fine cellulose, microfine cellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxy Methyl cellulose salts and other substituted and unsubstituted celluloses; starches; pregelatinized starches; inorganic diluents such as calcium carbonate and diphosphate and other diluents known to those skilled in the art. Still other suitable diluents include waxes, sugars and sugar alcohols such as mannitol and sorbitol, acrylate polymers and copolymers, and pectin, dextrin and gelatin.

  Other tableting excipients are binders such as gum acacia, pregelatinized starch, sodium alginate, glucose and other binders used in wet and dry granulation and direct compression tableting methods including. Excipients that may be present in the novel form solid composition of pravastatin sodium are further disintegrants such as sodium starch glycolate, crospovidone, low substituted hydroxypropylcellulose and others; lubricants such as stearin Magnesium and sodium and sodium stearyl fumarate; fragrances; sweeteners; preservatives; pharmaceutically acceptable pigments and glidants such as silicon dioxide.

  Capsule dosage forms comprise a solid composition within a capsule that may be made from gelatin or other encapsulating material. Tablets and powders can be coated. Tablets and powders can be coated with an enteric coating. Enteric coated powder forms include cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, polyvinyl alcohol phthalate, carboxymethylethylcellulose, copolymers of styrene and maleic acid, copolymers of methacrylic acid and methyl methacrylate, and as appropriate It has a coating comprising a material that can be utilized for various plasticizers and / or bulking agents. The coated tablet may have a coating on the tablet, or it may be a tablet comprising a powder or granule with an enteric coating.

Highly pure pravastatin sodium can also be administered in injectable dosage forms as solutes or suspended solids in sterile solutions or suspensions. Suitable carriers for sterile injectable forms include water and oil.
The following examples illustrate its implementation in some of the embodiments of the present invention, but such examples should not be considered as limiting the scope of the invention. Other aspects will be apparent to one skilled in the art from consideration of the specification and examples. It is intended that the specification, including examples, be considered exemplary only and that the spirit and scope of the invention be indicated by the appended claims.

Example
Example 1. Pravastatin purified culture (100 L) was acidified to about 2.5 to about 5.0 by addition of sulfuric acid. The acidified culture was extracted with i-butyl acetate (3 × 50 L). The yield of i-butyl acetate extract was found to be 95% by HPLC analysis calibrated against the internal standard of the culture. The combined i-butyl acetate layers were subsequently extracted with water (35 L) brought to about pH 7.5 to about pH 11.0 by the addition of concentrated ammonium hydroxide.

The resulting pravastatin aqueous solution was subsequently reacidified to a pH of about 2.0 to about 4.0 by addition of 5 M sulfuric acid and back extracted with i-butyl acetate (8 L). Resulting pravastatin acetate i- butyl solution was partially dried over Perlite and Na ₂ SO _4. The pravastatin solution was decanted and then filtered from the desiccant and decolorized with activated carbon (1.7 g). The solution was subsequently filtered and transferred to a flask equipped with a gas inlet with the activated carbon removed.

  Ammonia gas was introduced into the headspace above the rapidly stirred solution. Precipitated crystals of pravastatin ammonium carbonate are recovered by filtration and washed with i-butyl acetate followed by acetone, so that about one-half as determined by HPLC equipped with a UV absorptiometer measuring at λ = 238 nm. A pravastatin ammonium salt of 94% purity was produced.

Pravastatin ammonium salt was further purified by crystals from a saturated ammonium chloride solution as follows. Pravastatin salt containing 162 g of active substance was dissolved in water (960 ml) and diluted at 35-40 ° C. with acetone (96 ml) and i-butyl acetate (96 ml). The solution is cooled to about 30-32 ° C. and pravastatin ammonium is induced to crystallize by the addition of solid NH ₄ Cl, which does not cause an apparent increase in crystal formation upon further addition It was continued until. After the addition of ammonium chloride, the solution was cooled to about 0-26 ° C. Pravastatin ammonium crystals were collected by filtration and washed with i-butyl acetate and acetone and subsequently dried at about 40 ° C. as before. The resulting pravastatin ammonium salt crystals (155.5 g) were obtained with a purity of about 98% as determined by HPLC applying the aforementioned conditions.

Pravastatin ammonium salt was further purified by another crystal below. Pravastatin ammonium salt (155.5 g of active substance) was dissolved in water (900 ml). Isobutanol (2 ml) was added and the pH was raised to about pH 10 to about pH 13.7 by addition of concentrated sodium hydroxide solution and the solution was stirred at ambient temperature for 30 minutes. This solution was neutralized to a pH of about 7 by the addition of sulfuric acid, and crystallization of pravastatin ammonium was induced by the addition of solid NH ₄ Cl. Crystals (150 g) were recovered by filtration and washed with acetone. Pravastatin ammonium was found to be about 99.3% by HPLC determination using the conditions described above.

  Pravastatin ammonium was subsequently replaced by the sodium salt as follows. The crystals of pravastatin ammonium salt were dissolved in water (1800 ml). I-Butyl acetate (10.5 L) was added. This solution was acidified to a pH between about pH 2 and about pH 4 by the addition of sulfuric acid, thereby returning pravastatin to its free acid. The i-butyl acetate layer containing pravastatin was washed with water (5 × 10 ml). Pravastatin is subsequently converted to its sodium salt and an i-butyl acetate solution in about 900-2700 ml of water with 8 M NaOH added halfway until a pH of about pH 7.4 to about pH 13 is reached. Was extracted back into another aqueous layer by stirring.

The pravastatin sodium salt solution was subsequently treated with an ion exchange resin to trap excess sodium cations. After separation, the aqueous layer was stirred for 30 minutes on IRC of H ⁺ ion exchange resin. Stirring was continued until a pH of about pH 7.4 to about pH 7.8 was reached.

  This solution was subsequently filtered to remove the resin and partially concentrated to a weight of 508 g under reduced pressure. Acetonitrile (480 ml) was added and stirred over activated carbon (5 g) to decolorize the solution. Pravastatin sodium is 90% by crystallization after adding acetone and acetonitrile to form a 1/3/12 water / acetone / acetonitrile mixture (5.9 L) with cooling to about −10 to about 0 ° C. Was obtained as crystals in a yield of. Pravastatin sodium was obtained in 65% overall yield, about 99.8% purity from the active material produced by the starting culture as measured by HPLC using the conditions described above.

Example 2.
According to the procedure of Example 1 except that recrystallization from a water / acetone / acetonitrile mixture is omitted, pravastatin sodium is obtained in about 99% purity and about 72% yield by lyophilization of a concentrated aqueous solution of pravastatin sodium. Obtained.

Example 3.
According to the procedure of Example 1, pravastatin sodium was obtained in a purity of about 99.8% and a yield of 68.4%, except that the pravastatin ammonium salt was further purified by repeating the crystallization of pravastatin ammonium salt once.

Example 4.
According to the procedure of Example 1, pravastatin sodium was obtained in about 99.6% purity and 53% yield, except that the pravastatin ammonium salt was further purified by repeating the crystallization of pravastatin ammonium salt twice.

Example 5.
Following the procedure of Example 1, the culture (100 L) was acidified to a pH of about 2.5 to about 5.0 by the addition of sulfuric acid. The acidified culture was extracted with i-butyl acetate (3 × 50 L). The combined i-butyl acetate layers were extracted with water (35 L) basified to a pH of about pH 7.5 to about pH 11.0 by addition of concentrated ammonium hydroxide.
Instead of acidifying the aqueous extract again and extracting with i-butyl acetate to obtain a more concentrated solution as done in Example 1, the aqueous extract was concentrated under reduced pressure to 140 g / L. The resulting concentrated solution was subsequently acidified to a pH of about pH 4.0 to about pH 7.5 by addition of 1M HCl.

  Ammonium chloride crystals (405 g) were subsequently added to the concentrated solution and pravastatin ammonium salt crystallized upon standing at ambient temperature. The crystals were subsequently isolated by filtration and washed with a saturated solution of ammonium chloride. Subsequently, the crystals were added to 40 ° C. water (1 L). After dissolution, the temperature was lowered to 30 ° C. and ammonium chloride (330 g) was added to the solution. The solution was subsequently stirred for 15 hours at ambient temperature and the crystals of pravastatin ammonium salt were recovered by filtration and washed with i-butyl acetate followed by acetone and dried. The resulting crystals were further purified by recrystallization followed by replacement with the sodium salt and isolated as described in Example 1. Pravastatin sodium was obtained with a purity of about 99.9% and a yield of 67.7%.

Example 6.
According to the procedure of Example 1, pravastatin sodium salt is 1/15 of water / 15% in total yield and 99.8% purity from the active substance produced by the starting culture as measured by HPLC. Crystallized from an acetone mixture.

Example 7.
According to the method of the first two paragraphs of Example 5, a concentrated aqueous extract (140 g / L) was obtained. The concentrated aqueous extract was divided into equal parts.

Example 8.
According to the procedure of Example 1, pravastatin ammonium salt was isolated from the culture medium, but the active substance was dissolved and crystallized after precipitation with ammonia gas.
The concentrated pravastatin i-butyl acetate solution (6500 L) was decolorized with activated carbon (6.5 kg). Subsequently, the solution was filtered to remove the activated carbon and transferred to a vessel equipped with a gas inlet. The solution contained 183.2 kg of active substance.
Pravastatin ammonium salt was precipitated using ammonia gas according to the procedure of Example 1.

The precipitated pravastatin ammonium salt was dissolved by adding water (1099 L) to the vessel in the presence of i-butyl acetate mother liquor.
Pravastatin ammonium salt was crystallized by adding ammonium chloride (412 kg) to the vessel. Ammonium chloride was added for 5 hours at 31 parts at 30-32 ° C. The suspension was stirred at 24-26 ° C. for 1 hour. The crystals are filtered, suspended in i-butyl acetate and filtered, then suspended in i-butyl acetate: acetone (2: 1) and filtered, followed by suspension in acetone and filtration. did. The crystals were dried under reduced pressure after washing with acetone.
The method produced pravastatin ammonium salt that was approximately 93% pure as determined by HPLC with UV detection at I = 238 nm.

Example 9.
According to the procedure of Example 1, pravastatin ammonium salt was isolated from the culture medium, but crystallization was used instead of precipitation with ammonia gas.
Concentrated i-butyl acetate solution of pravastatin (4150 ml) was decolorized with activated carbon (4.15 g). The solution was filtered to remove the activated carbon and transferred to a flask.
Water (300 ml) was added to the i-butyl acetate solution. The pH was adjusted to 9.36 using concentrated ammonia solution (27 ml).

Pravastatin ammonium salt was crystallized by adding ammonium chloride (121.5 g) to the flask. Ammonium chloride was added in additional amounts at 30-32 ° C. for 5 hours. The suspension was stirred at 24-26 ° C. for 15 hours. The crystals were filtered, suspended multiple times, washed and dried.
This method produced pravastatin ammonium salt of about 95% purity as determined by HPLC. The active substance crystallized was 42.7 g.

Example 10.
According to the procedure of Example 8, pravastatin ammonium salt was produced with a purity of about 93%.
The active substance (10 g) was dissolved at 35-40 ° C. in a mixture of water (60 ml): acetone (6 ml): isobutyl acetate (6 ml). The solution was cooled to 30-32 ° C. Ammonium chloride (22 g) was added further to this solution for 5 hours.
The suspension was cooled to 24-36 ° C. and it was stirred for 1 hour, followed by filtration of the pravastatin ammonium salt and washing with isobutyl acetate followed by acetone.
Pravastatin ammonium salt was dried at 40 ° C. The yield was 96%. The purity was 97%.

Example 11.
According to the procedure of Example 8, pravastatin ammonium salt was produced with a purity of about 93%.
The active substance (10 g) was dissolved in a mixture of water (60 ml): acetone (6 ml), isobutyl acetate (6 ml) at 35-40 ° C. The solution was cooled to 30-32 ° C. Sodium chloride (11.4 g) was further added to this solution for 3 hours.
Pravastatin sodium salt was filtered and washed with isobutyl acetate and then with acetone, followed by drying at 40 ° C.
The yield was 77%. The purity was 97%.

Example 12.
According to the procedure of Example 8, pravastatin ammonium salt was produced with a purity of about 93%.
The active substance (10 g) was dissolved in a mixture of water (60 ml): acetone (6 ml): isobutyl acetate (6 ml) at 35-40 ° C. The solution was cooled to 30-32 ° C. Lithium chloride (9.3 g) was used for crystallization salt folding.
The filtered pravastatin lithium salt was washed with isobutyl acetate and dried.
Pravastatin lithium salt was obtained in 89% yield at 96% purity.

Claims (8)

  A method for isolating and purifying pravastatin or a pharmaceutically acceptable salt thereof by salting out using an ammonium salt.   The method according to claim 1, wherein the salt used for the salting out is ammonium chloride.   The method of Claim 1 or 2 which further implements the process of reducing an impurity with a base.   The method according to claim 1, wherein the step of reducing impurities with an acid is further performed.   The method of claim 4, wherein the acid is sulfuric acid.   6. The method according to claim 4 or 5, wherein the pH in the step for reducing impurities by acid is 2-5.   The method according to claim 4, wherein impurities are reduced by decomposition.   The method according to claim 5, wherein impurities are reduced by decomposition.